Investigating mitochondrial and ER protein profiles of cells expressing SPTLC1 mutations
Scott Stimpson
Thesis submitted for the award of
Doctor of Philosophy
Supervisor: Dr. Simon Myers
Associate Supervisor: Prof. Jens Coorssen
Associate Supervisor: Assoc. Prof. Paul Witting
Neuro-Cell Biology Laboratory
Molecular Medicine Research Group
School of Science and Health
Western Sydney University
Australia
STATEMENT OF AUTHENTICATION
I Scott Stimpson declare that this thesis contains no material that has been accepted for the award of any other degree or diploma and that, to the best of my knowledge and belief, this thesis contains no material previously published or written by another person, except where due reference has been made in the text of this thesis.
August 2015
S.E. Stimpson BMedSci (Hons)
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ABSTRACT
Axonal degeneration is the final common path in many neurological disorders. It is
seen in its pure form in hereditary axonal neuropathies. The hereditary neuropathies
are the most common group of diseases. Subsets of neuropathies involving the
sensory neuron are known as hereditary sensory neuropathies (HSNs). Hereditary
sensory neuropathy type I (HSN-I) (the most common subtype of HSNs) is an autosomal dominant inherited disorder, characterised by the progressive degeneration of the dorsal root ganglion and with onset of clinical symptoms occurring between the second or third decade of life. Heterozygous mutations in the serine palmitoyltransferase (SPT) long chain subunit 1 (SPTLC1) have been identified as the cause of HSN-I.
In Paper I, we optimised an isolation method of mitochondria to allow the production of a full and in-depth proteomic profile to elucidate the molecular mechanisms
underlying mitochondrial (dys) function in HSN-I. Paper II, detailed examinations of a
small sub-set of proteins that were found to be altered in abundance within harvested
mitochondria from HSN-I mutant SPTLC1 cells. Comparison of mitochondrial protein
isolates from control and patient lymphoblasts, showed an increased abundance of
Ubiquinol Cytochrome C Reductase Core Protein 1, an electron-transport chain
protein, as well as the immunoglobulin, Ig Kappa Chain C. In, Paper III, endoplasmic
reticulum (ER) protein lysates from HSN-I patient and control lymphoblasts, were
examined leading to identification of changes in expression of five proteins; Hypoxia
Up regulated Protein 1, Chloride Intracellular Channel Protein 1, Ubiqutin-40s
Ribosomal Protein S27a, Coactosin and Ig Kappa chain C.
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Further investigations into mitochondrial and ER protein profiles were carried out in
Paper IV, which showed a number of proteins that were altered in their relative abundance using membrane and soluble isolation techniques. Further analyses of these identified changes were carried out and replicated in Paper V, which revealed and confirmed the changes in protein expression and abundance of proteins earlier identified in Papers I and II. Changes were identified in V144D mutations, as well as
C133W and C133Y mutations. All of which are implicated to be casual of HSN-I.
Lipid droplets and alterations of lipid metabolism are hallmarks of a variety autosomal
dominant neurodegenerative diseases, including Alzheimer’s and Parkinson’s
disease. Paper VI, revealed significant increases in the presence of lipid droplets in
HSN-I patient-derived lymphoblasts, indicating a potential connection between lipid
droplet formation and the molecular mechanisms of HSN-I.
In conclusion, this study has shown alteration in mitochondrial and ER protein profiles
in patient-derived lymphoblasts and in transfected neuronal cells expressing the
mutations V144D, C133W and C133Y. This investigation has contributed to the field
by identifying protein alterations which has yielded a more detailed and in-depth
analysis of the cellular and molecular mechanisms involved in HSN-I.
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THESIS STRUCTURE
The work presented in this thesis provides an investigation into mitochondrial and endoplasmic reticulum proteome changes caused by mutations in the SPTLC1 gene.
These investigations are provided as a series of six papers (listed below). These papers are either published (Paper I, II, III, IV & VI), or currently submitted to journals for peer-review (Paper V), and adverted to in the thesis text by their Roman numerals.
I. Stimpson, SE. Coorssen, JR. Myers, SJ. Optimal isolation of mitochondria for proteomic analyses. Analytical Biochemistry: Methods in Biological Sciences, 2015. doi:10.1016/j.ab.2015.01.005
II. Stimpson, SE. Coorssen, JR. Myers, SJ. Mitochondrial protein alterations in a familial peripheral neuropathy caused by mutations in the sphingolipid protein, SPTLC1. J Chem Biol, 2014. 8 (1):25-35. Doi: 10.1007/s12154-014-0125-x.
III. Stimpson, SE. Lauto, A. Coorssen, JR. Myers, SJ. Isolation and identification of ER associated proteins with unique expression changes specific to the V144D SPTLC1 mutations in HSN-I. Biochem and Anal Biochem. 2016; 5 (1)
IV. Stimpson, SE. Coorssen, JR. Myers, SJ. Proteome alterations associated with the V144D SPTLC1 mutation that causes Hereditary Sensory Neuropathy-I. Electronic J Biol, 2015; 11 (4): 176-186
V. Stimpson, SE. Coorssen, JR. Myers, SJ. Identifying unique protein alterations caused by SPTLC1 mutations in a transfected neuronal cell model. Proteomes. 2015 (Manuscript under Review).
VI. Marshall, LL*. Stimpson, SE*, Coorssen, JR and Myers, SJ. Increased lipid droplet accumulation associated with a peripheral sensory neuropathy. J Chem Biol. 2014; 7(2):67-76. Doi: 10.1007 (* Co-first authors).
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TABLE OF CONTENTS
Abbreviated list of contents………………………………………..……………………… vi Comprehensive list of contents………………….……………………..………………... vii
List of Figures………….………………………………………..……………..…….….. viii List of Tables ...…………………..…………………………..…………………...... viii Acknowledgements...... ix List of Abbreviations………………..……...... x
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TABLE OF CONTENTS 1.1 Hereditary Sensory Neuropathies ...... 1 1.1.1 What are hereditary sensory neuropathies? ...... 1 1.1.2 Clinical features of HSN-I ...... 5 1.1.3 Pathological features of HSN-I ...... 5 1.1.4 Genetics of HSN-I ...... 6 1.2 Intracellular Lipid Functions and Interactions ...... 7 1.2.1 The role of lipids in biological membranes ...... 7 1.2.2 Interactions of lipids with intracellular proteins and organelles ...... 9 1.2.3 Sphingolipids, their role in normal cellular homeostasis and in the neuronal cell ...... 11 1.3 Serine Palmitoyltransferase ...... 13 1.3.1 The structure and regulation of SPT and its subunits ...... 13 1.3.2 The role of the SPTLC1 protein in neurodegenerative disease ...... 14 1.4 Protein Functions and Interactions ...... 17 1.4.1 Altered protein expression and the disease state ...... 17 1.5 Mitochondria in neurodegenerative diseases ...... 19 1.5.1 Mitochondrial Dynamics ...... 19 1.5.2 Mitochondrial fusion and fission ...... 20 1.5.3 Mitochondrial changes in inherited peripheral neuropathies ...... 27 1.5.4 Examples of Mitochondrial changes in inherited peripheral ...... 29 Neuropathies ...... 29 1.5.5 Novel link to Mitochondria in HSN-I ...... 32 1.6 The Role of ER in neurodegenerative diseases ...... 33 1.6.1 Protein processing in the ER ...... 33 1.6.2 UPR: Unfolded protein response pathway ...... 37 1.7 Mitochondria-Associated Membranes ...... 41 1.7.1 MAM Calcium Regulation ...... 41 1.7.2 Autophagy formation at MAMs ...... 42 1.7.3 Lipid transportation via MAMs ...... 44 1.7.4 MAMs and Neurodegeneration ...... 46 1.8 Aims of this Thesis ...... 49 Paper I ...... 51 Paper II ...... 52 Paper III ...... 67
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Paper IV ...... 76 Paper V ...... 88 Paper VI ...... 121 General Discussion ...... 132 Future Directions ...... 149 References ...... 156
LIST OF FIGURES
Figure 1.1: Molecular structure of the plasma membrane...... 8 Figure 1.2: The pathway of sphingolipid metabolism...... 12 Figure 1.3: Theoretical model of SPT complex structure...... 14 Figure 1.4: Schematic model of mitochondrial fusion...... 22 Figure 1.5: Schematic diagram for the proposed new model of fission...... 24 Figure 1.6: Schematic diagram of mitosis-specific mitochondrial fission ...... 25 Figure 1.7: Overview of mitochondrial dynamics and homeostasis...... 26 Figure 1.8: Cotranslational targeting of secretory proteins to the ER...... 34 Figure 1.9: Posttranslational translocation of proteins into the ER...... 35 Figure 1.10: ER stress and the unfolded protein response...... 39
LIST OF TABLES
Table 1.1: Hereditary sensory neuropathies (HSNs) ...... 4 Table 1.2: The three subunits of SPT; SPTLC1, SPTLC2 and SPTLC3 ...... 14 Table 1.3: Neurodegenerative diseases that interfere with mitochondrial function ..31
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ACKNOWLEDGEMENTS
This thesis is the cumulative work of not only myself, but also of the numerous people
who have supported me and were supportive of me, during my degree. It has been
both whirlwind and arduous journey, of which I could not have travelled, if not for the
presence of my mentors and friends.
Firstly, I would like to thank my supervisors, Dr Simon Myers and Prof. Jens Coorssen.
Thank you both for all your time and effort throughout this entire project. Thanks also
go to Dr Chandra Malladi and Dr Elise Wright for their technical assistance on two-
dimensional gel electrophoresis and to Dr Cathy Luxford for your enduring guidance and support.
Secondly, I owe large “thank yous” to the following people for their moral support, intellectual discussions, technical assistance, motivation and friendship, Noor Jwad and Melissa Partridge. Thirdly, to Chris, thank you for always believing in me and supporting me through the good and bad times, for being my rock and guiding light.
To others whom I may have forgotten, I am sorry, but thank you for your contributions.
Finally, to my family, especially Mum and Dad, my gratitude extends beyond words.
Scott Stimpson August 2015
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ABBREVIATIONS
2DGE Two Dimensional Gel Electrophoresis
AD Alzheimer’s disease
ALS Amyotrophic Lateral Sclerosis
AMPIB Amresco Mitochondrial Protein Isolation Buffer
APP ß-Amyloid Precursor Protein
ATF4 Activating Transcription Factor 4
ATF6 Activating Transcription Factor 6
ATL3 Atlastin GTPase 3
ATG Autophagy-Related Proteins
ATP Adenosine Triphosphate
BiP Immunoglobulin Heavy Chain Binding Protein
C133W Cytosine to Tryptophan at Position 133
Ca2+ Calcium ion
CAG Cytosine-Adenine-Guanine
CDases Ceramidases
CH2 Collagen Homology Domain
CHO Chinese Hamster Ovary
CoA Coenzyme A
DCFP1 Double FYVE-Containing Protein 1
DNA Deoxyribonucleic Acid
DNMT1 DNA Methyl Transferase 1
DRG Dorsal Root Ganglion
DRP Dynamin-related Protein
DSBs Deoxy-Sphingoid Bases eIF2a Eukaryotic Translation Initiation Factor 2 Subunit a eIF5A Eukaryotic Translation Initiation Factor 5A-1
ER Endoplasmic Reticulum
ERAD Endoplasmic Reticulum Associated Degradation
FCCP Carbonyl Cyanide p-trifluoromethoxyphenylhydrazone
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Fis1 Mitochondrial Fission Protein 1
GED GTPase Effector Domain
GPI Glycosylphosphatidylinositol
GRB2 Growth Factor Receptor-Binding Protein 2
GTPase Guanine Triphosphatase hFis1 Mitochondrial Fission Protein 1
HIV-1 Human Immunodeficiency Virus
HSANs Hereditary Sensory and Autonomic Neuropathies
HSNs Hereditary Sensory Neuropathies
HSN-I Hereditary Sensory Neuropathy Type 1
HSN-II Hereditary Sensory Neuropathy Type 2
HSN-III Hereditary Sensory Neuropathy Type 3
HSN-IV Hereditary Sensory Neuropathy Type 4
HSN-V Hereditary Sensory Neuropathy Type 5
Hsp70 Heat Shock Protein 70
IKBKAP Inhibitor of Kappa Light Polypeptide Enhancer in B-cells, Kinase Complex Associated Protein
IL-1b Interleukin 1b
IMM Inner Mitochondrial Membrane
IMS Inner Membrane Space
IP3 1, 4, 5- Trisphosphate iPS Induced Pluripotent Stem Cells
IRE1 Inositol-Requiring Enzyme 1
LCB1 Long-Chain Base 1
MAM Mitochondrial Associated Membrane
Mff Mitochondrial Fusion Factor
Mfn2 Mitofusin 2
MIB Mitofusin Binding Protein
MiD49 Mitochondrial Dynamics Protein 49
MiD51 Mitochondrial Dynamics Protein 51
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MiEF1 Mitochondrial Elongation Factor 1 mTOR Mechanistic Target of Rapamycin
NGF Nerve Growth Factor
NGFB Nerve Growth Factor Beta
NLRs NOD-like Receptors
OCR Oxygen Consumption Rate
OMM Outer Mitochondrial Membrane
PDI Protein Disulphide Isomerase
PE Phosphatidylethanolamine
PERK Protein Kinase-Like ER Kinase
PINK1 PTEN-induced Putative Kinase 1
PKA Cyclic-AMP-Dependent Protein Kinase
PS Phosphatidylserine
PSD PS Decarboxylase
RALA Ras-Related Protein Ral-A
ROS Reactive Oxygen Species
RER Rough Endoplasmic Reticulum
RNase Endoribonuclease
ScaMc-1 Calcium Binding Mitochondrial Carrier Protein
SL Sphingolipids
SKs Sphingosine Kinases
SOD1 Superoxide Dismutase-1
S1P Sphingosine-1-Phosphate
SPT Serine Palmitoyltransferase
SPTLC1 Serine Palmitoyltransferase Long Chain Subunit 1
SPTLC2 Serine Palmitoyltransferase Long Chain Subunit 2
SPTLC3 Serine Palmitoyltransferase Long Chain Subunit 3
SR Sarcoplasmic Reticulum
SREBP Sterol Response Element Binding Protein
SRP Signal Recognition Particle
TCA Tricarboxylic Acid Cycle
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TEM Transmission Electron Micrograph
TRKA Tyrosine Kinase A Receptor
TT Transiently Transfect
UCH-L1 Ubiquitin C-Terminal Esterase L1
UPR Unfolded Protein Response Pathway
UVB Ultraviolet Light B
VDAC Voltage Dependent Anion Selective Channel Protein 1
VAPB Vesicle-Associated Membrane Protein B
V144D Valine to Aspartate at position 144
WNK With No Lysine Kinase
WNK1 Lysine Deficient Protein Kinase 1 XBP1 X-Box-Binding Protein
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1.1 Hereditary Sensory Neuropathies
1.1.1 What are hereditary sensory neuropathies?
Subsets of neuropathies involving the sensory neuron are categorised as hereditary
sensory neuropathies (HSNs) (Dyck et. al., 2005). These inherited nerve disorders in
which the sensory dysfunction of the neuron is most prevalent and involvement of the
autonomic system are referred to as hereditary sensory and autonomic neuropathies
(HSANs) (Dyck et. al., 2005). HSNs are associated with a range of clinical
presentations, pathologic alterations, electrophysiological abnormalities and
increasingly specific biochemical, molecular and/or genetic abnormalities, summarised in Table 1.1 (Dyck et. al., 2005). HSNs have also been described with sensorineural deafness, keratitis (inflamed corneas), ataxia (dysfunction of motor coordination), spasticity (altered skeletal muscle performance), dementia and mental retardation (Cavanagh, 1979; Janzer, 1986; Donaghy, 1978; Kherbaoui-Redouani,
2004). HSNs are a clinically and genetically heterogeneous group of disorders which are classified into five different HSN subtypes labelled HSN I-V. With the exception of
HSN type I, which has an autosomal dominant trait, HSN II-V are autosomal recessive traits (Verhoeven et. al., 2006). Molecular genetics research has shown that at least eight loci and six genes are associated with HSNs (Verhoeven et. al., 2006).
Hereditary sensory neuropathy type I (HSN-I) is the most common subtype of HSNs
(Dyck et. al., 2005). As stated HSN-I is autosomal dominant inheritance and is characterised by progressive degeneration of the dorsal root ganglion (DRG) and an onset of clinical symptoms between the second or third decade of life (Verhoeven et. al., 2004). HSN-I is rarely fatal but imposes lifelong disability with the disease initially manifesting with sensory loss in the feet, followed by distal muscle wasting and
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weakness and subsequent positive sensory phenomena (such as lancinating or
'shooting' pains). Heterozygous mutations in the serine palmitoyltransferase (SPT) long chain subunit 1 (SPTLC1) were identified as the pathogenic cause of HSN-I
(Bejaoui et. al., 2001; Dawkins et. al., 2001).
HSN-II, also known as Morvan disease, is an autosomal recessive, early onset and very severe disease with clinical symptoms appearing in early infancy (Verpoorten et. al., 2006). It is manifested with sensory loss affecting all modalities but with touch being most severely affected (Verpoorten et. al., 2006). A well-conserved 434- amino- acid open reading frame located within intron 8 of the WNK (With no-lysine-kinase) lysine deficient protein kinase 1 gene (WNK1) gene for HSN-II has been identified
(Rivie`re et. al., 2004; Roddier et. al., 2005).The mutations identified to date predict truncation of the protein, suggesting a complete loss of function (Verpoorten et. al.,
2006).
HSN-III, also known as Familial Dysautonomia or Riley–Day syndrome, is an autosomal recessive disorder that affects the development and survival of sensory, sympathetic and some parasympathetic neurons (Houlden et. al., 2004). HSN-III manifests a variety of symptoms, including decreased sensitivity to pain, vibration and temperature, cardiovascular instability, recurrent pneumonias, vomiting crises and gastrointestinal dysfunction (Houlden et. al., 2004). The HSN-III mutations were located to chromosome 9q31, with sequencing identifying two mutations causing HSN-
III with the major haplotype mutation located on intron 20 involving the inhibitor of
kappa light polypeptide enhancer in B-lymphocytes, kinase complex associated
protein (IKBKAP) (Houlden et. al., 2004).
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HSN-IV, Congenital Insensitivity to Pain with Anhidrosis, is an autosomal recessive
disorder manifesting with insensitivity to pain, recurrent febrile episodes, anhidrosis,
self-mutilating behaviour and mental retardation. The tyrosine kinase A receptor
(TRKA) gene was identified as causal for HSN-IV. Additional mutations were identified and found to be caused by a 1926- ins-T mutation in the TRKA gene (Houlden et. al.,
2004). The mechanism underlying the development of HSN-IV in families with TRKA mutations is currently unknown, but there are some in-vitro data implicating deficient
TRKA phosphorylation in neuronal and non-neuronal cells caused by mutations
(Houlden et. al., 2004).
HSN-V is a rare disorder, with very few reported cases. The disease is characterised by loss of deep pain perception and impaired temperature sensitivity, ulcers, and in some cases self-mutilation, with most other neurological functions including sweating intact (Einarsdottir et. al., 2004). The genetic background of HSN-V is still unclear; however Einarsdottir et. al., (2004) located a potential causative mutation on a conserved region of the nerve growth factor beta gene (NGF) that produces the HSN-
V phenotype (Einarsdottir et. al., 2004).
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Table 1.1 Hereditary sensory neuropathies (HSNs). HNSs are inherited nerve disorders in which the sensory dysfunction of the neuron is most prevalent with occasional involvement of the autonomic system. HSN-I is the most prevalent of these diseases. Adapted from (Verpoorten et. al., 2006)
HSN-I SPTLC1 9q22.2 Autosomal Adulthood Sensory loss in the feet, followed by distal Dominant muscle wasting and weakness and (Bejaoui et. al., 2001; subsequent positive sensory phenomena, Dawkins et. al., 2001) such as lancinating or 'shooting' pains.
HSN-II HSN2 12p13.3 Autosomal Childhood Distal sensory loss affecting all modalities, Recessive with touch most severely affected leading to (Verpoorten et. al., 2006) amputations. HSN-III IKBKAP 9q31 Autosomal Congenital Impairment of development and survival of Recessive sensory, sympathetic and some parasympathetic neurons manifests a variety of symptoms, including decreased sensitivity to pain, vibration and temperature, (Houlden et. al., 2004) cardiovascular instability, recurrent pneumonias, vomiting crises and gastrointestinal dysfunction HSN-IV TRKA 1q21-22 Autosomal Congenital Insensitivity to pain and temperature Recessive sensation with frequent bone and joint (Houlden et. al., 2004) fractures. HSN-V NGF 1p13.1 Autosomal Congenital Distal loss of pain and temperature sensation Recessive leading to ulcers and self-mutilation. (Einarsdottir, et. al., 2004)
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1.1.2 Clinical features of HSN-I
HSN-I is the most common and best characterised of the degenerative sensory disorders. Degeneration of the DRG and ventral horn neurons is typical with one report of amyloid-like material accumulation within the DRG (Denny-Brown, 1951). Loss of sensation results in painless injuries that when left untreated display slow wound healing and can develop into osteomyelitis, often requiring amputation (Dyck et. al.,
2005; Auer-Grumbach et. al., 2003).
As the disease progresses into advanced stages, motor involvement with distal muscle weakness and wasting becomes apparent and symptoms spread to the proximal limbs
(Auer-Grumbach et. al., 2008). These complications cause long-term disablement with economic and social repercussions. Currently there is no cure for HSN-I and treatment is entirely symptom-focused (Auger-Grumbach et. al., 2003).
1.1.3 Pathological features of HSN-I
The pathological features of HSN-I are associated with axonal degeneration ('dying back') of the peripheral sensory fibres (Dyck et. al., 2005). Significant reductions in all of the peripheral sensory fibres (small/large, myelinated/non-myelinated) in the distal extremities have been observed through post mortem and electrophysiological studies. Individual neurons undergo axonal atrophy sufficiently slowly that myelin wrinkling and remodelling occur and eventually these axons further degenerate into linear rows of myelin ovoids and balls (Dyck et. al., 2005). A decrease in corresponding cell bodies in the DRG and atrophy of the dorsal spinal tract together with lumbosacral spinal ganglion neurons, accompanies fibre loss (Denny-Brown, 1951). As the peripheral sensory axons progressively retract from their peripheral targets over time,
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it reaches their cell body in the DRG resulting in death of the neuronal cells (Dyck et.
al., 2005).
1.1.4 Genetics of HSN-I
As stated earlier, HSN-I is an autosomal dominant inherited disease with the genetic
locus being mapped to chromosome 9q22.1-q22.3 in 1996 using 4 large Australian
kindreds (Verhoeven et. al., 2006; Nicholson et. al., 1996). Further studies confirmed the region that was narrowed and located to a missense mutation in the open reading frame of SPTLC1 as the pathogenic cause of HSN-I. This mutation causes a change in a single base resulting in an aberrant amino acid being incorporated, ultimately changing the final protein structure. The most common mutation in HSN-I as seen in
eight Australian/English families was a single DNA base mutation 399T→ G in exon 5
of the SPTLC1 coding region, resulting in an amino acid substitution of cysteine to
tryptophan at position 133 (C133W) (Dawkins et. al., 2001). A second mutation in 431T
→ A was identified in two families and a third mutation in 398G → A observed in
another family which resulted in a substitution of valine to aspartate at position 144
(V144D) and cysteine to tyrosine at position 133, respectively (Dawkins et. al., 2001).
A fourth mutation was identified in twin sisters by Verhoeven et. al., (2004) at 387G →
A, changing glycine for alanine. Recently, three more mutations in, SPTLC2, DNA
methyl transferase 1 (DNMT1) and atlastin GTPase 3 (ATL3) have been observed to
cause HSN-I (Rotthier et al. 2010; Klein et al. 2011 and Kornak et al. 2014)
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1.2 Intracellular Lipid Functions and Interactions
1.2.1 The role of lipids in biological membranes
Biological membranes are lipid structures that define cells and cellular organelle structure. They divide the interior of eukaryotic cells into distinct compartments and provide surfaces for the localisation of metabolic enzymes, transport proteins, receptors and various substrates (Fenske et. al., 1995). Membranes are semipermeable barriers which regulate the transport of water, ions and other metabolites, thereby providing a means of controlling the internal cellular environment.
In 1972, Singer and Nicholson first proposed the fluid-mosaic model of biological membranes with the basic understanding of membrane structure changing little since
(Fenske et. al., 1995).
On average 98% of the molecules in membranes are lipids. The main classes of lipids found in eukaryotic biological membranes include the glycerophospholipids, the sphingolipids and cholesterol (Cullis and Hope, 1991; Saladin, 2007). The molecules are amphiphilic and arrange themselves into a liquid-crystalline bilayer with their hydrophilic phosphate-containing heads facing the water on each side of the membrane with their hydrophobic tails directed towards the centre of the membrane.
The phospholipids can drift laterally, able to spin on their axes and move their tails, keeping the membrane fluid (Saladin, 2007). The membrane also consists of cholesterol molecules, membrane-bound and transmembrane proteins as shown in
Figure 1.1. Historically, the lipid portion of the membrane was viewed as a convenient barrier and environment for enzymes (Fenske et. al., 1995). However, many studies have been undertaken to show that biological membranes contain a wide diversity of lipids, far more than are needed to perform structural functions, with these lipids
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requiring elaborate metabolic pathways for their synthesis and transport, suggesting
specific roles for the individual lipid components of membranes (Fenske et. al., 1995).
The bilayer serves as a matrix and support for a vast array of proteins involved in important functions of the cell, such as energy transduction, signal transduction, solute transport, DNA replication, protein targeting, cell-cell recognition and many more
(Dowhan, 1997). Phospholipids do not have a static role in these processes, rather they are active participants which influence the properties of the proteins associated with the membrane and serve as precursors to important cellular components
(Dowhan, 1997).
Figure 1.1 Molecular structure of the plasma membrane. The plasma membrane consists of a lipid bilayer with transmembrane proteins imbedded in its structure. On the external surface of the membrane glycolipids extend out acting as receptors to the external environment. Adapted from Saladin, (2007)
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1.2.2 Interactions of lipids with intracellular proteins and organelles
Lipids in eukaryotic membranes are fluid (Van Meer and Sprong, 2004). Lipids in the
ER are generally unsaturated, whereas the saturated lipids in the other membranes are “fluidised” by the presence of cholesterol (Van Meer and Sprong, 2004). Most membranes are composed mainly of phospholipids, glycosphingolipids and cholesterol. Phospholipids are loosely packed in bilayers, forming liquid disordered membranes. In contrast, sphingolipids have longer and more saturated acyl chains than phospholipids and exhibit stronger lateral cohesion, generating tightly packed regions (Van Meer and Sprong, 2004). Cholesterol preferentially interacts with sphingolipids and occupies the space between the acyl chains. The combination of sphingolipids and cholesterol in small domains is responsible for the formation of lipid rafts, important in transportation of proteins, influencing membrane fluidity and regulating neurotransmission and receptor trafficking (Van Meer and Sprong, 2004).
Several membrane proteins have been shown to reside in lipid rafts, either permanently or temporarily, and this association can be facilitated by covalent lipid modification of the protein molecule (Van Meer and Sprong, 2004). In fact, addition of a lipid chain (N-myristoylation, palmitoylation) to proteins increases their hydrophobicity and therefore their propensity to associate with cellular membranes
(Van Meer and Sprong, 2004). Lipid rafts on the plasma membrane can serve as scaffolding platforms where different receptors with similar affinity for the lipid domains meet and orchestrate various signalling events (Van Meer and Sprong, 2004). It has also been shown that lipids can exchange in the cell membrane to alter kinase signalling events (Myers and Stanley, 1999). Moreover, the existence of lipid domains also on the membrane of cell organelles seems to be responsible for appropriate
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protein-lipid sorting to specific cell compartments, thus enabling vesicles to arrive at the correct destination and facilitating secretion of cellular products (Van Meer and
Sprong, 2004).
Eukaryotic cells use the physical properties of their individual membrane lipid classes at specific steps in vesicle traffic. The local concentration of these lipids is regulated by a multitude of enzymes and translocators and appears to be one parameter in regulating the membrane flux through the various pathways (Van Meer and Sprong,
2004).
The occurrences of two glycosylphosphatidylinositol proteins (GPI) located in separate plasma membrane domains of different lipid composition, and with the finding of lipid- anchored proteins in cholesterol independent microdomains on the cytosolic surface, illustrate the dynamic organisation of biomembranes and how they cannot be explained by the mere notion of sphingolipid/cholesterol rafts (Van Meer and Sprong,
2004). A specific function of sphingolipids on cytosolic surfaces is suggested by the cytosolic protein FAPP2, which contains a glycolipid-binding domain that plays a role in regulating membrane flow between the Golgi and the plasma membrane (Van Meer and Sprong, 2004).
Cholesterol transport out of endosomes and lysosomes requires both the soluble cholesterol-binding protein NPC2 in the lumen and the putative cholesterol transporter
NPC1 in the membrane, which work together in concert (Van Meer and Sprong, 2004).
Lipids and proteins have been well-studied but a lot remains to be learnt about how they ‘act in concert’ in cell membranes (Van Meer and Sprong, 2004).
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1.2.3 Sphingolipids, their role in normal cellular homeostasis and in the neuronal cell
Sphingolipids (SLs) are a ubiquitous class of lipids present in all higher order
organisms. A large number of individual sphingolipid species exist, resulting from
differences in both the hydrophobic ceramide moiety and in the polar head group
(Buccoliero et. al., 2003). SLs form a complex family of molecules which all contain a
long-chain amino alcohol, known as the sphingoid base to which a variety of fatty acids
are attached via N-acylation (Buccoliero et. al., 2003). The polar head group consists of phosphorylcholine, sugar or sulfatide residues. Many different combinations of sphingoid long chain bases, fatty acids and head group moieties lead to a vast array of possible SL and glycosphingolipid (GSL) structures, which is seen in Figure 1.2
(Buccoliero et. al., 2003; Sabourdy et.al., 2008).
Complex sphingolipids such as ceramide, sphingomyelin, glucosylceramide,
galactosylceramide, sphingosine, sphingosylphospho-choline, psychosine and
sphingosine-1-phosphate (S1P), glycosphingolipids and phosphosphingolipids have
essential roles in many aspects of cell biology. Some of these role include,
inflammatory responses, cell proliferation and apoptosis to cell migration,
differentiation and senescence. Many sphingolipids carry out structural roles in cell
membranes, particularly the plasma membrane (Ogretmen et. al., 2004).
In the neuronal cell, SLs have been shown to participate in neuronal development, proliferation, survival, migration, differentiation and plasticity. SLs play an essential role in cell regeneration after cell damage has occurred (Colombaioni and Garcia-Gil,
2004). Neurotransmitter release has also been shown to be regulated by SLs, with
SLs binding to certain receptors that control ion channels, enzymes and intracellular
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calcium levels, all regulating the release of neurotransmitters. Gene expression is also
regulated by SLs allowing control of proliferation, differentiation and overall cell
regeneration. The involvement of SLs in numerous signalling pathways leading to the
negative cell regulation of differentiation causing neurite retraction has also been
identified (Colombaioni and Garcia-Gil, 2004).
Figure 1.2 The pathway of sphingolipid metabolism. Ceramide can be formed de novo (pink) or from hydrolysis of sphingomyelin (blue) or cerebrosides (green). Ceramide can also be phosphorylated by ceramide kinase to yield ceramide- 1-phosphate, or can serve as a substrate for the synthesis of sphingomyelin or glycolipids. Ceramide can be metabolised (orange) by ceramidases (CDases) to yield sphingosine, which in turn is phosphorylated by sphingosine kinases (SKs) to generate sphingosine-1-phosphate (S1P). Adapted from Ogretmen et. al., (2004)
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1.3 Serine Palmitoyltransferase
1.3.1 The structure and regulation of SPT and its subunits
SPT is a pyridoxal 5'-phosphate-dependent multimeric enzyme (Figure 1.3) that
catalyses the first step of the biosynthesis of sphingolipids (SL), ceramide and
sphingomyelin (Hornemann et. al., 2006; Breslow. D, 2013). SPT is a rate determining
enzyme in the de novo sphingolipid synthesis pathway. It is a key enzyme in regulation
of cellular sphingolipid content by the condensation of palmitoyl coenzyme A (CoA)
with L-serine to form 3-ketodihydrosphingosine. SPT is comprised of a combination of
two known subunits; SPTLC1, SPTLC2 (also known as LCB1 and LCB2 respectively)
and SPTLC3 which are summarised in Table 1.2 (Breslow. D, 2013).
SPT is a type 1 integral membrane protein with a single highly hydrophobic domain in
the amino-terminal region of the SPTLC1 subunit, which represents a transmembrane
domain that anchors the enzyme to the ER membrane (Wei et. al., 2007; Lowther et. al., 2012). SPTLC2 contains an active lysine residue required for PLP-binding while
SPTLC1 lacks this lysine and other key catalytic residues which has led to speculation as to whether this lysine residue plays a regulatory role in the SPT dimer (Yard et. al.,
2007; Lowther et. al., 2012). SPTLC1 and SPTLC2 subunits associate in a 1:1 ratio with the active site residing between the interfaces of these two subunits (Hanada,
2003). Both of these subunits are essential to produce the functionally active, heterodimeric SPT; however, both subunits are membrane-associated and this has encumbered their isolation and characterisation (Yard et. al., 2007; Lowther et. al.,
2012). Previously the third subunit, SPTLC3, was thought to be a functionally redundant isoform of SPTLC2. However, SPTLC3 has been shown to generate two
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new sphingoid base metabolites, C (16)-sphinganine and C (16)-sphingosine
(Hornemann et. al., 2009).
SPT activity is regulated at both a transcriptional and post-translational level. SPT is a ubiquitously expressed protein with its subunits expressed at varying levels (Wei et. al., 2007; Breslow. D, 2013). SPT has been shown to be upregulated in response to a variety of extracellular stimuli such as inflammatory cytokines, skin barrier requirements, ultraviolet light B (UVB), excess fatty acids and other factors that induce apoptosis (Hanada, 2003).
Figure 1.3 Theoretical model of SPT complex structure. A) SPTLC2 or SPTLC3 join SPTLC1 to form a dimer resulting in formation of the basic functional unit of SPT which then forms a final octameric state. B) Model of the octameric SPT complex with two SPTLC1–SPTLC2 and two SPTLC1–SPTLC3 dimers assembled to form an octameric circular structure. C) Cytosolic view of the SPT enzyme. Adapted from (Hornemann et. al., 2007).
Table 1.2 The three subunits of SPT; SPTLC1, SPTLC2 and SPTLC3 (Dawkins et. al., 2002; Verhoeven et. al., 2006; Hornemann et. al., 2006 and Hanada, 2003)
Subunit Chromosome Gene size (kb) Protein size (kDa)
SPTLC1 9q21.1-q22.3 85 55
SPTLC2 14q24.3 -q31 110 65
SPTLC3 20p12.1-12.3 1.7 63
1.3.2 The role of the SPTLC1 protein in neurodegenerative disease
SPT is the crucial enzyme in the complex metabolic pathway of sphingolipid metabolism, with mutations in the SPT subunits resulting in potential dysfunction and perturbations in sphingolipid synthesis and metabolism causing a variety of diseases, in particular HSN-I (Wei et. al., 2007).
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SPTLC1 encodes the long-chain base one (LCB1) subunit of SPT. Mutations in this gene identified in HSN-I occur at single amino acids that are highly conserved
throughout different species and are likely to interfere with the functionality and
structure of SPT (Verhoeven et. al., 2006). SPT mutations causing HSN-I are
autosomal dominant, and have a late age of onset with only missense mutations
occurring. This is most consistent with two current hypotheses for the mechanism of
HSN-I: either a gain of function or a dominant negative effect (Verhoeven et. al., 2006).
The 'gain of function' hypothesis implies that mutations confer one or more toxic
properties on SPTLC1. A 'gain of toxic function' is the accumulation of mutant protein,
that then forms an insoluble aggregate that eventually leads to cell death (Verhoeven
et. al., 2006). Alternatively, peripheral neurons may be sensitive to a perturbation of
the sphingolipid metabolism (decrease in functional levels) caused by a mutation-
induced reduction in SPT enzyme activity (Verhoeven et. al., 2006). This hypothesis
has been shown to be consistent with studies on C133W and V144D, demonstrating
that both mutations reduce normal SPT activity in various cell types, including cultured
patient lymphoblasts (Verhoeven et. al., 2006). A concomitant change in the
membrane lipid composition would be expected to be seen but data has been
contradictory. Initially, an increase in glucosylceramide synthesis was reported,
however a decrease in ceramide levels and sphingomyelin synthesis yielded no
change in the lipid composition (Verhoeven et. al., 2006).
Bejaoui et. al., (2002) and Dedov et. al., (2004) studied SPT activity using patient
lymphoblasts that endogenously expressed the SPTLC1 mutation and reported
greater than 50% reduction of SPT activity. While the mechanism by which SPT
activity is reduced is yet to be confirmed, Bejaoui et. al., (2002) observed that the
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mutation did not directly affect the stability of the protein translated and postulated that
it may directly interfere with enzyme function. As SPTLC1 mutations have a direct
effect on the activity of SPT this supports the dominant negative effect theory. Thus,
competition possibly arising between mutated and wild type SPTLC1 for interaction
with SPTLC2 may be a potential molecular mechanism, with the mutated SPTLC1
possessing a higher affinity than wild type (Bejaoui et. al., 2002). As seen by
Verhoeven et. al., (2006), the SPTLC1 mutation does not reduce SL levels despite
SPT activity being reduced by more than half. Therefore, it suggests that the remaining
50% of SPT activity is sufficient to maintain the normal sphingolipid homeostasis in these cells, presumably because the cell may have more total SPT activity than is required as seen by in-vivo SPT down-regulation (Dedov et. al., 2004).
In contrast, higher overexpression of SPTLC1-like subunit mutations in yeast and
Chinese hamster ovary (CHO) cells can result in cell death (Dedov et. al., 2004). As a
50% reduction in SPT activity appears to have little impact on sphingolipid metabolism, resulting normal cell proliferation and viability may explain why HSN-I is a late on-set disorder (Dedov et. al., 2004). It is possible that the expression of SPTLC1 and
SPTLC2 are not as balanced in ageing neurons with a dominant negative effect manifesting in these cells more profoundly than in lymphoblasts (Dedov et. al., 2004).
It is also plausible that subtle changes in sphingolipid metabolism are involved, such as the accumulation of abnormal metabolites from dysregulation of these pathways
(Dedov et. al., 2004). There may also be accumulation of mutant SPT leading to the formations of insoluble aggregates, as demonstrated to be the common pathogenic mechanism of other common neurodegenerative disorders such as Alzheimer’s or
Huntington's diseases (Dedov et.al., 2004). Although no SPTLC1 aggregates have yet
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been discovered in HSN-I, elevated levels of deoxysphingoid bases (Deoxy-LCBs)
arising from condensation of alanine and glycine with palmitoyl-CoA has been
observed in HSN-I transgenic mice (Penno et. al., 2010). Such evidence would support
the 'toxic gain of function' theory and may also explain the late-onset of the disease.
1.4 Protein Functions and Interactions
1.4.1 Altered protein expression and the disease state
In biological structures such as proteins, 'form follows function' and 'form is function'
(Lodish et. al., 2008). This highlights the importance of correctly synthesised protein primary structure and folding into the proper three-dimensional conformation, essential for correct protein functioning. Recent evidence has shown that a protein may fold into an alternative three-dimensional structure or have a resulting post-translational modification that arises from mutations or inappropriate covalent modifications (Lodish et. al., 2008). This misfolding or modification leads to a loss of normal function and often marks the protein for proteolytic degradation (Lodish et. al., 2008). When degradation is incomplete or not sufficient to keep up with the amount of misfolding occurring, then subsequent accumulation of the proteolytic fragments or misfolded proteins occurs. This has been shown to contribute to certain degenerative diseases that are characterised by the presence of insoluble protein plaques in various organs including the liver and brain (Lodish et. al., 2008). Diseases like Alzheimer's,
Parkinson’s and Huntington's disease are all neurodegenerative diseases that have manifested from the resulting increase in proteolytic fragments and misfolded proteins
(Lodish et. al., 2008).
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Between 60 and 80% of all dementia-related illness is due to Alzheimer's disease
(Goldman et. al., 2008). Alzheimer's disease is marked by the presence of increasing numbers of β-amyloid peptide-containing deposits in neuritic plaques within the neocortex, and is associated with increasing severity of dementia (Goldman et. al.,
2008). It has been assumed for many years that the plaques were themselves toxic; instead the soluble aggregates of β-amyloid in oligomeric forms (i.e. consisting of a small number of monomers) are the key pathogenic molecules (Goldman et. al., 2008).
Neurofibrillary tangles are intracellular aggregates of the microtubule-associated protein tau. The neurofibrillary tangles represent a nonspecific response to β-amyloid
(Goldman et. al., 2008). Tamboli et. al., (2005) have shown that glycosphingolipid inhibition reduces transport of β-amyloid precursor protein (APP) and β-amyloid, thus highlighting the importance of sphingolipids and potential treatment in the future.
Parkinson's disease is the second most common neurodegenerative disorder occurring in approximately 1 in 1000 in the general population and in 1% of persons older than 65 years (Goldman et. al., 2008). Both autosomal dominant and recessive genes have been implicated in classic Parkinson's disease (Goldman et. al., 2008).
The protein α-synuclein, which is the major constituent of the cytoplasmic inclusion known as the Lewy body, is critical in the disease pathogenesis (Goldman et. al.,
2008). Abnormal aggregation of the protein, either from mutations in the α-synuclein gene or excessive production of the normal protein due to gene duplications or triplications, is associated with the varying disease phenotypes observed in
Parkinson's (Goldman et.al., 2008).
Huntington's disease is an autosomal dominant disorder caused by unstable cytosine- adenine-guanine (CAG) repeat expansions on chromosome 4 (Schapira et. al., 2007).
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Afflicted individuals have 37 to 86 repeats, compared to normal individuals that have
11 to 34 repeats. The normal protein, huntingtin, serves a role in intracellular trafficking and membrane recycling (Schapira et. al., 2007). The trinucleotide CAG codes for glutamine, with an increase in the polyglutamine tract preventing normal protein turnover, thereby resulting in protein aggregation (Schapira et. al., 2007). The aberrant protein product is ubiquitinated (marked for degradation) but fails to be efficiently degraded, leading to the formation of intranuclear inclusions that may disrupt mitochondrial processes and other functions. The mutant huntingtin is cleaved into fragments, with these fragments playing a primary role in huntingtin toxicity, including transcriptional dysregulation (Schapira et. al., 2007). Mutant huntingtin associates with an array of proteins. These “huntingtin-interacting proteins” are involved with numerous important cellular functions, including transcription, trafficking, signalling and metabolism (Schapira et. al., 2007). It is therefore essential that proteins are folded correctly and that any misfolded proteins are successfully degraded and removed to maintain normal cellular function.
1.5 Mitochondria in neurodegenerative diseases
1.5.1 Mitochondrial Dynamics
Mitochondria are the most recognisable membrane-bound organelle, responsible for
several biological processes, such as oxidative phosphorylation, lipid metabolism,
tricarboxylic acid (TCA) cycle, iron-sulphur cluster formation and apoptosis. They are
interconnected and form a highly dynamic network shaped by constant fusion and
fission events. They are usually scattered throughout the cytoplasm of most cells, but
they often concentrate in specific areas of high energy utilisation. Their number and
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size vary with metabolic activity and cell type: mature erythrocytes have none; a
hepatocyte has up to 2500. They are 1-10 μm in size and may be elongated, spherical,
or pleomorphic. These very dynamic organelles show constant motion, fusion, and
division in cells (Ovalle and Nahireny, 2008).
1.5.2 Mitochondrial fusion and fission
Mitochondrial fission contributes not only to the proper distribution of mitochondria in
response to the local demand for ATP, but also to the elimination of damaged
mitochondrial fragments through mitophagy (autophagy for mitochondria).
Mitochondrial fusion facilitates the exchange of mtDNA and other vital components
between mitochondria for the maintenance of normal function (van de Bliek et. al.,
2013). Mitochondrial fusion and fission are controlled by four high molecular weight
GTPases conserved from yeast to mammals: mitofusins, Mfn1 and Mfn2, in
mitochondrial outer membrane (OMM) fusion; Opa1 (a gene product of optic atrophy
type I) in mitochondrial inner membrane (IMM) fusion and cristae organisation; and
Drp1 (Dynamin related protein 1) in mitochondrial fission, indicating that the
fundamental mechanisms controlling mitochondrial dynamics have been maintained
throughout evolution (Figure 1.4). The morphology of the mitochondria results from a
balance between these opposing processes (van de Bliek et. al., 2013).
1.5.2.1 Mitochondrial Fusion
Mitochondrial fusion between closely apposed mitochondria is a complex regulatory process involving multiple proteins which fuse both the OMM and IMM of each mitochondrion (van de Bliek et. al., 2013). Although fusion reactions between OMMs
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of apposed mitochondria and the subsequent fusion between IMMs are normally
highly synchronised, the two processes can be functionally uncoupled (Otera and
Mihara, 2011). Mfn1 and Mfn2 are anchored to the OMM with a large N-terminal
GTPase domain and a C-terminal coiled-coil domain that is exposed to the cytosol
and they mediate OMM fusion in a GTPase-dependent manner (Otera and Mihara,
2011).
Mfn1 and Mfn2 form homo- or hetero-protein complexes. These interactions between the mitofusins on opposing mitochondria serve to tether and fuse the OMMs (van de
Bliek et. al., 2013). Mitofusin 1 and 2 appear to have distinct roles within mitochondrial
OMM fusion; Mfn1 is thought to be responsible for the initial GTP-dependent OMM tethering (Figure 1.4) (Otera and Mihara, 2011). Mfn2 is enriched in the mitochondria- associated membranes (MAM) of the ER, where it interacts with Mfn1 and Mfn2 on the mitochondria to form interorganellar bridges (Otera and Mihara, 2011).
Opa1 is another key molecule essential for mitochondrial IMM fusion and cristae remodelling. There are eight Opa1 splice variants, which are all synthesised as precursor proteins with the mitochondrial localisation sequence in the N-termini and
the following hydrophobic stretches that are responsible for sorting the protein into the
IMM (Otera and Mihara, 2011). During mitochondrial import, the mitochondrial
localisation sequence of Opa1 precursors is removed by mitochondrial processing
peptidase to form L-forms (Ishihara et. al., 2006). These Opa1 L-forms are anchored
to the IMM with the GTPase domain exposed to the mitochondrial intermembrane
space (IMS), and are subsequently processed either in the IMS to produce S-forms by
the intermembrane space AAA protease (i-AAA protease) YME1L or in the matrix by
Afg3L2 and Paraplegin, depending on where the process site localises in the IMS.
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Prohibitins (PHB1 and PHB2) are IMM proteins that function as protein- and lipid-
scaffolds which are essential for cell development and proliferation. Studies by Zhang
and Chan (2007), have shown mutations in the GTPase domain leads to the
development of fragmented mitochondria, thus indicating that GTP hydrolysis is
essential for mitochondrial fusion activities. Opa1 is also reported to be involved in
maintenance of the cristae structure; knockdown of Opa1 induces disintegration of the
cristae structure concomitant with cytochrome c release and apoptosis induction
(Otera and Mihara, 2011)
Figure 1.4 Schematic model of mitochondrial fusion. A. Mitochondrial outer membrane fusion is facilitated by Mfn2 interaction that takes place in trans across two mitochondria. It is possible that Mfn2 mediates outer membrane fusion by binding to and forming clusters at mitochondrial tips (the ends of these tubular organelles). B. A proposed model of potential buckle and molecular zippering mechanism for Mfn2-mediated mitochondrial fusion. The Mfn2 dimer binds adjacent mitochondrial outer membranes with its hydrophobic paddle domain and fuses them through a conformational change, induced by GTP hydrolysis. Other regulatory proteins are also likely to be involved. C. Optic atrophy 1 (OPA1)- mediated inner membrane fusion. Mitochondrial matrix contents mix after inner membrane fusion. Adapted from (Knott et. al., 2008) 22 | Page
1.5.2.2 Mitochondrial Fission
The mitochondrial division process requires at least two conserved proteins, hFis1
(human mitochondrial fission protein 1) and Drp1, and several other proteins, including
MTP18 in mammals. Drp1 and hFis1 localise to discrete points at future sites of scission on the mitochondrial outer membrane (Marchi et. al., 2014). Drp1 is a cytosolic protein with an N-terminal GTPase domain thought to provide mechanical force with a dynamin-like middle domain and a GTPase effector domain (GED) located in the C-terminal region. Drp1 mainly localises in the cytosol, and during mitochondrial fission, translocates from the cytosol to prospective fission sites on the mitochondria
(Marchi et. al., 2014).
These spiral higher-order structures are thought to constrict and eventually sever the mitochondrial membrane by a GTP hydrolysis dependent mechanism. Intermolecular
interactions between the N-terminal GTPase domain and C-terminal GED are also
important for Drp1 self-assembly and functional regulation (Marchi et. al., 2014). In
mammals, Fis1 has also been identified in mitochondria and is thought to be involved
in recruiting Drp1 to mitochondria (Marchi et. al., 2014). Mitochondrial fission factor
(Mff) is able to recruit Drp1 to mitochondria and form a complex with Drp1 and promote mitochondrial fission (Figure 1.5). Mff linked to the plasma membrane results in Drp1 recruitment to exactly that membrane. Mitochondrial dynamics proteins 49 and 51
(MiD49 and MiD51 respectively) and MIEF1 (mitochondrial elongation factor 1) are also capable of recruiting Drp1 to mitochondria (Figure 1.5). Importantly, mitochondrial fission is blocked rather than promoted by sequestering Drp1. MIEF1/MiD51 are able to form two different protein complexes which, in an apparent mutually exclusive manner, bind to either Drp1 or Fis1 (Palmer et. al., 2011;Zhao et. al., 2011)
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Figure 1.5 Schematic diagram for the proposed new model of fission. Classical model: hFis1 acts as the mitochondrial receptor for Drp1 promoting fission. Extended model: hFis1, Mff and/or MIEF1/MiD51/Mid49 recruit Drp1 to the mitochondria. The Mff–Drp1 complex promotes mitochondrial fission. In contrast, the MIEF1/Mid49/51–Drp1 sequesters Drp1, inhibits Drp1 function and promotes fusion in an Mfn2-independent manner. In an apparently mutually exclusive manner, MIEF1/MiD51 can also form a complex with hFis1. The inhibitory effect of MIEF1/MiD51 on Drp1 function is reduced and hence mitochondrial fission is indirectly promoted by Fis1. Active Drp1: green. Inhibited Drp1: red. Adapted from (Dikov and Reichert, 2011).
Mitotic kinase Aurora A and the small Ras-like GTPase RALA also controls the
recruitment of Drp1 to mitochondria Kashatus et. al., (2011). Aurora A is known as a
serine/threonine kinase and has a pivotal role in many aspects of cell division, such
as mitotic entry, chromosome segregation and spindle assembly. On the other hand,
RALA, a small G protein belonging to the Ras superfamily, controls and participates
in cellular processes such as vesicle sorting, cell morphology and gene expression by
cycling between active GTP-bound and inactive GDP-bound conformations (Kashatus
et. al., 2011). Kashatus et. al., (2011) found that phosphorylated RALA accumulates
preferentially on mitochondria, they also demonstrated that Drp1 protein levels in the
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mitochondria are reduced both in cells with diminished levels of RALA thus inhibiting
mitochondrial fission and induces a more highly interconnected mitochondrial network
(Kashatus et. al., 2011). RALBP1, a multifunctional effector of RALA, has also been
shown to affect the recruitment of Drp1. RALBP1 and cyclinB–Cdk1 interact with each
other and phosphorylate Drp1 (Dikov and Reichert, 2011; Kashatus et. al., 2011). This
suggests that RALBP1, following recruitment to mitochondria by RALA, may form a
mitosis-specific complex with cyclin B–CDK1, and that this interaction potentiates
cyclin B–Cdk1 kinase activity towards Drp1 and promotes Drp1 oligomerisation and
subsequent mitochondrial fission (Figure 1.6) (Dikov and Reichert, 2011; Kashatus et.
al., 2011).
Figure 1.6 Schematic diagram of mitosis-specific mitochondrial fission. Mitosis-specific mitochondrial fission involves the phosphorylation of DRP1. Mitochondrial morphology is coordinated with the cell cycle. During metaphase, in which condensed chromosomes align in the middle of the cell, mitotic kinase Aurora A phosphorylates RALA, which relocates to mitochondria. This encourages the formation of a complex consisting of phosphorylated RALA, RALBP1and cyclin B–CDK1 on mitochondria, and mediates the phosphorylation of DRP1. Finally, oligomeric Drp1 wraps around the mitochondrial surface and mitochondrial division ensues. Adapted from (Yamano and Youle, 2011).
Despite the growing insight into mitochondrial fission, it remains unclear whether the
phosphorylation of Drp1 is always essential for membrane scission. If so, cyclin B–
CDK1 can phosphorylate Drp1 during mitosis, but other kinases would be required for
the fission activity of DRP1. Furthermore, cyclic-AMP-dependent protein kinase (PKA)
has been identified to phosphorylate different, but nearby, serine residues during
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starvation (Dikov and Reichert, 2011; Kashatus et. al., 2011). A surmounting body of
research indicates that mitochondrial division responds to cellular stimuli in highly
regulated ways where a central role is the modulation of Drp1, not only by
phosphorylation, but potentially by other post-translational modifications including
ubiquitylation, Sumoylation and S-nitrosylation (Figure 1.7) (Dikov and Reichert, 2011;
Kashatus et. al., 2011). Thus, post-translational modifications could play a major role
in the regulation of Drp1 activity and thereby controlling mitochondrial morphology
(Otera and Mihara, 2011).
Figure 1.7 Overview of mitochondrial dynamics and homeostasis. Mitochondrial morphology is maintained by fusion and fission. Excessive mitochondrial fission often causes the generation of depolarised mitochondria. Dysfunctional mitochondria return to the cell soma and are eliminated by the autophagy system, named mitophagy. Disruption of the mitochondrial dynamics or mitochondrial quality control system leads to the accumulation of dysfunctional mitochondria and causes a collapse of the cellular environment followed by cell death. Adapted from (Otera and Mihara, 2011).
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1.5.3 Mitochondrial changes in inherited peripheral neuropathies
Neurodegenerative diseases are a clinically heterogeneous group of chronic progressive illnesses with varying, but distinct, clinical manifestations. The genetic cause of disorders such as Huntington’s disease, amyotrophic lateral sclerosis (ALS),
Charcot-Marie-Tooth syndrome Type II (CMT II), Parkinson’s disease, Friedreich’s ataxia and Alzheimer’s disease have been well established. Despite their obvious differences in primary aetiologies, the role for mitochondrial dysfunction is evident in the pathogenesis of these diseases (Manfredi and Flint Beal, 2000; Kwong et. al., 2006 and Duffy et. al., 2011).
The nervous system is especially susceptible to mitochondrial dysfunction due to the high metabolic activity of neurons, in particular bioenergetic failure through the loss of
ATP production. Mitochondrial dysfunction in neurons can lead to a myriad of different effects such as apoptosis, oxidative stress, excitotoxicity and destructive rises in intracellular calcium levels that contribute to several pathologies of the nervous system
(Hollenbeck and Saxton, 2005). Mitochondrial transport is also intimately dependent upon the functional state of the cell and of mitochondria themselves, and is essential for neurons due to their long axonal processes and high demand for energy (Kwong et. al., 2006).
Mitochondrial membrane depolarisation and inhibition of ATP synthesis, has been shown to inhibit movement of organelles, with 80% of slightly depolarised mitochondria in DRG neurons move retrogradely, implying unhealthy mitochondria are returned to the cell body for repair or removal, reducing the number of mitochondria that are transported in the anterograde direction (Miller and Sheetz, 2004).
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Mitochondrial dysfunction is likely to play an important role in several major
neurodegenerative diseases. However, despite an ever-expanding body of literature
on the mitochondrial involvement in neurodegeneration, many crucial questions
remain to be answered. How do mutations in neurodegenerative genes result in mitochondrial dysfunction? In some cases mutant proteins localised in multiple cell
compartments (often including mitochondria) may cause dysfunction, such as aberrant
aggregates in cellular compartments, as observed in Huntington’s disease and
Alzheimer’s disease (Duffy et. al., 2011). In addition to how mutations cause
mitochondrial dysfunction, what are the consequences of mitochondrial dysfunction?
With the multiplicity of mitochondrial functions (including intermediate metabolism,
energy production and apoptosis) there are many, nonmutually exclusive, potential
avenues whereby damaged mitochondria can sabotage the survival of a neuronal cell
(Kwong et. al., 2006, Duffy et. al., 2011).
While extensive research efforts are currently underway to answer these questions,
the consequence of mitochondrial dysfunction in very large and metabolically active
cells such as peripheral sensory neurons are especially adverse (Duffy et. al., 2011).
Differential sensitivity of distal neuronal regions to dysfunctions in mitochondrial
energy productions, axonal transport of mitochondria, or a combination of both are
now thought to be a major cause for axonal degeneration in peripheral neuropathies
(Duffy et. al., 2011). Until further research is thoroughly conducted the precise
involvement of mitochondria in inherited peripheral neuropathies remains to be
elucidated.
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1.5.4 Examples of Mitochondrial changes in inherited peripheral
Neuropathies
Charcot-Marie-Tooth type IIA is an inherited peripheral neuropathy with both motor
and sensory involvement that exhibits dying back of the axon, with similar clinical
presentation to HSN-I. This disease has been identified as a gene mutation involved
in mitochondrial function, known as mitofusin 2 (Zuchner et. al., 2004). While the exact
cause of axonal degeneration of CMT type IIA is undefined, recent studies have
postulated that impairment in anterograde axonal transportation of mitochondria and
consequent bioenergetic failure at the distal ends of neurons may be responsible
(Zuchner et. al., 2004; Baloh et. al, 2007 and Warner and Hammans, 2009).
Mitochondrial membrane potential, oxidative respiration and ATP production have been found to be normal in neurones from patients with CMT type IIA. However observed mitochondrial aggregates were postulated to ineffectively attach to the motor proteins involved in mitochondrial transport (Baloh et. al., 2007).
While axonal degeneration may be caused by impairment of mitochondrial transportation, neuronal die back could also be caused by proteins not normally associated with mitochondria. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder, which is pathologically defined by the progressive loss of motor neuron groups in the spinal cord, brain stem and motor cortex. ALS clinically presents within lower motor neuron with signs of progressive weakening and wasting of voluntary muscles and upper motor neuron spasticity and hyper-reflexia (Duffy et. al., 2011).
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ALS results from mutations in the gene encoding superoxide dismutase 1 (SOD1), the ubiquitous enzyme that mediates conversion of a superoxide anion, derived from oxidative phosphorylation, into hydrogen peroxide, which is an imperative role in antioxidant defence. SOD1 was previously thought to be solely a cytoplasmic enzyme, but studies have shown that it partially localises to mitochondria, with mutant SOD1 observed to be more abundant in nervous tissue of transgenic mice expressing the
ALS-associated mutants G93A-SOD1, when compared to tissue derived from the liver or heart (Duffy et. al., 2011).
Proportions of mutant SOD1 have been shown to localise to the mitochondrial IMS, the site of reactive oxygen species (ROS) generation and with mutant SOD1 identified in proteinaceous aggregates. This evidence suggests that mutant SOD1 is preferentially recruited to the IMS, where it acts to increase production of toxic ROS
(Duffy et. al., 2011).
Mutant SOD1 associated with mitochondria has an increased propensity to form cross-
linked oligomers, similar to those formed by ß-amyloid protein in Alzheimer’s disease
(Duffy et. al., 2011). The cross-linking enables mutant SOD1 to bind to the IMM,
shifting the redox state of the mitochondria. The shift in the redox state causes a
change to an oxidising environment, impairing the activity of the respiratory
complexes, with the presence of oxidative stress; SOD1 becomes insoluble,
increasing the potential to form aggregation upon oxidation (Duffy et. al., 2011).
Studies have also predicted that the formation of mutant SOD1 aggregates in both the
mitochondrial matrix, and associating with the cytosolic-facing outer mitochondrial membrane, may induce stress in mitochondria, potentially damaging the mitochondrial import machinery, dramatically impairing protein import as well as disturbing ionic
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homeostasis and dynamic regulation of mitochondria (Liu et. al., 2004; Vijayvergiya
et. al., 2005). Other neurodegenerative diseases involving inference with
mitochondrial function are summarised in Table 1.3.
Functional changes in mitochondria are often accompanied by disturbance in
mitochondrial morphology with changes to membrane integrity, disintegration of
cristae and massive swelling of the mitochondria (Kwong et. al., 2006). Mitochondrial
morphological changes have also been recently associated with HSN-I (Myers. et. al.,
2014). Table 1.3 Neurodegenerative diseases that interfere with mitochondrial function. Mitochondrial dysfunction in neurodegenerative diseases are not all caused by mutations in mitochondrial-related proteins, such as ALS, AD and HD. Adapted from (Kwong et. al., 2006).
Disease Mutated Gene Mitochondrial Functions Product Outer membrane GTPase Charcot-Marie- Mitofusin 2 involved in mitochondrial fusion, Tooth type IIA regulation of apoptosis
Cytosolic ROS scavenging Amyotrophic SOD1 enzyme found to localise to IMS Lateral Sclerosis and matrix when mutated
Mutated protein and its product ß- Alzheimer’s Amyloid ß Precursor amyloid localises to mitochondria Disease Protein where they increase ROS production
Cytosolic protein that causes Parkinson’s α-synuclein mitochondrial dysfunction leading Disease to oxidative stress
Mutant protein binds the outer Huntington’s mitochondrial membrane and Huntingtin Disease reduces mitochondrial uptake of calcium
Mitochondrial protein involved in heme biosynthesis, formation of Friedreich Ataxia Frataxin iron sulphur clusters, and iron detoxification
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1.5.5 Novel link to Mitochondria in HSN-I
Despite the numerous studies of SPTLC1 mutations and the effect upon cellular
functions by sphingolipid metabolism, other mechanisms have not been studied in
detail. Investigations may provide the missing link in our understanding of the pathogenesis of HSN-I. Mitochondrial morphology and localisation in HSN-I patient-
derived lymphoblasts were examined by transmission electron micrographs (TEMs) and changes have been reported (Myers et. al., 2014). Mitochondria within the patient-
derived lymphoblasts carrying the C133W and V144D mutations cluster to the
perinuclear area of the cell with the ER seen to be flanking mitochondrial aggregations.
Mitochondrial morphology was also altered with the mitochondrial membrane
displaying discontinuous breakages. The inner matrix exhibited a swollen appearance
with a significant loss of cristae. Perturbation to the mitochondria appear to be mutant-
specific especially within the C133W mutant lymphoblasts.
These striking morphological changes occurring in the mitochondria have not been
previously explored. The discontinuous and abnormal appearance of the mitochondrial membranes is suggestive of membrane depolarisation, potentially leading to deficiencies in ATP production and transport. As a result, these changes may in turn have consequences on axonal transport of mitochondria in neurons, with
a potential for increased apoptosis resulting from the mitochondrial dysfunction, as
demonstrated in ALS. As mitochondria are extremely important in all cells, and with
known adverse effects of mitochondrial dysfunction in peripheral sensory
neuropathies, these changes in mitochondrial function may provide an exciting new
prospect in the understanding of the HSN-I molecular mechanisms.
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1.6 The Role of ER in neurodegenerative diseases
1.6.1 Protein processing in the ER
The mechanism by which secretory proteins are targeted to the ER during their translation (the Cotranslational pathway) is well understood (Figure 1.8). The signal sequences span approximately 20 amino acids, including a stretch of hydrophobic residues usually at the amino terminus of the polypeptide chain (Cooper and
Hausman, 2007). As the polypeptide chains emerge from the ribosome, signal sequences are recognised and bound by a signal recognition particle (SRP) consisting of six polypeptides and a small cytoplasmic RNA (7SL RNA). SRP binds the ribosome as well as the signal sequence inhibiting further translation and targeting the entire complex (the SRP, ribosome, and growing polypeptide chain) to the rough ER by binding to the SRP receptor on the ER membrane (Cooper and Hausman, 2007).
Binding to the receptor releases the SRP from both the ribosome and the signal sequence of the growing polypeptide chain. The ribosome then binds to a protein translocation complex in the ER membrane and the signal sequence is inserted into a membrane channel (Cooper and Hausman, 2007).
The translocation channels through the ER membrane are complexes of three transmembrane proteins, called the Sec61 proteins. Transfer of the ribosome from the
SRP to the Sec61 complex allows translation to resume and the growing polypeptide chain is transferred directly into the Sec61 channel and across the ER membrane
(Cooper and Hausman, 2007). As translocation proceeds, the signal sequence is cleaved by signal peptidase and the polypeptide is released into the lumen of the ER
(Cooper and Hausman, 2007).
33 | Page
Figure 1.8 Cotranslational targeting of secretory proteins to the ER. As the signal sequence emerges from the ribosome, it is recognised and bound by the SRP. The SRP escorts the complex to the ER membrane, where it binds to the SRP receptor. The SRP is released, the ribosome binds to a membrane translocation complex of Sec61 proteins and the signal sequence is inserted into a membrane channel. Translation resumes, and the growing polypeptide chain is translocated across the membrane. Cleavage of the signal sequence by signal peptidase releases the polypeptide into the lumen of the ER. Adapted from (Cooper and Hausman, 2007).
Many proteins are targeted to the ER after their translation is complete
(posttranslational translocation) rather than being transferred into the ER during
synthesis on membrane -bound ribosom es (Cooper and Hausman, 2007). These
proteins are synthesis ed on free cytosolic ribosomes and their posttranslational
incorporation into the ER does not require SRP (Fig ure 1.9 ). Cytosolic chaperones are
required to maintain the polypeptide chains in an unfolded conformation so t hey can
enter the Sec61 channel. BiP (immunoglobulin heavy chain-binding protein), is
required to “pull” the polypeptide chain through the channel and into the ER (Cooper
and Hausman, 2007).
34 | Page
Figure 1.9 Posttranslational translocation of proteins into the ER. Proteins destined for posttranslational import to the ER are synthesised on free ribosomes and maintained in an unfolded conformation by cytosolic chaperones. Their signal sequences are recognised by the Sec62/63 complex, which is associated with the Sec61 translocation channel in the ER membrane. The Sec63 protein is also associated with a chaperone protein (BiP), which acts as a molecular ratchet to drive protein translocation into the ER. Adapted from (Cooper and Hausman, 2007).
Protein chaperones facilitate protein folding in the ER, but amino acid posttranslational
modifications such as asparagine ( N) -linked glycosylation and disulphide bond
formation are also involved (Cooper and Hausman, 2007) . In contrast to the highly
reducing environment of the cytosol, where disulphide bonds do not typically form, the
lumen of the ER is very oxidising, resulting in the rapid formation of disulphide bonds
(Cooper and Hausman, 2007).
35 | Page
Proteins destined for secretion or residence within the lumen of the ER, Golgi
apparatus or lysosomes are translocated across the ER membrane and released into
the lumen of the ER. However, proteins destined for incorporation into membranes are
initially inserted into the ER membrane instead of being released into the lumen. From
the ER membrane, they proceed to their final destination through budding and
intracellular vesicular transport. (Cooper and Hausman, 2007).
The ER is also the site of protein folding, assembly of multisubunit proteins, disulphide
bond formation, the initial stages of glycosylation and the addition of glycolipid anchors
to some plasma membrane proteins. Indeed, the primary role of luminal ER proteins
is to catalyse the folding and assembly of newly translocated polypeptides (Cooper and Hausman, 2007).
As previously mentioned, proteins are translocated across the ER membrane as unfolded polypeptide chains while their translation is still in progress. These polypeptides, therefore, fold into their three-dimensional conformations within the ER,
assisted by the molecular chaperones that facilitate the folding of polypeptide chains
(Cooper and Hausman, 2007). Immunoglobulin Binding protein (BiP) binds to the unfolded polypeptide chain as it crosses the membrane and then mediates protein folding and the assembly of multisubunit proteins within the ER. Correctly assembled proteins are released from BiP and are available for transport to the Golgi apparatus.
Abnormally folded or improperly assembled proteins, however, remain bound to BiP and are consequently retained within the ER or degraded rather than being transported further along the secretory pathway (Cooper and Hausman, 2007).
36 | Page
1.6.2 UPR: Unfolded protein response pathway
As a membranous compartment associated with the critical cellular functions
mentioned, the ER is extremely sensitive to changes that affect its structure, integrity
and function. As such any changes in calcium homeostasis leading to calcium
depletion from the ER lumen, inhibitors of protein glycosylation, inhibitors of
disulphide-bond formation, virus infection, hypoxia, ischemia and growth factor
depletion can all disrupt protein synthesis, translation and folding, resulting in unfolded
or misfolded proteins. The accumulation of unfolded and/or misfolded proteins causes
an imbalance between the synthesis of new proteins and the ER’s ability to process
newly synthesised proteins, resulting in the failure of the ER to cope with the excess protein load, which is termed ER stress. Cells in turn activate an integrated intracellular signalling cascade, known as the unfolded protein response (UPR) to avert ER stress
(Rao and Bredesen, 2004).
UPR activation results initially with a transient attenuation in the rate of protein
synthesis. The next event is an upregulation of genes encoding chaperones and other
proteins that prevent polypeptide aggregation and participate in polypeptide folding,
followed by retro translocation and degradation of ER-localised proteins. These
cellular responses minimise the accumulation and aggregation of misfolded proteins
by increasing the capacity of the ER machinery for folding and degradation (Malhotra
and Kaufman, 2007).
During this phase, nascent-folding-competent polypeptides are maintained in a
soluble form by interaction with ER luminal chaperones. BiP/GRP78 (glucose-
regulated protein) are one of the most highly expressed ER resident chaperones. BiP
is a member of the heat-shock protein (Hsp70) family (Malhotra and Kaufman, 2007).
37 | Page
Currently there are three identified components of the UPR; the double stranded RNA-
activated protein kinase-like ER kinase (PERK), the eukaryotic translation initiation
factor 2 (eIF2a); the activating transcription factor 6 (ATF6) and the inositol-requiring
enzyme 1 (IRE1). All of these proteins associate with BiP in their inactive states
(Malhotra and Kaufman, 2007). It has been postulated that when ER homeostasis is
perturbed, BiP preferentially binds to and is sequestered by unfolded/misfolded
proteins that accumulate within the ER lumen. As a consequence, BiP dissociates from the UPR transducers to permit their signalling. Although the UPR pathway is simultaneously activated upon severe ER stress, the immediate response occurs through the PERK/eIF2a pathway (Malhotra and Kaufman, 2007).
PERK is a type I ER transmembrane protein kinase. Upon release from BiP, PERK
dimerises to promote self autophosphorylation and activation. Activated PERK phosphorylates eIF2a, thus attenuating the rate of general translation initiation (Shen et. al., 2004). In conjunction with PERK activation upon BiP release, the release of BiP from IRE1 allows its dimerisation and autophosphorylation to activate its site-specific endoribonuclease (RNase) activity. The RNase activity of IRE1 initiates splicing of a
26-base intron from the X-box-binding protein 1 (XBP1) mRNA, altering the C-terminus of the protein to create a potent transcription factor (Malhotra and Kaufman, 2007).
ATF6 is a type II transmembrane protein of the ER. BiP release allows ATF6 to transit to the cis-Golgi compartment, where it is cleaved by site-1 protease and site-2
protease (the same enzymes that process the sterol response element binding protein
(SREBP) upon cholesterol deprivation). The cleaved cytosolic N-terminal fragment of
ATF6 migrates to the nucleus and acts as an active transcription factor (together with
ATF4 and spliced XBP1) to increase the expression of the genes encoding proteins
38 | Page
that function to augment the ER protein folding capacity (Malhotra and Kaufman,
2007). In addition, UPR activated genes stimulate ER biogenesis to compensate for
the increased demand for the protein-folding machinery and accelerate endoplasmic
reticulum associated degradation (ERAD) to remove terminally misfolded proteins
(Malhotra and Kaufman, 2007).
If protein repair by the ER chaperones is unsuccessful, aberrant proteins are cleared
from the ER by ERAD. During ERAD, aberrant proteins are translocated back to the
cytosol and degraded by the ubiquitin-proteasome system (Li et. al., 2011). Disposal
by ERAD involves the retro translocation of aberrant proteins across the ER
membrane to the cytosol, where they are ubiquitinated prior to degradation by the
proteasome (Figure 1.10) (Li et. al., 2011). Proteins that are ubiquitinated are
degraded by cytosolic proteasomes, with the free amino acids reused in protein
synthesis (Li et. al., 2011).
Figure 1.10 ER stress and the unfolded protein response. Stress in the ER stimulates the activation of the three stress receptors; PERK, ATF6 and Ire1, which are involved in the unfolded protein response (UPR). PERK phosphorylates eIF2α which inhibits general protein translation, allowing eIF2α-independent translation of ATF4, which activates transcription of chaperones such as GRP78. ATF6 undergoes specific proteolysis in the Golgi apparatus which leads to activation. IRE1 catalyses the alternative splicing of XBP1 mRNA leading to expression of the active XBP1 transcription factor. Together the three arms of the UPR block protein translation, increase chaperone expression and enhance ER-associated protein degradative pathways. Adapted from (Fulda et. al., 2010). 39 | Page
Misfolded proteins, and associated ER stress, appears to be an implicated feature of
neurodegenerative diseases. Misfolded proteins, UPR and ER stress induced cell
death could thus be involved in the pathogenesis of several neurodegenerative
disorders. Since these neurodegenerative disorders may be caused by specific mutant
proteins that accumulate as misfolded proteins and escape degradation, it is likely that
ER stress plays an important pathogenetic role.
Studies have suggested the following: firstly, a role for presenilin-1 in the activation of
IRE1 and induction of the UPR; secondly, a role for BiP in binding and limiting the production of beta-amyloid peptide; and thirdly, reduced cytotoxicity of the ß-amyloid peptide in caspase-12 deficient mice, suggesting a link between the role of the UPR and ER stress in Alzheimer’s disease (Rao and Bredesen, 2004). Oxidative stress and protein misfolding play critical roles in the pathogenesis of these neurodegenerative diseases that are characterised by fibrillar aggregates composed of misfolded proteins
(Malhotra and Kaufman, 2007). At the cellular level, neuronal death or apoptosis may be mediated by oxidative stress and/or ER stress. Upregulation of ER stress markers has been demonstrated in post-mortem brain tissues and cell culture models of many neurodegenerative disorders, including Parkinson’s disease, Alzheimer’s disease,
ALS and expanded polyglutamine diseases such as Huntington’s disease and spinocerebellar ataxias (Malhotra and Kaufman, 2007).
It seems likely that the protein chaperone BiP uses ATP/ADP exchange that is
essential to preserve ER function and prevent activation of the UPR. A reduced
efficiency of ATP-dependent BiP-mediated chaperone function may predispose it to
unfolded protein accumulation in the ER, activate the UPR and contribute to cellular damage and degeneration (Malhotra and Kaufman, 2007).
40 | Page
1.7 Mitochondria-Associated Membranes
Cellular processes require the proper communication between mitochondria and the
ER, this communication is facilitated by the mitochondria-associated endoplasmic
reticulum membrane (MAM) (Vance, J 2014). The MAM is characterised by direct
apposition to a mitochondrion, a unique lipid profile, and the expression of a unique
set of proteins involved in Ca2+ signaling, phospholipid biosynthesis, protein folding,
and membrane tethering (Vance, J 2014). The association of the MAM with a
mitochondrion is partially cytoskeletal independent and dynamically changes when
cytosolic Ca2+ level become elevated. The MAM is the centre for intermembrane
transportation of phospholipids and Ca2+ transmission to mitochondria that activates the tricarboxylic acid cycle (TCA) (Vance, J 2014). However, MAM is also involved in the interorganellar transport of cholesterol, ceramides, ATP, as well as in proteasomal protein degradation and lipid droplet formation. Recently the importance of interorganellar communication in the pathophysiology of neurodegenerative disorders has come more to light (Paillusson, et. al., 2016).
1.7.1 MAM Calcium Regulation
Calcium exchange via ER–mitochondrial contacts is facilitated following its release
from ER stores via inositol 1, 4, 5- trisphosphate (IP3) receptors (Paillusson, et. al.,
2016). Ca2+ is required by mitochondria to generate ATP via the TCA. Some of the mitochondrial enzymes involved in ATP synthesis, such as some dehydrogenases, are regulated by Ca2+ (Vance, J 2014). The concentrations of Ca2+ that is required to
elicit a response are high, however close contacts with the ER allows Ca2+ to be
released from the ER and can be achieved with high local concentrations capable of
41 | Page
driving an effect (Paillusson, et. al., 2016). Excessive uptake of Ca2+ by mitochondria can lead to opening of the mitochondrial permeability transition pore and apoptotic signalling occurs. Phospholipid exchange also occurs within the MAMs, this is important because the enzymes involved in some lipid biosynthesis are present in both organelles and so exchange is required for their production (Vance, J 2014).
MAMs are also known to be a specialised type of lipid raft linking intracellular trafficking between the mitochondria and ER. Both ER and mitochondria are transported by kinesin 1 and cytoplasmic dynein molecular motors (Paillusson, et. al., 2016). Kinesin
1 drives anterograde transport within neurons through the axons. Attachment of mitochondria to kinesin 1 involves the outer mitochondrial membrane (OMM) protein
Miro, which acts as a Ca2+ sensor; elevated Ca2+ levels alters their transport. Miro has been shown to localise to MAM contact sites and areas of ER have been shown to be co-transported with mitochondria (Paillusson, et. al., 2016). Potentially some ER may be transported with mitochondria through axons and Miro may sense Ca2+ exchange between the two organelles to regulate this transport in response to physiological stimuli (Paillusson, et. al., 2016).
1.7.2 Autophagy formation at MAMs
Autophagy is the process whereby damaged proteins and organelles are cleared from the cell by sequestering them in a double membrane-bound vesicle termed the autophagosome (Krols et.al. 2016). Subsequent delivery to the lysosome allows proteasomal breakdown and recycling of the substrates. Mitochondria contain many autophagy-related (ATG) proteins and regulators and have been proposed as an origin for phagophore formation, Many ATG proteins have been found to accumulate
42 | Page
specifically at ER-mitochondria contact sites in conditions of starvation, and there is
mounting evidence that MAMs might be the site of autophagosome formation (Krols
et.al. 2016). Lipid transfer from the ER to mitochondria during autophagosome
biogenesis is crucial. In mouse cells lacking MFN2, autophagy induction was disturbed
due to a decrease in lipid transfer towards mitochondria as a consequence of ER-
mitochondrial uncoupling and lipids transferred from the ER accumulate in the OMM,
from where they are trafficked to the expanding phagophore (Krols et.al. 2016).
In addition, during the selective autophagy of mitochondria, known as mitophagy, ER-
mitochondrial contact sites appear to constitute a platform promoting mitophagosome.
Mitochondria destined for degradation recruit the E3 ubiquitin ligase, Parkin, to the
OMM through PTEN-induced putative kinase 1 (PINK1) kinase activity (Krols et.al.
2016). Ubiquitination of Parkin substrates subsequently recruits autophagy proteins.
Accumulation of Double FYVE-containing protein 1 (DFCP1) along mitochondria that
are labelled for degradation with Parkin suggests a role for the ER/MAM as a
membrane source for the mitophagosome (Krols et.al. 2016). Drp1-mediated fission,
is in part mediated by contacts with the ER suggesting that it promotes mitophagy by
breaking off pieces of mitochondria thus enhancing engulfment by the
mitophagosome. Depolarisation of the IMM via Ca2+ over-loading could be involved in
PINK1 and Parkin translocation to the OMM, activating mitophagy (Krols et.al. 2016).
The presence at the MAMs of several important players in metabolism, such as
mechanistic target of rapamycin (mTOR), suggest that ER-mitochondrial contact could represent a site of crosstalk between sensors of the cells energetic needs and the early players in autophagy (Vance, J 2014). Additionally, through interorganellar
communication, dysfunctional mitochondria may be detected at MAMs, followed by
43 | Page
mitophagy induction and targeted removal of these unhealthy mitochondria (Vance, J
2014).
1.7.3 Lipid transportation via MAMs
Significant alterations in membrane lipid content is usually not tolerated by cells or
organelles, thus lipid composition of organelle membranes are highly regulated. The synthesis of the majority of membrane phospholipids in eukaryotic cells occurs on ER membranes and these lipids are subsequently distributed (Krols et.al. 2016). However not much is understood about the molecular mechanisms that are involved in interorganellar protein transport including lipid trafficking (Vance, J 2014).
Spontaneous diffusion of phospholipids between organelles through the cytosol is an
energetically unfavourable process and therefore occurs at very slow rates (Krols et.al.
2016). Lipids are transported between organelles by well characterised vesicle mediated processes that are used for protein trafficking, but mitochondria are not known to be connected to the ER by vesicular trafficking pathways (Krols et.al. 2016).
Non-vesicular mechanisms are important for the trafficking of lipids between organelles. Although the cytosol contains several lipid transfer proteins that can mediate phospholipid transfer between membranes, the net interorganellar transfer of lipids mediated by these soluble transport proteins is yet to be demonstrated (Krols et.al. 2016).
Compared to other organelles, mitochondria, particularly their inner membranes, are enriched in phosphatidylethanolamine (PE) (Vance, J 2014). PE is synthesised in mammalian cells by two major pathways that operate in spatially distinct organelles; in the ER via the CDP–ethanolamine pathway, and in mitochondria by the
44 | Page
decarboxylation of phosphatidylserine (PS) (Vance, J 2014). The PS decarboxylation
pathway provides the majority of PE in at least some cell types. For PE production by
this pathway, PS must be imported from its site of synthesis in the ER/MAM to IMM where PS decarboxylase (PSD) is located (Vance, J 2014). Thus, two distinct pools of
PE can potentially be generated: one in the ER, the other in mitochondria. In
mammalian cells, the PE that is synthesised by PSD in mitochondria is essential for
the normal functioning of mitochondria and cell survival (Krols et.al. 2016). A reduction
in the PE content of mitochondria can profoundly alter their morphology and reduce
cell growth, oxygen consumption and ATP production via the mitochondrial electron
transport chain (Vance, J 2014). The mechanism by which PS is imported into
mitochondria has been extensively studied in mammalian cells. The two mammalian
base-exchange enzymes that synthesise PS (PS synthase-1 and -2) are highly
enriched in MAM compared to the ER. The rate-limiting step in PE production from PS
is the transfer of PS from the ER/MAM to the OMM (Krols et.al. 2016).
Sterols and sphingolipids are minor constituents of mitochondrial membranes. The
processes by which these lipids are transported to, and imported into, mitochondria
have yet to be fully elucidated. MAMs have been reported to be enriched in cholesterol
relative to that of the ER (Krols et.al. 2016). Some studies with MAMs and
mitochondria have suggested that the depletion of cholesterol from MAMs promotes
the association between them and mitochondria, thereby increasing the conversion of
PS to PE (Krols et.al. 2016). Another functionally significant, sphingolipid constituent
of mitochondria is ceramide which can induce mitochondria-mediated apoptosis by
permeabilising the OMM (Vance, J 2014). However, the origin of the mitochondrial
pool of ceramide is not entirely clear. Although a large amount of cellular ceramide is
45 | Page
probably synthesised in the ER, some ceramide appears to be made in the MAM, and some ceramide is also synthesised in mitochondria (Krols et.al. 2016). However, despite evidence that PS is imported into mitochondria via the MAM, more investigation is required to elucidate the exact role attributed to MAMs in the import of other lipids into mitochondria from the ER (Vance, J 2014).
1.7.4 MAMs and Neurodegeneration
A major function of mitochondria is the generation of energy as ATP through oxidative phosphorylation via the electron transport chain. Several disorders exhibit alterations in mitochondrial morphology and/or calcium homeostasis, both of which can be altered by MAMs (Vance, J 2014). Neurons are highly polarised cells that rely heavily upon energy generated by mitochondria, particularly mitochondria that are recruited to dendrites and axons at sites far removed from the cell bodies (Krols et.al. 2016). Thus, the distribution of mitochondria within axons and dendrites is crucial for determining neuronal survival since mitochondria and elements of the ER are present in these neuronal processes, it is likely that axons and dendrites contain zones of contact between MAM and mitochondria (Krols et.al. 2016). Impaired regulation of mitochondrial calcium homeostasis provides one potential link between neuronal dysfunction and disruption of MAM–mitochondria contact sites since mitochondrial calcium modulates neurotransmitter release at the synapse (Vance, J 2014).
Alterations in mitochondrial dynamics and morphology are common features of neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's disease (Krols et.al. 2016). Whether alterations in mitochondrial fragmentation
46 | Page
highlight a response to the disease pathogenesis or directly contribute to the disease has not yet been established. Nevertheless, loss of either fusion or fission of mitochondria can severely impair mitochondrial function and neuronal survival (Vance,
J 2014). Several neurodegenerative diseases are characterised by either an abnormally high or low level of the mitochondrial fission factor, DRP1. DRP1 activity and regulation of mitochondrial fragmentation have been linked to the stability of mitochondrial–MAM contact sites (Krols et.al. 2016).
Excessive mitochondrial fragmentation and slower axonal transport of mitochondria are prominent features of Charcot–Marie–Tooth disease type 2A, a peripheral neuropathy that is caused by mutations in the mitochondrial fusion protein MFN2
(Vance, J 2014). Similarly, the neuropathy, dominant optic atrophy, is caused by mutations in another mitochondrial fusion factor, OPA1, resulting in diminished mitochondrial fusion. Consequently, since MAMs have been implicated in regulating mitochondrial fusion and fission, reduced formation of contacts between MAMs and mitochondria might contribute to the pathogenesis of these disorders (Vance, J 2014).
ER–mitochondrial contact have been linked to ER stress and increased UPR. The ER
UPR is an intracellular signalling pathway that is activated by the accumulation of unfolded proteins in the ER, which then stimulates transcriptional responses to modulate the protein-folding capacity of the ER (Krols et.al. 2016). Two of the known tethering proteins involved in connecting ER with mitochondria, MFN2 and vesicle- associated membrane protein-associated protein B (VAPB), have roles in the UPR. A variety of ER chaperones involved in protein folding, such as BiP, calnexin, calreticulin,
ERp44, ERp57, and the Sigma 1 receptor, are also present in MAMs, and structural
47 | Page
uncoupling of ER from mitochondria induces ER stress (Krols et.al. 2016). Thus, crosstalk between ER and mitochondria at MAM contact sites may have a role in facilitating stress responses and the UPR (Krols et.al. 2016).
MAMs have also been linked to formation of the inflammasome. Tissue damage and cell stresses, such as those that occur in neurodegenerative diseases, are sensed by the innate immune system through recognition receptors (Krols et.al. 2016). One class of these is the NOD-like receptors (NLRs), which sense abnormal cytosolic changes.
Upon activation, some NLRs, including NLRP3, form multiprotein complexes function to initiate proteolytic maturation of the proinflammatory cytokine interleukin 1b (IL-1b)
(Krols et.al. 2016). Reactive oxygen species from mitochondria are one signal for activation of the NLRP3 inflammasome. Recently, ROS was shown to cause the relocation of NLRP to MAMs, and this may provide a mechanism whereby NLRP senses damage to mitochondria to activate the inflammasome (Krols et.al. 2016).
48 | Page
1.8 Aims of this Thesis
SPTLC1 mutations reduce the activity of SPT in HSN-I patient-derived lymphoblasts.
However, the mechanism by which this occurs is yet to be identified. Notably, conflicting evidence has arisen in the literature. While SPT is inhibited in the disease state and thus should reduce or stop sphingolipid production, some studies have reported no detectable changes in sphingolipid metabolism (Dedov et. al., 2004;
Verhoeven et. al., 2006). Why this occurs is still unknown but some studies suggest that the overall reduction in SPT is not enough to globally reduce sphingolipid metabolism, yet axonal degeneration is still observed in HSN-I (Penno et. al., 2010).
Recent studies suggest that these mutations cause a deleterious gain of function with the production of toxic sphingolipids (Hornemann et. al., 2009).
Myers et. al., 2014, have observed by transmission electron microscopy (TEM) in
HSN-I patient-derived lymphoblasts that the mitochondrial morphology is completely altered and that the ER, wraps these morphologically challenged mitochondria, showing physical contact between the two organelle membranes.
49 | Page
We hypothesise that SPTLC1 mutations in HSN-I cause alterations in mitochondrial and ER proteins, and potentially cause alterations to lipids within the cells.
To investigate this hypothesis, this study aims to:
• Isolate clean mitochondrial protein fractions and to separate them into soluble
and membrane fractions.
• Determine by 2-dimensional gel electrophoresis (2DGE) alterations in the
protein profiles as a result of the SPTLC1 mutations.
• Identify the altered proteins observed.
• Isolate and determine the exact protein-protein interactions occurring due to
the altered proteins.
• Transiently transfect neuronal cells to produce the SPTLC1 mutations and
then confirm changes observed in patient-derived lymphoblast cell model in a
neuronal cell
• Determine whether other cellular functions are altered due to the disease state
and protein alterations.
• Identify microsomal protein alterations and interactions caused by the disease
state.
A patient-derived lymphoblast cell model was chosen as they endogenously express the SPTLC1 mutation; furthermore lymphoblasts are easily isolated and cultured lending themselves as a useful cell culture system (Verhoeven et. al., 2006).
50 | Page
Paper I
Published in Analytical Biochemistry
Contributions
SES carried out all experimentation, data analysis and formatted data.
51 | Page
Analytical Biochemistry 475 (2015) 1–3
Contents lists available at ScienceDirect
Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
Notes & Tips Optimal isolation of mitochondria for proteomic analyses ⇑ ⇑ Scott E. Stimpson a,b, Jens R. Coorssen b,c, , Simon J. Myers a,b, a Neuro-Cell Biology Laboratory, School of Science and Health, University of Western Sydney, Campbelltown, NSW 2560, Australia b Molecular Medicine Research Group, School of Medicine, University of Western Sydney, Campbelltown, NSW 2560, Australia c Molecular Physiology Unit, School of Medicine, University of Western Sydney, Campbelltown, NSW 2560, Australia article info abstract
Article history: Considering the key role of mitochondria in cellular (dys)functions, we compared a standard isolation Received 31 October 2014 protocol, followed by lysis in urea/detergent buffer, with a commercially available isolation buffer that Received in revised form 23 December 2014 rapidly yields a mitochondrial protein fraction. The standard protocol yielded significantly better overall Accepted 8 January 2015 resolution and coverage of both the soluble and membrane mitochondrial proteomes; although the kit Available online 14 January 2015 was faster, it resulted in recovery of only approximately 56% of the detectable proteome. The quality of ‘‘omic’’ analysis depends on sample handling; for large-scale protein studies, well-resolved proteomes Keywords: are highly dependent on the purity of starting material and the rigor of the extraction protocol. Mitochondria Ó 2015 Elsevier Inc. All rights reserved. Proteomics Two-dimensional gel electrophoresis
Considering the key role of mitochondria in cellular (dys) In efforts to optimize ongoing proteomic analyses, we thought functions, we compared a standard isolation protocol, followed by to switch from well-established protocols for intact mitochondrial lysis in urea/detergent buffer, with a commercially available isola- isolation and protein extraction to the promise of more rapid pro- tion buffer that rapidly yields a mitochondrial protein fraction. The tein isolation using a commercially available kit. Thus, we carried standard protocol yielded significantly better overall resolution and out a detailed assessment of mitochondrial proteins harvested coverage of both the soluble and membrane mitochondrial proteo- using the Amresco Mitochondrial Protein Isolation Buffer (AMPIB) mes; although the kit was faster, it resulted in recovery of only relative to those extracted by 2DE sample buffer following stan- approximately 56% of the detectable proteome. The quality of any dard isolation of mitochondria by sucrose gradient fractionation. ‘‘omic’’ analysis depends on sample handling; for large-scale A comparison of resulting total mitochondrial proteomes indicated protein studies, well-resolved proteomes are highly dependent on 510 ± 3 resolved protein species in the AMPIB extracts relative to the purity of the starting material and the rigor of the extraction the extracts of isolated mitochondria, 583 ± 7 (Fig. 1A and D). Nota- protocol. In many neurodegenerative disorders, mitochondria are bly, however, there was 87% overlap between the datasets. To known to play key roles in disease etiology [1]; because these assess this further, we subjected isolated mitochondria to a well- intracellular powerhouses have critical roles in the maintenance established protocol to recover separate total membrane and solu- and stability of cellular homeostasis, there are catastrophic conse- ble protein fractions prior to protein extraction and analysis. The quences with mitochondrial dysfunction. To better understand resulting 2DE analyses were consistently of markedly lower quality the molecular mechanisms underlying mitochondrial function, we for the AMPIB extracts (Fig. 1B and C) relative to those using stan- routinely analyzed the proteome of this organelle [2] using high- dard 2DE sample buffer (Fig. 1E and F); there was a marked resolution two-dimensional gel electrophoresis (2DE)1 [3] stained increase in the resolution of proteins in the low- to mid-molecu- using a modified colloidal Coomassie blue protocol [4]. lar-weight region (i.e., < 75 kDa) across the full range of 3 to 10 pI using the standard method as opposed to AMPIB. Essentially, no membrane proteins of less than approximately 75 kDa and no ⇑ Corresponding authors at: Molecular Medicine Research Group, School of Medicine, University of Western Sydney, Campbelltown, NSW 2560, Australia. Fax: soluble proteins of less than approximately 25 kDa were resolved +61 4620 3890 (J.R. Coorssen). Neuro-Cell Biology Laboratory, School of Science and from the AMPIB isolates. To determine what low-molecular-weight Health, University of Western Sydney, Campbelltown, NSW 2560, Australia. Fax: proteins were absent from the AMPIB isolation methods, a few +61 4620 3025 (S.J. Myers). protein species were identified from membrane and soluble E-mail addresses: [email protected] (J.R. Coorssen), [email protected] fractions—from the membrane annexin V (20 kDa, 5 pI) and (S.J. Myers). 1 Abbreviations used: 2DE, two-dimensional gel electrophoresis; AMPIB, Amresco voltage-dependent anion channel 1 (30 kDa, 8.7 pI) and from the Mitochondrial Protein Isolation Buffer; ER, endoplasmic reticulum; PBS, phosphate- soluble 60-kDa heat shock protein (60 kDa, 6 pI) and inorganic buffered saline; EDTA, ethylenediaminetetraacetic acid; 2D, two-dimensional. http://dx.doi.org/10.1016/j.ab.2015.01.005 0003-2697/Ó 2015 Elsevier Inc. All rights reserved. 2 Notes & Tips / Anal. Biochem. 475 (2015) 1–3
Total Membrane Soluble A B C 3 pI 10 3 pI 10 3 pI 10
250 250 250 150 150 150
100 100 100
75 75 75 37 37 37
25 25 25 MW (kDa) MW (kDa) 20 20 MW (kDa) 20
15 15 15 10 10 10
510 ± 3 346 ±6 280 ± 4
D E F 3 pI 10 3 pI 10 3 pI 10 250 250 250 150 150 150
100 100 100
75 75 75 37 37 37
25 25 25 MW (kDa) MW (kDa) 20 MW (kDa) 20 20
15 15 15 10 10 10
583 ± 7 550 ± 9 576 ± 6
Fig.1. Average union two-dimensional gel images of mitochondrial protein isolates from human lymphoblasts. Protein fractions isolated using AMPIB: (A) total mitochondrial proteins; (B, C) membrane and soluble mitochondrial proteins. Protein fractions isolated using sucrose density gradients: (D) total mitochondrial proteins; (E, F) membrane and soluble mitochondrial proteins. Here, 100 lg of protein was used for each 2DE analysis (n =3)[3]. 2DE was carried out, and resulting gels were imaged and analyzed using Delta2D software as described previously [4,7,9]. MW, molecular weight. pyrophosphatase 2 (39 kDa, 7 pI). This is in contrast to the manu- 123 4 facturer’s information indicating that, following cell lysis, the cyto- MTCO2 solic fraction yielded by buffer extraction is separated by 60 kDa centrifugation from an enriched mitochondrial fraction to increase the likelihood of detecting low-abundance proteins. Immunoblot analysis (Fig. 2) revealed the high-molecular-weight protein TOMM 22 MTCO2 present in both AMPIB and sucrose gradient isolated mito- 15 kDa chondrial proteins; however, the low-molecular-weight protein TOMM 22 was present only in the sucrose gradient isolated pro- Fig.2. Representative immunoblot images of MTCO2 and TOMM 22 from AMPIB teins correlating with Fig. 1. This apparent loss of low-molecular- and sucrose gradient isolated mitochondrial proteins (n = 3). Lanes 1 and 2 represent AMPIB isolated mitochondrial proteins, and lanes 3 and 4 represent weight mitochondrial proteins from the AMPIB kit may result from sucrose gradient isolated mitochondrial proteins. MTCO2 was present in both the kit not isolating intact mitochondria; such disruption of mem- AMPIB and sucrose gradient mitochondrial fractions; however, the lower molecular branes and loss of soluble proteins in the multiple centrifugation weight protein TOMM 22 was present only in the sucrose gradient mitochondrial steps could be the cause. Endoplasmic reticulum (ER) is a source fraction. of contamination during subcellular fractionations; to ensure that both isolation methods were free from contamination, we carried (1 Â 106) were centrifuged at 1500g for 5 min at 4 °C, and the out immunoblot analyses with specific ER markers, such as calnex- resulting pellet was washed in 10 ml of cold 1Â phosphate-buffered in and BiP, with immunoblot analysis revealing that there was no saline (PBS). The supernatant was removed, and the cell pellet was contamination in either isolation method. It would appear that suspended in 1 ml of ice-cold 1Â PBS, transferred to a 1.5-ml micro- AMPIB yields quantitatively different extraction of mitochondrial centrifuge tube, and centrifuged at 1500g for 5 min at 4 °C. The (and perhaps some cytosolic) proteins from cell lysates; in contrast resulting pellet was resuspended in Mitochondrial Protein Isolation to what might be expected, this selective extraction is even more Buffer (400 ll for 1 Â 106 cells). The cells were homogenized on ice pronounced when starting with isolated mitochondria. However, by passaging 20 times through a 1cc syringe with a 26.5-gauge if total mitochondria isolated are to be quantitatively analyzed, needle, followed by centrifugation at 1000g for 10 min at 4 °C. then the AMPIB method would seem to have quite limited capacity The supernatant was collected and transferred to a fresh 1.5-ml in terms of genuinely reflecting the mitochondrial proteome. Our tube, and the pellet (e.g., whole cells and nuclei) was discarded. data unequivocally show that to obtain a full protein profile for The collected supernatant was centrifuged at 14,000g for 15 min mitochondria (i.e., either the total protein pool, the mitochondrial at 4 °C, and the resulting supernatant was collected and transferred membrane pool, or the soluble mitochondrial protein pool), a tra- into a new tube labeled ‘‘cytosolic proteins.’’ The residual pellet ditional subcellular fractionation method using sucrose density (containing mitochondrial proteins) was resuspended in Mitochon- gradient centrifugation is most optimal. drial Protein Isolation Buffer (1 ml for 1 Â 106 cells), which was In the Amresco mitochondrial protein isolation protocol then centrifuged at 14,000g for 1 min at 4 °C. The supernatant (according to the manufacturer’s instructions), briefly, cells was discarded, and the pellet was resuspended in 40 llof Notes & Tips / Anal. Biochem. 475 (2015) 1–3 3
Mitochondrial Protein Isolation Buffer (i.e., as per 1 Â 106 cells of 2Â PBS subsequently added. Membranes were collected at starting material). 125,000g for 3 h. The supernatant was collected, and the mem- In mitochondrial isolation using sucrose density gradient brane pellet was resuspended in 1Â PBS and spun at 125,000g centrifugation [5,6], briefly, cells (1 Â 106) were pelleted at 1500g for a further 3 h. Washed membranes were solubilized in 2D solu- for 5 min at 4 °C and then washed in 10 ml of ice-cold 1Â PBS prior bilization buffer containing 8 M urea, 2 M thiourea, and 4% (w/v) to suspension in 10 ml of ice-cold CaSRB buffer (10 mM NaCl, CHAPS. Soluble protein fractions for both isolation methods were
1.5 mM CaCl2, and 10 mM Tris–HCl, pH 7.5) and incubation on concentrated using 3-kDa cutoff Millipore Amicon Ultra Centrifu- ice for 10 min. Cells were homogenized using a Dounce gal filters and resuspended in 4 M urea. homogenizer (Kimble–Chase, USA), and 7 ml of 2.5Â MS buffer (210 mM mannitol, 70 mM sucrose, 5 mM ethylenediaminetetra- References acetic acid [EDTA], and 5 mM Tris–HCl, pH 7.6) was added to restore isotonicity. Homogenate was centrifuged at 700g for [1] P.J. Hollenbeck, W.M. Saxton, The axonal transport of mitochondria, J. Cell Sci. 118 (2005) 5411–5419. 5 min to remove nuclei and unbroken cells. The resulting superna- [2] S. Myers, C. Malladi, R. Hyland, T. Bautista, R. Boadle, P. Robinson, G. Nicholson, tant was centrifuged at 15,000g for 10 min to pellet the crude Mutations in the SPTLC1 protein cause mitochondrial structural abnormalities mitochondria. Sucrose gradients were made in 4-ml high-speed and endoplasmic reticulum stress in lymphoblasts, DNA Cell Biol. 33 (2014) 399–407. centrifuge tubes (Beckman Coulter, USA) by overlaying 1 ml of [3] M. Churchward, R.H. Butt, J. Lang, K. Hsu, J. Coorssen, Enhanced detergent 1.7 M sucrose buffer (1.7 M sucrose, 10 mM Tris base, and extraction for analysis of membrane proteomes by two-dimensional gel 0.1 mM EDTA, pH 7.6) with 1.6 ml of 1.0 M sucrose buffer (1.0 M electrophoresis, Proteome Sci. 3 (2005) 5. sucrose, 10 mM Tris base, and 0.1 mM EDTA, pH 7.6). The mito- [4] V.J. Gauci, M.P. Padula, J.R. Coorssen, Coomassie blue staining for high sensitivity gel-based proteomics, J. Proteomics 90 (2013) 96–106. chondrial pellet was resuspended in 1.6 ml of 1Â MS buffer, lay- [5] P. Bozidis, C.D. Williamson, A.M. Colberg-Poley, Isolation of endoplasmic ered on top of the sucrose gradient, and centrifuged at 40,000g reticulum, mitochondria, and mitochondria-associated membrane fractions for 30 min at 4 °C. The mitochondrial band in the middle of the gra- from transfected cells and from human cytomegalovirus-infected primary fibroblasts, Curr. Protoc. Cell Biol. chap. 3 (2007). unit 3.27. dient was gently removed using a 20-gauge needle, transferred to a [6] A.V. Vaseva, U.M. Moll, Identification of p53 in mitochondria, Methods Mol. 1.5-ml tube, and centrifuged at 16,000g for 15 min. The resulting Biol. 962 (2013) 75–84. pellet was resuspended in two-dimensional (2D) solubilization [7] R.H. Butt, M. Lee, S.A. Pershahid, P. Backlund, S. Wood, J.R. Coorssen, An initial proteomic analysis of human preterm labour: placental membranes, J. buffer containing 8 M urea, 2 M thiourea, 4% (w/v) CHAPS, and a Proteome Res. 5 (2006) 3161–3172. cocktail of protease inhibitors [7–9] for total proteome analysis [8] R.H. Butt, T.A. Pfeifer, A. Delaney, T.A. Grigliatti, W.G. Tetzlaff, J.R. Coorssen, or resuspended in PBS for further separation into membrane and Enabling coupled quantitative genomic and proteomic analyses from rat spinal cord samples, Mol. Cell. Proteomics 6 (2007) 1574–1588. soluble fractionations. [9] E.P. Wright, M.A. Partridge, M.P. Padula, V.J. Gauci, C.S. Malladi, J.R. Coorssen, In membrane and soluble protein fractionation, harvested mito- Top-down proteomics: enhancing 2D gel electrophoresis from tissue chondrial proteins from both the AMPIB and sucrose gradient were processing to high-sensitivity protein detection, Proteomics 14 (2014) 872– 889. separated into membrane and soluble protein fractions as [10] R.H. Butt, J.R. Coorssen, Postfractionation for enhanced proteomic analyses: described previously [10]. Briefly, isolated mitochondria were routine electrophoretic methods increase the resolution of standard 2D-PAGE, placed in 20 mM Hepes for 3 min on ice with an equal volume of J. Proteome Res. 4 (2005) 982–991.
Paper II
Published in Journal of Chemical Biology
Contributions
SES carried out all experimentation, data analysis and wrote first draft manuscript.
55 | Page
J Chem Biol (2015) 8:25–35 DOI 10.1007/s12154-014-0125-x
ORIGINAL ARTICLE
Mitochondrial protein alterations in a familial peripheral neuropathy caused by the V144D amino acid mutation in the sphingolipid protein, SPTLC1
Scott E. Stimpson & Jens R. Coorssen & Simon J. Myers
Received: 17 July 2014 /Accepted: 29 October 2014 /Published online: 14 November 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Axonal degeneration is the final common path in as the pathogenic cause of HSN-I. Ultrastructural analysis of many neurological disorders. Subsets of neuropathies involv- mitochondria from HSN-I patient cells has displayed unique ing the sensory neuron are known as hereditary sensory neu- morphological abnormalities that are clustered to the ropathies (HSNs). Hereditary sensory neuropathy type I perinucleus where they are wrapped by the endoplasmic re- (HSN-I) is the most common subtype of HSN with autosomal ticulum (ER). This investigation defines a small subset of dominant inheritance. It is characterized by the progressive proteins with major alterations in abundance in mitochondria degeneration of the dorsal root ganglion (DRG) with clinical harvested from HSN-I mutant SPTLC1 cells. Using mito- symptom onset between the second or third decade of life. chondrial protein isolates from control and patient lympho- Heterozygous mutations in the serine palmitoyltransferase blasts, and a combination of 2D gel electrophoresis, immuno- (SPT) long chain subunit 1 (SPTLC1) gene were identified blotting and mass spectrometry, we have shown the increased abundance of ubiquinol-cytochrome c reductase core protein : 1, an electron transport chain protein, as well as the immuno- S. E. Stimpson S. J. Myers globulin, Ig kappa chain C. The regulation of these proteins Neuro-Cell Biology Laboratory, University of Western Sydney, may provide a new route to understanding the cellular and Penrith, Australia molecular mechanisms underlying HSN-I. J. R. Coorssen Molecular Physiology, University of Western Sydney, Penrith, Keywords Hereditary sensory neuropathy type 1 . Serine Australia palmitoyltransferase long chain subunit 1 . Mitochondria . S. E. Stimpson : J. R. Coorssen : S. J. Myers Ubiquinol-cytochrome c reductase core protein 1 Molecular Medicine Research Group, University of Western Sydney, Penrith, Australia Introduction S. E. Stimpson : J. R. Coorssen : S. J. Myers School of Science and Health, University of Western Sydney, Penrith, Australia Subsets of neuropathies involving the sensory neuron are : known as hereditary sensory neuropathies (HSNs). HSNs J. R. Coorssen S. J. Myers are associated with a range of clinical presentations, path- School of Medicine, University of Western Sydney, Locked Bag 1797, Penrith, NSW 2751, Australia ologic alterations, electrophysiological abnormalities and increasingly specific biochemical or molecular genetic S. J. Myers (*) abnormalities [10]. University of Western Sydney, Office 21.1.05, Campbelltown Hereditary sensory neuropathy type I (HSN-I) is the most campus, Locked Bag 1797, Penrith, NSW 2751, Australia e-mail: [email protected] common subtype of the HSN [10]. With autosomal dominant inheritance, it is characterised by the progressive degeneration J. R. Coorssen (*) of the dorsal root ganglion (DRG) and an onset of clinical School of Medicine, University of Western Sydney, Office 30.2.15, symptoms between the second or third decade of life [25]. Campbelltown campus, Locked Bag 1797, Penrith, NSW 2751, Australia HSN-I is rarely fatal but imposes lifelong disability with the e-mail: [email protected] disease initially manifesting with sensory loss in the feet, 26 J Chem Biol (2015) 8:25–35 followed by distal muscle wasting and weakness, and subse- cells using an integrated cell biology and proteomic method- quent positive sensory phenomena such as lancinating or ‘shoot- ology. This combined approach yields a detailed profile of ing’ pains. Heterozygous mutations in the serine mitochondrial proteins using coupled 2DE and mass spectro- palmitoyltransferase (SPT) long chain subunit 1 (SPTLC1)were metric technologies. The resulting proteomic analyses identi- identified as the pathogenic cause of HSN-I [1, 6]. The associ- fied, for the first time, a statistically significant increase in the ated mutations in this gene occur at single amino acids which are abundance of ubiquinol-cytochrome c reductase core protein I highly conserved throughout different species and are therefore and the immunoglobulin protein, Ig kappa chain C protein. likely to interfere with SPT functionality and structure [25]. SPT is a pyridoxal 5′-phosphate-dependent multimeric en- zyme that catalyses the first step in the biosynthesis of Materials sphingolipids, ceramide and sphingomyelin [14]; mutations in the SPT subunits thus result in potential dysfunction and All cell culture stock solutions, including RPMI-1640, foetal perturbations in sphingolipid synthesis and metabolism linked bovine serum (FBS), penicillin (100 U/mL), streptomycin to a variety of diseases, in particular HSN-I [26]. As the rate- (100 μg/mL), L-glutamine (2 mM), 4(2-hydroxyethyl)-1- determining enzyme in the de novo sphingolipid synthesis piperazineethane sulfonic acid (HEPES) (1 M) and pathway, SPT is therefore a key enzyme in the regulation of phosphate-buffered saline (PBS), were purchased from Gibco cellular sphingolipid content by condensation of palmitoyl Invitrogen (Australia). Cell culture consumables were pur- coenzyme A (CoA) with L-serine to form 3- chased from BD Falcon (Greiner, USA). MTCO2, Tomm ketodihydrosphingosine. SPT is composed of three known 22, ubiquinol-cytochrome c reductase core protein 1, Ig kappa subunits: SPTLC1, SPTLC2 and SPTLC3 [12]. SPT is a type chain C and GAPDH primary antibodies were purchased from 1 integral membrane protein with a single highly hydrophobic Abcam (USA). Secondary HRP mouse antibodies and 4', 6- domain in the amino-terminal region that anchors the enzyme diamidino-2-phenylindole (DAPI) stains were purchased from to the endoplasmic reticulum (ER) membrane [18, 26, 29, 30]. Sigma-Aldrich (Australia). Neurodegenerative diseases are a clinically heterogeneous group of chronic progressive illnesses with varying, but dis- tinct, clinical manifestations. Many of the genetic causes of Methods such disorders, including Huntington’s disease, some forms of familial amyotrophic lateral sclerosis (ALS), Charcot-Marie- EBV-transformed lymphoblasts Tooth syndrome type II (CMT II), Parkinson’s disease, Friedreich’sataxiaandAlzheimer’s disease, are well Epstein-Barr virus (EBV)-transformed control and V144D established; notably, despite obvious differences in underlying HSN-I patient lymphoblasts were kindly provided by Prof. aetiologies, a role for mitochondrial dysfunction is evident in Garth Nicholson (Molecular Medicine Laboratory, Anzac Re- the pathogenesis of all these diseases [9, 17, 19]. search Institute, Sydney) [7]. Mitochondrial dysfunction in neurons can lead to a myriad of different effects such as apoptosis, oxidative stress, Lymphoblast cultures excitotoxicity and destructive rises in intracellular calcium levels that contribute to several pathologies of the nervous Lymphoblasts were cultured in RPMI-1640 media (Gibco), system [13]. Mitochondrial transport is also intimately depen- supplemented with FBS (10 % v/v), penicillin (1 U/mL), dent upon the functional state of the cell and of mitochondria streptomycin (1 μg/mL), L-glutamine (2 mM) and HEPES themselves. Functioning mitochondria are essential for neuro- (1 M) at 37 °C in a humidified atmosphere of 5 % CO2,using nal survival due to their long axonal processes and high de- T75 cm2 culture flasks (Greiner, Interpath). Prior to use in mand for energy [17]. Mitochondrial membrane depolarisation biochemical assays, lymphoblasts were collected by centrifu- and inhibition of ATP synthesis have been shown to alter gation at 1500×g (5 min at RT) and washed in PBS. Cell movement of organelles; 80 % of slightly depolarised mito- counts were obtained using the Countess Automated Cell chondria in DRG neurons undergo retrograde movement, im- Counter (Invitrogen, Australia). plying that unhealthy mitochondria are returned to the cell body for repair or removal, reducing the number of mitochondria that Isolation of mitochondrial proteins are transported in the anterograde direction [22]. Recently, it has been shown that mitochondria from HSN-I Briefly, mitochondria were isolated using a sucrose den- patient cells, expressing the V144D SPTLC1 mutant, have sity gradient [2, 24]. Lymphoblasts were first centrifuged exceptionally electron-dense cristae [23]. Considering this at 1500×g for 5 min, and the cells were then washed in finding, in this study, we have investigated altered protein 10 mL of ice-cold 1× PBS prior to suspension in 10 mL expression changes from the mitochondria of HSN-I patient ice-cold CaSRB buffer (10 mM NaCl, 1.5 mM CaCl, J Chem Biol (2015) 8:25–35 27
10 mM Tris–HCl, pH 7.5) and left on ice for 10 min. to ensure complete reduction and alkylation. The equilibrated Cells were homogenised using a Dounce homogenizer IPG strips were then resolved in the second dimension using (Kimble-Chase, USA), and 7 mL of 2.5× MS buffer the MiniProtean II (Bio-Rad). (210 mM mannitol, 70 mM sucrose, 5 mM ethylenedi- A 12.5 % T, 2.6 % C polyacrylamide gel was buff- aminetetraacetic acid (EDTA), 5 mM Tris–HCl, pH 7.6) ered with 375 mM Tris buffer (pH 8.8), 0.1 % (w/v) was added to restore isotonicity. Homogenate was centri- sodium dodecyl sulphate and polymerised with 0.05 % fuged at 700×g for 5 min to remove nuclei and unbroken (w/v) ammonium persulphate and 0.05 % (v/v) cells. The resulting supernatant was centrifuged at tetramethylethylenediamine (TEMED). A stacking gel 15,000×g for 10 min to pellet the crude mitochondria. containing a 5 % T, 2.6 % C polyacrylamide buffered Sucrose gradients were made in 4-mL high-speed centri- with 375 mM Tris buffer (pH 8.8), 0.1 % (w/v)sodium fuge tubes (Beckman Coulter, USA) by adding 1 mL of dodecyl sulphate (SDS) and included 0.1 % 1.7 M sucrose buffer (1.7 M sucrose, 10 mM Tris-base, bromophenol blue was added to the resolving gel. The 0.1 mM EDTA, pH 7.6) overlayed with 1.6 mL of 1.0 M IPG strips were placed onto the stacking gel and over- sucrose buffer (1.0 M sucrose, 10 mM Tris-base, 0.1 mM laid with 0.5 % (w/v) low melting agarose dissolved in EDTA, pH 7.6). The mitochondrial pellet was resuspend- 375 mM Tris (pH 8.8), with 0.1 % (w/v)SDS.Electro- ed in 1.6 mL of 1× MS buffer and overlayed on top of the phoresis was carried out at 4 °C using pre-chilled Tris- sucrose gradient and centrifuged at 40,000×g for 30 min. glycine-SDS electrode buffer; 150 V was initially used The mitochondrial band, in the middle of the gradient, to rapidly drive proteins out of the IPG strips and into was gently removed using a 20-G needle, transferred to a the stacking gel for 5–10 min, and the voltage reduced 1.5-mL tube and centrifuged at 16,000×g for 15 min. The to 90 V for 2–3 h. The gels were fixed with 10 % resulting pellet was resuspended in 2D solubilisation buff- methanol and 7 % acetic acid for 1 h. The gels were er containing 8 M urea, 2 M thiourea, 4 % (w/v)CHAPS washed three times with distilled water for 20 min. and a cocktail of protease inhibitors. The gels were stained with colloidal Coomassie Blue (0.1 % (w/v) CCB G-250, 2 % (v/v) phosphoric acid, 10 % Protein concentration (w/v)ammoniumsulphate,20%(v/v)methanol)for20h,with constant shaking at RT [11] and subsequently de-stained five Determination of total cellular protein was performed using times with 0.5 M NaCl, 15 min each. Imaging of CBB-stained the EZQ Protein Estimation Assay (Invitrogen, Australia) as gels on the FLA-9000 imager (FUJIFILM, Tokyo, Japan) was previously described by Churchward et al. [4]. carried out at 685/750 excitation/emission with a photomultiplier tube (PMT) setting of 600 V and pixel reso- 2D gel electrophoresis lution set to 100 μm[11]. Analysis of 2D gel images was performed using Delta 2D software with automated spot de- Protein concentration estimations (EZQ assay) were per- tection (local background region, 96; average spot size, 32; formed on patient and control mitochondrial protein fractions; and sensitivity in percentage, 20.0) (version 4.0.8; a total of 100 μg protein was used for each 2DE analysis. 2DE DECODON GmbH, Gerifswald, Germany). was carried out as previously described [3, 11, 27]; briefly, mitochondrial proteins were reduced and alkylated in solu- tions containing total protein extraction buffer (containing SDS-PAGE and immunoblotting 8 M urea, 2 M thiourea and 4 % CHAPS without ampholytes), total extraction buffer with 2 % ampholytes, TBP/ Control and patient mitochondrial protein fractions (25 μg dithiothreitol (DTT) disulphide reduction buffer (2.3 mM total protein) were subjected to SDS-PAGE on 12.5 % resolv- tributyl phosphine and 45 mM DTT) and alkylation buffer ing gels and transferred to PVDF membrane. The membranes (230 mM acrylamide monomer). were blocked with 5 % skim milk in TBS buffer containing After incubation, the treated samples were added to 7-cm 0.1 % Tween-20. Whole membranes were blocked and incu- non-linear pH 3–10 immobilised pH gradient (IPG) strips bated with anti-Tomm22, anti-ubiquinol-cytochrome c reduc- (Bio-Rad ReadyStrip) and left to rehydrate for 16 h at RT. tase core protein 1, anti-Ig kappa chain C, anti-GAPDH, and Isoelectric focusing was then carried out at 20 °C using the anti-MTCO2 at 1:1,000, for 16 h at 4 °C. The membrane was Protean IEF Cell (Bio-Rad, USA); the initial 15 min at 250 V then incubated with secondary horse radish peroxidase anti- followed by linear ramping to 4,000 V at 50 μA/gel for a body (1:2,000 dilution) for 1 h at RT. Blots were developed further 2 h. After 2 h, isoelectric focusing was continued at using an enhanced chemiluminescence (ECL) detection kit 4000 V (constant) for a total of 37,500 Vh. (Pierce Thermo Scientific, USA). The membrane was devel- After IEF, the IPG strips were incubated in IEF equilibra- oped on CL-Xposure Film (Thermo Fisher Scientific, USA) tion buffer with 2 % DTT and 350 mM acrylamide monomer using an AGFA X-ray developer. 28 J Chem Biol (2015) 8:25–35
Mass spectrometry 1:200), and incubated for 1 h at RT. DAPI (1 μg/μL) was added to the cell suspension, and after 2 min, the cells were 2D gels were analysed for uniquely present or absent protein centrifuged and washed two times with PBS. Aliquots spots in control versus V144D mutant (i.e. all-or-none chang- (300 μL) were added to six-well culture plates containing es). The protein spots of interest were excised from gels and coverslips coated in Histogrip (Invitrogen, USA) and centri- de-stained overnight with 1:1 absolute acetonitrile and 25 mM fuged at 500×g for 10 min. The coverslips were washed in ammonium bicarbonate. The gel pieces were then reduced and warm PBS, left overnight to dry and mounted onto glass slides alkylated in 10 mM dithiothreitol (DTT) and 15 mM idoacetic prior to confocal imaging. The LSM 5 confocal microscope acid (IAA) and subsequently incubated with trypsin solution comprising the LSM 5 exciter laser scanning microscope with (10 ng/μL, pH 7.4) for 16 h at 37 °C. The digested peptides Axiovert 200 M inverted optical microscope (Carl Zeiss, Jena, were concentrated in a speedy vac and resuspended in 0.1 % Germany) was used for the acquisition of immunofluores- formic acid for subsequent analysis. LC-MS/MS analysis was cence images. For all acquisitions, a plan-Apochromat 63×/ carried out on a nanoAquity UPLC (Waters Corp., Milford, 1.40 oil DIC objective was used with an excitation wavelength MA, USA) linked to a Xevo QToF mass spectrometer from of 405 and 543 nm. Fluorescence was detected in a bandwidth Waters (Micromass, UK). Three microlitres of digested pep- of 460–560 nm. Data was analysed using the Carl Zeiss Zen tides was loaded onto a nanoAquity C18 BEH130 column 2009 Software. (1.7 μm, 75 μm×150 mm) and then resolved and eluted from the column using a binary gradient program at a flow rate of Flow cytometry 0.4 μL/min: mobile phase A was 0.1 % formic acid in water, and mobile phase B was 0.1 % formic acid in acetonitrile. The For flow cytometry analyses, lymphoblasts were isolated as nano-UPLC gradient was as follows: 0 min, 99:1 A/B; 1 min, above; the cells were then suspended in 1 mL of 4 % parafor- 99:1 A/B; 31 min, 50:50 A/B; 33 min, 15:85 A/B; 36 min, maldehyde in PBS and incubated for 15 min at RT. Thereafter, 15:85 A/B; and 37 min, 99:1 A/B. The mass spectrometer was the cell suspension was centrifuged at 1,000×g for 5 min at RT operated in positive ESI mode with capillary voltage of and then resuspended in 0.3 % Triton X-100 for 15 min at 2.3 kV, cone voltage of 25 V and source temperature of 37 °C. Cells were incubated in primary antibody for 1 h at RT. 80 °C. Targeted MS/MS data or data-dependent acquisition After incubation, the cell suspension was centrifuged at (DDA) was acquired with collision energy ramping from 30 to 1,000×g for 5 min and the pellet resuspended in secondary 40 eVon the eight most intense peaks of MS mode with mass antibody, anti-mouse FITC (Millipore, 1:200), for 1 h at RT. ranges of 350–1,500 Da. The data were acquired using The cell suspension was washed two times in PBS and Masslynx software (version 4.1, Micromass, UK). analysed using the MACSQuant flow cytometer (Miltenyi The acquired DDA data from Masslynx were converted to Biotech). Live cells were gated, and the mean of fluorescence PKL files by Protein Lynx Global Server (Waters, UK). The was obtained per 10,000 cellular events with an excitation MS/MS data files were searched against SwissProt database wavelength of 488 nm and emission filter of 525/50 nm. Data with semi-trypsin as the enzyme. The following parameters was analysed with MACSQuant software. were used in Mascot for identification of the peptides: maxi- mum missed cleavage of 2; positive peptide charge of 2, 3 and 4; peptide mass tolerant of 0.5 Da in MS and MS/MS data base; fixed modification,carbamidomethyl (C); and variable Results modifications,oxidation (M). Expression of GAPDH and mitochondrial markers in HSN-I Immunofluorescence patient-derived lymphoblasts
Lymphoblasts (1×106 cells) were suspended in 1 mL of warm Quantitative immunoblotting was used to determine whether PBS. After centrifugation at 1,000×g for 5 min, at RT, the cell both Tomm 22 (translocase of the outer mitochondrial mem- pellet was resuspended in 4 % paraformaldehyde for 15 min. brane) and MTCO2 (cytochrome c oxidase subunit II) were Cells were then placed in 0.5 % TritonX-100 and incubated at expressed in protein lysates from isolated mitochondrial frac- 37 °C for 30 min. The cells were then centrifuged and blocked tions and in the total cell extracts from control and V144D in 5 % BSA solution at 37 °C for 30 min. After washing in mutant HSN-I patient-derived lymphoblasts (V144D cells) PBS, the cells were resuspended in primary antibody, MTCO2 (Fig. 1a, b). Analyses indicated that there was no statistically (Abcam, 1:50), ubiquinol-cytochrome c reductase core pro- significant change in expression of these proteins in either tein 1 and Ig kappa chain C (Abcam, 1:100) and incubated for protein sample (Fig. 1d), with values of 1.74 and 4.24 for 1 h at RT. The cells were subsequently washed and resuspend- control and V144D Tomm 22 mitochondrial fractions, and 5.6 ed in secondary antibody, anti-mouse rhodamine (Millipore, and 11.30, and 1.46 and 2.06 from control and V144D J Chem Biol (2015) 8:25–35 29
Fig. 1 Immunoblot analysis of Tomm 22, MTCO2 and GAPDH. Expression of GAPDH and mitochondrial markers in HSN-I patient-derived lymphoblasts. a Immunoblot of Tomm 22; b immunoblot of MTCO2; c immunoblot of GAPDH. Lanes 1 and 2 represent control mitochondrial proteins; lanes 3 and 4 represent control total proteins; lanes 5 and 6 represent V144D mitochondrial proteins; and lanes 7 and 8 represent V144D total proteins. Representative graphs showing no statistically significant (p>0.05) difference between mitochondrial control and patient lymphoblast lysates (n=3) of Tomm 22 (blue)andMTCO2 (red)(d)andGAPDH(e)are shown. All blots were normalised to GAPDH. Error bars depict SE of means
mitochondrial and total fractions respectively. As a compara- 583±7 and 571±6 detectable proteins in the control and tive housekeeping protein for quantitation, as well as a protein V144D cell mitochondria, respectively. Further analysis of loading control, analysis of GAPDH was carried out in order the total mitochondrial protein profiles from control and to establish relative protein expression levels (Fig. 1c). De- V144D cells revealed three consistent ‘all-or-none’ protein spite some variability, 3,167.06 and 1,973.15 for control and changes in the V144D cells relative to control lymphoblasts. V144D mitochondrial fractions and 7,766.75 and 7,091.37 for These protein species were located at pI/MW (kDa) coordi- control and V144D total fractions, there were no statistically nates of 5.7/55, 6.6/24 and 8.3/24 (Table 1). Subsequent LC/ significant changes in the expression of GAPDH in either the MS analysis identified these proteins to be ubiquinol- controls or V144D cells (Fig. 1e). cytochrome c reductase core protein 1 and Ig kappa chain C.
2D gel images of mitochondrial proteins from control Expression of ubiquinol-cytochrome c and Ig kappa and patient-derived lymphoblasts from HSN-I patient-derived lymphoblasts
Total isolated mitochondrial proteins were resolved and quan- Immunoblot analysis was performed on isolated mitochondria titatively assessed using a refined 2DE protocol (Fig. 2)[3, 11, and total cell lysates from control and V144D cells in order to 27]. Mitochondrial proteins from control and V144D cells further quantitatively assess changes in protein abundance. were resolved using mini gel format; image analysis indicated Blots were normalised to GAPDH as shown in Fig. 1c. These 30 J Chem Biol (2015) 8:25–35 J Chem Biol (2015) 8:25–35 31
Fig. 2 Representative images of 2D gels and regions of mitochondrial increase) respectively, relative to the stained controls (Fig. 5c). proteins from control and patient-derived lymphoblasts. a Control and There was an increase in Ig kappa peak intensity in the V144D V144D mitochondrial proteins; b resolved protein species having a vastly ∼ altered abundance as indicated (red arrow). The molecular weights are in cells (an 1.5-fold increase) with an observed peak width kilodaltons (kDa), and the IEF dimension is in pH units increase in the control cells.
data showed a concomitant increase in the amount of ubiquinol-cytochrome c (5.01 compared to 2.59) in the protein Discussion samples from V144D cells compared to the control samples (p<0.05;Fig. 3a, c). In contrast, immunoblotting confirmed a HSN-I is an autosomal dominant sensory neuropathy resulting significant decrease in the amount of Ig kappa protein in the in the dying back of the peripheral sensory neurons and a total lysates of V144D cells compared to controls (1.47 com- progressive degeneration of the dorsal root ganglia [21]. HSN- pared to 2.71) (p<0.05; Fig. 3b, d). I is caused by missense mutations in the SPTLC1 gene, but the actual cellular and molecular mechanisms underlying the dis- SPTLC1 mutations cause no change to intracellular ease remain poorly understood. A recent study has shown localisation mitochondrial ultrastructural changes to be linked with ER stress in HSN-I cells [23]. Using an integrated proteomic and In order to establish the intracellular localisation and abun- cell biology approach, we have identified a significant in- dance of ubiquinol-cytochrome c, immunofluorescence stud- crease in the amount of ubiquinol-cytochrome c reductase ies were performed on control and V144D cells. There were core protein 1 in the mitochondria from HSN-I (V144D) no detectable changes in intracellular localisation of MTCO2 patient-derived lymphoblasts relative to control lymphoblasts. or ubiquinol-cytochrome c protein in control and V144D Of further interest, there is a decreased amount of the immu- cells, whereby both proteins were peripherally localised. noglobulin protein, Ig kappa chain C, in the V144D cells as When the intracellular localisation of Ig kappa protein was well as a change in the pI of this protein. assessed, there was also no detectable change between the Mitochondria are the intracellular energy producing organ- control or V144D cells and the localisation of the protein was elles where substrates are metabolised to fuel oxidative phos- also unchanged in the periphery (Fig. 4). phorylation through the electron transport chain within their inner membrane [31]. The electron transport chain consists of Relative quantification of MTCO2, ubiquinol-cytochrome c four multimeric enzyme complexes. These complexes facili- and Ig kappa in V144D cells tate the flow of electrons from the reducing substrates to oxygen to build a proton gradient required for ATP generation Immunostained MTCO2, ubiquinol-cytochrome c and Ig kap- [5]. Ubiquinol-cytochrome c reductase core protein 1 (also pa control and V144D cells were analysed using fluorescence- known as cytochrome b-c1 complex subunit I) is a central assisted cell sorting (FACS) to determine the total fluores- component of the electron transport chain, catalysing the cence per cell (Fig. 5a, b). There was no overall shift or oxidization of ubiquinol (ubihydroquinone) and reduction of increase in the fluorescence histograms from control and cytochrome c [5]. V144D mutant cell populations with respect to the MTCO2 In order to test whether there were protein changes due protein. However, there was a marked increase in the relative to the HSN-I SPTLC1 mutation, we assessed the fluorescence intensity of ubiquinol-cytochrome c in the proteomes of mitochondria isolated from control and V144D cells compared to that of control lymphoblasts with V144D cells using high-resolution 2DE, to enable quan- an increase in relative fluorescence of 80±1.5 OD (a 2.2-fold titative assessments (Fig. 2a). Tomm 22 and MTCO2,
Table 1 Mass spectrometry
Spot Protein identified Accession Number of unique Sequence Mascot Predicted Predicted Mascot Mascot number number peptides matched coverage protein pI MW (kDa) pI MW (kDa) score
I Ubiquinol-cytochrome c P31930 4 18 % 208 5.7 55.00 5.9 53.3 reductase core protein 1 II Ig kappa chain C P01834 14 88 % 908 6.6 22.00 5.5 11.7 III Ig kappa chain C P01834 12 86 % 1280 8.3 22.00 5.5 11.7
Summary table of mascot protein identification. LC-MS/MS and Mascot Database searching identified ubiquinol-cytochrome c reductase core protein 1 and Ig kappa chain C from V144D patient-derived lymphoblast and Ig kappa chain C from control lymphoblasts isolated mitochondria 32 J Chem Biol (2015) 8:25–35
Fig. 3 Immunoblot blot analysis of ubiquinol-cytochrome c and Ig kappa chain C. Expression of ubiquinol-cytochrome c and Ig kappa chain C from HSN-I patient-derived lymphoblasts. a Immunoblot detection of ubiquinol-cytochrome c. b Immunoblot detection of Ig kappa chain C. Lanes 1 and 2 represent control mitochondrial proteins; lanes 3 and 4 represent control total proteins; lanes 5 and 6 represent V144D mitochondrial proteins; and lanes 7 and 8 represent V144D total proteins. c, d Representative graphs showing statistically significant (*p<0.05) difference between control patient lymphoblasts and the mutant V144D lymphoblasts of ubiquinol-cytochrome c and Ig kappa chain C respectively (n=3). Blots were normalised to GAPDH (Fig. 1c). Error bars depict SE of means
Fig. 4 Immunofluorescence of ubiquinol-cytochrome c, MTCO2 and Ig kappa chain C. SPTLC1 mutations cause no change to the intracellular localisation of ubiquinol-cytochrome c, MTCO2 and Ig kappa chain C. Representative confocal micrographs showing ubiquinol-cytochrome c, MTCO2 and Ig kappa chain C stained lymphoblasts (red)andDAPI nuclear stain (blue). Scale bar= 5 μm J Chem Biol (2015) 8:25–35 33
Fig. 5 Flow cytometry analysis of ubiquinol-cytochrome c, MTCO2 and of the relative fluorescence intensity of ubiquinol-cytochrome c (a), Ig kappa chain C. Relative quantification of ubiquinol-cytochrome c, MTCO2 (b) and Ig kappa chain C (c) in control and V144D patient- MTCO2 and Ig kappa chain C in HSN-I patient-derived lymphoblasts derived lymphoblasts. Red histogram represents the V144D patient lym- expressing the V144D mutant SPTLC1 genes. Flow cytometry analysis phoblasts, and blue histogram represents control lymphoblasts (n=3)
mitochondrial marker proteins, confirmed the quality of reductase core protein 1 in V144D cells relative to control the isolated mitochondrial fraction used for analysis. cells. There were no statistically significant changes in expres- Ubiquinol-cytochrome c reductase core protein 1 functions sion of these mitochondrial markers, suggesting that these to ensure that the electron transfer rate is optimal and that fast proteins turn over at a constant rate in both the control dissociation of electrons occurs following transfer [8]. These and V144D cells. Analysis of GAPDH from control and processes are essential to maintain the electron flow and to V144D cells also indicated no significant changes in this prevent any potential electron leaks or break down of the protein (Fig. 1e). There was a protein selectively detected respiratory chain [8]. Ubiquinol-cytochrome c reductase core in the V144D cells at pI 5.7 and molecular weight protein 1 is also involved in free radical generation, producing 55 kDa; this protein was undetectable in the control reactive oxygen species (ROS) within mitochondria. ROS protein profile (Fig. 2b). This protein proved to be production can disrupt the homeostasis and interactions within ubiquinol-cytochrome c reductase core protein 1. Quanti- the mitochondrial matrix resulting in the loss of the oxidative tative immune-blotting confirmed a significant (i.e. 2- phosphorylation, along with the disruption of mitochondrial fold) increase in the amount of ubiquinol-cytochrome c functions and physiology leading to cell death [5]. 34 J Chem Biol (2015) 8:25–35
Further analysis was performed using immunostaining there is also a potential immunological component to this (Fig. 4) and FACS (Fig. 5) to determine cellular localisation neurodegenerative disorder, characterised by significantly de- and expression of ubiquinol-cytochrome c reductase core creased amounts of the immunoglobulin protein, Ig kappa protein 1 and MTCO2. Immunostaining revealed no distinct chain C. The exact role of this protein and its relationship to difference between the localisation of ubiquinol-cytochrome c HSN-I will be the aim of further investigations. Are reduced reductase core protein 1 in the control versus V144D cells. levels of this protein responsible for apparent reductions in FACS analysis correlated with the previous expression data, cellular repair responses? Clearly, far more work is required to exhibiting a concomitant 2.2-fold increase in fluorescence fully elucidate the mechanisms underlying peripheral neurop- intensity of ubiquinol-cytochrome c reductase core protein 1 athies, but the novel findings arising from this first coupled, in the V144D cells relative to controls. In contrast, the quantitative proteomic cell biological analysis provide critical MTCO2 protein displayed no increase in fluorescence inten- new directions not even previously hypothesised. The find- sity in control versus patient (V144D) lymphoblasts. ings in this study, coupled with the findings of Marshall et al. Furthermore, in the comparison of the control and V144D [20] and Myers et al. [23], suggest that there may well be cell proteomes, we identified two other marked protein chang- underlying molecular alterations broadly common to es. Both of these proteins were located in the 24-kDa molec- neurodegenerations as a whole, linked to both mitochondria ular weight region but were located in different pI (6.6 and 8.3 and lipids. respectively) regions. Both proteins were identified as Ig kappa chain C. The cause of the apparent shift in pI is yet to Acknowledgments We are grateful to Prof. Garth Nicholson (Molecu- be determined; however, it seems likely due to an as yet lar Medicine Laboratory and Northcott Neuroscience Laboratory Anzac unidentified posttranslational modification (potentially a gly- Research Institute, Sydney) for providing all EBV-transformed lympho- blast lines used in this study. SES was supported by APA Research cosylation, phosphorylation or methylation, or any combina- Scholarship and the UWS School of Science and Health Postgraduate tion of the three). This is the first study to identify a change in research fund. SJM notes the continuing support of an anonymous private an immunoglobulin due to the SPTLC1 mutation causing foundation. JRC acknowledges the support of the UWS School of HSN-I. The Ig kappa light-chain constant region undergoes Medicine. little to no variation in human immunoglobulins and forms part of the five immunoglobulin classes produced in mature B cells [16]. While B cells inherently produce Ig kappa, we have References determined a statistically significant decrease in the amount of Ig kappa chain C in the total cell lysate of V144D cells relative 1. Bejaoui K, Wu C, Scheffler MD, Haan G, Ashby P, Wu L, De Jong P, to control lymphoblasts. Immunoglobulin light chains have Brown RH Jr (2001) SPTLC1 is mutated in hereditary sensory neuropathy, type 1. Nat Genet 27:261–262 been implicated in and are biomarkers of diseases such as 2. Bozidis P, Williamson CD, Colberg-Poley AM (2007) Isolation of multiple myeloma and primary systemic or amyloid light- endoplasmic reticulum, mitochondria, and mitochondria-associated chain (AL) amyloidosis [28]. In these diseases, it has been membrane fractions from transfected cells and from human shown that the free Ig kappa chain associates with cytomegalovirus-infected primary fibroblasts. Curr Protoc Cell Biol. doi:10.1002/0471143030.cb0327s37 sphingomyelin on the plasma membrane of the myeloma cells 3. Butt RH, Coorssen JR (2005) Postfractionation for enhanced prote- forming aggregates that are required for intercalation with omic analyses: routine electrophoretic methods increase the resolu- membranes [15]. This further suggests the important role tion of standard 2D-PAGE. J Proteome Res 4:982–991 sphingolipids play in these disease processes. 4. Churchward M, Butt RH, Lang J, Hsu K, Coorssen J (2005) Enhanced detergent extraction for analysis of membrane proteomes The novel findings in this study thus suggest a link to by two-dimensional gel electrophoresis. Proteome Sci 3:5 oxidative phosphorylation, via ubiquinol-cytochrome c reduc- 5. Crofts AR (2004) The cytochrome bc1 complex: function in the tase core protein 1 (perhaps through regulation of ROS pro- context of structure. Annu Rev Physiol 66:689–733 duction), and that ensuing interference with energy production 6. Dawkins JL, Hulme DJ, Brahmbhatt SB, Auer-Grumbach M, ultimately leads to axonal retraction that is the hallmark char- Nicholson GA (2001) Mutations in SPTLC1, encoding serine palmitoyltransferase, long chain base subunit-1, cause hereditary acteristic of hereditary sensory neuropathy. Clearly, if ROS sensory neuropathy type I. Nat Genet 27:309–312 production increases with the increased levels of ubiquinol- 7. 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Paper III
Published in Biochemistry and Analytical Biochemistry
Contributions
SES carried out all experimentation, data analysis and wrote first draft manuscript
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Research Article Open Access Isolation and Identification of ER Associated Proteins with Unique Expression Changes Specific to the V144D SPTLC1 Mutations in HSN-I Scott E Stimpson1,3,4, Antonio Lauto4,6, Jens R Coorssen2,3,4,5* and Simon J Myers1,3,4,5* 1Neuro-Cell Biology Laboratory, Western Sydney University, Australia 2Molecular Physiology, Western Sydney University, Australia 3Molecular Medicine Research Group, Western Sydney University, Australia 4School of Science and Health, Western Sydney University, Australia 5School of Medicine, Western Sydney University, Australia 6Biomedical Engineering and Neuroscience (BENS), Western Sydney University, Australia
Abstract Axonal degeneration is the final common path in many neurological disorders. Hereditary sensory neuropathies (HSN) are a group of neuropathies involving the sensory neurons. The most common subtype is autosomal dominant hereditary sensory neuropathy type I (HSN-I). Progressive degeneration of the dorsal root ganglion (DRG) neuron with an onset of clinical symptoms between the second or third decade of life characterises HSN-I. Mutations in the serine palmitoyltransferase (SPT) long chain subunit 1 (SPTLC1) gene cause HSN-I. The endoplasmic reticulum (ER) is a dynamic organelle that houses the SPTLC1 protein. Ultra structural analysis has shown the ER in the HSN-I mutant cells to wrap around dysfunctional mitochondria and tethers them to the perinucleus. This investigation establishes that the V144D mutant of SPTLC1 alters the expression of and potentially interacts with a set of proteins within the ER. Using ER protein lysates from HSN-I patient and control lymphoblasts: we have identified a change in regulation of five proteins; Hypoxia Up regulated Protein 1: Chloride intracellular channel protein 1: Ubiqutin-40s Ribosomal protein S27a: Coactosin and Ig Kappa chain C. The expression and regulation of these proteins may help to establish a link between the ER and the ‘dying back’ process of the DRG neuron.
Keywords: Hereditary sensory neuropathy type 1; Serine complex sphingolipid metabolic pathway consisting of 3 subunits; palmitoyltransferase long chain sub-unit 1; Endoplasmic reticulum; SPTLC1: SPTLC2 and SPTLC3 [2]. Mutations within SPTLC1 result Oxidative stress in potential dysfunction and perturbations in sphingolipid synthesis and metabolism causing HSN-I [2]. These mutations are single Abbreviations: HSN: Hereditary Sensory Neuropathies; HSN-I: amino acid changes in the SPTLC1 gene that encodes the long-chain Hereditary Sensory Neuropathy Type I; DRG: Dorsal Root ganglion; base one (LCB1) subunit [3]. HSN-I is the most common subtype of SPT: Serine palmitoyl transferase; SPTLC1: Serine palmitoyl transferase HSN [4]. HSN-I is an autosomal dominant disorder characterised by long chain subunit 1; ER: Endoplasmic Reticulum; LCB1: Long- degeneration of the DRG neuron and with a clinical onset between the Chain Base One; UPR: Unfolded Protein Response; UPS: Ubiquitin second or third decades of life. Proteasome System; ROS: Reactive Oxygen Species; ORP-150: Hypoxia up Regulated Protein 1; CLIC1: Chloride Intracellular Channel The ER carries out extensive quality control of proteins to enable Protein 1; RPS27a: Ubiqutin-40s Ribosomal Protein s27a; COTL1: normal cellular function. Disruption to the function of the ER or loss Coactosin; HRP: Horse Radish Peroxidase; IEF: Isoelectric Focusing; of its integrity leads to ER stress [5]. ER stress can be characterised TEMED: Tetramethylethylenediamine; PMT: Photomultiplier Tube; by the accumulation of unfolded proteins and changes in calcium 2DE: Two Dimensional Gel Electrophoresis; IAA: Idoacetic acid; LC/ homeostasis within the ER with stress activating the unfolded protein MS: Liquid Chromatography/Mass Spectrometry; ECL: Enhanced response (UPR) [6]. The UPR and its signalling components can change Chemiluminescence; FACS: Fluorescence’s Assisted Cell Sorting; the expression of specific proteins such as, those designated for the ER Grp170: Glucose related protein 170; G-actin: Globular actin; F-actin: chaperones; the enhancement of degradation of misfolded (mutant or Filamentous actin unfolded) proteins; and the inhibition of protein synthesis to decrease Introduction the load within the ER [6]. The ER is an intracellular organelle which supports and maintains a plethora of functions critical for cellular survival. The ER plays a crucial *Corresponding author: Simon J. Myers, Western Sydney University, role in many aspects of protein compartmentalisation which include Campbelltown campus, Locked Bag 1797, Penrith, Australia, Tel: +61 02 4620 membrane translocation, folding, post-translational modification, and 3383; E-mail: [email protected] transport of both membrane and soluble proteins [1]. In addition, the Jens Coorssen, Professor, Western Sydney University, Campbelltown campus, Locked ER is involved in the synthesis of phospholipids and steroids and also Bag 1797, Penrith, Australia, Tel: +61 4620 3802; E-mail: [email protected] 2+ in the regulation of Ca homeostasis. The ER is organised in a complex: Received: February 06, 2016; Accepted: February 15, 2016; Published February continuous network of tubules and sheets that includes the nuclear 18, 2016 envelope and extends throughout the cytosol into the cell periphery Citation: Stimpson SE, Lauto A, Coorssen JR, Myers SJ (2016) Isolation and [1]. Nascent polypeptide chains fold, acquire further modifications Identification of ER Associated Proteins with Unique Expression Changes Specific like glycosylation and disulphide bonds and often assemble with other to the V144D SPTLC1 Mutations in HSN-I. Biochem Anal Biochem 5: 248. subunits before traversing further along the secretory pathway with in doi:10.4172/2161-1009.1000248 the ER. These processes are both assisted and monitored by molecular Copyright: © 2016 Stimpson SE, et al. This is an open-access article distributed chaperones [1]. under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the SPT is an ER bound and key rate determining enzyme in the original author and source are credited.
Biochem Anal Biochem ISSN: 2161-1009 Biochem, an open access journal Volume 5 • Issue 1 • 1000248 Citation: Stimpson SE, Lauto A, Coorssen JR, Myers SJ (2016) Isolation and Identification of ER Associated Proteins with Unique Expression Changes Specific to the V144D SPTLC1 Mutations in HSN-I. Biochem Anal Biochem 5: 248. doi:10.4172/2161-1009.1000248
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Disturbed functions of the ubiquitin proteasome system (UPS): mM EDTA, 5 mM Tris-HCl, pH 7.6) was added to restore isotonicity. responsible for the degradation of cytosolic: ER and synaptic proteins: Homogenate was centrifuged at 700 x g for 5 min to remove nuclei and can contribute to ER stress [5]. A common occurrence in many unbroken cells. The resulting supernatant was centrifuged at 15,000 x g neurodegenerative disorders is the accumulation and deposits of for 10 mins to remove mitochondria. A sucrose gradient was made in misfolded proteins that affects various cell signalling systems: as well as 15 mL high speed centrifuge tubes (Beckman Coulter, USA) by adding neuronal connectivity and cell death such as in Alzheimer’s: Parkinson’s 2 mL of 2.0 M sucrose buffer (2.0 M sucrose, 10 mM Tris-base, 0.1 mM and Huntington’s disorders. EDTA, pH 7.6) overlayed with 3.0 mL of 1.5 M sucrose buffer (1.5 M sucrose, 10 mM Tris-base, 0.1 mM EDTA, pH 7.6) and 3.0 mL of 1.3 The activity of the UPS is mitigated in degenerative disorders by M sucrose (1.3 M sucrose, 10 mM Tris-base, 0.1 mM EDTA, pH 7.6) the protein aggregation or by enhanced oxidative stress with other toxic ER containing supernatant was loaded on top of the sucrose gradient products [5]. Dysfunctional UPS in turn causes increased accumulation and spun at 152,000 x g for 70 min. The ER band, the interface of the of proteins in the cell leading to ER stress and the aggravation of the 1.5 M and 1.3 M sucrose, was gently removed using a 20 G needle, disorder [5]. Additionally the effect of environmental toxins, reactive transferred to a 4 mL high speed centrifuge tubes (Beckman Coulter, oxygen species (ROS) and other signals that influence mitochondria USA) and centrifuged at 100,000 x g for 35 min. The resulting pellet lead to the activation of the caspase family of cysteine proteases causing was resuspended in 2D solubilisation buffer containing 8 M urea, 2 M cell death [7]. This negative cycle of increasing ‘stressors’ within the thiourea, 4% (w/v) CHAPS and a cocktail of protease inhibitors. cell often leads to the cells inability to function properly and eventually leading to cell death [7]. Protein concentration It has recently been shown that HSN-I patient cells, expressing Determination of total cellular protein was performed using the the V144D SPTLC1 mutant have a marked increase in ER stress in EZQ protein estimation assay (Invitrogen, Australia) as previously comparison to healthy control cells [8]. Considering this finding: in this described by Churchward [12]. study we have investigated altered protein changes in the ER membrane of these HSN-I patient cells. The resulting proteomic and cell biology Two dimensional gel electrophoresis analyses have identified for the first time, increases in the Hypoxia up Protein concentration estimations (EZQ assay) were performed on regulated Protein 1 (ORP-150), Chloride intracellular channel protein patient and control ER protein fractions; a total of 100 µg protein was 1 (CLIC1), Ubiqutin-40s ribosomal protein S27a (RPS27a), Coactosin used for each 2DE analysis. 2DE was carried out as previously described (COTL1) and Ig Kappa chain C protein expression in HSN-I in this [13-16]. Briefly, ER proteins were reduced and alkylated in solutions membrane. containing total protein extraction buffer (containing 8 M urea, 2 M Materials and Methods thiourea and 4% CHAPS without ampholytes), total extraction buffer with 2% ampholytes, TBP/DTT disulphide reduction buffer (2.3 mM All cell culture stock solutions, including RPMI-1640, Foetal Tributyl phosphine and 45 mM DTT) and alkylation buffer (230 mM bovine serum (FBS), Penicillin (100 U/mL), Streptomycin (100 µg/mL), acrylamide monomer). L-glutamine (2 mM), HEPES (1 M): and phosphate buffered saline The treated samples were added to 7 cm Non-Linear pH 3-10 IPG (PBS) were purchased from GIBCO Invitrogen (Australia). Cell culture consumables were purchased from BD Falcon (Greiner, USA). DAPI strips (Bio-Rad ReadyStrip), and rehydrated for 16 hrs at RT. Isoelectric stains were purchased from Sigma-Aldrich (Australia). focusing (IEF) was then carried out at 20°C using the Protean IEF Cell (Bio-Rad, USA). After IEF, IPG strips were then resolved in the second EBV Transformed lymphoblasts dimension using a 12.5% T, 2.6% C polyacrylamide gel buffered with EBV transformed control and V144D HSN-I patient lymphoblasts 375 mM Tris buffer (pH 8.8), 0.1% (w/v) sodium dodecyl sulphate and were kindly provided by Prof. Garth Nicholson (Molecular Medicine polymerised with 0.05% (w/v) ammonium persulphate and 0.05% (v/v) Laboratory: Anzac Research Institute: Sydney) [9]. tetramethylethylenediamine (TEMED). A stacking gel containing a 5% T, 2.6% C polyacrylamide buffered with 375 mM Tris buffer (pH 8.8), Lymphoblast cultures 0.1% (w/v) SDS and included 0.1% bromophenol blue was added to Lymphoblasts were cultured in RPMI-1640 media (GIBCO), the resolving gel. The IPG strips were placed onto the stacking gel and supplemented with FBS (10% v/v), Penicillin (1 U/mL), Streptomycin overlaid with 0.5% (w/v) low melting agarose dissolved in 375 mM Tris (1 µg/mL), L-glutamine (2 mM), and HEPES (1 M) at 37°C in a (pH 8.8): with 0.1% (w/v) SDS. Electrophoresis was carried out at 4°C; 2 150 V initially for 10 min then reduced to 90 V for 2.5 h. The gels were humidified atmosphere of 5% CO2, using T75 cm culture flasks (Greiner: Interpath). Prior to use in biochemical assays, lymphoblasts placed in fixative containing 10% methanol and 7% acetic acid for 1 were collected by centrifugation at 1,500 × g (5 min at RT) and washed hr. The gels were washed with distilled water for 20 min, 3 times and in PBS. Cell counts were obtained using the Countess Automated Cell subsequently stained with colloidal coomassie blue (0.1% (w/v) CCB Counter (Invitrogen, Australia). G-250, 2% (v/v) phosphoric acid, 10% (w/v) ammonium sulphate, 20% (v/v) methanol) for 20 hr, with constant shaking at RT [14], the gels Isolation of ER proteins were then de-stained 5 times with 0.5 M NaCl, 15 min each. Imaging of Briefly, ER proteins were isolated using a sucrose density gradient CBB-stained gels on the FLA-9000 imager (FUJIFILM, Tokyo, Japan) [10,11]. Lymphoblasts were first centrifuged at 1,500 × g for 5 min, was carried out at 685/750 excitation/emission with a photomultiplier and the cells were then washed in 10 mL of ice cold 1 × PBS prior to tube (PMT) setting of 600 V and pixel resolution set to 100 µm [14]. suspension in 10 mL ice cold CaSRB Buffer (10 mM NaCl, 1.5 mM Analysis of 2D gel images was performed using Delta 2D software with CaCl, 10 mM Tris-HCL, pH 7.5) and left on ice for 10 min. Cells were automated spot detection (Local background region, 96; Average spot homogenised using a Dounce homogenizer (Kimble-Chase, USA) size, 32 and sensitivity in percentage, 20.0) (version 4.0.8; DECODON and 7 mL of 2.5 × MS buffer (210 mM Mannitol, 70 mM sucrose, 5 GmbH, Greifswald, Germany).
Biochem Anal Biochem ISSN: 2161-1009 Biochem, an open access journal Volume 5 • Issue 1 • 1000248 Citation: Stimpson SE, Lauto A, Coorssen JR, Myers SJ (2016) Isolation and Identification of ER Associated Proteins with Unique Expression Changes Specific to the V144D SPTLC1 Mutations in HSN-I. Biochem Anal Biochem 5: 248. doi:10.4172/2161-1009.1000248
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Mass spectrometry and resuspended in secondary antibody: anti-mouse Rhodamine (Millipore, 1:200), and incubated for 1 hour at RT. DAPI (1 µg/µL) was 2D gels were analysed for uniquely present or absent protein spots added to the cell suspension for 2 min, the cells were centrifuged and in control versus V144D mutant, (i.e. all or none changes) as previously washed twice with PBS. Aliquots (300 µL) were added to 6 well culture described 25 and 18. Briefly, the protein species of interest were excised plates containing coverslips coated in Histogrip (Invitrogen, USA) from gels and de-stained overnight. The gel pieces were then reduced and centrifuged at 500 x g for 10 min. The coverslips were washed in and alkylated in 10 mM Dithiothreitol (DTT) and 15 mM Idoacetic warm PBS, left overnight to dry and mounted onto glass slides prior to acid (IAA), and subsequently incubated with trypsin solution (10 ng/ confocal imaging using the LSM 5 confocal microscope comprising the µL, pH 7.4) for 16 hours at 37°C. LC-MS/MS analysis was carried out LSM 5 exciter laser scanning microscope with Axiovert 200 M inverted on a nanoaquity UPLC (Waters Corp, Milford, MA, USA) linked to a optical microscope (Carl Zeiss: Jena: Germany). Xevo QToF mass spectrometer from Waters (Micromass: UK). The data were acquired using Masslynx software (Version 4.1, Micromass UK). Flow cytometry The MS/MS data files were searched against SwissProt databases with FACS analysis was carried out as previously described by 18. semi-trypsin as the enzyme. Lymphoblasts were isolated as above; the cells were then suspended SDS-PAGE and immunoblotting in 4% paraformaldehyde and incubated for 15 min at RT and then resuspended in 0.3% Triton X-100 for 15 min at 37°C. After incubation Control and patient ER protein fractions (25 µg total protein) the cell suspension was centrifuged at 1,000 x g for 5 min and the pellet were subjected to SDS-PAGE on 12.5% resolving gels and transferred resuspended in primary antibody for 1 hr at RT. Cell suspension was to PVDF membrane. The membranes were blocked with 5% skim centrifuged, washed in PBS and resuspended in secondary antibody, milk in TBS buffer containing 0.1% Tween-20. Whole membranes anti-mouse FITC (Millipore, 1:200) for 1 hr at RT. The cell suspension were blocked and incubated with anti-Calnexin (Cell Signalling was then analysed using the MACSQuant flow cytometer (Miltenyi Cat# 2679S, RRID:AB_2228381), anti-SPTLC1 (Santa Cruz Biotechnology Cat# sc-32916, RRID,AB_2195864), anti-Ig kappa Biotech). chain C (Abcam Cat# ab1050, RRID:AB_297240), anti-GAPDH 2D gel images of ER proteins from control and patient de- (Abcam Cat# ab9485, RRID:AB_307275), anti-ORP-150 (Abcam rived lymphoblasts Cat# ab124884, RRID:AB_10973544), anti-CLIC1 (Abcam Cat# ab77214, RRID:AB_1566060), anti-RPS27a (Abcam Cat# ab57646, Total isolated ER proteins were resolved using a refined 2DE RRID:AB_2180587) and anti-COTL1 (Proteintech Group Cat# 10781- protocol [13,14] (Figure 1). Control and V144D total ER proteins were 1-AP, RRID:AB_2084785) at 1:1000, for 16 h. The membrane was resolved using mini gel format. Standard spot counts indicated 656 ± 5 then incubated with secondary HRP antibodies (Sigma-Aldrich Cat# and 675 ± 3 protein species were resolved in control and V144D mutant A9044, RRID:AB_258431 and Cat# A0545, RRID:AB_257896) (1:2000 ER fractions respectively. Further analysis of the total ER protein dilution) for 1 hr at RT. Blots were developed using an enhanced profiles from control and HSN-I patient derived lymphoblasts revealed chemiluminescence (ECL) detection kit (Pierce Thermo Scientific, five ‘all or none’ protein changes in the V144D mutant and control USA). The membrane was developed on CL-Xposure Film (Thermo lymphoblasts (Figure 2). These proteins were located in the pI of 5.5, Fisher Scientific, U.S.A) using an AGFA X-ray developer. 5.6, 8.0, 5.8, 6.6 and 8.3 and molecular weight of 120, 30, 15, 17, 22 and Immunofluorescence 22 respectively (kDa) (Table 1). Subsequent LC/MS analysis identified the protein to be ORP-150, CLIC1, RPS27a, Coactosin (COTL1) and Ig Immunofluorescence was carried out as previously described by Kappa Chain C. Stimpson [16]. Briefly: Lymphoblasts (1 × 106 cells) were suspended in 4% paraformaldehyde for 15 min. Cells were then placed in 0.5% Expression of identified ER proteins from HSN-I patient-de- TritonX-100 and incubated at 37°C for 30 min. The cells were then rived lymphoblasts blocked in 5% BSA solution at 37°C for 30 min: then resuspended In order to determine protein expression changes, immunoblot in primary antibody, SPTLC1, ORP-150, CLIC1, RPS27a, COTL1, analysis was carried out on isolated ER and total cell lysates from and stained for 1 hr at RT. The cells were subsequently washed control and HSN-I patient derived lymphoblasts. These data showed a
Figure 1: Representative images of 2D gels following resolution of ER proteins from control and patient derived lymphoblasts. (A) Control ER proteins; (B) V144D ER proteins. The molecular weights are in kilodaltons (kDa) and the IEF dimension is in pH units.
Biochem Anal Biochem ISSN: 2161-1009 Biochem, an open access journal Volume 5 • Issue 1 • 1000248 Citation: Stimpson SE, Lauto A, Coorssen JR, Myers SJ (2016) Isolation and Identification of ER Associated Proteins with Unique Expression Changes Specific to the V144D SPTLC1 Mutations in HSN-I. Biochem Anal Biochem 5: 248. doi:10.4172/2161-1009.1000248
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Figure 2: Representative images of 2D gel regions of ER proteins from control and patient derived lymphoblasts. Resolved protein species having a vastly altered abundance is indicated (Red Arrow). The molecular weights are in kilodaltons (kDa) and the IEF dimension is in pH units.
Number of Spot Accession Sequence Mascot Protein Predicted Mw Mascot Mw Protein Identified Unique peptides Number Number Coverage Score Predicted pI (kDa) Mascot pI (kDa) matched Hypoxia Up I. QY94L1 42 49% 1741 5.5 120 5.2 112 Regulated Protein 1 Chloride Intracellular II. O00299 15 50% 448 5.6 30 5.1 27.3 Channel Protein 1 Ubiquitin-40s III. Ribosomal protein P62979 10 33% 182 8.0 15 9.7 18.3 S27a IV. Coactosin Q14019 19 73% 190 5.8 17 5.6 16 V. Ig Kappa Chain C P01834 9 71% 587 6.6 22 5.5 12 VI. Ig Kappa Chain C P01834 10 80% 985 8.3 22 5.5 12 Table 1: Summary table of mascot protein identification. LC-MS/MS and Mascot Database searching identified Hypoxia up Regulated Protein 1, Chloride Intracellular Channel Protein 1, Ubiquitin 40s ribosomal protein s27a, Coactosin and Ig Kappa Chain C from V144D patient derived lymphoblast control lymphoblasts isolated ER. concomitant increase in the amount of ORP-150, CLIC1, and COTL1 protein in control and V144D mutant HSN-I patient lymphoblasts. in the V144D mutant protein samples compared to the control samples However, whilst there appeared to be an increase in the abundance of ORP- with a p value of < 0.05 (Figures 3A-3J). RPS27a was slightly increased 150, CLIC1, RPS27a and COTL1 in the V144D (patient) lymphoblasts, in the V144D mutant; however this change was not statistically there was no change in intracellular localisation (Figure 4). significant. Whereas quantitative analysis of Ig kappa protein displayed Control and patient-derived lymphoblasts were immuno significant increase in the amount of Ig Kappa protein being expressed stained for ORP-150, CLICL1, RPS27a and COTL1 were analysed in the control total protein lysates compared to that of the protein by fluorescence assisted cell sorting (FACS) to determine the total isolated from the V144D mutant samples (p < 0.05) (Figure 3J). fluorescence per cell (Figure 5). There was a marked increase in the The intracellular localisation and abundance of SPTLC1, ORP-150, relative fluorescence intensity of all the proteins in the V144D cells CLIC1, RPS27a and COTL1 was established using immunofluorescence compared to that of control lymphoblasts with an increase in relative studies on control and patient-derived lymphoblasts. It was observed that fluorescence of 1.4, 1.25, 2.4, 1.5 OD (fold increase) respectively, there was no apparent change in intracellular localisation of the SPTLC1 relative to the stained controls.
Biochem Anal Biochem ISSN: 2161-1009 Biochem, an open access journal Volume 5 • Issue 1 • 1000248 Citation: Stimpson SE, Lauto A, Coorssen JR, Myers SJ (2016) Isolation and Identification of ER Associated Proteins with Unique Expression Changes Specific to the V144D SPTLC1 Mutations in HSN-I. Biochem Anal Biochem 5: 248. doi:10.4172/2161-1009.1000248
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1 2 3 4 5 6 7 8 A. ORP-150 150 kDa 1 2 3 4 5 6 7 8 B. CLIC1 30 kDa 1 2 3 4 5 6 7 8 C. RPS27a 16 kDa
D . 1 2 3 4 5 6 7 8 COTL1 16 kDa
Figure 3: Expression of five ER proteins from HSN-I patient-derived lymphoblasts. (A) Immunoblot detection of ORP-150. (B) Immunoblot detection of CLIC1. (C) Immunoblot detection of RPS27a. (D) Immunoblot detection of COTL1. (E) Immunoblot detection of Ig Kappa Chain C. Lanes 1 and 2 represent control ER proteins, 3 and 4 represent control total proteins, 5 and 6 represent V144D ER proteins, 7 and 8 represent V144D total proteins. Figures (F-J) are representative graph showing statistical significant (*) (p < 0.05) difference between control patient lymphoblasts compared to the mutant V144D lymphoblasts of ORP-150, CLIC1, RPS27a, COTL1 and Ig Kappa Chain C respectively (n=3). Blots normalised to GAPDH. Errors bar depict SE of means.
Figure 4: Representative immunofluorescence images of the intracellular localisation of five proteins. Representative confocal micrographs showing SPTLC1, CLIC1, ORP-150, RPS27a and COTL1 stained lymphoblasts (red) and DAPI nuclear stain (blue). Scale bar = 5 µm.
Biochem Anal Biochem ISSN: 2161-1009 Biochem, an open access journal Volume 5 • Issue 1 • 1000248 Citation: Stimpson SE, Lauto A, Coorssen JR, Myers SJ (2016) Isolation and Identification of ER Associated Proteins with Unique Expression Changes Specific to the V144D SPTLC1 Mutations in HSN-I. Biochem Anal Biochem 5: 248. doi:10.4172/2161-1009.1000248
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A. ORP-150 B. CLIC1
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Figure 5: Relative quantification of ORP-150, CLICL1, RPS27a and COTL1 HSN-I patient-derived lymphoblasts expressing the V144D mutant SPTLC1 genes. Flow cytometry analysis of the relative fluorescence intensity of (A) ORP-150, (B) CLICL1, (C) RPS27a and (D) COTL1 in control and V144D patient-derived lymphoblasts. (A) Blue histogram represents the V144D patient lymphoblasts and Red histogram represents control lymphoblasts. (n=3).
Discussion house keeping protein) analysis showed no significant changes in in the V144D compared to that of the control lymphoblasts (Figure 6F). SPT is an ER bound and key rate determining enzyme in sphingolipid metabolism. Mutations within the SPT subunits result in potential 2DE analysis revealed 5 proteins species only detected in the dysfunction with possible perturbations in sphingolipid synthesis and V144D ER fractions in the pI of 5.5, 5.6, 8.0, 5.8, 6.6 and molecular metabolism causing HSN-I [2]. The ER plays a crucial role in many weight of 120, 30, 15, 17, 24 respectively (kDa) (Figure 2). Subsequent aspects of protein compartmentalisation which include membrane LC/MS analysis identified these protein species to be ORP-150, CLIC1, translocation, protein folding, post-translational modifications of RPS27a, COTL1, and Ig Kappa Chain C (Table 1). A protein species proteins, transport of both membrane and soluble proteins, as well was detected in the control ER fractions in the pI region of 8.3 and as monitoring protein synthesis and degradation [1]. These processes molecular weight range of 24 kDa. This protein species was identified are both assisted and monitored by molecular chaperones. This as Ig Kappa Chain C. Quantitative immunoblot analysis was carried investigation has identified several proteins that change in expression out to determine the expression of these 5 proteins: it was shown that in the V144D SPTLC1 mutant lymphoblasts. Whilst this investigation ORP-150, CLIC1 and COTL1 had statistically significant increases in only analysed one patient derived lymphoblast sample: we have the V144D mutant (Figures 3A-3J), however RPS27a showed slight conducted further investigation using a tranfected neuronal model, increase in the V144D mutant ER fraction: but was not statistically data in follow-up manuscript, with the data here correlating with the significant (Figure 3I). transfect neuronal system, thus indicating the changes observed here Oxygen regulated proteins are overexpressed under conditions of are not due to patient-patient variations. The proteins that have major hypoxia. The heat shock protein, oxygen-regulated protein of 150 kDa increases in expression are ORP-150, CLIC1, COTL1, Ig Kappa Chain (ORP-150) also known as Glucose related protein 170 (Grp170), serves C, and with an increase in RPS27a in the ER. as an important molecular chaperone of the endoplasmic reticulum To elucidate if protein changes in the proteomes of the ER fractions during stress [17]. Notably, hypoxia mediated up-regulation of ORP- occur due to mutations in SPTLC1 causing HSN-I ER membranes 150 suppresses programmed cell death driven by oxygen deprivation were isolated from control and V144D patient lymphoblasts and lysed [18]. Neurons with increased ORP-150 expression demonstrated proteins were subjected to high resolution 2DE (Figure 1). Calnexin, a suppressed caspase-3-like activity [19]. marker for the ER: confirmed the quality of the isolated fraction used for analysis. In a study by 14, ER stress markers increased in V144D Chloride intracellular channel protein 1 (CLIC1), is small in size lymphoblasts: here we detected no statistically significant changes in and exists in both soluble cytoplasmic and integral membrane forms expression of calnexin, suggesting that this protein is constant in both [20]. CLIC1 exists usually in a soluble form in the cytoplasm and the control and V144D cells (Figures 6A-6F) Expression of the SPTLC1 nucleoplasm, but following stimuli undergoes major structural changes protein in the V144D fractions analysed, in comparison to the control and inserts in lipid membranes, where it acts as a chloride-selective and revealed no significant increase in expression (Figure 6E) indicating ion channel. Cell oxidation seems to be the most important stimulus a constant expression state of SPTLC1 in the diseased state. GAPDH (a controlling the transition of CLIC1 between these two forms [21].
Biochem Anal Biochem ISSN: 2161-1009 Biochem, an open access journal Volume 5 • Issue 1 • 1000248 Citation: Stimpson SE, Lauto A, Coorssen JR, Myers SJ (2016) Isolation and Identification of ER Associated Proteins with Unique Expression Changes Specific to the V144D SPTLC1 Mutations in HSN-I. Biochem Anal Biochem 5: 248. doi:10.4172/2161-1009.1000248
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Figure 6: Expression of ER resident proteins and GAPDH in HSN-I patient-derived lymphoblasts. (A) Immunoblot of Calnexin, (B) Immunoblot of SPTLC1, (C) Immunoblot of GAPDH. Lanes 1 and 2 represent control ER proteins, 3 and 4 represent control total proteins, 5 and 6 represent V144D ER proteins, 7 and 8 represent V144D total proteins. A representative graph showing no statistical significant (p > 0.05) difference between ER control and patient lymphoblasts lysates (n=3) of Calnexin (D), SPTLC1 (E) and GAPDH (F). Blots were normalised to GAPDH. Errors bar depict SE of means.
While many of the processes that require ubiquitin: are common to spectral analysis: both proteins were identified as Ig Kappa Chain C. all cell types, ubiquitin also has distinct roles in protein degradation. For Ig kappa Chain C was found to significantly increase in the control example; the ubiquitin proteasome system: protein ubiquitylation is also lymphoblasts compared to that of the V144D mutant lymphoblasts responsible for regulating cell signalling by controlling the endocytosis (Figure 3J). This finding correlates with our previous studies in the of plasma membrane receptors. Ubiquitin: is highly conserved and is V144D patient lymphoblasts [15]. involved in processes of signal transduction, endocytosis, and DNA repair [22]. Conclusion Cells are able to move and extend dynamically which is facilitated The novel findings in this study thus suggest a link to increased by actin dynamics. Coactosin (COTL1) is an actin binding protein, oxidative stress within the V144D lymphoblasts. Previous studies and has been shown to associate with F-actin [23]. Under normal [8,15] have shown there is an increase in both ER stress and potential cellular conditions, monomeric globular actin (G-actin) is in a state oxidative phosphorylation (via a potential change to ROS) changes of equilibrium with filamentous actin (F-actin), forming the actin in V144D. It is evident that there is an increase in oxidative stress cytoskeleton and is responsible for maintaining and modifying cell shape within the V144D patient lymphoblasts demonstrated by the increased in motility, phagocytosis, and cytokinesis [24]. The actin cytoskeleton is expression of ORP-150, CLIC1, COTL1 and RPS27a. While these regulated by numerous actin-binding proteins that interact with actin proteins are functionally independent from one and another: together and regulate the cytoskeleton in cells [23]. COTL1 was also found to they help establish a strong connection that mutations in SPTLC1 directly interact with the filamentous, F-actin but does not form a stable cause oxidative stress within the cell. This increase in oxidative stress complex with globular, G-actin [23]. could be linked to the increase in Ubiquinol Cytochrome C expression Immunostaining (Figure 3) and FACS (Figure 4) analyses yielded from the mitochondria of V144D mutant cells previously observed: the cellular localisation and expression of the following 5 proteins thus an increase in ORP-150 is observed to compensate and protect SPTLC1, ORP-150, CLIC1, RPS27a and COTL1 was established. It was the cell from an increase in ROS production. Actin function is observed that there was no apparent change in intracellular localisation highly regulated by the association of actin binding proteins. Studies of the SPTLC1 protein in control and V144D mutant HSN-I patient have shown that actin oxidation generally inhibits the association of lymphoblasts. ORP-150, CLIC1, RPS27a and COTL1 displayed an actin binding proteins with actin [25]. As COTL1 is an actin binding intracellular localisation change to the cellular periphery and increased partner its upregulation could be due to the increased oxidative stress abundance. FACS analysis of ORP-150, CLIC1, RPS27a and COTL1 upon the cellular cytoskeletal system. Oxidative stress can cause actin was used to determine the total fluorescence per cell, revealing a remodelling and potential axonal retraction in the neuron [22]. Under marked increase in the relative fluorescence intensity of all the proteins the conditions of stress the UPR is activated to ensure misfolded in the V144D cells compared to that of control lymphoblasts with an proteins are targets for destruction [22]. RPS27a has a major role in increase in relative fluorescence of 1.4, 1.25, 2.4, 1.5 OD (fold increase) targeting cellular proteins for destruction as such its apparent increase respectively: relative to the stained controls. in the V144D mutant demonstrates that there is a possible increase in misfolded protein either directly due to ER stress, oxidative stress or by It was identified that there were two other proteins with a marked another mechanism that affects protein conformation. The findings in change in protein expression. Both proteins were located at 24 kDa, this study, coupled with others [8,15] suggest that there is a probable but each had a different pI (6.6 and 8.3 respectively). Following mass underlying mechanism that is common to sensory neurodegenerations.
Biochem Anal Biochem ISSN: 2161-1009 Biochem, an open access journal Volume 5 • Issue 1 • 1000248 Citation: Stimpson SE, Lauto A, Coorssen JR, Myers SJ (2016) Isolation and Identification of ER Associated Proteins with Unique Expression Changes Specific to the V144D SPTLC1 Mutations in HSN-I. Biochem Anal Biochem 5: 248. doi:10.4172/2161-1009.1000248
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Results serine palmitoyltransferase is sufficient for normal sphingolipid metabolism and viability of HSN1 patient cells. Biochimica et biophysica acta 1688: 168-175.
Expression of GAPDH and ER markers in HSN-I patient-de- 10. Bozidis P, Williamson CD, Colberg-Poley AM (2007) Isolation of endoplasmic rived lymphoblasts reticulum, mitochondria, and mitochondria-associated membrane fractions from transfected cells and from human cytomegalovirus-infected primary In order to assess the levels of calnexin and SPTLC1 expression fibroblasts. Curr Protoc Cell Biol Chapter 3: Unit 3: 27. in isolated ER fractions and total cell lysates from control and V144D 11. Vaseva AV, Moll UM (2013) Identification of p53 in mitochondria. Methods Mol mutant HSN-I patient-derived lymphoblasts: immunoblotting was Biol 962: 75-84. carried out (Figures 6A-6F). Purity of the ER fraction was determined 12. Churchward MA, Butt RH, Lang JC, Hsu KK, Coorssen JR (2005) Enhanced by immunoblotting for membrane, mitochondrial and Golgi complex detergent extraction for analysis of membrane proteomes by two-dimensional markers: all of which were absent from the isolated ER fractions, gel electrophoresis. Proteome Sci 3: 5. indicating the isolated ER fraction was devoid of other organelles. 13. Butt RH, Coorssen JR (2005) Postfractionation for enhanced proteomic There were no expression differences observed between the control analyses: routine electrophoretic methods increase the resolution of standard and V144D samples in either isolated ER isolations or total cell lysates. 2D-PAGE. J Proteome Res 4: 982-991. Quantitation of the immunoblots of the isolated ER and total cell 14. Gauci VJ, Padula MP, Coorssen JR (2013) Coomassie blue staining for high lysate fractions from control and HSN-I patient-derived lymphoblasts sensitivity gel-based proteomics. J Proteomics 90: 96-106. (Figures 6D and 6E): confirmed that there was no statistically significant 15. Stimpson S, Coorssen J, Myers S, (2014) Mitochondrial protein alterations in a change in expression of these proteins. Expression analysis of GAPDH familial peripheral neuropathy caused by the V144D amino acid mutation in the (Figure 6C) established there was no statistically significant change in sphingolipid protein, SPTLC1. Journal of Chemical Biology 8: 25-35. the expression of GAPDH (Figure 6F). 16. Wright EP, Partridge MA, Padula MP, Gauci VJ, Malladi CS, et al. (2014) Top- down proteomics: enhancing 2D gel electrophoresis from tissue processing to Acknowledgements high-sensitivity protein detection. Proteomics 14: 872-889. We are grateful to Prof Garth Nicholson (Molecular Medicine Laboratory and 17. Behnke J, Hendershot LM (2014) The large Hsp70 Grp170 binds to unfolded Northcott Neuroscience Laboratory Anzac Research Institute: Sydney) for providing protein substrates in vivo with a regulation distinct from conventional Hsp70s. all EBV transformed lymphoblast lines used in this study. SS was supported by APA J Biol Chem 289: 2899-2907. Research Scholarship: and the UWS School of Science and Health Postgraduate research fund. SM notes the continuing support of an anonymous Private 18. Stojadinovic A, Hooke JA, Shriver CD, Nissan A, Kovatich AJ, et al. (2007) Foundation. JC acknowledges the support of the UWS School of Medicine. HYOU1/Orp150 expression in breast cancer. Med Sci Monit 13: BR231-239.
References 19. Wu YB, Li HQ, Ren MS, Li WT, Lv XY, et al. (2013) CHOP/ORP150 ratio in endoplasmic reticulum stress: a new mechanism for diabetic peripheral 1. Pendin D, Mcnew JA, Daga A (2011) Balancing ER dynamics: shaping, neuropathy. Cell Physiol Biochem 32: 367-379. bending, severing, and mending membranes. Curr Opin Cell Biol 2: 435-442. 20. Warton K, Tonini R, Fairlie WD, Matthews JM, Valenzuela SM, et al. (2002) 2. Wei J, Yerokun Y, Liepelt M, Momin A, Wang E, et al. (2007) 2-1 Serine Recombinant CLIC1 (NCC27) assembles in lipid bilayers via a pH-dependent Palmitoyltransferase. Sphingolipid Biology 25-27. two-state process to form chloride ion channels with identical characteristics to those observed in Chinese hamster ovary cells expressing CLIC1. J Biol Chem 3. Verhoeven K, Timmerman V, Mauko B, Pieber TR, De Jonghe P, et al. (2006) 277: 26003-26011. Recent advances in hereditary sensory and autonomic neuropathies. Curr Opin Neurol 19: 474-480. 21. Averaimo S, Milton RH, Duchen MR, Mazzanti M (2010) Chloride intracellular channel 1 (CLIC1): Sensor and effector during oxidative stress. FEBS Lett 584: 4. Dyck PJ, Thomas PK (2005) Dyck: Peripheral Neuropathy, 4th Edition, Mosby 2076-2084. Elsevier, Philadelphia. 22. Hallengren J, Chen PC, Wilson SM (2013) Neuronal ubiquitin homeostasis. 5. Lindholm D, Wootz H, Korhonen L (2006) ER stress and neurodegenerative Cell Biochem Biophys 67: 67-73. diseases. Cell Death Differ 1: 385-392. 23. Provost P, Doucet J, Stock A, Gerisch G, Samuelsson B, et al. (2001) Coactosin- 6. Rao RV, Bredesen DE (2004) Misfolded proteins, endoplasmic reticulum stress like protein, a human F-actin-binding protein: critical role of lysine-75. Biochem and neurodegeneration. Curr Opin Cell Biol 16: 653-662. J 359: 255-263.
7. Fulda S, Gorman AM, Hori O, Samali A (2010) Cellular stress responses: cell 24. Carlier MF, Laurent V, Santolini J, Melki R, Didry D, et al. (1997) Actin survival and cell death. Int J Cell Biol 2010: 1-23. depolymerizing factor (ADF/cofilin) enhances the rate of filament turnover: implication in actin-based motility. J Cell Biol 136: 1307-1322. 8. Myers S, Malladi C, Hyland R, Bautista T, Boadle R, et al. (2014) Mutantions in the SPTLC1 protein cause mitochondrial structual abnormalisites and 25. Farah ME, Sirotkin V, Haarer B, Kakhniashvili D, Amberg DC (2011) Diverse endoplasmic reticulum stress in lymphoblasts. DNA and Cell Biology 3: 7. protective roles of the actin cytoskeleton during oxidative stress. Cytoskeleton (Hoboken) 68: 340-354. 9. Dedov V, Dedova I, Merrill A, Nicholson G (2004) Activity of partially inhibited
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Biochem Anal Biochem ISSN: 2161-1009 Biochem, an open access journal Volume 5 • Issue 1 • 1000248
Paper IV
Published in the Electronic Journal of Biology
Contributions
SES carried out all experimentation, data analysis and wrote first draft manuscript.
76 | Page
Electronic Journal of Biology, 2015, Vol.11(4): 176-186
Proteome Alterations Associated With the V144D SPTLC1 Mutation That Causes Hereditary Sensory Neuropathy-I Scott E. Stimpson1, 3, 4, Jens R. Coorssen2,3,4,5,*, Simon J. Myers1,3,4,5,* 1 Neuro-Cell Biology Laboratory, Western Sydney University, Australia; 2 Molecular Physiology, Western Sydney University, Australia; 3 Molecular Medicine Research Group, Western Sydney University, Australia; 4 School of Science and Health, Western Sydney University, Australia; 5 School of Medicine, Western Sydney University, Australia. *Corresponding author. Tel: 61 02 4620 3383, 61 4620 3802; E-mail: [email protected]; [email protected] Citation: Stimpson SE, Coorssen JR, Myers SJ, Proteome Alterations Associated With the V144D SPTLC1 Mutation That Causes Hereditary Sensory Neuropathy-I. Electronic J Biol, 11:4 Received: November 16, 2015; Accepted: December 11, 2015; Published: December 17, 2015
Research Article
Abstract 1. Introduction
Background: Hereditary sensory neuropathy type Hereditary sensory neuropathy type I (HSN-I) I is the most common subtype and presents with is the most common subtype of the HSNs [1], clinical onset in the second to third decade of life with characterised by the progressive degeneration of progressive degeneration of the dorsal root ganglion the dorsal root ganglion (DRG). Onset of clinical neurons. Three different missense mutations in the symptoms is between the second and third decade gene encoding for serine palmitoyltransferase long of life [2]. Heterozygous mutations in the serine chain subunit 1 have been linked to HSN-I. Here palmitoyltransferase (SPT) long chain subunit 1 we quantitatively assess the proteomes and identify (SPTLC1) have been identified as the cause of HSN-I marked protein alterations in both mitochondria [3,4]. The associated mutations in this gene occur and endoplasmic reticulum from HSN-I patient at single amino acids which are highly conserved lymphoblasts which harbour the V144D mutation. throughout different species and are therefore likely to interfere with SPT functionality and structure [5]. Methods: Mitochondria and endoplasmic reticulum SPT is a pyridoxal 5'- phosphate dependent were fractionated and lysed from control and multimeric enzyme that catalyses the first step in patient-derived lymphoblasts. Protein samples were the biosynthesis of sphingolipids, ceramide and separated into total soluble and total membrane sphingomyelin [6]. Mutations in the SPT subunits fractions and analysed using a well-established top- thus result in potential dysfunction and perturbations down proteomic protocol. Altered protein species in sphingolipid synthesis and metabolism linked to were identified by LC MS/MS. a variety of diseases, in particular HSN-I [7]. As the rate determining enzyme in the de novo sphingolipid Results: Using a detailed proteomic approach, we synthesis pathway, SPT is therefore a key enzyme identified 36 proteins that were completely altered in in the regulation of cellular sphingolipid content by abundance in cells harbouring the V144D SPTLC1 condensation of palmitoyl coenzyme A (CoA) with mutation relative to normal controls. L-serine to form 3-ketodihydrosphingosine [8-10].
Conclusion: The data establish that major protein We recently noted altered protein expression in the alterations occur in both the endoplasmic reticulum, mitochondria and ER from HSN-I (SPTLC1 V144D) where the SPTLC1 protein resides, and in the mutant lymphoblasts [11,12]. In order to improve mitochondria from V144D patient lymphoblasts. characterisation, a more detailed (i.e. ‘deeper’) top These proteins potentially play a major role in disease down analysis of the total membrane and total soluble pathogenesis and may thus help to further elucidate proteomes from the mitochondria and ER of control the molecular mechanism(s) underlying hereditary and HSN-I (SPTLC1 V144D) patient lymphoblasts sensory neuropathy type I and might also prove to was carried out. be potential therapeutic targets. Numerous protein species were found to change Keywords: Mitochondria; Endoplasmic reticulum; markedly in abundance in the mitochondria and SPTLC1; HSN-I; Proteomics. ER from the HSN-I (SPTLC1 V144D) patient
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lymphoblasts; these proteins are involved in energy Analysis of the membrane and soluble mitochondrial metabolism, catalytic activity, protein transport, and ER protein profiles from control and HSN-I oxidative stress and the cytoskeleton. These protein (SPTLC1 V144D) patient lymphoblasts revealed alterations reflect the changing cellular events that protein species alterations, change in abundance underlie HSN-I. greater than or equal to 2.0 fold, in the V144D cells relative to control lymphoblasts. The analysis 2. Results revealed 36 protein species that were located at 2.1 Gel images of mitochondrial and ER varying pI / MW (kDa) coordinates. These proteins membrane and soluble proteins from control and were excised; digested and LC/MS/MS analysis patient derived lymphoblasts was carried out to identify these proteins (protein identifications are summarized in Tables 2-5). All membrane and soluble protein samples were well-resolved proteomes covering the entire MW and 2.2 Functions of the identified proteins in pI range of the gels. The total numbers of resolved mitochondrial and ER fractions protein species for mitochondrial and ER membrane soluble samples are summarised in Table 1. Proteins identified from mitochondrial and ER
Table 1. Total protein species resolved by 2D from mitochondrial and ER membrane and soluble fractions obtained from controls and HSN-I (SPTLC1 V144D) patient lymphoblast. Membrane Soluble Organelle Control V144D Control V144D Mitochondrial 550 ± 9 561 ± 9 576 ± 6 562 ± 7 Endoplasmic Reticulum 558 ± 6 577 ± 5 623 ± 4 627 ± 4
Table 2. Summary table of mascot protein identification. LC-MS/MS and Mascot Database searching identified a number of proteins from control and V144D lymphoblasts isolated mitochondrial membrane proteins. Unique Mascot Fold Spot Protein Accession Sequence Predicted Predicted Mascot Mascot peptides Protein Change in Number Identified Number Coverage pI Mw (kDa) pI Mw (kDa) matched Score V144D Succinate Dehydrogenase 1 Flavoprotein P31040 23 24% 617 6.5 70 7.06 73.7 6.1 Fold ↑ Subunit, Mitochondrial Aldehyde 2 Dehydrogenase P30837 20 26% 340 6.1 50 6.36 57.6 2.2 Fold ↑ X, Mitochondrial Calcium Binding Mitochondrial 3 Q6NUK1 19 22% 98 5.7 45 6.22 53.5 2.6 Fold ↓ Carrier Protein SCaMc-1 Cytochrome B-C1 Complex 4 P31930 28 38% 971 5.5 50 5.94 53.3 4.0 Fold ↑ Subunit 1, Mitochondrial Pyruvate Dehydrogenase E1 Component 5 P08559 7 13% 23 7.9 38 8.17 44 3.2 Fold ↑ Subunit Alpha, Somatic Form, Mitochondrial Voltage- Dependent 6 Anion-Selective P45880 10 26% 265 8.2 30 8.62 31 2.1 Fold ↓ Channel Protein 1 Ig Kappa Chain Absent in 7 P01834 18 70% 802 8.3 22 5.58 12 C V144D Only Ig Kappa Chain 8 P01834 15 63% 704 6.6 22 5.58 12 present in C V144D
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Table 3. Summary table of mascot protein identification. LC-MS/MS and Mascot Database searching identified a number of proteins from control and V144D lymphoblasts isolated mitochondrial soluble proteins. Unique Mascot Mascot Fold Spot Protein Accession Sequence Predicted Predicted Mascot peptides Protein Mw Change in Number Identified Number Coverage pI Mw (kDa) pI matched Score (kDa) V144D 1 Ezrin P15311 55 55% 1021 5.5 67 5.94 69.5 3.9 Fold ↓ Prolyl 2 4-hydroxylase P13674 40 58% 1188 5.4 60 5.70 61.3 3.0 Fold ↑ subunit Alpha 1 60 kDa heat 3 shock protein, P10809 26 31% 694 5.2 58 5.70 61.2 2.7 Fold ↑ Mitochondrial Dipeptidyl 4 Q9Y2B0 14 16% 534 7.9 43 8.35 44 2.6 Fold ↑ Peptidase 1 Pyruvate dehydrogenase E1 component 5 P08559 26 38% 634 6.4 49 6.54 52.6 2.5 Fold ↑ subunit alpha, somatic form, Mitochondrial Inorganic 6 Pyrophosphatase Q9H2U2 36 59% 1265 7.0 32 7.07 38.4 2.2 Fold ↑ 2, Mitochondrial 7 Pro-Cathepsin H P09668 21 48% 581 6.1 30 8.35 38 2.2 Fold ↑ 8 Peroxiredoxin-4 Q13162 36 69% 1350 5.6 25 5.86 30.7 2.1 Fold ↑ Absent 9 Ig Kappa Chain C P01834 31 88% 1354 8.3 22 5.58 12 V144D Only present 10 Ig Kappa Chain C P01834 27 76% 998 6.6 22 5.58 12 in V144D
Table 4. Summary table of mascot protein identification. LC-MS/MS and Mascot Database searching identified a number of proteins from control and V144D lymphoblasts isolated ER membrane proteins. Fold Unique Mascot Mascot Spot Accession Sequence Predicted Predicted Mascot Change Protein Identified peptides Protein Mw Number Number Coverage pI Mw (kDa) pI in matched Score (kDa) V144D Heterogeneous nuclear 1 O14979 8 14% 230 8.1 40 9.59 46.6 3.5 Fold ↓ ribonucleoprotein D-like Serine/Threonine- protein Phosphatase 2 P62140 11 40% 418 5.2 35 5.84 38 2.0 Fold ↑ PP1-Beta Catalytic Subunit 3 Apolipoprotein L2 Q9BQE5 11 42% 210 5.7 36 6.28 37.1 2.8 Fold ↓ UPF0568 Protein 4 Q9Y224 18 56% 315 6.1 25 6.19 28.2 3.3 Fold ↓ Cl4orf166 Elongation Factor 5 P24534 9 46% 243 4.1 24 4.50 25 4 Fold ↓ 1-Beta Serine/Arginine-rich 6 P84103 6 36% 152 5.8 17 9.64 19.5 2.9 Fold ↓ Splicing Factor 3 Only 7 Ig Kappa Chain C P01834 26 80% 1303 6.6 22 5.58 12 present in V144D Absent 8 Ig Kappa Chain C P01834 22 80% 1065 8.8 22 5.58 12 from V144D membrane and soluble fractions were grouped has been used to provide a visual analysis of protein based upon their biological functionality; a pie graph changes in the HSN-I (SPTLC1 V144D) disease state
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Table 5. Summary table of mascot protein identification. LC-MS/MS and Mascot Database searching identified a number of proteins from control and V144D lymphoblasts isolated ER soluble proteins. Unique Mascot Fold Spot Accession Sequence Predicted Predicted Mascot Mascot Protein Identified peptides Protein Change in Number Number Coverage pI Mw (kDa) pI Mw (kDa) matched Score V144D 1 Peroxiredoxin-2 P32119 19 65% 453 5.0 20 5.66 45.3 2.4 Fold ↑ Lymphocyte- 2 P33241 24 49% 901 3.5 50 4.69 37.4 3.2 Fold ↑ Specific Protein 1 Proteasome 3 Activator Complex Q06323 10 37% 357 6.0 35 5.78 28.9 4.2 Fold ↑ Subunit 1 Proteasome 4 Subunit Alpha P25788 18 41% 372 5.2 30 5.19 28.6 2.6 Fold ↑ Type-3 Protein CDV3 5 Q9UKY7 14 64% 578 6.2 60 6.06 27.3 2.3 Fold ↓ Homolog Chloride 6 intracellular O00299 38 68% 1174 5.2 30 5.09 27.2 2.7 Fold ↑ channel protein 1 Adenine 7 Phosphoribos P07741 24 72% 876 5.1 17 5.78 19.8 3.0 Fold ↓ yltransferase Eukaryotic Translation 8 P63241 26 70% 898 4.3 15 5.08 17 2.1 Fold ↑ Initiation Factor 5A-1 Only 9 Ig Kappa Chain C P01834 21 80% 1278 6.6 22 5.58 12 present in V144D Absent in 10 Ig Kappa Chain C P01834 29 86% 1609 8.3 22 5.58 12 V144D
(Figure 1). This analysis indicates that the majority of and further separated into total membrane and total the protein alterations in mitochondria are involved soluble protein fractions prior to high resolution top- in catalytic activity, cytoskeleton, transport, oxidative down proteomic analyses using 2DE [14-16]. These stress, calcium binding and energy metabolism. analyses revealed numerous protein changes in However, whist there is some overlap in terms of both the membrane and soluble protein fractions alterations to mitochondrial and ER proteins (Figure from the control and HSN-I (SPTLC1 V144D) patient 2), many of those identified from the ER function in lymphoblasts. Mitochondrial protein species that the areas of protein biosynthesis, apoptosis, cell changed in abundance were involved in catalytic proliferation, protein binding and lipid binding (Figure 1). activity, cytoskeleton, protein transport, oxidative stress, calcium binding and energy metabolism 3. Discussion (Figure 1). The proteins identified in the ER fractions SPT is the key rate determining enzyme in sphingolipid were involved in catalytic activity, cytoskeleton, and metabolism. Mutations within the SPTLC1 subunit lipid binding (Figure 1). While there were a number thus result in potential perturbations in sphingolipid of non-related protein species that changed in synthesis and metabolism that may be the underlying the mitochondria compared to the ER, there were causative effects of HSN-I [7]. In initial studies, we a number of similarities in biological processes, showed that a number of mitochondrial and ER most notably catalytic activity, cytoskeleton, protein proteins are altered in abundance, correlating with transport and oxidative stress [17-19] (Figure 2). the SPT mutations in patient derived cells [11-13]. Here we have carried out a more detailed top-down Mitochondria are known to play a role in proteomic analysis and identified 36 protein species neurodegeneration, and structural alterations have that change in abundance in the mitochondria and been characterised in V144D patient lymphoblasts ER of HSN-I patient cells. [20], with further studies identifying changes at the protein level within isolated mitochondria [11]. To identify potentially critical protein changes, The higher resolution analyses here provide more mitochondria and ER were first isolated from control detailed information still. Oxidative stress can and HSN-I (SPTLC1 V144D) patient lymphoblasts have an impact upon the cell, causing severe and
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Figure 1. Pie graph of the identified proteins function in mitochondrial and ER fractions. Representative pie graph of proteins identified in both membrane and soluble mitochondrial and ER fractions grouped into their biological functions.
Figure 2. Similarities in protein changes identified in Mitochondria and ER. Representative graph revealing four common protein changes occurring in the mitochondria and the ER.
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C. 3 pI 10
250 1 150 2 3 5 4 100 6
7 75 8 10 9
) 37
k D a ( 25
M W 20
15 10
D. 3 pI 10
250 1 150 2 3 100 5 4 6
75 8 7 10 9
37
kDa) 25
MW ( 20
15
10
Figure 3. Representative 2D gels images of mitochondrial membrane and soluble proteomes from control and patient derived lymphoblasts. (A) Gel of control mitochondrial membrane proteins. (B) Gel of V144D mitochondrial membrane proteins. (C) Gel of control mitochondrial soluble proteins. (D) Gel of V144D mitochondrial soluble proteins. Red circles represent identified protein species. The molecular weights are in kilodaltons (kDa) and the IEF dimension is in pH units.
Figure 4. Representative 2D gels images of ER membrane and soluble proteomes from control and patient derived lymphoblasts. (A) Gel of control ER membrane proteins (B) Gel of V144D ER membrane proteins. (C) Gel of control ER soluble proteins. (D) Gel of V144D ER soluble proteins. Red circles represent identified protein species. The molecular weights are in kilodaltons (kDa) and the IEF dimension is in pH units.
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extensive damage including protein aggregation and increased mitochondrial apoptotic process occurring impaired ion transport [21]. Previously, ubiquinol (2.6 and 2.2 fold increase respectively). cytochrome C subunit 1 was found to increase in abundance in V144D patient lymphoblasts [11]; here Eukaryotic translation initiation factor 5A-1 (eIF5A) this protein was found to increase in abundance has been shown to regulate the Bcl-2 binding protein (i.e. 4-fold), and this was accompanied by a 2.1- P53 and the P53 apoptosis pathway. In addition, fold increase in the abundance of Peroxiredoxin-4, eIF5A has a regulatory function in protein synthesis. a protein with antioxidant functions, that reduces the Its increase in abundance of 2.1 fold in the V144D build-up of hydrogen peroxide via a thiol-dependent mutant may possibly be the cells response to stabilise cycle [21]. These findings correlate with a potential uncontrolled protein misfolding due to ER stress [27]. increase in reactive oxygen species (ROS) within the Peroxiredoxin-2 was detected in the ER with an disease cells, which could lead to further disruption increase in abundance of 2.4 fold, peroxiredoxin-2 to mitochondria. just like peroxiredoxin-4 found in the mitochondrial fraction is an antioxidant [28], potentially increased Perturbations to energy production within in the mutant state in response to increased ROS neurons, having high metabolic demands, can production occurring within the ER and throughout have catastrophic consequences. Succinate the cell. dehydrogenase flavoprotein and pyruvate dehydrogenase E1 subunit are both part of the ER stress may decrease mRNA to reduce the protein electron transport chain, and both are increased in load upon the ER to help reduce the amount of abundance (i.e. 6.1 and 3.2 fold, respectively), likely misfolded proteins being produced [29], thus we see highlighting an energy metabolism issue within the reduced levels of the serine/threonine phosphatase mitochondria [22]. In addition to a potential need to PP-1, serine/arginine-rich splicing factor and increase energy output, increased levels of succinate Elongation factor 1-beta within HSN-I (SPTLC1 dehydrogenase could also potentially increase V144D) patient lymphoblasts. With known ER stress superoxide formation [22]. Whether these proteins occurring [20] the reduction in abundance of these apparently increased abundance is due to a direct proteins could be as a result of a compensatory effect need for increased energy or a compensatory effect reducing the load of protein synthesis occurring in due to an increase in ROS production and oxidative the stressed ER. stress disrupting the electron transport chains ability Lymphocyte specific protein 1 is an F-actin binding to produce energy remains unclear, but a destructive protein [30], it’s 3.2 fold increase in abundance, spiral would seem a distinct possibility. correlates with other cytoskeletal changes observed Ca2+ is also required for energy production within in the HSN-I (SPTLC1 V144D) patient lymphoblasts mitochondria, but increased Ca2+ levels can lead to suggesting that maintenance of the cytoskeleton is free radical generation [23]. The data identifies a being increased potentially due to increasing amount 2.6-fold decrease in the Ca2+ binding mitochondrial of ROS, known to cause actin remodelling and potential axonal retraction in the neuron [31]. carrier protein (ScaMc-1). This decrease might be a protective mechanism due to the already high Proteasome activator complex 1 (PSME1) and levels of ROS but will also cause a decrease in proteasome subunit alpha type 3 (PSMA3), degrade ATP production within the mitochondria. Voltage misfolded proteins, in an ubiquitin dependent dependent anion selective channel protein 1 (VDAC) process [32,33]. Both these proteins are increased allows mitochondrial influx/efflux of metabolites such in abundance, 4.2 and 2.6 fold respectively in the as ATP, and may also have a role in regulating Ca2+ in V144D patient derived lymphoblasts. Previous mitochondria [24]. A decrease in VDAC in the V144D studies have identified Ubiqutin-40s Ribosomal mutant, in conjunction with the reduction of SCaMc-1 Protein S27a [12], as such we see here the increase could result in an overall decrease in intracellular in proteasomes correlating a potential increase in the Ca2+ levels in mitochondria and thus decreased ATP number misfolded proteins directly due to ER stress, production, again strengthening the possibility for a oxidative stress or by another mechanism that affects destructive circle of cellular events. protein conformation.
Dipeptidyl peptidase 1, also known as Cathepsin Bcl-2 family proteins are regulators of mitochondrial C and Pro-cathepsin H has been shown to be pro- derived apoptosis. Bcl-2 proteins can illicit or inhibit apoptotic by cleaving Bid and Blc-2 family proteins cell death. Apolipoprotein L2 (ApoL2) has a potential released by mitochondria; greatly increasing the apoptotic role being a BH3- protein, localising to cascade of caspase apoptotic factors to be released mitochondria. This region, known as the ‘BH3- [25,26]. The abundance of these proteins are domain’, is essential for the apoptotic function of Bcl- increased in the mutant cells indicating a link to 2 autophagy, while the exact role of ApoL2 remains
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to be determined, a reduction in the V144D diseased lymphoblasts were kindly provided by Prof. Garth state may cause dysregulation of authopghy [34]. Nicholson (Molecular Medicine Laboratory, Anzac Research Institute, Sydney) [13]. Interestingly, the Chloride intracellular channel protein 1 was identified with a 2.7 fold increased abundance 6.2 Lymphoblast cultures in the HSN-I (SPTLC1 V144D) patient lymphoblasts. We have previously reported this proteins increased Lymphoblasts were cultured in RPMI-1640 media expression within the HSN-I (SPTLC1 V144D) patient (GIBCO), supplemented with FBS (10% v/v), Penicillin (1 U/mL), Streptomycin (1 µg/mL), lymphoblasts [12]. It acts as a chloride-selective ion o channel and usually exists in a soluble form in the L-glutamine (2 mM), and HEPES (1 mM) at 37 C in a humidified atmosphere of 5%2 CO , using T75 cytoplasm and nucleoplasm [35], but following stimuli 2 undergoes major structural changes and inserts in cm culture flasks (Greiner, Interpath). Prior to use in lipid membranes, where cell oxidation appears to biochemical assays, lymphoblasts were collected by be an important stimuli determining the transition centrifugation at 1,500 x g (5 min at RT) and washed of Chloride intracellular channel protein 1 between in PBS. Cell counts were obtained using the Countess these two forms [36]. Automated Cell Counter (Invitrogen, Australia).
It was identified that there were four other proteins with 6.3 Isolation of mitochondrial proteins a marked absence or presence in all mitochondrial Briefly, mitochondria were isolated using a sucrose and ER fractions. These protein species were density gradient [14,15]. Lymphoblasts were first located at 24 kDa, but each had a different pI (6.6 centrifuged at 1,500 x g for 5 min, and the cells and 8.3 respectively). Following mass spectral were then washed in 10 ml of ice cold 1X PBS prior analysis, these proteins identified as Ig Kappa Chain to suspension in 10 ml ice cold CaSRB Buffer (10 C. This finding correlates with our previous studies mM NaCl, 1.5 mM CaCl, 10 mM Tris-HCL, pH 7.5) in the HSN-I (SPTLC1 V144D) patient lymphoblasts and left on ice for 10 min. Cells were homogenised [11,12]. using a Dounce homogenizer (Kimble-Chase, USA) and 7 mL of 2.5X MS buffer (210 mM Mannitol, 70 4. Conclusion mM sucrose, 5 mM EDTA, 5 mM Tris-HCl, pH 7.6) This investigation has shown a correlation between was added to restore isotonicity. Homogenates were previous studies revealing an increase in proteins centrifuged at 700 x g for 5 min to remove nuclei induced by oxidative stress and mitochondrial and unbroken cells. The resulting supernatant was electron transport chain proteins. This study also centrifuged at 15,000 x g for 10 min to pellet the identified changes in calcium channel proteins, crude mitochondria. Sucrose gradients were made in cytoskeletal proteins, energy transport proteins. 4 mL high speed centrifuge tubes (Beckman Coulter, Some of these findings reflect previous studies USA) by adding 1 mL of 1.7 M sucrose buffer (1.7 M carried out, providing more evidence for a link of sucrose, 10 mM Tris-base, 0.1 mM EDTA, pH 7.6) increased misfolded proteins, oxidative stress, and overlayed with 1.6 mL of 1.0 M sucrose buffer (1.0 M cytoskeleton remodelling and potential changes sucrose, 10 mM Tris-base, 0.1 mM EDTA, pH 7.6). in Ca2+ signalling within the mitochondria. With The mitochondrial pellet was resuspended in 1.6 mL mounting discoveries into protein alterations in the of 1x MS buffer and overlayed on top of the sucrose V144D mutation it may provide a greater in-sight gradient and centrifuged at 40,000 x g for 30 min. into the molecular mechanisms that are occurring in The mitochondrial band, in the middle of the gradient, HSN-I. was gently removed using a 20 G needle, transferred to a 1.5 mL tube, and centrifuged at 16,000 x g for 5. Materials 15 min. The resulting pellet was resuspended in All cell culture stock solutions, including RPMI- 2D solubilisation buffer containing 8 M urea, 2 M 1640, Foetal Bovine Serum (FBS), Penicillin (100 U/ thiourea, 4% (w/v) CHAPS and a cocktail of protease mL), Streptomycin (100 µg/mL), L-glutamine (2 M), inhibitors. HEPES (1 M), and phosphate buffered saline (PBS) 6.4 Isolation of ER proteins were purchased from GIBCO Invitrogen (Australia). Cell culture consumables were purchased from BD Briefly, [14,15] Lymphoblasts were first centrifuged at Falcon (Greiner, USA). 1,500 x g for 5 min, and the cells were then washed in 10 ml of ice cold 1X PBS prior to suspension in 6. Methods 10 mL ice cold CaSRB Buffer (10 mM NaCl, 1.5 mM 6.1 EBV transformed lymphoblasts CaCl, 10 mM Tris-HCL, pH 7.5) and left on ice for 10 min. Cells were homogenised using a Dounce EBV transformed control and V144D HSN-I patient homogenizer (Kimble-Chase, USA) and 7 mL of 2.5 X
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MS buffer (210 mM Mannitol, 70 mM sucrose, 5 mM phosphine and 45 mM DTT) and alkylation buffer EDTA, 5 mM Tris-HCl, pH 7.6) was added to restore (230 mM acrylamide monomer). isotonicity. Homogenates were centrifuged at 700 x g for 5 min to remove nuclei and unbroken cells. The treated samples were added to 7 cm Non- The resulting supernatant was centrifuged at 15,000 Linear pH 3-10 IPG strips (Bio-Rad ReadyStrip), x g for 10 mins to remove mitochondria. A sucrose and rehydrated for 16 h at RT. Isoelectric focusing gradient was made in 15 mL high speed centrifuge (IEF) was then carried out at 20° C using the Protean tubes (Beckman Coulter, USA) by adding 2 mL of 2.0 IEF Cell (Bio-Rad, USA). After IEF, IPG strips were M sucrose buffer (2.0 M sucrose, 10 mM Tris-base, then resolved in the second dimension using a 0.1 mM EDTA, pH 7.6) overlayed with 3.0 mL of 1.5 12.5% T, 2.6% C polyacrylamide gel buffered with M sucrose buffer (1.5 M sucrose, 10 mM Tris-base, 375 mM Tris buffer (pH 8.8), 0.1% (w/v) sodium 0.1 mM EDTA, pH 7.6) and 3.0 mL of 1.3 M sucrose dodecyl sulphate and polymerised with 0.05% (1.3 M sucrose, 10 mM Tris-base, 0.1 mM EDTA, pH (w/v) ammonium persulphate and 0.05% (v/v) 7.6) ER containing supernatant was loaded on top of tetramethylethylenediamine (TEMED). A stacking gel the sucrose gradient and spun at 152,000 x g for 70 containing a 5% T, 2.6% C polyacrylamide buffered min. The ER band, the interface of the 1.5 M and 1.3 with 375 mM Tris buffer (pH 8.8), 0.1% (w/v) SDS M sucrose, was gently removed using a 20 G needle, and included 0.1% bromophenol blue was added to transferred to a 4mL high speed centrifuge tubes the resolving gel. The IPG strips were placed onto the (Beckman Coulter, USA) and centrifuged at 100,000 stacking gel and overlaid with 0.5% (w/v) low melting x g for 35 min. The resulting pellet was resuspended agarose dissolved in 375 mM Tris (pH 8.8), with 0.1% in 2D solubilisation buffer containing 8 M urea, 2 M (w/v) SDS. Electrophoresis was carried out at 4° C; thiourea, 4% (w/v) CHAPS and a cocktail of protease 150V initially for 10 min then reduced to 90V for 2.5 inhibitors. h. The gels were placed in fixative containing 10% methanol and 7% acetic acid for 1 h. The gels were 6.5 Membrane and soluble protein fractionation washed with distilled water for 20 min, 3 times and subsequently stained with colloidal coomassie blue Harvested mitochondrial and ER proteins were (0.1% (w/v) CCB G-250, 2% (v/v) phosphoric acid, separated into membrane and soluble protein 10% (w/v) ammonium sulphate, 20% (v/v) methanol) fractions as previously described [16]. Briefly; isolated for 20 h, with constant shaking at RT [18]; the gels proteins were placed in 20mM HEPES for 3 min on ice were the de-stained 5 times with 0.5 M NaCl, 15 min with an equal volume 2X PBS subsequently added. each. Imaging of CBB-stained gels on the FLA-9000 Membranes were collected at 125 000 x g for 3 h. imager (FUJIFILM, Tokyo, Japan) was carried out at The supernatant was collected and membrane pellet 685/750 excitation/emission with a photomultiplier resuspended in 1X PBS and spun at 125 000 x g for tube (PMT) setting of 600 V and pixel resolution a further 3 h. Washed membranes were solubilised set to 100 µm [18]. Analysis of 2D gel images in 2D solubilisation buffer containing 8 M urea, 2 M was performed using Delta 2D software (version thiourea, 4% (w/v) CHAPS. Soluble protein fractions 4.0.8; DECODON GmbH, Gerifswald, Germany) were concentrated using a 3 kDa cut-off Millipore with automated spot detection (Local Background Amicon Ultra Centrifugal filters and resuspended in Region: 96; Average Spot Size: 32 and sensitivity in 4M Urea. percentage: 20.0) (Figure 3 and 4). 6.6 Protein concentration 6.6.2 Mass spectrometry Determination of total cellular protein was performed using the EZQ Protein Estimation Assay (Invitrogen, For analysis a selection criteria was applied. For Australia) as previously described [17]. inclusion, changes in mean normalised spot volume (the abundance of resolved protein species) had 6.6.1 Two dimensional gel electrophoresis to be greater than or equal to a 2.0 fold increase or decrease between control and HSN-I (SPTLC1 Protein concentration estimations (EZQ assay) were V144D) patient lymphoblast, have a p-value <0.05 performed on patient and control mitochondrial and and be present in all replicate gels [18,19]. The ER protein fractions; a total of 100 µg protein was protein species of interest were excised from gels used for each 2DE analysis. 2DE was carried out as and de-stained overnight. The gel pieces were then previously described [11,16,18,19]; briefly, proteins reduced and alkylated in 10 mM Dithiothreitol (DTT) were reduced and alkylated in solutions containing and 15 mM Idoacetic acid (IAA), and subsequently total protein extraction buffer (containing 8M urea, incubated with trypsin solution (10 ng/µL, pH 7.4) for 2M thiourea and 4% CHAPS without ampholytes), 16 h at 37ºC. LC-MS/MS analysis was carried out total extraction buffer with 2% ampholytes, TBP/ on a nanoAquity UPLC (Waters Corp., Milford, MA, DTT disulphide reduction buffer (2.3 mM Tributyl USA) linked to a Xevo QToF mass spectrometer from
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Waters (Micromass, UK). The data were acquired References using Masslynx software (Version 4.1, Micromass [1] Dyck PJ, Thomas PK.( 2005). Dyck: Peripheral UK). The MS/MS data files were searched against Neuropathy, 4th Edition. Philadelphia, Mosby Elsevier. SwissProt databases with semi-trypsin as the [2] Verhoeven K, Coen K, De Vriendt E, et al. (2004). enzyme. The following parameters were used in SPTLC1 mutation in twin sisters with hereditary Mascot for identification of the peptides: maximum sensory neuropathy type I. Neurology. 62: 1001-2. missed cleavage of 2, positive peptide charge of 2, 3 and 4, peptide mass tolerant of 0.5 Da in MS and MS/ [3] Bejaoui K, Wu C, Scheffler MD, et al. (2001). SPTLC1 is mutated in hereditary sensory neuropathy, type 1. MS data base, fixed modification: carbamidomethyl Nat Genet. 27: 261-2 (C) and variable modifications: oxidation (M). [4] Dawkins JL, Hulme DJ, Brahmbhatt SB, Auer- 7. Competing Interests Grumbach M, Nicholson G A. (2001). Mutations in The authors have no competing interests. SPTLC1, encoding serine palmitoyltransferase, long chain base subunit-1, cause hereditary sensory neuropathy type I. Nat Gene. 27: 309-12. 8. Author Contributions [5] Verhoeven K, Timmerman V, Mauko B, et al. (2006). SES carried out all experimentation, data analysis Recent advances in hereditary sensory and autonomic and wrote an initial draft of the manuscript; JRC neuropathies. Curr Opin Neurol. 19: 474-80. participated in design of study, provided access to [6] Hornemann T, Richard S, Rutti M, Wei Y, Von- the proteomics facility in which the bulk of the work Eckardstein A. (2006) ‘Cloning and initial was carried out, and re-drafted substantial portions characterization of a new subunit for mammalian of the draft; SJM conceived the study, participated serine-palmitoyltransferase’. The Journal of biological in design of study, re –drafted substantial sections chemistry. 49: 37275-37281. of the drafts. All authors read and approved the final [7] Wei J, Yerokun Y, Liepelt M, et al. (2007). 2-1 manuscript. Serine Palmitoyltransferase. Sphingolipid Biology. Springerlink. 25-27. Acknowledgements [8] Mandon EC, Ehses I, Rother J, Van Echten G, We are grateful to Prof Garth Nicholson (Molecular Sandhoff K. (1992). Subcellular localization and Medicine Laboratory and Northcott Neuroscience membrane topology of serine palmitoyltransferase, Laboratory Anzac Research Institute, Sydney) for 3-dehydrosphinganine reductase, and sphinganine providing all EBV transformed lymphoblast lines N-acyltransferase in mouse liver. J Biol Chem. 267: used in this study. SES was supported by an APA 111-448. Research Scholarship, and the UWS School of [9] Yard B, Carter L, Johnson K, et al. (2007). The Science and Health Postgraduate research fund. structure of serine palmitoyltransferase; gateway SJM notes the continuing support of an anonymous to sphingolipid biosynthesis. Journal of molecular Private Foundation. JRC acknowledges the support biology. 370: 870-886. of the UWS School of Medicine. [10] Yasuda S, Nishijima M, Hanada K. (2003). Localization, topology, and function of the LCB1 List of Abbreviations subunit of serine palmitoyltransferase in mammalian cells. J Biol Chem. 278: 4176-83. 2DE, two dimensional gel electrophoresis; Apol2, Apolipoprotein L2.; DRG, dorsal root ganglion; DTT, [11] Stimpson SE, Coorssen JR, Myers SJ. (2014). Dithiothreitol; eIF2A, Eukaryotic translation factor 5A- Mitochondrial protein alterations in a familial 1; ER, endoplasmic reticulum; FBS, Foetal Bovine peripheral neuropathy caused by mutations in the Serum; HSN, Hereditary sensory neuropathies; sphingolipid protein, SPTLC1. J Chem Biol. HSN-I, Hereditary sensory neuropathy type I; [12] Stimpson, SE, Coorssen JR, Myers SJ. (2015). IAA, Idoacetic acid; IEF, Isoelectric focusing; kDa, Isolation and identification of ER associated Kilodaltons; LCB1, long-chain base one; LC/MS, proteins with unique expression changes specific liquid chromatography/ mass spectrometry; PBS, to the V144D SPTLC1 mutations in HSN-I. BMC phosphate buffered saline; PMT, photomultiplier Neuroscience. tube; PSME1, Proteasome activator complex [13] Dedov V, Dedova I, Merrill A, Nicholson G. subunit 1; PSMA3, Proteasome subunit alpha (2004). Activity of partially inhibited serine type-3; ROS, reactive oxygen species; SCaMc-1, palmitoyltransferase is sufficient for normal Calcium binding mitochondrial carrier protein 1; sphingolipid metabolism and viability of HSN1 patient SPT, serine palmitoyltransferase; SPTLC1, serine cells. Biochimica et biophysica acta.1688: 168-175. palmitoyltransferase long chain subunit 1; TEMED, [14] Bozidis P, Williamson CD, Colberg-Poley AM. (2007). tetramethylethylenediamine; VDAC, Voltage Isolation of endoplasmic reticulum, mitochondria, and dependent anion-selective channel protein 1. mitochondria-associated membrane fractions from
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transfected cells and from human cytomegalovirus- [26] Turk V, Stoka V, Vasiljeva O, et al. (2012). Cysteine infected primary fibroblasts.Curr Protoc Cell Biol. 27. cathepsins: from structure, function and regulation to new frontiers. Biochim Biophys Acta. : 68-88. [15] Vaseva AV, Moll UM. (2013). Identification of p53 in 1824 mitochondria. Methods Mol Biol. 962: 75-84. [27] Huang Y, Higginson DS, Hester L, Park MH, Snyder [16] Butt RH, Coorssen JR. (2005). Postfractionation for SH. (2007). Neuronal growth and survival mediated enhanced proteomic analyses: routine electrophoretic by eIF5A, a polyamine-modified translation initiation methods increase the resolution of standard 2D-PAGE. factor. Proc Natl Acad Sci U S A. 104: 4194-9. J Proteome Res. 4: 982-91. [28] Ogasawara Y, Ohminato T, Nakamura Y, Ishii K. [17] Churchward M, Butt RH, Lang J, Hsu K, Coorssen J. (2012). Structural and functional analysis of native (2005). Enhanced detergent extraction for analysis peroxiredoxin 2 in human red blood cells. Int J of membrane proteomes by two-dimensional gel Biochem Cell Biol. 44: 1072-7. electrophoresis. Proteome Sci. 3: 5. [29] Kawai T, Fan J, Mazan-Mamczarz K, Gorospe M. [18] Gauci VJ, Padula MP, Coorssen JR. (2013). (2004). Global mRNA stabilization preferentially linked Coomassie blue staining for high sensitivity gel-based to translational repression during the endoplasmic proteomics. J Proteomics. 90: 96-106. reticulum stress response. Mol Cell Biol. 24: 6773-87. [19] Wright EP, Partridge MA, Padula MP, et al. [30] Liu L, Cara DC, Kaur J, et al. (2005). LSP1 is an (2014). Top-down proteomics: Enhancing 2D gel endothelial gatekeeper of leukocyte transendothelial electrophoresis from tissue processing to high- migration. J Exp Med. 201: 409-18. sensitivity protein detection. Proteomics. 14: 872-89. [31] Hallengren J, Chen PC, Wilson SM. (2013). Neuronal [20] Myers S, Malladi C, Hyland R, et al. (2014). ubiquitin homeostasis. Cell Biochem Biophys. 67: 67-73. Mutantions in the SPTLC1 protein cause mitochondrial structual abnormalisites and [32] Johnston SC, Whitby FG, Realini C, Rechsteiner M, endoplasmic reticulum stress in lymphoblasts. DNA Hill CP. (1997). The proteasome 11S regulator subunit and Cell Biology. 33: 7. REG alpha (PA28 alpha) is a heptamer. Protein Sci. 6: 2469-73. [21] Tavender TJ, Bulleid NJ. (2010). Peroxiredoxin IV protects cells from oxidative stress by removing H2O2 [33] Shi Z, Li Z, Li ZJ, et al. (2014). Cables1 controls p21/ produced during disulphide formation. J Cell Sci. 123: Cip1 protein stability by antagonizing proteasome 2672-9. subunit alpha type 3. Oncogene. [22] Guzzo G, Sciacovelli M, Bernardi P, Rasola A. [34] Galindo-Moreno J, Iurlaro R, El Mjiyad N, et al. (2014). Inhibition of succinate dehydrogenase by the (2014). Apolipoprotein L2 contains a BH3-like domain mitochondrial chaperone TRAP1 has anti-oxidant and but it does not behave as a BH3-only protein. Cell anti-apoptotic effects on tumor cells. Oncotarget. 5: Death Dis. 5: e1275. 11897-908. [35] Warton K, Tonini R, Fairlie WD, et al. (2002). [23] Feissner RF, Skalska J, Gaum WE, Sheu SS. (2009). Recombinant CLIC1 (NCC27) assembles in lipid Crosstalk signaling between mitochondrial Ca2+ and bilayers via a pH-dependent two-state process ROS. Front Biosci (Landmark Ed). 14: 1197-218. to form chloride ion channels with identical characteristics to those observed in Chinese hamster [24] Brookes PS, Yoon Y, Robotham JL, Anders MW, Sheu SS. (2004). Calcium, ATP, and ROS: a mitochondrial ovary cells expressing CLIC1. J Biol Chem. 277: 26003-11. love-hate triangle. Am J Physiol Cell Physiol. 287: C817-33. [36] Averaimo S, Milton RH, Duchen MR, Mazzanti M. [25] Droga-Mazovec G, Bojic L, Petelin A, et al. (2008). (2010). Chloride intracellular channel 1 (CLIC1): Cysteine cathepsins trigger caspase-dependent cell Sensor and effector during oxidative stress. FEBS death through cleavage of bid and antiapoptotic Bcl-2. Lett. 584: 2076-84.
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Paper V
Presented in Proteomes format. Submitted to Proteomes
Contributions
SES carried out all experimentation, data analysis and wrote first draft manuscript.
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Title: Identifying unique protein alterations caused by SPTLC1 mutations in a transfected neuronal cell model.
Running title:
Scott E. Stimpson, 1, 3, 4 Jens R. Coorssen†2,3,4,5 and Simon J. Myers†1,3,4,5
University of Western Sydney 1Neuro-Cell Biology Laboratory 2Molecular Physiology 3Molecular Medicine Research Group 4School of Science and Health 5School of Medicine Locked Bag 1797, NSW 2751, Australia
† Co-corresponding authors: Dr. Simon Myers [To communicate with Editorial and Production Offices]
Address: University of Western Sydney, Office 21.1.05, Campbelltown campus, Locked Bag 1797, Penrith, NSW 2751, Australia
Phone: +61 02 4620 3383 Email: [email protected] Facsimile: +61 4620 3025
Professor Jens Coorssen
Address: University of Western Sydney, Office 30.2.15, Campbelltown campus, Locked Bag 1797, Penrith, NSW 2751, Australia
Phone: +61 4620 3802 Email: [email protected] Facsimile: +61 4620 3890
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ABSTRACT
Hereditary sensory neuropathy type I is an autosomal dominant disorder that affects the sensory neurons. Three missense mutations in serine palmitoyltransferase long chain subunit 1 cause hereditary sensory neuropathy type I. The endoplasmic reticulum, where the serine palmitoyltransferase long chain subunit 1 protein resides, and mitochondria are both altered in hereditary sensory neuropathy type I mutant cells.
Utilising a transfected neuronal cell line (ND15) we have identified and confirmed, as previously described in a lymphoblast model [6-7], altered protein expression levels of Ubiquinol Cytochrome C, Hypoxia Up regulated Protein 1, Chloride Intracellular Channel Protein 1, Ubiqutin-40s Ribosomal Protein S27a, and Coactosin. Additionally, a further 14 new proteins that exhibiting altered expression within V144D, C133W and C133Y mutants were identified. These data have shown that mutations in SPTLC1 alters the expression of a set of proteins that may help to establish a causal link between the mitochondria and ER and the ‘dying back’ process of dorsal root ganglion neurons observed in HSN-I.
Keywords: Hereditary sensory neuropathy type 1, ND15, Transient Transfection, V144D, C133W, C133Y.
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Background
Hereditary sensory neuropathy type I (HSN-I) is an autosomal dominant inherited neurodegenerative disorder. It is caused by the missense mutations in the open reading frame of serine palmitoyltransferase (SPT) long chain subunit 1 (SPTLC1) [1]. SPTLC1 mutations are amino acid substitutions of cysteine to tryptophan at position 133 (C133W), valine to aspartate at position 144 (V144D) and cysteine to tyrosine at position 133 (C133Y) [1]. SPT is an endoplasmic reticulum (ER)-bound, key rate-determining enzyme in the complex sphingolipid metabolic pathway [3]. HSN-I is characterised by a degeneration in the dorsal root ganglion (DRG) neuron and presents with a clinical onset between the second or third decades of life [2-5].
In previous studies we investigated altered protein profile changes in the mitochondria and ER of HSN-I patient cells (SPTLC1 V144D mutation) [6-8]. Changes in protein expression were identified in both the isolated mitochondria and ER subcellular fractions. Ubiquinol cytochrome C was most notably altered in expression within the mitochondria of the HSN-I patient cells. Within the ER fractions; Hypoxia up regulated Protein 1 (ORP-150), Chloride Intracellular Channel Protein 1 (CLIC1), Ubiqutin-40s Ribosomal Protein S27a (RPS27a), and Coactosin (COTL1) protein expression was increased within the HSN-I patient cells (SPTLC1 V144D mutation). In addition to these findings, a further 36 proteins were identified from both the mitochondria and ER of HSN-I patients cells using top-down proteomic analyses [6-8]. Of these 36 proteins, the observed alterations were in proteins related to oxidative stress and cytoskeleton.
Based on these earlier findings, this investigation utilised a ND15 cell line (hybrid of rat dorsal root ganglion neurone and a mouse neuroblastoma) which had been transiently transfected (TT) to overexpress the three SPTLC1 missense mutations; V144D, C133W and C133Y. The data obtained from this neuronal cell model confirmed previous results identified from the HSN-I patient lymphoblast, while notably identifying changes exhibited in the C133W and C133Y mutations. We have also identified an additional 14 proteins that are altered in abundance within the transfected ND15 cells. Together these findings offer a greater insight into the molecular mechanisms occurring in the three known mutations causing HSN-I.
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Materials
All cell culture stock solutions, including DMEM, Foetal Bovine Serum (FBS), Penicillin (100 U/mL), Streptomycin (100 µg/mL), L-glutamine (2 M), NEAA (1 M) and phosphate buffered saline (PBS) were purchased from GIBCO Invitrogen (Australia). Cell culture consumables were purchased from BD Falcon (Greiner, USA). ORP-150, CLIC1, RPS27a, Calnexin, Ubiquinol Cytochrome C, MTCO2, and GAPDH primary antibodies were purchased from Abcam (USA). SPTLC1 primary antibody was purchased from Santa Cruz Biotechnology (USA). COTL1 primary antibody was purchased from Protein SciTech (USA). Kif2A and GFP primary antibodies were purchased from Merck Millipore (USA). Secondary horse radish peroxidase (HRP) labelled anti-mouse antibodies and DAPI stains were purchased from Sigma-Aldrich (Australia).
Methods
ND15 Cultures
ND15 cell lines were cultured in DMEM media (GIBCO), supplemented with FBS (10 % v/v), Penicillin (1 U/mL), Streptomycin (1 µg/mL), L-glutamine (2 mM), and NEAA (1 mM) at 37 oC in a humidified
2 atmosphere of 5 % CO2, using T75 cm culture flasks (Greiner, Interpath). Prior to use in biochemical assays, ND15 cells were collected by centrifugation at 1,500 x g (5 min at RT) and washed in PBS. Cell counts were obtained using the Countess Automated Cell Counter (Invitrogen, Australia).
Transient Transfection
ND15 cells were transiently transfected (TT) with plasmid constructs (wild type, V144D, C133W and C133Y (GFP) using Lipofectamine 2000 (L2K; Invitrogen, USA). Cells were plated at a density of 2x 105 per well in 6 well plates. Transfections were carried out when cells were 90-95 % confluent (approximately 24 hours
o after plating, at 37 C and 5 % CO2). Per well, both DNA plasmid constructs and L2K reagent were diluted in 250 µL of Opti-MEM I Reduced Serum Media (Invitrogen, USA). DNA constructs were diluted to 16 μg/ml and L2K to 40 μL/ml. Within 5 min of each dilution, the DNA construct diluents and L2K diluents were combined and incubated for 30 min at 25 oC. After incubation, DNA-L2K complexes (500 μL) were then added into each well, as required. The cells were then incubated at 37 oC in a humidified atmosphere of
5 % CO2 for 6 h. Cells then had media replaced with fresh media and were cultured for a further 48 h before being assessed using the LSM 5 confocal microscope (comprising the LSM 5 exciter laser scanning microscope with Axiovert 200M inverted optical microscope [Carl Zeiss, Jena, Germany]).
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Protein Concentration
Determination of total cellular protein was carried out using the EZQ Protein Estimation Assay (Invitrogen, Australia) as previously described [9].
SDS-PAGE and Immunoblotting
Wild type and mutant protein fractions (25 µg total protein) were subjected to SDS-PAGE on 12.5% resolving gels and transferred to PVDF membrane. The membranes were blocked with 5% skim milk in TBS buffer containing 0.1% Tween-20 for 1 h and then incubated with anti-SPTLC1, anti-GAPDH, anti-ORP- 150, anti-CLIC1, anti-RPS27a, anti-COTL1, anti-MTCO2, anti-GFP and anti-Kif2A at 1:1000, for 16 h. Membrane were then incubated with secondary HRP antibody (1:2000 dilution) for 1 h at RT. Blots were developed using an enhanced chemiluminescence (ECL) detection kit (Pierce Thermo Scientific, USA). All membranes were developed on CL-Xposure Film (Thermo Fisher Scientific, U.S.A) using an AGFA X-ray developer.
Immunofluorescence
Immunofluorescence was carried out as previously described by [6]. Briefly, ND15 cells (1 x106 cells) were grown on sterile glass coverslips in 6-well plates 24 h prior to transfection. Forty-eight hours post transfection, 4% paraformaldehyde was added to the cells for 15 min. Cells were then placed in 0.5% Triton X-100 and incubated at 37 oC for 20 min. The cells were then blocked in 5% BSA solution at 37 oC for 30 min, then resuspended in primary antibody, SPTLC1, Kif2A, Cytochrome C, RPS27a, CLIC1, ORP-150, COTL1, Calnexin and MTCO2, and stained for 1 h at RT. The cells were subsequently washed and resuspended in secondary antibody, anti-mouse Rhodamine (Millipore, 1:200), and incubated for 1 h at RT. DAPI (1µg/µL) was added for 2 min and then the cells were washed twice with PBS. The coverslips were left overnight to dry and mounted onto glass slides prior to confocal imaging using the LSM 5 confocal microscope comprising the LSM 5 exciter laser scanning microscope with Axiovert 200M inverted optical microscope (Carl Zeiss, Jena, Germany).
Flow Cytometry
FACS analyses were carried out as previously described by [6]. ND15 cells were transfected as above and then cells were then suspended in 4% paraformaldehyde and incubated for 15 min at RT and then resuspended in 0.3% Triton X-100 for 15 min at 37 oC. After incubation the cell suspension was centrifuged at 1,000 x g for 5 min and the pellet resuspended in primary antibody for 1 h at RT. Cell suspension was
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centrifuged, washed in PBS and resuspended in secondary antibody, anti-mouse or anti-rabbit Rhodamine (Millipore, 1:200) for 1 h at RT. The cell suspension was then analysed using the MACSQuant flow cytometer (Miltenyi Biotech, Germany).
Two Dimensional Gel Electrophoresis
Protein concentration estimations (EZQ assay) were carried out on wild type and mutant protein fractions; a total of 100 µg protein was used for each 2DE analysis. 2DE was carried out as previously described [10- 12] and [6]; briefly, whole ND15 proteins were reduced and alkylated in solutions containing total protein extraction buffer (containing 8M urea, 2M thiourea and 4% CHAPS without ampholytes), total extraction buffer with 2% ampholytes, TBP/DTT disulphide reduction buffer (2.3 mM Tributyl phosphine and 45 mM DTT) and alkylation buffer (230 mM acrylamide monomer).
The treated samples were added to 7 cm Non-Linear pH 3-10 IPG strips (Bio-Rad ReadyStrip) and rehydrated for 16 h at RT. Isoelectric focusing (IEF) was then carried out at 20 °C using the Protean IEF Cell (Bio-Rad, USA). After IEF, IPG strips were then resolved in the second dimension using a 12.5% T, 2.6%, C polyacrylamide gel buffered with 375 mM Tris buffer (pH 8.8), 0.1% (w/v) sodium dodecyl sulphate and polymerised with 0.05% (w/v) ammonium persulphate and 0.05% (v/v) tetramethylethylenediamine (TEMED). A stacking gel containing a 5% T, 2.6% C polyacrylamide buffered with 375 mM Tris buffer (pH 8.8), 0.1% (w/v) SDS and included 0.1% bromophenol blue was added to the resolving gel. The IPG strips were placed onto the stacking gel and overlaid with 0.5% (w/v) low melting agarose dissolved in 375 mM Tris (pH 8.8), with 0.1% (w/v) SDS. Electrophoresis was carried out at 4 °C; 150V initially for 10 min, then reduced to 90 V for 2.5 h. The gels were placed in fixative containing 10% methanol and 7% acetic acid for 1 h. The gels were washed with distilled water for 20 min, 3 times and subsequently stained with colloidal coomassie blue (0.1% (w/v) CCB G-250, 2% (v/v) phosphoric acid, 10% (w/v) ammonium sulphate, 20% (v/v) methanol) for 20 h, with constant shaking at RT (Gauci et. al., 2013). Gels were the de-stained by washing 5 times with 0.5 M NaCl for 15 min each wash. Imaging of CBB-stained gels on the FLA-9000 imager (FUJIFILM, Tokyo, Japan) was carried out at 685/750 excitation/emission with a photomultiplier tube (PMT) setting of 600 V and pixel resolution set to 100 µm (Gauci et. al., 2013). Analysis of 2D gel images was performed using Delta 2D software with automated spot detection (Local Background Region: 96; Average Spot Size: 32 and sensitivity in percentage: 20.0) (version 4.0.8; DECODON GmbH, Gerifswald, Germany).
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Mass Spectrometry
For analyses a selection criteria was applied. For inclusion, changes in mean normalised spot volume (the abundance of resolved protein species) had to be greater than a 1.0 fold difference between samples from wild type versus V144D, C133W and C133Y mutants and be present in all replicate gels [11-12]. Briefly, the protein species of interest were excised from gels and de-stained overnight. The gel pieces were then reduced and alkylated in 10 mM dithiothreitol (DTT) and 15 mM Iodoacetic acid (IAA), and subsequently incubated in trypsin solution (10 ng/µL, pH 7.4) for 16 hours at 37 oC. LC-MS/MS analysis was carried out on a nanoAquity UPLC (Waters Corp., Milford, MA, USA) linked to a Xevo QToF mass spectrometer from Waters (Micromass, UK). The data were acquired using Masslynx software (Version 4.1, Micromass UK). The MS/MS data files were searched against SwissProt databases with semi-trypsin as the enzyme.
Calcium imaging
ND15 cells were grown for 24 h in 35 mm glass bottom size 0 dishes (MatTek, USA) and transfected as previously described. Molecular Probes Rhod-3 calcium imaging kit (Molecular Probes, USA) was used to stain the cells for imaging. Briefly, cells were incubated at RT in the dark for 1 h in 10 µM Rohd-3 AM, 2.5 mM probenecid and 1x Powerload. Cells were briefly washed in calcium-free PBS and incubated for a further 1 h at RT with 2.5 mM probenecid. To obtain low and high intracellular calcium images, non- transfected (NT) ND15 cells were infused with PBS without calcium, containing 5 mM EGTA and 2 µM ionomycin to facilitate intracellular calcium to efflux from the cell. High intracellular calcium images were obtained by infusing the cells in PBS containing calcium and 2 µM ionomycin. Cells were ready for imaging after a further two washes in calcium-free PBS and imaged on the LSM 5 confocal microscope comprising the LSM 5 exciter laser scanning microscope with Axiovert 200M inverted optical microscope (Carl Zeiss, Jena, Germany).
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Results
Expression of proteins identified in HSN-I transfected ND15 cells
In order to assess the level of expression of proteins previously reported as altered, total cellular protein fractions from wild type and mutant HSN-I TT ND15 cells were isolated and quantitative immunoblot analyses were carried out (Figure 1). Quantitation of isolated total cell lysate immunoblots of wild type and mutant ND15 cells (Figures 2 A-J) confirmed that there were statistically significant (p < 0.05) changes in expression of COTL1 (Figure 2G), Cytochrome C (Figure 2H) and ORP-150 (Figure 2I) in all mutants compared to the wild type. RPS27a (Figure 2F) was significantly increased in V144D and C133Y. Quantitation analysis showed there was no statistically significant increase in expression of CLIC1 (Figure 2E), however there was an increase in expression in all mutants compared to the wild type. Expression analysis determined that there were no statistically significant changes in the expressions of MTCO2 (Figure 2A), GFP (Figure 2D), SPTLC1 (Figure 2C), GAPDH (Figure 2J) and Kif2A (Figure 2B).
Intracellular localisation analyses of SPTLC1 and proteins within transiently transfected ND15 cells.
The intracellular localisation and abundance of the proteins SPTLC1, Kif2A, Cytochrome C, RPS27a, CLIC1, ORP-150, COTL1 and MTCO2 were established using immunostained wild type and mutant TT ND15 cells. There were no apparent changes in intracellular localisation of the SPTLC1 when transfected, with GFP- labelled SPTLC1 localising to the perinuclear region where the ER resides (Figure 3A). Kif2A is a microtubule associated protein distributed evenly across the cytoskeleton [14]. Kif2A displayed consistent cytoskeletal patterns throughout the cell in both wild type and mutants (Figure 3B). MTCO2 is classically found to be distributed across the mitochondrial inner membrane. MTCO2 was distributed evenly throughout the mitochondria of the cells, indicating no change in the localisation within the mitochondria of mutant cells compared to the wild type cells (Figure 3H).
Cytochrome C is typically located within the mitochondrial inner membrane [15]. Interestingly, Cytochrome C showed a more perinuclear clustering in the mutant cells compared to that of the wild type cells, where the proteins were found to be more evenly distributed throughout the periphery of the cells (Figure 3C). RPS27a is located within the cytoplasm and nucleoplasm of cells [16]. With these proteins being evenly distributed throughout the cells in the wild type and mutants indicating no clustering of ubiquinated proteins occurring (Figure 3D). CLIC1 exists in a soluble and membrane bound form, typically distributed evenly within the cells [17]. CLIC1 was localised in the cell periphery in the wild type and mutant cells. However the mutants displayed a larger localisation towards the perinuclear region,
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indicating that CLIC1 may be present more in the membrane form in the mutants. ORP-150 is a chaperon protein localised throughout the cell [18] and was found to be distributed throughout the wild type and mutant cells. ORP-150 was also found to be more abundant within the mutants compared to the wild type cells (Figure 3F). COTL1 is a cytoskeletal associated protein interacting with the cytoskeleton [19]. COTL1 exhibited an even cytoskeletal pattern through all the cells. Interestingly, there appeared to be a more perinuclear clustering and potential co-localisation occurring in the C133Y mutant in comparison to the wild type cells (Figure 3G).
FACS analyses of Wild Type, V144D-, C133W- and C133Y-transfected ND15 cells reveal changes in fluorescence intensity of CLIC1, Cytochrome C, ORP-150 and RPS27a.
Fluorescence assisted cell sorting (FACS) was used to determine the total fluorescence per cell of TT ND15 immunostained cells for the proteins SPTLC1, Kif2A, Cytochrome C, RPS27a, CLIC1, ORP-150, COTL1, MTCO2 and GFP (Figure 4). There was a marked increase in the relative fluorescence intensity of CLIC1 (Figure 4E), Cytochrome C (Figure 4C), ORP-150 (Figure 4F) and RPS27a (Figure 4D) in the mutant cells compared to that of wild type. There were no changes to SPTLC1 (Figure 4A), Kif2A (Figure 4B) and GFP (Figure 4I) between the wild type and mutants. These results correlate with the quantitative immunoblot data presented in Figure 2.
Resolution of total cellular proteins using 2D gels from SPTLC1-transfected ND15 Cells
Total isolated wild type and mutant ND15 proteins were resolved and quantitatively assessed using a refined two dimensional gel electrophoresis (2DE) protocol [10-11]. The samples resolved covering the entire MW and pI range in all triplicate gels (Figure 5). Standard spot counts indicated 674 ± 7, 669 ± 4, 663 ± 9 and 655 ± 5 protein species were resolved in wild type, V144D, C133W and C133Y mutant fractions respectively. LC/MS data coupled with Mascot Daemon searches of SwissProt database resulted in identification of a further 14 protein alterations in the three mutant samples relative to the wild type, as summarised in Table 1.
Alterations within the intracellular calcium levels of V144D-, C133W-, C133Y-transfected ND15 cells compared to that of wild type and non-transfected controls.
Wild type and mutant ND15 cells were analysed for total intracellular calcium using the Rhod-3 Am calcium stain (Figure 6). Cell images were analysed using ImageJ (NIH, USA) and corrected total cellular fluorescence obtained. Analyses revealed a marked decrease in intracellular calcium in C133W and C133Y
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mutant cells. V144D mutant cells however, showed an increase in intracellular calcium when compared against basal levels determined in wild type and non-transfected (NT) cells (Figure 7).
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Discussion:
Mutations in the SPTLC1 subunit are known to be causal in HSN-I. Molecular and cellular studies of cells over-expressing the SPTLC1 mutations have identified potential dysfunction in sphingolipid biosynthesis and metabolic activity [4]. This investigation has correlated the previous findings from the lymphoblast cell model [6-8, 13] with a neuronal model. In addition to the V144D mutation, we have also investigated changes within the C133W and C133Y mutations causing HSN-I.
Ubiquinol cytochrome C Reductase Core Protein 1 is a central component of the electron transport chain, catalysing the oxidisation of ubiquinol and reduction of cytochrome C [15]. Here, we have shown in both quantitative protein expression (Figure 2G) and FACS analyses (Figure 4C) that cytochrome C abundance was increased significantly in the TT ND15 cells containing the individual mutations. These finding thus strengthen the potential link to oxidative phosphorylation, via ubiquinol cytochrome C, and altered energy production ultimately leading to axonal degeneration.
Further quantitative analyses were carried out which confirmed that the protein expression of RPS27a, COTL1, and ORP-150 (Figure 2F-2I) were significantly increased in the V144D TT ND15 cells. These finding correlated with results previously identified in the lymphoblast model [6-7]. In addition, COTL1 was significantly increased in the C133W and C133Y mutation. ORP-150 and RPS27a were found to be increased significantly in the C133Y mutation, however these proteins were increased in comparison to the wild type in C133W. FACS (Figure 4) analyses confirmed the altered expression of RPS27a (Figure 4D), CLIC1 (Figure 4E), ORP-150 (Figure 4F), and COTL1 (Figure 4G) in the TT mutant cells compared to that of the wild type.
The novel findings in this study indicate links to dysfunction in oxidative phosphorylation, via Ubiquinol Cytochrome C Reductase Core Protein 1 in all three mutations causing HSN-I. The increased expression of Cytochrome C results in the interference of energy production and oxidative stress upon the ER, eventually causing axonal retraction, a characteristic hallmark of HSN-I. Additionally, Stress-70 mitochondrial protein levels were identified in the C133Y mutant as being increased 2.3 fold relative to the wild type (Figure 5). When mitochondria are under stress, Stress-70 protein levels increase compensating for increased oxidative damage and maintain normal protein import and synthesis [20]. Thus, if mitochondrial oxidative stress is increased (potentially via ROS production), further cellular damage would occur, ultimately leading to a demise in ER efficiency eventually resulting in ER stress.
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It is evident that there is an increase in oxidative and ER stress within the cells containing HSN-I mutations. This is demonstrated by the increased expression of ORP-150, CLIC1, COTL1 and RPS27a. ORP-150 is an important molecular chaperone of the ER during stress [18]. CLIC1 usually exists in a soluble form in the cytoplasm but during times of stress undergoes structural changes and inserts into lipid membranes [17]. COTL1 is an actin binding partner with upregulation in response to stress upon the cytoskeletal system [19]. Alterations in expression of these proteins strongly indicate that oxidative stress could be linked to increases ubiquinol cytochrome C. Thus, an increase in ORP-150 is potentially observed to compensate and protect the cell from an increase in ROS production, causing a shift in CLIC1 expression and stabilisation of the cytoskeleton via COTL1. RPS27a is responsible for targeting misfolded proteins for destruction, with an apparent increase highlighting potential increases in misfolded proteins and protein aggregation due to oxidative stress [16].
Further strengthening the connection between ER stress and HSN-I; peptidyl-prolyl cis-trans isomerase was found to be increased in abundance by 1.7-fold in the C133W mutant. This protein ensures newly synthesised proteins are folded into their correct conformation [21]. The 26s proteasome is responsible for regulating the proteome through degradation of ubiquitin-tagged substrates [22]. 26s proteasome regulatory subunit 8 was found to be increased in abundance by 1.7-fold in the V144D mutation [22]. The increase in abundance of these two proteins coupled with the increased expression of RPS27a and ORP- 150 highlights the possible increased oxidative stress affecting protein folding conformation.
Calcium is an important signalling molecule involved in the regulation of many cellular functions. Mitochondrial calcium uptake has been shown to lead to free radical production, with a delicate balance existing between moderate ROS production to modulate physiological signalling. Overproduction of ROS can ultimately lead to oxidative and ER stress [23-24]. Decreases in calcium are believed to be a cellular response to increased stress, serving as a mechanism to limit further damage and increase cell survival [24]. As part of this study we examined the intracellular levels of calcium in wild type, V144D, C133W, and C133Y. Whilst intracellular calcium is decreased within C133W and C133Y, calcium within the V144D mutation is increased. Hence, is the increased level of calcium a correlation of ER stress and mitochondrial dysfunction occurring, and the result of the V144D-mutation being unable to reduce intracellular calcium to compensate and protect the cell? Could this difference give insight into how the three mutations differ, ultimately causing HSN-I? Further investigation into intracellular and mitochondrial calcium levels is required to delineate the differences within the three mutations.
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Conclusion
This investigation has shown a correlation between previous studies revealing an increase in a mitochondrial electron transport chain protein, increases in proteins induced by oxidative stress and changes in the intracellular calcium levels in all three SPTLC1 mutations causing HSN-I. These findings provide further evidence for mitochondrial and ER dysfunction occurring as a result of mutations in SPTLC1. Further analysis is required, however, the novel findings provide critical new directions in understanding the underlying molecular and cellular alterations broadly common (and specific) to all mutations causing HSN-I and neurodegenerations as a whole.
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References
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Figure 1
Figure 1. Immunoblots of proteins identified in HSN-I-transfected ND15 cells. Representative Immunoblots of MTCO2, Kif2A, SPTLC1, GFP, CLIC1, RPS27a, COTL1, Cytochrome C, ORP-150, and GAPDH from wild type, V144D, C133W and C133Y transfected ND15 cells. 25 µg of protein loaded per lane (n=3).
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Figure 2
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. F
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J.
Figure 2. Expression of proteins and GAPDH in HSN-I-transfected ND15 cells. Representative graphs showing the difference between wild type and mutant ND15 proteins. (A) MTCO2, (B) Kif2A, (C) SPTLC1, (D) GFP, (E) CLIC1, (F) RPS27a, (G) COTL1, (H) Cytochrome C, (I) ORP-150, and (J) GAPDH (*) p <0.05 statistically significant increase (n=3). Errors bar depict SE of means. Blots were normalised to GAPDH.
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Figure 3
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Figure 3. Representative immunofluorescence images of the intracellular localisation of proteins in transfected ND15 cells. Representative confocal micrographs showing (A) SPTLC1, (B) Kif2A, (C) Cytochrome C, (D) RPS27a, (E) CLIC1, (F) ORP-150, (G) COTL1 and (H) MTCO2 stained transfected (green) ND15 cells (red) DAPI nuclear stain (blue). Scale bar = 5 µm
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Figure 4
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Figure 4. Relative quantification of wild type, V144D-, C133W- and C133Y-transfected ND15 cells. Flow cytometric analysis of the relative fluorescence intensity of (A) SPTLC1, (B) Kif2A (C) Cytochrome C (D) RPS27a (E) CLIC1 (F) ORP-150 (G) COTL1 (H) MTCO2 and (I) GFP. Gold histogram represents Wild Type, Blue histogram represents V144D, Green histogram represents C133W and Red histogram represents C133Y (n=3).
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Figure 5
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Figure 5. Representative images of 2D gels following resolution of total cellular proteins from HSN-I- transfected ND15 Cells. (A) wild Type proteins; (B) V144D proteins (C) C133W proteins and (D) C133Y proteins. The molecular weights are in kilodaltons (kDa) and the IEF dimension is in pH units.
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Table 1. Summary table of mascot protein identifications. LC-MS/MS and Mascot Database searching identified 14 altered proteins from V144D, C133W and C133Y transiently transfected ND15 cells.
Unique Spot Accession Sequence Mascot Predicted Mw Mascot Fold increase or decrease within Protein Identified peptides Predicted Number Number Coverage Protein Score (kDa) Mascot pI Mw (kDa) the mutant matched pI
V144D- 1.3 Fold 1. Protein Disulphide Isomerase P09103 35 42% 1468 4.2 90 4.77 57.01
V144D- 1.6 Fold 2. Alpha-Enolase P17182 52 67% 1901 6.5 65 6.37 47.11
3. Long-chain specific acyl-CoA P51174 20 50% 1188 8.3 69 8.53 48.2 V144D- 1.7 Fold dehydrogenase 4. 26s protease regulatory P62196 44 63% 1287 7.0 60 7.11 45.2 V144D- 1.7 Fold subunit 8 5. RPS27a P62983 18 42% 193 9.2 16 8.00 9.68 C133W- 2.6 Fold 6. Peptidyl-prolyl cis-trans P30416 39 41% 576 5.4 90 5.54 57.6 C133W- 1.7 Fold isomerase 7. Stress-70 protein, P38647 33 38% 884 4.3 75 5.81 73.70 C133Y- 2.3 Fold Mitochondrial 8. 10 kDa heat shock protein, Q64433 3 37% 90 7.6 15 7.93 10.96 C133Y- 3.5 Fold Mitochondrial 9. Voltage-dependent anion- Q60930 7 29% 229 7.9 30 7.44 32.34 C133Y- 1.6 Fold selective channel protein 2 C133Y- 2.1 Fold 10. Long-chain specific acyl-CoA P51174 45 43% 1117 8.2 50 8.53 48.37 dehydrogenase, Mitochondrial C133Y- 1.5 Fold 11. 26s Proteasome non-ATPase O35593 25 39% 580 5.8 30 6.06 34.77 regulatory subunit 14 C133Y- 2.1 Fold 12. STIP1 homology and U box- Q9WUD1 27 58% 366 5.9 35 5.71 35.34 containing protein 1 C133Y- 2.4 Fold 13. Eukaryotic Translation Initiator Q6ZWX6 47 64% 944 4.5 37 5.02 36.37 Factor 2 Subunit 1 C133Y- 2.7 Fold 14. Triosephosphate Isomerase P48500 34 79% 1162 8.5 26 6.89 27.4
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Figure 6 A.
B.
C.
D.
E. F. G.
Figure 6. Representative calcium images of wild Type, V144D-, C133W-, C133Y-, Non-Transfected, Low and High Calcium in ND15 cells. Representative confocal micrographs showing (A) Wild Type, (B) V144D, (C) C133W (D) C133Y (E) NT (F) Low Calcium (G) High Calcium intracellular calcium stained. GFP (Green) ND15 cells (red) N=25.
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Figure 7
* *
p-values Wild-Type Non-Transfected
V144D 0.27139 0.01551**
C133W 0.00001* 0.00023*
C133Y 0.00002* 0.00148*
Figure 7. Relative intensity of intracellular calcium of wild Type, V144D-, C133W-, C133Y-, Non- Transfected, Low and High Calcium in ND15 cells. A graph showing the difference between NT, wild type and mutant ND15 proteins (n=25). Table shows p-values of mutants compared to WT and to NT stained ND15 cells. (*) p <0.05 statistically significant decrease. (**) p <0.05 statistically significant increase. Errors bar depict SE of means.
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Paper VI
Published in Journal of Chemical Biology
Author Contributions
SES assisted in experimentation, data analysis and finalised preliminary draft manuscript.
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J Chem Biol (2014) 7:67–76 DOI 10.1007/s12154-014-0108-y
ORIGINAL ARTICLE
Increased lipid droplet accumulation associated with a peripheral sensory neuropathy
Lee L. Marshall & Scott E. Stimpson & Ryan Hyland & Jens R. Coorssen & Simon J. Myers
Received: 3 December 2013 /Accepted: 3 March 2014 /Published online: 23 March 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Hereditary sensory neuropathy type 1 (HSN-1) is dynamic organelles containing sphingolipids and membrane an autosomal dominant neurodegenerative disease caused by bound proteins surrounding a core of neutral lipids, and thus missense mutations in the SPTLC1 gene. The SPTLC1 protein mediate the intracellular transport of these specific molecules. is part of the SPT enzyme which is a ubiquitously expressed, Current literature suggests that there are increased numbers of critical and thus highly regulated endoplasmic reticulum lipid droplets and alterations of lipid metabolism in a variety bound membrane enzyme that maintains sphingolipid concen- of other autosomal dominant neurodegenerative diseases, in- trations and thus contributes to lipid metabolism, signalling, cluding Alzheimer’sandParkinson’s disease. This study es- and membrane structural functions. Lipid droplets are tablishes for the first time, a significant increase in the pres- ence of lipid droplets in HSN-1 patient-derived lymphoblasts, indicating a potential connection between lipid droplets and Lee L. Marshall and Scott E. Stimpson contributed equally to this work. the pathomechanism of HSN-1. However, the expression of L. L. Marshall : S. E. Stimpson : R. Hyland adipophilin (ADFP), which has been implicated in the regu- Neuro-Cell Biology Laboratory, University of Western Sydney, lation of lipid metabolism, was not altered in lipid droplets Locked Bag 1797, Penrith South DC, NSW 1797, Australia from the HSN-1 patient-derived lymphoblasts. This appears to J. R. Coorssen be the first report of increased lipid body accumulation in a Molecular Physiology, University of Western Sydney, Locked Bag peripheral neuropathy, suggesting a fundamental molecular 1797, Penrith South DC, NSW 1797, Australia linkage between a number of neurodegenerative diseases. : : : : L. L. Marshall S. E. Stimpson R. Hyland J. R. Coorssen . S. J. Myers Keywords Hereditary sensory neuropathy type 1 Serine Molecular Medicine Research Group, University of Western Sydney, palmitoyltransferase . Serine palmitoyltransferase long chain Locked Bag 1797, Penrith South DC, NSW 1797, Australia subunit 1 . Lipid droplets . Nile red . ADFP
L. L. Marshall : S. E. Stimpson : R. Hyland : J. R. Coorssen : S. J. Myers School of Science and Health, University of Western Sydney, Locked Introduction Bag 1797, Penrith South DC, NSW 1797, Australia Serine palmitoyltransferase (SPT) is a critical, ubiquitously J. R. Coorssen : S. J. Myers School of Medicine, University of Western Sydney, Locked Bag expressed, and highly regulated endoplasmic reticulum bound 1797, Penrith South DC, NSW 1797, Australia membrane enzyme that maintains cellular sphingolipid con- centrations [1–3]. SPT is a pyridoxal-5′-sphosphate (PLP)- * S. J. Myers ( ) dependent multimeric enzyme that catalyses the first and rate University of Western Sydney, Office 21.1.05, Campbelltown campus, Locked Bag 1797, Penrith South DC, NSW 1797, Australia limiting step in the de novo synthesis of sphingolipids [1, 2]. e-mail: [email protected] SPT is a multimeric enzyme composed of three similar subunits: serine palmitoyltransferase long chain subunit 1 * J. R. Coorssen ( ) (SPTLC1), SPTLC2, and SPTLC3. SPTLC2 and SPTLC3 University of Western Sydney, Office 30.2.15, Campbelltown campus, Locked Bag 1797, Penrith South DC, NSW 1797, Australia both contain a lysine residue at the active site required for e-mail: [email protected] PLP binding; therefore, these two subunits are essential for 68 J Chem Biol (2014) 7:67–76 activating the SPT enzyme [2, 4]. SPTLC1, however, lacks is normally than sufficient; indeed, this is suggested by this PLP binding site and other key catalytic residues, sug- in vivo SPT downregulation [10]. gesting that SPTLC1 plays a more regulatory role in the SPT The alternative theory is that mutations in the SPTLC1 complex [2, 3]. gene cause a gain of toxic function. Mutations in the The SPTLC1 gene is located on chromosome 9p22, and SPTLC1 gene are thought to induce a shift in the substrate positional cloning has identified three missense mutations specificity of the SPT enzyme resulting in the production of associated with Hereditary sensory neuropathy type 1 one or more toxic lipid species [1]. The production of two (HSN-1), an autosomal dominant sensory neuropathy affect- atypical deoxysphingoid bases (DSB) has been linked to the ing peripheral sensory neurons [1, 5]. These mutations result mutant SPT enzyme [1]. These DSB metabolites can neither in a single amino acid substitution of cysteine to tryptophan at be converted to sphingolipids nor degraded and therefore position 133 (C133W), cysteine to tyrosine at position 133 accumulate in the cell, producing a neurotoxic affect. This (C133Y), and valine to aspartic acid at position 144 (V144D) gain of toxic function occurs in many other autosomal dom- [6]. HSN-1 is the most common HSN subtype resulting in the inant, inherited neurodegenerative disorders, including progressive degeneration and dying back of neurons in the Alzheimer’s disease and Parkinson’sdisease[12, 13]. dorsal root ganglia. Despite its initial characterisation over Increases in the number of lipid droplets and changes in 50 years ago [7] and the identification of critical mutations lipid metabolism have also been identified in a variety of in the SPTLC1 gene, the molecular mechanisms underlying autosomal dominant neurodegenerative diseases, including disease development and progression still remain poorly un- Alzheimer’s and Parkinson’s[12–14]. Lipid droplets are or- derstood [8, 9]. ganelles which contribute to cellular homeostasis by regulat- There are currently two main hypotheses as to the ing lipid metabolism and the transport of proteins and lipids pathomechanism(s) in HSN-1, suggesting either a ‘gain of (including sphingolipids) throughout the cell [15–19]. Current toxic function’ of the SPT enzyme or a dominant negative research into Alzheimer’s disease emphasises that accumula- effect [1, 10, 11]. Peripheral neurons may be sensitive to the tion of lipid droplets and abnormalities in lipid metabolism perturbation of sphingolipid metabolism (i.e. decrease in func- may cause or exacerbate the disease phenotype [13]. tional levels) caused by a mutation-induced reduction in SPT Similarly, in Parkinson’s disease, the dysregulation of intra- enzyme activity [11]. This hypothesis has been shown to be cellular lipid droplet interactions and expressions, as well as consistent with recent studies on C133W and V144D, dem- changes in lipid metabolism, may also contribute to the dis- onstrating that both mutations reduce normal SPT activity in ease phenotype [12, 14]. various cell types, including cultured patient lymphoblasts Considering the potential linkages between central and [11]. A concomitant change in the membrane lipid composi- peripheral neurodegenerative conditions [13], including the tion would be expected to be seen but recent data has been accumulation of lipid droplets and alterations of lipid metab- contradictory. Initially, an increase in glucosylceramide syn- olism in other autosomal dominant neurodegenerative dis- thesis was reported; however, a decrease in ceramide levels eases [12], we have tested the hypothesis that an accumulation and sphingomyelin synthesis yielded no change in the overall of lipid droplets would also be associated with HSN-1 disease. sphingolipid composition [11]. The data confirm a significantly increased number of lipid Studies of SPT activity using patient lymphoblasts droplets in lymphoblasts from HSN-1 patients expressing the that endogenously expressed the SPTLC1 mutation re- C133W and V144D mutant SPTLC1 proteins. This appears to ported greater than 50 % reduction of SPT activity [8, be the first report of increased lipid body accumulation in an 10]. While the mechanism by which SPT activity is reduced is autosomal dominant sensory neuropathy. We discuss these yet to be confirmed, Bejaoui et al. [8] observed that the findings in terms of probable molecular mechanisms underly- mutation did not directly affect the stability of the protein as ing the development and progression of HSN-1. translated but may interfere with the function of the enzyme. As SPTLC1 mutations have a direct effect on the activity of SPT, this supports the dominant negative effect theory. Thus, Results competition possibly arising between mutated and wild type SPTLC1 for interaction with SPTLC2 may represent an un- SPTLC1 mutations do not alter cellular morphology derlying disease mechanism, with the mutated SPTLC1 despite increases in lipid droplet accumulation possessing a higher affinity then wild type [8]. Nonetheless, the SPTLC1 mutation does not reduce SL levels despite EBV transformed, patient-derived lymphoblasts endogenous- SPT activity being reduced by more than half [11]. ly express the mutant SPTLC1 enzymes associated with HSN- Therefore, the remaining 50 % of SPT activity may be 1[10]. Detailed analysis indicated no gross morphological sufficient to maintain normal sphingolipid homeostasis changes in lymphoblasts derived from healthy controls and in these cells, presumably because the total SPT activity HSN-1 patients expressing the C133W and V144D mutant J Chem Biol (2014) 7:67–76 69
SPTLC1 proteins (Fig. 1a). To establish that an increase could showed statistically significant increases in lipid droplet accu- be effectively detected, control lymphoblasts were also treated mulation compared to the healthy control lymphoblasts; rela- with oleic acid, which is known to result in the accumulation tive fluorescence increases amounted to 0.0353Æ 0.0018 OD of lipid droplets [20]. Confocal analysis of patient-derived (a 2.3-fold increase) and 0.0420Æ 0.0057 OD (a 2.7-fold lymphoblasts, stained with DAPI (4′,6-diamidino-2- increase) for the V144D and C133W mutations, respectively. phenylindolenucleus; nuclear stain) and Nile red (for lipid droplets), confirmed an accumulation of lipid droplets within Quantification of lipid droplets in HSN-1 patient-derived the cytoplasm of oleic acid-treated lymphoblasts compared to lymphoblasts cells from healthy untreated controls (Fig. 1b). Notably, an obvious increase in the number of lipid droplets was also seen In order to best characterise the relationship between increased in the HSN-1 patient-derived lymphoblasts expressing the lipid droplet accumulation and HSN-1, Nile red-stained C133W and V144D mutant SPTLC1 proteins compared to patient-derived lymphoblasts were analysed using flow cy- the healthy control lymphoblasts (Fig. 1b). Interestingly, tometry to determine the lipid droplet fluorescence per cell punctate Nile red (i.e. lipid droplet) staining appears largely (Fig. 2). Labelling with Nile red resulted in an overall right- localised to the ER which is also where the SPTLC1 protein is ward shift of 102 units in the fluorescence histograms relative bound. to unlabelled cells, indicating that no endogenous fluores- To quantitatively assess the increase in lipid droplet accu- cence affected the analysis. Control cells previously treated mulation caused by the HSN-1 mutant SPTLC1 genes, Nile with oleic acid showed a further rightward shift relative to red-stained patient-derived lymphoblasts were analysed using those that were untreated, and Nile red fluorescence in the fluorescence spectroscopy (Fig. 1c). The oleic acid-treated HSN-1 patient-derived cells yielded peak midpoints and dis- positive control lymphoblasts showed a statistically signifi- tributions comparable to the oleic acid-treated controls. cant increase in lipid droplets compared to untreated cells Overall, the oleic acid-treated control lymphoblasts, as well derived from healthy control subjects. Although proportional- as the HSN-1 patient-derived lymphoblasts showed statisti- ly lower, the HSN-1 patient-derived lymphoblasts also cally significant increases in total lipid droplet staining per cell
Fig. 1 SPTLC1 mutations cause a no change to gross morphology Control V144D C133W but yield increased lipid droplets. a Representative bright field micrographs showing gross morphology of health control and patient-derived lymphoblasts. b Representative confocal micrographs showing Nile red- stained lipid droplets (red)and DAPI nuclear stain (blue). Scale b (-) Control (+) Control (-) V144D (-) C133W bar =20μm. c Fluorescence spectroscopy of the Nile red- stained lipid droplets in patient- derived lymphoblasts (n=5 separate experiments). Plus symbol = oleic acid treatment; minus symbol = no oleic acid treatment. Asterisk indicates p<0.05, relative to control (−) c 0.12 0.1 0.08 0.06 * 0.04 * (per cell) 0.02 0 Pooled Control (-)Control (+) V144D C133W Blank Relative Fluorescence Intensity Fluorescence Relative
Patient Lymphoblasts 70 J Chem Biol (2014) 7:67–76
a Control (#) Control (+) Cellular Events Cellular Events
PI/PE-Cy5-A PI/PE-Cy5-A
Control (-) V144D (-) Events Cellular Cellular Events
PI/PE-Cy5-A PI/PE-Cy5-A
C133W (-) Cellular Events
b PI/PE-Cy5-A 120
100
80
60 * * 40 (per cell)
20
Relative Fluorescence Intensity 0 Pooled Blank Control (-) Control (+) V144D C133W Patient Lymphoblasts J Chem Biol (2014) 7:67–76 71
Fig. 2 Relative quantification of lipid droplets in HSN-1 patient-derived oxidase subunit 2 of complex IV, located in the inner mem- lymphoblasts expressing the C133W and V144D mutant SPTLC1 genes. brane of mitochondria. Fluorescence micrographs of patient- a Representative flow cytometry scatter plots of Nile red-stained lipid droplets in control and patient-derived lymphoblasts. Number symbol = derived lymphoblasts, stained with DAPI (nucleus) and anti- unstained, non-treated; plus symbol = oleic acid treated, stained; and MTCO2 antibody, confirmed normal healthy mitochondrial minus symbol = non-treated, stained lymphoblasts. b Flow cytometry structures within the cytoplasm (Fig. 4a). The mitochondrial analysis of the relative fluoresence intensity of Nile red-stained lipid marker MTCO2 was detected solely in the mitochondrial droplets in patient-derived lymphoblasts. Plus symbol = oleic acid treat- ment. Minus symbol = no oleic acid treatment (n=5 separate experi- fractions, and the lipid droplet marker, ADFP, only in the ments). Asterisk indicates p<0.05, relative to control (−) cytosolic fractions, suggesting that the mitochondria and lipid droplets do not co-localise and are thus less likely to interact.
compared to the untreated healthy controls; for the C133W Protein expression of ADFP, enriched from a lipid droplet and V144D, mutants this amounted to increases in relative fractionation fluorescence of 46.004Æ 1.563 OD (a 1.6-fold increase) and 40.89Æ 1.099 OD (a 1.8-fold increase), respectively, relative In order to identify whether an isolated untreated lipid droplet to the stained controls. fraction expressed SPTLC1 and ADFP, lipid droplets were isolated using a well-established protocol [20]whereby,from Expression of lipid droplet marker protein ADFP in HSN-1 an equivalent number of lymphoblasts lipid droplets were patient-derived lymphoblasts isolated and an equal concentration of protein from each lipid droplet fraction (7 μg) was loaded onto an SDS-PAGE and Adipophilin (ADFP) is a membrane-associated protein pres- assessed by western blot analysis. SPTLC1 was never detect- ent in mature lipid droplets, and is thus widely used as a ed in the isolated lipid droplet fraction; however, ADFP was marker for lipid droplets. Western blot analysis was performed consistently detected in the lipid droplet fractions from both for ADFP on total cell lysates from oleic acid-treated control the healthy control and the patient lymphoblasts. This and on untreated control and HSN-1 patient-derived lympho- highlighted the successful isolation of an enriched lipid drop- blasts (Fig. 3a), revealing ADFP expression is below the level let fraction from untreated lymphoblasts, indicating that oleic of detection in the untreated samples. To confirm this, western acid treatment was not required for this level of analysis blot analysis was performed on total cell lysates of oleic acid- (Fig. 5). Most importantly, there was no change in the amount treated control and HSN-1 patient-derived lymphoblasts of ADFP in control vs. patient lipid droplets. (Fig. 3c). This western blot analyses show that ADFP protein expression is relatively abundant in the oleic acid-treated healthy control and patient lymphoblasts, thus highlighting Discussion the need for oleic acid treatment for total cell lysate analysis. Further western blot analyses of SPTLC1 (Fig. 3b)and HSN-1 is an autosomal dominant neuropathy resulting in the ADFP (Fig. 3c) were carried out, normalising to the house- progressive degeneration and dying back of the peripheral keeping protein glyceraldehyde-3-phosphate dehydrogenase sensory neurons in the dorsal root ganglia [5]. HSN-1 is caused (GAPDH) to establish relative protein expression levels of by missense mutations in the SPTLC1 gene; however, the SPTLC1 and ADFP from cells treated with oleic acid. actual cellular and molecular mechanisms underlying the dis- Despite some variability in expression levels, there were no ease still remain poorly understood [10]. This study quantified statistically significant changes in the expression of SPTLC1 increases in lipid droplets in HSN-1 patient-derived lympho- (Fig. 3e) or ADFP (Fig. 3f) in the HSN-1 patient-derived blasts, and also determined that there was no association be- lymphoblasts compared to those derived from healthy control tween these inclusions and mitochondria, the latter being often subjects. also effected in neurodegenerative diseases [12, 13]. Increased numbers of lipid droplets and changes in Lipid droplets show no co-localisation with mitochondria lipid metabolism have been seen in a variety of autoso- mal dominant neurodegenerative disorders such as Previous ultrastructural analysis has shown lipid droplet pres- Alzheimer’s disease and Parkinson’s disease [12, 13]. ence in close proximity to the ER and mitochondria mem- Current research into Alzheimer’s disease emphasises branes in the patient lymphoblasts (Myers, S.J, unpublished that accumulation of lipid droplets and abnormalities in observations). In order to determine if the lipid droplets co- lipid metabolism may cause or exacerbate the disease localise with mitochondria, oleic acid treated-healthy control phenotype [13]. Parkinson’s disease research has identi- and HSN-1 patient-derived lymphoblasts were fractionated fied a deregulation of intracellular lipid droplet interac- into mitochondrial and cytosolic fractions and immunoblotted tions and expressions, and also changes in lipid metab- for ADFP and MTCO2; the latter is part of Cytochrome c olism, which may contribute to the disease phenotype. 72 J Chem Biol (2014) 7:67–76
Fig. 3 Expression of lipid droplet 1234 marker protein ADFP in HSN-1 a ADFP patient-derived lymphoblasts. a 48 kDa Immunoblot detection of ADFP in oleic acid-treated and oleic 1 2 3 4 5 6 7 8 9 acid-untreated total cell lysates; 1 SPTLC1 represents treated controls; 2, b 55 kDa untreated controls; 3,untreated V144D mutant; and 4, untreated ADFP C133W mutant. Immunoblots of c total protein lysates from oleic 48 kDa acid-treated cells probed for SPTLC1 (b), ADFP (c), and d GAPDH GAPDH (d); 1–3 represents 40 kDa treated controls; 4–6,treated V144D mutants; and 7–9,treated e f C133W mutants. Histograms of 3.5 9 the immunoblotting results of 3 8 oleic acid-treated controls and of 7 2.5 SPTLC1 (e) and ADFP (f), 6 normalised to GAPDH (n=3) 2 5 1.5 4 3
1 (Arbitary Units) (Arbitary Units) 2 Relative Optical Density Relative Optical Density 0.5 1 0 0 Control V144D C133W Control V144D C133W SPTLC1 ADFP
The data presented here establish a significant increase increase in lipid droplet numbers within individual patient- in lipid droplets in lymphoblasts from patients with derived lymphoblasts (Fig. 2). HSN-1, a peripheral neurodegenerative disorder. With a ADFP is a lipid droplet-associated membrane protein, and breadth of research identifying accumulation of lipid has thus been used as a marker for these inclusions [19]. droplets and changes in lipid metabolism in other auto- Immunoblotting revealed that only the oleic acid-treated sam- somal dominant neurodegenerative diseases, it is now ples showed detectable ADFP protein from total cell lysates reasonable to suggest that the increased presence of (Fig. 3a). Analysis of SPTLC1 from oleic acid-treated healthy lipid droplets may cause or exacerbate the HSN-1 dis- control and patient-derived lymphoblasts revealed no signifi- ease phenotype. Indeed, such alterations suggest a more cant difference in expression (Fig. 3f), comparable to the central connection with the pathomechanism(s) underly- results of another study that found no change in the levels of ing a host of neurodegenerative disease, both central SPTLC1 in HSN-1 patient lymphoblasts [10]. By both con- and peripheral. focal and immunoblotting analyses, the mitochondrial mem- Bright field micrographs indicated no gross morphological brane marker MTCO2 was detected only in the mitochondrial changes between healthy control and HSN-1 patient-derived fraction, and not in the cytoplasmic fraction isolated from lymphoblasts; however, confocal micrographs of Nile red oleic acid-treated healthy control patient-derived lympho- stained lipid droplets showed an increase of these inclusions blasts (Fig. 4b). In contrast, immunoblotting of the lipid within the oleic acid-treated positive controls compared to the droplet marker ADFP identified it only in the cytosolic frac- healthy controls and patient-derived lymphoblasts (Fig. 1). tion, and not in the mitochondrial fraction, suggesting no co- Therefore, oleic acid-induced lipid droplet formation [21] localisation between the mitochondrial and lipid droplet was used as a positive control. More importantly the results membranes. indicated a significant quantitative increase of lipid droplets Immunoblotting for ADFP in lipid droplet fractions within the patient-derived lymphoblasts compared to the isolated from healthy control and HSN-1 patient-derived healthy controls. This increase in lipid droplet abundance in lymphoblasts expressing the C133W and V144D mutant HSN-1 patient-derived lymphoblasts was confirmed using SPTLC1 proteins indicated no detectable changes in pro- fluorescence spectroscopy. The confocal and fluorescence tein expression. ADFP is currently thought to be involved spectroscopy analyses thus indicated a possible connection in lipid homeostasis and lipolysis by protecting triacylglyc- between increased lipid droplet numbers and the cellular erol, within the lipid droplets, from cytosolic lipases [17, mechanism of HSN-1. The data confirmed using Nile red 19]. These results suggest that the increase in lipid drop- staining and flow cytometric analysis indicating a significant lets within the HSN-1 patient lymphoblasts, without a J Chem Biol (2014) 7:67–76 73
a Control V144D C133W
b 1 2 3 4 5 6 7 8 9
Mitochondria MTCO2 60 kDa
MTCO2 Cytosolic 60 kDa
c 1 2 3 4 5 6 7 8 9 ADFP Mitochondria 48 kDa
ADFP Cytosolic 48 kDa
Fig. 4 Mitochondria show no co-localisation with lipid droplets. a cytosolic fractions, 1–3 represents oleic acid-treated controls; 4–6, treated Representative confocal micrographs showing MTCO2 stained mito- V144D mutants; and 7–i, treated C133W mutants. c Immunoblot detec- chondria (red) and DAPI nuclear stain (blue). Scale bar =20μm. b tion of ADFP at 48 kDa from oleic acid-treated mitochondrial and Immunoblot detection of MTCO2 at 60 kDa from mitochondrial and cytosolic fractions change in ADFP, may cause abnormalities in lipid metab- lymphoblasts may well indicate a deregulation of lipid metab- olism or be indicative of a protein trafficking defect that olism which may exacerbate the HSN-1 phenotype. The data involve elements of the cytoskeleton [17]. Previously, thus also suggest a more common and/or central role for these abnormalities in lipid metabolism have been linked to a molecular alterations in specific types of central and periph- variety of other autosomal dominant neurodegenerative eral neurodegeneration. diseases, including Alzheimer’s disease and Parkinson’s disease, suggesting that changes in lipid metabolism may either cause or exacerbated the disease phenotype. Therefore, comparable changes in lipid metabolism, along Materials with a trafficking defect, may also cause or exacerbate the HSN-1 disease phenotype, a peripheral neurodegenerative All cell culture stock solutions, including RPMI-1640, fetal disorder. Further analyses are warranted to determine if bovine serum (FBS), penicillin (100 U/mL), streptomycin there is a deregulation of lipid metabolism within the (100 μg/mL), L-glutamine (2 mM), HEPES (1 M), and phos- HSN-1 patient lymphoblasts and to identify its link with phate buffered saline (PBS) were purchased from Gibco HSN-1 disease and other autosomal dominant neurode- Invitrogen (Australia). Cell culture consumables were pur- generative diseases. chased from BD Falcon (Greiner, USA). MTCO2 and This is the first study to investigate lipid droplet formation GAPDH primary antibodies were purchased from Abcam in an autosomal dominant sensory neuropathy. These data (USA); SPTLC1 primary antibody was purchased from indicate a possible connection between increased lipid droplet Santa Cruz Biotechnology (USA). ADFP primary antibody, abundance and the cellular mechanism underlying HSN-1. secondary HRP Mouse and Rabbit antibodies, oleic acid, Nile The increase in lipid droplets without a parallel increase in red, and DAPI stains were purchased from Sigma-Aldrich the expression of ADFP in the HSN-1 patient-derived (Australia). 74 J Chem Biol (2014) 7:67–76
1 2 3 4 5 6 7 8 9
ADFP 48 kDa
SPTLC1 55 kDa
Fig. 5 Expression of ADFP in an enriched lipid droplet fraction. Immunoblot detection of ADFP and SPTLC1 proteins from untreated lipid droplet enriched fractions, 1–3, untreated controls; 4–6, untreated V144D mutants; and 7–9, untreated C133W mutants (n=3)
Methods of 320 nM Nile red (Sigma, Australia) in Hank’s Buffered Salt Solution (HBSS; prewarmed to 37 °C) and incubated EBV-transformed lymphoblasts in the dark for 15 min at RT. For mitochondrial analysis the cell suspension was permiabilised with a 0.3 % Triton Epstein-Barr Virus (EBV)-transformed control and HSN-1 X-100 for 10 min at 37 °C. The cells were centrifuged at patient lymphoblasts were graciously provided by Prof. 1,000×g for 5 min, resuspended in 1 mL of 1 % BSA in Garth Nicholson (Molecular Medicine Laboratory, Anzac PBS, and incubated for 30 min at 37 °C. The cells were Institute, Sydney) [10]. subsequently centrifuged at 1,000×g for 5 min, resuspend- ed in 200 μL of a solution of 1 % BSA in PBS that also Lymphoblast cultures contained primary antibody (MTCO2 at 1:50 dilution), and incubated for 1 h at RT. The cells were then centrifuged Lymphoblasts were cultured in RPMI-1640 media (Gibco), at 1,000×g for 5 min, washed in 1 mL of PBS, centri- supplemented with FBS (10 % v/v), penicillin (1 U/mL), fuged again, and resuspended in 200 uL of a solution of streptomycin (1 μg/mL), L-glutamine (2 mM), and HEPES 1 % BSA in PBS that also contained secondary antibody
(1 M) at 37 °C in a humidified atmosphere of 5 % CO2, (1:200 dilution, anti-mouse, rhodamine conjugated) for 1 h using T75 cm2 culture flasks (Greiner, Interpath). Prior to at RT. Thereafter, the cell suspension was centrifuged at use in biochemical assays, lymphoblasts were collected by 1,000×g for 5 min at RT, resuspended in 1 mL of DAPI centrifugation at 1,000×g (3 min at RT) and washed in PBS. (10 mg/mL stock) in PBS, and incubated for 2 min at RT. Normalisation of cell count and viability was obtained The cell suspension was then washed twice in 1 mL of using the Countess Automated Cell Counter (Invitrogen, PBS then suspended in 2 mL of PBS, aliquoted into Australia). individual wells on 6-well plates, and centrifuged at 500×g (10 min, at RT) to pellet the cells onto the cover Oleic acid treatment slips at the bottom of each well. The PBS and non- adherent cells were then aspirated from the wells and the For positive controls, lymphoblasts from normal healthy do- cover slips allowed to dry for 20 min at RT. Thereafter, nors were treated (in culture) with a 400 μM solution of oleic the cover slips were mounted using 20 μL of DAKO acid (Sigma, Australia) for 24 h prior to isolation. This is a solution (Dako, Australia) and sealed with nail polish prior standardised and routine protocol to induce lipid droplet for- to assessment using a LSM-5 Exciter Confocal mation [21]. Microscope (Carl Zeiss, Australia).
Fluorescence microscopy Fluorescence detection
For fluorescence microscopy analyses, glass cover slips were For fluorescence detection, lymphoblasts were isolated as prepared in advance (i.e. overnight). The cover slips were above; the cells were then suspended in 1 mL of 4 % parafor- dipped 20 times into a 1:50 dilution of HistoGrip maldehyde in PBS, and incubated for 15 min at RT. (Invitrogen, Australia) in pure acetone. The cover slips were Thereafter, the cell suspension was centrifuged at 1,000×g then dipped 10 times in dH2O to wash off excess HistoGrip for 5 min at RT, and the resulting pellet suspended in 1 mL and dried overnight. Lymphoblasts were collected by centri- of 320 nM Nile red (Sigma, Australia), (10 mM stock) in fugation at 1,000×g for 5 min at RT, and resuspended in PBS. Hank’s Buffered Salt Solution (HBSS), and incubated in the Cell suspensions (containing 1×106 to 2×106 cells) were dark for 15 min at RT. After incubation, the cell suspension centrifuged at 1,000×g for 5 min at RT, resuspended in was centrifuged at 1,000×g for 5 min and resuspended in 1 mL 1 mL of 4 % paraformaldehyde in PBS and incubated for of PBS at RT. The cell suspension was then analysed using a 15 min at RT. BMG Polar Star Omega Fluorescence Plate Reader (BMG For lipid droplet analysis, the cell suspension was cen- Labtech, Germany) and a MACS Quant Flow Cytometry trifuged at 1,000×g for 5 min at RT, resuspended in 1 mL (Miltenyi Biotech, Australia). J Chem Biol (2014) 7:67–76 75
Isolation of mitochondrial and cytosolic proteins pipette, leaving only the white top layer containing lipid droplets. Briefly, mitochondria were isolated using a Mitochondrial Protein Isolation kit (Amresco Scientific, USA). Total protein preparation Lymphoblasts were first centrifuged at 1,000×g for 5 min, and the cells were then washed in 10 ml of ice cold 1X PBS For a positive control, normal healthy donor lymphoblasts prior to suspension in 1 ml ice cold 1X PBS. Cells were were treated with a 400 μM solution of oleic acid (Sigma- transferred to a 1.5 ml microcentrifuge tube, centrifuged at Aldrich, Australia) in standard media, 24 h prior to isolation. 1,000×g (4 °C for 5 min) and then suspended in the Lymphoblasts were collected by centrifugation at 1,000×g for Mitochondrial Protein Isolation Buffer (including 1X 3 min at RT and resuspended in 30 mL of PBS. The cell Protease Inhibitor cocktail). Cells were then homogenized suspension was then centrifuged at 500×g for 3 min at RT, on ice by 20 passages through a 26 ½G needle attached to a resuspended in 1 mL of PBS, and 10 μL was aliquoted for cell 1cm3 syringe prior to centrifugation at 1,000×g (4 °C for counts. The cell suspension was then centrifuged at 500×g for 10 min). The supernatant was collected, transferred to a fresh 3 min at RT, resuspended in 300 μLofNDRM(non-detergent 1.5-ml tube, and centrifuged at 14,000×g (15 min at 4 °C). resistant membrane lysis buffer; 10 mM Tris-HCl pH 8.0, The supernatant was collected and transferred into a new tube; 150 mM NaCl, 1 % Triton X-100, and 1× protease inhibitors this fraction contained the cytosolic proteins. The pellet, con- in PBS), and lysed for 20 min on ice. Following lysis, the cell taining mitochondrial proteins, was suspended in 1 ml lysate was centrifuged at 18,000×g for 15 min at 4 °C. The Mitochondrial Protein Isolation Buffer and centrifuged at supernatant containing total cell proteins were collected and 14,000×g (1 min at 4 °C). The supernatant was discarded transferred to fresh 1.5 mL microcentrifuge tubes ready for and the pellet suspended in 100 μl of Mitochondrial Protein protein concentration and analysis. The pellet containing Isolation Buffer. whole cells and detergent resistant membranes were resus- pended in 200 μL PBS containing 1× protease inhibitors. Lipid droplet isolation Protein concentration Lipid droplets were isolated essentially as previously described [20]. Briefly, lymphoblasts were collected by Determination of total cellular protein was performed using centrifugation at 1,000×g (5minat4°C)toyieldan the bicinchoninic acid (BCA) protein assay (Sigma-Aldrich, equivalent number of lymphoblasts, suspended in 30 mL Australia). of dissociation buffer (25 mM Tris-HCl pH 7.4, 1 mM EGTA, 1 mM EDTA, and 100 mM KCl). This cell SDS-PAGE and immunoblotting suspension was then centrifuged at 1,000×g (5 min at 4 °C); the pellet was then suspended in 10 mL of Cell lysates and lipid droplet fractions (50 μg total protein) dissociation buffer and transferred to 15-mL tubes. The were subjected to SDS-PAGE on 15 % resolving gels and cell suspension was then centrifuged at 1,000×g (5 min transferred to PVDF membrane. The membranes were at 4 °C), the cells suspended in 750 μL of dissociation blocked with 5 % skim milk in TBS buffer containing 0.1 % buffer containing 1× protease inhibitors, and transferred Tween-20. The blocked membranes were incubated with anti- to 1.5 mL microcentrifuge tubes. The cell suspension ADFP, anti-SPTLC1, and anti-MTCO2 at 1:1,000 and anti- was then placed on ice and homogenised using a 26 ½ GAPDH at 1:5,000, for 16 h. The membrane was then incu- G needle, as described above. The resulting cell lysate bated with secondary horse radish peroxidase antibody was then centrifuged at 15,000×g (10minat4°C)to (1:2,000 dilution) for 1 h at RT. Blots were developed using remove whole cells, nuclei, and large organelles. The an enhanced chemiluminescence (ECL) detection kit (WEST- supernatant was collected and transferred into new ZOL, Biotech, Korea). 1.5 mL microcentrifuge tubes. An equal volume of 1.08 M sucrose solution (approximately 300 μL) was Acknowledgments We are grateful to Prof Garth Nicholson (Molecu- added to the supernatant and the total volume then trans- lar Medicine Laboratory and Northcott Neuroscience Laboratory Anzac ferred to an ultracentrifuge tube. The supernatant/sucrose Research Institute, Sydney) for providing all EBV-transformed lympho- μ blast lines [10] used in this study. LLM was supported by a UWS mixture was overlayed sequentially with 700 Lof Honours Scholarships, the School of Science and Health Honours support 0.27 M sucrose, 500 μL 0.135 M sucrose, and 500 μL and an anonymous private foundation; SES was supported by APA of top solution (25 mM Tris-HCl pH 7.4, 1 mM EGTA, Research Scholarship, the UWS School of Science and Health Postgrad- and 1 mM EDTA). The gradient was then centrifuged at uate research fund; RH was supported by a UWS Postgraduate Research Award and anonymous private foundation. SJM notes the support of an 149,711×g (60 min at 4 °C). The middle and bottom anonymous Private Foundation. JRC acknowledges the support of the layers were subsequently removed using a fine glass UWS School of Medicine. 76 J Chem Biol (2014) 7:67–76
References palmitoyltransferase, long chain base subunit-1, cause hereditary sensory neuropathy type I. Nat Genet 27:309–312 10. Dedov V, Dedova I, Merrill A, Nicholson G (2004) Activity 1. Penno A, Reilly M, Houlden H, Laura M, Rentsch K, Niederkofler V, of partially inhibited serine palmitoyltransferase is sufficient Stoeckli E, Nicholson G, Eichler F, Brown R, Von-Eckardstein A, for normal sphingolipid metabolism and viability of HSN1 Hornemann T (2010) Hereditary sensory neuropathy type 1 is caused patient cells. Biochim Biophys Acta 1688(2):168–175 by the accumulation of two neurotoxic sphingolipids. J Biol Chem 11. Verhoeven K, Timmerman V, Mauko B, Pieber TR, De Jonghe P, 285(15):11178–11187 Auer-Grumbach M (2006) Recent advances in hereditary sensory and 2. Hornemann T, Richard S, Rutti M, Wei Y, Von-Eckardstein A (2006) autonomic neuropathies. Curr Opin Neurol 19:474–480 Cloning and initial characterization of a new subunit for mammalian 12. Cole N, Murphy D, Grider T, Rueter S, Brasaemle D, Nussbaum R serine-palmitoyltransferase. J Biol Chem 281(49):37275–37281 (2002) Lipid droplet binding and oligomerization properties of the 3. Yard B, Carter L, Johnson K, Overton I, Dorward M, Liu H, Parkinson’s disease protein alpha-synuclein. J Biol Chem 277(8): McMahon S, Oke M, Puech D, Barton G, Naismith J, Campopiano 6344–6352 D (2007) The structure of serine palmitoyltransferase; gateway to 13. Lane R, Farlow M (2005) Lipid homeostasis and apolipoprotein E in sphingolipid biosynthesis. J Mol Biol 370(5):870–886 the development and progression of Alzheimer’s disease. J Lipid Res 4. Han G, Gupta S, Gable K, Niranjanakumari S, Moitra P, Eichler F, 46(5):949–968 Brown R, Harmon J, Dunn T (2009) Identification of small subunits 14. Gitler A, Chesi A, Geddie M, Strathearn K, Hamamichi S, Hill K, of mammalian serine palmitoyltransferase that confer distinct acyl- Caldwell K, Caldwell G, Cooper A, Rochet J, Lindquist S (2008) α- CoA substrate specificities. Proc Natl Acad Sci U S A 106(20):8186– Synuclein is part of a diverse and highly conserved interaction 8191 network that includes PARK9 and manganese toxicity. Nat Genet 5. McCampbell A, Broom D, Truong D, Allchorne A, Gable K, Cutler 41(3):308–315 RG, Mattson M, Woolf C, Frosch M, Harmon J, Dunn T, Brown R 15. Beller M, Thiel K, Thul P, Jackle H (2010) Lipid droplets: a dynamic (2005) Mutant SPTLC1 dominantly inhibits serine organelle moves into focus. FEBS Lett 584(11):2176–2182 palmitoyltransferase activity in vivo and confers an age-dependent 16. Farese R, Walther T (2009) Lipid droplets finally get a little R-E-S-P- neuropathy. Hum Mol Genet 14(22):3507–3521 E-C-T. Cell 139(5):855–860 6. Hornemann T, Penno A, Richard S, Nicholson G, Van-Dijk F, 17. Ducharme N, Bickel P (2008) Minireview: lipid droplets in lipogen- Rotthier A, Timmerman V, Von-Eckardstein A (2009) A systematic esis and lipolysis. Endocrinology 149(3):942–949 comparison of all mutations in hereditary sensory neuropathy type I 18. Zehmer J, Huang Y, Peng G, Pu J, Anderson R, Liu P (2009) A role (HSAN I) reveals that the G387A mutation is not disease associated. for lipid droplets in inter-membrane lipid traffic. Proteomics 9(4): Neurogenetics 10(2):135–143 914–921 7. Houlden H, King R, Blake J, Groves M, Love S, Woodward C, 19. Hodges B, Wu C (2010) Proteomic insights into an expanded Hammans S, Nicoll J, Lennox G, O'Donovan DG, Gabriel C, cellular role for cytoplasmic lipid droplets. J Lipid Res 51(2): Thomas PK, Reilly MM (2006) Clinical, pathological and genetic 262–273 characterization of hereditary sensory and autonomic neuropathy 20. Lay, S.L, Hajduch, E, Linday, M.R, Liepvre, X.L, Thiele, C, Ferre, P, type 1 (HSAN I). Brain 129:411–425 Parton, R.G, Kurzchalia, T, Simons, K & Dugail, I. 2006. 8. Bejaoui Y, Uchida Y, Yasuda S, Ho M, Nishijima M, Brown RH Jr, Cholesterol-Induced Caveolin Targeting to Lipid Droplets in Holleran WM, Hanada K (2002) Hereditary sensory neuropathy type Adipocytes: A Role for Caveolar Endocytosis. Traffic, vol. 7, pp. 1 mutations confer dominant negative effects on serine 549–561. Journal of Biological Chemistry.Vol.280,No.52,pp. palmitoyltransferase, critical for sphingolipid synthesis. J Clin 42841–42847 Invest 110:1301–1308 21. Xu G, Sztalryd C, Lu X, Tansey JT, Gan J, Dorward H, Kimmel AR, 9. Dawkins JL, Hulme DJ, Brahmbhatt SB, Auer-Grumbach M, Londos C (2005) Post-translational regulation of adipose differenti- Nicholson GA (2001) Mutations in SPTLC1, encoding serine ation related protein by the ubiquitin/proteasome pathway
General Discussion
The research presented in this thesis has been undertaken to provide greater insight into the molecular and cellular mechanisms of the neurodegenerative disorder, HSN-
I, with the identification of significantly altered protein profiles within isolated mitochondria and ER fractions from patient derived lymphoblasts. There is an increasing number of studies highlighting the essential and critical role mitochondria play in many disease processes and in particular neurodegenerative diseases. As stated earlier, HSN-I is one such disease that exhibits altered mitochondrial morphology in patient derived lymphoblasts carrying mutations in SPTLC1, although the mechanism by which this occurs is yet to be fully elucidated. Previous studies have suggested that a reduction in SPT could not account for the global reduction of sphingolipid metabolism, yet axonal degeneration is still observed in HSN-I (Penno et. al., 2010). Other studies have proposed that these mutations cause a deleterious gain of function with the production of toxic sphingolipids (Hornemann et. al., 2009).
Recently, investigations utilising transmission electron microscopy (TEM) have shown that the ER wraps around morphologically-challenged mitochondria in a perinuclear fashion (Myers et. al., 2014). From these studies, we hypothesised that SPTLC1 mutations in HSN-I cause alterations to both mitochondrial and ER proteins, and alters cellular lipids. These alterations can cause perturbations in neuronal homeostasis as neurons are sensitive to changes in homeostasis (particularly energy production).
It is known that mitochondria are membrane-bound organelles responsible for numerous essential biological processes (including, but not limited to, oxidative phosphorylation, lipid metabolism, and apoptosis). They are inter-connected, forming highly dynamic networks throughout the cytoplasm of most cells, but are often
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concentrated in specific areas of high energy utilisation. In order to investigate the
functionality of mitochondria and their critical role within the disease HSN-I, it was imperative to isolate these organelles at the highest quality and purity. Thus, comparative analyses were undertaken to determine the most efficient and optimal isolation method of mitochondria, specifically comparing a commercial kit against traditional sucrose methods. We carried out detailed assessments of mitochondrial proteins harvested using the Amresco Mitochondrial Protein Isolation Buffer (AMPIB) relative to those extracted by 2DE sample buffer following isolation of mitochondria by sucrose gradient fractionation.
Analyses of the profiles obtained from total mitochondrial proteomes indicated there was greater than 87% overlap between the two isolation methods. To further assess these methods, isolated mitochondria were subjected to a well-established protocol to recover separate total membrane and soluble protein fractions. 2DE analyses showed consistently lower quality proteome isolates from the AMPIB extracts relative to those extracted using standard sucrose fractionation in 2DE sample buffer. A marked increase in protein resolution within the low to mid molecular weight region was observed using the standard isolation method as opposed to AMPIB. This data unequivocally showed that to obtain a full protein profile for mitochondria the traditional subcellular fractionation method using sucrose density gradient centrifugation yielded higher resolution of protein profiles, allowing isolated mitochondria to be quantitatively analysed and thus more accurately reflect the mitochondrial proteome.
Successful optimisation of mitochondrial isolation then enabled the investigation and comparison of total protein profiles of mitochondria isolated from both control and
V144D patient lymphoblasts using 2DE. We observed one protein that was selectively
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detected in the V144D cells at pI 5.7 and molecular weight 55 kDa which was undetectable in the control protein profiles. This protein was subsequently determined to be ubiquinol-cytochrome c reductase core protein 1. We were able to confirm this significant increase in the amount of ubiquinol cytochrome c reductase core protein 1 in V144D cells relative to control cells using quantitative immune-blotting. Additionally, comparison of control and V144D cell proteomes revealed two additional marked protein changes. Both of these proteins were located in the 24-kDa molecular weight region although were isolated in different pI regions (6.6 and 8.3 respectively). Both proteins were subsequently identified as Ig kappa chain C.
As described earlier, mitochondria function as intracellular energy producing organelles, where substrates are metabolised to fuel oxidative phosphorylation through the electron transport chain within their inner membranes. Specific protein complexes within the mitochondria facilitate the flow of electrons from the reducing substrates to oxygen and concomitantly build a proton gradient required for ATP generation (Crofts, AR 2004). Ubiquinol-cytochrome c reductase core protein 1 is a central component of this electron transport chain, catalysing the oxidisation of ubiquinol (ubihydroquinone) and reduction of cytochrome c. These processes are essential in maintaining the electron flow and preventing potential electron leaks and/or break down of the respiratory chain. (Crofts, AR 2004). Ubiquinol cytochrome c reductase core protein 1 is known to be involved in free radical generation, primarily by the production of reactive oxygen species (ROS), within mitochondria. ROS production has been shown to disrupt the homeostasis of and interactions within the mitochondrial matrix resulting in the perturbation of oxidative phosphorylation, leading to the disruption of mitochondrial function and physiology and resulting in cell death
(Drose et. al., 2014).
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The novel findings in this study provide a strong link between alterations in oxidative
phosphorylation, via ubiquinol cytochrome c reductase core protein 1 (possibly
through regulation of ROS production), and the ensuing interference of energy
production. These alterations are hypothesised to ultimately lead to axonal retraction
that is the characteristic hallmark of HSN-I. If ROS production increased as a result of increased levels of ubiquinol cytochrome c reductase core protein 1, such as the levels observed in mitochondria derived from HSN-I patient cells, this could contribute to the progression of damage attributed to the observed axonal retraction.
These initial analyses did not detect alterations in known antioxidant, chaperone or
other repair proteins that would typically be expected to be modified or up-regulated
in response to such perturbations in ROS production. The possibility that mutations
within the SPTLC1 protein hindered such a response needs to be investigated. It will be important to further investigate whether this potential decrease or lack of regulation of ROS production may have been a cause for the observed morphological alterations within the mitochondria from the HSN-I affected cells.
This study identified a change in an immunoglobulin potentially due to the SPTLC1 mutation causing HSN-I. The Ig kappa light-chain constant region undergoes little to no variation in human immunoglobulins and forms part of the five immunoglobulin classes produced in mature B cells. We have determined a statistically significant decrease in the amount of Ig kappa chain C in the total cell lysates isolated from
V144D cells relative to control lymphoblast cell lysates. While the mechanism leading
to the apparent shift in pI is yet to be fully elucidated, it seems likely to be due to an
as yet unidentified post-translational modification (potentially glycosylation,
phosphorylation or methylation, or possibly some combination of the three).
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Immunoglobulin light chains have been implicated in, and are biomarkers of, diseases
such as multiple myeloma and primary systemic or amyloid light chain (AL)
amyloidosis (Yamamoto et. al., 2010). In these diseases, it has been shown that the
free Ig kappa chain associates with sphingomyelin on the plasma membrane of the
myeloma cells forming aggregates that are required for intercalation with membranes
(Hutchinson et. al., 2010). This suggests an important role for sphingolipids in these
disease processes. The exact role of Ig kappa chain C and its relationship to HSN-I could form the basis of a future investigation, as reduced levels of this protein may be responsible for apparent reductions in cellular repair responses observed within cells from HSN-I patients.
The ER is an intracellular organelle and is critical for cellular survival. The ER plays a crucial role in many aspects of protein maturation that includes membrane translocation, folding, post-translational modification and transport of both membrane and soluble proteins (Pendin et. al., 2011). In addition, the ER is involved in the
synthesis of phospholipids and steroids and also in the regulation of Ca2+
homeostasis. The results presented earlier in this study indicated that if altered protein
profiles were observable within mitochondria isolated from HSN-I patient
lymphoblasts, then there could be subsequent detectable protein alterations within the
ER isolated from the same cells. As stated previously, the ER has been shown to be
wrapped around the damaged mitochondria in HSN-I lymphoblasts and thus we
hypothesised that there would also be significantly altered expression of proteins
caused by the SPTLC1 mutations.
Mutations within SPTLC1 result in potential dysfunction and perturbations in
sphingolipid synthesis and metabolism causing HSN-I (Wei et. al., 2007). Disruption
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to the function of the ER or loss of its integrity leads to ER stress (Lindholm et. al.,
2006), and ER stress is characterised by the accumulation of unfolded proteins and
changes in calcium homeostasis within the ER termed the unfolded protein response
(UPR) (Rao et. al., 2004).
To elucidate if protein changes in the proteomes of the ER fractions occur due to mutations in SPTLC1 causing HSN-I, ER membranes were isolated from control and
V144D patient lymphoblasts and proteins from the lysed cells were subjected to 2DE.
Analysis of the amount of the SPTLC1 protein fractions from cells carrying the
V144D mutation compared with the control cell fractions showed no significant difference in expression, indicating a constant expression of SPTLC1 in the diseased state.
2DE analyses revealed five protein species present only in the ER fractions of cells carrying the V144D mutation, with subsequent LC/MS analyses identifying these protein species to be Hypoxia Up Regulated Protein 1 (ORP-150), Chloride
Intracellular Channel Protein 1 (CLIC1), Ubiqutin-40s Ribosomal Protein S27a
(RPS27a), Coactosin (COTL1) and Ig Kappa Chain C. Quantitative immunoblot analyses of these isolated proteins were then carried out to determine their expression levels and we were able to determine that ORP-150, CLIC1 and COTL1 were increased significantly in the V144D mutant cells when compared to the control.
Oxygen-regulated proteins are known to be overexpressed under conditions of hypoxia. The heat shock protein, ORP-150 serves as an important molecular chaperone of the ER during stress (Behnke and Hendershot, 2014). Notably, hypoxia- mediated up-regulation of ORP-150 suppresses programmed cell death driven by oxygen deprivation (Stojadinovic et. al., 2007). CLIC1 is a small protein and exists in
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both soluble cytoplasmic and integral membrane forms (Warton et. al., 2002). CLIC1
exists primarily in a soluble form within the cytoplasm and nucleoplasm, but following
cell oxidation undergoes major structural changes and becomes inserted in lipid
membranes, where it acts as a chloride-selective ion channel (Averaimo et. al., 2010).
The ability of cells to move and extend dynamically is facilitated by actin dynamics.
Coactosin (COTL1) is an actin binding protein and has been shown to associate with
F-actin (Provost et. al., 2001). These findings, coupled with the alterations observed in the isolated mitochondrial proteomes, provide a strong evidence for increased
oxidative stress within the V144D lymphoblasts. It is evident that there is an increase
in oxidative stress within the V144D patient lymphoblasts demonstrated by the
increased expression of ORP-150, CLIC1, COTL1 and RPS27a. While these proteins are functionally independent from each other, together they help establish a strong connection to mutations in SPTLC1 causing oxidative stress within the cell. This
increase in oxidative stress could be linked to the increase in ubiquinol cytochrome c
expression from the mitochondria, thus an increase in ORP-150 is observed to
compensate and protect the cell from an increase in ROS production.
Actin function is highly regulated by the association of actin binding proteins. Studies
have shown that actin oxidation generally inhibits the association of actin binding
proteins with actin (Farah et. al., 2011). As COTL1 is an actin binding partner its
upregulation could be due to the increased oxidative stress upon the cellular
cytoskeletal system. Oxidative stress can cause actin remodelling and potential axonal
retraction in the neuron (Hallengren et. al., 2013). Under the conditions of stress the
UPR is activated to ensure misfolded proteins are targets for destruction (Hallengren et. al., 2013). RPS27a has a major role in targeting cellular proteins for destruction as
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such its apparent increase in the V144D mutant demonstrates that there is a possible
increase in misfolded proteins either directly due to ER stress, oxidative stress or by
another mechanism that affects protein conformation. The findings presented in this
study suggest that there is a strong underlying mechanism of oxidative damage
occurring in the mitochondria of the V144D lymphoblasts.
Given the evidence that altered protein species were identified within the total mitochondrial and ER proteomes it was clear that greater, more in-depth analyses of these two organelles were required to examine any further alterations. We then carried out detailed, high resolution top-down proteomic analyses, with mitochondria and ER initially isolated from control and V144D lymphoblasts that were subjected to further separations into total membrane and total soluble protein fractions prior to analyses by 2DE.
These analyses revealed a further 36 protein changes in both the membrane and soluble protein fractions from the control and V144D lymphoblasts. Mitochondrial protein species that changed in abundance were identified as being involved in catalytic activity, cytoskeleton, protein transport, oxidative stress, calcium binding and energy metabolism. The changed expression of the proteins identified within the ER fractions were involved in catalytic activity, cytoskeleton and lipid binding.
While there were a number of nonrelated protein species that were found to be altered in the mitochondria compared to the ER, there were a number of similarities in biological processes, most notably catalytic activity, cytoskeleton, protein transport and oxidative stress.
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In the previous analyses on total mitochondrial proteomes we showed that ubiquinol
cytochrome c subunit 1 increased in abundance in V144D lymphoblasts. We again
determined it to be increased in abundance in the ER fraction and this was
accompanied by an increase in the abundance of Peroxiredoxin-4, a protein with
antioxidant functions that reduces the build-up of hydrogen peroxide via a thiol-
dependent cycle (Travender and Bullied, 2010). These findings again strengthen the
correlation of potential increase in ROS within the V144D lymphoblasts that could lead
to further disruption of mitochondrial homeostasis.
Ca2+ is also required for energy production within mitochondria, but increased Ca2+
levels can lead to free radical generation (Feissner et. al., 2009). The in-depth analysis
identified a decrease in the Ca2+ binding mitochondrial carrier protein (ScaMc-1). This
decrease might be a protective mechanism due to the already (potentially) high levels
of ROS but will also cause a decrease in ATP production within the mitochondria.
Voltage dependent anion selective channel protein 1 (VDAC) allows mitochondrial
influx/efflux of metabolites such as ATP, and may also have a role in regulating Ca2+
in mitochondria (Brooks et. al., 2004). A decrease in VDAC in the V144D mutant, in
conjunction with the reduction of SCaMc-1 could result in an overall decrease in
intracellular Ca2+ levels in mitochondria and thus decreased ATP production, again
strengthening the possibility for perturbation of their mitochondrial homeostasis.
Dipeptidyl peptidase 1, also known as Cathepsin C and Pro-cathepsin H has been
shown to be proapoptotic by cleaving Bid and Blc-2 family proteins released by
mitochondria; greatly increasing the cascade of caspase apoptotic factors released
(Droga-Mazovec et. al., 2008). The abundance of these proteins is increased in the mutant cells indicating a link to increased occurrence of mitochondrial apoptotic
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processes (Turk et. al., 2012). Eukaryotic translation initiation factor 5A-1 (eIF5A) has been shown to regulate the Bcl-2 binding protein P53 and the P53 apoptosis pathway
(Huang et. al., 2007). In addition, eIF5A has a regulatory function in protein synthesis.
The increase in abundance of elF5A in the V144D mutant may possibly be the result of the cell’s response to stabilise uncontrolled protein misfolding due to ER stress
(Ogasawara et. al., 2012). Interestingly, CLIC1 was also identified with an increased abundance in the V144D lymphoblasts. We previously identified this change in the isolated total ER proteome. Cell oxidation appears to be the important stimuli determining the transition of CLIC1 between soluble and membrane bound forms
(Averaimo et. al., 2010). Four other proteins were identified with a marked absence or presence in all mitochondrial and ER fractions.
These analyses show a correlation between previous investigations on the total proteome revealing an increase in proteins induced by oxidative stress and mitochondrial electron transport chain proteins. We also identified further changes in calcium channel proteins, cytoskeletal proteins, and energy transport proteins. This provided more evidence for a link of increased misfolded proteins, oxidative stress, and cytoskeleton remodelling and potential changes in Ca2+ signalling within the mitochondria.
The investigations so far have revealed many important changes that are occurring in the V144D mutation in the patient derived lymphoblasts. While these alterations are significant this model has its limitations. As HSN-I is a neurodegenerative disease, we wanted to extend these investigations and examine a neuronal cell model. To carry out these analyses we utilised a ND15 cell line (hybrid of rat dorsal root ganglion neurone and a mouse neuroblastoma) which was transiently transfected (TT) to
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overexpress not only the V144D mutation but two other known missense mutations
that cause HSN-I; C133W and C133Y. The data obtained from this neuronal cell model
confirmed our previous results identified from the lymphoblast model, while notably
identifying changes exhibited in the C133W and C133Y mutations. We also identified
an additional 14 proteins that were altered in abundance within the transfected ND15
cells
Quantitative protein expression and FACS analyses enabled identification of
alterations in ubiquinol cytochrome c abundance. The ubiquinol cytochrome c levels were increased significantly in the TT ND15 cells containing the individual mutations.
These findings further strengthened the potential link between oxidative phosphorylation, via ubiquinol cytochrome c, and altered energy production ultimately leading to axonal degeneration. Further to this finding, quantitative analyses confirmed that the protein expressions of RPS27a, COTL1, and ORP-150 were significantly increased in the V144D TT ND15 cells compared to the levels measured from non- transfected ND15 cell controls.
Interestingly, Stress-70 mitochondrial protein levels were identified in the C133Y mutant as being increased relative to the wild type. When mitochondria are under stress, Stress-70 protein levels increase compensating for increased oxidative damage and maintain normal protein import and synthesis. Thus, if mitochondrial oxidative stress is increased (potentially via ROS production), further cellular damage would occur, ultimately leading to a demise in ER efficiency eventually resulting in ER stress.
Alterations in expression of these proteins strongly indicated that oxidative stress could be linked to increases in ubiquinol cytochrome c. Thus, an increase in ORP-150
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could be potentially explained to compensate and protect the cell from an increase in
ROS production, causing a shift in CLIC1 expression and stabilisation of the
cytoskeleton via COTL1. RPS27a is responsible for targeting misfolded proteins for
destruction, with an apparent increase highlighting potential increases in misfolded
proteins and protein aggregation due to oxidative stress (Hallengren et. al., 2013).
Further strengthening the connection between ER stress and HSN-I; peptidyl-prolyl
cis-trans isomerase was found to be increased in abundance in the C133W mutant.
This protein ensures newly synthesised proteins are folded into their correct
conformation. The 26s proteasome is responsible for regulating the proteome through
degradation of ubiquitin-tagged substrates (Shaw, PE 2002). 26s proteasome
regulatory subunit 8 was found to be increased in abundance in the cells containing
the V144D mutation. The increase in abundance of these two proteins coupled with
the increased expression of RPS27a and ORP-150 highlights the possible increased oxidative stress affecting protein folding conformation.
As described previously, mitochondrial calcium uptake has been shown to lead to free
radical production, with a delicate balance existing between moderate ROS production
to modulate physiological signalling (Glancy and Balaban, 2012). Overproduction of
ROS can ultimately lead to oxidative and ER stress. A decrease in intracellular calcium
is believed to be a cellular response to increased stress, serving as a mechanism to
limit further damage and increase cell survival (Feissner et. al., 2009). As part of this
study we examined the intracellular levels of calcium in wild type, V144D, C133W, and
C133Y ND15 cells. Intracellular calcium was found to be decreased within cells
containing C133W and C133Y mutations, whilst intracellular calcium within the cells
with the V144D mutation was increased.
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Although this investigation has revealed substantial amounts of information not
previously known about intracellular calcium levels in SPTLC1 mutant cells, the
following questions remain unanswered; is the increased level of calcium due to a correlation of ER stress and mitochondrial dysfunction occurring, and the result of the
V144D mutation being unable to reduce intracellular calcium to compensate and protect the cell? Could this difference give insight into how the three mutations differ, ultimately causing HSN-I? While much remains to be elucidated, the findings
described within this thesis broadens our knowledge of what is occurring as a
consequence of the individual known mutations that cause HSN-I.
Increases in the number of lipid droplets and changes in lipid metabolism have been
identified in a variety of autosomal dominant neurodegenerative diseases, including
Alzheimer’s and Parkinson’s (Cole et. al., 2002; Lane and Farkow, 2005). Considering the potential accumulation of lipid droplets and alterations of lipid metabolism in HSN-
I, we set out to determine if an accumulation of lipid droplets was associated with HSN-
I.
Confocal micrographs of Nile red-stained lipid droplets identified an increase of these
inclusions within the oleic acid-treated positive controls compared to the healthy
control and patient-derived lymphoblast cells. A significant increase of lipid droplets
was observed within the patient-derived lymphoblasts compared to the healthy control cells. This increase in lipid droplet abundance in HSN-I patient-derived lymphoblasts provided a possible connection between increased lipid droplet numbers and the cellular mechanism of HSN-I.
Immunoblotting revealed that only the oleic acid treated samples showed detectable
ADFP protein from total cell lysates. Analysis of SPTLC1 from oleic acid treated
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healthy control and patient-derived lymphoblasts revealed no significant difference in expression. ADFP is thought to be involved in lipid homeostasis and lipolysis by protecting triacylglycerol, within the lipid droplets, from cytosolic lipases (Ducharme and Bickel, 2008; Hodges and Wu, 2010). These results suggest that the increase in lipid droplets within the HSN-I patient lymphoblasts, without a change in ADFP, may cause abnormalities in lipid metabolism or be indicative of a protein-trafficking defect that involved elements of the cytoskeleton. The increase in lipid droplets without a parallel increase in the expression of ADFP in the HSN-I patient-derived lymphoblasts may well indicate a deregulation of lipid metabolism which may exacerbate this phenotype.
Normal cellular processes require the proper communication between mitochondria and the ER, which is facilitated by the mitochondria-associated endoplasmic reticulum membrane (MAM) (Vance, J 2014). MAM is involved in the inter-organellar transport of cholesterol, ceramides, ATP, as well as in proteasomal protein degradation and lipid droplet formation. Recently the importance of inter-organellar communication in the pathophysiology of neurodegenerative disorders has been identified (Paillusson, et. al., 2016).
An interesting example of a MAMs resident reactive oxygen species (ROS) generating protein is the p66Shc protein. Under physiological conditions, this growth factor adaptor protein is involved in signal transduction via the RAS protein (Wieckowski et. al., 2009). However, under oxidative stress, p66Shc can participate in the signalling pathway leading to apoptosis. The p66Shc protein contains an N-terminal proline-rich collagen homology domain (CH2) containing a serine phosphorylation site (Ser36) that is important for its ‘proapoptotic’ properties as well as a functional region that is responsible for its interaction with cytochrome c (Wieckowski et. al., 2009). Studies
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have revealed that mitochondrial p66Shc is present in the IMS where it interacts with
cytochrome c and, as a redox enzyme producing hydrogen peroxide (Wieckowski et.
al., 2009).
As we have identified numerous proteins that are upregulated during times of oxidative stress it would be interesting to know if the p66Shc protein was also altered in HSN-I.
If this protein is indeed altered it would further strengthen the correlation between ROS production occurring from the cytochrome c subunit of the mitochondria and subsequent apoptotic events.
Calcium exchange via ER–mitochondrial contacts is facilitated following its release from ER stores via inositol 1, 4, 5- trisphosphate (IP3) receptors (Paillusson, et. al.,
2016). Ca2+ is required by mitochondria to generate ATP via the TCA. Some of the
mitochondrial enzymes involved in ATP synthesis, such as some dehydrogenases,
are regulated by Ca2+ (Vance, J 2014). Excessive uptake of Ca2+ by mitochondria can
lead to opening of the mitochondrial permeability transition pore and apoptotic
signalling occurs. During hypoxic conditions, the ER and MAM resident proteins Ero1-
La and ERp44 interact and modulate ER-mitochondria Ca2+ levels. In addition, elevated levels of Ero1-La may promote activation of the UPR system with a consequent increase in ROS production (Gilady et. al., 2010).
Disorganisation of the ER mitochondrial interface is relevant to the progression of
Alzheimer's Disease (AD) (Marchi et. al., 2014). Up-regulation of MAM associated proteins were observed in sections of brains from human AD patients, and both amyloid β-peptide and the over-expression of presenilin 2, which is mutated in some cases of familial AD, elevates the number of ER mitochondria contact sites, favouring
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Ca2+ transfer between the two organelles (Marchi et. al., 2014). A link between Ca2+ signalling and ER mitochondria connections also appears in the context of ER stress- mediated apoptosis. The RNA dependent protein kinase (PKR)-like ER kinase
(PERK), a key ER stress sensor in the unfolded protein response, is enriched at MAMs and is important for maintaining ER mitochondrial contact sites (Marchi et. al., 2014).
Our in-depth analyses identified decreases in ScaMc-1 and VDAC, both of which are involved in regulating Ca2+. These decreases might be a protective mechanism due to the already high levels of ROS but will also cause a decrease in ATP production within the mitochondria. However this decrease may also be part of ER-stress-mediated processes that could be occurring during the cross talk between the ER and mitochondria. This is strengthened by the identification of UPR proteins that are altered in the disease state.
A clear picture can be seen of direct communication via MAM with ER and mitochondria altering Ca2+ regulation, generating ROS and upregulating the UPR pathway. It is also clear that both of these organelles are altered in their morphology and function in all mutant forms of HSN-I. However while we know that mutant SPTLC1 protein is causing this change the exact mechanism of action is still unclear. It may be that the ER-bound SPT complex is directly connecting the two organelles via MAMs.
Sterols and sphingolipids are minor constituents of mitochondrial membranes. The processes by which these lipids are transported to, and imported into, mitochondria may occur via MAMs (Krols et.al. 2016). Ceramide can induce mitochondria mediated apoptosis by permeabilising the OMM, with a large amount of cellular ceramide being synthesised in the ER, MAM and mitochondria (Krols et.al. 2016, Vance, J 2014).
Given this knowledge it is possible that the mutant SPTLC1, being involved in the
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sphingolipids synthesis pathway, is altering the homeostasis within the MAM that is
then causing a chain of events that results in changes in Ca2+ signalling and generation
of ROS. Another possibility is the toxic deoxy-sphingolipids that are known to be produced as a result of the mutation may also be impairing the communication between the ER and mitochondria as seen in other neurodegenerations where protein aggregations cause a shift in the cellular homeostasis.
Overall, a detailed picture is emerging that MAMs have a large impact on how
organelles communicate and maintain their functions and may help to bridge the
connection of how SPTLC1 mutation cause axonal degeneration. Alteration in one or
more of the organelles causes downstream perturbations that ultimately lead to impaired signalling, toxic product formation, unregulated protein synthesis and disruptions to energy production that terminate in the loss of neuronal function eventually causing axonal degeneration and cell death.
In conclusion, the results presented within this study provide novel and important information, insights and directionality to ultimately uncover the mechanism(s) within peripheral neuropathies. These findings suggest that there may be underlying molecular alterations broadly common to neurodegenerative diseases as a whole, linked to mitochondria, ER and lipids, that maybe facilitated by MAMs.
With mounting evidence into the nature of the protein alterations in HSN-I we have provided new and novel in-sights into the molecular and cellular mechanisms that are occurring within these diseased cells which could ultimately aid in identification of disease-specific bio-markers and facilitate future drug development to assist patient treatment, care and a cure.
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Future Directions
The research presented in this thesis has identified significant alterations within the
protein profiles of purified mitochondria and ER fractions isolated from HSN-I cells.
Uncovering the essential and critical roles of both mitochondria and the ER in
neurodegenerative disease processes is undoubtedly of the utmost importance. We
have also highlighted the potential communication network via MAMs between
mitochondria and ER and how mutant SPTLC1 may cause mitochondrial disturbance.
Many cellular events such as intracellular signalling of important pathways, including the synthesis of cholesterol and phospholipids, calcium homeostasis, ROS generation and activity occur within MAMs (Giorgi et. al., 2009). There are techniques available to isolate mitochondria, as we have determined in this investigation, however, only few specifically isolate MAMs. The isolation of MAMs containing the unique regions of ER membranes attached to the outer mitochondrial membrane requires it to be without
contamination from other organelles such as pure mitochondria.
For Example, Wieckowski et. al., 2009 used a procedure to study the proposed role of
MAMs in cellular responses to oxidative stress connected with the phosphorylation of
p66Shc protein. They indicated that a significant portion of p66Shc is present in the
MAM fraction. Briefly, Wieckowski et. al., 2009 designed the procedure for MAM
isolation by first separating the cellular contaminants from a crude mitochondrial
fraction and then further purifying this via differential centrifugation resulting in isolation of a highly purified MAM fraction.
Once the purified MAM fraction has been obtained a detailed proteomic analysis can then be carried out. The methods employed in this thesis have yielded high quality, in
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depth analyses and these protocols should be utilised to assess total proteomes to
determine the spectrum of proteins altered with the MAM. It would be of great interest
to further examine other proteins upregulated during times of stress, in particular
proteins affected by ROS activation. In addition to proteins that might be upregulated,
the possibility exists that SPT may be present. Considering SPT is housed within the
ER and that the ER and mitochondria communicate via the MAM this would be a highly unique finding and would give much greater insight into how an ER-bound protein facilitates such alterations.
Calcium homeostasis occurs through ‘cross-talk’ between the ER and mitochondria at
MAMs (Giorgi et al., 2015; Marchi et. al., 2014). The Ca2+ signalling apparatus consists
of the Sarcoplasmic Reticulum (SR) Ca2+ release channel, the Troponin protein
complex that mediates the Ca2+ effect to the myofibrillar structures leading to contraction, the Ca2+ pump responsible for its reuptake into the SR, and Calsequestrin,
the Ca2+ storage protein in the SR (Hofer, AM 2005). When calcium signalling is
stimulated in a cell, Ca2+ enters the cytoplasm from one of two general sources: it is
released from intracellular stores, or it enters the cell across the plasma membrane
(Putney et. al., 2001). We have shown that intracellular calcium levels are increased
in cells carrying the V144D mutation and decreased in cells carrying the C133W and
C133Y mutations. Thus isolating MAMs from cells carrying the different mutations and investigating the calcium “cross-talking” between the ER and mitochondria due to
SPTLC1 mutations should be explored.
We have tested the intracellular calcium levels of the TT ND15 neuronal cell models using the technique of fluorescent-based imaging. However future investigations could incorporate other sensitive assays, such as ratiometric analysis of intracellular calcium
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levels using Fura-2 AM (Thermofisher). Ratiometric methods are based on the use of
a ratio between two fluorescence intensities allowing for correction of artefacts due to
bleaching, changes in focus, and variations in laser intensity. This would ensure a more accurate assessment of calcium levels within TT ND15 cells as well as measuring the levels within the patient-derived lymphoblasts.
In addition to intracellular calcium levels, the expression of SR protein should be assessed to see if there are any changes within the calcium storage and channel proteins that are potentially causing mitochondrial alterations in the SPTLC1 mutant cells.
While an assessment of calcium signalling is a good assessment of mitochondrial function, it can also be measured in four additional ways: basal respiration rate, ATP-
linked respiration, proton leak, and reserve capacity (Choi et. al., 2009). Bioenergetic
capacity can be used to determine whether a response to stress is dysregulated,
therefore testing the capacity of mitochondria in cells carrying the SPTLC1 mutation
to respond to increasing energy demand under basal conditions as measured by
oxygen consumption rate (OCR). If the energetic reserves are depleted under normal
conditions due to the SPTLC1 mutation(s), respiration will fail and cell death occurs
(Choi et. al., 2009). Thus, measuring the bioenergetic reserve capacity may be an
effective way to assess or predict the ability of cells to manage and overcome stress,
such as that encountered during acute oxidative insults. This can be done by exposing
the cells to oligomycin (complex V inhibitor), FCCP (carbonyl cyanide p-
trifluoromethoxyphenylhydrazone) and antimycin A sequentially. The OCR will
increase in a concentration-dependent manner in an effort to counteract the increased
energy demand caused by the oxidative damage (Choi et. al., 2009). The OCR would
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reveal if there is increase in the leak of protons (thereby decreasing mitochondrial efficiency) and therefore increasing energy demand (Choi et. al., 2009).
Also of interest would be to assess the expression of the individual mitochondrial complexes (I-V). While we have determined that a subunit of the complex III is altered, determining if the expression of other complexes are altered would be the obvious next stage for investigation. Using the antibody preparation called OXPHOS (a cocktail containing 5 antibodies (Abcam, USA) would allow a comparison of the level of
expression of the mitochondrial complexes (I-V) in normal cells and in cells carrying
SPTLC1 mutation.
We have also shown that there is increased oxidative stress occurring within cells expressing the SPTLC1 mutations, possibly via ROS production. An assay that can be carried out to assess if/what species are being produced is called MitoSox Red
(Thermofisher Scientific). MitoSox Red is a fluorescent dye that becomes highly oxidised when it comes into contact with superoxide, resulting in an increase in fluorescence. Additionally, Amplex Red (Thermofisher Scientific) is another dye that when reduced by hydrogen peroxide molecules produce a red fluorescent product. If these assays reveal ROS is produced, then further assessment would need to be undertaken to determine the source of the ROS production and to ensure that any
ROS generated is not part of normal cellular functions. While the determination of a
ROS species being present is informative, this would need to be further investigated as to how this may alter proteins within the mitochondria. Additional assessment could use a ROS-scavenging molecule to see if the reduction in ROS changes protein expression, especially with regard to the proteins identified in this investigation.
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Although the methodology for quantifying protein abundance has been in use for many years, methods for assessing and analysing mRNA expression are becoming essential (Greenbaum et. al., 2003). The quantification of both mRNA and protein is
not an exercise in redundancy; measurements taken from mRNA and protein levels
are complementary and both provide a more complete understanding of how the cell
works. Also, as mRNA is translated into protein, it can be assumed that there should
be a correlation between the level of mRNA and that of protein (Greenbaum et. al.,
2003). mRNA profiles should be analysed from patient-derived lymphoblasts cells and
would add to the assessment of the expression of the major proteins described in this study. Of particular interest would be the analysis of the mRNA expression for the
proteins ubiquinol cytochrome c, ORP-150, RPS27a, Coactosin and CLIC1, to assess whether there is evidence for any changes in mRNA expression due to the mutant
SPTLC1 protein. A technique that could be used to assess mRNA levels and profiles with high throughput is RT-PCR. The information obtained from such future investigations would strengthen and build upon the numerous protein expression profiles occurring in HSN-I detailed within this thesis.
The proper functioning of quality control systems like autophagy is essential to maintain cellular homeostasis. Autophagy dysfunction has been implicated in many neurodegenerative disorders (Martinez-Vincente, M. 2015; Menzies et. al., 2015).
Dysfunction can occur at several steps of the autophagy machinery and can contribute
to the formation of intracellular aggregates and ultimately to neuronal death (Menzies
et. al., 2015). While studies have shown that protein aggregation does not occur in
HSN-I, two atypical neurotoxic deoxy-sphingoid bases (DSBs) are produced in HSN-I
(Penno et. al., 2010; Ernst et. al., 2015). Dysfunctional mitochondria release pro-death
proteins resulting in activation of cell death pathways. Aberrant mitochondria must be
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removed to limit damage to the cell. It would be of interest to determine if normal
autophagy processes are occurring in SPTLC1 mutant cells and whether the deoxy-
sphingoid bases potentially interact or impair the autophagosome/mitophagy
formation and thus removal of dysfunctional mitochondria.
In addition to potentially altering autophagy processes, the neurotoxic DSBs have
been shown to interact and affect the cytoskeleton of neurites (Penno et. al., 2010).
We have shown that Coactosin is upregulated within the SPTLC1 mutant cells. As
such, examination of whether this change is caused by the increased formation of
DSBs or a combination of increased oxidative stress, neurotoxic DSBs accumulation
and ER stress should be explored.
Production of induced pluripotent stem (iPS) cells by cellular reprogramming somatic cells can be achieved by ectopic expression of specific transcription factors (Singh et. al., 2009). The reprogramming factors are introduced into cultured somatic cells and the cells are then grown under embryonic stem cell conditions. After approximately 3 weeks, iPS cells emerge. These induced pluripotent stem cells may be differentiated into various cell types (Singh et. al., 2009).
In attempting to answer the question of ‘why mutation of the SPTLC1 protein subunit
causes HSN-I’, the next step could be to generate human DRGs. Somatic cells can
be harvested from HSN-I patients harbouring SPTLC1 mutations and be differentiated
into DRGs. Such patient-derived iPS cells would provide greater insight into the
molecular and cellular mechanisms of the neurodegenerative disease process,
enabling the study of patient-specific SPTLC1 mutations and testing of candidate
drugs. This would be essential in understanding the cellular mechanisms of how HSN-
I affects human DRGs (Singh et. al., 2009).
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Ultimately, any future investigations into the molecular and cellular mechanisms of the neurodegenerative disease processes of HSN-I must expand on the findings presented within this thesis and include investigations into the pathogenic mechanisms of SPTLC1 mutations and the links to mitochondrial and ER localisation,
MAMs and the subsequent disturbances and axonal degeneration that is a defining characteristic of HSN-I.
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