University of Florida Thesis Or Dissertation Formatting

NOVEL PROTEOLYTIC PROTEIN AND PEPTIDE BIOMARKERS FOR TRAUMATIC BRAIN INJURY

GEORGE ANIS SARKIS

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2017

To the souls and memory of my Father and Mother. To my Wife and Children thank you for your continued support, encouragement, and love throughout this process

ACKNOWLEDGMENTS

First and foremost, I would like to thank God, Jesus Christ and Mary Mother of

God. You have given me the power to believe in my passion and pursue my dreams. I could never have done this without the faith I have in you, the Almighty.

I thank my mother’s and father’s souls. I want to thank my family and friends who have stood by me as I have pursued my passion for learning. I thank my advisors,

Professor Richard A. Yost and Professor. Kevin K.W. Wang, for giving me the opportunity to work with their research teams, and to all the team members. Besides my advisors, I would like to thank the rest of my committee, Professor Benjamin W. Smith,

Professor Kenneth B. Wagener and Professor Kari B. Basso, for their insightful comments and encouragement, but also for the hard question, which incented me to widen my research from various perspectives.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 8

LIST OF FIGURES ...... 9

LIST OF ABBREVIATIONS ...... 13

ABSTRACT ...... 17

CHAPTER

1 BACKGROUND AND SIGNIFICANCE ...... 19

Introduction ...... 19 Military Based TBI ...... 21 Sports-Based TBI ...... 22 Current Methods of TBI Diagnosis ...... 23 Biomarker as Diagnosis Tools ...... 24 Biomarker Classifications ...... 26 The Detection of Biomarkers in Sports and Military-Related Injuries ...... 26 TBI Protein Biomarker...... 27 Mass Spectrometry Based Peptidomics ...... 30

2 EXPERIMENTAL METHODS AND ANALYTICAL DESIGNS ...... 35

SDS-Gel Electrophoresis Combined with Immunoblotting ...... 35 Sample Preparation for Electrophoresis ...... 35 Gel Electrophoresis ...... 36 Coomassie Brilliant Blue Staining of Gels ...... 37 Electrotransfer ...... 37 Immunoblotting-Antibody Incubation and Detection ...... 38 Ultrafiltration Method ...... 39 Mass Spectrometry ...... 39 Reversed-Phase Liquid Chromatography Tandem Mass Spectrometry ...... 39 Velos Pro Dual-Pressure Linear Ion Trap Mass Spectrometer ...... 41

3 IN VITRO DIGESTION OF PURIFIED HUMAN BRAIN PROTEINS AND NAÏVE MOUSE BRAIN LYSATE ...... 45

The Biomarker Neurogranin ...... 45 The Biomarker Vimentin ...... 47 Sample Preparation ...... 47 Naïve Mouse Brain Lysate Preparation ...... 48 In Vitro Digestion of Purified Protein-Naïve Mouse Brain Lysate ...... 49 Analysis of Digested Samples ...... 49 Neurogranin ...... 49 Vimentin ...... 50 Naïve Mouse Brain Lysate ...... 50 Results and Discussion...... 51 Purified Neurogranin Protein ...... 51 Purified Vimentin Protein ...... 52 Naïve Mouse Brain Lysate ...... 53 Neurogranin ...... 53 Vimentin ...... 54 Conclusions ...... 54

4 NEURO-CELL CULTURE CYTOTOXIC CHALLENGES TO EXAMINE BRAIN PROTEOLYTIC PEPTIDES FORMATION ...... 80

Introduction ...... 80 Cytotoxic Challenges ...... 81 Cell Culture and Lysis ...... 84 Results and Discussion...... 85 The Primary Cortical Mixed Neuron-Astroglia (CTX)/ Neuro2a (N2a) cell line . 85 U-251 Cell Line, Human Glioblastoma Astrocytoma Cell ...... 86 Conclusions ...... 87

5 MOUSE MODELS OF TRAUMATIC BRAIN INJURY ...... 99

Introduction ...... 99 In Vivo Models of Severe Traumatic Brain Injury ...... 102 In Vivo Models of Mild Traumatic Brain Injury ...... 103 Mouse Brain Tissue Collection and Preparation ...... 103 Results and Discussion...... 104 Immunoblotting Analysis for Cortex ...... 104 Immunoblotting Analysis for Hippocampus...... 104 Mass Spectrometry Analysis ...... 105 Conclusions ...... 105

6 ARCHIVED HUMAN TBI BIOFLUID CEREBROSPINAL FLUID ...... 116

Human Bio-Samples Procurement ...... 116 Cerebrospinal Fluid Collection Protocol ...... 117 Sample Preparation ...... 118 Results and Discussion...... 119 Conclusion ...... 120

7 SUMMARY AND FUTURE WORK ...... 128

Summary ...... 128 Future Work ...... 129

LIST OF REFERENCES ...... 131

BIOGRAPHICAL SKETCH ...... 142

LIST OF TABLES

Table page 1-1 Glasgow coma scale showing different cases of TBI...... 33

1-2 Possible TBI markers (existing biomarkers) and the peptidomic markers derived from this thesis work ...... 34

3-1 Neurogranin (NRGN) antibodies used ...... 56

3-2 List of peptides extracted from Proteome Discoverer 2.1 resulting from digestion of NRGN using calpain-1 ...... 57

LIST OF FIGURES

Figure page 2-1 Schematic diagram for SDS-PAGE-Immunoblotting (George Anis Sarkis)...... 42

2-2 Novel ultrafiltration-based TBI proteolytic peptidome enrichment, coupled to LC-MS for separation and identification of peptide ID...... 43

2-3 Schematic diagram for LTQ XL (Thermo Fisher Scientific)...... 43

2-4 Schematic diagram for LTQ-Velos Pro-Orbitrap (Thermo Fisher Scientific)...... 44

3-1 Digestion of purified NRGN protein with calpain-1 and caspase-3...... 58

3-2 Purified NRGN in vitro digestion with calpain-1, caspase-3 and -6...... 58

3-3 Schematic representation for the list of NRGN peptides generated from in vitro digestion of purified NRGN protein using calpain-1 protease (N=7)...... 59

3-4 MS/MS spectrum for the N-terminal peptide PGANAAAAKIQA (m/z 542.16) released from the digested human purified NRGN protein using calpain-1...... 60

3-5 MS/MS spectrum for the C-terminal peptide KSGERGRKGPGPGGPG (m/z 747.38) released from the digested human purified NRGN protein using calpain-1...... 61

3-6 Schematic representation for the list of NRGN peptides generated from in vitro digestion of purified NRGN protein using caspase-6 protease (N=5)...... 62

3-7 MS/MS spectrum for the peptide MDCCTE (m/z 408.22) released from the digested human purified NRGN protein using caspase-6...... 63

3-8 Digestion of VIM purified protein with calpain-1, caspase-3 and -6...... 64

3-9 Schematic representation for the list of VIM peptides generated from in vitro digestion of purified VIM protein using calpain-1 protease, filtered using 10 kDa MWCO (N=3)...... 65

3-10 MS/MS spectrum for the N-terminal peptide MSTRSVSSSSYRRMFGGP (m/z 677.02) released from the digested human purified VIM protein using calpain-1...... 66

3-11 MS/MS spectrum for the C-terminal peptide DGQVINETSQHHDD (m/z 798.05) released from the digested human purified VIM protein using calpain-1...... 67

3-12 Schematic representation for the list of VIM peptides generated from in vitro digestion of purified VIM protein using caspase-3 protease, filtered using 10 kDa MWCO (N=3)...... 68

3-13 MS/MS spectrum for the peptide TIGRLQDEIQNMKE (m/z 564.58) released from the digested human purified VIM protein using caspase-3...... 69

3-14 In vitro digestion of mouse brain lysate using calpain-1, caspase-3 and -6...... 70

3-15 Schematic representation for the list of peptides of NRGN protein generated from in vitro digestion of mouse brain lysate using calpain-1 protease, filtered using 10 kDa MWCO (N=5)...... 71

3-16 MS/MS spectrum for the N-terminal peptide DPGANAAAAKIQ (m/z 563.85) released from the mouse brain digested with calpain-1...... 72

3-17 MS/MS spectrum for the C-terminal peptide KGPGPGGPGGAGGARGGA (m/z 460.00) released from the mouse brain digested with calpain-1...... 73

3-18 Schematic representation for the list of peptides of NRGN protein generated from in vitro digestion of mouse brain lysate using caspase-3 protease, filtered using 10 kDa MWCO (N=5)...... 74

3-19 MS/MS spectrum for the peptide CGRKGPGPGGPGGAGGARGGAGGGPSGD (m/z 761.51) released from the mouse brain digested with caspase-3...... 75

3-20 Schematic representation for the list of peptides of VIM protein generated from in vitro digestion of mouse brain lysate using calpain-1 protease, filtered using 10 kDa MWCO (N=3)...... 76

3-21 MS/MS spectrum for the N-terminal peptide MSTRSVSSSSYRRMFGGSGTSSRPSSNRSYV (m/z 1131.65) released from the mouse brain digested with calpain-1...... 77

3-22 Schematic representation for the list of peptides of VIM protein generated from in vitro digestion of mouse brain lysate using caspase-3 protease, filtered using 10 kDa MWCO (N=3)...... 78

3-23 MS/MS spectrum for the peptide EIQELQAQIQE (m/z 664.32) released from the mouse brain digested with caspase-3...... 79

4-1 NRGN proteolysis breakdown in primary cortical mixed neuron-astroglia mixed culture (CTX) after subjecting to cytotoxin challenges (24 hr)...... 89

4-2 NRGN proteolysis breakdown in primary cortical mixed neuron-astroglia mixed culture (CTX) after subjecting to cytotoxin challenges ...... 90

4-3 Schematic representation for the list of NRGN peptides generated from N2a subjected to A23187 filtered using 10 kDa MWCO (N=5)...... 91

4-4 MS/MS spectrum for the peptide PGANAAAAKIQASFRGHMARKKIKSGECGRKGPGPGGPGGAGGARGG (m/z 1486.75) released from the N2a subjected to A23187...... 92

4-5 Schematic representation for the list of NRGN peptides generated from CTX subjected to STS (caspase cleavage only) filtered using 10 kDa MWCO (N=5)...... 93

4-6 MS/MS spectrum for the peptide ILDIPLDD (m/z 913.50) released from the CTX subjected to STS...... 93

4-7 Schematic representation for the list of NRGN peptides generated from CTX subjected to EDTA filtered using 10 kDa MWCO (N=5)...... 94

4-8 MS/MS spectrum for the peptide IPLDDPGANAAAAKIQASFRGHMARKKIKSGE (m/z 1117.93) released from the CTX subjected to EDTA...... 95

4-9 Vimentin and GFAP proteolysis in human glioblastoma U-251 cells subjected to various pro-necrotic and pro-apoptotic challenges...... 96

4-10 Schematic representation for the list of VIM peptides generated from U-251 cell line subjected to A23187 filtered using 10 kDa MWCO (N=5)...... 97

4-11 MS/MS spectrum for the N-terminal peptide STSRSLYSSSPGGAYVTRSSAVRLRSSV (m/z 973.50) released from the U- 251 cell line subjected to A23187...... 98

5-1 Ipsilateral cortex profile at different time points after CCI and rCHI injury in mice...... 107

5-2 Ipsilateral hippocampus profile at different time points after CCI and rCHI injury in mice...... 108

5-3 Schematic representation for the list of NRGN peptides generated from cortex CCI (day 1) lysate samples, filtered using 10 kDa MWCO (N=3)...... 109

5-4 MS/MS spectrum of the NRGN peptide PGANAAAAKIQASFRGHMARKKIKSGECGRKGPGG (m/z 1245.87) released from ipsilateral cortex CCI (day 1) injury in mice...... 110

5-5 Schematic representation for the list of NRGN peptides generated from cortex CCI (day 7) lysate samples, filtered using 10 kDa MWCO (N=3)...... 111

5-6 MS/MS spectrum of the NRGN peptide DDDILDIPLDDPGANAAAAKIQASFR (m/z 904.30) released from ipsilateral cortex CCI (day 7) injury in mice...... 112

5-7 Schematic representation for the list of VIM peptides generated from cortex CCI (day 1) lysate samples, filtered using 10 kDa MWCO (N=3)...... 113

5-8 MS/MS spectrum of the N-terminal VIM peptide LGSALRPSTSRSLY (m/z 585.63) released from ipsilateral cortex CCI (day 1) injury in mice...... 114

5-9 MS/MS spectrum of the C-terminal VIM peptide NLESLPLVDTHSKRTLLIKTVETRDGQVINE (m/z 1227.03) released from ipsilateral cortex CCI (day 1) injury in mice...... 115

6-1 NRGN and 9 kDa NRGN-BDP in human CSF within 24 h of severe TBI...... 121

6-2 Schematic representation for the list of NRGN peptides generated from filtered (10 kDa MWCO) TBI CSF samples...... 122

6-3 MS/MS spectrum of NRGN peptide in human TBI CSF (24 hr)...... 123

6-4 MS/MS spectrum of NRGN peptide in human TBI CSF (24 hr) ILDIPLDDPGANAAAAKIQASFRGHMARKKIKSGERGRKGPGPGGPGGA..... 124

6-5 Vim and 35, 26 kDa VBDP in human CSF within 24 h of severe TBI...... 125

6-6 Schematic representation for the list of VIM peptides generated from filtered (10 k Da MWCO) TBI CSF samples...... 126

6-7 MS/MS spectrum of the C-terminal VIM peptide in human TBI CSF (24 h)...... 127

7-1 Schematic representation for calpain-1 and caspase cleavage sites. A) NRGN protein. B) VIM protein...... 130

LIST OF ABBREVIATIONS

AD Alzheimer's Disease

APP Amyloid Precursor Protein

ATP Adenosine Triphosphate

BCIP 5-Bromo-4-Chloro-3-Indolyl Phosphate

BDP Breakdown Product

BEH Ethylene Bridged Hybrid

BNP B-type Natriuretic Peptide

BRCA1 Breast Cancer 1

CaM Calcium-Modulated protein

Caspase Cysteine-aspartic protease, or

Cysteine-dependent aspartate-directed protease

CBB Coomassie Brilliant Blue

CCI Controlled Cortical Impact

Cdk5 Cyclin-dependent kinase 5

CID Collision-Induced Dissociation

CK Creatine Kinase

CNS Central Nervous System

CSF Cerebrospinal Fluid

CT Computed Tomography

CTE Chronic Traumatic Encephalopathy

CTX Primary Cortical Mixed Neuron-Astroglia

DAG Diacylglycerol

DAI Diffuse Axonal Injury

DDA Data-Dependent Acquisition

DMEM Dulbecco's Modified Eagle Medium

DTT Dithiothreitol

ECF Extracellular Fluid

EDTA Ethylenediaminetetraacetic acid

EGFR Epidermal Growth Factor Receptor

EGTA Ethylene Glycol-bis (β-aminoethyl ether)-N,N,N',N'-Tetraacetic Acid

FBS Fetal Bovine Serum fMRI Functional Magnetic Resonance Imaging

GCS Glasgow Coma Scale

GFAP Glial Fibrillary Acidic Protein

HCD Higher Energy Collision

HF Heart Failure

HPLC-MS High-Performance Liquid Chromatography-Mass Spectrometry

IED Improvised Explosive Device

IRB Institutional Review Board

ISF Interstitial Fluid

KA Kainic acid

MBP Myelin Basic Protein

MCI Mild Cognitive Impairment

MMP-9 Matrix Metalloproteinase-9

MRI Magnetic Resonance Imaging mTBI mild Traumatic Brain Injury

MWCO Molecular Weight Cut-Off

N2a Neuro2a cells

NBT Nitro Blue Tetrazolium chloride

NF-H Neurofilament Heavy Protein

NF-L Neurofilament Light Protein

NMDA N-methyl-D-Aspartate Receptor

NRGN Neurogranin

NSE Neuron Specific Enolase

NT-proBNP N-terminal-pro-Brain Natriuretic Peptide

PA Phosphatidic Acid

PCS Post Concussive Syndrome

PDGF Platelet-Derived Growth Factor

PDHS Plasma-Derived Horse Serum

PKC Protein kinase C

PTSD Post-Traumatic Stress Disorder

PVDF PolyVinyliDene Fluoride rCHI Repeated Closed Head Injury

RP-nLC- Reversed-Phase nano-Liquid Chromatography Tandem MS/MS Mass Spectrometry

RSLC-MS/MS Rapid Separation Liquid Chromatography Tandem

Mass Spectrometry

RT-PCR9 Real-Time Polymerase Chain Reaction

SBDP αII-Spectrin Breakdown Product

SCUBE1 Signal peptide-Cub-Epidermal growth factor domain-containing protein

SDS-PAGE Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis

SEM Secondary Electron Multiplier

STS Staurosporine

TAI Traumatic Axonal Imaging

TBI Traumatic Brain Injury

TBST Tris-Buffered Saline Tween

U-251 Human Glioblastoma cells

UCHL-1 Ubiquitin Carboxyl-Terminal Esterase L1.

UPLC Ultra Performance Liquid Chromatography

VIM Vimentin

WBC White Blood Cells

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

NOVEL PROTEOLYTIC PROTEIN AND PEPTIDE BIOMARKERS FOR TRAUMATIC BRAIN INJURY

George Anis Sarkis

May 2017

Chair: Richard A. Yost Major: Chemistry

Traumatic brain injury (TBI), a complex injury, brings about a wide range of symptoms and consequences. An estimated 1.6-2.1 million TBI cases occur annually in the United States. The need for more sensitive and readily accessible diagnostic tools for TBI led to the search for novel biomarkers, especially for mild TBI (mTBI). Based on this work, and that of others, TBI is a neurological disorder which triggers a cellular proteolytic activation cascade (including calpain, caspase, matrix metalloproteinases and cathepsins), resulting in brain-specific protein degradation.

Proteolytic attack of these brain proteins produces truncated protein fragments, and often simultaneously releases small endogenous peptides of low molecular weight

(MW ~1,000– 5,000 Da). The utility of several reported protein biomarkers in mTBI, especially concussion, has not been completely established. It is conceivable that the blood-brain barrier might not be compromised or might have resealed quickly following mTBI, thus hindering the release of larger proteins or protein fragments into the circulating blood. Thus, it is of interest to examine small (less than 5 kDa) proteolytic peptides derived from brain proteins as possible TBI biomarker candidates. The

peptidomics analysis employed a novel ultrafiltration-based endogenous peptide isolation and enrichment coupled with two analytical methods: nano-liquid chromatography/mass spectrometry-based peptide identification (RP-nLC-LTQ-XL, RS- nLC-LTQ-Velos pro-Orbitrap), and SDS-gel electrophoresis and immunoblotting probed with target-specific antibodies.

A combination of purified proteins, brain lysate, neuron-glial cell culture injury models, animal models of TBI, human TBI and control cerebrospinal fluid was used.

From these samples, a list of unique peptides derived from diverse brain cell types or subcellular structures was compiled. The post-synaptic protein neurogranin and intermediate filament protein vimentin found in astroglia and neurons, as well as their proteolytic breakdown products and peptide, were scrutinized. The data indicated increased leakage of neurogranin and vimentin, and their breakdown products, from the

CSF compartment of TBI cases, compared to healthy control. Thus, these proteolytic breakdown products and peptides could be considered as diagnostic biomarker candidates and potential “theranostic” tools in clinical trials for new TBI drug development.

CHAPTER 1 BACKGROUND AND SIGNIFICANCE

Introduction

Traumatic brain injury is a complex injury with a wide range of symptoms and consequences for those who suffer from this type of injury. TBI is triggered by biomechanical forces that acutely impact the brain, resulting in macro- or microstructural damages and acute and/or chronic brain dysfunctions1. An estimated 1.6 - 2.1 million

TBI cases occur annually in the United States, with about half of the cases predominantly occurring in youngsters (ages 0-4 years) and adolescents (ages 15-19 years) due to risky behavior2. The other at-risk group comprises adults (ages 65 years and older). Of these traumatic brain injuries: 52,000 are fatal; 275,000 are hospitalized; and 1,365,000 (about 80%) are treated then released from an emergency department3.

Similar figures are seen in Europe and Asia. TBI accounts for 30.5% of all injury-related deaths in the United States3. The extent of a TBI can range from mild to severe. Mild

TBI (mTBI) is considered to be 75-80% of head-related traumas, including concussions sustained during a contact sport. Implying that if the condition is misdiagnosed, it can lead to serious long-term consequences like coma or death. TBI is now recognized as not only an acute trauma event that needs to be treated and managed, but it is also increasingly recognized that both severe/moderate TBI and mild TBI (e.g., from contact sports-related concussion) may lead to persistent post-concussive syndrome (PCS)

(e.g., cognitive/neuropsychological impairments and other comorbidities)1. Furthermore, in about 20-25% of the moderate to severe TBI cases, such as blunt force trauma to the head, cause damage to the underlying areas of the skull. TBI has a wide range of physical and psychological effects. Some of the symptoms of TBI can be observed

immediately following the traumatic event, while others may not be seen until after a few days or weeks. The severity and symptoms of TBI depend on many factors, one of which is the type of head injury sustained. This can either be a closed head injury resulting from a blow to the head, a sudden violent motion causing the brain to hit against the skull, or it could be an open head injury when an object penetrates the skull4. Other elements include the extent of physiological recovery, functions impacted, resources available to improve recovery, and areas of function not affected by TBI5.

Due to the complexity of the injury, TBI can have a broad range of effects and symptoms depending on the severity of the injury, such as headache unconsciousness; dizziness; mood swings; concussions; blurred vision; trouble with memory; loss of coordination; or even death6.

TBI is associated with a complex metabolic, biochemical and cellular disruption.

These could include ischemia7, diffusion hypoxia8, mitochondrial dysfunction9, and increased energy needs/metabolic disruption (from excitotoxicity, seizure activity, and spreading depolarization)10, axonal and dendritic injury11, neuroinflammation12, neuronal cell death13, and neurodegeneration (including the formation of chronic traumatic encephalopathy)14. TBI is also linked to altered protein expression levels, which can be visualized in altered levels of gene expression in rats 24-hours post-TBI using a CNS- specific GeneChip and real-time PCR. Glutamate receptor subunit genes were observed to be altered, which is extremely important because increased levels of glutamate in the synapse is neurotoxic and can activate specific receptors, which produce increased levels of Na+ and Ca2+15. Excess Ca2+ in the cytosol activates the protein calpain (a calcium-dependent system), which results in the cleavage of

cytoskeletal proteins16. In addition, TBI has been reported to induce different protein modifications such as oxidative modifications17, protein aggregation/translocation18, and phosphorylations19. Apoptosis-linked caspases are also activated following TBI20. Other proteases have been identified to be activated following TBI, including matrix metalloproteases (e.g., MMP-9)21, and lysosomal proteases (e.g. cathepsin B and D)22.

The pathways affected by TBI are complex, therefore further studies are required to better elucidate the pathology of TBI to improve and develop new treatment methods.

Military Based TBI

Since October 2001, over 1.6 million American soldiers have returned from Iraq and Afghanistan, many of whom have been diagnosed with a TBI. In the timeframe, from 2000-2015, there have been an estimated 333,169 cases of TBI in returning servicemen (prevalence of about 15.2-22.8%). The most common military-related TBI is mild (mTBI), and about 80% of the mTBI cases result from blast exposure to an improvised explosive device (IED)23. However, due to the lack of any physical evidence of damage to the head or brain following mTBI, these types of injuries in the military are difficult to diagnose and tend to be overlooked, despite the long-term effects being unknown24. There is evidence that some individuals experience persistent cognitive and behavioral changes following mild neurotrauma, and there are examples of World War I soldiers who developed “commotio cerebri” a mysterious condition with symptoms very similar to TBI-associated symptoms, such as headaches, memory loss, and depression24. In addition, there is evidence that suggests mTBI can produce long-term gray and white matter atrophy, accelerate neurodegeneration and increase the risk to the development of Alzheimer’s, Parkinson’s, and motor neuron disease24. Military- related TBI can result from several different causes (falls, athletics, physical training

practices, motor-vehicle accidents, etc.). It is difficult to diagnose these injuries. The need for more examination of those returning servicemen with these types of injuries and symptoms is necessary.

Sports-Based TBI

Sports-related TBI is a major problem for those who participate in contact sports where blows to the head are common, especially in children and young adults. Annually in the United States, about 473,947 emergency department visits for TBI are made by children ages 0-143. From 2001-2005, children and young adults (ages 5-18 years) made up for 2.4 million sports-related emergency department visits, and of these visits,

6% (about 135,000 cases) involved a concussion25. In US high schools, football is the leading cause of sports-related concussions, accounting for 60% of this type of TBI26.

Concussions (also known as a mild traumatic brain injury, mTBI), result from any activity, which causes the brain to move or shake violently inside the skull25. A concussion which may seem mild or inconsequential has been shown to change the normal functionality of the brain and frequently presents symptoms weeks after the incident, even after the initial symptoms have subsided26. At the cellular level, TBI can cause microscopic changes within the axon of a neural cell causing it to twist and shear27. This breakage and disruption often cause neuronal death which in serious cases cause personality changes, confusion, depression, and other TBI-related symptoms seen in those suffering from mild-severe TBI27. TBI has recently garnered much attention from the American population, due to its high prevalence among retired

(and many active) National Football League (NFL) players. According to a recent study using advanced scanning technology, it has been shown that more than 40% of retired

NFL players showed signs of a TBI26. In fact, according to the latest study conducted at

Johns Hopkins University, former NFL players show signs of brain atrophy in areas like the right hippocampus when compared to controls12. TBI has been identified as a common precursor to the degenerative brain disease, chronic traumatic encephalopathy

(CTE)28. In one study on the brain tissue examined, 59 out of 62 deceased NFL players analyzed at Boston University tested positive for CTE29. The analysis consisted of MRI brain imaging of the athletes while being given concentration and memory tests29. The

MRI measured damage to the brain’s white matter, which is a region that connects different areas of the brain, and 43% of participants showed levels below a healthy adult29. Of the retired athletes, 30% showed evidence on traditional MRI scans of axon disruption in cell signaling, and 50% showed significant problems in executive function29. Due to the limitations in scanning technology, the players have access to after a head injury, as well as the subjective nature of concussion (mTBI) tests, NFL players have been shown to be at an elevated risk of mild-severe TBI.

Current Methods of TBI Diagnosis

Despite the recent advances in understanding and visualizing TBI and its effects on the brain, there is still no truly objective measure of accurately diagnosing TBI, especially in the case of concussions, or mTBI. Today, most patients diagnosed with a

TBI rely solely on the physician’s analysis of the of the patient’s symptoms30, which may or may not be followed up with further examination31. Although there are several tests and medical imaging devices available, much of the latest technology has limitations in detecting TBI30. Usually, the examination of a possible TBI starts with the physician obtaining the patient’s score on the Glasgow Coma Scale (GCS)32(Table 1-1) which rates consciousness on the patient’s ability to open their eyes, their speech, and movement33. However, this test is not designed for diagnosing mTBI, which may not

affect any of these functions. The score given can be influenced by many other factors due to its subjective nature34. If a physician believes further examination should be done, a computed tomography (CT) scan is most often the next test due to its availability and inexpensiveness, as well as its speed compared to other imaging technologies, such as magnetic resonance imaging (MRI)33. Despite its advantages, less than 10% of patients considered having minor head injuries receive a positive result on a CT scan33. In addition, the CT signal during a scan can be displaced by metal, bone, or any high concentration of contrast, which can degrade the image quality33. CTs also pose an increased radiation exposure risk, if repeated CT scans are performed35. While CT scans are generally performed 24-hour after injury, MRI’s are typically done 48-72-hour post-injury and show better detection of axonal injury, small contusions, neuronal damage, and white matter shearing, compared to CT scans, which miss 10-20% of the abnormalities seen on MRI33. However, the majority of mTBI patients show no abnormality on an MRI image33. Recently, more technology has been developed to better aid in the objective diagnosis of mild-severe TBI, such as the quantitative encephalogram, visual tracking, and serum based protein markers30.

Biomarker as Diagnosis Tools

A biomarker plays an important role in biomedical research and medical treatment, as it is a measured substance that can manifest a sign of biological process or of a disease. A biomarker can provide crucial information regarding the disease state to an individual by physiological tests such as tissues, blood, and body serum tests36. A variety of advanced technology exams such as electrocardiographic study, CT, MRI

(e.g. functional magnetic resonance imaging-fMRI), and traumatic axonal imaging (TAI) can be used also providing vital information36. According to the National Cancer

Institute, the biomarker is defined as an indication of a typical or untypical process, as well as a manifestation of a state of health and well-being38. Furthermore, National

Institute of Health states that a biomarker can assess pathogenic activities, drug responses, or a treatment process36. It is noteworthy that there are some types of biomarkers called nonspecific biomarkers, hyperthermia, elevated WBC, sedimentation rate, or other experimental measurements. Even though they do not indicate any specific disease, these nonspecific biomarkers are important in the process of analyzing and evaluating with other indices39. The ideal biomarker can detect a disease early and accurately. It has the accessibility of being collected into testing samples easily and quickly as well as the exhibition of an expected progression or result from a disease condition, and the significant difference in biomarker levels between an untreated subject and a healthy control40. The fact is that neuroimaging biomarkers are widely used in treating neurological disorders; they can aid physicians and scientists in visualizing the conformational abnormalities of moderate to severe TBI41. In addition, visible identification of skull fractures, intracranial lesions, and intracranial hematomas, which can be detected by CT, would certainly ease the process of prognosis and diagnosis in patients37. However, a study of neuroimaging has shown that these conventional imaging techniques may not be able to detect properly the mild injuries.

Instead, mild TBI biomarkers could be significantly measured by pathophysiological changes on the microscopic level41. Moreover, it has shown that the increase of proteins

S100B and GFAP in serum and cerebrospinal fluid in severe astroglial injury, respectively, can help distinguish mild and severe TBI42. Literature supports that this

evidence as prognostic biomarkers would improve the clinical results significantly because of its practical effectiveness41.

Biomarker Classifications

It is worthy to note that biomarkers can be categorized into two main subtypes: prognostic and diagnostic biomarkers. Their functions in prognosis and diagnosing heart failure (HF) disease are very noteworthy. Many scientists have proven that the presence of B-type natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) can aid the process of diagnosing a patient. In addition to those noted biomarkers, emerging studies have figured out a variety of novel pathophysiological indicators. Those indicators can potentially lead to the development of therapeutic treatments in HF, including mid-regional pro-adrenomedullary, troponin, and soluble ST243. The importance of prognostic biomarkers is to detect a potential threat of a disease, monitor disease progression, evaluate overall outcomes, and then provide a guide for treatment44. In fact, a prognostic marker can be a facilitator in selecting patients therapy but not necessarily be able to predict the response44. For example, in the clinical practice of breast and ovarian cancer, the mutation of BRCA1 gene could be helpful in predicting the response of a patient to chemotherapy45. In addition, a study shows that an elevated serum concentration of SCUBE1 (Signal peptide-Cub-Epidermal growth factor domain-containing protein 1) which signals the coagulation is mostly related to the severity levels of trauma.

The Detection of Biomarkers in Sports and Military-Related Injuries

A novel study conducted by the University of Wisconsin-Platteville has shown there are changes in serum concentration biomarkers, such as S100 calcium-binding protein B (S100B) and neuron-specific enolase (NSE), in athletes during American

football. The values increase significantly after the competition, which can facilitate in determining if there is a head injury due to sub-concussive impacts46. In a similar manner in boxing, a previous study proves there are increases in S100B, NSE, and creatine kinase (CK) when an athlete received a direct hit to the head47. TBI is also commonly found in active military personnel and veterans. Thus, investigations in identifying a variety of biomarkers will give an indication of the clinical symptoms of

PTSD (post-traumatic stress disorder )48. Traumatic stress exposure is considered a biomarker that would reflect the symptoms of PTSD. The marker would somehow give a precise response to a sustained injury from a specific exposure39. Furthermore, worth noting, is that there are biomarkers called vulnerability and risk markers that facilitate the identification of people who are likely to be susceptible to PTSD. These markers are able to predict the risk even before a traumatic exposure39. These markers in PTSD certainly help give indications for prognosis, diagnosis, treatment planning, and potentially a new approach to the disease48.

TBI Protein Biomarker

In the case of TBI, physical force has an impact on brain tissue by causing cell and subcellular structural injury and compromising membrane integrity. At the same time, some proteins are susceptible to over-activated proteases, resulting in the formation of protein breakdown products and peptides. Thus, this leads to the release of the full-length protein itself and/or protein-BDPs from damaged neurons, subcellular structures (e.g. axons, dendrites) into the interstitial fluid (ISF)/extracellular fluid (ECF), and eventually reaching the circulation by diffusion from the cerebrospinal fluid (CSF) compartment to blood20.

There are examples of the released brain-specific proteins. A novel study showed elevation in serum concentration of S100B, NSE, CK and Ubiquitin C-terminal hydrolase-L1 (UCHL-1) of the athletes during an NFL football game and a boxing match, after receiving a direct hit to the head. Which indicates head injury due to sub- concussive impacts46,49. UCH-L1 is a protein that mainly resides in the neuronal cell body cytoplasm, and was first found to be released in CSF and serum of severe TBI patients27,31,50. The use of CSF UCH-L1 levels appeared to improve clinical outcome predictors of mortality following non-penetrating TBI. A new study also found UCH-L1 is useful in long-term prognosis of severe TBI31. In addition, UCH-L1 was found to be released into serum/plasma samples in mTBI subjects by two independent studies51. It has been suggested that UCH-L1, together with GFAP, form a basis of tandem biomarker representing the two dominant cell types in the brain52,53. Interestingly, serum levels of both UCH-L1 and GFAP also appear elevated in professional breacher trainees who were exposed to repeated explosive discharges54. Puvenna et al. found significant UCH-L1 elevation in serum among athletes after concussions53. UCH-L1 is considered as an index of neuronal cell body injury biomarker. The S100b protein is an astroglial 11 kDa calcium-binding protein. It is perhaps the most investigated brain injury biomarker, as preclinical animal TBI model data shows that S100b has been studied on various levels of TBI severity55–58. S100b is a sensitive biomarker for predicting post- concussive syndrome development among mild TBI patients59–61. S100b can also be released from adipose tissue and cardiac/skeletal muscles, thus its levels are also elevated in orthopedic trauma without head injury27. Despite these confounds, S100b is actually a sensitive biomarker for predicting CT scan abnormality and post-concussive

syndrome development among mild TBI patients37,60,61. Lastly, this subgroup of protein markers appears to be released as intact proteins.

Examples of brain protein breakdown products as TBI biomarkers: Glial fibrillary acidic protein (GFAP) is emerging as the top TBI biomarker. GFAP biomarker levels are elevated after 3 to 34 hours in biofluids such as CSF and serum/plasma following severe TBI55,62,63, and in serum/plasma samples after moderate to mild TBI64. GFAP, in the form of either the GFAP intact protein (50 kDa) or as a breakdown product (GFAP-

BDPs; 44-38 kDa), is predominantly released from injured brain tissue into biofluids such as CSF and serum/plasma shortly following TBI62,65. In parallel with human TBI studies, GFAP elevations in CSF have been identified in various rat models of severe

TBI (control cortical impact, penetrating brain injury blast overpressure wave brain injury)55,66,67 as well as in serum /plasma samples in mild TBI models57,58. There is additional evidence that the post-TBI elevation of GFAP is severely dependent67. Lastly,

GFAP levels are also linked to CT-pathological alterations and patient outcome64,68,69.

Other TBI biomarker candidates like αII-spectrin breakdown products (SBDPs) of 150,

145 and 120 kDa, N-terminal fragment (SNTF) (~140 kDa)70–73, myelin basic protein

(MBP) can be found in CSF fluids74–77. Neurofilament light protein (NF-L) and neurofilament–heavy protein (NF-H; including phospho-NF-H or pNF-H)78–81, are also known to be sensitive to proteases such as calpain79, Tau is also fragmented by calpain and/or caspase, producing distinct BDPs in brain injury82, which is considered to be a

TBI biomarker candidate. CSF Tau levels are also elevated in a rat cortical impact model74,83. In addition, serum Tau was recently reported to be elevated from 1 to 14 days following both single and repeated closed head injury (rCHI) in mice20. Tau is of

particular interest as it, and its phosphorylated forms have been implicated as

Alzheimer’s Disease biomarkers84. In addition, Tau and P-Tau have been linked to chronic traumatic encephalopathy, a chronic neurodegenerative condition believed to be generated by repetitive mTBI24,29. The important feature of this subgroup of biomarkers is that they are subjected to brain injury-induced proteolytic modifications.

Mass Spectrometry Based Peptidomics

Peptidomics is defined as the complete quantitative and qualitative identification of all peptides in a biological sample. It is an emerging sub-branch of proteomics.

Proteomics, which identifies the entire complement of proteins in a biological sample, differs from peptidomics in that proteomics targets complete, intact proteins (50 or more amino acids), while peptidomics aims to identify endogenously produced protein fragments or peptides (2-50 amino acids)85. Although both proteomics and peptidomics use similar techniques for protein analysis, peptidomics allows for a more thorough view of the proteome present within the sample. Peptidomics can identify protein fragments that have undergone some modification, such as enzyme degradation or proteolytic cleavage85. Peptidomics is a rapidly growing field, enabled by modern advanced separation and identification computational technologies86. Recent technologies such as ultra -performance liquid chromatography-mass spectrometry (UPLC-MS) or ultra- performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) have been implemented in various areas of proteomic and peptidomics-based research; which includes new drug discovery, bioinformatics, food protein digestion mapping, identifying hormones and signaling molecules, and identifying peptide biomarkers of disease85,87. These advances in technology allow for quick and accurate identification of peptides present within a sample, offering researchers the ability to identify smaller

peptides that often are difficult to detect using typical proteomic analysis methods88.

Workflow consisting of sample preparation, fractionation (separation) of protein/peptide samples, identification the samples by mass spectrometry, data processing, and validation are similar. Peptidomics offers the ability to detect smaller protein fragments and identify their structure, allowing detection of modifications such as proteolytic cleavages; post-translational modifications; and the presence of different forms of the same peptide62. An average proteomic study can identify typically larger proteins those between 10 to 200 kDa. Nevertheless, it is often the case that the smaller peptide (0.5 -

15 kDa), or a modified version of a protein, is central to the biological process being observed89. Peptidomics, however, in conjunction with mass spectrometry, can serve as an analysis method able to identify the precise forms of a peptide present in a given sample. Peptidomics can also identify the exact amino acid sequences of the peptide, as well as any post-translational modification that resulted from the biochemical process in question, allowing for the detection of both known and novel peptides89. Body fluids such as plasma or cerebrospinal fluid contain higher concentrations of proteins compared to peptides and, therefore, require peptide enrichment methods in order to identify and quantify the peptide fragments that can often be lost behind the larger proteins during identification 89. In relation to TBI, peptidomics has the advantage over proteomics due to the blood-brain barrier. When an injury occurs, the resultant proteins and peptides from the injury are both presents in the brain. Taking samples of CSF allows for the detection of TBI biomarkers. However, CSF samples only contain the substances that are able to cross the blood-brain barrier. Peptides can cross the blood- brain barrier, while larger proteins cannot; making the analysis of the CSF samples with

peptidomics the method of choice for TBI. The use of peptidomics allows for comprehensive analysis of peptide biomarkers and breakdown products of proteins from the brain. Particularly, quantitative peptidomics methods can be used to compare the levels of specific peptides between two samples90.

Table 1-1. Glasgow coma scale showing different cases of TBI. Eye Opening (E) Verbal Response (V) Motor Response (M) Score Total Score

N/A N/A Obeys commands 6 = E+V+M

N/A Oriented Localizes to painful 5 Mild 13-15 stimuli Moderate 9-12 Severe 3-8 Spontaneously Confused Withdraws from pain 4

In response to speech Inappropriate Abnormal flexion 3

In response to Pain Incomprehensible Sounds Abnormal extension 2

Does not open Makes no sounds Makes no 1 movements

Table 1-2. Possible TBI markers (existing biomarkers) and the peptidomic markers derived from this thesis work TBI biomarker Biomarker full name Origin pathobiological Existing evidence Peptidomic candidates mechanisms from others GFAP Glial fibrillary acidic protein Astrogliosis, astroglial injury Intact protein, BDP Peptidomic

S100b Astrocyte calcium-binding Astroglia/BBB damage Intact S100b S100b protein marker

UCH-L1 Ubiquitin C-terminal hydrolase- Neuronal cell body injury Intact protein L1 Tau Tau protein and Axonal Intact protein, BDP Peptidomic phosphorylated Tau injury/Tauopathy/CTE SBDPs αII-Spectrin breakdown Axonal injury; Brain cell αII-Spectrin BDP Peptidomic (SBDP150, product (SBDP150) or αII- Necrosis-Apoptosis Spectrin N-terminal fragment SBDP145, (SNTF) SBDP120, SNTF) MBP Myelin basic protein De-myelination Intact protein, BDP Peptidomic

NSE Neuron-specific enolase Neural Intact protein Peptidomic

NF-L, NF-M, Neurofilament proteins-Heavy Axonal Intact protein Peptidomic NF-H (pNF-H) (NF-H) including phosphorylated NF-H (pNF-H), - medium (NF-M), -light (NF-L)

NRGN Neurogranin Postsynaptic protein Intact protein Peptidomic

VIM Vimentin Intermediate filament (IF) None Peptidomic

CHAPTER 2 EXPERIMENTAL METHODS AND ANALYTICAL DESIGNS

SDS-Gel Electrophoresis Combined with Immunoblotting

Western blot is a powerful technique used in molecular and cell biology91 and is considered a gold standard for the separation and identification of mixtures of specific proteins with different molecular weights92. These proteins are extracted from cells, animal tissues, or human cerebrospinal fluid (CSF) based on its ability to bind to specific antibodies. This analytical technique is divided into different stages described below.

Sample Preparation for Electrophoresis

For the purpose of my research, the sample is a purified protein, lysis of cell culture or tissue, or human CSF. Protein is extracted from the cells or animal tissue using lysis buffer, followed by the use of either Bio-Rad DC Protein Assay Reagents A,

B, and S (Bio-Rad, Hercules, CA, USA), or Pierce 660 nm Protein Assay Reagent

(Thermo Scientific, Waltham, MA, USA). The protein concentration of the lysate (cell culture or tissue) is determined in order to perform even loading. To separate the mixture of proteins that are present in the lysate and CSF samples, western blot utilizes polyacrylamide gel electrophoresis (PAGE)91,93. It is difficult for the protein to travel through the gel while maintaining its tertiary structure, so the proteins should be denatured to their corresponding primary structure. This is done by adding the Laemmli loading buffer. It contains sodium dodecyl sulfate (SDS), 2-mercaptoethanol, glycerol, bromophenol blue, and Tris-HCl. SDS is a strong detergent which denatures the tertiary protein structure by disrupting the bonds (hydrogen, disulfide) which lead to the formation of the primary protein’s structure. Moreover, the SDS coats the protein with a uniform negative charge, which veils the characteristic charges on the R-groups. SDS

binds quite consistently to the linear proteins (around 1.4g SDS/ 1g protein), meaning that the charge of the protein is now approximately proportional to its molecular weight, which aids the protein to travel through the gel94. The reducing agent 2-mercaptoethanol reduces the disulfide bonds and prevents oxidation of cysteine amino acid present in the sample, forcing the protein to travel to higher molecular weight. Glycerol is denser than water and makes the sample go to the bottom of the gel wells instead of flowing out and mixing with the buffer in the upper reservoir. This increase in density and viscosity of the samples resulting in easier loading into the wells of the gel.

Bromophenol blue is a small anionic dye that aids in the visualization of the migration of the proteins through the gel.

Gel Electrophoresis

Electrophoresis is an analytical technique used for the separation of DNA, RNA or proteins in a mixture, under the influence of an applied electric field, according to molecular size. The protein samples, which contain an overall negative charge, travel through the small pores of gel to the positive node. The speed at which each soluble protein travels is related to its molecular weight, and inversely proportional to the lengths95.The smaller the MW, the faster the migration through the gel. The electrophoretic separation of proteins in polyacrylamide gels refers to sodium dodecyl sulfate polyacrylamide gel electrophoresis or SDS-PAGE. Based on their protein concentration assay, equal amounts of protein samples are loaded into the wells of the gel to ensure even loading. Along with the samples, full and low range molecular weight marker (225 kDa to 12 kDa full-range rainbow, RPN800E, and 38 kDa to 3.5 kDa low- range rainbow, RPN755E, from GE Healthcare, Bio-Sciences, Pittsburgh, PA, USA) is loaded in order to visualize the position of each protein upon completion. As these

markers pass through the gel, colored bands are left in the gel to signify specific kDa values. Using these markers, the molecular weight of the protein of interest can be approximated. The polyacrylamide gels, Novex 18% tris-glycine gel (1.00 mm X 10 well) or Novex Wedge Well 10-20% tris-glycine gel (1.00 mm X 12 well), are specifically used because they cover the range of molecular weights (6-200 kDa). The gel is placed in a buffer electrophoresis unit containing a tris-glycine buffer (25 mM Tris (2-Amino-2-

(hydroxymethyl) propane-1,3-diol), 192 mM glycine, 0.1% SDS). The gel is placed inside the unit with the negative electrode closer to the wells, and the positive electrode towards the bottom of the tank. The process of gel electrophoresis involves the power supply (Bio-Rad Power Pac 200), set at 200V, sending two amps of current through the gel for 60 minutes, which induces an electric field on the samples. The samples, which contain an overall negative charge, begin to migrate downwards to the positive node

(Figure 2-1).

Coomassie Brilliant Blue Staining of Gels

In order to visualize the bands on the PAGE gel, Coomassie Brilliant Blue (CBB)

R-250 staining solution (0.1% CBB R250, 10% acetic acid, 40% methanol in Milli-Q water) is added to the gel for an hour. It then follows a destaining step of the gel for 3 to

5 minutes with a destaining solution (10% acetic acid, 40% methanol in Milli-Q water), until protein bands are well visible.

Electrotransfer

In order to analyze the proteins that were separated by gel electrophoresis, they are transferred from the gel to a solid structure like polyvinylidene fluoride (PVDF)96.

The gel was placed in deionized water for five minutes, then placed in the transfer buffer

(25 mM Tris, 192 mM glycine, 20% methanol) for an additional five minutes to prepare

for transfer to the Invitrogen iBlot Dry Blotting system (Carlsbad, CA, USA). The transferring buffer aids in the electric field passing through the gel and PVDF, cause the proteins to transfer across the membrane. The transferring process uses the program

P3 (20 V for seven minutes) on the iBlot machine. It is significant not to allow the PVDF membrane to dry throughout (Figure 2-1).

Immunoblotting-Antibody Incubation and Detection

Blocking is important to prevent antibodies from binding to nonspecific areas on the membrane. Non-fat dried commercial milk powder (5%) was dissolved in tris- buffered saline tween (TBST) (20 mM Tris pH 7.5,15 mM NaCl 0.02 % Tween 20)97.

The PVDF was removed from the iBlot blotting system and placed in this blocking solution for thirty minutes. Depending on the proteins of interest, specific primary antibodies were mixed with 5% milk in a ratio of 1:500 or 1:1000 (Anti-Neurogranin or

Anti-Vimentin). This solution replaced the blotting solution and was left overnight at 4

°C. Before replacing the primary with the secondary antibody solution the following day, the membrane was washed three times with TBST to remove any unbound primary antibody. The secondary antibody stems from the species in which the primary antibody originated, from94 for example, Goat Anti-Rabbit IgG AP Conjugate or Goat Anti-Mouse

IgG AP Conjugate (H + L). After two hours of incubation in the secondary antibody solution, the PVDF was washed three times with TBST to remove all unbound secondary antibodies. Immunoreactive bands were then observed by adding 5-bromo-

4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) followed by alkaline phosphatase AP, whereupon visible purple precipitation (indigo dye) appears.

The PVDF was removed from the developer, washed with Milli-Q water three times and allowed to air dry98. The dried membrane was scanned with Expression 8836XL

(Epson) and the UN-SCAN-IT software (version 6.1, Silk Scientific Corporation).

Quantitative evaluation of the protein levels was performed with the computer-based densitometric NIH Image J (version 1.6) software (Figure 2-1).

Ultrafiltration Method

This is a novel analytical technique that aids in filtering proteins and peptides that are smaller than or equal to the molecular weight cut-off unit, which ranges from 3 kDa to 100 kDa. For my research purpose, the peptides of interest have a molecular weight less than 10 kDa, therefore 10 kDa molecular weight cut-off (MWCO) membrane filters were used (Sartorius Stedim Biotech, Gottingen, Germany, VS0102);. A sample of 250

μL was loaded into the ultrafiltration system and centrifuged at 4 °C for 5-20 min, 5000 x g, the 200 μL of the retentate was subjected to separation and identification through western blot analysis, while the 50 μL filtrate was concentrated using speed vacuum

(Thermo Scientific) to a volume of 5 µL. The samples were reconstituted with 5 µL

LCMS water with 0.1% formic acid and were analyzed using NanoAcquity UPLC system

(Waters, Milford, MA) coupled with LTQ XL (Thermo Scientific), or Ultimate 3000;

(Thermo Scientific) interfaced with a hybrid ion trap–Orbitrap high-resolution tandem mass spectrometer (Velos-Pro; Thermo Scientific) (Figure 2-2).

Mass Spectrometry

Reversed-Phase Liquid Chromatography Tandem Mass Spectrometry

Separation of proteins was performed on NanoAcquity UPLC (Waters, Milford,

MA) by reversed phase chromatography. 2 μL of each sample was loaded onto a nanoACQUITY UPLC symmetry C18 trap column, 100 Å, 5 µm, 180 µm x 20 mm followed by separation on an Acquity UPLC BEH (Ethylene Bridged Hybrid) C18 column, 130 Å, 1.7 µm, 100 μm X 100 mm. The mobile phase consisted of solvent A

(water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid)

(Honeywell, Muskegon, MI). Separation was achieved within a run time of 115 min at a flow rate of 300 nL/min. The first linear gradient was from 1% to 40% B over 90 min, the second linear gradient was from 40% to 100% B over 5 min and held for 5 min before returning to initial mobile-phase composition (1% B). Tandem mass spectra were collected on LTQ-XL (Thermo, San Jose, CA, USA) (Figure 2-3) using a data- dependent acquisition method in Xcalibur 4.0 (Thermo). The analysis was set up for a full scan recorded between m/z 200 – 2000, and an MS/MS scan to generate product ion spectra to determine amino acid sequence in consecutive instrument scans of the ten most abundant peaks in the spectrum MS (scan event 1) with dynamic exclusion enabled. Dynamic exclusion temporarily puts a mass into an exclusion list after its

MS/MS spectrum is acquired, providing the opportunity to collect MS/MS information on the second most intense ions from the full-scan spectrum MS (scan event 1). All MS/MS spectra were analyzed using Proteome Discoverer 2.1 (Thermo), SEQUEST HT

(version: 2.1.1.21). Database search engines were set up to search with no enzyme (for calpain-1) and with caspase enzyme (for caspase-3 and -6), Homo sapiens .fasta file

(92,271 sequences and 36,831,723 residues) and Mus musculus (35,126 sequences and 17,948,970 residues) version 2016_07. The search was achieved using the average mass for matching the precursor with a fragment ion mass tolerance of 0.8 Da and a parent ion tolerance of 0.8 Da. Carbamidomethylation of cysteine was selected as a static modification, while the oxidation of methionine was selected as a dynamic modification, using the output from SEQUEST HT.

Velos Pro Dual-Pressure Linear Ion Trap Mass Spectrometer

Velos pro Orbitrap is a hybrid mass spectrometer consisting of a linear ion trap

(LTQ) and an Orbitrap mass analyzer. The LTQ can store selected masses or mass ranges, isolate ions, and fragment ions that are necessary for MS/MS and MSn experiments, and then send them either to the Orbitrap for further high-resolution mass spectra acquisition or to a secondary electron multiplier (SEM) detector. The linear ion trap is a unique ion preparation and injection system for Orbitrap MS because it has a greate ion storage capacity (Figure 2-4).

Protein sample separation was performed on rapid separation liquid chromatography (RSLC) – tandem MS (RSLC-MS/MS) with a nanoflow liquid chromatography system (Ultimate3000; Thermo Scientific) interfaced with a hybrid ion trap Orbitrap high-resolution tandem mass spectrometer (Velos-Pro; Thermo Scientific) operated in data-dependent acquisition (DDA) mode. Briefly, 1 μL of each sample was injected onto a capillary column (C18 Onyx Monolithic, 0.10 × 150 mm Phenomenex) at a flow rate of 300 nl/ min. The electrospray was set at 1.2 kV using a dynamic nanospray probe with fused silica non-coated emitters (20 μM ID with 10 μM ID tip

PicoTip Emitter from New Objective). Chromatographic separation was carried out using a 90 min linear gradient (Mobile Phase A: 0.1% formic acid in MS-grade water, mobile phase B: 0.1% formic acid in MS-grade acetonitrile), from 3% B to 35% B over

60 min, then increasing to 95%B over 5 min. MS/MS spectra were acquired at 30 000 resolution using both collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) using data-dependent top 15 (ddMS2-top15).

Figure 2-1. Schematic diagram for SDS-PAGE-Immunoblotting (George Anis Sarkis).

Figure 2-2. Novel ultrafiltration-based TBI proteolytic peptidome enrichment, coupled to LC-MS for separation and identification of peptide ID.

Figure 2-3. Schematic diagram for LTQ XL (Thermo Fisher Scientific).

Figure 2-4. Schematic diagram for LTQ-Velos Pro-Orbitrap (Thermo Fisher Scientific).

CHAPTER 3 IN VITRO DIGESTION OF PURIFIED HUMAN BRAIN PROTEINS AND NAÏVE MOUSE BRAIN LYSATE

The Biomarker Neurogranin

Neurogranin (NRGN) is a neural protein that participates in synaptic signaling between axon terminals of the brain. The signaling that takes place requires the use of calcium to transmit neurotransmitters between the synaptic terminals and involves calmodulin (CaM)99 and protein kinase C (PKC) pathway100.

Originally discovered in rat brain101, neurogranin has been found to be abundant in areas of the brain, such as the telencephalon, which include sections of the hippocampus and cerebral cortex. More specifically, NRGN is concentrated in the perikarya and dendrites of neurons in these areas of the brain101. It is a small protein consisting of only 78 amino acids (7.61 kDa).

NRGN regulates the continuous flux of Ca2+ present in the synaptic cleft.

Calcium, in turn, is extremely important because it aids in neuron electric signaling102.

When calcium is needed, NRGN binds to calmodulin (CaM), which in turn begins a protein kinase C pathway. Increased complexes of neurogranin and calmodulin translate into an increase in calcium ions available to flow through the synaptic cleft.

Without changes in calcium ion concentration, exchange of electrical impulses and signals would not be able to take place. It shows the complex and elaborate ways that the body regulates needed cellular activity.

Calmodulin is a major factor in this flux of calcium concentrations as well. CaM is a calcium binding protein that becomes activated when bound to a protein like NRGN.

The complex that is formed is responsible for the increase in Ca2+ concentration in the

axons of the brain. However, it has been found that the binding between NRGN and

CaM can be inhibited if NRGN becomes phosphorylated by the protein kinase C. The sites for phosphorylation of NRGN by PKC were found to be within the binding site of

CaM. This would confirm the idea that only one can be bound to NRGN, either PKC or

CaM101. Serine-36 of NRGN is an important amino acid because it is here where NRGN becomes phosphorylated, and in turn, inhibited from binding to CaM103. The location of

PKC is usually near the membranes of the cells. This is similar to the location of NRGN, which is mostly located near the postsynaptic membranes. This close proximity increases the binding of the enzyme-substrate complex. One possible reason for NRGN being located near the membrane is because of its affinity to phosphatidic acid (PA).

This acid is generated near the postsynaptic membrane and thus might lead to a buildup of NRGN in these synapses. Another possible reason could stem from how diacylglycerol (DAG) can be metabolically transformed to the PA, which leads to an increase in NRGN concentration103.

Not only does the phosphorylation of NRGN alter the flux of Ca2+ concentrations between the neurons, it also regulates the activation of other kinases. Reduced amounts of CaM result in weakened activations of the CaM-dependent kinase II pathway, along with targets such as extracellular signal–regulated kinase (ERK) and the cAMP response element-binding protein (CREB). All of which affect learning and memory within the brain.

The neurodegenerative Alzheimer’s disease is linked to increased concentrations of certain proteins like neurogranin and the Tau protein104. It has been shown that levels of neurogranin are higher in cerebral spinal fluid in patients with Alzheimer’s than

patients without the disease105. It was also found that patients who demonstrated Mild

Cognitive Impairment (MCI) showed above-normal levels of this protein106. Peptidomic analysis demonstrated the presence of altered NRGN peptide structures in CSF samples taken from Schizophrenic patients107.This altered neurogranin could be useful as biomarkers for the early detection of the disease.

The Biomarker Vimentin

Vimentin is one of the major intermediate filament proteins that encode for the constituents of the cytoskeleton of a cell108. The vimentin protein creates the scaffolding necessary for the cell or tissue development, and thus vimentin could also be a useful marker for cell and tissue maturation.

Vimentin (466 amino acids,53.6 kDa) is present in human cells, and those of other species. It is found in the human brain in the regions such as the cerebellum, hippocampus, and the entorhinal cortex109, and is involved, inter alia, in cellular signaling.

Vimentin is a significant structural component of the CNS system and the removal of the protein can affect the integrity and stability of the neuronal cells110. Mice without vimentin showed many phenotypic alterations compared to mice with the protein111. The concentration of vimentin was found to increase in mechanically induced brain damage in mice109. Moreover, vimentin acts as a reliable indicator of the presence of astrocyte reactions as the protein is overexpressed by astrocytes following a TBI112.

Sample Preparation

In my research, I used the proteases calpain-1, caspase-3 and -6. These proteases are linked to proteolysis and neuronal degeneration observed in TBI patients, where the cytosolic protease calpain has cleaved a number of brain proteins. This

repeated degradation results in the sequential protein breakdown products (PBDPs) seen in human CSF samples from TBI patients. Animal models of TBI exhibit similar behavior. However, the exact mechanism of the proteolysis observed is still not fully understood. For calpain-1 the cleavage site, or sites, are not known, nor are the correct amino acid sequences of the protein breakdown products. My goal in using in vitro digestion with calpain/caspase was to mimic proteolysis of proteins that take place in the brain upon TBI, by analyzing PBDPs and proteolytic peptides by western blot and mass spectrometry techniques. Control or digested samples of brain lysate were subjected to LMW cut-off centrifugal ultrafiltration (10 kDa MWCO). The retentate was analyzed using the western blot technique, while the filtrate was evaluated using ultra- performance liquid chromatography tandem mass spectrometry.

Naïve Mouse Brain Lysate Preparation

Three to four-month-old male C57BL/6J naïve animals were euthanized with a lethal dose of pentobarbital. The whole brain was removed and flash-frozen using liquid nitrogen without any surgery. All of the collected brains were then stored at -80 °C until further use. The frozen brain samples were homogenized to a fine powder using mortar and pestle, settled in a dry ice bath. The powdered brain samples were lysed with 1%

Triton X-100 lysis buffer containing Tris-HCl, (pH 7.4), 5 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM dithiothreitol (DTT). The brain samples were allowed to lyse for

90-120 minutes at 4 °C. Following the lysis, the samples were centrifuged to remove any debris, and protein assay was performed using a Bio-Rad Protein Assay Kit to determine protein concentration in each sample for even loading in western blot.

In Vitro Digestion of Purified Protein-Naïve Mouse Brain Lysate

In order to mimic the proteolytic event after TBI, in vitro protease digestion of 4

μg purified neuro-recombinant human neurogranin protein as His Tag N-Terminus fusion protein, approx. 10 kDa (Abcam ab181939, UK) and 5 μg purified recombinant human vimentin protein Tag-free fusion protein, approx. 54.5 kDa (Origene TP723475,

USA), was performed using the purified protease calpain-1 (Millipore, 2.58 μg/μL) in a buffer containing 100 mM Tris/HCL (pH 7.4), 20 mM DTT (at protease to the substrate ratio of 1:50 and 1:25). Due to calpain-1 being a calcium-activated enzyme, 10 mM

CaCl2 was added to these samples, which were then incubated at room temperature for an hour.

The same amounts of 4 μg neurogranin and 5 μg vimentin proteins were each digested separately with human recombinant caspase-3 (BD Pharmingen, San Jose,

CA, USA) or recombinant caspase-6 (Enzo, Farmingdale, NY, USA), under the same conditions as calpain-1 but without addition of CaCl2, (at a protease to substrate ratio of

1:50), then incubated at room temperature for four hours. The protease reaction was stopped by the addition of 25 µM calpain inhibitor SNJ-1945 (a gift from Senju

Pharmaceuticals, Kobe, Japan) or 50 µM pan-caspase inhibitor Z-D-DCB (R&D,

Minneapolis, MN, USA). Similarly, 10 µg of naïve brain lysate was digested using calpain-1/caspase-3, 6 with the same methods used for purified protein.

Analysis of Digested Samples

Neurogranin

Digested purified protein samples were separated using SDS-PAGE and identified by staining the gel with Coomassie Brilliant Blue (CBB) R-250 using the staining procedure113. Confirmation experiments were performed by staining the PVDF

membranes for five minutes with CBB R-250, then destained to allow the visualization of the separated bands. The digested samples were mixed with an 8X sample buffer in the ratio 1:1 and directly probed using western blot by different antibodies (Table 3-1).

The digested solution was concentrated using speed vacuum to 5 µL, reconstituted with

5 µL LCMS water-0.1% formic acid, then loaded to RP-nLC MS/MS for separation and identification in order to accurately identify the peptide fragments produced by this digestion, as well as their exact amino acid sequence. A list of peptides generated by proteolysis of NRGN using calpain-1 and caspase-6 were extracted from Proteome

Discover 2.1. (Thermo). No data were recovered from the proteolysis experiments using caspase-3, perhaps because NRGN not a substrate for caspase-3.

Vimentin

Samples of purified protein digested and separated using SDS-PAGE were visualized by staining the PVDF membrane with Coomassie Brilliant Blue staining.

Digested samples were mixed with an 8X sample buffer in the ratio 1:1 and directly probed using western blot anti-vimentin (ab8978, Abcam, USA). Further digested protein samples were subjected to ultrafiltration (10 kDa) and separated by RP-nLC

MS/MS.

Naïve Mouse Brain Lysate

Equivalent amounts of digested brain lysate samples were mixed with an 8X sample buffer in the ratio 1:1 and separated using SDS-PAGE then probed with neurogranin antibodies. A 50 µL aliquot digested brain lysate samples was ultra-filtered using 10 kDa MWCO, 20 µL ultra-filtrate was concentrated to 5 µL and reconstituted in

LCMS water-0.1% formic acid to 10 μL and loaded into the RP-nLC MS/MS for separation and identification. The acquired data were processed using Proteome

Discoverer 2.1, SEQUEST HT version 2.1.1.21 (Thermo). Database search engines were set up to search with no enzyme (for calpain-1) and with caspase enzyme (for caspase-3 and -6), Homo sapiens neurogranin .fasta file (1 sequence and 78 residues), for the purified protein sample, while for the brain lysate, Mus musculus (1 sequence and 78 residues) version 2016_07 were used.

Results and Discussion

Purified Neurogranin Protein

Proteolytic peptides are considered as promising biomarkers for TBI. From calpain-1 digestion experiments, it was observed that the amount (as measured by the color intensity of the indigo dyes precipitated on the western blot PVDF membrane at the position of the band due to the relevant protein) of the intact NRGN protein

(apparent molecular weight on gel 15 kDa, calculated molecular weight 7.61 kDa) diminished over time, and the amount of breakdown product (apparent molecular weight on gel 9 kDa) increased. This was identified using Coomassie Brilliant Blue staining of

SDS-PAGE-gel for the digested NRGN samples. Caspase-3 digestion showed no significant effect on NRGN (Figure 3-1A). The gel staining results were confirmed using densitometric quantification (Figure 3-1B). NRGN and its BDPs were investigated using

SDS-PAGE western blot and probed using anti-neurogranin (EMD AB5620). NRGN exhibited the same BDP (9 kDa) with the calpain-1 and caspase-6 proteases. Caspase-

3 generated an insignificant amount of the 9 kDa BDP (Figure 3-2A). Densitometry showed elevated levels of the BDPs produced by the action of calpain-1 and caspase-6

(Figure 3-2C).

From the mass spectrometry analysis, it was demonstrated that the undigested purified NRGN protein shows no reasonable peptide or minor peptides relating to

protein turnover. The proteolytic peptides sequences of the digested protein with calpain-1 are listed in Table 3-2. The schematic diagram shown in Figure 3-3 shows the two main fragments resulting from calpain-1 cleavage, a representative MS/MS spectrum for the N-terminal peptide PGANAAAAKIQA is shown in Figure 3-4, and the

C-terminal peptide KSGERGRKGPGPGGPG is shown in Figure 3-5.

The larger the number of matches between the m/z values given by the MS/MS measurements and the m/z values for a particular peptide in the database, the greater the robustness of the identification.

From the schematic diagram (Figure 3-3), the mass spectra of the N-terminal peptide and that of the C- terminal peptide (Figures 3-4, 3-5), it appears that the two main fragments resulting from calpain-1 cleavages are between amino acid residues

24-42 and 47-64, the sequence from 24 to 64 has MW equivalent to 4 kDa. This supports the immunoblotting analysis that gave a value of 9 kDa BDP (real molecular weight of peptide is half the apparent value on western).

From the diagram shown in Figure 3-6, it appears that the cleavage sites for caspase-6 are between amino acid residues 1-22, supporting the hypothesis of caspase cleavage sites as cysteine class, which cleave after aspartate, glutamate and phosphoserine residues114. A representative MS/MS spectrum for the peptide generated from caspase-6 cleavage is shown in Figure 3-7.

Purified Vimentin Protein

Calpain-1 digestion for vimentin purified protein showed a 35 kDa BDP, while caspase-3 showed two BDPs (40, 31 kDa) and caspase-6 showed three BDPs (40, 31 and a minor one at 24 kDa). These observations were made for calpain-1 digestion using Coomassie Brilliant Blue staining of the PVDF membrane loaded and separated

with digested VIM samples (Figure 3-8A). In addition, the digested samples were separated with SDS-PAGE-western blot and probed using anti-vimentin (Abcam

EPR3776), along with the caspase-3 and -6 digestions (Figure 3-8B).

Mass spectrometry confirmed that the undigested purified VIM protein shows no acceptable peptide except for a few peptides that resulted from protein degradation.

The proteolytic peptide sequences of the digested VIM protein with calpain-1 are shown in Figure 3-9. Representative MS/MS spectra for the N- and C-terminal peptides derived from calpain-1 digestion are shown in Figures 3-10 and 3-11, respectively. Figure 3-12 shows the proteolytic peptide sequences of the digested VIM protein with caspase-3. A representative MS/MS spectrum for the peptide generated from caspase-3 digestion is shown in Figure 3-13.

Naïve Mouse Brain Lysate

Neurogranin

For further confirmation of the NRGN-BDP with calpain-1, the endogenously digested mouse brain lysate was separated using SDS-PAGE western blot and was consequently probed using anti-neurogranin (EMD AB5620). NRGN-BDP (9 kDa) was seen with the calpain-1 only, but it was hardly observed with caspase-3, or -6 (Figure 3-

14A). Quantification indicated the elevation BDP of calpain-1 (Figure 3-14B).

Proteolytic peptide sequences of the digested mouse brain with calpain-1 and caspase-3 are shown in Figures 3-15 and 3-18, respectively, showing the two major cleavage sites of calpain-1 and the peptide which resulted from the caspase-3 cleavage of the NRGN protein. The MS/MS spectrum of the N-terminal peptide DPGANAAAAKIQ

(amino acid residues 25-41), charge +2, m/z 563.85 and that of C-terminal peptide

KGPGPGGPGGAGGARGGA (amino acid residues 57-74), charge +3, m/z 460.00

resulted from digestion of the mouse brain with calpain-1 (Figure 3-16). The MS/MS spectrum generated from caspase-3 proteolysis is

CGRKGPGPGGPGGAGGARGGAGGGPSGD (Figure 3-19).

Vimentin

A schematic representation of the two regions generated from in vitro calpain-1 digestion for the mouse brain lysate is shown in Figure 3-20. The MS/MS spectrum of the proteolytic N-terminal peptide sequence that ensued from the digested mouse brain with calpain-1 is given in Figure 3-21; the fragmentation pattern of this peptide is clear.

Figure 3-22 shows the proteolytic peptide sequences for in vitro proteolysis of mouse brain lysate using caspase-3. Figure 3-23 shows a representative MS/MS spectrum for the peptide generated from caspase-3 digestion.

Conclusions

Purified human protein and mouse brain digestion with calpain-1 was used as a template to mimic the proteolysis after TBI. It could be concluded from MS studies of the calpain-1 digestion of human purified protein and mouse brain lysate that the cleavage sites of NRGN start at amino acid residue 23 or 24 and end at 64 or 65. A consideration of the m/z values for the peptides from the NRGN digestion leads to an average mass of the core peptides of approximately 4 kDa, which supports the value 9 kDa obtained from the western blot, taking into account the 9 kDa is an apparent molecular weight.

This suggests that the NRGN peptides, which I observed, might also be found circulating in the human biofluid, e.g. CSF, after brain trauma and could be effective TBI biomarker, this will be discussed in Chapter 6.

From MS results for VIM, the calpain-1 digestion cleaves the protein and leads to two terminal clusters of peptides. The N-terminal cluster approximately starts from

amino acid residue 1 and ends at 87. The C-terminal cluster starts from approximately

405 to the end of the protein sequence. This conclusion is supported by western blot experiments, which showed a 35 kDa band. As with NRGN, the m/z values for the peptides from the VIM digestion leads to an average mass of the core peptides of approximately 37 kDa, which supports the value approximately 35 kDa obtained from the western blot.

From my data, the caspase cleavage sites of NRGN cannot be clearly established. It is possible that NRGN is not a major natural substrate for caspases.

However, the caspases cleave the VIM between amino acid residues 150 and 380.

Table 3-1. Neurogranin (NRGN) antibodies used Neurogranin Antigen Epitope Mouse Mab, Rabbit pAb Vendor (Catalog No.) Lot#

Recombinant full-length NRGN Rabbit pAb EMD AB5620 2760630 (rat)

Recombinant full-length NRGN Rabbit pAb Abcam ab23570 GR291420-1 (rat)

(C-terminal) 66-78 of rat NRGN Rabbit pAb EMD 07-425 2689690 VARGGAGGGPSGD (Upstate 07425)

Phospho-NRGN (at Ser-36) in Rabbit pAb EMD- 07430 2475672 CaM-IQ binding motif (32-47) KIQASFRGHMARKKI

Internal Rabbit pAb Abcam GR251202-3 45-55 KIKSGERGRKG (ab99269)

N-terminal 17-27 Rabbit pAb UF (homemade) DIPLDDPGAN KW1

Internal 53-62 Rabbit pAb UF (homemade) RKGPGPGGPG KW2

Table 3-2. List of peptides extracted from Proteome Discoverer 2.1 resulting from digestion of NRGN using calpain-1 Sample Starting Ending Sequence Theoretical Theoretical Observed XCorr Confidence #charge AA # AA # MH+ [Da] m/z m/z Sequest Sequest HT HT NG+Calp 24 34 PGANAAAAKIQ 1011.56 506.58 507.15 3.26 High 2 (S1) NG+Calp 24 34 PGANAAAAKIQ 1011.56 506.58 507.15 3.26 High 2 (S2) NG+Calp 24 35 PGANAAAAKIQA 1083.32 542.11 542.16 3.78 High 2 (S3) NG+Calp 24 35 PGANAAAAKIQA 1082.60 542.11 542.16 4.02 High 2 (S4) NG+Calp 24 35 PGANAAAAKIQA 1082.60 542.11 542.04 4.02 High 2 (S5) NG+Calp 30 42 AAKIQASFRGHMA 1387.08 463.54 463.02 2.15 High 3 (S5) NG+Calp 31 42 AKIQASFRGHMA 1317.86 439.56 439.87 2.44 High 3 (S4 and 5) NG+Calp KSGERGRKGPGPGG 47 64 1623.23 541.28 541.74 2.38 High 3 (S6) PGGA NG+Calp KSGERGRKGPGPGG 47 64 1623.23 541.28 541.74 1.18 High 3 (S7) PGGA NG+Calp KSGERGRKGPGPGG 47 62 1493.79 747.40 747.38 1.99 High 2 (S6 and 7) PG S is number of replicates (N=7)

Figure 3-1. Digestion of purified NRGN protein with calpain-1 and caspase-3. A) Coomassie Brilliant Blue staining of the gel of the digested and separated sample using SDS-PAGE. B) Densitometric quantification of the intact and breakdown products of NRGN protein using image J and prism GraphPad software for the gel. Error bars represent the standard deviation of the mean (N=3).

Figure 3-2. Purified NRGN in vitro digestion with calpain-1, caspase-3 and -6. A) Immunoblotting analysis using mouse monoclonal (EMD-AB5620) (1:500). B) Immunoblotting analysis using rabbit polyclonal KW-1 (1:50). C) Densitometric quantification of the intact and breakdown products of NRGN protein for blot A. Error bars represent the standard deviation of the mean (N=3).

Figure 3-3. Schematic representation for the list of NRGN peptides generated from in vitro digestion of purified NRGN protein using calpain-1 protease (N=7). The red bars led to the conclusion that the regions of NRGN which were proteolysed by calpain-1, are the sequences shown in red.

Figure 3-4. MS/MS spectrum for the N-terminal peptide PGANAAAAKIQA (m/z 542.16) released from the digested human purified NRGN protein using calpain-1. A) MS/MS spectrum for the NRGN peptide PGANAAAAKIQA (amino acid residues 24-35), charge +2, monoisotopic m/z 542.16, displaying the fragment ions for this peptide. B) Identified b+ and y+ type ions for the NRGN peptide shown in red and blue identified from the database search results.

Figure 3-5. MS/MS spectrum for the C-terminal peptide KSGERGRKGPGPGGPG (m/z 747.38) released from the digested human purified NRGN protein using calpain-1. A) MS/MS spectrum for the NRGN peptide KSGERGRKGPGPGGPG (amino acid residues 47-62), charge +2, monoisotopic m/z 747.38, displaying the fragment ions for this peptide. B) Identified b+ and y+ type ions for the NRGN peptide shown in red and blue identified from the database search results.

Figure 3-6. Schematic representation for the list of NRGN peptides generated from in vitro digestion of purified NRGN protein using caspase-6 protease (N=5). The red bars led to the conclusion that the regions of NRGN which were proteolysed by caspase-6, are the sequences shown in red.

Figure 3-7. MS/MS spectrum for the peptide MDCCTE (m/z 408.22) released from the digested human purified NRGN protein using caspase-6. A) MS/MS spectrum for the NRGN peptide MDCCTE (amino acid residues 1-6), charge +2, monoisotopic m/z 408.22, displaying the fragment ions for this peptide. B) Identified b+ and y+ type ions for the NRGN peptide shown in red and blue identified from the database search results.

Figure 3-8. Digestion of VIM purified protein with calpain-1, caspase-3 and -6. A) Coomassie Brilliant Blue staining of the PVDF membrane of the digested and separated sample using SDS-PAGE. B) Western blot of digested purified VIM protein probed with anti-vimentin to display VIM breakdown products.

Figure 3-9. Schematic representation for the list of VIM peptides generated from in vitro digestion of purified VIM protein using calpain-1 protease, filtered using 10 kDa MWCO (N=3). The red bars led to the conclusion that the regions of VIM which were proteolysed by calpain-1, are the sequences shown in red.

Figure 3-10. MS/MS spectrum for the N-terminal peptide MSTRSVSSSSYRRMFGGP (m/z 677.02) released from the digested human purified VIM protein using calpain-1. A) MS/MS spectrum for the VIM peptide MSTRSVSSSSYRRMFGGP (amino acid residues 1-18), charge +3, monoisotopic m/z 677.02, displaying the fragment ions for this peptide. B) Identified b+ and y+ type ions for the VIM peptide shown in red and blue identified from the database search results.

Figure 3-11. MS/MS spectrum for the C-terminal peptide DGQVINETSQHHDD (m/z 798.05) released from the digested human purified VIM protein using calpain- 1. A) MS/MS spectrum for the VIM peptide DGQVINETSQHHDD (amino acid residues 451-464), charge +2, monoisotopic m/z 798.05, displaying the fragment ions for this peptide. B) Identified b+ and y+ type ions for the VIM peptide shown in red and blue identified from the database search results.

Figure 3-12. Schematic representation for the list of VIM peptides generated from in vitro digestion of purified VIM protein using caspase-3 protease, filtered using 10 kDa MWCO (N=3). The red bars led to the conclusion that the regions of VIM which were proteolysed by calpain-1, are the sequences shown in red.

Figure 3-13. MS/MS spectrum for the peptide TIGRLQDEIQNMKE (m/z 564.58) released from the digested human purified VIM protein using caspase-3. A) MS/MS spectrum for the VIM peptide TIGRLQDEIQNMKE (amino acid residues 361-374), charge +3, monoisotopic m/z 564.58, displaying the fragment ions for this peptide. B) Identified b+ and y+ type ions for the VIM peptide shown in red and blue identified from the database search results.

Figure 3-14. In vitro digestion of mouse brain lysate using calpain-1, caspase-3 and -6. A) Western blot displaying NG- BDP at 9 kDa, probed with anti-neurogranin B) Densitometric quantification of the intact and breakdown product of NRGN protein. Error bars represent the standard deviation of the mean (N=3).

Figure 3-15. Schematic representation for the list of peptides of NRGN protein generated from in vitro digestion of mouse brain lysate using calpain-1 protease, filtered using 10 kDa MWCO (N=5). The red bars led to the conclusion that the regions of NRGN which were proteolysed by calpain-1, are the sequences shown in red.

Figure 3-16. MS/MS spectrum for the N-terminal peptide DPGANAAAAKIQ (m/z 563.85) released from the mouse brain digested with calpain-1. A) MS/MS spectrum for the NRGN peptide DPGANAAAAKIQ (amino acid residues 25- 41), charge +2, monoisotopic m/z 563.85, displaying the fragment ions for this peptide. B) Identified b+ and y+ type ions for the NRGN peptide shown in red and blue identified from the database search results.

Figure 3-17. MS/MS spectrum for the C-terminal peptide KGPGPGGPGGAGGARGGA (m/z 460.00) released from the mouse brain digested with calpain-1. A) MS/MS spectrum for the NRGN peptide KGPGPGGPGGAGGARGGA (amino acid residues 57-74), charge +3, monoisotopic m/z 460.00, displaying the fragment ions for this peptide. B) Identified b+ and y+ type ions for the NRGN peptide shown in red and blue identified from the database search results.

Figure 3-18. Schematic representation for the list of peptides of NRGN protein generated from in vitro digestion of mouse brain lysate using caspase-3 protease, filtered using 10 kDa MWCO (N=5).The red bars led to the conclusion that the regions of NRGN which were proteolysed by caspase-3, are the sequences shown in red.

Figure 3-19. MS/MS spectrum for the peptide CGRKGPGPGGPGGAGGARGGAGGGPSGD (m/z 761.51) released from the mouse brain digested with caspase-3. A) MS/MS spectrum for the NRGN peptide CGRKGPGPGGPGGAGGARGGAGGGPSGD (amino acid residues 45-78), charge +3, monoisotopic m/z 761.51, displaying the fragment ions for this peptide. B) Identified b+ and y+ type ions for the NRGN peptide shown in red and blue identified from the database search results.

Figure 3-20. Schematic representation for the list of peptides of VIM protein generated from in vitro digestion of mouse brain lysate using calpain-1 protease, filtered using 10 kDa MWCO (N=3). The red bars led to the conclusion that the regions of VIM which were proteolysed by calpain-1, are the sequences shown in red.

Figure 3-21. MS/MS spectrum for the N-terminal peptide MSTRSVSSSSYRRMFGGSGTSSRPSSNRSYV (m/z 1131.65) released from the mouse brain digested with calpain-1. A) MS/MS spectrum for the VIM peptide MSTRSVSSSSYRRMFGGSGTSSRPSSNRSYV (amino acid residues 1-31), charge +3, monoisotopic m/z 1131.65, displaying the fragment ions for this peptide. B) Identified b+ and y+ type ions for the VIM peptide shown in red and blue identified from the database search results.

Figure 3-22. Schematic representation for the list of peptides of VIM protein generated from in vitro digestion of mouse brain lysate using caspase-3 protease, filtered using 10 kDa MWCO (N=3). The red bars led to the conclusion that the regions of VIM which were proteolysed by caspase-3, are the sequences shown in red.

Figure 3-23. MS/MS spectrum for the peptide EIQELQAQIQE (m/z 664.32) released from the mouse brain digested with caspase-3. A) MS/MS spectrum for the VIM peptide EIQELQAQIQE (amino acid residues 241-251), charge +2, monoisotopic m/z 664.32, displaying the fragment ions for this peptide. B) Identified b+ and y+ type ions for the VIM peptide shown in red and blue identified from the database search results.

CHAPTER 4 NEURO-CELL CULTURE CYTOTOXIC CHALLENGES TO EXAMINE BRAIN PROTEOLYTIC PEPTIDES FORMATION

Introduction

Cell culturing is a fast and reproducible model of TBI that can be subjected to conditions that resemble those of the brain after a TBI-like injury in order to analyze the cellular effects of TBI and replicate trials of new potential treatments. My primary research involved the Neuro2a (N2a) cell line, which is a fast-growing neuroblastoma cell line derived from a spontaneous tumor in an albino strain A mouse. N2a cell morphology has been described as amoeboid-like115. In addition to having the properties of a neuronal stem cell, it has many applications in the study of brain diseases, such as Alzheimer’s Disease (AD) and traumatic brain injury (TBI)116. N2a cells can be differentiated into neuron-like cells after a few days, complete with axonal structures and neurofilaments116. Antigen expression genes for acetylcholinesterase and tubulin have been identified in this cell line. The ability along this cell line to develop neuronal structures, like axons, and produce important components of the neuron cytoskeleton, such as tubulin proteins, make it an ideal cell type to study the cellular changes that occur within the brain following injury. Furthermore, including axonal shearing that leads to neuronal cell death and the breakdown of important structural proteins, of which the BDP of the β-tubulin protein following cytotoxic challenges was successfully identified.

The rat primary cortical mixed neuron-astroglia (CTX) cell cultures were also used to study the cellular consequences of brain injury. The CTX cell originated from rat brain tissue and was derived by transfecting primary astrocytes from the frontal cortex

of one-day-old rats with a DNA construct containing oncogenic characteristics117,118.

CTX cells are phenotypically similar to type one-cell astrocytes. Astrocytes are the most numerous cell types in the central nervous system and are responsible for providing structural and metabolic support to neurons. One critical function of astrocytes is the regulation of intracellular/extracellular glutamate levels119. Elevated levels of glutamate are hypothesized to be linked to brain injury120 and have been identified in patients suffering from TBI. Therefore, CTX cells provide the ability to study these changes in neurotransmitter concentrations, as well as manipulate important glutamate receptors found in nerve cells, such as the N-methyl-D-aspartate receptor121. A similar cell line, the U-251 cell, a human derived glioblastoma astrocytoma cell, was also examined. The line was derived from a malignant glioblastoma tumor found in a human brain122. This cell type possesses both pleomorphic and astrocytoma morphology and contains the

PDGF (platelet-derived growth factor) receptor and the tyrosine kinase, EGFR123. Both growth factor elements contribute to the fast-growing, cancerous nature of this cell type.

The fast-growing nature of the U-251 cell, as well as its ability to differentiate into cells with neuron-like morphology, make it an accurate model of human neurons124. In addition, the U-251 cell displays a fibroblastic growth pattern containing critical components of the cytoskeleton, such as the important vimentin protein used in cell structure and anchoring the cell in place125. Vimentin has been observed to be enzymatically targeted following brain injury, and therefore, its presence in the U-251 cell provides an excellent opportunity of studying these changes126.

Cytotoxic Challenges

A major consequence of TBI is an unusually high intracellular concentration of ions, with calcium ions being the most significant127. This influx of Ca2+ results in the

activation of certain proteases and these proteases are responsible for the breakdown of proteins essential for a neuronal cell’s structural and functional integrity. For instance, the enzyme calpain-1 (a calcium-dependent protease) targets elements such as the cytoskeletal protein’s spectrin, tubulin, and tau, as well as the cell adhesion molecules responsible for cell membrane stability and cell-to-cell adhesion127. However, the extent to which calpain-1 is responsible for neuron degradation and cell death is not well understood128. In order to replicate this process and to better understand the cellular processes and protein modifications, I treated my cells with the calcium ionophore,

A23187 (25 μM; Sigma, St Louis, MO, USA). The calcium ionophore A23187 is a mobile ion carrier that forms stable complexes with divalent ions (Mn2+>Ca2+& Mg2+>Sr2+>Ba2+ ) and has been shown to increase necrosis in cell cultures129. A23187 induces an influx of

Ca2+ by allowing the ion to cross the cell membrane130. This increase in cellular Ca2+ directly models the processes occurring during real brain injury. In order to assess the direct impact of the calcium-activated calpain-1, I inhibited the protease within the same group of cell cultures. In the treated cells, I used SNJ 1945 25 µM ( a gift from Senju

Pharmaceuticals, Kobe, Japan), a water-soluble calpain-1 inhibitor that has been shown to significantly decrease the neurodegenerative effects of calpain-1131. I added Z-D-

DCB (R&D, Minneapolis, MN, USA) to designated wells containing the cells. ZD is a cell-permeable caspase inhibitor that contains a specific peptide recognition sequence

(DEVD) that directly inhibits caspase-3. Protein separation and mass spectrometry gave the identity of the calpain proteolytic peptide derived from my cell cultures (CTX, U-251, and N2a).

Necrosis, or the premature death of cells and tissues, is a common consequence of neurodegenerative diseases such as Alzheimer’s Disease, and trauma such as

TBI132. During necrosis, a group of caspase enzymes were activated and were responsible for critical regulatory processes involved in cell death133. A specific caspase, caspase-3, has been identified as the main enzyme involved in the cleavage of amyloid-beta 4A precursor protein, a process found to be associated with cell death in Alzheimer’s Disease. To stimulate neuronal cell death as well as induce caspase-3 activation, I administered a treatment of staurosporine (STS) (2 μM, Sigma) a known inducer of apoptosis. Although the exact mechanism in which STS activates apoptosis is unknown, it has shown that administration of STS results in caspase-3 activation.

In order to study the direct effects of higher intracellular calcium levels observed in TBI-affected individuals, I administered specific amounts of ethylene glycol bis(β- aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), or ethylenediaminetetraacetic acid

(EDTA), as well as N-methyl-D-aspartate (NMDA). EGTA, EDTA, and NMDA are able to affect calcium levels in different ways. EGTA and EDTA are chelating agents, which form bonds with metal ions such as Ca2+, and in so doing, prevent some Ca2+ ions from entering the cell. NMDA results in the opening of the ion channel and thus allows extracellular calcium to move into the cell. Due to calcium-activated enzymes and related cellular effects, the ability to understand the cellular changes that occur due to calcium levels is crucial to understanding the consequences of TBI and related injuries.

To my cultures (N2a, CTX, U-251) I administered one of the cytotoxic challenges

A23187, STS, EGTA (or EDTA) and NMDA either alone or in combination with SNJ or

ZD.

Cell Culture and Lysis

Primary rat cortical mixed neuron-astroglia cultures were prepared from embryonic day 18 Sprague–Dawley rat fetuses and plated on poly-L-lysine coated 12- well plates (Erie Scientific, Portsmouth, NH, USA). The cells were allowed to grow in an atmosphere of 10% CO2 at 37 °C for 3 days and then treated with 1 mM 4-amino-6- hydrazino-7-D-ribofuranosyl-7H-pyrrolo-(2, 3-D)-pyrimidine-5-carboxamide (ARC) for 2 days. The ARC was removed, and fresh 10% PDHS in DMEM was added, after which the cells were grown for an additional 10 to 14 days in 12-well plates117, and then were subjected to treatment in neurobasal media.

N2a cells were purchased from (ATTC, Virginia, USA) and were grown according to the supplier’s instructions, in DMEM media supplemented with fetal bovine serum

(FBS) (10%). For cytotoxic challenges, a serum-free media was used in addition to the untreated controls.

The following conditions were used: calcium ionophore A23187 (25 μM, Sigma,

St. Louis, MO, USA) as a calpain-1 dominated pro-necrosis challenge for 24 hours; STS

(2 μM, Sigma) as caspase/calpain-dependent apoptosis inducer; and 5 mM EDTA as calpain-independent apoptosis inducer for 24 hours. An excitotoxic challenge glutamate analog 5 mM NMDA was used only for CTX culture, for 24 hours. For pharmacological intervention, cultures were pretreated one hour before the A23187, STS, EDTA, and

NMDA challenge with either the calpain-1 inhibitor SNJ-1945 at 25 µM or the pan- caspase inhibitor Z-D-DCB at 50 µM.

After subjecting the cell culture with the respective conditions, the cells were subjected to a lysis buffer containing 50 mM Tris (pH 7.4), 5 mmol/L EDTA, 1% (v/v) triton X-100, 1 mmol/L DTT, and a mini complete protease inhibitor cocktail tablet

(Roche Biochemicals). Lysates were centrifuged at 10,000 x g for five minutes at 4 °C to remove insoluble debris, and then stored at -80 °C. Protein concentrations of the cell lysates were determined via Bio-Rad DC Protein Assay (Bio-Rad, Hercules, CA, USA).

The cell media were collected separately, centrifuged to remove any debris, ultra- filtered using 10 kDa MWCO, and loaded to RP-nLC MS/MS. The centrifuged cell lysate was analyzed by western blot.

Results and Discussion

The Primary Cortical Mixed Neuron-Astroglia (CTX)/ Neuro2a (N2a) cell line

This section is related to the cell-based model to mimic the proteolytic effect in

TBI events. CTX/N2a cells were treated in a serum-free media with cytotoxin challenges for 24 hours (A23187, STS, EDTA, and NMDA for CTX only), with or without preincubation (1 hour) of either calpain-1 inhibitor (SNJ 1945, 25 µM) or caspase inhibitor (Z-D-DCB, 50 µM). The cell lysates were examined with anti-neurogranin (EMD

AB5620) using western blot technique and internal control β-tubulin to ensure even loading.

The level of intact NRGN in presence of A23187 was seen to be negligible, while

SNJ partially inhibited the action of the calcium ionophore. In the presence of STS, the level of NRGN decrease considerably, while SNJ and ZD had noticeable protective action (Figure 4-1A). EDTA caused a noticeable decrease in the intact NRGN that indicated caspase-3 activation, while with the caspase-3 inhibitor ZD the amount of

NRGN stayed the same as the control. NMDA successfully degraded the intact protein and the effect was suppressed when using SNJ (thus excitotoxic-induced NRGN cleavage appears to be calpain-mediated) as shown in Figure 4-1B. A densitometric quantification was used to confirm the decrease in the intact protein (Figure 4-1C). After

comparing the cell results with in vitro digestion of purified protein and brain lysate, it was readily observed that the BDP could be released into cell media, thus not found in the cell lysate.

The experiment was repeated with A23187 and STS without inhibitors with two different treatment times of 6 hours and 24 hours. It was observed that no decrease was noted in the intact NRGN protein after 6 hours, while there was a significant decrease after 24 hours (Figure 4-2).

The cell media of the A23187, STS and EDTA treatments were filtered with 10 kDa MWCO and analyzed with RP-nLC-MS/MS. Database search engines were set up to search with no enzyme (for calpain-1) and with caspase enzyme (for caspase-3),

UniProt-Rattus norvegicus (57,208 sequences and 27,260,515 residues) for CTX cells.

UniProt-Mus musculus (35,126 sequences and 17,948,970 residues) for N2a cells. A list of peptides extracted from Proteome Discoverer 2.1 is shown in Figures 4-3, 4-5 and

4-7 respectively. Figures 4-4, 4-6 and 4-8, respectively, show MS/MS spectra for the three conditions mentioned above.

U-251 Cell Line, Human Glioblastoma Astrocytoma Cell

This section is related to the cell-based model to mimic the proteolytic effect in

TBI events. U-251 cells were treated in serum-free media with cytotoxin challenges for

24 hours (A23187, STS, EDTA), with or without preincubation (1 hour) of either calpain-

1 inhibitor (SNJ 1945, 25 µM) or caspase inhibitor (ZD-CB, 50 µM). The cell lysate was examined with anti-vimentin (Ab92547) using the immunoblotting technique, and positive control anti-GFAP cocktail (BD Pharmingen MAB 556330), to show the effect of each challenge (Figure 4-4). The cell media of the A23187 treatment was filtered with

10 kDa MWCO and analyzed with RP-nLC-MS/MS. Database search engines were set

up to search with no enzyme (for calpain-1) and with caspase enzyme (for caspase-3),

Homo sapiens (92,271 sequences and 36,831,723 residues). A list of peptides extracted from Proteome Discoverer 2.1 is shown in the diagram (Figure 4-10). MS/MS spectrum for the N-terminal peptide STSRSLYSSSPGGAYVTRSSAVRLRSSV is shown in Figure 4-11.

Conclusions

Cytotoxin challenge in cell-based systems, combined with the use of protease inhibitors, is an advanced experimental model I used to better assess the origins of proteolytic peptides generated from brain-enriched proteins.

Regarding NRGN with the primary cortical mixed neuron-astroglia cell treatments, I observed using the western blot a decrease in the intact NRGN with the calcium ionophore calpain activator, while the caspase activator showed minimal significant effect. Unfortunately, no BDPs were observed, and this might be due to the release of the traces of BDPs into the cell media. Mass spectrometry analyses using

RP-nLC MS/MS could provide the information about the BDPs lacking in the western blots from CTX/N2a cell media.

As indicated in cellular NRGN digestion by calpain-1 (in A23187 treatment-N2a cells), the cleavage sites start at amino acid residue 23 or 24 and end at 64 or 65. This agrees with the results given above, that the calpain-1 cleavage sites deduced earlier for the purified protein and the mouse brain digestion start at amino acid residues 23, 24 or 25 and end at 64, 65 or 67.

In addition, I extensively characterized the formation of vimentin breakdown products due to cell injury/death linked protease activation. First, vimentin was truncated at both the C- and N-terminals by cytosolic protease calpain to vimentin breakdown

products (VBDPs) of 44 kDa then VBDP-35K following pro-necrotic (A23187) and pro- apoptotic (staurosporine) challenges to human glioblastoma astrocytoma cell. On the other hand, with a pro-apoptotic challenge (EDTA) where only caspases were activated, but not calpain, vimentin was fragmented to generate C-terminal VBDP at 26 kDa.

Figure 4-1. NRGN proteolysis breakdown in primary cortical mixed neuron-astroglia mixed culture (CTX) after subjecting to cytotoxin challenges (24 hr). A) Western blot exhibiting treatment with A23187, calpain-1 activator, and STS calpain-1 and caspase activator. B) Western blot exhibiting treatment with EDTA, caspase-3 activator, and NMDA calpain-1 activator, probed with Anti Neurogranin and β-tubulin. C) Densitometric quantification of the intact and breakdown products of NRGN protein. Error bars represent the standard deviation of the mean (N=3).

Figure 4-2. NRGN proteolysis breakdown in primary cortical mixed neuron-astroglia mixed culture (CTX) after subjecting to cytotoxin challenges (6 and 24 hr).Western blot exhibiting treatment with A23187, calpain-1 activator, and STS calpain-1 and caspase activator, for 6 and 24 hr, probed with Anti Neurogranin and αII-Spectrin as a positive control.

Figure 4-3. Schematic representation for the list of NRGN peptides generated from N2a subjected to A23187 filtered using 10 kDa MWCO (N=5).The red bars led to the conclusion that the regions of NRGN which were proteolysed by A23187, are the sequences shown in red.

Figure 4-4. MS/MS spectrum for the peptide PGANAAAAKIQASFRGHMARKKIKSGECGRKGPGPGGPGGAGGARGG (m/z 1486.75) released from the N2a subjected to A23187. A) MS/MS spectrum for the NRGN peptide PGANAAAAKIQASFRGHMARKKIKSGECGRKGPGPGGPGGAGGARGG (amino acid residues 24-70), charge +3, monoisotopic m/z 1486.75, displaying the fragment ions for this peptide. B) Identified b+ and y+ type ions for the NRGN peptide shown in red and blue identified from the database search results.

Figure 4-5. Schematic representation for the list of NRGN peptides generated from CTX subjected to STS (caspase cleavage only) filtered using 10 kDa MWCO (N=5). The red bars led to the conclusion that the regions of NRGN which were proteolysed by STS, are the sequences shown in red.

Figure 4-6. MS/MS spectrum for the peptide ILDIPLDD (m/z 913.50) released from the CTX subjected to STS. A) MS/MS spectrum for the NRGN peptide ILDIPLDD (amino acid residues 16-23), charge +1, monoisotopic m/z 913.50, displaying the fragment ions for this peptide. B) Identified b+ and y+ type ions for the NRGN peptide shown in red and blue identified from the database search results.

Figure 4-7. Schematic representation for the list of NRGN peptides generated from CTX subjected to EDTA filtered using 10 kDa MWCO (N=5). The red bars led to the conclusion that the regions of NRGN which were proteolysed by EDTA, are the sequences shown in red.

Figure 4-8. MS/MS spectrum for the peptide IPLDDPGANAAAAKIQASFRGHMARKKIKSGE (m/z 1117.93) released from the CTX subjected to EDTA. A) MS/MS spectrum for the NRGN peptide IPLDDPGANAAAAKIQASFRGHMARKKIKSGE (amino acid residues 18-32), charge +3, monoisotopic m/z 1117.93, displaying the fragment ions for this peptide. B) Identified b+ and y+ type ions for the NRGN peptide shown in red and blue identified from the database search results.

Figure 4-9. Vimentin and GFAP proteolysis in human glioblastoma U-251 cells subjected to various pro-necrotic and pro- apoptotic challenges. A) Western blot displaying treatment with A23187, calpain-1 activator, and STS calpain-1 and caspase activator, EDTA, caspase-3 activator, probed with anti-vimentin. B) Western blot displaying treatment with A23187, calpain-1 activator, STS calpain-1 and caspase activator, EDTA, caspase-3 activator, probed with Anti-GFAP. C) Densitometric quantification of the intact and breakdown products of vimentin protein. Error bars represent the standard deviation of the mean (N=3).

Figure 4-10. Schematic representation for the list of VIM peptides generated from U-251 cell line subjected to A23187 filtered using 10 kDa MWCO (N=5).The red bars led to the conclusion that the regions of VIM which were proteolysed by A23187, are the sequences shown in green.

Figure 4-11. MS/MS spectrum for the N-terminal peptide STSRSLYSSSPGGAYVTRSSAVRLRSSV (m/z 973.50) released from the U- 251 cell line subjected to A23187. A) MS/MS spectrum for the VIM peptide STSRSLYSSSPGGAYVTRSSAVRLRSSV (amino acid residues 46-73), charge +3, monoisotopic m/z 973.50, displaying the fragment ions for this peptide. B) Identified b+ and y+ type ions for the VIM peptide shown in red and blue identified from the database search results.

CHAPTER 5 MOUSE MODELS OF TRAUMATIC BRAIN INJURY

Introduction

Studies show that the use of animal models of TBI for research is prevalent, with the purpose of mimicking head trauma mechanisms in humans. It is expected that these models will help scientists and clinicians develop appropriate clinical therapies and treatments134. Mouse models are often used in TBI research because of the affordable cost, accessibility, and easy measurement135. They have crucial roles in determining the underlying mechanisms and appraising the assurance and efficiency of developing treatments, which eventually would help later clinical trials134. Another significant purpose of mouse models of TBI research is to facilitate the separation and categorization of different injury mechanisms with which clinicians are struggling to determine134. Genetically engineered mice are commonly used as TBI animal models, because they can be analyzed by neuropathological and neurobehavioral metrics as parameters, in order to better understand a variety of injuries and determine whether it is mild, moderate, or severe brain injury134. Researchers state that, although mouse models aid scientists in understanding the pathological mechanisms of head trauma, they experiment in a controlled environment. Therefore, there are some limitations of

134 equivalence in mechanisms and outcomes between humans and mouse models .

In order to assess the proteolytic effects of TBI within a living system, or in vivo, I used two mouse animal models which mimicked two types of TBI: controlled cortical impact (CCI) and repeated closed head injury (rCHI). CCI consists of more severe focal open-head injuries that mimic severe TBI in humans. This type of model replicates the penetrating head injury that directly caused brain deformation in animal models by using

a mechanically induced piston. This could also be referred to as the rigid percussion model which is useful in the biomechanical aspects of head trauma research135.The mechanical factors in CCI experiments are well controlled, including the velocity, force and depth of impact, and the intensity of resulting structural tissue changes, thus eventually helping to measure the outcome136. CCI takes advantage of these mechanical factors to regulate physical damage, dependable after effect and no repeated acceleration-deceleration events137. After the dura of the animal models was impacted with a certain force, along with other experimental designs, the results of intracranial hypertension and cellular functional deficiencies resulting from the conformational changes could be observed135. Apparently, cellular disruptions resulting from the damage potentially led to the disorders of diffuse axonal injury (DAI) and neuronal degeneration. This was primarily detected in the myelinated nerve cells of the cerebrum and folia of cerebellum areas135. Essentially, this type of experiment could result in the animal going into a coma. It was found that the damage caused by CCI presented a more locally concentrated injury compared with other models135, thus it linked with the manifestations of cortical contusion and extravasation of blood in the subarachnoid area137. Research shows that the use of craniotomy within CCI models induces the disruption of parenchyma tissues. Therefore, it stimulates the alterations of immune and glial cells or gliosis that can mistakenly be seen with mild TBI137. Clinical symptoms caused by CCI consist of coma, abnormalities in cerebral fluid accumulations, blood flows, and pressure, as well as neuronal alterations in endocrine and metabolism135. Obtaining these data through CCI models would certainly assist clinical TBI research in understanding the mechanisms and pathways of neurological

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deficits through molecular and genetic interpretation135. Correspondingly, it would allow the development of the brain injury healing process.

On the other hand, repeated closed head injury (rCHI) mimics mild concussive injury. The repetitive head injuries in humans are often seen in the closed head TBI and sports-related concussion. The closed head injury allows the free movement of the head after the collision. The difference from CCI, in animal models, is that CHI does not undergo a craniotomy or invasive cerebral damage but electronic magnetic impact system138. A study shows that none of the CHI mouse models experience skull fractures, cortical contusions, edema, or cerebral hemorrhage139. Nevertheless, they manifest intraventricular bleeding and cognitive deficits, including loss of consciousness, as well as hypertension but low arterial oxygen concentration139. In addition, the researchers also identified cellular alterations of neurofilament and microtubule in the brain, thus relating to behavioral insufficiency such as depression, anxiety, learning and navigating abilities139. The use of rCHI animal models is highly valuable in helping the transition of preclinical studies to clinical settings. In both mild and severe TBI, several complex metabolic, biochemical, and cellular disruptions occur.

These result in neuronal damage such as diffuse axonal injury (DAI), neuronal degeneration, and the overall disruption of normal brain function. At the protein level, there is evidence that TBI significantly alters protein concentrations as well as cause unusual post-translational modifications, such as phosphorylation that has been identified on the protein tau (a protein associated with Alzheimer's disease). In addition, trauma to the head results in axonal shearing and this has been identified as a direct contributor to the unregulated influx of ions into the axon. This influx of ions (K+ and

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Na+) into the axon results from an increase in the concentration of intra-axonal Ca2+.

The increase in calcium has been linked to several processes thought to contribute to the neuronal degeneration observed in TBI patients. Activation of calcium-dependent calpain-1 results in proteolysis of key brain proteins, excess glutamate released can be toxic to the cell at high levels and destroy the structural components of neurons like microtubules and cytoskeletons138,140. In particular, the increase of calcium ions entering into the cells after damage stimulates the pathway of cysteine, which breakdowns calpastatin a calpain-1 inhibitor. It follows the accumulation of calpain-1, a cysteine protease, causing the deterioration of the neuronal cytoskeleton network141. It was also noted that mitochondria were also affected directly by the trauma damage, which causes the influx of a large amount of Ca2+ into the cells. The event leads to mitochondrial swelling followed by mitochondrial breakdown. As a result, the disruption of cellular metabolism and ion concentration led to the dispersion of caspases, which degrade the injured axons, and apoptosis of proteins141.

In Vivo Models of Severe Traumatic Brain Injury

A controlled cortical impact (CCI) device was used to model TBI139. CB57BL/6 mice (male, 3 to 4 months old, Charles River Laboratories, Raleigh, NC, USA) were used. The mice were sedated with 4% isoflurane in oxygen as a carrier gas for four minutes, then anesthesia of 2% to 3% isoflurane. In the wake of achieving a profound plane of anesthesia, mice were seated in a stereotactic frame in a procumbent position and insured by ear and incisor bars. A midline cranial incision was made and a unilateral (ipsilateral) craniotomy (3 mm diameter) was performed adjacent to the central suture, and midway between the bregma and the lambda. The dura mater was kept intact over the cortex. Brain trauma was induced using a PSI TBI-0310 Impactor

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(Precision Systems and Instrumentation, LLC, Natick, MA, USA), by affecting the right cortex (ipsilateral cortex) with 2 mm diameter impactor tip at a velocity of 5 m/s, 1.8 mm compression depth and a 240 ms dwell time (compression duration). Sham-injured control animals underwent identical surgical procedures but did not receive an impact injury.

In Vivo Models of Mild Traumatic Brain Injury

Repeated closed head injury (rCHI) was performed on 3-4 month old male

C57BL/6J mice. The mice were provided bedding, nesting material, food and water, and were kept at ambient temperatures controlled at 20-22 °C with constant 12 hr light/12 hr dark cycles. The mice underwent the same pre-impact anesthesia procedure as did the

CCI mice, but no surgical craniotomy was performed. Brain trauma was produced using a PSI TBI-0310 Impactor (Precision Systems and Instrumentation, LLC, USA) by impacting the sagittal suture midway with a commercial rubber impactor tip (1 cm in thickness, 9 mm in diameter) at a velocity of 5.0 m/s, 4 mm compression depth and a

240 ms dwell time (compression duration). The center of impact corresponded to the central sagittal suture midway between coronal and lambdoid sutures. An impact was given and repeated 1 and 7 days after the initial injury.

Mouse Brain Tissue Collection and Preparation

At the different post-TBI time points (1 and 7 days), the animals were anesthetized and killed by decapitation. The injured cortex and /hippocampus of TBI- affected animals and similar areas for naïve animals were collected. The tissue samples were pulverized to a fine powder with a small mortar and pestle settled over dry ice. The powdered brain tissue was then lysed for 90 minutes at 4 °C with lysis buffer similar to the cell method. Protein concentrations of lysates were determined via Bio-Rad DC

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Protein Assay. Tissue lysates were then centrifuged at 10,000 x g for 10 minutes at 4

°C. The supernatants were snap-frozen and stored at -80 °C until used. Equivalent amounts of protein lysate (usually 20 µg) were separated by SDS-PAGE and then western blot analyzed. Separately, I performed ultrafiltration of the lysate using 10 kDa

MWCO to identify the peptides using tandem mass spectrometry.

Results and Discussion

Immunoblotting Analysis for Cortex

The brain lysate of the cortex was separated with SDS-PAGE immunoblotting and probed with anti-neurogranin (Abcam 99269-internal epitope) (Figure 5-1A), validated with densitometric quantification (Figure 5-1B) and anti-vimentin (Ab92547)

(Figure 5-1D), using β-tubulin as an internal control to ensure even loading.

The experiment was repeated for the neurogranin biomarker using a different antibody (EMD AB5620-a full-length NRGN) (Figure 5-1D). The 9 kDa BDP was observed on days one and seven after CCI, but only on day seven after rCHI. For vimentin, the 35 kDa BDP, corresponding to the calpain-1 cleavage, was observed at day seven after CCI and days one and seven after rCHI. The 26 kDa BDP corresponding to caspase cleavage was observed on days one and seven for both CCI and rCHI.

Immunoblotting Analysis for Hippocampus

The brain lysate of the hippocampus was separated with SDS-PAGE immunoblotting and probed with anti-neurogranin (Abcam 99269-internal epitope)

(Figure 5-2A), validated with densitometric quantification (Figure 5-2B) and anti-vimentin

(Ab92547) (Figure 5-2D), using β-tubulin as an internal control to ensure even loading.

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The experiment was repeated for the neurogranin biomarker using a different antibody (EMD AB5620-a full-length NRGN) (Figure 5-2D). The 9 kDa BDP was detected on day seven after the controlled cortical impact and day one and seven after closed head injury. The experiment was repeated for the neurogranin biomarker using a different antibody (EMD AB5620-a full-length NRGN). For vimentin, the 44 kDa BDP corresponding to the calpain/caspase cleavage was observed on days one and seven after CCI and rCHI (Figure 5-2D).

Mass Spectrometry Analysis

Database search engines were set up to search with no enzyme (for calpain-1),

Mus musculus (35,126 sequences and 17,948,970 residues) version 2016_07. NRGN peptides were identified from the database search using Proteome Discoverer 2.1 for the filtered (10 kDa) lysate cortex CCI-D1 and D7 samples and are recorded in Figures

5-3 and 5-5 respectively. From Figure 5-3, it was deduced that the major proteolytic peptide sequence starts at amino acid residue 22 and ends at amino acid 69. From

Figure 5-5, CCI-D7, it was observed that the cleavage of NRGN only occurred between amino acids 13 and 38. This suggests that the calpain-1 activity decreased with time.

MS/MS spectra of these peptides are shown in Figures 5-4 and 5-7 respectively. In a similar manner, VIM peptides were identified for the filtered lysate cortex CCI-D1

(Figure 5-7) and representative MS/MS spectra for the N- and C-terminals are shown in

Figures 5-8 and 5-9 respectively.

Conclusions

In this chapter, I used two mouse models, which mimicked severe (CCI) and mild

(rCHI) cases of TBI. Cortex and hippocampus lysate and lysate filtrate of this model were used to assess brain protein proteolysis, using both immunoblotting and mass

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spectrometry respectively. It was observed from the immunoblotting analysis that NRGN

9 kDa BDP(s) appeared at least one or more time points of both CCI and rCHI. As was shown in previous sections, mass spectrometry proved a powerful analytical tool for determining the peptide sequences for NRGN and VIM from the TBI mouse cases. The results support the same ideas about the cleavage sites for calpain-1 protease, starting at amino acid residue 22, 23 or 24 and ending at 64, 65 or 69. The caspase cleavage is still not well understood.

In addition, the formation of VIM breakdown products was observed mainly in the lysate of cortex due to cytosolic protease calpain-1, observed at 35 kDa and those from the caspase proteolysis were observed at 26 kDa.

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Figure 5-1. Ipsilateral cortex profile at different time points after CCI and rCHI injury in mice. A) Western blot displaying the BDP of NRGN using an internal epitope antibody with internal control β-actin. B) Densitometric quantification of the intact and BDP of the NRGN protein. Error bars represent the standard deviation of the mean (N=3). C) Western blot displaying BDP of NRGN using a full-length NRGN epitope (EMD AB5620). (D) Western blot demonstrating BDP of VIM using anti-vimentin (Ab92547).

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Figure 5-2. Ipsilateral hippocampus profile at different time points after CCI and rCHI injury in mice. A) Western blot displaying the BDP of NRGN using an internal epitope antibody with internal control β-actin. B) Densitometric quantification of the intact and BDP of the NRGN protein. Error bars represent the standard deviation of the mean (N=3). C) Western blot displaying BDP of NRGN using a full-length NRGN epitope (EMD AB5620). D) Western blot demonstrating BDP of VIM using anti-vimentin (Ab92547).

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Figure 5-3. Schematic representation for the list of NRGN peptides generated from cortex CCI (day 1) lysate samples, filtered using 10 kDa MWCO (N=3). The red bars led to the conclusion that the regions of NRGN which were proteolysed by CCI (D1), are the sequences shown in red.

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Figure 5-4. MS/MS spectrum of the NRGN peptide PGANAAAAKIQASFRGHMARKKIKSGECGRKGPGG (m/z 1245.87) released from ipsilateral cortex CCI (day 1) injury in mice. A) MS/MS spectrum for the NRGN peptide PGANAAAAKIQASFRGHMARKKIKSGECGRKGPGG (amino acid residues 24-63), charge +3, monoisotopic m/z 1245.87, displaying the fragment ions for this peptide. B) Identified b+ and y+ type ions for the NRGN peptide shown in red and blue identified from the database search results.

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Figure 5-5. Schematic representation for the list of NRGN peptides generated from cortex CCI (day 7) lysate samples, filtered using 10 kDa MWCO (N=3). The red bars led to the conclusion that the regions of NRGN which were proteolysed by CCI (D7), are the sequences shown in red.

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Figure 5-6. MS/MS spectrum of the NRGN peptide DDDILDIPLDDPGANAAAAKIQASFR (m/z 904.30) released from ipsilateral cortex CCI (day 7) injury in mice. A) MS/MS spectrum for the NRGN peptide DDDILDIPLDDPGANAAAAKIQASFR (amino acid residues 13-38), charge +3, monoisotopic m/z 904.30, displaying the fragment ions for this peptide. B) Identified b+ and y+ type ions for the NRGN peptide shown in red and blue identified from the database search results.

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Figure 5-7. Schematic representation for the list of VIM peptides generated from cortex CCI (day 1) lysate samples, filtered using 10 kDa MWCO (N=3). The red bars led to the conclusion that the regions of VIM which were proteolysed by CCI (D1), are the sequences shown in red.

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Figure 5-8. MS/MS spectrum of the N-terminal VIM peptide LGSALRPSTSRSLY (m/z 585.63) released from ipsilateral cortex CCI (day 1) injury in mice. A) MS/MS spectrum for the VIM peptide LGSALRPSTSRSLY (amino acid residues 40- 54), charge +3, monoisotopic m/z 585.63, displaying the fragment ions for this peptide. B) Identified b+ and y+ type ions for the VIM peptide shown in red and blue identified from the database search results.

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Figure 5-9. MS/MS spectrum of the C-terminal VIM peptide NLESLPLVDTHSKRTLLIKTVETRDGQVINE (m/z 1227.03) released from ipsilateral cortex CCI (day 1) injury in mice. A) MS/MS spectrum for the VIM peptide NLESLPLVDTHSKRTLLIKTVETRDGQVINE (amino acid residues 426-456), charge +3, monoisotopic m/z 1227.03, displaying the fragment ions for this peptide. B) Identified b+ and y+ type ions for the VIM peptide shown in red and blue identified from the database search results.

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CHAPTER 6 ARCHIVED HUMAN TBI BIOFLUID CEREBROSPINAL FLUID

Human Bio-Samples Procurement

Cerebrospinal fluid (CSF) is a clear bodily fluid that is located around the brain and the spinal cord. The production of CSF is aided and regulated by the choroid plexus. The choroid plexus is located in the ventricles of the brain, primarily the lateral, third and fourth ventricles. The cells of the choroid plexus do not directly produce CSF; rather, they direct the filtration of blood through its specialized cuboidal cells. Through the creation of electrical charge differences in the cuboidal cells, ions like sodium, chloride, and bicarbonate are moved into the ventricle. This newly formed osmotic difference causes water molecules to leave the blood capillaries, and move into the ventricles142. Mostly consisting of water, CSF also contains proteins and sugars due to the blood filtration in the brain. CSF is then free to move throughout the ventricles and, eventually, surrounds the brain and spinal cord. As it surrounds the brain, it pools into the subarachnoid space. CSF is constantly being generated by the body, around 600 ml are made a day143.

Cerebrospinal fluid can transport chemicals, proteins, and waste throughout the central nervous system142; thus, observing the contents of a CSF sample can give valuable insight into neurological diseases and brain trauma144.

Cerebrospinal fluid can be collected by lumbar puncture, a cisternal puncture or a ventriculostomy145. To perform the lumbar puncture (spinal tap) a long, thin needle is inserted between two lumbar vertebrae, and fluid is withdrawn from the thecal sac146.

On the cisternal puncture, the collecting needle is inserted below the occipital bone,

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which is more dangerous because of the close proximity to the brain stem147.

Ventriculostomy generates a hole inside a cerebral ventricle for withdrawing the fluid.

Analyzing proteins in the brain is not possible without extensive and life- threatening procedures; therefore analyzing the contents of CSF is vital in detecting certain proteins after a TBI. TBI initiates a multitude of reactions, including the activation of calpain-1 which leads to proteolysis of neurons and other proteins like alpha II spectrin82. Spectrin is a cytoskeletal protein located in dendrites and axon terminals148.

There is evidence that increased breakdown of the spectrin is positively correlated with the increasing severity of traumatic brain injuries149. This example of the breakdown of spectrin reveals the vital relationship CSF has with TBI. Control samples of CSF compared to samples exposed to TBI are different with respect to the breakdown of alpha II spectrin82. The ability to detect and measure this breakdown shows that a TBI has taken place. This relationship can be used to detect other breakdowns of proteins as well. This opens up the opportunity to detect other TBI biomarkers or diseases by the analysis of CSF.

Cerebrospinal Fluid Collection Protocol

The normal control samples CSF (n=20) were purchased from Bioreclamation

(Westbury, NY, USA). Archived de-identified CSF samples (n=30) from a severe TBI study were collected from consenting adult subjects presenting to the emergency department of the Ben Taub General Hospital, Baylor College of Medicine (Houston,

TX, USA). The study protocol was approved by the Baylor College of Medicine IRB, for subjects sustaining blunt trauma to the head with a Glasgow coma scale of 8. CSF samples were collected for up to 10 days or until an Intra ventriculostomy was no longer clinically indicated. The cerebrospinal fluid was sampled from the buretrol of the CSF

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drainage system by a qualified and trained hospital employee according to the hospital’s standard procedures. Alternatively, timed CSF samples (10 ml) with a total collection time not exceeding 1 hour were diverted to 15 mL conical polypropylene centrifuge tubes (BD Falcon, San Jose, CA, USA). These CSF samples (5 to 10 ml) were then centrifuged at 4,000 x g with a tabletop centrifuge at room temperature for 5 to 7 minutes to remove loose cells and debris. A volume of 1 ml aliquots of cleared CSF

(supernatant) was pipetted into a 2 ml cryogenic tube, snap-frozen and stored at -80 0C in an ultra-low freezer until further use. For this study, timed CSF samples collected within 24 hours from injury were used117.

Sample Preparation

In my study, 250 μL CSF from pooled control and TBI patients were used. The filtrates from 10 kDa MWCO filters (Sartorius Stedim Biotech, Gottingen, Germany) were centrifuged at 10,000 x g for 25 minutes. The 50 μL filtrates were concentrated using a speed vacuum (Thermo, Asheville, NC) to volumes of about 5 μL. The samples were reconstituted in LCMS water containing 0.1% formic acid to 10 μL. These reconstituted samples were analyzed by two methods. The first method used an RSLC tandem MS instrument, with a Nanoflow liquid chromatography system (Ultimate 3000,

Thermo Scientific) interfaced with a hybrid ion trap-Orbitrap, high-resolution tandem mass spectrometer (Velos-Pro, Thermo Scientific). The second method used RP-nLC

MS/MS (Water Acquity coupled with LTQ XL). The data were processed from both instruments using Proteome Discoverer 2.1 (Thermo), SEQUEST HT (version 2.1.1.21).

Database search engines were set up to search those with no enzyme Homo sapiens

.fasta file (92,271 sequences and 36,831,723 residues) version 2016_07. By conducting mass spectrometry analyses with both systems, I obtained high-resolution results from

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the Orbitrap system and daily accessible data from our LTQ XL machine. As for the 200

µL retentate, containing high molecular weight proteins, western blots were conducted to observe specific proteins in the mixture.

Results and Discussion

Experiments on the human biofluid were conducted using an archived bank of

CSF (n=30) from severe TBI cases and commercially control samples (n=20). CSF samples were subjected to western blot and probed with anti-neurogranin (EMD

AB5620), Figure 6-1A, and the scatter plot in Figure 6-1B shows the quantification results. Anti-phospho-Neurogranin (EMD 07-430), Figure 6-1C, and quantified using a scatter plot (Figure 6-1D). The filtrate of the control and human TBI CSF samples (10 kDa MWCO) was acidified with 0.1 % LCMS formic acid and loaded to RS-LC MS/MS, for identification of the proteolytic peptides. Database search engines were set up to search with no enzyme (for calpain-1), Homo sapiens (92,271 sequences and

36,831,723 residues). The data were processed using Proteome Discoverer 2.1. A list of NRGN peptides identified from the database search is shown in Figure 6-2. The

MS/MS spectrum of the peptide,

ILDIPLDDPGANAAAAKIQASFRGHMARKKIKSGERGRKGPGPGGPGGA, indicating the core peptide of NRGN, charge +7, m/z 713.6407 (Figure 6-3). The fragment ions that identify the peptide from the database were recorded. The MS/MS spectrum of the peptide in question, showing the fragmentation of each amino acid, is shown in Figure

6-4.

The CSF samples were examined by western blot analysis using anti-vimentin

(Abcam Ab92547) (Figure 6-5A), and quantification is represented using the scatter plot. Here the 35 kDa and 26 kDa BDP were observed, indicating calpain-1 and

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caspase cleavages respectively (Figure 6-5B). A list of VIM peptides identified from the database search is shown in Figure 6-6. The MS/MS spectrum of the C-terminal peptide is shown in Figure 6-7.

Conclusion

It is observed from the scatter plot that there is increased leakage of both intact

NRGN and 9 kDa NRGN-BDP from the CSF compartment in TBI cases compared to normal control samples. Thus, NRGN and its 9 kDa BDP might be a good candidate biomarker. Neurogranin is in fact, highly sensitive to TBI-induced proteolysis and could become a new TBI disease tracker diagnostic marker. In addition, the use of TBI proteolytic biomarkers could be used as drug development tools, i.e. TBI therapeutics to protect the brain should in principle attenuate the levels of such TBI-induced proteolytic biomarker levels. Thus, NRGN and vimentin proteolytic biomarkers could be considered

“theranostic tools” with the utilities in augmenting the clinical trials for new TBI drug development.

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Figure 6-1. NRGN and 9 kDa NRGN-BDP in human CSF within 24 h of severe TBI. A) Representative blot probed with (EMD AB5620) showing NG and NG-BDP in human control and TBI CSF samples. B). Scatter plot for control (n=20), and TBI CSF (n=30) (** p < 0.01). C) Western blot probed with phospho neurogranin (EMD 07-430) showing intact NRGN and 7 k BDP. D) Scatter plot for control (n=20), and TBI CSF (n=30) (** p < 0.01).

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Figure 6-2. Schematic representation for the list of NRGN peptides generated from filtered (10 kDa MWCO) TBI CSF samples. The red bars led to the conclusion that the regions of NRGN which were proteolysed from TBI cases are the sequences shown in red.

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Figure 6-3. MS/MS spectrum of NRGN peptide in human TBI CSF (24 hr). A) MS/MS spectrum for the NRGN peptide ILDIPLDDPGANAAAAKIQAS(*)FRGHMARKKIKSGERGRKGPGPGGPGGA (amino acid residues 18-64), charge +7, monoisotopic m/z 713.64075, displaying the fragment ions for this peptide. B) Identified b+ and y+ type ions for the NRGN peptide shown in red and blue identified from the database search results. Ser-36 is phosphorylated (*)

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Figure 6-4. MS/MS spectrum of NRGN peptide in human TBI CSF (24 hr) ILDIPLDDPGANAAAAKIQASFRGHMARKKIKSGERGRKGPGPGGPGGA (amino acid residues 18-64), displaying the fragmentation pattern of amino acids

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Figure 6-5. Vim and 35, 26 kDa VBDP in human CSF within 24 h of severe TBI. A) Two representative blots probed with Anti-Vimentin (Abcam Ab92547) showing Vim and VBDP in human control and TBI CSF samples. B) Scatter plot for control (n=20), and TBI CSF (n=30) showing the control intact and BDPs and TBI intact and BDPs (** p < 0.01).

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Figure 6-6. Schematic representation for the list of VIM peptides generated from filtered (10 k Da MWCO) TBI CSF samples. The red bars led to the conclusion that the regions of VIM which were proteolysed from TBI cases are the sequences shown in red.

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Figure 6-7. MS/MS spectrum of the C-terminal VIM peptide in human TBI CSF (24 h). A) MS/MS spectrum for the VIM peptide LLEGEESRISLPLPNFSSLNLR (amino acid residues 403-424), charge +4, monoisotopic m/z 661.82, displaying the fragment ions for this peptide. B) Identified b+ and y+ type ions for the VIM peptide shown in red and blue identified from the database search result.

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CHAPTER 7 SUMMARY AND FUTURE WORK

Summary

I approached this work in a stepwise fashion, starting from the simplest possible system and working my way up to the complex system CSF of human TBI patients.

I started with the in vitro investigation of the calpain-1 and caspases-3, and -6 proteolyses of purified NRGN and VIM proteins. Next, I studied the lysate from the naïve brain of mice, and following that, I proceeded the cell lines derived from N2a mice neuroblastoma and primary cortical mixed neuron-astroglia (NRGN) and human glioblastoma cell line (VIM). Following that mouse model, the brains of mice subjected to two forms of TBI which were CCI (severe) and rCHI (mild). Finally, CSF from healthy controls and the TBI patients was investigated.

Throughout, western blot and UPLC-mass spectrometry were the techniques chosen for the peptidomics analysis. The series of peptides formed at each stage of the research differed according to the system studied, which can be smaller in proteolysis of purified protein, mouse brain and CCI, rCHI cases due to over-digestion, while the human CSF was the final point showing exactly the formation of the required endogenous peptides.

Finally, it was observed from all the experiments done in my dissertation that

NRGN appeared to be predominantly cleaved by calpain in human TBI (assessed with

CSF samples). In addition to NGRN-BDP (apparent molecular weight 9 kDa) observed by immunoblot and the 10 kDa MWCO filtered samples, I repeatedly observed core peptides of NGRN starting at around amino acid 18 and ending at 64, although actual

NGRN peptides observed tend to be variable in length (Figure 7-1A). On the other

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hand, VIM appeared to be highly vulnerable to calpain and/or caspase proteolysis that caused N- and C-terminal truncation, producing major 35 kDa and 26 kDa VIM-BDP as observed in human CSF. Again, mass spectrometry based analysis of human CSF sample filtrate identified a new N- and C-terminal peptides of the protein starting at around amino acid 83 and ending at 397 (Figure 7-1B),

As proof of concept, I studied two brain proteins in depth, NGRN and VIM, the

BDPs and peptides generated by both proteins are elevated in human TBI samples compared to control CSF.

Finally, my data indicated that neurogranin and vimentin proteolytic breakdown products and peptides could be considered as diagnostic biomarker candidates and potential “theranostic” tools in augmenting the clinical trials for new TBI drug development.

Future Work

I propose for my future work a project to design a novel technique to detect

NRGN breakdown products in whole blood of TBI patients, building an antibody that targets the core peptide which I discovered in CSF from TBI patients, and using this antibody in building a new, easily available and competitive portable device that quickly and accurately diagnose concussion. Such a medical device will be of considerable importance to the military, National Football League and those living in rural America. I also propose to synthesize a calpain inhibitor which can cross the blood–brain barrier and thus reduce the proteolysis consequent to TBI.

Furthermore, I plan to identify the VIM cleavage sites using a reliable N-terminal sequencing protocol and, based on the novel peptides which I discovered, to build a more robust VIM antibody for the detection of its proteolytic breakdown products.

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Figure 7-1. Schematic representation for calpain-1 and caspase cleavage sites. A) NRGN protein. B) VIM protein.

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BIOGRAPHICAL SKETCH

George Anis Sarkis was born in Alexandria, Egypt, on October 27, 1978. He grew up with his parents and older brother. He attended Boghossian Armenian School from nursery until high school. After graduating from the Faculty of Science, Alexandria

University Special Chemistry Department with First Class Honors, he obtained his

Master of Science degree in Chemistry.

In the fall of 2014, George started at the University of Florida in the research group of Dr. Richard Yost, working in Dr. Kevin Wang’s, research lab, McKnight Brain

Institute, Department of Psychiatry. George completed his doctorate in May 2017.

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