Health Science Campus
FINAL APPROVAL OF DISSERTATION Doctor of Philosophy in Biomedical Sciences
The Role of Calcium and Mitochondria in the Etiology and Treatment of Three Different Disease Paradigms
Submitted by: Christine Brink
In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences
Examination Committee
Major Advisor: David Giovannucci, Ph.D.
Academic Linda Dokas, Ph.D. Advisory Committee: Joseph Margiotta, Ph.D.
Andrew Beavis, Ph.D.
Ana Marie Oyarce, Ph.D.
L. John Greenfield, M.D., Ph.D.
Senior Associate Dean College of Graduate Studies Michael S. Bisesi, Ph.D.
Date of Defense: December 7, 2007
The Role of Calcium and Mitochondria in the Etiology and Treatment of Three
Different Disease Paradigms
Christine A. Brink
University of Toledo, Health Science Campus
2007
Acknowledgements
First, I would like to thank my advisor, Dr. David Giovannucci, for his
encouragement and support throughout my graduate training. His advice and
understanding have been essential to my success over the past 5 years. I will
always be grateful for the education he has provided to me, and the compassion
he has shown toward me.
Next, I would like to thank Jenny Giovannucci for her illustrations and
encouragement. I would also like to thank Rebecca Pierson for providing the
primary cortical neuronal cultures, and countless words of advice. Additionally, I
would like to thank Christian Peters for his counsel and assistance throughout
our training. In addition, I would like to thank my committee members, Dr.
Greenfield, Dr. Dokas, Dr. Margiotta, Dr. Beavis, and Dr. Oyarce, for their
contributions and guidance.
Lastly, I would like to express my unending gratitude toward my family. My
husband’s patience and encouragement have been indispensable during this time. Also, the love and support of my parents were critical in preparing me to take on this incredible and fascinating journey.
i Abbreviations Used
IP3 1,4,5,triphosphate
GABA γ-aminobutyric acid
Allo Allopregnanolone
AD Alzheimer’s disease
Aβ Amyloid-β
APP Amyloid precursor protein
ALS Amyotrophic lateral sclerosis
Ca2+ Calcium
CRAC Calcium release activated channel
CCE Capacitative calcium entry
FCCP Carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone
CAI Carboxyamidotriazole
Icrac Current from CRAC channel
CIF Cytochrome c efflux inducing factor
DMEM Delbeco’s Modified Eagle Medium
EC50 Effective concentration at 50%
ER Endoplasmic reticulum
EDTA Ethylenediamine-tetraacetic acid disodium salt
EGTA Ethleneglycol-O, O’ bis(2aminoethyl)N,N,N’,N’-tetraacetic acid
GFAP Glial fibrillary acidic protein
HVA High voltage activated
ii HN Hypothalamic neurohypophysis
HPA Hypothalamic pituitary axis
ISS Intercellular saline solution
LDP Long term depression
LTP Long term potentiation
LVA Low voltage activated
HEDTA N-(2-hydroxyethlenediamine-N, N’, N’-triacetic acid trisodium salt
PIP2 Phosphatidylinositol biphosphate
PLC Phospholipase C
PSS Physiological saline solution
PKC Protein kinase C
SR Sarcoplasmic reticulum
SERCA Sarcoplasmic/endoplasmic reticulum calcium ATPase
SCID Severe combined immunodeficency syndrome
SNARES Soluble N-ethylmalemide sensitive factor protein receptor
SOC Store operated channel
TMRM Tetramethyl-rhodamine methyl ester perchlorate
TRP Transient receptor protein
VGCC Voltage gated calcium channel
iii
Table of Contents
Preliminaries
Page
Acknowledgments i
Abbreviations Page ii
Table of Contents iv
Chapters
Page
I Introduction 1
II Methods 6
III Background and Literature Review 15
IV Results 39
V Results 56
VI Results 78
VII Conclusions 103
Bibliography 106
Abstract 139
iv Chapter I: Introduction
Calcium (Ca2+) is an important cellular messenger involved in a variety of critical messaging pathways. From cell proliferation to apoptosis, Ca2+ is involved in multiple facets of cell health. Even in the most primitive prokaryotes maintaining the Ca2+ gradient is important, and cytosolic free Ca2+ concentrations are controlled (Case, 2007) . In eukaryotes, Ca2+’s importance is demonstrated early in development, as it controls fertilization and cellular differentiation (Lory et al., 2006). After development, Ca2+ plays a critical role through its control of gene transcription, and is responsible for a myriad of messages including: control of movement, synaptic plasticity, wound healing, insulin secretion and a host of other important functions (Berridge et al., 1998). Consequently, to control all these signaling pathways, Ca2+ uses a diverse range of signaling mechanisms to convey the proper signal for the desired outcome.
To achieve specificity of the signals, cells have devised a multitude of means to separate the messages. This is accomplished by a variety of types, locations, amplitudes, kinetics and specific sources of Ca2+ signals. For example,
Ca2+ can enter the cytosol through the plasma membrane from a variety of types of Ca2+ channels or transporters, or Ca2+ can be released from internal stores through the action of second messengers like inositol 1,4,5-triphosphate (IP3) on the intracellular release channel, the IP3 Receptor (Broad et al., 1999).
Localization of Ca2+ signals is often manifested as microdomains classified by the types of channels involved. Specific examples of these are: sparklets from voltage-operated channels, sparks from ryanodine receptors, blinks which are
1 decreases in ER Ca2+ due to release, and puffs which are responsible for Ca2+ waves induced by IP3 receptors (Berridge, 2006). In addition, there are two types
of Ca2+ waves, intracellular and intercellular, and each of these wave types is
responsible for different signals (Bootman et al., 2001). There are also a
multitude of types of Ca2+ channels that help with specificity of the Ca2+
message. These include: voltage-gated channels (L-type, T-type, R-type, N-
type), pumps and exchangers, Ca2+ ATPases, receptor operated channels,
second messenger operated channels, and channels on mitochondria and
internal stores. Additionally, each of these sources of Ca2+ signaling may be
linked to specific physiological outcomes.
However, because Ca2+ signaling is so critical and utilizes such diverse pathways, disturbing the critical balance of Ca2+ levels can be devastating to cell health and physiological cell function. Correspondingly, many diseases involve an interruption or unbalance in a Ca2+-signaling pathway. For example,
Alzheimer’s Disease (AD), cancer, diabetes, and Parkinson’s disease, have all
been linked to a possible deregulation of Ca2+; and in some cases altering the
Ca2+ signaling has been considered a possible treatment mechanism. Ca2+ is
known to be involved in regulation of the cell cycle. For example, Kao et al (1990)
demonstrated that chelating cytosolic Ca2+ blocked the transition from
metaphase to anaphase in three different cell types, showing that altering
physiological Ca2+ can alter the cell-cycle. Additionally, Guderman and Roelle
(2006) found evidence suggesting that disruption of Ca2+ signaling was a key
event in the abnormal growth of some small cell lung cancers. Furthermore,
2 many neurodegenerative disorders have focused on Ca2+ dysregulation having a
role in the death of neurons. Ca2+ is a major player in the pathways of apoptosis,
having a role in both the intrinsic and extrinsic pathways of apoptosis, including
those induced by ROS, UV-fragmentation of DNA, and cytotoxic drugs
(Hajnoczky et al., 2003). Apoptosis is suspected to play a role in neuronal death
in diseases such as AD. Additionally, there are multiple diseases and disorders that involve defects of specific Ca2+ channels. For example, genetic defects in
voltage gated Ca2+ channels can result in disorders involving the musculature,
neurological, cardiac and vision systems. However, in order to exploit Ca2+
signaling pathways for treatment, a better understanding of the pathways must
be established.
One important element in understanding Ca2+ signaling is the role of
mitochondria. The ability of mitochondria to uptake Ca2+ was first discovered in
the 1960’s (Babcock et al., 1997). Mitochondria sequester Ca2+ through the
mitochondrial uniporter, a highly selective pore on the mitochondria that utilizes
the membrane potential of mitochondria to drive the uptake of positively charged
Ca2+ ions (Kirichok et al., 2004). The mitochondrial membrane potential is established through the electron gradient created by the proton-motive force, a mechanism in which positively charged hydrogen ions are moved from inside the inner mitochondrial membrane to outside the membrane. Thus, this process creates a negative charge on the inside of the mitochondrial membrane, which is the driving force for influx of positively charged Ca2+ ions. Therefore, the
3 mitochondrial membrane potential is essential for the maintenance of Ca2+
signaling in mitochondria.
Furthermore, mitochondria are intimately involved in shaping many Ca2+
signals (Duchen, 2000; Rizzuto et al., 2000). However, the role of mitochondria in
Ca2+ signaling pathways was not fully appreciated until the discovery of Ca2+ microdomains, discussed previously. For example, the role of mitochondria in capacitative Ca2+ entry has recently been identified. When Ca2+ is released from
internal stores, store operated channels (SOC) on the plasma membrane are
activated, causing them to open and influx more Ca2+. High levels of Ca2+ around the SOC can deactivate the channels, causing them to close. Mitochondria, however, can take up the excess Ca2+ around the SOC keeping the levels low
enough for the channel to remain open, allowing more Ca2+ to enter the cell.
Furthermore, mitochondrial Ca2+ can also be used to signal cellular events like apoptosis. Activation of the mitochondrial permeability transition pore (PTP) from
Ca2+ overload or other apoptotic signals can cause the initiation of the
mitochondrial apoptosis pathway, which will be further discussed later in this
paper.
In this body of work, I investigated to role of Ca2+ and mitochondria in
three different disease paradigms. The first is capacitative Ca2+ entry and the
mechanism of a drug that alters this pathway to treat some types of cancer. The
second is the toxic effects of a peptide, Aβ, thought to be involved in AD. Finally,
I investigated the mechanisms of Ca2+ entry in primary cortical neurons after
acute response to the neurosteroid allopregnanolone and the neurotransmitter
4 GABA, and the possible role of this pathway in catamenial epilepsy. Recent evidence has suggested a possible role of Ca2+ in the neurosteroid regulation of
GABA receptors indicating a role for Ca2+ in modulating epilepsy. Tus I dissected
the role of Ca2+and mitochondria in three separate disease states in order to
develop a better understanding of Ca2+ signaling and mitochondrial health in
disease etiology and treatment. I hypothesize that in each of these three disease
models, mitochondrial health and calcium signaling are ultimately involved in the
disease etiology.
5 Chapter II – Materials and Methods
This section contains material and methods for all reported data for this body of
work. Please refer to this section for all information regarding experimental
techniques.
Cell Preparations
HEK Cell Culture
HEK-293 cells stably expressing the M3 muscarinic receptor were cultured in Delbecco’s modified eagle medium (DMEM) supplemented with 10% fetal calf
serum and penicillin (200,000 U/mL) and streptomycin (200,000 U/mL). These
are non-malignant cells.
Preparation of single nerve endings
Isolated nerve terminals were prepared from male Sprague Dawley rats
(~250 g) following CO2 asphyxiation and decapitation. The neural and intermediate lobes were separated from the anterior lobe of the pituitary under physiological saline solution (PSS) (for ingredients see solution section). The neurointermediate lobe was removed from the neural lobe, and isolated terminals were prepared by trituration in 150 µl of buffer that contained (in mM) 270 sucrose, 0.01 Ethleneglycol-O, O’ bis(2aminoethyl)N,N,N’,N’-tetraacetic acid
(EGTA), 10 Tris-HEPES, pH 7.2. Terminals were allowed to adhere to clean
6 glass coverslips that formed the bottom of a culture/recording dish and used or
maintained in culture for up to 48hrs.
Primary culture of pituicytes
The isolated posterior pituitary was cut into approximately 6-8 small pieces
in PSS. Equal parts of thrombin and chicken plasma were used to make clots on
clean glass coverslips placed in a 6 well dish and a tissue piece was inserted into
each clot. After 3 to 5 minutes, DMEM containing 1% penicillin/streptomycin, and
10% fetal calf serum were added. Pituicytes grew out from the clot, and were maintained for 2 weeks at 37˚C, changing the media every 2-3 days. To confirm that our cultures were primarily pituicytes we fixed the cells and preformed immunocytochemistry with an antibody against glial fibrillary acidic protein
(GFAP) in combination with trypan blue staining (for total cell number) and found
>99% of the viable cells stained with trypan blue also had the GFAP marker.
Cortical Cell Culture
Primary cortical cells were prepared by Rebecca Pierson. Cortices from
E16 rat pups were removed and placed in Hank’s Balanced Salt Solution
(HBSS). All non-cortical tissue was removed and cortices were kept on ice.
Tissue was spun for 5 minutes, supernatant was removed and 0.25% trypsin-
EDTA was added and incubated for 11 minutes to break up the tissue. Trypsin
was removed and tissue was rinsed 3 times with S minimal essential media
(SMEM). After the third rinse cells were suspended in complete SMEM (SMEM
7 with 10% horse serum and 10% fetal bovine serum). Cells were plated at 1 x 106 cells/mL, then placed in an incubator and kept at 37ºC and 5% CO2. Cells were
grown for 2 weeks before imaging experiments were preformed. For neurons chronically treated with allopregnanolone, 1nM allopregnanolone was applied to
the cell SMEM at day 12 in culture and imaging experiments were preformed at
day 14 in culture.
Imaging
Wide Field Fluorescence Microscopy
Ca2+ measurements were obtained using a pressure driven perfusion
system. Wide fluorescence imaging was preformed in a Nikon TE2000-S inverted
fluorescence microscope (Nikon, Melville NY) equipped with a monochrometer-
based imaging system (TILL Photonics, Martinsried Germany) and a Nikon 40X
SuperFluor oil-immersion objective lens, NA = 1.3.
Confocal microscopy
Time lapsed 4-D images were acquired using a Leica TCS SP5
broadband confocal microscope (Leica, Mannheim, Germany) system coupled to
a DMI 6000CS inverted microscope equipped with Argon-488 and diode pumped
solid state-561 laser sources. Confocal z-series were obtained with a 20x
objective NA = 0.07 excited with 488 nm or 561 nm lasers. Acquisition of emitted
light was optimized using a tunable SP detector. Pituicytes and nerve endings
8 were loaded with tetramethylrhodamine methyl ester perchlorate (TMRM) at 1μM for 15 minutes at room temperature in the dark. Fluorescently tagged Aβ was added to media 15 minutes before imaging began. Cells were imaged every 15 minutes for 12 hours. During imaging, cells were kept at 37°C in a 5% CO2
environment using a stage top environmental Luden chamber. Analysis was achieved using Leica software and IGOR PRO.
Dyes and Loading
Cytosolic Calcium Measurements
2+ 2+ The ratiometric [Ca ]c indicator dye Fura-2 AM was used to measure Ca
influx in pituicytes and nerve endings. Both were loaded in PSS containing 1μM
Fura for 30 minutes at room temperature in the dark. Following loading cells were
placed back in dye free PSS and placed in a perfusion chamber on the same
inverted microscope as previously described. (Various stimuli were applied to the
cells to induce Ca2+ influx.) The cells were excited with 340nm and 380nm light
and emission light was collected at 540nm. The ratio of 340/380 was determined
using T.I.L.L. Vision software.
Mitochondrial Calcium measurements
Cells were grown on clean 25 mm glass coverslips, which formed the
bottom of a perfusion chamber. Cells were loaded with 2 μM Rhod-2 AM, Rhod-
FF AM, Rhod-5N AM mixture for 30 minutes in the dark at room temperature.
9 Cells were then permeabilized for 3 minutes at 37˚C in an intracellular saline solution (ISS) containing 10 μM digitonin, but with no added Ca2+ (for ingredients
of ISS see solutions section). Intracellular Ca2+ solutions were obtained by
adding N-(2-hydroxyethlenediamine-N, N’, N’-triacetic acid trisodium salt
(HEDTA)/ Ca2+. EGTA (250μM) was added to the “zero” Ca2+ solution, pH 6.8.
The intracellular Ca2+ solutions contained between 3-3000 mM Ca2+. All fluorescence data was converted to ΔF/F0=100[(F-F0)/F0], were F is the recorded
fluorescence and F0 is the average of the first 15 frames of data. Full frame
images were collected at 1-second intervals for at least 400 seconds and
changes are expressed as the percentage of increase compared with F0.
TMRM digital imaging analysis
Cells were loaded with either 12.5 nM or 1.5 μM TMRM in PSS for 15
minutes at 37˚C in the dark. Cells were washed with PSS and kept at room
temperature for 30 minutes before measuring to allow the dye to concentrate in
the mitochondria. Following loading, coverslips were mounted in chambers and
monitored by digital fluorescence imaging at 1Hz.
JC-1 optical measurement
Cells plated on 25 mM coverglass were incubated in phosphate-buffered
saline (PBS) supplemented with 2μg/mL JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-
tetraethylbenzimidazolylcarbocyanine iodide) for 30 minutes at room temperature
in the dark. Cells were then rinsed with PSS and kept at room temperature for 30
10 minutes to allow for dye to accumulate in the mitochondria. Coverslips were then
mounted in chambers and monitored by digital fluorescence imaging.
Solutions
Physiological Saline Solution: (PSS) containing 140 mM NaCl, 5mM KCl, 1mM
MgCl2, 2.2 mM CaCl2, 10mM HEPES, 10mM glucose, pH 7.2.
Internal Saline Solutions: (ISS) contained 130mM KCl, 10mM NaCl, 1mM
K3PO4, 1mM ATP, 0.02 mM ADP, 2mM succinate, 20mM HEPES, 2mM MgCl2
(adjusted to buffer Ca2+). Intracellular Ca2+ solutions were obtained by adding
HEDTA/ Ca2+. EGTA (250μM) was added to the “zero” Ca2+ solution, pH 6.8.
Hank’s Balanced Salt Solution: (HBSS) contained 7.5% Sodium Bicarbonate,
1% 10mg/mL Gentamicin and 3.574g HEPES, pH 7.3.
SMEM: contained Minimum Essential Medium with 2.703g glucose, 10mM l- glutamine, and 5% penicillin/streptomycin.
Preparation of beta-amyloid peptides
Aβ oligomers (Aβ25-35, Aβ35-25 from Sigma or Fluorescent Aβ1-42 from
AnaSpec) were prepared by reconstituting the lyophilized power with ddH20,
11 aliquoted and left at room temperature overnight (10-12 hrs) before being stored
at –20°C in the dark.
CAI: CAI (carboxyamidotriazole) stock was made up in DMSO and was diluted in
PSS to final concentration. CAI was a gift from the Drug Synthesis and Chemistry
Branch, National Cancer Institute (Bethesda, MD).
Neurosteroids: Allopregnanolone and inactive allopregnanolone (Sigma) were
made up at a stock concentration of 1μM in DMSO and was diluted to final concentration in PSS.
GABA: γ-aminobutyric acid (GABA) was made up in DMSO at 100μM and was
diluted in PSS to final concentration.
Plate reader
Mitochondrial Calcium Analysis
HEK-293 cells were plated in 24 well plates and grown to confluency, then
loaded with 2 μM Rhod-2 AM, Rhod-FF AM, Rhod-5N AM, and 15 nM
Mitotracker Green. Cells were then permeabilized with 10 μM digitonin in 0 Ca2+
ISS for 3 minutes at 37ºC. These conditions were optimized using digital fluorescence imaging (See previous section). Cells were then rinsed and placed in an ISS solution with a known Ca2+ amount from 3-3000 μM Ca2+. A Fluostar
12 Optima multi-well plate reader (BMG Labtechnologies) was used to detect the
fluorescence. Cells were excited at 550 and 485 and emissions were monitored
at 590 and 520.
TMRM Plate Reader analysis
HEK-293 cells were loaded with 1.5 µM TMRM in PSS for 15 minutes at
room temperature in the dark. Cells were then washed with fresh dye-free PSS
and spun down at 300 g for 5 minutes. Cells were the resuspended in 24 mL
PSS and aliquoted into a 24 well plate at 1 mL per well and allowed to rest for 30 minutes prior to monitoring by fluorescence plate reader. Cells were excited with
544nM light and emission was measured at 595 nM. Wells were measured at 1- minute intervals for 10 minutes prior to treatment. Wells were treated with 0 μM
CAI (control) 10 μM CAI of 20 μM carbonyl cyanide 4-
trifluoromethoxyphenylhydrazone (FCCP) and monitoring was resumed for an
additional 30 minutes.
JC-1 Plate reader analysis
Cells were incubated in phosphate-buffered saline (PBS) supplemented
with 2μg/mL JC-1 for 30 minutes at room temperature in the dark. Cells were
then centrifuged at 1200 g for 5 minutes and resuspended in dye free
physiological saline solutions (PSS) containing 140 mM NaCl, 5mM KCl, 1mM
MgCl2, 2.2 mM CaCl2, 10mM HEPES, 10mM glucose, pH 7.2. The cells were
then placed in 48 well culture plates (Corning plates 3548). A Fluostar Optima
13 multi-well plate reader (BMG Labtechnologies) was used to detect the
fluorescence emission at 590 nM and 520 nM in response to alternating 550 nM and 485 nM excitation, respectively.
Statistics
All data was analyzed with student t-test using GraphPad Prism 4, expressed as mean ± SEM. Significance was set as p<0.05
14 Chapter III: Background and Significance
This work focuses on three different Ca2+ signaling mechanisms, with an emphasis on their role in disease etiologies and mitochondrial involvement. First,
I reviewed capacitative Ca2+ entry and the role of this pathway in cell proliferation and the corresponding relationship to cancer. Secondly, I have discussed Ca2+ signaling at the synapse and the role of glial cells in modulating Ca2+ signals in neurons, as the synapse is the primary target in AD. I have also discussed the role of Ca2+ in neurodegeneration through excitotoxicity and apoptosis.
Additionally, I addressed voltage gated Ca2+ channels and their role in long-term potentiation and long-term depression in neurons. Thirdly, I have discussed the possible role of voltage gated Ca2+ channels in modulating GABA receptor subunit expression and the relationship of this to disease as they may play a role in catamenial epilepsy. Lastly, I have discussed the involvement of mitochondria in each of these signaling pathways or disease pathologies.
Capacitative Calcium Entry
Capacitative Ca2+ entry (CCE) was first described by Putney in 1986 as the continuous loading and discharge of an intracellular Ca2+ store (Putney,
2007). Currently CCE is believed to be a mechanism used by cells to replenish
Ca2+ stores when levels get too low. The Ca2+ store (endoplasmic reticulum, ER or sarcoplasmic reticulum, SR for example) extends some unknown signal to a channel on the plasma membrane (referred to as a store operated channel or
SOC), which allows the entry of Ca2+ into the cytosol from the extracellular
15 space. Entering Ca2+ is bound to mobile and immobile buffers or sequestered
into the ER or other Ca2+ sinks like the mitochondria in the area of the SOC.
When Ca2+ levels within the ER or Ca2+ store are replenished, the cytosolic Ca2+
levels rise and deactivate the SOC via Ca2+ dependent inactivation.
Evidence supporting this theory began when Takemura (1989) showed
that IP3 is not necessary for CCE by introducing the use of thapsigargin.
Thapsigargin allows the release of Ca2+ from internal stores without activating
IP3. Thapsigargin poisons the SERCA (sarcoplasmic/endoplasmic reticulum
calcium ATPase) pump of the Ca2+ store. The SERCA pump is a Ca2+ ATPase
on the ER/SR membrane, which binds calcium and transports it into the lumen of
the ER/SR. When it is poisoned by thapsigargin, it cannot pump Ca2+ into the
ER/SR. Thus, by treating cells with thapsigargin and observing CCE, Takemura
demonstrated that the release of Ca2+ from internal stores alone was enough to
2+ activate CCE; IP3 was not necessary. Additionally, this revealed that the Ca
entry was from across the plasma membrane (as experiments done in the
absence of external Ca2+ did not show SOC) and not a cycle of uptake and
release of Ca2+ from internal stores, as originally proposed.
The next step was to identify how the Ca2+ was entering the cell. The Ca2+
2+ current created by CCE is known as Icrac (Ca release activated current) and was discovered by Hoth and Penner (1992). Hoth and Penner showed a highly selective Ca2+ current that was vastly different from other Ca2+ currents
suggesting the molecular structure was also different from other known Ca2+
16 channels. Once the current was identified, investigations looking at the proteins
responsible for the SOC and mechanisms of activation of this channel began.
Initial investigations pointed to the TRP family of proteins. Hardie and
Minke (1993) were first to identify that the TRP channel was a Ca2+ channel
through a Drosophila mutant. In Drosophilia the TRP channel is involved in Ca2+
influx in response to light entering the retina. In the mutant Drosophilia, it was
found that a defect in the TRP gene caused a deficiency in the Ca2+ influx that
lead to a transient and unsustained response to light (Parekh and Putney, 2005).
Because it was discovered that TRP was activated downstream of PLC, and SOC entry was also known to be activated downstream of PLC, investigation of a possible link between SOC and TRP channels began (Parekh and Putney
2005). Three major subfamilies of TRP channels have been identified in mammals: TRPC (canonical), TRPV (vanniliod), TRPM (melastatin) and a
diverse list of functions for these channels have been established including a role
as thermal and noxious receptors and mechanoreceptors (Huang, 2004). TRPC has the greatest homology to Drosophilia and have been the most studied in
terms of a relationship to SOC and Icrac. TRP channels have 6 transmembrane
domains and probably assemble in homo or hetero tetramers to form cation
selective channels (Nilius and Voets, 2005). Most are permeable to Ca2+ and provide Ca2+ influx. Some have been shown to reside on intracellular membranes
(Huang, 2004).
TRPC have been shown to be activated by stimulation of isoforms of PLC.
They have been shown to be responsible for agonist-stimulated Ca2+ influx in
17 most cell types (Kiselyov et al., 2005). Most experiments studying CCE have
used either over expression of TRP channels or blocking endogenous TRP
channels. Expression in Sf9 insect cells demonstrated a greater Ca2+ influx after
thapsigargin exposure. However, it was eventually shown that TRP channels in
Drosophila are not related to SOC, but require downstream lipid products of PLC, like PIP2 (Parekh and Putney 2005). Consequently, their role in CCE is not fully understood. However, new evidence suggests a role for another protein, Orai1.
Orai was discovered by Feske et al (2006) when one case of hereditary severe combined immune deficiency (SCID) was found to have a mutation in
Orai1. Furthermore, Feske et al (2006) showed that this mutation affected store-
operated Ca2+ entry in T-cells, thereby affecting the immune response. Orai1 is
located in the plasma membrane, making it an ideal candidate for the store
operated channel. In a study by Yeromin et al (2006), point mutations in the S1-
S2 loop of Orai transformed the Icrac current into an outward rectifying current or a
non-conduction channel depending on the mutation. Similarly, Prakriya (2006)
showed that point mutations in Orai in the highly conserved transmembrane
region caused a decrease in Ca2+ influx and an increase in monocovalent cation
current for Icrac. These studies indicated Orai has a role in the current involved in
CCE, and perhaps may be the SOC channel itself.
Several theories for activation of the SOC have been proposed including a
diffusible message, conformational coupling or exocytosis (Putney, 2007). One
factor that has been identified as a possible diffusible messenger for CCE is CIF
(Ca2+ influx factor). CIF, discovered by Randriamampita and Tsien (1993) may be
18 a viable candidate for this job; however, the identity of the protein is still
unknown. Additionally, there are several studies pointing to a role of Stim1 in
CCE. First, knock-down experiments by Liou et al (2005) and Roos et al (2005)
of Stim1 (not Stim2) resulted in a reduction (or nearly complete inhibition) of Ca2+ influx by CCE. Immunocytochemistry experiments have shown that Stim1 co- localizes with both ER and plasma membrane. Additionally, Liou et al (2005) have demonstrated that Stim1 can rearrange into puncta accumulated near the plasma membrane after Ca2+ store depletion, while mutant Stim1 did not respond
to Ca2+ depletion. This indicates that Stim1 may be functioning as the Ca2+ sensor activating the SOC at the plasma membrane.
Further evidence that Stim is the Ca2+ sensor involves the EF hand region of Stim. This region of the protein is predicted to be in the lumen of the ER and is therefore hypothesized to be the Ca2+ sensor region, monitoring the Ca2+ levels
in the Ca2+ stores (Soboloff et al., 2006). Mutations studies in the EF hand region
caused Stim to be localized into puncta before CCE activation, and Ca2+
depletion had no further effect on localization. Additionally, the mutation caused
an increase in Ca2+ influx if the stores were filled, but failed to promote SOC influx in response to store depletion (Roos et al 2005).
Moreover, Soboloff et al (2006) and Zhang et al (2006) have identified
Stim and Orai to be necessary for CRAC channel function, and co-expression of
these proteins causes a synergistic effect to form a large Icrac current. Yeromin
(2006) showed that thapsigargin treatment enhanced interaction between Orai and Stim. Additionally, Prakriya et al. (2006) demonstrated that over expression
19 of both increased Icrac. Currently, the mechanism of CCE is believed to be that
Stim1 is activated when ER Ca2+ is low; Ca2+ is then released from internal
stores and Stim1 redistributes to interact directly with Orai1, which allows Ca2+
influx through the Orai channel at the plasma membrane allowing Ca2+ to
accumulate in the cytosol and to be taken up by the internal stores.
CCE and Proliferation
Calcium has the ability to regulate both cell death and cell proliferation in
many cell types. As cancer involves cells that are growing unnaturally fast,
targeting Ca2+ pathways involved in cell proliferation or apoptosis may have a
potential therapeutic benefit in the treatment of cancer. Many studies have
demonstrated that CCE plays a role in regulating cell proliferation. First, Ca2+’s
2+ importance in mitosis was demonstrated when caged Ca and caged IP3 were used to elevate cytosolic Ca2+ artificially in some cell types (Lu and Means,
1993). The induction of anaphase was observed as confirmed by the presence of nuclear membrane breakdown and condensation of chromosomes. Conversely, when chelators such as EGTA reduced intracellular Ca2+, onset of anaphase was
blocked, as noted by lack of nuclear membrane breakdown and condensation of chromosomes (Kao et al., 1990). These results support the idea that Ca2+ may have a role in both normal and pathological cell cycle events. Additionally, the
2+ use of caged IP3 suggests the possible role of CCE specifically in Ca ’s control
of cell proliferation.
20 CCE has been shown to affect proliferation in various cell types. Waldron
et al (1994) used thapsigargin (known to trigger CCE) in DDT-MF2, a hamster
smooth muscle cell, to cause growth-arrest in these cells and then used serum to
restore cell growth. Waldron showed that SERCA pump expression was
necessary for cell re-entry into cell cycle and cell division. Golovina (1999;
Golovina et al., 2001) has shown that maintenance of the sarcoplasmic reticulum
Ca2+ levels are essential for cell proliferation and enhancement of CCE increased proliferation in human pulmonary artery smooth muscle cells. Chao (1992)
2+ 2+ showed that release of Ca from IP3 dependent Ca stores in fibroblasts by thapsigargin could activate MAP kinase activity, known to be involved in cell proliferation. Additionally, Ciapa et al. (1994) has shown that bursts of IP3 resulting in Ca2+ oscillations are necessary for final cell division.
T-lymphocytes, or T-cells, are a highly proliferating cell type. Domletsch
and Lewis (1994) were the first to show that Ca2+ stores and store operated Ca2+
channels were involved in generating Ca2+ oscillations in T-cells. Subsequently,
Dolmetsch et al. (1998) showed that these oscillations controlled gene
expression and cell differentiation by altering the frequency of the oscillations.
Under normal conditions, T-cells are activated by CCE when an antigen binds to
2+ a T-cell antigen receptor and causes the generation of IP3, which releases Ca from internal stores and causes Ca2+ influx through the plasma membrane
(Dolmetsch et al. 1998). Increases in cytosolic Ca2+ concentration can activate
transcription factors such as NFAT, NFkB, JNK1, MEK2 and CREB, which
control several diverse pathways including cell proliferation, cell differentiation,
21 and cell death (Feske et al., 2001). Feske et al showed that in T-cells from a
SCID patient, there was a defect in transmembrane Ca2+ influx after thapsigargin
treatment. These cells also exhibited defects in NFAT translocation to the nuclear
envelope as well as gene expression changes compared to T-cells from a normal
patient. Artificially increasing cytosolic Ca2+ increased NFAT translocation in
patient T-cells. Additionally, effects of CCE as measured by thapsigargin were
also seen in fibroblasts from the SCID patient.
Mitochondria and CCE
In addition to other homeostatic mechanisms, mitochondria have been
shown to be an integral part of Ca2+ signaling. Moreover, mitochondria are an
important modulator of the CCE pathway. As mentioned before, high amounts of
Ca2+ at the SOC can shut down the channel. Under normal conditions, this may
mean that the internal stores have been replenished. However, evidence has
shown that mitochondria are involved in CCE signaling. For example, Park
(2001) has shown in pancreatic acinar cells, that there is a population of
mitochondria that respond to store operated Ca2+ influx. Lawrie (1996) also
showed that the location of mitochondria is important in their regulation of Ca2+
influx in endothelial cells.
Glitch (2002) used RBL-1 cells, a rat basophil leukemia cell line, to show that IP3 failed to activate SOC entry unless mitochondria were in an energetic
state. In this same cell line, Gilabert et al. (2001) also showed that Ca2+
dependent slow-inactivation of Ca2+ influx in CCE is regulated by mitochondria.
22 This ability of mitochondria to regulate Ca2+ influx was also shown by Malli et al
(2003). Malli also demonstrated that when mitochondrial Ca2+ influx was inhibited, ER Ca2+ stores refilling was prevented in an endothelial cell line.
Hajnoczky et al (1999) used imaging experiments in hepatocytes to show similarly that blocking mitochondrial Ca2+ uptake effected ER Ca2+ uptake after
IP3 activation. Furthermore, Bakowski and Parekh (2007) found that pyruvic acid, which is a rate-limiting substrate in mitochondrial respiration, increased SOC entry by decreasing the inactivation of SOC. These experiments illustrate that mitochondria affect two distinct aspects of CCE, sensitivity to Ca2+ release from internal stores and Ca2+ dependent inactivation of Ca2+ influx through SOC channels.
Additionally, the role of mitochondria in CCE Ca2+ signaling has been directly demonstrated. Hoth (2000) used patch-clamping techniques in T-cells to show the role of mitochondria in controlling Icrac. Additionally, they demonstrated that inhibiting mitochondria reduced intracellular Ca2+ and NFAT translocation.
Nunez (2006) showed that blocking mitochondria with FCCP inhibited not only
SOC entry, but also inhibited cell proliferation in Jurkat cells and a human colon cancer cell line. Additionally, Nunez showed that treating the cells with salicylate decreased mitochondrial membrane potential and decreased cell proliferation in the cancer cell line. This evidence clearly shows a potential therapeutic target for both CCE and mitochondria involved in CCE.
23
Calcium Signaling at the Synapse
Calcium signaling at the synapse is unique, yet maintains the diverse
range of signals previously discussed in non-excitable cells. The synapse
contains three main parts, a pre-synaptic element, a post-synaptic element and
the surrounding glial cells. Here I will discuss two types of Ca2+ signaling specific
to the synapse, neurotransmitter release and long-term potentiation (LTP). Next,
I will discuss neurodegeneration and the role of glia in excitotoxicity and Ca2+ signaling in neurons. Then I will review another type of Ca2+ regulated cell death
related to neurodegeneration, apoptosis. Lastly, I will review the role of
mitochondria in regulating neurotransmitter release and apoptosis.
Calcium and Neurotransmitter Release
To begin, one of the unique Ca2+ signaling pathways in neurons is
neurotransmitter release. Neurotransmitter release is regulated by a family of
proteins known as SNARES (soluble N-ethylmalemide sensitive factor protein
receptor). There are two subgroups in this protein family, t-snares (or q-snares),
which are on the plasma membrane or intracellular organelle target membranes,
and v-snares (or r-snares), which are on the vesicle to be released. A group of v-
snares and t-snares will interact to form a SNARE protein complex. Different cells and compartments within cells have different groups of these proteins. For example, specific sets of snares are important for the release of different types of neurotransmitters (Sudhof, 1995), while others are important for transporting
24 GLUT-4 in skeletal muscle cells (Slot et al., 1997). While it was thought that Ca2+
was somehow involved in this signaling, it was not known how until Walch-
Solimena et al (1993) discovered the protein synaptotagmin. Synaptotagmin has
conserved C2A and C2B domains that can bind up to 5 Ca2+ ions (Walch-
Solimena et al., 1993). When Ca2+ enters the presynaptic axon, it binds to
synaptotagmin and the SNARE protein complex initiates the fusion of the vesicle
with the plasma membrane and the contents of the vesicle are released into the
synaptic cleft and subsequently activate receptors at the post-synaptic
membrane.
Other Unique Aspects of Calcium Signaling in Neurons
Neurons, like many other cells, utilize patterned Ca2+ signaling to activate
transcription factors. The Ca2+ dependent activation or deactivation of specific neuronal genes contributes to long-term potentiation (LTP) and long-term
depression (LTD) respectively. During these processes Ca2+ enters the cell at
specific microdomains or with specific kinetics to activate transcription factors
that can modulate LTP and LTD to regulate synaptic plasticity. Calcium entry
through NMDA receptors promotes CREB signaling by initiating phosphorylation
of CREB and CREB dependent gene-expression (Riccio and Ginty, 2002). CREB
is a nuclear transcription factor that is responsible for expression of genes with a
CRE promoter (Carrasco and Hidalgo, 2006). CREB activation from Ca2+ entry through NMDA receptors at post-synaptic microdomains has been implicated in synaptic plasticity. Calcium entry at NMDA receptors has also been shown to
25 activate ERK, and this has been implicated as a requirement for some forms of
LTP (Carrasco et al., 2002). For example, certain long term spatial memory tasks
are ERK dependent as demonstrated by Thomas and Huganir (2004). NMDA is
not the only place for Ca2+ entry that activates transcription factors; Ca2+ entry at
L-type Ca2+ channels, a voltage gated Ca2+ channel has also been shown to
activate CREB, and also ERK and NFAT (Carrasco et al. 2002). NFAT is a
cytoplasmic transcription factor that is Ca2+ dependent via calcinurin (Carrasco et
al. 2002). Unlike CREB signaling, Ca2+ activates NFAT by removing the phosphorylation, which allows translocation of NFAT to the nucleus. NFAT can
2+ also be activated by Ca released from internal stores after IP3 stimulation. In
the next section I will review voltage gated Ca2+ channels and their relationship to
GABA receptors.
Voltage Gated Calcium Channels
There are three main groups of voltage gated Ca2+ channels (VGCC):
Cav1 (L-type), Cav2 (P, N, and R-type), and Cav3 (T-type). The letter
designations are based on the channels biophysical and pharmacological
features (Bidaud et al., 2006). Channels are also identified by their voltage
sensitivity as high voltage activated (HVA) or low voltage activated (LVA). We will
be focusing on the L-type channel. L-type channels were initially identified by
their sensitivity to dihydropyridine (Kanngiesser et al., 1988). There are four main
groups of L-type channels: Cav1.1 (found in skeletal muscle), Cav1.2 and Cav1.3
(found in neurons, cardiac muscle, smooth muscle, fibroblasts, and pancreatic
26 cells), and Cav1.4 (found in retina) (Lipscombe et al., 2004). The Cav1.1 are
essential for muscular control and movement, while the Cav1.4 are important in
vision and visual discrimination. I will focus on Cav1.2 and Cav1.3 and their roles
in neuronal Ca2+ signaling.
The Cav1.2 and Cav1.3 differ in their activation sensitivity: Cav1.2 is an
HVA, while Cav1.3 is LVA (Lipscombe et al., 2002). Tanabe et al. (1987) cloned
the first channel and this information was then used to screen for other subunits.
The L-type channel is best known for it’s role in excitation coupling to muscle
contraction in skeletal muscle (Beam et al., 1989). However, important functions
of the L-type VGCC in neurons include initiation of neurotransmitter release,
control of neuronal firing patterns, neurite outgrowth, and gene expression
(Lipscombe et al., 2002); (Kim et al., 1998).
The initial criteria for identification of L-type channels included activation
by strong depolarization, high sensitivity to dihydropyridine and its agonists and
antagonists, slow activation kinetics, Ca2+ dependent inactivation, and large
single channel conductance. However, many deviations from this criteria have
since been discovered including low voltage activation channels (Cav1.3),
differences in inhibition by certain antagonists, variation in activation kinetics, and others (Lipscombe et al., 2002; Lipscombe et al., 2004). These differences may be due to splice variants and post-translational modifications.
The L-type channel is composed of four subunits, α-1, α-2δ, β, and γ (Kim
et al., 1998). There are 10 genes that encode for the α1 subunit with over 1000
theoretical variants (Gray et al., 2007). There are 6 known isoforms (Kim , Chang
27 et al. 1998). The α1 subunit expression controls temporal and spatial expression
of the channel (Kim, Chang et al. 1998). It is the pore forming unit of the channel
(Bidaud et al., 2006). Additionally, the α1 subunit is the binding site for dihydropyridine (Kim, Chang et al. 1998). Alternative splicing occurs at sites that are important for controlling channel activity and, therefore, can affect channel biophysics, density, targeting, and coupling to downstream pathways (Gray et al.,
2007). The other subunits are encoded by 4 genes for Cavβ, 3 for Cavαδ, and
eight for Cavγ (Lipscombe et al., 2002); although not all the γ variants associate
with the other VGCC subunits.
Calcium Signaling Modulation of GABA Receptor Expression
As previously discussed, Ca2+ signaling in neurons can affect gene
transcription and expression. Recent evidence has shown that Ca2+ and
depolarization of neurons can affect the expression of GABA receptor subunits.
For example, Gault (1997) demonstrated that treatment of cerebellar granular
neurons in depolarizing media (elevated potassium) caused an eight-fold
increase in the expression of the GABA receptor subunit delta, and this effect
was reduced when cells were returned to non-depolarizing media. Depolarization can occur by activation of L-type VGCC. Additionally, studies using modulators of
PKC (Ca2+-phospolipid-dependent protein kinase) demonstrated that increase in
PKC activity caused a decrease in GABA receptor activity. In addition,
Leidenheimer and Chapell (1997) showed that the effects of PKC could modulate
28 the effects of the positive GABA receptor modulator, THDOC, a neurosteroid.
The PKC pathway can be activated by Ca2+ entry at L-type VGCC
More recent studies have linked both GABA and L-type VGCC to these
effects. GABA has been shown to cause an increase in intracellular Ca2+ in various cell types and different points in development. For example, Rivera et al.
(2005) demonstrated that early in development intracellular chloride is increased and GABA is depolarizing. Chavas et al. (2004) used cerebellar intraneurons to demonstrate that GABA agonists could induce a Ca2+ rise until postnatal day 20.
Pellistri et al. (2005) then showed that the Ca2+ rise in these cells was blocked by both bicuculline and nimodipine, a GABA receptor blocker and L-type VGCC
blocker respectively. Additionally, in whole brain cultures, Lyons et al. (2001)
demonstrated that when nifedipine blocked the GABA induced Ca2+ increase, the
down regulation of GABA receptors was also blocked. Additionally they showed
that this down regulation of GABA receptors was not seen with application of
high potassium alone, indicating that both GABA and Ca2+ influx were required
for the down regulation of GABA receptors.
Calcium and Neurodegeneration
Whereas Ca2+ has the ability to regulate cell growth and function, Ca2+
also has been linked to various forms of neurodegeneration (neuronal death).
Calcium can activate oxygenases, increasing oxidative stress altering
mitochondrial Ca2+ levels and energy metabolism (Mattson, 2007). Ca2+ can also
activate either directly or indirectly, calpains and caspases leading to degradation
29 of cytoskeletal proteins, membrane receptors and metabolic proteins (Stefanis,
2005). Additionally, Ca2+ can trigger apoptosis altering mitochondrial Ca2+ signaling by a process that will be discussed later. Calcium is also involved in excitotoxicity. Excitotoxicity is a process by which excessive glutamate release and glutamate receptor overactivation induce excessive cytosolic Ca2+ and
neuronal death (Mattson, 2007). Mitochondria are involved in excitotoxicity
because they help to buffer Ca2+ in the cytosol. Pivovarova et al (2004) has
shown that NMDA stimulation leads to an increase in cytosolic Ca2+ leading to a
subpopulation of mitochondria undergoing swelling and eventually releasing
apoptotic proteins. The role of mitochondria in neurodegeneration will be
discussed in more detail later. Astrocytes, however, may have a more significant
role in regulating glutamate and mediating excitotoxicity.
Calcium and Apoptosis
Apoptosis was first described by Carl Voght in 1842, but the name was introduced by John Kerr in 1972 to describe the formation of “apoptotic bodies” from a cell (Lawen, 2003). Characteristics of apoptosis include cell shrinkage, rounding up and loss of contacts with neighboring cells, ER swelling and formation of vesicles and vacuoles, chromatic condensation and fragmentation and cells breaking up to form “apoptotic bodies” containing the cellular contents
(Lawen 2003). While apoptosis occurs without the leakage of cellular contents, necrosis, another type of cell death, can cause leakage of the cellular contents
30 and inflammation. Excitotoxicity can lead to both necrotic and apoptotic death in neurons (Haeberlein, 2004).
Apoptotic cell death is involved in many different types of diseases including neurodegenerative disorders like AD, Parkinson’s disease, AIDS, DNA mutations in p53 and Bcl-2, auto-immune diseases, and cellular stressors and toxins such as oxidative stress and alcohol (Lawen 2003). There are two major pathways for apoptosis, the extrinsic pathway and the intrinsic pathway. The extrinsic pathway is activated by channels and proteins on the plasma membrane, which may or may not also activate the intrinsic pathway. The intrinsic pathway involves the mitochondria and will be discussed in more detail later. Calcium is involved in both pathways and there is evidence suggesting that apoptosis induced by reactive oxygen species, UV-irradiation, drugs, cytokines, and ischemia-reperfusion require Ca2+ (Hajnoczky et al., 2003).
Glial-Neuronal Crosstalk
Astrocytes have been shown to have roles in neurogenesis, neuroprotection and neurodegeneration. For example, Song et al (2002) demonstrated that adult astrocytes in hippocampus promote neurogenesis by directing stem cells to become neurons. Watts (2004) used electron microscopy to show glial cells engulfing axons during development, demonstrating a role in axon pruning. Additionally, under physiological conditions, astrocytes have a neuroprotective effect by controlling glutamate transmission and regulating glial glutamate transporters (Gegelashvili et al., 2007). Much of what is known about
31 the bi-directional signaling of glia and neurons in the CNS has been learned by
monitoring Ca2+ in glial cells (Metea and Newman, 2006). Recent evidence has
shown that glial cells play an integral role in synaptic signaling. For example,
Muller cells, a specialized glial cell in the mammalian retina, generate transient increases in Ca2+ in response to light, and these are modulated by neuronal
activity (Newman, 2005).
Additionally, Pfrieger (1997) demonstrated that glial cells are important in
the development of fully functional neuronal synapses in culture. Furthermore,
glial cells of the posterior pituitary are involved in the modulation of hormone
release from nerve endings (Hatton 2002). Moreover, hormones released by the
nerve endings of the posterior pituitary can have effects on glial cells with
consequences for neuronal activity. In addition, in the supraoptic nucleus, glia
surrounding the synaptic area appear to control glutamate uptake and activation of presynaptic glutamate receptors, having a role in neurotransmitter release
(Piet et al., 2004). The role of glia in modulation of neuronal glutamate uptake may also have an important role in neuronal excitotoxicity, as will be discussed later.
Glial Role in Excitotoxicity
Glutamate transporters are abundant on astrocytes and can regulate the
concentration of glutamate present in the synaptic cleft (Tilleux and Hermans,
2007). However, under pathological conditions, impaired glutamate uptake by
astrocytes can lead to overactivation of post-synaptic glutamate receptors
32 leading to uncontrolled Ca2+ influx and eventually neuronal death (Tilleux and
Hermans 2007). Additionally, Zhao et al. (2004) has shown that microglia can
influence the efficiency of glial glutamate uptake. Astrocytes, which are highly sensitive to changes in the local environment, can have glutamate uptake affected by neighboring cells or diffusible factors (Tilleux and Hermans 2007).
This is obviously a factor in the process of neurodegeneration in ALS
(amyotrophic lateral sclerosis). For example, Rao and Weiss (2004) showed that
ROS (reactive oxygen species) produced in neurons affected the surrounding
astrocytes causing deficiencies in their glutamate uptake causing an increase in
stress to neurons leading to a viscous cycle ending in neuronal death. Van
Damme et al (2007) showed more specifically that the SOD1 mutation, a
common mutation in familial ALS, in astrocytes caused a reduction in the GluR2
receptor subunit in motor neurons making them more susceptible to Ca2+ influx. It
has been demonstrated that ALS patients have elevated glutamate levels in their
cerebrospinal fluid in addition to a severe reduction of GLT1, an astrocytic
glutamate transporter (Barbeito et al., 2004). Many ALS patients have been
shown to have gliosis surrounding the motor neurons (Barbetio et al. 2004).
Inflammatory changes in reactive astrocytes can modulate the release of
glutamate, and the use of COX-2 inhibitors to reduce inflammation has been
shown to delay onset of disease (Drachman et al., 2002), indicating a role for
reactive astrocytes in the disease mechanism. Excitotoxicity has been indicated
in the pathology of several other neurodegenerative disorders; however, it is not the only pathway for cell death that involves Ca2+.
33
Mitochondria and Apoptosis
Mitochondria play a role in both the activation and prevention of apoptosis.
Additionally, they may also be involved in determining if cell death will occur via
apoptosis or necrosis. Apoptosis is characterized by maintenance of ATP levels,
as it is required for activation of some caspases and DNA fragmentation.
Therefore, if there is acute disruption of mitochondrial ATP production, necrosis
will occur; however, if there is a chronic decrease is ATP production, as is
common in neurodegenerative disorders and aging, apoptosis is the more
common path for cell death (Haeberlein 2004). There are several proteins
involved in the intrinsic apoptotic pathway. The most well known family of
proteins associated with mitochondria and apoptosis are the Bcl family of
proteins, which provide both pro-apoptotic and anti-apoptotic signals.
The Bcl proteins are separated into subgroups based on their structure:
the Bcl-2 like survival factors, the Bax like death factors, and the BH-3 only death
proteins (Borner, 2003). Bcl-2 and Bcl-xL are the most important anti-apoptotic
proteins (Lawen, 2003). It has been shown that these proteins do not increase
proliferation, but actually prevent apoptosis by delaying the cells entry into the s
phase of the cell cycle (Vaux et al., 1988). Additionally, Bcl-2 has been shown to
decrease endoplasmic reticulum Ca2+ levels (Vanden Berghe et al., 2003) and
Bcl-xL has been identified as a requirement for neuronal survival (Motoyama in
(Cory and Adams, 2002). The two other subgroups of Bcl proteins are Bax and
BH-3 only proteins, and they are both pro-apoptotic protein groups. These
34 proteins are thought to act by binding to the pro-survival proteins and rendering
them inactive (Cory and Adams 2002). Bid, a BH-3 only protein, is a protein that
promotes cell death by activating Bax and Bak, which are thought to provoke or
contribute to the permeabilization of the mitochondrial membrane (Cory and
Adams 2002).
Once the mitochondrial membrane is permeabilized other, pro-apoptotic
proteins can be released into the cytosol where they can initiate caspases and promote more downstream pathways of apoptosis. Among these proteins are: cytochrome c, Diablo/Smac, Omi/HtrA2, AIF, and endonuclease G (Haeberlein
2004, Hajnozky 2003). Cytochrome C release is important in neuronal apoptosis activation from a variety of cellular stressors. Additionally, is it associated with
Fig. 1 Mitochondria and Apoptosis
Fig. 1 Model of part of the intrinsic apoptosis pathway. This depicts
some of the proteins associated with mitochondrial apoptosis pathways.
35 striatal damage in Huntington’s disease (Kiechle et al., 2002). Once cytochrome
c is released it interacts with apaf-1 and caspase-9 to create the apoptosome,
which can then activate caspase-3, an effector caspase (Cory and Adams 2002).
Endonuclease G, once released, can translocate to the nucleus and initiate caspase-independent DNA fragmentation. AIF (apoptosis inducing factor) is also translocated to the nucleus and initiates caspase-independent nuclear damage.
AIF has been shown to be induced after various neurotoxic stimuli and models of traumatic brain injury (Haeberlein 2004). The release of these proteins may work in concert to activate apoptosis (Haeberlein 2004). Smac/Diablo and Omi/HtrA2 are believed to bind to inhibitors of apoptosis (IAP’s) thus promoting caspase auto-activation (Haeberlein 2004 and Cory and Adams 2002).
Mitochondria and Calcium Signaling and the Synapse
Mitochondria play an important role in regulating Ca2+ signaling in
neurons. In a study by Wang et al (2003) disrupting the distribution of
mitochondria in neurons caused an increase in cytosolic Ca2+ and a subsequent decrease in mitochondrial Ca2+ levels, thus, indicating that mitochondria respond to Ca2+ microdomains in neurons and can shape specific neuronal Ca2+
signaling. The involvement of mitochondria in neurotransmission was initially
suggested by Parducz and Joo (1976), when they used electron microscopy to
show mitochondria from “stimulated” nerve endings had an increase in
accumulated Ca2+ compared to mitochondria from “unstimulated” nerve endings.
36 Giovannucci et al. (1999) provided support for the idea that mitochondria can modulate neurosecretion when they showed that poisoning mitochondria enhanced the secretory response in chromaffin cells. Additionally, a genetic mutation discovered in the nerve terminals of Milton Drosophilia, showed impaired synaptic transmission. Stowers et al. (2002) demonstrated that this mutation was responsible for the disruption of mitochondrial translocation to the synapse. Furthermore, the Bcl-2 family protein, Bcl-xL, a mitochondrial associated protein, has been demonstrated to modulate synaptic transmission
(Jonas et al., 2003).
The Role of Mitochondria in LTP/LTD
While I have previously discussed that L-type VGCC have a role in LTP and LTD, mitochondria also have demonstrated a role in LTP/LTD and synaptic plasticity. For example, Calabresi et al. (2001) used inhibitors of mitochondrial complex II to produce LTP of NMDA mediated synapse excitation. Additionally,
Weeber et al. (2002) and Levy et al. (2003) showed that inhibiting mitochondrial porins (ion conducting pores on the mitochondrial membrane) and mitochondrial membrane potential caused deficits in long and short term synaptic plasticity.
Specifically, deficits in fear conditioning and spatial learning tasks were observed. Also, Gincel et al. (2000) demonstrated that glutamate could modulate function of VDAC (a mitochondrial porin protein).
37
Role of Dysfunctional Mitochondria in Neurological Disorders
Modulation of mitochondria complexes and porins has a role in diseases
including Huntington’s disease and epilepsy. The mitochondrial toxin 3-
nitroprotionic acid (inhibitor on mitochondrial complex II) has been shown to
evoke seizures and can mimic the pathology of Huntington’s disease in rats and
humans (Calabresi et al., 2001). AMPA receptor antagonists have been shown to
inhibit the occurrence of seizures induced by 3-nitroprotionic acid in rats
(Urbanska et al., 1999), indicating a possible role for Ca2+. Additionally, the mitochondrial porin VDAC has been demonstrated to be altered after seizure
(Jiang et al., 2007) and mitochondrial dysfunction and ultrastructural damage was observed after kainic induced seizure in rats (Chuang et al., 2004). Currently mitochondria and mitochondrial-associated proteins are being considered as a possible therapeutic target for seizure disorders (Henshall et al., 2000).
38 Chapter IV
What is the mechanism by which CAI (Carboxyamidotriazole) alters Capacitative
Calcium Entry?
Background and Lit Review
In this section, I investigated the mechanism of carboxyamidotriazole’s
(CAI) effect on CCE using digital fluorescence imaging and fluorescent dyes. CAI
is a drug currently in Phase II clinical trials for treatment of various types of
cancer (Hussain et al., 2003). I investigated the effects of CAI on mitochondrial
membrane potential using two different fluorescent indicators of mitochondrial
membrane potential. I have shown that CAI has an effect on mitochondrial
membrane potential in a time and concentration dependent manner. Next, I
examined CAI’s effect on mitochondrial Ca2+ uptake. Again I used fluorescent
dyes to show that CAI causes mitochondria to uptake less Ca2+. Thus, I have shown that CAI’s effect on CCE involves, at least in part, an increase in mitochondrial membrane potential and consequent decrease in mitochondrial
Ca2+ uptake.
Cancer and CCE
In cancer, the uncontrolled growth of cells can be caused by an increase
in proliferation of cells, or a decrease or defect in the ability of cells to undergo
apoptosis. Due to the importance of Ca2+ in both proliferation and apoptosis, Ca2+ signaling pathways have been the target of many treatment possibilities.
39 Two approaches to treatment via Ca2+ pathways have been utilized. One
is termed “prodrug” treatment. In this case a non-specific Ca2+ pump inhibitor is
expressed with a tumor-specific enzyme to target the drug to the cancer
(Monteith et al., 2007). An example of this type of treatment option is the use of
thapsigargin, an inhibitor of the SERCA pump combined with prostate-specific
antigen (PSA) a prostate cancer specific protease (Denmeade and Isaacs,
2005). In this case the thapsigargin is inactive until it reaches the target cancer
cells where it is activated and can potentially affect Ca2+ signaling. The second
approach involves direct targeting of specific isoforms of Ca2+ channels or pumps
that have been associated with a particular cancer. One potential area for this
type of treatment is the TRP channels.
Though the role of TRP channels in CCE is not fully understood, there is a
plethora of evidence that they are involved in Ca2+ deregulation in many types of
cancer. Specifically, the over-expression of TRPV6, and TRPM8 have been demonstrated in cancers including prostate, breast, colon, lung, thyroid, and ovarian carcinomas (Prevarskaya et al., 2007). Additionally, the down regulation of TRPM1 has been indicated as a prognostic marker for melanoma metastasis
(Bodding, 2007). TRPV6 has been indicated as being highly selective for Ca2+
(Luo et al., 2001); however patch-clamp recordings have not been able to confirm these findings (Bodding 2007). Never the less, the TRP family of proteins
remains a possible therapeutic target for many types of cancer.
Mitochondria are another potential target for cancer treatment. This is
because of the role mitochondria play in apoptosis. Though the known
40 involvement of mitochondria in malignancies is rare, all of the anti-apoptotic Bcl-2
family members could potentially have oncogene potential (Cory et al., 2003).
Additionally, some pro-apoptotic proteins have potential as possible therapeutic
targets. Thus, a drug or treatment that could affect both Ca2+ signaling pathways
involved in proliferation and mitochondrial pathways of apoptosis would have a
higher probability of being effective for multiple causes of cancer.
CAI (Carboxyamidotriazole)
CAI (carboxyamidotriazole) is a substituted carboxyamido-imidazole with a
halogenated benzophenone tail. It was first discovered by Kohn and Liotta (1990)
and identified as a novel antiproliferative and antimetastasis agent. Kohn and
others then began to examine the mechanism behind CAI’s beneficial effects.
They found that CAI inhibited Ca2+ influx, arachidonic acid release and cellular
proliferation (Kohn et al., 1994). Additionally studies by Enfissi et al. (2004)
showed that CAI blocked Ca2+ entry due to release of Ca2+ from internal stores.
In addition to the evidence of CAI’s effect on Ca2+ signaling, Perabo et al. (2004)
demonstrated that CAI induced apoptosis and decreased Bcl-2 expression in
bladder cancer cells. Bcl-2 is an anti-apoptotic member of the Bcl protein family, and is associated with mitochondria. Guo et al (2006) also showed an increase in
apoptosis and down regulation of Bcl-2 in human breast cancer cells.
The induction of apoptosis and alterations in expression of Bcl-2 indicate a
possible role for the mitochondria. As previously mentioned, mitochondria are a
major player in apoptosis, and they have a role in regulating CCE. Clearly this
41 evidence indicates a possible role of mitochondria in the mechanism of action of
CAI. Additionally, evidence form Glitch et al. (2002) showed that mitochondria
need to be in an energetic state or IP3 fails to activate SOC entry. Malli et al.
(2003) and Gilabert and Parekh (2000) also showed that mitochondria have the
ability to regulate Ca2+ influx in CCE. In fact, mitochondria affect both sensitivity
to Ca2+ release from internal stores and Ca2+ dependent inactivation of Ca2+ influx through SOC channels. Therefore, it is possible to manipulate CCE by altering the release of Ca2+ from the internal stores, blocking the CCE channels,
or inhibit the ability of mitochondria to uptake Ca2+, thereby causing the channel to shut down early.
As previously discussed, CCE appears to be involved in cellular
proliferation (Golovina, 1999). This means that manipulating CCE may allow scientists to manipulate cellular proliferation in disease. CAI is a compound currently in phase II clinical trial as an anti-cancer compound. It has been shown
to inhibit cellular proliferation in human cancer patients, with fewer side effects than other anti-cancer compounds (Haverstick et al., 2000). However, the mechanism by which it has an effect is not known. CAI has many effects on Ca2+
regulation, not all involved with CCE, however, NCCE is not thought to be
involved in cellular proliferation, and CCE has been shown to have involvement
in cellular proliferation. It is important to determine what the effects of CAI are on
CCE and what aspect of the pathway CAI affects.
In this chapter I will investigate CAI’s effect on CCE, and what aspect of
CCE CAI may be affected. First, initial work by Mignen shows that CAI does
42 cause a dose dependent decrease in CCE after CAI treatment. He also shows that CAI inhibits [3H] thymidine incorporation in HEK-293 cells indicating a role in proliferation (Mignen et al., 2005). Based on this information, the question remains is CAI having an affect directly on the SOC or is there an affect on the mitochondria, causing the SOC to shut off early (See Model Fig 2).
Fig. 2 Model of CAI’s Possible Effects on CCE
Fig. 2 Model of CAI’s possible effects on CCE. I hypothesize that CAI will have an effect on mitochondria, causing them to be less polarized and take up less calcium. This would cause a build up of calcium and deactivation of the SOC.
I will show that CAI affects mitochondria, causing them to have a less negative membrane potential, as measured with JC-1 and TMRE. Additionally, the mitochondria treated with CAI take up less Ca2+ than control mitochondria. Thus,
CAI inhibits the ability of mitochondria to uptake Ca2+, allowing Ca2+ to build up around the store-operated channels and causing them to shut down prematurely.
43 Results
Effects of CAI on amplitude of CCE
First, to show that CCE had an effect on CCE, Mignen used HEK-293
cells loaded with Fura-2 AM to measure changes in cytosolic Ca2+. To initiate
CCE, Ca2+ stores were depleted with Thapsigargin (1 μM) in an external solution
containing no Ca2+. After the internal stores were depleted, an external solution
with 2 mM Ca2+ was added back to the cells. The change in cytosolic Ca2+ after the addition of external Ca2+ was attributed to the store-operated channels.
Control cells were compared to those treated with CAI for time points between 10
seconds and 5 minutes at doses ranging between 0.1 μM and 10 μM. A dose
dependent effect was observed with the CAI causing a decrease in the amount of
Ca2+ entering the cytosol after application of external Ca2+. After 5 minutes of CAI
treatment, the half-maximal inhibition was observed with 0.5 μM CAI and
maximal inhibition with 2 μM CAI, while after 10 seconds 10 μM CAI reduced
CCE to 40% of control. Concentrations over 10 μM were not used due to
potentially toxic effects (Enfissi 2004).
CAI’s effect on HEK-293 cell proliferation
Additionally, to show that CAI had an effect on cell proliferation in HEK-
293 cells, Mignen assessed cell proliferation by measuring the incorporation of
[3H] thymidine. Cells were incubated for 24 hours in the presence of 10% FCS.
External medium was then supplemented with various concentrations of CAI and
incubated for a further 48 hours. CAI blocked the incorporation of [3H] thymidine
into HEK-293 cells with an IC50 of 1.6 μM whereas vehicle had no effect. This is
44 consistent with what has been previously shown in other cell types, that CAI
blocks proliferation of cells (Mignen et al. 2005).
CAI depolarizes the mitochondrial inner-membrane potential in a time
dependent manner.
As previously described, mitochondria play an integral role in the
mechanism of CCE. By buffering the amount of cytosolic Ca2+ available around
the store-operated channels mitochondria prolong the open state of the channels
and delay the Ca2+ dependent deactivation of the channel. Therefore, I monitored
CAI’s effect on mitochondrial health and their ability to uptake Ca2+. To determine the time dependent effects of CAI, I used a multi-well fluorescence plate reader in combination with the mitochondrial selective ratiometric dye JC-1. A description of the dye can be found in chapter two. The ratio of the red to green fluorescence of JC-1 is used to monitor mitochondrial membrane potential without effects due to mitochondrial swelling, which can occur when mitochondria become unhealthy.
45 Fig. 3 Effects of CAI on Mitochondrial Membrane Potential
CAI Dose response for JC-1 A B Effects of CAI on Mitochondrial Membrane Potential 2.5 1.0
0.9 2.0
0.8 Control 1.5 0.7 CAI
FCCP 0.6 F590/F520 F590/F520 1.0
0.5 0.5 0.4 0.0
-1 1 3 C 10 10 10 0 10 20 30 [CAI] (µM) Confocal Images of JC-1 Loaded Cells Time (min)
control CAI FCCP
Fig. 3 A. HEK-293 cells were loaded with JC-1 and treated with various concentrations of CAI. JC-1 fluorescence levels were collected with a multi- well plate reader and a dose response was calculated. B. A time course of the effects of CAI on mitochondrial membrane potential. Cells were loaded with JC-1 and analyzed with the plate reader. C. Visualization of mitochondrial membrane potential with confocal microscopy. Cells were loaded with JC-1 then treated with DMSO (control) CAI (10μM) or FCCP (20μM).
HEK-293 cells were loaded with JC-1 and then treated with 10 μM CAI in suspension. The ratio at 590-520 was calculated using the plate reader at intervals of 5 minutes for up to 35 minutes following application of vehicle (0.1%
46 DMSO), 20 μM FCCP or 10 μM CAI (Fig 3B). CAI significantly altered
mitochondrial membrane potential in a time dependent manner. These results
were confirmed using confocal imaging in HEK-293 cells loaded with JC-1 and
treated for 20 minutes with vehicle, FCCP or CAI. The distinct red punctate
signaling seen in the vehicle treated cells is replaced with a diffuse green staining
in cells treated with FCCP, and this is mimicked in cells treated with CAI (Fig.
3C), consistent with the plate reader results. Additionally, the plate reader was
used to determine a dose response for CAI’s effect of mitochondrial membrane
potential (Fig 3A). These results are consistent with the dose response for CAI’s
effect on CCE preformed by Mignen.
While the ratio-metric nature of JC-1 provides several qualitative
advantages of this dye, there is some evidence that JC-1 is not as quantitative as other mitochondrial membrane potential dyes. Therefore, to obtain quantitative information of the effects of CAI on mitochondrial membrane potential, I used the dye TMRM. TMRM provides information on mitochondrial membrane potential in two ways. First, for digital fluorescence imaging experiments, a low concentration
(12.5 nM) of TMRM was used to observe the spontaneous changes in mitochondrial membrane potential as well as the possible redistribution of dye
(Fig 4A). In this case, ROI’s were placed on structures identified as mitochondria.
Additionally, these experiments were performed using a high concentration of
47
Fig. 5 Effects of CAI as Measured by TMRM A B A1 A2 B1 B2 C
4500
4000
A3 TMRM Fluorescence 3500 B3 200 300
TMRM Fluorescence 280 TMRM Fluorescence 3000 180 260
240 2500 220 160
200 2000 180 140
160
1500 140 120
0 20 40 60 80 100 120 0 50 100 150 200 0 10 20 30 40 Time (Sec) Time (Sec) Time (min)
Fig. 4 A. Cells loaded with TMRM at 12.5nM and treated with FCCP. Images of loaded cells before (A1) and after (A2) FCCP treatment. A3 Example traces of ROI’s of mitochondria. FCCP was administered at 60 seconds. B. Cells loaded with 1μM TMRM. At this concentration dye quenches in the mitochondria and as they become depolarized the dye leaks out and the cytosolic signal increases. Images before (B1) and after (B2) FCCP treatment. B3 Example trace of cytosolic signal. FCCP was administered at 125 seconds. C. Plate reader experiement in which cells loaded with 1μM TMRM and treated with CAI at time 10 min. Open circles are CAI treated.
TMRM. In this case the dye is taken up into the mitochondria in larger quantities where it then quenches (gives off less fluorescence due to interference). As the mitochondria loose their negative membrane potential, the dye leaks out and the cytosol gives off a higher fluorescence (Fig 4B). Both of these methods indicated that CAI altered mitochondrial membrane potential significantly different from control, but not significantly different from the positive control, 20 μM FCCP. I performed the plate reader experiments (similar to those previously described 48 with JC-1) using a high concentration (1µM) of TMRM. These results also
confirmed the information obtained with the JC-1, demonstrating that CAI caused
a significant increase in the depolarization of mitochondria (Fig 4C).
CAI inhibits mitochondrial calcium import.
Because of the observed effects of CAI on mitochondrial membrane
potential, and the fact that mitochondrial Ca2+ uptake depends on the
mitochondrial membrane potential, I examined the effects of CAI on
mitochondrial Ca2+ influx. To determine if the effects of CAI on mitochondrial
membrane potential were in fact reducing the ability of mitochondria to uptake
Ca2+, HEK-293 cells were permeabilized with digitonin. This was done to bypass any possible effects of CAI directly on the store-operated channels or any other
aspect of Ca2+ signaling. Cells were loaded with a mixture of Rhod dyes, (Rhod-2
AM, Rhod-FF AM, and Rhod-5N AM) to be able to measure Ca2+ changes with a
greater dynamic range. To negate the possible effects of CAI on other aspects of
CCE or Ca2+ signaling, we permeabilized the cells with 10 μM digitonin for 3
minutes at 37ºC in a intracellular solution containing no added Ca2+ (Bruce 2004)
(See Fig 5).
49 Fig. 5 Model of Digitonin Permeabilized HEK-293 Cells
e d
c
b
300 Ca
20% a δF
50 s
abc d e
Fig. 5 Example of cells loaded with Rhod dyes and permeabilized with digitonin. After permeabilization, an ISS with 300μM calcium was applied. Trace represents an ROI (region of interest) placed punctuate fluorescent area thought to be a mitochondria or group of mitochondria.
Under these conditions the plasma membrane was permeablized, while
the mitochondrial membrane remained intact, as determined by JC-1.
Additionally, the permeabilization reduced the background cytosolic Rhod signal,
indicating that the plasma membrane was in fact permeabilized (Fig. 5a). (Cells loaded with Rhod had a strong fluorescence signal in the mitochondria and a minimal fluorescence signal in the cytosol. Cells that continued to exhibit cytosolic fluorescence after digitonin treatment were not included in the data.)
50 ROI’s were placed on fluorescent punctate within permeabilized cells.
Identification of these puncta as mitochondria was confirmed by co-labeling with
MitoTracker Green-FM, a dye that selectively loads into mitochondria.
MitoTracker dyes are not Ca2+ sensitive and the fluorescence is not altered by
Ca2+ changes. An added advantage of this approach was that Rhod signal could be normalized to the MitoTracker signal. The validity of the technique was confirmed using FCCP and observing a consequent reduction in fluorescence.
After permeabilization was established, cells were exposed to intracellular solutions containing a set amount of Ca2+ ranging from 3 μM to 3 mM. The change in Rhod fluorescence was monitored and maximal fluorescence change was used to determine significance.
For multi-well plate reader experiments, cells were co-loaded with Mito-
Tracker Green and Rhod dyes and the ratio of 590/520 nM fluorescence was
determined (Fig 6A). I chose to focus on 30, 100 and 300 μM Ca2+ due to the
established EC50 for control cells. Control cells showed an EC50 of ~190 μM for
2+ mitochondrial Ca uptake. Cells pretreated with 10 μM CAI showed an EC50 of
~180 μM Ca2+ (Fig 6C). Though this change is small, it indicates an attenuation in
the dose response curve for mitochondrial Ca2+ influx in cells treated with CAI.
Next, I returned to the digital fluorescence imaging experiments to observe the kinetics of mitochondria in cells treated with CAI. I found a significant effect of
CAI at 30 and 300 μM Ca2+, but not 100 μM Ca2+ in maximal fluorescence
change. However, as can be seen in sample traces, I also observed two different
51 Fig. 6 Effect of CAI on Mitochondrial Calcium Uptake
2+ 2+ 2+ 2+ A Effects of CAI on [Ca ]m B Change in [Ca ]m in Response to Applied Ca [Ca ]o 1.00 types of kinetic effects of CAI. In some cases, there is3000 a decrease in fluorescence
0.95 after initial Ca2+ uptake. This decrease in fluorescence may indicate that Control 1000 0.90 CAI mitochondria are undergoing activation of the mitochondrial300 transition pore. The F590/F520
100 mitochondrial0.85 transition pore is a large20 % pore that is thought to open30 in response to dF 25 s 3 0 5 10 15 20 25 30 35 Time (min) Time (min) Relationship of [Ca2+] to Change in [Ca2+] Effect of CAI Treatment on Ca2+ Response C o m D 140
120 Control CAI
100 CAI 10 % 80 dF 200 s 30 60
40 20
% Change in Fluorescence 20 10 0
0 1 2 3 4 % Change in Fluorescence 0 10 10 10 10 10 Control CAI [Ca2+] (µM) m
Fig. 6 A Cells were co-loaded with Rhod dyes and MitoTracker Green and permeabilized (as described in Methods). Analysis of fluorescence was preformed by a multi well plate reader. Cells treated with CAI showed a decrease in mitochondrial calcium uptake after treatment. Cells were treated with CAI and calcium at time 0. B Sample traces of mitochondrial calcium uptake for treatment with ISS with indicated amount of calcium. C Dose response for mitochondria after treatment with indicated amount of calcium and difference for CAI treated cells. D. Statistical analysis of difference in calcium uptake by mitochondria treated with CAI and sample traces. Two different type of responses were seen in cells treated with CAI, the last trace suggest some cells may be undergoing activation of the permeability transition pore. * indicated p<0.001.
52 mitochondrial apoptosis activation. Thus, if the pore is opening in response to
CAI treatment, one might observe leaking of the dye and a decrease in
fluorescence. More studies are needed to confirm this possible observation.
Discussion
CCE has been shown to be involved in cell proliferation in a number of cell
types. CAI, a compound in phase II clinical trial as an anti-cancer drug, has been
shown to decrease cell proliferation. Because this drug has been shown to be
useful in several types of cancer with fewer side effects than other drugs, it is
important to understand the mechanism of the drugs action. Because CCE is a
complicated pathway that involves several important and diverse areas,
unraveling the mechanism of CAI is complicated.
Mignen et al (2005) has shown that CAI had an affect on cell proliferation in HEK-293 cells causing a decrease in proliferation. Additionally, I have shown
that CAI had a negative effect on mitochondrial membrane potential which
correlated to a decrease in mitochondrial Ca2+ uptake compared to control. This
evidence indicates that CAI has an effect on mitochondria causing them to
become depolarized. This depolarization decreases the amount of Ca2+ the mitochondria can buffer around the store-operated channels causing them to deactivate early, causing a reduction in CCE. This reduction in CCE is thought to be responsible for the decrease in cell proliferation that Mignen and others have shown.
53 I have clearly shown that CAI has an effect on mitochondria causing a
significant effect on membrane potential and Ca2+ uptake. This effect is
somewhat unexpected given that CAI has few side effects and mitochondria are
so ubiquitous. This raises the question that there might be a difference between
mitochondria of cancer cells, and mitochondria of healthy cells. Are mitochondria
in cancer cells more susceptible to the effects of CAI? Could this make
mitochondria a potential target for cancer treatment? Additionally, the effects on
mitochondria membrane potential indicate that ATP levels could be disrupted
after CAI treatment. ATP levels are important in determining cell death pathways
(Haeberlein 2004). If ATP levels are affected, do they play a role in CAI’s ability
to decrease proliferation? Or are cell death pathways activated? Percabo and
Guo (2004) demonstrated that CAI caused an increase in apoptosis and
alterations in mitochondrial associated apoptosis proteins. May CAI’s effect on
mitochondria activate apoptosis in addition to disruption of CCE? Obviously more
studies are needed to assess these questions.
Lastly, due to the possible effect of CAI on ATP it is also important to
determine in the decrease in proliferation is truly a decrease in the number of cells proliferating or an effect of cell death via apoptosis or necrosis. As previously discussed, others have shown that CAI can increase apoptosis and
decrease mitochondrial associated anti-apoptotic cells. This indicates that it is
possible that mitochondria may play an additional role in the mechanism of CAI
by inducing apoptosis. I have shown that there seems to be two types of kinetic
responses in cells treated with CAI. In the second type of response,
54 mitochondrial fluorescence had a dramatic decrease after an initial increase. This may indicate that mitochondria are undergoing permeability transition, or opening of the mitochondrial permeability transition pore. This event has been indicated in the mitochondrial apoptotic pathway and represents an area for further investigation.
55 Chapter V:
Are There Differential Effects of Amyloid-Beta on Cytosolic Calcium and
Mitochondrial Health on Glial Cells and Nerve Terminals of the Hypothalamic-
Neurohypophysis?
In this section, I examined the differential effects of Alzheimer’s disease
(AD) on the hypothalamo-neurohypophysial axis (HN), an area of the brain not affected by AD. While many neurodegenerative diseases have demonstrated a deregulation of Ca2+ signaling, the mechanism of Ca2+ signaling in neurodegeneration is not fully understood. I have investigated the role of Ca2+ signaling in AD by comparing the effects of the toxic peptide Aβ on the HN to what has been shown in the literature for other brain areas that are affected in
AD. Additionally, I have focused on the effects at the synapse, because the synapse has been shown to be preferentially affected in AD (Selkoe 2002).
Alzheimer’s Disease
Alzheimer’s disease (AD) was first described by Dr. Alzheimer in 1906. It is a fatal disease that is the seventh leading cause of death in America today
(www.alz.org). AD is the most common form of dementia affecting memory, cognitive abilities, social behavior, and sometimes causing paranoia and depression. AD is the most common neurodegenerative disorder; there are two types of AD, early onset occurs before age 65, and is typically caused by a hereditary genetic mutation and traditional AD occurring in adults over 65. The
56 cause of the disease is still unknown, and while there are treatments for many of
the symptoms of AD, there is no cure, and AD is still a terminal disease.
While the cause of AD is still unknown, research has centered around the
two hallmarks of AD, plaques and tangles. Tangles are found within the neuron and composed of a hyperphosphorylated tau protein. Genetic evidence,
however, has identified the protein involved in plaques to be a more likely
candidate for causing, or being involved in the cause of AD. Plaques are seen in
most adults over 40; however, AD patients have an unusually large amount of
plaques, combined with other symptoms (Okamura et al., 2004). The main
constituent of plaques is a 40-42 amino acid peptide called amyloid-beta (Aβ). Aβ
comes from the native protein amyloid precursor protein (APP).
APP Processing
APP is a type 1 membrane bound protein found in brain and other tissues
(Canevari et al., 2004). The physiological function of APP is still unknown
(Kamenetz et al., 2003) though there is evidence for roles in G protein coupled receptor signaling, neurogenesis and regulation of excitatory synapses (Caille et al., 2004). Mouse knockouts of APP appear without noticeable phenotype, however it is thought that APP like proteins (APLP1 and APLP2) may compensate for the loss of APP (Caille et al., 2004). There are two main pathways for APP processing, a non-amyloidgenic pathway, and an amyloidgenic pathway. In the non-amyloidgenic pathway APP is cleaved by α-
secretase within the region of the Aβ peptide. The c-terminal fragment can be
57
Fig. 7 APP
β α γ
α β
γ γ
Fig. 7 Model of APP processing by the α, β, and γ secretases. The red box represents the β-Amyloid peptide. Cleavage by β and γ secretases gives the peptide.
further cleaved by β-secretase releasing a p3 fragment. The role of these protein fragments is not known, but they are not known to be toxic or involved in AD. The amyloidgenic pathway is responsible for releasing Aβ from APP by β-secretase and γ-secretase activity. The γ-secretase can cleave the Aβ at either the 40 or 42 residues (Canevari et al., 2004). The Aβ1-42 peptide is thought to be the more toxic form and is most abundant in plaques. The Aβ peptide is sticky and forms monomers, oligomers and fibrils. While the fibrils comprise the main component
58 of plaques, there is new evidence that oligomers of Aβ may be involved in earlier toxicity in AD (Takahashi et al., 2004).
Recently proteins responsible for secretase activity or modifying secretase activity have been identified. Although little information regarding α-secretase activity is known, the proteins responsible for β-secretase have been identified.
BACE1 and BACE2 are aspartic proteases that have been shown to be responsible for β-secretase activity. While the physiological function of BACE is still unknown, BACE has been shown to be increased in AD. However, BACE knockout mice show a normal phenotype (Canevari et al., 2004). The identity of
γ-secretase is also still unknown; however, it is believed that it is an enzymatic complex, which includes presenilins 1 and 2, nicastrin and possibly other proteins
(Li et al., 2003). Presenilin-1 is an integral membrane protein with 6-8 membrane-spanning regions (Begley et al., 1999). Mutations causing familial AD have been associated with the presenilin proteins as well as with the APP protein itself. Many transgenic mouse models of AD are based on mutations of APP. The most common is the “Swiss” mutation, TG2576, with a mutated APP 670/671
(Takahashi et al., 2004). Other mouse models of AD include multiple mutations on either APP, presenilin proteins, or tau associated mutations or combinations thereof.
Another important aspect of APP processing concerns the clearance of
Aβ. The only protein to be shown to cleave Aβ in vivo models (Li et al., 2003) is
Neprilysin. Neprilysin is a 90-110 kDa plasma membrane glycoprotein protein from the M13 family (Wang et al., 2003). Neprilysin (also known as CALLA10
59 because it was previously identified as Common Acute Lymphoblastic Leukemia
Antigen 10) is present both pre and post synaptically in brain regions vulnerable
to plaque formation in AD (Wang et al., 2003). Neprilysin can also hydrolyze and
inactive several other proteins, including enkephalin, ANP, endothelin and
substance P among others (Shirotani et al., 2001). Neprilysin has also been
shown to be damaged by 4-hydroxylnonenal, a by-product of lipid peroxidation, and has been proposed by Wang (2003) as a possible factor for Aβ accumulation
in AD. This is based on evidence that neprilysin mRNA and protein amount are
decreased in AD patients in areas of high plaque accumulation (hippocampus
and temporal gyrus) compared to controls (Yasojima et al., 2001). Other proteins
have been implicated as possible players in Aβ clearance. Insulysin, for example,
has been shown to cleave Aβ in vitro (Mukherjee et al., 2000) but not in vivo.
Aβ at the Synapse
Aβ has been shown to be toxic to neurons, but the nature of that toxicity
has yet to be elucidated. Evidence indicates that the synapse may be involved in
the earliest events in AD (Selkoe 2002). The synapse is the primary site of Ca2+ dysregulation in AD (Mattson 2003). In a study by Lacor et al. (2007) hippocampal cultures treated with Aβ were found to have a significant decrease in spine density and abnormal spine morphology. Takahashi et al. (2002) also saw abnormal synaptic morphology in AD brain. Stern et al. (2004) showed that
Aβ plaque formation in Tg2576 mice (an AD mouse model) caused significant impairment in synaptic function. Furthermore, Kamenetz et al. (2003) showed
60 that neuronal activity modulates formation and secretion of Aβ in hippocampus
and that Aβ depressed excitatory synaptic transmission.
Previous studies in cortical and hippocampal cultures have determined Aβ
treatment to be toxic to neurons, astrocytes, and synaptosomes. Fu et al. (2006)
used MTT assay to identify a loss of viability in primary cortical neurons after Aβ
treatment. Kelly and Ferreira (2006) and Ekinci et al. (1999) have determined
that Aβ treatment can cause dysfunction of Ca2+ regulation in both cortical and
hippocampal neurons. Aβ treatment has also been shown to affect both
mitochondrial dysfunction and Ca2+ deregulation in hippocampal astrocytes
(Abramov et al., 2003). Mantha et al. (2006) demonstrated that Aβ treatment
caused an increase in resting Ca2+ levels in synaptosomes. In addition,
mitochondrial respiration (Selkoe, 2002) and assays of mitochondrial enzyme
activity (Mantha et al., 2006) have been shown to be altered by Aβ treatment.
Based on this evidence I decided to look at the effects of Aβ on two elements of the synapse, astrocytes and nerve terminals of the posterior pituitary.
Posterior Pituitary as a model system to Study AD
One interesting aspect of AD that has not been investigated is the
differential effects of the disease on different areas of the brain. The
hippocampus appears to be affected early in the disease, while other areas remain unaffected until the late stages in AD. For example, although changes in the hypothalamic-pituitary-adrenal axis (HPA) have been reported in some late stage AD patients, Petrie et al (1999) and Hatzinger et al. (2007) both indicate
61 these changes resulted from upstream-mediated stressors, primarily from the hippocampus. Additionally, Touma et al. (2004) concluded that hypersecretion of glucocorticoid from the pituitary in TgCRND8 mice (an APP transgenic model of
AD) was also caused by changes in hippocampus. Consequently, the HN, unlike the hippocampus, is not a major area of neurodegeneration in AD. However, why the HN does not seem to be affected has not been investigated. In this work, I examined the possible role of Aβ in the differential effects of AD at the HN utilizing the unique architecture of the posterior pituitary.
The posterior pituitary is largely comprised of astrocytic glial cells called
pituicytes, neurosecretory terminals of neurons from the supraoptic nucleus, and
endothelial cells of the vasculature system. These nerve terminals release either
oxytocin (OT) or vasopressin (VP) into the bloodstream. OT is involved in uterine
contractions during labor and milk ejection during lactation; it also has
implications in aggression (Huber et al., 2005). VP is involved in regulation of
blood volume during dehydration and hemorrhagic shock; and was also recently
implicated in aggression (Huber et al., 2005). Correspondingly, OT and VP have
effects on tissues in mammilary glands, uterus, kidneys and vascular beds
(Hatton, 2002) and the amygdala (Huber et al., 2005).
The astrocytes of the posterior pituitary have roles in both maintenance of
nerve endings under basal conditions and maintenance of active states (Hatton,
2002). To sustain normal conditions, the pituicytes are flat and engulf the nerve
terminals separating them from the vasculature. The astrocytes may also release
large amounts of taurine onto the neurons, which inhibits the release of VP via
62 glycine receptors on the neurons (Rosso et al., 2004). During lactation or
dehydration, when the neurons are active, the glial cells retract from the basal
lamina and the nerve terminals, exposing the terminals to the vasculature
system. Subsequently, terminals become enlarged as they release their peptide
into the blood stream (Hatton, 2002). The release of VP can also cause a rise in
Ca2+ in astrocytes from internal Ca2+ stores that cause the release of glutamate,
which can prolong the activity of the terminals (Hatton, 2002).
To facilitate these studies, we used the neural lobe of the pituitary as a
tractable simplified model of hypothalamic tissue. The neural lobe contains
primarily, astrocytic glial cells called pituicytes, nerve terminals of supraoptic
magnocellular neurosecretory neurons that have their cell bodies in the
hypothalamus, and endothelial cells of the micro-vasculature. Therefore utilizing cultures from the posterior pituitary allows one to look at neuronal endings and
glia in a more simplified environment (Fig. 8A). In the current study I exploited the
unique architecture of the posterior pituitary to prepare pure cultures of isolated
central nerve terminals or their associated astrocytes to clarify the earliest Aβ-
mediated modifications of neuronal and glial health. In an effort to begin to
address the underlying causes of the HN resistance to early manifestation of AD,
I investigated differences, primarily focusing on Aβ-mediated mitochondrial
dysfunction and Ca2+ deregulation, in the HN in contrast to that reported by
others from cortex and hippocampus. I hypothesize that Aβ will preferentially
effect the glial cells of the HN sparing the nerve endings from the toxic effects of
Aβ (Fig. 8B).
63 Fig. 8 Possible Effects of Aβ on Cells of the HPA
1 Norm a l re d ox 2 Ph a se I 3 Ph a se II microenvironment Compromised Neuro- redox balance degeneration AB AB THI O LS Ca 2+
NO, RNS, ROS APP, neurotoxic peptides?
Fig. 8 Model of possible effects of Aβ on glia and nerve endings of the posterior pituitary. 1. Under normal conditions the glial cells are supportive of the synapse. 2. I hypothesize that during Aβ exposure the pituicytes will be preferentially affected. 3. Is there neurodegeneration of this model of a HPA synapse? I hypothesize that Aβ will have a differential effect on the cells of the posterior pituitary compared to cells from other brain areas, and that this differential effect may explain the differential effect of AD on this brain area.
In this study, I began by investigating the effects of Aβ treatment on mitochondrial membrane potential at several time points. I found a differential effect of Aβ on pituicytes and nerve terminals. Next, I examined the effect of Aβ on cytosolic Ca2+ levels. Consistent with the mitochondrial membrane potential results, I found a differential effect of Aβ on nerve endings and pituicytes.
However, when measuring the change in cytosolic Ca2+ after stimulus, I found both nerve endings and pituicytes displayed a higher influx of Ca2+ in response to stimulus after exposure to Aβ for 24 hours. Lastly, I observed the distribution of
Aβ using a fluorescently tagged Aβ peptide. Again, I found a differential effect of
Aβ on nerve endings and pituicytes. While the nerve endings showed no signs of taking up the peptide, the pituicytes exhibited uptake of the peptide after 3 hours.
64 I hypothesized that these differential effects of Aβ may play a role in the lack of
toxicity observed in the HN in AD.
Results
Mitochondrial Membrane Potential
As previously discussed, Aβ has been shown to affect mitochondrial
membrane potential in both isolated mitochondria and mitochondria of intact
neurons and glia. Therefore, I explored Aβ’s effect on mitochondrial membrane
potential in neurosecretosomes and pituicytes following 6, 12, 24 or 48 hours of
continuous Aβ treatment. To measure mitochondrial membrane potential I used
the potentiometric dye called JC-1, a positively charged dye that accumulates in
the mitochondria. For an explanation of how the dye works see methods section.
Pituicytes cultures were prepared by removing the posterior pituitary, placing it in
a clot on a clean coverslip and grown in complete DMEM for 14 days (see
methods). At this time the pituicytes grew out from the explants and were
identified by immunocytochemistry for GFAP (glial fibulary acidic protein as
shown in Fig 9A). Pituicytes grown 14 days in culture, were then treated with
25μM Aβ25-35 in DMEM for the indicated time point. Pituicytes were loaded with
JC-1 2μg/μL for 30 min in the dark at room temperature. As shown in Fig 9B, control cells demonstrated pituicytes with punctate red signal and minimal green background signal. However, after 24 hours Aβ treatment, pituicytes
65 Fig. 9 Effects of Aβ on Mitochondrial Membrane Potential of Pituicytes
A B E 125
100
75 *
C D 50 * * % of Control 25 *
0 Con 6hrs 12hrs 24hrs 48hrs FCCP
Fig. 9 Effects of Aß on mitochondria membrane potential in pituicytes. Differential interference contrast image of live pituicytes (A) and Immunocytochemistry of cultures for GFAP (B). Representative pictures of control (C) Aß 25 µM treated for 24 hrs (D) and FCCP (E). Membrane potentials as measured by JC-1 for pituicytes compared to control values. FCCP was used as a positive control. Significance indicated by * p< 0.001.
demonstrated much less punctate red signal and significantly higher green signal as shown in Fig 9C, thus indicating that the mitochondrial membrane potential was less depolarized after 24 hour treatment. The protonophore FCCP, a mitochondrial uncoupler, also caused a decrease in red signal and increase in diffuse green signal as can be seen in Fig 9D. The time course of Aβ treatment is illustrated in Fig 9E. As shown, Aβ treatment caused a significant biphasic decrease in JC-1 ratio values in a time dependent manner compared to control, indicating a significant decrease in mitochondrial membrane potential after 12 hours (0.2214 ± 0.01) 24 hours (0.35 ± 0.03) and 48 hours (0.64 ± 0.01) compared to control (p<0.0001). The levels at 24 hours were comparable to
66 those induced by FCCP (p< 0.001, 0.49 ± 0.05). The 6 hour treatment time point
was not significantly different from control (p= 0.08, 0.96 ± 0.01).
Next, to determine the effects of Aβ on mitochondrial membrane potential
in nerve endings, I utilized the same JC-1 dye in the neurosecretosome
preparation. In this case Aβ treatment was examined after 24 and 48 hours. As
shown in Fig. 10A, control nerve endings, in culture for 24 hours exhibited
punctate red signaling, indicative of healthy mitochondria. However, as shown in
Fig. 10C and 10D, nerve endings treated with 25 μM Aβ for 24 or 48 hours
respectively, also demonstrated punctate red signaling, contrary to what was
seen in the pituicytes. Fig 10D is again an example of FCCP treated nerve endings, illustrating uncoupled mitochondria. The comparison of JC-1 ratios in control versus Aβ treated nerve endings for 24 and 48 hours is demonstrated in
Fig 10E. As shown in Fig 10E, control nerve endings at 24 hours showed similar fluorescence intensity as treated nerve endings (1.11 ± 0.08, p= 0.37) as did control and treated at 48 hours (0.91 ± 0.07 p= 0.45). Similar to pituicytes, as shown in Figure 10D, FCCP treatment caused a rapid and significant decrease in mitochondrial membrane potential (0.26 ± 0.01, p< 0.001). Because of the semi- quantitative nature of JC-1 (as explained in the previous chapter), these experiments were repeated using TMRM (data not shown). TMRM is a fluorescent indicator of mitochondrial membrane potential; similar to JC-1, but it is a more quantitative dye. Results with this dye indicated a significant increase
67 Fig. 10 Effects of Aβ on Mitochondrial Membrane Potential in Nerve Endings Control Treated 125 A B FCCP 100
75
CD 50
% of% Control * 25
0
Fig. 10 Effects of Aß on mitochondrial membrane potential in nerve endings. Representative images of control (B) and Aß 25 µM treated nerve endings at 24 hrs (C) and 48 hrs (D). E. Relative measure measurements of membrane potentials by JC-1 for treated nerve endings compared to their time matched controls. FCCP was used as a positive control. * indicates p< 0.001.
in fluorescent signal in treated nerve endings. This indicates that the Aβ treated mitochondria retained a more negative membrane potential, in contrast to what was determined in the Aβ treated pituicytes and what others have shown in cortical synaptosomes (Begley et al., 1999).
Cytosolic Calcium Measurements
Mitochondria are known to buffer physiologically relevant cytosolic Ca2+
levels in the pituitary nerve endings (Giovannucci 1997). However, the ability of
mitochondria to uptake Ca2+ is dependent on the depolarization state of the
mitochondrial membrane. Therefore, I hypothesized that the decrease in
mitochondrial membrane potential in pituicytes following 24 hours Aβ treatment
would deregulate cytosolic Ca2+ signaling. Again, we treated pituicytes with 25μM
68 Aβ for 24 hours and found that treated pituicytes had a significant increase in
resting Ca2+ levels (686.9 ± 6 ratio units) compared to control pituicytes (658.6 ±
8 ratio units, p= 0.004 ) as measured with Fura-2 AM. To investigate the effects of Aβ on evoked rises in Ca2+ in pituicytes, the physiological agonist vasopressin
(AVP) was used at 100nM and 1μM. AVP was applied for 50 seconds to activate cytosolic Ca2+ increase in pituicytes; two concentrations of agonist were used to
activate varying levels of Ca2+. The maximal increase in fluorescence from baseline (resting fluorescence) after stimulus was used to estimate the increase
Ca2+. Surprisingly, none of the treatments showed a significant difference from control in the amplitude of Ca2+ influx. However, others have reported
heterogeneity of signal for pituicyte clusters versus isolated pituicytes (Venance
et al., 1997), (Troadec et al., 1999). I also observed two distinct populations that
differed in responses to stimulus, although in the current study we found no
consistent correlation of signal amplitude and cell confluency.
To distinguish between the two populations, the data was binned in
increments of 20 ratio units change (Ca2+ increase from baseline after stimulus),
plotted as histograms and Gaussian peak detection algorithm functions were
applied to assess the distribution of responses to AVP stimulus. This analysis
demonstrated that there were two populations of responses to AVP treatment.
These populations were identified as “low responders” and “high responders”.
69 Fig. 11 Effect of Aβ on Cytosolic Calcium in Pituicytes
A a1 C Control Treated 2000 100 μM AVP
1000
B b1 Fura Ratio
0 100 μM Low 100 μM High 1 μM Low 1 μM High
1 μM AVP
Fig. 11 Sample traces of cytosolic calcium signal in pituicytes loaded with Fura- 2 after indicated stimulus for 50 seconds (A-B). (All resting fluorescence was normalized to 0.) Bar graph of low responders and high responders for AVP stimulated pituicytes (C).
Cells with maximal changes fewer than 850 units were termed “low responders”,
and cells with maximal changes above 850 units were termed “high responders”.
We chose this cut off point based on the distribution of responses indicated by
Gaussian fits. Examples of these groups are shown in sample traces in Fig 11 A-
B. We compared “low responders” versus “high responders” for each AVP
stimulation condition using student t-tests. It was determined that three of the
four conditions showed a significant difference in maximal Ca2+ increase
following AVP stimulus. Both high and low responders were significantly
increased in treated pituicytes compared to control after 1 μM AVP stimulus (low
459.6 ± 18 ratio units, high 1246 ± 27 ratio units). In contrast, following 100 nM
70 AVP stimulus, only the high responders showed a significant difference in treated
versus control (high 100 μM 1214 ± 60 ratio units, low 1 373.3 ± 29 ratio units,
high 1 987.1 ± 52 ratio units, low 1630 ± 61ratio units; p<0.01). (Control 405.2 ±
23 ratio units, Treated 405.2 ± 22 ratio units p= 0.9) Data is shown in Fig. 11C.
These same experiments were repeated in the nerve endings. I
hypothesized that because there was no change in mitochondrial membrane potential, there would be no change in cytosolic Ca2+ handling. Surprisingly, there was an observed significant difference in the resting levels of Ca2+. Treated
nerve endings actually had lower resting Ca2+ consistent with the mitochondrial
membrane potential determined by TMRM (Control 718.4 ± 25 ratio units,
Treated 617.7 ± 25 ratio units, p= 0.01). Next, I wanted to assess Aβ’s effect on
evoked Ca2+ rise in the nerve endings. To accomplish this I used a PSS solution supplemented with elevated potassium (50mM) to activate voltage gated Ca2+ channels. High potassium solution was applied for 50 seconds to induce a rise in cytosolic Ca2+. In nerve endings treated with Aβ a significantly greater rise in
Ca2+ was induced compared to control (Control 800.6 ± 68 ratio units, Treated
1020 ± 56 ratio units, p= 0.01). Example traces are shown in Fig. 12A; data is
shown in Fig. 12B. Thus, in contrast to pituicytes the resting Ca2+ was decreased
in nerve endings, and the amplitude of the Ca2+ rise after stimulus was
significantly increased in treated nerve endings. These results were similar to
that previously demonstrated for cortical synaptosomes (Begley et al., 1999),
(MacManus et al., 2000).
71
Fig. 12 Effects of Aβ on Cytosolic Calcium in Nerve Endings
A 1250 Control B * Treated 1000
750 100 * 25 500
250 Fura Ratio 0 Resting Change
Fig. 12 Sample traces of calcium influx in nerve endings loaded with Fura-2 after high K PSS for 50 sec (A). (Resting fluorescence normalized to 0.) Bar graph of data for resting fluorescence and maximal change in fluorescence after stimulus with high K in control and treated nerve endings (B).
Fluorescent Aβ Uptake
As a result of these differential effects of Aβ, I wanted to investigate the pituicytes and nerve endings handling of Aβ. Fluorescently tagged Aβ peptide
(Fl-Aβ) (AnaSpec) was used to treat the cells. Fl-Aβ was applied to the culture media, just as the cells had been treated with Aβ in the previous experiments.
Confocal microscopy was preformed in the continuous presence of the Fl-Aβ.
Internalization of the peptide was observed in pituicytes over a 12 hour period.
The fluorescent signal was maintained for at least 48 hours. The time course of
Fl-Aβ internalization was obtained using a Luden Chamber and 4-D confocal imaging. We observed a rise in fluorescent signal
72 Fig. 13 Uptake of Fluorescent Aβ A B
15 min 45 min CD
2 hrs 3 hrs
Fig. 13 Uptake of fluorescent AB in pituicytes loaded with TMRM (red). Projected confocal images at indicated time after Fl-AB treatment (green) (A-D). Image stacks were collected every 15 minutes for 12 hours.
within 1 hour (Fig. 13A) and over a period of 3 hours (Fig 13D) after which the signal stabilized. To visualize the cytosol, pituicytes were loaded with a high concentration of TMRM. (For an explanation of high concentration loading of
TMRM versus low concentration loading see Methods sections.) Images were 73 taken every 15 minutes. There was no significant effect of Aβ seen with TMRM,
however photobleaching was observed within the first hour of analysis making
the results difficult to interpret (Data not shown). Additionally, I attempted to
locate subcellular compartmentalization of the peptide. To accomplish this I used
fluorescent markers for mitochondria and lysosomes; however, no co-localization
was found after 48 hours.
Subsequently, I applied Fl-Aβ peptide to the nerve endings, again in the
same manner as previously described. In contrast to what was observed in the
pituicytes, the nerve endings showed no indication of internalization of the peptide at time point up to 48 hours. Images were collected using wide field fluorescence imaging (Fig 14). This differential uptake may explain the differential effects of Aβ on mitochondrial membrane potential and cytosolic Ca2+.
Fig. 14 Nerve Endings Treated with Fluorescent Aβ
Fig. 14 Bright field image (A) and fluorescence image of NE treated with Fl-AB. No uptake was found in NE.
74 Discussion
In these experiments, I have shown that Aβ treatment differentially affects
nerve endings and pituicytes. Where as pituicytes showed a time dependent
biphasic decrease in mitochondrial membrane potential, the nerve endings
exhibited no change as measured by JC-1 and a trend to be more polarized as
measured by TMRM. Consistent with these results, Aβ treatment was shown to
alter cytosolic resting Ca2+ levels. While the pituicytes showed an increase in
resting Ca2+ after Aβ treatment, the nerve endings showed a decrease in resting
Ca2+ levels after Aβ treatment. Surprisingly, when the change in cytosolic Ca2+ after stimulus was measured, I observed an elevated Ca2+ increase in both nerve
endings and pituicytes after Aβ treatment. Lastly, using a fluorescently tagged Aβ
peptide I demonstrated that pituicytes could take up the peptide after 3 hours, while nerve endings did not show any indication of uptaking of the peptide up to
48 hours. This evidence suggests two important findings: first the differential effect of Aβ on the pituicytes and the nerve endings of the HN, second the contrasting effects of Aβ on nerve endings of the HN compared to what has been shown in other brain areas.
For example, the effect of Aβ on pituicytes was not unexpected given that
others have shown similar effect. For example, Abramov et al. (2004)
demonstrated that glia cells in co-culture demonstrated a less polarized
mitochondrial membrane potential after Aβ treatment. However, the effect on
nerve terminals was unexpected. Studies with synaptosomes from cortical tissue
have shown mitochondria to be affected by Aβ (Begley et al., 1999). Because
75 synaptosome preparations and the nerve terminal preparation of the HN are similar, it is surprising to see differential results between these two preparations.
One possible explanation for this dissimilarity is that mitochondria of nerve
endings from specific brain areas are more or less susceptible to toxicity by Aβ.
Additionally, the cytosolic Ca2+ results are similarly surprising. While
resting Ca2+ levels are consistent with the mitochondrial membrane potential
data, they again differ from what has been demonstrated in synaptosomes.
(Mungarro-Menchaca et al., 2002). To further demonstrate the role of
mitochondria in the change in cytosolic Ca2+, I measured mitochondrial Ca2+
changes using Rhod dyes. No significant changes were found for nerve endings
treated with Aβ, while pituicytes treated with Aβ showed a significant decrease in
mitochondrial Ca2+ levels (data not shown). This indicated that the effects of Aβ
on cytosolic Ca2+ may not be related to mitochondria, but may be caused by
another effect of Aβ.
This possibility was further demonstrated by the effects of Aβ on the
elevation of cytosolic Ca2+ after stimulation. In both pituicytes and nerve endings,
there is a significantly higher increase in cytosolic Ca2+ in treated cells and nerve
endings compared to control. These results are clearly unexpected and may
indicate that Aβ is affecting the nerve endings in a way that does not directly involve the mitochondria.
A second possible explanation for the differential affects of Aβ is that the
nerve endings of the HN handle Aβ differently than other neurons. To further
explore this possibility, I implemented a fluorescently tagged Aβ peptide to
76 investigate how the peptide may be affecting the pituicytes and nerve endings.
Again, I observed a differential effect of Aβ. While the pituicytes exhibited uptake
of the peptide after 3 hours, which was sustained for at least 96 hours. The nerve endings did not show any indication that there was any uptake of the peptide up
to 48 hours. Additionally, if it is shown that Aβ is taken up by cortical or
hippocampal neurons this may help to explain why the neurons of the HN are not
affect in AD. These observations may be a key to explaining the differential effect
of AD on the HN.
77 Chapter VI:
How Does Allopregnanolone Treatment Affect Calcium Signaling in Cortical
Neurons?
Allopregnanolone, a progesterone metabolite, has been demonstrated to
have an anti-convulsant effect in women (Herzog, 1995). Changes in
allopregnanolone levels during menses may be involved in the etiology of a type
of epilepsy called catamenial epilepsy which will be discussed later in this
section. Recent evidence in NT2-N cells suggests that allopregnanolone may
alter the expression of proteins involved in Ca2+ signaling pathways (Pierson
2005), suggesting that altered Ca2+ homeostasis may be involved. Consequently,
due to the role of mitochondria on seizure activity, and mitochondria’s role in the
shaping of Ca2+ signals involving LTP and LTD, assessing the effects of
allopregnanolone on mitochondrial function is also important. Because
allopregnanolone is a known GABAR modulator and GABAR have been a
primary site for epilepsy treatment, it is also important to investigate the possible role of GABAR in the anti-convulsant effects of allopregnanolone. I hypothesized
that GABA and allopregnanolone activate an efflux of chloride through the
2+ GABAAR initiating a depolarization and Ca influx through VGCC. This excess
cytosolic calcium may lead to the changes in GABAAR subunit composition
demonstrated by Pierson (2005) and may alter seizure susceptibility.
Mitochondria may play a role in this process by altering the Ca2+ signaling. I have
examined the ability of allopregnanolone to cause a Ca2+ influx in primary cortical
neurons. Additionally, I have examined to effects of chronic allopregnanolone
78 treatment on mitochondrial membrane potential and cytosolic calcium levels in
order to tease out the role of Ca2+ and mitochondrial health in catamenial epilepsy.
Catamenial Epilepsy
The word catamenial is derived from the Greek word, “katamenious” meaning monthly; and originally, catamenial epilepsy referred to the cyclical nature of seizures attributed to the cycles of the moon (Foldvary-Schaefer and
Falcone, 2003). Now, catamenial epilepsy refers to a woman with epilepsy whose seizures are in some way influenced by sex hormones secreted during
Fig. 15 Model of Hormone Fluctuations During the Menstral Cycle
Mensus Follicular Phase Luteal Phase
Ovulation
Fig 15 Model of hormone fluctuations during the mentrual cycle. The black line represents estradiol, the blue line represents progesterone, and the red line represents allopregnanolone.
79 the menstrual cycle. Sir Charles Locock first described the relationship in 1857
(Foldvary-Schaefer and Falcone, 2003). The percentage of women whom
experience catamenial epilepsy is thought to be between 10 and 70% (Reddy,
2004). This large range is likely because the basis for determining catamenial
epilepsy is often unclear as some women experience an increase of seizures at
different times during their cycle (Pierson et al., 2005).
The menstrual cycle consists of 3 main stages, with specific changes in
hormone levels (Fig. 15). First is menses, a 1-4 day period when the body rids
itself of the unfertilized ovum. At this stage, estradiol (a form of estrogen) is low, progesterol (a form of progesterone) is low and allopregnanolone (a progesterone metabolite) is also low. Next is the follicular phase, an 8-10 day period when the ovum is becoming mature and preparing to be released from the ovaries. During this period, estradiol levels rise where as progesterol and allopregnanolone levels remain low. At this point there is ovulation and the luteal phase begins. This is typically a 14 day period when estradiol levels decrease, but not to the low levels observed during menses. Conversely, progesterol and allopregnanolone levels increase (Reddy 2004, (Scharfman and MacLusky,
2006).
The three common patterns of catamenial epilepsy described by Herzog
et al. (1997) are classified by changes in seizure frequency during these three
stages of the menstrual cycle. These are: 1. perimenstural, an increase in
seizure frequency during menses, 2. periovulatory, an increase in seizures during
ovulation, and 3. inadequate luteal phase, which occurs in women who
80 experience an inadequate luteal phase (one lasting less then 14 days). In these
women, seizures are common in all phases except the mid-follicular phase. It is
thought that the cyclical changes in hormones, both increases and withdrawals
are responsible for these patterns of catamenial epilepsy (Reddy 2004).
Hormones
Estrogen
The three main types of estrogens are esterone, estradiol and esteriol
(Reddy 2004, Foldvary-Schaefer and Falcone, 2003). As previously mentioned, estradiol increases in the follicular phase and decreases slightly in the luteal phase then decreases more during menses. Estrogens have been shown to be
proconvulsant in both animals and humans, and are thought to act through
NMDA receptors (Reddy 2004). Estradiol was shown to facilitate kindling
seizures in animal models (Reddy 2004). Estrogen has been shown to increase
or exacerbate seizures when given intravenously to woman premenstrually
(Reddy 2004). Chronic exposure in rats to estradiol increased the number and
density of dendritic spines and excitatory synapses in the hippocampus of rats,
likely through activation of the NMDA receptors (Reddy, 2004). Clearly, estrogen
increases may play a role in seizure frequency in women.
Progesterone and Allopregnanolone
The three main types or progesterones are progestins, progestagens, and
progesterone (Scharfman and MacLusky, 2006). Progesterones have been
shown to be anti-convulsant, but the mechanism responsible for this effect is
unknown. One of the issues involved is that progesterone can be converted into
81 dihydroprogesterone, which is further metabolized into allopregnanolone (5α- pregnan-3α-ol-20-one). Allopregnanolone is an endogenous neurosteroid and a potent positive allosteric modulator of the GABAA receptor (GABAAR).
Progesterone has been linked to the etiology of catamenial epilepsy since
Laidlaw (1956) first suggested that seizure exacerbation was seen after rapid decline in progesterone, and natural and synthetic progestrin therapy have been beneficial for some women with catamenial epilepsy (Herzog, 1995), is it unknown if these effects are from progesterone or the metabolite allopregnanolone.
There is evidence that the anti-seizure effects or progesterone are not related to the progesterone receptor. First, the effects are rapid. This is not consistent with the actions of the progesterone receptor which acts by altering gene transcription (Reddy 2004). Additionally, the effects are not blocked by
RU486 a progesterone receptor antagonist (Mohammad et al., 1998). Also,
Herzog found that progesterone’s therapeutic activity required to conversion of progesterone to a 5α reduced metabolite, such and allopregnanolone (Herzog and Frye, 2003). Allopregnanolone was also shown to block the seizure facilitating effects of pregnanolone sulfate (Kapur, 2003). Allopregnanolone is converted from progesterone in both peripheral tissue and in the brain
(Corpechot 1993). The physiological concentration during the luteal phase is 2-
4nM (Reddy, 2003) Allopregnanolone is a specific modulator of the GABAAR; it does not act through the progesterone receptor.
82 Neurosteroid Effects on the GABAAR
As previously mentioned, allopregnanolone is a known GABAR
modulator. GABA is the major inhibitory neurotransmitter in the central nervous
system. It binds to GABAAR, GABAB receptors (GABABR) and GABAC receptors
(GABACR) usually leading to an inhibition of cell depolarization. In developing
neurons GABAAR, and GABACR have been shown to cause depolarization. The
GABAAR and GABACR are ionotropic channels; they form a chloride specific
channel (Macdonald and Olsen, 1994). They are inhibitory when chloride goes
into the cell and excitatory when chloride leaves the cell through the channel.
GABABR are metabotropic receptors, acting through a G-protein coupled second messenger or the receptor can be coupled to Ca2+ of potassium channels (Bettler
et al., 2004). GABA dysfunction at both receptors is thought to be involved in disease states, including epilepsy, addiction, depression, and schizophrenia
(Bettler et al. 2004).
Some research into how allopregnanolone and neurosteroids may affect
seizure susceptibility has indicated a role for GABAAR subunit subtype
expression. First, Pierson et al. (2005) used rt-PCR to show that progesterone
treatment caused an increase in α2 subunits of the GABAAR at both 2 and 7 days
of chronic treatment, while γ3 was decreased after 2 days, and α5 and γ3 were
both increased after 7 days. Smith et al. (2007) showed that allopregnanolone caused an increase in α4 and δ subunits. The importance of changes in GABAR subunits is discussed later in this section. Pierson et al. (2005) also showed changes in α4 after both progesterone and estrogen treatments. Both α4 and δ
83 subunits have been shown to alter GABAAR pharmacology and synaptic currents
(Smith et al. 2007). Wafford et al. (1996) showed that withdrawal of progesterone
caused an increase in α4 subunit expression and that this related to a
pharmacological change. Most of these studies, however, were done in cell lines,
and the effects of neurosteroids vary within brain areas, and even receptor
subunit configuration (Belelli and Lambert, 2005). Thus, while there is evidence
that these neurosteroids can have an effect on GABAAR, more information is
needed.
GABAAR
GABAAR are composed of 5 subunits making up a chloride pore. Each subunit has 4 transmembrane domains and different subunits have binding sites
for various agonists, antagonists and modulators. The channel typically has 3
open states, 10 closed sites and a desensitized state (Macdonald and Olsen,
1994; Enz and Cutting, 1999). However, these gating properties vary based on
subunit subtype composition, which will be discussed later. The desensitized
state is important in drug tolerance. There are several agonists and antagonists
as well as positive and negative modulators of the GABAAR. There are specific
binding sites for barbiturates, benzodiazepines, picrotoxin, and anesthetic
steroids. Selective agonists include GABA and muscimol, and bicuculline is a
competitive antagonist at this same site. Non-competitive antagonists include
picrotoxin, PTZ and TBPS (Macdonald and Olsen 1994). Benzodiazepines,
barbiturates, and anesthetic steroids are all agonists of the channel with there
84 own binding sites. There are also other modulators of the GABAAR that do not
have known binding sites. Allopregnanolone, for example, is a positive allosteric
modulator of the GABAAR, which will be discussed later in this section (Reddy,
2003).
As previously mentioned, there are several subunits of the GABAAR, and there are different subtypes of the subunits. The different subtypes of subunits give GABAAR different physiological and pharmacological properties. The
different subunits include: α1-6, β1-4, γ1-3, δ, ε, π, and σ (Pierson et al., 2005).
GABAAR are typically composed of 2α subunits, 2β subunits, and 1γ subunit. The subunits differ through development, in various cell types and brain regions, and with environmental exposure. Different combinations of subunit subtypes may be responsible for susceptibility to diseases and drug sensitivity and tolerance. For example, the α1 subtype confers binding of type 1 benzodiazepines, while α2, 3, and 5 confer type 2 benzodiazepine binding (Pritchett and Seeburg, 1991). There are many other examples of the specificity of subtypes. Αlpha subunits typically confer benzodiazapine and steroid modulation, while β and γ subunits have only been shown to affect benzodiazepine efficacy.
GABABBR
GABABR induce a slow inhibitory post-synaptic current because, unlike
GABAAR which open a chloride sensitive pore, GABABR are coupled to either G
2+ proteins, Ca channels or potassium channels. GABABR has only two subunits,
GABAB1 and GABAB2 and both are required for a functional channel. In fact,
85 without the GABAB2 subunit the GABAB1 subunit will remain at the endoplasmic
reticulum; GABAB2 is required to traffic the receptor to the plasma membrane
were it can interact with a G-protein or Ca2+ or potassium channel (Bettler et al.,
2004). GABAB1 has 7 transmembrane domains like other G-protein coupled
receptors. Baclofen (β-chlorophenyl-GABA) is the only available medication for
GABABR, but other antagonists include saclofen. Transcription factors like
CREB2 and ATF-4 are also able to interact directly with the GABABR (Bettler,
Kaupmann et al. 2004). There are isoforms of the GABAB1 subunit; the most
abundant are GABAB1 and GABAB2 (Bettler, Kaupmann et al. 2004). There are no
known splice variants of the GABAB2R.
Relationship between GABAR and L-type VGCC
Given the amount of evidence pointing to a role for the GABAR in epilepsy
and allopregnanole’s known activity at the GABAR, the question remained if the
GABAR and calcium signaling could be related. In fact, a bidirectional
relationship between GABAR and L-type VGCC has been recently suggested. In
a study by Katsura et al. (2007) chronic exposure to benzodiazepines, GABAR
modulators, resulted in a change in L-type VGCC subunit expression and ultimately caused a change in Ca2+ influx in response to depolarization in these
cells. Conversely, L-type VGCC blockers have been shown to alter the effects of
withdrawal from GABAR modulators. El Ganouni (2004) has shown that
nifendipine, an L-type VGCC blocker, prevented anxiogenic-like effects caused
by diazepam (a GABAR modulator) withdrawal when administered both acutely
86 or concurrently. Gupta et al. (1996) also showed that nifendipine, as well as
nimodipine and verapamil (also L-type VGCC blockers) showed dose dependent blockade of all withdrawal symptoms (including audiogenic seizures) of
lorazepam, another benzodiazepine. Clearly, these data indicate that GABAR
modulators and L-type VGCC have some relationship that should be further
investigated.
Fig. 16 Model of Possible Mechanism for Ca2+ Entry After Allopregnanole Treatment Cl- Allo
VGCC GABAA R
Ca2+ Ca2+ signaling Alteration of pathway GABAR subunit
Nucleus
Fig. 16 A model of the hypothesized effect of allopregnanole treatment on cortical neurons. First allopreganone activates an efflux of chloride from 2+ the GABAAR causing a depolarization and influx of Ca and after chronic allopregananole exposure mitochondria may alter Ca2+ signaling ultimately changing transcription and GABAR subunit composition.
I hypothesized that allopregnanolone activates an efflux of chloride
2+ through the GABAAR initiating a depolarization and Ca influx through VGCC.
This excess cytosolic calcium may lead to the changes in GABAAR subunit
87 composition demonstrated by Peirson (2005) which may alter seizure
succeptibility or drug efficacy. Mitochondria may play a role in this process by altering the Ca2+ signaling (Fig 16). In this study, I have examined the ability of
allopregnanolone to cause a Ca2+ influx in primary coritical neurons. Additionally,
I have examined the effects of chronic allopregnanolone treatment on mitochondrial membrane potential and cytosolic calcium levels in order to tease
out the role of Ca2+ and mitochondrial health in catamenial epilepsy.
Results
Dose Response for Allopregnanolone and GABA
To perform these and subsequent experiments, I used cortical neurons
prepared from embryonic rats and kept in culture for 14 days. Cells were loaded
with 2μM Fura-2 in the dark at room temperature for 30 minutes. Neurons were
identified by morphology (large cell body with processes) as determined by
Fig. 17 Immunocytochemistry of Cortical Neuron Cultures
MAP-2 GFAP
Fig. 17 Immunocytochemistry of cortical cultures. Both neurons and glia are present in the cultures. Data was collected only from cells with morphology similar to what was identified by MAP-2 staining. 88 immunocytochemistry of MAP-2 (Fig 17A) and other cells types were excluded from analysis in this study. Specifically, glial cells were identified by morphology as demonstrated by GFAP staining (Fig 17B).
After cells were loaded they were mounted on the bottom of a perfusion chamber. (See Methods for more details of this procedure.) Cells were then acutely treated with allopregnanolone or GABA for 5 seconds and Ca2+ changes were monitored by changes in the Fura-2 fluorescence ratio. The acute treatment of GABA or allopregnanolone caused a rapid increase in cytosolic Ca2+ that
Fig. 18 Effect of Acute Alloprenanolone or GABA on Cytosolic Calcium
A 1600 1500
1400
1300
1200
Fura-2 Ratio Fura-2 1100
1000
0 50 100 150 600 B Allo C 700 500
600 400 500
400 300
300 200 Fura-2 Ratio Fura-2 Ratio Fura-2 200 100 100 0
-9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Gaba (μM) Allo (μM)
Fig. 18 A sample trace of cortical neuron response to acute allopregnanolone treatment. Primary cortical neurons were loaded with Fura-2AM and treated with a 5 second application of indicated compound. B Dose response curve for GABA. EC50 was determined to be 30µM. C Dose response curve for allopregnanolone. EC50 was determined to be 100nM.
89 typically returned to baseline within 100 seconds (Fig 18A). To quantify this
change, I measured the maximal increase in fluorescence ratio induced by GABA
or allopregnanolone for a variety of concentrations (GABA: 1nM, 30nM, 1μM, 10
μM, 30 μM, 300 μM, 1mM and 3mM. Allopregnanolone: 1pM, 100pM, 1nM,
100nM, 1 μM, 10 μM, and 30 μM) and used IGOR softwear to create a dose response curve for condition. I then used the dose response curve to determine
the EC50 for each treatment (GABA: 30μM Allopregnanolone: 100nM ) (Fig 18)
which were both within physiological ranges for each compound. Unless
otherwise stated, the EC50 concentrations were used for all acute treatment
experiments.
To be sure that the GABA and allopregnanolone evoked Ca2+ rise was not
a non-specific effect, I used two controls, cholesterol and an inactive form of
allopregnanolone. Cholesterol was chosen because the structure is similar to
allopregnanolone, and the inactive allopregnanolone is a common control for
allopregnanolone. As shown in Fig. 19 acute treatment with cholesterol had no
effect on cytosolic Ca2+ levels (N=20), nor did the inactive allopregnanolone
(N=19). These results indicate that the evoked Ca2+ rise is specific for
allopregnanolone and GABA.
90
Fig. 19 Cortical Neurons Calcium Response to Cholesterol AB
1020
1000 1050 980 1000 960 950 940 Fura Ratio 900
920 Fura Ratio 850 900
0 20 40 60 80 100 120 800
Time (min) 0 20 40 60 80 100 Cholesterol Allo Time (min) Inactive allo Allo
Fig. 19 Rat cortical neurons were acutely treated with cholesterol or inactive allopregnanolone to test for non-specific affects on calcium uptake. No responses were seen with either compound. Active allopregnanolone was used as a control at (A) 90 second and (B) 75 seconds.
The Role of Internal Calcium Stores
After determining that acute treatment with allopregnanolone or GABA could induce a rise in Ca2+, I next wanted to identify the source of the cytosolic
Ca2+ increase. First, I investigated the possible role of internal Ca2+ stores,
2+ because GABABR are coupled to G proteins that can induce the release of Ca from internal stores and activate store operated Ca2+ entry. I began by using an inhibitor of internal Ca2+ store release 2-APB. This was shown to inhibit the rise of Ca2+ induced by ATP, which is known to activate Ca2+ release from internal stores. Cells were pre-treated for 30 minutes with 2-APB and then acutely treated with allopregnanolone or GABA while in the presence of 2-APB. For both conditions there was no effect of 2-APB on Ca2+ increase (GABA control 224.8 ±
91 29.46 treated 249.4 ± 54.59; Allo control 236.4 ± 29.46 treated 277.7 ± 18.31)
(Fig. 20C). Additionally, to determine if external Ca2+ was required, cells were
stimulated in the presence of 0 Ca2+ external solutions (0 Ca2+ PSS). (See
methods for complete description of 0 Ca2+ PSS.) Cells were put in 0 Ca2+ PSS for 2-5 minutes before acute treatment with GABA or allopregnanolone. In both cases there was no observed Ca2+ increase when cells were in 0 Ca2+ PSS
(GABA N=28; Allo N=18) (Fig 20A-B). Cells were treated with 100μM ATP as a control as it does not require external Ca2+ to cause cytosolic Ca2+ increase. This
evidence indicated that the Ca2+ increase was likely from an external source.
Fig. 20 Role of Internal Calcium Stores A B 1600 1200
1400 1100 1200 1000 Fura Ratio Fura Ratio 1000 900
800
0 50 100 150 ATP Data 1 0 50 100 150 200 TIme TIme 450 400 ATP 350 300 250 200 Fura Ratio 150 100 50 0 Allo Allo 2-APB GABA GABA 2 APB
Fig. 20 Cultered cortical neurons were acutely treated with GABA at 25 sec (A) or allopregnanolone at 25 sec (B) in the presence of 0 calcium. No response was observed. Cells were treated with ATP as a control at (A) 125 seconds (B) 100 seconds. C. Cells were treated with 2-APB. No effect on calcium entry was observed.
92 Role of L-Type Voltage Gated Calcium Channels
To determine if L-type Ca2+ channels could be the source of Ca2+ influx, I
utilized the L-type VGCC blocker nifedipine. I investigated the role of these
channels because they are fast activating and abundant on neurons, making
them a good candidate for the rapid Ca2+ increase seen after acute treatment.
Additionally, L-type VGCC blockers have been shown to affect changes in Ca2+ signaling associated with effects of GABA modulators (Rabbani and Little, 1999), and allopregnanolone is a GABA modulator. Cells were pretreated with nifedipine for 3 minutes before stimulation with acute allopregnanolone or GABA. In the presence of nifedipine, no Ca2+ increase was observed (GABA N=23; Allo N=42)
(Fig 21). This indicates that the L-type VGCCs may be involved in the Ca2+ influx induced by GABA or allopregnanolone acute treatment.
Fig. 21 Effect of Nifedipine and Bumetanide on Calcium Influx
B A 1200
1100 500
450 1000
400 900
350 Fura-2 Ratio Fura-2 Ratio 800 300
0 50 100 150 TIme 0 50 100 150 200 TIme GABA ATP GABA ATP
Fig. 21 Nifedipine, a blocker of L-type VGCC successfully blocked the calcium increase induced by acute GABA or allopregnanolone treatment. (A) Cortical Neurons in the presence of nifedipine were treated with 30μM GABA at 20 seconds. Cells were then treated with 100μM ATP at 110 seconds as a control. (B) Bumetanide, a blocker of KCCl1 transporter also blocked the acute effects of allopregnanolone or GABA treatment. In this figure, cells were treated with 30μM GABA in the presence of bumetanide, and with 100μM ATP as a control at 70 seconds. 93 GABA Depolarization
These results were puzzling, because allopregnanolone is not known to
have a direct effect on L-type VGCCs; but somehow these channels appeared to
be activated after acute treatment. To address this question we used a blocker
for the NKCCl1 chloride exchanger, bumetanide. If GABA and allopregnanolone
acute treatment were causing an efflux of chloride and subsequent depolarization
of the cell to activate L-type VGCCs, then disrupting the chloride gradient would
inhibit this effect. Bumetanide disrupts the chloride gradient by inhibiting the
NKCCl1 chloride exchanger. Cells were pretreated with bumetanide for 30
minutes after being loaded with Fura-2. In the continued presence of
bumetanide, neither GABA nor allopregnanolone were able to induce a Ca2+ increase in cortical neuron cultures (GABA N=30; Allo N=21) (Fig. 21B). This indicates that the acute treatment of GABA or allopregnanolone causes a depolarization through the efflux of chloride, inducing a calcium influx through the
VGCC, however because bumetanide diminishes the chloride gradient, this does not indicate what channel is involved in the chloride efflux.
GABA Channel Blockers
Because allopregnanolone is a GABAAR modulator, and previous data
has shown affects of allopregnanolone on GABAAR subunit expression (Peirson
et al 2005) I first looked at the possible role of GABAAR in the depolarization of
the neurons. As described previously, there are three types of GABA channels,
GABAA, GABAB, and GABAC. To investigate the role of the GABAAR, I used a
94 GABAAR antagonist bicuculine. Bicuculine requires an initial activation of the
GABAAR to block the channel, so we pretreated the cells for either 5 or 100 seconds. In both cases we saw no effect of bicuculine to block the Ca2+ increase
induced by GABA or allopregnanolone (GABA 224.8 ± 14.22 control 176.1 ±
21.43 treated; Allo 236.4 ± 29.46 control 308.4 ± 43.60 treated) (Fig 22A). Next, I
looked at the GABAB channel using saclofen, a GABAB antagonist. Again cells
were pretreated for 30 minutes before acute treatment with GABA or
allopregnanolone. Again, there was no effect of saclofen on the Ca2+ increase
(GABA 224.8 ± 14.22 control 220.6 ± 28.9 treated; Allo 236.4 ± 29.46 control
324.2 ± 64.97 treated) (Fig 22B). Lastly, I used the GABAAR and GABACR blocker picrotoxin. Once more there was no change in Ca2+ increase after acute
treatment (data not shown). Because the channel often has to be opened to see
an effect of picrotoxin, I also tried acute treatment of GABA before application of
picrotoxin, then a second acute treatment of GABA or allopregnanolone. This
also had no effect on Ca2+ increase (data not shown).
Fig. 22 Role of GABA Channel Blockers Data 1 B A Data 1 400 400
300 300
200 200
100 Fura-2 Ratio Fura-2 Ratio 100
0 Allo Con Allo Bic Gaba Con Gaba Bic 0 Allo Allo SAC GABA GABA SAC
Fig. 22 Blockers of GABAAR (A) bicucline or GABABR (B) saclofen had no effect on the increase in cytosolic calcium induced by either allopregnanolone or GABA. 95 Effect of Chronic Treatment
Once I established that allopregnanolone can induce a Ca2+ increase, I next wanted to examine the effects of chronic allopregnanolone treatment on
Ca2+ signaling. Based on previous work by Pierson (2005), chronic treatment was established to be 48 hours treatment with 1nM allopregnanolone. After 48 hours the allopregnanolone was washed off and cells were loaded with Fura-2 just as in the previous experiments. Because of the role of mitochondria in Ca2+ signaling and synaptic plasticity, I first looked at the effects of chronic allopregnanolone treatment on mitochondria. We used the mitochondrial membrane potential dye
JC-1 to observe differences in mitochondrial membrane potential after chronic treatment. I found that the chronically treated neurons exhibited a significantly different membrane potential compared to control (Fig 23) (p<0.001, 2910 ±
72.49 control 872.4. ± 58.96 treated) This effect could be involved in altering the
Ca2+ signaling induced by acute GABA or allopregnanolone treatment.
Fig. 23 Effect of Chronic Allopregnanolone on Mitochondrial Membrane PotentialData 1 3000
2000 TMRM
1000
0 Control Chronic Treated
Fig. 23 Cortical cells treated with 0.1% DMSO (Control) or 1μM allopregnanolonefor 48 hours then loaded with TMRM 12.5nM. Cells treated with allopregnanolone showed a significacntly lower TMRM fluorescence then control cells. * indicates p<0.001. 96 I next utilized the Ca2+ indicator dye Fura-2 to determine changes in resting Ca2+ levels, and induced Ca2+ levels. First, I demonstrated a significant
change in resting Ca2+ levels in chronically treated cells. A significantly higher
resting Ca2+ was observed in neurons chronically treated with allopregnanolone.
This effect was expected given that mitochondrial membrane potential was
significantly less negative. Because there are two channels involved in the Ca2+ increase after allopregnanolone stimulation, I used a high potassium stimulation to investigate changes in VGCC. I found no significant difference in the Ca2+ influx induced by high potassium. Next, I used Ca2+ increase induced by acute
allopregnanolone or GABA stimulation. I chose three different concentrations of
GABA or allopregnanolone for the acute treatment: a low concentration (1pM
allopregnanolone, 10 μM GABA), middle (100nM allopregnanolone, 30μM
GABA), and high concentration (10μM allopregnanolone, 300μM GABA). These concentrations were chosen based on data from the dose response curves, the low concentration was at the bottom of the curve, the high concentration is at the top of the curve, and the middle concentration is the EC50. Chronically treated
cells showed a significantly lower Ca2+ increase after acute GABA treatment (Fig
24B) (GABA: low p=0.0042, 303.6 ± 42.41, 78.44 ± 13.29; middle p<0.0001,
224.8 ± 14.22, 74.67 ± 14.73; high p<0.0001, 680.0 ± 61.90 222.0. ± 42.93;
allopregnanolone: middle p<0.0001, 236.4 ± 29.46, 27.88 ± 2.083; high p<0.001,
459.6 ± 42.71, 33.14 ± 5.352). Additionally, chronically treated cells exhibited a
significantly lower Ca2+ rise induced by acute allopregnanolone treatment (Fig
24A); and the low concentration had no indication of a rise in Ca2+ in the
97 chronically treated cells. These results indicate that chronic allopregnanolone
treatment caused a change in some aspect of the mechanism of Ca2+ increase
induced by acute GABA or allopregnanolone treatment.
Fig. 24 Effect of Chronic Allopregnanolone Treatment on Calcium Influx
A Data 1 B Allo GABAData 1 600 750 500
400 500 300 * 200 Fura-2 Ratio Fura-2 Ratio 250 100 * *** 0 0 1pM 100nM 10μM 10μM 30μM 300μM 100 nM Chronic 1 pM Chronic 10 nM Chronic 10 µM Chronic 30 µM Chronic 300 µM Chronic Fig. 24 Chronic Treatment of allopregnanolone caused significantly less calcium increase after GABA or allopregnanolone stimulation. Primary cortical neurons were treated for 48 hours with 1μM allopregnanolone, then loaded with Fura-2 AM as before and calcium influx was induced by acute treatment of GABA or allorepgnanolone at the indicated concentration. Chronically treated cells had no calcium increase with 1pM acute allopregnanolone stimulation. * indicates p<0.0001.
Discussion
In this study, I have shown that GABA and allopregnanolone can induce a
cytosolic Ca2+ increase in a dose dependent manner in neurons after 14 days in
culture. I have shown that this effect is specific, as cholesterol does not cause
the same Ca2+ increase. However, chronic (48 hour) treatment with allopregnanolone causes the amount of Ca2+ increase to be diminished for both
98 acute GABA and acute allopregnanolone treatment. Additionally, the Ca2+ is not coming from internal stores, but from external solution. Nifedipine blocks the Ca2+
increase indicating that the L-type VGCC are likely the plasma membrane
channel that Ca2+ is entering the cell through. Lastly, I have shown that inhibiting
the NKCCl1 transporter also eliminates the Ca2+ increase induced by GABA and
allopregnanolone. This indicates that these compounds are causing chloride to
exit the cell leading to depolarization of the cell and activation of the VGCC.
In the first experiments, I treated cortical neurons with various
concentrations of GABA or allopregnanolone and measured the increase in
cytosolic Ca2+ with Fura-2. I used this information to create dose response curves
using IGOR software and identified the EC50 for both compounds. The EC50 was used for all future experiments unless were otherwise indicated. To test for any non-specific response to steroids, I determined whether cholesterol could induce a Ca2+ increase. Cholesterol had no effect on cytosolic Ca2+ levels as indicated
by Fura-2. This indicates that GABA and allopregnanolone are having a stero-
specific effect on the cortical neurons to induce a cytosolic Ca2+ increase. While
GABA has been shown to be depolarizing in some immature neurons (Grobin et
al., 2006), this has not been shown in “mature” neurons. Cortical neurons in
culture for 14 days are considered “mature”, so this response is novel.
Additionally, the mechanism involved in the cytosolic Ca2+ increase, including the
location of Ca2+ entry was previously unknown.
Next, I looked at the effect that chronic allopregnanolone treatment had on the Ca2+ increase induced by acute treatment. I compared three different
99 concentrations, a low middle and high concentration, for each acute treatment
conditions after chronic allopregnanolone treatment. I found that the chronic
treatment caused a decrease in Ca2+ influx after acute GABA or allopregnanolone treatment, except in the case of the chronically treated low
dose acute allopregnanolone treatment, which had no Ca2+ influx. These results
indicate that chronic exposure to allopregnanolone, as may be the case for a
woman during her menstrual cycle, may alter GABA's effect on chronic neurons.
This may alter a woman’s seizure susceptibility at certain times during her cycle.
In order to investigate this further, we must first understand the mechanism of the
induced Ca2+ increase.
Therefore, I set out to identify the source of the Ca2+ increase by first
investigating the role of internal Ca2+ stores versus Ca2+ influx from external
solution. I used 2-APB, an inhibitor of internal Ca2+ release, and found that it had
no effect on acute GABA or allopregnanolone treatment. Because 2-APB has
been shown to have different effect in different cell types, I also examined the
location of Ca2+ entry using cells in 0 Ca2+ external solutions. In both cases,
when cells were in 0 Ca2+ external solution there was no increase in cytosolic
Ca2+ after acute treatment. This indicates that the Ca2+ increase is coming from
the external solutions through a plasma membrane channel and not from internal
Ca2+ stores.
There are several sources for Ca2+ entry at the plasma membrane;
however, the knowledge that GABA can be depolarizing led us to begin our
search with the L-type VGCC. I used the L-type VGCC blocker nifedipine to
100 investigate the role of these channels after the acute treatment of GABA or
allopregnanolone. I found that in both cases nifedipine blocked all Ca2+ entry. To
be sure the cells would still respond to stimulus I used ATP to induce Ca2+ release from the internal stores and observed an increase in intracellular Ca2+.
This data combined with the 0 external Ca2+ data indicated that in fact the Ca2+ entry was from external solution through the VGCC.
This indicated that the chronic treatment of GABA and allopregnanolone
was causing the GABA channels to allow chloride to exit the cell leading to a
depolarization. To test this I used bumetanide, a blocker of the NKCC1
transporter. This transporter is electro-neutral and is responsible for entry of
sodium, potassium and chloride into the cell. Its activity is believed to be responsible for maintaining the chloride gradient (Pieraut et al., 2007). Use of
bumetanide disrupts the chloride gradient so that it will not move through plasma
membrane channels in either direction. Bumetanide, like nifedipine, blocked the
Ca2+ influx induced by acute GABA and allopregnanolone treatment. However,
because bumetanide disrupts the chloride gradient, the effect could be from any
number of chloride selective ion channels.
To further investigate the source of chloride efflux, I began treating cells
with various GABA channel blockers. The GABAbR blocker saclofen had no
effect on Ca2+ influx; however, because of the fast kinetics of the response
GABAbR are an unlikely source of the chloride efflux, due to the fact that
GABAbR are metabotropic receptors and have slower kinetics. More interesting,
2+ however, is that the GABAaR blocker bicuculine also had no effect on the Ca
101 increase. This was surprising given the evidence that allopregnanolone can
effect many of the GABAaR subunits, including subunits that have known effects
on drug susceptibility and synaptic currents. It is possible that the effects are due
to GABAcR, which are similar in function and structure to the GABAaR, but have different pharmacology including that they are not blocked by bicuculline (Enz and Cutting, 1999). Furthermore, there is evidence that allopregnanolone may have a direct effect on GABAcR (Li et al., 2006). Additionally there are other
chloride sensitive channels and transporters on the plasma membrane of cortical
neurons. Conversely, it is not known if allopregnanolone has any effects on these
channels.
102 Chapter VII
With these experiments I have demonstrated that Ca2+ signaling and
mitochondrial health are important players in the etiology of three different
disease states. While the importance of Ca2+ signaling in cell growth and
apoptosis was known, the first set of experiments illustrated that modulating Ca2+ signaling by altering mitochondrial health may be an effective treatment in some types of cancer. Furthermore, identifying a drug that can specifically target mitochondria in cancer cells may be an effective treatment with minimal side effects for some cancer types. In the second set of experiments, a known neurotoxin, Aβ, exhibited differential effects on mitochondria and Ca2+ signaling
in different cell types. While Ca2+ and mitochondria have been thought to be
involved in pathology of AD, this differential effect and differential uptake of the
peptide may explain why the hypothalamus is not affected in early AD. More importantly, this may indicate a possible treatment or preventative mechanisms
for AD. In the last set of experiments, I demonstrated that chronic
allopregnanolone treatment can alter Ca2+ signaling and mitochondrial health.
These changes may be responsible for the changes observed by Pierson (2005)
in GABAR subunit composition and ultimately effect seizure susceptibility. More
studies are needed to fully understand the Ca2+ signaling pathway involved and how this pathway may be exploited for possible treatment of catamenial epilepsy.
Through these examples, I have shown that understanding the role of calcium
signaling and mitochondrial function in disease are important tools in
understanding the underlying mechanism of disease pathology.
103
Conclusions:
1. CAI acute treatment causes mitochondria in HEK-293 cells to be less
polarized in a time and concentration dependent manner.
2. CAI acute treatment affects the ability of mitochondria to sequester Ca2+;
and this effect is independent of any effects of CAI on Ca2+ channels at
the plasma membrane.
3. Amyloid-ß has an effect on mitochondrial membrane potential in pituicytes
causing them to become less polarized; however, there is no effect of Aβ
on the mitochondrial membrane potential in nerve terminals of the
posterior pituitary. .
4. Amyloid-ß causes a significant decrease in the resting Ca2+ levels of nerve
endings, and a significant increase in the resting Ca2+ levels of pituicytes
5. Amyloid-ß treatment causes a significantly higher maximal change in Ca2+
after stimulus in both pituicytes and nerve endings.
6. Fluorescently tagged Aβ was taken up by pituicytes in culture; however, it
was not taken up by nerve endings within 48 hours.
7. Acute treatment of primary rat cortical neurons with either GABA or
allopregnanolone cause a rapid rise in cytosolic Ca2+ levels in a
concentration dependent manner.
8. The Ca2+ rise induced by acute GABA or allopregnanolone treatment was
blocked by both nifedipine and bumetanide, but not by bicuculine,
saclofen, picrotoxin or 2-APB.
104 9. Chronic treatment of cortical neurons with 1μM allopregnanolone caused a
significant decrease in the maximal rise in cytosolic Ca2+ after stimulus
with GABA or allopregnanolone acutely.
10. Chronic treatment of cortical neurons with 1μM allopregnanolone caused a
significant decrease in mitochondrial membrane polarization compared to
control.
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138 Abstract
Calcium signals are integral steps in pathways for a large variety of important
cellular functions ranging from cell growth and proliferation to apoptotic cell
death. The mechanism and subcellular location of calcium entry or rise in
cytosolic calcium is also important in determining what type of message the
calcium is activating. Additionally, the control of calcium levels and signaling
pathways is being investigated in treatments several types of diseases. Another
area of investigation in importance in studying calcium signaling is mitochondria.
Mitochondria are vital organelles with essential roles including the production of
ATP, regulation of calcium signals and are the main intracellular initiator of
apoptosis. Monitoring mitochondrial calcium and membrane potential can provide clues in overall cell health and mechanism of cytosolic calcium entry. In this
project I have used both imaging techniques and high through put assays to
monitor calcium and mitochondrial membrane potential in two models of disease
states and to investigate the mechanism of a possible treatment.
139