1 METABOLIC CONTROL OF NEURONAL ACTIVATION AND EPILEPSY Alexander Ksendzovsky, MD PhD Candidate Department of Molecular Physiology and Biological Physics Chief Resident Department of Neurosurgery | University of Virginia Surgical Neurology Branch | National Institutes of Health PhD Mentor (NIH): Kareem Zaghloul, MD, PhD PhD Mentor (UVA): Jaideep Kapur, MD, PhD PhD Committee Chair: Avril Somlyo, PhD Committee: Jeff Elias, MD Mark Beenhakker, PhD Brant Isakson, PhD Dissertation June 5, 2019
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METABOLIC CONTROL OF NEURONAL ACTIVATION AND EPILEPSY
Alexander Ksendzovsky, MD
PhD Candidate
Department of Molecular Physiology and Biological Physics
Chief Resident
Department of Neurosurgery | University of Virginia
Surgical Neurology Branch | National Institutes of Health
PhD Mentor (NIH): Kareem Zaghloul, MD, PhD
PhD Mentor (UVA): Jaideep Kapur, MD, PhD
PhD Committee Chair: Avril Somlyo, PhD
Committee: Jeff Elias, MD
Mark Beenhakker, PhD
Brant Isakson, PhD
Dissertation
June 5, 2019
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TABLE OF CONTENTS
I. Abstract ............................................................................................................................. 3
II. Introduction ...................................................................................................................... 5
III. “Chronic activation leads to neuronal glycolysis through the AMPK/HIF1a pathway” ................................................................................................................... 14
IV. “A feedforward mechanism for epilepsy regulated by lactate dehydrogenase A” .... 55
V. Special Methods ............................................................................................................... 85
a. "Modeling epilepsy in a dish: mixed cortical cells cultured on a microelectrode array” ................................................................................................................................... 85
b. "A novel mouse model of cobalt-induced focal cortical epilepsy” …............................................................................................................................... 105
VI. Conclusions ....................................................................................................................... 115
VII. Future Directions ............................................................................................................. 117
VIII. Acknowledgements ...........................................................................................................122
IX. References ......................................................................................................................... 124
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I. Abstract
The fundamental role of metabolism in the regulation of neuronal activation is not well
understood. Glycolysis is thought to support active neurons as a supplement to mitochondrial respiration
in times of high metabolic demand which occurs through the astrocyte neuron lactate shuttle (ANLS).
Recent evidence, however, strongly refutes this claim and argues that acute neuronal stimulation directly
leads to neuronal glucose utilization through glycolysis, which becomes the primary source of ATP. Due
to this lack of clarity, the role of metabolism in epilepsy formation is also unknown. In the present work,
we explore the neuronal metabolic phenotype during times of high metabolic demand from chronic
stimulation. We extend these findings toward understanding metabolism’s role in regulating epilepsy.
In our first aim we used a novel model of chronic activation and resected human tissue to
demonstrate that chronic neuronal stimulation leads to neuronal metabolic reprogramming from aerobic
respiration to glycolysis through the upregulation of neuronal LDHA. Our results challenge the
prevailing ANLS hypothesis, which holds that the majority of metabolism occurs via supporting
astrocytes during times of high neuronal metabolic demand. The second aim of our study was to
describe the molecular pathway that regulates the transition from aerobic respiration to glycolysis during
chronic neuronal stimulation. Drawing from similarities of high energy demands during hypoxia, we
hypothesized that the AMPK/ HIF1a hypoxia pathway plays a role in regulating neuronal metabolism
during chronic stimulation. Using this model, we confirmed that neuronal metabolic reprogramming to
glycolysis is mediated by the AMPK/ HIF1a hypoxia pathway. For our third aim, we applied insights
gained from the neuronal metabolic phenotype during times of chronic stimulation from our first two
aims to more clearly elucidate the etiology of epilepsy formation. We showed that LDHA, regulated by
upstream HIF1a, leads to epileptiform activity in culture and in an animal model.
Collectively, the work presented here lays the foundation of an overarching hypothesis for
metabolically driven pathogenesis of epilepsy. We believe a feedforward loop exists wherein chronic
seizure activity shifts neurons into glycolysis through AMPK/HIF1a mediated upregulation of LDHA,
4
which further pushes neurons to become hyperexcitable and subsequently elicit more seizures.
5
II. Introduction Neuronal glucose utilization
The brain represents only 2% of human body mass but consumes more than 20% of the daily
energy requirement [1]. When performing cognitive tasks there is a wide variation in brain energy
consumption across cortical tissue as signals propagate from one node to another. On a macroscopic
scale, changes in cerebral blood flow accompanying brain activity account in part for the uncanny ability
of the brain to adapt to instantaneous shifts in metabolic demand [2]. On a more cellular level, where the
majority of these changes occur, intertwined biochemical and molecular pathways work together to
maintain a tightly regulated metabolic homeostasis.
The seemingly fundamental concept of neuronal metabolism, to this day, is poorly understood.
Current explanations for neuronal glucose utilization are controversial and have been based on
incomplete or inadequate underlying evidence. Over the last three decades the prevailing theory has
been the Astrocyte Neuron Lactate Shuttle (ANLS) hypothesis. A main part of this hypothesis rests on
understanding the cellular location of the metabolic processing required to provide neurons with the
adenosine tri-phosphate (ATP) necessary to perform homeostatic and, more importantly, dynamic
metabolic functions. According to the ANLS hypothesis, neurons receive a large amount of substrate
necessary for metabolism from neighboring astrocytes, which metabolize glucose into lactate via
astrocytic lactate dehydrogenase A (LDHA). Astrocytic lactate is subsequently shuttled into neurons for
further metabolism into pyruvate and eventually the tricarboxylic acid (TCA) cycle [3-5] (Figure 1).
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Figure 1: Astrocyte Neuron Lactate Shuttle (Adapted from Rho et al.) [5]. Glucose is provided to astrocytes either
endogenously through breakdown of glycogen or through peripheral circulation via glucose transporters. Glucose is then
converted to pyruvate and then lactate through astrocytic lactate dehydrogenase A (LDHA). Lactate is shuttled to neurons
through MCT transporters which is then converted back to pyruvate by neuronal lactate dehydrogenase B (LDHB). Several
of these enzymes are potential points for inhibition for decreased ATP production [5, 6].
The brain’s metabolic response to neuronal activation is even less clear. Early observations of a
mismatch between oxygen and glucose utilization during physiological [7, 8] and pathological (i.e.
1Surgical Neurology Branch, National Institute of Neurologic Disorders and Stroke,
National Institute of Health, Bethesda, Maryland
2Department of Neurology, University of Virginia Health System
University of Virginia, Charlottesville, Virginia
3Neuroscience Department, University of Virginia Health System
University of Virginia, Charlottesville, Virginia
4Department of Neurological Surgery, University of Virginia Health System
University of Virginia, Charlottesville, Virginia
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Abstract
Introduction
Despite previous efforts, there remains no currently accepted mouse model for focal cortical epilepsy, which
accounts for a significant burden of disease. In this study we present a novel mouse model of cobalt-induced
chronic focal cortical epilepsy. We describe seizure and clinical outcomes and elaborate on the nature and pattern
of seizure propagation.
Methods
We analyzed four separate treatment groups, including five mice with cobalt implanted into the prefrontal cortex,
sixteen mice injected with homocysteine (HT) and five mice concurrently implanted with cobalt and injected with
HT. Animals were continuously monitored with video-electroencephalography. CLARITY was used to evaluate
neuronal activation in a fourth group of five transgenic c-fos mice that housed a doxycycline-controlled promoter
responsible for expressing fluorescent protein in activated neurons.
Results
Animals implanted with cobalt and injected with HT showed increasing seizure behavior scores and seizure
frequency throughout the monitoring period. This contrasted with other groups that showed significant seizure
reduction after 1-2 weeks. All animals in the concurrent cobalt with HT group went into status epilepticus after
injection, which was staged and characterized. We believe induction of SE with HT is necessary to produce
chronic focal epilepsy in mice. In all four groups, seizures illustrated similar patterns of propagation on EEG. This
was further visualized in the c-fos mice demonstrating perilesional neuronal activation spreading to the ipsilateral
then contralateral motor cortex and finally to bilateral hippocampi.
Conclusion
In this study, we establish a chronic model of focal cortical epilepsy using cobalt wire implantation and
homocysteine injection. This model can be used to probe mechanisms and novel treatments for focal cortical
epilepsy.
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Introduction
A murine model of disease can provide robust insight into its molecular, genetic and electrophysiological
properties. Despite prior efforts, there remains no currently accepted mouse model for focal cortical epilepsy
which accounts for more than 60% of epileptic seizures [83-85]. A working model of focal cortical epilepsy can
potentially unearth tremendous insights into this high disease burden.
In this study we present a novel mouse model of cobalt-induced chronic focal cortical epilepsy. Seizure
are critically evaluated across three experimental groups of mice that were either implanted with cobalt, received
homocysteine injection or both. We describe seizure and clinical outcomes and elaborate on the nature and pattern
of seizure propagation using a transgenic c-fos mouse model.
Methods
Animals
The use of animals in this protocol was approved by the University of Virginia Animal Care and Use
Committee, followed all regulatory requirements and guidelines, and was conducted in a facility that is accredited
by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), International.
To study cobalt-induced seizures we used four separate timing paradigms. The first included five adult
C57 wild-type (WT) mice implanted with cobalt wire (500mm diameter) into the prefrontal cortex. We also
implanted four intracranial recording electrodes for video-electroencephalography (video-EEG) monitoring. We
followed the mice with continuous video-EEG monitoring for 30 days and subsequently sacrificed, perfused and
processed them for histologic evaluation. We implanted a second group of 16 C57 WT mice with monitoring
electrodes, injected an intraperitoneal (841mg/kg) dose of HT and monitored for seizure activity for 30 days. A
third group of five C57 WT mice was implanted with a prefrontal cobalt wire and monitoring electrodes, injected
with homocysteine (841mg/kg) on post-operative day seven and followed with continuous video-EEG for 45
days. After monitoring we harvested the brains for histological analysis. Finally, we evaluated a fourth group of
seven transgenic c-fos (cfos-tTA/cfos-shEGFP, Jackson Laboratories, Bar Harbor, ME) mice for neuronal GFP
expression and thus seizure propagation. The c-fos mice had a doxycycline-mediated c-fos promoter region which
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allows for green fluorescence upon neuronal activation. This group was taken off doxycycline 24 hours before
cobalt placement and were monitored by video-EEG for a total of 48 hours.
Surgical procedure
The same surgical procedure was performed across all mice. We implanted A 500µm cobalt wire under
stereotactic guidance anterolateral to the left bregma and 1mm deep to the outer table. Two stainless steel
recording electrodes were placed 1mm posterior and 1mm on either side of the bregma just under the inner table
of the skull. We placed a left hippocampal depth electrode along with a reference electrode into the posterior
fossa. Electrodes were fixed with dental cranioplasty and animals were connected to continuous video-EEG
monitoring.
Clinical and seizure monitoring
We evaluated seizures for behavioral score (Table 1), duration, location of initiation and pattern of
propagation, latency to first and last seizure, total number of seizures and seizure frequency. We used a modified
Lothman scale [86] to monitor animals receiving homocysteine for status epilepticus. Status epilepticus was
analyzed for duration, duration and progression through each stage [86], frequency, total time, clinical morbidity
and mortality and power spectrum. We used a Fast Fourier Transformation to perform spectral analysis of power
in frequency bands during status epilepticus. The specifications of this analysis can be found in Phelan et al.
(2015) [87].
Clarity
C-fos mouse brains underwent modified CLARITY for evaluation of cellular fluorescence but without
electrophoresis to clear lipid molecules [74]. For lipid separation tissue was incubated with sodium dodecyl
sulfate (SDS) and imaged using 2-photon microscopy.
Results
Characteristics and activation pattern of acute cobalt-induced seizures
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We used seven transgenic c-fos mice to visualize seizure onset and to describe the pattern of seizure
propagation. In this group, the average total number of seizures was 7.7±3.02 with an average duration of
30.35±5s. Time to first and last seizure was 0.53±0.17hrs and 41.65±4.03hrs, respectively. The average
behavioral score was 2.7±0.17 (Table 2). There was no difference between average number of seizures, seizure
duration or behavioral score during the first two days of monitoring (Figure 2).
Electrographic analysis revealed a distinct pattern of seizure propagation. High frequency spiking activity
began in ipsilateral cortical electrodes and spread to the contralateral cortex and then hippocampus. This pattern
was observed across all mice and most seizures (Figure 1A, B). Two-photon microscopy of fluorescently labeled,
cleared tissue showed neuronal activation in a similar pattern (Figure 1 C - H). Neurons surrounding the cobalt
lesion showed intense fluorescence (Figure 1E), which continued to the ipsilateral primary motor area and
contralateral primary motor area (less intense) (Figure 1F). Some fluorescence was noted in the subiculum and
CA1 of the hippocampus (Figure 1H). There was no thalamic activation during seizure propagation (figure 1G).
The anterior olfactory nucleus served as an internal positive control and the lack of neuronal activation seen
around the lesion caused by a stainless-steel hippocampal recording electrode served as an internal negative
control (Figure 1H).
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Figure 1. Pattern of seizure initiation and propagation in transgenic c-fos mice. A and B. Intracranial EEG tracing for 2
separate seizures showing seizure initiation occurring first in the ipsilateral cortex (CTXi) then in the contralateral cortex
(CTXc) and then in the two hippocampal leads simultaneously (in same location within left hippocampus – twisted together).
C. Three-dimensional z-stack reconstruction of 300µm cleared tissue shows significant perilesional neuronal activation with
spread to bilateral motor cortices. E and F. Sections through the cobalt lesion (e) and 1mm posterior to the lesion (f) showing
anatomic distribution of neuronal activation in mice with cobalt-induced seizures. Neuronal activation is evident around the
site of the cobalt lesion and in bilateral primary motor cortices. Anterior olfactory nucleus neurons serve as an internal
control. G. Section through the thalamus of the same mouse showing lack of neuronal activation, questioning the thalamus’
involvement in seizure propagation in cobalt-induced focal cortical epilepsy. H. Section through the hippocampus of the
same mouse showing hippocampal activation in subiculum and CA1 on the right without neuronal activation in the dentate
gyrus. Of note, there is no perilesional neuronal activation around the site of the stainless steel (SS) hippocampal depth
electrodes, which serves as a negative internal control.
Figure 2. Comparison of seizure activity one and two days after cobalt wire placement in c-fos mice. A-C. There was
no statistically significant difference in the average amount of daily seizures, average behavioral scores and average seizure
duration on day 1 or day 2 after cobalt implantation.
Natural history of cobalt-induced seizures
To evaluate the long-term natural history of cobalt-wire implantation (without homocysteine), five WT
mice underwent cobalt wire implantation to the left premotor area. Two mice died after two days and underwent
separate analysis. The average total amount of seizures in the sacrificed group was 19.6±9.8 with an average
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duration of 15.19±1.7s and behavior score of 1.72±0.26. The mice that died had a total of 13.98±9.8 seizures in
two days which lasted longer (47.8±21.1) and had higher behavioral scores (2.127±0.37) than in the sacrificed
group (Table 2). These animals presumably succumbed to their seizures. Over a month of monitoring, seizure
number and behavioral scores decreases. Mice stopped having seizures two weeks after implantation. There was
no change in seizure duration over time (Figure 3A). The electrographic nature of the seizures mimicked the acute
c-fos animal cohort above.
Natural history of homocysteine-induced seizures
To evaluate the effects of homocysteine injection alone we treated 16 WT mice with 841mg/kg
homocysteine without cobalt implantation. Seizures were experience by 87.5% of mice and lasted an average
54.17 seconds. The mean number of seizures within 24hrs after injection was two, over 74.41 seconds with a
mean behavioral score of 5. After 24 hours only 55% of animals experienced seizures which lasted 30.9 seconds
and had a behavioral score of 3.5. All animals stopped seizing by day six (Figure 3B).
Cobalt and homocysteine-induced focal cortical epilepsy
Given the presumed additive effects of HT and cobalt we tested the combination of the two on five WT
animals. In these mice we implanted a 500µM cobalt wire into the left premotor area and injection homocysteine
(HT) (841mg/kg) seven days later. These animals were monitored for 45 days. The average pre- and post-HT
seizure number, duration and behavioral scores were: 2.4±1.69 and 25±9.7; 17.28±8.1 and 23.79±5.9; and
1.72±.077 and 2.5±0.7. Average latency to last seizure after HT therapy was 27±3.49 days (Table 2).
For the duration of the monitoring period after HT treatment, weekly seizure frequency and seizure
behavior scores increased (Figure 3C). This was in contrast to the cobalt group without HT and the animals who
received HT alone, suggesting that concurrent cobalt implantation and HT treatment was necessary to produce
chronic focal epilepsy. The animals who received HT in conjunction with cobalt had more seizures (25 vs 19.6)
which lasted longer (23.79s vs 15.19s) and had higher behavioral scores (2.5 vs 1.72 than the other two groups
(Table 2). The seizure onset zone and pattern of spread was the same as in the c-fos animals above.
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Figure 3. Comparative analysis of seizure characteristics in 3 treated groups. A. Cobalt only group. Weekly seizure
behavior scores, duration and frequency were monitored in mice implanted with a cobalt wire alone. There was a reduction in
weekly seizure frequency and behavior two weeks after implantation. There is no significant change in seizure duration
during the recording period. B. HT only group. Animals injected with homocysteine alone (without cobalt implantation) had
a significant reduction in seizure duration at week 2. No animals had seizures six days after implantation. C. Cobalt with
concurrent HT. Animals implanted with cobalt and injected with HT had an increase in seizure frequency and duration each
week with no change in behavior scores. These mice continued to have seizures throughout the entire monitoring period. This
is in contrast to the two previous groups and suggests that the addition of HT is necessary to induce chronic epilepsy in a
cobalt model of focal cortical epilepsy.
Characteristic of cobalt and homocysteine-induced status epilepticus
All five mice implanted with cobalt who received HT went into status epilepticus after HT administration.
The total duration of SE (from beginning of stage 1 to end of stage 4) was 1.74±0.2 hrs (Figure 4A-E). SE stage 1
lasted for an average of 9.3±3.2 minutes and consisted of high frequency intermittent spikes (Figure 4A). SE
stage 2 began with an initial seizure and lasted 30.1±8.4 minutes (Figure 4B). SE stages 3 and 4 consisted of
continuous high frequency seizure activity lasting 4.5±0.28min and 1.19±0.014hrs, respectively (Figure 4 C, D).
Power spectral analysis revealed 2-20Hz oscillations lasting for a total of 1.7hrs on average. This was consistent
with previous literature (Figure 4F) [76].
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Figure 4. Status epilepticus analysis in concurrent cobalt and HT group. A. SE stage 1 lasted for an average of 9.3±3.2
minutes and consisted of high frequency intermittent spikes. B. SE stage 2 began with the the initial seizure and lasted
30.1±8.4 minutes. C. SE stage 3 was a transitional stage and lasted for 4.5±0.28min. D. SE stage 4 consisted of continuous
high frequency seizure activity lasting 1.19±0.014hrs. E. Average time spent in each stage of status epileptics. The most time
was spent in SE stage 4. F) Power spectral analysis revealed 2-20Hz oscillations lasting for a total of 1.7hrs on average,
consistent with previous literature.
Discussion
Cobalt-induced epilepsy is not a novel concept and dates back over 50 years across several animal models
[76, 88-92]. From 1970 to 1992 cobalt implantation was used to produce focal seizures to examine novel medical
therapies. This model was instrumental in the discovery of carbamazepine [93, 94]. Interestingly, all
investigations noted seizure cessation after two weeks of implantation thus limiting the longevity of these models.
The cobalt model was reintroduced by Chang et al. in 2004. After three weeks of video-EEG monitoring
they noted significant seizure reduction and eventual seizure arrest after 18 days [95]. The present data confirms
that after two weeks of only cobalt implantation there is a significant reduction in seizure frequency and
behavioral score. This finding suggested that cobalt alone is limited to an acute model of focal epilepsy (Figure
3). Chang et al. subsequently showed that cobalt leaches into the brain parenchyma causing perilesional necrosis
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without otherwise widespread change [95]. It was hypothesized that this focal necrosis was likely responsible for
the focal nature of seizure initiation found in these animals. We confirmed focal perilesional seizure onset in our
animals, which was visualized in the c-fos group (Figure 1).
In 2011 Kim et al. used the cobalt model to evaluate the thalamus’ role in seizure propagation. Similar to
our approach, they implanted a frontal cortical cobalt wire and monitored for 30 days. Unlike our natural history
group and previous studies, however, they observed spiking activity 28 days after implantation [76]. This led the
investigators to believe that despite there being no clinical seizures, neurons around the cobalt lesion were still
hypeexcitable. Following the 1988 concept of homocysteine-induced SE in cobalt mice [96], Kim et al. injected a
cohort of cobalt mice with HT and noted convulsive seizures immediately after injection [76]. They did not
monitor these animals longitudinally, however, to see if chronic seizures developed.
In accordance with prevailing literature but in contrast to Kim et al., our data confirmed a significant
seizure reduction after two weeks in animals implanted with cobalt alone. We also noted seizure reduction only
six days after HT injection without cobalt implantation (Figure 3A,B). The combination of cobalt and HT,
however allowed our animals to develop consistent and chronic focal cortical seizures over the course of a month
(Figure 3C). Comparative analysis across groups suggests that both cobalt and homocysteine are necessary but
not individually sufficient to induce a chronic epileptic condition in cobalt-implanted mice. Furthermore, SE was
only observed in the cobalt with HT group suggesting the necessity of SE in production of chronic epilepsy
(Figure 4).
In all four groups, seizures followed similar patterns of propagation on EEG. This pattern was visualized
in transgenic c-fos mice showing perilesional neuronal activation spreading to the ipsilateral then contralateral
motor cortex and finally to bilateral hippocampi (Figure 1). EEG and c-fos neuronal activation confirmed focal
cortical epilepsy insofar as seizures propagated from the perilesional cobalt area.
In this study, we establish a chronic model of focal cortical epilepsy using cobalt wire implantation and
homocysteine injection. We describe the seizure characteristics and their pattern of propagation. This model can
be used in future studies to probe for mechanisms and potential treatments of focal cortical epilepsy.
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VI. Conclusions
The fundamental role of metabolism in the regulation of neuronal activation has long been
debated with several competing theories. Due to this lack of clarity, the role of metabolism in epilepsy is
also unknown. The present work describes the neuronal metabolic phenotype during chronic stimulation
and extends these findings toward understanding a more integrated pathogenesis of epilepsy.
In our first aim we used a novel model of chronic activation and resected human tissue to
demonstrate that chronic neuronal stimulation leads to neuronal metabolic reprogramming from aerobic
respiration to glycolysis through the upregulation of neuronal LDHA. Our results challenge the
prevailing ANLS hypothesis, which holds that the majority of metabolism occurs via supporting
astrocytes during times of high neuronal metabolic demand. The second aim of our study was to
describe the molecular pathway that regulates the transition from aerobic respiration to glycolysis during
chronic neuronal stimulation. Drawing from similarities of high energy demands during hypoxia, we
hypothesized that the AMPK/HIF1a hypoxia pathway plays a role in regulating neuronal metabolism
during chronic stimulation. Using our low Mg2+, we confirmed that neuronal metabolic reprogramming
to glycolysis is mediated by the AMPK/HIF1a hypoxia pathway. For our third aim, we applied insight
gained from the neuronal metabolic phenotype during times of chronic stimulation from our first two
aims to more clearly elucidate the etiology of epilepsy formation. We showed that LDHA, regulated by
upstream HIF1a, leads to epileptiform activity in culture and in an animal model.
The above three aims lay the foundation of an overarching hypothesis for metabolically driven
pathogenesis of epilepsy. We envision a feedforward loop in which chronic seizure activity shifts
neurons into glycolysis through AMPK/HIF1a mediated upregulation of LDHA, which pushes neurons
to become hyperexcitable and subsequently elicit more seizures.
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Figure 1. Representative schematic of the feedforward loop that drives metabolic control of epilepsy. As neurons are
chronically activated or seize, they upregulate LDHA expression and thus glycolysis (top arrows) through the AMPK/HIF1a
pathway (middle arrows) which is activated by a high AMP:ATP ratio. AMP leads to phosphorylation of AMPK, which
leads to stabilization of HIF1a. HIF1a translocates into the nucleus as a transcription factor to upregulate LDHA
transcription and protein expression and thus glycolysis. HIF1a-regulated LDHA expression goes on to further cause
pathologic activation in neurons (bottom arrow).
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VII. Future Directions
LDHA is responsible for cobalt-induced chronic seizures
Our research findings suggest an important role for LDHA in regulating neuronal firing and
potentiating seizures. However, we limited our cobalt model to observation in the acute period. In future
studies, we anticipate exploring the role of LDHA in cobalt-induced chronic seizures. We hypothesize
that chronic focal seizures are regulated by LDHA as well. In order to test this hypothesis, we will use
tamoxifen-dependent LDHA knockout mice. These mice will be implanted with cobalt and induced with
homocysteine as previously described in Section V in the manuscript “A mouse model of cobalt-induced
focal cortical epilepsy.” Mice will be monitored with continuous video-EEG for 30 days. LDHA will be
knocked down at different time points (24 hours, 48 hours, 3 days, 1 week, and 2 weeks) and seizure
frequency, severity, and timing will be recorded. We believe that LDHA inhibition will reduce seizure
burden in the cobalt model, lending further credence to its role in seizure formation.
Mechanisms underlying LDHA’s regulation of neuronal hyperexcitability
The mechanism by which LDHA modulates neuronal membrane potential is also unclear and
will provide motivation for our future research. The KATP channel provides a feasible target that may be
responsible for LDHA’s modulation of neuronal firing. The KATP channel is distributed widely
throughout the central nervous system [77-79]. Under normal conditions, this channel remains
constitutively inhibited by ATP. The channel maintains negative membrane potential in neurons by
staying open and hyperpolarizing the cell membrane in times of high energy demand [80, 81]. However,
shifts in neuronal metabolic phenotype can alter the efficacy of this channel [81]. Higher rates of ATP
production through glycolysis could potentially inhibit KATP channels. Moreover, lactate could play a
direct role in neuronal membrane potential modulation by directly inhibiting KATP channels, as described
in ventromedial hypothalamic neurons [82].
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In a series of future experiments, we plan to use similar models to the present work to explore
the role of the KATP channel in modulating electrical activity and metabolism. We plan to upregulate
LDHA using a lentivirus model on an MEA. We will monitor neuronal activity while selectively
activating the KATP channel with diazoxide, nicorandil, or P1075 [97]. If the KATP channel modulates
LDHA-dependent neuronal bursting, then we expect to observe decreased burst activity with channel
activation. In a similar setting, we will also inhibit the KATP channel with glyburide [97]. This will likely
potentiate burst activity in the context of elevated LDHA. In separate experiments, we anticipate using
genetic alterations of the KATP channel to further test this hypothesis. The KATP channel is an octamer
comprised of four pore-forming Kir6.2 subunits and four modulatory SUR subunits [98]. These subunits
are responsible for ATP’s inhibitory effects. We plan to use Kir6.2-/- neurons obtained from Kir6.2-/-
KO mice [98] to further test KATP channel’s role in LDHA induced seizures. We expect to observe
minimal neuronal bursting with LDHA upregulation in Kir6.2-/- neurons. Given that lactate itself may
play a role in modulating the KATP channel, we plan to modify intracellular lactate levels of cultured
neurons by inhibiting the MCT2 lactate transporter. AR-C155858 is a potent MCT2 inhibitor that binds
to intracellular MCT sites [99]. We plan to combine AR-C155858 with our culture model to determine
whether this results in increased neuronal bursting. We will combine this small molecule inhibitor with
our current LDHA lentivirus model and the proposed Kir6.2-/- cells to determine if lactate modulates the
KATP channel’s effect on neuronal bursting.
Finally, we plan to create tamoxifen-dependent Kir6.2-/- conditional knockout mice to use in
conjunction with our cobalt model to test the KATP channel’s regulation of chronic seizures after cobalt
implantation. In a similar experiment to our conditional LDHA KO mouse model, Kir6.2-/- will be
knocked down at 24 hours, 48 hours, 3 days, 1 week and 2 weeks to determine KATP channel’s role in
LDHA-induced seizures from cobalt.
LDHA plays a role in seizures associated with IDH-1 mutated low-grade gliomas
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Given the central role of LDHA in modulating metabolic regulation of neuronal activation and
epilepsy, we believe that LDHA is also involved in mechanisms underlying epilepsy associated with
isocitrate dehydrogenase-1 (IDH-1) mutated low-grade gliomas. The incidence of seizures in patients
with low-grade, IDH-1 mutated, primary brain tumors is extremely high and reaches 80-90%. Isocitrate
dehydrogenase is the second enzymatic step in the TCA cycle and typically catalyzes the conversion of
isocitrate to a-ketogluterate [100]. A mutation in this enzyme inhibits astrocytic oxidative
phosphorylation and likely drives tumor astrocytes into glycolysis. Furthermore, a-ketoglutarate is
necessary for hydroxyl-mediated degradation of HIF1a. As described above, HIF1a leads to LDHA
upregulation [100]. We believe the metabolic shift in IDH-1 mutated astrocytes leads to further
metabolic shifts in neighboring neurons similar to the shifts observed in chronically activated neurons.
In a preliminary clinical study, we used subdural electrodes to monitor five patients with IDH-1
mutated tumors for seizure localization. Briefly, patients underwent a two-stage operation for
intracranial grid-electrode placement and then tumor and epilepsy focus resection. Similar to the
technique described in Section III we evaluated cortical tissue based on overlying electrographic activity
and compared epileptic to non-epileptic peritumoral cortex for LDHA staining. In five participants, we
observed significantly higher LDHA staining in epileptic tissue compared to non-epileptic tissue (Figure
1).
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Figure 1. (a - b) Temporal lobe of a participant with intracranial electrodes placed over a low grade IDH-1 mutated tumors
for seizure monitoring. During the monitoring period, we identified electrodes that were or were not involved in seizure
activity. In this example, electrode 27 (green) was not involved in seizures while electrode 21 (red) was involved in seizures.
The underlying tissue was resected as part of the planned surgical procedure and analyzed as the epileptic or non-epileptic
specimen for this participant. (c) Tissue underlying the epileptic (red) and non-epileptic (green) tissue from the same
participant were sectioned. Representative 400x400 µm regions of interest were analyzed for IDH, NeuN (not shown) and
LDHA using semi-automated segmentation. Epileptic tissue demonstrates significant LDHA staining compared to non-
epileptic tissue, while the neuronal marker NeuN was approximately the same between the two specimens (not shown).
Mutant IDH staining was highly positive within the tumor (as expected) but decreased in tissue away from the tumor. (d) We
normalized LDHA staining to NeuN to account for differences in neuronal density across specimens. Normalized LDHA
staining, averaged across ten 400x400 µm regions of interest in each participant, is significantly elevated across participants
in epileptic compared to non-epileptic tissue (n = 5 participants; *p < .05, unpaired t-test; mean ± SEM).
4x
4x4x
4x
20x
20x
20x
20xEpileptic Non-epileptic
Epile
ptic
Non
-epi
lept
ic
IDH1 (R132H) mutation LDHA
IDH1 (R132H) mutation
a) b)
c) d)
TUMOR
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Given that the IDH-1 mutation lies in astrocytes and not neurons, we believe the underlying
mechanism for LDHA upregulation in peritumoral neurons arises from interacting with IDH-1 mutated
astrocytes or secreted cytokines. As described above, the IDH-1 mutation pushes astrocytes into
glycolysis and thus increases lactate release. Increased lactate release from tumor cells could directly
play a role in stimulating peritumoral bursting through KATP channel inhibition. This would
subsequently lead to neuronal LDHA expression in a similar fashion to the aforementioned feed-forward
epileptic-metabolic loop. In order to test this hypothesis, we plan to use IDH-1 mutated GL-261 glioma
cells grown in a transwell above mixed cortical cultures on an MEA. In preliminary transwell
experiments, we demonstrated that IDH-mutated GL-261 tumor cells increase neuronal bursting. We
plan to use this model to explore the role for LDHA in peritumoral neuronal activation and to unearth
the mechanism underlying LDHA expression and neuronal bursting (Figure 2).
Figure 2. Preliminary data from transwell experiment. IDH-mutated GL-261 were grown in a transwell above mixed cortical
cells grown on an MEA. Bursting activity was measured for 10 days. Cortical neurons with IDH-1 mutated GL-261 cells in
their respective transwell had significantly increased bursting than neurons associated with wild-type GL-261. These data
suggest that the IDH-1 mutation increases neuronal firing.
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VIII. Acknowledgements As the saying goes, “it takes a village to raise a child.” I came into the MPBP PhD program as a
third-year neurosurgery resident and a research infant. Over the last four years I have had the
tremendous fortune to be raised by mentors, colleagues, and friends across two huge academic
institutions. I would like to thank my mentors, Dr. Jaideep Kapur at UVA and Dr. Kareem Zaghloul at
NIH for taking this challenge on with me. I have learned a tremendous amount from both of them.
Mostly, however, I thank them for their patience with me. They embody what I always have and will
continue to strive for in my professional career – to be a true teacher, clinician, and scientist. I would
like to thank my PhD committee (Dr. Avril Somlyo, Dr. Brant Isakson, Dr. Mark Beenhakker and Dr.
Jeff Elias). I can remember initial looks of confusion when I presented my research objectives during
my first committee meeting. With their guidance, their faces changed over the years as I began to
achieve more concrete and consistent results. They always pushed me to better “my story,” which came
together into what is presented today.
I would also like to thank the leadership of the combined NIH/UVA neurosurgery residency
program: Dr. John Heiss, Dr. Mark Shaffrey and Dr. John Jane Jr. Five years ago I approached them
with a crazy scheme to leave neurosurgery residency with a PhD. Much to my surprise, they agreed to
let me do it. Since then, they never doubted my potential success and supported me throughout.
Furthermore, the NIH and UVA neurosurgery faculty were unanimously behind this experience as well.
They provided the intellectual and emotional support needed to see this endeavor through to the finish. I
certainly could not have done this without the support of my co-residents, especially my co-chiefs (Dr.
Dan Raper, Dr. James Nguyen, and Dr. Peter Christiansen). We came into the pit of residency together
and since the first day I could rely on them. For the completion of this thesis they took on the clinical
burden of our shared neurosurgery service at times when I needed to be in the lab. For this I am forever
grateful. From our neurosurgery program I would also like to thank Kaitlyn Benson, Camille Butler, and
Karen Saulle. They probably worked just as hard on the administrative portion of making this program
123
happen as I did on the actual research. This certainly could not have happened without their help.
From a laboratory perspective, I could not have achieved any of this work without learning the
myriad of techniques presented herein. In my UVA laboratory, I would like to thank John Williamson,
Pravin Wagley, and Dr. Suchitra Joshi. My first experience reading EEGs (in mice and humans) was
with this group and I will use this knowledge throughout my future research and clinical practice. At
NIH, I would like to thank Stuart Walbridge, Marcelle Altshuler, Joe Steiner, Muzna Bachani, Sara
Inati, and Nancy Edwards. Stuart has been teaching me how to do research since before I entered this
program. He harbors decades of experience and knowledge and was more than willing to pass it on. As a
testament to his lasting friendship, Stuart continued to perform certain aspects of animal experiments
that I did not find “appealing” until the very end. To Marcelle and Muzna, I certainly could not have
completed these experiments without your help. After I left NIH, they continued my work and have
taken it farther than I could have imagined. They will go on to accomplish great feats in medicine and
research and I am honored to have shared this time and these experiences with them.
Finally, and most importantly, I want to thank my family (my mother Nora, my father Pavel, my
sister Sofia, and my girlfriend Alyson). Throughout my life, they supported and encouraged me in every
decision I have made. During times of accomplishment they reminded me to stay humble and during
times of failure they reminded me to stay confident. They have always inspired me to do better. Thanks
to the village that raised me.
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