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A new model of neurodegeneration: integrating molecular, electrochemical, and neuronal behavior into a novel paradigm

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A new model of neurodegeneration: integrating molecular, electrochemical, and neuronal behavior into a novel paradigm

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Content A NEW MODEL OF NEURODEGENERATION:
INTEGRATING MOLECULAR, ELECTROCHEMICAL, AND NEURONAL BEHAVIOR
INTO A NOVEL PARADIGM
by
Robin Talamas
A Thesis Presented to the
FACULTY OF THE USC MANN SCHOOL OF PHARMACY
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
PHARMACEUTICAL SCIENCES
December 2024

ii
Acknowledgements
I would like to thank my advisor, Dr. Daryl Davies, for providing his insights into my
work and for giving me the opportunity to explore this area of research.
I would like to thank my mother for all of the emotional support that she has provided me
as I worked to complete my Master’s Degree.
I would like to thank my fellow members of the Davies, with special mention to
Samantha Skinner and Isis Janilkarn-Urena, for helping me to feel like part of a team.
I would like to thank the fellow members of my cohort, who helped me to develop a
sense of community during my time with USC.
And I would also like to thank my friend, Zachary Manning. I could not have done this
without your help.

iii
Table of Contents
Acknowledgements......................................................................................................................... ii
List of Figures................................................................................................................................ iv
Abstract............................................................................................................................................v
Chapter 1: Introduction....................................................................................................................1
1.1 Hippocampal Dysfunction .............................................................................................1
1.2 Neuronal Pathways ........................................................................................................2
1.3 Current Unknowns.........................................................................................................2
1.4 Creating a Coherent Model............................................................................................3
Chapter 2: Ion Transport Gradients .................................................................................................4
Chapter 3: Hippocampal Molecular Pathway ................................................................................10
3.1 Stress and Isolation ......................................................................................................10
3.2 Disruptions in the Urea Cycle and Metabolism...........................................................13
3.3 ATP in the Synapse......................................................................................................16
Chapter 4: Neural Pathways...........................................................................................................23
4.1 A Primary Neural Pathway ..........................................................................................23
4.2 Signaling Anticipation and Reward .............................................................................27
4.3 Rewiring of the VTA ...................................................................................................32
Chapter 5: Future Investigations....................................................................................................36
5.1 Insulin Resistance and the Brain..................................................................................36
5.2 Influence of the Gut-Brain Axis ..................................................................................37
5.3 Liver Secretion of FGF21 ............................................................................................37
Chapter 6: Conclusions..................................................................................................................39
Bibliography ..................................................................................................................................41

iv
List of Figures
Figure 1: Transport Dynamics Across the Cell Membrane .............................................................4
Figure 2: Ion Transport in the Synapse............................................................................................7
Figure 3: Interactions of the TCA and Urea Cycles ......................................................................15
Figure 4: Adenosine Signaling and Synaptic Function..................................................................19
Figure 5: Neural Pathway Regulating Behavior............................................................................24
Figure 6: MOR-CRFR1 Synaptic Modulation...............................................................................28
Figure 7: KOR-mediated Blockade of GABA Signaling ..............................................................30
Figure 8: CRFR1-CRFR2 Action in the VTA ...............................................................................34

v
Abstract
Neurodegenerative diseases such as Alzheimer’s and Parkinson’s are complex disorders that are
poorly understood. They incorporate a broad range of dysfunctions, including the formation of
Tau and Amyloid aggregates in the hippocampus. This results in changes in behavior and
impairment to cognitive function. However, given the complexity of these disorders, it is
difficult to characterize the active mechanisms and how they contribute to disease progression.
This thesis aims to provide a new model that can better describe the cellular and neurological
changes associated with neurodegenerative disorders. This includes an investigation into the
molecular mechanisms that contribute to impaired neuronal function. In particular, this thesis
proposes a neural pathway that connects multiple regions of the brain in order to characterize
changes in behavior. This thesis integrates current research into neurodegeneration in order to
synthesize a model that can guide future research.

1
Chapter 1: Introduction
Neurodegenerative diseases such as Alzheimer’s and Parkinson’s are complex disorders
characterized by loss of neuronal populations and the accumulation of cytotoxic
proteinopathies.1,2 These conditions are progressive and are often associated with comorbid
psychiatric disorders, such as addiction, anxiety, and other behavioral disorders.2,3,4,5 These
disorders affect complex molecular and signaling pathways within the brain, making it difficult
to characterize their pathologies. This presents a significant challenge in the treatment of these
disorders, as novel therapeutics cannot be developed without knowledge of the mechanisms
involved. Furthermore, little is known about the development of these disorders; this is
especially true for accumulating cytotoxic proteinopathies, as these proteins serve otherwise
unknown functions under physiological conditions. Given the complexity of these systems, a
broader understanding is necessary in order to characterize these disorders, and with mounting
evidence of comorbidities and overlapping mechanisms, the need for a coherent and
comprehensive model becomes more and more pressing. By understanding the mechanisms of
cellular dysfunction in the affected regions, it is possible to develop a model which recognizes
the coordinated actions of these regions and their contributions to neuropsychiatric pathology.
1.1 Hippocampal Dysfunction
Cellular dysfunction in the brain is observed in animals experiencing behavioral
impairments. Animal models of behavior are common techniques used in researching the
progression of psychiatric and neurological disorders as well as identifying risk factors in their
development.6,7 In particular, single-housing models of social isolation (SI) is a common method
for studying the development of anxiety and cognitive decline in mice.6,8,9,10 Previous
experiments have demonstrated that SI-treated mice experience cellular disruptions in the

2
hippocampus that were consistent with models of stress and chronic corticosterone
administration, implicating corticosterone as a key mediating factor.7,10,11 These experiments
have demonstrated various alterations in the cellular metabolic function of the hippocampus,
including disruptions to urea metabolism as well as aberrations in neurotransmitter signaling.
1.2 Neuronal Pathways
The complex role of the hippocampus in behavioral and neurological disorders points to
its intricate connectivity with other regions of the brain, in which dysfunction at this region
radiates outwards to other parts of the brain. Notably, investigations into behavioral deficits such
as anxiety and impaired social interaction have shown interactions between the hippocampus and
the ventral tegmental area (VTA) as well as with bed nucleus of the stria terminalis
(BNST).12,13,14 The VTA is notable for its involvement in stress and addiction as well as for
being a primary region of dopaminergic activity,15,16,17 whereas the BNST is a complex region of
diverse subnuclei with distinct connections across the brain.18,19 Understanding the roles and
connectivity of these regions as well as changes during disease is necessary to recognize the
impacts had by cellular dysfunction and how such dysfunction influences behavior. From this, it
is possible to expand the prospective model of neuropsychiatric disorders into a more
comprehensive framework, one that encapsulates a variety of pathologies.
1.3 Current Unknowns
Various unknowns still exist within the proposed model due to a lack of evidence to
describe causal interactions between regions and signaling pathways. Because of the complex
mechanisms and pathologies underlying neurodegenerative and psychiatric disorders, it is
difficult to characterize the roles played by regions outside of the brain. Hormones which are
secreted into circulation play a key role in maintaining healthy brain function, a key example

3
being liver-secreted FGF21 which declines in concentration with age and thereby presenting a
possible link to age-related cognitive decline.20 In addition to this is the complex behavior of the
human gut microbiome. Although the gut microbiome has been recognized as having a major
role in neurodevelopment and behavior, there are still many unknowns as to its interactions with
the brain. Recently, it has been reported that depletion of the gut microbiota was able to impair
social behavior in mice by altering signaling of corticosterone and oxytocin in the brain,12,13 thus
providing a tentative link between cognitive dysfunction and environmental factors such as diet
as well as alcohol-associated dysbiosis. However, the exact links between these regions and their
interactions remain unclear. To that end, it is paramount to characterize the activity of the brain
and how it is influenced by hormonal signaling. From this understanding, a clear link between
these regions can be deduced.
1.4 Creating a Coherent Model
The goal of this thesis is to present a model framework to describe the functions of the
hippocampus and its connected regions, including the VTA and the BNST, in modulating
behavior and thereby provide directions for current and future research. A clear, comprehensive
model of neurodegeneration and its related disorders can provide researchers with a direct
pathway towards developing novel therapeutics. Such a model should identify distinct changes in
the molecular and signaling pathways of the brain, namely in the hippocampus and its connected
regions. Beginning with changes in ion transport across the cell membrane, this thesis will
investigate dysfunction in the hippocampus and its subsequent role in a connected neural
pathway. From this framework, this thesis will describe key points for future research into
neurodegeneration.

4
Chapter 2: Ion Transport Gradients
Before realizing a clear model of neurodegeneration, it is necessary to characterize
cellular behavior and how it changes under inflammatory conditions. Aberrant signaling
behavior is a prominent feature in multiple models of neuroinflammation, including AD and
stroke, so it is important to understand the intracellular changes under these conditions.8,21,22
These changes reflected significant disruptions in inhibitory gamma-Aminobutyric Acid
(GABA) signaling, which included altered neuronal sensitivity and an increase in astrocytic
GABA synthesis and release.6,8,21 Astrocytes are both GABA sensitive and GABA secreting
Figure 1: Transport Dynamics Across the Cell Membrane
Stoichiometry of ion-dependent transport across the cell membrane. Movement of GABA and
Glutamate are mediated by GABA Transporters (GATs) and Excitatory Amino Acid
Transporters (EAATs), respectively. Ion concentrations are listed next to respective transporters.
Sodium and Calcium concentrations are maintained by the Sodium-Calcium Exchanger (NCE).
Created using BioRender.com

5
cells, allowing them to respond to changes in the extracellular environment and fine-tune
synaptic signaling.22,23 This process is primarily mediated by the activity of GABA Transporters
(GATs) and Excitatory Amino Acid Transporters (EAATs), which allow for the uptake of
GABA and glutamate from the synaptic space and limit their activity.22,24 Transporters such as
GATs use ion-dependent transport to carry neurotransmitters across the cell membrane and are
characterized by the Nernst Equation, which calculates the membrane potential at which ion
transport reaches equilibrium.24,25
1) Nernst Equation: 𝐸𝑟𝑒𝑣 = −
26.7
2𝑧𝑁𝑎 + 𝑧𝐶𝑙
𝐿𝑛[(
𝐶𝑙𝑖
−
𝐶𝑙𝑜
−
) (
𝐺𝐴𝐵𝐴𝑖
𝐺𝐴𝐵𝐴𝑜
) (
𝑁𝑎𝑖
+
𝑁𝑎𝑜
+
)
2
]
However, these ion gradients are shown to be disrupted in neuroinflammatory disorders,
with a notable increase in cellular calcium concentrations.8,26 Intracellular calcium becomes
cytotoxic at elevated concentrations,26,27 therefore prompting its efflux from the cell via the
Sodium-Calcium Exchanger (NCE).22,24 Because sodium concentration is relevant to the
transport of both GABA and glutamate, this counter transport activity subsequently changes their
respective equilibrium values and transport behavior.
21,22,24 However, because astrocytes are
responsible for the uptake of multiple signaling molecules, it becomes difficult to describe
changes in cellular transport across multiple channels. To that end, it was necessary to prepare a
modified version of the Nernst Equation.22,24,25 This version of the formula organizes ion
movements across the respective transporters as though acting as a single concerted mechanism.
This technique balances movement across the three transporters by using a net zero change in
Sodium concentration, as it is involved in the activity of each of them.22,24 This paradigm can be
used to better describe interactions between membrane transporters in response to extreme
changes in ion concentrations, such as increases in intracellular calcium. Three formulae were

6
developed to describe the different combinations of GABA and glutamate efflux/influx: GABAGlutamate Efflux, Glutamate-Dominant Efflux, and GABA-Dominant Efflux.
2) GABA-Glutamate: 𝐸𝑟𝑒𝑣 = −
26.7
4
𝐿𝑛[(
𝐶𝑎𝑖
2+
𝐶𝑎𝑜
2+
)
5
(
𝐺𝑙𝑢𝑖
−
𝐺𝑙𝑢𝑜
−
)
3
(
𝐶𝑙𝑖
−
𝐶𝑙𝑜
−
)
3
(
𝐺𝐴𝐵𝐴𝑖
𝐺𝐴𝐵𝐴𝑜
)
3
(
𝐻𝑖
+
𝐻𝑜
+
)
3
(
𝐾𝑜
+
𝐾𝑖
+)
3
]
3) Glutamate-Dominant: 𝐸𝑟𝑒𝑣 = −
26.7
4
𝐿𝑛[(
𝐶𝑎𝑖
2+
𝐶𝑎𝑜
2+
)
3
(
𝐺𝑙𝑢𝑖
−
𝐺𝑙𝑢𝑜
−
)
5
(
𝐶𝑙𝑜
−
𝐶𝑙𝑖
−
)
3
(
𝐺𝐴𝐵𝐴𝑜
𝐺𝐴𝐵𝐴𝑖
)
3
(
𝐻𝑖
+
𝐻𝑜
+
)
5
(
𝐾𝑜
+
𝐾𝑖
+)
5
]
4) GABA-Dominant: 𝐸𝑟𝑒𝑣 = −
26.7
1
𝐿𝑛[(
𝐶𝑎𝑖
2+
𝐶𝑎𝑜
2+
)
3
(
𝐺𝑙𝑢𝑜
−
𝐺𝑙𝑢𝑖
−
)(
𝐶𝑙𝑖
−
𝐶𝑙𝑜
−
)
6
(
𝐺𝐴𝐵𝐴𝑖
𝐺𝐴𝐵𝐴𝑜
)
6
(
𝐻𝑜
+
𝐻𝑖
+)(
𝐾𝑖
+
𝐾𝑜
+
)]
For the majority of astrocytes, intracellular glutamate remains at a relatively low
concentration of 0.3 mM.24 This has been suspected to be due to high activity of Glutamine
Synthetase in astrocytes, which provides a vital step in the recycling of neurotransmitters from
the extracellular space. However, a distinct population of hippocampal astrocytes has been found
to closely mediate synaptic plasticity through the rapid and localized release of cellular
glutamate.28 These astrocytes were determined to have the necessary cellular machinery for
vesicular glutamate release similar to neurons, demonstrating increased transcription of genes
associated with synaptic structure, ion transport, and exocytosis. These astrocytes were found to
play a significant role in synaptic long-term potentiation (LTP) as well as pathological processes
such as seizures and addiction.28,29 As such, it is necessary to include this subset within the
following calculations. However, because these astrocytes demonstrate a specialized molecular
profile, it is unlikely that the intracellular glutamate concentration will match with those
observed in most astrocyte populations. For this reason, the following calculations will be made
under the assumption that this astrocyte population possesses intracellular glutamate
concentrations similar to neurons (10 mM).30
Using Equations 2, 3, and 4 above, the reversal potentials can be determined for each
efflux mode. Under physiological conditions, these patterns would not be frequently observed as

7
part of astrocyte behavior, as the calculated reversal potentials would greatly exceed the
astrocyte resting potential of -80 mV.24 However, efflux can be predicted to become much more
prevalent under conditions of cytotoxic intracellular calcium, in which concentrations rise
beyond an estimated five-fold increase (>500 nM).26 More specifically, the reversal potential for
GABA-Dominant Efflux shows a significant drop from 12.9 mV to -116 mV in standard
Figure 2: Ion Transport in the Synapse
Mediation of astrocytes in synaptic signaling. GABA (orange) and Glutamate (purple) are
transported across the astrocyte membrane to modulate availability in the synapse. Astrocytes
perform Glutamate-Dominant (left) or GABA-Dominant (right) efflux depending on intracellular
ion concentrations. Stoichiometry of each transporter in the efflux pattern is shown. Created
using BioRender.com

8
astrocytes, far below the astrocyte resting potential. This aberrant efflux behavior has been
implicated in multiple neuroinflammatory conditions in which increases in intracellular calcium
have been observed.8,21,26 Conversely, astrocytes predicted to have a higher cellular glutamate
concentration have been estimated to be more prone towards GABA-Glutamate and GlutamateDominant Efflux, with reversal potentials dropping to a respective -35.4 mV and -39.1 mV
during increased cellular calcium. Furthermore, Glutamate-Dominant Efflux is predicted to
become more active under increased extracellular GABA, suggesting a compensatory
mechanism against astrocytic GABA secretion. In the context of the hippocampus, this presents
a possible means of balancing excitatory and inhibitory signaling under cellular stress,
maintaining normal synaptic activity and avoiding impairments in memory.
However, this behavior is no longer apparent in the case of AD, in which efflux of
astrocytic GABA appears predominant.8,21,22 This suggests an additional change to cellular ion
concentration in AD that impairs astrocyte efflux of glutamate. This is most likely due to an
elevation in intracellular chloride, which helps to mediate GABA transport and signaling.22
Chloride concentration gradients play little role in the activity of EAAT or NCE, meaning that
changes in concentration primarily affect GABA transport via GAT.22,24 Chloride concentration
gradients are maintained by the activity of transporters KCC2 and NKCC1, which have been
found to show changes in function due to stress and Amyloid exposure, increasing intracellular
chloride concentration as a result.31,32 Under a modest change of 30 mM to 40 mM, this is
sufficient to reduce the reversal potential of GABA-Dominant Efflux from -22.4 mV to -68.5
mV in glutamate-secreting astrocytes, well below the reversal potentials for glutamate-based
efflux. Additionally, these disruptions in cellular concentrations present a significant risk to
neuronal function, as ionotropic GABA signaling is largely dependent on chloride influx.31

9
Under moderate increases, this effect may blunt GABAergic inhibition, desensitizing the neuron
to astrocytic GABA as a result. However, under extreme increases from 5 mM to 15 mM, this
can be predicted to shift the chloride reversal potential from hyperpolarizing (-82.5 mV) to
potentially depolarizing (-53.2 mV). From these estimations, it is possible to predict a clear
change in cellular behavior, one which becomes increasingly prevalent in the research of
neurodegeneration.

10
Chapter 3: Hippocampal Molecular Pathway
3.1 Stress and Isolation
To understand the overlap between psychiatric and neurodegenerative disorders, it is
important to recognize the signs of neurodegeneration in behavioral models. Social Isolation (SI)
is an established model for the development of cognitive impairment in mice.6,8,9,10 In particular,
socially isolated mice have shown distinct behavioral changes such as anxiety and impaired
memory as well as neurological changes including neuroinflammation, impaired metabolic
energy, and even the accumulation of hyperphosphorylated Tau fibrils, a prominent marker in
Alzheimer’s Disease (AD).8,9,10,33
The hippocampus is critical for the consolidation of long-term
memory and is one of the primary regions affected by AD.6,8,9,10 Previous studies using SI
models have investigated cellular dysfunction within the hippocampus.6,8,9,10 These studies have
demonstrated a consistent pattern of cellular dysfunction within the hippocampus, including
impaired GABAergic signaling as well as a phosphorylation of Tau protein at the PHF-1
region.
6,8,10,33 Notably, this pattern is consistent with other mouse models of stress, including
models of Repeated Acute Stress as well as injection of corticosterone into the
Hippocampus.7,10,11
These effects are further compounded by chronic alcohol consumption. The combination
of stress and alcohol consumption yielded greater changes in plasma corticosterone and GABA
dysfunction.34,35,36 This is distinct from conditions in which stress alone was not sufficient to
induce significant changes, indicating that alcohol consumption provides a cumulative effect.34,36
The role of alcohol abuse in the development of AD is evidenced by alcohol-induced increases in
Tau phosphorylation and Amyloid-Beta content in the hippocampus.35,37 Additionally, chronic
alcohol consumption reveals the involvement of glycogen synthase kinase 3B (GSK-3B), an

11
ethanol-sensitive protein which induces Tau phosphorylation at the PHF-1 region.37,38,39 GSK-3B
showed increased activation in the above models of stress, suggesting a causal effect between
stress-induced elevations in corticosterone and Tau phosphorylation.7,11,39 Furthermore, changes
in GSK-3B activation following alcohol exposure were observed in the hippocampus as well as
in the prefrontal cortex (PFC) and the nucleus accumbens, with activation being associated with
increases in alcohol-seeking behavior and anxiety following abstinence.38,40,41 Using this
information, a clear relationship presents itself between GSK-3B and development of
neurodegeneration and alcohol abuse.
GSK-3B has various functions within the cell, but its most recognized role is as part of
the Beta-Catenin destruction complex. Beta-Catenin is a signaling molecule which induces gene
transcription in response to external stimuli but is limited by its degradation by the destruction
complex.42,43 In addition to this, Beta-Catenin is also involved in what is called the Noncanonical
Pathway, in which Beta-Catenin triggers a signaling cascade which releases calcium stored
within the endoplasmic reticulum into the cell.43 Under high intracellular concentrations, calcium
becomes cytotoxic, requiring efflux from the cell as well as negative feedback to prevent further
increase. This behavior is consistent with the existing data, as increased hippocampal calcium
influx was reported in mouse models of stress.8,44, Furthermore, it has been observed that DKK1,
a natural inhibitor of the Wnt-Beta-Catenin signaling pathway, is induced by corticosterone,
further implicating Beta-Catenin in mediating the rise in cellular calcium.42,43,45 This negative
feedback is likely to be mediated by calcium-induced proteolysis of GSK-3B. Activation of
GSK-3B is controlled by serine phosphorylation at the S9 site, with GSK-3B being deactivated
upon phosphorylation.42,43 However, Calpain has been identified as a candidate protease for this

12
site, indicating that calcium activation of Calpain can indirectly induce GSK-3B activity by
preventing phosphorylation.46,47,48
Overactivity of GSK-3B poses significant risk to cell survival, diminishing the cell’s
antioxidant capacity through degradation of NRF2.49,50 NRF2 is commonly regarded as a “master
regulator” for the cell’s antioxidant response as well as induction of mitochondrial function.51,52
Under normal conditions, NRF2 is readily degraded after modification by KEAP1, thereby
limiting its activity in the cell. However, this process is prevented by the presence of reactive
oxygen species, allowing NRF2 to induce an antioxidant response.52 GSK-3B, however, has been
found to induce degradation independent of KEAP1, thereby reducing the cell’s antioxidant
capacity as well as its sensitivity to reactive oxygen species.49,50 Furthermore, GSK-3B helps to
impair neuronal GABAergic signaling through degradation of Gephyrin, a scaffolding protein at
the postsynaptic site.48,53 Gephyrin scaffolding binds GABA-A receptors and closely groups
them at the synapse. However, SI mice were observed to have reduced tonic as well as
spontaneous inhibition by GABA, which was associated with reduced GABA-A receptor
clustering.6 This indicates that, consistent with the reported data, long term overactivity of GSK3B significantly disrupts neuronal antioxidant capacity as well as signaling behavior.
The effects of GSK-3B on GABA sensitivity underscores a broader disruption in
GABAergic signaling with significant implications for neuronal health. While impaired GABA
signaling is a common hallmark of memory impairments in AD and is reported in SI mouse
models, the dynamics of this change are more complex and allude to a broader cellular
dysfunction.8,21,54,55 In one experiment using a similar model of social isolation, SI mice
experienced an increase in tonic inhibition but not spontaneous inhibition, contrary to previous
reports in which both were reduced.6,8 This was identified as being due to an increased release of

13
astrocytic GABA, with hippocampal astrocytes demonstrating elevated intracellular Calcium as
well as increased expression of MAO-B, the primary enzyme involved in astrocytic GABA
synthesis.8 Furthermore, this effect was found to be dependent on two transporters: TRPA1, a
Calcium channel which was upregulated in the astrocytes of SI mice,8,44 as well as BEST1, a
Calcium-sensitive Chloride channel which has been regarded as a primary mechanism of
astrocytic GABA efflux.8,21 Similar changes in GABA synthesis were also observed in astrocyte
cultures treated with Amyloid-Beta as well as in mice subjected to chronic ethanol
consumption.35,36,56 Additionally, mice treated with chronic corticosterone showed an increase in
peroxide content in the hippocampus that was alleviated by TRPA1 inhibition.9.44 Contrary to
this, however, TRPA1 content in the hippocampus was reduced by chronic corticosterone
content, suggesting a negative feedback mechanism in response to the increased intracellular
calcium.8 This would provide clearer context for the reported decrease in GABA sensitivity in
the hippocampus, as prolonged SI would require negative feedback. This presents a key role for
GSK-3B in this model, as activation of GSK-3B would mediate GABA desensitization through
breakdown of Gephyrin.
3.2 Disruptions in the Urea Cycle and Metabolism
This pattern of aberrant GABA signaling presents a pathology with significant
implications for neuronal health in the hippocampus, as indicated by reported increases in
intracellular calcium and oxidative stress. In a mouse model of ischemic stroke, overproduction
of astrocytic GABA was observed in cortical regions affected by ischemia, with elevated
astrocytic GABA as well as increased expression of MAO-B and inducible Nitric Oxide
Synthase (iNOS).21 The latter, iNOS, is an enzyme that has been identified as involved in the
development of anxiety and depressive symptoms in SI and corticosterone-treated mice.57,58

14
Statistical analysis showed that there was a negative correlation between astrocytic GABA and
neuronal glucose metabolism in these regions.21 This association was further confirmed by sitespecific manipulation of MAO-B, which demonstrated MAO-B to be both necessary and
sufficient to impair neuronal glucose metabolism.21 Additionally, MAO-B poses significant risk
to cell survival through both its byproducts, which are ammonia and hydrogen peroxide, as well
as its substrate, putrescine, which is converted from ornithine in the Urea Cycle.56,59,60 This effect
was observed both in amyloid-beta-treated astrocyte cultures as well as in post-mortem
hippocampal samples, as the expression of several enzymes involved in the Urea Cycle had been
elevated.22,56 These samples showed increased concentrations of ammonia, which is the starting
substrate for the Urea Cycle and has been demonstrated to induce amyloidogenesis in
astrocytes.56,60
From this, activity of the Urea Cycle can be identified as a key factor in constructing a
model of neurodegeneration. As observed from previous studies, multiple enzymes involved in
the Urea Cycle were up-regulated in models of AD and ischemic stroke, and that selective
knockdown of these enzymes was able to restore cellular and cognitive function and improved
autophagy of amyloid-beta.21,56 Several studies have linked hyperammonemia with neurological
dysfunction, as treatment of astrocytes with ammonia resulted in increased production of
Amyloid Precursor Protein (APP) as well as a reduced rate of APP degradation.56,60 This
indicates a causal relationship between dysfunction of the Urea Cycle and amyloidogenesis, with
amyloid-beta seemingly produced as a response to increases in ammonia concentration.
Although the function of Amyloid-Beta under physiological conditions is not well understood, it
has been identified as interacting with multiple neuronal and non-neuronal surface proteins,61,62

15
suggesting that it acts to initiate a global shift in cellular machinery under hyperammonemic
conditions.
Figure 3: Interactions of the TCA and Urea Cycles
Representation of the TCA (left) and Urea (right) cycles within the cell. Pathways interact at the
regeneration of fumarate from aspartate with side reactions resulting in the production of
neurotransmitters GABA and Glutamate. Efflux of these neurotransmitters in disease states
results in dysregulation of both processes, impairing energy production and ammonia processing.
Created using BioRender.com
These shifts in Urea Cycle activity have further implications for cellular metabolism
through the TCA Cycle and the cell’s generation of energy, with the pathways intersecting

16
through the reactions involving aspartate and fumarate.56,63 In addition to fumarate being a
primary metabolite in the TCA Cycle, fumarate is also regenerated from aspartate as a byproduct
of the Urea Cycle.56,63 This reaction is mediated by Aspartate Transaminase, which
simultaneously metabolizes the excitatory neurotransmitter glutamate for use in the TCA
Cycle.63,64 Multiple investigations into neurodegeneration have reported significant decreases in
cellular energy, including decreased glucose metabolism,56 depleted ATP concentrations,6,65,66,
and down-regulations of Complexes I, II, and IV of the Electron Transport Chain (ETC).9,67
Notably, Complex II also demonstrated reduced capacity in comparison to Complexes I and
IV.67 Complex II, also known as Succinate Dehydrogenase, is directly involved in the TCA
Cycle, as it mediates the conversion of succinate to fumarate and is a rate-limiting step in the
production of ATP.63,67 Additionally, in the 5xFAD model of AD, neuronal concentrations of
both fumarate and aspartate were found to be reduced in the hippocampus of 5xFAD mice.63 This
effect was limited to metabolites of the TCA Cycle, as the concentrations of GABA and
glutamate synthesized from these reactions were found to vary between experimental models.63
As such, dysfunction to the Urea Cycle can be observed having a parallel, even causal
relationship with impaired production of cellular energy, with a consequent effect on cognitive
function.
3.3 ATP in the Synapse
The importance of energy homeostasis in the brain represents a significant risk factor in
the development of cognitive dysfunction, as reductions to ATP generation and increased efflux
of adenosine were observed in AD models.68,69 Changes in ATP production present an additional
risk to neurological function through the involvement of adenosine as an extracellular signaling
molecule: multiple classes of purine receptors are expressed on the cell surface, including the A1,

17
A2A, P1, and P2 receptors.68,70 These receptors are activated in response to extracellular
adenosine and other purines which have been effluxed from the cell, which can occur under
various conditions.68,70,71,72 Notably, this phenomenon has been observed to be further induced in
the hippocampus under pathological conditions such as anxiety, ischemia, and traumatic brain
injury, with hippocampal astrocytes having been identified as a major candidate for a primary
source.71,72,73 Furthermore, efflux of adenosine was influenced by changes in intracellular
calcium, suggesting a causal mechanism between elevated calcium as a result of cellular stress
and the subsequent release of adenosine as an inflammatory response.72,73,74 Under normal
physiological conditions, this reaction is likely to serve as a signaling mechanism in response to
stress, as the release of astrocytic adenosine was found to display an anxiolytic effect in animal
models of stress and resulted in increased anxious behavior when efflux was prevented.73
Conversely, however, this phenomenon appears to be a contributing factor under chronic models
of inflammation, including multiple models of AD.
The involvement of purine receptors in AD pathology has been largely recognized as
both a potential mechanism as well as a therapeutic target for disorder, with observed
correlations between the progression of AD and the activity of purine receptors.70,75, Of these
receptors, the receptor P2X7 has been identified as a promising candidate,70,75,76 with P2X7
activation being linked to the formation of Amyloid-Beta plaques.75,77,78 Moreover, activation of
the P2X7 receptor has been linked to increased activity of GSK-3B, which has also been linked
to cellular dysfunction as well Amyloid secretion.77,79 This association has led to the hypothesis
that the Amyloid-Beta peptide acts as an agonist for the receptor and induces neuroinflammation
through activation of glial cells.75,76 However, this proposal has been called into question, as
recent investigations have failed to demonstrate that the amyloid-beta peptide acts as a direct

18
P2X7 agonist.76 As such, Amyloid-Beta is more likely to indirectly mimic the effects of P2X7
activation, as incubation with amyloid-beta was able to induce release of cellular ATP as well as
the subsequent activation of P2X receptors.80 While this demonstrates the involvement of P2X
receptors in the pathogenesis of AD, it also illustrates their role as part of a larger signaling
cascade rather than as a direct target or mechanism. Therefore, while the P2X7 receptor presents
a means through which the ATP-mediated signaling cascade may be perpetuated, it fails to
demonstrate how this process is initiated, nor does it adequately illustrate the mechanism by
which Amyloid-Beta affects this process.
An alternative candidate would be the A2AR receptor, which has been shown to be
significantly upregulated in the brains of AD patients.81,82 Overexpression of the A2AR receptor
in the Hippocampus is an age-associated phenomenon that is further exacerbated in AD.82,83,84
This has prompted the investigation of A2AR as a potential biomarker for AD as well as a novel
therapeutic target,81 as memory impairments observed in AD mice were ameliorated by
pharmacological blockade of A2AR.82,85 Conversely, induction of A2AR was found to exacerbate
memory deficits in the same model, with overexpression being shown to induce age-associated
cognitive deficits in young mice.82,84 This suggests a causal mechanism between upregulation of
the A2AR receptor with age and the development of AD pathology. However, the A2AR receptor
also appears to play a significant role in injury, as A2AR activation was found to be linked to
models of neurological dysfunction including traumatic brain injury and kainate-induced
convulsions.86,87,88 A2AR mediated neurotoxicity under kainate-induced convulsions through
regulation of glutamate release and homeostasis, suggesting that the contribution of A2AR to AD
pathology is cumulative with injury.87,88,89 Moreover, the neurodegenerative effects of A2AR
activation were associated by increased activation of GSK-3B and subsequent Tau

19
phosphorylation at the PHF-1 region,86 strongly suggesting that A2AR is a key factor in AD
pathogenesis.
Figure 4: Adenosine Signaling and Synaptic Function
Schematic of A2AR activation in the synapse. Efflux of adenosine (red) via ENT is induced by
TrkB-BDNF signaling. A2AR activation induces presynaptic release of glutamate (purple) and
inhibits astrocytic uptake by GLT-1. A2AR activation sensitizes GR activation by corticosterone
(orange) against inhibition by BDNF. Combined GR activation and excitotoxicity induce
calpain-mediated cleavage of GSK-3B and TrkB, altering signaling within the cell. Created using
BioRender.com
A2AR activation plays an important role in synaptic activity and memory encoding.88,90 In
the dentate gyrus (DG), which is the primary region of input for the hippocampus, A2AR

20
activation was found to mediate LTP via retrograde signaling.88 Repeated burst firing in the DG
was accompanied by an increase in amplitude of excitatory postsynaptic currents (EPSCs) at the
affected synapse. This effect was found to be dependent on the presynaptic expression of A2AR
and the postsynaptic expression of tyrosine receptor kinase B (TrkB).88 TrkB is the primary
receptor of brain-derived neurotrophic factor (BDNF), a neurotrophin which promotes neuronal
cell survival and has been observed to be decreased in the brains of AD patients.91,92 The role of
A2AR in mediating LTP was determined to be through a retrograde signaling pathway in which
activation of postsynaptic TrkB induced the release of adenosine into the synaptic cleft with a
subsequent A2AR-induced release of glutamate.88 This pathway was also found to be involved in
the initiation and neurodegenerative effects of kainate-induced seizures, as kainate treatment
resulted in an upregulation of A2AR and a consequential increase in glutamate release.87,88
Furthermore, A2AR has been shown to play an important role in astrocytic glutamate
homeostasis, which is necessary for the clearance of neurotransmitters from the synapse.
Knockout of astrocytic A2AR was found to induce schizophrenia-like symptoms in mice through
upregulation of Glutamate Transporter-1 (GLT1), increasing the rate of glutamate clearance and
producing cognitive and locomotor impairments.89 This demonstrates that A2AR plays an
important role in mediating glutamate signaling in the synapse and that aberrant A2AR activity is
sufficient to induce a pathological state.
In the case of AD, A2AR demonstrates a significant increase in activation, which has been
demonstrated by increased expression in the hippocampus of aged adults and AD patients.81,82,83
Similar changes in A2AR expression were observed in multiple models of AD, in which blockade
of A2AR alleviated cognitive deficits and restored cellular survival and LTP in the
hippocampus.82,84,85 Furthermore, upregulation of A2AR induced in a transgenic mouse model of

21
AD was found to further exacerbate AD pathology, with greater impairments in memory and
increased Tau phosphorylation at the AT8 region. However, these changes were modest, with no
changes observed in Amyloid content or in phosphorylation of other Tau regions.84 This suggests
that, while A2AR activation plays a significant role in AD pathogenesis, the effect already
approaches its maximum in AD models. This relationship is further supported by the role of
BDNF in its neuroprotective effects against Amyloid toxicity. As previously observed, BDNF
acts as part of a retrograde signaling pathway with A2AR to induce LTP.88 However, although
BDNF exerted a neuroprotective effect in cultured neurons, this effect was found to be
independent of A2AR, suggesting a decoupling of the BDNF-TrkB-A2AR pathway.93 This was
consistent with reports finding that Amyloid interferes with BDNF signaling through the TrkB
receptor, as neuron cultures incubated with Amyloid-Beta demonstrated a shift from the fulllength TrkB receptor to its truncated non-functional isoform (TrkB.T).92,93 Given the
involvement of TrkB in the neurodegenerative effects of kainate-induced convulsions,87,88 this
represents a potential compensatory mechanism against inflammatory processes.
This shift in TrkB function indicates an intracellular signaling pathway which is induced
by Amyloid-Beta. A partial mediator of this effect was the increased transcription of the TrkB.T
isoform in response to excitotoxic conditions, which is a characteristic of neuroinflammatory and
neurodegenerative disorders.92,94 The increased expression of TrkB.T was found to exert an
inhibitory effect on the full length TrkB, further diminishing normal BDNF-TrkB signaling.94
Conversely, full length TrkB was subjected to proteolytic cleavage by Calpain, which was
induced by Amyloid-Beta.92,93,94 Activation of Calpain has been previously described as part of
the cellular response to stress and the subsequent activation of GSK-3B, indicating that TrkB
modification acts in parallel to this pathway.46,47,48 This is further confirmed by reported

22
interactions between TrkB and the Glucocorticoid Receptor (GR), which demonstrate a mutually
antagonistic relationship.91,95,96 Animals subjected to chronic stress displayed a widespread shift
from the full length to the truncated TrkB isoform across multiple brain regions.96 Furthermore,
BDNF was found to induce site-specific phosphorylation of GR, which was necessary for
synaptic maintenance and learning.95,97 This phosphorylation was disrupted under chronic stress
and resulted in impaired memory and increased mortality in AD models without affecting
Amyloid-Beta deposition.95 These effects were further amplified by overexpression of A2AR, as
it induced transcriptional activity of GR in the nucleus and sensitized the hippocampus to GR
agonism.98,99 In the context of synaptic activity, this suggests that A2AR activates GR as a
negative feedback mechanism against BDNF-TrkB signaling. Additionally, this provides a
substantial link between the cellular effects of chronic stress and the pathogenesis of AD, as both
are mediated by the age-associated increase in A2AR activation. And although the direct
relationship between Amyloid-Beta and A2AR activation remains unclear, it presents a novel
image of Amyloid-Beta deposition as part of the brain’s natural inflammatory response.

23
Chapter 4: Neural Pathways
4.1 A Primary Neural Pathway
Changes to cellular function in the hippocampus radiate outwards onto connected regions
of the brain, thereby causing complex changes in behavior. Understanding the links between
these connected regions is a necessary point in developing a clear model of neurodegeneration,
as the complex interactions between them shed light on how these disorders neuronal damage
local to the hippocampus rather than its signaling to other regions of the brain. Therefore, the
implications on behavior cannot be properly understood without the context of hippocampal
connectivity. Previous studies investigating the neurological action of the gut microbiome on
social behavior found significant roles by the hippocampus in conjunction with the hypothalamus
and the bed nucleus of the stria terminalis (BNST).12,13 This demonstrated a causal relationship
between depletion of the gut microbiome and elevated plasma corticosterone, resulting in
impaired social behavior.100 Similar studies into the ventral tegmental area (VTA) found similar
behavioral impairments, though these studies had investigated the role of oxytocin, specifically
onto the dopaminergic neurons of the VTA.13,14 Both induction of corticosterone signaling and
ablation of oxytocin signaling were observed to be sufficient to induce the observed social
impairments, while impairments caused by depletion of the gut microbiome could be reversed by
ameliorating these signaling mechanisms.100,101 Given previous findings that both corticosterone
and oxytocin were shown to induce neuronal spiking in the hippocampus13,100,101,102 as well as
projections by the VTA onto the hippocampus, it is likely that corticosterone and oxytocin act as
redundant signaling mechanisms in social behavior.
This redundant signaling is likely to have multiple factors which coordinate and regulate
the intensities of each pathway. One such factor is expected to be FGF21. FGF21 plays an

24
Figure 5: Neural Pathway Regulating Behavior
A diagram mapping the connections in the proposed neural pathway. Above pathway shows
signaling cascade by CORT and OXT. Dotted lines represent brain regions that are involved in
both pathways. FGF21: Fibroblast Growth Factor 21; CORT: Corticosterone; OXT: Oxytocin;
VTA: Ventral Tegmental Area; DR: Dorsal Raphe Nuclei; BNSTDM: Bed Nucleus of the Stria
Terminalis, Dorsomedial; BNSTAL: Bed Nucleus of the Stria Terminalis, Anterolateral; BNSTPR:
Bed Nucleus of the Stria Terminalis, Principal; BNSTFU: Bed Nucleus of the Stria Terminalis,
Fusiform; BNSTOV: Bed Nucleus of the Stria Terminalis, Oval; CeA: Central Amygdala; NAcc:
Nucleus Accumbens; PVNCRF: Paraventricular Nucleus, CRF-Secreting; PVNOXT:
Paraventricular Nucleus, Oxytocin-Secreting. Created using BioRender.com

25
interesting role in this signaling pathway because it induces the transcription of both
corticosterone103 and oxytocin,104 thereby increasing and synchronizing their respective
signaling. In particular, FGF21 and corticosterone appear to express a bi-directional feedback, in
which FGF21 induces the synthesis of corticosterone from the adrenal glands which
subsequently induces FGF21 synthesis in the liver.103 In addition, transcription of both oxytocin
and FGF21 appear to be NRF2 dependent, indicating that induction of NRF2 is able to induce
both upstream and downstream effectors of this pathway.104 The importance of FGF21 in the
induction and coordination of this neuronal pathway provides a possible mechanism for agedependent cognitive decline, as levels of FGF21 in the plasma, liver, and brain are reported to
experience an age-related decrease.20
As previously commented, a recent study demonstrated a joint action between the
hippocampus and the BNST modulating social behavior under conditions of microbial depletion
as well as induction of corticosterone signaling.100 Notably, however, observations regarding
blockade of corticosterone signaling onto the BNST was more nuanced: whereas social behavior
was restored in microbe-depleted mice, the opposite effect was observed in mice with intact
microbiomes, indicating paradoxical changes in activity at this region. It is important to note that
the BNST is a complicated region of the brain, with distinct subnuclei projecting to diverse
regions of the brain, including the VTA, the amygdala, and the hypothalamus, giving it
involvement in various signaling pathways.18 This is important to note, as manipulation of
corticosterone signaling was performed through injection of a viral vector into the BNST.100 As
such, transfection was likely distributed across the subnuclei of the BNST, thereby affecting the
different signaling pathways and yielding paradoxical results.

26
Fortunately, many of the BNST subnuclei have already been well characterized.18,105,106
Two important regions are the dorsomedial105 and anterolateral areas106 of the BNST. These
regions are of particular interest because of their mutual outputs onto the parvocellular neurons
of the PVN,105,106 which controls secretion of CRF and initiates the Hypothalamic-PituitaryAdrenal (HPA) axis.107 The HPA axis regulates the secretion of glucocorticoid hormones into
circulation, which is a major contributor to the cellular dysfunction observed in models of
chronic stress. Furthermore, the HPA axis has been demonstrated to be subject to dysregulation
in age-associated increases A2AR expression, which further exacerbates the effects of
corticosterone in the synapse.98 The dorsolateral and anterolateral areas of the BNST possess a
high number of inhibitory GABAergic projections,18,105,106 indicating that their signaling onto the
PVN serves as an important negative feedback mechanism onto the HPA. Importantly, both of
these regions receive input from the hippocampus and the VTA105,108,109 and display mutual
innervations with the dorsal raphe nucleus,105,110 indicating a coordinated signaling. Notably,
however, the dorsomedial nucleus of the BNST also projects onto the magnocellular neurons of
the PVN,105 thereby halting further secretion of the oxytocin signaling and limiting the negative
feedback onto the HPA axis.
An additional point of interest in the BNST is the principal nucleus, which receives input
from CRF secreted by the PVN and sends projections backwards to the upstream anterolateral
area.18,111 This region is of particular interest because of its strong dual-expression of the kappaopioid receptor receptor (KOR)18,112 and of CRF receptor type 2 (CRFR2).18,19,113 KOR is the
primary receptor to dynorphins, an endogenous opioid which is associated with stress-induced
behavior as well as the reinforcement of drug seeking and depression-like behaviors.114,115,116
Interestingly, CRFR2 was also found to be involved in response to aversive stimuli, as blockade

27
of CRFR2 was able to ameliorate stress response due to opioid withdrawal117 as well as improve
resilience against the development of PTSD-like symptoms under repeated negative stimuli.19
Additionally, CRFR2 was found to mediate KOR signaling, inducing conditioned-place aversion
(CPA) similar to treatment with KOR agonists. However, CRFR2 was indicated to be a
downstream signal to KOR, as blockade of KOR prevented CRFR2-induced aversion but not
vice versa.114 Furthermore, the role of CRFR2 in development of anxiety and PTSD-like
symptoms presents further complications, as conflicting reports conclude that the expression of
CRFR2, specifically in the BNST, reduces anxiety and improves response to stress.118,119
These apparent contradictions are likely due to differences between the anticipation of
and response to a negative stimulus. Activation of CRFR2 improves anxiety scores in tests such
as the elevated plus maze and open field tests,119 which measure behavior in anticipation to
danger. This is distinct to previously mentioned models such as opioid withdrawal and CPA, in
which negative associations are already encoded. This distinction is further emphasized between
models of PTSD, in which blockade of CRFR2 was found to improve symptoms in response to
repeated negative stimuli,19 whereas the opposite relationship was observed after incubation of
animals with a predator-associated odor.118 This presents the CRFR2 signaling of the principal
nucleus as a candidate for valence detection. Furthermore, it demonstrates a mechanism of
consolidating memory of negative stimuli, as it prolongs activation of the HPA axis through
inhibition of the upstream anterolateral area.18,111
4.2 Signaling Anticipation and Reward
This pathway presents a novel and concise model to better understand the connected roles
of these distinct regions as well as their involvement in pathological conditions, including
neurodegenerative diseases and disorders such as addiction or trauma. However, as previously

28
stated, this pathway appears to differentiate between the anticipation of a stimulus and the
stimulus itself,19,114,118,119 suggesting that further modulation occurs elsewhere in the brain before
feeding back into the above regions. In particular, this includes two additional regions of the
Figure 6: MOR-CRFR1 Synaptic Modulation
Co-localization of CRFR1 and MOR receptors at the Central Amygdala and the Anterolateral
Area. Release of CRF (blue) by the Oval and Fusiform Nuclei induces presynaptic release of
Glutamate (purple) with increases in postsynaptic excitation. Release of enkephalin (green) by
the Oval Nucleus eliminates postsynaptic excitation by inhibition of CRFR1 receptors. Created
using BioRender.com
BNST: the oval and fusiform nuclei.120 Both regions receive innervation by the nucleus of the
tractus solitarius (NTS),105,120 which is a major source of norepinephrine in the brain. However,

29
in a cue-conditioned model of drug delivery, release of norepinephrine was associated with the
cued stimulus followed by release of dopamine onto the oval nucleus upon drug
administration.121,122 This demonstrates a sequential signaling process in the BNST in which the
anticipation of reward precedes the rewarding stimulus, thereby enabling distinction between the
two. Additionally, of the oval and fusiform nuclei, the oval nucleus displays greater outputs onto
the fusiform than vice versa.120 This could help to serve as a negative feedback during the
anticipatory phase, whereas subsequent dopamine release onto the oval nucleus would create a
shift in balance between the two.
Understanding the interactions between these nuclei requires identification of their
mutual signaling targets. Both the fusiform and oval nuclei have been observed to have
projections onto the central amygdala and the anterolateral area of the BNST,120 the latter being a
core region in the previously described pathway. The central amygdala and the anterolateral area
share a distinct similarity in their receptor distributions, as both were found to have a significant
co-localization of the CRF-receptor type 1 (CRFR1) and the mu-opioid receptor (MOR).123 The
latter is the primary receptor for another class of endogenous opioids, enkephalins, and is
identified as the main mechanism of action for addictive opiates such as heroin and morphine.124
In both the central amygdala and the anterolateral area, approximately 63-66% of CRFR1-
expressing dendrites were found to co-localize with MOR, with a primary placement at synapses
which receive excitatory input.123 This suggests that both CRFR1 and MOR express postsynaptic roles in modulating the response to excitatory signals in these regions. However, these
roles were likely contradictory to each other, as presynaptic CRFR1 was largely found in the
axons of excitatory synapses whereas MOR was expressed in those that were inhibitory. Taken
together, this suggests that CRFR1 plays a role in increasing sensitivity to glutamate and other

30
excitatory signals whereas MOR subsequently desensitizes the cell. This reflects the sequential
activation of the fusiform and oval nuclei,121,122 as CRF is expressed in both regions while
enkephalin is mainly expressed in the oval nucleus.
Interestingly, mutual interactions between the anterolateral area and the central amygdala
appear to play an important role in this pathway: both the anterolateral and dorsomedial areas of
Figure 7: KOR-mediated Blockade of GABA Signaling
Synaptic signaling from the Central Amygdala onto the Anterolateral Area. Inhibitory signaling
is blocked by release of dynorphin (pink) onto presynaptic KOR receptors. Negative feedback is
restored upon cessation of dynorphin and release of GABA (orange). Created using
BioRender.com

31
the BNST project onto the central amygdala,105,106 which itself sends projections backwards to
the anterolateral area.125 The mutual inhibition by the anterolateral area and the central amygdala
potentially serves as a negative feedback loop, preventing overexcitation of either by the oval
and fusiform nuclei.120 However, these signals are not entirely symmetrical, as inhibitory
projections from the central amygdala onto the anterolateral area were found to express KOR at
the presynaptic site which prevented the release of GABA, thereby blocking inhibitory
signaling.118 This offsets the mutual inhibition of the two regions by preventing negative
feedback from the central amygdala onto the anterolateral area but not vice versa, placing
heavier restriction on the former. This effect may also be mediated by the oval and fusiform
nuclei, as both areas significantly express dynorphin112 in addition to their above roles in
glutamate sensitivity. This presents an additional means of differentiation between anticipation
and reward: similar to its role in the principal nucleus of the BNST,114 dynorphin signaling in the
anterolateral area may help in distinguishing anticipation, as its deactivation and the
simultaneous release of enkephalin would work in tandem to restore negative feedback.
The central amygdala as well as the fusiform nucleus both project onto the nucleus
accumbens,120,126 emphasizing the importance of dopamine signaling in this pathway. The
nucleus accumbens receives dopamine input from the VTA and has been identified as a key
region in reinforcement of reward as well as addiction.15 As a mediator in reward signaling,
inhibitory input into the nucleus accumbens during the anticipatory phase could represent a
priming in this pathway, as the subsequent disinhibition may produce a rebound in activity that is
cumulative to input from the VTA. This is observable on a cellular level, as reductions in
astrocytic glutamate release were a necessary condition for synaptic reinstatement of cocaineseeking behavior.29 This is supported by the proposed roles of dynorphin and enkephalin in

32
switching from the anticipatory to the reward phases as well as their respective involvements in
the reinforcement of addictive behavior. To this end, the nucleus accumbens modulates this
pathway via its GABAergic projections from the nucleus accumbens shell to the dorsomedial
nucleus,127,128,129 in which inhibition of the nucleus accumbens during the anticipatory phase
consequently disinhibits the dorsomedial nucleus. This demonstrates a priming mechanism for
signaling onto the PVN, as both the anterolateral and dorsomedial areas see increased activation
during the anticipatory phase. This also demonstrates a strict control over the pathway, as
dopamine release by the VTA restores negative feedback between the anterolateral area and the
central amygdala, bringing activation of this pathway to an abrupt halt. Recognizing this nuanced
behavior in signal timing is important for modeling the interactions between brain regions under
neurodegeneration.
4.3 Rewiring of the VTA
From this information, the VTA can be identified as having a critical role in modulating
this pathway, as the release of dopamine from the VTA appears to initiate a shift in equilibrium
between opposing signals. This is further emphasized by plasticity of the VTA on the cellular
level, as the VTA shows a bimodal response to CRF input, as excitation of the VTA in response
to CRF was diminished over increasing time and concentration. This effect was identified to be
due to differential action between presynaptic CRFR1 and CRFR2 receptors, with an initial
increase in glutamate release mediated by CRFR1 which was subsequently attenuated by
CRFR2, which induced the release of GABA onto the presynaptic site.130 As a source of CRF
which projects onto the VTA, this has particular implications for the dorsomedial nucleus,105,112
as output from the VTA onto the hippocampus and the nucleus accumbens send competing
excitatory and inhibitory signals back towards the dorsomedial nucleus.105,109,129 As a primary

33
source of dopamine and a key region in drug addiction, the impact to the VTA cannot be
understated: in animals which self-administered cocaine, the attenuating effect of CRFR2 was
diminished, as administration of a CRFR2 agonist saw a diminished effect on both glutamate and
GABA tone.130 This indicates a significant change on the cellular level of the VTA’s ability to
regulate its response to CRF during the anticipatory phase, potentially sensitizing this signaling
pathway to drug-associated cues.
A further indicator to this was the response of the VTA under conditions of stress: in
contrast to healthy mice, animals which experienced both cocaine self-administration as well as
yohimbine-induced distress saw a shift in CRFR2 activity from attenuating to sensitizing.130 This
is a stark difference because the role of the CRFR2 receptor is essentially reversed in this
condition, potentially amplifying the excitatory effect of CRF over a longer period. The cause of
this shift was identified as a response to adenosine signaling, as blockade of the A1 receptor was
able to mitigate sensitization by CRFR2 despite showing no impact in previous test conditions.
This suggests that, under conditions of stress and drug-associated cues, there is an increase in
extracellular adenosine that is not observable under other conditions, and that this increase
mediates a decrease in GABAergic tone through activation of the A1 receptor. This increase in
extracellular adenosine may have been mediated by efflux of ATP from nearby astrocytes, as
observed in the hippocampus. Notably, the increase in glutamate signaling by CRFR2 was
ablated by blockade of the GABA-B receptor. Because of the drop in GABA tone by A1-CRFR2
interactions, the blockade of presynaptic GABA receptors would be anticipated to have the
opposite effect, further preventing the release of glutamate. This suggests a non-neuronal
mechanism, likely nearby astrocyte cells, as GABA-B receptor-mediated increase in intracellular
Calcium could potentially result in efflux of ATP.131 Astrocytic involvement is further

34
Figure 8: CRFR1-CRFR2 Action in the VTA
Differential effects of CRFR1 and CRFR2 receptors in the VTA. CRFR1-induced release of
Glutamate (purple) is attenuated by CRFR2-mediated release of GABA (orange) onto the
presynaptic site. Attenuation by CRFR2 is blocked by astrocytic release of adenosine (red) onto
the A1 receptor. Stress-induced release of adenosine by astrocytes is mediated by activation of
the CRFR2 and GABA-B receptors. Created with BioRender.com
implicated by evidence of a cocaine-induced decrease in number of cells in the VTA expressing
S100B, an astrocyte-specific marker, as well as increased colocalization of S100B and CRFR2,
suggesting increased astrocyte sensitivity to CRF.132
The changes in CRF signaling as well as the changes to CRFR116,17,130 and
CRFR2130,132,133 activity represent a distinct time-dependent mechanism for the development of

35
dependence. Acute withdrawal after prolonged chronic ethanol exposure was shown to attenuate
CRF-induced increases in excitatory signaling onto the VTA while producing a significant
increase in glutamatergic tone.16 Moreover, this increase in glutamatergic tone was found to be
CRFR1 dependent, thereby coinciding with observed changes in CRFR2 activity under stress.130
Blockade of CRFR1 in the VTA was shown to limit binge-like drinking as well as prevent
reinstatement of drug-seeking behavior after stress.16,17 However, as with previous observations,
this effect was prevented by simultaneous blockade of CRFR2.17,130 This change in activity
appears to extend beyond the VTA into the nucleus accumbens and the BNST, with excitability
in these regions increasing after social isolation and chronic ethanol exposure, respectively.133,134
In the nucleus accumbens, release of dopamine after depolarization is significantly increased
following social isolation, with blockade of local CRFR1 being unable to attenuate dopamine
release in contrast to control rats, indicating a decrease in sensitivity.133 Notably, a similar
behavior was observed in the central amygdala, as CRFR1 was found to be an important
mediator of GABAergic signaling during binge-like drinking and withdrawal. This is consistent
with the proposed role of CRFR1 in sensitizing the central amygdala to excitatory input. Taken
together, this reinforces the overlapping mechanisms in the pathology of addiction and social
isolation, as disruptions to CRF molecular signaling result in aberrant behavior. Furthermore, it
creates a concrete image of the impact of stress on neuropsychiatric disorders, which can thus be
used to model the progression of neurodegenerative disorders in response to environmental
stressors.

36
Chapter 5: Future Investigations
5.1 Insulin Resistance in the Brain
Insulin plays a significant role in the brain as it helps to modulate cellular energy and cell
survival. Conversely, insulin resistance has been shown to act as a mechanism for the
development of cognitive impairment, in particular during the early stages of AD.100,135,136 This
has been identified as being mediated by Insulin Receptor Substrate 1 (IRS1), a signaling protein
which mediates the intracellular response to insulin and shows reduced activation in multiple
models of AD.135,136 Multiple factors involved in the pathology of AD have been observed to
reduce the activation of IRS1, including incubation of Beta-Amyloid as well as phosphorylation
by GSK-3B, both of which were recognized in the sections above as playing a key role in the
pathogenesis of AD.137,138
This is notable as it demonstrates an overlapping pathology between animal models of
stress and diabetes, as diabetic models of mice showed similar patterns of Tau phosphorylation
to SI and corticosterone-treated mice.11,139 Conversely, administration of intranasal insulin was
able to rescue activation of IRS1 and reduce activity of GSK-3B through induction of the PI3KAKT axis,140 suggesting a negative feedback mechanism between the two proteins. This is not
the first example of impaired energy homeostasis in the brain in AD pathology, as was observed
with reductions to ATP generation and the efflux of adenosine as a signaling mechanism for
neuroinflammation.68,69,70 Additionally, IRS1 can be inferred to have a distinct role in the
pathogenesis of AD, notably through Tau phosphorylation, as the expression of Tau protein is
necessary for IRS1 signaling.141 As both proteins have been shown to be targets of GSK-3B
activity, this presents a potential pattern of enzyme activity within the cell during the early stages
of AD.34,138 However, the role of this shift in activity under physiological conditions, as well as

37
the interactions between Tau expression and insulin signaling in the brain, both remain largely
unclear.
5.2 Influence of the Gut Brain Axis
As previously commented, studies into the gut microbiome demonstrate a causal
mechanism between dysbiosis and impaired social behavior, disrupted neuroendocrine signaling,
and aberrant activity in the hippocampus, the VTA, and the BNST.12,13,18 These effects are
mediated by excess concentrations of plasma corticosterone and diminished secretion of
oxytocin from the PVN of the hypothalamus, which serve as part of a redundant signaling
mechanism to activate the proposed primary pathway.12,13 This phenomenon was found to occur
in animal models of microbial depletion as well as in genetic models in which the animal in
which the microbiome had been disrupted but not otherwise depleted. This presents a distinct
chain of events in which an environmental or physiological factor induces specific gut dysbiosis
which hampers endocrine signaling to the brain and results in aberrant neurological and
behavioral responses. More specifically, these symptoms were found to result from the loss of
two specific taxa of bacteria: L. reuteri and E. faecalis, both of which are Lactic Acid
Bacteria.142,143 This raises important questions into the behavior of Lactic Acid Bacteria in the
gut microbiome and its contribution to neurological health. Additionally, this raises questions
regarding the effects of conditions known to impact the gut microbiome, such as alcohol
consumption, which creates various shifts in microbial content while also disrupting the
intestinal environment.144,145
5.3 Liver Secretion of FGF21
Similar to the microbiome, liver secretion of FGF21 plays an important role in
maintaining cognitive health, in particular through the coordination of corticosterone and

38
oxytocin signaling to the brain.103,104 FGF21 has been shown to exert strong neuroprotective
properties in the hippocampus while also suppressing alcohol consumption by modulating
activity of the basolateral amygdala.20,104,146 Conversely, FGF21 secretion decreases with age,
presenting a possible link to age-related cognitive decline.20 Additionally, FGF21 has been found
to mediate the inactivation of GSK-3B,147 which has been previously addressed as being a key
mediator in multiple pathologies in AD models, including Tau hyperphosphorylation and
inactivation of IRS1.27,138 The action of FGF21 in the brain presents an important means in
regulating metabolic function under cellular stress. However, the role of FGF21 in maintaining
neurological health, as well as the broader interactions between the brain and liver function,
requires deeper investigation.

39
Chapter 6: Conclusions
Neurodegenerative diseases such as Alzheimer’s and Parkinson’s are still poorly
understood, as their complex pathologies make it difficult to characterize the mechanisms behind
their progression. Similarly, much is unknown about the psychiatric disorders which are often
comorbid with them, presenting a significant obstacle in the treatment of patients afflicted with
one or multiple of these conditions. These disorders affect complex signaling pathways within
the brain, which results in a series of abnormalities cascading throughout the interconnected
regions. As such, the pathologies of these disorders must be understood in the global context of
how the individual regions influence each other in both healthy and in disease states.
The goal of this thesis has been to propose a viable model of neural activity across
multiple regions of the brain, including the hippocampus, the VTA, and the BNST, which
encapsulates a broad range of behaviors and neurological disorders. This model demonstrates
that the Hippocampus undergoes a series of complex changes in neuronal signaling, namely in
the release of astrocytic GABA responding to elevated intracellular calcium. This is an
instigating factor in the disruption of cellular energetics and the dismantling of the TCA and
Urea Cycles. These effects cascade downstream onto the BNST and the VTA, which mediate a
diverse network of interconnected regions involved in regulating behavior. The VTA plays an
important role in this network, providing a substantial driving force through the release of
dopamine and is shown to be inducible by several key factors. Moreover, it displays a distinct set
of plastic behaviors which are observable across various psychiatric disorders. This signaling is
further mediated by the diverse subsections of BNST, which helps to coordinate anticipation and
reward signaling through the timed release of dynorphin and enkephalin.

40
This model presents a significant step forward in understanding the brain, having
characterized not only neurodegeneration and addiction but also the brain’s responses to stress,
reward, and social interaction. Because of its complex structure, much is still unknown regarding
the various functions of the brain and their contributions to neuropsychiatric disorders.
Treatment for these disorders largely consists of pharmacological interventions which broadly
affect the brain’s signaling pathways, thereby resulting in diminished efficacy and a high
likelihood of side effects. Recent progress has evolved to include novel therapeutics such as
electrical stimulation of the brain as well as peptide-based drugs that are designed to target
specific proteins. However, these advances are limited by the available understanding of the
brain. This requires a more comprehensive understanding of the metabolic and signaling
pathways and their interactions at different regions. The model proposed by this thesis aims to
provide a foundation for future research into the brain’s complex processes.

41
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Abstract (if available)

Abstract Neurodegenerative diseases such as Alzheimer’s and Parkinson’s are complex disorders that are poorly understood. They incorporate a broad range of dysfunctions, including the formation of Tau and Amyloid aggregates in the hippocampus. This results in changes in behavior and impairment to cognitive function. However, given the complexity of these disorders, it is difficult to characterize the active mechanisms and how they contribute to disease progression. This thesis aims to provide a new model that can better describe the cellular and neurological changes associated with neurodegenerative disorders. This includes an investigation into the molecular mechanisms that contribute to impaired neuronal function. In particular, this thesis proposes a neural pathway that connects multiple regions of the brain in order to characterize changes in behavior. This thesis integrates current research into neurodegeneration in order to synthesize a model that can guide future research.

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Asset Metadata

Creator Talamas, Robin (author)

Core Title A new model of neurodegeneration: integrating molecular, electrochemical, and neuronal behavior into a novel paradigm

Contributor Electronically uploaded by the author (provenance)

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Degree Conferral Date 2024-12

Publication Date 01/15/2025

Defense Date 01/14/2025

Publisher Los Angeles, California (original), University of Southern California (original), University of Southern California. Libraries (digital)

Tag Addiction,Alzheimer's,bed nucleus of the stria terminalis,hippocampus,neurodegeneration,OAI-PMH Harvest

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Language English

Advisor Davies, Daryl (committee chair), Asatryan, Liana (committee member), Seidler, Paul (committee member)

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