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Ethanol induced modulation of microglial P2X7 receptor expression and its role in neuroinflammation

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Ethanol induced modulation of microglial P2X7 receptor expression and its role in neuroinflammation

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ETHANOL INDUCED MODULATION OF MICROGLIAL P2X7 RECEPTOR
EXPRESSION AND ITS ROLE IN NEUROINFLAMMATION

by

Sushmitha Gururaj

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)

August 2012

Copyright 2012 Sushmitha Gururaj
ii

ACKNOWLEDGEMENTS

I would like to take this opportunity to express my gratitude towards the people whose
contributions have guided my journey through graduate school and my research. First and
foremost, I would like to thank my thesis advisor, Dr.Daryl Davies. His teaching and
contributions, along with his good-natured support, have been instrumental to my
progress.

I owe my deepest gratitude to Dr.Liana Asatryan for being my mentor through my
research endeavors. I cannot thank her enough for her constant guidance and
encouragement at every stage of the research project. I would also like to thank Dr.Zoltan
Tokes and Dr.Vijay Kalra for serving on my committee and providing me with valuable
insights.

I am thankful to all my fellow lab members whose help and camaraderie have made
learning in the laboratory an enjoyable process, and contributed to my personal growth.

Finally, I am eternally grateful to my parents and family members for their unconditional
support and enthusiasm and for making this journey possible.

iii

TABLE OF CONTENTS

Acknowledgements ii
List of Figures v
Abstract vi
Chapter 1: Introduction
1.1 Alcoholism as a disease: Need to understand the 1
mechanisms of alcohol-induced brain damage
1.2 P2X7 receptors in AUDs
1.2.1 P2X7 receptors in neuroinflammation and 3
neurodegeneration
1.2.2 Alcohol induced neuroinflammation and 6
neurodegeneration: A possible role of P2X7Rs?

Chapter 2: Aims
2.1 Aim 1: Test the effect of ethanol exposure on the 12
expression of the P2X7 receptor in different brain
regions of C57BL/6J mice.
2.1.1 Rationale 12
2.1.2 Methods 14
2.1.3 Results 16
2.1.4 Discussion 20
2.1.5 Conclusions 22
2.2 Aim 2: Test the effects of ethanol on P2X7 receptor 24
expression in BV2 microglia.
2.2.1 Rationale 24
2.2.2 Methods 26
2.2.3 Results 28
2.2.4 Discussion 30
2.2.5 Conclusions 32

2.3 Aim 3: Investigate the role of microglial P2X7 33
receptors in alcohol induced neuroinflammation
through the release of IL-1β
2.3.1 Experiment 1: Ethanol as a neuroinflammatory 35
agent through P2X7R-mediated IL-1 release in
BV2 microglia
iv

2.3.1.1 Rationale 35
2.3.1.2 Methods 37
2.3.1.3 Results 38
2.3.1.4 Discussion 39
2.3.2 Experiment 2: Generation of BV2 microglial cell 42
line with P2X7R knockdown using recombinant
lentivirus mediated shRNA transfer: use in ethanol studies
2.3.2.1 Rationale 42
2.3.2.2 Methods 43
2.3.2.3 Results 44
2.3.2.4 Discussion 45
2.3.3 Conclusions 46
Chapter 3: Future Perspectives 48

Bibliography 51

v

LIST OF FIGURES

Figure 1 Schematic diagram of the P2X7 receptor 4

Figure 2 Two-bottle choice paradigm: Effects of ethanol 17
(10E) exposure on P2X7R protein expression
levels in different brain regions of C57BL/6J mice.

Figure 3 Single bottle, no choice- short term study in 18
C57BL/6J mice.

Figure 4 Single bottle, no choice long-term study in 19
C57BL/6J mice.

Figure 5 Acute ethanol (EtOH) exposure (24 h) 28
increased P2X7R protein levels in BV2 cells.

Figure 6 Chronic ethanol (EtOH) exposure (7d) increased 29
P2X7R protein levels in BV2 cells.

Figure 7 Ethanol (EtOH) induces an increase in IL-1β 39
release in the presence of LPS and ATP from
BV2 microglia.

Figure 8 A schematic representation of the production 41
and release of IL-1β in the presence of ethanol.

Figure 9 A representative Western blot for P2X7 and 45
β-Actin showing knockdown potentials of
2 rLV shRNAs targeting P2X7R sequences.

vi

ABSTRACT

The present work builds upon a central hypothesis that ATP-gated purinergic P2X7
receptors (P2X7Rs) play an important role in causing ethanol-induced
neuroinflammation and neurodegeneration. A distinctive set of properties set the
P2X7R apart from other members of the P2X superfamily, including activation by
pathologically significant levels of ATP and structural uniqueness that allows
participation in a multitude of signal transduction pathways. Further, P2X7Rs are mainly
localized to microglia in the brain and building evidence suggests that they play a critical
role in the activation of these cells. Recently published studies have suggested that
P2X7R signaling in microglia contributes to neurodegenerative pathologies through a
neuroinflammatory response involving the production of pro-inflammatory cytokines
such as Interleukin -1β (IL-1β). Despite the fact that alcohol abuse induces microglial
activation and neuroinflammation similar to neurodegenerative pathologies, the role of
the P2X7R in alcohol-induced neuroinflammation and brain damage has not been
investigated so far.

Three Specific Aims were set forth as initial steps to test the central hypothesis with the
objective of taking us closer to understanding the mechanism by which alcohol-induced
changes in P2X7R expression and function can cause or modulate the resulting
inflammatory cascade, which may further cause or modulate the resultant alcohol-
induced brain damage. Aim 1 tested the effects of ethanol exposure on P2X7R expression
vii

in different brain regions of C57BL mice using two different drinking paradigms:
continuous access two-bottle choice and continuous access single bottle no choice. Using
Western immunoblotting, these studies demonstrated that ethanol exposure had a
differential effect on P2X7R expression in brain regions depending on the amount and
duration of ethanol exposure. Low level of ethanol exposure (10 E) for a short period (10
d) caused an up-regulation of P2X7R in alcohol sensitive brain regions, whereas
exposure to higher ethanol levels (20 E) for a longer period of time (3months) reversed
the effect to down-regulation of P2X7R. Aim 2 tested effects of ethanol on P2X7R
expression levels in the BV2 murine microglial cell line (in vitro) using Western
immunoblotting. These studies found that ethanol up-regulated microglial P2X7R
expression in both the acute (24 h) and chronic (7 d) models. Aim 3 studies tested the
effects of ethanol on the P2X7R-mediated release of IL-1β in BV2 microglial cells in the
absence and presence of an inflammatory stimulus, LPS. Using ELISA measurements,
the studies demonstrated that ethanol causes an up-regulation in LPS-induced IL-1β
production and release upon ATP-activation of P2X7Rs.

Collectively, our findings from the three aims supported our central hypothesis and
suggested that ethanol-induced modulation of microglial P2X7R expression mediates a
neuroinflammatory response involving IL-1β release, the latter being a known mediator
of neurodegeneration. The confirmation of our hypothesis prompted efforts toward an
effective way to prevent ethanol-induced neuroinflammatory effects. Thus, Aim 3 also
included preliminary studies to inhibit P2X7R expression in BV2 microglia using a
viii

lentiviral-mediated shRNA transfer strategy. We were able to achieve successful
knockdown with one of the two shRNAs that we generated, and attempts to use this
shRNA construct to establish a stable BV2 cell line with long-term P2X7 knockdown are
currently being made. This could be used in future studies as a tool to investigate the
downstream effects of P2X7R inhibition and identify the signaling mechanisms by which
ethanol delivers its toxic neurological effects.

1

CHAPTER 1
INTRODUCTION

1.1 Alcoholism as a disease: Need to understand the mechanisms of alcohol-induced
brain damage
Alcohol abuse and misuse have a major national impact in the United States, affecting
nearly 18 million people, causing over 100,000 deaths, and costing upward of $235
billion annually (Bouchery et al., 2011;; Harwood, 2000). The health related risks of
alcohol abuse are a significant cause for concern, considering that excessive alcohol
consumption is associated with a wide range of health abnormalities that are referred to
as alcohol use disorders (AUDs). This includes cardiovascular disease (Costanzo et al.,
2010), chronic pancreatitis (Yadav and Whitcomb, 2010), alcoholic liver disease (Rubin
and Lieber, 1968), hepatitis (Szabo et al., 2010), skin conditions (Kazakevich et al.,
2011), and damage to the central and peripheral nervous systems (Muller et al., 1985).
Noteworthy, studies indicate that 50% of detoxified alcoholics have measurable cognitive
impairments, whereas over 75% of chronic alcoholics have significant brain damage at
autopsy (Vetreno et al., 2011). These observations have led to an association with a
spectrum of neurological disorders ranging from alcohol-related brain damage (ARBD)
to more severe conditions such as hepatic encephalopathy, Wernicke encephalopathy-
Korsakoff syndrome (WKS), Marchiafava–Bignami disease and central pontine
myelinolysis. Post-mortem human studies revealed neuropathological changes during
WKS such as a reduction in the brain volume due to white matter loss and thinning of the
2

corpus callosum (Zahr et al., 2011a). Gliosis is known to make a significant contribution
to these regional volume deficiencies in the thalamus, forebrain and cerebellum, making
the signaling pathways of the glia of great interest.

The gravity of these problems speaks of a vital unmet need for effective ways to manage
the consequences of chronic alcohol consumption. An improved understanding of the
molecular mechanisms and targets associated with chronic ethanol action in the brain is
necessary to develop better means and strategies for the treatment of AUDs. Our group at
the University of Southern California is currently investigating therapeutic targets for
AUDs with a specific focus on the most recently cloned family of ligand gated ion
channels- the purinergic P2X family of adenosine tri-phosphate (ATP) gated ion
channels. This work stems from previous findings that ethanol can modulate the function
of various P2XR family members when tested in vitro. Specifically, P2X2, P2X3 and
P2X4Rs were identified by our lab and others to be sensitive in concentration studies of
ethanol done in recombinant systems (Davies et al., 2005; Davies et al., 2002; Xiong et
al., 2000). These findings have set the stage for us to examine the role of other members
of the P2XR family in alcohol induced responses. As such, the potential effects of alcohol
on the P2X7 receptor (P2X7R) expression and function and the contribution of these
effects to the P2X7R’s role in chronic alcohol-induced brain damage pose interesting
questions that are yet to be answered.

3

1.2. P2X7 receptors in AUDs
1.2.1. P2X7 receptors in neuroinflammation and neurodegeneration
P2X7Rs are reported to express in a significantly wide array of organs and tissues in
rodents and humans, including the brain, thymus, spleen, liver, heart, prostate, pancreas,
testis, skeletal muscle, lung, and placenta (Collo et al., 1997; Rassendren et al., 1997).
Cell type specific studies in the rat brain have shown P2X7Rs to be localized mainly to
microglia (Collo et al., 1997; Ferrari et al., 1997b) , astrocytes (Ballerini et al., 1996;
Kukley et al., 2001), Schwann cells (Colomar and Amédée, 2001), and oligodendrocytes
(James and Butt, 2002).

The functional role of P2X7Rs is attributed to the localization and the unique properties
of these receptors among the members of the P2X superfamily. This includes the novel
finding that activation of this receptor subtype occurs at much higher concentrations of
ATP (millimolar range) as compared to other P2XR family members (micromolar range).
Moreover, continued exposure to ATP causes a transition from the ion channel modality
to a reversible pore formation in the plasma membrane that is large enough to facilitate
the passage of solutes up to the size of 900 Da (North, 2002). This property entails a role
for P2X7Rs during pathological events that are normally accompanied by an increased
production of ATP. Finally, the unusual elongated carboxyl terminus of the P2X7R
accounts for its ability to interact with several intracellular signaling proteins, implicating
the receptor in physiological as well as pathological signaling events (Fig.1, (North,
2002; Surprenant et al., 1996)).
4

The role of P2X7Rs in the areas of inflammation and glia-neuron communication has
recently attracted much attention. For example, P2X7Rs are known to be critically
involved in microglial activation, thereby contributing to the microglial role in
inflammatory responses to CNS insults (Boumechache et al., 2009; Monif et al., 2010).
Importantly, P2X7Rs were shown to modulate the release of IL-1β, a recognized
mediator of neurodegeneration (Bernardino et al., 2008; Clark et al., 2010; Honore et al.,
2009; Takenouchi et al., 2008). It is considered that ATP induced activation of the
P2X7R is an essential second signal in the triggering of the release of mature IL-1β from
microglial cells (Ferrari et al., 1997a; Takenouchi et al., 2009). Thus, given the
localization of P2X7Rs on immune cells and the role of ATP as a secondary stimulus in
Fig.1. Left panel - Schematic representation of a single, typical P2X7 receptor subunit showing the elongated
carboxyl terminus. First and second trans-membrane domains are labeled TM1 and TM2. Right panel – First
crystal structure of a P2XR: the related P2X4R in zebrafish which demonstrates the trimeric assembly of a
functional P2XR.

5

the generation and release of IL-1β, it is plausible that P2X7Rs are involved in a
neuroinflammatory process that could precede and assist in the development of
neurodegenerative conditions.

The possible neurodegenerative function of the P2X7R has been a growing focus of
interest over the past decade. Primary evidence was obtained in the form of findings that
direct administration of IL-1β into the CNS results in local inflammatory responses and
neuronal degradation (Wood, 2003). Additional support can be obtained from research
that has implicated the P2X7Rs in specific neurodegenerative pathologies. The presence
of P2X7Rs has been demonstrated within the beta-amyloid plaques, suggesting a role in
the processes of neurodegeneration in Alzheimer’s disease (AD, (McLarnon et al., 2006;
Parvathenani et al., 2003)). Increased immunoreactivity associated with activated
microglia consistent with the signs of neuronal damage has been found in the epileptic
brain (Rappold et al., 2006). Further, P2X7Rs appear to play an emerging role in
amyotrophic lateral sclerosis (ALS, (Volonte et al., 2011)).

Recent evidence also suggests the possible involvement of P2X7Rs in addictive diseases.
Consistent with this, recent work has demonstrated a strong association between the
spinal microglia-expressed P2X7R and the development of morphine tolerance (Zhou et
al., 2010). P2X7Rs are also mediators of TNFα and CC-chemokine ligand 3 secretion
(Kataoka et al., 2009; Suzuki et al., 2004; Zou et al., 2012), production of superoxide
(Parvathenani et al., 2003) and nitric oxide (NO, (Gendron et al., 2003)), and matrix
6

metalloproteinase 9 (MMP-9, (Choi et al., 2010; Shin et al., 2010)). In this context,
P2X7Rs have been implicated in the pathophysiology of neurodegenerative and
neuropsychiatric disorders (e.g. depression), chronic inflammation and pain, and
induction of spinal long-term potentiation (Skaper et al., 2010). Taken together, these
findings suggest that P2X7R signaling is an important contributor to neuroinflammation
mediated neurodegeneration, hence making it possible to envision a role for the P2X7
receptor in alcohol induced neurodegeneration.

1.2.2. Alcohol induced neuroinflammation and neurodegeneration: A possible role of
P2X7Rs?
The fact that alcoholics demonstrate dementia, much like neurodegenerative disorders,
gives us cause to believe that the underlying mechanisms for both might be related
(Blanco and Guerri, 2007; Zahr et al., 2011a). The mechanism behind ethanol-induced
neurodegeneration is not well understood, but several explanations have been proposed.
These include excitotoxicity associated with excessive neurotransmitter release, oxidative
stress leading to free radical damage, and cell death through an enhanced inflammatory
response. Notably, recent observations, with regard to neurodegenerative diseases,
provide important clues to the mechanisms that could mediate alcohol’s toxic effects on
brain cells. One such clue is the consistently shown association between P2X7R up-
regulation and neurodegenerative pathology. A well-established example is that of AD,
where enhanced microglial P2X7R expression has been demonstrated in animal models
of several hallmarks of AD, such as transgenic mice over-expressing mutant amyloid
7

precursor protein (APP) and amyloid plaques (Parvathenani et al., 2003; Sanz et al.,
2009). Consistent with these observations, increased expression of P2X7R was shown in
adult microglia derived from postmortem AD brains compared with that from non-
demented brains (McLarnon et al., 2006). In an induced-disease rodent model for prion
disease, Takenouchi et al. demonstrated that the mRNA level of P2X7R gradually
increased in the brain during disease progression (Takenouchi et al., 2007). More
recently, increases in the expression and function of P2X7R at synaptic terminals were
demonstrated in the brain of mice modeled for Huntington’s disease (HD), suggesting
that ATP-induced synaptic dys-regulation and neuronal cell death caused by the altered
expression of the P2X7R contributes to HD pathogenesis (Diaz-Hernandez et al., 2009).

A second finding to take into account is that microglial cells play an integral role in the
generation of the pro-inflammatory response to different insults including alcohol
(Alfonso-Loeches et al., 2010; Aschner et al., 1999; Blanco and Guerri, 2007). In
agreement with this notion, recent studies found an increased number of microglia in key
brain regions of post-mortem samples from human alcoholics (He and Crews, 2008).
Also, microglia activation was demonstrated in experimental rodent models of alcohol
abuse (McClain et al., 2011a; Qin and Crews, 2012a). Studies suggest that
neuroinflammation arising from microglia may lead to neuronal loss and gliosis, as was
found upon autopsy in chronic alcoholics (He and Crews, 2008; Kelley and Dantzer,
2011a).
8

Finally, the increased presence of ATP upon secretion from the damaged cells or tissue at
an inflammatory site can be considered as conducive to an up-regulated P2X7R signaling
response to inflammation caused by an insult (Burnstock, 2002). Given the latter and the
predominant expression of P2X7Rs by glial cells, combined with the findings on the
increased pro-inflammatory milieu and microglial presence in the degenerative and
alcoholic brains respectively, a possible role for P2X7Rs in chronic alcohol induced brain
damage can be suggested. Despite this body of evidence, the role of P2X7Rs in alcohol-
induced processes of neuroinflammation and neurodegeneration has not been
investigated. My thesis project represents a first step in the investigations focusing on
P2X7R’s contribution to the damaging neurological effects of alcohol.

9

CHAPTER 2
AIMS

The foundation for my thesis project is based on the central hypothesis that P2X7Rs play
an important role in causing alcohol-induced neuroinflammation and
neurodegeneration. Several lines of evidence support this hypothesis. First, available
literature suggests that P2X7Rs are implicated in the development of pro-inflammatory
immune response during chronic inflammation, pain, neurodegenerative and
neuropsychiatric disorders (Skaper et al., 2010). Functional studies have shown that
microglial activation and signaling through P2X7R expression is a central mechanism by
which this neuro-inflammatory response occurs during neurodegenerative pathologies
(McLarnon et al., 2006; Monif et al., 2010; Skaper et al., 2010). Substantial evidence
exists for microglial P2X7Rs being the principal mediators of ATP-dependent IL-1β
release, a recognized agent of neurodegeneration, upon lipopolysaccharide (LPS)
challenge (Ferrari et al., 1997b; Labasi et al., 2002; Solle et al., 2001). Secondly, the
production of pro-inflammatory cytokines like IL-1β is known to cause long-term
variations in alcohol induced behaviours and neurodegeneration (Achur et al., 2010).
Despite the intertwining of the two concepts that suggests an important role for P2X7Rs
in alcohol-induced neuroinflammatory responses, it has not been studied to this date. My
project is an attempt to start filling this gap in knowledge and to use biochemical and
molecular biology tools to define the underlying mechanisms of alcohol-induced brain
damage.
10

The following Specific Aims were proposed as initial steps to test the central hypothesis,
with the objective of taking us closer to understanding the mechanism by which alcohol-
induced changes in P2X7R expression and function can cause or modulate the resulting
inflammatory cascade, which may further cause or modulate the resultant alcohol-
induced brain damage.

Aim 1: Test the effect of ethanol exposure on the expression of the P2X7 receptor in
different brain regions of C57BL/6J mice. Studies of this first step were designed and
conducted to measure in vivo changes in the pattern of expression of P2X7Rs in the brain,
induced by ethanol. I used C57BL/6J mice that were part of acute and chronic drinking
paradigms previously established in our lab. These paradigms were as follows:
Continuous access two-bottle choice with access to 10% ethanol (10E), single-bottle
(20% - 20E) no choice, short-term (10 days) and single-bottle (20E) no choice, long-term
(3 months) drinking models. Different brain regions (Hypothalamus, Amygdala, Pre-
frontal cortex (PFC), Hippocampus, Striatum, Midbrain, Thalamus and Cerebellum) were
dissected and P2X7R protein expression levels were analyzed using Western
immunoblotting.

Aim 2: Test the effects of ethanol on P2X7 receptor expression in BV2 microglia.
The results from Aim 1 served as motivation to set up more focused studies at the cellular
level, directed at testing the effects of ethanol on P2X7R expression in microglia. For aim
2 studies, I used the BV2 murine microglial cell line that endogenously expresses
11

P2X7Rs. Thus, utilizing this in vitro cell system, Aim 2 studies explored the modulation
of P2X7R expression in response to ethanol exposure under acute (24 h) and chronic (7
d) conditions. Treatment conditions for the cells constituting varying concentrations of
ethanol were optimized and western immunoblotting was used to measure the consequent
variations in P2X7R expression.

Aim 3: Investigate the role of microglial P2X7 receptors in ethanol-induced
neuroinflammation. Aims 1 and 2 demonstrated ethanol-induced changes in P2X7R
expression in the mouse brain regions and BV2 microglia, which led to the hypothesis
that P2X7Rs play an important role in ethanol -induced neuroinflammation. Aim 3
studies used the in vitro BV2 cell system to test for the effects of ethanol on P2X7R-
mediated release of the pro-inflammatory cytokine, IL-1β. These studies were performed
in the presence of two key players in P2X7R signaling- ATP and LPS. To further
delineate the role of P2X7Rs in ethanol-induced neuroinflammation, Aim 3 studies also
used state of the art gene knockdown strategy - lentivirus-mediated shRNA delivery, to
knockdown P2X7R expression in BV2 microglial cells. Preliminary data from these
experiments has also been included as part of Aim 3.

The rationale and background for each of these aims and the interpretation of the results
obtained are discussed in subsequent pages.

12

2.1. Aim 1: Test the effect of ethanol exposure on the expression of the P2X7
receptor in different brain regions of C57BL/6J mice.
2.1.1. Rationale
The need to study alcohol-induced variations in P2X7R expression in the brain stems
from growing evidence that suggests a role for P2X7Rs in neurodegenerative pathologies.
Recent research in this direction has shown that the expression and function of P2X7Rs
are increased in the brain in animal models of AD (Parvathenani et al., 2003; Sanz et al.,
2009), prion disease and HD (Takenouchi et al., 2007), Parkinsons disease (PD) and ALS
(Glass et al., 2010), and acute and chronic phases of Temporal lobe epilepsy (Dona et al.,
2009). Together, these findings indicate that P2X7R up-regulation in the brain plays a
role in the development of neurodegenerative pathologies. There is similarity between
neurodegenerative conditions and chronic alcohol abuse in that both are known to cause
brain dementia (Blanco and Guerri, 2007; Zahr et al., 2011b). However, the role of
P2X7Rs in alcohol induced neurodegeneration is as yet unknown.

Alterations in pro-inflammatory signaling in response to ethanol are thought to mediate
the damaging effects of ethanol in the brain. These mechanisms include the release of
pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6 (Tiwari et al., 2009), the
regulation of reactive proteins iNOS, COX-2, and the involvement of NFκB, MAPKs and
JAK-STAT pathways (Suk, 2007). Several groups have examined region-specific
ethanol-induced variations in these signaling responses and found that certain brain
13

regions are implicated more than others. A study in ethanol-treated rats observed a
significant elevation in the levels of TNF-α and IL-1β, which is indicative of enhanced
neuroinflammation, in the cerebral cortex and hippocampus (Tiwari et al., 2009). In the
ARBD induced Wernicke encephalopathy (WKS), over-expression of IL-1β, IL-6, COX-
2, and TNF-α was found to confer selective vulnerability to neuronal loss and
accompanying gliosis in diencephalic regions such as the thalamus and mammillary
bodies (Neri et al., 2011). In entorhinal cortex–hippocampus slice cultures prepared from
mice on a 5 month ethanol diet, ethanol activation of NF-κB led to enhanced glutamate
neurotoxicity and the production of inflammatory mediators such as IL-1β, COX2, and
iNOS (Blanco et al., 2004; Zou and Crews, 2005). In contrast, another study found that
chronic alcohol consumption down-regulated NF-κB in the PFC of the human brain
(Okvist et al., 2007).

The P2X7Rs have been established as central to many of these pro-inflammatory
signaling responses. Especially well characterized is the role of P2X7Rs in the activation
of the inflammasome and the subsequent release of pro-inflammatory cytokines IL-1β
and IL-18 (Qu et al., 2007; van de Veerdonk et al., 2011). Together, these findings led to
the hypothesis that ethanol induces changes in P2X7R expression in those brain
regions that are prone to alcohol’s neuroinflammatory effects. In addition, building
evidence suggests that the response to alcohol depends on the length and amount of
alcohol exposure. Therefore, Aim 1 studies investigated changes in P2X7R expression
14

profile in different brain regions using short and long-term ethanol exposures. The brain
regions included in my investigation were: hypothalamus, amygdala, prefrontal cortex,
hippocampus, midbrain, striatum, thalamus and cerebellum.

2.1.2. Methods
 Alcohol exposure paradigms
1. Continuous access, two-bottle choice drinking model.
The 24-hour access model is widely used to assess changes in drinking behaviors (Crabbe
et al., 1993; Middaugh et al.,1999; Yoneyama et al., 2008) and has been recently
established in our laboratory (Yardley et al., 2012). We used a modification of this
behavioral model to suit the biochemical study of P2X7R expression in the brain. Six
female C57BL/6J mice at the age of 8-10 weeks were individually housed 7 days prior to
the study with continuous access to 2 inverted bottles (fitted with metal sippers)
containing tap water. Food (rodent chow) was distributed near both bottles to avoid food
associated tube preferences. At the start of the experiment, three of the six mice had one
bottle replaced with a bottle containing 10% v/v ethanol solution (10E) in tap water. The
remaining three mice were matched as controls for alcohol intake. Fresh fluids were
provided twice a week when cages were changed. Every morning, the daily fluid intake
(to the nearest 0.1 ml) was recorded from both bottles by measuring the level of the
meniscus on the graduated drinking tube. Mice were allowed to drink for 10 days after
the drinking stabilized. Body weights were measured for each mouse to calculate the
intake of ethanol that averaged at 12 g/kg/day.
15

2. Single-bottle no-choice drinking model.
Many of the progressive changes in innate and acquired immunity that accompany the
administration of alcohol are described in previous literature using a single bottle-no
choice alcohol model (Edsen-Moore et al., 2008; Song et al., 2002). Similar to these
earlier described paradigms, twelve C57BL/6J male mice at the age of 8-10 weeks were
single housed a few days prior to the experiment and provided with food and a single
inverted bottle containing tap water. At the start of the experiment, six of the mice had
the bottle with water replaced by a bottle containing 20% v/v ethanol solution (20E) in
tap water. The remaining six served as matched controls for the study. The mice were
allowed continued access to either ethanol (20E) or water (control) for a short-term (2
weeks) or long-term (3 months) period.
 Harvesting of brain tissue
Briefly after ethanol exposure, the mice were sacrificed by CO
2
and the brains were
dissected into different regions, namely the hypothalamus, amygdala, pre-frontal cortex,
hippocampus, striatum, midbrain, thalamus and cerebellum. The brain samples were
lysed in ice-cold RIPA buffer supplemented with protease inhibitors. Whole cell lysates
were obtained by subsequent centrifugation at 10 000 r/min for 10 min at 4 °C and stored
in -20
o
C for further analyses.
 Western Blotting
Protein concentrations were determined using the BCA (bicinchoninic acid) protein assay
with bovine serum albumin as a standard. Ten micrograms of protein extracts were
separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and
16

were transferred to Polyvinylidene fluoride (PVDF) membranes. Membranes were
blocked with a 5% solution of non-fat dry milk dissolved in a Tris-HCl-buffered solution
(TBS-Tween, pH 7.5) and were then probed with a rabbit, anti-P2X7 primary antibody
(1:1000, Alomone Labs, Israel) followed by secondary anti-rabbit antibody (1:10000
dilution). Bands were visualized using enhanced chemi-luminescence (Pierce
Biotechnology).
 Data Analysis
Scion Software (Scion Corporation, Frederick, MD) was used for densitometry of the
protein bands and data was represented in bar graphs as mean ± standard error mean
(SEM). Significant differences between different experimental conditions (i.e. mouse
groups) were determined using Student’s t-test and significance was set at P < 0.05.

2.1.3. Results
Ethanol exposure causes changes in P2X7R expression in alcohol-sensitive brain regions
depending on the alcohol exposure mode, amount and length.
Two-bottle choice model. Changes in P2X7R protein levels were measured in eight brain
regions of the female C57BL/6J mice that had access to 10%v/v ethanol for ten days,
yielding 12 g/kg/day ethanol consumption. Western immunoblotting showed that P2X7R
expression increased in the hypothalamus (Hyp), amygdala (Amy), hippocampus (Hipp)
and midbrain (Midbr) of the ethanol-drinking mice as compared to the control water
drinking counterparts (Fig.1). On the other hand, it was decreased in the striatum (Striat),
thalamus (Thal) and cerebellum (Cer). Of these changes, only the increases in Amy, Hipp
17

and Midbr were found to reach statistical significance (* P < 0.05). Interestingly, the
significant increases in P2X7R expression were localized to the brain regions that are
linked to alcohol’s effect on cognition and memory (hippocampus) and the reward
pathway (amygdala, midbrain (Crews et al., 2000; Rodd et al., 2004)). This is consistent
with the fact that alcohol causes cognitive difficulties and is involved in the reward
circuit, as seen in alcoholics displaying impaired memory and reasoning ability and
engaging in alcohol seeking behavior that could lead to eventual addiction.

Fig.2. Two-bottle choice paradigm: Effects of ethanol (10E) exposure on P2X7R protein expression levels
in different brain regions of C57BL/6J mice. There was a significant increase in P2X7R protein levels in
amygdala, hippocampus and midbrain, while changes in other brain regions did not reach significance (* P <
0.05). Data represent densitometry analysis of Western blots, n=3.

18

Single bottle, no choice model – short-term. In contrast to the 2-bottle choice paradigm,
ethanol consumption for 2 weeks in a single-bottle no choice model (20% v/v) yielded
considerably higher levels of ethanol consumption (25-30 g/kg/day) and significantly
reduced P2X7R expression levels in the Hyp, Hipp, Striat and Midbr (Fig.2). P2X7R
levels also decreased in Amy, PFC but did not reach statistical significance. There was no
change in P2X7R levels in the Thal and Cer (Fig. 2).

Fig.3. Single bottle, no choice- short term study in C57BL/6J mice. Ethanol (20E) exposure for 2 weeks
resulted in a significant decrease in P2X7R protein expression levels in hypothalamus, hippocampus, striatum
and midbrain, while changes in other brain regions did not reach significance (* P < 0.05). Data represent
densitometry analysis of Western blots, n=6.

0
0.2
0.4
0.6
0.8
1
1.2
1.4
Hyp Amyg PFC Hipp Striat MidBr Thal Cer
P2X7R Levels, Normalized on β-Actin
Density (Optional Units)
Water
20 E
*
*
*
*
19

Single bottle, no choice model – long-term. When the same amount of ethanol was
consumed by the mice in the single bottle, no choice model (20%v/v) for a period of 3
months, significantly reduced P2X7R protein expression was observed in all tested brain
regions except the thalamus and cerebellum (Fig.3). The down-regulation of P2X7R
expression in both the short and long term studies in the single bottle, no choice paradigm
seems to indicate that an increase in the amount of ethanol consumed can cause a reversal
in its effect on P2X7R expression, as compared to the up-regulation observed in the two
bottle choice paradigm.

Fig.4. Single bottle, no choice long-term study in C57BL/6J mice. Ethanol (20E) exposure for 3 months
resulted in a significant decrease in P2X7R protein expression levels in all brain regions except thalamus and
cerebellum. (P < 0.05 compared to “Water”). Data represent densitometry analysis of Western blots, n=6.

0
0.2
0.4
0.6
0.8
1
1.2
1.4
Hyp Amy PFC Hipp Striat Midbr Thal Cereb
P2X7R Levels, Normalized on β-Actin
Density (Optionl Units)
Water
20 E
*
20

2.1.4. Discussion
There are several outcomes from these studies that used different alcohol drinking
paradigms. First, the findings suggest that ethanol affects P2X7R expression in specific
brain regions and these regions, namely the hypothalamus, amygdala, hippocampus, PFC,
striatum and midbrain, undergo ethanol induced alterations. Existing knowledge indicates
that these regions are involved in the neurological effects of alcohol. In fact, a prior study
in alcoholic patients found volumetric alterations in specific regions including the
hippocampus, midbrain and left amygdala (Makris et al., 2008). Several lines of evidence
have consistently shown that the adolescent hippocampus is particularly vulnerable to
alcohol-induced impairments and damage and that excessive alcohol consumption
reduces hippocampal volume, interfering with memory and learning (Crews et al., 2000;
Sircar et al., 2009). Magnetic resonance imaging (MRI) studies to examine cognitive
impairment have confirmed volume differences between AD patients and healthy
controls for the hippocampus (Pennanen et al., 2004). Interestingly, a study that used a
transgenic mouse model induced to express human Il-1β showed that sustained elevation
of hIL-1β and the accompanying increase in other pro-inflammatory molecules in the
hippocampus drives mnemonic memory deficits (Moore et al., 2009). Together, these
findings suggest that an alcohol-induced pro-inflammatory response due to a change in
P2X7R expression levels in the hippocampus may play a role in the cognitive deficits in
alcoholics. Further, alterations of the P2X7R in the midbrain, amygdala and PFC agree
with the well-known roles of these regions in ethanol-conditioned effects and addiction
(Rodd et al., 2004). Ethanol-craving is mediated by signaling centers located in the
21

midbrain, which send messages to downstream targets including the striatum, anterior
cingulate cortex, amygdala, and prefrontal cortex in response to an alcohol-associated
stimulus (Wrase et al., 2002). This is supported by findings that electrolytic lesions in the
amygdala disrupted expression of ethanol conditioned placement preference in mice
(CPP, (Gremel and Cunningham, 2009). Increased P2X7R expression in the midbrain is
supported by studies that show that this region seems to have a particularly higher
abundance of microglia than other regions of the brain (Lawson et al., 1990), the
activation of which is known to play a pivotal role in the pathogenesis of
neurodegenerative conditions, notably PD (Hunot and Hirsch, 2003).

Second, findings from the present study indicate that changes in P2X7R expression are
dependent on the concentration of ethanol and the length of ethanol exposure. That is,
lower overall levels of ethanol consumption (10E-12 g/kg/day) in the two-bottle choice
model resulted in increases in P2X7R expression in several brain regions, whereas higher
levels of ethanol consumption (20E-25 g/kg/day) resulted in significant decreases in
P2X7R protein expression. Long-term exposure (3 months) to ethanol increased the
extent of these changes in comparison to short-term exposure (2 weeks). Overall, the
findings were in agreement with the existing knowledge on the opposite effects of
chronic and acute alcohol exposure with regard to immune response and inflammatory
cytokine production (Mandrekar et al., 2009). In this latter work, it was reported that
acute (24 h) exposure of primary circulating macrophages to an intoxicating dose of
22

ethanol (25 mM which is equal to 0.1g/dl comparable to legal limit of blood alcohol
concentration, 0.08g/dl) resulted in reduced TNF-α in response to LPS treatment whereas
chronic seven-day exposure to ethanol resulted in the opposite change.

Alcohol consumption is known to have both beneficial and harmful (impaired immune
responses) effects on the cardiovascular system based on the duration and amount of
alcohol exposure. It is likely that alcohol has a similar impact on the CNS. However, the
mechanisms behind this biphasic response to alcohol are not well understood. These
findings demonstrating that acute and chronic ethanol exposures differentially affected
the P2X7R expression in the mouse brain and the predominant expression of these
receptors in microglia suggest that P2X7Rs are involved in the mechanisms that drive
alcohol’s biphasic effects on the innate immune system.

Collectively, the findings suggest that ethanol induces changes in the expression of
P2X7Rs; these changes occur in alcohol-sensitive brain regions and depend on the
duration and amount of alcohol exposure.

2.1.5. Conclusions
Building evidence suggests that P2X7 receptor expression in the brain and the resulting
production of pro-inflammatory cytokines is a likely mechanism by which
23

neurodegeneration occurs in several neurodegenerative disorders including AD, multiple
sclerosis, PD, HD and ALS. Alcohol use has been documented to cause a similar pro-
inflammatory response in certain alcohol-sensitive regions of the brain, but there is
insufficient evidence to confirm the role of the P2X7R in alcohol-induced brain damage.
Investigations conducted in my Aim 1 studies addressed this gap in knowledge by testing
if ethanol exposure altered P2X7R expression in different regions of the mouse brain and
if so, whether the alterations were dependent on the amount and duration of ethanol
exposure. By using the two-bottle choice model and the single bottle, no choice model to
administer different amounts of ethanol (10E and 20E, respectively) for different
durations of time (short term and long term), I was able to show that the amount and
duration of ethanol exposure differentially affected P2X7R expression in specific brain
regions that are known to drive the decreased cognitive abilities of an alcoholic. Up-
regulation of P2X7R expression was seen when a relatively low amount of ethanol (10E)
was administered in the acute two bottle, choice study, whereas a higher amount of
ethanol (20E) down-regulated P2X7R expression in both the acute and chronic single
bottle, no choice studies. The significant changes in expression were seen in specific
brain regions implicated in the cognitive deficits (hippocampus) and the addictive
behaviors (midbrain, amygdala, PFC) of alcoholism. Thus, the findings support our
hypothesis that ethanol causes significant changes in P2X7R expression in alcohol-
sensitive regions of the mouse brain. Finally, the findings suggest that changes in
P2X7R expression are dependent on the amount of ethanol intake and the duration of
ethanol intake.
24

2.2. Aim 2: Test the effects of ethanol on P2X7 receptor expression in BV2
microglia.
2.2.1. Rationale
As presented in the previous section, ethanol exerts modulatory effects on P2X7R
expression in certain brain regions in a concentration and time dependent manner.
However, current knowledge on the specific cell type(s) that contribute to the changes in
P2X7R expression caused by alcohol exposure is lacking. Based on the predominant
localization of P2X7Rs in microglia and the role of the latter in the development of
neuroinflammation, studies of Aim 2 focused on the effects of ethanol on P2X7R
expression in microglia. For this Aim, I conducted studies that tested the hypothesis that
ethanol exposure causes modulation of P2X7R expression in microglia. I predicted
that microglia are the predominantly contributing cell type to the changes in P2X7R
expression levels observed in Aim 1. Therefore, Aim 2 studies tested whether ethanol
induced changes in P2X7R expression in microglial cells.

Several findings from a body of evidence implicating activated microglia in alcohol-
induced brain damage formed the basis for this study. First, alcohol induces brain
microglia activation. In their classical role as immune cells, microglia are established
producers of inflammatory mediators. Microglial activation and the resultant production
of pro-inflammatory mediators and cytokines are hallmarks of the brain inflammatory
response to CNS insults, including alcohol. Importantly, the release of pro- as well as
anti-inflammatory cytokines (TNFα, IL-1, IL-6, IL-10, TGF-β) was linked to long-term
25

changes in alcohol-induced behaviors and neurodegeneration (Crews et al., 2006; Crews
and Nixon, 2009; Qin et al., 2008). In agreement with this, increased levels of pro-
inflammatory cytokines associated with activated microglia were demonstrated in an
intermittent binge alcohol exposure model (Ward et al., 2009) and in postmortem human
alcoholic brains (He, 2008). Findings from McClain et al provide further evidence that
ethanol triggers long lasting partial activation of microglia in the mouse brain (McClain
et al., 2011b). In addition, an in vitro study showed that ethanol induces morphological
activation of microglia, resulting in the secretion of inflammatory mediators and
cytokines (Fernandez-Lizarbe et al., 2009). Secondly, P2X7R expression drives the
activation of microglia. Several studies suggest that P2X7R expression and microglial
activation work in concert to trigger signaling cascades that enable the brain to cope with
any form of inflammatory stimulus (Ferrari et al., 1997b; Suzuki et al., 2004). Existing
knowledge is that P2X7R signaling is the primary cause for the activation of microglia,
hence playing an essential role in the neuroinflammatory process (Boumechache et al.,
2009; Monif et al., 2010). In fact, it was recently demonstrated that P2X7R over-
expression in primary hippocampal cultures was sufficient to drive microglial activation
and proliferation (Monif et al., 2009). Together, these findings suggest that the study of
P2X7R expression in microglia under exposure to alcohol could be important to
elucidating the means by which alcohol exerts its toxic effects.

The in vitro studies conducted over the course of this aim used the BV2 murine
microglial cell line. This cell line was selected because BV2 cells endogenously express
26

P2X7Rs (Brautigam et al., 2005; Gendron et al., 2003). Henn et al characterized the BV2
cells for their suitability as a substitute for primary microglia or in vivo animal studies,
and the results showed an astounding 90% and 50% overlap with LPS induced genes in
primary microglia and the rodent hippocampus in vivo, respectively (Henn et al., 2009).
Therefore the BV2 in vitro system allowed me to investigate the direct effects of ethanol
on the expression of P2X7R in microglia while controlling for the amount of and
exposure time to ethanol.

2.2.2. Methods
 Culture of murine BV2 microglial cells
The BV2 murine microglial cell line was a kind gift from Dr. Baljit Khakh (UCLA). The
culture of the BV2 cells was done as described in the seminal paper by Giulian and Baker
in the mid-1980s, with a few modifications (Giulian and Baker, 1986). They were
cultured in 25 ml culture flasks in Dulbecco's modified Eagle's medium: Nutrient Mixture
F-12 Ham (DMEM/F-12) (Gibco RBL, Rockville, MD) supplemented with 5% fetal
bovine serum (U.S. Biotechnologies Inc., Parkerford, PA) and 1% penicillin/streptomycin
(Gibco RBL, Rockville, MD) in a humidified atmosphere of 5% CO
2
and 95% air at
37°C.
 Treatments
1. 24-hour ethanol treatment: acute study
The cultured BV2 cells were plated into six welled plates in 2 mls/well of the growth
medium, at a density that allowed the cells to retain their resting amoeboid morphology.
27

On the next day, the medium was changed to Phenol-Red free DMEM (Gibco RBL,
Rockville, MD) supplemented with 0.5% bovine albumin and 1%
penicillin/streptomycin. Ethanol at 0 (control), 25, 50 or 100 mM was added and the
plates were incubated at 37 °C, 5% CO2, and 95% relative humidity in a sealed chamber,
in the presence of two open petri-dishes containing 200 mM ethanol in sterile phosphate
buffered saline (PBS) to saturate the environment of the cells with ethanol vapors. After a
24 hour period of ethanol exposure, the cells were lysed with ice-cold RIPA buffer
supplemented with protease inhibitors. Cell lysates were obtained by subsequent
centrifugation at 10 000 r/min for 10 min at 4 °C and stored in -20
o
C.
2. Seven day ethanol treatment: chronic study
In this chronic treatment model, BV2 cells received exposure to 0, 25 or 100 mM ethanol
added to the cell medium every other day for the duration of seven days. All experimental
conditions were maintained identical to the acute study. The three sets were lysed and
collected as described earlier at the end of the seven day period of ethanol exposure.
 Western Blotting
The BV2 cell lysates were measured for protein concentrations using the BCA
(bicinchoninic acid) protein assay with bovine serum albumin as a standard. The
calculated amounts of protein extract were subjected to SDS-PAGE, transferred to PVDF
membranes and probed for P2X7R expression, as described earlier in section 2.1.2.
 Data Analysis
Densitometry using Scion Software yielded quantitative differences between the various
treatment groups, which were analyzed and presented as described earlier in 2.1.2.
28

2.2.3. Results
Acute and chronic ethanol exposure increased P2X7R expression in the BV2 microglial
cells in a concentration dependent manner.
Acute 24-hour study. The BV2 murine microglial cells were exposed to three
concentrations of ethanol (25, 50 and 100 mM) for 24 hours, and P2X7R expression was
measured using western immunoblotting technique. The results suggest that 24 hour
ethanol treatment induced a concentration-dependent increase in P2X7R expression (Fig.
4). Of the three concentrations, the 50 mM and 100 mM concentrations induced
statistically significant increases in P2X7R expression as compared to the control
(**P<0.01, *P<0.05).

Fig.5. Acute ethanol (EtOH) exposure (24 h) increased P2X7R protein levels in BV2 cells. (A) A
representative Western blot of BV2 lysates. (B) Bar graph of the data showing that the increase was significant
for 50 mM (30%, ** P < 0.01) and 100 mM (50%, * P < 0.05) ethanol, but not 25 mM ethanol. Data represent
densitometry analysis of blots and are mean ± SEM from 3-6 experiments.

A.

B.

P2X7
-Actin
0 25 50 100
EtOH Concentration, mM
P2X7
-Actin
0 25 50 100
EtOH Concentration, mM
0 25 50 100
0.0
0.5
1.0
1.5
2.0
**
*
EtOH Concentration (mM)
P2X7R Levels, Normalized on -Actin
Density (Optional Units)
29

Chronic seven-day study. Seven day exposure to ethanol (25 and 100 mM) induced an
increase in P2X7R protein expression (Fig.5). Similar to the findings of the acute study,
this increase was concentration dependent. Compared to the non-significant effect of 25
mM ethanol in the acute study, the effect caused by 25 mM ethanol in the chronic study
was significant (**P<0.01). The extent of the increase in P2X7R levels caused by 100
mM ethanol was similar to that caused by the acute ethanol exposure (*P<0.05).

Fig.6. Chronic ethanol (EtOH) exposure (7d) increased P2X7R protein levels in BV2 cells. A bar graph of
the data showing that the increase was significant for 25 mM (30%, * P < 0.01) and 100 mM (50%, * P < 0.01)
ethanol. Data represent densitometry analysis of blots and are mean ± SEM from 3-6 experiments.

0.0
0.5
1.0
1.5
2.0
2.5
0 25 100
P2X7R Levels, Normalized β-Actin
OD (Optional Units)
EtOH Concentration (mM)
**
**
30

2.2.4. Discussion
The in vitro studies from Aim 2 studies revealed that acute as well as chronic exposure to
ethanol increased P2X7R expression in microglial cells in a concentration-dependent
manner. The P2X7R has an established role in the activation of microglia and as a
mediator of inflammatory responses in the brain, due to which the finding that ethanol
up-regulates P2X7R expression in microglia suggests that these receptors play a role in
alcohol’s effects in the brain. This result is in line with previously published studies that
have found ethanol to deliver its toxic effects on brain cells by various means, one of
which is the generation of pro-inflammatory responses through the activation of
microglial cells (Alfonso-Loeches et al., 2010; Floreani et al., 2010; Qin and Crews,
2012b). Ethanol’s effects on the microglial inflammatory signaling pathways in the brain
occur primarily through the over-stimulation of pro-inflammatory cytokines such as Il-
1β, IL-6, IL-18, TNF-α, MCP-1 (He and Crews, 2008) and P2X7R expression has been
shown to mediate the post-translational processing of precursors of several of these
cytokines, notably IL-1β and IL-18, in human monocytes (Mehta et al., 2001; Perregaux
et al., 2000). Thus, it can be rationally postulated that P2X7R up-regulation is a possible
mechanism by which the consumption of high amounts of alcohol elicits a
neuroinflammatory response. In addition, other studies have shown that ethanol targets
other signaling pathways which are linked to P2X7R function such as stimulation of
nuclear factor of activated T cells (NFAT), the transcription factor NF-κB (Budagian et
al., 2003), and JNK (Humphreys et al., 2000). It is possible that ethanol alters these signal
31

transduction pathways through the up-regulation of P2X7Rs, and thus contributes to the
cell damage that occurs in the alcoholic brain.

As with studies in Aim 1, I investigated if the observed effects were dependent on the
amount and duration of ethanol exposure. An overall proportional relationship was found
between the expression of P2X7R and the amount of ethanol that the cells were exposed
to. Moreover, the results seem to indicate that chronic exposure to a lower concentration
of 25 mM ethanol is sufficient to induce significant up-regulation of microglial P2X7Rs,
whereas an equivalent effect upon acute exposure is only produced at higher
concentrations such as 50 and 100 mM of ethanol. Of note, 25 mM ethanol concentration
is equivalent to 0.1 g/dl, which is comparable to the legally permissible limit of blood
alcohol concentration and the intoxicating dose in humans - 0.08 g/dl. Furthermore, we
expected a greater degree of increase in P2X7R expression caused by exposure to higher
ethanol concentration (100 mM) in the chronic study compared to the extent found in the
acute study. However, there was no observed difference in this study. This could be due
to the duration of the chronic study, where the media conditions limit the metabolic rate
of the cultured cells. Thus, our findings of Aims 1 and 2 have consistently shown that the
effects of ethanol are time and concentration dependent in vivo and in vitro respectively,
suggesting that they are important factors to be considered in examining the extent of
neurological damage caused by alcohol. More importantly, chronic exposure to the
intoxicating ethanol concentration was found to cause long-lasting increases in P2X7R
expression levels suggesting a role for these receptors in microglia activation by ethanol.
32

Moreover, the in vitro up-regulation of P2X7R expression is consistent with a subset of
the in vivo findings from Aim 1, wherein the acute two-bottle choice study with 10% v/v
ethanol induced up-regulation of overall P2X7R expression in certain brain regions.
Thus, drawing upon our findings from Aims 1 and 2, the P2X7R up-regulation observed
on acute ethanol exposure can be further delineated to involve microglial activation and
function. However, it is important to note that the in vivo and in vitro findings are not
entirely comparable owing to the complexity of the in vivo system, given the
contributions from multiple cell types and the little experimental control over the
underlying signaling mechanisms.

2.2.5. Conclusions
Together, the outcomes of the present study support the hypothesis that the P2X7Rs are
involved in the activation of microglia in response to ethanol. Furthermore, this work
is consistent with the proposal that ethanol modulates, specifically up-regulates,
P2X7R expression in microglia. In addition, the results concur with the findings from
Aim 1 in that the alteration in P2X7R expression is dependent on the concentration
and duration of ethanol exposure. This study was based on the knowledge that alcohol
and P2X7Rs can activate microglia, thereby stimulating the production of pro-
inflammatory mediators and subsequently causing alcohol-induced neuroinflammation. I
used a BV2 microglial cell line to carry out treatments with varying concentrations of
ethanol for acute and chronic time-lines. The findings suggest that ethanol -induced up-
regulation of P2X7R expression could be driving the excessive production of a subset of
33

pro-inflammatory cytokines observed in the CNS after ethanol exposure. Obtaining
information in this regard would confirm that the P2X7R is indeed a primary contributor
to the neuro-inflammation that has been found to precede alcohol-induced brain damage.
Based on these findings, Aim 3 studies were designed to examine the functional role of
P2X7Rs in the microglial response to ethanol.

2.3. Aim 3: Investigate the role of microglial P2X7 receptors in alcohol induced
neuroinflammation through the release of IL-1β.
Results from Aim 2 suggested that ethanol induced an up-regulation in P2X7R
expression in microglia. Further, the findings suggested that this action led to microglial
activation toward a possible alcohol-induced neuroinflammation. Taken together, the
findings are consistent with a direct role for P2X7Rs in mediating alcohol induced
neuroinflammation. However, this hypothesis is yet to be investigated. This is the
purpose of the studies of Aim 3.

An important lead for Aim 3 investigations came from evidence that alcohol-induced
neuroinflammation contributes to the development of neurodegenerative pathologies
through the release of pro-inflammatory cytokines (Crews and Nixon, 2009). A body of
evidence exists in support of this viewpoint, showing that the release of a host of pro- and
anti-inflammatory cytokines (TNFα, IL-1, IL-6, IL-10, TGFβ) can induce long-term
changes in alcohol-induced behaviors and neurodegeneration (Crews et al., 2006; Crews
and Nixon, 2009; Qin et al., 2008). Specifically, pro-inflammatory markers such as
34

monocyte chemo-attractant protein 1 (MCP-1 or CCL-2) have been detected in
postmortem human alcoholic brains (He and Crews, 2008). Further, in an intermittent
binge alcohol exposure model, alcohol increased TNF-α, IL-1β, IL-6 and iNOS levels,
effects which were attenuated with Cox-2 inhibition or Toll like receptor-4 (TLR4)
knockout, known inhibitors of microglial activation (Alfonso-Loeches et al., 2010;
Fernandez-Lizarbe et al., 2009). Of these pro-inflammatory cytokines, Il-1 has been
well-documented for its neurodegenerative role; as modulators of IL-1 release, TLR4s
have gained importance in ethanol-induced neurotoxicity (Alfonso-Loeches et al., 2010),
reduction of neurogenesis and depressive behavior (Kelley and Dantzer, 2011b). Taken
together, the findings suggest that microglial activation mediates pro-inflammatory
cytokine production as a mechanism of response to alcohol.

Given that alcohol induces a neuroinflammatory response mediated by the increase in
P2X7R driven activation of microglia, I hypothesized that microglial P2X7R-stimulated
IL- Iβ release contributes to alcohol-induced neuroinflammation. To begin to test this
hypothesis, I used the BV2 murine microglial cell line. For Aim 3, the goals were two
fold - 1) Test the effect of ethanol on P2X7-mediated IL-1  release, 2) Use the shRNA
knockdown strategy to inhibit the P2X7 channel as an attempt to attenuate the
neuroinflammatory response caused by ethanol.

35

2.3.1. Experiment 1: Ethanol as a neuroinflammatory agent through P2X7R-
mediated IL-1 release in BV2 microglia
2.3.1.1. Rationale
In the brain, IL-1β is expressed and released mainly by microglia, although astrocytes
and invading macrophages are reported to contribute to IL-1β production at some time
after the insult (Mabuchi et al., 2000; Pearson et al., 1999). The study of microglial
P2X7Rs as important mediators of IL-1 release in the CNS is validated by several
studies. Experiments in P2X7R knockout mice have conclusively identified that the
P2X7R is responsible for ATP-dependent IL-1β release upon LPS challenge (Ferrari et
al., 1997b; Labasi et al., 2002). Furthermore, IL-1 production in response to LPS was
reduced in P2X7R knockout mice as compared to WT mice (Mingam et al., 2008).
Interestingly, under an insult, microglia-specific expression of IL-1β is seen to be rapidly
up-regulated (Pearson et al., 1999). Given this data and the finding that ethanol up-
regulates microglial P2X7R expression, I hypothesized that ethanol exposure leads to
increased P2X7R mediated IL-1β production and release from microglia.

In order to mimic the inflammatory microenvironment of the microglia fully, this study
incorporated exogenously applied ATP and bacterial endotoxin LPS. In pathological
conditions, the ATP that is released from the damaged or lysed cells (Dubyak and el-
Moatassim, 1993) causes an up-regulation of the P2X7R, rendering ATP as an
excitotoxic agent when released into the extracellular space during ischemia (Braun et al.,
1998) and mechanical stress resulting in tissue injury (Petruzzi et al., 1994). Consistent
36

with this idea, application of exogenous ATP in vitro has been found to be toxic to
primary neuronal cultures and cause necrosis and apoptosis (Amadio et al., 2002). The
other key player, LPS, is an endotoxin found in the cell wall of Gram-negative bacteria
and is used to elicit an inflammatory response that is similar to that induced by tissue
injury or infection, involving microglial activation and pro-inflammatory cytokine
production. LPS is a ligand for TLR-4 (Hoshino et al., 1999), and their interaction is
thought to drive pro-inflammatory mechanisms, which has made LPS by far the most
frequently used model stimulus for inflammatory signaling and pharmacology
experiments. The response of peripheral macrophages to the LPS stimulus is well-
described (Rosenberger et al., 2000), and at least some data exists for stimulation of
acutely isolated adult microglia (Baker and Manuelidis, 2003).

Together, LPS and ATP comprise the best characterized in vitro setup for Il-1β
production and release. IL-1β is expressed as an inactive 31 kDa precursor pro-IL-1β that
is cleaved by caspase-1 to an active mature 17 kDa molecule which is then released from
the cells (Thornberry et al., 1992). Studies have consistently shown that LPS and ATP act
as the primary and secondary signal, respectively, to activate P2X7Rs to trigger
processed IL-1β release (Brough et al., 2002; Ferrari et al., 2006; Sanz and Di Virgilio,
2000). While LPS alone is necessary and sufficient for the pro-IL-1  synthesis, ATP is
considered to act as a secondary signal for the post-translational processing and
accelerated release of the mature IL-1  into the extracellular medium through a caspase-1
dependent mechanism, in response to an inflammatory stimulus (Perregaux and Gabel,
37

1994; Simi et al., 2007; Solle et al., 2001). These studies seem to collectively suggest that
the processing of premature pro-IL-1  to the mature, active IL-1  appears to require the
ATP activation of the P2X7 receptor, while the pro-Il-1 synthesis itself is LPS-
dependent. A comprehensive study was done by Bernardino et al, showing that co-
stimulation with LPS and P2X7 agonists activates microglial cells and is associated with
neurotoxicity mediated by significant IL-1 up-regulation (Bernardino et al., 2008).
Based on the results of my work presented above, coupled with the aforementioned
findings, Experiment 1 is proposed to test whether ethanol treatment of BV2 microglia
results in an increase in LPS and ATP-induced Il-1  production and release, mediated
through the P2X7R.

2.3.1.2. Methods
 Cell cultures
BV2 microglial cells were grown as described in Aim 2. The cells were then seeded into
6-welled culture plates at an appropriate density and allowed to grow for 24 hours prior
to the experiment.
 Treatments
BV2 cells were exposed to ethanol at 0, 25 and 100 mM for 20-24 h prior to addition of
LPS (1 µM) or P2X7R ligand ATP (1 mM) in Phenol-red free DMEM. Treatments
continued for 20 hours. A subset of the cells received the combination treatment of LPS
and ATP, with ATP being added at the end of the LPS treatment period to enhance IL-1β
38

expression, and incubation was continued for 3 more hours. The cells were lysed after 2-3
PBS washes and cell lysates were processed and collected.
 IL-1β ELISA
IL-1β levels in the extracellular medium of the BV2 microglial cells was measured using
commercially available mouse IL-1β ELISA kits according to the manufacturer’s
instructions (Pierce, Redford, IL).
 Statistical analysis
The ELISA measurements were used to determine significant variations between
different treatment conditions upon analysis using the student’s t-test, with significance
set at P < 0.05. The values in the figures and text are presented as mean ± SEM.

2.3.1.3. Results
To test the hypothesis that ethanol induces a neuro-inflammatory response in microglial
cells through the up-regulation of P2X7Rs, we measured the levels of Il-1β secreted by
the cells into the extracellular medium in the presence of LPS and ATP. The results are as
presented in Fig. 6. The basal IL-1β level, as observed in the controls, was low while
ATP and LPS treatments for 20 h increased its release four- and fourteen-fold
respectively. Unexpectedly, ATP incubation for 3 h after LPS treatment did not further
stimulate IL-1β levels. Ethanol demonstrated a modest effect with the tendency of
increasing basal as well as LPS-stimulated and agonist-stimulated IL-1β release. More
importantly, 100 mM ethanol exposure induced a significant enhancement of IL-1β
secretion induced by the combination of LPS and ATP.
39

Fig.7. Ethanol (EtOH) induces an increase in IL-1β release in the presence of LPS and ATP from BV2
microglia. ATP (1 mM) and LPS (1uM) induced IL-1β secretion. 100 mM Ethanol stimulated LPS-induced IL-
1β secretion in the presence of P2X7R activation with ATP. * P < 0.05 compared to control. # P < 0.05
compared to LPS and ATP. Data represent mean ± SEM of ELISA analysis from 3 experiments.

2.3.1.4. Discussion
The primary finding of Aim 3 investigations is that ethanol leads to the generation of a
pro-inflammatory excitotoxic insult through microglial cells in the presence of specific
stimuli mimicking the inflammatory environment. The results suggest that the insult is
dependent on the activation of the microglial P2X7R and the consequent IL-1β release.
The measurements of IL-1β release revealed that basal levels differed from the levels in
the presence of ATP alone, but much lesser than the approximate fourteen fold increase
that was seen in the presence of LPS alone or when incubated with their combination.
0
5
10
15
ATP,
1 mM
Control
IL-1 , ng/mg prot
#
EtOH, 0 mM
EtOH, 25 mM
EtOH, 100 mM
LPS,
1 M
LPS, 1 M
ATP, 1mM
*
*

40

Further, an increase in the concentration of ethanol to 100 mM led to a significant
increase in the LPS+ATP-induced IL-1β level. Taken together, the aforementioned
findings suggest that LPS and ATP are key players in the ethanol-induced IL-1β pro-
inflammatory response. Firstly, LPS induced a much higher increase in IL-1β levels than
ATP. This could be attributed to LPS’s role as a primary signal in the production of IL-
1β. As discussed in the rationale section, this is consistent with prior studies that have
shown the insufficiency of ATP alone and that LPS is necessary and sufficient for IL-1β
production (Le Feuvre et al., 2002; Perregaux and Gabel, 1994). However, there is
inconsistency in that we measure this increase in the absence of ATP, which is
considered necessary for the release of mature IL-1β (Sanz and Di Virgilio, 2000).
Conversely, the increase, albeit smaller, induced by ATP in the absence of LPS suggests
that there is an alternative signaling mechanism causing the production of immature IL-
1β. Secondly, the high elevation in IL-1β levels seen when the cells were co-incubated
with LPS and ATP agrees with findings from other groups that have demonstrated
massive Il-1β mediated excitotoxicity through P2X7R-mediated activation of the
inflammasome (Fantuzzi and Dinarello, 1999). The inflammasome is an oligomeric
assembly that is responsible for the maturation of the pro-IL-1β through a caspase-1
dependent signaling cascade (Martinon et al., 2002). A schematic representation of these
events in the presence of LPS and ATP are shown in Figure 8, where ethanol-induced
expression and ATP-activation of P2X7R is shown to cause the release of LPS-stimulated
production of pro-IL-1β through the inflammasome mediated activation of caspase-1.
While the scope of this study did not allow an investigation into the mechanisms behind
41

the production and release of the ELISA measured IL-1β, one can postulate that LPS
induced synthesis of pro-IL-1β was followed by the ATP activation of the P2X7R, which
mediates the inflammasome dependent maturation and release of the processed IL-1β into
the extracellular medium. More focused studies are necessary to compare IL-1β transcript
levels with the amount that is processed and released from the cells, in order to
understand the signal transductions that implement our observations.

Fig.8. A schematic representation of the production and release of Il-1β in the presence of ethanol.

Finally, our results show that high concentrations of ethanol further elevate IL-1β levels.
Drawing from our Aim 2 finding that acute ethanol exposure up-regulates P2X7R
expression in microglia, it is possible to state that the P2X7R up-regulation is directly
42

responsible for the observed increase in IL-1β levels. Taken together, our findings are in
agreement with existing knowledge showing that P2X7R stimulation is mandatory for Il-
1β release linked to inflammation-associated neurodegenerative events (Bernardino et al.,
2008). Alcohol-induced increase in expression of P2X7R can lead to the release of
threatening concentrations of IL-1β into the extracellular milieu, which primes
surrounding brain cells for damage. Il-1β is known to mediate neurodegeneration through
direct effects on neurons, such as the induction of superoxides, ROS and nitrogen species
(Fink et al., 1999), and indirect effects on astrocytes, including increased glutamate
release (Bezzi et al., 2001).

2.3.2. Experiment 2: Generation of BV2 microglial cell line with P2X7R knockdown
using recombinant lentivirus mediated shRNA transfer: use in ethanol studies
2.3.2.1. Rationale
Several lines of evidence indicate that pharmacological blockade of the P2X7R pathway
ameliorates neuropathology in animal models of neurodegenerative diseases (Takenouchi
et al., 2010). For example, P2X7R antagonist Brilliant blue-G (BBG) reduced the levels
of P2X7R expression, attenuated gliosis, diminished the leakiness of blood-brain barrier
and was neuro-protective in an animal model for AD (Ryu and McLarnon, 2008).
Additionally, in vivo administration of BBG prevented neuronal apoptosis in the mouse
model of HD (Diaz-Hernandez et al., 2009). Similarly, P2X7R inhibition has been
shown to exhibit beneficial effects on other animal models of CNS diseases including
spinal cord injury and multiple sclerosis (Matute et al., 2007; Peng et al., 2009). Knock-
43

out strategies involving P2X7-deleted mice in an AD model demonstrated the abatement
of the accumulation of IL-1 when intra-hippocampal injections of Amyloid-  were
administered (Sanz et al., 2009). These suggest that inhibiting or antagonizing P2X7Rs
may reduce neurotoxicity caused by alcohol. Drawing parallels from these studies, we
used shRNA mediated strategies of P2X7R-specific knockdown in the BV2 microglia.
Obtaining concrete results in this regard would confer clinical relevance upon the
P2X7R, since a control over decrease in IL-1 release would facilitate future efforts
toward directing a putative neuro-protective mechanism. To enhance the efficiency of the
future experiments by our group, I began to construct a BV2 based cell line that shows
long-term P2X7R knockdown. This will be accomplished using a recombinant lentiviral
(rLV) shRNA transfer approach.

2.3.2.2. Methods
 Infection of BV2 cells with rLV encoding shRNAs
rLV encoding scrambled (scr-shRNA) and P2X7R target sequences (P2X7- shRNAs – S1
or S2) were generated in the Lentiviral Core of School of Pharmacy. Infections of BV2
cells grown at a suitable density in 12-well plates for 24 hours were initiated by addition
of 10
5
IFU/ml of rLV shRNAs. Infections were carried out in the presence of polybrene
(2 μg/ml) to increase infection efficiency. After 48 hours of infection, the cells were
observed for GFP fluorescence and lysed in ice-cold RIPA buffer supplemented with
protease inhibitors. Whole cell lysates were obtained by subsequent centrifugation at 10
44

000 g for 10 min at 4 °C. The changes in P2X7R protein expression were assessed on the
lysates.
 Western Blotting
Protein concentrations were determined using the BCA (bicinchoninic acid) protein assay
and ten micrograms of the protein extract was separated, transferred, blocked and probed
for P2X7 expression as described earlier. In order to demonstrate specificity, we also
probed the cell lysates for P2X4R expression using a rabbit, anti-P2X4 primary antibody
(1:1000, Alomone Labs, Israel) followed by secondary anti-rabbit antibody (1:10000
dilution). Bands were visualized using enhanced chemi-luminescence (Pierce
Biotechnology).

2.3.2.3. Results
As presented below, I found that one of the two P2X7-shRNAs that were tested- the S2
shRNA, successfully attenuated P2X7R expression in a concentration dependent manner
(Fig. 7). Ten µl of the S2 almost completely knocked down the P2X7R protein levels.
Notably, S1 was less effective compared to S2. The validity of these results was
confirmed in that the non-targeted scr-shRNA did not significantly alter the P2X7R
protein levels. In addition, I tested the specificity of the shRNAs by probing the cell
extracts for P2X4R expression in addition to P2X7R expression. As shown, there was no
effect of the shRNA on the expression of P2X4R, indicating that the effects of S1 and S2
were indeed P2X7R-specific. Based on these observations, I initiated the culture of S2-
transduced BV2 cells growing them under selective antibiotic power. After several
45

passages, the lysates will be tested for P2X7R levels to determine whether the
knockdown is sustained.

Fig.9. A representative Western blot for P2X7 and β-Actin showing knockdown potentials of 2 rLV
shRNAs targeting P2X7R sequences. rLVs were generated at the Lentiviral Core of USC School of Pharmacy.

2.3.2.4. Discussion
Findings from Aims 1 and 2 suggested that the modulation of P2X7R expression could
play a key role in the exertion of ethanol’s toxic effects. Aim 3 studies were undertaken
to examine the hypothesis that the P2X7R contributes to alcohol-induced
neuroinflammation through the release of the pro-inflammatory cytokine, IL-1β.
Findings from Aim 3, Experiment 1 findings supported this hypothesis and provided
evidence to suggest that P2X7R-mediated release of the pro-inflammatory cytokine IL-1β
is part of the ethanol-induced pro-inflammatory cascade in the brain cells. To begin to
establish a mechanistic link between microglial P2X7Rs and alcohol-induced
46

neuroinflammation, I constructed a cell line with P2X7R knockdown using BV2 cells.
My mentors and I felt that this approach was more feasible over pharmacological
inhibition of P2X7Rs using oxATP or BBG for a few reasons. First, ethanol studies
require long-term treatment conditions. Long-lasting inhibition may not be possible with
the use of pharmacological inhibition. Second, the pharmacological agents may have
other non-specific or toxic effects which will be diminished with the use of the cell line.
Our preliminary studies stemmed from this rationale and we obtained encouraging results
that the shRNA strategy successfully induces a targeted and significant decrease in
P2X7R expression. Further studies are in progress to establish a reliable strategy for long-
term inhibition of microglial P2X7R function, in an effort to abate the neurological
damage caused by ethanol.

2.3.3. Conclusions
Aim 3 experiments focused on the functional role of the P2X7R in response to ethanol.
We investigated the mechanism by which P2X7R activation leads to the mediation of
neuroinflammation, which is thought to occur as a precedent of alcohol-induced brain
damage. Drawing upon the in vitro findings from Aim 2, our first step used the BV2
microglial cell line to test for the ethanol-induced P2X7R-mediated production of the
pro-inflammatory cytokine, IL-1β, which is a known mediator of neurodegeneration. The
findings suggest that there are two key players involved in the production and release of
IL-1β, LPS and ATP, in whose presence alcohol induced an elevation in the IL-1β levels.
Given the earlier finding that ethanol induced an up-regulation of P2X7R in the BV2
47

cells, this suggested that P2X7R-mediated IL-1β release is a possible mechanism of
alcohol induced neuroinflammation. Next, I attempted to initiate the generation of a cell
line that has stably knocked down levels of P2X7Rs using an rLV mediated P2X7R-
specific shRNA transfer strategy. The initial successful inhibition of P2X7R protein
expression suggests at the possibility to ameliorate the toxic effects of alcohol through
P2X7R-targeted strategies. Further studies are necessary to look at the downstream
effects of the inhibition since they would provide additional evidence on the extent of
P2X7R’s contributions to alcohol-induced brain cell damage.

48

CHAPTER 3
FUTURE PERSPECTIVES

Although beyond the scope of my present investigation, there are several exciting
directions of research for this project. First, I found that there are several modifications
that could be integrated into the previously described drinking paradigms under Aim 1,
which would shed further light on the mechanism of action of ethanol in the CNS. A
chronic model with a withdrawal component at the end of the ethanol-administration
period might provide important clues regarding the extent of damage caused by ethanol
during and after consumption. In addition, repeated alcohol withdrawal events may
account for significant brain damage resulting from hyperactivity and changes in
neurotransmission, increased oxidative stress and neuroinflammation (Crabbe et al.,
1993). A chronic intermittent exposure model with withdrawal events between drinking
periods would be an interesting approach to carry out biochemical investigations focused
on alterations in P2X7R signaling. Further, focused behavioral studies following such
investigations might be useful in demonstrating the resultant cognitive and behavioral
deficits caused by ethanol-induced, P2X7R-mediated brain cell damage. Encouraging
results in this direction may eventually assist in establishing a P2X7R-knockout mouse
model, which would provide an exciting gateway to study alcohol-induced brain damage
and other AUDs.

49

Second, P2X7R expression is not confined to microglia in the CNS (Collo et al., 1997).
Other key targets include astrocytes and neurons. As such, these targets may represent
potential contributors to ethanol-induced P2X7R-mediated neuroinflammation and
neurodegeneration as well, and initial studies focused on P2X7R expression and function
in these cell types in response to ethanol would be of assistance. Further, co-cultures of
different glial subtypes, or of glia and neurons, would be an exciting approach to study
cytokine signaling mediated by P2X7R activation when ATP is released as a
neurotransmitter in response to an ethanol insult.

Finally, an immediate future challenge is to use the shRNA strategy described in Aim 3,
Experiment 2 to generate a BV2 cell line with stable long-lasting P2X7R knockdown and
demonstrating that this strategy can be successfully used to ameliorate the P2X7R-
mediated inflammatory damage induced by ethanol. Future studies could extend the
investigation by testing if P2X7R inhibition causes lowering of pro-inflammatory
cytokine levels such as IL-1β. Testing the levels of IL-1β following the establishment of
stable, long-term P2X7R knockdown would aid further research toward mitigating the
detrimental neuroinflammatory effects of alcohol. In addition, the identification of
downstream players in the P2X7R signal transduction pathway using gene expression
manipulation and in vivo loss-of-function studies may represent possible directions for
future investigations. Functional studies in this regard could provide clues to these
downstream players’ contribution to P2X7R-mediated neurodegeneration, and this would
50

aid in the abatement of other deleterious signaling functions of the P2X7 receptor in
response to ethanol.

51

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Abstract (if available)

Abstract The present work builds upon a central hypothesis that ATP-gated purinergic P2X7 receptors (P2X7Rs) play an important role in causing ethanol-induced neuroinflammation and neurodegeneration. A distinctive set of properties set the P2X7R apart from other members of the P2X superfamily, including activation by pathologically significant levels of ATP and structural uniqueness that allows participation in a multitude of signal transduction pathways. Further, P2X7Rs are mainly localized to microglia in the brain and building evidence suggests that they play a critical role in the activation of these cells. Recently published studies have suggested that P2X7R signaling in microglia contributes to neurodegenerative pathologies through a neuroinflammatory response involving the production of pro-inflammatory cytokines such as Interleukin -1β (IL-1β). Despite the fact that alcohol abuse induces microglial activation and neuroinflammation similar to neurodegenerative pathologies, the role of the P2X7R in alcohol-induced neuroinflammation and brain damage has not been investigated so far. ❧ Three Specific Aims were set forth as initial steps to test the central hypothesis with the objective of taking us closer to understanding the mechanism by which alcohol-induced changes in P2X7R expression and function can cause or modulate the resulting inflammatory cascade, which may further cause or modulate the resultant alcohol-induced brain damage. Aim 1 tested the effects of ethanol exposure on P2X7R expression in different brain regions of C57BL mice using two different drinking paradigms: continuous access two-bottle choice and continuous access single bottle no choice. Using Western immunoblotting, these studies demonstrated that ethanol exposure had a differential effect on P2X7R expression in brain regions depending on the amount and duration of ethanol exposure. Low level of ethanol exposure (10 E) for a short period (10 d) caused an up-regulation of P2X7R in alcohol sensitive brain regions, whereas exposure to higher ethanol levels (20 E) for a longer period of time (3months) reversed the effect to down-regulation of P2X7R. Aim 2 tested effects of ethanol on P2X7R expression levels in the BV2 murine microglial cell line (in vitro) using Western immunoblotting. These studies found that ethanol up-regulated microglial P2X7R expression in both the acute (24 h) and chronic (7 d) models. Aim 3 studies tested the effects of ethanol on the P2X7R-mediated release of IL-1β in BV2 microglial cells in the absence and presence of an inflammatory stimulus, LPS. Using ELISA measurements, the studies demonstrated that ethanol causes an up-regulation in LPS-induced IL-1β production and release upon ATP-activation of P2X7Rs. ❧ Collectively, our findings from the three aims supported our central hypothesis and suggested that ethanol-induced modulation of microglial P2X7R expression mediates a neuroinflammatory response involving IL-1β release, the latter being a known mediator of neurodegeneration. The confirmation of our hypothesis prompted efforts toward an effective way to prevent ethanol-induced neuroinflammatory effects. Thus, Aim 3 also included preliminary studies to inhibit P2X7R expression in BV2 microglia using a lentiviral-mediated shRNA transfer strategy. We were able to achieve successful knockdown with one of the two shRNAs that we generated, and attempts to use this shRNA construct to establish a stable BV2 cell line with long-term P2X7 knockdown are currently being made. This could be used in future studies as a tool to investigate the downstream effects of P2X7R inhibition and identify the signaling mechanisms by which ethanol delivers its toxic neurological effects.

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Asset Metadata

Creator Gururaj, Sushmitha (author)

Core Title Ethanol induced modulation of microglial P2X7 receptor expression and its role in neuroinflammation

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 06/01/2012

Defense Date 05/03/2012

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

Tag ethanol,neurodenegeration,neuroinflammation,OAI-PMH Harvest,P2X7 receptor,purinergic

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Tokes, Zoltan A. (committee chair), Davies, Daryl L. (committee member), Kalra, Vijay K. (committee member)

Creator Email sgururaj@usc.edu,sushmitagururaj@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-43372

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Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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