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Effects of age-related glial activation on neurite outgrowth

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Effects of age-related glial activation on neurite outgrowth

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EFFECTS OF AGE-RELATED GLIAL ACTIVATION ON NEURITE
OUTGROWTH

by

Angela May Wong

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR BIOLOGY)

May 2007

Copyright 2007 Angela May Wong
ii

Dedication

To my parents Keith and Shirley and my brother Alvin.
iii
Acknowledgments

I would like to thank my adviser, Caleb Finch, and my mentors, Todd
Morgan and Irina Rozovsky for their guidance and helpful input throughout my
projects. I would also like to acknowledge Min Wei, Nilay Patel, Zhong Xie, Qiong
Wu, Jason Arimoto, and other current and former members of the Finch lab for their
help in the lab. I would also like to thank my committee members, Steven Finkel,
John Tower, and Christian Pike for their support, comments, and critiques of my
research and this dissertation. In addition, I would like to thank my friends and
colleagues in the Davies lab and Finkel lab for keeping me sane throughout graduate
school. Finally, I would like to acknowledge the Molecular and Computational
Biology Department for making graduate school an enjoyable experience and
teaching me about topics outside of my research focus which will benefit me in my
future endeavors.
iv
Table of Contents

Dedication ii

Acknowledgments iii

List of Tables vi

List of Figures vii

Abstract ix

Chapter 1: Background and Introduction 1
Glial cells and their functions 1
Age-related glial activation and inflammation 2
Caloric restriction attenuates glial activation 5
Effects of ovarian steroids on glia and neurons 7
Conclusions 10

Chapter 2: Macrosialin increases during normal brain aging and its
attenuation by caloric restriction 12
Abstract 12
Introduction 13
Materials and Methods 15
Results 18
Discussion 23

Chapter 3: Progressive changes in regulation of apolipoproteins E and J in
glial cultures during postnatal development and aging 26
Abstract 26
Introduction 27
Materials and Methods 28
Results 30
Discussion 36

Chapter 4: Inflammation decreases neurite sprouting in vitro 40
Abstract 40
Introduction 41
Materials and Methods 43
Results 45
Discussion 47

Chapter 5: Progesterone antagonizes E2-induced compensatory sprouting 52
v
Abstract 52
Introduction 53
Materials and Methods 56
Results 63
Discussion 75

Chapter 6: Summary 83

Bibliography 88

Appendix: Anti-inflammatory mechanisms of dietary restriction
in slowing aging processes 113
vi
List of Tables

Table 4.1 NO ( µM) levels in media collected from mixed glial/neuron co-
cultures after lipopolysaccharide treatments. 45

Table 5.1 Circulating plasma estrogen and progesterone levels after
hormone or sham pellet replacement. 58

Table 5.2 Effects of estrogen and progesterone on uterine and body
weights from 3-month old female rats from this study. 58

Table 5.3 Summary of in vivo data from ipsilateral (lesioned) hippocampus. 76

Table 5.4 Summary of in vivo data from contralateral (unlesioned)
hippocampus. 77

Table 6.1 Progestins in common birth control methods. 87
vii
List of Figures

Figure 2.1 Age-related increases of macrosialin are attenuated by caloric
restriction. 19

Figure 2.2 Macrosialin protein is induced by oxLDL. 20

Figure 2.3 Inflammatory stimuli activates BV-2 cells and induces
macrosialinmRNA and protein. 22

Figure 3.1 Apolipoproteins (Apo) E and J secretion by mixed glia
originated from neonatal (3-5 day), young (3 month), and old
(24 month) rats. 31

Figure 3.2 Cultured young adult mixed glial cells immunostained for ApoJ
or ApoE. 33

Figure 3.3 Quantification of ApoJ in situ hybridization in neonatal, young
and old mixed glial cultures. 34

Figure 3.4 Effects of cytokine treatments on ApoE and ApoJ secretion. 35

Figure 3.5 Proposed mechanism for cytokine actions on ApoE and ApoJ
secretion in adult mixed glial cultures. 38

Figure 4.1 Representative micrographs from mixed glia-neuron co-cultures
treated with vehicle or LPS for 1, 2, and 5 days. 46

Figure 4.2 Effects of LPS on neurite outgrowth in mixed glial/neuron
co-cultures. 46

Figure 4.3 ApoE and ApoJ secretions from mixed glial/neuron co-cultures
1, 2, and 5 days after vehicle or LPS treatment. 47

Figure 5.1 Diagram of lesions made to neuronal processes (perforant path
fibers) extending from the entorhinal cortex to the hippocampus
in the entorhinal cortex lesions (ECL) model. 57

Figure 5.2 P4 antagonizes E2-induced compensatory sprouting. 64

Figure 5.3 P4 blocks E2 inhibition of GFAP in the molecular layer. 65

Figure 5.4 E2 decreases microglial activation after ECL. 67
viii

Figure 5.5 Addition of P4 to culture medium induces neurite outgrowth
in astrocyte-neuron co-cultures but not mixed glia-neuron
co-cultures. 68

Figure 5.6 Effects of progesterone on estrogen-induced neurite outgrowth. 70

Figure 5.7 NeuN-positive cells in 0.250 mm
2
areas adjacent to wound zone. 71

Figure 5.8 Hormone treatments do not affect neuron numbers (NSE-positive
cells) in wounded mixed glial-neuron co-cultures in 0.250 mm
2

areas adjacent to wound zone. 72

Figure 5.9 OX42-positive microglial cells in wounded mixed glial-neuron
co-cultures is decreased in E2-treated co-cultures. 73

Figure 5.10 The progesterone receptor antagonist ORG31710 partially
reverses P4 inhibition of E2-induced neurite outgrowth in mixed
glial-neuron co-cultures. 75

ix

Abstract
Declines in cognitive function are common during aging even in the absence
of disease. Increased glial activation and inflammation during normal brain aging
are implicated in neuron atrophy which may lead to cognitive impairments.
Mechanisms underlying glial activation and their consequences on synaptic plasticity
are explored in this thesis.
Macrosialin, a marker for activated for activated microglial cells, increases
with age in various brain regions. These age-related increases could be attenuated
with caloric restriction. In vitro studies showed that oxidized low density
lipoproteins and inflammatory stimuli regulate macrosialin expression.
Apolipoproteins (Apo) E and J increase in several disease models and normal
brain aging. However, basal Apo J secretion decreases with age in mixed glial cells
originated from neonatal, young, and old animals. ApoE levels did not change
significantly. While neonatal mixed glia were unaffected by the inflammatory
cytokines, IL-1 β, IL-6, and TNF α, adult mixed glia had different responses to IL-6
versus IL-1 β and TNF α. IL-6 increased ApoE secretion while IL-1 β and TNF α
increased ApoJ secretion.
Neuroinflammation is implicated in neuron atrophy so an in vitro glial-
neuron co-culture model was used to study the effects of glial cells activated by the
inflammatory stimulus, lipopolysaccharide (LPS), on neurite sprouting. Activated
glial cells secreted elevated levels of nitric oxide which persisted after LPS removal.
x
Neurite sprouts decreased on LPS-treated glia. ApoE secretion decreased while
ApoJ secretion increased in LPS-stimulated glia which may be an important
mechanism underlying decreased neurite sprouting in the presence of inflammation.
Finally, effects of the ovarian steroid, progesterone (P4), on compensatory
sprouting were studied in the in vivo entorhinal cortex lesioning (ECL) and in vitro
“wounding-in-a-dish” models. Both P4 and 17 β-estradiol (E2) affect glial cells and
inflammation. E2 has been shown to induce compensatory sprouting in vivo and in
vitro, but interactions between P4 and E2 are not known. This study showed that P4
antagonizes E2-induced neurite sprouting in the presence of microglia. However,
when microglial levels are low, P4 induces neurite outgrowth and synergizes with E2
in vitro. This may have important implications when interpreting the data on the
effects of hormone replacement therapy on cognition.
1
CHAPTER 1

BACKGROUND AND INTRODUCTION

Glial cells and their functions
Neuroglia, which translates to “nerve-glue”, was a word coined by Rudolph
Virchow in the mid-1800s to describe the interstitial substance in the brain that
“binds” the nervous system elements together. Rudolph characterized neuroglia as a
form of connective tissue. Cells within this interstitial region were called connective
tissue corpuscles or elements of the neuroglia [Kettenmann and Ransom, 2005].
Today, these cells are simply known as glial cells. Unlike the origin of their name,
these cells are not just passive bystanders that hold the brain together. They are
active and their many functions will be discussed later in the introduction.
Macroglial cells are the predominant glial cell type and consist primarily of
astrocytes and oligodendrocytes. Microglial cells, derived from bone marrow
macrophages that migrate to the brain, comprise about 10% of the total glial cell
population [Kettenmann and Ransom, 2005].
The main function of glial cells is to support the neurons in the brain.
Astrocytes provide both positive and negative influences on neurons [Emsley et al.,
2004]. The positive effects include providing neurotrophic factors and nutrients to
neurons [Pellerin and Magistretti, 2004], modifying synaptic transmission [Ullian et
al., 2001], maintaining the blood brain barrier (BBB), and providing structure to the
brain. However, when astrocyte cells become reactive, they exert negative
2
influences in neurons such as participating in the mechanical and chemical barrier to
inhibit nerve degeneration and neurite outgrowth [Emsley et al., 2004].
The main function of microglial cells is to monitor the central nervous system
by preventing damage to the brain from extracellular chemical changes, debris
accumulation, and possible pathogen invasion [Conde and Streit, 2006]. Like
astrocytes, microglia exert positive and negative influences on neurons. Although
microglia scavenge material that may cause damage to neurons, chronic activation of
microglia results in the production of inflammatory molecules and reactive oxygen
and nitrogen species that may be harmful to neurons [Xie et al., 2002].

Age-related glial activation and inflammation
Glial activation occurs during normal brain aging in the absence of
neuropathology [Finch and Morgan, 2003]. Many of these in vivo changes persist in
in vitro primary glial cell cultures originated from aging rodent brains [Rozovsky et
al., 1998]. Therefore, the mechanisms underlying age-related glial activation can be
studied using in vitro cell culture models. Characteristics of glial activation include
changes in their morphology and gene expression. It is hypothesized that these
changes are responsible for age-related neuron atrophy and declines in cognitive
functions [Rozovsky et al., 2005].
Activation of astrocytes include cellular hypertrophy and increases in
expression of proteins such as glial fibrillary acidic protein (GFAP), apolipoprotein
E (ApoE), and ApoJ [Morgan et al., 1999]. Age-related increases in reactive
3
astrocytes are not the result of proliferation [Geinisman et al., 1978; Lindsey et al.,
1979]. The exact role of activated astrocytes during aging is not well understood
because reactive astrocytes serve multiple roles in central nervous system (CNS)
injury models [Sofroniew, 2005]. Some of these roles include the formation of the
glial scar to restrict inflammation to the damaged areas repair and maintenance of the
BBB, regulation of tissue fluid levels to prevent excessive edema, and protection of
neuronal and oligodendrocyte functions [Bush et al., 1999; Faulkner et al., 2004a].
However the harmful actions of reactive astrocytes include the production of nitric
oxide and reactive oxygen species that may damage neurons [Ikeda and Murase,
2004] and the possible inhibition of axon regeneration [Bush et al., 1999; Faulkner et
al., 2004b].
GFAP is commonly used as a marker for activated astrocytes. Age-related
increases of GFAP are associated with decreased neurite outgrowth [Rozovsky et al.,
2005]. Astrocytes originated from GFAP null mice spinal cord or cerebral cortex
better support neuronal survival and neurite outgrowth [Menet et al., 2000; Menet et
al., 2001] by modifying expressions of the extracellular cell matrix protein, laminin,
and adhesion molecules, fibronectin and N-cadherin [Menet et al., 2001]. In vitro,
GFAP levels in enriched astrocyte-neuron co-cultures has been decreased by
antisense mRNA [Lefrancois et al., 1997], RNA interference [Rozovsky et al., 2005],
and estrogen [Rozovsky et al., 2002]. All of these manipulations of GFAP levels
result in increased neurite outgrowth due to the association of neurons to
4
extracellular laminin secreted by astrocytes [Lefrancois et al., 1997; Rozovsky et al.,
2002; Rozovsky et al., 2005].
Resting ramified microglial cells are characterized by an elongated cell body
with two or more primary processes extending from both cell poles. Each primary
process contains more secondary branches [Ling and Wong, 1993]. Upon activation,
microglial cells change morphology to become amoeboid [Ling and Wong, 1993;
Rozovsky et al., 1998]. Activated microglial cells are phagocytic and increase
expression of inflammatory molecules such as major histocompatibility complex II
antigens and complement receptor-3 [Conde and Streit, 2006; Morgan et al., 1999].
These features are also common to tissue macrophages.
Activated microglial cells are prominent in neurodegenerative diseases such
as Alzheimer disease (AD) [Akiyama et al., 2000; Finch et al., 2002] and Parkinson
disease [Czlonkowska et al., 1996; Kim and Joh 2006; Ouchi et al., 2005]. Even in
the absence of disease, microglial activation occurs during normal brain aging [Finch
et al., 2002; Morgan et al., 1999]. In vitro, microglial cells activated by
lipopolysaccharide (LPS) or amyloid-beta (1-42) plus interferon- γ produce
peroxynitrite, which is toxic to neurons [Xie et al., 2002]. Conditioned media from
N-11 microglial cells activated with LPS or advanced glycation endproducts (AGEs)
contain soluble products that are neurotoxic, inhibit neurite outgrowth, and cause
neurite retraction of the differentiated neuroblastoma cell line Neuro2a. In addition,
nitric oxide and its metabolites produced by activated microglia mediated
neurotoxicity and morphological changes in the Neuro2a cells [Munch et al., 2003].
5
Attenuation of microglial activation has been shown to be neuroprotective in
several rodent neurodegenerative disease models such as Alzheimer disease [Craft et
al., 2004; Gasparini et al., 2005; Klegeris and McGeer, 2005] and Parkinson’s
disease [Dehmer et al., 2004; Klegeris and McGeer, 2005; Sanchez-Pernaute et al.,
2004; Tripanichkul et al., 2006; Wu et al., 2002]. However, this inhibition is not
always desirable. Transgenic diabetic (db/db) mice subjected to middle cerebral
artery occlusion had delayed and reduced microglial action, however, this
compromised inflammatory response resulted in greater tissue damage [Zhang et al.,
2004; Kumari et al., 2006]. Therefore, it appears that some degree of microglial
activation at the appropriate time is necessary, however, prolonged activation of
microglial cells is harmful to healthy cells.

Caloric restriction attenuates age-related glial activation and inflammation
Caloric restriction (CR) extends life-span and slows age-related processes, in
several species such as worms, yeast, flies, rodents, and monkeys [Finch and Ruvkin,
2000; Lakowski and Hekimi, 1998; Jazwinski, 2000; Jiang et al., 2000; Fernandes et
al., 1976; Bodkin et al., 2003]. CR animals have lower body weight, body
temperature, plasma glucose, plasma insulin, and fertility. CR also elevates
glucocorticoids which is anti-inflammatory [Patel and Finch, 2002]. In addition, CR
attenuates oxidative damage [Sohal, 2002; Sohal and Weindruch, 1996; Hunt et al.,
2006] and preserves metabolic energetics [Hunt et al., 2006].
6
CR has also been proposed to be hormetic in which it is a lower level stressor
that induces stress responses to protect against later deleterious age-related changes
[Masoro, 2006]. In addition, Hipkiss has proposed that persistent glycolysis in ad lib
animals increases levels of methylglyoxal, a cytotoxic agent that glycates and cross-
links proteins and damges lipids and DNA. Occassional glycolysis in CR animals
upregulates glyoxalase, carnosine synthetase, and ornithine decarboxylase which
protects against damage induced by methylglyoxal [Hipkiss, 2006].
Microarray analysis suggests that normal aging in the brain is accompanied
with increases in inflammation and stress responses, however protein turnover and
neurotrophic factors are decreased. CR reverses these responses [Lee et al., 2000;
Morgan et al., 2007; Prolla, 2002]. To further confirm the effects of CR, several of
these genes have been studied in rodent models of aging. Age-related increases
GFAP in the hippocampus and corpus callosum were attenuated by CR in rodents
[Major et al., 1997; Morgan et al., 1999]. ApoE and ApoJ, which increase in aged
rodent corpus callosum and global pallidus, are restored to young control levels after
CR [Morgan et al., 1999]. Similar age-related changes in microglial activation and
proliferation were observed using the major histocompatibility complex class II
antigens and complement receptor 3 [Morgan et al., 1999]. (For more information
on the anti-inflammatory actions of CR, see Appendix A.)
The gene expression data suggests that CR would be beneficial in preventing
age-related cognitive decline, however results from rodent cognitive tests are
inconsistent. This may be due to differences in CR diets, age at which animals were
7
tested, and behavioral task used. Reports that show a positive effect of CR on
cognition examined long-term potentiation (LTP) [Eckles-Smith et al., 2000], tasks
involving balance and coordination [Means et al., 1993], or Y maze learning [Wu et
al., 2003]. However, CR consistently has either no effect [Bellush et al., 1996;
Hansalik et al., 2006; Means et al., 1993] or possibly a negative effect [Yanai et al.,
2004] on performance in the Morris water maze. Further studies are needed to
understand how caloric restriction affects molecular mechanisms behind these tasks.

Effects of ovarian steroids on glia and neurons
Menopause is the cessation of menstrual cycling in women. Common
complaints from women experiencing menopause include hot flashes, mood swings,
vaginal atrophy, and memory loss [Devi et al., 2005; Schnatz et al., 2005]. Of
particular interest to this project is the loss of memory, which suggests that ovarian
steroids have powerful effects on the brain.
Replacement of ovarian hormones, estrogen and progesterone, in hormone
replacement therapy (HRT) may be beneficial in preventing cognitive decline and
neurodegenerative disease. Many observational studies have reported benefits of
HRT on cognitive decline [Carlson et al., 2001] and memory [Yonker et al., 2006];
however, several studies indicated that estrogen replacement therapy or HRT had no
significant effect on cognition [Wolf et al., 2005]. Findings from the Women’s
Health Initiative Memory Study (WHIMS), a randomized clinical trial, concluded
that HRT had detrimental effects on cognitive function and dementia [Espeland et
8
al., 2004; Rapp et al., 2003; Shumaker et al., 2003; Shumaker et al., 2004].
However, none of these studies addressed how HRT affects more specific effects of
cognitive functions such as memory. In an ancillary study to WHIMS, Resnick et al.
found that long-term HRT (conjugated equine estrogens plus medroxyprogesterone
acetate) had a negative effect on verbal memory, slight positive effect on figural
memory, and no effect on other cognitive functions such as attention and working
memory, spatial ability, and fine motor speed [Resnick et al., 2006].
Numerous animal studies have examined the effects of estrogen on cognition.
Estrogen replacement in ovariectomized rodents improves performance in working
memory tasks [Frick et al., 2002;], however estrogen had no effect on performance
of tasks with configural association task [Gibbs and Gabor, 2003]. Studies in aged
ovariectomized rhesus monkeys confirmed that estrogen replacement improves
cognitive function. Estrogen-treated monkeys performed better on a spatial working
memory task and a sample recognition memory task [Rapp et al., 2003].
Effects of progesterone on cognition are less understood than estrogen. Duff
and Hampson showed that postmenopausal women taking estrogen and a progestin
performed similar to estrogen users in working memory tasks [Duff and Hampson,
2000]. In rodents, progesterone-treated ovariectomized aged rats performed worse in
a working memory task than animals not given progesterone [Bimonte-Nelson et al.,
2004]. In contrast, progesterone improved object recognition [Walf et al., 2006] in
rats. Traumatic brain injury models suggest that progesterone treatment in male rats
facilitates recovery of cognition [Djebaili et al., 2004; Roof et al., 1994]. In addition,
9
progesterone reversed estrogen-induced improvements in spatial memory tasks
[Bimonte-Nelson et al., 2006]. Harburger reported that progesterone antagonism of
estrogen-induced improvements in spatial memory consolidation depended on the
dose of progesterone [Harburger et al., 2006]. Further studies are needed to
understand the effects of progesterone on the brain and cognition.
Estrogen has beneficial effects on neurodegenerative disease models such as
Alzheimer disease (AD) [Vegeto et al., 2006] and Parkinson disease (PD) [Callier et
al., 2000; D’Astous et al., 2004; Morale et al., 2006]. In the AD mouse model
APP23, 17 β-estradiol inhibits microglial activation and inflammation [Vegeto et al.,
2006]. Estrogen neuroprotection in the 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine (MPTP) mouse model of PD involves the attenuation of reactive
astrocytes and microglial cells [Morale et al., 2006].
Progesterone appeared to antagonize dopaminergic effects in PD patients
[Strijks et al., 1999]. Progesterone also antagonized estrogen-reduction of MPTP-
induced dyskinesia in a PD monkey model [Gomez-Mancilla and Bedard, 1992].
Several studies have shown that progesterone is beneficial in animal models of
traumatic brain injury [Stein, 2001] by reducing edema [Wright et al., 2001;
Grossman et al., 2004] and gliosis [Pettus et al., 2005]. Effects of progesterone on
AD are not well understood, although its metabolite allopregnanolone increased
neurogenesis in an AD mouse model [Brinton and Wang, 2006].
Both estrogen and progesterone act on glial cells and neurons. Estrogen has
been shown to be neuroprotective [Hoffman et al., 2006], modulate GFAP levels in
10
astrocytes [Rozovsky et al., 2002; Rozovsky et al., 2005; Stone et al., 1998; Stone et
al., 2000], and inhibit microglial activation [Bruce-Keller et al., 2000; Vegeto et al.,
2006]. Progesterone also influences neuroprotection [Hoffman et al., 2006; Singh,
2006], alters astrocytic GFAP [Djebaili et al., 2005; Garcia-Estrada et al., 1999;
Melcangi et al., 1996], and affects microglial activation [Drew and Chavis, 2000;
Lieb et al., 2003]. The influence of progesterone on estrogen-induced effects may be
more important since progesterone is rarely administered alone. Therefore, it is
necessary to study how estrogen and progesterone interact in the different brain cell
types.

Conclusions
Glial activation occurs during normal brain aging. Changes in the expression
of several markers for reactive astrocytes and activated microglia are not necessarily
global throughout the brain [Morgan et al., 1999]. Some of these changes may be
attenuated by CR [Major et al., 1990; Morgan et al., 1999]. Although aging is
associated with cognitive impairments, the consequences of CR attenuating age-
related glial activation on neurons is not clearly understood since behavioral test
results are mixed. This underscores the need to understand mechanisms of age-
related glial activation and inflammation and their interactions with neurons which
will ultimately affect cognition.
Glial activation and inflammation can also be modulated by the ovarian
steroids, estrogen and progesterone. Although it is not clear whether HRT is
11
beneficial on age-related cognitive decline and dementia, both estrogen and
progesterone clearly affect glial cells and neurons. Further studies are needed to
understand whether HRT can be used as a potential therapeutic treatment for age-
related cognitive decline.

12

CHAPTER 2
MACROSIALIN INCREASES DURING NORMAL BRAIN AGING AND ITS
ATTENUATION BY CALORIC RESTRICTION
(Published in Neuroscience Letters, 390:76-80)

ABSTRACT
During normal brain aging, microglia develop an activated phenotype
characterized by morphologic changes and induction of inflammatory molecules
such CD11b and MHC II. We show that macrosialin (CD68), a macrophage-specific
protein, is increased by aging in select brain regions of male C57BL/6NNia mice. In
corpus callosum and striatum, macrosialin mRNA and protein increased ≥50% (24-
vs. 4-months); hippocampus and cerebellum were unchanged. Caloric restriction
(CR) attenuated these age-related increases. Since age-related increases in oxidative
damage and inflammation are also attenuated by CR, we examined their role in
regulating macrosialin. Treatment of BV-2 microglial cells with oxidized low-
density lipoproteins (oxLDL) induced macrosialin protein by 50%. Moreover,
macrosialin mRNA and protein were induced in response to lipopolysaccharide
(LPS) plus interferon- γ (IFN- γ) which activates inflammatory pathways in BV-2
cells. These findings implicate oxidized lipoproteins and inflammation as factors in
increased macrosialin expression during aging.
13

INTRODUCTION
Macrosialin, the mouse homolog of the human CD68, is a member of the
lysosomal/endosomal-associated membrane glycoprotein (LAMP) family and is
expressed primarily in monocytic cells [Holness & Simmons, 1993; Holness et al,
1993]. Macrosialin is also a member of the scavenger receptor family proteins
which recognize a wide range of anionic macromolecules such as oxidatively-
modified lipoproteins, apoptotic cells, and cell surface antigens of microorganisms.
In macrophages, macrosialin is mainly localized in lysosomes and endosomes, and
rapidly exchanges with a smaller subfraction of macrosialin on the cell surface
[Kurushima et al., 2000]. Its localization in late endosomes and predominance in
phagocytic macrophages implicates macrosialin in phagocytosis [de Beer et al.,
2003; Kobayashi et al., 1998]. For example, no macrosialin expression is detected in
resting vascular endothelial cells, but it can be induced by cholesterol treatment
which transdifferentiates vascular endothelia to phagocytic cells with macrophage
characteristics [Rong et al., 2003]. Macrosialin is also induced in mouse peritoneal
macrophages by oxidized low-density lipoproteins (oxLDL) [de Beer et al., 2003,
Yoshida et al., 1998].
Although implicated in phagocytosis, the exact function(s) of macrosialin is
not known. Under normal, physiological conditions, scavenger receptors function to
clear cellular debris; however, in Alzheimer’s and other neurodegenerative diseases,
scavenger receptors may mediate the recruitment and activation of macrophage cells.
14
Although less studied than other scavenger receptors, macrosialin may have similar
functions [Yoshida et al., 1998]. Scavenger receptors may contribute to the disease
pathology by inducing the microglial production of reactive oxygen species and
inflammation [El Khoury et al., 2003; Yoshida et al., 1998]. However, little is
known about macrosialin expression and function in the brain.
In two transgenic mouse models for Alzheimer disease (AD) (APP23 and
Tg2576), macrosialin immunostaining was localized to activated microglial cells
around amyloid plaques [Bornemann et al., 2001; Sasaki et al., 2002]. In human AD
brains, macrosialin is localized in cells near amyloid plaques and extraneuronal
neurofibrillary tangles [Kobayashi et al., 1998]. AD-related pathology is also
associated with increased oxidative damage and inflammation [Akiyama et al., 2000;
Butterfield et al., 2001; Perry et al., 2002].
In contrast to AD, glial activation and oxidative damage during normal brain
aging are more subtle. Microglia are activated in the absence of amyloid plaques in
many brain regions, particularly in myelinated pathways of aging rodents, primates,
and humans [Finch et al., 2002; Morgan et al., 1999; Peters, 2002; Streit, 2002].
These changes in rodents are attenuated by life-long caloric restriction which extends
life span and slows many aging processes including the accumulation of oxidative
damage [Finch et al., 2002; Patel and Finch, 2003; Prolla and Mattson, 2001; Sohal
and Weindruch, 1996]. Thus, we hypothesized that normal oxidative and/or
inflammatory mechanisms during aging in the brain may regulate macrosialin.
15

MATERIALS AND METHODS
Animals
Male C57BL/6NNia mice were purchased from Harlan Sprague Dawley.
Animal procedures followed the National Institute of Health Guide for the Care and
Use of Laboratory Animals (NIH Publications No. 80-23) revised 1996. Ad libitum
(AL) fed mice were maintained on the NIH-31 diet consisting of 4% fat. Caloric
restricted (CR)-mice, fed 40% less calories than AL mice, were maintained on the
NIH-31/NIA Fortified Diet (2.6% fat) from 4 to 24 months. Mice were anesthetized
with isofluorane and perfused with saline. Brains were removed and stored at –70
o
C.
Brain (20 µm) sections were mounted onto polylysine-coated slides.

In situ hybridization
Sections were fixed in buffered 4% paraformaldehyde (30 minutes), rinsed
with phosphate-buffered saline (PBS), and incubated with acetic anhydride in 0.1 M
triethanolamine, and dehydrated in an ethanol series. In situ hybridization on fresh
frozen sections for macrosialin used anti-sense
35
S-labeled cRNA to the 0.7 kb
region (264 to 982 nt.) of mouse macrosialin mRNA. Controls included sense cRNA
probe and RNase treatment. After high stringency washing (50% formamide; 63
o
C)
and dehydration, slides were exposed to XAR-5 X-ray film (Eastman Kodak,
Rochester, NY) and dipped with NTB2 emulsion (1-3 weeks) and counterstained
with Cresyl Violet (Sigma).
16

Immunocytochemistry
Frozen brain sections were fixed in 4% buffered paraformaldehyde.
Endogenous peroxidase activity was quenched with 0.3% H2O2. Cell membranes
were permeabilized in 1% NP-40 (Sigma). Sections were blocked in a solution
containing normal rat serum (Sigma) and incubated (18 hours, 4oC) with rat-anti-
mouse CD68 (clone FA-11) (Serotec, Oxford, England). Vectastain ABC (Vector
Laboratories, Burlingame, CA) followed by diaminobenzidine substrate (Vector
Laboratories) to visualize CD68 (macrosialin) immunoreactivity.

Image Analysis
In situ hybridization and immunostaining were quantified by image
densitometry (IPLAB Spectrum software, Signal Analytics Corp) by outlining
regions of interest and estimating the percent of the area covered by the
immunoreactive product within the defined region as previously described (Morgan
et al., 1999).

Cell Culture
BV-2 microglial cells (Blasi et al., 1990) were maintained in Dulbecco’s
modified Eagle’s medium-Ham’s F12 (DMEM/F12) 50:50 Mixture (Mediatech, Inc.,
Herndon, VA) supplemented with 10% fetal bovine serum, 50 U/ml penicillin, 0.05
mg/ml streptomycin, and 2 mM L-glutamine in a humidified 95%/5% (v/v) mixture
17
of air and CO
2
at 37
o
C. Cells were replated 24 hours before treatment in T-25 flasks
at 500,000 cells/flask. BV-2 cells were activated with 100 ng/ml lipopolysaccharide
(LPS) (Sigma) plus 10 ng/ml interferon- γ (IFN- γ) (R&D Systems, Minneapolis, MN)
or 30 µg/ml oxidized low density lipoproteins [de Beer et al., 2003].

Northern Blots
Total RNA was isolated using TRI Reagent (Sigma) [Chomczynski, 1993].
10 µg of total RNA were electrophoresed and blotted to Biodyne B nylon
membranes (Millipore, Bedford, MA), followed by hybridization to
32
P-macrosialin
cRNA (described above).

Western Blots
Cells were lysed in 25 mM morpholinoethanesulfonic acid (pH 6.5), 150 mM
NaCl, 1% Triton X-100, 60 mM octylglucoside on ice, 10 minutes, 13,000xg spin for
10 minutes. 10 µg of the supernatants were used for western immunoblots [de Beer
et al., 2003].

Statistical Analysis
Statistically significant differences between means were determined by
ANOVA, followed by Fisher post hoc tests (StatView 5.0, SAS Institute Inc., Cary,
NC). P values less than 0.05 were considered significant.
18

RESULTS
Macrosialin increases with age and its attenuation by caloric restriction
Macrosialin mRNA and protein increased with regional specificity in 24- vs.
4-month old mice. Macrosialin mRNA increased 400% in the corpus callosum and
40% in the striatum, but did not change in the hilus (Figure 2.1A). Protein levels
generally paralleled the mRNA, but the changes were smaller. The corpus callosum
displayed widespread macrosialin immunoreactivity as parallel arrays in microglia
adjacent to nerve bundles. In striatum, immunostaining in 24-month AL mice was
largely at the periphery of the corticostriatal bundles (arrow in Figure 2.1B inset).
Macrosialin immunoreactivity increased in the corpus callosum of aged mice by
75%, whereas the trend in the striatum was not significant (Figure 2.1B).
Macrosialin staining in most other regions [hippocampus (dentate gyrus, CA1, CA3,
and hilus), cerebral cortex and cerebellum] was too low for reliable quantification.
Caloric restriction during the adult life span attenuated age-related increases in
macrosialin mRNA and protein in the corpus callosum (Figures 2.1A and 2.1B).

Oxidized low density lipoproteins induce macrosialin protein
In peritoneal macrophages, macrosialin expression is regulated by
atherogenic diets and by inflammation and can be induced by oxidized low density
lipoproteins (oxLDL) [de Beer et al., 2003]. To examine effects of oxLDL on
macrosialin, BV-2 microglial cells [Blasi et al., 1990] were treated with 30 µg/ml
19
oxLDL [de Beer et al., 2003] or native LDL. By 48 hours, oxLDL, but not native
LDL, induced macrosialin protein by 50% (Figure 2.2). Neither oxLDL nor native
LDL altered macrosialin mRNA (not shown).

Figure 2.1 Age-related increases of macrosialin (CD68) expression are attenuated by
caloric restriction. Effects of age and CR on macrosialin mRNA (A) and protein (B)
expression in several brain regions of C57BL/6 mice. Macrosialin mRNA
expression was analyzed by in situ hybridization. Protein expression was analyzed
by Immunocytochemistry. Signal intensity was expressed as a percentage of the 4-
month AL group. Bars represent mean +/- SEM for 10 animals per group. *p<0.05
relative to 4-month AL, ** p<0.01 relative to 4-month AL, ^p<0.0001 relative to 4-
month AL, #p<0.05 relative to 24-month AL, ##p<0.01 relative to 24-month AL.
(C-E) Macrosialin immunoreactivity in the corpus callosum and corticostriatal
bundles (insets) of (C) 4-month AL, (D) 24-month AL, and (E) 24-month CR C57
BL/6 mice. Arrow identifies macrosialin immunostaining at periphery of
corticostriatal bundle in 24-month AL mice. Bars = 100 micrometers for
micrographs, 30 micrometers for insets.
20

Figure 2.2 Macrosialin protein is induced by oxLDL. Macrosialin protein levels in
BV-2 cells treated with 30 µg/ml LDL or oxLDL as analyzed by Western blots.
Results are representative of data from duplicates of three experiments. Normalized
values are expressed as a percentage of the control. *p<0.05 relative to control.
21
Macrosialin mRNA and protein is induced by lipopolysaccharide
As another approach, responses to lipopolysaccharide (LPS), a cell wall
component of gram-negative bacteria, a potent inflammatory agent which also
induces protein oxidation in BV-2 cells [Mehlhase et al., 2000] was also examined.
IFN- γ (10 ng/ml; R&D Systems; Minneapolis, MN) was added with 100 ng/ml LPS
(Sigma; St. Louis, MO) to maximize responses (Figure 2.3A). Although LPS alone
had no effect on macrosialin mRNA, LPS plus IFN- γ induced mRNA 50% by 24
hours (Figure 2.3B) and protein by 40 hours (Figure 2.3C). LPS plus IFN- γ also
induces nitric oxide by 24 hours [data not shown; Xie et al., 2002]. However, nitric
oxide did not alter macrosialin expression, since treatment of BV-2 cells with sodium
nitroprusside, a nitric oxide generator, did not alter macrosialin mRNA or protein
(data not shown).
Moreover, macrosialin may not mediate nitric oxide production in response
to LPS [Aramaki et al., 1999]. Therefore, the LPS induction of macrosialin and
nitric oxide production may be independent events.

22

Figure 2.3 Inflammatory stimuli activates BV-2 cells and induces macrosialin
mRNA and protein. (A) Nitrite levels after a 24 hour incubation with
lipopolysaccharide plus interferon- γ assessed by the Griess reagent. (B, C) LPS +
IFN- γ induces changes in macrosialin mRNA and protein in BV-2 cells. (B)
Macrosialin mRNA expression were determined by Northern blot analysis. (C)
Macrosialin protein levels were determined by Western blot analysis. Normalized
values are expressed as a percentage of the control. Results are the average of
triplicates from 3-4 experiments. *p<0.05 relative to control, ** p<0.001 relative to
control. Below the graph is a representation of one of the Western blots used to
quantify the data.
23

DISCUSSION
These findings show that macrosialin expression increases during normal
brain aging. We hypothesize that oxidized lipoproteins and/or inflammation are
possible regulators of macrosialin expression during aging. Macrosialin changes
show brain regional selectivity, with the greatest induction of mRNA and protein in
the white matter of the corpus callosum and striatum. These age changes were
attenuated by CR, which increases lifespan in experimental animal models with
corresponding attenuation of the load of oxidatively-modified proteins and lipids
[Forster et al., 2000]. These results extend the age-related activation of microglia in
the corpus callosum and in corticostriatal tracts by the surface membrane receptors,
CD11b and MHC II, [Finch et al., 2002; Morgan et al., 1999] to the scavenger
receptor macrosialin.
The age-related increases of macrosialin we observed in the corpus callosum
may be important to changes in myelinated pathways during normal brain aging.
Magnetic resonance imaging diffusion tensor imaging studies on adult humans
consistently show that white matter is structurally altered within the corpus callosum
[Abe et al., 2002; Salat et al., 2005]. Over four years of follow-up, elderly men
showed 0.9% annual rate of decrease in the corpus callosum size which was
correlated with a decline in word recognition by the Stroop test. In addition, the size
of the corpus callosal splenium correlated with performance in the Mini-Mental State
Examination, a test of cognitive function [Sullivan et al., 2002].
24
We propose that the altered gross structure of white matter is due, in part, to
the increased gliosis during aging. The number of glia (astrocytes and microglia)
increases in the corpus callosum of aging mice [Sturrock, 1980]. This can also be
extended to monkeys where there was an increased density of activated microglia in
the corpus callosum of cognitively impaired old monkeys [Sloane et al., 1999]. In
addition, we have shown that the regions with greatest activation of astrocytes and
microglia were the corpus callosum and striatum [Morgan et al., 1999]. The present
study adds macrosialin to the expanding list of age-related markers of glial activation
in white matter.
Several studies have associated brain oxidative damage to cognitive
impairments. The cognitively impaired old monkeys which had an increase in
activated microglial cells had increased inducible nitric oxide synthase and nitrated
proteins in white matter. Most of the nitrated proteins were associated with
myelinated axons [Sloane et al., 1999]. Another study in mice showed an increase
relationship between markers for brain oxidative stress and performance in tests to
assess neuromuscular or synaptic functions [Navarro et al, 2002]. We have also
detected increased nitrotyrosine immunostaining in aging corpus callosum of mice
(unpublished data).
CD68 is also increased in degenerating white-matter brain diseases. For
example, multiple sclerosis patients typically show increased CD68 expression in
lesions [De Groot et al., 2001; Kivisakk et al., 2004]. In two less common human
acute demyelinating diseases, acute hemorrhagic leucoencephalitis and acute
25
disseminated encephalomyelitis, CD68-positive cells correlate with damaged axons
adjacent to veins and venules [Ghosh et al.,2004]. The increase of macrosialin
expression in white matter during ‘normal aging’ reported here indicates the
sensitivity of this microglial response to mild aging changes in white matter.
The present findings add to the understanding of macrosialin (CD68)
expression in brain diseases by showing that macrosialin is induced during normal
brain aging independent of disease. These white matter-rich regions are also areas
prone to oxidative damage and changes in the structural integrity of myelin. Future
studies will determine the role of increased microglial activation, as indicated by
markers such as macrosialin, in age-related cognitive decline.
26

CHAPTER 3
PROGRESSIVE CHANGES IN REGULATION OF APOLIPOPROTEINS E
AND J IN GLIAL CULTURES DURING POSTNATAL DEVELOPMENT
AND AGING
(Published in Neuroscience Letters, 371:199-204)

ABSTRACT
Apolipoprotein (Apo) E and ApoJ (clusterin), carriers of lipids and
cholesterol in the central nervous system, are implicated in age-related
neurodegenerative diseases such as Alzheimer disease. ApoE and ApoJ are
primarily secreted by glial cells. We have found that mixed glial cultures originated
from neonatal rat cerebral cortex differentially regulate these proteins compared to
glial cultures derived from adult rats. Basal secretion of ApoJ was two-fold greater
in glial cultures derived from neonatal versus adults. Cytokine responses also
differed by donor age. In adult glia, IL-6 increased ApoE secretion, but slightly
decreased ApoJ. Both IL-1 β and TNF α increased ApoJ secretion from adult glia,
but had little effect on ApoE secretion. In contrast, ApoJ secretion from neonatal
glia did not change in response to IL-1 β, IL-6, or TNF α while ApoE secretion was
slightly increased by IL-6. Differences in ApoE and ApoJ secretion may contribute
to age-related cognitive decline and must be considered when using neonatal glia to
study neurodegenerative disease models.
27

INTRODUCTION
Apolipoprotein (Apo) E and ApoJ, the primary apolipoproteins in human
cerebrospinal fluid, are secreted by glial cells as distinct lipoparticles [Ladu et al.,
2000]. While ApoE-lipoparticles are rich in cholesterol and phospholipids, ApoJ-
lipoparticles contain less phospholipids and very little cholesterol [DeMattos et al.,
2001].
Both ApoE and ApoJ are believed to be involved in neuronal differentiation
[Lorent et al., 1995; O’Bryan et al., 1993] and neuronal sprouting after lesions
[Lampert-Etchells et al., 1991; May et al., 1990; Page et al., 1998; Poirier et al.,
1991; Schauwecker et al., 1998; White et al., 2001]. ApoE has been localized to
astrocytes [Mahley, 1988; Poirier et al., 1991; Stone et al., 1997] and microglia
[Stone et al., 1997]. ApoJ is produced primarily in astrocytes and some neurons
[May and Finch 1992; Messmer-Joudrier et al., 1996]. Microglia have not been
reported to contain ApoJ mRNA and protein, however cultured microglia secrete
very little ApoJ (unpublished observations).
ApoE and ApoJ can be detected during development [Mouchel et al., 1995;
O’Bryan et al., 1993]. Their expressions are increased during normal brain aging
[Pasinetti et al., 1999] and in neurodegenerative diseases [May et al., 1989; May et
al. 1990]. Both have been found in senile plaques and neurofibrillary tangles of AD
patients and rodent models [Choi-Miura et al., 1992; Kida et al., 1994; Namba et al.,
1991; Terai et al., 2001]. Both apolipoproteins can bind to A β and influence its
28
aggregation [Strittmatter et al., 1993a; Strittmatter et al., 1993; Wisniewski et al.,
1993]. Of particular interest is ApoJ since its binding to A β results in the formation
of soluble toxic A β oligomers [Oda et al., 1995].

MATERIALS AND METHODS
Cell Culture
Mixed glial cultures were originated from neonatal (3-5 days), young (3
months), and old (24 months) F344 rats as described previously [McCarthy and
DeVellis, 1980; Morgan et al., 1995; Rozovsky et al., 1998; Xie et al., 2003].
Briefly, glial cells were isolated from the cerebral cortex after mechanical
dissociation, plated onto plastic culture dishes coated with poly-D-lysine (Sigma),
and maintained in DMEM-Ham’s F-12 culture medium supplemented with 10% fetal
bovine serum, 100 U/ml penicillin, and 50 U/ml streptomycin. Medium was
renewed every 2-3 days until confluence. Cells were replated in 24-well plates or at
20,000 cells/well or 4-chamber slides at 200,000 cells/chamber. Basal ApoE and
ApoJ secretion levels were measured from media 48 hours after cells were replated.
For cytokine treatments, cells were incubated with 10 ng/ml IL-1 β, IL-6, or TNF α
for 48 hours (all from R&D Systems).

Western Blots
ApoE and ApoJ in conditioned media were assayed against standards of
purified human ApoJ or recombinant human ApoE3 (Chemicon International,
29
Temecula, CA). Blots were blocked in 3% milk powder diluted with PBS prior to
primary antibody incubation with ApoE (Academic Biosciences Inc.) or ApoJ (Santa
Cruz Biotechnology, Santa Cruz, CA). Signals were quantified using video
densitometry (IPLab Spectrum software, Signal Analytics Corp).

Immunocytochemistry
Methanol-fixed cells were rehydrated with PBS and permeabilized with 1%
NP-40. Cells were blocked in a solution contained goat serum (Sigma) and 0.1%
Triton X-100 before incubation with ApoJ or ApoE antibody. Vectastain ABC
(Vector Laboratories, Burlingame, CA) followed by diaminobenzidine substrate
(Vector Laboratories) to visualize ApoJ or ApoE immunoreactivity.

In situ hybridization
Cells were fixed in buffered 4% paraformaldehyde (30 minutes), rinsed with
phosphate-buffered saline (PBS), and incubated with acetic anhydride in 0.1 M
triethanolamine, and dehydrated in an ethanol series. In situ hybridization on fresh
frozen sections for ApoJ used anti-sense
35
S-labeled cRNA as previously described
[Stone et al., 1998]. Controls included sense cRNA probe and RNase treatment.
After high stringency washing (50% formamide; 63
o
C) and dehydration, slides were
exposed to XAR-5 X-ray film (Eastman Kodak, Rochester, NY) and dipped with
NTB2 emulsion (1-3 weeks) and counterstained with Cresyl Violet (Sigma).

30
Image Analysis
For in situ hybridization analysis, grain clusters from 50-60 individual cells
were quantified using IPLab Imaging Software (Signal Analytics Corp) as previously
described (Morgan et al., 1999).

Statistical Analysis
Statistically significant differences between means were determined by
ANOVA, followed by Neuman-Keuls post hoc tests (StatView 5.0, SAS Institute
Inc., Cary, NC). P values less than 0.05 were considered significant.

RESULTS
Basal secretions of ApoJ and ApoE
Basal secretion of ApoE was 35% lower in neonatal than adult glia (not
significant, Figure 3.1A), whereas basal secretion of ApoJ was greater in neonatal
compared to adults (Figure 3.1B). ApoE and ApoJ represent <1% of the proteins in
the media. Silver-stained gels did not reveal major age differences (data not shown),
confirming that this effect is specifically due to age-related changes. Levels of
secreted ApoJ in the media was higher than ApoE at all ages (J:E, 15:1 in neonates;
3:1 in adults) (Figure 3.1C).
31

Figure 3.1 Apolipoproteins (Apo) E and J secretion by mixed glia originated from
neonatal (3-5 day), young (3 month), and old (24 month) rats. (A) ApoE and (B)
ApoJ accumulation in IPTS-supplemented serum-free medium after 24 hours as
analyzed by Western blot analysis using recombinant human ApoE or human
plasma ApoJ as standards. (C) Ratio of ApoJ:ApoE (ng/ng) in the same samples.
Data are averages of 7 (ApoE) or 10 (ApoJ) experiments (2-3 wells/experiment) +/-
S.E.M. *p<0.05, neonatal vs. adult cultures.
32

Cellular expression of ApoJ and ApoE
Cellular ApoJ immunoreactivity was much lower than ApoE (Figure 3.2)
although both mRNAs were detected by in situ hybridization in nearly all cells of
neonatal and adult cultures (data not shown). This difference in cell content has also
been reported in hepatoma cells in association with the shorter intracellular half-life
and faster secretion of ApoJ than ApoE [Burkey et al., 1991; Schmitt et al., 1999].
Previous reports have documented modest neuronal expression of ApoJ [Pasinetti et
al., 1999; Senut et al., 1992] and ApoE [Harris et al., 2004] while secretion of ApoJ
and ApoE from cultured astrocytes are robust. Therefore, most of the brain ApoJ
and ApoE are attributed to glia [Ladu et al., 2000; Shanmugaratnam et al., 1997].
ApoE and ApoJ secretion from adult glia are ca. 0.5 ng/24 h/1000 cells (5 x 10
12

molecules/24 h/cell), consisted with data from DeMattos et al. [DeMattos et al.,
2001].
33

Figure 3.2 Cultured young adult mixed glial cells immunostained for ApoJ or ApoE.
(A) ApoJ is retained by few cells in mixed glial cultures, however most of the cells
are positive for cellular ApoE (B and C). (C) Phase-contrast image to show mixed
glial confluency. Similar results were observed for all three age cultures, suggesting
that differences are not due to alteration in secretion. Representative images for one
of 3-4 experiments. Scale bar = 10 micrometers

34
Donor age progressively decreased ApoJ mRNA per cell (Figure 3.3). This
age pattern agrees with the observed ApoJ secretion pattern in culture (Figure 3.1).
Recent gene array analysis show that cultured neonatal astrocytes have two-fold
higher levels of ApoJ mRNA compared to adult [Nakagawa and Schwartz, 2004]
which supports this data. In contrast, the opposite adult age trend occurs in vivo.
Both ApoJ and ApoE mRNA increase in aging rat striatum and corpus callosum
[Morgan et al., 1999; Pasinetti et al., 1994] while ApoJ immunoreactivity increases
in cortical neurons [Senut et al., 1992]. This difference in ApoJ age patterns was
unexpected since age-related increases in expression of GFAP [Morgan et al., 1999;
Rozovsky et al., 1998] and IL-6 [Xie et al., 2003; Ye and Johnson, 1999] persist in
vitro.

Figure 3.3 Quantification of ApoJ in situ hybridization in neonatal, young and old
mixed glial cultures. Results are representative of data from one of three separate
experiments. Data is represented as average +/- S.E.M. *p<0.05 relative to old.
**p<0.0001 relative to adult cultures.

35

Figure 3.4 Effects of cytokine treatments on ApoE and ApoJ secretion. (A) ApoE
and (B) ApoJ accumulation 24 hours after treatment with human IL-1b, human
TNFa, or recombinant IL-6 in IPTS-supplemented serum-free medium. Data
normalized to respective age control. (C) ApoJ data presented as percentage of
neonatal control. Data are averages of 3-4 experiments (2-3 wells/experiment) +/-
S.E.M. *p<0.05 relative to respective age control.

36
Response of ApoE and ApoJ to cytokines
Treatment of neonatal glia with IL-1 β, IL-6, or TNF α did not significantly
alter ApoE or ApoJ secretion (Figures 3.4A and B). Adult glial cells were more
responsive to these cytokines. IL-6 increased ApoE secretion from adult glia while
there was a trend for decreased ApoJ secretion. However, IL-1 β and TNF α
increased ApoJ secretion, but had no effect on ApoE. The IL-1 β- and TNFα-
induced levels of ApoJ secreted by adult reached neonatal basal secretions after the
data normalized to the neonatal control (Figure 3.4C). It is hypothesized that
neonatal glia are secreting ApoJ at maximum rates while basal adult secretions
decrease during maturation. A putative ceiling effect could explain the loss of
neonatal ApoJ response to the cytokines.

DISCUSSION
Differences between neonatal and adult-derived glia have been reported for
neuroinflammation-related glial characteristics. Neonatal glia had greater LPS-
induced neurotoxicity and NO production [Xie et al., 2003; Young et al., 1995] but
less A β –induced secretion of the chemoattractant MCP-1 [Wyss-Coray et al., 2003].
Involvement of ApoJ in inflammation extends to apoptosis in tumor cells. ApoJ
expression is relatively high in intestinal neoplastic cells and decreased ApoJ levels
in tumor cells is correlated with apoptosis [Chen et al., 2003]. TNF α was identified
as a serum factor that induced cell-death in tumors [Green et al., 1976]. Downstream
activation of NF- κB appears to be down-regulated by ApoJ-mediated stabilization of
37
I κB in neuroblastomas. TNF α treatment of MCF-7 cells, a breast cancer cell line,
decreased ApoJ secretion and induced apoptosis [O’Sullivan et al., 2003]. The
nuclear form of ApoJ, but the secreted form is implicated in apoptosis [Leskov et al.,
2003]. In adult glia, TNFa increased ApoJ secretion but did not increase intracellular
ApoJ without indications of cell loss or mitochondrial function as (MTT assay) (data
not shown).
These indications of increased responsiveness of ApoJ secretion to IL-1 β, IL-
6, and TNF α during maturation may be the first example a postnatal acquired glial
function. The apparent switch of ApoJ from constitutive expression during
development to a regulated mode in adult glia is concurrent with a 50% reduction in
basal secretion in vitro. Both ApoE and ApoJ are rapidly induced in response to
focal pathway lesions or traumatic injury and remain elevated for weeks [Lampert-
Etchells et al., 1991; May et al., 1992; Page et al., 1998; Poirier et al.., 1991;
Schauwecker et al., 1998; White et al., 2001]. The induction of IL-1 β, IL-6, and
TNF α is notably faster, within 8 hours of injury [Taupin et al., 1993; Yan et al.,
1992] and precedes the elevations of ApoE and ApoJ around the lesion site by 24
hours. Thus, the increased ApoE and ApoJ in the glia surrounding amyloid plaques
in AD-transgenic mice could be stimulated by IL-1 β and TNF α around the plaques
[Apelt et al., 2001; Mehlhorn et al, 2000].
38

Figure 3.5 Proposed mechanism for cytokine actions on ApoE and ApoJ secretions
in adult mixed glial cells.
39

In summary, we did not find the age increases of glial secretion of ApoJ and
ApoE as expected from previous reports in vivo [Morgan et al., 1999]. Maturation
represented the largest age change (neonatal versus adult), with increased
responsiveness of ApoE and ApoJ expression in adult cultures to inflammatory
cytokines. It appears as though there is a switch from a constitutively-on expression
of ApoJ in neonatal cultures to low basal expression in adult glia that could be
rapidly induced by IL-1 β and TNF α. In contrast, IL-6 down-regulated ApoJ
secretion and increased ApoE levels. This suggests that NF-kB- and Jak/STAT-
activating pathways have opposite effects on ApoE and ApoJ secretion (Figure 3.5).
The complex regulation of the two key brain-apolipoproteins by inflammatory
cytokines in glial cultures has direct implications for AD-pathology. These results
suggest that the interaction between neurodegeneration and lipid transport may be
mediated through cytokines. Furthermore, the muted response of neonatal glial to
cytokines documents physiological differences between neonatal and adult glial
cultures.
40

CHAPTER 4
INFLAMMATION DECREASES NEURITE SPROUTING IN VITRO

ABSTRACT
Glial activation increases during normal brain aging. These changes are
accompanied by declines in motor and cognitive functions. Activated glial cells
produce inflammatory molecules which have been shown to contribute to neuron
atrophy. We have used an in vitro glial-neuron co-culture model to study effects of
inflammation on neurite sprouting. The inflammatory stimuli lipopolysaccharide
(LPS) induces glial production of nitric oxide and decreases glial support of initial
neurite sprouts. In addition, LPS increases glial secretion of apolipoprotein J (ApoJ),
but decreases apolipoprotein E (ApoE) secretion, which are important in mediating
glial responses to neurons. This study which provides a direction connection
between inflammation and neuron atrophy will be a useful model to study
mechanisms underlying glial activated neuron atrophy.
41
INTRODUCTION
Glial activation during normal brain aging is characterized by changes in
morphology and increases in inflammatory gene expression. Microarray studies
performed on various regions of mice brains (neocortex, cerebellum, and
hippocampus) have shown an increase in the transcription of inflammatory genes and
oxidative stress during aging [Lee et al., 2000; Prolla, 2002; Blalock et al., 2003]. In
the hippocampus, these changes were associated with decreased performances on the
Morris water maze and object memory task [Blalock et al., 2003]. Therefore, it is
hypothesized that age-related glial activation may be a cause underlying cause of
neuron atrophy and cognitive decline during aging.
Rodent models have been used to study the effects of inflammation on
behavioral tests. Injection of the gram-negative cell wall protein lipopolysaccharide
(LPS) into the global pallidus of Fischer 344 rats induced microglial activation at the
site of injection and locomotor deficits immediately after the injection. Young (3
months), but not middle-aged (16 months) rats recovered 4 weeks after the injection
[Zhang et al., 2005].
Peripheral administration of cytokines has been shown to influence cognition
and behavior [Wilson et al., 2002]. Interleukin-1 β (IL-1 β) administered peripherally
in mice exhibit locomotor deficits [Anisman and Merali, 1999], decreased
exploratory behavior [Spadaro and Dunn, 1990], and decreased responses to tasks
involving food rewards [Crestani et al., 1999]. In addition, mice administered IL-1 β
exhibited decreased performance in the Morris water maze [Oitzl et al., 1993].
42
These effects may be mediated by the actions of IL-1 β on glial cells and neurons
[Wilson et al., 2002]. Other studies have shown that IL-1 β activates glial cells
[Benveniste, 1992; Schubert and Rudolfini, 1998] and direct IL-1 β injection into the
brain increases neuronal damage after an insult [Rothwell, 1999; Stroemer and
Rothwell, 1998].
Systemic maternal inflammation has also been examined in rodents.
Offspring from mothers injected with saline or LPS at gestation day 17 showed very
little differences in behavioral effects as analyzed by exploratory behavior, sensory
and motor function, and learning and memory tasks [Golan et al., 2006]. Prenatal
exposure to interleukin-6 (IL-6) increased neuronal loss and astrogliosis (GFAP
mRNA and protein) in the hippocampus. This was associated with impairments in
the Morris water maze [Samuelsson et al., 2006]. Therefore, it is inconclusive
whether prenatal exposure to inflammatory stimuli is a risk factor for later
neurodegenerative disease. Since the two studies also differ in the time of injection,
it may be necessary to determine the crucial time period for prenatal exposure to
inflammation to cause disease.
We have developed an in vitro model to study the effects of glial
inflammation on initial neurite sprouting. LPS was administered to mixed glial cells
(astrocytes plus microglia) prior to and/or at the time the neurons were plated on the
glial bed to determine what effects inflammation would have on neurite outgrowth.
In addition to the production of nitric oxide, apoliproprotein E (ApoE) and ApoJ
secretion were also analyzed since both of these proteins are induced during aging
43
[Pasinetti et al., 1999] and in neurodegenerative disease [May et al., 1989; May et
al., 1990]. ApoE, and possibly ApoJ, are involved in neurite outgrowth [Stone et al.,
1998] so changes in these proteins may be relevant in our model. This model allows
us to study inflammatory-induced changes to glial cells that influence how they
support neurons.

MATERIALS AND METHODS
Cell Culture
Mixed glial cultures were originated from neonatal (3-5 days) F344 rats as
described previously [Rozovsky et al., 1998]. Briefly, glial cells were isolated from
the cerebral cortex after mechanical dissociation, plated in plastic T75 culture flasks,
and maintained in DMEM-Ham’s F-12 culture medium supplemented with 10% fetal
bovine serum, 100 U/ml penicillin, and 50 U/ml streptomycin. Medium was
renewed every 2-3 days until confluence. Mixed glial cultures were replated onto 4-
chamber slides coated with poly-D-lysine 2 days before plating E18 cortical neurons
on the glial monolayer. 100 ng/ml lipopolysaccharide (LPS; Sigma), dissolved in
0.9% NaCl buffer, were added to cultures 24 hours prior to or immediately after
plating of neurons. 0.9% NaCl served as the vehicle control. Co-cultures were fixed
1, 2, or 5 days after addition of neurons using ice-cold methanol for 5 minutes.
Media was collected for analysis with Griess reagent [Huygen, 1970; Green et al.,
1982] or slot blots.

44
Immunocytochemistry
Co-cultures were immunostained for polyclonal GFAP (1:500; DAKO Corp;
Carpinteria, CA) and monoclonal microtubule associated protein-5 (MAP-5; Sigma;
1:50). Products were detected using secondary goat antibodies conjugated to Alexa
488 (mouse) or 594 (rabbit) (Molecular Probes Invitrogen; 1:400).
After micrographs were captured, the two fluorescent channels were digitally
separated. MAP-5 neurites were analyzed using IPLab Spectrum image analysis
software as described previously [Rozovsky et al., 2005]. Briefly, a semi-automated
program defined the edge profile of the neurites, excluding the cell bodies. The
filtered image was skeletonized to generate single pixel lines. Total neurite length
was calculated from the single pixel lines. Measurements were obtained from 10
random areas (0.4 mm
2
).

Slot Blots
ApoE and ApoJ in conditioned media were run on a slot blot apparatus and
membranes were dried overnight. Blots were blocked in 3% milk powder diluted
with PBS prior to primary antibody incubation with ApoE (Academic Biosciences
Inc.) or ApoJ (Santa Cruz Biotechnology, Santa Cruz, CA). Signals were quantified
using video densitometry (IPLab Spectrum software, Signal Analytics Corp).
45

RESULTS
Activation of glial cells by lipopolysaccharide
Glial activation by LPS was confirmed by the production of nitric oxide
using the Griess reagent [Huygen, 1970; Green et al., 1982]. As expected, LPS
treatment at the time of neuron addition resulted in higher secretion of nitric oxide
(Table 4.1). In addition, increased nitric oxide secretion still persisted in LPS
pretreated glia despite the LPS washout when neurons were added (Table 4.1).
Table 4.1 Nitric oxide (mM) production
1 Day 2 Day 5 Day
No Pretreatment
No Post Treatment
1.5 ± 0.2 1.9 ± 0.1 2.0 ± 0.1
LPS Pretreatment
No Post Treatment
6.4 ± 0.2 7.1 ± 0.4 6.6 ± 0.1
No Pretreatment
LPS Post Treatment
13.4 ± 0.3 36.5 ± 0.4 61.9 ± 4.3
LPS Pretreatment
LPS Post Treatment
16.5 ± 0.7 31.2 ± 0.8 54.5 ±1.5

Table 4.1 NO ( µM) levels in media collected from mixed glial/neuron co cultures
after LPS treatments. Measurements were made using the Griess reagent. NO is
increased even in LPS-pretreated cultures where LPS was washed out after the
addition of neurons. Mean +/- S.E.M., N=3.

Initial neurite sprouting is decreased in glial-neuron co-cultures treated with LPS
In general, neurite outgrowth (MAP5 immunoreactivity) increased with time
(Figure 4.1). 1 day after neurons were plated, LPS exposure decreased the ability of
mixed glial cells to support neurite sprouts by 25 to 50% (Figure 4.2). LPS
pretreatment alone no longer affected neurite sprouts after 2 days. By 5 days, only
the pre- and post treatment of LPS significantly decreased neurite sprouts.
46

Fig.4.1 Representative micrographs from mixed glia-neuron co-cultures treated with
vehicle or LPS for 1, 2, and 5 days. MAP5 immunoreactivity (green) is
representative of neurite outgrowth. GFAP immunopositive cells represent the glial
cell bed. Scale bar denotes 30 micrometers.

Fig 4.2 Effects of LPS on neurite outgrowth in mixed glial/neuron co cultures. The
area covered by MAP5-positive neurites in a 0.5 mm
2
field was performed using
video densitometry. Data is expressed as % ROI (region of interest). Mean +/-
S.E.M., N=3 chambers (10 fields/chamber). *p<0.0001 from vehicle. **p<0.0005
from vehicle. #p<0.001 from vehicle.
47

Effects of acute LPS pretreatment on glial secretion of ApoE and ApoJ
LPS has been previously reported to decrease ApoE, but increase ApoJ in
mixed glial cultures [Saura et al., 2003]. As expected, in co-cultures treated with
LPS at the time neurons were plated, LPS also decreased ApoE and increased ApoJ
secretions from the co-cultures (Figure 4.3). 24 hour LPS treatment prior to plating
of neurons did not significantly change ApoE or ApoJ secretions (data not shown).

Fig. 4.3 ApoE and ApoJ secretions from mixed glial/neuron co-cultures 1, 2, and 5
days after vehicle or LPS treatment. Signal intensities were measured using video
densitometry. Data is expressed as average signal intensity +/- S.E.M. *p<0.05.
**p<0.01. #p<0.0005. ##p<0.0001.

DISCUSSION
Inflammation induces changes in glial cells that can affect their ability to
support neurite outgrowth. We have used LPS to induce the inflammatory response
in mixed glia (astrocytes plus microglia) and shown that LPS-activated mixed glial
cell support less neurite outgrowth than those treated with vehicle. In addition, we
can show that exposure of LPS prior to plating neuron is enough to maintain an
48
activated phenotype (increased NO production) and reduce neurite outgrowth. Acute
LPS treatment decreases ApoE, but increases ApoJ secretions. The important of
these lipoproteins on neurons will be discussed later.
Normal brain aging is accompanied by increased glial activation [Morgan et
al., 1999; Wong et al., 2005] and expression of inflammatory genes [Lee et al., 2000;
Prolla 2002]. These processes occur even in the absence of a neurodegenerative
disease such as Alzheimer disease or Parkinson disease. Age-related increases glial
activation and neuroinflammation are believed to be an underlying cause of cognitive
decline. Although this study used mixed glial cells from neonatal rats (3-5 days), our
lab has previously shown that aged (24 mo.) astrocytes support less neurite
outgrowth than young (3 mo.) astrocytes, and this age-related effect can be reversed
by knocking down GFAP levels [Rozovsky et al., 2002].
Studies in humans have tried to associate age-related cognitive decline with
various serum inflammatory markers. Increased serum C-reactive protein (sCRP)
levels, which was used as an indicator or low-grade chronic inflammation, have
been associated with cognitive impairment as measured by the baseline memory
(delayed recall) performance and mini-mental state examination (MMSE)
[Dimopoulos et al., 2006; Ravaglia et al., 2005]. In addition, Dimopoulos et al.
found that levels of CRP, intracellular adhesion molecule-1, and vascular cell
adhesion molecule-1 were higher in patients with dementia [Dimoupoulos et al.,
2006]. However, another study found impaired MMSE performance associated with
49
serum levels of the inflammatory protein α1-antichymotrypsin, but not CRP [Dik et
al., 2005].
Molecular mechanisms underlying age-related cognitive decline have been
studied in rodents. In a study that compares young (3 mo.), middle-aged (12 mo.),
and old (24 mo.) FBNF1 female rats, young animals performed better than older
animals in the Morris water task and transverse patterning discriminations, two tests
that analyze hippocampal-dependent cognition. These age-related behavioral
changes were accompanied by decreased hippocampal volumes believed to be
caused by decreased neurogenesis [Driscoll et al., 2006].
Animals that overexpress inflammatory proteins have been used to further
dissect mechanisms of single inflammatory proteins on cognitive decline.
Transgenic mice that overexpress TNF- α in the brain have impaired learning during
the Morris water maze test which was attributed to decreased nerve growth factor
(NGF) and increased Neuropeptide Y (NPY) in the hippocampus [Fiore et al.,
2000]. NGF is a trophic factor that promotes neurite growth [Collins and Crutcher,
1985; Campenot, 1987] and may possibly improve cognition [Martinez-Serrano et
al., 1995; Nabeshima et al., 1994; Scali et al., 1994; Sinson et al., 1995]. NPY also
promotes neurite outgrowth [White and Mansfield, 1996] and has been shown to
influence cognition in rodents [Bouchard et al., 1997; Flood et al., 1989; Thorsell et
al., 2000]. Young (5 mo.) transgenic rats that overexpress NPY have been shown to
have impaired spatial learning in the Morris water maze [Thorsell et al., 2000].
50
ApoE is involved in compensatory sprouting following entorhinal cortex
lesions [Champagne et al., 2005; Stone et al., 1998]. In addition to its effects of
neurite sprouting in response to lesions, ApoE can also modulate the inflammatory
response. In cultured mixed glial cells, ApoE suppressed inducible nitric oxide
synthase (iNOS) and cyclo-oxygenase-2 (COX-2) production induced by oligomeric
A β (aka. ADDLs). However, ApoE had no effect on ADDLs-induced IL-1beta and
induced IL-1beta in the absence of ADDLs [Guo et al., 2004]. Therefore, in addition
to decreased glial support for neurite outgrowth, LPS suppression of ApoE may
result in increased inflammation and oxidative damage.
Some of the immunomodulatory effects of ApoE may be isoform-dependent.
This may have relevance to AD pathology since ApoE4 is associated with AD risk
[Roses 1996]. Activation of microglial cells by the secreted form of β-amyloid
precursor protein was blocked by pretreatment with ApoE3, but not ApoE4 [Barger
and Harmon 1997].
ApoJ is a multifunctional protein that has been implicated in cell death,
chaperone activity, and most importantly complement-mediated cell lysis. Like
ApoE, ApoJ has immunomodulatory actions in the brain. Our lab has recently
shown that ApoJ activates rat microglia in vivo and in vitro [Xie et al., 2005]. ApoJ-
activated microglia in vitro increased levels of reactive nitrogen species such as
peroxynitrite [Xie et al., 2002] that resulted in neuronal cell death [Xie et al., 2005].
In this study, LPS induction of ApoJ may be involved in activating microglial cells
which produces toxic molecules that damages neurons.
51
We have previously shown that interleukin-6 (IL-6) induces ApoE secretion
in adult mixed glial cells. ApoJ secretion was induced by IL-1 β or tumor necrosis
factor-alpha (TNF- α). This suggests that ApoE is regulated by the Jak/STAT
pathway while ApoJ is regulated by NF-kB [Patel et al., 2004]. LPS, IL-1, and TNF-
α can inhibit IL-6 signalling of the Jak/STAT pathway [Heinrich et al., 2003].
LPS-activated glial cells produce reactive oxygen and are less able to support
neurite outgrowth in vitro. We can now show that this may be partially due to the
effects of LPS on glial secretions of ApoE and ApoJ, two apolipoproteins that have
immumomodulatory functions. This will have important implications in
understanding how age-related inflammation contributes to cognitive decline and
neurogenerative disease pathology.
52
CHAPTER 5
PROGESTERONE ANTAGONIZES ESTRADIOL-INDUCED
COMPENSATORY SPROUTING

ABSTRACT
Estrogen and progesterone (P4), the ovarian steroids that comprise hormone
replacement therapy (HRT), have multiple effects on the brain. 17 β-estradiol (E2),
the major circulating estrogen, induces neurite outgrowth in vitro and in vivo.
Effects of P4 and its interactions with E2 are not as well understood. These
hormones were studied using the in vivo entorhinal cortex lesioning model, which
models the neuronal damage and degeneration observed in Alzheimer disease. E2
replacement enhanced ECL-induced neurite sprouting in the hippocampus compared
to sham replacement. This compensatory sprouting was attenuated by P4.
Compensatory sprouting in response to E2 was accompanied with decreased
astroglial glial fibrillary acidic protein (GFAP) levels in the hippocampal molecular
layer and reduced levels of microglial major histocompatibility complex class II 1-A
antigen (OX6) in the hilus. Using the in vitro “wounding-in-a-dish” model with co-
cultures of astrocytes and neurons, E2 and P4 induced neurite outgrowth. However
in co-cultures of mixed glia (astrocytes + 30% microglia) and neurons, P4 inhibited
neurite outgrowth and neuron survival even in the presence of E2. These results
suggest that the activities of P4, which inhibits the beneficial actions of E2, must be
considered when interpreting results of HRT on age-related cognitive decline.
53

INTRODUCTION
Hormone replacement therapy (HRT) is commonly used to alleviate the
symptoms of menopause. HRT consists of a formulation of estrogens that may be
opposed with a progestogen. Common HRT therapies include Premarin (0.625 mg
conjugated equine estrogens), the transdermal estradiol patch Climera (0.05 mg
estradiol/patch), and Prempro (0.625 mg conjugated equine estrogens plus 2.5 mg
medroxyprogesterone acetate). Meta-analyses on observational studies suggested
that HRT may reduce the risk of age-related cognitive function decline and dementia
[Hogervorst et al., 2000; LeBlanc et al., 2001], however factors that may bias the
data include health of HRT users, socioeconomic status, duration of HRT use, and
HRT formulation [Hogervorst et al., 2000]. The Women’s Health Initiative Memory
Study (WHIMS), a randomized clinical trial that used Premarin or Prempro,
concluded that HRT had detrimental effects on cognitive function and dementia
[Espeland et al., 2004; Rapp et al., 2003; Shumaker et al., 2003; Shumaker et al.,
2004]. However, it still not clear how HRT affects specific aspects of cognitive
function such as memory. An ancillary study to WHIMS reported that long-term (4-
5 yrs) HRT (Prempro) had a negative effect on verbal memory, slight positive effect
on figural memory, and no effect on attention, working memory, spatial ability, and
fine motor speed [Resnick et al., 2006].
Estrogen has protective effects on the brain in several rodent
neurodegenerative disease models of Parkinson’s disease, stroke, and Alzheimer
54
disease [Quesada and Micevych, 2004; Fan et al., 2003; van Groen and Kadish,
2005]. In addition, our lab has shown that ovariectomy in female Fischer 344 rats,
which lowers estrogen levels, decreases fiber outgrowth and compensatory neuronal
sprouting after a brain lesion [Stone et al., 2000]. Effects of estrogen on neurons and
neurite outgrowth also involve interactions with glial cells such as astrocytes and
microglia. Estrogen regulates glial gene expression of genes such as glial fibrillary
acidic protein (GFAP) [Rozovsky et al., 2002a; Rozovsky et al., 2002b; Stone et al.,
1998; Stone et al., 2000] and apolipoprotein E (ApoE) [Rozovsky et al., 2002a;
Stone et al., 1997; Stone et al., 2000] which influence neurite sprouting.
Estrogen and progesterone fluctuations during the rodent estrous cycle also
influence the morphology of neuronal processes in the CA1 region of the
hippocampus [Woolley and McEwen, 1993], a region of the brain important for
learning and memory. During proestrous, when circulating estrogen and
progesterone levels are high, CA1 neurons have increased spines on their processes
[Woolley et al., 1990] and increased density of synapses [Woolley and McEwen,
1992] compared to estrous, when levels of both estrogen and progesterone are low.
Ovariectomized female rats have decreased dendritic spine densities and synapse
densities which can be reversed by estrogen replacement [Woolley and McEwen,
1993; Woolley and McEwen, 1992]. Effects of progesterone on dendritic spine
densities are biphastic. Progesterone initially increases estrogen-induced spine
densities (2 to 6 hours after progesterone treatment), however progesterone later (18
hours after treatment) decreases estrogen-induced spine density to levels similar to
55
untreated, ovariectomized controls [Woolley and McEwen, 1993]. In addition,
treatment of intact female rats with RU486, progesterone receptor antagonist, during
proestrous blocked the decreased spine density observed during estrous, suggesting
that progesterone plays an important role in decreasing dendritic spine densities
during the stage between proestrous and estrous [Woolley and McEwen, 1993].
Although the literature on estrogen is vast, the actions of progesterone (P4)
are not as well understood. P4 has been shown to be beneficial in human clinical
trials [Wright et al., 2006] and rodent models of traumatic brain injury [Garcia-
Ovejero et al., 2005]. P4 reduced edema in rats with bilateral contusions; however,
these animals also had increased microglial activation. Numbers of reactive
astrocytes and neuron survival were not significantly affected by P4 [Grossman et
al., 2004]. In a hippocampal slice culture that models nerve fiber degeneration and is
used to study mechanisms of synaptic remodeling within the hippocampus after
injury, P4 inhibited estrogen-induced-neurite sprouting [Teter et al., 1999]. In vitro,
P4 inhibited nitric oxide synthesis in microglia activated with lipopolysaccharide
[Drew and Chavis, 2000; Lieb et al., 2003]. However, in another study using
different indicators of microglial activation, P4 had no effect on LPS-induced
superoxide release and phagocytic activity [Bruce-Keller et al., 2000]. Therefore, P4
effects on the brain may differ depending on the model and endpoint that is studied.
To better understand the effects of P4 and its interactions with estrogen in the
brain, we have used the in vivo entorhinal cortex lesioning (ECL) and in vitro
“wounding-in-a-dish” models [Stone et al., 1998; Stone et al., 2000; Rozovsky et al.,
56
2002b] to study how these hormones influence neurite sprouting and glial cells
during this phenomenon. In addition, we have included mixed glia (microglia plus
astrocytes) in the in vitro model to examine the contribution microglial cells have in
steroid regulation of neurite sprouting.

MATERIALS AND METHODS
Animals
2-month old, ovariectomized female Sprague-Dawley rats were purchased
from Harlan (Indianapolis, IN). Two weeks after overiectomy, slow-release E2 or
sham vehicle pellets (Innovative Research of America; Sarasota, FL) were inserted
subcutaneously. Two weeks after administration of E2 (0.72 mg/pellet; 30 day
release) or sham vehicle pellets, all animals were lesioned on the left side of the
brain so that neuronal processes extending from the entorhinal cortex to the
hippocampus were damaged (Figure 5.1). This rodent model mimics human
Alzheimer disease (AD) damage where loss of neurons in the entorhinal cortex also
removes input to the hippocampus [Geddes et al., 1985]. This neuron loss is
implicated in memory decline during AD. The ECL model also shows similar
molecular changes in compensatory neurite sprouting from remaining hippocampal
neurons that are similar to changes observed in AD brain tissue (Geddes et al., 1985].
P4 (50 mg/pellet; 15 day release) or sham vehicle pellets (Innovation
Research of America) were inserted subcutaneously immediately after lesions. All
animals were weighed and sacrificed two weeks later. After perfusing animals with
57
saline, brains (n=8 animals/group) were removed and fixed in 4% paraformaldehyde
at 4
o
C for 1 day, cryoprotected in 30% sucrose for 3 days, and sectioned at 18 µm.
Uteri were removed, blotted, and weighed to estimate ovarian function.

Figure 5.1 Diagram of lesions made to neuronal processes (perforant path fibers)
extending from the entorhinal cortex to the hippocampus in the entorhinal cortex
lesions (ECL) model. Diagram from M. Wei was adapted from Crispino et al., 1999.

Circulating levels of E2 and P4 were measured from a pilot study using a
similar group of animals and pellets from the same lot used for this study to estimate
the amount of hormones released (Table 5.1). Estrogen and progesterone levels were
quantified by radioimmunoassay after extraction and celite chromatography [Slater
et al., 2001]. Circulating E2 levels were >2 fold higher in animals given E2
replacement and are within estrogen levels during proestrus. P4 levels were 5-fold
58
higher in animals administered the P4 pellet and are within the range of diestrus
levels.

Table 5.1 Circulating estrogen and progesterone levels
Treatment
Group
E2
(pg/ml)
SE P4
(ng/ml)
SE
Sham Vehicle 12.0 5.4 2.5 0.9
E2 47.3 11.3 2.3 0.7
P4 3.8 0.9 16.1 4.3
E2+P4 29.3 6.1 14.6 2.7

Table 5.1 Circulating estrogen and progesterone levels after hormone or sham pellet
replacement. Hormone measurements obtained from a similar cohort of animals (3-
month Sprague-Dawley female rats) using the same pellet lots. N=8
animals/treatment group.

Uterine weights were also obtained to assess hormone activity and ovarian
function (Table 5.2). Animals given estrogen pellets had > 8-fold higher uterine
weights than animals given sham E2 pellets (p<0.0001). In contrast, P4 treatment
had no significant effect on uterine weight (p=0.8393, sham pellet vs. P4; p=0.8221,
E2 vs. E2+P4).

Table 5.2 Uterine and body weights
Treatment Group Uterine Weight (g) ± SE Body Weight (g) ± SE
Sham Pellet 38.7 ± 2.4 289.8 ± 3.8
E2 360.7 ± 33.2* 227.2 ± 3.4*
P4 45.9 ± 3.4 291.7 ± 2.7
E2+P4 368.8 ± 33.6* 224.2 ± 2.5*

Table 5.2 Effects of estrogen and progesterone on uterine and body weights from 3-
month old female rats from this study. Each uterus was drained of fluid, blotted on a
filter pad, and weighed. Animals were weighed before they were anesthetized and
perfused. N=8 animals/treatment group. *(p<0.0001 from sham vehicle and P4
treatment groups).

59
To ensure that the animals in this study were in good health, body weights
were obtained at the end of the study (Table 5.2). All animals were active and had
no obvious pathology. Animals given E2 pellets weighed 20% less than animals
receiving the sham E2 pellets (p<0.0001). Estrogen replacement in rodents
decreases body weight gain after ovariectomy [Albert et al., 1991]. Although food
intake was not measured in this study, estrogen replacement has shown to decrease
food intake in ovariectomized rodents [Varma et al., 1999]. P4 had no effect on
body weight (p=0.6711, sham pellet vs. P4; p=0.5046, E2 vs. E2+P4) although
addition of P4 to estrogen replacement also decreased food intake in ovariectomized
rodents [Varma et al., 1999].

Holmes Stain
Neurons and their processes were silver-stained [Holmes and Young, 1942].
Briefly, after hydration, sections were incubated in 1% silver nitrate for 2 hours in
the dark. Sections were then impregnated in 9% boric acid buffer, 8% borax buffer,
1% silver nitrate, and 10% pyridine overnight at 37
o
C. Sections were reduced in
10% sodium sulfate with 1% hydroquinone. Sections were then incubated in 0.2%
gold chloride for 6 minutes, 2% oxalic acid for 8 minutes, and 5% sodium thiosulfate
for 5 minutes.
60

Immunohistochemistry
Sections were thawed, permeabilized with NP-40, and treated with H
2
O
2
to
quench endogenous peroxidase activity. After treatment with normal goat serum,
tissues were incubated overnight at 4
o
C with anti-GFAP (DAKO Corp.; Carpinteria,
CA) or OX6 (Serotec, Oxford, U.K.). After incubation with secondary antibody, the
Vectastain ABC Kit (Vector Laboratories, Burlingame, CA) was used with
diaminobenzidine to visualize the signal. Specificity of antibodies was confirmed by
omitting primary or secondary antibody.

Cell Culture
Primary mixed glial cells were originated from cerebral cortices of 1- to 3-
day-old F344 rat pups [Rozovsky, et al., 1998]. Cells were plated in T-75 flasks and
maintained in DMEM/F12 media (Cellgro, Herndon, VA) supplemented with 10%
fetal bovine serum (HyClone, Logan, UT), 100 U/ml penicillin, 50U/ml
streptomycin (Invitrogen, Carlsbad, CA), and 2 mM L-glutamine (Life Technologies
Inc. Rockville, MD) at 37
o
C, 5% CO
2
. Media were refreshed every 2-3 days until
confluence (10-12 days). Astrocyte cultures were purified from mixed glia by
shaking off microglia and oligodendrocytes. Astrocyte and mixed glial cultures were
replated onto 4-chamber slides coated with poly-D-lysine 2 days before plating E18
cortical neurons on the glial monolayer. Co-cultures were maintained for 3 days
before wounding and hormone treatments.
61

Wounding and hormone treatment
Astrocyte-neuron or mixed glia-neuron co-cultures were given scratch
wounds with a plastic pipette tip [McMillian, et al., 1994]. Immediately after
wounding, 0.1 nM E2 (Sigma) and/or P4 (Steraloids, Newport, RI) was added in
DMEM high glucose medium without phenol red (Invitrogen) supplemented with
penicillin/streptomycin, 15 mM Hepes (Sigma), 1 mM sodium pyruvate (Sigma), and
B27 supplement (Invitrogen) . Ethanol was used as the vehicle control (0.08%, final
concentration). 48 hours later, cells were fixed in ice-cold methanol.

Immunocytochemistry and image analysis
For neurite outgrowth studies, co-cultures were immunostained for
polyclonal GFAP (1:500) and monoclonal microtubule associated protein-5 (MAP-5;
Sigma; 1:50). Secondary goat antibodies conjugated to Alexa 488 (mouse) or 594
(rabbit) (Molecular Probes Invitrogen; 1:400) were used to detect MAP-5 or GFAP
respectively. The numbers and lengths of MAP-5-positive neurites extending into
the wound zone were measured. Neurite length was measured using IPLab Spectrum
image analysis software. Measurements were obtained from 10 random areas (0.4
mm
2
) in the wound zone.
Unwounded and wounded co-cultures were immunostained for monoclonal
NeuN (Chemicon International, Inc, Temecula, CA; 1:50) and detected with goat-
anti-mouse Alexa 488. Numbers of NeuN-positive cells were counted from 6-8
62
areas (0.2 mm
2
) in unwounded and wounded zones. For studies with neuron-specific
enolase (NSE) and activated caspase-3 (Csp-3), wounded co-cultures were
immunostained with monoclonal NSE (DAKO Corp.; 1:100) and polyclonal cleaved
Csp-3 (Cell Signaling Technology; 1:500). Secondary goat antibodies conjugated to
Alexa 488 and 594 (detailed above) were used to detect the products. Numbers of
cells positive for NSE and NSE/Csp-3 double immunolabeled cells were counted
from 6 – 0.2 mm
2
areas adjacent to wound zones.
For detection of microglial cells, wounded mixed glial-neuron co-cultures
were immunostained with OX-42 (Serotec; 1:50) and GFAP. Secondary goat
antibodies conjugated to Alexa 488 and 594 (described above) were used to detect
OX-42 and GFAP. Numbers of cells positive for OX-42 or GFAP were counted
from 6 – 0.2 mm
2
areas adjacent to wound sites.

Statistical Analysis
Analysis of variance (ANOVA) and Fisher post-hoc analysis were used to
analyze differences between treatment groups (Statview, SAS Institute, Inc., Cary
NC). Statistical guidance provided by Dr. Wendy Mack (University of Southern
California).
63

RESULTS
Effects of P4 on the Entorhinal Cortex Lesioning (ECL) Model
Animals were treated with P4 and E2 to determine if P4 would alter E2-
induction of neurite outgrowth in the ECL model. Holmes fiber staining of neurons
and their processes was used to assess neurite outgrowth (Figure 5.2). E2 increased
neurite outgrowth 2-fold 14 d after brains were lesioned. P4 attenuated this
induction by 30% in the hippocampus. P4 alone had a modest 20% increase over
sham pellet animals, however, this change is small compared to E2-treated animals
(Figure 5.2B). Although the contralateral hippocampus served as a control for
lesions, E2 still induced a modest 20% increase in width of processes compared to
other treatment groups (Figure 5.2B).
64

Figure 5.2 P4 attenuates E2-induced compensatory sprouting. A, Representative
images of Holmes fiber stained sections in the ipsilateral (lesioned) molecular layer.
Scale bar represents 100 micrometers. B, Average fiber band width in the ipsilateral
and contralateral molecular layers. Data is expressed as % molecular layer width +/-
SEM. N=8 animals/treatment group, 3 sections/animals. *(p<0.0001 from other
groups in respective hippocampal side). **(p<0.05 from other groups in respective
hippocampal side). #(p<0.0001, ipsilateral vs. contralateral).

Effects of P4 on glial activation
Increased astrocytic GFAP has been experimentally linked to impaired
neuronal sprouting during aging [Rozovsky et al., 2005]. E2 regulates GFAP RNA
and protein in vitro [Rozovsky et al., 2002] and in vivo [McAsey et al., 2006]. In
cortical stab wounds, P4 can attenuate local GFAP induction around the needle track
[Garcia-Estrada et al., 1993; Garcia-Estrada et al., 1999]. However, effects of P4 on
GFAP in the ECL model are not known. P4 alone did not alter GFAP
immunoreactivity (Figure 5.3). Although E2 attenuated GFAP immunoreactivity by
65
25% compared sham pellet animals, P4 blocked this attenuation comparable to sham
pellet levels (Figure 5.3B).

Figure 5.3 P4 blocks E2 inhibition of GFAP in the molecular layer. A, GFAP-
stained sections in the ipsilateral molecular layer. Scale bar represents 100
micrometers. B, Area covered by GFAP immunoreactivity in the ipsilateral and
contralateral hippocampi. Data is expressed as percent of sham pellet group +/-
SEM. N=8 animals/treatment group, 3 sections/animal. *(p<0.005 from other
treatments in respective hippocampal side). **(p<0.01 from other treatments in
respective hippocampal side).

Increases in GFAP are associated with impairments in neurite sprouting
[Rozovsky et al., 2005]. E2 attenuates GFAP and is associated with increased
neurite sprouting [Figure 5.2 and Figure 5.3; Rozovsky et al., 2005]. Addition of P4
blocks E2 attenuation of GFAP, which results in impaired neurite sprouting seen in
ovariectomized, sham pellet-treated animals.
Microglial cells influence compensatory sprouting following ECL [Eyupoglu
et al., 2004]. Although activated microglia were absent from the dentate molecular
layer 14 days post-lesion [observed data not shown; Gall et al., 1979], microglial
66
activation in the hilus persists up to 14 days after ECL [Dietrich et al., 2000; Gall et
al., 1979] although this region does not directly contribute to sprouting observed in
the dentate molecular layer. It would appear that microglial activation is not directly
affecting neurite sprouting, however microglial activation in the molecular layer
peaks 3 days post-lesion before gradually returning to control levels by 30 days post-
lesion [Hailer et al., 1997; Hailer et al., 1999]. Since E2 and P4 can attenuate
microglial activation [Bruce-Keller et al., 2000; Drew and Chavis, 2000; Lieb et al.,
2003], we examined the effects of E2 and P4 on microglial activation after ECL
using an antibody for OX6, which recognizes the I-A antigen of the major
histocompatibility complex (MHC) class II. At 14d postlesion, OX6
immunoreactivity was not detected in the dentate molecular layer (data not shown).
However, OX6 was detected in the hilus (Figure 5.4A). E2 attenuated OX6
immunoreactivity by 30% compared to sham pellet animals (Figure 5.4B). In
addition, OX6 immunoreactivity was increased by 30% by P4 in lesioned areas
(Figure 5.4B). This suggests that P4 increases lesioned-induced microglial
activation while microglial activation induced by lesions is inhibited by E2.
67

Figure 5.4 E2 decreases microglial activation after ECL. A, OX6 immunoreactivity
in the ipsilateral hilus. Scale bar represents 100 micrometers. B, Area covered by
OX6 immunoreactivity in the ipsilateral and contralateral hilus. Data is expressed as
percent of sham pellet group +/- SEM. N=8 animals/treatment group, 3
sections/animal. *(p<0.05 from other treatments in respective hippocampal side).
**(p<0.05 from sham pellet in respective hippocampal side). #(p<0.01, ipsilateral
vs. contralateral).

P4 induces neurite outgrowth in astrocyte-neuron, but not mixed glia-neuron
cocultures
To better resolve the complex interactions between neurons and glia, the
effects of P4 were studied in the in vitro “wounding-in-a-dish” model with neurons
co-cultured with astrocytes or mixed glia (astrocytes plus microglia). Astrocyte
cultures contained ~3% microglia versus ~30% microglia in mixed glia (Figure
5.5A). 48 hours after wounding and treating the cultures with hormones, only
astrocyte-neuron co-cultures treated with P4 had an increased number of neurites
extending into the wound zone (Figure 5.5B and 5.5C). The 100 nM P4 treatment
68
was equivalent to circulating P4 levels in animals administed P4 pellets which was
determined to be physiologically equivalent to diestrous levels (Table 5.1). Higher
doses of progesterone (1 mM) did not show any further increases in neurite number
(data not shown). Progesterone treatment alone also increased neurite lengths at all
doses used (data not shown) in astrocyte-neuron co-cultures. However, neither
neurite number (Figure 5.5) nor neurite length were affected by P4 (1 nm to 1 µm) in
mixed glia-neuron co-cultures.

Figure 5.5 Addition of P4 to culture medium induces neurite outgrowth in astrocyte-
neuron cocultures but not mixed glia-neuron cocultures. A, Representative
micrographs of an enriched astrocyte vs. mixed glial culture prior to plating of
neurons. Scale bar denotes 50 micrometers. B, Average number of neurites
extending into the wound zone per 0.500 mm
2
region. #(p<0.0001 from all other
treatment groups). C, 48 hours after wounding, neurite outgrowth in astrocyte-
neuron or mixed glia-neuron co-cultures. Scale bar represents 30 micrometers.
Asterisks indicate wound zone.
69

Effects of P4 on E2-induced neurite outgrowth
Since E2 can induce neurite outgrowth in vitro with astrocyte-neuron co-
cultures, we examined mixed glial-neuron co-cultures to study the combined
interactions of neurons, astrocytes, and microglia. E2 induced neurite outgrowth
(neurite numbers and neurite length) into the wound area from neurons co-cultured
with astrocytes or mixed glia (Figure 5.6), however P4 was only effective in
increasing neurite numbers in astrocyte-neuron co-cultures (Figure 5.6). P4
treatment in astrocyte-neuron co-cultures also induced a 3-fold percentage increase
in neurite lengths greater than 40 µm which was similar to E2 treatment (Figure
5.6A). In addition, P4 further increased E2-induced neurite numbers, in astrocyte-
neuron co-cultures (Figure 5.6B) although the neurite lengths were similar to
cultures treated with E2 or P4 alone (Figure 5.6A). However, in co-cultures with
mixed glia (astrocytes + microglia), neurite numbers and lengths are inhibited in the
presence of P4 (Figure 5.6A and Figure 5.6B). The mixed glia/neuron co-culture
data supports the in vivo Holmes fiber staining data (Figure 5.2). More specifically,
the neurite length data shows that while E2 induces a 5-fold increase in neurites
greater than 20 µm compared to vehicle, P4 and E2+P4 treatments induce a more
modest 2-fold increase in neurites greater than 20 µm compared to vehicle (Figure
5.6A) which is a very similar to the Holmes data in the ipsilateral hippocampus
(Figure 5.2B). The similarities between the in vivo and in vitro data confirm that the
70
mixed glia-neuron co-cultures are a good model to study interactions between
neurons and glial cells in the ECL model.

Figure 5.6 Effects of progesterone on estrogen-induced neurite outgrowth. A,
Combined histogram of neurite lengths in astrocyte-neuron or mixed glia-neuron co-
cultures. Data represents 3 combined experiments. B, Average neurite number
extending into wound zone per 0.500 mm
2
region. Data is represented as average of
3 experiments +/- SEM. *(p<0.0001 from other treatments in neurite category).
**(p<0.05 from vehicle in neurite category). #(p<0.0001 from vehicle). ##(p<0.05
between indicated groups).

Effect of P4 on neurons
The absence of neurite outgrowth observed in mixed glial-neuron co-cultures
in the presence of P4 may reflect neuronal damage or survival or a population of
neurons that are not sprouting. Neurons were stained with neuron nuclei (NeuN) to
examine whether the hormone treatments affected neuron numbers. While intact
71
differentiated neurons stain positive for NeuN, the loss of NeuN immunoreactivity
may represent a damaged neuron that could undergo later degeneration [Collombet et
al., 2006; Unal-Cevik et al., 2004]. In unwounded cultures, hormone treatments did
not affect the number of NeuN-positive neurons (data not shown). However, in
wounded mixed glia-neuron co-cultures, P4 decreased NeuN-positive neurons at
areas adjacent to the wound site by 50% (Figure 5.7). Numbers of NeuN-positive
neurons in astrocyte-neuron co-cultures were not affected by P4 (Figure 5.7).

Figure 5.7 NeuN-positive cells in 0.250 mm
2
areas adjacent to wound zone. Data is
represented as average of 3 experiments +/- SEM. *(p<0.0005 from vehicle and E2
in respective co-culture model). **(p<0.0001 from vehicle and E2 in respective co-
culture model).

Since the NeuN data suggests that neurons may be damaged or lost in
wounded mixed glial-neuron co-cultures in the presence of P4, neurons were also
stained with antibodies to neuron-specific enolase (NSE; DAKO Corporation,
72
Carpinteria, CA) and active caspase-3 (Csp-3; Cell Signaling Technology, Danvers,
MA) to determine if the hormone treatments affected neuron numbers or neuronal
apoptosis in the mixed glia-neuron co-cultures. The active caspase-3 stain did not
appear to be specific since most of the cells appeared were labeled by the antibody
(data not shown). Numbers of all NSE-positive cells were counted adjacent to the
wound site. Total NSE cells did not differ between treatment groups (Figure 5.8).
Therefore, P4 does not appear to affect neuron numbers, but it may damage or alter
neurons which may result in a loss of NeuN immunoreactivity.

Figure 5.8 Hormone treatments do not affect neuron numbers (NSE-positive cells)
in wounded mixed glial-neuron co-cultures in 0.250 mm
2
areas adjacent to wound
zone. Data is represented as average of 3 experiments +/- SEM.

Effect of hormone treatments on microglial cells in mixed glial-neuron co-cultures
To determine whether E2, P4, or E2+P4 affect microglial cells in our co-
culture system, microglial cells were labeled with OX42 in wounded mixed glial-
neuron co-cultures. Astrocyte-neuron co-cultures contain few microglial cells so
only mixed glial-neuron co-cultures wer examined. Numbers of OX42-positive cells
were normalized to total glial cells (OX42+GFAP) in 0.250 mm
2
areas adjacent to
73
wound zones to determine percentage of microglial cells in the field. These fields
were similar to those where neurite sprouting was observed and where neurons were
counted. E2 reduced the percentage of OX42-positive cells by 70% (Figure 5.9B).
P4 had no effect on its own compared to vehicle. P4 also blocked E2-suppression of
OX42-postive cells (Figure 5.9). These trends appear to be inversely reciprocal with
neurite outgrowth trends (Figure 5.6A and B), suggesting that microglial cells may
mediate P4 inhibition of E2-induced neurite outgrowth.

Figure 5.9 OX42-positive microglial cells in wounded mixed glial-neuron co-
cultures is decreased in E2-treated co-cultures. A, Representative micrographs of
mixed glial-neuron co-cultures stained with antibodies to GFAP (red) and OX42
(green). Scale bar represents 100 micrometers. Asterisks indicate wound zone. B,
Percent of OX42-positive microglia adjacent to wound zone. Data is represented as
the average percentage of OX42 cells/(OX42+GFAP) cells from 3 experiments +/-
SEM. #(p<0.0001 from other treatments).
74

P4 inhibition of E2-induced neurite outgrowth is partially reversed by a
progesterone receptor antagonist
The progesterone receptor (PR) antagonist ORG31710 was used to determine
if the antagonism of P4 on E2-induced neurite outgrowth occurs via the progesterone
receptor. ORG31710 binds PR to prevent P4 from binding PR [Sanchez-Criado et
al., 2000; Thomas et al., 2006]. ORG31710 alone or in combination with P4 did not
affect neurite number or neurite length compared to vehicle (Figure 5.10). In the
presence of E2, ORG31710 reduced numbers of neurites sprouting into the wound by
20%, but had no effect on neurite length compared to E2 treatment alone (Figure
5.10). Other antiprogestins such as RU486 can act as noncompetitive inhibitors of
estrogen receptors [Hodgen et al., 1994; McDonnell and Goldman, 1994]. When
both E2 and P4 were present, ORG31710 partially blocked the P4 inhibition of E2-
induced neurite outgrowth. Neurite lengths were similar to the E2 and
E2+ORG31710 treatments (Figure 5.10B), however, E2+P4+ORG31710 decreased
neurite numbers by 40% compared to the E2 treatment group (Figure 5.10A).
Similar trends on neurite number and length were observed with another
progesterone receptor antagonist, RU486 (data not shown).
75

Figure 5.10 The progesterone receptor antagonist ORG31710 partially reverses P4
inhibition of E2-induced neurite outgrowth in mixed glial-neuron co-cultures. (A)
ORG31710 increases numbers of neurites extending into the wound zone in E2+P4
treated co-cultures. Data is represented as the average of 3 experiments +/- SEM.
(B) ORG31710 does not affect E2-induction of longer neurites. Data is represented
as the combined histogram of neurites collected from 3 experiments. *(p<0.0001
from other treatments). **(p<0.0001 from vehicle, E2+P4, ORG, and ORG+P4 in
neurite category).

DISCUSSION
Progesterone (P4) attenuates the beneficial effects of 17 β-estradiol (E2) on
lesion-induced neurite sprouting and glial activation in vivo and in vitro. Although
total neuron numbers in vitro were unaffected by the hormones, P4 in the presence of
activated microglia may alter neurons. However, in the absence of microglia, P4
increases neurite outgrowth and synergizes with E2 to increase neurite outgrowth in
neurons co-cultured with enriched astrocytes.
We used the entorhinal cortex lesioning model (ECL), which mimics damage
observed in Alzheimer disease, to study the effects of progesterone on the brain and
compensatory neurite sprouting in vivo. Results are been summarized in Tables 5.3
and 5.4. E2 induces compensatory neurite sprouting and attenuates glial activation
76
after brain lesions (Table 5.3). However, while P4 has a modest effect on neurite
sprouting and no effect on astroglial GFAP expression, P4 may increase lesion-
induced microglial activation (Table 5.3). In addition, P4 antagonizes E2-induced
compensatory sprouting sprouting and blocks E2-attenuation of lesion-induced glial
activation (Table 5.3).
Table 5.3 In Vivo Data from the Ipsilateral Hippocampus
E2

P4 E2+P4

Holmes Fiber
Stain (Neuron Processes)
+100%* +20%
#
+35%
#

GFAP (Astrocytes) -25% ** 0 0
OX6 (Microglia) -30% ^ +30%
#
-15%

Table 5.3 Summary of in vivo data from ipsilateral (lesioned) hippocampus. Data is
shown as relative percent change from sham pellet control. *(p<0.0001 from other
treatments). **(p<0.01 from other treatments). #(p<0.05 from other treatments).
^(p<0.05 from sham pellet control).

As a control for lesion-induced changes, the unlesioned contralateral
hippocampus shows only a modest increase in neurite sprouting in response to E2
(Table 5.4). E2 also reduced astroglial GFAP and OX6 immunoreactivity (Table
5.4). In contrast to the lesioned hippocampus, P4 or E2+P4 had no effect on neurite
sprouting or glial activation (Table 5.4).
77

Table 5.4 In Vivo Data from the Contralateral Hippocampus
E2

P4 E2+P4

Holmes Fiber
Stain (Neuron Processes)
+20%** 0 0
GFAP (Astrocytes) -30% * 0 0
OX6 (Microglia) -30%
#
0 0

Table 5.4 Summary of in vivo data from contralateral (unlesioned) hippocampus.
Data is shown as relative percent change from sham pellet control. *(p<0.005 from
other treatments). **(p<0.05 from other treatments). #(p<0.05 from sham pellet
control).

One potential in vivo mechanism that the hormones may influence are
synaptic proteins. Although estradiol increased levels of the synaptic proteins
syntaxin, synaptophysin, and spinophilin in the CA1 regions of female rhesus
macaques, P4 had no effect on these proteins compared to spayed controls [Choi et
al., 2003]. P4 has also been documented to antagonize E2 responses. Treatment
with estradiol and P4 in rats after 18 hours resulted in spine densities comparable to
6 day ovariectomized rats [Woolley and McEwen, 1993]. P4 also antagonizes E2-
induction of synaptic proteins [Choi et al., 2003] and neuroprotection [Rosario et al.,
2006].
The in vitro “wounding-in-a-dish” model was used to study mechanisms
underlying P4 actions on neurons and glia. In co-cultures of mixed glia and neurons,
P4 antagonizes E2-induction of neurite outgrowth (neurite number and neurite
length) which supports our in vivo results. However, P4 and E2+P4 treatments
induce a modest increase in neurites greater than 20 µm (Figure 5.6A), which is
78
very similar to the in vivo Holmes data (Figure 5.2B and Table 5.3). This suggests
that the in vitro mixed glia-neuron co-culture model is a good model to study the
complex interactions between microglia, astrocytes, and neurons in the ECL model.
Progesterone receptors antagonists ORG31710 and RU486 reverse P4 antagonism of
E2-induced neurite outgrowth, therefore, this antagonism is dependent on the
progesterone receptor. P4 antagonism of neurite outgrowth is accompanied by
increased numbers of OX42-positive microglial cells adjacent to the wound zone
which may affect neurons.
The neuron-specific markers, NeuN and NSE, were used to determine if
reduced neurite sprouting in mixed glial-neuron co-cultures in the presence of P4 are
due to reduced neuron survival or numbers. NeuN has been documented to be
specific to mature neurons [Sarnat et al., 1998] while NSE expression has been
detected in the early stages of the neuronal differentiation process [Schmechel et al.,
1980]. In mixed glial-neuron co-cultures, P4 decreases NeuN-positive neurons, but
has no effect on NSE-positive neurons. These two neuronal markers have produced
contrasting results in aging rat spinal cord. NeuN immunoreactivity decreased in 32
month old rats while NSE immunoreactivity remained constant [Portiansky et al.,
2006]. Although this significance of this finding is not known, it is clear from this
report and others [Uval-Cevic et al., 2004] that loss of NeuN immunoreactivity does
not necessarily represent loss or death of neurons. Therefore, it is possible that P4
may be affecting the integrity of the neurons or inhibiting E2-induced
neuroprotection.
79
In contrast to the mixed glial/neuron co-cultures, P4 alone also increases
neurite sprouts in the wound zone in enriched astrocyte-neuron co-cultures. In
addition, E2 and P4 synergize to increase neurite sprouting. This shows that
astrocytes and microglial cells have different responses to P4. It is also possible that
P4 may alter the interaction between astrocytes and microglial cells. Although both
E2 and P4 may support astroglial-support of neurite sprouting, lesion-induced
microglial activation may alter astrocytes so they are less able to support neurite
sprouting. Since E2 attenuates lesion-induced microglial activation in vivo and in
vitro, astroglial-supported neurite sprouting is not inhibited. This further strengthens
our hypothesis that microglial cells mediate inhibitory responses of P4 on neurite
outgrowth since the enriched astrocytes-neuron co-cultures support neurite
outgrowth in response to P4 when microglial cells are removed.
Increased numbers of microglia positive for OX42 (complement receptor
type-3; CR3) were detected adjacent to wound zones in wounded mixed glial-neuron
co-cultures. CR3 mediates NO production by activated microglial cells [Chien et al.,
2005]. Therefore, one possible mechanism by which microglial cells are inhibiting
neurite outgrowth and possibly damaging cells in the in vitro culture is by the
production of nitric oxide (NO). NO reacts with superoxide to form peroxynitrite
which is toxic to neurons [Xie et al., 2002]. To determine if P4 affects NO
production, media from wounded mixed glial-neuron co-cultures were analyzed with
the Griess reagent reaction. E2 and P4 did not consistently alter NO secretion in
80
wounded co-cultures (data not shown). Future studies may determine if NO is
involved in P4 inhibition of E2-induced neurite outgrowth.
E2-induced synaptic sprouting involves glial secretion of apolipoprotein E
(ApoE) [Stone et al., 1998]. To determine if P4 inhibition of E2-induced neurite
sprouting involved this pathway, media from wounded glial-neuron co-cultures were
examined for ApoE secretion using western blot analysis. Although ApoE protein
was induced in wounded co-cultures compared to unwounded co-cultures, E2 and P4
did not alter ApoE secretion in wounded co-cultures (data not shown). Secretion of
ApoJ, another apolipoprotein that may be involved in neurite sprouting after lesions
[Lampert-Etchells et al., 1991; Poirier et al., 1991], was also unaltered by E2 and P4
treatments in wounded co-cultures (data not shown). Since neonatal glial cells were
used in our co-culture system, it is possible that the lack of ApoE and ApoJ response
to E2 and P4 may be due to the lack of a glial function that will be acquired after the
postnatal stage. In a previous study, ApoE and ApoJ secretion from neonatal mixed
glia were unaffected by the proinflammatory cytokines IL-1 β, IL-6 or TNF- α,
however mixed glia originated from adults responded to cytokine treatments [Patel et
al., 2004]. Future studies with adult glia will determine if E2 and P4 will affect
ApoE and ApoJ secretion in wounded glial-neuron co-cultures.
Since synaptic plasticity underlies cognitive function, our results suggest that
progesterone may not be beneficial to cognition. Results from the literature have
been mixed. In the Morris water maze (MWM), P4 has a positive influence
performance [Galea et al., 2000; Markham et al., 2002] or no effect [Chesler and
81
Juraska 2000; El-Bakri et al., 2004]. Results of estrogen plus progesterone are also
mixed. Chesler and Juraska have reported that hormone combination impairs MWM
performance compared to ovariectomized females [Chesler and Juraska 2000],
however Markham et al. reported that estrogen plus progesterone improves MWM
acquisition compared to ovariectomized controls [Markham et al., 2002]. Although
both studies administered the hormones prior to training, the studies differed in the
route of administration and time the hormones were given prior to MWM training.
Without knowing the circulating concentrations of the hormones, it is difficult to
make further meaningful comparisons between studies.
Our results demonstrate the complex nature of steroid effects on different
brain cells. Therefore, it is necessary to understand how glia and neurons respond to
estrogen and progesterone at different physiological ranges to explain some of the
discrepancies between the reports. Steroid hormones clearly alter brain physiology
and future studies will determine how this will affect cognition.
82
CHAPTER 6
SUMMARY

Increased neuroinflammation and decreased circulating ovarian hormones
may play a role in age-related increase of cognitive decline. Glial cells, such as
astrocytes and microglia, actively support neuronal functions. Studies presented here
show that glial cells mediate neuroinflammation that consequently affects neurite
sprouting and outgrowth.
Normal brain aging is accompanied by increased inflammation and oxidative
damage. Previous studies using various glial molecular markers have shown
increases in inflammatory genes [Morgan et al., 1999] and oxidative damage [Sloane
et al., 1999]. Using the microglial marker macrosialin (CD68), we can show that
increases in inflammation are a cause of oxidative damage during normal aging. In
addition, microglial cells are a major contribuitor of inflammation and oxidative
damage during normal brain aging. The dietary intervention caloric restriction,
which also increases lifespan, attenuates neuroinflammation and oxidative damage.
Attenuation of macrosialin in caloric restricted aged mice confirmed this
observation.
Mixed glial cells originated from neonatal animals are inherently different
from adult glial cells. Although this may appear obvious, many in vitro studies using
glial cultures are derived from neonatal animals. As we have shown, ApoJ secretion
is much higher in neonatal mixed glial cultures than in adult mixed glial cultures. In
83
addition, neonatal mixed glial cultures did not significantly respond to cytokine
treatment whereas the pro-inflammation cytokines IL-6, IL-1 β, and TNF- α
influenced ApoE and ApoJ secretions from adult cultures. The higher basal ApoJ
secretion level and blunted response to cytokines in neonatal cultures may be due to
its stage in development when there is a significant loss of neuronal cells and
neurons forming synapses with appropriate targets [Garner et al., 2006; Kamiyama et
al., 2006; Zecevic et al., 1989]. Removal of neurons during development involves
microglial cells [Marin-Teva et al., 2004]. Although the adult brain has synaptic
plasticity, this process is less prevalent in the absence of injury or disease.
ApoE and ApoJ are both upregulated after injury and in neurodegenerative
disease. In vivo, both ApoE and ApoJ increase in the aged brain [Morgan et al.,
1999]. Despite this, ApoE and ApoJ are not regulated by the same pathway. IL-6
induced ApoE secretion from adult mixed glial cultures while ApoJ secretion was
induced by IL-1 β or TNF- α. We hypothesize that ApoE secretion is regulated by the
Jak/STAT pathway, while ApoJ is controlled by NF-kB.
The regulation of these two apolipoproteins is important to neurite outgrowth.
In an in vitro mixed glia-neuron co-culture model, an inflammatory stimulus,
lipopolysaccharide (LPS) decreased initial neurite sprouting. Results were most
dramatic at early time points (1 and 2 days after co-cultures were established). There
may be a gradual recovery by 5 days that progress further at later time points. This
model shows that inflammation activates glial cells to create an environment
unfavorable to neurite sprouting and accelerates neurodegeneration and age-related
84
processes. Since LPS has no effect on neurons in the absence of glia, it is the glial
cells that mediate neurite sprouting.
Activation of glial cells also resulted in the production of nitric oxide,
increased ApoJ secretion, but decreased ApoE secretion, which are all important
mechanisms by which glial cells are interacting with neurons. The production of
nitric oxide is used as an indicator of glial activation, however nitric oxide is also
neurotoxic to neurons via the production of peroxynitrite [Xie et al., 2002]. ApoJ
also plays an important role in mediating inflammation since our lab has previously
shown that ApoJ activates microglial cells to produce nitric oxide which results in
neurotoxicity [Xie et al., 2005]. In addition to modulating inflammation, ApoE has a
role in the promotion of neurite outgrowth [Stone et al., 1998]. Therefore, LPS-
induction nitric oxide production and ApoJ secretion combined with suppression of
ApoE creates an environment that is detrimental to neurons and inhibitory to support
neurite sprouting.
One possible pharmacological intervention for modulating age-related
neuroinflammation is hormone replacement therapy (HRT). Unopposed conjugated
equine estrogens are administed to women who have had their uterus removed. In
women with an intact uterus, a progestin such as medroxyprogesterone acetate
(MPA), is typically added to the regimen to prevent abnormal growth of the
endometrial lining by estrogen. Although the current literature conflicts about
whether HRT (CEE or CEE plus progestin) is beneficial for maintaining cognitive
85
function, reducing the risk and damage of diseases such as Alzheimer disease (AD),
it is clear that estrogens and progestins affect the brain.
This underscores the need to understand how these hormones affect the
different cell types of the brain. Using an in vivo entorhinal cortex lesioning model
in ovariectomized female rats which mimics damage from AD, we have confirmed
that replacement with 17beta-estradiol (E2) was beneficial in promoting
compensatory sprouting into the denervated hippocampal molecular layer, reducing
microglial activation, and possibly reducing astrogliosis. Progesterone (P4) alone
had effect on compensatory sprouting, reactive astrocytes, or microglial activation
compared to placebo animals. Since progestins are coadministered with estrogen in
HRT, the interaction between E2 and P4 is an important considering. In our model,
P4 attenuated E2-induced compensatory sprouting and reversed E2-reduction of
astrogliosis and microglial activation.
The in vitro “wound-in-a-dish” model with co-cultures of enriched astrocytes
(~3% microglial contamination) or mixed glia (30% microglia) and neurons was
used to study mechanisms of E2 and P4 actions on glial support of support of neurite
outgrowth. E2 promoted glial support of neurite outgrowth (assessed by neurite
number and neurite length) in neurons co-cultured with enriched astrocytes or mixed
glia. However, the effects of P4 differed between astrocyte and mixed glial co-
cultures. P4 increased neurite outgrowth in astrocyte-neuron co-cultures, but had no
effect on mixed glial-neuron co-cultures. In addition, while P4 synergized with E2
to increase neurite outgrowth in astrocyte-neuron co-cultures, P4 antagonized E2 in
86
mixed glial-neuron co-cultures. This antagonism was dependent on the progesterone
receptor.
The underlying mechanisms of P4 antagonism of E2 in the mixed glial co-
cultures was studied using two neuronal markers, neuronal nuclei (NeuN) and
neuron-specific enolase (NSE). The mature neuronal marker NeuN decreased in the
presence of P4 while the earlier neuronal differentiation marker NSE did not change.
This could represent changes in neuronal damage, neuroprotection, neurogenesis, or
neural dedifferentiation. Although E2 decreased microglial activation adjacent to the
wound site, P4 or E2+P4 had no effect on the number of activated microglial cells
compared to vehicle. Therefore, it appears that microglial activation decreases
neurite outgrowth in the “wounding-in-a-dish” co-culture model.
This study suggests that although E2 may be beneficial in promoting
compensatory sprouting and neurite outgrowth after a lesion, the addition of P4
attenuates this benefit. Although we were able to show that circulating E2 and P4
levels were physiological, many in vivo studies do not measure or present their
hormone levels. This makes it difficult to interpret how the different studies relate to
each other since one cannot determine whether hormone levels are physiological or
whether the contradictory data is due to hormone variations between the studies.
It is clear that estrogen and progesterone have powerful effects on glial cells
and neurons. Although this study has important implications for post-menopausal
women who are considering HRT, this study also affects younger women. Many
women use birth control which contain a progestin. Some of the progestins (Table
87
6.1) used in birth control are chemically different from progesterone or MPA so one
would expect that these progestins would have different effects on the brain
[Stanczyk, 2003]. Further studies are needed to determine how these progestins will
affect the brain.

Table 6.1 Progestins in Birth Control
Progestin Structural
Relationship
Progestin
Classification
Alesse 28 Levonorgestrel Testosterone 13-Ethylgonane
Loestrin Norethindrone
Acetate
Testosterone Estrane
Depo-Provera Medroxyprogesterone
Acetate (MPA)
Progesterone Acetylated
pregnane
Ortho Tri-Cyclen Norgestimate Testosterone 13-Ethylgonane
Yasmin Drospirenone Testosterone Non-ethinylated

Table 6.1 Progestins in common birth control methods. Progestins could be
structurally related to progesterone or testosterone [Stanczyk, 2003]. MPA is
considered an aceylated pregnane since it a methyl group at carbon 10 and is
acetylated. Subdivisions of progestins related to testosterone were based on presence
of ethyl group on carbon 13 (13-ethylgonane vs. non-ethinylated) and whether
ethinylated progestins were estranes (18 carbons) [Stanczyk, 2003]. Formulation of
birth control obtained from www.pharmacy-and-drugs.com/Birth_control/.

These studies suggest that age-related neuroinflammation and oxidative
damage can be influenced by diet and hormones. Modulating these responses in
glial cells affects the health of neurons and their ability to produce new extensions
for forming synapses. Future studies will determine how to alter diet and
pharmacological treatment to prevent or delay cognitive decline associated with age
or neurodegenerative disease.
88
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APPENDIX
Anti-inflammatory mechanisms of dietary restriction in slowing aging processes
Todd E. Morgan, Angela M. Wong, and Caleb E. Finch
(Published in Interdisciplinary Topics in Gerontology, 35:83-97)

INTRODUCTION
Dietary restriction (DR) remains the most powerful and general manipulation
of aging processes in laboratory animals. Evidence is now overwhelming that DR
increases life span by slowing the Gompertz mortality rate acceleration. The
Gompertz analysis of DR was first made by Benjamin Berg in 1976 [1]. This
fundamental effect of DR has been amply verified [2] (p 508) [3].
Corresponding to slowed mortality rates; most spontaneous degenerative
changes in aging are attenuated. In rodents, the age-related increases of tumors and
organ-specific pathology is delayed by DR, according to the genotype [2, 4-7]. In the
widely used F344 rats, for example, chronic renal disease, which may be the major
cause of morbidity, is strikingly reduced [8]. Cardiomyopathy of F344 rats is also
strongly associated with the severity of kidney degeneration, but the mechanisms
may be different [9]. Nonetheless, we must confront the puzzle in F344 rats that
about 25% of old DR rats have no gross organ pathology at death [8]. We suggest
the possibility of metabolic instability during DR below in lesion-free aging rodents.
114

DR attenuates neuroinflammatory aspects of aging
The first indication that DR is neuroprotective for aging came from a study
25 years ago [10]. In some rodent colonies, hind limb paralysis becomes increasingly
common during aging in association with degeneration of spinal motor neurons
(radiculoneuropathy) [10, 11]. The degeneration of myelin sheaths in spinal roots
arises after sporadic axonal atrophy and is associated with segmental demyelination
and local ballooning [12-14]. Hind-limb paralysis was markedly attenuated by DR
[10, 11].
Hindlimb paralysis varies widely between colonies and is unfamiliar to
current researchers of aging. The greatest incidence reported, 100%, was observed in
colonies before the era of modern husbandry (SPF, specific-pathogen free). In the
NIA contract colony at Charles River Laboratory, 1978-1983 rats (9 genotypes, both
sexes) had a 25% incidence, mean age at lesion of 31 months; the incidence in mice
(12 genotypes), was <0.1% [15]. These major differences are puzzling and not easily
attributed to improved husbandry and health. Early rodent colonies before 1970 often
carried a much higher load of infections than the present SPF colonies.
While most attention has been given to the biochemical, metabolic, and
genomic effects of DR, evidence is growing for the importance of system-wide anti-
inflammatory effects of DR in attenuating aging [16-18]. Our laboratory is focused
on neuroinflammatory changes of aging in rodents, primates, and humans. These
generalized aging changes arise in the absence of specific neurodegeneration [16,
115
17]. In aging rodents, the main brain aging changes are glial activation (microglia
and astrocytes)
1
[17] and synaptic atrophy [19-24]. These changes are progressive
during middle-age into old age and arise in the absence of disease. The type and
extent of change are selective and differ extensively between even closely connected
brain systems. The opposing glial and synaptic changes span a range of about 50%,
but are much larger than changes in cell number. In fact, several exacting studies
have looked for but did not detect age changes in the total numbers of neurons [25]
or glia [26]. Thus, in aging rodent and perhaps in humans, the main brain aging
changes represent a type of plasticity that remodels cell cytoarchitectonic
relationships without cell death. DR has a remarkable ability to attenuate these
changes. White matter myelinated tracts are a robust example of the plasticity of
neuroinflammatory aging.

1. Age-related microglial activation: white matter degeneration
Macroscopically, magnetic resonance imaging (MRI) studies on aging
humans and monkeys show subtle structural changes in the corpus callosum,
striatum and other white matter-rich tracts [27-29]. These changes may be caused by
the focal degeneration of myelin sheaths and differ by brain region. The later-
myelinated regions are more susceptible to demyelination during normal aging and
Alzheimer disease [30].

1
Microglia are bone-marrow derived monocytes which are constantly repopulated in
adult brains. Astrocytes are of neural crest origins and share the same stem cell
precursors as neurons.
116
White matter aging is accompanied by increased microglial activation [31,
32], but cause and effect are unclear. Aging rodent models show robust increases in
markers of microglial activation, e.g. CR3 (complement receptor) and MHC class II
antigens (antigen presentation by macrophages) [33, 34]. These changes are
attenuated by DR [33]. Most recently, we found that the scavenger receptor
macrosialin (CD68), a member of the lysosomal/endosomal-associated membrane
glycoprotein family, shows the greatest age-related increase in the corpus callosum
of C57BL/6NNia mice; again, this is attenuated by DR, Figure 1 [35]. Because
macrosialin (CD68) is increased in peripheral macrophages by oxidized lipids
(oxLDL) [36] and because oxidized lipids generally promote inflammation [37, 38],
we hypothesize that the oxidation of white matter lipids is a factor in microglia
activation. In fact, we showed that oxLDLs induced CD68 in BV-2 microglial cells
[35]. Moreover, CD68 is induced by inflammatory stimuli (lipopolysaccharide plus
interferon-gamma) in BV-2 cells [35]. Therefore, CD68 serves as an inflammatory
marker as well as an indicator of oxidative damage during normal brain aging.
117

Fig. 1A. Fig. 1B. Fig. 1C.

Figure 1. The age-related increase of macrosialin (CD68) expression is attenuated
by DR. Macrosialin immunoreactivity in the corpus callosum and corticostriatal
bundles (insets) of (A) 4-month AL, (B) 24-month AL, and (C) 24-month CR
C57BL/6NNia mice. Arrow identifies macrosialin immunostaining at periphery of a
corticostriatal bundle in 24-month AL mice. Bars = 100 µm for micrographs, 30 µm
for insets. Reprinted from Neuroscience Letters, Epub, Wong et al., “Macrosialin
increases during normal brain aging are attenuated by caloric restriction” (2005),
with permission from Elsevier.

Because DR clearly attenuates age-related increases in inflammatory genes
such as CD68, CR3, and MHC class II antigens, we hypothesize that DR will protect
against age-related demyelinating events. An ongoing study of DR in rhesus
monkeys has not given definitive information for technical reasons. After 11-13
years of DR, middle-aged (< 24 years old) and old monkeys (>24 years old) had
smaller putamen volumes than ad lib fed animals [39]. However, there was no initial
MRI data to establish the baseline (before or at the beginning of DR). Thus, it is
unresolved if the smaller putamen volumes in DR animals resulted from DR, or if the
volume differences were present at the beginning of the study.
118

2. Age-related astrocytic activation: GFAP
Astrocytes are an important source of neurotrophic factors, axonal guidance
molecules, and extracellular matrix molecules crucial for neuron survival and
sprouting. In response to injury or disease, astrocytes take on an activated phenotype
that is characterized by cell hypertrophy and upregulation of the intermediate
filament proteins, glial fibrillary acid protein (GFAP) and vimentin, as well as,
inflammatory mediators and extracellular matrix molecules [40, 41]. However during
normal aging astrocytes become activated with concomitant increases of GFAP &
vimentin in the absence of overt pathology [42-44]. This age-related astrocytic
activation [45, 46] contributes to age-related increased inflammatory and oxidative
damage [44, 47], decreased neurogenesis [48] and synaptic atrophy [19].
We are investigating the hypothesis that the increase of GFAP expression is a
primary cause in synaptic atrophy and impaired synaptogenesis during normal aging
[46]. We have developed a heterochronic cell culture model to test this hypothesis. In
brief, test neurons (E18 cortex) are seeded on monolayers of primary cultures of
astrocytes from young adult or aging rat cerebral cortex. The old-derived astrocytes
retain the high GFAP per cell [49] as observed in vivo [33]. Moreover, the E18
neurite outgrow poorly on old-derived astrocytes. These age impairments in
neurotrophic support are rapidly reversed by down-regulating GFAP by siRNA [46].
The mechanism involves an inverse relationship between GFAP expression and
secretion of laminin, a critical component of the extracellular matrix that guides
119
neurite outgrowth. Additional support for the critical role of GFAP comes from
studies on mice lacking both GFAP and vimentin which have improved synaptic
regeneration and increased neurogenesis [50, 51].
Just as age-related microglial activation is reduced by DR (discussed above),
DR is also effective at attenuating many of the genotypic and phenotypic changes
that astrocytes undergo during aging. The age-related increase of GFAP is attenuated
by DR [43, 44, 52] and this occurs at the transcriptional level [33, 53]. Microarray
profiling confirmed the effects of DR on GFAP [17, 44]. Although neuropathologists
have long used GFAP as a marker of neurodegeneration, our work clearly shows that
the age increase of GFAP arises in the absence of neuron cell death and may be an
upstream factor in synaptic atrophy during aging. Because of the concurrent
activation of microglial inflammatory markers, we provisionally consider that GFAP
is embedded in a neuroinflammatory network. The beneficial effect of DR on glial
activation may underlie DR’s ability to attenuate age-related declines in synaptic
plasticity and neurogenesis [54-58]. Ongoing studies are evaluating if DR improves
the neurotrophic support of aging glia.

3. Age-related neurodegenerative disease: experimental rodent models.
DR also protects against neurodegenerative processes in experimental rodent
models. For example, DR protects neurons from many toxins, including MPTP [59],
kainic acid [60], 3-nitropropionic acid & malonate [60, 61]. AD-like changes do not
arise in aging rodents, possibly because the rodent ß-peptide has several amino acid
120
substitutions that decrease its aggregation into fibrillar amyloids that are
characteristic of AD [62]. However, mice carrying human transgenes for early onset
familial AD develop fibrillar amyloids and various other specific AD-like
neuropathological changes during aging. We recently showed that DR attenuated
brain deposits of brain amyloid by 50% within the short time of several months
[63](Fig. 2A). These changes were accompanied by a reduction in GFAP in
astrocytes surrounding the plaque (Figure 2B). We demonstrated these beneficial
effects of DR in two transgenic mouse models of AD, APPswe/ind & APP+PS1
[63]. These effects of DR also extend to a third genotype, Tg2576 [64].
121
Fig. 2A Fig. 2B

Figure 2. DR reduces Aß number and Aß-associated astrocyte activation relative to
ad libitum (AL) feeding. (A) Plaque size and total Aß plaques were reduced by DR
in APPswe/ind, *P<0.05, N=7-8). (B) Sholl analysis of concentric rings around Aß
plaques (inset) showed reduced GFAP immunoreactivity nearest to plaques in DR
vs. AL (*P<0.05). Reprinted from Neurobiology of Aging, Volume 26, Patel et al.,
“Caloric restriction attenuates Abeta-deposition in Alzheimer transgenic models”,
p997 & p998, (2005), with permission from Elsevier.

Low energy diets in humans are being considered as an approach to lowering
AD risk, because in retrospective studies, AD victims tended to have higher calorie
intake [65, 66]. Of course, it is much harder to establish causality of diet in humans,
because individuals who adopt special diets also often pursue other health promoting
activities, such as exercise which may protect against cognitive declines in normal
aging [67, 68].
Another example of DR providing age-related neuroprotective activities is in
the experimental model of retinal ischemia/reperfusion [69]. As observed in cortical
& hippocampal regions (see above), microglia and astrocytes become progressively
activated in the aged retina [69]. Further glial activation occurs when the aged retina
is subjected to ischemia/reperfusion with concomitant neuronal damage. In this
model of ischemia/reperfusion with individual eyes, DR attenuated retinal glial
122
activation and neuronal damage [69]. In fact, these authors suggest that the beneficial
effects of DR are directly related to its effect on glial activation supporting the
hypothesis that the anti-inflammatory actions of DR on glia may mediate
neuroprotection.

DR attenuates inflammatory processes
1. Microarray profiling highlights DR anti-inflammatory effects
The broad scope of inflammatory gene expression during brain aging has
become clear through the numerous publications utilizing microarray gene
expression profiling [44, 70, 71]. These studies showed that inflammation-related
genes increased during aging. Importantly, DR attenuated the age-related increase in
inflammatory genes [44]. In fact, DR prevented the age-related increased expression
of 65% of those genes involved in the inflammatory response in the neocortex [44]
suggesting this is a primary mechanism underlying the beneficial effect DR has on
brain aging processes.

2. Suppression of inflammation in acute DR
Inflammatory responses are attenuated by DR throughout the body [17, 72].
We begin with examples from skin. In the classic pharmacologic model of foot-pad
edema induced by subcutaneous injection, DR shortened the inflammatory responses
in young mice on DR for 8 weeks [73]. In clinical studies, dermatitis was also
improved by eight weeks on a low energy diet with micronutrient supplements. All
123
patients responded to some degree, with the reductions of edema, oozing, and skin
sloughing (excoriation) being correlated with weight loss [74]. Inhibition of
keratinocyte proliferation, an observed effect of DR in young mice, e.g. [75], may
contribute to the reduced excoriation.
In humans, serum C-reactive protein (CRP) was 80% lower in a self-selected
group that has maintained DR for eight years [76]. CRP is an acute phase protein
secreted by liver in humans [77], which is an important host defense molecule by
binding to gram-negative bacteria and enhancing their clearance by phagocytozing
macrophages. However CRP also has major importance in vascular disease as a risk
indicator and for its potential direct role in lipid accumulations by macrophages
(foam cells) in atheromas. Serum CRP is elevated during obesity and, not
surprisingly, short-term weight reduction decreased serum CRP by 30% [78, 79].
Here we confront the complexities of weight reduction. DR could enhance the host
defense by lowering blood glucose [80-82], yet DR diminishes CRP and possibly
other defenses.
Changes in gene expression in liver during short-term DR (3-30 weeks) have
been profiled by microarrays in several studies [83-87]. Agreement is emerging,
despite differences in the choice of rodent genotypes, duration of DR, and
microarray technologies. Short-term DR induces and represses many mRNAs in liver
that mediate increased gluconeogenesis, increased protein and fatty acid catabolism,
and decreased synthesis of cholesterol, fatty acids, and triglycerides [84, 87]. The
Krebs’ cycle (tricarboxylic acid cycle) drives these changes, with increased shunting
124
of pyruvate to oxaloacetate in the liver by increased activity of pyruvate carboxylase
[88]. The increased oxaloacetate feeds into gluconeogenesis after conversion by
malate dehydrogenase, which is also increased by DR. Besides transcriptional
changes in these genes, levels of activity in some enzymes are allosterically
regulated, e.g., pyruvate carboxylase is activated by acetyl-CoA, which is increased
by the ß-oxidation of fatty acids liberated during lipolysis. Acute phase response
mRNAs are also decreased, including serum amyloid A4 and several complement
system factors (mannose binding lectin, C4 binding protein, C9) [84]. DNA repair is
up-regulated (Rad511), as are CYP450 family genes that mediate detoxification and
decrease DNA damage. Again, there is impressive overlap of genes associated with
lipid metabolism and vascular disease.
Overall, these 50-100 mRNA changes are a small subset (<1%) of all the
genes active in the liver. The race is on to find transcription factors that are shared
key regulators of these gene subsets. The effects of DR on many diseases of aging
with inflammatory components gives a basis to look for transcription factors that
could modulate inflammatory gene subsets implicated in AD, cancer, diabetes, and
vascular disease [17, 72]. In liver, Corton and colleagues [84] have shown that the
transcription factors PPAR, LXR and RXR, which regulate many genes during DR,
also have major roles in inflammation. Additional experimental models include
PPAR knockout mice and drugs antagonists, which induce mRNA changes that
overlap with DR to some extent (see below).

125
Mechanisms underlying anti-inflammatory actions of DR
1. Glucocorticoids
DR increases blood glucocorticoids by 20% or more [18, 73, 89]. The
increased glucocorticoids are a homeostatic response to increase the catabolism of
fatty acids for energy (gluconeogenesis), while decreasing the synthesis of fatty acids
and cholesterol. If the energy deficit is prolonged, protein catabolism is also
increased. DR also decreases tissue content of oxidatively damaged proteins and
lipids, which are always present with enough food intake and which accumulate
during aging. Importantly, glucocorticoids have broad anti-inflammatory effects are
mediated by the direct interaction between the glucocorticoid receptor and the
transactivation domain of NF-kappaB which serves as a key transcription factor in
the regulation of inflammation [90, 91]. Because chronically elevated
glucocorticoids are also broadly associated with neuronal damage and neuronal
death, it is paradoxic that DR is neuroprotective [18].

2. Glucose and advanced glycation end products (AGE)
DR lowers blood glucose by about 10-15%. Blood glucose levels directly
influence the formation of oxidation products, as was outlined two decades ago in
Cerami’s hypothesis of glucose as a mediator of aging [92]. Glucose and other
reducing sugars react spontaneously (nonenzymatically) with free amino groups of
proteins (e.g. -NH
2
of lysine) to form an initial ‘‘glycation’’ product by the Amadori
reaction, which is assayed as furosine. Then, Amadori glycation products become
126
oxidized to ‘‘glycoxidation products’’, assayed as pentosidine, which are also
referred to as advanced glycation end-products (AGEs) [93]. DR inhibits
glycoxidation during aging in rodent skin, whereas diabetes and end-stage renal
disease accelerate glycoxidation [93-95].
AGE adducts are recognized by macrophage scavenger receptors, the
‘RAGE’ (receptors for advanced glycation endproducts) of monocytes
(macrophages, microglia) and other cells. RAGEs are also activated by the amyloid
ß-peptide and other stress-associated proteins (S100/calgranulins). A working
hypothesis is that AGEs and RAGEs mediate feed-forward loops of oxidative stress
and inflammation that increase by-stander molecular damage in atherosclerosis,
Alzheimer, and other chronic inflammatory diseases [96, 97]. RAGE activation, in
turn, enhances proinflammatory pathways that release cytokines (e.g. IL-6) and
leukocyte adhesion factors (e.g. MCP-1 and VCAM-1), and that induces the
enzymatic synthesis of ROS through NAD(P)H oxidases (e.g. gp91phox) and
mitochondrial electron transport. Lastly, RAGE activation may stimulate feed-
forward vicious cycles by autoinduction in the same cell [98-100]. RAGE
downstream signaling pathways include PIP-3 kinase, NFkappaB and JAK/stat.
Feedback loops include the induction of RAGE by TNFalpha through production of
ROS, mediated by NFkappaB [101]. RAGE-dependent processes are also implicated
in Alzheimer disease.
The lower glucose may also be a risk factor in sudden death. Recall the
puzzle that some DR rats died without evidence of gross pathology. We suggest the
127
precedent of the sudden “dead-in-bed syndrome” of humans. Transient
hypoglycemia is implicated in sudden death from cardiac arrest in type 1 diabetics
(insulin-deficient), who have 3-fold more unexpected death than healthy young
[102].

3. Peroxisome proliferators-activated receptors (PPARs)
As discussed earlier, the nuclear hormone super family, peroxisome
proliferators-activated receptors (PPARs), may play a critical role in mediating many
of the transcriptional effects of DR in peripheral systems. Indeed, in rat kidney
PPAR mRNA, protein and DNA binding activities are decreased with age and these
changes are attenuated with DR [103]. While the PPARs show wide distribution
among glia and neurons in the brain [104] the effects of age or DR have not been
documented. Although PPARs are best known for their precise transcriptional
control of metabolic events, certain subtypes (in particular, PPARgamma) mediate
inflammatory processes, e.g., [105-107]. Regarding the brain, PPAR stimulation
reduces neuroinflammation, both in vivo [108, 109] and in vitro [105, 110, 111].
Thus, PPAR mediation of the anti-inflammatory effects of DR in the brain seems
likely.

Conclusion
DR attenuates many age-related inflammatory events in the CNS and
periphery of experimental animal models in concert with increasing lifespan. In the
128
aging brain, DR suppresses the activation of microglia and astrocytes which are
associated with demyelination, synaptic atrophy and neurodegeneration. These
events are believed to be the underlying causes of age-related cognitive decline.
Rodent models suggest that DR may also protect against age-related
neurodegenerative diseases involving inflammation such as AD and
ischemia/reperfusion.
Even short-term DR can attenuate inflammation and affect metabolic and
DNA repair pathways. Mechanisms by which DR suppresses peripheral
inflammation include the elevation of glucocorticoids, lowering of glucose, and
activation of PPARs. Although the effects of DR are less understood in the brain,
common pathways are emerging that link many normal aging inflammatory
processes with age-related diseases such as AD, cancer, diabetes, and cardiovascular
disease.
129

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Abstract (if available)

Abstract Declines in cognitive function are common during aging even in the absence of disease. Increased glial activation and inflammation during normal brain aging are implicated in neuron atrophy which may lead to cognitive impairments. Mechanisms underlying glial activation and their consequences on synaptic plasticity are explored in this thesis.

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Asset Metadata

Creator Wong, Angela May (author)

Core Title Effects of age-related glial activation on neurite outgrowth

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Molecular Biology

Publication Date 02/14/2007

Defense Date 12/13/2006

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

Tag aging,brain,caloric restriction,glia,hormone replacement therapy,OAI-PMH Harvest

Language English

Advisor Finch, Caleb E. (committee chair), Pike, Christian J. (committee member), Tower, John G. (committee member)

Creator Email angelamaywong@yahoo.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m253

Unique identifier UC1195126

Identifier etd-Wong-20070214 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-165273 (legacy record id),usctheses-m253 (legacy record id)

Legacy Identifier etd-Wong-20070214.pdf

Dmrecord 165273

Document Type Dissertation

Rights Wong, Angela May

Type texts

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

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu