HUR REGULATION IN CENTRAL NERVOUS SYSTEM DISORDERS
by
CRYSTAL GEORE WHEELER
L. BURT NABORS, COMMITTEE CHAIR
SCOTT R. BARNUM
PETER H. KING
HAROLD W. SONTHEIMER
LINDA O. WADICHE
A DISSERTATION
Submitted to the graduate faculty of The University of Alabama at Birmingham,
in partial fulfillment of the requirement for the degree of
Doctor of Philosophy
BIRMINGHAM, ALABAMA
2013
Copyright by
Crystal Geore Wheeler
2013
HUR REGULATION IN CENTRAL NERVOUS SYSEM DISORDERS
CRYSTAL GEORE WHEELER
DEPARTMENT OF NEUROBIOLOGY
ABSTRACT
In inflammatory diseases of the central nervous system (CNS), astrocytes play a
major role in the systemic response to disease by either enhancing or limiting the
inflammatory response through secretion of growth factors or cytokines. HuR RNA
binding protein regulates many genes involved in inflammation. Inflammatory diseases
such as brain tumors, Amylotrophic Lateral Sclerosis (ALS), and Multiple Sclerosis
(MS), have been linked to HuR, by our laboratory and others. To determine whether
astrocytic overexpression of HuR would regulate inflammation in diseases of the CNS,
we designed a transgenic mouse in which the HuR gene is overexpressed. In the first
study, we used this transgenic mouse model to understand how HuR regulates the disease
course of Experimental Autoimmune Encephalomyelitis (EAE), a mouse model to study
MS. In the second study, we implanted GL261 brain tumor cells in the transgenic mouse
to examine how an overexpression of HuR affects the peripheral immune cells in a
syngeneic tumor mouse model. Creation of this mouse model supplies us with a new tool
to understand how HuR regulation through astrocytes impacts multiple disease models.
iii
DEDICATION
I dedicate this dissertation to my family, my grandmother and grandfather, Leila Mae
Jones and Robert Charles Jones Sr. who made sure that we valued education and hard
work. For all the sacrifices you made to make sure I had everything I needed to succeed
which is your love and support.
To my mother and father, Rosie Mae Kidd and Archie Kidd Jr., although you are not here
to share this with me I know that you are watching from above and are pleased. Thank
you for your unconditional love.
To my sisters, Angela Dean Wheeler and Erica Trenè Wheeler, for all that you have
endured so that I could complete this. People come and go in your life but we will always
have each other no matter what. Thank you for putting up with my insanity and not
killing me in my sleep.
To my nephew, Christion Michael Ward, whose laughter brings me so much joy. “Tee
Tee” wants you to know there are no limits to what you can do. You can do anything, be
anything and go anywhere you want to go.
To my aunts, uncles and cousins, for all the encouragement I truly thank you.
iv
ACKNOWLEDGEMENTS
I would like to acknowledge and thank my mentor, L. Burt Nabors for his
guidance. He has been a definite light on my path. Dr. Nabors kind hearted demeanor
permeates all who meet him. I could not have been more fortunate than to be accepted to
his lab. Under his tutelage, I have further honed my scientific and interpersonal skills.
I would like to thank and acknowledge Dr. Peter King for his guidance. Working with
him has given me a breadth of knowledge. I consider him my co-mentor along with Dr.
Nabors. Thank you for taking the time to help develop my skills as a scientist.
I would like to acknowledge the other members of our lab. Sherry Yang has been so
much more to me that our technician. She has been a wonderful friend whom without her
help I would not have been able to complete my work. Dr. Natalia Filippova is our
dynamic post doctorate who is very knowledgeable and capable and has guided and
inspired me to be a better scientist. Dr. Xiaosi Han has been a joy to work with.
I want to acknowledge my dissertation committee. Thank you for being so understanding
and encouraging throughout my graduate career.
I want to thank all of my friends who have been my champions and support in this effort.
I could not have completed this without all of you. I am truly blessed to have such
wonderful people in my life.
v
TABLE OF CONTENTS
Page
ABSTRACT .....................................................................................................................iii
DEDICATION .................................................................................................................iv
ACKNOWLEDGEMENTS .............................................................................................v
LIST OF TABLES ...........................................................................................................vii
LIST OF FIGURES .........................................................................................................viii
LIST OF ABBREVIATIONS ..........................................................................................x
INTRODUCTION ...........................................................................................................1
HuR ..........................................................................................................................1
Astrocytic Function ..................................................................................................5
Multiple Sclerosis .....................................................................................................7
Glioblastoma Multiforme .........................................................................................10
Central Hypothesis ...................................................................................................14
SEX HORMONE-DEPENDENT ATTENUATION OF EAE IN
A TRANSGENIC MOUSE WITH ASTROCYTIC EXPRESSION
OF THE RNA REGULATOR HUR................................................................................15
HUR REGULATION OF THE NOTCH LIGAND JAGGED-1
IN BRAIN CANCER AND INFLAMMATION.............................................................30
SUMMARY AND DISCUSSION ...................................................................................56
LIST OF GENERAL REFERENCES .............................................................................60
APPENDIX
A IACUC APPROVAL FORM ...............................................................................75
B IACUC APPROVAL FORM................................................................................76
vi
LIST OF TABLES
Tables
Page
SEX HORMONE-DEPENDENT ATTENUATION OF
EAE INA TRANSGENIC MOUSE WITH
ASTROCYTIC EXPRESSIONOF THE RNA
REGULATOR HUR
1. EAE clinical parameters in wild type, HuR-Tg female and
HuR-Tg male mice .........................................................................................29
HUR REGULATION OF THE NOTCH LIGAND JAGGED-1
IN BRAIN CANCER AND INFLAMMATION
1. Antibodies and conjugated fluorochrome for FACs analysis
of PBMCs and spleen.....................................................................................54
2. Cell markers for cellular compartments. ........................................................55
SUMMARY AND DISCUSSION
1. CNS distribution of HuR transgene ...............................................................70
2. EAE clinical parameters in ovariectomized wild type
and HuR Tg mice ...........................................................................................71
vii
LIST OF FIGURES
Figures
Page
INTRODUCTION
1. HuR Protein Schematic ..................................................................................13
SEX HORMONE-DEPENDENT ATTENUATION OF EAE IN
A TRANSGENIC MOUSE WITH ASTROCYTIC EXPRESSION
OF THE RNA REGULATOR HUR
1. Generation of the HuR transgenic mouse ......................................................26
2. EAE phenotype is attenuated in HuR-Tg mice ..............................................27
HUR REGULATION OF THE NOTCH LIGAND JAGGED-1 IN
BRAIN CANCER
1. Jagged-1 3’ UTR AREs .................................................................................48
2. HuR associates with Jagged-1 .......................................................................49
3. Knockdown of HuR protein reduces the levels of jagged-1 ..........................50
4. Polyribosome redistribution of Jagged-1 in U251 control
viii
and shHuR cells ............................................................................................51
5. Example of the gating strategy for isolation of MDSC population
of cells from PBMCs and spleen. ..................................................................52
6. FACS of PBMC and spleen cells from Wt and Tg mice ...............................53
SUMMARY AND DISCUSSION
1. EAE in males with or without estrogen .........................................................72
2. Microarray comparison of female Tg and WT spinals cords
show similar gene expression patterns ...........................................................73
3. Validated Target from the microarray .............................................................74
ix
LIST OF ABBREVIATIONS
A
adenine
ARE’s
adenine- and uridine-rich elements
AUF1
ARE/poly(U)-binding/degradation factor-1
BBB
blood brain barrier
CARN1
coactivator-associated arginine methyltransferase 1
CDI
cumulative disease index
CFA
complete Freund’s adjuvant
CNS
central nervous system
CSF
cerebral spinal fluid
ELAV
Embryonic Lethal Abnormal Visual
E2
estradiol
eIF4E
eukaryotic initiation factor 4E
x
ER
estrogen receptor
IL-3
interleukin 3
GBM
glioblastoma multiforme
GFAP
glial fibrillary acidic protein
GM-CSF
granulocyte macrophage colony stimulating factor
Hif-1α
hypoxia inducible factor-1α
Hsp70
heat shock protein
IL-1β
Interleukin
INF-γ
interferon
Jag1
Jagged-1
KDa
kilodalton
KSRP
K homology-type splicing regulatory protein
LPS
lipopolysaccharide
mRNA
messenger RNA
MS
Multiple Sclerosis
NIC
Notch intracellular domain
PFA
paraformaldehyde
xi
PPMS
primary progressive multiple sclerosis
RBP
RNA binding protein
rpS6
ribosomal protein S6
RRM
RNA recognition motifs
RRMS
relapsing remitting multiple sclerosis
SIRT1
Silencing information regulator 2-1
SPMS
secondary progressive multiple sclerosis
TIA
T-cell restricted intercellular antigen
TNFα
tumor necrosis factor α
TTP
tristetraproline
TTR-RBPs
turnover and translation regulatory mRNA-binding proteins
U
uridine
UTR
untranslated region
VEGF
vascular endothelial growth factor
WHO
World Health Organization
xii
1
INTRODUCTION
HuR
A hallmark of human disease is gene dysregulation. Gene regulation is a highly
ordered multistep process that includes epigenetics, post-transcriptional events including
transcription, messenger RNA (mRNA) splicing, mRNA stability and mRNA targeting,
translation and post-translational events (Kapeli & Yeo, 2012; Kooistra & Helin, 2012).
Post transcriptional regulation has increasingly been studied as it is governed by RNA
and protein interactions. Cis-acting elements within the mRNA affect RNA fate. These
elements regulate the stability of the 5’ cap, internal ribosomal entry sites (IRES),
splicing in the coding region, and targeting/localization. In addition, the trans-acting
elements, the RNA binding proteins (RBP), also determine mRNA fate. Numerous RNA
binding proteins have been discovered that can associate with mRNA and modulate its
half-life, localization and access to the polyribosome or the exosome (Day & Tuite, 1998;
Lal et al., 2004).
The Hu family of RNA binding proteins regulates many developmental and
proliferative genes in the central nervous system (CNS). The Hu family are the human
homologues of the Drosophila Melanogaster, embryonic lethal abnormal visual (ELAV)
family of RNA binding proteins, that are required for development and maintenance of
the fly nervous system (Good, 1995). Interestingly, the human homologue of the ELAV
family was recognized by antibodies from patients with small cell carcinoma-associated
1
paraneoplastic neurologic syndrome (Dropcho & King, 1994; Good, 1995). The
mammalian Hu RBPs consist of four family members, HuR (HuA), HuB (Hel-N1), HuC
(PLE21), and HuD. Three of the four members, are expressed primarily in neurons, while
HuR, a 36 kilodalton (KDa) protein, is expressed in all cells types in the body (Good,
1995; Lagnado, Brown, & Goodall, 1994; Ma, Cheng, Campbell, Wright, & Furneaux,
1996; L. B. Nabors, Furneaux, & King, 1998). Our laboratory has focused on HuR
because of its regulation of multiple genes involved in the disease course and
reoccurrence of glioblastoma multifome (GBM).
Structurally, HuR consists of three RNA recognition motifs (RRM 1-3) (C. Y.
Chen, Xu, & Shyu, 2002) and a hinge region between RRM2 and RRM3 as illustrated in
figure 1 (King, Levine, Fremeau, & Keene, 1994). It has been proposed that RRM1 and
RRM2 act cooperatively to bind RNA as typical RBPs. RRM3 has been suggested to
stabilize the RNA-protein complex and the poly A tail in conjunction with other
proteins. The hinge region has been shown to contain a nuclear export and localization
signal (Atasoy, Watson, Patel, & Keene, 1998).
HuR is proposed to regulate mRNA stability and translation (Hinman & Lou,
2008). HuR is localized mainly in the nucleus but shuttles between the nucleus and
cytoplasm to stabilize mRNA transcripts (Peng, Chen, Xu, & Shyu, 1998). HuR has been
demonstrated to stabilize labile mRNA transcripts such as early response gene, cytokines,
growth factors, lymphokines, hormones, and cancer causing oncogenic transcripts
(Atasoy et al., 1998; C. Y. Chen et al., 2002). In the nucleus, HuR binds to RNA
transcripts and chaperones the mRNA to the cytoplasm, where it associates with the
polyribosome and undergoes translation (Gallouzi et al., 2000). HuR binds the 3’ UTR of
2
the mRNA recognizing AU-rich RNA elements (ARE) regions (Myer, Fan, & Steitz,
1997). AREs are adenylate (A) and uridylate (U) sequences that are categorized based on
how the sequences are positioned in the 3’ UTR. Class I ARE’s have 1-3 copies of
AUUUA motifs scattered throughout the 3’ UTR and are coupled to a U-rich region.
Class II AREs have at least two overlapping UUAUUUA(U/A)(U/A) in a U rich region.
Class III AREs do not have AUUUUA but are usually U rich (C.-Y. A. Chen & Shyu,
1995).
It has been estimated that approximately 8% of human genes contain AREs and
therefore maybe regulated by HuR (Bakheet, Frevel, Williams, Greer, & Khabar, 2001).
HuR has been shown to have many mRNA partners that it regulates through the 3’ UTR.
These include c-fos, vascular endothelial growth factor (VEGF), tumor necrosis factor α
(TNFα), c-myc, cyclin A, cyclin B1, cyclin D1, cyclooxygenase 2 (COX-2), hypoxia
inducible factor-1α (Hif-1α), estrogen receptor (ER), and p21 (Joseph, Orlian, &
Furneaux, 1998; Pryzbylkowski, Obajimi, & Keen, 2008). Thus, understanding HuR has
important implications for understanding multiple diseases involving the dysregulation of
these genes.
There are several additional turnover and translation regulatory mRNA-binding
proteins (TTR-RBPs) that regulate mRNAs by binding to the same 3’ UTR recognition
element. Other RNA binding proteins that destabilize mRNA transcripts include
ARE/poly(U)-binding/degradation factor-1 (C. Y. Chen et al., 2002), heat shock protein
70 (Hsp70), K homology-type splicing regulatory protein (KSRP), T-cell restricted
intercellular antigen (TIA), and tristetraproline (TTP). In the complex regulation of
transcription and translation, HuR has been shown to oligomerize and homodimerize
3
when binding and regulating mRNA transcripts depending on ARE interaction length
where it can compete with these other factors through cooperative or competitive binding
and subcellular expression (Eberhardt, Doller, Akool, & Pfeilschifter, 2007; FialcowitzWhite et al., 2007).
HuR is post-translationally modified covalently by phosphorylation and
methylation which can affect its interaction with regulation of mRNA (Abdelmohsen et
al., 2007; Liu et al., 2009; Yu et al., 2011). For example, silencing information regulator
2-1 (SIRT1) mRNA is stabilized by HuR, but upon stimulation by lipopolysaccharide
(LPS), HuR is phosphorylated by check point kinase 2 (CHK2) and disassociates from
SIRT1 (Abdelmohsen et al., 2007). In vitro studies have shown that HuR can be
methylated by coactivator-associated arginine methyltransferase 1 (CARN1) during LPS
induced stress which may play a role in HuR mRNA stabilization (Li et al., 2002).
HuR upregulation has been linked to many cancers and a poor prognosis. The
hypothesis is that HuR upregulation leads to an increase in brain, breast, colorectal and
prostate cancer. HuR regulates many genes, such TNF-α, IL-8, cox-2, and TLR-4
involved in inflammation (Ford, Marcus, & Lum, 1999; Lin, Hershberg, & Small, 2006;
L. B. Nabors, Gillespie,Yancey G., Harkins, Lualhati , and King,Peter H., 2001).
Overexpression of HuR in macrophages and myeloid tissues has been shown to reduce
the inflammatory response ex- and in vivo (Katsanou et al., 2005). Through regulation of
genes significant in neoplastic and inflammatory conditions, HuR has emerged as a novel
molecular target of drug design (Chae et al., 2009).
4
Astrocytic Function
The immune privileged CNS is composed of many cell types, mainly vascular
endothelial cells, neurons and glial cells. Vascular endothelial cells line the capillaries
that traverse the CNS and help form the blood brain barrier (BBB) that limits the
molecules that can enter the brain from peripheral blood (Wolburg, Noell, Mack,
Wolburg-Buchholz, & Fallier-Becker, 2009). Neurons are cells that use chemical and
electrical signals called action potentials to convey information in the CNS (Kandel,
2013). The glial cells consist of oligodendrocytes, microglia, ependymal cells and
astrocytes. Oligodendrocytes are glial cells that wrap the axon of neurons with a
specialized insulating material called myelin. Myelin allows the electrical signals to
travel between neurons and not loss signal strength (Baumann & Pham-Dinh, 2001;
Pfrieger & Slezak, 2012). Microglia are resident immune cells of the CNS. These
specialized glial cells secrete chemokines and cytokines and survey the CNS
microenvironment like macrophages by phagocytosing ,processing and presenting
antigen to immune cells. Ependymal cells are ciliated epithelial-like cells that line the
ventricles to maintain and direct the flow of cerebral spinal fluid (CSF) (Pfrieger &
Slezak, 2012).
The most abundant cells, called astrocytes by Michael von Lenhossek, due to their
stellate appearance are important for development, maintenance, and homeostasis in the
neural environment. Astrocytes are found in both grey and white matter and are further
classified based on their shape into protoplasmic, mainly found in the grey matter and
5
fibrous, in white matter. Staining of astrocytes by the Golgi method and the drawings by
Santiago Ramòn y Cajal illustrating the physical relationship of astrocytes to neurons and
capillaries led to hypothesis about the function of astrocytes in the brain (Oberheim,
Goldman, & Nedergaard, 2012; Parpura et al., 2012).
Astrocytes have traditionally been thought to function as support cells for
neuronal activity. In healthy tissue, astrocytes are known to provide structural support,
remove ions and neurotransmitters, and supply neurons and other glial cells with
necessary trophic and metabolic nutrients (Henn & Hamberger, 1971; Panatier et al.,
2006). Recent work suggests that astrocytes play a more active role by communicating
with other astrocytes through gap junctions and neurons by glutamate release and calcium
signaling (Cooper, Cornell-Bell, Chernjavsky, Dani, & Smith, 1990; Cornell-Bell,
Finkbeiner, Cooper, & Smith, 1990; Nadarajah, Thomaidou, Evans, & Parnavelas, 1996).
Astrocytes are proposed to have numerous functions in developing adult CNS. In
development, astrocytes help form domains of the brain by axonal guidance and pruning
of neurons. Astrocytes are required in the adult brain for neuronal survival and synapse
formation, and contribute to the tripartite synapse (Araque, Parpura, Sanzgiri, & Haydon,
1999; Sofroniew & Vinters, 2010). Astrocytes are also an integral part of the blood brain
barrier. Astrocytic endfeet help form and maintain tight junctions that restrict access to
the brain microenvironment (Abbott, Ronnback, & Hansson, 2006). Astrocytes also play
a critical role in the innate immunity of the brain when activated by pathogens or disease
states. Through expression of chemokines and cytokines, astrocytes cause the BBB to
become leaky and recruit T cells into the CNS. Astrocytes can phagocytose and act as
antigen presenting cells (APCs) by expressing MHC II (Farina, Aloisi, & Meinl, 2007;
6
Mayo, Quintana, & Weiner, 2012). The study of the astrocytic role in innate immunity
still needs much research but is poised to be a key target for manipulation in regulating
inflammation in disease states. Two diseases that are particularly important that involve
astrocytes and a focus of my dissertation studies are Multiple Sclerosis (MS) and
Glioblastoma Multiforme (GBM).
Multiple Sclerosis
Multiple sclerosis (MS) is a chronic disease characterized by inflammation,
axonal degeneration and white matter loss in the CNS. The causes of MS are thought to
involve genetic susceptibility and an environmental trigger. More than 2 million people
suffer from MS worldwide (Runia, van Pelt-Gravesteijn, & Hintzen, 2012). MS has a
heterogeneous disease course, 85% of people suffering from MS have relapsing remitting
MS (RRMS) form of MS that has full recovery between acute attacks, but ultimately
leads into a secondary progressive MS (SPMS) phase where each relapse leads to
diminished recovery. A smaller group of MS patients suffer from primary progressive
MS (PPMS) in which the disease steadily gets worse with each acute attack of symptoms
without remission (Noonan et al., 2010).
MS first presents with any variety of neurological symptoms, although generally
in the optic nerve, spinal cord or brain stem. In the early stages, MS is undetected until
loss of white matter is seen on magnetic resonance imaging (MRI) (Trojano, Paolicelli,
Bellacosa, & Cataldo, 2003). As the disease progresses, MS symptoms consist of
cognitive defects, fatigue, and loss of motor control and or sensory perception but vary
7
depending on the location of the lesion. Lesions are often found in the optic nerves,
periventricular white matter, brain stem, cerebellum, and spinal cord white matter, and
they often surround one or several medium-sized vessels (Popescu & Lucchinetti, 2012).
MS lesions are characterized by an infiltration of T cells and macrophage, myelin
loss, oligodendrocyte degeneration, accumulation of immunoglobulins and complement.
There is no cure for MS and treatment options are few and often have adverse side
effects. One medication, gilenya, is an oral drug that keeps lymphocytes from leaving the
lymph nodes. Injectable interferon- beta 1α reduces relapse rates. Glatiramer injections
shift reactive lymphocytes population from inflammatory to anti-inflammatory.
Intravenous immune globulin improved disability scores and relapse rates in RRMS.
Mitoxantrone hydrochloride, a pan immunosuppressor and cortical steroids inhibit
lymphocyte production and reduce pro-inflammatory cytokines. Another treatment is
plasma exchange, in which the blood is filter of immune factors and returned to the
patient. None of these treatments stop the progression of the disease (Habek, Barun,
Puretic, & Brinar, 2010; Loma & Heyman, 2011; Murray, 2009; Noseworthy,
Lucchinetti, Rodriguez, & Weinshenker, 2000; Pierson, Simmons, Castelli, & Goverman,
2012).
In examining the pathogenesis leading to MS, animal models are useful to
understand aspects of the disease course. No one animal model exactly replicates MS in
humans. However several animal models use techniques, such as genetics, viral
induction, toxins, and immunizations that mimic various aspects MS. The most well
studied model is the experimental autoimmune encephalomyelitis (EAE) immunization
model that uses CNS antigens or passive transfer of encephalitogenic T cells to immunize
8
the animal. This model can be used in a number of species such as mouse, rat and
monkey (Constantinescu, Farooqi, O'Brien, & Gran, 2011). To induce EAE, animals are
immunized with myelin proteins emulsified in complete Freund’s adjuvant (CFA) or
immunized by myelin specific T cells. EAE is not always induced through immunization,
and it is concluded that other factors contribute to disease susceptibility and disease
presentation (Batoulis, Recks, Addicks, & Kuerten, 2011). Rodent models are frequently
used because they are reproducible and have the lowest animal model costs.
EAE in rodents is accessed by observation of ascending flaccid paralysis. Clinical
scoring is as follows: 0 for no disease, 1 for tail involvement, 2 for hind limb
involvement, 3 for hind limb paralysis, 4 for fore limb involvement, 5 for fore limb
paralysis and 6 for moribund. EAE in mice is a CD4 T cell mediated response. CD4 cells
are activated in the periphery and hone to the CNS where they cross the blood brain
barrier (Stump et al., 2002). The T cells are then reactivated by myelin protein epitopes
presented by dendritic cells and more naïve T cells are recruited. Astrocytes,
macrophage, and microglia are activated and release a host of chemokines and cytokines,
such as tumor necrosis factor (TNF-α), Interleukin (IL-1β), (interferon) INF-γ and
granulocyte macrophage colony stimulating factor (Varnum-Finney, Brashem-Stein, &
Bernstein, 2003) in the initial inflammatory response that breaks down myelin. The
second wave of inflammation is started when the naïve T cells enter the CNS and antigen
presenting cells activate them with other epitopes of myelin (Chastain, Duncan, Rodgers,
& Miller, 2011; Pierson et al., 2012). Though some studies show conflicting results, the
CD4 T cell response involves subsets of T cells and cytokine expression that depending
9
on whether the lesion is in the brain or spinal cord may have opposite effects on
inflammation (Batoulis et al., 2011).
The EAE animal model has drawbacks in that it does not fully replicate MS, with
differences in disease pathogenesis, B cells have been shown to modulate disease and in
the rodent EAE model there is no B cell involvement. MS is primarily a sporadic disease
but EAE is induced, it does not occur spontaneously. The pathology is mainly in the
spinal cord in the EAE model but in MS, lesions can be found in any area with white
matter, including the brain (see above). Rodent EAE exhibits only flaccid paralysis but in
MS symptoms are varied from motor and cognitive deficits to sensory loss (Batoulis et
al., 2011; Harp, Lovett-Racke, Racke, Frohman, & Monson, 2008; Noseworthy et al.,
2000). EAE models do replicate aspects of MS, namely the T cell inflammatory response,
topography of lesions, relapsing remitting and chronic progressive phenotypes, and
demyelination and axonal damage (Gold & Heesen, 2006). Even with their limitation,
EAE animal models have been instrumental in the development of many of the drug
therapies (Constantinescu et al., 2011).
MS is a multifaceted disease that causes a variety of symptoms. The research field
is working to understand the disease from many angles. In elucidating how transcription
and translation including mRNA stability and RBPs play a role in the demyelination,
remyelination, and inflammatory response could yield opportunities for more effective
drug therapies that can improve quality of life for MS patients.
Glioblastoma Multiforme
10
Another disease involving astrocytes and inflammation is GBM. Gliomas are
CNS cancers of glial cell origin. Gliomas account for 30% of all primary brain tumors
and 80% of all malignant tumors. Gliomas are graded according to their severity by the
World Health Organization (WHO) with a score of I–IV. Grade I gliomas, pilocytic
astrocytomas, are usually benign, grow slowly, do not infiltrate into normal tissue and
have a good prognosis. Grade II gliomas, diffuse astrocytomas, are slightly more
aggressive because they will filtrate into normal tissue and will often reoccur. Many
Grade II gliomas will transform into higher grade tumors over time. Grade III, anaplastic
astrocytomas and Grade IV gliomas, glioblastoma multifome (GBM) are the malignant
gliomas. Anaplastic astrocytomas are mitotic and dedifferentiated. GBMs are very
aggressive, mitotically active, necrosis-prone, and have the poorest prognosis. Over 50%
of all malignant gliomas are GBMs. Only 1/3 of GBM patients survive more than a year
and less than 5% survive more than 5 years (CTBRUS, 2012; Jones & Holland, 2011).
In the past 50 years treatment options for GBMs have increased survival only
modestly. Treatments include surgery to remove as much tumor as possible but because
of the infiltration this proves difficult. Therefore radiation therapy after surgery is
standard for GBM. Chemotherapy after radiation has shown promise in survival benefit.
Other drug therapies used after surgery and radiation therapy are carmustine wafers that
are placed at the site of resection and the alkylating agent temozolimide administered
after radiation. Sadly with all the current treatments, GBMs usually reoccur (L. B. Nabors
et al., 2012).
Many therapeutics have targeted aspects of the signaling pathways involved in
GBM aggressive growth and invasive behavior. Many signaling pathways have been
11
implicated in the regulation of GBMs. These include receptor tyrosine kinase (RTK)
pathways like epidermal growth factor (EGF) and platelet derived growth factor (Gard,
Burrell, Pfeiffer, Rudge, & Williams, 1995), Ras, phosphoinositide 3-kinase (PI3K),
sonic hedgehog (SHH), and Notch signaling pathways (Kanu et al., 2009). Understanding
how GBMs maintain such a stronghold in the CNS is paramount to designing better
therapies to improve survival rate and quality of life. Animal models have also provided
tools to understand the molecular events that underlie GBMs tenacity. There are several
animal models to study GBMs. One of the most characterized is the GL261 syngeneic
mode. This model was created by implanting 3-methylcholantrene in the brain of a
C57BL/6 mouse and serial transplantation of small tumor pieces to maintain the tumor.
Although it is not a spontaneous tumor model, it is reproducible and when used in
C57BL/6 there is no immune response (Maes & Van Gool, 2011).
The poor prognosis of GBM and the lack of effective drug therapies to extend
quality and quantity of life make understanding the disease of utmost importance. Drug
therapy resistance is a key factor in the lack of progress in making life better for these
patients. Understanding how cell mechanics at the transcriptional and translational level
can open avenues to extend drug design and a longer healthier life for those with GBM.
12
Figure 1. HuR Protein Schematic. HuR protein consists of 3 RNA recognition motifs
and a hinge region the contains shuttling sequence that allows HuR to move back and
forth between the nucleus and the cytoplasm.
13
Central Hypothesis
Astrocytes play a major role in CNS diseases by supplying metabolic and
nutritive support, and maintaining homeostasis in the microenvironment. Astrocytes play
an active role in communications with cells of the CNS and the periphery. Understanding
how this complex cell type manages these diverse duties can help gain insight to
understanding disease processes and how to affect better outcomes for patients suffering
these diseases. RNA binding proteins (RBPs) are very important in regulating gene
expression in cells. RBPs offer an exquisite level of regulation that allows a gene product
to be expressed differently. The RBP HuR regulates many genes involved in
inflammatory conditions affecting the CNS. Expanding our molecular understanding of
how the astrocyte manages to be such a versatile cellular component of the CNS will help
define its role in disease status. My overall hypothesis for this dissertation is: Astrocytic
HuR contributes to neurological disease by altered expression of CNS specific targets
with resultant changes in cellular behavior.
14
15
SEX HORMONE-DEPENDENT ATTENUATION OF EAE IN A TRANSGENIC
MOUSE WITH ASTROCYTIC EXPRESSION OF THE RNA REGULATOR HUR
.
by
CRYSTAL WHEELER, L. BURT NABORS, SCOTT BARNUM, XIUHUA YANG,
XIANZHEN HU, TRENTON R. SCHOEB, DONGQUAN CHEN, AGNIESZKA A.
ARDELT, PETER H. KING
Journal of Neuroimmunology 246: 34-37
Copyright
2012
by
Elsevier B.V.
Used by permission
Format adapted for dissertation
ABSTRACT
In experimental autoimmune encephalomyelitis (EAE) and other
neurodegenerative diseases, astrocytes play an important role in promoting or attenuating
the inflammatory response through induction of different cytokines and growth factors.
HuR plays a major role in regulating many of these factors by modulating RNA stability
and translational efficiency. Here, we engineered transgenic mice to express HuR in
astrocytes using the human glial fibrillary acidic protein promoter and found that female
transgenic mice had significantly less clinical disability and histopathological changes in
the spinal cord. Ovariectomy prior to EAE induction abrogated the protective effect. Our
findings support a role for the astrocyte and posttranscriptional regulation in hormonallymediated attenuation of EAE.
16
INTRODUCTION
HuR, an RNA binding protein, modulates the stability and translational effciency
of many growth factor and cytokine mRNAs by binding to adenine- and uridine-rich
elements (ARE) in the 3’ untranslated region (UTR) (Brennan and Steitz, 2001; Barreau
et al., 2006). This level of gene regulation plays an important role in initiating or
terminating inflammatory responses as it can quickly alter expression levels of critical
mediators such as TNF-α, COX-2, and interferon-γ (Anderson, 2010). Since the astrocyte
expresses a large range of ARE-containing cytokine and chemokine mRNAs that can
prolong or attenuate inflammation (Dong and Benveniste, 2001; Nair et al., 2008), we
sought to determine the impact of HuR transgenic expression on experimental
autoimmune encephalitis (EAE). We found significant clinical and histological
attenuation of EAE in female and to a lesser extent male HuR transgenic (Tg) mice.
Reversal of protection in female mice following ovariectomy indicates the attenuation
was hormonally influenced. These findings shed new light on the mechanisms and cell
types that contribute to the protective effects of estradiol (E2) or progesterone in EAE.
MATERIALS AND METHODS
Mice
17
The UAB Transgenic Mouse Facility microinjected fertilized eggs of C57BL/6
(B6) mice with a Flag-tagged HuR cDNA (Nabors et al., 2003) construct downstream
from the human GFAP promoter (Brenner et al., 1994).Mice were genotyped using tail
clip DNA and the following oligonucleotides: upstream 5’TGGACTACAAGGACGACGAT-3’, and downstream 5’CGTCTTTGATCACCTCTGAGC-3’. Mice from 8–14 weeks of age were used for
experiments. All animal studies were performed with approval from the UAB
Institutional Animal Care and Use Committee.
Induction of EAE
For active EAE, control and HuR-Tg mice were immunized with myelin
oligodendrocyte glycoprotein (MOG) peptide 35–55 as described (Hu et al., 2010). Onset
and progression of EAE symptoms were monitored daily (30 days) using a standard
clinical scale (Hu et al., 2010). For each mouse, a cumulative disease index (CDI) was
calculated from the sum of the daily clinical scores observed between day 7 and day 30.
For ovariectomy, mice were sedated using a mixture of ketamine and xylazine. Ovaries
were surgically removed and mice were allowed to recover 7 days before induction of
EAE.
RNA analysis, histology and immunohistochemistry
All mice were sacrificed by CO2 inhalation. CNS tissues were removed and
frozen in OCT (for immunohistochemistry) or liquid N2 (for RNA analysis). RNA was
extracted and reversed transcribed using a reverse transcription kit (Applied Biosystems).
18
PCR was performed with the primers described above. For immunohistochemistry, 8
micrometer transverse sections were cut and briefly fixed with 4% paraformaldehyde
(PFA). Sections were blocked, permeabilized and stained with GFAP (DAKO) at 1:1000
and FLAG rabbit polyclonal (Sigma) at 1:5000 overnight at room temperature. Sections
were stained with secondary antibodies, Alexa.uor 488 and Alexa.uor 594 (Invitrogen) at
1:1000 and DAPI. For EAE histology, mice were sacrificed 30 days post MOG peptide
injection. Spinal columns were decalcified and all tissue was paraffin embedded. Five
micrometer sections from the cervical, thoracic and lumbar spinal cord were cut and
stained with hematoxylin and eosin or Luxol fast blue and periodic acid-Schiff. The
extent of inflammation, demyelination, and axonal degeneration was scored based on
previously published methods (Hu et al., 2010). Briefly, lesions were evaluated on a 0–4
scoring system for inflammation (lymphocyte accumulation and neutrophil infiltration),
demyelination, and axonal degeneration without knowledge of the experimental group.
Severity scores were calculated as the mean over all segments of the products of the
intensity scores multiplied by the extent scores for each lesion characteristic.
RESULTS
Generation of the HuR-Tg mouse
19
We used a cDNA containing HuR with an N-terminal Flag epitope (Nabors et al.,
2003) and cloned it into a plasmid containing 2.4 kb of the human glial fibrillary acidic
protein (GFAP) promoter (Fig. 1A) (Brenner et al., 1994). A positive transgenic line
(HuR-Tg) was identified by PCR genotyping with a unique primer to the FLAG epitope
and a HuR-specific downstream primer. Transgene mRNA expression in spinal cord and
brain tissue was confirmed by RT-PCR. Using an anti-FLAG antibody, we detected
extensive expression of FLAG-HuR in spinal cord astrocytes (Fig. 1B, upper panel). A
predominantly nuclear pattern was observed which is consistent with the cellular
distribution of endogenous HuR (Brennan and Steitz, 2001). Colocalization of FLAG and
GFAP immunoreactivity was confirmed with confocal imaging (Fig. 1B, lower panel).
The transgene was equally distributed throughout the cervical, thoracic and lumbar
regions and no gender-specific differences were observed (not shown).
Active EAE is attenuated clinically and histologically in HuR-Tg mice
Animals were injected with MOG peptide and then assessed for disease severity
using a standard scoring system (Hu et al., 2010). We observed a significant and genderdependent attenuation of clinical phenotype in HuR-Tg mice compared to littermate
controls (Fig. 2 and Table 1). In keeping with previously published work, we did not
observe a gender effect in wild-type mice (Okuda et al., 2002). Female HuR-Tg mice
showed significantly delayed disease onset (18.4 vs. 13.4 days for wild-type, p=0.0035)
and a lower maximum clinical score (2.6 v. 3.8 in wild-type, p=0.0012).No clinical signs
were detected in 25% of female transgenic mice whereas 100% of littermate controls and
20
HuR-Tg males developed disease. HuR-Tg males had a significantly lower maximum
clinical score (p=0.01) and a non-significant trend toward delayed onset and lower
cumulative disease index. Ovariectomy of female HuR-Tg mice prior to EAE induction
abrogated the protective effect (Fig. 2B). We next evaluated spinal cords of HuR-Tg and
wild-type mice for histological correlation of this attenuated phenotype (Fig. 2C and D).
Spinal cord sections obtained from wild-type mice 30 days after disease induction had
significant cellular infiltration in the meninges and white matter with perivascular cuffing
and demyelination. For statistical analysis, we scored the degree of inflammation,
demyelination, and axonal degeneration in spinal cords of HuR-Tg mice and littermate
controls using a scale of 0 to 4 based on previously published methods (Hu et al., 2010).
For female transgenic mice, there was a significant reduction in the scores of all three
histological parameters compared to littermate controls. Male HuR-Tg mice also
displayed a significant attenuation in inflammation and axonal degeneration, but not in
the degree of demyelination.
DISCUSSION
We have described a novel transgenic model whereby expression of the RNA
binding protein, HuR, in spinal cord astrocytes produces clinical and histological
attenuation of EAE in female, and to a lesser extent, male mice. Reversal of protection
after ovariectomy strongly suggests that the effect was modulated by E2, progesterone or
both. A possible link between E2 and EAE attenuation was observed in pregnant animals
21
and later confirmed in experiments involving exogenous administration of E2 (Offner
and Polanczyk, 2006). Progesterone has also been associated with clinical attenuation of
EAE (Garay et al., 2007; Yates et al., 2010). Although the protective effect of E2 is
dependent on E2 receptor (ER)-á, the identity of effector cell(s) remained unknown until
a recent study implicated astrocytes (Offner and Polanczyk, 2006; Spence et al., 2011). In
that study, investigators observed a loss of protection in EAE when ER-á was
conditionally knocked out in astrocytes (Spence et al., 2011). Astrocytes are
immunocompetent cells with pleiotropic effects on CNS inflammation and
neuroprotection. Similar to peripheral immune cells, astrocytes can express a broad range
of chemokines and cytokines that promote or attenuate inflammation and neuronal injury
(Dong and Benveniste, 2001; Nair et al., 2008; Sofroniew and Vinters, 2010). HuR
regulates mRNAs of many of these factors by binding AREs in the 3’ UTR and uridinerich elements in the 5’ UTR (Brennan and Steitz, 2001; Barreau et al., 2006;
Abdelmohsen et al., 2008). HuR modulates two distinct but related levels of
posttranscriptional regulation: mRNA turnover and translational efficiency (including
internal ribosome entry site-mediated translation) (Kullmann et al., 2002;Meng et al.,
2005; Abdelmohsen et al., 2008; Yeh et al., 2008; Durie et al., 2011). Depending on the
mRNA target and cellular context, HuR can positively or negatively regulate each level
distinctly or in combination. The mRNA target(s) affected by the HuR transgene in our
model remains to be characterized. Studies are also ongoing to further characterize the
hormonal influence of disease protection (E2 versus progesterone), the role of
testosterone, and whether attenuation of male HuR-Tg mice can be enhanced with E2 or
progesterone.
22
In summary, we describe a novel mouse model which links transgenic HuR
expression in astrocytes to hormonally dependent attenuation of EAE. This finding
provides future direction for understanding the molecular mechanisms and targets for
astrocyte-mediated protection in EAE.
23
ACKNOWLEDGEMENTS
This work was supported by a VA Merit Review, and NIH R01 NS064133 (PHK), R01
CA112397 (LBN), R01 NS46032 (SRB), the UAB Transgenic and Microarray Facilities
(P30 CA013148-39), and the UAB Neuroscience Blueprint Core (NS57098). Histology
services were provided by the UAB Comparative Pathology Laboratory.
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25
Fig. 1. Generation of the HuR transgenic mouse. A) HuR cDNA with an N-terminal Flag
epitope was cloned into the gfa2 cassette containing ~2.2 kb fragment of the human
GFAP promoter (Brenner et al., 1994). Arrows indicate PCR primers that were used for
RT-PCR analysis of brain cortex (Ctx) and spinal cord (SC) RNA shown below. RT,
reverse transcriptase. B) Upper panel: immunfluorescence of a lumbar spinal cord section
from a HuR-Tg mouse using anti-Flag antibody (red) and GFAP (green) antibodies with
a DAPI counter stain (blue). Lower panels: confocal microscopy with the same
antibodies showing colocalization of GFAP and Flag immunoreactivity in a HuR-Tg
mouse but not a littermate control.
26
Fig. 2. EAE phenotype is attenuated in HuR-Tg mice. A) Clinical assessment of HuR-Tg
females (Tg-F), males (Tg-M) and littermate controls (WT) after induction of EAE with
MOG peptide. Mice were examined for a total of 30 days. Numbers of mice in each
group are shown in parentheses. Results shown are the daily mean clinical score for all
groups of mice from three to four independent experiments. See Table 1 for further
details. B) Clinical assessment of EAE induction in female HuR-Tg or wild-type mice
who received ovariectomy 1 week prior to MOG inoculation. C) Representative spinal
cord sections from a female HuR-Tg and wild-type mouse at 30 days post MOG
immunization. Spinal cord sections stained with luxol fast blue and periodic acid-Schiff
(upper panels) show reduced demyelination in HuR-Tg female mouse compared to a
27
wild-type (areas of demyelination indicated by arrowheads). H&E staining of the boxed
regions (lower panels) demonstrate reduced in.ammation in the HuR-Tg mouse compared
to WT. D) Scoring of spinal cord sections (scale, 0–4; based on 5 ìm thick sections from
cervical, thoracic and lumbar regions) for in.ammation (InF), axonal degeneration (AxD),
demyelination (DeMy). F, female; M, Male. The number of mice examined is shown in
parentheses. **p<0.008, *p<0.03.
28
Table 1. EAE clinical parameters in wild type, HuR-Tg female and HuR-Tg male mice.
CDIa
Disease
Onsetb
Disease
Incidencec
Max Clinical
Scored
WT (n=23)
56.1
13.4d
100
3.8
HuR-Tg female (n=12)
30.2*
18.4d*
75
2.6*
HuR-Tg male (n=13)
47.3
14.1d
100
3.4**
a
Cumulative Disease Index is the mean of the sum of daily clinical scores observed
between days 7 and 30.
b
Disease onset is defined as the first day of two consecutive days with a clinical score of
two or more. Onset was significantly delayed in HuR-Tg female mice compared to wild
type mice.
c
Disease incidence is defined as the percent of mice that displayed any clinical signs of
disease.
d
Maximum mean clinical score is the mean of the highest daily clinical score for each
mouse. HuR-Tg female and male mice had significantly lower scores compared to wild
type mice.
**P<0.004.
*P=0.01
29
30
HUR REGULATION OF THE NOTCH LIGAND JAGGED-1 IN BRAIN CANCER
by
CRYSTAL WHEELER, XIUHUA YANG, NATALIA FILIPPOVA, BURT NABORS
In preparation
Format adapted for dissertation
30
ABSTRACT
Posttranscriptional regulation of labile gene expression has become increasingly
important in understanding the mechanisms involved in many cancers including
glioblastomas. The mRNA binding protein HuR has been shown to regulate many
chemokines and cytokines by stabilizing mRNA transcripts with resultant changes in
protein expression. The Notch signaling pathway has been shown to be upregulated in
many cancers. Jagged-1 signaling through Notch has been implicated in hematopoietic
cell development. In development of the nervous system, Notch signaling is upregulated
early but suppressed in the latter stage.
We demonstrate Jagged-1 is a Notch ligand overexpressed in glioma. HuR binds robustly
to the Jagged-1 mRNA and silencing of HuR accelerates the degradation of Jagged-1
RNA and shifts its distribution in the polyribosome fractions with a loss of protein
expression. Using a syngeneic intracranial model we compare the distribution of
circulation cellular elements influenced by Notch in wildtype and HuR transgenic mice.
We observe a trend toward myeloid cell distribution changes in the transgenic model.
31
INTRODUCTION
HuR RNA binding protein regulates many labile genes via the AU rich elements
(ARE) located in the 3’ UTR. HuR is one of a family of RNA binding proteins that are
expressed mainly in neurons. HuR is the only family member that is ubiquitously
expressed (Lagnado et al. 1994; Ma et al. 1996; Nabors et al. 1998). HuR shuttles
between the nucleus and the cytoplasm and has been shown to regulate translation as well
as transcription. As a RNA binding protein that stabilizes ARE containing substrates,
overexpression of HuR is noted in neoplastic and inflammatory disease states. Many
cancers exhibit an over expression of HuR which usually corresponds to a poorer
prognosis. In Glioblastoma Multiforme (GBMs), the highest grade glioma HuR is
overexpressed. Studies have shown that HuR stabilizes many genes that allow the cancer
to proliferate, evade the immune system, metastasize and elude cell death signals
(Abdelmohsen and Gorospe 2010).
Notch signaling is abnormal in many cancers. The Notch pathway is best known for its
role in development and determining cell fate. The Notch family consists of 4 receptors,
Notch 1-4 and 5 ligands, Delta1-3 and Jagged-1-2. In canonical signaling, cell to cell
contact allows the Notch ligand to bind the receptor and liberation of the Notch
intracellular domain (NIC) by ADAM protease at the cell membrane. The NIC is
ubiquitinylated, further cleaved by γ-secretase and translocated to the nucleus. Once in
the nucleus, the NIC binds to the repressor resulting in the expression of genes (Osborne
and Minter 2007).
32
Of the Notch ligands, Jagged-1 (Jag1) has a large 3’ UTR with several AREs suggesting
an opportunity for posttranscriptional regulation by HuR (fig. 1A). Jagged-1 has been
shown to be upregulated in GBMs and necessary for tumor proliferation and survival
(Purow et al. 2005). In addition, Jagged-1 plays a role in the cellular immune response of
myeloid derived suppressor cells (MDSCs) in disease states such as cancer (Saleem and
Conrad).
MDSCs are composed of several different cell types. Characterized by murine cell
surface markers CD11b and Gr1+, these cells are suppressors of T cells and comprised of
progenitors to dendritic cells, macrophages and granulocytes. MDSCs are further
subdivided into polymorphonuclear MDSCs and monocytic MDSCs (Gabrilovich et al.
2012). The Notch signing pathway regulation of MDSCs differentiation and expansion is
not fully understood and controversial (Orkin and Zon 2008; Saleem and Conrad 2011).
Here we show Jagged-1 associates and is regulated by Hur in glioma cells and in a novel
transgenic animal, overexpression of HuR in astrocytes changes the immune cell
compartments.
33
MATERIALS AND METHODS
Mice
The UAB Transgenic Mouse Facility microinjected fertilized eggs of C57BL/6
(B6) mice with a Flag-tagged HuR cDNA (Nabors et al. 2003) construct downstream
from the human GFAP promoter (Brenner et al. 1994).Mice were genotyped using tail
clip DNA and the following oligonucleotides: upstream 5’TGGACTACAAGGACGACGAT-3’, and downstream 5’CGTCTTTGATCACCTCTGAGC-3’. Mice from 10–12 weeks of age were used for
experiments. All animal studies were performed with approval from the UAB
Institutional Animal Care and Use Committee.
Intracranial mouse glioma model
C57BL/6 mice 10–12 week old were anesthetized with isoflurane. A midline
scalp incision was made and a burr hole was made on the left between Bregma and the
coronal suture. Gl261 tumor cells (1.25 X 105 cells/dose X 2) resuspended in
methylcellulose solution were stereotactically injected using a 250-ul Hamilton syringe
with a 30-gauge needle mounted in a Stoelting stereotaxic apparatus. The needle was
advanced vertically through the burr hole to a depth of 2.5 mm and then slowly retracted
120 s after injection: the incision was then closed with veterinary glue and the mouse was
injected with buprenorphine, 0.05-0.1 mg/kg / carprofen, 5 mg/kg IP. Mice were returned
to warmed sterile cages and were provided with sterile lab chow. Mice were weighed
34
daily for 18 days or until mouse lost 20% body weight. Mice were sacrificed after
sedation with isoflurane by cardiac puncture.
Cloning of Jagged-1 3’ UTR
cDNA from several tumor cell lines underwent RT-PCR with primers upstream 5’
to Jagged-1 3’ UTR Forward: 5’GACTTGGAAAGTGCCCAGAG 3’and 3’ downstream
Reverse: 5’ CAGTTCCAGCTTCACAGCAG 3’.
Isolation of PBMCs, Spleen lymphocytes and FACs analysis
Whole blood from cardiac puncture was lysed for red blood cells and the
remaining cells were washed and stained for Kidneys were removed and processed for
Fluorescent Activated Cell Sorting (FACS). Spleens were removed and sheared, RBCs
were lysed and the remaining cells were washed and prepared for FACS analysis. Cells
were stained for 30 minutes with Gr1, CD4, CD3, CD45, CD11b, Ly6C, Ly6g, and
F4/80. (BD Biosciences) Cell were read on a LSRII and analyzed with FLoJo Software.
Student t test was performed to evaluate significance.
Polyribosome Isolation
Cells were grown to 80% to 90% confluency and treated for 15 minutes at 37°C and 5%
CO2 with 100 mg/mL of cycloheximide in complete media (1× Dulbecco's modied
35
Eagle's medium/F12K 50/50, 7% FBS). Cells were washed with ice cold PBS with
cycloheximide (100 mg/mL) and trypsinized with 0.25% Trypsin/2.21 mmol/L EDTA
and washed with ice-cold 1× PBS containing cycloheximide (100 mg/mL) and pelleted
and resuspended in 300 µl of PEB lysis buffer (0.3 m NaCl, 15 mm MgCl2, 15 mm TrisHCl, pH 7.6, 1% Triton X-100, 1 mg/ml heparin, 0.1 mg/ml cycloheximide) and lysed on
ice for 10 min. Lysates were centrifuged at 10,000 × g for 10 min. Approximately 2.5 mg
of cytoplasmic lysate was layered on top of a linear 10–50% (w/v) sucrose gradient and
centrifuged in a Beckman SW41 rotor at 38,000 × g for 120 min at 4 °C. Polysome
profiles were obtained by absorbance measurements at 260 nm during fraction collection
(1 ml each). Equal volumes from each fraction were used for Western blotting.
RNA analysis
Clones were treated with actinomycin D, and RNA was collected at different
intervals over a 6-hour time period. Levels of mRNA were determined with qRT-PCR at
each interval and expressed as a percentage of target mRNA present at time 0. Total RNA
was extracted, purified, and quantified as previously described (Nabors et al. 2003).
Western Blotting, Immunoprecipitation, and RNA Immunopreciptiation
Cytosolic or whole cell lysates from cultured cells were prepared in the presence
of protease inhibitors and sodium orthovanadate using the M-PER kit (Pierce Endogen).
One hundred micrograms of cell extract were incubated with 1 µl of antibody overnight
at 4 °C. The following antibodies were used GAPDH (Santa Cruz Biotechnology) at
36
1:1000, anti-Jagged-1(Santa Cruz Biotechnology), and equivalent amounts of control IgG
mouse IgG (Santa Cruz Biotechnology). One-fifth of the supernatant served as a loading
control. Protein G beads were added, and the antibody-antigen complex was then
precipitated, washed, eluted in 1× Laemmli sample buffer, and subjected to SDS-PAGE
electrophoresis followed by Western blot analysis. After IP, RNA was eluted from
protein G beads using the RNeasy kit and analyzed by qRT-PCR mRNA. Standard realtime PCR amplification curves were generated (r2 > 0.98) for Jagged-1 mRNA or
GAPDH controls using the threshold cycle (Ct) method. Jagged-1 and GAPDH primers
and probe were obtained from Assays on Demand (Applied Biosystems). All qRT-PCR
analyses were performed on an ABI 7900 PCR instrument (Applied Biosystems).
UV Crosslinking
Nuclear extracts were prepared from U251 MG cells using the Nu-Per kit (Pierce
Endogen, Rockford, IL) and the protein concentration was determined with the BCA
protein assay kit. The UV crosslinking was performed as previously described (Nabors et
al. 2003). The samples were electrophoresed on a 4–15% tris gradient gel (BioRad,
Hercules, CA), dried and exposed on a phosphorimager. For immunoprecipitation, antiHuR IgG or control IgG was added to the UV-crosslinked sample in immunoprecipitation
buffer as previously described (Nabors et al. 2003).
Generation of shHuR lentivirus
37
To generate the pLVTHM-shHUR vector shHuR primers were annealed and cloned into
the Mlu1 and Cla1 sites of the pLVTHM vector. The control pLVTHM plasmid was
obtained from Addgene, plasmid # 12247. Viral particles were packaged by as previously
described (Liu et al. 2009). A total of 106 U251 cells were infected with 2 ml of viral
supernatant in the final volume of 4 mL. At 48 h post infection, cells were washed with
DMEM-F12 media. Protein extracts and RNA from control and shHuR cells were
obtained and analyzed after 4 weeks of cells culture expansion as previously described
(Filippova et al. 2011)
RESULTS
Jagged-1 is overexpressed in GBM
Previous studies have shown that Jagged-1 protein is present in GBMs (Purow et
al. 2005). We examined a panel of glioma and control brain for Jagged-1 RNA
expression. The expression was consistently elevated in GBM samples compared to
control brain and lower grade astrocytomas. RNA was quantified by TAQMAN and
results are illustrated in figure 1B. Jagged-1 RNA contains a large 3’UTR that contains
sequence elements determined to be binding sites for the RNA-binding protein, HuR
(fig.1). We next examined tumor tissue samples for the presence of the Jagged-1 3’UTR
by RT-PCR. As demonstrated in figure 2A, the Jagged-1 3’UTR is robustly present in
GBM.
HuR protein associates with Jagged-1 mRNA
38
We initially wanted to ensure that the 3’UTR of Jagged-1 interacts with HuR. We
cloned the 3’ UTR of Jagged-1 using primers that spanned the 3’ UTR Forward:
GACTTGGAAAGTGCCCAGAG Reverse: CAGTTCCAGCTTCACAGCAG. Using
the Jagged-1 3’UTR as a probe illustrated in figure 2B, we performed a UV crosslinking
experiment in which U251 cell lysates were incubated with a radiolabeled Jagged-1
3’UTR and UV crosslinked such that any proteins that bind to the RNA would be
crosslinked. Immunoprecipitation of the crosslinked protein with a HuR specific antibody
showed that HuR and the radiolabeled Jagged-1 probe interact (Fig 2C). To determine
this is a specific interaction, a negative control reaction was also performed with normal
mouse IgG and the crosslinked extract with incubated with RNase. Figure 2C illustrates
proteinase K digest of the cross link reaction in lane 1, the crosslinked reaction in lane 2,
the IgG IP in lane 3 and the HuR specific IP in lane 4. To validate this interaction, we
performed RNA immunoprecipitation using a HuR specific antibody and cellular extracts
from U251. IP followed by qRT-PCR of the reaction suggested that of the total RNA
associated with HuR antibody, there was a greater than 10 fold increase in Jagged-1
compared to the control GAPDH. This strengthens the data supporting an association
between HuR protein and Jagged-1 RNA (Fig. 2D).
Knockdown of HuR impacts Jagged-1 RNA kinetics
Next we examine if modulating HuR protein levels would affect Jagged-1 protein
or RNA levels. We created shHuR clones that knock down the endogenous HuR and
probed for Jag1 (Filippova et al. 2011). In the western blot in figure 3A, compared to
39
scramble control clones, shHuR clones knock down the expression of HuR and Jagged-1
protein when the same blot was probed for Jagged-1 expression. To begin to understand
how HuR is regulating the protein levels of Jagged-1 we next looked at the kinetics of the
mRNA transcript. At 4 and 6 hours post actinomycin D treatment, Jagged-1 mRNA
levels are less in the knock down of HuR compared to control (Fig 3B). This data
suggests that HuR regulates Jagged-1 mRNA by stabilizing the transcript.
Silencing of HuR effects Jagged-1 RNA and polyribosomal distribution
To test if HuR regulates Jagged-1 not only by stabilizing the mRNA but also
affecting its translation we performed a polyribosome fractionation and analysis. U251
and shHuR cells were treated with cycloheximide to inhibit protein synthesis, lysed, spun
through a sucrose gradient and fractions collected after mass spectrophotometry that was
continuously charted. In figure 4A is the representative chart that has the fractions
marked and illustrate the peaks of the monosome and polysomal fractions. Below the
charts are western blots of fractions probed for Jagged-1, eif4E, rpS6 and HuR. (Fig. 4B)
When HuR is knocked down, more Jagged-1 protein is bound in the initial fractions
compared to control and there is less rpS6 protein in all fractions suggesting less
translating polysomes when HuR is knocked down. These data lead to the conclusion that
knocking down HuR reduces translating polysomes indicating that HuR regulation of
Jagged-1 also occurs at the level of translation.
Myeloid derived cells distribution is altered in syngeneic intracranial tumor model
40
Notch ligands to include jagged-1 are implicated in maintenance of cell
populations and chemotaxis (Lewis et al. 1988; Artavanis-Tsakonas et al. 1999; Brabletz
et al. 2011). In addition HuR is a known regulator of multiple chemotactic factors (Tran
et al. 2003; Annabi et al. 2007). We hypothesize that cellular responses to glioma from
the brain microenvironment could influence circulating cellular elements. To test this, we
implanted GL261 glioma in WT and HuR Tg animals.
We next wanted to study the cellular compartments of the peripheral blood and
the spleen from animals with GL261 tumors. Animals with implanted tumors were
sacrifice and PBMCs and cells from the spleen were isolated and sorted by FACS
analysis according the schematic in Figure 5. Cells were gated by FSC and SSC. The
cellular subpopulation was gated according to expression of the fluorophore conjugated
(Tables 1) antibodies. From the cell subset, CD3+, CD4+, CD11+ CD45+ cells were
gated to express the T cell and myeloid cell populations. CD45+ cells were further gated
to quantify granulocytes and lymphocytes. CD11b+ myeloid cells were further gated to
study the myelocytes, and CD11b+GR1+ MDSCs. Subsets of the MDSCs were gated by
Ly6c+ and Ly6g+ cells which represent the MO-MDSC and PMN-MDSC populations.
Comparing Wt to Tg animals PBMCs, Tg animals trend to express fewer, CD3+ and
CD4+ T cells, myeloid cells, and MDSCs but more monocytes, lymphocytes and
leukocytes. (Fig 6) Comparing Wt to Tg animals spleen MDSCs, Tg mice trend to
express more monocytic cells. (Fig 6) Although not statistically significant, comparing
Wt animals to Tg, there appears to be a switch in the number of myeloid cells.
41
DISCUSSION
The RNA binding protein, HuR, regulates many genes with cytokine and
chemokine functions involved in inflammation and cancer such as COX-2, VEGF,
iNOS, TGF-β, EGF HIF-1α, ΤNF -α IL-6 and IL-8 (Nabors et al. 2003; Sheflin et al.
2004; Galban et al. 2008). The development and maintenance of cell populations along
with chemotactic influences are increasingly recognized as important roles for theses
secreted factors. A newly identified regulator of cellular differentiation is the Notch
pathway. Notch signaling has a broad array of functions but appears critical for the
development of the hemopoetic system and promoting lineage choice (Lehar et al. 2005).
Notch signaling occurs through a ligand receptor interaction thus it may be regulated at
many levels including extracellular, cytoplasmic, and nuclear (Mumm and Kopan 2000).
Here we show that HuR interacts with the Notch ligand Jagged-1 which contains
a large 3’UTR with many AREs. We confirm that Jagged-1 mRNA is elevated in GBM
compared to non-cancerous brain tissue and low grade tumors (Purow et al. 2005). UV
crosslinking and RNA immunoprecipitation experiments illustrated an interaction
between HuR protein and Jagged-1 RNA. HuR regulates Jagged-1 protein expression in
U251 human glioma clones. When HuR is knocked down, the levels of Jagged-1 protein
are reduced. Kinetic assays suggest that HuR regulation of Jagged-1 protein is regulated
in part by stabilizing Jagged-1 mRNA. These data strongly suggest that HuR regulated
Jagged-1 RNA stability via the 3’ UTR.
Notch signaling through Jagged-1 has been found to be instrumental in not only
the development of the hematopoietic system but in maintenance of stem cells and
42
lineage choices of particularly lymphoid cells (Varnum-Finney et al. 1998; VarnumFinney et al. 2003). The expansion of cell populations is now recognized as a common
event in disease states such as autoimmune inflammatory conditions and cancer.
Myeloid-derived suppressor cells (MDSC) are a broad group of cells capable of
accumulating in the circulation, spleen, or sites of inflammation or cancer (OstrandRosenberg and Sinha 2009). It is thought that disease state generated secretion of soluble
factors such as cytokines and chemokines are responsible for this effect. MDSCs are able
to alter the host immune response by down regulation of immune surveillance and antitumor immunity (Gabrilovich et al. 2007). These are hallmark features of GBM.
Raychaudhuri and colleagues have demonstrated the accumulation of MDSCs in the
peripheral blood of patients newly diagnosed with GBM specifically the neutrophilic
(CD15+CD33+HLADR-), monocytic (CD14+CD33+HLADR-, and lineage-negative
(CD15-CD14-CD33+HLADR-) cells (Raychaudhuri et al. 2011).
The evolving recognition of the interactions and reactions between cancer cells
and the host tissue environment as a promoter of the malignant phenotype is only recently
being studied. The situation with malignant glioma is likely one of the most complex of
these interactions. Not only is the extracellular matrix of the CNS unique but the location
is one of immunological privilege. The tumor environment is a complex and many cell
types play a multiple roles in tumor progression, suppression, and the overall pathology.
The glioma tumor mass is composed of microglia, astrocytes, endothelial cells, immune
cells, neural precursor cells, pericytes and fibroblasts. The tumor mass secretes a number
of cytokines and chemokines that allow peripheral immune cells to be recruited into the
tumor microenvironment and change the normal behavior of the other cell types. In this
43
manner, the host tumor can cause systemic changes that further compound efforts to treat
the diseased tissue (Charles et al. 2011).
The complexity of the tumor environment, secreted factors either by the tumor or
by non-transformed normal tissue cells in response to cancer and immnunological
response hold the potential to influence cancer behavior. This effort is seen primarily at
the level of Jagged-1 protein production utilizing a novel transgenic animal with HuR
specifically overexpressed in brain astrocytes, we are able to quantify circulating cellular
elements in response to an intracranial tumor. Several questions are left to be answered.
In future studies we want to study the cells recruited to the tumor mass and detect any
differences between the Wt and Tg animals. We would like to significantly validate the
trends we see in the PBMC and spleen compartments. We want to know if blocking
Jagged-1 signaling would change the distribution of the immune cell panel.
Understanding how HuR regulated Jagged-1 and how this regulation affects MDSCs in
response to intracranial tumors could give insight into a method to decrease tumor
evasion of immune responses.
44
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Abdelmohsen, K. and M. Gorospe (2010). "Posttranscriptional regulation of cancer traits
by HuR." Wiley Interdiscip Rev RNA 1(2): 214-229.
Annabi, B., J. C. Currie, et al. (2007). "Inhibition of HuR and MMP-9 expression in
macrophage-differentiated HL-60 myeloid leukemia cells by green tea polyphenol
EGCg." Leuk Res 31(9): 1277-1284.
Artavanis-Tsakonas, S., M. D. Rand, et al. (1999). "Notch signaling: cell fate control and
signal integration in development." Science 284(5415): 770-776.
Brabletz, S., K. Bajdak, et al. (2011). "The ZEB1/miR-200 feedback loop controls Notch
signalling in cancer cells." EMBO J 30(4): 770-782.
Brenner, M., W. C. Kisseberth, et al. (1994). GFAP promoter directs astrocyte-specific
expression in transgenic mice. 14: 1030-1037.
Charles, N. A., E. C. Holland, et al. (2011). "The brain tumor microenvironment." Glia
59(8): 1169-1180.
Filippova, N., X. Yang, et al. (2011). "The RNA-binding protein HuR promotes glioma
growth and treatment resistance." Mol Cancer Res 9(5): 648-659.
Gabrilovich, D. I., V. Bronte, et al. (2007). "The terminology issue for myeloid-derived
suppressor cells." Cancer Res 67(1): 425; author reply 426.
Gabrilovich, D. I., S. Ostrand-Rosenberg, et al. (2012). "Coordinated regulation of
myeloid cells by tumours." Nat Rev Immunol 12(4): 253-268.
Galban, S., Y. Kuwano, et al. (2008). "RNA-binding proteins HuR and PTB promote the
translation of hypoxia-inducible factor 1alpha." Mol Cell Biol 28(1): 93-107.
Lagnado, C. A., C. Y. Brown, et al. (1994). "AUUUA is not sufficient to promote
poly(A) shortening and degradation of an mRNA: the functional sequence within AUrich elements may be UUAUUUA(U/A)(U/A)." Mol Cell Biol 14(12): 7984-7995.
45
Lehar, S. M., J. Dooley, et al. (2005). "Notch ligands Delta 1 and Jagged1 transmit
distinct signals to T-cell precursors." Blood 105(4): 1440-1447.
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responses of neutrophils and monocytes from chronic dialysis patients." Clin Nephrol
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Angiogenic Factor Up-Regulation in Malignant Glioma Cells: A Role for RNA
Stabilization and HuR. Cancer Research. 63: 4181-4187.
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Purow, B. W., R. M. Haque, et al. (2005). Expression of Notch-1 and Its Ligands, DeltaLike-1 and Jagged-1, Is Critical for Glioma Cell Survival and Proliferation. 65: 23532363.
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Raychaudhuri, B., P. Rayman, et al. (2011). "Myeloid-derived suppressor cell
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47
A
B
Figure 1. Jagged-1 3’ UTR AREs and Jagged-1 RNA expression in normal and tumor
brain samples. A. 3’UTR of Jagged-1 mRNA transcript contains many A/U rich elements
(circled in red, upper panel). B. RNA expression of Jagged-1 is elevated in GBM brain
tissue.
48
A
B
C
D
Figure 2. HuR associates with Jagged-1. A. Cloning of the Jagged-1 3’ UTR from tissue
samples. RT-PCR of RNA with primers to the Jagged-1 3’ UTR (upper top panel). B. An
illustration of RNA probe design for the UV crosslinking experiment. C. HuR binds to
Jagged-1 probe but not IgG control. (lower left panel). D. RNA immunoprecipitation of
Jagged-1 and GAPDH RNA with HuR antibody. HuR bind over 10-fold more of the
Jagged-1 RNA compared to GAPDH.
49
A
B
Figure 3. Knockdown of HuR protein reduces the levels of jagged-1 protein and
decreases the ½ life of jagged-1 mRNA in shHuR clones. A. Western blot of protein
extracts of U251 glioma cells expressing shHuR illustrating knock down of HuR protein
also reduces the expression of Jagged-1 protein compared to the scramble control clones.
B. Jagged-1 RNA ½ life is decreased when HuR is knocked down.
50
Figure 4. Polyribosome redistribution of Jagged-1 in U251 control and shHuR cells.
Polyribosome fractionation and analysis of U251 glioma cells showed a redistribution of
Jagged-1 protein in fractions compared to control cells. eIF4E and rpS6 are controls for
monosomes and translating polysomes.
51
Figure 5. Example of gating strategy for isolation of MDSC population of cells from
PBMCs and Spleen. Cells were gated using FSC and SSC. The cell population was then
gated for subpopulations based on their fluorescence of cell markers listed in table 1.
52
PBMCs
A
Wt
Tg
100
50
C
D
3
C +T
D c
M 4+ ell
ye T s
lo ce
M id lls
ye ce
N loc lls
eu y
t t
M rop es
on h
oc ils
yt
M MD es
O
PM -M SC
s
N DS
C
M
Le D s
G uk SC
ra oc s
Ly nul yte
m oc s
ph yt
oc es
yt
es
0
Spleen
B
Wt
Tg
100
50
C
D
3
C +T
D c
M 4+ ell
ye T s
lo ce
M id lls
ye ce
N loc lls
eu y
t t
M rop es
on h
oc ils
yt
M MD es
O S
PM -M C
s
N DS
C
M
Le D s
G uk SC
ra oc s
Ly nul yte
m oc s
ph yt
oc es
yt
es
0
Figure 6. FACS of PBMC and spleen cells from Wt and Tg mice. Tg mice have fewer T
cells, myeloid cells and MDSCs compared to Wt mice PBMCs and spleen cells. Tg mice
have more monocytes, leukocytes and CD45+ lymphocytes compared to Wt mice
PBMCs. Tg mice spleen cells have lower number of leukocytes and CD45+ lymphocytes
and increased number of granulocytes.
53
Table 1. Antibodies and conjugated fluorochrome for FACs analysis of PBMCs and
spleen.
Antigen
Flurochrome
CD3
PE-Cy7
CD4
PerCP-Cy5.5
CD11b
Alexa 700
CD45
APC-Cy7
F4/80
FITC
Gr1
V450
Ly6c
PE
Ly6g
APC
54
Table 2. Cell markers for cellular compartments.
Cell Surface Markers
Cell Compartment
CD3+ cells
CD3+ Tcells
CD4+ cells
CD4+ Tcells
CD11b+ cells
Myeloid cells
CD11b+SSChi cells
Myelocytes
GR1+hi
Neutrophils
GR1+int
Monocytes
CD11b+GR1+
MDSCs
CD11b+GR-1+ Ly6c+cells
MO-MDSCs
CD11b+GR-1+Ly6g+cells
PMN-MDSCs
CD45+cells
Leukocytes
CD45+ SSChi
Granulocytes
CD45+ SSClow
Lymphocytes
55
SUMMARY AND DISCUSSION
The generation of a transgenic mouse with astrocyte-specific overexpression of
the RNA-binding protein, HuR (Elav1) has allowed us to investigate the role of posttranscriptional gene regulation in an animal model of disease. The HuR transgene
demonstrated widespread expression in the CNS (Table 1) with robust expression in the
spinal cord and brainstem. Despite an increasing amount of biochemical and molecular
research characterizing the expression and function of HuR, there is limited effort to
extend this work to the level of whole animals and place it in the setting of neurological
disease. We have characterized the impact of the HuR transgenic animal in two
neurological diseases: demyelinating disease and primary brain cancer. In both disease
states, we hypothesized that the induction of a neurological condition would result in a
differential response that would segregate based on the expression of the HuR transgene.
The induction of demyelinating disease with the EAE model tested and supported
our hypothesis. The observation that disease severity differed in WT compared to Tg but
also that a gender difference existed was novel and unexpected. The difference was
quantifiable at a cellular level. The gender difference was more extensively examined
than we reported in Chapter 1. In an effort to reverse the gender specific phenotype, male
animals were treated with pregnancy levels of estrogen and female animals were
ovariectomized. Males were dosed with 2.5mg estrogen starting one week prior to and
throughout the 30 day EAE induction. We did not find a significant difference in male
56
mice treated with estrogen compared to Wt mice in disease onset or severity (Fig. 1). In
Table 2, the clinical parameters for the ovariectomized female are shown. Removing
estrogen from the females abrogated the protective effect of the transgene in the Tg
females mice compared to Wt as shown in chapter 1, figure 2B supporting the
observation of a gender difference and suggesting a sex hormone regulation impact on
HuR.
Despite significant differences in phenotype and cell assays, an extensive effort to
quantify molecular differences did not identify causative genes. Microarray comparisons
between the spinal cords of female Wt and Tg animals show similar gene expression
patterns (Nicholas, Lukas, Jafri, Faoro, & Salgia, 2006). We performed microarray
analysis of spinal cords from naïve and EAE-induced mice. For the latter analysis, spinal
cords were harvested 14 days after MOG inoculation which represents the onset of
clinical signs. A heatmap of the results was obtained through hierarchical clustering (Fig.
2). The list of gene targets used for this map was generated by loose criteria of P<0.05
and fold-change greater than two for naïve versus EAE comparisons. No false discovery
rate was applied in order to show as many genes (516 in total) affected in the two
comparisons. We observed two major branches clustered vertically, indicating a clear
separation between the non-disease group (the left six chips) and the EAE group (the
right six chips). However, the transgene did not introduce enough separation from its
corresponding disease or non-disease controls. This occurred when clustering was done
either by individual chips or by the average intensity of all three chips. To validate the
microarray data, we performed qRT-PCR on 9 targets that emerged from this comparison
(Fig. 3). The fold-change (naïve versus EAE) of these targets ranged from 6 to 136. There
57
was no significant difference in target mRNA levels between wild-type and Tg mice
either naïve or EAE-induced. These findings validate the array data and suggest that the
transgene effect is either at a posttranscriptional level or not significant enough to be
shown in Affymetrix-based ST-arrays with 3 chips in each group.
The EAE model generated a substantial CNS inflammatory event with clear and
consistent differences based on gender. The clinical score and cell assays were
supportive, however, a molecular difference based on the transgenes expression was not
identified. Potential explanations we considered are the difference may exist at the
protein level and not the RNA level given the effects of this RNA binding protein on
more on protein expression and the targets we were focused on, the inflammatory
cytokines and chemokines are not the correct group to study.
The use of the intracranial glioma model provided a second avenue to examine
differences between wildtype and transgenic animals and to further examine the role of
cellular mediators of disease states that may be influenced by expression of HuR. The
results presented in chapter 2 support a strong interaction between HuR and the Notch
ligand, Jagged-1. In addition, at the molecular level we were able to demonstrate a
regulatory impact of HuR on Jagged-1. The effort to extend this finding to the animal
model suggests an impact but the current experimental results are weak. Additional cell
based experiments to examine the effect of Jagged-1 secretion and jagged-1 levels after
HuR silencing are important. Also potential co-culture experiments to examine the
impact of Jagged-1 on myeloid cell expansion could have focused our animal studies. In
summary, the HuR transgenic mice have provided a novel opportunity to study RNA
regulation as a contributor to neurological disease. A clear impact of the transgene was
58
noted in the EAE model but a molecular explanation was not identified. This contrasted
with the brain cancer model where a strong molecular role was seen but less apparent in
the animal model.
59
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Table 1. CNS distribution of HuR transgene
Females1
1
Males
Cortex
+
+
Hypothalamus
++
++
Medulla
+++
++
Midbrain
++
++
Olfactory bulb
++
++
Pons
++
++
Spinal cord
++++
++++
Score represents the average of 5 high powered fields from two transgenic mice.
+,0-2cells; ++, 3-10; +++, 11-20++++
70
Table 2. EAE clinical parameters in ovariectomized wild type and HuR Tg mice
CDIA
Disease
Onset
B
Disease
IncidenceC
Max Clinical
ScoreD
WT (n=9)
63.3
12.1d
100
3.9
HuR Tg female (n=14)
61.3
12.6d
100
3.9
A
Cumulative Disease Index is the mean of the sum of daily clinical scores observed
between days 7 and 30.
B
Disease onset is defined as the first day of two consecutive days with a clinical score of
two or more.
C
Disease incidence is defined as the percent of mice that displayed any clinical signs of
disease.
D
Maximum mean clinical score is the mean of the highest daily clinical score for each
mouse.
71
WT Placebo (2)
TG Placebo (4)
WT E2 (4)
TG E2 (2)
Clinical Score
4
2
35
30
25
20
15
10
5
0
Days Post-immunization
Figure 1. . EAE in males with or without estrogen. EAE phenotype is attenuated early in
disease in male WT supplemented with E2. Clinical assessment of HuR-Tg and littermate
controls (WT) after induction of EAE with MOG peptide. Mice were examined for a total
of 30 days. Numbers of mice in each group are shown in parentheses.
72
Figure 2. Microarray comparison of female Tg and WT spinals cords show similar gene
expression patterns. Heat map of 516 genes showing greater than 2-fold change (p<0.05)
for naïve versus EAE comparison in wild-type (W) and transgenic (T) female mice.
Brackets indicate… W, wild-ype; T, transgenic.
73
40
Naive
EAE
20
0
15
Naive
EAE
EAE
0
74
EA
E
IL1b
A
E
80
Tr
an
sg
en
ic
5
ro
l
Tr
an
sg
en
ic
C
on
tr
ol
EA
Tr
E
an
sg
en
ic
EA
E
60
GAPDH Ratio
on
t
ro
l
Tr
an
sg
en
ic
C
on
tr
ol
EA
Tr
E
an
sg
en
ic
EA
E
C
Tr
an
sg
en
ic
C
on
tr
ol
EA
Tr
E
an
sg
en
ic
EA
E
0
c
Naive
GAPDH Ratio
10
tr
ol
E
6
EAE
ol
8
GAPDH Ratio
CCL2
Tr
an
sg
en
i
Clec5a
20
C
on
t
0
Naive
GAPDH Ratio
10
30
C
on
tr
EAE
EA
E
Naive
c
ccl6
A
E
30
ol
E
2
ro
l
Tr
an
sg
en
ic
C
on
tr
ol
EA
Tr
E
an
sg
en
ic
EA
E
0
C
on
tr
4
C
on
tr
ol
5
GAPDH Ratio
GAPDH Ratio
EAE
c
ol
20
C
on
t
Tr
an
sg
en
ic
C
on
tr
ol
EA
Tr
E
an
sg
en
ic
EA
E
C
on
tr
ol
Naive
GAPDH Ratio
GAPDH Ratio
40
C
on
0
C
on
tr
ro
l
Tr
an
sg
en
ic
C
on
tr
ol
EA
Tr
E
an
sg
en
ic
EA
E
C
on
t
10
Tr
an
sg
en
i
EA
E
A
E
GAPDH Ratio
Chl313
Tr
an
sg
en
i
c
ol
E
Tr
an
sg
en
i
C
on
tr
c
ol
Tr
an
sg
en
i
C
on
tr
15
200
ccl5
150
100
Naive
50
EAE
0
300
cxcl10
200
100
Naive
EAE
0
ccr1
10
60
TIMP
40
Naive
20
EAE
0
Figure 3. Validated Target from the microarray. Chemokines and cytokines that were
validated from the RNA array by qRT-PCR show significance between Naïve (white
bars) and EAE (black bars) animals but not between control and HuR transgenic animals.
APPENDIX A
75
APPENDIX B
76