Faculty Research Fellowship Application for Summer 2007
Title: “Neurotrophic properties of microglia: implications for neuronal survival and
regeneration.”
Principal Investigator: Annemarie Shibata, Ph.D.
Abstract: Proper development and function of the central nervous system (CNS) is
regulated by cellular interactions between neuronal and glial cells during periods of
homeostasis and following injury or disease. Microglia, resident glial cells of the CNS,
direct immune responses in the CNS and play an important role in neuronal
differentiation, survival, and function. Brain microglia mediate inflammatory responses
that facilitate both neurotrophic (those that support the survival of neurons and glia) and
neurotoxic (those that damage and destroy neurons and glia) processes in the CNS.
Substantial information demonstrates that activated microglia can be neurotoxic,
however, much less is known about neurtrophic properties of microglia and the molecular
mechanisms underlying those neurotrophic properties.
This proposal presents
experiments utilizing an in vitro model system to test the hypothesis that microglia can
elicit enhanced neurotrophic responses in neurons through activation of specific
intracellular signaling pathways. Results from these studies will help to determine
whether microglial neurotrophic properties provide a mechanism for survival, repair and
regeneration of neurons following CNS trauma or disease.
I. and II.
Statement and Significance of the Problem
Historically, immune responses in the CNS are considered to be tightly regulated.
Restriction of immune activity in the CNS or “immune privilege” is likely an
evolutionarily adaptation that has been selected over time since many inflammatory
reactions lead to injury or death of “bystander” or healthy cells (Streilein, 1995). The
idea of CNS “immune privilege” is supported by data showing that neuronal parenchymal
cells express low levels of major histocompatiblity complex (MHC) class I and II
antigens (Matsumoto et al. 1986, Wucherpfennig, 1994), that CNS cells secrete local
factors that suppress immune responses (Streilein, 1993) and that transplantation of
allografts into CNS parenchyma show prolonged survival demonstrating a suppression of
immune responses in the brain (Rao et al., 1989; Poltorak and Freed, 1991). Increased
activation of microglia, associated with the release of proinflammatory cytokines,
reactive oxygen and nitrogen species, complement proteins and proteinases may
contribute to neuronal and glial degeneration in diseases such as stroke, Alzheimer’s
disease (AD), multiple sclerosis (MS), Parkinson’s disease (PD), and acquired immune
deficiency syndrome dementia complex (AIDS-DC) (reviewed by Kim and de Vellis,
2005). Such studies suggest that activation of immune responses in the brain are only
neurotoxic and contribute to the death of neurons and glia during disease.
Conversely, a growing body of work suggests that immune responses of microglia can
promote neuronal survival and function. Microglia phagocytose and remove cellular
debris during development and following ischemic or traumatic injury (Carson, 2002;
Danton and Dietrich, 2003; Freeman, 2006). Microglia secrete factors that recruit
leukocytes and present antigen to stimulate T cell responses at sites of bacterial or viral
infection (Nelson et al., 2002). Stimulated microglia enhance the survival of retinal axons
following injury (Shaked et al., 2004; Schwartz and Kipnis, 2004). In cell culture
experiments, microglial conditioned media protects neurons from excitotoxic injury
(Watanabe et al., 2000) while microglial-neuronal contact has been associated with an
increase in neuronal survival (Zietlow et al., 1999). Several studies have shown that
microglial cells secrete neurotrophic factors that promote neuronal differentiation and
survival (Aarun et al., 2003; Kim and de Vellis, 2005). Taken together, these studies
suggest that microglia are multifunctional immune cells with not only neurotoxic activity
but also neuroprotective activity.
While activation of microglia is commonly considered to be detrimental to neuronal
survival, accumulating evidence demonstrating that microglia support neuronal survival,
differentiation and regeneration calls for continued investigation of causal molecular
mechanisms. Little is known about the mechanisms regulating the neuroprotective
properties of microglia and the mechanisms underlying the behavioral response of
neurons to these microglial signals. The significance of microglia protective mechanisms
and whether these mechanisms are potential therapeutic targets warrants additional
investigation. This study is directed at investigating the intracellular signaling
mechanisms underlying neuroprotective properties of microglia and the subsequent
neuronal response.
Experiments are directed at dissecting the specific factors and
signaling cascades employed by microglia to express their neuroprotective phenotype.
Identifying the mechanisms used by microglia to aid in CNS protection will help
determine whether manipulating microglial function is a potential target for
neuroprotection in CNS disease or following CNS damage.
III.
Summary of Pertinent Literature
Central nervous system (CNS) immune activity plays a fundamental role in the proper function of
the nervous system. Microglia and infiltrating T cell lymphocytes mediate inflammatory
responses that facilitate both neurotrophic (those that support the survival of neurons and glia)
and neurotoxic (those that damage and destroy neurons and glia) processes in the CNS. The
importance of immune responses in the brain was first indicated by studies investigating the role
of microglia, the resident immune cells of the CNS, in the initiation and progression of
neurological disease. A significant body of work has elucidated much about the neurotoxic
phenotype of activated microglia. In Alzheimer’s disease (AD), multiple sclerosis (MS), and
Parkinson’s disease (PD), activated microglia and macrophages localize around areas of
pathologic lesions. Microglia activated in specific CNS disease states or following traumatic
injury secrete inflammatory cytokines, chemokines, arachidonic acid, reactive oxygen and
nitrogen species, and growth inhibiting proteins such as protaglandins (reviewed by Kim and de
Vellis, 2005). Similarly, activated microglia following stroke has been shown to secrete
neurotoxins enhancing neuronal death (Danton and Dietrich, 2003). In summary, dysregulaton of
microglial activity has been associated with neuronal and glial cell death in several models of
CNS disease and injury (Minegar et al., 2002; Kim and de Vellis, 2005). However, the ability of
microglia to act as scavengers, to affect intracellular killing of microbial pathogens and control
the production and/or regulation of secretory factors may also provide a pivotal protection
mechanism for the CNS. Consequently, it has been hypothesized that microglia are in principle,
neuroprotective cells (Hanisch, 2002).
Primarily as a host-defense reaction, the immune-inflammatory response of microglia can act to
clear debris resulting from neuronal injury and eliminate foreign invaders. Microglia may play a
role in differentiation of neural stem cells in the developing and mature brain. Primary microglial
cultures from fetal mouse brain release soluble factors that direct migration and promote
differentiation of neuronal precursor cells although the exact nature of the secreted factors has not
been determined (Aarun et al., 2003). Recent work by Morgan et al. 2004 demonstrated that
conditioned media from unstimulated microglia does contain neurotrophic factors that support the
proliferation of primary cerebellar granular neurons in vitro. Stimulation of microglia with
NMDA enhances their neuroprotective activity (Mitrasinovic et al., 2005). Neurotrophins are
potent survival signaling molecules that direct neuronal differentiation, survival and outgrowth.
A number of in vitro and in vivo studies have shown the production of neurotrophic factors from
microglia and other immune cells. For example, microglial-conditioned media contains nerve
growth factor (NGF), neurotrophin 3 (NT3 or Ntf3), and brain-derived neurotrophic factor
(BDNF) (Kim and de Vellis, 2005). Production of BDNF has been associated with activation of
the protein kinase C signaling cascade in rat microglial culture (Nakajima et al., 2001).
Additionally, microglia secrete several growth factors and cytokines such as TNFα, IL-1α, IL-1β,
IL-6, GDNF, and TGFβ. These signaling molecules have been shown to protect neurons from
NMDA receptor mediated excitotoxicity (Lai and Todd, 2006). In vivo studies have shown that
specifically stimulated microglia, macrophages, and infiltrating T cells protect neuronal axons
from secondary degeneration following injury, degrade inhibitory proteins that restrict neuronal
survival and regrowth, and induce neurotrophin production (Hauben et al., 2001; Barouch and
Schwartz, 2002; Shaked et al., 2004; Schwartz and Kipnis, 2004, Ziv et al., 2006).
In combination, these studies provide an enticing view of the multifunctional roles of microglia in
the CNS. Elucidating the intracellular mechanisms regulating neurotrophic and neurotoxic
function in microglia is essential for understanding the differences between these two distinct
phenotypes. While little is known about the mechanisms controlling neurotrophic function in
microglia, some data provide plausible directions for future studies. Previously published work
by the primary investigator demonstrates that macrophages (immune cells of the same lineage as
microglia) enhance neuronal process outgrowth, MAPK intracellular signaling and synaptic
function following stimulation with specific exogenous cues (Shibata et al., 2003). Unstimulated
microglial conditioned media enhances the growth and survival of cerebellar granule cells
(CGCs) by activating MAPK and PI3K/Akt signaling pathways in CGCs (Morgan et al, 2004).
Microglia activated in ischemia model systems exhibit enhanced p38 and MAPK pathway
activation, although this activation is associated with both neurotoxic and neuroprotective
function (Lai and Todd, 2006). Additional work with ceramide-stimulated microglia suggests
that activation of signaling pathways without upregulation of gene transcription selectively
stimulates BDNF and not cytokine release from microglia (Lai and Todd, 2006). These studies
demonstrate that regulation of microglia signaling pathways determines in large part their final
phenotype.
Taken together, these data suggest that microglia posses modifiable neurotoxic and neurotrophic
properties, making them potential targets for neuroprotective therapies. While there is a large
body of work describing the neurotoxic properties of microglia during disease and following
trauma, the neuroprotective role of microglia remains poorly understood. The following studies
are designed to help define the mechanisms by which microglia influence neuronal growth,
differentiation and survival. Understanding the molecular mechanisms by which microglia
influence neuronal behavior will provide insight into the intrinsic neuroprotective role of immune
activity in the CNS.
IV.
Research Hypothesis and Specific Aims (Questions)
This study proposes to test the hypothesis that microglia, the resident immune cells of the
brain, can elicit enhanced neurotrophic responses in neurons through the release of
neurotrophins and activation of specific intracellular signaling pathways.
Specific Aim 1: To develop an in vitro model system to evaluate neurotrophic properties
of microglia.
Specific Aim 2: To determine whether the neurotrophins NGF, NT3 and/or BDNF are
secreted by microglia in the in vitro model system through the activation of protein
kinase C (PKC), mitogen-activated protein kinase (MAPK), and/or the PI3K/Akt
pathway.
V. Design and Methods
Projects in the laboratory are in their infancy since my work began at Creighton University in
August of 2006. During the remainder of the Fall semester and Spring of 2007, an in vitro model
system to evaluate the ability of unstimulated and stimulated microglia to enhance neuronal
survival and differentiation will be established. Dissociated cell culture systems are powerful
models that can be used to evaluate the responses of target cells at the cellular and molecular
levels. Similar in vitro model systems have been established within the laboratory to examine the
neurotrophic role of stimulated macrohages. Results showed that unstimulated and, to a greater
extent, stimulated macrophages possess neuroprotective properties (Shibata et al., 2003).
Stimulated macrophages show an increase in neurotrophin production, especially for BDNF, and
an increase in MAPK activation and protein synthesis in neurons was observed (Shibata et al.,
2003). As brain macrophages, microglia are likely to use similar mechanisms for their
neuroprotective function. Recent work from other laboratories has shown that specific
stimulation of the microglial cell line BV-2 reduced N-methyl- D- aspartate (NMDA) mediated
neurotoxicity (Mitrasinovic et al., 2005). The signaling mechanisms underlying this effect are
unknown. Consequently, this proposal addresses the hypothesis that, dependent on the
stimulae, resident immune cells of the brain, or microglia, can elicit enhanced neurotrophic
responses in neurons through the release of neurotrophins and activation of specific
intracellular signaling pathways.
A series of three specific aims are designed to dissect the proposed hypothesis. The first specific
aim will be accomplished in the Spring and early Summer of 2007. The second specific aim will
begin to be addressed in the later part of Summer of 2007. Briefly, the experimental design and
methods are as follows:
Specific Aim 1: To develop an in vitro model system to evaluate neurotrophic properties of
unactivated and activated microglia.
Design: The experimental design used to address this specific aim requires the establishment of
co-culture assay systems of primary neuronal cultures and cultured microglial cell lines. A
summary of the steps involved establishing the in vitro model system is described below.
Isolation and culture of cortical neurons: Cultures of primary rat cortical neurons have been
previously established in the tissue culture facility of the Biology Department at Creighton
University. Cultures show a high percentage of healthy, differentiated neurons and similar
cultures will be used for all experiments relating to this protocol (Schmuck and Shibata, 2004).
Isolation and cultivation of rat cortical neurons follows the procedures most recently described by
Meberg and Miller (2003). Cortical cells are resuspended in neurobasal culture media and plated
onto poly-D-lysine coated plates and 0.5 X 106 cells per well in 2 well chamber tec slides
(immunocytochemistry and co-culture experiments) or 0.5 x 106 cells per well for 24 well plates
(for protein analysis and signal transduction studies) and maintained at 37oC, 5% CO2. After 3-4
days in culture, cytosine arabinofuranoside (10 µM) is added to cultures and to halt astrocytic
proliferation. Purity of cultures is confirmed using monoclonal antibodies against neurons
(MAP-2) and astroglia (GFAP) with usually >95% of cells positive for anti-MAP2.
Isolation and culture of microglia: Culturing of microglia involve the proliferation of microglial
cell lines. MLS-9 is a rat microglia cell line and is the preferred cell line since neurons are
isolated from rats. Primary microglia from postnatal 3-4 day old rat pups can be separated from
other CNS cell types on a Percoll gradient and then cultured. The MLS-9 cell line is more likely
to provide consistent results, is easily grown, does not require the sacrifice of animals, has been
shown to express cellular markers similar to primary microglia, and provides the number of cells
necessary for the multiple experiments proposed. Microglial cells will be seeded onto poly-Dlysine coated coverslips or onto transwell inserts (BD Falcon, Fisher Scientific) at 0.5 x 106 cells
per well for co-culture experiments, immunocytochemistry, protein analysis and signal
transduction assays. Microglial cultures are maintained in supplemented neurobasal media at
37oC, 5% CO2. Purity of microglia cultures will be determined by immunocytochemistry
experiments demonstrating the expression of the microglia marker OX-42.
Microglial and neuronal co-culture and activation: Following 3-4 days in culture after plating,
microglia coverslips or transwell inserts will be placed into neuronal cultures for 12 hours.
Following equilibration, half of the culture media will be replaced with control culture media or
culture media containing a final concentration of 5 or 100 µM NMDA (Tocris Cookson, Ballwin,
MO). Previous work has shown that 5µM NMDA is not neurotoxic while 100 µM NMDA is
neurotoxic. Further, previous studies have shown that treatment microglia can reduce the
neurotoxic effects of 100 µM NMDA (Mitrasinovic et al., 2005). As a negative control, NMDA
treatments without the presence of microglia will be assessed for neurotoxic effects on neuronal
cultures. Immunoreactivity of microglia for ED1 will confirm the presence of activated
microglia. NMDA mediated neurotoxicity is an appropriate neurotoxic signal because glutamate
is a prevalent neurotransmitter in the CNS. Regulated release of glutamate controls proper CNS
excitatory signaling while excessive release of is involved in the death of neurons.
Neurotrophic assays and interpretation of data: Co-culture experiments will be analyzed for
increased neuronal survival, expression of mature neuronal proteins and morphological changes,
cell death by necrosis and apoptosis. Cell viability will be assessed using double-staining method
using fluorescein diacetate (FDA) and propidium iodide (Kingham et al., 1999). Viability can
also be assessed with MTT conversion assays and plased read at 490nm on an ELISA plate
reader. Neuronal cultures will be assessed for necrosis by measuring the release of lactate
dehydrogenase and apoptosis by Hoechst 33342 staining (Kinghanm et al., 1999). All co-culture
experiments will be performed in triplicate. Western blots and immunocytochemistry will be
used to determine changes in neuronal markers (NeuN, Tau, MAP-2). Treatment effects in
immunocytochemistry experiments will evaluate the number of neurons, length of neuronal axons
(anti-Tau) and dendrites (anti-MAP2). Results will be normalized to allow the analysis of data
from separate experiments.
compare multiple groups.
Statistical analysis will be preformed by one-way ANOVA to
Following completion of Specific Aim 1, the established co-culture system will be used to begin
to address the mechanisms of microglial neuroprotective properties. Specific Aim 2 will be focus
of work performed at the end of the Summer and into the Fall of 2007.
Specific Aim 2: To determine whether the neurotrophins NGF, NT3 and/or BDNF are
secreted by microglia in the in vitro model system through the activation of protein kinase C
(PKC), mitogen-activated protein kinase (MAPK), and/or the PI3K/Akt pathway.
Design: Evaluation of underlying mechanisms of microglial neurotrophin function observed in
our co-culture system will measure neurotrophin protein synthesis and mRNA upregulation in
control and activated microglia. Evaluation of underlying signal transduction mechanims will
involve application of specific intracellular signaling pathway blockers to the co-culture paradigm
and analysis of activation of signaling proteins by Western Blot analysis.
Assays for neurotrophin synthesis by microglia: Microglial conditioned media will be aquired
from co-culture experiments demonstrating enhanced neurotrophic properties and analyzed for
NGF, NT3, and BDNF using specific antibodies from R& D Systems (Minneapolis, MN)
following sandwich ELISA procedures recommended by the manufacturer. In general, ELISA
assays employ the capture of NGF, NT3 and BDNF protein from culture media with anti-NGF,
NT3 and BDNF antibodies. Detection of captured antibody requires the binding of a biotinylated
affinity purified antibody.
Antibodies for these neurotrophins can also be used in
immunodepletion assays to determine whether the absence of these factors in neuronal cultures
with and without application of NMDA is necessary for neurotrophic effects observed.
Experiments will be performed in triplicate as described previously (Shibata et al., 2003). Real
time quantitative RT-PCR will be used to determine whether mRNA expression for NGF, NT3,
and BDNF increases in control and activated microglia in our co-culture experiments. Total
cellular RNA will be isolated and levels of NGF, NT3, and BDNF expression will be determined
as previously described (Shibata et al., 2003).
Assays for intracellular signal transduction in microglia: Co-culture experiments will be
performed in triplicate with and without the addition of intracellular signaling pathway inhibitors.
The following inhibitors will be used to specifically inhibit the following pathways: PD98059 (30
µM) inhibits MAPK pathway activation, LY294002 (40 µM) inhibits PI3K/Akt (Shibata et al.,
2002), calphostin C inhibits PKC activation (Kobayashi et al., 1989). Further direct evaluation of
activated signal transduction pathways involves isolation of microglia lysates and Western Blot
analysis. Briefly, control and activated microglia will be removed from co-culture experiments
and isolated for the assessment of activated signal transduction proteins. Microglia will be lysed
with Igepal lysis buffer supplemented with proteinase inhibitors. Lysates will be clarified and
separated in sodium dodecyl sulfate (SDS)/10% polyacrylamide gels and electrophorectically
transferred to PVDF membranes in Tris-glycine buffer with 20% methanol. After blocking the
membrane, the blots will be incubated with primary antibodies to phospho 44/42 (ERK),
phosphoryated PKC, and phosphorylated Akt and a secondary horseradish peroxidase-linked
antimouse IgG. Activation of these proteins will be determined by probing wetser blots with
these phosphospecific antibodies and re-probing with anti-ERK, PKC, and Akt antibodies. All
antibodies will be purchased from New England Biolabs (Beberly, MA). Blot will be visualized
by chemiluminescence.
Data Analysis: Data will be analyzed using GraphPad Prism software with one-way ANOVA
and Newman-Keuls post-test (for multiple comparisons) to test differences between groups,
between activation paradigms, and neuronal culture conditions. If tests are significant, pair-wise
comparisons will be conducted to determine differences.
Given the scope of the project proposed in a newly developed laboratory, it is anticipated that the
results from these experiments will result in a publishable body of work by the Spring of 2008.
If experiments yield interesting novel results prior to that estimated deadline, smaller publications
will certainly be assembled for rapid publication. Future experiments will involve the
collaboration with investigators at Creighton University and Purdue Univeristy to assess the
regulation of genetic mechanisms responsible for microglial neurotrophic properties. The goal of
this research proposal and future work is to increase the understanding of the molecular
mechanisms governing intrinsic neurotrophic capabilities in the CNS and to elucidate potential
targets for the promotion of neurotrophic capabilities following injury or during
neurodegenerative processes in the CNS. NIMH and independent neuroscience development
grants are potential future sources of funding for this research given acquisition of preliminary
data.
VI. References
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VII. Appendices
A. Biographical Sketch
Name
Annemarie Shibata Ph.D.
Position
Assistant Professor, Biology Department, Creighton University
Education and Training:
Institution and Location
Creighton University,
Omaha, NE
Degree
B.S. (Magna Cum Lauda)
Year
1992
Field of Study
Biology
Colorado State University,
Ft. Collins, CO
Ph.D.
1997
Anatomy and
Neurobiology
Research and Professional Experience:
4/97-6/99
Postdoctoral Research Associate, Eppley Institute for Research in
Cancer and Allied Disease, University of Nebraska Medical Center
(UNMC), Omaha, NE. (Laboratory Supervisor, Dr. Thomas
Smitgall)
7/99-5/01
Postdoctoral Research Associate and Chief, Neuroregeneration Program,
Center for Neurovirology and Neurodegenerative Disorders (CNND),
Department of Pathology and Microbiology, UNMC, Omaha, NE
(Laboratory Supervisor, Dr. Howard Gendelman)
8/02-5/04
Resident Assistant Professor, Biology Department, Creighton University,
Omaha, Nebraska (Department Chair, Dr. John Schalles)
8/06-present Assistant Professor, Biology Department, Creighton University, Omaha,
Nebraska (Department Chair, Dr. MaryAnn Vinton)
Grant Support:
12/94-3/97
NIH Predoctoral Fellowship FBM092, Inhibitory Regulation of Dendritic
and Axonal Growth Cones
4/97-5/98
NIH Research Training Fellowship, received from the Eppley Institute for
Research in Cancer and Allied Disease
6/98-3/2000 NIH Postdoctoral Fellowship 1 F32 Ca 79194-01, Regulation of Small
GTPases by the c-Fes Tyrosine Kinase
Teaching Experience:
Jan 2001
Developmental Biology, 400 level undergraduate course, Biology
Department, Creighton University, Omaha, NE
Fall 2001
Comparative Vertebrate Anatomy, 400 level undergraduate course,
Biology Department, Creighton University, Omaha, NE
Spring 2002 Cellular and Developmental Neuroscience, 500 level undergraduate
course, Biology Department, University, Omaha, NE
Fall 2002,
Spring 2003
General Biology Laboratory, development of instructional material,
Biology Department, Creighton University, Omaha, NE
Fall 2002 to
2004
Introduction to Neurobiology, 500 level undergraduate course, Biology
Department, Creighton University, Omaha, NE
Teaching Experience:
Fall 2003
Neuroanatomy, OTD 341 graduate level course, Occupational Therapy
Department, School of Pharmacy and Allied Health, Creighton University,
Omaha, NE
Fall 2003,
Introduction to Immunology, 400 level undergraduate course, Biology
Spring 2005 to Department, Creighton University, Omaha, NE
present
Fall 2004,
Directed independent research with undergraduate students and
Spring 2005 to development of a tissue culture facility, Biology Department, Creighton
present
University, Omaha, NE
Spring 2005 to Human Biology, 100 level non-majors course, Biology
present
Department, Creighton University, Omaha NE
Honors and Awards:
1988-1992
Centennial Scholarship, Creighton University , Omaha , NE
1994
Research Scholarship, Program of Molecular, Cellular and Integrated
Neurosciences, Colorado State University
1995 & 1996 Travel Award, Program of Molecular, Cellular and Integrated
Neurosciences, Colorado State University
2000
Invited Speaker, Mini-Medical School, University of Nebraska Medical
Center, Omaha NE
Selected Publications:
Kater S and Shibata A (1994) The unique and shared properties of neuronal growth
cones that enable navigation and specific pathfinding. J. Physiology (Paris) 88: 155-163.
Li M, Shibata A, Li C, Braun PE, McKerracher L, Roder J, Kater SB, and David S
(1996) Myelin-associated glycoprotein influences axon/neurite growth and growth cone
motility. J. Neuroscience Res. 46:404-414.
Shibata A, Wright MV, and Kater SB (1998) Unique response of differentiating neuronal
growth cones to the inhibitory cues presented by oligodendrocytes. J. Cell Biology 142:
191-202.
Smithgall T, Rogers JA, Peters KL, Briggs SD, Lionberger LM, Cheng H, Shibata A,
Scholtz B, Schreiner S, and Dunham N (1998) The c-Fes family of protein-tyrosine
kinases. Crit Rev Oncog. 9(1):43-62.
Ahmad I, Acharya HR, Rogers JA, Shibata A, Smithgall TE, and Dooley CM (1999)
The role of NeuroD as differentiation factor in the mammalian retina. J. Molecular
Neuroscience 2:165-178.
Shibata A, Laurent CE, Smithgall TE. The c-Fes protein-tyrosine kinase accelerates
NGF-induced differentiation of PC12 cells through a PI3K-dependent mechanism (2003)
Cell Signal. Mar;15(3):279-88.
Shibata A, Zelivyanskaya M, Limoges J, Carlson K.A., Branecki C, Bishu S, Xiong H.,
and Gendelman H.E. Peripheral nerve induced macrophage neurotropic secretory
function: Regulation of neuronal process outgrowth, intracellular signaling pathways and
synaptic physiology (2003) J. Neuroimmunology 142(1-2):112-29.
Zheng J, Niemann D, Bauer MS, Lopez AL, Erichsen DA, Ryan LA, Gendelman L,
Shibata A, Gendelman HE. The regulation of neurotrophic factor activities during HIV-1
infection and immune activation of mononuclear phagocytes. (submitted to Neurotoxicity
Res.)
Abstracts:
Fliege R, Barnell W, Tong S, Shibata A, Conway T (1992) Oxidative glucose
metabolism via the Enter-Doudoroff pathway in wildtype E. Coli via pqq-dependent
glucose dehydrogenase. Gen. Meet. Am. Soc. Microbio Abst. 92. 128:277.
Wright MV, Shibata A, Kater SB (1994) Reciprocal induction of collapse between
neuronal growth cones and oligodendrocyte processes. Soc. Neurosci. Abst. 20:659.
Kater SB, Kuhn TB, Shibata A, Wright MV, Williams CV (1995) A single second
messenger can serve opposing roles in growth cone regulation. IBRO World Congress of
Neurosci.
Kater SB, Braun PE, David D, McKerracher L, Shibata A (1996) Myelin-associated
glycoprotein (MAG) induced collapse of hippocampal axonal growth cones. Soc.
Neurosci. Abstr. 22:1466.
Li M, Shibata A, Li C, Braun PE, McKerracher L, Roder J, Kater SB, David S (1996)
Myelin associated glycoprotein (MAG) inhibits neurite/axon outgrowth. Soc. Neurosci.
Abstr. 22:315.
Shibata A, Wright MV, Kater SB (1996) Differentiation of neuronal processes
determines their response to inhibitory environmental cues. Soc. Neurosci. Abst.
22:1466.
Shibata A, Smithgall TE (1999) Fes accelerates NGF-induced neuronal differentiation
through a PI3K-dependent intracellular signaling pathway. Ann. Meet. Oncogenes and
Tumor Suppressorss, 15:188.
Shibata A, Branecki CE, Gendelman HE, Limoges J (2000) Immune competent
macrophages produce neurotrophic factors. World Alzheimer Congress.
Shibata A, Branecki C, Limoges J, Gendelman HE. (2000) Mononuclear Phagocytes
Produce Multiple Growth Factors that Aid in Neuronal Differentiation and Survival. Soc.
Neurosci. Abst
Lopez, A. L., Cotter, R. L., Erichsen, D. A., Peng, H., Gendelman, L., Shibata, A.,
Gendelman, H.E. and Zheng, J. (2002). The regulation of neurotrophic factor activities
following HIV-1 infection and immune activation of mononuclear phagocytes. 9th
conference on Retroviruses and Opportunistic Infections, Seattle, WA, Feb. 24-28.
Schmuck, T.L., Shibata A (2005) Characterization of Primary Cells in Newly
Established Tissue Culture Facility. Biology Department Student Research
Colloquium. Creighton University, Omaha ,NE.
B. Budget Justification ($500.00 total allowance)
The allowance for project supplies does not cover the costs necessary for the purchase of all items
needed to address the specific aim addressed by this proposal. Selected items needed for
experiments within Specific Aim 2 are listed below:
Experimental
Item
BDNF Duo Set
Additional items:
NT3 Duo Set
NGF Duo Set
Company
Application
R&D
Systems
ELISA assay
Specific Aim 2
R&D
Systems
R&D
Systems
ELISA assay
Specific Aim 2
ELISA assay
Specific Aim 2
Estimated Cost
(without educational discount)
$645.00
$645.00
$645.00