Impact of anti-viral immunity on neural stem/progenitor cell activity and implications for CNS

175 Pages·2017·5.94 MB·English

by  Apurva Kulkarni

Duquesne University
Duquesne Scholarship Collection
Electronic Theses and Dissertations
Summer 1-1-2017
Impact of anti-viral immunity on neural stem/
progenitor cell activity and implications for CNS
development
Apurva Kulkarni
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Kulkarni, A. (2017). Impact of anti-viral immunity on neural stem/progenitor cell activity and implications for CNS development
(Doctoral dissertation, Duquesne University). Retrieved fromhttps://dsc.duq.edu/etd/175
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IMPACT OF ANTI-VIRAL IMMUNITY ON NEURAL STEM/PROGENITOR CELL
ACTIVITY AND IMPLICATIONS FOR CNS DEVELOPMENT

A Dissertation
Submitted to the Graduate School of Pharmaceutical Sciences

Duquesne University

In partial fulfillment of the requirements for
the degree of Doctor of Philosophy

By
Apurva Kulkarni

August 2017
Copyright by
Apurva Kulkarni

2017
IMPACT OF ANTI-VIRAL IMMUNITY ON NEURAL STEM/PROGENITOR CELL

ACTIVITY AND IMPLICATIONS FOR CNS DEVELOPMENT

By

Apurva Kulkarni

Approved 5th June 2017

Lauren A. O’Donnell, Ph.D. Rehana K. Leak, Ph.D.
Associate Professor of Pharmacology Associate Professor of Pharmacology
Graduate School of Pharmaceutical Sciences Graduate School of Pharmaceutical Sciences
Duquesne University, Pittsburgh, PA Duquesne University, Pittsburgh, PA
(Committee Chair) (Committee Member)

Wilson S. Meng, Ph.D. John A. Pollock, Ph.D.
Associate Professor of Pharmaceutics Professor
Graduate School of Pharmaceutical Sciences Bayer School of Natural and Environmental
Duquesne University, Pittsburgh, PA Sciences, Department of Biological Sciences
(Committee Member) Duquesne University, Pittsburgh, PA
(Committee Member)

Michel Modo, Ph.D. James K. Drennen, III, Ph.D.
Associate Professor Associate Dean and Associate Professor
Department of Radiology of Pharmaceutics
University of Pittsburgh, Pittsburgh, PA Graduate School of Pharmaceutical
(Committee Member) Sciences
Duquesne University, Pittsburgh, PA

J. Douglas Bricker, Ph.D.
Dean, Mylan School of Pharmacy and the
Graduate School of Pharmaceutical Sciences
Duquesne University, Pittsburgh, PA
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ABSTRACT

IMPACT OF ANTI-VIRAL IMMUNITY ON NEURAL STEM/PROGENITOR CELL
ACTIVITY AND IMPLICATIONS FOR CNS DEVELOPMENT

By
Apurva Kulkarni
August 2017

Dissertation supervised by Lauren A. O’Donnell
Viral infection and inflammation in the central nervous system (CNS) can cause
neuropathology, particularly in the prenatal and neonatal stages. Severe damage to the CNS may
result from cytopathic effects of viral infection or from the immune response that may lyse virally-
infected cells or release inflammatory mediators to mediate viral clearance. Neural stem/progenitor
cells (NPSCs) are multipotent cells in the CNS that are often disrupted by neurotropic viral
infections. They may be directly infected by the virus or respond to inflammatory cytokines
released from resident as well as infiltrating immune cells. This bystander effect may affect NSPC
differentiation and proliferation depending on the milieu of inflammatory mediators. Interferon
gamma (IFN), a potent antiviral cytokine required for the control and clearance of many CNS
infections, can differentially affect cell survival and cell cycle progression depending upon the cell
type and the profile of activated intracellular signaling molecules. Here, we show that IFN
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inhibits proliferation of primary NSPCs through dephosphorylation of the tumor suppressor
Retinoblastoma protein (pRb), which is dependent on activation of Signal Transducers and
Activators of Transcription-1 (STAT1) signaling pathways. We observed inhibition of
proliferation in wild type NSPCs (WT/NSPCs) as well as a decrease in neurosphere growth. IFN
restricted cell cycle progression by inhibiting the G1- to S-phase transition. Cell cycle restriction
was associated with decreases in the G1–phase specific cyclin E/CDK2 proteins and in pRb
phosphorylation at serine 795 (S795). Together, these results indicate that the NSPC cell cycle was
restricted in the late G1-phase. In STAT1-deficient (STAT1-KO) NSPCs, the effects of IFN on
NSPC proliferation were lost, demonstrating that IFN signaling is STAT1-dependent. These data
define a mechanism by which IFN could contribute to a reduction in NSPC proliferation in
inflammatory conditions. Furthermore, this was the first study to implicate the pRb protein in
mediating anti-proliferative effects of IFN on NSPCs.
The cellular tropism of neurotropic viruses varies, with NSPCs being targeted by some viruses and
spared by others. During a viral infection, microglia are typically the first immune cells to become
activated in the brain. Microglia may contribute to the anti-viral program generated against the
virus and/or alter other neural cells through the release of inflammatory mediators. Evidence in
neonatal brains suggest that microglia can also influence NSPC numbers and differentiation under
basal conditions. However, whether microglia affect NSPCs during an anti-viral immune response
is an outstanding question. To evaluate the effects of microglial activation on NSPCs, we used a
mouse model for measles virus (MV) infection in neurons. In this model, MV infection is restricted
to mature CNS neurons expressing the human isoform of CD46, a receptor for MV. NSPCs and
microglia are spared from infection. In order to examine the interactions between infected neurons,
microglia, and NSPCs, primary microglia were co-cultured with MV-infected CD46+ neurons and
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the conditioned medium was used to treat primary NSPCs in culture. We found that factors
released from the infected neuron/microglia co-cultures increased BrdU-incorporation and
neuronal differentiation in NSPCs. Thus, even though the NSPCs are not infected in this model,
the cells respond by generating young neurons that could serve as potential replacements for the
mature neurons damaged by the virus. These studies provide a novel model system for identifying
the signals that microglia use to communicate between infected neurons and responding NSPCs.

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ACKNOWLEDGEMENTS

I thank Lauren A. O’Donnell, my advisor and mentor, for giving me the opportunity to
work with her, for her constant support and allowing me grow as a researcher. Her advice and
encouragement has been pivotal to my journey as a graduate student. Her enthusiasm and drive for
research was ‘infectious’.
I would like to thank my committee members Drs. John A. Pollock, Wilson Meng and
Michael Modo for their support and invaluable guidance. I would especially like to thank Dr.
Rehana Leak for being such a huge support for my research and her generosity in allowing me to
use her lab equipment at my liberty. Her immense understanding of neuroanatomy and statistical
methods among many other expertise have been invaluable. I would also like to thank Dr. Jane
Cavanaugh for her generosity in allowing me to use the EVOS microscope and the cell storage
facility. I also thank Dr. Glenn Rall for the CD46 mice that he gifted our lab without which this
project would not have been possible. I would also like to thank Denise Butler-Buccilli, Christine
Close and the animal care facility staff for their assistance in maintaining the animal colonies. I
have been fortunate to have an excellent faculty at Mylan School of Pharmacy who have been
helpful with not just the curriculum but also my research. I would like to thank Dr. James Drennen
and the Graduate School of Pharmaceutical Sciences for their continued encouragement
throughout the program.
I would like to thank Priya Ganesan, Manisha Chandwani, Dr. Anil Pattisapu, Taylor
Scully, Gaurav Rajani and Dr. Kristen Fantetti for their scientific inputs and their personal support.
I would also like to thank Dipy Vasa, Mike Wasko, and Mayur Parmar for being not just friends
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and colleagues but for always being there to support me. I thank Jackie Farrer, Mary Caruso and
Deb Wilson, our administrative staff for their support throughout my time at Duquesne University.
I derive all my inspiration from my parents, grandparents and in-laws, who have been with
me through the thick and thin of my life. I would like to acknowledge the immense contribution
of my parents, Yatin and Charusheela Kulkarni whose unconditional love has kept me going
through all the ups and downs. Lastly, I thank my amazing wife and my best friend, Ruchi Shah.
Your love, support and sacrifice made everything that has been achieved possible.

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TABLE OF CONTENTS
Page
Abstract………………………………………………………………………………………………………………………. iv
Acknowledgements…………………………………………………………………………………………………….. vii
Chapter 1: Literature Review……….………………………………………………………………………………… 1
NSPCs in the developing and adult CNS………………………………………………………. 1
Viral infections in the CNS…………………………………………………………………………… 5
Human Cytomegalovirus (HCMV) …………………………………………………………. 5
Herpes Simplex Virus-1 (HSV-1) ……………………………………………………………. 6
Zika Virus (ZIKV) …………………………………………………………………………………… 7
West Nile Virus (WNV) …………………………………………………………………………. 8
Japanese Encephalitis Virus (JEV) …………………………………………………………. 9
Borna Disease Virus (BDV) ……………………………………………………………………. 9
Measles Virus (MV) ………………………………………………………………………………. 10
Effects of antiviral immunity on NSPC activity…………………………………………….. 11
Herpes Simplex Virus-1 (HSV-1) …………………………………………………………….. 14
Cytomegalovirus (CMV) ………………………………………………………………………… 15
Zika Virus (ZIKV) …………………………………………………………………………………….. 17
Measles Virus (MV) ……………………………………………………………………………….. 18

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