The Pennsylvania State University The Graduate School Department of Neural and Behavioral Sciences A REGENERATIVE RESPONSE OF ENDOGENOUS NEURAL STEM CELLS TO PERINATAL HYPOXIC/ISCHEMIC BRAIN DAMAGE A Thesis in Neuroscience by Ryan J. Felling © 2006 Ryan J. Felling Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2006 ii The thesis of Ryan J. Felling was reviewed and approved* by the following: Steven W. Levison Professor of Neurology and Neurosciences Thesis Advisor Co-Chair of Committee Teresa L. Wood Associate Professor of Neural and Behavioral Sciences Co-Chair of Committee Sarah K. Bronson Assistant Professor of Cell and Molecular Biology Charles Palmer Professor of Pediatrics James R. Connor Professor and Vice-Chair Department of Neurosurgery; Director, G.M. Leader Family Laboratory for Alzheimer's Disease Research Robert J. Milner Professor of Neural and Behavioral Sciences Head of Neuroscience Graduate Program *Signatures are on file in the Graduate School iii ABSTRACT Hypoxic/ischemic (H/I) insults are the leading cause of neurologic injury during the perinatal period, affecting 2-4 per 1000 term births as well as a high percentage of premature infants. The ensuing sequelae are devastating and include cerebral palsy, epilepsy and cognitive deficits. Despite astounding advances in perinatal care, the incidence of cerebral palsy has changed little over the last 50 years. This demands that we pursue alternative therapeutic strategies that will reduce the significant morbidity associated with perinatal H/I encephalopathy. The revelation that the brain retains populations of neural stem cells throughout life offers the promise of endogenous regeneration following brain injury. Already extensive data have demonstrated that new neurons are born after cerebral ischemia and migrate to sites of injury. The existence of multipotential, self-renewing neural stem and progenitors, however, suggests that this response could be much more significant. This thesis offers data indicative of a mobilization of this population following perinatal H/I. Using a clonal assay to quantify neural stem and progenitors (NSPs), we demonstrated almost a doubling in the numbers of NSPs following perinatal H/I. This was accompanied by the generation of larger neurospheres and increased symmetric proliferative divisions in vitro upon EGF and FGF-2 stimulation. Furthermore, a higher proportion of the clonal neurospheres retained the capacity to generate all 3 neural cell types upon differentiation. These effects were preceded by an upregulation of genes important in the regulation of NSP maintenance and proliferation, including notably the EGF receptor and Notch1. iv Further investigation demonstrated that the larger size of neurospheres following perinatal H/I was due to increased proliferation, specifically in response to EGF stimulation. These effects as well as the induction of Notch1, were dependent upon signals from mature cells as demonstrated by an in vitro paradigm of H/I injury. Other components of the Notch signaling cascade were also induced specifically within the NSP niche of the subventricular zone (SVZ). Pharmacological inhibition of this pathway using a well-characterized inhibitor of γ-secretase that prevents release of the intracellular mediator, the Notch intracellular domain (NICD) effectively reduced both the increase in numbers of NSPs as well as the increase in tripotency of the neurospheres. These data provide evidence of a mobilization of the NSP population in response to perinatal H/I. A better understanding of the molecular mechanisms underlying these results will identify therapeutic targets for enhancing this response. It is no longer acceptable to settle for just managing neurologic injuries such as perinatal H/I encephalopathy; we must pursue strategies to repair the brain after such damage. This thesis demonstrates the basis for endogenous regeneration of the CNS following brain injury and describes several potential molecules that may be used to manipulate this response. v TABLE OF CONTENTS LIST OF FIGURES .....................................................................................................ix LIST OF TABLES.......................................................................................................xi ACKNOWLEDGEMENTS.........................................................................................xii Chapter 1 Literature Review.......................................................................................1 Stem Cells......................................................................................................1 General considerations ...........................................................................1 Neural stem cells ....................................................................................4 Maintenance of NSCs....................................................................................8 Mitotic spindle orientation .....................................................................9 Asymmetric inheritance of cell fate determinants..................................11 Notch Signaling......................................................................................12 EGF Receptor.........................................................................................13 Emx2 ......................................................................................................14 Pax6........................................................................................................15 Bmi-1......................................................................................................16 Enhanced neurogenesis following ischemic injury .......................................17 Proliferation of SVZ cells following ischemic injury ............................18 Migration of new cells following ischemic injury .................................20 Differentiation of new cells after ischemic injury..................................21 Perinatal hypoxic/ischemic encephalopathy..................................................23 Clinical Background...............................................................................23 Effects of perinatal H/I on the SVZ .......................................................24 Specific Aims of the Thesis...........................................................................25 Chapter 2 Summary of Results ...................................................................................26 NSPs are more abundant in the rodent SVZ following perinatal H/I............26 Perinatal H/I promotes increased Notch signaling in the SVZ......................30 Changes observed in NSPs following perinatal H/I depend on Notch signaling .................................................................................................33 Changes in the NSP niche underlie the effects of perinatal H/I....................34 Chapter 3 Discussion of Results .................................................................................36 Neural stem cells are more abundant following perinatal H/I.......................36 NSPs respond to the injured environment .....................................................37 The NSP population actively expands following perinatal H/I.....................38 Increased EGF signaling is an important contributor to NSP expansion following perinatal H/I ...........................................................................39 vi Notch signaling is an important contributor to NSP expansion following perinatal H/I............................................................................................40 Realizing Cajal’s vision – reflections on the future of regenerative medicine .................................................................................................42 Chapter 4 Conclusions and Future Directions ............................................................44 Chapter 5 Neural stem/progenitor cells initiate a regenerative response to perinatal hypoxia/ischemia ...................................................................................47 Introduction ...................................................................................................47 Methods .........................................................................................................49 Perinatal hypoxia/ischemia model .........................................................49 Tissue fixation and BrdU labeling .........................................................50 Antibodies and Immunohistochemistry .................................................51 In situ hybridization ...............................................................................53 Primary neurosphere propagation ..........................................................54 Secondary neurosphere propagation ......................................................55 Neurosphere quantitation .......................................................................56 Neurosphere immunohistochemistry......................................................57 RNA isolation.........................................................................................58 Real-time PCR........................................................................................59 Statistics .................................................................................................60 Results ...........................................................................................................60