Effect of gene knockout and NMDA receptor modulation on cell survival and neuronal stem cell fate in in vitro and in vivo models of excitotoxicity.
In this project, we set out to analyse the processes of proliferation, survival and maturation of neuronal precursor cells within the adult mouse hippocampus under physiological and pathological conditions. Injuries to the brain, such as cerebral ischemia, are characterised by neuronal cell death within the affected brain tissue but to date little treatment is available in order to minimise the extent of neuronal damage or to recover the affected tissue. Over-activation of glutamate receptors, termed excitotoxicity, in particular mediated by NMDA (Nmethyl- D-aspartate) receptor subtypes, results in a cascade of events within the cells resulting in apoptotic and necrotic cell death. Interestingly, insults to the brain like cerebral ischemia or traumatic brain injury (TBI) subsequently lead to an increase in proliferation of neuronal precursor cells within specific neurogenic niches of the brain; the hippocampal dentate gyms (DG) and the subventricular zone (SVZ) of the lateral ventricle (LV). However, their fate seems to be limited by cellular demise. The underlying mechanisms of injury-induced up-regulation of precursor cell proliferation and subsequent cell death of the newly generated cells are still poorly understood. Therefore, in order to use adult generated neuronal precursor cells as an endogenous pool, for example for cell replacement after injury to the brain, a better understanding about the processes of adult neurogenesis is necessary.
In this study, we established highly reproducible in vivo and in vitro models of excitotoxic injury specifically to the hippocampal CA1 subfield which resulted in delayed and transient up-regulation of precursor cell proliferation in the subgranular zone (SGZ) of the DG at 6 - 9 days following injury. We were able to demonstrate a positive correlation between the extent of injury and the number of newly generated cells exclusively during this peak time point of neurogenesis. Using the established in vivo model, we further showed an involvement of NR1/MR2B-containing NMDA receptors in cell proliferation. Application of the NR2B-subunit specific antagonist Ifenprodil led to significantly elevated numbers of newly generated cells indicating that NR2Bcontaining receptors regulate neurogenesis in a negative manner.
In addition, we provided here the first evidence that deficiency in the BH3-only proteins Bim or Puma, which belong to the B cell lymphoma 2 (Bcl-2) family and are key players of the intrinsic apoptosis signalling pathway, is sufficient to protect newly generated cells in the DG from subsequent apoptotic cell death under physiological conditions. However, using our in vivo model of excitotoxicity, the lack of Puma failed to protect the newly proliferated cells, suggesting that cell death of neuronal precursor cells is differentially regulated under physiological and pathological conditions.
Moreover, we could demonstrate that Bim, but not Puma, is involved in the progression of neuronal maturation of the progenitor cells. Here, the deficiency of the bim gene accelerated neuronal development. We propose that this process might be independent from the role of Bim in apoptosis and might possibly be mediated by sequestration of the anti-apoptotic protein Bcl-2.
These studies provide new insights in the mechanisms of adult hippocampal proliferation, survival and maturation of neuronal precursor cells under physiological and pathological conditions. The modulation of these processes in order to increase the generation and/or survival of new neurons will be the first step to use the potential of those cells for endogenous cell replacement following brain injury.
First SupervisorProfessor Dr. Jochen HM. Prehn
Second SupervisorDr. Brian P. Kirby
CommentsA thesis submitted to the Royal College of Surgeons in Ireland for the degree of Doctor of Philosophy from the National University of Ireland in 2009.
Published CitationBunk EC, Effect of gene knockout and NMDA receptor modulation on cell survival and neuronal stem cell fate in in vitro and in vivo models of excitotoxicity [PhD] Thesis. Dublin: Royal College of Surgeons in Ireland; 2009
- Doctor of Philosophy (PhD)