Control of CNS neuronal survival

All animal cells require extracellular signals to avoid undergoing apoptosis. In my thesis research, I investigated the nature of the identity and sources of extracellular signals that normally promote central nervous system (CNS) neuron survival. In the peripheral nervous system (PNS), neuron survival is promoted by single well-defined neurotrophic factors that are secreted by their target tissues in limiting amounts. In contrast, the identities and sources of neurotrophic factors for CNS neurons remain largely unclear despite several decades of research. In my thesis work, I took two different approaches to investigate this question. First, because astrocytes have been known for over 30 years to secrete a strongly neurotrophic activity for hippocampal neurons, I used biochemistry to characterize the nature of this activity. I found that astrocyte-conditioned medium (ACM) contained at least 4 different neurotrophic activities that are each weakly active but together strongly promote hippocampal neuron survival. Using a biochemical purification approach that I helped to develop, we have recently highly enriched one of these activities and then used mass spectrometry to identify an exciting short list of several candidate proteins that are presently being tested.; Because cortical neurons are a complex mixture of many different cell types, I next helped to develop a novel cell purification procedure that enabled me to obtain highly purified populations of corticospinal motor neurons from the postnatal rat brain. This method takes advantage of the ability of the non-toxic cholera toxin beta subunit (CTB), after injection into the axon pathway of a neuronal population of interest, to be retrogradely trafficked back into the neuron somas and then trafficked back to the cell surface where it is available for anti-CTB antibody binding by immunopanning. I demonstrated the efficacy of this method on two different populations of CNS neurons, retinal ganglion cells and corticospinal motor neurons (CSMNs).; I then focused the rest of my thesis work on elucidating signals that promote survival of the purified CSMNs because these neurons specifically die in the paralytic neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS), and identifying a neurotrophic factor has the potential to finally provide a treatment for this devastating disease. I found that BDNF was the only known candidate neurotrophic factor that supported their survival, but BDNF was not sufficient to maintain the long-term survival of CSMNs. To identify new candidates, I performed gene profiling on the purified populations of CSMNs to identify which trophic factor receptor mRNAs were most highly expressed. Surprisingly I found that the most highly expressed trophic receptor mRNAs were primarily receptors for secreted proteins that our lab has recently identified to be highly expressed by brain vascular cells, endothelial cells and pericytes. Two of the first factors I tested included IGF-2, which is made by vascular cells in the developing brain (probably by pericytes), and pleiotrophin, which is made by endothelial cells. I found that both IGF-2 and pleiotrophin have significant survival activity for the purified motor neurons and, in some preliminary experiments, I have found that these two factors combined with BDNF are able to promote the survival of more than half of the CSMNs. Thus the identities and sources of neurotrophic factors may be significantly different for CNS neurons than for PNS neurons. In particular, endothelial cells may be an important source of survival signals for CNS neurons. In conclusion, my findings suggest a novel role for CNS endothelial cells in promoting CNS neuron survival and identify two novel neurotrophic factors for corticospinal motor neurons which may be useful in promoting their survival in ALS and regeneration of their axons after spinal cord injury.