Ariel B. Abraham

3rd Year Graduate Student

Department: Pharmacological Sciences

Graduate Program: Genetics

Advisor: Dr. Emily Chen

Abstract:

Title: The Role of NMDA-Rs in NPC Differentiation

The main objective of my research is to determine the factors that induce the efficient and reproducible differentiation of neural stem and progenitor cells (NPCs) into neurons in vitro and in vivo. One of the factors that may influence cell fate is the expression of NMDA receptors, which are glutamate receptors that play a role in synaptic plasticity, brain development, and a variety of neurological diseases. NR1 is one of the essential subunits of the NMDA receptor, while NR2B is expressed during development and NR2A is expressed in adulthood. Recently, Tashiro and colleagues were able to show that NMDA receptors influence the cell specific integration of new neurons in the dentate gyrus. [Nature, 442(7105):929-33] By using a cre/lox system in which a viral vector was injected into the dentate gyrus of floxed NR1 mice they were able to demonstrate a decrease in the number of NR1KO new neurons when compared to wild type new neurons. The implications of these findings are substantial because they suggest that NPCs residing in the neurogenic niche of the dentate gyrus have an NMDA dependant survival mechanism. This leads to a broader question with respect to NPCs, NMDA signaling, and neurogenesis. Does NMDA expression and activity influence the neurogenic fate of NPCs during neurogenesis, and if so, what exactly is the role/mechanism of NMDA receptor signaling that leads to their effects on NPCs? To determine the effect of NMDA-Rs on NPC differentiation, I isolated embryonic NPCs using a neurosphere formation assay and attempted nucleofection of the NPCs with plasmids expressing NMDA-Rs. Embryonic day 12.5 brains were obtained from a C57 pregnant mouse and were grown at 50,000 cells/mL in neurobasal media with 20ng/mL EGF and 10ng/mL FGFb. Following neurosphere formation, the spheres were transfected with a plasmid expressing GFP alone using nucleofection. Nucleofection was initially performed according to the manufacturer’s protocols, but was later refined to determine which conditions were optimal for nucleofection, including DNA levels and passage time. Nucleofected spheres were given 48 hours for GFP expression and then analyzed by FACS to determine the efficiency of nucleofection. FACS analysis indicated that optimal nucleofection of the neurospheres produces GFP expression levels between 45 and 50%, 48 hours following nucleofection. Using the same optimal conditions for nucleofection, neurospheres were then nucleofected with NMDA-R subunits tagged with GFP; these included NR1-GFP, NR2A-GFP, and NR2B-GFP [Barria and Malinow, Neuron, 35(2):345-53 and Neuron 48(2):289-301]. FACS analysis of these spheres indicated that NR1 and NR2A expression was low (<5%), while NR2B was only slightly higher. Therefore, while optimizing the nucleofection of neurospheres with GFP alone was straight forward, it appears that the nucleofection of spheres with NMDA-R subunits is more complex and will require further optimization. Possible changes that may influence the efficiency of nucleofection include changes to the neurosphere formation assay as well as neurosphere size and passaging time. Continued work with these plasmids will allow us to determine whether specific combinations of NMDA-Rs affect neurogenesis in vitro. Future experiments with long term expression of GFP tagged NMDA-Rs will allow us to clarify the role, if any, of NMDA-Rs on NPC activity and neurogenesis in vitro.