of Work: The complex and poorly understood process of lineage progression from neural stem cells to neuronal and glial phenotypes has come under increasing study using a variety of in vitro strategies. An important issue to be resolved is how neural stem cells are regulated to either self-renew or differentiate. In vivo these cells line the ventricles of the developing central nervous system (CNS) with variable numbers radiating processes to the pial surface (radial glial form of neural stem cell). The cells integrate signals derived from both carrier proteins in the cerebrospinal fluid filling the ventricles and other cells in the neuroepithelium lining the ventricles. Signals conveyed by carrier proteins and via cell-cell interactions, which could serve to regulate cell lineage progression, have not been elucidated. We have developed a novel strategy to isolate identified subpopulations of neural stem cells (NSCs) and differentiaing progenitors from the CNS during neurogenesis and gliogenesis. The strategy involves labeling of live cells with reagents identifying surface gangliosides, which are conserved throughout vertebrate evolution, and epitopes characteristic of cells undergoing apoptosis in conjunction with fluorescence-activated cell sorting (FACS). This FACS strategy permits prospective cellular and molecular studies of neural stem cells for the first time. Previously, we identified basic fibroblast growth factor (bFGF) and one of its primary receptors, fibroblast growth factor receptor 1 (FGFR-1), as being expressed by the majority of NSCs. Further work has revealed that NSCs also express another FGFR (FGFR-3). Antisense knockdown of FGFR-1 or FGFR-3 attenuated NSC proliferation and self-renewal as undifferentiated progeny and instead promoted their progression along the neuronal lineage. Antisense knockdown of both FGFR-1 and FGFR-3 eliminated NSC proliferation and primarily induced apoptosis. Under these conditions some NSCs differentiated directly into neurons without dividing. These results together with others summarized in the FY 2003 Annual Report establish bFGF-mediated signaling via both FGFR-1 and FGFR-3 as being critical in maintaining NSC proliferation without overt differentiation. bFGF activation of FGFR-1 and FGFR-3 triggers receptor dimerization followed by autophosphorylation and the recruitment of multiple second messenger signal transduction pathways. We have now identified six such pathways by virtue of their involvement in bFGF/FGFR-1/FGFR-3-regulation of NSC cytosolic Ca2+ levels. A major part of the growth factor regulation of Ca2+ levels involves activation of a Ca2+ entry component. During FY 2004 we focused on identifying the Ca2+ entry component. Since NSCs do not express voltage-gated Ca2+ channels, which emerge among neural stem cell-derived progeny undergoing neuronal lineage progression, Ca2+ entry likely involves channels whose activation is largely independent of voltage changes. Channels with these properties have been identified in Drosophila photoreceptors and subsequently throughout phylogeny. A mutation in the Drosophila photoreceptor channel curtailed the physiological receptor potential in response to light, reducing it to a transient receptor potential (TRP). TRP-type cation channels are widely expressed in vertebrate tissues. At least 41 TRPs have been identified and fitted into a still-growing number of families according to their structural homologies. We have used PCR and immunocytochemistry to reveal that NSCs express five of the seven canonical TRP channels (TRPC1-4, 6). We have focused on the role of TRPC1 in NSC proliferation. TRPC1 protein co-immunoprecipitates with FGFR-1 protein in membranes derived from NSC progeny. This suggests that FGFR-1 regulation of TRPC1 may be facilitated by their close proximity. Antisense knockdown of TRPC1 decreases immunodetectable TRPC1 expressiion in NSC-derived progeny without affecting the expressions of either TRPC3 or 6. The decrease in TRPC1 expression coincides with a decrease in bFGF-mediated Ca2+ responses and Ca2+ currents. These results indicate that TRPC1 contributes to the Ca2+ entry component associated with proliferation without inducing either apoptosis or differentiation. Similar results on bFGF-mediated Ca2+ respones and NSC proliferation were found using inorganic and organic antagonists of TRP-type channels. Thus, bFGF activation of FGFR-1 and FGFR-3 catalyzes second messenger pathways that stimulate Ca2+ entry via channels that contain TRPC1. However, NSC proliferation was not eliminated by antisense targeting of TRPC1. Other TRPCs may also be targets of bFGF/FGFR-1/FGFR-3-mediated regulation. This will be pursued in future experiments. Two pilot studies have produced results that prompt continued investigation. In one study it was found that NSC-derived progeny self-renewing without overty differentiating expressed two functional receptors for neurotransmitters. Activation of these receptors rapidly attenuated or eliminated bFGF/FGFR-1/FGFR-3-mediated regulation of Ca2+ levels. In the continued presence of one or the other transmitter NSC-derived progeny progressed along either a neuronal or glial lineage. In some way neurotransmitter receptor activation, which involves G protein-coupled receptors (GPCR), inactivates bFGF-mediated signaling. Such ?transinactivation? between GPCRs and receptor tyrosine kinases like those of FGFR-1/FGFR-3 has been described in several phenotypes. One possible mechanism involves GPCR activation of protein tyrosine phosphatase(s), which could dephosphorylate activated FGFR-1/FGFR-3, thereby reducing the strength of the signals necessary to sustain NSC proliferation without differentiation. The mechanisms underlying the switch from growth factor to neurotransmitter signaling and their role in NSC lineage progression are a challenge for future study. In another study, C6 glioma cells were found to express most of the TRPC family of cation channels, as well as FGFR-1 receptors. Block of TRPC channel activity reduced C6 cell proliferation in a dose-dependent manner and led to apoptosis. The latter occurred at the Go/G1, S and G2/M stages, indicating a critical role for Ca2+ entry in all phases of the cell cycle. These results reveal that some of the same mechanisms underlying NSC proliferation support C6 and other CNS glioma growth in vitro. Both FGFRs and TRPCs may be therapeutic targets for drugs designed to reduce C6 tumors. This line is also worth pursuing since it is both insightful and clinically relevant.
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