Modulation of Post-Ischemic Astrogliosis by Human Glial Progenitor Cells

University Of Rochester, Rochester NY

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Abstract

There is an abundant pool of glial progenitor cells (GPCs) dispersed throughout adult human brain tissue. This proposal seeks to define the niche for astrogliogenesis in the human white matter, with an emphasis on defining the molecular basis for reactive astrocytosis. Absent autocrine and paracrine influences, adult GPCs are not restricted to any given neural lineage. Rather, the environment regulates their differentiated fate, suggesting that an ischemic environment might specifically direct GPCs to astrocytic fate in reactive astrocytosis. On this basis, we investigated the gene expression patterns of adult human GPCs derived from normal brain tissue. We identified a set of parallel and interacting ligand-receptor interactions, as well as their cognate signaling pathways, that may to determine whether a given GPC remains undifferentiated, or instead develops into an astrocyte, oligodendrocyte, or neuron. We now propose to define those pathways involved in both normal and post-ischemic astrocytosis from adult human glial progenitors. By this means, we expect to identify genetic and pharmacological targets by which to suppress reactive astrocytosis following ischemic injury, while sustaining our ability to mobilize progenitors to desired lineages. To this end, we shall focus on several pathways that appear differentially expressed in isolated GPCs. These include receptor tyrosine phosphatase-p/^ (RTPZ), which may play a central role in modulating li-catenin trafficking, its chondroitin sulfate proteoglycan ligands, and two interacting receptor systems, the FGFR3 tyrosine kinase, and the BMP4-dependent serine/threonine kinases. Specifically, weask: 1. Does the inhibition of RTPZ signaling suppress astrocytosis in vitro? Is this effect mediated by impeding B-catenin translocation? Can RTPZ inhibition suppress glial scar formation in vivo? What are the functional effects of suppressing post-ischemic gliosis through RTPZ inhibition? 2. Can reactive astrocytosis and glial scar formation after stroke be prevented by inhibiting BMP4-signaled astrocyte induction? What BMP inhibitors are best for this purpose? 3. How does the RTPZ pathway interact with FGFR3 and BMP signaling to instruct astrocytic fate? 4. Does the expression of CSPGs by GPCs produce an environment biased to astrocytosis, through CSPG-dependent activation of RTPZ? Can astrocytosis be suppressed through CSPG inactivation? Can concurrent RTPZ suppression further attenuate gliosis? What are the functional consequences of these strategies, in particular after MCA occlusion? The implications of this work are profound, not only in regards to stroke and trauma, but more broadly with regards to diabetic and hypertensive encephalopathies, which share an hypoxia-triggered disruption of the normal niches for cell genesis in the brain. In each of these disorders, reactive astrocytosis culminates in the sclerotic pathology that typifies terminally non-regenerative brain and spinal cord. Our goal is too prevent this fate, and by so doing preserve the cellular plasticity and regenerative capabilities of the healthy CNS. PHS 398 (Rev. 09/04) Page 145 Form Page 2 POT NS050315 Principal investigator (Last, First, Middle): Nedergaard, Maiken/Goldman, Steven Projects MODULATION OF POST-ISCHEMIC ASTROGLIOSIS BY HUMAN GLIAL PROGENITOR CELLS I. RESPONSE TO REVIEWERS I would like to thank the referees for their helpful critiques of my proposal, Modulation of post-ischemic astrogliosis by human glial progenitor cells. By way of review, this application proposes to assess the molecular basis for reactive astrocytosis, with particular attention to signaling pathways we have identified in adult human glial progenitor cells (GPCs), that appear to be important to GPC mobilization and astrocyte differentiation therefrom. In brief, the application was criticized as being insufficiently detailed in regards to some of the proposed methodologies and experimental designs, and too expansive in terms of the number of experiments proposed. In addition, some concern was voiced as to the proposed use of RNAi reagents whose validation we had not made explicit. More general concerns were also stated as to insufficient integration across the individual projects of this program project, which prompted us to describe in greater detail the interactions among our group. In response to the referees' specific concerns: Critique 1 Aim 1 proposes to test the effects of RTPZ modulation after MCA occlusion. It was criticized as providing insufficient definition of the stroke bed or its boundaries, and therefore insufficiently described histological endpoints for assessing treatment-associated effects. To this end, we have added a paragraph to the Methods section which better defines the histological criteria and scoring procedures that we are using. Aim 2 proposes to assess the role of BMP signaling and antagonists thereof in modulating post- ischemic astrocytic fate. It was criticized for insufficient delineation of the numbers of animals necessary of each experiment. The referee also noted that we provided no justification for the extensive in vivo testing proposed. We have now added clear estimates of our anticipated experimental sample sizes for all 4 aims of this application, with numbers based on data that we previously obtained using noggin to suppress astrocyte production in the adult VZ(Chmielnicki et al., 2004b) (Appendix 8). The BMP inhibitors assessed - neuralin, BAMBI, DAN, gremlin and noggin - have different BMP ligand specificities and binding efficiencies, and different local bioavailabilities, due to variable degrees of heparin binding. As a result, we intend to test each of them in vivo, both by adenoviral over-expression and lentiRNAi knock-down. I have to assume, until we learn otherwise, that the results of one BMP inhibitor will not necessarily predict the results using another. In regards to reagent availability, we have already constructed all of the adenoviral over-expression constructs, and expect to have all of the lentiviral shRNAis constructed within the next few months; to date, we have made lentiRNAis for neuralin and BAMBI, besides those made and validated for RTPZ and its interactants, syndecanS and CASK, and are now generating similar knock-down vectors for gremlin and DAN. I have added a table to the last page of the Methods that includes all of the vectors needed in this application, and the preparation status and current availability of each. Aim 3 addresses the interaction of RTPZ and BMP signaling with that effected through FGFR3, and tests the hypothesis that FGFR3 blocks astrocytic production and differentiation by inhibiting RTPZ and BMP signals, thereby potentiating the oligodendrocytic lineage bias imparted by RTPZ inhibition). The aim was criticized fro being too expansive, in that it includes many discrete experiments, with unbiased cell counts and formal stereological assessment and reconstruction being required for each. Yet the required throughput for cell counting, stereology and imaging is well within the capabilities of my group. We have published several unrelated studies (Appendices 6, 8 and 9; also(Louissaint et al., 2002)), that employ the same methods proposed here of scoring BrdU-tagged cells double or triple labeled with other markers, then quantifying the labeling indices as a function of treatment, time point and region. Prior papers from my lab (Chmielnicki et al., 2004b; Windrem et al., 2004) (Appendices 6 and 8) have been individually comparable in effort to what is proposed here in Aim 2. We have two BioQuant imaging systems in the lab, each run full-time by technicians under the direction of Martha Windrem and Abdel Benraiss - both investigators who moved with mefromNew York to Rochester - each of whom has considerable experience in these methods. My estimates of what we're capable of doing are thus predicated on past experience, running similar types of samples, with the same image acquisition and analysis systems, used by the same group of experienced investigators. The reviewer also requests a table presenting the individual treatment groups and assessment endpoints, and expresses concern as to the sufficiency of our sample sizes, as well as the detail of our experimental designs, especially in regards to the in vivo experiments within each aim. Accordingly, I have 146