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 ondefining the molecular basis for reactive astrocytosis. Absent autocrine and paracrine influences, adult GPCsare 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 reactiveastrocytosis. On this basis, we investigated the gene expression patterns of adult human GPCs derived fromnormal brain tissue. We identified a set of parallel and interacting ligand-receptor interactions, as well astheir cognate signaling pathways, that may to determine whether a given GPC remains undifferentiated, orinstead develops into an astrocyte, oligodendrocyte, or neuron. We now propose to define those pathwaysinvolved 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 astrocytosisfollowing 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 includereceptor 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 tyrosinekinase, and the BMP4-dependent serine/threonine kinases. Specifically, weask: 1. Does the inhibition of RTPZ signaling suppress astrocytosis in vitro? Is this effect mediated byimpeding B-catenin translocation? Can RTPZ inhibition suppress glial scar formation in vivo? What are thefunctional effects of suppressing post-ischemic gliosis through RTPZ inhibition? 2. Can reactive astrocytosisand glial scar formation after stroke be prevented by inhibiting BMP4-signaled astrocyte induction? WhatBMP inhibitors are best for this purpose? 3. How does the RTPZ pathway interact with FGFR3 and BMPsignaling to instruct astrocytic fate? 4. Does the expression of CSPGs by GPCs produce an environmentbiased to astrocytosis, through CSPG-dependent activation of RTPZ? Can astrocytosis be suppressedthrough CSPG inactivation? Can concurrent RTPZ suppression further attenuate gliosis? What are thefunctional 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 broadlywith regards to diabetic and hypertensive encephalopathies, which share an hypoxia-triggered disruption ofthe normal niches for cell genesis in the brain. In each of these disorders, reactive astrocytosis culminates inthe sclerotic pathology that typifies terminally non-regenerative brain and spinal cord. Our goal is too preventthis 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 2POT NS050315 Principal investigator (Last, First, Middle): Nedergaard, Maiken/Goldman, Steven ProjectsMODULATION OF POST-ISCHEMIC ASTROGLIOSIS BY HUMAN GLIAL PROGENITOR CELLSI. RESPONSE TO REVIEWERSI would like to thank the referees for their helpful critiques of my proposal, Modulation of post-ischemicastrogliosis by human glial progenitor cells. By way of review, this application proposes to assess themolecular basis for reactive astrocytosis, with particular attention to signaling pathways we have identified inadult human glial progenitor cells (GPCs), that appear to be important to GPC mobilization and astrocytedifferentiation therefrom. In brief, the application was criticized as being insufficiently detailed in regards tosome of the proposed methodologies and experimental designs, and too expansive in terms of the number ofexperiments proposed. In addition, some concern was voiced as to the proposed use of RNAi reagents whosevalidation we had not made explicit. More general concerns were also stated as to insufficient integrationacross the individual projects of this program project, which prompted us to describe in greater detail theinteractions 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 asproviding insufficient definition of the stroke bed or its boundaries, and therefore insufficiently describedhistological endpoints for assessing treatment-associated effects. To this end, we have added a paragraph tothe 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 ofeach experiment. The referee also noted that we provided no justification for the extensive in vivo testingproposed. We have now added clear estimates of our anticipated experimental sample sizes for all 4 aims ofthis application, with numbers based on data that we previously obtained using noggin to suppress astrocyteproduction 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, anddifferent local bioavailabilities, due to variable degrees of heparin binding. As a result, we intend to test each ofthem in vivo, both by adenoviral over-expression and lentiRNAi knock-down. I have to assume, until we learnotherwise, that the results of one BMP inhibitor will not necessarily predict the results using another. In regardsto reagent availability, we have already constructed all of the adenoviral over-expression constructs, andexpect to have all of the lentiviral shRNAis constructed within the next few months; to date, we have madelentiRNAis 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 addeda table to the last page of the Methods that includes all of the vectors needed in this application, and thepreparation status and current availability of each.
Aim 3 addresses the interaction of RTPZ and BMP signaling with that effected through FGFR3, andtests the hypothesis that FGFR3 blocks astrocytic production and differentiation by inhibiting RTPZ and BMPsignals, thereby potentiating the oligodendrocytic lineage bias imparted by RTPZ inhibition).
The aim wascriticized fro being too expansive, in that it includes many discrete experiments, with unbiased cell counts andformal stereological assessment and reconstruction being required for each. Yet the required throughput forcell counting, stereology and imaging is well within the capabilities of my group. We have published severalunrelated studies (Appendices 6, 8 and 9; also(Louissaint et al., 2002)), that employ the same methodsproposed here of scoring BrdU-tagged cells double or triple labeled with other markers, then quantifying thelabeling 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 isproposed here in Aim 2. We have two BioQuant imaging systems in the lab, each run full-time by techniciansunder the direction of Martha Windrem and Abdel Benraiss - both investigators who moved with mefromNewYork to Rochester - each of whom has considerable experience in these methods. My estimates of what we'recapable of doing are thus predicated on past experience, running similar types of samples, with the sameimage acquisition and analysis systems, used by the same group of experienced investigators. The reviewer also requests a table presenting the individual treatment groups and assessmentendpoints, and expresses concern as to the sufficiency of our sample sizes, as well as the detail of ourexperimental designs, especially in regards to the in vivo experiments within each aim. Accordingly, I have 146
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