Numerous developmental studies have documented the roles played by astroglial cells in the support of neuronal migration and neurite outgrowth. These studies have taken on an unexpected new dimension in recent years by observations of apparently related phenomena accompanying the neurodegenerative events of AIzheimer's disease (AD). One hallmark of AD is the extracellular deposition of a potentially neurotoxic peptide, (beta-amyloid, which is generated by an alternate cleavage of the larger membrane-associated APP. The relevance of this protein to glial-neuronal developmental interactions is that the normally-cleaved and secreted form of APP is homologous with protease nexin II (PNII), one of a family of nexins - glial-derived secreted molecules that, by binding to and suppressing neuronal surface proteases, mediate neurite outgrowth during development. Both reactive gliosis and abortive sprouting of surviving neurons are routinely observed in affected areas of AD brains. These findings prompt consideration of the possibility that a recapitulation of astroglial secretion of neurite-promoting factors, including PMII/APP and laminin, may prompt abortive sprouting, leading to additional cycles of neuronal degeneration. Neurons undergoing abortive sprouting attempts may generate increased amounts of amyloidogenic fragments of APP. We have developed a neuronal cell culture model to test these and related hypotheses. Preliminary studies demonstrate that treatment of undifferentiated neuronal cells with astroglial conditioned medium (CM) or purified APP induces neuritogenesis and no toxicity. By contrast, CM- or APP-treatment of long-term differentiated (""""""""aged"""""""") neuronal cells, provided the differentiating agent (dbcAMP) is reduced or eliminated, induces abortive sprouting and degeneration. CM and APP are benign in these aged cultures when sprouting is blocked by maintaining dbcAMP at high concentrations. Our previous studies demonstrate that hyperactivation of calpain (calcium-activated protease) and protein kinase C (PKC) in these cells induces ALZ-5O immunoreactivity. We will determine whether CM- and APP-induced neurotoxicity is accompanied by ALZ-50 induction, and whether beta-amyloid generation by neurons involves calpain and PKC hyperactivation by the intracellular delivery of specific calpain and PKC activator and inhibitors. The potential role of aberrantly phosphorylated tau (generating ALZ-50) in neurodegeneration will be examined by antisense oligonucleotide-mediated suppression of tau synthesis prior to CM- and APP-treatment, and determining whether neurodegeneration is lessened; these analyses will provide information on whether tau contributes to, or is a by-product of, neurodegeneration. The hypothesis that neurodegeneration is a consequence of recapitulation of sprouting will be further explored by antisense oligonucleotide- mediated down-regulation of the growth associated protein, GAP-43, which is required for sprouting in these cells. Potentially crucial interactions with extracellular matrix components will be examined by inclusion of laminin in alternate cultures. The studies contained within this proposal are not designed to be comprehensive in terms of including all glial-derived growth factors expressed either constituitively or during development and regeneration. Neither do they attempt to elucidate the initiating event(s) in AD. However, by exploring the possibility that glial cells exacerbate AD neuropathology, it is hoped that novel insights towards therapeutic approaches may evolve.
