Keith A. Crutcher - US grants

neural plasticity; aging; animal old age;

What factors regulate neuronal plasticity in the mature brain and spinal cord? This question is at the center of research that seeks to promote the ability of the central nervous system (CNS) to recover from injury resulting from stroke, trauma or degenerative disease. That axonal growth and synapse formation can occur in the mature CNS has been established beyond doubt. The mechanisms underlying such growth, however, are not clear. Damage to the basal forebrain of the rat (brain regions that are known to be affected in Alzheimer's disease) results in the growth of vascular autonomic fibers (sympathetic axons) into the hippocampal formation. This example of axonal growth within the mature mammalian brain is preceded by an increase in the amount of Nerve Growth Factor (NGF) in the hippocampal tissue. However, there is disagreement in the literature concerning the degree, specificity and duration of the increase in NGF depending on whether a biological or immunological assay was used to detect NGF. One of the goals of the research in this application is to identify the causes of these discrepancies by applying both types of assays to the same tissue sample. In addition to testing the role that endogenous NGF has in this sprouting response, experiments are proposed to determine whether exogenous NGF delivered into the rat brain, either by way of infusion or by implanting polymer containing NGF, can elicit or later such growth. Also, the possibility that changes in substrate-bound growth factors play a role in sympathetic ingrowth will be tested by using sections of the brain as substrates for neurite growth in tissue culture. These experiments include determining whether septal denervation makes hippocampal tissue more permissive as a substrate for neurite outgrowth in tissue culture as well as electron microscopic studies to identify the tissue elements that regenerating axons are associated with in such cultures. Recent results indicate that the poor regenerative growth normally observed in mature CNS tissue may be due to the presence of growth-inhibiting factors associated with CNS white matter. Additional experiments are proposed to examine the growth-promoting ability of gray and white matter regions of the mature and developing rat CNS using the same tissue culture assay. All of the experiments are designed to identify the conditions that permit or prevent axonal growth within the mature mammalian brain.

What factors regulate neuronal plasticity in the mature brain and spinal cord? This question is at the center of research that seeks to promote the ability of the central nervous system (CNS) to recover from injury resulting from stroke, trauma or degenerative disease. Axonal growth and synapse formation can occur in the mature CNS. The mechanisms underlying such growth, however, are not clear. Damage to the basal forebrain of the rat (brain regions that are known to be affected in Alzheimer's disease) results in the growth of vascular autonomic fibers (sympathetic axons) into the hippocampal formation, a response that is significantly reduced in the aged animal. This example of axonal growth within the young rat is preceded by an increase in the amount of Nerve Growth Factor (NGF) in the region where growth occurs. However, we have demonstrated that global infusion of exogenous NGF is not sufficient to induce sympathetic ingrowth in the presence of septal fibers suggesting that local elevation of NGF may be required to elicit sprouting. Two of the goals over the next five years are to determine whether local availability of NGF within the hippocampal formation determines the topography of sympathetic ingrowth and whether septal lesions alter the growth-promoting ability of hippocampal tissue sections when used as a substrate in culture. A third goal is to determine whether the age-related decline in sympathetic sprouting is due to reduced trophic factor induction following septal denervation and/or alterations in tissue substrate properties. This arises from our finding that there is no age-related decline in total NGF activity in the rat hippocampal formation. The fourth goal is to determine the responsivity of aged sympathetic neurons to exogenous NGF to test the hypothesis that reduced sprouting is partly due to reduced responsiveness of aged sympathetic neurons to NGF. All of the experiments are designed to reveal information regarding the factors that regulate axonal growth within the mature and aging nervous system.

DESCRIPTION (provided by applicant): Although abundant new information has recently been collected on the genetic and environmental risk factors associated with Alzheimer's disease, there is still no consensus on the critical pathway leading to neuropathology. The "amyloid hypothesis", which dominates much current research effort, has both strengths and weaknesses. One aspect of the postulated role for amyloid that is particularly contentious is the extent to which it is a direct cause of neuronal degeneration, as opposed to participating in a cascade of events that ultimately leads to neuronal dysfunction and death. Other genetic risk factors for proteins involved in AD include apolipoprotein E (apoE) and there is accumulating evidence that apoE4 may directly contribute to AD neuropathology. In particular, apoE4 has been shown to exhibit neurotoxic effects in vitro. This toxicity may be associated with proteolytic generation of a shortened form of apoE (truncated apoE), which is more abundant in AD brain tissue. In addition, several lines of evidence suggest that a C-terminal fragment of apoE binds to, and co-localizes with, amyloid. The work proposed here will examine the hypothesis that proteolytic fragments of apoE contribute to both neuropathology and amyloid deposition. A combination of immunohistochemical, biochemical, and tissue culture studies will be used to: examine the extent of apoE proteolysis in human brain and apoE transgenic mouse brain; identify the cellular source of apoE and its proteolysis in the CNS; study the role of specific receptors in apoE neurotoxicity; study the effect of C-terminal apoE on ABeta aggregation and activity; and localize apoE fragments at sites of AD pathology. The goal is to pursue evidence in support or against the major hypothesis that proteolysis of apoE contributes to AD pathology.

[unreadable] DESCRIPTION (provided by applicant): Axonal regeneration has now been demonstrated to occur in regions of the CNS where it was formerly thought to be impossible. These dramatic examples of successful regrowth have come from studies in which the influence of both inhibitors and promoters of neurite growth have been examined. The importance of neuronal growth state on regeneration success has recently been demonstrated in studies of optic nerve regeneration. Collectively, these results point to the importance of factors that normally stimulate growth in overcoming the non-permissive features of CNS white matter. The work proposed here will take advantage of techniques that have been developed in this laboratory over several years that permit assessment of the role of NGF in stimulating axonal growth within the CNS. Transgenic mice in which NGF is over-expressed under the control of an astrocyte-specific promoter (GFAP), will be used for both in vitro and in vivo studies of axonal growth in CNS white matter. In vivo studies of sympathetic axonal growth within CNS regions of such mice will be carried out to determine the extent to which ectopic expression of NGF modifies the growth of these aberrant fibers. In addition, tissue section culture studies will be carried out to determine whether NGF, either applied exogenously or expressed transgenically, will modify the generally non-permissive nature of CNS white matter in supporting axonal growth. Finally, the role of p75, the low-affinity NGF receptor, in modifying axonal growth of NGF-responsive neurons in CNS white matter will be studied through a series of transplant and tissue culture approaches using transgenic mice in which p75 is deficient in the presence of NGF overexpression. The primary goal is to study the role of neurotrophic factors in stimulating axonal regeneration within CNS white matter. [unreadable] [unreadable]

Considerable progress has been made in identifying factors that play important roles in regulating neuronal plasticity, both during development and in maturity. The PI has been actively studying one example of such plasticity, i.e., the growth of mature sympathetic axons into the injured CMS, using a variety of in vivo and in vitro manipulations. A dramatic example of "natural" plasticity is the response of uterine sympathetic nerves to changes in circulating levels of sex hormones. Uterine sympathetic nerves show constant phases of degeneration and regeneration during the natural estrous cycle, reflecting fluctuations in circulating levels of estrogen. Remarkably, the uterine sympathetic terminal plexus degenerates completely during normal pregnancy and regenerates after parturition. Failure of this "natural" plasticity occurs in pathological situations such as pre-eclampsia, which can be fatal to both mother and fetus. The cause(s) of pre- eclampsia are unknown, but persistent sympathetic innervation may contribute by inducing vasoconstriction. In the case of uterine leiomyomas (fibroids), which are sex hormone-sensitive benign myometrial tumors that occur in up to 77% of women of reproductive age, sympathetic nerves are absent. In fact, fibroids resemble the pregnant myometrium in both the absence of autonomic nerves and the enrichment of extracellular matrix (ECM) components. The mechanisms underlying plasticity in uterine sympathetic nerves are not fully understood, but available evidence indicates that such plasticity reflects changes in the balance between growth-promoting and growth-inhibiting substances in the target, as well as hormone-induced changes in the receptivity of uterine-projecting sympathetic neurons. Preliminary studies indicate that uterine substrate- bound signals are likely to contribute to plasticity in uterine sympathetic nerves.The goals of the present proposal are: (1) to establish the contribution of substrate-bound signals to the "natural" plasticity of the uterine sympathetic innervation; (b) to explore the role of substrate-bound signals in regulating sympathetic innervation in pre-eclampsia and uterine leiomyoma; (3) and to assess the interaction of sex hormones and neurotrophins with uterine substrate-bound signals in regulating innervation. The proposed work is a collaborative effort to be conducted primarily at the Institute de Investigaciones Biol6gicas Clemente Estable in Montevideo, Uruguay with Dr. Monica Brauer as an extension of NIH grant #R01NS044972.