Project 5 involves a multilevel molecular genetic, cytochemical and functional approach focused on understanding the role of neurotrophic factors in the NGF and TGFbeta family in the central nervous system. In particular, we will evaluate the involvement of these factors in the maintenance of cholinergic projections and for other functions in hippocampus and cortex cerebri, as well as the possibilities to use neurotrophic factors in the NGF and TGFbeta family to reverse age-related neurodegenerative disorders. Experiments in project 5 can be subdivided as follows: (1) Cloning of new members of the TGFbeta family, emphasis on GDNF-related molecules and GDNF receptors. Accumulating evidence suggests that GDNF is an important neurotrophic factor for dopaminergic and cholinergic neurons. It is important to clone and characterize GDNF receptors, and it is very likely that GDNF, being a distant member of the TGFbeta family constitutes the first found member of a new TGFbeta sub- family. (2) Characterization of neurotrophic factors using the in vitro ganglia bioassay system; Known members of the TGFbeta family and, in particular any new GDNF-related molecules discovered in project 5 or by other scientists will be characterized in the highly specific 5-ganglia in vitro bioassay system with predictive value for in vivo effects. (3) Mapping the cellular distribution of neurotrophic factor: Using primarily in situ hybridization, but also immunohistochemistry, we will map the precise cellular localization of GDNF and any GDNF-related factors and/or GDNF receptors in the adult CNS. Additionally factors and receptors in the NGF family will be mapped in further detail. (4) Transient and permanent neurotrophic factor expression systems: GDNF, related molecules and receptors as well as NGF will be expressed by transient systems to manufacture the proteins and by permanent expression systems to be used as sources of trophic factor in collaboration with project 4. (5) Distribution and behavioral effects of neurotrophic factors injected into adult and aged normal animals: This part will evaluate in detail the spatial and temporal distribution of neurotrophic factors injected into the CSF or into brain tissue. This information is crucial for interpretation of behavioral studies which will be carried out after acute and chronic injections of neurotrophins, GDNF and novel GDNF-related factors. (6) High-resolution magnetic resonance imaging: Using new dedicated equipment enabling imaging of magnetic resonance spectroscopic information, we will monitor circulatory and metabolic changes in the brain over time in animals following intracranial injections of neurotrophic factors. (7) Functional effects of novel neurotrophic factors: Novel GDNF-related neurotrophic factors found by project 5 or by other scientists will be made using recombinant technology and used for functional studies in collaboration with project 4, (8) Studies of human post-mortem brain tissue: Postmortem brain tissue from patients with Alzheimer's disease and age-matched controls will be studied using in situ hybridization technology proven to work in such tissue to search for any possible changes in the expression of GDNF, related molecules or receptors, or neurotrophins and their receptors in Alzheimer's disease.
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