Glial cell line-derived neurotrophic factor. : Expression patterns, neuronal transport, regulation, effects, and receptor dependence

Abstract: Background: The mammalian nervous system depends upon trophic proteins for survival, growth and adult maintenance. These trophic factors have also been considered as potential therapeutic agents for treating human neurodegenerative disorders. In Parkinson's disease, there is a loss of midbrain dopaminergic (DA) neurons and a subsequent reduction of DA in caudate and putamen, which leads to significant motor symtomatology. In 1993, a new trophic factor was discovered for midbrain DA neurons, which had marked positive effects in vitro (Lin et al., 1993). This factor, termed glial cell line-derived neurotrophic factor (GDNF) is a distant member of the TGF-B superfamily and is the first of a new subfamily of such factors. Two additional members of this subfamily have recently been cloned and have been termed Neurturin (NTN) and Persephin (PSP). More recently, cell surface binding proteins which are part of the receptor complex for these three trophic factors have been cloned and have been termed GFRa-l, 2, and 3, respectively. The signaling mechanism for GDNF and NTN is thought to involve initial association with GFR a-l and GFRa-2, respectively with subsequent activation of the tyrosine kinase receptor c-Ret, although this hypothesis has been validated only in vitro. Aims: In the first part of my thesis, I sought to define the localization and ontogeny of GDNF expression and how expression might be regulated. In the second part, I studied effects of GDNF on peripheral and central neurons, using ganglia explant cultures and the l-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) mouse model of Parkinson's disease. I also studied if a specific retrograde transport system for GDNF was present in midbrain DA circuits. In the last part of my thesis, I attempted to study the importance of GFRa-1 for the effects of GDNF, by generating a GFRa-1 null mutation. Results: The first study in my thesis was to characterized GDNF gene mRNA expression during development and adulthood using in situ hybridization (paper 1). Based on the results from the initial characterizations of GDNF gene expression during both development and adulthood, the following experiments were undertaken: 1. Studying if the GDNF gene was regulated in adulthood using pilocarpine-induced status epilepticus in the rat (paper II). These experiments clearly indicated that GDNF might play a crucial role in the protection of neurons against excitotoxic stress. 2. Testing the function of the GDNF protein on other types of cells in an in vitro explant system (paper Ill). Our results from these explant studies, taken together with the expression data, indicated that GDNF was also a potent factor for other classes of neurons in various autonomic ganglia. 3. Examining if there were protective and regenerative effects of the GDNF protein on the nigrostriatal system. This is the system that degenerates in humans with Parkinson's disease. To investigate this, adult MPTP-ex posed mice were treated with GDNF. We showed that GDNF could both partially regenerate and protect adult dopaminergic neurons (paper IV). 4. Determining if GDNF acted directly upon the dopaminergic neurons as a "classic target derived factor," or indirectly, by using retrograde transport studies (paper V). In these experiments we showed that GDNF, initially placed in the striatum, can be retrogradely transported to midbrain DA neuron soma via a specific receptor-mediated mech anisms. 5. Determining the importance of GFRa-1 for the effects of GDNF in vivo during development. It has previously been shown that null mutations of GDNF (Moore et al. 1996, Pichel et al. 1996; Sanchez et al., 1996) and Ret (Schuchardt 1994) have a similar phenotype, charactenzed by absence of, or severe malformation, of the kidneys and loss of enteric ganglia. We demonstrated that null mutations of GFRa-l have a similar phenotype (paper Vl) documenting the critical role of this binding protein for the actions of GDNF. Andreas C. Tomac, 1998