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, 199
