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Project 2  -  Brain degeneration & Parkinson's Disease

Background: Dopamine (DA) is an essential neurotransmitter mediating a variety of brain function. Therefore DA deficit leads to neural diseases such as Parkinson’s disease (PD) while up-regulation of dopamine signaling is thought to underlie drug addition (Nestler, 2001; Bonci et al, 2003; Dawson & Dawson, 2003). Parkinson’s disease is a devastating neurodegenerative disease, a key pathological feature of which is the progressive and selective loss of dopaminergic (DA) neurons in the substantia nigra (Olanow & Tatton, 1999; de Silva et al, 2000; Dekker et al, 2003). In spite of extensive studies, it is not known why DA neurons selectively degenerate.

      Recent genetic discoveries have established that at least familial forms of PD are directly associated with mutations of certain genes such as a-synuclein (a-Syn) and parkin (Dawson & Dawson, 2003; Dauer & Przedborski, 2003). These mutated PD genes cause selective DA cell death, although the encoded proteins are ubiquitously expressed in the brain (Lotharius & Brundin, 2002; Lykkebo & Jensen, 2002). Therefore, there are likely to be dopaminergic specific factors such as the synthesis/vesicular packaging/degradatioin of DA, underlying selective DA cell death. Among the proposed underlying causes, oxidative damage is thought to play an important role (Lotharius & Brundin, 2002; Lykkebo & Jensen, 2002) even though the mechanism by which the oxidative stress is initiated is poorly understood.

Why Drosophila neuronal cultures? Parkinson’s disease is complicated and develops in such a slow fashion that we can not easily use human subjects to study its molecular and cellular pathogenesis. An alternative is to use model animals, suitable for molecular and cellular manipulations. Several cellular and transgenic models of a-Syn-induced neurodegeneration have been developed. However, no mammalian transgenic model has completely recapitulated PD. The fruit fly Drosophila melanogaster has proven to be useful in studies of the mechanisms underlying neurodegenerative diseases (Muqit & Feany, 2002; Bonini & Fortini, 2003) due to its powerful and sophisticated genetics. Indeed, an intriguing PD model has been developed by engineering transgenic flies that express the human a-Syn protein (Feany & Bender, 2000). These animals exhibit typical anatomical and behavioral symptoms of PD, including an age-dependent loss of DA neurons in addition to filamentous inclusion of Lewy bodies and locomotor dysfunction. We have also observed age-dependent DA cell loss in neuronal cultures prepared from a-Syn transgenic flies. Therefore, Drosophila neuronal cultures provide a unique model system for PD research, since it can take advantage of sophisticated molecular genetic approaches in addition to easy of the pharmacological and biochemical manipulations. In addition, the simplicity of neural circuits in the primary cultures enables us to directly access molecular and sub-cellular changes induced by genetic and pharmacological manipulations without the involvement of unknown effects of the complicated circuits. All of these factors strongly suggest that Drosophila neuronal cultures will be an excellent model system to study the mechanisms underlying selective loss of dopaminergic neurons in PD. In this study, therefore, we will use Drosophila primary neuronal cultures as an in vitro model to examine role of dopamine in the selective neurondegeneration.

Project Discription: The goal of this project is to investigate the mechanisms underlying dopamine’s ability to induce selective neurodegeneration. First, we will examine whether high dopamine in the cytoplasm is enough to cause the degeneration by (a) depletion of dopamine synthesis and (b) overexpression of tyrosine hyroxylase (TH) in dopaminergic (DA) neurons. Depletion of DA is expected to be protective against a-synuclein-mediated neurodegeneration while TH overexpression is cytotoxic. If successful, the results will support our hypothesis that elevated cytoplasmic dopamine is a main factor mediating a-synuclein-induced neurodegeneration. The subsequent study will aim to find the mechanism(s) of a-synuclein-induced disruption of DA homeostasis.

Specific Aim I: To examine dopamine’s ability to mediate selective neuronal degeneration

                      in a-Syn transgenic fly neuronal cultures.

      PD genes such as -Syn are ubiquitously expressed in the brain, but mutations of these proteins cause selective DA cell death (Feany & Bender, 2000; Auluck et al, 2001; Erikson et al, 2003). Consistent with this finding, the general architecture of the fly brain, except DA cell loci, was not affected when human a-Syn was pan-neurally expressed (Feany & Bender, 2000; Auluck et al, 2001). Furthermore neurons synthesizing another important biogeneic amine, serotonin, did not degenerate (Feany & Bender, 2000). Therefore, there are likely to be dopaminergic specific factors that are involved in the selective DA cell loss. Dopamine is an essential neurotransmitter mediating a variety of brain functions and hence long-term deprivation of DA causes severe neurological defects observed in PD patients. Paradoxically, dopamine itself can become a source of oxidative stress and consequently contribute to selective loss of DA neurons in PD (Lotharius & Brundin, 2002). In this study, we hypothesize that abnormally high dopamine in the cytoplasm is enough to induce DA cell death. There are tow ways to test this ‘dopamine hypothesis.’ First, inhibition of DA synthesis is expected to lead to the protection of DA cell death mediated by a mutated a-synuclein (a-Syn A30P). Second, overexpression of TH (tyrosine hydroxylase) in DA neurons will induce a greater degeneration.

Specific Aim II: To determine whether a-Syn disrupts cytoplasmic DA homeostasis by

                       interfering synaptic packaging of DA.

      Dopamine is a highly reactive molecule and is able to form several cytotoxic molecules including superoxide anions, DA-quinone species and hydroxyl radicals. Therefore, dopamine itself can become a source of oxidative stress and consequently contribute to the selective DA cell death in PD (Lotharius & Brundin, 2002), suggesting that regulation of cytoplasmic DA at a non-toxic level is an important prerequisite for the cell survival. Indeed, depletion of DA production by a TH inhibitor a-MT resulted in suppression of a-Syn’s ability to induce DA cell death in cultured human neurons (Xu et al, 2002). However, the mechanism by which the oxidative stress is initiated is poorly understood despite of extensive studies to understand the selective vulnerability of DA cells. Recently, Lotharius et al (2002) reported that mutated a-Syn led to impairment in vesicular DA storage and consequently altered dopamine homeostasis in a human mesencephalic cell line. This is consistent with the notion that dopamine in synaptic vesicles is not reactive and thus not toxic to the cell due to the acidic environment of the vesicle (Lotharius & Brundin, 2002). The goal of this specific aim is to examine whether a-Syn disrupts DA packaging into synaptic vesicles, which will result in the increase in cytoplasmic DA concentration.
In this study, we will employ three different approaches: amperometry, pharmacology and molecular genetics. Carbon fiber-based amperometry (Chow & von Ruden, 1995; Koh & Hille, 1999; Kim et al, 2000) will allow us to directly monitor synaptically released DA. We will also use pharmacological probes such as reserpine to interfere synaptic storage of DA, which will result in the decrease of DA concentration stored in synaptic vesicles. Genetically, vesicular monoamine transporter (dVMAT) will be overexpressed to facilitate DA segregation into the synaptic vesicle, leading to neuroprotection against a-Syn cytotoxicity. The results will reveal potential mechanism(s) underlying a-Syn-mediated disruption of dopamine homeostasis.