6-Hydroxydopamine (6-OHDA)-induced loss of dopamine (DA) neurons has served to produce an animal model of DA neuron loss in Parkinson's disease. We report here the use of 6-OHDA to produce an in vitro model of this phenomena using dissociated cultures prepared from neonatal rat mesencephalon. Cultures were exposed to 6-OHDA (40-100 lM, 15 min) in an antioxidant medium, and DA and GABA neurons evaluated by immunocytochemistry. 6-OHDA induced morphological and biochemical signs of cell death in DA neurons within 3 h, followed by loss of tyrosine hydroxylase immunoreactive neurons within 2 days. In substantia nigra (SN) cultures, DA neurons were much more affected by 6-OHDA than were GABA neurons. In contrast, DA neurons from the ventral tegmental area were only lost at higher, non-specific concentrations of 6-OHDA. The effects of 6-OHDA on nigral DA neurons were blocked by inhibitors of high affinity DA transport and by z-DEVD-fmk (150 lM), a caspase inhibitor. Glial cell line-derived neurotrophic factor (GDNF) treatment reduced TUNEL labeling 3 h after 6-OHDA exposure, but did not prevent loss of DA neurons at 48 h. Thus, 6-OHDA can selectively destroy DA neurons in post-natal cultures of SN, acting at least in part by initiating caspase-dependent apoptosis, and this effect can be attenuated early but not late by GDNF.

We previously reported that the vesicular monoamine transporter 2 (VMAT2) is physically and functionally coupled with Hsc70 as well as with the dopamine synthesis enzymes tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase, providing a novel mechanism for dopamine homeostasis regulation. Here we expand those findings to demonstrate that Hsc70 physically and functionally interacts with TH to regulate the enzyme activity and synaptic vesicle targeting. Co-immunoprecipitation assays performed in brain tissue and heterologous cells demonstrated that Hsc70 interacts with TH and aromatic amino acid decarboxylase. Furthermore, in vitro binding assays showed that TH directly binds the substrate binding and carboxyl-terminal domains of Hsc70. Immunocytochemical studies indicated that Hsc70 and TH co-localize in midbrain dopaminergic neurons. The functional significance of the Hsc70-TH interaction was then investigated using TH activity assays. In both dopaminergic MN9D cells and mouse brain synaptic vesicles, purified Hsc70 facilitated an increase in TH activity. Neither the closely related protein Hsp70 nor the unrelated Hsp60 altered TH activity, confirming the specificity of the Hsc70 effect. Overexpression of Hsc70 in dopaminergic MN9D cells consistently resulted in increased TH activity whereas knockdown of Hsc70 by short hairpin RNA resulted in decreased TH activity and dopamine levels. Finally, in cells with reduced levels of Hsc70, the amount of TH associated with synaptic vesicles was decreased. This effect was rescued by addition of purified Hsc70. Together, these data demonstrate a novel interaction between Hsc70 and TH that regulates the activity and localization of the enzyme to synaptic vesicles, suggesting an important role for Hsc70 in dopamine homeostasis.Dopaminergic neurons within the substantia nigra and ventral tegmental area are the primary sources of the catecholamine neurotransmitter dopamine (DA).2 Despite the fact that dopaminergic neurons account for less than 0.01% of all neurons, they play a significant role in brain function (1-3). Consequently, DA homeostasis is crucial for the preservation and regulation of physiological functions such as locomotion, cognition, neuroendocrine secretion, and motivated behaviors (4). Thus, it is not surprising that disruptions in the DA system have been implicated in several neurological and psychiatric disorders, including Parkinson disease, depression, schizophrenia, attention deficit hyperactivity disorder, Tourette syndrome, and drug addiction (4 -8).The DA life cycle consists of a series of highly regulated molecular events that are ultimately responsible for controlling DA homeostasis. Synthesis of DA occurs in the presynaptic terminals via two enzymatic reactions. First, tyrosine is converted into L-3,4-dihydroxyphenylalanine through the actions of the rate-limiting enzyme tyrosine hydroxylase (TH) (9, 10). Subsequently, aromatic amino acid decarboxylase (AADC) converts L-DOPA into DA (11). When synthesized, DA is packaged and stored withi...

No abstract

Parkinson's disease is a progressive neurodegenerative disorder, caused in part by the loss of dopamine (DA) neurons in the substantia nigra (SN). Neurotrophic factors have been shown to increase the basal survival of DA neurons in vitro, as well as to protect the neurons from some toxins under certain in vitro conditions and in animal models. Although these factors have often been tested individually, they have rarely been studied in combinations. We therefore examined the effect of such combinations after acute exposure to the toxin 1-methyl-4-phenylpyridinium (MPP(+) ) using dissociated postnatal rat midbrain cultures isolated from SN and ventral tegmental area (VTA). We found that significant loss of DA neurons in the SN occurred with an LC50 of between 1 and 10 μm, whereas the LC50 of DA neurons from the VTA was approximately 1000-fold higher. We did not observe neuroprotection against MPP(+) by individual exposure to glial cell-line derived neurotrophic factor (GDNF), brain derived neurotrophic factor (BDNF), transforming growth factor beta (TGFβ), basic fibroblast growth factor (FGF-2) or growth/differentiation factor 5 (GDF5) at concentrations of 100 or 500 ng/mL. Combinations of two, three or four neurotrophic factors were also ineffective. However, when the SN cultures were exposed to a combination of all five neurotrophic factors, each at a concentration of 100 ng/mL, we observed a 30% increase in DA neuron survival in the presence of 10 and 500 μm MPP(+) . These results may be relevant to the use of neurotrophic factors as therapeutic treatments for Parkinson's disease.