Neurotoxicity and behavioral deficits associated with Septin 5 accumulation in dopaminergic neurons.

Septin 5, a parkin substrate, is a vesicle- and membrane-associated protein that plays a significant role in inhibiting exocytosis. The regulatory function of Septin 5 in dopaminergic (DAergic) neurons of substantia nigra (SN), maintained at relatively low levels, has not yet been delineated. As loss of function mutations of parkin are the principal cause of a familial Parkinson's disease, a prevailing hypothesis is that the loss of parkin activity results in accumulation of Septin 5 which confers neuron-specific toxicity in SN-DAergic neurons. In vitro and in vivo models were used to support this hypothesis. In our well-characterized DAergic SN4741 cell model, acute accumulation of elevated levels of Septin 5, but not synphilin-1 (another parkin substrate), resulted in cytotoxic cell death that was markedly reduced by parkin co-transfection. A transgenic mouse model expressing a dominant negative parkin mutant accumulated moderate levels of Septin 5 in SN-DAergic neurons. These mice acquired a progressive l-DOPA responsive motor dysfunction that developed despite a 25% higher than normal level of striatal dopamine (DA) and no apparent loss of DAergic neurons. The phenotype of this animal, increased striatal dopamine and reduced motor function, was similar to that observed in parkin knockout animals, suggesting a common DAergic alteration. These data suggest that a threshold level of Septin 5 accumulation is required for DAergic cell loss and that l-DOPA-responsive motor deficits can occur even in the presence of elevated DA.

Experimental strategy to identify genes susceptible to oxidative stress in nigral dopaminergic neurons.

Neuropathological evidence from both human and experimental models of Parkinson's disease (PD) firmly supports a significant role for oxidative stress (OS) in the death of dopaminergic (DA) neurons in substantia nigra. Largely unknown are the genes underlying selective susceptibility of nigral DA neuron to OS and how they effect nigral DA cell death. The major barriers to high-throughput identification of candidate genes are the paucity of nigral DA neurons as well as the dilution effect of non-DA cells both in primary cultures and brain tissues. To overcome these barriers, we have developed a DA cell line model, SN4741, appropriate for cDNA microarray analysis. Candidate genes were selected from both the microarray analysis and the molecular implication of their pathological mechanisms (i.e., decreased mitochondrial complex I activity and proteasomal dysfunction) of PD. Subsequent secondary validation tests were devised to characterize genes including clone #45 that may underlie selective vulnerability of nigral DA neuron to OS.

Oxidative stress regulated genes in nigral dopaminergic neuronal cells: correlation with the known pathology in Parkinson's disease.

Oxidative stress (OS) is a primary pathogenic mechanism of nigral dopaminergic (DA) cell death in Parkinson's disease (PD). Oxidative damage, Lewy body formation and decreased mitochondrial complex I activity are the consistent pathological findings in PD. In nigral DA neurons, however, it is unknown whether any gene expressional changes induced by OS contribute to the typical PD pathology. Here, using microarray analysis, we identified several groups of genes in the nigral DA cell line, SN4741 [J. Neurosci. 19 (1999) 10; J. Neurochem. 76 (2001) 1010], that were regulated by OS. Approximately 36 significantly regulated genes that encode functional molecules of nuclear subunits of mitochondrial complex I, exocytosis and membrane trafficking proteins, markers for OS and oxidoreductases, regulatory molecules of apoptosis and unidentified EST clones were further analysed. OS modulated the expression of specific genes, of which physiological dysfunctions have been implicated in PD. For instance, the expression of the nuclear-encoded subunits of mitochondrial complex I, B8 and B17, were significantly down-regulated by OS, possibly contributing to selective defect in mitochondrial complex I activity in PD. Furthermore, syntaxin 8 and heme oxygenase-1 (HO-1) are most dramatically up-regulated by OS in DA cells. Syntaxin 8 is a SNARE protein, regulating lipid vesicle docking and fusion as well as early endosome membrane recycling. Lipid membranes are significantly oxidative-damaged in PD. HO-1 is an important cytoplasmic constituent of Lewy bodies, a pathological hallmark of idiopathic PD. Thus, our findings provide novel molecular probes that may be useful in unraveling the molecular mechanism(s) of OS-induced pathogenesis in PD. Further functional characterization of the affected genes including ESTs can help elucidate the underlying molecular pathology as well as develop biomarkers for monitoring degenerating DA neurons in PD.

Amyloid precursor protein gene disruption attenuates degeneration of substantia nigra compacta neurons following axotomy.

Our past work has shown that the C-terminal fragment of amyloid precursor protein (APP) translocated to the nucleus in neurons destined for delayed excitotoxic degeneration. To test whether nuclear APP fragments also play a role in the progressive loss of dopaminergic (DA) substantia nigra compacta (SNc) neurons, we performed unilateral medial forebrain bundle (MFB) transection on APP wild type (WT) and on mice with disruption of the APP gene (KO). In WT mice immunoreactivity for APP C-terminal, beta-amyloid and Alz90 epitopes appeared in the nuclei of axotomized DA neurons at 3 days post-lesion (dpl), persisted at 7 dpl and was absent in 14 dpl mice. APP N-terminal immunoreactivity was restricted to the cytosol at all time points, precluding the possibility of full length APP in the nucleus. Nuclear localization of APP epitopes was absent in neurons of the contralateral SNc or in neurons of the ipsilateral ventral tegmental area and SN reticulata. The presence of APP C-terminal and Alz90 domains was confirmed by Western blotting performed on the nuclear fraction of the SN ipsilateral to the axotomy. Quantitative morphometric analysis revealed that WT mice demonstrated earlier and more profound loss of tyrosine hydroxylase+SNc neurons than did KO mice. These data showed that a novel nuclear C-terminal fragment appeared coincident with SNc neuron degeneration, and that APP deficiency correlated with significant neuroprotection in vivo.

APP knockout attenuates microglial activation and enhances neuron survival in substantia nigra compacta after axotomy.

Focal microglial activation and progressive dopaminergic neurodegeneration in substantia nigra compacta (SNc) have characterized Parkinson's disease (PD). We have hypothesized that the microglial response may be provoked by molecular signals from chronically stressed SNc neurons. To test whether amyloid precursor protein (APP) could serve as such a signal, we evaluated microglial activation in SN after unilateral transection of the medial forebrain bundle (MFB) in mice either wild-type (WT) or null (KO) for APP. WT and KO mice displayed comparable microglial response at the MFB transection site. In WT mice microglial activation was first apparent in the ipsilateral SN at 3 days postlesion (dpl), marked by morphological change and increased isolectin immunoreactivity. The microglial response intensified at 7 dpl and persisted in the medial nigra through 14 dpl. In contrast, in KO mice activated microglia appeared predominantly at 7 dpl, with little activation at 3 dpl and none at 14 dpl. Neuron number in affected WT SNc at 14 dpl was significantly reduced compared with loss in affected KO SNc. The delayed and limited local microglial activation and increased neuron survival in response to distal axotomy of SNc neurons in APP KO mice are consistent with the important role APP in neuronal stress responses in vivo.

Marked dopaminergic cell loss subsequent to developmental, intranigral expression of glial cell line-derived neurotrophic factor.

Glial cell line-derived neurotrophic factor (GDNF) shows potent neuroprotective as well as neurorestorative actions on the adult neurons impacted in animal models of Parkinson's disease (PD). Long-term pharmaco-physiological effects of GDNF on developing dopaminergic (DA) neurons have not yet been explored because of technical difficulties in producing prolonged cell type-specific delivery of this neurotrophic factor in mammalian embryonic brain. The current studies used our previously characterized 9.0-kb tyrosine hydroxylase promoter to produce transgenic mice with neuronal cell type-specific expression of GDNF in substantia nigra pars compacta (SNc) and locus coeruleus (LC). These mice were used to test the parsimonious hypothesis that increased developmental expression of GDNF in SNc and LC would significantly enhance the number of postmitotic adult neurons. To our surprise, adult transgenic mice carrying the TH9.0kb-GDNF hybrid gene showed dramatic reductions in both the numbers and the volumes of SNc-DA and LC-noradrenergic (NA) neurons by quantitative morphometric analysis. The decrease in the number of DA neurons was apparent as early as postnatal day 2, the period before the major naturally occurring apoptotic cell death in midbrain. Aged transgenic mice exhibited no further significant deficits in motor behaviors. These data suggest that continuous, early developmental GDNF expression exerts physiological effects on newly differentiated, immature dopamine neurons that differ from those observed on more mature and adult DA neurons. Further elucidation of the mechanisms underlying differential GDNF actions will greatly improve the pharmacological efficacy of GDNF in fetal neural transplantation as well as adult neuronal gene therapy in PD patients.