Hong S. Chun

Dopaminergic cell death induced by MPP+, oxidant and specific neurotoxicants shares the common molecular mechanism

Recent etiological study in twins (Tanner et al. 1999) strongly suggests that environmental factors play an important role in typical, non‐familial Parkinsons disease (PD), beginning after age 50. Epidemiological risk factor analyses of typical PD cases have identified several neurotoxicants, including MPP+ (the active metabolite of MPTP), paraquat, dieldrin, manganese and salsolinol. Here, we tested the hypothesis that these neurotoxic agents might induce cell death in our nigral dopaminergic cell line, SN4741 (Son et al. 1999) through a common molecular mechanism. Our initial experiments revealed that treatment with both MPP+ and the other PD‐related neurotoxicants induced apoptotic cell death in SN4741 cells, following initial increases of H2O2‐related ROS activity and subsequent activation of JNK1/2 MAP kinases. Moreover, we have demonstrated that during dopaminergic cell death cascades, MPP+, the neurotoxicants and an oxidant, H2O2 equally induce the ROS‐dependent events. Remarkably, the oxidant treatment alone induced similar sequential molecular events: ROS increase, activation of JNK MAP kinases, activation of the PITSLRE kinase, p110, by both Caspase‐1 and Caspase‐3‐like activities and apoptotic cell death. Pharmacological intervention using the combination of the antioxidant Trolox and a pan‐caspase inhibitor Boc‐(Asp)‐fmk (BAF) exerted significant neuroprotection against ROS‐induced dopaminergic cell death. Finally, the high throughput cDNA microarray screening using the current model identified downstream response genes, such as heme oxygenase‐1, a constituent of Lewy bodies, that can be the useful biomarkers to monitor the pathological conditions of dopaminergic neurons under neurotoxic insult.

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 Parkinsons 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.

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 Parkinsons 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.

Identification of potential compounds promoting BDNF production in nigral dopaminergic neurons : clinical implication in Parkinson's disease

Parkinsons disease (PD) is characterized by the selective loss of dopamine (DA) neurons in the substantia nigral brain region. Currently, there is no cure or treatment that prevents such neuronal loss. Brain-derived neurotrophic factor (BDNF) has been found to support the survival of DA neurons in animal models and in primary cell cultures. However, the large molecular size of BDNF, coupled with the blood brain barrier, prevents its delivery to DA neurons to promote cell survival in the PD brain. The nigral DA neurons have the ability to produce BDNF for neuroprotection via either autocrine or paracrine mechanisms. Low mol. wt compounds were tested to see whether they could increase the production of BDNF in the DA neurons. The compounds tested include neurotransmitters, neuropeptides, intracellular signaling agents, known neuroprotective agents and growth factors. Our results demonstrate that salicyclic acid, cGMP analog, okadaic acid, IBMX, dipyridamole and glutamate significantly enhance BDNF production in DA neuronal cells.

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 Parkinsons 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. GLIA 38:174–178, 2002.

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.

Experimental Strategy to Identify Genes Susceptible to Oxidative Stress in Nigral Dopaminergic Neurons

Neuropathological evidence from both human and experimental models of Parkinsons 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 (1–3). 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.

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 Parkinsons 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.