Dopaminergic neuron-specific deletion of p53 gene is neuroprotective in an experimental Parkinson's disease model.

p53, a stress response gene, is involved in diverse cell death pathways and its activation has been implicated in the pathogenesis of Parkinson's disease (PD). However, whether the neuronal p53 protein plays a direct role in regulating dopaminergic (DA) neuronal cell death is unknown. In this study, in contrast to the global inhibition of p53 function by pharmacological inhibitors and in traditional p53 knock-out (KO) mice, we examined the effect of DA specific p53 gene deletion in DAT-p53KO mice. These DAT-p53KO mice did not exhibit apparent changes in the general structure and neuronal density of DA neurons during late development and in aging. However, in DA-p53KO mice treated with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we found that the induction of Bax and p53 up-regulated modulator of apoptosis (PUMA) mRNA and protein levels by MPTP were diminished in both striatum and substantia nigra of these mice. Notably, deletion of the p53 gene in DA neurons significantly reduced dopaminergic neuronal loss in substantia nigra, dopaminergic neuronal terminal loss at striatum and, additionally, decreased motor deficits in mice challenged with MPTP. In contrast, there was no difference in astrogliosis between WT and DAT-p53KO mice in response to MPTP treatment. These findings demonstrate a specific contribution of p53 activation in DA neuronal cell death by MPTP challenge. Our results further support the role of programmed cell death mediated by p53 in this animal model of PD and identify Bax, BAD and PUMA genes as downstream targets of p53 in modulating DA neuronal death in the in vivo MPTP-induced PD model. We deleted p53 gene in dopaminergic neurons in late developmental stages and found that DA specific p53 deletion is protective in acute MPTP animal model possibly through blocking MPTP-induced BAX and PUMA up-regulation. Astrocyte activation measured by GFAP positive cells and GFAP gene up-regulation in the striatum shows no difference between wt and DA-p53 ko mice.

Inhibition of Drp1 mitochondrial translocation provides neural protection in dopaminergic system in a Parkinson's disease model induced by MPTP.

Accumulating evidence suggest mitochondria-mediated pathways play an important role in dopaminergic neuronal cell death in Parkinson's disease (PD). Drp1, a key regulator of mitochondrial fission, has been shown to be activated and translocated to mitochondria under stress, leading to excessive mitochondria fission and dopaminergic neuronal death in vitro. However, whether Drp1 inhibition can lead to long term stable preservation of dopaminergic neurons in PD-related mouse models remains unknown. In this study, using a classical MPTP animal PD model, we showed for the first time Drp1 activation and mitochondrial translocation in vivo after MPTP administration. Inhibition of Drp1 activation by a selective peptide inhibitor P110, blocked MPTP-induced Drp1 mitochondrial translocation and attenuated dopaminergic neuronal loss, dopaminergic nerve terminal damage and behavioral deficits caused by MPTP. MPTP-induced microglial activation and astrogliosis were not affected by P110 treatment. Instead, inhibition of Drp1 mitochondrial translocation diminished MPTP-induced p53, BAX and PUMA mitochondrial translocation. This study demonstrates that inhibition of Drp1 hyperactivation by a Drp1 peptide inhibitor P110 is neuroprotective in a MPTP animal model. Our data also suggest that the protective effects of P110 treatment might be mediated by inhibiting the p53 mediated apoptotic pathways in neurons through inhibition of Drp1-dependent p53 mitochondrial translocation.

Effect of the sonic hedgehog receptor smoothened on the survival and function of dopaminergic neurons.

OBJECTIVE: To determine the influence of the sonic hedgehog (shh) pathway and its receptor smoothened (smo), on the survival and functionality of dopaminergic (DA) neurons. BACKGROUND: During early development, shh induces the differentiation of DA neurons. However, it is unknown whether shh signaling is required in the maturation or maintenance of DA neurons during later development and adulthood due to the lethality of traditional shh knockout models. METHODS: We utilized the cre-loxP system to achieve late developmental stage and cell type-specific deletion of the shh receptor, smo, in DA neurons by crossing DATcre (dopamine transporter) mice with Smo(loxP/loxP) mice. We assessed for differences between knockout (ko) and wildtype (wt) mice using combined histochemistry, gene expression analysis, and behavioral evaluation. Number and size of DA neurons in ventral midbrain and the DA neural terminal density in striatum were measured using unbiased stereological quantification. The survival of DA neurons under neurotoxin challenge was examined in the unilateral 6-hydroxydopamine (6-OHDA) Parkinson's disease animal model and the more subtle function under challenge of the dopaminergic system was examined by methamphetamine single- and repeated challenge in wt and ko mice. RESULTS: Tyrosine hydroxylase (TH) positive neuronal counts and neuronal size in substantia nigra (SN) and ventral tegmental area (VTA) showed no difference between wt and DAT-Smo ko mice in young (5months) or aged (22months) mice. There was also no difference in the striatal DA projections between wt and ko mice in both age groups. In unilateral striatal 6-OHDA lesions modeling Parkinson's disease, using stereotaxic injection of 6-OHDA intrastriatally led to loss of dopaminergic neurons in SN and diminished TH positive projections in striatum. However, there was no differences in survival of DA neurons between wt and ko mice. DAT-Smo ko mice demonstrated hyperactivity compared to wt mice at 5months, but showed no difference in activity at 22months. When injected with a one-time bolus of methamphetamine (METH), despite the higher basal locomotion activity, DAT-Smo ko mice showed a diminished response to a single METH challenge. In METH sensitization testing, ko mice showed decreased sensitization compared to wt mice without evidence of a delayed shift in dynamics of sensitization. Gene expression analysis showed decreased gene expression of smo, Gli 1 (known target gene of smo) and BDNF (brain-derived neurotrophic factor) in the SN. Gene expression was not altered in striatum for the genes examined in this study including dopamine receptor genes, neurotropic genes such as Glial cell line-derived neurotrophic factor (GDNF), and bone morphogenetic protein 7 (BMP7). CONCLUSION: Our study showed the smo receptor function is not required for the maturation and survival of DA neurons during late development, aging or under stress challenge. However, smo function has an influence on behavior in young adult mice and in responses of mice to a drug that modulates DA neurochemistry through regulation of gene expression in DA neurons. Since young adult DAT-smo ko mice show hyperactivity and altered response to a psychostimulant drug (METH), this may indicate the involvement of the shh pathway in the development of functional changes that manifest as alterations in DA pathway dynamics.

Using iPSC-derived human DA neurons from opioid-dependent subjects to study dopamine dynamics.

INTRODUCTION: The dopaminergic (DA) system plays important roles in addiction. However, human DA neurons from drug-dependent subjects were not available for study until recent development in inducible pluripotent stem cells (iPSCs) technology. METHODS: In this study, we produced DA neurons differentiated using iPSCs derived from opioid-dependent and control subjects carrying different 3' VNTR (variable number tandem repeat) polymorphism in the human dopamine transporter (DAT or SLC6A3). In addition, the effects of valproic acid (VPA) exposures on iPSC-derived human DA neurons are also examined. RESULTS: We present the first evidence suggesting that the 3' VNTR polymorphism in the hDAT gene affects DAT expression level in iPSC-derived human DA neurons. In human DA neurons, which provide an appropriate cellular milieu, VPA treatment alters the expression of several genes important for dopaminergic neuron function including DAT, Nurr1, and TH; this might partly explain its action in regulating addictive behaviors. VPA treatment also significantly increased DA D2 receptor (Drd2) expression, especially in the opioid-dependent iPSC cell lines. CONCLUSIONS: Our data suggest that human iPSC-derived DA neurons may be useful in in vitro experimental model to examine the effects of genetic variation in gene regulation, to examine the underlying mechanisms in neurological disorders including drug addiction, and to serve as a platform for therapeutic development.

The shh signaling pathway is upregulated in multiple cell types in cortical ischemia and influences the outcome of stroke in an animal model.

Recently the sonic hedgehog (shh) signaling pathway has been shown to play an important role in regulating repair and regenerative responses after brain injury, including ischemia. However, the precise cellular components that express and upregulate the shh gene and the cellular components that respond to shh signaling remain to be identified. In this study, using a distal MCA occlusion model, our data show that the shh signal is upregulated both at the cortical area near the injury site and in the adjacent striatum. Multiple cell types upregulate shh signaling in ischemic brain, including neurons, reactive astrocytes and nestin-expressing cells. The shh signaling pathway genes are also expressed in the neural stem cells (NSCs) niche in the subventricular zone (SVZ). Conditional deletion of the shh gene in nestin-expressing cells both at the SVZ niche and at the ischemic site lead to significantly more severe behavioral deficits in these shh iKO mice after cortical stroke, measured using an automated open field locomotion apparatus (Student's t-test, p<0.05). In contrast, animals given post-stroke treatment with the shh signaling agonist (SAG) demonstrated less deficits in behavioral function, compared to vehicle-treated mice. At 7 days after stroke, SAG-treated mice showed higher values in multiple horizontal movement parameters compared to vehicle treated mice (Student's t-test, p<0.05) whereas there were no differences in pre-stroke measurements, (Student's t-test, p>0.05). In summary, our data demonstrate that shh signaling plays critical and ongoing roles in response to ischemic injury and modulation of shh signaling in vivo alters the functional outcome after cortical ischemic injury.