AMP-activated protein kinase is activated in Parkinson’s disease models mediated by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

The selective loss of dopaminergic neurons in the substantia nigra pars compacta is a feature of Parkinson’s disease (PD). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity is the most common experimental model used to investigate the pathogenesis of PD. Administration of MPTP in mice produces neuropathological defects as observed in PD and 1-methyl-4-pyridinium (MPP⁺) induces cell death when neuronal cell cultures are used. AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis. In the present study, we demonstrated that AMPK is activated by MPTP in mice and MPP⁺ in SH-SY5Y cells. The inhibition of AMPK by compound C resulted in an increase in MPP⁺-induced cell death. We further showed that overexpression of AMPK increased cell viability after exposure to MPP⁺ in SH-SY5Y cells. Based on these results, we suggest that activation of AMPK might prevent neuronal cell death and play a role as a survival factor in PD.     

IDH2 deficiency promotes mitochondrial dysfunction and dopaminergic neurotoxicity: implications for Parkinson’s disease

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and its pathogenesis is under intense investigation. Substantial evidence indicates that mitochondrial dysfunction and oxidative stress play central roles in the pathophysiology of PD, through activation of mitochondria-dependent apoptotic molecular pathways. Several mitochondrial internal regulating factors act to maintain mitochondrial function. However, the mechanism by which these internal regulating factors contribute to mitochondrial dysfunction in PD remains elusive. One of these factors, mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2), has been implicated in the regulation of mitochondrial redox balance and reduction of oxidative stress-induced cell injury. Here we report that IDH2 regulates mitochondrial dysfunction and cell death in MPP⁺/MPTP-induced DA neuronal cells, and in a mouse model of PD. Down-regulation of IDH2 increased DA neuron sensitivity to MPP⁺; lowered IDH2 levels facilitated induction of apoptotic cell death due to elevated mitochondrial oxidative stress. Deficient IDH2 also promoted loss of DA SNpc neurons in an MPTP mouse model of PD. Interestingly, Mito-TEMPO, a mitochondrial ROS-specific scavenger, protected degeneration of SNpc DA neurons in the MPTP model of PD. These findings demonstrate that IDH2 contributes to degeneration of the DA neuron in the neurotoxin model of PD and establish IDH2 as a molecular target of potential therapeutic significance for this disabling neurological illness.     

Tat-Fused Recombinant Human SAG Prevents Dopaminergic Neurodegeneration in a MPTP-Induced Parkinson’s Disease Model

Excessive reactive oxygen species (ROS) generated from abnormal cellular process lead to various human diseases such as inflammation, ischemia, and Parkinson’s disease (PD). Sensitive to apoptosis gene (SAG), a RING-FINGER protein, has anti-apoptotic activity and anti-oxidant activity. In this study, we investigate whether Tat-SAG, fused with a Tat domain, could protect SH-SY5Y neuroblastoma cells against 1-methyl-4-phenylpyridinium (MPP⁺) and dopaminergic (DA) neurons in the substantia nigra (SN) against 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP) toxicity. Western blot and immunohistochemical analysis showed that, unlike SAG, Tat-SAG transduced efficiently into SH-SY5Y cells and into the brain, respectively. Tat-SAG remarkably suppressed ROS generation, DNA damage, and the progression of apoptosis, caused by MPP⁺ in SH-SY5Y cells. Also, immunohistochemical data using a tyrosine hydroxylase antibody and cresyl violet staining demonstrated that Tat-SAG obviously protected DA neurons in the SN against MPTP toxicity in a PD mouse model. Tat-SAG-treated mice showed significant enhanced motor activities, compared to SAG- or Tat-treated mice. Therefore, our results suggest that Tat-SAG has potential as a therapeutic agent against ROS-related diseases such as PD.     

Involvment of Cytosolic and Mitochondrial GSK-3β in Mitochondrial Dysfunction and Neuronal Cell Death of MPTP/MPP+-Treated Neurons

Aberrant mitochondrial function appears to play a central role in dopaminergic neuronal loss in Parkinson''s disease (PD). 1-methyl-4-phenylpyridinium iodide (MPP⁺), the active metabolite of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is a selective inhibitor of mitochondrial complex I and is widely used in rodent and cell models to elicit neurochemical alterations associated with PD. Recent findings suggest that Glycogen Synthase Kinase-3β (GSK-3β), a critical activator of neuronal apoptosis, is involved in the dopaminergic cell death. In this study, the role of GSK-3β in modulating MPP⁺-induced mitochondrial dysfunction and neuronal death was examined in vivo, and in two neuronal cell models namely primary cultured and immortalized neurons. In both cell models, MPTP/MPP⁺ treatment caused cell death associated with time- and concentration-dependent activation of GSK-3β, evidenced by the increased level of the active form of the kinase, i.e. GSK-3β phosphorylated at tyrosine 216 residue. Using immunocytochemistry and subcellular fractionation techniques, we showed that GSK-3β partially localized within mitochondria in both neuronal cell models. Moreover, MPP⁺ treatment induced a significant decrease of the specific phospho-Tyr216-GSK-3β labeling in mitochondria concomitantly with an increase into the cytosol. Using two distinct fluorescent probes, we showed that MPP⁺ induced cell death through the depolarization of mitochondrial membrane potential. Inhibition of GSK-3β activity using well-characterized inhibitors, LiCl and kenpaullone, and RNA interference, prevented MPP⁺-induced cell death by blocking mitochondrial membrane potential changes and subsequent caspase-9 and -3 activation. These results indicate that GSK-3β is a critical mediator of MPTP/MPP⁺-induced neurotoxicity through its ability to regulate mitochondrial functions. Inhibition of GSK-3β activity might provide protection against mitochondrial stress-induced cell death.     

Naringin protects the nigrostriatal dopaminergic projection through induction of GDNF in a neurotoxin model of Parkinson's disease

This study investigated the effect of naringin, a major flavonoid in grapefruit and citrus fruits, on the degeneration of the nigrostriatal dopaminergic (DA) projection in a neurotoxin model of Parkinson's disease (PD) in vivo and the potential underlying mechanisms focusing on the induction of glia-derived neurotrophic factor (GDNF), well known as an important neurotrophic factor involved in the survival of adult DA neurons. 1-Methyl-4-phenylpyridinium (MPP⁺) was unilaterally injected into the medial forebrain bundle of rat brains for a neurotoxin model of PD in the presence or absence of naringin by daily intraperitoneal injection. To ascertain whether naringin-induced GDNF contributes to neuroprotection, we further investigated the effects of intranigral injection of neutralizing antibodies against GDNF in the MPP⁺ rat model of PD. Our observations demonstrate that naringin could increase the level of GDNF in DA neurons, contributing to neuroprotection in the MPP⁺ rat model of PD, with activation of mammalian target of rapamycin complex 1. Moreover, naringin could attenuate the level of tumor necrosis factor-α in microglia increased by MPP⁺-induced neurotoxicity in the substantia nigra. These results indicate that naringin could impart to DA neurons the important ability to produce GDNF as a therapeutic agent against PD with anti-inflammatory effects, suggesting that naringin is a beneficial natural product for the prevention of DA degeneration in the adult brain.     

The Peptidyl-prolyl Isomerase Pin1 Up-regulation and Proapoptotic Function in Dopaminergic Neurons: RELEVANCE TO THE PATHOGENESIS OF PARKINSON DISEASE*

Parkinson disease (PD) is a chronic neurodegenerative disease characterized by a slow and progressive degeneration of dopaminergic neurons in substantia nigra. The pathophysiological mechanisms underlying PD remain unclear. Pin1, a major peptidyl-prolyl isomerase, has recently been associated with certain diseases. Notably, Ryo et al. (Ryo, A., Togo, T., Nakai, T., Hirai, A., Nishi, M., Yamaguchi, A., Suzuki, K., Hirayasu, Y., Kobayashi, H., Perrem, K., Liou, Y. C., and Aoki, I. (2006) J. Biol. Chem. 281, 4117–4125) implicated Pin1 in PD pathology. Therefore, we sought to systematically characterize the role of Pin1 in PD using cell culture and animal models. To our surprise we observed a dramatic up-regulation of Pin1 mRNA and protein levels in dopaminergic MN9D neuronal cells treated with the parkinsonian toxicant 1-methyl-4-phenylpyridinium (MPP⁺) as well as in the substantia nigra of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. Notably, a marked expression of Pin1 was also observed in the substantia nigra of human PD brains along with a high co-localization of Pin1 within dopaminergic neurons. In functional studies, siRNA-mediated knockdown of Pin1 almost completely prevented MPP⁺-induced caspase-3 activation and DNA fragmentation, indicating that Pin1 plays a proapoptotic role. Interestingly, multiple pharmacological Pin1 inhibitors, including juglone, attenuated MPP⁺-induced Pin1 up-regulation, α-synuclein aggregation, caspase-3 activation, and cell death. Furthermore, juglone treatment in the MPTP mouse model of PD suppressed Pin1 levels and improved locomotor deficits, dopamine depletion, and nigral dopaminergic neuronal loss. Collectively, our findings demonstrate for the first time that Pin1 is up-regulated in PD and has a pathophysiological role in the nigrostriatal dopaminergic system and suggest that modulation of Pin1 levels may be a useful translational therapeutic strategy in PD.     

Angiotensin type 1 receptor antagonist losartan, reduces MPTP-induced degeneration of dopaminergic neurons in substantia nigra

Background

Recent attention has focused on understanding the role of the brain-renin-angiotensin-system (RAS) in stroke and neurodegenerative diseases. Direct evidence of a role for the brain-RAS in Parkinson's disease (PD) comes from studies demonstrating the neuroprotective effect of RAS inhibitors in several neurotoxin based PD models. In this study, we show that an antagonist of the angiotensin II (Ang II) type 1 (AT₁) receptor, losartan, protects dopaminergic (DA) neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity both in primary ventral mesencephalic (VM) cultures as well as in the substantia nigra pars compacta (SNpc) of C57BL/6 mice (Fig. 1).

Results

In the presence of exogenous Ang II, losartan reduced MPP⁺ (5 μM) induced DA neuronal loss by 72% in vitro. Mice challenged with MPTP showed a 62% reduction in the number of DA neurons in the SNpc and a 71% decrease in tyrosine hydroxylase (TH) immunostaining of the striatum, whereas daily treatment with losartan lessened MPTP-induced loss of DA neurons to 25% and reduced the decrease in striatal TH⁺ immunostaining to 34% of control.

Conclusion

Our study demonstrates that the brain-RAS plays an important neuroprotective role in the MPTP model of PD and points to AT₁ receptor as a potential novel target for neuroprotection.     

Selective degeneration of dopaminergic neurons by MPP+ and its rescue by D2 autoreceptors in Drosophila primary culture

Drosophila melanogaster is widely used to study genetic factors causing Parkinson's disease (PD) largely because of the use of sophisticated genetic approaches and the presence of a high conservation of gene sequence/function between Drosophila and mammals. However, in Drosophila, little has been done to study the environmental factors which cause over 90% of PD cases. We used Drosophila primary neuronal culture to study degenerative effects of a well‐known PD toxin MPP⁺. Dopaminergic (DA) neurons were selectively degenerated by MPP⁺, whereas cholinergic and GABAergic neurons were not affected. This DA neuronal loss was because of post‐mitotic degeneration, not by inhibition of DA neuronal differentiation. We also found that MPP⁺‐mediated neurodegeneration was rescued by D2 agonists quinpirole and bromocriptine. This rescue was through activation of Drosophila D2 receptor DD2R, as D2 agonists failed to rescue MPP⁺‐toxicity in neuronal cultures prepared from both a DD2R deficiency line and a transgenic line pan‐neuronally expressing DD2R RNAi. Furthermore, DD2R autoreceptors in DA neurons played a critical role in the rescue. When DD2R RNAi was expressed only in DA neurons, MPP⁺ toxicity was not rescued by D2 agonists. Our study also showed that rescue of DA neurodegeneration by Drosophila DD2R activation was mediated through suppression of action potentials in DA neurons.     

2′,3′-Dideoxycytidine Protects Dopaminergic Neurons in a Mouse Model of Parkinson’s Disease

DNA polymerase-β (DNA pol-β) plays a crucial role in the pathogenesis of Parkinson’s disease (PD). The aim of this study was to investigate the neuroprotective effects of a DNA polymerase-β inhibitor 2′,3′-dideoxycytidine (DDC) in PD models. In the in vitro studies, primary cultured neurons were challenged with 1-methyl-4-phenylpyridinium ion (MPP⁺). The expression of DNA pol-β was assessed using western blot. The neuroprotective effect of DNA pol-β knockdown and DNA pol-β inhibitor DDC was determined using cell viability assay and caspase-3 activity assay. We found that MPP⁺ induced neuronal death and the activation of caspase-3 in a dose-dependent manner. The expression of DNA pol-β increased after the neurons were exposed to MPP⁺. DNA pol-β siRNA or DNA pol-β inhibitor DDC attenuated neuronal death induced by MPP⁺. In the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD, MPTP treatment triggered behavioral deficits and nigrostriatal lesions. Pretreatment with DDC attenuated MPTP-induced behavioral deficits, dopaminergic neuronal death and striatal dopamine depletion in the MPTP mouse model. These results indicate that DNA pol-β inhibitors may present a novel promising therapeutic option for the neuroprotective treatment of PD.

     

Regulation of Histone Acetylation by Autophagy in Parkinson Disease

Parkinson disease (PD) is the most common age-dependent neurodegenerative movement disorder. Accumulated evidence indicates both environmental and genetic factors play important roles in PD pathogenesis, but the potential interaction between environment and genetics in PD etiology remains largely elusive. Here, we report that PD-related neurotoxins induce both expression and acetylation of multiple sites of histones in cultured human cells and mouse midbrain dopaminergic (DA) neurons. Consistently, levels of histone acetylation are markedly higher in midbrain DA neurons of PD patients compared to those of their matched control individuals. Further analysis reveals that multiple histone deacetylases (HDACs) are concurrently decreased in 1-methyl-4-phenylpyridinium (MPP⁺)-treated cells and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mouse brains, as well as midbrain tissues of human PD patients. Finally, inhibition of histone acetyltransferase (HAT) protects, whereas inhibition of HDAC1 and HDAC2 potentiates, MPP⁺-induced cell death. Pharmacological and genetic inhibition of autophagy suppresses MPP⁺-induced HDACs degradation. The study reveals that PD environmental factors induce HDACs degradation and histone acetylation increase in DA neurons via autophagy and identifies an epigenetic mechanism in PD pathogenesis.     

A Wnt1 regulated Frizzled-1/β-Catenin signaling pathway as a candidate regulatory circuit controlling mesencephalic dopaminergic neuron-astrocyte crosstalk: Therapeutical relevance for neuron survival and neuroprotection

Background

Dopamine-synthesizing (dopaminergic, DA) neurons in the ventral midbrain (VM) constitute a pivotal neuronal population controlling motor behaviors, cognitive and affective brain functions, which generation critically relies on the activation of Wingless-type MMTV integration site (Wnt)/β-catenin pathway in their progenitors. In Parkinson's disease, DA cell bodies within the substantia nigra pars compacta (SNpc) progressively degenerate, with causes and mechanisms poorly understood. Emerging evidence suggests that Wnt signaling via Frizzled (Fzd) receptors may play a role in different degenerative states, but little is known about Wnt signaling in the adult midbrain. Using in vitro and in vivo model systems of DA degeneration, along with functional studies in both intact and SN lesioned mice, we herein highlight an intrinsic Wnt1/Fzd-1/β-catenin tone critically contributing to the survival and protection of adult midbrain DA neurons.

Results

In vitro experiments identifie Fzd-1 receptor expression at a mRNA and protein levels in dopamine transporter (DAT) expressing neurons, and demonstrate the ability of exogenous Wnt1 to exert robust neuroprotective effects against Caspase-3 activation, the loss of tyrosine hydroxylase-positive (TH⁺) neurons and [³H] dopamine uptake induced by different DA-specific insults, including serum and growth factor deprivation, 6-hydroxydopamine and MPTP/MPP⁺. Co-culture of DA neurons with midbrain astrocytes phenocopies Wnt1 neuroprotective effects, whereas RNA interference-mediated knockdown of Wnt1 in midbrain astrocytes markedly reduces astrocyte-induced TH⁺ neuroprotection. Likewise, silencing β-catenin mRNA or knocking down Fzd-1 receptor expression in mesencephalic neurons counteract astrocyte-induced TH⁺ neuroprotection. In vivo experiments document Fzd-1 co-localization with TH⁺ neurons within the intact SNpc and blockade of Fzd/β-catenin signaling by unilateral infusion of a Fzd/β-catenin antagonist within the SN induces reactive astrocytosis and acutely inhibits TH⁺ neuron survival in ipsilateral SNpc, an effect efficiently prevented by pharmacological activation of β-catenin signaling within the SNpc.

Conclusion

These results defining a novel Wnt1/Fzd-1/β-catenin astrocyte-DA autoprotective loop provide a new mechanistic inside into the regulation of pro-survival processes, with potentially relevant consequences for drug design or drug action in Parkinson's disease.     

Protective effects of PEP-1-Catalase on stress-induced cellular toxicity and MPTP-induced Parkinson’s disease

Parkinson’s disease (PD) is a neurodegenerative disability caused by a decrease of dopaminergic neurons in the substantia nigra (SN). Although the etiology of PD is not clear, oxidative stress is believed to lead to PD. Catalase is antioxidant enzyme which plays an active role in cells as a reactive oxygen species (ROS) scavenger. Thus, we investigated whether PEP-1-Catalase protects against 1-methyl-4-phenylpyridinium (MPP⁺) induced SH-SY5Y neuronal cell death and in a 1-methyl-4-phenyl-1,2,3,6-trtrahydropyridine (MPTP) induced PD animal model. PEP-1-Catalase transduced into SH-SY5Y cells significantly protecting them against MPP⁺-induced death by decreasing ROS and regulating cellular survival signals including Akt, Bax, Bcl-2, and p38. Immunohistochemical analysis showed that transduced PEP-1-Catalase markedly protected against neuronal cell death in the SN in the PD animal model. Our results indicate that PEP-1-Catalase may have potential as a therapeutic agent for PD and other oxidative stress related diseases. [BMB Reports 2015; 48(7): 395-400]     

Proliferating Cell Nuclear Antigen Binds DNA Polymerase-β and Mediates 1-Methyl-4-Phenylpyridinium-Induced Neuronal Death

The mechanisms leading to dopaminergic neuronal loss in the substantia nigra of patients with Parkinson disease (PD) remain poorly understood. We recently reported that aberrant DNA replication mediated by DNA polymerase-β (DNA pol-β) plays a causal role in the death of postmitotic neurons in an in vitro model of PD. In the present study, we show that both proliferating cell nuclear antigen (PCNA) and DNA pol-β are required for MPP⁺-induced neuronal death. PCNA binds to the catalytic domain of DNA pol-β in MPP⁺-treated neurons and in post-mortem brain tissues of PD patients. The PCNA-DNA pol-β complex is loaded into DNA replication forks and mediates DNA replication in postmitotic neurons. The aberrant DNA replication mediated by the PCNA-DNA pol-β complex induces p53-dependent neuronal cell death. Our results indicate that the interaction of PCNA and DNA pol-β contributes to neuronal death in PD.     

Embelin averts MPTP-induced dysfunction in mitochondrial bioenergetics and biogenesis via activation of SIRT1

Parkinson's disease (PD) is a chronic neurodegenerative disease characterized by the death of dopamine neurons of Substantia nigra pars compacta (SNpc) leading to motor deficits. Amongst the mechanisms proposed, mitochondrial dysfunction, reduced complex-I and PGC1α levels were found to correlate with the pathology of PD. As embelin is a natural product with structural resemblance to ubiquinone, exhibits mitochondrial uncoupling and antioxidant effects, in the present study, we sought to examine its role in the mechanisms mediating PD. Results indicate that embelin protects from MPP⁺-induced oxidative stress and apoptosis in a time and dose-dependent manner in N27 dopaminergic cells. Cells treated with embelin exhibited increased levels of pAMPK, SIRT1 and PGC1α leading to enhanced mitochondrial biogenesis. Though treatment of cells with MPP⁺ also increased pAMPK levels, but, SIRT1 and PGC1α levels decreased substantially, possibly due to the block in the mitochondrial electron transport chain and reduced NAD/NADH levels. The mitochondrial uncoupling effects of embelin leading to increased NAD/NADH levels followed by enhanced SIRT1, PGC1α and mitochondrial biogenesis were found to confer embelin mediated protection as treatment of cells with SIRT1 inhibitor or siRNA nullified this effect. Embelin (10 mg/kg) also conferred protection in vivo in MPTP mouse model of PD, wherein, MPTP-induced loss of TH staining, reduced striatal dopamine and markers of mitochondrial biogenesis pathway were averted by embelin.     

Insulin-like growth factor 1 protects human neuroblastoma cells SH-EP1 against MPP<Superscript>+</Superscript>-induced apoptosis by AKT/GSK-3β/JNK signaling

Parkinson’s disease (PD) is primarily caused by severe degeneration and loss of dopamine neurons in the substantia nigra pars compacta. Thus, preventing the death of dopaminergic neurons is thought to be a potential strategy to interfere with the development of PD. In the present work, we studied the effect of insulin-like growth factor-1 (IGF-1) on 1-methyl-4-phenylpyridinium (MPP⁺)-induced apoptosis in human neuroblastoma SH-EP1 cells. We found that the PI3K/AKT pathway plays a central role in IGF-mediated cell survival against MPP⁺ neurotoxicity. Furthermore, we demonstrated that the protective effect of AKT is largely dependent on the inactivation of GSK-3β, since inhibition of GSK-3β by its inhibitor, BIO, could mimic the protective effect of IGF-1 on MPP⁺-induced cell death in SH-EP1 cells. Interestingly, the IGF-1 potentiated PI3K/AKT activity is found to negatively regulate the JNK related apoptotic pathway and this negative regulation is further shown to be mediated by AKT-dependent GSK-3β inactivation. Thus, our results demonstrated that IGF-1 protects SH-EP1 cells from MPP⁺-induced apoptotic cell death via PI3K/AKT/GSK-3β pathway, which in turn inhibits MPP⁺-induced JNK activation.     

Changes in Neuronal Dopamine Homeostasis following 1-Methyl-4-phenylpyridinium (MPP+) Exposure

1-Methyl-4-phenylpyridinium (MPP⁺), the active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, selectively kills dopaminergic neurons in vivo and in vitro via a variety of toxic mechanisms, including mitochondrial dysfunction, generation of peroxynitrite, induction of apoptosis, and oxidative stress due to disruption of vesicular dopamine (DA) storage. To investigate the effects of acute MPP⁺ exposure on neuronal DA homeostasis, we measured stimulation-dependent DA release and non-exocytotic DA efflux from mouse striatal slices and extracellular, intracellular, and cytosolic DA (DA_cyt) levels in cultured mouse ventral midbrain neurons. In acute striatal slices, MPP⁺ exposure gradually decreased stimulation-dependent DA release, followed by massive DA efflux that was dependent on MPP⁺ concentration, temperature, and DA uptake transporter activity. Similarly, in mouse midbrain neuronal cultures, MPP⁺ depleted vesicular DA storage accompanied by an elevation of cytosolic and extracellular DA levels. In neuronal cell bodies, increased DA_cyt was not due to transmitter leakage from synaptic vesicles but rather to competitive MPP⁺-dependent inhibition of monoamine oxidase activity. Accordingly, monoamine oxidase blockers pargyline and l-deprenyl had no effect on DA_cyt levels in MPP⁺-treated cells and produced only a moderate effect on the survival of dopaminergic neurons treated with the toxin. In contrast, depletion of intracellular DA by blocking neurotransmitter synthesis resulted in ∼30% reduction of MPP⁺-mediated toxicity, whereas overexpression of VMAT2 completely rescued dopaminergic neurons. These results demonstrate the utility of comprehensive analysis of DA metabolism using various electrochemical methods and reveal the complexity of the effects of MPP⁺ on neuronal DA homeostasis and neurotoxicity.     

Commitment of 1-Methyl-4-phenylpyrinidinium Ion-induced Neuronal Cell Death by Proteasome-mediated Degradation of p35 Cyclin-dependent Kinase 5 Activator

The dysfunction of proteasomes and mitochondria has been implicated in the pathogenesis of Parkinson disease. However, the mechanism by which this dysfunction causes neuronal cell death is unknown. We studied the role of cyclin-dependent kinase 5 (Cdk5)-p35 in the neuronal cell death induced by 1-methyl-4-phenylpyrinidinium ion (MPP⁺), which has been used as an in vitro model of Parkinson disease. When cultured neurons were treated with 100 μm MPP⁺, p35 was degraded by proteasomes at 3 h, much earlier than the neurons underwent cell death at 12–24 h. The degradation of p35 was accompanied by the down-regulation of Cdk5 activity. We looked for the primary target of MPP⁺ that triggered the proteasome-mediated degradation of p35. MPP⁺ treatment for 3 h induced the fragmentation of the mitochondria, reduced complex I activity of the respiratory chain without affecting ATP levels, and impaired the mitochondrial import system. The dysfunction of the mitochondrial import system is suggested to up-regulate proteasome activity, leading to the ubiquitin-independent degradation of p35. The overexpression of p35 attenuated MPP⁺-induced neuronal cell death. In contrast, depletion of p35 with short hairpin RNA not only induced cell death but also sensitized to MPP⁺ treatment. These results indicate that a brief MPP⁺ treatment triggers the delayed neuronal cell death by the down-regulation of Cdk5 activity via mitochondrial dysfunction-induced up-regulation of proteasome activity. We propose a role for Cdk5-p35 as a survival factor in countering MPP⁺-induced neuronal cell death.Parkinson disease (PD)³ is the second most common neurodegenerative disease, characterized pathologically by degenerated dopaminergic neurons and ubiquitin-positive aggregates known as Lewy bodies (1). Most cases of PD are sporadic, but a small proportion of patients with PD have the familial form. Several causative genes have been identified for familial PDs, including α-synuclein (2), ubiquitin C-terminal hydrolase L1 (UCH-L1) (3), and parkin, an ubiquitin ligase E3 of the ubiquitin-proteasome system (4), implicating the impairment of the ubiquitin-proteasome pathway in the pathogenesis of PD. However, the mechanisms underlying the involvement of the ubiquitin-proteasome system in the development of PD are not yet understood.The 1-methyl-4-phenylpyrinidinium ion (MPP⁺), a toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is a neurotoxin used widely to induce dopaminergic neuronal cell death in in vitro models of PD (5). Previous studies have indicated that MPP⁺ induces neuronal cell death via several pathways, including the inhibition of complex I activity of the respiratory chain in mitochondria, leading to energy depletion, protein peroxidation, and DNA damage by producing reactive oxygen species and the induction of cytotoxic glutamate secretion (6, 7). However, the precise molecular pathway resulting in neuronal cell death remains to be identified.Cyclin-dependent kinase 5 (Cdk5) is a member of the Cdk serine/threonine kinase family. Cdk5 plays a role in a variety of neuronal activities including neuronal migration during central nervous system development (8, 9), synaptic activity in matured neurons (10), and neuronal cell death in neurodegenerative diseases (11, 12). Generally, when Cdk5 are activated by their respective activator cyclins, they function in cell cycle progression. However, unlike those cell cycle Cdk5, the kinase activity of Cdk5 is detected mainly in post mitotic neurons. This is because Cdk5 activators p35 and p39 are expressed predominantly in neurons (13, 14). The amount of p35 is the major determinant of Cdk5 activity, and it is normally a short-lived protein degraded by the ubiquitin-proteasome pathway (15, 16). However, in stressed neurons, the calcium-activated protease calpain cleaves p35 to the more stable and active form, p25 (17–21). Hyperactivated or mislocalized Cdk5-p25 has been implicated in the pathogenesis of numerous neurodegenerative disorders including PD and Alzheimer disease. In the case of PD, Cdk5 and p35 are found in the Lewy bodies of the dopaminergic neurons of the brain (22, 23). Cdk5 is activated by p25 and is required for cell death in mouse models of PD induced with MPTP (24) or 6-hydroxydopamine (25). It has been shown that Cdk5-p25 in MPTP-treated neurons phosphorylates the survival factor, myocyte enhancer factor 2 (MEF2), to inactivate it, leading to cell death (26, 27). However, further studies are required to clarify the involvement of p35 metabolism in the PD pathway.Contrary to its role in cell death progression, recent studies have also suggested a survival function for Cdk5 in maintaining survival signals or counteracting apoptotic signals. For example, Cdk5 inhibits c-Jun phosphorylation by c-Jun-N-terminal protein kinase 3, which is activated by UV irradiation (28). Cdk5 also promotes the survival of neurons by activating Akt through the well known neuregulin/phosphatidylinositol 3-kinase (PI3K) survival pathway, which leads to the down-regulation of proapoptotic factors (29). Cdk5 attenuates cell death either by up-regulating Bcl-2 through the phosphorylation of ERK (30) or by phosphorylating Bcl-2 to maintain its neuroprotective effect (31). However, whether Cdk5 acts as the anti-apoptotic factor in the PD model of neuronal cell death has not been determined.Here, we studied the role of Cdk5-p35 in the cell death of neurons treated with MPP⁺. We found that p35 was proteolysed in cultured neurons by either calpain or proteasomes depending on the concentration of MPP⁺ used. The proteasomal MPP⁺-induced degradation of p35 occurred earlier and at lower MPP⁺ concentrations than did its cleavage by calpain. MPP⁺ up-regulated the overall proteasome activity in the neurons by impairing the mitochondrial protein import system. A brief MPP⁺ treatment for up to ∼3 h was sufficient to induce delayed cell death at 24 h. The overexpression of p35 suppressed this MPP⁺-induced cell death, and depletion of p35 increased cell death. Together, these results implicate a role for Cdk5-p35 as a survival factor in MPP⁺-treated neurons.