Lingling Lu

Silencing α-Synuclein Gene Expression Enhances Tyrosine Hydroxylase Activity in MN9D Cells

Abstractα-Synuclein has been implicated in the pathogenesis of Parkinson’s disease (PD). Previous studies have shown that α-synuclein is involved in the regulation of dopamine (DA) metabolism, possibly by down-regulating the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in DA biosynthesis. In this study, we constructed α-synuclein stably silenced MN9D/α-SYN− cells by vector mediated RNA interference and examined its effects on DA metabolism. We found that there were no significant differences in TH protein and mRNA levels between MN9D, MN9D/α-SYN− and MN9D/CON cells, suggesting that silencing α-synuclein expression does not affect TH gene expression. However, significant increases in phosphorylated TH, cytosolic 3, 4-dihydroxyphenylalanine (l-DOPA) and DA levels were observed in MN9D/α-SYN− cells. Our data show that TH activity and DA biosynthesis were enhanced by down-regulation of α-synuclein, suggesting that α-synuclein may act as a negative regulator of cytosolic DA. With respect to PD pathology, a loss of functional α-synuclein may result in increased DA levels in neurons that may lead to cell injury or even death.

GBA deficiency promotes SNCA/α-synuclein accumulation through autophagic inhibition by inactivated PPP2A

Loss-of-function mutations in the gene encoding GBA (glucocerebrosidase, β, acid), the enzyme deficient in the lysosomal storage disorder Gaucher disease, elevate the risk of Parkinson disease (PD), which is characterized by the misprocessing of SNCA/α-synuclein. However, the mechanistic link between GBA deficiency and SNCA accumulation remains poorly understood. In this study, we found that loss of GBA function resulted in increased levels of SNCA via inhibition of the autophagic pathway in SK-N-SH neuroblastoma cells, primary rat cortical neurons, or the rat striatum. Furthermore, expression of the autophagy pathway component BECN1 was downregulated as a result of the GBA knockdown-induced decrease in glucocerebrosidase activity. Most importantly, inhibition of autophagy by loss of GBA function was associated with PPP2A (protein phosphatase 2A) inactivation via Tyr307 phosphorylation. C2-ceramide (C2), a PPP2A agonist, activated autophagy in GBA-silenced cells, while GBA knockdown-induced SNCA accumulation was reversed by C2 or rapamycin (an autophagy inducer), suggesting that PPP2A plays an important role in the GBA knockdown-mediated inhibition of autophagy. These findings demonstrate that loss of GBA function may contribute to SNCA accumulation through inhibition of autophagy via PPP2A inactivation, thereby providing a mechanistic basis for the increased PD risk associated with GBA deficiency.

α-Synuclein amino terminus regulates mitochondrial membrane permeability.

Parkinsons disease (PD) is a common neurodegenerative movement disorder affecting an increasing number of elderly. Various studies have shown that mitochondrial dysfunction and abnormal protein aggregation are two major contributors to the progression of PD. The N terminus of α-synuclein (α-Syn/N), which adopts an α-helical conformation upon lipid binding, is essential for membrane interaction; yet its role in mitochondria remains poorly defined. A functional characterization of the α-Syn N-terminal domain and investigation of its effect on mitochondrial membrane permeability were undertaken in this study. α-Syn/N and α-Syn/delN (amino acids 1-65 and 61-140, respectively) constructs were overexpressed in dopaminergic MN9D cells and primary cortical neurons. A decrease in cell viability was observed in cells transfected with α-Syn/N but not α-Syn/delN. In addition, an α-Syn/N-induced increase in the level of intracellular reactive oxygen species, alteration in mitochondrial morphology, and decrease in mitochondrial membrane potential were accompanied by the activation of mitochondrial permeability transition pores (mPTP). These changes were also associated with a decline in mitochondrial cardiolipin content and interaction with the voltage-dependent anion channel and adenine nucleotide translocator in the mitochondrial membrane. The activation of mPTPs and reduction in cell viability were partially reversed by bongkrekic acid, an inhibitor of adenine nucleotide translocator (ANT), suggesting that the interaction between α-Syn and ANT promoted mPTP activation and was toxic to cells. BKA treatment reduced interaction of α-Syn/N with ANT and VDAC. These results suggest that the N terminus of α-Syn is essential for the regulation of mitochondrial membrane permeability and is a likely factor in the neurodegeneration associated with PD.

Loss of PINK1 function decreases PP2A activity and promotes autophagy in dopaminergic cells and a murine model.

Parkinsons disease (PD) is the most common neurodegenerative movement disorder. Mutations in PTEN-induced kinase 1 (PINK1) are a frequent cause of recessive PD. Autophagy, a pathway for clearance of protein aggregates or impaired organelles, is a newly identified mechanism for PD development. However, it is still unclear what molecules regulate autophagy in PINK1-silenced cells. Here we report that autophagosome formation is promoted in the early phase in response to PINK1 gene silencing by lentivirus transfer vectors expressed in mouse striatum. Reduced PP2A activity and increased phosphorylation of PP2A at Y307 (inactive form of PP2A) were observed in PINK1-knockdown dopaminergic cells and striatum tissues. Treatment with C2-ceramide (an agonist of PP2A) reduced autophagy levels in PINK1-silenced MN9D cells, which suggests that PP2A plays an important role in the PINK1-knockdown-induced autophagic pathway. Furthermore, phosphorylation of Bcl-2 at S87 increased in PINK1-silenced cells and was negatively regulated by additional treatment with C2-ceramide, which indicates that Bcl-2 may be downstream of PP2A inactivation in response to PINK1 dysfunction. Immunoprecipitation also revealed dissociation of the Bcl-2/Beclin1 complex in PINK1-silenced cells, which was reversed by additional treatment with C2-ceramide, and correlated with changes in level of autophagy and S87 phosphorylation of Bcl-2. Finally, Western blots for cleaved caspase-9 and flow cytometry results for active caspase-3 revealed that PP2A inactivation is involved in the protective effect of autophagy on PINK1-silenced cells. Our findings show that downregulation of PP2A activity in PINK1-silenced cells promotes the protective effect of autophagy through phosphorylation of Bcl-2 at S87 and blockage of the caspase pathway. These results may have implications for identifying the mechanism of PD.

DJ-1 upregulates tyrosine hydroxylase gene expression by activating its transcriptional factor Nurr1 via the ERK1/2 pathway

Loss-of-function DJ-1 mutations have been linked to autosomal recessive early-onset Parikinsonism. However, the putative function of DJ-1 is not completely understood. Previous studies indicate that DJ-1 overexpression results in upregulation of the tyrosine hydroxylase gene. The mechanism by which DJ-1 affects tyrosine hydroxylase expression remains elusive. In the present study, we show that DJ-1 overexpression induces ERK1/2 activation, along with increased tyrosine hydroxylase expression. The L166P DJ-1 mutant, which has been identified as being responsible for familial Parkinsonism, did not have this effect. Moreover, suppression of ERK1/2 phosphorylation by the pharmacological inhibitor U0126 partially abolished the regulating effect of DJ-1 on tyrosine hydroxylase. Nurr1, a transcriptional factor for tyrosine hydroxylase, can be phosphorylated by ERK1/2 and translocate to the nucleus, where it is activated. Thus, we measured nuclear translocation of Nurr1. Confocal microscopy and Western blotting revealed that Nurr1 translocated to the nucleus and was activated by overexpression of wild-type DJ-1, but not of its L166P mutant. Knockdown of Nurr1 gene expression abolished the effect on tyrosine hydroxylase induced by DJ-1. Taken together, these data suggest that DJ-1 upregulates tyrosine hydroxylase expression by activating its transcription factor Nurr1 via the ERK1/2 pathway.

Voltage-dependent anion channel involved in the α-synuclein-induced dopaminergic neuron toxicity in rats

Inclusion bodies containing the neural protein α-synuclein (α-syn) are observed in several neurodegenerative diseases, including Parkinsons disease (PD). Furthermore, over-expression of α-syn in rat brain partly mimics the neuropathological and behavioral features of PD by triggering the degeneration of dopaminergic neurons in the substantia nigra (SN). Mitochondrial dysfunction is also central to PD pathogenesis, and α-syn is found in the mitochondria. However, the precise mechanisms of α-syn-induced neurotoxicity remain elusive. To examine the potential mechanisms of α-syn-induced neurodegeneration, we over-expressed α-syn in the SN of rats using a recombinant adeno-associated viral vector (rAAV-syn). Immunohistochemical and immunogold labeling results indicated that α-syn was successfully over-expressed in the SN and striatum after vector injection. The number of tyrosine hydroxylase-positive (dopaminergic) neurons was significantly reduced in rats injected with rAAV-syn when compared with control rats. Compared with control rats, the density of α-syn-conjugated gold particles was greater in the axons, cytoplasm, nuclei, and notably also in the mitochondria of SN neurons in rAAV-syn-injected rats. In addition, SN neurons transfected with rAAV-syn exhibited swollen mitochondria with discontinuous outer membranes and internal vacuole-like structures, strongly suggesting α-syn-induced mitochondrial dysfunction. Mitochondria in rAAV-syn-injected rats were also observed in autophagosomes. α-Syn co-immunoprecipitated with voltage-dependent anion channel 1 (VDAC1), a component of the mitochondrial permeability transition pore (mPTP) that induces mitochondrial uncoupling and apoptosis. Over-expression of α-syn may cause the degeneration of dopaminergic neurons through an interaction with mitochondrial VDAC1, which leads to mPTP activation, mitochondrial uncoupling, and cell death.

Piperine induces autophagy by enhancing protein phosphotase 2A activity in a rotenone-induced Parkinson’s disease model

Parkinsons disease (PD) is the second most common neurodegenerative disorder, but there are few treatments currently available. The autophagy pathway plays an important role in the pathogenesis of PD; modulating this pathway is considered to be a promising treatment strategy. Piperine (PIP) is a Chinese medicine with anti-inflammatory and antioxidant effects. The present study investigated the neuroprotective effects of PIP on rotenone-induced neurotoxicity in SK-N-SH cells, primary rat cortical neurons, and in a mouse model. Mice were administered rotenone (10mg/kg) for 6 weeks; PIP (25mg/kg, 50mg/kg) was subsequently administered for 4 weeks. We found that PIP treatment attenuated rotenone-induced motor deficits, and rescued the loss of dopaminergic neurons in the substantia nigra. PIP increased cell viability and restored mitochondrial functioning in SK-N-SH cells and primary neurons. In addition, PIP induced autophagy by inhibiting mammalian target of rapamycin complex 1(mTORC1) via activation of protein phosphotase 2A (PP2A). However, inhibiting PP2A activity with okadaic acid reduced these protective effects, suggesting that PP2A is a target of PIP. These findings demonstrate that PIP exerts neuroprotective effects in PD models via induction of autophagy, and may be an effective agent for PD treatment.

DJ-1/PARK7, But Not Its L166P Mutant Linked to Autosomal Recessive Parkinsonism, Modulates the Transcriptional Activity of the Orphan Nuclear Receptor Nurr1 In Vitro and In Vivo.

Although mutations of DJ-1 have been linked to autosomal recessive Parkinsonism for years, its physiological function and the pathological mechanism of its mutants are not well understood. We report for the first time that exogenous application of DJ-1, but not its L166P mutant, enhances the nuclear translocation and the transcriptional activity of Nurr1, a transcription factor essential for dopaminergic neuron development and maturation, both in vitro and in vivo. Knockdown of DJ-1 attenuates Nurr1 activity. Further investigation showed that signaling of Raf/MEK/ERK MAPKs is involved in this regulatory process and that activation induced by exogenous DJ-1 is antagonized by U0126, an ERK pathway inhibitor, indicating that DJ-1 modulates Nurr1 activity via the Raf/MEK/ERK pathway. Our findings shed light on the novel function of DJ-1 to enhance Nurr1 activity and provide the first insight into the molecular mechanism by which DJ-1 enhances Nurr1 activity.

Piperlongumine restores the balance of autophagy and apoptosis by increasing BCL2 phosphorylation in rotenone-induced Parkinson disease models

ABSTRACT Parkinson disease (PD) is the second most common neurodegenerative disorder after Alzheimer disease and is caused by genetics, environmental factors and aging, with few treatments currently available. Apoptosis and macroautophagy/autophagy play critical roles in PD pathogenesis; as such, modulating their balance is a potential treatment strategy. BCL2 (B cell leukemia/lymphoma 2) is a key molecule regulating this balance. Piperlongumine (PLG) is an alkaloid extracted from Piper longum L. that has antiinflammatory and anticancer effects. The present study investigated the protective effects of PLG in rotenone-induced PD cell and mouse models. We found that PLG administration (2 and 4 mg/kg) for 4 wk attenuated motor deficits in mice and prevented the loss of dopaminergic neurons in the substantia nigra induced by oral administration of rotenone (10 mg/kg) for 6 wk. PLG improved cell viability and enhanced mitochondrial function in primary neurons and SK-N-SH cells. These protective effects were exerted via inhibition of apoptosis and induction of autophagy through enhancement of BCL2 phosphorylation at Ser70. These results demonstrate that PLG exerts therapeutic effects in a rotenone-induced PD models by restoring the balance between apoptosis and autophagy. Abbreviations: 6-OHDA, 6-hydroxydopamine; ACTB, actin, beta; BafA1, bafilomycin A1; BAK1, BCL2-antagonist/killer 1; BAX, BCL2-associated X protein; BCL2, B cell leukemia/lymphoma2; BECN1, Beclin 1, autophagy related; CoQ10, coenzyme Q10; COX4I1/COX IV, cytochrome c oxidase subunit 4I1; CsA, cyclosporine A; ED50, 50% effective dose; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; HPLC, high-performance liquid chromatography; JC-1, tetraethylbenz-imidazolylcarbocyanine iodide; LC3, microtubule-associated protein 1 light chain3; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LDH, lactate dehydrogenase; l-dopa, 3, 4-dihydroxyphenyl-l-alanine; MAPK8/JNK1, mitogen-activated protein kinase 8; MMP, mitochondrial membrane potential; mPTP, mitochondrial permeability transition pore; mRFP, monomeric red fluorescent protein; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NFE2L2/NRF2, nuclear factor, erythroid derived 2, like 2; PD, Parkinson disease; PLG, piperlongumine; pNA, p-nitroanilide; PI, propidium iodide; PtdIns3K, phosphatidylinositol 3-kinase; PtdIns3P, phosphatidylinositol-3-phosphate; PTX, paclitaxel; Rap, rapamycin; SQSTM1/p62, sequestosome 1; TH, tyrosine hydroxylase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; WIPI2, WD repeat domain, phosphoinositide interacting 2; ZFYVE1/DFCP1, zinc finger, FYVE domain containing 1.

Pink1 interacts with α -synuclein and abrogates α -synuclein-induced neurotoxicity by activating autophagy

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases, characterized by degeneration of dopaminergic neurons in the substantia nigra. α-synuclein (α-syn) and PTEN-induced putative kinase (PINK)1 are two critical proteins associated with the pathogenesis of PD. α-syn induces mitochondrial deficits and apoptosis, PINK1 was found to alleviate α-syn-induced toxicity, but the mechanistic details remain obscure. Here, we show that PINK1 interacts with α-syn mainly in the cytoplasm, where it initiates autophagy. This interaction was dependent on the kinase activity of PINK1 and was abolished by deletion of the kinase domain or a G309D point mutation, an inactivating mutation in the kinase domain. Interaction between PINK1 and α-syn stimulated the removal of excess α-syn, which prevented mitochondrial deficits and apoptosis. Our findings provide evidence for a novel mechanism underlying the protective effects of PINK1 against α-syn-induced neurodegeneration and highlight a novel therapeutic target for PD treatment.