The ubiquitin ligase E6-AP promotes degradation of α-synuclein

Parkinson's disease (PD) is a common neurodegenerative disorder caused mainly because of the loss of dopaminergic neurons in the substantia nigra. Protein inclusions called Lewy bodies are the most common pathological hallmark of PD and other synucleinopathies. Because the main component of these inclusions is α-synuclein, aggregation of this protein is thought to be a key pathogenic event in this disease. In the present investigation we report that E6 associated protein (E6-AP), a HECT (homologous to E6-AP C-terminus) domain ubiquitin ligase is a component of Lewy bodies in post-mortem PD brain. In the cell culture model, we demonstrate that endogenous E6-AP colocalizes with α-synuclein in juxtanuclear aggregates. E6-AP is also recruited to the centrosome upon inhibition of the proteasome function suggesting its involvement in the degradation of misfolded proteins. Over-expression of E6-AP enhances the degradation of wild type as well as the mutant forms of α-synuclein in a proteasome-dependent manner. E6-AP also promotes the degradation of the more toxic oligomeric forms of α-synuclein. Our data suggests that E6-AP is involved in the clearance of α-synuclein.     

Increased Dopaminergic Neuron Sensitivity to 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP) in Transgenic Mice Expressing Mutant A53T α-Synuclein

Familial Parkinson’s disease (PD) has been linked to point mutations and duplication of the α-synuclein gene and mutant α-synuclein expression increases the vulnerability of neurons to exogenous insults. In this study, we analyzed the levels of dopamine and its metabolites in the olfactory bulb (OB), and nigrostriatal regions of transgenic mice expressing human, mutant A53T α-synuclein (α-syn tg) and their non-transgenic (ntg) littermates using a sub-toxic, moderate dose of MPTP to determine if mutant human α-synuclein sensitizes the central dopaminergic systems to oxidative stress. We observed that after a single, sub-lethal MPTP injection, dopamine levels were reduced in striatum and SN in both the α-syn tg and ntg mice. In the olfactory bulb, a region usually resistant to MPTP toxicity, levels were reduced only in the α-syn tg mice. In addition, we identified a significant increase in dopamine metabolism in the α-syn transgenic, but not ntg mice. Finally, MPTP treatment of α-syn tg mice was associated with a marked elevation in the oxidative product, 3-nitrotyrosine that co-migrated with α-synuclein. Cumulatively, the data support the hypothesis that mutant α-synuclein sensitizes dopaminergic neurons to neurotoxic insults and is associated with greater oxidative stress. The α-syn tg line is therefore useful to study the genetic and environmental inter-relationship in PD.     

Aggregate formation and toxicity by wild-type and R621C synphilin-1 in the nigrostriatal system of mice using adenoviral vectors

Synphilin-1 was described as a protein interacting with α-synuclein and is commonly found in Lewy bodies, the pathological hallmark of Parkinson's disease (PD). Our group has previously described and characterized in vitro a mutation in the synphilin-1 gene (R621C) in PD patients. Providing the first characterization of synphilin-1 expression in an animal model, we here used adenoviral gene transfer to study the effects of wild-type (WT) and R621C synphilin-1 in dopaminergic neurons in mouse brain. As synphilin-1 is commonly used to trigger aggregation of α-synuclein in cell culture, we investigated not only non-transgenic C57Bl/6 mice but also A30P-α-synuclein transgenic animals. Both WT synphilin-1 and R621C synphilin-1 led to the formation of Thioflavine-S positive inclusions in C57Bl/6 mice and degeneration of dopaminergic neurons in the substantia nigra. R621C synphilin-1 induced more aggregate formation than WT synphilin-1 in A30P-α-synuclein transgenic mice, consistent with the role of the R621C mutation as a susceptibility factor for PD. Synphilin-1 expression may be used to improve current mouse models of PD, as it induced both the formation of aggregates and degeneration of dopaminergic neurons, two core characteristics of PD that have not been well reproduced with expression of α-synuclein.     

p53-dependent neuronal cell death in a DJ-1-deficient zebrafish model of Parkinson's disease

Mutations in DJ-1 lead to early onset Parkinson's disease (PD). The aim of this study was to elucidate further the underlying mechanisms leading to neuronal cell death in DJ-1 deficiency in vivo and determine whether the observed cell loss could be prevented pharmacologically. Inactivation of DJ-1 in zebrafish, Danio rerio, resulted in loss of dopaminergic neurons after exposure to hydrogen peroxide and the proteasome inhibitor MG132. DJ-1 knockdown by itself already resulted in increased p53 and Bax expression levels prior to toxin exposure without marked neuronal cell death, suggesting subthreshold activation of cell death pathways in DJ-1 deficiency. Proteasome inhibition led to a further increase of p53 and Bax expression with widespread neuronal cell death. Pharmacological p53 inhibition either before or during MG132 exposure in vivo prevented dopaminergic neuronal cell death in both cases. Simultaneous knockdown of DJ-1 and the negative p53 regulator mdm2 led to dopaminergic neuronal cell death even without toxin exposure, further implicating involvement of p53 in DJ-1 deficiency-mediated neuronal cell loss. Our study demonstrates the utility of zebrafish as a new animal model to study PD gene defects and suggests that modulation of downstream mechanisms, such as p53 inhibition, may be of therapeutic benefit.     

Neuroprotective Effects of Protocatechuic Aldehyde against Neurotoxin-Induced Cellular and Animal Models of Parkinson’s Disease

Protocatechuic aldehyde (PAL) has been reported to bind to DJ-1, a key protein involved in Parkinson’s disease (PD), and exerts potential neuroprotective effects via DJ-1 in SH-SY5Y cells. In this study, we investigated the neuroprotective pharmacological effects of PAL against neurotoxin-induced cell and animal models of PD. In cellular models of PD, PAL markedly increased cell viability rates, mitochondrial oxidation-reduction activity and mitochondrial membrane potential, and reduced intracellular ROS levels to prevent neurotoxicity in PC12 cells. In animal models of PD, PAL reduced the apomorphine injection, caused turning in 6-OHDA treated rats, and increased the motor coordination and stride decreases in MPTP treated mice. Meanwhile, in an MPTP mouse model, PAL prevented a decrease of the contents of dopamine (DA) and its metabolites in the striatum and TH-positive dopaminergic neuron loss in the substantia nigra (SN). In addition, PAL increased the protein expression of DJ-1 and reduced the level of α-synuclein in the SN of MPTP lesioned mice. PAL also increased the spine density in hippocampal CA1 neurons. The current study demonstrates that PAL can efficiently protect dopaminergic neurons against neurotoxin injury in vitro and in vivo, and that the potential mechanisms may be related to its effects in increasing DJ-1, decreasing α-synuclein and its growth-promoting effect on spine density.     

Accumulation and clearance of alpha-synuclein aggregates demonstrated by time-lapse imaging

Aggregates of α-synuclein are the pathological hallmark of sporadic Parkinson's disease (PD), and mutations in the α-synuclein gene underlie familial forms of the disease. To characterize the formation of α-synuclein aggregates in living cells, we developed a new strategy to visualize α-synuclein by fluorescence microscopy: α-synuclein was tagged with a six amino acid PDZ binding motif and co-expressed with the corresponding PDZ domain fused to enhanced green fluorescent protein (EGFP). In contrast to the traditional approach of α-synuclein-EGFP fusion proteins, this technique provided several-fold higher sensitivity; this allowed us to compare α-synuclein variants and perform time-lapse imaging. A C-terminally truncated α-synuclein variant showed the highest prevalence of aggregates and toxicity, consistent with stabilization of the α-synuclein monomer by its C-terminus. Time-lapse imaging illustrated how cells form and accumulate aggregates of α-synuclein. A substantial number of cells also reduced their aggregate load, primarily through formation of an aggresome, which could itself be cleared from the cell. The molecular chaperone Hsp70 not only prevented the formation of aggregates, but also increased their reduction and clearance, underlining the therapeutic potential of similar strategies. In contrast to earlier assumptions build-up, reduction and clearance of α-synuclein aggregation thus appear a highly dynamic process.     

Dual effects of α-synuclein on neurotoxicity induced by low dosage of rotenone are dependent on exposure time in dopaminergic neuroblastoma cells

This study was designed to investigate the effects of α-synuclein on toxicity induced by long-term exposure to relatively low concentrations of rotenone.Compared with the control groups,the inhibition of cell viability which overexpressed α-synuclein(SH-SY5Y-Syn) improved after 1 and 2 weeks of rotenone treatment.The complex I activity was greater and the mitochondrial membrane swelling intensity was reduced after 1 and 2 weeks of treatment,which indicated that α-synuclein,at least in part,resists the rotenone-induced oxidative stress.The results indicate that α-synuclein has a dual effect on toxicity of rotenone according to exposure time in human SH-SY5Y cells.     

Phenylbutyrate up-regulates the DJ-1 protein and protects neurons in cell culture and in animal models of Parkinson disease

Parkinson disease is caused by the death of midbrain dopamine neurons from oxidative stress, abnormal protein aggregation, and genetic predisposition. In 2003, Bonifati et al. (23) found that a single amino acid mutation in the DJ-1 protein was associated with early-onset, autosomal recessive Parkinson disease (PARK7). The mutation L166P prevents dimerization that is essential for the antioxidant and gene regulatory activity of the DJ-1 protein. Because low levels of DJ-1 cause Parkinson, we reasoned that overexpression might stop the disease. We found that overexpression of DJ-1 improved tolerance to oxidative stress by selectively up-regulating the rate-limiting step in glutathione synthesis. When we imposed a different metabolic insult, A53T mutant α-synuclein, we found that DJ-1 turned on production of the chaperone protein Hsp-70 without affecting glutathione synthesis. After screening a number of small molecules, we have found that the histone deacetylase inhibitor phenylbutyrate increases DJ-1 expression by 300% in the N27 dopamine cell line and rescues cells from oxidative stress and mutant α-synuclein toxicity. In mice, phenylbutyrate treatment leads to a 260% increase in brain DJ-1 levels and protects dopamine neurons against 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) toxicity. In a transgenic mouse model of diffuse Lewy body disease, long-term administration of phenylbutyrate reduces α-synuclein aggregation in brain and prevents age-related deterioration in motor and cognitive function. We conclude that drugs that up-regulate DJ-1 gene expression may slow the progression of Parkinson disease by moderating oxidative stress and protein aggregation.     

Identification of bilateral changes in TID1 expression in the 6-OHDA rat model of Parkinson's disease

Parkinson's disease (PD) is a common neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra and the aggregation of α-synuclein into Lewy bodies. Existing therapies address motor dysfunction but do not halt progression of the disease. A still unresolved question is the biochemical pathway that modulates the outcome of protein misfolding and aggregation processes in PD. The molecular chaperone network plays an important defensive role against cellular protein misfolding and has been identified as protective in experimental models of protein misfolding diseases like PD. Molecular mechanisms underlying chaperone-neuroprotection are actively under investigation. Current evidence implicates a number of molecular chaperones in PD including Hsp25, Hsp70 and Hsp90, however their precise involvement in the neurodegenerative cascade is unresolved. The J protein family (DnaJ or Hsp40 protein family) has long been known to be important in protein conformational processes.We assessed sensory and motor function of control and PD rats and then evaluated the brain region-specific expression levels of select J proteins by Western analysis. Surprisingly, we observed a widespread 26 kDa breakdown product of the J protein, TID1, (tumorous imaginal discs, mtHsp40 or DnaJ3) in a 6-hydroxydopamine (6-OHDA) rat model of PD in which food handling, gait symmetry and sensory performance were impaired. Greater behavioral deficits were associated with lower TID1 expression. Furthermore, direct application of either 6-OHDA or MPP+ (1-methyl-4-phenylpyridinum) to CAD (CNS-derived catecholinaminergic neuronal cell line) cell cultures, reduced TID1 expression levels.Our results suggest that changes in cellular TID1 are a factor in the pathogenesis of PD by impeding functional and structural compensation and exaggerating neurodegenerative processes. In contrast, no changes were observed in CSPα, Hsp40, Hsp70, Hsc70 and PrP(C) levels and no activation of caspase3 was observed. This study links TID1 to PD and provides a new target for therapeutics that halts the PD progression.     

DJ-1 up-regulates glutathione synthesis during oxidative stress and inhibits A53T alpha-synuclein toxicity

DJ-1 is the third gene that has been linked to Parkinson disease. Mutations in the DJ-1 gene cause early onset PD with autosomal recessive inheritance. To clarify the mechanism of DJ-1 protection, we have overexpressed the gene in cultured dopaminergic cells that were then subjected to chemical stress. In the rat dopaminergic cell line, N27, and in primary dopamine neurons, overexpression of wild type DJ-1 protected cells from death induced by hydrogen peroxide and 6-hydroxydopamine. Overexpressing the L166P mutant DJ-1 had no protective effect. By contrast, knocking down endogenous DJ-1 with antisense DJ-1 rendered cells more susceptible to oxidative damage. We have found that DJ-1 improves survival by increasing cellular glutathione levels through an increase in the rate-limiting enzyme glutamate cysteine ligase. Blocking glutathione synthesis eliminated the beneficial effect of DJ-1. Protection could be restored by adding exogenous glutathione. Wild type DJ-1 reduced cellular reactive oxygen species and reduced the levels of protein oxidation caused by oxidative stress. By a separate mechanism, overexpressing wild type DJ-1 inhibited the protein aggregation and cytotoxicity usually caused by A53T human alpha-synuclein. Under these circumstances, DJ-1 increased the level of heat shock protein 70 but did not change the glutathione level. Our data indicate that DJ-1 protects dopaminergic neurons from oxidative stress through up-regulation of glutathione synthesis and from the toxic consequences of mutant humanalpha-synuclein through increased expression of heat shock protein 70. We conclude that DJ-1 has multiple specific mechanisms for protecting dopamine neurons from cell death.     

Methionine sulfoxide reductase A protects dopaminergic cells from Parkinson's disease-related insults

Parkinson's disease (PD) is a neurologic disorder characterized by dopaminergic cell death in the substantia nigra. PD pathogenesis involves mitochondrial dysfunction, proteasome impairment, and alpha-synuclein aggregation, insults that may be especially toxic to oxidatively stressed cells including dopaminergic neurons. The enzyme methionine sulfoxide reductase A (MsrA) plays a critical role in the antioxidant response by repairing methionine-oxidized proteins and by participating in cycles of methionine oxidation and reduction that have the net effect of consuming reactive oxygen species. Here, we show that MsrA suppresses dopaminergic cell death and protein aggregation induced by the complex I inhibitor rotenone or mutant alpha-synuclein, but not by the proteasome inhibitor MG132. By comparing the effects of MsrA and the small-molecule antioxidants N-acetylcysteine and vitamin E, we provide evidence that MsrA protects against PD-related stresses primarily via methionine sulfoxide repair rather than by scavenging reactive oxygen species. We also demonstrate that MsrA efficiently reduces oxidized methionine residues in recombinant alpha-synuclein. These findings suggest that enhancing MsrA function may be a reasonable therapeutic strategy in PD.     

The role of JNK pathway in familial Parkinson's disease

Parkinson's disease is a neurodegenerative disorder characterized by a dramatic loss of dopaminergic neurons in the substantia nigra. Among the many pathogenic mechanisms thought to contribute to the demise of these cells in sporadic cases of PD, oxidative stress has taken center stage due to extensive experimental evidence showing that dopamine- or MPTP-derived reactive oxygen species and oxidized dopamine metabolites may trigger toxicity through mitochondrial inhibition or deleterious modifications of biomolecules. In familial forms of PD, however, the involvement of toxic protein aggregation (synuclein), impairment of ubiquitin-proteosome system (parkin. and loss of antioxidative properties (DJ-1) has gained attention. Recently, JNK pathway has come to light that could link malfunction of mutated DJ-1, parkin, PINK1 and alpha-synuclein to the oxidative stress-triggered apoptosis, finally ascribing a common pathogenic mechanism to both the sporadic and familial forms of PD.     

Relationship between alpha synuclein phosphorylation, proteasomal inhibition and cell death: relevance to Parkinson's disease pathogenesis

Alpha synuclein can be phosphorylated at serine129 (P-S129), and the presence of highly phosphorylated α-synuclein in Lewy bodies suggests changes to its phosphorylation status has an important pathological role. We demonstrate that the kinase(s) responsible for α-synuclein S129 phosphorylation is constitutively active in SH-SY5Y cells and involves casein kinase 2 activity. Increased oxidative stress or proteasomal inhibition caused significant elevation of P-S129 α-synuclein levels. Under these conditions, similar increases in P-S129 α-synuclein were found in both sodium dodecyl sulphate lysates and Triton extracts indicating the phosphorylated protein was soluble and did not lead to aggregation. The rate of S129 phosphorylation was increased in response to proteasomal inhibition indicating a higher activity of the relevant kinase. Cells expressing the phosphorylation mimic, S129D α-synuclein increased cell death and enhanced sensitivity to epoxomycin exposure. Proteasomal inhibition markedly decreased S129D α-synuclein turnover suggesting proteasomal inhibition leads to the accumulation of P-S129 α-synuclein through an increase in the kinase activity and a decrease in protein turnover resulting in increased cell death. We conclude that S129 phosphorylation is toxic to dopaminergic cells and both the levels of S129 phosphorylated protein and its toxicity are increased with proteasomal inhibition emphasising the interdependence of these pathways in Parkinson's disease pathogenesis.     

Sestrin2 Protects Dopaminergic Cells against Rotenone Toxicity through AMPK-Dependent Autophagy Activation

Dysfunction of the autophagy-lysosomal pathway (ALP) and the ubiquitin-proteasome system (UPS) was thought to be an important pathogenic mechanism in synuclein pathology and Parkinson''s disease (PD). In the present study, we investigated the role of sestrin2 in autophagic degradation of α-synuclein and preservation of cell viability in a rotenone-induced cellular model of PD. We speculated that AMP-activated protein kinase (AMPK) was involved in regulation of autophagy and protection of dopaminergic cells against rotenone toxicity by sestrin2. The results showed that both the mRNA and protein levels of sestrin2 were increased in a TP53-dependent manner in Mes 23.5 cells after treatment with rotenone. Genetic knockdown of sestrin2 compromised the autophagy induction in response to rotenone, while overexpression of sestrin2 increased the basal autophagy activity. Sestrin2 presumably enhanced autophagy in an AMPK-dependent fashion, as sestrin2 overexpression activated AMPK, and genetic knockdown of AMPK abrogated autophagy induction by rotenone. Restoration of AMPK activity by metformin after sestrin2 knockdown recovered the autophagy activity. Sestrin2 overexpression ameliorated α-synuclein accumulation, inhibited caspase 3 activation, and reduced the cytotoxicity of rotenone. These results suggest that sestrin2 upregulation attempts to maintain autophagy activity and suppress rotenone cytotoxicity through activation of AMPK, and that sestrin2 exerts a protective effect on dopaminergic cells.     

Increase in anti-apoptotic molecules,nucleolin, and heat shock protein 70, against upregulated LRRK2 kinase activity

Leucine-rich repeat kinase 2 (LRRK2) is involved in Parkinson’s disease (PD) pathology. A previous study showed that rotenone treatment induced apoptosis, mitochondrial damage, and nucleolar disruption via up-regulated LRRK2 kinase activity, and these effects were rescued by an LRRK2 kinase inhibitor. Heat-shock protein 70 (Hsp70) is an anti-oxidative stress chaperone, and overexpression of Hsp70 enhanced tolerance to rotenone. Nucleolin (NCL) is a component of the nucleolus; overexpression of NCL reduced cellular vulnerability to rotenone. Thus, we hypothesized that rotenone-induced LRRK2 activity would promote changes in neuronal Hsp70 and NCL expressions. Moreover, LRRK2 G2019S, the most prevalent LRRK2 pathogenic mutant with increased kinase activity, could induce changes in Hsp70 and NCL expression. Rotenone treatment of differentiated SH-SY5Y (dSY5Y) cells increased LRKK2 levels and kinase activity, including phospho-S935-LRRK2, phospho-S1292-LRRK2, and the phospho-moesin/moesin ratio, in a dose-dependent manner. Neuronal toxicity and the elevation of cleaved poly (ADP-ribose) polymerase, NCL, and Hsp70 were increased by rotenone. To validate the induction of NCL and Hsp70 expression in response to rotenone, cycloheximide (CHX), a protein synthesis blocker, was administered with rotenone. Post-rotenone increased NCL and Hsp70 expression was repressed by CHX; whereas, rotenone-induced kinase activity and apoptotic toxicity remained unchanged. Transient expression of G2019S in dSY5Y increased the NCL and Hsp70 levels, while administration of a kinase inhibitor diminished these changes. Similar results were observed in rat primary neurons after rotenone treatment or G2019S transfection. Brains from G2019S-transgenic mice also showed increased NCL and Hsp70 levels. Accordingly, LRRK2 kinase inhibition might prevent oxidative stress-mediated PD progression.

Abbreviations: 6-OHDA: 6-hydroxydopamine; CHX: cycloheximide; dSY5Y: differentiated SH-SY5Y; g2019S tg: g2019S transgenic mouse; GSK/A-KI: GSK2578215A kinase inhibitor; HSP70: heat shock protein 70; LDH: lactose dehydrogenase; LRRK2: leucine rich-repeat kinase 2; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; myc-GS LRRK2: myc-tagged g2019S LRRK2; NCL: nucleolin; PARP: poly(ADP-ribose) polymerase; PD: Parkinson’s disease; PINK1: PTEN-induced putative kinase 1; pmoesin: phosphorylated moesin at t558; ROS: reactive oxygen species     

DJ-1 Protects Dopaminergic Neurons against Rotenone-Induced Apoptosis by Enhancing ERK-Dependent Mitophagy

Loss-of-function mutations in the gene encoding the multifunctional protein, DJ-1, have been implicated in the pathogenesis of early-onset familial Parkinson's disease (PD), suggesting that DJ-1 may act as a neuroprotectant for dopaminergic (DA) neurons. Enhanced autophagy may benefit PD by clearing damaged organelles and protein aggregates; thus, we determined if DJ-1 protects DA neurons against mitochondrial dysfunction and oxidative stress through an autophagic pathway. Cultured DA cells (MN9D) overexpressing DJ-1 were treated with the mitochondrial complex I inhibitor, rotenone. In addition, rotenone was injected into the left substantia nigra of rats 4 weeks after injection with a DJ-1 expression vector. Overexpression of DJ-1 protected MN9D cells against apoptosis, significantly enhanced the survival of nigral DA neurons after rotenone treatment in vivo, and rescued rat behavioral abnormalities. Overexpression of DJ-1 enhanced rotenone-evoked expression of the autophagic markers, beclin-1 and LC3II, while transmission electron microscopy and confocal imaging revealed that the ultrastructural signs of autophagy were increased by DJ-1. The neuroprotective effects of DJ-1 were blocked by phosphoinositol 3‐kinase and the autophagy inhibitor, 3-methyladenine, and by the ERK pathway inhibitor, U0126. Confocal imaging revealed that the size of p62-positive puncta decreased significantly in DJ-1 overexpression of MN9D cells 12 h after rotenone treatment, suggesting that DJ-1 reveals the ability to clear aggregated p62 associated with PD. Factors that control autophagy, including DJ-1, may inhibit rotenone-induced apoptosis and present novel targets for therapeutic intervention in PD.     

The Parkinson disease-associated A30P mutation stabilizes α-synuclein against proteasomal degradation triggered by heme oxygenase-1 over-expression in human neuroblastoma cells

Proteosomal degradation of proteins is one of the major mechanisms of intracellular protein turnover. Failure of the proteosome to degrade misfolded protein is implicated in the accumulation of α-synuclein in Parkinson's disease (PD). Heme oxygenase-1 (HO-1), an enzyme that converts heme to free iron, carbon monoxide (CO) and biliverdin (bilirubin precursor) is expressed in response to various stressors. HO-1 is up-regulated in PD- and Alzheimer's disease-affected neural tissues. In this study, we found that HO-1 over-expression engenders dose-dependent decreases in α-synuclein protein levels in human neuroblastoma M17 cells. When over-expression of HO-1 was silenced in HO-1 transfected cells, level of α-synuclein was restored. Likewise, treatment of HO-1 over-expressing cells with the HO-1 inhibitor, tin mesoporphyrin, the iron chelator deferoxamine or antagonist of CO-dependent cGMP activation, methylene blue, mitigated the HO-1-induced reduction in α-synuclein levels. Furthermore, when HO-1 over-expressing cells were treated with the proteosome inhibitors, lactacystin and MG132, level of α-synuclein was almost completely restored. In contrast to the effect on α-synuclein [wild-type (WT)] levels, HO-1 over-expression did not significantly impact PD-associated α-synuclein (A30P) levels in these cells. HO-1 also significantly reduced aggregation of α-synuclein (WT) but not that of A30P. Our results suggest that HO-1, which is expressed when neurons are exposed to toxic stimuli capable of inducing protein misfolding, triggers proteosomal degradation of proteins and prevents intracellular accumulation of protein aggregates and inclusions. Resistance to HO-1 induced proteosomal degradation may render the familial PD-associated A30P mutation prone to toxic intracellular aggregation.     

α - synuclein and Parkinson's disease: the first roadblock

α-synuclein gene mutations are major underlying genetic defects known in familial juvenile onset Parkinson's disease (PD), and α-synuclein is a major constituent of Lewy Bodies, the pathological hallmark of PD. The normal cellular function of α-synuclein has been elusive, and its exact etiological mechanism in causing dopaminergic neuronal death in PD is also not clearly understood. Very recent reports now indicate that mutant or simply over-expressed α-synuclein could cause damage by interfering with particular steps of neuronal membrane traffic. α-synuclein selectively blocks endoplamic reticulum-to-Golgi transport, thus causing ER stress. A screen in a yeast revealed that α-synuclein toxicity could be suppressed by over-expression of the small GTPase Ypt1/Rab1, and that over-expression of the latter rescues neuron loss in invertebrate and mammalian models of α-synuclein-induced neurodegeneration. α-synuclein may also serve a chaperone function for the proper folding of synaptic SNAREs that are important for neurotransmitter release. We discuss these recent results and the emerging pathophysiological interaction of α-synuclein with components of neuronal membrane traffic.     

p21-activated kinase 4 controls the aggregation of α-synuclein by reducing the monomeric and aggregated forms of α-synuclein: involvement of the E3 ubiquitin ligase NEDD4-1

Aggregation of misfolded alpha-synuclein (α-synuclein) is a central player in the pathogenesis of neurodegenerative diseases. Therefore, the regulatory mechanism underlying α-synuclein aggregation has been intensively studied in Parkinson’s disease (PD) but remains poorly understood. Here, we report p21-activated kinase 4 (PAK4) as a key regulator of α-synuclein aggregation. Immunohistochemical analysis of human PD brain tissues revealed an inverse correlation between PAK4 activity and α-synuclein aggregation. To investigate their causal relationship, we performed loss-of-function and gain-of-function studies using conditional PAK4 depletion in nigral dopaminergic neurons and the introduction of lentivirus expressing a constitutively active form of PAK4 (caPAK4; PAK4^S445N/S474E), respectively. For therapeutic relevance in the latter setup, we injected lentivirus into the striatum following the development of motor impairment and analyzed the effects 6 weeks later. In the loss-of-function study, Cre-driven PAK4 depletion in dopaminergic neurons enhanced α-synuclein aggregation, intracytoplasmic Lewy body-like inclusions and Lewy-like neurites, and reduced dopamine levels in PAK4^DAT-CreER mice compared to controls. Conversely, caPAK4 reduced α-synuclein aggregation, as assessed by a marked decrease in both proteinase K-resistant and Triton X100-insoluble forms of α-synuclein in the AAV-α-synuclein-induced PD model. Mechanistically, PAK4 specifically interacted with the NEDD4-1 E3 ligase, whose pharmacological inhibition and knockdown suppressed the PAK4-mediated downregulation of α-synuclein. Collectively, these results provide new insights into the pathogenesis of PD and suggest PAK4-based gene therapy as a potential disease-modifying therapy in PD.Subject terms: Parkinson''s disease, Cell death in the nervous system     

Association of DJ-1 with chaperones and enhanced association and colocalization with mitochondrial Hsp70 by oxidative stress

DJ-1 is a novel oncogene and causative gene for familial form of the Parkinson's disease (PD). DJ-1 has been shown to play a role in anti-oxidative stress by eliminating reactive oxygen species (ROS). The onset of PD is thought to be caused by oxidative stress and mitochondrial injury, which leads to protein aggregation that results in neuronal cell death. However, the mechanism by which DJ-1 triggers the onset of PD is still not clear. In this study, we analyzed association and localization of DJ-1 and its mutants with various chaperones. The results showed that DJ-1 and its mutants were associated with Hsp70, CHIP and mtHsp70/Grp75, a mitochondria-resident Hsp70, and that L166P and M26I mutants found in PD patients were strongly associated with Hsp70 and CHIP compared to wild-type and other DJ-1 mutants. DJ-1 and its mutants were colocalized with Hsp70 and CHIP in cells. Furthermore, association and colocalization of wildtype DJ-1 with mtHsp70 in mitochondria were found to be enhanced by treatment of cells with H₂O₂. These results suggest that translocation of DJ-1 to mitochondria after oxidative stress is carried out in association with chaperones.