Elevated α-synuclein caused by SNCA gene triplication impairs neuronal differentiation and maturation in Parkinson's patient-derived induced pluripotent stem cells

We have assessed the impact of α-synuclein overexpression on the differentiation potential and phenotypic signatures of two neural-committed induced pluripotent stem cell lines derived from a Parkinson''s disease patient with a triplication of the human SNCA genomic locus. In parallel, comparative studies were performed on two control lines derived from healthy individuals and lines generated from the patient iPS-derived neuroprogenitor lines infected with a lentivirus incorporating a small hairpin RNA to knock down the SNCA mRNA. The SNCA triplication lines exhibited a reduced capacity to differentiate into dopaminergic or GABAergic neurons and decreased neurite outgrowth and lower neuronal activity compared with control cultures. This delayed maturation phenotype was confirmed by gene expression profiling, which revealed a significant reduction in mRNA for genes implicated in neuronal differentiation such as delta-like homolog 1 (DLK1), gamma-aminobutyric acid type B receptor subunit 2 (GABABR2), nuclear receptor related 1 protein (NURR1), G-protein-regulated inward-rectifier potassium channel 2 (GIRK-2) and tyrosine hydroxylase (TH). The differentiated patient cells also demonstrated increased autophagic flux when stressed with chloroquine. We conclude that a two-fold overexpression of α-synuclein caused by a triplication of the SNCA gene is sufficient to impair the differentiation of neuronal progenitor cells, a finding with implications for adult neurogenesis and Parkinson''s disease progression, particularly in the context of bioenergetic dysfunction.Parkinson''s disease (PD), the second most common neurodegenerative disorder, is characterized by impairment of the motor system and associated non-motor clinical manifestations.¹ Age² and exposure to environmental toxins³ constitute the most important non-genetic risk factors in the development of sporadic disease. Neuronal loss is progressive, primarily (but not exclusively) dopaminergic, and accompanied by the accumulation of intracellular proteinaceous inclusions known as Lewy bodies and Lewy neurites.⁴ α-Synuclein (aSyn) is the main protein constituent of these inclusions⁵ and numerous findings attribute to it a central role in the pathogenesis of PD.^{6, 7, 8, 9} Both missense mutations (p.A30P, p.E46K, p.H50Q,¹⁰ p.G51D,¹¹ p.A53T, p.A53E¹²) and increased copy number (duplication¹³ or triplication¹⁴) of the SNCA gene encoding aSyn (PARK1/4 locus) cause early onset autosomal dominant PD. In addition, multiple genome-wide association studies have established that variations at the SNCA locus contribute significantly to the etiology of sporadic disease.^{15, 16, 17}The induced pluripotent stem cell (iPSC) technology offers a unique and valuable tool for defining the early mechanisms underlying PD and the development of early diagnostics and new therapeutics.^{18, 19, 20} Cell lines have been generated from fibroblasts obtained from patients with a variety of neurodegenerative diseases and neurons differentiated therefrom reproduce specific features of those diseases in vitro.²⁰ Comparisons between patient-derived and appropriately selected healthy control lines are feasible, but unfortunately phenotypic differences unrelated to the disease mechanisms arise due to the high clonal variability inherent in the generation of iPSCs and differences in the genetic background of the iPSC lines.^{21, 22, 23} Lines manipulated by single gene mutation have demonstrated the power of iPS technology for disease modeling^{18, 19, 20} with possible therapeutic potential.^{24, 25}We have examined in this study the effects of increased aSyn expression on the differentiation capacity and phenotypic signatures of two iPS clones derived from a patient with a triplication of the SNCA gene, and compared them with (i) lines generated by lentiviral infection of the patient cells by an shRNA construct targeting aSyn, and (ii) two control iPSC lines one from an unaffected age-matched sibling²⁶ and the other from an unrelated healthy individual.²⁷ All lines were differentiated by defined protocols into neurons that exhibited cardinal neuronal markers. These paradigms were used to assess differentiation capacity, cell survival, neurite outgrowth and electrophysiological properties. The results establish aSyn-dosage as an important modulator of developmental fitness of neuronal progenitor cells and support our previous findings from studies of PD patient fibroblasts²⁸ and neural-committed induced pluripotent stem cells (NiPSCs) (including the knockdown lines featured in this report)²⁹ exposed to toxins: (i) quantifiable reduction in viability under starvation and stress and (ii) decreased mitochondrial function and upregulated catabolism.     

Ubiquitination of α-synuclein and autophagy in Parkinson's disease

α-synuclein is mutated in Parkinson's disease (PD) and is found in cytosolic inclusions, called Lewy bodies, in sporadic forms of the disease. A fraction of α-synuclein purified from Lewy bodies is monoubiquitinated, but the role of this monoubiquitination has been obscure. We now review recent data indicating a role of α-synuclein monoubiquitination in Lewy body formation and implicating the autophagic pathway in regulating these processes. The E3 ubiquitin-ligase SIAH is present in Lewy bodies and monoubiquitinates α-synuclein at the same lysines that are monoubiquitinated in Lewy bodies. Monoubiquitination by SIAH promotes the aggregation of α-synuclein into amorphous aggregates and increases the formation of inclusions within dopaminergic cells. Such effect is observed even at low monoubiquitination levels, suggesting that monoubiquitinated α-synuclein may work as a seed for aggregation. Accumulation of monoubiquitinated α-synuclein and formation of cytosolic inclusions is promoted by autophagy inhibition and to a lesser extent by proteasomal and lysosomal inhibition. Monoubiquitinated α-synuclein inclusions are toxic to cells and recruit PD-related proteins, such as synphilin-1 and UCH-L1. Altogether, the new data indicate that monoubiquitination might play an important role in Lewy body formation. Decreasing α-synuclein monoubiquitination, by preventing SIAH function or by stimulating autophagy, constitutes a new therapeutic strategy for Parkinson's disease.

Addendum to: Rott R, Szargel R, Haskin J, Shani V, Shainskaya A, Manov I, Liani E, Avraham E, Engelender S. Monoubiquitination of α-synuclein by SIAH promotes its aggregation in dopaminergic cells. J Biol Chem 2007; Epub ahead of print.     

Fibrillar α-synuclein and huntingtin exon 1 assemblies are toxic to the cells

The aggregation of alpha-synuclein (α-syn) and huntingtin (htt) into fibrillar assemblies in nerve and glial cells is a molecular hallmark of Parkinson's and Huntington's diseases. Within the aggregation process, prefibrillar and fibrillar oligomeric species form. Prefibrillar assemblies rather than fibrils are nowadays considered cytotoxic. However, recent reports describing spreading of fibrillar assemblies from one cell to another, in cell cultures, animal models, and brains of grafted patients suggest a critical role for fibrillar assemblies in pathogenesis. Here we compare the cytotoxic effect of defined and comparable particle concentrations of on-assembly pathway oligomeric and fibrillar α-syn and Htt fragment corresponding to the first exon of the protein (HttEx1). We show that homogeneous populations of α-syn and HttEx1 fibrils, rather than their precursor on-assembly pathway oligomers, are highly toxic to cultured cells and induce apoptotic cell death. We document the reasons that make fibrils toxic. We show that α-syn and HttEx1 fibrils bind and permeabilize lipid vesicles. We also show that fibrils binding to the plasma membrane in cultured cells alter Ca(2+) homeostasis. Overall, our data indicate that fibrillar α-syn and HttEx1, rather than their precursor oligomers, are highly cytotoxic, the toxicity being associated to their ability to bind and permeabilize the cell membranes.     

Resistance to MPTP-neurotoxicity in α-synuclein knockout mice is complemented by human α-synuclein and associated with increased β-synuclein and Akt activation

Genetic and biochemical abnormalities of α-synuclein are associated with the pathogenesis of Parkinson's disease. In the present study we investigated the in vivo interaction of mouse and human α-synuclein with the potent parkinsonian neurotoxin, MPTP. We find that while lack of mouse α-synuclein in mice is associated with reduced vulnerability to MPTP, increased levels of human α-synuclein expression is not associated with obvious changes in the vulnerability of dopaminergic neurons to MPTP. However, expressing human α-synuclein variants (human wild type or A53T) in the α-synuclein null mice completely restores the vulnerability of nigral dopaminergic neurons to MPTP. These results indicate that human α-synuclein can functionally replace mouse α-synuclein in regard to vulnerability of dopaminergic neurons to MPTP-toxicity. Significantly, α-synuclein null mice and wild type mice were equally sensitive to neurodegeneration induced by 2'NH(2)-MPTP, a MPTP analog that is selective for serotoninergic and noradrenergic neurons. These results suggest that effects of α-synuclein on MPTP like compounds are selective for nigral dopaminergic neurons. Immunoblot analysis of β-synuclein and Akt levels in the mice reveals selective increases in β-synuclein and phosphorylated Akt levels in ventral midbrain, but not in other brain regions, of α-synuclein null mice, implicating the α-synuclein-level dependent regulation of β-synuclein expression in modulation of MPTP-toxicity by α-synuclein. Together these findings provide new mechanistic insights on the role α-synuclein in modulating neurodegenerative phenotypes by regulation of Akt-mediated cell survival signaling in vivo.     

Influence of microRNA deregulation on chaperone-mediated autophagy and α-synuclein pathology in Parkinson's disease

The presence of α-synuclein aggregates in the characteristic Lewy body pathology seen in idiopathic Parkinson''s disease (PD), together with α-synuclein gene mutations in familial PD, places α-synuclein at the center of PD pathogenesis. Decreased levels of the chaperone-mediated autophagy (CMA) proteins LAMP-2A and hsc70 in PD brain samples suggests compromised α-synuclein degradation by CMA may underpin the Lewy body pathology. Decreased CMA protein levels were not secondary to the various pathological changes associated with PD, including mitochondrial respiratory chain dysfunction, increased oxidative stress and proteasomal inhibition. However, decreased hsc70 and LAMP-2A protein levels in PD brains were associated with decreases in their respective mRNA levels. MicroRNA (miRNA) deregulation has been reported in PD brains and we have identified eight miRNAs predicted to regulate LAMP-2A or hsc70 expression that were reported to be increased in PD. Using a luciferase reporter assay in SH-SY5Y cells, four and three of these miRNAs significantly decreased luciferase activity expressed upstream of the lamp-2a and hsc70 3′UTR sequences respectively. We confirmed that transfection of these miRNAs also decreased endogenous LAMP-2A and hsc70 protein levels respectively and resulted in significant α-synuclein accumulation. The analysis of PD brains confirmed that six and two of these miRNAs were significantly increased in substantia nigra compacta and amygdala respectively. These data support the hypothesis that decreased CMA caused by miRNA-induced downregulation of CMA proteins plays an important role in the α-synuclein pathology associated with PD, and opens up a new avenue to investigate PD pathogenesis.     

Convergence of miRNA expression profiling, α-synuclein interacton and GWAS in Parkinson's disease

miRNAs were recently implicated in the pathogenesis of numerous diseases, including neurological disorders such as Parkinson''s disease (PD). miRNAs are abundant in the nervous system, essential for efficient brain function and play important roles in neuronal patterning and cell specification. To further investigate their involvement in the etiology of PD, we conducted miRNA expression profiling in peripheral blood mononuclear cells (PBMCs) of 19 patients and 13 controls using microarrays. We found 18 miRNAs differentially expressed, and pathway analysis of 662 predicted target genes of 11 of these miRNAs revealed an over-representation in pathways previously linked to PD as well as novel pathways. To narrow down the genes for further investigations, we undertook a parallel approach using chromatin immunoprecipitation-sequencing (ChIP-seq) analysis to uncover genome-wide interactions of α-synuclein, a molecule with a central role in both monogenic and idiopathic PD. Convergence of ChIP-seq and miRNomics data highlighted the glycosphingolipid biosynthesis and the ubiquitin proteasome system as key players in PD. We then tested the association of target genes belonging to these pathways with PD risk, and identified nine SNPs in USP37 consistently associated with PD susceptibility in three genome-wide association studies (GWAS) datasets (0.46≤OR≤0.63) and highly significant in the meta-dataset (3.36×10⁻⁴−3). A SNP in ST8SIA4 was also highly associated with PD (p = 6.15×10⁻³) in the meta-dataset. These findings suggest that several miRNAs may act as regulators of both known and novel biological processes leading to idiopathic PD.     

Differential expression of αB-crystallin causes maturationdependent susceptibility of oligodendrocytes to oxidative stress

Oligodendrocyte precursor cells (OPCs) are most susceptible to oxidative stress in the brain. However, the cause of differences in susceptibility to oxidative stress between OPCs and mature oligodendrocytes (mOLs) remains unclear. Recently, we identified in vivo that αB-crystallin (aBC) is expressed in mOLs but not in OPCs. Therefore, we examined in the present study whether aBC expression could affect cell survival under oxidative stress induced by hydrogen peroxide using primary cultures of OPCs and mOLs from neonatal rat brains. Expression of aBC was greater in mOLs than in OPCs, and the survival rate of mOLs was significantly higher than that of OPCs under oxidative stress. Suppression of aBC by siRNA transfection resulted in a decrease in the survival rate of mOLs under oxidative stress. These data suggest that higher susceptibility of OPCs than mOLs to oxidative stress is due, at least in part, to low levels of aBC expression. [BMB Reports 2013; 46(10): 501-506]     

Susceptibility to oxidative stress in Adélie and emperor penguin

The antioxidant defences in aerobic organisms represent the detoxification pathway against toxicity of reactive oxygen species (ROS). These highly reactive molecules are normally produced during the 4-electrons reduction of molecular oxygen to water coupled with oxidative phosphorylation, and during the activity of several enzymatic systems which produce ROS as intermediates. However, the endogenous generation of oxyradicals may be influenced by different environmental and biological factors, and the basal efficiency of antioxidant systems generally reflects the normal prooxidant pressure to which organisms are exposed. If the antioxidant capacity is exceeded (i.e. as a consequence of enhanced intracellular formation of ROS), a pathological condition, generally termed oxidative stress, may arise. In this preliminary work, susceptibility to oxidative stress has been compared in plasma of Adélie penguin (Pygoscelis adeliae), emperor penguin (Aptenodytes forsteri), south polar skua (Catharacta maccormicki) and snow petrel (Pagodroma nivea). Within the framework of the Italian Research Program in Antarctica, blood samples were collected during the austral summer 1998-1999 and the Total Oxyradical Scavenging Capacity (TOSC) analysed. The TOSC assay, measuring the capability of biological samples to neutralise different oxyradicals, has been recently standardised to provide a quantifiable value of biological resistance to toxicity of ROS. Penguins exhibited higher scavenging capacity towards peroxyl radicals than south polar skua and snow petrel. The greater resistance to toxicity of oxyradicals might suggest that penguins are naturally exposed to a higher basal prooxidant pressure in comparison to other analysed Antarctic birds.     

Nrf2-dependent induction of proteasome and Pa28αβ regulator are required for adaptation to oxidative stress

The ability to adapt to acute oxidative stress (e.g. H(2)O(2), peroxynitrite, menadione, and paraquat) through transient alterations in gene expression is an important component of cellular defense mechanisms. We show that such adaptation includes Nrf2-dependent increases in cellular capacity to degrade oxidized proteins that are attributable to increased expression of the 20 S proteasome and the Pa28αβ (11 S) proteasome regulator. Increased cellular levels of Nrf2, translocation of Nrf2 from the cytoplasm to the nucleus, and increased binding of Nrf2 to antioxidant response elements (AREs) or electrophile response elements (EpREs) in the 5'-untranslated region of the proteasome β5 subunit gene (demonstrated by chromatin immunoprecipitation (or ChIP) assay) are shown to be necessary requirements for increased proteasome/Pa28αβ levels, and for maximal increases in proteolytic capacity and stress resistance; Nrf2 siRNA and the Nrf2 inhibitor retinoic acid both block these adaptive changes and the Nrf2 inducers DL-sulforaphane, lipoic acid, and curcumin all replicate them without oxidant exposure. The immunoproteasome is also induced during oxidative stress adaptation, contributing to overall capacity to degrade oxidized proteins and stress resistance. Two of the three immunoproteasome subunit genes, however, contain no ARE/EpRE elements, and Nrf2 inducers, inhibitors, and siRNA all have minimal effects on immunoproteasome expression during adaptation to oxidative stress. Thus, immunoproteasome appears to be (at most) minimally regulated by the Nrf2 signal transduction pathway.     

Aggregated α-synuclein and complex I deficiency: exploration of their relationship in differentiated neurons

α-Synuclein becomes misfolded and aggregated upon damage by various factors, for example, by reactive oxygen species. These aggregated forms have been proposed to have differential toxicities and their interaction with mitochondria may cause dysfunction within this organelle that contributes to the pathogenesis of Parkinson''s disease (PD). In particular, the association of α-synuclein with mitochondria occurs through interaction with mitochondrial complex I and importantly defects of this protein have been linked to the pathogenesis of PD. Therefore, we investigated the relationship between aggregated α-synuclein and mitochondrial dysfunction, and the consequences of this interaction on cell survival. To do this, we studied the effects of α-synuclein on cybrid cell lines harbouring mutations in either mitochondrial complex I or IV. We found that aggregated α-synuclein inhibited mitochondrial complex I in control and complex IV-deficient cells. However, when aggregated α-synuclein was applied to complex I-deficient cells, there was no additional inhibition of mitochondrial function or increase in cell death. This would suggest that as complex I-deficient cells have already adapted to their mitochondrial defect, the subsequent toxic effects of α-synuclein are reduced.The pathological hallmark of Parkinson''s disease (PD) is the presence of α-synuclein aggregates, particularly within the substantia nigra (SN). These aggregations take the form of intracellular Lewy bodies, and also neuritic aggregations. However, both the effect of these inclusions on neuronal survival and the toxicity of different forms of α-synuclein are still debated. To aggregate α-synuclein must undergo a conformational change, however, the mechanism behind this change and subsequent aggregation in PD remains to be determined.Mutations within the α-synuclein gene (SNCA (MIM 163890)) were the first to be associated with autosomal dominant PD, while more recently genome-wide association studies have suggested that single-nucleotide polymorphisms in this gene are important for sporadic PD. A widely expressed protein α-synuclein is important for synaptic vesicle recycling and the modulation of dopamine transmission within SN neurons.^{1, 2, 3, 4, 5, 6, 7, 8} It interacts with curved cellular membranes including those of mitochondria suggesting a possible mode of its toxicity,^{9, 10, 11} and can be imported into mitochondria in an energy-dependent manner.⁹ The accumulation of α-synuclein within mitochondria leads to complex I impairment, decreased mitochondrial membrane potential (ΔΨ_m) and increased reactive oxygen species (ROS) production. The occurrence of these changes is also dependent on calcium homoeostasis.^{9, 12, 13}Mitochondrial dysfunction has also been heavily implicated in the pathogenesis of PD. Early studies showed a decrease in mitochondrial complex I in the SN of PD patients and studies involving the inhibition of this complex replicate many of the features of this disease. In addition, SN neurons show high levels of mitochondrial DNA deletions in old age,^{14, 15} which lead to respiratory deficiency, and the environment of the SN is believed to be particularly oxidative due to a number of processes, including the metabolism of dopamine. More recently a number of genes known to cause autosomal recessive forms of PD have been shown to encode proteins with functions associated with mitochondrial turnover (Parkin/Pink1 (MIM 602544, MIM 608309)) or oxidative stress (DJ-1 (MIM 602533)). However, the link between these two processes and the loss of dopaminergic neurons in PD remains to be elucidated.Several hypotheses have been suggested for what might cause α-synuclein to undergo the conformational change into more aggregate prone forms, from oxidative stress to gene mutations. Furthermore, the accumulation of mitochondrial DNA (mtDNA) mutations and dysfunctional mitochondria with advancing age are likely to have an effect on oxidative stress levels within the SN, which might contribute further to the misfolding and accumulation of this protein. Numerous studies have used rotenone and other toxins to induce mitochondrial dysfunction and monitor the accumulation of α-synuclein, despite the wealth of information that these studies provide they often do not reflect the subtleties of the slow accumulation of mitochondrial dysfunction within ageing SN neurons.Therefore, we investigated the relationship between mitochondria and aggregated α-synuclein, focussing on how these forms affect neurons with and without mitochondrial dysfunction. We wanted to understand how aggregated α-synuclein impacted on the survival of cells with mitochondrial dysfunction, to enable a deeper understanding of the effect of these two processes on neuronal survival. To investigate this we used cells with mutations in and partial inhibition of complexes I and IV.     

Intermediate filaments are important for astrocyte response to oxidative stress induced by oxygen–glucose deprivation and reperfusion

As a response to central nervous system injury, astrocytes become reactive. Two cellular hallmarks of reactive gliosis are hypertrophy of astrocyte processes and upregulation of intermediate filament (nanofilament) proteins glial fibrillary acidic protein (GFAP), vimentin, nestin, and synemin. Astrocytes in mice devoid of GFAP and vimentin (GFAP ^?/? Vim ^?/?) do not form cytoplasmic intermediate filaments. GFAP ^?/? Vim ^?/? mice develop larger infarcts after ischemic stroke (Li et al. in J Cereb Blood Flow Metab 28(3):468–481, 2008). Here, we attempted to analyze the underlying mechanisms using oxygen–glucose deprivation (OGD), an in vitro ischemia model, examining a potential link between astrocyte intermediate filaments and reactive oxygen species (ROS). We observed a reorganization of the intermediate filament network in astrocytes exposed to OGD. ROS accumulation was higher in GFAP ^?/? Vim ^?/? than wild-type astrocytes when exposed to OGD followed by reperfusion or when exposed to hydrogen peroxide. These results indicate that the elimination of ROS is impaired in the absence of the intermediate filament system. Compared to wild-type astrocytes, GFAP ^?/? Vim ^?/? astrocytes exposed to OGD and reperfusion exhibited increased cell death and conferred lower degree of protection to cocultured neurons. We conclude that the astrocyte intermediate filament system is important for the cell response to oxidative stress induced by OGD followed by reperfusion.     

Free radicals production and estimation of oxidative stress related to γ irradiation

The effectiveness of chemiluminescence (ChL) in vitro to measure free radicals generated as a result of metabolic disorganization caused by radiation sickness is evaluated. The results are correlated with those obtained by measuring superoxide dismutase (SOD) activity and lipid peroxide as levels of thiobarbituric acid reacting substances (TBARS). To this aim, livers from irradiated Wistar rats were removed immediately (day 0) after irradiation and also 7 and 14 d later. ChL results, expressed in arbitrary units (AU)/min/mg protein, were analyzed for irradiated samples and controls, for different doses at different times. Increased levels of ChL emission were observed not only on day 0, but also on days 7 and 14. On the other hand, SOD activity showed a decrease on the 7th d, and significantly higher lipid peroxide levels were observed in the assays performed on the 14th d, at all exposure doses. The correlation between temporal changes in the SOD activity, ChL emission, and higher TBARS levels a week later were evident from the data. These results indicate that the ChL technique proved to be useful in combination with other techniques currently used for evaluating radiation oxidative injury.     

Effects of curvature and composition on α-synuclein binding to lipid vesicles

Parkinson's disease is characterized by the presence of intracellular aggregates composed primarily of the neuronal protein α-synuclein (αS). Interactions between αS and various cellular membranes are thought to be important to its native function as well as relevant to its role in disease. We use fluorescence correlation spectroscopy to investigate binding of αS to lipid vesicles as a function of the lipid composition and membrane curvature. We determine how these parameters affect the molar partition coefficient of αS, providing a quantitative measure of the binding energy, and calculate the number of lipids required to bind a single protein. Specific anionic lipids have a large effect on the free energy of binding. Lipid chain saturation influences the binding interaction to a lesser extent, with larger partition coefficients measured for gel-phase vesicles than for fluid-phase vesicles, even in the absence of anionic lipid components. Although we observe variability in the binding of the mutant proteins, differences in the free energies of partitioning are less dramatic than with varied lipid compositions. Vesicle curvature has a strong effect on the binding affinity, with a >15-fold increase in affinity for small unilamellar vesicles over large unilamellar vesicles, suggesting that αS may be a curvature-sensing protein. Our findings provide insight into how physical properties of the membrane may modulate interactions of αS with cellular membranes.     

Parkinson's disease-associated mutations in α-synuclein and UCH-L1 inhibit the unconventional secretion of UCH-L1

Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) is an intracellular protein abundantly expressed in neurons, and a mutation in UCH-L1 has been identified in familial Parkinson’s disease. UCH-L1 has been detected in human cerebrospinal fluid, raising the possibility that UCH-L1 is secreted from neurons. In the present study, we showed that a portion of UCH-L1 is secreted from cultured cells. The secretion of D30K UCH-L1, which lacks ubiquitin binding activity, was decreased compared with that of wild-type UCH-L1, while the secretion of C90S UCH-L1, which lacks hydrolase activity, was not. Treatment with Brefeldin A, an inhibitor of vesicle transport from the endoplasmic reticulum to the Golgi, did not block the secretion of UCH-L1, indicating that UCH-L1 is secreted by an unconventional pathway. The UCH-L1 sequence from Leu-32 to Leu-39 is similar to the unconventional secretory signal sequence of engrailed 2, and substitution of the leucines within this region (L32S/L32A/L34S/L34A/L39S/L39A) reduced the secretion of UCH-L1. We found that the Parkinson’s disease-associated mutation I93M in UCH-L1 decreased the secretion of I93M UCH-L1. In addition, Parkinson’s disease-linked α-synuclein mutants reduced the secretion of endogenous UCH-L1. Our results indicate that the hydrolase activity is not necessary for the unconventional secretion of UCH-L1, and suggest that the ubiquitin binding activity and the sequence between Leu-32 and Leu-39 are involved in the secretion. Moreover, the secretion of UCH-L1 could be involved in the pathology of Parkinson’s disease.     

Proteomic analysis of dopamine and α-synuclein interplay in a cellular model of Parkinson's disease pathogenesis

Altered dopamine homeostasis is an accepted mechanism in the pathogenesis of Parkinson's disease. α-Synuclein overexpression and impaired disposal contribute to this mechanism. However, biochemical alterations associated with the interplay of cytosolic dopamine and increased α-synuclein are still unclear. Catecholaminergic SH-SY5Y human neuroblastoma cells are a suitable model for investigating dopamine toxicity. In the present study, we report the proteomic pattern of SH-SY5Y cells overexpressing α-synuclein (1.6-fold induction) after dopamine exposure. Dopamine itself is able to upregulate α-synuclein expression. However, the effect is not observed in cells that already overexpress α-synuclein as a consequence of transfection. The proteomic analysis highlights significant changes in 23 proteins linked to specific cellular processes, such as cytoskeleton structure and regulation, mitochondrial function, energetic metabolism, protein synthesis, and neuronal plasticity. A bioinformatic network enrichment procedure generates a significant model encompassing all proteins and allows us to enrich functional categories associated with the combination of factors analyzed in the present study (i.e. dopamine together with α-synuclein). In particular, the model suggests a potential involvement of the nuclear factor kappa B pathway that is experimentally confirmed. Indeed, α-synuclein significantly reduces nuclear factor kappa B activation, which is completely quenched by dopamine treatment.     

Synphilin-1 enhances α-synuclein aggregation in yeast and contributes to cellular stress and cell death in a Sir2-dependent manner

Background

Parkinson''s disease is characterized by the presence of cytoplasmic inclusions, known as Lewy bodies, containing both aggregated α-synuclein and its interaction partner, synphilin-1. While synphilin-1 is known to accelerate inclusion formation by α-synuclein in mammalian cells, its effect on cytotoxicity remains elusive.

Methodology/Principal Findings

We expressed wild-type synphilin-1 or its R621C mutant either alone or in combination with α-synuclein in the yeast Saccharomyces cerevisiae and monitored the intracellular localization and inclusion formation of the proteins as well as the repercussions on growth, oxidative stress and cell death. We found that wild-type and mutant synphilin-1 formed inclusions and accelerated inclusion formation by α-synuclein in yeast cells, the latter being correlated to enhanced phosphorylation of serine-129. Synphilin-1 inclusions co-localized with lipid droplets and endomembranes. Consistently, we found that wild-type and mutant synphilin-1 interacts with detergent-resistant membrane domains, known as lipid rafts. The expression of synphilin-1 did not incite a marked growth defect in exponential cultures, which is likely due to the formation of aggresomes and the retrograde transport of inclusions from the daughter cells back to the mother cells. However, when the cultures approached stationary phase and during subsequent ageing of the yeast cells, both wild-type and mutant synphilin-1 reduced survival and triggered apoptotic and necrotic cell death, albeit to a different extent. Most interestingly, synphilin-1 did not trigger cytotoxicity in ageing cells lacking the sirtuin Sir2. This indicates that the expression of synphilin-1 in wild-type cells causes the deregulation of Sir2-dependent processes, such as the maintenance of the autophagic flux in response to nutrient starvation.

Conclusions/Significance

Our findings demonstrate that wild-type and mutant synphilin-1 are lipid raft interacting proteins that form inclusions and accelerate inclusion formation of α-synuclein when expressed in yeast. Synphilin-1 thereby induces cytotoxicity, an effect most pronounced for the wild-type protein and mediated via Sir2-dependent processes.     

Expression of Pink1 with α-synuclein in the dopaminergic neurons of Drosophila leads to increases in both lifespan and healthspan

Overexpression of the gene coding for α-synuclein has been shown to be an inherited cause of Parkinson disease. Our laboratory has previously co-expressed the parkin and Pink1 genes to rescue α-synuclein-induced phenotypes within a Drosophila model. To further investigate the effect of Pink1 in this model, we performed longevity and behavioral studies using several drivers to express the α-synuclein and Pink1 genes. Our findings showed that overexpression of Pink1 and overexpression of Pink1 with α-synuclein resulted in an increased lifespan when driven with the TH-Gal4 transgene. This increase in longevity was accompanied by an increased healthspan, as measured by mobility over time, suggesting that this is an example of improved functional aging. Our results indicate that, in the dopaminergic cells targeted by TH-Gal4, increased expression of α-synuclein and Pink1 together have a synergistic effect, allowing for enhanced protection and increased survival of the organism.