Casein kinase II-mediated phosphorylation regulates alpha-synuclein/synphilin-1 interaction and inclusion body formation

Alpha-synuclein is a phosphoprotein that accumulates as a major component of Lewy bodies in the brains of patients with Parkinson disease. Synphilin-1, which is also present in Lewy bodies, binds with alpha-synuclein and forms cytoplasmic inclusions in transfected cells. Yet the molecular determinants of this protein-protein interaction are unknown. Here we report that casein kinase II (CKII) phosphorylates synphilin-1 and that the beta subunit of this enzyme complex binds to synphilin-1. Additionally, both CKII alpha and beta subunits are present within cytoplasmic inclusions in cells that overexpress synphilin-1. Notably, the interaction between synphilin-1 and alpha-synuclein is markedly dependent on phosphorylation. Inhibition of CKII activity by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole blocks the binding between these two proteins and significantly reduces the percentage of cells that contain eosinophilic cytoplasmic inclusions. Mutation of the major CKII phosphorylation site in alpha-synuclein (S129A) has no significant impact on the binding between alpha-synuclein and synphilin-1 or on the formation of synphilin-1/alpha-synuclein-positive inclusions. These data suggest that the CKII-mediated phosphorylation of synphilin-1 rather than that of alpha-synuclein is critical in modulating their tendency to aggregate into inclusions. These observations collectively indicate that a ubiquitous post-translational modification such as phosphorylation can regulate inclusion body formation in the context of alpha-synuclein and synphilin-1 interaction.     

Dyrk1A phosphorylates parkin at Ser‐131 and negatively regulates its ubiquitin E3 ligase activity

Mutations of parkin are associated with the occurrence of autosomal recessive familial Parkinson's disease (PD). Parkin acts an E3 ubiquitin ligase, which ubiquitinates target proteins and subsequently regulates either their steady‐state levels through the ubiquitin–proteasome system or biochemical properties. In this study, we identify a novel regulatory mechanism of parkin by searching for new regulatory factors. After screening human fetal brain using a yeast two hybrid assay, we found dual‐specificity tyrosine‐(Y)‐phosphorylation‐regulated kinase 1A (Dyrk1A) as a novel binding partner of parkin. We also observed that parkin interacts and co‐localizes with Dyrk1A in mammalian cells. In addition, Dyrk1A directly phosphorylated parkin at Ser‐131, causing the inhibition of its E3 ubiquitin ligase activity. Moreover, Dyrk1A‐mediated phosphorylation reduced the binding affinity of parkin to its ubiquitin‐conjugating E2 enzyme and substrate, which could be the underlying inhibitory mechanism of parkin activity. Furthermore, Dyrk1A‐mediated phosphorylation inhibited the neuroprotective action of parkin against 6‐hydroxydopamine toxicity in dopaminergic SH‐SY5Y cells. These findings suggest that Dyrk1A acts as a novel functional modulator of parkin. Parkin phosphorylation by Dyrk1A suppresses its E3 ubiquitin ligase activity potentially contributing to the pathogenesis of PD under PD‐inducing pathological conditions.

     

C. elegans model identifies genetic modifiers of alpha-synuclein inclusion formation during aging

Inclusions in the brain containing alpha-synuclein are the pathological hallmark of Parkinson's disease, but how these inclusions are formed and how this links to disease is poorly understood. We have developed a C. elegans model that makes it possible to monitor, in living animals, the formation of alpha-synuclein inclusions. In worms of old age, inclusions contain aggregated alpha- synuclein, resembling a critical pathological feature. We used genome-wide RNA interference to identify processes involved in inclusion formation, and identified 80 genes that, when knocked down, resulted in a premature increase in the number of inclusions. Quality control and vesicle-trafficking genes expressed in the ER/Golgi complex and vesicular compartments were overrepresented, indicating a specific role for these processes in alpha-synuclein inclusion formation. Suppressors include aging-associated genes, such as sir-2.1/SIRT1 and lagr-1/LASS2. Altogether, our data suggest a link between alpha-synuclein inclusion formation and cellular aging, likely through an endomembrane-related mechanism. The processes and genes identified here present a framework for further study of the disease mechanism and provide candidate susceptibility genes and drug targets for Parkinson's disease and other alpha-synuclein related disorders.     

Golgi fragmentation occurs in the cells with prefibrillar alpha-synuclein aggregates and precedes the formation of fibrillar inclusion

Amyloid-like fibrillar aggregates of intracellular proteins are common pathological features of human neurodegenerative diseases. However, the nature of pathogenic aggregates and the biological consequences of their formation remain elusive. Here, we describe (i) a model cellular system in which prefibrillar alpha-synuclein aggregates and fibrillar inclusions are naturally formed in the cytoplasm with distinctive kinetics and (ii) a tight correlation between the presence of prefibrillar aggregates and the Golgi fragmentation. Consistent with the structural abnormality of Golgi apparatus, trafficking and maturation of dopamine transporter through the biosynthetic pathway were impaired in the presence of alpha-synuclein aggregates. Reduction in cell viability was also observed in the prefibrillar aggregate-forming condition and before the inclusion formation. The fibrillar inclusions, on the other hand, showed no correlation with Golgi fragmentation and were preceded by these events. Furthermore, at the early stage of inclusion formation, active lysosomes and mitochondria were enriched in the juxtanuclear area and co-aggregate into a compact inclusion body, suggesting that the fibrillar inclusions might be the consequence of an attempt of the cell to remove abnormal protein aggregates and damaged organelles. These results support the hypothesis that prefibrillar alpha-synuclein aggregates are the pathogenic species and suggest that Golgi fragmentation and subsequent trafficking impairment are the specific consequence of alpha-synuclein aggregation.     

Protein aggregation propensity is a crucial determinant of intracellular inclusion formation and quality control degradation

Protein aggregation is linked to many pathological conditions, including several neurodegenerative diseases. The aggregation propensities of proteins are thought to be controlled to a large extent by the physicochemical properties encoded in the primary sequence. We have previously exploited a set of amyloid β peptide (Aβ42) variants exhibiting a continuous gradient of intrinsic aggregation propensities to demonstrate that this rule applies in vivo in bacteria. In the present work we have characterized the behavior of these Aβ42 mutants when expressed in yeast. In contrast to bacteria, the intrinsic aggregation propensity is gated by yeast, in such a way that this property correlates with the formation of intracellular inclusions only above a specific aggregation threshold. Proteins displaying solubility levels above this threshold escape the inclusion formation pathway. In addition, the most aggregation-prone variants are selectively cleared by the yeast quality control degradation machinery. Thus, both inclusion formation and proteolysis target the same aggregation-prone variants and cooperate to minimize the presence of these potentially dangerous species in the cytosol. The demonstration that sorting to these pathways in eukaryotes is strongly influenced by protein primary sequence should facilitate the development of rational approaches to predict and hopefully prevent in vivo protein deposition.     

DJ-1 is a redox-dependent molecular chaperone that inhibits alpha-synuclein aggregate formation

Parkinson's disease (PD) pathology is characterized by the degeneration of midbrain dopamine neurons (DNs) ultimately leading to a progressive movement disorder in patients. The etiology of DN loss in sporadic PD is unknown, although it is hypothesized that aberrant protein aggregation and cellular oxidative stress may promote DN degeneration. Homozygous mutations in DJ-1 were recently described in two families with autosomal recessive inherited PD (Bonifati et al. 2003). In a companion article (Martinat et al. 2004), we show that mutations in DJ-1 alter the cellular response to oxidative stress and proteasomal inhibition. Here we show that DJ-1 functions as a redox-sensitive molecular chaperone that is activated in an oxidative cytoplasmic environment. We further demonstrate that DJ-1 chaperone activity in vivo extends to alpha-synuclein, a protein implicated in PD pathogenesis.     

JNK phosphorylates synaptotagmin-4 and enhances Ca2+-evoked release

Ca2+ influx induced by membrane depolarization triggers the exocytosis of secretory vesicles in various cell types such as endocrine cells and neurons. Peptidyl growth factors enhance Ca2+-evoked release, an effect that may underlie important adaptive responses such as the long-term potentiation of synaptic transmission induced by growth factors. Here, we show that activation of the c-Jun N-terminal kinase (JNK) plays an essential role in nerve growth factor (NGF) enhancement of Ca2+-evoked release in PC12 neuroendocrine cells. Moreover, JNK associated with phosphorylated synaptotagmin-4 (Syt 4), a key mediator of NGF enhancement of Ca2+-evoked release in this system. NGF treatment led to phosphorylation of endogenous Syt 4 at Ser135 and translocation of Syt 4 from immature to mature secretory vesicles in a JNK-dependent manner. Furthermore, mutation of Ser135 abrogated enhancement of Ca2+-evoked release by Syt 4. These results provide a molecular basis for the effect of growth factors on Ca2+-mediated secretion.     

Rho-kinase phosphorylates PAR-3 and disrupts PAR complex formation

A polarity complex of PAR-3, PAR-6, and atypical protein kinase C (aPKC) functions in various cell polarization events. PAR-3 directly interacts with Tiam1/Taim2 (STEF), Rac1-specific guanine nucleotide exchange factors, and forms a complex with aPKC-PAR-6-Cdc42*GTP, leading to Rac1 activation. RhoA antagonizes Rac1 in certain types of cells. However, the relationship between RhoA and the PAR complex remains elusive. We found here that Rho-kinase/ROCK/ROK, the effector of RhoA, phosphorylated PAR-3 at Thr833 and thereby disrupted its interaction with aPKC and PAR-6, but not with Tiam2. Phosphorylated PAR-3 was observed in the leading edge, and in central and rear portions of migrating cells having front-rear polarity. Knockdown of PAR-3 by small interfering RNA (siRNA) impaired cell migration, front-rear polarization, and PAR-3-mediated Rac1 activation, which were recovered with siRNA-resistant PAR-3, but not with the phospho-mimic PAR-3 mutant. We propose that RhoA/Rho-kinase inhibits PAR complex formation through PAR-3 phosphorylation, resulting in Rac1 inactivation.     

Dyrk1A Phosphorylates p53 and Inhibits Proliferation of Embryonic Neuronal Cells

Down syndrome (DS) is associated with many neural defects, including reduced brain size and impaired neuronal proliferation, highly contributing to the mental retardation. Those typical characteristics of DS are closely associated with a specific gene group “Down syndrome critical region” (DSCR) on human chromosome 21. Here we investigated the molecular mechanisms underlying impaired neuronal proliferation in DS and, more specifically, a regulatory role for dual-specificity tyrosine-(Y) phosphorylation-regulated kinase 1A (Dyrk1A), a DSCR gene product, in embryonic neuronal cell proliferation. We found that Dyrk1A phosphorylates p53 at Ser-15 in vitro and in immortalized rat embryonic hippocampal progenitor H19-7 cells. In addition, Dyrk1A-induced p53 phosphorylation at Ser-15 led to a robust induction of p53 target genes (e.g. p21^CIP1) and impaired G₁/G₀-S phase transition, resulting in attenuated proliferation of H19-7 cells and human embryonic stem cell-derived neural precursor cells. Moreover, the point mutation of p53-Ser-15 to alanine rescued the inhibitory effect of Dyrk1A on neuronal proliferation. Accordingly, brains from embryonic DYRK1A transgenic mice exhibited elevated levels of Dyrk1A, Ser-15 (mouse Ser-18)-phosphorylated p53, and p21^CIP1 as well as impaired neuronal proliferation. These findings suggest that up-regulation of Dyrk1A contributes to altered neuronal proliferation in DS through specific phosphorylation of p53 at Ser-15 and subsequent p21^CIP1 induction.     

NHK-1 phosphorylates BAF to allow karyosome formation in the Drosophila oocyte nucleus

Accurate chromosome segregation in meiosis requires dynamic changes in chromatin organization. In Drosophila melanogaster, upon completion of recombination, meiotic chromosomes form a single, compact cluster called the karyosome in an enlarged oocyte nucleus. This clustering is also found in humans; however, the mechanisms underlying karyosome formation are not understood. In this study, we report that phosphorylation of barrier to autointegration factor (BAF) by the conserved kinase nucleosomal histone kinase-1 (NHK-1; Drosophila Vrk1) has a critical function in karyosome formation. We find that the noncatalytic domain of NHK-1 is crucial for its kinase activity toward BAF, a protein that acts as a linker between chromatin and the nuclear envelope. A reduction of NHK-1 or expression of nonphosphorylatable BAF results in ectopic association of chromosomes with the nuclear envelope in oocytes. We propose that BAF phosphorylation by NHK-1 disrupts anchorage of chromosomes to the nuclear envelope, allowing karyosome formation in oocytes. These data provide the first mechanistic insight into how the karyosome forms.     

Tyrosine-to-cysteine modification of human alpha-synuclein enhances protein aggregation and cellular toxicity

The deposition of alpha-synuclein and other cellular proteins in Lewy bodies in midbrain dopamine neurons is a pathological hallmark of Parkinson's disease. Nitrative and oxidative stress can induce alpha-synuclein protein aggregation, possibly initiated by the formation of stable cross-linking dimers. To determine whether enhanced dimer formation can accelerate protein aggregation and increase cellular toxicity, we have substituted cysteine for tyrosine at positions 39, 125, 133, and 136 in human wild-type (WT) alpha-synuclein, and in A53T and A30P mutant alpha-synuclein. To reduce the likelihood of cross-linking, phenylalanine was substituted for tyrosine at the same sites. We have found that overexpression of Y39C or Y125C mutant proteins leads to increased intracellular inclusions and apoptosis in a rat dopaminergic cell line (N27 cells) and in human embryonic kidney 293 cells. Expression of Y133C, Y136C, and all four Tyr-to-Phe mutations were not more cytotoxic than WT control. Exposure to oxidative stress increased Y39C and Y125C alpha-synuclein aggregation and toxicity. Dimers and oligomers were found in Triton X-100-soluble fractions from adenovirus-mediated overexpression of Y39C and Y125C in N27 cells. In contrast, WT beta-synuclein and all four Tyr-to-Cys mutant beta-synucleins did not cause protein aggregation and cell death. We conclude that cysteine substitution at critical positions in the alpha-synuclein molecule can increase dimer formation and accelerate protein aggregation and cellular toxicity of alpha-synuclein.     

QSAR analysis of pyrazolidine-3,5-diones derivatives as Dyrk1A inhibitors

Individuals with Down syndrome (DS) suffer from mental retardation. Overexpression and the resulting increased specific activity of Dyrk1A kinase located on chromosome 21 cause a learning and memory deficit in Dyrk1A transgenic mice. To search for therapeutic agents with Dyrk1A inhibition activity, previously we obtained HCD160 as a new hit compound for Dyrk1A inhibition. In the present study, we synthesized 34 HCD160 derivatives to investigate the quantitative structure–activity relationship (QSAR). This analysis could provide important information for novel drug discovery for treatment of DS related learning and memory deficits.     

Overexpression of Dyrk1A causes the defects in synaptic vesicle endocytosis

Trisomy 21-linked Dyrk1A (dual-specificity tyrosine phosphorylation-regulated kinase 1A) overexpression is implicated in pathogenic mechanisms underlying mental retardation in Down syndrome (DS). It is known to phosphorylate multiple substrates including endocytic proteins in vitro, but the functional consequence of Dyrk1A-mediated phosphorylation on endocytosis has never been investigated. Here, we show that overexpression of Dyrk1A causes defects in clathrin-mediated endocytosis and specifically, in the recruitment of endocytic proteins to clathrin-coated pits in fibroblasts. Synaptic vesicle endocytosis also significantly slowed down as a result of Dyrk1A overexpression in cultured hippocampal neurons. These effects are dependent on Dyrk1A kinase activity. The inhibitory effect of Dyrk1A on synaptic vesicle endocytosis was confirmed in neuronal cultures derived from transgenic mice overexpressing Dyrk1A at levels found in DS. Pharmacological blockade of Dyrk1A with epigallocatechin gallate rescued the endocytic phenotypes found in transgenic neurons. Together, our results suggest that aberrant Dyrk1A-mediated phosphorylation of the endocytic machinery perturbs synaptic vesicle endocytosis, which may contribute to synaptic dysfunctions and cognitive deficits associated with DS.     

Harmine is an ATP-competitive inhibitor for dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A)

Harmine is a β-carboline alkaloid. The compound is a potent inhibitor of dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A), a kinase implicated in Down syndrome. In this study, we show that harmine functions as an ATP-competitive inhibitor against Dyrk1A. Our conclusion is supported by kinetic analysis of harmine inhibition as well as by the characterization of a Dyrk1A mutation conferring significant resistance to harmine. The mutation, V306A, is located next to the highly conserved D307 residue in kinases known to coordinate the phosphate groups of ATP through a Mg²⁺ ion. The V306A mutation offers harmine resistance by differentially altering Dyrk1A affinity for harmine and ATP. The V306A mutation causes no apparent alteration to Dyrk1A activity except for the reduction in ATP affinity. This deficiency could be fully compensated by supplying ATP with a concentration in the physiological range. Our results reveal that harmine inhibits Dyrk1A activity by interacting with residues in the ATP-binding pocket and displacing ATP. Our results also suggest that harmine will be a good lead compound for further designing of selective ATP-competitive Dyrk1A inhibitors through exploration of the ATP-binding pocket of Dyrk1A.     

ERK binds, phosphorylates InsP3 type 1 receptor and regulates intracellular calcium dynamics in DT40 cells

Modulation on the duration of intracellular Ca(2+) transients is essential for B-cell activation. We have previously shown that extracellular-signal-regulated kinase (ERK) can phosphorylate inositol 1,4,5-trisphosphate receptor type 1 (IP(3)R1) at serine 436 and regulate its calcium channel activity. Here we investigate the potential physiological interaction between ERK and IP(3)R1 using chicken DT40 B-cell line in which different mutants are expressed. The interaction between ERK and IP(3)R1 is confirmed by co-immunoprecipitation and fluorescence resonance energy transfer (FRET) assays. This constitutive interaction is independent of either ERK kinase activation or IP(3)R1 phosphorylation status. Back phosphorylation analysis further shows that type 1 IP(3)R (IP(3)R1) is phosphorylated by ERK in anti-IgM-activated DT40 cells. Finally, our data show that the phosphorylation of Ser 436 in the IP(3)-binding domain of IP(3)R1 leads to less Ca(2+) release from endoplasmic reticulum (ER) microsomes and accelerates the declining of calcium increase in DT40 cells in response to anti-IgM stimulation.     

CpG demethylation enhances alpha-synuclein expression and affects the pathogenesis of Parkinson's disease

Background

Alpha-synuclein (SNCA) gene expression is an important factor in the pathogenesis of Parkinson''s disease (PD). Gene multiplication can cause inherited PD, and promoter polymorphisms that increase SNCA expression are associated with sporadic PD. CpG methylation in the promoter region may also influence SNCA expression.

Methodology/Principal Findings

By using cultured cells, we identified a region of the SNCA CpG island in which the methylation status altered along with increased SNCA expression. Postmortem brain analysis revealed regional non-specific methylation differences in this CpG region in the anterior cingulate and putamen among controls and PD; however, in the substantia nigra of PD, methylation was significantly decreased.

Conclusions/Significance

This CpG region may function as an intronic regulatory element for SNCA gene. Our findings suggest that a novel epigenetic regulatory mechanism controlling SNCA expression influences PD pathogenesis.