Yoshito Nagano

Tyrosine 125 of α-synuclein plays a critical role for dimerization following nitrative stress

Abstract α-Synuclein is a major component of Lewy bodies in Parkinson’s disease, dementia with Lewy bodies, and glial cytoplasmic inclusions in multiple system atrophy. Increasing evidence suggests that the nitration of tyrosine residues in α-synuclein induced by oxidative injury is involved in the formation of inclusions characteristic to these synucleinopathies. Exposure of α-synuclein to peroxynitrite induces nitration of tyrosine residues, thereby forming α-synuclein oligomers. However, the contribution of tyrosine residues to either the nitration or the oligomerization is currently unknown. The present study used recombinant wild-type and mutant α-synuclein proteins to investigate the role of each α-synuclein tyrosine residue in the in vitro formation of α-synuclein oligomers under nitrative stress. Confocal microscopic analysis revealed that wild-type α-synuclein protein was able to accumulate and form an inclusion-like structure in the cytoplasm of living cells upon introduction by streptolysin O. Authentic peroxynitrite induced nitration of tyrosine residues in α-synuclein protein, as well as dimerization of α-synuclein. The formation of both SDS- and heat-stable dimers suggests cross-linking between nitrated tyrosine residues. Nonetheless, dimerization of α-synuclein proteins lacking tyrosine 125 was significantly decreased compared with α-synuclein proteins lacking tyrosine residues at positions 39, 133, or 136. Presumably, tyrosine 125 plays a critical role for α-synuclein dimerization under nitrative stress.

Activation of Pyk2/RAFTK induces tyrosine phosphorylation of α-synuclein via Src-family kinases

α‐Synuclein (αS) is a neuronal protein that has been implicated in the pathogenesis of Parkinsons disease. The present report demonstrates that the protein tyrosine kinase Pyk2/RAFTK is involved in cell stress‐induced tyrosine phosphorylation of αS. Hyperosmotic stress induced tyrosine phosphorylation of αS via Pyk2/RAFTK at tyrosine residue 125. Pyk2/RAFTK‐mediated phosphorylation of αS was primarily achieved with Src‐family kinases. In addition, osmotic stress‐induced phosphorylation of αS was dependent on Pyk2/RAFTK activation. Accordingly, such results indicate that Pyk2/RAFTK lies upstream of Src‐family kinases in the signaling cascade by which osmotic stress induces tyrosine phosphorylation of αS.

Hypoxia-induced upregulation of endothelial small G protein RhoA and Rho-kinase/ROCK2 inhibits eNOS expression.

The small G protein RhoA and its downstream effector Rho-kinase/ROCK2 play an important role in regulation of various vasculature cellular functions. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) is an important mediator of vascular homeostasis and cerebral blood flow. Using the human endothelial cell line HUVEC, the present study investigated the role of RhoA and Rho-kinase in endothelial eNOS protein expression under hypoxic conditions as an in vitro model of ischemia. RhoA protein levels in HUVEC were low under normoxic conditions, but were significantly increased after 5h of hypoxia. Endothelial Rho-kinase expression was not detected until after 3h of hypoxia; such expression remained significantly increased after 5h. On the other hand, endothelial eNOS expression was similar after 3h of hypoxia, but was significantly decreased after 5h. The hypoxia-induced decrease in eNOS expression was significantly enhanced by expression of the constitutively active form of RhoA and significantly inhibited by suppression of RhoA expression by small interfering RNA. The hypoxia-induced decrease in eNOS expression was significantly inhibited when endogenous Rho-kinase activation was inhibited by Rho-binding domain expression. Furthermore, the hypoxia-induced decrease in eNOS expression was significantly enhanced by expression of the constitutively active form of Rho-kinase. Since expression and activation of RhoA and Rho-kinase inhibit eNOS expression in endothelial cells, attempts to down-regulate RhoA and Rho-kinase by multiple drugs, such as statins or Rho-kinase inhibitors, might provide endothelial and cardiovascular benefits through upregulation of eNOS.

Embryonic stem cell‐derived neuron models of Parkinson's disease exhibit delayed neuronal death

Establishment of a Parkinsons disease (PD) neuron model was attempted with mouse embryonic stem (ES) cells. ES cell lines over‐expressing mouse nuclear receptor‐related 1 (Nurr1), together with human wild‐type and alanine 30 → proline (A30P) and alanine 53 → threonine (A53T) mutant α‐synuclein were established and subjected to differentiation into dopaminergic neurons. The ES cell‐derived dopaminergic neurons expressing wild‐type or mutant α‐synuclein exhibited the fundamental characteristics consistent with dopaminergic neurons in the substantia nigra. The ES cell‐derived PD model neurons exhibited increased susceptibility to oxidative stress, proteasome inhibition, and mitochondrial inhibition. Cell viability of PD model neurons and the control neurons was similar until 28 days after differentiation. Nonetheless, after that time, PD model neurons gradually began to undergo neuronal death over the course of 1 month, showing cytoplasmic aggregate formation and an increase of insoluble α‐synuclein protein. Such delayed neuronal death was observed in a mutant α‐synuclein protein level‐dependent manner, which was slightly inhibited by a c‐jun N‐terminal kinase inhibitor and a caspase inhibitor. Such cell death was not observed when the same ES cell lines were differentiated into oligodendrocytes. The ES cell‐derived PD model neurons are considered as prospective candidates for a new prototype modelling PD that would allow better investigation of the underlying neurodegenerative pathophysiology.

Granulovacuolar Degenerations Appear in Relation to Hippocampal Phosphorylated Tau Accumulation in Various Neurodegenerative Disorders

Background Granulovacuolar degeneration (GVD) is one of the pathological hallmarks of Alzheimers disease (AD), and it is defined as electron-dense granules within double membrane-bound cytoplasmic vacuoles. Several lines of evidence have suggested that GVDs appear within hippocampal pyramidal neurons in AD when phosphorylated tau begins to aggregate into early-stage neurofibrillary tangles. The aim of this study is to investigate the association of GVDs with phosphorylated tau pathology to determine whether GVDs and phosphorylated tau coexist among different non-AD neurodegenerative disorders. Methods An autopsied series of 28 patients with a variety of neurodegenerative disorders and 9 control patients were evaluated. Standard histological stains along with immunohistochemistry using protein markers for GVD and confocal microscopy were utilized. Results The number of neurons with GVDs significantly increased with the level of phosphorylated tau accumulation in the hippocampal regions in non-AD neurodegenerative disorders. At the cellular level, diffuse staining for phosphorylated tau was detected in neurons with GVDs. Conclusions Our data suggest that GVDs appear in relation to hippocampal phosphorylated tau accumulation in various neurodegenerative disorders, while the presence of phosphorylated tau in GVD-harbouring neurons in non-AD neurodegenerative disorders was indistinguishable from age-related accumulation of phosphorylated tau. Although GVDs in non-AD neurodegenerative disorders have not been studied thoroughly, our results suggest that they are not incidental findings, but rather they appear in relation to phosphorylated tau accumulation, further highlighting the role of GVD in the process of phosphorylated tau accumulation.

HDAC6 maintains mitochondrial connectivity under hypoxic stress by suppressing MARCH5/MITOL dependent MFN2 degradation

Mitochondria undergo fusion and fission in response to various metabolic stresses. Growing evidences have suggested that the morphological change of mitochondria by fusion and fission plays a critical role in protecting mitochondria from metabolic stresses. Here, we showed that hypoxia treatment could induce interaction between HDAC6 and MFN2, thus protecting mitochondrial connectivity. Mechanistically, we demonstrated that a mitochondrial ubiquitin ligase MARCH5/MITOL was responsible for hypoxia-induced MFN2 degradation in HDAC6 deficient cells. Notably, genetic abolition of HDAC6 in amyotrophic lateral sclerosis model mice showed MFN2 degradation with MARCH5 induction. Our results indicate that HDAC6 is a critical regulator of MFN2 degradation by MARCH5, thus protecting mitochondrial connectivity from hypoxic stress.

Lack of binding observed between human α-synuclein and Bcl-2 protein family

Abstract α-Synuclein is a presynaptic protein of unknown function that has been implicated in the pathogenesis of Parkinsons disease. To gain insight into the function of α-synuclein, the present study examined the association between α-synuclein and the following Bcl-2 family proteins: Bcl-2; Bcl-XL; Bcl-associated death promoter (BAD); and Bcl-2-associated X-protein. The results of a binding assay using gluthathione S-transferase (GST) fusion α-synuclein protein and an immunoprecipitation assay revealed that wild-type or mutant (A30P and A53T) α-synuclein (~16 kDa) does not bind to any of these members of the Bcl-2 family. Furthermore, no binding was observed between α-synuclein and BAD, regardless of the phosphorylation state of the serine residue in BAD. In contrast, α-synuclein was observed to bind to synphilin-1. Although α-synuclein has been reported to bind to BAD, modification of α-synuclein might be required for such binding to occur.

Interactions of Synphilin‐1 with phospholipids and lipid membranes

Synphilin‐1 is an α‐synuclein binding protein that is involved in the pathogenesis of Parkinsons disease. The present study investigated the phospholipid‐binding capacity of Synphilin‐1. The C‐terminus of Synphilin‐1 was found to selectively bind to acidic phospholipids, including phosphatidic acid, phosphatidylserine, and phosphatidylglycerol, but not to naturally charged phospholipids. Synphilin‐1 was targeted to cytoplasmic lipid droplets in mammalian cells. The amino acid sequence 610–640 was found to represent the primary determinant site for phospholipid binding. Moreover, the R621C mutation identified in Parkinsons disease abolished Synphilin‐1 association with lipid droplets. The lipophilicity of Synphilin‐1 might prove relevant to its physiologic function.

Phosphatidylinositol-4,5-bisphosphate is enriched in granulovacuolar degeneration bodies and neurofibrillary tangles

Among the pathological findings in Alzheimers disease (AD), the temporal and spatial profiles of granulovacuolar degeneration (GVD) bodies are characteristic in that they seem to be related to those of neurofibrillary tangles (NFTs), suggesting a common mechanism underlying the pathogenesis of these structures. Flotillin‐1, a marker of lipid rafts, accumulates in lysosomes of tangle‐bearing neurones in AD patients. In addition, recent reports have shown that GVD bodies accumulate at the nexus of the autophagic and endocytic pathways. The aim of this study was to elucidate the distribution of the lipid component of lipid rafts, phosphatidylinositol‐4,5‐bisphosphate [PtdIns(4,5)P2], in AD and other neurodegenerative disorders.

Dural arteriovenous fistula presenting with progressive dementia and parkinsonism

We report a rare case of progressive parkinsonism and cognitive dysfunction due to dural arteriovenous fistula (DAVF). A 69-year-old man, with a history of hypertension and diabetes, was admitted to our hospital because of parkinsonism and dementia. Susceptibility-weighted imaging (SWI) revealed a thrombus in the superior sagittal sinus (SSS) and marked dilation of the medullary vein suggestive of the presence of comorbid DAVF. A single-photon emission CT (SPECT) showed widespread hypoperfusion in the bilateral frontal lobes. Selective cerebral angiography revealed a DAVF in SSS. These symptoms were significantly ameliorated following transvenous embolisation of the venous sinus at the shunting point. Reversible parkinsonism and dementia after embolisation was correlated with decreased dilation of medullary vein on SWI and improved cerebral blood flow on SPECT in the frontal lobes. Differentiation of parkinsonian and dementia symptoms due to DAVF from those associated with neurodegenerative disease is of great importance because DAVF-associated deficits may be reversed by endovascular therapy.