␣-Synuclein (a-Syn) is a major component of fibrillar aggregates in Lewy bodies (LBs), a characteristic hallmark of Parkinson disease. Almost 90% of a-Syn deposited in LBs is phosphorylated at Ser-129. However, the role of Ser-129-phosphorylated a-Syn in the biogenesis of LBs remains unclear. Here, we investigated the metabolism of Ser-129-phosphorylated a-Syn. In SH-SY5Y cells, inhibition of protein phosphatase 2A/1 by okadaic acid, and inhibition of the proteasome pathway by MG132 or lactacystin accumulated Ser-129-phosphorylated a-Syn. However, these inhibitions did not alter the amounts of total a-Syn within the observation time. Inhibition of the autophagy-lysosome pathway by 3-methyladenine or chloroquine accumulated Ser-129-phosphorylated a-Syn in parallel to total a-Syn during longer incubations. Experiments using cycloheximide showed that Ser-129-phosphorylated a-Syn diminished rapidly (t1 ⁄ 2 ‫؍‬ 54.9 ؎ 6.4 min), in contrast to the stably expressed total a-Syn. The short half-life of Ser-129-phosphorylated a-Syn was blocked by MG132 to a greater extent than okadaic acid. In rat primary cortical neurons, either MG132, lactacystin, or okadaic acid accumulated Ser-129-phosphorylated a-Syn. Additionally, we did not find that phosphorylated a-Syn was ubiquitinated in the presence of proteasome inhibitors. These data show that Ser-129-phosphorylated a-Syn is targeted to the proteasome pathway in a ubiquitin-independent manner, in addition to undergoing dephosphorylation. The proteasome pathway may play a role in the biogenesis of Ser-129-phosphorylated a-Syn-rich LBs. Sporadic Parkinson disease (sPD)3 is characterized pathologically by a loss of dopaminergic neurons in the substantia nigra pars compacta and the presence of intracytoplasmic inclusions called Lewy bodies (LBs) and Lewy neurites (LNs) in surviving neurons. ␣-Synuclein (a-Syn) is a major component of fibrillar aggregates in LBs and LNs. Accumulating lines of evidence have shown that prefibrillar intermediates of a-Syn, such as soluble oligomers or protofibrils, play a toxic role in degeneration of dopaminergic neurons, and mature fibrils of a-Syn contribute toward this toxicity to a lesser extent (1-4). Therefore, the process of a-Syn aggregation eventually forming LBs is proposed to play a causative role in neuronal degeneration of PD (5, 6). Immunohistochemical and biochemical studies have revealed that ϳ90% of a-Syn deposited in LBs is phosphorylated at serine 129 (Ser-129) (7,8). In contrast, the portion of phosphorylated a-Syn in normal brains is known to be only about 4% (7) or less than the limits of quantification of the assays used (8). This discrepancy implicates a pathogenic role of Ser-129-phosphorylated a-Syn in the biogenesis of LBs (7, 9). One possibility is that the Ser-129-phosphorylation promotes the aggregation-prone property of a-Syn. To elucidate this issue, several in vitro studies have been performed. However, the accelerating effect of phosphorylation on fibril formation of a-Syn is controversial at present (7, 10). Anoth...

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra (SN) and the appearance of fibrillar aggregates of insoluble ␣-synuclein (␣-syn) called Lewy bodies (LBs). Approximately 90% of ␣-syn deposited in LBs is phosphorylated at serine 129 (Ser129). In contrast, only 4% of total ␣-syn is phosphorylated in normal brain, suggesting that accumulation of Ser129-phosphorylated ␣-syn is involved in the pathogenesis of PD. However, the role of Ser129 phosphorylation in ␣-syn neurotoxicity remains unclear. In this study, we coexpressed familial PD-linked A53T ␣-syn and G-protein-coupled receptor kinase 6 (GRK6) in the rat SN pars compacta using recombinant adeno-associated virus 2. Coexpression of these proteins yielded abundant Ser129-phosphorylated ␣-syn and significantly exacerbated degeneration of dopaminergic neurons when compared with coexpression of A53T ␣-syn and GFP. Immunohistochemical analysis revealed that Ser129-phosphorylated ␣-syn was preferentially distributed to swollen neurites. However, biochemical analysis showed that the increased expression of Ser129-phosphorylated ␣-syn did not promote accumulation of detergentinsoluble ␣-syn. Coexpression of catalytically inactive K215R mutant GRK6 failed to accelerate A53T ␣-syn-induced degeneration. Furthermore, introducing a phosphorylation-incompetent mutation, S129A, into A53T ␣-syn did not alter the pace of degeneration, even when GRK6 was coexpressed. Our study demonstrates that authentically Ser129-phosphorylated ␣-syn accelerates A53T ␣-syn neurotoxicity without the formation of detergent-insoluble ␣-syn, and suggests that the degenerative process could be constrained by inhibiting the kinase that phosphorylates ␣-syn at Ser129.

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We examined the feasibility of using gelatin hydrogels as carrier sheets for the transplantation of cultivated corneal endothelial cells. The mechanical properties, transparency, and permeability of gelatin hydrogel sheets were compared with those of atelocollagen sheets. Immunohistochemistry (ZO-1, Na(+)/K(+)-ATPase, and N-cadherin), hematoxylin and eosin staining, and scanning electron microscopy were performed to assess the integrity of corneal endothelial cells that were cultured on gelatin hydrogel sheets. The gelatin hydrogel sheets displayed greater transparency, elastic modulus, and albumin permeability compared to those of atelocollagen sheets. The corneal endothelial cells on gelatin hydrogel sheets showed normal expression levels of ZO-1, Na(+)/K(+)-ATPase, and N-cadherin. Hematoxylin and eosin staining revealed the formation of a continuous monolayer of cells attached to the gelatin hydrogel sheet. Scanning electron microscopy observations showed that the corneal endothelial cells were arranged in a regular, mosaic, and polygonal pattern with normal cilia. These results indicate that the gelatin hydrogel sheet is a promising material to transport corneal endothelial cells during transplantation.

The deformation and failure phenomena of materials are the results of stress-driven, thermally activated processes at the atomic scale. Molecular-dynamics (MD) simulations can only span a very limited time range which hinders one from gaining full view of the deformation physics. Inspired by the Eshelby transformation formalism, we present here a transformation "strain-boost" method for accelerating atomistic simulations, which is often more efficient and robust than the bond-boost hyperdynamics method [R. A. Miron and K. A. Fichthorn, J. Chem. Phys. 119, 6210 (2003)] for exploring collective stress-driven processes such as dislocation nucleation, that have characteristic activation volumes larger than one atomic volume. By introducing an adaptive algorithm that safely maximizes the boost factor, we directly access the finitetemperature dynamical process of dislocation nucleation in compressed Cu nanopillar over time scale comparable to laboratory experiments. Our method provides stress-and temperature-dependent activation enthalpy, activation entropy and activation volume for surface-dislocation nucleation with no human guidance about crystallography or deformation physics. Remarkably, the accelerated MD results indicate that harmonic transition-state theory and the empirical Meyer-Neldel compensation rule give reasonable approximations of the dislocation nucleation rate. The deformation and failure phenomena of materials are the results of stress-driven, thermally activated processes at the atomic scale. Molecular-dynamics ͑MD͒ simulations can only span a very limited time range which hinders one from gaining full view of the deformation physics. Inspired by the Eshelby transformation formalism, we present here a transformation "strain-boost" method for accelerating atomistic simulations, which is often more efficient and robust than the bond-boost hyperdynamics method ͓R. A. Miron and K. A. Fichthorn, J. Chem. Phys. 119, 6210 ͑2003͔͒ for exploring collective stress-driven processes such as dislocation nucleation, that have characteristic activation volumes larger than one atomic volume. By introducing an adaptive algorithm that safely maximizes the boost factor, we directly access the finite-temperature dynamical process of dislocation nucleation in compressed Cu nanopillar over time scale comparable to laboratory experiments. Our method provides stress-and temperature-dependent activation enthalpy, activation entropy and activation volume for surface-dislocation nucleation with no human guidance about crystallography or deformation physics. Remarkably, the accelerated MD results indicate that harmonic transition-state theory and the empirical Meyer-Neldel compensation rule give reasonable approximations of the dislocation nucleation rate. Disciplines Engineering | Materials Science and Engineering Comments