Benoit I. Giasson

Intracytoplasmic proteinaceous inclusions, primarily composed of tau or α-synuclein (α-syn), are predominant pathological features of Alzheimer's disease (AD) and Parkinson's disease (PD), respectively. However, the coexistence of these pathological aggregates is identified in many neurodegenerative disorders, including spectrum disorders of AD and PD. Whereas α-syn can spontaneously polymerize into amyloidogenic fibrils,in vitro, tau polymerization requires an inducing agent. The current study presents a human-derived cellular model, in which recombinant, preformed α-syn fibrils cross-seed intracellular tau to promote the formation of neurofibrillary tangle-like aggregates. These aggregates were hyperphosphorylated, Triton insoluble, and thioflavin-S positive, either comingling with endogenously expressed α-syn aggregates or induced by only exogenously applied recombinant α-syn fibrils. Furthermore, filamentous, amyloidogenic tau took over the cellular soma, displacing the nucleus and isolating or displacing organelles, likely preventing cellular function. Although a significant proportion of wild-type tau formed these cellular inclusions, the P301L mutation in tau increased aggregation propensity resulting from α-syn seeds to over 50% of total tau protein. The role of phosphorylation on the development of these tau aggregates was investigated by coexpressing glycogen synthase kinase 3 β or microtubule-associated protein/microtubule affinity-regulating kinase 2. Expression of either kinase inhibited the formation of α-syn-induced tau aggregates. Analyses of phosphorylation sites suggest that multiple complex factors may be associated with this effect and that Triton-soluble versus Triton-insoluble tau may be independently targeted by kinases. The current work not only provides an exceptional cellular model of tau pathology, but also examines α-syn-induced tau inclusion formation and provides novel insights into hyperphosphorylation observed in disease.

α‐Synuclein (α‐syn) is the major component of pathological inclusions characteristic of several neurodegenerative disorders, such as Parkinson's disease. The major posttranslational modification of α‐syn is phosphorylation at S129, and previous studies estimate that approximately 90% of α‐syn in proteinaceous, pathological inclusions is phosphorylated at this site. α‐Syn can be phosphorylated by polo‐like kinases (PLKs) 1–3 and casein kinases (CK) 1 and 2; however, the kinases associated with the hyperphosphorylation of aggregated α‐syn are still under debate. Using a high‐efficiency cellular model of α‐syn aggregate formation, we found that selective inhibitors for CK2 and PLKs each partially inhibited S129 phosphorylation of soluble (nonaggregated) α‐syn, but only PLK inhibitors modestly attenuated the phosphorylation of aggregated α‐syn. In addition, none of the kinase inhibitors used had a substantial effect on the propensity of α‐syn to aggregate. Overexpression of all PLKs promoted robust phosphorylation of soluble α‐syn, but none altered the propensity of α‐syn to aggregate. Overexpression of only PLK2 increased phosphorylation of aggregated α‐syn at S129, which likely is due to increased phosphorylation of soluble α‐syn, which then was incorporated into aggregates. Overexpression of PLK1 and treatment with BI2536 resulted in a significant reduction of phosphorylated, aggregated α‐syn protein, beyond that of BI2536 treatment alone. These studies suggest that phosphorylation of α‐syn is independent of α‐syn aggregate formation, that PLK1 is involved in the phosphorylation of aggregated α‐syn at S129 in this system, and that mechanisms resulting in hyperphosphorylation of aggregated α‐syn appear to be independent of those responsible for the phosphorylation of soluble α‐syn.

J. Neurochem. (2010) 115, 36–46. Mutations in leucine‐rich repeat kinase‐2 (LRRK2) are the most common cause of late‐onset Parkinson disease. Previously, we showed that the G2019S pathogenic mutation can cause a dramatic increase (approximately 10‐fold) in kinase activity, far above other published studies. A notable experimental difference was the use of Mn‐ATP as a substrate. Therefore, the effects of metal cation‐ATP cofactors on LRRK2 kinase activity were investigated. It is shown, using several divalent metal cations, that only Mg²⁺ or Mn²⁺ can support LRRK2 kinase activity. However, for wild‐type, I2020T, and R1441C LRRK2, Mn²⁺ was significantly less effective at supporting kinase activity. In sharp contrast, both Mn²⁺ and Mg²⁺ were effective at supporting the activity of G2019S LRRK2. These divergent effects associated with divalent cation usage and the G2019S mutation were predominantly because of differences in catalytic rates. However, LRRK2 was shown to have much lower (approximately 40‐fold) ATP K_m for Mn‐ATP compared with Mg‐ATP. Consequently, sub‐stoichiometric concentrations of Mn²⁺ can act to inhibit the kinase activity of wild‐type, but not G2019S LRRK2 in the presence of Mg²⁺. From these findings, a new model is proposed for a possible function of LRRK2 and the consequence of the G2019S LRRK2 pathogenic mutation.

A variety of mutations in the gene encoding DJ‐1 protein cause autosomal recessive early‐onset parkinsonism. Recently, a novel pathogenic homozygous DJ‐1 missense mutation resulting in the L10P amino acid substitution was reported. In a separate study, a novel homozygous mutation resulting in the deletion of DJ‐1 residue P158 was also reported to be causative of disease. The specific effects of the novel L10P and P158DEL mutations on protein function have not been studied. Here, L10P and P158DEL DJ‐1 proteins were assessed for protein stability, dimerization, solubility, subcellular localization, and protective function in comparison with WT and the L166P DJ‐1 pathogenic variant. It was discovered that, compared with WT protein, L10P, L166P, and P158DEL DJ‐1 variants exhibited dramatically reduced protein stabilities. Degradation of each of the pathogenic mutants appeared to be mediated in part by the proteasome. Interestingly, unlike L166P DJ‐1, the L10P and P158DEL DJ‐1 variants retained the ability to dimerize with WT DJ‐1 protein; however, neither of these mutants was able to form homodimers. Additionally, the L10P, L166P, and P158DEL DJ‐1 variants exhibited altered profiles on size‐exclusion chromatography and demonstrated reduced solubilities in comparison with WT protein, and the latter aberration could be exacerbated in the presence of MG‐132. Furthermore, cells stably expressing L10P DJ‐1 were more vulnerable to treatments with proteasome inhibitors, suggesting that L10P DJ‐1 may be toxic to cells under conditions of proteasome stress. Taken together, these findings suggest that diverse aberrant mechanisms, including alterations in protein stability and protein folding, are associated with the pathogenicity of the L10P and P158DEL DJ‐1 variants.

Mutations or deletions inPARKIN/PARK2,PINK1/PARK6, andDJ-1/PARK7lead to autosomal recessive parkinsonism. InDrosophila, deletions inparkinandpink1result in swollen and dysfunctional mitochondria in energy-demanding tissues. The relationship betweenDJ-1and mitochondria, however, remains unclear. We now report thatDrosophilaand mouse mutants inDJ-1show compromised mitochondrial function with age. Flies deleted forDJ-1manifest similar defects aspink1andparkinmutants: male sterility, shortened lifespan, and reduced climbing ability. We further found poorly coupled mitochondria in vitro and reduced ATP levels in fly and mouseDJ-1mutants. Surprisingly, up-regulation ofDJ-1can amelioratepink1, but notparkin,mutants inDrosophila; cysteine C104 (analogous to C106 in human) is critical for this rescue, implicating the oxidative functions of DJ-1 in this property. These results suggest thatDJ-1is important for proper mitochondrial function and acts downstream of, or in parallel to,pink1. These findings linkDJ-1,pink1, andparkinto mitochondrial integrity and provide the foundation for therapeutics that link bioenergetics and parkinsonism.

J. Neurochem. (2010) 113, 402–417. Abstract Various mutations in the PARK2 gene which encodes the protein, parkin, are causal of a disease entity‐termed autosomal recessive juvenile parkinsonism. Parkin can function as an E3 ubiquitin‐protein ligase, mediating the ubiquitination of specific targeted proteins and resulting in proteasomal degradation. Parkin is thought to lead to parkinsonism as a consequence of a loss in its function. In this study, immunoblot analyses of brain extracts from Balb/c, C57BL/6, C3H, and 129S mouse strains demonstrated significant variations in immunoreactivity with anti‐parkin monoclonal antibodies (PRK8, PRK28, and PRK109). This resulted partly from differences in the steady‐state levels of parkin protein across mouse strains. There was also a complete loss of immunoreactivity for PRK8 and PRK28 antibodies in C3H mice due to was because of a homologous nucleotide mutation resulting in an E398Q amino acid substitution. In cultured cells, parkin harboring this mutation had a greater tendency to aggregate, exhibited reduced interaction with the E2 ubiquitin‐conjugating enzymes, UbcH7 and UbcH8, and demonstrated loss‐of‐function in promoting the proteosomal degradation of a specific putative substrate, synphilin‐1. In situ, C3H mice displayed age‐dependent increased levels of brain cortical synphilin‐1 compared with C57BL/6, suggesting that E398Q parkin in these mice is functionally impaired and that C3H mice may be a suitable model of parkin loss‐of‐function similar to patients with missense mutations.

J. Neurochem. (2010) 113, 374–388. Intracytoplasmic alpha‐synuclein (α‐syn) amyloidogenic inclusions are a major pathological feature of Parkinson’s disease, dementia with Lewy body disease and multiple systems atrophy. The mechanisms involved in the formation and inhibition of these aggregates are areas of intense investigation. The present study characterizes a novel cellular model for the study of α‐syn aggregation, incorporating nucleation‐dependent aggregation and a new function for calcium phosphate precipitation. Cultured cells were readily induced to develop large, cytoplasmic α‐syn filamentous aggregates that were hyperphosphorylated, often ubiquitinated and thioflavin positive. These cellular aggregates formed in the majority of transfected cells and recruited approximately half of endogenously expressed α‐syn. Using this system, we examined single‐point mutations that inhibit α‐syn amyloid formation in vitro. Three mutations (V66P, T72P and T75P) significantly hindered α‐syn aggregation in this cell model. The T75P mutant, which could abrogate amyloid formation of wild‐type α‐syn in vitro, did not prevent wild‐type α‐syn cellular aggregates. These studies suggest that the propensity of α‐syn to form cellular aggregates may be more pronounced than in isolated in vitro studies. This novel high‐efficiency cellular model of α‐syn aggregation is a valuable system that may be used to further understand α‐syn aggregation and allow for the generation of future therapeutics.