Intracellular Accumulation of α-Synuclein Aggregates Promotes S-Nitrosylation of MAP1A Leading to Decreased NMDAR-Evoked Calcium Influx and Loss of Mature Synaptic Spines.

Cortical synucleinopathies, including dementia with Lewy bodies and Parkinson's disease dementia, collectively known as Lewy body dementia, are characterized by the aberrant aggregation of misfolded α-synuclein (α-syn) protein into large inclusions in cortical tissue, leading to impairments in proteostasis and synaptic connectivity and eventually resulting in neurodegeneration. Here, we show that male and female rat cortical neurons exposed to exogenous α-syn preformed fibrils accumulate large, detergent-insoluble, PS129-labeled deposits at synaptic terminals. Live-cell imaging of calcium dynamics coupled with assessment of network activity reveals that aberrant intracellular accumulation of α-syn inhibits synaptic response to glutamate through NMDARs, although deficits manifest slowly over a 7 d period. Impairments in NMDAR activity temporally correlated with increased nitric oxide synthesis and S-nitrosylation of the dendritic scaffold protein, microtubule-associated protein 1A. Inhibition of nitric oxide synthesis via the nitric oxide synthase inhibitor l-NG-nitroarginine methyl ester blocked microtubule-associated protein 1A S-nitrosylation and normalized NMDAR-dependent inward calcium transients and overall network activity. Collectively, these data suggest that loss of synaptic function in Lewy body dementia may result from synucleinopathy-evoked nitrosative stress and subsequent NMDAR dysfunction.SIGNIFICANCE STATEMENT This work shows the importance of the redox state of microtubule-associated protein 1A in the maintenance of synaptic function through regulation of NMDAR. We show that α-syn preformed fibrils promote nitric oxide synthesis, which triggers S-nitrosylation of microtubule-associated protein 1A, leading to impairment of NMDAR-dependent glutamate responses. This offers insight into the mechanism of synaptic dysfunction in Lewy body dementia.

α-Synuclein mutation impairs processing of endomembrane compartments and promotes exocytosis and seeding of α-synuclein pathology.

Neuronal loss in Parkinson's disease (PD) is associated with impaired proteostasis and accumulation of α-syn microaggregates in dopaminergic neurons. These microaggregates promote seeding of α-synuclein (α-syn) pathology between synaptically linked neurons. However, the mechanism by which seeding is initiated is not clear. Using human pluripotent stem cell (hPSC) models of PD that allow comparison of SNCA mutant cells with isogenic controls, we find that SNCA mutant neurons accumulate α-syn deposits that cluster to multiple endomembrane compartments, specifically multivesicular bodies (MVBs) and lysosomes. We demonstrate that A53T and E46K α-syn variants bind and sequester LC3B monomers into detergent-insoluble microaggregates on the surface of late endosomes, increasing α-syn excretion via exosomes and promoting seeding of α-syn from SNCA mutant neurons to wild-type (WT) isogenic controls. Finally, we show that constitutive inactivation of LC3B promotes α-syn accumulation and seeding, while LC3B activation inhibits these events, offering mechanistic insight into the spread of synucleinopathy in PD.

Cardiolipin exposure on the outer mitochondrial membrane modulates α-synuclein.

Neuronal loss in Parkinson's disease (PD) is associated with aberrant mitochondrial function and impaired proteostasis. Identifying the mechanisms that link these pathologies is critical to furthering our understanding of PD pathogenesis. Using human pluripotent stem cells (hPSCs) that allow comparison of cells expressing mutant SNCA (encoding α-synuclein (α-syn)) with isogenic controls, or SNCA-transgenic mice, we show that SNCA-mutant neurons display fragmented mitochondria and accumulate α-syn deposits that cluster to mitochondrial membranes in response to exposure of cardiolipin on the mitochondrial surface. Whereas exposed cardiolipin specifically binds to and facilitates refolding of α-syn fibrils, prolonged cardiolipin exposure in SNCA-mutants initiates recruitment of LC3 to the mitochondria and mitophagy. Moreover, we find that co-culture of SNCA-mutant neurons with their isogenic controls results in transmission of α-syn pathology coincident with mitochondrial pathology in control neurons. Transmission of pathology is effectively blocked using an anti-α-syn monoclonal antibody (mAb), consistent with cell-to-cell seeding of α-syn.

Tumor cell kill by c-MYC depletion: role of MYC-regulated genes that control DNA double-strand break repair.

MYC regulates a myriad of genes controlling cell proliferation, metabolism, differentiation, and apoptosis. MYC also controls the expression of DNA double-strand break (DSB) repair genes and therefore may be a potential target for anticancer therapy to sensitize cancer cells to DNA damage or prevent genetic instability. In this report, we studied whether MYC binds to DSB repair gene promoters and modulates cell survival in response to DNA-damaging agents. Chromatin immunoprecipitation studies showed that MYC associates with several DSB repair gene promoters including Rad51, Rad51B, Rad51C, XRCC2, Rad50, BRCA1, BRCA2, DNA-PKcs, XRCC4, Ku70, and DNA ligase IV. Endogenous MYC protein expression was associated with increased RAD51 and KU70 protein expression of a panel of cancer cell lines of varying histopathology. Induction of MYC in G(0)-G(1) and S-G(2)-M cells resulted in upregulation of Rad51 gene expression. MYC knockdown using small interfering RNA (siRNA) led to decreased RAD51 expression but minimal effects on homologous recombination based on a flow cytometry direct repeat green fluorescent protein assay. siRNA to MYC resulted in tumor cell kill in DU145 and H1299 cell lines in a manner independent of apoptosis. However, MYC-dependent changes in DSB repair protein expression were not sufficient to sensitize cells to mitomycin C or ionizing radiation, two agents selectively toxic to DSB repair-deficient cells. Our results suggest that anti-MYC agents may target cells to prevent genetic instability but would not lead to differential radiosensitization or chemosensitization.

Efficacy of combining GMX1777 with radiation therapy for human head and neck carcinoma.

PURPOSE: Rapidly metabolizing tumor cells have elevated levels of nicotinamide phosphoribosyltransferase, an enzyme involved in NAD(+) biosynthesis, which serves as an important substrate for proteins involved in DNA repair. GMX1777, which inhibits nicotinamide phosphoribosyltransferase, was evaluated in two human head and neck cancer models in combination with radiotherapy. EXPERIMENTAL DESIGN: Effects of GMX1777-mediated radiosensitization were examined via metabolic and cytotoxicity assays in vitro; mechanism of action, in vivo antitumor efficacy, and radiosensitization were also investigated. RESULTS: IC(50) values of GMX1777 for FaDu and C666-1 cells were 10 and 5 nmol/L, respectively, which interacted synergistically with radiotherapy. GMX1777 induced a rapid decline in intracellular NAD(+) followed by ATP reduction associated with significant cytotoxicity. These metabolic changes were slightly increased with the addition of radiotherapy, although poly(ADP-ribose) polymerase activity was significantly reduced when GMX1777 was combined with radiotherapy, thereby accounting for the synergistic cytotoxicity of these two modalities. Systemic GMX1777 administration with local tumor radiotherapy caused complete disappearance of FaDu and C666-1 tumors for 50 and 20 days, respectively. There was also significant reduction in tumor vascularity, particularly for the more sensitive FaDu model. [(18)F]FDG-positron emission tomography/computed tomography images showed reduction in [(18)F]FDG uptake after GMX1777 administration, showing decreased glucose metabolism in vivo. CONCLUSIONS: Our data represent the first report showing that GMX1777 plus radiotherapy is an effective therapeutic strategy for head and neck cancer, mediated via pleiotropic effects of inhibition of DNA repair and tumor angiogenesis, while sparing normal tissues. Therefore, GMX1777 combined with radiotherapy definitely warrants clinical evaluation in human head and neck cancer patients.