Pathological α-syn aggregation is mediated by glycosphingolipid chain length and the physiological state of α-syn in vivo.

GBA1 mutations that encode lysosomal ß-glucocerebrosidase (GCase) cause the lysosomal storage disorder Gaucher disease (GD) and are strong risk factors for synucleinopathies, including Parkinson's disease and Lewy body dementia. Only a subset of subjects with GBA1 mutations exhibit neurodegeneration, and the factors that influence neurological phenotypes are unknown. We find that α-synuclein (α-syn) neuropathology induced by GCase depletion depends on neuronal maturity, the physiological state of α-syn, and specific accumulation of long-chain glycosphingolipid (GSL) GCase substrates. Reduced GCase activity does not initiate α-syn aggregation in neonatal mice or immature human midbrain cultures; however, adult mice or mature midbrain cultures that express physiological α-syn oligomers are aggregation prone. Accumulation of long-chain GSLs (≥C22), but not short-chain species, induced α-syn pathology and neurological dysfunction. Selective reduction of long-chain GSLs ameliorated α-syn pathology through lysosomal cathepsins. We identify specific requirements that dictate synuclein pathology in GD models, providing possible explanations for the phenotypic variability in subjects with GCase deficiency.

Neuron-specific mitochondrial oxidative stress results in epilepsy, glucose dysregulation and a striking astrocyte response.

Mitochondrial superoxide (O2-) production is implicated in aging, neurodegenerative disease, and most recently epilepsy. Yet the specific contribution of neuronal O2- to these phenomena is unclear. Here, we selectively deleted superoxide dismutase-2 (SOD2) in neuronal basic helix-loop-helix transcription factor (NEX)-expressing cells restricting deletion to a subset of excitatory principle neurons primarily in the forebrain (cortex and hippocampus). This resulted in nSOD2 KO mice that lived into adulthood (2-3 months) with epilepsy, selective loss of neurons, metabolic rewiring and a marked mitohormetic gene response. Surprisingly, expression of an astrocytic gene, glial fibrillary acidic protein (GFAP) was significantly increased relative to WT. Further studies in rat primary neuron-glial cultures showed that increased mitochondrial O2-, specifically in neurons, was sufficient to upregulate GFAP. These results suggest that neuron-specific mitochondrial O2- is sufficient to drive a complex and catastrophic epileptic phenotype and highlights the ability of SOD2 to act in a cell-nonautonomous manner to influence an astrocytic response.

SNARE proteins rescue impaired autophagic flux in Down syndrome.

Down syndrome (DS) is a chromosomal disorder caused by trisomy of chromosome 21 (Ts21). Unbalanced karyotypes can lead to dysfunction of the proteostasis network (PN) and disrupted proteostasis is mechanistically associated with multiple DS comorbidities. Autophagy is a critical component of the PN that has not previously been investigated in DS. Based on our previous observations of PN disruption in DS, we investigated possible dysfunction of the autophagic machinery in human DS fibroblasts and other DS cell models. Following induction of autophagy by serum starvation, DS fibroblasts displayed impaired autophagic flux indicated by autophagolysosome accumulation and elevated p62, NBR1, and LC3-II abundance, compared to age- and sex-matched, euploid (CTL) fibroblasts. While lysosomal physiology was unaffected in both groups after serum starvation, we observed decreased basal abundance of the Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptor (SNARE) family members syntaxin 17 (STX17) and Vesicle Associated Membrane Protein 8 (VAMP8) indicating that decreased autophagic flux in DS is due at least in part to a possible impairment of autophagosome-lysosome fusion. This conclusion was further supported by the observation that over-expression of either STX17 or VAMP8 in DS fibroblasts restored autophagic degradation and reversed p62 accumulation. Collectively, our results indicate that impaired autophagic clearance is a characteristic of DS cells that can be reversed by enhancement of SNARE protein expression and provides further evidence that PN disruption represents a candidate mechanism for multiple aspects of pathogenesis in DS and a possible future target for therapeutic intervention.

Toxicant-mediated redox control of proteostasis in neurodegeneration.

Disruption in redox signaling and control of cellular processes has emerged as a key player in many pathologies including neurodegeneration. As protein aggregations are a common hallmark of several neuronal pathologies, a firm understanding of the interplay between redox signaling, oxidative and free radical stress, and proteinopathies is required to sort out the complex mechanisms in these diseases. Fortunately, models of toxicant-induced neurodegeneration can be utilized to evaluate and report mechanistic alterations in the proteostasis network (PN). The epidemiological links between environmental toxicants and neurological disease gives further credence into characterizing the toxicant-mediated PN disruptions observed in these conditions. Reviewed here are examples of mechanistic interaction between oxidative or free radical stress and PN alterations. Additionally, investigations into toxicant-mediated PN disruptions, specifically focusing on environmental metals and pesticides, are discussed. Finally, we emphasize the need to distinguish whether the presence of protein aggregations are contributory to phenotypes related to neurodegeneration, or if they are a byproduct of PN deficiencies.

Cholestatic liver disease results increased production of reactive aldehydes and an atypical periportal hepatic antioxidant response.

Cholangiopathies such as primary sclerosing cholangitis (PSC) are chronic liver diseases characterized by increased cholestasis, biliary inflammation and oxidative stress. The objective of this study was to elucidate the impact of cholestatic injury on oxidative stress-related factors. Using hepatic tissue and whole cell liver extracts (LE) isolated from 11-week old C57BL/6J (WT) and Mdr2KO mice, inflammation and oxidative stress was assessed. Concurrently, specific targets of carbonylation were assessed in LE prepared from murine groups as well as from normal and human patients with end-stage PSC. Identified carbonylated proteins were further evaluated using bioinformatics analyses. Picrosirius red staining revealed extensive fibrosis in Mdr2KO liver, and fibrosis colocalized with increased periportal inflammatory cells and both acrolein and 4-HNE staining. Western blot analysis revealed elevated periportal expression of antioxidant proteins Cbr3, GSTµ, Prdx5, TrxR1 and HO-1 but not GCLC, GSTπ or catalase in the Mdr2KO group when compared to WT. From immunohistochemical analysis, increased periportal reactive aldehyde production colocalized with elevated staining of Cbr3, GSTµ and TrxR1 but surprisingly not with Nrf2. Mass spectrometric analysis revealed an increase in carbonylated proteins in the Mdr2KO and PSC groups compared to respective controls. Gene ontology and KEGG pathway analysis of carbonylated proteins revealed a propensity for increased carbonylation of proteins broadly involved in metabolic processes as well more specifically in Rab-mediated signal transduction, lysosomes and the large ribosomal subunit in human PSC. Western blot analysis of Rab-GTPase expression revealed no significant differences in Mdr2KO mice when compared to WT livers. In contrast, PSC tissue exhibited decreased levels of Rabs 4, 5 and increased abundance of Rabs 6 and 9a protein. Results herein reveal that cholestasis induces stage-dependent increases in periportal oxidative stress responses and protein carbonylation, potentially contributing to pathogenesis in Mdr2KO. Furthermore, during early stage cholestasis, there is cell-specific upregulation of some but not all, antioxidant proteins.

Acute Maneb Exposure Significantly Alters Both Glycolysis and Mitochondrial Function in Neuroblastoma Cells.

The pesticides paraquat (PQ) and maneb (MB) have been described as environmental risk factors for Parkinson's disease (PD), with mechanisms associated with mitochondrial dysfunction and reactive oxygen species generation. A combined exposure of PQ and MB in murine models and neuroblastoma cells has been utilized to further advance understanding of the PD phenotype. MB acts as a redox modulator through alkylation of protein thiols and has been previously characterized to inhibit complex III of the electron transport chain and uncouple the mitochondrial proton gradient. The purpose of this study was to analyze ATP-linked respiration and glycolysis in human neuroblastoma cells utilizing the Seahorse extracellular flux platform. Employing an acute, subtoxic exposure of MB, this investigation revealed a MB-mediated decrease in mitochondrial oxygen consumption at baseline and maximal respiration, with inhibition of ATP synthesis and coupling efficiency. Additionally, MB-treated cells showed an increase in nonmitochondrial respiration and proton leak. Further investigation into mitochondrial fuel flex revealed an elimination of fuel flexibility across all 3 major substrates, with a decrease in pyruvate capacity as well as glutamine dependency. Analyses of glycolytic function showed a substantial decrease in glycolytic acidification caused by lactic acid export. This inhibition of glycolytic parameters was also observed after titrating the MB dose as low as 6 µM, and appears to be dependent on the dithiocarbamate functional group, with manganese possibly potentiating the effect. Further studies into cellular ATP and NAD levels revealed a drastic decrease in cells treated with MB. In summary, MB significantly impacted both aerobic and anaerobic energy production; therefore, further characterization of MB's effect on cellular energetics may provide insight into the specificity of PD to dopaminergic neurons.

Taurine treatment prevents derangement of the hepatic γ-glutamyl cycle and methylglyoxal metabolism in a mouse model of classical homocystinuria: regulatory crosstalk between thiol and sulfinic acid metabolism.

Cystathionine ß-synthase-deficient homocystinuria (HCU) is a poorly understood, life-threatening inborn error of sulfur metabolism. Analysis of hepatic glutathione (GSH) metabolism in a mouse model of HCU demonstrated significant depletion of cysteine, GSH, and GSH disulfide independent of the block in trans-sulfuration compared with wild-type controls. HCU induced the expression of the catalytic and regulatory subunits of γ-glutamyl ligase, GSH synthase (GS), γ-glutamyl transpeptidase 1, 5-oxoprolinase (OPLAH), and the GSH-dependent methylglyoxal detoxification enzyme, glyoxalase-1. Multiple components of the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated antioxidant-response regulatory axis were induced without any detectable activation of Nrf2. Metabolomic analysis revealed the accumulation of multiple γ-glutamyl amino acids and that plasma ophthalmate levels could serve as a noninvasive marker for hepatic redox stress. Neither cysteine, nor betaine treatment was able to reverse the observed enzyme inductions. Taurine treatment normalized the expression levels of γ-glutamyl ligase C/M, GS, OPLAH, and glyoxalase-1, and reversed HCU-induced deficits in protein glutathionylation by acting to double GSH levels relative to controls. Collectively, our data indicate that the perturbation of the γ-glutamyl cycle could contribute to multiple sequelae in HCU and that taurine has significant therapeutic potential for both HCU and other diseases for which GSH depletion is a critical pathogenic factor.-Maclean, K. N., Jiang, H., Aivazidis, S., Kim, E., Shearn, C. T., Harris, P. S., Petersen, D. R., Allen, R. H., Stabler, S. P., Roede, J. R. Taurine treatment prevents derangement of the hepatic γ-glutamyl cycle and methylglyoxal metabolism in a mouse model of classical homocystinuria: regulatory crosstalk between thiol and sulfinic acid metabolism.

The burden of trisomy 21 disrupts the proteostasis network in Down syndrome.

Down syndrome (DS) is a genetic disorder caused by trisomy of chromosome 21. Abnormalities in chromosome number have the potential to lead to disruption of the proteostasis network (PN) and accumulation of misfolded proteins. DS individuals suffer from several comorbidities, and we hypothesized that disruption of proteostasis could contribute to the observed pathology and decreased cell viability in DS. Our results confirm the presence of a disrupted PN in DS, as several of its elements, including the unfolded protein response, chaperone system, and proteasomal degradation exhibited significant alterations compared to euploid controls in both cell and mouse models. Additionally, when cell models were treated with compounds that promote disrupted proteostasis, we observed diminished levels of cell viability in DS compared to controls. Collectively our findings provide a cellular-level characterization of PN dysfunction in DS and an improved understanding of the potential pathogenic mechanisms contributing to disrupted cellular physiology in DS. Lastly, this study highlights the future potential of designing therapeutic strategies that mitigate protein quality control dysfunction.

Antioxidant effects of whey protein on muscle C2C12 cells.

In the present study, the in vitro scavenging activity of sheep whey protein against free radicals, as well as its reducing power were determined and compared with that of beef protein, soy protein and cow whey protein. Moreover, the possible protective effects of sheep whey protein from tert-butyl hydroperoxide (tBHP)-induced oxidative stress in muscle C2C12 cells were determined by assessing oxidative stress markers by flow cytometry and spectrophotometry. The results showed that sheep whey protein scavenged DPPH, ABTS(+) and OH radicals with IC50 values of 3.1, 4.1 and 1.8 mg of protein/ml. Moreover, the reducing power activity assessed with potassium ferricyanide of sheep whey protein was 1.3mg/ml. As regards to the antioxidant effects in muscle cell line, sheep whey protein at 0.78, 1.56, 3.12 and 6.24 mg of protein/ml increased GSH levels up to 138%, lowered TBARS levels up to 25% and decreased ROS levels up to 41.4%.