Reactive oxygen species production by forward and reverse electron fluxes in the mitochondrial respiratory chain.

Reactive oxygen species (ROS) produced in the mitochondrial respiratory chain (RC) are primary signals that modulate cellular adaptation to environment, and are also destructive factors that damage cells under the conditions of hypoxia/reoxygenation relevant for various systemic diseases or transplantation. The important role of ROS in cell survival requires detailed investigation of mechanism and determinants of ROS production. To perform such an investigation we extended our rule-based model of complex III in order to account for electron transport in the whole RC coupled to proton translocation, transmembrane electrochemical potential generation, TCA cycle reactions, and substrate transport to mitochondria. It fits respiratory electron fluxes measured in rat brain mitochondria fueled by succinate or pyruvate and malate, and the dynamics of NAD(+) reduction by reverse electron transport from succinate through complex I. The fitting of measured characteristics gave an insight into the mechanism of underlying processes governing the formation of free radicals that can transfer an unpaired electron to oxygen-producing superoxide and thus can initiate the generation of ROS. Our analysis revealed an association of ROS production with levels of specific radicals of individual electron transporters and their combinations in species of complexes I and III. It was found that the phenomenon of bistability, revealed previously as a property of complex III, remains valid for the whole RC. The conditions for switching to a state with a high content of free radicals in complex III were predicted based on theoretical analysis and were confirmed experimentally. These findings provide a new insight into the mechanisms of ROS production in RC.

Postmortem Neocortical 3H-PiB Binding and Levels of Unmodified and Pyroglutamate Aß in Down Syndrome and Sporadic Alzheimer's Disease.

Individuals with Down syndrome (DS) have a genetic predisposition for amyloid-ß (Aß) overproduction and earlier onset of Aß deposits compared to patients with sporadic late-onset Alzheimer's disease (AD). Positron emission tomography (PET) with Pittsburgh Compound-B (PiB) detects fibrillar Aß pathology in living people with DS and AD, but its relationship with heterogeneous Aß forms aggregated within amyloid deposits is not well understood. We performed quantitative in vitro 3H-PiB binding assays and enzyme-linked immunosorbent assays of fibrillar (insoluble) unmodified Aß40 and Aß42 forms and N-terminus truncated and pyroglutamate-modified AßNpE3-40 and AßNpE3-42 forms in postmortem frontal cortex and precuneus samples from 18 DS cases aged 43-63 years and 17 late-onset AD cases aged 62-99 years. Both diagnostic groups had frequent neocortical neuritic plaques, while the DS group had more severe vascular amyloid pathology (cerebral amyloid angiopathy, CAA). Compared to the AD group, the DS group had higher levels of Aß40 and AßNpE3-40, while the two groups did not differ by Aß42 and AßNpE3-42 levels. This resulted in lower ratios of Aß42/Aß40 and AßNpE3-42/AßNpE3-40 in the DS group compared to the AD group. Correlations of Aß42/Aß40 and AßNpE3-42/AßNpE3-40 ratios with CAA severity were strong in DS cases and weak in AD cases. Pyroglutamate-modified Aß levels were lower than unmodified Aß levels in both diagnostic groups, but within group proportions of both pyroglutamate-modified Aß forms relative to both unmodified Aß forms were lower in the DS group but not in the AD group. The two diagnostic groups did not differ by 3H-PiB binding levels. These results demonstrate that compared to late-onset AD cases, adult DS individuals with similar severity of neocortical neuritic plaques and greater CAA pathology have a preponderance of both pyroglutamate-modified AßNpE3-40 and unmodified Aß40 forms. Despite the distinct molecular profile of Aß forms and greater vascular amyloidosis in DS cases, cortical 3H-PiB binding does not distinguish between diagnostic groups that are at an advanced level of amyloid plaque pathology. This underscores the need for the development of CAA-selective PET radiopharmaceuticals to detect and track the progression of cerebral vascular amyloid deposits in relation to Aß plaques in individuals with DS.

Low-dose bafilomycin attenuates neuronal cell death associated with autophagy-lysosome pathway dysfunction.

We have shown previously that the plecomacrolide antibiotics bafilomycin A1 and B1 significantly attenuate cerebellar granule neuron death resulting from agents that disrupt lysosome function. To further characterize bafilomycin-mediated cytoprotection, we examined its ability to attenuate the death of naïve and differentiated neuronal SH-SY5Y human neuroblastoma cells from agents that induce lysosome dysfunction in vitro, and from in vivo dopaminergic neuron death in C. elegans. Low-dose bafilomycin significantly attenuated SH-SY5Y cell death resulting from treatment with chloroquine, hydroxychloroquine amodiaquine and staurosporine. Bafilomycin also attenuated the chloroquine-induced reduction in processing of cathepsin D, the principal lysosomal aspartic acid protease, to its mature 'active' form. Chloroquine induced autophagic vacuole accumulation and inhibited autophagic flux, effects that were attenuated upon treatment with bafilomycin and were associated with a significant decrease in chloroquine-induced accumulation of detergent-insoluble alpha-synuclein oligomers. In addition, bafilomycin significantly and dose-dependently attenuated dopaminergic neuron death in C. elegans resulting from in vivo over-expression of human wild-type alpha-synuclein. Together, our findings suggest that low-dose bafilomycin is cytoprotective in part through its maintenance of the autophagy-lysosome pathway, and underscores its therapeutic potential for treating Parkinson's disease and other neurodegenerative diseases that exhibit disruption of protein degradation pathways and accumulation of toxic protein species.

Cortical pyroglutamate amyloid-ß levels and cognitive decline in Alzheimer's disease.

Posterior cingulate cortex (PCC) accumulates amyloid-ß (Aß) early in Alzheimer's disease (AD). The relative concentrations of full-length Aß and truncated, pyroglutamate-modified Aß (NpE3) forms, and their correlations to cognitive dysfunction in AD, are unknown. We quantified AßNpE3-42, AßNpE3-40, Aß1-42, and Aß1-40 concentrations in soluble (nonfibrillar) and insoluble (fibrillar) pools in PCC from subjects with an antemortem clinical diagnosis of no cognitive impairment, mild cognitive impairment, or mild-moderate AD. In clinical AD, increased PCC concentrations of Aß were observed for all Aß forms in the insoluble pool but only for Aß1-42 in the soluble pool. Lower Mini-Mental State Exam and episodic memory scores correlated most strongly with higher concentrations of soluble and insoluble Aß1-42. Greater neuropathology severity by Consortium to Establish a Registry for Alzheimer's Disease and National Institute on Aging-Reagan pathologic criteria was associated with higher concentrations of all measured Aß forms, except soluble AßNpE3-40. Low concentrations of soluble pyroglutamate Aß across clinical groups likely reflect its rapid sequestration into plaques, thus, the conversion to fibrillar Aß may be a therapeutic target.

Rotenone inhibits autophagic flux prior to inducing cell death.

Rotenone, which selectively inhibits mitochondrial complex I, induces oxidative stress, α-synuclein accumulation, and dopaminergic neuron death, principal pathological features of Parkinson's disease. The autophagy-lysosome pathway degrades damaged proteins and organelles for the intracellular maintenance of nutrient and energy balance. While it is known that rotenone causes autophagic vacuole accumulation, the mechanism by which this effect occurs has not been thoroughly investigated. Treatment of differentiated SH-SY5Y cells with rotenone (10 µM) induced the accumulation of autophagic vacuoles at 6 h and 24 h as indicated by Western blot analysis for microtubule associated protein-light chain 3-II (MAP-LC3-II). Assessment of autophagic flux at these time points indicated that autophagic vacuole accumulation resulted from a decrease in their effective lysosomal degradation, which was substantiated by increased levels of autophagy substrates p62 and α-synuclein. Inhibition of lysosomal degradation may be explained by the observed decrease in cellular ATP levels, which in turn may have caused the observed concomitant increase in acidic vesicle pH. The early (6 h) effects of rotenone on cellular energetics and autophagy-lysosome pathway function preceded the induction of cell death and apoptosis. These findings indicate that the classical mitochondrial toxin rotenone has a pronounced effect on macroautophagy completion that may contribute to its neurotoxic potential.

Oxidative stress and autophagy in the regulation of lysosome-dependent neuron death.

Lysosomes critically regulate the pH-dependent catabolism of extracellular and intracellular macromolecules delivered from the endocytic/heterophagy and autophagy pathways, respectively. The importance of lysosomes to cell survival is underscored not only by their unique ability effectively to degrade metalloproteins and oxidatively damaged macromolecules, but also by the distinct potential for induction of both caspase-dependent and -independent cell death with a compromise in the integrity of lysosome function. Oxidative stress and free radical damage play a principal role in cell death induced by lysosome dysfunction and may be linked to several upstream and downstream stimuli, including alterations in the autophagy degradation pathway, inhibition of lysosome enzyme function, and lysosome membrane damage. Neurons are sensitive to lysosome dysfunction, and the contribution of oxidative stress and free radical damage to lysosome dysfunction may contribute to the etiology of neurodegenerative disease. This review provides a broad overview of lysosome function and explores the contribution of oxidative stress and autophagy to lysosome dysfunction-induced neuron death. Putative signaling pathways that either induce lysosome dysfunction or result from lysosome dysfunction or both, and the role of oxidative stress, free radical damage, and lysosome dysfunction in pediatric lysosomal storage disorders (neuronal ceroid lipofuscinoses or NCL/Batten disease) and in Alzheimer's disease are emphasized.