Copper nanoparticles exert size and concentration dependent toxicity on somatosensory neurons of rat.

Metal nanoparticles, due to their unique properties and important applications in optical, magnetic, thermal, electrical, sensor devices and cosmetics, are beginning to be widely manufactured and used. This new and rapidly growing field of technology warrants a thorough examination of the material's bio-compatibility and safety. Ultra-small particles may adversely affect living cells and organisms since they can easily penetrate the body through skin contact, inhalation and ingestion. Retrograde transport of copper nanoparticles from nerve endings on the skin can reach the somatosensory neurons in dorsal root ganglion (DRG). Since copper nanoparticles have industrial and healthcare applications, we determined the concentration and size-dependant effects of their exposure on survival of DRG neurons of rat in cell culture. The neurons were exposed to copper nanoparticles of increasing concentrations (10-100 muM) and sizes (40, 60 and 80 nm) for 24 h. Light microscopy, histochemical staining for copper, lactate dehydrogenase (LDH) assay for cell death, and MTS [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay for cell viability were performed to measure the resultant toxicity and cell survival. DRG neurons exposed to copper nanoparticles displayed vacuoles and detachment of some neurons from the substratum. Neurons also exhibited disrupted neurite network. LDH and MTS assays revealed that exposure to copper nanoparticles had significant toxic effect with all the sizes tested when compared to unexposed control cultures. Further analysis of the results showed that copper nanoparticles of smaller size and higher concentration exerted the maximum toxic effects. Rubeanic acid staining showed intracellular deposition of copper. These results demonstrate that copper nanoparticles are toxic in a size- and concentration-dependent manner to DRG neurons.

Mitochondrial import and accumulation of alpha-synuclein impair complex I in human dopaminergic neuronal cultures and Parkinson disease brain.

Alpha-synuclein, a protein implicated in the pathogenesis of Parkinson disease (PD), is thought to affect mitochondrial functions, although the mechanisms of its action remain unclear. In this study we show that the N-terminal 32 amino acids of human alpha-synuclein contain cryptic mitochondrial targeting signal, which is important for mitochondrial targeting of alpha-synuclein. Mitochondrial imported alpha-synuclein is predominantly associated with the inner membrane. Accumulation of wild-type alpha-synuclein in the mitochondria of human dopaminergic neurons caused reduced mitochondrial complex I activity and increased production of reactive oxygen species. However, these defects occurred at an early time point in dopaminergic neurons expressing familial alpha-synuclein with A53T mutation as compared with wild-type alpha-synuclein. Importantly, alpha-synuclein that lacks mitochondrial targeting signal failed to target to the mitochondria and showed no detectable effect on complex I function. The PD relevance of these results was investigated using mitochondria of substantia nigra, striatum, and cerebellum of postmortem late-onset PD and normal human brains. Results showed the constitutive presence of approximately 14-kDa alpha-synuclein in the mitochondria of all three brain regions of normal subjects. Mitochondria of PD-vulnerable substantia nigra and striatum but not cerebellum from PD subjects showed significant accumulation of alpha-synuclein and decreased complex I activity. Analysis of mitochondria from PD brain and alpha-synuclein expressing dopaminergic neuronal cultures using blue native gel electrophoresis and immunocapture technique showed the association of alpha-synuclein with complex I. These results provide evidence that mitochondrial accumulated alpha-synuclein may interact with complex I and interfere with its functions.

Accumulation of amyloid precursor protein in the mitochondrial import channels of human Alzheimer's disease brain is associated with mitochondrial dysfunction.

Mitochondrial dysfunction is one of the major intracellular lesions of Alzheimer's disease (AD). However, the causative factors involved in the mitochondrial dysfunction in human AD are not well understood. Here we report that nonglycosylated full-length and C-terminal truncated amyloid precursor protein (APP) accumulates exclusively in the protein import channels of mitochondria of human AD brains but not in age-matched controls. Furthermore, in AD brains, mitochondrially associated APP formed stable approximately 480 kDa complexes with the translocase of the outer mitochondrial membrane 40 (TOM40) import channel and a super complex of approximately 620 kDa with both mitochondrial TOM40 and the translocase of the inner mitochondrial membrane 23 (TIM23) import channel TIM23 in an "N(in mitochondria)-C(out cytoplasm)" orientation. Accumulation of APP across mitochondrial import channels, which varied with the severity of AD, inhibited the entry of nuclear-encoded cytochrome c oxidase subunits IV and Vb proteins, which was associated with decreased cytochrome c oxidase activity and increased levels of H2O2. Regional distribution of mitochondrial APP showed higher levels in AD-vulnerable brain regions, such as the frontal cortex, hippocampus, and amygdala. Mitochondrial accumulation of APP was also observed in the cholinergic, dopaminergic, GABAergic, and glutamatergic neuronal types in the category III AD brains. The levels of translocationally arrested mitochondrial APP directly correlated with mitochondrial dysfunction. Moreover, apolipoprotein genotype analysis revealed that AD subjects with the E3/E4 alleles had the highest content of mitochondrial APP. Collectively, these results suggest that abnormal accumulation of APP across mitochondrial import channels, causing mitochondrial dysfunction, is a hallmark of human AD pathology.