Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and PARK2/Parkin mutations cause autosomal recessive forms of Parkinson's disease. Upon a loss of mitochondrial membrane potential (ΔΨ m ) in human cells, cytosolic Parkin has been reported to be recruited to mitochondria, which is followed by a stimulation of mitochondrial autophagy. Here, we show that the relocation of Parkin to mitochondria induced by a collapse of ΔΨ m relies on PINK1 expression and that overexpression of WT but not of mutated PINK1 causes Parkin translocation to mitochondria, even in cells with normal ΔΨ m . We also show that once at the mitochondria, Parkin is in close proximity to PINK1, but we find no evidence that Parkin catalyzes PINK1 ubiquitination or that PINK1 phosphorylates Parkin. However, co-overexpression of Parkin and PINK1 collapses the normal tubular mitochondrial network into mitochondrial aggregates and/or large perinuclear clusters, many of which are surrounded by autophagic vacuoles. Our results suggest that Parkin, together with PINK1, modulates mitochondrial trafficking, especially to the perinuclear region, a subcellular area associated with autophagy. Thus by impairing this process, mutations in either Parkin or PINK1 may alter mitochondrial turnover which, in turn, may cause the accumulation of defective mitochondria and, ultimately, neurodegeneration in Parkinson's disease.autophagy | Parkinson's disease | phosphatase and tensin homolog-induced putative kinase 1 T he common neurodegenerative disorder Parkinson's disease (PD) occasionally can be inherited (1, 2). Parkinson disease 6/ phosphatase and tensin homolog (PTEN)-induced putative kinase-1 (PARK6/PINK1) is among the gene products associated with familial PD (2, 3). This 581-amino acid polypeptide is localized to the mitochondria and has only a single recognized functional domain, a serine/threonine kinase with a high degree of homology to that of the Ca 2+ /calmodulin kinase family. Overexpression of WT PINK1 rescues abnormal mitochondrial morphology that has been described in Drosophila carrying Pink1 mutations (4, 5), a finding that supports the notion that the mutated allele gives rise to a loss-of-function phenotype. Loss-offunction mutations in the gene encoding PARK2/Parkin (an E3 ubiquitin ligase) also can cause an autosomal recessive form of familial PD (2, 6). Parkin is thought to operate within the same molecular pathway as PINK1 to modulate mitochondrial dynamics (4, 5, 7). This possibility is intriguing, because Parkin has been reported to be essentially cytosolic (8, 9). However, we have shown that PINK1 spans the outer mitochondrial membrane, with its kinase domain facing the cytoplasm (10). These details of PINK1 topology are relevant to the reported Parkin/PINK1 genetic interaction because they place the only known functional domain of PINK1 in the same subcellular compartment as Parkin.However, the role played by Parkin, PINK1, or both in mitochondrial dynamics is still uncertain. Perhaps, the beginning of an answer to th...

Familial Parkinson disease is associated with mutations in ␣-synuclein (␣-syn), a presynaptic protein that has been localized not only to the cytosol, but also to mitochondria. We report here that wild-type ␣-syn from cell lines, and brain tissue from humans and mice, is present not in mitochondria but rather in mitochondria-associated endoplasmic reticulum (ER) membranes (MAM), a structurally and functionally distinct subdomain of the ER. Remarkably, we found that pathogenic point mutations in human ␣-syn result in its reduced association with MAM, coincident with a lower degree of apposition of ER with mitochondria, a decrease in MAM function, and an increase in mitochondrial fragmentation compared with wild-type. Although overexpression of wild-type ␣-syn in mutant ␣-synexpressing cells reverted the fragmentation phenotype, neither overexpression of the mitochondrial fusion/MAM-tethering protein MFN2 nor inhibition/ablation of the mitochondrial fission protein DRP1 was able to do so, implying that ␣-syn operates downstream of the mitochondrial fusion/fission machinery. These novel results indicate that wild-type ␣-syn localizes to the MAM and modulates mitochondrial morphology, and that these behaviors are impaired by pathogenic mutations in ␣-syn. We believe that our results have far-reaching implications for both our understanding of ␣-syn biology and the treatment of synucleinopathies.

Neuronal mitochondrial morphology abnormalities occur in models of familial amyotrophic lateral sclerosis (ALS) associated with SOD1 and TDP43 mutations. These abnormalities have been linked to mitochondrial axonal transport defects, but the temporal and spatial relationship between mitochondrial morphology and transport alterations in these two distinct genetic forms of ALS has not been investigated in vivo. To address this question, we crossed SOD1 (wild-type SOD1(WT) and mutant SOD1(G93A)) or TDP43 (mutant TDP43(A315T)) transgenic mice with mice expressing the fluorescent protein Dendra targeted to mitochondria in neurons (mitoDendra). At different time points during the disease course, we studied mitochondrial transport in the intact sciatic nerve of living mice and analyzed axonal mitochondrial morphology at multiple sites, spanning from the spinal cord to the motor terminals. Defects of retrograde mitochondrial transport were detected at 45 days of age, before the onset of symptoms, in SOD1(G93A) and TDP43(A315T) mice, but not in SOD1(WT). At later disease stages, also anterograde mitochondrial transport was affected in both mutant mouse lines. In SOD1(G93A) mice, mitochondrial morphological abnormalities were apparent at 15 days of age, thus preceding transport abnormalities. Conversely, in TDP43(A315T) mice, morphological abnormalities appeared after the onset of transport defects. Taken together, these findings demonstrate that neuronal mitochondrial transport and morphology abnormalities occur in vivo and that they are common denominators of different genetic forms of the ALS. At the same time, differences in the temporal and spatial manifestation of mitochondrial abnormalities between the two mouse models of familial ALS imply that different molecular mechanisms may be involved.

Intracellular ␤-amyloid 42 (A␤42) accumulation is increasingly recognized as an early event in the pathogenesis of Alzheimer's disease (AD). We have developed a doxycycline-inducible adenoviral-based system that directs intracellular A␤42 expression and accumulation into the endoplasmic reticulum of primary neuronal cultures in a regulated manner. A␤42 exhibited a perinuclear distribution in cell bodies and an association with vesicular compartments. Virally expressed intracellular A␤42 was toxic to neuronal cultures 24 hr after induction in a dose-dependent manner. A␤42 expression prompted the rapid induction of stress-inducible Hsp70 protein in neurons, and virally mediated Hsp70 overexpression rescued neurons from the toxic effects of intracellular A␤ accumulation. Together, these results implicate the cellular stress response as a possible modulator of A␤-induced toxicity in neuronal cultures.