Pathologic accumulation of ␣-synuclein is a feature of human parkinsonism and other neurodegenerative diseases. This accumulation may be counteracted by mechanisms of protein degradation that have been investigated in vitro but remain to be elucidated in animal models. In this study, lysosomal clearance of ␣-synuclein in vivo was indicated by the detection of ␣-synuclein in the lumen of lysosomes isolated from the mouse midbrain. When neuronal ␣-synuclein expression was enhanced as a result of toxic injury (i.e. treatment of mice with the herbicide paraquat) or transgenic protein overexpression, the intralysosomal content of ␣-synuclein was also significantly increased. This effect was paralleled by a marked elevation of the lysosome-associated membrane protein type 2A (LAMP-2A) and the lysosomal heat shock cognate protein of 70 kDa (hsc70), two essential components of chaperonemediated autophagy (CMA). Immunofluorescence microscopy revealed an increase in punctate (lysosomal) LAMP-2A staining that co-localized with ␣-synuclein within nigral dopaminergic neurons of paraquat-treated and ␣-synuclein-overexpressing animals. The data provide in vivo evidence of lysosomal degradation of ␣-synuclein under normal conditions and, quite importantly, under conditions of enhanced protein burden. In the latter, increased lysosomal clearance of ␣-synuclein was mediated, at least in part, by CMA induction. It is conceivable that these neuronal mechanisms of protein clearance play an important role in neurodegenerative processes characterized by abnormal ␣-synuclein buildup.Several lines of clinical and experimental evidence support a pathogenetic role of ␣-synuclein in Parkinson disease (PD) 2 and other neurodegenerative disorders. The precise mechanisms by which this endogenously expressed protein becomes involved in pathologic processes have yet to be fully elucidated. However, data from both clinical and laboratory studies indicate that enhanced ␣-synuclein expression is itself capable of triggering a parkinsonian syndrome in humans and PD-like pathology in animal models. Indeed, multiplication mutations of the ␣-synuclein gene that result in increased expression of the wild-type protein are causally associated with autosomal dominant parkinsonism (1, 2). From the experimental standpoint, evidence of a gain of toxic function of elevated ␣-synuclein includes the observation of neurodegeneration and neuronal inclusions in the substantia nigra of rats and monkeys that overexpress ␣-synuclein after viral-mediated neuronal transduction (3, 4).An important corollary to the concept that enhanced ␣-synuclein may have deleterious consequences is that intraneuronal mechanisms of protein homeostasis, such as degradation pathways, could well play a key role in maintaining "non-toxic" levels of ␣-synuclein. Although ␣-synuclein clearance is likely to occur through different mechanisms, recent reports have underscored the important contribution of lysosomal pathways of protein degradation and, in particular, chaperone-mediated autopha...

Parkinson’s disease associated mutations in leucine rich repeat kinase 2 (LRRK2) impair mitochondrial function and increase the vulnerability of induced pluripotent stem cell (iPSC)-derived neural cells from patients to oxidative stress. Since mitochondrial DNA (mtDNA) damage can compromise mitochondrial function, we examined whether LRRK2 mutations can induce damage to the mitochondrial genome. We found greater levels of mtDNA damage in iPSC-derived neural cells from patients carrying homozygous or heterozygous LRRK2 G2019S mutations, or at-risk individuals carrying the heterozygous LRRK2 R1441C mutation, than in cells from unrelated healthy subjects who do not carry LRRK2 mutations. After zinc finger nuclease-mediated repair of the LRRK2 G2019S mutation in iPSCs, mtDNA damage was no longer detected in differentiated neuroprogenitor and neural cells. Our results unambiguously link LRRK2 mutations to mtDNA damage and validate a new cellular phenotype that can be used for examining pathogenic mechanisms and screening therapeutic strategies.

The protein α-synuclein is involved in the pathogenesis of Parkinson's disease and other neurodegenerative disorders. Its toxic potential appears to be enhanced by increased protein expression, providing a compelling rationale for therapeutic strategies aimed at reducing neuronal α-synuclein burden. Here, feasibility and safety of α-synuclein suppression were evaluated by treating monkeys with small interfering RNA (siRNA) directed against α-synuclein. The siRNA molecule was chemically modified to prevent degradation by exo- and endonucleases and directly infused into the left substantia nigra. Results compared levels of α-synuclein mRNA and protein in the infused (left) vs. untreated (right) hemisphere and revealed a significant 40–50% suppression of α-synuclein expression. These findings could not be attributable to non-specific effects of siRNA infusion since treatment of a separate set of animals with luciferase-targeting siRNA produced no changes in α-synuclein. Infusion with α-synuclein siRNA, while lowering α-synuclein expression, had no overt adverse consequences. In particular, it did not cause tissue inflammation and did not change (i) the number and phenotype of nigral dopaminergic neurons, and (ii) the concentrations of striatal dopamine and its metabolites. The data represent the first evidence of successful anti-α-synuclein intervention in the primate substantia nigra and support further development of RNA interference-based therapeutics.

Parkinson disease (PD) is a multi-factorial neurodegenerative disorder with loss of dopaminergic neurons in the substantia nigra and characteristic intracellular inclusions, called Lewy bodies. Genetic predisposition, such as point mutations and copy number variants of the SNCA gene locus can cause very similar PD-like neurodegeneration. The impact of altered α-synuclein protein expression on integrity and developmental potential of neuronal stem cells is largely unexplored, but may have wide ranging implications for PD manifestation and disease progression. Here, we investigated if induced pluripotent stem cell-derived neuronal precursor cells (NPCs) from a patient with Parkinson’s disease carrying a genomic triplication of the SNCA gene (SNCA-Tri). Our goal was to determine if these cells these neuronal precursor cells already display pathological changes and impaired cellular function that would likely predispose them when differentiated to neurodegeneration. To achieve this aim, we assessed viability and cellular physiology in human SNCA-Tri NPCs both under normal and environmentally stressed conditions to model in vitro gene-environment interactions which may play a role in the initiation and progression of PD. Human SNCA-Tri NPCs displayed overall normal cellular and mitochondrial morphology, but showed substantial changes in growth, viability, cellular energy metabolism and stress resistance especially when challenged by starvation or toxicant challenge. Knockdown of α-synuclein in the SNCA-Tri NPCs by stably expressed short hairpin RNA (shRNA) resulted in reversal of the observed phenotypic changes. These data show for the first time that genetic alterations such as the SNCA gene triplication set the stage for decreased developmental fitness, accelerated aging, and increased neuronal cell loss. The observation of this “stem cell pathology” could have a great impact on both quality and quantity of neuronal networks and could provide a powerful new tool for development of neuroprotective strategies for PD.

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