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At present there is a significant unmet need for clinically available treatments for Parkinson’s disease (PD) patients to stably restore balance to dopamine network function, leaving patients with inadequate management of symptoms as the disease progresses. Gene therapy is an attractive approach to impart a durable effect on neuronal function through introduction of genetic material to reestablish dopamine levels and/or functionally recover dopaminergic signaling by improving neuronal health. Ongoing clinical gene therapy trials in PD are focused on enzymatic enhancement of dopamine production and/or the restoration of the nigrostriatal pathway to improve dopaminergic network function. In this review, we discuss data from current gene therapy trials for PD and recent advances in study design and surgical approaches.

Convergent evidence implicates impaired mitochondrial function and α-Synuclein accumulation as critical upstream events in the pathogenesis of Parkinson's disease, but comparatively little is known about how these factors interact to provoke neurodegeneration. We previously showed that α-Synuclein knockdown protected rat substantia nigra dopaminergic neurons from systemic exposure to the mitochondrial complex I inhibitor rotenone. Here we show that motor abnormalities prior to neuronal loss in this model are associated with extensive α-Synuclein-dependent cellular thiol oxidation. In order to elucidate the underlying events in vivo we constructed novel transgenic zebrafish that co-express, in dopaminergic neurons: (i) human α-Synuclein at levels insufficient to provoke neurodegeneration or neurobehavioral abnormalities; and (ii) genetically-encoded ratiometric fluorescent biosensors to detect cytoplasmic peroxide flux and glutathione oxidation. Live intravital imaging of the intact zebrafish CNS at cellular resolution showed unequivocally that α-Synuclein amplified dynamic cytoplasmic peroxide flux in dopaminergic neurons following exposure to the mitochondrial complex I inhibitors MPP+ or rotenone. This effect was robust and clearly evident following either acute or prolonged exposure to each inhibitor. In addition, disturbance of the resting glutathione redox potential following exogenous hydrogen peroxide challenge was augmented by α-Synuclein. Together these data show that α-Synuclein is a critical determinant of the redox consequences of mitochondrial dysfunction in dopaminergic neurons. The findings are important because the mechanisms underlying α-Synuclein-dependent reactive oxygen species fluxes and antioxidant suppression might provide a pharmacological target in Parkinson's disease to prevent progression from mitochondrial dysfunction and oxidative stress to cell death., Graphical abstract Image 1, Highlights • Extensive neuronal thiol oxidation in a rat PD model is α-Synuclein-dependent. • Peroxide flux and glutathione oxidation can be imaged in live transgenic zebrafish. • α-Synuclein amplifies cytosolic peroxide flux in dopaminergic neurons. • α-Synuclein exacerbates dynamic disturbances of the glutathione redox potential. • The underlying molecular mechanisms may provide therapeutic targets in PD.

In the study of neurodegenerative diseases, it is imperative to study the cellular and molecular changes associated with pathogenesis in the relevant cell type, central nervous system neurons. The unique compartmentalized morphology and bioenergetic needs of primary neurons present complications for their study in culture. Recent microculture techniques utilizing microfluidic culture devices allows for environmental separation and analysis of neuronal cell bodies and neurites in culture. Here, we present our protocol for culture of primary neurons in microfluidic devices and their chronic treatment with the Parkinson’s disease (PD) relevant toxicant rotenone. In addition, we present a method for reuse of devices for culture. This culture methodology presents advantages for evaluating early pathogenic cellular and molecular changes in neurons in a compartment-specific manner.

Mitochondrial dysfunction has been implicated in Parkinson's disease (PD) neuropathology. Mic60, also known as mitofilin, is a protein of the inner mitochondrial membrane and a key component of the mitochondrial contact site and cristae junction organizing system (MICOS). Mic60 is critical for maintaining mitochondrial membrane structure and function. We previously demonstrated that mitochondrial Mic60 protein is susceptible to both covalent modification and loss in abundance following exposure to dopamine quinone. In this study, we utilized neuronally-differentiated SH-SY5Y and PC12 dopaminergic cell lines to examine the effects of altered Mic60 levels on mitochondrial function and cellular vulnerability in response to PD-relevant stressors. Short hairpin RNA (shRNA)-mediated knockdown of endogenous Mic60 protein in neuronal SH-SY5Y cells significantly potentiated dopamine-induced cell death, which was rescued by co-expressing shRNA-insensitive Mic60. Conversely, in PC12 and SH-SY5Y cells, Mic60 overexpression significantly attenuated both dopamine- and rotenone-induced cell death as compared to controls. Mic60 overexpression in SH-SY5Y cells was also associated with increased mitochondrial respiration, and, following rotenone exposure, increased spare respiratory capacity. Mic60 knockdown cells exhibited suppressed respiration and, following rotenone treatment, decreased spare respiratory capacity. Mic60 overexpression also affected mitochondrial fission/fusion dynamics. PC12 cells overexpressing Mic60 exhibited increased mitochondrial interconnectivity. Further, both PC12 cells and primary rat cortical neurons overexpressing Mic60 displayed suppressed mitochondrial fission and increased mitochondrial length in neurites. These results suggest that altering levels of Mic60 in dopaminergic neuronal cells significantly affects both mitochondrial homeostasis and cellular vulnerability to the PD-relevant stressors dopamine and rotenone, carrying implications for PD pathogenesis.

Dysregulation of mitochondrial biogenesis is implicated in the pathogenesis of neurodegenerative diseases such as Parkinson's disease (PD). However, it is not clear how mitochondrial biogenesis is regulated in neurons, with their unique compartmentalized anatomy and energetic demands. This is particularly relevant in PD because selectively vulnerable neurons feature long, highly arborized axons where degeneration initiates. We previously found that exposure of neurons to chronic, sublethal doses of rotenone, a complex I inhibitor linked to PD, causes early increases in mitochondrial density specifically in distal axons, suggesting possible upregulation of mitochondrial biogenesis within axons. Here, we directly evaluated for evidence of mitochondrial biogenesis in distal axons and examined whether PD-relevant stress causes compartmentalized alterations. Using BrdU labeling and imaging to quantify replicating mitochondrial DNA (mtDNA) in primary rat neurons (pooled from both sexes), we provide evidence of mtDNA replication in axons along with cell bodies and proximal dendrites. We found that exposure to chronic, sublethal rotenone increases mtDNA replication first in neurites and later extending to cell bodies, complementing our mitochondrial density data. Further, isolating axons from cell bodies and dendrites, we discovered that rotenone exposure upregulates mtDNA replication in distal axons. Utilizing superresolution stimulated emission depletion (STED) imaging, we identified mtDNA replication at sites of mitochondrial–endoplasmic reticulum contacts in axons. Our evidence suggests that mitochondrial biogenesis occurs not only in cell bodies, but also in distal axons, and is altered under PD-relevant stress conditions in an anatomically compartmentalized manner. We hypothesize that this contributes to vulnerability in neurodegenerative diseases. SIGNIFICANCE STATEMENT Mitochondrial biogenesis is crucial for maintaining mitochondrial and cellular health and has been linked to neurodegenerative disease pathogenesis. However, regulation of this process is poorly understood in CNS neurons, which rely on mitochondrial function for survival. Our findings offer fundamental insight into these regulatory mechanisms by demonstrating that replication of mitochondrial DNA, an essential precursor for biogenesis, can occur in distal regions of CNS neuron axons independent of the soma. Further, this process is upregulated specifically in axons as an early response to neurodegeneration-relevant stress. This is the first demonstration of the compartmentalized regulation of CNS neuronal mitochondrial biogenesis in response to stress and may prove a useful target in development of therapeutic strategies for neurodegenerative disease.

Extensive convergent evidence collectively suggests that mitochondrial dysfunction is central to the pathogenesis of Parkinson's disease (PD). Recently, changes in the dynamic properties of mitochondria have been increasingly implicated as a key proximate mechanism underlying neurodegeneration. However, studies have been limited by the lack of a model in which mitochondria can be imaged directly and dynamically in dopaminergic neurons of the intact vertebrate CNS. We generated transgenic zebrafish in which mitochondria of dopaminergic neurons are labeled with a fluorescent reporter, and optimized methods allowing direct intravital imaging of CNS dopaminergic axons and measurement of mitochondrial transport in vivo. The proportion of mitochondria undergoing axonal transport in dopaminergic neurons decreased overall during development between 2days post-fertilization (dpf) and 5dpf, at which point the major period of growth and synaptogenesis of the relevant axonal projections is complete. Exposure to 0.5-1.0mM MPP(+) between 4 and 5dpf did not compromise zebrafish viability or cause detectable changes in the number or morphology of dopaminergic neurons, motor function or monoaminergic neurochemistry. However, 0.5mM MPP(+) caused a 300% increase in retrograde mitochondrial transport and a 30% decrease in anterograde transport. In contrast, exposure to higher concentrations of MPP(+) caused an overall reduction in mitochondrial transport. This is the first time mitochondrial transport has been observed directly in CNS dopaminergic neurons of a living vertebrate and quantified in a PD model in vivo. Our findings are compatible with a model in which damage at presynaptic dopaminergic terminals causes an early compensatory increase in retrograde transport of compromised mitochondria for degradation in the cell body. These data are important because manipulation of early pathogenic mechanisms might be a valid therapeutic approach to PD. The novel transgenic lines and methods we developed will be useful for future studies on mitochondrial dynamics in health and disease.

In Parkinson's disease, oxidative stress is implicated in protein misfolding and aggregation, which may activate the unfolded protein response by the endoplasmic reticulum (ER). Dopamine (DA) can initiate oxidative stress via H(2)O(2) formation by DA metabolism and by oxidation into DA quinone. We have previously shown that DA quinone induces oxidative protein modification, mitochondrial dysfunction in vitro, and dopaminergic cell toxicity in vivo and in vitro. In this study, we used cysteine- and lysine-reactive fluorescent dyes with 2D difference in-gel electrophoresis, mass spectrometry, and peptide mass fingerprint analysis to identify proteins in PC12 cell mitochondrial-enriched fractions that were altered in abundance following DA exposure (150 muM, 16 h). Quantitative changes in proteins labeled with fluorescent dyes indicated increases in a subset of proteins after DA exposure: calreticulin, ERp29, ERp99, Grp58, Grp78, Grp94 and Orp150 (149-260%), and decreased levels of aldolase A (39-42%). Changes in levels of several proteins detected by 2D difference in-gel electrophoresis were confirmed by western blot. Using this unbiased proteomics approach, our findings demonstrated that in PC12 cells, DA exposure leads to a cellular response indicative of ER stress prior to the onset of cell death, providing a potential link between DA and the unfolded protein response in the pathogenesis of Parkinson's disease.

Disruption of the dynamic properties of mitochondria (fission, fusion, transport, degradation, and biogenesis) has been implicated in the pathogenesis of neurodegenerative disorders, including Parkinson's disease (PD). Parkin, the product of gene PARK2 whose mutation causes familial PD, has been linked to mitochondrial quality control via its role in regulating mitochondrial dynamics, including mitochondrial degradation via mitophagy. Models using mitochondrial stressors in numerous cell types have elucidated a PINK1-dependent pathway whereby Parkin accumulates on damaged mitochondria and targets them for mitophagy. However, the role Parkin plays in regulating mitochondrial homeostasis specifically in neurons has been less clear. We examined whether a stressor linked to neurodegeneration, glutamate excitotoxicity, elicits Parkin–mitochondrial translocation and mitophagy in neurons. We found that brief, acute exposure to glutamate causes Parkin translocation to mitochondria in neurons, in a calcium- and N-methyl-d-aspartate (NMDA) receptor-dependent manner. In addition, we found that Parkin accumulates on endoplasmic reticulum (ER) and mitochondrial/ER junctions following excitotoxicity, supporting a role for Parkin in mitochondrial–ER crosstalk in mitochondrial homeostasis. Despite significant Parkin–mitochondria translocation, however, we did not observe mitophagy under these conditions. To further investigate, we examined the role of glutamate-induced oxidative stress in Parkin–mitochondria accumulation. Unexpectedly, we found that glutamate-induced accumulation of Parkin on mitochondria was promoted by the antioxidant N-acetyl cysteine (NAC), and that co-treatment with NAC facilitated Parkin-associated mitophagy. These results suggest the possibility that mitochondrial depolarization and oxidative damage may have distinct pathways associated with Parkin function in neurons, which may be critical in understanding the role of Parkin in neurodegeneration.

Recent studies delineate a pathway involving familial Parkinson’s disease (PD)-related proteins PINK1 and Parkin, in which PINK1-dependent mitochondrial accumulation of Parkin targets depolarized mitochondria towards degradation through mitophagy. The pathway has been primarily characterized in cells less dependent on mitochondria for energy production than neurons. Here we report that in neurons, unlike other cells, mitochondrial depolarization by carbonyl cyanide m-chlorophenyl hydrazone did not induce Parkin translocation to mitochondria or mitophagy. PINK1 overexpression increased basal Parkin accumulation on neuronal mitochondria, but did not sensitize them to depolarization-induced Parkin translocation. Our data suggest that bioenergetic differences between neurons and cultured cell lines contribute to these different responses. In HeLa cells utilizing usual glycolytic metabolism, mitochondrial depolarization robustly triggered Parkin–mitochondrial translocation, but this did not occur in HeLa cells forced into dependence on mitochondrial respiration. Declining ATP levels after mitochondrial depolarization correlated with the absence of induced Parkin–mitochondrial translocation in both HeLa cells and neurons. However, intervention allowing neurons to maintain ATP levels after mitochondrial depolarization only modestly increased Parkin recruitment to mitochondria, without evidence of increased mitophagy. These data suggest that changes in ATP levels are not the sole determinant of the different responses between neurons and other cell types, and imply that additional mechanisms regulate mitophagy in neurons. Since the Parkin–m itophagy pathway is heavily dependent on bioenergetic status, the unique metabolic properties of neurons likely influence the function of this pathway in the pathogenesis of PD.

The unique energy demands of neurons require well-orchestrated distribution and maintenance of mitochondria. Thus, dynamic properties of mitochondria, including fission, fusion, trafficking, biogenesis, and degradation, are critical to all cells, but may be particularly important in neurons. Dysfunction in mitochondrial dynamics has been linked to neuropathies and is increasingly being linked to several neurodegenerative diseases, but the evidence is particularly strong, and continuously accumulating, in Parkinson's disease (PD). The unique characteristics of neurons that degenerate in PD may predispose those neuronal populations to susceptibility to alterations in mitochondrial dynamics. In addition, evidence from PD-related toxins supports that mitochondrial fission, fusion, and transport may be involved in pathogenesis. Furthermore, rapidly increasing evidence suggests that two proteins linked to familial forms of the disease, parkin and PINK1, interact in a common pathway to regulate mitochondrial fission/fusion. Parkin may also play a role in maintaining mitochondrial homeostasis through targeting damaged mitochondria for mitophagy. Taken together, the current data suggests that mitochondrial dynamics may play a role in PD pathogenesis, and a better understanding of mitochondrial dynamics within the neuron may lead to future therapeutic treatments for PD, potentially aimed at some of the earliest pathogenic events.

Dopamine oxidation has been previously demonstrated to cause dysfunction in mitochondrial respiration and membrane permeability, possibly related to covalent modification of critical proteins by the reactive dopamine quinone. However, specific mitochondrial protein targets have not been identified. In this study, we utilized proteomic techniques to identify proteins directly conjugated with (14)C-dopamine from isolated rat brain mitochondria exposed to radiolabeled dopamine quinone (150 microM) and differentiated SH-SY5Y cells treated with (14)C-dopamine (150 microM). We observed a subset of rat brain mitochondrial proteins that were covalently modified by (14)C-dopamine, including chaperonin, ubiquinol-cytochrome c reductase core protein 1, glucose regulated protein 75/mitochondrial HSP70/mortalin, mitofilin, and mitochondrial creatine kinase. We also found the Parkinson's disease associated proteins ubiquitin carboxy-terminal hydrolase L1 and DJ-1 to be covalently modified by dopamine in both brain mitochondrial preparations and SH-SY5Y cells. The susceptibility of the identified proteins to covalent modification by dopamine may carry implications for their role in the vulnerability of dopaminergic neurons in Parkinson's disease pathogenesis.