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Age at onset of amyotrophic lateral sclerosis (ALS) is highly variable (eg, 27–74 years in carriers of the G4C2-expansion in C9orf72). It might be influenced by environmental and genetic factors via the modulation of DNA methylation (DNAm) at CpG-sites. Hence, we combined an epigenetic and genetic approach to test the hypothesis that some common single nucleotide polymorphisms (SNPs) at CpG-sites (CpG-SNPs) could modify ALS age of onset. Our genome-wide DNAm analysis suggested three CpG-SNPs whose DNAm levels are significantly associated with age of onset in 249 ALS patients (q C9orf72-carriers (n = 333; P = 0.025), suggesting that each A-allele delays onset by 1.6 years. Analysis of Genotype-Tissue Expression data revealed that the protective A-allele is linked with the reduced expression of CTSS in cerebellum (P = 0.00018), which is a critical brain region in the distributed neural circuits subserving motor control. CTSS encodes cathepsin S protein playing a key role in antigen presentation. In conclusion, we identified a 16 Kb locus tagged by rs4970944 as a modifier of ALS age of onset. Our findings support the role of antigen presenting processes in modulating age of onset of ALS and suggest potential drug targets (eg, CTSS). Future replication studies are encouraged to validate the link between the locus tagged by rs4970944 and age of onset in independent ALS cohorts, including different ethnic groups.

Polygenic inheritance plays a central role in Parkinson disease (PD). A priority in elucidating PD etiology lies in defining the biological basis of genetic risk. Unraveling how risk leads to disruption will yield disease-modifying therapeutic targets that may be effective. Here, we utilized a high-throughput and hypothesis-free approach to determine biological processes underlying PD using the largest currently available cohorts of genetic and gene expression data from International Parkinson’s Disease Genetics Consortium (IPDGC) and the Accelerating Medicines Partnership-Parkinson’s disease initiative (AMP-PD), among other sources. We applied large-scale gene-set specific polygenic risk score (PRS) analyses to assess the role of common variation on PD risk focusing on publicly annotated gene sets representative of curated pathways. We nominated specific molecular sub-processes underlying protein misfolding and aggregation, post-translational protein modification, immune response, membrane and intracellular trafficking, lipid and vitamin metabolism, synaptic transmission, endosomal–lysosomal dysfunction, chromatin remodeling and apoptosis mediated by caspases among the main contributors to PD etiology. We assessed the impact of rare variation on PD risk in an independent cohort of whole-genome sequencing data and found evidence for a burden of rare damaging alleles in a range of processes, including neuronal transmission-related pathways and immune response. We explored enrichment linked to expression cell specificity patterns using single-cell gene expression data and demonstrated a significant risk pattern for dopaminergic neurons, serotonergic neurons, hypothalamic GABAergic neurons, and neural progenitors. Subsequently, we created a novel way of building de novo pathways by constructing a network expression community map using transcriptomic data derived from the blood of PD patients, which revealed functional enrichment in inflammatory signaling pathways, cell death machinery related processes, and dysregulation of mitochondrial homeostasis. Our analyses highlight several specific promising pathways and genes for functional prioritization and provide a cellular context in which such work should be done. Electronic supplementary material The online version of this article (10.1007/s00401-020-02181-3) contains supplementary material, which is available to authorized users.

Massive genetic analysis identifies critical pathways and cell types involved in pathogenesis of amyotrophic lateral sclerosis., Despite the considerable progress in unraveling the genetic causes of amyotrophic lateral sclerosis (ALS), we do not fully understand the molecular mechanisms underlying the disease. We analyzed genome-wide data involving 78,500 individuals using a polygenic risk score approach to identify the biological pathways and cell types involved in ALS. This data-driven approach identified multiple aspects of the biology underlying the disease that resolved into broader themes, namely, neuron projection morphogenesis, membrane trafficking, and signal transduction mediated by ribonucleotides. We also found that genomic risk in ALS maps consistently to GABAergic interneurons and oligodendrocytes, as confirmed in human single-nucleus RNA-seq data. Using two-sample Mendelian randomization, we nominated six differentially expressed genes (ATG16L2, ACSL5, MAP1LC3A, MAPKAPK3, PLXNB2, and SCFD1) within the significant pathways as relevant to ALS. We conclude that the disparate genetic etiologies of this fatal neurological disease converge on a smaller number of final common pathways and cell types.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Despite the considerable progress in unraveling the genetic causes of amyotrophic lateral sclerosis (ALS), we do not fully understand the molecular mechanisms underlying the disease. We analyzed genome-wide data involving 78,500 individuals using a polygenic risk score approach to identify the biological pathways and cell types involved in ALS. This data-driven approach identified multiple aspects of the biology underlying the disease that resolved into broader themes, namely neuron projection morphogenesis, membrane trafficking, and signal transduction mediated by ribonucleotides. We also found that genomic risk in ALS maps consistently to GABAergic cortical interneurons and oligodendrocytes, as confirmed in human single-nucleus RNA-seq data. Using two-sample Mendelian randomization, we nominated five differentially expressed genes (ATG16L2, ACSL5, MAP1LC3A, PLXNB2, and SCFD1) within the significant pathways as relevant to ALS. We conclude that the disparate genetic etiologies of this fatal neurological disease converge on a smaller number of final common pathways and cell types.

Fibroblasts from an amyotrophic lateral sclerosis patient with simultaneous mutations in the MATR3 gene and KIF5A gene were isolated and reprogrammed into induced pluripotent stem cells via a non-integrating Sendai viral vector. The generated iPSC clones demonstrated normal karyotype, expression of pluripotency markers, and the capacity to differentiate into three germ layers. The unique presence of two simultaneous mutations in ALS-associated genes represent a novel tool for the study of ALS disease mechanisms.

The genetic basis of Lewy body dementia (LBD) is not well understood. Here, we performed whole-genome sequencing in large cohorts of LBD cases and neurologically healthy controls to study the genetic architecture of this understudied form of dementia and to generate a resource for the scientific community. Genome-wide association analysis identified five independent risk loci, whereas genome-wide gene-aggregation tests implicated mutations in the gene GBA. Genetic risk scores demonstrate that LBD shares risk profiles and pathways with Alzheimer’s and Parkinson’s disease, providing a deeper molecular understanding of the complex genetic architecture of this age-related neurodegenerative condition.

Globally, 50 million people live with dementia, with Alzheimer disease (AD) being responsible for two-thirds of the total cases. As ageing is the main risk factor for dementia-related neurodegeneration, changes in the timing or nature of the cellular hallmarks of normal ageing might be key to understanding the events that convert normal ageing into neurodegeneration. Cellular senescence is a candidate mechanism that might be important for this conversion. Under persistent stress, as occurs in ageing, both postmitotic cells — including neurons — and proliferative cells — such as astrocytes and microglia, among others — can engender a state of chronic cellular senescence that is characterized by the secretion of pro-inflammatory molecules that promote the functional decline of tissues and organs. Ablation of senescent cells has been postulated as a promising therapeutic venue to target the ageing phenotype and, thus, prevent or mitigate ageing-related diseases. However, owing to a lack of evidence, it is not possible to label cellular senescence as a cause or a consequence of neurodegeneration. This Review examines cellular senescence in the context of ageing and AD, and discusses which of the processes — cellular senescence or AD — might come first. Ageing is the main risk factor for Alzheimer disease (AD), but the mechanisms connecting ageing to this disease remain incompletely understood. In this Review, Saez-Atienzar and Masliah examine whether cellular senescence may have a role in linking ageing and AD.

Polygenic inheritance plays a central role in Parkinson disease (PD). A priority in elucidating PD etiology lies in defining the biological basis of genetic risk. Unraveling how risk leads to disruption will yield disease-modifying therapeutic targets that may be effective. Here, we utilized a high-throughput and hypothesis-free approach to determine biological pathways underlying PD using the largest currently available cohorts of genetic data and gene expression data from International Parkinson’s Disease Genetics Consortium (IPDGC) and the Accelerating Medicines Partnership - Parkinson’s disease initiative (AMP-PD), among other sources. We placed these insights into a cellular context. We applied large-scale pathway-specific polygenic risk score (PRS) analyses to assess the role of common variation on PD risk in a cohort of 457,110 individuals by focusing on a compilation of 2,199 publicly annotated gene sets representative of curated pathways, of which we nominate 46 pathways associated with PD risk. We assessed the impact of rare variation on PD risk in an independent cohort of whole-genome sequencing data, including 4,331 individuals. We explored enrichment linked to expression cell specificity patterns using single-cell gene expression data and demonstrated a significant risk pattern for adult dopaminergic neurons, serotonergic neurons, and radial glia. Subsequently, we created a novel way of building de novo pathways by constructing a network expression community map using transcriptomic data derived from the blood of 1,612 PD patients, which revealed 54 connecting networks associated with PD. Our analyses highlight several promising pathways and genes for functional prioritization and provide a cellular context in which such work should be done.

Parkinson’s disease (PD) is a common neurodegenerative motor disorder characterized in part by neuropathological lesions in the nigrostriatal pathway. While most cases of PD are sporadic in nature, several inherited monogenic syndromes exist that overlap clinically and pathologically with sporadic PD. Of these, loss of function mutations in DNAJC6 , which encodes the protein Auxilin, cause an aggressive form of juvenile onset PD. Auxilin and its homologues are known to play a role in clathrin-mediated trafficking, which is crucial for cellular function in all eukaryotes and plays a specialized role in synaptic transmission in higher organisms. Auxilin is the major neuronal uncoating protein for clathrin-coated vesicles required for delivery of cargo from the plasma membrane and trans -Golgi network to intracellular destinations. However, how mutations in Auxilin cause PD is currently not understood. To address this problem, we generated a novel mouse model carrying an endogenous pathogenic Auxilin mutation. When bred to homozygosity, this mutation induced neurological phenotypes that phenocopy clinical features observed in patients, including motor impairments reminiscent of bradykinesia and gait problems. Mapping the interactome of Auxilin confirmed clathrin and synaptic clathrin adaptor protein interactions and also identified novel Golgi-resident interactors. Critically, all tested pathogenic mutations in Auxilin retained clathrin adaptor protein binding but lost interaction with clathrin itself. These observations describe a mechanism by which impaired clathrin-mediated trafficking in R857G Auxilin mice, both at the Golgi and the synapse, results in neuropathological lesions in the nigrostriatal pathway. Collectively, these results provide novel insights for PD pathogenesis in Auxilin mutation carriers, reinforcing a key role for clathrin-mediated trafficking in PD, and expand our understanding of the cellular function of Auxilin. Highlights Auxilin interacts with Golgi-resident clathrin adaptor protein GGA2 Auxilin is involved with uncoating of CCVs at the Golgi and synapse Impaired clathrin-mediated trafficking underlies PD-like phenotypes in R857G Auxilin mice

Summary: Parkinson’s disease causes the most profound loss of the aldehyde dehydrogenase 1A1-positive (ALDH1A1+) nigrostriatal dopaminergic neuron (nDAN) subpopulation. The connectivity and functionality of ALDH1A1+ nDANs, however, remain poorly understood. Here, we show in rodent brains that ALDH1A1+ nDANs project predominantly to the rostral dorsal striatum, from which they also receive most monosynaptic inputs, indicating extensive reciprocal innervations with the striatal spiny projection neurons (SPNs). Functionally, genetic ablation of ALDH1A1+ nDANs causes severe impairments in motor skill learning, along with a reduction in high-speed walking. While dopamine replacement therapy accelerated walking speed, it failed to improve motor skill learning in ALDH1A1+ nDAN-ablated mice. Altogether, our study provides a comprehensive whole-brain connectivity map and reveals a key physiological function of ALDH1A1+ nDANs in motor skill acquisition, suggesting the motor learning processes require ALDH1A1+ nDANs to integrate diverse presynaptic inputs and supply dopamine with dynamic precision. : Wu et al. show that aldehyde dehydrogenase 1A1-positive nigrostriatal dopaminergic neurons, which display the most profound degeneration in Parkinson’s disease, are essential in the acquisition of skilled movements in rodent models. The proper synaptic inputs to these neurons are critical for providing the timely dopamine release required in the learning process. Keywords: aldehyde dehydrogenase 1a1, ALDH1A1, Parkinson’s disease, dopamine, dopaminergic neurons, substantia nigra, striatum, brain mapping, motor skill learning

Aldehyde dehydrogenase 1A1 (ALDH1A1), a retinoic acid (RA) synthase, is selectively expressed by the nigrostriatal dopaminergic (nDA) neurons that preferentially degenerate in Parkinson’s disease (PD). ALDH1A1–positive axons mainly project to the dorsal striatum. However, whether ALDH1A1 and its products regulate the activity of postsynaptic striatal neurons is unclear. Here we show that μ–type opioid receptor (MOR1) levels were severely decreased in the dorsal striatum of postnatal and adult Aldh1a1 knockout mice, whereas dietary supplement of RA restores its expression. Furthermore, RA treatment also upregulates striatal MOR1 levels and signaling and alleviates L-DOPA–induced dyskinetic movements in pituitary homeobox 3 (Pitx3)–deficient mice that lack of ALDH1A1–expressing nDA neurons. Therefore, our findings demonstrate that ALDH1A1–synthesized RA is required for postsynaptic MOR1 expression in the postnatal and adult dorsal striatum, supporting potential therapeutic benefits of RA supplementation in moderating L-DOPA–induced dyskinesia.

Background PD is a complex polygenic disorder. In recent years, several genes from the endocytic membrane-trafficking pathway have been suggested to contribute to disease etiology. However, a systematic analysis of pathway-specific genetic risk factors is yet to be performed. Objectives To comprehensively study the role of the endocytic membrane-trafficking pathway in the risk of PD. Methods Linkage disequilibrium score regression was used to estimate PD heritability explained by 252 genes involved in the endocytic membrane-trafficking pathway including genome-wide association studies data from 18,869 cases and 22,452 controls. We used pathway-specific single-nucleotide polymorphisms to construct a polygenic risk score reflecting the cumulative risk of common variants. To prioritize genes for follow-up functional studies, summary-data based Mendelian randomization analyses were applied to explore possible functional genomic associations with expression or methylation quantitative trait loci. Results The heritability estimate attributed to endocytic membrane-trafficking pathway was 3.58% (standard error = 1.17). Excluding previously nominated PD endocytic membrane-trafficking pathway genes, the missing heritability was 2.21% (standard error = 0.42). Random heritability simulations were estimated to be 1.44% (standard deviation = 0.54), indicating that the unbiased total heritability explained by the endocytic membrane-trafficking pathway was 2.14%. Polygenic risk score based on endocytic membrane-trafficking pathway showed a 1.25 times increase of PD risk per standard deviation of genetic risk. Finally, Mendelian randomization identified 11 endocytic membrane-trafficking pathway genes showing functional consequence associated to PD risk. Conclusions We provide compelling genetic evidence that the endocytic membrane-trafficking pathway plays a relevant role in disease etiology. Further research on this pathway is warranted given that critical effort should be made to identify potential avenues within this biological process suitable for therapeutic interventions. © 2019 International Parkinson and Movement Disorder Society.

We have explored the mechanisms underlying ethanol-induced mitochondrial dynamics disruption and mitophagy. Ethanol increases mitochondrial fission in a concentration-dependent manner through Drp1 mitochondrial translocation and OPA1 proteolytic cleavage. ARPE-19 (a human retinal pigment epithelial cell line) cells challenged with ethanol showed mitochondrial potential disruptions mediated by alterations in mitochondrial complex IV protein level and increases in mitochondrial reactive oxygen species production. In addition, ethanol activated the canonical autophagic pathway, as denoted by autophagosome formation and autophagy regulator elements including Beclin1, ATG5-ATG12 and P-S6 kinase. Likewise, autophagy inhibition dramatically increased mitochondrial fission and cell death, whereas autophagy stimulation rendered the opposite results, placing autophagy as a cytoprotective response aimed to remove damaged mitochondria. Interestingly, although ethanol induced mitochondrial Bax translocation, this episode was associated to cell death rather than mitochondrial fission or autophagy responses. Thus, Bax required 600mM ethanol to migrate to mitochondria, a concentration that resulted in cell death. Furthermore, mouse embryonic fibroblasts lacking this protein respond to ethanol by undergoing mitochondrial fission and autophagy but not cytotoxicity. Finally, by using the specific mitochondrial-targeted scavenger MitoQ, we revealed mitochondria as the main source of reactive oxygen species that trigger autophagy activation. These findings suggest that cells respond to ethanol activating mitochondrial fission machinery by Drp1 and OPA1 rather than bax, in a manner that stimulates cytoprotective autophagy through mitochondrial ROS.

Over the last two decades, we have witnessed a revolution in the field of Parkinson’s disease (PD) genetics. Great advances have been made in identifying many loci that confer a risk for PD, which has subsequently led to an improved understanding of the molecular pathways involved in disease pathogenesis. Despite this success, it is predicted that only a relatively small proportion of the phenotypic variability has been explained by genetics. Therefore, it is clear that common heritable components of disease are still to be identified. Dissecting the genetic architecture of PD constitutes a critical effort in identifying therapeutic targets and although such substantial progress has helped us to better understand disease mechanism, the route to PD disease-modifying drugs is a lengthy one. In this review, we give an overview of the known genetic risk factors in PD, focusing not on individual variants but the larger networks that have been implicated following comprehensive pathway analysis. We outline the challenges faced in the translation of risk loci to pathobiological relevance and illustrate the need for integrating big-data by noting success in recent work which adopts a broad-scale screening approach. Lastly, with PD genetics now progressing from identifying risk to predicting disease, we review how these models will likely have a significant impact in the future.

BACKGROUND AND PURPOSE Huntington's disease is a neurodegenerative process associated with mitochondrial alterations. Inhibitors of the electron–transport channel complex II, such as 3-nitropropionic acid (3NP), are used to study the molecular and cellular pathways involved in this disease. We studied the effect of 3NP on mitochondrial morphology and its involvement in macrophagy. EXPERIMENTAL APPROACH Pharmacological and biochemical methods were used to characterize the effects of 3NP on autophagy and mitochondrial morphology. SH-SY5Y cells were transfected with GFP-LC3, GFP-Drp1 or GFP-Bax to ascertain their role and intracellular localization after 3NP treatment using confocal microscopy. KEY RESULTS Untreated SH-SY5Y cells presented a long, tubular and filamentous net of mitochondria. After 3NP (5 mM) treatment, mitochondria became shorter and rounder. 3NP induced formation of mitochondrial permeability transition pores, both in cell cultures and in isolated liver mitochondria, and this process was inhibited by cyclosporin A. Participation of the mitochondrial fission pathway was excluded because 3NP did not induce translocation of the dynamin-related protein 1 (Drp1) to the mitochondria. The Drp1 inhibitor Mdivi-1 did not affect the observed changes in mitochondrial morphology. Finally, scavengers of reactive oxygen species failed to prevent mitochondrial alterations, while cyclosporin A, but not Mdivi-1, prevented the generation of ROS. CONCLUSIONS AND IMPLICATIONS There was a direct correlation between formation of mitochondrial permeability transition pores and autophagy induced by 3NP treatment. Activation of autophagy preceded the apoptotic process and was mediated, at least partly, by formation of reactive oxygen species and mitochondrial permeability transition pores. LINKED ARTICLE This article is commented on by Gonzalez-Polo et al., pp. 60–62 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.02203.x

Oxidative stress causes cellular damage by (i) altering protein stability, (ii) impairing organelle function, or (iii) triggering the formation of 4-HNE protein aggregates. The catabolic process known as autophagy is an antioxidant cellular response aimed to counteract these stressful conditions. Therefore, autophagy might act as a cytoprotective response by removing impaired organelles and aggregated proteins. In the present study, we sought to understand the role of autophagy in the clearance of 4-HNE protein aggregates in ARPE-19 cells under rotenone exposure. Rotenone induced an overproduction of reactive oxygen species (ROS), which led to an accumulation of 4-HNE inclusions, and an increase in the number of autophagosomes. The latter resulted from a disturbed autophagic flux rather than an activation of the autophagic synthesis pathway. In compliance with this, rotenone treatment induced an increase in LC3-II while upstream autophagy markers such as Beclin- 1, Vsp34 or Atg5-Atg12, were decreased. Rotenone reduced the autophagosome-to-lysosome fusion step by increasing tubulin acetylation levels through a ROS-mediated pathway. Proof of this is the finding that the free radical scavenger, N-acetylcysteine, restored autophagy flux and reduced rotenone-induced tubulin hyperacetylation. Indeed, this dysfunctional autophagic response exacerbates cell death triggered by rotenone, since 3-methyladenine, an autophagy inhibitor, reduced cell mortality, while rapamycin, an inductor of autophagy, caused opposite effects. In summary, we shed new light on the mechanisms involved in the autophagic responses disrupted by oxidative stress, which take place in neurodegenerative diseases such as Huntington or Parkinson diseases, and age-related macular degeneration.

We have used the human neuroblastoma cell line SH-SY5Y overexpressing Bcl-xL (SH-SY5Y/Bcl-xL) to clarify the effects of this mitochondrial protein on the control of mitochondrial dynamics and the autophagic processes which occur after the inhibition of leucine-rich repeat kinase 2 (LRRK2) with GSK2578215A. In wild type (SH-SY5Y/Neo) cells, GSK2578215A (1nM) caused a disruption of mitochondrial morphology and an imbalance in intracellular reactive oxygen species (ROS) as indicated by an increase in dichlorofluorescein fluorescence and 4-hydroxynonenal. However, SH-SY5Y/Bcl-xL cells under GSK2578215A treatment, unlike the wild type, preserved a high mitochondrial membrane potential and did not exhibit apoptotical chromatins. In contrast to wild type cells, in SH-SY5Y/Bcl-xL cells, GSK2578215A did not induce mitochondrial translocation of neither dynamin related protein-1 nor the proapoptotic protein, Bax. In SH-SY5Y/Neo, but not SH-SY5Y/Bcl-xL cells, mitochondrial fragmentation elicited by GSK2578215A precedes an autophagic response. Furthermore, the overexpression of Bcl-xL protein restores the autophagic flux pathway disrupted by this inhibitor. SH-SY5Y/Neo, but not SH-SY5Y/Bcl-xL cells, responded to LRRK2 inhibition by an increase in the levels of acetylated tubulin, indicating that this was abrogated by Bcl-xL overexpression. This hyperacetylation of tubulin took place earlier than any of the above-mentioned events suggesting that it is involved in the autophagic flux interruption. Pre-treatment with tempol prevented the GSK2578215A-induced mitochondrial fragmentation, autophagy and the rise in acetylated tubulin in SH-SY5Y/Neo cells. Thus, these data support the notion that ROS act as a second messenger connexion between LRRK2 inhibition and these deleterious responses, which are markedly alleviated by the Bcl-xL-mediated ROS generation blockade.

Synaptojanin 1 (SJ1) is a major presynaptic phosphatase that couples synaptic vesicle endocytosis to the dephosphorylation of PI(4,5)P2, a reaction needed for the shedding of endocytic factors from their membranes. While the role of SJ1’s 5-phosphatase module in this process is well established, the contribution of its Sac phosphatase domain, whose preferred substrate is PI4P, remains unclear. Recently a homozygous mutation in its Sac domain was identified in early-onset Parkinsonism patients. We show that mice carrying this mutation developed neurological manifestations similar to those of human patients. Synapses of these mice displayed endocytic defects and a striking accumulation of clathrin coated intermediates strongly implicating Sac domain’s activity in endocytic protein dynamics. Mutant brains had elevated auxilin (PARK19) and parkin (PARK2) levels. Moreover, dystrophic axonal terminal changes were selectively observed in dopaminergic axons in the dorsal striatum. These results strengthen evidence for a link between synaptic endocytic dysfunction and Parkinson’s disease.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been associated with Parkinson's disease, and its inhibition opens potential new therapeutic options. Among the drug inhibitors of both wild-type and mutant LRRK2 forms is the 2-arylmethyloxy-5-subtitutent-N-arylbenzamide GSK257815A. Using the well-established dopaminergic cell culture model SH-SY5Y, we have investigated the effects of GSK2578215A on crucial neurodegenerative features such as mitochondrial dynamics and autophagy. GSK2578215A induces mitochondrial fragmentation of an early step preceding autophagy. This increase in autophagosome results from inhibition of fusion rather than increases in synthesis. The observed effects were shared with LRRK2-IN-1, a well-described, structurally distinct kinase inhibitor compound or when knocking down LRRK2 expression using siRNA. Studies using the drug mitochondrial division inhibitor 1 indicated that translocation of the dynamin-related protein-1 has a relevant role in this process. In addition, autophagic inhibitors revealed the participation of autophagy as a cytoprotective response by removing damaged mitochondria. GSK2578215A induced oxidative stress as evidenced by the accumulation of 4-hydroxy-2-nonenal in SH-SY5Y cells. The mitochondrial-targeted reactive oxygen species scavenger MitoQ positioned these species as second messengers between mitochondrial morphologic alterations and autophagy. Altogether, our results demonstrated the relevance of LRRK2 in mitochondrial-activated pathways mediating in autophagy and cell fate, crucial features in neurodegenerative diseases.

In the present study, the activation of autophagy and its interaction with the mitochondrial fission machinery was investigated in an experimental model of Parkinson's disease. The addition of 50µM 6-hydroxydopamine (6-OHDA) to the dopaminergic cell line SH-SY5Y profoundly stimulated formation of autophagosomes within 12h. Under these conditions, mitochondrial fission was also activated in a sustained manner, but this occurred at earlier time points (after 3h). Upon 6-OHDA treatment, dynamin-related protein 1 (Drp1) transiently translocated to mitochondria, with increased levels of mitochondrial Drp1 being observed after 3 and 9h. Pharmacological inhibition of Drp1, through treatment with the mitochondrial-division inhibitor-1 (mdivi-1), resulted in the abrogation of mitochondrial fission and in a decrease of the number of autophagic cells. In addition, 6-OHDA failed to induce the expression of the proapoptotic protein Bax in total cellular extracts although it did induce its migration to mitochondria. In our model, Bax migrated later than Drp1. However, Drp1 inhibition did not block Bax migration. These results show that reactive oxygen species but not quinone derivates act as mediators of autophagy at an early stage of the process. 6-OHDA induces hydrogen peroxide production, which was placed upstream of mitochondrial fission, given that mdivi-1 did not abrogate this increase. Furthermore, the 6-OHDA-induced activation of autophagy was also suppressed by addition of the free radical scavengers TEMPOL and MnTBAP. This effect could be reproduced by the addition of hydrogen peroxide, but not with aged 6-OHDA. To our knowledge, this is the first detailed study highlighting the various mediators that are implicated in mitochondrial alterations and autophagy of cells in response to 6-OHDA.

Retinal pigment epithelium has a crucial role in the physiology and pathophysiology of the retina due to its location and metabolism. Oxidative damage has been demonstrated as a pathogenic mechanism in several retinal diseases, and reactive oxygen species are certainly important by-products of ethanol (EtOH) metabolism. Autophagy has been shown to exert a protective effect in different cellular and animal models. Thus, in our model, EtOH treatment increases autophagy flux, in a concentration-dependent manner. Mitochondrial morphology seems to be clearly altered under EtOH exposure, leading to an apparent increase in mitochondrial fission. An increase in 2′,7′-dichlorofluorescein fluorescence and accumulation of lipid peroxidation products, such as 4-hydroxy-nonenal (4-HNE), among others were confirmed. The characterization of these structures confirmed their nature as aggresomes. Hence, autophagy seems to have a cytoprotective role in ARPE-19 cells under EtOH damage, by degrading fragmented mitochondria and 4-HNE aggresomes. Herein, we describe the central implication of autophagy in human retinal pigment epithelial cells upon oxidative stress induced by EtOH, with possible implications for other conditions and diseases.