Heterozygous PNPT1 Variants Cause Spinocerebellar Ataxia Type 25.

OBJECTIVE: Dominant spinocerebellar ataxias (SCA) are characterized by genetic heterogeneity. Some mapped and named loci remain without a causal gene identified. Here we applied next generation sequencing (NGS) to uncover the genetic etiology of the SCA25 locus. METHODS: Whole-exome and whole-genome sequencing were performed in families linked to SCA25, including the French family in which the SCA25 locus was originally mapped. Whole exome sequence data were interrogated in a cohort of 796 ataxia patients of unknown etiology. RESULTS: The SCA25 phenotype spans a slowly evolving sensory and cerebellar ataxia, in most cases attributed to ganglionopathy. A pathogenic variant causing exon skipping was identified in the gene encoding Polyribonucleotide Nucleotidyltransferase PNPase 1 (PNPT1) located in the SCA25 linkage interval. A second splice variant in PNPT1 was detected in a large Australian family with a dominant ataxia also mapping to SCA25. An additional nonsense variant was detected in an unrelated individual with ataxia. Both nonsense and splice heterozygous variants result in premature stop codons, all located in the S1-domain of PNPase. In addition, an elevated type I interferon response was observed in blood from all affected heterozygous carriers tested. PNPase notably prevents the abnormal accumulation of double-stranded mtRNAs in the mitochondria and leakage into the cytoplasm, associated with triggering a type I interferon response. INTERPRETATION: This study identifies PNPT1 as a new SCA gene, responsible for SCA25, and highlights biological links between alterations of mtRNA trafficking, interferonopathies and ataxia. ANN NEUROL 2022;92:122-137.

LRRK2 impairs PINK1/Parkin-dependent mitophagy via its kinase activity: pathologic insights into Parkinson's disease.

Mutations of LRRK2, encoding leucine-rich repeat kinase 2 (LRRK2), are the leading cause of autosomal dominant Parkinson's disease (PD). The most frequent of these mutations, G2019S substitution, increases kinase activity, but it remains unclear how it causes PD. Recent studies suggest that LRRK2 modulates mitochondrial homeostasis. Mitochondrial dysfunction plays a key role in the pathogenesis of autosomal recessive PD forms linked to PARK2 and PINK1, encoding the cytosolic E3 ubiquitin-protein ligase Parkin and the mitochondrial kinase PINK1, which jointly regulate mitophagy. We explored the role of LRRK2 and its kinase activity in PINK1/Parkin-dependent mitophagy. LRRK2 increased mitochondrial aggregation and attenuated mitochondrial clearance in cells coexpressing Parkin and exposed to the protonophore carbonylcyanide m-chlorophenylhydrazone. Förster resonance energy transfer imaging microscopy showed that LRRK2 impaired the interactions between Parkin and Drp1 and their mitochondrial targets early in mitophagy. The inhibition of LRRK2 kinase activity by a 'kinase-dead' LRRK2 mutation or with a pharmacological inhibitor (LRRK2-IN-1) restored these interactions. The monitoring of mitophagy in human primary fibroblasts with the novel dual-fluorescence mtRosella reporter and a new hypothermic shock paradigm revealed similar defects in PD patients with the G2019S LRRK2 substitution or PARK2 mutations relative to healthy subjects. This defect was restored by LRRK2-IN-1 treatment in LRRK2 patients only. Our results suggest that PD forms due to LRRK2 and PARK2 mutations involve pathogenic mechanisms converging on PINK1/Parkin-dependent mitophagy.

Characterization of Recessive Parkinson Disease in a Large Multicenter Study.

Studies of the phenotype and population distribution of rare genetic forms of parkinsonism are required, now that gene-targeting approaches for Parkinson disease have reached the clinical trial stage. We evaluated the frequencies of PRKN, PINK1, and DJ-1 mutations in a cohort of 1,587 cases. Mutations were found in 14.1% of patients; 27.6% were familial and 8% were isolated. PRKN was the gene most frequently mutated in Caucasians, whereas PINK1 mutations predominated in Arab-Berber individuals. Patients with PRKN mutations had an earlier age at onset, and less asymmetry, levodopa-induced motor complications, dysautonomia, and dementia than those without mutations. ANN NEUROL 2020;88:843-850.

Autophagy in neurodegeneration: New insights underpinning therapy for neurological diseases.

In autophagy long-lived proteins, protein aggregates or damaged organelles are engulfed by vesicles called autophagosomes prior to lysosomal degradation. Autophagy dysfunction is a hallmark of several neurodegenerative diseases in which misfolded proteins or dysfunctional mitochondria accumulate. Excessive autophagy can also exacerbate brain injury under certain conditions. In this review, we provide specific examples to illustrate the critical role played by autophagy in pathological conditions affecting the brain and discuss potential therapeutic implications. We show how a singular type of autophagy-dependent cell death termed autosis has attracted attention as a promising target for improving outcomes in perinatal asphyxia and hypoxic-ischaemic injury to the immature brain. We provide evidence that autophagy inhibition may be protective against radiotherapy-induced damage to the young brain. We describe a specialized form of macroautophagy of therapeutic relevance for motoneuron and neuromuscular diseases, known as chaperone-assisted selective autophagy, in which heat shock protein B8 is used to deliver aberrant proteins to autophagosomes. We summarize studies pinpointing mitophagy mediated by the serine/threonine kinase PINK1 and the ubiquitin-protein ligase Parkin as a mechanism potentially relevant to Parkinson's disease, despite debate over the physiological conditions in which it is activated in organisms. Finally, with the example of the autophagy-inducing agent rilmenidine and its discrepant effects in cell culture and mouse models of motor neuron disorders, we illustrate the importance of considering aspects such a disease stage and aggressiveness, type of insult and load of damaged or toxic cellular components, when choosing the appropriate drug, timepoint and duration of treatment.

Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1.

Mutations in the PTEN-induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson's disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser(65)) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1-dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub-family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser(111)) in response to PINK1 activation. Using phospho-specific antibodies raised against Ser(111) of each of the Rabs, we demonstrate that Rab Ser(111) phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient-derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser(111) phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser(111) phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser(65). We further show mechanistically that phosphorylation at Ser(111) significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser(111) may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase-mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson's disease.

Loss of VPS13C Function in Autosomal-Recessive Parkinsonism Causes Mitochondrial Dysfunction and Increases PINK1/Parkin-Dependent Mitophagy.

Autosomal-recessive early-onset parkinsonism is clinically and genetically heterogeneous. The genetic causes of approximately 50% of autosomal-recessive early-onset forms of Parkinson disease (PD) remain to be elucidated. Homozygozity mapping and exome sequencing in 62 isolated individuals with early-onset parkinsonism and confirmed consanguinity followed by data mining in the exomes of 1,348 PD-affected individuals identified, in three isolated subjects, homozygous or compound heterozygous truncating mutations in vacuolar protein sorting 13C (VPS13C). VPS13C mutations are associated with a distinct form of early-onset parkinsonism characterized by rapid and severe disease progression and early cognitive decline; the pathological features were striking and reminiscent of diffuse Lewy body disease. In cell models, VPS13C partly localized to the outer membrane of mitochondria. Silencing of VPS13C was associated with lower mitochondrial membrane potential, mitochondrial fragmentation, increased respiration rates, exacerbated PINK1/Parkin-dependent mitophagy, and transcriptional upregulation of PARK2 in response to mitochondrial damage. This work suggests that loss of function of VPS13C is a cause of autosomal-recessive early-onset parkinsonism with a distinctive phenotype of rapid and severe progression.

Parkin deficiency modulates NLRP3 inflammasome activation by attenuating an A20-dependent negative feedback loop.

Neuroinflammation and mitochondrial dysfunction, key mechanisms in the pathogenesis of Parkinson's disease (PD), are usually explored independently. Loss-of-function mutations of PARK2 and PARK6, encoding the E3 ubiquitin protein ligase Parkin and the mitochondrial serine/threonine kinase PINK1, account for a large proportion of cases of autosomal recessive early-onset PD. PINK1 and Parkin regulate mitochondrial quality control and have been linked to the modulation of innate immunity pathways. We report here an exacerbation of NLRP3 inflammasome activation by specific inducers in microglia and bone marrow-derived macrophages from Park2-/- and Pink1-/- mice. The caspase 1-dependent release of IL-1ß and IL-18 was, therefore, enhanced in Park2-/- and Pink1-/- cells. This defect was confirmed in blood-derived macrophages from patients with PARK2 mutations and was reversed by MCC950, which specifically inhibits NLRP3 inflammasome complex formation. Enhanced NLRP3 signaling in Parkin-deficient cells was accompanied by a lack of induction of A20, a well-known negative regulator of the NF-κB pathway recently shown to attenuate NLRP3 inflammasome activity. We also found an inverse correlation between A20 abundance and IL-1ß release, in human macrophages challenged with NLRP3 inflammasome inducers. Overall, our observations suggest that the A20/NLRP3-inflammasome axis participates in the pathogenesis of PARK2-linked PD, paving the way for the exploration of its potential as a biomarker and treatment target.

What genetics tells us about the causes and mechanisms of Parkinson's disease.

Parkinson's disease (PD) is a common motor disorder of mysterious etiology. It is due to the progressive degeneration of the dopaminergic neurons of the substantia nigra and is accompanied by the appearance of intraneuronal inclusions enriched in α-synuclein, the Lewy bodies. It is becoming increasingly clear that genetic factors contribute to its complex pathogenesis. Over the past decade, the genetic basis of rare PD forms with Mendelian inheritance, representing no more than 10% of the cases, has been investigated. More than 16 loci and 11 associated genes have been identified so far; genome-wide association studies have provided convincing evidence that polymorphic variants in these genes contribute to sporadic PD. The knowledge acquired of the functions of their protein products has revealed pathways of neurodegeneration that may be shared between inherited and sporadic PD. An impressive set of data in different model systems strongly suggest that mitochondrial dysfunction plays a central role in clinically similar, early-onset autosomal recessive PD forms caused by parkin and PINK1, and possibly DJ-1 gene mutations. In contrast, α-synuclein accumulation in Lewy bodies defines a spectrum of disorders ranging from typical late-onset PD to PD dementia and including sporadic and autosomal dominant PD forms due to mutations in SCNA and LRRK2. However, the pathological role of Lewy bodies remains uncertain, as they may or may not be present in PD forms with one and the same LRRK2 mutation. Impairment of autophagy-based protein/organelle degradation pathways is emerging as a possible unifying but still fragile pathogenic scenario in PD. Strengthening these discoveries and finding other convergence points by identifying new genes responsible for Mendelian forms of PD and exploring their functions and relationships are the main challenges of the next decade. It is also the way to follow to open new promising avenues of neuroprotective treatment for this devastating disorder.

The endoplasmic reticulum-mitochondria interface is perturbed in PARK2 knockout mice and patients with PARK2 mutations.

Mutations in PARK2, encoding the E3 ubiquitin protein ligase Parkin, are a common cause of autosomal recessive Parkinson's disease (PD). Loss of PARK2 function compromises mitochondrial quality by affecting mitochondrial biogenesis, bioenergetics, dynamics, transport and turnover. We investigated the impact of PARK2 dysfunction on the endoplasmic reticulum (ER)-mitochondria interface, which mediates calcium (Ca2+) exchange between the two compartments and is essential for Parkin-dependent mitophagy. Confocal and electron microscopy analyses showed the ER and mitochondria to be in closer proximity in primary fibroblasts from PARK2 knockout (KO) mice and PD patients with PARK2 mutations than in controls. Ca2+ flux to the cytosol was also modified, due to enhanced ER-to-mitochondria Ca2+ transfers, a change that was also observed in neurons derived from induced pluripotent stem cells of a patient with PARK2 mutations. Subcellular fractionation showed the abundance of the Parkin substrate mitofusin 2 (Mfn2), which is known to modulate the ER-mitochondria interface, to be specifically higher in the mitochondrion-associated ER membrane compartment in PARK2 KO tissue. Mfn2 downregulation or the exogenous expression of normal Parkin restored cytosolic Ca2+ transients in fibroblasts from patients with PARK2 mutations. In contrast, a catalytically inactive PD-related Parkin variant had no effect. Overall, our data suggest that Parkin is directly involved in regulating ER-mitochondria contacts and provide new insight into the role of the loss of Parkin function in PD development.

Probes of ubiquitin E3 ligases enable systematic dissection of parkin activation.

E3 ligases represent an important class of enzymes, yet there are currently no chemical probes for profiling their activity. We develop a new class of activity-based probe by re-engineering a ubiquitin-charged E2 conjugating enzyme and demonstrate the utility of these probes by profiling the transthiolation activity of the RING-in-between-RING (RBR) E3 ligase parkin in vitro and in cellular extracts. Our study provides valuable insight into the roles, and cellular hierarchy, of distinct phosphorylation events in parkin activation. We also profile parkin mutations associated with patients with Parkinson's disease and demonstrate that they mediate their effect largely by altering transthiolation activity. Furthermore, our probes enable direct and quantitative measurement of endogenous parkin activity, revealing that endogenous parkin is activated in neuronal cell lines (≥75%) in response to mitochondrial depolarization. This new technology also holds promise as a novel biomarker of PINK1-parkin signaling, as demonstrated by its compatibility with samples derived from individuals with Parkinson's disease.

De novo tetrahydrobiopterin biosynthesis is impaired in the inflammed striatum of parkin(-/-) mice.

Parkinson's disease (PD), the second-most prevalent neurodegenerative disease, is primarily characterized by neurodegeneration in the substantia nigra pars compacta, resulting in motor impairment. Loss-of-function mutations in parkin are the major cause of the early onset familial form of the disease. Although rodents deficient in parkin (parkin(-/-) ) have some dopaminergic system dysfunction associated with central oxidative stress and energy metabolism deficiencies, these animals only display nigrostriatal pathway degeneration under inflammatory conditions. This study investigated the impact of the inflammatory stimulus induced by lypopolisaccharide (LPS) on tetrahydrobiopterin (BH4) synthesizing enzymes (de novo and salvage pathways), since this cofactor is essential for dopamine synthesis. The mitochondrial content and architecture was investigated in the striatum of LPS-exposed parkin(-/-) mice. As expected, the LPS (0.33 mg/kg; i.p.) challenge compromised spontaneous locomotion and social interaction with juvenile parkin(-/-) and WT mice. Moreover, the genotype impacted the kinetics of the investigation of the juvenile. The inflammatory scenario did not induce apparent changes in mitochondrial ultrastructure; however, it increased the quantity of mitochondria, which were of smaller size, and provoked the perinuclear distribution of the organelle. Furthermore, the BH4 de novo biosynthetic pathway failed to be up-regulated in the LPS challenge, a well-known stimulus for its activation. The LPS treatment increased sepiapterin reductase (SPR) expression, suggesting compensation by the salvage pathway. This might indicate that dopamine synthesis is compromised in parkin(-/-) mice under inflammatory conditions. Finally, this scenario impaired the striatal expression of the transcription factor BDNF, possibly favoring cell death.

Functional interplay between Parkin and Drp1 in mitochondrial fission and clearance.

Autosomal recessive early-onset Parkinson's disease is most often caused by mutations in the genes encoding the cytosolic E3 ubiquitin ligase Parkin and the mitochondrial serine/threonine kinase PINK1. Studies in Drosophila models and mammalian cells have demonstrated that these proteins regulate various aspects of mitochondrial physiology, including organelle transport, dynamics and turnover. How PINK1 and Parkin orchestrate these processes, and whether they always do so within a common pathway remain to be clarified. We have revisited the role of PINK1 and Parkin in mitochondrial dynamics, and explored its relation to the mitochondrial clearance program controlled by these proteins. We show that PINK1 and Parkin promote Drp1-dependent mitochondrial fission by mechanisms that are at least in part independent. Parkin-mediated mitochondrial fragmentation was abolished by treatments interfering with the calcium/calmodulin/calcineurin signaling pathway, suggesting that it requires dephosphorylation of serine 637 of Drp1. Parkinson's disease-causing mutations with differential impact on mitochondrial morphology and organelle degradation demonstrated that the pro-fission effect of Parkin is not required for efficient mitochondrial clearance. In contrast, the use of Förster energy transfer imaging microscopy revealed that Drp1 and Parkin are co-recruited to mitochondria in proximity of PINK1 following mitochondrial depolarization, indicating spatial coordination between these events in mitochondrial degradation. Our results also hint at a major role of the outer mitochondrial adaptor MiD51 in Drp1 recruitment and Parkin-dependent mitophagy. Altogether, our observations provide new insight into the mechanisms underlying the regulation of mitochondrial dynamics by Parkin and its relation to the mitochondrial clearance program mediated by the PINK1/Parkin pathway.

ER-stress-associated functional link between Parkin and DJ-1 via a transcriptional cascade involving the tumor suppressor p53 and the spliced X-box binding protein XBP-1.

Parkin and DJ-1 are two multi-functional proteins linked to autosomal recessive early-onset Parkinson's disease (PD) that have been shown to functionally interact by as-yet-unknown mechanisms. We have delineated the mechanisms by which parkin controls DJ-1. Parkin modulates DJ-1 transcription and protein levels via a signaling cascade involving p53 and the endoplasmic reticulum (ER)-stress-induced active X-box-binding protein-1S (XBP-1S). Parkin triggers the transcriptional repression of p53 while p53 downregulates DJ-1 protein and mRNA expressions. We show that parkin-mediated control of DJ-1 is fully p53-dependent. Furthermore, we establish that p53 lowers the protein and mRNA levels of XBP-1S. Accordingly, we show that parkin ultimately upregulates XBP-1 levels. Subsequently, XBP-1S physically interacts with the DJ-1 promoter, thereby enhancing its promoter trans-activation, mRNA levels and protein expression. This data was corroborated by the examination of DJ-1 in both parkin- and p53-null mice brains. This transcriptional cascade is abolished by pathogenic parkin mutations and is independent of its ubiquitin-ligase activity. Our data establish a parkin-dependent ER-stress-associated modulation of DJ-1 and identifies p53 and XBP-1 as two major actors acting downstream of parkin in this signaling cascade in cells and in vivo. This work provides a mechanistic explanation for the increase in the unfolded protein response observed in PD pathology, i.e. that it is due to a defect in parkin-associated control of DJ-1.

The endoplasmic reticulum/mitochondria interface: a subcellular platform for the orchestration of the functions of the PINK1-Parkin pathway?

Mitochondrial dysfunction is a hallmark of both idiopathic and familial Parkinson's disease (PD). Mutations in the PARK2 and PARK6 genes, coding for the cytosolic E3 ubiquitin protein ligase Parkin and the mitochondrial serine/threonine kinase PINK1 [phosphatase and tensin homologue (PTEN)-induced putative kinase 1], lead to clinically similar early-onset Parkinsonian syndromes. PINK1 and Parkin cooperate within a conserved pathway to preserve mitochondrial quality through the regulation of a variety of processes, including mitochondrial dynamics, transport, bioenergetics, biogenesis and turnover. The molecular mechanisms behind the orchestration of this plethora of functions remain poorly understood. In the present review, we emphasize the functional overlap between the PINK1-Parkin pathway and the endoplasmic reticulum (ER)-mitochondria interface, a subcellular compartment critically involved in neurodegeneration. We discuss how this compartment may constitute a hub for the spatiotemporal organization of the activities of the PINK1-Parkin pathway.

Genotype-phenotype correlation in PRKN-associated Parkinson's disease.

Bi-allelic pathogenic variants in PRKN are the most common cause of autosomal recessive Parkinson's disease (PD). 647 patients with PRKN-PD were included in this international study. The pathogenic variants present were characterised and investigated for their effect on phenotype. Clinical features and progression of PRKN-PD was also assessed. Among 133 variants in index cases (n = 582), there were 58 (43.6%) structural variants, 34 (25.6%) missense, 20 (15%) frameshift, 10 splice site (7.5%%), 9 (6.8%) nonsense and 2 (1.5%) indels. The most frequent variant overall was an exon 3 deletion (n = 145, 12.3%), followed by the p.R275W substitution (n = 117, 10%). Exon3, RING0 protein domain and the ubiquitin-like protein domain were mutational hotspots with 31%, 35.4% and 31.7% of index cases presenting mutations in these regions respectively. The presence of a frameshift or structural variant was associated with a 3.4 ± 1.6 years or a 4.7 ± 1.6 years earlier age at onset of PRKN-PD respectively (p < 0.05). Furthermore, variants located in the N-terminus of the protein, a region enriched with frameshift variants, were associated with an earlier age at onset. The phenotype of PRKN-PD was characterised by slow motor progression, preserved cognition, an excellent motor response to levodopa therapy and later development of motor complications compared to early-onset PD. Non-motor symptoms were however common in PRKN-PD. Our findings on the relationship between the type of variant in PRKN and the phenotype of the disease may have implications for both genetic counselling and the design of precision clinical trials.

Parkin depletion delays motor decline dose-dependently without overtly affecting neuropathology in α-synuclein transgenic mice.

BACKGROUND: Mutations of the gene encoding the major component of Lewy bodies (LB), α-synuclein (α-syn), cause autosomal dominant forms of Parkinson's disease (PD), whereas loss-of-function mutations of the gene encoding the multifunctional E3 ubiquitin-protein ligase Parkin account for autosomal recessive forms of the disease. Parkin overproduction protects against α-syn-dependent neurodegeneration in various in vitro and in vivo models, but it remains unclear whether this process is affected by Parkin deficiency. We addressed this issue, by carrying out more detailed analyses of transgenic mice overproducing the A30P variant of human α-syn (hA30Pα-syn) and with two, one or no parkin knockout alleles. RESULTS: Longitudinal behavioral follow-up of these mice indicated that Parkin depletion delayed disease-predictive sensorimotor impairment due to α-syn accumulation, in a dose-dependent fashion. At the end stage of the disease, neuronal deposits containing fibrillar α-syn species phosphorylated at S129 (PS129α-syn) were the predominant neuropathological feature in hA30Pα-syn mice, regardless of their parkin expression. Some of these deposits colocalized with the LB markers ubiquitin and α-syn truncated at D135 (α-synD135), indicating that PS129α-syn is subjected to secondary posttranslational modification (PTM); these features were not significantly affected by parkin dysfunction. CONCLUSIONS: These findings suggest that Parkin deficiency acts as a protective modifier in α-syn-dependent neurodegeneration, without overtly affecting the composition and characteristics of α-syn deposits in end-stage disease.

Developmental transcriptional control of mitochondrial homeostasis is required for activity-dependent synaptic connectivity.

During neuronal circuit formation, local control of axonal organelles ensures proper synaptic connectivity. Whether this process is genetically encoded is unclear and if so, its developmental regulatory mechanisms remain to be identified. We hypothesized that developmental transcription factors regulate critical parameters of organelle homeostasis that contribute to circuit wiring. We combined cell type-specific transcriptomics with a genetic screen to discover such factors. We identified Telomeric Zinc finger-Associated Protein (TZAP) as a temporal developmental regulator of neuronal mitochondrial homeostasis genes, including Pink1 . In Drosophila , loss of dTzap function during visual circuit development leads to loss of activity-dependent synaptic connectivity, that can be rescued by Pink1 expression. At the cellular level, loss of dTzap/TZAP leads to defects in mitochondrial morphology, attenuated calcium uptake and reduced synaptic vesicle release in fly and mammalian neurons. Our findings highlight developmental transcriptional regulation of mitochondrial homeostasis as a key factor in activity-dependent synaptic connectivity.

Does the Expression and Epigenetics of Genes Involved in Monogenic Forms of Parkinson's Disease Influence Sporadic Forms?

Parkinson's disease (PD) is a disorder characterized by a triad of motor symptoms (akinesia, rigidity, resting tremor) related to loss of dopaminergic neurons mainly in the Substantia nigra pars compacta. Diagnosis is often made after a substantial loss of neurons has already occurred, and while dopamine replacement therapies improve symptoms, they do not modify the course of the disease. Although some biological mechanisms involved in the disease have been identified, such as oxidative stress and accumulation of misfolded proteins, they do not explain entirely PD pathophysiology, and a need for a better understanding remains. Neurodegenerative diseases, including PD, appear to be the result of complex interactions between genetic and environmental factors. The latter can alter gene expression by causing epigenetic changes, such as DNA methylation, post-translational modification of histones and non-coding RNAs. Regulation of genes responsible for monogenic forms of PD may be involved in sporadic PD. This review will focus on the epigenetic mechanisms regulating their expression, since these are the genes for which we currently have the most information available. Despite technical challenges, epigenetic epidemiology offers new insights on revealing altered biological pathways and identifying predictive biomarkers for the onset and progression of PD.

Modelling α-Synuclein Aggregation and Neurodegeneration with Fibril Seeds in Primary Cultures of Mouse Dopaminergic Neurons.

To model α-Synuclein (αS) aggregation and neurodegeneration in Parkinson's disease (PD), we established cultures of mouse midbrain dopamine (DA) neurons and chronically exposed them to fibrils 91 (F91) generated from recombinant human αS. We found that F91 have an exquisite propensity to seed the aggregation of endogenous αS in DA neurons when compared to other neurons in midbrain cultures. Until two weeks post-exposure, somal aggregation in DA neurons increased with F91 concentrations (0.01-0.75 µM) and the time elapsed since the initiation of seeding, with, however, no evidence of DA cell loss within this time interval. Neither toxin-induced mitochondrial deficits nor genetically induced loss of mitochondrial quality control mechanisms promoted F91-mediated αS aggregation or neurodegeneration under these conditions. Yet, a significant loss of DA neurons (~30%) was detectable three weeks after exposure to F91 (0.5 µM), i.e., at a time point where somal aggregation reached a plateau. This loss was preceded by early deficits in DA uptake. Unlike αS aggregation, the loss of DA neurons was prevented by treatment with GDNF, suggesting that αS aggregation in DA neurons may induce a form of cell death mimicking a state of trophic factor deprivation. Overall, our model system may be useful for exploring PD-related pathomechanisms and for testing molecules of therapeutic interest for this disorder.

Modeling native and seeded Synuclein aggregation and related cellular dysfunctions in dopaminergic neurons derived by a new set of isogenic iPSC lines with SNCA multiplications.

Triplication of the SNCA gene, encoding the protein alpha-Synuclein (αSyn), is a rare cause of aggressive and early-onset parkinsonism. Herein, we generated iPSCs from two siblings with a recently described compact SNCA gene triplication and suffering from severe motor impairments, psychiatric symptoms, and cognitive deterioration. Using CRISPR/Cas9 gene editing, each SNCA copy was inactivated by targeted indel mutations generating a panel of isogenic iPSCs with a decremental number from 4 down to none of functional SNCA gene alleles. We differentiated these iPSC lines in midbrain dopaminergic (DA) neuronal cultures to characterize αSyn aggregation in native and seeded conditions and evaluate its associated cellular dysfunctions. Utilizing a new nanobody-based biosensor combined with super-resolved imaging, we were able to visualize and measure αSyn aggregates in early DA neurons in unstimulated conditions. Calcium dysregulation and mitochondrial alterations were the first pathological signs detectable in early differentiated DA neuronal cultures. Accelerated αSyn aggregation was induced by exposing neurons to structurally well-characterized synthetic αSyn fibrils. 4xSNCA DA neurons showed the highest vulnerability, which was associated with high levels of oxidized DA and amplified by TAX1BP1 gene disruption. Seeded DA neurons developed large αSyn deposits whose morphology and internal constituents resembled Lewy bodies commonly observed in Parkinson's disease (PD) patient brain tissues. These findings provide strong evidence that this isogenic panel of iPSCs with SNCA multiplications offers a remarkable cellular platform to investigate mechanisms of PD and validate candidate inhibitors of native and seeded αSyn aggregation.