Mic19 depletion impairs endoplasmic reticulum-mitochondrial contacts and mitochondrial lipid metabolism and triggers liver disease.

Endoplasmic reticulum (ER)-mitochondria contacts are critical for the regulation of lipid transport, synthesis, and metabolism. However, the molecular mechanism and physiological function of endoplasmic reticulum-mitochondrial contacts remain unclear. Here, we show that Mic19, a key subunit of MICOS (mitochondrial contact site and cristae organizing system) complex, regulates ER-mitochondria contacts by the EMC2-SLC25A46-Mic19 axis. Mic19 liver specific knockout (LKO) leads to the reduction of ER-mitochondrial contacts, mitochondrial lipid metabolism disorder, disorganization of mitochondrial cristae and mitochondrial unfolded protein stress response in mouse hepatocytes, impairing liver mitochondrial fatty acid ß-oxidation and lipid metabolism, which may spontaneously trigger nonalcoholic steatohepatitis (NASH) and liver fibrosis in mice. Whereas, the re-expression of Mic19 in Mic19 LKO hepatocytes blocks the development of liver disease in mice. In addition, Mic19 overexpression suppresses MCD-induced fatty liver disease. Thus, our findings uncover the EMC2-SLC25A46-Mic19 axis as a pathway regulating ER-mitochondria contacts, and reveal that impairment of ER-mitochondria contacts may be a mechanism associated with the development of NASH and liver fibrosis.

Hypoxia-reprogramed megamitochondrion contacts and engulfs lysosome to mediate mitochondrial self-digestion.

Mitochondria are the key organelles for sensing oxygen, which is consumed by oxidative phosphorylation to generate ATP. Lysosomes contain hydrolytic enzymes that degrade misfolded proteins and damaged organelles to maintain cellular homeostasis. Mitochondria physically and functionally interact with lysosomes to regulate cellular metabolism. However, the mode and biological functions of mitochondria-lysosome communication remain largely unknown. Here, we show that hypoxia remodels normal tubular mitochondria into megamitochondria by inducing broad inter-mitochondria contacts and subsequent fusion. Importantly, under hypoxia, mitochondria-lysosome contacts are promoted, and certain lysosomes are engulfed by megamitochondria, in a process we term megamitochondria engulfing lysosome (MMEL). Both megamitochondria and mature lysosomes are required for MMEL. Moreover, the STX17-SNAP29-VAMP7 complex contributes to mitochondria-lysosome contacts and MMEL under hypoxia. Intriguingly, MMEL mediates a mode of mitochondrial degradation, which we termed mitochondrial self-digestion (MSD). Moreover, MSD increases mitochondrial ROS production. Our results reveal a mode of crosstalk between mitochondria and lysosomes and uncover an additional pathway for mitochondrial degradation.

SIAH proteins regulate the degradation and intra-mitochondrial aggregation of PINK1: Implications for mitochondrial pathology in Parkinson's disease.

Parkinson's disease (PD) is characterized by degeneration of neurons, particularly dopaminergic neurons in the substantia nigra. PD brains show accumulation of α-synuclein in Lewy bodies and accumulation of dysfunctional mitochondria. However, the mechanisms leading to mitochondrial pathology in sporadic PD are poorly understood. PINK1 is a key for mitophagy activation and recycling of unfit mitochondria. The activation of mitophagy depends on the accumulation of uncleaved PINK1 at the outer mitochondrial membrane and activation of a cascade of protein ubiquitination at the surface of the organelle. We have now found that SIAH3, a member of the SIAH proteins but lacking ubiquitin-ligase activity, is increased in PD brains and cerebrospinal fluid and in neurons treated with α-synuclein preformed fibrils (α-SynPFF). We also observed that SIAH3 is aggregated together with PINK1 in the mitochondria of PD brains. SIAH3 directly interacts with PINK1, leading to their intra-mitochondrial aggregation in cells and neurons and triggering a cascade of toxicity with PINK1 inactivation along with mitochondrial depolarization and neuronal death. We also found that SIAH1 interacts with PINK1 and promotes ubiquitination and proteasomal degradation of PINK1. Similar to the dimerization of SIAH1/SIAH2, SIAH3 interacts with SIAH1, promoting its translocation to mitochondria and preventing its ubiquitin-ligase activity toward PINK1. Our results support the notion that the increase in SIAH3 and intra-mitochondrial aggregation of SIAH3-PINK1 may mediate α-synuclein pathology by promoting proteotoxicity and preventing the elimination of dysfunctional mitochondria. We consider it possible that PINK1 activity is decreased in sporadic PD, which impedes proper mitochondrial renewal in the disease.

Can We Treat Neurodegenerative Proteinopathies by Enhancing Protein Degradation?

Neurodegenerative proteinopathies are defined as a class of neurodegenerative disorders, with either genetic or sporadic age-related onset, characterized by the pathological accumulation of aggregated protein deposits. These mainly include Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD) as well as frontotemporal lobar degeneration (FTLD). The deposition of abnormal protein aggregates in the brain of patients affected by these disorders is thought to play a causative role in neuronal loss and disease progression. On that account, the idea of improving the clearance of pathological protein aggregates has taken hold as a potential therapeutic strategy. Among the possible approaches to pursue for reducing disease protein accumulation, there is the stimulation of the main protein degradation machineries of eukaryotic cells: the ubiquitin proteasomal system (UPS) and autophagy lysosomal pathway (ALP). Of note, several clinical trials testing the efficacy of either UPS- or ALP-active compounds are currently ongoing. Here, we discuss the main gaps and controversies emerging from experimental studies and clinical trials assessing the therapeutic efficacy of modulators of either the UPS or ALP in neurodegenerative proteinopathies, to gather whether they may constitute a real gateway from these disorders. © 2022 International Parkinson and Movement Disorder Society.

ATAD3B is a mitophagy receptor mediating clearance of oxidative stress-induced damaged mitochondrial DNA.

Mitochondrial DNA (mtDNA) encodes several key components of respiratory chain complexes that produce cellular energy through oxidative phosphorylation. mtDNA is vulnerable to damage under various physiological stresses, especially oxidative stress. mtDNA damage leads to mitochondrial dysfunction, and dysfunctional mitochondria can be removed by mitophagy, an essential process in cellular homeostasis. However, how damaged mtDNA is selectively cleared from the cell, and how damaged mtDNA triggers mitophagy, remain mostly unknown. Here, we identified a novel mitophagy receptor, ATAD3B, which is specifically expressed in primates. ATAD3B contains a LIR motif that binds to LC3 and promotes oxidative stress-induced mitophagy in a PINK1-independent manner, thus promoting the clearance of damaged mtDNA induced by oxidative stress. Under normal conditions, ATAD3B hetero-oligomerizes with ATAD3A, thus promoting the targeting of the C-terminal region of ATAD3B to the mitochondrial intermembrane space. Oxidative stress-induced mtDNA damage or mtDNA depletion reduces ATAD3B-ATAD3A hetero-oligomerization and leads to exposure of the ATAD3B C-terminus at the mitochondrial outer membrane and subsequent recruitment of LC3 for initiating mitophagy. Furthermore, ATAD3B is little expressed in m.3243A > G mutated cells and MELAS patient fibroblasts showing endogenous oxidative stress, and ATAD3B re-expression promotes the clearance of m.3243A > G mutated mtDNA. Our findings uncover a new pathway to selectively remove damaged mtDNA and reveal that increasing ATAD3B activity is a potential therapeutic approach for mitochondrial diseases.

SUMOylation in α-Synuclein Homeostasis and Pathology.

The accumulation and aggregation of α-synuclein are central to Parkinson's disease (PD), yet the molecular mechanisms responsible for these events are not fully understood. Post-translational modifications of α-synuclein regulate several of its properties, including degradation, interaction with proteins and membranes, aggregation and toxicity. SUMOylation is a post-translational modification involved in various nuclear and extranuclear processes, such as subcellular protein targeting, mitochondrial fission and synaptic plasticity. Protein SUMOylation increases in response to several stressful situations, from viral infections to trauma. In this framework, an increasing amount of evidence has implicated SUMOylation in several neurodegenerative diseases, including PD. This review will discuss recent findings in the role of SUMOylation as a regulator of α-synuclein accumulation, aggregation and toxicity, and its possible implication in neurodegeneration that underlies PD.

Physiological and pathological roles of LRRK2 in the nuclear envelope integrity.

Mutations in LRRK2 cause autosomal dominant and sporadic Parkinson's disease, but the mechanisms involved in LRRK2 toxicity in PD are yet to be fully understood. We found that LRRK2 translocates to the nucleus by binding to seven in absentia homolog (SIAH-1), and in the nucleus it directly interacts with lamin A/C, independent of its kinase activity. LRRK2 knockdown caused nuclear lamina abnormalities and nuclear disruption. LRRK2 disease mutations mostly abolish the interaction with lamin A/C and, similar to LRRK2 knockdown, cause disorganization of lamin A/C and leakage of nuclear proteins. Dopaminergic neurons of LRRK2 G2019S transgenic and LRRK2 -/- mice display decreased circularity of the nuclear lamina and leakage of the nuclear protein 53BP1 to the cytosol. Dopaminergic nigral and cortical neurons of both LRRK2 G2019S and idiopathic PD patients exhibit abnormalities of the nuclear lamina. Our data indicate that LRRK2 plays an essential role in maintaining nuclear envelope integrity. Disruption of this function by disease mutations suggests a novel phosphorylation-independent loss-of-function mechanism that may synergize with other neurotoxic effects caused by LRRK2 mutations.

The NMDA receptor activation by d-serine and glycine is controlled by an astrocytic Phgdh-dependent serine shuttle.

Astrocytes express the 3-phosphoglycerate dehydrogenase (Phgdh) enzyme required for the synthesis of l-serine from glucose. Astrocytic l-serine was proposed to regulate NMDAR activity by shuttling to neurons to sustain d-serine production, but this hypothesis remains untested. We now report that inhibition of astrocytic Phgdh suppressed the de novo synthesis of l-and d-serine and reduced the NMDAR synaptic potentials and long-term potentiation (LTP) at the Schaffer collaterals-CA1 synapse. Likewise, enzymatic removal of extracellular l-serine impaired LTP, supporting an l-serine shuttle mechanism between glia and neurons in generating the NMDAR coagonist d-serine. Moreover, deletion of serine racemase (SR) in glutamatergic neurons abrogated d-serine synthesis to the same extent as Phgdh inhibition, suggesting that neurons are the predominant source of the newly synthesized d-serine. We also found that the synaptic NMDAR activation in adult SR-knockout (KO) mice requires Phgdh-derived glycine, despite the sharp decline in the postnatal glycine levels as a result of the emergence of the glycine cleavage system. Unexpectedly, we also discovered that glycine regulates d-serine metabolism by a dual mechanism. The first consists of tonic inhibition of SR by intracellular glycine observed in vitro, primary cultures, and in vivo microdialysis. The second involves a transient glycine-induce d-serine release through the Asc-1 transporter, an effect abolished in Asc-1 KO mice and diminished by deleting SR in glutamatergic neurons. Our observations suggest that glycine is a multifaceted regulator of d-serine metabolism and implicate both d-serine and glycine in mediating NMDAR synaptic activation at the mature hippocampus through a Phgdh-dependent shuttle mechanism.

Lipid and immune abnormalities causing age-dependent neurodegeneration and Parkinson's disease.

This article describes pathogenic concepts and factors, in particular glycolipid abnormalities, that create cell dysfunction and synaptic loss in neurodegenerative diseases. By phenocopying lysosomal storage disorders, such as Gaucher disease and related disorders, age- and dose-dependent changes in glycolipid cell metabolism can lead to Parkinson's disease and related dementias. Recent results show that perturbation of sphingolipid metabolism can precede or is a part of abnormal protein handling in both genetic and idiopathic Parkinson's disease and Lewy body dementia. In aging and genetic predisposition with lipid disturbance, α-synuclein's normal vesicular and synaptic role may be detrimentally shifted toward accommodating and binding such lipids. Specific neuronal glycolipid, protein, and vesicular interactions create potential pathophysiology that is amplified by astroglial and microglial immune mechanisms resulting in neurodegeneration. This perspective provides a new logic for therapeutic interventions that do not focus on protein aggregation, but rather provides a guide to the complex biology and the common sequence of events that lead to age-dependent neurodegenerative disorders.

SUMOylation and ubiquitination reciprocally regulate α-synuclein degradation and pathological aggregation.

α-Synuclein accumulation is a pathological hallmark of Parkinson's disease (PD). Ubiquitinated α-synuclein is targeted to proteasomal or lysosomal degradation. Here, we identify SUMOylation as a major mechanism that counteracts ubiquitination by different E3 ubiquitin ligases and regulates α-synuclein degradation. We report that PIAS2 promotes SUMOylation of α-synuclein, leading to a decrease in α-synuclein ubiquitination by SIAH and Nedd4 ubiquitin ligases, and causing its accumulation and aggregation into inclusions. This was associated with an increase in α-synuclein release from the cells. A SUMO E1 inhibitor, ginkgolic acid, decreases α-synuclein levels by relieving the inhibition exerted on α-synuclein proteasomal degradation. α-Synuclein disease mutants are more SUMOylated compared with the wild-type protein, and this is associated with increased aggregation and inclusion formation. We detected a marked increase in PIAS2 expression along with SUMOylated α-synuclein in PD brains, providing a causal mechanism underlying the up-regulation of α-synuclein SUMOylation in the disease. We also found a significant proportion of Lewy bodies in nigral neurons containing SUMO1 and PIAS2. Our observations suggest that SUMOylation of α-synuclein by PIAS2 promotes α-synuclein aggregation by two mutually reinforcing mechanisms. First, it has a direct proaggregatory effect on α-synuclein. Second, SUMOylation facilitates α-synuclein aggregation by blocking its ubiquitin-dependent degradation pathways and promoting its accumulation. Therefore, inhibitors of α-synuclein SUMOylation provide a strategy to reduce α-synuclein levels and possibly aggregation in PD.

AF-6 Protects Against Dopaminergic Dysfunction and Mitochondrial Abnormalities in Drosophila Models of Parkinson's Disease.

Afadin 6 (AF-6) is an F-actin binding multidomain-containing scaffolding protein that is known for its function in cell-cell adhesion. Interestingly, besides this well documented role, we recently found that AF-6 is a Parkin-interacting protein that augments Parkin/PINK1-mediated mitophagy. Notably, mutations in Parkin and PINK1 are causative of recessively inherited forms of Parkinson's disease (PD) and aberrant mitochondrial homeostasis is thought to underlie PD pathogenesis. Given the novel role of AF-6 in mitochondrial quality control (QC), we hypothesized that AF-6 overexpression may be beneficial to PD. Using the Drosophila melanogaster as a model system, we demonstrate in this study that transgenic overexpression of human AF-6 in parkin and also pink1 null flies rescues their mitochondrial pathology and associated locomotion deficit, which results in their improved survival over time. Similarly, AF-6 overexpression also ameliorates the pathological phenotypes in flies expressing the Leucine Rich Repeat Kinase 2 (LRRK2) G2019S mutant, a mutation that is associated with dominantly-inherited PD cases in humans. Conversely, when endogenous AF-6 expression is silenced, it aggravates the disease phenotypes of LRRK2 mutant flies. Aside from these genetic models, we also found that AF-6 overexpression is protective against the loss of dopaminergic neurons in flies treated with rotenone, a mitochondrial complex I inhibitor commonly used to generate animal models of PD. Taken together, our results demonstrate that AF-6 protects against dopaminergic dysfunction and mitochondrial abnormalities in multiple Drosophila models of PD, and suggest the therapeutic value of AF-6-related pathways in mitigating PD pathogenesis.

Glycation potentiates α-synuclein-associated neurodegeneration in synucleinopathies.

α-Synuclein misfolding and aggregation is a hallmark in Parkinson's disease and in several other neurodegenerative diseases known as synucleinopathies. The toxic properties of α-synuclein are conserved from yeast to man, but the precise underpinnings of the cellular pathologies associated are still elusive, complicating the development of effective therapeutic strategies. Combining molecular genetics with target-based approaches, we established that glycation, an unavoidable age-associated post-translational modification, enhanced α-synuclein toxicity in vitro and in vivo, in Drosophila and in mice. Glycation affected primarily the N-terminal region of α-synuclein, reducing membrane binding, impaired the clearance of α-synuclein, and promoted the accumulation of toxic oligomers that impaired neuronal synaptic transmission. Strikingly, using glycation inhibitors, we demonstrated that normal clearance of α-synuclein was re-established, aggregation was reduced, and motor phenotypes in Drosophila were alleviated. Altogether, our study demonstrates glycation constitutes a novel drug target that can be explored in synucleinopathies as well as in other neurodegenerative conditions.

The Threshold Theory for Parkinson's Disease.

Parkinson's disease (PD) is recognized by the accumulation of α-synuclein within neurons. In contrast to the current ascending theory where α-synuclein would propagate from neuron to neuron, we now propose the threshold theory for PD based on evidence of parallel degeneration of both central nervous system (CNS) and peripheral nervous system (PNS) in PD. The functional threshold is lower for the emergence of early symptoms before the classical motor symptoms of PD. This is due to the larger functional reserve of the midbrain dopamine and integrated basal ganglia motor systems to control movement. This threshold theory better accounts for the current neurobiology of PD symptom progression compared to the hypothesis that the disease ascends from the PNS to the CNS as proposed by Braak's hypothesis.

Ubiqutination via K27 and K29 chains signals aggregation and neuronal protection of LRRK2 by WSB1.

A common genetic form of Parkinson's disease (PD) is caused by mutations in LRRK2. We identify WSB1 as a LRRK2 interacting protein. WSB1 ubiquitinates LRRK2 through K27 and K29 linkage chains, leading to LRRK2 aggregation and neuronal protection in primary neurons and a Drosophila model of G2019S LRRK2. Knocking down endogenous WSB1 exacerbates mutant LRRK2 neuronal toxicity in neurons and the Drosophila model, indicating a role for endogenous WSB1 in modulating LRRK2 cell toxicity. WSB1 is in Lewy bodies in human PD post-mortem tissue. These data demonstrate a role for WSB1 in mutant LRRK2 pathogenesis, and suggest involvement in Lewy body pathology in sporadic PD. Our data indicate a role in PD for ubiquitin K27 and K29 linkages, and suggest that ubiquitination may be a signal for aggregation and neuronal protection in PD, which may be relevant for other neurodegenerative disorders. Finally, our study identifies a novel therapeutic target for PD.

The PINK1, synphilin-1 and SIAH-1 complex constitutes a novel mitophagy pathway.

PTEN-induced putative kinase 1 (PINK1) and parkin are mutated in familial forms of Parkinson's disease and are important in promoting the mitophagy of damaged mitochondria. In this study, we showed that synphilin-1 interacted with PINK1 and was recruited to the mitochondria. Once in the mitochondria, it promoted PINK1-dependent mitophagy, as revealed by Atg5 knockdown experiments and the recruitment of LC3 and Lamp1 to the mitochondria. PINK1-synphilin-1 mitophagy did not depend on PINK1-mediated phosphorylation of synphilin-1 and occurred in the absence of parkin. Synphilin-1 itself caused depolarization of the mitochondria and increased the amount of uncleaved PINK1 at the organelle. Furthermore, synphilin-1 recruited seven in absentia homolog (SIAH)-1 to the mitochondria where it promoted mitochondrial protein ubiquitination and subsequent mitophagy. Mitophagy via this pathway was impaired by synphilin-1 knockdown or by the use of a synphilin-1 mutant that is unable to recruit SIAH-1 to the mitochondria. Likewise, knockdown of SIAH-1 or the use of a catalytically inactive SIAH-1 mutant abrogated mitophagy. PINK1 disease mutants failed to recruit synphilin-1 and did not activate mitophagy, indicating that PINK1-synphilin-1-SIAH-1 represents a new parkin-independent mitophagy pathway. Drugs that activate this pathway will provide a novel strategy to promote the clearance of damaged mitochondria in Parkinson's disease.

Synphilin-1 attenuates mutant LRRK2-induced neurodegeneration in Parkinson's disease models.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal-dominant Parkinsonism with pleomorphic pathology including deposits of aggregated protein and neuronal degeneration. The pathogenesis of LRRK2-linked Parkinson's disease (PD) is not fully understood. Here, using co-immunoprecipitation, we found that LRRK2 interacted with synphilin-1 (SP1), a cytoplasmic protein that interacts with α-synuclein and has implications in PD pathogenesis. LRRK2 interacted with the N-terminus of SP1 whereas SP1 predominantly interacted with the C-terminus of LRRK2, including kinase domain. Co-expression of SP1 with LRRK2 increased LRRK2-induced cytoplasmic aggregation in cultured cells. Moreover, SP1 also attenuated mutant LRRK2-induced toxicity and reduced LRRK2 kinase activity in cultured cells. Knockdown of SP1 by siRNA enhanced LRRK2 neuronal toxicity. In vivo Drosophila studies, co-expression of SP1 and mutant G2019S-LRRK2 in double transgenic Drosophila increased survival and improved locomotor activity. Expression of SP1 protects against G2019S-LRRK2-induced dopamine neuron loss and reduced LRRK2 phosphorylation in double transgenic fly brains. Our findings demonstrate that SP1 attenuates mutant LRRK2-induced PD-like phenotypes and plays a neural protective role.

Convergence of Parkin, PINK1, and α-Synuclein on Stress-induced Mitochondrial Morphological Remodeling.

Mutations in PARKIN (PARK2), an ubiquitin ligase, cause early onset Parkinson disease. Parkin was shown to bind, ubiquitinate, and target depolarized mitochondria for destruction by autophagy. This process, mitophagy, is considered crucial for maintaining mitochondrial integrity and suppressing Parkinsonism. Here, we report that under moderate mitochondrial stress, parkin does not translocate to mitochondria to induce mitophagy; rather, it stimulates mitochondrial connectivity. Mitochondrial stress-induced fusion requires PINK1 (PARK6), mitofusins, and parkin ubiquitin ligase activity. Upon exposure to mitochondrial toxins, parkin binds α-synuclein (PARK1), and in conjunction with the ubiquitin-conjugating enzyme Ubc13, stimulates K63-linked ubiquitination. Importantly, α-synuclein inactivation phenocopies parkin overexpression and suppresses stress-induced mitochondria fission, whereas Ubc13 inactivation abrogates parkin-dependent mitochondrial fusion. The convergence of parkin, PINK1, and α-synuclein on mitochondrial dynamics uncovers a common function of these PARK genes in the mitochondrial stress response and provides a potential physiological basis for the prevalence of α-synuclein pathology in Parkinson disease.

The alanine-serine-cysteine-1 (Asc-1) transporter controls glycine levels in the brain and is required for glycinergic inhibitory transmission.

Asc-1 (SLC7A10) is an amino acid transporter whose deletion causes neurological abnormalities and early postnatal death in mice. Using metabolomics and behavioral and electrophysiological methods, we demonstrate that Asc-1 knockout mice display a marked decrease in glycine levels in the brain and spinal cord along with impairment of glycinergic inhibitory transmission, and a hyperekplexia-like phenotype that is rescued by replenishing brain glycine. Asc-1 works as a glycine and L-serine transporter, and its transport activity is required for the subsequent conversion of L-serine into glycine in vivo. Asc-1 is a novel regulator of glycine metabolism and a candidate for hyperekplexia disorders.