CHCHD2 P14L, found in amyotrophic lateral sclerosis, exhibits cytoplasmic mislocalization and alters Ca2+ homeostasis.

CHCHD2 and CHCHD10, linked to Parkinson's disease and amyotrophic lateral sclerosis-frontotemporal dementia (ALS), respectively, are mitochondrial intermembrane proteins that form a heterodimer. This study aimed to investigate the impact of the CHCHD2 P14L variant, implicated in ALS, on mitochondrial function and its subsequent effects on cellular homeostasis. The missense variant of CHCHD2, P14L, found in a cohort of patients with ALS, mislocalized CHCHD2 to the cytoplasm, leaving CHCHD10 in the mitochondria. Drosophila lacking the CHCHD2 ortholog exhibited mitochondrial degeneration. In contrast, human CHCHD2 P14L, but not wild-type human CHCHD2, failed to suppress this degeneration, suggesting that P14L is a pathogenic variant. The mitochondrial Ca2+ buffering capacity was reduced in Drosophila neurons expressing human CHCHD2 P14L. The altered Ca2+-buffering phenotype was also observed in cultured human neuroblastoma SH-SY5Y cells expressing CHCHD2 P14L. In these cells, transient elevation of cytoplasmic Ca2+ facilitated the activation of calpain and caspase-3, accompanied by the processing and insolubilization of TDP-43. These observations suggest that CHCHD2 P14L causes abnormal Ca2+ dynamics and TDP-43 aggregation, reflecting the pathophysiology of ALS.

Parkinson disease-associated Leucine-rich repeat kinase regulates UNC-104-dependent axonal transport of Arl8-positive vesicles in Drosophila.

Some Parkinson's disease (PD)-causative/risk genes, including the PD-associated kinase leucine-rich repeat kinase 2 (LRRK2), are involved in membrane dynamics. Although LRRK2 and other PD-associated genes are believed to regulate synaptic functions, axonal transport, and endolysosomal activity, it remains unclear whether a common pathological pathway exists. Here, we report that the loss of Lrrk, an ortholog of human LRRK2, leads to the accumulation of the lysosome-related organelle regulator, Arl8 along with dense core vesicles at the most distal boutons of the neuron terminals in Drosophila. Moreover, the inactivation of a small GTPase Rab3 and altered Auxilin activity phenocopied Arl8 accumulation. The accumulation of Arl8-positive vesicles is UNC-104-dependent and modulated by PD-associated genes, Auxilin, VPS35, RME-8, and INPP5F, indicating that VPS35, RME-8, and INPP5F are upstream regulators of Lrrk. These results indicate that certain PD-related genes, along with LRRK2, drive precise neuroaxonal transport of dense core vesicles.

Ubiquitination at the lysine 27 residue of the Parkin ubiquitin-like domain is suggestive of a new mechanism of Parkin activation.

The mitochondrial kinase PTEN-induced kinase 1 (PINK1) and cytosolic ubiquitin ligase (E3) Parkin/PRKN are involved in mitochondrial quality control responses. PINK1 phosphorylates ubiquitin and the Parkin ubiquitin-like (Ubl) domain at serine 65 and promotes Parkin activation and translocation to damaged mitochondria. Upon Parkin activation, the Ubl domain is ubiquitinated at lysine (K) 27 and K48 residues. However, the contribution of K27/K48 ubiquitination toward Parkin activity remains unclear. In this study, ubiquitination of K56 (corresponding to K27 in the human), K77 (K48 in the human) or both was blocked by generating Drosophila Parkin (dParkin) mutants to examine the effects of Parkin Ubl domain ubiquitination on Parkin activation in Drosophila. The dParkin, in which K56 was replaced with arginine (dParkin K56R), rescued pupal lethality in flies by co-expression with PINK1, whereas dParkin K77R could not. The dParkin K56R exhibited reduced abilities of mitochondrial fragmentation and motility arrest, which are mediated by degrading Parkin E3 substrates Mitofusin and Miro, respectively. Pathogenic dParkin K56N, unlike dParkin K56R, destabilized the protein, suggesting that not only was dParkin K56N non-ubiquitin-modified at K56, but also the structure of the Ubl domain for activation was largely affected. Ubiquitin attached to K27 of the Ubl domain during PINK1-mediated Parkin activation was likely to be phosphorylated because human Parkin K27R weakened Parkin self-binding and activation in trans. Therefore, our findings suggest a new mechanism of Parkin activation, where an activation complex is formed through phospho-ubiquitin attachment on the K27 residue of the Parkin Ubl domain.

Syntaxin 17, an ancient SNARE paralog, plays different and conserved roles in different organisms.

Mammalian syntaxin 17 (Stx17) has several roles in processes other than membrane fusion, including in mitochondrial division, autophagosome formation and lipid droplet expansion. In contrast to conventional syntaxins, Stx17 has a long C-terminal hydrophobic region with a hairpin-like structure flanked by a basic amino acid-enriched C-terminal tail. Although Stx17 is one of the six ancient SNAREs and is present in diverse eukaryotic organisms, it has been lost in multiple lineages during evolution. In the present study, we compared the localization and function of fly and nematode Stx17s expressed in HeLa cells with those of human Stx17. We found that fly Stx17 predominantly localizes to the cytosol and mediates autophagy, but not mitochondrial division. Nematode Stx17, on the other hand, is predominantly present in mitochondria and facilitates mitochondrial division, but is irrelevant to autophagy. These differences are likely due to different structures in the C-terminal tail. Non-participation of fly Stx17 and nematode Stx17 in mitochondrial division and autophagy, respectively, was demonstrated in individual organisms. Our results provide an insight into the evolution of Stx17 in metazoa. This article has an associated First Person interview with the first author of the paper.

Analysis of Dopaminergic Functions in Drosophila.

Dopaminergic (DA) neurons regulate various physiological functions, including motor function, emotion, learning, sleep, and arousal. Degeneration of DA neurons in the substantia nigra of the midbrain causes motor disturbance in Parkinson's disease (PD). Studies on familial PD have revealed that a subset of PD genes encode proteins that regulate mitochondrial function and synaptic dynamics. Drosophila is a powerful model of PD, whereby genetic interactions of PD genes with well-conserved cellular signaling can be evaluated. Morphological changes in mitochondria, along with dysfunction and degeneration of DA neurons, have been reported in many studies using Drosophila PD models. In this chapter, we will describe imaging methods to visualize mitochondria in DA neurons and to evaluate spontaneous neural activity of DA neurons in the Drosophila brain.

Cytosolic and Mitochondrial Ca2+ Imaging in Drosophila Dopaminergic Neurons.

The ATP-producing organelle mitochondrion controls cellular or synaptic Ca2+ concentrations through temporal uptake of Ca2+ outside of the mitochondria. Although intracellular Ca2+ influx occurs during neuronal activity, a persistently higher concentration of intracellular Ca2+ is neurotoxic. Healthy mitochondria ensure rapid Ca2+ uptake, which is necessary for proper neuronal activity. Mitochondrial Ca2+ buffering activity decreases in aged or sick neurons. In this chapter, we will introduce our protocol for evaluating Ca2+ buffering activity through the mitochondria during neuronal activity of dopaminergic neurons.

Reduced astrocytic reactivity in human brains and midbrain organoids with PRKN mutations.

Parkin (encoded by PRKN) is a ubiquitin ligase that plays an important role in cellular mitochondrial quality control. Mutations in PRKN cause selective dopaminergic cell loss in the substantia nigra and are presumed to induce a decrease in mitochondrial function caused by the defective clearance of mitochondria. Several studies have demonstrated that parkin dysfunction causes mitochondrial injury and astrocytic dysfunction. Using immunohistochemical methods, we analyzed astrocytic changes in human brains from individuals with PRKN mutations. Few glial fibrillary acidic protein- and vimentin-positive astrocytes were observed in the substantia nigra in PRKN-mutated subjects compared with subjects with idiopathic Parkinson's disease. We also differentiated patient-specific induced pluripotent stem cells into midbrain organoids and confirmed decreased numbers of glial fibrillary acidic protein-positive astrocytes in PRKN-mutated organoids compared with age- and sex-matched controls. Our study reveals PRKN-mutation-induced astrocytic alteration and suggests the possibility of an astrocyte-related non-autonomous cell death mechanism for dopaminergic neurons in brains of PRKN-mutated patients.

Identifying Therapeutic Agents for Amelioration of Mitochondrial Clearance Disorder in Neurons of Familial Parkinson Disease.

Parkinson disease (PD) is a neurodegenerative disorder caused by the progressive loss of midbrain dopaminergic neurons, and mitochondrial dysfunction is involved in its pathogenesis. This study aimed to establish an imaging-based, semi-automatic, high-throughput system for the quantitative detection of disease-specific phenotypes in dopaminergic neurons from induced pluripotent stem cells (iPSCs) derived from patients with familial PD having Parkin or PINK1 mutations, which exhibit abnormal mitochondrial homeostasis. The proposed system recapitulates the deficiency of mitochondrial clearance, ROS accumulation, and increasing apoptosis in these familial PD-derived neurons. We screened 320 compounds for their ability to ameliorate multiple phenotypes and identified four candidate drugs. Some of these drugs improved the locomotion defects and reduced ATP production caused by PINK1 inactivation in Drosophila and were effective for idiopathic PD-derived neurons with impaired mitochondrial clearance. Our findings suggest that the proposed high-throughput system has potential for identifying effective drugs for familial and idiopathic PD.

A Cell-Based High-Throughput Screening Identified Two Compounds that Enhance PINK1-Parkin Signaling.

Early-onset Parkinson's disease-associated PINK1-Parkin signaling maintains mitochondrial health. Therapeutic approaches for enhancing PINK1-Parkin signaling present a potential strategy for treating various diseases caused by mitochondrial dysfunction. We report two chemical enhancers of PINK1-Parkin signaling, identified using a robust cell-based high-throughput screening system. These small molecules, T0466 and T0467, activate Parkin mitochondrial translocation in dopaminergic neurons and myoblasts at low doses that do not induce mitochondrial accumulation of PINK1. Moreover, both compounds reduce unfolded mitochondrial protein levels, presumably through enhanced PINK1-Parkin signaling. These molecules also mitigate the locomotion defect, reduced ATP production, and disturbed mitochondrial Ca2+ response in the muscles along with the mitochondrial aggregation in dopaminergic neurons through reduced PINK1 activity in Drosophila. Our results suggested that T0466 and T0467 may hold promise as therapeutic reagents in Parkinson's disease and related disorders.

Light-driven activation of mitochondrial proton-motive force improves motor behaviors in a Drosophila model of Parkinson's disease.

Mitochondrial degeneration is considered one of the major causes of Parkinson's disease (PD). Improved mitochondrial functions are expected to be a promising therapeutic strategy for PD. In this study, we introduced a light-driven proton transporter, Delta-rhodopsin (dR), to Drosophila mitochondria, where the mitochondrial proton-motive force (Δp) and mitochondrial membrane potential are maintained in a light-dependent manner. The loss of the PD-associated mitochondrial gene CHCHD2 resulted in reduced ATP production, enhanced mitochondrial peroxide production and lower Ca2+-buffering activity in dopaminergic (DA) terminals in flies. These cellular defects were improved by the light-dependent activation of mitochondrion-targeted dR (mito-dR). Moreover, mito-dR reversed the pathology caused by the CHCHD2 deficiency to suppress α-synuclein aggregation, DA neuronal loss, and elevated lipid peroxidation in brain tissue, improving motor behaviors. This study suggests the enhancement of Δp by mito-dR as a therapeutic mechanism that ameliorates neurodegeneration by protecting mitochondrial functions.

Mutations in CHCHD2 cause α-synuclein aggregation.

Mutations in CHCHD2 are linked to a familial, autosomal dominant form of Parkinson's disease (PD). The gene product may regulate mitochondrial respiratory function. However, whether mitochondrial dysfunction induced by CHCHD2 mutations further yields α-synuclein pathology is unclear. Here, we provide compelling genetic evidence that mitochondrial dysfunction induced by PD-linked CHCHD2 T61I mutation promotes α-synuclein aggregation using brain autopsy, induced pluripotent stem cells (iPSCs) and Drosophila genetics. An autopsy of an individual with CHCHD2 T61I revealed widespread Lewy pathology with both amyloid plaques and neurofibrillary tangles that appeared in the brain stem, limbic regions and neocortex. A prominent accumulation of sarkosyl-insoluble α-synuclein aggregates, the extent of which was comparable to that of a case with α-synuclein (SNCA) duplication, was observed in CHCHD2 T61I brain tissue. The prion-like activity and morphology of α-synuclein fibrils from the CHCHD2 T61I brain tissue were similar to those of fibrils from SNCA duplication and sporadic PD brain tissues. α-Synuclein insolubilization was reproduced in dopaminergic neuron cultures from CHCHD2 T61I iPSCs and Drosophila lacking the CHCHD2 ortholog or expressing the human CHCHD2 T61I. Moreover, the combination of ectopic α-synuclein expression and CHCHD2 null or T61I enhanced the toxicity in Drosophila dopaminergic neurons, altering the proteolysis pathways. Furthermore, CHCHD2 T61I lost its mitochondrial localization by α-synuclein in Drosophila. The mislocalization of CHCHD2 T61I was also observed in the patient brain. Our study suggests that CHCHD2 is a significant mitochondrial factor that determines α-synuclein stability in the etiology of PD.

Parkinson's disease-associated iPLA2-VIA/PLA2G6 regulates neuronal functions and α-synuclein stability through membrane remodeling.

Mutations in the iPLA2-VIA/PLA2G6 gene are responsible for PARK14-linked Parkinson's disease (PD) with α-synucleinopathy. However, it is unclear how iPLA2-VIA mutations lead to α-synuclein (α-Syn) aggregation and dopaminergic (DA) neurodegeneration. Here, we report that iPLA2-VIA-deficient Drosophila exhibits defects in neurotransmission during early developmental stages and progressive cell loss throughout the brain, including degeneration of the DA neurons. Lipid analysis of brain tissues reveals that the acyl-chain length of phospholipids is shortened by iPLA2-VIA loss, which causes endoplasmic reticulum (ER) stress through membrane lipid disequilibrium. The introduction of wild-type human iPLA2-VIA or the mitochondria-ER contact site-resident protein C19orf12 in iPLA2-VIA-deficient flies rescues the phenotypes associated with altered lipid composition, ER stress, and DA neurodegeneration, whereas the introduction of a disease-associated missense mutant, iPLA2-VIA A80T, fails to suppress these phenotypes. The acceleration of α-Syn aggregation by iPLA2-VIA loss is suppressed by the administration of linoleic acid, correcting the brain lipid composition. Our findings suggest that membrane remodeling by iPLA2-VIA is required for the survival of DA neurons and α-Syn stability.

Syntaxin 17 regulates the localization and function of PGAM5 in mitochondrial division and mitophagy.

PGAM5, a mitochondrial protein phosphatase that is genetically and biochemically linked to PINK1, facilitates mitochondrial division by dephosphorylating the mitochondrial fission factor Drp1. At the onset of mitophagy, PGAM5 is cleaved by PARL, a rhomboid protease that degrades PINK1 in healthy cells, and the cleaved form facilitates the engulfment of damaged mitochondria by autophagosomes by dephosphorylating the mitophagy receptor FUNDC1. Here, we show that the function and localization of PGAM5 are regulated by syntaxin 17 (Stx17), a mitochondria-associated membrane/mitochondria protein implicated in mitochondrial dynamics in fed cells and autophagy in starved cells. In healthy cells, loss of Stx17 causes PGAM5 aggregation within mitochondria and thereby failure of the dephosphorylation of Drp1, leading to mitochondrial elongation. In Parkin-mediated mitophagy, Stx17 is prerequisite for PGAM5 to interact with FUNDC1. Our results reveal that the Stx17-PGAM5 axis plays pivotal roles in mitochondrial division and PINK1/Parkin-mediated mitophagy.

Regulation of membrane dynamics by Parkinson's disease-associated genes.

Parkinson's disease (PD), the second most common neurodegenerative disease after Alzheimer's disease, develops sporadically, and its cause is unknown. However, 5-10% of PD cases are inherited as monogenic diseases, which provides a chance to understand the molecular mechanisms underlying neurodegeneration. Over 20 causative genes have already been identified and are being characterized. These PD-associated genes are broadly classified into two groups: genes involved in mitochondrial functions and genes related to membrane dynamics such as intracellular vesicle transport and the lysosomal pathway. In this review, we summarize the latest findings on the mechanism by which members of the latter group of PD-associated genes regulate membrane dynamics, and we discuss how mutations of these genes lead to dopaminergic neurodegeneration.

Monitoring Mitochondrial Changes by Alteration of the PINK1-Parkin Signaling in Drosophila.

Mitochondrial quality control is a key process in tissues with high energy demands, such as the brain and muscles. Recent studies using Drosophila have revealed that the genes responsible for familial forms of juvenile Parkinson's disease (PD), PINK1 and Parkin regulate mitochondrial function and motility. Cell biological analysis using mammalian cultured cells suggests that the dysregulation of mitophagy by PINK1 and Parkin leads to neurodegeneration in PD. In this chapter, we describe the methods to monitor mitochondrial morphology in the indirect flight muscles of adult Drosophila and Drosophila primary cultured neurons and the methods to analyze the motility of mitochondria in the axonal transport of living larval motor neurons.

Reduced TDP-43 Expression Improves Neuronal Activities in a Drosophila Model of Perry Syndrome.

Parkinsonian Perry syndrome, involving mutations in the dynein motor component dynactin or p150Glued, is characterized by TDP-43 pathology in affected brain regions, including the substantia nigra. However, the molecular relationship between p150Glued and TDP-43 is largely unknown. Here, we report that a reduction in TDP-43 protein levels alleviates the synaptic defects of neurons expressing the Perry mutant p150G50R in Drosophila. Dopaminergic expression of p150G50R, which decreases dopamine release, disrupts motor ability and reduces the lifespan of Drosophila. p150G50R expression also causes aggregation of dense core vesicles (DCVs), which contain monoamines and neuropeptides, and disrupts the axonal flow of DCVs, thus decreasing synaptic strength. The above phenotypes associated with Perry syndrome are improved by the removal of a copy of Drosophila TDP-43 TBPH, thus suggesting that the stagnation of axonal transport by dynactin mutations promotes TDP-43 aggregation and interferes with the dynamics of DCVs and synaptic activities.

Loss of Parkinson's disease-associated protein CHCHD2 affects mitochondrial crista structure and destabilizes cytochrome c.

Mutations in CHCHD2 have been identified in some Parkinson's disease (PD) cases. To understand the physiological and pathological roles of CHCHD2, we manipulated the expression of CHCHD2 in Drosophila and mammalian cells. The loss of CHCHD2 in Drosophila causes abnormal matrix structures and impaired oxygen respiration in mitochondria, leading to oxidative stress, dopaminergic neuron loss and motor dysfunction with age. These PD-associated phenotypes are rescued by the overexpression of the translation inhibitor 4E-BP and by the introduction of human CHCHD2 but not its PD-associated mutants. CHCHD2 is upregulated by various mitochondrial stresses, including the destabilization of mitochondrial genomes and unfolded protein stress, in Drosophila. CHCHD2 binds to cytochrome c along with a member of the Bax inhibitor-1 superfamily, MICS1, and modulated cell death signalling, suggesting that CHCHD2 dynamically regulates the functions of cytochrome c in both oxidative phosphorylation and cell death in response to mitochondrial stress.

Evidence that phosphorylated ubiquitin signaling is involved in the etiology of Parkinson's disease.

The ubiquitin (Ub) kinase PINK1 and the E3 Ub ligase Parkin, two gene products associated with young-onset Parkinson's disease (PD), participate in mitochondrial quality control. The phosphorylation of mitochondrial polyUb by PINK1, which is activated in a mitochondrial membrane potential (ΔΨm)-dependent manner, facilitates the mitochondrial translocation and concomitant enzymatic activation of Parkin, leading to the clearance of phospho-polyUb-tagged mitochondria via mitophagy. Thus, Ub phosphorylation is a key event in PINK1-Parkin-mediated mitophagy. Here, we examined the role of phospho-Ub signaling in the pathogenesis of PD using fly PD models, human brain tissue and dopaminergic neurons derived from induced pluripotent stem cells (iPSCs) containing Parkin or PINK1 mutations, as well as normal controls. We report that phospho-Ub signaling is highly conserved between humans and Drosophila, and that phospho-Ub signaling and the relocation of axonal mitochondria upon ΔΨm reduction are indeed compromised in human dopaminergic neurons containing Parkin or PINK1 mutations. Moreover, phospho-Ub signaling is prominent in tyrosine hydroxylase-positive neurons compared with tyrosine hydroxylase-negative neurons, suggesting that PINK1-Parkin signaling is more required for dopaminergic neurons. These results shed light on the particular vulnerability of dopaminergic neurons to mitochondrial stress.

Vps35 in cooperation with LRRK2 regulates synaptic vesicle endocytosis through the endosomal pathway in Drosophila.

Mutations of the retromer component Vps35 and endosomal kinase LRRK2 are linked to autosomal dominant forms of familial Parkinson's disease (PD). However, the physiological and pathological roles of Vps35 and LRRK2 in neuronal functions are poorly understood. Here, we demonstrated that the loss of Drosophila Vps35 (dVps35) affects synaptic vesicle recycling, dopaminergic synaptic release and sleep behavior associated with dopaminergic activity, which is rescued by the expression of wild-type dVps35 but not the PD-associated mutant dVps35 D647N. Drosophila LRRK2 dLRRK together with Rab5 and Rab11 is also implicated in synaptic vesicle recycling, and the manipulation of these activities improves the Vps35 synaptic phenotypes. These findings indicate that defects of synaptic vesicle recycling in which two late-onset PD genes, Vps35 and LRRK2, are involved could be key aspects of PD etiology.

Live Imaging of Axonal Transport in the Motor Neurons of Drosophila Larvae.

Axonal transport, which is composed of microtubules, motor proteins and a variety of types of cargo, is a prominent feature of neurons. Monitoring these molecular dynamics is important to understand the biological processes of neurons as well as neurodegenerative disorders that are associated with axonal dysfunction. Here, we describe a protocol for monitoring the axonal transport of motor neurons in Drosophila larvae using inverted fluorescence microscopy.