Mechanism of human PINK1 activation at the TOM complex in a reconstituted system.

Loss-of-function mutations in PTEN-induced kinase 1 (PINK1) are a frequent cause of early-onset Parkinson's disease (PD). Stabilization of PINK1 at the translocase of outer membrane (TOM) complex of damaged mitochondria is critical for its activation. The mechanism of how PINK1 is activated in the TOM complex is unclear. Here, we report that co-expression of human PINK1 and all seven TOM subunits in Saccharomyces cerevisiae is sufficient for PINK1 activation. We use this reconstitution system to systematically assess the role of each TOM subunit toward PINK1 activation. We unambiguously demonstrate that the TOM20 and TOM70 receptor subunits are required for optimal PINK1 activation and map their sites of interaction with PINK1 using AlphaFold structural modeling and mutagenesis. We also demonstrate an essential role of the pore-containing subunit TOM40 and its structurally associated subunits TOM7 and TOM22 for PINK1 activation. These findings will aid in the development of small-molecule activators of PINK1 as a therapeutic strategy for PD.

Development and characterization of phospho-ubiquitin antibodies to monitor PINK1-PRKN signaling in cells and tissue.

The selective removal of dysfunctional mitochondria, a process termed mitophagy, is critical for cellular health and impairments have been linked to aging, Parkinson disease, and other neurodegenerative conditions. A central mitophagy pathway is orchestrated by the ubiquitin (Ub) kinase PINK1 together with the E3 Ub ligase PRKN/Parkin. The decoration of damaged mitochondrial domains with phosphorylated Ub (p-S65-Ub) mediates their elimination though the autophagy system. As such p-S65-Ub has emerged as a highly specific and quantitative marker of mitochondrial damage with significant disease relevance. Existing p-S65-Ub antibodies have been successfully employed as research tools in a range of applications including western blot, immunocytochemistry, immunohistochemistry, and enzyme-linked immunosorbent assay. However, physiological levels of p-S65-Ub in the absence of exogenous stress are very low, therefore difficult to detect and require reliable and ultrasensitive methods. Here we generated and characterized a collection of novel recombinant, rabbit monoclonal p-S65-Ub antibodies with high specificity and affinity in certain applications that allow the field to better understand the molecular mechanisms and disease relevance of PINK1-PRKN signaling. These antibodies may also serve as novel diagnostic or prognostic tools to monitor mitochondrial damage in various clinical and pathological specimens.Abbreviations: AD: Alzheimer disease; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ELISA: enzyme-linked immunosorbent assay; HEK293E cell: human embryonic kidney E cell; ICC: immunocytochemistry; IHC: immunohistochemistry: KO: knockout; LoB: limit of blank; LoD: limit of detection; LoQ: limit of quantification; MEF: mouse embryonic fibroblast; MSD: Meso Scale Discovery; n.s.: non-significant; nonTg: non-transgenic; PBMC: peripheral blood mononuclear cell; PD: Parkinson disease; p-S65-PRKN: phosphorylated PRKN at serine 65; p-S65-Ub: phosphorylated Ub at serine 65; Ub: ubiquitin; WT: wild-type.

Discovery and characterization of noncanonical E2-conjugating enzymes.

E2-conjugating enzymes (E2s) play a central role in the enzymatic cascade that leads to the attachment of ubiquitin to a substrate. This process, termed ubiquitylation, is required to maintain cellular homeostasis and affects almost all cellular process. By interacting with multiple E3 ligases, E2s dictate the ubiquitylation landscape within the cell. Since its discovery, ubiquitylation has been regarded as a posttranslational modification that specifically targets lysine side chains (canonical ubiquitylation). We used Matrix-Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry to identify and characterize a family of E2s that are instead able to conjugate ubiquitin to serine and/or threonine. We used structural modeling and prediction tools to identify the key activity determinants that these E2s use to interact with ubiquitin as well as their substrates. Our results unveil the missing E2s necessary for noncanonical ubiquitylation, underscoring the adaptability and versatility of ubiquitin modifications.

Design and high-throughput implementation of MALDI-TOF/MS-based assays for Parkin E3 ligase activity.

Parkinson's disease (PD) is a progressive neurological disorder that manifests clinically as alterations in movement as well as multiple non-motor symptoms including but not limited to cognitive and autonomic abnormalities. Loss-of-function mutations in the gene encoding the ubiquitin E3 ligase Parkin are causal for familial and juvenile PD. Among several therapeutic approaches being explored to treat or improve the prognosis of patients with PD, the use of small molecules able to reinstate or boost Parkin activity represents a potential pharmacological treatment strategy. A major barrier is the lack of high-throughput platforms for the robust and accurate quantification of Parkin activity in vitro. Here, we present two different and complementary Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF/MS)-based approaches for the quantification of Parkin E3 ligase activity in vitro. Both approaches are scalable for high-throughput primary screening to facilitate the identification of Parkin modulators.

CURTAIN-A unique web-based tool for exploration and sharing of MS-based proteomics data.

To facilitate analysis and sharing of mass spectrometry (MS)-based proteomics data, we created online tools called CURTAIN (https://curtain.proteo.info) and CURTAIN-PTM (https://curtainptm.proteo.info) with an accompanying series of video tutorials (https://www.youtube.com/@CURTAIN-me6hl). These are designed to enable non-MS experts to interactively peruse volcano plots and deconvolute primary experimental data so that replicates can be visualized in bar charts or violin plots and exported in publication-ready format. They also allow assessment of overall experimental quality by correlation matrix and profile plot analysis. After making a selection of protein "hits", the user can analyze known domain structure, AlphaFold predicted structure, reported interactors, relative expression as well as disease links. CURTAIN-PTM permits analysis of all identified PTM sites on protein(s) of interest with selected databases. CURTAIN-PTM also links with the Kinase Library to predict upstream kinases that may phosphorylate sites of interest. We provide examples of the utility of CURTAIN and CURTAIN-PTM in analyzing how targeted degradation of the PPM1H Rab phosphatase that counteracts the Parkinson's LRRK2 kinase impacts cellular protein levels and phosphorylation sites. We also reanalyzed a ubiquitylation dataset, characterizing the PINK1-Parkin pathway activation in primary neurons, revealing data of interest not highlighted previously. CURTAIN and CURTAIN-PTM are free to use and open source, enabling researchers to share and maximize the impact of their proteomics data. We advocate that MS data published in volcano plot format be reported containing a shareable CURTAIN weblink, thereby allowing readers to better analyze and exploit the data.

Development and characterization of phospho-ubiquitin antibodies to monitor PINK1-PRKN signaling in cells and tissue.

The selective removal of dysfunctional mitochondria, a process termed mitophagy, is critical for cellular health and impairments have been linked to aging, Parkinson disease, and other neurodegenerative conditions. A central mitophagy pathway is orchestrated by the ubiquitin (Ub) kinase PINK1 together with the E3 Ub ligase PRKN/Parkin. The decoration of damaged mitochondrial domains with phosphorylated Ub (p-S65-Ub) mediates their elimination though the autophagy system. As such p-S65-Ub has emerged as a highly specific and quantitative marker of mitochondrial damage with significant disease relevance. Existing p-S65-Ub antibodies have been successfully employed as research tools in a range of applications including western blot, immunocytochemistry, immunohistochemistry, and ELISA. However, physiological levels of p-S65-Ub in the absence of exogenous stress are very low, therefore difficult to detect and require reliable and ultrasensitive methods. Here we generated and characterized a collection of novel recombinant, rabbit monoclonal p-S65-Ub antibodies with high specificity and affinity in certain applications that allow the field to better understand the molecular mechanisms and disease relevance of PINK1-PRKN signaling. These antibodies may also serve as novel diagnostic or prognostic tools to monitor mitochondrial damage in various clinical and pathological specimens.

Whole proteome copy number dataset in primary mouse cortical neurons.

The functional diversity of neurons is specified through their proteome resulting in elaborate and tightly regulated protein interaction networks and signalling that regulates neuronal processes. Dysregulation of these dynamic networks in development or in adulthood lead to neurodevelopmental or neurological disorders respectively. Over the past few decades, mass spectrometry has become a powerful tool for quantifying and resolving any proteome, including complex tissues such as the brain proteome, with technological advances leading to higher levels of resolution and throughput than traditional biochemical techniques. In this article, we provide a proteomic reference dataset that has been generated to identify proteins and quantify their level of expression in primary mouse cortical neurons. It represents a summary analysis of previously published data in (Antico et al., 2021). Mouse cortical neurons were isolated from E16.5 C57Bl/6J mice and cultured for 21 days in vitro (DIV). We employed the mitochondrial uncouplers AntimycinA/Oligomycin (AO) to induce mitochondrial depolarisation that is a well-established paradigm to assess mitophagic signalling. Total lysates from mouse primary cortical neurons were subjected to label-free quantitative proteomic analysis using both data dependent acquisition (DDA) and data independent acquisition (DIA) modes. DDA proteomic analysis identified a total dataset of 9367 proteins in mouse cortical neurons and absolute abundance of proteins was calculated as copy numbers per cell. DDA dataset was also processed to generate a reference spectral library to fit in and quantify MS spectra generated in DIA mode. Quantitative DIA analysis identified more than 6000 protein groups and statistical comparison of the two analysed groups (untreated and AO-treated) revealed that the neuronal proteome was largely unchanged post mitochondrial depolarisation for 5 hours. To our knowledge, these files represent the most comprehensive DDA and DIA reference datasets of fully functional maturated mouse primary cortical neurons and serve as a valuable resource for further investigating the role of specific proteins involved in neurobiology and neurological disorders such as Alzheimer's disease (AD), Parkinson's disease (PD) and Autism Spectrum Disorders (ASD).

Role of Autophagy Pathway in Parkinson's Disease and Related Genetic Neurological Disorders.

The elucidation of the function of the PINK1 protein kinase and Parkin ubiquitin E3 ligase in the elimination of damaged mitochondria by autophagy (mitophagy) has provided unprecedented understanding of the mechanistic pathways underlying Parkinson's disease (PD). We provide a comprehensive overview of the general importance of autophagy in Parkinson's disease and related disorders of the central nervous system. This reveals a critical link between autophagy and neurodegenerative and neurodevelopmental disorders and suggests that strategies to modulate mitophagy may have greater relevance in the CNS beyond PD.

Mapping of a N-terminal α-helix domain required for human PINK1 stabilization, Serine228 autophosphorylation and activation in cells.

Autosomal recessive mutations in the PINK1 gene are causal for Parkinson's disease (PD). PINK1 encodes a mitochondrial localized protein kinase that is a master-regulator of mitochondrial quality control pathways. Structural studies to date have elaborated the mechanism of how mutations located within the kinase domain disrupt PINK1 function; however, the molecular mechanism of PINK1 mutations located upstream and downstream of the kinase domain is unknown. We have employed mutagenesis studies to define the minimal region of human PINK1 required for optimal ubiquitin phosphorylation, beginning at residue Ile111. Inspection of the AlphaFold human PINK1 structure model predicts a conserved N-terminal α-helical extension (NTE) domain forming an intramolecular interaction with the C-terminal extension (CTE), which we corroborate using hydrogen/deuterium exchange mass spectrometry of recombinant insect PINK1 protein. Cell-based analysis of human PINK1 reveals that PD-associated mutations (e.g. Q126P), located within the NTE : CTE interface, markedly inhibit stabilization of PINK1; autophosphorylation at Serine228 (Ser228) and Ubiquitin Serine65 (Ser65) phosphorylation. Furthermore, we provide evidence that NTE and CTE domain mutants disrupt PINK1 stabilization at the mitochondrial Translocase of outer membrane complex. The clinical relevance of our findings is supported by the demonstration of defective stabilization and activation of endogenous PINK1 in human fibroblasts of a patient with early-onset PD due to homozygous PINK1 Q126P mutations. Overall, we define a functional role of the NTE : CTE interface towards PINK1 stabilization and activation and show that loss of NTE : CTE interactions is a major mechanism of PINK1-associated mutations linked to PD.

PTEN-induced kinase 1 (PINK1) and Parkin: Unlocking a mitochondrial quality control pathway linked to Parkinson's disease.

Dissection of the function of two Parkinson's disease-linked genes encoding the protein kinase, PTEN-induced kinase 1 (PINK1) and ubiquitin E3 ligase, Parkin, has illuminated a highly conserved mitochondrial quality control pathway found in nearly every cell type including neurons. Mitochondrial damage-induced activation of PINK1 stimulates phosphorylation-dependent activation of Parkin and ubiquitin-dependent elimination of mitochondria by autophagy (mitophagy). Structural, cell biological and neuronal studies are unravelling the key steps of PINK1/Parkin-dependent mitophagy and uncovering new insights into how the pathway is regulated. The emerging role for aberrant immune activation as a driver of dopaminergic neuron degeneration after loss of PINK1 and Parkin poses new exciting questions on cell-autonomous and noncell-autonomous mechanisms of PINK1/Parkin signalling in vivo.

Global ubiquitylation analysis of mitochondria in primary neurons identifies endogenous Parkin targets following activation of PINK1.

How activation of PINK1 and Parkin leads to elimination of damaged mitochondria by mitophagy is largely based on cell lines with few studies in neurons. Here, we have undertaken proteomic analysis of mitochondria from mouse neurons to identify ubiquitylated substrates of endogenous Parkin. Comparative analysis with human iNeuron datasets revealed a subset of 49 PINK1 activation­dependent diGLY sites in 22 proteins conserved across mouse and human systems. We use reconstitution assays to demonstrate direct ubiquitylation by Parkin in vitro. We also identified a subset of cytoplasmic proteins recruited to mitochondria that undergo PINK1 and Parkin independent ubiquitylation, indicating the presence of alternate ubiquitin E3 ligase pathways that are activated by mitochondrial depolarization in neurons. Last, we have developed an online resource to search for ubiquitin sites and enzymes in mitochondria of neurons, MitoNUb. These findings will aid future studies to understand Parkin activation in neuronal subtypes.

Parkinson's: A Disease of Aberrant Vesicle Trafficking.

Parkinson's disease (PD) is a leading cause of neurodegeneration that is defined by the selective loss of dopaminergic neurons and the accumulation of protein aggregates called Lewy bodies (LBs). The unequivocal identification of Mendelian inherited mutations in 13 genes in PD has provided transforming insights into the pathogenesis of this disease. The mechanistic analysis of several PD genes, including α-synuclein (α-syn), leucine-rich repeat kinase 2 (LRRK2), PTEN-induced kinase 1 (PINK1), and Parkin, has revealed central roles for protein aggregation, mitochondrial damage, and defects in endolysosomal trafficking in PD neurodegeneration. In this review, we outline recent advances in our understanding of these gene pathways with a focus on the emergent role of Rab (Ras analog in brain) GTPases and vesicular trafficking as a common mechanism that underpins how mutations in PD genes lead to neuronal loss. These advances have led to previously distinct genes such as vacuolar protein-sorting-associated protein 35 (VPS35) and LRRK2 being implicated in a common signaling pathway. A greater understanding of these common nodes of vesicular trafficking will be crucial for linking other PD genes and improving patient stratification in clinical trials underway against α-syn and LRRK2 targets.

PINK1-dependent phosphorylation of Serine111 within the SF3 motif of Rab GTPases impairs effector interactions and LRRK2-mediated phosphorylation at Threonine72.

Loss of function mutations in the PTEN-induced kinase 1 (PINK1) kinase are causal for autosomal recessive Parkinson's disease (PD) whilst gain of function mutations in the LRRK2 kinase cause autosomal dominant PD. PINK1 indirectly regulates the phosphorylation of a subset of Rab GTPases at a conserved Serine111 (Ser111) residue within the SF3 motif. Using genetic code expansion technologies, we have produced stoichiometric Ser111-phosphorylated Rab8A revealing impaired interactions with its cognate guanine nucleotide exchange factor and GTPase activating protein. In a screen for Rab8A kinases we identify TAK1 and MST3 kinases that can efficiently phosphorylate the Switch II residue Threonine72 (Thr72) in a similar manner as LRRK2 in vitro. Strikingly, we demonstrate that Ser111 phosphorylation negatively regulates the ability of LRRK2 but not MST3 or TAK1 to phosphorylate Thr72 of recombinant nucleotide-bound Rab8A in vitro and demonstrate an interplay of PINK1- and LRRK2-mediated phosphorylation of Rab8A in transfected HEK293 cells. Finally, we present the crystal structure of Ser111-phosphorylated Rab8A and nuclear magnetic resonance structure of Ser111-phosphorylated Rab1B. The structures reveal that the phosphorylated SF3 motif does not induce any major changes, but may interfere with effector-Switch II interactions through intramolecular H-bond formation and/or charge effects with Arg79. Overall, we demonstrate antagonistic regulation between PINK1-dependent Ser111 phosphorylation and LRRK2-mediated Thr72 phosphorylation of Rab8A indicating a potential cross-talk between PINK1-regulated mitochondrial homeostasis and LRRK2 signalling that requires further investigation in vivo.

Therapeutic approaches to enhance PINK1/Parkin mediated mitophagy for the treatment of Parkinson's disease.

The discovery of rare familial monogenic forms of early-onset Parkinson's disease has led to the identification of a mitochondrial quality control process as a key player in this disease. Loss-of-function mutations in the genes encoding PINK1 or Parkin result in insufficient removal of dysfunctional mitochondria through autophagy, a process termed mitophagy. Understanding the mechanism of this process and the function of its two key players, PINK1 and Parkin, has led to the discovery of new therapeutic approaches. Small molecule activators of mitophagy, either activating PINK1 or Parkin directly or inhibiting Parkin's counterplayer, the ubiquitin-specific protease USP30, are in preclinical development. To enable clinical success of future small molecule mitophagy enhancers, biomarkers for mitochondrial integrity and mitophagy are being developed. Only a few years after the discovery of mitophagy deficits in Parkinson's disease, research of the underlying mechanisms, drug discovery of modulators for this mechanism and identification of biomarkers provide new avenues towards the development of disease-modifying therapies.

Are PARKIN patients ideal candidates for dopaminergic cell replacement therapies?

Parkinson's is a heterogeneous, complex condition. Stratification of Parkinson's subtypes will be essential to identify those that will benefit most from a cell replacement therapy. Foetal mesencephalic grafts can alleviate motor symptoms in some Parkinson's patients. However, on-going synucleinopathy results in the grafts eventually developing Lewy bodies, and they begin to fail. We propose that Parkinson's patients with PARKIN mutations may benefit most from a cell replacement therapy because (a) they often lack synucleinopathy, and (b) their neurodegeneration is often confined to the nigrostriatal pathway. While patients with PARKIN mutations exhibit clinical signs of Parkinson's, post-mortem studies to date indicate the majority lack Lewy bodies suggesting the nigral dopaminergic neurons are lost in a cell autonomous manner independent of α-synuclein mechanisms. Furthermore, these patients are usually younger, slow progressing and typically do not suffer from complex non-nigral symptoms that are unlikely to be ameliorated by a cell replacement therapy. Transplantation of dopaminergic cells into the putamen of these patients will provide neurons with wild-type PARKIN expression to re-innervate the striatum. The focal nature of PARKIN-mediated neurodegeneration and lack of active synucleinopathy in most young-onset cases makes these patients ideal candidates for a dopaminergic cell replacement therapy. Strategies to improve the outcome of cell replacement therapies for sporadic Parkinson's include the use of adjunct therapeutics that target α-synuclein spreading and the use of genetically engineered grafts that are resistant to synucleinopathy.

Phosphorylation of Parkin at serine 65 is essential for its activation in vivo.

Mutations in PINK1 and Parkin result in autosomal recessive Parkinson's disease (PD). Cell culture and in vitro studies have elaborated the PINK1-dependent regulation of Parkin and defined how this dyad orchestrates the elimination of damaged mitochondria via mitophagy. PINK1 phosphorylates ubiquitin at serine 65 (Ser65) and Parkin at an equivalent Ser65 residue located within its N-terminal ubiquitin-like domain, resulting in activation; however, the physiological significance of Parkin Ser65 phosphorylation in vivo in mammals remains unknown. To address this, we generated a Parkin Ser65Ala (S65A) knock-in mouse model. We observe endogenous Parkin Ser65 phosphorylation and activation in mature primary neurons following mitochondrial depolarization and reveal this is disrupted in ParkinS65A/S65A neurons. Phenotypically, ParkinS65A/S65A mice exhibit selective motor dysfunction in the absence of any overt neurodegeneration or alterations in nigrostriatal mitophagy. The clinical relevance of our findings is substantiated by the discovery of homozygous PARKIN (PARK2) p.S65N mutations in two unrelated patients with PD. Moreover, biochemical and structural analysis demonstrates that the ParkinS65N/S65N mutant is pathogenic and cannot be activated by PINK1. Our findings highlight the central role of Parkin Ser65 phosphorylation in health and disease.

Basal Mitophagy Occurs Independently of PINK1 in Mouse Tissues of High Metabolic Demand.

Dysregulated mitophagy has been linked to Parkinson's disease (PD) due to the role of PTEN-induced kinase 1 (PINK1) in mediating depolarization-induced mitophagy in vitro. Elegant mouse reporters have revealed the pervasive nature of basal mitophagy in vivo, yet the role of PINK1 and tissue metabolic context remains unknown. Using mito-QC, we investigated the contribution of PINK1 to mitophagy in metabolically active tissues. We observed a high degree of mitophagy in neural cells, including PD-relevant mesencephalic dopaminergic neurons and microglia. In all tissues apart from pancreatic islets, loss of Pink1 did not influence basal mitophagy, despite disrupting depolarization-induced Parkin activation. Our findings provide the first in vivo evidence that PINK1 is detectable at basal levels and that basal mammalian mitophagy occurs independently of PINK1. This suggests multiple, yet-to-be-discovered pathways orchestrating mammalian mitochondrial integrity in a context-dependent fashion, and this has profound implications for our molecular understanding of vertebrate mitophagy.

The Anthelmintic Drug Niclosamide and Its Analogues Activate the Parkinson's Disease Associated Protein Kinase PINK1.

Mutations in PINK1, which impair its catalytic kinase activity, are causal for autosomal recessive early-onset Parkinson's disease (PD). Various studies have indicated that the activation of PINK1 could be a useful strategy in treating neurodegenerative diseases, such as PD. Herein, it is shown that the anthelmintic drug niclosamide and its analogues are capable of activating PINK1 in cells through the reversible impairment of the mitochondrial membrane potential. With these compounds, for the first time, it is demonstrated that the PINK1 pathway is active and detectable in primary neurons. These findings suggest that niclosamide and its analogues are robust compounds for the study of the PINK1 pathway and may hold promise as a therapeutic strategy in PD and related disorders.

Ubiquitin and Parkinson's disease through the looking glass of genetics.

Biochemical alterations found in the brains of Parkinson's disease (PD) patients indicate that cellular stress is a major driver of dopaminergic neuronal loss. Oxidative stress, mitochondrial dysfunction, and ER stress lead to impairment of the homeostatic regulation of protein quality control pathways with a consequent increase in protein misfolding and aggregation and failure of the protein degradation machinery. Ubiquitin signalling plays a central role in protein quality control; however, prior to genetic advances, the detailed mechanisms of how impairment in the ubiquitin system was linked to PD remained mysterious. The discovery of mutations in the α-synuclein gene, which encodes the main protein misfolded in PD aggregates, together with mutations in genes encoding ubiquitin regulatory molecules, including PTEN-induced kinase 1 (PINK1), Parkin, and FBX07, has provided an opportunity to dissect out the molecular basis of ubiquitin signalling disruption in PD, and this knowledge will be critical for developing novel therapeutic strategies in PD that target the ubiquitin system.

Kinetin Riboside and Its ProTides Activate the Parkinson's Disease Associated PTEN-Induced Putative Kinase 1 (PINK1) Independent of Mitochondrial Depolarization.

Since loss of function mutations of PINK1 lead to early onset Parkinson's disease, there has been growing interest in the discovery of small molecules that amplify the kinase activity of PINK1. We herein report the design, synthesis, serum stability, and hydrolysis of four kinetin riboside ProTides. These ProTides, along with kinetin riboside, activated PINK1 in cells independent of mitochondrial depolarization. This highlights the potential of modified nucleosides and their phosphate prodrugs as treatments for neurodegenerative diseases.