LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-κB to the nucleus.

Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling; however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here, we show that the NF-κB pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-κB to the nucleus. TNF treatment induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-κB is locally activated and transported to the nucleus by mitochondria, leading to an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 furthermore contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN. Overall, our study reveals a role for mitochondria in amplifying TNF-mediated NF-κB activation, both serving as a signaling platform, as well as a transport mode for activated NF-κB to the nuclear.

Role of GBA variants in Lewy body disease neuropathology.

Rare and common GBA variants are risk factors for both Parkinson's disease (PD) and dementia with Lewy bodies (DLB). However, the degree to which GBA variants are associated with neuropathological features in Lewy body disease (LBD) is unknown. Herein, we assessed 943 LBD cases and examined associations of 15 different neuropathological outcomes with common and rare GBA variants. Neuropathological outcomes included LBD subtype, presence of a high likelihood of clinical DLB (per consensus guidelines), LB counts in five cortical regions, tyrosine hydroxylase immunoreactivity in the dorsolateral and ventromedial putamen, ventrolateral substantia nigra neuronal loss, Braak neurofibrillary tangle (NFT) stage, Thal amyloid phase, phospho-ubiquitin (pS65-Ub) level, TDP-43 pathology, and vascular disease. Sequencing of GBA exons revealed a total of 42 different variants (4 common [MAF > 0.5%], 38 rare [MAF < 0.5%]) in our series, and 165 cases (17.5%) had a copy of the minor allele for ≥ 1 variant. In analysis of common variants, p.L483P was associated with a lower Braak NFT stage (OR = 0.10, P < 0.001). In gene-burden analysis, presence of the minor allele for any GBA variant was associated with increased odds of a high likelihood of DLB (OR = 2.00, P < 0.001), a lower Braak NFT stage (OR = 0.48, P < 0.001), a lower Thal amyloid phase (OR = 0.55, P < 0.001), and a lower pS65-Ub level (ß: -0.37, P < 0.001). Subgroup analysis revealed that GBA variants were most common in LBD cases with a combination of transitional/diffuse LBD and Braak NFT stage 0-II or Thal amyloid phase 0-1, and correspondingly that the aforementioned associations of GBA gene-burden with a decreased Braak NFT stage and Thal amyloid phase were observed only in transitional or diffuse LBD cases. Our results indicate that in LBD, GBA variants occur most frequently in cases with greater LB pathology and low AD pathology, further informing disease-risk associations of GBA in PD, PD dementia, and DLB.

Cathepsin B p.Gly284Val Variant in Parkinson's Disease Pathogenesis.

Parkinson's disease (PD) is generally considered a sporadic disorder, but a strong genetic background is often found. The aim of this study was to identify the underlying genetic cause of PD in two affected siblings and to subsequently assess the role of mutations in Cathepsin B (CTSB) in susceptibility to PD. A typical PD family was identified and whole-exome sequencing was performed in two affected siblings. Variants of interest were validated using Sanger sequencing. CTSB p.Gly284Val was genotyped in 2077 PD patients and 615 unrelated healthy controls from the Czech Republic, Ireland, Poland, Ukraine, and the USA. The gene burden analysis was conducted for the CTSB gene in an additional 769 PD probands from Mayo Clinic Florida familial PD cohort. CTSB expression and activity in patient-derived fibroblasts and controls were evaluated by qRT-PCR, western blot, immunocytochemistry, and enzymatic assay. The CTSB p.Gly284Val candidate variant was only identified in affected family members. Functional analysis of CTSB patient-derived fibroblasts under basal conditions did not reveal overt changes in endogenous expression, subcellular localization, or enzymatic activity in the heterozygous carrier of the CTSB variant. The identification of the CTSB p.Gly284Val may support the hypothesis that the CTSB locus harbors variants with differing penetrance that can determine the disease risk.

Mitophagy alterations in Alzheimer's disease are associated with granulovacuolar degeneration and early tau pathology.

INTRODUCTION: The cytoprotective PTEN-induced kinase 1 (PINK1)-parkin RBR E3 ubiquitin protein ligase (PRKN) pathway selectively labels damaged mitochondria with phosphorylated ubiquitin (pS65-Ub) for their autophagic removal (mitophagy). Because dysfunctions of mitochondria and degradation pathways are early features of Alzheimer's disease (AD), mitophagy impairments may contribute to the pathogenesis. METHODS: Morphology, levels, and distribution of the mitophagy tag pS65-Ub were evaluated by biochemical analyses combined with tissue and single cell imaging in AD autopsy brain and in transgenic mouse models. RESULTS: Analyses revealed significant increases of pS65-Ub levels in AD brain, which strongly correlated with granulovacuolar degeneration (GVD) and early phospho-tau deposits, but were independent of amyloid beta pathology. Single cell analyses revealed predominant co-localization of pS65-Ub with mitochondria, GVD bodies, and/or lysosomes depending on the brain region analyzed. DISCUSSION: Our study highlights mitophagy alterations in AD that are associated with early tau pathology, and suggests that distinct mitochondrial, autophagic, and/or lysosomal failure may contribute to the selective vulnerability in disease.

Heterozygous PINK1 p.G411S increases risk of Parkinson's disease via a dominant-negative mechanism.

SEE GANDHI AND PLUN-FAVREAU DOI101093/AWW320 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: It has been postulated that heterozygous mutations in recessive Parkinson's genes may increase the risk of developing the disease. In particular, the PTEN-induced putative kinase 1 (PINK1) p.G411S (c.1231G>A, rs45478900) mutation has been reported in families with dominant inheritance patterns of Parkinson's disease, suggesting that it might confer a sizeable disease risk when present on only one allele. We examined families with PINK1 p.G411S and conducted a genetic association study with 2560 patients with Parkinson's disease and 2145 control subjects. Heterozygous PINK1 p.G411S mutations markedly increased Parkinson's disease risk (odds ratio = 2.92, P = 0.032); significance remained when supplementing with results from previous studies on 4437 additional subjects (odds ratio = 2.89, P = 0.027). We analysed primary human skin fibroblasts and induced neurons from heterozygous PINK1 p.G411S carriers compared to PINK1 p.Q456X heterozygotes and PINK1 wild-type controls under endogenous conditions. While cells from PINK1 p.Q456X heterozygotes showed reduced levels of PINK1 protein and decreased initial kinase activity upon mitochondrial damage, stress-response was largely unaffected over time, as expected for a recessive loss-of-function mutation. By contrast, PINK1 p.G411S heterozygotes showed no decrease of PINK1 protein levels but a sustained, significant reduction in kinase activity. Molecular modelling and dynamics simulations as well as multiple functional assays revealed that the p.G411S mutation interferes with ubiquitin phosphorylation by wild-type PINK1 in a heterodimeric complex. This impairs the protective functions of the PINK1/parkin-mediated mitochondrial quality control. Based on genetic and clinical evaluation as well as functional and structural characterization, we established p.G411S as a rare genetic risk factor with a relatively large effect size conferred by a partial dominant-negative function phenotype.

(Patho-)physiological relevance of PINK1-dependent ubiquitin phosphorylation.

Mutations in PINK1 and PARKIN cause recessive, early-onset Parkinson's disease (PD). Together, these two proteins orchestrate a protective mitophagic response that ensures the safe disposal of damaged mitochondria. The kinase PINK1 phosphorylates ubiquitin (Ub) at the conserved residue S65, in addition to modifying the E3 ubiquitin ligase Parkin. The structural and functional consequences of Ub phosphorylation (pS65-Ub) have already been suggested from in vitro experiments, but its (patho-)physiological significance remains unknown. We have generated novel antibodies and assessed pS65-Ub signals in vitro and in cells, including primary neurons, under endogenous conditions. pS65-Ub is dependent on PINK1 kinase activity as confirmed in patient fibroblasts and postmortem brain samples harboring pathogenic mutations. We show that pS65-Ub is reversible and barely detectable under basal conditions, but rapidly induced upon mitochondrial stress in cells and amplified in the presence of functional Parkin. pS65-Ub accumulates in human brain during aging and disease in the form of cytoplasmic granules that partially overlap with mitochondrial, lysosomal, and total Ub markers. Additional studies are now warranted to further elucidate pS65-Ub functions and fully explore its potential for biomarker or therapeutic development.

A specific subset of E2 ubiquitin-conjugating enzymes regulate Parkin activation and mitophagy differently.

Loss-of-function mutations in the genes encoding PINK1 and Parkin (also known as PARK2) are the most common causes of recessive Parkinson's disease. Both together mediate the selective degradation of mitochondrial proteins and whole organelles via the proteasome and the autophagy-lysosome pathway (mitophagy). The mitochondrial kinase PINK1 activates and recruits the E3 ubiquitin ligase Parkin to de-energized mitochondria. However, the cognate E2 co-enzymes of Parkin in this ubiquitin-dependent pathway have not been investigated. Here, we discovered a total of four E2s that either positively or negatively regulate the activation, translocation and enzymatic functions of Parkin during mitochondrial quality control. UBE2D family members and UBE2L3 redundantly charged the RING-HECT hybrid ligase Parkin with ubiquitin, resulting in its initial activation and translocation to mitochondria. UBE2N, however, primarily operated through a different mechanism in order to mediate the proper clustering of mitochondria, a prerequisite for degradation. Strikingly, in contrast to UBE2D, UBE2L3 and UBE2N, depletion of UBE2R1 resulted in enhanced Parkin translocation and clustering upon mitochondrial uncoupling. Our study uncovered redundant, cooperative or antagonistic functions of distinct E2 enzymes in the regulation of Parkin and mitophagy that might suggest a putative role in Parkinson's disease pathogenesis.

Structural and Functional Impact of Parkinson Disease-Associated Mutations in the E3 Ubiquitin Ligase Parkin.

Mutations in the PARKIN/PARK2 gene that result in loss-of-function of the encoded, neuroprotective E3 ubiquitin ligase Parkin cause recessive, familial early-onset Parkinson disease. As an increasing number of rare Parkin sequence variants with unclear pathogenicity are identified, structure-function analyses will be critical to determine their disease relevance. Depending on the specific amino acids affected, several distinct pathomechanisms can result in loss of Parkin function. These include disruption of overall Parkin folding, decreased solubility, and protein aggregation. However pathogenic effects can also result from misregulation of Parkin autoinhibition and of its enzymatic functions. In addition, interference of binding to coenzymes, substrates, and adaptor proteins can affect its catalytic activity too. Herein, we have performed a comprehensive structural and functional analysis of 21 PARK2 missense mutations distributed across the individual protein domains. Using this combined approach, we were able to pinpoint some of the pathogenic mechanisms of individual sequence variants. Similar analyses will be critical in gaining a complete understanding of the complex regulations and enzymatic functions of Parkin. These studies will not only highlight the important residues, but will also help to develop novel therapeutics aimed at activating and preserving an active, neuroprotective form of Parkin.

UBE2E ubiquitin-conjugating enzymes and ubiquitin isopeptidase Y regulate TDP-43 protein ubiquitination.

Trans-activation element DNA-binding protein of 43 kDa (TDP-43) characterizes insoluble protein aggregates in distinct subtypes of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. TDP-43 mediates many RNA processing steps within distinct protein complexes. Here we identify novel TDP-43 protein interactors found in a yeast two-hybrid screen using an adult human brain cDNA library. We confirmed the TDP-43 interaction of seven hits by co-immunoprecipitation and assessed their co-localization in HEK293E cells. As pathological TDP-43 is ubiquitinated, we focused on the ubiquitin-conjugating enzyme UBE2E3 and the ubiquitin isopeptidase Y (UBPY). When cells were treated with proteasome inhibitor, ubiquitinated and insoluble TDP-43 species accumulated. All three UBE2E family members could enhance the ubiquitination of TDP-43, whereas catalytically inactive UBE2E3(C145S) was much less efficient. Conversely, silencing of UBE2E3 reduced TDP-43 ubiquitination. We examined 15 of the 48 known disease-associated TDP-43 mutants and found that one was excessively ubiquitinated. This strong TDP-43(K263E) ubiquitination was further enhanced by proteasomal inhibition as well as UBE2E3 expression. Conversely, UBE2E3 silencing and expression of UBPY reduced TDP-43(K263E) ubiquitination. Moreover, wild-type but not active site mutant UBPY reduced ubiquitination of TDP-43 C-terminal fragments and of a nuclear import-impaired mutant. In Drosophila melanogaster, UBPY silencing enhanced neurodegenerative TDP-43 phenotypes and the accumulation of insoluble high molecular weight TDP-43 and ubiquitin species. Thus, UBE2E3 and UBPY participate in the regulation of TDP-43 ubiquitination, solubility, and neurodegeneration.

Activation of the E3 ubiquitin ligase Parkin.

The PINK1 (phosphatase and tensin homologue-induced putative kinase 1)/Parkin-dependent mitochondrial quality control pathway mediates the clearance of damaged organelles, but appears to be disrupted in Parkinson's disease (PD) [Springer and Kahle (2011) Autophagy 7, 266-278]. Upon mitochondrial stress, PINK1 activates the E3 ubiquitin (Ub) ligase Parkin through phosphorylation of the Ub-like (UBL) domain of Parkin and of the small modifier Ub itself at a conserved residue [Sauvé and Gehring (2014) Cell Res. 24, 1025-1026]. Recently resolved partial crystal structures of Parkin showed a 'closed', auto-inhibited conformation, consistent with its notoriously weak enzymatic activity at steady state [Wauer and Komander (2013) EMBO J. 32, 2099-2112; Riley et al. (2013) Nat. Commun. 4, 1982; Trempe et al. (2013) Science 340, 1451-1455; Spratt et al. (2013) Nat. Commun. 4, 1983]. It has thus become clear that Parkin must undergo major structural rearrangements in order to unleash its catalytic functions. Recent published findings derived from X-ray structures and molecular modelling present a complete structural model of human Parkin at an all-atom resolution [Caulfield et al. (2014) PLoS Comput. Biol. 10, e1003935]. The results of the combined in silico simulations-based and experimental assay-based study indicates that PINK1-dependent Ser65 phosphorylation of Parkin is required for its activation and triggering of 'opening' conformations. Indeed, the obtained structures showed a sequential release of Parkin's intertwined domains and allowed docking of an Ub-charged E2 coenzyme, which could enable its enzymatic activity. In addition, using cell-based screening, select E2 enzymes that redundantly, cooperatively or antagonistically regulate Parkin's activation and/or enzymatic functions at different stages of the mitochondrial autophagy (mitophagy) process were identified [Fiesel et al. (2014) J. Cell Sci. 127, 3488-3504]. Other work that aims to pin-point the particular pathogenic dysfunctions of Parkin mis-sense mutations have been recently disseminated (Fabienne C. Fiesel, Thomas R. Caulfield, Elisabeth L. Moussaud-Lamodiere, Daniel F.A.R. Dourado, Kotaro Ogaki, Owen A. Ross, Samuel C. Flores, and Wolfdieter Springer, submitted). Such a structure-function approach provides the basis for the dissection of Parkin's regulation and a targeted drug design to identify small-molecule activators of this neuroprotective E3 Ub ligase.

Phosphorylation by PINK1 releases the UBL domain and initializes the conformational opening of the E3 ubiquitin ligase Parkin.

Loss-of-function mutations in PINK1 or PARKIN are the most common causes of autosomal recessive Parkinson's disease. Both gene products, the Ser/Thr kinase PINK1 and the E3 Ubiquitin ligase Parkin, functionally cooperate in a mitochondrial quality control pathway. Upon stress, PINK1 activates Parkin and enables its translocation to and ubiquitination of damaged mitochondria to facilitate their clearance from the cell. Though PINK1-dependent phosphorylation of Ser65 is an important initial step, the molecular mechanisms underlying the activation of Parkin's enzymatic functions remain unclear. Using molecular modeling, we generated a complete structural model of human Parkin at all atom resolution. At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions. Evidently, Parkin has to undergo major structural rearrangements in order to unleash its catalytic activity. As a spark, we have modeled PINK1-dependent Ser65 phosphorylation in silico and provide the first molecular dynamics simulation of Parkin conformations along a sequential unfolding pathway that could release its intertwined domains and enable its catalytic activity. We combined free (unbiased) molecular dynamics simulation, Monte Carlo algorithms, and minimal-biasing methods with cell-based high content imaging and biochemical assays. Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain. This motion propagates through further opening conformations that allow binding of an Ub-loaded E2 co-enzyme. Subsequent spatial reorientation of the catalytic centers of both enzymes might facilitate the transfer of the Ub moiety to charge Parkin. Our structure-function study provides the basis to elucidate regulatory mechanisms and activity of the neuroprotective Parkin. This may open up new avenues for the development of small molecule Parkin activators through targeted drug design.

Knockdown of transactive response DNA-binding protein (TDP-43) downregulates histone deacetylase 6.

TDP-43 is an RNA/DNA-binding protein implicated in transcriptional repression and mRNA processing. Inclusions of TDP-43 are hallmarks of frontotemporal dementia and amyotrophic lateral sclerosis. Besides aggregation of TDP-43, loss of nuclear localization is observed in disease. To identify relevant targets of TDP-43, we performed expression profiling. Thereby, histone deacetylase 6 (HDAC6) downregulation was discovered on TDP-43 silencing and confirmed at the mRNA and protein level in human embryonic kidney HEK293E and neuronal SH-SY5Y cells. This was accompanied by accumulation of the major HDAC6 substrate, acetyl-tubulin. HDAC6 levels were restored by re-expression of TDP-43, dependent on RNA binding and the C-terminal protein interaction domains. Moreover, TDP-43 bound specifically to HDAC6 mRNA arguing for a direct functional interaction. Importantly, in vivo validation in TDP-43 knockout Drosophila melanogaster confirmed the specific downregulation of HDAC6. HDAC6 is necessary for protein aggregate formation and degradation. Indeed, HDAC6-dependent reduction of cellular aggregate formation and increased cytotoxicity of polyQ-expanded ataxin-3 were found in TDP-43 silenced cells. In conclusion, loss of functional TDP-43 causes HDAC6 downregulation and might thereby contribute to pathogenesis.

TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR.

TDP-43 is linked to neurodegenerative diseases including frontotemporal dementia and amyotrophic lateral sclerosis. Mostly localized in the nucleus, TDP-43 acts in conjunction with other ribonucleoproteins as a splicing co-factor. Several RNA targets of TDP-43 have been identified so far, but its role(s) in pathogenesis remains unclear. Using Affymetrix exon arrays, we have screened for the first time for splicing events upon TDP-43 knockdown. We found alternative splicing of the ribosomal S6 kinase 1 (S6K1) Aly/REF-like target (SKAR) upon TDP-43 knockdown in non-neuronal and neuronal cell lines. Alternative SKAR splicing depended on the first RNA recognition motif (RRM1) of TDP-43 and on 5'-GA-3' and 5'-UG-3' repeats within the SKAR pre-mRNA. SKAR is a component of the exon junction complex, which recruits S6K1, thereby facilitating the pioneer round of translation and promoting cell growth. Indeed, we found that expression of the alternatively spliced SKAR enhanced S6K1-dependent signaling pathways and the translational yield of a splice-dependent reporter. Consistent with this, TDP-43 knockdown also increased translational yield and significantly increased cell size. This indicates a novel mechanism of deregulated translational control upon TDP-43 deficiency, which might contribute to pathogenesis of the protein aggregation diseases frontotemporal dementia and amyotrophic lateral sclerosis.

In Silico Investigation of Parkin-Activating Mutations Using Simulations and Network Modeling.

Complete loss-of-function mutations in the PRKN gene are a major cause of early-onset Parkinson's disease (PD). PRKN encodes the Parkin protein, an E3 ubiquitin ligase that works in conjunction with the ubiquitin kinase PINK1 in a distinct quality control pathway to tag damaged mitochondria for autophagic clearance, i.e., mitophagy. According to previous structural investigations, Parkin protein is typically kept in an inactive conformation via several intramolecular, auto-inhibitory interactions. Here, we performed molecular dynamics simulations (MDS) to provide insights into conformational changes occurring during the de-repression of Parkin and the gain of catalytic activity. We analyzed four different Parkin-activating mutations that are predicted to disrupt certain aspects of its auto-inhibition. All four variants showed greater conformational motions compared to wild-type protein, as well as differences in distances between domain interfaces and solvent-accessible surface area, which are thought to play critical roles as Parkin gains catalytic activity. Our findings reveal that the studied variants exert a notable influence on Parkin activation as they alter the opening of its closed inactive structure, a finding that is supported by recent structure- and cell-based studies. These findings not only helped further characterize the hyperactive variants but overall improved our understanding of Parkin's catalytic activity and nominated targets within Parkin's structure for potential therapeutic designs.

Identification and structural characterization of small molecule inhibitors of PINK1.

Mutations in PINK1 and Parkin cause early-onset Parkinson's Disease (PD). PINK1 is a kinase which functions as a mitochondrial damage sensor and initiates mitochondrial quality control by accumulating on the damaged organelle. There, it phosphorylates ubiquitin, which in turn recruits and activates Parkin, an E3 ubiquitin ligase. Ubiquitylation of mitochondrial proteins leads to the autophagic degradation of the damaged organelle. Pharmacological modulation of PINK1 constitutes an appealing avenue to study its physiological function and develop therapeutics. In this study, we used a thermal shift assay with insect PINK1 to identify small molecules that inhibit ATP hydrolysis and ubiquitin phosphorylation. PRT062607, an SYK inhibitor, is the most potent inhibitor in our screen and inhibits both insect and human PINK1, with an IC50 in the 0.5-3 µM range in HeLa cells and dopaminergic neurons. The crystal structures of insect PINK1 bound to PRT062607 or CYC116 reveal how the compounds interact with the ATP-binding pocket. PRT062607 notably engages with the catalytic aspartate and causes a destabilization of insert-2 at the autophosphorylation dimer interface. While PRT062607 is not selective for PINK1, it provides a scaffold for the development of more selective and potent inhibitors of PINK1 that could be used as chemical probes.

The UFMylation pathway is impaired in Alzheimer's disease.

Background: Alzheimer's disease (AD) is characterized by the presence of neurofibrillary tangles made of hyperphosphorylated tau and senile plaques composed of beta-amyloid. These pathognomonic deposits have been implicated in the pathogenesis, although the molecular mechanisms and consequences remain undetermined. UFM1 is an important, but understudied ubiquitin-like protein that is covalently attached to substrates. This UFMylation has recently been identified as major modifier of tau aggregation upon seeding in experimental models. However, potential alterations of the UFM1 pathway in human AD brain have not been investigated yet. Methods: Here we used frontal and temporal cortex samples from individuals with or without AD to measure the protein levels of the UFMylation pathway in human brain. We used multivariable regression analyses followed by Bonferroni correction for multiple testing to analyze associations of the UFMylation pathway with neuropathological characteristics, primary biochemical measurements of tau and additional biochemical markers from the same cases. We further studied associations of the UFMylation cascade with cellular stress pathways using Spearman correlations with bulk RNAseq expression data and functionally validated these interactions using gene-edited neurons that were generated by CRISPR-Cas9. Results: Compared to controls, human AD brain had increased protein levels of UFM1. Our data further indicates that this increase mainly reflects conjugated UFM1 indicating hyperUFMylation in AD. UFMylation was strongly correlated with pathological tau in both AD-affected brain regions. In addition, we found that the levels of conjugated UFM1 were negatively correlated with soluble levels of the deUFMylation enzyme UFSP2. Functional analysis of UFM1 and/or UFSP2 knockout neurons revealed that the DNA damage response as well as the unfolded protein response are perturbed by changes in neuronal UFM1 signaling. Conclusions: There are marked changes in the UFMylation pathway in human AD brain. These changes are significantly associated with pathological tau, supporting the idea that the UFMylation cascade might indeed act as a modifier of tau pathology in human brain. Our study further nominates UFSP2 as an attractive target to reduce the hyperUFMylation observed in AD brain but also underscores the critical need to identify risks and benefits of manipulating the UFMylation pathway as potential therapeutic avenue for AD.

NLRP3 inflammasome activation and altered mitophagy are key pathways in inclusion body myositis.

Background: Inclusion body myositis (IBM) is the most prevalent muscle disease in adults for which no current treatment exists. The pathogenesis of IBM remains poorly defined. Inflammation and mitochondrial dysfunction are the most common histopathological findings. In this study, we aimed to explore the interplay between inflammation and mitochondrial dysfunction in IBM patients, highlighting sex differences. Methods: We included 38 IBM patients and 22 age- and sex-matched controls without myopathy. Bulk RNA sequencing, Meso Scale Discovery ELISA, western blotting, histochemistry and immunohistochemistry were performed on frozen muscle samples from the study participants. Results: We demonstrated activation of the NLRP3 inflammasome in IBM muscle samples, with the NLRP3 inflammasome pathway being the most upregulated. On muscle histopathology, there is increased NRLP3 immunoreactivity in both inflammatory cells and muscle fibers. Mitophagy is critical for removing damaged mitochondria and preventing the formation of a vicious cycle of mitochondrial dysfunction-NLRP3 activation. In the IBM muscle samples, we showed altered mitophagy, most significantly in males, with elevated levels of p-S65-Ubiquitin, a mitophagy marker. Furthermore, p-S65-Ubiquitin aggregates accumulated in muscle fibers that were mostly type 2 and devoid of cytochrome-c-oxidase reactivity. Type 2 muscle fibers are known to be more prone to mitochondrial dysfunction. NLRP3 RNA levels correlated with p-S65-Ubiquitin levels in both sexes but with loss of in muscle strength only in males. Finally, we identified sex-specific molecular pathways in IBM, with females having activation of pathways that could offset some of the pathomechanisms of IBM. Conclusions: NLRP3 inflammasome is activated in IBM, along with altered mitophagy particularly in males, which is of potential therapeutic significance. These findings suggest sex-specific mechanisms in IBM that warrant further investigation.

Alterations of PINK1-PRKN signaling in mice during normal aging.

The ubiquitin kinase-ligase pair PINK1-PRKN identifies and selectively marks damaged mitochondria for elimination via the autophagy-lysosome system (mitophagy). While this cytoprotective pathway has been extensively studied in vitro upon acute and complete depolarization of mitochondria, the significance of PINK1-PRKN mitophagy in vivo is less well established. Here we used a novel approach to study PINK1-PRKN signaling in different energetically demanding tissues of mice during normal aging. We demonstrate a generally increased expression of both genes and enhanced enzymatic activity with aging across tissue types. Collectively our data suggest a distinct regulation of PINK1-PRKN signaling under basal conditions with the most pronounced activation and flux of the pathway in mouse heart compared to brain or skeletal muscle. Our biochemical analyses complement existing mitophagy reporter readouts and provide an important baseline assessment in vivo, setting the stage for further investigations of the PINK1-PRKN pathway during stress and in relevant disease conditions.

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.

Structural and Functional Characterization of the Most Frequent Pathogenic PRKN Substitution p.R275W.

Mutations in the PINK1 and PRKN genes are the most frequent genetic cause of early-onset Parkinson disease. The pathogenic p.R275W substitution in PRKN is the most frequent substitution observed in patients, and thus far has been characterized mostly through overexpression models that suggest a possible gain of toxic misfunction. However, its effects under endogenous conditions are largely unknown. We used patient fibroblasts, isogenic neurons, and post-mortem human brain samples from carriers with and without PRKN p.R275W to assess functional impact. Immunoblot analysis and immunofluorescence were used to study mitophagy activation, and mitophagy execution was analyzed by flow cytometry of the reporter mitoKeima. The functional analysis was accompanied by structural investigation of PRKN p.R275W. We observed lower PRKN protein in fibroblasts with compound heterozygous p.R275W mutations. Isogenic neurons showed an allele-dose dependent decrease in PRKN protein. Lower PRKN protein levels were accompanied by diminished phosphorylated ubiquitin and decreased MFN2 modification. Mitochondrial degradation was also allele-dose dependently impaired. Consistently, PRKN protein levels were drastically reduced in human brain samples from p.R275W carriers. Finally, structural simulations showed significant changes in the closed form of PRKN p.R275W. Our data suggest that under endogenous conditions the p.R275W mutation results in a loss-of-function by destabilizing PRKN.