Loss of mitochondrial function often leads to neurodegeneration and is thought to be one of the underlying causes of neurodegenerative diseases such as Parkinson's disease (PD). However, the precise events linking mitochondrial dysfunction to neuronal death remain elusive. PTEN-induced putative kinase 1 (PINK1) and Parkin (Park), either of which, when mutated, are responsible for early-onset PD, mark individual mitochondria for destruction at the mitochondrial outer membrane. The specific molecular pathways that regulate signaling between the nucleus and mitochondria to sense mitochondrial dysfunction under normal physiological conditions are not well understood. Here, we show that Drosophila Clueless (Clu), a highly conserved protein required for normal mitochondrial function, can associate with Translocase of the outer membrane (TOM) 20, Porin and PINK1, and is thus located at the mitochondrial outer membrane. Previously, we found that clu genetically interacts with park in Drosophila female germ cells. Here, we show that clu also genetically interacts with PINK1, and our epistasis analysis places clu downstream of PINK1 and upstream of park. In addition, Clu forms a complex with PINK1 and Park, further supporting that Clu links mitochondrial function with the PINK1-Park pathway. Lack of Clu causes PINK1 and Park to interact with each other, and clu mutants have decreased mitochondrial protein levels, suggesting that Clu can act as a negative regulator of the PINK1-Park pathway. Taken together, these results suggest that Clu directly modulates mitochondrial function, and that Clu's function contributes to the PINK1-Park pathway of mitochondrial quality control.

Neddylation is a posttranslational modification that plays important roles in regulating protein structure and function by covalently conjugating NEDD8, an ubiquitin-like small molecule, to the substrate. Here, we report that Parkinson's disease (PD)-related parkin and PINK1 are NEDD8 conjugated. Neddylation of parkin and PINK1 results in increased E3 ligase activity of parkin and selective stabilization of the 55 kDa PINK1 fragment. Expression of dAPP-BP1, a NEDD8 activation enzyme subunit, in Drosophila suppresses abnormalities induced by dPINK1 RNAi. PD neurotoxin MPP(+) inhibits neddylation of both parkin and PINK1. NEDD8 immunoreactivity is associated with Lewy bodies in midbrain dopaminergic neurons of PD patients. Together, these results suggest that parkin and PINK1 are regulated by neddylation and that impaired NEDD8 modification of these proteins likely contributes to PD pathogenesis.

CNOT1 is a member of the CCR4-NOT complex, which is a master regulator, orchestrating gene expression, RNA deadenylation, and protein ubiquitination. We report on 39 individuals with heterozygous de novo CNOT1 variants, including missense, splice site, and nonsense variants, who present with a clinical spectrum of intellectual disability, motor delay, speech delay, seizures, hypotonia, and behavioral problems. To link CNOT1 dysfunction to the neurodevelopmental phenotype observed, we generated variant-specific Drosophila models, which showed learning and memory defects upon CNOT1 knockdown. Introduction of human wild-type CNOT1 was able to rescue this phenotype, whereas mutants could not or only partially, supporting our hypothesis that CNOT1 impairment results in neurodevelopmental delay. Furthermore, the genetic interaction with autism-spectrum genes, such as ASH1L, DYRK1A, MED13, and SHANK3, was impaired in our Drosophila models. Molecular characterization of CNOT1 variants revealed normal CNOT1 expression levels, with both mutant and wild-type alleles expressed at similar levels. Analysis of protein-protein interactions with other members indicated that the CCR4-NOT complex remained intact. An integrated omics approach of patient-derived genomics and transcriptomics data suggested only minimal effects on endonucleolytic nonsense-mediated mRNA decay components, suggesting that de novo CNOT1 variants are likely haploinsufficient hypomorph or neomorph, rather than dominant negative. In summary, we provide strong evidence that de novo CNOT1 variants cause neurodevelopmental delay with a wide range of additional co-morbidities. Whereas the underlying pathophysiological mechanism warrants further analysis, our data demonstrate an essential and central role of the CCR4-NOT complex in human brain development.Master regulators controlling development include, but are not limited to, paired box (PAX) proteins, 1 SRY-related HMG-box (SOX) proteins, 2 and the relatively unknown CCR4-NOT protein complex. 3 Although the full spectrum of functional diversity for the CCR4-NOT complex has not yet been established, it has already become apparent that it is active on all levels of gene expression, from accessibility of the DNA to translation and degradation of mRNA. 4

Mutations in the Parkin gene (PARK2) have been linked to a recessive form of Parkinson's disease (PD) characterized by the loss of dopaminergic neurons in the substantia nigra. Deficiencies of mitochondrial respiratory chain complex I activity have been observed in the substantia nigra of PD patients, and loss of Parkin results in the reduction of complex I activity shown in various cell and animal models. Using co-immunoprecipitation and proximity ligation assays on endogenous proteins, we demonstrate that Parkin interacts with mitochondrial Stomatin-like protein 2 (SLP-2), which also binds the mitochondrial lipid cardiolipin and functions in the assembly of respiratory chain proteins. SH-SY5Y cells with a stable knockdown of Parkin or SLP-2, as well as induced pluripotent stem cell-derived neurons from Parkin mutation carriers, showed decreased complex I activity and altered mitochondrial network morphology. Importantly, induced expression of SLP-2 corrected for these mitochondrial alterations caused by reduced Parkin function in these cells. In-vivo Drosophila studies showed a genetic interaction of Parkin and SLP-2, and further, tissue-specific or global overexpression of SLP-2 transgenes rescued parkin mutant phenotypes, in particular loss of dopaminergic neurons, mitochondrial network structure, reduced ATP production, and flight and motor dysfunction. The physical and genetic interaction between Parkin and SLP-2 and the compensatory potential of SLP-2 suggest a functional epistatic relationship to Parkin and a protective role of SLP-2 in neurons. This finding places further emphasis on the significance of Parkin for the maintenance of mitochondrial function in neurons and provides a novel target for therapeutic strategies.

The identification of genetic variants that predispose individuals to cardiovascular disease and a better understanding of their targets would be highly advantageous. Genome-wide association studies have identified variants that associate with QT-interval length (a measure of myocardial repolarization). Three of the strongest associating variants (single-nucleotide polymorphisms) are located in the putative promotor region of CNOT1, a gene encoding the central CNOT1 subunit of CCR4-NOT: a multifunctional, conserved complex regulating gene expression and mRNA stability and turnover. We isolated the minimum fragment of the CNOT1 promoter containing all three variants from individuals homozygous for the QT risk alleles and demonstrated that the haplotype associating with longer QT interval caused reduced reporter expression in a cardiac cell line, suggesting that reduced CNOT1 expression might contribute to abnormal QT intervals. Systematic siRNA-mediated knockdown of CCR4-NOT components in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) revealed that silencing CNOT1 and other CCR4-NOT genes reduced their proliferative capacity. Silencing CNOT7 also shortened action potential duration. Furthermore, the cardiac-specific knockdown of Drosophila orthologs of CCR4-NOT genes in vivo (CNOT1/Not1 and CNOT7/8/Pop2) was either lethal or resulted in dilated cardiomyopathy, reduced contractility or a propensity for arrhythmia. Silencing CNOT2/Not2, CNOT4/Not4 and CNOT6/6L/twin also affected cardiac chamber size and contractility. Developmental studies suggested that CNOT1/Not1 and CNOT7/8/Pop2 are required during cardiac remodeling from larval to adult stages. To summarize, we have demonstrated how disease-associated genes identified by GWAS can be investigated by combining human cardiomyocyte cell-based and whole-organism in vivo heart models. Our results also suggest a potential link of CNOT1 and CNOT7/8 to QT alterations and further establish a crucial role of the CCR4-NOT complex in heart development and function.This article has an associated First Person interview with the first author of the paper.

Highlights d Flies in microgravity exhibit cardiac constriction, remodeling, and diminished output d Heart defects correlate with reduced sarcomeric and extracellular matrix gene expression d Proteosome gene or protein expression is upregulated, suggesting proteostasis imbalance

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Background - KCNMA1 encodes the α-subunit of the large-conductance Ca 2+ -activated K + channel, K Ca 1.1, and lies within a linkage interval for atrial fibrillation (AF). Insights into the cardiac functions of K Ca 1.1 are limited and KCNMA1 has not been investigated as an AF candidate gene. Methods - The KCNMA1 gene was sequenced in 118 patients with familial AF. The role of K Ca 1.1 in normal cardiac structure and function was evaluated in humans, mice, zebrafish, and fly. A novel KCNMA1 variant was functionally characterized. Results - A complex KCNMA1 variant was identified in one kindred with AF. To evaluate potential disease mechanisms, we first evaluated the distribution of K Ca 1.1 in normal hearts using immunostaining and immunogold electron microscopy. K Ca 1.1 was seen throughout the atria and ventricles in humans and mice, with strong expression in the sinus node. In an ex vivo murine sinoatrial node preparation, addition of the K Ca 1.1 antagonist, paxilline, blunted the increase in beating rate induced by adrenergic receptor stimulation. Knockdown of the K Ca 1.1 ortholog, kcnma1b , in zebrafish embryos resulted in sinus bradycardia with dilatation and reduced contraction of the atrium and ventricle. Genetic inactivation of the Drosophila K Ca 1.1 ortholog, slo , systemically or in adult stages, also slowed the heartbeat and produced fibrillatory cardiac contractions. Electrophysiological characterization of slo -deficient flies revealed bursts of action potentials, reflecting increased events of fibrillatory arrhythmias. Flies with cardiac-specific overexpression of the human KCNMA1 mutant also showed increased heart period and bursts of action potentials, similar to the K Ca 1.1 loss-of-function models. Conclusions - Our data point to a highly conserved role of K Ca 1.1 in sinus node function in humans, mice, zebrafish and fly and suggest that K Ca 1.1 loss of function may predispose to AF.