Unrestrained poly-ADP-ribosylation provides insights into chromatin regulation and human disease.

ARH3/ADPRHL2 and PARG are the primary enzymes reversing ADP-ribosylation in vertebrates, yet their functions in vivo remain unclear. ARH3 is the only hydrolase able to remove serine-linked mono(ADP-ribose) (MAR) but is much less efficient than PARG against poly(ADP-ribose) (PAR) chains in vitro. Here, by using ARH3-deficient cells, we demonstrate that endogenous MARylation persists on chromatin throughout the cell cycle, including mitosis, and is surprisingly well tolerated. Conversely, persistent PARylation is highly toxic and has distinct physiological effects, in particular on active transcription histone marks such as H3K9ac and H3K27ac. Furthermore, we reveal a synthetic lethal interaction between ARH3 and PARG and identify loss of ARH3 as a mechanism of PARP inhibitor resistance, both of which can be exploited in cancer therapy. Finally, we extend our findings to neurodegeneration, suggesting that patients with inherited ARH3 deficiency suffer from stress-induced pathogenic increase in PARylation that can be mitigated by PARP inhibition.

A genetic modifier suggests that endurance exercise exacerbates Huntington's disease.

Polyglutamine expansions in the huntingtin gene cause Huntington's disease (HD). Huntingtin is ubiquitously expressed, leading to pathological alterations also in peripheral organs. Variations in the length of the polyglutamine tract explain up to 70% of the age-at-onset variance, with the rest of the variance attributed to genetic and environmental modifiers. To identify novel disease modifiers, we performed an unbiased mutagenesis screen on an HD mouse model, identifying a mutation in the skeletal muscle voltage-gated sodium channel (Scn4a, termed 'draggen' mutation) as a novel disease enhancer. Double mutant mice (HD; Scn4aDgn/+) had decreased survival, weight loss and muscle atrophy. Expression patterns show that the main tissue affected is skeletal muscle. Intriguingly, muscles from HD; Scn4aDgn/+ mice showed adaptive changes similar to those found in endurance exercise, including AMPK activation, fibre type switching and upregulation of mitochondrial biogenesis. Therefore, we evaluated the effects of endurance training on HD mice. Crucially, this training regime also led to detrimental effects on HD mice. Overall, these results reveal a novel role for skeletal muscle in modulating systemic HD pathogenesis, suggesting that some forms of physical exercise could be deleterious in neurodegeneration.

Behavioural Characterisation of Macrod1 and Macrod2 Knockout Mice.

Adenosine diphosphate ribosylation (ADP-ribosylation; ADPr), the addition of ADP-ribose moieties onto proteins and nucleic acids, is a highly conserved modification involved in a wide range of cellular functions, from viral defence, DNA damage response (DDR), metabolism, carcinogenesis and neurobiology. Here we study MACROD1 and MACROD2 (mono-ADP-ribosylhydrolases 1 and 2), two of the least well-understood ADPr-mono-hydrolases. MACROD1 has been reported to be largely localized to the mitochondria, while the MACROD2 genomic locus has been associated with various neurological conditions such as autism, attention deficit hyperactivity disorder (ADHD) and schizophrenia; yet the potential significance of disrupting these proteins in the context of mammalian behaviour is unknown. Therefore, here we analysed both Macrod1 and Macrod2 gene knockout (KO) mouse models in a battery of well-defined, spontaneous behavioural testing paradigms. Loss of Macrod1 resulted in a female-specific motor-coordination defect, whereas Macrod2 disruption was associated with hyperactivity that became more pronounced with age, in combination with a bradykinesia-like gait. These data reveal new insights into the importance of ADPr-mono-hydrolases in aspects of behaviour associated with both mitochondrial and neuropsychiatric disorders.

Biallelic ADPRHL2 mutations in complex neuropathy affect ADP ribosylation and DNA damage response.

ADP ribosylation is a reversible posttranslational modification mediated by poly(ADP-ribose)transferases (e.g., PARP1) and (ADP-ribosyl)hydrolases (e.g., ARH3 and PARG), ensuring synthesis and removal of mono-ADP-ribose or poly-ADP-ribose chains on protein substrates. Dysregulation of ADP ribosylation signaling has been associated with several neurodegenerative diseases, including Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease. Recessive ADPRHL2/ARH3 mutations are described to cause a stress-induced epileptic ataxia syndrome with developmental delay and axonal neuropathy (CONDSIAS). Here, we present two families with a neuropathy predominant disorder and homozygous mutations in ADPRHL2 We characterized a novel C26F mutation, demonstrating protein instability and reduced protein function. Characterization of the recurrent V335G mutant demonstrated mild loss of expression with retained enzymatic activity. Although the V335G mutation retains its mitochondrial localization, it has altered cytosolic/nuclear localization. This minimally affects basal ADP ribosylation but results in elevated nuclear ADP ribosylation during stress, demonstrating the vital role of ADP ribosylation reversal by ARH3 in DNA damage control.

Serine-linked PARP1 auto-modification controls PARP inhibitor response.

Poly(ADP-ribose) polymerase 1 (PARP1) and PARP2 are recruited and activated by DNA damage, resulting in ADP-ribosylation at numerous sites, both within PARP1 itself and in other proteins. Several PARP1 and PARP2 inhibitors are currently employed in the clinic or undergoing trials for treatment of various cancers. These drugs act primarily by trapping PARP1 on damaged chromatin, which can lead to cell death, especially in cells with DNA repair defects. Although PARP1 trapping is thought to be caused primarily by the catalytic inhibition of PARP-dependent modification, implying that ADP-ribosylation (ADPr) can counteract trapping, it is not known which exact sites are important for this process. Following recent findings that PARP1- or PARP2-mediated modification is predominantly serine-linked, we demonstrate here that serine ADPr plays a vital role in cellular responses to PARP1/PARP2 inhibitors. Specifically, we identify three serine residues within PARP1 (499, 507, and 519) as key sites whose efficient HPF1-dependent modification counters PARP1 trapping and contributes to inhibitor tolerance. Our data implicate genes that encode serine-specific ADPr regulators, HPF1 and ARH3, as potential PARP1/PARP2 inhibitor therapy biomarkers.

Linking the FTO obesity rs1421085 variant circuitry to cellular, metabolic, and organismal phenotypes in vivo.

Variants in FTO have the strongest association with obesity; however, it is still unclear how those noncoding variants mechanistically affect whole-body physiology. We engineered a deletion of the rs1421085 conserved cis-regulatory module (CRM) in mice and confirmed in vivo that the CRM modulates Irx3 and Irx5 gene expression and mitochondrial function in adipocytes. The CRM affects molecular and cellular phenotypes in an adipose depot-dependent manner and affects organismal phenotypes that are relevant for obesity, including decreased high-fat diet-induced weight gain, decreased whole-body fat mass, and decreased skin fat thickness. Last, we connected the CRM to a genetically determined effect on steroid patterns in males that was dependent on nutritional challenge and conserved across mice and humans. Together, our data establish cross-species conservation of the rs1421085 regulatory circuitry at the molecular, cellular, metabolic, and organismal level, revealing previously unknown contextual dependence of the variant's action.

MacroD1 Is a Promiscuous ADP-Ribosyl Hydrolase Localized to Mitochondria.

MacroD1 is a macrodomain containing protein that has mono-ADP-ribose hydrolase enzymatic activity toward several ADP-ribose adducts. Dysregulation of MacroD1 expression has been shown to be associated with the pathogenesis of several forms of cancer. To date, the physiological functions and sub-cellular localization of MacroD1 are unclear. Previous studies have described nuclear and cytosolic functions of MacroD1. However, in this study we show that endogenous MacroD1 protein is highly enriched within mitochondria. We also show that MacroD1 is highly expressed in human and mouse skeletal muscle. Furthermore, we show that MacroD1 can efficiently remove ADP-ribose from 5' and 3'-phosphorylated double stranded DNA adducts in vitro. Overall, we have shown that MacroD1 is a mitochondrial protein with promiscuous enzymatic activity that can target the ester bonds of ADP-ribosylated phosphorylated double-stranded DNA ends. These findings have exciting implications for MacroD1 and ADP-ribosylation within the regulation of mitochondrial function and DNA-damage in vivo.

Validating the RedMIT/GFP-LC3 Mouse Model by Studying Mitophagy in Autosomal Dominant Optic Atrophy Due to the OPA1Q285STOP Mutation.

Background: Autosomal dominant optic atrophy (ADOA) is usually caused by mutations in the essential gene, OPA1. This encodes a ubiquitous protein involved in mitochondrial dynamics, hence tissue specificity is not understood. Dysregulated mitophagy (mitochondria recycling) is implicated in ADOA, being increased in OPA1 patient fibroblasts. Furthermore, autophagy may be increased in retinal ganglion cells (RGCs) of the OPA1Q285STOP mouse model. Aims: We developed a mouse model for studying mitochondrial dynamics in order to investigate mitophagy in ADOA. Methods: We crossed the OPA1Q285STOP mouse with our RedMIT/GFP-LC3 mouse, harboring red fluorescent mitochondria and green fluorescent autophagosomes. Colocalization between mitochondria and autophagosomes, the hallmark of mitophagy, was quantified in fluorescently labeled organelles in primary cell cultures, using two high throughput imaging methods Imagestream (Amnis) and IN Cell Analyzer 1000 (GE Healthcare Life Sciences). We studied colocalization between mitochondria and autophagosomes in fixed sections using confocal microscopy. Results: We validated our imaging methods for RedMIT/GFP-LC3 mouse cells, showing that colocalization of red fluorescent mitochondria and green fluorescent autophagosomes is a useful indicator of mitophagy. We showed that colocalization increases when lysosomal processing is impaired. Further, colocalization of mitochondrial fragments and autophagosomes is increased in cultures from the OPA1Q285STOP/RedMIT/GFP-LC3 mice compared to RedMIT/GFP-LC3 control mouse cells that were wild type for OPA1. This was apparent in both mouse embryonic fibroblasts (MEFs) using IN Cell 1000 and in splenocytes using ImageStream imaging flow cytometer (Amnis). We confirmed that this represents increased mitophagic flux using lysosomal inhibitors. We also used microscopy to investigate the level of mitophagy in the retina from the OPA1Q285STOP/RedMIT/GFP-LC3 mice and the RedMIT/GFP-LC3 control mice. However, the expression levels of fluorescent proteins and the image signal-to-background ratios precluded the detection of colocalization so we were unable to show any difference in colocalization between these mice. Conclusions: We show that colocalization of fluorescent mitochondria and autophagosomes in cell cultures, but not fixed tissues from the RedMIT/GFP-LC3, can be used to detect mitophagy. We used this model to confirm that mitophagy is increased in a mouse model of ADOA. It will be useful for cell based studies of diseases caused by impaired mitochondrial dynamics.

A Wars2 Mutant Mouse Model Displays OXPHOS Deficiencies and Activation of Tissue-Specific Stress Response Pathways.

Mutations in genes essential for mitochondrial function have pleiotropic effects. The mechanisms underlying these traits yield insights into metabolic homeostasis and potential therapies. Here we report the characterization of a mouse model harboring a mutation in the tryptophanyl-tRNA synthetase 2 (Wars2) gene, encoding the mitochondrial-localized WARS2 protein. This hypomorphic allele causes progressive tissue-specific pathologies, including hearing loss, reduced adiposity, adipose tissue dysfunction, and hypertrophic cardiomyopathy. We demonstrate the tissue heterogeneity arises as a result of variable activation of the integrated stress response (ISR) pathway and the ability of certain tissues to respond to impaired mitochondrial translation. Many of the systemic metabolic effects are likely mediated through elevated fibroblast growth factor 21 (FGF21) following activation of the ISR in certain tissues. These findings demonstrate the potential pleiotropy associated with Wars2 mutations in patients.

Use of FGF-21 as a Biomarker of Mitochondrial Disease in Clinical Practice.

Recent work has suggested that fibroblast growth factor-21 (FGF-21) is a useful biomarker of mitochondrial disease (MD). We routinely measured FGF-21 levels on patients who were investigated at our centre for MD and evaluated its diagnostic performance based on detailed genetic and other laboratory findings. Patients' FGF-21 results were assessed by the use of age-adjusted z-scores based on normalised FGF-21 values from a healthy population. One hundred and fifty five patients were investigated. One hundred and four of these patients had molecular evidence for MD, 27 were deemed to have disorders other than MD (non-MD), and 24 had possible MD. Patients with defects in mitochondrial DNA (mtDNA) maintenance (n = 32) and mtDNA rearrangements (n = 17) had the highest median FGF-21 among the MD group. Other MD patients harbouring mtDNA point mutations (n = 40) or mutations in other autosomal genes (n = 7) and those with partially characterised MD had lower FGF-21 levels. The area under the receiver operating characteristic curve for distinguishing MD from non-MD patients was 0.69. No correlation between FGF-21 and creatinine, creatine kinase, or cardio-skeletal myopathy score was found. FGF-21 was significantly associated with plasma lactate and ocular myopathy. Although FGF-21 was found to have a low sensitivity for detecting MD, at a z-score of 2.8, its specificity was above 90%. We suggest that a high serum concentration of FGF-21 would be clinically useful in MD, especially in adult patients with chronic progressive external ophthalmoplegia, and may enable bypassing muscle biopsy and directly opting for genetic analysis. Availability of its assay has thus modified our diagnostic pathway.

Novel gene function revealed by mouse mutagenesis screens for models of age-related disease.

Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss.