Highlights d NR supplementation in aged subjects augments the skeletal muscle NAD + metabolome d NR supplementation does not affect skeletal muscle mitochondrial bioenergetics d NR supplementation reduces levels of circulating inflammatory cytokines

Abbreviations: ACC, acetyl-CoA carboxylase; AMP, adenosine monophosphate; AMPK, adenosine monophosphate activated protein kinase; CCCP, carbonyl cyanide m-chlorophenyl hydrazine; CISD1, CDGSH iron sulfur domain 1; DRP1, dynamin-related protein 1; GFP, green fluorescence protein; MFF, mitochondrial fission factor; MFN-1/2, mitofusin-1/2; mtFIS1 101-152 , mitochondrial targeting sequence of FIS1 (amino acids 101-152); NDP52, nuclear dot protein 52; OPA1, dynamin-like 120 kDa protein; OPTN, optineurin; OXPHOS, oxidative phosphorylation; PINK1, PTEN-induced kinase 1; SQSTM1/p62, sequestosome-1; TBK1, TANK-binding kinase 1; Ub, ubiquitin; UBA UBQLN1 , his-halo-ubiquilin1 UBA domain tetramer; ULK1, unc-51 like autophagy activating kinase 1. AbstractMitophagy is a key process regulating mitochondrial quality control. Several mechanisms have been proposed to regulate mitophagy, but these have mostly been studied using stably expressed non-native proteins in immortalized cell lines. In skeletal muscle, mitophagy and its molecular mechanisms require more thorough investigation. To measure mitophagy directly, we generated a stable skeletal muscle C2C12 cell line, expressing a mitophagy reporter construct (mCherry-green fluorescence protein-mtFIS1 101-152 ). Here, we report that both carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment and adenosine monophosphate activated protein kinase (AMPK) activation by 991 promote mitochondrial fission via phosphorylation of MFF and induce mitophagy by ~20%. Upon CCCP treatment, but not 991, ubiquitin phosphorylation, a read-out of PTEN-induced kinase 1 (PINK1) activity, and Parkin E3 ligase activity toward CDGSH iron sulfur domain 1 (CISD1) were increased. Although the PINK1-Parkin signaling pathway is active in response to CCCP treatment, we observed no change in markers of mitochondrial protein content. Interestingly, our data shows that TANK-binding kinase 1 (TBK1) phosphorylation is increased after both CCCP and 991 treatments, suggesting TBK1 activation to be independent of both PINK1 and Parkin. Finally, we confirmed in non-muscle cell lines that TBK1 phosphorylation occurs in the absence of PINK1 and is regulated by AMPKdependent signaling. Thus, AMPK activation promotes mitophagy by enhancing mitochondrial fission (via MFF phosphorylation) and autophagosomal engulfment (via TBK1 activation) in a PINK1-Parkin independent manner.

Background Patients with rheumatoid arthritis (RA) experience extra-articular manifestations including osteoporosis and muscle wasting, which closely associate with severity of disease. Whilst therapeutic glucocorticoids (GCs) reduce inflammation in RA, their actions on muscle and bone metabolism in the context of chronic inflammation remain unclear. We utilised the TNF-tg model of chronic polyarthritis to ascertain the impact of therapeutic GCs on bone and muscle homeostasis in the context of systemic inflammation. Methods TNF-tg and wild-type (WT) animals received either vehicle or the GC corticosterone (100 μg/ml) in drinking water at onset of arthritis. Arthritis severity and clinical parameters were measured, serum collected for ELISA and muscle and bone biopsies collected for μCT, histology and mRNA analysis. In vivo findings were examined in primary cultures of osteoblasts, osteoclasts and myotubes. Results TNF-tg mice receiving GCs showed protection from inflammatory bone loss, characterised by a reduction in serum markers of bone resorption, osteoclast numbers and osteoclast activity. In contrast, muscle wasting was markedly increased in WT and TNF-tg animals receiving GCs, independently of inflammation. This was characterised by a reduction in muscle weight and fibre size, and an induction in anti-anabolic and catabolic signalling. Conclusions This study demonstrates that when given in early onset chronic polyarthritis, oral GCs partially protect against inflammatory bone loss, but induce marked muscle wasting. These results suggest that in patients with inflammatory arthritis receiving GCs, the development of interventions to manage deleterious side effects in muscle should be prioritised. Electronic supplementary material The online version of this article (10.1186/s13075-019-1962-3) contains supplementary material, which is available to authorized users.

Tel: + 44(0)121 414 3917, email: g.g.lavery@bham.ac.uk 26 27 2012), reduction of blood glucose, hepatic steatosis, and neuropathy on high fat diet 64 (Trammell et al. 2016), improvement of cardiac function in genetic cardiomyopathy (Diguet et 65 al., 2018), and prevention of cortical neuronal degeneration (Vaur et al., 2017). Depletion of 66 the enzyme nicotinamide phosphoribosyltransferase (NAMPT), rate-limiting for NAD + 67 biosynthesis, in mouse skeletal muscle severely diminishes NAD + levels and induces 68 sarcopenia. Oral repletion of NAD + with NR in this model rescued pathology in skeletal 69 muscle in a cell-autonomous manner (Frederick et al., 2016). However, recent data in mice 70 tracing NAD + fluxes questioned whether oral NR has the ability to access muscle (Liu et al., 2018). Thus, whether oral NR can augment the human skeletal muscle NAD + metabolome is 72 currently unknown. 73A decline in NAD + availability and signalling appears to occur as part of the aging process in 74 many species (Gomes et al., 2013; Mouchiroud et al., 2013), though there is a paucity of 75 data to confirm that this is the case in human aging. NR and nicotinamide mononucleotide 76 (NMN) are reported to extend life span (Zhang et al., 2016) and enhance metabolism in 77 aged mice (Mills et al., 2016). To date, NR supplementation studies in humans have been 78 reported, focussing on cardiovascular (Martens et al., 2018), systemic metabolic (Dollerup et 79 al., 2018), and safety (Conze et al, 2019) end-points, but have not addressed advanced 80 aging, tissue metabolomic changes, or effects on muscle metabolism and function. 81Herein, we set out to study if oral NR is available to aged human skeletal muscle and 82 whether potential effects on muscle metabolism can be detected. We conducted a 21-day 83 NR supplementation intervention in a cohort of 70 -80 year old men in a randomized, 84 double-blind, placebo-controlled crossover trial. We demonstrate that NR augments the 85 skeletal muscle NAD + metabolome inducing a gene expression signature suggestive of 86 downregulation of energy metabolism pathways, but without affecting muscle mitochondrial 87 bioenergetics or metabolism. Additionally, NR suppresses specific circulating inflammatory 88 cytokines levels. In an additional study, we used 31 P magnetic resonance spectroscopy 89 (MRS) and show that NAD + decline is not associated with chronological aging per se in 90 either human muscle or brain. 91 92 5 RESULTS 93Oral NR is safe and well tolerated in aged adults 94Twelve aged (median age 75 years) and marginally overweight (median BMI 26.6 kg/m 2 ; 95 range 21 -30), but otherwise healthy men were recruited and orally supplemented with NR 1 96 g per day for 21-days in a randomized, double-blind, placebo-controlled crossover design 97 with 21-days washout period between phases. Baseline characteristics of participants are 98 included in Suppl Table 1. NR chloride (Niagen ®) and placebo were provided as 250 mg 99 capsules (ChromaDex, Inc.) and subjects were instructed to take ...

The selective removal of damaged mitochondria, also known as mitophagy, is an important mechanism that regulates mitochondrial quality control. Evidence suggests that mitophagy is adversely affected in aged skeletal muscle, and this is thought to contribute toward the age-related decline of muscle health. While our knowledge of the molecular mechanisms that regulate mitophagy are derived mostly from work in non-muscle cells, whether these mechanisms are conferred in muscle under physiological conditions has not been thoroughly investigated. Recent findings from our laboratory and those of others have made several novel contributions to this field. Herein, we consolidate current literature, including our recent work, while evaluating how ubiquitin-dependent mitophagy is regulated both in muscle and non-muscle cells through the steps of mitochondrial fission, ubiquitylation, and autophagosomal engulfment. During ubiquitin-dependent mitophagy in non-muscle cells, mitochondrial depolarization activates PINK1-Parkin signaling to elicit mitochondrial ubiquitylation. TANK-binding kinase 1 (TBK1) then activates autophagy receptors, which in turn, tether ubiquitylated mitochondria to autophagosomes prior to lysosomal degradation. In skeletal muscle, evidence supporting the involvement of PINK1-Parkin signaling in mitophagy is lacking. Instead, 5′-AMP-activated protein kinase (AMPK) is emerging as a critical regulator. Mechanistically, AMPK activation promotes mitochondrial fission before enhancing autophagosomal engulfment of damaged mitochondria possibly via TBK1. While TBK1 may be a point of convergence between PINK1-Parkin and AMPK signaling in muscle, the critical question that remains is: whether mitochondrial ubiquitylation is required for mitophagy. In future, improving understanding of molecular processes that regulate mitophagy in muscle will help to develop novel strategies to promote healthy aging.