👤 David J Cutler

Muscle satellite cells (SCs), essential for skeletal muscle regeneration, decline in number and function with age, contributing to sarcopenia. A fully defined viscoelastic hydrogel that preserves SC-myofiber interactions and supports tunable densities of fibronectin-derived RGD ligands was used to investigate age-related defects in extracellular matrix sensing by SCs. Elevating RGD density increased the number of activating and proliferating SCs on myofibers from young mice, whereas SCs from aged mice were unresponsive. Loss of FGF receptor 1 signaling in SCs from aged mice abrogated the coordinated Syndecan-4 and Integrin-β1 matrix response observed in SCs from young mice. Activating Integrin-β1 promoted asymmetric division and self-renewal in SCs from young mice whereas combined FGFR1 and Integrin-β1 signaling drove symmetric expansion. In SCs from aged mice, FGFR1 dysfunction disrupted this balance, impairing asymmetric division, but constitutive FGFR1 activation restored receptor co-localization, self-renewal, and fibronectin responsiveness. Therefore, FGFR1 integrates matrix and growth factor signals, suggesting that targeting the FGFR1-Integrin-β1 axis may enhance SC regenerative potential in aging organisms. Show less

Calorie restriction (CR; calorie intake reduced by ∼20%-40% below ad libitum, AL, intake) potentiates skeletal muscle insulin sensitivity during old age by incompletely understood mechanisms. We aimed to identify CR-induced changes in muscle insulin signaling that may explain this enhanced sensitivity. We examined how CR (65% of AL intake for 8-weeks) alters muscle insulin action and signaling in aged rats (24-month old) of both sexes. We assessed insulin-stimulated glucose uptake (ISGU) in muscle together with deep phosphoproteomic profiling. CR enhanced ISGU in both sexes, with higher ISGU in females regardless of diet. We identified 590 diet-responsive phosphosites, indicating extensive CR-induced remodelling of muscle phosphorylation, particularly within structural and contractile pathways. Strikingly, 70% of these sites were sex-specific. Numerous insulin-responsive sites were identified (193 in females; 107 in males) with 60 overlapping sites. The magnitude of the insulin-effects among all significantly regulated sites correlated between sexes. S1443 phosphorylation on EH domain-binding protein 1-like protein-1 (Ehbp1l1; a potential regulator of Rab proteins that control GLUT4 glucose transporter trafficking) was insulin-responsive in both sexes but only associated to ISGU in females. Personalized phosphoproteomic analysis also identified insulin-responsive sites on Leiomodin-1 (Lmod1) that correlated with ISGU across individuals. Both Lmod1 and Ehbp1l1 have strong genetic association with glycemic traits in humans, reinforcing their translational relevance. This study revealed sex-dependent and sex-independent phosphosignaling mechanisms that associate with muscle insulin responsiveness as well as hundreds of sex-specific, CR-responsive phosphosites. These findings provide a rich resource for future research on CR and insulin sensitivity. Show less

Atrial fibrillation (AF) is a prevalent and morbid abnormality of the heart rhythm with a strong genetic component. Here, we meta-analyzed genome and exome sequencing data from 36 studies that included 52,416 AF cases and 277,762 controls. In burden tests of rare coding variation, we identified novel associations between AF and the genes MYBPC3, LMNA, PKP2, FAM189A2 and KDM5B. We further identified associations between AF and rare structural variants owing to deletions in CTNNA3 and duplications of GATA4. We broadly replicated our findings in independent samples from MyCode, deCODE and UK Biobank. Finally, we found that CRISPR knockout of KDM5B in stem-cell-derived atrial cardiomyocytes led to a shortening of the action potential duration and widespread transcriptomic dysregulation of genes relevant to atrial homeostasis and conduction. Our results highlight the contribution of rare coding and structural variants to AF, including genetic links between AF and cardiomyopathies, and expand our understanding of the rare variant architecture for this common arrhythmia. Show less

To broaden our understanding of bradyarrhythmias and conduction disease, we performed common variant genome-wide association analyses in up to 1.3 million individuals and rare variant burden testing in 460,000 individuals for sinus node dysfunction (SND), distal conduction disease (DCD) and pacemaker (PM) implantation. We identified 13, 31 and 21 common variant loci for SND, DCD and PM, respectively. Four well-known loci (SCN5A/SCN10A, CCDC141, TBX20 and CAMK2D) were shared for SND and DCD, while others were more specific for SND or DCD. SND and DCD showed a moderate genetic correlation (r Show less

The inflammatory bowel diseases (IBD) ulcerative colitis (UC) and Crohn's disease (CD) cause significant morbidity and are increasing in prevalence among all populations, including African Americans. More than 200 susceptibility loci have been identified in populations of predominantly European ancestry, but few loci have been associated with IBD in other ethnicities. We performed 2 high-density, genome-wide scans comprising 2345 cases of African Americans with IBD (1646 with CD, 583 with UC, and 116 inflammatory bowel disease unclassified) and 5002 individuals without IBD (controls, identified from the Health Retirement Study and Kaiser Permanente database). Single-nucleotide polymorphisms (SNPs) associated at P < 5.0 × 10 We detected SNPs at HLA-DRB1, and African-specific SNPs at ZNF649 and LSAMP, with associations of genome-wide significance for UC. We detected SNPs at USP25 with associations of genome-wide significance for IBD. No associations of genome-wide significance were detected for CD. In addition, 9 genes previously associated with IBD contained SNPs with significant evidence for replication (P < 1.6 × 10 We performed a genome-wide association study of African Americans with IBD and identified loci associated with UC in only this population; we also replicated IBD, CD, and UC loci identified in European populations. The detection of variants associated with IBD risk in only people of African descent demonstrates the importance of studying the genetics of IBD and other complex diseases in populations beyond those of European ancestry. Show less

Retinal degeneration and visual impairment are the first signs of juvenile neuronal ceroid lipofuscinosis caused by CLN3 mutations, followed by inevitable progression to blindness. We investigated retinal degeneration in Cln3(Δex1-6) null mice, revealing classic 'fingerprint' lysosomal storage in the retinal pigment epithelium (RPE), replicating the human disease. The lysosomes contain mitochondrial F0-ATP synthase subunit c along with undigested membranes, indicating a reduced degradative capacity. Mature autophagosomes and basal phagolysosomes, the terminal degradative compartments of autophagy and phagocytosis, are also increased in Cln3(Δex1) (-6) RPE, reflecting disruption to these key pathways that underpin the daily phagocytic turnover of photoreceptor outer segments (POS) required for maintenance of vision. The accumulated autophagosomes have post-lysosome fusion morphology, with undigested internal contents visible, while accumulated phagosomes are frequently docked to cathepsin D-positive lysosomes, without mixing of phagosomal and lysosomal contents. This suggests lysosome-processing defects affect both autophagy and phagocytosis, supported by evidence that phagosomes induced in Cln3(Δex1) (-) (6)-derived mouse embryonic fibroblasts have visibly disorganized membranes, unprocessed internal vesicles and membrane contents, in addition to reduced LAMP1 membrane recruitment. We propose that defective lysosomes in Cln3(Δex1) (-) (6) RPE have a reduced degradative capacity that impairs the final steps of the intimately connected autophagic and phagocytic pathways that are responsible for degradation of POS. A build-up of degradative organellar by-products and decreased recycling of cellular materials is likely to disrupt processes vital to maintenance of vision by the RPE. Show less

The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are a group of inherited childhood-onset neurodegenerative disorders characterized by the lysosomal accumulation of undigested material within cells. To understand this dysfunction, we analysed trafficking of the cation-independent mannose 6-phosphate receptor (CI-MPR), which delivers the digestive enzymes to lysosomes. A common form of NCL is caused by mutations in CLN3, a multipass transmembrane protein of unknown function. We report that ablation of CLN3 causes accumulation of CI-MPR in the trans Golgi network, reflecting a 50% reduction in exit. This CI-MPR trafficking defect is accompanied by a fall in maturation and cellular activity of lysosomal cathepsins. CLN3 is therefore essential for trafficking along the route needed for delivery of lysosomal enzymes, and its loss thereby contributes to and may explain the lysosomal dysfunction underlying Batten disease. Show less

The neuronal ceroid lipofuscinoses (NCLs) are common neurodegenerative disorders of childhood and are classified as lysosomal storage diseases since affected cells exhibit lysosomes containing ceroid and lipofuscin-like material. CLN3 is the most widely conserved NCL gene, suggesting that it has a basic eukaryotic cell function; its loss might be expected to cause the earliest onset and/or most severe disease. However, mutations in CLN3 are linked to juvenile NCL (JNCL), the latest onset and mildest form of NCL in children. We sought to explain this paradox. Almost all patients with JNCL are homozygous or heterozygous for an intragenic 1 kb deletion within CLN3, hitherto presumed to be a null mutation. We hypothesized that the 1 kb mutation may allow CLN3 residual function. We confirmed the presence of CLN3 transcripts in JNCL patient cells. When RNA silencing was used to deplete these transcripts in cells from JNCL patients, the lysosomes significantly increased in size, confirming the presence of functional protein in these cells. Consistently, overexpression of mutant CLN3 transcript caused lysosomes to decrease in size. We modelled the JNCL mutant transcripts and those corresponding to mouse models for Cln3 in Schizosaccharomyces pombe and confirmed that most transcripts retained significant function as we predicted. Therefore, we concluded that the common mutant CLN3 protein does indeed retain significant function and that JNCL is a mutation-specific disease phenotype. This finding has important consequences for recognition and diagnosis of disease caused by mutations in CLN3 and for the development of therapy for JNCL. Show less

Despite its widespread use to assess fibrosis, liver biopsy has several important drawbacks, including that is it semi-quantitative, invasive, and limited by sampling and observer variability. Non-invasive serum biomarkers may more accurately reflect the fibrogenetic process. To identify potential biomarkers of fibrosis, we compared serum protein expression profiles in patients with chronic hepatitis C (CHC) virus infection and fibrosis. Twenty-one patients with no or mild fibrosis (METAVIR stage F0, F1) and 23 with advanced fibrosis (F3, F4) were retrospectively identified from a pedigreed database of 1600 CHC patients. All samples were carefully phenotyped and matched for age, gender, race, body mass index, genotype, duration of infection, alcohol use, and viral load. Expression profiling was performed in a blinded fashion using a 2D polyacrylamide gel electrophoresis/LC-MS/MS platform. Partial least squares discriminant analysis and likelihood ratio statistics were used to rank individual differences in protein expression between the 2 groups. Seven individual protein spots were identified as either significantly increased (alpha2-macroglobulin, haptoglobin, albumin) or decreased (complement C-4, serum retinol binding protein, apolipoprotein A-1, and two isoforms of apolipoprotein A-IV) with advanced fibrosis. Three individual proteins, haptoglobin, apolipoprotein A-1, and alpha2-macroglobulin, are included in existing non-invasive serum marker panels. Biomarkers identified through expression profiling may facilitate the development of more accurate marker algorithms to better quantitate hepatic fibrosis and monitor disease progression. Show less

The neuronal ceroid lipofuscinoses (NCLs) are severe inherited neurodegenerative disorders affecting children. In this disease, lysosomes accumulate autofluorescent storage material and there is death of neurons. Five types of NCL are caused by mutations in lysosomal proteins (CTSD, CLN1/PPT1, CLN2/TTPI, CLN3 and CLN5), and one type is caused by mutations in a protein that recycles between the ER and ERGIC (CLN8). The CLN6 gene underlying a variant of late infantile NCL (vLINCL) was recently identified. It encodes a novel 311 amino acid transmembrane protein. Antisera raised against CLN6 peptides detected a protein of 30 kDa by Western blotting of human cells, which was missing in cells from some CLN6 deficient patients. Using immunofluorescence microscopy, CLN6 was shown to reside in the endoplasmic reticulum (ER). CLN6 protein tagged with GFP at the C-terminus and expressed in HEK293 cells was also found within the ER. Investigation of the effect of five CLN6 disease mutations that affect single amino acids showed that the mutant proteins were retained in the ER. These data suggest that CLN6 is an ER resident protein, the activity of which, despite this location, must contribute to lysosomal function. Show less