Rapamycin-induced autophagy aggravates pathology and weakness in a mouse model of VCP-associated myopathy

Pathological phenotypes in inclusion body myopathy (IBM) associated with Paget disease of the bone (PDB), frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) (IBMPFD/ALS) include defective autophagosome and endosome maturation that result in vacuolation, weakness and muscle atrophy. The link between autophagy and IBMPFD/ALS pathobiology has been poorly understood. We examined the AKT-FOXO3 and MTOR pathways to characterize the regulation of autophagy in IBMPFD/ALS mouse muscle. We identified a defect in MTOR signaling that results in enhanced autophagosome biogenesis. Modulating MTOR signaling may therefore be a viable therapeutic target in IBMPFD/ALS

Provisional practice recommendation for the management of myopathy in VCP-associated multisystem proteinopathy

Valosin-containing protein (VCP)-associated multisystem proteinopathy (MSP) is a rare genetic disorder with abnormalities in the autophagy pathway leading to various combinations of myopathy, bone diseases, and neurodegeneration. Ninety percent of patients with VCP-associated MSP have myopathy, but there is no consensus-based guideline. The goal of this working group was to develop a best practice set of provisional recommendations for VCP myopathy which can be easily implemented across the globe. As an initiative by Cure VCP Disease Inc., a patient advocacy organization, an online survey was initially conducted to identify the practice gaps in VCP myopathy. All prior published literature on VCP myopathy was reviewed to better understand the different aspects of management of VCP myopathy, and several working group sessions were conducted involving international experts to develop this provisional recommendation. VCP myopathy has a heterogeneous clinical phenotype and should be considered in patients with limb-girdle muscular dystrophy phenotype, or any myopathy with an autosomal dominant pattern of inheritance. Genetic testing is the only definitive way to diagnose VCP myopathy, and single-variant testing in the case of a known familial VCP variant, or multi-gene panel sequencing in undifferentiated cases can be considered. Muscle biopsy is important in cases of diagnostic uncertainty or lack of a definitive pathogenic genetic variant since rimmed vacuoles (present in ~40% cases) are considered a hallmark of VCP myopathy. Electrodiagnostic studies and magnetic resonance imaging can also help rule out disease mimics. Standardized management of VCP myopathy will optimize patient care and help future research initiatives

Current and future approaches to classify VUSs in LGMD-related genes

Next-generation sequencing (NGS) has revealed large numbers of genetic variants in LGMD-related genes, with most of them classified as variants of uncertain significance (VUSs). VUSs are genetic changes with unknown pathological impact and present a major challenge in genetic test interpretation and disease diagnosis. Understanding the phenotypic consequences of VUSs can provide clinical guidance regarding LGMD risk and therapy. In this review, we provide a brief overview of the subtypes of LGMD, disease diagnosis, current classification systems for investigating VUSs, and a potential deep mutational scanning approach to classify VUSs in LGMD-related genes

Clinical classification of variants in the valosin-containing protein gene associated with multisystem proteinopathy

BACKGROUND AND OBJECTIVES: Pathogenic variants in the valosin-containing protein ( METHODS: A 6-item clinical score was developed to evaluate the phenotypic level of evidence to support the pathogenicity of the novel variants. Each item is allocated a value, a score ranging from 0.5 to 5.5 points. A receiver-operating characteristic curve was used to identify a cutoff value of 3 to consider a variant as high likelihood disease associated. The scoring system results were confronted with results of in vitro ATPase activity assays and with in silico analysis. RESULTS: All variants were missense, except for one small deletion-insertion, 18 led to amino acid changes within the N and D1 domains, and 13 increased the enzymatic activity. The clinical score coincided with the functional studies in 17 of 19 variants and with the in silico analysis in 12 of 19. For 12 variants, the 3 predictive tools agreed, and for 7 variants, the predictive tools disagreed. The pooled data supported the pathogenicity of 13 of 19 novel VCP variants identified in the study. DISCUSSION: This study provides data to support pathogenicity of 14 of 19 nove

Comparisons of ELISA and Western blot assays for detection of autophagy flux

We analyzed autophagy/mitophagy flux in vitro (C2C12 myotubes) and in vivo (mouse skeletal muscle) following the treatments of autophagy inducers (starvation, rapamycin) and a mitophagy inducer (carbonyl cyanide m-chlorophenylhydrazone, CCCP) using two immunodetection methods, ELISA and Western blotting, and compared their working range, accuracy, and reliability. The ELISAs showed a broader working range than that of the LC3 Western blots (Table 1). Table 2 showed that data value distribution was tighter and the average standard error from the ELISA was much smaller than those of the Western blot, directly relating to the accuracy of the assay. Test-retest reliability analysis showed good reliability for three individual ELISAs (interclass correlation, ≥ 0.7), but poor reliability for three individual Western blots (interclass correlation, ≤ 0.4) (Table 3). Keywords: Autophagy, Mitophagy, ELISA, Western blot, Skeletal muscl

Client processing is altered by novel myopathy-causing mutations in the HSP40 J domain

The misfolding and aggregation of proteins is often implicated in the development and progression of degenerative diseases. Heat shock proteins (HSPs), such as the ubiquitously expressed Type II Hsp40 molecular chaperone, DNAJB6, assist in protein folding and disaggregation. Historically, mutations within the DNAJB6 G/F domain have been associated with Limb-Girdle Muscular Dystrophy type 1D, now referred to as LGMDD1, a dominantly inherited degenerative disease. Recently, novel mutations within the J domain of DNAJB6 have been reported in patients with LGMDD1. Since novel myopathy-causing mutations in the Hsp40 J domain have yet to be characterized and both the function of DNAJB6 in skeletal muscle and the clients of this chaperone are unknown, we set out to assess the effect of these mutations on chaperone function using the genetically tractable yeast system. The essential yeast Type II Hsp40, Sis1, is homologous to DNAJB6 and is involved in the propagation of yeast prions. Using phenotypic, biochemical, and functional assays we found that homologous mutations in the Sis1 J domain differentially alter the processing of specific yeast prion strains, as well as a non-prion substrate. These data suggest that the newly-identified mutations in the J domain of DNAJB6 cause aberrant chaperone function that leads to the pathogenesis in LGMDD1