The myotubularin-amphiphysin 2 complex in membrane tubulation and centronuclear myopathies.

Myotubularin (MTM1) and amphiphysin 2 (BIN1) are two proteins mutated in different forms of centronuclear myopathy, but the functional and pathological relationship between these two proteins was unknown. Here, we identified MTM1 as a novel binding partner of BIN1, both in vitro and endogenously in skeletal muscle. Moreover, MTM1 enhances BIN1-mediated membrane tubulation, depending on binding and phosphoinositide phosphatase activity. BIN1 patient mutations induce a conformational change in BIN1 and alter its binding and regulation by MTM1. In conclusion, we identified the first molecular and functional link between MTM1 and BIN1, supporting a common pathological mechanism in different forms of centronuclear myopathy.

Mitochondrial dysfunction in osteoarthritis is associated with down-regulation of superoxide dismutase 2.

OBJECTIVE: Superoxide dismutase 2 (SOD2) is down- regulated in osteoarthritis (OA). This study was undertaken to investigate the functional effects of this down-regulation in the context of oxidative damage and mitochondrial dysfunction. METHODS: Lipid peroxidation in articular cartilage from OA patients and from lesion-free control subjects with femoral neck fracture was assessed by measuring malondialdehyde levels using the thiobarbituric acid reactive substances assay. Long-range polymerase chain reaction amplification and a mitochondrial DNA (mtDNA) strand break assay were used to investigate the presence of somatic large-scale mtDNA rearrangements in cartilage. Microscale oxygraphy was used to explore possible changes in mitochondrial respiratory activity between OA and control chondrocytes. RNA interference was used to determine the effects of SOD2 depletion on lipid peroxidation, mtDNA damage, and mitochondrial respiration. RESULTS: OA cartilage had higher levels of lipid peroxidation compared to control cartilage, and lipid peroxidation was similarly elevated in SOD2-depleted chondrocytes. SOD2 depletion led to a significant increase in mtDNA strand breaks in chondrocytes, but there was no notable difference in the level of strand breaks between OA and control chondrocytes. Furthermore, only very low levels of somatic, large-scale mtDNA rearrangements were identified in OA cartilage. OA chondrocytes showed less spare respiratory capacity (SRC) and higher proton leak compared to control chondrocytes. SOD2-depleted chondrocytes also showed less SRC and higher proton leak. CONCLUSION: This is the first study to analyze the effects of SOD2 depletion in human articular chondrocytes in terms of changes to oxidation and mitochondrial function. The findings indicate that SOD2 depletion in chondrocytes leads to oxidative damage and mitochondrial dysfunction, suggesting that SOD2 down-regulation is a potential contributor to the pathogenesis of OA.

Matrix metalloproteinase 13 expression in response to double-stranded RNA in human chondrocytes.

OBJECTIVE: To investigate the mechanism of matrix metalloproteinase 13 (MMP-13) expression in chondrocytes via pattern-recognition receptors (PRRs) for double-stranded RNA (dsRNA). METHODS: Differential expression of PRRs was determined by real-time reverse transcription-polymerase chain reaction (RT-PCR) of RNA from patients with osteoarthritis (OA) and patients with femoral neck fracture (as normal control). Isolated human articular chondrocytes and the chondrosarcoma cell line SW-1353 were activated with poly(I-C) of different molecular weights as a dsRNA mimic, and changes in gene and protein expression were monitored by real-time RT-PCR and immunoblotting, respectively. RESULTS: The dsRNA signaling moieties Toll-like receptor 3 (TLR-3), retinoic acid-inducible gene 1 (RIG-1), and nucleotide-binding oligomerization domain-like receptor X1 were all differentially expressed in OA cartilage compared to normal cartilage, as determined by gene expression screening. Depletion of the dsRNA-sensing receptors TLR-3, RIG-1, or melanoma differentiation-associated gene 5 (MDA-5) suppressed the induction of MMP13 messenger RNA (mRNA) expression by poly(I-C), regardless of its mode of delivery. In addition, depletion of the downstream transcription factor interferon regulatory factor 3 resulted in reduced induction of MMP13 mRNA expression by poly(I-C). CONCLUSION: Signaling by dsRNA in chondrocytes requires a range of PRRs, including TLR-3, RIG-1, and MDA-5, for the full-induction of MMP13, thus providing tight regulation of a gene critical for maintenance of cartilage integrity. Our data add to the understanding of MMP13 regulation, which is essential before such mechanisms can be exploited to alleviate the cartilage destruction associated with OA.

Differential physiological roles for BIN1 isoforms in skeletal muscle development, function and regeneration.

Skeletal muscle development and regeneration are tightly regulated processes. How the intracellular organization of muscle fibers is achieved during these steps is unclear. Here, we focus on the cellular and physiological roles of amphiphysin 2 (BIN1), a membrane remodeling protein mutated in both congenital and adult centronuclear myopathies (CNM), that is ubiquitously expressed and has skeletal muscle-specific isoforms. We created and characterized constitutive muscle-specific and inducible Bin1 homozygous and heterozygous knockout mice targeting either ubiquitous or muscle-specific isoforms. Constitutive Bin1-deficient mice died at birth from lack of feeding due to a skeletal muscle defect. T-tubules and other organelles were misplaced and altered, supporting a general early role for BIN1 in intracellular organization, in addition to membrane remodeling. Although restricted deletion of Bin1 in unchallenged adult muscles had no impact, the forced switch from the muscle-specific isoforms to the ubiquitous isoforms through deletion of the in-frame muscle-specific exon delayed muscle regeneration. Thus, ubiquitous BIN1 function is necessary for muscle development and function, whereas its muscle-specific isoforms fine tune muscle regeneration in adulthood, supporting that BIN1 CNM with congenital onset are due to developmental defects, whereas later onset may be due to regeneration defects.

The MTM1-UBQLN2-HSP complex mediates degradation of misfolded intermediate filaments in skeletal muscle.

The ubiquitin proteasome system and autophagy are major protein turnover mechanisms in muscle cells, which ensure stemness and muscle fibre maintenance. Muscle cells contain a high proportion of cytoskeletal proteins, which are prone to misfolding and aggregation; pathological processes that are observed in several neuromuscular diseases called proteinopathies. Despite advances in deciphering the mechanisms underlying misfolding and aggregation, little is known about how muscle cells manage cytoskeletal degradation. Here, we describe a process by which muscle cells degrade the misfolded intermediate filament proteins desmin and vimentin by the proteasome. This relies on the MTM1-UBQLN2 complex to recognize and guide these misfolded proteins to the proteasome and occurs prior to aggregate formation. Thus, our data highlight a safeguarding function of the MTM1-UBQLN2 complex that ensures cytoskeletal integrity to avoid proteotoxic aggregate formation.

Amphiphysin 2 Orchestrates Nucleus Positioning and Shape by Linking the Nuclear Envelope to the Actin and Microtubule Cytoskeleton.

Nucleus positioning is key for intracellular organization, cell differentiation, and organ development and is affected in many diseases, including myopathies due to alteration in amphiphysin-2 (BIN1). The actin and microtubule cytoskeletons are essential for nucleus positioning, but their crosstalk in this process is sparsely characterized. Here, we report that impairment of amphiphysin/BIN1 in Caenorhabditis elegans, mammalian cells, or muscles from patients with centronuclear myopathy alters nuclear position and shape. We show that AMPH-1/BIN1 binds to nesprin and actin, as well as to the microtubule-binding protein CLIP170 in both species. Expression of the microtubule-anchoring CAP-GLY domain of CLIP170 fused to the nuclear-envelope-anchoring KASH domain of nesprin rescues nuclear positioning defects of amph-1 mutants. Amphiphysins thus play a central role in linking the nuclear envelope with the actin and microtubule cytoskeletons. We propose that BIN1 has a direct and evolutionarily conserved role in nuclear positioning, altered in myopathies.