SIL1 deficiency causes degenerative changes of peripheral nerves and neuromuscular junctions in fish, mice and human.

BACKGROUND: Marinesco-Sjögren Syndrome (MSS) is a rare neuromuscular condition caused by recessive mutations in the SIL1 gene resulting in the absence of functional SIL1 protein, a co-chaperone for the major ER chaperone, BiP. As BiP is decisive for proper protein processing, loss of SIL1 results in the accumulation of misshaped proteins. This accumulation likely damages and destroys cells in vulnerable tissues, leading to congenital cataracts, cerebellar ataxia, vacuolar myopathy and other MSS phenotypes. Whether the peripheral nervous system (PNS) is affected in MSS has not been conclusively shown. METHODS: To study PNS vulnerability in MSS, intramuscular nerves fibres from MSS patients and from SIL1-deficient mice (woozy) as well as sciatic nerves and neuromuscular junctions (NMJ) from these mice have been investigated via transmission electron microscopic and immunofluorescence studies accompanied by transcript studies and unbiased proteomic profiling. In addition, PNS and NMJ integrity were analyzed via immunofluorescence studies in an MSS-zebrafish model which has been generated for that purpose. RESULTS: Electron microscopy revealed morphological changes indicative of impaired autophagy and mitochondrial maintenance in distal axons and in Schwann cells. Moreover, changes of the morphology of NMJs as well as of transcripts encoding proteins important for NMJ function were detected in woozy mice. These findings were in line with a grossly abnormal structure of NMJs in SIL1-deficient zebrafish embryos. Proteome profiling of sciatic nerve specimens from woozy mice revealed altered levels of proteins implicated in neuronal maintenance suggesting the activation of compensatory mechanisms. CONCLUSION: Taken together, our combined data expand the spectrum of tissues affected by SIL1-loss and suggest that impaired neuromuscular transmission might be part of MSS pathophysiology.

Proteome Profiling and Ultrastructural Characterization of the Human RCMH Cell Line: Myoblastic Properties and Suitability for Myopathological Studies.

Studying (neuro)muscular disorders is a major topic in biomedicine with a demand for suitable model systems. Continuous cell culture (in vitro) systems have several technical advantages over in vivo systems and became widely used tools for discovering physiological/pathophysiological mechanisms in muscle. In particular, myoblast cell lines are suitable model systems to study complex biochemical adaptations occurring in skeletal muscle and cellular responses to altered genetic/environmental conditions. Whereas most in vitro studies use extensively characterized murine C2C12 cells, a comprehensive description of an equivalent human cell line, not genetically manipulated for immortalization, is lacking. Therefore, we characterized human immortal myoblastic RCMH cells using scanning (SEM) and transmission electron microscopy (TEM) and proteomics. Among more than 6200 identified proteins we confirm the known expression of proteins important for muscle function. Comparing the RCMH proteome with two well-defined nonskeletal muscle cells lines (HeLa, U2OS) revealed a considerable enrichment of proteins important for muscle function. SEM/TEM confirmed the presence of agglomerates of cytoskeletal components/intermediate filaments and a prominent rough ER. In conclusion, our results indicate RMCH as a suitable in vitro model for investigating muscle function-related processes such as mechanical stress burden and mechanotransduction, EC coupling, cytoskeleton, muscle cell metabolism and development, and (ER-associated) myopathic disorders.

Myopathy in Marinesco-Sjögren syndrome links endoplasmic reticulum chaperone dysfunction to nuclear envelope pathology.

Marinesco-Sjögren syndrome (MSS) features cerebellar ataxia, mental retardation, cataracts, and progressive vacuolar myopathy with peculiar myonuclear alterations. Most MSS patients carry homozygous or compound heterozygous SIL1 mutations. SIL1 is a nucleotide exchange factor for the endoplasmic reticulum resident chaperone BiP which controls a plethora of essential processes in the endoplasmic reticulum. In this study we made use of the spontaneous Sil1 mouse mutant woozy to explore pathomechanisms leading to Sil1 deficiency-related skeletal muscle pathology. We found severe, progressive myopathy characterized by alterations of the sarcoplasmic reticulum, accumulation of autophagic vacuoles, mitochondrial changes, and prominent myonuclear pathology including nuclear envelope and nuclear lamina alterations. These abnormalities were remarkably similar to the myopathy in human patients with MSS. In particular, the presence of perinuclear membranous structures which have been reported as an ultrastructural hallmark of MSS-related myopathy could be confirmed in woozy muscles. We found that these structures are derived from the nuclear envelope and nuclear lamina and associate with proliferations of the sarcoplasmic reticulum. In line with impaired function of BiP secondary to loss of its nucleotide exchange factor Sil1, we observed activation of the unfolded protein response and the endoplasmic-reticulum-associated protein degradation-pathway. Despite initiation of the autophagy-lysosomal system, autophagic clearance was found ineffective which is in agreement with the formation of autophagic vacuoles. This report identifies woozy muscle as a faithful phenocopy of the MSS myopathy. Moreover, we provide a link between two well-established disease mechanisms in skeletal muscle, dysfunction of chaperones and nuclear envelope pathology.

Plastoquinone-9 biosynthesis in cyanobacteria differs from that in plants and involves a novel 4-hydroxybenzoate solanesyltransferase.

PQ-9 (plastoquinone-9) has a central role in energy transformation processes in cyanobacteria by mediating electron transfer in both the photosynthetic as well as the respiratory electron transport chain. The present study provides evidence that the PQ-9 biosynthetic pathway in cyanobacteria differs substantially from that in plants. We identified 4-hydroxybenzoate as being the aromatic precursor for PQ-9 in Synechocystis sp. PCC6803, and in the present paper we report on the role of the membrane-bound 4-hydroxybenzoate solanesyltransferase, Slr0926, in PQ-9 biosynthesis and on the properties of the enzyme. The catalytic activity of Slr0926 was demonstrated by in vivo labelling experiments in Synechocystis sp., complementation studies in an Escherichia coli mutant with a defect in ubiquinone biosynthesis, and in vitro assays using the recombinant as well as the native enzyme. Although Slr0926 was highly specific for the prenyl acceptor substrate 4-hydroxybenzoate, it displayed a broad specificity with regard to the prenyl donor substrate and used not only solanesyl diphosphate, but also a number of shorter-chain prenyl diphosphates. In combination with in silico data, our results indicate that Slr0926 evolved from bacterial 4-hydroxybenzoate prenyltransferases catalysing prenylation in the course of ubiquinone biosynthesis.

Identification of Cellular Pathogenicity Markers for SIL1 Mutations Linked to Marinesco-Sjögren Syndrome.

Background and objective: Recessive mutations in the SIL1 gene cause Marinesco-Sjögren syndrome (MSS), a rare neuropediatric disorder. MSS-patients typically present with congenital cataracts, intellectual disability, cerebellar ataxia and progressive vacuolar myopathy. However, atypical clinical presentations associated with SIL1 mutations have been described over the last years; compound heterozygosity of SIL1 missense mutations even resulted in a phenotype not fulfilling the clinical diagnostic criteria of MSS. Thus, a read-out system to evaluate reliably the pathogenicity of amino acid changes in SIL1 is needed. Here, we aim to provide suitable cellular biomarkers enabling the robust evaluation of pathogenicity of SIL1 mutations. Methods: Five SIL1 variants including one polymorphism (p.K132Q), three known pathogenic mutations (p.V231_I232del, p.G312R, and p.L457P) and one ambiguous missense variant (p.R92W) were studied along with the wild-type proteins in Hek293 in vitro models by cell biological assays, immunoprecipitation, immunoblotting, and immunofluorescence as well as electron microscopy. Moreover, the SIL1-interactomes were interrogated by tandem-affinity-purification and subsequent mass spectrometry. Results: Our combined studies confirmed the pathogenicity of p.V231_I232del, p.G312R, and p.L457P by showing instability of the proteins as well as tendency to form aggregates. This observation is in line with altered structure of the ER-Golgi system and vacuole formation upon expression of these pathogenic SIL1-mutants as well as the presence of oxidative or ER-stress. Reduced cellular fitness along with abnormal mitochondrial architecture could also be observed. Notably, both the polymorphic p.K132Q and the ambiguous p.R92W variants did not elicit such alterations. Study of the SIL1-interactome identified POC1A as a novel binding partner of wild-type SIL1; the interaction is disrupted upon the presence of pathogenic mutants but not influenced by the presence of benign variants. Disrupted SIL1-POC1A interaction is associated with centrosome disintegration. Conclusions: We developed a combination of cellular outcome measures to evaluate the pathogenicity of SIL1 variants in suitable in vitro models and demonstrated that the p. R92W missense variant is a polymorphism rather than a pathogenic mutation leading to MSS.

Biochemical and pathological changes result from mutated Caveolin-3 in muscle.

BACKGROUND: Caveolin-3 (CAV3) is a muscle-specific protein localized to the sarcolemma. It was suggested that CAV3 is involved in the connection between the extracellular matrix (ECM) and the cytoskeleton. Caveolinopathies often go along with increased CK levels indicative of sarcolemmal damage. So far, more than 40 dominant pathogenic mutations have been described leading to several phenotypes many of which are associated with a mis-localization of the mutant protein to the Golgi. Golgi retention and endoplasmic reticulum (ER) stress has been demonstrated for the CAV3 p.P104L mutation, but further downstream pathophysiological consequences remained elusive so far. METHODS: We utilized a transgenic (p.P104L mutant) mouse model and performed proteomic profiling along with immunoprecipitation, immunofluorescence and immunoblot examinations (including examination of α-dystroglycan glycosylation), and morphological studies (electron and coherent anti-Stokes Raman scattering (CARS) microscopy) in a systematic investigation of molecular and subcellular events in p.P104L caveolinopathy. RESULTS: Our electron and CARS microscopic as well as immunological studies revealed Golgi and ER proliferations along with a build-up of protein aggregates further characterized by immunoprecipitation and subsequent mass spectrometry. Molecular characterization these aggregates showed affection of mitochondrial and cytoskeletal proteins which accords with our ultra-structural findings. Additional global proteomic profiling revealed vulnerability of 120 proteins in diseased quadriceps muscle supporting our previous findings and providing more general insights into the underlying pathophysiology. Moreover, our data suggested that further DGC components are altered by the perturbed protein processing machinery but are not prone to form aggregates whereas other sarcolemmal proteins are ubiquitinated or bind to p62. Although the architecture of the ER and Golgi as organelles of protein glycosylation are altered, the glycosylation of α-dystroglycan presented unchanged. CONCLUSIONS: Our combined data classify the p.P104 caveolinopathy as an ER-Golgi disorder impairing proper protein processing and leading to aggregate formation pertaining proteins important for mitochondrial function, cytoskeleton, ECM remodeling and sarcolemmal integrity. Glycosylation of sarcolemmal proteins seems to be normal. The new pathophysiological insights might be of relevance for the development of therapeutic strategies for caveolinopathy patients targeting improved protein folding capacity.

Tracking Effects of SIL1 Increase: Taking a Closer Look Beyond the Consequences of Elevated Expression Level.

SIL1 acts as a co-chaperone for the major ER-resident chaperone BiP and thus plays a role in many BiP-dependent cellular functions such as protein-folding control and unfolded protein response. Whereas the increase of BiP upon cellular stress conditions is a well-known phenomenon, elevation of SIL1 under stress conditions was thus far solely studied in yeast, and different studies indicated an adverse effect of SIL1 increase. This is seemingly in contrast with the beneficial effect of SIL1 increase in surviving neurons in neurodegenerative disorders such as amyotrophic lateral sclerosis and Alzheimer's disease. Here, we addressed these controversial findings. Applying cell biological, morphological and biochemical methods, we demonstrated that SIL1 increases in various mammalian cells and neuronal tissues upon cellular stress. Investigation of heterozygous SIL1 mutant cells and tissues supported this finding. Moreover, SIL1 protein was found to be stabilized during ER stress. Increased SIL1 initiates ER stress in a concentration-dependent manner which agrees with the described adverse SIL1 effect. However, our results also suggest that protective levels are achieved by the secretion of excessive SIL1 and GRP170 and that moderately increased SIL1 also ameliorates cellular fitness under stress conditions. Our immunoprecipitation results indicate that SIL1 might act in a BiP-independent manner. Proteomic studies showed that SIL1 elevation alters the expression of proteins including crucial players in neurodegeneration, especially in Alzheimer's disease. This finding agrees with our observation of increased SIL1 immunoreactivity in surviving neurons of Alzheimer's disease autopsy cases and supports the assumption that SIL1 plays a protective role in neurodegenerative disorders.

In-depth phenotyping of lymphoblastoid cells suggests selective cellular vulnerability in Marinesco-Sjögren syndrome.

SIL1 is a ubiquitous protein of the Endoplasmic Reticulum (ER) acting as a co-chaperone for the ER-resident chaperone, BiP. Recessive mutations of the corresponding gene lead to vulnerability of skeletal muscle and central nervous system in man (Marinesco-Sjögren syndrome; MSS) and mouse. However, it is still unclear how loss of ubiquitous SIL1 leads to selective vulnerability of the nervous system and skeletal muscle whereas other cells and organs are protected from clinical manifestations. In this study we aimed to disentangle proteins participating in selective vulnerability of SIL1-deficient cells and tissues: morphological examination of MSS patient-derived lymphoblastoid cells revealed altered organelle structures (ER, nucleus and mitochondria) thus showing subclinical vulnerability. To correlate structural perturbations with biochemical changes and to identify proteins potentially preventing phenotypical manifestation, proteomic studies have been carried out. Results of proteomic profiling are in line with the morphological findings and show affection of nuclear, mitochondrial and cytoskeletal proteins as well as of such responsible for cellular viability. Moreover, expression patterns of proteins known to be involved in neuromuscular disorders or in development and function of the nervous system were altered. Paradigmatic findings were confirmed by immunohistochemistry of splenic lymphocytes and the cerebellum of SIL1-deficient mice. Ataxin-10, identified with increased abundance in our proteome profile, is necessary for the neuronal survival but also controls muscle fiber apoptosis, thus declaring this protein as a plausible candidate for selective tissue vulnerability. Our combined results provide first insights into the molecular causes of selective cell and tissue vulnerability defining the MSS phenotype.

Sil1-Mutant Mice Elucidate Chaperone Function in Neurological Disorders.

Chaperone dysfunction leading to the build-up of misfolded proteins could frequently be linked to clinical manifestations also affecting the nervous system and the skeletal muscle. In addition, increase in chaperone function is beneficial to antagonize protein aggregation and thus represents a promising target for therapeutic concepts for many genetic and acquired chaperonopathies. However, little is known on the precise molecular mechanisms defining the cell and tissue abnormalities in the case of impaired chaperone function as well as on underlying effects in the case of compensatory up-regulation of chaperones. This scarcity of knowledge often arises from a lack of appropriate animal models that mimic closely the human molecular, cellular, and histological characteristics. Here, we introduce the Sil1-mutant woozy mouse as a suitable model to investigate molecular and cellular mechanisms of impaired ER-chaperone function affecting the integrity of nervous system and skeletal muscle. The overlapping clinical findings in man and mouse indicate that woozy is a good copy of a human phenotype called Marinesco-Sjögren syndrome. We confirm the presence of ER-stress and expand the biochemical knowledge of altered nuclear envelope in muscle, a hallmark of SIL1-disease. In addition, our data suggest that impaired excitation-contraction coupling might be part of the SIL1-pathophysiology. Our results moreover indicate that divergent expression of pro- and anti-survival proteins is decisive for Purkinje cell survival. By summarizing the current knowledge of woozy, we focus on the suitability of this animal model to study neuroprotective co-chaperone function and to investigate the involvement of co-chaperones in the predisposition of other disorders such as diabetic neuropathy.

Cellular Signature of SIL1 Depletion: Disease Pathogenesis due to Alterations in Protein Composition Beyond the ER Machinery.

SIL1 acts as nucleotide exchange factor for the endoplasmic reticulum chaperone BiP. Mutations of SIL1 cause Marinesco-Sjögren syndrome (MSS), a neurodegenerative disorder. Moreover, a particular function of SIL1 for etiopathology of amyotrophic lateral sclerosis (ALS) was highlighted, thus declaring the functional SIL1-BiP complex as a modifier for neurodegenerative disorders. Thereby, depletion of SIL1 was associated with an earlier manifestation and in strengthened disease progression in ALS. Owing to the absence of appropriate in vitro models, the precise cellular pathophysiological mechanisms leading to neurodegeneration in MSS and triggering the same in further disorders like ALS are still elusive. We found that SIL1 depletion in human embryonic kidney 293 (HEK293) cells led to structural changes of the endoplasmic reticulum (ER) including the nuclear envelope and mitochondrial degeneration that closely mimic pathological alterations in MSS and ALS. Functional studies revealed disturbed protein transport, cytotoxicity with reduced proliferation and viability, accompanied by activation of cellular defense mechanisms including the unfolded protein response, ER-associated degradation pathway, proteolysis, and expression of apoptotic and survival factors. Our data moreover indicated that proteins involved in cytoskeletal organization, vesicular transport, mitochondrial function, and neurological processes contribute to SIL1 pathophysiology. Altered protein expression upon SIL1 depletion in vitro could be confirmed in Sil1-deficient motoneurones for paradigmatic proteins belonging to different functional classes. Our results demonstrate that SIL1-depleted HEK293 cells are an appropriate model to identify proteins modulated by SIL1 expression level and contributing to neurodegeneration in MSS and further disorders like ALS. Thereby, our combined results point out that proteins beyond such involved ER-related protein processing are affected by SIL1 depletion.