VAPB and C9orf72 mutations in 1 familial amyotrophic lateral sclerosis patient

Previously, we have reported amyotrophic lateral sclerosis (ALS) families with multiple mutations in major ALS-associated genes. These findings provided evidence for an oligogenic basis of ALS. In our present study, we screened a cohort of 755 sporadic ALS patients, 111 familial ALS patients (97 families), and 765 control subjects of Dutch descent for mutations in vesicle-associated membrane protein B (VAPB). We have identified 1 novel VAPB mutation (p.V234I) in a familial ALS patient known to have a chromosome 9 open reading frame 72 (C9orf72) repeat expansion. This p.V234I mutation was absent in control subjects, located in a region with high evolutionary conservation, and predicted to have damaging effects. Taken together, these findings provide additional evidence for an oligogenic basis of ALS.     

ALS‐Associated Endoplasmic Reticulum Proteins in Denervated Skeletal Muscle: Implications for Motor Neuron Disease Pathology

Alpha‐motoneurons and muscle fibres are structurally and functionally interdependent. Both cell types particularly rely on endoplasmic reticulum (ER/SR) functions. Mutations of the ER proteins VAPB, SigR1 and HSP27 lead to hereditary motor neuron diseases (MNDs). Here, we determined the expression profile and localization of these ER proteins/chaperons by immunohistochemistry and immunoblotting in biopsy and autopsy muscle tissue of patients with amyotrophic lateral sclerosis (ALS) and other neurogenic muscular atrophies (NMAs) and compared these patterns to mouse models of neurogenic muscular atrophy. Postsynaptic neuromuscular junction staining for VAPB was intense in normal human and mouse muscle and decreased in denervated Nmd^2J mouse muscle fibres. In contrast, VAPB levels together with other chaperones and autophagy markers were increased in extrasynaptic regions of denervated muscle fibres of patients with MNDs and other NMAs, especially at sites of focal myofibrillar disintegration (targets). These findings did not differ between NMAs due to ALS and other causes. G93A‐SOD1 mouse muscle fibres showed a similar pattern of protein level increases in denervated muscle fibres. In addition, they showed globular VAPB‐immunoreactive structures together with misfolded SOD1 protein accumulations, suggesting a primary myopathic change. Our findings indicate that altered expression and localization of these ER proteins and autophagy markers are part of the dynamic response of muscle fibres to denervation. The ER is particularly prominent and vulnerable in both muscle fibres and alpha‐motoneurons. Thus, ER pathology could contribute to the selective build‐up of degenerative changes in the neuromuscular axis in MNDs.     

FUS, TARDBP, and SOD1 mutations in a Taiwanese cohort with familial ALS

The cause of familial amyotrophic lateral sclerosis (FALS) has been attributed to mutations in several genes. The authors analyzed these genes, including SOD1, FUS, VAPB, ANG, TDP-43, FIG4, and CHMP2B, in a cohort of 15 index patients of Han Chinese descent with adult-onset FALS. Seven different mutations in eight patients, including three in SOD1 (G85R, T137R, and G138E), two in exon 15 of FUS (H517D and R521H), and two in exon 6 of TARDBP (M337V and N378D) were identified. Among them, T137R SOD1, G138E SOD1, H517D FUS, and N378D TARDBP were novel. No mutation was found in VAPB, ANG, FIG4, or CHMP2B genes. Mutations in SOD1, FUS, and TARDBP account for 20%, 13.3%, and 20% of FALS, respectively. This study defined the distribution and frequency of mutations of FALS in a Taiwanese Han Chinese population, which not only broadens the spectrum of the mutations causing FALS, but also further highlights the importance of FUS and TARDBP in the pathogenesis of amyotrophic lateral sclerosis (ALS).     

Role of Vesicle-Associated Membrane Protein-Associated Proteins (VAP) A and VAPB in Nuclear Egress of the Alphaherpesvirus Pseudorabies Virus

The molecular mechanism affecting translocation of newly synthesized herpesvirus nucleocapsids from the nucleus into the cytoplasm is still not fully understood. The viral nuclear egress complex (NEC) mediates budding at and scission from the inner nuclear membrane, but the NEC is not sufficient for efficient fusion of the primary virion envelope with the outer nuclear membrane. Since no other viral protein was found to be essential for this process, it was suggested that a cellular machinery is recruited by viral proteins. However, knowledge on fusion mechanisms involving the nuclear membranes is rare. Recently, vesicle-associated membrane protein-associated protein B (VAPB) was shown to play a role in nuclear egress of herpes simplex virus 1 (HSV-1). To test this for the related alphaherpesvirus pseudorabies virus (PrV), we mutated genes encoding VAPB and VAPA by CRISPR/Cas9-based genome editing in our standard rabbit kidney cells (RK13), either individually or in combination. Single as well as double knockout cells were tested for virus propagation and for defects in nuclear egress. However, no deficiency in virus replication nor any effect on nuclear egress was obvious suggesting that VAPB and VAPA do not play a significant role in this process during PrV infection in RK13 cells.     

Expanding the phenotypes of the Pro56Ser VAPB mutation: proximal SMA with dysautonomia

The phenotype of 16 members of a family affected by a late-onset, dominant, progressive, motor and autonomic disorder is described. The VAPB (Pro56Ser) mutation was detected in Brazilian families with different phenotypes of motor neuron disorders. In this family, proximal and axial muscle weakness and atrophy, associated with abdominal protrusion, defined the motor phenotype. Death occurred in 10-15 years due to respiratory insufficiency. Tone and tendon reflexes were decreased and a distal tremor was common. Sensation was preserved. Autonomic abnormalities were also present, including choking, chronic intestinal constipation, sexual dysfunction, and sudomotor abnormalities, and on nerve morphology there was involvement of unmyelinated fibers. Electromyography disclosed ongoing denervation and reinnervation. Isolated dysfunction of motor and autonomic neurons is unusual among the spinal muscular atrophies. On this basis, this condition seems to represent a new category of disease.     

ALS pathogenesis: Recent insights from genetics and mouse models

For the vast majority of cases of amyotrophic lateral sclerosis (ALS) the etiology remains unknown. After the discovery of missense mutations in the gene coding for the Cu/Zn superoxide dismutase 1 (SOD1) in subsets of familial ALS, several transgenic mouse lines have been generated with various forms of SOD1 mutants overexpressed at different levels. Studies with these mice yielded complex results with multiple targets of damage in disease including mitochondria, proteasomes, and secretory pathways. Many unexpected discoveries were made. For instance, the toxicity of mutant SOD1 seems unrelated to copper-mediated catalysis but rather to formation of misfolded SOD1 species and aggregates. Transgenic studies revealed a potential role of wtSOD1 in exacerbating mutant SOD1-mediated disease. Another key finding came from chimeric mouse studies and from Cre-lox mediated gene deletion experiments which have highlighted the importance of non-neuronal cells in the disease progression. Involvement of cytoskeletal components in ALS pathogenesis is supported by several mouse models of motor neuron disease with neurofilament abnormalities and with genetic defects in microtubule-based transport. Recently, the generation of new animal models of ALS has been made possible with the discovery of ALS-linked mutations in other genes encoding for alsin, dynactin, senataxin, VAPB, TDP-43 and FUS. Following the discovery of mutations in the TARDBP gene linked to ALS, there have been some reports of transgenic mice with high level overexpression of WT or mutant forms of TDP-43 under strong gene promoters. However, these TDP-43 transgenic mice do not exhibit all pathological features the human ALS disease. Here, we will describe these new TDP-43 transgenic mice and discuss their validity as animal models of human ALS.     

Intracellular organelles in health and kidney disease

Subcellular organelles consist of smaller substructures called supramolecular assemblies and these in turn consist of macromolecules. Various subcellular organelles have critical functions that consist of genetic disorders of organelle biogenesis and several metabolic disturbances that occur during non-genetic diseases e.g. infection, intoxication and drug treatments. Mitochondrial damage can cause renal dysfunction as ischemic acute renal injury, chronic kidney disease progression. Moreover, mitochondrial dysfunction is an early event in aldosterone-induced podocyte injury and cardiovascular disease due to oxidative stress in chronic kidney disease. Elevated production of reactive oxygen species could be able to activate NLRP3 inflammasome representing new deregulated biological machinery and a novel therapeutic target in hemodialysis patients. Peroxisomes are actively involved in apoptosis and inflammation, innate immunity, aging and in the pathogenesis of age related diseases, such as diabetes mellitus and cancer. Peroxisomal catalase causes alterations of mitochondrial membrane proteins and stimulates generation of mitochondrial reactive oxygen species. High concentrations of hydrogen peroxide exacerbate organelles and cellular aging. The importance of proper peroxisomal function for the biosynthesis of bile acids has been firmly established. Endoplasmic reticulum stress-induced pathological diseases in kidney cause glomerular injury and tubulointerstitial injury. Furthermore, there is a link between oxidative stress and inflammations in pathological states are associated with endoplasmic reticulum stress. Proteinuria and hyperglycemia in diabetic nephropathy may induce endoplasmic reticulum stress in tubular cells of the kidney. Due to the accumulation in the proximal tubule lysosomes, impaired function of these organelles may be an important mechanism leading to proximal tubular toxicity.