Spinal cord neurone loss and foot placement changes in a rat knock-in model of amyotrophic lateral sclerosis Type 8.

Amyotrophic lateral sclerosis is an age-dependent cell type-selective degenerative disease. Genetic studies indicate that amyotrophic lateral sclerosis is part of a spectrum of disorders, ranging from spinal muscular atrophy to frontotemporal dementia that share common pathological mechanisms. Amyotrophic lateral sclerosis Type 8 is a familial disease caused by mis-sense mutations in VAPB. VAPB is localized to the cytoplasmic surface of the endoplasmic reticulum, where it serves as a docking point for cytoplasmic proteins and mediates inter-organelle interactions with the endoplasmic reticulum membrane. A gene knock-in model of amyotrophic lateral sclerosis Type 8 based on the VapBP56S mutation and VapB gene deletion has been generated in rats. These animals display a range of age-dependent phenotypes distinct from those previously reported in mouse models of amyotrophic lateral sclerosis Type 8. A loss of motor neurones in VapBP56S/+ and VapBP56S/P56S animals is indicated by a reduction in the number of large choline acetyl transferase-staining cells in the spinal cord. VapB-/- animals exhibit a relative increase in cytoplasmic TDP-43 levels compared with the nucleus, but no large protein aggregates. Concomitant with these spinal cord pathologies VapBP56S/+ , VapBP56S/P56S and VapB-/- animals exhibit age-dependent changes in paw placement and exerted pressures when traversing a CatWalk apparatus, consistent with a somatosensory dysfunction. Extramotor dysfunction is reported in half the cases of motor neurone disease, and this is the first indication of an associated sensory dysfunction in a rodent model of amyotrophic lateral sclerosis. Different rodent models may offer complementary experimental platforms with which to understand the human disease.

Age-dependent dynamics of neuronal VAPBALS inclusions in the adult brain.

Amyotrophic Lateral Sclerosis (ALS) is a relentlessly progressive and fatal disease, caused by the degeneration of upper and lower motor neurons within the brain and spinal cord in the ageing human. The dying neurons contain cytoplasmic inclusions linked to the onset and progression of the disease. Here, we use a Drosophila model of ALS8 (VAPP58S) to understand the modulation of these inclusions in the ageing adult brain. The adult VAPP58S fly shows progressive deterioration in motor function till its demise 25 days post-eclosion. The density of VAPP58S-positive brain inclusions is stable for 5-15 days of age. In contrast, adding a single copy of VAPWT to the VAPP58S animal leads to a large decrease in inclusion density with concomitant rescue of motor function and lifespan. ER stress, a contributing factor in disease, shows reduction with ageing for the disease model. Autophagy, rather than the Ubiquitin Proteasome system, is the dominant mechanism for aggregate clearance. We explored the ability of Drosophila Valosin-containing protein (VCP/TER94), the ALS14 locus, which is involved in cellular protein clearance, to regulate age-dependent aggregation. Contrary to expectation, TER94 overexpression increased VAPP58S punctae density, while its knockdown led to enhanced clearance. Expression of a dominant positive allele, TER94R152H, further stabilised VAPP58S puncta, cementing roles for an ALS8-ALS14 axis. Our results are explained by a mechanism where autophagy is modulated by TER94 knockdown. Our study sheds light on the complex regulatory events involved in the neuronal maintenance of ALS8 aggregates, suggesting a context-dependent switch between proteasomal and autophagy-based mechanisms as the larvae develop into an adult. A deeper understanding of the nucleation and clearance of the inclusions, which affect cellular stress and function, is essential for understanding the initiation and progression of ALS.

Yeast Models of Amyotrophic Lateral Sclerosis Type 8 Mimic Phenotypes Seen in Mammalian Cells Expressing Mutant VAPBP56S.

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disease that results in the loss of motor neurons and can occur sporadically or due to genetic mutations. Among the 30 genes linked to familial ALS, a P56S mutation in VAPB, an ER-resident protein that functions at membrane contact sites, causes ALS type 8. Mammalian cells expressing VAPBP56S have distinctive phenotypes, including ER collapse, protein and/or membrane-containing inclusions, and sensitivity to ER stress. VAPB is conserved through evolution and has two homologs in budding yeast, SCS2 and SCS22. Previously, a humanized version of SCS2 bearing disease-linked mutations was described, and it caused Scs2-containing inclusions when overexpressed in yeast. Here, we describe a yeast model for ALS8 in which the two SCS genes are deleted and replaced with a single chromosomal copy of either wild-type or mutant yeast SCS2 or human VAPB expressed from the SCS2 promoter. These cells display ER collapse, the formation of inclusion-like structures, and sensitivity to tunicamycin, an ER stress-inducing drug. Based on the phenotypic similarity to mammalian cells expressing VAPBP56S, we propose that these models can be used to study the molecular basis of cell death or dysfunction in ALS8. Moreover, other conserved ALS-linked genes may create opportunities for the generation of yeast models of disease.

Overexpression of mTOR in Leukocytes from ALS8 Patients.

BACKGROUND: The mutated VAPBP56S (vesicle B associated membrane protein - P56S) protein has been described in a Brazilian family and classified as Amyotrophic Lateral Sclerosis type 8 (ALS8). OBJECTIVE: We aimed to study altered biochemical and immunological parameters in cells from ALS8 patients to identify possible biomarkers or therapeutic targets. METHODS: Wild-type VAPB, VAPBP56S, mTOR, proinflammatory cytokines, and oxidant/reducing levels in serum, leucocytes, and cellular lysate from ALS8 patients and health Controls were performed by ELISA, fluorimetry, and spectrophotometry. RESULTS: Our results showed similar levels of mutant and wild-type VAPB in serum and intracellular lysate (p > 0.05) when ALS8 patients and Controls were compared. IL-1ß, IL-6, and IL-18 levels in patients and Controls showed no difference, suggesting an absence of peripheral inflammation (p > 0.05). Oxidative metabolic response, assessed by mitochondrial ROS production, and reductive response by MTT reduction, were higher in the ALS8 group compared to Controls (p < 0.05), although not characterizing typical oxidative stress in ALS8 patients. Total mTOR levels (phosphorylated or non-phosphorylated) of ALS8 patients were significantly lower in serum and higher in intracellular lysate than the mean equivalents in Controls (p < 0.05). A similar result was observed when we quantified the phosphorylated protein (p < 0.05). CONCLUSION: We demonstrate the possibility of using these biochemical and immunological parameters as potential therapeutic targets or biomarkers. Furthermore, by hypothesis, we suggest a hormetic response in which both VAPB forms could coexist in different proportions throughout life. The mutated VAPBP56S production would increase with aging and predominate over the wild-type VAPB levels, determining the onset of symptoms and aggravating the disease.

The history behind ALS type 8: from the first phenotype description to the discovery of VAPB mutation / A história por detrás da ELA tipo 8: da primeira descrição do fenótipo à descoberta da mutação VAPB

ABSTRACT Over the past 68 years, the Finkel type late-onset adult autosomal dominant spinal muscular atrophy (SMA) that is allelic with amyotrophic lateral sclerosis-8 (ALS8) gained a genotype-phenotype correlation among the motor neuron diseases through the work of groups led by Zatz and Marques Jr.

RESUMO Nos últimos 68 anos, a atrofia muscular espinhal (AME), autossômica dominante, de início tardio, em adultos, conhecida como doença de Finkel, que é alélica com esclerose lateral amiotrófica tipo 8 (ELA8), ganhou uma correlação fenotípica e genotípica dentre as doenças do neurônio motor, a partir da colaboração dos grupos de Zatz e Marques Jr.

Case Report: Cognitive Conversion in a Non-brazilian VAPB Mutation Carrier (ALS8).

Amyotrophic lateral sclerosis 8 (ALS8) is a predominantly lower motor neuron syndrome originally described in a Portuguese-Brazilian family, which originated from a common founder. ALS8 is caused by a VAPB mutation and extremely rare in Central Europe. We present a 51-year-old German man with ALS8 who had the P56S VAPB mutation independently of the founder effect. In the final 4 years of his life (disease duration 10 years), the patient had five MRI scans and four in-depth neuropsychological assessments. This paper addresses the course of the patient's cognitive status and relates cognitive performance to structural brain changes in order to determine whether this ALS8 case showed a different pattern of cognitive decline compared with sporadic ALS. The executive functions, verbal fluency, and memory of the patient and 17 age-, sex-, and education-matched controls were assessed on four different occasions. His cognitive performance and decline were investigated for abnormality using cross-sectional and longitudinal matched case-control analysis. We obtained five T1-weighted MRI, which we analyzed using voxel-wise non-parametric analysis with statistical non-parametric mapping in Matlab. Moreover, we conducted a single-subject correlation between cognitive performance and brain atrophy. The cognitive profile of the index patient featured executive dysfunction. Notably, his working memory and shifting ability declined from a healthy baseline to an impaired performance, leading to a transition from cognitively non-impaired (ALSni) to cognitively impaired (ALSci). The correlations we observed between cerebellar atrophy and verbal fluency in addition to fusiform gyrus atrophy and shifting are novel findings. We found that the conversion from ALSni to ALSci was associated with widespread cerebral atrophy, which extended beyond the primary motor and premotor cortex and affected, among others, the cerebellum and left fusiform gyrus. The index patients' cognitive profile resembles that of other ALS phenotypes, but the extensive atrophy beyond extra-motor areas has not yet been described.

VAMP associated proteins are required for autophagic and lysosomal degradation by promoting a PtdIns4P-mediated endosomal pathway.

Mutations in the ER-associated VAPB/ALS8 protein cause amyotrophic lateral sclerosis and spinal muscular atrophy. Previous studies have argued that ER stress may underlie the demise of neurons. We find that loss of VAP proteins (VAPs) leads to an accumulation of aberrant lysosomes and impairs lysosomal degradation. VAPs mediate ER to Golgi tethering and their loss may affect phosphatidylinositol-4-phosphate (PtdIns4P) transfer between these organelles. We found that loss of VAPs elevates PtdIns4P levels in the Golgi, leading to an expansion of the endosomal pool derived from the Golgi. Fusion of these endosomes with lysosomes leads to an increase in lysosomes with aberrant acidity, contents, and shape. Importantly, reducing PtdIns4P levels with a PtdIns4-kinase (PtdIns4K) inhibitor, or removing a single copy of Rab7, suppress macroautophagic/autophagic degradation defects as well as behavioral defects observed in Drosophila Vap33 mutant larvae. We propose that a failure to tether the ER to the Golgi when VAPs are lost leads to an increase in Golgi PtdIns4P levels, and an expansion of endosomes resulting in an accumulation of dysfunctional lysosomes and a failure in proper autophagic lysosomal degradation. Abbreviations: ALS: amyotrophic lateral sclerosis; CSF: cerebrospinal fluid; CERT: ceramide transfer protein; FFAT: two phenylalanines in an acidic tract; MSP: major sperm proteins; OSBP: oxysterol binding protein; PH: pleckstrin homology; PtdIns4P: phosphatidylinositol-4-phosphate; PtdIns4K: phosphatidylinositol 4-kinase; UPR: unfolded protein response; VAMP: vesicle-associated membrane protein; VAPA/B: mammalian VAPA and VAPB proteins; VAPs: VAMP-associated proteins (referring to Drosophila Vap33, and human VAPA and VAPB).