Studies of Hereditary Neurological Disease: Disease Mechanisms

National Institute Of Neurological Disorders And Stroke

Investigators

Linked publications, trials & patents

Abstract

Recently our research has focused on five hereditary motor neuron diseases: spinal and bulbar muscular atrophy (SBMA) due to polyglutamine expansion in the androgen receptor (AR), autosomal recessive spinal muscular atrophy (SMA) due to deficiency of the protein SMN, amyotrophic lateral sclerosis type 4 (ALS4) due to mutation in senataxin, amyotrophic lateral sclerosis caused by mutation in VAPB (ALS8), and amyotrophic lateral sclerosis and frontotemporal dementia caused by mutation in C9orf72. Specific research accomplishments include the following: (1) After seeing signs of fatty liver in SBMA patients, we set out to determine the prevalence and features of this finding. Two groups of participants with SBMA were evaluated. In the first group, 22 participants underwent laboratory analysis and liver imaging. In the second group, 14 participants were compared to 13 female carriers and 23 controls. Liver biopsies were done in 4 participants with SBMA. Evidence of fatty liver disease was detected by magnetic resonance spectroscopy in all participants with SBMA in the first group, with an average dome intrahepatic triacylglycerol of 27% (range 6%66%, ref #5.5%). Liver dome magnetic resonance spectroscopy measurements were significantly increased in participants with SBMA in the second group relative to age- and sex-matched controls, with average disease and male control measurements of 17% and 3%, respectively. Liver biopsies were consistent with simple steatosis in 2 participants and nonalcoholic steatohepatitis in 2 others. These observations expand the phenotypic spectrum of the disease and provide a potential biomarker that can be monitored in future studies. (2) RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. In recent years, miRNA-mediated gene regulation has shown potential for treatment of neurological disorders caused by a toxic gain of function mechanism. However, efficient delivery to target tissues has limited its application. Here we used a transgenic mouse model for spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR), to test gene silencing by a newly identified AR-targeting miRNA, miR-298. We overexpressed miR-298 using a recombinant adeno associated virus (rAAV) serotype 9 vector to facilitate transduction of non-dividing cells. A single tail-vein injection in SBMA mice induced sustained and widespread overexpression of miR-298 in skeletal muscle and motor neurons and resulted in amelioration of the neuromuscular phenotype in the mice. (3) R-loops are three-stranded nucleic acid structures found abundantly and yet often viewed as by products of transcription. Studying cells from patients with ALS4 and a mutation in senataxin, we discovered how R-loops promote transcription. In ALS4 patients, the senataxin mutation depletes R-loops with a consequent effect on gene expression. With fewer R-loops in ALS4 cells, the expression of BAMBI, a negative regulator of transforming growth factor b (TGF-b), is reduced; that then leads to the activation of the TGF-b pathway. We discovered that genome-wide R-loops influence promoter methylation of over 1,200 human genes. DNA methyl-transferase 1 favors binding to double stranded DNA over R-loops. Thus, in forming R-loops, nascent RNA blocks DNA methylation and promotes further transcription. Hence, our results show that nucleic acid structures, in addition to sequences, influence the binding and activity of regulatory proteins. (4) We evaluated a North American ALS patient using exome sequencing, and identified a P56S mutation in VAPB. The disease protein had similar subcellular localization and expression levels in the patient and control fibroblasts. Patient fibroblasts showed increased basal endoplasmic reticulum stress and dysfunction of nucleocytoplasmic transport as evidenced by impaired Ran trafficking. This finding indicates a cellular defect in ALS8 similar to other forms of hereditary motor neuron disease. (5) We collaborated in a study of ALS/FTD with C9orf72 repeat expansion mutation. The pathogenic mechanism of this repeat remains unclear. Using human induced motor neurons, it was found that repeat-expanded C9orf72 was haploinsufficient in ALS. The C9orf72 gene product interacted with endosomes and was required for normal vesicle trafficking and lysosomal biogenesis in motor neurons. Repeat expansion reduced C9orf72 expression, triggering neurodegeneration through two mechanisms: accumulation of glutamate receptors, leading to excitotoxicity, and impaired clearance of neurotoxic dipeptide repeat proteins derived from the repeat expansion. Thus, cooperativity between gain- and loss-of-function mechanisms led to neurodegeneration. Restoring C9orf72 levels or augmenting its function rescued patient neuron survival and ameliorated neurodegenerative processes in both gain- and loss-of-function C9orf72 mouse models. Thus, modulating vesicle trafficking was able to rescue neurodegeneration caused by the C9ORF72 repeat expansion. Coupled with rare mutations in ALS2, FIG4, CHMP2B, OPTN and SQSTM1, these results reveal mechanistic convergence on vesicle trafficking in ALS and FTD.