C9orf72 frontotemporal lobar degeneration is characterised by frequent neuronal sense and antisense RNA foci.

An expanded GGGGCC repeat in a non-coding region of the C9orf72 gene is a common cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis. Non-coding repeat expansions may cause disease by reducing the expression level of the gene they reside in, by producing toxic aggregates of repeat RNA termed RNA foci, or by producing toxic proteins generated by repeat-associated non-ATG translation. We present the first definitive report of C9orf72 repeat sense and antisense RNA foci using a series of C9FTLD cases, and neurodegenerative disease and normal controls. A sensitive and specific fluorescence in situ hybridisation protocol was combined with protein immunostaining to show that both sense and antisense foci were frequent, specific to C9FTLD, and present in neurons of the frontal cortex, hippocampus and cerebellum. High-resolution imaging also allowed accurate analyses of foci number and subcellular localisation. RNA foci were most abundant in the frontal cortex, where 51 % of neurons contained foci. RNA foci also occurred in astrocytes, microglia and oligodendrocytes but to a lesser degree than in neurons. RNA foci were observed in both TDP-43- and p62-inclusion bearing neurons, but not at a greater frequency than expected by chance. RNA foci abundance in the frontal cortex showed a significant inverse correlation with age at onset of disease. These data establish that sense and antisense C9orf72 repeat RNA foci are a consistent and specific feature of C9FTLD, providing new insight into the pathogenesis of C9FTLD

C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins

An expanded GGGGCC repeat in C9orf72 is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis. A fundamental question is whether toxicity is driven by the repeat RNA itself and/or by dipeptide repeat proteins generated by repeat-associated, non-ATG translation. To address this question we developed in vitro and in vivo models to dissect repeat RNA and dipeptide repeat protein toxicity. Expression of pure repeats in Drosophila caused adult-onset neurodegeneration attributable to poly-(glycine-arginine) proteins. Thus, expanded repeats promoted neurodegeneration through neurotoxic proteins. Expression of individual dipeptide repeat proteins with a non-GGGGCC RNA sequence showed both poly-(glycine-arginine) and poly-(proline-arginine) proteins caused neurodegeneration. These findings are consistent with a dual toxicity mechanism, whereby both arginine-rich proteins and repeat RNA contribute to C9orf72-mediated neurodegeneration

Frontotemporal dementia caused by CHMP2B mutation is characterised by neuronal lysosomal storage pathology

Mutations in the charged multivesicular body protein 2B (CHMP2B) cause frontotemporal dementia (FTD). We report that mice which express FTD-causative mutant CHMP2B at physiological levels develop a novel lysosomal storage pathology characterised by large neuronal autofluorescent aggregates. The aggregates are an early and progressive pathology that occur at 3 months of age and increase in both size and number over time. These autofluorescent aggregates are not observed in mice expressing wild-type CHMP2B, or in non-transgenic controls, indicating that they are a specific pathology caused by mutant CHMP2B. Ultrastructural analysis and immuno- gold labelling confirmed that they are derived from the endolysosomal system. Consistent with these findings, CHMP2B mutation patient brains contain morphologically similar autofluorescent aggregates. These aggregates occur significantly more frequently in human CHMP2B mutation brain than in neurodegenerative disease or age-matched control brains. These data suggest that lysosomal storage pathology is the major neuronal pathology in FTD caused by CHMP2B mutation. Recent evidence suggests that two other genes associated with FTD, GRN and TMEM106B are important for lysosomal function. Our identification of lysosomal storage pathology in FTD caused by CHMP2B mutation now provides evidence that endolysosomal dysfunction is a major degenerative pathway in FTD

Frontotemporal dementia causative CHMP2B impairs neuronal endolysosomal traffic-rescue by TMEM106B knockdown

Mutations in the endosome-associated protein CHMP2B cause frontotemporal dementia and lead to lysosomal storage pathology in neurons. We here report that physiological levels of mutant CHMP2B causes reduced numbers and significantly impaired trafficking of endolysosomes within neuronal dendrites, accompanied by increased dendritic branching. Mechanistically, this is due to the stable incorporation of mutant CHMP2B onto neuronal endolysosomes, which we show renders them unable to traffic within dendrites. This defect is due to the inability of mutant CHMP2B to recruit the ATPase VPS4, which is required for release of CHMP2B from endosomal membranes. Strikingly, both impaired trafficking and the increased dendritic branching were rescued by treatment with antisense oligonucleotides targeting the well validated frontotemporal dementia risk factor TMEM106B, which encodes an endolysosomal protein. This indicates that reducing TMEM106B levels can restore endosomal health in frontotemporal dementia. As TMEM106B is a risk factor for frontotemporal dementia caused by both C9orf72 and progranulin mutations, and antisense oligonucleotides are showing promise as therapeutics for neurodegenerative diseases, our data suggests a potential new strategy for treating the wide range of frontotemporal dementias associated with endolysosomal dysfunction

The integration site of the APP transgene in the J20 mouse model of Alzheimer’s disease

Background: Transgenic animal models are a widely used and powerful tool to investigate human disease and develop therapeutic interventions. Making a transgenic mouse involves random integration of exogenous DNA into the host genome that can have the effect of disrupting endogenous gene expression. The J20 mouse model of Alzheimer’s disease (AD) is a transgenic overexpresser of human APP with familial AD mutations and has been extensively utilised in preclinical studies and our aim was to determine the genomic location of the J20 transgene insertion. Methods: We used a combination of breeding strategy and Targeted Locus Amplification with deep sequencing to identify the insertion site of the J20 transgene array. To assess RNA and protein expression of Zbtb20, we used qRT-PCR and Western Blotting. Results: We demonstrate that the J20 transgene construct has inserted within the genetic locus of endogenous mouse gene Zbtb20 on chromosome 16 in an array, disrupting expression of mRNA from this gene in adult hippocampal tissue, while expression of Zbtb20 protein remains unchanged. We note that the endogenous mouse App gene also lies on chromosome 16, although 42 Mb from the Zbtb20 locus. CONCLUSIONS: These data will be useful for future studies utilising this popular model of AD, particularly those investigating gene interactions between the J20 APP transgene and other genes present on Mmu16 in the mouse