Broad clinical phenotypes associated with TAR-DNA binding protein (TARDBP) mutations in amyotrophic lateral sclerosis

The finding of TDP-43 as a major component of ubiquitinated protein inclusions in amyotrophic lateral sclerosis (ALS) has led to the identification of 30 mutations in the transactive response-DNA binding protein (TARDBP) gene, encoding TDP-43. All but one are in exon 6, which encodes the glycine-rich domain. The aim of this study was to determine the frequency of TARDBP mutations in a large cohort of motor neurone disease patients from Northern England (42 non-superoxide dismutase 1 (SOD1) familial ALS (FALS), nine ALS-frontotemporal dementia, 474 sporadic ALS (SALS), 45 progressive muscular atrophy cases). We identified four mutations, two of which were novel, in two familial (FALS) and two sporadic (SALS) cases, giving a frequency of TARDBP mutations in non-SOD1 FALS of 5% and SALS of 0.4%. Analysis of clinical data identified that patients had typical ALS, with limb or bulbar onset, and showed considerable variation in age of onset and rapidity of disease course. However, all cases had an absence of clinically overt cognitive dysfunction

Bioenergetic status modulates motor neuron vulnerability and pathogenesis in a zebrafish model of spinal muscular atrophy

Degeneration and loss of lower motor neurons is the major pathological hallmark of spinal muscular atrophy (SMA), resulting from low levels of ubiquitously-expressed survival motor neuron (SMN) protein. One remarkable, yet unresolved, feature of SMA is that not all motor neurons are equally affected, with some populations displaying a robust resistance to the disease. Here, we demonstrate that selective vulnerability of distinct motor neuron pools arises from fundamental modifications to their basal molecular profiles. Comparative gene expression profiling of motor neurons innervating the extensor digitorum longus (disease-resistant), gastrocnemius (intermediate vulnerability), and tibialis anterior (vulnerable) muscles in mice revealed that disease susceptibility correlates strongly with a modified bioenergetic profile. Targeting of identified bioenergetic pathways by enhancing mitochondrial biogenesis rescued motor axon defects in SMA zebrafish. Moreover, targeting of a single bioenergetic protein, phosphoglycerate kinase 1 (Pgk1), was found to modulate motor neuron vulnerability in vivo. Knockdown of pgk1 alone was sufficient to partially mimic the SMA phenotype in wild-type zebrafish. Conversely, Pgk1 overexpression, or treatment with terazosin (an FDA-approved small molecule that binds and activates Pgk1), rescued motor axon phenotypes in SMA zebrafish. We conclude that global bioenergetics pathways can be therapeutically manipulated to ameliorate SMA motor neuron phenotypes in vivo

Antisense RNA foci in the motor neurons of C9ORF72-ALS patients are associated with TDP-43 proteinopathy

GGGGCC repeat expansions of C9ORF72 represent the most common genetic variant of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We and others have proposed that RNA transcribed from the repeat sequence is toxic via sequestration of RNA-binding factors. Both GGGGCC-repeat (sense) and CCCCGG-repeat (antisense) molecules are detectable by fluorescence in situ hybridisation as RNA foci, but their relative expression pattern within the CNS and contribution to disease has not been determined. Blinded examination of CNS biosamples from ALS patients with a repeat expansion of C9ORF72 showed that antisense foci are present at a significantly higher frequency in cerebellar Purkinje neurons and motor neurons, whereas sense foci are present at a significantly higher frequency in cerebellar granule neurons. Consistent with this, inclusions containing sense or antisense derived dipeptide repeat proteins were present at significantly higher frequency in cerebellar granule neurons or motor neurons, respectively. Immunohistochemistry and UV-crosslinking studies showed that sense and antisense RNA molecules share similar interactions with SRSF2, hnRNP K, hnRNP A1, ALYREF, and hnRNP H/F. Together these data suggest that, although sense and antisense RNA molecules might be expected to be equally toxic via their shared protein binding partners, distinct patterns of expression in various CNS neuronal populations could lead to relative differences in their contribution to the pathogenesis of neuronal injury. Moreover in motor neurons, which are the primary target of pathology in ALS, the presence of antisense foci (χ2, p 2, p = 0.75) correlated with mislocalisation of TDP-43, which is the hallmark of ALS neurodegeneration. This has implications for translational approaches to C9ORF72 disease, and furthermore interacting RNA-processing factors and transcriptional activators responsible for antisense versus sense transcription might represent novel therapeutic targets

TMEM106B is a genetic modifier of frontotemporal lobar degeneration with C9orf72 hexanucleotide repeat expansions

Hexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9orf72) have recently been linked to frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis, and may be the most common genetic cause of both neurodegenerative diseases. Genetic variants at TMEM106B influence risk for the most common neuropathological subtype of FTLD, characterized by inclusions of TAR DNA-binding protein of 43 kDa (FTLD-TDP). Previous reports have shown that TMEM106B is a genetic modifier of FTLD-TDP caused by progranulin (GRN) mutations, with the major (risk) allele of rs1990622 associating with earlier age at onset of disease. Here, we report that rs1990622 genotype affects age at death in a single-site discovery cohort of FTLD patients with C9orf72 expansions (n = 14), with the major allele correlated with later age at death (p = 0.024). We replicate this modifier effect in a 30-site international neuropathological cohort of FTLD-TDP patients with C9orf72 expansions (n = 75), again finding that the major allele associates with later age at death (p = 0.016), as well as later age at onset (p = 0.019). In contrast, TMEM106B genotype does not affect age at onset or death in 241 FTLD-TDP cases negative for GRN mutations or C9orf72 expansions. Thus, TMEM106B is a genetic modifier of FTLD with C9orf72 expansions. Intriguingly, the genotype that confers increased risk for developing FTLD-TDP (major, or T, allele of rs1990622) is associated with later age at onset and death in C9orf72 expansion carriers, providing an example of sign epistasis in human neurodegenerative disease

Sequestration of multiple RNA recognition motif-containing proteins by C9orf72 repeat expansions

GGGGCC repeat expansions of C9orf72 represent the most common genetic variant of amyotrophic lateral sclerosis and frontotemporal degeneration, but the mechanism of pathogenesis is unclear. Recent reports have suggested that the transcribed repeat might form toxic RNA foci that sequester various RNA processing proteins. Consensus as to the identity of the binding partners is missing and whole neuronal proteome investigation is needed. Using RNA fluorescence in situ hybridization we first identified nuclear and cytoplasmic RNA foci in peripheral and central nervous system biosamples from patients with amyotrophic lateral sclerosis with a repeat expansion of C9orf72 (C9orf72 + ), but not from those patients without a repeat expansion of C9orf72 (C9orf72) or control subjects. Moreover, in the cases examined, the distribution of foci-positive neurons correlated with the clinical phenotype (t-test P5 0.05). As expected, RNA foci are ablated by RNase treatment. Interestingly, we identified foci in fibroblasts from an asymptomatic C9orf72 + carrier. We next performed pulldown assays, with GGGGCC5, in conjunction with mass spectrometry analysis, to identify candidate binding partners of the GGGGCC repeat expansion. Proteins containing RNA recognition motifs and involved in splicing, messenger RNA nuclear export and/or translation were significantly enriched. Immunohistochemistry in central nervous system tissue from C9orf72 + patients with amyotrophic lateral sclerosis demonstrated co-localization of RNA foci with SRSF2, hnRNP H1/F, ALYREF and hnRNP A1 in cerebellar granule cells and with SRSF2, hnRNP H1/F and ALYREF in motor neurons, the primary target of pathology in amyotrophic lateral sclerosis. Direct binding of proteins to GGGGCC repeat RNA was confirmed in vitro by ultraviolet-crosslinking assays. Co-localization was only detected in a small proportion of RNA foci, suggesting dynamic sequestration rather than irreversible binding. Additional immunohistochemistry demonstrated that neurons with and without RNA foci were equally likely to show nuclear depletion of TDP-43 (2 P = 0.75) or poly-GA dipeptide repeat protein inclusions (2 P = 0.46). Our findings suggest two non-exclusive pathogenic mechanisms: (i) functional depletion of RNA-processing proteins resulting in disruption of messenger RNA splicing; and (ii) licensing of expanded C9orf72 pre-messenger RNA for nuclear export by inappropriate association with messenger RNA export adaptor protein(s) leading to cytoplasmic repeat associated non-ATG translation and formation of potentially toxic dipeptide repeat protein.

The Spectrum of C9orf72-mediated Neurodegeneration and Amyotrophic Lateral Sclerosis

The discovery that a hexanucleotide repeat expansion in C9orf72 is the most numerous genetic variant of both amyotrophic lateral sclerosis and frontotemporal dementia has opened a rapidly growing field, which may provide long hoped for advances in the understanding and treatment of these devastating diseases. In this review we describe the various phenotypes, clinical and pathological, associated with expansion of C9orf72, which go beyond amyotrophic lateral sclerosis and frontotemporal dementia to include neurodegeneration more broadly. Next we take a step back and summarize the current understanding of the C9orf72 expansion and its protein products at a molecular level. Three mechanisms are prominent: toxicity mediated directly by RNA transcribed from the repeat; toxicity mediated by dipeptide repeat proteins translated from the repeat sequence; and haploinsufficiency resulting from reduced transcription of the C9orf72 exonic sequence. A series of exciting advances have recently described how dipeptide repeat proteins might interfere with the normal role of the nucleolus in maturation of RNA binding proteins and in production of ribosomes. Importantly, these mechanisms are unlikely to be mutually exclusive. We draw attention to the fact that clinical and pathological similarities to other genetic variants without a repeat expansion must not be overlooked in ascribing a pathogenic mechanism to C9orf72-disease. Finally, with a view to impact on patient care, we discuss current practice with respect to genetic screening in patients with and without a family history of disease, and the most promising developments towards therapy that have been reported to date

Microglia in Motor Neuron Disease

Motor Neuron Disease (MND) is a fatal neurodegenerative condition, which is characterised by the selective loss of the upper and lower motor neurons. At the sites of motor neuron injury, accumulations of activated microglia, the primary immune cells of the central nervous system, are commonly observed in both human post-mortem studies and animal models of MND. Microglial activation has been found to correlate with many clinical features and importantly, the speed of disease progression in humans. Both anti-inflammatory and pro-inflammatory microglial responses have been shown to influence disease progression in humans and models of MND. As such, microglia could both contribute to and protect against inflammatory mechanisms of pathogenesis in MND. While murine models have characterised the microglial response to MND, these studies have painted a complex and often -contradictory picture, indicating a need for further characterisation in humans. This review examines the potential role microglia play in MND, in human and animal studies. Both the pro-inflammatory and anti-inflammatory responses will be addressed, throughout the course of disease, followed by the potential of microglia as a target in the development of disease modifying treatments for MND