J. Robin Highley

Pathological TDP‐43 distinguishes sporadic amyotrophic lateral sclerosis from amyotrophic lateral sclerosis with SOD1 mutations

Amyotrophic lateral sclerosis (ALS) is a common, fatal motor neuron disorder with no effective treatment. Approximately 10% of cases are familial ALS (FALS), and the most common genetic abnormality is superoxide dismutase‐1 (SOD1) mutations. Most ALS research in the past decade has focused on the neurotoxicity of mutant SOD1, and this knowledge has directed therapeutic strategies. We recently identified TDP‐43 as the major pathological protein in sporadic ALS. In this study, we investigated TDP‐43 in a larger series of ALS cases (n = 111), including familial cases with and without SOD1 mutations.

Clinico-pathological features in amyotrophic lateral sclerosis with expansions in C9ORF72

Intronic expansion of the GGGGCC hexanucleotide repeat within the C9ORF72 gene causes frontotemporal dementia and amyotrophic lateral sclerosis/motor neuron disease in both familial and sporadic cases. Initial reports indicate that this variant within the frontotemporal dementia/amyotrophic lateral sclerosis spectrum is associated with transactive response DNA binding protein (TDP-43) proteinopathy. The amyotrophic lateral sclerosis/motor neuron disease phenotype is not yet well characterized. We report the clinical and pathological phenotypes associated with pathogenic C9ORF72 mutations in a cohort of 563 cases from Northern England, including 63 with a family history of amyotrophic lateral sclerosis. One hundred and fifty-eight cases from the cohort (21 familial, 137 sporadic) were post-mortem brain and spinal cord donors. We screened DNA for the C9ORF72 mutation, reviewed clinical case histories and undertook pathological evaluation of brain and spinal cord. Control DNA samples (n = 361) from the same population were also screened. The C9ORF72 intronic expansion was present in 62 cases [11% of the cohort; 27/63 (43%) familial, 35/500 (7%) cases with sporadic amyotrophic lateral sclerosis/motor neuron disease]. Disease duration was significantly shorter in cases with C9ORF72-related amyotrophic lateral sclerosis (30.5 months) compared with non-C9ORF72 amyotrophic lateral sclerosis/motor neuron disease (36.3 months, P < 0.05). C9ORF72 cases included both limb and bulbar onset disease and all cases showed combined upper and lower motor neuron degeneration (amyotrophic lateral sclerosis). Thus, clinically, C9ORF72 cases show the features of a relatively rapidly progressive, but otherwise typical, variant of amyotrophic lateral sclerosis associated with both familial and sporadic presentations. Dementia was present in the patient or a close family member in 22/62 cases with C9ORF72 mutation (35%) based on diagnoses established from retrospective clinical case note review that may underestimate significant cognitive changes in late disease. All the C9ORF72 mutation cases showed classical amyotrophic lateral sclerosis pathology with TDP-43 inclusions in spinal motor neurons. Neuronal cytoplasmic inclusions and glial inclusions positive for p62 immunostaining in non-motor regions were strongly over-represented in the C9ORF72 cases. Extra-motor pathology in the frontal cortex (P < 0.0005) and the hippocampal CA4 subfield neurons (P < 0.0005) discriminated C9ORF72 cases strongly from the rest of the cohort. Inclusions in CA4 neurons were not present in non-C9ORF72 cases, indicating that this pathology predicts mutation status.

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

Expansion of GGGGCC repeats in C9orf72 causes familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, but the underlying mechanism is unclear. Using RNA pulldown and immunohistochemistry in ALS biosamples, Cooper-Knock et al. identify proteins that bind to the repeat expansions. Disrupted RNA splicing and/or nuclear export may underlie C9orf72-ALS pathogenesis.

Molecular pathology and genetic advances in amyotrophic lateral sclerosis: an emerging molecular pathway and the significance of glial pathology

Research into amyotrophic lateral sclerosis (ALS) has been stimulated by a series of genetic and molecular pathology discoveries. The hallmark neuronal cytoplasmic inclusions of sporadic ALS (sALS) predominantly comprise a nuclear RNA processing protein, TDP-43 encoded by the gene TARDBP, a discovery that emerged from high throughput analysis of human brain tissue from patients with frontotemporal dementia (FTD) who share a common molecular pathology with ALS. The link between RNA processing and ALS was further strengthened by the discovery that another genetic locus linking familial ALS (fALS) and FTD was due to mutation of the fused in sarcoma (FUS) gene. Of potentially even greater importance it emerges that TDP-43 accumulation and inclusion formation characterises not only most sALS cases but also those that arise from mutations in several genes including TARDBP (predominantly ALS cases) itself, C9ORF72 (ALS and FTD cases), progranulin (predominantly FTD phenotypes), VAPB (predominantly ALS cases) and in some ALS cases with rare genetic variants of uncertain pathogenicity (CHMP2B). “TDP-proteinopathy” therefore now represents a final common pathology associated with changes in multiple genes and opens the possibility of research by triangulation towards key common upstream molecular events. It also delivers final proof of the hypothesis that ALS and most FTD cases are disorders within a common pathology expressed as a clinico-anatomical spectrum. The emergence of TDP-proteinopathy also confirms the view that glial pathology is a crucial facet in this class of neurodegeneration, adding to the established view of non-nerve cell autonomous degeneration of the motor system from previous research on SOD1 fALS. Future research into the mechanisms of TDP-43 and FUS-related neurodegeneration, taking into account the major component of glial pathology now revealed in those disorders will significantly accelerate new discoveries in this field, including target identification for new therapy.

Novel FUS/TLS Mutations and Pathology in Familial and Sporadic Amyotrophic Lateral Sclerosis

OBJECTIVE To determine the frequency of and clinicopathologic phenotypes associated with FUS/TLS mutations in a large cohort of amyotrophic lateral sclerosis (ALS) cases from the north of England. DESIGN Genetic screening project with neuropathologic examination of postmortem tissue in selected cases. The clinical details of selected cases are also presented. SETTING Neurology departments of 2 university teaching hospitals in the north of England. PARTICIPANTS The 15 exons of FUS/TLS were sequenced in an initial cohort of 42 familial ALS (FALS) and 117 sporadic ALS (SALS) cases. Exons 14 and 15 were subsequently screened in a larger cohort of 431 SALS cases. Regions mutated in ALS cases were also screened in 293 controls. MAIN OUTCOME MEASURE Evaluation of gene-sequencing chromatographs and detailed histopathologic analysis of the central nervous system. RESULTS Four heterozygous mutations, 1 of which is novel, were identified in 6 patients with ALS (4 with FALS and 2 with SALS). Two of the substitutions were not found to be present in controls, and neuropathology in these cases revealed neuronal and/or glial cytoplasmic inclusions positive for the FUS/TLS protein. One of these cases is also the first reported SALS case with an FUS/TLS mutation. The other 2 substitutions identified were also identified in control cases. Neuropathology in these cases revealed typical SALS pathology, suggesting that they are likely to represent benign polymorphisms. CONCLUSIONS FUS/TLS mutations represented approximately 5% of FALS cases screened. A FUS/TLS mutation was also identified in a single SALS case. Subsequent screening of this region in a larger cohort of SALS cases, however, did not reveal any additional mutations.

The size and fiber composition of the anterior commissure with respect to gender and schizophrenia

BACKGROUND In light of evidence for deviations in asymmetry and alterations in the anatomy of the corpus callosum in schizophrenia, this study examined the anterior commissure in post mortem brains (n = 14 female control patients, 15 male control patients, 11 female schizophrenic patients, 15 male schizophrenic patients). METHODS Measures were made of the cross-sectional area of the anterior commissure in the midsagittal plane. In addition, the fiber density and fiber number were measured in a subset of cases (n = 10 female control subjects, 10 male control subjects, 8 female schizophrenic patients, 9 male schizophrenic patients), using the Palmgren silver stain and stereological methods. RESULTS In control subjects, fiber numbers were greater (p = .024) in women than men. In schizophrenia, the cross-sectional area was unaffected, but for fiber density there was a significant gender x diagnosis interaction (p = .026), corresponding to a reduction in female, but not male patients with schizophrenia. CONCLUSIONS The reduction in density of fibers in the anterior commissure is consistent with an alteration of interhemispheric connectivity in schizophrenia, but the restriction of the finding to women emphasizes the relevance of gender to understanding the nature of the hemispheric interaction.

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.

Sex-dependent effects of schizophrenia: an MRI study of gyral folding, and cortical and white matter volume

Alterations, sometimes sex-dependent, in volumes and gyral structure of areas of cerebral cortex have been reported in schizophrenia. Such changes imply an anomaly of connectivity. The gyrification, percentage of tissue volume attributed to white matter, cortical volume and white matter volume were measured from magnetic resonance images in males and females with (n = 61) and without (n = 42) schizophrenia. The frontal, temporal and an amalgam of occipital and parietal lobes were examined in both hemispheres. There was no effect of schizophrenia on the gyrification of the brain. For the volume of occipito-parietal white matter, females with schizophrenia had bilaterally lower volumes, while males with schizophrenia had greater volumes than controls. It is concluded that the changes in connectivity underlying the pathogenesis of schizophrenia are sex-specific and expressed in occipito-parietal white matter.

C9ORF72 GGGGCC Expanded Repeats Produce Splicing Dysregulation which Correlates with Disease Severity in Amyotrophic Lateral Sclerosis.

Objective An intronic GGGGCC-repeat expansion of C9ORF72 is the most common genetic variant of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. The mechanism of neurodegeneration is unknown, but a direct effect on RNA processing mediated by RNA foci transcribed from the repeat sequence has been proposed. Methods Gene expression profiling utilised total RNA extracted from motor neurons and lymphoblastoid cell lines derived from human ALS patients, including those with an expansion of C9ORF72, and controls. In lymphoblastoid cell lines, expansion length and the frequency of sense and antisense RNA foci was also examined. Results Gene level analysis revealed a number of differentially expressed networks and both cell types exhibited dysregulation of a network functionally enriched for genes encoding ‘RNA splicing’ proteins. There was a significant overlap of these genes with an independently generated list of GGGGCC-repeat protein binding partners. At the exon level, in lymphoblastoid cells derived from C9ORF72-ALS patients splicing consistency was lower than in lines derived from non-C9ORF72 ALS patients or controls; furthermore splicing consistency was lower in samples derived from patients with faster disease progression. Frequency of sense RNA foci showed a trend towards being higher in lymphoblastoid cells derived from patients with shorter survival, but there was no detectable correlation between disease severity and DNA expansion length. Significance Up-regulation of genes encoding predicted binding partners of the C9ORF72 expansion is consistent with an attempted compensation for sequestration of these proteins. A number of studies have analysed changes in the transcriptome caused by C9ORF72 expansion, but to date findings have been inconsistent. As a potential explanation we suggest that dynamic sequestration of RNA processing proteins by RNA foci might lead to a loss of splicing consistency; indeed in our samples measurement of splicing consistency correlates with disease severity.

Concurrence of multiple sclerosis and amyotrophic lateral sclerosis in patients with hexanucleotide repeat expansions of C9ORF72

Background Crossover in the pathogenic mechanisms of amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS) has been described but is poorly understood. A GGGGCC hexanucleotide repeat expansion of C9ORF72 has recently been identified in a significant proportion of patients with ALS. Methods In approximately 650 patients diagnosed with ALS from the North of England we identified seven patients who initially presented with MS. DNA obtained from five patients with MS-ALS and 215 patients with MS alone was screened for the C9ORF72 expansion. Post-mortem material was examined from two patients with MS-ALS. Gene expression profiling was performed on lymphoblastoid cells and levels of CXCL10 were measured in cerebrospinal fluid (CSF) from patients with ALS with and without the C9ORF72 expansion and controls. Results Concurrence of MS and ALS is higher than expected in our population. The C9ORF72 expansion was identified in 80% of patients with MS-ALS but not in those with MS alone. In the presence of preceding MS, C9ORF72-ALS was more rapidly progressive. MetaCore analysis identified alteration of the NF-кB pathway in C9ORF72-ALS and non-C9ORF72-ALS. NF-кB activation is associated with increased expression of the neuroprotective cytokine CXCL10 but, in C9ORF72-ALS, CXCL10 is downregulated and CSF levels are reduced. Conclusions We propose that MS-associated neuroinflammation may affect penetrance and progression of the C9ORF72 expansion. In particular, the NF-кB pathway is activated in MS and appears to be dysfunctional in C9ORF72-ALS. Aberrant downregulation of CXCL10 may explain the predisposition of C9ORF72 expansion carriers to develop ALS in the context of MS and NF-кB activation, and offers a potential therapeutic target.