Joanna J. Bury

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.

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.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that lacks a predictive and broadly applicable biomarker. Continued focus on mutation-specific upstream mechanisms has yet to predict disease progression in the clinic. Utilising cellular pathology common to the majority of ALS patients, we implemented an objective transcriptome-driven approach to develop noninvasive prognostic biomarkers for disease progression. Genes expressed in laser captured motor neurons in direct correlation (Spearman rank correlation, p < 0.01) with counts of neuropathology were developed into co-expression network modules. Screening modules using three gene sets representing rate of disease progression and upstream genetic association with ALS led to the prioritisation of a single module enriched for immune response to motor neuron degeneration. Genes in the network module are important for microglial activation and predict disease progression in genetically heterogeneous ALS cohorts: Expression of three genes in peripheral lymphocytes - LILRA2, ITGB2 and CEBPD - differentiate patients with rapid and slowly progressive disease, suggesting promise as a blood-derived biomarker. TREM2 is a member of the network module and the level of soluble TREM2 protein in cerebrospinal fluid is shown to predict survival when measured in late stage disease (Spearman rank correlation, p = 0.01). Our data-driven systems approach has, for the first time, directly linked microglia to the development of motor neuron pathology. LILRA2, ITGB2 and CEBPD represent peripherally accessible candidate biomarkers and TREM2 provides a broadly applicable therapeutic target for ALS.

Amyotrophic lateral sclerosis (ALS) is characterized by motor neurone loss resulting in muscle weakness, spasticity and ultimately death. 5-10% are caused by inherited mutations, most commonly C9ORF72, SOD1, TARDBP and FUS. Rarer genetic causes of ALS include mutation of optineurin (mt OPTN). Furthermore, optineurin protein has been localized to the ubiquitylated aggregates in several neurodegenerative diseases, including ALS. This study: (i) investigated the frequency of mt OPTN in ALS patients in England; (ii) characterized the clinical and neuropathological features of ALS associated with a mt OPTN; and (iii) investigated optineurin neuropathology in C9ORF72-related ALS (C9ORF72-ALS). We identified a heterozygous p.E322K missense mutation in exon 10 of OPTN in one familial ALS patient who additionally had a C9ORF72 mutation. This patient had bulbar, limb and respiratory disease without cognitive problems. Neuropathology revealed motor neurone loss, trans-activation response DNA protein 43 (TDP-43)-positive neuronal and glial cytoplasmic inclusions together with TDP-43-negative neuronal cytoplasmic inclusions in extra motor regions that are characteristic of C9ORF72-ALS. We have demonstrated that both TDP-43-positive and negative inclusion types had positive staining for optineurin by immunohistochemistry. We went on to show that optineurin was present in TDP-43-negative cytoplasmic extra motor inclusions in C9ORF72-ALS cases that do not carry mt OPTN. We conclude that: (i) OPTN mutations are associated with ALS; (ii) optineurin protein is present in a subset of the extramotor inclusions of C9ORF72-ALS; (iii) It is not uncommon for multiple ALS-causing mutations to occur in the same patient; and (iv) studies of optineurin are likely to provide useful dataregarding the pathophysiology of ALS and neurodegeneration.

Type 2 diabetes mellitus (T2D), characterised by peripheral insulin resistance, is a risk factor for dementia. In addition to its contribution to small and large vessel disease, T2D may directly damage cells of the brain neurovascular unit. In this study, we investigated the transcriptomic changes in cortical neurones, and associated astrocytes and endothelial cells of the neurovascular unit, in the ageing brain. Neurone, astrocyte, and endothelial cell-enriched mRNA, obtained by immuno-laser capture microdissection of temporal cortex (Brodmann area 21/22) from 6 cases with self-reported T2D in the Cognitive Function and Ageing Study neuropathology cohort, and an equal number of age and sex-matched controls, was assessed by microarray analysis. Integrated Molecular Pathway Level Analysis was performed using the Kyoto Encyclopaedia of Genes and Genomes database on significantly differentially expressed genes, defined as P < 0.05 and fold-change ± 1.2. Hub genes identified from Weighted Gene Co-expression Network Analysis were validated in neurones using the NanoString nCounter platform. The expression and cellular localisation of proteins encoded by selected candidate genes were confirmed by immunohistochemistry. 912, 2202, and 1227 genes were significantly differentially expressed between cases with self-reported T2D and controls in neurones, astrocytes, and endothelial cells respectively. Changes in cortical neurones included alterations in insulin and other signalling pathways, cell cycle, cellular senescence, inflammatory mediators, and components of the mitochondrial respiratory electron transport chain. Impaired insulin signalling was shared by neurovascular unit cells with, additionally, apoptotic pathway changes in astrocytes and dysregulation of advanced glycation end-product signalling in endothelial cells. Transcriptomic analysis identified changes in key cellular pathways associated with T2D that may contribute to neuronal damage and dysfunction. These effects on brain cells potentially contribute to a diabetic dementia, and may provide novel approaches for therapeutic intervention.