Alice Brockington

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.

Mutations in CHMP2B in Lower Motor Neuron Predominant Amyotrophic Lateral Sclerosis (ALS)

Background Amyotrophic lateral sclerosis (ALS), a common late-onset neurodegenerative disease, is associated with fronto-temporal dementia (FTD) in 3–10% of patients. A mutation in CHMP2B was recently identified in a Danish pedigree with autosomal dominant FTD. Subsequently, two unrelated patients with familial ALS, one of whom also showed features of FTD, were shown to carry missense mutations in CHMP2B. The initial aim of this study was to determine whether mutations in CHMP2B contribute more broadly to ALS pathogenesis. Methodology/Principal Findings Sequencing of CHMP2B in 433 ALS cases from the North of England identified 4 cases carrying 3 missense mutations, including one novel mutation, p.Thr104Asn, none of which were present in 500 neurologically normal controls. Analysis of clinical and neuropathological data of these 4 cases showed a phenotype consistent with the lower motor neuron predominant (progressive muscular atrophy (PMA)) variant of ALS. Only one had a recognised family history of ALS and none had clinically apparent dementia. Microarray analysis of motor neurons from CHMP2B cases, compared to controls, showed a distinct gene expression signature with significant differential expression predicting disassembly of cell structure; increased calcium concentration in the ER lumen; decrease in the availability of ATP; down-regulation of the classical and p38 MAPK signalling pathways, reduction in autophagy initiation and a global repression of translation. Transfection of mutant CHMP2B into HEK-293 and COS-7 cells resulted in the formation of large cytoplasmic vacuoles, aberrant lysosomal localisation demonstrated by CD63 staining and impairment of autophagy indicated by increased levels of LC3-II protein. These changes were absent in control cells transfected with wild-type CHMP2B. Conclusions/Significance We conclude that in a population drawn from North of England pathogenic CHMP2B mutations are found in approximately 1% of cases of ALS and 10% of those with lower motor neuron predominant ALS. We provide a body of evidence indicating the likely pathogenicity of the reported gene alterations. However, absolute confirmation of pathogenicity requires further evidence, including documentation of familial transmission in ALS pedigrees which might be most fruitfully explored in cases with a LMN predominant phenotype.

Vascular endothelial growth factor and the nervous system

Vascular endothelial growth factor (VEGF) is an angiogenic factor essential for the formation of new blood vessels during embryogenesis and in many pathological conditions. A new role for VEGF as a neurotrophic factor has recently emerged. In the developing nervous system, VEGF plays a pivotal role not only in vascularization, but also in neuronal proliferation, and the growth of coordinated vascular and neuronal networks. After injury to the nervous system, activation of VEGF and its receptors may restore blood supply and promote neuronal survival and repair. There is a growing body of evidence that VEGF is essential for motor neurone survival, and that aberrant regulation of VEGF may play a role in the degeneration of neurones in diseases such as amyotrophic lateral sclerosis.

Expression of vascular endothelial growth factor and its receptors in the central nervous system in amyotrophic lateral sclerosis.

Vascular endothelial growth factor (VEGF) prolongs survival in the mutant SOD1 transgenic mouse model of amyotrophic lateral sclerosis (ALS), whereas dysregulation of VEGF through deletion of its hypoxia-regulatory element causes motor neuron degeneration in mice. We investigated the expression of VEGF and its major agonist receptors in the normal central nervous system and in patients with ALS. Immunohistochemistry demonstrated similar expression patterns of VEGF and VEGF receptor 2 (VEGFR2) in the spinal cord with finely punctate staining of the neuropil and strong expression in anterior horn cells (AHCs). Granular staining on the surface of some AHCs, similar to that seen with synaptic markers, suggested synaptic labeling. VEGFR2 staining was reduced in the neuropil of ALS cases (p = 0.018) associated with a reduction of synaptophysin but not SNAP25 expression. A greater proportion of AHCs in ALS cases showed low expression of VEGF (p = 0.006) and VEGFR2 (p = 0.009) compared with controls. Expression of VEGF and VEGFR2 was confirmed by Western blotting and quantitative reverse transcriptase-polymerase chain reaction (QPCR). The similar expression patterns of VEGF and VEGFR2 suggests autocrine/paracrine effects on spinal motor neurons, and the reduction in their expression seen in ALS cases would support the hypothesis that, as in mouse models of the disease, reduced VEGF signaling may play a role in the pathogenesis of ALS.

Meta-analysis of vascular endothelial growth factor variations in amyotrophic lateral sclerosis: increased susceptibility in male carriers of the −2578AA genotype

Background: Targeted delivery of the angiogenic factor, vascular endothelial growth factor (VEGF), to motor neurons prolongs survival in rodent models of amyotrophic lateral sclerosis (ALS), while mice expressing reduced VEGF concentrations develop motor neuron degeneration reminiscent of ALS, raising the question whether VEGF contributes to the pathogenesis of ALS. An initial association study reported that VEGF haplotypes conferred increased susceptibility to ALS in humans, but later studies challenged this initial finding. Methods and findings: A meta-analysis was undertaken to critically reappraise whether any of the three common VEGF gene variations (−2578C/A, −1154G/A and −634G/C) increase the risk of ALS. Over 7000 subjects from eight European and three American populations were included in the analysis. Pooled odds ratios were calculated using fixed-effects and random-effects models, and four potential sources of heterogeneity (location of disease onset, gender, age at disease onset and disease duration) were assessed. After correction, none of the genotypes or haplotypes was significantly associated with ALS. Subgroup analysis by gender revealed, however, that the −2578AA genotype, which lowers VEGF expression, increased the risk of ALS in males (OR = 1.46 males vs females; 95% CI = 1.19 to 1.80; p = 7.8 10E-5), even after correction for publication bias and multiple testing. Conclusions: This meta-analysis does not support the original conclusion that VEGF haplotypes increase the risk of ALS in humans, but the significant association of the low-VEGF −2578AA genotype with increased susceptibility to ALS in males reappraises the link between reduced VEGF concentrations and ALS, as originally revealed by the fortuitous mouse genetic studies.

Pattern of spread and prognosis in lower limb-onset ALS

Abstract Our objective was to establish the pattern of spread in lower limb-onset ALS (contra- versus ipsi-lateral) and its contribution to prognosis within a multivariate model. Pattern of spread was established in 109 sporadic ALS patients with lower limb-onset, prospectively recorded in Oxford and Sheffield tertiary clinics from 2001 to 2008. Survival analysis was by univariate Kaplan-Meier log-rank and multivariate Cox proportional hazards. Variables studied were time to next limb progression, site of next progression, age at symptom onset, gender, diagnostic latency and use of riluzole. Initial progression was either to the contralateral leg (76%) or ipsilateral arm (24%). Factors independently affecting survival were time to next limb progression, age at symptom onset, and diagnostic latency. Time to progression as a prognostic factor was independent of initial direction of spread. In a regression analysis of the deceased, overall survival from symptom onset approximated to two years plus the time interval for initial spread. In conclusion, rate of progression in lower limb-onset ALS is not influenced by whether initial spread is to the contralateral limb or ipsilateral arm. The time interval to this initial spread is a powerful factor in predicting overall survival, and could be used to facilitate decision-making and effective care planning.

Downregulation of genes with a function in axon outgrowth and synapse formation in motor neurones of the VEGFδ/δ mouse model of amyotrophic lateral sclerosis

BackgroundVascular endothelial growth factor (VEGF) is an endothelial cell mitogen that stimulates vasculogenesis. It has also been shown to act as a neurotrophic factor in vitro and in vivo. Deletion of the hypoxia response element of the promoter region of the gene encoding VEGF in mice causes a reduction in neural VEGF expression, and results in adult-onset motor neurone degeneration that resembles amyotrophic lateral sclerosis (ALS). Investigating the molecular pathways to neurodegeneration in the VEGFδ/δ mouse model of ALS may improve understanding of the mechanisms of motor neurone death in the human disease.ResultsMicroarray analysis was used to determine the transcriptional profile of laser captured spinal motor neurones of transgenic and wild-type littermates at 3 time points of disease. 324 genes were significantly differentially expressed in motor neurones of presymptomatic VEGFδ/δ mice, 382 at disease onset, and 689 at late stage disease. Massive transcriptional downregulation occurred with disease progression, associated with downregulation of genes involved in RNA processing at late stage disease. VEGFδ/δ mice showed reduction in expression, from symptom onset, of the cholesterol synthesis pathway, and genes involved in nervous system development, including axonogenesis, synapse formation, growth factor signalling pathways, cell adhesion and microtubule-based processes. These changes may reflect a reduced capacity of VEGFδ/δ mice for maintenance and remodelling of neuronal processes in the face of demands of neural plasticity. The findings are supported by the demonstration that in primary motor neurone cultures from VEGFδ/δ mice, axon outgrowth is significantly reduced compared to wild-type littermates.ConclusionsDownregulation of these genes involved in axon outgrowth and synapse formation in adult mice suggests a hitherto unrecognized role of VEGF in the maintenance of neuronal circuitry. Dysregulation of VEGF may lead to neurodegeneration through synaptic regression and dying-back axonopathy.

Screening of the regulatory and coding regions of vascular endothelial growth factor in amyotrophic lateral sclerosis

Sirs, Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disorder characterized by progressive loss of motor neurones in the spinal cord, brainstem, and cerebral cortex, which in about 10% of cases is inherited in an autosomal dominant fashion. In 20% of these patients, mutations have been identified in the gene encoding the antioxidant defence protein, SOD1. However, in the majority of familial and sporadic ALS, the cause of selective degeneration of motor neurones is unknown. Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen essential for angiogenesis. VEGF expression is upregulated by hypoxia, a response mediated by elements in both the 5′ and 3′ regions of the gene. VEGF transcription is induced by binding of hypoxia inducible factor (HIF-1) to a hypoxia regulatory element (HRE) in the 5′ promoter, its mRNA is stabilized by binding of the protein HuR to an AU rich sequence in the 3′ UTR under hypoxic conditions, and its 5′ UTR contains functional internal ribosomal entry sites which maintain efficient translation in hypoxia [1]. In 2001, Oosthuyse et al. [2] found that deletion of the HRE of vegf in mice unexpectedly caused adult onset motor neurone degeneration with clinical and neuropathological features reminiscent of ALS, and reduced VEGF levels in the neural tissue. The mechanism underlying the phenotype of these VEGFmice is not certain. They have abnormalities of baseline spinal cord blood flow, suggesting that chronic ischaemia may underlie late onset neurodegeneration. Alternatively, VEGF acts as a neurotrophic factor in vitro and in vivo [2, 3], and motor neurone loss in VEGFmice may be due to inadequate trophic support. The relevance of this murine model to human ALS is currently unknown. The first aim of the present study was to screen the 5′ and 3′ regulatory regions, the coding regions and the intron/ exon boundaries of the VEGF gene in a population of patients with ALS from the North-East of England, for mutations which might impair regulation of VEGF production or alter the structure or function of the protein.We screened the entire 5′ and 3′ regions rather than the HRE alone, as hypoxic regulation of VEGF can occur independently of the HRE, and VEGF is induced by other stresses including hypoglycaemia and oxidative stress, mediated by elements throughout the regulatory regions. Our second aim was to determine if single nucleotide polymorphisms (SNPs) in theVEGF regulatory regionsmay be a genetic susceptibility factor for sporadicALS.Nineteen SNPs have been identified in the regulatory regions of VEGF, some of which have functional significance: alleles –2578A, −1154A, −634G and +936T are associated with lower VEGF production [4, 5]. Significant over-representation of these alleles amongst the ALS population would suggest that low VEGF production is a risk factor for human ALS. We have screened 100 ALS patients, and 100 unrelated neurologically normal controls from the North-East of England for mutations and polymorphisms in the 5′UTR and Alice Brockington and Janine Kirby are joint first authors

Screening of the transcriptional regulatory regions of vascular endothelial growth factor receptor 2 (VEGFR2) in amyotrophic lateral sclerosis

BackgroundVascular endothelial growth factor (VEGF) has neurotrophic activity which is mediated by its main agonist receptor, VEGFR2. Dysregulation of VEGF causes motor neurone degeneration in a mouse model of amyotrophic lateral sclerosis (ALS), and expression of VEGFR2 is reduced in motor neurones and spinal cord of patients with ALS.MethodsWe have screened the promoter region and 4 exonic regions of functional significance of the VEGFR2 gene in a UK population of patients with ALS, for mutations and polymorphisms that may affect expression or function of this VEGF receptor.ResultsNo mutations were identified in the VEGFR2 gene. We found no association between polymorphisms in the regulatory regions of the VEGFR2 gene and ALS.ConclusionMechanisms other than genetic variation may downregulate expression or function of the VEGFR2 receptor in patients with ALS.

156 Motor neurones subtypes resistant to degeneration in amyotrophic lateral sclerosis show distinct synaptic characteristics across species

A consistent clinical feature of amyotrophic lateral sclerosis (ALS) is the sparing of eye movements and the function of external sphincters, with corresponding preservation of motor neurones (MNs) in the brainstem oculomotor nuclei, and of Onufs nucleus in the sacral spinal cord. Studying the differences in properties of neurones that are vulnerable and resistant to the disease process in ALS may provide insights into the mechanisms of neuronal degeneration, and identify targets for therapeutic manipulation. We used microarray analysis to determine the differences in gene expression between oculomotor and spinal MNs, isolated from the spinal cord of neurologically normal controls, using laser capture microdissection. We have compared these transcriptional profiles to those of oculomotor nuclei and spinal cord from rat and mouse, obtained from the GEO omnibus database. We show that oculomotor neurones have a distinct transcriptional profile, with significant differential expression in isolated human MNs of 1757 named genes (q<0.001). Marked differences are seen, across the three species, in genes with a function in synaptic transmission, including several glutamate and GABA receptor subunits. We have confirmed the functional significance of this, using patch clamp recording in acute spinal and brainstem slices. We show significant differences in the inward calcium current induced by agonists at these receptors, which would predict an altered susceptibility to excitotoxicity.