James Neal

A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD

The chromosome 9p21 amyotrophic lateral sclerosis-frontotemporal dementia (ALS-FTD) locus contains one of the last major unidentified autosomal-dominant genes underlying these common neurodegenerative diseases. We have previously shown that a founder haplotype, covering the MOBKL2b, IFNK, and C9ORF72 genes, is present in the majority of cases linked to this region. Here we show that there is a large hexanucleotide (GGGGCC) repeat expansion in the first intron of C9ORF72 on the affected haplotype. This repeat expansion segregates perfectly with disease in the Finnish population, underlying 46.0% of familial ALS and 21.1% of sporadic ALS in that population. Taken together with the D90A SOD1 mutation, 87% of familial ALS in Finland is now explained by a simple monogenic cause. The repeat expansion is also present in one-third of familial ALS cases of outbred European descent, making it the most common genetic cause of these fatal neurodegenerative diseases identified to date.

Long-term effects of Aβ42 immunisation in Alzheimer's disease: follow-up of a randomised, placebo-controlled phase I trial

BACKGROUND Immunisation of patients with Alzheimers disease with full-length amyloid-beta peptide (Abeta(42)) can clear amyloid plaques from the brain. Our aim was to assess the relation between Abeta(42) immune response, degree of plaque removal, and long-term clinical outcomes. METHODS In June, 2003, consent for long-term clinical follow-up, post-mortem neuropathological examination, or both, was sought from 80 patients (or their carers) who had entered a phase I randomised, placebo-controlled trial of immunisation with Abeta(42) (AN1792, Elan Pharmaceuticals) in September, 2000. The follow-up study was completed in September, 2006. Plaques were assessed in terms of the percentage area of the cortex with Abeta immunostaining (Abeta load) and in terms of characteristic histological features reflecting plaque removal. Survival of all 80 individuals until severe dementia or death was assessed with a Cox proportional hazard model. FINDINGS 20 participants--15 in the AN1792 group, five in the placebo group--died before follow-up started. A further 22 patients--19 in the AN1792 group, three in the placebo group--died during follow-up. Nine of the deceased patients, all in the AN1792 group, had given consent for post-mortem analysis; one of these who did not die with Alzheimers disease was excluded. In the remaining eight participants who received immunisation and who were examined neuropathologically, mean Abeta load was lower than in an unimmunised control group that was matched for age at death (2.1% [SE 0.7] in treated participants vs 5.1% [0.9] in controls; mean difference 3.0%, 95% CI 0.6-5.4; p=0.02). Although there was considerable variation in Abeta load and degree of plaque removal among immunised participants, the degree of plaque removal varied significantly with mean antibody response attained during the treatment study period (Kruskal-Wallis p=0.02). Seven of the eight immunised patients who underwent post-mortem assessment, including those with virtually complete plaque removal, had severe end stage dementia before death. In the whole cohort, there was no evidence of improved survival (hazard ratio 0.93, 95% CI 0.43-3.11; p=0.86) or of an improvement in the time to severe dementia (1.18, 0.45-3.11; p=0.73) in the AN1792 group versus the placebo group. INTERPRETATION Although immunisation with Abeta(42) resulted in clearance of amyloid plaques in patients with Alzheimers disease, this clearance did not prevent progressive neurodegeneration.

The Membrane Attack Complex of Complement Causes Severe Demyelination Associated with Acute Axonal Injury

Complement is implicated in pathology in the human demyelinating disease multiple sclerosis and in animal models that mimic the demyelination seen in multiple sclerosis. However, the components of the complement system responsible for demyelination in vivo remain unidentified. In this study, we show that C6-deficient (C6−) PVG/c rats, unable to form the membrane attack complex (MAC), exhibit no demyelination and significantly reduced clinical score in the Ab-mediated experimental autoimmune encephalomyelitis model when compared with matched C6-sufficient (C6+) rats. In C6+ rats, perivenous demyelination appeared, accompanied by abundant mononuclear cell infiltration and axonal injury. Neither demyelination nor axonal damage was seen in C6− rats, whereas levels of mononuclear cell infiltration were equivalent to those seen in C6+ rats. Reconstitution of C6 to C6− rats yielded pathology and clinical disease indistinguishable from that in C6+ rats. We conclude that demyelination and axonal damage occur in the presence of Ab and require activation of the entire complement cascade, including MAC deposition. In the absence of MAC deposition, complement activation leading to opsonization and generation of the anaphylatoxins C5a and C3a is insufficient to initiate demyelination.

Spontaneous Classical Pathway Activation and Deficiency of Membrane Regulators Render Human Neurons Susceptible to Complement Lysis

This study investigated the capacity of neurons and astrocytes to spontaneously activate the complement system and control activation by expressing complement regulators. Human fetal neurons spontaneously activated complement through the classical pathway in normal and immunoglobulin-deficient serum and C1q binding was noted on neurons but not on astrocytes. A strong staining for C4, C3b, iC3b neoepitope and C9 neoepitope was also found on neurons. More than 40% of human fetal neurons were lysed when exposed to normal human serum in the presence of a CD59-blocking antibody, whereas astrocytes were unaffected. Significant reduction in neuronal cell lysis was observed after the addition of soluble complement receptor 1 at 10 microg/ml. Fetal neurons were stained for CD59 and CD46 and were negative for CD55 and CD35. In contrast, fetal astrocytes were strongly stained for CD59, CD46, CD55, and were negative for CD35. This study demonstrates that human fetal neurons activate spontaneously the classical pathway of complement in an antibody-independent manner to assemble the cytolytic membrane attack complex on their membranes, whereas astrocytes are unaffected. One reason for the susceptibility of neurons to complement-mediated damage in vivo may reside in their poor capacity to control complement activation.

Reduced C9orf72 protein levels in frontal cortex of amyotrophic lateral sclerosis and frontotemporal degeneration brain with the C9ORF72 hexanucleotide repeat expansion

An intronic G4C2 hexanucleotide repeat expansion in C9ORF72 is a major cause of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Several mechanisms including RNA toxicity, repeat-associated non-AUG translation mediated dipeptide protein aggregates, and haploinsufficiency of C9orf72 have been implicated in the molecular pathogenesis of this disorder. The aims of this study were to compare the use of two different Southern blot probes for detection of repeat expansions in an amyotrophic lateral sclerosis and frontotemporal lobar degeneration pathological cohort and to determine the levels of C9orf72 transcript variants and protein isoforms in patients versus control subjects. Our Southern blot studies identified smaller repeat expansions (250–1800 bp) that were only detectable with the flanking probe highlighting the potential for divergent results using different Southern blotting protocols that could complicate genotype–phenotype correlation studies. Further, we characterize a new C9orf72 antibody and show for the first time decreased C9orf72 protein levels in the frontal cortex from patients with a pathological hexanucleotide repeat expansion. These data suggest that a reduction in C9orf72 protein may be a consequence of the disease.

Inflammatory components in human Alzheimer's disease and after active amyloid-β42 immunization

Inflammatory processes are important in the pathogenesis of Alzheimers disease and in response to amyloid-β immunotherapy. We investigated the expression of multiple inflammatory markers in the brains of 28 non-immunized patients with Alzheimers disease and 11 patients with Alzheimers disease immunized against amyloid-β42 (AN1792): microglial ionized calcium-binding adaptor Iba-1, lysosome marker CD68, macrophage scavenger receptor A, Fcγ receptors I (CD64) and II (CD32); and also immunoglobulin IgG, complement C1q and the T lymphocyte marker CD3 using immunohistochemistry. The data were analysed with regard to amyloid-β and phospho-tau pathology, severity of cerebral amyloid angiopathy and cortical microhaemorrhages. In non-immunized Alzheimers disease cases, amyloid-β42 correlated inversely with CD32 and Iba-1, whereas phospho-tau correlated directly with all microglial markers, IgG, C1q and the number of T cells. In immunized Alzheimers disease cases, amyloid-β42 load correlated directly with macrophage scavenger receptor A-positive clusters and inversely with C1q. The severity of cerebral amyloid angiopathy and microhaemorrhages did not relate to any of the analysed markers. Overall, the levels of CD68, macrophage scavenger receptor A, CD64, CD32 and the number of macrophage scavenger receptor A-positive plaque-related clusters were significantly lower in immunized than non-immunized cases, although there was no significant difference in Iba-1 load, number of Iba-1-positive cells, IgG load, C1q load or number of T cells. Our findings indicate that different microglial populations co-exist in the Alzheimers disease brain, and that the local inflammatory status within the grey matter is importantly linked with tau pathology. After amyloid-β immunization, the microglial functional state is altered in association with reduced amyloid-β and tau pathology. The results suggest that, in the long term, amyloid-β immunotherapy results in downregulation of microglial activation and potentially reduces the inflammation-mediated component of the neurodegeneration of Alzheimers disease.

Deficiency of the complement regulator CD59a enhances disease severity, demyelination and axonal injury in murine acute experimental allergic encephalomyelitis

There is a growing body of evidence implicating complement and, in particular, the terminal pathway (membrane attack complex; MAC) in inducing demyelination in multiple sclerosis and experimental allergic encephalomyelitis. In this paper, we examined the disease course and pathological changes in mice deficient in the major regulator of MAC assembly, CD59a, during the course of acute experimental allergic encephalomyelitis induced by immunisation with recombinant myelin oligodendrocyte glycoprotein. Disease incidence and severity were significantly increased in CD59a-deficient mice. The extent of inflammation, demyelination and axonal injury were assessed in spinal cord cross-sections from CD59a-deficient and control mice, and all these parameters were enhanced in the absence of CD59a. Areas of myelin loss and axonal damage in CD59a-deficient mice were associated with deposits of MAC, firmly implicating MAC as a cause of the observed injury. These findings are relevant to some types of human demyelination, where abundant deposits of MAC are found in association with pathology.

C3-dependent mechanism of microglial priming relevant to multiple sclerosis

Microglial priming predisposes the brain to neurodegeneration and affects disease progression. The signal to switch from the quiescent to the primed state is unknown. We show that deleting the C3 convertase regulator complement receptor 1-related protein y (Crry) induces microglial priming. Mice that were double-knockout for Crry and either C3 or factor B did not show priming, demonstrating dependence on alternative pathway activation. Colocalization of C3b/iC3b and CR3 implicated the CR3/iC3b interaction in priming. Systemic lipopolysaccharide challenge overactivated primed microglia with florid expression of proinflammatory molecules, which were blocked by complement inhibition. Relevance for neurodegenerative disease is exemplified by human multiple sclerosis (MS) and by experimental autoimmune encephalomyelitis (EAE), a model of MS. In human MS, microglial priming was evident in perilesional white matter, in close proximity to C3b/iC3b deposits. EAE was accelerated and exacerbated in Crry-deficient mice, and was dependent on C activation. In summary, C3-dependent microglial priming confers susceptibility to other challenges. Our observations are relevant to progression in MS and other neurological diseases exacerbated by acute insults.

The membrane attack pathway of complement drives pathology in passively induced experimental autoimmune myasthenia gravis in mice

The human neuromuscular disease myasthenia gravis (MG) is characterized by the generation of autoantibodies reactive with nicotinic acetylcholine receptors (AChR) that cause loss of AChR from the neuromuscular end‐plate with resultant failure of neuromuscular transmission. A role for complement (C) in AChR loss has been suggested based upon morphological identification of C at the end‐plate in MG and from the effects of C inhibition in murine models. Here we provide further evidence implicating C, and specifically the membrane attack complex (MAC), in a mouse model of MG. Mice deficient in the C regulators Daf1 and/or Cd59a were tested in the model. Wild‐type mice were resistant to disease while mice deficient in Daf1 had mild disease symptoms with evidence of C activation and AChR loss at end‐plates. Cd59a‐deficient mice had very mild disease with some muscle inflammation and essentially undamaged end‐plates. In contrast, mice deficient in both C regulators developed a severe paralytic disease with marked muscle inflammation and loss of end‐plates. Inhibition of MAC assembly abrogated clinical disease in these double‐deficient mice, demonstrating conclusively that MAC formation was driving pathology in the model. These findings provoke us to suggest that current anti‐C therapeutics targeting MAC assembly will be beneficial in MG patients resistant to conventional therapies.

Decay-accelerating factor (CD55) is expressed by neurons in response to chronic but not acute autoimmune central nervous system inflammation associated with complement activation.

There is compelling evidence that a unique innate immune response in the CNS plays a critical role in host defense and clearance of toxic cell debris. Although complement has been implicated in neuronal impairment, axonal loss, and demyelination, some preliminary evidence suggests that the initial insult consequently activates surrounding cells to signal neuroprotective activities. Using two different models of experimental autoimmune encephalomyelitis, we herein demonstrate selective C1q complement activation on neuron cell bodies and axons. Interestingly, in brains with chronic but not acute experimental autoimmune encephalomyelitis, C3b opsonization of neuronal cell bodies and axons was consistently associated with robust neuronal expression of one of the most effective complement regulators, decay-accelerating factor (CD55). In contrast, levels of other complement inhibitors, complement receptor 1 (CD35), membrane cofactor protein (CD46), and CD59 were largely unaffected on neurons and reactive glial cells in both conditions. In vitro, we found that proinflammatory stimuli (cytokines and sublytic doses of complement) failed to up-regulate CD55 expression on cultured IMR32 neuronal cells. Interestingly, overexpression of GPI-anchored CD55 on IMR32 was capable of modulating raft-associated protein kinase activities without affecting MAPK activities and neuronal apoptosis. Critically, ectopic expression of decay-accelerating factor conferred strong protection of neurons against complement attack (opsonization and lysis). We conclude that increased CD55 expression by neurons may represent a key protective signaling mechanism mobilized by brain cells to withstand complement activation and to survive within an inflammatory site.