Vesicle associated membrane protein B (VAPB) is decreased in ALS spinal cord
Introduction
Research into the identification of new genes in both typical and atypical forms of familial ALS (FALS) has yielded at least eight genes that are either causal or may contribute to motor neurone degeneration, namely, superoxide dismutase 1 (SOD1), vesicle associated membrane protein (VAPB), alsin, senataxin (SETX), dynactin (DCTN1), vascular endothelial growth factor (VEGF), neurofilaments (NFH) and most recently the TAR DNA binding protein, TDP-43. Although the mutations responsible for the majority of cases of classical FALS have not been identified, new FALS loci on chromosomes 16, 18 and 9 may also be elucidated in the near future (Abalkhail et al., 2003, Ruddy et al., 2003, Sapp et al., 2003, Hand et al., 2002, Morita et al., 2006, Vance et al., 2006, Yan et al., 2006, Valdmanis et al., 2007). In addition, a number of other genes have been shown either to play an important part in maintaining the viability of motor neurones, such as insulin-like growth factor-1 (IGF1), heat shock protein 27 (HSPB1) and heat shock protein 22 (HSPB8), or to be fundamental to the disease process, such as caspases-1 and -3 and glial fibrillary acidic protein (GFAP). Identifying FALS genes has been a valuable approach to understanding factors that cause degeneration of the motor neurone. However, it is crucial to determine the relevance of these findings to sporadic cases of ALS (SALS), which will help to identify targets for therapeutic intervention. Our aim in this study was to investigate the involvement of 11 of these genes in the pathogenic events occurring in SALS by quantification of spinal cord mRNA in a large cohort of well characterised ALS cases and controls.
Following the identification of the first FALS gene, SOD1 (Rosen et al., 1993), which is now known to account for approximately 20% of cases of classical adult onset dominantly inherited ALS, research into the identification of new genes in both typical and atypical forms of FALS has been extremely productive. This has been important in elucidating the involvement of cytoskeletal proteins, antioxidant proteins, molecular chaperones, neurotrophic factors and cytoplasmic trafficking in the pathogenic processes occurring in ALS. More recently, mutations in vesicle associated membrane protein/synaptobrevin-associated membrane protein B (VAPB) have been detected in Brazilian families presenting predominantly with spinal muscular atrophy but also in one family with typical ALS, designated ALS8 (Nishimura et al., 2004a, Nishimura et al., 2004b). Mutations have also been reported in TDP-43 both in a single familial ALS kindred and sporadic cases of ALS (Sreedharan et al., 2008) which is particularly relevant to elucidating disease mechanisms since inclusions containing ubiquitinated TDP-43 are a key pathological feature of both forms of ALS. However, other groups have not detected TDP-43 mutations in SALS or identified association of common variants with disease (Gijselinck et al., 2008).
Other identified genes have mainly emerged from atypical forms of ALS. A point mutation in the p150 subunit of dynactin, important in retrograde axoplasmic flow, occurs in an atypical form of lower motor neurone disease with onset starting with vocal cord weakness and progressing to facial and limb weakness with bulbar involvement (Puls et al., 2003). Four additional mutations in the DCTN1 gene have been identified in patients with FALS, one with a putative diagnosis of sporadic ALS, as well as one in a family where both FALS and FTD occur (Munch et al., 2004, Munch et al., 2005). This involvement of dynactin is supported by observations in mice where mutations in dynein (Hafezparast et al., 2003) or affecting the dynein–dynactin complex cause progressive motor neurone disorders (LaMonte et al., 2002). Deletions in the tail domain of neurofilament heavy chain (NF-H) have been reported in 1% of sporadic cases and some familial cases (Figlewicz et al., 1994, Al-Chalabi et al., 1999) but these are also present in unaffected individuals and likely to represent a low penetrance risk factor. A slowly progressing recessive form of juvenile onset ALS, linked to chromosome 2q33, is caused by mutations in Alsin, a guanine exchange factor (Hadano et al., 2001, Yang et al., 2001) that may potentially be involved in cytoskeletal dynamics, signal transduction and intracellular trafficking. In addition, a slowly progressing dominant form of juvenile-onset ALS (ALS4) localised to chromosome 9q34 is associated with a mutation in SETX, a DNA/RNA helicase, which plays a role in mRNA processing and potentially in ensuring the production of error-free mRNA (Chance et al., 1998, Chen et al., 2004). Moreover, mutations in SETX can also cause Ataxia-Ocula Apraxia (AOA2) (Moreira et al., 2004) as well as ataxia with distal amyotrophy, and peripheral neuropathy (Duquette et al., 2005).
Mutations in the molecular chaperones, HSPB8 and HSPB1, have also been shown to be causal in two disorders associated with motor neuropathy, Distal Hereditary Motor Neuropathy (Type 2) and autosomal dominant axonal Charcot-Marie-Tooth disease (CMT2F), respectively (Irobi et al., 2004a, Irobi et al., 2004b, Evgrafov et al., 2004). The relevance of HSPB1 to motor neurone degeneration caused by mutations in SOD1 has been demonstrated by Maatkamp et al. (2004) in G93A mutant SOD1 mice where the depletion of HSPB1 precedes loss of motor neurones and the onset of clinical signs.
VEGF was first implicated in the pathophysiology of ALS with the discovery that ALS-like symptoms and neuropathology could be produced in mice after targeted deletion of the hypoxia response element (HRE) in the VEGF promoter (Oosthuyse et al., 2001) and, subsequently, an “at risk” haplotype in the VEGF promoter region was detected which conferred a 1.8-fold greater risk of ALS and was associated with reduced plasma levels of VEGF (Lambrechts et al., 2003). Further support for the importance of VEGF in motor neurone survival comes from studies in the G93A SOD1 mouse model of ALS where VEGF enhances survival (Zheng et al., 2004, Azzouz et al., 2004). IGF1 is a major neurotrophic factor, essential for normal development of the nervous system, which increases survival of motor neurones in cell culture and in vivo following sciatic nerve axotomy (Dore et al., 1997, Vergani et al., 1998). Increased survival of G93A SOD1 mouse has also been demonstrated using either viral delivery or continuous intrathecal infusion of IGF-1 into the lumbar spinal cord (Kaspar et al., 2003, Nagano et al., 2005).
Analysis of gene expression in short post-mortem delay tissue has revealed much about the processes activated in ALS spinal cord, involving apoptotic signalling (caspase-3 and -1 activation), gliosis (GFAP upregulation), inflammation, antioxidant and cytoprotective systems (Ishigaki et al., 2002, Jiang et al., 2005, Malaspina et al., 2000, Malaspina et al., 2001, Malaspina and de Belleroche, 2004), many of which have also been demonstrated in the G93A SOD1 mouse (Olsen et al., 2001, Hensley et al., 2002, Yoshihara et al., 2002). These and similar studies indicate the value of expression studies in defining active disease mechanisms despite the presence of varying degrees of cell loss. The two most robust findings relate to the induction of GFAP and activation of caspase-1, which have been used as reference genes in this study.
The involvement of caspase-1 in mutant SOD1-mediated cell death was first suggested by the finding that expression of a dominant negative inhibitor transgene of caspase-1 increased survival in G93A SOD1 transgenic mice (Friedlander et al., 1997). Caspase is induced early in disease progression in the SOD1 mouse model (Pasinelli et al., 2000, Li et al., 2000) and both activated caspase-1 and -3 have been detected in spinal cord neurones. More recently, microarray studies have demonstrated significant upregulation of caspases-1 and -3 in spinal cord of G93A SOD1 mice (Hensley et al., 2002, Yoshihara et al., 2002) as well as in post-mortem spinal cords (Martin, 1999, Li et al., 2000) and spinal motor neurones (Jiang et al., 2005) of ALS cases. GFAP accumulation is a hallmark of astrocytic reaction to CNS injury (Burbach et al., 2004) or diseases, such as ALS (e.g. Nagy et al., 1994), occurring as a result of reactive astrogliosis. Significant upregulation of GFAP has been reported in microarray studies of G93A SOD1 mouse spinal cord (Olsen et al., 2001, Yoshihara et al., 2002) and in a macroarray study monitoring gene expression in spinal cord from ALS cases (Malaspina et al., 2001).
In this study, we quantified spinal cord expression of 11 genes known to be causal in FALS or strongly implicated in the pathogenesis of disease using real-time quantitative PCR for mRNA analysis (RT-PCR). RT-PCR is regarded as the current “state of the art” technique and has been widely shown to be reliable and reproducible. We have collected a large cohort of consented cases and controls with short post-mortem intervals of which 58 cases (35 sporadic ALS, 4 familial ALS cases (1 with a SOD1 mutation and 3 lacking SOD1 mutations) and 19 control cases) have satisfied stringent criteria for intactness of mRNA. The ALS cases were well-documented, allowing correlation between mRNA levels and duration of disease, age at onset, bulbar involvement and other disease characteristics. From this study, one particular FALS candidate, VAPB, emerged which was significantly reduced in SALS and furthermore was selectively localised to motor neurones at the mRNA and protein level.
Section snippets
Subjects
Blocks (1 cm3) of lumbar spinal cord were rapidly dissected at autopsy from 74 cases and controls, of which 39 ALS patients (35 SALS and 4 FALS cases, one with a SOD1 mutation) and 19 control individuals satisfied RNA quality criteria (as described below). The dissected blocks of tissue were frozen immediately and stored at −80 °C until further use. Informed consent from the subjects and their relatives had been previously obtained. The study was approved by the Riverside Research Ethics
Subjects used for analysis
Tissue extracts of ALS and control lumbar spinal cord from a total of 58 cases satisfying criteria for RNA integrity were used for cDNA synthesis and RT-PCR. A summary of these cases is provided in Table 1. A large ALS cohort was used to allow stratification according to disease duration and signs at onset. The male to female ratio in the ALS group was 2.0, which is consistent with the normal prevalence ratio for ALS, and a similar gender ratio (1.7) was present in the control cohort. The age
Discussion
From the analysis of the expression of 11 candidate genes in ALS spinal cord, four transcripts, namely, VAPB, HSPB1, HSPB8 and caspase-1, were found to be significantly changed in a large cohort of ALS cases compared to control cases of similar post-mortem interval, age and gender distribution, using two independent reference genes, GAPDH and β-actin. The most remarkable finding emerging from this study is the change in VAPB. This is the first time that a deficit in VAPB, which is causal in a
Conflict of interest statement
The authors have no conflicts of interest.
Acknowledgements
We are grateful to the Motor Neurone Disease Association, The Henry Smith Charity and the American ALS Association for their contribution to funding this research.
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