Jacqueline de Belleroche

TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder characterized pathologically by ubiquitinated TAR DNA binding protein (TDP-43) inclusions. The function of TDP-43 in the nervous system is uncertain, and a mechanistic role in neurodegeneration remains speculative. We identified neighboring mutations in a highly conserved region of TARDBP in sporadic and familial ALS cases. TARDBPM337V segregated with disease within one kindred and a genome-wide scan confirmed that linkage was restricted to chromosome 1p36, which contains the TARDBP locus. Mutant forms of TDP-43 fragmented in vitro more readily than wild type and, in vivo, caused neural apoptosis and developmental delay in the chick embryo. Our evidence suggests a pathophysiological link between TDP-43 and ALS.

Familial amyotrophic lateral sclerosis is associated with a mutation in D-amino acid oxidase

We report a unique mutation in the D-amino acid oxidase gene (R199W DAO) associated with classical adult onset familial amyotrophic lateral sclerosis (FALS) in a three generational FALS kindred, after candidate gene screening in a 14.52 cM region on chromosome 12q22-23 linked to disease. Neuronal cell lines expressing R199W DAO showed decreased viability and increased ubiquitinated aggregates compared with cells expressing the wild-type protein. Similarly, lentiviral-mediated expression of R199W DAO in primary motor neuron cultures caused increased TUNEL labeling. This effect was also observed when motor neurons were cocultured on transduced astrocytes expressing R199W, indicating that the motor neuron cell death induced by this mutation is mediated by both cell autonomous and noncell autonomous processes. DAO controls the level of D-serine, which accumulates in the spinal cord in cases of sporadic ALS and in a mouse model of ALS, indicating that this abnormality may represent a fundamental component of ALS pathogenesis.

The C9ORF72 expansion mutation is a common cause of ALS+/-FTD in Europe and has a single founder.

A massive hexanucleotide repeat expansion mutation (HREM) in C9ORF72 has recently been linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we describe the frequency, origin and stability of this mutation in ALS+/−FTD from five European cohorts (total n=1347). Single-nucleotide polymorphisms defining the risk haplotype in linked kindreds were genotyped in cases (n=434) and controls (n=856). Haplotypes were analysed using PLINK and aged using DMLE+. In a London clinic cohort, the HREM was the most common mutation in familial ALS+/−FTD: C9ORF72 29/112 (26%), SOD1 27/112 (24%), TARDBP 1/112 (1%) and FUS 4/112 (4%) and detected in 13/216 (6%) of unselected sporadic ALS cases but was rare in controls (3/856, 0.3%). HREM prevalence was high for familial ALS+/−FTD throughout Europe: Belgium 19/22 (86%), Sweden 30/41 (73%), the Netherlands 10/27 (37%) and Italy 4/20 (20%). The HREM did not affect the age at onset or survival of ALS patients. Haplotype analysis identified a common founder in all 137 HREM carriers that arose around 6300 years ago. The haplotype from which the HREM arose is intrinsically unstable with an increased number of repeats (average 8, compared with 2 for controls, P<10−8). We conclude that the HREM has a single founder and is the most common mutation in familial and sporadic ALS in Europe.

Identification of PEX7 as the Second Gene Involved in Refsum Disease

Patients affected with Refsum disease (RD) have elevated levels of phytanic acid due to a deficiency of the peroxisomal enzyme phytanoyl-CoA hydroxylase (PhyH). In most patients with RD, disease-causing mutations in the PHYH gene have been identified, but, in a subset, no mutations could be found, indicating that the condition is genetically heterogeneous. Linkage analysis of a few patients diagnosed with RD, but without mutations in PHYH, suggested a second locus on chromosome 6q22-24. This region includes the PEX7 gene, which codes for the peroxin 7 receptor protein required for peroxisomal import of proteins containing a peroxisomal targeting signal type 2. Mutations in PEX7 normally cause rhizomelic chondrodysplasia punctata type 1, a severe peroxisomal disorder. Biochemical analyses of the patients with RD revealed defects not only in phytanic acid α-oxidation but also in plasmalogen synthesis and peroxisomal thiolase. Furthermore, we identified mutations in the PEX7 gene. Our data show that mutations in the PEX7 gene may result in a broad clinical spectrum ranging from severe rhizomelic chondrodysplasia punctata to relatively mild RD and that clinical diagnosis of conditions involving retinitis pigmentosa, ataxia, and polyneuropathy may require a full screen of peroxisomal functions.

Actions of Tremorgenic Fungal Toxins on Neurotransmitter Release

Abstract: The neurochemical effects of the tremorgenic mycotoxins Verruculogen and Penitrem A, which produce a neurotoxic syndrome characterised by sustained tremors, were studied using sheep and rat synaptosomes. The toxins were administered in vivo, either by chronic feeding (sheep) or intraperitoneal injection 45 min prior to killing (rat) and synaptosomes were subsequently prepared from cerebrocortical and spinal cord/medullary regions of rat and corpus striatum of sheep. Penitrem A (400 mg mycelium/kg) increased the spontaneous release of endogenous glutamate, GABA (γ‐aminobutyric acid) and aspartate by 213%, 455% and 277%, respectively, from cerebrocortical synaptosomes. Verruculogen (400 mg mycelium/kg) increased the spontaneous release of glutamate and aspartate by 1300% and 1200%, respectively, but not that of GABA from cerebrocortical synaptosomes. The spontaneous release of the transmitter amino acids or other amino acids was not increased by the tremorgens in spinal cord/medullary synaptosomes. Penitrem A pretreatment reduced the veratrine (75 μm) stimulated release of glutamate, aspartate and GABA from cerebrocortical synaptosomes by 33%, 46% and 11%, respectively and the stimulated release of glycine and GABA from spinal cord/ medulla synaptosomes by 67% and 32% respectively. Verruculogen pretreatment did not alter the veratrine‐induced release of transmitter amino acids from cerebrocortex and spinal cord/medulla synaptosomes. Penitrem A pretreatment increased the spontaneous release of aspartate, glutamate and GABA by 68%, 62% and 100%, respectively, from sheep corpus striatum synaptosomes but did not alter the synthesis and release of dopamine in this tissue. Verruculogen was shown to cause a substantial increase (300–400%) in the miniature‐end‐plate potential (m.e.p. p.) frequency at the locust neuromuscular junction. The response was detectable within 1 min, rose to a maximum within 5–7 min and declined to the control rate over a similar period. No change in the amplitude of the m.e.p. p. ‘s was observed. These effects of the tremorgens on transmitter release are interpreted in terms of their mode of action.

Vesicle associated membrane protein B (VAPB) is decreased in ALS spinal cord

The aim of this study was to quantify spinal cord expression of genes known to cause familial amyotrophic lateral sclerosis (FALS) or influence survival in a large cohort of sporadic cases of ALS (SALS), in order to determine their relevance to pathogenic mechanisms occurring in SALS. The expression of superoxide dismutase 1 (SOD1), vesicle associated membrane protein (VAPB), senataxin (SETX), dynactin (DCTN1), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF1), the small heat shock proteins, HSPB1 and HSPB8, and three genes activated during disease progression, caspases-1 and -3 and glial fibrillary acidic protein (GFAP), were quantified. Robust changes in the expression of four genes were found, VAPB mRNA levels were decreased in the spinal cord of ALS patients compared to controls (p<0.006), whilst HSPB1, HSPB8 and caspase-1 showed significant increases (1.5-2.3-fold). Expression of VAPB mRNA and protein was predominantly localised to large motor neurones further supporting the relevance of this finding to disease progression occurring in SALS.

Extracellular Release of the Atheroprotective Heat Shock Protein 27 Is Mediated by Estrogen and Competitively Inhibits acLDL Binding to Scavenger Receptor-A

We recently identified heat shock protein 27 (HSP27) as an estrogen receptor beta (ER&bgr;)-associated protein and noted its role as a biomarker for atherosclerosis. The current study tests the hypothesis that HSP27 is protective against the development of atherosclerosis. HSP27 overexpressing (HSP27o/e) mice were crossed to apoE−/− mice that develop atherosclerosis when fed a high-fat diet. Aortic en face analysis demonstrated a 35% reduction (P≤0.001) in atherosclerotic lesion area in apoE−/−HSP27o/e mice compared to apoE−/− mice, but primarily in females. Serum HSP27 levels were >10-fold higher in female apoE−/−HSP27o/e mice compared to males, and there was a remarkable inverse correlation between circulating HSP27 levels and lesion area in both male and female mice (r2=0.78, P≤0.001). Mechanistic in vitro studies showed upregulated HSP27 expression and secretion in macrophages treated with estrogen or acLDL. Moreover, exogenous HSP27 added to culture media inhibited macrophage acLDL uptake and competed for the scavenger receptor A (SR-A)—an effect that was abolished with the SR-A competitive ligand fucoidan and absent in macrophages from SR-A−/− mice. Furthermore, extracellular HSP27 decreased acLDL-induced release of the proinflammatory cytokine IL-1&bgr; and increased the release of the antiinflammatory cytokine IL-10. HSP27 is atheroprotective, perhaps because of its ability to compete for the uptake of atherogenic lipids or attenuate inflammation.

Protective effects of heat shock protein 27 in a model of ALS occur in the early stages of disease progression.

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disorder, characterised by progressive motor neuron degeneration and muscle paralysis. Heat shock proteins (HSPs) have significant cytoprotective properties in several models of neurodegeneration. To investigate the therapeutic potential of heat shock protein 27 (HSP27) in a mouse model of ALS, we conducted an extensive characterisation of transgenic mice generated from a cross between HSP27 overexpressing mice and mice expressing mutant superoxide dismutase (SOD1(G93A)). We report that SOD1(G93A)/HSP27 double transgenic mice showed delayed decline in motor strength, a significant improvement in the number of functional motor units and increased survival of spinal motor neurons compared to SOD1(G93A) single transgenics during the early phase of disease. However, there was no evidence of sustained neuroprotection affecting long-term survival. Marked down-regulation of HSP27 protein occurred during disease progression that was not associated with a reduction in HSP27 mRNA, indicating a translational dysfunction due to the presence of mutant SOD1 protein. This study provides further support for the therapeutic potential of HSPs in ALS and other motor neuron disorders.

Induction of the immediate early gene c-jun in human spinal cord in amyotrophic lateral sclerosis with concomitant loss of NMDA receptor NR-1 and glycine transporter mRNA.

The aetiology of the sporadic form of amyotrophic lateral sclerosis (ALS) is poorly understood although abnormalities in glutamate and glycine transport have been implicated which both could contribute to a neurodegenerative process mediated through the N-methyl-D-aspartate (NMDA) receptor. In this study we have used in situ hybridization to investigate whether any changes in the expression of NMDA receptors, the glycine transporter or glutamate-mediated injury responses are detectable in ALS. Two immediate early genes were investigated as markers of neuronal injury responses, c-jun and zif-268, both constitutively expressed in the spinal cord. Levels of c-jun mRNA were most abundant in intermediate grey and layer IX of the ventral horn containing motor neurones. This pattern was markedly changed in ALS with large increases (2-3 fold) in c-jun mRNA occurring in dorsal and ventral horn. The marked increase in c-jun mRNA was also substantiated by slot blot analysis of tissue homogenates of spinal cord and a parallel induction of zif-268 mRNA was also seen. NMDA receptor NR-1 mRNA was widely distributed in control spinal cord with the highest concentrations occurring in layers IX, X, intermediate grey and dorsal horn. The ALS cases showed a selective decrease in the level of NR-1 mRNA in the ventral region (50%) whilst no significant decrease was detected in the dorsal region. Quantitation of tissue homogenates with dorsal and ventral regions combined also yielded a significant decrease of 40% which supports the analysis from in situ hybridization densitometry.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathogenesis of Parkinson's disease: emerging role of molecular chaperones.

Several neurodegenerative diseases, including Parkinsons disease (PD) are associated with protein misfolding and the formation of distinct aggregates, resulting in a putative pathological protein load on the nervous system. A variety of factors cause proteins to aggregate, including aggregation-prone sequences, specific mutations, protein modifications and also dysregulation of the protein degradation machinery. Molecular chaperones are responsible for maintaining normal protein homeostasis within the cell by assisting protein folding and modulating protein-degrading pathways. Here, we review the fundamental mechanisms of neurodegeneration occurring in PD involving alpha-synuclein fibrillisation and aggregation, endoplasmic reticulum stress, ubiquitin proteasome systems, autophagy and lysosomal degradation. Molecular chaperones serve a neuroprotective role in many of these pathways, and we discuss recent evidence indicating that these proteins might provide the basis for new therapeutic approaches.