Consensus paper: pathological mechanisms underlying neurodegeneration in spinocerebellar ataxias.

Intensive scientific research devoted in the recent years to understand the molecular mechanisms or neurodegeneration in spinocerebellar ataxias (SCAs) are identifying new pathways and targets providing new insights and a better understanding of the molecular pathogenesis in these diseases. In this consensus manuscript, the authors discuss their current views on the identified molecular processes causing or modulating the neurodegenerative phenotype in spinocerebellar ataxias with the common opinion of translating the new knowledge acquired into candidate targets for therapy. The following topics are discussed: transcription dysregulation, protein aggregation, autophagy, ion channels, the role of mitochondria, RNA toxicity, modulators of neurodegeneration and current therapeutic approaches. Overall point of consensus includes the common vision of neurodegeneration in SCAs as a multifactorial, progressive and reversible process, at least in early stages. Specific points of consensus include the role of the dysregulation of protein folding, transcription, bioenergetics, calcium handling and eventual cell death with apoptotic features of neurons during SCA disease progression. Unresolved questions include how the dysregulation of these pathways triggers the onset of symptoms and mediates disease progression since this understanding may allow effective treatments of SCAs within the window of reversibility to prevent early neuronal damage. Common opinions also include the need for clinical detection of early neuronal dysfunction, for more basic research to decipher the early neurodegenerative process in SCAs in order to give rise to new concepts for treatment strategies and for the translation of the results to preclinical studies and, thereafter, in clinical practice.

Microsatellite evolution--evidence for directionality and variation in rate between species.

Microsatellite DNA sequences are rapidly becoming the dominant source of nuclear genetic markers for a wide range of applications, from genome mapping to forensic testing to population studies. If misinterpretation is to be avoided, it is vital that we understand fully the way in which microsatellite sequences evolve. We have therefore compared allele length distributions for 42 microsatellites in humans with their homologues in a range of related primates. We find a highly significant trend for the loci to be longer in humans, showing that microsatellites can evolve directionally and at different rates in closely related species.

Mutational bias provides a model for the evolution of Huntington's disease and predicts a general increase in disease prevalence.

Huntington's disease (HD) correlates with abnormal expansion in a block of CAG repeats in the Huntington's disease gene. We have investigated HD evolution by typing CAG alleles in several human populations and in a variety of primates. We find that human alleles have expanded from a shorter ancestral state and exhibit unusual asymmetric length distributions. Computer simulations are used to show that the human state can be derived readily from a primate ancestor, without the need to invoke natural selection. The key element is a simple length-dependent mutational bias towards longer alleles. Our model can explain a number of empirical observations, and predicts an ever-increasing incidence of HD.

APOE and ACE polymorphisms and dementia risk in the older population over prolonged follow-up: 10 years of incidence in the MRC CFA Study.

BACKGROUND: dementia risk conferred by apolipoprotein-E (APOE) and angiotensin-1-converting enzyme (ACE) polymorphisms have been reported for the MRC Cognitive Function and Ageing Study (CFAS) at 6-year follow-up. We concentrate on incident dementia risk over 10 years. METHODS: participants come from MRC CFAS, a multi-centre longitudinal population-based study of ageing in England and Wales. Three follow-up waves of data collection were used: 2, 6 and 10 years. Logistic regressions were undertaken to investigate associations between APOE (n = 955) and ACE (n = 856) alleles/genotypes and incident dementia. Two types of control groups were used: non-demented and highly functioning non-demented. Results were back-weighted. RESULTS: compared to APOE epsilon3, epsilon2 conferred protection of odds ratio (OR) = 0.3 (95% confidence interval, CI = 0.1-0.6) and epsilon4 risk of OR = 2.9 (95% CI = 1.7-4.9) for incident dementia. Compared to epsilon3/epsilon3, the epsilon3/epsilon4 and epsilon4/epsilon4 genotypes conferred risks of OR = 3.6 (95% CI = 1.8-7.3) and OR = 7.9 (95% CI = 1.6-39.2), respectively. The epsilon3/epsilon2 genotype protected against dementia (OR = 0.2, 95% CI = 0.1-0.7), and epsilon2/epsilon2 had a similar protective effect but with wide CIs (OR = 0.3, 95% CI = 0.1-1.7). Restricting the control group accentuated these differentials. The effects of ACE alleles/genotypes on incident dementia risk were small. CONCLUSIONS: APOE but not ACE is associated with late-onset incident dementia in the population. Using longer term follow-up with proper adjustment for attrition and incident cases increases estimates of risk.

Wild-type but not mutant huntingtin modulates the transcriptional activity of liver X receptors.

BACKGROUND: Huntington's disease is caused by expansion of a polyglutamine tract found in the amino-terminal of the ubiquitously expressed protein huntingtin. Well studied in its mutant form, huntingtin has a wide variety of normal functions, loss of which may also contribute to disease progression. Widespread transcriptional dysfunction occurs in brains of Huntington's disease patients and in transgenic mouse and cell models of Huntington's disease. METHODS: To identify new transcriptional pathways altered by the normal and/or abnormal function of huntingtin, we probed several nuclear receptors, normally expressed in the brain, for binding to huntingtin in its mutant and wild-type forms. RESULTS: Wild-type huntingtin could bind to a number of nuclear receptors; LXRalpha, PPARgamma, VDR and TRalpha1. Over-expression of huntingtin activated, while knockout of huntingtin decreased, LXR mediated transcription of a reporter gene. Loss of huntingtin also decreased expression of the LXR target gene, ABCA1. In vivo, huntingtin deficient zebrafish had a severe phenotype and reduced expression of LXR regulated genes. An LXR agonist was able to partially rescue the phenotype and the expression of LXR target genes in huntingtin deficient zebrafish during early development. CONCLUSION: Our data suggest a novel function for wild-type huntingtin as a co-factor of LXR. However, this activity is lost by mutant huntingtin that only interacts weakly with LXR.

Association analysis of dynamin-binding protein (DNMBP) on chromosome 10q with late onset Alzheimer's disease in a large caucasian UK sample.

A recent scan of single nucleotide polymorphisms (SNPs) in the region 40-107 Mb on chromosome 10q in a large Japanese case-control cohort identified six SNPs in or near the dynamin-binding protein gene (DNMBP) that were associated with late onset Alzheimer's disease (LOAD) in individuals lacking the APOE epsilon4 allele [Kuwano et al. (2006); Hum Mol Genet 15:2170-2182]. We genotyped these six SNPs in 1,212 unrelated Caucasian patients of UK origin with LOAD and 1,389 ethnically, gender and age matched control subjects. We did not observe a statistically significant association with the risk of LOAD for any of the six SNPs in the sample as a whole. When stratifying the sample by APOE one SNP (intergenic SNP rs11190302) was associated with LOAD in individuals lacking the epsilon4 allele (genotypic P = 0.027, allelic P = 0.066). However this association was in the opposite direction to that detected in the Japanese population. It remains to be determined whether DNMBP is associated with LOAD.

Association analysis of 528 intra-genic SNPs in a region of chromosome 10 linked to late onset Alzheimer's disease.

Late-onset Alzheimer's disease (LOAD) is a genetically complex neurodegenerative disorder. Currently, only the epsilon4 allele of the Apolipoprotein E gene has been identified unequivocally as a genetic susceptibility factor for LOAD. Others remain to be found. In 2002 we observed genome-wide significant evidence of linkage to a region on chromosome 10q11.23-q21.3 [Myers et al. (2002) Am J Med Genet 114:235-244]. Our objective in this study was to test every gene within the maximum LOD-1 linkage region, for association with LOAD. We obtained results for 528 SNPs from 67 genes, with an average density of 1 SNP every 10 kb within the genes. We demonstrated nominally significant association with LOAD for 4 SNPs: rs1881747 near DKK1 (P = 0.011, OR = 1.24), rs2279420 in ANK3 (P = 0.022, OR = 0.79), rs2306402 in CTNNA3 (P = 0.024, OR = 1.18), and rs5030882 in CXXC6 (P = 0.046, OR = 1.29) in 1,160 cases and 1,389 controls. These results would not survive correction for multiple testing but warrant attempts at confirmation in independent samples.

Chylomicron remnant clearance from the plasma is normal in familial hypercholesterolemic homozygotes with defined receptor defects.

The retinyl palmitate fat tolerance test was used to measure chylomicron remnant clearance in 10 normal subjects (apolipoprotein E [apo E] isotypes 3 or 4 only), 6 normolipidemic apo E2/2 homozygotes and 5 familial hypercholesterolemic homozygotes. Skin fibroblasts with fully upregulated LDL receptors from the latter subjects degraded rabbit 125I-beta VLDL in vitro at rates ranging from less than 10-48% of normal. Experiments in vivo revealed no significant differences between the normal and homozygous familial hypercholesterolemic (FHH) subjects in chylomicron remnant clearance assessed on the basis of "areas under the curves" for retinyl palmitate levels present in post-prandial serum, chylomicron remnants (Sf. less than 1,000), or chylomicrons (Sf. greater than 1,000). Remnant clearance was greatly decreased at all times in the apo E2/2 homozygotes, indicative of an important degree of flux control exerted by a receptor-mediated step involving apo E as ligand. The absence of any excess remnant accumulation in FHH subjects with varying "impairment" of LDL receptor-mediated degradation of apo E-containing lipoproteins, permits the conclusion that chylomicron remnants are initially cleared from the plasma by apo E-recognizing receptors which are genetically distinct from LDL receptors.

Polyalanine and polyserine frameshift products in Huntington's disease.

Codon reiteration disorders are caused by abnormal expansions of either polyglutamine or polyalanine tracts within the coding region of a protein. These mutations impair normal protein folding, resulting in aggregate formation in the affected tissues. Huntington's disease is the most common of the nine disorders caused by polyglutamine expansion mutations. The most extensively studied polyalanine expansion disorder is oculopharyngeal muscular dystrophy. There may be a link between diseases caused by polyglutamine and polyalanine expansion mutations as it has been shown that the expanded CAG/polyglutamine tract within the SCA3 gene can shift to the GCA[corrected]/polyalanine frame. Here, we show that this frameshifting phenomenon is more widespread and occurs in Huntington's disease. We have shown both +1 frameshift and +2 frameshift products (which may contain polyalanine or polyserine tracts, respectively) in human postmortem Huntington's disease brains and in a transgenic mouse model of Huntington's disease. Our data suggest that +1 and +2 frameshift products are generated at low levels. This may be relevant to the pathogenesis of human Huntington's disease, as we have previously shown that both polyserine and polyalanine-containing proteins are modifiers of mutant huntingtin toxicity, with low expression levels of polyalanine-containing proteins having a protective effect.

Fluorescence-Based Assays to Analyse Phosphatidylinositol 5-Phosphate in Autophagy.

Autophagosome formation is stimulated by VPS34-dependent PI(3)P formation and by alternative VPS34-independent pathways. We recently described that PI(5)P regulates autophagosome biogenesis and rescues autophagy in VPS34-inactivated cells, suggesting that PI(5)P contributes to canonical autophagy. Our analysis revealed a hitherto unknown functional interplay between PIKfyve and PIPK type II in controlling PI(5)P levels in the context of autophagy. Among phosphoinositides, visualization of PI(5)P in intact cells has remained difficult. While PI(5)P has been implicated in signaling pathways, chromatin organization, bacterial invasion, and cytoskeletal remodeling, our study is the first report showing PI(5)P localization on autophagosomes and early autophagosomal structures when autophagy is induced by nutrient deprivation (amino acids or glucose starvation). We provided a detailed analysis of PI(5)P distribution by the use of super-resolution structured illuminated microscopy. Here, we present a set of tools for detection of PI(5)P during autophagy by confocal microscopy, live-cell imaging, and super-resolution microscopy.

The genetics of Alzheimer's disease.

Alzheimer's disease (AD) is the major cause of dementia in the U.K. The clinical diagnosis of the specific disease resulting in dementia is unreliable and thus a definitive diagnosis of AD is best made in conjunction with post-mortem findings of amyloid plaques and neurofibrillary tangles. Alzheimer's disease is neuropathologically indistinguishable in the young and old, but has been divided arbitrarily into early- and late-onset disease using age cut-offs of 60 or 65 years. Twin and family studies suggest that genetic factors play a major role in its aetiology. This review considers the three loci which have been shown to be associated with early-onset AD: amyloid precursor protein, presenilin (PS)-1 and PS-2. Mutations in these genes seem to be associated with overproduction of the 42-amino acid form of beta-amyloid, suggesting that this may be a central pathological process in AD. The impact of the different apo E alleles on the risks for late- and early-onset AD is discussed and compared with other dementing conditions. Recent analyses suggest that there are likely to be other genes besides apo E which impact on late-onset AD risk. The possible roles in AD of the mitochondrial mutation at position 4336, the PS intron 8 polymorphism, and variants in the alpha 1-antichymotrypsin and VLDL-receptor genes, are considered.

Yoshinori Ohsumi's Nobel Prize for mechanisms of autophagy: from basic yeast biology to therapeutic potential.

On 3 October 2016, Japanese cell biologist Yoshinori Ohsumi was awarded the Nobel Prize in Physiology or Medicine 'for his discoveries of mechanisms for autophagy'; autophagy being an intracellular degradation pathway that helps maintain cytoplasmic homeostasis. This commentary discusses Ohsumi's Nobel prize-winning work in context, before explaining the clinical relevance of autophagy.

Identification of two new LDL-receptor mutations causing homozygous familial hypercholesterolemia in a South African of Indian origin.

South Africans of Indian origin have a high frequency of Familial Hypercholesterolemia (FH). Fibroblasts from a South African Indian FH homozygote, D, expressed about 30% of the normal number of LDL receptors. These receptors showed defective LDL binding. Sequence and haplotype analysis revealed that D had two different mutant LDL receptor alleles: FH Durban-1 is a point mutation [asp69(GAT) to tyr(TAT)] in ligand-binding repeat 2 and FH Durban-2 is a point mutation [glu119(GAG) to lys(AAG)] in ligand-binding repeat three of the LDL receptor. Single-strand conformational polymorphism analysis, which was used in the initial detection of these mutations, was also employed for subsequent population screening assays. These mutations were not detected in any of the South African Indian FH or hypercholesterolemic patients that were screened.

Stop signal response inhibition is not modulated by tryptophan depletion or the serotonin transporter polymorphism in healthy volunteers: implications for the 5-HT theory of impulsivity.

RATIONALE: Reduced serotonin neurotransmission is implicated in disorders of impulse control, but the involvement of serotonin in inhibitory processes in healthy human subjects remains unclear. OBJECTIVES: To investigate the effects of an acute manipulation of serotonin and genotype at a functional polymorphism in a gene coding for the serotonin transporter (5-HTT) on an established measure of response inhibition. METHODS: Serotonin function was reduced by the acute tryptophan depletion (ATD) procedure in a double-blind, crossover design in 42 healthy subjects. The Stop Signal Task (SST) was administered 5-7 h after drink administration. The influences of 5-HTT polymorphism, gender and trait impulsivity were investigated. RESULTS: ATD was associated with significant depletion of plasma tryptophan levels but did not increase the stop signal reaction time in comparison to the balanced (placebo) amino acid mixture. Subjects possessing the short allele of the 5-HTT polymorphism were not more impulsive on the SST than subjects homozygous for the long allele under placebo conditions and were not disproportionately sensitive to the effects of ATD. There was no effect of gender or trait impulsivity on ATD-induced change. CONCLUSIONS: We find no support for the involvement of brain serotonin neurotransmission in this form of inhibitory control in healthy human subjects.

Apolipoprotein E4 is only a weak predictor of dementia and cognitive decline in the general population.

BACKGROUND: Apolipoprotein E (APOE) polymorphisms are unequivocally associated with risk for Alzheimer's disease (AD). It is crucial to understand how this genetic factor affects dementia risk in the general population, as well as in narrowly diagnosed, selected, patient groups. METHODS: We assessed the cross sectional association between APOE genotype and dementia status in a community based sample, the MRC Cognitive Function and Ageing Study (MRC CFAS). In addition, we tested the effects of APOE genotypes on the differences in MMSE scores between the first and third assessment waves (about six years apart), an index of cognitive decline. RESULTS: The APOE epsilon4 allele conferred increased risk for dementia (OR=1.5, 95% CI=1.1 to 2.2) compared to epsilon3 in the MRC CFAS sample. Compared with APOE epsilon3/epsilon 3 subjects, those with the epsilon3/epsilon4 genotypes were not at significantly higher risk for dementia (OR=1.1, 95% CI=0.6 to 1.9), although epsilon4/epsilon4 subjects were (OR= 3.8, 95% CI=1.0 to 14.0). Risk estimates were not different between men and women. Notably, our risk estimates for dementia were significantly lower than those reported for a diagnosis of Alzheimer's disease. MMSE scores at wave 3 and the difference in MMSE between baseline and at the third assessment wave were not different across APOE genotypes. INTERPRETATION: The APOE epsilon4 allele is a weaker predictor for dementia in the general population than for AD. This may be because dementia can be caused by non-AD pathological processes and because most prevalent dementia occurs at an age when the APOE epsilon4 effect on AD risk (and therefore dementia) has started to decline.

Wild type Huntingtin reduces the cellular toxicity of mutant Huntingtin in mammalian cell models of Huntington's disease.

OBJECTIVES: Recent data suggest that wild type huntingtin can protect against apoptosis in the testis of mice expressing full length huntingtin transgenes with expanded CAG repeats. It is not clear if this protective effect was confined to particular cell types, or if wild type huntingtin exerted its protective effect in this model by simply reducing the formation of toxic proteolytic fragments from mutant huntingtin. METHODS: We cotransfected neuronal (SK-N-SH, human neuroblastoma) and non-neuronal (COS-7, monkey kidney) cell lines with HD exon 1 (containing either 21 or 72 CAG repeats) construct DNA and either full length wild type huntingtin or pFLAG (control vector). RESULTS: Full length wild type huntingtin significantly reduced cell death resulting from the mutant HD exon 1 fragments containing 72 CAG repeats in both cell lines. Wild type huntingtin did not significantly modulate cell death caused by transfection of HD exon 1 fragments containing 21 CAG repeats in either cell line. CONCLUSIONS: Our results suggest that wild type huntingtin can significantly reduce the cellular toxicity of mutant HD exon 1 fragments in both neuronal and non-neuronal cell lines. This suggests that wild type huntingtin can be protective in different cell types and that it can act against the toxicity caused by a mutant huntingtin fragment as well as against a full length transgene.

The ACE I allele is associated with increased risk for ruptured intracranial aneurysms.

Genetic and environmental factors play roles in the aetiology of ruptured intracranial aneurysms. Hypertension has been reported as a risk factor for intracranial aneurysm haemorrhage. We have tested if genotypes at the angiotensin converting enzyme (ACE) gene locus are associated with ruptured intracranial aneurysms. The insertion/deletion polymorphism in the ACE gene was genotyped in 258 subjects presenting in East Anglia with ruptured intracranial aneurysms (confirmed at surgery or angiographically) and 299 controls from the same region. ACE allele frequencies were significantly different in the cases and the controls (alleles chi(2)(1)=4.67, p=0.03). The I allele was associated with aneurysm risk (odds ratio for I allele v D allele = 1.3 (95% CI=1.02-1-65); odds ratio for II v DD genotype = 1.67 (95% CI=1.04-2.66)). The I allele at the ACE locus is over-represented in subjects with ruptured intracranial aneurysms. These data are supported by non-significant trends in the same direction in two previous smaller studies. Thus, this allele may be associated with risk for ruptured intracranial aneurysms.