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.

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.

Calpain inhibition mediates autophagy-dependent protection against polyglutamine toxicity.

Over recent years, accumulated evidence suggests that autophagy induction is protective in animal models of a number of neurodegenerative diseases. Intense research in the field has elucidated different pathways through which autophagy can be upregulated and it is important to establish how modulation of these pathways impacts upon disease progression in vivo and therefore which, if any, may have further therapeutic relevance. In addition, it is important to understand how alterations in these target pathways may affect normal physiology when constitutively modulated over a long time period, as would be required for treatment of neurodegenerative diseases. Here we evaluate the potential protective effect of downregulation of calpains. We demonstrate, in Drosophila, that calpain knockdown protects against the aggregation and toxicity of proteins, like mutant huntingtin, in an autophagy-dependent fashion. Furthermore, we demonstrate that, overexpression of the calpain inhibitor, calpastatin, increases autophagosome levels and is protective in a mouse model of Huntington's disease, improving motor signs and delaying the onset of tremors. Importantly, long-term inhibition of calpains did not result in any overt deleterious phenotypes in mice. Thus, calpain inhibition, or activation of autophagy pathways downstream of calpains, may be suitable therapeutic targets for diseases like Huntington's disease.

Inhibition of autophagy, lysosome and VCP function impairs stress granule assembly.

Stress granules (SGs) are mRNA-protein aggregates induced during stress, which accumulate in many neurodegenerative diseases. Previously, the autophagy-lysosome pathway and valosin-containing protein (VCP), key players of the protein quality control (PQC), were shown to regulate SG degradation. This is consistent with the idea that PQC may survey and/or assist SG dynamics. However, despite these observations, it is currently unknown whether the PQC actively participates in SG assembly. Here, we describe that inhibition of autophagy, lysosomes and VCP causes defective SG formation after induction. Silencing the VCP co-factors UFD1L and PLAA, which degrade defective ribosomal products (DRIPs) and 60S ribosomes, also impaired SG assembly. Intriguingly, DRIPs and 60S, which are released from disassembling polysomes and are normally excluded from SGs, were significantly retained within SGs in cells with impaired autophagy, lysosome or VCP function. Our results suggest that deregulated autophagy, lysosomal or VCP activities, which occur in several neurodegenerative (VCP-associated) diseases, may alter SG morphology and composition.

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.

Meta-analysis of 74,046 individuals identifies 11 new susceptibility loci for Alzheimer's disease.

Eleven susceptibility loci for late-onset Alzheimer's disease (LOAD) were identified by previous studies; however, a large portion of the genetic risk for this disease remains unexplained. We conducted a large, two-stage meta-analysis of genome-wide association studies (GWAS) in individuals of European ancestry. In stage 1, we used genotyped and imputed data (7,055,881 SNPs) to perform meta-analysis on 4 previously published GWAS data sets consisting of 17,008 Alzheimer's disease cases and 37,154 controls. In stage 2, 11,632 SNPs were genotyped and tested for association in an independent set of 8,572 Alzheimer's disease cases and 11,312 controls. In addition to the APOE locus (encoding apolipoprotein E), 19 loci reached genome-wide significance (P < 5 × 10(-8)) in the combined stage 1 and stage 2 analysis, of which 11 are newly associated with Alzheimer's disease.

Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012.

In 2009, the Nomenclature Committee on Cell Death (NCCD) proposed a set of recommendations for the definition of distinct cell death morphologies and for the appropriate use of cell death-related terminology, including 'apoptosis', 'necrosis' and 'mitotic catastrophe'. In view of the substantial progress in the biochemical and genetic exploration of cell death, time has come to switch from morphological to molecular definitions of cell death modalities. Here we propose a functional classification of cell death subroutines that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death and mitotic catastrophe. Moreover, we discuss the utility of expressions indicating additional cell death modalities. On the basis of the new, revised NCCD classification, cell death subroutines are defined by a series of precise, measurable biochemical features.

The Parkinson's disease protein LRRK2 impairs proteasome substrate clearance without affecting proteasome catalytic activity.

Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common known cause of Parkinson's disease (PD). The clinical features of LRRK2 PD are indistinguishable from idiopathic PD, with accumulation of α-synuclein and/or tau and/or ubiquitin in intraneuronal aggregates. This suggests that LRRK2 is a key to understanding the aetiology of the disorder. Although loss-of-function does not appear to be the mechanism causing PD in LRRK2 patients, it is not clear how this protein mediates toxicity. In this study, we report that LRRK2 overexpression in cells and in vivo impairs the activity of the ubiquitin-proteasome pathway, and that this accounts for the accumulation of diverse substrates with LRRK2 overexpression. We show that this is not mediated by large LRRK2 aggregates or sequestration of ubiquitin to the aggregates. Importantly, such abnormalities are not seen with overexpression of the related protein LRRK1. Our data suggest that LRRK2 inhibits the clearance of proteasome substrates upstream of proteasome catalytic activity, favouring the accumulation of proteins and aggregate formation. Thus, we provide a molecular link between LRRK2, the most common known cause of PD, and its previously described phenotype of protein accumulation.

A Multiple Indicators Multiple Causes (MIMIC) model of Behavioural and Psychological Symptoms in Dementia (BPSD).

INTRODUCTION: Although there is evidence for distinct behavioural sub-phenotypes in Alzheimer's disease (AD), their inter-relationships and the effect of clinical variables on their expression have been little investigated. METHODS: We have analysed a sample of 1850 probable AD patients from the UK and Greece with 10 item Neuropsychiatric Inventory (NPI) data. We applied a Multiple Indicators Multiple Causes (MIMIC) approach to investigate the effect of MMSE, disease duration, gender, age and age of onset on the structure of a four-factor model consisting of "psychosis", "moods", "agitation" and "behavioural dyscontrol". RESULTS: Specific clinical variables predicted the expression of individual factors. When the inter-relationship of factors is modelled, some previously significant associations are lost. For example, lower MMSE scores predict psychosis, agitation and behavioural dyscontrol factors, but psychosis and mood predict the agitation factor. Taking these associations into account MMSE scores did not predict agitation. CONCLUSIONS: The complexity of the inter-relations between symptoms, factors and clinical variables is efficiently captured by this MIMIC model.

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.

Apoptosis blocks Beclin 1-dependent autophagosome synthesis: an effect rescued by Bcl-xL.

Apoptotic cell death is mediated by caspase activation. Autophagy involves the sequestration of cytoplasmic contents into autophagosomes for traffic to lysosomes for degradation. Although autophagy is antiapoptotic, increased numbers of autophagosomes have been associated with forms of non-apoptotic cell death. Apoptosis and autophagy may be co-regulated in the same directions, as the antiapoptotic Bcl-2 and Bcl-xL proteins negatively regulate autophagy by binding to Beclin 1 (mammalian Atg6), and proapoptotic BH3-only proteins may reverse this effect by displacing these interactions. Here, we show that apoptosis can suppress autophagy. Apoptosis induced by the proapoptotic protein Bax reduced autophagy by enhancing caspase-mediated cleavage of Beclin 1 at D149. After cleavage, both N- and C-terminal Beclin 1 fragments change their localisations and these fragments do not interact normally with Vps34, which is required for autophagy. The cleavage of Beclin 1 is a critical event whereby caspases inhibit autophagy, as a non-cleavable Beclin 1 mutant restored autophagy in cells overexpressing Bax.

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.

Rapamycin and mTOR-independent autophagy inducers ameliorate toxicity of polyglutamine-expanded huntingtin and related proteinopathies.

The formation of intra-neuronal mutant protein aggregates is a characteristic of several human neurodegenerative disorders, like Alzheimer's disease, Parkinson's disease (PD) and polyglutamine disorders, including Huntington's disease (HD). Autophagy is a major clearance pathway for the removal of mutant huntingtin associated with HD, and many other disease-causing, cytoplasmic, aggregate-prone proteins. Autophagy is negatively regulated by the mammalian target of rapamycin (mTOR) and can be induced in all mammalian cell types by the mTOR inhibitor rapamycin. It can also be induced by a recently described cyclical mTOR-independent pathway, which has multiple drug targets, involving links between Ca(2+)-calpain-G(salpha) and cAMP-Epac-PLC-epsilon-IP(3) signalling. Both pathways enhance the clearance of mutant huntingtin fragments and attenuate polyglutamine toxicity in cell and animal models. The protective effects of rapamycin in vivo are autophagy-dependent. In Drosophila models of various diseases, the benefits of rapamycin are lost when the expression of different autophagy genes is reduced, implicating that its effects are not mediated by autophagy-independent processes (like mild translation suppression). Also, the mTOR-independent autophagy enhancers have no effects on mutant protein clearance in autophagy-deficient cells. In this review, we describe various drugs and pathways inducing autophagy, which may be potential therapeutic approaches for HD and related conditions.

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.

Interaction between the ADAM12 and SH3MD1 genes may confer susceptibility to late-onset Alzheimer's disease.

The neuropathology of Alzheimer's disease (AD) is characterized by intracellular neurofibrillary tangles and the extracellular deposition of beta-amyloid (Abeta) in senile plaques. Abeta has been shown to mediate neurodegenerative and inflammatory changes associated with amyloid plaques, although the pathological mechanism of Abeta remains largely unknown. Recent evidence suggests that the FISH adapter protein binds to, and potentially regulates, ADAM12 (a disintegrin and metalloprotease 12) to mediate a neurotoxic effect of Abeta. The ADAM12 gene lies on chromosome 10q26.3, and the gene encoding FISH, SH3MD1, lies within a region of linkage to late-onset AD (LOAD) on 10q25.1. This study investigates whether there is a relationship between variation in ADAM12 and SH3MD1 and susceptibility to LOAD in a sample of 1,051 AD cases and 1,269 matched controls. We observe significant interactions between variants in the two genes that may influence susceptibility to LOAD. The most significant statistical interaction is between rs3740473, a synonymous single nucleotide polymorphism (SNP) in SH3MD1 and rs11244787, an intronic SNP in ADAM12 (effect size = 2.1 for interaction term, P = 0.006).

Association studies of 23 positional/functional candidate genes on chromosome 10 in late-onset Alzheimer's disease.

Late-onset Alzheimer's disease (LOAD) is a common neurodegenerative disorder, with a complex etiology. APOE is the only confirmed susceptibility gene for LOAD. Others remain yet to be found. Evidence from linkage studies suggests that a gene (or genes) conferring susceptibility for LOAD resides on chromosome 10. We studied 23 positional/functional candidate genes from our linkage region on chromosome 10 (APBB1IP, ALOX5, AD037, SLC18A3, DKK1, ZWINT, ANK3, UBE2D1, CDC2, SIRT1, JDP1, NET7, SUPV3L1, NEN3, SAR1, SGPL1, SEC24C, CAMK2G, PP3CB, SNCG, CH25H, PLCE1, ANXV111) in the MRC genetic resource for LOAD. These candidates were screened for sequence polymorphisms in a sample of 14 LOAD subjects and detected polymorphisms tested for association with LOAD in a three-stage design involving two stages of genotyping pooled DNA samples followed by a third stage in which markers showing evidence for association in the first stages were subjected to individual genotyping. One hundred and twenty polymorphisms were identified and tested in stage 1 (4 case + 4 control pools totaling 366 case and 366 control individuals). Single nucleotide polymorphisms (SNPs) showing evidence of association with LOAD were then studied in stage 2 (8 case + 4 control pools totaling 1,001 case and 1,001 control individuals). Five SNPs, in four genes, showed evidence for association (P < 0.1) at stage 2 and were individually genotyped in the complete dataset, comprising 1,160 LOAD cases and 1,389 normal controls. Two SNPs in SGPL1 demonstrated marginal evidence of association, with uncorrected P values of 0.042 and 0.056, suggesting that variation in SGPL1 may confer susceptibility to LOAD.

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.

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.