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.

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.

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 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.

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.

Allelic variation in the promoter region of the LDL receptor gene: analysis of an African-specific variant in the FP2 cis-acting regulatory element.

DNA samples of 2303 individuals from nine different population groups were screened for variant -175g-->t in the promoter region of the low-density lipoprotein receptor (LDLR) gene. The -175g-->t variant detected at carrier frequencies of 3-10% in different African population groups was absent in the Caucasian and Asian (Chinese) individuals studied. In contrast to previous findings in Black South Africans where this polymorphism predominated in patients with familial hypercholesterolaemia (FH), it occurred at a significantly lower frequency in hypercholesterolaemics from the recently admixed Coloured population of South Africa compared with population-matched controls (P<0.0001). Haplotype and mutation analysis excluded the likelihood that this finding is due to association with a specific disease-related mutation in FH patients, although reversal of the positive association with FH observed in the Black population may, at least in part, be due to admixture linkage disequilibrium. Transient transfection studies in HepG2 cells demonstrated that the -175t allele is associated with a non-significant decrease ( approximately 7%) of LDLR transcription in the absence of sterols. The data presented in this study raise the possibility that the -175g-->t polymorphism may have subtle effects that become clinically important within certain genetic and/or environmental contexts.

Polyglutamine expansions cause decreased CRE-mediated transcription and early gene expression changes prior to cell death in an inducible cell model of Huntington's disease.

Huntington's disease (HD) is one of 10 known diseases caused by a (CAG)(n) trinucleotide repeat expansion that is translated into an abnormally long polyglutamine tract. We have developed stable inducible neuronal (PC12) cell lines that express huntingtin exon 1 with varying CAG repeat lengths under doxycycline (dox) control. The expression of expanded repeats is associated with aggregate formation, caspase-dependent cell death and decreased neurite outgrowth. Post-mitotic cells expressing mutant alleles were more prone to cell death compared with identical cycling cells. To determine early metabolic changes induced by this mutation in cell models, we studied changes in gene expression after 18 h dox induction, using Affymetrix arrays, cDNA filters and adapter-tagged competitive PCR (ATAC-PCR). At this time point there were low rates of inclusion formation, no evidence of mitochondrial compromise and no excess cell death in the lines expressing expanded compared with wild-type repeats. The expression profiles suggest novel targets for the HD mutation and were compatible with impaired cAMP response element (CRE)-mediated transcription, which we confirmed using CRE-luciferase reporter assays. Reduced CRE-mediated transcription may contribute to the loss of neurite outgrowth and cell death in polyglutamine diseases, as these phenotypes were partially rescued by treating cells with cAMP or forskolin.

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.

Mutation analysis of the spastin gene (SPG4) in patients with hereditary spastic paraparesis.

BACKGROUND: Hereditary spastic paraparesis is a genetically heterogeneous condition. Recently, mutations in the spastin gene were reported in families linked to the common SPG4 locus on chromosome 2p21-22. OBJECTIVES: To study a population of patients with hereditary spastic paraparesis for mutations in the spastin gene (SPG4) on chromosome 2p21-22. METHODS: DNA from 32 patients (12 from families known to be linked to SPG4) was analysed for mutations in the spastin gene by single strand conformational polymorphism analysis and sequencing. All patients were also examined clinically. RESULTS: Thirteen SPG4 mutations were identified, 11 of which are novel. These mutations include missense, nonsense, frameshift, and splice site mutations, the majority of which affect the AAA cassette. We also describe a nucleotide substitution outside this conserved region which appears to behave as a recessive mutation. CONCLUSIONS: Recurrent mutations in the spastin gene are uncommon. This reduces the ease of mutation detection as a part of the diagnostic work up of patients with hereditary spastic paraparesis. Our findings have important implications for the presumed function of spastin and schemes for mutation detection in HSP patients.

12-O-tetradecanoyl-phorbol-13-acetate down-regulates the Huntingtin promoter at Sp1 sites.

We have studied the effects of the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on Huntington's disease (HD) gene transcription in neuronal and non-neuronal cell lines, to investigate pathways regulating HD gene expression. TPA reduced transcription from the HD gene promoter in SK-N-SH (neuroblastoma) and HeLa cells but not in JEG3 (choriocarcinoma) cells. In SK-N-SH cells, the responsible cis-acting promoter sequences comprise the tandemly duplicated Sp1 sites in the region from -213 to -174, relative to the translation start site. The TPA-down-regulating region in HeLa cells was mapped to the sequence from -141 to -126. In conclusion, this demonstrates that HD gene transcription can be down-regulated in vitro in a cell-specific manner.

Bacterial and yeast chaperones reduce both aggregate formation and cell death in mammalian cell models of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative condition caused by expansions of more than 35 uninterrupted CAG repeats in exon 1 of the huntingtin gene. The CAG repeats in HD and the other seven known diseases caused by CAG codon expansions are translated into long polyglutamine tracts that confer a deleterious gain of function on the mutant proteins. Intraneuronal inclusions comprising aggregates of the relevant mutant proteins are found in the brains of patients with HD and related diseases. It is crucial to determine whether the formation of inclusions is directly pathogenic, because a number of studies have suggested that aggregates may be epiphenomena or even protective. Here, we show that fragments of the bacterial chaperone GroEL and the full-length yeast heat shock protein Hsp104 reduce both aggregate formation and cell death in mammalian cell models of HD, consistent with a causal link between aggregation and pathology.

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.

Intracellular green fluorescent protein-polyalanine aggregates are associated with cell death.

Eight diseases, exemplified by Huntington's disease and spinocerebellar ataxia type 1, are caused by CAG-repeat expansion mutations. The CAG repeats are translated into expanded polyglutamine tracts, which are associated with deleterious novel functions. While these diseases are characterized by intraneuronal aggregate formation, it is unclear whether the aggregates cause disease. We have addressed this debate by generating intracellular aggregates with green fluorescent protein (GFP) fused to 19-37 alanines. No aggregates were seen in cells expressing native GFP or GFP fused to seven alanines. Aggregate-containing cells expressing GFP fused to 19-37 polyalanines show high rates of nuclear fragmentation compared with cells expressing the same constructs without aggregates, or cells expressing GFP fused to seven alanines. This suggests an association between aggregate formation and cell death.

Alpha-synuclein overexpression promotes aggregation of mutant huntingtin.

Protein aggregates are a neuropathological feature of Huntington's disease and Parkinson's disease. Mutant huntingtin exon 1 with 72 CAG repeats fused to enhanced green fluorescent protein (EGFP) forms hyperfluorescent inclusions in PC12 cells. Inclusion formation is enhanced in cells co-transfected with EGFP-huntingtin-(CAG)(72) and alpha-synuclein, a major component of Parkinson's disease aggregates. However, alpha-synuclein does not form aggregates by itself, nor does it appear in huntingtin inclusions in vitro.

Effects of heat shock, heat shock protein 40 (HDJ-2), and proteasome inhibition on protein aggregation in cellular models of Huntington's disease.

Huntington's disease (HD), spinocerebellar ataxias types 1 and 3 (SCA1, SCA3), and spinobulbar muscular atrophy (SBMA) are caused by CAG/polyglutamine expansion mutations. A feature of these diseases is ubiquitinated intraneuronal inclusions derived from the mutant proteins, which colocalize with heat shock proteins (HSPs) in SCA1 and SBMA and proteasomal components in SCA1, SCA3, and SBMA. Previous studies suggested that HSPs might protect against inclusion formation, because overexpression of HDJ-2/HSDJ (a human HSP40 homologue) reduced ataxin-1 (SCA1) and androgen receptor (SBMA) aggregate formation in HeLa cells. We investigated these phenomena by transiently transfecting part of huntingtin exon 1 in COS-7, PC12, and SH-SY5Y cells. Inclusion formation was not seen with constructs expressing 23 glutamines but was repeat length and time dependent for mutant constructs with 43-74 repeats. HSP70, HSP40, the 20S proteasome and ubiquitin colocalized with inclusions. Treatment with heat shock and lactacystin, a proteasome inhibitor, increased the proportion of mutant huntingtin exon 1-expressing cells with inclusions. Thus, inclusion formation may be enhanced in polyglutamine diseases, if the pathological process results in proteasome inhibition or a heat-shock response. Overexpression of HDJ-2/HSDJ did not modify inclusion formation in PC12 and SH-SY5Y cells but increased inclusion formation in COS-7 cells. To our knowledge, this is the first report of an HSP increasing aggregation of an abnormally folded protein in mammalian cells and expands the current understanding of the roles of HDJ-2/HSDJ in protein folding.

Aberrant splicing in the presenilin-1 intron 4 mutation causes presenile Alzheimer's disease by increased Abeta42 secretion.

We previously described a splice donor site mutation in intron 4 of presenilin-1 (PSEN1) in two patients with autopsy-confirmed early-onset Alzheimer's disease (AD). Here we provide evidence that the intron 4 mutation is present in four additional unrelated early-onset AD cases, that the mutation segregates in an autosomal dominant manner and that all cases have one common ancestor. We demonstrate that the intron 4 mutation produces three different transcripts, two deletion transcripts (Delta4 and Delta4cryptic) and one insertion transcript (insTAC), by aberrant splicing. The deletion transcripts result in the formation of C-truncated (approximately 7 kDa) PSEN1 proteins while the insertion transcript produces a full-length PSEN1 with one extra amino acid (Thr) inserted between codons 113 and 114 (PSEN1 T113-114ins). The truncated proteins were not detectable in vivo in brain homogenates or lymphoblast lysates of mutation carriers. In vitro HEK-293 cells overexpressing Delta4, Delta4cryptic or insTACPSEN1 cDNAs showed increased Abeta42 secretion (approximately 3.4 times) only for the insertion cDNA construct. Increased Abeta42 production was also observed in brain homogenates. Our data indicate that in the case of intron 4 mutation, the AD pathophysiology results from the presence of the PSEN1 T113-114ins protein comparable with cases carrying dominant PSEN1 missense mutations.

Intracellular inclusions, pathological markers in diseases caused by expanded polyglutamine tracts?

The largest group of currently known trinucleotide repeat diseases is caused by (CAG)n repeat expansions. These (CAG)n repeats are translated into polyglutamine tracts from both mutant and wild type alleles. Genetic and transgenic mouse data suggest that the expanded polyglutamines cause disease by conferring a novel deleterious gain of function on the mutant protein. These mutations are associated with the formation of intracellular inclusions. This review will consider findings from necropsy studies of human patients and transgenic mouse models of these diseases, along with in vitro models, in order to try to synthesise the current understanding of these diseases and the evidence for and against inclusion formation as a primary mechanism leading to pathology.