Regulation of mammalian autophagy in physiology and pathophysiology.

(Macro)autophagy is a bulk degradation process that mediates the clearance of long-lived proteins and organelles. Autophagy is initiated by double-membraned structures, which engulf portions of cytoplasm. The resulting autophagosomes ultimately fuse with lysosomes, where their contents are degraded. Although the term autophagy was first used in 1963, the field has witnessed dramatic growth in the last 5 years, partly as a consequence of the discovery of key components of its cellular machinery. In this review we focus on mammalian autophagy, and we give an overview of the understanding of its machinery and the signaling cascades that regulate it. As recent studies have also shown that autophagy is critical in a range of normal human physiological processes, and defective autophagy is associated with diverse diseases, including neurodegeneration, lysosomal storage diseases, cancers, and Crohn's disease, we discuss the roles of autophagy in health and disease, while trying to critically evaluate if the coincidence between autophagy and these conditions is causal or an epiphenomenon. Finally, we consider the possibility of autophagy upregulation as a therapeutic approach for various conditions.

Self- versus parent-ratings of industriousness, affect, and life satisfaction in relation to academic outcomes.

BACKGROUND: Parents consult with schools on how to help their children succeed, but schools rarely consult with parents, even though most parents have considerable expertise concerning their children's thoughts, feelings, and abilities. AIMS: This study compares the prediction of academic achievement from self- and parent-ratings of feelings towards school (both positive and negative), life satisfaction, and the conscientiousness facet of industriousness for 357 adolescents. SAMPLE: The student sample consisted of 383 participants (194 boys) mostly aged between 12 and 14. The parent sample consisted of 374 participants, 83% of whom were mothers. METHOD: Self-report and other-report scales measuring the above-mentioned constructs were administered to students and parents. Hierarchical regression analysis was used to test hypotheses concerning the incremental validity of parent-ratings. RESULTS: Self-ratings explained 28.6% of the variance in grade point average (GPA) with parent-ratings explaining an additional 12.1%. The incremental effect was strongest for industriousness. CONCLUSION: These results suggest that parent-reports are often more accurate than adolescent self-reports, but that both methods of assessment make unique contributions to the explanation of variance in school grades. Parental understanding constitutes a relatively untapped reservoir of knowledge available to teachers, school counsellors and administrators, education policy makers, and beyond. It makes sense to ask parents about their children when assessing those individual differences that contribute to better educational outcomes.

Over-expression of BCL2 rescues muscle weakness in a mouse model of oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset muscular dystrophy caused by a polyalanine expansion mutation in the coding region of the poly-(A) binding protein nuclear 1 (PABPN1) gene. In unaffected individuals, (GCG)(6) encodes the first 6 alanines in a homopolymeric stretch of 10 alanines. In most patients, this (GCG)(6) repeat is expanded to (GCG)(8-13), leading to a stretch of 12-17 alanines in mutant PABPN1, which is thought to confer a toxic gain of function. Thus, OPMD has been modelled by expressing mutant PABPN1 transgenes in the presence of endogenous copies of the gene in cells and mice. In these models, increased apoptosis is seen, but it is unclear whether this process mediates OPMD. The role of apoptosis in the pathogenesis of different muscular dystrophies is unclear. Blocking apoptosis ameliorates muscle disease in some mouse models of muscular dystrophy such as laminin α-2-deficient mice, but not in others such as dystrophin-deficient (mdx) mice. Here we demonstrate that apoptosis is not only involved in the pathology of OPMD but also is a major contributor to the muscle weakness and dysfunction in this disease. Genetically blocking apoptosis by over-expressing BCL2 ameliorates muscle weakness in our mouse model of OPMD (A17 mice). The effect of BCL2 co-expression on muscle weakness is transient, since muscle weakness is apparent in mice expressing both A17 and BCL2 transgenes at late time points. Thus, while apoptosis is a major pathway that causes muscle weakness in OPMD, other cell death pathways may also contribute to the disease when apoptosis is inhibited.

Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease.

Huntington disease is one of nine inherited neurodegenerative disorders caused by a polyglutamine tract expansion. Expanded polyglutamine proteins accumulate abnormally in intracellular aggregates. Here we show that mammalian target of rapamycin (mTOR) is sequestered in polyglutamine aggregates in cell models, transgenic mice and human brains. Sequestration of mTOR impairs its kinase activity and induces autophagy, a key clearance pathway for mutant huntingtin fragments. This protects against polyglutamine toxicity, as the specific mTOR inhibitor rapamycin attenuates huntingtin accumulation and cell death in cell models of Huntington disease, and inhibition of autophagy has the converse effects. Furthermore, rapamycin protects against neurodegeneration in a fly model of Huntington disease, and the rapamycin analog CCI-779 improved performance on four different behavioral tasks and decreased aggregate formation in a mouse model of Huntington disease. Our data provide proof-of-principle for the potential of inducing autophagy to treat Huntington disease.

Molecular and phenotypic characterization of a mouse model of oculopharyngeal muscular dystrophy reveals severe muscular atrophy restricted to fast glycolytic fibres.

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by ptosis, dysphagia and proximal limb weakness. Autosomal-dominant OPMD is caused by a short (GCG)(8-13) expansions within the first exon of the poly(A)-binding protein nuclear 1 gene (PABPN1), leading to an expanded polyalanine tract in the mutated protein. Expanded PABPN1 forms insoluble aggregates in the nuclei of skeletal muscle fibres. In order to gain insight into the different physiological processes affected in OPMD muscles, we have used a transgenic mouse model of OPMD (A17.1) and performed transcriptomic studies combined with a detailed phenotypic characterization of this model at three time points. The transcriptomic analysis revealed a massive gene deregulation in the A17.1 mice, among which we identified a significant deregulation of pathways associated with muscle atrophy. Using a mathematical model for progression, we have identified that one-third of the progressive genes were also associated with muscle atrophy. Functional and histological analysis of the skeletal muscle of this mouse model confirmed a severe and progressive muscular atrophy associated with a reduction in muscle strength. Moreover, muscle atrophy in the A17.1 mice was restricted to fast glycolytic fibres, containing a large number of intranuclear inclusions (INIs). The soleus muscle and, in particular, oxidative fibres were spared, even though they contained INIs albeit to a lesser degree. These results demonstrate a fibre-type specificity of muscle atrophy in this OPMD model. This study improves our understanding of the biological pathways modified in OPMD to identify potential biomarkers and new therapeutic targets.

Doxycycline attenuates and delays toxicity of the oculopharyngeal muscular dystrophy mutation in transgenic mice.

The muscular dystrophies are a heterogeneous group of disorders for which there are currently no cures. Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant late-onset, progressive disease that generally presents in the fifth or sixth decade with dysphagia, ptosis and proximal limb weakness. OPMD is caused by the abnormal expansion of a (GCG)n trinucleotide repeat in the coding region of the poly-(A) binding protein nuclear 1 (PABPN1) gene. In unaffected individuals, (GCG)6 codes for the first six alanines in a homopolymeric stretch of ten alanines. In most individuals with OPMD this (GCG)6 repeat is expanded to (GCG)8-13, leading to a stretch of 12-17 alanines in mutant PABPN1. PABPN1 with an expanded polyalanine tract forms aggregates consisting of tubular filaments within the nuclei of skeletal muscle fibers. We have developed a transgenic mouse model of OPMD that manifests progressive muscle weakness accompanied by intranuclear aggregates and TUNEL-stained nuclei in skeletal muscle fibers. The onset and severity of these abnormalities were substantially delayed and attenuated by doxycycline treatment, which may exert its therapeutic effect by reducing aggregates and by distinct antiapoptotic properties. Doxycycline may represent a safe and feasible therapeutic for this disease.

Wild-type PABPN1 is anti-apoptotic and reduces toxicity of the oculopharyngeal muscular dystrophy mutation.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset, progressive disease caused by the abnormal expansion of a polyalanine tract-encoding (GCG)(n) trinucleotide repeat in the poly-(A) binding protein nuclear 1 (PABPN1) gene. OPMD is generally inherited as an autosomal dominant disorder and the polyalanine expansion mutation is thought to confer a toxic gain-of-function on mutant PABPN1 which forms aggregates within skeletal myocyte nuclei. Here we describe a novel beneficial function of wild-type PABPN1. Wild-type PABPN1 over-expression can reduce mutant PABPN1 toxicity in both cell and mouse models of OPMD. In addition, wild-type PABPN1 provides some protection to cells against pro-apoptotic insults distinct from the OPMD mutation such as staurosporine treatment and Bax expression. Conversely, PABPN1 knockdown (which itself is not toxic) makes cells more susceptible to apoptotic stimuli. The protective effect of wild-type PABPN1 is mediated by its regulation of X-linked inhibitor of apoptosis (XIAP) protein translation. This normal activity of PABPN1 is partially lost for mutant PABPN1; elevated levels of XIAP are seen in mice expressing a wild-type but not a mutant PABPN1 transgene. This raises the possibility that a compromise of the anti-apoptotic function of PABPN1 might contribute to the disease mechanism of OPMD.

Cdk5 phosphorylation of huntingtin reduces its cleavage by caspases: implications for mutant huntingtin toxicity.

Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded polyglutamine (polyQ) tract in the huntingtin (htt) protein. Mutant htt toxicity is exposed after htt cleavage by caspases and other proteases release NH(2)-terminal fragments containing the polyQ expansion. Here, we show htt interacts and colocalizes with cdk5 in cellular membrane fractions. Cdk5 phosphorylates htt at Ser434, and this phosphorylation reduces caspase-mediated htt cleavage at residue 513. Reduced mutant htt cleavage resulting from cdk5 phosphorylation attenuated aggregate formation and toxicity in cells expressing the NH(2)-terminal 588 amino acids (htt588) of mutant htt. Cdk5 activity is reduced in the brains of HD transgenic mice compared with controls. This result can be accounted for by the polyQ-expanded htt fragments reducing the interaction between cdk5 and its activator p35. These data predict that the ability of cdk5 phosphorylation to protect against htt cleavage, aggregation, and toxicity is compromised in cells expressing toxic fragments of htt.

Huntington's disease: from pathology and genetics to potential therapies.

Huntington's disease (HD) is a devastating autosomal dominant neurodegenerative disease caused by a CAG trinucleotide repeat expansion encoding an abnormally long polyglutamine tract in the huntingtin protein. Much has been learnt since the mutation was identified in 1993. We review the functions of wild-type huntingtin. Mutant huntingtin may cause toxicity via a range of different mechanisms. The primary consequence of the mutation is to confer a toxic gain of function on the mutant protein and this may be modified by certain normal activities that are impaired by the mutation. It is likely that the toxicity of mutant huntingtin is revealed after a series of cleavage events leading to the production of N-terminal huntingtin fragment(s) containing the expanded polyglutamine tract. Although aggregation of the mutant protein is a hallmark of the disease, the role of aggregation is complex and the arguments for protective roles of inclusions are discussed. Mutant huntingtin may mediate some of its toxicity in the nucleus by perturbing specific transcriptional pathways. HD may also inhibit mitochondrial function and proteasome activity. Importantly, not all of the effects of mutant huntingtin may be cell-autonomous, and it is possible that abnormalities in neighbouring neurons and glia may also have an impact on connected cells. It is likely that there is still much to learn about mutant huntingtin toxicity, and important insights have already come and may still come from chemical and genetic screens. Importantly, basic biological studies in HD have led to numerous potential therapeutic strategies.

The ubiquitin proteasome system in Huntington's disease and the spinocerebellar ataxias.

Huntington's disease and several of the spinocerebellar ataxias are caused by the abnormal expansion of a CAG repeat within the coding region of the disease gene. This results in the production of a mutant protein with an abnormally expanded polyglutamine tract. Although these disorders have a clear monogenic cause, each polyglutamine expansion mutation is likely to cause the dysfunction of many pathways and processes within the cell. It has been proposed that the ubiquitin proteasome system is impaired in polyglutamine expansion disorders and that this contributes to pathology. However, this is controversial with some groups demonstrating decreased proteasome activity in polyglutamine expansion disorders, some showing no change in activity and others demonstrating an increase in proteasome activity. It remains unknown whether the ubiquitin proteasome system is a feasible therapeutic target in these disorders. Here we review the conflicting results obtained from different assays performed in a variety of different systems. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).

Oculopharyngeal muscular dystrophy: potential therapies for an aggregate-associated disorder.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset, autosomal dominant disease caused by the abnormal expansion of a polyalanine tract within the coding region of poly(A) binding protein nuclear 1 (PABPN1). The resultant mutant PABPN1 forms aggregates within the nuclei of skeletal muscle fibres. The mechanism by which the polyalanine expansion mutation in PABN1 causes disease is unclear. However, the mutation is thought to confer a toxic gain-of-function on the protein. Despite controversy over the role of aggregates, it has been consistently shown that agents that reduce aggregate load in cell models of OPMD also reduce levels of cell death. Recently generated animal models of OPMD will help elucidate the mechanism of disease and allow the trial of potential therapeutics. Indeed, administration of known anti-aggregation drugs attenuated muscle weakness in an OPMD mouse model. This suggests that anti-aggregation therapies may be beneficial in OPMD.

Cystamine suppresses polyalanine toxicity in a mouse model of oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is caused by a trinucleotide repeat expansion mutation in the coding region of the gene encoding PABPN1 (polyadenylate-binding protein nuclear 1). Mutant PABPN1 with a polyalanine tract expansion forms aggregates within the nuclei of skeletal muscle fibers. There is currently no effective treatment. We have developed cell and mouse models of OPMD and have identified the aggregation of mutant PABPN1 and apoptosis as therapeutic targets. Here, we show that transglutaminase activity is increased in muscle from OPMD model mice. Elevated transglutaminase 2 expression enhances, whereas TG2 knockdown suppresses, the toxicity and aggregation of mutant PABPN1 in cells. Cystamine protects against the toxicity of mutant PABPN1 and exerts its effect via the inhibition of transglutaminase 2, as cystamine treatment is unable to further reduce the protective effect of transglutaminase 2 knockdown on mutant PABPN1 toxicity in cells. Cystamine also reduces the aggregation and toxicity of mutant PABPN1 in human cells. In a mouse model of OPMD, cystamine treatment reduced the elevated transglutaminase activity, attenuated muscle weakness, reduced aggregate load, and decreased apoptotic markers in muscle. Therefore, inhibitors of transglutaminase 2 should be considered as possible therapeutics for OPMD.

Trehalose, a novel mTOR-independent autophagy enhancer, accelerates the clearance of mutant huntingtin and alpha-synuclein.

Trehalose, a disaccharide present in many non-mammalian species, protects cells against various environmental stresses. Whereas some of the protective effects may be explained by its chemical chaperone properties, its actions are largely unknown. Here we report a novel function of trehalose as an mTOR-independent autophagy activator. Trehalose-induced autophagy enhanced the clearance of autophagy substrates like mutant huntingtin and the A30P and A53T mutants of alpha-synuclein, associated with Huntington disease (HD) and Parkinson disease (PD), respectively. Furthermore, trehalose and mTOR inhibition by rapamycin together exerted an additive effect on the clearance of these aggregate-prone proteins because of increased autophagic activity. By inducing autophagy, we showed that trehalose also protects cells against subsequent pro-apoptotic insults via the mitochondrial pathway. The dual protective properties of trehalose (as an inducer of autophagy and chemical chaperone) and the combinatorial strategy with rapamycin may be relevant to the treatment of HD and related diseases, where the mutant proteins are autophagy substrates.

Trehalose reduces aggregate formation and delays pathology in a transgenic mouse model of oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease that presents in the fifth or sixth decade with dysphagia, ptosis and proximal limb weakness. OPMD is caused by the abnormal expansion of a polyalanine tract within the coding region of polyA binding protein nuclear 1 (PABPN1). The resultant mutant PABPN1 forms aggregates within the nuclei of skeletal muscle fibres. We have previously described a transgenic mouse model of OPMD that recapitulates the human disease and develops progressive muscle weakness accompanied by the formation of aggregates in skeletal muscle nuclei. The chemical chaperone trehalose has been used effectively to alleviate symptoms in a mouse model of Huntington's disease and is thought to elicit its effect by binding and stabilizing partially folded polyglutamine proteins and inhibiting the formation of aggregates. Here, we show that trehalose reduces aggregate formation and toxicity of mutant PABPN1 in cell models. Furthermore, oral administration of trehalose attenuated muscle weakness, reduced aggregate formation and decreased the number of TUNEL-labelled nuclei in skeletal muscle in an OPMD transgenic mouse model. Thus, anti-aggregation therapy may prove effective in the treatment of human OPMD.

Deleterious and protective properties of an aggregate-prone protein with a polyalanine expansion.

Many aggregate-prone proteins, including proteins with long polyglutamine or polyalanine tracts, cause human diseases. Polyalanine proteins may also be present in the tissue of polyglutamine diseases as a result of frameshifting of the primary polyglutamine-encoding (CAG)n repeat mutation. We have generated a Drosophila model expressing green fluorescent protein tagged to 37 alanines that manifests both toxicity and inclusion formation in various tissues. Surprisingly, we show that this aggregate-prone protein with a polyalanine expansion can also protect against polyglutamine toxicity, which can be explained by induction of heat-shock response. A heat-shock response was also seen in an oculopharyngeal muscular dystrophy mouse model expressing an authentic polyalanine-expanded protein. We also show that long polyalanines can protect against a pro-apoptotic stimulus or the toxicity caused by the long polyalanines themselves. Thus, overexpression of an aggregate-prone protein without any normal functions can result in both pathogenic and protective effects in cell culture and in vivo.