Developing Therapies for Neurodegenerative Disorders: Insights from Protein Aggregation and Cellular Stress Responses.

As the world's population ages, neurodegenerative disorders are poised to become the commonest cause of death. Despite this, they remain essentially untreatable. Characterized pathologically both by the aggregation of disease-specific misfolded proteins and by changes in cellular stress responses, to date, therapeutic approaches have focused almost exclusively on reducing misfolded protein load-notably amyloid beta (Aß) in Alzheimer's disease. The repeated failure of clinical trials has led to despondency over the possibility that these disorders will ever be treated. We argue that this is in fact a time for optimism: Targeting various generic stress responses is emerging as an increasingly promising means of modifying disease progression across these disorders. New treatments are approaching clinical trials, while novel means of targeting aggregates could eventually act preventively in early disease.

Mammalian Autophagy: How Does It Work?

Autophagy is a conserved intracellular pathway that delivers cytoplasmic contents to lysosomes for degradation via double-membrane autophagosomes. Autophagy substrates include organelles such as mitochondria, aggregate-prone proteins that cause neurodegeneration and various pathogens. Thus, this pathway appears to be relevant to the pathogenesis of diverse diseases, and its modulation may have therapeutic value. Here, we focus on the cell and molecular biology of mammalian autophagy and review the key proteins that regulate the process by discussing their roles and how these may be modulated by posttranslational modifications. We consider the membrane-trafficking events that impact autophagy and the questions relating to the sources of autophagosome membrane(s). Finally, we discuss data from structural studies and some of the insights these have provided.

Autophagy as a promoter of longevity: insights from model organisms.

Autophagy is a conserved process that catabolizes intracellular components to maintain energy homeostasis and to protect cells against stress. Autophagy has crucial roles during development and disease, and evidence accumulated over the past decade indicates that autophagy also has a direct role in modulating ageing. In particular, elegant studies using yeasts, worms, flies and mice have demonstrated a broad requirement for autophagy-related genes in the lifespan extension observed in a number of conserved longevity paradigms. Moreover, several new and interesting concepts relevant to autophagy and its role in modulating longevity have emerged. First, select tissues may require or benefit from autophagy activation in longevity paradigms, as tissue-specific overexpression of single autophagy genes is sufficient to extend lifespan. Second, selective types of autophagy may be crucial for longevity by specifically targeting dysfunctional cellular components and preventing their accumulation. And third, autophagy can influence organismal health and ageing even non-cell autonomously, and thus, autophagy stimulation in select tissues can have beneficial, systemic effects on lifespan. Understanding these mechanisms will be important for the development of approaches to improve human healthspan that are based on the modulation of autophagy.

Publisher Correction: Autophagy as a promoter of longevity: insights from model organisms.

In the original article a Note added in proof was not included. This has now been amended.

Lack of Neuronal IFN-ß-IFNAR Causes Lewy Body- and Parkinson's Disease-like Dementia.

Neurodegenerative diseases have been linked to inflammation, but whether altered immunomodulation plays a causative role in neurodegeneration is not clear. We show that lack of cytokine interferon-ß (IFN-ß) signaling causes spontaneous neurodegeneration in the absence of neurodegenerative disease-causing mutant proteins. Mice lacking Ifnb function exhibited motor and cognitive learning impairments with accompanying α-synuclein-containing Lewy bodies in the brain, as well as a reduction in dopaminergic neurons and defective dopamine signaling in the nigrostriatal region. Lack of IFN-ß signaling caused defects in neuronal autophagy prior to α-synucleinopathy, which was associated with accumulation of senescent mitochondria. Recombinant IFN-ß promoted neurite growth and branching, autophagy flux, and α-synuclein degradation in neurons. In addition, lentiviral IFN-ß overexpression prevented dopaminergic neuron loss in a familial Parkinson's disease model. These results indicate a protective role for IFN-ß in neuronal homeostasis and validate Ifnb mutant mice as a model for sporadic Lewy body and Parkinson's disease dementia.

Diverse autophagosome membrane sources coalesce in recycling endosomes.

Autophagic protein degradation is mediated by autophagosomes that fuse with lysosomes, where their contents are degraded. The membrane origins of autophagosomes may involve multiple sources. However, it is unclear if and where distinct membrane sources fuse during autophagosome biogenesis. Vesicles containing mATG9, the only transmembrane autophagy protein, are seen in many sites, and fusions with other autophagic compartments have not been visualized in mammalian cells. We observed that mATG9 traffics from the plasma membrane to recycling endosomes in carriers that appear to be routed differently from ATG16L1-containing vesicles, another source of autophagosome membrane. mATG9- and ATG16L1-containing vesicles traffic to recycling endosomes, where VAMP3-dependent heterotypic fusions occur. These fusions correlate with autophagosome formation, and both processes are enhanced by perturbing membrane egress from recycling endosomes. Starvation, a primordial autophagy activator, reduces membrane recycling from recycling endosomes and enhances mATG9-ATG16L1 vesicle fusion. Thus, this mechanism may fine-tune physiological autophagic responses.

The poly(A)-binding protein nuclear 1 suppresses alternative cleavage and polyadenylation sites.

Alternative cleavage and polyadenylation (APA) is emerging as an important layer of gene regulation. Factors controlling APA are largely unknown. We developed a reporter-based RNAi screen for APA and identified PABPN1 as a regulator of this process. Genome-wide analysis of APA in human cells showed that loss of PABPN1 resulted in extensive 3' untranslated region shortening. Messenger RNA transcription, stability analyses, and in vitro cleavage assays indicated enhanced usage of proximal cleavage sites (CSs) as the underlying mechanism. Using Cyclin D1 as a test case, we demonstrated that enhanced usage of proximal CSs compromises microRNA-mediated repression. Triplet-repeat expansion in PABPN1 (trePABPN1) causes autosomal-dominant oculopharyngeal muscular dystrophy (OPMD). The expression of trePABPN1 in both a mouse model of OPMD and human cells elicited broad induction of proximal CS usage, linked to binding to endogenous PABPN1 and its sequestration in nuclear aggregates. Our results elucidate a novel function for PABPN1 as a suppressor of APA.

PSMC5 insufficiency and P320R mutation impair proteasome function.

The ubiquitin-proteasome system mediates the degradation of a wide variety of proteins. Proteasome dysfunction is associated with neurodegenerative diseases and neurodevelopmental disorders in humans. Here we identified mutations in PSMC5, an AAA ATPase subunit of the proteasome 19S regulatory particle, in individuals with neurodevelopmental disorders, which were initially considered as variants of unknown significance. We have now found heterozygotes with the following mutations: P320R (6 individuals), R325W, Q160A, and one nonsense mutation at Q69. We focused on understanding the functional consequence of PSMC5 insufficiency and the P320R mutation in cells and found that both impair proteasome function and activate apoptosis. Interestingly, the P320R mutation impairs proteasome function by weakening the association between the 19S regulatory particle and the 20S core particle. Our study supports that proteasome dysfunction is the pathogenic cause of neurodevelopmental disorders in individuals carrying PSMC5 variants.

Autophagy and aging.

Genetic inhibition of autophagy induces degenerative changes in mammalian tissues that resemble those associated with aging, and normal and pathological aging are often associated with a reduced autophagic potential. Pharmacological or genetic manipulations that increase life span in model organisms often stimulate autophagy, and its inhibition compromises the longevity-promoting effects of caloric restriction, Sirtuin 1 activation, inhibition of insulin/insulin growth factor signaling, or the administration of rapamycin, resveratrol, or spermidine. Here, we discuss the probable cause and effect relationship between perturbed autophagy and aging, as well as possible molecular mechanisms that may mediate the anti-aging effects of autophagy.

Autophagosome precursor maturation requires homotypic fusion.

Autophagy is a catabolic process in which lysosomes degrade intracytoplasmic contents transported in double-membraned autophagosomes. Autophagosomes are formed by the elongation and fusion of phagophores, which can be derived from preautophagosomal structures coming from the plasma membrane and other sites like the endoplasmic reticulum and mitochondria. The mechanisms by which preautophagosomal structures elongate their membranes and mature toward fully formed autophagosomes still remain unknown. Here, we show that the maturation of the early Atg16L1 precursors requires homotypic fusion, which is essential for subsequent autophagosome formation. Atg16L1 precursor homotypic fusion depends on the SNARE protein VAMP7 together with partner SNAREs. Atg16L1 precursor homotypic fusion is a critical event in the early phases of autophagy that couples membrane acquisition and autophagosome biogenesis, as this step regulates the size of the vesicles, which in turn appears to influence their subsequent maturation into LC3-positive autophagosomes.

Autophagy in major human diseases.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

The post-translational regulation of transcription factor EB (TFEB) in health and disease.

Transcription factor EB (TFEB) is a basic helix-loop-helix leucine zipper transcription factor that acts as a master regulator of lysosomal biogenesis, lysosomal exocytosis, and macro-autophagy. TFEB contributes to a wide range of physiological functions, including mitochondrial biogenesis and innate and adaptive immunity. As such, TFEB is an essential component of cellular adaptation to stressors, ranging from nutrient deprivation to pathogenic invasion. The activity of TFEB depends on its subcellular localisation, turnover, and DNA-binding capacity, all of which are regulated at the post-translational level. Pathological states are characterised by a specific set of stressors, which elicit post-translational modifications that promote gain or loss of TFEB function in the affected tissue. In turn, the resulting increase or decrease in survival of the tissue in which TFEB is more or less active, respectively, may either benefit or harm the organism as a whole. In this way, the post-translational modifications of TFEB account for its otherwise paradoxical protective and deleterious effects on organismal fitness in diseases ranging from neurodegeneration to cancer. In this review, we describe how the intracellular environment characteristic of different diseases alters the post-translational modification profile of TFEB, enabling cellular adaptation to a particular pathological state.

Identification of MOAG-4/SERF as a regulator of age-related proteotoxicity.

Fibrillar protein aggregates are the major pathological hallmark of several incurable, age-related, neurodegenerative disorders. These aggregates typically contain aggregation-prone pathogenic proteins, such as amyloid-beta in Alzheimer's disease and alpha-synuclein in Parkinson's disease. It is, however, poorly understood how these aggregates are formed during cellular aging. Here we identify an evolutionarily highly conserved modifier of aggregation, MOAG-4, as a positive regulator of aggregate formation in C. elegans models for polyglutamine diseases. Inactivation of MOAG-4 suppresses the formation of compact polyglutamine aggregation intermediates that are required for aggregate formation. The role of MOAG-4 in driving aggregation extends to amyloid-beta and alpha-synuclein and is evolutionarily conserved in its human orthologs SERF1A and SERF2. MOAG-4/SERF appears to act independently from HSF-1-induced molecular chaperones, proteasomal degradation, and autophagy. Our results suggest that MOAG-4/SERF regulates age-related proteotoxicity through a previously unexplored pathway, which will open up new avenues for research on age-related, neurodegenerative diseases.

The complexity of biological control systems: An autophagy case study.

Autophagy and YAP1-WWTR1/TAZ signalling are tightly linked in a complex control system of forward and feedback pathways which determine different cellular outcomes in differing cell types at different time-points after perturbations. Here we extend our previous experimental and modelling approaches to consider two possibilities. First, we have performed additional mathematical modelling to explore how the autophagy-YAP1 crosstalk may be controlled by posttranslational modifications of components of the pathways. Second, since analogous contrasting results have also been reported for autophagy as a regulator of other transduction pathways engaged in tumorigenesis (Wnt/ß-catenin, TGF-ß/Smads, NF-kB or XIAP/cIAPs), we have considered if such discrepancies may be explicable through situations involving competing pathways and feedback loops in different cell types, analogous to the autophagy-YAP/TAZ situation. Since distinct posttranslational modifications dominate those pathways in distinct cells, these need to be understood to enable appropriate cell type-specific therapeutic strategies for cancers and other diseases.

VCP/p97 regulates Beclin-1-dependent autophagy initiation.

Autophagy is an essential cellular process that removes harmful protein species, and autophagy upregulation may be able to protect against neurodegeneration and various pathogens. Here, we have identified the essential protein VCP/p97 (VCP, valosin-containing protein) as a novel regulator of autophagosome biogenesis, where VCP regulates autophagy induction in two ways, both dependent on Beclin-1. Utilizing small-molecule inhibitors of VCP ATPase activity, we show that VCP stabilizes Beclin-1 levels by promoting the deubiquitinase activity of ataxin-3 towards Beclin-1. VCP also regulates the assembly and activity of the Beclin-1-containing phosphatidylinositol-3-kinase (PI3K) complex I, thus regulating the production of PI(3)P, a key signaling lipid responsible for the recruitment of downstream autophagy factors. A decreased level of VCP, or inhibition of its ATPase activity, impairs starvation-induced production of PI(3)P and limits downstream recruitment of WIPI2, ATG16L and LC3, thereby decreasing autophagosome formation, illustrating an important role for VCP in early autophagy initiation.

Lysosome positioning and mTOR activity in Lowe syndrome.

Lowe syndrome is a rare, developmental disorder caused by mutations in the phosphatase, OCRL. A study in this issue of EMBO Reports shows that OCRL is required for microtubule nucleation and that mutations in this protein lead to an inability to activate mTORC1 signaling and consequent cell proliferation in the presence of nutrients. These defects are the result of impaired microtubule-dependent lysosomal trafficking to the cell periphery and are independent of OCRL phosphatase activity.

Polyglutamine tracts regulate beclin 1-dependent autophagy.

Nine neurodegenerative diseases are caused by expanded polyglutamine (polyQ) tracts in different proteins, such as huntingtin in Huntington's disease and ataxin 3 in spinocerebellar ataxia type 3 (SCA3). Age at onset of disease decreases with increasing polyglutamine length in these proteins and the normal length also varies. PolyQ expansions drive pathogenesis in these diseases, as isolated polyQ tracts are toxic, and an N-terminal huntingtin fragment comprising exon 1, which occurs in vivo as a result of alternative splicing, causes toxicity. Although such mutant proteins are prone to aggregation, toxicity is also associated with soluble forms of the proteins. The function of the polyQ tracts in many normal cytoplasmic proteins is unclear. One such protein is the deubiquitinating enzyme ataxin 3 (refs 7, 8), which is widely expressed in the brain. Here we show that the polyQ domain enables wild-type ataxin 3 to interact with beclin 1, a key initiator of autophagy. This interaction allows the deubiquitinase activity of ataxin 3 to protect beclin 1 from proteasome-mediated degradation and thereby enables autophagy. Starvation-induced autophagy, which is regulated by beclin 1, was particularly inhibited in ataxin-3-depleted human cell lines and mouse primary neurons, and in vivo in mice. This activity of ataxin 3 and its polyQ-mediated interaction with beclin 1 was competed for by other soluble proteins with polyQ tracts in a length-dependent fashion. This competition resulted in impairment of starvation-induced autophagy in cells expressing mutant huntingtin exon 1, and this impairment was recapitulated in the brains of a mouse model of Huntington's disease and in cells from patients. A similar phenomenon was also seen with other polyQ disease proteins, including mutant ataxin 3 itself. Our data thus describe a specific function for a wild-type polyQ tract that is abrogated by a competing longer polyQ mutation in a disease protein, and identify a deleterious function of such mutations distinct from their propensity to aggregate.

PI(5)P regulates autophagosome biogenesis.

Phosphatidylinositol 3-phosphate (PI(3)P), the product of class III PI3K VPS34, recruits specific autophagic effectors, like WIPI2, during the initial steps of autophagosome biogenesis and thereby regulates canonical autophagy. However, mammalian cells can produce autophagosomes through enigmatic noncanonical VPS34-independent pathways. Here we show that PI(5)P can regulate autophagy via PI(3)P effectors and thereby identify a mechanistic explanation for forms of noncanonical autophagy. PI(5)P synthesis by the phosphatidylinositol 5-kinase PIKfyve was required for autophagosome biogenesis, and it increased levels of PI(5)P, stimulated autophagy, and reduced the levels of autophagic substrates. Inactivation of VPS34 impaired recruitment of WIPI2 and DFCP1 to autophagic precursors, reduced ATG5-ATG12 conjugation, and compromised autophagosome formation. However, these phenotypes were rescued by PI(5)P in VPS34-inactivated cells. These findings provide a mechanistic framework for alternative VPS34-independent autophagy-initiating pathways, like glucose starvation, and unravel a cytoplasmic function for PI(5)P, which previously has been linked predominantly to nuclear roles.

cGMP via PKG activates 26S proteasomes and enhances degradation of proteins, including ones that cause neurodegenerative diseases.

Because raising cAMP enhances 26S proteasome activity and the degradation of cell proteins, including the selective breakdown of misfolded proteins, we investigated whether agents that raise cGMP may also regulate protein degradation. Treating various cell lines with inhibitors of phosphodiesterase 5 or stimulators of soluble guanylyl cyclase rapidly enhanced multiple proteasome activities and cellular levels of ubiquitinated proteins by activating protein kinase G (PKG). PKG stimulated purified 26S proteasomes by phosphorylating a different 26S component than is modified by protein kinase A. In cells and cell extracts, raising cGMP also enhanced within minutes ubiquitin conjugation to cell proteins. Raising cGMP, like raising cAMP, stimulated the degradation of short-lived cell proteins, but unlike cAMP, also markedly increased proteasomal degradation of long-lived proteins (the bulk of cell proteins) without affecting lysosomal proteolysis. We also tested if raising cGMP, like cAMP, can promote the degradation of mutant proteins that cause neurodegenerative diseases. Treating zebrafish models of tauopathies or Huntington's disease with a PDE5 inhibitor reduced the levels of the mutant huntingtin and tau proteins, cell death, and the resulting morphological abnormalities. Thus, PKG rapidly activates cytosolic proteasomes, protein ubiquitination, and overall protein degradation, and agents that raise cGMP may help combat the progression of neurodegenerative diseases.

Molecular definitions of autophagy and related processes.

Over the past two decades, the molecular machinery that underlies autophagic responses has been characterized with ever increasing precision in multiple model organisms. Moreover, it has become clear that autophagy and autophagy-related processes have profound implications for human pathophysiology. However, considerable confusion persists about the use of appropriate terms to indicate specific types of autophagy and some components of the autophagy machinery, which may have detrimental effects on the expansion of the field. Driven by the overt recognition of such a potential obstacle, a panel of leading experts in the field attempts here to define several autophagy-related terms based on specific biochemical features. The ultimate objective of this collaborative exchange is to formulate recommendations that facilitate the dissemination of knowledge within and outside the field of autophagy research.