Clearance Of Aggregate-prone Proteins Research Articles

DNA Methyltransferase 1 (DNMT1) Acts on Neurodegeneration by Modulating Proteostasis-Relevant Intracellular Processes.

The limited regenerative capacity of neurons requires a tightly orchestrated cell death and survival regulation in the context of longevity, as well as age-associated and neurodegenerative diseases. Subordinate to genetic networks, epigenetic mechanisms, such as DNA methylation and histone modifications, are involved in the regulation of neuronal functionality and emerge as key contributors to the pathophysiology of neurodegenerative diseases. DNA methylation, a dynamic and reversible process, is executed by DNA methyltransferases (DNMTs). DNMT1 was previously shown to act on neuronal survival in the aged brain, whereby a DNMT1-dependent modulation of processes relevant for protein degradation was proposed as an underlying mechanism. Properly operating proteostasis networks are a mandatory prerequisite for the functionality and long-term survival of neurons. Malfunctioning proteostasis is found, inter alia, in neurodegenerative contexts. Here, we investigated whether DNMT1 affects critical aspects of the proteostasis network by a combination of expression studies, live cell imaging, and protein biochemical analyses. We found that DNMT1 negatively impacts retrograde trafficking and autophagy, with both being involved in the clearance of aggregation-prone proteins by the aggresome–autophagy pathway. In line with this, we found that the transport of GFP-labeled mutant huntingtin (HTT) to perinuclear regions, proposed to be cytoprotective, also depends on DNMT1. Depletion of Dnmt1 accelerated perinuclear HTT aggregation and improved the survival of cells transfected with mutant HTT. This suggests that mutant HTT-induced cytotoxicity is at least in part mediated by DNMT1-dependent modulation of degradative pathways.

Detection of Autophagy in Caenorhabditis elegans.

Autophagy is a dynamic and catabolic process that results in the breakdown and recycling of cellular components through the autophagosomal-lysosomal pathway. Many autophagy genes identified in yeasts and mammals have orthologs in the nematode Caenorhabditis elegans. In recent years, gene inactivation by RNA interference (RNAi) and chromosomal mutations has been useful to probe the functions of autophagy in C. elegans, and a role for autophagy has been shown to contribute to multiple processes, such as the adaptation to stress, longevity, cell death, cell growth control, clearance of aggregation-prone proteins, degradation of P granules during embryogenesis, and apoptotic cell clearance. Here, we discuss some of these roles and describe methods that can be used to study autophagy in C. elegans. Specifically, we summarize how to visualize autophagy in embryos, larva, or adults, how to detect the lipidation of the ubiquitin-like modifier LGG-1 by western blot, and how to inactivate autophagy genes by RNAi.

Cytoplasmic dynein: a key player in neurodegenerative and neurodevelopmental diseases

Cytoplasmic dynein is the most important molecular motor driving the movement of a wide range of cargoes towards the minus ends of microtubules. As a molecular motor protein, dynein performs a variety of basic cellular functions including organelle transport and centrosome assembly. In the nervous system, dynein has been demonstrated to be responsible for axonal retrograde transport. Many studies have revealed direct or indirect evidence of dynein in neurodegenerative diseases such as amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, Alzheimer's disease, Parkinson's disease and Huntington's disease. Among them, a number of mutant proteins involved in various neurodegenerative diseases interact with dynein. Axonal transport disruption is presented as a common feature occurring in neurodegenerative diseases. Dynein heavy chain mutant mice also show features of neurodegenerative diseases. Moreover, defects of dynein-dependent processes such as autophagy or clearance of aggregation-prone proteins are found in most of these diseases. Lines of evidence have also shown that dynein is associated with neurodevelopmental diseases. In this review, we focus on dynein involvement in different neurological diseases and discuss potential underlying mechanisms.

Implication of Autophagy in Parkinson’s Disease

Autophagy is an intracellular catabolic mechanism mediated by lysosomes, which is responsible for most of the degradation and recycling of cytoplasmic components and intracellular dysfunctional or damaged organelles. Increasing evidences suggest that autophagic deregulation causes accumulation of abnormal proteins or damaged organelles, which is a characteristic of chronic neurodegenerative conditions, such as Parkinson's disease (PD), a multifactorial disorder, which is neuropathologically characterized by age-dependent neurodegeneration of dopaminergic neurons in the midbrain. Indeed, promoting the clearance of aggregate-prone proteins via pharmacological induction of autophagy has proved to be an useful mechanism for protecting cells against the toxic effects of these proteins in the context of neurodegenerative diseases and protecting neurons from apoptosis. This special issue is composed of seven excellent reviews addressing the analysis of different models of Parkinsonism in which autophagy plays a key role.

The p53-induced Gene Ei24 Is an Essential Component of the Basal Autophagy Pathway

Ei24 is a DNA damage response gene involved in growth suppression and apoptosis. The physiological function of Ei24, however, is poorly understood. Here we generated conditional knock-out mice of Ei24 and demonstrated that EI24 is an essential component of the basal autophagy pathway. Mice with neural-specific Ei24 deficiency develop age-dependent neurological abnormalities caused by massive axon degeneration and extensive neuron loss in brain and spinal cord. Notably, ablation of Ei24 leads to vacuolated oligodendroglial cells and demyelination of axons. Liver-specific depletion of Ei24 causes severe hepatomegaly with hepatocyte hypertrophy. Ei24 deficiency impairs autophagic flux, leading to accumulation of LC3, p62 aggregates, and ubiquitin-positive inclusions. Our study indicates that Ei24 is an essential autophagy gene and plays an important role in clearance of aggregate-prone proteins in neurons and hepatocytes.

428 Heparanase powers a chronic inflammatory circuit that promotes colitis-associated tumourigenesis

Autophagy refers to bulk degradation processes responsible for the turnover of long-lived proteins, disposal of damaged organelles, and clearance of aggregate prone proteins. Aberrant autophagy causes the formation of cytoplasmic inclusion bodies, leading to liver injury and neurodegeneration. However, details of abnormalities related to impaired autophagy are largely unknown. The efficiency of the autophagy pathway relies on cargo receptors to identify the ubiquitinated targets destined for the degradation pathway. For instance, p62 promotes the formation of protein aggregates and their association with the autophagosome. Recent studies showed that murine p62 contains a highly conserved LC3 recognition sequence (LRS). Structural analysis of the LC3–LRS complex revealed an interaction between Trp340 and Leu343 of p62 and two hydrophobic pockets (hp1 and hp2) on the ubiquitin fold of LC3. The LRS motif of NBR1, autophagy receptor, presents differences to this classical LRS motif with a tyrosine residue and an isoleucine residue substituting Trp and Leu, respectively. NMR studies of NBR1–LRS complexed with GABARAPL, another Atg8 homologue, indicated that the presence of tryptophan residue in the LRS motif increases the binding affinity, but other substitutions have little effect on the binding affinity due to enthalpy–entropy compensation. The aforementioned results indicate that each autophagic receptor has a unique interaction form. Most recently, it has been demonstrated that the selectivity of the autophagy receptor NDP52 for LC3C is crucial for innate immunity. Other than those listed above, many autophagy receptors and Atg8 homologue binding proteins are reported.In vivo experiments showed that cells expressing p62 mutants lacking LC3 binding ability accumulate ubiquitin-positive inclusion bodies, instead of autophagosomes, as in hepatitis and neurodegenerative diseases. These data demonstrate that cellular levels of p62 are tightly regulated by autophagy through direct interaction with LC3, and that selective turnover of p62 via autophagy prevents inclusion body formation.

MPP+ impairs autophagic clearance of alpha-synuclein by impairing the activity of dynein

Increasing evidence suggests that dynein has an important role in the clearance of misfolded proteins by autophagy. Here we show that treatment of cells with 1-methyl-4-phenylpyridinium (MPP) cause alpha-synuclein overexpression and aggregation, leading to the accumulation of autophagic vacuoles and the recruitment of LC3-II to these vacuoles in the cytoplasm. After MPP treatment, dynein expression decreased and was mainly aggregated at the periphery of cytoplasm and lost its colocalization with alpha-synuclein and lamp1, indicating that dynein lost its function in the aggresome formation and failed to return autophagosome and lysosomes to the center of the cell for degradation. We consider that dynein plays an important role in the autophagic clearance of aggregate-prone proteins.

Lithium Decreases IP 3 to Promote Autophagy

Lithium is widely known for its application in the treatment of bipolar and unipolar mood disorders. Sarkar et al. provide evidence that lithium may also be effective in the treatment of neurodegenerative disorders arising from toxicity of the accumulation of aggregate-prone proteins, such as mutant huntingtin and mutant α-synuclein, which are implicated in Huntington's disease and certain forms of Parkinson's disease. Stimulation of autophagy may be a mechanism to prevent the accumulation of these aggegrate-prone proteins, and Sarkar et al. show that lithium promotes autophagy, detected as an increase in the formation of autophagic vesicles and a decrease in the abundance of known autophagic substrates. Furthermore, lithium decreased the accumulation of mutant α-synuclein or huntingtin aggregates and the toxicity (cell death) of these proteins in inducible PC12 cell lines or COS-7 cell lines, an effect that was blocked by the autophagic inhibitor 3-methyladenine (3-MA). Lithium is a nonspecific inhibitor of several enzymes. The effect on autophagy appeared to be mediated by the inhibition of inositol monophosphatase (IMPase), because autophagy and clearance of mutant huntingtin and α-synuclein was also stimulated by inhibition of IMPase with a specific pharmacologic agent (L-690,330), whereas selective inhibition of glycogen synthase kinase-3β (GSK-3β, another lithium target) did not promote clearance of the aggregate-prone proteins. Both L-690,330 and lithium decreased inositol-1,4,5-trisphosphate (IP 3 ) concentrations. That lithium exerted its effects on autophagy through decreased IP 3 concentrations was validated by experiments in which IP 3 concentrations were increased before lithium treatment by addition of myo-inositol or prolyl endopeptidase inhibitor 2 (PEI). Increased IP 3 concentration promoted the accumulation of mutant huntingtin and cell death and blocked the effects of lithium to reduce these phenomena. Autophagy is also stimulated by inhibition of mTOR (mammalian target of rapamycin); however, lithium appeared to act independently from the pathway regulated by mTOR, because inhibition of mTOR with rapamycin combined with lithium had an additive effect on clearance of the aggregate-prone proteins and cell survival. The ability of lithium to stimulate autophagy and clearance of aggregate-prone proteins was also observed for other mood-stabilizing drugs that promote inositol depletion. Thus, a pathway involving IP 3 and inositol appears to be one mechanism regulating autophagy that may be exploited for therapeutic benefit in certain neurodegenerative diseases. S. Sarkar, A. Floto, Z. Berger, S. Imarisio, A. Cordenier, M. Pasco, L. J. Cook, D. C. Rubinsztein, Lithium induces autophage by inhibiting inositol monophosphate. J. Cell Biol. 170 , 1101-1111 (2005). [Abstract] [Full Text]

The roles of the ubiquitin-proteasome and autophagy–lysosome pathways in Huntington's disease and related conditions

Huntington's disease (HD) is one of nine known conditions caused by a CAG trinucleotide repeat expansion that is translated into a polyglutamine tract in the disease protein. Genetic and transgenic data suggest that the primary effect of these CAG/polyglutamine expansions is to confer a toxic gain-of-function on the mutant protein. The cellular pathology of all known polyglutamine diseases is characterised by the accumulation of the relevant mutant protein along with other proteins in aggregates (also known as inclusions) in neurons in the disease brain. Such aggregates are seen in neuronal nuclei and processes in HD. The role of aggregates in these diseases has been a subject of vigorous debate. However, irrespective of the nature(s) of the toxic species of huntingtin, it is desirable for cells to be able to control the levels of these toxic proteins and restrict their accumulation. In this paper we will review the ubiquitin-proteasome and autophagy–lysosome routes for protein degradation and how they may be involved in the clearance of aggregate-prone proteins, exemplified by mutant huntingtin exon 1. We will also consider the possibilities that these protein degradation pathways may be perturbed as a consequence of the mutations that form the aggregate-prone proteins.