Autophagy and polyglutamine diseases

AbstractIn polyglutamine diseases, an abnormally elongated polyglutamine tract results in protein misfolding and accumulation of intracellular aggregates. The length of the polyglutamine expansion correlates with the tendency of the mutant protein to aggregate, as well as with neuronal toxicity and earlier disease onset. Although currently there is no effective cure to prevent or slow down the progression of these neurodegenerative disorders, increasing the clearance of mutant proteins has been proposed as a potential therapeutic approach. The ubiquitin–proteasome system and autophagy are the two main degradative pathways responsible for eliminating misfolded and unnecessary proteins in the cell. We will review some of the studies that have proposed autophagy as a strategy to reduce the accumulation of polyglutamine-expanded protein aggregates and protect against mutant protein neurotoxicity. We will also discuss some of the currently known mechanisms that induce autophagy, which may be beneficial for the treatment of these and other neurodegenerative disorders

Fluorescence-Based Phenotypic Selection Allows Forward Genetic Screens in Haploid Human Cells

The isolation of haploid cell lines has recently allowed the power of forward genetic screens to be applied to mammalian cells. The interest in applying this powerful genetic approach to a mammalian system is only tempered by the limited utility of these screens, if confined to lethal phenotypes. Here we expand the scope of these approaches beyond live/dead screens and show that selection for a cell surface phenotype via fluorescence-activated cell sorting can identify the key molecules in an intracellular pathway, in this case MHC class I antigen presentation. Non-lethal haploid genetic screens are widely applicable to identify genes involved in essentially any cellular pathway

Mutation in VPS35 associated with Parkinson's disease impairs WASH complex association and inhibits autophagy

Endosomal protein sorting controls the localization of many physiologically important proteins and is linked to several neurodegenerative diseases. VPS35 is a component of the retromer complex, which mediates endosome-to-Golgi retrieval of membrane proteins such as the cation-independent mannose 6-phosphate receptor. Furthermore, retromer is also required for the endosomal recruitment of the actin nucleation promoting WASH complex. The VPS35 D620N mutation causes a rare form of autosomal-dominant Parkinson’s disease (PD). Here we show that this mutant associates poorly with the WASH complex and impairs WASH recruitment to endosomes. Autophagy is impaired in cells expressing PD-mutant VPS35 or lacking WASH. The autophagy defects can be explained, at least in part, by abnormal trafficking of the autophagy protein ATG9A. Thus, the PD-causing D620N mutation in VPS35 restricts WASH complex recruitment to endosomes, and reveals a novel role for the WASH complex in autophagosome formation

Transcriptional regulation of Annexin A2 promotes starvation-induced autophagy.

Autophagy is an important degradation pathway, which is induced after starvation, where it buffers nutrient deprivation by recycling macromolecules in organisms from yeast to man. While the classical pathway mediating this response is via mTOR inhibition, there are likely to be additional pathways that support the process. Here, we identify Annexin A2 as an autophagy modulator that regulates autophagosome formation by enabling appropriate ATG9A trafficking from endosomes to autophagosomes via actin. This process is dependent on the Annexin A2 effectors ARP2 and Spire1. Annexin A2 expression increases after starvation in cells in an mTOR-independent fashion. This is mediated via Jun N-terminal kinase activation of c-Jun, which, in turn, enhances the trans-activation of the Annexin A2 promoter. Annexin A2 knockdown abrogates starvation-induced autophagy, while its overexpression induces autophagy. Hence, c-Jun-mediated transcriptional responses support starvation-induced autophagy by regulating Annexin A2 expression levels.Openheimer Memorial TrustThis is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/ncomms904

PICALM modulates autophagy activity and tau accumulation.

Genome-wide association studies have identified several loci associated with Alzheimer's disease (AD), including proteins involved in endocytic trafficking such as PICALM/CALM (phosphatidylinositol binding clathrin assembly protein). It is unclear how these loci may contribute to AD pathology. Here we show that CALM modulates autophagy and alters clearance of tau, a protein which is a known autophagy substrate and which is causatively linked to AD, both in vitro and in vivo. Furthermore, altered CALM expression exacerbates tau-mediated toxicity in zebrafish transgenic models. CALM influences autophagy by regulating the endocytosis of SNAREs, such as VAMP2, VAMP3 and VAMP8, which have diverse effects on different stages of the autophagy pathway, from autophagosome formation to autophagosome degradation. This study suggests that the AD genetic risk factor CALM modulates autophagy, and this may affect disease in a number of ways including modulation of tau turnover.We are grateful for funding from a Wellcome Trust Principal Research Fellowship (D.C.R.), a Wellcome Trust/MRC Strategic Grant on Neurodegeneration (D.C.R., C.J.O’.K.), a Wellcome Trust Strategic Award to Cambridge Institute for Medical Research, Wellcome Trust Studentship (E.Z.), the Alzheimer’s disease Biomedical Research Unit and Addenbrooke’s Hospital, the Tau Consortium, a fellowship from University of Granada (A.L.R.), a V Foundation/Applebee’s Research Grant (D.S.W.) and NCI R01 CA 109281 (D.S.W.).This is the final published version. It is also available from Nature Publishing at http://www.nature.com/ncomms/2014/140922/ncomms5998/full/ncomms5998.html

Characterization of CHIP, a ubiquitin ligase involved in neurodegenerative disease

Neurosciencehttp://deepblue.lib.umich.edu/bitstream/2027.42/63937/1/zavodszky_eszter_2009.pd

Isolation of knockout clones deficient for components of the MHC-I antigen presentation pathway. A.

<p>Identifying knockout clones by PCR. A schematic representation of the β2m, tapasin and TAP2 knockout clones identified by PCR from 96 single cell clones from the HLA-B^low selected population. Screening a relatively low number of single cell clones is sufficient to identify knockout cells representing the relevant target genes found in a screen. <b>B.</b> The gene-trap insertions result in a loss of gene expression. The knockout clones were analyzed for HLA-A2, β2m, tapasin and TAP2 expression by RT-PCR. <b>C.</b> Knockout of genes involved in the MHC-I pathway impairs cell surface expression of MHC-I molecules. The β2m, HLA-A2, tapasin and TAP2 knockout clones were labeled for the indicated proteins and analyzed by flow cytometry.</p

A haploid genetic screen to identify genes required for cell surface expression of MHC-I molecules. A.

<p>Schematic of the screen. <b>B.</b> Selecting MHC-I^low cells by FACS. Mutagenised KBM7 cells were labeled for surface MHC-I and those cells defective for MHC-I presentation enriched by two sequential rounds of FACS. The FACS plots correspond to the stages of the screen outlined above them in A. <b>C.</b> The genetic screen identifies multiple genes known to be involved in the MHC-I antigen presentation pathway. Important genes within the MHC-I antigen presentation pathway are targeted by multiple independent retroviral integrations (red triangles).</p