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.

/pdf/inhibition-of-mtor-induces-autophagy-and-reduces-toxicity-of-32ebr5onpq.pdf

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

https://www.thelancet.com/pdfs/journals/lancet/PIIS0140-6736(07)60111-1.pdf

Huntington's disease.

Clathrin-mediated endocytosis is the endocytic portal into cells through which cargo is packaged into vesicles with the aid of a clathrin coat. It is fundamental to neurotransmission, signal transduction and the regulation of many plasma membrane activities and is thus essential to higher eukaryotic life. Morphological stages of vesicle formation are mirrored by progression through various protein modules (complexes). The process involves the formation of a putative FCH domain only (FCHO) initiation complex, which matures through adaptor protein 2 (AP2)-dependent cargo selection, and subsequent coat building, dynamin-mediated scission and finally auxilin- and heat shock cognate 70 (HSC70)-dependent uncoating. Some modules can be used in other pathways, and additions or substitutions confer cell specificity and adaptability.

/pdf/molecular-mechanism-and-physiological-functions-of-clathrin-2hpvjtbosd.pdf

Molecular mechanism and physiological functions of clathrin-mediated endocytosis

Isogenic strain construction and gene mapping in Candida albicans.

Large-scale genetic analysis of lethal phenotypes has elucidated the molecular underpinnings of many biological processes. Using the bacterial clustered regularly interspaced short palindromic repeats (CRISPR) system, we constructed a genome-wide single-guide RNA library to screen for genes required for proliferation and survival in a human cancer cell line. Our screen revealed the set of cell-essential genes, which was validated with an orthogonal gene-trap-based screen and comparison with yeast gene knockouts. This set is enriched for genes that encode components of fundamental pathways, are expressed at high levels, and contain few inactivating polymorphisms in the human population. We also uncovered a large group of uncharacterized genes involved in RNA processing, a number of whose products localize to the nucleolus. Last, screens in additional cell lines showed a high degree of overlap in gene essentiality but also revealed differences specific to each cell line and cancer type that reflect the developmental origin, oncogenic drivers, paralogous gene expression pattern, and chromosomal structure of each line. These results demonstrate the power of CRISPR-based screens and suggest a general strategy for identifying liabilities in cancer cells.

http://science.sciencemag.org/content/sci/350/6264/1096.full.pdf

Identification and characterization of essential genes in the human genome

The extraordinary virulence of the Ebola and Marburg filoviruses has spurred intensive research into the molecular mechanisms by which they multiply and cause disease. Carette et al. use a genome-wide genetic screen in human cells to identify factors required for entry of Ebola virus. The screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann–Pick C1 (NPC1). Cote et al. report the identification of a novel benzylpiperazine adamantane diamide-derived compound that inhibits EboV infection in cell culture, with NPC1 being the target. The unexpected role for the hereditary disease gene NPC1 in Ebola virus infection may facilitate the development of antifilovirus therapeutics. Infections by the Ebola and Marburg filoviruses cause a rapidly fatal haemorrhagic fever in humans for which no approved antivirals are available1. Filovirus entry is mediated by the viral spike glycoprotein (GP), which attaches viral particles to the cell surface, delivers them to endosomes and catalyses fusion between viral and endosomal membranes2. Additional host factors in the endosomal compartment are probably required for viral membrane fusion; however, despite considerable efforts, these critical host factors have defied molecular identification3,4,5. Here we describe a genome-wide haploid genetic screen in human cells to identify host factors required for Ebola virus entry. Our screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes6, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann–Pick C1 (NPC1)7. Cells defective for the HOPS complex or NPC1 function, including primary fibroblasts derived from human Niemann–Pick type C1 disease patients, are resistant to infection by Ebola virus and Marburg virus, but remain fully susceptible to a suite of unrelated viruses. We show that membrane fusion mediated by filovirus glycoproteins and viral escape from the vesicular compartment require the NPC1 protein, independent of its known function in cholesterol transport. Our findings uncover unique features of the entry pathway used by filoviruses and indicate potential antiviral strategies to combat these deadly agents.

/pdf/ebola-virus-entry-requires-the-cholesterol-transporter-4a0r642kc4.pdf

Ebola virus entry requires the cholesterol transporter Niemann–Pick C1

Huntington's disease (HD) is an inherited neurodegenerative disease caused by expansion of a polyglutamine tract in the huntingtin protein. Transcriptional dysregulation has been implicated in HD pathogenesis. Here, we report that huntingtin interacts with the transcriptional activator Sp1 and coactivator TAFII130. Coexpression of Sp1 and TAFII130 in cultured striatal cells from wild-type and HD transgenic mice reverses the transcriptional inhibition of the dopamine D2 receptor gene caused by mutant huntingtin, as well as protects neurons from huntingtin-induced cellular toxicity. Furthermore, soluble mutant huntingtin inhibits Sp1 binding to DNA in postmortem brain tissues of both presymptomatic and affected HD patients. Understanding these early molecular events in HD may provide an opportunity to interfere with the effects of mutant huntingtin before the development of disease symptoms.

https://science.sciencemag.org/content/sci/early/2002/05/02/science.1072613.full.pdf

Sp1 and TAFII130 Transcriptional Activity Disrupted in Early Huntington's Disease

Clathrin-coated pits assemble on a membrane and pinch off as coated vesicles. The released vesicles then rapidly lose their clathrin coats in a process mediated by the ATPase Hsc70, recruited by auxilin, a J-domain-containing cofactor. How is the uncoating process regulated? We find that during coat assembly small and variable amounts of auxilin are recruited transiently but that a much larger burst of association occurs after the peak of dynamin signal, during the transition between membrane constriction and vesicle budding. We show that the auxilin burst depends on domains of the protein likely to interact with lipid head groups. We conclude that the timing of auxilin recruitment determines the onset of uncoating. We propose that, when a diffusion barrier is established at the constricting neck of a fully formed coated pit and immediately after vesicle budding, accumulation of a specific lipid can recruit sufficient auxilin molecules to trigger uncoating.

https://www.jstor.org/stable/pdfplus/30049612.pdf

A burst of auxilin recruitment determines the onset of clathrin-coated vesicle uncoating

A single species, Candida albicans, causes half of all invasive fungal infections in humans. Unlike other fungal pathogens, this organism switches between growth as budding yeast and as pseudohyphal and hyphal filaments in host organs and in vitro. Both cell types play a role in invasive disease: while hyphal and pseudohyphal filaments penetrate host cells and tissues, yeast cells are likely to facilitate dissemination through the bloodstream and establishment of distant foci of infection. Many regulators of the yeast-to-hypha switch have emerged from intensive investigations of this morphogenetic step, but the hypha-to-yeast switch remains poorly understood. Using a forward genetic approach, a novel putative regulator involved in the hypha-to-yeast switch was identified, the C. albicans pescadillo homolog, PES1. In eukaryotes from yeast to human, pescadillo homologs are involved in cell cycle control and ribosome biogenesis, and are essential. We find a pescadillo homolog to act in fungal morphogenesis, specifically in lateral yeast growth on filamentous cells. We also find essentiality of PES1 in C. albicans to be dependent on cell type, because hyphal cells, but not yeast cells, tolerate its loss. PES1 is therefore critical for completion of the C. albicans life cycle, in which the fungus switches between filamentous and yeast growth. Consistent with these in vitro findings, PES1 is required for C. albicans virulence in an in vivo insect model of infection.

April M. Griffin

Papers

Ebola virus entry requires the cholesterol transporter Niemann–Pick C1

Sp1 and TAFII130 Transcriptional Activity Disrupted in Early Huntington's Disease

A burst of auxilin recruitment determines the onset of clathrin-coated vesicle uncoating

The Candida albicans pescadillo homolog is required for normal hypha-to-yeast morphogenesis and yeast proliferation.