The Down syndrome critical region protein TTC3 inhibits neuronal differentiation via RhoA and Citron kinase.

The Down syndrome critical region (DSCR) on Chromosome 21 contains many genes whose duplication may lead to the major phenotypic features of Down syndrome and especially the associated mental retardation. However, the functions of DSCR genes are mostly unknown and their possible involvement in key brain developmental events still largely unexplored. In this report we show that the protein TTC3, encoded by one of the main DSCR candidate genes, physically interacts with Citron kinase (CIT-K) and Citron N (CIT-N), two effectors of the RhoA small GTPase that have previously been involved in neuronal proliferation and differentiation. More importantly, we found that TTC3 levels can strongly affect the NGF-induced differentiation of PC12 cells, by a CIT-K-dependent mechanism. Indeed, TTC3 overexpression leads to strong inhibition of neurite extension, which can be reverted by CIT-K RNAi. Conversely, TTC3 knockdown stimulates neurite extension in the same cells. Finally, we find that Rho, but not Rho kinase, is required for TTC3 differentiation-inhibiting activity. Our results suggest that the TTC3–RhoA–CIT-K pathway could be a crucial determinant of in vivo neuronal development, whose hyperactivity may result in detrimental effects on the normal differentiation program

Calpain inhibition mediates autophagy-dependent protection against polyglutamine toxicity.

Over recent years, accumulated evidence suggests that autophagy induction is protective in animal models of a number of neurodegenerative diseases. Intense research in the field has elucidated different pathways through which autophagy can be upregulated and it is important to establish how modulation of these pathways impacts upon disease progression in vivo and therefore which, if any, may have further therapeutic relevance. In addition, it is important to understand how alterations in these target pathways may affect normal physiology when constitutively modulated over a long time period, as would be required for treatment of neurodegenerative diseases. Here we evaluate the potential protective effect of downregulation of calpains. We demonstrate, in Drosophila, that calpain knockdown protects against the aggregation and toxicity of proteins, like mutant huntingtin, in an autophagy-dependent fashion. Furthermore, we demonstrate that, overexpression of the calpain inhibitor, calpastatin, increases autophagosome levels and is protective in a mouse model of Huntington's disease, improving motor signs and delaying the onset of tremors. Importantly, long-term inhibition of calpains did not result in any overt deleterious phenotypes in mice. Thus, calpain inhibition, or activation of autophagy pathways downstream of calpains, may be suitable therapeutic targets for diseases like Huntington's disease.This is the published version of the manuscript. It is available online from NPG in Cell Death and Differentiaiton here: http://www.nature.com/cdd/journal/vaop/ncurrent/full/cdd2014151a.html

Huntington's disease: a clinical review

Huntington disease (HD) is a rare neurodegenerative disorder of the central nervous system characterized by unwanted choreatic movements, behavioral and psychiatric disturbances and dementia. Prevalence in the Caucasian population is estimated at 1/10,000-1/20,000. Mean age at onset of symptoms is 30-50 years. In some cases symptoms start before the age of 20 years with behavior disturbances and learning difficulties at school (Juvenile Huntington's disease; JHD). The classic sign is chorea that gradually spreads to all muscles. All psychomotor processes become severely retarded. Patients experience psychiatric symptoms and cognitive decline. HD is an autosomal dominant inherited disease caused by an elongated CAG repeat (36 repeats or more) on the short arm of chromosome 4p16.3 in the Huntingtine gene. The longer the CAG repeat, the earlier the onset of disease. In cases of JHD the repeat often exceeds 55. Diagnosis is based on clinical symptoms and signs in an individual with a parent with proven HD, and is confirmed by DNA determination. Pre-manifest diagnosis should only be performed by multidisciplinary teams in healthy at-risk adult individuals who want to know whether they carry the mutation or not. Differential diagnoses include other causes of chorea including general internal disorders or iatrogenic disorders. Phenocopies (clinically diagnosed cases of HD without the genetic mutation) are observed. Prenatal diagnosis is possible by chorionic villus sampling or amniocentesis. Preimplantation diagnosis with in vitro fertilization is offered in several countries. There is no cure. Management should be multidisciplinary and is based on treating symptoms with a view to improving quality of life. Chorea is treated with dopamine receptor blocking or depleting agents. Medication and non-medical care for depression and aggressive behavior may be required. The progression of the disease leads to a complete dependency in daily life, which results in patients requiring full-time care, and finally death. The most common cause of death is pneumonia, followed by suicide

Regulation of miR-146a by RelA/NFkB and p53 in STHdhQ111/HdhQ111 Cells, a Cell Model of Huntington's Disease

Huntington's disease (HD) is caused by the expansion of N-terminal polymorphic poly Q stretch of the protein huntingtin (HTT). Deregulated microRNAs and loss of function of transcription factors recruited to mutant HTT aggregates could cause characteristic transcriptional deregulation associated with HD. We observed earlier that expressions of miR-125b, miR-146a and miR-150 are decreased in STHdhQ111/HdhQ111 cells, a model for HD in comparison to those of wild type STHdhQ7/HdhQ7 cells. In the present manuscript, we show by luciferase reporter assays and real time PCR that decreased miR-146a expression in STHdhQ111/HdhQ111 cells is due to decreased expression and activity of p65 subunit of NFkB (RelA/NFkB). By reporter luciferase assay, RT-PCR and western blot analysis, we also show that both miR-150 and miR-125b target p53. This partially explains the up regulation of p53 observed in HD. Elevated p53 interacts with RelA/NFkB, reduces its expression and activity and decreases the expression of miR-146a, while knocking down p53 increases RelA/NFkB and miR-146a expressions. We also demonstrate that expression of p53 is increased and levels of RelA/NFkB, miR-146a, miR-150 and miR-125b are decreased in striatum of R6/2 mice, a mouse model of HD and in cell models of HD. In a cell model, this effect could be reversed by exogenous expression of chaperone like proteins HYPK and Hsp70. We conclude that (i) miR-125b and miR-150 target p53, which in turn regulates RelA/NFkB and miR-146a expressions; (ii) reduced miR-125b and miR-150 expressions, increased p53 level and decreased RelA/NFkB and miR-146a expressions originate from mutant HTT (iii) p53 directly or indirectly regulates the expression of miR-146a. Our observation of interplay between transcription factors and miRNAs using HD cell model provides an important platform upon which further work is to be done to establish if such regulation plays any role in HD pathogenesis

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 $\textit{in vivo}$ 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 $\textit{in vivo}$ 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.We thank the Wellcome Trust (Principal Research Fellowship to D.C.R. (095317/Z/11/Z), Wellcome Trust Strategic Grant to Cambridge Institute for Medical Research (100140/Z/12/Z)), National Institute for Health Research Biomedical Research Centre at Addenbrooke’s Hospital, and Addenbrooke’s Charitable Trust and Federation of European Biochemical Societies (FEBS Long-Term Fellowship to A.A.) for funding; R. Antrobus for mass spectrometry analysis; S. Luo for truncated HTT constructs; M. Jimenez-Sanchez and C. Karabiyik for assistance with the primary mouse cell cultures; and J. Lim and Z. Ignatova for reagents

Linking Proteomic and Transcriptional Data through the Interactome and Epigenome Reveals a Map of Oncogene-induced Signaling

Cellular signal transduction generally involves cascades of post-translational protein modifications that rapidly catalyze changes in protein-DNA interactions and gene expression. High-throughput measurements are improving our ability to study each of these stages individually, but do not capture the connections between them. Here we present an approach for building a network of physical links among these data that can be used to prioritize targets for pharmacological intervention. Our method recovers the critical missing links between proteomic and transcriptional data by relating changes in chromatin accessibility to changes in expression and then uses these links to connect proteomic and transcriptome data. We applied our approach to integrate epigenomic, phosphoproteomic and transcriptome changes induced by the variant III mutation of the epidermal growth factor receptor (EGFRvIII) in a cell line model of glioblastoma multiforme (GBM). To test the relevance of the network, we used small molecules to target highly connected nodes implicated by the network model that were not detected by the experimental data in isolation and we found that a large fraction of these agents alter cell viability. Among these are two compounds, ICG-001, targeting CREB binding protein (CREBBP), and PKF118–310, targeting β-catenin (CTNNB1), which have not been tested previously for effectiveness against GBM. At the level of transcriptional regulation, we used chromatin immunoprecipitation sequencing (ChIP-Seq) to experimentally determine the genome-wide binding locations of p300, a transcriptional co-regulator highly connected in the network. Analysis of p300 target genes suggested its role in tumorigenesis. We propose that this general method, in which experimental measurements are used as constraints for building regulatory networks from the interactome while taking into account noise and missing data, should be applicable to a wide range of high-throughput datasets.National Science Foundation (U.S.) (DB1-0821391)National Institutes of Health (U.S.) (Grant U54-CA112967)National Institutes of Health (U.S.) (Grant R01-GM089903)National Institutes of Health (U.S.) (P30-ES002109