Chemical interference with DSIF complex formation lowers synthesis of mutant huntingtin gene products and curtails mutant phenotypes.

Earlier work has shown that siRNA-mediated reduction of the SUPT4H or SUPT5H proteins, which interact to form the DSIF complex and facilitate transcript elongation by RNA polymerase II (RNAPII), can decrease expression of mutant gene alleles containing nucleotide repeat expansions differentially. Using luminescence and fluorescence assays, we identified chemical compounds that interfere with the SUPT4H-SUPT5H interaction and then investigated their effects on synthesis of mRNA and protein encoded by mutant alleles containing repeat expansions in the huntingtin gene (HTT), which causes the inherited neurodegenerative disorder, Huntington's Disease (HD). Here we report that such chemical interference can differentially affect expression of HTT mutant alleles, and that a prototypical chemical, 6-azauridine (6-AZA), that targets the SUPT4H-SUPT5H interaction can modify the biological response to mutant HTT gene expression. Selective and dose-dependent effects of 6-AZA on expression of HTT alleles containing nucleotide repeat expansions were seen in multiple types of cells cultured in vitro, and in a Drosophila melanogaster animal model for HD. Lowering of mutant HD protein and mitigation of the Drosophila "rough eye" phenotype associated with degeneration of photoreceptor neurons in vivo were observed. Our findings indicate that chemical interference with DSIF complex formation can decrease biochemical and phenotypic effects of nucleotide repeat expansions.

The Na+ pump Ena1 is a yeast epsin-specific cargo requiring its ubiquitylation and phosphorylation sites for internalization.

It is well known that in addition to its classical role in protein turnover, ubiquitylation is required for a variety of membrane protein sorting events. However, and despite substantial progress in the field, a long-standing question remains: given that all ubiquitin units are identical, how do different elements of the sorting machinery recognize their specific cargoes? Our results indicate that the yeast Na+ pump Ena1 is an epsin (Ent1 and Ent2 in yeast)-specific cargo and that its internalization requires K1090, which likely undergoes Art3-dependent ubiquitylation. In addition, an Ena1 serine and threonine (ST)-rich patch, proposed to be targeted for phosphorylation by casein kinases, was also required for its uptake. Interestingly, our data suggest that this phosphorylation was not needed for cargo ubiquitylation. Furthermore, epsin-mediated internalization of Ena1 required a specific spatial organization of the ST patch with respect to K1090 within the cytoplasmic tail of the pump. We hypothesize that ubiquitylation and phosphorylation of Ena1 are required for epsin-mediated internalization.

Lowe syndrome patient cells display mTOR- and RhoGTPase-dependent phenotypes alleviated by rapamycin and statins.

Lowe syndrome (LS) is an X-linked developmental disease characterized by cognitive deficiencies, bilateral congenital cataracts and renal dysfunction. Unfortunately, this disease leads to the early death of affected children often due to kidney failure. Although this condition was first described in the early 1950s and the affected gene (OCRL1) was identified in the early 1990s, its pathophysiological mechanism is not fully understood and there is no LS-specific cure available to patients. Here we report two important signaling pathways affected in LS patient cells. While RhoGTPase signaling abnormalities led to adhesion and spreading defects as compared to normal controls, PI3K/mTOR hyperactivation interfered with primary cilia assembly (scenario also observed in other ciliopathies with compromised kidney function). Importantly, we identified two FDA-approved drugs able to ameliorate these phenotypes. Specifically, statins mitigated adhesion and spreading abnormalities while rapamycin facilitated ciliogenesis in LS patient cells. However, no single drug was able to alleviate both phenotypes. Based on these and other observations, we speculate that Ocrl1 has dual, independent functions supporting proper RhoGTPase and PI3K/mTOR signaling. Therefore, this study suggest that Ocrl1-deficiency leads to signaling defects likely to require combinatorial drug treatment to suppress patient phenotypes and symptoms.

Kidney-differentiated cells derived from Lowe Syndrome patient's iPSCs show ciliogenesis defects and Six2 retention at the Golgi complex.

Lowe syndrome is an X-linked condition characterized by congenital cataracts, neurological abnormalities and kidney malfunction. This lethal disease is caused by mutations in the OCRL1 gene, which encodes for the phosphatidylinositol 5-phosphatase Ocrl1. While in the past decade we witnessed substantial progress in the identification and characterization of LS patient cellular phenotypes, many of these studies have been performed in knocked-down cell lines or patient's cells from accessible cell types such as skin fibroblasts, and not from the organs affected. This is partially due to the limited accessibility of patient cells from eyes, brain and kidneys. Here we report the preparation of induced pluripotent stem cells (iPSCs) from patient skin fibroblasts and their reprogramming into kidney cells. These reprogrammed kidney cells displayed primary cilia assembly defects similar to those described previously in cell lines. Additionally, the transcription factor and cap mesenchyme marker Six2 was substantially retained in the Golgi complex and the functional nuclear-localized fraction was reduced. These results were confirmed using different batches of differentiated cells from different iPSC colonies and by the use of the human proximal tubule kidney cell line HK2. Indeed, OCRL1 KO led to both ciliogenesis defects and Six2 retention in the Golgi complex. In agreement with Six2's role in the suppression of ductal kidney lineages, cells from this pedigree were over-represented among patient kidney-reprogrammed cells. We speculate that this diminished efficacy to produce cap mesenchyme cells would cause LS patients to have difficulties in replenishing senescent or damaged cells derived from this lineage, particularly proximal tubule cells, leading to pathological scenarios such as tubular atrophy.

The Information Content of Glutamine-Rich Sequences Define Protein Functional Characteristics.

The presence of abnormally expanded glutamine (Q) repeats within specific proteins (e.g., huntingtin) are the well-established cause of several neurogenerative diseases, including Huntington disease and spinocerebellar ataxias. However, the impact of "expanded Q" stretches on the protein function is not well-understood, mostly due to lack of knowledge about the physiological role of Q repeats and the mechanism by which these repeats achieve functional-specificity. Indeed, is intriguing that regions with such low complexity (low information content) can display exquisite functional specificity. Prompting the question: where is this information stored? Applying biochemical/structural constraints and statistical analysis of protein composition we identified Q-rich (QR) regions present in coiled coils of yeast transcription factors and endocytic proteins. Our analysis indicated the existence of non-Q amino-acids differentially enriched or excluded from QR regions in one protein group versus the other. Importantly, when the non-Q amino-acids from an endocytic protein were exchanged by the ones enriched in QR from transcription factors, the resulting protein was unable to localize to the plasma membrane and was instead found in the nucleus. These results indicate that while QR repeats can efficiently engage in binding, the non-Q amino-acids provide essential specificity information. We speculate that coupling low complexity regions with information-intensive determinants might be a strategy used in many protein systems involved in different biological processes.

Spt4 is selectively required for transcription of extended trinucleotide repeats.

Lengthy trinucleotide repeats encoding polyglutamine (polyQ) stretches characterize the variant proteins of Huntington's disease and certain other inherited neurological disorders. Using a phenotypic screen to identify events that restore functionality to polyQ proteins in S. cerevisiae, we discovered that transcription elongation factor Spt4 is required to transcribe long trinucleotide repeats located either in ORFs or nonprotein-coding regions of DNA templates. Mutation of SPT4 selectively decreased synthesis of and restored enzymatic activity to expanded polyQ protein without affecting protein lacking long-polyQ stretches. RNA-seq analysis revealed limited effects of Spt4 on overall gene expression. Inhibition of Supt4h, the mammalian ortholog of Spt4, reduced mutant huntingtin protein in neuronal cells and decreased its aggregation and toxicity while not altering overall cellular mRNA synthesis. Our findings identify a cellular mechanism for transcription through repeated trinucleotides and a potential target for countermeasures against neurological disorders attributable to expanded trinucleotide regions.

The flunitrazepam abuse prevention program at a general hospital in Taiwan: a descriptive study.

The Bureau of Controlled Drugs at Ministry of Health, Executive Yuan in Taiwan announced, on 1 April 2000, the schedules of controlled drugs with abuse potential and implemented a policy on 1 October 2000 to control them. Flunitrazepam (FM2), along with other two benzodiazepines (triazolam and brotizolam), is placed on Schedule III. The aim of the present study was to analyze the pattern of flunitrazepam prescriptions across all medical subspecialty departments at Taipei Medical University-Wan Fang Hospital (TMU-WFH), Taiwan. We analyzed 1170 prescriptions over 12 month period from 1 July 2000 to 31 May 2001. All prescription data were divided into three 4 month periods: period I was when the flunitrazepam prescription was not controlled, period II represented the time when flunitrazepam was placed on Schedule III and when physicians were required to use a special duplicated prescription form and period III was when the TMU-WFH started to set a stricter control for the prescription of flunitrazepam. The results indicated that the number of flunitrazepam prescriptions during period III had decreased significantly compared with period I (P < or = 0.05). Eventually, 45.7% of flunitrazepam-medicated patients were followed up monthly with a restriction of their flunitrazepam supply to no more than 14 days, 22.9% of patients were followed up fortnightly at clinics with a 14 day supply of flunitrazepam, 15.7% were followed up fortnightly with a 14 day restriction of flunitrazepam plus a non-flunitrazepan benzodiazepine supplement, 10.7% were referred to clinics within the Department of Psychiatry and 5% were switched from flunitrazepam to other drugs.