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.

Detection of K+ Efflux from Stimulated Cortical Neurons by an Aptamer-Modified Silicon Nanowire Field-Effect Transistor.

The concentration gradient of K+ across the cell membrane of a neuron determines its resting potential and cell excitability. During neurotransmission, the efflux of K+ from the cell via various channels will not only decrease the intracellular K+ content but also elevate the extracellular K+ concentration. However, it is not clear to what extent this change could be. In this study, we developed a multiple-parallel-connected silicon nanowire field-effect transistor (SiNW-FET) modified with K+-specific DNA-aptamers (aptamer/SiNW-FET) for the real-time detection of the K+ efflux from cultured cortical neurons. The aptamer/SiNW-FET showed an association constant of (2.18 ± 0.44) × 106 M-1 against K+ and an either less or negligible response to other alkali metal ions. The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) stimulation induced an outward current and hyperpolarized the membrane potential in a whole-cell patched neuron under a Na+/K+-free buffer. When neurons were placed atop the aptamer/SiNW-FET in a Na+/K+-free buffer, AMPA (13 µM) stimulation elevated the extracellular K+ concentration to ∼800 nM, which is greatly reduced by 6,7-dinitroquinoxaline-2,3-dione, an AMPA receptor antagonist. The EC50 of AMPA in elevating the extracellular K+ concentration was 10.3 µM. By stimulating the neurons with AMPA under a normal physiological buffer, the K+ concentration in the isolated cytosolic fraction was decreased by 75%. These experiments demonstrate that the aptamer/SiNW-FET is sensitive for detecting cations and the K+ concentrations inside and outside the neurons could be greatly changed to modulate the neuron excitability.

A recurrent WARS mutation is a novel cause of autosomal dominant distal hereditary motor neuropathy.

Distal hereditary motor neuropathy is a heterogeneous group of inherited neuropathies characterized by distal limb muscle weakness and atrophy. Although at least 15 genes have been implicated in distal hereditary motor neuropathy, the genetic causes remain elusive in many families. To identify an additional causal gene for distal hereditary motor neuropathy, we performed exome sequencing for two affected individuals and two unaffected members in a Taiwanese family with an autosomal dominant distal hereditary motor neuropathy in which mutations in common distal hereditary motor neuropathy-implicated genes had been excluded. The exome sequencing revealed a heterozygous mutation, c.770A > G (p.His257Arg), in the cytoplasmic tryptophanyl-tRNA synthetase (TrpRS) gene (WARS) that co-segregates with the neuropathy in the family. Further analyses of WARS in an additional 79 Taiwanese pedigrees with inherited neuropathies and 163 index cases from Australian, European, and Korean distal hereditary motor neuropathy families identified the same mutation in another Taiwanese distal hereditary motor neuropathy pedigree with different ancestries and one additional Belgian distal hereditary motor neuropathy family of Caucasian origin. Cell transfection studies demonstrated a dominant-negative effect of the p.His257Arg mutation on aminoacylation activity of TrpRS, which subsequently compromised protein synthesis and reduced cell viability. His257Arg TrpRS also inhibited neurite outgrowth and led to neurite degeneration in the neuronal cell lines and rat motor neurons. Further in vitro analyses showed that the WARS mutation could potentiate the angiostatic activities of TrpRS by enhancing its interaction with vascular endothelial-cadherin. Taken together, these findings establish WARS as a gene whose mutations may cause distal hereditary motor neuropathy and alter canonical and non-canonical functions of TrpRS.

Differential Releases of Dopamine and Neuropeptide Y from Histamine-Stimulated PC12 Cells Detected by an Aptamer-Modified Nanowire Transistor.

Silicon nanowire field-effect transistors modified with specific aptamers can directly detect the minute dopamine and neuropeptide Y released from cells. The binding of these molecules to the aptamers results in a conductance change of the transistor biosensor and illustrates the differential releasing mechanisms of these molecules stored in various vesicle pools.

High photosensitivity and broad spectral response of multi-layered germanium sulfide transistors.

In this paper, we report the optoelectronic properties of multi-layered GeS nanosheet (∼28 nm thick)-based field-effect transistors (called GeS-FETs). The multi-layered GeS-FETs exhibit remarkably high photoresponsivity of Rλ ∼ 206 A W(-1) under 1.5 µW cm(-2) illumination at λ = 633 nm, Vg = 0 V, and Vds = 10 V. The obtained Rλ ∼ 206 A W(-1) is excellent as compared with a GeS nanoribbon-based and the other family members of group IV-VI-based photodetectors in the layered-materials realm, such as GeSe and SnS2. The gate-dependent photoresponsivity of GeS-FETs was further measured to be able to reach Rλ ∼ 655 A W(-1) operated at Vg = -80 V. Moreover, the multi-layered GeS photodetector holds high external quantum efficiency (EQE ∼ 4.0 × 10(4)%) and specific detectivity (D* ∼ 2.35 × 10(13) Jones). The measured D* is comparable to those of the advanced commercial Si- and InGaAs-based photodiodes. The GeS photodetector also shows an excellent long-term photoswitching stability over a long period of operation (>1 h). These extraordinary properties of high photocurrent generation, broad spectral range, and long-term stability make the GeS-FET photodetector a highly qualified candidate for future optoelectronic applications.

Allele-selective suppression of mutant genes in polyglutamine diseases.

Polyglutamine (polyQ) diseases are heritable dominant neurological disorders, caused by abnormal CAG tri-nucleotide expansion in the coding sequence of affected genes. Extension of CAG repeats results in the production of aberrant gene products that are deleterious to neurons, such as transcripts with a CAG stem-loop secondary structure, and proteins containing a long stretch of polyQ residues. Thus, determining methods for the prevention or elimination of these mutant gene products from neuronal cells and translating this knowledge to clinical application are currently important goals in the fields of neurology and neurogenetics. Recently, several studies have revealed intriguing findings related to the allele-selective regulation of CAG-expanded genes, and have proposed novel designs to selectively diminish the mutant polyQ proteins. In this review, we focus on the genes, genetically engineered proteins, and oligonucleotides that show potential to modulate the expression of mutant genes. We also discuss their respective molecular functions at the levels of transcription, translation, and post-translation.

Effects on murine behavior and lifespan of selectively decreasing expression of mutant huntingtin allele by supt4h knockdown.

Production of protein containing lengthy stretches of polyglutamine encoded by multiple repeats of the trinucleotide CAG is a hallmark of Huntington's disease (HD) and of a variety of other inherited degenerative neurological and neuromuscular disorders. Earlier work has shown that interference with production of the transcription elongation protein SUPT4H results in decreased cellular capacity to transcribe mutant huntingtin gene (Htt) alleles containing long CAG expansions, but has little effect on expression of genes containing short CAG stretches. zQ175 and R6/2 are genetically engineered mouse strains whose genomes contain human HTT alleles that include greatly expanded CAG repeats and which are used as animal models for HD. Here we show that reduction of SUPT4H expression in brains of zQ175 mice by intracerebroventricular bolus injection of antisense 2'-O-methoxyethyl oligonucleotides (ASOs) directed against Supt4h, or in R6/2 mice by deletion of one copy of the Supt4h gene, results in a decrease in mRNA and protein encoded specifically by mutant Htt alleles. We further show that reduction of SUPT4H in mouse brains is associated with decreased HTT protein aggregation, and in R6/2 mice, also with prolonged lifespan and delay of the motor impairment that normally develops in these animals. Our findings support the view that targeting of SUPT4H function may be useful as a therapeutic countermeasure against HD.

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.

Fluorescence lifetime dynamics of enhanced green fluorescent protein in protein aggregates with expanded polyglutamine.

Protein aggregation is one of the characteristic steps in a number of neurodegenerative diseases eventually leading to neuronal death and thorough study of aggregation is required for the development of effective therapy. We apply fluorescence lifetime imaging for the characterization of the fluorescence dynamics of the enhanced green fluorescent protein (eGFP) in fusion with the polyQ-expanded polyglutamine stretch. At the expansion of polyQ above 39 residues, it has an inherent propensity to form amyloid-like fibrils and aggregates, and is responsible for Huntington's disease. The results of the experiments show that expression of the eGFP in fusion with the 97Q protein leads to the decrease of the eGFP fluorescence lifetime by approximately 300 ps. This phenomenon does not appear in Hsp104-deficient cells, where the aggregation in polyQ is prevented. We demonstrate that the lifetime decrease observed is related to the aggregation per se and discuss the possible role of refractive index and homo-FRET in these dynamics.