GAPDH binders as potential drugs for the therapy of polyglutamine diseases: Design of a new screening assay

AbstractProteins with long polyglutamine repeats form a complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which enhances aggregation and cytotoxicity in models of Huntington disease. The aim of this study was to develop a novel assay for the screening of anti-aggregation compounds with a focus on the aggregation-promoting capacity of GAPDH. The assay includes a pure Q58 polyglutamine fragment, GAPDH, and a transglutaminase that links the two proteins. The feasibility of the new assay was verified using two GAPDH binders, hydroxynonenal and −(−)deprenyl, and the benzothiazole derivative PGL-135 which exhibits anti-aggregation effect. All three substances were shown to reduce aggregation and cytotoxicity in the cell and in the fly model of Spinocerebellar ataxia

Human APP Gene Expression Alters Active Zone Distribution and Spontaneous Neurotransmitter Release at the Drosophila Larval Neuromuscular Junction

This study provides further insight into the molecular mechanisms that control neurotransmitter release. Experiments were performed on larval neuromuscular junctions of transgenic Drosophila melanogaster lines with different levels of human amyloid precursor protein (APP) production. To express human genes in motor neurons of Drosophila, the UAS-GAL4 system was used. Human APP gene expression increased the number of synaptic boutons per neuromuscular junction. The total number of active zones, detected by Bruchpilot protein puncta distribution, remained unchanged; however, the average number of active zones per bouton decreased. These disturbances were accompanied by a decrease in frequency of miniature excitatory junction potentials without alteration in random nature of spontaneous quantal release. Similar structural and functional changes were observed with co-overexpression of human APP and β-secretase genes. In Drosophila line with expression of human amyloid-β42 peptide itself, parameters analyzed did not differ from controls, suggesting the specificity of APP effects.These results confirm the involvement of APP in synaptogenesis and provide evidence to suggest that human APP overexpression specifically disturbs the structural and functional organization of active zone and results in altered Bruchpilot distribution and lowered probability of spontaneous neurotransmitter release

The Development Of Drosophila Melanogaster under Different Duration Space Flight and Subsequent Adaptation to Earth Gravity.

In prospective human exploration of outer space, the need to preserve a species over several generations under changed gravity conditions may arise. This paper demonstrates our results in the creation of the third generation of fruit fly Drosophila melanogaster (third-stage larvae) during the 44.5-day space flight (Foton-M4 satellite (2014, Russia)), then the fourth generation on Earth and the fifth generation again in conditions of the 12-day space flight (2014, in the Russian Segment of the ISS). The species preserves fertility despite a number of changes in the level of expression and content of cytoskeletal proteins, which are the key components of the cleavage spindle and the contractile ring of cells. The results of transcriptome screening and space analysis of cytoskeletal proteins show that the exposure to weightless conditions leads to the increased transcription of metabolic genes, cuticle components and the decreased transcription of genes involved in morphogenesis, cell differentiation, cytoskeletal organization and genes associated with the plasma membrane. "Subsequent" exposure to the microgravity for 12 days resulted in an even more significant increase/decrease in the transcription of the same genes. On the contrary, the transition from the microgravity conditions to the gravity of Earth leads to the increased transcription of genes whose products are involved in the morphogenesis, cytoskeletal organization, motility of cells and transcription regulation, and to the decreased transcription of cuticle genes and proteolytic processes

Relative content of actin in the membrane (MF) and cytoplasmic (CF) fractions.

<p>(A) and (B) Total actin. The total actin content in the MF from the 3LS and 3LS+12h groups exceeded the level in the MF from the 5LSS group by 45% and 51% (p<0.05) but was not significantly different in the CF. In the 3LS, 3LS+12h, 5LSF, and 5LFF groups, the total actin contents in the MF and CF were reduced by 24% and 46% (p<0.05), 27% and 58% (p<0.05), 19% and 36% (p<0.05), and 17% and 29% (p<0.05), respectively, compared with the 5LSS group. (C) and (D) Beta-actin. The beta-actin content in the MF from the 3LS and 3LS+12h groups exceeded the level of 5LSS group by 61% and 44% (p<0.05), respectively, but remained unchanged in the CF. The beta-actin content in the CF from the 3LF and 3LF+12h groups was reduced by 27% and 29% (p<0.05) compared with the CF of the 5LSS group but remained unchanged in the MF. In the 3LF+24h group, the beta-actin content in the MF exceeded the level in the MF of the 5LSS group by 28% (p<0.05) but remained unchanged in the CF.</p

Primer sequences and product sizes.

<p>Primer sequences and product sizes.</p

Relative mRNA content of genes (qPCR data) that encode proteins of microtubule cytoskeleton.

<p>(A), (B), (D), (E) The <i>Betatub85D</i> (A), <i>Msps</i> (B), <i>Cct5</i> (D) and <i>T-cp1eta</i> (E) mRNA contents were the same in all study groups, with the exception of the 74%, 71%, 76% and 72% decreases in the 5LFF group compared with the 5LSS group (p<0.05), respectively. (C) The <i>T-cp1</i> mRNA content in the 3LS and 3LS+12h groups exceeded the level of the 5LSS group by 152% and 90% (p<0.05), respectively, and by 91% and 41% (p<0.05) in the 3LF and 3LF+12h groups, respectively. The <i>T-cp1</i> mRNA content was the same as the control level in the 3LS+24h, 3LF+24h, and 5LSF groups. The <i>T-cp1</i> mRNA content in the 5LFF group was reduced by 49% compared with the 5LSS group (p<0.05).</p

Design and cyclogram of the experiment.

<p>The experimental groups are highlighted by the circles.</p

Relative mRNA content of genes (qPCR data) that encode actin isoforms.

<p>(A) The <i>Act57B</i> mRNA content was increased by 100% in the 3LS group (p<0.05), by 111% in the 3LS+12h group(p<0.05), and by 42% in the 3LS+24h group (p<0.05) compared with the 5LSS group. In 3LF group, <i>Act57B</i> mRNA was reduced by 24% (p<0.05), it was the same in the 3LF+12h group, and it exceeded the level of the 5LSS group by 130% in the 3LF+24h group (p<0.05). In the 5LSS, 5LFS, 5LSF, and 5LFF groups, the mRNA content was the same. (B) The <i>Act87E</i> mRNA content was the same in the 3LS group, was increased by 31% in the 3LS+12h group (p<0.05), and was decreased by 31% in the 3LS+24h group (p<0.05) compared with the 5LSS group. <i>Act87E</i> mRNA was reduced by 26% (p<0.05) in the 3LF group, was the same in the 3LF+12h group, and was increased by 54% in the 3LF+24h group (p<0.05) compared with the 5LSS group. Similar to <i>Act57B</i>, the mRNA content was the same in the 5LSS, 5LF, 5LSF, and 5LFF groups. (C) The <i>Act5C</i> mRNA content in the 3LS, 3LS+12h, 3LS+24h, 3LF, 3LF+12h and 3LF+24h groups, as well as the 5LFF group was reduced by 23%, 46%, 79%, 62%, 44%, 49% and 86% compared with the 5LSS group (p<0.05).</p

Distribution of differentially expressed genes among pairwise comparisons of the study groups.

<p>(A) DEG distribution according to the biological processes. (B) DEG distribution according to the cellular compartments. (C) DEG distribution according to the molecular functions.</p

Relative mRNA content of genes (qPCR data) that encode metabolic proteins.

<p>(A) The <i>Cyt-c</i> mRNA content in 3LS, 3LS+12h, 3LS+24h, 5LSS, 5LFS and 5LSF groups was the same. In the 3LF and 3LF+12h groups, the <i>Cyt-c</i> mRNA was reduced by 48% and 46% compared with the 5LSS group (p<0.05) but was restored to the control level in the 3LF+24h group. In the 5LFF group, the <i>Cyt-c</i> mRNA content was reduced by 48% compared with the 5LSS group (p<0.05). (B) The <i>Gapdh</i> mRNA content in the 3LS, 3LS+12h, 3LS+24h, 3LF+24h, 5LSS, 5LFS, 5LSF and 5LFF groups was the same. In the 3LF and 3LF+12h groups, <i>Gapdh</i> mRNA was reduced by 27% and 29% compared with the 5LSS group (p<0.05).</p