Wavreil, F. D. M. | bioRxiv Science

bioRxiv2026

Development of a novel VHH intrabody targeting the N17 region of huntingtin exon 1 protein that prevents inclusion body formation.

Huntingtons disease (HD) is a progressive neurodegenerative disease caused by a mutation in the exon 1 of the huntingtin (HTT) gene, which leads to an extended polyglutamine (polyQ) tract in the mutant protein. As a result, mutant huntingtin (mHTT) exon 1 fragments aggregate in cells, which disrupts proper neuronal function and eventually induces cell death. The selective reduction of these toxic mHTT fragments without disturbing the wild-type full-length HTT function would be a potential therapeutic strategy to treat and prevent HD. Intracellular antibodies (intrabodies) have emerged as an attractive strategy to specifically target disease-related proteins, with VHH intrabodies being of high interest as they are much smaller than single-chain variable fragments (scFv). Here, we describe the identification and development of VHH 1 as a lead candidate intrabody targeting the first 17 amino acids of the mHTT protein, using a humanized VHH page-display library to screen against mHTT(Q46) exon 1 to identify potential binders. Next, we further optimized VHH 1 into VHH 1a to improve cytoplasmic solubility. Using immortalized mouse striatal cells that express inducible untagged mHTT exon 1 fragments, we investigated the effects of the intrabody on soluble and insoluble mHTT species via microscopy and biochemical assays. We showed that the VHH 1a intrabody reduces the levels of insoluble mHTT species, thereby effectively interrupting the aggregation process. This study highlights the potential for VHH intrabodies to specifically target mHTT fragments, enabling therapeutic strategies to delay and prevent HD pathology. HighlightsO_LIThree binders were down-selected from a phage-display library to bind HTT N17 C_LIO_LIVHH 1a intrabody is the most efficient at reducing mutant HTT exon 1 aggregation C_LIO_LIVHH 1a acts on soluble HTT exon 1 oligomers to block the transition to inclusion body C_LI

neuroscience↗

bioRxiv2024

The evolutionary modifications of a GoLoco motif in the AGS protein facilitate micromere formation in the sea urchin embryo

The evolutionary introduction of asymmetric cell division (ACD) into the developmental program facilitates the formation of a new cell type, contributing to developmental diversity and, eventually, to species diversification. The micromere of the sea urchin embryo may serve as one of those examples: An ACD at the 16-cell stage forms micromeres unique to echinoids among echinoderms. We previously reported that a polarity factor, Activator of G-protein Signaling (AGS), plays a crucial role in micromere formation. However, AGS and its associated ACD factors are present in all echinoderms and across most metazoans. This raises the question of what evolutionary modifications of AGS protein or its surrounding molecular environment contributed to the evolutionary acquisition of micromeres only in echinoids. In this study, we learned that the GoLoco motifs at the AGS C-terminus play critical roles in regulating micromere formation in sea urchin embryos. Further, other echinoderms AGS or chimeric AGS that contain the C-terminus of AGS orthologs from various organisms showed varied localization and function in micromere formation. In contrast, the sea star or the pencil urchin orthologs of other ACD factors were consistently localized at the vegetal cortex in the sea urchin embryo, suggesting that AGS may be a unique variable factor that facilitates ACD diversity among echinoderms. Consistently, sea urchin AGS appears to facilitate micromere-like cell formation and accelerate the enrichment timing of the germline factor Vasa during early embryogenesis of the pencil urchin, an ancestral type of sea urchin. Based on these observations, we propose that the molecular evolution of a single polarity factor facilitates ACD diversity while preserving the core ACD machinery among echinoderms and beyond during evolution. HighlightsO_LIEvolutionary modifications of GoLoco motifs are critical for AGS function in micromere formation in the sea urchin embryo. C_LIO_LIThe chimeric AGS, which contains the C-terminus of AGS orthologs from various organisms, suggests that human LGN, pencil urchin AGS, and Drosophila Pins compensate for the activity of sea urchin AGS. C_LIO_LISea urchin AGS (SpAGS) regulates the localization of the conserved asymmetric cell division (ACD) machinery members at the vegetal cortex. C_LIO_LISpAGS is a variable factor facilitating ACD diversity during species diversification. C_LI

developmental biology↗