Chaurasia, S. | bioRxiv Science

bioRxiv2024

Ibudilast Protects Retinal Bipolar Cells from Excitotoxic Retinal Damage and Activates the mTOR Pathway

Ibudilast, an inhibitor of macrophage migration inhibitory factor (MIF) and phosphodiesterase (PDE), has been recently shown to have neuroprotective effects in a variety of neurologic diseases. We utilize a chick excitotoxic retinal damage model to investigate ibudilasts potential to protect retinal neurons. Using single cell RNA-sequencing (scRNA-seq), we find that MIF, putative MIF receptors CD74 and CD44, and several PDEs are upregulated in different retinal cells during damage. Intravitreal ibudilast is well tolerated in the eye and causes no evidence of toxicity. Ibudilast effectively protects neurons in the inner nuclear layer from NMDA-induced cell death, restores retinal layer thickness on spectral domain optical coherence tomography, and preserves retinal neuron function, particularly for the ON bipolar cells, as assessed by electroretinography. PDE inhibition seems essential for ibudilasts neuroprotection, as AV1013, the analogue that lacks PDE inhibitor activity, is ineffective. scRNA-seq analysis reveals upregulation of multiple signaling pathways, including mTOR, in damaged Muller glia (MG) with ibudilast treatment compared to AV1013. Components of mTORC1 and mTORC2 are upregulated in both bipolar cells and MG with ibudilast. The mTOR inhibitor rapamycin blocked accumulation of pS6 but did not reduce TUNEL positive dying cells. Additionally, through ligand-receptor interaction analysis, crosstalk between bipolar cells and MG may be important for neuroprotection. We have identified several paracrine signaling pathways that are known to contribute to cell survival and neuroprotection and might play essential roles in ibudilast function. These findings highlight ibudilasts potential to protect inner retinal neurons during damage and show promise for future clinical translation. Graphical AbstractO_ST_ABSMain PointsC_ST_ABS- Ibudilast, a MIF and PDE inhibitor, preserves the form and function of the retina, especially bipolar cells, during excitotoxic damage - Ibudilast upregulates multiple signaling pathways, including mTOR, in damaged Muller glia and bipolar cells O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/585556v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@18d9916org.highwire.dtl.DTLVardef@13db1b2org.highwire.dtl.DTLVardef@15d3d8dorg.highwire.dtl.DTLVardef@742b0c_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2021

The structural determinants of intra-protein compensatory substitutions

Compensating substitutions happen when one mutation is advantageously selected because it restores the loss of fitness induced by a previous deleterious mutation. How frequent such mutations occur in evolution and what is the structural and functional context permitting their emergence remain open questions. We built an atlas of intra-protein compensatory substitutions using a phylogenetic approach and a dataset of 1,630 bacterial protein families for which high-quality sequence alignments and experimentally derived protein structures were available. We identified more than 51,000 positions coevolving by the mean of predicted compensatory mutations. Using the evolutionary and structural properties of the analyzed positions, we demonstrate that compensatory mutations are scarce (typically only a few in the protein history) but widespread (the majority of proteins experienced at least one). Typical coevolving residues are evolving slowly, are located in the protein core outside secondary structure motifs, and are more often in contact than expected by chance, even after accounting for their evolutionary rate and solvent exposure. An exception to this general scheme are residues coevolving for charge compensation, which are evolving faster than non-coevolving sites, in contradiction with predictions from simple coevolutionary models, but similar to stem pairs in RNA. While sites with a significant pattern of coevolution by compensatory mutations are rare, the comparative analysis of hundreds of structures ultimately permits a better understanding of the link between the three-dimensional structure of a protein and its fitness landscape.

evolutionary biology↗