Probes for MTOR

The efficiency of recombinant Adeno-associated virus vectors (AAV) transducing host cells is very low, limiting their therapeutic potential in patients. There are several cellular pathways interacting and interfering with the journey of the AAV from the cell surface to the nucleus, opening the possibility to enhance AAV transduction by modifying these interactions. In this study, we explored the results of AAV hepatic transduction when different mTOR inhibitors.- rapamycin, MLN0128, RapaLink-1 -were used in preconditioned juvenile and adult mice. We confirmed rapamycin as an AAV hepatic transduction enhancer in juvenile and adult mice; however, RapaLink-1, a stronger mTOR inhibitor and a clear hepatic autophagy inducer, had no positive effect. Moreover, MLN0128 reduced AAV hepatic transduction. Therefore, our results show a complex interaction between the mTOR pathway and AAV-mediated hepatic transduction and indicate that mTOR inhibition is not a straightforward strategy for improving AAV transduction. More studies are necessary to elucidate the molecular mechanisms involve in the positive and negative effects of mTOR inhibitors on AAV transduction efficiency.

Cerebrovascular dysfunction is a hallmark feature of Alzheimer's disease (AD). One of the greatest risk factors for AD is the apolipoprotein E4 (E4) allele. The APOE4 genotype has been shown to negatively impact vascular amyloid clearance, however, its direct influence on the molecular integrity of the cerebrovasculature compared to other APOE variants (APOE2 and APOE3) has been largely unexplored. To address this, we employed a 10-plex tandem isobaric mass tag approach in combination with an ultra-high pressure liquid chromatography MS/MS (Q-Exactive) method, to interrogate unbiased proteomic changes in cerebrovessels from AD and healthy control brains with different APOE genotypes. We first interrogated changes between healthy control cases to identify underlying genotype specific effects in cerebrovessels. EIF2 signaling, regulation of eIF4 and 70S6K signaling and mTOR signaling were the top significantly altered pathways in E4/E4 compared to E3/E3 cases. Oxidative phosphorylation, EIF2 signaling and mitochondrial dysfunction were the top significant pathways in E2E2 vs E3/E3cases. We also identified AD-dependent changes and their interactions with APOE genotype and found the highest number of significant proteins from this interaction was observed in the E3/E4 (192) and E4/E4 (189) cases. As above, EIF2, mTOR signaling and eIF4 and 70S6K signaling were the top three significantly altered pathways in E4 allele carriers (i.e. E3/E4 and E4/E4 genotypes). Of all the cerebrovascular cell-type specific markers identified in our proteomic analyses, endothelial cell, astrocyte, and smooth muscle cell specific protein markers were significantly altered in E3/E4 cases, while endothelial cells and astrocyte specific protein markers were altered in E4/E4 cases. These proteomic changes provide novel insights into the longstanding link between APOE4 and cerebrovascular dysfunction, implicating a role for impaired autophagy, ER stress, and mitochondrial bioenergetics. These APOE4 dependent changes we identified could provide novel cerebrovascular targets for developing disease modifying strategies to mitigate the effects of APOE4 genotype on AD pathogenesis.

Zebrafish robustly regenerate fins, including their characteristic bony ray skeleton. Amputation activates intra-ray fibroblasts and dedifferentiates osteoblasts that migrate under a wound epidermis to establish an organized blastema. Coordinated proliferation and re-differentiation across lineages then sustains progressive outgrowth. We generate a single cell transcriptome dataset to characterize regenerative outgrowth and explore coordinated cell behaviors. We computationally identify sub-clusters representing most regenerative fin cell lineages, and define markers of osteoblasts, intra- and inter-ray fibroblasts and growth-promoting distal blastema cells. A pseudotemporal trajectory and in vivo photoconvertible lineage tracing indicate distal blastemal mesenchyme restores both intra- and inter-ray fibroblasts. Gene expression profiles across this trajectory suggest elevated protein production in the blastemal mesenchyme state. O-propargyl-puromycin incorporation and small molecule inhibition identify insulin growth factor receptor (IGFR)/mechanistic target of rapamycin kinase (mTOR)-dependent elevated bulk translation in blastemal mesenchyme and differentiating osteoblasts. We test candidate cooperating differentiation factors identified from the osteoblast trajectory, finding IGFR/mTOR signaling expedites glucocorticoid-promoted osteoblast differentiation in vitro. Concordantly, mTOR inhibition slows but does not prevent fin regenerative outgrowth in vivo. IGFR/mTOR may elevate translation in both fibroblast- and osteoblast-lineage cells during the outgrowth phase as a tempo-coordinating rheostat.