Del Boccio, P. | Search bioRxiv

bioRxiv2024

An in vivo 'turning model' reveals new RanBP9 interactions in lung macrophages.

The biological functions of the scaffold protein Ran Binding Protein 9 (RanBP9) remain elusive in macrophages or any other cell type where this protein is expressed together with its CTLH (C-terminal to LisH) complex partners. We have engineered a new mouse model, named RanBP9-TurnX, where RanBP9 fused to three copies of the HA tag (RanBP9-3xHA) can be turned into RanBP9-V5 tagged upon Cre-mediated recombination. We created this model to enable stringent biochemical studies at cell type specific level throughout the entire organism. Here, we have used this tool crossed with LysM-Cre transgenic mice to identify RanBP9 interactions in lung macrophages. We show that RanBP9-V5 and RanBP9-3xHA can be both co-immunoprecipitated with the known members of the CTLH complex from the same whole lung lysates. However, more than ninety percent of the proteins pulled down by RanBP9-V5 differ from those pulled-down by RanBP9-HA. The lung RanBP9-V5 associated proteome includes previously unknown interactions with macrophage-specific proteins as well as with players of the innate immune response, DNA damage response, metabolism, and mitochondrial function. This work provides the first lung specific RanBP9-associated interactome in physiological conditions and reveals that RanBP9 and the CTLH complex could be key regulators of macrophage bioenergetics and immune functions.

cell biology↗

bioRxiv2019

Inhibition of de novo ceramide biosynthesis affects aging phenotype in an in vitro model of neuronal senescence

Although aging is considered to be an unavoidable event, recent experimental evidence suggests that the process can be delayed, counteracted, if not completely interrupted. Aging is the primary risk factor for the onset and development of neurodegenerative conditions like Alzheimers disease, Parkinsons disease, and Amyotrophic Lateral Sclerosis. Intracellular calcium (Ca2+i) dyshomeostasis, mitochondrial dysfunction, oxidative stress, and lipid dysregulation are critical factors that contribute to senescence-related processes. Ceramides, a class of sphingolipids involved in a wide array of biological functions, are important mediators of cellular senescence, but their role in neuronal aging is still largely unexplored.\n\nIn this study, we investigated the effects of L-cycloserine (L-CS), an inhibitor of de novo ceramide biosynthesis, on the aging phenotype of cortical neurons that have been maintained in culture for 22 days, a setting employed as an in vitro model of cellular senescence. Our findings indicate that aged neurons display, when compared to control cultures, overt dysregulation of cytosolic and subcellular [Ca2+]i levels, mitochondrial dysfunction, increased reactive oxygen species generation, altered synaptic activity as well as the activation of neuronal death-related molecules. Treatment with L-CS (30 {micro}M) positively affected the senescent phenotype, a result accompanied by recovery of neuronal [Ca2+]i signaling, and reduction of mitochondrial dysfunction and reactive oxygen species generation.\n\nThe results suggest that the de novo ceramide biosynthesis may represent a critical intermediate in the molecular and functional cascade leading to neuronal senescence. Our findings also identify ceramide biosynthesis inhibitors as promising pharmacological tools to decrease age-related neuronal dysfunctions.

neuroscience↗