Saenz, J. | bioRxiv Science

bioRxiv2023

KAT2 paralogs prevent dsRNA accumulation and interferon signaling to maintain intestinal stem cells

Histone acetyltransferases KAT2A and KAT2B are paralogs highly expressed in the intestinal epithelium, but their functions are not well understood. In this study, double knockout of murine Kat2 genes in the intestinal epithelium was lethal, resulting in robust activation of interferon signaling and interferon-associated phenotypes including the loss of intestinal stem cells. Use of pharmacological agents and sterile organoid cultures indicated a cell-intrinsic double-stranded RNA trigger for interferon signaling. Acetyl-proteomics and dsRIP-seq were employed to interrogate the mechanism behind this response, which identified mitochondria-encoded double-stranded RNA as the source of intrinsic interferon signaling. Kat2a and Kat2b therefore play an essential role in regulating mitochondrial functions as well as maintaining intestinal health. Highlights of the workO_LIKat2a and Kat2b double knockout in the murine intestinal epithelium triggers activation of the interferon signaling pathway C_LIO_LIKat2a/Kat2b knockout leads to intestinal stem cell loss and other mucosal phenotypes consistent with interferon activation C_LIO_LIHistone PTM mass spec profiling reveals the first in vivo study showing H3K9ac-specific loss with Kat2a and Kat2b double knockout, yet without correlation to interferon signaling pathway genes C_LIO_LIComprehensive proteomic analysis identifies non-histone acetyl-lysine targets of KAT2 in the mouse intestine in vivo, including mitochondrial proteins C_LIO_LIMitochondrial function is compromised upon Kat2 loss C_LIO_LIdsRIP-seq identifies double-stranded RNA from the mitochondria as a trigger for the intrinsic immune response upon Kat2 double knockout C_LI

molecular biology↗

bioRxiv2022

A novel pH-sensitive reporter reveals the cocaine-regulated trafficking of dopamine transporters in neuronal processes

The dopamine transporter (DAT) mediated DA reuptake is a major molecular mechanism for termination of dopaminergic signaling in the brain. Psychoactive substances such as cocaine act by inhibition of plasma membrane DAT function as well as by altering its expression. The precise manner and mechanism by which cocaine regulates DAT trafficking, especially at neuronal processes, are poorly understood. We have now engineered a novel pH-sensitive reporter for DAT by conjugating pHluorin to the second exofacial loop of human DAT. We show that DAT-pHluorin can be used to study DAT localization and its dynamic trafficking at neuronal processes. Using DAT-pHluorin we show that unlike neuronal soma and dendrites, which contain majority of the DATs in weakly acidic intracellular compartments, axonal DATs at both shafts and boutons are primarily (75%) localized to the plasma membrane, while varicosities contain abundant intracellular DAT within acidic intracellular structures. Using this novel reporter, we show, for the first time, that cocaine exposure leads to a brief DAT internalization followed by membrane reinsertion that lasts for days. We further show that the cocaine-induced DAT trafficking is sensitive to the activities of Synaptojanin1 phosphatase. Thus, our study using the newly engineered DAT optical reporter reveals the previously unknown dynamics and molecular regulation for cocaine-regulated DAT trafficking in neuronal processes.

neuroscience↗