Fortuno, L. V. | bioRxiv Science

bioRxiv2025

Generation and characterization of a tamoxifen-inducible, Cre driver rat for transgene expression in microglia

Microglia are the resident immune cells of the central nervous system (CNS) and display diverse functions under both physiological and pathological conditions. The past decade has seen burgeoning interest in microglia function, with a variety of transgenic tools developed for specific genetic manipulation of microglia in various injury, disease, and developmental models. Although the majority of models have been developed in mice, the ability to manipulate microglia in rats provides additional advantages to studying microglial function in the brain especially related to complex behavior. Using BAC transgenesis, our lab has created a transgenic rat (Cx3cr1-CreERT2) that expresses a tamoxifen inducible Cre recombinase (CreERT2) under control of the microglial/macrophage specific fractalkine C-X3-C Motif Chemokine Receptor 1 (Cx3cr1) promoter. In mice, CreERT2 and other transgenes have been expressed in microglia using the Cx3cr1 promoter, however, this is the first demonstration in rats. Importantly, these rats exhibit similar cognitive behaviors compared to their wildtype (WT) controls. Microglial specificity of inducible Cre expression was confirmed by breeding the novel Cx3cr1-CreERT2+/- rat with a previously reported double floxed inverse open reading frame (DIO)-mCherry+/- reporter rat to show tamoxifen inducible mCherry expression that colocalizes with the microglial marker Iba1. In addition, we utilize flow cytometry to demonstrate time and Cre dependent differences in recombination of Cx3cr1+ cells in the spleen, peripheral blood, and brain at two- and eight-weeks post-tamoxifen treatment. Overall, we have created a novel transgenic rat model for researchers to employ in understanding microglial and peripheral immune cell function in rats.

neuroscience↗

bioRxiv2023

Excitotoxic glutamate levels cause the secretion of resident endoplasmic reticulum proteins

Dysregulation of synaptic glutamate levels can lead to excitotoxicity such as that observed in stroke, traumatic brain injury, and epilepsy. The role of increased intracellular calcium (Ca2+) in the development of excitotoxicity is well established. However, less is known regarding the impact of glutamate on endoplasmic reticulum (ER)-Ca2+-mediated processes such as proteostasis. To investigate this, we expressed a secreted ER Ca2+ modulated protein (SERCaMP) in primary cortical neurons to monitor exodosis, a phenomenon whereby ER calcium depletion causes the secretion of ER resident proteins that perform essential functions to the ER and the cell. Activation of glutamatergic receptors (GluRs) led to an increase in SERCaMP secretion indicating that normally ER resident proteins are being secreted in a manner consistent with ER Ca2+ depletion. Antagonism of ER Ca2+ channels attenuated the effects of glutamate and GluR agonists on SERCaMP release. We also demonstrate that endogenous proteins containing an ER retention sequence (ERS) are secreted in response to GluR activation supporting that neuronal activation by glutamate promotes ER exodosis. Ectopic expression of KDEL receptors attenuated the secretion of ERS-containing proteins caused by GluR agonists. Taken together, our data indicate that excessive GluR activation causes disruption of neuronal proteostasis by triggering the secretion of ER resident proteins through ER Ca2+ depletion and describes a new facet of excitotoxicity. SignificanceDuring excitotoxicity, the excessive activation of glutamate receptors causes elevated intracellular calcium (Ca2+) that promotes cellular dysfunction and death. While the role of cytosolic Ca2+ in excitotoxicity has been well-studied, the consequences of changes in endoplasmic reticulum (ER) Ca2+ during excitotoxicity remains unclear. The relatively high concentration of calcium in the ER is necessary for ER resident proteins to function prop out essential functions and maintain cellular proteostasis. We show here that excitotoxic conditions destabilize the ER proteome by triggering ER resident protein secretion. Stabilizing ER Ca2+ or overexpressing receptors that interact with ER resident proteins can prevent disruption of proteostasis associated with excitotoxicity. The present study provides a new link between excitotoxicity, ER Ca2+ homeostasis, and the ER proteome.

neuroscience↗