Post-stroke Intranasal (+)-Naloxone Delivery Reduces Microglial Activation and Improves Behavioral Recovery from Ischemic Injury

Peer reviewe

A target-agnostic screen identifies approved drugs to stabilize the endoplasmic reticulum-resident proteome

Endoplasmic reticulum (ER) dysregulation is associated with pathologies including neurodegenerative, muscular, and diabetic conditions. Depletion of ER calcium can lead to the loss of resident proteins in a process termed exodosis. To identify compounds that attenuate the redistribution of ER proteins under pathological conditions, we performed a quantitative high-throughput screen using the Gaussia luciferase (GLuc)-secreted ER calcium modulated protein (SERCaMP) assay, which monitors secretion of ER-resident proteins triggered by calcium depletion. We identify several clinically used drugs, including bromocriptine, and further characterize them using assays to measure effects on ER calcium, ER stress, and ER exodosis. Bromocriptine elicits protective effects in cell-based models of exodosis as well as in vivo models of stroke and diabetes. Bromocriptine analogs with reduced dopamine receptor activity retain similar efficacy in stabilizing the ER proteome, indicating a non-canonical mechanism of action. This study describes a strategic approach to identify small-molecule drugs capable of improving ER proteostasis in human disease conditions.Peer reviewe

The Function of KDEL Receptors as UPR Genes in Disease

The KDEL receptor retrieval pathway is essential for maintaining resident proteins in the endoplasmic reticulum (ER) lumen. ER resident proteins serve a variety of functions, including protein folding and maturation. Perturbations to the lumenal ER microenvironment, such as calcium depletion, can cause protein misfolding and activation of the unfolded protein response (UPR). Additionally, ER resident proteins are secreted from the cell by overwhelming the KDEL receptor retrieval pathway. Recent data show that KDEL receptors are also activated during the UPR through the IRE1/XBP1 signaling pathway as an adaptive response to cellular stress set forth to reduce the loss of ER resident proteins. This review will discuss the emerging connection between UPR activation and KDEL receptors as it pertains to ER proteostasis and disease states

Effects of Dietary Intake on Endoplasmic Reticulum Calcium Homeostasis

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 1,000-10,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for longitudinal in vivo studies. Our lab has recently developed a novel secreted ER calcium monitoring protein (GLuc-SERCaMP), to longitudinally monitor ER calcium levels in vivo by measuring small volumes of blood. Additionally, we describe a complementary tool to measure the unfolded protein response utilizing a Nano luciferase (NLuc) reporter. This work highlights the application of both reporters in vivo. Furthermore, towards the overarching goal of monitoring ER calcium homeostasis in a disease model, we describe this use of GLuc-SERCaMP in in vitro and in vivo models of diet-induced obesity. Disruption to ER calcium homeostasis has been implicated in obesity, however, the ability to directly monitor fluctuations to ER calcium has been limited with previous techniques. GLuc-SERCaMP release revealed ER calcium depletion in the presence of free fatty acid (FFA), palmitate. Consumption of a cafeteria diet or high fat pellets further demonstrated alterations to hepatic ER calcium homeostasis in rats, as evidenced by increased GLuc-SERCaMP release. Attenuation of GLuc-SERCaMP was observed during dantrolene administration. Taken together, our results further corroborate the influence of dietary intake on ER calcium homeostasis

NLuc inhibitor blocks NLuc activity using CTZ as a substrate

<p>(A) Schematic showing expected activities of NLuc and GLuc with CTZ and FMZ substrates. (B) Chemical structure of the NLuc inhibitor. (C) The NLuc Inhibitor blocks the enzymatic activity of secNLuc, but not GLuc, towards CTZ. The inhibitor was added to 8 μM CTZ substrate and light emission was measured (mean ± SD, n = 3).</p

Dual luciferase assay can be used to examine ER-calcium-dependent (GLuc-SERCaMP) and constitutive (secNLuc) secretion.

<p>(A) Schematic of AAV-constructs used to express secNLuc and GLuc-SERCaMP. Orange represents hMANF signal peptide, pink is hMANF C-terminal peptide, and purple is SV40 polyA tail. (B, C) Constitutive secretion and ER calcium-dependent secretion can be monitored in rat primary cortical neurons using the dual GLuc and secNLuc reporter approach. Neurons were transduced with AAV-secNLuc, AAV-GLuc-SERCaMP, or a combination of both viruses. Secreted levels of the reporter proteins were assessed after 8 hrs of Tg (0–300 nM) treatment using 5 μL of culture medium and 100 μL of the indicated substrate (mean ± SEM, n = 12). (D, E) Blood was collected from rats expressing AAV-secNLuc, AAV-GLuc-SERCaMP, or a combination of both, prior to and following 1 mg/kg Tg exposure. Plasma was assayed for GLuc-SERCaMP or secNLuc using CTZ plus NLuc inhibitor or FMZ, respectively. Raw luminescence values are shown (mean ± SEM, n = 4 per group, 2-way ANOVA, * p<0.05, Dunnett’s multiple comparison test vs. day 14; # p<0.05 Tukey’s multiple comparison test between viral groups). (F) Design of viral construct expressing both secNLuc and GLuc-SERCaMP from a single transgene using a self-cleaving 2A peptide. (G, H) SH-SY5Y cells transfected with pAAV-EF1α-secNLuc-2A-GLuc- SERCaMP were treated with 300 nM Tg for 8 hrs. (G) Medium was collected and assayed for secNLuc using FMZ (mean ± SD, n = 6; *p<0.01, two-tailed t-test). (H) Medium was collected and assayed for GLuc-SERCaMP activity using 8 μM CTZ + 5 μM NLuc inhibitor (mean ± SD, n = 6; *p<0.01, two-tailed t- test).</p

Longitudinal monitoring of <i>Gaussia</i> and Nano luciferase activities to concurrently assess ER calcium homeostasis and ER stress <i>in vivo</i>

<div><p>The endoplasmic reticulum (ER) is essential to many cellular processes including protein processing, lipid metabolism and calcium storage. The ability to longitudinally monitor ER homeostasis in the same organism would offer insight into progressive molecular and cellular adaptations to physiologic or pathologic states, but has been challenging. We recently described the creation of a <i>Gaussia</i> luciferase (GLuc)-based secreted ER calcium-modulated protein (SERCaMP or GLuc-SERCaMP) to longitudinally monitor ER calcium homeostasis. Here we describe a complementary tool to measure the unfolded protein response (UPR), utilizing a UPRE-driven secreted Nano luciferase (UPRE-secNLuc) to examine the activating transcription factor-6 (ATF6) and inositol-requiring enzyme 1 (IRE1) pathways of the UPR. We observed an upregulation of endogenous ATF6- and XBP1-regulated genes following pharmacologically-induced ER stress that was consistent with responsiveness of the UPRE sensor. Both GLuc and NLuc-based reporters have favorable properties for <i>in vivo</i> studies, however, they are not easily used in combination due to overlapping substrate activities. We describe a method to measure the enzymatic activities of both reporters from a single sample and validated the approach using culture medium and rat blood samples to measure GLuc-SERCaMP and UPRE-secNLuc. Measuring GLuc and NLuc activities from the same sample allows for the robust and quantitative measurement of two cellular events or cell populations from a single biological sample. This study is the first to describe the <i>in vivo</i> measurement of UPRE activation by sampling blood, using an approach that allows concurrent interrogation of two components of ER homeostasis.</p></div

UPRE-induced secNLuc can be used to monitor ER stress.

<p>(A) Schematic of AAV constructs used to express UPRE-dependent and control (minimal promoter, minP) secNLuc reporters. Tg treatment (100 nM) increases (B) 5X-UPRE-secNLuc activity (culture medium, 24 hrs, mean ± SD, n = 9) and endogenous BiP protein levels in the cell (cell lysates, 24 hrs, mean ± SD, n = 4). (C) Representative blot of BiP/actin as function of Tg treatment used for graph in (B). (D) Expression levels of UPR-responsive genes BiP, ERdj4, and ASNS (mRNA analysis, 8 hrs, mean ± SD n = 9) **p<0.01, ****p<0.0001, 1-way ANOVA, Dunnett’s multiple comparison test vehicle vs Tg). (E) TMP induces 5X-UPRE-secNLuc but has minimal effect on MinP-secNLuc (mean ± SD, n = 6 wells/transfection/TMP treatment ****p<0.0001, 2-way ANOVA). (F) Doxycycline-inducible XBP1 activates 5X-UPRE-secNLuc in HEK^DAX cells (mean ± SEM, n = 4 wells/transfection/doxycycline treatment ****p<0.0001, 2-way ANOVA, Sidak’s multiple comparison test).</p