Fig 1.
NLuc inhibitor blocks NLuc activity using CTZ as a substrate
(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).
Fig 2.
Dual luciferase assay can be used to examine ER-calcium-dependent (GLuc-SERCaMP) and constitutive (secNLuc) secretion.
(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).
Fig 3.
UPRE-induced secNLuc can be used to monitor ER stress.
(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 HEKDAX cells (mean ± SEM, n = 4 wells/transfection/doxycycline treatment ****p<0.0001, 2-way ANOVA, Sidak’s multiple comparison test).
Fig 4.
Dual luciferase assay to examine ER calcium dysregulation and UPRE-induced secNLuc in vitro and in vivo.
(A) Concurrent measurement of ER calcium homeostasis and ER stress by measuring extracellular luciferase in medium. SH-SY5Y-GLuc-SERCaMP stable cells were transfected with 5X-UPRE-secNLuc and treated with Tg (0–300 nM)) for 8 hrs (mean ± SEM, n = 4 wells/transfection/ treatment, *p<0.05, **p<0.01, ****p<0.0001, 2-way ANOVA, Dunnett’s multiple comparison test vs vehicle). (B) Effect of Tg on plasma levels of GLuc and secNLuc. Rats were intrahepatically injected with AAV-GLuc-SERCaMP and AAV-5X-UPRE-secNLuc. Luminescence was normalized to levels measured immediately prior to Tg injection (mean ± SEM, n = 4 rats, 1-way ANOVA, Dunnett’s multiple comparison test vs. Tg administration (days 14 and 28), *p<0.05, **p<0.01). (C) RT-qPCR analysis of UPR targets, BiP, Erjd4, and ASNS, from rat liver tissue 24 hrs post-Tg (1 mg/kg) or vehicle administration. Data expressed as fold change (2-ΔΔCt) relative to vehicle-treated rats (mean ± upper and lower limits (2-ΔΔCt±SD), n = 3 rats (Tg), n = 4 rats (vehicle), unpaired t-test (Tg vs vehicle) transformed from ΔCt values, *p<0.05, **p<0.01).