Sigma-1 receptor chaperones regulate the secretion of brain-derived neurotrophic factor

Abstract

The sigma-1 receptor (Sig-1R) is a novel endoplasmic reticulum (ER) molecular chaperone that regulates protein folding and degradation. The Sig-1R activation by agonists is known to improve memory, promote cell survival, and exert an antidepressant-like action in animals. Cutamesine (SA4503), a selective Sig-1R ligand, was shown to increase BDNF in the hippocampus of rats. How exactly the intracellular chaperone Sig-1R or associated ligand causes the increase of BDNF or any other neurotrophins is unknown. We examined here whether the action of Sig-1Rs may relate to the post-translational processing and release of BDNF in neuroblastoma cell lines. We used in vitro assays and confirmed that cutamesine possesses the bona fide Sig-1R agonist property by causing the dissociation of BiP from Sig-1Rs. The C-terminus of Sig-1Rs exerted robust chaperone activity by completely blocking the aggregation of BDNF and GDNF in vitro. Chronic treatment with cutamesine in rat B104 neuroblastoma caused a time- and dose-dependent potentiation of the secretion of BDNF without affecting the mRNA level of BDNF. Cutamesine decreased the intracellular level of pro-BDNF and mature BDNF whereas increased the extracellular level of mature BDNF. The pulse-chase experiment indicated that the knockdown of Sig-1Rs decreased the secreted mature BDNF in B104 cells without affecting the synthesis of BDNF. Our findings indicate that, in contrast to clinically used antidepressants that promote the transcriptional upregulation of BDNF, the Sig-1R agonist cutamesine potentiates the post-translational processing of neurotrophins. This unique pharmacological profile may provide a novel therapeutic opportunity for the treatment of neuropsychiatric disorders.

Fig. 1. Cutamesine is a bona fide agonist of the Sig-1R

A. Cutamesine dose-dependently dissociates Sig-1Rs from BiP. CHO cells stably expressing EYFP or EYFP-tagged Sig-1R (Sig-1R-EYFP) were grown in 6-cm dishes. Cutamesine (1 μM, n=9) or (+)pentazocine (PTZ; 0.3 μM, n=7) was applied to the culture medium followed by incubation at 37 °C for 30 min. EYFP or Sig-1R-EYFP were immunoprecipitated with polyclonal GFP-antibodies in total cell lysates of CHO cells. Co-immunoprecipitated BiP was detected by monoclonal anti-BiP antibodies. B. Cutamesine time-dependently dissociates Sig-1Rs from BiP. Cutamesine (1 μM) or (+)pentazocine (0.3 μM) was applied to the culture medium for up to 60 min. N=4. C. The long-lasting effect of (+)pentazocine or cutamesine promoting the dissociation of Sig-1Rs form BiP. BiP associated with Sig-1Rs was measured as described in A. D. Inhibition of the effect of cutamesine by the Sig-1R antagonist NE100. Cutamesine (1 μM for 30 min) and NE100 (10 μM for 35 min) were applied to the culture medium. BiP associated with Sig-1Rs was measured by immunoprecipitation.

Fig. 2. Prevention of BDNF and GDNF aggregation by the purified GST-fused C-terminus of the Sig-1R polypeptide

Protein aggregates formed at 43°C were monitored every 10 sec by a light scattering assay. Citrate synthase (CS) (A), BDNF (B), or GDNF (C) at 75 nM was incubated at 43 °C in the presence or absence of purified GST-fused C-terminus (a.a. 116-223) of the Sig-1R polypeptide (75 nM). GST-fused Sig-1R 166-223 was purified as described previously (Hayashi and Su, 2007).

Fig. 3. Cutamesine potentiates the BDNF secretion from neuroblastoma cells

A. The comparison of activity to secrete BDNF between two neuroblastoma cell lines. SH-SY5Y cells (n=2) and B104 cells (n=3) were cultured for indicated days without changing the medium. Concentrations of BDNF in the culture medium were measured by ELISA. B. Cutamesine treatment time-dependently enhances the secretion of BDNF. B104 cells were treated with 1 μM of cutamesine for up to 9 days. BDNF concentrations in the culture medium were measured by ELISA. Control and cutamesine-treated samples collected on the same day were compared by two-tailed Student t test. n=5–6. *p<0.05. C. Cutamesine treatment dose-dependently enhances the secretion of BDNF. The x-axis indicates the concentration of cutamesine (μM). B104 cells were cultured with cutamesine for 7 days. BDNF concentrations in the culture medium were measured by ELISA. Control and cutamesine-treated samples were compared by one-way ANOVA followed by Dunnett’s multiple comparison test. n=6. *p < 0.05, **p < 0.01. D. Blockade of the effect of cutamesine by the Sig-1R antagonist NE100. B104 cells were cultured with 1 μM of cutamesine and/or 1 μM of NE100 for 7 days. BDNF concentrations in the culture medium were measured by ELISA. Control and cutamesine-treated samples were compared by one-way ANOVA followed by Dunnett’s multiple comparison test. n=9. **p < 0.01. E. Effect of cutamesine on the BDNF mRNA level. B104 were treated with cutamesine (1 mM, 7 days) or vehicle (control), and BDNF mRNA in extracts were measured by RT-PCR. The level of BDNF mRNA was normalized to β-actin mRNA (N=5).

Fig. 4. Effect of cutamesine on protein levels of BDNF inside and outside cells

One day after transfection with human proBDNF cDNA, B104 cells were treated with 1 μM of cutamesine overnight. Pro- and mature BDNF were immunoprecipitated in both cell lysate (IN) and culture medium (OUT), and detected by immunoblotting. The level of Sig-1Rs and ERK (the loading control) were measured in total cell lysates (bottom). Images represent data from 3 repeated experiments. Mean intensities of mature BDNF bands (% of control) are: control=100.0±7.1, cutamesine=70.6±8.8 (IN); control=100.0±6.2, cutamesine=174.7±21.4 (OUT).

Fig. 5. Effect of knockdown of Sig-1Rs on the secretion of BDNF from B104 cells

A. Effect of Sig-1R knockdown on the BDNF protein levels inside and outside cells. B104 cells were transfected with proBDNF cDNA with or without Sig-1R siRNA and incubated for 2 days. CTR: control scramble siRNA. Pro- and mature BDNF inside (IN) and outside (OUT) cells were measured by immunoprecipitation as described in Fig. 5. The level of Sig-1Rs and ERK (the loading control) were measured in total cell lysates (bottom). Images represent data from 4 repeated experiments. Mean intensities of mature BDNF bands (% of CTR) are: CTR siRNA=100.0±4.2, Sig-1R siRNA=88.78±6.7 (IN); CTR siRNA=100.0±7.54, Sig-1R siRNA=65.8±12.7 (OUT). B. Pulse-chase experiment for monitoring synthesis and secretion of BDNF. B104 cells transfected with human BDNF cDNA for 1 day were pulse-labeled with ³⁵S-Met/Cys for 60 min followed by a chasing for 180 min. CTR: control scramble siRNA. BDNF were immunoprecipitated in both cell lysates (IN) and culture medium (OUT), and visualized by direct autoradiography. ERK and Sig-1Rs were measured in total cell lysates with immunoblotting. Images represent data from 3 repeated experiments. Mean intensities of mature BDNF bands (% of CTR) 180 min after chasing are: CTR siRNA=100.0±0.1, Sig-1R siRNA=113.6±27.9 (IN); CTR siRNA=100.0±8.9, Sig-1R siRNA=53.4±8.0 (OUT).