Innately activated TLR4 signal in the nucleus accumbens is sustained by CRF amplification loop and regulates impulsivity

Abstract

Cognitive impulsivity is a heritable trait believed to represent the behavior that defines the volition to initiate alcohol drinking. We have previously shown that a neuronal Toll-like receptor 4 (TLR4) signal located in the central amygdala (CeA) and ventral tegmental area (VTA) controls the initiation of binge drinking in alcohol-preferring P rats, and TLR4 expression is upregulated by alcohol-induced corticotropin-releasing factor (CRF) at these sites. However, the function of the TLR4 signal in the nucleus accumbens shell (NAc-shell), a site implicated in the control of reward, drug-seeking behavior and impulsivity and the contribution of other signal-associated genes, are still poorly understood. Here we report that P rats have an innately activated TLR4 signal in NAc-shell neurons that co-express the α2 GABA_A receptor subunit and CRF prior to alcohol exposure. This signal is not present in non-alcohol drinking NP rats. The TLR4 signal is sustained by a CRF amplification loop, which includes TLR4-mediated CRF upregulation through PKA/CREB activation and CRF-mediated TLR4 upregulation through the CRF type 1 receptor (CRFR1) and the MAPK/ERK pathway. NAc-shell Infusion of a neurotropic, non-replicating herpes simplex virus vector for TLR4-specific small interfering RNA (pHSVsiTLR4) inhibits TLR4 expression and cognitive impulsivity, implicating the CRF-amplified TLR4 signal in impulsivity regulation.

Keywords: Activated TLR4 signal; CRF; GABA(A) α2; HSV siRNA vectors; Impulsivity; PKA/CREB.

Fig. 1. P rats have elevated levels of TLR4 and GABA_A α2 and increased numbers of co-expressing cells in the NAc-shell

(A,B) Protein extracts of micropunches collected from the NAc-shell from NP (n=4) and P (n=7) rats were immunoblotted with TLR4 (A) or α2 (B) antibodies, stripped and reprobed with antibody to GAPDH used as gel loading control. The results were quantitated by densitometric scanning and expressed as densitometric units normalized to GAPDH ± SEM. Each lane represents a distinct animal. The TLR4 and α2 levels are elevated in P compared to NP rats. (*p≤0.05 by ANOVA). (C,D) Confocal microscopy and Z-stack imaging of double immunofluorescent staining of NAc-shell sections (n=5/group) with TLR4 (red) and GABA_A α2 (green) antibodies. Merged images reveal numerous α2+ neurons expressing TLR4 in the NAc-shell from P rats (C), but the numbers of TLR4+/α2+ cells are significantly lower in the NAc-shell from NP rats (D). Scale bars: 15 µm. The % co-staining cells are summarized in Table 1.

Fig. 2. The levels of CRF and numbers of TLR4+/CRF+ cells are elevated in the NAc-shell from P rats

(A) Protein extracts of the micropunches examined in Fig. 1 were immunoblotted with CRF monoclonal antibody (Santa Cruz Biotechnology, Cat. # sc-293187), stripped and re-probed with antibody to GAPDH and the results are expressed as densitometric units normalized to GAPDH ± SEM. As also shown in SI, Fig. S2A, the only detected band is ~20 kDa and its levels are significantly (*p≤0.05 by ANOVA) elevated in P as compared to NP rats. Similar results were obtained for the polyclonal antibody (Bioss Antibodies, Cat. # bs-0246R). B, C) Confocal microscopy and Z-stack imaging of double immunofluorescent staining of NAc-shell sections (n=5/group) with TLR4 (red) and CRF (Bioss Antibodies, Cat. # bs-0246R) (green) antibodies. Merged images reveal numerous CRF+ neurons expressing TLR4 in the NAc-shell from P rats (B), but the numbers of TLR4+/CRF+ cells are significantly lower in the NAc-shell from NP rats (C). Scale bars: 20 µm. The % co-staining cells are summarized in Table 1.

Fig. 3. CRF amplification loop sustains activated TLR4 signal; α2 contribution

(A) Protein extracts from mock- or TLR4-transfected SK-N-SH cells (n=5 each) were immunoblotted with pCREB antibody, and the blots were sequentially stripped and immunoblotted with antibodies to pPKA, pERK1/2, TLR4 and β-Actin used as gel loading control. The results were quantitated by densitometric scanning and expressed as densitometric units normalized to β-Actin ± SEM. The levels of pCREB, pPKA and TLR4, but not pERK1/2, are significantly higher in the TLR4- than mock-transfected SK-N-SH cells (**p <0.01 by ANOVA). (B) Protein extracts from mock- or TLR4-transfected SK-N-SH cells treated or not (n=5 each) with the PKA inhibitor H89 (10 µM) were immunoblotted with antibody to pCREB and the blots were sequentially stripped and immunoblotted with antibodies to CRF (Bioss Antibodies, Cat. # bs-0246R), CRFR1 and β-Actin. The results are expressed as densitometric units normalized to β-Actin ± SEM. The levels of pCREB, CRF and CRFR1 are significantly higher in the TLR4- than mock-transfected cells, and upregulation is inhibited by H89 (*p <0.05; **p<0.01; *** p<0.001 by ANOVA). (C) Protein extracts from mock- or α2-transfected Neuro2a cells in the presence or absence of amplicons that deliver TLR4 (siTLR4) or scrambled (siNC) siRNA (n=5 each) were immunoblotted with antibody to CRF (Bioss Antibodies, Cat. # bs-0246R), the blots were sequentially stripped and immunoblotted with antibodies to TLR4 and β-Actin. Results are expressed as densitometric units normalized to β-Actin ± SEM. The levels of CRF and TLR4 are significantly higher in the α2- than mock-transfected cells and upregulation is inhibited by siTLR4, but not siNC (*p≤0.05 by ANOVA). (D) Neuro2a cells mock- or α2-transfected in the presence or absence of amplicons for TLR4 siRNA (siTLR4), α2 siRNA [siLA2 (Liu et al., 2011)] or scrambled siRNA (siNC) were stained with pCREB antibody (red) and examined for nuclear localization (activation). DAPI (blue) was used as nuclear counterstain. pCREB nuclear staining was minimal in the mock-transfected cells (CTL) and it was almost entirely cytoplasmic. α2 transfection significantly increased the % cells with nuclear pCREB staining and nuclear localization was inhibited by siTLR4 and siLA2, but not siNC (*** p≤0.001 by ANOVA).

Fig. 4. CRF regulates TLR4 expression through CRFR1 and MAPK/ERK activation

(A) Protein extracts from mock- or CRF-transfected SK-N-SH cells (n=5 each) were immunoblotted with antibodies to CRF (Bioss Antibodies, Cat. # bs-0246R) or β-Actin and the results are expressed as densitometric units normalized to β-Actin ± SEM. The levels of CRF are significantly higher in the CRF- than mock-transfected cells (****P<0.0001 CTL vs. CRF); (B) Protein extracts from mock- or CRF-transfected SK-N-SH cells untreated or treated with the CRFR1 antagonist antalarmin (15nM) or the ERK1/2 specific inhibitor U0126 (20 µM) (n=5 each) were immunoblotted with antibodies to TLR4 or β-Actin and the results are expressed as densitometric units normalized to β-Actin ± SEM. The levels of TLR4 are significantly higher in the CRF- than mock-transfected cells and this increase is reduced by treatment with antalarmin (57.2±5.1%) or U0126 (42.8±3.8%). The levels of TLR4 in CRF-transfected cells treated with both inhibitors are virtually identical to those in the mock-transfected cells (100% inhibition) (*p<0.05; **p <0.01 by ANOVA). (C) Protein extracts from mock- or CRF-transfected SK-N-SH cells treated or not (n=5 each) with U0126 (20 µM) were immunoblotted with pERK1/2 antibody, and the blots were sequentially stripped and immunoblotted with antibodies to pPKA and β-Actin. Results are expressed as densitometric units normalized to β-Actin ± SEM. The pERK1/2 and pPKA levels are significantly higher in the CRF- than mock-transfected cells, and this increase is reduced by U0126 treatment (**p<0.01 by ANOVA).

Fig. 5. TLR4 is innately activated in the NAc-shell from P rats

Confocal microscopy and Z-stack imaging of double immunofluorescent staining of NAc-shell sections from P and NP rats (n=5/group) with TLR4 (red) and pCREB (green) antibodies or CRF (red) (Santa Cruz Biotechnology, Cat. # sc-293187) and pCREB (green) antibodies. Most of the TLR4+ and CRF+ cells in the NAc-shell from the P rats have co-localized nuclear staining with pCREB antibody. The numbers of TLR4+ and CRF+ cells with pCREB nuclear staining are significantly lower in the NAc-shell from NP rats (***p<0.001 by ANOVA). Scale bars: 20 µm. The % co-staining cells are summarized in Table 1.

Fig. 6. The CRF-associated TLR4 signal is innately activated in the NAc-shell from P rats

(A) Protein extracts of NAc-shell micropunches collected from P rats not previously exposed to alcohol but treated (n=6) or not (n=5) with antalarmin were immunoblotted with CRF antibody (Bioss Antibodies, Cat. # bs-0246R). The blots were stripped and sequentially blotted with antibodies to TLR4 and GAPDH and the results are expressed as densitometric units normalized to GAPDH ± SEM. Each lane represents a distinct animal. The levels of both CRF and TLR4 are significantly lower in the antalarmin treated than untreated rats (**p<0.01 by ANOVA). (B) Confocal microscopy and Z-stack imaging of double immunofluorescent staining of NAc-shell sections from P and NP rats (n=5/group) with CRF (red) (Santa Cruz Biotechnology, Cat. # sc-293187) and pERK1/2 (green) antibodies or TLR4 (red) and pERK1/2 (green) antibodies. TLR4+ and CRF+ cells in the NAc-shell from the P rats have co-localized nuclear staining with pERK1/2 antibody (21.7 ± 5.3% and 23.1 ± 7.8%, respectively) but co-localization is barely detectable in the NAc-shell from NP rats (0.93 ± 0.91% and 1.1 ± 0.98%, respectively) (*p<0.05 by ANOVA). Scale bars: 15 µm. The % co-staining cells are summarized in Table 1.

Fig. 7. pHSVsiTLR4 infusion in NAc-shell inhibits impulsivity associated with TLR4 inhibition

(A) Mean adjusted delay scores are significantly increased (impulsivity is decreased) in P rats infused with pHSVsiTLR4 (n=8) relative to those of P rats injected with pHSVsiNC (n=13). Impulsivity is decreased on day 7 post-surgery and returns to baseline levels on day 17. (*p≤ 0.05 by ANOVA). (B) pHSVsiTLR4 infusion in the NAc-shell inhibits TLR4 expression at days 3 after injection (*p≤0.05 by ANOVA), but inhibition is no longer significant on day 16 post injection and expression is not inhibited by pHSVsiNC (p≥0.05 by ANOVA).

Fig. 8. Schematic representation of TLR4 function in impulsivity and the transition to alcohol dependence

Current findings identify an innately activated TLR4 signal in the NAc-shell from P rats that is sustained by a CRF amplification loop and regulates impulsivity. The loop includes TLR4-mediated CRF upregulation through PKA/CREB activation and CRF-mediated TLR4 upregulation through CRFR1 and MAPK/ERK activation. The major contributor appears to be the CRF/CRFR1 system, because pERK1/2 is only seen in 25% of the TLR4+ and CRF+ neurons. Impulsivity is visualized as a major contributor to the initiation of alcohol drinking by previously non-alcohol exposed subjects (yellow). Dotted line represents potential involvement of α2 in the stimulation of the CRF-amplification loop in the NAc-shell. Together with previous findings that: (i) the TLR4 signal contributes to the initiation of alcohol drinking (binge drinking) at other brain sites (viz. CeA and VTA) (Liu et al., 2011), (ii) the TLR4 signal is upregulated by alcohol-induced CRF expression at these sites (June et al., 2015), and (iii) a TLR4/TH signal in the VTA is associated with increased levels of impulsivity (Aurelian et al., 2016), we posit the schematically represented emerging picture as a contributor to the transition to alcohol-dependence (grey).