Fig 1: CXCL12 activates the small GTPase Rac1 in cultured cortical neurons in a CXCR4- and Gαi-dependent manner.(A) Cultured cortical neurons (21 DIV) were treated with CXCL12 (20 nM) for the indicated time points and subjected to pulldown using PAK agarose beads. Immunoblotting revealed a significant activation of Rac1 at 5 and 15 min and a return to baseline at 30 min. Positive and negative controls were performed by incubating lysates with GTPγS and GDP, respectively. N = 3 (B) Pretreatment with the CXCR4 antagonist, AMD3100 (100 ng/mL; 20 min) attenuated CXCL12-induced activation of Rac1. Positive and negative controls were performed by incubating lysates with GTPγS and GDP, respectively. N = 3 (C) Pretreatment with the Gαi inhibitor pertussis toxin (PTX, 100 ng/mL; 18 hr) completely prevented activation of Rac1 by CXCL12. Positive and negative controls were performed by incubating lysates with GTPγS and GDP, respectively. N = 3.
Fig 2: CXCL12 phosphorylates downstream mediators of the Rac1 pathway and results in changes to actin polymerization in cortical neurons.(A) Cultured cortical neurons (21 DIV) were exposed to CXCL12 (20 nM) for the indicated time points and the phosphorylation status of PAK1, LIMK1, and cofilin was examined. CXCL12 treatment resulted in a time-dependent increase in the phosphorylation of all three proteins. N = 3, *p<0.05, **p<0.01. (B) Pretreatment with AMD3100 (100 ng/mL; 20 min) blocked CXCL12-induced phosphorylation of Rac1 downstream mediators. N = 3, **p<0.01, ***p<0.001. (C) Inhibition of Gαi signaling (via PTX) blocked the ability of CXCL12 to phosphorylate Rac1 downstream mediators. N = 3, *p<0.05, **p<0.01, ***p<0.001. (D) Following treatment with CXCL12, the protein phosphatase SSH1 is phosphorylated (and inactivated) in a time-dependent manner. N = 3, *p<0.05. (E) Separation of F and G-actin in cortical neurons revealed a shift in favor of F-actin following CXCL12 treatment. Latrunculin A (5 μM, 2 hr), a potent actin polymerization inhibitor, and jaspakinolide (5 μM, 2 hr), an inducer of actin polymerization, were used as internal controls for the assay. N = 3; *p<0.05.Figure 5—source data 1.Densitometry statistical analysis.
Fig 3: CXCL12 depends on Rac1 activation to rescue cognitive flexibility and dendritic spine density in the HIV-Tg rat.(A) Inhibition of Rac1 activity blocked the ability of CXCL12 to positively enhance cognitive flexibility in an attentional set-shifting task. N = 9 for veh+veh, N = 9 for veh+CXCL12, N = 8 for NSC+veh, and N = 8 for NSC+CXCL12. (B) NSC23766 co-treatment significantly attenuated the rate at which animals reached criterion on the shift to cue phase as assessed by linear regression. N = 9 for veh+veh, N = 9 for veh+CXCL12, N = 8 for NSC+veh, and N = 8 for NSC+CXCL12, F(3,32)=7.503, p=0.0006. (C) Blockade of Rac1 activation abrogated the effect of CXCL12 on overall dendritic spine density in HIV-Tg rats. N = 9 for veh+veh, N = 9 for veh+CXCL12, N = 8 for NSC+veh, and N = 8 for NSC+CXCL12, eight dendrites measured for each animal and averaged into single data point, **p<0.01, ***p<0.001. (D) CXCL12’s impact on thin spines was mitigated by co-treatment with NSC23766. N = 9 for veh+veh, N = 9 for veh+CXCL12, N = 8 for NSC+veh, and N = 8 for NSC+CXCL12, eight dendrites measured for each animal and averaged into single data point, **p<0.01, ***p<0.001. (E) NSC23766 prevented decreases in stubby spine density mediated by CXCL12 treatment. N = 9 for veh+veh, N = 9 for veh+CXCL12, N = 8 for NSC+veh, and N = 8 for NSC+CXCL12, eight dendrites measured for each animal and averaged into single data point, *p<0.05. (F) There was no significant relationship observed between overall dendritic spine density and trials to criterion on the set-shifting phase of the behavioral task. N = 12 animals, Pearson’s r = −0.2256, p=0.4808. (G) Thin spine density was negatively associated with the number of trials to criterion in the shift to cue phase of the behavioral task. N = 12 animals, Pearson’s r = −0.7787, p=0.0029.Figure 9—source data 1.HIV-Tg ±CXCL12/NSC23766 raw data and statistical analysis.
Fig 4: CXCL12 specifically modulates thin spine density via activation of Rac1.(A) Cultured cortical neurons (21 DIV) were treated with CXCL12 (20 nM, 3 hr), resulting in a specific increase in thin spine density and a decrease in stubby spine numbers. N = 9 coverslips/group, 4 dendrites measured/coverslip and averaged into single data point, 3 separate experiments, *p<0.05, ***p<0.001. (B) Pretreatment with the specific Rac1 inhibitor NSC23766 (100 μM, 15 min) completely blocked CXCL12-induced activation of Rac1 in cortical neurons. N = 3 (C) Subsequently, inhibition of Rac1 activation by NSC23766 prevented phosphorylation of downstream mediators by CXCL12. N = 3, *p<0.05, **p<0.01, ***p<0.001. (D) Inhibition of Rac1 activation by NSC23766 blocked the ability of CXCL12 to modulate overall dendritic spine density, as well as thin and stubby spine density. N = 9 coverslips/group, 4 dendrites measured/coverslip and averaged into single data point, 3 separate experiments, *p<0.05, ***p<0.001. (E) Cortical neurons (18 DIV) were infected with control or Rac1-shRNA viral particles and GFP-positive neurons were analyzed (21 DIV) following treatment with either vehicle or CXCL12. Knockdown of Rac1 inhibited CXCL12-mediated alterations in spine density and morphology. N = 9 coverslips/group, 4 dendrites measured/coverslip and averaged into single data point, 3 separate experiments, *p<0.05, **p<0.01, ***p<0.001.Figure 6—source data 1.In vitro raw data and statistical analysis.
Fig 5: Blockade of Rac1 activation prevents alterations in cognitive flexibility and dendritic spines by CXCL12.(A) Treatment with NSC23766 mitigated the ability of CXCL12 to increase the number of animals reaching criterion on the shift to cue phase. N = 12 animals/group, p=0.0498. (B) The rate at which animals reached criterion was significantly delayed with NSC23766 pretreatment as assessed via linear regression. N = 12 animals/group, F(3,32)=12.6622, p<0.001. (C) Blockade of Rac1 activation by NSC23766 prevents CXCL12-mediated upregulation of overall dendritic spine density in the mPFC. N = 12 animals/group, 8 dendrites measured for each animal and averaged into single data point, **p<0.01. (D) Additionally, NSC23766 prevented changes in thin spine density induced by CXCL12. N = 12 animals/group, 8 dendrites measured for each animal and averaged into single data point, ***p<0.001. (E) The ability of CXCL12 to decrease stubby spine density was blocked by NSC23766. N = 12 animals/group, 8 dendrites measured for each animal and averaged into single data point, **p<0.01. (F) As previously observed, overall dendritic spine density was negatively associated with trials to criterion on the set-shifting phase of the behavioral task. N = 23 animals, Pearson’s r = −0.7862, p=0.0127. (G) This relationship became even stronger when only thin spine density was considered. N = 23 animals, Pearson’s r = −0.8350, p=0.0051.Figure 8—source data 1.WT ±CXCL12/NSC23766 raw data and statistical analysis.
Supplier Page from OriGene Technologies for Rac1 Rat shRNA Plasmid (Locus ID 363875)