HIV induces synaptic hyperexcitation via cGMP-dependent protein kinase II activation in the FIV infection model

Abstract

Over half of individuals infected with human immunodeficiency virus (HIV) suffer from HIV-associated neurocognitive disorders (HANDs), yet the molecular mechanisms leading to neuronal dysfunction are poorly understood. Feline immunodeficiency virus (FIV) naturally infects cats and shares its structure, cell tropism, and pathology with HIV, including wide-ranging neurological deficits. We employ FIV as a model to elucidate the molecular pathways underlying HIV-induced neuronal dysfunction, in particular, synaptic alteration. Among HIV-induced neuron-damaging products, HIV envelope glycoprotein gp120 triggers elevation of intracellular Ca2+ activity in neurons, stimulating various pathways to damage synaptic functions. We quantify neuronal Ca2+ activity using intracellular Ca2+ imaging in cultured hippocampal neurons and confirm that FIV envelope glycoprotein gp95 also elevates neuronal Ca2+ activity. In addition, we reveal that gp95 interacts with the chemokine receptor, CXCR4, and facilitates the release of intracellular Ca2+ by the activation of the endoplasmic reticulum (ER)-associated Ca2+ channels, inositol triphosphate receptors (IP3Rs), and synaptic NMDA receptors (NMDARs), similar to HIV gp120. This suggests that HIV gp120 and FIV gp95 share a core pathological process in neurons. Significantly, gp95's stimulation of NMDARs activates cGMP-dependent protein kinase II (cGKII) through the activation of the neuronal nitric oxide synthase (nNOS)-cGMP pathway, which increases Ca2+ release from the ER and promotes surface expression of AMPA receptors, leading to an increase in synaptic activity. Moreover, we culture feline hippocampal neurons and confirm that gp95-induced neuronal Ca2+ overactivation is mediated by CXCR4 and cGKII. Finally, cGKII activation is also required for HIV gp120-induced Ca2+ hyperactivation. These results thus provide a novel neurobiological mechanism of cGKII-mediated synaptic hyperexcitation in HAND.

Fig 1. FIV envelope glycoprotein gp95, not capsid protein p26, increases neuronal Ca²⁺ activity.

(A) Representative traces of GCaMP5 fluorescence intensity and a summary graph of normalized average of total Ca²⁺ activity in control, 10-pM, 100-pM, and 1-nM gp95-treated neurons showing that 1-nM gp95 treatment significantly increases neuronal Ca²⁺ activity (n = number of neurons, **p < 0.01, one-way ANOVA, uncorrected Fischer’s LSD, F (3,195) = 5.204, p = 0.0018). (B) Average frequency and amplitude of Ca²⁺ activity in control and gp95-treated neurons showing that gp95 elevates both frequency and amplitude of Ca²⁺ activity (n = number of neurons, *p < 0.05 and **p < 0.01, unpaired two-tailed Student t tests). (C) FIV capsid protein 700-nM p26 treatment has no effect on neuronal Ca²⁺ activity (n = number of cells). A scale bar indicates 20 seconds. FIV, feline immunodeficiency virus; LSD, Least Significant Difference.

Fig 2. Cellular pathway of gp95-induced Ca²⁺ hyperactivity.

Representative traces of GCaMP5 fluorescence intensity and a summary graph of normalized average of total Ca²⁺ activity in (i) control neurons and neurons treated with (ii) 1-nM gp95, (iii) 200-nM AMD3100, (iv) 200-nM AMD3100 and 1-nM gp95, (v) 25-μM 2APB, (vi) 25-μM 2APB and 1-nM gp95, (vii) 10-μM Dantrolene, (viii) 10-μM Dantrolene and 1-nM gp95, (ix) 25-μM DL-APV, and (x) 25-μM DL-APV and 1-nM gp95, showing that the gp95-induced elevation of neuronal Ca²⁺ activity is dependent on CXCR4, IP3Rs, RyRs, and NMDARs (n = number of neurons, ****p < 0.0001, one-way ANOVA, uncorrected Fischer’s LSD, F (9,481) = 6.289). A scale bar indicates 20 seconds. AMD3100, bicyclam derivative plerixafor hydrochloride; IP3R, inositol triphosphate receptor; LSD, Least Significant Difference; NMDAR, NMDA receptor; RyR, Ryanodine receptor.

Fig 3. Gp95-induced Ca²⁺ hyperactivity is mediated by nNOS-cGKII activation.

(A) Representative traces of GCaMP5 fluorescence intensity and a summary graph of normalized average of total Ca²⁺ activity in (i) control neurons and neurons treated with (ii) 1-nM gp95, (iii) 2-μM NPA, (iv) 2-μM NPA and 1-nM gp95, (v) 1-μM RP, and (vi) 1-μM RP and 1-nM gp95 showing that nNOS activity and cGKII activity are required for gp95-induced Ca²⁺ hyperactivity (n = number of neurons, ****p < 0.0001, one-way ANOVA, uncorrected Fischer’s LSD, F (5,253) = 12.38). A scale bar indicates 20 seconds. (B) gp95 is unable to induce neurotoxic effects when the cGKII gene is deleted (n = number of neurons). A scale bar indicates 20 seconds. (C) Representative immunoblots and quantitative analysis of pIP3R(S1756) levels in each condition showing that gp95 treatment is able to increase pIP3Rs, which are dependent on cGKII activity (n = 4 experiments, *p < 0.05, one-way ANOVA, uncorrected Fischer’s LSD, F (3,20) = 3.795, p = 0.0264). (D) Representative immunoblots and quantitative analysis of pIP3R(S1756) levels in WT and cGKII KO neurons showing that gp95 treatment has no effect on IP3R phosphorylation (n = 4 experiments). cGKII, cGMP-dependent protein kinase II; IP3R, inositol triphosphate receptor; KO, knockout; LSD, Least Significant Difference; nNOS, neuronal nitric oxide synthase; NPA, N^ω-Propyl-L-arginine hydrochloride.

Fig 4. Gp95 increases surface expression of the AMPAR GluA1 subunit via cGKII activation.

(A) Representative immunoblots and quantitative analysis of the synaptosome fraction from cultured cortical neurons in each condition showing that gp95 is capable of elevating GluA1 S845 phosphorylation (pGluA1), which is mediated by cGKII (n = 3 experiments, *p < 0.05, one-way ANOVA, uncorrected Fischer’s LSD, F (3,22) = 3.884, p = 0.0228). (B) Representative immunoblots and quantitative analysis of the synaptosome fraction from cultured WT and cGKII KO cortical neurons showing that gp95 is unable to increase pGluA1 in cGKII KO neurons (n = 5 experiments). (C) Representative immunoblots and quantitative analysis of surface biotinylation in each condition showing that gp95 is able to increase surface GluA1 via cGKII activation (n = 6 experiments, *p < 0.05, one-way ANOVA, uncorrected Fischer’s LSD, F (3,20) = 3.839, p = 0.0254). (D) Representative immunoblots and quantitative analysis of surface biotinylation in WT and KO hippocampal neurons showing that cGKII is required for gp95-induced GluA1 surface trafficking (n = 5 experiments). (E) Representative traces of mEPSC recordings in control and gp95-treated neurons showing average mEPSC amplitude and frequency are significantly increased by gp95 treatment (n = number of neurons, *p < 0.05 and ****p < 0.0001, unpaired two-tailed Student t tests). AMPAR, AMPA receptor; cGKII, cGMP-dependent protein kinase II; KO, knockout; LSD, Least Significant Difference; mEPSC, miniature excitatory postsynaptic current.

Fig 5. Activity-dependent gp95 effects on AMPAR-mediated Ca²⁺ hyperactivity.

Representative traces of GCaMP5 fluorescence intensity and a summary graph of normalized average of total Ca²⁺ activity in (i) control neurons and neurons treated with (ii) 1-nM gp95, (iii) 10-μM CNQX, (iv) 10-μM CNQX and 1-nM gp95, (v) 20-μM NASPM, and (vi) 20-μM NASPM and 1-nM gp95, showing that CP-AMPARs are required for gp95-induced Ca²⁺ hyperactivity (n = number of neurons, ****p < 0.0001, one-way ANOVA, uncorrected Fischer’s LSD, F (7,179) = 12.1). A scale bar indicates 20 seconds. AMPAR, AMPA receptor; CNQX, 6-Cyano-7-nitroquinoxaline-2,3-dione; CP-AMPAR, Ca²⁺-permeable AMPAR; LSD, Least Significant Difference; NASPM, 1-naphthyl acetyl spermine.

Fig 6. Gp95-induced Ca²⁺ hyperactivity in feline cultured hippocampal neurons.

Representative traces of GCaMP5 fluorescence intensity and a summary graph of normalized average of total Ca²⁺ activity in (i) control neurons and neurons treated with (ii) 1-nM gp95, (iii) 200-nM AMD3100, (iv) 200-nM AMD3100 and 1-nM gp95, (v) 10-μM RP, (vi) 10-μM RP and 1-nM gp95, (vii) 10-μM CNQX, and (viii) 10-μM CNQX and 1-nM gp95, showing that the gp95-induced Ca²⁺ hyperactivity in cat hippocampal neurons is dependent on CXCR4, cGKII, and AMPARs (n = number of neurons, ****p < 0.0001, one-way ANOVA, uncorrected Fischer’s LSD, F (7,260) = 5.296). A scale bar indicates 20 seconds. AMD3100, bicyclam derivative plerixafor hydrochloride; AMPAR, AMPA receptor; cGKII, cGMP-dependent protein kinase II; CNQX, 6-Cyano-7-nitroquinoxaline-2,3-dione; LSD, Least Significant Difference.

Fig 7. cGKII activation is required for HIV gp120 and SDF-1-induced Ca²⁺ hyperactivity.

(A) Representative traces of GCaMP5 fluorescence intensity and a summary graph of normalized average of total Ca²⁺ activity in (i) control neurons and neurons treated with (ii) 1-nM gp120 (IIIB), (iii) 200-nM AMD3100 and 1-nM gp120 (IIIB), and (iv) 1-μM RP and 1-nM gp120 (IIIB), showing that an increase in Ca²⁺ activity by 1-nM CXCR4-tropic gp120 (IIIB) treatment is dependent on CXCR4 and cGKII (n = number of neurons, *p < 0.05, one-way ANOVA, uncorrected Fischer’s LSD, F (3,51) = 3.936, p = 0.0133). A scale bar indicates 20 seconds. (B) Representative traces of GCaMP5 fluorescence intensity and a summary graph of normalized average of total Ca²⁺ activity in (i) control neurons and neurons treated with (ii) 1-nM gp120 (JRFL), (iii) 200-nM AMD3100 and 1-nM gp120 (JRFL), and (iv) 1-μM RP and 1-nM gp120 (JRFL), showing that an increase in Ca²⁺ activity by 1-nM CCR5-tropic gp120 (JRFL) treatment is dependent on CXCR4 and cGKII (n = number of neurons, *p < 0.05, one-way ANOVA, uncorrected Fischer’s LSD, F (3,115) = 3.903, p = 0.0107). A scale bar indicates 20 seconds. (C) Representative traces of GCaMP5 fluorescence intensity and a summary graph of normalized average of total Ca²⁺ activity in (i) control neurons and neurons treated with (ii) 20-nM SDF-1, (iii) 1-μM RP and 20-nM SDF-1, and (iv) 200-nM AMD3100 and 20-nM SDF-1, showing that 20-nM SDF-1 induces cGKII- and CXCR4-dependent Ca²⁺ overactivation (n = number of neurons, ***p < 0.001, one-way ANOVA, uncorrected Fischer’s LSD, F (3,63) = 6.234, p = 0.0009). A scale bar indicates 20 seconds. AMD3100, bicyclam derivative plerixafor hydrochloride; cGKII, cGMP-dependent protein kinase II; LSD, Least Significant Difference; SDF-1, stromal cell-derived factor-1.

Fig 8. Model for gp95/120-induced activity-dependent synaptic hyperexcitation.

Both FIV gp95 and HIV gp120 stimulate CXCR4 and NMDARs, inducing activity-dependent synaptic dysfunction via cGKII activation. The gp95/120 stimulation of NMDARs activates nNOS, production of NO, leading to activation of soluble guanylyl cyclase and the production of cGMP, which in turn activates cGKII. Both the production of IP3 by the gp95/120 stimulation of CXCR4 and cGKII-induced phosphorylation of IP3Rs enhance ER Ca²⁺ release, contributing to Ca²⁺ hyperactivity. In addition, gp95-induced cGKII activation increases GluA1 phosphorylation, promoting elevation of surface AMPARs, which leads to the elevation of synaptic excitation. Therefore, the gp120/gp95-induced stimulation of cGKII is critical for synaptic hyperexcitation in HAND pathophysiology. AMD3100, bicyclam derivative plerixafor hydrochloride; AMPAR, AMPA receptor; cGKII, cGMP-dependent protein kinase II; cGMP, cyclic GMP; CNQX, 6-Cyano-7-nitroquinoxaline-2,3-dione; ER, endoplasmic reticulum; FIV, feline immunodeficiency virus; HAND, HIV-associated neurocognitive disorder; HIV, human immunodeficiency virus; IP3R, inositol triphosphate receptor; NMDAR, NMDA receptor; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NPA, N^ω-Propyl-L-arginine hydrochloride; PSD95, postsynaptic density 95.