Plasma gelsolin protects HIV-1 gp120-induced neuronal injury via voltage-gated K+ channel Kv2.1

Abstract

Plasma gelsolin (pGSN), a secreted form of gelsolin, is constitutively expressed throughout the central nervous system (CNS). The neurons, astrocytes and oligodendrocytes are the major sources of pGSN in the CNS. It has been shown that levels of pGSN in the cerebrospinal fluid (CSF) are decreased in several neurological conditions including HIV-1-associated neurocognitive disorders (HAND). Although there is no direct evidence that a decreased level of pGSN in CSF is causally related to the pathogenesis of neurological disorders, neural cells, if lacking pGSN, are more vulnerable to cell death. To understand how GSN levels relate to neuronal injury in HAND, we studied the effects of pGSN on HIV-1 gp120-activated outward K+ currents in primary rat cortical neuronal cultures. Incubation of rat cortical neurons with gp120 enhanced the outward K+ currents induced by voltage steps and resulted in neuronal apoptosis. Treatment with pGSN suppressed the gp120-induced increase of delayed rectifier current (IK) and reduced vulnerability to gp120-induced neuronal apoptosis. Application of Guangxitoxin-1E (GxTx), a Kv2.1 specific channel inhibitor, inhibited gp120 enhancement of IK and associated neuronal apoptosis, similar effects to pGSN. Western blot and PCR analysis revealed gp120 exposure to up-regulate Kv2.1 channel expression, which was also inhibited by treatment with pGSN. Taken together, these results indicate pGSN protects neurons by suppressing gp120 enhancement of IK through Kv2.1 channels and reduction of pGSN in HIV-1-infected brain may contribute to HIV-1-associated neuropathy.

Figure 1. pGSN protection of gp120-induced neuronal damage

(A) Dose-dependent toxicity of gp120 on cortical neurons. Exposure of neuronal cells to gp120 at and greater than 500pM resulted in maximal reduction in cell viability. (B) Neurons were treated with 500 pM gp120 and different concentrations of GSN for 24 h. pGSN significantly protected neurons from gp120-mediated damage at a concentration of 2.5 µg/ml. Heat (boiled) inactivated pGSN (indicated by a symbol §, the same as in other figures) failed to produce protective effect. Data represent mean ± SEM from at least 5 independent experiments and expressed as % of control. ** p <0.01 vs. control; ^# p<0.05 vs. gp120 (0 GSN).

Figure 2. Gp120 increases outward K⁺ currents

Cultures were treated without or with 50pM, 100pM gp120 respectively 24h. (A) Representative current traces evoked by voltage protocol (upper left, not proportional to the current traces in duration) from three different neurons treated without (Ctrl) or with 50pM, 100pM gp120 respectively for 24h. Note gp120 at 100pM increased outward K⁺ currents. (B) Instantaneous and steady-state current densities measured at +80mV from groups of neurons treated without (Ctrl, n=15) or with 50pM gp120 (n=13), 100pM gp120 (n=18). Note that gp120 increased both instantaneous and steady-state outward K⁺ currents at 100 pM. ** p<0.01 vs. control.

Figure 3. pGSN decrease of gp120 enhancement of neuronal outward K+ currents

(A) Representative current traces recorded from 6 different neurons treated with gp120 (100pM) alone, or gp120 (100pM) and different concentrations of pGSN (5, 50, 500, 500§), or untreated control. Whole-cell currents were generated by voltage steps from the holding potential of −80mV to +80mV in increments of 20mV. Both instantaneous and steady-state outward K⁺ currents were measured and current densities were calculated and displayed in (B). pGSN only inhibited gp120-induced steady-state K⁺ current. In contrast, heat denatured pGSN lose such an inhibitory ability. * p<0.05 vs. control, ** p<0.01 vs. control; ^## p<0.01 vs. gp120 alone.

Figure 4. pGSN inhibits gp120 enhancement of TEA-sensitive I_K, but not 4-AP-sensitive I_A

(A) shows representative total (upper), 4-AP-sensitive (middle) and TEA-sensitive (lower) outward K⁺ currents recorded from neurons treated with gp120 (100pM), gp120 (100pM)+pGSN(500ng/ml), pGSN (500ng/ml) or untreated controls(Ctrl). (B) and (C) are current density-voltage relationships, illustrating GSN inhibition of TEA-sensitive I_K. pGSN alone did not affect either the 4-AP- or TEA-sensitive outward K⁺ currents (p>0.05). For each type of outward K⁺ currents, 25 neurons were recorded in each treatment group. * p<0.05 vs. control, ** p<0.01 vs. control; ^## p<0.01 vs. gp120 group.

Figure 5. Involvement of Kv2.1 in pGSN inhibition of gp120 enhancement of TEA-sensitive I_K

Cultures were treated for 24 h with 100 pM gp120, 500 ng/ml pGSN or gp120 and pGSN. (A) Representative TEA sensitive currents recorded from neurons with treatments indicated. TEA sensitive I_K was recorded in the presence of 4-AP (5mM) alone or 4-AP and GxTx (50nM). Note that GxTx, a specific Kv2.1 channel blocker, abolished pGP120 enhancement of the TEA sensitive I_K. (B) a summary bar graph of current densities (n=25) showing GxTx inhibition of gp120 increase of TEA sensitive K⁺ currents generated by a voltage step from −80 to +80mV and GxTx per se decreased TEA sensitive K⁺ currents. **p<0.01 vs. control; ^##p<0.01 vs. gp120 group.

Figure 6. pGSN blocks gp120-induced shift of TEA sensitive K⁺ current inactivation curve

(A) The original current traces generated by inactivation and activation voltage protocols (upper) before and after bath perfusion of 200 pM gp120 (middle) or 200 pM gp120 and 1 µg/ml pGSN (lower) from two different neurons. Normalized data points were fitted with the Boltzmann equation: I/I_max=1/{1+exp[(V-V_1/2)/k]} and G/G_max=1-1/{1+exp[(V-V_1/2)]/k}. Each point represents the mean ± SEM, n=5. For inactivation, V_1/2 was −38.51 mV in the presence of gp120 compared with −51.64 mV in Control (B). V_1/2 was −52.61 mV for gp120+GSN compared with −51.14 mV for Control (C). For activation, V_1/2 values for TEA sensitive K⁺ currents were −41.93 mV in the presence of gp120 vs −30.59mV in Control (B); and −31.13 mV in gp120+GSN group vs −30.32 mV in Control group (C). These results showed that gp120 shifted inactivation curve to the right, which was inhibited by pGSN.

Figure 7. Inhibition of gp120-induced Kv2.1 channel expression by pGSN

Neuronal cultures were treated with 500 pM gp120 with or without 2.5µg/ml pGSN for 24h. Band density was analyzed by Image J and normalized by internal control. Data represents means ± SEM from 3 independent experiments. (A) gp120 increased the levels of Kv1.1 mRNA expression (p<0.05). Although increased levels of Kv2.1 and Kv4.2 mRNA expression did not reach the statistical significance (p>0.05 compare to control), the expression levels of Kv2.1 mRNA was down-regulated by pGSN compared to gp120 group (p<0.05). (B) gp120 significantly increased the levels of Kv1.1, Kv2.1 and Kv4.2 channel protein expression (p<0.01). pGSN significantly reduced only Kv2.1 channel protein expression (p<0.01). pGSN alone did not alter the expression levels of Kv channels. * p< 0.05 vs. control; **p<0.01 vs. control; ^#p<0.05 vs. gp120 group; ^##P<0.01 vs. gp120 group.

Figure 8. pGSN protects against gp120-induced neuronal damage via Kv2.1

Neurons were exposed to gp120 (500pM), pGSN (2.5µg/ml), gp120(500pM) and pGSN (2.5µg/ml), or heat-inactivated pGSN (2.5µg/ml) in the presence or absence co-incubated GxTx (12.5 or 25 nM) for 24h. (A) Cell viability determined by MTT assay exhibited that gp120 exposure reduced cell viability (p<0.01 compare to Control), which was attenuated by GSN or GxTx. Heat-inactivated pGSN (§) showed no protective effect. pGSN or GxTx alone did not alter cell viability. (B) left panel shows TUNEL staining results as a measure of apoptosis, with cell nuclei visualized using Dapi stain. 10 visual fields were analyzed for each of three independent experiments. Gp120 induced neuronal apoptosis that was attenuated by either pGSN or GxTx. These data suggest that pGSN protects against gp120-induced neuronal damage via Kv2.1. **p<0.01 vs. control; ^#p<0.05 vs. gp120 group; ^## p<0.01 vs. gp120 group. Scale bars denote 50µm.