Broad-spectrum effects of 4-aminopyridine to modulate amyloid beta1-42-induced cell signaling and functional responses in human microglia

Abstract

We investigated the modulating actions of the nonselective K(+) channel blocker 4-aminopyridine (4-AP) on amyloid beta (Abeta(1-42))-induced human microglial signaling pathways and functional processes. Whole-cell patch-clamp studies showed acute application of Abeta(1-42) (5 mum) to human microglia led to rapid expression of a 4-AP-sensitive, non-inactivating outwardly rectifying K(+) current (I(K)). Intracellular application of the nonhydrolyzable analog of GTP, GTPgammaS, induced an outward K(+) current with similar properties to the Abeta(1-42)-induced I(K) including sensitivity to 4-AP (IC(50) = 5 mm). Reverse transcriptase-PCR showed a rapid expression of a delayed rectifier Kv3.1 channel in Abeta(1-42)-treated microglia. Abeta(1-42) peptide also caused a slow, progressive increase in levels of [Ca(2+)](i) (intracellular calcium) that was partially blocked by 4-AP. Chronic exposure of human microglia to Abeta(1-42) led to enhanced p38 mitogen-activated protein kinase and nuclear factor kappaB expression with factors inhibited by 4-AP. Abeta(1-42) also induced the expression and production of the pro-inflammatory cytokines interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha, the chemokine IL-8, and the enzyme cyclooxygenase-2; 4-AP was effective in reducing all of these pro-inflammatory mediators. Additionally, toxicity of supernatant from Abeta(1-42)-treated microglia on cultured rat hippocampal neurons was reduced if 4-AP was included with peptide. In vivo, injection of Abeta(1-42) into rat hippocampus induced neuronal damage and increased microglial activation. Daily administration of 1 mg/kg 4-AP was found to suppress microglial activation and exhibited neuroprotection. The overall results suggest that 4-AP modulation of an Abeta(1-42)-induced I(K) (candidate channel Kv3.1) and intracellular signaling pathways in human microglia could serve as a therapeutic strategy for neuroprotection in Alzheimer's disease pathology.

Figure 1.

Aβ_1–42 induces an outwardly rectifying current (I_K) that is attributed to K⁺. A, A representative recording of the Aβ_1–42-induced outward K⁺ current (I_K) in response to a depolarizing step in human microglia. The trace is a typical leak current evoked in control solution with a depolarization step to +20 mV from a holding potential of −60 mV. Aβ_1–42 (5 μm) elicited a large I_K with the same step depolarization. The current recovered subsequent to washoff of Aβ_1–42. B, I_K, induced by Aβ_1–42 (5 μm), with sequential depolarizing steps applied from −60 mV to a maximum level of +20 mV in 10 mV increments. C, The I–V relationship constructed from the pulse protocol shown indicates that the outward current induced by acute Aβ_1–42 was outwardly rectifying with a threshold of −40 mV. The figure is a representative recording from one cell. D, Determination of the reversal potential of the I_K via analysis of tail currents. The protocol for tail current analysis consisted of applying a depolarizing step from −60 to +40 mV, followed by a secondary step to potentials varying from −100 to −20 mV. The resulting tail currents elicited with the secondary steps from −100 to −20 mV in a representative experiment are indicated by arrows. E, An I–V plot of tail current amplitudes versus step potential is shown, and results indicate that the reversal potential of the current is −78 mV, which is close to the equilibrium potential for K⁺.

Figure 2.

A, I_K is inhibited by the nonselective K⁺ channel inhibitor 4-AP. A representative recording from one cell is shown. The first trace is a typical current evoked in control solution with a depolarization step to +20 mV. Aβ_1–42 (5 μm) elicited I_K with the same step depolarization. Application of 4-AP (2 mm) in the presence of Aβ_1–42 reduced I_K to 52% of control. The current recovered subsequent to washoff of 4-AP with Aβ_1–42 maintained in bath solution. B, Typical profile of the intracellular GTPγS-induced outward current. Intracellular application of GTPγS (10 μm) via the electrode induced an outward current within minutes of rupture of the cell membrane in the whole-cell patch-clamp mode in response to a depolarizing step from −60 to +20 mV. Extracellular application of 4-AP (2 mm) reduced the outward K⁺ current to 65% of the control. Washoff of 4-AP allowed the current to recover. C, Concentration-dependent inhibition of the intracellular GTPγS-induced outward K⁺ current by 4-AP. 4-AP was applied in the extracellular bath solution, and amplitudes of the GTPγS-induced currents were measured in the presence of 4-AP and normalized to control amplitudes (C; current amplitude before 4-AP application). Results are a summary of the following: n = 4 cells for 1 mm; n = 6 cells for 2 mm; n = 10 cells for 5 mm; n = 4 cells for 10 mm; n = 3 cells for 20 mm.

Figure 3.

A, A representative RT-PCR experiment of Aβ_1–42 (5 μm) treatment for 10 min, 30 min, 1 h, and 2 h on Kv1.1, Kv1.2, Kv1.3, Kv1.5, Kv1.6, Kv2.1, and Kv3.1 channel expression from n = 3 independent experiments. G3PDH served as a reaction standard. B, Summary of relative mRNA levels of Kv channels induced by Aβ_1–42. Results are expressed as mean ± SEM from n = 3 independent experiments. One-way ANOVA and the Newman–Keuls multiple comparison test was used to evaluate statistical significance. *p < 0.05 and **p < 0.001, statistical significance from control. C, PTX (100 ng/ml) before treatment (2 h) attenuated the effects of Aβ_1–42 (5 μm; 30 min) to increase the expression of Kv3.1.

Figure 4.

A, Acute application of Aβ_1–42 induces a slow, progressive increase in [Ca²⁺]_i. A representative trace of the increase in [Ca²⁺]_i induced by Aβ_1–42 (5 μm) in Ca²⁺-PSS (n = 21 cells) is shown. Subsequent application of Aβ_1–42 in Ca²⁺-free PSS resulted in an immediate decrease in [Ca²⁺]_i to baseline levels. B, The standard PSS was first exchanged for Ca²⁺-free PSS. Acute application of Aβ_1–42 (5 μm) in Ca²⁺-free PSS did not elicit an increase in [Ca²⁺]_i (n = 23 cells). C, A representative trace of the effect of 4-AP on the Ca²⁺ influx pathway induced by Aβ_1–42 (n = 26 cells). Subsequent to the slow, progressive increase in [Ca²⁺]_i induced by acute Aβ_1–42 (5 μm) in Ca²⁺ PSS, application of 4-AP (2 mm) rapidly decreased [Ca²⁺]_i to baseline levels.

Figure 5.

Effects of 4-AP on Aβ_1–42-induced p38 MAPK and NF-κB activation. A, Left, Representative photomicrographs of phosphorylated p38 (phospho-p38)-stained microglia. Green and blue indicate staining for phospho-p38 MAPK- and DAPI-positive nuclei, respectively. Under control conditions, little or no phospho-p38 MAPK expression was observed. Aβ_1–42 (5 μm) treatment of microglia for 30 min induced an intense expression of phospho-p38 MAPK. Aβ_1–42 in the maintained presence of 4-AP (2 mm) inhibited expression of phospho-p38 MAPK. Application of 4-AP (2 mm) alone had no effect on phospho-p38 MAPK expression. Right, The percentage of phospho-p38 MAPK-positive microglia relative to total cells is shown. Data are means ± SEM from four independent experiments. *p < 0.001, significance compared with control; **p < 0.001, significance compared with Aβ_1–42. B, Left, Effects of 4-AP on Aβ_1–42-induced NF-κB activation. Representative photomicrographs of p65 (the active subunit of NF-κB)-stained microglia are shown. Green and blue indicate staining for p65- and DAPI-positive nuclei, respectively. Under control conditions, little or no p65 expression was observed. Aβ_1–42 (5 μm) treatment of microglia for 8 h induced an intense expression of p65. Aβ_1–42 in the maintained presence of 4-AP (2 mm) inhibited expression of p65. Application of 4-AP (2 mm) alone had no effect on p65 expression. Right, The percentage of p65-positive microglia relative to total cells is shown. Data are means ± SEM from five independent experiments. *p < 0.01, significance compared with control; **p < 0.01, significance compared with Aβ_1–42.

Figure 6.

Effects of 4-AP on Aβ_1–42-induced expression and production of pro-inflammatory mediators by human microglia. A, Expression of IL-1β, IL-6, IL-8, TNF-α, and COX-2 were examined in microglia incubated for 8 h with Aβ_1–42, 4-AP, Aβ_1–42 in the presence of 4-AP (2 mm), or medium alone. Stimulation of microglia with vehicle solution or Aβ_42–1 (5 μm) alone served as control. The results shown are a representative of seven independent experiments. The expression of G3PDH served as a reaction standard. B, Summary of relative mRNA levels of inflammatory mediators induced by Aβ_1–42, 4-AP, and combined Aβ_1–42 and 4-AP. Results are expressed as mean ± SEM from n = 7 independent experiments. One-way ANOVA and the Newman–Keuls multiple comparison post-test were performed to evaluate statistical significance. *p < 0.05, statistically significant from control; **p < 0.05, statistically significant from Aβ_1–42-stimulated levels. Effects of Aβ_1–42, 4-AP, and Aβ_1–42 in the maintained presence of 4-AP on pro-inflammatory cytokine secretion by human microglia using ELISA. C–F, Data are mean ± SEM of TNF-α from four independent experiments (C), IL-6 from three independent experiments (D), IL-1β from six independent experiments (E), and IL-8 from four independent experiments (F); each experiment was performed in duplicate. Human microglia were exposed to medium alone, Aβ_1–42 (5 μm), 4-AP (2 mm), Aβ_1–42 in the presence of 4-AP, or Aβ_42–1 for 48 h. One-way ANOVA and the Newman–Keuls multiple comparison post-test were performed to evaluate statistical significance. *p < 0.001, statistical significance from control; **p < 0.001, statistical significance from Aβ_1–42-stimulated levels.

Figure 7.

Effects of 4-AP on Aβ_1–42-induced COX-2-expressing microglia. A, Representative photomicrographs of COX-2-stained microglia. Green and blue indicate staining for COX-2- and DAPI-positive nuclei, respectively. Under control conditions, little or no COX-2 expression was evident. Treatment of microglia for 24 h with Aβ_1–42 (5 μm) induced an intense expression of COX-2. Aβ_1–42 in the presence of 4-AP (2 mm) treatment inhibited production of COX-2. 4-AP alone had no effect on basal levels of COX-2 production. B, The percentage of COX-2-positive microglia relative to total cells is shown under the different experimental conditions. Data are means ± SEM from six independent experiments. *p < 0.001, significance compared with control; and **p < 0.01, significance compared with Aβ_1–42.

Figure 8.

Effects of microglial conditioned medium on neuronal survival. A, Representative photomicrographs of DAPI-stained primary hippocampal neurons treated for 16 h with microglial conditioned medium [microglia stimulated for 48 h with Aβ_1–42 (5 μm), 4-AP (2 mm), each alone, or in combination]. Condensed (arrow) and fragmented (arrowhead) nuclei indicate damaged neurons. Scale bar, 20 μm. B, Summary of microglial-mediated neurotoxicity results from n = 5 independent experiments and corresponding control experiments (neurons treated with unconditioned medium) from n = 5 independent experiments. *p < 0.001, statistically significant from medium of unstimulated microglia; **p < 0.001, statistically significant from conditioned medium of Aβ_1–42-stimulated microglia.

Figure 9.

Effects of 4-AP on Aβ_1–42-induced hippocampal neuron degeneration and microglial activation in vivo. A, Representative photographs of tissue sections stained with NeuN antibody from the superior blade of dentate granule cell layer taken 7 d after injection with vehicle, Aβ_1–42 (1 nmol), Aβ_1–42 plus 4-AP (1 mg/kg, i.p.), and 4-AP and Aβ_42–1 (1 nmol). B, Representative photographs of tissue sections stained with ED1 from the superior blade of dentate granule cell layer taken from vehicle-injected rats, Aβ_1–42 (1 nmol), Aβ_1–42 plus 4-AP (1 mg/kg), and 4-AP or Aβ_42–1 (1 nmol) at 7 d after injection. C, Quantification of the effects of Aβ_1–42, 4-AP, and Aβ_1–42 in the presence of 4-AP on NeuN-positive neurons. Data are mean ± SEM (n = 4/group). *p < 0.05 versus vehicle; **p < 0.05 versus Aβ_1–42. D, Quantification of the effects of Aβ_1–42, 4-AP, and Aβ_1–42 in the presence of 4-AP on ED1-positive microglia. Data are mean ± SEM (n = 4/group). *p < 0.05 versus vehicle; **p < 0.05 versus Aβ_1–42. Scale bars, 50 μm.