Co-activation of selective nicotinic acetylcholine receptors is required to reverse beta amyloid-induced Ca2+ hyperexcitation

Abstract

Beta-amyloid (Aβ) peptide accumulation has long been implicated in the pathogenesis of Alzheimer's disease (AD). Hippocampal network hyperexcitability in the early stages of the disease leads to increased epileptiform activity and eventually cognitive decline. We found that acute application of 250 nM soluble Aβ42 oligomers increased Ca²⁺ activity in hippocampal neurons in parallel with a significant decrease in activity in Aβ42-treated interneurons. A potential target of Aβ42 is the nicotinic acetylcholine receptor (nAChR). Three major subtypes of nAChRs (α7, α4β2, and α3β4) have been reported in the human hippocampus. Simultaneous inhibition of both α7 and α4β2 nAChRs mimicked the Aβ42 effects on both excitatory and inhibitory neurons. However, inhibition of all 3 subtypes showed the opposite effect. Importantly, simultaneous activation of α7 and α4β2 nAChRs was required to reverse Aβ42-induced neuronal hyperexcitation. We suggest co-activation of α7 and α4β2 nAChRs is required to reverse Aβ42-induced Ca²⁺ hyperexcitation.

Keywords: Alzheimer's disease; Beta-amyloid; Co-activation; Disinhibition; Hyperexcitation; Nicotinic acetylcholine receptor.

Figure 1.. Soluble Aβ42 oligomers induce hyperexcitability in hippocampal cells.

(A) Representative traces of GCaMP5 fluorescence intensity in hippocampal cells and a summary graph of the normalized average of total Ca²⁺ activity in neurons treated with either sAβ42 (black) or oAβ42 (red) at concentrations of 100nM, 250nM and 500nM showing oAβ42 significantly increases Ca²⁺ activity at concentrations of 250nM and 500nM (n = number of neurons, ***p < 0.001 and ****p < 0.0001, two-tailed student’s t-test). (B) Average frequency and amplitude of Ca²⁺ activity in sAβ42 or oAβ42-treated neurons showing 250nM oAβ42 significantly elevates both frequency and amplitude of Ca²⁺ activity in cultured hippocampal neurons (n = number of neurons, ****p < 0.0001, two-tailed student’s t-test).

Figure 2.. Soluble Aβ42 oligomer-induced hyperexcitability is mediated by GABAergic disinhibition.

(A) Representative traces of GCaMP5 fluorescence intensity in hippocampal neurons and a summary graph of normalized average of total Ca²⁺ activity in each condition showing that a GABA_AR agonist, 25nM muscimol, abolishes oAβ42-induced hyperexcitability (n = number of neurons, ****p < 0.0001, one-way ANOVA, Tukey Test). (B) Representative traces of GCaMP6f fluorescence intensity and a summary graph of normalized average of total Ca²⁺ activity in hippocampal interneurons treated with 250nM sAβ42 (black) or 250nM oAβ42 (red) showing that oAβ42 treatment significantly reduces neuronal Ca²⁺ activity (n = number of neurons, **p < 0.01, two-tailed student’s t-test).

Figure 3.. Selective inhibition of α7 and α4β2 nAChRs mimics Aβ42-induced hyperexcitability in hippocampal pyramidal and inhibitory neurons.

(A) Representative traces of GCaMP6f fluorescence intensity and a summary graph of normalized average of total Ca²⁺ activity in each condition showing application of 50nM αBTX, an α7 nAChR antagonist and 1μM DhβE, an α4β2 nAChR antagonist, together significantly increases neuronal Ca²⁺ activity, similar to oAβ42 treatment. Opposingly, treatment of all three antagonists together, 50nM αBTX, 1μM DhβE, and 3μM αCTX AulB, an α3β4 antagonist, significantly decreases Ca²⁺ activity. Application of each antagonist individually, 50nM αBTX and 3μM αCTX AulB together, or 1μM DhβE and 3μM αCTX AulB together had no effect on Ca²⁺ activity (n = number of neurons, ***p < 0.001, ****p < 0.0001, Tukey Test). (B) Representative traces of GCaMP6f fluorescence intensity and a summary graph of normalized average of total Ca²⁺ activity in each condition showing application of 50nM αBTX and 1μM DhβE together significantly decreases Ca²⁺ activity in interneurons, similar to oAβ42 treatment. Opposingly, treatment of all three antagonists together, 50nM αBTX, 1μM DhβE, and 3μM αCTX AulB significantly increases Ca²⁺ activity in inhibitory cells. Application of each antagonist individually had no effect on Ca²⁺ activity (n = number of neurons, *p < 0.05, ***p < 0.001, one-way ANOVA, Tukey Test).

Figure 4.. Combination treatment of α7 and α4β2 nAChRs abolishes oAβ42-induced hyperexcitation in hippocampal pyramidal and inhibitory cells.

(A) Representative traces of GCaMP5 fluorescence intensity and a summary graph of normalized average of total Ca²⁺ activity in each condition showing that treatment of 1μM PNU-282987 (PNU), an α7 agonist, and 2μM RJR-2403 oxalate (RJR), an α4β2 agonist, together significantly decreases oAβ42-induced Ca²⁺ hyperactivity. Notably, activation of either α7 or α4β2 singularly does not decrease oAβ42-induced Ca²⁺ hyperactivity (n = number of neurons, *p < 0.05, **p < 0.01, ****p < 0.0001, one-way ANOVA, Tukey Test). (B) Representative traces of GCaMP6f fluorescence intensity and a summary graph of normalized average of total Ca²⁺ activity in each condition showing that treatment of 1μM PNU and 2μM RJR together significantly increases oAβ42-induced Ca²⁺ hypoactivity in interneurons (n = number of neurons, *p < 0.05, ****p < 0.0001, one-way ANOVA, Tukey Test).

Figure 5.. Carbachol is unable to rescue oAβ42-induced Ca²⁺ hyperactivity.

Representative traces of GCaMP5 fluorescence intensity and a summary graph of normalized average of total Ca²⁺ activity in each condition showing that application of a non-selective AChR agonist, 1μM carbachol, does not rescue oAβ42-induced Ca²⁺ hyperactivity and exacerbates Ca²⁺ activity in the presence or absence of oAβ42 (n = number of neurons, *p < 0.05, ****p < 0.0001, one-way ANOVA, Tukey Test).

Figure 6.. Soluble Aβ42 oligomer-induced hyperexcitability via selective inhibition of nAChRs in hippocampal interneurons.

Excitability of hippocampal excitatory neurons (E) is regulated by a balance between AMPAR (light green) and NMDAR (dark green)-mediated glutamatergic excitation and GABAergic inhibition via GABA_ARs (red). nAChRs are densely expressed in hippocampal inhibitory interneurons (I) and regulated by cholinergic inputs. oAβ42 (purple) selectively inhibits both α7 (black) and α4β2 (yellow) nAChR subtypes in interneurons but not α3β4 receptors (grey) in excitatory cells to reduce neuronal activity in interneurons, leading to hyperexcitation in hippocampal pyramidal neurons. Therefore, combination treatment of α7 and α4β2 nAChR agonists, PNU and RJR, in the hippocampus can be neuroprotective in AD.