Intraneuronal Aβ accumulation induces hippocampal neuron hyperexcitability through A-type K⁺ current inhibition mediated by activation of caspases and GSK-3

Abstract

Amyloid β-protein (Aβ) pathologies have been linked to dysfunction of excitability in neurons of the hippocampal circuit, but the molecular mechanisms underlying this process are still poorly understood. Here, we applied whole-cell patch-clamp electrophysiology to primary hippocampal neurons and show that intracellular Aβ₄₂ delivery leads to increased spike discharge and action potential broadening through downregulation of A-type K⁺ currents. Pharmacologic studies showed that caspases and glycogen synthase kinase 3 (GSK-3) activation are required for these Aβ₄₂-induced effects. Extracellular perfusion and subsequent internalization of Aβ₄₂ increase spike discharge and promote GSK-3-dependent phosphorylation of the Kv4.2 α-subunit, a molecular determinant of A-type K⁺ currents, at Ser-616. In acute hippocampal slices derived from an adult triple-transgenic Alzheimer's mouse model, characterized by endogenous intracellular accumulation of Aβ₄₂, CA1 pyramidal neurons exhibit hyperexcitability accompanied by increased phosphorylation of Kv4.2 at Ser-616. Collectively, these data suggest that intraneuronal Aβ₄₂ accumulation leads to an intracellular cascade culminating into caspases activation and GSK-3-dependent phosphorylation of Kv4.2 channels. These findings provide new insights into the toxic mechanisms triggered by intracellular Aβ₄₂ and offer potentially new therapeutic targets for Alzheimer's disease treatment.

Introduction

Alzheimer's disease (AD) is the most common form of dementia with a high prevalence among the aging population (Citron, 2010). Extracellular fibrillar amyloid β-protein (Aβ) accumulation and neurofibrillary tangles composed of hyperphosphorylated tau are considered the neuropathologic hallmarks of AD (Selkoe, 2001). Aβ originates from the amyloid β-precursor protein (APP) through sequential cleavage by the β- and the γ-secretase enzyme complex (Thinakaran and Koo, 2008). Most of the full-length Aβ peptide consists of the 40-amino-acid long Aβ (Aβ₄₀), whereas a small proportion (10%) is the 42-residues variant (Aβ₄₂), a species that because of its high hydrophobicity has been considered more prone to fibril formation and neurotoxicity than the Aβ₄₀ (Jarrett et al., 1993).

A plethora of neurophysiological studies have reported toxic activity of Aβ₄₂ oligomers on synaptic function and activity-dependent plasticity in the hippocampus and cerebral cortex building the notion that AD is a synaptopathy (Crimins et al., 2013, Klyubin et al., 2012, Selkoe, 2002, Shankar et al., 2008, Sheng et al., 2012). It is believed, indeed, that disruption in synaptic structure and function induced by Aβ₄₂ is one of the underlying mechanisms leading to the aberrant neuronal processing and network dysfunction that characterize AD cognitive impairment and memory loss (Mucke and Selkoe, 2012). Surprisingly, although, much less knowledge has been gained on other Aβ toxic effects on neuronal activity that might precede synaptic dysfunction and as such could be targeted for early therapeutic interventions against AD.

Emerging evidence indicates that changes in neuronal excitability could play a fundamental role in initiating early Aβ₄₂ pathology predisposing to and/or triggering subsequent synaptic dysfunction. In vitro and animal model studies have demonstrated that Aβ pathology associates with increased excitability of hippocampal neurons, leading to hypersynchronous network activity and higher risk for seizures (Born et al., 2014, Brown et al., 2011, Busche et al., 2012, Davis et al., 2014, Del Vecchio et al., 2004, Minkeviciene et al., 2009, Palop et al., 2007, Putcha et al., 2011). Epidemiologic studies have confirmed comorbidity between patients with AD and epilepsy, showing increased risk of seizures in AD patients (Amatniek et al., 2006, Hommet et al., 2008). This repertoire of cellular and clinical studies indicating potential commonalities in early AD and epilepsy opens new horizons in the understanding of early AD triggers and therapies. However, despite this evidence, the molecular mechanisms linking Aβ pathology to aberrant neuronal firing is still poorly understood. A missing link in associating Aβ effects to neuronal excitability is the identification of the source of toxic oligomers.

Early studies based on the observation of senile plaques in the extracellular space (Hardy and Selkoe, 2002) have pointed for extracellular Aβ₄₂ (eAβ₄₂) as the primary cause of AD. However, in studies on postmortem AD and mild cognitively impaired patients immunoreactivity of intracellular Aβ₄₂ (iAβ₄₂) has been identified in neurons of the hippocampus and entorhinal cortex (Gouras et al., 2000, Mori et al., 2002), the 2 main brain regions affected in early AD pathology. These findings have been corroborated by results from transgenic mouse models (Guzmán et al., 2014, Youmans et al., 2012; for review see; LaFerla et al., 2007), leading to the overall hypothesis of iAβ accumulation as an early triggering event in the AD progression preceding senile plaques (Kuo et al., 2001, LaFerla et al., 2007, Oakley et al., 2006). Intracellular Aβ-driven effects on neuronal firing might therefore be one of the earliest detectable triggers of the AD pathology preceding and predisposing to synaptic deficits.

Building on the growing interest in identifying early Aβ targets, we studied the effect of iAβ₄₂ on neuronal excitability in primary hippocampal neurons and in CA1 hippocampal neurons of triple-transgenic AD (3xTg-AD) mouse model that harbor the mutant genes for amyloid precursor protein (APPSwe), presenilin 1PS1M146V and for tauP301L (Oddo et al., 2003) and overexpresses intracellular Aβ₄₂.

We found that Aβ₄₂ internalization is required for inducing increase in intrinsic excitability in hippocampal pyramidal neurons, an effect that is mediated by inhibition of A-type K⁺ channels (Kv), and requires caspases activation and glycogen synthase kinase 3- (GSK-3) dependent phosphorylation of Kv4.2. Parallel changes in intrinsic excitability, A-type K⁺ currents, and Kv4.2 phosphorylation are found in CA1 hippocampal neurons derived from brain slices of 3xTg-AD mice.