Regular articleIntraneuronal Aβ accumulation induces hippocampal neuron hyperexcitability through A-type K+ current inhibition mediated by activation of caspases and GSK-3
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.
Section snippets
Animals
A colony of 3xTg-AD (harboring the PS1 M146V, APPSwe KM670/671NL, and tau P301L transgenes) and non-transgenic (B6129SF2/J, named non-Tg) mice were used for electrophysiological experiments in brain slices and for Western blot analysis. The colonies were established in-house from breeding pairs purchased by the Jackson Laboratory. All animal procedures were approved by the Ethics Committee of the Università Cattolica “S. Cuore” and were fully compliant with the Italian and European Union
Intracellular Aβ42 increases neuronal firing in primary mouse hippocampal neurons
To investigate whether iAβ42 affects intrinsic excitability, whole-cell patch-clamp recordings were performed on primary mouse hippocampal neurons in current-clamp mode. Action potential firing was examined by exposing cells to a series of 800 ms current pulses (1 every 5 minutes), whereas recombinant Aβ42 (200 nM) was intracellularly perfused through the patch pipette. The amplitude of current pulses ranged from −50 pA (hyperpolarizing, subthreshold) to 200 pA (depolarizing, suprathreshold) in
Discussion
The present study revealed that intracellularly injected Aβ42 downregulates A-type K+ currents and increases spike width of hippocampal pyramidal neurons, thereby increasing excitability. These effects require caspases and GSK-3 activation and correlate with an increase in phosphorylation of a molecular determinant of A-type K+ currents, the Kv4.2 subunit, at Ser-616, a predicted GSK-3 site. At an early AD stage (5 months old), the 3xTg-AD mouse model exhibits intracellular accumulation of Aβ
Disclosure statement
None of the authors has any actual or potential conflicts of interest to report. All animal procedures were approved by the Ethics Committee of the Catholic University and complied with the Italian Ministry of Health guidelines and with national laws (legislative decree 116/1992) and the European Union guidelines on animal research (No. 86/609/EEC).
Acknowledgements
This work was supported by grants from Università Cattolica (D1-2012 #70200969 to Claudio Grassi and Marcello D’Ascenzo), Alzheimer's Association (NIRG-14-321307; to Cristian Ripoli), the Institute for Translational Sciences at the University of Texas Medical Branch as part of a Clinical and Translational Science Award (UL1TR000071, Fernanda Laezza) from the National Center for Advancing Translational Sciences, National Institutes of Health (NIH NIMH R01 MH095995 to Fernanda Laezza).
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