Piccialli, Ilaria (2017) Characterizaton of the role played by NCX isoform 3 in a transgenic model of Alzheimer's disease by electrophysiological and biochemical studies. [Tesi di dottorato]

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Item Type: Tesi di dottorato
Resource language: English
Title: Characterizaton of the role played by NCX isoform 3 in a transgenic model of Alzheimer's disease by electrophysiological and biochemical studies
Creators:
CreatorsEmail
Piccialli, Ilariapiccialli.ilaria@gmail.com
Date: 10 December 2017
Number of Pages: 126
Institution: Università degli Studi di Napoli Federico II
Department: dep16
Dottorato: phd058
Ciclo di dottorato: 30
Coordinatore del Corso di dottorato:
nomeemail
Annunziato, Luciolannunzi@unina.it
Tutor:
nomeemail
Pannaccione, AnnaUNSPECIFIED
Date: 10 December 2017
Number of Pages: 126
Keywords: ALzheimer's disease, Na+/Ca2+ exchanger, ionic homeostasis
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/14 - Farmacologia
Date Deposited: 08 Jan 2018 11:24
Last Modified: 20 Mar 2019 12:05
URI: http://www.fedoa.unina.it/id/eprint/12173

Collection description

Alzheimer’s disease (AD), the most common neurodegenerative disorder is characterized by progressive memory loss and impairment of cognitive ability. Aβ1-42 deposition, the principal hallmark of AD, triggers several mechanisms, including dysregulation of ionic homeostasis, contributing to neuronal dysfunction and death. In particular, the dysregulation of intracellular calcium concentrations ([Ca2+]i) triggers a series of events including oxidative damage and activation of apoptotic machinery. Furthermore, the dysregulation of intracellular sodium concentrations ([Na+]i) affects neuronal excitability and contributes to epileptogenesis in AD. The Na+/Ca2+ exchanger (NCX) couples in a bidirectional manner the exchange of 3Na+ for 1Ca2+, thereby playing a relevant role in maintaining intracellular Na+ and Ca2+ homeostasis. For this reason, we investigated the role of NCX3 in Aβ1-42-induced ionic dysregulation in primary hippocampal neurons from Tg2576 mice, a transgenic animal model of AD. First, we validated primary hippocampal neurons from Tg2576 mice, here set up for the first time, as an in vitro model of AD by confirming the presence of Aβ1-42 oligomers through western blot experiments. In particular, we observed Aβ1-42 trimers, detectable as a ~ 12 kDa band, in Tg2576 primary hippocampal neurons, whereas they were absent in Wild Type (WT) neurons. Importantly, the same band has been detected in the hippocampus of 3-month-old Tg2576 mice. Patch clamp experiments revealed that NCX activity was progressively up-regulated in the reverse mode of operation in Tg2576 hippocampal neurons compared to WT, at 8 and 12 DIV, whereas no modulation occurred in the forward mode. Furthermore, silencing experiments with a specific siRNA directed against NCX3, revealed that this increase of NCX currents was mediated by only NCX isoform 3. However, as revealed by western blot analyses, the up-regulation of NCX3 activity was not accompanied by a significant increase of NCX3 protein expression. Interestingly, [Na+] detection with SBFI probe showed a significant increase of [Na+]i in Tg2576 hippocampal neurons at 12 DIV compared to WT, thus indicating that the up-regulation of NCX activity was Na+-dependent. Moreover, electrophysiological experiments revealed that NaV currents were progressively up-regulated in Tg2576 hippocampal neurons compared to WT at 8 and 12 DIV. To determine whether the up-regulation of NCX activity results in increased [Ca2+]i or rather in Ca2+ refilling into ER, we performed Fura-2 AM measurements to determine both [Ca2+]i and ER Ca2+ content. In particular, we found a significant reduction of [Ca2+]i in Tg2576 hippocampal neurons at 8 DIV compared to WT and a significant increase in ER Ca2+ content at 12 DIV. Western blot on Tg2576 mouse brain, revealed that NCX3 protein expression was significantly increased in the hippocampus of 3-month-old Tg2576 mice compared to WT. Importantly, immunohistochemical analyses confirmed western blot results. In fact, in both CA1 and CA3 hippocampal regions as well as within the corpus callosum of 3-month-old Tg2576 mice, the anti-NCX3 antibody revealed an increased NCX3 immunoreactivity signal, which was mainly confined along the processes of cells and dendrites of pyramidal cells. By contrast, we observed a significant reduction of NCX3 protein expression in the hippocampus of 8-month-old Tg2576 compared to WT, although an increase in the hippocampus of 8-month-old WT mice has been observed in comparison with 3-month-old WT mice. Importantly, immunohistochemical analyses confirmed western blot results. In fact, in both CA1 and CA3 hippocampal regions as well as within the corpus callosum of 8-month-old Tg2576 mice, NCX3 immunostaining appeared robustly decreased. Moreover, western blot experiments did not detect any modulation of NCX3 protein expression in the cerebral cortex of both 3 and 8-month-old Tg2576 mice compared to WT. On the other hand, a clear loss of intensity of immunoreactivity signal has been observed in cortical sections from both 3 and 8-month-old Tg2576 mice. All together, these results suggest that NCX3 up-regulation could represent a protective mechanism against Na+ disruption occurring in Tg2576 hippocampal neurons. Notably, this evidence points to a possible role of NCX3 in neuronal survival against hyperexcitability and subsequent epileptiform activity observed in AD. Moreover, the role of NCX3 in Ca2+ refilling into ER further supports its positive implication.

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