Alzheimer’s disease (AD) is pathologically characterized by amyloid beta (Aβ) depositions and hyperphosphorylated tau. A disintegrin and metalloproteinase 10 (ADAM10), is responsible for the α-secretase cleavage of Amyloid Precursor Protein (APP), that prevents Aβ generation. The synaptic localization and its activity towards APP are modulated by protein partners, which interact with the cytoplasmic tail of ADAM10. Considering this, we performed a two-hybrid screening that identified actin-binding protein “cyclase-associated protein 2” (CAP2) as a novel binding partner of ADAM10. CAPs are implicated in actin cytoskeleton remodelling and vesicle trafficking of non-neuronal cells, and microarray analysis of AD patient hippocampi specimens revealed a down-regulation of CAP2 in AD patients. Therefore, the aim of this project is to evaluate the role of the ADAM10/CAP2/actin complex in the amyloid cascade and in AD-related synaptic dysfunction. The main results of the project indicate that: - CAP2 is self-aggregating and interacts with both actin and ADAM10. - In the hippocampus of CAP2 knockout mice the synaptic levels of ADAM10 and of the NMDA receptor subunit GluN2A are significantly reduced, suggesting that the lack of CAP2 affects their localization. In particular, the CAP2/actin complex is essential for ADAM10 synaptic localization since it is involved in ADAM10 endocytosis. Additionally, we detect an altered spine density and morphology in hippocampal neurons of CAP2 knockout mice, and they show elevated levels of the toxic Aβ1-42 in their hippocampus. These data indicate the relevance of CAP2 in synaptic function and structural plasticity. - Regarding AD, a reduction in CAP2 protein levels and in its synaptic localization is detected in the hippocampi of AD patients and AD mice. In addition, alterations in the association of CAP2 to actin and ADAM10 are found. - CAP2 has a role in activity-dependent synaptic plasticity mediated by long-term potentiation, and in aberrant plasticity triggered by Aβ oligomers, which regulate CAP2 synaptic localization and association to its binding partners. In light of these results, we designed two potential therapeutic approaches to tackle the CAP2 pathological pathway in AD. First, we overexpressed CAP2 in an in vitro system modeling AD, and we found an increase in ADAM10 synaptic levels after overexpression of CAP2. Secondly, since the CAP2-actin complex is increased in AD pathogenesis and this complex seems to be important for ADAM10 synaptic localization, we designed a cell-permeable peptide (CPP) to interfere with the CAP2-actin association. The interference by the CPP treatment can affect ADAM10 synaptic levels and increases its activity towards APP. Overall these data indicate that CAP2 is a potential pharmacological target to increase ADAM10 synaptic localization as a novel AD therapeutic strategy.
La malattia di Alzheimer (AD) è caratterizzata da depositi di beta-Amiloide (Aβ) e di proteina tau iperfosforilata. L’enzima ADAM10 taglia la Proteina Precursore dell’Amiloide (APP) impedendo la formazione di Aβ. L’attività e la localizzazione sinaptica di ADAM10 sono modulate da proteine che interagiscono con la coda citoplasmatica di ADAM10. Ciò considerato, abbiamo condotto un “two-hybrid screening” che ha permesso l’identificazione di cyclase-associated protein 2 (CAP2) come nuovo partner di ADAM10. Le CAPs sono proteine che legano l’actina e sono coinvolte in rimodellamento del citoscheletro e trafficking vescicolare in cellule non neuronali. Un’analisi di microarray ha rivelato come in campioni autoptici da pazienti affetti da AD vi sia una riduzione dell’espressione di CAP2. Pertanto, lo scopo del presente progetto è valutare il ruolo del complesso ADAM10/CAP2/actina nella formazione di Aβ e nella disfunzione sinaptica legata all’AD. I principali risultati di questo progetto indicano che: - CAP2 è in grado di formare aggregati e di interagire sia con actina che ADAM10 - È nell’ippocampo di topi CAP2 knock-out, i livelli postsinaptici di ADAM10 e della subunità GluN2A del recettore NMDA siano drasticamente ridotti, indicando come possibile causa la mancanza di CAP2. Il complesso CAP2/actina influenza l’endocitosi di ADAM10, perciò la sua modulazione è fondamentale per la localizzazione sinaptica di ADAM10. L’analisi di neuroni ippocampali di topi CAP2 knock-out ha evidenziato un’alterazione della morfologia delle spine dendritiche, indicando come la mancanza di CAP2 possa influenzare la plasticità strutturale dei neuroni. Inoltre abbiamo rilevato un aumento dei livelli di Aβ1-42 nell’ippocampo di topi CAP2 knock-out. - Per quanto riguarda AD, nei campioni di ippocampo di pazienti affetti da AD, così come nell’ippocampo di un modello murino di AD, è riscontrabile riduzione di CAP2 e la sua localizzazione sinaptica. In aggiunta, il complesso ADAM10/CAP2/actina è alterato in questi stessi campioni. - CAP2 ha un ruolo importante nei fenomeni di plasticità sinaptica attività-dipendente e nella plasticità aberrante indotta da oligomeri di Aβ, processi in grado di modificare la localizzazione sinaptica di CAP2 e l’interazione con i suoi partner. Alla luce di questi risultati, abbiamo disegnato due approcci terapeutici per modulare la via di CAP2. Nel primo caso, abbiamo overespresso CAP2 in un modello in vitro di AD e abbiamo osservato un aumento della localizzazione sinaptica di ADAM10. In secondo luogo, poiché l’interazione CAP2/Actina è aumentata in AD e questo complesso è importante per la localizzazione sinaptica di ADAM10, abbiamo sviluppato dei peptidi permeabili alle cellule (CPP) in grado di interferire con l’interazione CAP2/Actina. Il trattamento con CPP determina un incremento dei livelli sinaptici di ADAM10 e ne aumenta l’attività nei confronti di APP. In conclusione, dal nostro studio emerge come CAP2 sia potenziale target farmacologico per AD.
UNDERSTANDING THE EARLY STAGES OF ALZHEIMER¿S DISEASE PATHOGENESIS: CAP2 AS A LINK BETWEEN SPINE DYSMORPHOGENESIS AND THE AMYLOID CASCADE / L.l. Vandermeulen ; cotutor: A.L. Catapano; tutor: M. Diluca ; coordinator: A. L. Catapano. DIPARTIMENTO DI SCIENZE FARMACOLOGICHE E BIOMOLECOLARI, 2019 Jun 24. 31. ciclo, Anno Accademico 2018. [10.13130/vandermeulen-lina-lorna_phd2019-06-24].
UNDERSTANDING THE EARLY STAGES OF ALZHEIMER¿S DISEASE PATHOGENESIS: CAP2 AS A LINK BETWEEN SPINE DYSMORPHOGENESIS AND THE AMYLOID CASCADE.
L.L. Vandermeulen
2019
Abstract
Alzheimer’s disease (AD) is pathologically characterized by amyloid beta (Aβ) depositions and hyperphosphorylated tau. A disintegrin and metalloproteinase 10 (ADAM10), is responsible for the α-secretase cleavage of Amyloid Precursor Protein (APP), that prevents Aβ generation. The synaptic localization and its activity towards APP are modulated by protein partners, which interact with the cytoplasmic tail of ADAM10. Considering this, we performed a two-hybrid screening that identified actin-binding protein “cyclase-associated protein 2” (CAP2) as a novel binding partner of ADAM10. CAPs are implicated in actin cytoskeleton remodelling and vesicle trafficking of non-neuronal cells, and microarray analysis of AD patient hippocampi specimens revealed a down-regulation of CAP2 in AD patients. Therefore, the aim of this project is to evaluate the role of the ADAM10/CAP2/actin complex in the amyloid cascade and in AD-related synaptic dysfunction. The main results of the project indicate that: - CAP2 is self-aggregating and interacts with both actin and ADAM10. - In the hippocampus of CAP2 knockout mice the synaptic levels of ADAM10 and of the NMDA receptor subunit GluN2A are significantly reduced, suggesting that the lack of CAP2 affects their localization. In particular, the CAP2/actin complex is essential for ADAM10 synaptic localization since it is involved in ADAM10 endocytosis. Additionally, we detect an altered spine density and morphology in hippocampal neurons of CAP2 knockout mice, and they show elevated levels of the toxic Aβ1-42 in their hippocampus. These data indicate the relevance of CAP2 in synaptic function and structural plasticity. - Regarding AD, a reduction in CAP2 protein levels and in its synaptic localization is detected in the hippocampi of AD patients and AD mice. In addition, alterations in the association of CAP2 to actin and ADAM10 are found. - CAP2 has a role in activity-dependent synaptic plasticity mediated by long-term potentiation, and in aberrant plasticity triggered by Aβ oligomers, which regulate CAP2 synaptic localization and association to its binding partners. In light of these results, we designed two potential therapeutic approaches to tackle the CAP2 pathological pathway in AD. First, we overexpressed CAP2 in an in vitro system modeling AD, and we found an increase in ADAM10 synaptic levels after overexpression of CAP2. Secondly, since the CAP2-actin complex is increased in AD pathogenesis and this complex seems to be important for ADAM10 synaptic localization, we designed a cell-permeable peptide (CPP) to interfere with the CAP2-actin association. The interference by the CPP treatment can affect ADAM10 synaptic levels and increases its activity towards APP. Overall these data indicate that CAP2 is a potential pharmacological target to increase ADAM10 synaptic localization as a novel AD therapeutic strategy.File | Dimensione | Formato | |
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phd_unimi_R11497.pdf
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