Alzheimer’s disease (AD) is a multifactorial neurodegenerative disease and the leading cause of dementia globally. Most of the available drugs only transiently relieve symptoms, and novel therapies have failed in clinical studies, except for the expensive monoclonal antibody Aducanumab (Villain et al. 2022) not yet approved by EMA. There is therefore a pressing need for the development of innovative therapeutic strategies. Proprotein Convertase Subtilisin-like Kexin type 9 (PCSK9), an enzyme that degrades LDL receptor and regulates plasma cholesterol (Cho), is also expressed in central nervous system. Clinical studies have shown increased levels of PCSK9 in the cerebrospinal fluid and the frontal cortex of AD patients (Zimetti et al. 2017, Picard et al. 2019). PCSK9 may play a role in AD pathophysiology including Cho dyshomeostasis (Adorni et al. 2019). For instance, recent findings demonstrated that PCSK9 alters Cho metabolism in astrocytes and neurons, reducing the expression of apoE-interacting receptors and leading to reduced neuronal Cho intake. In addition, PCSK9 overexpression worsened Aβ -induced neurotoxicity (Papotti et al. 2022). The aim of this study was to test the possible protective effects of PCSK9 on AD-related neurodegenerative processes in both in vitro and in vivo models. More specifically, we tested the pro-inflammatory effects of PCSK9 by incubating U373 human astrocytoma cells with Aβ fibrils and human recombinant PCSK9 in vitro. Then, we assessed the pathophysiological consequences of PCSK9 genetic ablation in 5XFAD mice, a well-established transgenic mouse model of AD. In vitro, our results demonstrated that PCSK9 significantly increases IL6, IL1β and TNFα mRNA levels and MCP-1 release in Aβ fibrils-treated U373 cells without influencing inflammasome gene expression. In vivo, PCSK9 ablation significantly improved hippocampal-dependent spatial memory and ameliorated search strategy in 5XFAD mice at the Morris Water Maze test; in addition, a reduction of corticohippocampal Aβ burden was observed, affecting neither plaque spatial/regional distribution and composition nor global BACE1 expression. Furthermore, PCSK9 loss attenuated the inflammatory state in several brain regions of 5XFAD mice decreasing microglial reactivity. Ex vivo analysis showed an altered expression of lipid metabolism-related genes, proinflammatory cytokines and total Cho levels in the brain of 5XFAD mice, that was not significantly affected by the loss of PCSK9. Instead, PCSK9 deletion decreased total Cho levels in the serum of both 5XFAD and control mice. Finally, knocking out PCSK9 had no significant impact on hydroxysterol levels in any experimental groups. In a side project, several newly synthesized lipophilic small molecules (MR-compounds) were tested on human hepatoma cells (HepG2) to evaluate their cytotoxicity and PCSK9 inhibition efficacy. Among them, three compounds were selected for in vivo studies on C57BL/6 mice to evaluate: tolerability with SHIRPA test; plasma lipid profile and biodistribution in liver and brain. Overall, these data support an involvement of PCSK9 in AD pathophysiology, indicating that: 1) PCSK9 enhances Aß-induced neuroinflammatory response in U373 cells; 2) PCSK9 deletion reduces amyloidosis and neuroinflammation improving cognitive performance in 5XFAD mice; 3) MR-compounds inhibit PCSK9 and reverse PCSK9-mediated reduction of Cho uptake in neuronal cells. The subacute application of selected MR compounds to C57BL/6 mice was well tolerated. Present research point to PCSK9 as a promising pharmacological target in AD worth of further investigation and therapeutic development.
La malattia di Alzheimer (AD) è una patologia neurodegenerativa multifattoriale e rappresenta la causa più comune di demenza a livello globale. La maggior parte dei farmaci disponibili allevia solo transitoriamente i sintomi, e nuove terapie hanno fallito negli studi clinici, ad eccezione del costoso anticorpo monoclonale Aducanumab (Villain et al. 2022) non ancora approvato dall'EMA. È quindi urgente sviluppare nuove strategie terapeutiche. La proproteina convertasi subtilisina/kexina di tipo 9 (PCSK9), un enzima che degrada il recettore LDL e regola il colesterolo plasmatico (Cho), è anche espresso nel sistema nervoso centrale. Studi clinici hanno dimostrato un aumento dei livelli di PCSK9 nel liquido cerebrospinale e nella corteccia frontale dei pazienti con AD (Zimetti et al. 2017, Picard et al. 2019). PCSK9 potrebbe svolgere un ruolo nella fisiopatologia dell'AD, alterando l’omeostasi del Cho (Adorni et al. 2019). Ad esempio, recenti scoperte hanno dimostrato che il PCSK9 altera il metabolismo di Cho in astrociti e neuroni, riducendo l'espressione dei recettori che interagiscono con apoE, portando a una riduzione dell’internalizzazione di Cho da parte dei neuroni. Inoltre, la sovraespressione di PCSK9 peggiora la neurotossicità indotta da Aβ (Papotti et al. 2022). I nostri risultati in vitro hanno dimostrato che PCSK9 aumenta significativamente i livelli di mRNA di IL6, IL1β e TNFα e il rilascio di MCP-1 in cellule U373 trattate con fibrille Aβ. In vivo, l'ablazione di PCSK9 nei topi 5XFAD migliora significativamente la memoria spaziale ippocampo-dipendente e le strategie di ricerca nel test del labirinto acquatico di Morris; inoltre, è stata osservata una riduzione dell’accumulo di proteine amiloide (Aβ) nelle regioni corticali e ippocampali, senza alterare né la distribuzione, né la composizione spaziale/regionale della placca, né l'espressione globale di BACE1. Inoltre, tali alterazioni sono accompagnate da un attenuato stato infiammatorio in diverse regioni cerebrali dei topi 5XFAD e dalla ridotta reattività microgliale. L’analisi ex vivo ha mostrato che l’espressione alterata di geni legati al metabolismo lipidico, citochine pro-infiammatorie e livelli totali di Cho nel cervello di topi 5XFAD, non sono significativamente influenzati dalla perdita di PCSK9. Invece, l’assenza di PCSK9 induce una riduzione dei livelli totali di Cho nel siero sia di topi 5XFAD che di quelli di controllo. Infine, l’assenza di PCSK9 non produce effetti significativi sui livelli di idrossisteroli a livello cerebrale. Inoltre, in un progetto parallelo, diverse piccole molecole lipofile di nuova sintesi (composti MR) sono state testate sulle cellule epatiche umane (HepG2) per valutare la loro citotossicità e l'efficacia dell'inibizione di PCSK9. Tra questi, tre composti sono stati selezionati per studi in vivo su topi C57BL/6J per valutare: tollerabilità con test SHIRPA; profilo lipidico plasmatico e biodistribuzione nel fegato e nel cervello. Complessivamente, i dati di questo studio supportano un coinvolgimento di PCSK9 nella patologia di AD, indicando che: 1) PCSK9 aumenta la risposta neuroinfiammatoria indotta da Aß nelle cellule U373; 2) la delezione di PCSK9 riduce l'amiloidosi e la neuroinfiammazione migliorando le capacità cognitive dei topi 5XFAD; 3) i composti MR inibiscono PCSK9 ripristinano i livelli di Cho neuronale. L'applicazione subacuta di componenti MR selezionati ai topi C57BL/6 era ben tollerata. La presente ricerca dimostra che PCSK9 rappresenta un obiettivo farmacologico promettente nell’AD ed è quindi oggetto di ulteriore indagini e di sviluppo terapeutico.
Definire il ruolo di PCSK9 nella fisiopatologia della malattia di Alzheimer: studio in vivo nel modello murino 5XFAD / Martina Bodria , 2024 May 08. 36. ciclo, Anno Accademico 2022/2023.
Definire il ruolo di PCSK9 nella fisiopatologia della malattia di Alzheimer: studio in vivo nel modello murino 5XFAD
BODRIA, MARTINA
2024
Abstract
Alzheimer’s disease (AD) is a multifactorial neurodegenerative disease and the leading cause of dementia globally. Most of the available drugs only transiently relieve symptoms, and novel therapies have failed in clinical studies, except for the expensive monoclonal antibody Aducanumab (Villain et al. 2022) not yet approved by EMA. There is therefore a pressing need for the development of innovative therapeutic strategies. Proprotein Convertase Subtilisin-like Kexin type 9 (PCSK9), an enzyme that degrades LDL receptor and regulates plasma cholesterol (Cho), is also expressed in central nervous system. Clinical studies have shown increased levels of PCSK9 in the cerebrospinal fluid and the frontal cortex of AD patients (Zimetti et al. 2017, Picard et al. 2019). PCSK9 may play a role in AD pathophysiology including Cho dyshomeostasis (Adorni et al. 2019). For instance, recent findings demonstrated that PCSK9 alters Cho metabolism in astrocytes and neurons, reducing the expression of apoE-interacting receptors and leading to reduced neuronal Cho intake. In addition, PCSK9 overexpression worsened Aβ -induced neurotoxicity (Papotti et al. 2022). The aim of this study was to test the possible protective effects of PCSK9 on AD-related neurodegenerative processes in both in vitro and in vivo models. More specifically, we tested the pro-inflammatory effects of PCSK9 by incubating U373 human astrocytoma cells with Aβ fibrils and human recombinant PCSK9 in vitro. Then, we assessed the pathophysiological consequences of PCSK9 genetic ablation in 5XFAD mice, a well-established transgenic mouse model of AD. In vitro, our results demonstrated that PCSK9 significantly increases IL6, IL1β and TNFα mRNA levels and MCP-1 release in Aβ fibrils-treated U373 cells without influencing inflammasome gene expression. In vivo, PCSK9 ablation significantly improved hippocampal-dependent spatial memory and ameliorated search strategy in 5XFAD mice at the Morris Water Maze test; in addition, a reduction of corticohippocampal Aβ burden was observed, affecting neither plaque spatial/regional distribution and composition nor global BACE1 expression. Furthermore, PCSK9 loss attenuated the inflammatory state in several brain regions of 5XFAD mice decreasing microglial reactivity. Ex vivo analysis showed an altered expression of lipid metabolism-related genes, proinflammatory cytokines and total Cho levels in the brain of 5XFAD mice, that was not significantly affected by the loss of PCSK9. Instead, PCSK9 deletion decreased total Cho levels in the serum of both 5XFAD and control mice. Finally, knocking out PCSK9 had no significant impact on hydroxysterol levels in any experimental groups. In a side project, several newly synthesized lipophilic small molecules (MR-compounds) were tested on human hepatoma cells (HepG2) to evaluate their cytotoxicity and PCSK9 inhibition efficacy. Among them, three compounds were selected for in vivo studies on C57BL/6 mice to evaluate: tolerability with SHIRPA test; plasma lipid profile and biodistribution in liver and brain. Overall, these data support an involvement of PCSK9 in AD pathophysiology, indicating that: 1) PCSK9 enhances Aß-induced neuroinflammatory response in U373 cells; 2) PCSK9 deletion reduces amyloidosis and neuroinflammation improving cognitive performance in 5XFAD mice; 3) MR-compounds inhibit PCSK9 and reverse PCSK9-mediated reduction of Cho uptake in neuronal cells. The subacute application of selected MR compounds to C57BL/6 mice was well tolerated. Present research point to PCSK9 as a promising pharmacological target in AD worth of further investigation and therapeutic development.File | Dimensione | Formato | |
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Descrizione: Tesi definitiva Bodria Martina
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