Discovery of MDM2-p53 and MDM4-p53 protein-protein interactions small molecule dual inhibitors

MDM2 and MDM4 are key negative regulators of p53, an important protein involved in several cell processes (e. g. cell cycle and apoptosis). Not surprisingly, the p53 tumor suppressor function is inactivated in tumors over -expressing these two proteins. Therefore, both MDM2 and MDM4 are considered important therapeutic targets for an effective reactivation of the p53 function. Herein, we present our studies on the development of spi-ropyrazoline oxindole small molecules able to inhibit MDM2/4-p53 protein-protein interactions (PPIs). Twenty-seven potential spiropyrazoline oxindole dual inhibitors were prepared based on in silico structural optimization studies of a hit compound with MDM2 and MDM4 proteins. The antiproliferative activity of the target com-pounds was evaluated in cancer cell lines harboring wild-type p53 and overexpressing MDM2 and/or MDM4. The most active compounds in SJSA-1 cells, 2q and 3b, induce cell death via apoptosis and control cell growth by targeting the G0/G1 cell cycle checkpoint in a concentration-dependent manner. The ability of the five most active spiropyrazoline oxindoles in dissociating p53 from MDM2 and MDM4 was analyzed by an immu-noenzymatic assay. Three compounds inhibited MDM2/4-p53 PPIs with IC50 values in the nM range, while one compound inhibited more selectively the MDM2-p53 PPI over the MDM4-p53 PPI. Collectively, these results show: i) 3b may serve as a valuable lead for obtaining selective MDM2-p53 PPI inhibitors and more efficient anti-osteosarcoma agents; ii) 2a, 2q and 3f may serve as valuable leads for obtaining dual MDM2/4 inhibitors and more effective p53 activators

Apolipoprotein E ε4 triggers neurotoxicity via cholesterol accumulation, acetylcholine dyshomeostasis, and PKCε mislocalization in cholinergic neuronal cells

16 páginas, 6 figuras, 2 tablasThe Apolipoprotein E (ApoE) has been known to regulate cholesterol and β-amyloid (Aβ) production, redistribution, and elimination, in the central nervous system (CNS). The ApoE ε4 polymorphic variant leads to impaired brain cholesterol homeostasis and amyloidogenic pathway, thus representing the major risk factor for Alzheimer's Disease (AD). Currently, less is known about the molecular mechanisms connecting ApoE ε4-related cholesterol metabolism and cholinergic system degeneration, one of the main AD pathological features. Herein, in vitro cholinergic neuron models were developed in order to study ApoE neuronal expression and investigate the possible interplay between cholesterol metabolism and cholinergic pathway impairment prompted by ε4 isoform. Particularly, alterations specifically occurring in ApoE ε4-carrying neurons (i.e. increased intracellular ApoE, amyloid precursor protein (APP) and Aβ levels, elevated apoptosis, and reduced cell survival) were recapitulated. ApoE ε4 expression was found to increase intracellular cholesterol accumulation, by regulating the related gene expression, while reducing cholesterol precursor acetyl-CoA, which in turn fuels the acetylcholine (ACh) synthesis route. In parallel, although the ACh intracellular signalling was activated, as demonstrated by the boosted extracellular ACh as well as increased IP3 and Ca2+, the PKCε activation via membrane translocation was surprisingly suppressed, probably explained by the cholesterol overload in ApoE ε4 neuron-like cells. Consequently, the PKC-dependent anti-apoptotic and neuroprotective roles results impaired, reliably adding to other causes of cell death prompted by ApoE ε4. Overall, the obtained data open the way to further critical considerations of ApoE ε4-dependent cholesterol metabolism dysregulation in the alteration of cholinergic pathway, neurotoxicity, and neuronal death.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. A.M.B. has a INVESTIGO contract from the Generalitat Valenciana (INVEST/2022/ 456). M.V. acknowledges the Spanish Ministry of Science and Innovation (grant PID2021-127600NB-I00).Peer reviewe

P2‐239: POTENTIAL DIAGNOSTIC VALUE OF RED BLOOD CELLS α‐SYNUCLEIN HETEROAGGREGATES IN ALZHEIMER'S DISEASE

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Proteine correlate a patologie neurodegenerative: accumulo eritrocitario ed intestinale in un modello animale di Alzheimer.

Le malattie neurodegenerative sono un gruppo eterogeneo di malattie del sistema nervoso centrale definite dalla progressiva ed inarrestabile degenerazione di neuroni e glia. Queste malattie, oltre ad essere caratterizzate dalla disfunzione e dalla perdita di neuroni in specifiche aree del sistema nervoso, si distinguono per la presenza di “proteine misfolded”, ovvero caratterizzate da uno scorretto ripiegamento, che si accumulano sia a livello centrale che a livello periferico. Sebbene queste malattie siano distinte da specifici segni e sintomi clinici, condividono evidenti somiglianze e sono state definite “proteinopatie”. La malattia di Alzheimer è una malattia neurodegenerativa, annoverata tra le più importanti “proteinopatie”, che lentamente, progressivamente ed irreversibilmente distrugge la memoria e le funzioni cognitive. Tra le modificazioni patologiche della malattia di Alzheimer è presente atrofia cerebrale, data dalla progressiva perdita di neuroni, in particolare neuroni colinergici, con conseguente riduzione dell’attività della colina acetiltransferasi e del neurotrasmettitore acetilcolina; inoltre si ha la presenza, a livello della corteccia cerebrale, di depositi di proteina beta-amiloide, che a sua volta forma placche extracellulari e lesioni vascolari cerebrali, e di grovigli neurofibrillari intracellulari costituiti da proteina tau. La proteina beta-amiloide è prodotta a partire dalla APP, amyloid precursor protein, che porta a due forme di beta-amiloide: beta-amiloide 1-40 è la forma più comune; beta-amiloide 1-42 è la forma meno abbondante, ma è quella associata alla patologia. La sovrapproduzione di beta-amiloide 1-42 porta all’associazione fra monomeri, che conduce alla formazione di placche amiloidi. La proteina tau è codificata dal gene MAPT ed esiste in varie isoforme. È una fosfoproteina ed alti livelli di fosforilazione sono stati identificati come chiave molecolare della formazione dei grovigli neurofibrillari. L’accumulo delle proteine misfolded sembra verificarsi non solo a livello centrale, ma anche a livello di tessuti periferici, quali sangue ed intestino. Tuttavia, si ritrovano ancora pochi studi di correlazione sull’accumulo di queste proteine tra i vari compartimenti tissutali. Lo scopo di questo lavoro di tesi è stato quello di analizzare i livelli di proteine amiloidogeniche quali beta-amiloide, tau e fosfo-tau in campioni di sangue, di cervello e di intestino in topi SAMP8, topi transgenici che sviluppano spontaneamente la malattia di Alzhaimer, e topi SAMR1, topi di controllo che non sviluppano la malattia. Il sangue, prelevato dai topi a diversi stadi della malattia, è stato sottoposto a centrifugazione per isolare i globuli rossi, cellule nelle quali sono state analizzate le proteine amiloidogenetiche. Il cervello e l’intestino sono stati prelevati post-mortem e sono stati conservati in tampone fosfato. Per verificare la presenza delle proteine beta-amiloide, tau e fosfo-tau, e per determinarne la quantità nei globuli rossi, nel cervello e nell’intestino sono state utilizzate rispettivamente la tecnica di separazione elettroforetica in SDS-PAGE seguita da Western blot ed il saggio immunoenzimatico ELISA. I dati raccolti sono stati sottoposti ad analisi statistica attraverso StatView, un programma che comprende l’analisi della varianza, il t-test e l’analisi di regressione lineare. Al fine di individuare potenziali biomarker periferici della malattia, i risultati ottenuti sono stati analizzati per stabilire una eventuale correlazione cervello-sangue e cervello-intestino, relativamente ai livelli di proteine beta-amiloide, tau e fosfo-tau e di omo-aggregati proteici sia in topi sani, SAMR1, che in topi malati, SAMP8. E' stato quindi possibile valutare il livello di proteine che si accumulano a livello centrale nella malattia di Alzheimer ed equipararlo, in alcuni casi, a quello delle stesse nel sangue e nell’intestino. Questi dati possono contribuire all’utilissima individuazione di nuovi biomarker periferici della malattia. Infatti, in clinica, attualmente, per la diagnosi della malattia di Alzheimer, vengono analizzati i biomarker centrali individuati con imaging diagnostico, oppure biomarker a livello del liquido cefalorachidiano, CFS. Tuttavia, l’imaging è molto costoso ed accessibile solo in centri specializzati e, anche se la raccolta di CSF è una pratica di routine nella neurologia clinica ed il costo per i test di biomarker è molto più basso rispetto a quello dell’imaging, il prelievo lombare di CSF è una procedura complicata, lunga ed invasiva. In conclusione, lo studio eseguito vuole essere un punto di partenza per appurare l’eventuale potenzialità dei livelli di proteine e di omo-aggregati a livello periferico come nuovi potenziali biomarker, nella malattia di Alzheimer, in quanto l’analisi dei biomarker a livello eritrocitario ed intestinale può rappresentare un approccio veloce, semplice, pratico, economic

Signal pathways in aging and neurodegeneration

The current Ph.D. thesis aimed to investigate the signal pathways in aging and neurodegeneration, particularly focusing on the localized and systemic amyloidosis and the corresponding amyloid proteins, thus contributing to novel biomarkers discovery and drug development. Particularly, taking advantage of biochemical and molecular and cellular biology techniques, the current Ph.D. thesis aimed to: 1) investigate misfolded proteins accumulation and inflammatory marker as peripheral prognostic and diagnostic biomarkers for localized amyloidosis, with particular regards to AD and more generally to NDs, establishing their role in reproducing the pathological alterations that occur in the brain also in peripheral tissues, firstly in an animal model of AD and then in NDs patients (i.e. AD and LBDs); 2) explore the influence of ApoE polymorphism on the development of AD-related pathogenic mechanisms, i.e. oxidative stress and misfolded protein accumulation as well as lipid assessment and proteasome activity, also in relation to the lifestyle, in peripheral cells of healthy subject genetically predisposed to develop AD, as putative peripheral prognostic biomarkers; 3) assess the influence of ApoE polymorphism on the cholinergic pathway in a cellular model of AD, i.e. cholinergic-like neurons, evaluating candidate novel central biomarkers and targets for new drug development; 4) compare the stability of TTR fibrils produced by in vitro mechanisms and the ex vivo ones, thus assessing the ability of in vitro models to reproduce the pathology as well as the putative role of kinetic stability of TTR as a biomarker for ATTR and testing TTR-stabilizers as promising drugs

Exploring the Role of Lipid-Binding Proteins and Oxidative Stress in Neurodegenerative Disorders: A Focus on the Neuroprotective Effects of Nutraceutical Supplementation and Physical Exercise

The human brain is primarily composed of lipids, and their homeostasis is crucial to carry on normal neuronal functions. In order to provide an adequate amount of lipid transport in and out of the central nervous system, organisms need a set of proteins able to bind them. Therefore, alterations in the structure or function of lipid-binding proteins negatively affect brain homeostasis, as well as increase inflammation and oxidative stress with the consequent risk of neurodegeneration. In this regard, lifestyle changes seem to be protective against neurodegenerative processes. Nutraceutical supplementation with antioxidant molecules has proven to be useful in proving cognitive functions. Additionally, regular physical activity seems to protect neuronal vitality and increases antioxidant defenses. The aim of the present review was to investigate mechanisms that link lipid-binding protein dysfunction and oxidative stress to cognitive decline, also underlining the neuroprotective effects of diet and exercise

Impact of ApoE Polymorphism and Physical Activity on Plasma Antioxidant Capability and Erythrocyte Membranes

The allele epsilon 4 (ε4) of apolipoprotein E (ApoE) is the strongest genetic risk factor for Alzheimer’s disease (AD). ApoE protein plays a pivotal role in the synthesis and metabolism of amyloid beta (Aβ), the major component of the extracellular plaques that constitute AD pathological hallmarks. Regular exercise is an important preventive/therapeutic tool in aging and AD. Nevertheless, the impact of physical exercise on the well-being of erythrocytes, a good model of oxidative stress and neurodegenerative processes, remains to be investigated, particularly depending on ApoE polymorphism. Herein, we evaluate the oxidative status, Aβ levels, and the membrane’s composition of erythrocytes in a cohort of human subjects. In our hands, the plasma antioxidant capability (AOC), erythrocytes membrane fluidity, and the amount of phosphatidylcholine (PC) were demonstrated to be significantly decreased in the ApoE ε4 genotype and non-active subjects. In contrast, erythrocyte Aβ content and lipid peroxidation increased in ε4 carriers. Regular physical exercise was associated with an increased plasma AOC and membrane fluidity, as well as to a reduced amount of erythrocytes Aβ. Altogether, these data highlight the influence of the ApoE genotype on erythrocytes’ well-being and confirm the positive impact of regular physical exercise

Intermittent hypoxia treatments cause cellular priming in human microglia

Obstructive sleep apnoea syndrome (OSAS) is a sleep-disordered breathing characterized by nocturnal collapses of the upper airway resulting in cycles of blood oxygen partial pressure oscillations, which lead to tissue and cell damage due to intermittent hypoxia (IH) episodes. Since OSAS-derived IH may lead to cognitive impairment through not fully cleared mechanisms, herein we developed a new in vitro model mimicking IH conditions to shed light on its molecular effects on microglial cells, with particular attention to the inflammatory response. The in vitro model was set-up and validated by measuring the hypoxic state, HIF-1α levels, oxidative stress by ROS production and mitochondrial activity by MTS assay. Then, the mRNA and protein levels of certain inflammatory markers (NF-κB and interleukin 6 (IL-6)) after different IH treatment protocols were investigated. The IH treatments followed by a normoxic period were not able to produce a high inflammatory state in human microglial cells. Nevertheless, microglia appeared to be in a state characterized by increased expression of NF-κB and markers related to a primed phenotype. The microglia exposed to IH cycles and stimulated with exogenous IL-1β resulted in an exaggerated inflammatory response with increased NF-κB and IL-6 expression, suggesting a role for primed microglia in OSAS-driven neuroinflammation

The Role of Amyloid-β, Tau, and α-Synuclein Proteins as Putative Blood Biomarkers in Patients with Cerebral Amyloid Angiopathy

Cerebral amyloid angiopathy (CAA) is a cerebrovascular disorder characterized by the deposition of amyloid-β protein (Aβ) within brain blood vessels that develops in elderly people and Alzheimer's disease (AD) patients. Therefore, the investigation of biomarkers able to differentiate CAA patients from AD patients and healthy controls (HC) is of great interest, in particular in peripheral fluids