Showing papers by "Sandra M. Cardoso published in 2021"

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

Abstract: In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Footprints of a microbial toxin from the gut microbiome to mesencephalic mitochondria

Abstract: This work was funded by Santa Casa da Misericordia de Lisboa, Portugal, through Mantero Belard Neurosciences Prize 2016 (MB-40-2016); by FMUCPEPITA (2018); by the European Regional Development Fund (ERDF), through the Centro 2020 Regional Operational Programme under project CENTRO-01-0145-FEDER-000012 (HealthyAging 2020) and through the COMPETE 2020 - Operational Programme for Competitiveness and Internationalization and by Portuguese national funds via FCT—Fundacao para a Ciencia e a Tecnologia under projects PTDC/MED-NEU/3644/2020, PINFRA/22184/2016/POCI-01-0145-FEDER-022184 and UIDB/04539/2020. EC was supported by fellowship MB-40-2016. IT was supported by IF/01061/2014 Investigator contract. JDM is supported by PhD fellowship PD/BD/146409/2019, DN-C is supported by PhD fellowship SFRH/BD

Structural and Functional Alterations in Mitochondria-Associated Membranes (MAMs) and in Mitochondria Activate Stress Response Mechanisms in an In Vitro Model of Alzheimer’s Disease

Abstract: Alzheimer's disease (AD) is characterized by the accumulation of extracellular plaques composed by amyloid-β (Aβ) and intracellular neurofibrillary tangles of hyperphosphorylated tau. AD-related neurodegenerative mechanisms involve early changes of mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) and impairment of cellular events modulated by these subcellular domains. In this study, we characterized the structural and functional alterations at MAM, mitochondria, and ER/microsomes in a mouse neuroblastoma cell line (N2A) overexpressing the human amyloid precursor protein (APP) with the familial Swedish mutation (APPswe). Proteins levels were determined by Western blot, ER-mitochondria contacts were quantified by transmission electron microscopy, and Ca2+ homeostasis and mitochondria function were analyzed using fluorescent probes and Seahorse assays. In this in vitro AD model, we found APP accumulated in MAM and mitochondria, and altered levels of proteins implicated in ER-mitochondria tethering, Ca2+ signaling, mitochondrial dynamics, biogenesis and protein import, as well as in the stress response. Moreover, we observed a decreased number of close ER-mitochondria contacts, activation of the ER unfolded protein response, reduced Ca2+ transfer from ER to mitochondria, and impaired mitochondrial function. Together, these results demonstrate that several subcellular alterations occur in AD-like neuronal cells, which supports that the defective ER-mitochondria crosstalk is an important player in AD physiopathology.

Derivatives of Tenuazonic Acid as Potential New Multi-Target Anti-Alzheimer’s Disease Agents

Abstract: Alzheimer’s disease (AD) is generally recognized as a multifactorial neurodegenerative pathology with an increasing impact on society. Tenuazonic acid (TA) is a natural compound that was recently identified as a potential multitarget ligand with anti-cholinesterase, anti-amyloidogenic and antioxidant activities. Using its structure as a chemical scaffold, we synthesized and evaluated new derivatives (1–5), including tenuazonic-donepezil (TA-DNP) hybrids (4 and 5) due to the clinical importance of the anti-AD drug donepezil. These novel compounds all achieved activity in the micromolar range towards all selected targets and demonstrated to be potentially orally absorbed. Moreover, a selected compound (1) was further investigated as a chelating agent towards copper (II), zinc (II) and iron (III) and showed good chelating ability (pFe = 16.6, pCu = 11.6, pZn = 6.0 at pH 7.4). Therefore, the TA motif can be considered an interesting building block in the search for innovative multi-functional anti-neurodegenerative drugs, as exemplified by hybrid 5, a promising non-cytotoxic lead compound adequate for the early stages of AD, and capable of ameliorating the oxidative status of SH-SY5Y human neuroblastoma cells.

Novel Donepezil-Arylsulfonamide Hybrids as Multitarget-Directed Ligands for Potential Treatment of Alzheimer's Disease.

Abstract: Alzheimer's disease (AD) is one of the most devastating neurodegenerative disorders, characterized by multiple pathological features. Therefore, multi-target drug discovery has been one of the most active fields searching for new effective anti-AD therapies. Herein, a series of hybrid compounds are reported which were designed and developed by combining an aryl-sulfonamide function with a benzyl-piperidine moiety, the pharmacophore of donepezil (a current anti-AD acetylcholinesterase AChE inhibitor drug) or its benzyl-piperazine analogue. The in vitro results indicate that some of these hybrids achieve optimized activity towards two main AD targets, by displaying excellent AChE inhibitory potencies, as well as the capability to prevent amyloid-β (Aβ) aggregation. Some of these hybrids also prevented Aβ-induced cell toxicity. Significantly, drug-like properties were predicted, including for blood-brain permeability. Compound 9 emerged as a promising multi-target lead compound (AChE inhibition (IC50 1.6 μM); Aβ aggregation inhibition 60.7%). Overall, this family of hybrids is worthy of further exploration, due to the wide biological activity of sulfonamides.

Macroautophagy and Mitophagy in Neurodegenerative Disorders: Focus on Therapeutic Interventions.

Abstract: Macroautophagy, a quality control mechanism, is an evolutionarily conserved pathway of lysosomal degradation of protein aggregates, pathogens, and damaged organelles. As part of its vital homeostatic role, macroautophagy deregulation is associated with various human disorders, including neurodegenerative diseases. There are several lines of evidence that associate protein misfolding and mitochondrial dysfunction in the etiology of Alzheimer's, Parkinson's, and Huntington's diseases. Macroautophagy has been implicated in the degradation of different protein aggregates such as Aβ, tau, alpha-synuclein (α-syn), and mutant huntingtin (mHtt) and in the clearance of dysfunctional mitochondria. Taking these into consideration, targeting autophagy might represent an effective therapeutic strategy to eliminate protein aggregates and to improve mitochondrial function in these disorders. The present review describes our current understanding on the role of macroautophagy in neurodegenerative disorders and focuses on possible strategies for its therapeutic modulation.

The Mitochondrial Ribosome: A World of Opportunities for Mitochondrial Dysfunction Toward Parkinson's Disease.

Abstract: Significance: Mitochondrial ribosomes (mitoribosomes) are organelles that translate mitochondrial messenger RNA in the matrix and, in mammals, have evolved to translate 13 polypeptides of the pathw...