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Decoding the regulation of microglia phenotype by microRNAs in Alzheimer's Disease
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Evaluation of neuroprotective and neurotoxic functions of different microglia phenotypes in Alzheimer’s disease onset and progression, using in vitro and in vivo models
Publication . Caldeira, Cláudia Alexandra Oliveira Lopes, 1973-; Brites, Dora, 1951-; Borralho, Adelaide Maria Afonso Fernandes
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by a progressive cognitive decline, and accumulation of amyloid-β (Aβ) in senile plaques that are associated with inflammatory molecules released by activated glial cells. Among glial cells, microglia that constitute the intrinsic defense system within the central nervous system, may become activated by Aβ aggregates and develop critical neuroprotective and neurotoxic functions with implications in AD onset and progression. Actually,inflammation has been associated with AD, although nonsteroidal anti-inflammatory drugs have not proven efficacy in halting the progression of the disease, reason why they are indicated as beneficial only in the very early stages of AD. Therefore, features of neuroinflammation and associated microglia phenotypes are still open questions in the understanding of AD pathogenesis and neurodegeneration. Another important issue is the association of AD with ageing and the observation of few microglia and accumulation of dystrophic/desensitized microglia in samples from AD old subjects, strongly suggesting their progressive degeneration and diminished replenishment. Investigation of the multiple activated states of microglia when stressed by Aβ, in particular the increased inflammatory status of microglia with aging, referred as primed reactive, or sensitized cell, or in opposite the proposed state of irresponsive aged microglia, are challenged issues once there are no appropriate procedures to isolate degenerative and senescent microglia for experimentation. Therefore, the global aim of this thesis was to explore how different microglia phenotypes and ageing may influence Alzheimer’s disease (AD) pathogenesis and neuroinflammation, by unraveling their associated neuroprotective and neurotoxic functions in in vitro and in vivo models. To achieve these objectives, we first (i) developed an experimental model to naturally age primary microglia, which allowed the evaluation of microglia defensive mechanisms (e.g. migration, phagocytosis, autophagy), of a panoply of inflammatory mediators and of senescence-associated markers, in an acutely isolated and activated microglia [2 days in vitro (2 DIV)] and an aged cell culture (16 DIV). With the characterization of such differently aged microglia, we (ii) progressed towards the assessment of their responsiveness when treated with 1000 nM of Aβ1-42 for 24 h. Finally, in an attempt to translate our in vitro research into the triple transgenic AD (3xTg-AD) mice model, we decided (iii) to explore the inflammatory status of the hippocampus and cortex in animals with 3-, 6- and 9 months-old, by assessing microglia phenotypes, as well as the expression of inflammatory cytokines and microRNAs. We observed in our first study, already published (Caldeira et al Front Cell Neurosci 2014), that (i) in vitro aged microglia switch from a predominant reactive phenotype into cells that although not showing decreased survival, revealed increased dormancy, with morphometric features characteristic of ramified morphology, together with compromised migration, impaired autophagy, reduced phagocytosis, decreased expression of inflamma-miRNAs, and increased presence of senescence-associated markers. In the second study, recently submitted to publication by invitation (Caldeira et al Front Aging Neurosci), using the ageing microglia model and Aβ1-42 treatment, we observed that (ii) Aβ treatment caused soma volume increase and process shortening compatible with activated microglia, in both 2 DIV and 16 DIV cells, together with impairment of neuroprotective functions, namely phagocytosis and migration abilities, as well as autophagy, in in vitro aged microglia. Interestingly, Aβ led to an increased expression of senescence-like associated markers in 2 DIV microglia, similarly to those of the aged cells. Age-dependent changes included the decrease in the expression of inflammatory mediators and surface receptors, together with the reduction of CD11b+ cells and gain of CD86+ microglia and downregulation of miR 155 and miR-124. Lastly, in our in vivo studies at the early-AD stage in the 3xTg-AD at 3 months-old, when Aβ accumulates intraneurally, we observed a downregulation of some activated microglia markers, as well as both typical M1 pro-inflammatory and M2 anti-inflammatory/damage resolution markers. Interestingly, miR-155 revealed to be early upregulated and its increase was sustained at 9 months-old, when extracellular Aβ accumulation is an AD hallmark. At this stage, increase of HMGB1 and decrease of both miR-146a and miR-124 expression is apparent. Curiously, when looking at miR-155 target gene expression we observed new immune-associated molecules that were differently expressed in the 3xTgAD animals by comparison with the wild type mice, both at 3-months and 9-months of age, which will be the subject of study in future works. We may then conclude that the aged in vitro microglia model is very suitable to unravel microglia phenotypic alterations that may explain different cell reactivity in neurodegenerative disorders associated with neuroinflammation and diverse states of disease progression, thus requiring diverse disease-modifying therapies depending on the inflammatory status. We further demonstrate that Aβ induce a heterogeneous population of microglia subtypes instead of only M1 and M2 polarization and that their distribution are age-dependent and influenced by microglia activation state. The increased expression of miR-155 in very early stages of AD in the 3xTg-AD animal model, to be confirmed in AD patients, may additionally reveal as a sensitive biomarker with predictive value if detected in the peripheral blood. The work developed in the present thesis contributed to better define microglial activation phenotypes, in particular the notion of “good” or “bad” states during AD pathogenesis, while identified new targets to be modulated and assessed as predictive biomarkers, with potential relevance for diagnosis and therapeutic tools for developing innovative medicines.
Role of microRNA in microglial phenotype during the progression of Alzheimer’s disease
Publication . Monteiro, Mafalda Aurélio; Fernandes, Adelaide Maria Afonso; Brites, Dora
Alzheimer’s disease (AD) is the most prevalent form of dementia and its impact in society has been aggravating throughout years. Due to its progressive nature and lack of marked treatment benefits, many efforts have been done to unveil AD pathogenesis seeking for novel therapeutic targets or biomarkers.
Current view on AD pathogenesis attributes significant importance to neuroinflammation, where microglia play a pivotal role. Under normal conditions, microglia exhibit a quiescent/vigilant state and perform the brain surveillance. After an injury, microglia initiate the immune defense of the brain and acquire a pro-inflammatory or anti-inflammatory phenotype depending on stimuli. After the inflammation resolution, the brain homeostasis is restored. Various conditions such as the presence of amyloid β-peptide (Aβ) and aging can deregulate microglial response, though it remains unclear how microglial deregulation affect the course of AD. Furthermore, it was established that some microRNAs (miRNAs or miRs) that are known to promote microglial quiescence (miR-124) or regulate microglial activation states (miR-155 and miR-146a) are deregulated in AD. However, it has not been established whether the deregulation of these miRNAs can influence microglial phenotype and response in AD, particularly concerning human microglia.
With this work, we proposed to analyze the temporal response of human CHME3 microglia when co-cultured with two Aβ-expressing human neuroblastoma cells, SH-SY5Y APP695 or SH-SY5Y APP695 Swe cells. We assessed microglia for miRNAs (miR-124, miR-155 and miR-146a) and their targets, as well as for pro-inflammatory (IL-1β, IL-6 and TNF-α), anti-inflammatory (TGFβ, IL-10 and Arginase 1) and immune (iNOS and MHC class II) markers, and additionally for phagocytic capacity and senescence.
We found that when CHME3 microglia are co-cultured with SH-SY5Y APP695 Swe cells they exhibit a more pronounced response than when co-cultured with other neuroblastoma cells. Indeed, in the presence of SH-SY5Y APP695 Swe cells CHME3 microglia initially exhibit a miR-124low/miR-155high/miR-146ahigh profile like activated cells but gradually switch to a miR-124high/miR-155low/miR-146alow profile typical of a gradual shift towards an alternative activated/deactivated phenotype that ultimately give rise to quiescent cells. The pro-inflammatory markers are robustly expressed in microglia during the whole time, but the expression of the anti-inflammatory markers is gradually enhanced suggesting an immunoregulatory response. With regards to immunity, microglia rapidly express the innate immune marker iNOS followed by a later induction of the adaptive immune marker MHC class II.
Altogether, we demonstrated that the CHME3 / SH-SY5Y APP695 Swe co-culture is the most adequate in vitro AD model to study human microglial response and possibly to assay new microglia-targeted therapeutic strategies.
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Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
EXPL/NEU-NMC/1003/2013