SNPs in microRNAs and susceptibility to Late-Onset Alzheimer´s disease

Case-control association study of SNPs in microRNAs and susceptibility to Late-Onset Alzheimer´s disease

Evaluation of neuroprotective and neurotoxic functions of different microglia phenotypes in Alzheimer’s disease onset and progression, using in vitro and in vivo models

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2017Alzheimer’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.Fundo Europeu de Desenvolvimento Regional (FEDER); EU Joint Programme - Neurodegenerative Disease Research (JPND), projeto JPco-ND/0003/2015; Grupo de Estudos de Envelhecimento Cerebral e Demência (GEECD), prémio Edgar Cruz e Silva 2012; Fundação Amadeu Dia

Cellular, subcellular, and molecular elements of cerebral malaria pathogenesis

A complex network of elements is responsible for cerebral malaria (CM) development, but interactions between these elements are still being explored. Annually, there are 212 million cases of malaria with 1-2% progressing to CM. Plasma microvesicles (MV) are increased in patients and mice with CM and blocking their release protects against CM. The miRNA content of circulating plasma MV and brain tissue during murine CM and non-CM was assessed using microarray and RT-qPCR techniques. Following infection, MV and brain miRNA were altered in CM mice, coinciding with neurological syndrome onset. Particularly, miR-146a and miR-193b were dysregulated in plasma MV from CM mice and play roles in apoptosis, cytokine regulation and inflammatory cell recruitment. Several other miRNA were dysregulated in the brains of CM mice and play roles in TGF- signalling, endocytosis, FoxO signalling and adherens junctions. Using confocal microscopy, we investigated monocyte, platelet, T cell, parasite and ICAM-1-positive cell accumulation in CM brains. These cells accumulate at vessel branch points, associated with haemorrhaging and the development of tissue hypoxia, consistent with previous findings. As monocytes were the most numerous cells in the brains of CM mice, we targeted them therapeutically. Treatment in vivo with immuno-modulatory particles and artesunate resulted in 88% protection when administered at CM onset, while monotherapies resulted in greatly reduced protection. All successfully treated mice displayed reduced clinical signs of CM and monocyte and MV numbers, and were immune to reinfection. This thesis reinforces existing findings regarding CM pathogenesis and highlights several novel candidates as players in CM development. We explore the potential of miRNA as biomarkers of the neurological syndrome. We show that monocytes displaying a Ly6Clo phenotype aggravate CM development and that therapy targeting this cell subset is a novel strategy to abrogate late-stage CM