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Presymptomatic and symptomatic ALS SOD1(G93A) mice differ in adenosine A1 and A2A receptor-mediated tonic modulation of neuromuscular transmission

Publication . Nascimento, Filipe; Sebastião, Ana M; Ribeiro, Joaquim A.

Amyotrophic lateral sclerosis (ALS) is a disease leading to neuromuscular transmission impairment. A2A adenosine receptor (A2AR) function changes with disease stage, but the role of the A(1) receptors (A1Rs) is unknown and may have a functional cross-talk with A2AR. The role of A1R in the SOD1(G93A) mouse model of ALS in presymptomatic (4-6 weeks old) and symptomatic (12-14 weeks old) phases was investigated by recording endplate potentials (EPPs), miniature endplate potentials (MEPPs), and quantal content (q.c.) of EPPs, from Mg(2+) paralyzed hemidiaphragm preparations. In presymptomatic mice, the A1R agonist, N (6)-cyclopentyladenosine (CPA) (50 nM), decreased mean EPP amplitude, MEPP frequency, and q.c. of EPPs, an effect quantitatively similar to that in age-matched wild-type (WT) mice. However, coactivation of A2AR with CGS 21680 (5 nM) prevented the effects of CPA in WT mice but not in presymptomatic SOD1(G93A) mice, suggestive of A1R/A2AR cross-talk disruption in this phase of ALS. DPCPX (50 nM) impaired CGS 21680 facilitatory action on neuromuscular transmission in WT but not in presymptomatic mice. In symptomatic animals, CPA only inhibited transmission if added in the presence of adenosine deaminase (ADA, 1 U/mL). ADA and DPCPX enhanced more transmission in symptomatic mice than in age-matched WT mice, suggestive of increase in extracellular adenosine during the symptomatic phase of ALS. The data documents that at the neuromuscular junction of presymptomatic SOD1(G93A) mice, there is a loss of A1R-A2AR functional cross-talk, while in symptomatic mice there is increased A1R tonic activation, and that with disease progression, changes in A1R-mediated adenosine modulation may act as aggravating factors during the symptomatic phase of ALS.

2015Journal article

Restricted access

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.

2017Doctoral thesis

Open access

Downregulated glia interplay and increased miRNA-155 as promising markers to track ALS at an early stage

Publication . Cunha, Carolina; Santos, Catarina; Gomes, Cátia; Fernandes, Adelaide; Marcal Correia, Alexandra; Sebastião, Ana M; Vaz, Ana R; Brites, Dora

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of unknown cause. Absence of specific targets and biomarkers compromise the development of new therapeutic strategies and of innovative tools to stratify patients and assess their responses to treatment. Here, we investigate changes in neuroprotective-neuroinflammatory actions in the spinal cord of SOD1 G93A mice, at presymptomatic and symptomatic stages to identify stage-specific biomarkers and potential targets. Results showed that in the presymptomatic stage, there are alterations in both astrocytes and microglia, which comprise decreased expression of GFAP and S100B and upregulation of GLT-1, as well as reduced expression of CD11b, M2-phenotype markers, and a set of inflammatory mediators. Reduced levels of Connexin-43, Pannexin-1, CCL21, and CX3CL1 further indicate the existence of a compromised intercellular communication. In contrast, in the symptomatic stage, increased markers of inflammation became evident, such as NF-κB/Nlrp3-inflammasome, Iba1, pro-inflammatory cytokines, and M1-polarizion markers, together with a decreased expression of M2-phenotypic markers. We also observed upregulation of the CX3CL1-CX3CR1 axis, Connexin-43, Pannexin-1, and of microRNAs (miR)-124, miR-125b, miR-146a and miR-21. Reduced motor neuron number and presence of reactive astrocytes with decreased GFAP, GLT-1, and GLAST further characterized this inflammatory stage. Interestingly, upregulation of miR-155 and downregulation of MFG-E8 appear as consistent biomarkers of both presymptomatic and symptomatic stages. We hypothesize that downregulated cellular interplay at the early stages may represent neuroprotective mechanisms against inflammation, SOD1 aggregation, and ALS onset. The present study identified a set of inflamma-miRNAs, NLRP3-inflammasome, HMGB1, CX3CL1-CX3CR1, Connexin-43, and Pannexin-1 as emerging candidates and promising pharmacological targets that may represent potential neuroprotective strategies in ALS therapy.

2017Journal article

Restricted access

Novel therapeutic approach to amyotrophic lateral sclerosis : targeting neurotoxic astrocytes before transplantation

Publication . Gomes, Cátia; Brites, Dora, 1951-; Kaspar, Brian; Salgado, António

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons (MNs). However, astrocytes are also major contributors in ALS pathogenesis, though the underlying mechanisms are still unclear. Using the brain cortex of the symptomatic ALS mouse model SOD1G93A (mSOD1), we identified markers of astrocyte reactivity and dysfunction, as well as neuronal loss and SOD1 accumulation. Such abnormal features were early manifested in the primary cortical astrocytes isolated from mSOD1 mice 7 day-old pups and cultured for 13 days in vitro (DIV). These astrocytes, showing neurotoxic potential, revealed a specific aberrant phenotype characterized by SOD1high/GFAPlow/GLT-1low/S100Bhigh/Cx43high/Ki-67high. Remarkably, in contrast with spinal astrocytes that revealed elevated miRNA(miR)-155, neonatal cortical astrocytes exhibited decreased miR-146a levels, also present in brain cortex homogenates of mSOD1 mice at either pre- or symptomatic stages. This finding highlights low miR-146a as a potential therapeutic target and ALS biomarker. To further assess astrocyte dysfunctional heterogeneity, spinal and cortical astrocytes were cultured for 5 and 13 DIV. We found that mSOD1 cortical astrocytes exhibited early and sustained aberrancies, as well as deregulated signaling pathways than their age-matched spinal astrocytes. Moreover, with only 5 DIV in culture, cortical astrocytes were able to reduce neurite outgrowth of mSOD1 NSC-34 MNs, a feature only replicated by 13 DIV spinal ones. Among miRNAs known to play a critical role in CNS inflammation, miR-155/miR-21/miR-146a showed discriminatory potential in being upregulated in spinal astrocytes and downregulated in cortical cells. However, when evaluated in extracellular vesicles (EVs) from 13 DIV spinal and cortical mSOD1 astrocytes, depleted miR-155/miR-21/miR-146a was a common feature in both. These data in the mSOD1 mouse model identify regional different therapeutic candidates in astrocytes, depending on their cortical or spinal origin, thus suggesting specific and combined pharmacological approaches to restore their neuroprotective role. To validate such results in human cells, we then used induced astrocytes (iAstrocytes) directly converted from familial and sporadic ALS male and female patients, as well as from matched non-diseased individuals (controls). All ALS patientiAstrocyes were neurotoxic, with reactive and inflammatory markers differently distributed among ALS iAstrocytes, which may have then discriminatory potential in patient stratification. Up-/down-regulation disparities were noticed for miR-146a and replicated in their derived small EVs. Its upregulation in samples showing decreased miR-146a levels recovered diverse iAstrocytes aberrancies and induced synaptic/axonal gene expression in NSC-34 MNs, reinforcing its relevance for developing novel medicines. Overall, our studies reveal that cortical and spinal astrocytes distinctively acquire ALS malfunction properties. Importantly, iAstrocytes are powerful tools to stratify patients, reflecting the ALS phenotypic variability. Results highlight miR-146a as a potential target to recover the neurosupportive role of astrocytes and evidence the requirement of personalized medicine in ALS.

2019Doctoral thesis

Open access