Excitotoxicity is a phenomenon that describes the toxic actions of excitatory neurotransmitters, primarily glutamate, where the exacerbated or prolonged activation of glutamate receptors starts a cascade of neurotoxicity that ultimately leads to the loss of neuronal function and cell death. In this process, the shift between normal physiological function and excitotoxicity is largely controlled by astrocytes since they can control the levels of glutamate on the synaptic cleft. This control is achieved through glutamate clearance from the synaptic cleft and its underlying recycling through the glutamate-glutamine cycle. The molecular mechanism that triggers excitotoxicity involves alterations in glutamate and calcium metabolism, dysfunction of glutamate transporters, and malfunction of glutamate receptors, particularly N-methyl-D-aspartic acid receptors (NMDAR). On the other hand, excitotoxicity can be regarded as a consequence of other cellular phenomena, such as mitochondrial dysfunction, physical neuronal damage, and oxidative stress. Regardless, it is known that the excessive activation of NMDAR results in the sustained influx of calcium into neurons and leads to several deleterious consequences, including mitochondrial dysfunction, reactive oxygen species (ROS) overproduction, impairment of calcium buffering, the release of pro-apoptotic factors, among others, that inevitably contribute to neuronal loss. A large body of evidence implicates NMDAR-mediated excitotoxicity as a central mechanism in the pathogenesis of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), and epilepsy. In this review article, we explore different causes and consequences of excitotoxicity, discuss the involvement of NMDAR-mediated

Reactive astrocytes in Amyotrophic Lateral Sclerosis (ALS) change their molecular expression pattern and release toxic factors that contribute to neurodegeneration and microglial activation. We and others identified a dysregulated inflammatory miRNA profile in ALS patients and in mice models suggesting that they represent potential targets for therapeutic intervention. Such cellular miRNAs are known to be released into the secretome and to be carried by small extracellular vesicles (sEVs), which may be harmful to recipient cells. Thus, ALS astrocyte secretome may disrupt cell homeostasis and impact on ALS pathogenesis. Previously, we identified a specific aberrant signature in the cortical brain of symptomatic SOD1-G93A (mSOD1) mice, as well as in astrocytes isolated from the same region of 7-day-old mSOD1 mice, with upregulated S100B/HMGB1/Cx43/vimentin and downregulated GFAP. The presence of downregulated miR-146a on both cases suggests that it can be a promising target for modulation in ALS. Here, we upregulated miR-146a with pre-miR-146a, and tested glycoursodeoxycholic acid (GUDCA) and dipeptidyl vinyl sulfone (VS) for their immunoregulatory properties. VS was more effective in restoring astrocytic miR-146a, GFAP, S100B, HMGB1, Cx43, and vimentin levels than GUDCA, which only recovered Cx43 and vimentin mRNA. The miR-146a inhibitor generated typical ALS aberrancies in wild type astrocytes that were abolished by VS. Similarly, pre-miR-146a transfection into the mSOD1 astrocytes abrogated aberrant markers and intracellular Ca2+ overload. Such treatment counteracted miR-146a depletion in sEVs and led to secretome-mediated miR-146a enhancement in NSC-34-motor neurons (MNs) and N9-microglia. Secretome from mSOD1 astrocytes increased early/late apoptosis and FGFR3 mRNA in MNs and microglia, but not when derived from pre-miR-146a or VS-treated cells. These last strategies prevented the impairment of axonal transport and synaptic dynamics by the pathological secretome, while also averted microglia activation through either secretome, or their isolated sEVs. Proteomic analysis of the target cells indicated that pre-miR-146a regulates mitochondria and inflammation via paracrine signaling. We demonstrate that replenishment of miR-146a in mSOD1 cortical astrocytes with pre-miR-146a or by VS abrogates their phenotypic aberrancies and paracrine deleterious consequences to MNs and microglia. These results propose miR-146a as a new causal and emerging therapeutic target for astrocyte pathogenic processes in ALS.

Glutamate uptake is a process mediated by sodium-dependent glutamate transporters, preventing glutamate spillover from the synapse. Typically, astrocytes express higher amounts of glutamate transporters, thus being responsible for most of the glutamate uptake; nevertheless, neurons can also express these transporters, albeit in smaller concentrations. When not regulated, glutamate uptake can lead to neuronal death. Indeed, the majority of the studies regarding glutamate transporters have focused on excitotoxicity and the subsequent neuronal loss. However, later studies have found that glutamate uptake is not a static process, evincing a possible correlation between this phenomenon and the efficiency of synaptic transmission and plasticity. In this review, we will focus on the role of the increase in glutamate uptake that occurs during long-term potentiation (LTP) in the hippocampus, as well as on the impairment of long-term depression (LTD) under the same conditions. The mechanism underpinning the modulatory effect of glutamate transporters over synaptic plasticity still remains unascertained; yet, it appears to have a more prominent effect over the N -methyl- D -aspartate receptor (NMDAR), despite changes in other glutamate receptors may also occur.

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Caffeine, a stimulant largely consumed around the world, is a non-selective adenosine receptor antagonist, and therefore caffeine actions at synapses usually, but not always, mirror those of adenosine. Importantly, different adenosine receptors with opposing regulatory actions co-exist at synapses. Through both inhibitory and excitatory high-affinity receptors (A 1 R and A 2 R, respectively), adenosine affects NMDA receptor (NMDAR) function at the hippocampus, but surprisingly, there is a lack of knowledge on the effects of caffeine upon this ionotropic glutamatergic receptor deeply involved in both positive (plasticity) and negative (excitotoxicity) synaptic actions. We thus aimed to elucidate the effects of caffeine upon NMDAR-mediated excitatory postsynaptic currents (NMDAR-EPSCs), and its implications upon neuronal Ca 2+ homeostasis. We found that caffeine (30-200 μM) facilitates NMDAR-EPSCs on pyramidal CA1 neurons from Balbc/ByJ male mice, an action mimicked, as well as occluded, by 1,3-dipropyl-cyclopentylxantine (DPCPX, 50 nM), thus likely mediated by blockade of inhibitory A 1 Rs. This action of caffeine cannot be attributed to a pre-synaptic facilitation of transmission because caffeine even increased paired-pulse facilitation of NMDA-EPSCs, indicative of an inhibition of neurotransmitter release. Adenosine A 2A Rs are involved in this likely pre-synaptic action since the effect of caffeine was mimicked by the A 2A R antagonist, SCH58261 (50 nM). Furthermore, caffeine increased the frequency of Ca 2+ transients in neuronal cell culture, an action mimicked by the A 1 R antagonist, DPCPX, and prevented by NMDAR blockade with AP5 (50 μM). Altogether, these results show for the first time an influence of caffeine on NMDA receptor activity at the hippocampus, with impact in neuronal Ca 2+ homeostasis.