Astrocyte Contribution to ALS

The first evidence of astrocyte alterations in ALS has derived from animal models in which mutant SOD1 was selectively expressed or deleted in these cells [30]. Pivotal experiments in chimeric animals bearing mixtures of normal cells and cells that express a human mutant SOD1 at levels sufficient to cause fatal MN disease reported that wild-type non-neuronal cells in the SOD1G37R and SOD1G85R chimeras delayed disease onset and prolonged mouse survival. In accordance, transgenic animals expressing mutant SOD1 in non-neuronal cells but not in MNs showed histological signs of neurodegeneration caused by the accumulation of ubiquitinated epitopes absent in age-matched wild-type littermates [30]. Some years later, similar experiments used Cre-Lox recombination to delete mutant SOD1 genes selectively in microglia or astrocytes. The reduction of mutant SOD1 in the astrocytes of SOD1G37R or SOD1G85R mice did not delay disease onset and early disease progression. Moreover, it significantly slowed the late disease course, extending mouse survival, suggesting that astrocyte dysregulation negatively controls the status of MNs at the late stage of ALS [31]. At the same time, they exhibit a more protective phenotype at the disease onset [18,28,31]. Furthermore, transplantation of wild-type astrocyte precursors in the cervical spinal cord of SOD1G93A mice slowed the disease progression and prolonged survival probability [32]. Oppositely, the transplantation of astrocyte precursors carrying the SOD1G93A gene mutation promoted local degeneration in the spinal cord and caused motor dysfunction in wild-type mice [33].

Moreover, mutant SOD1 astrocytes were able to induce in-vitro neurodegeneration both in ALS MNs from SOD1G93A, SOD1G37R, or SOD1G85R mice, as well as in healthy MNs, supporting the hypothesis of a gain of toxic functions of astrocytes in ALS [34]. Several studies demonstrated that MN viability was strongly impaired when wild-type or mutant MNs were co-cultured in direct contact with ALS astrocytes or exposed to ALS astrocyte-conditioned medium, thus encouraging the characterization of astrocyte secretome to clarify the contribution of astrocytes to disease progression [35,36,37,38,39]. Astrocytes differentiated from human fibroblasts carrying C9orf72 mutation-induced MN death in co-culture experiments [37,39]. C9orf72-astrocyte RNA sequencing showed several gene alterations, including genes involved in ionotropic glutamate receptor signalling (GRIA1, GRIA4), complement activation, ribosomal subunit assembly, nuclear RNA export, cell adhesion (L1CAM, TSP1, NTN1), synapse assembly (BDNF, NRG1, THBS2), cell-to-cell signalling (GPC6), regulation of sodium ion transport (SLC8A1, ATP1B2, NKAIN4), and potassium ion import (DLG1, ATP1B2) [39]. In addition, the mutant TDP-43 or mutant VCP expression in astrocytes increased mitochondrial and ER dysfunction and induced abnormal oxidative stress in MNs, thus causing MN death [36,38]. Overall, the current literature has revealed a remarkable astrocytic dysfunction in ALS, potentially underlying molecular mechanisms that could represent a target for therapeutic approaches [40,41,42].

Apart from the fundamental astrocyte physiological functions, their dysfunction can generate neurological disorders such as neurodegenerative or neurodevelopmental diseases, epilepsy, and astrogliomas [59]. In response to a damaging insult, astrocytes shift from rest to a highly reactive and proliferative phenotype with supportive characteristics to mend the damage by supplying trophic factors and reducing neuronal degeneration. In many neurodegenerative diseases, including ALS, this mechanism is impaired and leads to neurotoxic events [60]. Indeed, the influence of astrocytes is more complex and can be beneficial or detrimental depending on the disease and the pathological conditions [61]. Accordingly, distinctive molecular and functional profiles characterize the reactive astrocytes and their impact on diseases and produce unique astrocyte phenotypes [62]. Many studies have suggested that astrocytes can act as two distinct reactive categories: the A1 neurotoxic phenotype and the A2 neuroprotective [63–65]. However, scientists should consider this dual polarization with caution since more recent studies proposed moving beyond the A1–A2 classification since only a subset of transcripts related to the A1 and A2 states are upregulated in patient or mouse models of CNS disease astrocytes, and multidimensional data support the idea that A1 and A2 are just two of many potential transcriptomes of astrocytes [66–69].

Thus, astrocytes may play a fundamental role in shaping ALS genesis and progression by several mechanisms, such as neuroinflammation, mitochondrial dysfunction, oxidative stress, energy metabolism impairment, miRNAs and extracellular vesicle involvement, protein misfolding, autophagy dysfunction, neurotrophic factor dysregulation, and glutamate excitotoxicity.

Glutamate released by non-neuronal cells can represent an additional factor contributing to the increasing extracellular glutamate levels in ALS [209]. According to the emerging astrocyte roles, these cells should be both “producers” and “targets” of glutamate excitotoxicity in ALS.

Astrocytes as Producers of Excessive Glutamate in ALS

Astrocytes can actively contribute to defining the glutamate commitment in developing neuronal and glial damage during ALS progression by different mechanisms, as described below.

Excitatory Amino Acid Transporter 2. Abnormal glutamate availability represents one of the fundamental ALS-linked features. In 1995, EAAT2, expressed predominantly in astrocytes and responsible for about 90% of glutamate reuptake from the synapses, was found dysfunctional in the brain cortex and spinal cord astrocytes of ALS patients, causing impairment of the synapse glutamate clearance [174,210]. Many factors affect EAAT2 transcription, translation, and activity, such as oxidative stress, fatty acids, growth factors, or cytokines [212–215]. The reduced glutamate clearance leads to increased activation of the glutamate receptors of MNs with an abnormal influx of Ca2+, determining fatal changes in cell physiology and inducing ER stress, mitochondria overload, and cell death [216]. Overall, a significant reduction of EAAT2 in the motor cortex and spinal cord is one of the principal factors leading to glutamate excitotoxicity in ALS [220].

Exocytotic Glutamate Release. Ca2+-dependent astrocytic exocytosis is a long-lasting known occurrence in astrocytes [221,222]. The KCl-induced depolarization evoked glutamate release has also been reported in adult astrocytes and peri-synaptic astrocyte processes (gliosomes) prepared from rat brain cortex [229], thus supporting the astrocytic excitability properties. The presence of vesicle-associated proteins, including the vesicular glutamate transporters, suggested the presence of glutamate-containing vesicles in the astrocyte active zone and cytoplasm, which were evidenced by electron microscopy and proteomic studies [232–236]. Manfredi and colleagues analyzed Ca2+ homeostasis and exocytosis in SOD1G93A mouse-derived astrocytes. They found that ATP stimulation augmented [Ca2+]i due to excessive Ca2+ release from endoplasmic reticulum (ER) stores and based on altered Ca2+ accumulation in the ER in SOD1G93A astrocytes [238]. Astrocytic exocytosis inhibition in SOD1G93A astrocytes preserved MNs from death in astrocyte-MN co-cultures and delayed the disease onset in SOD1G93A mice without affecting disease progression, providing in vitro and in vivo evidence that astrocyte exocytosis contributes to ALS pathogenesis. Considering that the exocytotic mechanism represents one of the main processes for glutamate release by astrocytes, further studies are needed to gather compelling evidence that, besides altered neuronal release, astrocyte glutamate exocytosis is instrumental to ALS.

Purinergic P2X7 Receptors. The purinergic P2X7 receptor subtype (P2X7R) is a ligand-gated cation channel that provides another pathway for glutamate release from astrocytes [221]. The P2X7R is upregulated in microglia and astrocytes, resident in the spinal cord of ALS patients and SOD1G93A animals, thus leading to the hypothesis that ATP signalling may trigger cytotoxic events in astroglial cells, resulting in proximal motor neuron damage [249,250]. Extensive preclinical studies identified the P2X7Rs as playing two roles in ALS: neuroprotective and neurodegenerative, depending on the disease stage, being predominantly neuroprotective in early disease stages while becoming gradually detrimental during ALS progression [251–253]. Moreover, the P2X7R pharmacological modulation displayed controversial gender-dependent effects in SOD1G93A mice [254,255]. Gandelman and collaborators demonstrated that selective activation of the P2X7Rs in SOD1G93A astrocytes led to motor neuron death, most likely due to the release of toxic factors, including glutamate. Of note, supporting the role of ATP and the consequent P2X7R activation, depleting ATP with apyrase or blocking the P2X7R with the antagonist brilliant blue G (BBG) significantly prevent the SOD1G93A astrocyte-mediated MN death [257]. Considering the above evidence, astrocyte-expressed P2X7Rs play a role in ALS disease, contributing to oxidative stress, inflammatory signalling and glutamate-mediated neurotoxicity.

Cystine/Glutamate Antiporter System xc. The cystine/glutamate antiporter system xc (Sxc) is a membrane heterodimer crucial to sustaining astroglial glutamate release in several CNS regions. Increased Sxc-mediated glutamate release was observed even before the EAAT2 reduction, thus contributing to the early glutamate toxicity during the disease initiation in the SOD1G93A transgenic mouse model of ALS [259]. A recent study showed that the deletion of xCT (core protein of Sxc-) delayed the disease progression rate in the mutant SOD1G37R ALS mouse model [260], thus confirming its essential role in driving the disease. Of note, the oxidant environment upregulates xCT, causing an increase in extracellular glutamate levels that, in turn, induce Ca2+-mediated excitotoxicity [261]. Since oxidant species are present during ALS progression, this exchanger could enhance glutamate excitotoxicity during the disease progression.

Hemichannels. Connexins (Cx) and pannexins (Pn) are two membrane protein families forming hem-ichannels [262], creating connexons or gap junctions (GJs), allowing the exchange of molecules and ions as well as toxic substances, such as excitatory amino acids, with neighbouring cells and promoting Ca2+ overload [263,264]. Accumulating evidence suggests a connection between ALS and Cxs [279,280]. Astrocytic GJ Cx43 was strongly dysregulated in the anterior horns of the spinal cords of mu-tant SOD1G93A transgenic mice during disease progression and at the end stage, suggesting that the GJ disruption can aggravate MN death, contributing to glutamate excitotoxicity and ALS progression [281]. Keller and colleagues found an intimate connection between activated microglia and astrocytes via Cx43 at the end stage of ALS [282], thus supporting the idea that the altered Cx43 function affects microglia reactivity and the inflammatory response. As confirmation of this scenario, Cx43 overexpression was described in the SOD1G93A mouse model as well as in post-mortem motor cortex, spinal cord, and cerebrospinal fluid derived from ALS patients; accordingly, neuroprotection through Cx43 and Cx43 hemichannel blockers was shown to be beneficial [283].

Bestrophin-1, TWIK-Related Potassium Channel 1 and Volume-Regulated Anion Channels. Bestrophin-1 (Best-1) is an anionic channel activated by Ca2+. Its physiological activities include the release of molecules, such as glutamate, GABA, and chloride ions [289,290]. Best-1 is expressed in astrocytes and releases glutamate upon increased Ca2+ concentration, thus activating neuronal and non-neuronal NMDA receptors and, in turn, potentiating synaptic responses and modulating synaptic plasticity [291].

The TWIK-related potassium channel 1 (TREK-1) is a type of K2P channel with a double-pore-domain background potassium channel [294]. In astrocytes, TREK-1 controls cell excitability by maintaining the membrane negative potential [295], and it mediates the passive potassium conductance and release of glutamate from astrocytes upon heterodimerization [296]. Similarly, volume-regulated anion channels (VRACs) release massive amounts of glutamate from swollen astrocytes, which could increase the extracellular amino acid level and overstimulate glutamate receptors in surrounding cells [298].

Based on the role of these channels in contributing to glutamate excitotoxicity [301], it is reasonable to suppose their implication in ALS progression. However, to the best of our knowledge, no information is available.

Other Mechanisms Triggering Astrocytic Glutamate Excitotoxicity. A direct link between neuroinflammation and astrocyte-fostered glutamate excitotoxicity has been demonstrated [302]. In astroglia, the TNF-𝛼 interaction with its receptor TNFR1 induces a cascade of intracellular events leading to the generation of prostaglandin E2 that, in turn, activates intracellular Ca2+ elevation followed by glutamate exocytosis [303,304]. Moreover, TNF-𝛼 has a detrimental effect on astroglial glutamate uptake [305], downregulating EAAT2/GLT1 mRNA [306,307], thus inducing higher extracellular glutamate levels. TNF-𝛼 can also potentiate glutamate-mediated cytotoxicity by rapidly triggering the surface expression of Ca2+ permeable-AMPA and NMDA receptors while decreasing inhibitory GABAA receptors on neurons. Thus, the net effect of TNF-α alters the balance of excitation and inhibition, resulting in a higher synaptic excitatory/inhibitory ratio [302].

Similarly, interleukin (IL)-1β and TNF-𝛼 dose-dependently inhibited astrocyte gluta-mate uptake by a mechanism involving nitric oxide, whereas interferon (IFN)-gamma alone stimulated this activity [308]. Moreover, an IL-1β, TNF-α, and IFN-γ cytokine mixture enhanced the calcium-dependent glutamate release from astrocytes induced by NO [309].

Inward rectifying Kir4.1 channels in astrocytes mediate spatial potassium (K+) buffering, a clearance mechanism for excessive extracellular K+ in tripartite synapses, and it is also essential for glutamate and water homeostasis in synapses [310]. A reduction of Kir4.1 was observed in the brain and ventral spinal cord of asymptomatic animals [315], and altered Kir currents were observed in cultured SOD1G96A astrocytes [316,317]. Thus, the dysregulation of the Kir4.1 channels in astrocytes might contribute to glutamate excitotoxicity and is considered a novel and promising therapeutic astrocyte link [318].

Astrocytes as Target of Glutamate Excitotoxicity in ALS

Glutamate toxicity is highly relevant in ALS since it may contribute to disease progression via multiple pathways, including a direct effect on the MNs and a modulation of the astrocytic reactive phenotype and their secretome, thus providing paracrine signals to neighbouring cells. As described above, astrocytes can contribute to elevating glutamate excitotoxicity by several mechanisms and can sense glutamate becoming targets of their own released excitatory amino acid.

After being released, glutamate binds to several receptors, including the ionotropic N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methy-4-isoxazolepropionic acid (AMPA), kainate receptors, and the metabotropic mGluRs, contributing to physiological and pathological events depending on the amplitude of the glutamate exposure. To our knowledge, there is no evidence of a direct contribution of astrocytic NMDA receptors as glutamate targets affecting the astrocytes’ phenotype during ALS progression [319]. Also, no direct evidence exists for the other ionotropic glutamate receptors expressed by astrocytes in ALS [320]. Focusing on astrocytes as glutamate targets in ALS, it is fundamental to understand how excessive extracellular glutamate can change the astrocyte phenotype and reactivity, either by itself or with the contribution of other toxic molecules.

Supporting the crucial role of astrocytes as a target of glutamate, Zuo and colleagues recently showed that excessive extracellular glutamate can activate astrocyte C3 expression and promote the release of pro-inflammatory factors, such as TNF-α and IL-1β [324]. However, the pathways responsible for this activation have not been elucidated. ALS can exacerbate this scenario due to the presence of high extracellular glutamate levels and mutant astrocytes, which are more reactive to toxic stimuli. Rossi and colleagues corroborated the above hypotheses, showing that in vitro exposure to glutamate resulted in focal degeneration of astrocytes cultured from the spinal cord of SOD1G93A mice and not in WT-SOD1 mouse astrocytes [325]. The selective toxicity was triggered by activating specific astrocytic mGluRs [325], suggesting a possible mechanism for the glutamate-induced excitotoxicity in astrocytes. This evidence has also been recently confirmed by our research group [78].

Of note, astrocytes derived from an animal model of ALS carrying mutant SOD1 evidenced altered expressions and functions of mGluR5, which is involved in the activity and proliferation of astrocytes following damaging insults [338]. In accordance, primary astrocyte cultured from the brain cortex of SOD1G93A rats showed a higher expression of mGluR5 than wild-type rats and dysregulation of the cross-talk between mGluR5 and EAAT2, leading to a lower number of Ca2+ oscillations and reduced glutamate clearance in the synapses [325,339,340]. The altered mGluR5 function derives from the downregulation of protein kinase C epsilon isoform (PKCε). Indeed, the restoration of PKCε in mutant SOD1G93A astrocytes determined the normalization of Ca2+ oscillation and restoration of the dynamic mGluR5-dependent control of glutamate clearance by these cells. As a further proof-of-concept, PKCε silencing in wild-type astrocytes recapitulated the decreased Ca2+ oscillations observed in SOD1G93A astrocytes [340]. In ALS, the elevated expression of mGluR5 makes astrocytes highly vulnerable to glutamate, causing aberrant and persistent elevations of intracellular Ca2+ concentrations [345] and inducing cell death [325]. Recently, it has been demonstrated that high extracellular glutamate levels increase the Lipocalin-2 (Lcn-2) concentration in the astrocyte cytoplasm by inducing a dose-dependent release of the protein via mGluR3 activation [344]. Since Lcn-2 is a key regulator of neuroinflammation, this mechanism could also be important in ALS.

Conclusions

It is now clear that astrocytes indeed regulate different important aspects of ALS pathology, such as excitotoxicity, inflammation, oxidative stress, mitochondria function, and energy metabolism. It is as much crucial to understand how glutamate contributes to these pathological astrocytic mechanisms. We highlighted that astrocytes may participate in these processes by two distinct ways. Indeed, the excess of glutamate produced by astrocytes can affect heterologous cells, including astrocytes that will become the direct target, exacerbating, or even triggering, aberrant astrocyte activation during ALS progression. This latter aspect has yet to be widely investigated in ALS; therefore, it deserves future studies that may unveil alternative therapeutic strategies that focus on the selective interception of pro-death signals in a cell-type-specific way.

This entry is adapted from the peer-reviewed paper 10.3390/ijms242015430