InteractiveFly: GeneBrief

Excitatory amino acid transporter 1: Biological Overview | References

BIOLOGICAL OVERVIEW

In the mammalian CNS, glial cells expressing excitatory amino acid transporters (EAATs) tightly regulate extracellular glutamate levels to control neurotransmission and protect neurons from excitotoxic damage. Dysregulated EAAT expression is associated with several CNS pathologies in humans, yet mechanisms of EAAT regulation and the importance of glutamate transport for CNS development and function in vivo remain incompletely understood. Drosophila is an advanced genetic model with only a single high-affinity glutamate transporter termed Eaat1. Eaat1 expression in CNS glia was found to be regulated by the glycosyltransferase Fringe, which promotes neuron-to-glia signaling through the Delta-Notch ligand-receptor pair during embryogenesis. Eaat1 loss-of-function mutations were made and it was found that homozygous larvae could not perform the rhythmic peristaltic contractions required for crawling. No evidence was found for excitotoxic cell death or overt defects in the development of neurons and glia, and the crawling defect could be induced by postembryonic inactivation of Eaat1. Eaat1 fully rescued locomotor activity when expressed in only a limited subpopulation of glial cells situated near potential glutamatergic synapses within the CNS neuropil. Eaat1 mutants had deficits in the frequency, amplitude, and kinetics of synaptic currents in motor neurons whose rhythmic patterns of activity may be regulated by glutamatergic neurotransmission among premotor interneurons; similar results were seen with pharmacological manipulations of glutamate transport. These findings indicate that Eaat1 expression is promoted by Fringe-mediated neuron-glial communication during development and suggest that Eaat1 plays an essential role in regulating CNS neural circuits that control locomotion in Drosophila (Stacey, 2010).

Glutamate is the principal excitatory neurotransmitter in the mammalian CNS. Extracellular glutamate levels are tightly regulated for precise control of neurotransmission at glutamatergic synapses, and to prevent neuronal cell death from excitotoxicity. Certain astrocyte populations take up and recycle extracellular glutamate by expressing high-affinity, sodium-dependent excitatory amino acid transporters (EAATs) known as GLAST (alternate name EAAT1) and GLT-1 (alternate name EAAT2), the primary transporters for glutamate in the mammalian CNS (Matthias, 2003). GLAST mutations are found in patients with type 6 episodic ataxia (EA6), a rare form of the disease that variably involves seizures, migraine, cerebellar atrophy, and hemiplegia. Furthermore, expression of EAATs is dysregulated in amyotrophic lateral sclerosis, stroke, epilepsy, schizophrenia, and Alzheimer's and Huntington's diseases, among others. However, the mechanisms of EAAT regulation and the consequences of aberrant glutamate transport for CNS function and pathology remain to be fully understood (Stacey, 2010).

Drosophila provides an advanced genetic model system to study EAAT regulation and function in vivo because the differentiation of glial cell subtypes can be studied in an intact nervous system. Moreover, the importance of glutamate transport for CNS development and function can be assessed in vivo with complementary behavioral studies and electrophysiology. In Drosophila, many functional, morphological, and molecular features of glial cells are conserved with mammals, including the selective expression of the Eaat1 gene in a subpopulation of glial cells (Soustelle, 2002; Freeman, 2003). Drosophila Eaat1 has 41% and 35% amino acid identity, respectively, to human EAAT1 and EAAT2 (Besson, 1999). Eaat1 is the only high-affinity glutamate transporter in flies (Besson, 2000), and so studies of Eaat1 mutations are unlikely to be complicated by redundancy with the related protein Eaat2, which is a selective high-affinity transporter for taurine and aspartate (Besson, 2000; Besson, 2005). Previous research in adult flies has shown that reduction of Eaat1 in glia using RNA interference (RNAi) increases sensitivity to oxidative stress, and results in fewer dopaminergic neurons, degeneration of the brain neuropil, and decreased life span (Rival, 2004). However, understanding of the importance of this glutamate transporter for CNS development and function remains incomplete since RNAi approaches to study Eaat1 function did not reveal phenotypes at embryonic and larval stages (Rival, 2004, Rival, 2006; Stacey, 2010 and references therein).

Eaat1 expression in embryogenesis is shown in this study to be regulated by the glycosyltransferase Fringe (Fng), which has been shown to promote neuron-to-glia signaling through the Delta-Notch ligand-receptor pair. Eaat1 loss-of-function mutations were generated, and mutant larvae were found have severe defects of locomotion. The electrophysiological and genetic approaches provide evidence that Eaat1 acts in a limited subpopulation of CNS glial cells to influence glutamatergic neurotransmission controlling the rhythmic patterning of motor neuron activity. Thus, this study has identified cellular and molecular interactions during development that affect the emergence of a functionally distinct glial subtype capable of influencing glutamatergic neurotransmission in the CNS, and a discovered an essential role for the Eaat1 glial glutamate transporter in locomotor behavior (Stacey, 2010).

The major nerve tracts of the Drosophila ventral nerve cord (VNC), called commissures and longitudinal connectives, mark a dense neuropil of axon projections, dendrites, and synapses within the segmentally repeated embryonic and larval CNS. A subset of CNS glial cells expresses the gene CG31235, including the nine longitudinal glia (LG) found in each VNC hemi-segment. LG lie just dorsal to the longitudinal connectives and ensheath the neuropil. In building genetic tools to study these glia in vivo, it was found that a 3 kb promoter/enhancer of CG31235 can direct the expression of Gal4 or nuclear GFP (nGFP) transgenes to the nine LG, plus five additional glial cells in each VNC hemi-segment. in situ hybridization was used to examine the expression of Eaat1 transcripts in the VNC of CG31235-nGFP animals and it was noted that Eaat1 was expressed in glial cells, including a subset of LG. Onset of Eaat1 transcript expression occurred rather late in embryogenesis (stages 15-16), which is consistent with previous reports (Besson, 1999; Soustelle, 2002), and only narrowly precedes the initiation of spontaneous and uncoordinated muscle contractions (Stacey, 2010).

Using Eaat1-Gal4 to mark Eaat1-expressing cells in the VNC, it was found that virtually all of them also expressed CG31235-nGFP. The nine LG in each hemi-segment can be subdivided further because the anterior-most six of these cells express the transcrtiption factor Prospero (Pros). It was found that 84% (173/205) of Eaat1-Gal4 cells are also Pros positive, indicating that a large majority of Eaat1-expressing cells are of the anterior LG subtype. This subtype also expresses Glutamine synthetase 2 (Gs2). Glutamine synthetases convert glutamate to glutamine, which is synaptically inert and can be safely recycled back to neurons. Coexpression of Gs2 and Eaat1 in the anterior LG strongly suggests that this subtype of glial cell is well equipped for the uptake and metabolism of glutamate from CNS synapses in Drosophila, and could potentially modulate glutamatergic neurotransmission. Consistent with this, the presynaptic vesicular glutamate transporter VGlut, and the postsynaptic glutamate receptor KaiRIA (GluR-IID) are both expressed in the dorsal neuropil of the VNC of embryos and larvae, near the cell bodies of LG. To determine whether Eaat1-expressing LG infiltrate the neuropil and express Eaat1 near putative glutamatergic synapses in first instar (L1) larvae, Eaat1-Gal4 was used to drive expression of an Eaat1::GFP fusion protein (UAS-Eaat1::GFP), and colabelling was performed with either the membrane-targeted reporter mCD8-red fluorescent protein (RFP) or anti-VGlut to mark potential sites of glutamatergic presynaptic terminals in first instar larvae. Eaat1::GFP was broadly expressed among the RFP-labeled glial membranes and, relative to RFP, appeared to be enriched in glial membranes that had infiltrated the CNS neuropil. VGlut-positive puncta were located dorsally within the VNC neuropil, similar to the pattern observed previously in third instar larvae. Optical sections through the neuropil revealed extensive Eaat1::GFP labeling in close proximity to VGlut-positive puncta, consistent with the idea that glutamatergic transmission at CNS synapses in Drosophila could be influenced by the Pros-positive anterior LG subtype that express both the glutamate transporter Eaat1 and the glutamine synthetase Gs2 (Stacey, 2010).

For this study the first mutations were created in Drosophila Eaat1 as a means to better understand the importance of glutamate uptake for CNS development and function. Previous approaches using RNAi had uncovered no function for Eaat1 in larvae; this is likely due to insufficient knockdown at larval stages since this study has that found Eaat1 mutants have a severe crawling deficit. The requirement for this glutamate transporter was narrowed to a subpopulation of CNS glia, and it was found that the crawling defect could be induced by conditional inactivation of Eaat1 after embryogenesis. Consistent with the observations that several immunohistochemical markers of neurons and glia were unaffected in Eaat1 mutants, these results indicate that the crawling deficit is not secondary to developmental defects. Furthermore, no evidence was found for widespread cell death in Eaat1 mutant larvae, suggesting that the crawling deficit is not a consequence of neurotoxic damage induced by excess glutamate (Stacey, 2010).

Interestingly, Eaat1 mutant larvae withdrew and turned their heads normally in response to mechanical stimulus, raising the possibility that CNS neural circuits controlling rhythmic contractions required for larval crawling were specifically affected. The importance of glutamate-mediated neurotransmission in the CNS of Drosophila larvae has not been well characterized, though it has a well studied and essential role at the neuromuscular junction (DiAntonio, 2006). The function of neuromuscular synaptic transmission was not specifically tested in Eaat1 mutants, but several lines of evidence indicate the crawling deficit is primarily due to a requirement for Eaat1 within the CNS. First, electrophysiological recordings from motor neuron cell bodies within the CNS showed that Eaat1 mutants displayed decreased frequency of synaptic drive onto motor neurons. This mimics the effects observed with acute pharmacological manipulations of glutamate transport, and resembles a published report on mutants of the AMPA-type glutamate receptor KaiRIA (also known as GluR-IID or brec) (Featherstone, 2005). Second, Eaat1 has been reported to be absent from neuromuscular junctions in larvae (Rival, 2006). Third, crawling was rescued with a Gal4 driver expressed exclusively in CNS glia. Together, these results lead to a proposas that the crawling deficit in Eaat1 mutants is primarily due to a failure of specific glial cells in the CNS to efficiently remove excess glutamate from central synapses, leading to perturbed glutamatergic neurotransmission and reduced motor output. Since Drosophila motor neurons receive direct cholinergic input from premotor interneurons, the idea is currently favored that Eaat1 acts to influence the patterning of rhythmic motor neuron activity by modulating glutamatergic synapses onto these premotor interneurons, or perhaps synapses further upstream in the circuitry (Stacey, 2010).

Previous research has demonstrated that proteins essential for glutamatergic neurotransmission are expressed in the embryonic and larval CNS, such as VGlut, and the ionotropic glutamate receptor subunits KaiRIA, GluR-IA, GluR-IB, Nmdar1, and Nmdar2. AMPA-like receptors have already been implicated since KaiRIA (brec) mutants are paralyzed and have reduced spontaneous rhythmic current (SRC) frequency (Featherstone, 2005). In addition, noncompetitive NMDA receptor antagonists have also been shown to inhibit rhythmic locomotor activity in larvae, but the role of these NMDA-like receptors in CNS motor circuitry remains to be tested by genetic means. The metabotropic glutamate receptor mGluRA is unlikely to be involved since null mutants are viable and have only mild defects of synaptic plasticity and morphology at neuromuscular synapses (Stacey, 2010).

One might predict that loss of glial glutamate uptake in Eaat1 mutants would elevate extracellular glutamate levels and lead to hyperactivity of glutamate receptors. However, SRC frequency is reduced by loss-of-function mutations of either Eaat1 or the KaiRIA (brec) glutamate receptor subunit, implying that Eaat1 mutants have reduced glutamatergic signaling instead. Perhaps this is due to receptor desensitization in the presence of excess glutamate. Eaat1 mutant larvae remain capable of some movement, while brec null mutants are completely paralyzed (Featherstone, 2005). This difference may reflect reduced glutamate neurotransmission resulting from excess receptor desensitization, versus the complete loss of neurotransmission in animals lacking an essential receptor subunit (Stacey, 2010).

Currently, why pharmacological manipulations of glutamate transport affect SRC frequency and duration, while Eaat1 mutations also affect SRC amplitude, cannot be explained. It could reflect differences between acute, short-term treatment with DL-TBOA versus the genetic approach, in which loss of Eaat1 function may lead to additional compensatory changes induced by prolonged increases of extracellular glutamate; it is conceivable that reduced SRC frequency is an acute and proximate effect of Eaat1 mutations, and that the changes observed in SRC peak amplitude are induced secondarily (Stacey, 2010).

These findings complement genetic studies in mice that are complicated by functional redundancy of the glial glutamate transporters GLAST and GLT-1. Mice singly mutant for GLAST or GLT-1 are viable (Tanaka, 1997; Watase, 1998), but double-knock-out mice die in utero and exhibit multiple proliferation and migration defects of stem cells and/or neurons in cortex, hippocampus, and olfactory bulb (Matsugami, 2006). As in Drosophila, glutamate neurotoxicity was not readily apparent in double-mutant mice, but the neuroanatomical defects observed in mice may reflect added importance for glutamate- and activity-dependent processes in the development of mammalian nervous systems. Knock-out mice for only GLAST fail complex motor tasks (Watase, 1998). This might reflect a role for this transporter in Bergmann glia of the cerebellum, where loss of GLAST is associated with inappropriate innervation and neurotransmission at glutamatergic synapses onto Purkinje neurons (Watase, 1998; Marcaggi, 2003; Takayasu, 2005, Takayasu, 2006). The acute role in crawling that was uncovered by conditional inactivation of Eaat1 after embryogenesis supports the idea that, in Drosophila as in mice, glutamate transport strongly influences neurotransmission controlling motor function in vivo (Stacey, 2010).

This study found that the requirement for Eaat1 in locomotor behavior is limited to a subpopulation of glia marked by the CNS-specific driver CG31235-Gal4. At present, the tools available cannot distinguish the relative importance of glial cells located in the VNC versus the brain lobes. Nonetheless, Eaat1 is expressed in a limited subset of neuropil-associated glia in the VNC, including the anterior LG subtype, where it is coexpressed with the glutamate recycling enzyme Gs2 and its expression is regulated by the glycosyltransferase Fng. Fng sensitizes the Notch receptor on the anterior LG to stimulation from developing axons bearing the Delta ligand and thereby promotes neuron-to-glial signaling during embryogenesis. Anterior and posterior LG are derived from a common glioblast, and so, as a consequence of this interplay between neurons and glia, Fng provides a mechanism for the selective expression of Eaat1 in the anterior LG subtype. Thus, Fng promotes the emergence of a functionally distinct glial cell subtype that can take up glutamate and has the potential to modulate neurotransmission at central synapses (Stacey, 2010).

In vitro studies using cocultures of mammalian neurons and astrocytes have shown that factors secreted from neurons, and direct neuron-glial contact, can promote the expression of GLAST and/or GLT-1 in astrocytes. Studies in vivo provide evidence that the expression of glutamate transporters in the mammalian CNS is regulated by neuron-glial communication during development and also at mature stages. In the developing rodent cerebellum, for example, neuron-glial signaling through Notch and its ligand DNER regulates the maturation of Bergmann glia and promotes GLAST expression in these specialized astrocytes. Interestingly, the Fng ortholog Lunatic Fringe is expressed in Bergmann glia, but its involvement in neuron-glial interactions there remains unknown. In the mature hippocampus, direct neuron-glial contact and EphA4-ephrinA3 signaling regulate synaptic plasticity by controlling the expression of GLAST and GLT-1 and thereby regulating glutamate transport (Carmona, 2009; Filosa, 2009). Thus, mammals and insects both use neuron-glial communication to regulate glial glutamate transporter expression. The mechanism of regulation by Fng-mediated Notch signaling that this study has discovered in flies may be conserved in the mammalian CNS, and the Eaat1 mutants described in this study provide an important model to study the molecular pathogenesis of CNS diseases in humans that result from dysregulation of glutamate transport (Stacey, 2010).

Inhibiting glutamate activity during consolidation suppresses age-related long-term memory impairment in Drosophila

In Drosophila, long-term memory (LTM) formation requires increases in glial gene expression. Klingon (Klg), a cell adhesion molecule expressed in both neurons and glia, induces expression of the glial transcription factor, Repo. However, glial signaling downstream of Repo has been unclear. This study demonstrates that Repo increases expression of the glutamate transporter, EAAT1, and EAAT1 is required during consolidation of LTM. The expressions of Klg, Repo, and EAAT1 decrease upon aging, suggesting that age-related impairments in LTM are caused by dysfunction of the Klg-Repo-EAAT1 pathway. Supporting this idea, overexpression of Repo or EAAT1 rescues age-associated impairments in LTM. Pharmacological inhibition of glutamate activity during consolidation improves LTM in klg mutants and aged flies. Altogether, the results indicate that LTM formation requires glial-dependent inhibition of glutamate signaling during memory consolidation, and aging disrupts this process by inhibiting the Klg-Repo-EAAT1 pathway (Matsuno, 2019).

Changes in glial transcription due to neuronal activity have been studied previously, but a specific role of glial transcription in LTM has been less characterized. Expression of the glial transcription factor, Repo, increases shortly after spaced training, and this increase is required for LTM formation. This report has identified Eaat1 as a Repo-regulated glial gene required for LTM consolidation. Eaat1 encodes a glial glutamate transporter that removes glutamate from synaptic sites and transports it into astrocytes. Thus, the data indicate that glutamate signaling needs to be inhibited during LTM consolidation (Matsuno, 2019).

To identify Eaat1, a screen was performed for various genes regulating glial physiology for altered expression during LTM formation. Expression of Eaat1 and crammer was found to increase after spaced training. As Eaat1, but not crammer, is expressed exclusively in glia, focus was placed on Eaat1 as a likely Repo-regulated gene. Indeed, spaced-training-induced increases in EAAT1 depend on Repo and Klg activity. Interestingly, expression of the glial gene, genderblind, which encodes another glial glutamate transporter, required Repo activity for expression, but was not affected by spaced training, suggesting that other transcriptional regulatory factors besides Repo are likely necessary to differentially regulate genes required for memory consolidation from those required for other glial functions (Matsuno, 2019).

Because only screened selected genes were screened, it is possible that Repo induces the expression of other unidentified genes after spaced training. However, somewhat unexpectedly, it was found that overexpression of Eaat1 alone in glial cells is sufficient to rescue the LTM defects of klg and repo mutants. This indicates that the major function of the Klg/Repo signaling pathway is to induce glial expression of Eaat1. It further suggests that one function of astrocytes is to decrease glutamate signaling during LTM consolidation (Matsuno, 2019).

Combined with results from previous studies, this work identifies a putative pathway linking neuronal activity to glial inhibition of glutamate signaling. In flies, the homophilic cell adhesion molecule, Klingon, is expressed in both neurons and glia, and needs to be expressed in both cell types for normal LTM (Matsuno, 2015). Repo expression normally increases after spaced training, whereas it fails to do so in klg mutants, indicating that Klg-mediated neuron-glia communication is necessary for this increase (Matsuno, 2015). Thus, it is proposed that spaced training increases neuronal activity, which induces signaling to glia via the cell adhesion molecule Klg. This results in increased Repo activity in glia, which increases Eaat1 expression, and subsequently decreases glutamate signaling (Matsuno, 2019).

Previous work from various groups including has shown that glutamate signaling through NMDA-type receptors (NRs) is necessary for learning and memory. Overexpression of NRs in mice enhances learning and memory formation, and it has been shown that glial production of D-serine, a neuromodulator that functions as a coactivator of NRs, is necessary for short-lasting memory. In the current study, focus was placed on glutamate activity specifically during memory consolidation, instead of during initial learning and memory formation. Considering the current findings with those of previous studies, it is proposed that NR-dependent glutamate signaling needs to be initially high, during formation of short-lasting memories, but low during a later phase where short-lasting memories are consolidated into LTM. This suggests that glia play at least two roles in memory. They produce D-serine that contributes to high NR activity during memory formation and also produce EAAT1 after learning, which functions to reduce glutamate signaling during memory consolidation (Matsuno, 2019).

Age-related impairments in Drosophila memory do not consist of a general decrease in all forms of learning and memory, but instead consist of decreases in two specific phases of memory, MTM and LTM. The current results suggest that both these memory effects are caused by age-related glial dysfunction. Glia in young flies are able to produce sufficient amounts of D-serine for normal MTM, whereas D-serine amounts decrease 2-fold in aged flies. This decrease is responsible for age-related impairments in 1-h memory, because increasing glial production of D-serine, or directly feeding of D-serine to aged flies, rescues this impairment. Likewise, glial dysfunction is also responsible for age-related impairments in LTM because aged glia are unable to inhibit glutamate signaling during consolidation. Thus, in contrast to young flies, aged flies are unable to modulate glutamate activity during learning and consolidation, leading to defects in the two memory phases (Matsuno, 2019).

The model that EAAT1 inhibits glutamate activity during consolidation stems from EAAT1's role in clearing glutamate from synaptic sites and transporting it into astrocytes. This model is consistent with several mammalian studies that demonstrated decreased expression of astrocytic glutamate transporters upon aging, with a consequent reduction of glutamate uptake. Further supporting this model, it was found that feeding flies memantine or MK801, NMDA receptor antagonists, after spaced training, restores normal LTM in klg mutants and restores LTM in aged flies to youthful levels. This effect requires feeding after training during the consolidation phase. Similar results were obtained by feeding riluzole, a glutamate modulator, which decreases glutamate release and increases astrocytic glutamate uptake. Riluzole has also been reported to ameliorate age-related cognitive decline in mammals, suggesting that the mechanisms of AMI may be conserved between species. In contrast, this study found that D-serine feeding, which rescues age-related declines in short-lasting (1-h) memory, does not improve declines in LTM, but rather attenuates it. This is consistent with the model wherein declines in short-lasting memory and LTM are caused by distinct or opposing mechanisms and glutamate signaling needs to be suppressed during consolidation. Somewhat unexpectedly, it was also found that (s)-4C3HPG, the mGluR1 antagonist/mGluR2 agonist, also ameliorated age-related impairments in LTM. This result indicates that glutamate activity through both ionotropic and metabotropic glutamate receptors antagonizes memory consolidation (Matsuno, 2019).

Currently, it is unclear why glutamate signaling needs to be inhibited during consolidation, but a previous study has shown that Mg2+ block mutations in NMDA-type glutamate receptors (NRs) cause specific defects in LTM in Drosophila. Although Mg2+ block mutations have various effects, one effect is to increase NR activity. Increased NR activity results in increased activity of dCREB2b, an inhibitory isoform of CREB. CREB-dependent gene expression is required during consolidation of LTM, suggesting that consolidation may be preferentially sensitive to NR activity (Matsuno, 2019).

Alternatively, it is possible that neuronal activity needs to be inhibited globally during memory consolidation. Sleep is known to be important for LTM. Sleep deprivation during consolidation prevents LTM formation, whereas artificially inducing sleep after training has been reported to improve LTM. Thus a second possibility is suggested that inhibition of glutamate signaling after spaced training may be a brain-wide phenomenon that promotes consolidation by inducing the organism to sleep. Thus far, gross alterations in sleep duration in klg and repo mutants have not been detected, although this does not preclude minor disruptions in sleep quality that may not be detectable by motion-based sleep assays. Finally, a third possibility is envisioned wherein neuronal inhibition may be required as a neuroprotective mechanism that may be necessary to prevent cell death in neurons that were extensively stimulated during spaced training (Matsuno, 2019).

Mapping the glutamatergic neurons whose activity is inhibited during consolidation will be of great interest in the future. As aversive olfactory memories are formed and stored in the Drosophila MBs, it is possible that specific glutamatergic MB output neurons (MBONs) are inhibited during consolidation. Several glutamatergic MBONs are involved in feedback networks with the lobes of the MBs, suggesting that altering the activity of these neurons may modulate memory consolidation and memory-associated behavioral responses (Matsuno, 2019).

This study has demonstrate that increased expression of Eaat1 is required for LTM consolidation. Based on numerous results from other groups, it is hypothesized that Eaat1 functions to reduce glutamate signaling, and support for this model is provided by demonstrating that pharmacological inhibition of glutamate signaling during consolidation improves LTM under various conditions. However, due to technical limitations, it was not possible to actually measure glutamate concentrations at synapses during memory consolidation and it is not known where and how much glutamate signaling has to be inhibited for optimal LTM consolidation (Matsuno, 2019).

A circuit-dependent ROS feedback loop mediates glutamate excitotoxicity to sculpt the Drosophila motor system

Overproduction of reactive oxygen species (ROS) is known to mediate glutamate excitotoxicity in neurological diseases. However, how ROS burdens can influence neural circuit integrity remains unclear. This study investigated the impact of excitotoxicity induced by depletion of Drosophila Eaat1, an astrocytic glutamate transporter, on locomotor central pattern generator (CPG) activity, neuromuscular junction architecture, and motor function. Glutamate excitotoxicity triggers a circuit-dependent ROS feedback loop to sculpt the motor system. Excitotoxicity initially elevates ROS to inactivate cholinergic interneurons, consequently changing CPG output activity to overexcite motor neurons and muscles. Remarkably, tonic motor neuron stimulation boosts muscular ROS, gradually dampening muscle contractility to feedback-enhance ROS accumulation in the CPG circuit and subsequently exacerbate circuit dysfunction. Ultimately, excess premotor excitation of motor neurons promotes ROS-activated stress signaling to alter neuromuscular junction architecture. Collectively, these results reveal that excitotoxicity-induced ROS can perturb motor system integrity by a circuit-dependent mechanism (Peng, 2019).

Reactive oxygen species (ROS) are generated as the by-product of mitochondrial oxidative phosphorylation. In the central nervous system, under physiological conditions, high energy demand results in higher levels of ROS production relative to those in other body parts. In the past, endogenously generated ROS were recognized as signaling molecules that regulate a range of nervous system processes, including neuronal polarity, growth cone pathfinding, neuronal development, synaptic plasticity, and neural circuit tuning (Li, 2016; Oswald, 2018). By contrast, ROS overproduction and/or overwhelming the antioxidant machinery can generate ROS burdens, termed oxidative stress, in aging and diverse pathological conditions. In turn, excess ROS causes the malfunction and overactivation of ROS-regulated cell signaling pathways. Moreover, the highly oxidative properties of ROS are damaging to nucleotides, proteins, and lipids, eventually leading to neuronal dysfunction or demise. Hence, advancing understanding of the mechanisms underlying ROS-induced neurotoxicity should aid the development of potent therapeutic treatments for neurological disorders (Peng, 2019).

Glutamate acts as the major excitatory neurotransmitter that regulates nearly all activities of the nervous system, with a tight balance between glutamate release and reuptake keeping the micromolar concentration of extracellular glutamate low. In diseases, accumulation of extrasynaptic glutamate results in glutamate-mediated excitotoxicity to the nervous system. Dysfunction of Na+/K+-dependent excitatory amino acid transporters (EAATs) is a key element of glutamate-mediated excitotoxicity. In mammals, there are five EAAT subtypes, that is EAAT1 (GLAST), EAAT2 (GLT1), EAAT3 (EAAC1), EAAT4, and EAAT5. EAAT3, EAAT4 and EAAT5 are expressed in neurons, whereas EAAT1 and EAAT2 are mainly present in astrocytes, where they are enriched in astrocyte terminal processes that form tripartite synapses with neurons and where they take up approximately 90% of released glutamate. Glutamate-mediated excitotoxicity can trigger bulk Ca2+ influx into postsynaptic neurons via NMDA receptors, which causes mitochondrial Ca2+ overload, along with other cellular responses, and which subsequently generates excess amounts of ROS. Notably, it has emerged that dysregulation of neural circuit activity can initiate subsequent disruption of the integrity of other constituents in the same network, resulting in overall circuit dysfunction and even neurodegeneration. However, it still remains unclear whether and how excitotoxicity-induced ROS can influence the integrity of neural circuits (Peng, 2019).

Coordinated animal behaviors are linked to the activity of spinal cord central pattern generators (CPG), which are known to be specialized circuits that integrate inputs from the central brain and sensory neurons, and that subsequently generate rhythmic and patterned outputs to motor neurons. The Drosophila feed-forward locomotor circuit has served as an appropriate model for exploring the pathogenic network mechanisms that underlie neurodegenerative diseases, because it has a relatively simple neural circuitry compared to mammals yet retains conserved functions. This study explored whether glutamate-mediated excitotoxicity impacts locomotor CPG activity, neuromuscular junction (NMJ) architecture, and motor function. Interestingly, it was found that glutamate-mediated excitotoxicity due to depletion of Drosophila Eaat1, the sole Drosophila homolog of human EAAT2, can induce a circuit-dependent ROS feedback loop that impairs the proper activities of the locomotor CPG circuit and muscles, ultimately leading to motor neuron overexcitation, abnormal NMJ growth and strength, and compromised movement. Together, this work reveals a circuit-dependent mechanism for increasing ROS, which mediates glutamate excitotoxicity to sculpt the Drosophila locomotion network (Peng, 2019).

This work utilized a fly model of glutamate excitotoxicity induced by loss of Drosophila eaat1 to explore the impact of glutamate excitotoxicity on the integrity of the motor system. A circuit-dependent feedback mechanism is described for increasing ROS that mediates excitotoxicity to alter premotor circuit activity, NMJ architecture, and motor function. Glutamate excitotoxicity initially alters locomotor CPG activity and hence prolongs CPG output bursts onto motor neurons by ROS-mediated inactivation of the cholinergic interneurons constituting the CPG circuit. Then, tonic premotor stimulation triggers activity-dependent ROS overproduction in both motor neurons and muscles. In muscles, the increased ROS level gradually dampens muscle contractility and consequent sensory input back to the locomotor CPG circuit, with this feedback strengthening ROS accumulation within the CPG circuit to exacerbate circuit dysfunction. Thus, a positive feedback loop between ROS production in the CPG circuit and muscles is established. Finally, in motor neurons, the induced ROS activate JNK stress signaling to promote abnormal NMJ bouton outgrowth and strength. Apart from genetic rescue, pharmacological treatment with the antioxidant AD4 or the K+ channel blocker 4-AP can also significantly alleviate these motor-system deficits (Peng, 2019).

The locomotor CPG circuit for Drosophila larval feed-forward locomotion is positioned in the VNC and is activated by input from the central brain. Furthermore, acute treatment of dissected Drosophila larvae with non-competitive NMDA antagonists has been shown to reduce the initial output burst duration of the locomotor CPG and eventually abolishes all output activity (Cattaert, 2001), suggesting that glutamatergic transmission drives locomotor CPG activity and positively controls its output burst duration during larval movement. Consistent with these latter results, this study has shown that VNC-restricted expression of eaat1-venus using tsh-GAL4 could reverse the prolonged CPG output burst but not the reduced CPG output frequency in eaat1 mutants, indicating that central brain removal of eaat1 reduced burst frequency, whereas VNC removal markedly extended burst duration. The exact neuronal identity and network connections that build up the core components of the Drosophila larval locomotor CPG circuit remain unknown. Intriguingly, the phenotype of prolonged CPG output has also been reported under conditions in which the motor neuron inputs from proprioceptive sensory neurons or period-positive median segmental interneurons (PMSI) are limited. Moreover, RNAi-mediated knockdown of eaat1 extends PMSI-evoked inhibitory postsynaptic currents in motor neurons (MacNamee, 2016). An increase in extrasynaptic glutamate at the axonal synapses of PMSI was noticed when eaat1 is lost, raising an alternative possibility that excess extrasynaptic glutamate may desensitize GluClα to further diminish sensory inhibition feedback. However, this study found that reducing gluRIID but not gluClα in the eaat1 mutant background shortened prolonged CPG output. Hence, upon loss of eaat1, glutamate-mediated excitotoxicity mainly contributes to locomotor CPG circuit dysfunction. In this regard, the CPG outputs to motor neurons may be elongated and/or the potential inhibition from CPG output to PMSI or its upstream interneurons may be abrogated. Further experiments will be needed to unravel the detailed mechanism operating in the CPG circuit upon loss of eaat1 (Peng, 2019).

Targeted relief of the increased ROS in cholinergic interneurons by genetic approaches could significantly alleviate altered CPG activity arising from either eaat1 mutation or short-term exposure to H2O2, indicating that a subset of cholinergic interneurons, which presumably constitutes the locomotor CPG circuit, is vulnerable to and influenced by the ROS increase. Temporal rescue experiments further suggest that the effect of the ROS increase on circuit activity is acute and reversible. In support of the fact that ROS is known to reduce the inactivation of voltage-gated potassium channels in neurons, long-term food-mediated feeding of (or even short-term exposure to) the K+ channel blocker 4-AP led to a restoration of CPG activity in eaat1 mutants. Thus, temporal hypoexcitability of cholinergic interneurons most probably underlies ROS-induced locomotor CPG dysfunction upon eaat1 loss. Interestingly, immediate blockade of glutamatergic transmission shortens the burst duration of the CPG output (Cattaert, 2001). Under this scenario, it is expected that shortened rather than prolonged CPG burst durations should occur upon loss of eaat1. Thus, it is likely that the induced ROS may occur in a restricted way in a certain subset of cholinergic interneurons, resulting in uneven suppression of cholinergic transmission in the locomotor CPG circuit (Peng, 2019).

The GABA neurotransmitter has a crucial role in neuronal inhibition in the central nervous system through its actions on GABA receptors. Notably, regulation of GABAergic transmission by redox signaling is increasingly recognized. ROS, especially those derived from mitochondrial respiration, act to strengthen the neuronal inhibition mediated by GABAA receptors. Thus, it cannot be excluded that, if those ROS-vulnerable cholinergic interneurons also receive GABAergic input, the increased ROS may silence cholinergic transmission of the locomotor CPG circuit by strengthening GABA-mediated inhibition (Peng, 2019).

The pathological roles of ROS in the regulation of skeletal muscles have been studied extensively, and most targets of redox signaling in skeletal muscle participate in muscle contraction. For instance, excess ROS can modulate SR calcium ATPase (SERCA) and ryanodine receptor (RyR) activity, both of which control the Ca2+ homeostasis of sarcoplasmic reticulum. ROS exposure can also oxidize some myofilament proteins, such as myosin heavy chain and troponin C and, in turn, can impair their functions. Consistent with these findings, the ROS increase dampens the muscle contractility of Drosophila larvae. This study found that mitochondrial and cytosolic ROS levels increase upon excess motor neuron stimulation when eaat1 is lost. Moreover, while increasing ROS by dsod1 knockdown reduced muscle contractility and movement velocity, relieving excess ROS in eaat1 mutant muscles improved locomotion. In the motor system, muscles are not only recognized as the end executive tissues for body movement, but also have a crucial role in triggering proprioceptive sensory feedback input to the central circuit. Recent studies in Drosophila have also revealed that proprioceptive sensory feedback plays a vital role in tuning locomotor circuit activity in the homeostatic adjustment of Drosophila larval crawling and in a Drosophila model of amyotrophic lateral sclerosis (ALS). Unexpectedly, this study found that, upon glutamate excitotoxicity, ROS-induced muscle weakness can cause inefficient sensory feedback input to worsen the ROS burden and can negatively impact the functioning of the central locomotor network. Therefore, under pathological conditions, impaired muscle activity can serve as a key mediator for initiating the ROS feedback loop between the CPG circuit and muscles, which may contribute to network dysfunction in excitotoxicity-associated diseases (Peng, 2019).

Excitotoxicity-induced premotor circuit dysfunction elicits activity-dependent synaptic changes ROS are known to activate the JNK/AP-1 signaling pathway that regulates synaptic formation and strength in Drosophila. The mutation in Drosophila spinster (spin), which encodes a late endosome and lysosome protein, causes impaired lysosomal activity and a consequent ROS burden, leading to synaptic bouton outgrowth by activating the JNK signaling pathway. Intriguingly, c-FOS but not c-JUN is important for bouton outgrowth under spin loss. Similarly, it was found that the synaptic bouton phenotypes of eaat1 mutants are dependent on ROS and c-FOS activity. Interestingly, Milton (2011) have shown that 'constitutive' boosting of mitochondria-derived ROS under loss of dsod2 or after paraquat treatment can also promote bouton growth, but in that case it requires both c-FOS and c-JUN activities. By contrast, in eaat1 mutants, the altered CPG pattern possibly elicits a pulsed increase of mitochondrial ROS. Thus, it may be postulated that different resources and temporal generation of ROS may be responsible for engaging different cellular signaling processes. In support of this notion, in addition to mitochondria, NADPH oxidases provide another major source of ROS to control diverse cellular processes. Recently, DJ-1β, a Parkinson's disease-linked protein, has been identified as a redox sensor that mediates the mitochondrial ROS regulating activity-dependent synaptic plasticity at Drosophila NMJ. It will be interesting to further investigate the underlying mechanisms in detail (Peng, 2019).

Potential relevance of ROS-induced motor-circuit dysregulation for neurodegenerative diseases Downregulation of EAAT2 has been demonstrated in patients with Alzheimer's disease or amyotrophic lateral sclerosis (ALS), as well as in ALS rodent models. ALS is a fatal adult-onset disease that predominantly causes NMJ denervation, motor neuron degeneration, and compromised motor function. Spinal removal of mouse EAAT2 is sufficient to elicit motor neuron death. Transgenic expression of EAAT2or treatment with the small compound LDN/OSU-0212320, which mainly increases translation of EAAT2 mRNA, improves the motor performance of an ALS mouse model expressing hSOD1G93A. However, a recent clinical study testing ceftriaxone, an FDA-approved β-lactam antibiotic that can transcriptionally promote EAAT2 expression, in ALS patients concluded that this drug treatment had no therapeutic effect. Therefore, it is questionable whether increasing EAAT2 expression represents a feasible therapeutic strategy for ALS. It has been argued, however, that EAAT2 downregulation largely occurs at the posttranslational and not at the transcriptional level in ALS. There was no evidence for increased EAAT2 in patients treated with ceftriaxone, and ceftriaxone treatment only slightly increases protein levels of EAAT2 in hSOD1G93A mice. In addition, the efficacy of ceftriaxone in hSOD1G93A mice is not consistent among different studies. Hence, more investigations will be needed to strengthen evidence for the pathogenic contribution of EAAT2 dysfunction in ALS (Peng, 2019).

Oxidative stress is known as a hallmark of Alzheimer's disease, Parkinson's disease, and ALS. During aging, neurons are thought to be susceptible to excitotoxicity, and the nervous system and muscles are vulnerable to ROS accumulation because of high oxygen consumption demand. Administration of antioxidants can improve the motor function of hSOD1G93A mice and ALS patients. Nonetheless, how oxidative stress is produced in ALS and how this burden is involved in disease pathogenesis is not well understood. Interestingly, in this study, Drosophila Eaat1 depletion was shown to cause ALS-like characteristics, including motor neuron excitotoxicity, NMJ bouton abnormalities, muscle weakness, and compromised motor performance. In the future, it will be worth exploring whether ROS-induced motor circuit dysfunction might also participate in ALS progression and age-dependent motor system decline (Peng, 2019).

It has previously been shown that reduced excitability of proprioceptive sensory neurons and cholinergic interneurons is causative of locomotor CPG circuit dysfunction and compromised locomotion in Drosophila smn mutants, which are used as a Drosophila model of spinal muscular atrophy (SMA), a motor neuron disease of juveniles. Increasing neuronal excitability by 4-AP treatment reverses these motor system defects. Intriguingly, the current data show that long-term food-mediated feeding of (or even short-term exposure to) the K+ channel blocker 4-AP also rescued altered locomotor CPG activity in eaat1 mutants. Notably, although the precise mechanisms are unknown, 4-AP has been used to treat several motor system-related disorders such as spinal cord injury, Lambert-Eaton syndrome, and hereditary canine spinal muscular atrophy, and it is an FDA-approved therapy for multiple sclerosis. Thus, as supported by the current findings, it seems plausible that neuronal hypoexcitability may be a shared mechanism underlying the motor-system defects displayed in motor-related disorders (Peng, 2019).

Astrocytic glutamate transport regulates a Drosophila CNS synapse that lacks astrocyte ensheathment

Anatomical, molecular, and physiological interactions between astrocytes and neuronal synapses regulate information processing in the brain. The fruit fly Drosophila melanogaster has become a valuable experimental system for genetic manipulation of the nervous system and has enormous potential for elucidating mechanisms that mediate neuron-glia interactions. This study shows the first electrophysiological recordings from Drosophila astrocytes and characterizes their spatial and physiological relationship with particular synapses. Astrocyte intrinsic properties were found to be strongly analogous to those of vertebrate astrocytes, including a passive current-voltage relationship, low membrane resistance, high capacitance, and dye-coupling to local astrocytes. Responses to optogenetic stimulation of glutamatergic premotor neurons were correlated directly with anatomy using serial electron microscopy reconstructions of homologous identified neurons and surrounding astrocytic processes. Robust bidirectional communication was present: neuronal activation triggered astrocytic glutamate transport via excitatory amino acid transporter 1 (Eaat1), and blocking Eaat1 extended glutamatergic interneuron-evoked inhibitory postsynaptic currents in motor neurons. The neuronal synapses were always located within 1 mmicrom of an astrocytic process, but none were ensheathed by those processes. Thus, fly astrocytes can modulate fast synaptic transmission via neurotransmitter transport within these anatomical parameters (MacNamee, 2016).

Physiological requirement for the glutamate transporter dEAAT1 at the adult Drosophila neuromuscular junction

L-glutamate is the major excitatory neurotransmitter in the mammalian brain. Specific proteins, the Na+/K+-dependent high affinity excitatory amino acid transporters (EAATs), are involved in the extracellular clearance and recycling of this amino acid. Type I synapses of the Drosophila neuromuscular junction (NMJ) similarly use L-glutamate as an excitatory transmitter. However, the localization and function of the only high-affinity glutamate reuptake transporter in Drosophila, dEAAT1, at the NMJ was unknown. Using a specific antibody and transgenic strains, it was observed that dEAAT1 is present at the adult, but surprisingly not at embryonic and larval NMJ, suggesting a physiological maturation of the junction during metamorphosis. dEAAT1 is not localized in motor neurons but in glial extensions that closely follow motor axons to the adult NMJ. Inactivation of the dEAAT1 gene by RNA interference generated viable adult flies that were able to walk but were flight-defective. Electrophysiological recordings of the thoracic dorso-lateral NMJ were performed in adult dEAAT1-deficient flies. The lack of dEAAT1 prolonged the duration of the individual responses to motor nerve stimulation and this effect was progressively increased during physiological trains of stimulations. Therefore, glutamate reuptake by glial cells is required to ensure normal activity of the Drosophila NMJ, but only in adult flies (Rival, 2006).

A glutamate-dependent redox system in blood cells is integral for phagocytosis in Drosophila melanogaster

Glutamate transport is highly regulated as glutamate directly acts as a neurotransmitter and indirectly regulates the synthesis of antioxidants. Although glutamate deregulation has been repeatedly linked to serious human diseases such as HIV infection and Alzheimer's, glutamate's role in the immune system is still poorly understood. A putative glutamate transporter in Drosophila melanogaster, polyphemus (polyph), was found to play an integral part in the fly's immune response. Flies with a disrupted polyph gene exhibit decreased phagocytosis of microbial-derived bioparticles. When infected with S. aureus, polyph flies show an increase in both susceptibility and bacterial growth. Additionally, the expression of two known glutamate transporters, genderblind and excitatory amino acid transporter 1, in blood cells affects the flies' ability to phagocytose and survive after an infection. Consistent with previous data showing a regulatory role for glutamate transport in the synthesis of the major antioxidant glutathione, polyph flies produce more reactive oxygen species (ROS) as compared to wild-type flies when exposed to S. aureus. In conclusion, this study has demonstrated that a polyph-dependent redox system in blood cells is necessary to maintain the cells' immune-related functions. Furthermore, the model provides insight into how deregulation of glutamate transport may play a role in disease (Gonzales, 2013).

Decreasing glutamate buffering capacity triggers oxidative stress and neuropil degeneration in the Drosophila brain

L-glutamate is both the major brain excitatory neurotransmitter and a potent neurotoxin in mammals. Glutamate excitotoxicity is partly responsible for cerebral traumas evoked by ischemia and has been implicated in several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). In contrast, very little is known about the function or potential toxicity of glutamate in the insect brain. This study shows that decreasing glutamate buffering capacity is neurotoxic in Drosophila. The only Drosophila high-affinity glutamate transporter, dEAAT1, is selectively addressed to glial extensions that project ubiquitously through the neuropil close to synaptic areas. Inactivation of dEAAT1 by RNA interference led to characteristic behavior deficits that were significantly rescued by expression of the human glutamate transporter hEAAT2 or the administration in food of riluzole, an anti-excitotoxic agent used in the clinic for human ALS patients. Signs of oxidative stress included hypersensitivity to the free radical generator paraquat and rescue by the antioxidant melatonin. Inactivation of dEAAT1 also resulted in shortened lifespan and marked brain neuropil degeneration characterized by widespread microvacuolization and swollen mitochondria. This suggests that the dEAAT1-deficient fly provides a powerful genetic model system for molecular analysis of glutamate-mediated neurodegeneration (Rival, 2004).

Adult Drosophila head sections probed with a specific dEAAT1 antibody revealed the quasi-ubiquitous presence of the glutamate transporter in the brain neuropil, where synaptic contacts occur, and absence in the cortex, which contains neuronal and glial cell bodies. The highest protein level was detected in the protocerebrum bridge in the brain and in the optic lobes. Similarly, dEAAT1 was principally detected in the neuropil in the larval brain and ventral cord. High expression was also observed in the larval optic proliferative centers that give rise to the adult optic neuromers, in agreement with previous observations carried out by in situ hybridization (Rival, 2004).

These observations were further confirmed with a dEAAT1-GFP fusion protein expressed under the control of a dEAAT1-GAL4 driver. In dEAAT1-GAL4, UAS-dEAAT1-GFP larvae, GFP fluorescence was detected in the whole brain and ventral cord neuropil , but not in the cortex. In contrast, native GFP protein expressed with dEAAT1-GAL4 was detected in numerous large cell bodies scattered at the periphery of the CNS corresponding to dEAAT1-expressing cells, but not in the neuropil. This shows that GFP alone does not freely diffuse into the neuropil. Similarly, in adult dEAAT1-GAL4, UAS-dEAAT1-GFP flies, nearly all brain neuropil regions appeared GFP positive whereas the cell bodies were not. It is noted that dEAAT1-GFP seems to accumulate in small, intensely fluorescent spots that may correspond to synaptic clusters. In contrast, the nonspecific membrane marker mCD8-GFP was addressed to both cell bodies and neuropil extensions in adult and did not accumulate in specific spots (Rival, 2004).

To identify the cells expressing dEAAT1, an nls-lacZ reporter that targets β-galactosidase to the nucleus was expressed under the control of dEAAT1-GAL4 and colabeling experiments were performed. In the third-instar larval CNS, most dEAAT1 cells were found to be cortical cells that coexpress the glial marker Repo. dEAAT1 is also present in the midline glia, which express neither Repo nor the neuronal marker Elav. In the adult CNS, dEAAT1-expressing cells also colocalize with Repo except for a subset of cells with a small nucleus in the optic lobe. Therefore, dEAAT1 appears to be predominantly glial at all stages. Remarkably, the transporter protein is excluded from the cell bodies and selectively addressed to cytoplasmic extensions that project into the neuropil (Rival, 2004).

dEAAT1 was inactived by heritable gene silencing with double-stranded RNA. Two identical 1 kb fragments from the dEAAT1 cDNA were PCR amplified, ligated together in a sense-antisense orientation, and cloned downstream from UAS. The 1 kb fragment was complementary to the dEAAT1 cDNA from exons 2 to 9, a region that exhibits a low homology to dEAAT2, the other Drosophila EAAT gene that is a selective aspartate transporter. Two transformed lines were used in parallel as a control for possible position effects: UAS-dEAAT1-IR-X and UAS-dEAAT1-IR-II, inserted respectively on the first and second chromosome (Rival, 2004).

UAS-dEAAT1-IR and dEAAT1-GAL4 flies were crossed to induce synthesis of the double-stranded RNA selectively in the dEAAT1-expressing cells of the progeny. Viable flies were recovered that developed normally and did not have apparent behavioral defects before adult stages. When expressed with the dEAAT1-GAL4 driver, the dEAAT1-IR RNA led to a complete loss of endogenous dEAAT1 immunostaining in the whole adult brain and optic lobes except for some cells in the lamina. The maintained expression in cells of the lamina indicated that the promoter fragment included in the dEAAT1-GAL4 construct lacked regulatory elements to target these cells or, alternatively, that the RNAi mechanism did not work well in these cells (Rival, 2004).

The adult dEAAT1 RNAi flies exhibited striking neurological phenotypes that were already evident by casual observation. They could walk normally, indicating that the glutamatergic neuromuscular junctions (NMJs) were functional, but they flew poorly and did not try to escape when gently touched with a finger. Although these flies were generally hypoactive, they paradoxically overreacted when startled. This phenotype was quantified by a startle-induced negative geotaxis test previously adapted to analyze hyperexcitability behavior. Flies were placed in a column with a conic bottom end and suddenly tapped down. Wild-type Drosophila or control flies containing either the dEAAT1-GAL4 or UAS-dEAAT1-IR constructs alone immediately left the bottom of the column and generally reached the top rapidly. In contrast, the dEAAT1 RNAi flies became markedly overexcited and needed a longer delay before they started climbing. About 70% remained at the bottom, whirling erratically and falling down on each other for more than one minute. The RNAi flies that left the bottom generally stopped their course many times before climbing again, with only 3%-8% reaching the top within 1 min. This phenotype was apparently not due to neuromuscular dysfunction. First, electrophysiological recordings from the dEAAT1 RNAi fly thoracic muscles demonstrated that the NMJs were still active. Second, selective expression of the dEAAT1-IR in motor neuron-associated peripheral glia with the gliotactin-GAL4 driver did not induce similar behavioral impairments. This indicated that the hyperexcitability phenotype induced by dEAAT1 inactivation is most likely of central origin (Rival, 2004).

Since this behavior is specific and relatively easy to quantify, a number of genetic conditions and pharmacological agents were tested for their potential rescuing effects on the dEAAT1 RNAi. First, it was important to check that this phenotype is directly related to the lack of glutamate transport activity. Rescue experiments were performed by expressing hEAAT2, a distantly related (35% amino acid identity) homolog of dEAAT1 that could not be inactivated by the dEAAT1-IR double-stranded RNA. hEAAT2 (also known as GLT-1 in rat) is the major human glutamate transporter, widely expressed in brain astrocytes. Whereas expression of hEAAT2 alone had no effect on behavior, coexpression of hEAAT2 with dEAAT1-IR significantly improved the performances of the RNAi flies: 26% were now able to reach the top of the column and only 38% stayed at the bottom. Therefore, the hyperexcitability behavior is at least in part a consequence of glutamate transport deficiency and likely due to an excitotoxic accumulation of glutamate in the extracellular space (Rival, 2004).

The mechanisms of glutamate excitotoxicity in mammals involve increased mitochondrial calcium concentrations, resulting in oxidative stress due to the generation of intracellular free radicals. The effect of riluzole, an anti-excitotoxic agent known to inhibit selectively glutamate release from mammalian presynaptic terminals used in the clinic for ALS patients, was tested. dEAAT1 RNAi flies fed with riluzole presented significantly enhanced performance in the negative geotaxis test as compared to the control RNAi flies. Similar results were obtained with melatonin, an antioxidant and free radical scavenger. Oxidative stress has been implicated as a cause of aging in Drosophila. The adult dEAAT1 RNAi flies have a significant reduction in life span: they survive 10-13 days at 29°C whereas the control dEAAT1-GAL4, UAS-dEAAT1-IR-II, and UAS-dEAAT1-IR-X flies survive 20, 22, and 29 days, respectively. Sensitivity to the redox-cycling agent paraquat, a free radical generator, is a robust indicator of resistance to oxidative stress in Drosophila. Exposure of 1- to 3-day-old control flies to 10 mM paraquat decreased the survival from 90% to 50%. With the dEAAT1 RNAi flies, survival further dropped to 14% or 28%, depending on the UAS-dEAAT1-IR insertion line used. Collectively, these data strongly suggest that the lack of dEAAT1 generates oxidative stress in Drosophila (Rival, 2004).

The most drastic result of glutamate toxicity in mammals is neuronal degeneration and loss. Therefore an ultrastructural analysis was carried out of brain sections in RNAi and control flies. EM views of the dEAAT1 RNAi fly brains showed marked alterations in the whole neuropil. Neurites appeared contracted compared to control, and most neuronal processes contained abnormally electron-dense cytoplasmic material, which is characteristic of neurodegeneration. As a result, the neuropil of the RNAi flies appeared darker than the control tissues in the EM images. Numerous microvacuoles that are rare in the control are evident in the whole brain neuropil of the dEAAT1 RNAi flies. These microvacuoles are often surrounded by an electron-dense plasma membrane, suggesting that they may be formed after the degeneration of a neuronal projection. The mitochondria were often markedly swollen and distended, clearly revealing the internal crests. This was further attested by a quantitative analysis of mitochondria diameters in the two tissues. Interestingly, swollen mitochondria were often observed in mammalian brain under conditions of cellular stress and glutamate excitotoxicity, as in transgenic mouse models of ALS. In contrast, no significant effect of the lack of dEAAT1 was observed at this resolution in the cortex area where most cell bodies are located, except for the presence of myeloid-like cytoplasmic inclusions in some of the neuronal somas. In particular, nuclei with an apoptotic morphology were not observed. However, a statistically significant loss of neurons was found in three dopaminergic clusters when dEAAT1 expression was disrupted compared to control strains. Dopaminergic neurons are particularly sensitive to oxidative stress. This indicates that a moderate level of neuronal death also occurs in the absence of glutamate transporter in the Drosophila brain (Rival, 2004).

The ubiquitous and abundant expression of dEAAT1 in the neuropil suggests that glutamate plays important roles in the insect CNS and that its extracellular level has to be strictly regulated, as is the case in the mammalian brain. Accordingly, this study showed that inactivation of dEAAT1 by RNA interference induces dramatic physiological and morphological alterations in the adult. This was an unexpected finding, since glutamate was considered to date as a relatively minor neurotransmitter in the insect brain (Rival, 2004).

Although the dEAAT1-deficient flies are rather inactive, they paradoxically present a characteristic hyperexcitability behavior when startled. Several lines of evidence indicate that this is at least in part due to glutamate accumulation, leading to increased oxidative stress in the CNS. First, the behavior of the dEAAT1 RNAi flies is significantly rescued by the expression of the human glutamate transporter hEAAT2. Second, administration of the anti-excitotoxic agent riluzole or the free radical scavenger melatonin both have a significant rescuing effect. Third, the dEAAT1 RNAi flies have increased sensitivity to the free radical generator paraquat, suggesting that the lack of dEAAT1 triggers oxidative stress. Similarly in mammals, mitochondrial dysfunction and intracellular free radical generation are primary effects of glutamate excitotoxicity. Fourth, these flies have a shortened lifespan and present patent signs of neurodegeneration in the CNS, including a modest but significant loss of dopaminergic neurons. Degeneration was most prominent in the neuropil, suggesting that the neurites are principally affected. This result fits well with the observation that the dEAAT1 transporter is selectively addressed to the neuropil and excluded from the cell bodies that lay in the cortex. It can be noted that embryos and larvae of dEAAT1 RNAi flies did not present any degeneration phenotype, behavior impairments, or increased lethality, suggesting that these effects were age dependent and that the adult brain in flies is more sensitive to glutamate toxicity and oxidative stress than in earlier developmental stages (Rival, 2004).

In mammals, antisense knockdown experiments have shown that loss of glutamate uptake capability can lead to neuronal degeneration. GLT-1 knockout mice undergo lethal spontaneous seizures, increased susceptibility to brain injury, and much-shortened life span. These effects are evocative of the phenotypes observed in dEAAT1 RNAi flies. The mechanisms of glutamate neurotoxicity in Drosophila are not known. In mammals, Ca²⁺ influx in cells overstimulated by glutamate and subsequent Ca²⁺ uptake by mitochondria are major factors in excitotoxicity. Ca²⁺ entry can be mediated in particular by NMDA receptors. NMDA-like receptor subunits have been identified in Drosophila that are expressed in the CNS (Rival, 2004).

Glutamate transporter inactivation has also been detected in sporadic and familial ALS patients as well as in mouse models of this disease. Although the pathogenetic relevance of glutamate excitotoxicity in this disease is still a matter of debat, interference with glutamate-mediated toxicity is so far the only neuroprotective therapeutic strategy. The anti-excitotoxic agent riluzole indeed has an effect in prolonging survival in patients with ALS. Interestingly, this study observed a significant improvement in the behavior of the dEAAT1 RNAi flies when this drug was added to the food. Mitochondrial swelling and changes of morphology have been reported in the motor neurons of ALS patients and in mouse models of the disease. Swollen mitochondria are also apparent in the degenerating neuropil of dEAAT1 RNAi Drosophila. Therefore, the phenotype of dEAAT1 RNAi flies exhibits striking similarities with some of the symptoms associated with ALS (Rival, 2004).

In conclusion, decreasing glutamate buffering capacity in the Drosophila brain leads to various neurological defects, which suggest that extracellular glutamate accumulation is neurotoxic in insects. The dEAAT1 RNAi flies provide a new genetic model system to study the molecular mechanisms of glutamate-mediated neurotoxicity and its involvement in various neurodegenerative conditions (Rival, 2004).

Terminal glial differentiation involves regulated expression of the excitatory amino acid transporters in the Drosophila embryonic CNS

The Drosophila excitatory amino acid transporters dEAAT1 and dEAAT2 are nervous-specific transmembrane proteins that mediate the high affinity uptake of L-glutamate or aspartate into cells. This study demonstrates by colocalization studies that both genes are expressed in discrete and partially overlapping subsets of differentiated glia and not in neurons in the embryonic CNS. Expression of these transporters is disrupted in mutant embryos deficient for the glial fate genes gcm and repo. Conversely, ectopic expression of gcm in neuroblasts, which forces all nerve cells to adopt a glial fate, induces an ubiquitous expression of both EAAT genes in the nervous system.The dEAAT transcripts were detected in the midline glia in late embryos and dEAAT2 in a few peripheral neurons in head sensory organs. The results show that glia play a major role in excitatory amino acid transport in the Drosophila CNS and that regulated expression of the dEAAT genes contributes to generate the functional diversity of glial cells during embryonic development (Soustelle, 2002).

This report provides evidence that the two Drosophila excitatory amino acid transporters are selectively expressed in glial cells in the CNS. First, the dEAAT transcripts are not localized in motor neurons but in cells expressing either the lateral glia marker repo or the midline glia marker AA142. Second, dEAAT expression profiles are nearly abolished in lateral glia-deficient gcm or repo mutants, except for the midline glia expression in late embryos. Third, early ectopic expression of gcm in neuroblasts induced a marked and ubiquitous coexpression of both transporters. This last experiment indicates that the glial master gene gcm is sufficient to trigger expression of these transporters and suggests that dEAAT expression may be part of a default state of lateral glia differentiation. However, gcm can be dispensable for dEAAT transcription in some cases since the transporters were also detected in the midline glia and dEAAT2 in sensory neurons (Soustelle, 2002).

Several observations suggest that gcm is not a direct activator of dEAAT transcription in the lateral glia. In wild-type embryos, gcm is transitorily expressed to trigger the lateral glia fate and this occurs at a much earlier stage than the onset of dEAAT expression. Similarly, in the ectopic gcm experiment, it was observed that the onset of dEAAT expression occurred later than the onset of repo expression, at a stage when the embryonic CNS was more disorganized. In addition, the consensus sequence for gcm binding is absent in the 5' upstream region of the dEAAT genes, whereas 11 such sequences are present in the repo promoter. In repo mutants, glial determination is initiated but terminal differentiation is altered, leading to fewer glial cells, disrupted fasciculation of axons, and defects in ventral nerve cord condensation. The disruption of dEAAT patterns in repo mutants confirms that the onset of expression of these transporters coincides with terminal glial differentiation and not early gliogenesis. The dEAAT genes could be direct targets of repo but other regulatory factors are normally involved since numerous repo-positive cells do not express these transporters in wild-type embryos, like the peripheral glia and some CNS glial cells. This implies that cell-autonomous cues or additional regulatory signals originating from the developing neighbor neurons or glial cells are required to repress expression of these transporters in specific glia subsets (Soustelle, 2002).

In the vertebrate CNS, glutamate uptake is crucial to maintain low extracellular glutamate concentration and permit an efficient excitatory synaptic transmission. Different EAATs are found in neurons or glial cells, but the glial transporters are primarily responsible for transmitter recycling at synapses and protection of neurons against glutamate excitotoxicity. Mammalian astrocytes capture glutamate at synapses and rapidly convert it to glutamine with the glial-specific enzyme glutamine synthetase. According to the glutamate/glutamine cycle hypothesis, glutamine is then furnished to neurons where it is converted back to L-glutamate in nerve terminals by the neuron-specific enzyme glutaminase, thereby restoring the synaptic pool of neurotransmitter. For now, only two observations argue for the occurrence of such a metabolic cycle in the Drosophila CNS, i.e., the presence of a glutamine synthetase gene specifically expressed in the nervous system and the glial-specific expression of the dEAAT genes reported in this study (Soustelle, 2002).

The glial localization of the glutamate transporter dEAAT1 suggests that this protein could play a physiological role in neurotransmission at central glutamatergic synapses in Drosophila as is the case for their vertebrate counterparts. However, no dEAAT1 was detected at the glutamatergic NMJs or in motor neurons in embryos. At this stage, NMJs are not fully mature and the glial wrappings around the motor axons terminate about 5-10 microm before the synapse. During larval life, peripheral glia further elongate, and in third instar larvae, the distal motor endings of type I and II synapses are entirely wrapped by glial extensions. So it is quite possible that the onset of dEAAT1 expression occurs at a later stage at the NMJ. Recent observations suggest that this is likely to be the case (Soustelle, 2002).

The dEAAT transporters could also play nonsynaptic roles. The fact that dEAAT1 and dEAAT2 are expressed in the neuropile-associated longitudinal and midline glia in late embryo is intriguing and suggests that these transporters could participate in the specific function of these cells in axon guidance and enwrapping. Such a role has not been proposed previously for vertebrate EAATs. Interestingly, the EAAT protein GLAST was detected before synaptogenesis in the radial glial cells in the mouse spinal cord. These cells produce long processes along which developing neurons migrate and elongate their axons. In addition, the widespread expression of dEAAT1 in the CNS suggests that this transporter may contribute to neuron survival and CNS integrity by protecting nerve cells against excitotoxicity, as is the case with the vertebrate glial glutamate transporters. Some of the developmental and structural defects observed in the CNS in repo mutants could be consequent to the lack of dEAAT expression (Soustelle, 2002).

In conclusion, these results show that glia play a major role in excitatory amino acid transport in the Drosophila CNS and that regulated expression of the dEAAT genes most contributes to generate the functional diversity of glial cells during embryonic development. Further work will address the precise role of these transporters in nervous system development and function and the genetic mechanisms underlying the control of their expression during glial differentiation (Soustelle, 2002).

Search PubMed for articles about Drosophila Eaat1

Besson M. T., Soustelle, L. and Birman, S. (1999). Identification and structural characterization of two genes encoding glutamate transporter homologues differently expressed in the nervous system of Drosophila melanogaster. FEBS Lett 443: 97-104. PubMed ID: 9989583

Besson, M. T., Soustelle, L. and Birman, S. (2000). Selective high-affinity transport of aspartate by a Drosophila homologue of the excitatory amino-acid transporters. Curr. Biol. 10: 207-210. PubMed ID: 10704415

Besson, M. T., Ré D. B., Moulin, M. and Birman, S. (2005). High affinity transport of taurine by the Drosophila aspartate transporter dEAAT2. J. Biol. Chem. 280: 6621-6626. PubMed ID: 15611131

Carmona, M. A., et al. (2009). Glial ephrin-A3 regulates hippocampal dendritic spine morphology and glutamate transport. Proc. Natl. Acad. Sci. 106: 12524-12529. PubMed ID: 19592509

Cattaert, D. and Birman, S. (2001). Blockade of the central generator of locomotor rhythm by noncompetitive NMDA receptor antagonists in Drosophila larvae. J Neurobiol 48(1): 58-73. PubMed ID: 11391649

DiAntonio, A. (2006). Glutamate receptors at the Drosophila neuromuscular junction. Int. Rev. Neurobiol. 75: 165-179. PubMed ID: 17137928

Featherstone, D. E., et al. (2005). An essential Drosophila glutamate receptor subunit that functions in both central neuropil and neuromuscular junction. J. Neurosci. 25: 3199-3208. PubMed ID: 15788777

Filosa, A., et al. (2009). Neuron-glia communication via EphA4/ephrin-A3 modulates LTP through glial glutamate transport. Nat. Neurosci. 12: 1285-1292. PubMed ID: 19734893

Freeman, M. R., et al. (2003). Unwrapping glial biology: Gcm target genes regulating glial development, diversification, and function. Neuron 38: 567-580. PubMed ID: 12765609

Gonzalez, E. A., Garg, A., Tang, J., Nazario-Toole, A. E. and Wu, L. P. (2013). A glutamate-dependent redox system in blood cells is integral for phagocytosis in Drosophila melanogaster. Curr Biol 23: 2319-2324. PubMed ID: 24210616

Li, G., Gong, J., Lei, H., Liu, J. and Xu, X. Z. (2016). Promotion of behavior and neuronal function by reactive oxygen species in C. elegans. Nat Commun 7: 13234. PubMed ID: 27824033

MacNamee, S. E., Liu, K. E., Gerhard, S., Tran, C. T., Fetter, R. D., Cardona, A., Tolbert, L. P. and Oland, L. A. (2016). Astrocytic glutamate transport regulates a Drosophila CNS synapse that lacks astrocyte ensheathment. J Comp Neurol 524(10): 1979-1998. PubMed ID: 27073064

Marcaggi, P., Billups, D. and Attwell, D. (2003). The role of glial glutamate transporters in maintaining the independent operation of juvenile mouse cerebellar parallel fibre synapses. J. Physiol. 552: 89-107. PubMed ID: 12878755

Matsugami, T. R., et al. (2006). Indispensability of the glutamate transporters GLAST and GLT1 to brain development. Proc. Natl. Acad. Sci. 103: 12161-12166. PubMed ID: 16880397

Matsuno, M., Horiuchi, J., Tully, T. and Saitoe, M. (2009). The Drosophila cell adhesion molecule klingon is required for long-term memory formation and is regulated by Notch. Proc Natl Acad Sci U S A 106(1): 310-315. PubMed ID: 19104051

Matsuno, M., Horiuchi, J., Yuasa, Y., Ofusa, K., Miyashita, T., Masuda, T. and Saitoe, M. (2015). Long-term memory formation in Drosophila requires training-dependent glial transcription. J Neurosci 35(14): 5557-5565. PubMed ID: 25855172

Matsuno, M., Horiuchi, J., Ofusa, K., Masuda, T. and Saitoe, M. (2019). Inhibiting glutamate activity during consolidation suppresses age-related long-term memory impairment in Drosophila. iScience 15: 55-65. PubMed ID: 31030182

Matthias, K., et al. (2003). Segregated expression of AMPA-type glutamate receptors and glutamate transporters defines distinct astrocyte populations in the mouse hippocampus. J. Neurosci. 23: 1750-1758. PubMed ID: 12629179

MacNamee, S. E., Liu, K. E., Gerhard, S., Tran, C. T., Fetter, R. D., Cardona, A., Tolbert, L. P. and Oland, L. A. (2016). Astrocytic glutamate transport regulates a Drosophila CNS synapse that lacks astrocyte ensheathment. J Comp Neurol 524(10): 1979-1998. PubMed ID: 27073064

Milton, V. J., Jarrett, H. E., Gowers, K., Chalak, S., Briggs, L., Robinson, I. M. and Sweeney, S. T. (2011). Oxidative stress induces overgrowth of the Drosophila neuromuscular junction. Proc Natl Acad Sci U S A 108(42): 17521-17526. PubMed ID: 21987827

Oswald, M. C., Brooks, P. S., Zwart, M. F., Mukherjee, A., West, R. J., Giachello, C. N., Morarach, K., Baines, R. A., Sweeney, S. T. and Landgraf, M. (2018). Reactive oxygen species regulate activity-dependent neuronal plasticity in Drosophila. Elife 7. PubMed ID: 30540251

Peng, J. J., Lin, S. H., Liu, Y. T., Lin, H. C., Li, T. N. and Yao, C. K. (2019). A circuit-dependent ROS feedback loop mediates glutamate excitotoxicity to sculpt the Drosophila motor system. Elife 8. PubMed ID: 31318331

Rival, T., et al. (2004). Decreasing glutamate buffering capacity triggers oxidative stress and neuropil degeneration in the Drosophila brain. Curr. Biol. 14: 599-605. PubMed ID: 15062101

Rival, T., et al. (2006). Physiological requirement for the glutamate transporter dEAAT1 at the adult Drosophila neuromuscular junction. J. Neurobiol. 66: 1061-1074. PubMed ID: 16838372

Soustelle, L., Besson, M. T., Rival, T. and Birman, S. (2002). Terminal glial differentiation involves regulated expression of the excitatory amino acid transporters in the Drosophila embryonic CNS. Dev. Biol. 248: 294-306. PubMed ID: 12167405

Stacey, S. M., et al. (2010). Drosophila glial glutamate transporter Eaat1 is regulated by fringe-mediated notch signaling and is essential for larval locomotion. J. Neurosci. 30(43): 14446-57. PubMed ID: 20980602

Takayasu, Y., et al. (2005). Differential roles of glial and neuronal glutamate transporters in Purkinje cell synapses. J. Neurosci. 25: 8788-8793. PubMed ID: 16177048

Takayasu, Y., et al. (2006). Glial glutamate transporters maintain one-to-one relationship at the climbing fiber-Purkinje cell synapse by preventing glutamate spillover. J. Neurosci. 26: 6563-6572. PubMed ID: 16775144

Tanaka, K., et al. (1997). Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science 276: 1699-1702. PubMed ID: 9180080

Watase, K., et al. (1998). Motor discoordination and increased susceptibility to cerebellar injury in GLAST mutant mice. Eur. J. Neurosci. 10: 976-988. PubMed ID: 9753165

date revised: 12 September 2022

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