Glutamate, glutamate transporters, and circadian rhythm sleep

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Glutamate, glutamate transporters, and circadian rhythm sleep disorders in neurodegenerative diseases Suifen He, Xiuping Zhang, and Shaogang Qu ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00419 • Publication Date (Web): 28 Nov 2018 Downloaded from http://pubs.acs.org on November 30, 2018

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Glutamate, glutamate transporters, and circadian rhythm sleep disorders in neurodegenerative diseases Suifen He1, 2, 3 Xiuping Zhang4 Shaogang Qu*1, 2, 3

1Central

Laboratory, Shunde Hospital of Southern Medical University (The First People’s Hospital of Shunde Foshan), Foshan, Guangdong 528300, China;

2Department of Neurology, Shunde Hospital of Southern Medical University (The First People’s Hospital

of Shunde Foshan), Foshan, Guangdong 528300, China;

3Key Laboratory of Mental Health of the Ministry of Education, Southern Medical University, Guangzhou,

Guangdong 510515, China;

4Teaching

Center of Experimental Medicine, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong 510515, China.

Correspondence: Shaogang Qu ([email protected])

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ABSTRACT Glutamate, a primary excitatory neurotransmitter and an important intermediate in the cellular metabolism of the brain, has a wide spread influence in the sleep–wake regulatory system. Glutamate transporters, including vesicular glutamate transporters and excitatory amino acid transporters, serve as the main force controlling the extracellular concentration of glutamate in the brain. These are likely to be critical tools needed for the brain to modulate the sleep–wake cycle and are likely innervated by the circadian rhythm system in a day–night variant pattern. Because in the initial stages, nearly all patients with neurodegenerative diseases have rhythmic sleep disorders that become aggravated with disease development and often exhibit glutamate uptake dysfunction, we examined whether the above glutamate transporters could be used as potential targets to help address circadian rhythm sleep disorders in patients with neurodegenerative diseases. Therefore, in this review, we sought to analyze the principles governing glutamate transmission and discuss whether the circadian rhythm regulatory properties of these processes endow glutamate transporters with unique functions in the sleep–wake shift of the brain. We attempt to provide a theoretical framework in this field for future studies, to help in the exploration of potential therapeutic targets to delay or prevent the development of neurodegenerative diseases. Key Words: glutamate, glutamate transporters, ATP, adenosine, circadian rhythm sleep disorders, neurodegenerative diseases


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Introduction Sleep is one of the essential physiological functions for human survival. Circadian rhythm sleep disorders (CRSD) drive pathogenesis early in the course of neurodegenerative diseases and have long been symptomatic hallmarks of these diseases1. Disrupted sleep may increase the risk of Alzheimer disease (AD) via increased amyloid β (Aβ) production2. Accordingly, Aβ accumulation in the brain of patients with AD may result from an imbalance between Aβ production and clearance3,4; to a certain extent, this may be owing to CRSD. In addition, sleep loss leads to numerous consequences for the nervous system and can seriously impact other physiological functions and psychiatric states, such as depression and anxiety disorders5,6. Therefore, it is essential to decipher the pathogenic mechanism of CRSD, to explore potential therapeutic targets for neurodegenerative diseases. Sleep is regulated by circadian rhythms and sleep homeostasis7. Disruption of normal circadian rhythms leads to incorrect ordering of daily cellular metabolic cycles and consequent metabolic disorders and neurodegeneration8,9. To reveal the underlying mechanisms of sleep, much attention has been given to studying the functional mechanisms of neuropeptides and neurotransmitters in the brain, including melatonin10, orexin11,12, adenosine13,14, amino acid transmitters15,16, and monoaminergic and cholinergic transmitters17-19. Recently, accumulating evidence indicates that the backbone of the sleep– wake regulatory system mainly depends on the fast neurotransmitters, especially glutamate and γaminobutyric acid (GABA)20. Via their different distributions, proportions, and discharge profiles, glutamatergic and GABAergic neurons make different contributions to the generation and shift of sleep–wake states21. Based on the harmonious cooperation of these neurons, the brain can be flexible and efficient in controlling the shift of sleep–wake states, under normal physiological conditions. Glutamate is a pivotal compound involved in the intracellular metabolism of the brain22. Variation in the concentrations of extracellular glutamate exhibits significant circadian rhythm23. Because its availability is closely correlated with the arousal state, glutamatergic signaling must be strictly regulated to maintain the proper timing of sleep16,24. Therefore, we attempted to review the regulatory models of sleep–wake circuitry and the governing principles of glutamate transmission in the brain, to analyze how the circadian rhythm regulatory system could modulate the sleep–wake shift via glutamate transporters, namely, vesicular glutamate transporters (VGLUTs) and excitatory amino acid transporters (EAATs). Metabolism of glutamate in the brain Glutamate and GABA are metabolically interrelated and tightly coupled to the intermediary metabolism within cellular energy homeostasis. Because glutamate is the only direct precursor of GABA in the brain, here we mainly review the metabolism of glutamate. Glutamate cannot penetrate the blood–brain barrier25. According to some reports, glutamate in the brain is synthesized de novo in astrocytes, or it is indirectly converted from glucose by pyruvate dehydrogenase and astrocyte-specific enzyme pyruvate carboxylase26-30. Brancaccio and colleagues proved that extracellular glutamate originates specifically from astrocytes30. Recently, a novel intraneuronal glutamate biosynthetic pathway has been revealed, using single-cell mass spectrometry and isotopic labeling technology that UV-triggered glutamate synthesis derived from urocanic acid in neurons31. Thus, glutamate in the brain might be mainly generated by astrocytes32; however, the contribution of neurons cannot be ignored. VGLUTs in neurons or astrocytes are responsible for the uptake of intracellular glutamate into vesicles, wherein glutamate will then be released into the synaptic cleft via exocytosis. After its release, excessive glutamate in the synaptic cleft will either be up taken into cells via EAATs or its excitotoxicity will cause neuronal injury or death33. No enzyme systems in the extracellular fluid can metabolize glutamate, and glutamine synthetase (GS; an enzyme that catalyzes the adenosine triphosphate (ATP)dependent condensation of glutamate with ammonia to form glutamine) is selectively expressed in astrocytes; therefore, EAATs of astrocytes act as the main force to undertake most glutamate clearance and promote the metabolism of glutamate in astrocytes. After transport into astrocytes, large amounts of glutamate enter the glutamate–glutamine cycle, as follows: 1) glutamate is converted to glutamine by GS; 2) glutamine is exported via astrocytic sodium-coupled neutral amino acid transporters (SNATs); 3) extracellular glutamine is then imported into neurons where phosphate activated glutaminase can reconvert it to glutamate34-36. In addition, part of the remaining glutamate will be converted to GABA, which is the primary inhibitory transmitter in the central nervous system (CNS) as well as a component of glutathione. The other part of the remaining glutamate will be converted to α-ketoglutarate by amino transferases in the cytoplasm or by glutamate dehydrogenase in the mitochondria37,38, to finally provide alternate sources of adenosine triphosphate (ATP) that can be hydrolyzed to adenosine (Ado)14. Obviously, the metabolism of glutamate can greatly impact the generation of glutamine, GABA, ATP, and Ado.




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Function of glutamate in the modulation of sleep In the mammalian brain, glutamate is the primary excitatory neurotransmitter and it is indispensable for all motor, sensory, and autonomic processing39. In the glutamatergic system, glutamate can trigger the excitatory impulse by activating glutamate receptors on the postsynaptic membrane of glutamatergic neurons22. In the GABAergic system, glutamate can increase GABAergic inhibitory tone across the circuit by activating pre-synaptic glutamate receptors32. Therefore, the comprehensive effect of glutamate actually depends on the specific location of its receptors. In addition, the activities of glutamatergic neurons induce neuronal secretion of glutamate, which triggers nearby astrocytes to release ATP and Ado in a feedback loop (Figure 1)40. Although extracellular ATP could be rapidly degraded to Ado by ectonucleotidases, both ATP and Ado are sent to mediate heterosynaptic suppression by activating their receptors (P2YR, purinergic 2Y receptors; A1R, adenosine A1 receptors). In the modulating process of sleep homeostasis, ATP and Ado are considered to be critical gliotransmitters for transforming metabolic need into sleep demand13,41. Decreasing gliotransmission could reduce the activation of A1R and impair sleep homeostasis42,43. Moreover, astrocytic activation of A1R could decrease the surface expression of NMDAR (N-methyl-D-aspartate receptor, a kind of glutamate receptor) through an SFK (the Src family of tyrosine kinases)-dependent pathway on a long time scale44. Therefore, once extracellular Ado reaches a high concentration, the availability of glutamate will decrease gradually and fundamentally. Interestingly, astrocyte-derived ATP also acts as a key factor involved in the modulation of depressive-like behavior in adult mice45. This might help us to understand why patients with neurodegenerative disease generally have comorbid sleep disorders and depression46. Deficiency of the RNA-editing gene Adar leads to increased sleep in Drosophila, which might be owing to the dysfunction of glutamatergic synapses24. Interestingly, in Adar-deficient Drosophila, VGLUTs are upregulated, which might result in greater release of glutamate from neurons. This leads to subsequent overactivation of glutamate receptors (AMPAR, AMPA receptor; NMDAR). Generally speaking, frequent neural activity will induce greater ATP and Ado release from astrocytes, which in turn could mediate heterosynaptic suppression (Figure 1)40. That is to say, during continuous activity of neurons, Adar can break sleep-promoting glutamatergic signaling by restricting the expression of VGLUTs, so as to control the quantity of available glutamate that is released and reduce the activation of neuronal glutamate receptors24. Collectively, glutamate is a critical mediator in the sleep–wake regulatory system. We presume that modulating the release or uptake of glutamate could effectively alter the available glutamate and thus have a fundamental impact on sleep demand. Molecular mechanism of glutamate transport by VGLUTs and EAATs It is well known that both VGLUTs and EAATs are capable of glutamate uptake, but they have different transmembrane topologies47-49, as well as different molecular mechanisms of glutamate uptake. Glutamate accumulation in synaptic vesicles is achieved by VGLUTs coupled to the vesicular proton-pump ATPase, which can generate an electrochemical proton gradient to support VGLUTs’ uptake of glutamate50,51. This mechanism enables glutamate to enter the neurotransmitter pathway, but away from its metabolic pathway. At the expense of ATP generated through local glycolysis52, synaptosomes accumulate glutamine-derived glutamate or α-ketoglutarate-derived glutamate in the synaptic vesicles50,53. In neurons, the immunohistochemistry of VGLUTs shows that VGLUT2 is localized in glutamatergic synapses, with higher release probability than VGLUT154; VGLUT3 is primarily found in nonglutamatergic neurons55. The different localization of different VGLUT subtypes is generally responsible for the diverse modes of glutamate signaling. Although astrocytes possess a great deal of intracellular and cell-surface membranes and receptors, their long-distance communication abilities are very poor. Gliotransmission enables astrocytes adapt so as to suit local chemical signal computation, storage, and processing on different spatiotemporal scales. Yet the mechanism of vesicular gliotransmitter release remains unclear and forms the basis for much controversy56. As reported, VGLUTs in astrocytes have been detected at low expression levels using both single-cell real-time polymerase chain reaction (RT-PCR) and immunogold electron microscopy labeling57 but not transcriptome analysis58. Because astrocytes are always active during circadian night and release a substantial proportion of glutamate at night32, the above methods might yield different results if performed at different times of the day. EAATs are high-affinity sodium-dependent glutamate transporters, by which glutamate is cotransported with three sodium ions and one proton into the cell and countertransported with a potassium ion out of the cell59,60. Additionally, an uncoupled chloride (Cl) conductance is activated by


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binding glutamate and Na+, but the Cl− flux is independent of the rate and uncoupled to glutamate transport61-63. However, the Cl− flux determines the uptake activity of glutamate as well64. According to the Nernst equation, glutamate transporters could generate a one-million-fold concentration gradient of glutamate across the membrane at resting membrane potential59. In the mammalian brain, there are five subtypes of EAATs: EAAT1 (GLAST), EAAT2 (GLT-1), EAAT3 (EAAC1), EAAT4, and EAAT5. EAAT1 is mainly expressed by astrocytes and oligodendrocytes65,66. EAAT2 represents up to nearly 1% of total brain protein67 and is expressed at much higher levels in astrocytes than in other brain cells66,68. In contrast, EAAT3 is mainly located on the postsynaptic membrane of neurons69. EAAT4 is mainly expressed in cerebellar Purkinje cells70, and EAAT5 is found on retinal bipolar cells71. Notably, EAAT1 and EAAT2 in astrocytes make the greatest contribution to most synaptic glutamate clearance in the CNS. Therefore, downregulation of EAAT1 and EAAT2 could seriously hinder the clearance of glutamate from the synaptic cleft and thus delay the termination of excitation. Generally, VGLUTs are involved in vesicular storage of intracellular glutamate and modulation of glutamate release, whereas EAATs are responsible for uptake of extracellular glutamate from the synaptic cleft; therefore, EAATs can prevent excitatory neurotoxicity from excess glutamate or can perform local subtle regulation of the synaptic membrane on which they are located. Thus, we presume that VGLUTs and EAATs jointly determine the basic concentration of extracellular glutamate as well as mediate glutamatergic transmission of neuronal–glial circuits. Glutamatergic signaling versus circadian rhythm Using brain microdialysis technology, extracellular concentrations of different neurotransmitters have been found to follow circadian rhythm72-74. In the neostriatum of the awake rat, glutamate and GABA present higher concentrations during the night but lower levels in the daytime; perfusion with melatonin (for 19 consecutive hours) prevents reductions in both transmitters during the daytime23. Melatonin, produced by the pineal gland in vertebrates, is an important regulator of sleep and circadian rhythms, and it easily passes through the blood–brain barrier. Melatonin also has unique biochemical properties in redox activity. The diurnal change in endogenous secretion of melatonin can influence day–night variations in glutamate and GABA but not those in dopamine (DA) and its metabolites. Thus, melatonin could directly influence the electrical and metabolic activity of the suprachiasmatic nucleus (SCN)75 and could impact glutamatergic signaling in circadian rhythm. The mammalian SCN is the master pacemaker for circadian rhythms, in which more than 95% of neurons are GABAergic76. Without external stimulation, SCN neurons sustain circadian gene expression and electrical activity32. Although GABA, the principal neurotransmitter in the SCN, may act as the signal to induce rhythmic oscillations of core clock genes in the neurons, the clock gene Bmal1 in astrocytes may regulate the daily rhythms of GABA tone via modulating the expression of GABA transporter 3 (GAT3)77,78 (Figure 2), and thus alter circadian locomotor behavior as well as cognition in mice77. Moreover, high concentration of astrocytic glutamate can trigger the release of GABA via activation of pre-synaptic NR2C (an NMDAR subunit) in the dorsal SCN, and subsequently inhibit postsynaptic neurons32 (Figure 2). Selectively inhibiting NR2C not only impacts membrane potential but also impairs circadian neuronal rhythms of membrane potential, and clock gene expression32. Both types of cells can equally impart timekeeping information to the rest of the body32. This indicates that the pacemaker circuit, a combination of neurons and astrocytes, depends on the network of glutamatergic and GABAergic signaling systems32,78. That is to say, astrocytes in the SCN may communicate with neurons via glutamate and GABA, whereas neurons can sense local oscillations of astrocytically released glutamate via NR2C and can accept the signal of inhibitory GABA tone which can affect the oscillator. Thus, dysfunction in glutamatergic or GABAergic signaling systems may become the cause of CRSD. Experiments have showed that regulating EAAT1 expression can alter the sleep phenotype of Drosophila79. Mice subjected to sleep deprivation were found to have upregulated expression of GLT180. In addition, compensation of elevated DVGLUT (VGLUT in Drosophila) could restore normal sleep in Adar-deficient Drosophila24. It proves that glutamate transporters are closely related to the working concentration of glutamate in the brain, as well as the sleep–wake states. Considering that injection of glutamate into the SCN results in phase shifts of the circadian activity rhythm81, astrocytically released glutamate may act as a rhythmic signal to regulate the release of GABA from the presynaptic membrane32. Therefore, modulating the expression of astrocytic glutamate transporters may become an auxiliary approach to restore circadian sleep–wake states. CRSD in neurodegenerative diseases CRSD represent the most common incipient clinical symptom of neurodegenerative diseases82. Considering that patients with neurodegenerative diseases generally exhibit glutamate uptake dysfunction83-85 as well as sleep disorder86, their circadian clock genes might fail to control glutamatergic




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signaling with a normal paradigm. At the cellular level, the circadian clock is essentially comprised of a group of conserved clock proteins. These proteins constitute a transcriptional–translational feedback loop and mediate daily oscillations via the expression of circadian clock genes87. Transcriptomic studies of habitual short sleep duration reveal that hundreds of genes in cerebellar and brain stem samples from mice exhibit circadian oscillations88. Sleep loss also impacts mammalian target of rapamycindependent protein synthesis89,90 and subsequently impairs sleep-regulated functions91,92. These findings provide a potential link between sleep disturbance and adverse metabolic and inflammatory outcomes. These results could help to explain why disruption of normal circadian rhythms would impact numerous pivotal processes involved in neurodegeneration and why sleep disorders can also impair memory, cognitive functions, and mental health93. Given that the intracellular clocks of SCN astrocytes can remold the circadian behavioral rhythms of adult mice32, a promising strategy might be to adjust circadian rhythms via modulating the levels of astrocytic glutamate transporters in the SCN. However, the sleep–wake regulatory system is implicated in various brain regions and circuits20, thus, different subtypes of sleep disorder might result from the malfunction of different brain regions. Additionally, circadian clock genes have been proved to be expressed in most cells of the brain94,95, and they could drive oscillations in diverse biological processes that impact sleep, locomotor activity, blood hormone levels, and so on. Neurons and astrocytes in culture also exhibit circadian clock gene oscillations95. Therefore, disorders of circadian behavioral rhythms might also result from failure of the sleep–wake regulatory system to obtain the available reset instructions from the circadian pacemaker. In this case, precise regulation of patients’ glutamatergic signaling in specific regions may lead to personalized treatment and obtain better curative effects in CRSD. However, it is an extremely challenging task to locate at the specific lesion of different subtypes of sleep disorder for precise regulation, which may require creating innovative drug delivery approaches for the objective target. Moreover, different working- concentrations of glutamate may be required for various brain regions. For example, in the SCN, NR2C specifically mediates astrocyticneuronal glutamatergic signaling, and its activation requires a higher working-concentration of glutamate32. This suggests that the regulation of glutamatergic signaling may have greater inner complexity than previously thought. Conclusion Via shuttling between neurons and gliacytes, glutamate can either mediate excitatory neuronal activities directly or mediate inhibitory neuronal activities indirectly. The extracellular glutamate concentration is regulated by VGLUTs and EAATs together. On the one hand, neuronal VGLUTs directly impact the released quantity of glutamate from neurons whereas astrocytic EAATs are responsible for maintaining the concentration of extracellular glutamate below levels of excitatory toxicity. VGLUTs and EAATs serve as the main forces in determining the basic tone of glutamate in the synaptic cleft. On the other hand, neuronal EAATs and astrocytic VGLUTs appear to make greater contributions in the subtle regulation of local glutamatergic signaling transmission. Accordingly, to avoid excesses and shortfalls in daily sleep, VGLUTs and EAATs may cooperate closely in controlling glutamatergic signaling in a circadian paradigm. Thus, if the underlying mechanisms of these processes were completely revealed, regulation of glutamatergic signaling to control the sleep–wake shift might became a promising therapeutic approach in CRSD, which may also offer hope for delaying the progression of neurodegenerative diseases. However, it is challenging to design drugs that target and regulate glutamate transporters in the local lesion, to achieve appropriate concentrations of glutamate. Further effort is required to clarify the causal relationship between specific etiological mechanisms and their relative subtypes of CRSD. Currently, the best suggestion for patients with CRSD remains to seek medical intervention as early as possible.

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Circadian clock may modulate daily rhythm via glutamatergic signaling

Author contributions He S, Zhang X, and Qu S wrote and edited the manuscript. Notes The authors declare that there are no conflicts of interest regarding the publication of this paper. ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (grant nos. 31570716, 81870991, and U1603281 to Shaogang Qu), Clinical Research Startup Program of Southern Medical University by High-Level University Construction Funding of Guangdong Provincial Department of Education (grant no. LC2016PY055 to Shaogang Qu), and the Program for Changjiang Scholars and Innovative Research Team in University (grant no. IRT_16R37). REFERENCES 1 Musiek, E. S. & Holtzman, D. M. Mechanisms linking circadian clocks, sleep, and neurodegeneration. Science 354, 1004-1008, doi:10.1126/science.aah4968 (2016). 2

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FIGURE 1 Glutamate-induced heterosynaptic suppression of astrocytic-derived ATP and Ado. Following frequent neural activity, intracellular glutamate will be taken up into synaptosomes via VGLUTs and released into the synaptic cleft from the pre-synaptic membrane. Extracellular glutamate activates glutamate receptors on the postsynaptic membrane and immediately induces the excitatory impulse of the postsynaptic neuron. Meanwhile, glutamate released from the pre-synaptic membrane activates astrocytes. To feedback the neuronal activity, astrocytes release ATP and Ado for heterosynaptic suppression40, which is achieved by ATP and Ado acting on their receptors (i.e., P2YR and A1R). Ado activating A1R on the postsynaptic membrane increases the endocytosis of NMDAR via an SFKdependent pathway44, such that the excitatory effect of glutamate could be suppressed on a long time scale. Abbreviations: Pre, pre-synaptic terminal; Post, postsynaptic terminal; Glu, glutamate; NMDAR, N-methyl-D-aspartate receptor; EAAT, excitatory amino acid transporter; VGLUT, vesicular glutamate transporter. ATP, adenosine triphosphate; Ado, adenosine; P2YR, purinergic 2Y receptor; A1R, adenosine A1 receptor; SFK, the Src family of tyrosine kinases.


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FIGURE 2 Both glutamate and GABA are involved in rhythm transmission in the pacemaker circuit. The GABA inhibitory tune is coupled to the oscillator. In the SCN, NR2C on the postsynaptic membrane is responsible for sensing local oscillations of astrocytically released glutamate32. VGLUTs and EAATs in astrocytes may influence glutamate in the extracellular space. During the circadian night, high concentration of glutamate from astrocyte can activate NR2C to trigger synaptic release of GABA and thus regulate GABAergic inhibitory tone. Moreover, the clock gene Bmal1 in astrocytes may impact the extracellular GABA concentration by regulating the expression of GAT377. Abbreviations: EAAT, excitatory amino acid transporter; GAT3, GABA transporter 3; GABAAR, GABAA receptor; NR2C, an NMDAR subunit; VGLUT, vesicular glutamate transporter.


Glutamate, glutamate transporters, and circadian rhythm sleep

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