Evidence that N-acetylaspartylglutamate is the Astrocyte-Targeted Neurovascular Coupling Agent that Regulates Slow Tonic Control of Brain Blood Flow

N-acetylaspartylglutamate (NAAG) is the highest concentration dipeptide present in brain. It is found primarily in neurons but its function is unclear. NAAG is synthesized by neurons from N-acetylaspartate and glutamate (Glu), maintained at mM concentrations and is released non-synaptically to extracellular fluid (ECF). NAAG is a non-excitatory form of Glu, and is targeted to the metabotropic group II Glu receptor 3 (mGluR3) on the surface of astrocytes. After docking with the receptor, Glu is released by the action of NAAG peptidase. Previously, it was shown for the first time that an NAAG-peptidase inhibitor reduced global cerebral blood flow (CBF) in mouse brain but did not affect their physical performance. Recently, it has been demonstrated that there are two separate systems involved in neurovascular coupling by astrocytes, one is a rapid focal phasic response providing energy for stimulation-induced neuronal activity, and the other a slower global tonic response providing energy for ongoing metabolic activities. Many neurovascular coupling mechanisms are known that regulate phasic changes in CBF, but how the brain accomplishes tonic control is unknown. In this paper we bring together two separate lines of inquiry, the decades’ long search for the function of NAAG, and the more recent search for the mechanism of tonic neurovascular control. Herein, we present evidence that NAAG is the neurovascular coupling agent that regulates tonic changes in CBF via the astrocyte mGluR3NAAG peptidase connection. DOI : 10.14302/issn.2572-5424.jgm-16-1028 Corresponding author : Center for Biomedical Imaging and Neuromodulation, Nathan Kline Institute for Psychiatric Research, 140 Old Orangeburg Road, Orangeburg, NY, 10962, USA. Tel 845 398 5573, FAX: 845 398 5472 baslow@nki.rfmh.org Running title: NAAG regulates tonic cerebral blood flow


Introduction
The Question of the Function of NAAG The presence of high levels of N-acetylaspartate (NAA) and N-acetylaspartylglutamate (NAAG) in the mammalian brain, and of most of the enzymes that synthesize and hydrolyze them were discovered more than six decades ago. At that time it was also noted that these substances and their anabolic and catabolic enzymes were highly compartmentalized with most NAA and NAAG and their anabolic enzymes present in neurons, and their catabolic enzymes present in macroglia. These early findings have been reviewed [1,2]. Subsequently, in a review of the properties of metabotropic glutamate receptors (mGluR's) found in brain, it was reported that of the eight members of the three groups of mGluR's, that NAAG was a selective agonist for the group II mGluR3 receptor [3]. At the same time, two papers were published, one that demonstrated for the first time that the enzyme that hydrolyzed NAA was only expressed in oligodendrocytes [4], and the other that showed that the enzyme that hydrolyzed NAAG was expressed "exclusively in astrocytic glial cells" [5]. The "nagging" question of the function of NAAG was considered in 1997 [6] and revisited in 2015 [7].
The hypothesis that NAAG was intimately involved in neuron-astrocyte communication was first suggested in 1999 [5] and it was speculated that NAAG, via the action of its substrate-specific astrocytic peptidase, "may be an important mediary of neuronal-glial communication".
The expanded NAAG functional hypothesis was a logical step in the evolution of the concept in that it considered the entire NAA-NAAG metabolic sequence as a unique linked tri-cellular system and stated that "NAAG in the CNS may have a …primary role" in "neuronal-glial cellspecific signaling and communication" [8]. In 2005 it was observed for the first time that by inhibiting NAAG peptidase in vivo, the astrocytic enzyme that hydrolyzes NAAG, that there was a prolonged global reduction in the proton magnetic resonance blood oxygen leveldependent (BOLD) signal indicating a reduction in global cerebral blood flow (CBF), but with little or no effect on physical activity [9]. In 2006 the hypothesis was expanded to suggest that NAAG functioned "to control focal or regional hyperemia" by stimulating astrocytes to synthesize and release second messengers to the vascular system via cyclooxygenase-1 (COX-1) synthesized prostaglandins [10].
A number of recent studies support aspects of the original hypothesis. In 2013, it was demonstrated that the mGluR3 receptor was the predominant mGluR receptor present in mature murine and human astrocytes and that other mGluRs were very low or absent [11]. Thus, not only is NAAG uniquely targeted to the mGluR3 receptor, but it may be the only mGluR receptor on the surface of mature astrocytes, the cells that are integral to regulating near-field CBF [12].
Additional support for the hypothesis was developed in 2015, when the presence of a neuronal efflux transporter that transports both NAA and NAAG into ECF by a non-synaptic mechanism was reported [13], thus providing a possible mechanism for their continuous release to oligodendrocytes and astrocytes respectively.
The Bimodal Nature of Neurovascular Coupling The brain exhibits a remarkable feature in that there is a high degree of functional specialization in a relatively small organ. As a result there are many different small neuronal "neighborhoods", independent of neuron type or connectivity, that require different temporal allocations of CBF in order to supply sufficient quantities of glucose (Glc), O 2 and nutrients, and also to serve as a sink for waste products CO 2, H 2 O and generated heat. This is accomplished by an intricate system of chemical feedback signals between neurons, astrocytes and the vascular system. This cellular association has been termed the "neurovascular unit"  [14,15]. In this process, glutamate (Glu) plays an important role in activating astrocytes to initiate signaling to the vascular system [12]. In 2015, it was reported that there were two different types of NVC, one rapid and phasic in response to abrupt changes in neuron synaptic activity, inducing astrocyte Ca 2+ oscillations and eliciting immediate vascular responses.
The other, slow and tonic and independent of regional changes in neuronal synaptic activity, using resting intracellular Ca 2+ and continuous release of COX-1 generated second prostaglandin messengers [16]. Many neurovascular coupling mechanisms are known that can regulate phasic changes in CBF [14,15], but how the brain accomplished tonic control of CBF was reported to be unknown. In this analytical review, we bring together evidence of a signaling mechanism that matches the criteria for tonic regulation CBF and suggests that the function of neuronal release of NAAG in brain is to regulate tonic control of CBF.

Discussion
The Tri-Cellular Metabolism of NAA and NAAG Metabolism NAA and NAAG are among the highest concentration amino acids and dipeptides present in brain, and are almost exclusively found in neurons. Within neurons, the ratio of NAAG to NAA is lowest in gray matter (GM) and highest in white matter (WM) [1]. NAA is synthesized from L-aspartate (Asp) and acetyl Co-enzyme A (AcCoA) by NAA synthase [17] with Glc the source of acetate (Ac) in AcCoA. NAA is the only known precursor of NAAG, a non-excitatory form of Glu synthesized from NAA and Glu in neurons by NAAG synthase [18].
Importantly, because of their genesis, the rates of synthesis of both NAA and NAAG always reflect the rate of neuronal Glc oxidation, with about 1 molecule of NAAG synthesized for every 320 molecules of Glc oxidized [19]. This is shown in equation 1.
For their catabolism, they are exported to extracellular fluid (ECF) [20]. NAA is targeted to oligodendrocytes where it is hydrolyzed by aspartoacylase (ASPA) liberating Ac and Asp [21], (Eq 2), and NAAG is targeted to the mGluR3 receptor on the astrocyte surface where the Glu is cleaved by NAAG peptidase [22], (Eq 3.).

NAAG NAA + Glu
NAA is also a byproduct of astrocyte NAAG hydrolysis but astrocytes cannot further metabolize it. For its catabolism it must be liberated to ECF and hydrolyzed by oligodendrocyte ASPA. The unique tri-cellular metabolism of NAA and NAAG with two synthetic and two hydrolytic enzymes distributed between three cell types, and the NAAG-mGluR3-NAAG peptidase Glu release mechanism on the astrocyte surface has been called the "operating system" of the brain. This is because failure of several parts of the system in humans has been observed to lead to abnormal brain function [23]. The group II mGluR3 is also unique among mGluR's in that there is an astrocyte-targeted neurondedicated neurotransmitter (NAAG) and an associated specific enzyme (NAAG peptidase) for its hydrolysis [24]. involvement in rapid synaptic events [11]. A cartoon showing this association is presented in figure 1.
Mechanism of export of NAA and NAAG to ECF is non-synaptic and may also be associated with membrane depolarization Both NAA and NAAG are released to ECF upon neuron depolarization. This has been observed for NAA in rat microdialysis studies where neurons were depolarized by K + [25,26] and for NAA and NAAG in a rat brain slice superfusion study where depolarization was by electrical stimulation at 15 Hz, 20mA for 3 min and NAA and NAAG were observed to be released at the same relative % rate [27]. The release of NAA and NAAG is likely primarily non-synaptic since neurons do not express ASPA or NAAG peptidase that would be that mimic NAAG structure interfere with access of natural NAAG to the receptor-enzyme complex, disrupting this dynamic neurovascular energy supply mechanism [24]. Although discovered decades ago, the function of neuron synthesized NAAG remained a mystery. In an anesthetized mouse study, it was observed that a specific inhibitor of the mGluR3-associated NAAG peptidase, 2-(phosphonomethyl) pentanedioic acid (2-PMPA), resulted in a prolonged global reduction in BOLD of about 3% while vital signs were maintained, but had no effect on physical activity of awake mice in rotarod testing over 24 h [9]. This was the first evidence that NAAG was a global neurotransmitter involved in longterm tonic regulation of blood flow, but not in short-term stimulation-induced changes to meet rapid phasic requirements for increased energy. COX-1 is involved in the production of prostaglandins which, when released by astrocytes induce a hyperemic response [29]. In this study using a COX-1 inhibitor blocking second prostaglandin messengers to the vascular system from astrocytes, vibrissal stimulation in mice was still able to induce a phasic response. As a check, homozygous COX-1-null mice in this same study which did not synthesize prostanoids were observed to still retain this phasic response to stimulation. These authors concluded that phasic responses were clearly different from tonic responses and that the prostanoid products of COX-1 were critical in maintaining resting cerebrovascular tone.
The studies also demonstrated that the tonic regulation of blood flow based on the regular release of prostaglandins is decoupled from phasic responses.
Evidence that neuronal and/or astrocyte generated nitric • Neurons are the primary source of NAAG in the brain; NAAG can only be synthesized from NAA • NAAG is a non-excitatory form of Glu • NAAG is released to ECF continuously, and also upon neuron depolarization • NAAG is released to ECF extra-synaptically • NAAG is specifically targeted to the mGluR3 receptor on the astrocyte surface • The only mGluR receptor expressed in mature astrocytes is mGluR3 • The only enzyme that can hydrolyze NAAG, NAAG peptidase, is associated with the Gi/Go-protein coupled astrocytic mGluR3 receptor • Inhibiting astrocyte NAAG peptidase results in the rapid buildup of NAAG in ECF and a global decrease in CBF • mGluR3 activation stimulates astrocytes but does not trigger rapid Ca 2+ increases • The products of NAAG hydrolysis are Glu and NAA • Glu activates astrocytes to liberate gliotransmitter prostaglandins via COX-1 and communicate with the vascular system • The continuous release of NAAG to ECF by neurons is associated with resting state or tonic neurovascular coupling, but stimulation-induced release of NAAG may also participate in prolongation of phasic neurovascular coupling • In lower forms, the absence of NAA and NAAG, NAAG peptidase or mGluR3 is not lethal. Neurons are myelinated and can signal, and the animals appear to be little affected. However, in a single human case where NAA and NAAG are absent there are profound developmental defects. In figure 3 the proposed interaction of the NAAG system with additional elements that control phasic changes in blood flow is presented where it is shown that multiple neurons are involved and that synaptic firing is a key component.

Conclusions
The are rarely mentioned or considered in the numerous reviews of neurovascular coupling mechanisms [32,33].
In one case where the role of mGluR3 was considered, it was concluded that "it is unlikely that mGluR3 directly mediates NVC" [14]. In this paper, we show that contrary to such a conclusion, the NAAG system is a major component of NVC at all times and is involved in regulating the constant needs of neurons for energy to maintain cellular integrity even in the absence of synaptic firing and evoked astrocytic Ca 2+ signals.
Because the tonic system is independent of synaptic firing [16], it follows that it may be the most important regulator of CBF in GM during sleep when brain activity is reduced [33], as well as the primary regulator of CBF in WM, via release to astrocytes at nodes of Ranvier, where there are few synapses and NAAG is found in highest concentration [20].

Failure of the NAAG System may be Involved in Various
Brain Neuropathies We present evidence that NAAG functions as the global regulator of tonic regional blood flow to meet the ongoing housekeeping energy needs of neurons, estimated to be about 50% of their total energy requirements [16]. While NAAG release and its tonic function is independent of synaptic events, its generation is Glc oxidation-dependent and therefore therefore the ability of neurons to rapidly repolarize and be able to transmit a full range of meaningful frequencyencoded messages [35]. Chronic hypoxia has been linked to episodes of hallucinations and deficits in language production, cognition and memory. Currently, use of drugs that affect the tri-cellular metabolism of NAAG have been tested for their effects in animal models of disease, and as a result several have been advocated for possible treatment of human brain diseases and conditions including hypoxic and traumatic brain injury, stroke, diabetic neuropathy, amyotrophic lateral sclerosis, multiple sclerosis, Alzheimer's disease and schizophrenia [36,37,38]. Given a rationale for how NAAG may impact neuron physiology and their ability to communicate, a more focused approach to therapeutic interventions may now be possible.