2-PMPA, a NAAG Peptidase Inhibitor, Attenuates Magnetic Resonance BOLD Signals in Brain of Anesthetized Mice: Evidence of a Link Between Neuron NAAG Release and Hyperemia

Abstract

N-acetylaspartylglutamate (NAAG), a dipeptide derivative of N-acetylaspartate (NAA) and glutamate (Glu), is present in neurons. Upon neurostimulation, NAAG is exported to astrocytes where it activates a specific metabotropic Glu surface receptor (mGluR3), and is then hydrolyzed by an astrocyte-specific enzyme, NAAG peptidase, liberating Glu, which can then be taken up by the astrocyte. NAAG is a selective mGluR3 agonist, one of several mGluRs that, when activated, triggers Ca2+ waves that spread to astrocytic endfeet in contact with the vascular system, where a secondary release of vasoactive agents induces a focal hyperemic response providing increased oxygen and nutrient availability to the stimulated neurons. Changes in blood oxygen levels can be assessed in vivo using a blood oxygenation level-dependent (BOLD) magnetic resonance imaging technique that reflects a paramagnetic effect of deoxyhemoglobin. In this study we used the competitive NAAG peptidase inhibitor 2-(phosphonomethyl) pentanedioic acid (2-PMPA) as a probe to interrupt the NAAG-mGluR3- Glu-astrocyte Ca2+ activation sequence. Using this probe, we investigated the relationship between release of the endogenous neuropeptide NAAG and brain blood oxygenation levels, as measured by changes in BOLD signals. In an anesthetized mouse, using an overtly nontoxic dose of 2-PMPA of 250 mg/kg i.p., there was an initial global BOLD signal increase of about 3% above control, lasting about 4 min, followed by a decrease from control of about 4%, sustained over a 32.5-min period of the drug test procedure. Similar changes, but of reduced magnitude and duration, were observed at a dose of 167 mg/kg. The 2-PMPA-induced decreases in BOLD signals appear to indicate that blood deoxyhemoglobin is elevated when endogenous NAAG cannot be hydrolyzed, thus linking the efflux of NAAG from neurons and its hydrolysis by astrocytes to hyperemic oxygenation responses in brain.

Stimulus-induced Rotary Saturation (SIRS): A potential method for the detection of neuronal currents with MRI

“…Finally, there is some hope that pharmacologic preparation might help for neuronal current studies in animal models. Here agents are being sought which act only on astrocytes and effect the flow response but not electrophysiology (Baslow et al, 2005).…”

The Bimodal Nature of Neurovascular Coupling: Slow Tonic and Rapid Phasic Responses are Separately Controlled by Specific Astrocyte Metabotropic and Ionotropic Glutamate Receptors

“…Also, its dedicated metabotropic receptor, mGluR3 is a G-protein Gi/Go bound receptor negatively coupled to adenylate cyclase that does not trigger Ca 2+ increases in astrocytes, thus excluding its involvement in rapid synaptic events that trigger astrocyte Ca 2+ waves and release of other NVC agents [20]. In addition, evidence of a connection between NAAG and CBF was previously obtained by inhibiting astrocyte mGluR3-associated NAAG peptidase activity in mice with 2-(phosphonomethyl) pentanedioic acid (2-PMPA) and observing that there was a prolonged global drop in the BOLD signal of about 3% [21].…”

“…Thus, the BOLD signal varies inversely with RBC Hb levels, and the signal increases as CBF increases bringing a fresh supply of HbO 2 and reducing Hb levels. Therefore, the decrease in the BOLD signal in the case of inhibiting the action of NAAG peptidase was interpreted as a lack of increase in CBF and a sign that a normal NVC mechanism had been uncoupled to some degree by blocking the release of Glu at the astrocyte surface [21].…”

Evidence that N-acetyaspartylglutamate is the astrocyte-targeted neurovascular coupling agent that regulates slow tonic control of brain blood flow

“…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.…”

“…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].…”