Morris H. Baslow

Effect of N-acetylaspartic acid on the diffusion coefficient of water : A proton magnetic resonance phantom method for measurement of osmolyte-obligated water

N-acetyl-L-aspartic acid (NAA) is an amino acid present in the vertebrate brain that is synthesized and stored primarily in neurons, although it cannot be hydrolyzed in these cells. Nonetheless, neuronal NAA is dynamic and turns over more than once each day by cycling, via extracellular fluids (ECF), between neurons and catabolic compartments in oligodendrocytes. One important role of the NAA intercompartmental cycle appears to be osmoregulatory, and in this role it may be the primary mechanism for the removal of metabolic water, against a water gradient, from myelinated neurons. However, the number of water molecules that might be cotransported to ECF per NAA molecule released is as yet unclear. In this investigation, using a proton nuclear magnetic resonance method and diffusion measurements at two magnetic field strengths on water and NAA phantoms in vitro, the effect of NAA on the diffusion coefficient of water has been measured, and a ratio (K) of obligated water molecules per molecule of NAA has been determined. For NAA measured at 100mM and 3 Tesla K=24 and at 7 Tesla K=14. Based on these results, apparent K(NAA) varies inversely with field strength, and with a computed field strength factor of 2.55mmol water/unit Tesla, K(NAA) in the absence of any applied magnetic field strength would be 32.

2-PMPA, a NAAG peptidase inhibitor, attenuates magnetic resonance BOLD signals in brain of anesthetized mice

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.

Are Astrocytes the Missing Link Between Lack of Brain Aspartoacylase Activity and the Spongiform Leukodystrophy in Canavan Disease

Canavan disease (CD) is a genetic degenerative brain disorder associated with mutations of the gene encoding aspartoacylase (ASPA). In humans, the CD syndrome is marked by early onset, hydrocephalus, macroencephaly, psychomotor retardation, and spongiform myelin sheath vacuolization with progressive leukodystrophy. Metabolic hallmarks of the disease include elevated N-acetylaspartate (NAA) levels in brain, plasma and CSF, along with daily excretion of large amounts of NAA and its anabolic metabolite, N-acetylaspartylglutamate (NAAG). Of the observed neuropathies, the most important appears to be the extensive demyelination that interferes with normal neuronal signaling. However, finding the links between the lacks of ASPA activity in oligodendrocytes, the buildup of NAA in white matter (WM) and the mechanisms underlying the edematous spongiform leukodystrophy have remained elusive. In this analytical review we consider what those links might be and propose that in CD, the pathological buildup of NAA in limited WM extracellular fluid (ECF) is responsible for increased ECF osmotic–hydrostatic pressure and initiation of the demyelination process. We also hypothesize that NAA is not directly liberated by neurons in WM as it is in gray matter, and that its source in WM ECF is solely as a product of the catabolism of axon-released NAAG at nodes of Ranvier by astrocyte NAAG peptidase after it has docked with the astrocyte surface metabotropic glutamate receptor 3. This hypothesis ascribes for the first time a possible key role played by astrocytes in CD, linking the lack of ASPA activity in myelinating oligodendrocytes, the pathological buildup of NAA in WM ECF, and the spongiform demyelination process. It also offers new perspectives on the cause of the leukodystrophy in CD, and on possible treatment strategies for this inherited metabolic disease.

Stimulation‐induced transient changes in neuronal activity, blood flow and N‐acetylaspartate content in rat prefrontal cortex: a chemogenetic fMRS‐BOLD study

Brain activation studies in humans have shown the dynamic nature of neuronal N‐acetylaspartate (NAA) and N‐acetylaspartylglutamate (NAAG) based on changes in their MRS signals in response to stimulation. These studies demonstrated that upon visual stimulation there was a focal increase in cerebral blood flow (CBF) and a decrease in NAA or in the total of NAA and NAAG signals in the visual cortex, and that these changes were reversed upon cessation of stimulation. In the present study we have developed an animal model in order to explore the relationships between brain stimulation, neuronal activity, CBF and NAA. We use “designer receptor exclusively activated by designer drugs” (DREADDs) technology for site‐specific neural activation, a local field potential electrophysiological method for measurement of changes in the rate of neuronal activity, functional MRS for measurement of changes in NAA and a blood oxygenation level‐dependent (BOLD) MR technique for evaluating changes in CBF. We show that stimulation of the rat prefrontal cortex using DREADDs results in the following: (i) an increase in level of neuronal activity; (ii) an increase in BOLD and (iii) a decrease in the NAA signal. These findings show for the first time the tightly coupled relationships between stimulation, neuron activity, CBF and NAA dynamics in brain, and also provide the first demonstration of the novel inverse stimulation–NAA phenomenon in an animal model.

N-acetyl-L-histidine, a Prominent Biomolecule in Brain and Eye of Poikilothermic Vertebrates

N-acetyl-l-histidine (NAH) is a prominent biomolecule in brain, retina and lens of poikilothermic vertebrates. In fish lens, NAH exhibits an unusual compartmentalized metabolism. It is synthesized from l-histidine (His) and acetyl Co-enzyme A. However, NAH cannot be catabolized by lens cells. For its hydrolysis, NAH is exported to ocular fluid where a specific acylase cleaves His which is then actively taken up by lens and re-synthesized into NAH. This energy-dependent cycling suggested a pump mechanism operating at the lens/ocular fluid interface. Additional studies led to the hypothesis that NAH functioned as a molecular water pump (MWP) to maintain a highly dehydrated lens and avoid cataract formation. In this process, each NAH molecule released to ocular fluid down its gradient carries with it 33 molecules of bound water, effectively transporting the water against a water gradient. In ocular fluid the bound water is released for removal from the eye by the action of NAH acylase. In this paper, we demonstrate for the first time the identification of NAH in fish brain using proton magnetic resonance spectroscopy (MRS) and describe recent evidence supporting the NAH MWP hypothesis. Using MRS, we also document a phylogenetic transition in brain metabolism between poikilothermic and homeothermic vertebrates.

Response of the authors to the Letter by Silvia Mangia and Ivan Tkac

We agree with Mangia and Tkac that N-acetylaspartate (NAA) behavior during the brain activation and recovery is important, and we appreciate that they found our study interesting. We are also aware that contradicting reports have been published. Several of these studies are discussed in detail in our paper (Baslow et al. 2007). It is encouraging that the study most similar to ours (both in terms of the visual stimulation paradigm and the MR acquisition parameters) reported very similar downregulation of NAA on the order of 10% in normal controls (Sarchielli et al. 2005). The study at 7 T (Mangia et al. 2007) used similar visual stimuli and timing, and its sensitivity was clearly higher thanks to the higher main field. The most important difference from our study that we could identify was the echo time (TE). At the ultrashort TE used in Mangia et al. (2007), the spectrum becomes much more crowded due to metabolites with shorter relaxation times, broad macromolecule signals underlying the peaks, and mutual interactions. The influences of eddy currents and imperfect water and lipid suppression are also more profound. Of course, if many metabolites are to be studied simultaneously, short TE is required despite the increased technical difficulties. However, our goal was much more modest, concentrating exclusively on the NAA. This made it possible to use a very simple and robust approach of a point-resolved spectroscopy acquisition with long TE. Even though the absolute available signal is reduced, this smaller signal can be significantly more reproducible (Inglese et al. 2006). Mangia and Tkac refer to the difference spectrum in Mangia et al. (2007; Fig. 4 of the study) as a proof that NAA concentration remained constant during the visual stimulation. In our opinion, this is a very difficult conclusion to make without the detailed time course of the NAA signal, similar to those in Figs. 2 and 3 of the study. Figure 4 of the study is based on averages from second halves of the ON and OFF periods. Considering 10-min stimulation and subsequent 10-min recovery and using our rates obtained by linear regression (Baslow et al. 2007), these two time points would be expected to reach values of 88.75% (after 7.5 min of stimulation) and 91% (after 7.5 min of subsequent recovery toward the baseline), resulting in a difference of only 2.25%. The authors do not state any statistical descriptors or levels of significance for their time series but their Figs. 2 and 3 suggest that a difference so small may not be statistically significant. Furthermore, their line-broadening procedure (to account for the blood oxygenation level-dependent effect) was based on the minimization of the NAA difference signal, which implies that a stable NAA signal was regarded as an assumption rather than a hypothesis. Finally, their Fig. 4 reportedly combined data from short and long visual stimulation paradigms, while only the long stimulations are comparable to our results. This would likely further diminish the NAA difference spectrum. J Mol Neurosci (2008) 35:247–248 DOI 10.1007/s12031-008-9051-0

Rescuing Canavan disease: engineering the wrong cell at the right time

I would like to bring attention to an important recent study (Gessler et al. 2017) in which genetic engineering placed a genetic construct into the wrong cell at the right time and was thus able to rescue murine Canavan disease (CD). Hopefully, this letter will provide some additional context for evaluating this remarkable finding. CD is a rare human inborn error of metabolism in which oligodendrocyte aspartoacylase (ASPA) is inactive, with only several hundred human CD cases worldwide at any given time. Although the precise roles of ASPA and N-acetyl-L-aspartate (NAA), its natural substrate, are still debated, they are clearly important in that they are present in every human brain thus far examined, save one (Martin et al. 2001), and in almost every other vertebrate brain examined. CD is a spongiform leukodystrophy characterized by earlyonset megalocephaly and a progressive loss of function, generally leading to early mortality. Important characteristics of CD are the buildup of high concentrations of NAA in brain, formation of edematous vacuoles, and continuous presence of high concentrations of NAA in urine. ASPA and NAA are part of a unique tricellular metabolic system in the brain wherein NAA is made in neurons from acetyl Co-enzyme A and L-aspartate by NAA synthase and is then used to synthesize an L-glutamate (Glu) adduct, N-acetylaspartylglutamate (NAAG), via NAAG synthase (Baslow 2000). Neurons cannot further metabolize either of these substances and for hydrolysis are released to extracellular fluid (ECF). NAAG is targeted to the metabotropic glutamate receptor 3 (mGluR3) on the surface of astrocytes and upon docking is cleaved into Glu and NAA by NAAG peptidase. The NAA product cannot be further hydrolyzed by astrocytes, since they do not express ASPA (Baslow et al. 1999), and is thus released to oligodendrocytes, which do express this enzyme. This unique system requires coordinated functioning of four enzymes, two anabolic and two catabolic, distributed in three cell types and a specific receptor for its completion in order to maintain normal brain function. The absence of ASPA activity results in CD, and the absence of NAA synthase activity results in hypoacetylaspartia where neither NAA nor NAAG are produced (Waime et al. 2010). Both conditions display profound, clinically observed, negative consequences. While the function of this system is as yet unclear, a recent hypothesis suggests that the role of the NAAG-mGluR3-NAAG peptidase complex is to communicate a neuron’s ongoing requirements for energy and oxygen to astrocytes that in turn signal the vascular system to increase focal blood flow (Baslow and Guilfoyle 2016). Previous genetic engineering attempts to treat human CD have been aimed at oligodendrocytes, the cells that naturally express ASPA. As reported (Gessler et al. 2017), there has been a breakthrough in treatment of murine CD using a novel gene therapy approach that inserts the normal gene into astrocytes rather than into oligodendrocytes. This appears to be of major importance in rescuing CD in that ASPA activity is introduced at the end of the NAAG metabolic chain within the same cell and after it has activated the mGluR3 astrocyte receptor. It had been hypothesized that astrocytes were the cause of the spongiform leukodystrophy component in CD. This is because NAAG liberated at nodes of Ranvier in white matter would be hydrolyzed by astrocytes forming NAA, which in the case of CD could not be hydrolyzed by oligodendrocytes. Consequently, NAAwould build up in restricted ECF space at these nodes, penetrating between existing oligodendrocyte myelin layers and producing Communicated by: Ertan Mayatepek

Evidence that lithium Inhibits Export of N-Acetyl-L-Aspartate from Neurons: A Retrospective Study of Canavan Disease and Bipolar Disorder Patients

Evidence that lithium Inhibits Export of N-Acetyl-L-Aspartate from Neurons: A Retrospective Study of Canavan Disease and Bipolar Disorder Patients Lithium (Li) is an effective treatment for human bipolar disorder (BD) but whose precise mechanism and site of action are unknown. N-acetyl-L-aspartic acid (NAA) is an amino acid synthesized by and maintained at high steady-state levels within neurons from where it is exported to extracellular fluid (ECF) upon depolarization. NAA is the only precursor for N-acetylaspartylglutamate (NAAG), a neurotransmitter synthesized by neurons and also exported to ECF upon depolarization. The physiological function of NAA is as yet unclear but its unique tri-cellular metabolism between neurons, oligodendrocytes (NAA) and astrocytes (NAAG) is vital for normal brain function. Canavan disease (CD) is a rare inborn error in metabolism of NAA where oligodendrocyte aspartoacylase (ASPA) is inactive and NAA cannot be hydrolyzed resulting in its buildup in brain ECF and excretion in urine.

A Breakthrough in Understanding the Nature of Canavan Disease, a Human Spongiform Leukodystrophy due to Inborn Errors in the Gene Encoding for Aspartoacylase

Canavan disease (CD) is a rare early-onset progressive spongiform leukodystrophy in brain of both humans and animals and is due to mutations in the gene encoding for aspartoacylase (ASPA), the enzyme that hydrolyzes N-acetyl-L-aspartate (NAA) [1]. In humans, the effects of CD are generally much more profound than in rodents exhibiting this same genetic lesion. The gene for ASPA is an autosomal recessive and human or animal carriers of mutations do not appear to be affected. ASPA is expressed in oligodendrocytes and based on their large fractional cellular volume, these cells are the major source of ASPA in brain. However, ASPA has also been identified in microglia and in several other cellular brain compartments.