NAAG synthetase deficiency has only low influence on pathogenesis in a Canavan disease mouse model.

Canavan disease (CD) is a leukodystrophy caused by mutations in the N-acetylaspartate (NAA) hydrolase aspartoacylase (ASPA). Inability to degrade NAA and its accumulation in the brain results in spongiform myelin degeneration. NAA is mainly synthesized by neurons, where it is also a precursor of the neuropeptide N-acetylaspartylglutamate (NAAG). Hydrolysis of this peptide by glutamate carboxypeptidases is an additional source of extracellular NAA besides the instant neuronal release of NAA. This study examines to what extent NAA released from NAAG contributes to NAA accumulation and pathogenesis in the brain of Aspanur7/nur7 mutant mice, an established model of CD. Towards this aim, Aspanur7/nur7 mice with additional deficiencies in NAAG synthetase genes Rimklb and/or Rimkla were generated. Loss of myelin in Aspanur7/nur7 mice was not significantly affected by Rimkla and Rimklb deficiency and there was also no obvious change in the extent of brain vacuolation. Astrogliosis was slightly reduced in the forebrain of Rimkla and Rimklb double deficient Aspanur7/nur7 mice. However, only minor improvements at the behavioral level were found. The brain NAA accumulation in CD mice was, however, not significantly reduced in the absence of NAAG synthesis. In summary, there was only a weak tendency towards reduced pathogenic symptoms in Aspanur7/nur7 mice deficient in NAAG synthesis. Therefore, we conclude that NAAG metabolism has little influence on NAA accumulation in Aspanur7/nur7 mice and development of pathological symptoms in CD.

An enzymatic fluorimetric assay for determination of N-acetylaspartate.

N-acetylaspartate (NAA) is an abundant metabolite in the mammalian brain and a precursor of the neuropeptide N-acetylaspartylglutamate (NAAG). The physiological role of NAA is not fully understood and requires further studies. We here describe the development of a coupled enzymatic fluorimetric assay for the determination of NAA in biological samples. Deproteinized tissue extracts are first passed through a strong cation exchange column to remove aspartate. NAA in the sample is hydrolysed by aspartoacylase and released aspartate oxidized using l-aspartate oxidase. Generated H2O2 is measured with peroxidase in a fluorimetric assay using Ampliflu Red. The limit of detection and the lower limit of quantification are 1.0 µM (10 pmol/well) and 3.3 µM (33 pmol/well), respectively, with a linear range to 100 µM. Specificity of the assay was confirmed using samples from mice deficient in NAA synthase Nat8l that were spiked with NAA. Analysis of samples from aspartoacylase-deficient mice showed a 2 to 3-fold increase in brain NAA concentration, in line with previous reports. Mice lacking NAAG synthetases had a slightly reduced (-10%) brain NAA level. Thus, the new fluorimetric enzymatic assay is useful to perform sensitive and large scale quantification of NAA in biological samples without the need for expensive equipment.

Age-Dependent Increase in Schmidt-Lanterman Incisures and a Cadm4-Associated Membrane Skeletal Complex in Fatty Acid 2-hydroxylase Deficient Mice: a Mouse Model of Spastic Paraplegia SPG35.

PNS and CNS myelin contain large amounts of galactocerebroside and sulfatide with 2-hydroxylated fatty acids. The underlying hydroxylation reaction is catalyzed by fatty acid 2-hydroxylase (FA2H). Deficiency in this enzyme causes a complicated hereditary spastic paraplegia, SPG35, which is associated with leukodystrophy. Mass spectrometry-based proteomics of purified myelin isolated from sciatic nerves of Fa2h-deficient (Fa2h-/-) mice revealed an increase in the concentration of the three proteins Cadm4, Mpp6 (Pals2), and protein band 4.1G (Epb41l2) in 17-month-old, but not in young (4 to 6-month-old), Fa2h-/- mice. These proteins are known to form a complex, together with the protein Lin7, in Schmidt-Lanterman incisures (SLIs). Accordingly, the number of SLIs was significantly increased in 17-month-old but not 4-month-old Fa2h-/- mice compared to age-matched wild-type mice. On the other hand, the relative increase in the SLI frequency was less pronounced than expected from Cadm4, Lin7, Mpp6 (Pals2), and band 4.1G (Epb41l2) protein levels. This suggests that the latter not only reflect the higher SLI frequency but that the concentration of the Cadm4 containing complex itself is increased in the SLIs or compact myelin of Fa2h-/- mice and may potentially play a role in the pathogenesis of the disease. The proteome data are available via ProteomeXchange with identifier PXD030244.

Citrylglutamate synthase deficient male mice are subfertile with impaired histone and transition protein 2 removal in late spermatids.

Chromatin remodelling in spermatids is an essential step in spermiogenesis and involves the exchange of most histones by protamines, which drives chromatin condensation in late spermatids. The gene Rimklb encodes a citrylglutamate synthase highly expressed in testes of vertebrates and the increase of its reaction product, ß-citrylglutamate, correlates in time with the appearance of spermatids. Here we show that deficiency in a functional Rimklb gene leads to male subfertility, which could be partially rescued by in vitro fertilization. Rimklb-deficient mice are impaired in a late step of spermiogenesis and produce spermatozoa with abnormally shaped heads and nuclei. Sperm chromatin in Rimklb-deficient mice was less condensed and showed impaired histone to protamine exchange and retained transition protein 2. These observations suggest that citrylglutamate synthase, probably via its reaction product ß-citrylglutamate, is essential for efficient chromatin remodelling during spermiogenesis and may be a possible candidate gene for male subfertility or infertility in humans.

Absence of endogenous carnosine synthesis does not increase protein carbonylation and advanced lipoxidation end products in brain, kidney or muscle.

Carnosine and other histidine-containing dipeptides are expected to be important anti-oxidants in vertebrates based on various in vitro and in vivo studies with exogenously administered carnosine or its precursor ß-alanine. To examine a possible anti-oxidant role of endogenous carnosine, mice lacking carnosine synthase (Carns1-/-) had been generated and were examined further in the present study. Protein carbonylation increased significantly between old (18 months) and aged (24 months) mice in brain and kidney but this was independent of the Carns1 genotype. Lipoxidation end products were not increased in 18-month-old Carns1-/- mice compared to controls. We also found no evidence for compensatory increase of anti-oxidant enzymes in Carns1-/- mice. To explore the effect of carnosine deficiency in a mouse model known to suffer from increased oxidative stress, Carns1 also was deleted in the type II diabetes model Leprdb/db mouse. In line with previous studies, malondialdehyde adducts were elevated in Leprdb/db mouse kidney, but there was no further increase by additional deficiency in Carns1. Furthermore, Leprdb/db mice lacking Carns1 were indistinguishable from conventional Leprdb/db mice with respect to fasting blood glucose and insulin levels. Taken together, Carns1 deficiency appears not to reinforce oxidative stress in old mice and there was no evidence for a compensatory upregulation of anti-oxidant enzymes. We conclude that the significance of the anti-oxidant activity of endogenously synthesized HCDs is limited in mice, suggesting that other functions of HCDs play a more important role.

The PGRMC1 Antagonist AG-205 Inhibits Synthesis of Galactosylceramide and Sulfatide.

Sulfatide synthesis in the human renal cancer cell line SMKT-R3 was strongly inhibited in the presence of low µM concentrations of AG-205, a progesterone receptor membrane component 1 (PGRMC1) antagonist. This was also the case in Chinese hamster ovary (CHO) cells stably transfected with UDP-galactose: ceramide galactosyltransferase and cerebroside sulfotransferase, the two enzymes required for sulfatide synthesis. In CHO cells synthesizing galactosylceramide but not sulfatide, galactosylceramide was also strongly reduced, suggesting an effect at the level of galactolipid synthesis. Notably, AG-205 inhibited galactosylceramide synthesis to a similar extent in wild type CHO cells and cells that lack PGRMC1 and/or PGRMC2. In vitro enzyme activity assays showed that AG-205 is an inhibitor of UDP-galactose: ceramide galactosyltransferase, but not cerebroside sulfotransferase. This study shows that PGRMC1 is only one of several targets of AG-205 and should be used with caution, especially in studies using cells synthesizing galactosylceramide and sulfatide.

Mice deficient in the NAAG synthetase II gene Rimkla are impaired in a novel object recognition task.

N-acetylaspartylglutamate (NAAG) is an abundant neuropeptide in the mammalian nervous system, synthesized by two related NAAG synthetases I and II (NAAGS-I and -II) encoded by the genes Rimklb and Rimkla, respectively. NAAG plays a role in cognition and memory, according to studies using inhibitors of the NAAG hydrolase glutamate carboxypeptidase II that increase NAAG concentration. To examine consequences of reduced NAAG concentration, Rimkla-deficient (Rimkla-/- ) mice were generated. These mice exhibit normal NAAG level at birth, likely because of the intact Rimklb gene, but have significantly reduced NAAG levels in all brain regions in adulthood. In wild type mice NAAGS-II was most abundant in brainstem and spinal cord, as demonstrated using a new NAAGS-II antiserum. In the hippocampus, NAAGS-II was only detectable in neurons expressing parvalbumin, a marker of GABAergic interneurons. Apart from reduced open field activity, general behavior of adult (6 months old) Rimkla-/- mice examined in different tests (dark-light transition, optokinetic behavior, rotarod, and alternating T-maze) was not significantly altered. However, Rimkla-/- mice were impaired in a short-term novel object recognition test. This was also the case for mice lacking NAA synthase Nat8l, which are devoid of NAAG. Together with results from previous studies showing that inhibition of the NAAG degrading enzyme glutamate carboxypeptidase II is associated with a significant improvement in object recognition, these results suggest a direct involvement of NAAG synthesized by NAAGS-II in the memory consolidation underlying the novel object recognition task.

Risk assessment report of potential impurities in cetirizine dihydrochloride.

Recently, the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA) had reported on "unexpected" impurities in a couple of sartans, ranitidine, and metformin. These events led to a lot of discussion with regard to the risk assessment for the production process itself. Most of these discussions covered the field of nitrosamine impurities only, but that would be too short-sighted. One should expand that scope. It is impossible to synthesize a 100 % pure compound which holds true for all active pharmaceutical ingredients (APIs). Different synthetic routes result in different impurity profiles. Therefore, pharmacopoeias try to consider all possible impurities that can arise from different drug synthesis routes in one a single monograph for impurity assessment. However, API manufacturers cannot simply rely on the impurity profile reported in pharmacopoeias for the production of a high-quality product. They have to implement a whole risk assessment to rate the presence of impurities in the API. Here, a strategy to evaluate and minimize the load of potential risks of impurities during the manufacturing process of the drug substance cetirizine dihydrochloride within the frame of a detailed risk assessment report is demonstrated.

N-acetylaspartylglutamate synthetase II synthesizes N-acetylaspartylglutamylglutamate.

N-Acetylaspartylglutamate (NAAG) is found at high concentrations in the vertebrate nervous system. NAAG is an agonist at group II metabotropic glutamate receptors. In addition to its role as a neuropeptide, a number of functions have been proposed for NAAG, including a role as a non-excitotoxic transport form of glutamate and a molecular water pump. We recently identified a NAAG synthetase (now renamed NAAG synthetase I, NAAGS-I), encoded by the ribosomal modification protein rimK-like family member B (Rimklb) gene, as a member of the ATP-grasp protein family. We show here that a structurally related protein, encoded by the ribosomal modification protein rimK-like family member A (Rimkla) gene, is another NAAG synthetase (NAAGS-II), which in addition, synthesizes the N-acetylated tripeptide N-acetylaspartylglutamylglutamate (NAAG(2)). In contrast, NAAG(2) synthetase activity was undetectable in cells expressing NAAGS-I. Furthermore, we demonstrate by mass spectrometry the presence of NAAG(2) in murine brain tissue and sciatic nerves. The highest concentrations of both, NAAG(2) and NAAG, were found in sciatic nerves, spinal cord, and the brain stem, in accordance with the expression level of NAAGS-II. To our knowledge the presence of NAAG(2) in the vertebrate nervous system has not been described before. The physiological role of NAAG(2), e.g. whether it acts as a neurotransmitter, remains to be determined.

Molecular characterization of N-acetylaspartylglutamate synthetase.

The dipeptide N-acetylaspartyl-glutamate (NAAG) is an abundant neuropeptide in the mammalian brain. Despite this fact, its physiological role is poorly understood. NAAG is synthesized by a NAAG synthetase catalyzing the ATP-dependent condensation of N-acetylaspartate and glutamate. In vitro NAAG synthetase activity has not been described, and the enzyme has not been purified. Using a bioinformatics approach we identified a putative dipeptide synthetase specifically expressed in the nervous system. Expression of the gene, which we named NAAGS (for NAAG synthetase) was sufficient to induce NAAG synthesis in primary astrocytes or CHO-K1 and HEK-293T cells when they coexpressed the NAA transporter NaDC3. Furthermore, coexpression of NAAGS and the recently identified N-acetylaspartate (NAA) synthase, Nat8l, in CHO-K1 or HEK-293T cells was sufficient to enable these cells to synthesize NAAG. Identity of the reaction product of NAAGS was confirmed by HPLC and electrospray ionization tandem mass spectrometry (ESI-MS). High expression levels of NAAGS were restricted to the brain, spinal cord, and testis. Taken together our results strongly suggest that the identified gene encodes a NAAG synthetase. Its identification will enable further studies to examine the role of this abundant neuropeptide in the vertebrate nervous system.

Differential expression of (dihydro)ceramide synthases in mouse brain: oligodendrocyte-specific expression of CerS2/Lass2.

Synthesis of dihydroceramide is catalyzed by a family of (dihydro)ceramide synthases (CerS), first identified in yeast as longevity-assurance genes. Six members (CerS1-6; Lass1-6) of this gene family have been identified in mammals. We examined expression of CerS genes during postnatal development in mouse brain by means of Northern blot analysis, real-time RT-PCR, and in situ-hybridization. In situ-hybridization experiments showed that CerS1 was the predominant CerS in neurons throughout the brain. This observation is in line with the high levels of C18:0-ceramide in neurons and the substrate specificity of CerS1. A similar distribution, but lower expression levels, were found for CerS4 and CerS6. Only low or undetectable amounts of CerS1, CerS4 and CerS6 were, however, present in white matter. In contrast, CerS5 mRNA was detected in most cells within gray and white matter of all brain regions, suggesting ubiquitous expression of this palmitoyl-CoA specific CerS. Expression of CerS2 was transiently increased during the period of active myelination. Furthermore, expression of CerS2 was specifically localized to white matter tracts of the brain. Furthermore, CerS2 was the predominant CerS in Schwann cells of sciatic nerves. These data suggest that CerS2 is important for the synthesis of dihydroceramide used for synthesis of myelin sphingolipids.

Regulation of heme oxygenase-1 gene expression by anoxia and reoxygenation in primary rat hepatocyte cultures.

Heme oxygenase (HO) catalyzes the rate-limiting enzymatic step of heme degradation and regulates the cellular heme content. Gene expression of the inducible isoform of HO, HO-1, is upregulated in response to various oxidative stress stimuli. To investigate the regulatory role of anoxia and reoxygenation (A/R) on hepatic HO-1 gene expression, primary cultures of rat hepatocytes were exposed after an anoxia of 4 hr to normal oxygen tension for various lengths of time. For comparison, gene expression of the noninducible HO isoform, HO-2, and that of the heat-shock protein 70 (HSP70) were determined. During reoxygenation, a marked increase of HO-1 and HSP70 steady-state mRNA levels was observed, whereas no alteration of HO-2 mRNA levels occurred. Corresponding to HO-1 mRNA, an increase of HO-1 protein expression was determined by Western blot analysis. The anoxia-dependent induction of HO-1 was prevented by pretreatment with the transcription inhibitor, actinomycin D, but not by the protein synthesis inhibitor, cycloheximide, suggesting a transcriptional regulatory mechanism. After exposure of hepatocytes to anoxia, the relative levels of oxidized glutathione increased within the first 40 min of reoxygenation. Pretreament of cell cultures with the antioxidant agents, beta-carotene and allopurinol, before exposure to A/R led to a marked decrease of HO-1 and HSP70 mRNA expression during reoxygenation. An even more pronounced reduction of mRNA expression was observed after exposure to desferrioxamine. Taken together, the data demonstrate that HO-1 gene expression in rat hepatocyte cultures after A/R is upregulated by a transcriptional mechanism that may be, in part, mediated via the generation of ROS and the glutathione system.