Age-Associated Upregulation of Glutamate Transporters and Glutamine Synthetase in Senescent Astrocytes In Vitro and in the Mouse and Human Hippocampus.

Aging is marked by complex and progressive physiological changes, including in the glutamatergic system, that lead to a decline of brain function. Increased content of senescent cells in the brain, such as glial cells, has been reported to impact cognition both in animal models and human tissue during normal aging and in the context of neurodegenerative disease. Changes in the glutamatergic synaptic activity rely on the glutamate-glutamine cycle, in which astrocytes handle glutamate taken up from synapses and provide glutamine for neurons, thus maintaining excitatory neurotransmission. However, the mechanisms of glutamate homeostasis in brain aging are still poorly understood. Herein, we showed that mouse senescent astrocytes in vitro undergo upregulation of GLT-1, GLAST, and glutamine synthetase (GS), along with the increased enzymatic activity of GS and [3H]-D-aspartate uptake. Furthermore, we observed higher levels of GS and increased [3H]-D-aspartate uptake in the hippocampus of aged mice, although the activity of GS was similar between young and old mice. Analysis of a previously available RNAseq dataset of mice at different ages revealed upregulation of GLAST and GS mRNA levels in hippocampal astrocytes during aging. Corroborating these rodent data, we showed an increased number of GS + cells, and GS and GLT-1 levels/intensity in the hippocampus of elderly humans. Our data suggest that aged astrocytes undergo molecular and functional changes that control glutamate-glutamine homeostasis upon brain aging.

D-aspartate oxidase gene duplication induces social recognition memory deficit in mice and intellectual disabilities in humans.

The D-aspartate oxidase (DDO) gene encodes the enzyme responsible for the catabolism of D-aspartate, an atypical amino acid enriched in the mammalian brain and acting as an endogenous NMDA receptor agonist. Considering the key role of NMDA receptors in neurodevelopmental disorders, recent findings suggest a link between D-aspartate dysmetabolism and schizophrenia. To clarify the role of D-aspartate on brain development and functioning, we used a mouse model with constitutive Ddo overexpression and D-aspartate depletion. In these mice, we found reduced number of BrdU-positive dorsal pallium neurons during corticogenesis, and decreased cortical and striatal gray matter volume at adulthood. Brain abnormalities were associated with social recognition memory deficit at juvenile phase, suggesting that early D-aspartate occurrence influences neurodevelopmental related phenotypes. We corroborated this hypothesis by reporting the first clinical case of a young patient with severe intellectual disability, thought disorders and autism spectrum disorder symptomatology, harboring a duplication of a chromosome 6 region, including the entire DDO gene.

Regulation of l- and d-Aspartate Transport and Metabolism in Acinetobacter baylyi ADP1.

The regulated uptake and consumption of d-amino acids by bacteria remain largely unexplored, despite the physiological importance of these compounds. Unlike other characterized bacteria, such as Escherichia coli, which utilizes only l-Asp, Acinetobacter baylyi ADP1 can consume both d-Asp and l-Asp as the sole carbon or nitrogen source. As described here, two LysR-type transcriptional regulators (LTTRs), DarR and AalR, control d- and l-Asp metabolism in strain ADP1. Heterologous expression of A. baylyi proteins enabled E. coli to use d-Asp as the carbon source when either of two transporters (AspT or AspY) and a racemase (RacD) were coexpressed. A third transporter, designated AspS, was also discovered to transport Asp in ADP1. DarR and/or AalR controlled the transcription of aspT, aspY, racD, and aspA (which encodes aspartate ammonia lyase). Conserved residues in the N-terminal DNA-binding domains of both regulators likely enable them to recognize the same DNA consensus sequence (ATGC-N7-GCAT) in several operator-promoter regions. In strains lacking AalR, suppressor mutations revealed a role for the ClpAP protease in Asp metabolism. In the absence of the ClpA component of this protease, DarR can compensate for the loss of AalR. ADP1 consumed l- and d-Asn and l-Glu, but not d-Glu, as the sole carbon or nitrogen source using interrelated pathways. IMPORTANCE A regulatory scheme was revealed in which AalR responds to l-Asp and DarR responds to d-Asp, a molecule with critical signaling functions in many organisms. The RacD-mediated interconversion of these isomers causes overlap in transcriptional control in A. baylyi. Our studies improve understanding of transport and regulation and lay the foundation for determining how regulators distinguish l- and d-enantiomers. These studies are relevant for biotechnology applications, and they highlight the importance of d-amino acids as natural bacterial growth substrates.

Glutamate receptor, ionotropic, N­methyl D­aspartate­associated protein 1 promotes colorectal cancer cell proliferation and metastasis, and is negatively regulated by miR­296­3p.

N­methyl D­aspartate receptors (NMDARs) are closely associated with the development, growth and metastasis of cancer. Glutamate receptor, ionotropic, N­methyl D­aspartate­associated protein 1 (GRINA) is a member of the of the NMDAR family, and its aberrant expression is associated with gastric cancer. However, the role of GRINA in colorectal cancer (CRC) is not completely understood. In the present study, expression profiles of GRINA in several CRC databases were obtained and further verified using clinical CRC samples. The effects of GRINA overexpression on CRC progression both in vivo and in vitro were assessed. Briefly, cell proliferation was detected using MTT assay, and cell migration and invasion ability were evaluated by wound healing and Transwell assay. In addition, the molecular mechanism underlying the upregulated expression of GRINA in CRC was investigated. The regulatory association between GRINA and miR­296­3p was detected by luciferase assay, reverse transcription­quantitative PCR and western blotting. The results demonstrated that GRINA expression levels were significantly increased in tumor samples compared with those in healthy samples, and upregulated expression of GRINA was associated with a less favorable prognostic outcome in patients with CRC. GRINA overexpression significantly increased CRC cell proliferation, invasion and migration. Additionally, it was determined that GRINA was post­transcriptionally regulated by microRNA (miR)­296­3p. Together, the results of the present study suggested the potential importance of the miR­296­3p/GRINA axis and highlighted potential novel targets for the management of CRC.

Association between the aspartic acid D-repeat polymorphisms and osteoarthritis susceptibility: An updated systematic review and meta-analyses.

OBJECTIVES: Association between the D-repeat of asporin (ASPN) gene and osteoarthritis (OA) was still inconsistent. We performed this meta-analysis to systematically assess the D-repeat polymorphisms in OA susceptibility. METHODS: Relevant studies were enrolled by searching databases. Odd ratios (ORs) with 95% confidence intervals (95% CIs) were used for evaluating the association between ASPN gene and OA. Heterogeneity was calculated using the Q statistic, and three different subgroup analyses were performed on ethnicity, gender, and OA positions respectively. False discovery rate (FDR) was applied to regulate the multiple comparisons. RESULTS: Twelve qualified articles involving 5190 OA patients and 5167 healthy controls were included. With D13 polymorphism, Caucasian male patients have low OA susceptibility (P = .008, PFDR = .024, OR [95% CI] = 0.83 [0.73-0.95]). As to D14 polymorphism, all male patients (P = .0004, PFDR = .001, OR [95% CI] = 1.38 [1.15-1.64]), Asian male patients (P = .01, PFDR = .01, OR [95% CI] = 1.72 [1.11-2.66]), and Caucasian male patients (P = .005, PFDR = .001, OR [95% CI] = 1.32 [1.09-1.60]) have high OA susceptibility. In the pooled-population of KOA with D14 polymorphism, overall male patients (P = .03, PFDR = .045, OR [95% CI] = 1.35 [1.02-1.78]) and Asian male patients (P = .01, PFDR = .03, OR [95% CI] = 1.72 [1.11-2.66]) have high OA risk. With D16 polymorphism, Latin America patients may have high OA risk (P = .04, PFDR = .15, OR [95% CI] = 1.43 [1.02-2.01]). CONCLUSION: Our results suggest that D-repeat of ASPN gene is mainly associated with male patients. The D13 polymorphism plays a protective role for OA in Caucasians male individuals while D14 plays a risk factor for KOA in male patients.

Serine racemase is involved in d-aspartate biosynthesis.

d-Aspartate is found in the nervous and reproductive system and participates in various physiological roles. While several lines of evidence suggest that this amino acid has an endogenous origin, the enzyme responsible for mammalian d-Asp biosynthesis has not yet been identified. We show that mammalian serine racemase (SRR), the primary enzyme responsible for brain d-Ser production, catalyses Asp racemization via a two-base mechanism. We observed that overexpression of SRR in rat pheochromocytoma PC12 cells resulted in an increase in intracellular d-Asp compared with control cells, demonstrating that SRR functions as an Asp racemase in the cells. To investigate the impact of endogenous SRR on endogenous d-Asp levels in the cells, we generated SRR-knockout (SRR-KO) PC12 cells. The SRR-KO cells exhibited decreased intracellular d-Ser levels, but production levels of d-Asp were unaffected. In contrast, SRR-KO mice showed significantly decreased d-Asp levels in their frontal cortices and hippocampi, where SRR is normally highly expressed, while d-Asp levels in the cerebellum and testes remained unchanged. Our results indicate that SRR indeed acts as a d-Asp biosynthetic enzyme in some organs and/or tissues, and also provide evidences that there should be some additional enzyme for d-Asp synthesis in mammals.

Endonuclease G does not play an obligatory role in poly(ADP-ribose) polymerase-dependent cell death after transient focal cerebral ischemia.

Activation of poly(ADP-ribose) polymerase (PARP) and subsequent translocation of apoptosis-inducing factor contribute to caspase-independent neuronal injury from N-methyl-d-aspartate, oxygen-glucose deprivation, and ischemic stroke. Some studies have implicated endonuclease G in the DNA fragmentation associated with caspase-independent cell death. Here, we compared wild-type and endonuclease G null mice to investigate whether endonuclease G plays a role in the PARP-dependent injury that results from transient focal cerebral ischemia. Latex casts did not reveal differences in the cerebral arterial distribution territory or posterior communicating arterial diameter, and the decrease in laser-Doppler flux during middle cerebral artery occlusion was similar in wild-type and endonuclease G null mice. After 90 min of occlusion and 1 day of reperfusion, similar degrees of nuclear translocation of apoptosis-inducing factor and DNA degradation were evident in male wild-type and null mice. At 3 days of reperfusion, infarct volume and neurological deficit scores were not different between male wild-type and endonuclease G null mice or between female wild-type and endonuclease G null mice. These data demonstrate that endonuclease G is not required for the pathogenesis of transient focal ischemia in either male or female mice. Treatment with a PARP inhibitor decreased infarct volume and deficit scores equivalently in male wild-type and endonuclease G null mice, indicating that the injury in endonuclease G null mice remains dependent on PARP. Thus endonuclease G is not obligatory for executing PARP-dependent injury during ischemic stroke.

[Degradation of biomolecules: a comparative study of diagenesis of DNA and proteins in human bone tissue]. / Degradation von Biomolekülen: Eine vergleichende Studie zur Diagenese von DNA und Proteinen in humanem Knochengewebe.

Diagenesis of macromolecules is a not yet fully understood process that can be important for anthropological and forensic research. Trying to elucidate the diagenesis of DNA and proteins we investigated the process of fragmentation of DNA and razemisation of aspartic acid in human bone material. We created an in vitro-model of accelerated aging by incubating bone samples in hot water. A comparison of diagenesis of molecules in those artificially aged samples with altogether 30 historical bones from different regions and of different ages was carried out. The in vitro-model showed the expected positive correlation between the increase of razemisation of aspartic acid and DNA fragmentation, while there was a much lesser correlation when investigating historical bones. The in vitro-model showed the expected correlation between the increase of razemisation of aspartic acid and DNA fragmentation and to a much lesser extent in historical bones. This study shows that diagenesis is probably influenced by additional forces affecting different macromolecules in different ways.