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Human evolution is characterized by rapid brain enlargement and the emergence of unique cognitive abilities. Besides its distinctive cytoarchitectural organization and extensive inter-neuronal connectivity, the human brain is also defined by high rates of synaptic, mainly glutamatergic, transmission, and energy utilization. While these adaptations' origins remain elusive, evolutionary changes occurred in synaptic glutamate metabolism in the common ancestor of humans and apes via the emergence of GLUD2 , a gene encoding the human glutamate dehydrogenase 2 (hGDH2) isoenzyme. Driven by positive selection, hGDH2 became adapted to function upon intense excitatory firing, a process central to the long-term strengthening of synaptic connections. It also gained expression in brain astrocytes and cortical pyramidal neurons, including the CA1-CA3 hippocampal cells, neurons crucial to cognition. In mice transgenic for GLUD2 , theta-burst-evoked long-term potentiation (LTP) is markedly enhanced in hippocampal CA3-CA1 synapses, with patch-clamp recordings from CA1 pyramidal neurons revealing increased sNMDA receptor currents. D-lactate blocked LTP enhancement, implying that glutamate metabolism via hGDH2 potentiates L-lactate-dependent glia-neuron interaction, a process essential to memory consolidation. The transgenic (Tg) mice exhibited increased dendritic spine density/synaptogenesis in the hippocampus and improved complex cognitive functions. Hence, enhancement of neuron-glia communication, via GLUD2 evolution, likely contributed to human cognitive advancement by potentiating synaptic plasticity and inter-neuronal connectivity.

The human brain is characterized by the upregulation of synaptic, mainly glutamatergic, transmission, but its evolutionary origin(s) remain elusive. Here we approached this fundamental question by studying mice transgenic (Tg) for GLUD2 , a human gene involved in glutamate metabolism that emerged in the hominoid and evolved concomitantly with brain expansion. We demonstrate that Tg mice express the human enzyme in hippocampal astrocytes and CA1-CA3 pyramidal neurons. LTP, evoked by theta-burst stimulation, is markedly enhanced in the CA3-CA1 synapses of Tg mice, with patch-clamp recordings from CA1 pyramidal neurons revealing increased sNMDA currents. LTP enhancement is blocked by D-lactate, implying that GLUD2 potentiates L-lactate-mediated astrocyte-neuron interaction. Dendritic spine density and synaptogenesis are increased in the hippocampus of Tg mice, which exhibit enhanced responses to sensory stimuli and improved performance on complex memory tasks. Hence, GLUD2 likely contributed to human brain evolution by enhancing synaptic plasticity and metabolic processes central to cognitive functions., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

Glutamate dehydrogenase (GDH) interconverts glutamate to a-ketoglutarate and ammonia, interconnecting amino acid and carbohydrate metabolism. In humans, two functional GDH genes, GLUD1 and GLUD2 , encode for hGDH1 and hGDH2, respectively. GLUD2 evolved from retrotransposition of the GLUD1 gene in the common ancestor of modern apes. These two isoenzymes are involved in the pathophysiology of human metabolic, neoplastic, and neurodegenerative disorders. The 3D structures of hGDH1 and hGDH2 have been experimentally determined; however, no information is available about the path of GDH2 structure changes during primate evolution. Here, we compare the structures predicted by the AlphaFold Colab method for the GDH2 enzyme of modern apes and their extinct primate ancestors. Also, we analyze the individual effect of amino acid substitutions emerging during primate evolution. Our most important finding is that the predicted structure of GDH2 in the common ancestor of apes was the steppingstone for the structural evolution of primate GDH2s. Two changes with a strong functional impact occurring at the first evolutionary step, Arg443Ser and Gly456Ala, had a destabilizing and stabilizing effect, respectively, making this step the most important one. Subsequently, GDH2 underwent additional modifications that fine-tuned its enzymatic properties to adapt to the functional needs of modern-day primate tissues.

Using exome sequencing, we analyzed 196 participants of the Cretan Aging Cohort (CAC; 95 with Alzheimer's disease [AD], 20 with mild cognitive impairment [MCI], and 81 cognitively normal controls). The APOE ε4 allele was more common in AD patients (23.2%) than in controls (7.4%; p < 0.01) and the PSEN2 p.Arg29His and p.Cys391Arg variants were found in 3 AD and 1 MCI patient, respectively. Also, we found the frontotemporal dementia (FTD)-associated TARDBP gene p.Ile383Val variant in 2 elderly patients diagnosed with AD and in 2 patients, non CAC members, with the amyotrophic lateral sclerosis/FTD phenotype. Furthermore, the p.Ser498Ala variant in the positively selected GLUD2 gene was less frequent in AD patients (2.11%) than in controls (16%; p < 0.01), suggesting a possible protective effect. While the same trend was found in another local replication cohort (n = 406) and in section of the ADNI cohort (n = 808), this finding did not reach statistical significance and therefore it should be considered preliminary. Our results attest to the value of genetic testing to study aged adults with AD phenotype., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background and Objectives: Our goal was to study hereditary transthyretin-related amyloidosis (hATTR) in Crete, Greece., Methods: We aimed at ascertaining all hATTR cases in Crete, an island of 0.62 million people. For this, we evaluated patients with polyneuropathy, autonomic involvement, cardiomyopathy, and/or ophthalmopathy suggestive of hATTR, who presented to the physicians of this study or were referred to them by other physicians. Genetic analyses were performed on all patients suspected of suffering from hATTR. We included in our observational longitudinal cohort study all individuals, residents of Crete, who, during the study period (1993-2019), were found to carry a pathogenic TTR variant., Results: Over the past 27 years, 30 individuals (15 female patients, 15 male patients), from 12 apparently unrelated families, were diagnosed with hATTR, whereas evaluation of their offspring identified 5 asymptomatic TTR pathogenic variant carriers. The most prevalent TTR variant detected was p.Val50Met, affecting 19 patients (11 female patients, 8 male patients) and causing a rather consistent phenotype characterized by predominant polyneuropathy of early adult onset (median age of symptom onset: 30 years; range: 18-37 years). Specifically, patients affected by the p.Val50Met TTR variant experienced progressive sensorimotor disturbances, involving mainly the lower extremities, associated with autonomic and/or gastrointestinal dysfunction. The second most frequent TTR variant was p.Val114Ala, found in 10 patients (4 female patients, 6 male patients) who were affected at an older age (median age of symptom onset: 70 years; range: 54-78 years). This variant caused a predominantly cardiomyopathic phenotype, manifested by congestive heart failure and associated with peripheral neuropathy, carpal tunnel syndrome, and/or autonomic involvement. In these patients, cardiac amyloid deposition was detected on 99m-technetium pyrophosphate scintigraphy and/or heart biopsy. The third TTR variant (p.Arg54Gly) was found in a 50-year-old male patient with ophthalmopathy due to vitreous opacities and positive family history for visual loss. As 22 patients were alive at the end of the study, we calculated the hATTR prevalence in Crete to be 35 cases per 1 million inhabitants., Discussion: Our study revealed that the prevalence of hATTR in Crete is one of the world's highest. Three different pathogenic TTR variants causing distinct clinical phenotypes were identified in this relatively small population pool., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Introduction: Mammalian glutamate dehydrogenase (hGDH1 in human cells) interconverts glutamate to α-ketoglutarate and ammonia while reducing NAD(P) to NAD(P)H. During primate evolution, humans and great apes have acquired hGDH2, an isoenzyme that underwent rapid evolutionary adaptation concomitantly with brain expansion, thereby acquiring unique catalytic and regulatory properties that permitted its function under conditions inhibitory to its ancestor hGDH1. Although the 3D-structures of GDHs, including hGDH1, have been determined, attempts to determine the hGDH2 structure were until recently unsuccessful. Comparison of the hGDH1/hGDH2 structures would enable a detailed understanding of their evolutionary differences. This work aimed at the determination of the hGDH2 crystal structure and the analysis of its functional implications. Recombinant hGDH2 was produced in the Spodoptera frugiperda ovarian cell line Sf21, using the Baculovirus expression system. Purification was achieved via a two-step chromatography procedure. hGDH2 was crystallized, X-ray diffraction data were collected using synchrotron radiation and the structure was determined by molecular replacement. The hGDH2 structure is reported at a resolution of 2.9 Å. The enzyme adopts a novel semi-closed conformation, which is an intermediate between known open and closed GDH1 conformations, differing from both. The structure enabled us to dissect previously reported biochemical findings and to structurally interpret the effects of evolutionary amino acid substitutions, including Arg470His, on ADP affinity. In conclusion, our data provide insights into the structural basis of hGDH2 properties, the functional evolution of hGDH isoenzymes, and open new prospects for drug design, especially for cancer therapeutics., (© 2021 International Society for Neurochemistry.)

Glutamate dehydrogenase 1 (GDH1) contributes to glucose-stimulated insulin secretion in murine β-cells, but not to basic insulin release. The implications of these findings for human biology are unclear as humans have two GDH-specific enzymes: hGDH1 (GLUD1-encoded) and hGDH2 (GLUD2-encoded), a novel enzyme that is highly activated by ADP and L-leucine. Here we studied in vivo glucose homeostasis in transgenic (Tg) mice generated by inserting the GLUD2 gene and its putative regulatory elements into their genome. Using specific antibodies, we observed that hGDH2 was co-expressed with the endogenous murine GDH1 in pancreatic β-cells of Tg mice. Fasting blood glucose (FBG) levels were lower and of a narrower range in Tg (95% CI: 90.6-96.8 mg/dl; N = 26) than in Wt mice (95% CI: 136.2-151.4 mg/dl; N = 23; p < 0.0001), closely resembling those of healthy humans. GLUD2 also protected the host mouse from developing diabetes with advancing age. Tg animals maintained 2.6-fold higher fasting serum insulin levels (mean ± SD: 1.63 ± 0.15 ng/ml; N = 12) than Wt mice (0.63 ± 0.05 ng/ml; N = 12; p < 0.0001). Glucose loading (1 mg/g, given i.p.) induced comparable serum insulin increases in Tg and Wt mice, suggesting no significant GLUD2 effect on glucose-stimulated insulin release. L-leucine (0.25 mg/g given orally) induced a 2-fold increase in the serum insulin of the Wt mice, implying significant activation of the endogenous GDH1. However, L-leucine had little effect on the high insulin levels of the Tg mice, suggesting that, under the high ADP levels that prevail in β-cells in the fasting state, glutamate flux through hGDH2 is close to maximal. Hence, the present data, showing that GLUD2 expression in Tg mice improves in vivo glucose homeostasis by boosting fasting serum insulin levels, suggest that evolutionary adaptation of hGDH2 has enabled humans to achieve narrow-range euglycemia by regulating glutamate-mediated basal insulin secretion., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Our aim was to explore the burden of dementia in the Cretan Aging Cohort, comprised of 3140 persons aged ≥60 years (56.8% women, 5.8 ± 3.3 years formal education, 86.2% living in rural areas) who attended selected primary health-care facilities on the island of Crete, Greece. In the first study phase, a formal diagnosis of dementia had been reached in 4.0% of the participants. However, when selected 505 participants underwent thorough neuropsychiatric evaluation in the second phase of this study (344 with Mini-Mental State Examination [MMSE] <24 and 161 with MMSE ≥24), and results were extrapolated to the entire cohort, the prevalence of dementia and mild cognitive impairment was estimated at 10.8% (9.7%-11.9%) and 32.4% (30.8%-34.0%), respectively. Using both the field diagnostic data and the extrapolated data, the highest dementia prevalence (27.2%) was found in the 80- to 84-year-old group, who also showed the lowest educational level, apparently due to lack of schooling during World War II.

Human evolution is characterized by brain expansion and up-regulation of genes involved in energy metabolism and synaptic transmission, including the glutamate signaling pathway. Glutamate is the excitatory transmitter of neural circuits sub-serving cognitive functions, with glutamate-modulation of synaptic plasticity being central to learning and memory. GLUD2 is a novel positively-selected human gene involved in glutamatergic transmission and energy metabolism that underwent rapid evolutionary adaptation concomitantly with prefrontal cortex enlargement. Two evolutionary replacements (Gly456Ala and Arg443Ser) made hGDH2 resistant to GTP inhibition and allowed distinct regulation, enabling enhanced enzyme function under high glutamatergic system demands. GLUD2 adaptation may have contributed to unique human traits, but evidence for this is lacking. GLUD2 arose through retro-positioning of a processed GLUD1 mRNA to the X chromosome, a DNA replication mechanism that typically generates pseudogenes. However, by finding a suitable promoter, GLUD2 is thought to have gained expression in nerve and other tissues, where it adapted to their particular needs. Here we generated GLUD2 transgenic (Tg) mice by inserting in their genome a segment of the human X chromosome, containing the GLUD2 gene and its putative promoter. Double IF studies of Tg mouse brain revealed that the human gene is expressed in the host mouse brain in a pattern similar to that observed in human brain, thus providing credence to the above hypothesis. This expressional adaptation may have conferred novel role(s) on GLUD2 in human brain. Previous observations, also in GLUD2 Tg mice, generated and studied independently, showed that the non-redundant function of hGDH2 is markedly activated during early post-natal brain development, contributing to developmental changes in prefrontal cortex similar to those attributed to human divergence. Hence, GLUD2 adaptation may have influenced the evolutionary course taken by the human brain, but understanding the mechanism(s) involved remains challenging.

Glutamate dehydrogenase (GDH) is a hexameric enzyme that catalyzes the reversible conversion of glutamate to α-ketoglutarate and ammonia while reducing NAD(P)⁺ to NAD(P)H. It is found in all living organisms serving both catabolic and anabolic reactions. In mammalian tissues, oxidative deamination of glutamate via GDH generates α-ketoglutarate, which is metabolized by the Krebs cycle, leading to the synthesis of ATP. In addition, the GDH pathway is linked to diverse cellular processes, including ammonia metabolism, acid-base equilibrium, redox homeostasis (via formation of fumarate), lipid biosynthesis (via oxidative generation of citrate), and lactate production. While most mammals possess a single GDH1 protein (hGDH1 in the human) that is highly expressed in the liver, humans and other primates have acquired, via duplication, an hGDH2 isoenzyme with distinct functional properties and tissue expression profile. The novel hGDH2 underwent rapid evolutionary adaptation, acquiring unique properties that enable enhanced enzyme function under conditions inhibitory to its ancestor hGDH1. These are thought to provide a biological advantage to humans with hGDH2 evolution occurring concomitantly with human brain development. hGDH2 is co-expressed with hGDH1 in human brain, kidney, testis and steroidogenic organs, but not in the liver. In human cerebral cortex, hGDH1 and hGDH2 are expressed in astrocytes, the cells responsible for removing and metabolizing transmitter glutamate, and for supplying neurons with glutamine and lactate. In human testis, hGDH2 (but not hGDH1) is densely expressed in the Sertoli cells, known to provide the spermatids with lactate and other nutrients. In steroid producing cells, hGDH1/2 is thought to generate reducing equivalents (NADPH) in the mitochondria for the biosynthesis of steroidal hormones. Lastly, up-regulation of hGDH1/2 expression occurs in cancer, permitting neoplastic cells to utilize glutamine/glutamate for their growth. In addition, deregulation of hGDH1/2 is implicated in the pathogenesis of several human disorders.

Mammalian glutamate dehydrogenase1 (GDH1) (E.C. 1.4.1.3) is a mitochondrial enzyme that catalyzes the reversible oxidative deamination of glutamate to α-ketoglutarate and ammonia while reducing NAD+ and/or NADP+ to NADH and/or NADPH. It links amino acid with carbohydrate metabolism, contributing to Krebs cycle anaplerosis, energy production, ammonia handling and redox homeostasis. Although GDH1 was one of the first major metabolic enzymes to be studied decades ago, its role in cell biology is still incompletely understood. There is however growing interest in a novel GDH2 isoenzyme that emerged via duplication in primates and underwent rapid evolutionary selection concomitant with prefrontal human cortex expansion. Also, the anaplerotic function of GDH1 and GDH2 is currently under sharp focus as this relates to the biology of glial tumors and other neoplasias. Here we used antibodies specific for human GDH1 (hGDH1) and human GDH2 (hGDH2) to study the expression of these isoenzymes in human tissues. Results revealed that both hGDH1 and hGDH2 are expressed in human brain, kidney, testis and steroidogenic organs. However, distinct hGDH1 and hGDH2 expression patterns emerged. Thus, while the Sertoli cells of human testis were strongly positive for hGDH2, they were negative for hGDH1. Conversely, hGDH1 showed very high levels of expression in human liver, but hepatocytes were virtually devoid of hGDH2. In human adrenals, both hGDHs were densely expressed in steroid-producing cells, with hGDH2 expression pattern matching that of the cholesterol side chain cleavage system involved in steroid synthesis. Similarly in human ovaries and placenta, both hGDH1 and hGDH2 were densely expressed in estrogen producing cells. In addition, hGDH1, being a housekeeping enzyme, was also expressed in cells that lack endocrine function. Regarding human brain, study of cortical sections using immunofluorescence (IF) with confocal microscopy revealed that hGDH1 and hGDH2 were both expressed in the cytoplasm of gray and white matter astrocytes within coarse structures resembling mitochondria. Additionally, hGDH1 localized to the nuclear membrane of a subpopulation of astrocytes and of the vast majority of oligodendrocytes and their precursors. Remarkably, hGDH2-specific staining was detected in human cortical neurons, with different expression patterns having emerged. One pattern, observed in large cortical neurons (some with pyramidal morphology), was a hGDH2-specific labeling of cytoplasmic structures resembling mitochondria. These were distributed either in the cell body-axon or on the cell surface in close proximity to astrocytic end-feet that encircle glutamatergic synapses. Another pattern was observed in small cortical neurons with round dense nuclei in which the hGDH2-specific staining was found in the nuclear membrane. A detailed description of these observations and their functional implications, suggesting that the GDH flux is used by different cells to serve some of their unique functions, is presented below.

Human hGDH2 arose via duplication in the apes and driven by positive selection acquired enhanced catalytic ability under conditions inhibitory to its precursor hGDH1 (common to all mammals). To explore the biological advantage provided by the novel enzyme, we studied, by immunohistochemistry (IHC) and immunofluorescence (IF), hGDH1 and hGDH2 expression in the human brain. Studies on human cortical tissue using anti-hGDH1-specific antibody revealed that hGDH1 was expressed in glial cells (astrocytes, oligodendrocytes, and oligodendrocyte precursors) with neurons being devoid of hGDH1 staining. In contrast, an hGDH2-specific antiserum labeled both astrocytes and neurons. Specifically, hGDH2 immunoreactivity was found in the cytoplasm of large neuronal cells within coarse structures resembling mitochondria. These were distributed either in the perikaryon or in the cell periphery. Double immunofluorescence (IF) suggested that the latter represented hGDH2-labeled mitochondria of presynaptic nerve terminals. Hence, hGDH2 evolution bestowed large human neurons with enhanced glutamate metabolizing capacity, thus strengthening cortical excitatory transmission.

Mammalian glutamate dehydrogenase (GDH), a nuclear-encoded enzyme central to cellular metabolism, is among the most abundant mitochondrial proteins (constituting up to 10% of matrix proteins). To attain such high levels, GDH depends on very efficient mitochondrial targeting that, for human isoenzymes hGDH1 and hGDH2, is mediated by an unusually long cleavable presequence (N53). Here, we studied the mitochondrial transport of these proteins using isolated yeast mitochondria and human cell lines. We found that both hGDHs were very rapidly imported and processed in isolated mitochondria, with their presequences (N53) alone being capable of directing non-mitochondrial proteins into mitochondria. These presequences were predicted to form two α helices (α1: N 1-10; α2: N 16-32) separated by loops. Selective deletion of the α1 helix abolished the mitochondrial import of hGDHs. While the α1 helix alone had a very weak hGDH mitochondrial import capacity, it could direct efficiently non-mitochondrial proteins into mitochondria. In contrast, the α2 helix had no autonomous mitochondrial-targeting capacity. A peptide consisting of α1 and α2 helices without intervening sequences had GDH transport efficiency comparable with that of N53. Mutagenesis of the cleavage site blocked the intra-mitochondrial processing of hGDHs, but did not affect their mitochondrial import. Replacement of all three positively charged N-terminal residues (Arg3, Lys7 and Arg13) by Ala abolished import. We conclude that the synergistic interaction of helices α1 and α2 is crucial for the highly efficient import of hGDHs into mitochondria., (© 2016 The Author(s).)

Besides the housekeeping glutamate dehydrogenase1 (hGDH1), humans have acquired, via a recent duplication event, a hGDH2 isoenzyme with distinct functional properties and tissue expression profile. GDH catalyzes the reversible deamination of glutamate to α-ketoglutarate while reducing NAD(P) to NAD(P)H. As the generated NADPH can be used in bio-synthetic pathways, we studied here the expression of hGDH1 and hGDH2 in human steroidogenic tissues using specific antibodies. Results revealed high levels of hGDH1 and hGDH2 expression in steroid-producing cells in all tissues studied. While the cellular expression pattern of the two proteins was similar for the adrenal cortex, it was distinct for testis, ovaries and placenta. Functional analyses revealed that steroid hormones interacted differentially with the two isoenzymes. As synthesis of steroid hormones requires NADPH, expression of hGDH1 and hGDH2 in steroidogenic cells may serve their particular metabolic needs., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Manganese (Mn) is an essential trace element that serves as co-factor for many important mammalian enzymes. In humans, the importance of this cation is highlighted by the fact that low levels of Mn cause developmental and metabolic abnormalities and, on the other hand, chronic exposure to excessive amounts of Mn is characterized by neurotoxicity, possibly mediated by perturbation of astrocytic mitochondrial energy metabolism. Here we sought to study the effect of Mn on the two human glutamate dehydrogenases (hGDH1 and hGDH2, respectively), key mitochondrial enzymes involved in numerous cellular processes, including mitochondrial metabolism, glutamate homeostasis and neurotransmission, and cell signaling. Our studies showed that, compared to magnesium (Mg) and calcium (Ca), Mn exerted a significant inhibitory effect on both human isoenzymes with hGDH2 being more sensitive than hGDH1, especially under conditions of low ADP levels. Specifically, in the presence of 0.25 mM ADP, the Mn IC50 was 1.14 ± 0.02 mM and 1.54 ± 0.08 mM for hGDH2 and for hGDH1, respectively (p = 0.0001). Increasing Mn levels potentiated this differential effect, with 3 mM Mn inhibiting hGDH2 by 96.5% and hGDH1 by 70.2%. At 1 mM ADP, the Mn IC50 was 1.84 ± 0.02 mM and 2.04 ± 0.07 mM (p = 0.01) for hGDH2 and hGDH1, respectively, with 3 mM Mn inhibiting hGDH2 by 93.6% and hGDH1 by 70.9%. These results were due to the sigmoidal inhibitory curve of Mn that was more pronounced for hGDH2 than for hGDH1. Indeed, at 0.25 mM, the Hill coefficient value was higher for hGDH2 (3.42 ± 0.20) than for hGDH1 (1.94 ± 0.25; p = 0.0002) indicating that interaction of Mn with hGDH2 was substantially more co-operative than for hGDH1. These findings, showing an enhanced sensitivity of the hGDH2 isoenzyme to Mn, especially at low ADP levels, might be of pathophysiological relevance under conditions of Mn neurotoxicity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Glutamate Dehydrogenase (GDH) is central to the metabolism of glutamate, a major excitatory transmitter in mammalian central nervous system (CNS). hGDH1 is activated by ADP and L-leucine and powerfully inhibited by GTP. Besides this housekeeping hGDH1, duplication led to an hGDH2 isoform that is expressed in the human brain dissociating its function from GTP control. The novel enzyme has reduced basal activity (4-6% of capacity) while remaining remarkably responsive to ADP/L-leucine activation. While the molecular basis of this evolutionary adaptation remains unclear, substitution of Ser for Arg443 in hGDH1 is shown to diminish basal activity (< 2% of capacity) and abrogate L-leucine activation. To explore whether the Arg443Ser mutation disrupts hydrogen bonding between Arg443 and Ser409 of adjacent monomers in the regulatory domain ('antenna'), we replaced Ser409 by Arg or Asp in hGDH1. The Ser409Arg-1 change essentially replicated the Arg443Ser-1 mutation effects. Molecular dynamics simulation predicted that Ser409 and Arg443 of neighboring monomers come in close proximity in the open conformation and that introduction of Ser443-1 or Arg409-1 causes them to separate with the swap mutation (Arg409/Ser443) reinstating their proximity. A swapped Ser409Arg/Arg443Ser-1 mutant protein, obtained in recombinant form, regained most of the wild-type hGDH1 properties. Also, when Ser443 was replaced by Arg443 in hGDH2 (as occurs in hGDH1), the Ser443Arg-2 mutant acquired most of the hGDH1 properties. Hence, side-chain interactions between 409 and 443 positions in the 'antenna' region of hGDHs are crucial for basal catalytic activity, allosteric regulation, and relative resistance to thermal inactivation., (© 2014 International Society for Neurochemistry.)

Objectives: The coexistence of systemic lupus erythematosus (SLE) and multiple sclerosis (MS) in the same individual has rarely been described. Our objective was to report on the prevalence, clinical characteristics, and prognosis of cases fulfilling the criteria for both SLE and MS., Methods: We utilized existing patient cohorts from the Departments of Rheumatology and Neurology, University of Crete, and screened patients diagnosed with either SLE (n = 728) or MS (n = 819) for features of both diseases. The clinical, laboratory, and neuroimaging findings were assessed., Results: We identified nine patients who fulfilled the diagnostic criteria for both SLE and MS, corresponding to a prevalence rate of 1.0-1.2% in each cohort. All patients were women, with an average age at SLE diagnosis of 42.1 years (range: 34-56 years). The diagnosis of SLE preceded the development of MS in five patients, with a time lag ≤ 5 years in four of them. Initial presentation of MS included spinal symptoms in seven patients. All patients had features of mild SLE with predominantly cutaneous, mucosal, and musculoskeletal manifestations. Accordingly, therapeutic decisions were mainly guided by the severity of the neurological syndrome. During the median follow-up of 4 years (range: 1-10 years), three patients remained stable and the remaining experienced gradual deterioration in their neurological status. SLE remained quiescent in all patients while on standard immunomodulatory MS therapy., Conclusions: Occurrence of both diseases in the same individual is rare, corroborating data that suggest distinct molecular signatures. SLE and MS coexistence was not associated with a severe phenotype for either entity., (© 2013 Published by Elsevier Inc.)

Genome-wide association studies (GWAS) have been shown to be a powerful approach to identify risk loci for neurodegenerative diseases. Recent GWAS in Parkinson's disease (PD) have been successful in identifying numerous risk variants pointing to novel pathways potentially implicated in the pathogenesis of PD. Contributing to these GWAS efforts, we performed genotyping of previously identified risk alleles in PD patients and control subjects from Greece. We showed that previously published risk profiles for Northern European and American populations are also applicable to the Greek population. In addition, although our study was largely underpowered to detect individual associations, we replicated 5 of 32 previously published risk variants with nominal p values <0.05. Genome-wide complex trait analysis revealed that known risk loci explain disease risk in 1.27% of Greek PD patients. Collectively, these results indicate that there is likely a substantial genetic component to PD in Greece, similarly to other worldwide populations, that remains to be discovered., (Published by Elsevier Inc.)

Mammalian glutamate dehydrogenase (GDH) is an evolutionarily conserved enzyme central to the metabolism of glutamate, the main excitatory transmitter in mammalian CNS. Its activity is allosterically regulated and thought to be controlled by the need of the cell for ATP. While in most mammals, GDH is encoded by a single GLUD1 gene that is widely expressed (housekeeping; hGDH1 in the human), humans and other primates have acquired via retroposition a GLUD2 gene encoding an hGDH2 isoenzyme with distinct functional properties and tissue expression profile. Whereas hGDH1 shows high levels of expression in the liver, hGDH2 is expressed in human testis, brain and kidney. Recent studies have provided significant insight into the functional adaptation of hGDH2. This includes resistance to GTP control, enhanced sensitivity to inhibition by estrogens and other endogenous allosteric effectors, and ability to function in a relatively acidic environment. While inhibition of hGDH1 by GTP, derived from Krebs cycle, represents the main mechanism by which the flux of glutamate through this pathway is regulated, dissociation of hGDH2 from GTP control may provide a biological advantage by permitting enzyme function independently of this energy switch. Also, the relatively low optimal pH for hGDH2 is suited for transmitter glutamate metabolism, as glutamate uptake by astrocytes leads to significant mitochondrial acidification. Although mammalian GDH is a housekeeping enzyme, its levels of expression vary markedly among the various tissues and among the different types of cells that constitute the same organ. In this paper, we will review existing evidence on the cellular and subcellular distribution of GDH in neural and non-neural tissues of experimental animals and humans, and consider the implications of these findings in biology of these tissues. Special attention is given to accumulating evidence that glutamate flux through the GDH pathway is linked to cell signaling mechanisms that may be tissue-specific.

Glutamate dehydrogenase (GDH) is a crucial enzyme on the crossroads of amino acid and energy metabolism and it is operating in all domains of life. According to current knowledge GDH is present only in one functional isoform in most animals, including mice. In addition to this housekeeping enzyme (hGDH1 in humans), humans and apes have acquired a second isoform (hGDH2) with a distinct tissue expression profile. In the current study we have cloned both mouse and human GDH constructs containing FLAG and (His)6 small genetically-encoded tags, respectively. The hGDH1 and hGDH2 constructs containing N-terminal (His)6 tags were successfully expressed in Sf9 cells and the recombinant proteins were isolated to ≥95 % purity in a two-step procedure involving ammonium sulfate precipitation and Ni(2+)-based immobilized metal ion affinity chromatography. To explore whether the presence of the FLAG and (His)6 tags affects the cellular localization and functionality of the GDH isoforms, we studied the subcellular distribution of the expressed enzymes as well as their regulation by adenosine diphosphate monopotassium salt (ADP) and guanosine-5'-triphosphate sodium salt (GTP). Through immunoblot analysis of the mitochondrial and cytosolic fraction of the HEK cells expressing the recombinant proteins we found that neither FLAG nor (His)6 tag disturbs the mitochondrial localization of GDH. The addition of the small tags to the N-terminus of the mature mitochondrial mouse GDH1 or human hGDH1 and hGDH2 did not change the ADP activation or GTP inhibition pattern of the proteins as compared to their untagged counterparts. However, the addition of FLAG tag to the C-terminus of the mouse GDH left the recombinant protein fivefold less sensitive to ADP activation. This finding highlights the necessity of the functional characterization of recombinant proteins containing even the smallest available tags.

Background: Patients with clinically isolated syndrome (CIS) demonstrate brain hemodynamic changes and also suffer from difficulties in processing speed, memory, and executive functions., Objective: To explore whether brain hemodynamic disturbances in CIS patients correlate with executive functions., Methods: Thirty CIS patients and forty-three healthy subjects, matched for age, gender, education level, and FSIQ, were administered tests of visuomotor learning and set shifting ability. Cerebral blood volume (CBV), cerebral blood flow (CBF), and mean transit time (MTT) values were estimated in normal-appearing white matter (NAWM) and normal-appearing deep gray Matter (NADGM) structures, using a perfusion MRI technique., Results: CIS patients showed significantly elevated reaction time (RT) on both tasks, while their CBV and MTT values were globally increased, probably due to inflammatory vasodilation. Significantly, positive correlation coefficients were found between error rates on the inhibition condition of the visuomotor learning task and CBV values in occipital, periventricular NAWM and both thalami. On the set shifting condition of the respective task significant, positive associations were found between error rates and CBV values in the semioval center and periventricular NAWM bilaterally., Conclusion: Impaired executive function in CIS patients correlated positively with elevated regional CBV values thought to reflect inflammatory processes.

While the evolutionary changes that led to traits unique to humans remain unclear, there is increasing evidence that enrichment of the human genome through DNA duplication processes may have contributed to traits such as bipedal locomotion, higher cognitive abilities and language. Among the genes that arose through duplication in primates during the period of increased brain development was GLUD2, which encodes the hGDH2 isoform of glutamate dehydrogenase expressed in neural and other tissues. Glutamate dehydrogenase GDH is an enzyme central to the metabolism of glutamate, the main excitatory neurotransmitter in mammalian brain involved in a multitude of CNS functions, including cognitive processes. In nerve tissue GDH is expressed in astrocytes that wrap excitatory synapses, where it is thought to play a role in the metabolic fate of glutamate removed from the synaptic cleft during excitatory transmission. Expression of GDH rises sharply during postnatal brain development, coinciding with nerve terminal sprouting and synaptogenesis. Compared to the original hGDH1 (encoded by the GLUD1 gene), which is potently inhibited by GTP generated by the Krebs cycle, hGDH2 can function independently of this energy switch. In addition, hGDH2 can operate efficiently in the relatively acidic environment that prevails in astrocytes following glutamate uptake. This adaptation is thought to provide a biological advantage by enabling enhanced enzyme catalysis under intense excitatory neurotransmission. While the novel protein may help astrocytes to handle increased loads of transmitter glutamate, dissociation of hGDH2 from GTP control may render humans vulnerable to deregulation of this enzyme's function. Here we will retrace the cloning and characterization of the novel GLUD2 gene and the potential implications of this discovery in the understanding of mechanisms that permitted the brain and other organs that express hGDH2 to fine-tune their functions in order to meet new challenging demands. In addition, the potential role of gain-of-function of hGDH2 variants in human neurodegenerative processes will be considered.

Purpose: The multi-organ involvement of mitochondrial diseases means that patients are likely to be more vulnerable to sleep disturbances. We aimed to assess if early recognition and treatment of obstructive sleep apnea (OSA) in patients with Leigh disease may influence primary disease outcome., Methods: We describe a case of adult-onset Leigh disease presenting as severe brainstem encephalopathy of subacute onset. Based on the clinical symptoms that developed after the appearance of the neurological disease, an attended overnight polysomnography examination was performed., Results: A marked clinical recovery was seen after administration of high doses of thiamine, coenzyme Q, L-carnitine, and vitamins C and E, combined with effective treatment with continuous positive airway pressure for the underlying severe obstructive sleep apnea (OSA). The latter condition was diagnosed on the basis of suggestive symptoms that appeared a few weeks before the establishment of the neurological disease. The improvement in the neurological disease (based on clinical and brain MRI features) with the appropriate medical treatment also resulted in a significant improvement in the OSA., Conclusions: Early recognition and treatment of sleep apnea may not only improve sleep and overall quality of life but also ameliorate the deleterious effects of nocturnal desaturations on the neurological features. This may be crucial for disease outcome when added to the generally advised pharmacological therapy.

Introduction: Familial amyloid polyneuropathy is characterized by transthyretin (TTR) deposition in various tissues, including the kidneys. While deposition induces organ dysfunction, renal involvement in TTR-related amyloidosis could manifest from proteinuria to end-stage kidney failure. As proteinuria is considered result of glomerular filtration barrier injury we investigated whether TTR deposition affects either glomerular basement membrane (GBM) or podocytes., Materials and Methods: Immunohistochemistry, immunoblot and gene expression studies for nephrin, podocin and WT1 were run on renal tissue from human-TTRV30M transgenic mice hemizygous or homozygous for heat shock factor one (Hsf-1). Transmission electron microscopy was used for evaluation of podocyte foot process width (PFW) and GBM thickness in Hsf-1 hemizygous mice with or without TTRV30M or amyloid deposition., Results: Glomeruli of hsf-1 hemizygous transgenic mice showed lower nephrin and podocin protein levels but an increased podocyte number when compared to Hsf-1 homozygous transgenic mice. Nephrin, podocin and WT1 gene expression levels were unaffected by the Hsf-1 carrier status. TTRV30M deposition was associated with increased PFW and GBM thickness., Conclusions: Under the effect of Hsf-1 hemizygosity, TTRV30M deposition has deleterious effects on GBM thickness, PFW and slit diaphragm composition, without affecting nephrin and podocin gene expression.

Mammalian glutamate dehydrogenase is an allosterically regulated enzyme that is central to glutamate metabolism. It contributes to important cellular processes, including Krebs cycle anaplerotic mechanisms, energy production, and ammonia homeostasis. In addition to this housekeeping hGDH1, humans have acquired through duplication an hGDH2 isoenzyme expressed in neural tissues with distinct regulatory properties. There is increasing evidence that deregulation of human GDHs leads to human disorders. Thus, in hGDH1, regulatory mutations that attenuate GTP inhibition can result in the hyperinsulinism/hyperammonemia syndrome, which is often associated with epileptic seizures, mental retardation, and generalized dystonia. Also, transgenic overexpression of GLUD1 in neurons has resulted in age-dependent degeneration of the CA1 behippocampal region, associated with upregulation of α-synuclein and other proteins linked to major human movement disorders. With regard to hGDH2, a rare T1492G variation in the GLUD2 gene, resulting in substitution of Ala for Ser445 in the regulatory domain of hGDH2, interacts significantly with Parkinson's disease (PD) onset. In two independent Greek and one North American PD cohorts, Ser445Ala hemizygous males, but not heterozygous females, developed PD 6-13 years earlier than subjects with other genotypes. The Ala445-hGDH2 variant displays increased catalytic activity that is amenable to inhibition by estrogens. Enhanced glutamate oxidation by Ala445-hGDH2 is thought to accelerate nigral cell degeneration in hemizygous males, and inhibition of the overactive variant by estrogens may protect heterozygous females. Hence, deregulation of hGDH1 and hGDH2 may play a role in degenerative processes, so these observations identify novel targets for therapeutic intervention in human disorders., (Copyright © 2013 Wiley Periodicals, Inc.)

Glutamate dehydrogenase (GDH) uses ammonia to reversibly convert α-ketoglutarate to glutamate using NADP(H) and NAD(H) as cofactors. While GDH in most mammals is encoded by a single GLUD1 gene, humans and other primates have acquired a GLUD2 gene with distinct tissue expression profile. The two human isoenzymes (hGDH1 and hGDH2), though highly homologous, differ markedly in their regulatory properties. Here we obtained hGDH1 and hGDH2 in recombinant form and studied their Km for ammonia in the presence of 1.0 mM ADP. The analyses showed that lowering the pH of the buffer (from 8.0 to 7.0) increased the Km for ammonia substantially (hGDH1: from 12.8 ± 1.4 mM to 57.5 ± 1.6 mM; hGDH2: from 14.7 ± 1.6 mM to 62.2 ± 1.7 mM), thus essentially precluding reductive amination. Moreover, lowering the ADP concentration to 0.1 mM not only increased the K0.5 [NH4 (+)] of hGDH2, but also introduced a positive cooperative binding phenomenon in this isoenzyme. Hence, intra-mitochondrial acidification, as occurring in astrocytes during glutamatergic transmission should favor the oxidative deamination of glutamate. Similar considerations apply to the handling of glutamate by the proximal convoluted tubules of the kidney during systemic acidosis. The reverse could apply for conditions of local or systemic hyperammonemia or alkalosis.

Transthyretin related amyloidosis is a nosological entity that leads to disability, diminished quality of life, all stages of chronic kidney disease and eventually death. Podocytes are polarized, highly differentiated epithelial cells important for proper nephron function. In the present study we investigated whether deposited TTRVal30Met (TTRV30M) molecules could be localized within podocytes in situ under the effect of different housing conditions (i.e. specific pathogen free [SPF] vs. non-SPF). Murine renal glomeruli from human TTRV30M (hTTRV30M) transgenic mice were examined via direct and indirect immunofluorescence techniques for the presence of hTTRV30M, murine serum amyloid P, activated caspase-3 and NPHS1. Association strength and amount of colocalization for NPHS1-hTTRV30M, NPHS1-activated caspase-3, hTTRV30M-murine serum amyloid P were estimated. Localization of hTTRV30M in podocytes was demonstrated by immuno-electron microscopy. Renal hTTRV30M gene and NPHS1 gene expression levels were estimated. Non-SPF transgenic mice showed increased glomerular hTTRV30M deposition compared to their SPF counterparts. Furthermore increased podocytic localization of hTTRV30M was noticed in non-SPF mice. Glomerular caspase-3 activation was increased only in the non SPF housing conditions. Podocytic caspase-3 activation was increased in SPF and in non-SPF transgenic mice when compared to non transgenic controls. Environmental conditions influence glomerular deposition and podocytic localization of hTTRV30M. In this context increased caspase-3 activation occurred.

The dynamic susceptibility contrast magnetic resonance imaging perfusion technique was used to investigate possible hemodynamic changes in normal appearing white matter and deep gray matter (DGM) of 30 patients with clinically isolated syndrome (CIS) and 30 patients with relapsing-remitting multiple sclerosis. Thirty normal volunteers were studied as controls. Cerebral blood volume, cerebral blood flow (CBF), and mean transit time values were estimated. Normalization was achieved for each subject with respect to average values of CBF and mean transit time of the hippocampi's dentate gyrus. Measurements concerned three regions of normal white matter of normal volunteers, normal appearing white matter of CIS and patients with relapsing-remitting multiple sclerosis, and DGM regions, bilaterally. All measured normal appearing white matter and DGM regions of the patients with CIS had significantly higher cerebral blood volume and mean transit time values, while averaged DGM regions had significantly lower CBF values, compared to those of normal volunteers (P < 0.001). Regarding patients with relapsing-remitting multiple sclerosis, all measured normal appearing white matter and DGM regions showed lower CBF values than those of normal volunteers and lower cerebral blood volume and CBF values compared to patients with CIS (P < 0.001). These data provide strong evidence that hemodynamic changes--affecting both white and DGM--may occur even at the earliest stage of multiple sclerosis, with CIS patients being significantly different than relapsing-remitting multiple sclerosis patients., (Copyright © 2012 Wiley Periodicals, Inc.)

Human glutamate dehydrogenase (hGDH) exists in two highly homologous isoforms with a distinct regulatory and tissue expression profile: a housekeeping hGDH1 isoprotein encoded by the GLUD1 gene and an hGDH2 isoenzyme encoded by the GLUD2 gene. There is evidence that both isoenzymes are synthesized as pro-enzymes containing a 53 amino acid long N-terminal leader peptide that is cleaved upon translocation into the mitochondria. However, this GDH signal peptide is substantially larger than that of most nuclear DNA-encoded mitochondrial proteins, the leader sequence of which typically contains 17-35 amino acids and they often form a single amphipathic α-helix. To decode the structural elements that are essential for the mitochondrial targeting of human GDHs, we performed secondary structure analyses of their leader sequence. These analyses predicted, with 82% accuracy, that both leader peptides are positively charged and that they form two to three α-helices, separated by intermediate loops. The first α-helix of hGDH2 is strongly amphipathic, displaying both a positively charged surface and a hydrophobic plane. We then constructed GLUD2-EGFP deletion mutants and used them to transfect three mammalian cell lines (HEK293, COS 7 and SHSY-5Y). Confocal laser scanning microscopy, following co-transfection with pDsRed2-Mito mitochondrial targeting vector, revealed that deletion of the entire leader sequence prevented the enzyme from entering the mitochondria, resulting in its retention in the cytoplasm. Deletion of the first strongly amphipathic α-helix only was also sufficient to prevent the mitochondrial localization of the truncated protein. Moreover, truncated leader sequences, retaining the second and/or the third putative α-helix, failed to restore the mitochondrial import of hGDH2. As such, the first N-terminal alpha helical structure is crucial for the mitochondrial import of hGDH2 and these findings may have implications in understanding the evolutionary mechanisms that led to the large mitochondrial targeting signals of human GDHs., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Glutamate dehydrogenase (GDH), a mitochondrial enzyme with a key metabolic role, exists in the human in hGDH1 and hGDH2 isoforms encoded by the GLUD1 and GLUD2 genes, respectively. It seems that GLUD1 was retroposed to the X chromosome where it gave rise to GLUD2 via random mutations and natural selection. Of these, evolutionary Gly456Ala substitution dissociated hGDH2 from GTP control, while replacement of Arg443 by Ser drastically modified basal activity, heat stability, optimal pH, allosteric regulation and migration pattern in SDS-PAGE, thus suggesting an effect on enzyme's conformation. While GLUD2-specific transcripts have been detected in human brain, retina and testis, data on the endogenous hGDH2 protein are lacking. Given the housekeeping nature of hGDH1 and its high homology to hGDH2, the specific detection of hGDH2 in tissues presents a challenge. To develop an antibody specific for hGDH2, we considered that an epitope containing the Arg443Ser change was an attractive target. We accordingly used a peptide that corresponds to residues 436-447, with Ser at position 443, to immunize rabbits and succeeded in raising a polyclonal antibody specific for hGDH2. Western blots showed that human testis contained equal amounts of hGDH2 and hGDH1 and that both isoproteins localized to the mitochondrial fraction. In human brain, however, hGDH2 expression was lower than that of hGDH1. Immuno-histochemical studies on human testis and cerebral cortex, showed punctuate, organelle-like hGDH2 immuno-labeling in sertoli cells and in astrocytes, respectively, consistent with the mitochondrial localization of the enzyme. Similar studies in kidney revealed that hGDH2 is expressed in epithelial cells of the proximal convoluted tubule. As hGDH2 can metabolize glutamate at relatively low pH without the GTP constrain, it may function efficiently under conditions of relative acidification that prevail in astrocytes following glutamate uptake. Similarly, in the kidney, hGDH2 could contribute to enhanced excretion of ammonia under acidosis., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Mammalian glutamate dehydrogenase (GDH) is a housekeeping mitochondrial enzyme (hGDH1 in the human) that catalyses the reversible inter-conversion of glutamate to α-ketoglutarate and ammonia, thus interconnecting amino acid and carbohydrate metabolism. It displays an energy sensing mechanism, which permits enzyme activation under low cellular energy states. As GDH is at the crossroads of important metabolic pathways, a tight control of its activity is essential. Indeed, to fulfill its role in metabolism and cellular energetics, mammalian GDH has evolved into a highly regulated enzyme subject to allosteric modulation by diverse compounds. The recent emergence (<23 million years ago) in apes and humans of a hGDH2 isoenzyme with distinct regulatory properties, as well as, the detection of gain-of-function variants in hGDH1 and hGDH2 that affect the nervous system, have introduced additional complexities. The properties of the two highly homologous human GDHs were studied using purified recombinant hGDH1 and hGDH2 obtained by expression of the corresponding cDNAs in Sf21 cells. Results showed that, in contrast to hGDH1 that maintains substantial basal activity (35-40% of its maximal capacity), hGDH2 displays low basal activity (3-8% of maximal) that is remarkably responsive to activation by rising levels of ADP and/or l-leucine. This is primarily due to the Arg443Ser evolutionary change, which also made hGDH2 markedly sensitive to estrogens and neuroleptic drugs. In contrast to hGDH1, which is subject to potent GTP inhibition, hGDH2 has dissociated its function from this energy switch, being able to metabolize glutamate even when the Krebs cycle generates GTP levels sufficient to inactivate the housekeeping hGDH1. Our data also show that spermidine, a polyamine thought to reduce oxidative stress and to prolong survival, and EGCG, a green tea polyphenol, inhibit hGDH2 at lower concentrations than hGDH1. The implications of these findings in nerve tissue biology are discussed., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

There is a need in autoimmune diseases to uncover the mechanisms involved in the natural resolution of inflammation. In this article, we demonstrate that granulocytic myeloid-derived suppressor cells (G-MDSCs) abundantly accumulate within the peripheral lymphoid compartments and target organs of mice with experimental autoimmune encephalomyelitis prior to disease remission. In vivo transfer of G-MDSCs ameliorated experimental autoimmune encephalomyelitis, significantly decreased demyelination, and delayed disease onset through inhibition of encephalitogenic Th1 and Th17 immune responses. Exposure of G-MDSCs to the autoimmune milieu led to up-regulation of the programmed death 1 ligand that was required for the G-MDSC-mediated suppressive function both in vitro and in vivo. Importantly, myeloid-derived suppressor cells were enriched in the periphery of subjects with active multiple sclerosis and suppressed the activation and proliferation of autologous CD4(+) T cells ex vivo. Collectively, this study revealed a pivotal role for myeloid-derived suppressor cells in the regulation of multiple sclerosis, which could be exploited for therapeutic purposes.

The study investigates primary and secondary verbal memory and motor/executive functions (response inhibition and strategy shifting ability) in multiple sclerosis (MS) patients with clinically isolated syndrome (CIS). We studied 44 CIS patients and compared them to 49 patients with relapsing remitting MS (RR-MS) displaying mild disability and to a large cohort of age- and education level-matched healthy volunteers(n=230). Results showed that both CIS and RR-MS patients evidenced a disproportionate impairment in the immediate and delayed recall of the second (as compared to the first) of two short narratives of the Logical Memory WMS-III subtest, and reduced performance on the Memory for Digits-Forward. Performance of either group on the executive tasks was not impaired, showing evidence of a reversed speed-accuracy trade-off. Illness duration emerged as a significant predictor of memory and executive task performance. Clinical, psychoemotional, and brain imaging findings were also examined as potential correlates of memory deficits and disease progression among CIS patients. These findings may signify early-onset decline of specific cognitive functions in CIS, which merits regular follow-up assessments and monitoring of psychoemotional adaptation and everyday functioning.

Glutamate dehydrogenase (GDH) catalyzes the reversible inter-conversion of glutamate to α-ketoglutarate and ammonia. High levels of GDH activity is found in mammalian liver, kidney, brain, and pancreas. In the liver, GDH reaction appears to be close-to-equilibrium, providing the appropriate ratio of ammonia and amino acids for urea synthesis in periportal hepatocytes. In addition, GDH produces glutamate for glutamine synthesis in a small rim of pericentral hepatocytes. Hence, hepatic GDH can be either a source for ammonia or an ammonia scavenger. In the kidney, GDH function produces ammonia from glutamate to control acidosis. In the human, the presence of two differentially regulated isoforms (hGDH1 and hGDH2) suggests a complex role for GDH in ammonia homeostasis. Whereas hGDH1 is sensitive to GTP inhibition, hGDH2 has dissociated its function from GTP control. Furthermore, hGDH2 shows a lower optimal pH than hGDH1. The hGDH2 enzyme is selectively expressed in human astrocytes and Sertoli cells, probably facilitating metabolic recycling processes essential for their supportive role. Here, we report that hGDH2 is also expressed in the epithelial cells lining the convoluted tubules of the renal cortex. As hGDH2 functions more efficiently under acidotic conditions without the operation of the GTP energy switch, its presence in the kidney may increase the efficacy of the organ to maintain acid base equilibrium.

Background and Purpose: Although corticosteroid use in acute hemorrhagic stroke is not widely adopted, management with intravenous dexamethasone has been standard of care at the University Hospital of Heraklion, Crete with observed outcomes superior to those reported in the literature. To explore this further, we conducted a retrospective, multivariable-adjusted 2-center study., Methods: We studied 391 acute hemorrhagic stroke cases admitted to the University Hospital of Heraklion, Crete between January 1997 and July 2010 and compared them with 510 acute hemorrhagic stroke cases admitted to Massachusetts General Hospital, Boston, from January 2003 to September 2009. Of the Cretan cases, 340 received a tapering scheme of intravenous dexamethasone, starting with 16 to 32 mg/day, whereas the Boston patients were managed without steroids., Results: The 2 cohorts had comparable demographics and stroke severity on admission, although anticoagulation was more frequent in Boston. The in-hospital mortality was significantly lower on Crete (23.8%, n=340) than in Boston (38.0%, n=510; P<0.001) as was the 30-day mortality (Crete: 25.4%, n=307; Boston: 39.4%, n=510; P<0.001). Exclusion of patients on anticoagulants showed even greater differences (30-day mortality: Crete 20.8%; n=259; Boston 37.0%; n=359; P<0.001). The improved survival on Crete was observed 3 days after initiation of intravenous dexamethasone and was pronounced for deep-seated hemorrhages. After adjusting for acute hemorrhagic stroke volume/location, Glasgow Coma Scale, hypertension, diabetes mellitus, smoking, coronary artery disease and statin, antiplatelet, and anticoagulant use, intravenous dexamethasone treatment was associated with better functional outcomes and significantly lower risk of death at 30 days (OR, 0.357; 95% CI, 0.174-0.732)., Conclusions: This study suggests that intravenous dexamethasone improves outcome in acute hemorrhagic stroke and supports a randomized clinical trial using this approach.

Introduction: Pregabalin efficacy and safety as an adjunctive treatment for partial seizures was evaluated using an open-label, flexible-dose., Study Design: In 98 adults with refractory partial epilepsy taking 1-3 anti-epileptic drugs with ≥2 seizures during an 8-week baseline period., Methods: Pregabalin was increased to ≤600 mg/day during a 9-week dose optimization period with dosage maintained for 12 additional weeks. Primary endpoint was the percentage change in partial seizure frequency between the 8-week baseline and 12-week observation period., Results: Pregabalin treatment was associated with a significant reduction in partial seizure frequency: median percent change in partial seizure frequency from baseline to 12 weeks was -33% and -22% in patients with a baseline seizure frequency of ≤3 and >3 per 28 days, respectively. The 50% and 75% responder rates were 41.94% (95% CI: 31.91-51.96) and 30.11% (95% CI: 20.78-39.43), respectively. Nineteen percent of subjects were seizure-free throughout the last 12 weeks. Pregabalin administration resulted in a significant reduction in anxiety (mean reduction in Hospital Anxiety and Depression Scale scores of 1.68 units, 95% CI: -2.60 to -0.76). Most patients were much improved or very much improved on Patient Global Impression of Change (53.8%) and Clinical Global Impression of Change (53.8%). The most frequently self-reported adverse events (AEs) were mild or moderate somnolence (20.4%) and dizziness (5.1%) with a low AE discontinuation rate (5.1%)., Conclusions: The efficacy and side-effect profile of pregabalin were similar to previous pregabalin double-blind, controlled studies. Additionally, pregabalin, as an add-on treatment for partial epilepsy, exhibits significant anti-anxiety properties., (Copyright © 2011 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.)

Motor neuron disease is a devastating neurodegenerative condition, with the majority of sporadic, non-familial cases being of unknown etiology. Several epidemiological studies have suggested that occupational exposure to chemicals may be associated with disease pathogenesis. We report the case of a patient developing progressive motor neuron disease, who was chronically exposed to pesticides and organic solvents. The patient presented with leg spasticity and developed gradually clinical signs suggestive of amyotrophic lateral sclerosis, which was supported by the neurophysiologic and radiological findings. Our report is an evidence based case of combined exposure to organochlorine (DDTs), organophosphate pesticides (OPs) and organic solvents as confirmed by laboratory analysis in samples of blood and hair confirming systematic exposure. The concentration of non-specific dialkylphosphates metabolites (DAPs) of OPs in hair (dimethyphopshate (DMP) 1289.4 pg/mg and diethylphosphate (DEP) 709.4 pg/mg) and of DDTs (opDDE 484.0 pg/mg, ppDDE 526.6 pg/mg, opDDD 448.4 pg/mg, ppDDD+opDDT 259.9 pg/mg and ppDDT 573.7 pg/mg) were considerably significant. Toluene and n-hexane were also detected in blood on admission at hospital and quantified (1.23 and 0.87 μg/l, respectively), while 3 months after hospitalization blood testing was found negative for toluene and n-hexane and hair analysis was provided decrease levels of HCHs, DDTs and DAPs., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Whereas glutamate dehydrogenase in most mammals (hGDH1 in the human) is encoded by a single functional GLUD1 gene expressed widely, humans and other primates have acquired through retroposition an X-linked GLUD2 gene that encodes a highly homologous isoenzyme (hGDH2) expressed in testis and brain. Using an antibody specific for hGDH2, we showed that hGDH2 is expressed in testicular Sertoli cells and in cerebral cortical astrocytes. Although hGDH1 and hGDH2 have similar catalytic properties, they differ markedly in their regulatory profile. While hGDH1 is potently inhibited by GTP and may be controlled by the need of the cell for ATP, hGDH2 has dissociated its function from GTP and may metabolize glutamate even when the Krebs cycle generates GTP amounts sufficient to inactivate hGDH1. As astrocytes are known to provide neurons with lactate that largely derives from the Krebs cycle via conversion of glutamate to α-ketoglutarate, the selective expression of hGDH2 may facilitate metabolic recycling processes essential for glutamatergic transmission. As there is evidence for deregulation of glutamate metabolism in degenerative neurologic disorders, we sequenced GLUD1 and GLUD2 genes in neurologic patients and found that a rare T1492G variation in GLUD2 that results in substitution of Ala for Ser445 in the regulatory domain of hGDH2 interacted significantly with Parkinson's disease (PD) onset. Thus, in two independent Greek and one North American PD cohorts, Ser445Ala hemizygous males, but not heterozygous females, developed PD 6-13 years earlier than subjects with other genotypes. The Ala445-hGDH2 variant shows enhanced catalytic activity that is resistant to modulation by GTP, but sensitive to inhibition by estrogens. These observations are thought to suggest that enhanced glutamate oxidation by the Ala445-hGDH2 variant accelerates nigral cell degeneration in hemizygous males and that inhibition of the overactive enzyme by estrogens protects heterozygous females. We then evaluated the interaction of estrogens and neuroleptic agents (haloperidol and perphenazine) with the wild-type hGDH1 and hGDH2 and found that both inhibited hGDH2 more potently than hGDH1 and that the evolutionary Arg443Ser substitution was largely responsible for this sensitivity. Hence, the properties acquired by hGDH2 during its evolution have made the enzyme a selective target for neuroactive steroids and drugs, providing new means for therapeutic interventions in disorders linked to deregulation of this enzyme., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Mammalian glutamate dehydrogenase (GDH) is a housekeeping enzyme central to the metabolism of glutamate. Its activity is potently inhibited by GTP (IC(50) = 0.1-0.3 μM) and thought to be controlled by the need of the cell in ATP. Estrogens are also known to inhibit mammalian GDH, but at relatively high concentrations. Because, in addition to this housekeeping human (h) GDH1, humans have acquired via a duplication event an hGDH2 isoform expressed in human cortical astrocytes, we tested here the interaction of estrogens with the two human isoenzymes. The results showed that, under base-line conditions, diethylstilbestrol potently inhibited hGDH2 (IC(50) = 0.08 ± 0.01 μM) and with ∼18-fold lower affinity hGDH1 (IC(50) = 1.67 ± 0.06 μM; p < 0.001). Similarly, 17β-estradiol showed a ∼18-fold higher affinity for hGDH2 (IC(50) = 1.53 ± 0.24 μM) than for hGDH1 (IC(50) = 26.94 ± 1.07 μM; p < 0.001). Also, estriol and progesterone were more potent inhibitors of hGDH2 than hGDH1. Structure/function analyses revealed that the evolutionary R443S substitution, which confers low basal activity, was largely responsible for sensitivity of hGDH2 to estrogens. Inhibition of both human GDHs by estrogens was inversely related to their state of activation induced by ADP, with the slope of this correlation being steeper for hGDH2 than for hGDH1. Also, the study of hGDH1 and hGDH2 mutants displaying different states of activation revealed that the affinity of estrogen for these enzymes correlated inversely (R = 0.99; p = 0.0001) with basal catalytic activity. Because astrocytes are known to synthesize estrogens, these hormones, by interacting potently with hGDH2 in its closed state, may contribute to regulation of glutamate metabolism in brain.

Mammalian glutamate dehydrogenase (GDH) is an allosterically regulated enzyme that is expressed widely. Its activity is potently inhibited by GTP and thought to be controlled by the need of the cell for ATP. In addition to this housekeeping human (h) GDH1, humans have acquired (via a duplication event) a highly homologous isoenzyme (hGDH2) that is resistant to GTP. Although transcripts of GLUD2, the gene encoding hGDH2, have been detected in human neural and testicular tissues, data on the endogenous protein are lacking. Here, we developed an antibody specific for hGDH2 and used it to study human tissues. Western blot analyses revealed, to our surprise, that endogenous hGDH2 is more densely expressed in testis than in brain. At the subcellular level, hGDH2 localized to mitochondria. Study of testicular tissue using immunocytochemical and immunofluorescence methods revealed that the Sertoli cells were strongly labeled by our anti-hGDH2 antibody. In human cerebral cortex, a robust labeling of astrocytes was detected, with neurons showing faint hGDH2 immunoreactivity. Astrocytes and Sertoli cells are known to support neurons and germ cells, respectively, providing them with lactate that largely derives from the tricarboxylic acid cycle via conversion of glutamate to alpha-ketoglutarate (GDH reaction). As hGDH2 is not subject to GTP control, the enzyme is able to metabolize glutamate even when the tricarboxylic acid cycle generates GTP amounts sufficient to inactivate the housekeeping hGDH1 protein. Hence, the selective expression of hGDH2 by astrocytes and Sertoli cells may provide a significant biological advantage by facilitating metabolic recycling processes essential to the supportive role of these cells.

Paraneoplastic cerebellar degeneration is a rare neurological disorder that frequently precedes the detection of malignancy. Here, we report the case of a 60 year-old woman with locally advanced squamous cell carcinoma of the tongue who developed a subacute cerebellar syndrome associated with the presence of anti-CV2/CRMP5 antibodies in the cerebrospinal fluid, after achieving complete remission of the primary tumor and the involved cervical lymph nodes by chemoradiation. The patient's symptoms on presentation were dizziness and gait unsteadiness. On examination she showed dysarthria, nystagmus and limb and gait ataxia. The diagnosis of paraneoplastic cerebellar syndrome was made on the basis of the clinical findings and immunological testing that revealed the presence of anti-CV2/CRMP5 antibodies in the patient's cerebrospinal fluid. This syndrome, which is very rare in association with head and neck cancer, commonly precedes the detection of malignancy by a year or more and has been documented in only a few cases after completion of anticancer treatment.

Parkinson's disease (PD), a common neurodegenerative disorder characterized by progressive loss of dopaminergic neurons and their terminations in the basal ganglia, is thought to be related to genetic and environmental factors. Although the pathophysiology of PD neurodegeneration remains unclear, protein misfolding, mitochondrial abnormalities, glutamate dysfunction and/or oxidative stress have been implicated. In this study, we report that a rare T1492G variant in GLUD2, an X-linked gene encoding a glutamate dehydrogenase (a mitochondrial enzyme central to glutamate metabolism) that is expressed in brain (hGDH2), interacted significantly with age at PD onset in Caucasian populations. Individuals hemizygous for this GLUD2 coding change that results in substitution of Ala for Ser445 in the regulatory domain of hGDH2 developed PD 6-13 years earlier than did subjects with other genotypes in two independent Greek PD groups and one North American PD cohort. However, this effect was not present in female PD patients who were heterozygous for the DNA change. The variant enzyme, obtained by substitution of Ala for Ser445, showed an enhanced basal activity that was resistant to GTP inhibition but markedly sensitive to modification by estrogens. Thus, a gain-of-function rare polymorphism in hGDH2 hastens the onset of PD in hemizygous subjects, probably by damaging nigral cells through enhanced glutamate oxidative dehydrogenation. The lack of effect in female heterozygous PD patients could be related to a modification of the overactive variant enzyme by estrogens.

To investigate the possible association between essential tremor (ET) and Parkinson's disease (PD) we conducted a prospective clinical and epidemiological study in a population of similar genetic background. The first-degree relatives of 303 PD probands and 249 controls from Crete were evaluated for the presence of ET. In addition, the possible co-occurrence of ET and PD in the same family or in the same individual was investigated. Results showed that ET was present in the relatives of PD patients more often than in those of controls (OR:3.64, P < 0.001). The risk was even greater (OR: 4.48) when the affected proband had tremor-dominant or mixed PD. Female relatives and siblings of PD patients were more likely to have ET than male relatives and parents of PD patients (OR: 4.36 v/s 2.89 and 4.49 v/s 2.74, respectively). Twelve subjects had both ET and PD phenotypes. While this may have occurred by chance, a number of families were identified in which ET and PD were coinherited through the same parental line. Hence, in certain families ET and PD are genetically related probably sharing common hereditary predisposition., (2009 Movement Disorder Society.)

In all mammals, glutamate dehydrogenase (GDH), an enzyme central to the metabolism of glutamate, is encoded by a single gene (GLUD1 in humans) which is expressed widely (housekeeping). Humans and other primates also possess a second gene, GLUD2, which encodes a highly homologous GDH isoenzyme (hGDH2) expressed predominantly in retina, brain and testis. There is evidence that GLUD1 was retro-posed <23 million years ago to the X chromosome, where it gave rise to GLUD2 through random mutations and natural selection. These mutations provided the novel enzyme with unique properties thought to facilitate its function in the particular milieu of the nervous system. hGDH2, having been dissociated from GTP control (through the Gly456Ala change), is mainly regulated by rising levels of ADP/l-leucine. To achieve full-range regulation by these activators, hGDH2 needs to set its basal activity at low levels (<10% of full capacity), a property largely conferred by the evolutionary Arg443Ser change. Studies of structure/function relationships have identified residues in the regulatory domain of hGDH2 that modify basal catalytic activity and regulation. In addition, enzyme concentration and buffer ionic strength can influence basal enzyme activity. While mature hGDH1 and hGDH2 isoproteins are highly homologous, their predicted leader peptide sequences show a greater degree of divergence. Study of the subcellular sites targeted by hGDH2 in three different cultured cell lines using a GLUD2/EGFP construct revealed that hGDH2 localizes mainly to mitochondria and to a lesser extent to the endoplasmic reticulum of these cells. The implications of these findings for the potential role of this enzyme in the biology of the nervous system in health and disease are discussed.

The Roc domain of the Lrrk2 protein harbors two pathogenic mutations which cause autosomal dominant parkinsonism (R1441C and R1441G). A third putatively pathogenic variant (R1441H) has been identified in four probands of diverse ethnicity with parkinsonism. Herein we show that the R1441H substitutions lie on different haplotypes within our patients, confirming this codon as a mutational hotspot. The absence of this variant in control subjects and the presence of two other pathogenic variants at this amino acid position collectively support the contention that R1441H is a pathogenic substitution.

Mammalian glutamate dehydrogenase (GDH), an enzyme central to glutamate metabolism, is thought to localize to the mitochondrial matrix, although there are also suggestions for the extramitochondrial presence of this protein. Whereas GDH in mammals is encoded by the GLUD1 gene, humans and the great apes have, in addition, a GLUD2 gene showing a distinct expression pattern. The encoded hGDH1 and hGDH2 isoenzymes are highly homologous, but their leader sequences are more divergent. To explore their subcellular targeting, we constructed expression vectors in which hGDH1 or hGDH2 was fused with the enhanced green fluorescent protein (EGFP) and used these to transfect COS 7, HeLa, CHO, HEK293, or neuroblastoma SHSY-5Y cells. Confocal microscopy revealed GDH-EGFP fluorescence in the cytoplasm within coarse structures. Cotransfection experiments using organelle-specific markers revealed that hGDH1 or hGDH2 colocalized with the mitochondrial marker DsRed2-Mito and to a lesser extent with the endoplasmic reticulum marker DsRed2-ER. Western blots detected two GDH-EGFP specific bands: a ~90 kDa band and a ~95 kDa band associated with the mitochondria and the endoplasmic reticulum containing cytosol, respectively. Deletion of the signal sequence, while altering drastically the fluoresce distribution within the cell, prevented GDH from entering the mitochondria, with the ~90 kDa band being retained in the cytosol. In addition, the deletion eliminated the ~95 kDa band from cell lysates, thus confirming that it represents the full-length GDH. Hence, while most of the hGDHs translocate into the mitochondria (a process associated with cleavage of the signal sequence), part of the protein localizes to the endoplasmic reticulum, probably serving additional functions.

Glutamate dehydrogenase (GDH) in human exists in GLUD1 and GLUD2 gene-encoded isoforms (hGDH1 and hGDH2, respectively), differing in their regulation and tissue expression pattern. Whereas hGDH1 is subject to GTP control, hGDH2 uses for its regulation, a novel molecular mechanism not requiring GTP. This is based on the ability of hGDH2 to maintain a baseline activity of <10% of its capacity subject to full activation by rising ADP/L-leucine levels. Here we studied further the molecular mechanisms regulating hGDH2 function by creating and analyzing hGDH2 mutants harboring single amino acid substitutions in the regulatory domain (antenna, pivot helix) of the protein. Five hGDH2 mutants were obtained: two with an amino acid change (Gln441Arg, Ser445Leu) in the antenna, two (Lys450Glu, His454Tyr) in the pivot helix, and one (Ser448Pro) in the junction between the two structures. Functional analyses revealed that, while the antenna mutations increased basal enzyme activity without affecting its allosteric properties, the pivot helix mutations drastically reduced basal activity and impaired enzyme regulation. On the other hand, the Ser448Pro mutation reduced basal activity but did not alter allosteric regulation. Also, compared with wild-type hGDH2, the antenna mutants were relatively thermostable, whereas the pivot helix mutants were extremely heat labile. Hence, the present data further our understanding of the molecular mechanisms involved in the function and stability of hGDH2, an enzyme thought to be of importance for nerve tissue biology.