Cerebellum DOI 10.1007/s12311-014-0573-4

ORIGINAL PAPER

Glutamate and GABA-Metabolizing Enzymes in Post-mortem Cerebellum in Alzheimer’s Disease: Phosphate-Activated Glutaminase and Glutamic Acid Decarboxylase G. Sh. Burbaeva & I. S. Boksha & E. B. Tereshkina & O. K. Savushkina & T. A. Prokhorova & E. A. Vorobyeva

# Springer Science+Business Media New York 2014

Abstract Enzymes of glutamate and GABA metabolism in postmortem cerebellum from patients with Alzheimer’s disease (AD) have not been comprehensively studied. The present work reports results of original comparative study on levels of phosphate-activated glutaminase (PAG) and glutamic acid decarboxylase isoenzymes (GAD65/67) in autopsied cerebellum samples from AD patients and matched controls (13 cases in each group) as well as summarizes published evidence for altered levels of PAG and GAD65/67 in AD brain. Altered (decreased) levels of these enzymes and changes in links between amounts of these enzymes and other glutamate-metabolizing enzymes (such as glutamate dehydrogenase and glutamine synthetase-like protein) in AD cerebella suggest significantly impaired glutamate and GABA metabolism in this brain region, which was previously regarded as not substantially involved in AD pathogenesis.

Abbreviations AD Alzheimer’s disease APOE Apolipoprotein E AU Arbitrary units Aβ Amyloid beta peptide CK BB Creatine kinase BB GABA Gamma-aminobutyric acid GAD Glutamic acid decarboxylase GDH Glutamic acid dehydrogenase Glu Glutamic acid Gln Glutamine GSLP Glutamine synthetase-like protein HRP Horseradish peroxidase PAG Phosphate-activated glutaminase PFC Prefrontal cortex PMI Postmortem interval

Introduction Keywords Phosphate-activated glutaminase . Glutamic acid decarboxylase . GABA metabolism . Glutamate metabolism . Alzheimer’s disease . Autopsied cerebellum

G. S. Burbaeva : E. B. Tereshkina : O. K. Savushkina : T. A. Prokhorova : E. A. Vorobyeva Mental Health Research Center, Russian Academy of Medical Sciences, 34, Kashirskoye Shosse, Moscow, Russia 115522 I. S. Boksha (*) Laboratory of Neurochemistry, Mental Health Research Center, Russian Academy of Medical Sciences, 2-2, bldg.16, Zagorodnoe Shosse, Moscow, Russia 117152 e-mail: [email protected] I. S. Boksha e-mail: [email protected]

Glutamate (Glu) excitotoxicity—neurodegeneration, resulting from elevated Glu concentrations, associated with cascades of Ca2+ influx-induced events, including activation of phospholipases, endonucleases, and proteases—is not a single mechanism involving the Glu neurotransmitter system in Alzheimer’s disease (AD) pathogenesis [1]. Although it plays a certain crucial role, it probably occurs together with other pathological events; moreover, its reciprocal influence is known on the central players in AD pathogenesis, namely, amyloid beta peptide (Aβ) and apolipoprotein E (APOE) [2–4]. Glu metabolism is impaired in the brains of patients with AD, and this fact has been confirmed by direct measurements of Glu and glutamine (Gln) concentrations by 1Н-NMR [5]. Glu and Gln concentrations reflect the intensities of their enzymatic conversion, and levels of many key enzymes involved in the Glu/Gln cycle are altered in AD brain [6–8].

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In the present study, we focused on two enzymes, phosphate-activated glutaminase (PAG, EC3.5.1.2) and glutamic acid decarboxylase (GAD, EC 4.1.1.15). The first, PAG, is of special interest because of its multifunctionality [9–12] and the recently found link between Glu and APOE pathways [13], whereas the second, GAD, links two neurotransmitter systems (Glu and GABA), impairments in both of which are implicated in AD pathogenesis. Taking into account inconsistent data from determinations of levels of PAG and GAD isoenzymes in AD brain and the shortage of studies on these enzymes in human cerebellum, we have performed comparative measurements of GAD65/67 and PAG concentrations in postmortem cerebellum samples from AD patients and age-matched controls and evaluated the possible link with this pathology. For our study, we have chosen the cerebellum, a brain structure attracting increased interest during recent years due to the found correlation between cerebellar neurochemical alterations and clinical psychopathological symptoms in AD [14] and possible participation of the cerebellum in cognitive impairments [15]. In the present study, PAG and GAD concentrations are compared in AD cerebellum versus normal cerebellum controls. This brain structure has never been studied in this aspect, and we have also chosen it because the level of PAG in the cerebellum has been characterized as “significant” [16] and comparable with that in the prefrontal cortex (PFC), which we have investigated in our previous work [7]. Also, looking for possible correlates between levels of PAG, GAD, and other key enzymes of Glu metabolism, we have measured levels of glutamate dehydrogenase (GDH), glutamine synthetase-like protein (GSLP) as well as the energy metabolism enzyme creatine kinase BB (CK BB) in the same samples.

cocktail P8340—5 ml, with subsequent centrifugation at 1,000g, and the pellet containing nuclear fragments and cell debris was removed. The supernatants were matched in protein concentration (protein concentration was measured by the Lowry method), and SDS, 2-mercaptoethanol, and Bromophenol blue were added to the resulting samples (to final concentrations of 1, 10, and 0.004 %, respectively) with subsequent heating on a boiling water bath for 5 min. Then, the samples were subjected to Laemmli SDS-PAGE, wherein each lane was loaded with 10 μg of total protein, with subsequent enhanced chemiluminescence (ECL)-Western immunoblotting according with GE Healthcare chemicals and protocol using the following: commercially available polyclonal antibodies AB1511 (Chemicon), staining both GAD65/67 isoforms (at 1:10,000 working dilution); polyclonal rabbit antibodies to Cterminal peptide fragment of PAG (rat kidney PAG isoform), kindly provided by Prof. O.P. Ottersen and Dr. I.A. Turgner (Norway), as described in [7], (at 1:20,000 working dilution);monoclonal antibody to CK BB conjugated to horseradish peroxidase (HRP), as described in [17, 18]; polyclonal antibodies recognizing GDHI (as described in [19]);polyclonal antibodies to GSLP (as described in [7]); and secondary donkey anti-rabbit IgG H&L (HRP) (Abcam) (at 1:10,000 working dilution). Patterns of staining for CK BB, GDHI isoform, and GSLP were similar to those observed in PFC [7, 17] and are not shown here. After scanning of films exposed to chemiluminescence from nitrocellulose membranes and quantitative image processing, the amounts of PAG, GAD isoforms, GSLP, GDHI, and CK BB were evaluated and expressed in arbitrary units (AU). Amounts of all proteins were measured in triplicate. STATISTICA 6.0 (StatSoft) software (non-parametric module: Mann–Whitney U test, Spearman correlations) was used for data processing.

Materials and Methods Autopsied cerebellum cortex samples from the brain sample collection accumulated and stored at −80 °C in the Laboratory of Neurochemistry at MHRC were studied in the present work. All brain samples were obtained from the Psychiatric Moscow Hospital N1; the clinical diagnosis “Alzheimer’s disease” was confirmed by the brain slides’ inspection made in the Laboratory of Clinical Neuromorphology at MHRC (numerous Aβ deposits, neurofibrillary tangles, and typical brain atrophy are recorded in corresponding database). Thirteen cases from AD patients and 13 control samples were selected and matched for postmortem interval (4–6 h) and age (52–81 years in the control group, with median 70 years, and 57–83 years in AD group, with median 72 years). The groups contained eight women and five men in each. Tissue samples (50 mg) were homogenized in a Potter homogenizer (glass/Teflon) in 1 ml of 50 mM Tris-HCl buffer, pH 7.0, containing 0.35 M sucrose and Sigma protease

Results Phosphate-Activated Glutaminase Figure 1 demonstrates the most typical pattern of staining obtained after immunoblotting of cerebellum samples, and the diagram (Fig. 2) shows relative amounts of immunoreactive PAG (AU) in cerebellum samples from AD and control brain samples. Clearly, the majority of samples from AD brain contain less PAG than the control samples. Although the medians in the two groups are slightly different (21 AU in AD vs. 28 AU in control), the Mann–Whitney U test gives a significant between-group difference in PAG amount (U = 28.5; Z=−2.87; p=0.004). The difference in PAG amount between groups is probably due to the pathology (AD), since both groups were matched in age, postmortem interval (PMI) (p= 0.6 by Mann–Whitney U test), and numbers of men and

Cerebellum Fig. 1 Pattern of PAG staining in ECL immunoblotting. Cerebellum cortex samples: lanes 1 samples from AD cases, lanes 2 samples from controls. Molecular mass is given in the left in kilodaltons

women. Besides, the 16 male samples and 10 female samples did not differ in PAG amounts (Mann–Whitney U test, p= 0.70). Glutamic Acid Decarboxylase Figure 3 demonstrates the most typical pattern of staining obtained after electrophoresis followed by immunoblotting of cerebellum samples with subsequent treatment with antibodies recognizing both GAD65/67 isoforms. The presence of low molecular mass protein zones is seen in some samples that Fig. 2 PAG amounts in cerebellum samples from AD cases and controls as determined by ECL immunoblotting (in AU), solid symbols—samples from AD cases, open symbols–controls

are stained in immunoblots and caused by effects described in the literature [20]. However, these zones are not seen in every sample, and their link with AD is not obvious in the present study. The diagram (Fig. 4) shows the data on determination of relative amounts of immunoreactive GAD65 and GAD67 (in arbitrary units, AU) in control and AD cerebellum samples. Most of the samples from AD brain contain lesser amounts of both GAD isoforms than the controls. For both isoforms, the median levels are about 50 AU in the AD group. This level is twice lower than in controls (110 AU) for GAD67 and almost

Cerebellum Fig. 3 Pattern of GAD65/67 staining in ECL immunoblotting. Cerebellum cortex samples: lanes 1 samples from AD cases, lanes 2 samples from controls. Molecular mass is given on the left in kilodaltons

kDa 94

1 2 1 2 1 2 1 2 2 1 2 1 2 1 2 1 1 2 2 1 2

67 43

4-fold lower than in controls (190 AU) for GAD65. That is, the decrease in amount of GAD65 isoform responsible for synthesis of the neurotransmitter GABA in AD is greater than the decrease in amount of isoform GAD67 responsible for GABA basic metabolism. The between-group differences as evaluated by Mann–Whitney U test are significant: Z=−4, p=0.00004 for GAD67; Z=−3.7, p=0.0002 for GAD65. The levels of GAD67 and GAD65 are linked with positive correlation in the whole group (AD +controls): R =0.62, p=0.0006, although no correlation was found in controls or in AD group separately. Several Other Enzymes of Glu and Energy Metabolism Levels of other Glu-metabolizing enzymes—glutamic acid dehydrogenase, GDH isoform I, and glutamine synthetaselike protein, GSLP—have been also measured in the cerebellum in the present work (Fig. 5 demonstrates results of the measurements). The following positive correlations were found: between GAD67 and GDHI levels (in AD group, R=0.77, p=0.002); between GAD65 and PAG (in the whole studied group, R=0.58, p=0.002); between each GAD isoform and GSLP levels (in control group, R=0.69, p=0.01 for GAD67 and R=0.62, p=0.025 for GAD65). In the present work, the total amount of creatine phosphokinase (CK BB, energy metabolism enzyme) was measured and found to be significantly decreased in AD cerebellum samples as compared with controls (Fig. 5), (Mann–Whitney U test gives significant between-group difference Z=−3, p=0.002).

Discussion A number of neurochemical studies suggested that Glu plays a key neurotransmitter role in the cerebellum [21, 22]. High activity of the central Glu-producing enzyme, PAG, is found in mossy fiber and parallel-fiber terminals in rat cerebellum [16]. PAG is localized in neuronal mitochondria [23–25] and

participates in biosynthesis of Glu neurotransmitter (Fig. 6), i.e., it is involved in the Glu/glutamine shuttle, which is linked to neuron–glial Glu/glutamine cycling [6, 9–11]. There are brain PAG isoenzymes, such as kidney PAG and liver PAG isoforms, the latter is present in nuclear fraction and is not assigned yet to certain cell type [16]. Recently, the presence of non-neuronal (astrocytic) PAG isoform was also discovered: it is negligibly weakly recognized by antibodies to liver and kidney PAG isoforms, but has enzymatic activity; its role and regional localization in brain are not clear yet [26]. The fact that antibodies raised against C-terminal fragment of rat kidney PAG isoform recognize PAG not only in rat brain but also cross-react with PAG in human brain, has enabled comparative study of PAG level in human brain tissue [7, 27]. Although the PAG level in AD was studied by various methods in different brain structures [28–30], no studies are known devoted to PAG in human cerebellum. In the present work, we used antibodies to the C-terminus of rat kidney PAG isoform (the same antibodies as we used in [7]) to determine the amount of kidney PAG isoform in cerebellum cortex tissue from AD patients vs. controls. For this, we preliminarily separated the nuclear fraction to avoid possible interference with liver PAG isoform [16]. Demonstrated in the present work for the first time, significant decrease in level of kidney PAG—the key Glu synthesizing enzyme (Fig. 6) and the most abundant isoform predominantly involved in neurotransmitter Glu synthesis—in the cerebellum from patients with AD, may suggest for decreased PAG-mediated neurotransmitter Glu production in the cerebellum in AD, contributing to cognitive impairment and dementia (because Glu-ergic system is well-known to be responsible for cognitive functions). One cannot conclude however about bulk Glu production and concentration in AD cerebellum from the decreased kidney PAG level only. To do this, it would be necessary to involve in vivo MRI measurements of Glu level and to regard the system pathways of Glu synthesis/utilization as a whole (Fig. 6) in cerebellum tissue—these items would be addressed

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Fig. 5 GSLP, GDHI, and CK BB amounts in cerebellum samples from AD cases and controls as determined by ECL immunoblotting (in AU). Symbols: rhombus—GSLP, triangular—GDHI, circle—CK BB, solid symbols—samples from AD cases, open symbols—controls

Fig. 4 GAD65 and GAD67 amounts in cerebellum samples from AD cases and controls as determined by ECL immunoblotting (in AU), solid symbols—samples from AD cases, open symbols—controls

in other works. Moreover, in view of excitotoxicity of high bulk Glu levels and further findings of the present study (decrease in levels of GAD isoforms), one can even admit the elevated bulk Glu level in AD cerebellum. In fact, high bulk Glu concentrations are shown to decrease GAD protein content due to the activation of Ca2+-dependent proteases (as a

result of Ca2+-inflow through Glu-dependent channels and elevation of Ca2+ intracellular concentrations) and protease cleavage of GAD protein [31]. GAD is the central enzyme producing GABA via decarboxylation of Glu in the brain. Like PAG, GAD in human brain is represented by at least two isoforms, GAD65 and GAD67, with different localization and functions [32–34]. Note the presence of several isoforms for key enzymes in mammalian brain is a fundamental property of the nervous system, providing its ability to separate neurotransmission from basic metabolism, as well as maintaining the possibility to use alternative metabolic pathways [6]. Being encoded by GAD2 gene whose expression is induced under acute demand for GABA, GAD65 is mainly responsible for neurotransmission, and the enzyme is associated with synaptic membranes in nerve terminals [35]. GAD65 forms a complex with other proteins, particularly with GABA vesicular transporter, thus favoring the coupling of GABA synthesis and transport into synaptic vesicles [36], and its state cyclically alters from apoenzyme to holoenzyme according with GABA demand

Cerebellum Fig. 6 Scheme of glutamate (Glu) and GABA pathways adopted from [6]. Gln glutamine, AATc and AATm cytoplasmic and mitochondrial aspartate aminotransferases, α-KG αketoglutarate, PAG phosphateactivated glutaminase, GAD glutamic acid decarboxylase, GS glutamine synthetase, GDH glutamic acid dehydrogenase

GABA GAD Glu

PAG

PAG

Glu AATm

AATc

GDH

Gln

GDH

α-KG α-KG

Gln

Gln GS

Glu

Glu

[37, 38]. GAD67 is encoded by a constitutively expressed GAD1 gene and is more evenly distributed in cells, than GAD65. GAD67 catalyzes basic GABA formation for housekeeping neuronal functions that are not directly associated with neurotransmission, for instance, GABA can be used as an additional energy source or a trophic factor [38–40]. Knockout of GAD1 gene leads to extreme fear reactions, seizures, and epilepsy in laboratory animals, suggesting the crucial role of GAD67 for mammalian brain [41, 42]. Immunohistochemical localization of GAD65 and GAD67 in human brain (in the neocortex, hippocampus, basal ganglia, and cerebellum) has demonstrated neuronal localization of the isoenzymes: they were found in neuropil granules, axonal terminals, and in a subset of small to midsized neurons (GAD65 was preferentially associated with neuropil granules, while GAD67—with neuronal cell bodies) [43]. In rat (but not human) cerebellum, the distribution of both forms of GAD detected in most classes of GABA neurons was carefully studied and described [34]. Interestingly, a heterodimer GAD 65/67 has been revealed in rat cerebellum; although its specific cellular localization is unknown, association between GAD65 and GAD67 forms may have a physiological function [44, 45]. As judged from our data, the correlation found between GAD65 and GAD67 protein amounts suggests that there is a tendency to their coordinated synthesis in human cerebellum; however, since the correlation coefficient is not high, GAD65 and GAD67 seem present not in equimolar amounts, and hence, probably not all molecules form heterodimers in every cerebellum sample. Since disturbances of GABA-ergic system are observed in many nervous and mental disorders, many studies are known devoted to the regulation of GAD isoforms at gene and protein levels. GAD has been studied in detail particularly in AD, and, as in PAG studies, the results are mostly not related to the cerebellum, except studies using transgenic AD mouse models, wherein the authors observed increase—compared

with controls—in GAD activity in a cortex area enriched with Aβ deposits [46] and decrease in the number of hippocampal neurons immunochemically stained for GAD67 [47], but changes in GAD activity were not found in cerebellum [46], a structure free of Aβ deposits in the transgenic animals. Taking into account drastic neuronal loss, atrophy, and gliosis detected in cerebellum from patients with AD [48], the study on GAD in human cerebellum in AD seems interesting but not done before, whereas data on GAD activity and levels of GAD isoenzymes in other structures of autopsied brain in AD are known [43, 49–53]. To avoid artifacts that can influence the results of measurements of GABA and GAD levels in human autopsied brain [49, 54–56], in the present work, we matched the control and AD groups in age, PMI, and numbers of each gender representatives. In addition, taking into account different extent of association between GAD65 or GAD67 isoforms and membranes and other proteins [45], we used the protein extraction method from cerebellum tissue enabling the complete extraction of both GAD isoforms and obtaining them in the soluble state. Significant prominent decrease in both GAD isoforms in the cerebellum of patients with AD was found in the present work, wherein the amount of GAD65 isoform responsible for neurotransmitter GABA synthesis is decreased more drastically than the amount of housekeeping GAD67. This finding is in line with results of a recent paper describing a drastic decrease of GAD65 in AD brain (as determined by the same method, as we used, i.e., Western blotting) [43]. Thus, one can admit that intensity of processes associated with GABA-ergic neurotransmission as well as basic processes maintaining neurons and glia supply with GABA in AD cerebellum is significantly decreased. The decrease in amounts of both GAD isoforms revealed in our work agrees well with views on insufficiency of GABA-ergic-inhibiting system in AD [43, 57] and imbalance between excitation/

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inhibition processes as consequence. A method was proposed for GAD activity elevation by means of gene therapy: GAD “insufficiency” was proposed to compensate by means of vector construction (DNA sequence encoding GAD and thus enlarging GAD amounts) and introduction in brain of patients with epilepsy, Parkinson disease, or AD [57]. Further development of investigations in this direction is of interest and is associated with applications in clinical practice. Finally, some links found in the present study between Glu and GABA converting enzymatic systems should be noted. Besides PAG and GAD, we have measured levels of other Glu-metabolizing enzymes—glutamate dehydrogenase (GDHI) and glutamine synthetase-like protein (GSLP)—in cerebellum. The scheme on Fig. 6 shows the main Glu and GABA pathways. The correlations found between GAD67 and GDHI levels, between GAD65 and PAG, as well as between each GAD isoform and GSLP levels suggest a possible link between Glu metabolism (GDH, PAG, and GSLP) and GABA metabolism (GAD) in the cerebellum via co-regulation of these key enzymes. It would be interesting to study these links in PFC: they could be different. In fact, recent studies on GDH inhibitors [58, 59] have revealed that Glu metabolism and GABA transport in membranes prepared from cortical and cerebellar tissues are linked in different ways: in the case of cortex, GDH inhibitors suppress sodium-dependent Glu and GABA transport, but they do not inhibit sodiumdependent GABA transport in the cerebellum. Besides, the level of creatine phosphokinase (CK BB) measured in the present work in the cerebellum was found to be significantly lower in AD samples than in controls, suggesting drastic decrease in energy metabolism, which, however, is uniformly typical for all brain structures in AD [7, 17, 18]. The authors of a recent paper have revealed the link between neurochemical alterations in autopsied cerebellum cortex and AD clinical symptoms (recorded pre-mortem) [14]. They have found correlations between cerebellar monoamines’ turnover signs and agitated behavior and affective disturbances of patients with AD, the effect requiring further examination due to important, but underestimated role of cerebellum in the neurochemical pathophysiology of neuropsychiatric symptoms in AD. Thus, basing on the results obtained in present work and [14], the cerebellum, although generally not heavily burden with amyloid plaques [48], the most important hallmark of brain impairment in AD, demonstrates important disturbances in Glu, GABA, energy, and monoamines’ metabolism in AD. The cerebellum is linked functionally and anatomically with PFC [60], limbic structures, and brain nuclei, and its role in memory and learning is recognized [15, 61–65]. Taking into account this fact and substantially decreased levels of the key enzymes, we suppose that the discovered

disturbances of cerebellar metabolism can be linked (related) to AD pathogenesis. Acknowledgements The authors are indebted to Prof. O.P. Ottersen and Dr. I.A. Turgner for they kindly provided antibodies recognizing human PAG. Conflict of interest None of the authors (G. Sh. Burbaeva, I. S. Boksha, E. B. Tereshkina., O. K. Savushkina, T. A. Prokhorova, E. A. Vorobyeva) has any financial or other interests to disclose.

References 1. Fujiwara T, Morimoto K. A compound CP-31398 suppresses excitotoxicity-induced neurodegeneration. Biochem Biophys Res Commun. 2013;440(3):359–63. 2. Hoey SE, Buonocore F, Cox CJ, Hammond VJ, Perkinton MS, Williams RJ. AMPA receptor activation promotes nonamyloidogenic amyloid precursor protein processing and suppresses neuronal amyloid-β production. PLoS One. 2013;8(10):e78155. doi: 10.1371/journal.pone.0078155. 3. Wang ZC, Zhao J, Li S. Dysregulation of synaptic and extrasynaptic N-methyl-D-aspartate receptors induced by amyloid-β. Neurosci Bull. 2013;29(6):752–60. 4. Majláth Z, Toldi J, Vécsei L. The potential role of kynurenines in Alzheimer’s disease: pathomechanism and therapeutic possibilities by influencing the glutamate receptors. J Neural Transm. 2013. doi: 10.1007/s00702-013-1135-5. 5. Jones RS, Waldman AD. 1H-MRS evaluation of metabolism in Alzheimer’s disease and vascular dementia. Neurol Res. 2004;26(5):488–95. 6. Boksha IS. Coupling between neuronal and glial cells via glutamate metabolism in brain of healthy persons and patients with mental disorders. Biochemistry (Mosc). 2004;69(7):705–19. 7. Burbaeva GS, Boksha IS, Tereshkina EB, Savushkina OK, Starodubtseva LI, Turishcheva MS. Glutamate metabolizing enzymes in prefrontal cortex of Alzheimer’s disease patients. Neurochem Res. 2005;30(11):1443–51. 8. Olabarria M, Noristani HN, Verkhratsky A, Rodríguez JJ. Agedependent decrease in glutamine synthetase expression in the hippocampal astroglia of the triple transgenic Alzheimer’s disease mouse model: mechanism for deficient glutamatergic transmission? Mol Neurodegener. 2011;6:55–63. 9. Adeva MM, Souto G, Blanco N, Donapetry C. Ammonium metabolism in humans. Metabolism. 2012;61(11):1495–511. 10. Albrecht J, Norenberg MD. Glutamine: a Trojan horse in ammonia neurotoxicity. Hepatology. 2006;44(4):788–94. 11. Albrecht J, Sonnewald U, Waagepetersen HS, Schousboe A. Glutamine in the central nervous system: function and dysfunction. Front Biosci. 2007;1(12):332–43. 12. Bae N, Wang Y, Li L, Rayport S, Lubec G. Network of brain protein level changes in glutaminase deficient fetal mice. J Proteomics. 2013;27(80):236–49. 13. Dumanis SB, DiBattista AM, Miessau M, Moussa CE, Rebeck GW. APOE genotype affects the pre-synaptic compartment of glutamatergic nerve terminals. J Neurochem. 2013;124(1):4–14. 14. Vermeiren Y, Van Dam D, Aerts T, Engelborghs S, De Deyn PP. Brain region-specific monoaminergic correlates of neuropsychiatric symptoms in Alzheimer’s disease. J Alzheimer’s Dis. 2014 Mar 31. 15. O’Halloran CJ, Kinsella GJ, Storey E. The cerebellum and neuropsychological functioning: a critical review. J Clin Exp Neuropsychol. 2012;34(1):35–56.

Cerebellum 16. Holten AT, Gundersen V. Glutamine as a precursor for transmitter glutamate, aspartate and GABA in the cerebellum: a role for phosphate-activated glutaminase. J Neurochem. 2008;104(4): 1032–42. 17. Burbaeva GS, Savushkina OK, Dmitriev AD. Brain isoforms of creatine kinase in health and mental diseases: Alzheimer’s disease and schizophrenia. Vestn Ross Akad Med Nauk. 1999;1:20–4 [Russian]. 18. Burbaeva GSh, Savushkina OK, Boksha IS. Comparative study of creatine kinase BB decrease in brain of patients with Alzheimer’s disease and schizophrenia. In: Kekelidze T, Holtzman D, editors. Creatine kinase and brain energy metabolism. NATO Science Series, 342, Netherlands: IOS Press; 2003. p. 125–132. 19. Burbaeva GS, Turishcheva MS, Vorobyeva EB, Savushkina OK, Tereshkina EB, Boksha IS. Diversity of glutamate dehydrogenase in human brain. Prog Neuropsyhopharmacol Biol Psychiatry. 2002;26(3):427–35. 20. Buddhala C, Suarez M, Modi J, Prentice H, Ma Z, Tao R, et al. Calpain cleavage of brain glutamic acid decarboxylase 65 is pathological and impairs GABA neurotransmission. PLoS One. 2012;7(3): e33002. 21. Kaneko T, Itoh K, Shigemoto R, Mizuno N. Glutaminase-like immunoreactivity in the lower brain stem and cerebellum of the adult rat. Neuroscience. 1989;32(1):79–98. 22. Levi G, Gallo V. Glutamate as a putative transmitter in the cerebellum: stimulation by GABA of glutamic acid release from specific pools. J Neurochem. 1981;37(1):22–31. 23. Kvamme E, Roberg B, Torgner IA. Phosphate-activated glutaminase and mitochondrial glutamine transport in the brain. Neurochem Res. 2000;25(9–10):1407–19. 24. Kvamme E, Torgner IA, Roberg B. Kinetics and localization of brain phosphate activated glutaminase. J Neurosci Res. 2001;66(5): 951–8. 25. Roberg B, Torgner IA, Laake J, Takumi Y, Ottersen OP, Kvamme E. Properties and submitochondrial localization of pig and rat renal phosphate-activated glutaminase. Am J Physiol Cell Physiol. 2000;279(3):C648–57. 26. Kvamme E, Nissen-Meyer LS, Roberg BA, Torgner IA. Novel form of phosphate activated glutaminase in cultured astrocytes and human neuroblastoma cells, PAG in brain pathology and localization in the mitochondria. Neurochem Res. 2008;33(7):1341–5. 27. Shimmura C, Suzuki K, Iwata Y, Tsuchiya KJ, Ohno K, Matsuzaki H, et al. Enzymes in the glutamate glutamine cycle in the anterior cingulate cortex in postmortem brain of subjects with autism. Mol Autism. 2013;4(1):6–13. 28. Akiyama H, McGeer PL, Itagaki S, McGeer EG, Kaneko T. Loss of glutaminase-positive cortical neurons in Alzheimer’s disease. Neurochem Res. 1989;14(4):353–8. 29. Haug LS, Ostvold AC, Cowburn RF, Garlind A, Winblad B, Bogdanovich N, et al. Decreased inositol (1,4,5)-trisphosphate receptor levels in Alzheimer’s disease cerebral cortex: selectivity of changes and possible correlation to pathological severity. Neurodegeneration. 1996;5(2):169–76. 30. Lynn BC, Wang J, Markesbery WR, Lovell MA. Quantitative changes in the mitochondrial proteome from subjects with mild cognitive impairment, early stage and late stage Alzheimer’s disease. J Alzheimers Dis. 2010;19(1):325–39. 31. Monnerie H, Le Roux PD. Glutamate alteration of glutamic acid decarboxylase (GAD) in GABA ergic neurons: the role of cysteine proteases. Exp Neurol. 2008;213(1):145–53. 32. Turský T, Lassánová M. Inhibition of different molecular forms of brain glutamic acid decarboxylase (GAD) with ATP. J Neurochem. 1978;30(4):903–5. 33. Erlander MG, Tobin AJ. The structural and functional heterogeneity of glutamic acid decarboxylase: a review. Neurochem Res. 1991;16(3):215–26.

34. Esclapez M, Tillakaratne NJK, Kaufman DL, Tobin AJ, Houser CR. Comparative localization of two forms of glutamic acid decarboxylase and their mRNAs in rat brain supports the concept of functional differences between the forms. J Neurosci. 1994;14(3):1834–55. 35. Tian N, Petersen C, Kash S, Baekkeskov S, Copenhagen D, Nicoll R. The role of the synthetic enzyme GAD65 in the control of neuronal gamma-aminobutyric acid release. Proc Natl Acad Sci U S A. 1999;96:12911–6. 36. Wei J, Wu J-Y. Post-translational regulation of L-glutamic acid decarboxylase in the brain. Neurochem Res. 2008;33:1459–65. 37. Martin DL, Martin SB, Wu SJ, Espina N. Cofactor interactions and the regulation of glutamate decarboxylase activity. Neurochem Res. 1991;16(3):243–9. 38. Fenalti G, Law RH, Buckle AM, Langendorf C, Tuck K, Rosado CJ, et al. GABA production by glutamic acid decarboxylase is regulated by a dynamic catalytic loop. Nat Struct Mol Biol. 2007;14:280–6. 39. Kaufman DL, Houser CR, Tobin A. Two forms of the aminobutyric acid synthetic enzyme glutamate decarboxylase have distinct intraneuronal distribution and cofactor interaction. J Neurochem. 1991;56:720–3. 40. Owens D, Kriegstein A. Is there more to GABA than synaptic inhibition? Nat Rev Neurosci. 2002;3:715–27. 41. Asada H, Kawamura Y, Maruyama K. Mice lacking the 65 kDa isoform of glutamic acid decarboxylase (GAD65) maintain normal levels of GAD67 and GABA in their brains but are susceptible to seizures. Biochem Biophys Res Commun. 1996;229:891–5. 42. Asada H, Kawamura Y, Maruyama K. Cleft palate and decreased brain gamma-aminobutyric acid in mice lacking the 67-kDa isoform of glutamic acid decarboxylase. Proc Natl Acad Sci U S A. 1997;94(12):6496–9. 43. Schwab C, Yu S, Wong W, McGeer EG, McGeer PL. GAD65, GAD67, and GABAT immunostaining in human brain and apparent GAD65 loss in Alzheimer’s disease. J Alzheimers Dis. 2013;33(4):1073–88. 44. Dirkx Jr R, Thomas A, Li L, Lernmark A, Sherwin RS, De Camilli P, et al. Targeting of the 67-kDa isoform of glutamic acid decarboxylase to intracellular organelles is mediated by its interaction with the NH2terminal region of the 65-kDa isoform of glutamic acid decarboxylase. J Biol Chem. 1995;270:2241–6. 45. Kanaani J, Lissin D, Kash SF, Baekkeskov S. The hydrophilic isoform of glutamate decarboxylase, GAD67, is targeted to membranes and nerve terminals independent of dimerization with the hydrophobic membrane-anchored isoform, GAD65. J Biol Chem. 1999;274(52):37200–9. 46. Mitew S, Kirkcaldie MT, Dickson TC, Vickers JC. Altered synapses and gliotransmission in Alzheimer’s disease and AD model mice. Neurobiol Aging. 2013;34(10):2341–51. 47. Krantic S, Isorce N, Mechawar N, Davoli MA, Vignault E, Albuquerque M, et al. Hippocampal GABAergic neurons are susceptible to amyloid-β toxicity in vitro and are decreased in number in the Alzheimer’s disease TgCRND8 mouse model. J Alzheimers Dis. 2012;29(2):293–308. 48. Sjöbeck M, Englund E. Alzheimer’s disease and the cerebellum: a morphologic study on neuronal and glial changes. Dement Geriatr Cogn Disord. 2001;12(3):211–8. 49. Bowen DM, White P, Flack RH, Smith CB, Davison AN. Braindecarboxylase activities as indices of pathological change in senile dementia. Lancet. 1974;1(7869):1247–9. 50. Reinikainen KJ, Paljärvi L, Huuskonen M, Soininen H, Laakso M, Riekkinen PJ. A postmortem study of noradrenergic, serotonergic and GABAergic neurons in Alzheimer’s disease. J Neurol Sci. 1988;84(1):101–16. 51. Boissière F, Faucheux B, Duyckaerts C, Hauw JJ, Agid Y, Hirsch EC. Striatal expression of glutamic acid decarboxylase gene in Alzheimer’s disease. J Neurochem. 1988;71(2):767. 52. Perry EK, Gibson PH, Blessed G, Perry RH, Tomlinson BE. Neurotransmitter enzyme abnormalities in senile dementia. Choline

Cerebellum

53.

54.

55.

56.

57. 58.

59.

acetyltransferase and glutamic acid decarboxylase activities in necropsy brain tissue. J Neurol Sci. 1977;34(2):247–65. Gluck MR, Thomas RG, Davis KL, Haroutunian V. Implications for altered glutamate and GABA metabolism in the dorsolateral prefrontal cortex of aged schizophrenic patients. Am J Psychiatry. 2002;159(7):1165–11673. Yates CM, Butterworth J, Tennant MC, Gordon A. Enzyme activities in relation to pH and lactate in postmortem brain in Alzheimer-type and other dementias. J Neurochem. 1990;55(5):1624–30. Monfort JC, Javoy-Agid F, Hauw JJ, Dubois B, Agid Y. Brain glutamate decarboxylase in Parkinson’s disease with particular reference to a premortem severity index. Brain. 1985;108(2):301–13. Simpson MD, Cross AJ, Slater P, Deakin JF. Loss of cortical GABA uptake sites in Alzheimer’s disease. J Neural Transm. 1988;71(3): 219–26. Bland R, Fitzsimons H. Novel glutamic acid decarboxylase (GAD) chimera and methods of use. USA Patent US8071563 B2. 2009 Whitelaw BS, Robinson MB. Inhibitors of glutamate dehydrogenase block sodium-dependent glutamate uptake in rat brain membranes. Front Endocrinol (Lausanne). 2013;4:123. Bauer DE, Jackson JG, Genda EN, Montoya MM, Yudkoff M, Robinson MB. The glutamate transporter, GLAST, participates in a

60. 61.

62.

63.

64.

65.

macromolecular complex that supports glutamate metabolism. Neurochem Int. 2012;61(4):566–74. Middleton FA, Strick PL. Cerebellar projections to the prefrontal cortex of the primate. J Neurosci. 2001;21(2):700–12. Schmahmann JD. The role of the cerebellum in cognition and emotion: personal reflections since 1982 on the dysmetria of thought hypothesis, and its historical evolution from theory to therapy. Neuropsychol Rev. 2010;20(3):236–60. Timmann D, Daum I. Cerebellar contributions to cognitive functions: a progress report after two decades of research. Cerebellum. 2007;6(3):159–62. Arnsten AF, Rubia K. Neurobiological circuits regulating attention, cognitive control, motivation, and emotion: disruptions in neurodevelopmental psychiatric disorders. J Am Acad Child Adolesc Psychiatry. 2012;51(4):356–67. Konarski JZ, McIntyre RS, Grupp LA, Kennedy SH. Is the cerebellum relevant in the circuitry of neuropsychiatric disorders? J Psychiatry Neurosci. 2005;30(3):178–86. Chen J, Cohen ML, Lerner AJ, Yang Y, Herrup K. DNA damage and cell cycle event simplicate cerebellar dentate nucleus neurons as targets of Alzheimer’s disease. Mol Neurodegener. 2010;20(5): 60–71.