(Glo1) and Methylglyoxal (MG) in Behavior: Recent Advances and Mechanistic Insights

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REVIEW ARTICLE published: 19 November 2012 doi: 10.3389/fgene.2012.00250 Role of Glyoxalase 1 (Glo1) and methylglyoxal (MG) in behavior: recent advances and mechanistic insights

Margaret G. Distler 1 and Abraham A. Palmer 2,3*

1 Department of Pathology, University of Chicago, Chicago, IL, USA 2 Department of Human Genetics, University of Chicago, Chicago, IL, USA 3 Department of Psychiatry and Behavioral Neuroscience, University of Chicago, Chicago, IL, USA

Edited by: Glyoxalase 1 (GLO1) is a ubiquitous cellular enzyme that participates in the detoxification Maria Grigoroiu-Serbanescu, of methylglyoxal (MG), a cytotoxic byproduct of glycolysis that induces protein Medical University Bucharest, modification (advanced glycation end-products, AGEs), oxidative stress, and apoptosis. Romania The concentration of MG is elevated under high-glucose conditions, such as diabetes. As Reviewed by: Sabine Spijker, Research Institute such, GLO1 and MG have been implicated in the pathogenesis of diabetic complications. of Neurosciences, Netherlands Recently, findings have linked GLO1 to numerous behavioral phenotypes, including Britta Haenisch, German Center psychiatric diseases (anxiety, depression, schizophrenia, and autism) and pain. This review for Neurodegenerative highlights GLO1’s association with behavioral phenotypes, describes recent discoveries Diseases, Germany Samina Salim, University of that have elucidated the underlying mechanisms, and identifies opportunities for future Houston, USA research. *Correspondence: Abraham A. Palmer, Department of Keywords: anxiety-like behavior, pain, depression, restless-leg syndrome, GABA, CNV, advanced glycation Human Genetics, University of end-products, AGE Chicago, 920 E. 58th Street, CLSC 507D, Chicago, IL 60637,USA. e-mail: [email protected]

BACKGROUND with anxiety-like behavior (Hovatta et al., 2005; Reiner-Benaim Glyoxalase 1 (GLO1) is an enzyme in the glyoxalase sys- et al., 2007; Loos et al., 2009; Benton et al., 2011; Distler et al., tem, a metabolic pathway that detoxifies α-oxoaldehydes, par- 2012), depression (Benton et al., 2011), and neuropathic pain ticularly methylglyoxal (MG) (Thornalley, 1990, 1993, 2003a; (Jack et al., 2011, 2012; Bierhaus et al., 2012). New observa- Mannervik, 2008). MG is primarily formed by the degrada- tionsinratsusingoxidativestressmodels(Salim et al., 2010a,b) tion of the glycolytic intermediates, dihydroxyacetone phos- and the sleep-deprivation model of psychological stress (Vollert phate, and glyceraldehyde-3-phosphate (Thornalley, 1993). MG et al., 2011), both suggest role of GLO1 in the anxious pheno- levels rise under high-glucose conditions, such as diabetes type of rats (Salim et al., 2011). Additional associations have been (Brownlee, 2001). At supra-physiological levels, MG induces identified with habituation, locomotor activity, motor coordina- protein and nucleotide modification (advanced glycation end- tion, exploratory behavior, and learning and memory (Williams products, AGEs), reactive oxygen species (ROS), and apoptosis et al., 2009). In humans, genetic studies have implicated poly- (Brownlee, 2001; Thornalley, 2003b). AGEs cause dysfunction morphismsinGLO1inpanicdisorder(Politi et al., 2006), of affected proteins (Brownlee, 2001) and ligate the receptor for depression (Fujimoto et al., 2008), autism (Junaid et al., 2004; advanced glycation end-products (RAGE), triggering the produc- Barua et al., 2011), schizophrenia (Arai et al., 2010; Toyosima tion of ROS (Schleicher and Friess, 2007)andapoptosis(Loh et al., 2011), and restless legs syndrome (RLS) (Stefansson et al., et al., 2006). To combat MG’s cytotoxic effects, GLO1 enzy- 2007; Winkelmann et al., 2007, 2011; Kemlink et al., 2009). matically converts MG into the less reactive substance, d-lactate While the studies of RLS identify a haplotype block that includes (Thornalley, 2003b). In vitro, overexpression of Glo1 prevents GLO1, more attention has been paid to the neighboring gene MG accumulation (Shinohara et al., 1998); conversely, GLO1 BTBD9. The other human genetic studies utilized a candidate inhibition increases MG accumulation and decreased cellular via- gene approach and had small sample sizes; in most cases, those bility (Kuhla et al., 2006). Given their roles in AGE formation results have not been replicated. Aside from genome-wide asso- and cytotoxicity, GLO1 and MG have been implicated in dis- ciation studies (GWAS) of RLS, GLO1 has not been identified in eases where these effects are relevant to pathogenesis, such as GWASforotherpsychiatricorneurologicaltraits. diabetic complications (i.e., micro- and macro-vascular disease), Recently, two major studies have shed light on the mecha- cancer, and aging (Brownlee, 2001; Thornalley, 2003a; Ahmed nisms underlying GLO1’s behavioral effects. One identified MG and Thornalley, 2007; Morcos et al., 2008; Fleming et al., 2010; as an agonist at GABAA receptors (Distler et al., 2012), while the Thornalley and Rabbani, 2011). other identified MG modification of voltage-gated sodium chan- Surprisingly, an increasing number of studies have identified nels (Bierhaus et al., 2012). This article will focus on GLO1’s associations between GLO1 and behavioral phenotypes. In mice, behavioral correlates and the potential underlying mechanisms. several lines of evidence suggest that Glo1 expression is associated The basic biochemistry of GLO1 has been reviewed elsewhere

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(Thornalley, 1990, 1993, 2003b), as have its roles in cancer were available from public databases, including locomotor habit- (Thornalley, 2003a; Thornalley and Rabbani, 2011)anddiabetic uation in a novel environment (negative correlation), total complications (Brownlee, 2001; Ahmed and Thornalley, 2007; locomotor activity (positive correlation), motor coordination Schleicher and Friess, 2007; Jack and Wright, 2012; Rabbani and (positive correlation), rearing (positive correlation), anxiety- Thornalley, 2012). like behavior in the elevated plus maze and light-dark box tests (positive correlation), stretch-attends in tests of anxiety- GLO1’S BEHAVIORAL CORRELATES like behavior (negative correlation), and measures of learning ANXIETY and memory (Williams et al., 2009). These findings suggested Mouse genetic studies a role for Glo1 copy number in a broad range of behavioral Among the behavioral phenotypes associated with GLO1, phenotypes. anxiety-like behavior is the most commonly reported and widely The discovery of this CNV helped to explain the previously studied. In mice, numerous genetic studies have identified asso- reported differences in Glo1 expression among inbred strains ciations between Glo1 expression and anxiety-like behavior (Hovatta et al., 2005). In addition, it explained the differential (Hovatta et al., 2005; Kromer et al., 2005; Williams et al., 2009). GLO1 protein expression reported in the HAB and LAB lines: Hovatta et al. analyzed gene expression profiles in a panel of six LAB mice were subsequently shown to have the duplication, while inbred mouse strains, which were scored for anxiety-like behavior HAB mice do not (Hambsch et al., 2010). Although it is some- in the open field and light-dark box tests (Hovatta et al., 2005). what surprising that selection did not have the opposite result, They found a positive correlation between Glo1 expression and other factors, including differences in initial allelic frequencies, anxiety-like behavior. Several subsequent studies also identified linked alleles, and drift before or during inbreeding could have positive correlations between Glo1 expression and anxiety-like contributed to the fixation of the duplication in LAB but not behavior among inbred mouse strains (Reiner-Benaim et al., HAB mice. The effect of the Glo1 duplication on anxiety-like 2007; Loos et al., 2009; Benton et al., 2011). behavior was likely offset by numerous other alleles that collec- Importantly, Hovatta et al. established a causal role for Glo1 tively contributed to anxiety-like behavior in the selected lines. in anxiety-like behavior by using viral vectors to overexpress or HAB and LAB lines are known to harbor many genetic differ- knock down Glo1 in the anterior cingulate cortex (Hovatta et al., ences due to selection and inbreeding (Bunck et al., 2009; Ditzen 2005). Local Glo1 overexpression increased anxiety-like behav- et al., 2010; Czibere et al., 2011; Filiou et al., 2011; Tasan et al., ior, while local Glo1 knockdown decreased anxiety-like behavior 2011; Zhang et al., 2011). Indeed, selection studies are prone to (Hovatta et al., 2005). This experimental manipulation supported such confounding forces, especially when the selected lines are not a causal role for Glo1 in anxiety-like behavior. However, this study replicated. was limited by the use of viral vectors: expression was manipu- The discovery of the Glo1 CNV also raised the possibility that lated only in one brain region during adulthood. As such, it might Glo1 expression could simply be a marker for the presence of not have accurately modeled Glo1’s normal physiological role in the CNV rather than directly regulating anxiety-like behavior. anxiety-like behavior. Furthermore, viral vectors often produce Specifically, the CNV contained four genes, any of which could variable expression levels between animals (Kirik and Bjorklund, have affected behavioral phenotypes. Further, the CNV could 2003). have been linked to unidentified causal alleles or could have dis- Kromer et al. also identified a correlation between GLO1 and rupted neighboring genes. As such, the discovery of the CNV anxiety-like behavior in mice (Kromer et al., 2005). They selec- complicated observed associations between Glo1 expression and tively bred mice for high anxiety-like behavior (HAB) or low behavior. anxiety-like behavior (LAB) and subsequently inbred the mice to Therefore, in order to test the hypothesis that increased Glo1 generate two lines. The LAB line had higher GLO1 protein lev- expression was sufficient to replicated the behavioral differences els than the HAB line (Kromer et al., 2005). Therefore, Kromer associated with the CNV, targeted genetic manipulation of Glo1 et al. proposed that increased GLO1 was a biomarker for low copy number performed in which mice with a transgenic bacte- anxiety-like behavior, a result directionally opposite from stud- rial artificial chromosome (BAC) containing Glo1 were created to ies that used panels of inbred mouse strains (Hovatta et al., 2005; model the CNV (Distler et al., 2012). To isolate Glo1’s effect, the Reiner-Benaim et al., 2007; Loos et al., 2009; Benton et al., 2011) BAC was engineered so that only Glo1 was expressed. BAC trans- and those that used viral vectors to manipulate Glo1 expression genic mice overexpressed Glo1 and displayed increased anxiety- (Hovatta et al., 2005). In fact, the discrepancy among these stud- like behavior. This effect was observed in multiple founder lines ies was used as grounds to dispute a role for Glo1 in anxiety-like and on two genetic backgrounds: C57BL/6J (B6) and FVB/NJ behavior (Thornalley, 2006). (FVB). The effect of Glo1 overexpression on anxiety-like behav- A subsequent study identified a common copy number vari- ior was strongest in the lines with the highest copy numbers. ant (CNV) among inbred mice that caused a duplication of Unlike the viral vector studies (Hovatta et al., 2005), this study four genes, including Glo1 (Williams et al., 2009). Of the 72 demonstrated the physiological relevance of Glo1 in behavior, inbred strains examined, 23 carried the duplication. The dupli- because Glo1 was expressed under its endogenous promoter ele- cation was positively correlated with increased Glo1 expression ments, in its natural expression pattern, and throughout the (it was an expression QTL; eQTL) and increased anxiety-like animal’s life. Therefore, this study demonstrated that increased behavior (Williams et al., 2009). Increased Glo1 copy num- Glo1 copy number increased Glo1 expression and anxiety-like ber was also associated with other behavioral phenotypes that behavior.

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Human genetic studies of GLO1 and anxiety studies have not replicated this finding: there was no associa- In contrast to mouse studies, human genetic studies have not tion between SNPs in GLO1 and autism among Han Chinese identified robust associations between GLO1 and anxiety disor- patients (Wu et al., 2008), nor was there significant linkage or ders. In humans, there are two common GLO1 alleles (Kompf association between SNPs in GLO1 and autism among Finnish et al., 1975) that result from a single nucleotide polymor- patients (Rehnstrom et al., 2008). Furthermore, a family-based phism (SNP) at position 419 (Kim et al., 1995). Adenine (419A) and case-control study found that the 419A allele was more com- encodes a glutamate at amino acid 111 (111E), and cytosine mon among unaffected siblings of autistic patients, indicating (419C) encodes an alanine (111A). Politi et al. (2006)identi- that it might be protective (Sacco et al., 2007). Finally, several fied a weak, but significant increase in the risk of panic disorder human genome-wide association studies (GWAS) have failed to without agoraphobia among people carrying the 419A allele. identify associations between GLO1 polymorphisms and autism. Limitations, such as the small sample size and potential popu- At the very least, this suggests that the study of Junaid et al. over- lation stratification, made this result preliminary. A subsequent estimated the effect size of this allele, which must have been small study found no correlation between GLO1 expression and sus- enough to escape detection by larger GWAS. ceptibility to panic attacks (Eser et al., 2011), but suffered from Nevertheless, Junaid et al. suggested that the 419A GLO1 allele the same limitations, so cannot be said to disprove the origi- was associated with a functional change in GLO1 enzymatic activ- nal observation. Similarly, no published GWAS studies of panic ity. They reported that post-mortem brain tissue from autistic disorder have identified GLO1 (Erhardt et al., 2011). Despite dis- patients had reduced GLO1 enzymatic activity and increased AGE appointing findings from human genetic studies, there remains content compared to that from control patients (Junaid et al., a need for large, carefully controlled genetic studies in order 2004). In a later study, Baura et al. reported a correlation between to address whether differences in GLO1 affect anxiety among the 111E GLO1 isoform and reduced enzymatic activity (Barua humans. et al., 2011). However, Barua et al. did not directly examine enzymatic activity of the isoforms. Rather, they utilized cell extracts DEPRESSION derived from autistic and control patients. As such, differences There is strong evidence for shared genetic vulnerability to anx- in enzymatic activity and MG concentration could have resulted iety and depression (Cerda et al., 2010). Among a panel of from differences in GLO1 expression or other genetic differences. inbred mice, Benton et al. found a positive correlation between Therefore, further studies will help determine the functional GLO1 protein levels and baseline depression-like behavior using significance of the 419A/C SNP and its contribution to autism. the tail-suspension test (Benton et al., 2011), suggesting that Experimental studies in mice may help determine whether Glo1 could contribute to the common genetic etiology of anx- GLO1 is related to autism-like behaviors. Various behavioral iety and depression. However, a small human study of GLO1 assays have been proposed for measuring autism-like phenotypes and depression reported apparently conflicting data. Fujimoto in mice, including social interaction tests, ultrasonic vocalization et al. reported a negative correlation between GLO1 expres- measurements, assessment of repetitive and stereotyped behavior, sion and depression in humans (Fujimoto et al., 2008). GLO1 and reversal learning tests (Silverman et al., 2010). In future stud- mRNA expression was reduced in peripheral white blood cells ies, these assays may be used to study mutant mice with increased (WBC) of patients with major depressive and bipolar disorders or decreased Glo1 expression. (Fujimoto et al., 2008). GLO1 mRNA expression did not significantly differ between patients in remission and healthy controls, SCHIZOPHRENIA suggesting that reduced GLO1 expression was a state-dependent Recently, two studies have suggested a role for GLO1 in marker for depression (Fujimoto et al., 2008). However, this schizophrenia (Arai et al., 2010; Toyosima et al., 2011). In a study did not establish whether reduced GLO1 expression affected single schizophrenic patient, a frameshift mutation in GLO1 GLO1 protein levels or enzymatic activity, which would reflect was correlated with reduced GLO1 enzymatic activity (Toyosima the functional significance of differences in mRNA expression. et al., 2011). In a separate study, schizophrenic patients were Further, WBC mRNA may not adequately reflect mRNA expres- found to have increased AGE accumulation compared to control sion in the brain (Mehta et al., 2010). Corroborating findings subjects (Arai et al., 2010), suggesting reduced GLO1 func- from post-mortem brain samples would significantly strengthen tion. Nevertheless, small sample sizes and confounding vari- the association between GLO1 expression and depression (Mehta ables made these associations preliminary. Therefore, additional et al., 2010). Therefore, although data from mice implicate Glo1 human genetic studies and studies of GLO1 in mice will help in increasing depression-like behavior, further experiments are establish an association with schizophrenia. Several behavioral required to resolve the discrepancy with human data. assays of schizophrenia-like behavior have been proposed (Young et al., 2010), which could be used for studying mice with aberrant AUTISM Glo1 expression. Human studies have proposed an association between a cod- ing SNP in GLO1 (419A/C; rs2736654) and autism. Junaid et al. RESTLESS LEGS SYNDROME reported that the 419A allele was 1.5 times more common among Multiple genome-wide association studies have implicated a autistic patients than controls (Junaid et al., 2004). However, this haplotype block that includes GLO1 in restless legs syndrome study was limited by its small sample size and potential confound- (Stefansson et al., 2007; Winkelmann et al., 2007, 2011; Kemlink ing variables (e.g., population stratification). Indeed, subsequent et al., 2009). These studies have focused on BTBD9, another gene

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that, like GLO1, is in this largely non-recombinant haplotype, behavior in the elevated plus maze compared to vehicle-treated but this association could also reflect regulatory changes that mice (Hambsch et al., 2010). Chronic MG treatment also impact GLO1. reduced immobility in the tail suspension test, reflecting an antidepressant effect of MG (Hambsch et al., 2010). Distler et al. PAIN identified a similar anxiolytic effect of MG within minutes of Neuropathic pain is a feature of neuropathy, a common sequela a single intra-peritoneal injection (Distler et al., 2012), suggest- of diabetes (Vinik et al., 2000; Edwards et al., 2008). There is ing that MG acutely affects behavior. At high doses, acute MG an extensive literature on the relationship between Glo1 and administration was also shown to have sedative effects, including neuropathy which has been recently reviewed (Jack and Wright, locomotor depression, ataxia, hypothermia, and lethargy (Distler 2012). Recent studies have identified associations between the et al., 2012). Acute MG administration also caused hyperalgesia Glo1 CNV in mice and behavioral measures of neuropathic pain equivalent to that caused by diabetes in mice (Bierhaus et al., (Jack et al., 2011, 2012). After diabetes induction, A/J mice, 2012). Together, these studies indicated that MG concentration which carry a triplication of the Glo1 allele, were less sensi- regulates multiple behavioral phenotypes and that MG’s effects tive to mechanical pain than B6 mice, which have only a single are opposite to those of Glo1 overexpression. copy of Glo1 (Jack et al., 2011). Similarly, Glo1 copy number was associated with mechanical pain sensitivity after induction BEHAVIORAL EFFECTS OF PURE MG of diabetes in two closely related inbred strains, BALB/cJ and The behavioral experiments described above utilized crude BALB/cByJ. BALB/cByJ mice carry the Glo1 duplication, while MG preparations, which may contain contaminants, such as BALB/cJ mice do not (Williams et al., 2009). Similar to the pre- formaldehyde and methanol (Pourmotabbed and Creighton, vious report, increased levels of Glo1 protected against diabetic 1986; McLellan and Thornalley, 1992). We recently tested the hyperalgesia in BALB/cByJ mice compared to BALB/cJ mice (Jack behavioral effects of pure MG and found that it elicited similar et al., 2012). These results suggested that Glo1 mediates behav- pharmacological effects as crude MG. At a low dose, pure MG was ioral responses to pain associated with diabetic neuropathy. A anxiolytic in the open field test, and at a high dose, it caused loco- recent study has confirmed that GLO1 protects against neuro- motor depression, ataxia, and hypothermia (Distler et al., 2012). pathic pain associated with diabetes. Overexpression of human Notably, the doses of pure MG required for sedation, locomotor GLO1 reduced thermal hyperalgesia in diabetic mice (Bierhaus depression, ataxia, and hypothermia were similar to the doses of et al., 2012). Conversely, pharmacological inhibition of GLO1 crude MG, while pure MG elicited an anxiolytic effect at a lower and Glo1 knockdown exacerbated both mechanical and thermal dose than crude MG. It remains to be determined whether pure hyperalgesia in diabetic mice (Bierhaus et al., 2012). Together, MG affects other reported behavioral effects, such as depression these findings suggest a role for GLO1 in mediating behavioral and hyperalgesia. measures of pain, particularly that related to diabetic neuropathy. To date, no study has investigated the role of GLO1 in other MG’S MECHANISM OF ACTION IN BEHAVIOR: AGE FORMATION types of pain, such as nociceptive and central pain, which is an MG is best characterized as a cytotoxic agent that forms AGEs. important area of future investigation. This molecular action has been attributed to the pathogenesis of several diseases characterized by increased MG concentrations, GLO1’S MECHANISM OF ACTION IN BEHAVIOR including diabetic complications. For instance, increased MG lev- MG REGULATES BEHAVIOR els and subsequent AGE formation have been well-established There is robust evidence that GLO1 activity regulates MG con- pathogenic mechanisms in diabetes-associated microvascular centration (Thornalley, 1990, 1993, 2003b). Recent studies have disease (e.g., diabetic retinopathy) and macrovascular disease suggested this activity is critical to GLO1’s effects on behav- (e.g., atherosclerosis) (Brownlee, 2001). ior. In vitro, Glo1 overexpression prevented MG accumulation Recently, AGEs have been attributed to the development of under conditions of high glucose (Shinohara et al., 1998), and diabetic neuropathy and neuropathic pain (Jack and Wright, in C. elegans, Glo1 overexpression reduced basal MG concentra- 2012). Induction of diabetes in rats increased AGEs in the extra- tion (Morcos et al., 2008). Similarly, Glo1 overexpression reduced cellular matrix of sensory nerves (Duran-Jimenez et al., 2009). baseline MG concentration in the brains of mice (Distler et al., In particular, laminin and fibronectin were found to be mod- 2012) and plasma MG concentration in diabetic mice (Bierhaus ified by MG (Duran-Jimenez et al., 2009). Among the numer- et al., 2012). ous proteins likely modified by MG in diabetes (Rabbani and Given the strong association between GLO1 and MG con- Thornalley, 2012), a voltage-gated sodium channel, Nav1.8, was centration, it was hypothesized that GLO1 affects behavior by recently reported to be modified by MG and it was suggested that controlling levels of MG. This hypothesis suggested that direct this modification contribute to neuropathic pain (Bierhaus et al., administration of MG would alter behavior. Recent reports have 2012). Nav1.8 was MG-modified in peripheral sensory neurons demonstrated MG’s behavioral effects, including those related to of mice subjected to diabetes, MG administration, or Glo1 knock anxiety-like behavior (Hambsch et al., 2010; Distler et al., 2012), down. Knock down of Nav1.8 reduced MG-induced thermal depression (Zhang et al., 2011), motor coordination (Distler hyperalgesia. Finally, MG-modification of Nav1.8 dysregulated et al., 2012), and pain (Bierhaus et al., 2012). Hambsch et al. electrical signaling in sensory neurons. Specifically, prolonged administered MG to mice by intra-cereberoventricular injection incubation with MG (3–14 h) modified Nav1.8 and altered resting daily for 6 days; MG-treated mice displayed reduced anxiety-like membrane potential, current threshold, and voltage threshold.

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These findings suggested that AGEs promote protein dysfunction (Hambsch et al., 2010). This is characteristic of GABAA recep- under conditions of high MG concentrations, thus contributing tor activation, which decreases excitatory post-synaptic potentials to neuropathic pain. and suppresses LTP in CA1 slices (Seabrook et al., 1997). BecauseMG’sroleinAGEformationiswellcharacter- ized, it was thought that this mechanism was relevant to IMPLICATIONS OF MG’S ACTIVITY AT GABAA RECEPTORS other behavioral phenotypes, including anxiety-like behavior and MG’s role in activating GABAA receptors has important physio- depression (Hambsch, 2011). Hambsch et al. proposed that MG- logical implications. MG production increases during high gly- dependent AGE formation contributed to MG’s anxiolytic action colytic activity. MG can then be metabolized by GLO1, can form (Thornalley, 2006; Hambsch et al., 2010). They found increased AGEs or can activate GABAA receptors (Figure 1). Importantly, AGEs in the brains of mice treated chronically with MG by the effects at GABAA receptors occur at physiological concen- intracerebroventricular administration. Hambsch et al. utilized trations of MG. Similarly, changes in MG concentration that are a high dose of MG, corresponding to an average concentra- associated with diabetes can lead to changes in AGE formation at tion of 1.6 mM throughout the brain (assuming a brain volume a variety of targets, including Nav1.8, which appears to modulate of 0.45 mL). This study did not establish a causal relationship pain sensitivity. between AGE formation and anxiety-like behavior. Therefore, a One physiological function of MG’s activity at GABAA recep- role for AGE formation in anxiety-like behavior remains uncer- tors may be to maintain inhibitory tone under conditions of low tain. Further, the discovery that MG activates GABAA receptors GABA concentrations. In the synaptic cleft, the concentration of (discussed below) provides a compelling alternative mechanism GABA is in the millimolar range during synaptic release (Farrant for MG’s anxiolytic effect. and Nusser, 2005); in the extracellular space, GABA concentrations are in the submicromolar range (Vithlani et al., 2010). The MG’S MECHANISM OF ACTION IN BEHAVIOR: GABAA RECEPTOR concentration of MG in the brain is approximately 5 μM(Distler ACTIVATION et al., 2012), and these levels are likely similar in both the synapse A novel role for MG-independent of AGE formation was recently and extracellular space. Therefore, MG is less abundant than identified. MG was found to activate GABAA receptors at phys- GABA at the synapse; however, in the extracellular space MG is iological concentrations (Distler et al., 2012). In vivo,MG’s more abundant than GABA and likely acts as a partial agonist pharmacodynamic profile was similar to that of known GABAA (Distler et al., 2012). Thus, while it has yet to be well explored MG receptor agonists, which are anxiolytic at low doses and have seda- may have a complex role in the regulation of GABAergic signaling. tive effects at higher doses (Rudolph and Mohler, 2004; Carter While there are no data to directly support the possibility, et al., 2009; Kumar et al., 2009). In vitro, MG selectively acti- another physiological role for MG may be as a negative vated GABAA receptors and was characterized as a competitive regulator of excitatory signaling. Excitatory synaptic trans- partial agonist (Distler et al., 2012). There is a wealth of evi- mission is associated with increased glycolysis (Barros and dence that GABAA receptors play key roles in anxiety (Kent et al., Deitmer, 2010). In fact, 60–75% of neuronal glucose utiliza- 2002; Kalueff and Nutt, 2007). As such, MG’s activity at GABAA tion is estimated to be in glutamatergic neurons (Shulman receptors can account its anxiolytic effect. et al., 2004). Since neuronal excitation is coupled with gly- This mechanism is distinct from MG’s previously reported colysis, MG concentrations may increase in excitatory neu- cellular effects, specifically AGE formation and cytotoxicity. A rons. MG would then activate local GABAA receptors and thus low dose of MG was anxiolytic within minutes of peripheral serve as a negative feedback signal. In addition, it would also administration (Distler et al., 2012). This time course differs provide inhibitory tone to neighboring cells. Such an action from that of AGE formation, which requires hours to days (Lo could be neuroprotective, since excessive N-methyl-D-aspartate et al., 1994). Therefore, AGE formation is unlikely to mediate (NMDA) receptor activation by glutamate triggers excitotox- MG’s effect on anxiety-like behavior. MG’s anxiolytic effect was icity and apoptosis (Naegele, 2007; Henshall and Murphy, also independent of cytotoxicity. In vitro, MG concentrations of 2008). 100–1000 μM have been reported to induce neuronal cell death At the organismal level, MG concentration increases under (Di Loreto et al., 2004, 2008; Li et al., 2010). In contrast, an anx- conditions of high glucose load. Accordingly, it is possible iolytic dose of MG only increased the concentration of MG in that MG concentration may link metabolic state to neuronal the brain to approximately 6 μM(Distler et al., 2012). In addi- inhibitory tone and behavior, including anxiety. Specifically, high tion, MG requires hours to days to induce apoptosis (Di Loreto levels of glucose may be coupled to reduced anxiety through et al., 2008), which is inconsistent with the acute time course increased levels of MG. In humans, consumption of foods high in (∼10 min) of MG’s behavioral effects. Finally, there was no evi- fat and sugar is associated with emotional reward (Jacquier et al., dence of apoptosis in mice treated with an anxiolytic dose of MG 2012). In rats, fructose consumption increased levels of MG in (Distler et al., 2012). Therefore, MG’s effect at GABAA receptors rats (Wang et al., 2008), which could provide a direct link between occurs at lower concentrations and is temporally dissociable for the intake of high-sugar foods and emotional satiety. One notable its better-known effects, such as AGE formation and cytotoxic- exception to the association between increased metabolic load ity. MG’s GABAergic action may explain previous observations and reduced anxiety may be in diabetic patients, where elevated of MG’s electrophysiological effects. For example, Hambsch et al. MG level have consistently been observed (Shinohara et al., 1998; found that incubation with 10 μM MG reduced the magnitude Brownlee, 2001; Masterjohn et al., 2011). There is an increased of long-term potentiation (LTP) in hippocampal brain slices prevalence of anxiety among diabetic patients (Anderson et al.,

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FIGURE 1 | A model of the glyoxalase system’s role in GABAergic MG that is not catabolized can cross the plasma membrane, where it signaling. During glycolysis, triose phosphate intermediates can be accesses pre- or post-synaptic GABAA receptors where it causes GABAA converted to MG by non-enzymatic fragmentation. Excess MG is receptor activation, inward Cl− current, and membrane hyperpolarization, catabolized by the glyoxalase system (GLO1 and GLO2) to form d-lactate. which is hypothesized to alter behavior.

2002), whereas higher concentrations of MG would be expected McLean and Anderson, 2009). The etiology underlying differen- to decrease anxiety. Nevertheless, it remains possible that diabetic tial anxiety between males and females is not well understood, but patients experience increased anxiety in spite of increased MG it likely includes environmental and hormonal factors (McLean concentration, since diabetes is clearly associated with numerous and Anderson, 2009; Solomon and Herman, 2009; Nillni et al., physiological changes in addition to changes in MG concentra- 2011). tion and is correlated with numerous environmental and epi- demiological factors. Thus, the presence of increased anxiety in GLO1 COPY NUMBER AND ANXIETY-LIKE BEHAVIOR IN MICE diabetes patients is difficult to interpret. Another unresolved issue is that only the BAC transgenic lines with high Glo1 copy numbers, and correspondingly high Glo1 FUTURE DIRECTIONS expression, exhibited increased anxiety-like behavior (Distler GLO1’S EFFECTS IN FEMALES et al., 2012). The initial association between Glo1 copy num- Glo1’s effect on anxiety-like behavior has not been assessed in ber and anxiety-like behavior involved a duplication. In contrast, females. Virtually all of the studies that identified associations B6 and FVB BAC transgenic mice with two additional copies of between Glo1 expression and anxiety-like behavior utilized only Glo1 (equivalent to a homozygous duplication) did not signifi- male mice (Hovatta et al., 2005; Loos et al., 2009; Benton et al., cantly differ from wild-type mice in anxiety-like behavior (Distler 2011), although there are isolated exceptions (Williams et al., et al., 2012). This negative result could reflect inadequate power 2009). Studies that directly manipulated Glo1 overexpression by to detect a difference or could reflect a true lack of effect of a viral vectors (Hovatta et al., 2005) and BAC transgenes (Distler Glo1 duplication on anxiety-like behavior. The latter possibility et al., 2012) studied behavioral effects in male mice only. It could be due to differences in Glo1 expression despite equal copy would not be unprecedented if the behavioral effects of Glo1 numbers at the genomic level. Another possibility is that Glo1 are sex-specific; sex differences in anxiety have been described in overexpression has more robust anxiogenic effects on specific both humans and animal models (Palanza, 2001; Hamann, 2005; genetic backgrounds. Consistent with this explanation, Hovatta

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et al. reported that viral-vector-mediated Glo1 overexpression γ1–3, δ, ε, θ,andπ (Bollan et al., 2003). Future studies will be robustly increased anxiety-like behavior in 129S6/SvEvTac mice required to identify MG’s receptor subtype selectivity. This is rel- but not B6 mice (Hovatta et al., 2005). evant to MG’s behavioral effects, because different subunits and subunit compositions mediate different GABAergic effects. For EVOLUTIONARY SIGNIFICANCE instance, α2 subunits have been reported to mediate the anxi- The evolutionary significance of the Glo1 CNV is unknown. Glo1 olytic effects of benzodiazepines, and α5 subunits are thought CNVs are present in both laboratory and wild -caught mice, indi- to mediate their sedative effects (Rudolph and Knoflach, 2011). cating that the duplicated allele arose prior to the domestication MG’s activity at different receptor subtypes may contribute to of mice by mouse fanciers and later scientists (Williams et al., its endogenous role in the CNS. Future studies will be critical 2009). While Redon et al. (2006)reportedCNVsthatincluded in identifying the pharmacological and biophysical responses of GLO1 in humans, the underlying data for those particular CNVs specific GABAA receptor compositions to MG. are unconvincing. We are not aware of any other evidence that Future studies will also be important for assessing MG’s activ- GLO1 CNVs are common among humans or any other species; ity at synaptic and extra-synaptic GABAA receptors. Synaptic although it is possible they have simply escaped detection. GABAA receptors regulate phasic inhibitory signaling, while MG is byproduct of glycolysis that is generated in every organ- extra-synaptic GABAA receptors contribute to tonic inhibitory ism. Homologs of GLO1 exist in bacteria, fungi, plants, and signaling (Hablitz et al., 2009)andanxiety(Maguire et al., 2005). animals (Cooper, 1984; Yadav et al., 2008; Inoue et al., 2011), Future studies must directly measure MG’s activity at synaptic highlighting its ancient physiological importance. Because MG and extra-synaptic receptors in order to determine MG’s role in accumulation is cytotoxic, GLO1’s conservation is likely tied to phasic and tonic inhibitory signaling. its role in cellular survival. The extent to which MG’s action at GABAA receptors is conserved is unknown. GABA exists in bac- GLO1’S MECHANISM OF ACTION IN OTHER BEHAVIORAL PHENOTYPES teria, fungi, plants, and animals (Guthrie and Nicholson-Guthrie, As reviewed above, two major mechanisms have been ascribed to 1989; Rauh et al., 1990; Kumar and Punekar, 1997; Bouche and GLO1’s behavioral effects: regulating AGE formation and GABAA Fromm, 2004), though it is only thought to serve as a neurotrans- receptor activation. These mechanisms are proposed to mediate mitter among animals; GABAA receptors are prominent among GLO1’s role in pain and anxiety, respectively. However, the extent animals, including invertebrates (Rauh et al., 1990). Therefore, an to which each mechanism underlies the many other behavioral important future direction will be to define the evolution of the correlates of GLO1 remains an important area of future investi- relationship between MG and GABAA receptors in other species. gation. For instance, it is possible that GLO1 regulates depression ItispossiblethatMGactsatGABAA receptors in invertebrates; through its role in modulating GABAergic tone, given evidence application of 10–150 μM MG had an excitatory effect on the that deficits in the GABA system have been implicated in depres- sixth abdominal ganglion of the cockroach (Davies et al., 1986). sion (Gerner and Hare, 1981; Petty and Schlesser, 1981; Petty Although GABA is an inhibitory neurotransmitter in insects, it and Sherman, 1984; Crestani et al., 1999; Earnheart et al., 2007; is also excitatory in some cell types (Beg and Jorgensen, 2003; Shen et al., 2010; Hasler and Northoff, 2011; Luscher et al., 2011). Gisselmann et al., 2004). Similarly, the mechanism underlying GLO1’s putative involve- ment in autism remains unknown. Autism has been associated MG’S BIOPHYSICAL INTERACTION WITH GABAA RECEPTORS with high levels of MG and AGEs (Junaid et al., 2004); however, The discovery that MG activates GABAA receptors has created few studies have examined whether AGEs contribute to autism an exciting new direction of research. Specifically, studying MG’s (Boso et al., 2006). Alternatively, it is possible that GLO1’s role in biophysiocal interactions with GABAA receptors is liable to yield GABAergic signaling could contribute to autism since disruptions important results. Although MG and GABA are both small in GABAergic signaling have been identified in autism (Di Cristo, molecules, they are not structurally similar (Figure 2). Therefore, 2007; Rubenstein, 2011). Therefore, additional studies in humans it is unknown whether they share a binding site on GABAA recep- and mice are required to elucidate the mechanisms underlying tors or whether binding of one prevents binding of the other. GLO1’s associations with additional behavioral phenotypes. Structural studies are necessary to resolve this question. Similarly, GABAA receptors are pentamers comprised of var- GLO1 AS A TARGET FOR THERAPEUTIC AGENTS ious combinations of at least 16 possible subunits: α1–6, β1–3, GLO1 may be a useful target for pharmacological interven- tion. For instance, GLO1 inhibition was shown to increase MG concentration and reduce anxiety-like behavior in vivo (Distler et al., 2012). GLO1 inhibition would represent a novel mechanism of action among psychiatric drugs. Most current anxiolytic therapies target neurotransmitter systems, including the sero- tonin and GABA systems (Pollack, 2009). In contrast, GLO1 inhibitors would increase the production of MG, an endogenous GABAA receptor agonist. This could have a different and pos- sibly more favorable side-effect profile compared to agents that

FIGURE 2 | Molecular structures of GABA and MG. allosterically activate GABAA receptors. For instance, endogenous MG might have receptor subtype speciﬁcity, thus preventing

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off-target effects. Similarly, GLO1 inhibition may not increase was recently shown to exacerbate diabetic hyperalgesia (Bierhaus MG levels sufﬁciently to cause sedation or have abuse potential, et al., 2012), which could limit the use of GLO1 inhibitors, which are common concerns of current GABAA receptor agonists. especially in diabetic patients. Development of better GLO1 inhibitors and characterization of the side-effects of GLO1 inhibition are thus important goals for CONCLUSION future studies. Despite a controversial history, recent studies have demonstrated Studies of the adverse effects of GLO1 inhibition in vivo must roles for GLO1 in behavioral phenotypes, including anxiety- also pay particular attention to cytotoxicity. In vitro studies have like behavior and pain. Although animal studies support a role demonstrated that GLO1 inhibition can cause cell death (Kuhla for GLO1 in behavioral phenotypes, human genetic studies are et al., 2006). Over a short period of time, this is unlikely to less convincing. As such, the extent to which polymorphisms in occur in vivo, since GLO1 inhibition was shown to only mod- GLO1 regulate human diseases remains an important direction estly increase MG levels. Nevertheless, chronic GLO1 inhibition for future studies. MG can induce protein modiﬁcation and acts may increase MG to more toxic levels, causing AGE formation as an endogenous GABAA receptor agonist. These cellular func- or cell death. Since AGEs play a prominent role in diabetic com- tions likely contribute to MG’s behavioral effects. GLO1 inhibitors plications, GLO1 inhibitors may be a poor choice for patients may provide novel therapeutic tools for the treatment of CNS with diabetes (Brownlee, 2001). In particular, GLO1 inhibition disorders.

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Williams,R.T.,Lim,J.E.,Harr, This article was submitted to Frontiers Saha,K.,Tejada-Simon,M.V., Tasan, R. O., Bukovac, A., Peterschmitt, B.,Wing,C.,Walters,R.,Distler, in Behavioral and Psychiatric Genetics, a and Chugh, G. (2010b). Moderate Y.N.,Sartori,S.B.,Landgraf,R., M. G., et al. (2009). A common specialty of Frontiers in Genetics. treadmill exercise prevents oxidative Singewald, N., et al. (2011). Altered and unstable copy number vari- Copyright © 2012 Distler and stress-induced anxiety-like behav- GABA transmission in a mouse ant is associated with differences Palmer. This is an open-access arti- ior in rats. Behav. Brain Res. 208, model of increased trait anxiety. in Glo1 expression and anxiety- cle distributed under the terms of 545–552. Neuroscience 183, 71–80. like behavior. PLoS ONE 4:e4649. the Creative Commons Attribution Salim, S., Asghar, M., Taneja, M., Thornalley, P. J. (1990). The glyox- doi: 10.1371/journal.pone.0004649 License, which permits use, distribution Hovatta,I.,Chugh,G.,Vollert,C., alase system: new developments Winkelmann, J., Czamara, D., and reproduction in other forums, et al. (2011). Potential contribution towards functional characterization Schormair, B., Knauf, F., Schulte, provided the original authors and of oxidative stress and inflammation of a metabolic pathway fundamen- E. C., Trenkwalder, C., et al. source are credited and subject to to anxiety and hypertension. Brain tal to biological life. Biochem. J. 269, (2011). Genome-wide association any copyright notices concerning any Res. 1404, 63–71. 1–11. study identifies novel restless legs third-party graphics etc.

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