Screening for New Inhibitors of Glycine Transporter 1 and 2 by Means of MS Binding Assays.

A straightforward screening of a compound library comprising 2439 substances for the identification of new inhibitors for the neurotransmitter transporters GlyT1 and GlyT2 is described. Screening and full-scale competition experiments were performed using recently developed GlyT1 and GlyT2 MS Binding Assays. That way for both targets, GlyT1 and GlyT2, ligands were identified, which exhibited affinities (pKi values) in the low micromolar to sub-micromolar range. The majority of these binders exhibit new chemical scaffolds in the class of GlyT1 and GlyT2 inhibitors, which could be of interest for the development of new ligands with improved affinities for the target proteins. Additionally, compounds with excellent fluorescent properties were found for GlyT2, which renders them promising compounds for future fluorescence-based techniques. All in all, this study demonstrates that MS Binding Assays represent a powerful technology platform also well suited for the screening of compound libraries in a highly reliable and effective manner.

Risk factors for vitamin D deficiency in sickle cell disease.

Vitamin D deficiency (VDD), 25-OHD levels <20 ng/ml, is prevalent among patients with sickle cell disease (SCD) and is linked to acute and chronic pain and bone fracture in this population. There is limited literature regarding VDD-associated risk factors for SCD. We examined potential clinical and genomic parameters associated with VDD in 335 adults with SCD in a cross-sectional study. VDD was present in 65% of adult SCD patients, and 25-OHD levels independently and positively correlated with older age (P < 0·001) and vitamin D supplementation (P < 0·001). 25-OHD levels were higher in SCD patients over 40 years of age compared to the general African-American population. Both lower 25-OHD levels and increased pain frequency were associated with increased expression of SLC6A5 encoding glycine transporter-2 (GlyT2), a protein involved in neuronal pain pathways. Lower 25-OHD levels were also associated with increased expression of CYP3A4, and with decreased expression of GC (also termed DBP) and VDR, three genes involved in vitamin D metabolism. We conclude that vitamin D supplementation should be an almost universal feature of the care of young adults with SCD, and that further research is warranted into genomic factors that regulate vitamin D metabolism in SCD.

Physiology and anatomy of neurons in the medial superior olive of the mouse.

In mammals with good low-frequency hearing, the medial superior olive (MSO) computes sound location by comparing differences in the arrival time of a sound at each ear, called interaural time disparities (ITDs). Low-frequency sounds are not reflected by the head, and therefore level differences and spectral cues are minimal or absent, leaving ITDs as the only cue for sound localization. Although mammals with high-frequency hearing and small heads (e.g., bats, mice) barely experience ITDs, the MSO is still present in these animals. Yet, aside from studies in specialized bats, in which the MSO appears to serve functions other than ITD processing, it has not been studied in small mammals that do not hear low frequencies. Here we describe neurons in the mouse brain stem that share prominent anatomical, morphological, and physiological properties with the MSO in species known to use ITDs for sound localization. However, these neurons also deviate in some important aspects from the typical MSO, including a less refined arrangement of cell bodies, dendrites, and synaptic inputs. In vitro, the vast majority of neurons exhibited a single, onset action potential in response to suprathreshold depolarization. This spiking pattern is typical of MSO neurons in other species and is generated from a complement of Kv1, Kv3, and IH currents. In vivo, mouse MSO neurons show bilateral excitatory and inhibitory tuning as well as an improvement in temporal acuity of spiking during bilateral acoustic stimulation. The combination of classical MSO features like those observed in gerbils with more unique features similar to those observed in bats and opossums make the mouse MSO an interesting model for exploiting genetic tools to test hypotheses about the molecular mechanisms and evolution of ITD processing.

GlyT1 Inhibitor NFPS Exerts Neuroprotection via GlyR Alpha1 Subunit in the Rat Model of Transient Focal Cerebral Ischaemia and Reperfusion.

BACKGROUND/AIMS: Glycine is a strychnine-sensitive inhibitory neurotransmitter in the central nervous system (CNS), especially in the spinal cord, brainstem, and retina. The objective of the present study was to investigate the potential neuroprotective effects of GlyT1 inhibitor N [3-(4'-fluorophenyl)-3-(4'-phenylphenoxy) propyl] sarcosine (NFPS) in the rat model of experimental stroke. METHODS: In vivo ischaemia was induced by transient middle cerebral artery occlusion (tMCAO). The methods of Western Blotting, Nissl Staining and Morris water maze methods were applied to analyze the anti-ischaemia mechanism. RESULTS: The results showed that high dose of NFPS (H-NFPS) significantly reduced infarct volume, neuronal injury and the expression of cleaved caspase-3, enhanced Bcl-2/Bax, and improved spatial learning deficits which were administered three hours after transient middle cerebral artery occlusion (tMCAO) induction in rats, while, low dose of NFPS (L-NFPS) exacerbated the injury of ischaemia. These findings suggested that low and high dose of NFPS produced opposite effects. Importantly, it was demonstrated that H-NFPS-dependent neuronal protection was inverted by salicylate (Sal), a specific GlyR x0251;1 antagonist. Such effects could probably be attributed to the enhanced glycine level in both synaptic and extrasynaptic clefts and the subsequently altered extrasynaptic GlyRs and their subtypes. CONCLUSIONS: These data imply that GlyT1 inhibitor NFPS may be a novel target for clinical treatment of transient focal cerebral ischaemia and reperfusion which are associated with altered GlyR alpha 1 subunits.

Comparative evaluation of two glycine transporter 1 radiotracers [11C]GSK931145 and [18F]MK-6577 in baboons.

Glycine transporter type-1 (GlyT1) has been proposed as a target for drug development for schizophrenia. PET imaging with a GlyT1 specific radiotracer will allow for the measurement of target occupancy of GlyT1 inhibitors, and for in vivo investigation of GlyT1 alterations in schizophrenia. We conducted a comparative evaluation of two GlyT1 radiotracers, [(11) C]GSK931145, and [(18) F]MK-6577, in baboons. Two baboons were imaged with [(11) C]GSK931145 and [(18) F]MK-6577. Blocking studies with GSK931145 (0.3 or 0.2 mg/kg) were conducted to determine the level of tracer specific binding. [(11) C]GSK931145 and [(18) F]MK-6577 were synthesized in good yield and high specific activity. Moderately fast metabolism was observed for both tracers, with ∼ 30% of parent at 30 min post-injection. In the brain, both radiotracers showed good uptake and distribution profiles consistent with regional GlyT1 densities. [(18) F]MK-6577 displayed higher uptake and faster kinetics than [(11) C]GSK931145. Time activity curves were well described by the two-tissue compartment model. Regional volume of distribution (VT ) values were higher for [(18) F]MK-6577 than [(11) C]GSK931145. Pretreatment with GSK931145 reduced tracer uptake to a homogeneous level throughout the brain, indicating in vivo binding specificity and lack of a reference region for both radiotracers. Linear regression analysis of VT estimates between tracers indicated higher specific binding for [(18) F]MK-6577 than [(11) C]GSK931145, consistent with higher regional binding potential (BPND ) values of [(18) F]MK-6577 calculated using VT from the baseline scans and non-displaceable distribution volume (VND ) derived from blocking studies. [(18) F]MK-6577 appears to be a superior radiotracer with higher brain uptake, faster kinetics, and higher specific binding signals than [(11) C]GSK931145.

Distribution of glutamatergic, GABAergic, and glycinergic neurons in the auditory pathways of macaque monkeys.

Macaque monkeys use complex communication calls and are regarded as a model for studying the coding and decoding of complex sound in the auditory system. However, little is known about the distribution of excitatory and inhibitory neurons in the auditory system of macaque monkeys. In this study, we examined the overall distribution of cell bodies that expressed mRNAs for VGLUT1, and VGLUT2 (markers for glutamatergic neurons), GAD67 (a marker for GABAergic neurons), and GLYT2 (a marker for glycinergic neurons) in the auditory system of the Japanese macaque. In addition, we performed immunohistochemistry for VGLUT1, VGLUT2, and GAD67 in order to compare the distribution of proteins and mRNAs. We found that most of the excitatory neurons in the auditory brainstem expressed VGLUT2. In contrast, the expression of VGLUT1 mRNA was restricted to the auditory cortex (AC), periolivary nuclei, and cochlear nuclei (CN). The co-expression of GAD67 and GLYT2 mRNAs was common in the ventral nucleus of the lateral lemniscus (VNLL), CN, and superior olivary complex except for the medial nucleus of the trapezoid body, which expressed GLYT2 alone. In contrast, the dorsal nucleus of the lateral lemniscus, inferior colliculus, thalamus, and AC expressed GAD67 alone. The absence of co-expression of VGLUT1 and VGLUT2 in the medial geniculate, medial superior olive, and VNLL suggests that synaptic responses in the target neurons of these nuclei may be different between rodents and macaque monkeys.

Feedback in the brainstem: an excitatory disynaptic pathway for control of whisking.

Sensorimotor processing relies on hierarchical neuronal circuits to mediate sensory-driven behaviors. In the mouse vibrissa system, trigeminal brainstem circuits are thought to mediate the first stage of vibrissa scanning control via sensory feedback that provides reflexive protraction in response to stimulation. However, these circuits are not well defined. Here we describe a complete disynaptic sensory receptor-to-muscle circuit for positive feedback in vibrissa movement. We identified a novel region of trigeminal brainstem, spinal trigeminal nucleus pars muralis, which contains a class of vGluT2+ excitatory projection neurons involved in vibrissa motor control. Complementary single- and dual-labeling with traditional and virus tracers demonstrate that these neurons both receive primary inputs from vibrissa sensory afferent fibers and send monosynaptic connections to facial nucleus motoneurons that directly innervate vibrissa musculature. These anatomical results suggest a general role of disynaptic architecture in fast positive feedback for motor output that drives active sensation.

Quantification of the glycine transporter 1 in rhesus monkey brain using [18F]MK-6577 and a model-based input function.

BACKGROUND: Glycine transporter 1 (GlyT1) inhibitors have emerged as potential treatments for schizophrenia due to their potentiation of NMDA receptor activity by modulating the local concentrations of the NMDA co-agonist glycine. [18F]MK-6577 is a potent and selective GlyT1 inhibitor PET tracer. Although differences in ligand kinetics can be expected between non-human primates and humans, the tracer pre-clinical evaluation can provide valuable information supporting protocol design and quantification in the clinical space. The main objective of this work was to evaluate the in vivo kinetics of [18F]MK-6577 in rhesus monkey brain. Additionally, a method for estimating the tracer input function from the tracer brain tissue kinetics and venous sampling was validated. This technique was applied for determination of the dose-occupancy relationship of a GlyT1 inhibitor in monkey brain. METHODS: Compartmental and Logan graphical analysis were utilized for quantification of the [18F]MK-6577 binding using the measured tracer arterial input function. The stability of the tracer volume of distribution relative to scan length was assessed. The proposed model-based input function method takes advantage of the agreement between the tracer concentration in arterial and venous plasma from ~5 min. The approach estimates the initial peak of the input curve by adding a gamma like function term to the measured venous curve. The parameters of the model function were estimated by simultaneously fitting several brain time activity curves to a compartmental model. RESULTS: Good agreement was found between the model-based and the measured arterial plasma curve and the corresponding distribution volumes. The Logan analysis was the preferred method of analysis providing reliable and stable volume of distribution and occupancy results using a 90 and possibly 60 min scan length. CONCLUSION: The model-based input function method and Logan analysis are well suited for quantification of [18F]MK-6577 binding and GlyT1 occupancy in monkey brain.

The synthesis and preclinical evaluation in rhesus monkey of [¹8F]MK-6577 and [¹¹C]CMPyPB glycine transporter 1 positron emission tomography radiotracers.

Two positron emission tomography radiotracers for the glycine transporter 1 (GlyT1) are reported here. Each radiotracer is a propylsulfonamide-containing benzamide and was labeled with either carbon-11 or fluorine-18. [¹¹C]CMPyPB was synthesized by the alkylation of a 3-hydroxypyridine precursor using [¹¹C]MeI, and [¹8F]MK-6577 was synthesized by a nucleophilic aromatic substitution reaction using a 2-chloropyridine precursor. Each tracer shows good uptake into rhesus monkey brain with the expected distribution of highest uptake in the pons, thalamus, and cerebellum and lower uptake in the striatum and gray matter of the frontal cortex. In vivo blockade and chase studies of [¹8F]MK-6577 showed a large specific signal and reversible binding. In vitro autoradiographic studies with [¹8F]MK-6577 showed a large specific signal in both rhesus monkey and human brain slices and a distribution consistent with the in vivo results and those reported in the literature. In vivo metabolism studies in rhesus monkeys demonstrated that only more-polar metabolites are formed for each tracer. Of these two tracers, [¹8F]MK-6577 was more extensively characterized and is a promising clinical positron emission tomography tracer for imaging GlyT1 and for measuring GlyT1 occupancy of therapeutic compounds.

Adenoviral-mediated Cre expression effectively suppresses GlyT1 binding in the thalamic area of GlyT1 conditional knock-out mice.

To properly understand the function of genes of neurological interest, in vivo manipulation in the adult is essential, particularly when the target gene is involved in brain development. Moreover, since the physiological effects of target protein may be region-specific, targeting a distinct brain region could be required to dissect these effects in specific brain locations. Infection of somatic tissues of transgenic mice bearing loxP-flanked gene sequences with a viral vector expressing Cre recombinase provides a means of allowing flexible spatio-temporal control of target gene expression. Viral vector-mediated Cre expression could be used to mediate localized gene modulation in a specific brain region. In the present study this technology was applied to the glycine transporter type-1 (GlyT1) protein which is responsible for the uptake of synaptic glycine in the forebrain and has been implicated as a therapeutic target for the treatment of schizophrenia. Since GlyT1 is widely expressed in glial cells, we employed an adenoviral-based vector (Ad5) to deliver Cre protein, due to the preferentially transduction of glial cells by adenoviral vectors in rodent brain. We show significant reduced GlyT1 binding specifically in the thalamic area of conditional GlyT1 (GlyT1c) transgenic mice injected with Ad5-Cre virus, as measured by GlyT1 autoradiography. In conclusion, we demonstrated the validity of viral vector-mediated delivery of Cre to loxP targeted transgenic mice as a novel strategy to investigate target gene function in selected subregions of the adult brain, which provides a valuable technique to investigate gene function both in normal physiology and in disease models.

Differential distribution of activated spinal neurons containing glycine and/or GABA and expressing c-fos in acute and chronic pain models.

The inhibitory transmitters GABA and glycine play an important role in modulating pain transmission, both in normal and in pathological situations. In the present study we have combined in situ hybridization for identifying spinal neurons that use the transmitter(s) glycine and/or GABA (Gly/GABA neurons) with immunohistochemistry for c-fos, a marker for neuronal activation. This procedure was used with acute pain models induced by the injection of capsaicin or formalin; and chronic pain models using Complete Freund's Adjuvant (CFA, chronic inflammation), and the spared nerve injury (SNI) model (neuropathic pain). In all models Gly/GABA neurons were activated as indicated by their expression of c-fos. The pattern of Gly/GABA neuronal activation was different for every model, both anatomically and quantitatively. However, the averaged percentage of activated neurons that were Gly/GABA in the chronic phase (≥20h survival, 46%) was significantly higher than in the acute phase (≤2h survival, 34%). In addition, the total numbers of activated Gly/GABA neurons were similar in both phases, showing that the activation of non-Gly/GABA (presumed excitatory) neurons in the chronic phase decreased. Finally, morphine application equally decreased the total number of activated neurons and activated Gly/GABA neurons. This showed that morphine did not specifically activate Gly/GABA neurons to achieve nociceptive inhibition. The present study shows an increased activity of Gly/GABA neurons in acute and chronic models. This mechanism, together with mechanisms that antagonize the effects of GABA and glycine at the receptor level, may determine the sensitivity of our pain system during health and disease.

The glutamatergic compounds sarcosine and N-acetylcysteine ameliorate prepulse inhibition deficits in metabotropic glutamate 5 receptor knockout mice.

RATIONALE: Mice lacking metabotropic glutamate receptors 5 (mGluR5) exhibit reduced glutamatergic function and behavioral abnormalities, including deficits in prepulse inhibition (PPI) of the startle response that may be relevant to schizophrenia. Thus, these mice are an animal model that may be used for preclinical evaluation of potentially new classes of antipsychotic compounds. Recent clinical studies have suggested several compounds that modulate glutamatergic transmission through distinct mechanisms, such as potentiation of the N-methyl-D: -aspartate (NMDA) receptor glycine site, activation of group II mGluR, and activation of glutamate-cysteine antiporters, as being efficacious in the treatment of schizophrenia. OBJECTIVES: The aim of this work is to evaluate the effects of sarcosine (a selective inhibitor of the glycine transporter 1 [GlyT1]), LY379268 (a group II mGluR agonist), and N-acetylcysteine (a cysteine prodrug that indirectly activates cystine-glutamate antiporters to increase glutamate levels in the extrasynaptic space) on PPI deficits in mGluR5 knockout mice. RESULTS: Sarcosine and N-acetylcysteine, but not LY379268, ameliorated PPI deficits in mGluR5 knockout mice. The ability of N-acetylcysteine to restore PPI deficits was not blocked by the group II mGluR antagonist LY341495, indicating that the effects of N-acetylcysteine were not attributable to activation of group II mGluRs by glutamate. CONCLUSIONS: These findings provide evidence that the interactions between mGluR5 and NMDA receptors are involved in the regulation of PPI and suggest that activation of glutamate receptors, other than group II receptors, by increased endogenous glutamate transmission, may ameliorate the behavioral abnormalities associated with mGluR5 deficiency.

[Molecular bases of hereditary hyperekplexia]. / Bases moleculares de la hiperplexia hereditaria.

INTRODUCTION: Hereditary hyperekplexia is a rare clinical syndrome typically characterized by sudden and generalized startle in response to trivial but unexpected tactile or acoustic stimulations. Typically it is accompanied by a temporally but complete muscular rigidly, and usually it manifests shortly after birth. Some affected infants die suddenly from lapses in cardiorespiratory function. Mental development usually is normal. AIM: To summarize and update the molecular bases underlying the hereditary hyperekplexia syndrome. DEVELOPMENT: Approximately 30% of the individuals suffering hereditary hyperekplexia show mutations on a gene located on chromosome 5q32 with a dominant or recessive trait. This gene encodes the alpha subunit of the strychnine-sensitive glycine receptor, which plays a crucial role in inhibitory glycinergic neurotransmission that process sensory and motor information. About 70% of the patients with hyperekplexia do not show genetic defects in the glycine receptor gene; this suggested that additional genes might be affected in this disease. Recent studies have reveals that mutations in the neuronal glycine transporter GLYT2 are a second major cause of hyperekplexia. CONCLUSIONS: Hereditary hyperekplexia is a complex genetic disease in which several genes can be implicated, all of them directly or indirectly involved in inhibitory glycinergic neurotransmission. Two major proteins involved in hyperekplexia are the strychnine-sensitive glycine receptor (GlyR) and the neuronal glycine transporter GLYT2. Implication of secondary additional accompanying or interacting proteins in glycinergic terminals are not ruled out.

Glycine transporters and synaptic function.

Glycine is an inhibitory neurotransmitter that is mainly active in the caudal areas of the CNS. However, glycine also participates in excitatory neurotransmission since it is a co-agonist of the NMDA subtype of glutamate receptors. The concentration of glycine at synapses is mainly controlled by two sodium and chloride dependent transporters, GLYT1 and GLYT2, proteins that display a complementary distribution and activity in the nervous system. Our understanding of the physiological role of these transporters has advanced recently, thanks to the development of specific inhibitors and the generation of mice defective in the corresponding genes. In addition, the three-dimensional resolution of the structure of a bacterial homologue has shed light on the mechanisms of glycine transport. It is likely that this knowledge will prove to be useful for the development of drugs with antipsychotic, procognitive or analgesic properties.

Inhibition of the glycine transporter GlyT-1 potentiates the effect of risperidone, but not clozapine, on glutamatergic transmission in the rat medial prefrontal cortex.

Clinical studies suggest that the efficacy of the atypical antipsychotic drug (APD) risperidone (but not clozapine) can be augmented by adjunctive treatment with agonists at the glycine site of the N-methyl-D-aspartate (NMDA) receptor. By using intracellular recording, we have investigated the effect of the glycine transporter-1 (GlyT-1) inhibitor N [3-(4'-fluorophenyl)-3-(4'phenylphenylphenoxy) propyl] sarcosine (NFPS) on NMDA-induced currents in pyramidal cells of the medial prefrontal cortex (mPFC), both when given alone and in combination with either risperidone or clozapine. Both risperidone and clozapine enhanced the NMDA-induced currents. The concentration-response curves were biphasic, and the maximal effect of clozapine on the NMDA-induced currents was significantly larger than the maximal effect of risperidone. NFPS also significantly potentiated the NMDA-induced currents, when given alone. Moreover, NFPS (1 microM) augmented the effect of both the maximal (20 nM), and a submaximal (10 nM), concentration of risperidone. In contrast, NFPS did not potentiate either the effect of the maximal (100 nM) or a submaximal (80 nM) concentration of clozapine on the NMDA-induced currents. These data may explain the beneficial clinical results of using glycine reuptake antagonists as adjuvant treatment to risperidone. Our findings also suggest that risperidone and clozapine may affect NMDA receptor-mediated neurotransmission differently in the mPFC.

Glutamate-based therapeutic approaches: inhibitors of glycine transport.

A growing body of evidence suggests that activation of the glutamatergic system, particularly N-methyl-D-aspartate (NMDA) receptor function, may be a viable approach to the treatment of schizophrenia, and potentially other cognitive disorders. The excitotoxicity associated with direct NMDA receptor agonists limits their therapeutic potential, and the glycine modulatory site of the NMDA receptor has received growing interest as a therapeutic target. One approach to enhance NMDA receptor function is to increase the availability of the necessary co-agonist glycine at this modulatory site through inhibition of glycine reuptake from the synapse via glycine transporter-1 (GlyT1). Both preclinical and clinical evidence provide support for this approach, as do recent findings demonstrating the regulation of dopaminergic neurotransmission by GlyT1 inhibition. As a result, several groups have focused on the development of novel GlyT1 inhibitors. In addition, recent electrophysiological findings and data from transgenic mouse models suggest that GlyT1 might also play a role in terminating the actions of glycine at strychnine-sensitive glycine receptors, and therefore GlyT1 antagonists also have potential for the treatment of conditions where activation of inhibitory pathways in the central nervous system might be beneficial.

Glutamate release induced by activation of glycine and GABA transporters in spinal cord is enhanced in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a progressive and fatal neurodegenerative disease, involving both upper and lower motor neurons, the cause of which is obscure, although glutamate (GLU)-induced excitotoxicity has been suggested to play a major role. We studied the release of [3H]d-aspartate ([3H]d-ASP) and endogenous glutamate evoked by glycine (GLY) or GABA from spinal cord synaptosomes in mice expressing a mutant form of human SOD1 with a Gly93Ala substitution ([SOD1-G93A(+)]), a transgenic model of amyotrophic lateral sclerosis, in mice expressing the non-mutated form of human SOD1 [SOD1+], and in non-transgenic littermates [SOD1(-)/G93A(-)]. In parallel experiments, we also studied the release of [3H]GABA evoked by GLY and that of [3H]GLY evoked by GABA. Mutant mice were killed at advanced phase of pathology or during the pre-symptomatic period. In SOD1(-)/G93A(-) or SOD1(+) mice GLY evoked [3H]d-ASP and [3H]GABA release, while GABA caused [3H]d-ASP, but not [3H]GLY, release. The GLY-evoked release of [3H]d-ASP, but not that of [3H]GABA, and the GABA-evoked [3H]d-ASP release, but not that of [3H]GLY, were more pronounced in SOD1-G93A(+) than in SOD1(+) or SOD1(-)/G93A(-) mice. Furthermore, the excessive potentiation of [3H]d-ASP by GLY or GABA was already present in asymptomatic 30-40 day-old SOD1-G93A(+) mice. The releases of endogenous glutamate and GABA also were enhanced by GLY and the GLY-evoked release of endogenous glutamate, but not of endogenous GABA, was higher in SOD1-G93A(+) than in control animals. Potentiation of the spontaneous amino acid release is likely to be mediated by activation of a GLY or a GABA transporter, since the effect of GLY was counteracted by the GLY transporter blocker glycyldodecylamide but not by the GLY receptor antagonists strychnine and 5,7-dichlorokynurenate while the effect of GABA was diminished by the GABA transporter blocker SKF89976-A but not by the GABA receptor antagonists SR9531 and CGP52432. It is concluded that the glutamate release machinery seems excessively functional in SOD1-G93A(+) animals.