Calnexin-assisted biogenesis of the neuronal glycine transporter 2 (GlyT2).

The neuronal transporter GlyT2 is a polytopic, 12-transmembrane domain, plasma membrane glycoprotein involved in the removal and recycling of synaptic glycine from inhibitory synapses. Mutations in the human GlyT2 gene (SLC6A5) that cause deficient glycine transport or defective GlyT2 trafficking are the second most common cause of hyperekplexia or startle disease. In this study we examined several aspects of GlyT2 biogenesis that involve the endoplasmic reticulum chaperone calnexin (CNX). CNX binds transiently to an intermediate under-glycosylated transporter precursor and facilitates GlyT2 processing. In cells expressing GlyT2, transporter accumulation and transport activity were attenuated by siRNA-mediated CNX knockdown and enhanced by CNX overexpression. GlyT2 binding to CNX was mediated by glycan and polypeptide-based interactions as revealed by pharmacological approaches and the behavior of GlyT2 N-glycan-deficient mutants. Moreover, transporter folding appeared to be stabilized by N-glycans. Co-expression of CNX and a fully non-glycosylated mutant rescues glycine transport but not mutant surface expression. Hence, CNX discriminates between different conformational states of GlyT2 displaying a lectin-independent chaperone activity. GlyT2 wild-type and mutant transporters were finally degraded in the lysosome. Our findings provide further insight into GlyT2 biogenesis, and a useful framework for the study of newly synthesized GlyT2 transporters bearing hyperekplexia mutations.

Expression and purification of recombinant calpain-derived N-terminal peptides from glycine transporter GlyT2.

Glycine transporter GlyT2 contains an extended N-terminal domain which is about three times longer than the N-termini of its closest family members. We previously found that this domain could be separated from the transporter by proteolysis with calpain resulting in the generation of at least two GlyT2N derived peptides. In this work we analyzed the properties of these peptides using bio-informatics, by expressing them in mammalian cell lines and by overexpressing them in bacteria. When expressed in mammalian cell lines, these peptides show widespread localization in the cytoplasm. Their unusually high number of alanine, proline and glycine residues suggests that they posses significant disorder and conformational flexibility, which is supported by their thermal resistivity. Making use of these phenomena, we developed a simple purification method for obtaining pure recombinant GlyT2N derived calpain fragments without using an affinity tag. This procedure can be used to obtain peptide fragments in large amounts for structural, interaction studies or for determining their potential biological activity.

Transport activities and expression patterns of glycine transporters 1 and 2 in the developing murine brain stem and spinal cord.

Glycine serves as a neurotransmitter in spinal cord and brain stem, where it activates inhibitory glycine receptors. In addition, it serves as an essential co-agonist of excitatory N-methyl-d-aspartate receptors. In the central nervous system, extracellular glycine concentrations are regulated by two specific glycine transporters (GlyTs), GlyT1 and GlyT2. Here, we determined the relative transport activities and protein levels of GlyT1 and GlyT2 in membrane preparations from mouse brain stem and spinal cord at different developmental stages. We report that early postnatally (up to postnatal day P5) GlyT1 is the predominant transporter isoform responsible for a major fraction of the GlyT-mediated [(3)H]glycine uptake. At later stages (≥ P10), however, the transport activity and expression of GlyT2 increases, and in membrane fractions from adult mice both GlyTs contribute about equally to glycine uptake. These alterations in the activities and expression profiles of the GlyTs suggest that the contributions of GlyT1 and GlyT2 to the regulation of extracellular glycine concentrations at glycinergic synapses changes during development.

Early glycinergic axon contact with the Mauthner neuron during zebrafish development.

Glycinergic neurons are the major inhibitory neurons in the vertebrate central nervous system. In teleosts, they play important roles in the escape response by regulating the activity of the Mauthner (M-) cells. Here we studied the contact between glycinergic axons and the M-cells in early zebrafish embryos by double immunostaining with an anti-glycine antibody and the 3A10 antibody that labels M-cells. We also studied a transgenic line, Tg(GlyT2:GFP), in which GFP is expressed under the control of the promoter for the glycine transporter-2 gene. The initial contacts by ascending glycinergic axons on the M-soma were observed within 27h post-fertilization (hpf) on the lateral part of the ventral surface of the M-soma. Stochastic labeling of glycinergic neurons was then performed by injecting a GlyT2:GFP construct into early cleaving eggs. We identified the origin of the earliest glycinergic axons that contact the M-soma as commissural neurons, located in the anterior spinal cord, whose axons ascend along the lateral longitudinal fascicles with a short descending branch. We also found, in the fourth rhombomere, late-developed glycinergic commissural neurons whose axons contact anterior or posterior edge of both M-somas. This study provides the first example of the initial development of an inhibitory network on an identifiable neuron in vertebrates.

Calcium dependent modification of distal C-terminal sequences of glycine transporter GlyT1.

Glycine transporter GlyT1 plays important role in maintaining accurate glycine concentration in local brain microenvironment. Transporting efficiency of GlyT1 is strongly affected by the state of its distal C-terminus, which regulates transporter trafficking and cellular surface density. Using selected range of antibody epitopes against C-terminal region of GlyT1 we investigated its changes during calcium overload, the ubiquitous phenomena of several brain pathologies. We show that immunoreactivity against the last 12 amino acids of GlyT1C-terminal region exhibits robust calcium dependent decline, while the immunoreactivity of closely located region shows relatively small changes. Process is fully blocked by calcium chelation and inhibited by cysteine proteases inhibitors as well as inhibitors of protein kinase C. Distal GlyT1C-terminal end contains PDZ binding motif responsible for GlyT1 interaction with trafficking and clustering proteins. Its removal/modification could be part of the mechanism changing glycine homeostasis during physiological/pathological conditions characterized by elevated calcium.

Functional 'glial' GLYT1 glycine transporters expressed in neurons.

Glycine transporter 1 (GLYT1) and GLYT2 are the glycine transporters in CNS. While GLYT2 is largely expressed in glycinergic neurons, GLYT1 has long been considered to be exclusively present in glial cells. There is increasing evidence that significant amounts of the 'glial' transporter also exist on neurons, particularly on pre-synaptic nerve endings of glutamatergic neurons. The functions of 'neuronal GLYT1' may be manifold and are discussed in this review. Of major interest are the interactions between neuronal GLYT1 and glutamatergic receptors of the NMDA type the activity of which is modulated not only by astrocytic GLYT1 but also by neuronal GLYT1. Pathophysiological roles and therapeutic implications of neuronal GLYT1 are emerging from recent studies with genetically modified mice, particularly with animals lacking forebrain neuron-specific GLYT1 transporters. These mutant mice exhibit promnesic phenotypes reflecting enhancement of NMDA receptor function, as it occurs following administration of GLYT1 inhibitors. Inactivation of neuronal GLYT1 in the forebrain may represent an effective therapeutic intervention for the treatment of schizophrenia.

Altered mnemonic functions and resistance to N-METHYL-d-Aspartate receptor antagonism by forebrain conditional knockout of glycine transporter 1.

Converging evidence from pharmacological and molecular studies has led to the suggestion that inhibition of glycine transporter 1 (GlyT1) constitutes an effective means to boost N-methyl-d-aspartate receptor (NMDAR) activity by increasing the extra-cellular concentration of glycine in the vicinity of glutamatergic synapses. However, the precise extent and limitation of this approach to alter cognitive function, and therefore its potential as a treatment strategy against psychiatric conditions marked by cognitive impairments, remain to be fully examined. Here, we generated mutant mice lacking GlyT1 in the entire forebrain including neurons and glia. This conditional knockout system allows a more precise examination of GlyT1 downregulation in the brain on behavior and cognition. The mutation was highly effective in attenuating the motor-stimulating effect of acute NMDAR blockade by phencyclidine, although no appreciable elevation in NMDAR-mediated excitatory postsynaptic currents (EPSC) was observed in the hippocampus. Enhanced cognitive performance was observed in spatial working memory and object recognition memory while spatial reference memory and associative learning remained unaltered. These findings provide further credence for the potential cognitive enhancing effects of brain GlyT1 inhibition. At the same time, they indicated potential phenotypic differences when compared with other constitutive and conditional GlyT1 knockout lines, and highlighted the possibility of a functional divergence between the neuronal and glia subpopulations of GlyT1 in the regulation of learning and memory processes. The relevance of this distinction to the design of future GlyT1 blockers as therapeutic tools in the treatment of cognitive disorders remains to be further investigated.

Compartmentation of the cerebellar nuclei of the mouse.

The cerebellar nuclei integrate inhibitory input from Purkinje cells with excitatory input from mossy and climbing fiber collaterals and are the sole cerebellar output. Numerous studies have shown that the cerebellar cortex is highly compartmentalized into hundreds of genetically determined, reproducible topographic units--transverse zones and parasagittal stripes--that can be identified through the expression patterns of numerous molecules. The Purkinje cell stripes project to the cerebellar nuclei. However, there is no known commensurate topographic complexity in the cerebellar nuclei. Rather, conventional anatomical descriptions identify four major subdivisions--the medial, anterior and posterior interposed, and lateral nuclei--together with a few intranuclear subdivisions. To begin to address the apparent complexity gap, we have used a panel of antigens and transgenes to reveal a reproducible molecular heterogeneity in the mouse cerebellar nuclei. Based on the differential expression patterns, singly and in combination, a new cerebellar nuclear topographic map has been constructed. This reveals the subdivision of the cerebellar nuclei into at least 12 reproducible expression domains. We hypothesize that such heterogeneity is the counterpart of the zones and stripes of the cerebellar cortex.

Ptf1a, Lbx1 and Pax2 coordinate glycinergic and peptidergic transmitter phenotypes in dorsal spinal inhibitory neurons.

Inhibitory neurons in the dorsal horn synthesize a variety of neurotransmitters, including GABA, glycine and a set of peptides. Here we show that three transcription factors, Ptf1a, Pax2, and Lbx1, which have been reported to promote a GABAergic cell fate, also specify glycinergic and peptidergic transmitter phenotypes. First, Ptf1a appears to be a master regulator, as indicated by a requirement of Ptf1a for the expression of glycinergic marker GlyT2 and a set of peptides, including neuropeptide Y (NPY), nociceptin/orphanin FQ (N/OFQ), somatostatin (SOM), enkephalin (ENK), dynorphin (DYN) and galanin (GAL). Second, Pax2 is a downstream target of Ptf1a and controls subsets of transmitter phenotypes, including the expression of GlyT2, NPY, N/OFQ, DYN, and GAL, but is dispensable for SOM or ENK expression. Third, for Lbx1, due to neuronal cell loss at late stages, our analyses focused on early embryonic stages, and we found that Lbx1 is required for the expression of GlyT2, NPY, N/OFQ and is partially responsible for SOM expression. Our studies therefore suggest a coordinated and hierarchical specification of a variety of neurotransmitters in dorsal spinal inhibitory neurons.

Spinal antiallodynia action of glycine transporter inhibitors in neuropathic pain models in mice.

Neuropathic pain is refractory against conventional analgesics, and thus novel medicaments are desired for the treatment. Glycinergic neurons are localized in specific brain regions, including the spinal cord, where they play an important role in the regulation of pain signal transduction. Glycine transporter (GlyT)1, present in glial cells, and GlyT2, located in neurons, play roles in modulating glycinergic neurotransmission by clearing synaptically released glycine or supplying glycine to the neurons and thus could modify pain signal transmission in the spinal cord. In this study, we demonstrated that i.v. or intrathecal administration of GlyT1 inhibitors, cis-N-methyl-N-(6-methoxy-1-phenyl-1,2,3,4-tetrahydronaphthalen-2-yl methyl)amino methylcarboxylic acid (ORG25935) or sarcosine, and GlyT2 inhibitors, 4-benzyloxy-3,5-dimethoxy-N-[1-(dimethylaminocyclopently)-methyl]benzamide (ORG25543) and (O-[(2-benzyloxyphenyl-3-fluorophenyl)methyl]-L-serine) (ALX1393), or knockdown of spinal GlyTs by small interfering RNA of GlyTs mRNA produced a profound antiallodynia effect in a partial peripheral nerve ligation model and other neuropathic pain models in mice. The antiallodynia effect is mediated through spinal glycine receptor alpha3. These results established GlyTs as the target molecules for the development of medicaments for neuropathic pain. However, these manipulations to stimulate glycinergic neuronal activity were without effect during the 4 days after nerve injury, whereas manipulations to inhibit glycinergic neuronal activity protected against the development of allodynia in this phase. The results implied that the timing of medication with their inhibitors should be considered, because glycinergic control of pain was reversed in the critical period of 3 to 4 days after surgery. This may also provide important information for understanding the underlying molecular mechanisms of the development of neuropathic pain.

The transporters GlyT2 and VIAAT cooperate to determine the vesicular glycinergic phenotype.

The mechanisms that specify the vesicular phenotype of inhibitory interneurons in vertebrates are poorly understood because the two main inhibitory transmitters, glycine and GABA, share the same vesicular inhibitory amino acid transporter (VIAAT) and are both present in neurons during postnatal development. We have expressed VIAAT and the plasmalemmal transporters for glycine and GABA in a neuroendocrine cell line and measured the quantal release of glycine and GABA using a novel double-sniffer patch-clamp technique. We found that glycine is released from vesicles when VIAAT is coexpressed with either the neuronal transporter GlyT2 or the glial transporter GlyT1. However, GlyT2 was more effective than GlyT1, probably because GlyT2 is unable to operate in the reverse mode, which gives it an advantage in maintaining the high cytosolic glycine concentration required for efficient vesicular loading by VIAAT. The vesicular inhibitory phenotype was gradually altered from glycinergic to GABAergic through mixed events when GABA is introduced into the secretory cell and competes for uptake by VIAAT. Interestingly, the VIAAT ortholog from Caenorhabditis elegans (UNC-47), a species lacking glycine transmission, also supports glycine exocytosis in the presence of GlyT2, and a point mutation of UNC-47 that abolishes GABA transmission in the worm confers glycine specificity. Together, these results suggest that an increased cytosolic availability of glycine in VIAAT-containing terminals was crucial for the emergence of glycinergic transmission in vertebrates.