Reduced proliferation in the adult mouse subventricular zone increases survival of olfactory bulb interneurons

Neurogenesis in the adult brain is largely restricted to the subependymal zone (SVZ) of the lateral ventricle, olfactory bulb (OB) and the dentate subgranular zone, and survival of adult-born cells in the OB is influenced by factors including sensory experience. We examined, in mice, whether survival of adult-born cells is also regulated by the rate of precursor proliferation in the SVZ. Precursor proliferation was decreased by depleting the SVZ of dopamine after lesioning dopamine neurons in the substantia nigra compacta with 6-hydroxydopamine. Subsequently, we examined the effect of reduced SVZ proliferation on the generation, migration and survival of neuroblasts and mature adult-born cells in the SVZ, rostral migratory stream (RMS) and OB. Proliferating cells in the SVZ, measured by 5-bromo-2-deoxyuridine (BrdU) injected 2 hours prior to death or by immunoreactivity against Ki67, were reduced by 47% or 36%, respectively, 7 days after dopamine depletion, and by 29% or 31% 42 days after dopamine depletion, compared to sham-treated animals. Neuroblast generation in the SVZ and their migration along the RMS were not affected, neither 7 nor 42 days after the 6-hydroxydopamine injection, since the number of doublecortin-immunoreactive neuroblasts in the SVZ and RMS, as well as the number of neuronal nuclei-immunoreactive cells in the OB, were stable compared to control. However, survival analysis 15 days after 6-hydroxydopamine and 6 days after BrdU injections showed that the number of BrdU+ cells in the SVZ was 70% higher. Also, 42 days after 6-hydroxydopamine and 30 days after BrdU injections, we found an 82% increase in co-labeled BrdU+/γ-aminobutyric acid-immunoreactive cell bodies in the granular cell layer, while double-labeled BrdU+/tyrosine hydroxylase-immunoreactive cell bodies in the glomerular layer increased by 148%. We conclude that the number of OB interneurons following reduced SVZ proliferation is maintained through an increased survival of adult-born precursor cells, neuroblasts and interneurons.     

Postnatal Expression of GAD67

N-methyl-d-aspartate receptor blockade promotes apoptosis at postnatal day 7 (P7) and is linked to loss of glutamic acid decarboxylase 67 (GAD67) expression in older animals. To more fully appreciate this relationship we must first understand how GAD67 is regulated postnatally. Thus, the brains of P7, P14 and P21 rats were examined for expression of GAD67 protein and we found that levels of this GABAergic marker increased steadily with age, such that by P21 there was as much as a 6-fold increase compared to P7 animals and a 1.5- to 2-fold increase compared to P14 animals, depending on the region sampled. At P7, GAD67 was almost exclusively detected in puncta, with very few cell bodies displaying this marker. In contrast, at P14 and especially P21, both puncta and cell bodies were robustly labeled. Our data indicate that adult-like expression of GAD67 emerges quite late in the postnatal period.     

Rate and pattern of migration of lineally-related olfactory bulb interneurons generated postnatally in the subventricular zone of the rat

A spatially discrete region of the anterior part of the postnataltelescephalic subventricular zone, referred to as the SVZa generatesvast numbers of lineally-related neurons destined for the olfactorybulb (Luskin, 1993). The cells originating in the SVZa migrateto the olfactory bulb along a highly restricted pathway whichis in a direction orthogonal to the orientation of radial glialfibers. In this study we analysed the number, distribution,orientation and rate of migration of SVZa-derived cells as theyapproach the olfactory bulb. In order to track the SVZa-derivedcells, a retroviral lineage tracer, encoding the reporter geneE.coli ß-galactosidase (lacZ) was injected preciselyinto the rat SVZa at postnatal day 1 (Pl). The lacZ-positivecells were visualized 1, 2 and 3 days later by X-Gal histochemistryin cryostat sections. As the number of SVZa-derived cells inthe pathway increased with survival time, their distributionchanged systematically. The distribution pattern of lacZ-positivecells by 2 and 3 days postinjection suggested that some of theprogeny of infected progenitor cells were undergoing neurogenesisas they proceeded to the olfactory bulb; a large percentageof the lacZ-positive cells were substantially displaced fromthe SVZa injection site. To investigate whether lacZ-positivecells migrate in a directed fashion, their orientation preferencewas scored. For the majority of lacZ-positive cells (>94%),their leading process was directed toward the olfactory bulb,possibly reflecting a response to migratory cues present alongthe pathway. The estimated average rate of cell migration tothe olfactory bulb was 23 µm/h, which is approximatelytwice the speed of radially directed neuronal migration fromthe telencephalic ventricular zone to the cortical plate (O'Rourkeet al, 1992). Collectively, these results suggest that SVZa-derivedintemeurons en route to the olfactory bulb may employ a novelmode of tangential migration.     

Mechanism of migration of olfactory bulb interneurons

Neuroblasts in the subventricular zone of the walls of the lateral ventricle in the brain of young and adult rodents migrate into the olfactory bulb where they differentiate into local interneurons. These cells move closely associated with each other, forming chains without radial glial or axonal guidance. The migrating neuroblasts express PSA-NCAM on their surface and PSA residues are crucial for cell-cell interaction during chain migration. This migration occurs throughout the lateral wall of the lateral ventricle, where the precursors form an extensive network of chains. Cells remain organized as chains until they reach the olfactory bulb, where they disperse organized as chains until they reach the olfactory bulb, where they disperse radially as individual cells. Chain migration defines a novel form of neuronal precursor translocation which is based on homotypic interactions between cells.     

An analysis of the cross-reactivity of autoantibodies to GAD65 and GAD67 in diabetes

Background

Autoantibodies to GAD65 (anti-GAD65) are present in the sera of 70–80% of patients with type 1 diabetes (T1D), but antibodies to the structurally similar 67 kDa isoform GAD67 are rare. Antibodies to GAD67 may represent a cross-reactive population of anti-GAD65, but this has not been formally tested.

Methodology/Principal Findings

In this study we examined the frequency, levels and affinity of anti-GAD67 in diabetes sera that contained anti-GAD65, and compared the specificity of GAD65 and GAD67 reactivity. Anti-GAD65 and anti-GAD67 were measured by radioimmunoprecipitation (RIP) using ¹²⁵I labeled recombinant GAD65 and GAD67. For each antibody population, the specificity of the binding was measured by incubation with 100-fold excess of unlabeled GAD in homologous and heterologous inhibition assays, and the affinity of binding with GAD65 and GAD67 was measured in selected sera. Sera were also tested for reactivity to GAD65 and GAD67 by immunoblotting. Of the 85 sera that contained antibodies to GAD65, 28 contained anti–GAD67 measured by RIP. Inhibition with unlabeled GAD65 substantially or completely reduced antibody reactivity with both ¹²⁵I GAD65 and with ¹²⁵I GAD67. In contrast, unlabeled GAD67 reduced autoantibody reactivity with ¹²⁵I GAD67 but not with ¹²⁵I GAD65. Both populations of antibodies were of high affinity (>10¹⁰ l/mol).

Conclusions

Our findings show that autoantibodies to GAD67 represent a minor population of anti-GAD65 that are reactive with a cross-reactive epitope found also on GAD67. Experimental results confirm that GAD65 is the major autoantigen in T1D, and that GAD67 per se has very low immunogenicity. We discuss our findings in light of the known similarities between the structures of the GAD isoforms, in particular the location of a minor cross-reactive epitope that could be induced by epitope spreading.     

Smoking-mediated up-regulation of GAD67 expression in the human airway epithelium

Background

The production of gamma-amino butyric acid (GABA) is dependent on glutamate decarboxylases (GAD65 and GAD67), the enzymes that catalyze the decarboxylation of glutamate to GABA. Based on studies suggesting a role of the airway epithelial GABAergic system in asthma-related mucus overproduction, we hypothesized that cigarette smoking, another disorder associated with increased mucus production, may modulate GABAergic system-related gene expression levels in the airway epithelium.

Methods

We assessed expression of the GABAergic system in human airway epithelium obtained using bronchoscopy to sample the epithelium and microarrays to evaluate gene expression. RT-PCR was used to confirm gene expression of GABAergic system gene in large and small airway epithelium from heathy nonsmokers and healthy smokers. The differences in the GABAergic system gene was further confirmed by TaqMan, immunohistochemistry and Western analysis.

Results

The data demonstrate there is a complete GABAergic system expressed in the large and small human airway epithelium, including glutamate decarboxylase, GABA receptors, transporters and catabolism enzymes. Interestingly, of the entire GABAergic system, smoking modified only the expression of GAD67, with marked up-regulation of GAD67 gene expression in both large (4.1-fold increase, p < 0.01) and small airway epithelium of healthy smokers (6.3-fold increase, p < 0.01). At the protein level, Western analysis confirmed the increased expression of GAD67 in airway epithelium of healthy smokers compared to healthy nonsmokers (p < 0.05). There was a significant positive correlation between GAD67 and MUC5AC gene expression in both large and small airway epithelium (p < 0.01), implying a link between GAD67 and mucin overproduction in association with smoking.

Conclusions

In the context that GAD67 is the rate limiting enzyme in GABA synthesis, the correlation of GAD67 gene expression with MUC5AC expressions suggests that the up-regulation of airway epithelium expression of GAD67 may contribute to the increase in mucus production observed in association with cigarette smoking.

Trial registration

NCT00224198; NCT00224185

     

Molecular and electrophysiological characterization of GFP-expressing CA1 interneurons in GAD65-GFP mice

The use of transgenic mice in which subtypes of neurons are labeled with a fluorescent protein has greatly facilitated modern neuroscience research. GAD65-GFP mice, which have GABAergic interneurons labeled with GFP, are widely used in many research laboratories, although the properties of the labeled cells have not been studied in detail. Here we investigate these cells in the hippocampal area CA1 and show that they constitute ～20% of interneurons in this area. The majority of them expresses either reelin (70±2%) or vasoactive intestinal peptide (VIP; 15±2%), while expression of parvalbumin and somatostatin is virtually absent. This strongly suggests they originate from the caudal, and not the medial, ganglionic eminence. GFP-labeled interneurons can be subdivided according to the (partially overlapping) expression of neuropeptide Y (42±3%), cholecystokinin (25±3%), calbindin (20±2%) or calretinin (20±2%). Most of these subtypes (with the exception of calretinin-expressing interneurons) target the dendrites of CA1 pyramidal cells. GFP-labeled interneurons mostly show delayed onset of firing around threshold, and regular firing with moderate frequency adaptation at more depolarized potentials.     

Structural and functional analysis of cysteine residues in human glutamate decarboxylase 65 (GAD65) and GAD67

Previously, we have shown that brain glutamate decarboxylase (GAD) is greatly inhibited by sulfhydryl reactive reagent suggesting cysteine residue(s) may play an important role in GAD function. In this report, we determined the role of cysteine residues in the recombinant human 65-kDa GAD isoform (hGAD65) and 67-kDa GAD isoform (hGAD67), using a combination of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and site-directed mutagenesis. Here, we report that cysteine 446 (C446) in hGAD65 is important for its activity and is present as free sulfhydryl group. This conclusion is based on the following observations: (i) mutation of C446 in hGAD65 to alanine reduced hGAD65 activity by more than 90%, (ii) MALDI-TOF analysis of the non-reduced, trypsin-digested GAD65 revealed that C446 is present as a free sulfhydryl group as indicated by a peak at m/z (mass/charge) 647.3446 (peptide 443-448) and, when GAD65 was treated with sulfhydryl reagent, N-ethylmaleimide (NEM), the peak is shifted to m/z 772.3702,a mass increase of 125.1 daltons (Da) as a result of modification of cysteine by NEM. Parallel studies have also been conducted with hGAD67. Cysteine 455 was found to be important for GAD67 activity.     

Protein phosphorylation of human brain glutamic acid decarboxylase (GAD)65 and GAD67 and its physiological implications

Previously, we reported that protein phosphorylation plays an important role in regulating soluble l-glutamic acid decarboxylase (GAD) [Bao, J. (1995) J. Biol. Chem. 270, 6464-6467] and membrane-associated GAD activity [Hsu, C. C. (1999) J. Biol. Chem. 274, 24366-24371]. Here, we report the effect of phosphorylation on the two well-defined GAD isoforms, namely, GAD65 and GAD67, using highly purified preparations of recombinant human brain GAD65 and GAD67. GAD65 was activated by phosphorylation, while GAD67 was inhibited by phosphorylation. The effect of phosphorylation on GAD65 and GAD67 could be reversed by treatment with protein phosphatases. We further demonstrate that protein kinase A (PKA) and protein kinase C isoform epsilon are the protein kinases responsible for phosphorylation and regulation of GAD67 and GAD65, respectively. Direct phosphorylation of GAD65 and GAD67 was demonstrated by incorporation of [(32)P] from [gamma-(32)P]ATP into purified GAD65 and GAD67 and immunoblotting assay using anti-phosphoserine/threonine antibodies. We have identified one specific phosphorylation site, threonine 91 (T91), in hGAD67 that can be phosphorylated by PKA using MALDI-TOF. Site-directed mutation of T91 to alanine abolished PKA-mediated phosphorylation and inhibition of GAD activity. Furthermore, mutation of T91 to aspartic acid or glutamic acid mimics the effect of phosphorylation. A model depicting the effect of phosphorylation on GAD activity upon neuronal stimulation is also proposed.     

Wnt5a controls neurite development in olfactory bulb interneurons

Neurons born in the postnatal SVZ (subventricular zone) must migrate a great distance before becoming mature interneurons of the OB (olfactory bulb). During migration immature OB neurons maintain an immature morphology until they reach their destination. While the morphological development of these cells must be tightly regulated, the cellular pathways responsible are still largely unknown. Our results show that the non-canonical Wnt pathway induced by Wnt5a is important for the morphological development of OB interneurons both in vitro and in vivo. Additionally, we demonstrate that non-canonical Wnt signalling works in opposition to canonical Wnt signalling in neural precursors from the SVZ in vitro. This represents a novel role for Wnt5a in the development of OB interneurons and suggests that canonical and non-canonical Wnt pathways dynamically oppose each other in the regulation of dendrite maturation.     

Gastrointestinal phenotype of GAD67lacZ transgenic mice with early postnatal lethality

It has been proposed that gamma-aminobutyric acid (GABA) in the gut may function as a neurotransmitter, hormone and/or paracrine agent. Our aim was to examine transgenic mice of the GAD67-lacZ line with impaired postnatal growth and early postnatal lethality for gastrointestinal abnormalities. The gastrointestinal tract was dissected and processed for histology, immunohistochemistry, electron microscopy, western blotting and measurement of GAD activity. Homozygous mice of both sexes displayed an intestinal phenotype characterized by a fragile and haemorrhagic intestinal wall, a reduced number of villi, epithelial lesions and the occasional appearance of pseudostratified epithelium. The number of GABA-immunoreactive enteroendocrine cells and mucin-secreting goblet cells increased significantly relative to wild-type epithelium. The appearance of GABA-immunopositive neuronal perikarya and the lack of GABA-immunoreactive varicose fibres were observed in the enteric plexuses of transgenic mice. Tissue homogenates of transgenic mice showed higher levels of expression of GAD67 and GAD65 as compared with wild-type mice. Our results suggest that the possible reason underlying the growth impairment and postnatal lethality observed in GAD67 transgenic mice is a functional impairment of GABAergic enteric neurons and disintegration of intestinal epithelium.     

Two distinct mechanisms target GAD67 to vesicular pathways and presynaptic clusters

The inhibitory neurotransmitter γ-amino butyric acid (GABA) is synthesized by two isoforms of the enzyme glutamic acid decarboxylase (GAD): GAD65 and GAD67. Whereas GAD67 is constitutively active and produces >90% of GABA in the central nervous system, GAD65 is transiently activated and augments GABA levels for rapid modulation of inhibitory neurotransmission. Hydrophobic lipid modifications of the GAD65 protein target it to Golgi membranes and synaptic vesicles in neuroendocrine cells. In contrast, the GAD67 protein remains hydrophilic but has been shown to acquire membrane association by heterodimerization with GAD65. Here, we identify a second mechanism that mediates robust membrane anchoring, axonal targeting, and presynaptic clustering of GAD67 but that is independent of GAD65. This mechanism is abolished by a leucine-103 to proline mutation that changes the conformation of the N-terminal domain but does not affect the GAD65-dependent membrane anchoring of GAD67. Thus two distinct mechanisms target the constitutively active GAD67 to presynaptic clusters to facilitate accumulation of GABA for rapid delivery into synapses.     

MiR-30d Participates in Vincristine-Induced Neuropathic Pain by Down-Regulating GAD67

Neurochemical Research - Vincristine is a common chemotherapeutic agent in cancer treatment, while it often causes chemotherapy-induced&nbsp;peripheral&nbsp;neuropathy(CIPN), which brings...     

Early specification of ascidian larval motor neurons

In the tadpole larvae of the ascidian Halocynthia roretzi, six motor neurons, Moto-A, -B, and -C (a pair of each), are localized proximal to the caudal neural tube and show distinct morphology and innervation patterns. To gain insights into early mechanisms underlying differentiation of individual motor neurons, we have isolated an ascidian homologue of Islet, a LIM type homeobox gene. Earliest expression of Islet was detected in a pair of bilateral blastomeres on the dorsal edge of the late gastrula. At the neurula stage, this expression began to disappear and more posterior cells started to express Islet. Compared to expression of a series of motor neuron genes, it was confirmed that early Islet-positive blastomeres are the common precursors of Moto-A and -B, and late Islet-positive cells in the posterior neural tube are the precursors of Moto-C. Overexpression of Islet induced ectopic expression of motor neuron markers, suggesting that Islet is capable of regulating motor neuron differentiation. Since early expression of Islet colocalizes with that of HrBMPb, the ascidian homologue of BMP2/4, we tested a role of BMP in specification of the motor neuron fate. Overexpression of HrBMPb led to expansion of Lim and Islet expression toward the central area of the neural plate, and microinjection of mRNA coding for a dominant-negative BMP receptor weakened the expression of these genes. Our results suggest that determination of the ascidian motor neuron fate takes place at late gastrula stage and local BMP signaling may play a role in this step.     

Crossinhibitory activities of Ngn1 and Math1 allow specification of distinct dorsal interneurons

Distinct classes of neurons are generated from progenitor cells distributed in characteristic dorsoventral patterns in the developing spinal neural tube. We define restricted neural progenitor populations by the discrete, nonoverlapping expression of Ngn1, Math1, and Mash1. Crossinhibition between these bHLH factors is demonstrated and provides a mechanism for the generation of discrete bHLH expression domains. This precise control of bHLH factor expression is essential for proper neural development since as demonstrated in both loss- and gain-of-function experiments, expression of Math1 or Ngn1 in dorsal progenitor cells determines whether LH2A/B- or dorsal Lim1/2-expressing interneurons will develop. Together, the data suggest that although Math1 and Ngn1 appear to be redundant with respect to neurogenesis, they have distinct functions in specifying neuronal subtype in the dorsal neural tube.