Mouse Cerebellar Granule Cells Research Articles

Chronic Treatment with Anti-bipolar Drugs Down-Regulates Gene Expression of TRPC1 in Neurones.

In the brain, TRPC1 channels are abundantly expressed in neurones virtually in all regions; these proteins function as receptor-activated ion channels and are implicated in numerous processes, being specifically important for neurogenesis. Primary cultures of mouse cerebellar granule cell, cerebral cortical neurones, and freshly isolated neurones from in vivo brains were used to study effects of chronic treatment with anti-bipolar drugs [carbamazepine (CBZ), lithium salts and valproic acid] on gene expression of TRPC1. Expression of TRPC1 mRNA was identified with reverse transcription-polymerase chain reaction, whereas protein content was determined by Western blotting. Store-operated plasmalemmal Ca2+ entry (SOCE) was measured with fura-2 based microfluorimetry. Chronic treatment with each of the three drugs down-regulated mRNA and protein expression in cultured cerebellar granule cells in a time- and concentration-dependent manner. Similar effect was also observed in cultured cerebral cortical neurones treated with CBZ, lithium salts and valproic acid and in freshly isolated neurones from the brains of CBZ-treated animals. The amplitude of SOCE was substantially decreased in cerebellar granule cells chronically treated with each of the three drugs. Our findings indicate that down-regulation of TRPC1 gene expression and function in neurones may be one of the mechanisms of anti-bipolar drugs action.

Trimebutine, a small molecule mimetic agonist of adhesion molecule L1, contributes to functional recovery after spinal cord injury in mice.

ABSTRACTCuring spinal cord injury (SCI) in mammals is a daunting task because of the lack of permissive mechanisms and strong inhibitory responses at and around the lesion. The neural cell adhesion molecule L1CAM (L1) has been shown to favor axonal regrowth and enhance neuronal survival and synaptic plasticity but delivery of full-length L1 or its extracellular domain could encounter difficulties in translation to therapy in humans. We have, therefore, identified several small organic compounds that bind to L1 and stimulate neuronal survival, neuronal migration and neurite outgrowth in an L1-dependent manner. Here, we assessed the functions of two L1 mimetics, trimebutine and honokiol, in regeneration following SCI in young adult mice. Using the Basso Mouse Scale (BMS) score, we found that ground locomotion in trimebutine-treated mice recovered better than honokiol-treated or vehicle-receiving mice. Enhanced hindlimb locomotor functions in the trimebutine group were observed at 6 weeks after SCI. Immunohistology of the spinal cords rostral and caudal to the lesion site showed reduced areas and intensities of glial fibrillary acidic protein immunoreactivity in both trimebutine and honokiol groups, whereas increased regrowth of axons was observed only in the trimebutine-treated group. Both L1- and L1 mimetic-mediated intracellular signaling cascades in the spinal cord lesion sites were activated by trimebutine and honokiol, with trimebutine being more effective than honokiol. These observations suggest that trimebutine and, to a lesser extent under the present experimental conditions, honokiol have a potential for therapy in regeneration of mammalian spinal cord injuries.

α6-Containing GABAA Receptors Are the Principal Mediators of Inhibitory Synapse Strengthening by Insulin in Cerebellar Granule Cells.

Activity-dependent strengthening of central synapses is a key factor driving neuronal circuit behavior in the vertebrate CNS. At fast inhibitory synapses, strengthening is thought to occur by increasing the number of GABAA receptors (GABARs) of the same subunit composition to preexisting synapses. Here, we show that strengthening of mouse cerebellar granule cell GABAergic synapses occurs by a different mechanism. Specifically, we show that the neuropeptide hormone, insulin, strengthens inhibitory synapses by recruiting α6-containing GABARs rather than accumulating more α1-containing receptors that are resident to the synapse. Because α6-receptors are targeted to functionally distinct postsynaptic sites from α1-receptors, we conclude that only a subset of all inhibitory synapses are strengthened. Together with our recent findings on stellate cells, we propose a general mechanism by which mature inhibitory synapses are strengthened. In this scenario, α1-GABARs resident to inhibitory synapses form the hardwiring of neuronal circuits with receptors of a different composition fulfilling a fundamental, but unappreciated, role in synapse strengthening.

Glycohydrolases in the central nervous system: the role of GBA2 in the neuronal differentiation

Mammalian neurodevelopment is characterized by qualitative and quantitative changes in plasma membrane glycosphingolipids due to a fine regulation of their metabolic pathways. While the biosynthetic pathway is largely studied scant is the information available on the catabolic one. For this reason, I studied the activity of the main glycohydrolases expressed in the central nervous system during neuronal differentiation. To this purpose the activities of the principal glycohydrolases involved in glycosphingolipid catabolism have been evaluated in different experimental models such as brains and cerebella of mouse at different ages and neuronal cell cultures (immortalized mouse neuronal cell lines GN11 and GT1-7, primary cultures of mouse cerebellar granule cells and human neuroblastoma cells SH-SY5Y). The results obtained indicate that the process of neuronal differentiation is associated with a marked increase in the activities of all the glycohydrolases evaluated, in particular, the activity of the non-lysosomal β-glucosylceramidase GBA2 undergoes the most relevant increase representing the prevalent form of β-glucocerebrosidase in mature neurons. In order to evaluate the possible role of GBA2 in the neuronal differentiation, SH-SY5Y cells have been stably trasfected for GBA2 overexpression. Cells overexpressing GBA2 acquired a neuronal phenotype and showed a significant increase in ceramide levels. These results are in line with literature data that demonstrate the involvement of ceramide in the neuronal differentiation. Therefore, it is possible to hypothesize that the hydrolysis of glucosylceramide to ceramide, catalyzed by GBA2 at the plasma membrane level, has a functional role in the neuronal differentiation process. Collectively these findings suggest that GBA2 may represent a possible neuronal marker and demonstrate for the first time its direct involvement in the neuronal differentiation.

Casein kinase 1δ is an APC/C(Cdh1) substrate that regulates cerebellar granule cell neurogenesis.

Although casein kinase 1δ (CK1δ) is at the center of multiple signaling pathways, its role in the expansion of CNS progenitor cells is unknown. Using mouse cerebellar granule cell progenitors (GCPs) as a model for brain neurogenesis, we demonstrate that the loss of CK1δ or treatment of GCPs with a highly selective small molecule inhibits GCP expansion. In contrast, CK1δ overexpression increases GCP proliferation. Thus, CK1δ appears to regulate GCP neurogenesis. CK1δ is targeted for proteolysis via the anaphase-promoting complex/cyclosome (APC/C(Cdh1)) ubiquitin ligase, and conditional deletion of the APC/C(Cdh1) activator Cdh1 in cerebellar GCPs results in higher levels of CK1δ. APC/C(Cdh1) also downregulates CK1δ during cell-cycle exit. Therefore, we conclude that APC/C(Cdh1) controls CK1δ levels to balance proliferation and cell-cycle exit in the developing CNS. Similar studies in medulloblastoma cells showed that CK1δ holds promise as a therapeutic target.

Calretinin: from a "simple" Ca(2+) buffer to a multifunctional protein implicated in many biological processes.

The hexa-EF-hand Ca2+-binding protein calretinin (CR) is predominantly expressed in specific neurons of the central and peripheral nervous system. However, CR expression is also observed in non-neuronal cells, e.g., during embryonic development and in mesothelioma cells. Of the 6 EF-hand domains, 5 are functional; the first 4 domains form 2 pairs showing high cooperativity within a pair that results in non-linear modulation of intracellular Ca2+ signals by CR. EF-hand domain 5 has a low affinity and represents the identified interaction site with CR-binding partners present in mouse cerebellar granule cells. CR binding to other targets including the pore-forming α1 subunit of the Ca2+ channel CaV2.1, as well as to huntingtin indicates additional Ca2+ sensor functions besides the well-known Ca2+-buffering functions. The absence of CR in cerebellar granule cells of CR−/− mice results in increased excitability and altered firing of Purkinje cells and promotes cerebellar 160-Hz oscillations impairing motor coordination. The putative role of CR in neuroprotection is still highly discussed. Altogether, CR emerges as a multi-functional protein also associated with development, i.e., cell proliferation, differentiation, and cell death.

Insufficient ER-stress response causes selective mouse cerebellar granule cell degeneration resembling that seen in congenital disorders of glycosylation

BackgroundCongenital disorders of glycosylation (CDGs) are inherited diseases caused by glycosylation defects. Incorrectly glycosylated proteins induce protein misfolding and endoplasmic reticulum (ER) stress. The most common form of CDG, PMM2-CDG, is caused by deficiency in the cytosolic enzyme phosphomannomutase 2 (PMM2). Patients with PMM2-CDG exhibit a significantly reduced number of cerebellar Purkinje cells and granule cells. The molecular mechanism underlying the specific cerebellar neurodegeneration in PMM2-CDG, however, remains elusive.ResultsHerein, we report that cerebellar granule cells (CGCs) are more sensitive to tunicamycin (TM)-induced inhibition of total N-glycan synthesis than cortical neurons (CNs). When glycan synthesis was inhibited to a comparable degree, CGCs exhibited more cell death than CNs. Furthermore, downregulation of PMM2 caused more CGCs to die than CNs. Importantly, we found that upon PMM2 downregulation or TM treatment, ER-stress response proteins were elevated less significantly in CGCs than in CNs, with the GRP78/BiP level showing the most significant difference. We further demonstrate that overexpression of GRP78/BiP rescues the death of CGCs resulting from either TM-treatment or PMM2 downregulation.ConclusionsOur results indicate that the selective susceptibility of cerebellar neurons to N-glycosylation defects is due to these neurons’ inefficient response to ER stress, providing important insight into the mechanisms of selective neurodegeneration observed in CDG patients.

GluRδ2 Assembles Four Neurexins intoTrans-Synaptic Triad to Trigger Synapse Formation

Elucidation of molecular mechanisms of synapse formation is a prerequisite for the understanding of neural wiring, higher brain functions, and mental disorders. The trans-synaptic interaction of postsynaptic glutamate receptor δ2 (GluRδ2) and presynaptic neurexins (NRXNs) through cerebellin precursor protein 1 (Cbln1) mediates synapse formation in vivo in the cerebellum. Here, we asked how the trans-synaptic triad induces synapse formation. Native GluRδ2 existed as a tetramer in the membrane, whereas the N-terminal domain (NTD) of GluRδ2 formed a stable homodimer. When incubated with cultured mouse cerebellar granule cells (GCs), dimeric GluRδ2-NTD and Cbln1 exerted little effect on the accumulation of punctate immunostaining signals for Bassoon and vesicular glutamate transporter 1 in GC axons. However, tetramerized GluRδ2-NTD stimulated the accumulation of these presynaptic proteins in the axons. Analysis of Cbln1 mutants suggested that the binding sites of GluRδ2 and NRXN1β on Cbln1 are differential. Furthermore, there was no competition in the binding to Cbln1 between GluRδ2-NTD and the extracellular domain (ECD) of NRXN1β. Thus, GluRδ2 and Cbln1 interacted with each other rather independently of Cbln1-NRXN1β interaction and vice versa. Gel filtration and isothermal titration calorimetry analyses consistently showed that dimeric GluRδ2-NTD and hexameric Cbln1 assembled in the 1:1 ratio, whereas hexameric Cbln1 and the laminin-neurexin-sex hormone-binding globulin domain of NRXN1β-ECD assembled in the 1:2 ratio. Thus, the synaptogenic triad is assembled from tetrameric GluRδ2, hexameric Cbln1, and monomeric NRXN in the ratio of 1:2:4. These results suggest that GluRδ2 triggers synapse formation by clustering four NRXNs through triad formation.

IL-6 is increased in the cerebellum of autistic brain and alters neural cell adhesion, migration and synaptic formation

BackgroundAlthough the cellular mechanisms responsible for the pathogenesis of autism are not understood, a growing number of studies have suggested that localized inflammation of the central nervous system (CNS) may contribute to the development of autism. Recent evidence shows that IL-6 has a crucial role in the development and plasticity of CNS.MethodsImmunohistochemistry studies were employed to detect the IL-6 expression in the cerebellum of study subjects. In vitro adenoviral gene delivery approach was used to over-express IL-6 in cultured cerebellar granule cells. Cell adhesion and migration assays, DiI labeling, TO-PRO-3 staining and immunofluorescence were used to examine cell adhesion and migration, dendritic spine morphology, cell apoptosis and synaptic protein expression respectively.ResultsIn this study, we found that IL-6 was significantly increased in the cerebellum of autistic subjects. We investigated how IL-6 affects neural cell development and function by transfecting cultured mouse cerebellar granule cells with an IL-6 viral expression vector. We demonstrated that IL-6 over-expression in granule cells caused impairments in granule cell adhesion and migration but had little effect on the formation of dendritic spines or granule cell apoptosis. However, IL-6 over-expression stimulated the formation of granule cell excitatory synapses, without affecting inhibitory synapses.ConclusionsOur results provide further evidence that aberrant IL-6 may be associated with autism. In addition, our results suggest that the elevated IL-6 in the autistic brain could alter neural cell adhesion, migration and also cause an imbalance of excitatory and inhibitory circuits. Thus, increased IL-6 expression may be partially responsible for the pathogenesis of autism.

Interaction between very‐KIND Ras guanine exchange factor and microtubule‐associated protein 2, and its role in dendrite growth – structure and function of the second kinase noncatalytic C‐lobe domain

The kinase noncatalytic C-lobe domain (KIND) is a putative protein-protein interaction module. Four KIND-containing proteins, Spir-2 (actin-nuclear factor), PTPN13 (protein tyrosine phosphatase), FRMPD2 (scaffold protein) and very-KIND (v-KIND) (brain-specific Ras guanine nucleotide exchange factor), have been identified to date. Uniquely, v-KIND has two KINDs (i.e. KIND1 and KIND2), whereas the other three proteins have only one. The functional role of KIND, however, remains unclear. We previously demonstrated that v-KIND interacts with the high-molecular weight microtubule-associated protein 2 (MAP2), a dendritic microtubule-associated protein, leading to negative regulation of neuronal dendrite growth. In the present study, we analyzed the structure-function relationships of the v-KIND-MAP2 interaction by generating a series of mutant constructs. The interaction with endogenous MAP2 in mouse cerebellar granule cells was specific to v-KIND KIND2, but not KIND1, and was not observed for the KINDs from other KIND-containing proteins. The binding core modules critical for the v-KIND-MAP2 interaction were defined within 32 residues of the mouse v-KIND KIND2 and 43 residues of the mouse MAP2 central domain. Three Leu residues at amino acid positions 461, 474 and 477 in the MAP2-binding core module of KIND2 contributed to the interaction. The MAP2-binding core module itself promoted dendrite branching as a dominant-negative regulator of v-KIND in hippocampal neurons. The results reported in the present study demonstrate the structural and functional determinant underlying the v-KIND-MAP2 interaction that controls dendrite arborization patterns.

Genome-Wide Analysis of Alternative Splicing in Medulloblastoma Identifies Splicing Patterns Characteristic of Normal Cerebellar Development

Alternative splicing is an important mechanism for the generation of protein diversity at a post-transcriptional level. Modifications in the splicing patterns of several genes have been shown to contribute to the malignant transformation of different tissue types. In this study, we used the Affymetrix Exon arrays to investigate patterns of differential splicing between pediatric medulloblastomas and normal cerebellum on a genome-wide scale. Of the 1,262 genes identified as potentially generating tumor-associated splice forms, we selected 14 examples of differential splicing of known cassette exons and successfully validated 11 of them by reverse transcriptase PCR. The pattern of differential splicing of three validated events was characteristic for the molecular subset of sonic hedgehog (Shh)-driven medulloblastomas, suggesting that their unique gene signature includes the expression of distinctive transcript variants. Generally, we observed that tumor and normal fetal cerebellar samples shared significantly lower exon inclusion rates than normal adult cerebellum. We investigated whether tumor-associated splice forms were expressed in primary cultures of Shh-dependent mouse cerebellar granule cell precursors (GCP) and found that Shh caused a decrease in the cassette exon inclusion rate of five of the seven tested genes. Furthermore, we observed a significant increase in exon inclusion between postnatal days 7 and 14 of mouse cerebellar development, at the time when GCPs mature into postmitotic neurons. We conclude that inappropriate splicing frequently occurs in human medulloblastomas and may be linked to the activation of developmental signaling pathways and a failure of cerebellar precursor cells to differentiate.

AMPAR signaling mediating GABA AR δ subunit up-regulation in cultured mouse cerebellar granule cells

Depolarization of cerebellar granule cells in culture leads to up-regulation of the GABA A receptor δ subunit expression. To determine the signaling molecules involved, we examined the effects of protein kinase inhibitors and cyclic AMP-elevating compounds on basal and AMPAR agonist-induced δ mRNA expression in cerebellar granule cells. Treatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 or with pituitary adenylate activating polypeptide increased δ subunit expression by 70%. Selective activation of AMPA receptors with CPW-399 also increased δ mRNA expression (2–4-fold). CPW-399 induction of δ subunit mRNA was reduced by prior treatment with either the MEK1/2 inhibitor U0126 or protein kinase A (PKA) inhibitors KT 5720 and H89. These effects were additive and combined treatment with U0126 and H89 completely prevented induction of δ subunit expression above basal levels. These results suggest that the role of JNK and ERK1/2/PKA on maintainence of δ subunit expression is diammetrically opposite. While JNK activity negatively regulates δ subunit mRNA expression in unstimulated neurons, activity of ERK1/2 and PKA are required for full induction of GABA A receptor δ subunit expression in response to AMPA receptor stimulation.

AMPA receptors serum-dependently mediate GABA A receptor α1 and α6 subunit down-regulation in cultured mouse cerebellar granule cells

Depolarization of cultured mouse cerebellar granule cells with potassium or kainate results in developmentally arrested state that includes down-regulation of GABA A receptor α1, α6 and β2 subunit expression. These subunits are normally strongly expressed in cerebellar granule cells from second postnatal week throughout the adulthood. In the present study we demonstrate that selective activation of AMPA subtype of glutamate receptors down-regulates α1 and α6 subunit mRNA expression. Removal of AMPA agonist from culture medium restores expression of these subunits indicating reversibility of the down-regulation. In serum-free culture medium AMPA receptor activation did not down-regulate α1 or α6 subunit expression. Furthermore, the down-regulation was strongly attenuated when the cells were cultured in the presence of dialysed fetal calf serum. The results indicate that down-regulation of GABA A receptor α1 and α6 subunits by AMPA receptor activation is dependent on the presence of low molecular weight compounds present in fetal calf serum. In order to study mouse cerebellar granule cell maturation and/or regulation of GABA A receptor subunit expression in culture, the experiments should be performed in the absence of fetal calf serum.

Amyloid-β and aluminum ions enhance neuronal damage mediated by NMDA-activated glutamate receptors

Effects of N-methyl-D-aspartate (NMDA), aluminum and amyloid-β (Aβ) on mouse cerebellar granule cells were studied. In the presence of all three compounds, reactive oxygen species levels and cell necrosis were increased dramatically. Mg2+ ions and D-AP5, which are known to prevent ligand binding to NMDA-activated glutamate receptors, were effective in attenuating the neurotoxic effect induced by the presence of all three compounds. All substances tested induced activation of p42/44 MAPK (mitogen activated protein kinase) with no cumulative effects between them. We conclude that neurotoxicity induced by aluminum and Aβ appears at outer cell membranes and NMDA receptors take part in this process. Increase in excitotoxic effect of glutamate in the presence of aluminum and Aβ is suggested to be a factor which provokes Alzheimer’s disease in brain neurons.

Neuroligin-2 accelerates GABAergic synapse maturation in cerebellar granule cells

Neuroligins (NLGs) are postsynaptic cell adhesion molecules that are thought to function in synaptogenesis. To investigate the role of NLGs on synaptic transmission once the synapse is formed, we transfected neuroligin-2 (NLG-2) in cultured mouse cerebellar granule cells (CGCs), and recorded GABA A (γ-aminobutyric acid) receptor mediated miniature postsynaptic currents (mIPSCs). NLG-2 transfected cells had mIPSCs with faster decay than matching GFP expressing controls at young culture ages (days in vitro, DIV7–8). Down-regulation of NLG-2 by the isoform specific shRNA-NLG-2 resulted in an opposite effect. We and others have shown that the switch of α subunits of GABA ARs from α2/3 to α1 underlies developmental speeding of the IPSC decay in various CNS regions, including the cerebellum. To assess whether the reduced decay time of mIPSCs by NLG-2 is due to the recruitment of more α1 containing GABA ARs at the synapses, we examined the prolongation of current decay by the Zolpidem, which has been shown to preferentially enhance the activity of α1 subunit-containing GABA channel. The application of Zolpidem resulted in a significantly greater prolongation kinetics of synaptic currents in NLG-2 over-expressing cells than control cells, suggesting that NLG-2 over-expression accelerates synapse maturation by promoting incorporation of the α1 subunit-containing GABA ARs at postsynaptic sites in immature cells. In addition, the effect of NLG-2 on the speeding of decay time course of synaptic currents was abolished when we used CGC cultures from α1−/− mice. Lastly, to exclude the possibility that the fast decay of mIPSCs induced by NLG-2 could be also due to the impacts of NLG-2 on the GABA transient in synaptic cleft, we measured the sensitivity of mIPSCs to the fast-off competitive antagonists TPMPA. We found that TPMPA similarly inhibits mIPSCs in control and NLG-2 over-expressing CGCs both at young age (DIV8) and old age (DIV14) of cultures. However, we confirm our previous finding of a greater inhibition of mIPSCs in young (DIV8) than more mature (DIV14) cultures. Together, our results suggest that NLG-2 does not alter uniquantal GABA release, and the fast decay of mIPSC induced by NLG-2 is due to the differential expression of postsynaptic GABA A receptor subtypes. Taken all together, we propose that NLG-2 plays important functional role in inhibitory synapse development and maturation.

Autoinactivation of Neuronal AMPA Receptors via Glutamate-Regulated TARP Interaction

Neuronal AMPA receptors autoinactivate at high concentrations of glutamate, i.e., the current declines at glutamate concentrations above 10-100 microM. The mechanisms underlying this phenomenon are unclear. Stargazin-like TARPs are AMPA receptor auxiliary subunits that modulate receptor trafficking and channel properties. Here, we found that neuronal AMPA receptors and recombinant AMPA receptors coexpressed with stargazin autoinactivate at high concentrations of glutamate, whereas recombinant AMPA receptors expressed alone do not. The reduction of currents at high glutamate concentrations is not associated with a reduction of AMPA receptor number, but rather with the loss of stargazin-associated allosteric modulation of channel gating. We show that receptor desensitization promotes the dissociation of TARP-AMPA receptor complexes in a few milliseconds. This dissociation mechanism contributes to synaptic short-term modulation. The results demonstrate a mechanism for dynamic regulation of AMPA receptor activity to tune synaptic strength.

Mechanism of subunit switching from NR2B to NR2C in mouse cerebellar granule cell cultures

To understand brain functions, it is important to observe directly how multiple neural circuits are performing in living brains. However, due to tissue opaqueness, observable depth and spatiotemporal resolution are severely degraded in vivo. Here, we propose an optical brain clearing method for in vivo fluorescence microscopy, termed MAGICAL (magical additive glycerol improves clear alive luminance). MAGICAL enabled two-photon microscopy to capture vivid images with fast speed, at cortical layer V and hippocampal CA1 in vivo. Moreover, MAGICAL promoted conventional confocal microscopy to visualize finer neuronal structures including synaptic boutons and spines in unprecedented deep regions, without intensive illumination leading to phototoxic effects. Fluorescence emission spectrum transmissive analysis showed that MAGICAL improved in vivo transmittance of shorter wavelength light, which is vulnerable to optical scattering, thus unsuited for in vivo microscopy. These results suggest that MAGICAL would transparentize living brains via scattering reduction.

Dual regulation of NR2B and NR2C expression by NMDA receptor activation in mouse cerebellar granule cell cultures

In the developing cerebellum, switching of the subunit composition of NMDA receptors occurs in granule cells from NR2B-containing receptors to NR2C-containing ones. We investigated the mechanisms underlying switching of NR2B and NR2C subunit composition in primary cultures of mouse granule cells at the physiological KCl concentration (5 mM). Granule cells extensively extended their neuritic processes 48 h after having been cultured in serum-free medium containing 5 mM KCl. Consistent with this morphological change, NR2B mRNA and NR2C mRNA were down- and up-regulated, respectively, in the granule cells. This dual regulation of the two mRNAs was abrogated by blocking excitation of granule cells with TTX. This neuronal activity-dependent regulation of NR2B and NR2C mRNAs was abolished by the addition of selective antagonists of AMPA receptors and NMDA receptors. Furthermore, the dual regulation of NR2B and NR2C mRNAs in TTX-treated cells was restored by the addition of NMDA in the presence of the AMPA receptor antagonist, but not by that of AMPA in the presence of the NMDA receptor antagonist. Importantly, the NMDA receptor activation drove the NR2B/NR2C switching of NMDA receptors in the cell-surface membrane of granule cells. This investigation demonstrates that stimulation of NMDA receptors in conjunction with the AMPA receptor-mediated excitation of granule cells plays a key role in functional subunit switching of NMDA receptors in maturing granule cells at the physiological KCl concentration.

Novel compounds selectively enhance δ subunit containing GABAA receptors and increase tonic currents in thalamus

Inhibition in the brain is dominated by the neurotransmitter γ-aminobutyric acid (GABA); operating through GABAA receptors. This form of neural inhibition was presumed to be mediated by synaptic receptors, however recent evidence has highlighted a previously unappreciated role for extrasynaptic GABAA receptors in controlling neuronal activity. Synaptic and extrasynaptic GABAA receptors exhibit distinct pharmacological and biophysical properties that differentially influence brain physiology and behavior. Here we used a fluorescence-based assay and cell lines expressing recombinant GABAA receptors to identify a novel series of benzamide compounds that selectively enhance, or activate α4β3δ GABAA receptors (cf. α4β3γ2 and α1β3γ2). Utilising electrophysiological methods, we illustrate that one of these compounds, 4-chloro-N-[6,8-dibromo-2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide (DS1) potently (low nM) enhances GABA-evoked currents mediated by α4β3δ receptors. At similar concentrations DS1 directly activates this receptor and is the most potent known agonist of α4β3δ receptors. 4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide (DS2) selectively potentiated GABA responses mediated by α4β3δ receptors, but was not an agonist.Recent studies have revealed a tonic form of inhibition in thalamus mediated by the α4β2δ extrasynaptic GABAA receptors that may contribute to the regulation of thalamocortical rhythmic activity associated with sleep, wakefulness, vigilance and seizure disorders. In mouse thalamic relay cells DS2 enhanced the tonic current mediated by α4β2δ receptors with no effect on their synaptic GABAA receptors. Similarly, in mouse cerebellar granule cells DS2 potentiated the tonic current mediated by α6βδ receptors. DS2 is the first selective positive allosteric modulator of δ-GABAA receptors and such compounds potentially offer novel therapeutic opportunities as analgesics and in the treatment of sleep disorders. Furthermore, these drugs may be valuable in elucidating the physiological and pathophysiological roles played by these extrasynaptic GABAA receptors.

XRCC1 protects against the lethality of induced oxidative DNA damage in nondividing neural cells

XRCC1 is a critical scaffold protein that orchestrates efficient single-strand break repair (SSBR). Recent data has found an association of XRCC1 with proteins causally linked to human spinocerebellar ataxias—aprataxin and tyrosyl-DNA phosphodiesterase 1—implicating SSBR in protection against neuronal cell loss and neurodegenerative disease. We demonstrate herein that shRNA lentiviral-mediated XRCC1 knockdown in human SH-SY5Y neuroblastoma cells results in a largely selective increase in sensitivity of the nondividing (i.e. terminally differentiated) cell population to the redox-cycling agents, menadione and paraquat; this reduced survival was accompanied by an accumulation of DNA strand breaks. Using hypoxanthine–xanthine oxidase as the oxidizing method, XRCC1 deficiency affected both dividing and nondividing SH-SY5Y cells, with a greater effect on survival seen in the former case, suggesting that the spectrum of oxidative DNA damage created dictates the specific contribution of XRCC1 to cellular resistance. Primary XRCC1 heterozygous mouse cerebellar granule cells exhibit increased strand break accumulation and reduced survival due to increased apoptosis following menadione treatment. Moreover, knockdown of XRCC1 in primary human fetal brain neurons leads to enhanced sensitivity to menadione, as indicated by increased levels of DNA strand breaks relative to control cells. The cumulative results implicate XRCC1, and more broadly SSBR, in the protection of nondividing neuronal cells from the genotoxic consequences of oxidative stress.