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1.
Elife ; 122024 Feb 15.
Article in English | MEDLINE | ID: mdl-38358390

ABSTRACT

The transcription factor Bcl11b has been linked to neurodevelopmental and neuropsychiatric disorders associated with synaptic dysfunction. Bcl11b is highly expressed in dentate gyrus granule neurons and is required for the structural and functional integrity of mossy fiber-CA3 synapses. The underlying molecular mechanisms, however, remained unclear. We show in mice that the synaptic organizer molecule C1ql2 is a direct functional target of Bcl11b that regulates synaptic vesicle recruitment and long-term potentiation at mossy fiber-CA3 synapses in vivo and in vitro. Furthermore, we demonstrate C1ql2 to exert its functions through direct interaction with a specific splice variant of neurexin-3, Nrxn3(25b+). Interruption of C1ql2-Nrxn3(25b+) interaction by expression of a non-binding C1ql2 mutant or by deletion of Nrxn3 in the dentate gyrus granule neurons recapitulates major parts of the Bcl11b as well as C1ql2 mutant phenotype. Together, this study identifies a novel C1ql2-Nrxn3(25b+)-dependent signaling pathway through which Bcl11b controls mossy fiber-CA3 synapse function. Thus, our findings contribute to the mechanistic understanding of neurodevelopmental disorders accompanied by synaptic dysfunction.


The human brain contains billions of neurons working together to process the vast array of information we receive from our environment. These neurons communicate at junctions known as synapses, where chemical packages called vesicles released from one neuron stimulate a response in another. This synaptic communication is crucial for our ability to think, learn and remember. However, this activity depends on a complex interplay of proteins, whose balance and location within the neuron are tightly controlled. Any disruption to this delicate equilibrium can cause significant problems, including neurodevelopmental and neuropsychiatric disorders, such as schizophrenia and intellectual disability. One key regulator of activity at the synapse is a protein called Bcl11b, which has been linked to conditions affected by synaptic dysfunction. It plays a critical role in maintaining specific junctions known as mossy fibre synapses, which are important for learning and memory. One of the genes regulated by Bcl11b is C1ql2, which encodes for a synaptic protein. However, it is unclear what molecular mechanisms Bcl11b uses to carry out this role. To address this, Koumoundourou et al. explored the role of C1ql2 in mossy fibre synapses of adult mice. Experiments to manipulate the production of C1ql2 independently of Bcl11b revealed that C1ql2 is vital for recruiting vesicles to the synapse and strengthening synaptic connections between neurons. Further investigation showed that C1ql2's role in this process relies on interacting with another synaptic protein called neurexin-3. Disrupting this interaction reduced the amount of C1ql2 at the synapse and, consequently, impaired vesicle recruitment. These findings will help our understanding of how neurodevelopmental and neuropsychiatric disorders develop. Bcl11b, C1ql2 and neurexin-3 have been independently associated with these conditions, and the now-revealed interactions between these proteins offer new insights into the molecular basis of synaptic faults. This research opens the door to further study of how these proteins interact and their roles in brain health and disease.


Subject(s)
Mossy Fibers, Hippocampal , Synapses , Animals , Mice , Transcription Factors , Synaptic Vesicles , Tumor Suppressor Proteins , Repressor Proteins
2.
Front Cell Neurosci ; 17: 1253424, 2023.
Article in English | MEDLINE | ID: mdl-37881493

ABSTRACT

K+/Cl- cotransporter 2 (KCC2) is a major Cl- extruder in mature neurons and is responsible for the establishment of low intracellular [Cl-], necessary for fast hyperpolarizing GABAA-receptor mediated synaptic inhibition. Electrogenic sodium bicarbonate cotransporter 1 (NBCe1) is a pH regulatory protein expressed in neurons and glial cells. An interactome study identified NBCe1 as a possible interaction partner of KCC2. In this study, we investigated the putative effect of KCC2/NBCe1 interaction in baseline and the stimulus-induced phosphorylation pattern and function of KCC2. Primary mouse hippocampal neuronal cultures from wildtype (WT) and Nbce1-deficient mice, as well as HEK-293 cells stably transfected with KCC2WT, were used. The results show that KCC2 and NBCe1 are interaction partners in the mouse brain. In HEKKCC2 cells, pharmacological inhibition of NBCs with S0859 prevented staurosporine- and 4-aminopyridine (4AP)-induced KCC2 activation. In mature cultures of hippocampal neurons, however, S0859 completely inhibited postsynaptic GABAAR and, thus, could not be used as a tool to investigate the role of NBCs in GABA-dependent neuronal networks. In Nbce1-deficient immature hippocampal neurons, baseline phosphorylation of KCC2 at S940 was downregulated, compared to WT, and exposure to staurosporine failed to reduce pKCC2 S940 and T1007. In Nbce1-deficient mature neurons, baseline levels of pKCC2 S940 and T1007 were upregulated compared to WT, whereas after 4AP treatment, pKCC2 S940 was downregulated, and pKCC2 T1007 was further upregulated. Functional experiments showed that the levels of GABAAR reversal potential, baseline intracellular [Cl-], Cl- extrusion, and baseline intracellular pH were similar between WT and Nbce1-deficient neurons. Altogether, our data provide a primary description of the properties of KCC2/NBCe1 protein-protein interaction and implicate modulation of stimulus-mediated phosphorylation of KCC2 by NBCe1/KCC2 interaction-a mechanism with putative pathophysiological relevance.

3.
Int J Mol Sci ; 22(21)2021 Oct 28.
Article in English | MEDLINE | ID: mdl-34769111

ABSTRACT

Characterization of new pharmacological targets is a promising approach in research of neurorepair mechanisms. The G protein-coupled receptor 17 (GPR17) has recently been proposed as an interesting pharmacological target, e.g., in neuroregenerative processes. Using the well-established ex vivo model of organotypic slice co-cultures of the mesocortical dopaminergic system (prefrontal cortex (PFC) and substantia nigra/ventral tegmental area (SN/VTA) complex), the influence of GPR17 ligands on neurite outgrowth from SN/VTA to the PFC was investigated. The growth-promoting effects of Montelukast (MTK; GPR17- and cysteinyl-leukotriene receptor antagonist), the glial cell line-derived neurotrophic factor (GDNF) and of two potent, selective GPR17 agonists (PSB-16484 and PSB-16282) were characterized. Treatment with MTK resulted in a significant increase in mean neurite density, comparable with the effects of GDNF. The combination of MTK and GPR17 agonist PSB-16484 significantly inhibited neuronal growth. qPCR studies revealed an MTK-induced elevated mRNA-expression of genes relevant for neuronal growth. Immunofluorescence labelling showed a marked expression of GPR17 on NG2-positive glia. Western blot and RT-qPCR analysis of untreated cultures suggest a time-dependent, injury-induced stimulation of GPR17. In conclusion, MTK was identified as a stimulator of neurite fibre outgrowth, mediating its effects through GPR17, highlighting GPR17 as an interesting therapeutic target in neuronal regeneration.


Subject(s)
Acetates/pharmacology , Cyclopropanes/pharmacology , Leukotriene Antagonists/pharmacology , Neuronal Outgrowth/drug effects , Quinolines/pharmacology , Receptors, G-Protein-Coupled/metabolism , Sulfides/pharmacology , Animals , Animals, Newborn , Coculture Techniques , Drug Evaluation, Preclinical , Female , Male , Nerve Regeneration/drug effects , Neuronal Outgrowth/genetics , Rats
4.
Front Cell Neurosci ; 13: 389, 2019.
Article in English | MEDLINE | ID: mdl-31551711

ABSTRACT

Zika virus (ZIKV) infection of pregnant women and diaplazental transmission to the fetus is linked to the congenital syndrome of microcephaly in newborns. This neuropathology is believed to result from significant death of neuronal progenitor cells (NPC). Here, we examined the fate of neurons in the developing hippocampus, a brain structure which houses neuronal populations of different maturation states. For this purpose, we infected hippocampal slice cultures from immunocompetent newborn mice with ZIKV and monitored changes in hippocampal architecture. In neurons of all hippocampal subfields ZIKV was detected by immunofluorescence labeling and electron microscopy. This includes pyramidal neurons that maturate during the embryonic phase. In the dentate gyrus, ZIKV could be found in the Cajal-Retzius (CR) cells which belong to the earliest born cortical neurons, but also in granule cells that are predominantly generated postnatally. Intriguingly, virus particles were also present in the correctly outgrowing mossy fiber axons of juvenile granule cells, suggesting that viral infection does not impair region- and layer-specific formation of this projection. ZIKV infection of hippocampal tissue was accompanied by both a profound astrocyte reaction indicating tissue injury and a microglia response suggesting phagocytotic activity. Furthermore, depending on the viral load and incubation time, we observed extensive overall neuronal loss in the cultured hippocampal slice cultures. Thus, we conclude ZIKV can replicate in various neuronal populations and trigger neuronal death independent of the maturation state of infected cells.

5.
Cell Rep ; 23(10): 2976-2988, 2018 06 05.
Article in English | MEDLINE | ID: mdl-29874584

ABSTRACT

While mitochondrial dysfunction is emerging as key in Parkinson's disease (PD), a central question remains whether mitochondria are actual disease drivers and whether boosting mitochondrial biogenesis and function ameliorates pathology. We address these questions using patient-derived induced pluripotent stem cells and Drosophila models of GBA-related PD (GBA-PD), the most common PD genetic risk. Patient neurons display stress responses, mitochondrial demise, and changes in NAD+ metabolism. NAD+ precursors have been proposed to ameliorate age-related metabolic decline and disease. We report that increasing NAD+ via the NAD+ precursor nicotinamide riboside (NR) significantly ameliorates mitochondrial function in patient neurons. Human neurons require nicotinamide phosphoribosyltransferase (NAMPT) to maintain the NAD+ pool and utilize NRK1 to synthesize NAD+ from NAD+ precursors. Remarkably, NR prevents the age-related dopaminergic neuronal loss and motor decline in fly models of GBA-PD. Our findings suggest NR as a viable clinical avenue for neuroprotection in PD and other neurodegenerative diseases.


Subject(s)
Drosophila melanogaster/physiology , Induced Pluripotent Stem Cells/pathology , Mitochondria/pathology , NAD/metabolism , Neurons/metabolism , Neurons/pathology , Niacinamide/analogs & derivatives , Parkinson Disease/pathology , Animals , Autophagy , Disease Models, Animal , Dopaminergic Neurons/metabolism , Dopaminergic Neurons/pathology , Endoplasmic Reticulum Stress , Glucosylceramidase/metabolism , Humans , Mitochondria/metabolism , Mitochondria/ultrastructure , Mitochondrial Dynamics , Motor Activity , Niacinamide/metabolism , Parkinson Disease/physiopathology , Pyridinium Compounds , Unfolded Protein Response
6.
Front Mol Neurosci ; 11: 103, 2018.
Article in English | MEDLINE | ID: mdl-29674952

ABSTRACT

Structural and functional plasticity of synapses are critical neuronal mechanisms underlying learning and memory. While activity-dependent regulation of synaptic strength has been extensively studied, much less is known about the transcriptional control of synapse maintenance and plasticity. Hippocampal mossy fiber (MF) synapses connect dentate granule cells to CA3 pyramidal neurons and are important for spatial memory formation and consolidation. The transcription factor Bcl11b/Ctip2 is expressed in dentate granule cells and required for postnatal hippocampal development. Ablation of Bcl11b/Ctip2 in the adult hippocampus results in impaired adult neurogenesis and spatial memory. The molecular mechanisms underlying the behavioral impairment remained unclear. Here we show that selective deletion of Bcl11b/Ctip2 in the adult mouse hippocampus leads to a rapid loss of excitatory synapses in CA3 as well as reduced ultrastructural complexity of remaining mossy fiber boutons (MFBs). Moreover, a dramatic decline of long-term potentiation (LTP) of the dentate gyrus-CA3 (DG-CA3) projection is caused by adult loss of Bcl11b/Ctip2. Differential transcriptomics revealed the deregulation of genes associated with synaptic transmission in mutants. Together, our data suggest Bcl11b/Ctip2 to regulate maintenance and function of MF synapses in the adult hippocampus.

7.
Front Mol Neurosci ; 11: 30, 2018.
Article in English | MEDLINE | ID: mdl-29479305

ABSTRACT

Neuronal growth regulator 1 (NEGR1), a member of the immunoglobulin superfamily cell adhesion molecule subgroup IgLON, has been implicated in neuronal growth and connectivity. In addition, genetic variants in or near the NEGR1 locus have been associated with obesity and more recently with learning difficulties, intellectual disability and psychiatric disorders. However, experimental evidence is lacking to support a possible link between NEGR1, neuronal growth and behavioral abnormalities. Initial expression analysis of NEGR1 mRNA in C57Bl/6 wildtype (WT) mice by in situ hybridization demonstrated marked expression in the entorhinal cortex (EC) and dentate granule cells. In co-cultures of cortical neurons and NSC-34 cells overexpressing NEGR1, neurite growth of cortical neurons was enhanced and distal axons occupied an increased area of cells overexpressing NEGR1. Conversely, in organotypic slice co-cultures, Negr1-knockout (KO) hippocampus was less permissive for axons grown from EC of ß-actin-enhanced green fluorescent protein (EGFP) mice compared to WT hippocampus. Neuroanatomical analysis revealed abnormalities of EC axons in the hippocampal dentate gyrus (DG) of Negr1-KO mice including increased numbers of axonal projections to the hilus. Neurotransmitter receptor ligand binding densities, a proxy of functional neurotransmitter receptor abundance, did not show differences in the DG of Negr1-KO mice but altered ligand binding densities to NMDA receptor and muscarinic acetylcholine receptors M1 and M2 were found in CA1 and CA3. Activity behavior, anxiety-like behavior and sensorimotor gating were not different between genotypes. However, Negr1-KO mice exhibited impaired social behavior compared to WT littermates. Moreover, Negr1-KO mice showed reversal learning deficits in the Morris water maze and increased susceptibility to pentylenetetrazol (PTZ)-induced seizures. Thus, our results from neuronal growth assays, neuroanatomical analyses and behavioral assessments provide first evidence that deficiency of the psychiatric disease-associated Negr1 gene may affect neuronal growth and behavior. These findings might be relevant to further evaluate the role of NEGR1 in cognitive and psychiatric disorders.

8.
Glia ; 65(8): 1361-1375, 2017 08.
Article in English | MEDLINE | ID: mdl-28568893

ABSTRACT

The electrogenic sodium bicarbonate cotransporter NBCe1 (SLC4A4) expressed in astrocytes regulates intracellular and extracellular pH. Here, we introduce transforming growth factor beta (TGF-ß) as a novel regulator of NBCe1 transcription and functional expression. Using hippocampal slices and primary hippocampal and cortical astrocyte cultures, we investigated regulation of NBCe1 and elucidated the underlying signaling pathways by RT-PCR, immunoblotting, immunofluorescence, intracellular H(+ ) recording using the H(+ ) -sensitive dye 2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein, mink lung epithelial cell (MLEC) assay, and chromatin immunoprecipitation. Activation of TGF-ß signaling significantly upregulated transcript, protein, and surface expression of NBCe1. These effects were TGF-ß receptor-mediated and suppressed following inhibition of JNK and Smad signaling. Moreover, 4-aminopyridine (4AP)-dependent NBCe1 regulation requires TGF-ß. TGF-ß increased the rate and amplitude of intracellular H+ changes upon challenging NBCe1 in wild-type astrocytes but not in cortical astrocytes from Slc4a4-deficient mice. A Smad4 binding sequence was identified in the NBCe1 promoter and Smad4 binding increased after activation of TGF-ß signaling. The data show for the first time that NBCe1 is a direct target of TGF-ß/Smad4 signaling. Through activation of the canonical pathway TGF-ß acts directly on NBCe1 by binding of Smad4 to the NBCe1 promoter and regulating its transcription, followed by increased protein expression and transport activity.


Subject(s)
Astrocytes/metabolism , Gene Expression Regulation/physiology , Signal Transduction/physiology , Sodium-Bicarbonate Symporters/metabolism , Transforming Growth Factor beta/metabolism , 4-Aminopyridine/pharmacology , Aldehyde Dehydrogenase 1 Family , Animals , Benzamides/pharmacology , Cells, Cultured , Cerebral Cortex/cytology , Chloride-Bicarbonate Antiporters/pharmacology , Dioxoles/pharmacology , Female , Gene Expression Regulation/drug effects , Glial Fibrillary Acidic Protein/metabolism , Hippocampus/cytology , Hydrogen-Ion Concentration , Isoenzymes/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Potassium Channel Blockers/pharmacology , Retinal Dehydrogenase/metabolism , Signal Transduction/drug effects , Smad4 Protein/metabolism , Sodium-Bicarbonate Symporters/antagonists & inhibitors , Sodium-Bicarbonate Symporters/genetics , Transforming Growth Factor beta/genetics
9.
J Neurosci ; 36(18): 5084-93, 2016 05 04.
Article in English | MEDLINE | ID: mdl-27147660

ABSTRACT

UNLABELLED: The aggregation of amyloid-ß peptide (Aß) in brain is an early event and hallmark of Alzheimer's disease (AD). We combined the advantages of in vitro and in vivo approaches to study cerebral ß-amyloidosis by establishing a long-term hippocampal slice culture (HSC) model. While no Aß deposition was noted in untreated HSCs of postnatal Aß precursor protein transgenic (APP tg) mice, Aß deposition emerged in HSCs when cultures were treated once with brain extract from aged APP tg mice and the culture medium was continuously supplemented with synthetic Aß. Seeded Aß deposition was also observed under the same conditions in HSCs derived from wild-type or App-null mice but in no comparable way when HSCs were fixed before cultivation. Both the nature of the brain extract and the synthetic Aß species determined the conformational characteristics of HSC Aß deposition. HSC Aß deposits induced a microglia response, spine loss, and neuritic dystrophy but no obvious neuron loss. Remarkably, in contrast to in vitro aggregated synthetic Aß, homogenates of Aß deposits containing HSCs induced cerebral ß-amyloidosis upon intracerebral inoculation into young APP tg mice. Our results demonstrate that a living cellular environment promotes the seeded conversion of synthetic Aß into a potent in vivo seeding-active form. SIGNIFICANCE STATEMENT: In this study, we report the seeded induction of Aß aggregation and deposition in long-term hippocampal slice cultures. Remarkably, we find that the biological activities of the largely synthetic Aß aggregates in the culture are very similar to those observed in vivo This observation is the first to show that potent in vivo seeding-active Aß aggregates can be obtained by seeded conversion of synthetic Aß in a living (wild-type) cellular environment.


Subject(s)
Amyloid beta-Peptides/metabolism , Hippocampus/metabolism , Hippocampus/pathology , Plaque, Amyloid/metabolism , Plaque, Amyloid/pathology , Amyloid beta-Protein Precursor/metabolism , Amyloidosis/pathology , Animals , Female , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microglia/pathology , Neurites/pathology , Neurons/pathology , Organ Culture Techniques
10.
J Mol Neurosci ; 56(4): 773-781, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25645684

ABSTRACT

Endocannabinoid receptors CB1R and CB2R are present in the CNS and modulate synaptic activity. By using an in vitro model, two concentrations of CB1R agonist ACEA at 0.5 and 5 µM doses and CB1R antagonist AM251 at 1 and 10 µM doses were administered in organotypic slice cultures of mouse hippocampus, and their effects on neurons and glial cells were analyzed at different time points. Exposure to low concentrations of ACEA (0.5 µM) did not seem to affect tissue organization, neuronal morphology, or glial response. In contrast, at a higher concentration of ACEA, many neurons in the dentate gyrus exhibited strong caspase-3 immunoreactivity. After treatment with AM251, we observed an increase in caspase-3 immunoreactivity and a downregulation of CB1R expression. Results show that long-term hippocampal slice cultures respond to both CB1R activation and inactivation by changing neuronal protein expression patterns. In the present study, we demonstrate that CB1R agonist ACEA promotes alterations in the neuronal cytoskeleton as well as changes in CB1R expression in organotypic hippocampal slice cultures, and that CB1R antagonist AM251 promotes neuronal death and astroglial reaction.


Subject(s)
Cannabinoids/metabolism , Hippocampus/drug effects , Receptor, Cannabinoid, CB1/antagonists & inhibitors , Receptor, Cannabinoid, CB2/antagonists & inhibitors , Animals , Arachidonic Acids/pharmacology , Caspase 3/metabolism , Cells, Cultured , Cytoskeleton/metabolism , Hippocampus/metabolism , Mice , Mice, Inbred C57BL , Piperidines/pharmacology , Pyrazoles/pharmacology , Receptor, Cannabinoid, CB1/genetics , Receptor, Cannabinoid, CB1/metabolism , Receptor, Cannabinoid, CB2/genetics , Receptor, Cannabinoid, CB2/metabolism
11.
Proc Natl Acad Sci U S A ; 111(45): 16124-9, 2014 Nov 11.
Article in English | MEDLINE | ID: mdl-25349433

ABSTRACT

Extracellular soluble signals are known to play a critical role in maintaining neuronal function and homeostasis in the CNS. However, the CNS is also composed of extracellular matrix macromolecules and glia support cells, and the contribution of the physical attributes of these components in maintenance and regulation of neuronal function is not well understood. Because these components possess well-defined topography, we theorize a role for topography in neuronal development and we demonstrate that survival and function of hippocampal neurons and differentiation of telencephalic neural stem cells is modulated by nanoroughness. At roughnesses corresponding to that of healthy astrocytes, hippocampal neurons dissociated and survived independent from astrocytes and showed superior functional traits (increased polarity and calcium flux). Furthermore, telencephalic neural stem cells differentiated into neurons even under exogenous signals that favor astrocytic differentiation. The decoupling of neurons from astrocytes seemed to be triggered by changes to astrocyte apical-surface topography in response to nanoroughness. Blocking signaling through mechanosensing cation channels using GsMTx4 negated the ability of neurons to sense the nanoroughness and promoted decoupling of neurons from astrocytes, thus providing direct evidence for the role of nanotopography in neuron-astrocyte interactions. We extrapolate the role of topography to neurodegenerative conditions and show that regions of amyloid plaque buildup in brain tissue of Alzheimer's patients are accompanied by detrimental changes in tissue roughness. These findings suggest a role for astrocyte and ECM-induced topographical changes in neuronal pathologies and provide new insights for developing therapeutic targets and engineering of neural biomaterials.


Subject(s)
Alzheimer Disease/metabolism , Calcium Channels/metabolism , Cell Communication , Mechanotransduction, Cellular , Neurons/metabolism , Alzheimer Disease/mortality , Animals , Astrocytes/pathology , Cell Differentiation , Humans , Intercellular Signaling Peptides and Proteins , Male , Neurons/pathology , PC12 Cells , Peptides/pharmacology , Rats , Spider Venoms/pharmacology
12.
PLoS One ; 8(10): e77258, 2013.
Article in English | MEDLINE | ID: mdl-24167567

ABSTRACT

Ischemic and traumatic brain injury is associated with increased risk for death and disability. The inhibition of penumbral tissue damage has been recognized as a target for therapeutic intervention, because cellular injury evolves progressively upon ATP-depletion and loss of ion homeostasis. In patients, thiopental is used to treat refractory intracranial hypertension by reducing intracranial pressure and cerebral metabolic demands; however, therapeutic benefits of thiopental-treatment are controversially discussed. In the present study we identified fundamental neuroprotective molecular mechanisms mediated by thiopental. Here we show that thiopental inhibits global protein synthesis, which preserves the intracellular energy metabolite content in oxygen-deprived human neuronal SK-N-SH cells or primary mouse cortical neurons and thus ameliorates hypoxic cell damage. Sensitivity to hypoxic damage was restored by pharmacologic repression of eukaryotic elongation factor 2 kinase. Translational inhibition was mediated by calcium influx, activation of the AMP-activated protein kinase, and inhibitory phosphorylation of eukaryotic elongation factor 2. Our results explain the reduction of cerebral metabolic demands during thiopental treatment. Cycloheximide also protected neurons from hypoxic cell death, indicating that translational inhibitors may generally reduce secondary brain injury. In conclusion our study demonstrates that therapeutic inhibition of global protein synthesis protects neurons from hypoxic damage by preserving energy balance in oxygen-deprived cells. Molecular evidence for thiopental-mediated neuroprotection favours a positive clinical evaluation of barbiturate treatment. The chemical structure of thiopental could represent a pharmacologically relevant scaffold for the development of new organ-protective compounds to ameliorate tissue damage when oxygen availability is limited.


Subject(s)
Hypnotics and Sedatives/pharmacology , Neurons/metabolism , Peptide Elongation Factor 2/metabolism , Protein Biosynthesis/drug effects , Thiopental/pharmacology , Animals , Brain Injuries/drug therapy , Brain Injuries/metabolism , Brain Injuries/pathology , Brain Ischemia/drug therapy , Brain Ischemia/metabolism , Brain Ischemia/pathology , Cell Death/drug effects , Cell Hypoxia/drug effects , Cell Line , Elongation Factor 2 Kinase/metabolism , Humans , Mice , Neurons/pathology , Oxygen/metabolism
13.
J Pharmacol Exp Ther ; 347(3): 781-93, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24049063

ABSTRACT

Oxygen deprivation during ischemic or hemorrhagic stroke results in ATP depletion, loss of ion homeostasis, membrane depolarization, and excitotoxicity. Pharmacologic restoration of cellular energy supply may offer a promising concept to reduce hypoxic cell injury. In this study, we investigated whether carbimazole, a thionamide used to treat hyperthyroidism, reduces neuronal cell damage in oxygen-deprived human SK-N-SH cells or primary cortical neurons. Our results revealed that carbimazole induces an inhibitory phosphorylation of eukaryotic elongation factor 2 (eEF2) that was associated with a marked inhibition of global protein synthesis. Translational inhibition resulted in significant bioenergetic savings, preserving intracellular ATP content in oxygen-deprived neuronal cells and diminishing hypoxic cellular damage. Phosphorylation of eEF2 was mediated by AMP-activated protein kinase and eEF2 kinase. Carbimazole also induced a moderate calcium influx and a transient cAMP increase. To test whether translational inhibition generally diminishes hypoxic cell damage when ATP availability is limiting, the translational repressors cycloheximide and anisomycin were used. Cycloheximide and anisomycin also preserved ATP content in hypoxic SK-N-SH cells and significantly reduced hypoxic neuronal cell damage. Taken together, these data support a causal relation between the pharmacologic inhibition of global protein synthesis and efficient protection of neurons from ischemic damage by preservation of high-energy metabolites in oxygen-deprived cells. Furthermore, our results indicate that carbimazole or other translational inhibitors may be interesting candidates for the development of new organ-protective compounds. Their chemical structure may be used for computer-assisted drug design or screening of compounds to find new agents with the potential to diminish neuronal damage under ATP-limited conditions.


Subject(s)
Antithyroid Agents/pharmacology , Carbimazole/pharmacology , Cell Hypoxia/drug effects , Neurons/drug effects , Protein Synthesis Inhibitors , AMP-Activated Protein Kinases/metabolism , Adenosine Triphosphate/metabolism , Autoradiography , Blotting, Western , Calcium/metabolism , Caspases/metabolism , Cell Line , Cell Survival/drug effects , Cyclic AMP/metabolism , Elongation Factor 2 Kinase/metabolism , Energy Metabolism/drug effects , Humans , Phosphorylation
14.
Cell Mol Life Sci ; 70(22): 4399-410, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23793543

ABSTRACT

Borna disease virus (BDV) persistently infects neurons of the central nervous system of various hosts, including rats. Since type I IFN-mediated antiviral response efficiently blocks BDV replication in primary rat embryo fibroblasts, it has been speculated that BDV is not effectively sensed by the host innate immune system in the nervous system. To test this assumption, organotypical rat hippocampal slice cultures were infected with BDV for up to 4 weeks. This resulted in the secretion of IFN and the up-regulation of IFN-stimulated genes. Using the rat Mx protein as a specific marker for IFN-induced gene expression, astrocytes and microglial cells were found to be Mx positive, whereas neurons, the major cell type in which BDV is replicating, lacked detectable levels of Mx protein. In uninfected cultures, neurons also remained Mx negative even after treatment with high concentrations of IFN-α. This non-responsiveness correlated with a lack of detectable nuclear translocation of both pSTAT1 and pSTAT2 in these cells. Consistently, neuronal dissemination of BDV was not prevented by treatment with IFN-α. These data suggest that the poor innate immune response in rat neurons renders this cell type highly susceptible to BDV infection even in the presence of exogenous IFN-α. Intriguingly, in contrast to rat neurons, IFN-α treatment of mouse neurons resulted in the up-regulation of Mx proteins and block of BDV replication, indicating species-specific differences in the type I IFN response of neurons between mice and rats.


Subject(s)
Borna Disease/immunology , Borna disease virus/physiology , Immunity, Innate/physiology , Neurons/metabolism , Animals , Astrocytes/cytology , Astrocytes/metabolism , Borna Disease/metabolism , Borna Disease/virology , Hippocampus/cytology , Hippocampus/metabolism , Interferon-alpha/pharmacology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Microglia/cytology , Microglia/metabolism , Myxovirus Resistance Proteins/metabolism , Neurons/cytology , Neurons/virology , Phosphorylation , Rats , Rats, Inbred Lew , Rats, Sprague-Dawley , STAT1 Transcription Factor/metabolism , STAT2 Transcription Factor/metabolism , Up-Regulation/drug effects , Virus Replication
15.
Front Cell Neurosci ; 7: 24, 2013.
Article in English | MEDLINE | ID: mdl-23504389

ABSTRACT

In cells cultured from neocortex of newborn rats, phosphoinositide-3-kinases of class I regulate the DNA synthesis in a subgroup of astroglial cells. We have studied the location of these cells as well as the kinase isoforms which facilitate the S phase entry. Using dominant negative (dn) isoforms as well as selective pharmacological inhibitors we quantified S phase entry by nuclear labeling with bromodeoxyuridine (BrdU). Only in astroglial cells harvested from the marginal zone (MZ) of the neocortex inhibition of phosphoinositide-3-kinases reduced the nuclear labeling with BrdU, indicating that neocortical astroglial cells differ in the regulation of proliferation. The two kinase isoforms p110α and p110ß were essential for S phase entry. p110α diminished the level of the p27(Kip1) which inactivates the complex of cyclin E and CDK2 necessary for entry into the S phase. p110ß phosphorylated and inhibited glycogen synthase kinase-3ß which can prevent S-phase entry. Taken together, both isoforms mediated S phase in a subgroup of neocortical astroglial cells and acted via distinct pathways.

16.
Mol Cell Neurosci ; 56: 10-7, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23403072

ABSTRACT

Besides mediating most of the fast excitatory neurotransmission in the mammalian CNS, ionotropic glutamate receptors of the AMPA subtype (AMPARs) serve highly diverse functions in brain development controlling neuronal migration, synaptic growth, and synaptic maturation. Pioneering proteomic studies suggest that this functional diversity is met by a great molecular complexity in native AMPAR composition. Here, we have investigated the expression patterns of two recently identified AMPAR constituents, the cornichon homologues CNIH-2 and CNIH-3, and their assembly with the AMPAR core subunits GluA1-4 in developing rat brain. Unlike GluA1-4 expression, which is up-regulated during postnatal brain development, the two cornichon homologues show maximum mRNA and protein expression early after birth, which then decline towards adulthood. Despite rather reciprocal expression profiles, the overall ratio of CNIH-2/3 complexed with GluAs remains constant throughout development. Our data reveal an excess amount of AMPAR-free CNIH-2/3 early in development, which might serve the evolutionarily conserved role of cornichon as a cargo exporter. With progressing development, however, the amount of AMPAR-free CNIH-2/3 subsides, whereas the one being integrated into AMPAR complexes increases. Hence, the cornichon homologues CNIH-2/3 gain importance in their role as auxiliary subunits of native AMPARs during ontogeny, which reflects their functional evolution in phylogeny.


Subject(s)
Receptors, AMPA/metabolism , Animals , Brain/growth & development , Brain/metabolism , Gene Expression Regulation, Developmental , Phylogeny , Protein Binding , Protein Multimerization , Protein Subunits/genetics , Protein Subunits/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Wistar , Receptors, AMPA/genetics
17.
Proc Natl Acad Sci U S A ; 110(5): 1899-904, 2013 Jan 29.
Article in English | MEDLINE | ID: mdl-23319640

ABSTRACT

Infection of newborn rats with Borne disease virus (BDV) results in selective degeneration of granule cell neurons of the dentate gyrus (DG). To study cellular countermechanisms that might prevent this pathology, we screened for rat strains resistant to this BDV-induced neuronal degeneration. To this end, we infected hippocampal slice cultures of different rat strains with BDV and analyzed for the preservation of the DG. Whereas infected cultures of five rat strains, including Lewis (LEW) rats, exhibited a disrupted DG cytoarchitecture, slices of three other rat strains, including Sprague-Dawley (SD), were unaffected. However, efficiency of viral replication was comparable in susceptible and resistant cultures. Moreover, these rat strain-dependent differences in vulnerability were replicated in vivo in neonatally infected LEW and SD rats. Intriguingly, conditioned media from uninfected cultures of both LEW and SD rats could prevent BDV-induced DG damage in infected LEW hippocampal cultures, whereas infection with BDV suppressed the availability of these factors from LEW but not in SD hippocampal cultures. To gain further insights into the genetic basis for this rat strain-dependent susceptibility, we analyzed DG granule cell survival in BDV-infected cultures of hippocampal neurons derived from the F1 and F2 offspring of the crossing of SD and LEW rats. Genome-wide association analysis revealed one resistance locus on chromosome (chr) 6q16 in SD rats and, surprisingly, a locus on chr3q21-23 that was associated with susceptibility. Thus, BDV-induced neuronal degeneration is dependent on the host genetic background and is prevented by soluble protective factors in the disease-resistant SD rat strain.


Subject(s)
Borna disease virus/physiology , Dentate Gyrus/virology , Nerve Degeneration/virology , Neurons/virology , Animals , Animals, Newborn , Biological Factors/chemistry , Biological Factors/pharmacology , Brain-Derived Neurotrophic Factor/pharmacology , Cell Survival/drug effects , Chromosome Mapping , Chromosomes, Mammalian/genetics , Culture Media, Conditioned/chemistry , Culture Media, Conditioned/pharmacology , Dentate Gyrus/metabolism , Dentate Gyrus/pathology , Disease Resistance/genetics , Female , Hippocampus/metabolism , Hippocampus/pathology , Hippocampus/virology , Host-Pathogen Interactions , Male , Nerve Degeneration/genetics , Nerve Degeneration/prevention & control , Neurons/metabolism , Neurons/pathology , Polymorphism, Single Nucleotide , Rats , Rats, Inbred Lew , Rats, Sprague-Dawley , Solubility , Species Specificity , Tissue Culture Techniques
18.
J Virol ; 86(20): 11223-30, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22875966

ABSTRACT

Beta interferon (IFN-ß) is a major component of innate immunity in mammals, but information on the in vivo source of this cytokine after pathogen infection is still scarce. To identify the cell types responsible for IFN-ß production during viral encephalitis, we used reporter mice that express firefly luciferase under the control of the IFN-ß promoter and stained organ sections with luciferase-specific antibodies. Numerous luciferase-positive cells were detected in regions of La Crosse virus (LACV)-infected mouse brains that contained many infected cells. Double-staining experiments with cell-type-specific markers revealed that similar numbers of astrocytes and microglia of infected brains were luciferase positive, whereas virus-infected neurons rarely contained detectable levels of luciferase. Interestingly, if a mutant LACV unable of synthesizing the IFN-antagonistic factor NSs was used for challenge, the vast majority of the IFN-ß-producing cells in infected brains were astrocytes rather than microglia. Similar conclusions were reached in a second series of experiments in which conditional reporter mice expressing the luciferase reporter gene solely in defined cell types were infected with wild-type or mutant LACV. Collectively, our data suggest that glial cells rather than infected neurons represent the major source of IFN-ß in LACV-infected mouse brains. They further indicate that IFN-ß synthesis in astrocytes and microglia is differentially affected by the viral IFN antagonist, presumably due to differences in LACV susceptibility of these two cell types.


Subject(s)
Astrocytes/immunology , Encephalitis, California/immunology , Interferon-beta/biosynthesis , La Crosse virus/immunology , Microglia/immunology , Animals , Astrocytes/metabolism , Astrocytes/virology , Brain/metabolism , Brain/virology , Interferon-beta/genetics , Interferon-beta/immunology , Luciferases, Firefly/biosynthesis , Luciferases, Firefly/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microglia/metabolism , Microglia/virology , Neurons/immunology , Neurons/metabolism , Neurons/virology , Promoter Regions, Genetic
19.
Cell Mol Life Sci ; 69(7): 1179-91, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22068610

ABSTRACT

During development, axonal projections have a remarkable ability to innervate correct dendritic subcompartments of their target neurons and to form regular neuronal circuits. Altered axonal targeting with formation of synapses on inappropriate neurons may result in neurodevelopmental sequelae, leading to psychiatric disorders. Here we show that altering the expression level of the polysialic acid moiety, which is a developmentally regulated, posttranslational modification of the neural cell adhesion molecule NCAM, critically affects correct circuit formation. Using a chemically modified sialic acid precursor (N-propyl-D: -mannosamine), we inhibited the polysialyltransferase ST8SiaII, the principal enzyme involved in polysialylation during development, at selected developmental time-points. This treatment altered NCAM polysialylation while NCAM expression was not affected. Altered polysialylation resulted in an aberrant mossy fiber projection that formed glutamatergic terminals on pyramidal neurons of the CA1 region in organotypic slice cultures and in vivo. Electrophysiological recordings revealed that the ectopic terminals on CA1 pyramids were functional and displayed characteristics of mossy fiber synapses. Moreover, ultrastructural examination indicated a "mossy fiber synapse"-like morphology. We thus conclude that homeostatic regulation of the amount of synthesized polysialic acid at specific developmental stages is essential for correct synaptic targeting and circuit formation during hippocampal development.


Subject(s)
Homeostasis , Neural Cell Adhesion Molecules/metabolism , Sialic Acids/metabolism , Synapses/metabolism , Animals , Mice , Mice, Inbred C57BL
20.
Cell Tissue Res ; 344(1): 13-6, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21331522

ABSTRACT

The neurotropic Borna disease virus (BDV) is unusual in that it can persistently infect neurons of the central nervous system (CNS) without causing general cell death, reflecting its favourable adaptation to the brain. The activity-dependent enhancement of neuronal network activity is however disturbed after BDV infection, possibly by its effect on the protein kinase C signalling pathway. The best model for studying BDV, which has a non-cytolytic replication strategy in primary neurons, is the rat. Infection of adult rats results in a fatal immune-mediated disease, whereas BDV establishes persistent infection of the brain in newborn rats resulting in progressive neuronal cell loss in defined regions of the CNS. Our recently developed system of BDV-infected hippocampal slice cultures has clearly shown that the onset of granule cell loss begins after the formation of the mossy fibre projection. Quantitative analysis has revealed a significant increase in synaptic density on identified remaining granule cell dendrites at 6 weeks after infection, followed by a decline. Granule cells are the major target of entorhinal afferents. However, despite an almost complete loss of dentate granule cells during BDV infection, entorhinal axons persist in their correct layer, both in vivo and in slice cultures, possibly exploiting rewiring capabilities and thereby allowing new synapse formation with available targets. These morphological observations, together with electrophysiological and biochemical data, indicate that BDV is a suitable model virus for studying virus-induced morphological and functional changes of neurons and connectivity patterns.


Subject(s)
Borna Disease/pathology , Borna disease virus/physiology , Brain/virology , Neurons/virology , Animals , Brain/pathology , Cells, Cultured , Host-Pathogen Interactions , Neurons/pathology , Rats , Rats, Inbred Lew
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