ABSTRACT
Hematopoietic stem and progenitor cells (HSPCs) and leukocytes circulate between the bone marrow (BM) and peripheral blood following circadian oscillations. Autonomic sympathetic noradrenergic signals have been shown to regulate HSPC and leukocyte trafficking, but the role of the cholinergic branch has remained unexplored. We have investigated the role of the cholinergic nervous system in the regulation of day/night traffic of HSPCs and leukocytes in mice. We show here that the autonomic cholinergic nervous system (including parasympathetic and sympathetic) dually regulates daily migration of HSPCs and leukocytes. At night, central parasympathetic cholinergic signals dampen sympathetic noradrenergic tone and decrease BM egress of HSPCs and leukocytes. However, during the daytime, derepressed sympathetic noradrenergic activity causes predominant BM egress of HSPCs and leukocytes via ß3-adrenergic receptor. This egress is locally supported by light-triggered sympathetic cholinergic activity, which inhibits BM vascular cell adhesion and homing. In summary, central (parasympathetic) and local (sympathetic) cholinergic signals regulate day/night oscillations of circulating HSPCs and leukocytes. This study shows how both branches of the autonomic nervous system cooperate to orchestrate daily traffic of HSPCs and leukocytes.
Subject(s)
Cell Movement , Cholinergic Agents/pharmacology , Circadian Rhythm , Hematopoietic Stem Cells/physiology , Leukocytes/physiology , Parasympathetic Nervous System/physiology , Sympathetic Nervous System/physiology , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Bone Marrow Cells/physiology , Cell Adhesion , Cells, Cultured , Chemotaxis , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Endothelium, Vascular/physiology , Female , Glial Cell Line-Derived Neurotrophic Factor Receptors/physiology , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Leukocytes/cytology , Leukocytes/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Adrenergic, beta-2 , Receptors, Adrenergic, beta-3/physiology , Receptors, G-Protein-Coupled/physiologyABSTRACT
In central respiratory circuitry, synaptic excitation is responsible for synchronizing neuronal activity in the different respiratory rhythm phases, whereas chloride-mediated inhibition is important for shaping the respiratory pattern itself. The potassium chloride cotransporter KCC2, which serves to maintain low intraneuronal Cl- concentration and thus render chloride-mediated synaptic signaling inhibitory, exists in two isoforms, KCC2a and KCC2b. KCC2 is essential for functional breathing motor control at birth, but the specific contribution of the KCC2a isoform remains unknown. Here, to address this issue, we investigated the respiratory phenotype of mice deficient for KCC2a. In vivo plethysmographic recordings revealed that KCC2a-deficient pups at P0 transiently express an abnormally low breathing rate and a high occurrence of apneas. Immunostainings confirmed that KCC2a is normally expressed in the brainstem neuronal groups involved in breathing (pre-Bötzinger complex, parafacial respiratory group, hypoglossus nucleus) and is absent in these regions in the KCC2a-/- mutant. However, in variously reduced in vitro medullary preparations, spontaneous rhythmic respiratory activity is similar to that expressed in wild-type preparations, as is hypoglossal motor output, and no respiratory pauses are detected, suggesting that the rhythm-generating networks are not intrinsically affected in mutants at P0. In contrast, inhibitory neuromodulatory influences exerted by the pons on respiratory rhythmogenesis are stronger in the mutant, thereby explaining the breathing anomalies observed in vivo. Thus, our results indicate that the KCC2a isoform is important for establishing proper breathing behavior at the time of birth, but by acting at sites that are extrinsic to the central respiratory networks themselves.
Subject(s)
Neurons/metabolism , Symporters/metabolism , Animals , Brain Stem/metabolism , Medulla Oblongata/metabolism , Mice, Inbred C57BL , Mice, Transgenic , Parturition/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Respiratory Rate , Symporters/genetics , K Cl- CotransportersABSTRACT
To understand the developmental trajectories in early lymphocyte differentiation, we identified differentially expressed surface markers on lineage-negative lymphoid progenitors (LPs). Single-cell polymerase chain reaction experiments allowed us to link surface marker expression to that of lineage-associated transcription factors (TFs) and identify GFRA2 and BST1 as markers of early B cells. Functional analyses in vitro and in vivo as well as single-cell gene expression analyses supported that surface expression of these proteins defined distinct subpopulations that include cells from both the classical common LPs (CLPs) and Fraction A compartments. The formation of the GFRA2-expressing stages of development depended on the TF EBF1, critical both for the activation of stage-specific target genes and modulation of the epigenetic landscape. Our data show that consecutive expression of Ly6D, GFRA2, and BST1 defines a developmental trajectory linking the CLP to the CD19+ progenitor compartment.
Subject(s)
B-Lymphocytes/cytology , B-Lymphocytes/immunology , Cell Compartmentation , Lymphopoiesis , Stem Cells/cytology , ADP-ribosyl Cyclase/metabolism , Animals , Antigens, CD/metabolism , Antigens, Ly/metabolism , Bone Marrow/metabolism , Cell Lineage , Cell Membrane/metabolism , GPI-Linked Proteins/metabolism , Glial Cell Line-Derived Neurotrophic Factor Receptors/metabolism , Mice , Models, BiologicalABSTRACT
The neuron-specific K-Cl cotransporter KCC2 maintains the low intracellular chloride concentration required for the fast hyperpolarizing responses of the inhibitory neurotransmitters γ-aminobutyric acid (GABA) and glycine. The two KCC2 isoforms, KCC2a and KCC2b differ by their N-termini as a result of alternative promoter usage. Whereas the role of KCC2b in mediating the chloride transport is unequivocal, the physiological role of KCC2a in neurons has remained obscure. We show that KCC2a isoform can decrease the intracellular chloride concentration in cultured neurons and attenuate calcium responses evoked by application of the GABAA receptor agonist muscimol. While the biotinylation assay detected both KCC2 isoforms at the cell surface of cultured neurons, KCC2a was not detected at the plasma membrane in immunostainings, suggesting that the N-terminal KCC2a epitope is masked. Confirming this hypothesis, KCC2a surface expression was detected by the C-terminal KCC2 pan antibody but not by the N-terminal KCC2a antibody in KCC2b-deficient neurons. One possible cause for the epitope masking is the binding site of Ste20-related proline-alanine-rich kinase (SPAK) in the KCC2a N-terminus. SPAK, a known regulator of K-Cl cotransporters, was co-immunoprecipitated in a complex with KCC2a but not KCC2b isoform. Moreover, SPAK overexpression decreased the transport activity of KCC2a but not that of KCC2b, as revealed by rubidium flux assay in HEK293 cells. Thus, our data indicate that both KCC2 isoforms perform as chloride cotransporters in neuronal cells, while their N-terminal heterogeneity could play an important role in fine-tuning of the K-Cl transport activity.
Subject(s)
Neurons/physiology , Symporters/physiology , Amino Acid Sequence , Animals , Female , HEK293 Cells , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Isoforms/physiology , Rats , K Cl- CotransportersABSTRACT
Synaptic inhibition depends on a transmembrane gradient of chloride, which is set by the neuron-specific K+-Cl- co-transporter KCC2. Reduced KCC2 levels in the neuronal membrane contribute to the generation of epilepsy, neuropathic pain, and autism spectrum disorders; thus, it is important to characterize the mechanisms regulating KCC2 expression. In the present study, we determined the role of KCC2-protein interactions in regulating total and surface membrane KCC2 expression. Using quantitative immunofluorescence in cultured mouse hippocampal neurons, we discovered that the kainate receptor subunit GluK2 and the auxiliary subunit Neto2 significantly increase the total KCC2 abundance in neurons but that GluK2 exclusively increases the abundance of KCC2 in the surface membrane. Using a live cell imaging assay, we further determined that KCC2 recycling primarily occurs within 1-2 h and that GluK2 produces an â¼40% increase in the amount of KCC2 recycled to the membrane during this time period. This GluK2-mediated increase in surface recycling translated to a significant increase in KCC2 expression in the surface membrane. Moreover, we found that KCC2 recycling is enhanced by protein kinase C-mediated phosphorylation of the GluK2 C-terminal residues Ser-846 and Ser-868. Lastly, using gramicidin-perforated patch clamp recordings, we found that the GluK2-mediated increase in KCC2 recycling to the surface membrane translates to a hyperpolarization of the reversal potential for GABA (EGABA). In conclusion, our results have revealed a mechanism by which kainate receptors regulate KCC2 expression in the hippocampus.
Subject(s)
Cell Membrane/metabolism , Hippocampus/metabolism , Membrane Potentials/physiology , Neurons/metabolism , Receptors, Kainic Acid/metabolism , Symporters/metabolism , Animals , Cell Membrane/genetics , Cells, Cultured , Hippocampus/cytology , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Knockout , Neurons/cytology , Receptors, Kainic Acid/genetics , Symporters/genetics , K Cl- Cotransporters , GluK2 Kainate ReceptorABSTRACT
Despite the variety of physiological and target-related functions, little is known regarding the cellular complexity in the sympathetic ganglion. We explored the heterogeneity of mouse stellate and thoracic ganglia and found an unexpected variety of cell types. We identified specialized populations of nipple- and pilo-erector muscle neurons. These neurons extended axonal projections and were born among other neurons during embryogenesis, but remained unspecialized until target organogenesis occurred postnatally. Target innervation and cell-type specification was coordinated by an intricate acquisition of unique combinations of growth factor receptors and the initiation of expression of concomitant ligands by the nascent erector muscles. Overall, our results provide compelling evidence for a highly sophisticated organization of the sympathetic nervous system into discrete outflow channels that project to well-defined target tissues and offer mechanistic insight into how diversity and connectivity are established during development.
Subject(s)
Motor Neurons/physiology , Muscle, Smooth/physiology , Neurons/physiology , Nipples/physiology , Piloerection/physiology , Animals , Cell Differentiation/physiology , Female , Ganglia, Sympathetic/physiology , Glial Cell Line-Derived Neurotrophic Factor Receptors/metabolism , Homeodomain Proteins/metabolism , Male , Mice , Neurons/metabolism , Proto-Oncogene Proteins c-ret/metabolism , Tumor Suppressor Proteins/metabolismABSTRACT
The four members of the LRRTM family (LRRTM1-4) are postsynaptic adhesion molecules essential for excitatory synapse development. They have also been implicated in neuropsychiatric diseases. Here, we focus on LRRTM3, showing that two distinct LRRTM3 variants generated by alternative splicing regulate LRRTM3 interaction with PSD-95, but not its excitatory synapse-promoting activity. Overexpression of either LRRTM3 variant increased excitatory synapse density in dentate gyrus (DG) granule neurons, whereas LRRTM3 knockdown decreased it. LRRTM3 also controlled activity-regulated AMPA receptor surface expression in an alternative splicing-dependent manner. Furthermore, Lrrtm3-knockout mice displayed specific alterations in excitatory synapse density, excitatory synaptic transmission and excitability in DG granule neurons but not in CA1 pyramidal neurons. Lastly, LRRTM3 required only specific splice variants of presynaptic neurexins for their synaptogenic activity. Collectively, our data highlight alternative splicing and differential presynaptic ligand utilization in the regulation of LRRTMs, revealing key regulatory mechanisms for excitatory synapse development.
Subject(s)
Alternative Splicing , Cell Adhesion Molecules, Neuronal/metabolism , Excitatory Postsynaptic Potentials , Animals , CA1 Region, Hippocampal/cytology , CA1 Region, Hippocampal/growth & development , CA1 Region, Hippocampal/metabolism , Cell Adhesion Molecules, Neuronal/genetics , Cells, Cultured , Dentate Gyrus/cytology , Dentate Gyrus/growth & development , Dentate Gyrus/metabolism , HEK293 Cells , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurogenesis , Protein Transport , Pyramidal Cells/metabolism , Pyramidal Cells/physiology , Rats , Receptors, AMPA/metabolism , Synapses/metabolism , Synapses/physiologyABSTRACT
Many primary sensory neurons in mouse dorsal root ganglia (DRG) express one or several GFRα's, the ligand-binding receptors of the GDNF family, and their common signaling receptor Ret. GFRα2, the principal receptor for neurturin, is expressed in most of the small nonpeptidergic DRG neurons, but also in some large DRG neurons that start to express Ret earlier. Previously, GFRα2 has been shown to be crucial for the soma size of small nonpeptidergic nociceptors and for their target innervation of glabrous epidermis. However, little is known about this receptor in other Ret-expressing DRG neuron populations. Here we have investigated two populations of Ret-positive low-threshold mechanoreceptors that innervate different types of hair follicles on mouse back skin: the small C-LTMRs and the large Aß-LTMRs. Using GFRα2-KO mice and immunohistochemistry we found that, similar to the nonpeptidergic nociceptors, GFRα2 controls the cell size but not the survival of both C-LTMRs and Aß-LTMRs. In contrast to the nonpeptidergic neurons, GFRα2 is not required for the target innervation of C-LTMRs and Aß-LTMRs in the back skin. These results suggest that different factors drive target innervation in these three populations of neurons. In addition, the observation that the large Ret-positive DRG neurons lack GFRα2 immunoreactivity in mature animals suggests that these neurons switch their GFRα signaling pathways during postnatal development.
Subject(s)
Glial Cell Line-Derived Neurotrophic Factor Receptors/metabolism , Sensory Receptor Cells/cytology , Sensory Receptor Cells/metabolism , Signal Transduction , Skin/cytology , Animals , Cell Size , Female , Ganglia, Spinal/cytology , Gene Knockout Techniques , Glial Cell Line-Derived Neurotrophic Factor Receptors/deficiency , Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics , Hair Follicle/innervation , Male , Mice , Neurofilament Proteins/metabolism , Proto-Oncogene Proteins c-ret/metabolism , Receptors, G-Protein-Coupled/metabolism , Tyrosine 3-Monooxygenase/metabolismABSTRACT
KCC2 is the neuron-specific K+-Cl(-) cotransporter required for maintaining low intracellular Cl(-), which is essential for fast inhibitory synaptic transmission in the mature CNS. Despite the requirement of KCC2 for inhibitory synaptic transmission, understanding of the cellular mechanisms that regulate KCC2 expression and function is rudimentary. We examined KCC2 in its native protein complex in vivo to identify key KCC2-interacting partners that regulate KCC2 function. Using blue native-polyacrylamide gel electrophoresis (BN-PAGE), we determined that native KCC2 exists in a macromolecular complex with kainate-type glutamate receptors (KARs). We found that KAR subunits are required for KCC2 oligomerization and surface expression. In accordance with this finding, acute and chronic genetic deletion of KARs decreased KCC2 function and weakened synaptic inhibition in hippocampal neurons. Our results reveal KARs as regulators of KCC2, significantly advancing our growing understanding of the tight interplay between excitation and inhibition.
Subject(s)
Chlorides/metabolism , Hippocampus/metabolism , Neurons/metabolism , Receptors, Kainic Acid/metabolism , Symporters/metabolism , Animals , Female , Hippocampus/cytology , Homeostasis , Male , Mice, Inbred C57BL , Neurons/cytology , K Cl- CotransportersABSTRACT
Leucine-rich repeat transmembrane neuronal proteins (LRRTMs) form in mammals a family of four postsynaptic adhesion proteins, which have been shown to bind neurexins and heparan sulphate proteoglycan (HSPG) glypican on the presynaptic side. Mutations in the genes encoding LRRTMs and neurexins are implicated in human cognitive disorders such as schizophrenia and autism. Our analysis shows that in most jawed vertebrates, lrrtm1, lrrtm2, and lrrtm3 genes are nested on opposite strands of large conserved intron of α-catenin genes ctnna2, ctnna1, and ctnna3, respectively. No lrrtm genes could be found in tunicates or lancelets, while two lrrtm genes are found in the lamprey genome, one of which is adjacent to a single ctnna homolog. Based on similar highly positive net charge of lamprey LRRTMs and the HSPG-binding LRRTM3 and LRRTM4 proteins, we speculate that the ancestral LRRTM might have bound HSPG before acquiring neurexins as binding partners. Our model suggests that lrrtm gene translocated into the large ctnna intron in early vertebrates, and that subsequent duplications resulted in three lrrtm/ctnna gene pairs present in most jawed vertebrates. However, we detected three prominent exceptions: (1) the lrrtm3/ctnna3 gene structure is absent in the ray-finned fish genomes, (2) the genomes of clawed frogs contain ctnna1 but lack the corresponding nested (lrrtm2) gene, and (3) contain lrrtm3 gene in the syntenic position but lack the corresponding host (ctnna3) gene. We identified several other protein-coding nested gene structures of which either the host or the nested gene has presumably been lost in the frog or chicken lineages. Interestingly, majority of these nested genes comprise LRR domains.
Subject(s)
Evolution, Molecular , Membrane Proteins/genetics , Nerve Tissue Proteins/genetics , Phylogeny , Vertebrates/genetics , alpha Catenin/genetics , Alternative Splicing/genetics , Amino Acid Sequence , Animals , Chromosome Mapping , DNA Primers/genetics , Humans , Likelihood Functions , Models, Genetic , Mutation/genetics , Polymerase Chain Reaction , Sequence Alignment , Species Specificity , Synteny/geneticsABSTRACT
The neuronal K-Cl cotransporter KCC2 maintains the low intracellular chloride concentration required for the fast hyperpolarizing actions of inhibitory neurotransmitters in mature central nervous system (CNS). The KCC2 gene produces two isoforms, KCC2a and KCC2b, that differ in their N-termini. Increase of KCC2b in the cortex underlies the developmental shift in γ-aminobutyric acid (GABA)ergic responses, whereas the physiological role of KCC2a is still poorly characterized. The two KCC2 isoforms show equal distribution in mouse brainstem neurons at birth; however their postnatal expression patterns, and the subcellular localization of KCC2a, have not yet been described. Here, we compared the pattern of KCC2a and KCC2b expression in different regions of postnatal mouse CNS by immunohistochemistry by using isoform-specific antibodies. Tissue from KCC2a isoform-specific knockout mice was used as a negative control. KCC2b expression increased postnatally and was widely expressed in adult brain. KCC2a immunoreactivity was low or absent in most parts of the adult cortex, hippocampus, thalamus, and cerebellar cortex. Both isoforms were widely present in the developing and mature hypothalamus, a large part of the brainstem, and the spinal cord. A notable exception was the lack of KCC2a staining in the brainstem auditory system. At the subcellular level, the isoforms were only partially colocalized. In neuronal somas, KCC2b immunoreactivity was concentrated at the plasma membrane, whereas KCC2a signal was not. Moreover, although both isoforms were expressed in microtubule-associated protein (MAP)2-positive dendrites, they appeared in non-overlapping dendritic compartments. The results, together with those of previous studies, suggest that KCC2a and KCC2b have overlapping roles in neonatal neurons but presumably different roles in mature neurons.
Subject(s)
Brain Chemistry , Neurons/chemistry , Spinal Cord/chemistry , Symporters/analysis , Amino Acid Sequence , Animals , Animals, Newborn , Brain/metabolism , Brain Chemistry/physiology , Female , HEK293 Cells , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Neurons/metabolism , Protein Isoforms/analysis , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Spinal Cord/metabolism , Symporters/biosynthesis , Symporters/genetics , K Cl- CotransportersABSTRACT
Efferent signals from the vagus nerve are thought to mediate both basal and meal-induced gastric acid secretion, and provide trophic support of the mucosa. However, the underlying mechanisms are incompletely understood. Neurturin, signalling via glial cell line-derived neurotrophic factor (GDNF)-family receptor α2 (GFRα2), is essential for parasympathetic innervation of many target tissues but its role in gastric innervation is unknown. Here we show that most nerve fibres in wild-type mouse gastric mucosa, including all positive for gastrin-releasing peptide, are cholinergic. GFRα2-deficient (KO) mice lacked virtually all cholinergic nerve fibres and associated glial cells in the gastric (oxyntic and pyloric) mucosa but not in the smooth muscle, consistent with the selective expression of neurturin mRNA in the gastric mucosa. 2-Deoxyglucose and hexamethonium failed to affect acid secretion in the GFRα2-KO mice indicating the lack of functional innervation in gastric mucosa. Interestingly, basal and maximal histamine-induced acid secretion did not differ between wild-type and GFRα2-KO mice. Moreover, circulating gastrin levels in both fasted and fed animals, thickness of gastric mucosa, and density of parietal and different endocrine cells were similar. Carbachol-stimulated acid secretion was higher in GFRα2-KO mice, while atropine reduced basal secretion similarly in both genotypes. We conclude that cholinergic innervation of gastric mucosa depends on neurturin-GFRα2 signalling but is dispensable for gastrin secretion and for basal and maximal acid output. Basal acid secretion in the KO mice appears to be, at least partly, facilitated by constitutive activity of muscarinic receptors.
Subject(s)
Gastric Acid/metabolism , Gastric Mucosa/innervation , Gastrins/metabolism , Glial Cell Line-Derived Neurotrophic Factor Receptors/physiology , Animals , Cholinergic Neurons/physiology , Female , Gastrins/blood , Hydrogen-Ion Concentration , Male , Mice , Mice, Knockout , Neuroglia/physiology , Neurturin/physiologyABSTRACT
Neuronal LRRTM3 (leucine-rich repeat transmembrane 3) protein has been reported to promote amyloid-ß protein precursor (AßPP) processing and LRRTM3 is a candidate gene in late-onset Alzheimer's disease. To address the role of LRRTM3 in AßPP processing and amyloid-ß (Aß) production in vivo, we analyzed amyloidogenic processing of AßPP in the brains of LRRTM3-deficient mice and transgenic AßPP/PS1 mice with or without LRRTM3. We did not find differences between the genotypes in the levels of Aß or AßPP C-terminal fragments indicating that LRRTM3 is not an essential regulator of Aß production in adult mice. Moreover, Aß levels in primary cortical neurons were similar between the genotypes, indicating that LRRTM3 is not required for Aß generation in developing mice.
Subject(s)
Amyloid beta-Peptides/biosynthesis , Amyloid beta-Protein Precursor/biosynthesis , Cell Adhesion Molecules, Neuronal/deficiency , Membrane Proteins/deficiency , Nerve Tissue Proteins/deficiency , Amyloid beta-Peptides/genetics , Amyloid beta-Protein Precursor/genetics , Animals , Animals, Newborn , Cell Adhesion Molecules, Neuronal/genetics , Cell Adhesion Molecules, Neuronal/physiology , Cells, Cultured , Cerebral Cortex/chemistry , Cerebral Cortex/metabolism , Genotype , Membrane Proteins/genetics , Membrane Proteins/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/physiology , Peptide Fragments/biosynthesis , Peptide Fragments/deficiency , Peptide Fragments/geneticsABSTRACT
KCC2 is the neuron-specific member of the of K(+)-Cl(-) cotransporter gene family. It is also the only member of its family that is active under physiologically normal conditions, in the absence of osmotic stress. By extruding Cl(-) from the neuron under isotonic conditions, this transporter maintains a low concentration of neuronal Cl(-), which is essential for fast inhibitory synaptic transmission by GABA and glycine in the mature nervous system. The other members of this K(+)-Cl(-) cotransporter gene family are exclusively swelling-activated. Here we demonstrate that a 15 aa region near the end of the C terminus, unique to KCC2 (termed the ISO domain), is required for KCC2 to cotransport K(+) and Cl(-) out of the neuron under isotonic conditions. We made this discovery by overexpressing chimeric KCC2-KCC4 cDNA constructs in cultured hippocampal neurons prepared from Sprague Dawley rat embryos and assaying neuronal Cl(-) through gramicidin perforated patch-clamp recordings. We found that when neurons had been transfected with a chimeric KCC2 that lacked the unique ISO domain, hyperpolarizing responses to GABA were abolished. This finding indicates that the ISO domain is required for neuronal Cl(-) regulation. Furthermore, we discovered that when KCC2 lacks the ISO domain, it still retains swelling-activated transport, which demonstrates that there are exclusive molecular determinants of isotonic and swelling-induced K(+)-Cl(-) cotransport in neurons.
Subject(s)
Symporters/physiology , Synaptic Transmission/physiology , gamma-Aminobutyric Acid/physiology , Animals , Biological Transport, Active/physiology , Cell Size , Chlorides/metabolism , DNA/genetics , DNA/isolation & purification , Female , Hippocampus/cytology , Image Processing, Computer-Assisted , Immunohistochemistry , Ion Transport , Microscopy, Confocal , Oocytes/metabolism , Patch-Clamp Techniques , Potassium/metabolism , Pregnancy , Rats , Rats, Sprague-Dawley , Xenopus , K Cl- CotransportersABSTRACT
The K-Cl cotransporter KCC2 plays a crucial role in the functional development of GABA(A)-mediated responses rendering GABA hyperpolarizing in adult neurons. We have previously shown that BDNF upregulates KCC2 in immature neurons through the transcription factor Egr4. The effect of BDNF on Egr4 and KCC2 was shown to be dependent on the activation of ERK1/2. Here we demonstrate that the trophic factor neurturin can also trigger Egr4 expression and upregulate KCC2 in an ERK1/2-dependent manner. These results show that Egr4 is an important component in the mechanism for trophic factor-mediated upregulation of KCC2 in immature neurons involving the activation of specific intracellular pathways common to BDNF and Neurturin.
Subject(s)
Early Growth Response Transcription Factors/biosynthesis , MAP Kinase Signaling System/physiology , Neurons/metabolism , Neurturin/physiology , Symporters/biosynthesis , Up-Regulation/physiology , Animals , Animals, Newborn , Cells, Cultured , Early Growth Response Transcription Factors/physiology , Hippocampus/metabolism , Mice , Mitogen-Activated Protein Kinases/physiology , Organ Culture Techniques , Symporters/physiology , K Cl- CotransportersABSTRACT
A major event in the maturation of CNS GABAergic transmission is the qualitative change in GABA(A)-mediated responses from depolarizing to hyperpolarizing. In cortical regions, this is attributed to the increased expression of potassium chloride cotransporter 2b (KCC2b), the main isoform of the neuron-specific K-Cl cotransporter KCC2. We have previously shown that transcription factor early growth response 4 (Egr4) can activate the KCC2b promoter. Here we demonstrate that in immature hippocampal neurons BDNF robustly induces ERK1/2 (extracellular signal-regulated kinase 1/2)-dependent Egr4 expression and rapid Egr4-dependent activation of the KCC2b promoter. The subsequent increase in KCC2b mRNA contributes to the expression of total KCC2 protein levels. These results indicate that Egr4 is an important component in the mechanism of BDNF-dependent KCC2 gene regulation via the ERK1/2 pathway in immature neurons.
Subject(s)
Brain-Derived Neurotrophic Factor/biosynthesis , Early Growth Response Transcription Factors/physiology , Symporters/biosynthesis , Animals , Binding Sites , Cells, Cultured , Hippocampus/metabolism , Mice , Mitogen-Activated Protein Kinase 1/physiology , Mitogen-Activated Protein Kinase 3/physiology , Neurons/metabolism , Promoter Regions, Genetic , RNA, Messenger/biosynthesis , Signal Transduction , Symporters/genetics , Transcription, Genetic , K Cl- CotransportersABSTRACT
The neuron-specific K-Cl cotransporter KCC2 maintains the low intracellular chloride concentration required for the fast hyperpolarizing actions of inhibitory neurotransmitters. The KCC2 gene codes for two isoforms, KCC2a and KCC2b, which differ in their N termini. The relative expression and cellular distribution of the two KCC2 protein isoforms are unknown. Here, we characterize an antibody against the KCC2a isoform and show that a previously described antibody against KCC2 is specific for the KCC2b isoform (Hubner, C. A., Stein, V., Hermans-Borgmeyer, I., Meyer, T., Ballanyi, K., and Jentsch, T. J. (2001) Neuron 30, 515-524). Immunostaining of dissociated hippocampal cultures confirms that both KCC2 isoforms are neuron-specific. Immunoblot analysis indicates that KCC2b is the major KCC2 isoform in the adult brain, whereas in the neonatal mouse central nervous system, half of total KCC2 protein is KCC2a. At this stage, the two KCC2 isoforms are largely colocalized and show similar patterns of distribution in the brain. When coexpressed in HEK293 cells, KCC2a and KCC2b proteins form heteromeric complexes. Moreover, the two isoforms can be coimmunoprecipitated from the neonatal brain, suggesting the presence of endogenous KCC2a-KCC2b heteromers. Consistent with this, native gel analysis shows that a substantial part of endogenous KCC2 isoforms in the neonatal brain constitute dimers.
Subject(s)
Gene Expression Regulation/physiology , Hippocampus/metabolism , Symporters/biosynthesis , Animals , Cell Line , Dimerization , Hippocampus/cytology , Humans , Mice , Mice, Mutant Strains , Neurotransmitter Agents/metabolism , Protein Isoforms/biosynthesis , K Cl- CotransportersABSTRACT
Recent years have witnessed a steep increase in studies on the diverse roles of neuronal cation-chloride cotransporters (CCCs). The versatility of CCC gene transcription, posttranslational modification, and trafficking are on par with what is known about ion channels. The cell-specific and subcellular expression patterns of different CCC isoforms have a key role in modifying a neuron's electrophysiological phenotype during development, synaptic plasticity, and disease. While having a major role in controlling responses mediated by GABA(A) and glycine receptors, CCCs also show close interactions with glutamatergic signaling. A cross-talk among CCCs and trophic factors is important in short-term and long-term modification of neuronal properties. CCCs appear to be multifunctional proteins that are also involved in shaping neuronal structure at various stages of development, from stem cells to synaptogenesis. The rapidly expanding work on CCCs promotes our understanding of fundamental mechanisms that control brain development and functions under normal and pathophysiological conditions.
Subject(s)
Chlorides/metabolism , Neurons/physiology , Symporters/metabolism , Animals , Biological Evolution , Cations/metabolism , Gene Expression , Humans , Nervous System Diseases/genetics , Nervous System Diseases/metabolism , Neuronal Plasticity/physiology , Symporters/classification , Symporters/genetics , Synapses/physiologyABSTRACT
Delineating the molecular basis of synapse development is crucial for understanding brain function. Cocultures of neurons with transfected fibroblasts have demonstrated the synapse-promoting activity of candidate molecules. Here, we performed an unbiased expression screen for synaptogenic proteins in the coculture assay using custom-made cDNA libraries. Reisolation of NGL-3/LRRC4B and neuroligin-2 accounts for a minority of positive clones, indicating that current understanding of mammalian synaptogenic proteins is incomplete. We identify LRRTM1 as a transmembrane protein that induces presynaptic differentiation in contacting axons. All four LRRTM family members exhibit synaptogenic activity, LRRTMs localize to excitatory synapses, and artificially induced clustering of LRRTMs mediates postsynaptic differentiation. We generate LRRTM1(-/-) mice and reveal altered distribution of the vesicular glutamate transporter VGLUT1, confirming an in vivo synaptic function. These results suggest a prevalence of LRR domain proteins in trans-synaptic signaling and provide a cellular basis for the reported linkage of LRRTM1 to handedness and schizophrenia.
Subject(s)
Genetic Testing/methods , Membrane Proteins/metabolism , Neurons/cytology , Synapses/metabolism , Animals , Cell Differentiation/physiology , Cells, Cultured , Cloning, Molecular , Cricetinae , Cricetulus , Disks Large Homolog 4 Protein , Embryo, Mammalian , Gene Expression , Gene Expression Regulation/physiology , Gene Library , Guanylate Kinases , Hippocampus/cytology , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Luminescent Proteins/genetics , Membrane Potentials/genetics , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mice , Mice, Knockout , PDZ Domains/physiology , Patch-Clamp Techniques/methods , Presynaptic Terminals/metabolism , Rats , Transfection/methods , Vesicular Glutamate Transport Protein 1/genetics , Vesicular Glutamate Transport Protein 1/metabolismABSTRACT
The neuron-specific K-Cl cotransporter (KCC2) maintains a low intracellular Cl(-) concentration in neurons and is necessary for fast hyperpolarizing responses to GABA and glycine. The mammalian KCC2 gene (alias Slc12a5) generates two neuron-specific isoforms by using alternative promoters and first exons. Expression of the major isoform, KCC2b, is strongly upregulated during neuronal maturation, and is modulated by neuronal activity, trauma, and neurotrophic factors. In the present study, we have focused on the regulatory influence of the upstream stimulating factors USF1 and USF2 via an E-box control element in the KCC2b promoter (E-boxKCC2b). Electrophoretic mobility shift assay in cell lines and chromatin immunoprecipitation in neurons demonstrated binding of endogenous USF1 and USF2 to the E-box(KCC2b) element. Mutation of the E-boxKCC2b site resulted in reduced KCC2b promoter activity in cell lines and cortical neurons. Overexpression of a dominant-negative form of USF confirmed the involvement of endogenous USF proteins in the regulation of the KCC2b gene. The results suggest that binding of USF proteins to the E-boxKCC2b may contribute to the upregulation of KCC2b gene expression in developing brain.