Conditional activation of the N598R point mutation in the NR1 subunit of the NMDA receptor in knock-in mice

The NMDA-type glutamate receptor is involved in synaptic plasticity. NMDA receptors are heterooligomers of the NR1 together with any of the NR2 (NR2A-D) subunit variants. Introduction of the point mutation N598R within the channel-lining segment M2 of NR1 abolishes voltage-dependent Mg²⁺-block and Ca²⁺-permeability of the NMDA receptor channel. The NR1 N598R mouse model, which had been obtained earlier in the group of Dr. Ralf Schoepfer, carries the N598R point mutation and a floxed neo cassette within intron 18 of the NR1 gene. This NR1 (N598Rneo) allele is essentially inactive. However, Cre-mediated excision of the floxed neo cassette yields an active NR1 (N598R) allele. The presented work has two parts: 1.) Analysis of mice with global expression of the NR1 N598R mutation. Global activation of the mutation - through breeding with the 'Deleter' mouse - results in lethality shortly after birth in mice carrying one mutant and one wildtype NR1 allele. Thus, the NR1 N598R mutation is dominant negative lethal. The impact of the NR1 N598R mutation on the formation of the whisker-related somatotopic map in the brainstem of newborn mice was investigated. A comparative histochemical study involving mice with five different genotypes is presented. A part of this work has been published in abstract form (Rudhard et al., 2000). 2.) Generation of a Cre knock-in mouse line, in which Cre is co-expressed with the kainate receptor subunit 1 (KA1). The targeting vector was assembled in yeast by homologous recombination with a YAC carrying the KA1 gene. The following elements were inserted in the 3'UTR of the native KA1 gene: internal ribosome entry site (IRES), followed by an EGFP-Cre translation unit, followed by flirted selection markers. Chimeric mice have been generated (performed by Eurogentec, Belgium) and are being bred for germline transmission for further analysis

Absence of Whisker-Related Pattern Formation in Mice with NMDA Receptors Lacking Coincidence Detection Properties and Calcium Signaling

Precise refinement of synaptic connectivity is the result of activity-dependent mechanisms in which coincidence-dependent calcium signaling by NMDA receptors (NMDARs) under control of the voltage-dependent Mg2+ block might play a special role. In the developing rodent trigeminal system, the pattern of synaptic connections between whisker-specific inputs and their target cells in the brainstem is refined to form functionally and morphologically distinct units (barrelettes). To test the role of NMDA receptor signaling in this process, we introduced the N598R mutation into the native NR1 gene. This leads to the expression of functional NMDARs that are Mg2+ insensitive and Ca2+impermeable. Newborn mice expressing exclusively NR1 N598R-containing NMDARs do not show any whisker-related patterning in the brainstem, whereas the topographic projection of trigeminal afferents and gross brain morphology appear normal. Furthermore, the NR1 N598R mutation does not affect expression levels of NMDAR subunits and other important neurotransmitter receptors. Our results show that coincidence detection by, and/or Ca2+ permeability of, NMDARs is necessary for the development of somatotopic maps in the brainstem and suggest that highly specific signaling underlies synaptic refinement

Subcellular localisation of recombinant α- and γ-synuclein

α-Synuclein, a protein implicated in neurodegenerative diseases and of elusive physiological function owes its name to an observed presence in presynaptic and nuclear compartments. However, its nuclear localisation has remained controversial. We expressed synuclein–eGFP fusion proteins in organotypic rat hippocampal slice cultures and murine hippocampal primary neurons using a Sindbis virus expression system. Recombinant full-length α-synuclein accumulated in presynaptic locations, mimicking its native distribution. Expression of deletion mutant α-synuclein revealed that presynaptic targeting depended on the presence of its N-terminal and core region. This domain also causes nuclear exclusion of the α-synuclein fusion protein. In contrast, the C-terminal domain of α-synuclein directs fusion proteins into the nuclear compartment. The related protein γ-synuclein contains a similar N-terminal and core domain as α-synuclein. However, γ-synuclein lacks a C-terminal domain that causes nuclear localisation of the fusion protein, suggesting that the two synucleins might have different roles relating to the cell nucleus

Disruption of erythroid K-Cl cotransporters alters erythrocyte volume and partially rescues erythrocyte dehydration in SAD mice

K-Cl cotransport activity in rbc is a major determinant of rbc volume and density. Pathologic activation of erythroid K-Cl cotransport activity in sickle cell disease contributes to rbc dehydration and cell sickling. To address the roles of individual K-Cl cotransporter isoforms in rbc volume homeostasis, we disrupted the Kcc1 and Kcc3 genes in mice. As rbc K-Cl cotransport activity was undiminished in Kcc1–/– mice, decreased in Kcc3–/– mice, and almost completely abolished in mice lacking both isoforms, we conclude that K-Cl cotransport activity of mouse rbc is mediated largely by KCC3. Whereas rbc of either Kcc1–/– or Kcc3–/– mice were of normal density, rbc of Kcc1–/–Kcc3–/– mice exhibited defective volume regulation, including increased mean corpuscular volume, decreased density, and increased susceptibility to osmotic lysis. K-Cl cotransport activity was increased in rbc of SAD mice, which are transgenic for a hypersickling human hemoglobin S variant. Kcc1–/–Kcc3–/– SAD rbc lacked nearly all K-Cl cotransport activity and exhibited normalized values of mean corpuscular volume, corpuscular hemoglobin concentration mean, and K+ content. Although disruption of K-Cl cotransport rescued the dehydration phenotype of most SAD rbc, the proportion of the densest red blood cell population remained unaffected

NKCC1-dependent GABAergic excitation drives synaptic network maturation during early hippocampal development.

A high intracellular chloride concentration in immature neurons leads to a depolarizing action of GABA that is thought to shape the developing neuronal network. We show that GABA-triggered depolarization and Ca2+ transients were attenuated in mice deficient for the Na-K-2Cl cotransporter NKCC1. Correlated Ca2+ transients and giant depolarizing potentials (GDPs) were drastically reduced and the maturation of the glutamatergic and GABAergic transmission in CA1 delayed. Brain morphology, synaptic density, and expression levels of certain developmental marker genes were unchanged. The expression of lynx1, a protein known to dampen network activity, was decreased. In mice deficient for the neuronal Cl-/HCO3- exchanger AE3, GDPs were also diminished. These data show that NKCC1-mediated Cl- accumulation contributes to GABAergic excitation and network activity during early postnatal development and thus facilitates the maturation of excitatory and inhibitory synapses

Raising cytosolic Cl− in cerebellar granule cells affects their excitability and vestibulo-ocular learning

Cerebellar cortical throughput involved in motor control comprises granule cells (GCs) and Purkinje cells (PCs), both of which receive inhibitory GABAergic input from interneurons. The GABAergic input to PCs is essential for learning and consolidation of the vestibulo-ocular reflex, but the role of GC excitability remains unclear. We now disrupted the Kcc2 K-Cl cotransporter specifically in either cell type to manipulate their excitability and inhibition by GABA A-receptor Cl - channels. Although Kcc2 may have a morphogenic role in synapse development, Kcc2 disruption neither changed synapse density nor spine morphology. In both GCs and PCs, disruption of Kcc2, but not Kcc3, increased [Cl -] i roughly two-fold. The reduced Cl - gradient nearly abolished GABA-induced hyperpolarization in PCs, but in GCs it merely affected excitability by membrane depolarization. Ablation of Kcc2 from GCs impaired consolidation of long-term phase learning of the vestibulo-ocular reflex, whereas baseline performance, short-term gain-decrease learning and gain consolidation remained intact. These functions, however, were affected by disruption of Kcc2 in PCs. GC excitability plays a previously unknown, but specific role in consolidation of phase learning.Fil: Seja, Patricia. Leibniz-Institut für Molekulare Pharmakologie; Alemania. Max-Delbruck-Centrum für Molekulare Medizin; AlemaniaFil: Schonewille, Martijn. Erasmus MC; Países BajosFil: Spitzmaul, Guillermo Federico. Leibniz-Institut für Molekulare Pharmakologie; Alemania. Max-Delbruck-Centrum für Molekulare Medizin; AlemaniaFil: Badura, Aleksandra. Erasmus MC; Países BajosFil: Klein, Ilse. Universitat Hamburg; AlemaniaFil: Rudhard, York. Universitat Hamburg; AlemaniaFil: Wisden, William. Imperial College London; Reino UnidoFil: Hübner, Christian A. Universitat Hamburg; Alemania. Universitat Jena; AlemaniaFil: De Zeeuw, Chris I. Erasmus MC; Países Bajos. Charite-Universitatsmedizin Berlin; AlemaniaFil: Jentsch, Thomas J. Leibniz-Institut für Molekulare Pharmakologie; Alemania. Universitat Hamburg; Alemania. Charite-Universitatsmedizin Berlin; Alemani

Discovery of inhibitors of microglial neurotoxicity acting through multiple mechanisms using a stem-cell-based phenotypic assay.

Stem cells, through their ability to both self-renew and differentiate, can produce a virtually limitless supply of specialized cells that behave comparably to primary cells. We took advantage of this property to develop an assay for small-molecule-based neuroprotection using stem-cell-derived motor neurons and astrocytes, together with activated microglia as a stress paradigm. Here, we report on the discovery of hit compounds from a screen of more than 10,000 small molecules. These compounds act through diverse pathways, including the inhibition of nitric oxide production by microglia, activation of the Nrf2 pathway in microglia and astrocytes, and direct protection of neurons from nitric-oxide-induced degeneration. We confirm the activity of these compounds using human neurons. Because microglial cells are activated in many neurological disorders, our hit compounds could be ideal starting points for the development of new drugs to treat various neurodegenerative and neurological diseases

Behavioral deficits and subregion-specific suppression of LTP in mice expressing a population of mutant NMDA receptors throughout the hippocampus

The NMDA receptor (NMDAR) subunit GluN1 is an obligatory component of NMDARs without a known functional homolog and is expressed in almost every neuronal cell type. The NMDAR system is a coincidence detector with critical roles in spatial learning and synaptic plasticity. Its coincidence detection property is crucial for the induction of hippocampal long-term potentiation (LTP). We have generated a mutant mouse model expressing a hypomorph of the Grin1N598R allele, which leads to a minority (about 10%) of coincidence detection-impaired NMDARs. Surprisingly, these animals revealed specific functional changes in the dentate gyrus (DG) of the hippocampal formation. Early LTP was expressed normally in area CA1 in vivo, but was completely suppressed at perforant path-granule cell synapses in the DG. In addition, there was a pronounced reduction in the amplitude of the evoked population spike in the DG. These specific changes were accompanied by behavioral impairments in spatial recognition, spatial learning, reversal learning, and retention. Our data show that minor changes in GluN1-dependent NMDAR physiology can cause dramatic consequences in synaptic signaling in a subregion-specific fashion despite the nonredundant nature of the GluN1 gene and its global expression

CaMKII translocation requires local NMDA receptor-mediated Ca(2+) signaling

Excitatory synaptic transmission and plasticity are critically modulated by N-methyl-D-aspartate receptors (NMDARs). Activation of NMDARs elevates intracellular Ca(2+) affecting several downstream signaling pathways that involve Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Importantly, NMDAR activation triggers CaMKII translocation to synaptic sites. NMDAR activation failed to induce Ca(2+) responses in hippocampal neurons lacking the mandatory NMDAR subunit NR1, and no EGFP-CaMKIIα translocation was observed. In cells solely expressing Ca(2+)-impermeable NMDARs containing NR1(N598R)-mutant subunits, prolonged NMDA application elevated internal Ca(2+) to the same degree as in wild-type controls, yet failed to translocate CaMKIIα. Brief local NMDA application evoked smaller Ca(2+) transients in dendritic spines of mutant compared to wild-type cells. CaMKIIα mutants that increase binding to synaptic sites, namely CaMKII-T286D and CaMKII-TT305/306VA, rescued the translocation in NR1(N598R) cells in a glutamate receptor-subtype-specific manner. We conclude that CaMKII translocation requires Ca(2+) entry directly through NMDARs, rather than other Ca(2+) sources activated by NMDARs. Together with the requirement for activated, possibly ligand-bound, NMDARs as CaMKII binding partners, this suggests that synaptic CaMKII accumulation is an input-specific signaling event