Impaired NMDA receptor function in mouse olfactory bulb neurons by tetracycline-sensitive NR1(N598R) expression

High Ca2+ permeability and its control by voltage-dependent Mg2+ block are defining features of NMDA receptors. These features are lost if the principal NR1 subunit carries an asparagine (N) to arginine (R) substitution in a critical channel site at NR1 position 598. NR1(R) expression from a single allele in gene-targeted NR1+/R mice is lethal soon after birth, precluding analysis of altered synaptic functions later in life. We therefore employed the forebrain specific small alpha, GreekCaMKII promoter to drive tTA-mediated tetracyclin sensitive transcription of transgenes for NR1(R) and for lacZ as reporter. Transgene expression was observed in cortex, striatum, hippocampus, amygdala and olfactory bulb and was mosaic in all these forebrain regions. It was highest in olfactory bulb granule cells, in most of which Ca2+ permeability and voltage-dependent Mg2+ block of NMDA receptors were reduced to different extents. This indicates significant impairment of NMDA receptor function by NR1(R) in presence of the wild-type NR1 complement. Indeed, even though NR1(R) mRNA constituted only 18% of the entire NR1 mRNA population in forebrain, the transgenic mice died during adolescence unless transgene expression was suppressed by doxycycline. Thus, glutamate receptor function can be altered in the mouse by regulated NR1(R) transgene expression

A highly sensitive novel immunoassay specifically detects low levels of soluble Aβ oligomers in human cerebrospinal fluid

Introduction: Amyloid β-protein oligomers play a key role in Alzheimer’s disease (AD), but well-validated assays that routinely detect them in cerebrospinal fluid (CSF) are just emerging. We sought to confirm and extend a recent study using the Singulex Erenna platform that reported increased mean CSF oligomer levels in AD. Methods: We tested four antibody pairs and chose one pair that was particularly sensitive, using 1C22, our new oligomer-selective monoclonal antibody, for capture. We applied this new assay to extracts of human brain and CSF. Results: A combination of 1C22 for capture and 3D6 for detection yielded an Erenna immunoassay with a lower limit of quantification of approximately 0.15 pg/ml that was highly selective for oligomers over monomers and detected a wide size-range of oligomers. Most CSFs we tested had detectable oligomer levels but with a large overlap between AD and controls and a trend for higher mean levels in mild cognitive impairment (MCI) than controls. Conclusion: Aβ oligomers are detectable in most human CSFs, but AD and controls overlap. MCI CSFs may have a modest elevation in mean value by this assay. Electronic supplementary material The online version of this article (doi:10.1186/s13195-015-0100-y) contains supplementary material, which is available to authorized users

Studies on conditional gene expression in the brain

This manuscript summarizes our recent attempts to regulate in vitro and in vivo the expression of genes encoding components and regulators of the postsynaptic machinery along with marker genes such as lacZ and GFP. In particular, we studied tTA-dependent regulation and utilized Cre in combination with reversible silencing by intron engineering of dominant negative alleles. We further present a "knockin" approach for on-site artificial regulation of chromosomal genes

An acute functional screen identifies an effective antibody targeting amyloid-β oligomers based on calcium imaging

Soluble amyloid β oligomers (AβOs) are widely recognized neurotoxins that trigger aberrant signaling in specific subsets of neurons, leading to accumulated neuronal damage and memory disorders in Alzheimer’s disease (AD). One of the profound downstream consequences of AβO-triggered events is dysregulation of cytosolic calcium concentration ([Ca2+]i), which has been implicated in synaptic failure, cytoskeletal abnormalities, and eventually neuronal death. We have developed an in vitro/in vivo drug screening assay to evaluate putative AβO-blocking candidates by measuring AβO-induced real-time changes in [Ca2+]i. Our screening assay demonstrated that the anti-AβO monoclonal antibody ACU3B3 exhibits potent blocking capability against a broad size range of AβOs. We showed that picomolar concentrations of AβOs were capable of increasing [Ca2+]i in primary neuronal cultures, an effect prevented by ACU3B3. Topical application of 5 nM AβOs onto exposed cortical surfaces also elicited significant calcium elevations in vivo, which was completely abolished by pre-treatment of the brain with 1 ng/mL (6.67 pM) ACU3B3. Our results provide strong support for the utility of this functional screening assay in identifying and confirming the efficacy of AβO-blocking drug candidates such as the human homolog of ACU3B3, which may emerge as the first experimental AD therapeutic to validate the amyloid oligomer hypothesis

Respiration and parturition affected by conditional overexpression of the Ca2+ -activated K+ channel subunit, SK3

In excitable cells, small-conductance Ca2+-activated potassium channels (SK channels) are responsible for the slow after-hyperpolarization that often follows an action potential. Three SK channel subunits have been molecularly characterized. The SK3 gene was targeted by homologous recombination for the insertion of a gene switch that permitted experimental regulation of SK3 expression while retaining normal SK3 promoter function. An absence of SK3 did not present overt phenotypic consequences. However, SK3 overexpression induced abnormal respiratory responses to hypoxia and compromised parturition. Both conditions were corrected by silencing the gene. The results implicate SK3 channels as potential therapeutic targets for disorders such as sleep apnea or sudden infant death syndrome and for regulating uterine contractions during labor. SK channels are potassium-selective, voltage-independent, and activated by increases in the levels of intracellular Ca2+, such as what occurs during an action potential (1, 2). We have characterized three mammalian SK subunits (hSK1, rSK2, and rSK3) by molecular cloning. All three form SK channels with similar Ca2+ sensitivity and gating kinetics; constitutive association of calmodulin accomplishes Ca2+ gating with an intracellular domain of the channel alpha subunits (3, 4). To investigate the physiological role of murine SK3, we site-specifically inserted a tetracycline-based genetic switch into the 5 untranslated region of the gene so that subunit expression could be abolished by dietary doxycycline (dox) administration without interfering with the normal profile of SK3 expression