A Novel H+ Conductance in Eosinophils: Unique Characteristics and Absence in Chronic Granulomatous Disease

Efficient mechanisms of H+ ion extrusion are crucial for normal NADPH oxidase function. However, whether the NADPH oxidase—in analogy with mitochondrial cytochromes—has an inherent H+ channel activity remains uncertain: electrophysiological studies did not find altered H+ currents in cells from patients with chronic granulomatous disease (CGD), challenging earlier reports in intact cells. In this study, we describe the presence of two different types of H+ currents in human eosinophils. The “classical” H+ current had properties similar to previously described H+ conductances and was present in CGD cells. In contrast, the “novel” type of H+ current had not been described previously and displayed unique properties: (a) it was absent in cells from gp91- or p47-deficient CGD patients; (b) it was only observed under experimental conditions that allowed NADPH oxidase activation; (c) because of its low threshold of voltage activation, it allowed proton influx and cytosolic acidification; (d) it activated faster and deactivated with slower and distinct kinetics than the classical H+ currents; and (e) it was ∼20-fold more sensitive to Zn2+ and was blocked by the histidine-reactive agent, diethylpyrocarbonate (DEPC). In summary, our results demonstrate that the NADPH oxidase or a closely associated protein provides a novel type of H+ conductance during phagocyte activation. The unique properties of this conductance suggest that its physiological function is not restricted to H+ extrusion and repolarization, but might include depolarization, pH-dependent signal termination, and determination of the phagosomal pH set point

Béta-amiloid peptidek aggregációja és kölcsönhatása fehérjékkel; új neuroprotektív vegyületek alkalmazása az Alzheimer-kór megelőzésére = Beta-amyloid aggregation and interaction with proteins; novel neuroprotective compounds for prevention of Alzheimer's disease

Új, standardizálható módszert dolgoztunk ki toxikus �béta-amiloid (Abéta) 1-42 peptid oligomerek előállítására, a preparált oligomereket fiziko-kémiai módszerekkel jellemeztük. Két új neuroprotektív peptidmimetikum vegyületcsaládot találtunk, ezek az anyagok megvédik a neuronokat az Alzheimer-kór (AK) állatmodelljében az Abéta neurotoxikus hatásától. Mindkét vegyületcsoportot szabadalmilag védjük, mint az AK potenciális gyógyszerjelölt vegyületeit. Új ex vivo módszert dolgoztunk ki az Abéta peptidek toxicitásának mérésére (patkány hippocampus szelet, MTT-teszt), a módszer alkalmas az új neuroprotektív vegyületeink aktivitásmérésére is. Az ex vivo hippocampus szeleteket sikerrel alkalmaztuk a neuronális plaszticitás (LTP) mérésére, az Abéta-toxicitás meghatározására, multielektród array (MEA) technikával. In vivo, egysejt-elvezetéses elektrofiziológiai mérésekkel bizonyítottuk az új peptidmimetikumaink neuroprotektív hatását. Proteomikai módszerekkel azonosítottuk az Abéta peptidekkel kölcsönhatásba lépő fehérjéket, ezek elsősorban plazmamembrán, ill. intraneuronális fehérjék (mitokondrium, endoplazmás reticulum, mikrotubuláris rendszer). Az intraneuronális fehérjék és az Abéta peptidek kölcsönhatásai kulcsszerepet játszhatnak az AK patogenezisében. Igazoltuk, hogy a Zn2+ ionok toxikus Abéta-aggregátumok képződését indukálják. Az AK transzgén állatmodelljén bizonyítottuk, hogy a Zn-kelátorok (pl. Perindopril) neuroprotektív hatásúak. Új AK-állatmodellt dolgoztunk ki az Abéta oligomerek icv bevitelével. | A new method was introduced for the preparation of toxic beta-amyloid (Abeta) 1-42 oligomers, these assemblies were characterized with physicochemical methods. Two families of novel neuroprotective peptidomimetics were found, these substances protect neurons against the toxic effect of Abeta in tg mouse models of Alzheimer’s disease (AD). Both groups of the novel substances will be patented as putative drug candidates for AD treatment. A new ex vivo method was introduced for toxicity measurement of Abeta peptides (rat hippocampal slices, MTT-assay); this method proved to be suitable for activity measurement of the novel neuroprotective substances. Hippocampal slices were successfully used for measurement of neuronal plasticity (LTP) for demonstrating neurotoxicity of Abeta aggregates, applying multielectrode array (MEA) technique. The neuroprotective effect of our novel peptidomimetics was demonstrated also in vivo, using one-cell electrophysiology. Proteomic methods were used for identification of proteins interacting with Abeta peptides; these are mainly plasma membrane and intraneuronal (mitochondrial, endoplasmatic reticular and microtubular) proteins. Interaction of intracellular proteins with Abeta may play key role in AD pathogenesis. The role of Zn2+ ions in formation of toxic Abeta-aggregates was demonstrated. Zn2+-chelators (e.g. Perindopril) were neuroprotective in a tg-mouse model of AD. A new AD rat model was introduced using icv administration of synthetic Abeta oligomers

A Claudin-9–Based Ion Permeability Barrier Is Essential for Hearing

Hereditary hearing loss is one of the most common birth defects, yet the majority of genes required for audition is thought to remain unidentified. Ethylnitrosourea (ENU)–mutagenesis has been a valuable approach for generating new animal models of deafness and discovering previously unrecognized gene functions. Here we report on the characterization of a new ENU–induced mouse mutant (nmf329) that exhibits recessively inherited deafness. We found a widespread loss of sensory hair cells in the hearing organs of nmf329 mice after the second week of life. Positional cloning revealed that the nmf329 strain carries a missense mutation in the claudin-9 gene, which encodes a tight junction protein with unknown biological function. In an epithelial cell line, heterologous expression of wild-type claudin-9 reduced the paracellular permeability to Na+ and K+, and the nmf329 mutation eliminated this ion barrier function without affecting the plasma membrane localization of claudin-9. In the nmf329 mouse line, the perilymphatic K+ concentration was found to be elevated, suggesting that the cochlear tight junctions were dysfunctional. Furthermore, the hair-cell loss in the claudin-9–defective cochlea was rescued in vitro when the explanted hearing organs were cultured in a low-K+ milieu and in vivo when the endocochlear K+-driving force was diminished by deletion of the pou3f4 gene. Overall, our data indicate that claudin-9 is required for the preservation of sensory cells in the hearing organ because claudin-9–defective tight junctions fail to shield the basolateral side of hair cells from the K+-rich endolymph. In the tight-junction complexes of hair cells, claudin-9 is localized specifically to a subdomain that is underneath more apical tight-junction strands formed by other claudins. Thus, the analysis of claudin-9 mutant mice suggests that even the deeper (subapical) tight-junction strands have biologically important ion barrier function

Overlapping Activities of Two Neuronal Splicing Factors Switch the GABA Effect from Excitatory to Inhibitory by Regulating REST

Summary: A truncating mutation in the mouse Srrm4 gene, which encodes a neuronal splicing factor, causes alternative splicing defects selectively in the ear. The mechanism by which splicing is preserved in the brain of these mice is not known. Here, we show that SRRM3 limits the Srrm4 mutation-associated defects to the ear and that, in cortical neurons, overlapping SRRM3-SRRM4 activity regulates the development of interneuronal inhibition. In vitro, SRRM3 and SRRM4 regulate the same splicing events, but a mutation in mouse Srrm3 causes tremors and mild defects in neuronal alternative splicing, demonstrating unique SRRM3 roles in vivo. Mice harboring mutations in both Srrm3 and Srrm4 die neonatally and exhibit severe splicing defects. In these mice, splicing alterations prevent inactivation of the gene repressor REST, which maintains immature excitatory GABAergic neurotransmission by repressing K-Cl cotransporter 2. Thus, our data reveal that SRRM3 and SRRM4 act redundantly to regulate GABAergic neurotransmission by inactivating REST. : Nakano et al. report that the paralogous proteins SRRM3 and SRRM4 regulate the alternative splicing of overlapping groups of exons in the CNS and that the SRRM3-SRRM4-regulated splicing alterations change GABAergic neurotransmission from excitatory to inhibitory during development by inactivating the gene repressor REST and derepressing its target gene Kcc2. Keywords: alternative splicing, gene expression regulation, neuronal development, GABAergic neurotransmission, SRRM3, SRRM4, REST, KCC2, GABA-A receptor, calcium imagin

Conserved Cysteine Residues Provide a Protein-Protein Interaction Surface in Dual Oxidase (DUOX) Proteins*

Intramolecular disulfide bond formation is promoted in oxidizing extracellular and endoplasmic reticulum compartments and often contributes to protein stability and function. DUOX1 and DUOX2 are distinguished from other members of the NOX protein family by the presence of a unique extracellular N-terminal region. These peroxidase-like domains lack the conserved cysteines that confer structural stability to mammalian peroxidases. Sequence-based structure predictions suggest that the thiol groups present are solvent-exposed on a single protein surface and are too distant to support intramolecular disulfide bond formation. To investigate the role of these thiol residues, we introduced four individual cysteine to glycine mutations in the peroxidase-like domains of both human DUOXs and purified the recombinant proteins. The mutations caused little change in the stabilities of the monomeric proteins, supporting the hypothesis that the thiol residues are solvent-exposed and not involved in disulfide bonds that are critical for structural integrity. However, the ability of the isolated hDUOX1 peroxidase-like domain to dimerize was altered, suggesting a role for these cysteines in protein-protein interactions that could facilitate homodimerization of the peroxidase-like domain or, in the full-length protein, heterodimeric interactions with a maturation protein. When full-length hDUOX1 was expressed in HEK293 cells, the mutations resulted in decreased H2O2 production that correlated with a decreased amount of the enzyme localized to the membrane surface rather than with a loss of activity or with a failure to synthesize the mutant proteins. These results support a role for the cysteine residues in intermolecular disulfide bond formation with the DUOX maturation factor DUOXA1

Mutation of the Cyba gene encoding p22phox causes vestibular and immune defects in mice

In humans, hereditary inactivation of either p22phox or gp91phox leads to chronic granulomatous disease (CGD), a severe immune disorder characterized by the inability of phagocytes to produce bacteria-destroying ROS. Heterodimers of p22phox and gp91phox proteins constitute the superoxide-producing cytochrome core of the phagocyte NADPH oxidase. In this study, we identified the nmf333 mouse strain as what we believe to be the first animal model of p22phox deficiency. Characterization of nmf333 mice revealed that deletion of p22phox inactivated not only the phagocyte NADPH oxidase, but also a second cytochrome in the inner ear epithelium. As a consequence, mice of the nmf333 strain exhibit a compound phenotype consisting of both a CGD-like immune defect and a balance disorder caused by the aberrant development of gravity-sensing organs. Thus, in addition to identifying a model of p22phox-dependent immune deficiency, our study indicates that a clinically identifiable patient population with an otherwise cryptic loss of gravity-sensor function may exist. Thus, p22phox represents a shared and essential component of at least 2 superoxide-producing cytochromes with entirely different biological functions. The site of p22phox expression in the inner ear leads us to propose what we believe to be a novel mechanism for the control of vestibular organogenesis

A Mutation in the Mouse <i>Ttc26</i> Gene Leads to Impaired Hedgehog Signaling

<div><p>The phenotype of the spontaneous mutant mouse hop-sterile (hop) is characterized by a hopping gait, polydactyly, hydrocephalus, and male sterility. Previous analyses of the hop mouse revealed a deficiency of inner dynein arms in motile cilia and a lack of sperm flagella, potentially accounting for the hydrocephalus and male sterility. The etiology of the other phenotypes and the location of the <i>hop</i> mutation remained unexplored. Here we show that the <i>hop</i> mutation is located in the <i>Ttc26</i> gene and impairs Hedgehog (Hh) signaling. Expression analysis showed that this mutation led to dramatically reduced levels of the Ttc26 protein, and protein-protein interaction assays demonstrated that wild-type Ttc26 binds directly to the Ift46 subunit of Intraflagellar Transport (IFT) complex B. Although IFT is required for ciliogenesis, the Ttc26 defect did not result in a decrease in the number or length of primary cilia. Nevertheless, Hh signaling was reduced in the hop mouse, as revealed by impaired activation of Gli transcription factors in embryonic fibroblasts and abnormal patterning of the neural tube. Unlike the previously characterized mutations that affect IFT complex B, <i>hop</i> did not interfere with Hh-induced accumulation of Gli at the tip of the primary cilium, but rather with the subsequent dissociation of Gli from its negative regulator, Sufu. Our analysis of the hop mouse line provides novel insights into Hh signaling, demonstrating that Ttc26 is necessary for efficient coupling between the accumulation of Gli at the ciliary tip and its dissociation from Sufu.</p></div

The hop mouse exhibits patterning defects and hearing impairment.

<p>(<b>A</b>) Representative images of <i>hop/+</i> and <i>hop/hop</i> mice at postnatal day 4. (<b>B</b>) Comparison of Alcian Blue-stained fore and hind limbs of <i>hop/+</i> and <i>hop/hop</i> mice. Extra digits are indicated by arrows. (<b>C</b>) Statistical analysis of FoxA2-positive cells in the lumbar neural tubes of <i>hop/+</i> and <i>hop/hop</i> mice (E10.5). Each symbol represents the average number of FoxA2⁺ cells per focal plane in a single embryo (for each embryo, 12 focal planes in 4 sections were analyzed, Mann-Whitney test: *<i>P</i> = 0.01). (<b>D</b>) Immunostaining of the lumbar neural tube of <i>hop/+</i> and <i>hop/hop</i> mice (E10.5) with antibodies against the V3 progenitor marker Nkx2.2 (white contrast), the floor plate marker FoxA2 (red), and the motor neuron protein HB9 (white contrast). The <i>hop/hop</i> genotype is associated with reduced FoxA2 expression and ventralization of the Nkx2.2- and HB9-expressing cells. Scale bar: 50 µm. (<b>E</b>) Representative ABR waveforms for 3–4 week-old <i>hop/+</i> and <i>hop/hop</i> mice. Broadband click stimuli were applied at the indicated sound pressure levels (in dB). (<b>F</b>) Statistical analysis of ABR thresholds measured in 3–4 week-old <i>hop/+</i> and <i>hop/hop</i> mice. Broadband click stimuli between 30 and 90 dB sound pressure level (SPL) were used. Each symbol represents the value for a single mouse (Mann-Whitney test: ***<i>P</i><0.0001).</p

The <i>Ttc26</i> gene of the hop mouse contains a nonsense mutation.

<p>(<b>A</b>) Schematic representation of genomic positions of genes that both fall within the 16-mega base pair (Mbp) interval to which the <i>hop</i> mutation was mapped and had a known association with the ciliome. (<b>B</b>) Comparison of the 15^th exon of the <i>Ttc26</i> gene in wild-type and <i>hop/hop</i> mice. Horizontal lines represent introns, and black and white rectangles represent the coding and non-coding regions of exons, respectively. A deoxycytidine nucleotide (C) of wild-type <i>Ttc26</i> (upper chromatogram) is replaced with a deoxyadenosine (A) in the hop mouse, as indicated by an arrow in the lower chromatogram. The point mutation changes the tyrosine (Tyr) at position 430 of Ttc26 to a stop codon (Stop), as shown in the amino-acid sequence lines. (<b>C</b>) Schematic representation of the Ttc26 protein. Blue boxes indicate the predicted TPR motifs. The bracket indicates the C-terminal 125-amino acid region of the protein that is predicted to be missing in <i>hop/hop</i> cells. The asterisk indicates the position of the epitope that is recognized by the anti-Ttc26 antibody. (<b>D</b>) Immunoblot analysis of Ttc26 expression in transfected HEK293 cells, airway epithelial cells (AEC) and testis of wild type (wt) and <i>hop/hop</i> (hop) mice. HEK293 cells were transfected with the indicated Ttc26-encoding construct or an empty expression vector. Arrowheads indicate the positions of the 64, 49, and 37 kDa standards. The antibodies used for immunoblotting are indicated next to the upper and lower panels.</p

The <i>hop</i> mutation does not impair ciliogenesis or ciliary localization of the Ttc26-interacting protein Ift46.

<p>(<b>A</b>) Color test of protein-protein interactions in yeast transformed with the indicated combination of Ift46 and Ttc26^wt or Ttc26^hop. Blue staining of the yeast colony (upper patch) is indicative of a protein-protein interaction; a lack thereof indicates the absence of a protein-protein interaction (lower patch). (<b>B</b>) Immunoprecipitation analysis of the Ttc26^wt-Ift46 interaction. HEK293 cells were transfected with the indicated combinations of HA-tagged Ift46 and flag (FL)-tagged Ttc26^wt or Ttc26^hop. The two upper panels show immunoblot analysis of tagged proteins pulled down with an anti-flag antibody, and the two lower panels show immunoblot analysis of input controls. (<b>C</b>) Percentages of ciliated <i>hop/+</i> and <i>hop/hop</i> MEFs following 2 days of serum starvation. MEFs were isolated from 4 embryos per genotype, and 150 MEFs per embryo were analyzed. (<b>D</b>) Statistical analysis of cilium length in the primary cultures of serum-starved <i>hop/+</i> and <i>hop/hop</i> MEFs (mean ± SEM, n = 200 cilia per genotype, unpaired <i>t</i>-test with Welch correction, ***<i>P</i><0.0001). (<b>E,F</b>) Immunofluorescence analysis of (<b>E</b>) Ttc26 and (<b>F</b>) Ift46 expression in the cilia of <i>hop/+</i> and <i>hop/hop</i> MEFs. The axoneme was visualized by immunolabeling of acetylated-α-tubulin (AαT).</p