No abstract

In Drosophila, Pumilio (Pum) is important for neuronal homeostasis as well as learning and memory. We have recently characterized a mammalian homolog of Pum, Pum2, which is found in discrete RNAcontaining particles in the somatodendritic compartment of polarized neurons. In this study, we investigated the role of Pum2 in developing and mature neurons by RNA interference. In immature neurons, loss of Pum2 led to enhanced dendritic outgrowth and arborization. In mature neurons, Pum2 down-regulation resulted in a significant reduction in dendritic spines and an increase in elongated dendritic filopodia. Furthermore, we observed an increase in excitatory synapse markers along dendritic shafts. Electrophysiological analysis of synaptic function of neurons lacking Pum2 revealed an increased miniature excitatory postsynaptic current frequency. We then identified two specific mRNAs coding for a known translational regulator, eIF4E, and for a voltage-gated sodium channel, Scn1a, which interacts with Pum2 in immunoprecipitations from brain lysates. Finally, we show that Pum2 regulates translation of the eIF4E mRNA. Taken together, our data reveal a previously undescribed role for Pum2 in dendrite morphogenesis, synapse function, and translational control.

Ca 2؉ enters the stereocilia of hair cells through mechanoelectrical transduction channels opened by the deflection of the hair bundle and is exported back to endolymph by an unusual splicing isoform (w/a) of plasma-membrane calcium-pump isoform 2 (PMCA2). Ablation or missense mutations of the pump cause deafness, as described for the G283S mutation in the deafwaddler (dfw) mouse. A deafness-inducing missense mutation of PMCA2 (G293S) has been identified in a human family. The family also was screened for mutations in cadherin 23, which accentuated hearing loss in a previously described human family with a PMCA2 mutation. A T1999S substitution was detected in the cadherin 23 gene of the healthy father and affected son but not in that of the unaffected mother, who presented instead the PMCA2 mutation. The w/a isoform was overexpressed in CHO cells. At variance with the other PMCA2 isoforms, it became activated only marginally when exposed to a Ca 2؉ pulse. The G293S and G283S mutations delayed the dissipation of Ca 2؉ transients induced in CHO cells by InsP3. In organotypic cultures, Ca 2؉ imaging of vestibular hair cells showed that the dissipation of stereociliary Ca 2؉ transients induced by Ca 2؉ uncaging was compromised in the dfw and PMCA2 knockout mice, as was the sensitivity of the mechanoelectrical transduction channels to hair bundle displacement in cochlear hair cells.hereditary hearing loss ͉ mutations ͉ calcium homeostasis ͉ calcium transport T he receptive organelle of sensory hair cells in the mammalian cochlea, the stereocilia bundle, protrudes from the cell's apical surface. Inner hair cells transduce mechanical vibrations into electrical signals that are eventually transmitted to the brain to be transformed into hearing signals (1), whereas outer hair cells (OHCs) (2) amplify the vibrations of the basilar membrane (3). Mechanical stimuli that are detected as excitatory deflect a hair bundle, thus increasing tension in the tip link, a filament stretched between the tops of stereocilia. This tension is conveyed to mechanosensitive transduction (MET) channels that open to allow ions into the cell (4). The apical surface of hair cells is bathed in endolymph, which is rich in K ϩ but low in Na ϩ and Ca 2ϩ (5). K ϩ carries most the transduction current, but MET channels are Ca 2ϩ selective, i.e., Ca 2ϩ influx is significant even at the low Ca 2ϩ levels of the endolymph, which are much lower than those of other extracellular fluids (6-10): 20-23 M in the rodent cochlea (11, 12), 200-250 M in the vestibular system, possibly because of the presence there of calcium carbonate crystals (13,14). Approximately 10% of the MET current may actually be carried by Ca 2ϩ ions (15). Ca 2ϩ entering through MET channels is rapidly sequestered by buffers in the stereocilia (16,17) and is shuttled back to endolymph by the plasma membrane Ca 2ϩ pump (PMCA) (18,19), which is very concentrated in the stereocilia membrane (Ϸ2,000 per squared micrometer) (15, 19-21). The PMCA is assumed to increase Ca 2ϩ in the immediate pro...

Progressive hearing loss is common in the human population, but we have few clues to the molecular basis. Mouse mutants with progressive hearing loss offer valuable insights, and ENU (N-ethyl-N-nitrosourea) mutagenesis is a useful way of generating models. We have characterised a new ENU-induced mouse mutant, Oblivion (allele symbol Obl), showing semi-dominant inheritance of hearing impairment. Obl/+ mutants showed increasing hearing impairment from post-natal day (P)20 to P90, and loss of auditory function was followed by a corresponding base to apex progression of hair cell degeneration. Obl/Obl mutants were small, showed severe vestibular dysfunction by 2 weeks of age, and were completely deaf from birth; sensory hair cells were completely degenerate in the basal turn of the cochlea, although hair cells appeared normal in the apex. We mapped the mutation to Chromosome 6. Mutation analysis of Atp2b2 showed a missense mutation (2630C→T) in exon 15, causing a serine to phenylalanine substitution (S877F) in transmembrane domain 6 of the PMCA2 pump, the resident Ca2+ pump of hair cell stereocilia. Transmembrane domain mutations in these pumps generally are believed to be incompatible with normal targeting of the protein to the plasma membrane. However, analyses of hair cells in cultured utricular maculae of Obl/Obl mice and of the mutant Obl pump in model cells showed that the protein was correctly targeted to the plasma membrane. Biochemical and biophysical characterisation showed that the pump had lost a significant portion of its non-stimulated Ca2+ exporting ability. These findings can explain the progressive loss of auditory function, and indicate the limits in our ability to predict mechanism from sequence alone.

Arnica montana (Arnica m.) is used for its purported anti-inflammatory and tissue healing actions after trauma, bruises, or tissue injuries, but its cellular and molecular mechanisms are largely unknown. This work tested Arnica m. effects on gene expression using an in vitro model of macrophages polarized towards a “wound-healing” phenotype. The monocyte-macrophage human THP-1 cell line was cultured and differentiated with phorbol-myristate acetate and Interleukin-4, then exposed for 24h to Arnica m. centesimal (c) dilutions 2c, 3c, 5c, 9c, 15c or Control. Total RNA was isolated and cDNA libraries were sequenced with a NextSeq500 sequencer. Genes with significantly positive (up-regulated) or negative (down-regulated) fold changes were defined as differentially expressed genes (DEGs). A total of 20 DEGs were identified in Arnica m. 2c treated cells. Of these, 7 genes were up-regulated and 13 were down-regulated. The most significantly up-regulated function concerned 4 genes with a conserved site of epidermal growth factor-like region (p<0.001) and three genes of proteinaceous extracellular matrix, including heparin sulphate proteoglycan 2 (HSPG2), fibrillin 2 (FBN2), and fibronectin (FN1) (p<0.01). Protein assay confirmed a statistically significant increase of fibronectin production (p<0.05). The down-regulated transcripts derived from mitochondrial genes coding for some components of electron transport chain. The same groups of genes were also regulated by increasing dilutions of Arnica m. (3c, 5c, 9c, 15c), although with a lower effect size. We further tested the healing potential of Arnica m. 2c in a scratch model of wound closure based on the motility of bone marrow-derived macrophages and found evidence of an accelerating effect on cell migration in this system. The results of this work, taken together, provide new insights into the action of Arnica m. in tissue healing and repair, and identify extracellular matrix regulation by macrophages as a therapeutic target.