VGLUT3-p.A211V variant fuses stereocilia bundles and elongates synaptic ribbons.

DFNA25 is an autosomal-dominant and progressive form of human deafness caused by mutations in the SLC17A8 gene, which encodes the vesicular glutamate transporter type 3 (VGLUT3). To resolve the mechanisms underlying DFNA25, we studied phenotypes of mice harbouring the p.A221V mutation in humans (corresponding to p.A224V in mice). Using auditory brainstem response and distortion product otoacoustic emissions, we showed progressive hearing loss with intact cochlear amplification in the VGLUT3A224V/A224V mouse. The summating potential was reduced, indicating the alteration of inner hair cell (IHC) receptor potential. Scanning electron microscopy examinations demonstrated the collapse of stereocilia bundles in IHCs, leaving those from outer hair cells unaffected. In addition, IHC ribbon synapses underwent structural and functional modifications at later stages. Using super-resolution microscopy, we observed oversized synaptic ribbons and patch-clamp membrane capacitance measurements showed an increase in the rate of the sustained releasable pool exocytosis. These results suggest that DFNA25 stems from a failure in the mechano-transduction followed by a change in synaptic transfer. The VGLUT3A224V/A224V mouse model opens the way to a deeper understanding and to a potential treatment for DFNA25. KEY POINTS: The vesicular glutamate transporter type 3 (VGLUT3) loads glutamate into the synaptic vesicles of auditory sensory cells, the inner hair cells (IHCs). The VGLUT3-p.A211V variant is associated with human deafness DFNA25. Mutant mice carrying the VGLUT3-p.A211V variant show progressive hearing loss. IHCs from mutant mice harbour distorted stereocilary bundles, which detect incoming sound stimulation, followed by oversized synaptic ribbons, which release glutamate onto the afferent nerve fibres. These results suggest that DFNA25 stems from the failure of auditory sensory cells to faithfully transduce acoustic cues into neural messages.

Polyploidy can drive rapid adaptation in yeast.

Polyploidy is observed across the tree of life, yet its influence on evolution remains incompletely understood. Polyploidy, usually whole-genome duplication, is proposed to alter the rate of evolutionary adaptation. This could occur through complex effects on the frequency or fitness of beneficial mutations. For example, in diverse cell types and organisms, immediately after a whole-genome duplication, newly formed polyploids missegregate chromosomes and undergo genetic instability. The instability following whole-genome duplications is thought to provide adaptive mutations in microorganisms and can promote tumorigenesis in mammalian cells. Polyploidy may also affect adaptation independently of beneficial mutations through ploidy-specific changes in cell physiology. Here we perform in vitro evolution experiments to test directly whether polyploidy can accelerate evolutionary adaptation. Compared with haploids and diploids, tetraploids undergo significantly faster adaptation. Mathematical modelling suggests that rapid adaptation of tetraploids is driven by higher rates of beneficial mutations with stronger fitness effects, which is supported by whole-genome sequencing and phenotypic analyses of evolved clones. Chromosome aneuploidy, concerted chromosome loss, and point mutations all provide large fitness gains. We identify several mutations whose beneficial effects are manifest specifically in the tetraploid strains. Together, these results provide direct quantitative evidence that in some environments polyploidy can accelerate evolutionary adaptation.

Actin Filaments Regulate Exocytosis at the Hair Cell Ribbon Synapse.

Exocytosis at the inner hair cell ribbon synapse is achieved through the functional coupling between calcium channels and glutamate-filled synaptic vesicles. Using membrane capacitance measurements, we investigated whether the actin network regulates the exocytosis of synaptic vesicles at the mouse auditory hair cell. Our results suggest that actin network disruption increases exocytosis and that actin filaments may spatially organize a subfraction of synaptic vesicles with respect to the calcium channels. Significance statement: Inner hair cells (IHCs), the auditory sensory cells of the cochlea, release glutamate onto the afferent auditory nerve fibers to encode sound stimulation. To achieve this task, the IHC relies on the recruitment of glutamate-filled vesicles that can be located in close vicinity to the calcium channels or more remotely from them. The molecular determinants responsible for organizing these vesicle pools are not fully identified. Using pharmacological tools in combination with membrane capacitance measurements, we show that actin filament disruption increases exocytosis in IHCs and that actin filaments most likely position a fraction of vesicles away from the calcium channels.

[Communicating about the flare-up and its treatment]. / Bien communiquer sur la poussée et ses traitements.

Relapsing-remitting multiple sclerosis progresses by relapse. It is therefore necessary to know how to identify this phenomenon in order to be able to provide the best possible support to patients. The term "relapse" is used to characterize the period of a few days to a few weeks during which an attack of inflammation of the myelin occurs. Depending on the area affected, the symptoms will be different. To qualify as a relapse, the patient must have had new, permanent symptoms for at least 24 hours without fever or signs of infection and within 30 days of the last relapse.

Intrapulmonary effects of setting parameters in portable intrapulmonary percussive ventilation devices.

BACKGROUND: Despite potential benefits of intrapulmonary percussive ventilation (IPV) in various respiratory diseases, the impact of setting parameters on the mechanical effects produced by IPV in the lungs is unknown. We hypothesized that changing the parameters on IPV would modulate these effects. This in vitro study aimed at comparing the changes in intrapulmonary effects resulting from changes in parameters in 3 portable IPV devices (IMP2, Impulsator, and Pegaso). METHODS: Parameters were set in 72 combinations of frequency (90-250 cycles/min), inspiratory to expiratory (I/E) time ratio (from 1/2 to 3/1), and pressure (10-60 cm H(2)O). Four resulting effects were recorded on a test lung via a pneumotachometer: the expiratory to inspiratory flow ratio (E/I flow ratio), the PEEP, the ventilation, and the percussion. Percussion was assessed by the end-slope of the pressure curve. Analysis of variance was used for data analysis. RESULTS: E/I flow ratio increased with increasing I/E time ratio (P < .001). The Pegaso produced the lowest E/I flow ratio. PEEP raised 6 cm H(2)O in both IMP2 and Impulsator, and 17 cm H(2)O in the Pegaso with increasing frequency (P < .01), pressure, and I/E time ratio (P < .001). In all devices, ventilation increased with increasing pressure and decreasing frequency (P < .001). Percussion increased with increasing frequency and decreasing I/E time ratio (P < .001), and with increasing pressure when I/E time ratio was 1/1 or less. The Pegaso provided the poorest percussion. CONCLUSIONS: This study suggests that changing the parameters considerably modulates the mechanical effects produced by portable IPV devices in the lungs. Increasing frequency increased PEEP and percussion, but decreased ventilation. Increasing I/E time increased PEEP and E/I flow ratio, and decreased percussion. Finally, increasing pressure increased PEEP and ventilation. The Pegaso produced the highest PEEP, least percussion, and smallest change in E/I flow ratio.

MEN signaling: daughter bound pole must escape her mother to be fully active.

Budding yeast divide asymmetrically and must therefore align the position of the mitotic spindle with the plane of division. The success of this process is monitored by a checkpoint-signaling mechanism. Two recent papers in Molecular Cell reveal an important new facet of this signal transduction pathway.

dUTPase activity is critical to maintain genetic stability in Saccharomyces cerevisiae.

We identified a viable allele (dut1-1) of the DUT1 gene that encodes the dUTPase activity in Saccharomyces cerevisiae. The Dut1-1 protein possesses a single amino acid substitution (Gly82Ser) in a conserved motif nearby the active site and exhibits a greatly reduced dUTPase activity. The dut1-1 single mutant exhibits growth delay and cell cycle abnormalities and shows a strong spontaneous mutator phenotype. All phenotypes of the dut1-1 mutant are suppressed by the simultaneous inactivation of the uracil DNA N-glycosylase, Ung1. However, the ung1 dut1-1 double mutant accumulates uracil in its genomic DNA. The viability of the dut1-1 mutant is greatly impaired by the simultaneous inactivation of AP endonucleases. These data strongly suggest that the phenotypes of the dut1-1 mutant result from the incorporation of dUMPs into DNA subsequently converted into AP sites. The analysis of the dut1-1 strain mutation spectrum showed that cytosines are preferentially incorporated in front of AP sites in a Rev3-dependent manner during translesion synthesis. These results point to a critical role of the Dut1 protein in the maintenance of the genetic stability. Therefore, the normal cellular metabolism, and not only its byproducts, is an important source of endogenous DNA damage and genetic instability in eukaryotic cells.

Origin of endogenous DNA abasic sites in Saccharomyces cerevisiae.

Abasic (AP) sites are among the most frequent endogenous lesions in DNA and present a strong block to replication. In Saccharomyces cerevisiae, an apn1 apn2 rad1 triple mutant is inviable because of its incapacity to repair AP sites and related 3'-blocked single-strand breaks (M. Guillet and S. Boiteux, EMBO J. 21:2833, 2002). Here, we investigated the origin of endogenous AP sites in yeast. Our results show that the deletion of the UNG1 gene encoding the uracil DNA glycosylase suppresses the lethality of the apn1 apn2 rad1 mutant. In contrast, inactivation of the MAG1, OGG1, or NTG1 and NTG2 genes encoding DNA glycosylases involved in the repair of alkylation or oxidation damages does not suppress lethality. Although viable, the apn1 apn2 rad1 ung1 mutant presents growth delay due to a G(2)/M checkpoint. These results point to uracil as a critical source of the formation of endogenous AP sites in DNA. Uracil can arise in DNA by cytosine deamination or by the incorporation of dUMP during replication. Here, we show that the overexpression of the DUT1 gene encoding the dUTP pyrophosphatase (Dut1) suppresses the lethality of the apn1 apn2 rad1 mutant. Therefore, this result points to the dUTP pool as an important source of the formation of endogenous AP sites in eukaryotes.

Use of yeast for detection of endogenous abasic lesions, their source, and their repair.

Apurinic/apyrimidinic (AP) sites are expected to be one of the most frequent endogenous lesions in DNA. AP sites are potentially lethal and mutagenic. Data shows that the simultaneous inactivation of two AP endonucleases (Apn1 and Apn2) and of the nuclease Rad1-Rad10 causes cell death in Saccharomyces cerevisiae. We suggest that the essential function of Apn1, Apn2, and Rad1-Rad10 is to repair endogenous AP sites and related 3'-blocked single strand breaks. This data led us to conclude that the burden of endogenous AP sites is not compatible with life in absence of DNA repair. This chapter describes two genetic assays to investigate origin, repair, and biological consequences of endogenous AP sites in yeast. The first assay relies on genetic crosses and tetrad analysis and uses the apn1 apn2 rad1 triple mutant. The apn1 apn2 rad1 triple mutant is unviable; however, it can form microcolonies. By means of genetic crosses, apn1 apn2 rad1 x quadruple mutants are generated. The size of the colonies formed by each quadruple mutant is compared to that of the apn1 apn2 rad1 triple mutant. Three classes of genes (x) were identified: (i) genes whose inactivation aggravates the phenotype (reduces microcolony size), such as RAD9, RAD50, RAD51, RAD52, MUS81, and MRE11; (ii) genes whose inactivation alleviates the phenotype, such as UNG1, NTG1, and NTG2; and (iii) genes whose inactivation is neutral, such as MAG1 or OGG1. The second assay uses the apn1 apn2 rad14 triple mutant, which is viable but exhibits a spontaneous mutator phenotype. This mutant was used in a colethal screen. This assay allowed the identification of mutation in DNA repair genes such as RAD1 or RAD50, as well as a mutation in the DUT1 gene coding for the dUTPase, which has impact on the formation of AP sites in DNA. A model that summarizes our present and puzzling data on the origin and repair of endogenous AP sites is also presented.

Remodeling of the Inner Hair Cell Microtubule Meshwork in a Mouse Model of Auditory Neuropathy AUNA1.

Auditory neuropathy 1 (AUNA1) is a form of human deafness resulting from a point mutation in the 5' untranslated region of the Diaphanous homolog 3 (DIAPH3) gene. Notably, the DIAPH3 mutation leads to the overexpression of the DIAPH3 protein, a formin family member involved in cytoskeleton dynamics. Through study of diap3-overexpressing transgenic (Tg) mice, we examine in further detail the anatomical, functional, and molecular mechanisms underlying AUNA1. We identify diap3 as a component of the hair cells apical pole in wild-type mice. In the diap3-overexpressing Tg mice, which show a progressive threshold shift associated with a defect in inner hair cells (IHCs), the neurotransmitter release and potassium conductances are not affected. Strikingly, the overexpression of diap3 results in a selective and early-onset alteration of the IHC cuticular plate. Molecular dissection of the apical components revealed that the microtubule meshwork first undergoes aberrant targeting into the cuticular plate of Tg IHCs, followed by collapse of the stereociliary bundle, with eventual loss of the IHC capacity to transmit incoming auditory stimuli.

An uncommon phenotype with familial central hypogonadism caused by a novel PROP1 gene mutant truncated in the transactivation domain.

CONTEXT: PROP1 gene mutations are usually associated with childhood onset GH and TSH deficiencies, whereas gonadotroph deficiency is diagnosed at pubertal age. OBJECTIVES: We report a novel PROP1 mutation revealed by familial normosmic hypogonadotropic hypogonadism. We performed in vitro transactivation and DNA binding experiments to study functional consequences of this mutation. SETTING: Three brothers were followed in the Department of Endocrinology of a French university hospital. PATIENTS: These patients from a consanguineous kindred were referred for cryptorchidism and/or delayed puberty. RESULTS: Initial investigations revealed hypogonadotropic hypogonadism. One of the patients had psychomotor retardation, intracranial hypertension, and minor renal malformations. The brothers reached normal adult height and developed GH and TSH deficiencies after age 30. A novel homozygous nonsense mutation (W194X) was found in the PROP1 gene, indicating that the protein is truncated in its transactivation domain. Transfection studies confirmed the deleterious effect of this mutation, whose transactivation capacity was only 34.4% of that of the wild-type. Unexpectedly altered DNA-binding properties suggested that the C-terminal end of the factor plays a role in protein-DNA interaction. CONCLUSIONS: PROP1 mutations should be considered among the growing number of genetic causes of initially isolated hypogonadotropic hypogonadism. This report extends the phenotype variability associated with PROP1 mutations.

Abasic sites in DNA: repair and biological consequences in Saccharomyces cerevisiae.

Apurinic/apyrimidinic (AP) sites are one of the most frequent spontaneous lesions in DNA. They are potentially mutagenic and lethal lesions that can block DNA replication and transcription. In addition, cleavage of AP sites by AP endonucleases or AP lyases generates DNA single-strand breaks (SSBs) with 5'- or 3'-blocked ends, respectively. Therefore, we suggest that AP sites and 3'- or 5'-blocked SSBs, we name "honorary AP sites", constitute a single class of lesions. In this review, we describe the different mechanisms used by the budding yeast Saccharomyces cerevisiae to remove or tolerate AP sites and related SSBs. In wild-type cells, AP sites are primarily repaired by the base excision repair (BER) pathway, with the nucleotide excision repair (NER) pathway as a back up activity. BER is initiated by one of the two AP endonucleases, Apn1 or Apn2. Three DNA N-glycosylases/AP lyases, Ntg1, Ntg2 and Ogg1, can also incise AP sites in DNA. Rad27, a structure specific endonuclease, is involved in the repair of 5'-blocked ends, whereas Apn1, Apn2 and Rad1-Rad10 are involved in the removal of 3'-blocked ends using their 3'-phosphodiesterase and 3'-flap endonuclease activities, respectively. AP sites can stall DNA replication forks, as well as they block in vitro DNA synthesis by DNA polymerase delta. Restart of stalled forks can occur through a recombination-associated pathway initiated by the Mus81-Mms4 endonuclease or mutagenic translesion DNA synthesis (TLS). The mutagenic bypass of AP sites is a two-polymerases affair with an inserter DNA polymerase (Poldelta, Poleta or Rev1) and an extender DNA polymerase (Polzeta). Under normal growth conditions, inactivation of Apn1, Apn2 and Rad1-Rad10 causes cell death. Therefore, the burden of spontaneous AP sites is not compatible with life, in the absence of excision repair pathways. These results in yeast demonstrate that AP sites are critical endogenous DNA damages that cause genetic instability and by analogy could be associated with degenerative pathologies in human.

Regulation of prolactin, GH, and Pit-1 gene expression in anterior pituitary by Pitx2: An approach using Pitx2 mutants.

The transcription factor Pitx2 is required for the morphogenesis of anterior structures such as the eye, teeth, and anterior pituitary. We investigated the functional properties of Pitx2 missense mutants previously reported in Axenfeld-Rieger syndrome, using reporter genes under the control of pituitary target gene [human (h)PRL, hGH, hPit-1] promoters transfected in nonpituitary and pituitary cell lines. The five mutants appeared to be transcriptionally defective despite conserved DNA-binding in CV1 cells. In addition, one mutation, R91P, almost completely blocked the wt-Pitx2-induced activation of the target promoters, prevented the Pitx2/Pit-1 synergistic activation of the hPRL promoter, and was able to counteract the Pitx1-driven transactivation effects. The dominant negative properties of this mutant were further established in cells endogenously expressing Pitx2 because transfection of R91P in GH4C1 somatolactotroph cells resulted in a dose-dependent inhibition of basal activities of the pituitary promoters. These results, which show that Pitx2 mutants are defective in activating pituitary target genes, confirm the critical role of this homeodomain factor in the differentiated functions of the pituitary somatolactotroph cells. Furthermore, these results might form the basis for future experiments because dominant negative forms of Pitx2 such as R91P might provide instructive tools to further delineate the detailed mechanisms mediating Pitx2 functions in cell proliferation and differentiation.

A 3-year causative study of pompholyx in 120 patients.

OBJECTIVE: To assess the relative frequency of the different causes of pompholyx evoked in the literature. DESIGN: Prospective survey. SETTING: Clinical outpatient setting. PATIENTS: A total of 120 consecutive patients with pompholyx referred to our department from 2000 through 2003. MAIN OUTCOME MEASURES: Systematic investigation of different causes of pompholyx: fungal intertrigo, hyperhidrosis, atopy, contact eczema, and internal reactions with systematic provocation tests to metals, balsam of Peru, and food allergen when suspected. RESULTS: The present study found the following causes of pompholyx in the 120 patients: mycosis (10.0%); allergic contact pompholyx (67.5%), with cosmetic and hygiene products as the main factor (31.7%), followed by metals (16.7%); and internal reactivation from drug, food, or haptenic (nickel) origin (6.7%). The remaining 15.0% of patients were classified as idiopathic patients, but all were atopic. (Percentages do not total 100 because of rounding.) CONCLUSIONS: Our data confirm the existence of reactional pompholyx to interdigital-plantar intertrigos and endogenous reactions to metals or other allergens, but they mainly point at the unexpected importance of a so-called contact pompholyx in which cosmetic and hygiene products play a preponderant role compared with metals. The great frequency of atopic conditions, even if idiopathic pompholyx is not inferred as an equivalent of atopy, should lead to further causative investigations before undertaking more expensive or extensive treatments of refractory pompholyx.

Endogenous DNA abasic sites cause cell death in the absence of Apn1, Apn2 and Rad1/Rad10 in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, mutations in APN1, APN2 and either RAD1 or RAD10 genes are synthetic lethal. In fact, apn1 apn2 rad1 triple mutants can form microcolonies of approximately 300 cells. Expression of Nfo, the bacterial homologue of Apn1, suppresses the lethality. Turning off the expression of Nfo induces G(2)/M cell cycle arrest in an apn1 apn2 rad1 triple mutant. The activation of this checkpoint is RAD9 dependent and allows residual DNA repair. The Mus81/Mms4 complex was identified as one of these back-up repair activities. Furthermore, inactivation of Ntg1, Ntg2 and Ogg1 DNA N-glycosylase/AP lyases in the apn1 apn2 rad1 background delayed lethality, allowing the formation of minicolonies of approximately 10(5) cells. These results demonstrate that, under physiological conditions, endogenous DNA damage causes death in cells deficient in Apn1, Apn2 and Rad1/Rad10 proteins. We propose a model in which endogenous DNA abasic sites are converted into 3'-blocked single-strand breaks (SSBs) by DNA N-glycosylases/AP lyases. Therefore, we suggest that the essential and overlapping function of Apn1, Apn2, Rad1/Rad10 and Mus81/Mms4 is to repair 3'-blocked SSBs using their 3'-phosphodiesterase activity or their 3'-flap endonuclease activity, respectively.