Chromatin remodeling protein CHD4 regulates axon guidance of spiral ganglion neurons in developing cochlea.

The chromodomain helicase binding protein 4 (CHD4) is an ATP-dependent chromatin remodeler. De-novo pathogenic variants of CHD4 cause Sifrim-Hitz-Weiss syndrome (SIHIWES). Patients with SIHIWES show delayed development, intellectual disability, facial dysmorphism, and hearing loss. Many cochlear cell types, including spiral ganglion neurons (SGNs), express CHD4. SGNs are the primary afferent neurons that convey sound information from the cochlea, but the function of CHD4 in SGNs is unknown. We employed the Neurog1(Ngn1) CreERT2 Chd4 conditional knockout animals to delete Chd4 in SGNs. SGNs are classified as type I and type II neurons. SGNs lacking CHD4 showed abnormal fasciculation of type I neurons along with improper pathfinding of type II fibers. CHD4 binding to chromatin from immortalized multipotent otic progenitor-derived neurons was used to identify candidate target genes in SGNs. Gene ontology analysis of CHD4 target genes revealed cellular processes involved in axon guidance, axonal fasciculation, and ephrin receptor signaling pathway. We validated increased Epha4 transcripts in SGNs from Chd4 conditional knockout cochleae. The results suggest that CHD4 attenuates the transcription of axon guidance genes to form the stereotypic pattern of SGN peripheral projections. The results implicate epigenetic changes in circuit wiring by modulating axon guidance molecule expression and provide insights into neurodevelopmental diseases.

Chromodomain helicase DNA binding protein 4 in cell fate decisions.

Loss of spiral ganglion neurons (SGNs) in the cochlea causes hearing loss. Understanding the mechanisms of cell fate transition accelerates efforts that employ directed differentiation and lineage conversion to repopulate lost SGNs. Proposed strategies to regenerate SGNs rely on altering cell fate by activating transcriptional regulatory networks, but repressing networks for alternative cell lineages is also essential. Epigenomic changes during cell fate transitions suggest that CHD4 represses gene expression by altering the chromatin status. Despite limited direct investigations, human genetic studies implicate CHD4 function in the inner ear. The possibility of CHD4 in suppressing alternative cell fates to promote inner ear regeneration is discussed.

Semaphorin3A/PlexinA3 association with the Scribble scaffold for cGMP increase is required for apical dendrite development.

The development of the apical dendrite from the leading process of the bipolar pyramidal neuron might be directed by spatially organized extrinsic cues acting on localized intrinsic determinants. The extracellular cues regulating apical dendrite polarization remain elusive. We show that leading process and apical dendrite development are directed by class III Semaphorins and mediated by a localized cGMP-synthesizing complex. The scaffolding protein Scribble that associates with the cGMP-synthesizing enzyme soluble guanylate cyclase (sGC) also associates with the Semaphorin3A (Sema3A) co-receptor PlexinA3. Deletion or knockdown of PlexinA3 and Sema3A or disruption of PlexinA3-Scribble association prevents Sema3A-mediated cGMP increase and causes defects in apical dendrite development. These manipulations also impair bipolar polarity and leading process establishment. Local cGMP elevation or sGC expression rescues the effects of PlexinA3 knockdown or PlexinA3-Scribble complex disruption. During neuronal polarization, leading process and apical dendrite development are directed by a scaffold that links Semaphorin cue to cGMP increase.

Modular and Distinct Plexin-A4/FARP2/Rac1 Signaling Controls Dendrite Morphogenesis.

Diverse neuronal populations with distinct cellular morphologies coordinate the complex function of the nervous system. Establishment of distinct neuronal morphologies critically depends on signaling pathways that control axonal and dendritic development. The Sema3A-Nrp1/PlxnA4 signaling pathway promotes cortical neuron basal dendrite arborization but also repels axons. However, the downstream signaling components underlying these disparate functions of Sema3A signaling are unclear. Using the novel PlxnA4KRK-AAA knock-in male and female mice, generated by CRISPR/cas9, we show here that the KRK motif in the PlxnA4 cytoplasmic domain is required for Sema3A-mediated cortical neuron dendritic elaboration but is dispensable for inhibitory axon guidance. The RhoGEF FARP2, which binds to the KRK motif, shows identical functional specificity as the KRK motif in the PlxnA4 receptor. We find that Sema3A activates the small GTPase Rac1, and that Rac1 activity is required for dendrite elaboration but not axon growth cone collapse. This work identifies a novel Sema3A-Nrp1/PlxnA4/FARP2/Rac1 signaling pathway that specifically controls dendritic morphogenesis but is dispensable for repulsive guidance events. Overall, our results demonstrate that the divergent signaling output from multifunctional receptor complexes critically depends on distinct signaling motifs, highlighting the modular nature of guidance cue receptors and its potential to regulate diverse cellular responses.SIGNIFICANCE STATEMENT The proper formation of axonal and dendritic morphologies is crucial for the precise wiring of the nervous system that ultimately leads to the generation of complex functions in an organism. The Semaphorin3A-Neuropilin1/Plexin-A4 signaling pathway has been shown to have multiple key roles in neurodevelopment, from axon repulsion to dendrite elaboration. This study demonstrates that three specific amino acids, the KRK motif within the Plexin-A4 receptor cytoplasmic domain, are required to coordinate the downstream signaling molecules to promote Sema3A-mediated cortical neuron dendritic elaboration, but not inhibitory axon guidance. Our results unravel a novel Semaphorin3A-Plexin-A4 downstream signaling pathway and shed light on how the disparate functions of axon guidance and dendritic morphogenesis are accomplished by the same extracellular ligand in vivo.

Neuropilin 2 Signaling Mediates Corticostriatal Transmission, Spine Maintenance, and Goal-Directed Learning in Mice.

The striatum represents the main input structure of the basal ganglia, receiving massive excitatory input from the cortex and the thalamus. The development and maintenance of cortical input to the striatum is crucial for all striatal function including many forms of sensorimotor integration, learning, and action control. The molecular mechanisms regulating the development and maintenance of corticostriatal synaptic transmission are unclear. Here we show that the guidance cue, Semaphorin 3F and its receptor Neuropilin 2 (Nrp2), influence dendritic spine maintenance, corticostriatal short-term plasticity, and learning in adult male and female mice. We found that Nrp2 is enriched in adult layer V pyramidal neurons, corticostriatal terminals, and in developing and adult striatal spiny projection neurons (SPNs). Loss of Nrp2 increases SPN excitability and spine number, reduces short-term facilitation at corticostriatal synapses, and impairs goal-directed learning in an instrumental task. Acute deletion of Nrp2 selectively in adult layer V cortical neurons produces a similar increase in the number of dendritic spines and presynaptic modifications at the corticostriatal synapse in the Nrp2-/- mouse, but does not affect the intrinsic excitability of SPNs. Furthermore, conditional loss of Nrp2 impairs sensorimotor learning on the accelerating rotarod without affecting goal-directed instrumental learning. Collectively, our results identify Nrp2 signaling as essential for the development and maintenance of the corticostriatal pathway and may shed novel insights on neurodevelopmental disorders linked to the corticostriatal pathway and Semaphorin signaling.SIGNIFICANCE STATEMENT The corticostriatal pathway controls sensorimotor, learning, and action control behaviors and its dysregulation is linked to neurodevelopmental disorders, such as autism spectrum disorder (ASD). Here we demonstrate that Neuropilin 2 (Nrp2), a receptor for the axon guidance cue semaphorin 3F, has important and previously unappreciated functions in the development and adult maintenance of dendritic spines on striatal spiny projection neurons (SPNs), corticostriatal short-term plasticity, intrinsic physiological properties of SPNs, and learning in mice. Our findings, coupled with the association of Nrp2 with ASD in human populations, suggest that Nrp2 may play an important role in ASD pathophysiology. Overall, our work demonstrates Nrp2 to be a key regulator of corticostriatal development, maintenance, and function, and may lead to better understanding of neurodevelopmental disease mechanisms.

Floor plate-derived neuropilin-2 functions as a secreted semaphorin sink to facilitate commissural axon midline crossing.

Commissural axon guidance depends on a myriad of cues expressed by intermediate targets. Secreted semaphorins signal through neuropilin-2/plexin-A1 receptor complexes on post-crossing commissural axons to mediate floor plate repulsion in the mouse spinal cord. Here, we show that neuropilin-2/plexin-A1 are also coexpressed on commissural axons prior to midline crossing and can mediate precrossing semaphorin-induced repulsion in vitro. How premature semaphorin-induced repulsion of precrossing axons is suppressed in vivo is not known. We discovered that a novel source of floor plate-derived, but not axon-derived, neuropilin-2 is required for precrossing axon pathfinding. Floor plate-specific deletion of neuropilin-2 significantly reduces the presence of precrossing axons in the ventral spinal cord, which can be rescued by inhibiting plexin-A1 signaling in vivo. Our results show that floor plate-derived neuropilin-2 is developmentally regulated, functioning as a molecular sink to sequester semaphorins, preventing premature repulsion of precrossing axons prior to subsequent down-regulation, and allowing for semaphorin-mediated repulsion of post-crossing axons.

Evaluation of salvage male transobturator sling placement following recurrent stress urinary incontinence after failed transobturator sling.

OBJECTIVE: To evaluate the outcome of patients treated with a salvage AdVance male sling after a failed primary transobturator sling placement. METHODS: Retrospective review of patients treated at our center with a primary and subsequent salvage AdVance sling. Success was defined as a dry safety pad or no pads (cured), or >50% improvement in pads used per day and patient satisfaction (improved). Early primary sling failures (<6 months) were compared with late (≥6 months) failures with regard to continence outcomes. RESULTS: We identified 18 patients who underwent a salvage AdVance sling placement at our institution. Overall success was 72% at 6 months postoperatively and 56% at a mean follow-up of 17.5 months, including 50% and 39% of patients who were dry at those same time periods. Patients failing late after their primary sling (n = 8) enjoyed improved outcomes with salvage sling placement compared with patients who failed early (n = 10) after the primary sling. At 6 months, more patients in the late primary failure group were cured (75% vs. 30%; P = .031). These improved cure rates remained significant through final follow-up with cure rates of 63% and 20%, respectively (P = .041). CONCLUSION: Salvage AdVance male sling is a viable treatment option after a failed primary sling procedure, especially in patients who demonstrated a prolonged efficacy period before primary sling failure.

Vertebrate spinal commissural neurons: a model system for studying axon guidance beyond the midline.

For bilaterally symmetric organisms, the transfer of information between the left and right side of the nervous system is mediated by commissures formed by neurons that project their axons across the body midline to the contralateral side of the central nervous system (CNS). After crossing the midline, many of these axons must travel long distances to reach their targets, including those that extend from spinal commissural neurons. Owing to the highly stereotyped trajectories of spinal commissural neurons that can be divided into several segments as these axons project to their targets, it is an ideal system for investigators to ask fundamental questions related to mechanisms of short- and long-range axon guidance, fasciculation, and choice point decisions at the midline intermediate target. In addition, studies of patterning genes of the nervous system have revealed complex transcription factor codes that function in a combinatorial fashion to specify individual classes of spinal neurons including commissural neurons. Despite these advances and the functional importance of spinal commissural neurons in mediating the transfer of external sensory information from the peripheral nervous system (PNS) to the CNS, only a handful of studies have begun to elucidate the mechanistic logic underlying their long-range pathfinding and the characterization of their synaptic targets. Using in vitro assays, in vivo labeling methodologies, in combination with both loss- and gain-of-function experiments, several studies have revealed that the molecular mechanisms of long-range spinal commissural axon pathfinding involve an interplay between classical axon guidance cues, morphogens and cell adhesion molecules. For further resources related to this article, please visit the WIREs website.

Distinct cytoplasmic domains in Plexin-A4 mediate diverse responses to semaphorin 3A in developing mammalian neurons.

Guidance receptor signaling is crucial for neural circuit formation and elicits diverse cellular events in specific neurons. We found that signaling from the guidance cue semaphorin 3A diverged through distinct cytoplasmic domains in its receptor Plexin-A4 to promote disparate cellular behavior in different neuronal cell types. Plexin-A4 has three main cytoplasmic domains--C1, Hinge/RBD, and C2--and interacts with family members of the Rho guanine nucleotide exchange factor FARP proteins. We show that growth cone collapse occurred in Plexin-A4-deficient dorsal root ganglion sensory neurons reconstituted with Plexin-A4 containing either the Hinge/RBD or C2 domain, whereas both of the Hinge/RBD and C1 domains were required for dendritic arborization in cortical neurons. Although knockdown studies indicated that both the collapse and arborization responses involved FARP2, mutations in the cytoplasmic region of Plexin-A4 that reduced its interaction with FARP2 strongly inhibited semaphorin 3A-induced dendritic branching but not growth cone collapse, suggesting that different degrees of interaction are required for the two responses or that developing axons have an indirect path to FARP2 activation. Thus, our study provided insights into the multifunctionality of guidance receptors, in particular showing that the semaphorin 3A signal diverges through specific functions of the modular domains of Plexin-A4.

Neuropilin2 regulates the guidance of post-crossing spinal commissural axons in a subtype-specific manner.

BACKGROUND: Spinal commissural axons represent a model system for deciphering the molecular logic that regulates the guidance of midline-crossing axons in the developing central nervous system (CNS). Whether the same or specific sets of guidance signals control the navigation of molecularly distinct subtypes of these axons remains an open and largely unexplored question. Although it is well established that post-crossing commissural axons alter their responsiveness to midline-associated guidance cues, our understanding of the repulsive mechanisms that drive the post-crossing segments of these axons away from the midline and whether the underlying guidance systems operate in a commissural axon subtype-specific manner, remains fragmentary at best. RESULTS: Here, we utilize axonally targeted transgenic reporter mice to visualize genetically distinct dorsal interneuron (dI)1 and dI4 commissural axons and show that the repulsive class 3 semaphorin (Sema3) guidance receptor Neuropilin 2 (Npn2), is selectively expressed on the dI1 population and is required for the guidance of post-crossing dI1, but not dI4, axons. Consistent with these observations, the midline-associated Npn2 ligands, Sema3F and Sema3B, promote the collapse of dI1, but not dI4, axon-associated growth cones in vitro. We also identify, for the first time, a discrete GABAergic population of ventral commissural neurons/axons in the embryonic mouse spinal cord that expresses Npn2, and show that Npn2 is required for the proper guidance of their post-crossing axons. CONCLUSIONS: Together, our findings indicate that Npn2 is selectively expressed in distinct populations of commissural neurons in both the dorsal and ventral spinal cord, and suggest that Sema3-Npn2 signaling regulates the guidance of post-crossing commissural axons in a population-specific manner.

Determination of selected heavy metal concentrations and distribution in a southwestern stream using macrophytes.

Since the reduction of the arsenic standard from 50 to 10 µg L(-1) by the US Environmental Protection Agency in 2006 many small town and rural water municipalities were left with the task of preventing or mitigating arsenic contamination of drinking water supplies. In this study macrophytes and sediments were used to determine the concentration and distribution of heavy metals (As, Cd, Cu, Pb, and Zn) within the primary source of drinking water (Gallinas River watershed) to the residents of Las Vegas, New Mexico. Sampling was done in the spring and fall at four sites, two above the city and two below, and samples were analyzed using ICP-MS. Results showed significantly higher (p<.05) metal concentrations in plant roots than shoots for most metals. Spearman's correlation showed positive correlations (r>.3) between plant and sediment concentrations of Cd, Pb, Zn, As, and a negative correlation for Cu. The site above waste water treatment plant (AWWTP) had the highest plant tissue concentrations of Cd, Pb, Zn, and As. All of these concentrations attained critical toxicity levels exceeding sediment quality guidelines. High concentration factor values and levels of metals detected in macrophyte tissues indicate that heavy metals within sediments in the Gallinas River occur in bioavailable forms. Correlations between plant and sediment metal concentrations indicate that metal concentrations in macrophyte tissues are a good reflection of metal concentrations within the sediment in the Gallinas River.

Teratogenic versus mutagenic abnormalities in chironomid larvae exposed to zinc and lead.

Before chironomid mouthpart deformities can be utilized as indicators of aquatic metal pollution with certainty, it must first be established that deformities are teratogenic and not mutagenic. A laboratory experiment was conducted to assess this question using Zn and Pb as causative agents. Parent populations were reared in sediments spiked with zinc (Zn) or lead (Pb) and their resulting offspring (F1 generation) were reared in clean sediments. The proportions of mouthpart deformities in C. tentans larvae were compared via logistic regression, accounting for time of exposure, between parent and offspring populations. Results indicate that 14% of chironomids from Zn-spiked sediment contained deformed menta and/or mandibles. However, the F1-Zn generation displayed a deformity of 1.7%. Larvae reared in Pb-spiked sediments displayed a deformity frequency of 9% and the F1 generations (F1-Pb a and F1-Pb b) had deformity proportion of 7 and 6%, respectively. We concluded that the deformities caused by Zn stress were morphological because the resulting F1 deformity frequencies declined to control levels. However, deformities caused by Pb appear to be genetic since F1 deformity percentages did not differ from the parent deformity frequency. Because larvae reared in Zn- and Pb-spiked sediments were larger than larvae reared in uncontaminated sediments, we could not conclude that Zn and Pb in the sediments stunted the development of C. tentans.

Effects of exposure to a combination of zinc- and lead-spiked sediments on mouthpart development and growth in Chironomus tentans.

Exposures to either zinc or lead in contaminated sediments have been shown to induce characteristic deformities in larval chironomids. This study examined the effects of exposure to lead and zinc in combination on Chironomus tentans larvae. Proportions of mouthpart deformities in populations of larvae reared in sediments containing nominal combinations of lead and zinc were tested for additive, synergistic, and antagonistic interactions using logistic regression. Metal body burdens, body size measurements, and survival were used to evaluate toxicity and developmental impacts. Results demonstrate zinc and lead mixtures produce fewer deformities than the individual metal, so their interaction may be characterized as antagonistic. However, exposure to the metal mixtures also caused delayed development and failure to hatch. The apparent decline in deformities may be an artifact of higher mortalities or developmental effects. This research provides better understanding of some of the problems and considerations for use of chironomid population deformity proportions in bioassessments for sediment metal contamination.

Survival of Salmonella enterica in freshwater and sediments and transmission by the aquatic midge Chironomus tentans (Chironomidae: Diptera).

Survival of a nalidixic acid-resistant strain of Salmonella enterica serovar Typhimurium mr-DT-104 in water and sediments was tested using artificially contaminated aquaria. Water samples remained culture positive for salmonella for up to 54 days. Sediment samples were culture positive up to 119 days. In addition, potential mechanisms for spreading salmonella in the environments by chironomid larvae and adults were tested. We evaluated the acquisition of mr-DT-104 by chironomids from contaminated aquatic sediments and subsequent spread to uncontaminated sediments. Larval chironomids raised in contaminated sediments became culture positive, and the bacteria were carried over to adults after emergence. Contamination of clean sediments by chironomid larvae was not demonstrated. These findings clearly suggest that mr-DT-104 serovar organisms can survive in aquatic sediments for at least several months. Uptake of salmonellae by chironomid larvae and adults suggests that they are possible vectors of mr-DT-104 in both aquatic and terrestrial environments, although the role of larval defecation in movement of bacteria to new sediments was not demonstrated.

Morphological abnormalities in Chironomus tentans exposed to cadmium-and copper-spiked sediments.

The induction of mouthpart deformities and the developmental response with exposure to sediments spiked with three concentrations (9, 39, and 61 microgg(-1) Cd dry wt.) of cadmium (Cd) and three concentrations (30, 125, and 215 microgg(-1) Cu dry wt.) of copper (Cu) were investigated. Mouthpart deformity proportions in Chironomus tentans larvae were compared between metal-spiked and control populations and between parent and offspring (F1) populations. Cd- and Cu-treated sediments induced deformities (low Cd=13%, medium Cd=7%, high Cd=4%, low Cu=6%, medium Cu=9%, high Cu=6%) at significantly higher proportions than control (3%) sediments. No negative developmental response was determined. Larval sizes in metal-treated aquaria and control aquaria were not significantly different. F1 larvae from parents reared in medium and high Cu had significantly lower deformity rates than their parents. Our research adds to the growing evidence implicating heavy metals in general, and Cd and Cu specifically, as teratogenic agents.

Aspectos epidemiológicos do acidente ofídico no Vale do Ribeira, Säo Paulo, 1985 a 1989 / Epidemiologic aspects of snakebites in Vale do Ribeira, Säo Paulo, 1985-1989

Estudo da incidência dos acidentes por serpentes peçonhentas nos 12 municípios do Vale do Ribeira, estado de Säo Paulo, baseado na avaliaçäo de 840 casos que ocorreram de janeiro de 1985 a dezembro de 1989. A incidência global no Vale mostrou-se muito maior (68,7 a 84,2/100.000 habitantes) do que a conhecida para o estado de Säo Paulo (6,8 a 7,4/100.000 habitantes). Maior ocorrência foi observada nos primeiros e nos últimos meses do ano, e os municípios de maior incidência foram os de Juquiá (159,4/100.000 habitantes) e Eldorado (131,4/100.000 habitantes). As pessoas do sexo masculino foram as mais acometidas (79 por cento) assim como os lavradores (52,5 por cento). A maioria dos casos foi atendida precocemente (57,7 por cento dentro de duas horas após o acidente). A semelhança do que ocorre em todo o estado de Säo Paulo, o quadro clínico provocado pelo envenenamento foi, em geral, leve e a letalidade foi baixa (0,2 por cento).

Control de enfermedades transmisibles en la amazonía peruana / Control of communicable diseases at peruvian amazony

Manual relativo al diagnóstico, tratamiento y control de las enfermedades más prevalecientes en la amazonía. Se describen 31 cuadros mórbidos, y un capítulo sobre vacunación como actividad preventiva