Autism-associated CHD8 controls reactive gliosis and neuroinflammation via remodeling chromatin in astrocytes.

Reactive changes of glial cells during neuroinflammation impact brain disorders and disease progression. Elucidating the mechanisms that control reactive gliosis may help us to understand brain pathophysiology and improve outcomes. Here, we report that adult ablation of autism spectrum disorder (ASD)-associated CHD8 in astrocytes attenuates reactive gliosis via remodeling chromatin accessibility, changing gene expression. Conditional Chd8 deletion in astrocytes, but not microglia, suppresses reactive gliosis by impeding astrocyte proliferation and morphological elaboration. Astrocyte Chd8 ablation alleviates lipopolysaccharide-induced neuroinflammation and septic-associated hypothermia in mice. Astrocytic CHD8 plays an important role in neuroinflammation by altering the chromatin landscape, regulating metabolic and lipid-associated pathways, and astrocyte-microglia crosstalk. Moreover, we show that reactive gliosis can be directly mitigated in vivo using an adeno-associated virus (AAV)-mediated Chd8 gene editing strategy. These findings uncover a role of ASD-associated CHD8 in the adult brain, which may warrant future exploration of targeting chromatin remodelers in reactive gliosis and neuroinflammation in injury and neurological diseases.

Transsynaptic cerebellin 4-neogenin 1 signaling mediates LTP in the mouse dentate gyrus.

Five decades ago, long-term potentiation (LTP) of synaptic transmission was discovered at entorhinal cortex→dentate gyrus (EC→DG) synapses, but the molecular determinants of EC→DG LTP remain largely unknown. Here, we show that the presynaptic neurexin­ligand cerebellin-4 (Cbln4) is highly expressed in the entorhinal cortex and essential for LTP at EC→DG synapses, but dispensable for basal synaptic transmission at these synapses. Cbln4, when bound to cell-surface neurexins, forms transcellular complexes by interacting with postsynaptic DCC (deleted in colorectal cancer) or neogenin-1. DCC and neogenin-1 act as netrin and repulsive guidance molecule-a (RGMa) receptors that mediate axon guidance in the developing brain, but their binding to Cbln4 raised the possibility that they might additionally function in the mature brain as postsynaptic receptors for presynaptic neurexin/Cbln4 complexes, and that as such receptors, DCC or neogenin-1 might mediate EC→DG LTP that depends on Cbln4. Indeed, we observed that neogenin-1, but not DCC, is abundantly expressed in dentate gyrus granule cells, and that postsynaptic neogenin-1 deletions in dentate granule cells blocked EC→DG LTP, but again did not affect basal synaptic transmission similar to the presynaptic Cbln4 deletions. Thus, binding of presynaptic Cbln4 to postsynaptic neogenin-1 renders EC→DG synapses competent for LTP, but is not required for establishing these synapses or for otherwise enabling their function.

Netrin-1 Confines Rhombic Lip-Derived Neurons to the CNS.

During brainstem development, newborn neurons originating from the rhombic lip embark on exceptionally long migrations to generate nuclei important for audition, movement, and respiration. Along the way, this highly motile population passes several cranial nerves yet remains confined to the CNS. We found that Ntn1 accumulates beneath the pial surface separating the CNS from the PNS, with gaps at nerve entry sites. In mice null for Ntn1 or its receptor DCC, hindbrain neurons enter cranial nerves and migrate into the periphery. CNS neurons also escape when Ntn1 is selectively lost from the sub-pial region (SPR), and conversely, expression of Ntn1 throughout the mutant hindbrain can prevent their departure. These findings identify a permissive role for Ntn1 in maintaining the CNS-PNS boundary. We propose that Ntn1 confines rhombic lip-derived neurons by providing a preferred substrate for tangentially migrating neurons in the SPR, preventing their entry into nerve roots.

Synergistic integration of Netrin and ephrin axon guidance signals by spinal motor neurons.

During neural circuit assembly, axonal growth cones are exposed to multiple guidance signals at trajectory choice points. While axonal responses to individual guidance cues have been extensively studied, less is known about responses to combination of signals and underlying molecular mechanisms. Here, we studied the convergence of signals directing trajectory selection of spinal motor axons entering the limb. We first demonstrate that Netrin-1 attracts and repels distinct motor axon populations, according to their expression of Netrin receptors. Quantitative in vitro assays demonstrate that motor axons synergistically integrate both attractive or repulsive Netrin-1 signals together with repulsive ephrin signals. Our investigations of the mechanism of ephrin-B2 and Netrin-1 integration demonstrate that the Netrin receptor Unc5c and the ephrin receptor EphB2 can form a complex in a ligand-dependent manner and that Netrin-ephrin synergistic growth cones responses involve the potentiation of Src family kinase signaling, a common effector of both pathways.

Complete Loss of Netrin-1 Results in Embryonic Lethality and Severe Axon Guidance Defects without Increased Neural Cell Death.

Netrin-1 regulates cell migration and adhesion during the development of the nervous system, vasculature, lung, pancreas, muscle, and mammary gland. It is also proposed to function as a dependence ligand that inhibits apoptosis; however, studies disagree regarding whether netrin-1 loss-of-function mice exhibit increased cell death. Furthermore, previously studied netrin-1 loss-of-function gene-trap mice express a netrin-1-ß-galactosidase protein chimera with potential for toxic gain-of-function effects, as well as a small amount of wild-type netrin-1 protein. To unambiguously assess loss of function, we generated netrin-1 floxed and netrin-1 null mouse lines. Netrin-1(-/-) mice die earlier and exhibit more severe axon guidance defects than netrin-1 gene-trap mice, revealing that complete loss of function is more severe than previously reported. Netrin-1(-/-) embryos also exhibit increased expression of the netrin receptors DCC and neogenin that are proposed dependence receptors; however, increased apoptosis was not detected, inconsistent with netrin-1 being an essential dependence receptor ligand in the embryonic spinal cord.

Neogenin may functionally substitute for Dcc in chicken.

Dcc is the key receptor that mediates attractive responses of axonal growth cones to netrins, a family of axon guidance cues used throughout evolution. However, a Dcc homolog has not yet been identified in the chicken genome, raising the possibility that Dcc is not present in avians. Here we show that the closely related family member neogenin may functionally substitute for Dcc in the developing chicken spinal cord. The expression pattern of chicken neogenin in the developing spinal cord is a composite of the distribution patterns of both rodent Dcc and neogenin. Moreover, whereas the loss of mouse neogenin has no effect on the trajectory of commissural axons, removing chicken neogenin by RNA interference results in a phenotype similar to the functional inactivation of Dcc in mouse. Taken together, these data suggest that the chick neogenin is functionally equivalent to rodent Dcc.

Superior semicircular canal dehiscence: positive predictive value of high-resolution CT scanning.

Patients with superior dehiscence (SCD) syndrome present with vertigo and oscillopsia evoked by loud sounds and changes in middle ear or intracranial pressure. The first objective of this retrospective cohort study is to demonstrate that thin-section computed tomography (CT) scans reformatted in the plane of the superior semicircular canal (SSC) overestimate this anomaly compared to pathologic studies. The second objective of this study is to re-evaluate the positive predictive value of temporal bone scanning. All temporal bone CT scans with 0.55-mm collimation and reconstruction in the SSC plane performed over a 1-year period were analysed at a tertiary referral centre. CT-positive cases had their clinical data reviewed and patients were re-examined, if available. A total of 581 temporal bone CT-scans were analysed. A dehiscent-appearing superior canal was seen in 4.0% of studies while published pathologic studies report that only 0.5% of temporal bones SSCs have a dehiscence (P < 0.001). Of the 21 patients with positive temporal bone CTs, only 1 presented with sufficient clinical dues to identify the syndrome. Three additional patients did not have symptoms consistent with the diagnosis, but had surgery for a dehiscence of the tegmen mastoideum. When our findings are added to published data, the positive predictive value of temporal bone CT-scanning drops from 93 to 57%. The prevalence of dehiscent-appearing superior canal on thin-section temporal bone scanning with reformation in the SSC plane is much higher than anticipated by pathologic studies. Even with 0.55 mm-collimated helical CT and reformation in the SSC plane, the risk of overdiagnosis is present.

Trio mediates netrin-1-induced Rac1 activation in axon outgrowth and guidance.

The chemotropic guidance cue netrin-1 promotes neurite outgrowth through its receptor Deleted in Colorectal Cancer (DCC) via activation of Rac1. The guanine nucleotide exchange factor (GEF) linking netrin-1/DCC to Rac1 activation has not yet been identified. Here, we show that the RhoGEF Trio mediates Rac1 activation in netrin-1 signaling. We found that Trio interacts with the netrin-1 receptor DCC in mouse embryonic brains and that netrin-1-induced Rac1 activation in brain is impaired in the absence of Trio. Trio(-/-) cortical neurons fail to extend neurites in response to netrin-1, while they are able to respond to glutamate. Accordingly, netrin-1-induced commissural axon outgrowth is reduced in Trio(-/-) spinal cord explants, and the guidance of commissural axons toward the floor plate is affected by the absence of Trio. The anterior commissure is absent in Trio-null embryos, and netrin-1/DCC-dependent axonal projections that form the internal capsule and the corpus callosum are defective in the mutants. Taken together, these findings establish Trio as a GEF that mediates netrin-1 signaling in axon outgrowth and guidance through its ability to activate Rac1.

Isolated vertigo and dizziness of vascular origin.

INTRODUCTION: Vertigo of vascular origin is usually limited to migraine, transient ischemic attacks, and ischemic or hemorrhagic stroke. Excluding migraines, focal neurologic deficits usually allow a fast identification of the impending neural damage and permit a rapid transfer to a stroke team. Few authors have considered that a vascular origin should be thought of in cases of positional vertigo or vertigo not associated with neurologic signs (ie, isolated vertigo) in patients with vascular disease risk factors. OBJECTIVES: (1) To present a case series of vertigo and dizziness without vertigo of probable vascular origin and (2) to perform an evidence-based review regarding the incidence of isolated vertigo and dizziness of vascular origin. DESIGN: Retrospective nonrandomized case series. SETTING: Tertiary referral centre. METHOD: (1) All patients from a vertigo clinic found to have vertigo of potential vascular origin had a complete neuro-otologic examination and were investigated by magnetic resonance angiography. (2) A MEDLINE literature search was performed using specific search terms to identify pertinent publications concerning this pathology. They were reviewed and graded according to the quality of their evidence. RESULTS: We identified nine cases of vertigo and dizziness without vertigo of probable vascular origin. We identified 27 818 potentially pertinent articles. A detailed review yielded 13 studies for further analysis. Although isolated vertigo has frequently been reported as the only symptom in case series of cerebellar infarcts, only one article discussed its incidence. CONCLUSION: Vascular origin should be considered in cases of positional vertigo and isolated vertigo or dizziness when the etiology remains unclear. A single study has determined that 52% of patients with isolated vertigo of unclear etiopathology have posterior circulation anomalies.

Development of enzymatic probes of oxidative and nitrosative DNA damage caused by reactive nitrogen species.

Chronic inflammation is associated with a variety of human diseases, including cancer, with one possible mechanistic link involving over-production of nitric oxide (NO*) by activated macrophages. Subsequent reaction of NO* with superoxide in the presence of carbon dioxide yields nitrosoperoxycarbonate (ONOOCO2-), a strong oxidant that reacts with guanine in DNA to form a variety of oxidation and nitration products, such 2'-deoxy-8-oxoguanosine. Alternatively, the reaction of NO and O2 leads to the formation of N2O3, a nitrosating agent that causes nucleobase deamination to form 2'-deoxyxanthosine (dX) and 2'-deoxyoxanosine (dO) from dG; 2'-deoxyinosine (dI) from dA; and 2'-deoxyuridine (dU) from dC, in addition to abasic sites and dG-dG cross-links. The presence of both ONOOCO2- and N2O3 at sites of inflammation necessitates definition of the relative roles of oxidative and nitrosative DNA damage in the genetic toxicology of inflammation. To this end, we sought to develop enzymatic probes for oxidative and nitrosative DNA lesions as a means to quantify the two types of DNA damage in in vitro DNA damage assays, such as the comet assay and as a means to differentially map the lesions in genomic DNA by the technique of ligation-mediated PCR. On the basis of fragmentary reports in the literature, we first systematically assessed the recognition of dX and dI by a battery of DNA repair enzymes. Members of the alkylpurine DNA glycosylase family (E. coli AlkA, murine Aag, and human MPG) all showed repair activity with dX (k(cat)/Km 29 x 10(-6), 21 x 10(-6), and 7.8 x 10(-6) nM(-1) min(-1), respectively), though the activity was considerably lower than that of EndoV (8 x 10(-3) nM(-1) min(-1)). Based on these results and other published studies, we focused the development of enzymatic probes on two groups of enzymes, one with activity against oxidative damage (formamidopyrimidine-DNA glycosylase (Fpg); endonuclease III (EndoIII)) and the other with activity against nucleobase deamination products (uracil DNA glycosylase (Udg); AlkA). These combinations were assessed for recognition of DNA damage caused by N2O3 (generated with a NO*/O2 delivery system) or ONOOCO2- using a plasmid nicking assay and by LC-MS analysis. Collectively, the results indicate that a combination of AlkA and Udg react selectively with DNA containing only nitrosative damage, while Fpg and EndoIII react selectively with DNA containing oxidative base lesions caused by ONOOCO2-. The results suggest that these enzyme combinations can be used as probes to define the location and quantity of the oxidative and nitrosative DNA lesions produced by chemical mediators of inflammation in systems, such as the comet assay, ligation-mediated polymerase chain reaction, and other assays of DNA damage and repair.

Subannular ventilation tubes: retrospective study.

OBJECTIVE: To evaluate the efficiency of a subannular tube insertion technique in a group of pediatric patients with adhesive otitis or severe atelectasis of the tympanic membrane. DESIGN: Retrospective nonrandomized case series. SETTING: Tertiary referral centre. MAIN OUTCOME MEASURES: The main outcomes of this study are tube duration according to the type of tube used, the complication rate, and the audiometric gain associated with this procedure. RESULTS: The study group consisted of 190 patients (316 tubes) aged between 3 and 19 years (average 9 years old) and operated on between 1993 and 1999 by four pediatric otolaryngologists. The average follow-up was 53 months. The tubes remained in place for an average of 21.8 months, with fluoroplastic tubes lasting 17.8 months and Goode T tubes lasting 23.8 months. When used in children between 5 and 9 years of age and in cases of adhesive otitis, Goode T tubes showed statistically significantly better results than fluoroplastic tubes. The complications of this technique were otorrhea (17.7%), perforation (7.9%), a plugged tube (7.0%), and cholesteatoma (1.6%). The 5- to 9-year-old group and the reintervention group of patients showed statistically higher complication rates compared with all other groups. Sixty-four patients (128 tubes) were eligible for audiogram analysis, which showed a gain of 13.4 dB (speech reception threshold). CONCLUSIONS: The technique of subannular tube insertion is a safe and effective method for long-term middle ear ventilation in cases of adhesive otitis or severely atelectatic tympanic membrane or for patients with pathology related to dysfunction of the eustachian tube. It offers an alternative to repeated short-term tube insertions for otitis media with effusion or recurrent acute otitis media.

Evaluating the reproducibility of sinus lavages with a saline solution administered directly in the maxillary sinus of patients after endoscopic sinus surgery.

INTRODUCTION: Chronic sinusitis is recognized as having a strong inflammatory component, and failures of endoscopic sinus surgery (ESS) are frequently attributed to persistent inflammation. A test that would allow rhinologists to evaluate the inflammatory state of a patient's sinuses would be helpful to evaluate cases refractory to therapy, determine appropriate medical therapy, and monitor the response to therapy. OBJECTIVES: The goal of this preliminary research is to assess the optimal method of collection and the reproducibility and specificity of sinus lavages. METHOD: Twelve patients who had undergone ESS were recruited. They were divided into two groups according to the persistence of their symptoms and the recurrence of acute sinusitis after ESS. The subjects were seen twice. Three successive lavages were collected from each maxillary sinus and were analyzed by cell count. RESULTS: Intrasession cell counts were most reproducible (Spearman rank correlation .7 for eosinophils and .6 for neutrophils) for the second lavage. Intersession cell counts were highly reproducible for eosinophils (r = .7) for the second lavage. The two-tailed t-test did not reveal any statistically significant differences between the good and the poor outcome groups. CONCLUSION: Assessment of eosinophil cell counts on sinus lavage is a feasible and reproducible method to evaluate the inflammatory state of a patient's sinuses in patients who have undergone ESS.