Complement factor B is essential for the proper function of the peripheral auditory system.

Sensorineural hearing loss is associated with dysfunction of cochlear cells. Although immune cells play a critical role in maintaining the inner ear microenvironment, the precise immune-related molecular mechanisms underlying the pathophysiology of hearing loss remain unclear. The complement cascade contributes to the regulation of immune cell activity. Additionally, activation of the complement cascade can lead to the cellular opsonization of cells and pathogens, resulting in their engulfment and elimination by phagocytes. Complement factor B (fB) is an essential activator protein in the alternative complement pathway, and variations in the fB gene are associated with age-related macular degeneration. Here we show that mice of both sexes deficient in fB functional alleles (fB-/-) demonstrate progressive hearing impairment. Transcriptomic analysis of auditory nerves from adult mice detected 706 genes that were significantly differentially expressed between fB-/- and wild-type control animals, including genes related to the extracellular matrix and neural development processes. Additionally, a subset of differentially expressed genes was related to myelin function and neural crest development. Histological and immunohistochemical investigations revealed pathological alterations in auditory nerve myelin sheathes of fB-/- mice. Pathological alterations were also seen in the stria vascularis of the cochlear lateral wall in these mice. Our results implicate fB as an integral regulator of myelin maintenance and stria vascularis integrity, underscoring the importance of understanding the involvement of immune signaling pathways in sensorineural hearing loss.

A Faculty-Led, Community-Building Program That Enhances Student-Faculty Connections within Biology.

Connections between students and faculty on campus may influence students' sense of belonging, and a greater sense of belonging has a positive effect on student success. We developed a low-cost, faculty-led program of community-building events and implemented the program in the biology department at a small liberal-arts institution with the goal of improving students' sense of community. Student responses to surveys indicated that the majority of students felt connected to faculty and students in the department; however, Black or African American students initially felt a lower level of connection to faculty than did white students. After implementing our series of community-building events, students surveyed reported high levels of satisfaction with the events. Furthermore, there was a trend toward a higher percentage of Black or African American students than white students reporting that they were more likely to reach out to faculty after participating in the community-building events. Thus, our low-cost program improved connections between students and faculty in the biology department. Collectively, our results suggest that academic departments can implement community-building programs to improve students' sense of belonging.

Cellular inhibitor of apoptosis 2 (cIAP2) restricts neuroinflammation during experimental autoimmune encephalomyelitis.

BACKGROUND: Immune activation, neuroinflammation, and cell death are the hallmarks of multiple sclerosis (MS), which is an autoimmune demyelinating disease of the central nervous system (CNS). It is well-documented that the cellular inhibitor of apoptosis 2 (cIAP2) is induced by inflammatory stimuli and regulates adaptive and innate immune responses, cell death, and the production of inflammatory mediators. However, the impact of cIAP2 on neuroinflammation associated with MS and disease severity remains unknown. METHODS: We used experimental autoimmune encephalomyelitis (EAE), a widely used mouse model of MS, to assess the effect of cIAP2 deletion on disease outcomes. We performed a detailed analysis on the histological, cellular, and molecular levels. We generated and examined bone-marrow chimeras to identify the cIAP2-deficient cells that are critical to the disease outcomes. RESULTS: cIAP2-/- mice exhibited increased EAE severity, increased CD4+ T cell infiltration, enhanced proinflammatory cytokine/chemokine expression, and augmented demyelination. This phenotype was driven by cIAP2-deficient non-hematopoietic cells. cIAP2 protected oligodendrocytes from cell death during EAE by limiting proliferation and activation of brain microglia. This protective role was likely exerted by cIAP2-mediated inhibition of the non-canonical NLRP3/caspase-8-dependent myeloid cell activation during EAE. CONCLUSIONS: Our findings suggest that cIAP2 is needed to modulate neuroinflammation, cell death, and survival during EAE. Significantly, our data demonstrate the critical role of cIAP2 in limiting the activation of microglia during EAE, which could be explored for developing MS therapeutics in the future.

RelB acts as a molecular switch driving chronic inflammation in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a primary brain tumor characterized by extensive necrosis and immunosuppressive inflammation. The mechanisms by which this inflammation develops and persists in GBM remain elusive. We identified two cytokines interleukin-1ß (IL-1) and oncostatin M (OSM) that strongly negatively correlate with patient survival. We found that these cytokines activate RelB/p50 complexes by a canonical NF-κB pathway, which surprisingly drives expression of proinflammatory cytokines in GBM cells, but leads to their inhibition in non-transformed astrocytes. We discovered that one allele of the gene encoding deacetylase Sirtuin 1 (SIRT1), needed for repression of cytokine genes, is deleted in 80% of GBM tumors. Furthermore, RelB specifically interacts with a transcription factor Yin Yang 1 (YY1) in GBM cells and activates GBM-specific gene expression programs. As a result, GBM cells continuously secrete proinflammatory cytokines and factors attracting/activating glioma-associated microglia/macrophages and thus, promote a feedforward inflammatory loop.

RelB controls adaptive responses of astrocytes during sterile inflammation.

In response to brain injury or infections, astrocytes become reactive, undergo striking morphological and functional changes, and secrete and respond to a spectrum of inflammatory mediators. We asked whether reactive astrocytes also display adaptive responses during sterile IL-1ß-induced neuroinflammation, which may limit tissue injury associated with many disorders of the central nervous system. We found that astrocytes display days-to-weeks long specific tolerance of cytokine genes, which is coordinated by NF-κB family member, RelB. However, in contrast to innate immune cells, astrocytic tolerance does not involve epigenetic silencing of the cytokine genes. Establishment of tolerance depends on persistent higher levels of RelB in tolerant astrocytes and its phosphorylation on serine 472. Mechanistically, this phosphorylation prevents efficient removal of RelB from cytokine promoters by IκBα and helps to establish tolerance. Importantly, ablation of RelB from astrocytes in mice abolishes tolerance during experimental neuroinflammation in vivo.

Noise-Induced Dysregulation of Quaking RNA Binding Proteins Contributes to Auditory Nerve Demyelination and Hearing Loss.

Noise exposure causes auditory nerve (AN) degeneration and hearing deficiency, though the proximal biological consequences are not entirely understood. Most AN fibers and spiral ganglion neurons are ensheathed by myelinating glia that provide insulation and ensure rapid transmission of nerve impulses from the cochlea to the brain. Here we show that noise exposure administered to mice of either sex rapidly affects myelinating glial cells, causing molecular and cellular consequences that precede nerve degeneration. This response is characterized by demyelination, inflammation, and widespread expression changes in myelin-related genes, including the RNA splicing regulator Quaking (QKI) and numerous QKI target genes. Analysis of mice deficient in QKI revealed that QKI production in cochlear glial cells is essential for proper myelination of spiral ganglion neurons and AN fibers, and for normal hearing. Our findings implicate QKI dysregulation as a critical early component in the noise response, influencing cochlear glia function that leads to AN demyelination and, ultimately, to hearing deficiency.SIGNIFICANCE STATEMENT Auditory glia cells ensheath a majority of spiral ganglion neurons with myelin, protect auditory neurons, and allow for fast conduction of electrical impulses along the auditory nerve. Here we show that noise exposure causes glial dysfunction leading to myelin abnormality and altered expression of numerous genes in the auditory nerve, including QKI, a gene implicated in regulating myelination. Study of a conditional mouse model that specifically depleted QKI in glia showed that QKI deficiency alone was sufficient to elicit myelin-related abnormality and auditory functional declines. These results establish QKI as a key molecular target in the noise response and a causative agent in hearing loss.

Macrophage-Mediated Glial Cell Elimination in the Postnatal Mouse Cochlea.

Hearing relies on the transmission of auditory information from sensory hair cells (HCs) to the brain through the auditory nerve. This relay of information requires HCs to be innervated by spiral ganglion neurons (SGNs) in an exclusive manner and SGNs to be ensheathed by myelinating and non-myelinating glial cells. In the developing auditory nerve, mistargeted SGN axons are retracted or pruned and excessive cells are cleared in a process referred to as nerve refinement. Whether auditory glial cells are eliminated during auditory nerve refinement is unknown. Using early postnatal mice of either sex, we show that glial cell numbers decrease after the first postnatal week, corresponding temporally with nerve refinement in the developing auditory nerve. Additionally, expression of immune-related genes was upregulated and macrophage numbers increase in a manner coinciding with the reduction of glial cell numbers. Transient depletion of macrophages during early auditory nerve development, using transgenic CD11bDTR/EGFP mice, resulted in the appearance of excessive glial cells. Macrophage depletion caused abnormalities in myelin formation and transient edema of the stria vascularis. Macrophage-depleted mice also showed auditory function impairment that partially recovered in adulthood. These findings demonstrate that macrophages contribute to the regulation of glial cell number during postnatal development of the cochlea and that glial cells play a critical role in hearing onset and auditory nerve maturation.

Contributions of Mouse and Human Hematopoietic Cells to Remodeling of the Adult Auditory Nerve After Neuron Loss.

The peripheral auditory nerve (AN) carries sound information from sensory hair cells to the brain. The present study investigated the contribution of mouse and human hematopoietic stem cells (HSCs) to cellular diversity in the AN following the destruction of neuron cell bodies, also known as spiral ganglion neurons (SGNs). Exposure of the adult mouse cochlea to ouabain selectively killed type I SGNs and disrupted the blood-labyrinth barrier. This procedure also resulted in the upregulation of genes associated with hematopoietic cell homing and differentiation, and provided an environment conducive to the tissue engraftment of circulating stem/progenitor cells into the AN. Experiments were performed using both a mouse-mouse bone marrow transplantation model and a severely immune-incompetent mouse model transplanted with human CD34+ cord blood cells. Quantitative immunohistochemical analysis of recipient mice demonstrated that ouabain injury promoted an increase in the number of both HSC-derived macrophages and HSC-derived nonmacrophages in the AN. Although rare, a few HSC-derived cells in the injured AN exhibited glial-like qualities. These results suggest that human hematopoietic cells participate in remodeling of the AN after neuron cell body loss and that hematopoietic cells can be an important resource for promoting AN repair/regeneration in the adult inner ear.

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration.

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Neural stem/progenitor cell properties of glial cells in the adult mouse auditory nerve.

The auditory nerve is the primary conveyor of hearing information from sensory hair cells to the brain. It has been believed that loss of the auditory nerve is irreversible in the adult mammalian ear, resulting in sensorineural hearing loss. We examined the regenerative potential of the auditory nerve in a mouse model of auditory neuropathy. Following neuronal degeneration, quiescent glial cells converted to an activated state showing a decrease in nuclear chromatin condensation, altered histone deacetylase expression and up-regulation of numerous genes associated with neurogenesis or development. Neurosphere formation assays showed that adult auditory nerves contain neural stem/progenitor cells (NSPs) that were within a Sox2-positive glial population. Production of neurospheres from auditory nerve cells was stimulated by acute neuronal injury and hypoxic conditioning. These results demonstrate that a subset of glial cells in the adult auditory nerve exhibit several characteristics of NSPs and are therefore potential targets for promoting auditory nerve regeneration.