Utilization of RNA sequencing to investigate olfactory dysfunction in chronic rhinosinusitis without nasal polyps: A pilot study.

Objectives: Prior research on olfactory dysfunction in chronic rhinosinusitis (CRS) has focused on patients with polyps and suggests that direct inflammation of the olfactory cleft mucosa plays a contributory role. The purpose of this study was to evaluate gene expression in superior turbinate mucosal specimens, comparing normosmic and dysosmic CRS patients without polyps (CRSsNP). Methods: Tissue samples were obtained from the superior turbinates of patients with CRSsNP at the time of endoscopic sinus surgery. Samples subsequently underwent RNA sequencing and functional analysis to investigate biological pathways associated with differentially expressed genes between dysosmic (n = 7) and normosmic (n = 4) patients. Results: Differential gene expression analysis comparing dysosmic and normosmic CRSsNP patients showed upregulation of 563 genes and downregulation of 327 genes. Using stringent criteria for multiple comparisons, one upregulated gene (Immediate Early Response 3 [IER3]) had an false discovery rate (FDR) correction adjusted P value considered statistically significant (P < 0.001, fold change 2.69). Reactome functional analysis revealed eight biological pathways significantly different between dysosmic and normosmic patients (P < 0.05, FDR correction) including IL-4 and IL-13 signaling, IL-10 signaling, and rhodopsin-like receptors. Conclusions: RNA sequencing of the superior turbinates in patients with CRSsNP can provide valuable information regarding biological pathways and genes involved in olfactory dysfunction. This study supports literature suggesting that Type 2 inflammation may play a role in olfactory dysfunction in at least some patients with CRSsNP. This study also prompts questions regarding the role of IL-10, rhodopsin-like receptors, and IER3 in the pathogenesis of olfactory dysfunction.

Gene expression and cellular changes in injured myocardium of Ciona intestinalis.

Ciona intestinalis is an invertebrate animal model system that is well characterized and has many advantages for the study of cardiovascular biology. The regulatory mechanisms of cardiac myocyte proliferation in Ciona are intriguing since regeneration of functional tissue has been demonstrated in other organs of Ciona in response to injury. To identify genes that are differentially expressed in response to Ciona cardiac injury, microarray analysis was conducted on RNA from adult Ciona hearts with normal or damaged myocardium. After a 24- or 48-h recovery period, total RNA was isolated from damaged and control hearts. Initial results indicate significant changes in gene expression in hearts damaged by ligation in comparison to control hearts. Ligation injury shows differential expression of 223 genes as compared to control with limited false discovery (5.8%). Among these 223 genes, 117 have known human orthologs of which 68 were upregulated and 49 were downregulated. Notably, Fgf9/16/20, insulin-like growth factor binding protein and Ras-related protein Rab11b were significantly upregulated in injured hearts, whereas expression of a junctophilin ortholog was decreased. Histological analyses of injured myocardium were conducted in parallel to the microarray study which revealed thickened myocardium in injured hearts. Taken together, these studies will connect differences in gene expression to cellular changes in the myocardium of Ciona, which will help to promote further investigations into the regulatory mechanisms of cardiac myocyte proliferation across chordates.

Epicardial deletion of Sox9 leads to myxomatous valve degeneration and identifies Cd109 as a novel gene associated with valve development.

Epicardial-derived cells (EPDCs) are involved in the regulation of myocardial growth and coronary vascularization and are critically important for proper development of the atrioventricular (AV) valves. SOX9 is a transcription factor expressed in a variety of epithelial and mesenchymal cells in the developing heart, including EPDCs. To determine the role of SOX9 in epicardial development, an epicardial-specific Sox9 knockout mouse model was generated. Deleting Sox9 from the epicardial cell lineage impairs the ability of EPDCs to invade both the ventricular myocardium and the developing AV valves. After birth, the mitral valves of these mice become myxomatous with associated abnormalities in extracellular matrix organization. This phenotype is reminiscent of that seen in humans with myxomatous mitral valve disease (MVD). An RNA-seq analysis was conducted in an effort to identify genes associated with this myxomatous degeneration. From this experiment, Cd109 was identified as a gene associated with myxomatous valve pathogenesis in this model. Cd109 has never been described in the context of heart development or valve disease. This study highlights the importance of SOX9 in the regulation of epicardial cell invasion-emphasizing the importance of EPDCs in regulating AV valve development and homeostasis-and reports a novel expression profile of Cd109, a gene with previously unknown relevance in heart development.

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.

The Stria Vascularis in Mice and Humans Is an Early Site of Age-Related Cochlear Degeneration, Macrophage Dysfunction, and Inflammation.

Age-related hearing loss, or presbyacusis, is a common degenerative disorder affecting communication and quality of life for millions of older adults. Multiple pathophysiologic manifestations, along with many cellular and molecular alterations, have been linked to presbyacusis; however, the initial events and causal factors have not been clearly established. Comparisons of the transcriptome in the lateral wall (LW) with other cochlear regions in a mouse model (of both sexes) of "normal" age-related hearing loss revealed that early pathophysiological alterations in the stria vascularis (SV) are associated with increased macrophage activation and a molecular signature indicative of inflammaging, a common form of immune dysfunction. Structure-function correlation analyses in mice across the lifespan showed that the age-dependent increase in macrophage activation in the stria vascularis is associated with a decline in auditory sensitivity. High-resolution imaging analysis of macrophage activation in middle-aged and aged mouse and human cochleas, along with transcriptomic analysis of age-dependent changes in mouse cochlear macrophage gene expression, support the hypothesis that aberrant macrophage activity is an important contributor to age-dependent strial dysfunction, cochlear pathology, and hearing loss. Thus, this study highlights the SV as a primary site of age-related cochlear degeneration and aberrant macrophage activity and dysregulation of the immune system as early indicators of age-related cochlear pathology and hearing loss. Importantly, novel new imaging methods described here now provide a means to analyze human temporal bones in a way that had not previously been feasible and thereby represent a significant new tool for otopathological evaluation.SIGNIFICANCE STATEMENT Age-related hearing loss is a common neurodegenerative disorder affecting communication and quality of life. Current interventions (primarily hearing aids and cochlear implants) offer imperfect and often unsuccessful therapeutic outcomes. Identification of early pathology and causal factors is crucial for the development of new treatments and early diagnostic tests. Here, we find that the SV, a nonsensory component of the cochlea, is an early site of structural and functional pathology in mice and humans that is characterized by aberrant immune cell activity. We also establish a new technique for evaluating cochleas from human temporal bones, an important but understudied area of research because of a lack of well-preserved human specimens and difficult tissue preparation and processing approaches.

Increased Proteoglycanases in Pulmonary Valves after Birth Correlate with Extracellular Matrix Maturation and Valve Sculpting.

Increased mechanical forces on developing cardiac valves drive formation of the highly organized extracellular matrix (ECM) providing tissue integrity and promoting cell behavior and signaling. However, the ability to investigate the response of cardiac valve cells to increased mechanical forces is challenging and remains poorly understood. The developmental window from birth (P0) to postnatal day 7 (P7) when biomechanical forces on the pulmonary valve (PV) are altered due to the initiation of blood flow to the lungs was evaluated in this study. Grossly enlarged PV, in mice deficient in the proteoglycan protease ADAMTS5, exhibited a transient phenotypic rescue from postnatal day 0 (P0) to P7; the Adamts5-/- aortic valves (AV) did not exhibit a phenotypic correction. We hypothesized that blood flow, initiated to the lungs at birth, alters mechanical load on the PV and promotes ECM maturation. In the Adamts5-/- PV, there was an increase in localization of the proteoglycan proteases ADAMTS1, MMP2, and MMP9 that correlated with reduced Versican (VCAN). At birth, Decorin (DCN), a Collagen I binding, small leucine-rich proteoglycan, exhibited complementary stratified localization to VCAN in the wild type at P0 but colocalized with VCAN in Adamts5-/- PV; concomitant with the phenotypic rescue at P7, the PVs in Adamts5-/- mice exhibited stratification of VCAN and DCN similar to wild type. This study indicates that increased mechanical forces on the PV at birth may activate ECM proteases to organize specialized ECM layers during cardiac valve maturation.

Sfrp4 expression in thyroxine treated calvarial cells.

AIMS: Evidence suggests alterations of thyroid hormone levels can disrupt normal bone development. Most data suggest the major targets of thyroid hormones to be the Htra1/Igf1 pathway. Recent discovery by our group suggests involvement of targets WNT pathway, specifically overexpression of antagonist Sfrp4 in the presence of exogenous thyroid hormone. MAIN METHODS: Here we aimed to model these interactions in vitro using primary and isotype cell lines to determine if thyroid hormone drives increased Sfrp4 expression in cells relevant to craniofacial development. Transcriptional profiling, bioinformatics interrogation, protein and function analyses were used. KEY FINDINGS: Affymetrix transcriptional profiling found Sfrp4 overexpression in primary cranial suture derived cells stimulated with thyroxine in vitro. Interrogation of the SFRP4 promoter identified multiple putative binding sites for thyroid hormone receptors. Experimentation with several cell lines demonstrated that thyroxine treatment induced Sfrp4 expression, demonstrating that Sfrp4 mRNA and protein levels are not tightly coupled. Transcriptional and protein analyses demonstrate thyroid hormone receptor binding to the proximal promoter of the target gene Sfrp4 in murine calvarial pre-osteoblasts. Functional analysis after thyroxine hormone stimulation for alkaline phosphatase activity shows that pre-osteoblasts increase alkaline phosphatase activity compared to other cell types, suggesting cell type susceptibility. Finally, we added recombinant SFRP4 to pre-osteoblasts in combination with thyroxine treatment and observed a significant decrease in alkaline phosphatase positivity. SIGNIFICANCE: Taken together, these results suggest SFRP4 may be a key regulatory molecule that prevents thyroxine driven osteogenesis. These data corroborate clinical findings indicating a potential for SFRP4 as a diagnostic or therapeutic target for hyperostotic craniofacial disorders.

Peripheral Auditory Nerve Impairment in a Mouse Model of Syndromic Autism.

Dysfunction of the peripheral auditory nerve (AN) contributes to dynamic changes throughout the central auditory system, resulting in abnormal auditory processing, including hypersensitivity. Altered sound sensitivity is frequently observed in autism spectrum disorder (ASD), suggesting that AN deficits and changes in auditory information processing may contribute to ASD-associated symptoms, including social communication deficits and hyperacusis. The MEF2C transcription factor is associated with risk for several neurodevelopmental disorders, and mutations or deletions of MEF2C produce a haploinsufficiency syndrome characterized by ASD, language, and cognitive deficits. A mouse model of this syndromic ASD (Mef2c-Het) recapitulates many of the MEF2C haploinsufficiency syndrome-linked behaviors, including communication deficits. We show here that Mef2c-Het mice of both sexes exhibit functional impairment of the peripheral AN and a modest reduction in hearing sensitivity. We find that MEF2C is expressed during development in multiple AN and cochlear cell types; and in Mef2c-Het mice, we observe multiple cellular and molecular alterations associated with the AN, including abnormal myelination, neuronal degeneration, neuronal mitochondria dysfunction, and increased macrophage activation and cochlear inflammation. These results reveal the importance of MEF2C function in inner ear development and function and the engagement of immune cells and other non-neuronal cells, which suggests that microglia/macrophages and other non-neuronal cells might contribute, directly or indirectly, to AN dysfunction and ASD-related phenotypes. Finally, our study establishes a comprehensive approach for characterizing AN function at the physiological, cellular, and molecular levels in mice, which can be applied to animal models with a wide range of human auditory processing impairments.SIGNIFICANCE STATEMENT This is the first report of peripheral auditory nerve (AN) impairment in a mouse model of human MEF2C haploinsufficiency syndrome that has well-characterized ASD-related behaviors, including communication deficits, hyperactivity, repetitive behavior, and social deficits. We identify multiple underlying cellular, subcellular, and molecular abnormalities that may contribute to peripheral AN impairment. Our findings also highlight the important roles of immune cells (e.g., cochlear macrophages) and other non-neuronal elements (e.g., glial cells and cells in the stria vascularis) in auditory impairment in ASD. The methodological significance of the study is the establishment of a comprehensive approach for evaluating peripheral AN function and impact of peripheral AN deficits with minimal hearing loss.

Two distinct types of nodes of Ranvier support auditory nerve function in the mouse cochlea.

The auditory nerve (AN) of the inner ear is the primary conveyor of acoustic information from sensory hair cells to the brainstem. Approximately 95% of peripheral AN fibers are myelinated by glial cells. The integrity of myelin and the glial-associated paranodal structures at the node of Ranvier is critical for normal AN activity and axonal survival and function in the central auditory nervous system. However, little is known about the node of Ranvier's spatiotemporal development in the AN, how the aging process (or injury) affects the activity of myelinating glial cells, and how downstream alterations in myelin and paranodal structure contribute to AN degeneration and sensorineural hearing loss. Here, we characterized two types of Ranvier nodes-the axonal node and the ganglion node-in the mouse peripheral AN, and found that they are distinct in several features of postnatal myelination and age-related degeneration. Cellular, molecular, and structure-function correlations revealed that the two node types are each critical for different aspects of peripheral AN function. Neural processing speed and synchrony is associated with the length of the axonal node, while stimulus level-dependent amplitude growth and action potentials are associated with the ganglion node. Moreover, our data indicate that dysregulation of glial cells (e.g., satellite cells) and degeneration of the ganglion node structure are an important new mechanism of age-related hearing loss.

PDE5 inhibition rescues mitochondrial dysfunction and angiogenic responses induced by Akt3 inhibition by promotion of PRC expression.

Akt3 regulates mitochondrial content in endothelial cells through the inhibition of PGC-1α nuclear localization and is also required for angiogenesis. However, whether there is a direct link between mitochondrial function and angiogenesis is unknown. Here we show that Akt3 depletion in primary endothelial cells results in decreased uncoupled oxygen consumption, increased fission, decreased membrane potential, and increased expression of the mitochondria-specific protein chaperones, HSP60 and HSP10, suggesting that Akt3 is required for mitochondrial homeostasis. Direct inhibition of mitochondrial homeostasis by the model oxidant paraquat results in decreased angiogenesis, showing a direct link between angiogenesis and mitochondrial function. Next, in exploring functional links to PGC-1α, the master regulator of mitochondrial biogenesis, we searched for compounds that induce this process. We found that, sildenafil, a phosphodiesterase 5 inhibitor, induced mitochondrial biogenesis as measured by increased uncoupled oxygen consumption, mitochondrial DNA content, and voltage-dependent anion channel protein expression. Sildenafil rescued the effects on mitochondria by Akt3 depletion or pharmacological inhibition and promoted angiogenesis, further supporting that mitochondrial homeostasis is required for angiogenesis. Sildenafil also induces the expression of PGC-1 family member PRC and can compensate for PGC-1α activity during mitochondrial stress by an Akt3-independent mechanism. The induction of PRC by sildenafil depends upon cAMP and the transcription factor CREB. Thus, PRC can functionally substitute during Akt3 depletion for absent PGC-1α activity to restore mitochondrial homeostasis and promote angiogenesis. These findings show that mitochondrial homeostasis as controlled by the PGC family of transcriptional activators is required for angiogenic responses.

Age-dependent alterations of Kir4.1 expression in neural crest-derived cells of the mouse and human cochlea.

Age-related hearing loss (or presbyacusis) is a progressive pathophysiological process. This study addressed the hypothesis that degeneration/dysfunction of multiple nonsensory cell types contributes to presbyacusis by evaluating tissues obtained from young and aged CBA/CaJ mouse ears and human temporal bones. Ultrastructural examination and transcriptomic analysis of mouse cochleas revealed age-dependent pathophysiological alterations in 3 types of neural crest-derived cells, namely intermediate cells in the stria vascularis, outer sulcus cells in the cochlear lateral wall, and satellite cells in the spiral ganglion. A significant decline in immunoreactivity for Kir4.1, an inwardly rectifying potassium channel, was seen in strial intermediate cells and outer sulcus cells in the ears of older mice. Age-dependent alterations in Kir4.1 immunostaining also were observed in satellite cells ensheathing spiral ganglion neurons. Expression alterations of Kir4.1 were observed in these same cell populations in the aged human cochlea. These results suggest that degeneration/dysfunction of neural crest-derived cells maybe an important contributing factor to both metabolic and neural forms of presbyacusis.

Transcriptomic characterization of signaling pathways associated with osteoblastic differentiation of MC-3T3E1 cells.

Bone remodeling involves the coordinated actions of osteoclasts, which resorb the calcified bony matrix, and osteoblasts, which refill erosion pits created by osteoclasts to restore skeletal integrity and adapt to changes in mechanical load. Osteoblasts are derived from pluripotent mesenchymal stem cell precursors, which undergo differentiation under the influence of a host of local and environmental cues. To characterize the autocrine/paracrine signaling networks associated with osteoblast maturation and function, we performed gene network analysis using complementary "agnostic" DNA microarray and "targeted" NanoString nCounter datasets derived from murine MC3T3-E1 cells induced to undergo synchronized osteoblastic differentiation in vitro. Pairwise datasets representing changes in gene expression associated with growth arrest (day 2 to 5 in culture), differentiation (day 5 to 10 in culture), and osteoblast maturation (day 10 to 28 in culture) were analyzed using Ingenuity Systems Pathways Analysis to generate predictions about signaling pathway activity based on the temporal sequence of changes in target gene expression. Our data indicate that some pathways involved in osteoblast differentiation, e.g. Wnt/ß-catenin signaling, are most active early in the process, while others, e.g. TGFß/BMP, cytokine/JAK-STAT and TNFα/RANKL signaling, increase in activity as differentiation progresses. Collectively, these pathways contribute to the sequential expression of genes involved in the synthesis and mineralization of extracellular matrix. These results provide insight into the temporal coordination and complex interplay between signaling networks controlling gene expression during osteoblast differentiation. A more complete understanding of these processes may aid the discovery of novel methods to promote osteoblast development for the treatment of conditions characterized by low bone mineral density.

The acute transcriptome response of the midbrain/diencephalon to injury in the adult mummichog (Fundulus heteroclitus).

Adult fish produce new cells throughout their central nervous system during the course of their lives and maintain a tremendous capacity to repair damaged neural tissue. Much of the focus on understanding brain repair and regeneration in adult fish has been directed at regions of the brainstem and forebrain; however, the mesencephalon (midbrain) and diencephalon have received little attention. We sought to examine differential gene expression in the midbrain/diencephalon in response to injury in the adult fish using RNA-seq. Using the mummichog (Fundulus heteroclitus), we administered a mechanical lesion to the midbrain/diencephalon and examined differentially expressed genes (DEGs) at an acute recovery time of 1 h post-injury. Comparisons of whole transcriptomes derived from isolated RNA of intact and injured midbrain/diencephalic tissue identified 404 DEGs with the vast majority being upregulated. Using qPCR, we validated the upregulation of DEGs pim-2-like, syndecan-4-like, and cd83. Based on genes both familiar and novel regarding the adult brain response to injury, these data provide an extensive molecular profile giving insight into a range of cellular processes involved in the injury response of a brain regenerative-capable vertebrate.

Maternal cardiac messenger RNA expression of extracellular matrix proteins in mice during pregnancy and the postpartum period.

IMPACT STATEMENT: This study provides the first comprehensive analysis of extracellular matrix protein (ECM) gene expression combined with echocardiographic analyses of heart functional parameters in the murine heart during pregnancy and the early postpartum period. Our findings show regulation of all Timp, selected Mmps, and Col1a1, Col3a1, and Col8a1 mRNA levels with reproductive status, with the greatest number of significant changes occurring in the early postpartum period. Left ventricle cardiac diastolic parameters were the first to change during pregnancy and remained elevated postpartum, whereas systolic parameters were increased in late pregnancy and began to recover during the first week postpartum. These novel findings indicate that although some ECM genes are elevated during late pregnancy, that the postpartum period is a time of robust altered ECM gene expression. These studies provide a basis for examining ECM proteins and their activities in the normal pregnant and postpartum heart and in models of postpartum cardiomyopathy.

Smoking-induced control of miR-133a-3p alters the expression of EGFR and HuR in HPV-infected oropharyngeal cancer.

PURPOSE: Human papillomavirus (HPV) infected oropharyngeal squamous cell carcinoma (OPSCC) patients have a better prognosis compared to HPV(-) counterparts. However, a subset of HPV(+) patients with a smoking history fail to respond to the standard of care treatments such as radiation and chemotherapy. To understand the underlying mechanism driving HPV(+) OPSCC patient resistance to treatment and recurrence, we sought to identify and characterize the differentially expressed miRNAs and their target genes in HPV(+) smokers and non-smokers. EXPERIMENTAL DESIGN: MicroRNA expression analysis was performed using Nanostring in tumor tissues isolated from a prospective cohort of HPV(+) smoking (n = 9) and HPV(+) (n = 13) non-smoking OPSCC patients. Identified miRNAs of interest were further validated using qRT-PCR in cigarette smoke extract (CSE) treated HPV(+) and E6/E7 overexpressing HPV(-) cells. RESULTS: In comparison to OPSCC HPV(+) non-smokers, 38 miRNAs were significantly altered in the HPV(+) smoker patients cohort and out of that 9 were downregulated. Altered miRNA expression was also detected in the serum and metastatic lymph nodes of HPV(+) smokers versus non-smokers. Expression of miR-133a-3p was significantly downregulated in OPSCC smokers, HPV(+) cells and E6/E7 overexpressing HPV(-) cells treated with CSE. Reduction of miR-133a-3p induced the upregulation of miR-133a-3p target mRNAs EGFR and HuR. CONCLUSIONS: Our results indicate that miR-133a-3p is a target of smoking-induced changes in HPV(+) patients and alters the expression of EGFR and HuR which may promote HPV associated oropharyngeal cancer. Therefore, future treatment strategies for HPV(+) OPSCC smokers should focus on EGFR inhibition and the development of selective therapies to target HuR.

Use of Proteomic Imaging Coupled With Transcriptomic Analysis to Identify Biomolecules Responsive to Cochlear Injury.

Exposure to noise or ototoxic agents can result in degeneration of cells in the sensory epithelium and auditory nerve, as well as non-sensory cells of the cochlear lateral wall. However, the molecular mechanisms underlying this pathology remain unclear. The purpose of this study was to localize and identify proteins in the cochlea that are responsive to noise or ototoxic exposure using a complementary proteo-transcriptomic approach. MALDI imaging of cochlear sections revealed numerous protein signals with distinct cochlear localization patterns in both cochlear injury models, of which six were chosen for further investigation. A query of proteomic databases identified 709 candidates corresponding to m/z values for the six proteins. An evaluation of mRNA expression data from our previous studies of these injured models indicated that 208 of the candidates were affected in both injury models. Downstream validation analyses yielded proteins with confirmatory distributions and responses to injury. The combined analysis of MALDI imaging with gene expression data provides a new strategy to identify molecular regulators responsive to cochlear injury. This study demonstrates the applicability of MALDI imaging for investigating protein localization and abundance in frozen sections from animals modeling cochlear pathology.

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.

Serum Proteins Associated with Emphysema Progression in Severe Alpha-1 Antitrypsin Deficiency.

Computed tomography (CT) lung density is an accepted biomarker for emphysema in alpha-1 antitrypsin deficiency (AATD), although concerns for radiation exposure limit its longitudinal use. Serum proteins associated with emphysema, particularly in early disease, may provide additional pathogenic insights. We investigated whether distinct proteomic signatures characterize the presence and progression of emphysema in individuals with severe AATD and normal forced expiratory volume in 1 second (FEV1). QUANTitative lung CT UnMasking emphysema progression in AATD (QUANTUM-1) is a multicenter, prospective 3-year study of 49 adults with severe AATD and FEV1 post-bronchodilator values (Post-BD) ≥ 80% predicted. All participants received chest CT, serial spirometry, and contributed to the serum biobank. Volumetric imaging display and analysis (VIDA) software defined the baseline 15th percentile density (PD15) which was indexed to CT-derived total lung capacity (TLC). We measured 317 proteins using a multiplexed immunoassay (Myriad Discovery MAP® panel) in 31 individuals with a complete dataset. We analyzed associations between initial PD15/TLC, PD15/TLC annual decline, body mass index (BMI), and protein levels using Pearson's product moment correlation. C-reactive protein (CRP), adipocyte fatty acid-binding protein (AFBP), leptin, and tissue plasminogen activator (tPA) were found to be associated with baseline emphysema and all but leptin were associated with emphysema progression after adjustments were made for age and sex. All 4 proteins were associated with BMI after further adjustment for multiple comparisons was made. The relationship between these proteins and BMI, and further validation of these findings in replicative cohorts require additional studies.

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.