dissection-adult-mouse-stria-vascularis-for-single-nucleus-sequencing

Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining

Endocochlear potential, which is generated by the stria vascularis, is essential to maintain an environment conducive to appropriate hair cell ...

National Institutes of Health

The stria vascularis (SV) generates the endocochlear potential (EP) in the inner ear and is necessary for proper hair cell mechanotransduction and hearing. While channels belonging to SV cell types are known to play crucial roles in EP generation, relatively little is known about gene regulatory networks that underlie the ability of the SV to generate and maintain the EP. Using single cell and single nucleus RNA-sequencing, we identify and validate known and rare cell populations in the SV. Furthermore, we establish a basis for understanding molecular mechanisms underlying SV function by identifying potential gene regulatory networks as well as druggable gene targets. Finally, we associate known deafness genes with adult SV cell types. This work establishes a basis for dissecting the genetic mechanisms underlying the role of the SV in hearing and will serve as a basis for designing therapeutic approaches to hearing loss related to SV dysfunction.

Single Cell and Single Nucleus RNA-Seq Reveal Cellular Heterogeneity and Homeostatic Regulatory Networks in Adult Mouse Stria Vascularis.

Human pathogenic variants of  are associated with clinically heterogeneous phenotypes, including recessive nonsyndromic deafness DFNB86, dominant nonsyndromic deafness DFNA65, seizure accompanied by deafness, a variety of isolated seizure phenotypes and DOORS syndrome, characterized by deafness, onychodystrophy, osteodystrophy, intellectual disability and seizures. Thirty-five pathogenic variants of human  associated with deafness have been reported. However, functions of TBC1D24 in the inner ear and the pathophysiology of TBC1D24-related deafness are unknown. In this study, a novel splice-site variant of  c.965 + 1G > A in compound heterozygosity with c.641G > A p.(Arg214His) was found to be segregating in a Pakistani family. Affected individuals exhibited, either a deafness-seizure syndrome or nonsyndromic deafness. In human temporal bones, TBC1D24 immunolocalized in hair cells and spiral ganglion neurons, whereas in mouse cochlea,  expression was detected only in spiral ganglion neurons. We engineered mouse models of  p.(Asp70Tyr) and  p.(Ser178Leu) nonsyndromic deafness and syndromic forms of deafness p.(His336Glnfs*12) that have the same pathogenic variants that were reported for human . Unexpectedly, no auditory dysfunction was detected in  mutant mice, although homozygosity for some of the variants caused seizures or lethality. We provide some insightful supporting data to explain the phenotypic differences resulting from equivalent pathogenic variants of mouse  and human .

Mouse Models of Human Pathogenic Variants of  Associated with Non-Syndromic Deafness DFNB86 and DFNA65 and Syndromes Involving Deafness.

The stria vascularis (SV) in the cochlea generates and maintains the endocochlear potential, thereby playing a pivotal role in normal hearing. Knowing transcriptional profiles and gene regulatory networks of SV cell types establishes a basis for studying the mechanism underlying SV-related hearing loss. While we have previously characterized the expression profiles of major SV cell types in the adult mouse, transcriptional profiles of rare SV cell types remained elusive due to the limitation of cell capture in single-cell RNA-Seq. The role of these rare cell types in the homeostatic function of the adult SV remain largely undefined. In this study, we performed single-nucleus RNA-Seq on the adult mouse SV in conjunction with sample preservation treatments during the isolation steps. We distinguish rare SV cell types, including spindle cells and root cells, from other cell types, and characterize their transcriptional profiles. Furthermore, we also identify and validate novel specific markers for these rare SV cell types. Finally, we identify homeostatic gene regulatory networks within spindle and root cells, establishing a basis for understanding the functional roles of these cells in hearing. These novel findings will provide new insights for future work in SV-related hearing loss and hearing fluctuation.

Characterization of rare spindle and root cell transcriptional profiles in the stria vascularis of the adult mouse cochlea.

The stria vascularis generates the endocochlear potential and is involved in processes that underlie ionic homeostasis in the cochlear endolymph, both which play essential roles in hearing. The histological hallmark of Meniere's disease (MD) is endolymphatic hydrops, which refers to the bulging or expansion of the scala media, which is the endolymph-containing compartment of the cochlea. This histologic hallmark suggests that processes that disrupt ion homeostasis or potentially endocochlear potential may underlie MD. While treatments exist for vestibular symptoms related to MD, effective therapies for hearing fluctuation and hearing loss seen in MD remain elusive. Understanding the potential cell types involved in MD may inform the creation of disease mouse models and provide insight into underlying mechanisms and potential therapeutic targets. For these reasons, we compare published datasets related to MD in humans with our previously published adult mouse stria vascularis single-cell and single-nucleus RNA-Seq datasets to implicate potentially involved stria vascularis (SV) cell types in MD. Finally, we provide support for these implicated cell types by demonstrating co-expression of select candidate genes for MD within SV cell types.

Identification of Potential Meniere's Disease Targets in the Adult Stria Vascularis.

Hearing loss and impaired fertility are common human disorders each with multiple genetic causes. Sometimes deafness and impaired fertility, which are the hallmarks of Perrault syndrome, co-occur in a person. Perrault syndrome is inherited as an autosomal recessive disorder characterized by bilateral mild to severe childhood sensorineural hearing loss with variable age of onset in both sexes and ovarian dysfunction in females who have a 46, XX karyotype. Since the initial clinical description of Perrault syndrome 70 years ago, the phenotype of some subjects may additionally involve developmental delay, intellectual deficit and other neurological disabilities, which can vary in severity in part dependent upon the genetic variants and the gene involved. Here, we review the molecular genetics and clinical phenotype of Perrault syndrome and focus on supporting evidence for the eight genes (CLPP, ERAL1, GGPS1, HARS2, HSD17B4, LARS2, RMND1, TWNK) associated with Perrault syndrome. Variants of these eight genes only account for approximately half of the individuals with clinical features of Perrault syndrome where the molecular genetic base remains under investigation. Additional environmental etiologies and novel Perrault disease-associated genes remain to be identified to account for unresolved cases. We also report a new genetic variant of CLPP, computational structural insight about CLPP and single cell RNAseq data for eight reported Perrault syndrome genes suggesting a common cellular pathophysiology for this disorder. Some unanswered questions are raised to kindle future research about Perrault syndrome.

New insights into Perrault syndrome, a clinically and genetically heterogeneous disorder.

To identify genes implicated in sudden sensorineural hearing loss (SSNHL) and localize their expression in the cochlea to further explore potential pathogenic mechanisms and therapeutic targets.

Utilizing Single Cell RNA-Sequencing to Implicate Cell Types and Therapeutic Targets for SSNHL in the Adult Cochlea.

Corticosteroids, oral or transtympanic, remain the mainstay for inner ear diseases characterized by hearing fluctuation or sudden changes in hearing, including sudden sensorineural hearing loss (SSNHL), Meniere's disease (MD), and autoimmune inner ear disease (AIED). Despite their use across these diseases, the rate of complete recovery remains low, and results across the literature demonstrates significant heterogeneity with respect to the effect of corticosteroids, suggesting a need to identify more efficacious treatment options. Previously, our group has cross-referenced steroid-responsive genes in the cochlea with published single-cell and single-nucleus transcriptome datasets to demonstrate that steroid-responsive differentially regulated genes are expressed in spiral ganglion neurons (SGN) and stria vascularis (SV) cell types. These differentially regulated genes represent potential druggable gene targets. We utilized multiple gene target databases (DrugBank, Pharos, and LINCS) to identify orally administered, FDA approved medications that potentially target these genes. We identified 42 candidate drugs that have been shown to interact with these genes, with an emphasis on safety profile, and tolerability. This study utilizes multiple databases to identify drugs that can target a number of druggable genes in otologic disorders that are commonly treated with steroids, providing a basis for establishing novel repurposing treatment trials.

Repurposable Drugs That Interact with Steroid Responsive Gene Targets for Inner Ear Disease.

Hereditary deafness and retinal dystrophy are each genetically heterogenous and clinically variable. Three small unrelated families segregating the combination of deafness and retinal dystrophy were studied by exome sequencing (ES). The proband of Family 1 was found to be compound heterozygous for NM_004525.3: LRP2: c.5005A > G, p.(Asn1669Asp) and c.149C > G, p.(Thr50Ser). In Family 2, two sisters were found to be compound heterozygous for LRP2 variants, p.(Tyr3933Cys) and an experimentally confirmed c.7715 + 3A > T consensus splice-altering variant. In Family 3, the proband is compound heterozygous for a consensus donor splice site variant LRP2: c.8452_8452 + 1del and p.(Cys3150Tyr). In mouse cochlea, Lrp2 is expressed abundantly in the stria vascularis marginal cells demonstrated by smFISH, single-cell and single-nucleus RNAseq, suggesting that a deficiency of LRP2 may compromise the endocochlear potential, which is required for hearing. LRP2 variants have been associated with Donnai-Barrow syndrome and other multisystem pleiotropic phenotypes different from the phenotypes of the four cases reported herein. Our data expand the phenotypic spectrum associated with pathogenic variants in LRP2 warranting their consideration in individuals with a combination of hereditary hearing loss and retinal dystrophy.

Shoujun Gu

Auditory Development and Restoration Program,
National Institute on Deafness and Other Communication Disorders,
Auditory Development and Restoration Program, National Institute on Deafness and Other Communication Disorders

National Institutes of Health

Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining