Atypical Auditory Brainstem Response and Protein Expression Aberrations Related to ASD and Hearing Loss in the Adnp Haploinsufficient Mouse Brain.

Autism is a wide spread neurodevelopmental disorder with growing morbidity rates, affecting more boys than girls worldwide. Activity-dependent neuroprotective protein (ADNP) was recently recognized as a leading gene accounted for 0.17% of autism spectrum disorder (ASD) cases globally. Respectively, mutations in the human ADNP gene (ADNP syndrome), cause multi-system body dysfunctions with apparent ASD-related traits, commencing as early as childhood. The Adnp haploinsufficient (Adnp+/-) mouse model was researched before in relations to Alzheimer's disease and autism. Adnp+/- mice suffer from deficient social memory, vocal and motor impediments, irregular tooth eruption and short stature, all of which corresponds with reported phenotypes in patients with the ADNP syndrome. Recently, a more elaborated description of the ADNP syndrome was published, presenting impediments such as hearing disabilities in > 10% of the studied children. Irregular auditory brainstem response (ABR) has been connected to ASD-related cases and has been suggested as a potential hallmark for autism, allowing diagnosis of ASD risk and early intervention. Herein, we present detriment hearing in the Adnp+/- mice with atypical ABR and significant protein expression irregularities that coincides with ASD and hearing loss studies in the brain.

Reduced changes in protein compared to mRNA levels across non-proliferating tissues.

BACKGROUND: The quantitative relations between RNA and protein are fundamental to biology and are still not fully understood. Across taxa, it was demonstrated that the protein-to-mRNA ratio in steady state varies in a direction that lessens the change in protein levels as a result of changes in the transcript abundance. Evidence for this behavior in tissues is sparse. We tested this phenomenon in new data that we produced for the mouse auditory system, and in previously published tissue datasets. A joint analysis of the transcriptome and proteome was performed across four datasets: inner-ear mouse tissues, mouse organ tissues, lymphoblastoid primate samples and human cancer cell lines. RESULTS: We show that the protein levels are more conserved than the mRNA levels in all datasets, and that changes in transcription are associated with translational changes that exert opposite effects on the final protein level, in all tissues except cancer. Finally, we observe that some functions are enriched in the inner ear on the mRNA level but not in protein. CONCLUSIONS: We suggest that partial buffering between transcription and translation ensures that proteins can be made rapidly in response to a stimulus. Accounting for the buffering can improve the prediction of protein levels from mRNA levels.

The GPSM2/LGN GoLoco motifs are essential for hearing.

The planar cell polarity (PCP) pathway is responsible for polarizing and orienting cochlear hair cells during development through movement of a primary cilium, the kinocilium. GPSM2/LGN, a mitotic spindle-orienting protein associated with deafness in humans, is a PCP effector involved in kinocilium migration. Here, we link human and mouse truncating mutations in the GPSM2/LGN gene, both leading to hearing loss. The human variant, p.(Trp326*), was identified by targeted genomic enrichment of genes associated with deafness, followed by massively parallel sequencing. Lgn (ΔC) mice, with a targeted deletion truncating the C-terminal GoLoco motifs, are profoundly deaf and show misorientation of the hair bundle and severe malformations in stereocilia shape that deteriorates over time. Full-length protein levels are greatly reduced in mutant mice, with upregulated mRNA levels. The truncated Lgn (ΔC) allele is translated in vitro, suggesting that mutant mice may have partially functioning Lgn. Gαi and aPKC, known to function in the same pathway as Lgn, are dependent on Lgn for proper localization. The polarization of core PCP proteins is not affected in Lgn mutants; however, Lgn and Gαi are misoriented in a PCP mutant, supporting the role of Lgn as a PCP effector. The kinocilium, previously shown to be dependent on Lgn for robust localization, is essential for proper localization of Lgn, as well as Gαi and aPKC, suggesting that cilium function plays a role in positioning of apical proteins. Taken together, our data provide a mechanism for the loss of hearing found in human patients with GPSM2/LGN variants.

Genomic characterization and therapeutic utilization of IL-13-responsive sequences in asthma.

Epithelial responses to the cytokine interleukin-13 (IL-13) cause airway obstruction in asthma. Here we utilized multiple genomic techniques to identify IL-13-responsive regulatory elements in bronchial epithelial cells and used these data to develop a CRISPR interference (CRISPRi)-based therapeutic approach to downregulate airway obstruction-inducing genes in a cell type- and IL-13-specific manner. Using single-cell RNA sequencing (scRNA-seq) and acetylated lysine 27 on histone 3 (H3K27ac) chromatin immunoprecipitation sequencing (ChIP-seq) in primary human bronchial epithelial cells, we identified IL-13-responsive genes and regulatory elements. These sequences were functionally validated and optimized via massively parallel reporter assays (MPRAs) for IL-13-inducible activity. The top secretory cell-selective sequence from the MPRA, a novel, distal enhancer of the sterile alpha motif pointed domain containing E-26 transformation-specific transcription factor (SPDEF) gene, was utilized to drive CRISPRi and knock down SPDEF or mucin 5AC (MUC5AC), both involved in pathologic mucus production in asthma. Our work provides a catalog of cell type-specific genes and regulatory elements involved in IL-13 bronchial epithelial response and showcases their use for therapeutic purposes.

Absent from DNA and protein: genomic characterization of nullomers and nullpeptides across functional categories and evolution.

Nullomers and nullpeptides are short DNA or amino acid sequences that are absent from a genome or proteome, respectively. One potential cause for their absence could be their having a detrimental impact on an organism. RESULTS: Here, we identify all possible nullomers and nullpeptides in the genomes and proteomes of thirty eukaryotes and demonstrate that a significant proportion of these sequences are under negative selection. We also identify nullomers that are unique to specific functional categories: coding sequences, exons, introns, 5'UTR, 3'UTR, promoters, and show that coding sequence and promoter nullomers are most likely to be selected against. By analyzing all protein sequences across the tree of life, we further identify 36,081 peptides up to six amino acids in length that do not exist in any known organism, termed primes. We next characterize all possible single base pair mutations that can lead to the appearance of a nullomer in the human genome, observing a significantly higher number of mutations than expected by chance for specific nullomer sequences in transposable elements, likely due to their suppression. We also annotate nullomers that appear due to naturally occurring variants and show that a subset of them can be used to distinguish between different human populations. Analysis of nullomers and nullpeptides across vertebrate evolution shows they can also be used as phylogenetic classifiers. CONCLUSIONS: We provide a catalog of nullomers and nullpeptides in distinct functional categories, develop methods to systematically study them, and highlight the use of variability in these sequences in other analyses.

Neonatal AAV gene therapy rescues hearing in a mouse model of SYNE4 deafness.

Genetic variants account for approximately half the cases of congenital and early-onset deafness. Methods and technologies for viral delivery of genes into the inner ear have evolved over the past decade to render gene therapy a viable and attractive approach for treatment. Variants in SYNE4, encoding the protein nesprin-4, a member of the linker of nucleoskeleton and cytoskeleton (LINC), lead to DFNB76 human deafness. Syne4-/- mice have severe-to-profound progressive hearing loss and exhibit mislocalization of hair cell nuclei and hair cell degeneration. We used AAV9-PHP.B, a recently developed synthetic adeno-associated virus, to deliver the coding sequence of Syne4 into the inner ears of neonatal Syne4-/- mice. Here we report rescue of hair cell morphology and survival, nearly complete recovery of auditory function, and restoration of auditory-associated behaviors, without observed adverse effects. Uncertainties remain regarding the durability of the treatment and the time window for intervention in humans, but our results suggest that gene therapy has the potential to prevent hearing loss in humans with SYNE4 mutations.

DNA methylation dynamics during embryonic development and postnatal maturation of the mouse auditory sensory epithelium.

The inner ear is a complex structure responsible for hearing and balance, and organ pathology is associated with deafness and balance disorders. To evaluate the role of epigenomic dynamics, we performed whole genome bisulfite sequencing at key time points during the development and maturation of the mouse inner ear sensory epithelium (SE). Our single-nucleotide resolution maps revealed variations in both general characteristics and dynamics of DNA methylation over time. This allowed us to predict the location of non-coding regulatory regions and to identify several novel candidate regulatory factors, such as Bach2, that connect stage-specific regulatory elements to molecular features that drive the development and maturation of the SE. Constructing in silico regulatory networks around sites of differential methylation enabled us to link key inner ear regulators, such as Atoh1 and Stat3, to pathways responsible for cell lineage determination and maturation, such as the Notch pathway. We also discovered that a putative enhancer, defined as a low methylated region (LMR), can upregulate the GJB6 gene and a neighboring non-coding RNA. The study of inner ear SE methylomes revealed novel regulatory regions in the hearing organ, which may improve diagnostic capabilities, and has the potential to guide the development of therapeutics for hearing loss by providing multiple intervention points for manipulation of the auditory system.

Single cell analysis of the inner ear sensory organs.

The inner ear is composed of a complex mixture of cells, which together allow organisms to hear and maintain balance. The cells in the inner ear, which undergo an extraordinary process of development, have only recently begun to be studied on an individual level. As it has recently become clear that individual cells, previously considered to be of uniform character, may differ dramatically from each other, the need to study cell-to-cell variation, along with distinct transcriptional and regulatory signatures, has taken hold in the scientific community. In conjunction with high-throughput technologies, attempts are underway to dissect the inter- and intra-cellular variability of different cell types and developmental states of the inner ear from a novel perspective. Single cell analysis of the inner ear sensory organs holds the promise of providing a significant boost in building an omics network that translates into a comprehensive understanding of the mechanisms of hearing and balance. These networks may uncover critical elements for trans-differentiation, regeneration and/or reprogramming, providing entry points for therapeutics of deafness and vestibular pathologies.