AAV-ie-K558R mediated cochlear gene therapy and hair cell regeneration.

The cochlea consists of multiple types of cells, including hair cells, supporting cells and spiral ganglion neurons, and is responsible for converting mechanical forces into electric signals that enable hearing. Genetic and environmental factors can result in dysfunctions of cochlear and auditory systems. In recent years, gene therapy has emerged as a promising treatment in animal deafness models. One major challenge of the gene therapy for deafness is to effectively deliver genes to specific cells of cochleae. Here, we screened and identified an AAV-ie mutant, AAV-ie-K558R, that transduces hair cells and supporting cells in the cochleae of neonatal mice with high efficiency. AAV-ie-K558R is a safe vector with no obvious deficits in the hearing system. We found that AAV-ie-K558R can partially restore the hearing loss in Prestin KO mice and, importantly, deliver Atoh1 into cochlear supporting cells to generate hair cell-like cells. Our results demonstrate the clinical potential of AAV-ie-K558R for treating the hearing loss caused by hair cell death.

Espin distribution as revealed by super-resolution microscopy of stereocilia.

Auditory hair cells are the mechanical sensors of sound waves in the inner ear, and the stereocilia, which are actin-rich protrusions of different heights on the apical surfaces of hair cells, are responsible for the transduction of sound waves into electrical signals. As a crucial actin-binding and bundling protein, espin is able to cross-link actin filaments and is therefore necessary for stereocilia morphogenesis. Using advanced super-resolution stimulated emission depletion microscopy, we imaged espin expression at the sub-diffraction limit along the whole length of the stereocilia in outer hair cells and inner hair cells in order to better understand espin's function in the development of stereocilia.

Critical role of spectrin in hearing development and deafness.

Inner ear hair cells (HCs) detect sound through the deflection of mechanosensory stereocilia. Stereocilia are inserted into the cuticular plate of HCs by parallel actin rootlets, where they convert sound-induced mechanical vibrations into electrical signals. The molecules that support these rootlets and enable them to withstand constant mechanical stresses underpin our ability to hear. However, the structures of these molecules have remained unknown. We hypothesized that αII- and ßII-spectrin subunits fulfill this role, and investigated their structural organization in rodent HCs. Using super-resolution fluorescence imaging, we found that spectrin formed ring-like structures around the base of stereocilia rootlets. These spectrin rings were associated with the hearing ability of mice. Further, HC-specific, ßII-spectrin knockout mice displayed profound deafness. Overall, our work has identified and characterized structures of spectrin that play a crucial role in mammalian hearing development.

AAV-ie enables safe and efficient gene transfer to inner ear cells.

Hearing loss is the most common sensory disorder. While gene therapy has emerged as a promising treatment of inherited diseases like hearing loss, it is dependent on the identification of gene delivery vectors. Adeno-associated virus (AAV) vector-mediated gene therapy has been approved in the US for treating a rare inherited eye disease but no safe and efficient vectors have been identified that can target the diverse types of inner ear cells. Here, we identify an AAV variant, AAV-inner ear (AAV-ie), for gene delivery in mouse inner ear. Our results show that AAV-ie transduces the cochlear supporting cells (SCs) with high efficiency, representing a vast improvement over conventional AAV serotypes. Furthermore, after AAV-ie-mediated transfer of the Atoh1 gene, we find that many SCs trans-differentiated into new HCs. Our results suggest that AAV-ie is a useful tool for the cochlear gene therapy and for investigating the mechanism of HC regeneration.

Molecular Mechanism for Ligand Recognition and Subtype Selectivity of α2C Adrenergic Receptor.

Adrenergic G-protein-coupled receptors (GPCRs) mediate different cellular signaling pathways in the presence of endogenous catecholamines and play important roles in both physiological and pathological conditions. Extensive studies have been carried out to investigate the structure and function of ß adrenergic receptors (ßARs). However, the structure of α adrenergic receptors (αARs) remains to be determined. Here, we report the structure of the human α2C adrenergic receptor (α2CAR) with the non-selective antagonist, RS79948, at 2.8 Å. Our structure, mutations, modeling, and functional experiments indicate that a α2CAR-specific D206ECL2-R409ECL3-Y4056.58 network plays a role in determining α2 adrenergic subtype selectivity. Furthermore, our results show that a specific loosened helix at the top of TM4 in α2CAR is involved in receptor activation. Together, our structure of human α2CAR-RS79948 provides key insight into the mechanism underlying the α2 adrenergic receptor activation and subtype selectivity.

Diverse Supramolecular Nanofiber Networks Assembled by Functional Low-Complexity Domains.

Self-assembling supramolecular nanofibers, common in the natural world, are of fundamental interest and technical importance to both nanotechnology and materials science. Despite important advances, synthetic nanofibers still lack the structural and functional diversity of biological molecules, and the controlled assembly of one type of molecule into a variety of fibrous structures with wide-ranging functional attributes remains challenging. Here, we harness the low-complexity (LC) sequence domain of fused in sarcoma (FUS) protein, an essential cellular nuclear protein with slow kinetics of amyloid fiber assembly, to construct random copolymer-like, multiblock, and self-sorted supramolecular fibrous networks with distinct structural features and fluorescent functionalities. We demonstrate the utilities of these networks in the templated, spatially controlled assembly of ligand-decorated gold nanoparticles, quantum dots, nanorods, DNA origami, and hybrid structures. Owing to the distinguishable nanoarchitectures of these nanofibers, this assembly is structure-dependent. By coupling a modular genetic strategy with kinetically controlled complex supramolecular self-assembly, we demonstrate that a single type of protein molecule can be used to engineer diverse one-dimensional supramolecular nanostructures with distinct functionalities.