Rescue of Hearing by Gene Delivery to Inner-Ear Hair Cells Using Exosome-Associated AAV.

Adeno-associated virus (AAV) is a safe and effective vector for gene therapy for retinal disorders. Gene therapy for hearing disorders is not as advanced, in part because gene delivery to sensory hair cells of the inner ear is inefficient. Although AAV transduces the inner hair cells of the mouse cochlea, outer hair cells remain refractory to transduction. Here, we demonstrate that a vector, exosome-associated AAV (exo-AAV), is a potent carrier of transgenes to all inner ear hair cells. Exo-AAV1-GFP is more efficient than conventional AAV1-GFP, both in mouse cochlear explants in vitro and with direct cochlear injection in vivo. Exo-AAV shows no toxicity in vivo, as assayed by tests of auditory and vestibular function. Finally, exo-AAV1 gene therapy partially rescues hearing in a mouse model of hereditary deafness (lipoma HMGIC fusion partner-like 5/tetraspan membrane protein of hair cell stereocilia [Lhfpl5/Tmhs-/-]). Exo-AAV is a powerful gene delivery system for hair cell research and may be useful for gene therapy for deafness.

Adult-born dentate neurons are recruited in both spatial memory encoding and retrieval.

Adult neurogenesis occurs in the dentate gyrus (DG) of the hippocampus, which is a key structure in learning and memory. Adult-generated granule cells have been shown to play a role in spatial memory processes such as acquisition or retrieval, in particular during an immature stage when they exhibit a period of increased plasticity. Here, we demonstrate that immature and mature neurons born in the DG of adult rats are similarly activated in spatial memory processes. By imaging the activation of these two different neuron generations in the same rat and by using the immediate early gene Zif268, we show that these neurons are involved in both spatial memory acquisition and retrieval. These results demonstrate that adult-generated granule cells are involved in memory beyond their immaturity stage.

Overlapping and distinct pRb pathways in the mammalian auditory and vestibular organs.

Retinoblastoma gene (Rb1) is required for proper cell cycle exit in the developing mouse inner ear and its deletion in the embryo leads to proliferation of sensory progenitor cells that differentiate into hair cells and supporting cells. In a conditional hair cell Rb1 knockout mouse, Pou4f3-Cre-pRb(-/-), pRb(-/-) utricular hair cells differentiate and survive into adulthood whereas differentiation and survival of pRb(-/-) cochlear hair cells are impaired. To comprehensively survey the pRb pathway in the mammalian inner ear, we performed microarray analysis of (pRb(-/-) cochlea and utricle. The comparative analysis shows that the core pathway shared between pRb(-/-) cochlea and utricle is centered on E2F, the key pathway that mediates pRb function. A majority of differentially expressed genes and enriched pathways are not shared but uniquely associated with pRb(-/-) cochlea or utricle. In pRb(-/-) cochlea, pathways involved in early inner ear development such as Wnt/ß-catenin and Notch were enriched, whereas pathways involving in proliferation and survival are enriched in pRb(-/-) utricle. Clustering analysis showed that the pRb(-/-) inner ear has characteristics of a younger control inner ear, an indication of delayed differentiation. We created a transgenic mouse model (ER-Cre-pRb(flox/flox)) in which Rb1 can be acutely deleted postnatally. Acute Rb1 deletion in the adult mouse fails to induce proliferation or cell death in inner ear, strongly indicating that Rb1 loss in these postmitotic tissues can be effectively compensated for, or that pRb-mediated changes in the postmitotic compartment result in events that are functionally irreversible once enacted. This study thus supports the concept that pRb-regulated pathways relevant to hair cell development, encompassing proliferation, differentiation and survival, act predominantly during early development.

Diverse expression patterns of LIM-homeodomain transcription factors (LIM-HDs) in mammalian inner ear development.

LIM-homeodomain transcription factors (LIM-HDs) are essential in tissue patterning and differentiation. But their expression patterns in the inner ear are largely unknown. Here we report on a study of twelve LIM-HDs, by their tempo-spatial patterns that imply distinct yet overlapping roles, in the developing mouse inner ear. Expression of Lmx1a and Isl1 begins in the otocyst stage, with Lmx1a exclusively in the non-sensory and Isl1 in the prosensory epithelia. The second wave of expression at E12.5 includes Lhx3, 5, 9, Isl2, and Lmx1b in the differentiating sensory epithelia with cellular specificities. With the exception of Lmx1a and Lhx3, all LIM-HDs are expressed in ganglion neurons. Expression of multiple LIM-HDs within a cell type suggests their redundant function.

The α1 subunit of nicotinic acetylcholine receptors in the inner ear: transcriptional regulation by ATOH1 and co-expression with the γ subunit in hair cells.

Acetylcholine is a key neurotransmitter of the inner ear efferent system. In this study, we identify two novel nAChR subunits in the inner ear: α1 and γ, encoded by Chrna1 and Chrng, respectively. In situ hybridization shows that the messages of these two subunits are present in vestibular and cochlear hair cells during early development. Chrna1 and Chrng expression begin at embryonic stage E13.5 in the vestibular system and E17.5 in the organ of Corti. Chrna1 message continues through P7, whereas Chrng is undetectable at post-natal stage P6. The α1 and γ subunits are known as muscle-type nAChR subunits and are surprisingly expressed in hair cells which are sensory-neural cells. We also show that ATOH1/MATH1, a transcription factor essential for hair cell development, directly activates CHRNA1 transcription. Electrophoretic mobility-shift assays and supershift assays showed that ATOH1/E47 heterodimers selectively bind on two E boxes located in the proximal promoter of CHRNA1. Thus, Chrna1 could be the first transcriptional target of ATOH1 in the inner ear. Co-expression in Xenopus oocytes of the α1 subunit does not change the electrophysiological properties of the α9α10 receptor. We suggest that hair cells transiently express α1γ-containing nAChRs in addition to α9α10, and that these may have a role during development of the inner ear innervation.

Gene expression profiling identifies Hes6 as a transcriptional target of ATOH1 in cochlear hair cells.

ATOH1 is a basic Helix-Loop-Helix transcription factor crucial for hair cell (HC) differentiation in the inner ear. In order to identify ATOH1 target genes, we performed a genome-wide expression profiling analysis in cells expressing ATOH1 under the control of a tetracycline-off system and found that HES6 expression is induced by ATOH1. We performed in situ hybridisation and showed that the rise and fall of Hes6 expression closely follow that of Atoh1 in cochlear HC. Moreover, electrophoretic mobility shift assays and luciferase assays show that ATOH1 activates HES6 transcription through binding to three clustered E boxes of its promoter.

Essential role of retinoblastoma protein in mammalian hair cell development and hearing.

The retinoblastoma protein pRb is required for cell-cycle exit of embryonic mammalian hair cells but not for their early differentiation. However, its role in postnatal hair cells is unknown. To study the function of pRb in mature animals, we created a new conditional mouse model, with the Rb gene deleted primarily in the inner ear. Progeny survive up to 6 months. During early postnatal development, pRb(-/-) hair cells continue to divide and can transduce mechanical stimuli. However, adult pRb(-/-) mice exhibit profound hearing loss due to progressive degeneration of the organ of Corti. We show that pRb is required for the full maturation of cochlear outer hair cells, likely in a gene-specific manner, and is also essential for their survival. In addition, lack of pRb results in cell division in postnatal auditory supporting cells. In contrast, many pRb(-/-) vestibular hair cells survive and continue to divide in adult mice. Significantly, adult pRb(-/-) vestibular hair cells are functional, and pRb(-/-) mice maintain partial vestibular function. Therefore, the functional adult vestibular pRb(-/-) hair cells, derived from proliferation of postnatal hair cells, are largely integrated into vestibular pathways. This study reveals essential yet distinct roles of pRb in cochlear and vestibular hair cell maturation, function, and survival and suggests that transient block of pRb function in mature hair cells may lead to propagation of functional hair cells.

Proliferation of functional hair cells in vivo in the absence of the retinoblastoma protein.

In mammals, hair cell loss causes irreversible hearing and balance impairment because hair cells are terminally differentiated and do not regenerate spontaneously. By profiling gene expression in developing mouse vestibular organs, we identified the retinoblastoma protein (pRb) as a candidate regulator of cell cycle exit in hair cells. Differentiated and functional mouse hair cells with a targeted deletion of Rb1 undergo mitosis, divide, and cycle, yet continue to become highly differentiated and functional. Moreover, acute loss of Rb1 in postnatal hair cells caused cell cycle reentry. Manipulation of the pRb pathway may ultimately lead to mammalian hair cell regeneration.

Islet-1 expression in the developing chicken inner ear.

The cell types of the inner ear originate from the otic placode, a thickened layer of ectoderm adjacent to the developing hindbrain. The placode invaginates and forms the otic pit, which pinches off as a small vesicle called the otocyst. Presumptive cochleovestibular neurons delaminate from the anterior ventral part of the otocyst and form the cochleovestibular ganglion of the inner ear. Here we show that the LIM/homeodomain protein islet-1 is expressed in cells of the ventral part of the otic placode and that this ventral expression is maintained at the otic pit and the otocyst stages. Auditory and vestibular neurons originate from this islet-1-positive zone of the otocyst, and these neurons maintain islet-1 expression until adulthood. We also demonstrate that islet-1 becomes up-regulated in the presumptive sensory epithelia of the inner ear in regions that are defined by the expression domains of BMP4. The up-regulation of islet-1 in developing inner ear hair and supporting cells is accompanied by down-regulation of Pax-2 in these cell types. Islet-1 expression in hair and supporting cells persists until early postnatal stages, when the transcriptional regulator is down-regulated in hair cells. Our data is consistent with a role for islet-1 in differentiating inner ear neurons and sensory epithelia cells, perhaps in the specification of cellular subtypes in conjunction with other LIM/homeodomain proteins.

Distribution of alpha-amino-3-hydroxy-5-methyl-4 isoazolepropionic acid and N-methyl-D-aspartate receptor subunits in the vestibular and spiral ganglia of the mouse during early development.

We investigated the distribution of the glutamate receptor subunits, alpha-amino-3-hydroxy-5-methyl-4 isoazolepropionic acid (AMPA) GluR2 and GluR2/R3, and N-methyl-D-aspartate (NMDA) NR1, and the timing of their appearance during early development of the mouse vestibular and spiral ganglia. NMDA NR1 was the first to be expressed, in the statoacoustic ganglion neurons on E11. GluR2/R3 immunoreactivity was detected in these neurons on E12. This signal probably corresponded exclusively to GluR3, as no signal was obtained for GluR2 alone at this stage. The appearance of these proteins began much earlier than previously reported. GluR2 staining was observed later, on E14 in the vestibular neurons and on E17 in the spiral neurons. The sequence in which these three glutamate receptors appeared suggested possible differences in their roles in the establishment of neuronal circuitry in the inner ear sensory epithelia. The production of NR1 and GluR2/R3 began during the early period of neuron growth and fasciculation. GluR2 appeared later and its expression paralleled synaptogenesis in the vestibular sensory epithelia and in the organ of Corti.