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For the development of new therapies, proof-of-concept studies in large animal models that share clinical features with their human counterparts represent a pivotal step. For inherited retinal dystrophies primarily involving photoreceptor cells, the efficacy of gene therapy has been demonstrated in canine models of stationary cone dystrophies and progressive rod-cone dystrophies but not in large models of progressive cone-rod dystrophies, another important cause of blindness. To address the last issue, we evaluated gene therapy in the retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1)-deficient dog, a model exhibiting a severe cone-rod dystrophy similar to that seen in humans. Subretinal injection of AAV5 (n = 5) or AAV8 (n = 2) encoding the canine Rpgrip1 improved photoreceptor survival in transduced areas of treated retinas. Cone function was significantly and stably rescued in all treated eyes (18-72% of those recorded in normal eyes) up to 24 months postinjection. Rod function was also preserved (22-29% of baseline function) in four of the five treated dogs up to 24 months postinjection. No detectable rod function remained in untreated contralateral eyes. More importantly, treatment preserved bright- and dim-light vision. Efficacy of gene therapy in this large animal model of cone-rod dystrophy provides great promise for human treatment.

A detrimental perceptive consequence of damaged auditory sensory hair cells consists in a pronounced masking effect exerted by low-frequency sounds, thought to occur when auditory threshold elevation substantially exceeds 40 dB. Here, we identified the submembrane scaffold protein Nherf1 as a hair-bundle component of the differentiating outer hair cells (OHCs). Nherf1 −/− mice displayed OHC hair-bundle shape anomalies in the mid and basal cochlea, normally tuned to mid-and high-frequency tones, and mild (22-35 dB) hearing-threshold elevations restricted to midhigh sound frequencies. This mild decrease in hearing sensitivity was, however, discordant with almost nonresponding OHCs at the cochlear base as assessed by distortion-product otoacoustic emissions and cochlear microphonic potentials. Moreover, unlike wild-type mice, responses of Nherf1 −/− mice to high-frequency (20-40 kHz) test tones were not masked by tones of neighboring frequencies. Instead, efficient maskers were characterized by their frequencies up to two octaves below the probe-tone frequency, unusually low intensities up to 25 dB below probe-tone level, and growth-ofmasker slope (2.2 dB/dB) reflecting their compressive amplification. Together, these properties do not fit the current acknowledged features of a hypersensitivity of the basal cochlea to lower frequencies, but rather suggest a previously unidentified mechanism. Low-frequency maskers, we propose, may interact within the unaffected cochlear apical region with midhigh frequency sounds propagated there via a mode possibly using the persistent contact of misshaped OHC hair bundles with the tectorial membrane. Our findings thus reveal a source of misleading interpretations of hearing thresholds and of hypervulnerability to low-frequency sound interference.off-frequency detection | Usher syndrome | hearing impairment | Nherf2 | tail hypersensitivity M ammalian hearing displays remarkable sensitivity, fine temporal acuity, and exquisite frequency selectivity, which contribute to auditory scene analysis and speech intelligibility. The first steps of sound processing, i.e., sound wave detection and neuronal encoding in the cochlea, are performed by two populations of hair cells, the inner hair cells (IHCs) and the outer hair cells (OHCs). These cells are sandwiched between the underlying basilar membrane (BM) and the overlying tectorial membrane (TM) (SI Appendix, Fig. S1A). IHCs are the genuine sensory cells that transduce the sound stimuli into electrical signals in the primary auditory neurons. OHCs are mechanical effectors that amplify the sound-evoked movements of the cochlear partition, sharpen its frequency selectivity, and produce waveform distortions (1, 2). A pure-tone stimulus entering the cochlea elicits a traveling wave that propagates along the BM from the cochlear base toward its apex, increasing in amplitude until it peaks at a characteristic place, where the mechanical properties of the cochlea are best tuned to the stimulus frequency. Beyond this characteristic place, the ampl...

Mutations in the myosin VIIa gene cause Usher syndrome type IB (USH1B), characterized by deaf-blindness. A delay of opsin trafficking has been observed in the retinal photoreceptor cells of myosin VIIa-deficient mice. We identified spectrin βV, the mammalian β-heavy spectrin, as a myosin VIIa- and rhodopsin-interacting partner in photoreceptor cells. Spectrin βV displays a polarized distribution from the Golgi apparatus to the base of the outer segment, which, unlike that of other β spectrins, matches the trafficking route of opsin and other phototransduction proteins. Formation of spectrin βV-rhodopsin complex could be detected in the differentiating photoreceptors as soon as their outer segment emerges. A failure of the spectrin βV-mediated coupling between myosin VIIa and opsin molecules thus probably accounts for the opsin transport delay in myosin VIIa-deficient mice. We showed that spectrin βV also associates with two USH1 proteins, sans (USH1G) and harmonin (USH1C). Spectrins are supposed to function as heteromers of α and β subunits, but fluorescence resonance energy transfer and in vitro binding experiments indicated that spectrin βV can also form homodimers, which likely supports its αII-independent βV functions. Finally, consistent with its distribution along the connecting cilia axonemes, spectrin βV binds to several subunits of the microtubule-based motor proteins, kinesin II and the dynein complex. We therefore suggest that spectrin βV homomers couple some USH1 proteins, opsin and other phototransduction proteins to both actin- and microtubule-based motors, thereby contributing to their transport towards the photoreceptor outer disks.

The remarkable hearing capacities of mammals arise from various evolutionary innovations. These include the cochlear outer hair cells and their singular feature, somatic electromotility, i.e., the ability of their cylindrical cell body to shorten and elongate upon cell depolarization and hyperpolarization, respectively. To shed light on the processes underlying the emergence of electromotility, we focused on the βV giant spectrin, a major component of the outer hair cells' cortical cytoskeleton. We identified strong signatures of adaptive evolution at multiple sites along the spectrin-βV amino acid sequence in the lineage leading to mammals, together with substantial differences in the subcellular location of this protein between the frog and the mouse inner ear hair cells. In frog hair cells, spectrin βV was invariably detected near the apical junctional complex and above the cuticular plate, a dense F-actin meshwork located underneath the apical plasma membrane. In the mouse, the protein had a broad punctate cytoplasmic distribution in the vestibular hair cells, whereas it was detected in the entire lateral wall of cochlear outer hair cells and had an intermediary distribution (both cytoplasmic and cortical, but restricted to the cell apical region) in cochlear inner hair cells. Our results support a scenario where the singular organization of the outer hair cells' cortical cytoskeleton may have emerged from molecular networks initially involved in membrane trafficking, which were present near the apical junctional complex in the hair cells of mammalian ancestors and would have subsequently expanded to the entire lateral wall in outer hair cells.unconventional spectrins | inner ear | F-actin cytoskeleton | cortical lattice | phylogenetics T he response of the mammalian auditory organ (cochlea) to acoustic stimuli in an extended frequency range (including high frequencies) has remarkable properties including very high sensitivity and exquisitely sharp tuning (1-3). These properties are the consequence of an evolutionary process that involved major morphological and functional changes. One of them is the emergence, in the cochlea, of the outer hair cells, a unique type of specialized sensory cells that display somatic electromotility, i.e., they undergo periodic length changes in response to the oscillation of their membrane potential evoked by the sound wave (they shorten upon depolarization and elongate upon hyperpolarization) (SI Appendix, Fig. S1 A-C). This process has endowed the mammalian auditory organ with a singular mechanism of spectral analysis of the acoustic stimulus through frequencyselective mechanical amplification (1-3), whereas spectral analysis in other vertebrates (fish, amphibians, reptiles, and birds) primarily relies on electrical tuning of the hair cells (4, 5).An intriguing question is how the emergence of somatic electromotility is related with the evolution of individual proteins involved in this process. The electromotility of outer hair cells critically depends on the presence, in th...

Ciliopathies manifest from sensory abnormalities to syndromic disorders with multi-organ pathologies, with retinal degeneration a highly penetrant phenotype. Photoreceptor cell death is a major cause of incurable blindness in retinal ciliopathies. To identify drug candidates to maintain photoreceptor survival, we performed an unbiased, high-throughput screening of over 6,000 bioactive small molecules using retinal organoids differentiated from induced pluripotent stem cells (iPSC) of rd16 mouse, which is a model of Leber congenital amaurosis (LCA) type 10 caused by mutations in the cilia-centrosomal gene CEP290. We identified five non-toxic positive hits, including the lead molecule reserpine, which maintained photoreceptor development and survival in rd16 organoids. Reserpine also improved photoreceptors in retinal organoids derived from induced pluripotent stem cells of LCA10 patients and in rd16 mouse retina in vivo. Reserpine-treated patient organoids revealed modulation of signaling pathways related to cell survival/death, metabolism, and proteostasis. Further investigation uncovered dysregulation of autophagy associated with compromised primary cilium biogenesis in patient organoids and rd16 mouse retina. Reserpine partially restored the balance between autophagy and the ubiquitin-proteasome system at least in part by increasing the cargo adaptor p62, resulting in improved primary cilium assembly. Our study identifies effective drug candidates in preclinical studies of CEP290 retinal ciliopathies through cross-species drug discovery using iPSC-derived organoids, highlights the impact of proteostasis in the pathogenesis of ciliopathies, and provides new insights for treatments of retinal neurodegeneration.

Cone photoreceptors mediate color perception and daylight vision through intricate synaptic circuitry. In most mammalian retina, cones are greatly outnumbered by rods and exhibit interdependence for functional maintenance and survival. Currently, we have limited understanding of cone-specific molecular components that mediate response to extrinsic signaling factors or are involved in communication with rods and other retinal cells. To fulfill this gap, we compared the recently-published transcriptomes of developing S-cone-like photoreceptors from the Nrl−/− mouse retina with those of rods and identified candidate genes responsible for cone cell functions and communication. We generated an in silico expression profile of 823 genes that encode candidate transmembrane and secretory proteins and are up-regulated in Nrl−/− cone photoreceptors compared to wild type cones. In situ hybridization analysis validated high expression of seven of the selected candidate genes in the Nrl−/− retina. To examine their relevance to cone function, we performed in vivo knockdown of Epha10 in the Nrl−/− retina and demonstrated aberrant morphology and mislocalization of the photoreceptor cell bodies. Thus, the receptor tyrosine kinase Ephrin type-A receptor 10 appears to influence cone morphogenesis. Our studies reveal novel cone-enriched genes involved in interaction of cones with other retinal cell types and provide a framework for examining molecular pathways associated with intercellular communication.

Ciliopathies manifest from sensory abnormalities to syndromic disorders with multiorgan pathologies, with retinal degeneration a highly penetrant phenotype. Photoreceptor cell death is a major cause of incurable blindness in retinal ciliopathies. To identify drug candidates to maintain photoreceptor survival, we performed an unbiased, high-throughput screening of over 6,000 bioactive small molecules using retinal organoids differentiated from induced pluripotent stem cells (iPSC) of rd16 mouse, which is a model of Leber congenital amaurosis (LCA)10 caused by mutations in the cilia-centrosomal gene CEP290. We identified five non-toxic positive hits, including the lead molecule reserpine, which improved photoreceptor survival in rd16 organoids. Reserpine also maintained photoreceptors in retinal organoids derived from induced pluripotent stem cells of LCA10 patients and in rd16 mouse retina in vivo. Reserpine-treated patient organoids revealed modulation of signaling pathways related to cell survival/death, metabolism, and proteostasis. Further investigation uncovered misregulation of autophagy associated with compromised primary cilium biogenesis in patient organoids and rd16 mouse retina. Reserpine partially restored the balance between autophagy and the ubiquitin-proteasome system, at least in part by increasing the cargo adaptor p62 and improving primary cilium assembly. Our study identifies effective drug candidates in preclinical studies of CEP290 retinal ciliopathies through cross-species drug discovery using iPSC-derived organoids, highlights the impact of proteostasis in the pathogenesis of ciliopathies, and provides new insights for treatments of retinal neurodegeneration.