Heffer, A. | bioRxiv Science

bioRxiv2025

A 3D iPSC retina model reveals non-cell-autonomous and non-neuronal mechanism of photoreceptor degeneration in a lysosomal storage disorder.

Disruption of photoreceptor-retinal pigment epithelium (RPE) interface with loss of photoreceptor outer segments (POSs) in the retina is a pathological hallmark of several neurodegenerative and retinal diseases including lysosomal storage disorders like CLN3 disease. However, the retina is a functional composite in vivo; and in vitro stem cell models of retina that enable investigation of the photoreceptor-RPE interface in healthy and diseased retina are lacking. Here, we developed a 3D human pluripotent stem cell (hPSC)-derived retina model to investigate the photoreceptor-RPE interface in healthy and disease tissue. Using this 3D hPSC retina model, we demonstrated that the most common disease causing CLN3 mutation (CLN3{Delta}ex7-8) leads to reduced levels of acid ceramidase (AC) and consequently altered sphingolipid metabolism and signaling and POS loss in CLN3 disease. Consistent with the 3D hPSC retina model, altered sphingolipid metabolism and signaling coincided with POS loss in a large animal model of CLN3 disease, CLN3 miniswine. Therapeutically, recombinant human acid ceramidase (rhAC) targeted both altered sphingolipid metabolism and retina degeneration in the CLN3 hPSC retina model and the CLN3 miniswine eye. These findings demonstrate a proof-of-concept that rhAC can rescue disease phenotype in a large animal model of CLN3 disease and suggest that rhAC could be a therapeutic approach for CLN3 disease. One Sentence SummaryAcid ceramidase deficiency and consequently altered sphingolipid signaling promotes disease phenotype(s) in a lysosomal storage disorder, CLN3 disease.

neuroscience↗

bioRxiv2024

Notch1 is required to maintain supporting cell identity and vestibular function during maturation of the mammalian balance organs

The inner ear houses two sensory modalities: the hearing organ, located in the cochlea, and the balance organs, located throughout the vestibular regions of the ear. Both hearing and vestibular sensory regions are composed of similar cell types, including hair cells and associated supporting cells. Recently, we showed that Notch1 is required for maintaining supporting cell survival postnatally during cochlear maturation. However, it is not known whether Notch1 plays a similar role in the balance organs of the inner ear. To characterize the role of Notch during vestibular maturation, we conditionally deleted Notch1 from Sox2-expressing cells of the vestibular organs in the mouse at P0/P1. Histological analyses showed a dramatic loss of supporting cells accompanied by an increase in type II hair cells without cell death, indicating the supporting cells are converting to hair cells in the maturing vestibular regions. Analysis of 6-week old animals indicate that the converted hair cells survive, despite the reduction of supporting cells. Interestingly, measurements of vestibular sensory evoked potentials (VsEPs), known to be generated in the striolar regions of the vestibular afferents in the maculae, failed to show a response, indicating that NOTCH1 expression is critical for striolar function postnatally. Consistent with this, we find that the specialized type I hair cells in the striola fail to develop the complex calyces typical of these cells. These defects are likely due to the reduction in supporting cells, which have previously been shown to express factors critical for the striolar region. Similar to other mutants that lack proper striolar development, Notch1 mutants do not exhibit typical vestibular behaviors such as circling and head shaking, but do show difficulties in some vestibular tests, including the balance beam and forced swim test. These results indicate that, unlike the hearing organ in which the supporting cells undergo cell death, supporting cells in the balance regions retain the ability to convert to hair cells during maturation, which survive into adulthood despite the reduction in supporting cells. Significance StatementNotch signaling regulates the cell fate choices between hair cells and supporting cells during inner ear development. However, little is known about how Notch functions in the mammalian vestibular sensory organs once cell fate has been determined. Here, we examine the role of Notch1 in the maturing balancing organs. We show that deletion of Notch1 results in vestibular physiological and behavioral dysfunction by 3 months of age. Histological analyses reveal supporting cells are converting to type II hair cells in the utricle, and despite a loss of supporting cells, the hair cells survive to adulthood. Additionally, the striolar type I hair cells important for generating a VsEP response are decreased in number and not innervated properly. These results show that Notch continues to function in maintaining supporting cell identity in the vestibular organs postnatally, which may be important in strategies for hair cell regeneration.

developmental biology↗