Characterization and AAV-mediated CRB gene augmentation in human-derived CRB1KO and CRB1KOCRB2+/- retinal organoids.

The majority of patients with mutations in CRB1 develop either early-onset retinitis pigmentosa as young children or Leber congenital amaurosis as newborns. The cause for the phenotypic variability in CRB1-associated retinopathies is unknown, but might be linked to differences in CRB1 and CRB2 protein levels in Müller glial cells and photoreceptor cells. Here, CRB1KO and CRB1KOCRB2+/- differentiation day 210 retinal organoids showed a significant decrease in the number of photoreceptor nuclei in a row and a significant increase in the number of photoreceptor cell nuclei above the outer limiting membrane. This phenotype with outer retinal abnormalities is similar to CRB1 patient-derived retinal organoids and Crb1 or Crb2 mutant mouse retinal disease models. The CRB1KO and CRB1KOCRB2+/- retinal organoids develop an additional inner retinal phenotype due to the complete loss of CRB1 from Müller glial cells, suggesting an essential role for CRB1 in proper localization of neuronal cell types. Adeno-associated viral (AAV) transduction was explored at early and late stages of organoid development. Moreover, AAV-mediated gene augmentation therapy with AAV.hCRB2 improved the outer retinal phenotype in CRB1KO retinal organoids. Altogether, these data provide essential information for future gene therapy approaches for patients with CRB1-associated retinal dystrophies.

AAV-mediated gene augmentation therapy of CRB1 patient-derived retinal organoids restores the histological and transcriptional retinal phenotype.

Retinitis pigmentosa and Leber congenital amaurosis are inherited retinal dystrophies that can be caused by mutations in the Crumbs homolog 1 (CRB1) gene. CRB1 is required for organizing apical-basal polarity and adhesion between photoreceptors and Müller glial cells. CRB1 patient-derived induced pluripotent stem cells were differentiated into CRB1 retinal organoids that showed diminished expression of variant CRB1 protein observed by immunohistochemical analysis. Single-cell RNA sequencing revealed impact on, among others, the endosomal pathway and cell adhesion and migration in CRB1 patient-derived retinal organoids compared with isogenic controls. Adeno-associated viral (AAV) vector-mediated hCRB2 or hCRB1 gene augmentation in Müller glial and photoreceptor cells partially restored the histological phenotype and transcriptomic profile of CRB1 patient-derived retinal organoids. Altogether, we show proof-of-concept that AAV.hCRB1 or AAV.hCRB2 treatment improved the phenotype of CRB1 patient-derived retinal organoids, providing essential information for future gene therapy approaches for patients with mutations in the CRB1 gene.

Pre-ataxic loss of intrinsic plasticity and motor learning in a mouse model of SCA1.

Spinocerebellar ataxias are neurodegenerative diseases, the hallmark symptom of which is the development of ataxia due to cerebellar dysfunction. Purkinje cells, the principal neurons of the cerebellar cortex, are the main cells affected in these disorders, but the sequence of pathological events leading to their dysfunction is poorly understood. Understanding the origins of Purkinje cells dysfunction before it manifests is imperative to interpret the functional and behavioural consequences of cerebellar-related disorders, providing an optimal timeline for therapeutic interventions. Here, we report the cascade of events leading to Purkinje cells dysfunction before the onset of ataxia in a mouse model of spinocerebellar ataxia 1 (SCA1). Spatiotemporal characterization of the ATXN1[82Q] SCA1 mouse model revealed high levels of the mutant ATXN1[82Q] weeks before the onset of ataxia. The expression of the toxic protein first caused a reduction of Purkinje cells intrinsic excitability, which was followed by atrophy of Purkinje cells dendrite arborization and aberrant glutamatergic signalling, finally leading to disruption of Purkinje cells innervation of climbing fibres and loss of intrinsic plasticity of Purkinje cells. Functionally, we found that deficits in eyeblink conditioning, a form of cerebellum-dependent motor learning, precede the onset of ataxia, matching the timeline of climbing fibre degeneration and reduced intrinsic plasticity. Together, our results suggest that abnormal synaptic signalling and intrinsic plasticity during the pre-ataxia stage of spinocerebellar ataxias underlie an aberrant cerebellar circuitry that anticipates the full extent of the disease severity. Furthermore, our work indicates the potential for eyeblink conditioning to be used as a sensitive tool to detect early cerebellar dysfunction as a sign of future disease.

Sleep Deprivation Does not Change the Flash Electroretinogram in Wild-type and Opn4-/-Gnat1-/- Mice.

Sleep deprivation reduces the response of neuronal activity in the suprachiasmatic nucleus (SCN) and the phase shift in circadian behaviour to phase shifting light pulses, and thus seems to impair the adaptation of the circadian clock to the external light-dark cycle. The question remains where in the pathway of light input to the SCN the response is reduced. We therefore investigated whether the electroretinogram (ERG) changes after sleep deprivation in wild-type mice and in Opn4-/-Gnat1-/- mutant male mice. We found that the ERG is clearly affected by the Opn4-/-Gnat1-/- mutations, but that the ERG after sleep deprivation does not differ from the baseline response. The difference between wild-type and mutant is in accordance with the lack of functional rod and melanopsin in the retina of the mutant mice. We conclude that the decrease in light responsiveness of the SCN after sleep deprivation is probably not caused by changes at the retinal level, but rather at the postsynaptic site within the SCN, reflecting affected neurotransmitter signalling.

Defining Phenotype, Tropism, and Retinal Gene Therapy Using Adeno-Associated Viral Vectors (AAVs) in New-Born Brown Norway Rats with a Spontaneous Mutation in Crb1.

Mutations in the Crumbs homologue 1 (CRB1) gene cause inherited retinal dystrophies, such as early-onset retinitis pigmentosa and Leber congenital amaurosis. A Brown Norway rat strain was reported with a spontaneous insertion-deletion (indel) mutation in exon 6 of Crb1. It has been reported that these Crb1 mutant rats show vascular abnormalities associated with retinal telangiectasia and possess an early-onset retinal degenerative phenotype with outer limiting membrane breaks and focal loss of retinal lamination at 2 months of age. Here, we further characterized the morphological phenotype of new-born and adult Crb1 mutant rats in comparison with age-matched Brown Norway rats without a mutation in Crb1. A significantly decreased retinal function and visual acuity was observed in Crb1 mutant rats at 1 and 3 months of age, respectively. Moreover, in control rats, the subcellular localization of canonical CRB1 was observed at the subapical region in Müller glial cells while CRB2 was observed at the subapical region in both photoreceptors and Müller glial cells by immuno-electron microscopy. CRB1 localization was lost in the Crb1 mutant rats, whereas CRB2 was still observed. In addition, we determined the tropism of subretinal or intravitreally administered AAV5-, AAV9- or AAV6-variant ShH10Y445F vectors in new-born control and Crb1 mutant rat retinas. We showed that subretinal injection of AAV5 and AAV9 at postnatal days 5 (P5) or 8 (P8) predominantly infected the retinal pigment epithelium (RPE) and photoreceptor cells; while intravitreal injection of ShH10Y445F at P5 or P8 resulted in efficient infection of mainly Müller glial cells. Using knowledge of the subcellular localization of CRB1 and the ability of ShH10Y445F to infect Müller glial cells, canonical hCRB1 and hCRB2 AAV-mediated gene therapy were explored in new-born Crb1 mutant rats. Enhanced retinal function after gene therapy delivery in the Crb1 rat was not observed. No timely rescue of the retinal phenotype was observed using retinal function and visual acuity, suggesting the need for earlier onset of expression of recombinant hCRB proteins in Müller glial cells to rescue the severe retinal phenotype in Crb1 mutant rats.