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Eye size is a key parameter of visual function, but the precise mechanisms of eye size control remain poorly understood. Here, we discovered that the lipogenic transcription factor sterol regulatory element-binding protein 2 (SREBP2) has an unanticipated function in the retinal pigment epithelium (RPE) to promote eye size in postnatal mice. SREBP2 transcriptionally represses low density lipoprotein receptor-related protein 2 (Lrp2), which has been shown to restrict eye overgrowth. Bone morphogenetic protein 2 (BMP2) is the downstream effector of Srebp2 and Lrp2, and Bmp2 is suppressed by SREBP2 transcriptionally but activated by Lrp2. During postnatal development, SREBP2 protein expression in the RPE decreases whereas that of Lrp2 and Bmp2 increases as the eye growth rate reduces. Bmp2 is the key determinant of eye size such that its level in mouse RPE inversely correlates with eye size. Notably, RPE-specific Bmp2 overexpression by adeno-associated virus effectively prevents the phenotypes caused by Lrp2 knock out. Together, our study shows that rapid postnatal eye size increase is governed by an RPE-derived signaling pathway, which consists of both positive and negative regulators of eye growth.

Adeno-associated viral vectors (AAVs) have become popular for gene therapy, given their many advantages, including their reduced inflammatory profile compared with that of other viruses. However, even in areas of immune privilege such as the eye, AAV vectors are capable of eliciting host-cell responses. To investigate the effects of such responses on several ocular cell types, we tested multiple AAV genome structures and capsid types using subretinal injections in mice. Assays of morphology, inflammation, and physiology were performed. Pathological effects on photoreceptors and the retinal pigment epithelium (RPE) were observed. Muller glia and microglia were activated, and the proinflammatory cytokines TNF-α and IL-1β were up-regulated. There was a strong correlation between cis-regulatory sequences and toxicity. AAVs with any one of three broadly active promoters, or an RPE-specific promoter, were toxic, while AAVs with four different photoreceptor-specific promoters were not toxic at the highest doses tested. There was little correlation between toxicity and transgene, capsid type, preparation method, or cellular contaminants within a preparation. The toxic effect was dose-dependent, with the RPE being more sensitive than photoreceptors. Our results suggest that ocular AAV toxicity is associated with certain AAV cis-regulatory sequences and/or their activity and that retinal damage occurs due to responses by the RPE and/or microglia. By applying multiple, sensitive assays of toxicity, AAV vectors can be designed so that they can be used safely at high dose, potentially providing greater therapeutic efficacy.

Haploinsufficiency of progranulin (PGRN) is a leading cause of frontotemporal lobar degeneration (FTLD). Loss of PGRN leads to lysosome dysfunction during aging. TMEM106B, a gene encoding a lysosomal membrane protein, is the main risk factor for FTLD with PGRN haploinsufficiency. But how TMEM106B affects FTLD disease progression remains to be determined. Here, we report that TMEM106B deficiency in mice leads to accumulation of lysosome vacuoles at the distal end of the axon initial segment in motor neurons and the development of FTLD‐related pathology during aging. Ablation of both PGRN and TMEM106B in mice results in severe neuronal loss and glial activation in the spinal cord, retina, and brain. Enlarged lysosomes are frequently found in both microglia and astrocytes. Loss of both PGRN and TMEM106B results in an increased accumulation of lysosomal vacuoles in the axon initial segment of motor neurons and enhances the manifestation of FTLD phenotypes with a much earlier onset. These results provide novel insights into the role of TMEM106B in the lysosome, in brain aging, and in FTLD pathogenesis., Loss of the FTLD risk factor TMEM106B causes lysosome vacuolization in motor neuron axons and FTLD‐related phenotypes in mice. Loss of both TMEM106B and the FTLD protein PGRN causes severe autophagy‐lysosome defects, glial activation, neuronal loss and early death in mice.

Significance Adeno-associated viral (AAV) gene therapy is becoming an important therapeutic modality, especially for ocular diseases, due to its efficiency of gene delivery and relative lack of pathogenicity. However, AAV sometimes can cause inflammation and toxicity. We explored such effects using injections into the mouse eye. We found a strong correlation of toxicity and inflammation with the use of promoters that were broadly active, or specifically active in the retinal pigment epithelium. AAVs with photoreceptor-specific promoters were found to be nontoxic at all doses tested. These studies reveal that safer vectors can be designed if assays for relevant and specific cell types are developed and tested with a range of vectors with different genomic elements., Adeno-associated viral vectors (AAVs) have become popular for gene therapy, given their many advantages, including their reduced inflammatory profile compared with that of other viruses. However, even in areas of immune privilege such as the eye, AAV vectors are capable of eliciting host-cell responses. To investigate the effects of such responses on several ocular cell types, we tested multiple AAV genome structures and capsid types using subretinal injections in mice. Assays of morphology, inflammation, and physiology were performed. Pathological effects on photoreceptors and the retinal pigment epithelium (RPE) were observed. Müller glia and microglia were activated, and the proinflammatory cytokines TNF-α and IL-1β were up-regulated. There was a strong correlation between cis-regulatory sequences and toxicity. AAVs with any one of three broadly active promoters, or an RPE-specific promoter, were toxic, while AAVs with four different photoreceptor-specific promoters were not toxic at the highest doses tested. There was little correlation between toxicity and transgene, capsid type, preparation method, or cellular contaminants within a preparation. The toxic effect was dose-dependent, with the RPE being more sensitive than photoreceptors. Our results suggest that ocular AAV toxicity is associated with certain AAV cis-regulatory sequences and/or their activity and that retinal damage occurs due to responses by the RPE and/or microglia. By applying multiple, sensitive assays of toxicity, AAV vectors can be designed so that they can be used safely at high dose, potentially providing greater therapeutic efficacy.

To investigate the in vitro and in vivo release characteristics of Tacrolimus (FK506) from an episcleral drug-delivery implant.For in vitro experiments, Tacrolimus-loaded implants (0.5 mL; at concentrations of 0.25, 0.5, and 1.0 mg/mL) were immersed in a balanced salt solution. Samples of the surrounding liquid were aspirated at different times over a 96-h period. For in vivo experiments, the experimental group received an implant loaded with Tacrolimus (0.5 mg/mL; 0.5 mL); the control group was given a subconjunctival injection of 0.5 mL Tacrolimus (0.5 mg/mL). On postoperative days 3, 7, 14, 28, and 56, 3 animals were sacrificed, and their eyes were enucleated. Tacrolimus concentrations were determined by liquid chromatographic-tandem mass spectrometry. Ocular toxicity was evaluated by slit-lamp photography, fundus photography, intraocular pressure (IOP), and histology.The implants released Tacrolimus in a biphasic pattern for 96 h in the in vitro study. The release kinetics were not dependent on the drug concentrations. The in vivo study showed statistically significant differences between the 2 treatment groups. Tacrolimus levels were particularly high in the conjunctiva, iris, ciliary body, cornea, sclera, choroid, and retina in the experimental group, while concentrations were low and only lasted for 1 week in the controls. Slit-lamp photography, fundus photography, IOP, and histology showed no evidence of toxic effects.The episcleral drug-delivery implant mechanically released Tacrolimus through the apertures of capsules and, consequently, may be a promising drug vehicle for the treatment of immune-mediated ocular disorders.