Evaluating therapeutic potential of NR2E3 doses in the rd7 mouse model of retinal degeneration.

Retinitis Pigmentosa is a leading cause of severe vision loss. Retinitis Pigmentosa can present with a broad range of phenotypes impacted by disease age of onset, severity, and progression. This variation is influenced both by different gene mutations as well as unique variants within the same gene. Mutations in the nuclear hormone receptor 2 family e, member 3 are associated with several forms of retinal degeneration, including Retinitis Pigmentosa. In our previous studies we demonstrated that subretinal administration of one Nr2e3 dose attenuated retinal degeneration in rd7 mice for at least 3 months. Here we expand the studies to evaluate the efficacy and longitudinal impact of the NR2E3 therapeutic by examining three different doses administered at early or intermediate stages of retinal degeneration in the rd7 mice. Our study revealed retinal morphology was significantly improved 6 months post for all doses in the early-stage treatment groups and for the low and mid doses in the intermediate stage treatment groups. Similarly, photoreceptor function was significantly improved in the early stage for all doses and intermediate stage low and mid dose groups 6 months post treatment. This study demonstrated efficacy in multiple doses of NR2E3 therapy.

Seeing the Future: A Review of Ocular Therapy.

Ocular diseases present a unique challenge and opportunity for therapeutic development. The eye has distinct advantages as a therapy target given its accessibility, compartmentalization, immune privilege, and size. Various methodologies for therapeutic delivery in ocular diseases are under investigation that impact long-term efficacy, toxicity, invasiveness, and delivery range. While gene, cell, and antibody therapy and nanoparticle delivery directly treat regions that have been damaged by disease, they can be limited in the duration of the therapeutic delivery and have a focal effect. In contrast, contact lenses and ocular implants can more effectively achieve sustained and widespread delivery of therapies; however, they can increase dilution of therapeutics, which may result in reduced effectiveness. Current therapies either offer a sustained release or a broad therapeutic effect, and future directions should aim toward achieving both. This review discusses current ocular therapy delivery systems and their applications, mechanisms for delivering therapeutic products to ocular tissues, advantages and challenges associated with each delivery system, current approved therapies, and clinical trials. Future directions for the improvement in existing ocular therapies include combination therapies, such as combined cell and gene therapies, as well as AI-driven devices, such as cortical implants that directly transmit visual information to the cortex.

Preclinical dose response study shows NR2E3 can attenuate retinal degeneration in the retinitis pigmentosa mouse model RhoP23H+/.

Retinitis pigmentosa (RP) is a heterogeneous disease and the main cause of vision loss within the group of inherited retinal diseases (IRDs). IRDs are a group of rare disorders caused by mutations in one or more of over 280 genes which ultimately result in blindness. Modifier genes play a key role in modulating disease phenotypes, and mutations in them can affect disease outcomes, rate of progression, and severity. Our previous studies have demonstrated that the nuclear hormone receptor 2 family e, member 3 (Nr2e3) gene reduced disease progression and loss of photoreceptor cell layers in RhoP23H-/- mice. This follow up, pharmacology study evaluates a longitudinal NR2E3 dose response in the clinically relevant heterozygous RhoP23H mouse. Reduced retinal degeneration and improved retinal morphology was observed 6 months following treatment evaluating three different NR2E3 doses. Histological and immunohistochemical analysis revealed regions of photoreceptor rescue in the treated retinas of RhoP23H+/- mice. Functional assessment by electroretinogram (ERG) showed attenuated photoreceptor degeneration with all doses. This study demonstrates the effectiveness of different doses of NR2E3 at reducing retinal degeneration and informs dose selection for clinical trials of RhoP23H-associated RP.

Alzheimer's Disease: Models and Molecular Mechanisms Informing Disease and Treatments.

Alzheimer's Disease (AD) is a complex neurodegenerative disease resulting in progressive loss of memory, language and motor abilities caused by cortical and hippocampal degeneration. This review captures the landscape of understanding of AD pathology, diagnostics, and current therapies. Two major mechanisms direct AD pathology: (1) accumulation of amyloid ß (Aß) plaque and (2) tau-derived neurofibrillary tangles (NFT). The most common variants in the Aß pathway in APP, PSEN1, and PSEN2 are largely responsible for early-onset AD (EOAD), while MAPT, APOE, TREM2 and ABCA7 have a modifying effect on late-onset AD (LOAD). More recent studies implicate chaperone proteins and Aß degrading proteins in AD. Several tests, such as cognitive function, brain imaging, and cerebral spinal fluid (CSF) and blood tests, are used for AD diagnosis. Additionally, several biomarkers seem to have a unique AD specific combination of expression and could potentially be used in improved, less invasive diagnostics. In addition to genetic perturbations, environmental influences, such as altered gut microbiome signatures, affect AD. Effective AD treatments have been challenging to develop. Currently, there are several FDA approved drugs (cholinesterase inhibitors, Aß-targeting antibodies and an NMDA antagonist) that could mitigate AD rate of decline and symptoms of distress.

Deletion of transcription factor AP-2ß from the developing murine trabecular meshwork region leads to progressive glaucomatous changes.

Glaucoma is one of the leading causes of irreversible blindness and can result from abnormalities in anterior segment structures required for aqueous humor outflow, including the trabecular meshwork (TM) and Schlemm's canal (SC). Transcription factors such as AP-2ß play critical roles in anterior segment development. Here, we show that the Mgp-Cre knock-in (Mgp-Cre.KI) mouse can be used to target the embryonic periocular mesenchyme giving rise to the TM and SC. Fate mapping of male and female mice indicates that AP-2ß loss causes a decrease in iridocorneal angle cells derived from Mgp-Cre.KI-expressing populations compared to controls. Moreover, histological analyses revealed peripheral iridocorneal adhesions in AP-2ß mutants that were accompanied by a decrease in expression of TM and SC markers, as observed using immunohistochemistry. In addition, rebound tonometry showed significantly higher intraocular pressure (IOP) that was correlated with a progressive significant loss of retinal ganglion cells, reduced retinal thickness, and reduced retinal function, as measured using an electroretinogram, in AP-2ß mutants compared with controls, reflecting pathology described in late-stage glaucoma patients. Importantly, elevated IOP in AP-2ß mutants was significantly reduced by treatment with latanoprost, a prostaglandin analog that increases unconventional outflow. These findings demonstrate that AP-2ß is critical for TM and SC development, and that these mutant mice can serve as a model for understanding and treating progressive human primary angle-closure glaucoma.

Progressive Loss of Retinal Ganglion Cells in Activating Protein-2ß Neural Crest Cell Knockout Mice.

Purpose: Our lab has shown that conditionally disrupting the transcription factor activating protein 2ß (Tfap2b) gene, responsible for the activating protein-2ß (AP-2ß) transcription factor, exclusively in cranial neural crest cells (AP-2ß NCC KO), leads to anterior segment dysgenesis and a closed angle phenotype. The purpose of the current study is to determine if there is a progressive loss of retinal ganglion cells (RGCs) in the mutant over time and whether this loss was associated with macroglial activity changes and elevated intraocular pressure (IOP).Methods: Using the Cre-loxP system, we generated a conditional knockout of Tfap2b exclusively in cranial NCC (AP-2ß NCC KO). Immunohistochemistry was performed using anti-Brn3a, anti-GFAP and anti-Vimentin antibodies. IOP was measured using a tonometer and the data was analyzed using GraphPad Prism software. Brn3a and DAPI positive cells were counted using Image-J and statistical analysis was performed with GraphPad Prism software.Results: Our findings revealed that while no statistical difference in Brn3a expression was observed between wild-type and mutant mice at postnatal day (P) 4 or P10, at P40 (p < .01) and P42 (p < .0001) Brn3a expression was significantly reduced in the mutant retina at the region of the ONH. There was also increased expression of glial fibrillary acidic protein (GFAP) by Müller cells in the AP-2ß NCC KO mice at P35 and P40, indicating the presence of neuroinflammation. Moreover, increased IOP was observed starting at P35 and continuing at P40 and P42 (p < .0001 for all three ages examined).Conclusions: Together, these findings suggest that the retinal damage observed in the KO mouse becomes apparent by P40 after increased IOP was observed at P35 and progressed over time. The AP-2ß NCC KO mouse may therefore be a novel experimental model for glaucoma.

Corneal development: Role of the periocular mesenchyme and bi-directional signaling.

The cornea is a highly specialized transparent tissue located at the anterior most surface of the eye. It consists of three main layers, the outer stratified squamous epithelium, the inner endothelium, and the intermediate stroma. Formation of these layers during development involves a complex interaction between ectodermal-derived structures, such as the overlying head ectoderm with the periocular mesenchyme (POM), the latter of which is comprised of neural crest cells (NCC) and mesoderm-derived progenitor cells. Regulation of corneal epithelial development, including both epithelial cell fate and stratification, has been shown to depend on numerous bi-directional mesenchymal-epithelial signaling pathways. In this review we pay particular attention to the genes and signaling pathways that involve the POM.

AP-2ß is required for formation of the murine trabecular meshwork and Schlemm's canal.

Previously, we have shown that Tfap2b, the gene encoding transcription factor AP-2ß, is needed for normal mouse eye development. Specifically, targeted loss of Tfap2b in neural crest cells (NCCs)1 and their derivatives, particularly the periocular mesenchyme (POM), resulted in anterior segment defects affecting the cornea and angle tissue. These defects were further associated with an increase in intraocular pressure (IOP). The present study investigates the underlying changes in embryonic and postnatal POM cell development and differentiation caused by loss of AP-2ß in the NCCs, particularly in the structures that control aqueous outflow, using Wnt1Cre+/-; Tfap2b-/lox; tdTomatolox/+ mice (AP-2ß neural crest cell knockout or AP-2ß NCC KO). Toluidine blue-stained sections and ultrathin sections stained with uranyl acetate and lead citrate were used to assess morphology and ultrastructure, respectively. Immunohistochemistry of KO and control eyes was performed at embryonic day (E) 15.5, E18.5, postnatal day (P) 1, P7 and P14 using phospho-histone H3 (PH3), α-smooth muscle actin (α-SMA), myocilin and endomucin antibodies, as well as a TUNEL assay. Conditional deletion of AP-2ß in the NCC-derived POM resulted in defects that appeared during both embryogenesis and postnatal stages. Fate mapping of the knockout cells in the mutants revealed that the POM migrated appropriately into the eye during embryogenesis. However, during postnatal stages a significant reduction in POM proliferation in the angle region was observed in the mutants compared to controls. This was accompanied by a lack of expression of appropriate trabecular meshwork and Schlemm's canal markers. This is the first study to show that AP-2ß is required for development and differentiation of the trabecular meshwork and Schlemm's canal. Together, these defects likely contributed to the elevated intraocular pressure (IOP) previously reported in the AP-2ß NCC KO mice.

Relationship between neural crest cell specification and rare ocular diseases.

Development of the eye is closely associated with neural crest cell migration and specification. Eye development is extremely complex, as it requires the working of a combination of local factors, receptors, inductors, and signaling interactions between tissues such as the optic cup and periocular mesenchyme (POM). The POM is comprised of neural crest-derived mesenchymal progenitor cells that give rise to numerous important ocular structures including those tissues that form the optic cup and anterior segment of the eye. A number of genes are involved in the migration and specification of the POM such as PITX2, PITX3, FOXC1, FOXE3, PAX6, LMX1B, GPR48, TFAP2A, and TFAP2B. In this review, we will discuss the relevance of these genes in the development of the POM and how mutations and defects result in rare ocular diseases.

Conditional deletion of AP-2ß in mouse cranial neural crest results in anterior segment dysgenesis and early-onset glaucoma.

Anterior segment dysgenesis (ASD) encompasses a group of developmental disorders in which a closed angle phenotype in the anterior chamber of the eye can occur and 50% of patients develop glaucoma. Many ASDs are thought to involve an inappropriate patterning and migration of the periocular mesenchyme (POM), which is derived from cranial neural crest cells (NCCs) and mesoderm. Although, the mechanism of this disruption is not well understood, a number of transcriptional regulatory molecules have previously been implicated in ASDs. Here, we investigate the function of the transcription factor AP-2ß, encoded by Tfap2b, which is expressed in NCCs and their derivatives. Wnt1-Cre-mediated conditional deletion of Tfap2b in NCCs resulted in post-natal ocular defects typified by opacity. Histological data revealed that the conditional AP-2ß NCC knockout (KO) mutants exhibited dysgenesis of multiple structures in the anterior segment of the eye including defects in the corneal endothelium, corneal stroma, ciliary body and disruption in the iridocorneal angle with adherence of the iris to the cornea. We further show that this phenotype leads to a significant increase in intraocular pressure and a subsequent loss of retinal ganglion cells and optic nerve degeneration, features indicative of glaucoma. Overall, our findings demonstrate that AP-2ß is required in the POM for normal development of the anterior segment of the eye and that the AP-2ß NCC KO mice might serve as a new and exciting model of ASD and glaucoma that is fully penetrant and with early post-natal onset.