Genetic background determines severity of Loxl1-mediated systemic and ocular elastosis in mice.

Pseudoexfoliation syndrome (PEX) is a systemic, age-related disorder characterized by elastosis and extracellular matrix deposits. Its most significant ocular manifestation is an aggressive form of glaucoma associated with variants in the gene encoding lysyl oxidase-like 1 (LOXL1). Depending upon the population, variants in LOXL1 can impart risk or protection for PEX, suggesting the importance of genetic context. As LOXL1 protein levels are lower and the degree of elastosis is higher in people with PEX, we studied Loxl1-deficient mice on three different genetic backgrounds: C57BL/6 (BL/6), 129S×C57BL/6 (50/50) and 129S. Early onset and high prevalence of spontaneous pelvic organ prolapse in BL/6 Loxl1-/- mice necessitated the study of mice that were <2 months old. Similar to pelvic organ prolapse, most elastosis endpoints were the most severe in BL/6 Loxl1-/- mice, including skin laxity, pulmonary tropoelastin accumulation, expansion of Schlemm's canal and dilation of intrascleral veins. Interestingly, intraocular pressure was elevated in 50/50 Loxl1-/- mice, depressed in BL/6 Loxl1-/- mice and unchanged in 129S Loxl1-/- mice compared to that of control littermates. Overall, the 129S background was protective against most elastosis phenotypes studied. Thus, repair of elastin-containing tissues is impacted by the abundance of LOXL1 and genetic context in young animals.

Lysyl oxidase-like 1-antisense 1 (LOXL1-AS1) lncRNA differentially regulates gene and protein expression, signaling and morphology of human ocular cells.

Pseudoexfoliation glaucoma (PEXG) is characterized by dysregulated extracellular matrix (ECM) homeostasis that disrupts conventional outflow function and increases intraocular pressure (IOP). Prolonged IOP elevation results in optic nerve head damage and vision loss. Uniquely, PEXG is a form of open angle glaucoma that has variable penetrance, is difficult to treat and does not respond well to common IOP-lowering pharmaceuticals. Therefore, understanding modulators of disease severity will aid in targeted therapies for PEXG. Genome-wide association studies have identified polymorphisms in the long non-coding RNA lysyl oxidase-like 1-antisense 1 (LOXL1-AS1) as a risk factor for PEXG. Risk alleles, oxidative stress and mechanical stretch all alter LOXL1-AS1 expression. As a long non-coding RNA, LOXL1-AS1 binds hnRNPL and regulates global gene expression. In this study, we focus on the role of LOXL1-AS1 in the ocular cells (trabecular meshwork and Schlemm's canal) that regulate IOP. We show that selective knockdown of LOXL1-AS1 leads to cell-type-specific changes in gene expression, ECM homeostasis, signaling and morphology. These results implicate LOXL1-AS1 as a modulator of cellular homeostasis, altering cell contractility and ECM turnover, both of which are well-known contributors to PEXG. These findings support LOXL1-AS1 as a key target for modifying the disease.

Increased Susceptibility and Intrinsic Apoptotic Signaling in Neurons by Induced HDAC3 Expression.

Purpose: Inhibition or targeted deletion of histone deacetylase 3 (HDAC3) is neuroprotective in a variety neurodegenerative conditions, including retinal ganglion cells (RGCs) after acute optic nerve damage. Consistent with this, induced HDAC3 expression in cultured cells shows selective toxicity to neurons. Despite an established role for HDAC3 in neuronal pathology, little is known regarding the mechanism of this pathology. Methods: Induced expression of an HDAC3-mCherry fusion protein in mouse RGCs was accomplished by transduction with AAV2/2-Pgk-HDAC3-mCherry. Increased susceptibility to optic nerve damage in HDAC3-mCherry expressing RGCs was evaluated in transduced mice that received acute optic nerve crush surgery. Expression of HDAC3-FLAG or HDAC3-mCherry was induced by nucleofection or transfection of plasmids into differentiated or undifferentiated 661W tissue culture cells. Immunostaining for cleaved caspase 3, localization of a GFP-BAX fusion protein, and quantitative RT-PCR was used to evaluate HDAC3-induced damage. Results: Induced expression of exogenous HDAC3 in RGCs by viral-mediated gene transfer resulted in modest levels of cell death but significantly increased the sensitivity of these neurons to axonal damage. Undifferentiated 661W retinal precursor cells were resilient to induced HDAC3 expression, but after differentiation, HDAC3 induced GFP-BAX recruitment to the mitochondria and BAX/BAK dependent activation of caspase 3. This was accompanied by an increase in accumulation of transcripts for the JNK2/3 kinases and the p53-regulated BH3-only gene Bbc3/Puma. Cell cycle arrest of undifferentiated 661W cells did not increase their sensitivity to HDAC3 expression. Conclusions: Collectively, these results indicate that HDAC3-induced toxicity to neurons is mediated by the intrinsic apoptotic pathway.

Targeting HDAC3 in the DBA/2J spontaneous mouse model of glaucoma.

High intraocular pressure (IOP) is the most common risk factor associated with glaucoma in humans. While lowering IOP is effective at reducing the rate of retinal ganglion cell (RGC) loss, to date, no treatment exists to directly preserve these cells affected by damage to the optic nerve. Recently, histone deacetylase-3 (HDAC3) has become a potential therapeutic target because it plays an important role in the early nuclear atrophic events that precede RGC death. Conditional knockout or inhibition of HDAC3 prevents histone deacetylation, heterochromatin formation, apoptosis, and eventual RGC loss following acute optic nerve injury. Using these approaches to repress HDAC3 activity, we tested whether targeting HDAC3 protects RGCs from ganglion cell-specific BRN3A expression loss, total somatic cell loss, and optic nerve degeneration in the DBA/2J mouse model of spontaneous glaucoma. Targeted ablation of Hdac3 activity did not protect RGCs from axonal degeneration or somatic cell death in the DBA/2J mouse model of glaucoma. However, inhibition of HDAC3 activity using RGFP966 conferred mild protection against somatic cell loss in the ganglion cell layer in aged DBA/2J mice. Further experimentation is necessary to determine whether other class I HDACs may serve as potential therapeutic targets in chronic models of glaucoma.

Identification and activity of the functional complex between hnRNPL and the pseudoexfoliation syndrome-associated lncRNA, LOXL1-AS1.

Individuals with pseudoexfoliation (PEX) syndrome exhibit various connective tissue pathologies associated with dysregulated extracellular matrix homeostasis. PEX glaucoma is a common, aggressive form of open-angle glaucoma resulting from the deposition of fibrillary material in the conventional outflow pathway. However, the molecular mechanisms that drive pathogenesis and genetic risk remain poorly understood. PEX glaucoma-associated single-nucleotide polymorphisms are located in and affect activity of the promoter of LOXL1-AS1, a long non-coding RNA (lncRNA). Nuclear and non-nuclear lncRNAs regulate a host of biological processes, and when dysregulated, contribute to disease. Here we report that LOXL1-AS1 localizes to the nucleus where it selectively binds to the mRNA processing protein, heterogeneous nuclear ribonucleoprotein-L (hnRNPL). Both components of this complex are critical for the regulation of global gene expression in ocular cells, making LOXL1-AS1 a prime target for investigation in PEX syndrome and glaucoma.

Targeting HDAC3 Activity with RGFP966 Protects Against Retinal Ganglion Cell Nuclear Atrophy and Apoptosis After Optic Nerve Injury.

PURPOSE: HDAC3 regulates nuclear atrophy as an early response to axonal injury in retinal ganglion cells (RGCs) following optic nerve crush (ONC). Since conditional knockout of Hdac3 prevents nuclear atrophy post ONC, HDAC3 selective inhibition with RGFP966 through localized and systemic dosing of RGFP966 is necessary for application to acute and chronic models of optic nerve injury. METHODS: C57BL/6 mice were injected intravitreally with 1-10 µM RGFP966 immediately following ONC, and retinas were analyzed at 5, 7, and 14 days for metrics of nuclear atrophy and cell loss. Mice were similarly assessed after intraperitoneal (IP) injections with RGFP966 doses of 2-10 mg/kg, and eyes were harvested at 5, 14, and 28 days after ONC. H&E and BrdU staining were used to analyze toxicity to off-target tissues after 14 days of daily treatment with RGFP966. RESULTS: A single intravitreal injection of RGFP966 prevented histone deacetylation, heterochromatin formation, apoptosis, and DNA damage at 5 and 7 days post ONC. After IP injection, RGFP966 bioavailability in the retina reached peak concentration within 1 h after injection and then rapidly declined. A single IP injection of 2-10 mg/kg RGFP966, significantly prevented histone deacetylation. Repeated IP injections of 2 mg/kg RGFP966 over the course of 2 and 4 weeks post ONC prevented RGC loss. There were no significant toxic or antiproliferative effects to off-target tissues in mice treated daily for 14 days with RGFP966. CONCLUSION: Inhibition of HDAC3 activity with systemic dosing of RGFP966 prevents apoptosis-related histone deacetylation and attenuates RGC loss after acute optic nerve injury.

AAV2-Mediated Transduction of the Mouse Retina After Optic Nerve Injury.

Purpose: Gene therapy of retinal ganglion cells (RGCs) has promise as a powerful therapeutic for the rescue and regeneration of these cells after optic nerve damage. However, early after damage, RGCs undergo atrophic changes, including gene silencing. It is not known if these changes will deleteriously affect transduction and transgene expression, or if the therapeutic protein can influence reactivation of the endogenous genome. Methods: Double-transgenic mice carrying a Rosa26-(LoxP)-tdTomato reporter, and a mutant allele for the proapoptotic Bax gene were reared. The Bax mutant blocks apoptosis, but RGCs still exhibit nuclear atrophy and gene silencing. At times ranging from 1 hour to 4 weeks after optic nerve crush (ONC), eyes received an intravitreal injection of AAV2 virus carrying the Cre recombinase. Successful transduction was monitored by expression of the tdTomato reporter. Immunostaining was used to localize tdTomato expression in select cell types. Results: Successful transduction of RGCs was achieved at all time points after ONC using AAV2 expressing Cre from the phosphoglycerate kinase (Pgk) promoter, but not the CMV promoter. ONC promoted an increase in the transduction of cell types in the inner nuclear layer, including Müller cells and rod bipolar neurons. There was minimal evidence of transduction of amacrine cells and astrocytes in the inner retina or optic nerve. Conclusions: Damaged RGCs can be transduced and at least some endogenous genes can be subsequently activated. Optic nerve damage may change retinal architecture to allow greater penetration of an AAV2 virus to transduce several additional cell types in the inner nuclear layer.

Role of HDACs in optic nerve damage-induced nuclear atrophy of retinal ganglion cells.

Optic neuropathies are characterized by retinal ganglion cell (RGC) death, resulting in the loss of vision. In glaucoma, the most common optic neuropathy, RGC death is initiated by axonal damage, and can be modeled by inducing acute axonal trauma through procedures such as optic nerve crush (ONC) or optic nerve axotomy. One of the early events of RGC death is nuclear atrophy, and is comprised of RGC-specific gene silencing, histone deacetylation, heterochromatin formation, and nuclear shrinkage. These early events appear to be principally regulated by epigenetic mechanisms involving histone deacetylation. Class I histone deacetylases HDACs 1, 2, and 3 are known to play important roles in the process of early nuclear atrophy in RGCs, and studies using both inhibitors and genetic ablation of Hdacs also reveal a critical role in the cell death process. Select inhibitors, such as those being developed for cancer therapy, may also provide a viable secondary treatment option for optic neuropathies.

Histone deacetylase 3 (HDAC3) plays an important role in retinal ganglion cell death after acute optic nerve injury.

BACKGROUND: Optic nerve damage initiates a series of early atrophic events in retinal ganglion cells (RGCs) that precede the BAX-dependent committed step of the intrinsic apoptotic program. Nuclear atrophy, including global histone deacetylation, heterochromatin formation, shrinkage and collapse of nuclear structure, and the silencing of normal gene expression, comprise an important obstacle to overcome in therapeutic approaches to preserve neuronal function. Several studies have implicated histone deacetylases (HDACs) in the early stages of neuronal cell death, including RGCs. Importantly, these neurons exhibit nuclear translocation of HDAC3 shortly after optic nerve damage. Additionally, HDAC3 activity has been reported to be selectively toxic to neurons. RESULTS: RGC-specific conditional knockout of Hdac3 was achieved by transducing the RGCs of Hdac3fl/fl mice with an adeno-associated virus serotype 2 carrying CRE recombinase and GFP (AAV2-Cre/GFP). Controls included similar viral transduction of Rosa26fl/fl reporter mice. Optic nerve crush (ONC) was then performed on eyes. The ablation of Hdac3 in RGCs resulted in significant amelioration of characteristics of ONC-induced nuclear atrophy such as H4 deacetylation, heterochromatin formation, and the loss of nuclear structure. RGC death was also significantly reduced. Interestingly, loss of Hdac3 expression did not lead to protection against RGC-specific gene silencing after ONC, although this effect was achieved using the broad spectrum inhibitor, Trichostatin A. CONCLUSION: Although other HDACs may be responsible for gene expression changes in RGCs, our results indicate a critical role for HDAC3 in nuclear atrophy in RGC apoptosis following axonal injury. This study provides a framework for studying the roles of other prevalent retinal HDACs in neuronal death as a result of axonal injury.