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

Abstract

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.

Keywords: Epigenetics; Glaucoma; HDAC3; Optic nerve; RGFP966; Retinal ganglion cell.

Figure 1.

Conditional knockout of Hdac3 in N6 DBA/2J mice does not confer protection against RGC cell loss. (A.) N6 DBA/2J-Hdac3^fl/fl (Hdac3 cKO) and N6 DBA/2J-Hdac3^+/+ mice develop spontaneous ocular hypertension with age. A control cohort of naïve N6 DBA/2J-Hdac3^+/+ (Control) mice were assessed at 4 months of age for intraocular pressure (IOP), and no significant difference was observed between this group and 6 month old WT and Hdac3 cKO treatment groups. Hdac3^+/+ and Hdac3^cKO DBA/2J mice exhibited similar increases in mean IOPs at 10 months of age (p=0.1334), which were significantly higher than 6-month cohorts (****p<0.00001). Box plots indicate the minimum, maximum, mean, and upper and lower quartiles of the data presented. 4 month controls: n=36 eyes, Hdac3^+/+: n=38 eyes, Hdac3^cKO: n=44 eyes. Conditional knockout of Hdac3 in the ganglion cell layer of N6 DBA/2J Gpnmb^−/− and Tyrp1^−/− mice does not protect against cell loss. (B.-D.) Fluorescence images of whole mounted (B.) 4 month Hdac3^+/+ control, (C.) 10 month Hdac3^+/+, and (D.) 10 month Hdac3 cKO retinas labeled with RGC marker BRN3A (green) and nuclear label DAPI (blue). Scale bar=20 μm. (E.) 10 month Hdac3^+/+ and Hdac3^cKO retinas had significantly fewer BRN3A positive cells per field (****p<0.00001) in comparison to 4 month controls. (F.) Additionally, 10 month Hdac3^+/+ and Hdac3^cKO retinas had significantly fewer DAPI positive cells per field (****p<0.00001) in comparison to 4 month controls. Bar graphs indicate mean +/− SEM. Bonferroni correction for significance was set to p=7.92×10⁻⁵ (α=0.05). For BRN3A positive cells/field, 4 month controls: n= 414 fields (20 eyes), 10 month Hdac3^+/+: n= 227 fields (11 eyes), 10 month Hdac3^cKO: n=290 fields (16 eyes). For DAPI positive cells/field, 4 month Hdac3^+/+ controls: n= 310 fields (18 eyes), Hdac3^+/+: n= 324 fields (15 eyes), Hdac3^cKO: n= 618 fields (28 eyes). Bonferroni correction: p=0.00007 (alpha=0.05). (G.-J.) Conditional knockout of Hdac3 in the ganglion cell layer of N6 DBA/2J Gpnmb^−/− and Tyrp1^−/− mice does not prevent optic nerve degeneration. Optic nerve cross-sections were stained for phosphorylated neurofilament (pNEFL) (green) and DAPI (blue), imaged using consistent exposure time across the cohort, and scored for level of degeneration by 3 masked observers. Scores were then averaged to give consensus scores. (G.-I.) Images that had consistent pNEFL staining (green) throughout the nerve were considered mildly degenerated or healthy (G.), images with regions of reduced or no staining were considered moderately degenerated (H.), and images with a predominant lack of staining were considered severely degenerated (I.). (J.) Optic nerve scoring indicated a significant increase in degeneration in both Hdac3^+/+ (n=33 optic nerves) (χ² value=23.32) and Hdac3 cKO (n=37 optic nerves) (χ² value=23.33) optic nerves after 10 months of elevated IOP (****p<0.00001) in comparison to 4 month controls (n=35 optic nerves). (Scale bar=100μm)

Figure 2.

Inhibition of HDAC3 in DBA/2J.BALB^Rgcs1 mice offers mild protection against somatic cell loss, but not BRN3A expression or optic nerve degeneration. (A.) DBA/2J.BALB^Rgcs1 mice develop spontaneous elevated IOP. DBA/2J.BALB^Rgcs1 mice were age-matched and assigned to either receiving vehicle or 2mg/kg RGFP966 IP injection every 3 days from 6 to 10 months of age. IOPs were recorded in a 4-month old DBA/2J cohort, and the experimental mice before and after their treatment period from 6–10 months of age showing an age-related increase in pressure in the RGFP966 treated group (****p<0.00001). There was no significant difference in IOP between vehicle- and RGFP966-treated mice at 10 months of age (p=0.2213). Box plots indicate the minimum, maximum, mean, and upper and lower quartiles of the data presented. 4 month controls: n=8 eyes, vehicle: n=20 eyes, RGFP966: n=22 eyes. (B.-F.) Systemic injection of 2mg/kg RGFP966 every 3 days protects against total cell loss in the DBA/2J.BALB^Rgcs1 mouse model of glaucoma. DBA/2J.BALB^Rgcs1 mice were age-matched and assigned to either receiving vehicle or 2mg/kg RGFP966 IP injection every 3 days from 6 to 10 months of age. At 10 months of age, animals were euthanized and retinal whole mounts were analyzed for RGC loss. (B.-D.) Whole mounted retinas were stained for nuclear BRN3A (green) and DAPI (blue). Scale bar=20 μm. (E., F.) Cell counts indicated that animals treated with either vehicle or RGFP966 had significantly fewer BRN3A and DAPI labeled cells per field in comparison to 4-month control animals (****p<0.00001). At 10 months of age, RGFP966 treated mice did not have significantly more BRN3A positive cells present in comparison to vehicle treated mice. However, RGFP966 treated mice did have significantly more DAPI labeled cells present in comparison to vehicle treated mice (****p<0.00001) Bar graphs indicate mean +/− SEM. For BRN3A positive cells/field, 4 month controls: n=138 fields (6 eyes), 10 month vehicle treated: n= 397 fields (17 eyes), 10 month RGFP966 treated: n= 504 fields (21 eyes). For DAPI positive cells/field, 4 month controls: n=186 fields (8 eyes), 10 month vehicle treated: n=371 fields (16 eyes), 10 month RGFP966 treated: n= 512 fields (22 eyes) Bonferroni correction: p=0.0001 (alpha=0.05). (G.-J.) Systemic injection of 2mg/kg RGFP966 every 3 days does not prevent optic nerve degeneration in the DBA/2J.BALB^Rgcs1 mouse model of glaucoma. (G.-I.) Masked optic nerve scoring was done, and optic nerves were scored based on phosphorylated neurofilament (P-NEFL) immunofluorescence: (G.) mild degeneration, (H.) moderate degeneration, and (I.) severe degeneration. (J.) Analysis of optic nerve scoring indicated that 4-month old control optic nerves (n=8 optic nerves) were significantly healthier when compared to vehicle (n=19 optic nerves) (****p<0.00001, χ² value=89.11) and RGFP966 treatment (n=20 optic nerves) (****p<0.00001, χ² value=78.67) optic nerves at 10 months of age. RGFP966 treated mice had significantly more degenerated optic nerves when compared to vehicle treated mice (***p<0.001, χ² value=13.96) (Scale bar=100μm).