GSK3 inhibition reduces ECM production and prevents age-related macular degeneration-like pathology

Abstract

Malattia Leventinese/Doyne Honeycomb Retinal Dystrophy (ML/DHRD) is an age-related macular degeneration (AMD)-like retinal dystrophy caused by an autosomal dominant R345W mutation in the secreted glycoprotein, fibulin-3 (F3). To identify new small molecules that reduce F3 production from retinal pigmented epithelium (RPE) cells, we knocked-in a luminescent peptide tag (HiBiT) into the endogenous F3 locus which enabled simple, sensitive, and high throughput detection of the protein. The GSK3 inhibitor, CHIR99021 (CHIR), significantly reduced F3 burden (expression, secretion, and intracellular levels) in immortalized RPE and non-RPE cells. Low-level, long-term CHIR treatment promoted remodeling of the RPE extracellular matrix (ECM), reducing sub-RPE deposit-associated proteins (e.g., amelotin, complement component 3, collagen IV, and fibronectin), while increasing RPE differentiation factors (e.g., tyrosinase, and pigment epithelium derived factor). In vivo, treatment of 8 mo R345W^+/+ knockin mice with CHIR (25 mg/kg i.p., 1 mo) was well tolerated and significantly reduced R345W F3-associated AMD-like basal laminar deposit number and size, thereby preventing the main pathological feature in these mice. This is the first demonstration of small molecule-based prevention of AMD-like pathology in ML/DHRD mice and may herald a rejuvenation of interest in GSK3 inhibition for the treatment of neurodegenerative diseases, including, potentially AMD itself.

Keywords: AMD; CHIR99021; DHRD; EFEMP1; F3; ML; fibulin-3; glycogen synthase kinase 3.

Figure 1:. Design and validation of fibulin-3 (F3) HiBiT tagging in ARPE-19 cells using CRISPR.

(A) Schematic of CRISPR editing of exon 2 of the F3 gene to knockin a 2xFLAG-VS-HiBiT sequence immediately proceeding the signal sequence cleavage site (upwards arrow). (B) Successful editing was verified by gDNA amplification of exon 2. An additional band corresponding to insertion of the 87 bp 2x FLAG VS HiBiT tag was identified with a calculated editing efficiency of ~15%. (C) F3 HiBiT tagging results in a single extracellular protein species of correct molecular weight (~55 kDa) as identified by immunoprecipitation (FLAG beads) followed by elution and HiBiT blotting. (D) siRNA verifies that >95% of the HiBiT signal can be attributed to F3 gene translation. n = 3 independent experiments performed in biological triplicates, **** p ≤ 0.0001, t-test vs. non-targeting siRNA.

Figure 2:. Structurally diverse glycogen synthase kinase 3 (GSK3) inhibitors reduce F3 transcripts and extracellular/intracellular levels without causing toxicity.

(A, B) A series of chemically unrelated GSK3 inhibitors significantly reduce (A) extracellular and (B) intracellular HiBiT F3 levels after 72 h of treatment. n = 3 independent experiments, mean of each plotted here, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, one sample t-test vs. hypothetical unchanged value of 1 (vehicle-treated). (C) CHIR99021-dependent HiBiT assay results in (A) were confirmed at the protein level by HiBiT blotting. Representative image of n ≥ 3 independent experiments. (D) Seventy-two hour CHIR99021 treatment reduces F3 mRNA expression. Representative data of n = 3 independent experiments, average ± S.D. of technical triplicates. (E) Treatment with CHIR does not elevate release of cytosolic lactate dehydrogenase (LDH). n = 3 independent experiments performed in biological triplicate, ns = not significant, t-test vs. vehicle-treated levels.

Figure 3:. Genetic knockdown of GSK3 isoforms also reduces HiBiT F3 levels, paralleling pharmacologic GSK3 inhibitor effects.

(A) Knockdown (96 h) of either GSK3α or GSK3β significantly lowers F3 production in ARPE-19 cells and the effects of knocking down both α and β isoforms are additive. n = 3 independent experiments performed in biological triplicate, *** p ≤ 0.001, **** p ≤ 0.0001, t-test vs. either vehicle-treated levels or single isoform knockdowns as indicated. (B) Protein-level knockdown effects were verified by western and HiBiT blotting. Representative images of n = 3 independent experiments.

Figure 4:. Low level, prolonged CHIR99021 treatment reduces F3 production while avoiding triggering TCF4-dependent Wnt activation.

(A) Seventy-two-hour CHIR99021 treatment dose-dependently reduces HiBiT F3 extracellular levels without necessarily triggering TCF4-dependent green fluorescent protein (GFP) expression (B). n = 3 independent experiments performed in at least biological triplicate, with representative data presented. *** p ≤ 0.001, t-test vs. vehicle treatment. Scale bar ~ 200 μm (C) One-week treatment with CHIR99021 (1 μM) significantly lowers HiBiT F3 extracellular levels without activating TCF4-dependent firefly luciferase (FLuc). n = 3 independent experiments performed in biological replicates. * p ≤ 0.05, *** p ≤ 0.001, t-test vs. vehicle treatment. (E) Prolonged low level CHIR99021 does not increase cell death as indicated by LDH release. n = 3 independent experiments performed in biological replicates. ns = not significant, t-test vs. vehicle treatment.

Figure 5:. Low level, one-week CHIR99021 treatment reduces extracellular matrix (ECM) and collagen-associated transcripts while upregulating RPE differentiation-associated genes.

(A) Bulk RNAseq analysis of ARPE-19 cells treated with CHIR99021 indicates significant transcript reduction (≥ 2-fold) of genes implicated in ECM formation and sub-RPE deposit formation while upregulating (≥ 2-fold) RPE differentiation genes. n = 3 independent experiments combined to produce these data, * padj < 0.05 using a negative binomial distribution model. (B) Pathway enrichment of significantly altered genes from RNAseq data set using Gene Ontology enrichment analysis.

Figure 6:. CHIR99021 reduces R345W F3-associated matrix metalloproteinase 2 (MMP2) alterations in culture.

(A) ARPE-19 cells expressing HiBiT R345W F3 demonstrate significantly elevated apical and basal MMP2 activity levels compared to HiBiT WT F3 cells after one week on transwells. n = 3 independent experiments, performed in sextuplet biological replicates. **** p ≤ 0.0001, t-test vs. WT cells. (B) Treatment (one-week, 1 μM) with CHIR99021 significantly decreases HiBiT WT and R345W F3 both apically and basally in transwell format (C) while simultaneously significantly decreasing MMP2 activity. n = 3 independent experiments, performed in triplicate biological replicates. ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, t-test vs. each respective vehicle-treated control.

Figure 7:. One-month CHIR99021 treatment does not affect retinal function or gross structure.

(A,B) Eight-month-old R345W^+/+ C57BL/6 mice were injected intraperitonially (i.p.) with vehicle (PBS) or CHIR99021 for one month (every weekday). Scotopic electroretinogram (ERG) readings demonstrated no difference between groups in either a-wave (outer retina, A) or b-wave (inner retina, B), values not significant by an ANOVA test. (C) After completion of ERG evaluation, mice were sacrificed and their eye prepared for H&E histology, which demonstrate no observable differences between the two groups. n = 4 mice/treatment group, 2 male, 2 female. Scale bar = 2 μm.

Figure 8:. CHIR99021 significantly reduces the formation of basal laminar deposits (BLamD) in vivo.

(A, B) After one month of vehicle (A) or CHIR99021 (B) treatment, nine-month-old mice were evaluated for BLamD (arrows) by transmission microscopy (TEM). Representative fields of view (FOV) of twenty-two fields are presented for vehicle or CHIR99021 treatment. (C, D) A masked observer systematically quantified number of FOV containing any BLamD (C), **** p ≤ 0.0001, chi-square test, as well as the average size of the BLamD, if at all present (D), ** p ≤ 0.01, t-test vs. vehicle-treated samples.