The role of AMPK related kinase 5 (ARK5/NUAK1) in lamina cribrosa fibrosis in glaucoma

Purpose: Glaucoma, the leading cause of blindness globally is a chronic optic neuropathy characterised by structural fibrotic changes in the lamina cribrosa (LC), leading to a progressive retinal ganglion cells death and irreversible vision impairment and blindness. TGF-β is a known driver of these pathological changes in the LC extracellular matrix (ECM). At a cellular level, oxidative stress, mitochondrial dysfunction and altered bioenergetics are evident in glaucoma. AMPK related kinase 5 (ARK5) is a member of the AMPK class of serine/threonine protein kinases that play an important role in cellular energy metabolism. The aims of this study were to establish differential expression of ARK5 in glaucomatous LC fibroblasts, to examine the effects of the known ARK5 inhibitors HTH-01-015 on ARK5 and ECM gene expression as well as analysing the effect of ARK5 inhibition using miR-211 and miR-145 in the modulation of TGF-β1 induced ECM gene expression and fibrosis in LC cells. We also aimed to identify IGF-1 as an upstream activator of ARK5 and to investigate the effect of HTH-01-015 on IGF-1 induced cellular proliferation. Methods: LC cells from human glaucomatous (GLC) donor eyes and normal (NLC) age-matched controls were cultured. Differential gene expression levels were measured in normal and glaucoma LC cells using quantitative real time qRT-PCR and total protein expression levels were measured using Western blot analyses. Proliferation rates in NLC and GLC cells were measured using MTS assays. miRNA transfections were performed using HiPerFect transfection reagent, and SliGlow fluorescence was used to analyse transfection efficiency. Results: ARK5 expression was significantly elevated in GLC cells compared to NLC cells. TGFβ1 significantly increased ARK5 expression in the NLC and GLC cells. Treatment with 10µM HTH-01-015 significantly downregulated ARK5 and ECM gene expression in GLC cells versus untreated GLC cells. We identified IGF-1 is an upstream activator of ARK5. Treatment with 10 µM HTH-01-015 significantly downregulated IGF-1 induced cellular proliferation rates. There was no significant difference between miR-211 levels in NLC versus GLC cells. TGFβ1 treatment significantly reduced expression of miR-211 in NLC and GLC cells. Transfection with miR-211 increased miR-211 levels in LC cells treated with TGFβ-1. Transfection with miR-211 mimic significantly downregulated ARK5 expression levels in TGFβ-1 treated LC cells and ECM genes collagen 1A1 and fibronectin. miR-145 was significantly upregulated in GLC cells versus NLC cells. Treatment with TGFβ1 significantly increased the expression of miR-145 in NLC cells but did not alter the miR-145 expression levels in GLC cells. Transfection with miR-145 mimic significantly increased miR-145 expression levels in LC cells treated with TGF-β1. Transfection with miR-145 significantly downregulated ARK5 expression in LC cells treated with TGF-β1. Conclusion: ARK5 is a pro-fibrotic kinase that is dysregulated in glaucoma and its expression is upregulated by TGF-β1. ARK5 inhibition using HTH-01-015 and miR-211 reduced pro-fibrotic ECM gene expression and cellular proliferation. Halting the pro-fibrotic activity and metabolism of GLC cells by downregulating ARK5 expression is an exciting new therapeutic approach for glaucoma.