Kruppel-like factor (KLF4) knockout in retinal ganglion cells improves visual function in Experimental Autoimmune Encephalomyelitis (EAE) mice model of optic neuritis

Abstract

Purpose : To rescue visual loss and optic neuropathy in the EAE mouse model using ssAAV2Cre-GFP mediated knockout (KO) of KLF4^fl/fl, a transcriptional repressor of axon growth in RGC optic nerves.

Methods : EAE was induced in KLF4^fl/fl and littermates (n=20) by subdermal injection of 0.1 ml homologous spinal cord emulsion in complete Freund's adjuvant (CFA). Following a significant drop in PERG amplitudes compared to CFA mice, KLF4^fl/fl EAE mice received an intravitreal injection of ssAAV-Cre-GFP in both eyes, EAE littermates received scAAV-mCherry injection. CFA mice injected with scAAV-mCherry were no disease controls. Visual function was assessed by PERG and SD-OCT to evaluate the thickness of inner retina. Tuj1 labeled RGC numbers were evaluated by immunofluorescence. Axonal regeneration was tested by Gap43 immunoblotting of retina and optic nerve (ON) samples and TEM analysis of ON axons.

Results : PERG Analysis. PERG amplitudes decreased significantly in EAE mice compared to CFA controls at 9 months post sensitization (MPS) (P<0.05). PERG amplitudes of CFA control mice at 9MPS and 19 MPS, did not show significant difference, PERG amplitudes of EAE littermates at 9MPS and 19MPS (9MPI) showed a slight decrease (16.7+1.7, 15.1+1.5, P>0.05), however the KLF4 KO in EAE KLF4^fl/fl mice improved amplitudes significantly (17.8+1.4 at 9MPS, 24+1.2 at 19 MPS, p=0.005). OCT Analysis. RGC+IPL thickness of CFA control mice at 9MPS and 19MPS did not show significant difference, likewise RGC+IPL thickness of EAE littermates 9MPS and 19MPS (9MPI) did not show any significant difference however, the EAE KLF4^fl/fl mice showed significant improvement in RGC+IPL thickness following KLF4 KO (64.5+1.7 vs 68.9+0.3, P=0.04). RGC counts. Tuj1+ve RGCs were significantly higher in EAE-KLF4 KO mice compared to EAE mCherry mice (p=7.01e-15). Axon Regeneration Analysis. Western blots showed increased expression of axonal regenerative marker GAP43 in EAE-KLF4 KO retina and ONs compared to CFA and EAE mCherry groups. TEM analysis of ON axons showed a significantly higher low caliber axons in EAE KLF4 KO mice compared to CFA controls.

Conclusions : Targeted deletion of the KLF4 in RGCs and optic nerve axons improved axonal regeneration and improved visual function in optic neuritis of EAE mice.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.