June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Investigating TFEB-regulated transcriptional program in ATP Binding Cassette Subfamily A Member 4 (ABCA4) knockout retinal pigment epithelium
Author Affiliations & Notes
  • Mallika Valapala
    Indiana University, Bloomington, Indiana, United States
  • Hsuan-Yeh Pan
    Indiana University, Bloomington, Indiana, United States
  • Manas R Biswal
    University of South Florida Taneja College of Pharmacy, Tampa, Florida, United States
  • Footnotes
    Commercial Relationships   Mallika Valapala None; Hsuan-Yeh Pan None; Manas Biswal None
  • Footnotes
    Support  Brightfocus foundation
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 2107. doi:
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      Mallika Valapala, Hsuan-Yeh Pan, Manas R Biswal; Investigating TFEB-regulated transcriptional program in ATP Binding Cassette Subfamily A Member 4 (ABCA4) knockout retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2023;64(8):2107.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Impaired autophagy and lysosomal function is connected to the pathogenesis of age-related macular degeneration (AMD). Recent studies suggest that restoring autophagy and lysosomal function enhance cellular clearance and preventing RPE degeneration. We investigated the molecular targets that co-regulate lysosomal function and autophagy in the RPE. Transcription factor EB (TFEB) is a master regulator of lysosomal function and autophagy. We studied the expression and regulation of TFEB in the ABCA4 KO to enhance lysosomal function and autophagy

Methods : One eye of the 6 week and 6-month-old ABCA4 KO mouse was injected with AAV-TFEB by subretinal injection and the contralateral eye was injected with AAV-GFP control virus. The expression of the transgene in the ABCA4 KO RPE was examined by and immunostaining of the RPE flat mounts and sections. qRT-PCR analysis was performed to reveal the expression of the following TFEB-regulated coordinated lysosomal expression and regulation (CLEAR) network genes: Transcription Factor EB (TFEB), Cathepsin D (CTSD), Cathepsin B (CTSB), Lysosomal-associated membrane protein 1 (LAMP1), Sequestosome 1(SQSTM1), Microtubule Associated Protein 1 Light Chain 3 Beta (MAP1LC3B). Immunoblotting was performed to evaluate the expression of TFEB, LC3 and SQSTM1.

Results : Immunostaining of retinal flat mounts and sections revealed the expression of GFP-tagged TFEB in the RPE of ABCA4 KO mice, 3 months post injection. QRT-PCR analysis reported an upregulation in the expression of TFEB and TFEB-regulated CLEAR network genes in the AAV-TFEB-injected ABCA4 KO RPE compared to the control RPE. Immunoblotting revealed an increase of LC3II and a decrease in the expression of SQSTM1 in the AAV-TFEB-injected RPE compared to the control RPE.

Conclusions : AAV-TFEB subretinal injection induces the expression of TFEB and TFEB-regulated CLEAR network genes in the ABCA4 KO RPE. We also observed an increase in expression of LC3II and a decrease in the expression of SQSTM1, which indicates an induction of autophagy. These results suggest that the TFEB-regulated transcriptional program plays a key role in the regulation of cellular clearance in the ABCA4 KO RPE.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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