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other·ophthalmology, retina, ocular immunology, antioxidants, animal science·PMC10031543

DAPL1 deficiency in mice impairs antioxidant defenses in the RPE and leads to retinal degeneration with AMD-like features

Redox Biology · 13 authors, 4 centres

AI SUMMARY

FIDELITY 94%

POPULATIONDapl1 knockout mice (Dapl1−/−) and wild-type (WT) C57BL/6J mice; also ARPE-19 and D407 RPE cell lines

INTERVENTIONDapl1 deficiency (knockout), AAV9-mediated DAPL1 overexpression in RPE, MITF overexpression (transgenic or AAV9), E2F4 or MYC modulation (siRNA or overexpression), sodium iodate (NaIO3) injection to induce oxidative stress

COMPARISONWild-type mice vs. Dapl1−/− mice; AAV9-DAPL1 vs. AAV9 empty vector; Dapl1−/− vs. Dapl1−/−;Mitf+/− vs. Dapl1−/−;Dct-Mitf; control siRNA vs. specific siRNAs

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TL;DR

This study demonstrates that DAPL1 deficiency in mice impairs the antioxidant defense system in retinal pigment epithelium (RPE) cells, leading to age-related retinal degeneration with features resembling dry age-related macular degeneration (AMD). Mechanistically, DAPL1 binds E2F4 to suppress MYC expression, thereby upregulating MITF and its downstream targets NRF2 and PGC1α, which are critical for antioxidant protection. These findings identify the DAPL1/E2F4-MYC-MITF-NRF2/PGC1α axis as a potential therapeutic target for AMD and other retinal degenerative diseases.

Full summary

4,454 CHARS

**Background:** Oxidative stress is a major contributor to age-related macular degeneration (AMD), a leading cause of irreversible blindness. The retinal pigment epithelium (RPE) is critical for retinal homeostasis and antioxidant defense. Death-associated protein like-1 (DAPL1) has been linked to AMD through genetic association, but its functional role in the RPE and retinal degeneration was unknown. This study investigates whether DAPL1 deficiency impairs antioxidant defenses in the RPE and leads to retinal degeneration in mice, and explores the underlying molecular mechanisms.

**Methods:** The study used Dapl1 knockout (Dapl1−/−) mice, wild-type (WT) C57BL/6J mice, and various genetic crosses including Mitf mutant mice (Mitfmi-vga9) and Dct-Mitf transgenic mice. Retinal function was assessed by scotopic electroretinography (ERG) at 2 and 18 months of age. Retinal structure was evaluated by histology (H&E staining) and immunofluorescence for Rhodopsin, Opsin, ZO-1, and OTX2. Oxidative stress was measured using the CM-H2DCFDA probe for ROS detection. In vivo oxidative damage was induced by intraperitoneal injection of sodium iodate (NaIO3) at 30 or 50 mg/kg. AAV9-mediated gene transfer was used to overexpress DAPL1 or MITF specifically in RPE cells. In vitro studies used ARPE-19 and D407 RPE cell lines with lentiviral overexpression or siRNA knockdown of DAPL1, MITF, MYC, and E2F4. Protein interactions were assessed by co-immunoprecipitation. Cell viability was measured by CCK8 assay, and ROS levels by DHE staining.

**Key Results:** (1) Dapl1−/− mice showed no retinal differences at 2 months, but by 18 months exhibited significantly reduced ERG a- and b-wave amplitudes (P<0.01), thinner ONL and INL (P<0.05), increased ROS in the RPE layer (P<0.01), and disrupted RPE morphology (multinucleated cells, damaged junctions) compared to age-matched WT mice. (2) After NaIO3 injection (50 mg/kg), 2-month-old Dapl1−/− mice had more severe RPE damage, higher ROS levels (P<0.01), thinner ONL (P<0.01), lower Rhodopsin/Opsin expression (P<0.01), and more TUNEL-positive cells (P<0.01) than WT mice. (3) AAV9-mediated DAPL1 overexpression in the RPE of Dapl1−/− mice restored HA-tagged DAPL1 and P21 levels, and after NaIO3 injection, preserved ONL thickness (P<0.001), increased Rhodopsin/Opsin (P<0.01), and reduced TUNEL positivity (P<0.01) compared to AAV9 empty vector. (4) In vitro, DAPL1 overexpression in ARPE-19 and D407 cells increased MITF, NRF2, and PGC1α protein levels (P<0.01). siRNA knockdown of MITF in DAPL1-overexpressing cells reduced cell viability (CCK8) and increased ROS after glucose oxidase (GOX) treatment (P<0.05). (5) In vivo, MITF, NRF2, and PGC1α protein levels were decreased in 2-month-old Dapl1−/− RPE compared to WT (P<0.01). Dapl1−/−;Mitf+/− mice (with reduced MITF) showed greater sensitivity to NaIO3 (30 mg/kg) than Dapl1−/−;Mitf+/+ mice, with thinner ONL (P<0.05), lower Rhodopsin/Opsin (P<0.05), and more TUNEL-positive cells (P<0.05). Conversely, transgenic overexpression of MITF in Dapl1−/− mice (Dapl1−/−;Dct-Mitf) reduced ROS (P<0.05), preserved ONL thickness (P<0.05), increased Rhodopsin/Opsin (P<0.05), and decreased TUNEL (P<0.05) after NaIO3. (6) MYC protein was increased in Dapl1−/− RPE and decreased in DAPL1-overexpressing cells (P<0.01). MYC overexpression reduced MITF levels, while MYC knockdown increased MITF (P<0.01). Overexpressing MYC in DAPL1-overexpressing cells decreased MITF without affecting DAPL1. (7) Co-immunoprecipitation showed DAPL1 binds E2F4 but not E2F1. Knockdown of E2F4 decreased MYC, and overexpression of E2F4 increased MYC (P<0.01). Deletion of the PKC phosphorylation domain (aa24-28) of DAPL1 abolished binding to E2F4 and failed to suppress MYC or increase MITF.

**Clinical Implications:** This study identifies DAPL1 as a critical antioxidant regulator in the RPE through the DAPL1/E2F4-MYC-MITF-NRF2/PGC1α axis. DAPL1 deficiency leads to age-related retinal degeneration with features resembling dry AMD, including RPE dysfunction, oxidative stress, and photoreceptor loss. The findings provide a mechanistic link between DAPL1 genetic variants and AMD susceptibility. AAV-mediated DAPL1 or MITF gene therapy may represent a novel therapeutic strategy to enhance RPE antioxidant capacity and protect against retinal degeneration. The pathway also offers potential targets for pharmacological intervention to prevent or slow AMD progression.

PICO

PPOPULATION

Dapl1 knockout mice (Dapl1−/−) and wild-type (WT) C57BL/6J mice; also ARPE-19 and D407 RPE cell lines

IINTERVENTION

Dapl1 deficiency (knockout), AAV9-mediated DAPL1 overexpression in RPE, MITF overexpression (transgenic or AAV9), E2F4 or MYC modulation (siRNA or overexpression), sodium iodate (NaIO3) injection to induce oxidative stress

OOUTCOME

Electroretinogram (ERG) a- and b-wave amplitudes, outer nuclear layer (ONL) thickness, inner nuclear layer (INL) thickness, ROS levels (CM-H2DCFDA probe), RPE cell morphology (ZO-1 staining), cell viability (CCK8), apoptosis (TUNEL), protein expression (western blot) of DAPL1, MITF, NRF2, PGC1α, MYC, E2F4, Rhodopsin, Opsin, GFAP

STUDY TYPE

other

SPECIALTY

ophthalmology

SUMMARISED BY

AI pipeline

FIDELITY CHECK

94% · A

ORIGINAL PAPER

PMC10031543

DAPL1 deficiency in mice impairs antioxidant defenses in the RPE and leads to retinal degeneration with AMD-like features

Redox Biology

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ABSTRACT

Abstract The decreased antioxidant capacity in the retinal pigment epithelium (RPE) is the hallmark of retinal degenerative diseases including age-related macular degeneration (AMD). Nevertheless, the exact regulatory mechanisms underlying the pathogenesis of retinal degenerations remain largely unknown. Here we show in mice that deficiencies in Dapl1 , a susceptibility gene for human AMD, impair the antioxidant capacity of the RPE and lead to age-related retinal degeneration in the 18-month-old mice homozygous for a partial deletion of Dapl1 . Dapl1 -deficiency is associated with a reduction of the RPE's antioxidant capacity, and experimental re-expression of Dapl1 reverses this reduction and protects the retina from oxidative damage. Mechanistically, DAPL1 directly binds the transcription factor E2F4 and inhibits the expression of MYC, leading to upregulation of the transcription factor MITF and its targets NRF2 and PGC1α, both of which regulate the RPE's antioxidant function. When MITF is experimentally overexpressed in the RPE of DAPL1 deficient mice, antioxidation is restored and retinas are protected from degeneration. These findings suggest that the DAPL1-MITF axis functions as a novel regulator of the antioxidant defense system of the RPE and may play a critical role in the pathogenesis of age-related retinal degenerative diseases. Keywords: Oxidative stress, MITF, Pigment cell, AMD, AAV

CITATION EXPORT

Xiaoyin Ma, Huaicheng Chen, Shuhui Jian et al.. DAPL1 deficiency in mice impairs antioxidant defenses in the RPE and leads to retinal degeneration with AMD-like features. Redox Biology. (2023). https://doi.org/10.1016/j.redox.2023.102675

AUTHORS

13 · 4 CENTRES

Xiaoyin Ma, Huaicheng Chen, Shuhui Jian, Junhao He, Youjia Liu, Shuxian Han, Lifu Chang, Pingping Li, Ying-ao Chen, Xiaoyan Liu, Xiaojuan Hu, Yu Chen, Ling Hou

aLaboratory of Developmental Cell Biology and Disease, School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, China
bState Key Laboratory of Ophthalmology, Optometry and Vision Science, Wenzhou Medical University, Wenzhou, 325003, China
cDepartment of Ophthalmology, The Fourth Affiliated Hospital, Zhejiang University School of Medicine, Yiwu, Zhejiang, 322000, China
dThe Affiliated Eye Hospital of Nanjing Medical University, Nanjing, 210029, China