SARM1 deficiency promotes rod and cone photoreceptor cell survival in a model of retinal degeneration

Abstract

Retinal degeneration is the leading cause of incurable blindness worldwide and is characterised by progressive loss of light-sensing photoreceptors in the neural retina. SARM1 is known for its role in axonal degeneration, but a role for SARM1 in photoreceptor cell degeneration has not been reported. SARM1 is known to mediate neuronal cell degeneration through depletion of essential metabolite NAD and induction of energy crisis. Here, we demonstrate that SARM1 is expressed in photoreceptors, and using retinal tissue explant, we confirm that activation of SARM1 causes destruction of NAD pools in the photoreceptor layer. Through generation of rho ^-/- sarm1 ^-/- double knockout mice, we demonstrate that genetic deletion of SARM1 promotes both rod and cone photoreceptor cell survival in the rhodopsin knockout (rho ^-/- ) mouse model of photoreceptor degeneration. Finally, we demonstrate that SARM1 deficiency preserves cone visual function in the surviving photoreceptors when assayed by electroretinography. Overall, our data indicate that endogenous SARM1 has the capacity to consume NAD in photoreceptor cells and identifies a previously unappreciated role for SARM1-dependent cell death in photoreceptor cell degeneration.

Figure 1.. SARM1 is expressed in the photoreceptor cells of the retina.

(A) Data extracted from Human proteome map showing relative expression of SARM1, Rhodopsin (RHO), and RPE65 in different tissues. (B) qRT-PCR analysis of SARM1 transcript levels in the neural retina and RPE/choroid of wild-type C57BL/6J mice (**P ≤ 0.01, by t test, n = 5 mice). (C) Schematic of a wild-type and rho^−/− retina over time. (D) Haemotoxylin and eosin staining of paraffin-embedded sections from wild-type and rho^−/− mice on the C57BL/6J background at 12 wk of age (40× magnification). (E) qRT-PCR analysis of SARM1 transcript levels in the neural retina of wild-type and rho^−/− mice on the C57BL/6J background at 12 wk of age (***P ≤ 0.001, by t test, n = 6 mice). (F, G) Western blot analysis and (G) quantification of SARM1 expression in neural retina of wild-type n = 5, rho^−/− n = 5, and sarm1^−/− n = 3 mice (***P ≤ 0.001, by t test).

Figure 2.. Activation of SARM1 results in NAD depletion and mediates photoreceptor cell death.

(A) Schematic diagram of the SARM1 plasmid constructs used for transfection. (B, C, D) MTS metabolic assay, (C) LDH cytotoxicity assay and (D) bright-field images (10× magnification) of 661W cells transfected with 1 μg/ml of empty vector (EV), full-length SARM1 (FL), and dN190-SARM1 (dN190) for 24 h (*P ≤ 0.05, ***P ≤ 0.001 by t test, n = 3). (E) Graphical representation of retinal explant preparation. (F) NADH fluorescent lifetime imaging (FLIM) quantification in WT and sarm1^−/− retinal explants treated with 50 μM CCCP for 24 h, showing the relative fold values when normalized to vehicle-treated WT and sarm1^−/− retinal explants, respectively (**P ≤ 0.01 by t test, n = 4). (G) Colour-coded FLIM images of the retinal explants from WT and sarm1^−/− retinal explants treated with 50 μM CCCP for 24 h.

Figure 3.. Photoreceptor loss in rho^−/− model of retinal degeneration is delayed with SARM1 deficiency.

(A, B, C) Haemotoxylin and eosin staining of paraffin-embedded sections from rho^−/−, rho^−/−sarm1^+/−, and rho^−/−sarm1^−/− mice at 3, 6, 9, and 12 wk of age with (B) quantification of the number of photoreceptor rows in the ONL and (C) quantification of the inner and outer segment length using ImageJ (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, by ANOVA with Tukey’s post hoc test; n = 3–8 mice per group). (D) Table illustrating percentage drop in ONL photoreceptor layers and inner and outer segment length from 9 to 12 wk of age in the rho^−/−, rho^−/−sarm1^+/−, and rho^−/−sarm1^−/− mice.

Figure S1.. Photoreceptor ONL and segment length are comparable between SARM1-deficient and WT mice.

Linked to Fig 3. (A, B, C) H&E staining of paraffin-embedded sections from WT and sarm1^−/− mice at 6 and 12 wk of age with (B) quantification of the number of photoreceptor rows in the ONL and (C) quantification of the inner and outer segment length using ImageJ (n = 3–4 mice per group).

Figure 4.. SARM1 deficiency delays thinning of the outer retina in the rho^−/− model.

(A, B) Optical coherence tomography (OCT) images taken in vivo from rho^−/−, rho^−/−sarm1^+/−, and rho^−/−sarm1^−/− mice at 3, 6, 9, and 12 wk of age with (B) quantification of the ONL to the outer segment (OS) width (marked with red line in the OCT images) using ImageJ (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, by ANOVA with Tukey’s post hoc test; n = 5–8 mice per group). (C) Correlation graph of number of photoreceptors rows in the H&E images versus the ONL to OS width in the OCT images (Pearson’s r = 0.9879).

Figure S2.. Optical coherence tomography (OCT) analysis is comparable between SARM1-deficient and WT mice.

Linked to Fig 4. (A, B) OCT images taken in vivo from WT and sarm1^−/− mice at 6 and 12 wk of age with (B) quantification of the ONL to outer segment (OS) width (marked with red line in the OCT images) using ImageJ (n = 4 mice per group).

Figure 5.. SARM1 deficiency delays cone photoreceptor loss and maintains local (NAD) and visual function by electroretinogram (ERG).

(A) Immunohistochemistry of retinal cryosections from rho^−/−, rho^−/−sarm1^+/−, and rho^−/−sarm1^−/− mice at 3, 6, 9, and 12 wk of age stained with the cone-specific marker peanut agglutinin (PNA) and the photoreceptor marker recoverin (red, PNA; green, recoverin; blue, DAPI; 40× magnification). (B) Quantification by Mean fluorescence intensity using ImageJ of Z-stack images of retinal flat-mounts from WT, rho^−/−, and rho^−/−sarm1^−/− mice at 9 and 12 wk of age stained with PNA (40× magnification) (**P ≤ 0.01 by t test; n = 3–8 mice) with representative images shown alongside. (C, D) NADH FLIM quantification of NADH photons emitted per square micrometre and (D) colour-coded images of retinal explants isolated from 4-wk-old rho^−/− and rho^−/−sarm1^−/− mice (*P ≤ 0.05 by t test; n = 3 mice). (E, F) b-wave measurements from dark-adapted single-flash ERGs (F) and 10-Hz flicker ERGs in sarm1^−/− (9–12 wk), and rho^−/− and rho^−/−sarm1^−/− mice at 9 and 12 wk of age (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 by t test; n = 8–18 eyes) with representative ERG traces shown on the right-hand side.

Figure 6.. Graphical Abstract for SARM1 induction of photoreceptor cell death.