PRMT5 mediates FoxO1 methylation and subcellular localization to regulate lipophagy in myogenic progenitors

Abstract

Development is regulated by various factors, including protein methylation status. While PRMT5 is well known for its roles in oncogenesis by mediating symmetric di-methylation of arginine, its role in normal development remains elusive. Using Myod1^Cre to drive Prmt5 knockout in embryonic myoblasts (Prmt5^MKO), we dissected the role of PRMT5 in myogenesis. The Prmt5^MKO mice are born normally but exhibit progressive muscle atrophy and premature death. Prmt5^MKO inhibits proliferation and promotes premature differentiation of embryonic myoblasts, reducing the number and regenerative function of satellite cells in postnatal mice. Mechanistically, PRMT5 methylates and destabilizes FoxO1. Prmt5^MKO increases the total FoxO1 level and promotes its cytoplasmic accumulation, leading to activation of autophagy and depletion of lipid droplets (LDs). Systemic inhibition of autophagy in Prmt5^MKO mice restores LDs in myoblasts and moderately improves muscle regeneration. Together, PRMT5 is essential for muscle development and regeneration at least partially through mediating FoxO1 methylation and LD turnover.

Keywords: CP: Developmental biology; CP: Molecular biology; PRMT; PTM; SCs; autophagy; myogenesis; posttranslational modification; protein arginine methyltransferase; satellite cells.

Figure 1.. Conditional KO of Prmt5 in embryonic myoblasts (Prmt5^MKO) reduces muscle mass, impairs muscle function, and leads to pre-term death in adult mice

(A and B) Growth curve (A) and total body weight gain over 6 weeks (B) in 4-week-old WT and Prmt5^MKO mice (n = 7/group). (C) Survival of adult WT and Prmt5^MKO mice; all KO mice died before reaching 18 weeks old (n = 15). (D) Lean and fat mass of 8-week-old WT and Prmt5^MKO mice (n = 6) measured by EchoMRI body composition analyzer. (E) Weight of soleus (SOL), extensor digitorum longus (EDL), tibialis anterior (TA), gastrocnemius (GAS), and quadriceps (QU) muscles in adult WT and Prmt5^MKO mice (n = 3). (F) Myofiber morphology outlined by sarcolemma dystrophin immunofluorescence of TA muscle sections from WT and Prmt5^MKO mice (n = 4). Scale bar, 100 μm. (G) Frequency distribution of TA muscle myofiber cross-sectional area (CSA) in adult WT and Prmt5^MKO mice (n = 4). (H and I) Average myofiber CSA (H) and total myofiber number (I) per TA muscle of WT and Prmt5^MKO mice (n = 4). (J–L) Running time (J), maximum speed (K), and running distance (L) of adult WT and Prmt5^MKO mice measured by treadmill exhaustion test (n = 5–7). (M) Grip strength test normalized to body weight in adult WT and Prmt5^MKO mice (n = 5–8). (N and O) Absolute contractile force as a function of frequency of electric stimulation (left) and maximal absolute force (right) measured in the EDL (N) and SOL (O) muscles isolated from adult WT and Prmt5^MKO mice (n = 4). (P and Q) Specific force as a function of frequency (left) and maximal specific force (right) of EDL (P) and SOL (Q) muscles isolated from adult WT and Prmt5^MKO mice (n = 4). Values are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by t tests. See also Figure S1.

Figure 2.. Prmt5 KO in embryonic myoblasts (Prmt5^MKO) delay postnatal myogenesis and muscle growth

(A and B) immunofluorescence of Pax7 (A) and MyoG (B) in the left panels and corresponding quantification of cells, in right panels, of WT and Prmt5^MKO TA muscle sections at P7 (n = 3) and P14 (n = 5). Myofiber membranes are stained with dystrophin and nuclei are stained by DAPI sarcolemma. Scale bar, 10 μm. (C) eMyHC immunofluorescence (left) and quantification of eMyHC⁺ myofibers (right) in TA muscle of WT and Prmt5^MKO mice at P7 (n = 4–5). Scale bar, 10 μm. (D and E) immunoblotting analysis showing the protein contents of PRMT5, MyoD, eMyHC, and tubulin at P7 (D) and P14 (E) in skeletal muscles of WT and Prmt5^MKO mice. KD, kilodalton size marker. (F) Body weights of P7 and P14 WT and Prmt5^MKO mice (P7, n = 3; P14, n = 5). Values are expressed as mean ± SEM. *p < 0.05, **p < 0.01 by t test. See also Figure S3.

Figure 3.. Prmt5 KO in embryonic myoblast (Prmt5^MKO) impedes cell proliferation and promotes premature differentiation, hampering embryonic and fetal myogenesis

(A) Gross morphology (left) and body weight (right) of newborn mice (P1) in WT and Prmt5^MKO mice (n = 3–4). (B) Pax7⁺ and Ki67⁺ immunofluorescence (left) and quantification (right) in hindlimb cross sections of newborn WT and Prmt5^MKO mice (n = 3–4). Scale bar, 10 μm. (C) MyoG immunofluorescence (left) and quantification of MyoG⁺ cells per area (right) in hindlimb muscle sections of P1 WT and Prmt5^MKO mice (n = 3–4). Scale bar, 10 μm. (D) Gross morphology of E18.5 WT and Prmt5^MKO embryos. (E) H&E staining (left) and quantification of E18.5 hindlimb (right) of WT and Prmt5^MKO mice. (F and G) Pax7 (F) and MyoG (G) immunofluorescence images (left) and corresponding quantification of positive cells per myofiber (right) in WT and Prmt5^MKO E18.5 hindlimb sections (n = 3). Scale bar, 10 μm. (H) Pax7 and Ki67 immunofluorescence (left) and percentage of proliferating (Ki67⁺) cells among Pax7⁺ myoblasts (right). Primary myoblasts were treated with vehicle or 4-OH-TMX (4-hydroxyl-tamoxifen) (to induce deletion of Prmt5). Scale bar, 10 μm. (I) Schematic outline of EdU incorporation assay to assess proliferating SCs on single myofibers isolated from adult WT and Prmt5^MKO mice. (J) Pax7 immunofluorescence cells and EdU click labeling (left), along with the quantification (percentages of EdU⁺ cells among Pax7⁺ cells) grown on WT and Prmt5^MKO myofibers (right) (n = 3). Scale bar, 10 μm. Values are expressed as mean ± SEM. *p < 0.05, **p < 0.01 by t test. See also Figure S2.

Figure 4.. Prmt5 KO diminishes LD abundance and oxidative respiration of SCs, disrupting their cell fate homeostasis

(A) Oil red O staining (left) and quantification (right) of LD content in control (MeOH) and Prmt5 KO (4-OH) myoblasts. Scale bar, 50 μm. (B) Pax7 and Bodipy immunofluorescence (left) and quantification of Bodipy intensity (right) in control (MeOH) and Prmt5 KO (4-OH) myoblasts. Scale bar, 10 mm. (C) Pax7 and Bodipy immunofluorescence (left) and quantification of Bodipy intensity (right) in SCs after proliferating for 72 h on cultured single myofibers isolated from WT and Prmt5^MKO mice. Scale bar, 10 μm. (D) qPCR analysis of lipogenic marker (Pnpla2) and adipogenesis markers (Dgat1, Fabp4) in control (MeOH) and Prmt5 KO (4-OH) myoblasts (n = 4). (E–G) Pax7 and MyoD fluorescence to distinguish cell fate status in WT and Prmt5^MKO SCs (E), quantification of SCs per cluster (F), and proportions of self-renewing (Pax7⁺MyoD⁻), proliferating (Pax7⁺MyoD⁺), and differentiating (Pax7⁻MyoD⁺) cells (G) after culturing on single myofibers for 72 h (n = 3). Scale bar, 10 μm. (H) Seahorse measurement of oxygen consumption rate (OCR) in control (MeOH) and Prmt5 KO (4-OH) myoblasts in response to sequential addition of oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and rotenone/antimycin A (n = 3). (I) Average OCR for basal respiration (BR), proton leak (PL), ATP respiration (ATP R), maximal respiration (Max R), spare capacity, and non-mitochondrial respiration (NR) based on H (n = 3). SC, spare capacity. Values are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001 by t test. See also Figure S4.

Figure 5.. Prmt5 KO activates autophagy in satellite cells

(A) Western blotting showing levels of PRMT5, LC3B, and GAPDH in control (MeOH) and Prmt5 KO (4-OH) myoblasts in the absence or presence of hydroxychloroquine (HCQ), along with quantified levels (normalized to GAPDH) of LC-II/I, as an indicator of autophagy (right). KD, kilodalton size marker. (B) qPCR analysis of autophagy makers in control (MeOH) and Prmt5 KO (4-OH) myoblasts (n = 4). (C) Pax7 and LC3B immunofluorescence (left) and the relative intensity of LC3B (right) in Pax7⁺ cells grown on freshly isolated single myofibers from WT and Prmt5^MKO mice (n = 4). Scale bar, 10 μm. Values are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by t test. See also Figure S5.

Figure 6.. PRMT5 mediates methylation of FoxO1 to alter its subcellular localization

(A) Western blot images showing subcellular fraction of control (MeOH) and Prmt5 KO (4-OH) myoblasts and cytoplasmic retention of FoxO1 in Prmt5 KO cells. KD, kilodalton size marker. (B) Pax7 and FoxO1 immunofluorescence (left) of SCs on freshly isolated myofibers from WT and Prmt5^MKO mice, along with quantification of FoxO1 subcellular location (right) (n = 5). Scale bar, 10 μm. (C) Control (MeOH) and Prmt5 KO (4-OH) myoblasts were immunoprecipitated with SYM10 and PRMT5 antibody and blotted with FoxO1, PRMT5, and GAPDH antibodies. KD, kilodalton size marker. (D and E) Quantification of methylated FoxO1 (D) and PRMT5-FoxO1 binding (E) (n = 3). (F) Control (MeOH) and Prmt5 KO (4-OH) myoblasts were immunoprecipitated with SYM10 after subcellular fractionation and blotted with PRMT5, FoxO1, GAPDH, and histone 3 antibodies. KD, kilodalton size marker. (G and H) Quantification of methylated FoxO1 in the nucleus (G) and cytoplasm (H) (n = 4). (I and J) Proximity ligation assay (PLA) on control (MeOH) and Prmt5 KO (4-OH) myoblasts using SYM10 and FoxO1 (I), or PRMT5 and FoxO1 (J) antibody pairs. The presence of red punta indicates potential protein modification or interaction. Scale bar, 10 μm. Values are expressed as mean ± SEM. *p < 0.05, **p < 0.01 by t test. See also Figure S6.

Figure 7.. Pharmacological inhibition of autophagy alleviates defects of Prmt5-KO myoblasts

(A and B) Pax7 and Bodipy immunofluorescence (A), along with quantification of Bodipy intensity (B) in control (MeOH) and Prmt5 KO (4-OH) myoblasts treated with or without HCQ. Scale bar, 10 μm. (C) qPCR analysis of autophagy marker (LC3B) and lipid metabolism markers (Dgat1) in control (MeOH) and Prmt5 KO (4-OH) myoblasts treated with or without HCQ (n = 3). (D) Immunostaining of Pax7 and Bodipy in SCs after being cultured for 72 h on myofibers from WT and Prmt5^MKO treated with or without HCQ (n = 3). Scale bar, 10 μm. (E) Experimental design for vehicle or HCQ treatment in adult WT and Prmt5^MKO mice to examine muscle regeneration. (F) H&E images of WT and Prmt5^MKO TA muscle cross sections at 7 DPI after vehicle or HCQ treatment. Scale bar, 100 μm. (G and H) Immunostaining of eMyHC and laminin (G) along with quantification of myofiber size (H) in WT and Prmt5^MKO TA muscle cross sections at 7 DPI after vehicle or HCQ treatment (n = 6). Scale bar, 10 μm. Values are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by t test.