AARS2-catalyzed lactylation induces follicle development and premature ovarian insufficiency

Abstract

Lactate, a metabolite which is elevated in various developmental and pathological processes, exerts its signal through alanyl tRNA synthetases (AARS)-catalyzed protein lactylation. Herein, we report that elevated lactate and gain-of-function mitochondrial AARS (AARS2) mutations-induced hyper-lactylation promotes premature ovarian insufficiency (POI). Serum lactate is elevated in POI patients. POI-driving AARS2 mutations gain lactyltransferase activity. AARS2 lactylates and inactivates carnitine palmitoyl transferase 2 (CPT2), resulting in FFA accumulation that activates peroxisome proliferator-activated receptor γ (PPARγ), and potentiates follicle-stimulating hormone (FSH) to initiate follicle development. These, in synergy with the anabolites accumulation effects of AARS2, promoted lactylation-induced PDHA1 inactivation promote granular cell (GC) proliferation and primordial follicle development. GC-specific AARS2 overexpression does not affect primordial follicle number but speed up follicle depletion. AARS2 ablation or lactylation-inhibiting β-alanine treatments can prevent folliculogenesis and POI traits in mouse. These findings reveal that lactate signal drives follicle development, and inhibiting lactate signal could treat/prevent POI.

Fig. 1. Lactate and fatty acid levels are elevated in POI patients.

Serum lactate and fatty acids levels were measured in 42 POI patients and 61 age-matched healthy individuals (A, C), and lactate and fatty acids levels were plotted against the functional ovarian reserve indicator AMH (B, D). The following symbols have been used for indicating statistical analysis throughout the Figures: *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001, NS not significant.

Fig. 2. GC-specific AARS2 overexpression induces mouse POI.

A–B AARS2 regulates estrous cyclicity. Representative estrous cyclicity of C57, AARS2 GOE, Aars2 GKO, and AARS2 ZOE mice during 15 consecutive days since postnatal 8 weeks (A) and the cycle phase times (n = 16 for AARS2 GOE and 14 for Aars2 GKO and AARS2 ZOE) represented as mean ± SEM (B). C AARS2 affects the litter size. The cumulative number of pups in C57, AARS2 GOE, Aars2 GKO, and AARS2 ZOE was analyzed (n = 6). D–E Levels of FSH (D) and E2 (E) in adult AARS2 GOE and WT mice (16–20 weeks old, n = 14) were measured. F AARS2 does not affect oocyte number. The number of oocytes in C57, AARS2 GOE, and Aars2 GKO mice (n = 6, one ovary from each mouse) was measured at PD1. See also Fig. S2. G AARS2 affects ovarian follicle number. The ovarian follicles in AARS2 GOE, Aars2 GKO, and C57 mice were quantified (n = 6) at PD5, 8, and PD3-, 5-, and 7-weeks. Numbers of primordial (Pri), primary (Pr), type 4 (T4), type 5 (T5), type 6 (T6), and type 7 (T7) follicles were counted (mean ± SEM). See also Fig. S2.

Fig. 3. POI-causing AARS2 mutants gain lactyltransferase activities.

A–B R199C gained lactyltransferase activity. Lac-K336 levels (A) and Lac-K457/8 levels (B) of PDHA1-Flag and CPT2-Flag that were expressed from AARS2^−/−COV434, and from AARS2^−/− COV434 that co-expressed AARS2, N104Y, or R199C were detected. C–D R199C has stronger lactyltransferase activity than AARS2. The abilities to form Lac-K336 in purified PDHA1 (C) and Lac-K457/8 in purified CPT2 (D) of AARS2 and R199C were detected in vitro. E–F All POI-causing AARS2 mutants gained lactyltransferase activity. Lac-K336 (E) and Lac-K457/8 (F) levels were detected in immunoprecipitated PDHA1-Flag and CPT2-Flag purified from COV434, AARS2^−/− COV434, and AARS2^−/− COV434 cells ectopically expressing AARS2, F50C, T328K, or A77V. G POI-causing AARS2 mutations cluster around substrate-binding pocket. Disease-causing sites are marked in human AARS2 structure. POI-associated mutations are shown as spheres and labeled in red, cardiomyopathy-associated mutations are labeled in green, and leukodystrophy-associated mutations are labeled in violet. Aminoacylation subdomain (35–312 aa, gray) is in gray, tRNA recognition subdomain (313–477 aa) is in green, linker (478–529 aa) between tRNA recognition and editing domain is in blue, and editing domain (530–783 aa) and C-terminal domain (784-985) are in magenta and cyan, respectively. Backbone of docked tRNA and alanyl-adenylate are shown in orange and rainbow, respectively. H R199C has stronger binding ability for lactate. Isothermal Titration Calorimetry (ITC) was employed to measure the binding affinity of lactate to AARS2 (balk line) and R199C (red line). I–J R199C inhibits CPT2 and PDHA1 more strongly than AARS2. CPT2 (I) and PDC (J) specific activities were detected in purified CPT2 and cell lysates from COV434, AARS2^−/− COV434, and AARS2^−/− COV434 that co-expressed AARS2 or R199C, respectively (n = 3).

Fig. 4. R199C is a stronger follicle development inducer than AARS2.

A–F AARS2 inhibits the TCA cycle and accumulates anabolites in GCs. Heatmap of annotated metabolites in TCA cycle (A), glycolysis (B), and PPP (C). The inhibition of the TCA cycle is further confirmed by measuring OCR (D), ATP (E), and lactate production (F) in GCs of C57 and GOE mice. G–L R199C has a strong ability to reprogram GC metabolism. The metabolites changes in TCA cycle (G), glycolysis (H), PPP (I), and OCR (J), ATP (K), and lactate (L) were compared between AARS2^−/− COV434 cells and AARS2^−/− COV434 cells expressing either AARS2 or R199C. M AARS2 activates mTORC1 signaling in COV434 cells. p-S6K and p-4EBP were detected in COV434 cells, AARS2^−/− COV434 cells, AARS2^−/− COV434 cells expressing AARS2, and AARS2 mutants. N AARS2 promotes cell proliferation in COV434 cells. CCK-8 assay was employed to measure growth of COV434, AARS2^−/− COV434 cells, and AARS2^−/− COV434 expressing AARS2 or R199C. O AARS2 activates mTORC1 signaling in mouse GCs. p-S6K and p-4EBP were detected in C57 and AARS2 GOE mouse GCs. P AARS2 promotes cell proliferation in mouse GCs. CCK-8 assay was employed to measure growth of GCs from C57 and AARS2 GOE mice. Q AARS2 promotes mouse follicular development. Representative images of PD8-C57 and AARS2 GOE mice ovarian sections, stained for DAPI, FOXL2 (right panel). The secondary follicles in 6 mice ovaries were counted (left panel) (scale bars, 200 μm (top panel), 20 μm (bottom panel)).

Fig. 5. Lactate signal activates PPARγ to potentiate FSH action.

A–B AARS2 induces PPARγ in mouse GCs. The levels of PPARγ were compared between C57 and AARS2 GOE (A) and between C57 and Aars2 GKO (B) mouse GCs. C PPARγ was detected in COV434 cells, AARS2^−/− COV434 cells and in AARS2^−/− COV434 cells expressing either AARS2 or AARS2 mutations. D AARS2 CPT2-dependently induces PPARγ. The abilities of AARS2 and R199C to induce PPARγ were detected in AARS2^−/− COV434 cells and AARS2^−/− COV434 cells with silenced CPT2 through shRNAs. E Lactate induces PPARγ in cells. The PPARγ levels were detected in COV434 cells and AARS2^−/− COV434 cells with or without methyl L-lactate treatments. F Lactate induces PPARγ in mouse ovaries. The PPARγ levels were stained in ovarian sections of C57 and Aars2 GKO mice that were intraperitoneally injected with either PBS or 20 mg/kg lactate (scale bars, 200 μm (left panel), 50 μm (right panel)). G CPT2 lactylation accounts for lactate’s effects on inducing PPARγ. The PPARγ levels were detected in COV434 cells and CPT2 KD COV434 cells that were transfected with CPT2 and lactylation null CPT2^K457/8R. H THI increases and GW9662 decreases PPARγ signaling in mouse ovaries. Mean fluorescence intensity (MFI) of PPARγ signaling in ovarian sections of mice intraperitoneal injected with 30% DMSO, THI, or GW9662 (n = 6) was analyzed and represented as mean ± SEM. See also Fig. S6. I, J PPARγ inhibition diminished, and PPARγ activated FSH to promote mouse follicle development. Ovarian secondary follicles were counted in WT mice intraperitoneal injected with indicated FSH together with either 30% DMSO or GW9662 dissolved in 30% DMSO (I), and with either 30% DMSO or THI dissolved in 30% DMSO (J). Data are represented as mean ± SEM, n = 6. See also Fig. S6. K AARS2 overexpression promotes mouse follicle development. Ovarian secondary follicles were counted in WT and AARS2 GOE mice that were intraperitoneal injected with indicated FSH together with either 30% DMSO and GW9662 dissolved in 30% DMSO. Data are represented as mean ± SEM, n = 6. See also Fig. S6. L AARS2 promotes follicle growth in vitro. The maximum cross-sectional area of in vitro cultured WT and AARS2 GOE mouse follicles, with presence of gradient FSH, and under absence or presence of GW9662 was quantified. Data are represented as mean ± SEM, n = 6. See also Fig. S6. M Lactate promotes follicle development in mouse ovaries in an AARS2-dependent manner. Ovarian secondary follicles of WT and Aars2 GKO mice with or without intraperitoneal lactate injection were counted. Data are represented as mean ± SEM, n = 6. See also Fig. S6. N Lactate potentiates FSH to initiate follicle development in an AARS2-dependent manner. Ovarian secondary follicles of WT and Aars2 GKO mice that were with or without FSH, or lactate together with FSH, were quantified, n = 6. See also Fig. S6. O Lactate AARS2-dependently promotes follicles growth in vitro. The maximum cross-sectional area of WT and AARS2 GOE mice follicles cultured under with or without lactate in the culture media was quantified, n = 6. See also Fig. S6. P Lactate CPT2-dependently increases EdU incorporation. EdU positive cells in WT and CPT2 KD COV434 cells with or without lactate treatment were analyzed, n = 6. See also Fig. S6.

Fig. 6. High lactate signal promotes follicles depletion.

A AARS2 overexpression induces meiotic maturation defects. Spindle morphologies and chromosome alignment of in vitro C57 and AARS2 GOE mouse oocytes were monitored at 12 and 16 h after culturing. DNA was stained with DAPI, and spindle was stained with α-tubulin (scale bars, 20 μm). B–C SO treatment decreases lactate levels. Lactate concentration in COV434 cells (B, n = 3) and in mouse ovary (C, n = 6) were detected with or without the LDH inhibitor SO treatment. D–F Lactylation activates mTORC1 and GC proliferation. The effects of SO on p-S6K and p-4EBP (D) and growth (E, n = 3) were detected in COV434 cells and AARS2-overexpressing COV434 cells, and in C57 and AARS2 GOE mice GCs (F). G Lactylation regulates follicle development. The FOXL2 levels in ovaries of C57 and AARS2 GOE mice intraperitoneally injected with PBS or SO were quantified (right, n = 6). H–I α-CHCA treatment decreased mitochondrial lactate. Mitochondrial lactate was detected in COV434 cells (H) and Mouse ovaries (I) with or without α-CHCA treatment. J–L Lactylation activates mTORC1 and GC proliferation. The effects of α-CHCA on p-S6K and p-4EBP (J) and growth (K, n = 3) were detected in COV434 cells and AARS2-overexpressing COV434 cells, and in C57 and AARS2 GOE mice GCs (L). M Lactylation regulates follicle development. The FOXL2 levels in ovaries of C57 and AARS2 GOE mice intraperitoneally injected with DMSO or α-CHCA were quantified (right, n = 6). N–O Lactate AARS2-dependently activates GC mTORC1. p-S6K and p-4EBP were detected in GCs of C57 and Aars2 GKO mice (N) and in COV434 cells and AARS2^−/− COV434 cells (O), which were cultured with or without methyl L-lactate. P Lactate AARS2-dependently promotes GC proliferation. Proliferation of COV434 cells and AARS2^−/− COV434 cells was measured with or without methyl L-lactate in the culture media.

Fig. 7. Downregulating lactate signal prevents POI traits in AARS2 GOE mice.

A β-alanine inhibits AARS2- and R199C-catalyzed PDHA1 and CPT2 lactylation in vitro. In vitro AARS2- and R199C-catalyzed PDHA1 (left) and CPT2 (right) lactylation were detected when β-alanine was present and absent in the reaction mix. B–C β-alanine inhibits AARS2- and R199C-induced mTORC1 signaling. p-S6K and p-4EBP (B) and cell proliferation (C, n = 3) were detected in COV434 cells and AARS2-overexpressing COV434 cells that were cultured with or without β-alanine. D–E AARS2 deletion abrogated β-alanine to inhibit GC mTORC1 and proliferation. The β-alanine effects on p-S6K and p-4EBP (D), and proliferation (E, n = 3) of COV434 cells and AARS2^−/− COV434 cells were detected. F–H β-alanine inhibits follicle development. The β-alanine effects on p-S6K and p-4EBP (F), GC proliferation (G, n = 3), and number of secondary follicles (H, n = 6) of C57 and AARS2 GOE mice were detected. See also Fig. S7. I–K β-alanine AARS2-dependently inhibits follicle development. The effects of β-alanine on p-S6K and p-4EBP (I), GC proliferation (J, n = 3), and number of secondary follicles of C57 and Aars2 GKO mice (K, n = 6) were detected. See also Fig. S7. L–N β-alanine relieves POI traits in AARS2 GOE mice. The effects of intraperitoneally injected β-alanine on estrous cyclicity (L) and the cycle phase times (M) of C57 and AARS2 GOE mice were detected, n = 6. (N) β-alanine increases AARS2 GOE mice litter size. Effects of intraperitoneally injected β-alanine on the cumulative number of pups per mother of C57 and AARS2 GOE mice were measured, n = 6. O β-alanine reserves primordial follicles and decreases developed follicles of AARS2 GOE mice. The follicle number of C57 and AARS2 GOE mice with or without intraperitoneal β-alanine injection was counted at PD3-, 5-, and 7-weeks, n = 6. See also Figs. S7 and S8.