Fig 1: Impairment of Mitochondrial Ca2+ Buffering Due to Reduced MCU and MICU2 Levels in C9ORF72 MNs(A and B) (A) Representative image of Rhod 2AM-loaded neurons and (B) representative Ca2+ traces.(C) Ca2+ uptake in the mitochondria is lower in C9ORF72 MNs compared with the C9-Ed MNs and healthy control MNs on glutamate stimulation; n = 15–62 neurons from two to three independent differentiations.(D) Mitochondrial Ca2+ uptake in response to caffeine is significantly reduced in C9ORF72 MNs (***p < 0.001); n = 8–23 neurons from two to three independent differentiations.(E) Representative Ca2+ traces recorded in response to glutamate stimulation and CCCP.(F) Amplitude of Ca2+ released from mitochondria is significantly lower in C9ORF72 MNs (**p < 0.01, ***p < 0.001); n = 27–39 neurons from two independent differentiations.(G and H) Immunoblotting of MCU (G) shows reduced levels in C9ORF72 MNs (H) (*p < 0.05, **p < 0.01, ***p < 0.001).(I–K) (I) RNA-seq showing MICU2 levels are downregulated in C9ORF72 iPS-derived MNs compared with C9-Ed and (J) immunoblotting confirming reduced expression of MICU2 in C9-02 and (K) qPCR confirming reduced MICU2 expression (*p < 0.05, **p < 0.01, ***p < 0.001). Data are shown as mean ± SEM with one-way ANOVA and Sidak's post hoc tests.See also Figure S5.
Fig 2: YTHDF3 promotes translation of MCU protein by binding to the MCU mRNA dependently of METTL3-mediated m6A modification. (A–C), RIP analysis of the interaction of YTHDF3 (A) or YTHDF1 (B) or YTHDF2 (C) with MCU mRNA using total cell lysates of HAECs with or without METTL3 knockdown under mock-infected or HCMV-infected condition. Enrichment of MCU mRNA with an antibody against YTHDF3 or YTHDF1 or YTHDF2 was measured by qRT-PCR and normalized to input. Western blotting was performed as indicated. (D) MCU RNA enrichment by HA RIP qPCR. Cells were transfected with HA-YTHDF3, HA-?YTH, or HA-YTH, then exposed to HCMV infection. Magnetic beads coated with 5ug of antibody against HA were incubated with indicated cell lysates. (E) qRT-PCR analysis of MCU mRNA in HAECs with or without YTHDF3 knockdown under mock-infected or HCMV-infected condition. (F) Western blotting of MCU protein expression in HAECs with or without YTHDF3 knockdown under mock-infected or HCMV-infected condition. (G) Luciferase assays analysis of changes in translation efficiency of luciferase-MCU 3'-UTR mRNA by knockdown of YTHDF1-3. (H) Western blotting analysis to identify whether the impact of YTHDF3 on MCU protein expression was dependent on METTL3. Cells were transfected with Flag-YTHDF3 with or without METTL3 knockdown. All data are expressed as the mean ± SD of triplicate experiments. p values were calculated with student’s t-test, **p < 0.01 and ***p < 0.001. ns: no significant difference.
Fig 3: TARDBP MNs Show Reductions in Mitochondrial Ca2+ Uptake with Increased Expression of MICU1(A) Ca2+ traces after stimulation with glutamate followed by perfusion with CCCP.(B) Mitochondrial Ca2+ release after CCCP is significantly lower in TARDBP MNs (***p < 0.001); n = 22–75 neurons from two to three independent differentiations.(C) RNA-seq shows upregulation of MICU1 and validation by qPCR (*p < 0.05).(D) Immunoblotting shows no significant differences in the expression of MCU between healthy controls and TDP-43 MNs.(E and F) MitoTracker imaging shows higher intensity in TDP-43I383T (E), but no significant difference in TDP-43M337V (F). Data are presented as mean ± SEM with one-way ANOVA and Sidak's post hoc tests.See also Figure S6.
Fig 4: The mechanism diagram of vitamin D3 inhibiting HCMV-induced apoptosis in HAECs. HCMV infection upregulates the expression of METTL3 and YTHDF3. METTL3 methylates MCU at three m6A residues in the 3'-UTR, thereby promoting the binding of RNA-binding protein YTHDF3 to MCU-methylated mRNA and promoting the translation of MCU mRNA. Increased MCU protein expression is responsible for HCMV-induced apoptosis. Vitamin D3 inhibits METTL3 expression via the VDR/AMPK pathway, thereby suppressing the MCU m6A modification and MCU-induced apoptosis.
Fig 5: Vitamin D3 reduces MCU expression via the VDR/AMPK pathway and MCU contributes to HCMV-induced apoptosis. (A) Representative Western blotting graphs verify that vitD3 reduces HCMV-induced increase in MCU (phosphorylated and total MCU) expression via the VDR/AMPK pathway. (B) Statistical histogram representing a densitometric analysis performed to quantify the relative intensity of the total MCU expressive bands detected by Western blotting in (A). (C) Statistical histogram representing a densitometric analysis performed to quantify the ratio of p-MCU to total MCU in (A). (D) Representative Annexin-V/PI double-labeled flow cytometric assay shows that MCU inhibition attenuates the apoptosis induced by HCMV. Left, representative images of flow cytometry; right, statistical charts. (E) Representative Western blotting graphs verify that MCU inhibition attenuates the expression of apoptosis-associated proteins. HAECs were exposed to HCMV or not with or without Ru360 (5 µM) or spermine (10 µM). gB, a HCMV gene used to quantify HCMV infection. (F) Statistical histogram representing a densitometric analysis performed to quantify the relative intensity of the indicated bands detected by Western blotting in (E). All data are expressed as the mean ± SD of triplicate experiments. Student’s t-test, *p < 0.05, **p < 0.01, and ***p < 0.001. ns: no significant difference.
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