Fig 1: Autofluorescent lesions in the homozygous Cln3Δex7/8 mouse retinal OPL/INL were immunoreactive for mitochondrial ATP synthase F0 sub-complex subunit C.Immunoreactivity of subunit C, a marker known to accumulate in the lysosomes and ceroids/lipofuscins in JNCL [7, 8], was previously reported in the ONL, INL, and RGC of 10 month old homozygous Cln3Δex7/8 mice on an outbred 129Sv/Ev/CD1 mixed background [2]. However, CD1 background itself carried retinal degenerative genetic loci [9]. Here we observed subunit C immunoreactivity in the photoreceptor IS, OPL, INL and IPL in both WT and homozygous Cln3Δex7/8 mice (22-24 month old) on a C57BL/6J background which did not harbor the retinal degenerative Rd1 or Rd8 mutation. Specifically, autofluorescent lesions (green) prominent in the OPL and INL of the homozygous Cln3Δex7/8 mouse retina was subunit C immunoreactive. Due to the spectral properties of the autofluorescent lesions in the homozygous Cln3Δex7/8 mouse retina (see Fig. 1), autofluorescence was collected with a 487.9 nm laser and a 525/25 nm emission filter. Subunit C IHC-IF was performed on retinal cyro-sections stained with a rabbit polyclonal anti-subunit C antibody (Abcam ab181243) followed by an Alexa Fluor Plus 647-labeled secondary antibody against rabbit IgG(H+L) (Thermo Fisher Scientific A32733) and confocal fluorescent images were acquired with a 638.6 nm laser and a 700/75 nm emission filter. (A) Representative DIC and confocal fluorescent images of homozygous Cln3Δex7/8 mouse retina show autofluorescent lesions (green channel; pointed by yellow arrows) in the OPL/INL immunoreactive for subunit C (red, pseudo-color). (B) Representative DIC and confocal fluorescent images of WT mouse retina show lack of autofluorescent lesions (green) in the OPL/INL despite subunit C immunoreactivity in other areas (red, pseudo-color). (C) Control fluorescent images of homozygous Cln3Δex7/8 mouse retina acquired and processed exactly as in (A) except that anti-subunit C was omitted during immunostaining. Note that bringing up signals in the red channel revealed autofluorescence of the autofluorescent lesions rather than subunit C immunoreactivity in the OPL/INL. (D) Control fluorescent images of WT mouse retina acquired and processed exactly as in (B) except that anti-subunit C was omitted during immunostaining.
Fig 2: Functional analysis of Oligomycin A-Csub binding in PTP inhibition in primary human cardiac fibroblasts. (A) Immunofluorescence assay for mitochondrial localization of mutants: FLAG-tag (green), ATP5A as mitochondrial marker (red), and nuclei (blue, DAPI). Scale bar in white is 10 µm. (B) PTP activity measurement (left, analysis of PTP activity; right, representative kinetics) with mutants expressed in HCF and compared with pCMV6 and ATP5G1WT. (C) PTP activity measurement (left, analysis of PTP activity; right, representative kinetics) with mutants expressed in HCF with or without Oligomycin A pre-treatment (10 µM, 30 min). Graphs show mean ± SEM. Statistical differences were analyzed by one-way ANOVA. ***, p < 0.001; ****, p < 0.0001, n.s. not statistically significant.
Fig 3: PFKFB3 inhibition in CLN7 patient-derived neural precursor cells restores mitochondrial condensation.a Schematic representation of the locations of the CLN7 mutations found in patient 380 (Pa380, c.881 C > A; pT294K) and patient 474 (Pa474, c.1393 C > T; p.R465W). b iPSC characterization in Pa474 with the pluripotency markers OCT4, SOX2, Nanog, and Tra-1-60. Scale bar, 50 µm. c Characterization of differentiated neurons derived from iPSC in Pa474. Scale bar, 50 µm. d NPCs characterization in Pa380, Pa474, and a healthy, age-matched control individual. Scale bar, 50 µm. e Immunocytochemical analysis of SCMAS abundance in NPCs derived from Pa474 iPSC. Data are the mean ± SEM values from n = 4 (control), n = 3 (Pa474) independent samples (two-tailed Student’s t test). Scale bar, 50 µm. f Mitochondrial ROS analysis in NPCs. Data are the mean ± SEM values from n = 3 independent samples (two-tailed Student’s t test). g Immunocytochemical analysis of mitochondrial marker ATP5A in NPCs derived from Pa380, Pa474, and healthy-matched control patients. Scale bar, 50 µm. The right panel shows a representative pixel intensity profile of ATP5A across the maximal axis of the cell that departs from the nucleus. h Representative image of NPCs derived from Pa474 iPSC incubated with AZ67 for 24 h, fixed and subjected to immunocytochemical analysis for ATP5A. Scale bars, 60 µm (upper images of each condition) and 20 µm (lower images of each condition). The right panel shows a representative pixel intensity profile of ATP5A across the maximal axis of the cell that departs from the nucleus. Source data are provided as a Source Data file.
Supplier Page from Abcam for Anti-ATP synthase C antibody [EPR13907]