Fig 2: OMI/HTRA2 protein level is higher in AD than control brains. a, b Immunohistochemical detection of OMI/HTRA2 in human postmortem brain sections of frontal cortex. Representative image of sections from non-demented controls (a) and AD (b) subjects. Staining: OMI/HTRA2 (brown): anti-OMI/HTRA2 rabbit primary antibody, biotinylated horse anti-rabbit antibody, and ABC-Elite HRP. Counterstaining: hematoxylin (blue). Scale bar = 20 µm. c Quantitative analyses showing the number of cells that present either a diffuse or punctuate staining pattern of OMI/HTRA2 in postmortem brain tissues. d, e Quantification of activated OMI/HTRA2 protein levels in postmortem brain homogenates (BH) of AD subjects (n = 6) and non-demented controls (n = 6), by sandwich ELISA. d Box plot graph showing the overall level of activated OMI/HTRA2 in AD compared to controls, measured in all the extracts from the three brain regions and expressed as ng/mg of total protein. e Graph showing the level of activated OMI/HTRA2 in each brain region separately, expressed as percentage of the control levels. f, gOMI/HTRA2 gene expression was quantified in the postmortem brain samples from the MFG, SPG, and STG brain regions. f Graph showing the normalized transcript levels of OMI/HTRA2 in each of the analyzed brain regions. g Negative correlation of OMI/HTRA2 transcript levels and activated OMI/HTRA2 protein levels, expressed as % of the mean of the controls (Ctrl). Activated OMI/HTRA2 was quantified in medial frontal gyrus (MFG), superior temporalis gyrus (STG), and superior parietal gyrus (SPG) brain regions. The total activated OMI/HTRA2 refers to the sum of its protein levels in all extracts. AD, dark gray squares; non-demented controls, light gray squares. Mean values ± S.E.M. are shown (two asterisks, p < 0.01; one asterisk, p < 0.05)

Fig 3: OPA1 proteolytic cleavage by mdivi-1 treatment associated with altered expression of mitochondrial proteases. (A) Representative Western blot and densitometric quantification showing that mdivi-1 (100 µmol/L, 24 h) induced proteolytic cleavage of L-OPA1 under the autophagy-modifying conditions induced by Atg7 overexpression, p62 siRNA knockdown, and p62 siRNA knockdown on Atg7 overexpression in cardiomyocytes. Ponceau S protein staining of the membrane after transfer was used as a loading control to confirm approximately equal loading across the gel (n = 4). (B) Representative Western blot showing that mdivi-1 (100 µmol/L) induced temporal changes in the expression of different mitochondrial proteases, ATPases, and GTPases in cardiomyocytes at different time points (3, 6, 12, or 24 h). Densitometric quantification showing that mdivi-1 treatment induced temporal changes in (C) OMA1, (D) YME1L1, (E) HtrA2, (F) LonP1, (G) ClpP1, (H) paraplegin, (I) VCP, and (J) Rab24. HSP60 was used as a house keeping protein for mitochondria and Ponceau S protein staining of the membrane after transfer was used as a loading control to confirm approximately equal loading across the gel (n = 4). Bars represent mean ± SEM. P values were determined by one-way ANOVA followed by Tukey's post hoc test.NS = non-significant.

Fig 4: Positive correlation between OMI/HTRA2 and ACHE gene and protein expression. OMI/HTRA2 and ACHE gene and protein expression were quantified in AD subjects’ (n = 6) and non-demented controls’ (n = 6) postmortem brain, in medial frontal gyrus (MFG), superior temporalis gyrus (STG), and superior parietal gyrus (SPG) brain regions. a–d Graphs showing the positive correlation between OMI/HTRA2 and ACHE gene expression, in the average of the three analyzed brain regions (a) and in each region separately (b–d); e graph showing the positive correlation between activated OMI/HTRA2 protein level and AChE-R protein level in the STG region. ACHE = acetylcholinesterase gene; AChE-R = “Read-through” AChE splice variant protein

Fig 5: Positive correlation of activated OMI/HTRA2 with ChAT protein expression and activity. ChAT protein expression and activity were quantified in AD subjects’ (n = 6) and non-demented controls’ (n = 6) postmortem brain medial frontal gyrus (MFG), superior temporalis gyrus (STG), and superior parietal gyrus (SPG) regions, within the soluble (s), ionic (i), and membrane-bound (m) protein extracts, and then correlated to the activated OMI/HTRA2 levels in the same regions. a Graph showing the positive correlation of OMI/HTRA2 level and total ChAT protein level in the MFG brain region. b–d Graphs showing the positive correlation of OMI/HTRA2 level with the total ChAT (b), m-ChAT (c), and s-ChAT (d) enzymatic activity in the MFG brain region. ChAT = choline acetyltransferase