Fig. 1.

Changes in hPreP activity/expression in brain mitochondria of AD temporal lobe. Degradation of biotin-Aβ (A, B) and F₁β presequence peptide (C). hPreP proteins extracted from the cortical mitochondria of the indicated AD and non-AD brains (A, B) were incubated with biotin-labeled Aβ (0.25 μg), and then subjected to immunoblotting with Extravidin peroxidase conjugated IgG and detection with ECL to reveal immunoreactive biotin Aβ. C) For degradation of F₁β substrate, mitochondrial hPreP proteins were incubated with pF₁β followed by the immunoblot with antibody to pF₁β. Densitometry of the combined immunoreactive Aβ (A, B) or F₁β bands (C) using NIH Image program is shown. D) Determination of hPreP proteolytic activity and hPreP-dependent degradation of Aβ in brain mitochondrial fraction. Biotin-labeled Aβ was completely degraded by the isolated human mitochondrial matrix hPreP protein (MA-hPreP) (lanes 2 and 6). The oPh (ortho-phenantroline, an inhibitor of PreP proteolytic activity) blocked the degradation of biotin Aβ (lanes 3 and 7). The immuno-inactivation assay confirmed hPreP-induced degradation of Aβ. When isolated human mitochondrial matrix hPreP protein (MA-hPreP) was pre-incubated with antibody against hPreP, degradation of Aβ_1-40 (lane 4 & 8) was almost completely inhibited, whereas incubation of pF₁β presequence antibodies did not affect Aβ degradation (lanes 5 and 9). E) Immunoblotting of brain mitochondria from AD and non-AD brains for hPreP. The upper panel denotes representative immunoblots for human PreP and Hsp60. Hsp60 was used as a mitochondrial marker and protein loading controls. The lower panel shows densitometry of hPreP immunoreactive bands from the indicated brain mitochondria. NS: no statistical significance between these groups (p > 0.05). Mitochondria were isolated from 12 AD brains and 8 non-AD brains. All experiments were performed in triplicates.