Figure 4

CathD-deficient brains show increased HMW α-synuclein-positive oligomers in formic acid extracts. (A) Western blot analyses carried out under reducing conditions of formic acid extracts obtained from previously generated SDS pellets of wild-type (+) and ctsd-/- (-) mouse brains (see Fig. 3 above). Immunoblots were probed with high sensitivity, polyclonal aSyn Abs, hSA-2 and mSA-1, and anti-β-actin Ab, as indicated. Under these conditions, HMW aSyn-positive smears and trace amounts of monomeric, 16 kDa aSyn (aSyn, FL) are detectable in these formic acid extracts.(B) Quantification of aSyn-reactive species by densitometry examining the ratio of HMW smears (blue bars) and 16 kDa monomer (empty bars) versus the signal of β-actin-positive bands. Note, an ~15 per cent increase in HMW oligomers of aSyn is detected in formic acid extracts of ctsd-/- brain by independent analysis of both mSA-1 (B) and hSA-2 immunoblots. (C) NP-40 and SDS extracts probed with anti-ubiquitin consistently reveal HMW immunoreactive smears in SDS brain extracts of ctsd-/- mice. Representative examples of twelve mouse brains are shown (n = 4, ctsd+/+; n = 8, ctsd-/-).