Nilotinib-induced autophagic changes increase endogenous parkin level and ubiquitination, leading to amyloid clearance

Abstract

Alzheimer's disease (AD) is a neurodegenerative disorder associated with amyloid accumulation and autophagic changes. Parkin is an E3 ubiquitin ligase involved in proteasomal and autophagic clearance. We previously demonstrated decreased parkin solubility and interaction with the key autophagy enzyme beclin-1 in AD, but tyrosine kinase inhibition restored parkin-beclin-1 interaction. In the current studies, we determined the mechanisms of nilotinib-induced parkin-beclin-1 interaction, which leads to amyloid clearance. Nilotinib increased endogenous parkin levels and ubiquitination, which may enhance parkin recycling via the proteasome, leading to increased activity and interaction with beclin-1. Parkin solubility was decreased and autophagy was altered in amyloid expressing mice, suggesting that amyloid stress affects parkin stability, leading to failure of protein clearance via the lysosome. Isolation of autophagic vacuoles revealed amyloid and parkin accumulation in autophagic compartments but nilotinib decreased insoluble parkin levels and facilitated amyloid deposition into lysosomes in wild type, but not parkin(-/-) mice, further underscoring an essential role for endogenous parkin in amyloid clearance. These results suggest that nilotinib boosts the autophagic machinery, leading to increased level of endogenous parkin that undergoes ubiquitination and interacts with beclin-1 to facilitate amyloid clearance. These data suggest that nilotinib-mediated autophagic changes may trigger parkin response via increased protein levels, providing a therapeutic strategy to reduce Aβ and Tau in AD.

Key message: Parkin solubility (stability) is decreased in AD and APP transgenic mice. Nilotinib-induced autophagic changes increase endogenous parkin level. Increased parkin level leads to ubiquitination and proteasomal recycling. Re-cycling decreases insoluble parkin and increases parkin-beclin-1 interaction. Beclin-1-parkin interaction enhances amyloid clearance.

Conflict of interest statement

The authors have read the manuscript and declare no conflict of interest whatsoever.

Figures

Fig. 1. Nilotinib increases parkin level and induces protein clearance

A) WB of total brain lysates on 4–12% SDS-NuPAGE gel showing the levels of parkin (1st blot), LC3-II (2nd blot) and Beclin-1 (3rd blot) relative to actin in C57BL/6 mice treated with Nilotinib; and B) graphs represent densitometry analysis. C) Parkin E3 ubiquitin ligase function in B35 neuroblastoma cells treated with DMSO or Nilotinib for 24hr, insert shows parkin input and IP after normalization. D) WB of immunoprecipitated ubiquitin (1st blot, input) probed with parkin antibody (2nd blot) on 4–12% SDS-NuPAGE gel. E) WB of immunoprecipitated parkin probed with ubiquitin antibody (2nd blot) on 4–12% SDS-NuPAGE gel. F) Parkin levels as measured by ELISA in B35 expressing Aβ42 and treated with Nilotinib. G). Proteasome activity via Chymotrypsin-like assays in human neuroblastoma cells (n=12) ± Nilotinib. H) Parkin ELISA in AVs isolated from 1 year old male C57BL/6 or parkin−/− mice injected 10mg/kg for 3 weeks. * significantly different to control or as indicated, Mean±SEM, ANOVA with Neumann Keuls multiple comparison, p<0.05.

Fig. 2. Nilotinib is a non-specific TKI

WB in brain lysates in A) 1-year old male C57BL/6 mice treated IP with 10mg/kg Nilotinib for 3 weeks showing T412 Abl (1st blot) and phospho-tyrosine proteins (2nd blot) relative to actin, and B) WB in rat neuroblastoma B35 cells transfected with Abl cDNA or shRNA (2nd blot) showing total parkin level (1st blot), and immunoprecipitated parkin probed with ubiquitin antibody (2nd blot) relative to actin on 4–12% SDS-NuPAGE gel. C) Densitometry of total parkin levels. D). ELISA of Aβ1–42 levels in B35 cells co-transfected with either Aβ1–42 and Abl or Aβ1–42 and Abl shRNA. Staining of 20μm thick hippocampal sections in C57BL/6 mice treated IP with 10mg/Kg Nilotinib or DMSO for 3 weeks showing E) T412 Abl, F) parkin (insert is PRK8 staining in parkin−/− cortex showing antibody specificity. and G) parkin and T412 Abl co-localization in DMSO treated mice. H) T412 Abl, I) parkin and insert parkin staining in neuronal processes, J) parkin and T412 Abl co-localization in Nilotinib treated mice. * significantly different to control or as indicated, Mean±SEM, ANOVA with Neumann Keuls multiple comparison, p<0.05.

Fig. 3. Nilotinib-induced parkin activation clears brain amyloid

A) WB of post-mortem cortical extracts of AD patients (N=12 AD and 7 control) on 10% SDS Nu-PAGE and B) Graph is densitometry and ratio of Abl and p-Abl and parkin. C) WB analysis on 4–12% SDS Nu-PAGE gels of brain extracts from Tg-APP treated with Nilotinib or DMSO showing APP, Abl, p-Abl and CTFs and MAP-2 as control (n=11). D) Graph represents ELISA levels of soluble and insoluble mouse parkin in the brain of 8–12 months old Tg-APP mice (n=9) injected (IP) with 10mg/kg (daily for 3 weeks). Graph represents ELISA levels of soluble and insoluble E) Aβ1–42 and F) Aβ1–40 in the brain of 8–12 months old Tg-APP mice (n=9) injected (IP) with 10mg/kg once a day for 3 weeks. G) Graph represents ELISA levels of mouse p-Tau in the brain of 8–12 months old Tg-APP mice (n=9). Graph represents ELISA levels of H). soluble and insoluble human Aβ1–42, and I) ELISA levels of mouse p-Tau in the brain of mice (N=9) injected (IP) with 10mg/kg (3 weeks). * Significantly different to control or as indicated, Mean±SEM, ANOVA with Neumann Keuls multiple comparison, p<0.05.

Fig. 4. Nilotinib promotes autophagic clearance of amyloid

WB of brain extracts on 4–12% Nu-Page SDS gels A) in lentiviral Aβ1–42 in WT mice±Nilotinib showing, Abl, p-Abl, LC3, parkin, and beclin-1 relative to MAP-2 (N=9) and B) in Tg-APP±Nilotinib showing, parkin, beclin-1 and LC3 relative to tubulin. C). WB of brain extracts on 4–12% Nu-Page SDS gels in lentiviral Aβ1–42 in WT and parkin−/− mice±Nilotinib showing parkin, LC3, and beclin-1 relative to actin (N=9). D) Human Aβ1–42 ELISA before and after Nilotinib treatment in B35 rat neuroblastoma cells (N=12) in media, soluble and insolublelysates in the presence and absence of shRNA beclin-1. E) ELISA level of human Aβ1–42 and F) mouse p-Tau in autophagic vacuoles isolated from lentiviral Aβ1–42 expressing WT and parkin−/− mice (N=5).* Significantly different or as indicated, # significantly different to Aβ1–42 expressing WT, Mean±SEM, ANOVA with Neumann Keuls multiple comparison, p<0.05.

Fig. 5. Nilotinib increases parkin level and decreases plaque load

Staining of 20μm brain sections shows plaque formation within various brain regions in different A-C) Tg-APP+DMSO and E-G) Nilotinib group after 3-week treatment. Staining of 20μm thick brain sections shows D) thioflavin-S staining in Tg-APP+DMSO and Tg-APP+Nilotinib. Staining of 20μm thick brain sections shows I). parkin, J). Aβ1–42 and K) merged figure in hippocampus of Tg-APP mice after 3 weeks of DMSO treatment, and L). parkin, M). Aβ1–42 and N) merged figure in hippocampus of Tg-APP mice after 3 weeks of Nilotinib treatment. O). parkin, P). Aβ1–42 and Q) merged figure in cortex of Tg-APP mice after 3 weeks of DMSO treatment, and R). parkin, S). Aβ1–42 and T) merged figure in cortex of Tg-APP mice after 3 weeks of Nilotinib treatment. Intracellular Aβ1–42 within the U). hippocampus of lentiviral Aβ1–42 injected mice, inset higher magnification, and V) and Nilotinib clearance of intracellular Aβ1–42 (inset is higher magnification). Staining of 20μm brain sections shows intracellular Aβ1–42 within the W). cortex of lentiviral Aβ1–42 injected mice, inset higher magnification, and X) and Nilotinib clearance of intracellular Aβ1–42, inset is higher magnification.

Fig. 6. Nilotinib eliminates plaques in lentiviral Aβ1–42 injected WT but not parkin−/− mice

Staining of 20μm brain sections shows plaque formation within various brain regions in different A-C) lentiviral Aβ1–42+DMSO WT mice and D-F) Nilotinib group after 3-week treatment. G-I) lentiviral Aβ1-42+DMSO in parkin−/− mice and J-L) Nilotinib group after 3-week treatment. Transmission EM shows autophagic defects in different lentiviral Aβ1–42+DMSO WT brains within M). hippocampus showing distrophic neurons, N) cortex showing accumulation of autophagic vacuoles, O). hippocampus showing enlarged lysosomes. And lentiviral Aβ1–42+Nilotinib WT brains within P). hippocampus, Q) cortex showing clearance of autophagic vacuoles, R). hippocampus. And lentiviral Aβ1–42±Nilotinib in parkin−/− brains within S&V). hippocampus showing distrophic neurons, T&W) cortex showing accumulation of autophagic vacuoles, U&X). hippocampus showing accumulation of autophagic vacuoles.