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, 20 (11), 2091-102

Parkin Mediates Beclin-Dependent Autophagic Clearance of Defective Mitochondria and Ubiquitinated Abeta in AD Models

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Parkin Mediates Beclin-Dependent Autophagic Clearance of Defective Mitochondria and Ubiquitinated Abeta in AD Models

Preeti J Khandelwal et al. Hum Mol Genet.

Abstract

Intraneuronal amyloid-β (Aβ) may contribute to extracellular plaque deposition, the characteristic pathology of Alzheimer's disease (AD). The E3-ubiquitin ligase parkin ubiquitinates intracellular proteins and induces mitophagy. We previously demonstrated that parkin reduces Aβ levels in lentiviral models of intracellular Aβ. Here we used a triple transgenic AD (3xTg-AD) mouse, which over-expresses APP(Swe), Tau(P301L) and harbor the PS1(M146V) knock-in mutation and found that lentiviral parkin ubiquitinated intracellular Aβ in vivo, stimulated beclin-dependent molecular cascade of autophagy and facilitated clearance of vesicles containing debris and defective mitochondria. Parkin expression decreased intracellular Aβ levels and extracellular plaque deposition. Parkin expression also attenuated caspase activity, prevented mitochondrial dysfunction and oxidative stress and restored neurotransmitter synthesis. Restoration of glutamate synthesis, which was independent of glial-neuronal recycling, depended on mitochondrial activity and led to an increase in γ-amino butyric acid levels. These data indicate that parkin may be used as an alternative strategy to reduce Aβ levels and enhance autophagic clearance of Aβ-induced defects in AD. Parkin-mediated clearance of ubiquitinated Aβ may act in parallel with autophagy to clear molecular debris and defective mitochondria and restore neurotransmitter balance.

Figures

Figure 1.
Figure 1.
Western blot, ELISA and mass spectroscopy show parkin decrease of ubiquitinated Aβ levels in 3xTg-AD mice. Immunhistochemistry of 20 μm thick cortical brain sections of (A) contralateral and (B) parkin-injected (ipsilateral) hemispheres from 3xTg-AD mice. (C) Higher magnification of parkin stained cells in the cortex. (D) Stereological counting of parkin-positive cells in the ipsilateral compared with contralateral hemispheres. (E) Western blot analysis and (F) quantification of parkin, amyloid precursor protein APP and its secreted fragments (75) and CTFs in brain lysates from contra and ipsilateral hemispheres. (G) Detergent and FA-extracted Aβ40 and Aβ42 levels in contralateral and ipsilateral (parkin injected) 3xTg-AD mouse brain. (H) Western blot of immunoprecipitated brain lysates and (I) spectrograms of immunoprecipitated brain lysates identified and sequenced by MALDI-TOF mass spectroscopy. (J) Table showing the identity of sequenced peptides. Asterisk is significantly different to contralateral, P< 0.05, t-test. Data are mean ± SD; n= 8 for western blot and ELISA, n = 3 for MADLI-TOF.
Figure 2.
Figure 2.
Beclin-dependent autophagic molecular steps and EM reveal parkin meditation of autophagic clearance in 3xTg-AD mice. (A) Western blot analysis and (B) quantification of autophagic pathway proteins, including beclin-1, Atg7, Atg12 and LC3-B levels in contra and ipsilateral hemispheres. Electron microscope (EM) scans of the cortex in 3xTg-AD mice showing accumulation of vesicles in the cytosol and fragmented ER (C and D) and defective mitochondria and molecular debris in vesicles (E). Cortical sections of parkin-injected hemispheres showing (F) clear cytosol and distinct nuclei, (G) mitochondria and healthy ER and (H) vacuoles packed with molecular and cellular debris in a clear and healthy looking cytosol. ER, endoplasmic reticulum; mit, mitochondria. (I) Histogram represents caspase-3 and 9 activities. Asterisk is significantly different to contralateral, P< 0.05, t-test. Data are mean ± SD; n = 8 for ELISA. n = 3 for EM.
Figure 3.
Figure 3.
Histology shows that parkin-mediated autophagy reduces intracellular Aβ levels and extracellular Aβ plaque. (A) 6E10-3, 3′-diaminobenzidine (DAB) staining of 20 μm thick brain sections under ×4 magnification showing the contralateral and ipsilateral (parkin-injected) hemispheres. Arrows indicate the CA1 hippocampus region showing immunoreactivity to Aβ. (B and C) Higher magnification of inserts showing Aβ immunoreactivity (arrows) in deep cortical layers. (D and E) 6E10-DAB staining of entorhinal cortex and (F and G) human-specific Aβ42 immunostainning of hippocampus brain sections. (H and I) Human-specific Aβ42 immunostainning of the cortex. (J and K) Brain sections show thioflavin-s-positive staining of the entorhinal cortex. (L and M) Silver staining of hippocampus at ×4, and (N and (O higher magnification (×20) view of hippocampal slices. (P and Q) Silver staining of sections of deep cortical layers. Sections stained with GFAP in the (R and S) cortex and (T and U) the entorhinal cortex.
Figure 4.
Figure 4.
High-frequency [1H]-13C NMR spectroscopy suggests that parkin-facilitated clearance of defective mitochondria attenuates oxidative stress and restores TCA cycle activity. High-frequency [1H]-13C NMR spectroscopy showing (A) histograms that represent metabolic flux of 13C label from glucose into Succ C2/C3 and Lac C3; (B) metabolic pool size of Succ and Lac; (C) metabolic flux into Glu C4, (D) Gln C4 and (E) GABA C2; (F) metabolic pool size of Glu, Gln and GABA. (G) Schematic representation of neuronal and astrocytic compartments, depicting Glu–Gln cycle, cytosolic and mitochondrial metabolism and the effects of Aβ on cell compartmentalization. Aβ decreases 13C flux into the mitochondria and stimulates Lac production resulting in oxidative stress. Aβ decreases TCA-derived neurotransmitters Glu and GABA and depresses the Glu–Gln cycle. Parkin decreases the levels of intracellular Aβ, enhances 13C flux into mitochondrial TCA cycle and decreases Lac production, thus alleviating oxidative stress. Parkin reverses Aβ-induced decrease in Glu and GABA levels via amelioration of TCA cycle activity, which is independent of Glu–Gln cycle, restoring synaptic neurotransmitter balance. TCA, tricarboxylic acid; Glu, glutamate; Gln, glutamine; GABA, γ-amino butyric acid; Succ, succinate; Lac, lactate. Asterisk is significantly different to contralateral or as indicated, P< 0.05, t-test. Data are mean ± SD; n= 4.

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