Abl Activation Reduces Parkin Activity and Arrests Autophagy

Research Article

Austin Neurol & Neurosci. 2016; 1(2): 1010.

Abl Activation Reduces Parkin Activity and Arrests Autophagy

Lonskaya I¹, Hebron M¹, Chen W1,2, Feng Y¹, Schachter JB³ and Moussa C¹*

¹Department of Neurology, Laboratory for Dementia and Parkinsonism, Georgetown University Medical Center, USA

²Department of Traditional Chinese Medicine, Xuanwu Hospital, Capital Medical University, China

³Neuroscience Discovery, Merck Research Laboratories, USA

*Corresponding author: Charbel E Moussa, Department of Neurology, Laboratory for Dementia and Parkinsonism, Georgetown University School of Medicine, USA

Received: August 09, 2016; Accepted: October 26, 2016; Published: October 31, 2016


The non-receptor tyrosine kinase Abelson (Abl) is activated in neurodegeneration and regulates Parkin activity via unknown mechanisms. Parkin plays a critical role in autophagic clearance of toxic protein accumulation. To study the effects of Abl activation on Parkin, lentiviral Tau or Aβ42 were expressed in the hippocampus of 1-year old Tet-off mice that conditionally express active Abl (AblPP/tTA). Abl activation reduces endogenous Parkin activity and arrests autophagic flux in brains accumulating Tau and Aβ42. Our analysis suggests that Abl activation regulates Parkin activity via ubiquitination. Abl activation also decreases Parkin stability, leading to accumulation of insoluble protein. Since Parkin expression promotes autophagic clearance of p-Tau and Aβ42 and protects against cell death, these new data suggest that Abl inhibition is an alternative strategy to activate Parkin and reduce protein accumulation in neurodegenerative diseases.

Keywords: Abl; Parkin; Autophagy; Tau; Aβ42


Abelson (Abl) encodes a protein tyrosine kinase that is distributed in the nucleus and the cytoplasm and is involved in a wide range of functions [1]. Abl activation is implicated in a number of neurodegenerative diseases [2-5]. Phosphorylated Abl is detected with both neuritic plaques and Neuro Fibrillary Tangles (NFTs) in the hippocampus and entorhinal cortex in Alzheimer’s Disease (AD) [3,5-7]. In AD mouse models β-amyloid (Aβ) activates Abl and Tau hyper-phosphorylation (p-Tau), while Abl inhibition reduces Aβ and p-Tau and reverses cognitive decline [8,9]. Intracranial injection of Aβ fibrils into the mouse hippocampus up-regulates Abl [9]. In primary neuronal culture, Abl inhibition prevents Aβ fibrillation and cell death [10].

Some studies suggest that Abl activation directly phosphorylates Parkin to alter Parkin’s E3 ubiquitin ligase function [2,4,11], thereby altering Parkin regulation of autophagy and proteasome activity [12- 15]. Mutations in the gene coding for Parkin (Park2) are associated with autosomal recessive early onset Parkinson Disease (PD) [16]. Parkin stability is reduced in the nigrostriatum of sporadic PD [4,17] and temporal lobe of AD brains [17-19]. Relevant to AD pathology, Aβ accumulation leads to formation of undigested autophagic vacuoles. Parkin expression enhances autophagic clearance of Aβ and resolves undigested autophagic vacuole accumulation [18,20,21]. Pharmacologic inhibition of Abl increases Parkin activity and reduces Aβ and p-Tau in models of neurodegeneration [22-25].

In the current studies we evaluated the role of Abl activation on Parkin modification, including phosphorylation, ubiquitination and activity. We used lentiviral delivery of either human four repeat (4R) Tau or Aβ42 into the hippocampus of 1-year old conditional mice (AblPP/tTA) that express active Abl under a neuron-specific promotor (CamKIIα) regulated by doxycycline (Tet-off) [26]. We determined the effects of Abl activation on Parkin modification and autophagic p-Tau and Aβ42 clearance after 6 weeks of Abl activation via Doxycycline (Dox) withdrawal. The results indicate that Abl activation reduces Parkin ubiquitination, and thereby reduces Parkin activity. This leads to a decrease in Parkin solubility that is reminiscent of Parkin modification in PD and AD.

Materials and Methods

Stereotaxic injection

We used lentiviral gene delivery of 4R human Tau or Aβ42 into the CA1 hippocampus of 1 year old AblPP/tTA mice [26]. Peter Davies’s group generated conditional mice under a neuron-specific promotor (CamKIIα) regulated by Dox (Tet-off) to express active Abl [26]. AblPP/tTA mouse colony is viable and expresses active T412 Abl when taken off-dox for 6 weeks. Stereotaxic surgery was performed on AblPP/tTA mice to inject 1x109 m.o.i (Multiplicity of Infection) lentiviral Tau or Aβ42 with and without 1x109 m.o.i lentiviral Parkin or LacZ into hippocampus as previously described [21,27-29]. Males and females were divided into group 1 on-Dox and group 2 off-Dox (control chow) for 3 weeks and stereotaxic surgery was performed, leading to gene expression in the entire mouse hemisphere [21]. Mice were sacrificed 3 weeks post-injection (total period of Dox withdrawal was 6 weeks). All experiments were conducted in full compliance with the recommendations of Georgetown University Animal Care and Use Committee (GUAUC).

Cell Culture and transfection

Human neuroblastoma M17 cells were grown in 24 well dishes (Falcon) and transiently transfected with 3μg human wild type or mutant T240R Parkin cDNA for 24 hours. Cells were then either transfected with 3μg Abl shRNA for 24hr or treated with 10?m Nilotinib (AMN-107, Shellacked Chemical, LLC, USA) or DMSO (1μL) for 24 hours and harvested after a total of 48 hours of transfection for Western blot and immunoprecipitation.

Wetsern blot (WB) analysis

The brain was isolated and tissues will be homogenized in 1x STEN buffer (50 mM Tris (pH 7.6), 150 mM NaCl, 2 mM EDTA, 0.2 % NP-40, 0.2 % BSA, 20 mM PMSF and protease cocktail inhibitor), centrifuged at 10,000 x g for 20 min at 40C and the supernatant containing the soluble protein fraction was collected. To extract insoluble Aβ42, the pellet was re-suspended in 30% formic acid and centrifuged at 10,000 g for 20 min at 4°C and the supernatant was collected. Extracts were analyzed by Western Blot (WB) on SDS NuPAGE 4-12% Bis-Tris gel (Invitrogen, NP0301BOX). β-actin was probed (1:1000) with polyclonal antibody (ThermoScientific, PA121167). Total Abl was probed with (1:500) rabbit polyclonal antibody (Thermo Fisher) and p-Abl (Y214) with (1:500) rabbit polyclonal antibody (Millipore). Total Parkin was probed and immunoprobed (1:1000) with PRK8 antibody (Pierce), and Parkin phospho-serine65 was probed (1:500) with Rabbit polyclonal antibodies (Abcam). A rabbit polyclonal (Pierce) anti-LC3 (1:1000) antibody and lysosomal fractions were probed with (1:1000) rabbit polyclonal Lysosmal associated membrane protein-2 (LAMP2) antibodies (Abcam) were used. Rabbit anti-ubiquitin (Santa Cruz Biotechnology) antibody (1:1000) and total phospho-tyrosine antibody (1:2000) 4G10 (EMD Millipore) were used. Total tau was probed (1:1000) with Tau-5 monoclonal antibody (Chemicon, Temecula, CA, USA), and phosphorylated tau was probed (1:1000) with epitopes against polyclonal serine-396 (Chemicon, Temecula, CA, USA), polyclonal AT8 (1:1000) Serine-199/202 (Biosource, Carlsbad, CA, USA), polyclonal AT180 (1:1000) threonine-231 (Biosource, Carlsbad, CA, USA) and monoclonal (1:1000) human specific (HT7) antibody (Thermo Scientific). WBs were quantified by densitometry using Quantity One 4.6.3 software (Bio Rad).


Mouse brains were homogenized in 1XSTEN buffer and the soluble fraction was isolated as indicated above. The lysates were pre-cleaned with immobilized recombinant protein G agarose (Pierce #20365), and centrifuged at 2500 × g for 3 min at 4°C. The supernatant was recovered and quantified by protein assay and a total of 100 mg protein was incubated for 1 h at 4°C with primary 1:100 mouse anti-Parkin (PRK8) antibodies in the presence of sepharose G and an IgG control with primary antibodies. The immunoprecipiates were collected by centrifugation at 2500 × g for 3 min at 4°C, washed 5× in PBS, with spins of 3 min, 2500 × g using detergent-free buffer for the last washing step and the proteins were eluted according to Pierce instructions (Pierce #20365). After IP, the samples were sizefractionated on 4–12% SDS-NuPAGE and transferred onto 20 μm nitrocellulose membranes.

Immuno histo chemitry (IHC) of brain sections

Animals were deeply anesthetized with a mixture of Xylazine and Ketamine (1:8), washed with 1X saline for 1 min and then perfused with 4% Para Formaldehyde (PFA) for 15-20 min. Brains were quickly dissected out and immediately stored in 4% PFA for 24h at 40C, and then transferred to 30% sucrose at 40C for 48h. Tissues were cut using a cryostat at 40C into 20 μm thick sections and stored at -200C. Mouse monoclonal (6E10) antibody (1:100) with DAB were used (Covance) followed by DAB staining. Tau staining was performed with HT7 (1:1000), AT180 (1:5000) and AT8 (1:3000) followed by DAB staining according to manufacturer’s instructions (Sigma). Cupric silver staining that detects degenerating fibers and neurons was also performed according to manufacturer’s protocol (FD Neurotechnologies, Baltimore,MD). Stereological methods were applied by a blinded investigator using unbiased stereology analysis (Stereologer, Systems Planning and Analysis, Chester, MD) as previously described [21,27-29].

Subcellular fractionation for isolation of autophagic compartments

To determine autopahgic flux in vivo, 0.5g of animal brains were homogenized at low speed (Cole-Palmer homogenizer, LabGen 7, 115 Vac) in 1xSTEN buffer and centrifuged at 1,000g for 10 minutes to isolate the supernatant from the pellet. The pellet was re-suspended in 1xSTEN buffer and centrifuged once to increase the recovery of lysosomes. The pooled supernatants were centrifuged at 100,000 rpm for 1h at 4°C to extract the pellet containing Autophagic Vacuoles (AVs) and lysosomes. The pellet was re-suspended in 10 ml (0 .33 g/ml) 50% Metrizamide and 10 ml in cellulose nitrate tubes. A discontinuous Metrizamide gradient was constructed in layers from bottom to top as follows: 6 ml of pellet suspension, 10 ml of 26%; 5 ml of 24%; 5 ml of 20%; and 5 ml of 10% Metrizamide [30]. After centrifugation at 10,000 rpm for 1 hour at 40C, the fraction floating on the 10% layer (Lysosome) and the fractions banding at the 24%/20% (AV 20) and the 20%/10% (AV10) Metrizamide inter-phases were collected by a syringe and examined via p-Tau and Aβ1-42 specific ELISA.

Aβ42 and p-Tau and α-synuclein enzyme-linked immuno sorbent assay (ELISA)

Specific p-Tau ser396 (Invitrogen, KHB7031), AT8 (Invitrogen, KHB7041), AT180 (Invitrogen, KHB7031), human A Aβ42 (Invitrogen, KHB3442,) and α-Synuclein (Invitrogen, KHB0061) ELISA were performed according to manufacturer’s protocol. Caspase-3 activity assays were performed according to manufacturer’s protocol as we previously described [21,27-29].

Transmission electron miscroscope (EM)

Brain tissues were fixed in (1:4, v:v) 4% PFA-picric acid solution and 25% glutaraldehyde overnight, then washed 3× in 0.1 M cacodylate buffer and osmicated in 1% osmium tetroxide/1.5% potassium ferrocyanide for 3h, followed by another 3× wash in distilled water. Samples were treated with 1% uranyl acetate in maleate buffer for 1 h, washed 3× in maleate buffer (pH 5.2), then exposed to a graded cold ethanol series up to 100% and ending with a propylene oxide treatment. Samples were embedded in pure plastic and incubated at 600C for 1–2 days. Blocks were sectioned on a Leica ultracut microtome at 95 nm, picked up onto 100 nm formvar-coated copper grids and analyzed using a Philips Technai Spirit transmission EM.

Statistical analysis

All statistical analysis was performed using a GraphPad Prism, version 5.0 (GraphPad software, Inc, San Diego, CA). The number (N) indicates the number of independent experiments (cell culture) or number of individual animals. Asterisks designate significantly different as indicated, all data are presented with Mean ± SEM, with actual p-values obtained using ANOVA with Neumann Keuls multiple comparison.

Parkin activity and solubility

To evaluate Abl effects on endogenous Parkin we determined Parkin solubility via fractionation of soluble (supernatant) and insoluble (pellet re-suspended in 4M urea) Parkin and performed WB with anti-total Parkin (PRK8) antibodies. To determine whether Parkin level correlates with its enzymatic activity, we immunoprecipitated Parkin (1:100) with anti-Parkin antibody (PRK8) from mouse brain lysates and measured its E3 ubiquitin ligase activity using E3LITE customizable ubiquitin ligase kit (LifeSensors, Cat# UC101). E3LITE measures the mechanisms of E1-E2-E3 activity in the presence of different ubiquitin chains as we previously described [21,27-29]. UbcH7 as an E2 that provides maximum activity with Parkin E3 ligase and add E1 and E2 in the presence of recombinant ubiquitin, K0 or K48 or K63 to determine the lysine-linked type of ubiquitin. E3 was added to an ELISA microplate that captures polyubiquitin chains formed in the E3-dependent reaction, which is initiated with ATP at RT for 60 minutes and read on a chemiluminescense plate reader.


Abl activation increases Aβ42, α-synuclein and p-tau levels

Lentiviral expression of human Aβ42 in AblPP/tTA mice brains results in a significant increase in soluble and insoluble Aβ42 (Figure 1A, N=4, p<0.05) with -Dox (Abl induced) compared to +Dox (Abl not induced). Co-expression of human Parkin with Aβ42 significantly reduced Aβ42 with +Dox, but induction of Abl expression by Dox withdrawal abrogated Parkin’s effects on Aβ42 (Figure 1A, N=4, p<0.01). No Aβ42 was detected in LacZ injected mice (indicated as -Aβ42 in figures). The concentration of p-Tau epitopes, including Ser 396, AT8 and AT180 were significantly increased when Aβ42 was expressed with –Dox (Figure 1B, N=4) compared to +Dox. p-Tau was also increased with Aβ42 +Dox compared to LacZ +Dox. Parkin expression significantly reduced p-Tau levels (Figure 1B, N=4) when Aβ42 was expressed with Dox, but Abl overexpression (-Dox) blocked Parkin’s effects on p-Tau levels. Western blot analysis supports ELISA measurement as shown in Suppl Figure 1.