Exp Neurobiol 2014; 23(4): 365-371
Published online December 31, 2014
© The Korean Society for Brain and Neural Sciences
Eun-Jin Bae1,2, Cheolsoon Lee2,3, He-Jin Lee2,3, Seokjoong Kim4 and Seung-Jae Lee1,2,5,*
1Department of Biomedical Science and Technology, 2Institute of Biomedical Science and Technology,
3Department of Anatomy, School of Medicine, Konkuk University, Seoul 143-701,
4ToolGen, Inc., Biotechnology Incubating Center, Seoul National University, Seoul 305-390,
5College of Veterinary Medicine, Konkuk University, Seoul 143-701, Korea
Correspondence to: *To whom correspondence should be addressed.
TEL: 82-2-447-5685, FAX: 82-2-447-5683
Parkinson's disease is a multifactorial disorder with several genes linked to the familial types of the disease.
Keywords: ATP13A2, PARK9, Lysosome, Parkinson’s disease, alpha-synuclein, SH-SY5Y cell
Parkinson's disease (PD) is the second most prevalent neurodegenerative disease, with clinical characteristics of resting tremor, muscle tone rigidity, bradykinesia, and postural instability . However, patients with PD also suffer from wide-range of non-motor symptoms, including psychiatric, autonomic, sensory, and sleep abnormalities . These symptoms become diverse and severe as the disease progresses, indicating broad spectrum pathology in the central nervous system as well as the peripheral nervous system [3,4,5,6]. Pathologically, PD is characterized by the loss of specific neurons, such as dopaminergic neurons in the substantia nigra pars compacta, and the occurrence of proteinacious inclusion bodies known as Lewy bodies and Lewy neurites . These inclusion bodies are composed of numerous proteins and vesicles, among which amyloid fibril aggregates of a neuronal protein α-synuclein are the major constituents .
Progress in PD research has been driven by human genetic studies that identified about 20 genes that are associated with PD [9,10]. Among these genes, SNCA, which encodes α-synuclein, has been not only linked to several inherited forms of PD  but also identified as the most consistent and strongest genetic risk factor for sporadic PD from genome-wide association studies .
Several genes linked to PD have been suggested to function in the lysosomal degradation pathway and in formation of α-synuclein pathology [42,43,44]. Here, we generated a human neuroblastoma cell line lacking ATP13A2 and investigated the role of this protein in the general lysosomal function and in α-synuclein metabolism.
The following antibodies were used in this study: ATP13A2 polyclonal antibody (Abcam, ab135674, Cambridge, MA; 1:1,000), β-actin monoclonal antibody AC-15 (Sigma-Aldrich, A5441, St. Louis, MO; 1:10,000), p62 monoclonal antibody (BD Transduction Laboratories, c2384-0B, Swampscott, MA; 1:1,000), ubiquitin polyclonal antibodies (Dako, #z0458, Glostrup, Denmark, and Chemicon, Temecula, CA; 1:1,000), α-synuclein monoclonal antibody (BD Biosciences, #610787, San Diego, CA; 1:1,500), α-synuclein monoclonal antibody Ab274 (1:1,500), α-synuclein monoclonal antibody Ab62 (1:1,000), HRP-conjugated goat anti-mouse IgG (H+L) (Bio-Rad Laboratories, 172-1011, Hercules, CA; 1:3,000), and HRP-conjugated goat anti-rabbit IgG (H+L) (Bio-Rad Laboratories; 1:3,000).
Fluorescein-conjugated dextran (10,000 molecular weight; D-1821), TO-PRO-3 iodide (T3605), and LysoTracker Red DND-99 (L-7528) were purchased from Invitrogen (Carlsbad, CA).
SH-SY5Y cells (ATCC, CRL-2266, Manassas, VA) were transfected with plasmids encoding zinc-finger nuclease and a magnetic reporter (ToolGen, Seoul, Korea) by using electroporation. After incubation for 48 h, cells were trypsinized and mixed with magnetic bead-conjugated antibody against H-2Kk (MACSelect Kk microbeads, Miltenyi Biotech, Germany). The mixture was applied to a MACS LS column (Miltenyi Biotech). A single cell isolated from eluates was maintained until the clonal colony was picked from the culture dish. Nonsense mutations in the
SH-SY5Y human neuroblastoma cell lines were subcultured as described previously . Cells were maintained every 2 days at 37℃ in humidified air with 5% CO2 in Dulbecco's modified eagle's medium (DMEM) (HyClone, SH30243.01, Logan, UT) containing 10% fetal bovine serum (HyClone, SH30396.03), 100 units/mL penicillin, and 100 units/mL streptomycin (Gibco, 15140-122, Grand Island, NY). To differentiate SH-SY5Y cells, cells were maintained in the presence of 50 µM all-
After washing with ice-cold phosphate-buffered saline (PBS) twice, cells were lysed in extraction buffer (1% Triton X-100 and 1% (v/v) protease inhibitor cocktail (Sigma) in PBS). Cell lysates were incubated on ice for 10 min and centrifuged at 16,000 × g for 10 min. The Triton X-100 insoluble fraction was resuspended in 1× Laemmli sample buffer and sonicated briefly.
Western blotting was performed as previously described . Images were obtained and quantified using a Luminescent Image Analyzer (LAS-3000) and MultiGauge version 3.0 software (Fujifilm, Tokyo, Japan).
To analyze the accumulation of acidic compartments, SH-SY5Y cells were incubated with 75 nM LysoTracker solution diluted in growth medium. After incubation for 1 h at 37℃ in a CO2 incubator, cells were washed with ice-cold PBS and fixed in a 4% paraformaldehyde (PFA) solution. To analyze the degradation ratio of internalized dextran, cells were incubated with 20 µg/mL of fluorescein isothiocyanate (FITC)-labeled dextran (Invitrogen) for 2 h. After washing with DMEM, cells were incubated with fresh growth medium for 30 min and fixed with a 4% PFA solution. The fluorescence intensity was measured using Olympus FV1000 software. The extent of degradation of internalized dextran-FITC was calculated using the equation (Ftime0-Ftime30)/Ftime0, where Ftime0 and Ftime30 are the integrated fluorescence intensities at 0 and 30 min, respectively.
ELISA was performed as previously described . 96-well ELISA plates (Nalgene Nunc International, Rochester, NY) were coated with 1 mg/mL capture antibody (Ab62) in 50 mM carbonate buffer (pH 9.6) at 4℃ overnight. After washing with PBS with 0.05% Tween 20 (PBST), the plate was incubated with SuperBlock T20 (PBS) Blocking Buffer (Thermo Scientific, Rockford, IL) at room temperature (RT) with shaking for 1 h, and washed five times in PBST. Samples and standards were incubated at RT for 2.5 h with shaking. After washing with PBST, 1 µg/mL biotinylated Ab62 in blocking buffer was added to each well. After 1.5 h incubation at RT with shaking, the plates were washed with PBST. Avidin-conjugated peroxidase (ExtrAvidin, Sigma) was incubated for 1 h at RT. After washing with PBST, 100 µL of 3,3',5,5'-tetramethylbenzidine solution (Sigma) was added to each well and plate was incubated for 15 min with shaking. To stop the reaction, 50 µL of 2N H2SO4 was added to each well. The absorbance was measured at 450 nm.
Values shown in the figures are means±S.E.M. To analyze the statistical significance, p values were calculated by means of paired, two-tailed Student's
To generate a human neuronal cell line deficient in
To examine the role of ATP13A2 in lysosomal functions, we performed the following analyses. First, we measured the steady state levels of lysosomal substrate proteins, p62 and polyubiquitinated proteins. The levels of these proteins were not altered significantly in the knockout cells (Fig. 2A, B). Second, we measured the amounts of cytoplasmic acidic compartments using lysotracker, a protonophilic fluorescent dye. It has been shown that production of acidic compartments were elevated in cells with lysosomal dysfunction, perhaps as a compensatory mechanism .
Previous studies suggested that ATP13A2 and α-synuclein exert their pathogenic actions in the same pathway . Lysosomal functions were postulated to play a role in the cooperation between these two proteins in PD pathogenesis. To examine the functional link between these proteins, we measured the steady state levels of α-synuclein in the control and
A large number of previous studies suggested that ATP13A2 deficiency caused lysosomal dysfunction and α-synuclein accumulation in both in vitro and in vivo models [29,30,33,34]. The relationship between ATP13A2 and α-synuclein seems to be conserved throughout the evolutionary stages from yeast through nematode to mammals . The lack of effect of
Although many previous studies showed the role of ATP13A2 in lysosomal functions and metal homeostasis, our study suggests that functional manifestation of ATP13A2 deficiency varies depending on the cell types. SH-SY5Y cells are commonly used in the field of neurological diseases, particularly frequently among PD researchers for their catecholaminergic phenotypes. Based on the results of the current study, we conclude that SH-SY5Y cells are not a suitable model system to study the loss-of-function effects of ATP13A2 on lysosomal functions, however, can be useful as a negative control to study lysosomal dysfunction caused by genetic modifications. We do not exclude the possibility that SH-SY5Y/