advances.sciencemag.org/cgi/content/full/6/18/eaba1193/DC1
Supplementary Materials for
Intracellular delivery of Parkin rescues neurons from accumulation of damaged
mitochondria and pathological α-synuclein
Eunna Chung, Youngsil Choi, Jiae Park, Wonheum Nah, Jaehyung Park, Yukdong Jung, Joonno Lee, Hyunji Lee, Soyoung Park, Sunyoung Hwang, Seongcheol Kim, Jongseok Lee, Dongjae Min, Junghwan Jo,
Shinyoung Kang, Minyong Jung, Phil Hyu Lee, H. Earl Ruley, Daewoong Jo*
*Corresponding author. Email: [email protected]
Published 29 April 2020, Sci. Adv. 6, eaba1193 (2020)
DOI: 10.1126/sciadv.aba1193
This PDF file includes:
Supplementary Materials and Methods Table S1 Figs. S1 to S8
Supplementary Materials and Methods
Antibodies used for immune-based analysis
Myc-Mfn1 (Addgene, 23212), Flag-Peal-R (Origene, RC208054), α-Synuclein antibody (Abcam,
ab138501), GFAP (Abcam, ab53554), CD68 (Abcam, ab53444), S100β (Abcam, ab52642) and
NeuN antibody (Abcam, ab134014). Immuno-based analysis (e.g., immunoprecipitation,
immunostaining) was performed as described in the main method section.
In vivo and ex vivo imaging of Cy5-iCP-Parkin
iCP-Parkin was dialyzed against 50 mM HEPES, 50 mM Na2CO3- NaHCO3, pH 8.0 and labeled
using a Cy5 labeling kit (Jena Bioscience) according to the manufacturer’s instructions. Unbound
Cy5 was removed by dialysis and protein and Cy5 fluorescence were determined by the
bicinchoninic acid (BCA) protein assay and a microplate reader (Synergy H1, Biotek Instruments),
respectively, from which fluorescein/protein molar ratio (F/P) was calculated. Mice were kept on
the imaging stage under anesthesia with 2.5% isoflurane gas in oxygen flow (2 L/min) and imaged
using emission and excitation filters (excitation/emission: 640/700 nm) with an IVIS Spectrum
system (IVIS200, PerkinElmer). Ex vivo imaging was performed using the IVIS Spectrum system
3 hrs after I.V. injection, under the same conditions used for in vivo imaging.
Isolation of total RNA and quantitative RT-PCR
Total RNA was extracted with Ribospin (GeneAll), and cDNA was synthesized from total RNA
(2 µg) using a HyperscriptTM
First Strand Synthesis Kit (GeneAll). Aliquots of cDNA were used
as templates for the real-time qRT-PCR procedure. Relative quantities of mRNA expression were
analyzed using real-time PCR (CFX96 Touch™ Real-Time PCR Detection System, (Bio-Rad).
The SsoAdvanced Universal Reagents (Bio-Rad) was used according to the manufacturer’s
instructions. The primer sequences are described as follows: hRPLP0 (h36B4) forward (5ʹ-
TGCATCAGTACCCCATTCTATCA-3ʹ) with reverse (5ʹ-
AAGGTGTAATCCGTCTCCACAGA-3ʹ); hPGC1α (PPARGC1A) forward (5ʹ-
CTCAAAGACCCCAAAGGATG-3ʹ) with reverse (5ʹ- TGGAATATGGTGATCGGGAA-3ʹ);
hTFAM forward (5ʹ-AGCTCAGAACCCAGATGC-3ʹ) with reverse (5ʹ-
CCACTCCGCCCTATAAGC-3ʹ); hNRF1 forward (5ʹ- GGCTACCATAGAAGCACATG-3ʹ)
with reverse (5ʹ-GAAGAAGGCGAGTCTTCATC-3ʹ); hNRF2 (GABPA) forward (5ʹ-
ACATCAATGAACCAATAGGC-3ʹ) with reverse (5ʹ- CCTTGGTCAAATAAACTTCG-3ʹ).
PD animal models
AAV-α-Synuclein-induced PD rat model. Anesthetized male Sprague-Dawley rats (~230
g) were injected with 2 µL of the WT α-Synuclein AAV/DJ vector (titer: 1.4 x 1013
GC/mL) at a
rate of 0.5 µL/min into the substantia nigra at the following coordinates (relative to bregma):
anterior-posterior (AP) = -5.3 mm, medial-lateral (ML) = -2.0 mm and dorsal-ventral (DV) = -7.2
mm (from the dura) with a flat skull position. The control group was injected with saline.
Animal studies were performed in accordance with the guidelines of the Institutional Review
Board of the Cellivery R&D Institute, Cellivery Therapeutics, Inc. Male Sprague-Dawley rats
(~230 g) were housed in groups of 3 in standard cages and 2-3 per cage following surgery.
Animals were maintained at 22 ± 2℃ on a 12:12 hrs. dark/light cycle with free access to food and
water.
MPTP-induced PD mouse model. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP,
Sigma-Aldrich, St. Louis, MO) was dissolved in saline solution. For an acute model, the MPTP
solution was administered to C57BL/6 mice (8 weeks, male) 3 times (15 mg/kg, i.p.) or 4 times
(20 mg/kg, i.p.) per day (2 hr interval) for 3 consecutive days. Alternatively, MPTP (20 mg/kg, i.p.)
was administered to mice once per day for 5 consecutive days. Mice used as controls received an
equivalent volume of saline in an identical manner. For sub-chronic model, MPTP (20 mg/kg, i.p.)
was administered to mice 4 times on day 1 followed for 34 days (day 2-35) before iCP-Parkin
treatment.
Motor function analysis
Forced swim test. Mice were individually placed in a glass cylinder (30 cm height * 20
cm width * 20 cm depth) filled with water. Mice normally swim almost continuously whereas
lesioned mice float immobile for varying lengths of time. The total duration of immobility in 1
min. was recorded a blinded observer.
Gait test. For the gait test (footprint analysis), mice were trained to run in an open field
runway. The forepaws and hindpaws of the mice were painted with nontoxic black color, and the
mice were individually placed at one end of a sheet of paper (10 cm width * 50 cm height). After
drying, the footprint sheets were scanned and analyzed.
Wire test. For the wire test, mice were individually placed on a horizontal grid. Each
mouse slowly moved along the grid by grabbing the grid with its forepaws and hindpaws. The
time that elapsed until the mouse completely released its grasp and fell was recorded.
Beam test. Motor coordination was assessed as the ability of animals to walk along a
narrow beam suspended between a start platform and its home cage. The test recorded the time to
cross the beam (2 x 100 cm), and the number of forelimb and hindlimb foot faults (defined as any
foot slip off the top surface of the beam or any limb usage on the side of the beam) over a period
of 120 seconds. Each animal was tested 3 times.
Determination of urine dopamine levels
Urine samples were collected using metabolic cages. Dopamine levels in urine samples were
assessed using an ELISA kit (Dopamine High Sensitive ELISA, Eagle Bioscience, Inc). The assay
and data analysis were carried out according to the manufacturer’s instructions.
Supplementary Tables
Table S1. Ranges of each critical factor comparing selected CPPs and synthetic aMTD
sequences.
Supplementary Figures
Fig. S1. The 3rd
generation hydrophobic CPP (named as aMTD), which satisfied all 6 critical
factors, had the highest cell-permeability. Fluorescence was measured using fluorescence-
activated cell sorting (FACS) for quantitative cell-permeability after incubating RAW264.7 cells
with FITC-labeled recombinant proteins that contain different peptides. (A) Comparison of cell-
permeability between aMTD and unsatisfying peptide (uPs). (B) Comparison of cell-permeability
among the 1st
(MTM), 2nd
(MTD85) and 3rd
(aMTD910) generation hydrophobic CPPs. (C) Amino
acid sequences of peptides used in the quantitative cell-permeability studies. All recombinant
proteins contain the same nonfunctional cargo [solubilization domain A (SDA)] with different
peptides in the N-terminus of the cargo.
Fig. S2. Delivery of iCP-Parkin to deep brain tissues. (A) Dose-dependent uptake of FITC-
labeled iCP-Parkin in C2C12 cells. (B) Time-dependent uptake of FITC-labeled iCP-Parkin in
C2C12 cells. (C) Cell permeability of iCP-Parkin in non-neuronal cells (NHA astrocyte cell)
measured by flow cytometry and confocal laser scanning microscopy. (D) Distribution of Cy5-
labeled iCP-Parkin at 3 and 5 hr post-injection timepoints. Fluorescence data are presented as the
mean ± S.E.M, and the p values were determined by Student’s t-test (n = 4). Ex vivo imaging of
brains after injection with Cy5-labeled iCP-Parkin for 3 hrs. (E) Fluorescent micrographs showing
the in vivo tissue distribution of iCP-Parkin in mice. Tissue sections were analyzed 2 hrs after i.v.
injection of 10 and 50 mg/kg Parkin recombinant proteins. (F and G) Immunohistochemistry for
iCP-Parkin in mouse cortex, striatum and substantia nigra after i.v. injection of Parkin
recombinant protein. Anti-Parkin antibody with anti-GFAP antibody for astrocytes (F) or anti-
CD68 for microglia (G) were used, Scale bar = 10 μm. (H) Immunohistochemistry for iCP-Parkin
in mouse substantia nigra after i.v. injection of Parkin recombinant protein. Anti-TH antibody
were used for the detection of dopaminergic neurons. Scale bar = 10 μm.
Fig. S3. iCP-Parkin is activated in a PINK1-independent manner. (A) Western blot analysis
for analyzing phosphorylation of iCP-Parkin by PINK1 in a test tube. After reaction with the
indicated protein combination, protein was separated using Phos-tag SDS-PAGE and
immunoblotted with anti-pSer65
-Parkin antibody and anti-Parkin antibody to detect both Parkin
and iCP-Parkin. * iCP-Parkin (64 kDa), ** Parkin (53 kDa), # Phosphorylated form,
## Non-
phosphorylated form. The representative data are presented, and the tests were carried out with 3
repetitions. (B) Western blot analysis of ubiquitination of wild-type (WT) Parkin and iCP-Parkin
by PINK1 in a test tube. The representative data are presented, and the tests were carried out with
3 repetitions. (C) Western blot analysis of PINK1-mediated iCP-Parkin phosphorylation. CCCP
was used for PINK1 activation, which was confirmed with an anti-PINK1 antibody. Note that
CCCP increases iCP-Parkin phosphorylation by overexpressing PINK1. The representative data
are presented, and the tests were carried out with 3 repetitions. (D) Western blot analysis for
analyzing PINK1-dependent iCP-Parkin phosphorylation in PINK1 knockout (KO) HAP1 cells.
The representative data are presented, and the tests were carried out with 3 repetitions. (E)
Analysis of the cytoprotective effect of iCP-Parkin in PINK1 WT and KO HAP1 cells using the
ATP Glo assay. Data are represented as the mean ± S.E.M with Student’s t-test (n=3).
Fig. S4. iCP-Parkin colocalizes with PINK1 and suppresses neuronal poisoning. (A) Confocal
laser scanning microscopy demonstrating the colocalization of iCP-Parkin with endogenous
PINK1 on the mitochondria. Green, red and gray letters represent iCP-Parkin detected with anti-
iCP-Parkin antibody, endogenous PINK1 with anti-PINK1 antibody and mitochondria with
Mitotracker Deep Red FM, respectively. (B and C) Immunoprecipitation and western blot analysis
for analyzing the ubiquitination of Mfn1 (B) and Mfn2 (C) by iCP-Parkin using cell lysate from
HeLa cells transfected with the indicated constructs and treated with MG132 (20 μM) in the
presence or absence of iCP-Parkin. (D) Auto-ubiquitination assay for quantify ubiquitination
activities of iCP-Parkin and C444S (n=3). (E) Immunoprecipitation and Western blot analysis for
analyzing the ubiquitination of Mfn2 by iCP-Parkin and C444S. (F) Confocal laser scanning
microscopy for detecting mitophagy modified by iCP-Parkin and C444S under CCCP treatment in
HeLa cells. A graph indicating the quantification of mitophagy by iCP-Parkin and C444S (n=5).
Scale bar = 10 μm. (G) Western blot analysis for detecting mitochondrial proteins, Tom20 and
Tim23 in lysates from HeLa cells treated with CCCP (40 μM), CCCP with iCP-Parkin or Non-
CP-Parkin (10 and 20 μM). The representative data are presented, and the tests were carried out in
duplicate. (H) Western blot analysis for detecting mitochondrial proteins, Tom20, Tim23, MFN1
and MFN2 in lysates from CCCP or CCCP + iCP-Parkin treated HeLa cells. The representative
data are presented, and the tests were carried out in quadruplicate. (I) Quantification of the
relative mRNA levels of PGC-1α, TFAM, NRF1, NRF2 in HeLa cells treated with either 30 μM
CCCP or 30 μM iCP-Parkin determined by real-time quantitative PCR and normalized to the
expression levels of ribosomal protein lateral stalk subunit P0 (Rplp0), a housekeeping gene (n=3).
(J) iCP-Parkin also recovers cellular ROS levels decreased by MPP+ (n=3). (K) iCP-Parkin
recovers ATP levels decreased by CCCP in a dose-dependent manner (n=3). (L) Determination of
iCP-Parkin EC50 using Annexin V/7-AAD apoptosis detection assay in SH-SY5Y cells treated
with 2 mM MPP+ (The minimum n number for this test is 3).
(M) Western blot analysis with
apoptotic biomarkers in SH-SY5Y cells, showing suppression of the pro-apoptotic marker
expression (p53, p-p53, cytochrome C and Cleaved caspase-3) or promotion of the anti-apoptotic
protein (Bcl2) by iCP-Parkin under MPP+
treatment. (N) Western blot analysis for iCP-Parkin in
the presence of CCCP in Parkin KO HAP1 and HeLa cells. The representative data are presented,
and the tests were carried out in triplicate except N (duplicate). Data in D is the mean ± S.D. with
Student’s t-test. Data in F, I, J, K and L are the mean ± S.E.M with Student’s t-test. Scale bar =
10 μm.
Fig. S5. Neuro-protective effect of iCP-Parkin in α-Synuclein-overexpressing SH-SY5Y cells
is due to its activity of E3 ubiquitin ligase α-Synuclein. (A and B) Immunoprecipitation assay
for Pael-R ubiquitination by iCP-Parkin in HeLa (A) and WT HAP1 (B) cells. The representative
data are presented, and the tests regarding (A) were carried out in duplicate and the tests regarding
(B) was repeated in both PINK1 KO and WT HAP1 cells. (C) Fluorescence cell images showing
the involvement of autophagy in clearing aggregated α-Synuclein. Scale bar = 20 μm. (D)
Representative western blot image showing a significant decrease in cleaved Caspase-3 in RA-
differentiated TagGFP2-α-Synuclein-SH-SY5Y cells at 24 hrs. The graph indicates the relative
fold of band intensity (n = 3). (E) Western blot analysis showing decrease in cleaved Caspase-3 in
RA-differentiated TagGFP2-α-Synuclein-overexpressing SH-SY5Y cells at 8 hrs. C444S
decreases cleaved Caspase-3 less than iCP-Parkin. The representative data are presented, and the
tests were carried out in triplicate. Differential interference contrast (DIC) cell images showing
cell viability in RA-differentiated TagGFP2-α-Synuclein SH-SY5Y cells at 24 hrs. (F) Western
blot analysis showing a decrease in pathological α-Synuclein forms such as phosphorylated (p-
Ser129
) α-Synuclein by iCP-Parkin in the insoluble fraction. C444S decreases pathological α-
Synuclein forms less than iCP-Parkin. The representative data are presented, and the tests were
carried out in triplicate. The semi-quantified graph of western blot data showing pSer129
α-
Synuclein and α-Synuclein presented in western blot analysis (n=3). (G) CytoTox-Glo analysis
showing a decrease in cytotoxicity by iCP-Parkin at 24 hrs. C444S decreases cytotoxicity less
than iCP-Parkin (n=3). (H) Dot blot analysis showing the modified levels of pathological α-
Synuclein forms, such as oligomeric and filamentous α-Synuclein by iCP-Parkin or Non-CP-
Parkin in the soluble fraction at 8 hrs. The representative data are presented, and the tests were
carried out in duplicate. (I) Western blot analysis for detecting phosphorylated (pSer129
) α-
Synuclein and α-Synuclein in the soluble fraction. Representative data showed decreased levels of
only phosphorylated (pSer129
) α-Synuclein by dose-dependent iCP-Parkin for 24 hrs following
treatment with rotenone (repetition in triplicate). (J) Western blot analysis for detecting
phosphorylated (pSer129
) α-Synuclein and α-Synuclein in the insoluble and soluble fractions.
Representative data showed decreased levels of phosphorylated (pSer129
) α-Synuclein and α-
Synuclein by dose-dependent iCP-Parkin for 24 hrs following treatment with rotenone (3-time
repetition). (K-L) Western blot data (K) and its quantified graph (L) showing the modified
induction of phosphorylated (pSer129
) α-Synuclein by iCP-Parkin for 4 hrs following treatment
with neurotoxins rotenone with or without the ubiquitin-proteasome inhibitor. The graph indicates
the relative fold of band intensity (n = 4). Data in D and F are expressed as the mean ± S.E.M
with Student’s t-test. Data in G and L is expressed as the mean ± S.D. with Student’s t-test.
Fig. S6. Dose and interval optimization for long-term iCP-Parkin administration (4 weeks) in
the 6-OHDA- and α-Synuclein-induced mouse and rat PD models. (A) Schematic diagram of
the experimental protocols in mice. For protocol 1, 10-30 mg/kg iCP-Parkin was i.v. injected 1
time per week for 4 weeks from 2 weeks after injecting 6-OHDA (4 μg/head) into the right side of
the medial forebrain bundle (MFB). For protocols 2 and 3, 10 mg/kg iCP-Parkin was i.v. injected 2
and 3 times a week for 4 weeks from 2 weeks. (B) Rota-rod test (n=5-8). (C) Western blot analysis
of TH expression (at least n=2). (D) Western blot analysis of COX4 and VDAC1 in the left (L) and
right (R) sides of brain (n=2). (E) Protocol for iCP-Parkin administration in the 6-OHDA-induced
rat PD model. 6-OHDA was injected into the striatum on the right side of the brain. (F) Rota-rod
test (n=3-6). (G) Protocol for the AAV-α-Synuclein rat model. iCP-Parkin (30 or 50 mg/kg) was i.v.
injected 1 time at 8 weeks after AAV-α-Synuclein was injected into the right side of the brain. (H)
Beam test. Relative motor activity is based on the value of the diluent control group set at 100%
(n=3-5). Data in B, F and H are presented as the mean ± S.E.M, and the p values were determined
by one-way ANOVA with post hoc Tukey’s test.
Fig. S7. iCP-Parkin recovers behavioral and biochemical defects in MPTP-induced PD
mouse models. (A-D) Efficacy of iCP-Parkin in the acute MPTP-induced PD mouse model. (A)
Schematic diagram of an acute protocol. iCP-Parkin (30 mg/kg) was intraperitoneally injected 1
time per day for 5 consecutive days after i.p. injections of 15 mg/kg MPTP (3 times per day for 3
days). (B) Gait test. Representative gait images (left panel) and quantitative graphs showing the
length of stride (middle panel) and sway (right panel). (C) Urine dopamine levels (n=3). (D)
Swim test. After measuring the time actively swimming in water, raw data were converted to
relative motor activity based on the swim time of the diluent group set to 100% (n=10). (E-G)
Efficacy of iCP-Parkin in the acute MPTP-induced PD mouse model. (E) Schematic diagram of
the experimental protocol. iCP-Parkin (30 mg/kg) was i.v. injected 1 time per day for 5
consecutive days starting 4 days after induction with MPTP. (F) Immunostaining of TH
expression the substantia nigra. Scale bar = 100 μm. (G) Western blot analysis showing TH
expression levels in brain. (H-J) Efficacy of iCP-Parkin in a subacute MPTP-induced mouse PD
model. (H) Schematic diagram of the experimental protocol. iCP-Parkin (30 mg/kg) was i.v.
injected 1 time per day for 5 consecutive days starting 4 days after MPTP induction. (I) Western
blot analysis showing TH expression in whole brain. (J) Wire test (n=4-6). (K and L) Efficacy of
iCP-Parkin in a subchronic MPTP-induced PD mouse model. (K) Schematic diagram of the
experimental protocol. iCP-Parkin (30 mg/kg) was i.v. injected 1 time per day for 5 consecutive
days starting 35 days after MPTP induction. (L) Rota-rod test (n=3-5). Data in B, C, D, J and L
are the mean ± S.D. with Student’s t-test.
Fig. S8. iCP-Parkin recovers motor and pathological defects in the AAV-α-Synuclein-
induced PD mouse model. (A-E) Efficacy of iCP-Parkin in the AAV-α-Synuclein-induced PD
mouse model. (A) Schematic diagram of the experimental protocol in the AAV-α-Synuclein-
induced PD mouse model. iCP-Parkin (30 mg/kg) was i.v. injected 3 times per week for 4 weeks
from 8 weeks after injecting AAV-α-Synuclein into the right side of the brain. (B) Dopaminergic
neurons were reduced in the group injected with AAV-α-Synuclein compared with the normal
group and confirmed the protection of dopaminergic neurons in the group injected with iCP-
Parkin in the striatum. Scale bar = 100 μm. (C) Confocal laser scanning microscopy for detecting
α-Synuclein (green) in NeuN-positive neuronal cells (red) in the striatum. Scale bar = 20 μm. (D)
Confocal laser scanning microscopy for detecting α-Synuclein aggregate (green) using thioflavin
S staining in the striatum. Scale bar = 20 μm. (E) Confocal laser scanning microscopy for
detecting α-Synuclein (green) in NeuN-positive neuronal cells (red) in the substantia nigra. White
dashed rectangles in the third column (scale bar = 20 μm) indicate the areas shown in the high
magnified views (scale bar = 10 μm) in the fourth column. (F-H) Efficacy of iCP-Parkin in the
AAV-α-Synuclein-induced PD mouse model. (F) Schematic diagram of the experimental protocol
in the AAV-α-Synuclein-induced PD mouse model. iCP-Parkin (10 mg/kg) was i.v. injected 3
times per week for 4 weeks from 8 weeks after injecting AAV-α-Synuclein into the right side of
the brain. (G) Confocal laser scanning microscopy for detecting neuro-inflammatory responses
(green) with glial fibrillary acidic protein (GFAP) antibody in the substantia nigra. Scale bar = 100
μm. (H) iCP-Parkin suppresses the accumulation of pathological α-Synuclein. Confocal laser
scanning microscopy for detecting aggregated α-Synuclein (green) by thioflavin-S staining in the
striatum. Scale bar = 100 μm.