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: ceo@cellivery.com

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.