Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Aug 17;50(8):1-13.
doi: 10.1038/s12276-018-0124-z.

Cell-penetrating artificial mitochondria-targeting peptide-conjugated metallothionein 1A alleviates mitochondrial damage in Parkinson's disease models

Young Cheol Kang  1 , Minuk Son  1 , Sora Kang  1 , Suyeol Im  1 , Ying Piao  2   3 , Kwang Suk Lim  4 , Min-Young Song  2   5 , Kang-Sik Park  2 , Yong-Hee Kim  4 , Youngmi Kim Pak  6   7
Affiliations

Cell-penetrating artificial mitochondria-targeting peptide-conjugated metallothionein 1A alleviates mitochondrial damage in Parkinson's disease models

Young Cheol Kang et al. Exp Mol Med. .

Abstract

An excess of reactive oxygen species (ROS) relative to the antioxidant capacity causes oxidative stress, which plays a role in the development of Parkinson's disease (PD). Because mitochondria are both sites of ROS generation and targets of ROS damage, the delivery of antioxidants to mitochondria might prevent or alleviate PD. To transduce the antioxidant protein human metallothionein 1A (hMT1A) into mitochondria, we computationally designed a cell-penetrating artificial mitochondria-targeting peptide (CAMP). The recombinant CAMP-conjugated hMT1A fusion protein (CAMP-hMT1A) successfully localized to the mitochondria. Treating a cell culture model of PD with CAMP-hMT1A restored tyrosine hydroxylase expression and mitochondrial activity and reduced ROS production. Furthermore, injection of CAMP-hMT1A into the brain of a mouse model of PD rescued movement impairment and dopaminergic neuronal degeneration. CAMP-hMT1A delivery into mitochondria might be therapeutic against PD by alleviating mitochondrial damage, and we predict that CAMP could be used to deliver other cargo proteins to the mitochondria.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Schematic structure of in silico-designed CAMP peptide.
a Amino-acid sequence of the CAMP peptide. The cleavage site predicted by MitoProtII is marked with an underscore. b α-Helical structure of CAMP predicted by COOT. c Helical wheel projection of CAMP. Positively charged amino acids in TAT are presented in black, and those in the MTS are blue
Fig. 2
Fig. 2. Mitochondrial localization of CAMP-hMT1A and CAMP-EGFP by transfection.
SK-Hep1 cells were stably transfected with pcDNA3.1-myc/His (mock), CAMP-hMT1A-6xHis, EGFP, or CAMP-EGFP. a Confocal microscopy images showing the localization of CAMP fusion proteins. Cells were stained with MitoTracker (MitoT, red) and immunostained with anti-6xHis or EGFP antibodies (green) before microscopy (×400, scale bar = 10 μm). Co-localization of red and green signal appears yellow in merged images. b Western blot analysis. Cell or mitochondrial lysates were analyzed by western blot with 6xHis-tag to detect CAMP-hMT1A and β-actin and HSP60 antibodies for loading and mitochondria controls, respectively
Fig. 3
Fig. 3. Preparation of CAMP-hMT1A recombinant protein.
CAMP-hMT1A protein was expressed in E. coli in the presence of 1 mM ZnSO4 and purified by Ni-NTA affinity chromatography. a Coomassie blue staining. M molecular weight marker. b Western blot with 6xHis-tag and metallothionein (MT1A) antibodies to detect CAMP-hMT1A. c Mass spectrum analysis. The spectrum of the identified peptide sequence is presented
Fig. 4
Fig. 4. Transfer of CAMP-hMT1A protein into mitochondria.
a Western blot of TAT-hMT1A, CAMP-hMT1A, and CAMP-EGFP recombinant proteins using 6xHis-tag antibody. b Time-dependent uptake of recombinant proteins. SK-Hep1 cells expressing DsRed2-mito (red) were treated with 2 μM CAMP-hMT1A, TAT-hMT1A, or CAMP-EGFP for 3, 24, or 48 h. Cells were immunostained with 6xHis-tag or EGFP antibodies (green) before confocal microscopy (×400, scale bar = 10 μm). The yellow merged images show mitochondrial localization of the fusion proteins. c Western blot of CAMP-hMT1A in mitochondria isolated from cells treated with 2 μM CAMP-hMT1A for 24 h. The upper and lower bands of CAMP-hMT1A represent the precursor and mature forms, respectively. β-Actin and HSP60 antibodies were used for loading and mitochondria control, respectively. d Schematic representation of the precursor and mature forms of CAMP-hMT1A. The processing site is indicated by an arrow
Fig. 5
Fig. 5. CAMP-hMT1A increases mitochondrial function in neuronal cells without cytotoxicity.
a Confocal microscopy images of CAMP-hMT1A in the mitochondria of SH-SY5Y neuronal cells (×400, scale bar = 10 μm). Cells were treated with 2 μM CAMP-hMT1A protein for 24 h, stained with MitoTracker (MitoT, red), and immunostained with 6xHis-tag antibody (green). b Western blot of tyrosine hydroxylase (TH) levels in CAMP-MT1A- or PBS-treated cells. β-Actin was detected as a loading control. c Time- and dose-dependent effects of CAMP-hMT1A on complex 1 of OXPHOS, NADH dehydrogenase activity. d Dose-dependent effects of CAMP-hMT1A on intracellular ATP content. e The oxygen consumption rate (OCR) of isolated mitochondria for state 2 (–ADP) and state 3 (+1.5 mM ADP) respiration were measured, with mitochondrial complex 1 inhibitors MPP+ (1 mM) and rotenone (1 μM) used as positive controls. The data are presented as the mean ± SEM, n = 3
Fig. 6
Fig. 6. Recovery of mitochondrial activity and TH expression by CAMP-hMT1A in cellular PD models.
a Intracellular ATP content of SH-SY5Y cells treated with 1 mM MPP+ for 24 h, followed by treatment with 4 μM CAMP-hMT1A or TAT-hMT1A (n = 4). b–e Dose-dependent effects of CAMP-hMT1A in MPP+-treated SH-SY5Y cells: b intracellular ATP (n = 12); c complex 1 of OXPHOS, NADH dehydrogenase activity (n = 4); d superoxide level as determined by MitoSOX (n = 6); e TH expression as determined by western blot with β-actin loading control. f Effects of CAMP-hMT1A on complex 1 activity in cells treated with 1 μM rotenone for 24 h (n = 3). The data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. CTL; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. MPP+-treated
Fig. 7
Fig. 7. Sensorimotor function and TH expression are restored by stereotaxic injection of CAMP-hMT1A into SN of MPTP-injected mice.
a Timeline of the MPTP-induced PD model experiment. CAMP-hMT1A (3 μg) or PBS was stereotaxically injected unilaterally into the left substantia nigra. b Rotarod performance test (n = 7). c Western blot and quantification of band intensities of TH in the left substantia nigra (SN) or the striatum (ST) (n = 3). β-Actin was the loading control. d Immunohistochemistry of TH and relative number of TH-positive cells in the SN (n = 4) (×4, scale bar = 1 mm). e Immunohistochemistry of TH and relative density of TH-positive fibers in the striatum (×4, scale bar = 1 mm) (n = 4, *stereotaxic injection site). The data are presented as the mean ± SEM. ipsi ipsilateral side, contra contralateral side; *P < 0.05, ***P < 0.001 vs. control. ###P < 0.001 vs. MPTP-treated
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Poewe W, et al. Parkinson disease. Nat. Rev. Dis. Prim. 2017;3:17013. doi: 10.1038/nrdp.2017.13. - DOI - PubMed
    1. Kalia LV, Lang AE. Parkinson’s disease. Lancet. 2015;386:896–912. doi: 10.1016/S0140-6736(14)61393-3. - DOI - PubMed
    1. Gomez-Sanchez R, Bravo-San Pedro JM, Gegg ME, Gonzalez-Polo RA, Fuentes JM. Mitochondria: key organelle in Parkinson’s disease. Park. Dis. 2016;2016:6230370. - PMC - PubMed
    1. Lin MT, Beal MF. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature. 2006;443:787–795. doi: 10.1038/nature05292. - DOI - PubMed
    1. Schapira AH. Mitochondria in the aetiology and pathogenesis of Parkinson’s disease. Lancet Neurol. 2008;7:97–109. doi: 10.1016/S1474-4422(07)70327-7. - DOI - PubMed

Publication types

MeSH terms

Substances