Impaired Mitophagy and Protein Acetylation Levels in Fibroblasts from Parkinson’s Disease Patients

Abstract

Parkinson’s disease (PD) is a chronic and progressive neurodegenerative disorder. While most PD cases are idiopathic, the known genetic causes of PD are useful to understand common disease mechanisms. Recent data suggests that autophagy is regulated by protein acetylation mediated by histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities. The changes in histone acetylation reported to be involved in PD pathogenesis have prompted this investigation of protein acetylation and HAT and HDAC activities in both idiopathic PD and G2019S leucine-rich repeat kinase 2 (LRRK2) cell cultures. Fibroblasts from PD patients (with or without the G2019S LRRK2 mutation) and control subjects were used to assess the different phenotypes between idiopathic and genetic PD. G2019S LRRK2 mutation displays increased mitophagy due to the activation of class III HDACs whereas idiopathic PD exhibits downregulation of clearance of defective mitochondria. This reduction of mitophagy is accompanied by more reactive oxygen species (ROS). In parallel, the acetylation protein levels of idiopathic and genetic individuals are different due to an upregulation in class I and II HDACs. Despite this upregulation, the total HDAC activity is decreased in idiopathic PD and the total HAT activity does not significantly vary. Mitophagy upregulation is beneficial for reducing the ROS-induced harm in genetic PD. The defective mitophagy in idiopathic PD is inherent to the decrease in class III HDACs. Thus, there is an imbalance between total HATs and HDACs activities in idiopathic PD, which increases cell death. The inhibition of HATs in idiopathic PD cells displays a cytoprotective effect.

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Abbreviations

AA:

anacardic acid

Ac-K:

acetylated lysine

ATG:

autophagy-related

BAF. A1:

bafilomycin A1

CBP:

CREB-binding protein

CCCP:

carbonyl cyanide 3-chlorophenylhydrazone

COX IV:

cytochrome c oxidase subunit 4

CsA:

cyclosporin A

DiOC6(3):

3,3′-dihexyloxacarbocyanine iodide

DRP1:

dynamin related protein 1

EBSS:

Earle’s balanced salt solution

ERK1/2:

extracellular signal-regulated kinase 1/2

GAPDH:

glyceraldehyde-3-phosphate dehydrogenase

GNAT:

GCN5-related N-acetyltransferase

GFP:

green fluorescent protein

H3:

histone 3

H3K14:

histone 3 lysine 14

H4:

histone 4

H4K5K8K12:

histone 4 lysine 5 lysine 8 lysine 12

H4K16:

histone 4 lysine 16

HAT:

histone acetyltransferase

HDAC:

histone deacetylase

HFs:

human fibroblasts

hMOF:

human male absent of first

IPD:

idiopathic Parkinson’s disease

JNK1:

Jun-N-terminal kinase 1

LC3:

light-chain microtubule-associated protein

LONP1:

lon peptidase 1

LRRK2:

leucine-rich repeat kinase 2

MAPK:

mitogen-activated protein kinase

MMP:

mitochondrial membrane potential

MPP+ :

1-methyl-4-phenylpyridinium iodide

MPTP:

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride

MTG:

MitoTracker green

mTOR:

mammalian target of rapamycin

NAD+ :

nicotinamide adenine dinucleotide

NAM:

nicotinamide

PCAF:

p300/CREB-binding protein-associated factor

PD:

Parkinson’s disease

PI:

propidium iodide

PINK1:

PTEN-induced putative kinase 1

ROS:

reactive oxygen species

RT:

room temperature

SIRT:

sirtuin

TIP60:

tat-interactive protein 60

TMRM:

tetramethylrhodamine methyl ester perchlorate

TSA:

trichostatin A

WB:

western blotting

WT:

wild-type

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Acknowledgments

We are grateful to the patients and donors without whom this work would not have been possible. The authors thank M. P. Delgado-Luceño and Dr. J.A. Tapia-Garcia. The authors also thank FUNDESALUD for helpful assistance.

Funding

SMS.Y-D was supported by Isabel Gemio Foundation. M. N-S was funded by “Ramon y Cajal Program (RYC-2016-20883) Spain. M. R-A. and E. U-C were supported by a FPU predoctoral fellowship (FPU13/01237 and FPU16/00684, respectively) from Ministerio de Educación, Cultura y Deporte, Spain. R. G-S. was supported by a Marie Sklodowska-Curie Individual Fellowship (IF-EF) (655027) from the European Commission. JM. B-S. P. was funded by La Ligue Contre le Cancer. JM. F. received research support from the Instituto de Salud Carlos III, CIBERNED (CB06/05/004) and Instituto de Salud Carlos III, FIS, (PI15/00034). RA. G-P. was supported by a “Contrato destinado a la retención y atracción del talento investigador, TA13009“ from Junta de Extremadura, and received a research support from the Instituto de Salud Carlos III, FIS, (PI14/00170). JM.C. was funded by a Parkinson’s UK project grant (G-1406). This work was also supported by “Fondo Europeo de Desarrollo Regional” (FEDER) from the European Union.

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Correspondence to Rosa A. González-Polo or José M. Fuentes.

Electronic supplementary material

Figure S1
figure7

Determination of G2019S LRRK2 mutation and expression levels of SIRT2 and SIRT6. A/ Restriction enzyme of LRRK2 exon 41. Bfm I hydrolyses the exon 41 harboring the G2019S mutation into 2 bands (300 and 200 base pairs (bp)) confirming that the mutation is heterozygous. SIRT2 expression levels. B/ mRNA expression of SIRT2 by qPCR. Data are the normalized mean ± SD of three independent experiments. C-E/ Detection of two isoforms of SIRT2 by immunoblotting, SIRT2 isoform I (43 kDa) (C, D) and SIRT2 isoform II (39 kDa) (C, E). The densitometry of each isoform is normalized to GAPDH. The results correspond to the relative mean ± SD of three independent experiments, *p < 0.05 in comparison to Co, (Student’s t-test). F-H/ SIRT6 expression levels. F/ mRNA expression of SIRT6 by qPCR. Data are the normalized mean ± SD of three independent experiments, **p < 0.01 versus Co, (Student’s t-test). G, H/ Assessment of SIRT6 expression and its quantification to referenced GAPDH. Data are the normalized means ± SD of three independent experiments, **p < 0.01 up to Co, (Student’s t-test). (PNG 319 kb)

Figure S2
figure8

Ac-K proteins and mRNA expression levels of the HAT family. A, B/ Immunofluorescence intensity of labeled nuclear Ac-K proteins (red) in HFs, the nuclei were stained with Hoechst 33,342 (blue). Original magnification: 40X, scale bar corresponds to 10 μm. B/ Represents the quantification of the fluorescence intensity (n = 60 cells/condition). Data are the mean ± SD of two independent experiments, **p < 0.01, ***p˂0.001 in comparison to Co, (Student’s t-test). p300 (C) and PCAF (D) are members of the p300/CBP and GNAT families, respectively. Members of the MYST family are hMOF (E) and TIP60 (F). Their mRNA expression levels were assessed by qPCR, and the results represent the relative mean ± SD of at least three independent experiments, *p˂0.05, **p˂0.01, ***p˂0.001 versus Co, (Student’s t-test). G/ Immunofluorescence intensity of labeled cytoplasmic Ac-K proteins (red) in HFs treated with TSA (1 μM) during 4 h. Scale bar corresponds to 10 μm. (PNG 340 kb)

Figure S3
figure9

mRNA expression levels of class I and II HDACs. A-C/ Class I HDACs includes HDAC1 (A), HDAC2 (B) and HDAC3 (C). D, E/ Class II HDACs includes HDAC4 (D) and HDAC6 (E). Their mRNA expression levels were assessed by qPCR, and the results correspond to the relative mean ± SD of three independent experiments, *p˂0.05, **p˂0.01, ***p˂0.001 versus Co, (Student’s t-test). (PNG 104 kb)

Figure S4
figure10

Cell viability with HDAC inhibitors. A, B/ HFs were treated with TSA (0–100 μM) or NAM (0–100 mM) for 24 h. Cell viability was assessed by the colorimetric test MTT. Data correspond to the normalized mean percentage of untreated cells, *p˂0.05, **p˂0.01, ***p˂0.001, (Student’s t-test). C, D/ Cells were treated with TSA (1 μM) or NAM (1 mM) for 4 h. Histone 3 acetylated on lysine 14 (Ac-H3K14) was detected by immunoblotting and was normalized to total Histone 3 (H3). E/ Cells were treated overnight with EX-527 (1 μM). Cells were stained with propidium iodide (PI), and the percentage of PI+ cells was evaluated by flow cytometry, (n = 10.000 events). Data are the mean percentage ± SD of three independent experiments, *p˂0.05, **p˂0.01, (Student’s t-test). (PNG 236 kb)

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Yakhine-Diop, S.M.S., Niso-Santano, M., Rodríguez-Arribas, M. et al. Impaired Mitophagy and Protein Acetylation Levels in Fibroblasts from Parkinson’s Disease Patients. Mol Neurobiol 56, 2466–2481 (2019). https://doi.org/10.1007/s12035-018-1206-6

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Keywords

  • Acetylation
  • Histone acetyltransferases
  • Histone deacetylases
  • LRRK2
  • Mitophagy