The parkinsonian LRRK2 R1441G mutation shows macroautophagy-mitophagy dysregulation concomitant with endoplasmic reticulum stress

Abstract

Autophagy is a mechanism responsible for the degradation of cellular components to maintain their homeostasis. However, autophagy is commonly altered and compromised in several diseases, including neurodegenerative disorders. Parkinson’s disease (PD) can be considered a multifactorial disease because environmental factors, genetic factors, and aging are involved. Several genes are involved in PD pathology, among which the LRRK2 gene and its mutations, inherited in an autosomal dominant manner, are responsible for most genetic PD cases. The R1441G LRRK2 mutation is, after G2019S, the most important in PD pathogenesis. Our results demonstrate a relationship between the R1441G LRRK2 mutation and a mechanistic dysregulation of autophagy that compromises cell viability. This altered autophagy mechanism is associated with organellar stress including mitochondrial (which induces mitophagy) and endoplasmic reticulum (ER) stress, consistent with the fact that patients with this mutation are more vulnerable to toxins related to PD, such as MPP+.

Graphical abstract

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Availability of data and material

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Code availability

Not applicable.

Abbreviations

Baf. A1:

Bafilomycin A1 (Baf. A1)

CCCP:

Carbonyl cyanide 3-chlorophenylhydrazone

EBSS:

Earle’s balanced salt solution

ER:

Endoplasmic reticulum

LRRK2:

Leucine-rich repeat kinase 2

LTR:

Lysotracker red

MAMs:

Mitochondria-associated ER membranes

MPP+ :

1-Methyl-4-phenylpiridinium

PD:

Parkinson’s disease

PINK1:

PTEN-induced putative kinase 1

ROS:

Reactive oxygen species

TOMM20:

Translocase of outer mitochondrial membrane 20

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Acknowledgements

The authors thank M.P. Delgado-Luceño and FUNDESALUD for helpful assistance and are grateful to the patients and donors for their participation.

Funding

This research was supported by the “Instituto de Salud Carlos” III CIBERNED (CB06/05/0041 and PI14/00170) and partially supported by the “Fondo Europeo de Desarrollo Regional” (FEDER) from the European Union. S.M.S.Y-D was supported by CIBERNED. S. C-C and E.U-C are supported by a FPU fellowship (FPU19/04435 and FPU16/00684, respectively) from the Ministerio de  Ciencia, Innovación y Universidades, Spain. G. M-C is supported by University of Extremadura (ONCE Foundation). M. B-B is supported by a collaboration grant from the Ministerio de Educación y Formación Profesional, Spain. G. D-G is supported by the Consejería de Educación y Empleo-SEXPE-Fondo Social Europeo (TE-0031-19). M. P-B is a recipient of a fellowship from the “Plan Propio de Iniciación a la  Investigación, Desarrollo Tecnológico e Innovación (University of Extremadura)”. E.A-C is supported by a grant (IB18048) from the Junta de Extremadura, Spain. M.N-S and J.M-B. S-P were funded by the “Ramon y Cajal” Program (RYC-2016–20883 and RYC-2018–025099, respectively), Spain.

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RAGP, JMF, and MNS designed the study and wrote the publication with help of JMBSP, MRA, and SMSYD. SMSYD, MRA, and SCC carried out most the lab experiments. GMC, EUC, MBB, GDG, MPB, and EAC provided help with the experiments. VC provided technical assistance with electron microscopy studies. AA and ALM provided the fibroblast model from patient biopsies. All authors have read and approved the final manuscript.

Corresponding authors

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

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All patients gave written consent, and all procedures were approved by the “Comité Ético de Investigación Clínica del Área Sanitaria de Gipuzkoa,” Spain.

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The authors declare no competing interests.

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Mireia Niso-Santano, José M. Fuentes, and Rosa A. González-Polo are senior co-authors

Supplementary Information

Below is the link to the electronic supplementary material.

10565_2021_9617_MOESM1_ESM.pdf

Supplementary Figure S1 Characterization and pools selection. (a) PCR products of the different cell lines after digestion with the endonuclease Bsh 1236 I. Line 1: negative control (Neg1) of the restriction analysis, using water instead of enzyme, line 2: negative control 2 (Neg2), using water instead of PCR product, lines 3-6: healthy controls without LRRK2 R1441G mutation (C1-4), lines 7-10: PD patients controls without LRRK2 R1441G mutation (Cα1-4 ), lines 11-13: Healthy subjects with LRRK2 R1441G mutation (Rα1-3), lines 14-17: PD patients heterozygous for LRRK2 R1441G (RG1-4). (b, c) Autophagy characterization of cell lines from subjects with or without R1441G LRRK2. The lysates were obtained from fibroblasts of healthy subjects (C), idiopathic PD patients (Cα), healthy subjects with R1441G LRRK2 mutation (Rα) and PD patients with R1441G LRRK2 mutation. (a) Shows the representative blot of LC3 protein. GAPDH is the loading control. (b) represents the densitometry of LC3-II/GAPDH, in arbitrary units (PDF 1213 KB)

10565_2021_9617_MOESM2_ESM.pdf

Supplementary Figure S2 Analysis of lysosomal and autophagic proteins. (a) LAMP2 determination by immunofluorescence. Control and R1441G human fibroblasts were treated or not with Baf. A1 (100 nM) or EBSS for 4 hours. Next, the cells were fixed and labeled against the LAMP2 protein as described in Materials and Methods. Panel a shows representative images from three independent experiments of each cell line for the conditions studied. The scale bar represents the length of 10 μm. (b-g) Characterization of lysosomal proteins by western blot. Cell lysates were obtained from control (Co) and R1441G fibroblasts (RG) treated or not with Baf. A1 (100 nM) or EBSS for 4 hours and equal amounts of proteins were separated in 12% polyacrylamide gels as described in the Materials and Methods section. Panels (b, d, e) show representative western blots targeting LAMP1 and LAMP2 (b), CTSB (d) and CTSC (e) proteins in three independent experiments. β-Actin was used as a loading control. Panels (c, f, g) show the densitometry histograms associated with the experiments represented in b, d and e, respectively. The asterisks show significant differences between cell types, while the pound signs reflect differences related to the treatment (* or #, p < 0.05; ** or ##, p < 0.01). (h, i) Analysis of p-S6 protein. Cell lysates were obtained from control (Co) and R1441G fibroblasts (RG) treated or not with Baf. A1 (100 nM) or EBSS for 4 hours and equal amounts of proteins were separated in 12% polyacrylamide gels as described in the Materials and Methods section. Panel h shows representative western blot and panel i shows the densitometry histogram. β-Actin was used as a loading control. The pound sign reflect differences related to the treatment (###, p < 0.001). (j-m) Characterization of autophagic proteins by western blot. Cell lysates from control (Co) and R1441G fibroblasts (RG) treated or not with Baf. A1 (100 nM) or EBSS for 4 hours were obtained and equal amounts of proteins were loaded in 12% polyacrylamide gels as described in the Materials and Methods section. Panel j shows representative western blots of p-mTOR, ATG5 and Beclin1 proteins of three independent experiments. β-Actin was used as a loading control. The panels show the densitometry histograms associated for p-mTOR (k), ATG5 (l) and BECN1 (m) proteins of three independent experiments. The asterisks show significant differences between cell types, while the pound signs reflect differences related to the treatment (** p < 0.01; #, p < 0.05) (PDF 101 KB)

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Supplementary Figure S3 Determination of mitochondrial status and mitochondrial proteins as well as mitolysosome formation in the presence of CCCP. (a-e) Densitometric analyses of selected mitochondrial proteins. The control (Co) and R1441G fibroblasts (RG) were treated and collected as in Fig. 2 (c-e). The panels show the densitometries of the representative western blot shown in Fig. 2 (c-e) for the TOMM20 (a), PHB1 (b), VDAC1 (c), TIMM23 (d) and COXIV (e) proteins, respectively. The asterisks show significant differences (* p < 0.05; ** p < 0.01). (f) Electron microscopy (EM) analysis of mitochondrial content. The cell pellet from the control and R1441G fibroblasts was fixed and processed for EM as described in the Materials and Methods section. The arrows and colored structures in the image point to the recognizable mitochondria. (g) Analysis of mitochondrial reactive oxygen species (ROS) through MitoSOX labeling. The control and R1441G fibroblasts were treated or not with rotenone (1 μM) for 4 hours. Next, the cells were labeled with the MitoSOX staining and processed according to the Materials and Methods section. Panel shows representative immunofluorescence images. h, Study of mitochondria and lysosome colocalization by fluorescence microscopy. The control (Co) and R1441G fibroblasts (RG) were treated for four hours with CCCP (10 μM). Next, the cells were stained and processed as in Fig. 2 (h, i). Panel (h) shows representative images of each of the cell groups under the conditions studied. The scale bar represents a length of 10 μm. (i) The graphs represent the average of the Mander coefficient for the proportion of LTR on the TOMM20 label plus the standard deviation after the treatment with CCCP. (j, k). Effect of Baf. A1 in TOMM20 and TIMM23 proteins levels. Cell lysates were obtained from control (Co) and R1441G fibroblasts (RG) treated or not with Baf. A1 (100 nM) for 4 hours and equal amounts of proteins were separated in 12% polyacrylamide gels as described in the Materials and Methods section. Panel j shows the densitometry histogram associated with the representative western blot targeting TIMM23 and TOMM20 proteins in three independent experiments showed in panel k. β-Actin was used as a loading control. The asterisks show significant differences between cell types, while the pound signs reflect differences related to the treatment (* or #, p < 0.05) (PDF 88 KB)

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Yakhine-Diop, S.M.S., Rodríguez-Arribas, M., Canales-Cortés, S. et al. The parkinsonian LRRK2 R1441G mutation shows macroautophagy-mitophagy dysregulation concomitant with endoplasmic reticulum stress. Cell Biol Toxicol (2021). https://doi.org/10.1007/s10565-021-09617-w

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Keywords

  • Autophagy
  • MAMs
  • Mitochondrial dysfunction
  • Neurodegeneration
  • Parkinson disease