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The Upshot of LRRK2 Inhibition to Parkinson’s Disease Paradigm

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Abstract

Mutations in leucine-rich repeat kinase 2 gene (LRRK2) are implicated in autosomal dominant familial and sporadic Parkinson’s disease (sPD). Given its relative frequency in PD and its putative function in several cellular pathways that are known to be impaired in the disease, we wanted to tackle LRRK2 physiological role and to address its potential as a PD therapeutic target. We investigated the impact of pharmacological inhibition of LRRK2 kinase activity in control and PD cell function. We provide evidence that physiologically LRRK2, through its kinase activity, regulates mitochondrial fission events and facilitates autophagic degradation by modulating lysosomal cellular localization. Upon LRRK2 inhibition, normal fission decreases, leading to the elongation of mitochondrial network which contributes to a poor degradation of deficient mitochondria. Moreover, LRRK2 inhibition promotes lysosomal perinuclear clustering, through Rab7 that further hinders autophagosomes degradation. These events induce a decrease in the autophagic flow, which contributed directly to a decreased proteolytic degradation of damaged mitochondria. These data resembled the results observed in sPD cells. Interestingly, the LRRK2 kinase activity is increased in sPD cells, and despite its inhibition recovers mitochondrial cellular localization, it did not improve microtubule network-dependent trafficking. Our results provide novel insights into the multiple mechanisms that dictate the association between LRRK2 and mitophagy in sPD, and contribute with new findings that could have important therapeutic implications.

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Acknowledgments

Work in our laboratory is supported by Portuguese Foundation for Science and Technology (FCT-MCTES, Portugal) (funds from PTDC/SAU-NEU/102710/2008 to SM Cardoso and PEst-C/SAU/LA0001/2011). AR Esteves is supported by Post-Doctoral Fellowship and D Santos is supported by PhD Fellowship from FCT-MCTES, Portugal.

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Authors and Affiliations

  1. CNC—Centro de Neurociências e Biologia Celular, Universidade de Coimbra, Coimbra, Portugal

    A. R. Esteves, M. G-Fernandes, D. Santos & S. M. Cardoso

  2. Hospitais da Universidade de Coimbra, 3000, Coimbra, Portugal

    C. Januário

  3. Institue of Biology, Faculty of Medicine, Universidade de Coimbra, Coimbra, Portugal

    S. M. Cardoso

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  1. A. R. Esteves
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  2. M. G-Fernandes
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  3. D. Santos
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  4. C. Januário
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  5. S. M. Cardoso
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Correspondence to S. M. Cardoso.

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Supplementary Fig. 1

IN-1 does not affect cellular proliferation. MTT reduction ability assay of CT and sPD cells in the presence of several IN-1 concentrations. Data is reported as the fold increase over untreated CT cybrids. N = 3. (GIF 1329 kb)

High Resolution Image (TIFF 552 kb)

Supplementary Fig. 2

Mitochondria are ubiquitinated and signaled for degradation in sPD cells. a Representative imunoblot of mitochondrial and cytosolic Parkin of CT and sPD cybrids. b Densitometric analysis of Parkin levels. Data is reported as the fold increase over untreated CT cybrids. The blots were reprobed for Hsp60 and GAPDH to confirm equal protein loading and to confirm fraction purity. N = 3, *p < 0.05 significantly different relatively to untreated CT cybrid. c Co-immunoprecipitation of Mitofusin1 and ubiquitin in CT and PD cybrids. d Determination of Mitofusin 1/ubiquitin physical interaction. Data is reported as the fold increase over untreated CT cybrids. N = 3 *p < 0.05 significantly different relatively to untreated CT cybrid. (GIF 1819 kb)

High Resolution Image (TIFF 1848 kb)

Supplementary Figure 3

The essential activator of autophagy, Beclin1 is not affected upon exposure to IN-1. a Representative immunoblot for Beclin1 from CT and PD cybrids after treatment with or without IN-1. b Densitometric analysis of endogenous levels of Beclin1. Data is reported as the fold increase over untreated CT cybrids. The blots were reprobed for α-Tubulin to confirm equal protein loading. N = 3. (GIF 1450 kb)

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Supplementary Figure 4

a The number of LC3B dots was quantified with ImageJ. Data is reported as absolute values. *p < 0.05, **p < 0.01 and ***p < 0.001, significantly different when compared to untreated CT cybrid cells; && p < 0.01, significantly different when compared to NL-treated CT cybrid cells. b Lamp1 fluorescence intensity was calculated after morphometric quantification of cells stained as the ones shown here. Data is reported as absolute values. *p < 0.05, ***p < 0.001, significantly different when compared to untreated CT cybrid cells; ##p < 0.01, significantly different when compared to untreated PD cybrid cells. (GIF 1381 kb)

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Supplementary Figure 5

HDAC6 content is not altered in the presence of IN-1. a Representative immunoblot of HDAC6. b Densitometric analysis of HDAC6. Data is reported as the fold increase over untreated CT cybrids. The blots were reprobed for α-Tubulin to confirm equal protein loading. N = 4. (GIF 1422 kb)

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Esteves, A.R., G-Fernandes, M., Santos, D. et al. The Upshot of LRRK2 Inhibition to Parkinson’s Disease Paradigm. Mol Neurobiol 52, 1804–1820 (2015). https://doi.org/10.1007/s12035-014-8980-6

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  • DOI: https://doi.org/10.1007/s12035-014-8980-6

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