Dopaminergic Neuronal Loss, Reduced Neurite Complexity and Autophagic Abnormalities in Transgenic Mice Expressing G2019S Mutant LRRK2

Brain Mind Institute, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
PLoS ONE (Impact Factor: 3.23). 04/2011; 6(4):e18568. DOI: 10.1371/journal.pone.0018568
Source: PubMed


Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant familial Parkinson's disease (PD) and also contribute to idiopathic PD. LRRK2 mutations represent the most common cause of PD with clinical and neurochemical features that are largely indistinguishable from idiopathic disease. Currently, transgenic mice expressing wild-type or disease-causing mutants of LRRK2 have failed to produce overt neurodegeneration, although abnormalities in nigrostriatal dopaminergic neurotransmission have been observed. Here, we describe the development and characterization of transgenic mice expressing human LRRK2 bearing the familial PD mutations, R1441C and G2019S. Our study demonstrates that expression of G2019S mutant LRRK2 induces the degeneration of nigrostriatal pathway dopaminergic neurons in an age-dependent manner. In addition, we observe autophagic and mitochondrial abnormalities in the brains of aged G2019S LRRK2 mice and markedly reduced neurite complexity of cultured dopaminergic neurons. These new LRRK2 transgenic mice will provide important tools for understanding the mechanism(s) through which familial mutations precipitate neuronal degeneration and PD.

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    • "Unbiased stereological estimation of the number of dopaminergic neurons was performed using the optical fractionator probe of the StereoInvestigator software (MicroBrightField Biosciences), as described (37,38). Every fifth serial coronal section of 40 μm thickness, covering the entire substantia nigra region was immunostained with rabbit anti-TH antibody and counterstained with cresyl violet stain. "
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    ABSTRACT: Mutations in the vacuolar protein sorting 35 homolog (VPS35) gene at the PARK17 locus, encoding a key component of the retromer complex, were recently identified as a new cause of late-onset, autosomal dominant Parkinson's disease (PD). Here we explore the pathogenic consequences of PD-associated mutations in VPS35 using a number of model systems. VPS35 exhibits a broad neuronal distribution throughout the rodent brain, including within the nigrostriatal dopaminergic pathway. In the human brain, VPS35 protein levels and distribution are similar in tissues from control and PD subjects, and VPS35 is not associated with Lewy body pathology. The common D620N missense mutation in VPS35 does not compromise its protein stability or localization to endosomal and lysosomal vesicles, or the vesicular sorting of the retromer cargo, sortilin, SorLA and cation-independent mannose 6-phosphate receptor, in rodent primary neurons or patient-derived human fibroblasts. In yeast we show that PD-linked VPS35 mutations are functional and can normally complement VPS35 null phenotypes suggesting that they do not result in a loss-of-function. In rat primary cortical cultures the overexpression of human VPS35 induces neuronal cell death and increases neuronal vulnerability to PD-relevant cellular stress. In a novel viral-mediated gene transfer rat model, the expression of D620N VPS35 induces the marked degeneration of substantia nigra dopaminergic neurons and axonal pathology, a cardinal pathological hallmark of PD. Collectively, these studies establish that dominant VPS35 mutations lead to neurodegeneration in PD consistent with a gain-of-function mechanism, and support a key role for VPS35 in the development of PD.
    Human Molecular Genetics 04/2014; 23(17). DOI:10.1093/hmg/ddu178 · 6.39 Impact Factor
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    • "Alternatively, LRRK2 modestly phosphorylates OPA1 in vitro and the levels of mature S-OPA1 are reduced in G2019S PD brains. Therefore, the functional interaction of LRRK2 with dynamin GTPases is rather complex but provides initial mechanistic support of a role for LRRK2 in regulating membrane dynamics important for mediating endocytosis and mitochondrial function (14,24–27,32,36–38). Future studies are warranted to clarify the physiological or pathological interaction of LRRK2 with dynamin GTPases in animal models. "
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    ABSTRACT: Mutations in LRRK2 cause autosomal dominant Parkinson's disease (PD). LRRK2 encodes a multi-domain protein containing GTPase and kinase domains, and putative protein-protein interaction domains. Familial PD mutations alter the GTPase and kinase activity of LRRK2 in vitro. LRRK2 is suggested to regulate a number of cellular pathways although the underlying mechanisms are poorly understood. To explore such mechanisms it has proved informative to identify LRRK2-interacting proteins, some of which serve as LRRK2 kinase substrates. Here, we identify common interactions of LRRK2 with members of the dynamin GTPase superfamily. LRRK2 interacts with dynamin 1-3 that mediate membrane scission in clathrin-mediated endocytosis and with dynamin-related proteins that mediate mitochondrial fission (Drp1) and fusion (mitofusins and OPA1). LRRK2 partially co-localizes with endosomal dynamin-1 or with mitofusins and OPA1 at mitochondrial membranes. The subcellular distribution and oligomeric complexes of dynamin GTPases are not altered by modulating LRRK2 in mouse brain, whereas mature OPA1 levels are reduced in G2019S PD brains. LRRK2 enhances mitofusin-1 GTP binding, whereas dynamin-1 and OPA1 serve as modest substrates of LRRK2-mediated phosphorylation in vitro. While dynamin GTPase orthologs are not required for LRRK2-induced toxicity in yeast, LRRK2 functionally interacts with dynamin-1 and mitofusin-1 in cultured neurons. LRRK2 attenuates neurite shortening induced by dynamin-1 by reducing its levels, whereas LRRK2 rescues impaired neurite outgrowth induced by mitofusin-1 potentially by reversing excessive mitochondrial fusion. Our study elucidates novel functional interactions of LRRK2 with dynamin superfamily GTPases that implicate LRRK2 in the regulation of membrane dynamics important for endocytosis and mitochondrial morphology.
    Human Molecular Genetics 11/2013; 23(8). DOI:10.1093/hmg/ddt600 · 6.39 Impact Factor
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    • "Tissues from 2 months old LRRK2WT or LRRK2G2019S transgenic mice [16] were quickly dissected and frozen. Subcellular fractionation was conducted as described in [31]. "
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    ABSTRACT: The leucine-rich repeat kinase 2 (LRRK2) gene was found to play a role in the pathogenesis of both familial and sporadic Parkinson's disease (PD). LRRK2 encodes a large multi-domain protein that is expressed in different tissues. To date, the physiological and pathological functions of LRRK2 are not clearly defined. In this study we have explored the role of LRRK2 in controlling vesicle trafficking in different cellular or animal models and using various readouts. In neuronal cells, the presence of LRRK2(G2019S) pathological mutant determines increased extracellular dopamine levels either under basal conditions or upon nicotine stimulation. Moreover, mutant LRRK2 affects the levels of dopamine receptor D1 on the membrane surface in neuronal cells or animal models. Ultrastructural analysis of PC12-derived cells expressing mutant LRRK2(G2019S) shows an altered intracellular vesicle distribution. Taken together, our results point to the key role of LRRK2 to control vesicle trafficking in neuronal cells.
    PLoS ONE 10/2013; 8(10):e77198. DOI:10.1371/journal.pone.0077198 · 3.23 Impact Factor
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