Smith, W. W. et al. Leucine-rich repeat kinase 2 (LRRK2) interacts with parkin, and mutant LRRK2 induces neuronal degeneration. Proc. Natl Acad. Sci. USA 102, 18676-18681

Department of Psychiatry, Division of Neurobiology, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 01/2006; 102(51):18676-81. DOI: 10.1073/pnas.0508052102
Source: PubMed


Parkinson's disease (PD) is a disorder of movement, cognition, and emotion, and it is characterized pathologically by neuronal degeneration with Lewy bodies, which are cytoplasmic inclusion bodies containing deposits of aggregated proteins. Most PD cases appear to be sporadic, but genetic forms of the disease, caused by mutations in alpha-synuclein, parkin, and other genes, have helped elucidate pathogenesis. Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal-dominant Parkinsonism with clinical features of PD and with pleomorphic pathology including deposits of aggregated protein. To study expression and interactions of LRRK2, we synthesized cDNAs and generated expression constructs coding for human WT and mutant LRRK2 proteins. Expression of full-length LRRK2 in cells in culture suggests that the protein is predominately cytoplasmic, as is endogenous protein by subcellular fractionation. Using coimmunoprecipitation, we find that LRRK2, expressed in cells in culture, interacts with parkin but not with alpha-synuclein, DJ-1, or tau. A small proportion of the cells overexpressing LRRK2 contain protein aggregates, and this proportion is greatly increased by coexpression of parkin. In addition, parkin increases ubiquitination of aggregated protein. Also, mutant LRRK2 causes neuronal degeneration in both SH-SY5Y cells and primary neurons. This cell model may be useful for studies of PD cellular pathogenesis and therapeutics. These findings suggest a gain-of-function mechanism in the pathogenesis of LRRK2-linked PD and suggest that LRRK2 may be involved in a pathogenic pathway with other PD-related proteins such as parkin, which may help illuminate both familial and sporadic PD.

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Available from: Zhong Pei, Jan 05, 2015
    • "Human recombinant, E1, E2a, E3-deleted (second generation), serotype 5 adenoviruses (Ad5) were produced and purified as previously described (Dusonchet et al., 2011). Codon-optimized full-length WT and G2019S human LRRK2 cDNAs, tagged at the N-terminus with a 3×FLAG epitope tag (kindly provided by Dr Christopher Ross, Johns Hopkins University (Smith et al., 2005)) were cloned into a modified pDC511 shuttle plasmid 3′ of a human synapsin-1 promoter and synthetic intron (Ng and Graham, 2002; Young and Neher, 2009). The D1994N mutation was introduced into the pDC511-G2019S LRRK2 sequence through site-directed mutagenesis using the QuickChange XL site-directed mutagenesis kit (Stratagene). "
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    ABSTRACT: Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant Parkinson's disease (PD). LRRK2 contains functional GTPase and kinase domains. The most common G2019S mutation enhances the kinase activity of LRRK2 in vitro whereas G2019S LRRK2 expression in cultured neurons induces toxicity in a kinase-dependent manner. These observations suggest a potential role for kinase activity in LRRK2-associated PD. We have recently developed a novel rodent model of PD with progressive neurodegeneration induced by the adenoviral-mediated expression of G2019S LRRK2. In the present study, we further characterize this LRRK2 model and determine the contribution of kinase activity to LRRK2-mediated neurodegeneration. Recombinant human adenoviral vectors were employed to deliver human wild-type, G2019S or kinase-inactive G2019S/D1994N LRRK2 to the rat striatum. LRRK2-dependent pathology was assessed in the striatum, a region where LRRK2 protein is normally enriched in the mammalian brain. Human LRRK2 variants are robustly expressed throughout the rat striatum. Expression of G2019S LRRK2 selectively induces the accumulation of neuronal ubiquitin-positive inclusions accompanied by neurite degeneration and the altered distribution of axonal phosphorylated neurofilaments. Importantly, the introduction of a kinase-inactive mutation (G2019S/D1994N) completely ameliorates the pathological effects of G2019S LRRK2 in the striatum supporting a kinase activity-dependent mechanism for this PD-associated mutation. Collectively, our study further elucidates the pathological effects of the G2019S mutation in the mammalian brain and supports the development of kinase inhibitors as a potential therapeutic approach for treating LRRK2-associated PD. This adenoviral rodent model provides an important tool for elucidating the molecular basis of LRRK2-mediated neurodegeneration. Copyright © 2015. Published by Elsevier Inc.
    Neurobiology of Disease 02/2015; 77. DOI:10.1016/j.nbd.2015.02.019 · 5.08 Impact Factor
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    • "Furthermore, dopamine neuron loss has been demonstrated in invertebrate organisms and through virally mediated G2019S LRRK2 expression in rodent brain (Lee et al. 2010; Dusonchet et al. 2011). Numerous studies on primary neuronal cultures and intact rodent brain have demonstrated that LRRK2 is important in maintaining neurite length and branching (Smith et al. 2005; MacLeod et al. 2006; West et al. 2007) and may be important in regulating neurite outgrowth, possibly via phosphorylation of ERM (ezrin/radixin/moesin) proteins and consequent maintenance of F-actin homeostasis in filopodia necessary for proper neurite outgrowth (Jaleel et al. 2007; Parisiadou et al. 2009). Expression of pathogenic LRRK2 variants results in neurite dystrophy or loss and ultimately leads to cell death. "
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    ABSTRACT: Mutations in the catalytic Roc-COR and kinase domains of leucine-rich repeat kinase 2 (LRRK2) are a common cause of familial Parkinson's disease (PD). LRRK2 mutations cause PD with age-related penetrance and clinical features identical to late-onset sporadic PD. Biochemical studies support an increase in LRRK2 kinase activity and a decrease in GTPase activity for kinase domain and Roc-COR mutations, respectively. Strong evidence exists that LRRK2 toxicity is kinase-dependent leading to extensive efforts to identify selective and brain-permeable LRRK2 kinase inhibitors for clinical development. Cell and animal models of PD indicate that LRRK2 mutations affect vesicular trafficking, autophagy, protein synthesis and cytoskeletal function. Although some of these biological functions are affected consistently by most disease-linked mutations, others are not and it is currently unclear how mutations that produce variable effects on LRRK2 biochemistry and function all commonly result in the degeneration and death of dopamine neurons. LRRK2 is typically present in Lewy bodies and its toxicity in mammalian models appears to be dependent on the presence of α-synuclein, which is elevated in human iPS-derived dopamine neurons from patients harboring LRRK2 mutations. Here, we summarize biochemical and functional studies of LRRK2 and its mutations and focus on aberrant vesicular trafficking and protein synthesis as two leading mechanisms underlying LRRK2-linked disease.This article is protected by copyright. All rights reserved.
    Journal of Neurochemistry 09/2014; 131(5). DOI:10.1111/jnc.12949 · 4.28 Impact Factor
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    • "LRRK2 or PARK8 is a protein kinase expressed preferentially in DAergic neurons (Han et al., 2008). It interacts with parkin in the cytosol and overexpression of either WT LRRK2 or gain-of-function mutants results in DAergic neurodegeneration (Smith et al., 2005). Together, these proteins either interact with parkin or have similar function to parkin. "
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    ABSTRACT: Manganese (Mn), is a trace metal required for normal physiological processes in humans. Mn levels are tightly regulated, as high levels of Mn result in accumulation in the brain and cause a neurological disease known as manganism. Manganism shares many similarities with Parkinson's disease (PD), both at the physiological level and the cellular level. Exposure to high Mn-containing environments increases the risk of developing manganism. Mn is absorbed primarily through the intestine and then released in the blood. Excessive Mn is secreted in the bile and excreted in feces. Mn enters and exits cells through a number of non-specific importers localized on the cell membrane. Mutations in one of the Mn exporters, SLC30A10 (solute carrier family 30, member 10), result in Mn induced toxicity with liver impairments and neurological dysfunction. Four PD genes have been identified in connection to regulation of Mn toxicity, shedding new light on potential links between manganism and PD.
    Frontiers in Genetics 08/2014; 5:265. DOI:10.3389/fgene.2014.00265
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