NGFN-PLUS
Functional characterization of LRRK2 in mammalian cells and tissues
| Coordinator: | PD Dr. Marius Ueffing | |
| Institution: | Helmholtz Zentrum München / TU München | |
| Homepage: | www.helmholtz-muenchen.de |
Parkinson Disease (PD) is a progressive neurogenerative disease which impairs motor skills. It is characterised by the loss of dopaminergic neurons in the substantia nigra, a structure in the midbrain. Besides the sporadic form of PD (idiopathic PD), characterised by a complex etiology, familiar forms exist which are caused by single gene defects. One of these genes is the leucine rich repeat kinase 2 (LRRK2). Mutations within LRRK2 show a dominant inheritance and explain up to 5% of the familiar forms of PD (Zimprich et al., 2004). The LRRK2 protein has a complex domain composition and belongs to the family of protein kinases. Protein kinases play a fundamental role in the regulation of complex cellular responses by transferring phosphate groups onto their substrate proteins. Recent studies point towards an increase of LRRK2 kinase activity caused by PD-associated mutations (West et al., 2005, Gloeckner et al., 2006). The most common PD-associated LRRK2 mutation, glycine to serine at position 2019 (G2019S), is localised within the activation segment of the kinase domain. The kinase domain of LRRK2 shows similarity to mitogen activated protein kinase kinase kinases (MAPKKK) which play a central role in mediating cellular stress events. In addition to its kinase domain, LRRK2 contains a Ras-like (Ras of complex proteins, roc) domain, which exhibits GTPase activity (West et al., 2007). Ras-like GTPases are part of MAP-kinase signalling pathways. For this reason, the aim of the work supported by the NGFN is to systematically address the potential function of LRRK2 in cellular signalling. This involves systematic as well as candidate-driven approaches to search for LRRK2 substrates. A systematic screen recently identified the structure protein moesin as a LRRK2 substrate, in vitro. As part of the NGFN-supported work, a candidate-driven approach revealed that MKKs, kinases which are involved in cellular stress-signalling, serve as LRRK2 substrates, in vitro (Gloeckner et al., 2009).
Cell culture experiments revealed that an increase in LRRK2 activity exhibits cytotoxic effects (Smith et al., 2006, West et al., 2007). Established cell cultures as well as primary neuron cultures (i.e. dopaminergic neurons) will now be used to get a more detailed knowledge of the LRRK2-function in neurons and the mechanisms underlying the cytotoxic effects of LRRK2 mutations. Furthermore, experiments are planned to systematically analyse the interaction network of LRRK2 on the basis of protein complexes formed by LRRK2. Analysing the LRRK2 interactome is of special interest because LRRK2 is a large protein of 280 kDa and contains domains like the leucine rich repeats or a WD40 propeller domain, the latter belonging to domain families known to mediate protein interactions and often found in scaffolding proteins (reviewed in Mata et al., 2006). The interactome analysis will be done with an affinity-based enrichment of the LRRK2 protein complexes based on a tandem affinity purification technique described in (Gloeckner et al., 2007) followed by mass-spectrometric analysis of their compositions.
In consequence, in collaboration with the pharmaceutical industry, the knowledge gained by the experiments will be used to identify targets for a drug treatment of PD.
References:
Gloeckner et al. (2006), Hum. Mol. Genet., 15:223-232
Gloeckner et al. (2007), Proteomics, 7:4228-4234
Gloeckner et al. (2009), J. Neurochem., in press
Jaleel et al. (2007), Biochem. J., 405:307-317
Mata et al. (2006), Trends Mol. Med., 12:76-82
Smith et al. (2006), Nat. Neurosci., 9:1231-1233
West el al. (2005), Proc. natl. Acad. Sci., 102:16842-16847
West et al. (2007), Hum. Mol. Genet., 16:223-232
Zimprich et al. (2004), Neuron, 44:601-607
Functional analysis of PD-associated LRRK2 mutations
Cell culture experiments revealed that an increase in LRRK2 activity exhibits cytotoxic effects (Smith et al., 2006, West et al., 2007). Established cell cultures as well as primary neuron cultures (i.e. dopaminergic neurons) will now be used to get a more detailed knowledge of the LRRK2-function in neurons and the mechanisms underlying the cytotoxic effects of LRRK2 mutations. Furthermore, experiments are planned to systematically analyse the interaction network of LRRK2 on the basis of protein complexes formed by LRRK2. Analysing the LRRK2 interactome is of special interest because LRRK2 is a large protein of 280 kDa and contains domains like the leucine rich repeats or a WD40 propeller domain, the latter belonging to domain families known to mediate protein interactions and often found in scaffolding proteins (reviewed in Mata et al., 2006). The interactome analysis will be done with an affinity-based enrichment of the LRRK2 protein complexes based on a tandem affinity purification technique described in (Gloeckner et al., 2007) followed by mass-spectrometric analysis of their compositions.
In consequence, in collaboration with the pharmaceutical industry, the knowledge gained by the experiments will be used to identify targets for a drug treatment of PD.
References:
Gloeckner et al. (2006), Hum. Mol. Genet., 15:223-232
Gloeckner et al. (2007), Proteomics, 7:4228-4234
Gloeckner et al. (2009), J. Neurochem., in press
Jaleel et al. (2007), Biochem. J., 405:307-317
Mata et al. (2006), Trends Mol. Med., 12:76-82
Smith et al. (2006), Nat. Neurosci., 9:1231-1233
West el al. (2005), Proc. natl. Acad. Sci., 102:16842-16847
West et al. (2007), Hum. Mol. Genet., 16:223-232
Zimprich et al. (2004), Neuron, 44:601-607
Functional analysis of PD-associated LRRK2 mutations