The Parkinson disease protein leucine-rich repeat kinase 2 transduces death signals via Fas-associated protein with death domain and caspase-8 in a cellular model of neurodegeneration - PubMed (original) (raw)

The Parkinson disease protein leucine-rich repeat kinase 2 transduces death signals via Fas-associated protein with death domain and caspase-8 in a cellular model of neurodegeneration

Cherry Cheng-Ying Ho et al. J Neurosci. 2009.

Abstract

Neurodegenerative illnesses such as Parkinson and Alzheimer disease are an increasingly prevalent problem in aging societies, yet no therapies exist that retard or prevent neurodegeneration. Dominant missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson disease (PD), but the mechanisms by which mutant forms of LRRK2 disrupt neuronal function and cause cell death remain poorly understood. We report that LRRK2 interacts with the death adaptor Fas-associated protein with death domain (FADD), and that in primary neuronal culture LRRK2-mediated neurodegeneration is prevented by the functional inhibition of FADD or depletion of caspase-8, two key elements of the extrinsic cell death pathway. This pathway is activated by disease-triggering mutations, which enhance the LRRK2-FADD association and the consequent recruitment and activation of caspase-8. These results establish a direct molecular link between a mutant PD gene and the activation of programmed cell death signaling, and suggest that FADD/caspase-8 signaling contributes to LRRK2-induced neuronal death.

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Figures

Figure 1.

Figure 1.

Parkinson disease mutations enhance the interaction between LRRK2 and FADD. A, Domain structure and Parkinson disease mutations of LRRK2. LRR, leucine-rich repeat; Roc, Ras of complex GTPase; COR, C-terminal of Roc. Five dominantly inherited PD-causing missense mutations are indicated. B, LRRK2 interacts with death adaptor proteins of the extrinsic pathway. 293T cells coexpressing GFP-LRRK2 and V5-tagged death adaptor proteins were subjected to anti-GFP immunoprecipitation followed by anti-GFP and anti-V5 immunoblotting. C, LRRK2 does not associate with death receptors. 293T cells coexpressing GFP-LRRK2 with GST-tagged cytoplasmic domains of death receptors were subjected to GST pull-down followed by immunoblotting. D, Enhanced association of FADD with LRRK2 PD mutants. 293T cells were cotransfected with wild-type (WT) or PD mutant GFP-LRRK2 and V5-tagged FADD. Anti-GFP immunoprecipitates were analyzed by anti-V5 and anti-GFP immunoblots. Ratios indicate the binding of FADD to LRRK2 relative to WT-LRRK2. E, PD mutations fail to enhance the association of LRRK2 with RIP1. 293T cells were cotransfected with V5-tagged RIP1 and WT or PD mutant GFP-LRRK2 and were assessed as in D. F, PD mutations fail to enhance the association of LRRK2 with TRADD. 293T cells were cotransfected with V5-tagged TRADD and WT or PD mutant GFP-LRRK2 and were assessed as in D. G, Endogenous LRRK2-FADD complex formation in mouse brain. Whole-brain lysates from 1-year-old wild-type mice were subjected to immunoprecipitation with anti-FADD (clone 7A2). Copurified LRRK2 was determined with anti-LRRK2 immunoblotting. H, Blocking LRRK2 kinase function prevents the enhanced FADD association with LRRK2 disease mutants. 293T cells expressing V5-tagged FADD and GFP-tagged WT or kinase-dead (KD) LRRK2 were immunoprecipitated and immunoblotted as in D.

Figure 2.

Figure 2.

LRRK2-induced neuronal death requires FADD. A, A schematic depicts the domain structure of FADD, the isolated death domain (FADD-DD), and the leucine-zipper-DD (LZ-FADD-DD) in which the death domain is dimerized through the addition of a leucine zipper. B, FADD interacts with LRRK2 via its DD. GFP-LRRK2 was coexpressed with V5-tagged full-length FADD, FADD-DED, or FADD-DD in 293T cells, and was immunoprecipitated with anti-GFP. Copurified FADD or FADD domains were detected by anti-V5 immunoblotting. C, LRRK2-FADD interaction is enhanced by dimerization of FADD-DD. The interaction between GFP-LRRK2 and monomeric (DD) or dimeric (LZ) FADD-DD was assessed by anti-HA after immunoprecipitation with anti-GFP in 293T cells. D, FADD-DD is a poor inhibitor of LRRK2 neurotoxicity. Mouse cortical neurons were transfected with LRRK2 + lacZ (Ctrl) or LRRK2 + FADD-DD. A GFP reporter was cotransfected in each case. Transfected neurons displaying apoptotic nuclear morphology were counted 48 h after transfection using DAPI. Data are the mean ±SEM from three individual experiments of triplicate coverslips (n.s., nonsignificant; ANOVA with Tukey's post hoc test). E, Dimeric FADD-DD effectively blocks LRRK2 neurotoxicity. Mouse cortical neurons expressing LRRK + lacZ (Ctrl) or LRRK2 + LZ-FADD-DD were assessed as in D (***p < 0.001).

Figure 3.

Figure 3.

LRRK2-induced neuronal death is caspase-8 (Casp8) dependent. A, FADD recruits caspase-8 to LRRK2. 293T cells were transfected with GFP-LRRK2, V5-FADD, and caspase-8 inactive mutant (C360S), as indicated. GFP-LRRK2 was immunoprecipitated, and copurified FADD and caspase-8 were detected by V5 and caspase-8 antibodies, respectively. B, Knockdown of caspase-8 blocks LRRK2-induced neurotoxicity. Mouse cortical neurons were incubated with Penetratin1-linked scrambled control (Ctrl) or caspase-8 siRNA 24 h before transfection with GFP-tagged wild-type (WT) or mutant LRRK2. Data are the mean ±SEM from three individual experiments of triplicate coverslips (*p < 0.05; **p < 0.01; ***p < 0.001). A representative immunoblot of caspase-8 levels after treatment with Penetratin1-linked siRNA is shown (inset). C, Knockdown of caspase-9 (Casp9) fails to prevent LRRK2-induced neuronal death. Caspase-9 RNAi, transfection, and neuronal death were performed and assessed as in B. D, Caspase-8 is selectively activated in brain tissue from patients with LRRK2 PD. Striatal lysates were analyzed by caspase-8 (clone 1C12), caspase-9, and caspase-1 (clone A19) immunoblotting. Caspase-2 was undetectable using three commercial antibodies (data not shown). The locations of the pro-caspase isoforms and their corresponding cleavage products are indicated (*nonspecific immunoreactive bands; #potential cleavage products with higher than expected mass: 37 and 35 kDa).

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