No abstract

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) cause late-onset Parkinson's disease indistinguishable from idiopathic disease. The mechanisms whereby missense alterations in the LRRK2 gene initiate neurodegeneration remain unknown. Here, we demonstrate that seven of 10 suspected familial-linked mutations result in increased kinase activity. Functional and disease-associated mutations in conserved residues reveal the critical link between intrinsic guanosine triphosphatase (GTPase) activity and downstream kinase activity. LRRK2 kinase activity requires GTPase activity, whereas GTPase activity functions independently of kinase activity. Both LRRK2 kinase and GTPase activity are required for neurotoxicity and potentiate peroxide-induced cell death, although LRRK2 does not function as a canonical MAP-kinase-kinase-kinase. These results suggest a link between LRRK2 kinase activity and pathogenic mechanisms relating to neurodegeneration, further supporting a gain-of-function role for LRRK2 mutations.

INTRODUCTION Parkinson’s disease (PD) is the second most common neurodegenerative disorder that leads to slowness of movement, tremor, rigidity and in the later stages of PD, cognitive impairment. Pathologically PD is characterized by the accumulation of α-synuclein in Lewy bodies and neurites. There is degeneration of neurons throughout the nervous system with the degeneration of dopamine neurons in the substantia nigra pars compacta leading to the major symptoms of PD. RATIONALE In the brains of PD patients, pathologic α-synuclein seems to spread from cell-to-cell via self-amplification, propagation, and transmission in a stereotypical and topographical pattern among neighboring cells and/or anatomically connected brain regions. The spread or transmission of pathologic α-synuclein is emerging as potentially important driver of PD pathogenesis. The underlying mechanisms and molecular entities responsible for the transmission of pathologic α-synuclein from cell-to-to cell are not known, but the entry of pathologic α-synuclein into neurons is thought to occur, in part through an active clathrin-dependent endocytic process. RESULTS Using recombinant α-synuclein pre-formed fibrils (PFF) as a model system to study the transmission of misfolded α-synuclein from neuron to neuron, we screened a library encoding transmembrane proteins for α-synuclein-biotin PFF binding candidates via detection by streptavidin-AP (alkaline phosphatase) staining. Three positive clones were identified that bind α-synuclein PFF and include lymphocyte-activation gene 3 (LAG3), neurexin 1β and amyloid beta precursor-like protein 1 (APLP1). Of these three transmembrane proteins, LAG3 demonstrated the highest ratio of selectivity for α-synuclein PFF over the α-synuclein monomer. α-Synuclein PFF binds to LAG3 in a saturable manner (Kd = 77 nM), while the α-synuclein monomer does not bind to LAG3. Co-immunoprecipitation also suggests that pathological α-synuclein PFF specifically binds to LAG3. Tau PFF, β-amyloid oligomer and β-amyloid PFF do not bind LAG3 indicating that LAG3 is specific for α-synuclein PFF. The internalization of α-synuclein PFF involves LAG3 since deletion of LAG3 reduces the endocytosis of α-synuclein PFF. LAG3 colocalizes with the endosomal GTPases, Rab5 and Rab7 and co-endocytoses with pathologic α-synuclein. Neuron-to-neuron transmission of pathologic α-synuclein and the accompanying pathology and neurotoxicity is substantially attenuated by deletion of LAG3 or by LAG3 antibodies. The lack of LAG3 also substantially delayed α-synuclein PFF induced loss of dopamine neurons, as well as biochemical and behavioral deficits in vivo. CONCLUSION We discovered that pathologic α-synuclein transmission and toxicity is initiated by binding to LAG3 and that neuron-to-neuron transmission of pathological α-synuclein involves the endocytosis of exogenous α-synuclein PFF by the engagement of LAG3 on neurons. Depletion of LAG3 or antibodies to LAG3 substantially reduce the pathology set in motion by the transmission of pathologic α-...

Apoptosis-inducing factor (AIF), a mitochondrial oxidoreductase, is released into the cytoplasm to induce cell death in response to poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) activation. How PARP-1 activation leads to AIF release is not known. Here we identify PAR polymer as a cell death signal that induces release of AIF. PAR polymer induces mitochondrial AIF release and translocation to the nucleus. PAR glycohydrolase, which degrades PAR polymer, prevents PARP-1-dependent AIF release. Cells with reduced levels of AIF are resistant to PARP-1-dependent cell death and PAR polymer cytotoxicity. These results reveal PAR polymer as an AIF-releasing factor that plays important roles in PARP-1-dependent cell death.excitotoxicity ͉ poly(ADP-ribose) polymerase

Excessive activation of the nuclear enzyme, poly(ADP-ribose) polymerase-1 (PARP-1) plays a prominent role in various of models of cellular injury. Here, we identify poly(ADP-ribose) (PAR) polymer, a product of PARP-1 activity, as a previously uncharacterized cell death signal. PAR polymer is directly toxic to neurons, and degradation of PAR polymer by poly(ADP-ribose) glycohydrolase (PARG) or phosphodiesterase 1 prevents PAR polymer-induced cell death. PARP-1-dependent, NMDA excitotoxicity of cortical neurons is reduced by neutralizing antibodies to PAR and by overexpression of PARG. Neuronal cultures with reduced levels of PARG are more sensitive to NMDA excitotoxicity than WT cultures. Transgenic mice overexpressing PARG have significantly reduced infarct volumes after focal ischemia. Conversely, mice with reduced levels of PARG have significantly increased infarct volumes after focal ischemia compared with WT littermate controls. These results reveal PAR polymer as a signaling molecule that induces cell death and suggests that interference with PAR polymer signaling may offer innovative therapeutic approaches for the treatment of cellular injury.excitotoxicity ͉ poly(ADP-ribose) glycohydrolase ͉ poly(ADP-ribose) polymerase ͉ stroke P oly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear protein that is involved in the DNA base excision repair system, where it is potently activated by DNA strand nicks and breaks (1, 2). Using NAD ϩ as a substrate, PARP-1 builds up homopolymers of ADP ribose units on various nuclear proteins including histones, DNA polymerases, topoisomerases, DNA ligase-2, transcription factors (3, 4), and PARP-1 itself (5, 6). Although the exact physiologic function of PARP-1 is not completely understood, in some tissues it plays an important role in DNA repair and genomic stability (5,7,8). Poly(ADP-ribose) (PAR) catabolism and metabolism is a dynamic process, with PAR glycohydrolase (PARG) playing the major role in the degradation of the polymer (9).Recent studies using pharmacologic inhibition of PARP or genetic KO of PARP-1 indicate that PARP-1 plays a dramatic and significant role in cellular injury after stroke, trauma, ischemiareperfusion of the heart, spleen, skeletal muscle, and retina, arthritis, ␤-islet cytotoxicity͞diabetes mellitus, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease, experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis, endotoxic shock, multiple-system organ failure, and liver damage (for review, see refs. 1 and 10). PARP-1 activation also plays a prominent role in NMDA excitotoxicity, because PARP-1 KO mice are remarkably resistant both in vitro and in vivo to the excitotoxic effects of glutamate and NMDA (11,12). A cell-suicide hypothesis has been proposed (1,2,13,14) to explain the actions of PARP-1 in mediating cell death. However, studies in mice lacking PARG suggest that PAR polymer formed during the activation of PARP-1 might play a role in PARP-1-dependent cell death. PARG KO mice die at embryoni...

The association of LRRK2 with a variety of membrane and vesicular structures, membrane-bound organelles, and microtubules suggests an affinity of LRRK2 for lipids or lipid-associated proteins and may suggest a potential role in the biogenesis and/or regulation of vesicular and membranous intracellular structures within the mammalian brain.

Parkinson's disease (PD) is a common neurodegenerative disease caused by genetic and environmental factors. We analyzed induced pluripotent stem cell (iPSC)-derived neural cells from PD patients and presymptomatic individuals carrying mutations in the PINK1 and LRRK2 genes, and healthy control subjects. We measured several aspects of mitochondrial responses in the iPSC-derived neural cells including production of reactive oxygen species, mitochondrial respiration, proton leakage and intraneuronal movement of mitochondria. Cellular vulnerability associated with mitochondrial function in iPSC-derived neural cells from PD patients and at-risk individuals could be rescued with coenzyme Q10, rapamycin or the LRRK2 kinase inhibitor GW5074. Analysis of mitochondrial responses in iPSC-derived neural cells from PD patients carrying different mutations provides insights into convergence of cellular disease mechanisms between different familial forms of PD and highlights the importance of oxidative stress and mitochondrial dysfunction in PD.

Mitochondrial dynamics and mitophagy have been linked to cardiovascular and neurodegenerative diseases. Here, we demonstrate that the mitochondrial division dynamin Drp1 and the Parkinson's disease-associated E3 ubiquitin ligase parkin synergistically maintain the integrity of mitochondrial structure and function in mouse heart and brain. Mice lacking cardiac Drp1 exhibited lethal heart defects. In Drp1KO cardiomyocytes, mitochondria increased their connectivity, accumulated ubiquitinated proteins, and decreased their respiration. In contrast to the current views of the role of parkin in ubiquitination of mitochondrial proteins, mitochondrial ubiquitination was independent of parkin in Drp1KO hearts, and simultaneous loss of Drp1 and parkin worsened cardiac defects. Drp1 and parkin also play synergistic roles in neuronal mitochondrial homeostasis and survival. Mitochondrial degradation was further decreased by combination of Drp1 and parkin deficiency, compared with their single loss. Thus, the physiological importance of parkin in mitochondrial homeostasis is revealed in the absence of mitochondrial division in mammals.