New Substrate for Parkinson’s Protein Is Picky About Phosphate

Mutations in leucine-rich repeat kinase-2 (LRRK2) are the leading cause of familial Parkinson’s disease, yet scientists understand little about how this gene influences neuronal function or contributes to neurodegeneration. In the September 20 Neuron, researchers led by Bart De Strooper and Patrik Verstreken of K.U. Leuven, Belgium, identify EndophilinA (EndoA), a protein important for synaptic vesicle endocytosis, as a LRRK2 substrate. Experiments with human and fly analogues of LRRK2 suggest that the kinase phosphorylates EndoA, causing it to detach from the plasma membrane. Curiously, the data indicate that synaptic vesicle formation requires “Goldilocks” regulation: not too much EndoA phosphorylation—but not too little. While some researchers in the field are wondering whether the interpretation is “just right,” the findings may help explain how PD could result from both gain and loss of LRRK2 function.

Studies in hippocampal neurons suggested a role for LRRK2 in synaptic vesicle recycling (Shin et al., 2008; Piccoli et al., 2011), as did recent work by Bingwei Lu’s lab at Stanford University School of Medicine, Palo Alto, California, using Drosophila LRRK mutants (Lee et al., 2010). (Flies have just one LRRK, whereas mammals have LRRK1 and LRRK2.) In the present study, first author Samer Matta and colleagues further characterized Lu’s loss-of-function LRRK flies (Lee et al., 2007). Compared to wild-type flies, the mutants formed fewer synaptic vesicles and released lower amounts of neurotransmitter during nerve stimulation. Expression of wild-type human LRRK2 rescued the defects, alleviating some concern that fly and human LRRK homologues may function differently.

EndoA came to the fore when the researchers found that vesicle release and neurotransmission occurred normally in LRRK loss-of-function flies that had just one copy of the EndoA gene. Manipulating other genes involved in vesicle endocytosis failed to restore neurotransmission. Matta and colleagues went on to find that purified human LRRK2 or Drosophila LRRK phosphorylated EndoA in vitro and in Chinese hamster ovary (CHO) cells. Mass spectrometry revealed that LRRK phosphorylates serine 75 (S75) in EndoA’s BAR domain, which binds to membranes.

Other experiments suggested that LRRK-dependent S75 phosphorylation is functionally meaningful. When serine 75 was changed to aspartic acid to mimic constitutive phosphorylation, this EndoA mutant could no longer bind or bend membranes in vitro as does normal EndoA. A phospho-dead version of the membrane protein retained those capabilities, however. The researchers checked these results in vivo using flies expressing kinase-dead LRRK or the G2019S gain-of-function LRRK2. They found more EndoA in membranes of flies expressing kinase-dead LRRK, and less in membranes of G2019S mutants. This suggests that phosphorylation of EndoA promotes its detachment from membranes.

Why might LRRK2-driven EndoA phosphorylation matter to neurons? The scientists measured neurotransmission with electrophysiological recordings at neuromuscular junctions, and visualized endocytosis of synaptic vesicles by fluorescence and electron microscopy. As it turns out, both neurotransmitter release and endocytosis were impaired if there was too little or too much EndoA phosphorylation. The data support a model where LRRK controls an EndoA phosphorylation cycle that drives vesicle recycling, suggest the authors. "EndoA needs to be dephosphorylated to stick to the membrane, but also must be phosphorylated to properly detach [when vesicle uptake is needed]," Matta told Alzforum. “There is a balance between phosphorylated and non-phosphorylated endophilin. Shifting the balance too far either way is not good.”

Overall, other scientists found the study provocative, though some had concerns about the dual regulation model. Mark Cookson of the National Institute on Aging (NIA), Bethesda, Maryland, said it is hard to understand how too much and too little EndoA phosphorylation could lead to the same effect, i.e., reduced synaptic endocytosis. This seems inconsistent with mouse data, he said. Transgenic mice expressing G2019S LRRK2 have impaired dopamine neurotransmission (Melrose et al., 2010), whereas LRRK2 knockout mice do not (Hinkle et al., 2012; Tong et al., 2010). G2019S knock-in and lack of LRRK2 also seem to produce different phenotypes, at least in the kidney: The knockouts have abnormally large lysosomes in kidney proximal tubule cells, whereas these cells appear normal in G2019S knock-in mice (Herzig et al., 2011).

Plus, since LRRK2 is expressed widely throughout neurons, “it would be remarkable if it had a selective effect on endocytosis,” noted Robert Edwards of the University of California, San Francisco, in an e-mail to Alzforum. “If [LRRK2] has many other effects, then the influence on endocytosis could be indirect.” How these events lead to neurodegeneration also remains unclear.

Huaibin Cai, also at the NIA, said fly data should be interpreted with caution. Mammalian LRRK2 is mainly expressed in soma and dendrites, not at axon terminals (Mandemakers et al., 2012), which argues against a role for LRRK2 in presynaptic vesicle release, Cai noted.

G2019S LRRK2 is the most common familial PD mutation, accounting for some 5 percent of familial PD and 2 percent of sporadic PD cases (Gilks et al., 2005; Nichols et al., 2005). Even so, “it is an outlier in that it is the only LRRK2 mutation known to increase the protein’s kinase activity,” Cookson said. “Do other LRRK2 mutations also affect EndoA? Do they require EndoA for their detrimental effects?” The authors plan to test additional LRRK2 mutations in follow-up studies, Matta said.—Esther Landhuis

Comments

  1. We are grateful for the interest in our paper describing EndoA as a LRRK2 target in synaptic recycling, and would like to respond to comments raised on Alzforum regarding our work.

    The model we propose, where both too much and too little LRRK2-dependent EndoA1 phosphorylation would result in reduced endocytosis, explains our data. It is also in line with results other groups obtained specifically with rat neurons. Shin et al., 2008 report that the rate of endocytosis is reduced both when LRRK2 is knocked down using shRNA or by expressing the kinase active LRRK2G2019S. We would like to note that some confusion exists about the effects of different mutations in LRRK2 causing PD. The G2019S mutation is clearly a gain of function, enhancing phosphorylation. The others have unclear effects on LRRK2 kinase function. Our model, in which fine-tuning of activity is important, provides at least an interesting new perspective on the discussion of whether gain or loss of function of the kinase activity is critically involved in the disease.

    With regard to kidney phenotypes and potential other pathways in which LRRK2 might be involved, we agree that LRRK2 is likely to phosphorylate other substrates in addition to EndoA and that this even might be cell-type dependent. As such we expect that LRRK2 may impinge on other pathways; however, we anticipate that in these pathways, as well, LRRK2 will have a “tuning” effect. We are quite confident that our studies in neurons are important to unravel the role of LRRK2 in the central nervous system, and that the disturbed neurotransmission phenotype we study is relevant for the understanding of the neurobiological role of LRRK2 in general. Such studies might well turn out to be relevant for Parkinson’s disease. We recommend the News and Views article by Heutinck and Verhage. It clearly explains our strategy, which focuses first on the synaptic role of LRRK2, especially since little is known about the function of this protein.

    It is correct that LRRK2 is broadly expressed in neurons; however, this feature per se does not make a role of LRRK2 in endocytosis necessarily indirect. Again, we think that LRRK2 acts in other pathways. Given the mild phenotypes in the deficient flies, mice or other species, it likely acts in a regulating and fine tuning way as we describe here for EndoA. Our work indicates beyond doubt that fruit fly LRRK or mammalian LRRK2 in vivo and in vitro are both necessary and sufficient for EndoA(1) phosphorylation.

    EndoA(1) has been firmly implicated in the actual formation and uncoating of synaptic vesicles (Verstreken et al., 2003 ; Milosevic et al., 2011). The most parsimonious explanation follows that LRRK2 acts via EndoA in endocytosis. The remarkable observation that EndoA1-3 mouse triple knockouts result in neurodegeneration, yet other mouse knockouts in endocytic genes do not (P. De Camilli personal communication) requires further investigation, also in relation to other clinical LRRK2 mutants than the G2019S that we used in our studies.

    We take issue with the statement that the fly LRRK data ought to be interpreted with caution because mammalian LRRK2 would not be present at presynaptic terminals. In Mandemakers et al., 2012, we do not report that LRRK2 is absent from synapses. Although it is not enriched, LRRK2 is present at synapses and is detected in the synaptic vesicle fraction after subcellular fractionation. Synaptic localization of LRRK2 has previously been shown by many others (i.e. Piccoli et al., 2011, Stafa et al., 2012). Mammalian LRRK2 rescues all the synaptic endocytosis defects that we report in Drosophila, indicating the mammalian protein is able to act in the pathways affected by loss of fly LRRK. Noteworthy, our loss of function studies in LRRK null mutant flies are performed in the absence of potential compensation of LRRK1 that may confound interpretation of synaptic phenotypes in mammalian systems. Finally, other researchers have found synaptic transmission defects in LRRK2 deficient mammalian neurons that are in line with the data we reported (Piccoli et al., 2011; Shin et al., 2008).

    Given the extensive conservation of endocytic proteins and mechanisms across species (Lloyd et al., 2000; Littleton et al., 2000), our observation that human LRRK2 can phosphorylate fly and human EndoA1 indicates conservation of this regulatory mechanism, as well. We would like to remind Alzforum’s readers of the power of Drosophila genetics to unravel fundamental, conserved biological pathways. The field of neurodegeneration is replete with examples where salient novel features were originally discovered in small genetic models such as the fruit fly and the worm.

    In conclusion we hope that future work by our lab and others will rigorously test our hypothesis on the role of LRRK2 in synaptic vesicle endocytosis and how this pathway contributes to the development of PD.

    References:

    Shin N, Jeong H, Kwon J, Heo HY, Kwon JJ, Yun HJ, Kim CH, Han BS, Tong Y, Shen J, Hatano T, Hattori N, Kim KS, Chang S, Seol W. LRRK2 regulates synaptic vesicle endocytosis. Exp Cell Res. 2008 Jun 10;314(10):2055-65. Epub 2008 Mar 5 PubMed.

    Verstreken P, Koh TW, Schulze KL, Zhai RG, Hiesinger PR, Zhou Y, Mehta SQ, Cao Y, Roos J, Bellen HJ. Synaptojanin is recruited by endophilin to promote synaptic vesicle uncoating. Neuron. 2003 Nov 13;40(4):733-48. PubMed.

    Milosevic I, Giovedi S, Lou X, Raimondi A, Collesi C, Shen H, Paradise S, O'Toole E, Ferguson S, Cremona O, De Camilli P. Recruitment of endophilin to clathrin-coated pit necks is required for efficient vesicle uncoating after fission. Neuron. 2011 Nov 17;72(4):587-601. PubMed.

    Mandemakers W, Snellinx A, O'Neill MJ, de Strooper B. LRRK2 expression is enriched in the striosomal compartment of mouse striatum. Neurobiol Dis. 2012 Dec;48(3):582-93. Epub 2012 Jul 29 PubMed.

    Piccoli G, Condliffe SB, Bauer M, Giesert F, Boldt K, De Astis S, Meixner A, Sarioglu H, Vogt-Weisenhorn DM, Wurst W, Gloeckner CJ, Matteoli M, Sala C, Ueffing M. LRRK2 controls synaptic vesicle storage and mobilization within the recycling pool. J Neurosci. 2011 Feb 9;31(6):2225-37. PubMed.

    Stafa K, Trancikova A, Webber PJ, Glauser L, West AB, Moore DJ. GTPase activity and neuronal toxicity of Parkinson's disease-associated LRRK2 is regulated by ArfGAP1. PLoS Genet. 2012;8(2):e1002526. PubMed.

    Lloyd TE, Verstreken P, Ostrin EJ, Phillippi A, Lichtarge O, Bellen HJ. A genome-wide search for synaptic vesicle cycle proteins in Drosophila. Neuron. 2000 Apr;26(1):45-50. PubMed.

    Littleton JT. A genomic analysis of membrane trafficking and neurotransmitter release in Drosophila. J Cell Biol. 2000 Jul 24;150(2):F77-82. PubMed.

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References

Paper Citations

  1. Shin N, Jeong H, Kwon J, Heo HY, Kwon JJ, Yun HJ, Kim CH, Han BS, Tong Y, Shen J, Hatano T, Hattori N, Kim KS, Chang S, Seol W. LRRK2 regulates synaptic vesicle endocytosis. Exp Cell Res. 2008 Jun 10;314(10):2055-65. Epub 2008 Mar 5 PubMed.
  2. Piccoli G, Condliffe SB, Bauer M, Giesert F, Boldt K, De Astis S, Meixner A, Sarioglu H, Vogt-Weisenhorn DM, Wurst W, Gloeckner CJ, Matteoli M, Sala C, Ueffing M. LRRK2 controls synaptic vesicle storage and mobilization within the recycling pool. J Neurosci. 2011 Feb 9;31(6):2225-37. PubMed.
  4. Lee SB, Kim W, Lee S, Chung J. Loss of LRRK2/PARK8 induces degeneration of dopaminergic neurons in Drosophila. Biochem Biophys Res Commun. 2007 Jun 29;358(2):534-9. PubMed.
  5. Melrose HL, Dächsel JC, Behrouz B, Lincoln SJ, Yue M, Hinkle KM, Kent CB, Korvatska E, Taylor JP, Witten L, Liang YQ, Beevers JE, Boules M, Dugger BN, Serna VA, Gaukhman A, Yu X, Castanedes-Casey M, Braithwaite AT, Ogholikhan S, Yu N, Bass D, Tyndall G, Schellenberg GD, Dickson DW, Janus C, Farrer MJ. Impaired dopaminergic neurotransmission and microtubule-associated protein tau alterations in human LRRK2 transgenic mice. Neurobiol Dis. 2010 Dec;40(3):503-17. PubMed.
  6. Hinkle KM, Yue M, Behrouz B, Dächsel JC, Lincoln SJ, Bowles EE, Beevers JE, Dugger B, Winner B, Prots I, Kent CB, Nishioka K, Lin WL, Dickson DW, Janus CJ, Farrer MJ, Melrose HL. LRRK2 knockout mice have an intact dopaminergic system but display alterations in exploratory and motor co-ordination behaviors. Mol Neurodegener. 2012;7:25. PubMed.
  7. Tong Y, Yamaguchi H, Giaime E, Boyle S, Kopan R, Kelleher RJ, Shen J. Loss of leucine-rich repeat kinase 2 causes impairment of protein degradation pathways, accumulation of alpha-synuclein, and apoptotic cell death in aged mice. Proc Natl Acad Sci U S A. 2010 May 25;107(21):9879-84. PubMed.
  8. Herzig MC, Kolly C, Persohn E, Theil D, Schweizer T, Hafner T, Stemmelen C, Troxler TJ, Schmid P, Danner S, Schnell CR, Mueller M, Kinzel B, Grevot A, Bolognani F, Stirn M, Kuhn RR, Kaupmann K, van der Putten PH, Rovelli G, Shimshek DR. LRRK2 protein levels are determined by kinase function and are crucial for kidney and lung homeostasis in mice. Hum Mol Genet. 2011 Nov 1;20(21):4209-23. PubMed.
  9. Mandemakers W, Snellinx A, O'Neill MJ, de Strooper B. LRRK2 expression is enriched in the striosomal compartment of mouse striatum. Neurobiol Dis. 2012 Dec;48(3):582-93. Epub 2012 Jul 29 PubMed.
  10. Gilks WP, Abou-Sleiman PM, Gandhi S, Jain S, Singleton A, Lees AJ, Shaw K, Bhatia KP, Bonifati V, Quinn NP, Lynch J, Healy DG, Holton JL, Revesz T, Wood NW. A common LRRK2 mutation in idiopathic Parkinson's disease. Lancet. 2005 Jan 29-Feb 4;365(9457):415-6. PubMed.
  11. Nichols WC, Pankratz N, Hernandez D, Paisán-Ruíz C, Jain S, Halter CA, Michaels VE, Reed T, Rudolph A, Shults CW, Singleton A, Foroud T, . Genetic screening for a single common LRRK2 mutation in familial Parkinson's disease. Lancet. 2005 Jan 29-Feb 4;365(9457):410-2. PubMed.

Further Reading

Papers

  1. Piccoli G, Condliffe SB, Bauer M, Giesert F, Boldt K, De Astis S, Meixner A, Sarioglu H, Vogt-Weisenhorn DM, Wurst W, Gloeckner CJ, Matteoli M, Sala C, Ueffing M. LRRK2 controls synaptic vesicle storage and mobilization within the recycling pool. J Neurosci. 2011 Feb 9;31(6):2225-37. PubMed.

Primary Papers

  1. Matta S, Van Kolen K, da Cunha R, van den Bogaart G, Mandemakers W, Miskiewicz K, De Bock PJ, Morais VA, Vilain S, Haddad D, Delbroek L, Swerts J, Chávez-Gutiérrez L, Esposito G, Daneels G, Karran E, Holt M, Gevaert K, Moechars DW, De Strooper B, Verstreken P. LRRK2 controls an EndoA phosphorylation cycle in synaptic endocytosis. Neuron. 2012 Sep 20;75(6):1008-21. PubMed.
  2. Heutink P, Verhage M. Neurodegeneration: new road leads back to the synapse. Neuron. 2012 Sep 20;75(6):935-8. PubMed.

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