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RESEARCH ARTICLE

LRRK2 functions in synaptic vesicle endocytosis through a kinase-dependent mechanism

Amaia M. Arranz1,2,*, Lore Delbroek3,*, Kristof Van Kolen3,`, Marco R. Guimaraes3, Wim Mandemakers1,2,Guy Daneels3, Samer Matta1,2, Sara Calafate3, Hamdy Shaban3, Pieter Baatsen1,2, Pieter-Jan De Bock4,5,Kris Gevaert4,5, Pieter Vanden Berghe2,6, Patrik Verstreken1,2, Bart De Strooper1,2 and Diederik Moechars3

ABSTRACT

Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated

with Parkinson’s disease, but the precise physiological function of

the protein remains ill-defined. Recently, our group proposed a

model in which LRRK2 kinase activity is part of an EndoA

phosphorylation cycle that facilitates efficient vesicle formation at

synapses in the Drosophila melanogaster neuromuscular junctions.

Flies harbor only one Lrrk gene, which might encompass the

functions of both mammalian LRRK1 and LRRK2. We therefore

studied the role of LRRK2 in mammalian synaptic function and

provide evidence that knockout or pharmacological inhibition of

LRRK2 results in defects in synaptic vesicle endocytosis, altered

synaptic morphology and impairments in neurotransmission. In

addition, our data indicate that mammalian endophilin A1 (EndoA1,

also known as SH3GL2) is phosphorylated by LRRK2 in vitro at T73

and S75, two residues in the BAR domain. Hence, our results

indicate that LRRK2 kinase activity has an important role in the

regulation of clathrin-mediated endocytosis of synaptic vesicles and

subsequent neurotransmission at the synapse.

KEY WORDS: LRRK2, Endophilin A1, Endocytosis

INTRODUCTIONLeucine-rich repeat kinase 2 (LRRK2) is a large multidomain

protein harboring a kinase, GTPase and several protein-binding

domains (Cookson and Bandmann, 2010). Mutations in this protein

are linked to familial as well as sporadic forms of Parkinson’s

disease (Paisan-Ruız et al., 2004, Zimprich et al., 2004). For

example, the G2019S mutation, which causes elevated kinase

activity, is responsible for 4% of familial Parkinson’s disease

worldwide (Healy et al., 2008). Interestingly, this mutation was

also found in some apparently sporadic cases. Several other

mutations (e.g. R1441C/H/G and I2020T) in the GTPase and

kinase domains have also been shown to segregate with

Parkinson’s disease (Zimprich et al., 2004; Healy et al., 2008).

Despite the apparent clinical association between LRRK2

mutations and Parkinson’s disease, it remains enigmatic how

such mutations lead to disease progression and the biological

function of LRRK2 itself also still needs to be clarified further.

Although several proteins have been identified as kinase

substrates of LRRK2, none of these substrates have been

independently reported in vivo. Substrates are involved in

different processes, such as neuronal survival, cytoskeletal

rearrangement, autophagy and apoptosis (Berwick and Harvey,

2011; Gillardon, 2009; Plowey and Chu, 2011; Martin et al.,

2014). LRRK2 has also been shown to regulate

neurotransmission, by interacting with synaptic proteins that

modulate clathrin-mediated endocytosis of synaptic vesicles,

such as Rab5b, snapin and N-ethylmaleimide-sensitive factor

(NSF) (Piccoli et al., 2011; Shin et al., 2008; Yun et al., 2013;

Piccoli et al., 2014). Moreover, we revealed an essential role

for LRRK in synaptic vesicle endocytosis at Drosophila

melanogaster neuromuscular junctions through phosphorylation

of endophilin A (EndoA) (Matta et al., 2012). EndoA is an

evolutionary conserved protein involved in clathrin-mediated

endocytosis (Ringstad et al., 1997). It drives the formation of

synaptic vesicles by sensing or inducing plasma membrane

curvature (Gallop et al., 2006; Masuda et al., 2006) and

facilitates clathrin uncoating once synaptic vesicles are

generated (Milosevic et al., 2011; Verstreken et al., 2002).

Consequently, impairment of EndoA function results in severe

deficits in synaptic vesicle endocytosis (Gad et al., 2000;

Milosevic et al., 2011; Schuske et al., 2003; Verstreken et al.,

2002). Our study identified EndoA as a substrate of LRRK and

demonstrated that LRRK has an essential role in synaptic vesicle

endocytosis in Drosophila by phosphorylating EndoA at S75

(Matta et al., 2012).

Given that flies express only one LRRK protein, which might

encompass the function of both mammalian LRRK1 and

LRRK2, we specifically assessed the role of LRRK2 in

synaptic vesicle endocytosis in mammals. We demonstrate that

LRRK2 knockout leads to impairments in clathrin-mediated

synaptic vesicle endocytosis and neurotransmission and the

presence of endocytic intermediates and abnormal vesicle

morphology and number, similar to Lrrk knockout in

Drosophila. Furthermore, we show that mammalian EndoA1

(SH3GL2), a neuron-specific variant of EndoA (Giachino et al.,

1997), is a kinase substrate of LRRK2, confirming the

conservation between Drosophila and the mammalian system.

Finally, we show that LRRK2 phosphorylates EndoA1 not only on

residue S75, identical to the residue phosphorylated in Drosophila,

but on T73 as well. Taken together, our findings advance

the understanding of the evolutionary conserved biological

1VIB Center for the Biology of Disease, 3000 Leuven, Belgium. 2KU Leuven,Center for Human Genetics and Leuven Research Institute for Neuroscience andDisease (LIND), 3000 Leuven, Belgium. 3Janssen Pharmaceutical Companies ofJohnson and Johnson, Department of CNS Research, Turnhoutseweg 30, 2340Beerse, Belgium. 4Department of Medical Protein Research, VIB, 9000 Gent,Belgium. 5Department of Biochemistry, Ghent University, 9000 Gent, Belgium.6Laboratory for Enteric NeuroScience (LENS), TARGID, KULeuven, 3000 Leuven,Belgium.*These authors contributed equally to this work

`Author for correspondence (kvkolen@its.jnj.com)

Received 15 June 2014; Accepted 26 November 2014

� 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 541–552 doi:10.1242/jcs.158196

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function of LRRK2 and its pivotal role in clathrin-mediatedendocytosis.

RESULTSKnockout of LRRK2 impairs mammalian synapticvesicle endocytosisOur group recently reported that knockout of LRRK, theDrosophila melanogaster orthologue of LRRK1 and LRRK2,causes an impairment in clathrin-mediated endocytosis of

synaptic vesicles, indicating that there is a role for this proteinin synaptic vesicle formation (Matta et al., 2012). To determinewhether this role in endocytosis is contained within the LRRK2

protein, we performed dynamic assays of endocytosis in wild-type and LRRK2-knockout primary striatal neurons by usingsypHy, a fusion construct of synaptophysin and super ecliptic

pHluorin. At the onset of stimulus (20 Hz for 15 s), exocytosiscaused a rapid increase in sypHy fluorescence (Fig. 1Aii)which, after cessation of stimulus, slowly returned to baseline(Fig. 1Aiii). As shown in Fig. 1B, the decay of fluorescence

was delayed in LRRK2-knockout cells when compared to wild-type controls. The fluorescence decay, which reflectsendocytosis, was assessed by fitting the trace with a single

exponential time constant (tdecay). Quantification shows thattdecay is significantly increased in LRRK2-knockout neurons(wild-type, 68.9665.07; LRRK2 knockout, 89.2963.92;

mean6s.e.m., P,0.0001, Fig. 1C), indicating that endocytosisof synaptic vesicles is impaired in these cells. However,no significant differences in exocytosis were observed under

these conditions (wild-type, 10.0060.69; LRRK2 knockout,9.3060.63; Fig. 1D). These results demonstrate that the role ofLRRK2 in synaptic vesicle endocytosis, a mechanism previously

identified in Drosophila Lrrk mutants, is conserved inmammalian neurons.

LRRK2-knockout synapses present endocytic intermediatesand a reduced number of synaptic vesicles withaltered morphologyTo gain insight into whether the endocytic delay observed inmammalian LRRK2-knockout neurons could also be observedat a morphological level, we performed transmission electron

microscopy (TEM) of wild-type (Fig. 2A) and LRRK2-knockout(Fig. 2B–E) striatal brain sections. Ultrastructural analysis ofknockout synapses revealed a phenotype different from control

littermates: a decreased number of synaptic vesicles (Fig. 2B),which were in general more heterogeneous in size with the presenceof vesicles with a larger diameter (Fig. 2C, asterisks). Abnormal

endocytic intermediates (Fig. 2D, asterisks), including clathrin-coated endocytic intermediates (Fig. 2E), were also evident in someknockout nerve terminals. Quantification of TEM micrographsshowed that the mean number of synaptic vesicles per mm2

synapse was substantially lower in knockout than in controlsynapses (Fig. 2F, wild-type, 194.865.1; knockout, 146.064.6;mean6s.e.m.; P,0.0001). In addition, the average synaptic vesicle

diameter was increased in LRRK2 knockouts compared to controls(Fig. 2G, wild-type, 47.5960.47 nm; knockout 50.1560.48 nm,P,0.001) and vesicles with a larger diameter were more abundant

in knockout than in control synapses (Fig. 2H). These observationsare reminiscent of the morphological changes we previouslydescribed in Drosophila, where synaptic vesicle size was

increased in LRRK-knockout boutons (Matta et al., 2012).Interestingly, a reduction in the number of synaptic vesicles andaccumulation of clathrin-coated vesicular profiles were also

Fig. 1. Knockout of LRRK2 causes defects insynaptic vesicle endocytosis. (A) Representativesnapshots taken from 2 Hz recordings of SypHy-expressing wild-type (WT) and LRRK2-knockout (KO)neurons at rest (i), at maximal stimulation (ii) andtowards the end of recovery (iii). Scale bar: 20 mm.(B–D) Slower endocytic recovery in LRRK2 KO neurons.(B) Following the onset of stimulus (300 action potentialsat 20 Hz), exocytosis of sypHy caused a rapidincrease in fluorescence, followed by an exponentialdecay after the cessation of stimulation, which wasdelayed in LRRK2 KO cells. Grey lines indicate theduration of the 20 Hz stimulus. Snapshot timepoints asshown in Ai–iii are indicated by red circles.(C) Quantification of the decay of fluorescence. Timeconstant tdecay shows that endocytosis is delayed inLRRK2 KO neurons (**P,0.01, Student’s t-test).(D) Quantification of the increase in fluorescence. Timeconstant tupstroke shows that exocytosis is not altered inLRRK2 KO cells (ns, not significant). Data in C and Dare expressed as mean6s.e.m. derived from at leastthree independent experiments and corrected fordifferences in endocytosis in D.

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observed in endophilin triple-knockout mouse synapses (Milosevic

et al., 2011) and in fly endoA-knockout boutons (Guichet et al.,2002; Verstreken et al., 2002), suggesting that the morphologicaldefects observed in LRRK2 knockouts and mouse endophilin triple-knockouts might be due to overlapping mechanisms.

Synaptic transmission is impaired in LRRK2-knockoutmammalian neuronsThe previous findings prompted us to hypothesize that LRRK2 mighthave an important role in synaptic function. To determine whetherknockout of LRRK2 affects neurotransmission, we measured the

number of spontaneous excitatory postsynaptic currents (sEPSCs) inwild-type (n519) and LRRK2-knockout (n522) hippocampalneurons. All neurons in culture appeared healthy and, under basal

conditions, the number of sEPSCs was similar in wild-type andLRRK2-knockout cells (Fig. 3A). To measure sEPSCs coming fromnewly formed synaptic vesicles, sucrose (50 mM) was added to thebath solution as it stimulates the release of the readily releasable pool

through Ca2+-independent mechanical stress (Rosenmund andStevens, 1996). In sucrose-treated wild-type cells, the number ofsEPSCs was increased compared to non-treated wild-type cells

(control, 47.74614.03; sucrose, 100.8469.76; mean6s.e.m.). Inknockout cells, however, sucrose treatment failed to increase the

number of sEPSCs (control, 32.0568.07; sucrose, 43.1469.76;

Fig. 3B). The effect of sucrose on the number of sEPSCs wascalculated by determination of the sucrose versus control ratios(wild-type, 4.0761.38; knockout, 1.6960.37; Fig. 3C). These dataindicate that LRRK2 modulates synaptic function, as knockout of

LRRK2 causes impairment in neurotransmission following therelease of the readily releasable pool.

To investigate this further, we verified whether the diminished

response to sucrose observed in LRRK2-knockout neurons is dueto impairments at the pre- or post-synaptic level. When analyzingthe frequency of sEPSCs, which is well known to be dependent

on the presynaptic machinery (Marinelli et al., 2003), weobserved differences between wild-type and LRRK2-knockoutneurons. Under basal conditions, the frequency was similar in

wild-type and LRRK2-knockout neurons, whereas the presence ofsucrose stimulated neurotransmitter release and significantlyincreased the sEPSC frequency in wild-type (control,26.3264.27; sucrose, 46.6466.48) but not in LRRK2-knockout

neurons (control, 35.4464.01; sucrose, 30.0064.67, Fig. 3D).Analysis of sucrose versus control ratios (wild-type, 2.0860.33;knockout, 0.9760.18; Fig. 3E) confirmed the increase in

frequency in wild-type neurons, but not in LRRK2-knockoutneurons. This indicates that LRRK2 has a role in the presynaptic

Fig. 2. Altered synaptic vesicle morphology and numberin LRRK2-knockout synapses. (A) Wild-type (WT)synapse. (B,C) LRRK2-knockout (KO) synapses revealing adecrease in the number of synaptic vesicles (B) andheterogeneity in vesicle size (C) with the presence of someabnormally large vesicles (asterisks in C). (D,E) LRRK2 KOsynapses occasionally showed aberrantly formed endocyticintermediates (asterisks in D) or were entirely occupied byless densely packed clathrin-coated vesicles (E). The inset inE is a high-magnification view showing the clathrin-coatedvesicular profiles in more detail. Scale bars: 200 nm.(F) Quantification of the vesicle density as number ofsynaptic vesicles (SVs) per mm2 (**** P,0.0001, Student’st-test). (G) Quantification of the average synaptic vesiclediameter (nm). Each data point represents the mean synapticvesicle diameter for a single terminal. At least 20 synapticvesicles were measured in each terminal (*** P,0.001,Student’s t-test). (H) Frequency distribution of the synapticvesicle diameter (10 nm bins). Vesicles exceeding 80 nm indiameter were four times more abundant in KO synapses (20vesicles in KO versus five vesicles in WT). Data in F and Gare expressed as mean6s.e.m. and derived from at leastthree independent experiments.

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mechanisms that replenish the synaptic vesicle pool to governhigh frequency neurotransmission, consistent with a role inendocytosis and vesicle recycling. To evaluate whether LRRK2

also affects the postsynaptic mechanisms, we measured thesEPSC peak amplitude, of which changes are thought to reflectchanges in the response of the postsynaptic receptors (Sebe

et al., 2003; Thompson et al., 1993). As shown in Fig. 3F, therewere no differences in peak amplitude between control andsucrose condition in wild-type (control, 225.9062.84; sucrose,

225.2762.47) or LRRK2-knockout cells (control, 219.4461.97;sucrose, 218.2961.37). Calculation of the sucrose versuscontrol ratio (Fig. 3G) confirmed that the peak amplituderemained unchanged (wild-type, 1.0960.17; knockout,

0.9960.06).Taken together, these data indicate that LRRK2 modulates

neurotransmission at the presynaptic level. We believe these

effects are specific to loss of LRRK2 as we observed similardefects in neurotransmission when wild-type neurons were

treated with LRRK2-IN-1, a well-defined LRRK2 kinaseinhibitor (Deng et al., 2011). Treating wild-type cells withdifferent concentrations of LRRK2-IN-1 attenuated the effect of

sucrose on the number and frequency of sEPSCs, when comparedto DMSO-treated cells (supplementary material Fig. S1). Again,no effect on peak amplitude was observed (supplementary

material Fig. S1). These data indicate that the presynapticfunction of LRRK2 is dependent on its kinase activity.

LRRK2 knockout mimics the effects of dynasore treatmentOur data suggest that LRRK2 has a presynaptic role inneurotransmission by affecting synaptic vesicle endocytosis. Tostrengthen the previous results, we compared the effect of

LRRK2 knockout to the effect of independently blockingendocytosis. To block endocytosis we treated wild-type(control, n515; dynasore, n514) and LRRK2-knockout

(control, n512; dynasore, n514) hippocampal neurons withdynasore (40 mM for 5 min, Fig. 4A), a cell-permeable small

Fig. 3. Knockout of LRRK2 affects neurotransmission byimpairing presynaptic function. (A) Representative whole-cell patch-clamp recordings from wild-type (WT) and LRRK2-knockout (KO) hippocampal neurons in control conditions andin the presence of sucrose (50 mM). (B) In control conditions,the number of sEPSCs was similar in both genotypes, whereasit was significantly increased in WT, but not in LRRK2 KOneurons in the presence of sucrose. (C) Quantification of foldchange in the number of sEPSCs indicates that sucroseinduces a significant change in WT, but not in LRRK2 KOneurons. (D) The frequency (Hz) of sEPSCs was similar in bothgenotypes in control conditions, whereas it was significantlyincreased in WT, but not in LRRK2 KO neurons in the presenceof sucrose. (E) Quantification of fold change in frequency ofsEPSCs indicates that sucrose induces a significant change inWT, but not in LRRK2 KO neurons. (F) The mean peakamplitude in WT and LRRK2 KO neurons in control conditionsand in the presence of sucrose. Although differences in currentamplitudes are observed between WTand LRRK2 KO cultures,the presence of sucrose does not affect amplitude in eithergenotype. (G) Analysis of fold change in peak amplitudeconfirmed that sucrose had no effect in both genotypes. Dataare expressed as mean6s.e.m. (*P,0.05, Student’s t-test).

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molecule that is a noncompetitive inhibitor of dynamin 1(Douthitt et al., 2011; Macia et al., 2006).

Similar to previous data, the presence of sucrose caused anincrease in the number of sEPSCs in vehicle pretreated wild-type(control, 24.8066.35; sucrose, 62.73617.21; mean6s.e.m.),but not in LRRK2-knockout neurons (control, 45.09614.00;

sucrose, 51.09612.89, Fig. 4B). Pretreatment with dynasoreabolished the effect of sucrose in wild-type neurons (control,

119.57631.76; sucrose, 147.36634.02), but did not causedifferences in LRRK2-knockout neurons (control, 97.56615.00;

sucrose, 139.75630.78, Fig. 4B). Calculation of the sucroseversus control ratios (wild-type control, 3.7760.66; wild-typedynasore-treated, 1.7260.25; knockout control, 1.5560.23;knockout dynasore-treated, 1.8260.40; Fig. 4C) confirmed

these results. To ensure that we were looking at presynapticchanges, the frequency and peak amplitude of sEPSCs were

Fig. 4. LRRK2 knockout mimics the effects of dynasore treatment. (A) Representative whole-cell patch-clamp recordings from wild-type (WT) or LRRK2-knockout (KO) hippocampal neurons in control (0.2% DMSO) conditions or upon treatment with dynasore (40 mM). (B) In control conditions, the presence ofsucrose (50 mM) significantly increased the number of sEPSCs in WT, but not in LRRK2 KO neurons. Although treatment with dynasore increased thenumber of sEPSCs in both genotypes, it abolished the effect of sucrose in WT neurons. (C) Quantification of fold change in number of sEPSCs indicates thattreatment of WT neurons with dynasore mimics the lack of effect of sucrose observed in LRRK2 KO cells. (D) The frequency of sEPSCs was significantlyincreased in WT, but not in LRRK2 KO neurons in the presence of sucrose in control conditions. Although treatment with dynasore increased the frequency ofsEPSCs in both genotypes, it abolished the effect of sucrose in WT neurons. (E) Quantification of fold change in the frequency of sEPSCs indicates thattreatment of WT neurons with dynasore mimics the lack of effect of sucrose observed in LRRK2 KO cells. (F,G) Treatment with dynasore did not affect peakamplitude in either genotype. Data are expressed as mean6s.e.m. *P,0.05 versus WT DMSO treated, two-tailed Student’s t-test.

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analyzed. In control conditions, the frequency of sEPSCs wasincreased in the presence of sucrose in wild-type (control,

18.9864.05; sucrose, 35.3766.43) but not in LRRK2-knockoutneurons (control, 29.6664.74; sucrose, 33.1065.70, Fig. 4D).Such increase was not present in dynasore-treated cells of eithergenotype (wild-type control, 48.3066.52; wild-type with sucrose,

54.1765.61; knockout control, 40.6066.38; knockout withsucrose, 46.1567.66, Fig. 4D). Analysis of the sucrose versuscontrol frequency ratios further strengthened these observations

(wild-type control, 2.6360.47; wild-type dynasore-treated,1.3360.18; knockout control, 1.1160.17; knockout dynasore-treated, 1.1860.10, Fig. 4E). Peak amplitude was not altered

in the presence of sucrose (wild-type control, 222.6962.76;wild-type with sucrose, 226.1863.68; knockout control,220.5663.24; knockout with sucrose 222.2764.50, Fig. 4F)

and treatment with dynasore did not affect this parameter(wild-type control, 219.1362.63; wild-type with sucrose,220.4863.19; knockout control, 223.6262.55; knockout withsucrose, 222.4262.50; Fig. 4F). Analysis of the sucrose versus

control ratios confirmed this observation (wild-type control,1.1460.05; wild-type dynasore-treated, 1.0560.03; knockoutcontrol, 1.1560.25; knockout dynasore-treated, 0.9660.05,

Fig. 4G).

Human LRRK2 phosphorylates mammalian EndoA1 in vitroWe revealed a clear phenotype in LRRK2-knockout neurons,with impairments in clathrin-mediated synaptic vesicle

endocytosis and neurotransmission, and the presence ofendocytic intermediates and abnormal vesicle morphology and

number. Recently, we reported a similar phenotype in Drosophila

as the result of changes in LRRK-dependent phosphorylation ofEndoA and we specifically identified the serine residue located atposition 75 as a LRRK-dependent phosphorylation site (Matta

et al., 2012). EndoA is an evolutionary conserved membrane-interacting protein that is widely known to play a role inendocytosis (Milosevic et al., 2011; Verstreken et al., 2002).

When comparing the sequences of EndoA in Homo sapiens, Mus

musculus, Rattus norvegicus and Drosophila melanogaster, wenoticed that the S75 residue is highly conserved across species

(Fig. 5C). Hence, we questioned whether LRRK2 can alsophosphorylate EndoA in mammals. To determine whetherhuman LRRK2 phosphorylates the mammalian EndoA1, the

neuron-specific EndoA isoform (Giachino et al., 1997; Milosevicet al., 2011), we performed an in vitro [33P]ATP phosphorylationassay with the following recombinant proteins: wild-typeEndoA1, EndoB1, which lacks the S75 residue and EndoA1

S75A, in which the serine residue is mutated to an alanineresidue. As shown in Fig. 5A, there is a time-dependent increasein phosphorylation of EndoA1 and, to a lesser extent, in the

EndoA1 S75A mutant. Even less 33P incorporation was observedin EndoB1. Consistently, quantification of the phosphorylatedversus total substrate ratios indicated that phosphorylation was

less efficient when the S75 residue was mutated into an alanineresidue or was not present (Fig. 5B). An in vitro [33P]ATP

Fig. 5. LRRK2 phosphorylates EndoA1 in vitro at S75. (A) Autoradiographs of in vitro LRRK2 kinase reactions using human wild-type endophilin A1(EndoA1), S75A mutated endophilin A1 (EndoA1 S75A) and endophilin B1 (EndoB1) as substrates for the indicated times. Total protein was determined bywestern blotting. (B) Quantification of the ratios of [33P]ATP signal in A shows decreased phosphorylation levels in EndoA1 S75A and EndoB1 compared toEndoA1. Data are expressed as mean6s.e.m. and are representative of at least three independent experiments. ***P,0.001, two-way ANOVA with Bonferronipost-tests. (C) Alignment of fruit fly, rat, mouse and human EndoA1, EndoA1 S75A and EndoB1 around S75 indicates that S75 is widely conserved acrossspecies, whereas T73 is only conserved in mammals.

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phosphorylation assay with recombinant wild-type, kinasehyperactive G2019S or kinase deficient D1994A LRRK2 as

kinases and recombinant EndoA1 as a substrate shows thatEndoA1 phosphorylation was enhanced using G2019S LRRK2,but almost completely abolished with the D1994A LRRK2variant (supplementary material Fig. S2A,B). To address the

specificity of LRRK2-dependent EndoA1 phosphorylation wecompared LRRK2 and GSK3b as EndoA1 kinases in vitro. In thisphosphorylation assay GSK3b, unlike LRRK2, was not able to

phosphorylate EndoA1, although its autophosphorylation wasmore prominent in comparison to LRRK2 (supplementarymaterial Fig. S2C,D). These observations provide further

evidence that EndoA1 is not a universal in vitro substrate forthe majority of kinases, supporting the specificity of the LRRK2-driven phenomenon.

The previous analysis indicated that LRRK2 phosphorylatesEndoA1 at S75, but suggested this is not the only LRRK2-dependent phosphorylation site in EndoA1. Thus, wehypothesized that T73, which is also conserved in the

mammalian protein, but not present in Drosophila EndoA normammalian EndoB1 (Fig. 5C) might be another LRRK2-dependent phosphorylation site, as it is positioned in the same

functional domain as S75 (Fig. 6A,B). In order to test whetherEndoA1 has a dual LRRK2-dependent phosphorylation site atT73 and S75, we performed an additional in vitro [33P]ATP

phosphorylation assay with wild-type EndoA1 and the T73A,S75A and T73A/S75A mutant proteins as substrates. Data in

Fig. 6C confirm our hypothesis and show that LRRK2-mediatedphosphorylation of EndoA1 was diminished to the same extent by

mutating the T73 or the S75 residue (Fig. 6D). As expected,phosphorylation of the double mutant EndoA1 was alsosignificantly decreased in comparison to wild-type EndoA1.However, phosphorylation of double mutant EndoA1 was not

significantly different from phosphorylation of T73A or S75AEndoA1. We speculate that this is because mutating either sitechanges the conformation of the protein, meaning LRRK2 is

unable to phosphorylate the other residue. Although our dataindicate that the T73/S75 site is crucial for LRRK2-mediatedphosphorylation of EndoA1, we cannot rule out other

phosphorylation events in vitro.

DISCUSSIONIn the present study, we reveal a role for LRRK2 at themammalian synapse and provide evidence that the function ofLRRK in the regulation of endocytosis in flies overlaps with therole of mammalian LRRK2 in this process. We show that

knockout of LRRK2 impairs clathrin-mediated synaptic vesicleendocytosis and neurotransmission. In line with this, previouswork by our group provided evidence for the involvement

of LRRK in synaptic vesicle endocytosis in Drosophila

melanogaster. We identified Drosophila EndoA as a substrateof LRRK2 and through rescue experiments using Drosophila

EndoA mutants, we demonstrated that LRRK-dependent EndoAphosphorylation regulates EndoA membrane affinity and

Fig. 6. LRRK2 phosphorylates EndoA1 in vitro at T73 and S75. (A,B) Three-dimensional model of a mouse EndoA1 dimer highlighting the location of T73and S75 in the BAR domain. (A) Top view of a mouse EndoA1 dimer with helix1 appendages in green. Arrow indicates viewing direction from B. (B) A front viewof the same EndoA1 dimer is shown. (C) Autoradiographs of in vitro LRRK2 kinase reactions using human wild-type, S75A, T73A and T73A/S75A mutantendophilin A1 as substrates during the indicated time points. Total protein was determined by western blotting. (D) Quantification of the ratios of the [33P]ATPsignal and total protein in C shows that phosphorylation is significantly decreased in all mutant forms of EndoA1 when compared to wild-type (WT) EndoA1 after60 min of incubation. Data are expressed as mean6s.e.m. and representative of at least three independent experiments. **P,0.01; ***P,0.001, two-wayANOVA with Bonferroni post-tests.

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consequently modulates synaptic endocytosis (Matta et al., 2012).Here, we show that, like the Drosophila variant, mammalian

EndoA1 is also phosphorylated by LRRK2, suggesting that thesignaling cascade regulating synapse function translates from flyto mammalian neurons.

We provide evidence for a role for LRRK2 in endocytosis in

mammalian cells. Our data indicate that knockout of LRRK2causes a significant impairment in clathrin-mediated endocytosisin striatal neurons, in agreement with our observations in

Drosophila LRRK-knockout neurons and previous studies inhippocampal neurons treated with short hairpin RNA (shRNA)(Matta et al., 2012; Shin et al., 2008). Impaired rates of

endocytosis have also been observed in experiments withregulators of endocytosis such as amphiphysin, endophilin,SPIN90 and dynamin (Di Paolo et al., 2002; Kim et al., 2005;

Milosevic et al., 2011; Newton et al., 2006; Ferguson et al.,2007). Clathrin-mediated endocytosis is a dynamic process inwhich numerous proteins act as effectors and/or regulators. Thefunction of many of these proteins depends on interactions with

other proteins and thus, the removal of a specific protein canimpair the cascade of protein–protein interactions andconsequently affect endocytosis. Therefore, it is plausible that

the lack of LRRK2 alters clathrin-mediated endocytosis byimpairing the function of one or more endocytic proteins.

Recently, it has been described that LRRK2 could also

modulate synaptic vesicle exocytosis, either by regulatingpresynaptic vesicle release or by interacting with snapin(Piccoli et al., 2011; Yun et al., 2013). Yun and colleagues

claimed that LRRK2-dependent phosphorylation of snapindecreased the extent of exocytotic release in hippocampalneurons as measured by using vGlut-phluorin assays. However,an earlier study from the same group failed to show an effect of

LRRK2 knockdown on synaptic vesicle exocytosis (Shin et al.,2008). Our data in striatal neurons are in line with this study (Shinet al., 2008), as we do not observe any effect on synaptic vesicle

exocytosis in LRRK2-knockout neurons. The use of differentneuronal populations (hippocampal vs striatal) and differentsystems to measure exocytosis (vGlut-phluorin versus sypHy)

could possibly explain these divergences.Our findings that LRRK2 affects endocytosis prompted us to

hypothesize that LRRK2 consequently might have an importantrole in neurotransmission. Indeed, we observe that knockout of

LRRK2 leads to impaired presynaptic function upon sucrosestimulation. As all sEPSCs measured after sucrose perfusion arecoming from newly formed vesicles, it is tempting to speculate

that this impairment in neurotransmission is driven by defects insynaptic vesicle endocytosis. Hence, we propose that slowedsynaptic vesicle endocytosis in LRRK2-knockout neurons might

be the cause of impaired neurotransmission, especially duringintense neuronal activity, where the vesicle replenishment iscrucial for effective neurotransmitter release. In fact, we found

similar effects in synaptic transmission when cells are treatedwith dynasore, a compound that has been demonstrated to inhibitendocytosis through dynamin-dependent and -independentmechanisms (Macia et al., 2006; Park et al., 2013). Hence, as

results in LRRK2-knockout neurons are similar to results seenin dynasore-treated neurons, it is likely that these results arealso due to the effect of LRRK2 knockout on endocytosis.

Interestingly, treatment with dynasore increased the number ofsEPSCs detected in both genotypes in the presence and absenceof sucrose. This could be explained by an increase in transmitter

release, as dynasore has been described to elevate resting

intra-terminal Ca2+ concentrations, causing an increase in theprobability of transmitter release (Chung et al., 2010; Douthitt

et al., 2011).In addition, pharmacological inhibition of LRRK2 kinase

activity with LRRK2-IN-1 causes similar impairments insynaptic transmission, indicating that such impairments are, at

least partially, dependent on the LRRK2 kinase domain.However, one needs to keep in mind possible off-target effectsof LRRK2-IN-1, as it has been previously shown that in

LRRK2-knockout neurons the effect of LRRK2-IN-1 mimics theeffect of LRRK2 ablation (Luerman et al., 2014). Further studiesin LRRK2-knockout neurons would be needed to shed light on

possible off-target effects of LRRK2-IN-1 on synaptic vesicleendocytosis. Interestingly, synaptic transmission is also impairedin mouse triple-knockout neurons of endophilin, an important

regulator of synaptic vesicle endocytosis (Milosevic et al.,2011). In line with our data, an in vivo study in LRRK2mutant animals has suggested an involvement of LRRK2 inneurotransmitter release (Tong et al., 2009). Our data further

suggest that this function of LRRK in flies is mediated byLRRK2 and not LRRK1 in the mammalian system, as LRRK2-IN-1 does not inhibit LRRK1 kinase function (Kramer et al.,

2012). Future studies in LRRK1-knockout neurons couldstrengthen this hypothesis further.

Defects in clathrin-mediated endocytosis can also be observed

at the ultrastructural level. LRRK2-knockout synapses show adecreased number of synaptic vesicles that are heterogeneous insize and present various aberrant endocytic intermediates,

including clathrin-coated intermediates. Similar phenotypes areobserved after inhibition of dynamin function using dynasore(Newton et al., 2006) and in Drosophila mutants of endocyticproteins such as EndoA, AP-180/lap, StonedB and Intersectin/

Dap160 (Dickman et al., 2005; Koh et al., 2004; Verstreken et al.,2002; Verstreken et al., 2003; Zhang et al., 1998; Ferguson et al.,2007; Guichet et al., 2002). These observations are also similar to

the morphological changes described in Lrrk Drosophila mutants,where synaptic vesicle diameter was increased in knockoutboutons (Matta et al., 2012). Furthermore, a reduction in the

number of synaptic vesicles and an accumulation of clathrin-coated vesicular profiles are also observed in endophilin triple-knockout mice (Milosevic et al., 2011). As the ultrastructuraldefects observed in these mice are very similar in LRRK2 and

endophilin-knockout synapses, one could speculate that LRRK2regulates endophilin function during endocytosis in mammals.Interestingly, a decreased number of synaptic vesicles was also

reported in a knockout model of a-synuclein, another proteininvolved in Parkinson’s disease (Cabin et al., 2002) that isreported to have a role in clathrin-mediated endocytosis (Ben

Gedalya et al., 2009). In addition, endophilin has also beenreported to bind with high affinity to Parkin, another proteinlinked to Parkinson’s disease (Trempe et al., 2009). Therefore,

one could speculate that LRRK2, a-synuclein and Parkin functionin a common pathway at the synapse and disturbances of thispathway have a role in Parkinson’s disease. Interestingly, defectsin synaptic transmission are frequently and consistently observed

in both dominant and recessive forms of Parkinson’s disease(Kitada et al., 2009; Nakamura and Edwards, 2007; Tong et al.,2009), although the molecular mechanisms remain unknown.

Further studies are now needed to determine whether thisintriguing common synaptic function of LRRK2 and otherParkinson’s disease risk factors contributes to Parkinson’s

disease pathology associated with these genes.

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In summary, our data reveal a clear phenotype in LRRK2-knockout neurons, with slowed clathrin-mediated endocytosis,

impaired neurotransmission and ultrastructural abnormalities. Asmentioned before, similar phenotypes are observed in bothDrosophila EndoA and Drosophila Lrrk mutants and inendophilin-knockout mice (Matta et al., 2012; Milosevic et al.,

2011; Verstreken et al., 2002). As such phenotypes in Lrrk

Drosophila mutants are the result of changes in LRRK-dependentphosphorylation of EndoA (Matta et al., 2012), we aimed at

studying whether LRRK2 also phosphorylates mammalianEndoA1. In this study, we provide in vitro evidence thatEndoA1 is phosphorylated by LRRK2 on at least two sites, T73

and S75. It is possible that additional serine and/or threonineresidues in EndoA1 are phosphorylated by LRRK2, because theT73A/S75A mutant protein is still phosphorylated by LRRK2 in

vitro. The observation that GSK3b did not phosphorylate EndoA1suggests that EndoA1 is only phosphorylated by specific kinases,including LRRK2. The fact that LRRK2 does not phosphorylateEndoB1, indicates that LRRK2 has a discrete number of selective

substrates.Both T73 and S75 are located in the EndoA1 BAR domain,

which is known for its involvement in membrane curvature

(Gallop et al., 2006) and is highly conserved across species.Interestingly, recent evidence indicates that the phosphorylationof S75 constitutes a regulatory mechanism for guiding the

curvature state of EndoA1 and could be used to control differenttypes of membrane curvature in vivo (Ambroso et al., 2014). Thisphenomenon was also observed in our previous study (Matta

et al., 2012). Hence, we suggest that LRRK2-dependentphosphorylation of this site regulates the membrane affinity ofEndoA1 and consequently its role in clathrin-mediatedendocytosis. However, it will be essential to provide evidence

that LRRK2 also phosphorylates EndoA1 in vivo. Furtherresearch will need to be conducted to resolve this issue.Whether phosphorylation of T73 has a similar function to that

of S75 phosphorylation remains to be determined. Compared toautophosphorylation of LRRK2, phosphorylation of EndoA1appears to be low. However, LC-MS/MS data show that whereas

in EndoA1 only T73 and S75 were detected as phosphosites, inLRRK2 more than 20 phosphosites were detected (supplementarymaterial Table S1), explaining the difference in 33P incorporation.Furthermore, it needs to be kept in mind that the amount of

phosphorylation is not directly correlated with functionality in

vivo. Besides driving the formation of synaptic vesicles throughsensing or inducing plasma membrane curvature (Gallop et al.,

2006; Masuda et al., 2006), EndoA1 has another importantfunction in synaptic vesicle endocytosis, that is, facilitatingclathrin uncoating once synaptic vesicles have been generated

(Milosevic et al., 2011; Verstreken et al., 2002). Morphologicalphenotypes found in LRRK2-knockout synapses suggest that bothEndoA1 functions might be affected. Nevertheless, further work

is needed to determine precisely how EndoA1 is affected byLRRK2 in vivo. For example, similar to what was performedbefore in Drosophila (Matta et al., 2012), it would be interestingto use phosphomimetic or phosphodeficient EndoA1 mutants to

investigate whether expression of these mutants would rescue orworsen the endocytosis deficiency phenotype that is observed inLRRK2-knockout neurons. This would further strengthen our

hypothesis that LRRK2-dependent EndoA1 phosphorylation isfunctionally important in synaptic vesicle endocytosis.

In this study, we look at effects of LRRK2-knockout and show

that this causes impairments in neurotransmission. However,

G2019S, one of the most prevalent LRRK2 mutations inParkinson’s disease, is known to elevate kinase activity

(Greggio and Cookson, 2009). Interestingly, transfection studiesin human cells have shown that kinase-activating as well askinase-dead mutations have similar toxic effects (Cookson andBandmann, 2010). Similarly, Shin and colleagues report that

overexpression of LRRK2 slows down synaptic vesicleendocytosis (Shin et al., 2008). This is in accordance withprevious work from our group, where we showed that in

Drosophila both too much and too little LRRK2 causeimpairments in synaptic vesicle endocytosis (Matta et al.,2012). Therefore, it is tempting to speculate that LRRK2 is part

of a carefully balanced system, in which both too much and toolittle protein activity can cause defects in the synaptic vesiclecycle. More research is needed to validate this speculation and it

would be interesting to investigate whether our experiments yieldsimilar results in neurons derived from G2019S LRRK2 knock-inmodels.

In conclusion, we unravel an essential role of LRRK2, and in

particular its kinase domain, in the regulation of clathrin-mediated endocytosis of synaptic vesicles and subsequentneurotransmission in mammals. We show that a mechanism

that was previously identified in Drosophila melanogaster ishighly conserved across species and relevant in mammaliansystems.

MATERIALS AND METHODSAnimalsLRRK2-knockout C57Bl6/J mice (Charles River, France) and LRRK2-

knockout Long Evans rats (SAGE Laboratories, USA) were housed under

specific pathogen-free conditions and were used in accordance with

the University of Leuven and Janssen Pharmaceutica Animal Ethics

Committee. For the endocytosis assay and electrophysiology

experiments, wild-type and homozygous LRRK2-knockout animals

were used. Pregnant females were killed by decapitation and embryos

were isolated. For electron microscopy analysis, LRRK2-knockout

animals and wild-type littermates were used. Animals were killed with

CO2 when needed.

SypHy endocytosis assayStriatal cultures were generated from E17-19 embryos from wild-type

and LRRK2-knockout Long Evans rats (SAGE Laboratories, USA). At

DIV11 primary neurons were co-transfected with sypHy, a fusion

construct of synaptophysin and super ecliptic pHluorin (Granseth et al.,

2006) and pRFP-c-RS (Origine) using Lipofectamin-2000 (Invitrogen,

Gent, Belgium). SypHy is a pH-sensitive fluorescent reporter that, by

analogy with the original synaptopHluorin (synaptobrevin-pHluorin,

(Miesenbock et al., 1998; Sankaranarayanan and Ryan 2000) is quenched

in the acidic intracellular space of the synaptic vesicles and will only

become fluorescent upon exposure to the more basic pH of the

extravesicular space. In these experiments, only RFP-positive boutons

were assayed for endocytosis in a custom-build stimulation chamber on

the stage of a Zeiss Axiovert 200M equipped with a mono-chromator

(Poly V) and a cooled CCD camera (PCO, Imago QE), both from TILL

Photonics (Grafelfing, Germany). The assay was carried out as described

previously (Kim et al., 2005; Sankaranarayanan and Ryan 2000). Briefly,

cells were submerged in 500 ml of HBS buffer (10 mM HEPES,

136 mM NaCl, 2.5 mM KCl, 10 mM D-glucose, 1.3 mM MgCl2,

25 mM NaHCO3, 2 mM CaCl2, pH 7.3). SypHy was excited at

475 nm and its fluorescence emission collected at 525 nm using a 406,

1.1 NA water immersion objective. Images were acquired every half

second for 200 s using TillVision software (TILL Photonics). At frame

15, cells were stimulated for 15 s at 20 Hz. Quantitative measurements of

the fluorescence intensity at individual boutons were obtained by

averaging a selected area of pixel intensities using custom written

macros in Igor Pro (Wavemetrics). Net fluorescence changes (DF) were

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obtained by subtracting the average intensity of the first 15 frames (F0)

from the intensity of each frame (Ft) for individual boutons and

normalized to F0 (DF/F0). Both the fluorescence upstroke and decay

were fitted with a single exponential t (tupstroke and tdecay, respectively).

Data are expressed as mean6s.e.m. and statistical significance was

assessed with an unpaired two-tailed Student’s t-test (GraphPad Prism).

Electron microscopyLRRK2-knockout mice (Charles River, France) and wild-type littermates

(n53 per genotype) were transcardially perfused with 2.5%

glutaraldehyde, 2% paraformaldehyde in 0.1 M cacodylate buffer.

Brains were dissected and post-fixed in the same solution overnight at

4 C. Coronal brain sections (300 mm thick) were cut on a vibratome

(Leica, Buffalo Grove, IL) and rectangular pieces of tissue comprising

the striatum were dissected. The tissue was post-fixed with 1% OsO4,

1.5% K4Fe(CN)6 in 0.1 M cacodylate buffer, rinsed, stained with 3%

uranyl acetate and dehydrated in graded ethanols and propyleneoxide,

followed by embedding in EMbed812. Ultrathin sections (70–90 nm)

were obtained using an ultramicrotome (Leica), mounted on copper grids

and treated with uranyl acetate and lead citrate before being imaged using

a JEM-1400 transmission electron microscope (Jeol, Zaventem,

Belgium), equipped with a 11Mpixel Olympus SIS Quemesa camera.

Images were taken at a 300006 magnification, and morphometry and

measurements were performed with ImageJ software. For analysis of

synaptic vesicle numbers, 14830 vesicles in total from 112 wild-type and

115 knockout synapses from the six brain samples (n53 per genotype)

were counted. Synapses were selected based on the presence of an active

zone and all synaptic vesicles present within the synaptic vesicle cluster

were counted. For analysis of synaptic vesicle diameters, a total of 1043

wild-type and 1040 knockout vesicles from the same brain samples used

for the synaptic vesicle counts were measured. Statistical significance

was assessed by Student’s t-test (GraphPad Prism) and data are expressed

as mean6s.e.m.

ElectrophysiologyFor patch-clamp experiments, hippocampal neurons were derived from

E17–E19 embryos from wild-type and LRRK2-knockout C57Bl6/J mice

(Charles River, France). Cells were plated in a density of 50,000 cells per

ml on cover glasses. Cultures were maintained in neurobasal medium

with B27 supplement (Life Technologies, Gent, Belgium) for 7–12 days,

after which recordings were performed in bath solution (140 mM NaCl,

2.4 mM KCl, 10 mM HEPES, 10 mM glucose, 2.5 mM CaCl2, 1.3 mM

MgCl2, pH 7.3). Pyramidal neurons were voltage clamped under an

Axioskop 2FS upright microscope (Carl Zeiss, Nossegem, Belgium) at a

holding potential of 270 mV using HEKA software. Electrodes (thick-

walled borosilicate glass capillaries with an outer diameter of 1.5 mm

and an inner diameter of 0.86 mm, Harvard Apparatus) were pulled with

a P97 Flaming/Brown Micropipette puller (Sutter Instruments).

Micropipette tips were heated with a micro forge to produce a smooth

surface, with a diameter and resistance of 1 mm and 3–5 MV. Total

sEPSCs were recorded in the absence of TTX to collect all possible

synaptic activities including action-potential-mediated vesicle release.

Events were recorded with intracellular solution (146 mM K-gluconate,

17.80 mM HEPES, 4 mM Mg-ATP, 0.30 mM Na2-ATP, 1 mM EGTA

and 12 mM phosphocreatine, pH 7.3) (Rost et al., 2010) for 10 s with an

EPC10 USB Patch Clamp Amplifier (HEKA). Liquid junctional potential

and series resistance were automatically compensated before each

recording. To further ensure the recorded cells were indeed neurons,

firing patterns were routinely determined at the beginning of each

experiment by introducing depolarization steps. Directly after obtaining a

whole-cell patch configuration, a 1-min recording of baseline activities

was made. After recording baseline activities, bath solution was replaced

by hypertonic solution (bath solution with 50 mM sucrose) by perfusion.

Sucrose is known to increase exocytosis, and consequently endocytosis,

leading to an increase in instantaneous frequency of sEPSCs (Geppert

et al., 1994; Rosenmund and Stevens, 1996). After 2 min in hypertonic

solution, new recordings were made and all measured sEPSCs are

thought to come from newly endocytosed vesicles. For dynasore

experiments, cells were incubated in bath solution containing 40 mM

dynasore for 5 min before recordings were made. After the first

recordings, neurons were incubated in hypertonic solution with added

40 mM dynasore. After 2 min, new recordings were made. All

experiments were performed at room temperature. All measurements

were analyzed using Igor Pro software (Wavemetrics). Data are

expressed as mean6s.e.m. from n cells. Statistical significance was

assessed by two-tailed Student’s t-test (GraphPad Prism).

In vitro phosphorylation assayRecombinant GST-tagged LRRK2 protein (Life Technologies) and GST-

tagged substrates (Abnova, Taipei, Taiwan, or Dario Alessi, University of

Dundee, Dundee, UK) were incubated in kinase buffer (50 mM Tris-HCl

pH 7.5, 1 mM EGTA, 10 mM MgCl2, 2 mM DTT, 0.01% Tween-20)

in the presence of 1 mM ATP (with 0.5 mCi [33P]ATP, Perkin Elmer,

Zaventem, Belgium) during the indicated time points at 37 C. Reactions

were stopped by adding SDS-PAGE sample buffer. Proteins were

separated by electrophoresis on 4–12% NuPAGE gradient gels and

electroblotted onto nitrocellulose membranes. Phosphorylation of

substrates was quantified using Typhoon (GE Healthcare, Belgium)

and total protein levels were determined by western blotting using an

anti-GST antibody (Sigma-Aldrich, Diegem, Belgium). Data are

expressed as mean6s.e.m. Statistical significance was assessed by two-

way ANOVA with Bonferroni post-test (GraphPad Prism).

Phosphopeptide analysis by LC-MS/MSLRRK2 (recombinant protein; Life Technologies) was used in an in vitro

phosphorylation assay in the presence or absence of human SH3GL2.

Protein samples were separated by SDS-PAGE and stained with

Coomassie Brilliant Blue G-250. Excised gel bands were washed with

water, followed by 50% acetonitrile and acetonitrile before vacuum

drying. Bands were cut in two and one half was fully submerged in

digestion buffer (50 mM NH4HCO3 in 10% acetonitrile) with 0.1 mg

trypsin (Promega) for in-gel digestion. Digestion proceeded overnight at

37 C, following which the generated peptide mixtures were acidified with

formic acid. The other half of the protein band was first N-propionylated

in-gel before digestion with trypsin, as in this way, side-chains of lysines

are blocked (indicated in the supplementary material Table S1 as

,prop*.) and no longer recognized by trypsin, which then only cleaves

C-terminal to arginines. All peptide mixtures were then analyzed by

LC-MS/MS using a LTQ Orbitrap XL mass spectrometer (Thermo

Scientific). The generated MS/MS spectra were searched in the human

subsection of the Swiss-Prot database by the Mascot algorithm. For

phosphopeptide enrichment, TiO2 Mag Sepharose beads (GE Healthcare)

were used prior to LC-MS/MS analysis.

AcknowledgementsWe thank Lucia Chavez-Gutierrez for her advice on the 3D modeling andmembers of the Patrik Verstreken, Bart De Strooper and Janssen CNS Researchlaboratories for comments.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConception and design of the study was performed by P.V., B.D.S. and D.M.Experiments were performed by L.D. (phosphorylation assay), A.M.A.(TEM andsypHy assay),M.R.G. (electrophysiology),W.M. (sypHy assay), G.D.(phosphorylationassay), S.M. (sypHy assay), S.C. (electrophysiology), H.S. (electrophysiology), P.V.B.(sypHy assay), P.B. (TEM), P.-J.D.B. (LC-MS/MS analysis) and K.G. (LC-MS/MSanalysis). All authors were involved in data interpretation. Preparation of themanuscript was performed by L.D., A.M.A. and K.V.K.

FundingThe research was supported by a research and development grant [grant number100508] from the Agency for Innovation by Science and Technology(IWT-Flanders); the European Research Council (ERC); the Fonds voorWetenschappelijk Onderzoek (FWO); the KULeuven; and Flemish Institute forBiotechnology (VIB); and a Methusalem grant of the KULeuven/Flemish

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Government to B.D.S. B.D.S. is supported by the Bax-Vanluffelen chair forAlzheimer’s Disease.

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.158196/-/DC1

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