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Received 24 Jan 2014 | Accepted 2 May 2014 | Published 6 Jun 2014

DOI: 10.1038/ncomms5028

iPSC-derived neurons from GBA1-associated Parkinsons disease patients show autophagic defects and impaired calcium homeostasis

David C. Schndorf1,2, Massimo Aureli3,*, Fiona E. McAllister4,*, Christopher J. Hindley5,*, Florian Mayer6, Benjamin Schmid1,2, S. Pablo Sardi7, Manuela Valsecchi3, Susanna Hoffmann1,2, Lukas Kristoffer Schwarz1,2, Ulrike Hedrich2,8, Daniela Berg1,2, Lamya S. Shihabuddin7, Jing Hu6, Jan Pruszak5,9, Steven P. Gygi4, Sandro Sonnino3, Thomas Gasser1,2 & Michela Deleidi1,2

Mutations in the acid b-glucocerebrosidase (GBA1) gene, responsible for the lysosomal storage disorder Gauchers disease (GD), are the strongest genetic risk factor for Parkinsons disease (PD) known to date. Here we generate induced pluripotent stem cells from subjects with GD and PD harbouring GBA1 mutations, and differentiate them into midbrain dopaminergic neurons followed by enrichment using uorescence-activated cell sorting. Neurons show a reduction in glucocerebrosidase activity and protein levels, increase in glucosylceramide and a-synuclein levels as well as autophagic and lysosomal defects.

Quantitative proteomic proling reveals an increase of the neuronal calcium-binding protein 2 (NECAB2) in diseased neurons. Mutant neurons show a dysregulation of calcium homeostasis and increased vulnerability to stress responses involving elevation of cytosolic calcium. Importantly, correction of the mutations rescues such pathological phenotypes. These ndings provide evidence for a link between GBA1 mutations and complex changes in the autophagic/lysosomal system and intracellular calcium homeostasis, which underlie vulnerability to neurodegeneration.

1 Department of Neurodegenerative Diseases, German Center for Neurodegenerative Diseases (DZNE), Tbingen 72076, Germany. 2 Hertie-Institute for Clinical Brain Research, University of Tbingen, Tbingen 72076, Germany. 3 Department of Medical Biotechnology and Translational Medicine, University of Milan, Milan 20090, Italy. 4 Department of Cell Biology, Harvard Medical School, Boston, Massacchusetts 02115, USA. 5 Emmy Noether-Group for Stem Cell Biology, Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg, Freiburg 79104, Germany. 6 Werner Reichardt Center for Integrative Neuroscience (CIN), University of Tbingen, Tbingen 72076, Germany. 7 Genzyme, a Sano Company, Framingham, Massachusetts 01701, USA. 8 Department of Epileptology, Hertie Institute for Clinical Brain Research, University of Tbingen and German Center for Neurodegenerative Diseases, Tbingen 72076, Germany. 9 Center for Biological Signaling Studies (BIOSS), University of Freiburg, Freiburg 79104 Germany. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to M.D. (email:mailto:[email protected]

Web End [email protected] ) or to T.G. (email: mailto:[email protected]

Web End [email protected] ).

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5028

Parkinsons disease (PD) is the second most common neurodegenerative disorder, with 41% of the population over 65 years of age affected and 44% affected by the age

of 85 years. Several clues to understanding disease pathogenesis come from mutations in genes, which have been linked to familial forms of PD or identied as risk factors. Among these, heterozygous mutations in the acid b-glucocerebrosidase (GBA1) gene, which encodes the lysosomal enzyme b-glucocerebrosidase (GCase), have been linked to a higher risk of developing PD and other synucleinopathies1,2. GCase is a lysosomal enzyme that catalyses the hydrolysis of glucosylceramide (Glc-Cer) to ceramide and glucose. Homozygous mutations of GBA1 are responsible for Gauchers disease (GD), which is the most prevalent lysosomal storage disorder, characterized by decreased activity of GCase and subsequent accumulation of Glc-Cer in several organs including the brain. Different clinical forms are described depending on the absence (non-neuronopathic forms, GD type 1) or presence (neuronopathic forms, GD type 2 and 3) of neurological symptoms. GD type 1 is the most common type and is often associated with PD and parkinsonism, thus challenging such classication3. To date, over 300 mutations have been identied in GBA1 and linked to GD. In recent years, GD and PD have been connected on account of the clinical observation of parkinsonism and Lewy Body (LB) pathology in patients with GD3. Compared with the general population, patients with GD type 1 have a 20-fold increased lifetime risk of developing parkinsonism4, whereas individuals carrying heterozygous GBA1 mutations have a ve times greater risk of developing PD than non-carrier individuals1. The pathological mechanisms through which the mutant enzyme causes PD still remain elusive and both gain- and loss-of-function theories have been postulated. Such hypotheses are not mutually exclusive and include increased a-synuclein (a-syn) protein aggregation, sphingolipid accumulation and impaired trafcking as potential mechanisms involved in the pathogenesis of neurodegeneration5,6.

Recent progress in the induced pluripotent stem cell (iPSC) technology has allowed the generation of functional dopaminergic (DA) neurons from familial and sporadic PD subjects79, offering a unique opportunity to study the role of genetic mutations and risk factors in the pathogenesis of PD. Neurons from PD patients carrying heterozygous GBA1 mutations have not yet been generated, and it is unknown whether such cells recapitulate the pathological phenotypes observed in vivo.

In this study, we demonstrate that iPSC-derived neurons from GD and PD individuals carrying GBA1 mutations display relevant disease phenotypes, including a signicant reduction of GCase enzymatic activity and protein levels as well as increased levels of a-syn and Glc-Cer. Furthermore, mutant neurons show defects in the autophagic/lysosomal system and impaired intracellular calcium homeostasis. Importantly, such phenotypes are reverted upon gene correction, thus supporting a direct link between GBA1 mutations and disease-relevant phenotypes.

ResultsGeneration of iPSCs and isogenic gene-corrected controls. Skin broblasts were obtained from two healthy controls, four PD patients carrying heterozygous GBA1 mutations (RecNcil/wt; L444P/wt; N370S/wt) and four GD patients (type 1: N370S/ N370S; type 3: L444P/L444P) (Supplementary Table 1). Fibro-blasts were reprogrammed by transduction with SOX2, OCT4, KLF4 and c-MYC retroviruses. After 4 to 5 weeks, iPSC colonies were isolated and further characterized (Supplementary Table 2). All generated iPSCs showed human embryonic stem cell (hESC)-like morphology and expressed alkaline phosphatase and

the pluripotency markers SSEA4, TRA-1-81, TRA-1-60, OCT4, NANOG and LIN28 (Supplementary Figs 1 and 2a). iPSCs were genetically matched with their parental cells by PCR-based DNA ngerprinting analysis, maintained euploid karyotypes over several passages in vitro and showed effective silencing of the exogenous transgenes and upregulation of endogenous hESC markers (Supplementary Fig. 2bd). All lines tested were efciently differentiated to mesoderm, endoderm and ectoderm in vitro (Supplementary Fig. 3a) and formed teratomas consisting of the three germ layers when injected into the kidney capsule and testis of immune-compromised mice (Supplementary Fig. 3b).

Correction of GBA1 mutations in iPSCs was performed by zinc-nger nuclease (ZFN)-mediated homologous recombination. Three iPSC lines (PD1-2, PD2-2 and PD4-2) were selected for gene-targeting experiments. iPSCs were nucleofected with ZFNs, which were designed to introduce a double-strand brake adjacent to the N370S and L444P mutations, along with a wild-type (wt)-GBA1 construct harbouring a neomycin resistance cassette (Fig. 1a). Single resistant colonies were selected, and corrected sequences at the mutation site were conrmed by DNA and RNA sequencing (Fig. 1b). Karyotype analysis revealed that no translocation had occurred in the clones after ZFN-mediated insertion.

Differentiation of iPSCs to mDA neurons. iPSCs from controls, GBA-PD and GD subjects were differentiated to mDA neurons by using a oor-plate-based neural differentiation protocol10. This early neural patterning protocol induces 70% FOXA2 /

OTX2 oor plate precursors by day 11 (ref. 10 and Supplementary Fig. 4). By day 40, all iPSC lines generated b-TubIII neurons and cells expressing tyrosine hydroxylase (TH). At DIV 65, 1520% of the cells were TH and co-expressed markers of mDA phenotype (FOXA2, NURR1, GIRK2,

VMAT2) (Fig. 2). No signicant differences in differentiation potential were observed among control, GBA-PD, gene-corrected and GD lines (Supplementary Fig. 5a). Differentiated neurons showed a punctate staining of synapsin 1, indicating the presence of synaptically mature neurons (Supplementary Fig. 5b).

Enrichment of neuronal populations from differentiated iPSCs. To enrich neuronal populations from differentiated iPSC cultures, we included a neuronal cell isolation step using uorescence-activated cell sorting (FACS)1114. At DIV 6570, differentiated neurons from iPSCs were puried based on a surface marker code of CD24high CD29 CD184 CD44

CD15 (Fig. 3a). Analysis of post-FACS cell cultures revealed highly enriched neuronal populations (6.1-fold enrichment compared with unsorted cells), which expressed mature DA neuronal markers and could be kept in vitro for 3 weeks after sorting (Fig. 3b,c and Supplementary Fig. 6). Whole-cell patch clamp recordings showed that single neurons were able to re action potentials (AP) repetitively. Importantly, around 50% of the patched neurons derived from iPSCs showed spontaneous ring activity (Supplementary Fig. 7a), whereas in 93% of the recorded cells AP could be elicited by current injections into the soma (Supplementary Fig. 7b). The mean resting membrane potential of all recorded neurons was 53.11.5 mV. In

addition, recorded cells showed sodium and potassium currents (Supplementary Fig. 7c). Importantly, differentiated neuronal cultures released DA (Supplementary Fig. 7d).

The polysialoganglioside content of iPSC-derived neurons. Gangliosides are sphingolipids (GSL) particularly abundant in the neuronal plasma membrane playing an important role in the pathogenesis of neurological disorders. With respect to

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5028 ARTICLE

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Figure 1 | Gene correction of GBA-PD iPSCs. (a) Structure of the GBA1 gene on chromosome 1 and schematic overview of gene correction using ZFNs. Black boxes represent exons. (b) Mutated and gene-corrected (GC) iPSCs after sequencing (upper lanes: iPSCs with heterozygous mutations; lower lanes: iPSCs with homozygous wt-GBA1 sequences as indicated by arrows).

synucleinopathies, alterations in the neuronal GSL composition cause impaired a-syn membrane binding and enhanced aggregation in the cytoplasm15. To study the link between GCase and a-syn and to investigate the pathogenesis of neurodegeneration in sphingolipidoses, it is therefore crucial to utilize cell models that resemble the GSL composition and pattern of adult human brain. The content and pattern of gangliosides markedly change during brain development16 and the ganglioside concentration is B10 times higher in the brain than in other organs17. The total brain ganglioside content is very low at the embryonic and fetal stage and then increases several folds with a shift from gangliosides of the Lac series, GM3 and GD3, to monosialo-, during development, and polysialo-species in the adult brain16,17. The GSL content and pattern are therefore useful markers to follow neural development both in vivo and in vitro18,19. To investigate whether iPSCs recapitulate such developmental changes during in vitro differentiation and whether iPSC-derived neurons have the GSL composition of developmentally mature neurons, we metabolically labelled gangliosides by feeding broblasts, iPSCs and iPSC-derived neurons (before and after FACS enrichment) from control, GBA-PD and GD type 3 subjects with isotopically tritium labelled [13H]sphingosine19. After a 48-hour chase, gangliosides were puried from the total lipid extract, separated by HPTLC and analysed by radio-imaging20. As expected, the ganglioside pattern of broblasts was characterized by the presence of GM3 and a minor quantity of GD3 (Fig. 4a). Minor differences in the ganglioside pattern and in the GM3/ GD3 ratio among broblast cell lines were detected (Fig. 4a),

which have been described to be age and patient specic21. In iPSC cultures, GM3 still represented the main ganglioside, but low amounts of GM2, GM1 and GD1a components were identied (Fig. 4a). By contrast, differentiated iPSCs contained all the polysialogangliosides found in the CNS, even though high levels of GM3 and GD3 were still present16 (Fig. 4a and Supplementary Fig. 8). After FACS enrichment, the levels of GD3 and GM3 were found to be signicantly reduced compared with the total GSL composition and the levels of GM1, GD1a, GD1b and GT1b were in a percentage distribution similar to that found in the adult human and rodent brain (Fig. 4a and Supplementary Fig. 9)22. Importantly, the total ganglioside content in FACS-enriched iPSC-derived neurons was about 10 times higher than that in broblasts: the radioactivity associated with gangliosides ranged from 41,250 to 57,500 dpm mg 1 cell protein in broblasts (control, GD and GBA-PD) and from 330,650 to 415,000 dpm mg 1 cell protein in sorted neurons.

Glycohydrolases in human iPSCs and enriched neurons. The developmental changes of GSL content and patterns can be largely attributed to switches in the expression and activity of several enzymes involved in their synthesis and catabolism16. We therefore investigated the activities of enzymes involved in the catabolic pathway of GSL during human iPSC differentiation. Control iPSCs were differentiated for 6570 days in vitro, neurons enriched by FACS and the activities of the main hydrolases involved in the metabolism of glycoconjugates (conduritol B

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5028

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Figure 2 | Differentiation of iPSCs into mDA neurons. Immunostaining of indicated differentiated iPSC cultures at DIV65. Cells were stained for TH (red) and b-TubIII, FOXA2, NURR1, GIRK2 and VMAT2 (all green). Cell nuclei were counterstained with Hoechst (blue). (Bars, 20 mm).

epoxide (CBE)-sensitive b-glucosidase, non-lysosomal b-glucosyl ceramidase (GBA2), b-galactosidase and b-hexosaminidase) were investigated. Two different cellular compartment-specic bglucosidase activities were analysed: the lysosomal bglucocerebrosidase GCase and the non-lysosomal beta-glucosyl ceramidase GBA2 (ref. 23). By using CBE, a specic inhibitor of GCase, it was possible to evaluate the activity associated with GBA2, whereas, by application of the specic inhibitor N-(5-adamantane-1-yl-methoxy-pentyl)-deoxynojirimycin (AMP-DNM)24, it was possible to determine the b-glucosidase activity due to GCase. We found that the activity of all tested enzymes was signicantly increased in iPSC-derived neurons compared with iPSCs and broblasts, suggesting an important role of such enzymes in differentiation and maintenance of GSL composition in neurons (Fig. 4b and Supplementary Fig. 10a). Quantitative RT-PCR and immunoblot revealed an increase in GBA1, GBA2, b-galactosidase and b-hexosaminidase mRNA and protein levels in differentiated cultures compared with iPSCs (Fig. 4c,d).

Increased Glc-Cer and a-syn levels in GBA1 mutant neurons. Recent studies using iPSC-derived neurons from GD patients showed that DA neurons accumulate glycosphingolipids25,26. To examine whether neurons from PD patients carrying heterozygous GBA1 mutations also display such phenotypes, we carried out liquid chromatography (LC) and tandem mass spectrometry (MS) (LC-MS/MS) analysis on differentiated iPSCs from controls, GBA-PD and GD patients. Both homozygous and heterozygous GBA1 mutant iPSC-derived neurons showed elevated levels of Glc-Cer compared with control cells (Fig. 5a). Importantly, gene-corrected isogenic controls showed reduced Glc-Cer levels compared with the parental lines (Fig. 5a).

A direct link between GCase and a-syn accumulation has previously been shown25,27,28. However, it has not yet been investigated whether heterozygous GBA1 mutations are linked to increased levels of a-syn. We therefore sought to examine the effects of GBA1 mutations on a-syn accumulation in

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5028 ARTICLE

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Figure 3 | FACS enriches for neuronal populations. (a) Gating strategy used for neuronal cell sorting. Live cells as determined by FSC/SSC gating were selected for CD184-APC-negative/low surface expression and subsequently separated for CD24-PE versus CD15/CD29/CD44 expression, detected in the FITC channel as indicated in the dot plots. Post-sort reanalysis shows representative samples of both fractions immediately after FACS enrichment, which were then plated for comparative in vitro analysis (phase contrast images. Bars, 50 mm). (b,c) Immunocytochemistry at DIV2 post-sort for TH (green) and the neuronal marker MAP2 (red) performed on the neuronal (b) and mixed (c) populations of a representative control and GBA-PD line.

Nuclei were counterstained with Hoechst (blue). FACS enriches for both TH and neuronal marker positivity in the neuronal population compared with the non-neuronal population. (Bars, 50 mm.)

differentiated iPSCs. First, we examined the effect of loss of GCase activity in control iPSC-derived neurons. We found that CBE treatment induced an increase in a-syn protein levels in control iPSC-derived neurons (Fig. 5b). Next, iPSCs from controls, GBA-PD and GD subjects were differentiated to mDA neurons, and a-syn levels were measured by western blot. A signicant increase in a-syn protein levels was found in GBA-PD neurons carrying the L444P allele and in GD neurons compared with controls (Fig. 5c,e). It is known that differences in expression levels of a-syn are inuenced by individual genetic background, which could explain the high variability of a-syn expression in the general population29. Thus, we measured a-syn levels in neuronal cultures from GBA-PD iPSCs and corresponding isogenic gene-corrected controls. Interestingly, we found a signicant reduction in a-syn levels in

all gene-corrected neurons (RecNcil, L444P, N370S) compared with their parental lines (Fig. 5d,f).

Reduced GCase in GBA1 mutant neurons. The GCase activity was signicantly reduced in GBA-PD and GD broblasts compared with healthy control cells (Supplementary Fig. 11). Next, control, GBA-PD, isogenic gene-corrected controls and GD iPSCs were differentiated for 65 days and neuronal cultures were enriched by FACS. We found that the GCase enzymatic activity was signicantly reduced in GBA-PD and GD neurons compared with controls (Fig. 6a). Importantly, isogenic gene-corrected lines showed a signicantly higher average of enzymatic activity compared with non-corrected lines (Fig. 6a). Quantitative analysis of intracellular GCase protein levels in cells of

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5028

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Figure 4 | Ganglioside and glycohydrolase changes during human iPSC differentiation recapitulate human brain development. (a) Ganglioside composition of human broblasts, iPSCs, differentiated iPSCs and enriched iPSC-derived neurons (after FACS) from control (line C1-1), GBA-PD (line PD1-1) and GD (line GD1-1) subjects. Cell lipids were metabolically labelled with [1-3H]sphingosine and visualized by digital autoradiography. (b) Activities of glycohydrolases (GCase, GBA2, b-galactosidase, b-hexosaminidase) in control iPSCs and iPSC-derived enriched neurons at DIV65. Enzyme activities are expressed as pmoles mg 1 h 1. Data are represented as mean s.d.; experiments were independently repeated three times in triplicate. *Po0.01;

Students t-test. (c) GBA1, GBA2, b-galactosidase and HEXA and HEXB gene expression levels in control iPSCs and iPSC-derived enriched neurons at DIV65. Data are represented as mean s.d.; experiments were independently repeated three times in triplicate. *Po0.05; **Po0.01, Students t-test.

(d) Representative western blots showing GCase, GBA2, b-galactosidase and HEXA and HEXB protein levels in control iPSCs and corresponding iPSC-derived neurons (DIV65).

different patients by western-blotting revealed that protein levels of the enzyme were also reduced in GBA-PD and GD patients compared with control individuals (Fig. 6b,c). ZFN-mediated gene correction rescued the levels of GCase (Fig. 6b,c).

Decreased glycohydrolase activities in GBA1 mutant neurons. We next analysed the activity of additional glycohydrolases in iPSC-derived neurons from controls, GBA-PD, isogenic gene-corrected controls and GD subjects. Activity levels of the lysosomal enzymes b-galactosidase and b-hexosaminidase, as well as

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5028 ARTICLE

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Figure 5 | Glc-Cer and a-syn levels are increased in GBA-PD and GD iPSC-derived neurons. (a) LC-MS/MS analysis showing the level of Glc-Cer in control, GBA-PD, isogenic controls and GD iPSC-derived neurons. Data are represented as mean s.d.; n 3, *Po0.01, One-way ANOVA. (b) Control

iPSCs (line C1-1) were differentiated for 65 days and exposed to the GCase inhibitor CBE (25 mM) for 48 h. Representative western blot showing increased levels of a-syn in CBE-treated iPSC-derived neurons compared to untreated neurons. (c,d) Representative western blots showing the protein levels of a-syn in control, GBA-PD, gene-corrected isogenic controls and GD iPSC-derived neurons. (e) Optical densities of a-syn bands normalized by the averaged value of GAPDH and expressed as a percentage of the control (CTRL) line value. Data are represented as mean s.d.; experiments were

independently repeated ve times. *Po0.01, one-way ANOVA. (f) Average a-syn protein levels normalized by the averaged value of GAPDH and expressed as a percentage of the respective isogenic mutant lines. Data are represented as mean s.d.; experiments were independently repeated ve

times. *Po0.01, Students t-test.

the activity of the non-lysosomal b-glucosylceramidase GBA2, were examined in differentiated iPSC-neurons after FACS enrichment. Our analysis revealed a signicant decrease in the activity of GBA2 and b-galactosidase in GBA-PD and GD neurons compared with healthy controls, whereas activity levels of bhexosaminidase remained unchanged (Fig. 6a). Interestingly, gene correction rescued such changes in RecNcil- and L444P-associated PD lines (Fig. 6a). Gene expression levels of GBA2 and b-galactosidase in patient neurons were unchanged compared with control neurons (Supplementary Fig. 10b).

An increase in the activity of GBA2, b-hexosaminidase and bgalactosidase has recently been described in GD broblasts and leukocytes30,31. We therefore examined the enzyme activities in respective broblast lines and found a signicant increase in GBA2 activity in GBA-PD (L444P/wt) and GD (L444P/L444P) broblasts, while GD broblasts (L444P/L444P) showed in addition a signicant increase in b-galactosidase activity compared with controls (Supplementary Fig. 11).

Glycohydrolase activities in the CSF of GBA-PD patients. Recent studies showed that GCase activity is lower in the cerebrospinal uid (CSF) and brain samples of patients with PD or dementia with LB than that in healthy individuals3234. To determine whether changes in iPSC-derived neurons were accompanied by changes in the glycohydrolase enzymatic activity in the CSF, we measured the activity of several glycohydrolases (GCase, GBA2, b-galactosidase, b-hexosaminidase, a-fucosidase, a- and b-mannosidase) in the CSF of PD patients carrying heterozygous RecNcil, L444P or N370S GBA1 mutations, idiopathic PD patients as well as age-matched unaffected controls. Enzyme activity levels were normalized against total

protein levels. The CSF of patients used for iPSC generation in this study was included in the analysis. No signicant differences between idiopathic PD patients and controls were observed for all tested enzymes (Fig. 7); however, in GBA-PD patients the activities of GCase, GBA2 and b-galactosidase were signicantly reduced compared with unaffected controls (Fig. 7). In addition, we found a signicant reduction in the enzyme activity of GCase, GBA2, a- and b-mannosidase in GBA-PD patients compared with idiopathic PD patients (Fig. 7).

Lysosomal and autophagic defects in GBA1 mutant neurons. To extend these ndings, we analysed the autophagic/lysosomal machinery in GBA1 mutant neurons. Immunouorescent staining for LAMP1, a lysosomal marker, revealed a signicant increase in number and size of LAMP1 particles in patient iPSC-derived neurons compared with healthy individuals, suggesting an accumulation of lysosomes (Fig. 8a,b and Supplementary Fig. 12a). To examine macroautophagy, we rst investigated the autophagosome content by immunostaining for endogenous light chain type 3 protein (LC3), a marker of autophagosomes. Our analysis revealed a signicant increase of LC3 vesicles in GBA-PD and GD neurons compared with control neurons (Supplementary Fig. 12b,d). These results were conrmed by immunoblot, which showed an increase in basal levels of LC3-II in GBA-PD and GD-derived neurons (Fig. 8c,d). Inhibition of lysosomal degradation by leupeptin and ammonium chloride (NH4Cl) revealed that the autophagic ux was signicantly reduced in GBA-PD and GD neurons (Fig. 8e). To examine autophagosome-lysosome fusion, we assessed the co-localization of LC3 and LAMP1 markers in iPSC-derived neurons. We observed a decreased degree of co-localization

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5028

GCase (CBE-sensitive glucosidase) GBA2 (AMP-DNM-sensitive glucosidase)

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L444P/wt

L444P/wt GC

N370S/wt

N370S/wt GC

L444P/L444P

N370S/N370S

CTRL

RecNcil/wt

L444P/wt

L444P/wt GC

N370S/wt

0 RecNcil/wt GC

N370S/wt GC

L444P/L444P

N370S/N370S

Galactosidase Hexosaminidase

140,000 120,000 100,000

80,000 60,000 40,000 20,000

GCase

*** *

pmoles mg1h1

* **

* * * **

CTRL

0 RecNcil/wt GC

RecNcil/wt

L444P/wt

L444P/wt GC

N370S/wt

N370S/wt GC

L444P/L444P

N370S/N370S

CTRL

RecNcil/wt GC

RecNcil/wt

L444P/wt

L444P/wt GC

N370S/wt

N370S/wt GC

L444P/L444P

N370S/N370S

1.2

GCase OD

(% GAPDH)

10.80.60.40.2 0

C1-1

C1-2

C2-1

PD1-1

PD1-2

PD2-1

PD2-2

PD3-1

PD3-2

PD4-1

PD4-2

** * *

60 kDa

36 kDa

GAPDH

GCase 60 kDa

36 kDa

CTRL

RecNcil/wt

L444P/wt

N370S/wt

PD1-1

PD1-2

PD1-1

PD1-2

PD1-2 GC1

PD2-1

PD2-2

PD2-2 GC1

GD1-1

GD1-2

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PD4-2

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GD2-1

GD2-2

GCase

GAPDH

60 kDa

36 kDa

GCase

GAPDH

60 kDa

36 kDa

GAPDH

0.8 2

1.5

0.5

GCase OD (% GAPDH)

* *

0.6

0.4

0.2

RecNcil/wt

1.5

0.5

RecNcil/wt GC

0 L444P/wt

L444P/wt GC

L444P/L444P

0 N370S/wt

N370S/wt GC

N370S/N370S

Figure 6 | GBA-PD and GD iPSC-derived neurons show decreased glycohydrolase enzyme activities and protein levels. (a) Glycohydrolase enzyme activities (GCase, GBA2, b-galactosidase, b-hexosaminidase) were measured in enriched neurons from control, GBA-PD, isogenic gene-corrected control and GD iPSC-derived neurons at DIV65. Enzyme activities are expressed as pmoles mg 1 h 1. Data are represented as mean s.d.; experiments

were independently repeated three times in triplicate. *Po0.05; **Po0.01; ***Po0.001 compared with controls, one-way ANOVA. (b,c) Representative western blots of GCase protein levels in control, GBA-PD, corresponding gene-corrected and GD iPSC-derived neurons. Data are represented asmean s.d.; experiments were independently repeated three times. *Po0.05; **Po0.01, one-way ANOVA and Students t-test.

between LC3 vacuoles and LAMP1 vacuoles in GBA-PD and GD neurons, which suggests an impaired autophagosome-lyso-some fusion in GBA1 mutant neurons. (Supplementary Fig. 12c,e).

Impaired calcium homeostasis in GBA1 mutant neurons. We next used quantitative mass spectrometry (MS)-based proteomics to search for proteins whose levels are affected by GBA1

mutations in iPSC-derived neurons. Two independent iPSC lines harbouring the L444P allele and three independent control lines were differentiated and enriched by FACS. We extracted proteins and performed quantitative proteomic proling by isotopic labelling (tandem mass tag, TMT) and MS analysis (Table 1, Supplementary Fig. 13a,b and Supplementary Data 1). In total, 7,069 proteins were identied from enriched neurons at a protein false-discovery rate (FDR) o1%. We calculated the differences in protein abundances between GBA-PD neurons and control lines,

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GCase (CBE-sensitive

1,000 500 450 400 350 300 250 200 150 100

180,000 160,000 140,000 120,000 100,000

80,000 60,000 40,000 20,000

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-Glucosidase) GBA2 (AMP-DNM-sensitive

-Glucosidase)

pmoles mg1h1

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0 CTRL iPD GBA-PD

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Galactosidase

pmoles mg1h1

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pmoles mg1h1

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pmoles mg1h1

mg ml1

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CTRL iPD GBA-PD CTRL iPD GBA-PD

Mannosidase Protein concentration

pmoles mg1h1

14,000 10.90.80.70.60.50.40.30.20.1 0

Figure 7 | Glycohydrolase enzyme activities in the CSF of unaffected individuals, GBA-PD and idiopathic PD patients. CSF glycohydrolase activities were measured in patients with idiopathic PD (iPD), GBA-PD patients and age-matched unaffected individuals (CTRL). Enzyme activities were normalized to total protein concentrations and expressed as pmoles mg 1 h 1. Data are represented as mean s.e.m.; CTRL, n 14; iPD, n 15; GBA-PD, n 15.

Experiments were performed in triplicate. *Po0.05; **Po0.01, MannWhitney U test.

tested for statistical signicance by t-test analysis and ltered for proteins whose levels changed by at least 1.2-fold (Table 1). To validate the MS proteomic data, immunoblotting of selected proteins was performed on three independent differentiated iPSC cultures (Supplementary Fig. 13a). Among the differentially expressed proteins, we found that the neuronal calcium-binding protein 2 (NECAB2) was signicantly upregulated (1.9-fold) in lines harbouring the L444P allele (Table 1 and Supplementary Fig. 13a). Calcium-induced excitotoxicity plays a key role in the pathogenesis of several neurodegenerative diseases including PD35. To further investigate the potential role of calcium in the vulnerability of neurons harbouring GBA1 mutations, we examined calcium homeostasis using the calcium dye Fura-2 acetoxymethyl ester and compared calcium levels in control and diseased neurons. Interestingly, we found a signicant increase in the basal calcium levels in GBA-PD and GD iPSC-derived

neurons compared with controls (Fig. 9a). We therefore investigated the effects of caffeine, a RyR agonist that increases the calcium release from the calcium-sensitive internal stores, namely the endoplasmic reticulum (ER), into the cytoplasm. Caffeine induced an increase in cytosolic calcium, which was signicantly greater in GBA-PD (L444P/wt) and GD neurons compared with control neurons (Fig. 9b and Supplementary Fig. 14a). Although we also found a consistent trend for increased calcium levels in GBA-PD (RecNcil/wt) neurons, this increase was not statistically signicant (Fig. 9b). However, intracellular calcium levels in response to caffeine stimulation were comparable between isogenic gene-corrected neurons (both L444P/wt and RecNcil/wt) and control neurons (Fig. 9b and Supplementary Fig. 14a), suggesting a role of GBA1 mutations in regulating intracellular calcium concentration. Our data therefore indicate that RyR-mediated calcium release is increased in the

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Hoechst -TubIII TH LAMP1 Merge

C2-1

PD1-2

PD1-2 GC2

GD2-1

1.2

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in TH+ cells ( m)

** * *

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number in TH+ cells

* *

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+ + + + +

+ + + +

16 kDa 14 kDa

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LC3-I LC3-II

+ + + + +

-Actin

43.5 3

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8 *

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21.5

LC3II flux

** **

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N370S/wt GC

L444P/L444P

Figure 8 | GBA-PD and GD iPSC-derived neurons show alterations in the autophagic/lysosomal system. (a) Immunostaining of differentiated iPSC cultures at DIV65 under basal conditions. Cells were stained for TH (green), b-TubIII (magenta) and LAMP1 (red). Cell nuclei were counterstained with Hoechst (blue). High magnication insets are shown in the right lane. (Bars, 20 mm and 10 mm in the insets.) (b) Average size and number of LAMP1 particles in TH neurons were quantied by ImageJ. Data are represented as mean s.e.m.; experiments were independently repeated three times in

triplicate. *Po0.01, **Po0.001, one-way ANOVA. (c) Western blot analysis for LC3 in iPSC-derived neuronal cultures at DIV65, untreated ( ) or treated

with 200 mM leupeptin and 20 mM NH4Cl for 4 h ( ). (d) Quantication of the basal levels of LC3-II relative to b-actin. Data are represented as

mean s.e.m.; experiments were independently repeated three times. *Po0.01; **Po0.001, one-way ANOVA. (e) Quantication of LC3 ux normalized to

actin. Data are represented as mean s.e.m.; experiments were independently repeated three times in triplicate. *Po0.01; **Po0.001, one-way ANOVA.

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Table 1 | Identication of proteins with changed expression levels in iPSC-derived enriched neurons carrying GBA1 mutations.

Protein Gene symbol

Description N. peptides

Fold change

Q7Z6G3 NECAB2 N-terminal EF-hand calcium-binding protein 2

4 1.9

Q15173-2 PPP2R5B Serine/threonine-protein phosphatase 2 A 56 kDa regulatory subunit beta isoform

1 1.8

Q8NF91-4 SYNE1 Isoform 4 of Nesprin-1 1 1.7 Q8ND56-2 LSM14A Isoform 2 of Protein

LSM14 homolog A

5 1.7

2 1.4

Q9Y2Y0-2 ARL2BP Isoform 2 of ADP-ribosylation factor-like protein 2-binding protein

45 1.3

Q9BR76 CORO1B Coronin-1B 5 1.3 Q8NC44 FAM134A Protein FAM134A 1 2.1

Q9HBL7 C9orf46 Transmembrane proteinC9orf46

P62979 RPS27A Ubiquitin-40S ribosomal protein S27a

1 1.5

10 1.2

Summary of the identied proteins from quantitative mass spectrometry proteomics that were signicantly (Po0.05, Students t-test) up- or downregulated in puried neurons from GBA-PD iPSCs compared with control neurons.

Q9NUJ1 ABHD10 Abhydrolase domain-containing protein 10, mitochondrial

presence of GBA1 mutations. We next examined the vulnerability of mutant neurons to ER stress responses by application of either A23187 (05 mM), a calcium ionophore that induces ER stress by increasing the cytosolic calcium concentration, or rotenone (0200 nM), an inhibitor of complex I of the mitochondrial electron transport chain, which induces mitochondrial ROS production and elevation of intracellular levels of calcium36,37. Neuronal vulnerability was assessed by measuring cellular release of lactate dehydrogenase (LDH). Our results show that neurons carrying GBA1 mutations were more sensitive to A23187 (12.5 mM) and rotenone (50200 nM) than control cells (Fig. 9c and Supplementary Fig. 15a). Immunocytochemistry and cell counts of TH cells conrmed the increased vulnerability of mutant neurons (Fig. 9d and Supplementary Fig. 15b). Importantly, such a phenotype was rescued by gene correction. To gain insight into the functional role of NECAB2, we knocked down NECAB2 in iPSC-derived neurons using lentiviral-mediated short hairpin RNA (shRNA). The gene expression and protein levels of NECAB2 were reduced by B80% in knocked down iPSC-derived neurons (Fig. 9e,f). To further examine the functional role of NECAB2, iPSC-derived neurons were exposed to A23187 and neurotoxicity was assessed by immunostaining for TH. We found that knockdown of NECAB2 resulted in a statistically signicant increase of calcium-mediated neurotoxicity (Fig. 9g,h). In addition, calcium-imaging experiments revealed an increase in caffeine-induced cytosolic calcium levels after NECAB2 knockdown (Supplementary Fig. 14b).

DiscussionHeterozygous GBA1 mutations have been recognized as the strongest risk factor for PD and LB disorders1. In this study, we describe the generation and characterization of a collection of iPSC lines from GD patients, PD subjects harbouring heterozygous GBA1 mutations, as well as unaffected individuals.

The cells used for iPSC generation in this study were obtained from donors harbouring L444P, RecNcil and N370S alleles. L444P and N370S point mutations are the most frequent GD-causing alleles that have been linked to PD1,38, whereas the RecNcil is a complex GBA-pseudogene recombinant allele composed of three point mutations, namely the 6433 T-C transition (L444P), 6468 G-C transition (A456P) and 6482 G-C transition (silent mutation). To identify PD phenotypes related to GBA1 mutations, isogenic gene-corrected iPSC lines were generated for each mutation. iPSCs were efciently differentiated to mDA neurons without signicant differences and enriched by FACS.

For the rst time, we report that iPSC neuronal differentiation recapitulates the developmental changes observed during human brain development and that enriched iPSC-derived neurons show the polysialoganglioside content of adult human brain. To examine the shift of the ganglioside pattern along in vitro differentiation of human iPSCs, we utilized a radioactive labelling method and compared the ganglioside composition of broblasts, iPSCs and iPSC-derived neurons before and after FACS enrichment. Unsorted iPSC-derived neurons showed an increased complexity of the ganglioside pattern compared with broblasts and iPSCs; however, they still had a high content of GM3, which is usually found at high levels in other cell types. Interestingly, unsorted neurons showed high levels of GD3, which is mainly associated with proliferating neural stem cells16. By contrast, despite the presence of low quantities of GM3 and GM2, the gangliosidic pattern of puried neurons mirrored the composition of human brain with GM1, GD1a, GD1b, GT1b as major compounds and GD3, GD2 and GQ1b in less abundance39. These ndings show that using unsorted differentiated iPSCs, which are heterogeneous cell populations, signicantly limits the analysis of disease-related pathways. This underlines the importance of cell sorting to reduce background noise in such experimental approaches12,40. Taken together, these data indicate that enriched iPSC-derived neurons represent a valuable tool to study the role of GSLs in neurodegeneration as well as in sphingolipidoses and disorders of brain development. In this context, we also show that the glycosyltransferases involved in the catabolic pathways of GSLs (GCase, GBA2, b-galactosidase, b-hexosaminidase) are upregulated on human iPSC neuronal differentiation. Activity and protein levels of these enzymes were almost absent in undifferentiated iPSCs, whereas mature neurons expressed high levels of such enzymes. This may suggest a key role of these glycohydrolases in brain development and maintenance of GSL pattern and composition in the adult human brain.

GD patients with parkinsonism and PD patients carrying GBA1 mutations show classic PD pathology with widespread asyn inclusions41. The exact mechanism is unknown, but GBA1 mutants may contribute to the enhanced aggregation of a-syn via a direct interaction5. In support of such a gain of function hypothesis, post-mortem studies in PD and LB disease, along with GD or GBA1 mutation carriers, have shown the presence of GCase in LBs and Lewy neurites42. Recent data suggest that loss of GCase activity may also lead to a-syn accumulation as a secondary effect of lysosomal dysfunction25,27 and that the accumulation of Glc-Cer due to GCase deciency induces increased a-syn oligomerization, which in turn decreases GCase activity25,43. By utilizing iPSC-derived neurons carrying heterozygous GBA1 mutations and their isogenic gene-corrected controls, we show that GBA1 mutations are directly linked to the increase of a-syn levels. For the rst time, we report an increase of

Glc-Cer in iPSC-derived neurons from PD patients carrying heterozygous GBA1 mutations, which may in turn inuence a-syn levels and aggregation.

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0.6

1.4

Fura2 fluorescence

0.3

0.2

0.1

Fura2 fluorescence (F/F)

0.0

0.5

0.4

* * *

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* **

1.0

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CTRL L444P/wt L444P/wt GC L444P/L444P

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Relative LDH release

CTRL N370S/wt N370S/wt GC N370S/N370S

* *

* * *

0 0 1 2.5 5 0 1 2.5 5A23187 (M)

0 0 1 2.5 5A23187 (M)

A23187 (M)

(relative to untreated)

100

80 60 40 20

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TH+ cells

Relative mRNA levels

(fold to scrb)

* *

0.8

0.6

NECAB2

1 2

43 kDa

0.4

***

CTRL RecNcil/wt

L444P/L444P

0 N370S/wt GC

0.2

42 kDa

N370S/wt

N370S/N370S

0 scrb shRNA

NECAB2 shRNA

-Actin

scrb shRNA

TH+ cells

(relative to scrb)

100

80 60 40 20

* *

A23187 (1 M)

NECAB2 shRNA Hoechst/-TubIII/TH Hoechst/-TubIII/TH

RecNcil/wt

L444P/L444P

0 N370S/wt

N370/N370S

Figure 9 | Calcium homeostasis is dysregulated in GBA-PD and GD iPSC-derived neurons. (a) Cytosolic calcium levels were evaluated in iPSC-derived neurons at basal conditions by measuring the 340/380 nm ratio of Fura-2 acetoxymethyl ester. Data are represented as mean s.e.m.; experiments were

independently repeated three times in triplicate. *Po0.01; Students t-test. (Number of cells analysed: CTRL, n 58; RecNcil/wt, n 36; RecNcil/wt GC,

n 107; L444P/wt, n 82; L444P/wt GC, n 33; L444P/L444P, n 62). (b) Caffeine (10 mM) was applied to iPSC-derived neurons, and the peak

cytosolic calcium concentration was measured as a change in Fura-2 uorescence. Data are represented as mean s.e.m.; experiments were independently

repeated three times in triplicate. *Po0.01; **Po0.001, Students t-test. (Number of cells analysed: CTRL, n 58; RecNcil/wt, n 36; RecNcil/wt GC,

n 107; L444P/wt, n 82; L444P/wt GC, n 33; L444P/L444P, n 62). (c) iPSC-derived neurons were treated with A23187 (05 mM) for 4 h and

cytotoxicity was evaluated by released LDH activity. LDH release values were calculated as the percentage of untreated cells lysed by incubation with Triton X-100. Data are represented as mean s.e.m.; experiments were independently repeated four times in triplicate. *Po0.05, one-way ANOVA, control lines

versus mutated lines; dPo0.05, one-way ANOVA, mutated lines versus isogenic gene corrected controls. (d) Quantication of TH cells treated with A23187 (1 mM) for 4 h expressed as a percentage of DMSO-treated controls. Data are represented as mean s.d.; experiments were independently

repeated three times in triplicate. *Po0.01, one-way ANOVA. (e) Knockdown efciency of NECAB2 determined by qRT-PCR and normalized to scrambled non-targeting shRNA. Data are represented as mean s.d.; experiments were independently repeated three times in triplicate. ***Po0.0001, Students t-

test. (f) Representative western blot for NECAB2 showing knockdown efciency. 1, scrambled non-targeting shRNA; 2, NECAB2 shRNA. (g) Quantication of TH cells in GBA-PD and GD iPSC-derived neuronal cultures infected with scrambled non-targeting shRNA or NECAB2 shRNA lentivirus and treated with A23187 (1 mM for 4 h). Values were calculated as a percentage of scrambled shRNA-treated cells. Data are represented as mean s.e.m.;

experiments were independently repeated three times in triplicate. *Po0.01, Students t-test. (h) Representative images showing GBA-PD iPSC-derived DA neurons infected with scrambled non-targeting shRNA or NECAB2 shRNA lentivirus and treated with A23187 (1 mM for 4 h). Cells were stained for b-TubIII (green) and TH (red). Nuclei were counterstained with Hoechst (blue). (Bar, 20 mm.)

12 NATURE COMMUNICATIONS | 5:4028 | DOI: 10.1038/ncomms5028 | http://www.nature.com/naturecommunications

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Our data show that GBA1 mutations are linked to defects in the autophagic/lysosomal system characterized by an impairment of the autophagic ux with consequences for autophagosome clearance. Although we have observed an accumulation of lysosomes, the fusion between autophagosomes and lysosomes and the lysosomal hydrolytic function were impaired. This may explain the increased levels of a-syn in GBA1 mutant neurons and a-syn pathology in GBA-PD patients. Interestingly, the reduction in the glycohydrolase lysosomal activity in iPSC-derived neurons was accompanied by a reduction in the enzymatic activity of these enzymes in the CSFs of GBA-PD patients compared with control individuals. PD and parkinsonism have been described in many lysosomal storage diseases, suggesting that impairment of the endolysosomal compartment is strictly correlated with the onset of neuronal degeneration44. In this context, GBA1 mutations may alter the lysosomal function via several pathways. The intracellular lipid content strongly inuences autolysosome formation as well as a-syn aggregation15,45. In contrast, increased levels of a-syn may cause lysosomal dysfunction in GBA1 mutant neurons via a positive feedback loop25. Furthermore, our data in isogenic controls, along with previous reports46, suggest the existence of a crosstalk between different enzymes involved in sphingolipid metabolism. Interestingly, we found that GBA2 enzyme activity was signicantly decreased in iPSC-derived neurons harbouring GBA1 mutations. GBA2 is a non-lysosomal enzyme with glucosylceramidase function localized at the ER and plasma membrane47. It is encoded by the GBA2 gene and is highly expressed in the brain, testis and liver48. Notably, GBA2 mutations cause a form of autosomal-recessive cerebellar ataxia and have been recently linked to motor neuron defects in hereditary spastic paraplegia49,50. Mutant GCase in the ER leads to ER stress and induces the unfolded protein response in skin broblasts from GD patients and mutation carriers51. Since GBA2 is also located in the ER, such perturbations may affect its activity. Even though the mechanisms responsible for the observed impairment of GBA2 function remain unclear, our data show that the decrease in GBA2 enzymatic activity was reverted on ZFN-mediated GBA1 gene correction, suggesting a crosstalk between GCase and GBA2 activity. In favour of this hypothesis, a recent report shows that GBA2 activity depends on GCase, whereas GCase activity is independent of GBA2 (ref. 52). Further studies are needed to better elucidate such crosstalk and its role in disease onset and progression. GBA-PD and GD parental broblast lines showed an upregulation of the enzymatic activity of GBA2 and b-galactosidase compared with control cells.

Such discrepancy can be attributed to the different sphingolipid metabolism in broblasts and neurons. As described above, neurons have high glycohydrolase enzymatic activity and a unique GSL content and distribution. Alternatively, even though only low passage broblasts were used in this study, such ndings may also be due to the changes of the GSL pattern observed during subculture progression21. These data further support the importance of using iPSC-derived neurons as the ideal tool to model neurological diseases and in particular neurodegeneration associated with sphingolipidoses.

In this study, we have generated a proteome prole of differentiated iPSCs. This is the rst report of multiplexed quantitative proteomics being performed on a highly enriched neuronal fraction. As little as 15 mg for each cell line was used for quantitative proteomics using TMT labelling, leading to the identication of over 7,000 proteins from 12 LC-mass spectrometry runs. Such analysis enabled the identication of a protein, namely NECAB2, whose levels were increased in human iPSC-derived neurons harbouring mutations in the GBA1 gene. NECAB2 belongs to the family of neuronal calcium-binding

proteins containing an N-terminal EF-hand domain (NECAB). It is highly expressed in the striatopallidal structures where it interacts with the adenosine A2A receptor53,54. Interestingly, a recently published microarray data set for putative novel mDA neuron-related transcription factors and membrane-associated proteins has shown that NECAB1 is a marker for mDA neurons, and it is highly expressed in the dorsal ventral tegmental area and dorsal substantia nigra55. Adult mDA neurons are autonomous pacemakers and rely on the activation of L-type Ca(V)1.3 calcium channels, leading to elevated intracellular calcium concentrations56. In addition, mDA neurons lack signicant intrinsic calcium-buffering properties, which may explain their selective vulnerability to neurodegeneration57. A recent study demonstrates that the levels of calcium-binding proteins are reduced in cell bodies of neurons in areas selectively vulnerable to degeneration in PD. However, the surviving cells upregulate such proteins suggesting a compensatory mechanism58. Interestingly, we found increased basal levels of calcium and an increased calcium release from the ER on caffeine stimulation in GBA-PD and GD neurons. Such ndings are in line with previous observations in pharmacological models of neuronopathic GD showing that Glc-Cer accumulation leads to overactivation of the ryanodine receptor59.

The dysregulation of calcium homeostasis in diseased neurons was accompanied by an increased vulnerability to ER stress responses involving elevation of cytosolic calcium by the calcium ionophore A23187 and rotenone. Recently, mitochondrial impairment has been linked to GCase dysfunction providing a further rationale for the increased susceptibility to rotenone described in this study60. Importantly, increased neurotoxicity was observed in GBA1 mutant neurons exposed to A23187 on NECAB2 knockdown. These data demonstrate the calcium-buffering properties of NECAB2 and support the hypothesis that the increase of NECAB2 in neurons harbouring GBA1 mutations represents a compensatory mechanism in such vulnerable neurons.

In summary our studies showed that both GD and PD iPSC-derived neurons have increased levels of a-syn and Glc-Cer as well as complex changes in the autophagic/lysosomal system and calcium homeostasis, which may underlie the selective vulnerability of mDA neurons to degeneration in PD. Taken together, our results suggest possible targets for the development of disease-modifying drugs for patients with familial and idiopathic PD.

Methods

Sample collection. Informed consent was obtained from all patients involved in our study before cell donation or CSF collection. The Ethics Committee of the Medical Faculty and the University Hospital Tbingen previously approved this consent form. PD was diagnosed according to UK Brain Bank criteria61. The open-reading frame (11 exons) of the GBA1 gene was entirely sequenced in cases and controls. See Supplementary Table 3 for primer sequences. For CSF studies, control individuals underwent spinal taps for exclusion of vascular events or inammatory conditions. Any clinical sign of a neurodegenerative disorder led to exclusion from the study. Dermal broblasts were cultured in broblast medium, which consisted of DMEM (Biochrom) supplemented with 10% fetal calf serum, 1% penicillin/ streptomycin/glutamine, 1% nonessential amino acids, 1% sodium pyruvate (all PAA) and 0.5 mM b-mercaptoethanol (Invitrogen). GD broblasts were obtained from the Telethon Genetic Biobank Network (02FFF0491979, 02FFF0352006, 02FFF0201995, 02FFF0361999).

Generation of iPSCs and gene correction. Fibroblasts were infected with a mix of retroviruses encoding OCT4, SOX2, KLF4 and c-MYC. Five days after infection, cells were split into six-well plates pre-seeded with irradiated mouse embryonic broblasts (MEF, GlobalStem). Seven days after infection the medium was changed to human embryonic stem cell (hESC) medium consisting of KO-DMEM (Gibco) supplemented with 20% KO-Serum Replacement (Invitrogen), 2 mM Glutamax (Invitrogen), 50 mM b-mercaptoethanol (Invitrogen), non-essential amino acids

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5028

(Cambrex) and 10 ng ml 1 b-FGF (Peprotech). Individual colonies were isolated and clonally expanded on MEFs.

For gene correction, iPSCs were dissociated into single cells using Accutase (PAA). In total, 1.5 106 cells were diluted in hESC medium and collected by

centrifugation. Cells were transfected with 0.83 mg of each ZFN construct (Sigma), as well as 3.3 mg linearized targeting vector, using the Stem cell Nucleofection Kit and the Amaxa Nucleofector I (both Lonza), according to the manufacturers instructions (program B16). The transfected cells were replated onto MEF-coated plates in hESC medium supplemented with 10mM ROCK inhibitor Y27632 (Ascent

Scientic). From day 2 on, medium was changed on a daily basis. After colony formation (57 days), 250 mg ml 1 G418 (Biochrom) was added to select for homologous recombined clones. Resistant colonies were transferred to 12-well plates and expanded on MEFs for further characterization. To conrm gene correction, genomic DNA was extracted from the gene-targeted clones and sequenced at the corresponding locus. Final characterization was achieved by extraction of RNA using the RNeasy kit (Qiagen), transcription into cDNA and sequencing of the cDNA. See Supplementary Table 4 for primer sequences.

Karyotyping. Karyotyping was performed by G-banding to examine the integrity of the chromosomes by the Cytogenetics Laboratory at the University of Tbingen.

In vitro differentiation of human iPSCs. To test in vitro pluripotency, embryoid bodies (EBs) were generated from iPSCs by culturing the colonies in hESC medium in the absence of b-FGF. After 5 days, EBs were transferred to gelatin-coated plates and subsequently cultured for another 14 days in N2/B27 supplemented with10 mM SB431542 (Tocris) (ectoderm) or knockout DMEM (Invitrogen) supplemented with 20% FBS, 1 mM L-glutamine (L-glut), 1% non-essential amino acids,0.1 mM b-mercaptoethanol and 1% penicillin/streptomycin (endoderm/mesoderm). After 2 weeks, cultures were xed and stained as indicated.

Teratoma assay. Teratoma assays were performed by Applied Stem Cell (Menlo Park)62. In brief, 106 iPSCs were injected into the kidney capsule and testis of Fox Chase SCID-beige mice. Animals were killed B612 weeks after injection and haematoxylin-eosin (H&E) stainings were used to analyse harvested tissues.

Differentiation of iPSCs into midbrain DA neurons. iPSCs were differentiated into DA neurons following a oor plate-based mDA neuron induction protocol10, with minor modications. iPSC colonies were dissociated into a single-cell suspension using Accutase and 10 mM ROCK inhibitor Y-27632. Cells were collected by centrifugation and seeded at a density of 1.5 105 cells per well on

Matrigel (BD Biosciences)-coated 12-well plates. Differentiation was based on exposure to LDN193189 (100 nM, Stemgent) from days 011, SB431542 (10 mM,

Tocris) from days 05, SHH C25II (100 ng ml 1, R&D), purmorphamine (2 mM, EMD) and FGF8 (100 ng ml 1, Peprotech) from days 17 and CHIR99021 (CHIR;

3 mM, Stemgent) from days 313. Cells were grown for 11 days on Matrigel in knockout serum replacement medium (KSR) containing KO-DMEM, 15% knockout serum replacement, 2 mM L-glutamine and 10 mM b-mercaptoethanol.

KSR medium was gradually shifted to N2 medium starting on day 5 of differentiation. On day 11, media was changed to Neurobasal/B27/L-glut containing medium (NB/B27; Invitrogen) supplemented with CHIR (until day 13), BDNF (brain-derived neurotrophic factor, 20 ng ml 1; R&D), ascorbic acid(0.2 mM, Sigma), GDNF (glial cell line-derived neurotrophic factor, 20 ng ml 1; R&D), TGFb3 (transforming growth factor type b3, 1 ng ml 1; R&D), dibutyryl cAMP (dbcAMP, 0.5 mM; Sigma) and DAPT (10 mM; Tocris,) for 9 days. On day 21, cells were dissociated using Accutase and replated on dishes pre-coated with poly-L-ornithine (PLO; 15 mg ml 1) and laminin (1 mg ml 1) in differentiation medium (NB/B27 BDNF, ascorbic acid, GDNF, dbcAMP, TGFb3 and DAPT).

Quantitative Real-Time PCR. Total RNA was extracted with an RNeasy kit and quantitative RTPCR reaction was performed using the SYBR GREEN Kit (Qiagen). The expression level of each gene was normalized to endogenous HMBS levels. Fold-change in gene expression was calculated using the 2 DDCT method, based on biological reference samples and housekeeping genes for normalization.

All the results were obtained from the evaluation of three technical replicates of three independent experiments. See Supplementary Table 5 for primer sequences.

Flow cytometry. Cells were harvested and gently dissociated using TrypLE (Life Technologies) to obtain a single-cell suspension of 0.52 106 cells ml 1 and

resuspended in Ca2 /Mg2 free phosphate buffered saline (PBS; PAA Laboratories) with 2% FBS and distributed into 1.5 ml microcentrifuge tubes. Centrifugation steps were conducted using a refrigerated table microcentrifuge (Peqlab Perfect Spin 24 R) at 2,400 r.p.m. (542 g) for 35 min. Surface antigens were labelled by incubating with the primary antibodies for 30 min in the dark at room temperature. The following conjugated primary antibodies were used in the study: CD15-FITC, CD29-FITC, CD44-FITC, CD24-PE, CD184-APC. See Supplementary Table 6 for the list of antibodies. All antibodies were carefully titrated. The stained cells were analysed on a BD Accuri C6 benchtop cytometer

equipped with FL1 (533/30), FL2 (585/40) and FL4 (675/25) bandpass lters. Data were analysed and are presented using BD CFlow software version 1.0.227.4. Neuronal cell sorting was performed on a Beckman Coulter MoFlo instrument using a 100 mm nozzle and sheath pressure of 20 to 25 psi (ref. 11). Excluding debris and the minor amount of cells observed to be dead after harvesting, a primary gate based on forward and side scatter was set to select the overall population of interest. FSC-peak (height) versus FSC-integral (area) gating was applied to exclude doublets for cell sorting. Gates for detecting positive staining were set against unstained controls for surface antigens. Where appropriate, compensation was applied according to single-stained controls of the same cell type included in each individual experiment.

Electrophysiology. For whole-cell patch clamp experiments, neurons derived from iPSCs were plated on 35 mm petri-dishes (Greiner-bio-one). Recordings of cultured neurons were performed at room temperature using an Axopatch 200B amplier (Molecular devices). Data were digitized using a DigiData 1320A (Molecular devices). During electrophysiological recordings, cells were kept in extracellular solution containing (in mM): 142 NaCl, 8.09 KCl, 6 MgCl2, 1 CaCl2, 10 glucose, 10 HEPES and a nal pH of 7.4. Patch pipettes were pulled from borosilicate glass (Science products) using a Sutter P97 Puller (Sutter Instruments Company). Their resistance ranged from 3 to 5 MO. Patch pipettes were lled with intracellular solution containing (in mM): 5 KCl, 4 ATP-Mg, 10 phosphocreatine, 0.3 GTP-Na, 10 HEPES, 125 K-gluconate, 2 MgCl2, 10 EGTA with a nal pH of 7.2 and an osmolarity of 290 mosmol. The sampling rate was 50 kHz and data were low-pass ltered at10 kHz. Series resistance (o20 MO) was monitored during the experiment. Cells showing unstable series resistance or resting membrane potential were discarded.

HPLC analysis. Differentiated neuronal cultures at DIV65 of differentiation were washed once with HBSS and then treated with 56 mM KCl in HBSS for 30 min; control cultures were exposed to HBSS. Supernatants were collected, snap-frozen and stored at 80 C until analysis. Concentrations of DA were determined by

column liquid chromatography with electrochemical detection. The HPLC system (HTEC-500, Eicom Corp.) including a pulse-free microow pump, a degasser and an amperometric detector equipped with a glassy-carbon electrode, operating at

0.45 V versus an Ag/AgCl electrode, was used. Samples were injected by use of a CMA/200 Refrigerated Microsampler (CMA/Microdialysis). The chromatograms were recorded and integrated by a computerized data acquisition system (DataApex). DA was separated on a 150 2.1 i.d. mm column (CA5-ODS, Eicom

Corp.). The mobile phase consisted of 0.1 M phosphate buffer at pH 6.0, 0.13 mM EDTA, 2.3 mM sodium-1-octanesulfonate and 20% methanol. The detection limit (signal-to-noise ratio 3) for DA was estimated to 0.5 fmol in 15 ml (0.03 nM)

injected onto the column. Well to well variations were adjusted by concentration of cleared protein lysates.

Glc-Cer and Gal-Cer analysis by mass spectrometry. Quantitative analysis of Glc-Cer was carried out by LC-MS/MS. In brief, frozen cell pellets were suspended in 50 ml of water and extracted with 1 ml of a solution of acetonitrile: methanol: water (97:2:1, v v 1 v 1) at room temperature. Extracts were injected onto an

Atlantis HILIC silica column (Waters Corp.) for separation of Glc-Cer and Gal-Cer, and respective molecules were detected using MRM mode tandem mass spectrometry with an AB Sciex API-5000 mass spectrometer. Analytes were quantitated against standard curves using standards obtained from Matreya, LLC (Pleasant Gap).

Enzymatic activity assays. Total cell associated GCase activities were analysed using 4-methylumbelliferyl-b-D-glucopyranoside (MUB-Glc), b-galactosidase and b-hexosaminidase activities were assayed using the articial substrates 4-methylumbelliferyl b-D-galactopyranoside (MUB-Gal) and 4-methylumbelliferyl N-acetyl-b-D-glucosaminide (MUG) (all Glycosynth)30. Differentiated iPSCs at DIV65-70 were enriched by FACS and plated into Matrigel-coated 48-well assay plates in differentiation medium (500 ml per well). Media was changed every 3 days.

Seven days after plating, cells were washed twice with PBS, harvested and lysed in water containing complete protease inhibitor cocktails (Roche). Total cell protein was measured using the Micro BCA assay reagent (Pierce). Cell lysates were transferred to a 96-well microplate and enzymatic assays were performed in triplicates. The activities of b-galactosidase and b-hexosaminidase were measured in McIlvaine Buffer (0.1 M Citrate/0.2 M Phosphate) pH 5.2 using 0.5 mM MUBGal and 1 mM MUG, respectively. For CBE-sensitive GCase and GBA2 assays, the cells were pre-incubated for 30 min at room temperature in McIlvaine Buffer(0.1 M Citrate/0.2 M Phosphate) pH 6.0 containing 5 nM AMP-DNM N-(5-adamantane-1-yl-methoxy-pentyl)-deoxynojirimycin)24 and 1 mM CBE (conduritol B epoxide, Calbiochem), respectively. GCase and GBA2 activities were also determined using 150 nM AMP-DNJ (Miglustat, Zavesca) as an inhibitor (no signicant differences were found compared with AMP-DNM). No enzymatic activity was detected after the concomitant use of CBE and AMP-DNM or AMPDNJ. After incubation with the inhibitors, MUB-Glc was added at a nal concentration of 3 mM. The reaction mixtures were incubated at 37 C under gentle shaking. The uorescence was recorded after transferring 20 ml of the

14 NATURE COMMUNICATIONS | 5:4028 | DOI: 10.1038/ncomms5028 | http://www.nature.com/naturecommunications

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5028 ARTICLE

reaction mixtures to a microplate and adding 180 ml of 0.25 M glycine, pH 10.7. Data were calculated as pmoles of converted substrate mg 1 of cell protein h 1.

Sphingolipid analyses. Fibroblasts, undifferentiated iPSCs, differentiated iPSCs, sorted neurons and granule cells obtained from the cerebellum of 8-day-old Harlan SpragueDawley rats at DIV 8 were plated on poly-L-ornithine (PLO)/laminin-coated 48 wells. Two days (broblasts, undifferentiated iPSCs and primary granule cells, unsorted differentiated iPSCs) or seven days (sorted neurons) after plating, cells were incubated with 3 10 8 [1-3H]-

D-erythro-C18/C20-sphingosine in the appropriate culture medium (2-h pulse followed by a 48-h chase). Fresh brains from three 2-month-old C57BL/6j mice and three Harlan SpragueDawley rats were weighted, minced with a razor blade, homogenized in iced water (500 mg of fresh tissue ml 1), sonicated and then lyophilized. Lipids were extracted from cells and brains with chloroform/methanol/water in the ratio 2:1:0.1 by volume and subjected to a two-phase partitioning. Radioactive gangliosides were separated by mono- or two-dimensional HPTLC carried out with the solvent systems chloroform/methanol/0.2% aqueous CaCl2, 50: 42: 11 or 55: 45: 1063. Brain endogenous gangliosides were recognized using Ehrlichs reagent64. Radioactive lipids were identied and quantied with a Beta-Imager 2000 instrument (Biospace) using an acquisition time of about 48 h. The use of animal models was approved by the Italian Government (D.M. 33/2004).

Immunouorescence. Cells were xed in 4% paraformaldehyde for 10 min, rinsed with PBS and blocked by 10% normal goat or donkey serum in PBST (PBS 0.1%

TritonX-100) for 60 min62. Alkaline phosphatase staining was performed using the Alkaline Phosphatase Kit (Millipore), according to the manufacturers protocol. For LC3, LAMP1 and LAMP2 analysis, cells were co-stained for b-TubIII and TH, and images were acquired with a Zeiss LSM 510 (Carl Zeiss) confocal microscope using a 63 1.4NA plan-apochromat oil objective. Images were processed in

ImageJ (NIH) and brightness/contrast was adjusted equally. The particle size and number in b-TubIII and TH cells were quantied with the analyse particles

plug-in in ImageJ (NIH). Quantication was carried out on at least 50 cells per condition from independent experiments. Co-localization of LC3/LAMP1 in TH neurons was quantied in threshold images with the JACoP plug-in of the ImageJ software. See Supplementary Table 6 for the list of antibodies used.

Protein extraction and western blotting. Proteins were extracted using RIPA protein extraction buffer (50 mM Tris pH 8, 150 mM NaCl, 0.5% natriumdeoxycholate, 1% NP40, 0.1% SDS) containing protease and phosphatase inhibitors (Roche) on ice. In total, 1530 mg of the protein lysate was loaded on a 412% Bis-

Tris Gel (NuPAGE, Invitrogen) and transferred on a PVDF membrane (Millipore). Blots were incubated with primary antibodies overnight at 4 C on a shaker platform and were then probed with anti-mouse (Dianova), anti-rabbit (Dako) or anti-goat (JacksonLab) IgG-HRP (H L) secondary antibody (all 1:10.000) for 1 h at

room temperature. Visualization was done by using Amersham ECL Western Blotting Detection Reagent (GE Healthcare). Densitometry analyses on the immunoblots from multiple experiments were performed by ImageJ software. See Supplementary Table 6 for the list of antibodies used. Full-length images of immunoblots are shown in Supplementary Figs 1618.

Autophagy studies. Where indicated, cells were treated with NH4Cl (20 mM) and leupeptin (200 mM) (EMD, Millipore) for 4 h and then xed or lysed for western blot analysis. LC3-II levels were quantied by densitometry and normalized for b-actin. LC3 ux was quantied by dividing levels of LC3-II after lysosomal inhibitor treatment for 4 h by the level of LC3-II without treatment.

Calcium imaging. Cells were loaded with Fura-2 acetoxymethyl ester (Sigma) for 30 min at RT in recording medium (NaCl 140 mM, KCl 4 mM, CaCl2 2 mM, MgCl2 1 mM, Glucose 4 mM, HEPES 10 mM). KCl (60 mM) and caffeine (10 mM, Sigma) were prepared in recording medium. For baseline recordings, scans were acquired every 10 s for 5 min. Fluorescence measurements were obtained on an epiuorescence inverted microscope (Zeiss Axio Observer). All imaging data were collected and analysed using AxioVision (rel. 4.8) software.

Cytotoxicity assays. After 6365 days in vitro, cells were plated on PLO/laminin-coated 96-well plates at a density of 30.000 cells per well. Two days later, cells were incubated with A23187 (1 to 5 mM, Tocris), or rotenone (50 to 200 nM, Sigma) in

N2 medium for 4 h and 24 h, respectively. Dimethyl sulfoxide (DMSO) was used as vehicle and control conditions. The LDH assay (Promega) was performed as per the manufacturers instructions. For knockdown experiments, cytotoxicity assays were performed 72 h after non-targeting shRNA or NECAB2 shRNA lentiviral infection.

Proteomic analysis. Cells were resuspended in 100 ml lysis buffer (8 M urea, 5 mM DTT, 50 mM HEPES pH 8.5, 1 Roche Protease Inhibitors, 1 Roche Phos

phoStop phosphatase inhibitors) and cell debris was removed by centrifugation (10 min, 9,000 g). The claried lysate was allowed to incubate at room temperature

for 30 min for reduction of disulphide bonds. Cysteines were then alkylated with 15 mM iodoacetamide (45 min, RT, dark). Excess iodoacetamide was quenched with DTT (15 min, RT, dark). The proteins were precipitated using methanol-chloroform and then resuspended in a small volume of 8 M urea, 50 mM HEPES pH 8.5. Before digestion, they were diluted to 1 M urea, 50 mM HEPES pH 8.5. The protein concentrations were measured using the BCA assay (Pierce). Samples were digested overnight at 37 C with trypsin, which was added at a 1:100 proteaseto-protein ratio. This reaction was subsequently quenched with 1% formic acid, the sample desalted using C18 solid-phase extraction (Sep-Pak, Waters) and then dried by vacuum-centrifugation. To perform tandem mass tag labelling, desalted peptides were resuspended in 35 ml of 100 mM HEPES pH 8.5 and 8 ml of acetonitrile. TMT reagents (0.8 mg) were dissolved in anhydrous acetonitrile (40 ml) of which 4 ml was added to the peptides. The mixture was incubated at room temperature for 1 h. The labelling reaction was then quenched by the addition of hydroxylamine to a nal concentration of 0.3% (v/v). Each of the TMT-labelled samples were combined at a 1:1:1:1:1:1:1 ratio, dried, desalted and fractionated using high pH reversed-phase (HPRP). Following fractionating the samples were acidied to 1% formic acid (v/v), dried, desalted using StageTips, dried and then reconstituted in 5% acetonitrile and 5% formic acid. LC-MS/MS was performed on an LTQ Orbitrap Elite mass spectrometer (Thermo-Fisher Scientic)65. The total LC-MS run length for each sample was 180 min consisting of a 150 min gradient from 6 to 33% acetonitrile in0.125% formic acid. A recently developed MS3 method was used to overcome the interference problem in acquisition of TMT data66.

Data analysis. Mass spectrometry data were processed using an in-house software pipeline67. Raw les were converted to mzXML les and searched using the Sequest algorithm68 against a composite database containing all sequences from the human International Protein Index (IPI) database (version 3.6) in forward and reverse orientations. Database searching matched MS/MS spectra with fully tryptic peptides from this composite database with a precursor ion tolerance of 20 p.p.m. and a product ion tolerance of 1 Da. Carbamidomethylation of cysteine residues( 57.02146 Da) and TMT tags on peptide N-termini ( 229.162932 Da) were set

as static modications. Oxidation of methionines ( 15.99492 Da) was set as a

variable modication. Linear discriminant analysis was used to lter peptide spectral matches to a 1% FDR, as described previously67. For analysis of TMT data, the reporter ion counts of the peptide spectral matches for each protein were summed, as described previously66,69. Statistical signicance was calculated by t-test analysis adjusted using the Benjamini and Hochberg method to control for false-discovery rate and ltered for proteins whose levels changed by at least1.2-fold. Expression changes were considered signicantly up- or downregulated when the P-value was r0.05.

Cerebrospinal uid enzyme assays. Total protein amounts were measured using a DC protein assay in triplicate according to the manufacturers instructions (Biorad). b-Glucosidase activities were analysed by using 4-methylumbelliferyl-b-

D-glucopyranoside (MUB-Glc). b-Galactosidase and b-hexosaminidase activities were assayed by using the articial substrates 4-Methylumbelliferyl b-D-galactopyranoside (MUB-Gal) and 4-Methylumbelliferyl N-acetyl-b-D-glucosaminide (MUG). b-Mannosidase, a-mannosidase and a-fucosidase activities were determined using 4-methylumbelliferyl-b-D-mannopyranoside (MUB-b-Man), 4-methylumbelliferyl-a-D-mannopyranoside (MUB-a-Man) and 4-methylumbelliferyl-a-L-fucopyranoside, respectively (MUB-Fuc) (all Glycosynth). Same amounts of CSF protein was transferred to a 96-well microplate and enzymatic assays performed in triplicate. Determination of the b-galactosidase, b-hexosaminidase, b-mannosidase, a-mannosidase and a-fucosidase activities were performed in McIlvaine Buffer (0.1 M Citrate/0.2 M Phosphate) pH 5.2 using0.5 mM MUB-Gal and 3 mM MUG, 3 mM MUB-b-Man, 3 mM MUB-a-Man and 3 mM MUB- Fuc, respectively. For CBE-sensitive b-glucosidase and GBA2 assays, cells were pre-incubated for 30 min at room temperature in McIlvaine Buffer(0.1 M Citrate/0.2 M Phosphate) pH 6.0 with 5 nM AMP-DNM and 1 mM CBE, respectively. The activity of the CBE-sensitive b-glucosidase was determined in the presence of 0.5% Triton X-100. After 30 min, MUB-Glc was added at a nal concentration of 6 mM. After 30 to 180 min of incubation at 450 r.p.m. stirring and 37 C, uorescence was detected by transferring 20 ml of the reaction mixtures to a microplate and adding 180 ml of 0.25 M glycine, pH 10.7.

Lentivirus-mediated shRNA. The veried Mission lentiviral plasmids encoding turbo GFP (SHC003), non-targeting shRNA (SHC016) and NECAB2 shRNA (TRCN0000056234, TRCN000056236) in pLKO.1-puro vector backbone were purchased from Sigma-Aldrich. Lentiviral stocks were prepared by calcium phosphate transfection and the packaging vectors pLP1, pLP2 and pLP/VSVG using the ViraPower HiPerform T-REx Gateway Expression System (Invitrogen). In brief, 80% conuent HEK 293T cells were transfected with 3 mg of plasmid DNA and9 mg of the ViraPower packaging mix using the TransIT-LT1 Transfection Reagent (Mirus). The following day, the medium was replaced with fresh culture medium and cells were incubated at 37 C in a humied 5% CO2 incubator. After 48 h, medium was changed and the virus-containing supernatant was harvested, ltered through a 0.45 mm PVDF lter membrane (Millipore) and concentrated by centrifugation in a Vivaspin column (Sartorius Stedim Biotech) at 4,000 r.p.m. at 4 C

NATURE COMMUNICATIONS | 5:4028 | DOI: 10.1038/ncomms5028 | http://www.nature.com/naturecommunications

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5028

for 15 min. Supernatants were collected over 48 to 60 h and titred used at an MOI of ve to infect cells.

Statistical analysis. The Statistical Package GraphPad Prism version 4.00 (GraphPad Software, San Diego California USA) was used to analyse the data. Statistical testing involved a two-tailed paired or unpaired Students t-test, One-way Anova with Bonferronis multiple comparison test or MannWhitney U test, as appropriate. Data are expressed as mean s.e.m. (or s.d.) as indicated.

Signicance was considered for Po0.05.

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16 NATURE COMMUNICATIONS | 5:4028 | DOI: 10.1038/ncomms5028 | http://www.nature.com/naturecommunications

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5028 ARTICLE

Acknowledgements

We thank Daniela M. Arduino for helping with the autophagy studies; Ann-Kathrin Hauser for the sequencing analysis; Dr Marie Follo and Klaus Geiger of the Core Facility, University Medical Center Freiburg for cell sorting; Lindsay Sweet, Genzyme Corporation for LC-MS measurements. Gauchers disease samples were obtained from the Cell Line and DNA Biobank from Patients affected by Genetic Diseases (G. Gaslini Institute)Telethon Genetic Biobank Network (Project No. GTB07001). We thank the PRIDE team for assistance in the submission of the mass spectrometry proteomics data. This work was supported by the Marie Curie Career Integration Grant MC CIG304108 (M.D.), Fritz Thyssen Foundation grant 10.13.1.155 (M.D.), German Research Foundation (D.F.G.) grant KR2119/8-1 (T.G.), Eva-Theers-Stiftung (T.G. and M.D.) and by German Research Foundation (D.F.G.) grant PR1132/3-1 (J.P.). C.H. and M.D. were each supported by an Alexander von Humboldt-Foundation Postdoctoral Fellowship.

Author contributions

D.C.S. performed the experiments and analysed the data; M.A. performed the experiments and analysed the data, participated in writing the manuscript; F.E.M. performed the MS experiments and analysed the data, participated in writing the manuscript; C.J.H. performed the FACS experiments and analysed the data; F.M. performed experiments; B.S. performed experiments; S.P.S. provided LC/MS data; M.V. performed experiments; S.H. performed experiments; L.K.S. performed experiments; U.H. performed experiments; D.B. oversaw patient sample collection; L.S. provided LC/MS data; J.H. designed calcium imaging experiments; J.P. performed experiments, designed the FACS experiments and participated in writing the manuscript; S.P.G. carried out mass spectrometry; S.S. participated in designing experiments and writing the manuscript; T.G. supervised part of the study and wrote the manuscript; M.D. designed and coordinated the study, performed experiments, analysed the data and wrote the manuscript.

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Additional information

Accession codes The mass spectrometry proteomics data have been deposited in ProteomeXchange via the PRIDE partner repository70 under the accession code PXD000866.

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

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Competing nancial interests: The authors declare no conict of interest.

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How to cite this article: Schndorf, D. C. et al. iPSC-derived neurons from GBA1-associated Parkinsons disease patients show autophagic defects and impaired calcium homeostasis. Nat. Commun. 5:4028 doi: 10.1038/ncomms5028 (2014).

NATURE COMMUNICATIONS | 5:4028 | DOI: 10.1038/ncomms5028 | http://www.nature.com/naturecommunications

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Abstract

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Mutations in the acid β-glucocerebrosidase (GBA1) gene, responsible for the lysosomal storage disorder Gaucher's disease (GD), are the strongest genetic risk factor for Parkinson's disease (PD) known to date. Here we generate induced pluripotent stem cells from subjects with GD and PD harbouring GBA1 mutations, and differentiate them into midbrain dopaminergic neurons followed by enrichment using fluorescence-activated cell sorting. Neurons show a reduction in glucocerebrosidase activity and protein levels, increase in glucosylceramide and α-synuclein levels as well as autophagic and lysosomal defects. Quantitative proteomic profiling reveals an increase of the neuronal calcium-binding protein 2 (NECAB2) in diseased neurons. Mutant neurons show a dysregulation of calcium homeostasis and increased vulnerability to stress responses involving elevation of cytosolic calcium. Importantly, correction of the mutations rescues such pathological phenotypes. These findings provide evidence for a link between GBA1 mutations and complex changes in the autophagic/lysosomal system and intracellular calcium homeostasis, which underlie vulnerability to neurodegeneration.

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Title

iPSC-derived neurons from GBA1-associated Parkinson's disease patients show autophagic defects and impaired calcium homeostasis

Author

Schöndorf, David C; Aureli, Massimo; Mcallister, Fiona E; Hindley, Christopher J; Mayer, Florian; Schmid, Benjamin; Sardi, S Pablo; Valsecchi, Manuela; Hoffmann, Susanna; Schwarz, Lukas Kristoffer; Hedrich, Ulrike; Berg, Daniela; Shihabuddin, Lamya S; Hu, Jing; Pruszak, Jan; Gygi, Steven P; Sonnino, Sandro; Gasser, Thomas; Deleidi, Michela

Pages

4028

Publication year

2014

Publication date

Jun 2014

Publisher

Nature Publishing Group

e-ISSN

20411723

Source type

Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms5028

ProQuest document ID

1532971965

iPSC-derived neurons from GBA1-associated Parkinson's disease patients show autophagic defects and impaired calcium homeostasis

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