Stem Cell Reports Article Patient-Specific iPSC-Derived Astrocytes Contribute to Non-Cell-Autonomous Neurodegeneration in Parkinson’s Disease Angelique di Domenico,1,2,14 Giulia Carola,1,2,14 Carles Calatayud,1,2,3 Meritxell Pons-Espinal,1,2 Juan Pablo Muñoz,4 Yvonne Richaud-Patin,3,5 Irene Fernandez-Carasa,1,2 Marta Gut,6 Armida Faella,1,2 Janani Parameswaran,1,2 Jordi Soriano,7,8 Isidro Ferrer,2,9 Eduardo Tolosa,9,10 Antonio Zorzano,4 Ana Maria Cuervo,11 Angel Raya,3,5,12,15,* and Antonella Consiglio1,2,13,15,* 1Department of Pathology and Experimental Therapeutics, Bellvitge University Hospital-IDIBELL, Hospitalet de Llobregat, Barcelona 08908, Spain 2Institute of Biomedicine of the University of Barcelona (IBUB), Barcelona 08028, Spain 3Center of Regenerative Medicine in Barcelona (CMRB), Hospital Duran i Reynals, Hospitalet de Llobregat, Barcelona 08908, Spain 4Institute for Research in Biomedicine (IRB), Carrer Baldiri Reixac 10, Barcelona 08028, Spain 5Centre for Networked Biomedical Research on Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN), Madrid 28029, Spain 6Centre Nacional d’Anàlisi Genòmica (CNAG-CRG), Parc Cientı́fic de Barcelona, Barcelona 08028, Spain 7Departament de Fı́sica de la Matèria Condensada, Universitat de Barcelona, Barcelona 08028, Spain 8Universitat de Barcelona Institute of Complex Systems (UBICS), Barcelona 08028, Spain 9Centre for Networked Biomedical Research on Neurodegenerative Diseases (CIBERNED), Madrid 28049, Spain 10Department of Neurology, Hospital Clinic de Barcelona, Institut d’Investigacions Biomédiques August Pi i Sunyer (IDIBAPS), University of Barcelona (UB), Barcelona 08036, Spain 11Albert Einstein College of Medicine, Bronx, NY 10461, USA 12Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona 08010, Spain 13Department of Molecular and Translational Medicine, University of Brescia, Brescia 25121, Italy 14Co-first author 15Co-senior author *Correspondence: araya@cmrb.eu (A.R.), consiglio@ub.edu (A.C.) https://doi.org/10.1016/j.stemcr.2018.12.011 SUMMARY Parkinson’s disease (PD) is associated with the degeneration of ventral midbrain dopaminergic neurons (vmDAns) and the accumulation of toxic a-synuclein. A non-cell-autonomous contribution, in particular of astrocytes, during PD pathogenesis has been suggested by observational studies, but remains to be experimentally tested. Here, we generated induced pluripotent stem cell-derived astrocytes and neurons from familial mutant LRRK2 G2019S PD patients and healthy individuals. Upon co-culture on top of PD astrocytes, control vmDAns displayed morphological signs of neurodegeneration and abnormal, astrocyte-derived a-synuclein accumulation. Conversely, control astrocytes partially prevented the appearance of disease-related phenotypes in PD vmDAns. We additionally identified dysfunc- tional chaperone-mediated autophagy (CMA), impaired macroautophagy, and progressive a-synuclein accumulation in PD astrocytes. Finally, chemical enhancement of CMA protected PD astrocytes and vmDAns via the clearance of a-synuclein accumulation. Our find- ings unveil a crucial non-cell-autonomous contribution of astrocytes during PD pathogenesis, and open the path to exploring novel ther- apeutic strategies aimed at blocking the pathogenic cross talk between neurons and glial cells. INTRODUCTION involved in several cellular functions, including autophagy (Cookson, 2016; Orenstein et al., 2013; Su et al., 2015). Parkinson’s disease (PD) is the second most prevalent neuro- Correlations between mutant LRRK2 and several path- degenerative disease after Alzheimer’s disease, affecting 7 ogenic mechanisms linked to PD progression have been to 10 million people worldwide (Global Burden of Disease previously reported, including alterations in autophagy Study Collaborators, 2015). PD is characterized by a signifi- and consequent accumulation of a-syn (Cookson, cant loss of ventral midbrain dopaminergic neurons 2017). Neuronal mutant LRRK2 toxicity was found to (vmDAns) in the substantia nigra pars compacta. The pres- depend on LRRK2 levels and a-syn accumulation as ence of intracellular protein aggregates of a-synuclein opposed to kinase activity or inclusion bodies in induced (a-syn) in the surviving vmDAns has been reported in post- pluripotent stem cell (iPSC)-derived neurons (Skibinski mortem PD tissue (Greenamyre and Hastings, 2004). Most et al., 2014). During PD pathogenesis, mutant LRRK2 PD cases are sporadic (85%), but familial mutations are was found to directly bind LAMP2A, the receptor respon- accountable for 15% of patients (Lill, 2016). Mutations in sible for chaperone-mediated autophagy (CMA) nor- the gene encoding leucine-rich repeat kinase 2 (LRRK2), mally used by both LRRK2 and a-syn for degradation causing an autosomal dominant form of PD, account for (Orenstein et al., 2013). This binding blocks the proper 5% of familial cases and 2% of sporadic cases (Gilks et al., functioning of the CMA translocation complex, resulting 2005; Nichols et al., 2005). LRRK2 is a highly complex in defective CMA, leading to the accumulation of a-syn protein with both GTPase and protein kinase domains and cell death. Stem Cell Reports j Vol. 12 j 213–229 j February 12, 2019 j ª 2018 The Authors. 213 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). iPSCs derived from healthy individuals and patients have Table 1. Summary of the Healthy Controls and Patients Used in accelerated advances in developing genuinely human This Study experimental models of diseases (Zeltner and Studer, Age at Isogenic 2015). In the case of PD, previous studies by our groups Code Status Sex Biopsy Mutation Control and others have generated iPSCs from patients with PD SP09 control M 66 no associated with LRRK2 mutations, and described the appearance of disease-specific phenotypes in iPSC-derived SP11 control M 52 no neurons, including impaired axonal outgrowth and defi- SP17 control F 48 no cient autophagic vacuole clearance (Heman-Ackah et al., 2017; Nguyen et al., 2011; Sanchez-Danes et al., 2012; SP06 Parkinson’s M 44 LRRK2 disease G2019S Skibinski et al., 2014). Moreover, dopaminergic (DA) neu- rons from LRRK2-mutant iPSCs displayed alterations in SP12 Parkinson’s F 63 LRRK2 CMA that were, at least in part, responsible for the disease G2019S abnormal accumulation of a-syn observed in these cells, SP13 Parkinson’s F 68 LRRK2 LRRK2 G2019S which predated any morphological signs of neurodegener- disease G2019S corrected ation (Orenstein et al., 2013). Studies investigating PD pathogenesis have been mostly of CMA, we were able to prevent the appearance of focused on the mechanisms underlying vmDAn degenera- PD-related phenotypes in patients’ astrocytes. Overall, tion and death. However, there is evidence of astrocytes our findings represent a direct indication that dysfunc- accumulating a-syn during PD through postmortem anal- tional astrocytes play a crucial role during PD pathogen- ysis (Braak et al., 2007; Wakabayashi et al., 2000). Altered esis and may have broad implications for future a-syn released by axon terminals in the surrounding synap- intervention in early stages of PD. ses is taken up by astrocytes, supporting the hypothesis of the spread of a-syn through neuron-astrocyte interactions (Braak et al., 2007; Lee et al., 2010). Overexpression of RESULTS mutant SNCA in primary astrocytes altered their normal functioning and impaired proper blood-brain barrier con- Generation and Characterization of iPSC-Derived trol and glutamate homeostasis, and eventually resulted Patient-Specific Astrocytes in a significant loss of vmDAns (Gu et al., 2010). In a study To establish an in vitro human cellular model for dissecting using human brain homogenates from PD patients with the interplay between neurons and astrocytes in PD, we Lewy bodies, a-syn was found to be taken up and spread first derived astrocyte-like cells from iPSCs, using a previ- from astrocytes to neurons, leading to neuronal death ously published protocol (Serio et al., 2013). Specifically, (Cavaliere et al., 2017). As a result, a role of astrocyte astrocyte cultures were successfully established from iPSC dysfunction in PD pathogenesis is emerging (Booth et al., lines from three PD patients carrying the G2019S mutation 2017). in the LRRK2 gene (PD SP06, PD SP12, and PD SP13) and In the present study, we generated patient-specific iPSC- two healthy age-matched controls (Ctrl SP09 and Ctrl derived astrocytes and vmDAns from PD patients with the SP17) (see Table 1 and Tables S1 and S2 for a summary of LRRK2 G2019S mutation and healthy individuals. We the iPSC lines used, and Experimental Procedures for de- consistently generated a population of human vmDAns tails on their origin). Immunocytochemistry (ICC) detec- in vitro that expressed postmitotic dopaminergic markers tion of key astrocyte markers showed robust expression and fired action potentials. Subsequently, we co-cultured of CD44, glial fibrillary acidic protein (GFAP), and S100 healthy iPSC-derived vmDAns with iPSC-derived astro- calcium-binding protein b (S100b), as well as of the excit- cytes expressing the mutated form of LRRK2 associated atory amino acid transporter 2 (EAAT2, also known as with PD. In co-culture experiments, we detected a signifi- GLT1), in all human iPSC-derived astrocytes (Figure 1A). cant decrease in the number of vmDAns in the presence of No evident contamination by other cell types, such as LRRK2-PD astrocytes, which correlated with the abnormal neurons or oligodendroglial progenitors, was found as accumulation of astrocyte-derived a-syn. Conversely, con- assessed by immunostaining with anti-MAP2 or NG2 trol astrocytes were able to partially rescue disease-related antibody, respectively (Figures 1A and 1B). The astrocytic phenotypes in LRRK2-PD vmDAns during co-culture. A identity was further confirmed by quantitative RT-PCR of more in-depth investigation revealed impaired autopha- GFAP and additional astrocyte-specific genes, including gic machinery, as well as progressive accumulation of MLC1, SOX9, ALDH1L1, AQP4, DIO2, and SLC4A4, which endogenous a-syn in PD astrocytes, compared with con- were expressed in Ctrl and PD astrocytes, and in human trol astrocytes. By treating the cells with an activator primary astrocytes, but not in iPSCs (Figures S1A–S1C). 214 Stem Cell Reports j Vol. 12 j 213–229 j February 12, 2019 Figure 1. iPSC-Derived Patient-Specific Astrocyte Generation and Characterization (A) Representative ICC images of astrocytes from two Ctrl iPSC lines (Ctrl SP09 and Ctrl SP17) and three PD iPSC lines (PD SP12, PD SP13, and PD SP06) staining positive for CD44 (astrocytic precursor marker), GFAP (general astrocytes), S100b (mature astrocytes), and GLT1 (excitatory amino acid transporter 2), and negative for TUJ1 (immature neurons), MAP2 (mature neurons), and NG2 (oligodendrocytes) expression. Number of independent astrocyte lines generated from iPSC per patient = 3. Number of independent experiments per astrocyte line generated = 3. Scale bar, 100 mm. (B) Astrocyte cultures are composed of approximately 95% astrocytes, 4% neurons, and 1% other (n = 3). (C) Heatmap showing sample similarities taking the rlog transformed data and Euclidean distances between samples. iPSC-derived astrocyte (Ctrl SP09 and PD SP12) samples cluster closer to the human primary astrocytes than the corresponding iPSC group (n = 2). (D) Functional ATP production luminescence (counts normalized to controls) in both Ctrl (SP09 and SP17) and PD (SP13, SP12, and SP06) astrocytes (n = 3). Data are expressed as mean ± SEM, unpaired two-tailed Student’s t test. To validate astrocyte cell type identity, we also tested both Ctrl and PD iPSC-derived astrocytes was closer to the expression of astrocyte-specific genes revealed by the that of human primary astrocytes than to that of their Human Astrocyte RNA-Seq database (www.brainrnaseq. corresponding iPSC line, thus confirming their identity org/) in our iPSC-derived astrocytes, through RNA (Figure 1C). We next determined the functional matura- sequencing. We found that the transcriptomic profile of tion of iPSC-derived astrocytes by confirming their capacity Stem Cell Reports j Vol. 12 j 213–229 j February 12, 2019 215 Figure 2. vmDAn Generation, Characterization, and Co-culture Setup (A and B) Representative immunofluorescence images of Ctrl SP11 vmDAn after (A) 35 or (B) 50 days of neuronal differentiation. iPSC-derived neural cultures express markers specific for neurons (MAP2), DAns (TH), and midbrain-type DAns (FOXA2 and GIRK2). Scale bar, 20 mm. (C) Percentage of differentiated cells that stained positive for TH and double positive for TH and FOXA2 and TH and GIRK2 after 50 days of differentiation (n = 4). (legend continued on next page) 216 Stem Cell Reports j Vol. 12 j 213–229 j February 12, 2019 to produce ATP (Figure 1D) and propagate intercellular neuronal network comprising MAP2-positive cells was Ca2+ waves (Figures S1D–S1H). Indeed, by using the formed upon co-culture (Figure 2H). Fluo-4 AM Ca2+ indicator, recordings from Ctrl and PD astrocytes showed a heterogeneous pattern of Ca2+ fluctua- Control vmDAns Show Morphological Signs of tions under basal conditions, revealing their func- Neurodegeneration when Co-cultured with PD tionality. All together, these data support the successful Astrocytes generation of highly pure populations of functionally We then examined the effects of astrocytes expressing equivalent astrocyte-like cells, which represent a contin- mutated LRRK2 on the survival of Ctrl iPSC-derived uous source of human Ctrl and PD astrocytes for subse- vmDAns upon co-culture (Figure 3A). After 2 weeks of quent analyses. co-culture with PD astrocytes, Ctrl vmDAns displayed morphological alterations, including shortened neurites, Generation of vmDAns and Setup of Neuron-Astrocyte and significantly decreased cell survival compared with Co-culture System co-cultures with Ctrl astrocytes (Figures S2A–S2C). These To investigate whether astrocytes contribute to PD patho- alterations were much more evident after 4 weeks of genesis, we established a co-culture system of iPSC-derived co-culture, when Ctrl vmDAns cultured on top of PD astrocytes and iPSC-derived vmDAns. PD vmDAns were astrocytes showed extensive signs of neurodegenerative generated from iPSC lines from two PD patients carrying phenotypes (fewer and shorter neurites, and abundance the G2019S mutation in the LRRK2 gene (iPSC lines PD of beaded-necklace neurites) and severely compromised SP12 and PD SP13), whereas Ctrl vmDAns were obtained cell survival (less than 25% of control), compared with from two independent iPSC clones (Ctrl SP11 and Ctrl co-cultures with control astrocytes (Figures 3B–3F). The SP11#4) from a healthy age-matched control (see Tables fact that the numbers of vmDAns did not change signifi- 1, S1, and S2 for a summary of the iPSC lines used). To cantly during the co-culture with control astrocytes, but differentiate iPSCs toward vmDAns, we used a combina- progressively declined when co-cultured with PD astro- tion of two previously published (Chambers et al., 2009; cytes, strongly suggests that vmDAns were lost under the Kriks et al., 2011) midbrain floor-plate differentiation pro- latter conditions as a result of neurodegeneration, rather tocols that was comparably effective in all iPSC lines than a blockade in vmDAn differentiation or maturation. analyzed. Under these conditions, 20% of cells in the Viability tests of both Ctrl and PD astrocytes at 2 and cultures were committed to DA neuron fate by day 35 of 4 weeks of co-culture revealed highly similar values, indi- differentiation, as judged by the expression of tyrosine cating that neurodegenerative signs displayed by Ctrl hydroxylase (TH) and forkhead box protein A2 (FOXA2) vmDAns were not caused by a dying PD astrocyte (Fig- (Figure 2A). By day 50 of differentiation, the percentage ure S2D). Interestingly, vmDAn neurodegeneration upon of TH+ neurons reached 30%, most of which also co-culture with PD astrocytes was specific to this type of expressed the A9-domain-specific marker G-protein- neuron, because non-dopaminergic neurons (TH/MAP2+) activated inward rectifier potassium channel 2 (GIRK2), did not significantly change in numbers or morphology and displayed spontaneous action potential firing (Figures after co-culture with Ctrl or PD astrocytes (Figures S2E– S2B–S2D). For co-culture experiments with control astro- S1J). All together, these results indicate a neurotoxic capac- cytes, we dissociated vmDAn cultures after 35 days of dif- ity of PD astrocytes toward vmDAns, with no effects on ferentiation and plated them onto a confluent layer of other neuronal types concomitantly present in cultures. Ctrl iPSC-derived astrocytes (Figure 2E). After 4 weeks of co-culture, we found that astrocyte-neuron glutamate Control vmDAns Accumulate a-syn when Co-cultured exchange was present through glutamate transporter 1 with PD Astrocytes (GLT1) expression (Figure 2F) and neuronal synapse for- Given the relevance of a-syn in the context of PD patho- mation (Figure 2G). Accordingly, an overall healthy genesis (Braak et al., 2007), we sought to examine whether (D) Calcium wave flux recording over 30 min with calcium tracer Fluo-8 AM of vmDAns at day 50 (n = 3). (E) Diagram of co-culture system. (F) Representative ICC images of 4-week co-culture staining positive for Ctrl SP11 vmDAns (TH), Ctrl SP17 astrocytes (S100b), excitatory amino acid transporter 2 (GLT1), and nuclear DAPI. Scale bar, 20 mm. (G) Representative ICC images of presynaptic markers a-syn and synapsin-1 of a Ctrl SP11 vmDAn (TH) on the top of Ctrl SP11 astrocytes after 4 weeks in co-culture. Scale bar, 10 mm. (H) Representative ICC images of Ctrl SP11 vmDAns (TH) and mature neurons (MAP2) on the top of Ctrl SP09 astrocytes (GFAP) during a 4-week co-culture period. Scale bar, 20 mm. Boxed area on the left in (F), (G), and (H) is shown on the right. Stem Cell Reports j Vol. 12 j 213–229 j February 12, 2019 217 (legend on next page) 218 Stem Cell Reports j Vol. 12 j 213–229 j February 12, 2019 vmDAns co-cultured with PD astrocytes abnormally accu- different concentrations (Figure S3A). Exposure of Ctrl mulated a-syn. a-syn was barely detectable in the cyto- vmDAns to PD astrocyte-conditioned medium for 1 week, plasm of Ctrl vmDAns cultured alone (data not shown) or even at low concentrations, resulted in a-syn accumula- when co-cultured with Ctrl astrocytes (Figure 3G). In tion, morphological alterations suggestive of neurodegen- contrast, Ctrl vmDAns accumulated high levels of a-syn eration, and decreased cell survival (Figures S3B–S3H), throughout the neurites and cell body after 4 weeks of indicating that PD astrocytes secrete a molecule(s) that is co-culture with PD astrocytes (Figures 3H and 3I). Notably, toxic to vmDAns. Direct uptake by vmDAns of a-syn while Ctrl astrocytes had undetectable levels of a-syn (Fig- from conditioned medium was tested by exposing Ctrl ure 3J), PD astrocytes displayed high levels of a-syn (Figures vmDAns to medium collected from PD a-syn-FLAG-tagged 3K and 3L), raising the intriguing possibility that a-syn astrocytes (Figures S3I–S3J), suggesting that the neurotoxic from PD astrocytes might be transferred to Ctrl vmDAns. effect of PD astrocytes on vmDAns is, at least in part, medi- To directly address if this was the case, we genetically engi- ated by secretion of a-syn. neered two iPSC lines (representing one PD patient and one healthy control) using CRISPR/Cas9 technology so that the Control Astrocytes Partially Rescue endogenous a-syn would be tagged with a FLAG peptide Neurodegeneration of PD vmDAns (a-syn-FLAG iPSC lines; Figures S2K and S2L and Tables We have previously shown that vmDAns derived from S2 and S3). a-syn-FLAG-tagged astrocytes were generated PD-iPSCs show signs of neurodegeneration (including and fully characterized (Figure S2M). As expected, PD reduced numbers of neurites and neurite arborization, as a-syn-FLAG tagged astrocytes accumulated abnormally well as accumulation of abnormal a-syn in the soma) after high levels of a-syn, which co-localized with anti-FLAG 50 days of culture, which are not evident in Ctrl vmDAns staining (Figure 3M). More importantly, the co-culture of (Sanchez-Danes et al., 2012). To test whether the neurode- Ctrl vmDAns on top of a-syn-FLAG-tagged PD astrocytes generation could be rescued or prevented by healthy for 4 weeks resulted in FLAG-tagged a-syn accumulation astrocytes, we co-cultured PD vmDAns with Ctrl or PD in neurons, demonstrating the direct transfer of astrocytic astrocytes. After a 4-week co-culture, PD vmDAns showed a-syn to neurons (Figure 3N). a partially recovered neurite number and complex In addition to co-culturing cells with direct glia-neuron neurite arborization when co-cultured on control astro- contact, we tested the effect of culturing Ctrl vmDAns cytes, compared with co-cultures with PD astrocytes (Fig- with medium conditioned by Ctrl or PD astrocytes at ures 4A–4E). This rescue was partial, since PD vmDAns Figure 3. Ctrl Neurons Show Signs of Neurodegeneration and Accumulate a-syn when Co-cultured with PD Astrocytes (A) Scheme representing co-culture system of Ctrl neurons on the top of Ctrl or PD astrocytes for 4 weeks. (B and C) Representative ICC images of tyrosine hydroxylase (TH, black) from co-cultures of Ctrl SP11 neurons with (B) Ctrl SP09 astrocytes and (C) PD SP13 astrocytes for 4 weeks. Images on the right show a magnification of the area boxed in the left images; scale bars, (left) 20 mm and (right) 0.2 mm. (D) Percentage of TH+/DAPI of Ctrl SP11 and Ctrl SP11#4 neurons when co-cultured with Ctrl SP09, Ctrl SP17, or PD SP12, PD SP13, and PD SP06 astrocytes for 4 weeks (n = 3 per combination). (E and F) (E) Neurite length quantification and (F) number of branches of Ctrl SP11 TH-positive neurons and Ctrl SP11#4 TH-positive neurons when co-cultured on Ctrl SP09, Ctrl SP17, and Ctrl SP11 astrocytes or PD SP12, PD SP13, and PD SP06 astrocytes for 4 weeks (n = 3); 30 neurons counted per experiment. (G and H) Representative ICC images of Ctrl SP11 vmDAns co-cultured with (G) Ctrl SP09 and (H) PD SP13 astrocytes after 4 weeks and stained for TH (vmDAn), a-syn, and DAPI. Arrows indicate the selected cell for which an insert is shown at higher magnification. Scale bar, 0.2 mm. (I) Quantitative analysis of the percentage of vmDAns stained positive for a-syn when Ctrl SP11 and Ctrl SP11#4 neurons were co-cultured with Ctrl SP09, Ctrl SP17, or PD SP12, PD SP13, and PD SP06 astrocytes for 4 weeks (n = 3). (J and K) Representative ICC images of (J) Ctrl SP09 or (K) PD SP13 astrocytes co-cultured with Ctrl SP11 vmDAns for 4 weeks, stained for TH (vmDAn), GFAP (astrocytes), a-syn, and DAPI. Arrows indicate the selected cell for which an insert is shown at higher magnification. Scale bar, 20 mm. (L) Quantitative analysis of the percentage of astrocytes stained positive for a-syn when Ctrl SP11 and Ctrl SP11#4 neurons were co-cultured with Ctrl SP09, Ctrl SP17, or PD SP12, PD SP13, and PD SP06 astrocytes for 4 weeks (n = 3). (M) Scheme representing the generation of CRISPR/Cas9 edited a-syn-FLAG astrocyte line. Representative image of a-syn-FLAG PD SP12 astrocyte (GFAP) showing perfect a-syn (red) and FLAG (green) co-localization. Scale bar, 20 mm. (N) Representative ICC image depicting astrocyte-derived FLAG (green) inside of a TH-positive Ctrl SP11 neuron (red) during a 4-week co-culture period with PD SP12 a-syn-FLAG astrocytes (n = 3). Dashed line shows the outline of the cell. Scale bar, 10 mm. Data are expressed as mean ± SEM, unpaired two-tailed Student’s t test, ***p < 0.001. Stem Cell Reports j Vol. 12 j 213–229 j February 12, 2019 219 (legend on next page) 220 Stem Cell Reports j Vol. 12 j 213–229 j February 12, 2019 co-cultured with control astrocytes did not reach the levels decreased survival of vmDAns and evident morphological of cell survival and complex neurite arborization seen in alterations (Figures S4H–S4I), including fewer and shorter co-cultures of Ctrl vmDAns and Ctrl astrocytes (compare neurites compared with vmDAns cultured on top of GFP- Figures 4A and 4C–4E with 3B and 4D–4F). Moreover, transfected astrocytes, indicating that the expression of co-culture with Ctrl astrocytes also prevented the accumu- pathogenic LRRK2 in Ctrl astrocytes is deleterious for the lation of a-syn in PD vmDAns that was evident in co-cul- survival of dopaminergic neurons. tures with PD astrocytes (Figures 4A, 4B, and 4F). Notably, For the converse experiment, we generated isogenic PD most Ctrl astrocytes when co-cultured with PD vmDAns astrocytes lacking the LRRK2 G2019S mutation by adopted a flat morphology with moderate levels of a-syn; CRISPR/Cas9-mediated gene editing of PD iPSCs (iPSC however, some harbored a hypertrophic morphology line PD SP13, from here on referred to as PD iso), and fully with retracted processes that accumulated very high levels characterized these cells (Figures S5A–S5E). Abnormal of a-syn (Figures 4G and 4H), suggesting that reactive astro- a-syn accumulation did not occur in gene-corrected astro- cytes may contribute to the clearance of vmDAn a-syn cytes, in contrast with their isogenic mutant counterparts accumulation. Culture of PD vmDAns with medium (Figures S5F and S5G). Moreover, co-culturing gene- conditioned by Ctrl astrocytes also rescued cell survival, corrected astrocytes with Ctrl vmDAns for 4 weeks pre- morphological alterations, and a-syn accumulation (Fig- vented the accumulation of a-syn and decrease in neuron ures S3K–S3S), indicating that direct neuronal-glial contact survival observed when Ctrl vmDAns were co-cultured was not necessary for the neuroprotective effect. with PD astrocytes (Figures S5H–S5J), further supporting We next investigated the causative role of the genetic that the expression of mutant LRRK2 in astrocytes is path- background of patient-specific astrocyte cells by ectopically ogenic to Ctrl vmDAns. expressing mutated LRRK2 G2019S in Ctrl astrocytes. In these experiments, Ctrl astrocytes were transfected with a Dysfunctional Chaperone-Mediated Autophagy and plasmid expressing V5-tagged LRRK2 G2019S, or with a Progressive a-syn Accumulation in PD Astrocytes Ctrl plasmid expressing GFP, and analyzed 7 days after Since PD astrocytes displayed higher levels of a-syn transfection for the presence of a-syn. Astrocytes trans- compared with controls, we next investigated possible dif- fected with LRRK2 G2019S exhibited diffuse cytoplasmic ferences in a-syn turnover in these cells. Degradation of accumulations of a-syn (Figure S4A), which were not a-syn in lysosomes occurs in large extent through CMA present in GFP-transfected cells (Figure S4B). The transfec- (Cuervo et al., 2004; Martinez-Vicente et al., 2008). To tion efficiencies (30%–40%, as evaluated by co-staining for investigate possible changes in CMA in PD astrocytes, we V5/GFAP or GFP/GFAP) were comparable under both con- first stained at 6 and 14 days for both a-syn and LAMP2A, ditions (Figure S4C). Next, we co-cultured Ctrl vmDAns for the receptor for CMA (Figures 5A and S6A). Ctrl astrocytes 4 weeks with LRRK2 G2019S-transfected Ctrl astrocytes, or showed LAMP2A in the perinuclear area (perinuclear lyso- with GFP-transfected astrocytes as a control, and we found somal positioning occurs during CMA activation; Kiffin a-syn accumulation in 50% of the TH+ neurons only in et al., 2004) and barely detectable levels of a-syn at both co-cultures with LRRK2 G2019S-transfected astrocytes 6 and 14 days (Figures 5A, 5B, and S6A). In contrast, PD (Figures S4D–S4G). Overall in these cultures we found astrocytes displayed LAMP2A-positive vesicles all around Figure 4. PD Neurons Restore Arborized Morphology and Accumulate Less a-syn when Co-cultured with Ctrl Astrocytes (A and B) Representative ICC images of PD SP12 vmDAns during 4-week co-cultures with (A) Ctrl SP09 or (B) PD SP13 astrocytes stained for TH (vmDAns), a-syn, GFAP (astrocytes), and DAPI. Images on the right show a magnification of the area boxed in the left images. Arrows indicate the selected cell for which an insert is shown at higher magnification with a-syn accumulation. Scale bar, 20 mm. (C) Quantitative analysis of the percentage of PD SP12 vmDAns remaining after 4-week co-culture with Ctrl SP09, PD SP13, and PD SP12 astrocytes (n = 3). (D and E) (D) Neurite length quantification and (E) number of branches of PD SP12 TH-positive neurons when co-cultured on PD SP12 or PD SP13 astrocytes for 4 weeks compared with the wild-type condition Ctrl SP11 neurons on Ctrl SP09, Ctrl SP17, and Ctrl SP11 astrocytes for 4 weeks (n = 3); 40 neurons counted per experiment. (F) Quantitative analysis of the percentage of PD SP12 vmDAns that stained positive for a-syn when co-cultured on the top of Ctrl SP09, Ctrl SP17, Ctrl SP11, PD SP13, and PD SP12 astrocytes for 4 weeks (n = 3). (G) Immunofluorescence analysis of PD SP12 neurons on the top of Ctrl SP09 astrocytes stained for TH, GFAP, a-syn, and DAPI. Images on the right show a magnification of the area boxed in the left image. Arrows in the inset shows a-syn accumulation inside Ctrl SP09 astrocyte processes. Inset scale bar, 20 mm. (H) Quantitative analysis of the percentage of astrocytes that stained positive for a-syn after being cultured with PD SP12 neurons for 4 weeks. Ctrl astrocytes were derived from SP09, SP11, and SP17 iPSCs, while PD astrocytes were derived from SP12 and SP13 iPSCs (n = 3). Data are expressed as mean ± SEM, unpaired two-tailed Student’s t test, **p < 0.01, ***p < 0.001). Stem Cell Reports j Vol. 12 j 213–229 j February 12, 2019 221 Figure 5. Altered CMA and a-syn Accumulation in LRRK2-PD Astrocytes (A) Representative ICC images of CMA receptor (LAMP2A), astrocyte marker GFAP, a-syn, and nuclear marker DAPI in Ctrl SP09 and PD SP13 astrocytes at 6 and 14 days. Scale bar, 20 mm. Smaller white circles represent perinuclear area, whereas larger green circle represents non-perinuclear area. (B) Percentage of astrocytes with LAMP2A-positive puncta positioning outside of perinuclear area and percentage of astrocytes that stained positive for a-syn. Astrocyte lines used in the experiment were Ctrl SP09, Ctrl SP17, PD SP12, and SP13 (n = 3). (C) Representative ICC image of positive co-localization of LAMP2A and a-syn in PD SP13 astrocytes. Scale bar, 10 mm. (legend continued on next page) 222 Stem Cell Reports j Vol. 12 j 213–229 j February 12, 2019 the cell body as early as 6 days, which continued to be pre- and S6C). KFREQ-Dendra is present in the cytosol (diffuse sent after 14 days (Figures 5A, 5B, and S6A). Moreover, fluorescent pattern) but as it is delivered to lysosomes via abnormal accumulation of a-syn was confirmed in PD CMA it changes to a fluorescent punctate pattern. Ctrl astrocytes after 14 days of culture, compared with Ctrl as- astrocytes displayed these puncta, indicative of functional trocytes (Figures 5A–5E). Interestingly, this accumulation CMA, whereas the signal in PD astrocytes remained was not present after 6 days of culture, suggesting progres- diffused in the cytosol, suggestive of an inactive CMA. sive a-syn accumulation over the 14-day time period. Since PD astrocytes displayed higher levels of a-syn Co-localization analyses of a-syn with the LAMP2A recep- compared with Ctrl astrocytes, we next investigated tor revealed a positive co-localization that was more possible differences in a-syn turnover in these cells. a-syn evident in PD astrocytes (Figures 5C and S6B). CMA sub- has previously been shown to undergo degradation both strates are usually rapidly internalized and degraded inside by the ubiquitin/proteasome system and by autophagy lysosomes, but we have previously described a similar (Cuervo et al., 2004; Webb et al., 2003); therefore a-syn persistent association of a-syn with LAMP2A-positive lyso- flux in the presence of lysosomal and proteasome inhibi- somes in PD models due to blockage in a-syn translocation tors (leupeptin and lactacystin, respectively) was evaluated inside lysosomes (Orenstein et al., 2013). These findings in Ctrl and PD astrocytes at 14 days (Figures 5I, 5J, and suggest, thus, a similar CMA blockage in the PD astrocytes S7D). An increase of 40% in a-syn levels was found in at the receptor level. Also supportive of reduced a-syn Ctrl astrocytes after leupeptin treatment, while this in- degradation, western blot analysis confirmed a higher crease was not found in PD astrocytes analyzed under the monomeric protein level of a-syn in PD astrocytes same conditions, indicating an impaired flux. No changes compared with controls (Figures 5D, 5E, and S7A). By using were found in either Ctrl or PD astrocytes after lactacystin an antibody that detects specifically oligomeric a-syn, we treatment (Figures 5I, 5J, and S7D). These findings suggest were able to detect other pathogenic forms of a-syn in PD major alterations in a-syn proteostasis due to poor degrada- astrocytes, which were similar to those of PD postmortem tion by lysosomal systems in PD astrocytes. brain tissue (Figure S7B). To investigate the contribution of the defect in CMA to Impaired Macroautophagy in PD Astrocytes the progressive accumulation of a-syn in PD astrocytes, Cells often respond to blockage in CMA by upregulating we next performed a knockdown of LAMP2A using lentivi- other autophagic pathways such as macroautophagy (Mas- ral-mediated short hairpin RNA (shRNA) targeting and sey et al., 2006; Schneider et al., 2015). However, altered silencing the LAMP2A spliced transcript (shLAMP2A), or macroautophagy has also been reported in the context of an shRNA targeting the Luciferase gene (shLuc) as a control PD (Sanchez-Danes et al., 2012; Winslow et al., 2010). To (Figure 5F). The shLuc control astrocytes displayed an investigate the status of macroautophagy, the endo/lyso- expected low level of a-syn, whereas after shLAMP2A trans- somal marker LAMP1, autophagosome marker LC3, astro- duction, there was a statistically significant (p < 0.001) cyte marker GFAP, and nuclear DAPI were used during 2.5-fold increase in a-syn puncta, comparable to the levels ICC on all astrocyte lines at both 6 and 14 days. In Ctrl observed in PD astrocytes (Figure 5G). Knockdown of astrocytes, there was lysosomal LAMP1 staining in the LAMP2A did not change a-syn puncta levels in PD astro- perinuclear area and very few visible autophagic vacuoles cytes, further suggesting defective CMA for a-syn in these (LC3-positive vesicles) at both 6 and 14 days (Figures 6A, cells. CMA activity was monitored using a photoactivatable 6B, and S6D). In PD astrocytes, as for LAMP2A, LAMP1- CMA reporter, KFREQ-Dendra (Koga et al., 2011), in all positive vesicles lost the preferable perinuclear distribution astrocyte lines at 52 hr after photoactivation (Figures 5H and were found throughout the entire cell (Figures 6A, 6B, (D and E) (D) Western blot of a-syn and a-tubulin as a loading control and (E) quantification in Ctrl SP09 and PD SP13 astrocytes after 14 days in culture (n = 4). (F) Representative ICC images of Ctrl SP09 and PD SP13 astrocytes after 14 days of transduction with either LV-shLAMP2A or LV-shLuciferase (as a control) stained for a-syn, GFP, and DAPI. Boxed areas highlight the region for which high magnification images are shown. Scale bars, 20 and 10 mm, respectively. (G) Percentage of a-syn puncta area per cell in Ctrl SP09 and PD SP13 astrocytes transduced with LV-shLuciferase or LV-shLAMP2A (n = 3). (H) KFERQ-DENDRA (CMA reporter) in Ctrl SP09 and PD SP13 astrocytes 52 hr after photo-switching with UV light (n = 3). Images in the insets at the bottom are a magnification of the boxed area. (I and J) (I) Western blot of a-syn and b-actin as a loading control and (J) quantification of a-syn flux ratio normalized to b-actin in Ctrl SP09 and PD SP13 after the addition of inhibitors of lysosomal proteolysis (leupeptin [LEU], 100 mM) for 12 hr and proteasomal degradation (lactacystin [LAC], 5 mM) for 2 hr (n = 3). Data are expressed as mean ± SEM, unpaired two-tailed Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001. Stem Cell Reports j Vol. 12 j 213–229 j February 12, 2019 223 Figure 6. Dysfunctional Macroautophagy in LRRK2-PD Astrocytes (A) Representative ICC images of lysosomal protein marker LAMP1 and autophagosome marker LC3 in Ctrl SP09 and PD SP13 astrocytes (GFAP) at 6 and 14 days. Smaller white circles represent perinuclear area, whereas larger green circle represents non-perinuclear area. Scale bar, 20 mm. (B) Percentage of astrocytes with LAMP1-positive puncta positioning outside of perinuclear area and percentage of astrocytes that stained positive for LC3-II. Astrocyte lines used in the experiment were Ctrl SP09 and SP13 (n = 3). (C–E) (C) Western blot of LC3-II protein levels and b-actin as loading control with corresponding quantification of (D) the LC3-II basal expression and (E) LC3-II flux with or without lysosomal inhibitors NH4Cl and leupeptin (L/N) for 2 hr in Ctrl SP09 and PD SP13 astrocytes (n = 3). (F–H) (F) Western blot of p62 protein levels and b-actin as loading control with corresponding quantification of (G) the P62 basal expression and (H) P62 flux without inhibitors or with inhibitors leupeptin for 12 hr and lactacystin for 2 hr in Ctrl SP09 and PD SP13 astrocytes (n = 3). Data are expressed as mean ± SEM, unpaired two-tailed Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001. and S6D). In addition, there was a marked increase in auto- insights into the mechanism behind the accumulated phagic vacuoles starting as early as 6 days that continued LC3-II levels in PD astrocytes, both Ctrl and PD astrocytes increasing through the 14-day time point (Figures 6A, 6B, were treated with leupeptin and NH4Cl, inhibitors of lyso- and S6D). Most of the accumulated LC3-positive vesicles somal proteolysis, to inhibit LC3-II degradation. Under in PD astrocytes did not co-localize with LAMP1 lysosomes these conditions, PD astrocytes exhibited lower increase (Figures S6E and S6F), suggesting that they were autopha- in LC3-II levels compared with controls, suggesting an gosomes that persisted in these cells due to their poor impairment of the autophagy flux in PD astrocytes (Figures clearance by lysosomes. 6E and S7C). Moreover, we found higher p62 levels in PD In agreement with the immunofluorescence studies, astrocytes at baseline compared with controls, and lower western blot analyses detected higher basal levels of flux ratio in the presence of inhibitor (Figures 6F–6H and LC3-II in PD astrocytes compared with Ctrls (Figures 6C, S7E). Overall these findings suggest that reduced function 6D, and S7C). To monitor the autophagy flux and to gain in both autophagic pathways, CMA and macroautophagy, 224 Stem Cell Reports j Vol. 12 j 213–229 j February 12, 2019 Figure 7. CMA Activator Drug Treatment Rescues a-syn Accumulation in Neurons Cultured with PD Astrocytes (A and B) Representative ICC images of (A) Ctrl and (B) PD astrocytes after 2 weeks in culture without treatment stained for LAMP2A, a-syn, GFAP, and DAPI. Orthogonal views reveal positive co-localization of a-syn to LAMP2A in PD untreated sample. Images on the right show a magnification of the area boxed in the left images. Dashed circles outline the perinuclear area of the cell. Scale bars, 100 and 20 mm in merge and zoom, respectively. (C) Representative ICC images of PD astrocytes after 20 mg of CA drug treatment stained for LAMP2A, a-syn, GFAP, and DAPI. Scale bars, 100 and 20 mm in merge and zoom, respectively. (D and E) (D) Quantitative analysis of PD astrocytes (SP13 and SP12) stained positive for a-syn either not treated or treated with CA; (E) quantitative analysis of the same astrocytes under the same conditions, showing LAMP2A puncta in the perinuclear area (n = 3). (F) Representative ICC images of 4-week Ctrl SP11 vmDAns co-cultured on PD SP13 astrocytes (left) or treated with CA for 2 weeks (right). Samples were stained for GFAP, TH, a-syn, and DAPI. Scale bars, 20 mm. (legend continued on next page) Stem Cell Reports j Vol. 12 j 213–229 j February 12, 2019 225 contribute to the altered a-syn proteostasis observed in PD iPSC-based co-culture model, in this study we describe a astrocytes. role for PD astrocytes in midbrain neuronal cell death. Spe- cifically, in a 4-week co-culture system, we found morpho- Restoration of a-syn Proteostasis in PD Astrocytes logical alterations resembling those of neurodegeneration, Alone and during Co-culture of Control Neurons with such as short and few neurites as well as beaded necklace- PD Astrocytes like neurites, typically observed in neurons upon transport Intracellular accumulation of a-syn has been shown to alterations (Fu et al., 2005; Garrido et al., 2011), and contribute to cellular toxicity in PD and to further disrupt increased neuronal loss in Ctrl neurons co-cultured with functioning of cellular proteostasis systems (reviewed in PD astrocytes. We interpret these altered phenotypes as Abeliovich and Gitler, 2016). We next investigated whether representing PD astrocyte-induced vmDAn neurodegener- a-syn accumulation in PD astrocytes could be ameliorated ation. An alternative explanation could be that PD astro- by enhancing lysosomal activity. PD astrocytes were cytes impinged on the differentiation and/or maturation treated with a CMA activator (CA), which operates through of DAn progenitors in our iPSC-derived co-culture system. the release of the endogenous inhibition of the retinoic However, while we cannot formally rule out this possibil- receptor-a signaling pathway over CMA (Anguiano et al., ity, two lines of evidence strongly argue against it playing 2013). Cells were treated with a concentration of 20 mM a significant role in the phenotypes described here. First, for 5 days and levels of a-syn were analyzed by immunoflu- we used vmDA neural differentiation cultures at 35 days orescence (Figure 7). LAMP2A-positive lysosomes in PD as- of differentiation for our co-culture experiments. At trocytes treated with the CA (Figures 7C–7E) recovered the this time, most vmDAns are already committed in fate perinuclear distribution observed in Ctrl cells (Figure 7A) (TH+/FOXA2+, see Figure 2A), but are still at a stage of matu- compared with when not treated (Figure 7B), suggesting re- ration that does not compromise their survival upon cell activation of CMA in these cells. Consistent with higher dissociation and plating on top of the astrocyte cultures. CMA activity, CA-treated cells had significantly lower Second, the numbers of vmDAns at different time points a-syn content than untreated cells (Figures 7C–7E). along the co-culture experiments showed progressive In addition, we treated PD astrocytes when in co-culture decline in co-cultures with PD astrocytes, but no significant with Ctrl neurons (Figure 7F). Under untreated conditions, changes when co-cultured with Ctrl astrocytes (Figure S2C). Ctrl neurons accumulate astrocytic a-syn and show signs of These results indicate that few new TH+ neurons are gener- neurodegeneration. Interestingly, the treatment with CA ated during co-culture, and further support that the cleared out a-syn not only in astrocytes, but also in decreased numbers of vmDAns observed upon co-culture vmDAns, partially restored neuron survival, and decreased with PD astrocytes are a consequence of vmDAn degenera- the number of TH-positive cells with a degenerative tion. Importantly, the altered phenotypes were specifically morphology (Figures 7G–7L). These findings suggest that observed in the subpopulation of midbrain dopaminergic although multiple protein degradation pathways fail to neurons, as numbers of MAP2+/TH neurons did not efficiently degrade a-syn in PD cells, reactivation of one change significantly upon co-culture with Ctrl or PD astro- of these pathways, in our case CMA, is able to restore func- cytes. In accordance with this, it has been already shown tional a-syn proteostasis. that a-syn toxicity was responsible for nigrostriatal neuronal cell death in midbrain cultures (Petrucelli et al., 2002), a relevant finding regarding the particular vulnera- DISCUSSION bility of nigral neurons in PD. However, it remains to be tested whether, in prolonged culture, PD astrocytes also Astrocytes from three PD patients carrying the G2019S mu- impair the survival of TH populations. tation in the LRRK2 gene and two healthy individuals were Postmortem brain tissue of PD patients revealed a-syn successfully generated using a previously published proto- accumulation in astrocytes (Wakabayashi et al., 2000). col and fully characterized. By implementing a patient (G–I) (G) Quantitative analysis of the percentage of vmDAns remaining after being co-cultured with PD SP12 or PD SP13 (without treatment or treated with CA) for 4 weeks. Percentage of the (H) vmDAns or (I) astrocytes that stained positive for a-syn 4 weeks after the same co-culture conditions (n = 6). (J) Representative ICC images of 4-week Ctrl SP11 vmDAns co-cultured on PD SP12 astrocytes with or without CA treatment for 2 weeks. Samples were stained for TH, a-syn, and DAPI. Arrows indicate the absence of a-syn accumulation in the selected TH-positive cell. Scale bars, 20 mm. (K and L) (K) Neurite length quantification and (L) number of branches of 4-week Ctrl SP11 vmDAns when co-cultured on PD SP12 or PD SP13 astrocytes with or without CA treatment for 2 weeks (n = 4); 20 neurons counted per experiment. Data are expressed as mean ± SEM, unpaired two-tailed Student’s t test, *p<0.05; ***p < 0.001. 226 Stem Cell Reports j Vol. 12 j 213–229 j February 12, 2019 It has been previously described that astrocytes accumulate Ctrl cells. Although a-syn was cleared, restoration during neuronal-derived a-syn as a mechanism of neuroprotection a co-culture with Ctrl neurons was only partial in terms (Booth et al., 2017). Indeed, in our study we found that Ctrl of neurite length and number, suggesting that the neurode- astrocytes accumulate a-syn when co-cultured with PD neu- generation observed could also be due to other non-a-syn- rons and partially rescued the morphological phenotype of related factors secreted by PD astrocytes. neurodegeneration and clearance of neuronal a-syn. This Overall, our findings propose a specific role for astrocytes behavior suggests a neuroprotective effect via inflamma- in mediating dopaminergic cell death during PD. PD-spe- tory-mediated activation of the Ctrl astrocytes. In addition, cific phenotypes specifically related to dysfunctions in the by using a CRISPR/Cas9 gene-edited cell line tagging the pathways of protein degradation have been observed in endogenous SNCA locus with an FLAG tag, our results reveal PD astrocytes and not in Ctrl astrocytes. Dysfunctional that PD astrocytes also accumulate and transfer a-syn to the CMA, progressive a-syn accumulation, and glia-to-neuron surrounding neurons, suggesting that astrocytes actively transfer found in our PD astrocytes are all aspects that can contribute to the distribution of a-syn. compromise neuronal survival during PD pathogenesis. Taking into account that our PD astrocytes come from Future studies will identify whether additional factors other patients carrying the LRRK2 G2019S mutation, we investi- than a-syn are being secreted by (or lacking in) PD gated whether disease-specific phenotypes related to the astrocytes, and thus contributing to triggering vmDA mutation were present. The a-syn accumulation in our neuronal cell death. iPSC-based technology allows for the co-culture system indicated a disruption in the way a-syn proper recapitulation of patient-specific disease-related phe- is usually degraded in PD astrocytes. Degradation of a-syn notypes, which will aid in the discovery of new therapies. has been shown to occur by both proteasome and autopha- gic pathways, and conversely, high levels of a-syn have EXPERIMENTAL PROCEDURES been demonstrated to be toxic for both systems (Tanaka et al., 2001; Webb et al., 2003; Winslow et al., 2010). Here Experimental procedures are also provided in Supplemental we found that lysosomal degradation of a-syn was severely Information. inhibited in PD astrocytes. We have previously described in PD neurons that one of the early events in the dysfunction iPSC-Derived Astrocyte Generation and Culture of the proteostasis systems in these cells is the disruption of The parental iPSC lines used in our studies were previously gener- CMA by mutant LRRK2 binding to the LAMP2A, thus ated and fully characterized (Sanchez-Danes et al., 2012). The gen- causing the accumulation of a-syn (Orenstein et al., eration and use of human iPSCs in this work were approved by the 2013). Here we demonstrate that CMA is also altered in Spanish competent authorities (Commission on Guarantees con- PD astrocytes and confirm that a-syn degradation by cerning the Donation and Use of Human Tissues and Cells of the CMA in these cells was almost completely abolished. Carlos III National Institute of Health). iPSCs were differentiated The increase in intracellular levels of a-syn, due to its into astrocytes following a previously published protocol (Serio et al., 2013). See Supplemental Information for more details. poor degradation in PD astrocytes by CMA, may contribute to precipitating malfunctioning of other proteostasis mechanisms, such as the proteasome and macroautoph- iPSC-Derived vmDAn Generation Four different iPSCs, two PD (SP12 and SP13) and two Ctrl (SP11 agy. In fact, we demonstrated that macroautophagy was and SP11#4), were differentiated into dopaminergic neurons using also markedly impaired in these cells, by showing higher a combination of two previously published protocols for midbrain basal levels of autophagic vacuoles (LC3-II) and the auto- induction (Chambers et al., 2009; Kriks et al., 2011). Detailed phagic cargo p62, and reduced autophagic flux (for both methods are provided in Supplemental Information. LC3-II and p62). The lower co-localization between the ICC autophagosomal and lysosomal markers observed in PD ICC on cell cultures was performed as described in Supplemental astrocytes suggests that the reduced autophagic flux is Information. due to a defect in autophagosome/lysosome fusion, similar Statistical Analysis to that previously described in PD neurons. Statistical analyses of the obtained data were performed using two- Taking into account the coordinate functioning of the tailed unequal variance Student’s t tests and ANOVA (*p < 0.05, proteolytic systems, and the fact that CMA disruption **p < 0.01, ***p < 0.001), and the mean and standard error of the seems to occur early during the development of PD pathol- mean were plotted using Prism (Mac OS X). Number of indepen- ogy, we attempted to restore normal a-syn proteostasis by dent experiments (n) is indicated in each figure legend. enhancing CMA activity. Our findings in cells treated with a chemical activator of CMA suggest that upregulation SUPPLEMENTAL INFORMATION of CMA is still possible in these cells and that this interven- Supplemental Information includes Supplemental Experimental tion is sufficient to return levels of a-syn close to those in Procedures, seven figures, and three tables and can be found Stem Cell Reports j Vol. 12 j 213–229 j February 12, 2019 227 with this article online at https://doi.org/10.1016/j.stemcr.2018. Booth, H.D.E., Hirst, W.D., and Wade-Martins, R. (2017). The role 12.011. of astrocyte dysfunction in Parkinson’s disease pathogenesis. Trends Neurosci. 40, 358–370. AUTHOR CONTRIBUTIONS Braak, H., Sastre, M., and Del Tredici, K. (2007). Development of Conceptualization, A.C. and A.R.; Methodology, I.F., J.P.M., A.Z., alpha-synuclein immunoreactive astrocytes in the forebrain paral- A.M.C., and J.S.; Investigation, Y.R.P, A.D., G.C., C.C., M.P.-E., lels stages of intraneuronal pathology in sporadic Parkinson’s M.G., I.F.-C., J.P., and A.F.; Validation, A.C. and A.R.; Writing – disease. Acta Neuropathol. 114, 231–241. Original Draft, A.D.; Writing – Review & Editing, A.C. and A.R.; Cavaliere, F., Cerf, L., Dehay, B., Ramos-Gonzalez, P., De Giorgi, F., Visualization, A.D.; Resources, A.Z., A.M.C., J.S., E.T., A.C., and Bourdenx, M., Bessede, A., Obeso, J.A., Matute, C., Ichas, F., et al. A.R.; Funding Acquisition, A.C.; Supervision, A.C. (2017). In vitro alpha-synuclein neurotoxicity and spreading among neurons and astrocytes using Lewy body extracts from ACKNOWLEDGMENTS Parkinson disease brains. Neurobiol. Dis. 103, 101–112. Chambers, S.M., Fasano, C.A., Papapetrou, E.P., Tomishima, M., The authors are indebted to the patients with PD who have partic- Sadelain, M., and Studer, L. (2009). Highly efficient neural conver- ipated in this study. The authors thank Chrysanthi Blithikioti for sion of human ES and iPS cells by dual inhibition of SMAD helping with some co-culture experiments and ICC, Neus Bayó- signaling. Nat. Biotechnol. 27, 275–280. Puxan for her advice on western blotting, Jose Miquel Andres Vaquero (CMRB) for performing flow cytometry analysis, and Da- Cookson, M.R. (2016). Cellular functions of LRRK2 implicate vesic- vid Maynar for excellent artwork. We are thankful to Mark Cook- ular trafficking pathways in Parkinson’s disease. Biochem. Soc. son for the LRRK2 G2019S plasmid (Addgene Plasmid No. Trans. 44, 1603–1610. 29401). We are grateful to the Advanced Fluorescence Microscopy Cookson, M.R. (2017). Mechanisms of mutant LRRK2 neurodegen- Unit of the Institute of Biomedicine of the University of Barcelona eration. Adv. Neurobiol. 14, 227–239. (especially to Elena Rebollo Arredondo). Research from the au- Cuervo, A.M., Stefanis, L., Fredenburg, R., Lansbury, P.T., and thors’ laboratories is supported by the European Research Council Sulzer, D. (2004). Impaired degradation of mutant alpha-synuclein (ERC) (2012-StG-311736-PD-HUMMODEL), the Spanish Ministry by chaperone-mediated autophagy. Science 305, 1292–1295. of Economy and Competitiveness (MINECO) (FIS2016-78507- Fu, L., Gao, Y.S., Tousson, A., Shah, A., Chen, T.L., Vertel, B.M., and C2-2-P, SAF2015-69706-R, and BFU2016-80870-P), Instituto de Sztul, E. (2005). Nuclear aggresomes form by fusion of PML-associ- Salud Carlos III (ISCIII/FEDER) (Red de Terapia Celular [TerCel] ated aggregates. Mol. Biol. Cell 16, 4905–4917. RD16/0011/0024), AGAUR (2014-SGR-878 and 2017-SGR-899), and CERCA Program/Generalitat de Catalunya. 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