Astrocytic atrophy as a pathological feature of Parkinson’s...

来自 : 发布时间：2024-05-08

Astrocytic atrophy as a pathological feature of Parkinson鈥檚 disease with LRRK2 mutation AbstractThe principal hallmark of Parkinson鈥檚 disease (PD) is the selective neurodegeneration of dopaminergic neurones. Mounting evidence suggests that astrocytes may contribute to dopaminergic neurodegeneration through decreased homoeostatic support and deficient neuroprotection. In this study, we generated induced pluripotent stem cells (iPSC)-derived astrocytes from PD patients with LRRK2(G2019S) mutation and healthy donors of the similar age. In cell lines derived from PD patients, astrocytes were characterised by a significant decrease in S100B and GFAP-positive astrocytic profiles associated with marked decrease in astrocyte complexity. In addition, PD-derived astrocytes demonstrated aberrant mitochondrial morphology, decreased mitochondrial activity and ATP production along with an increase of glycolysis and increased production of reactive oxygen species. Taken together, our data indicate that astrocytic asthenia observed in patient-derived cultures with LRRK2(G2019S) mutation may contribute to neuronal death through decreased homoeostatic support, elevated oxidative stress and failed neuroprotection. IntroductionParkinson鈥檚 disease is the second most common neurodegenerative disorder with unknown aetiology1. Age is the principal risk factor for PD, which affects around 1% of people older than 65 years2. The progressive death of dopaminergic neurones in the substantia nigra pars compacta (SNpc) and the appearance of protein deposits in a form of Lewy bodies (LB) mainly composed by 伪-synuclein (伪-syn) represent two major histopathological hallmarks of PD3,4,5. Although the disease is mostly idiopathic, 10% of the cases appear related to specific mutations in different genes. The G2019S mutation in Leucine Rich Repeat Kinase 2 (LRRK2) gene is the most common cause of the familial PD6. This mutation leads to an idiopathic phenotype of the disease albeit, in certain cases, with the absence of LB7. The G2019S mutation is the most frequent pathogenetic mutation in the overall LRRK2-PD population8. This mutation occurs in the kinase domain of LRRK2, leading to an increase in the activity of the enzyme9, which has been shown to affect mitochondrial functionality, cytoskeletal dynamics, response to reactive oxygen species (ROS) production, and autophagy10,11.Fibroblasts from PD patients carrying the G2019S mutation showed abnormal mitochondrial morphology12. Similarly, overexpression of wild-type LRRK2 in SH-SY5Y neuroblastoma cells caused mitochondrial fragmentation, which was further enhanced when the R1441C and G2019S mutations were expressed13. Overexpression of LRRK2G2019S mutation in SH-SY5Y cells causes mitochondrial uncoupling, leading to membrane depolarisation and increased oxygen consumption14. The LRRK2G2019S mutation also delays the digestion of dysfunctional mitochondria and the initiation of mitophagy15.Numerous studies have established a connection between LRRK2 and both microtubules (MTs) and filamentous actin (F-actin). A high-throughput screening performed to reveal LRRK2 interactome identified proteins involved in actin filament assembly, organisation, rearrangement, and maintenance, suggesting that the biological function of LRRK2 is linked to cytoskeletal dynamics16. The same study demonstrated that LRRK2 binds to F-actin and modulates F-actin assembly in mouse primary dopaminergic neurones in vitro. This suggests that morphological changes and abnormalities in neurites outgrowth and branching may be consequences of LRRK2-modulation of cytoskeletal dynamics.Thus, analysis of PD pathogenesis has been mostly focused on the mechanisms underlying ventral midbrain dopaminergic neurones (vmDAn) degeneration and death. Neuronal survival, however, is defined by multiple neuroprotective mechanisms expressed in astrocytes, the principal homoeostatic and defensive cells of the central nervous system17,18,19. Astrocytes density in the SN is relatively low20, which may strain their ability to adequately support and protect neurones. In PD, in contrast to other 伪-synucleopathies, astrocytes do not mount reactive astrogliosis21, an evolutionary conserved defensive response; rather, astrocytes become dysfunctional and lose their protective capabilities22. Astroglial atrophy, asthenia and loss of homoeostatic and protective function contribute to several neurodegenerative and psychiatric diseases23. A recent study demonstrated that the treatment of LRRK2G2019S transgenic mice with 伪-syn increases the expression of endoplasmic reticulum (ER) stress proteins in astrocytes thus affecting neurites length and neuronal viability, supporting the idea that ER stress in PD astrocytes can aggravate neuronal damage24.In this study, we have generated and characterised human iPS-derived astrocytes (hiA) from PD patients carrying LRRK2G2019S mutation. These PD astrocytes display an atrophic morphology with decreased complexity, as well as altered mitochondrial functionality that results in higher basal protein oxidation. As a consequence, PD astrocytes show reduced mitochondrial metabolism and increased glycolytic activity. Overall, we suggest that LRRK2G2019S mutation in astrocytes induces mitochondrial unbalance, leading to cell autonomous and non-autonomous damage that ultimately translates to or exacerbates neurodegeneration. Our results highlight an improvement of astroglial functionality as a relevant therapeutic target.ResultsGeneration of patient-derived astrocytes from dermal fibroblastsSkin fibroblasts from two patients with LRRK2G2019S mutation and two healthy donors (Supplementary Table 1) were reprogrammed and differentiated to mature astrocytes. Fibroblast were reprogrammed using the episomal Sendai viral vector bearing the Yamanaka factors Klf4-Oct3/4-Sox2 (KOS), L-Myc and Klf4 (see Supplementary Fig. 1 for protocol details). Fibroblasts were expanded in Geltrex until they formed colonies positive for the pluripotent markers Sox2, Oct4 and Nanog (Supplementary Fig. 2). To further potentiate the formation of iPSC, colonies were picked and expanded for 2鈥? days in human recombinant laminin-521 (LN521). LN521 is normally expressed in the human embryo at the inner cell mass and replicates the human stem cell niche in vitro stabilising pluripotent gene expression. At this stage and before neural induction, iPSC can differentiate to the three germ layer as evidenced by the expression of Neurone-specific class III 尾-tubulin (Tuj1 for ectoderm), smooth muscle actin (SMA for mesoderm) and alpha-Fetoprotein (AFP for endoderm) (Supplementary Fig. 2). To induce the differentiation to NSC, neural rosettes were cultured with a 50%:50% mixture of laminin 211 (LN211) and laminin 111 (LN111) coating. Unlike LN521, these two laminins are mostly expressed in extra-embryonic membranes and promote cell differentiation. In our culture conditions, NSCs were differentiated to astrocyte progenitor cells in 21 days. Subsequently, astroglial precursors were further differentiated into mature astrocytes (see Material and Methods) while maintaining the coating with LN211/LN111 (50%:50%). After 60鈥?5 days of maturation, cells were fixed and stained with the astrocyte marker GFAP. Maturation efficiency was evaluated by cytofluorimetry assay (Supplementary Fig. 3) demonstrating 95%鈥?8% of astrocyte differentiation. Astrocyte differentiation was also confirmed by immunofluorescence with antibodies to GFAP and S100B, whereas expression of MAP2 and 尾-III tubulin (for neurones) and NG2 (for non-astrocyte glia) was absent or minimal (Fig. 1a). The hiA also expressed the functional markers EAAT2 (glutamate transporter) and CD49f, with undetectable differences between healthy subjects and PD donors (Fig. 1b). All generated lines from the four donors (healthy and PD) displayed neither genetic nor structural variations in somatic and sex chromosomes as demonstrated in Supplementary Fig. 4.Fig. 1: Astrocytic marker expression in hiA.a hiA were maturated for 60 days in LN211/LN111 and fixed for immunofluorescence. All cell cultures, from healthy (Ctrl1-2) and patient (PD1-2) donors, were positive for GFAP and S100尾 expression, whereas neuronal (MAP2 and 尾-III-Tub) and non-astrocyte-glial (NG2) markers were nearly absent. White staining shows nuclei labelling by DAPI. b hiA co-immunostaining of GFAP with CD49f and EAAT2 in healthy (Ctrl) and patient (PD) donors. The picture is representative of two Ctrls and two PD cell lines. Scale bar is 25鈥壩糾. Photographs are representative of at least five experiments.Full size imageMorphology of PD astrogliaWe observed a striking difference in the morphology between healthy and PD astrocytes (Fig. 2a鈥揷). The surface area and perimeter of GFAP-positive profiles of PD-derived astrocytes were, respectively, 60% and 45% smaller when compared to healthy cells, as measured by high-content screening (Fig. 2d鈥揺). Similar data (decrease in surface area and perimeter by 69% and 50%; data not shown) were obtained by manual measurements using the image software Fiji. Astrocytes from PD patients showed a lower complexity with significant reduction in number or complete absence of primary and secondary processes (Fig. 2f), as evidenced by high-content screening analysis (35% lower than healthy astrocytes), suggesting a decreased structural capacity for supporting neurones. Decreased complexity of PD-derived astrocytes was also confirmed by Sholl analysis. As shown in Fig. 2g, astrocytes derived from PD donors exhibit 61% less intersections. Morphological atrophy in PD cultures was not detected in fibroblasts; moreover, we did not detect differences during the iPSC colony formation, but only after astrocyte differentiation (data not shown), suggesting a specificity for the astrocytic phenotype.Fig. 2: Cell morphology analysis of PD astroglia.Cells from healthy (Ctrl1-2 in a) and patients (PD1-2 in b, c) donors were fixed after 60 days of maturation and stained with GFAP antibodies. Images in c illustrate a higher magnification of a subfield in b. Morphological analysis was performed by high-content screening (d鈥?b>f) and Sholl analysis (g) as described in Materials and Methods. d鈥?b>f histograms showing astrocyte area (as a means of squared 渭m in d), perimeter (as a means of linear 渭m in e) and complexity (as a means of arbitrary units-au- of shape P2A in f). In g is expressed the total number of intersections with concentric rings of Sholl grid (5鈥壩糾 apart) after GFAP immunostaining of controls and PD astrocytes. Data are presented as mean values鈥壜扁€塖EM with n鈥?鈥?.Full size imageFunctional characterisation of PD astrocytesWe next characterised functional properties of hiA. Astroglial function and reactivity are tightly integrated with the dynamics of cytosolic and mitochondrial Ca2+ concentrations controlling bioenergetics; abnormal astrocytic Ca2+ signalling is increasingly recognised as a key process in neurodegenerative conditions25,26,27. Thus, we analysed Ca2+ dynamics in PD astroglia. Neither healthy nor PD astrocytes generated spontaneous Ca2+ transients under our experimental conditions and we found no difference in resting cytoplasmic Ca2+ concentration ([Ca2+]i) between healthy and PD hiA (Fig. 3a鈥揵). Application of 100鈥壩糓 ATP (an archetypal activator of astroglial Ca2+ signalling) evoked transient [Ca2+]i elevation (Fig. 3c鈥揹) confirming the presence of functional purinergic receptors coupled to astrocytic Ca2+ signalling machinery. In PD astrocytes we observed a tendency (which did not reach the level of significance) of reduction in amplitude and integral of Ca2+ transients in response to ATP (Fig. 3c鈥揹). It has to be noted however, that control hiA lines tested in this study displayed marked differences in the amplitude of ATP-induced Ca2+ transients. We further tested mitochondrial membrane polarisation by imaging the quenching of the mitochondrial membrane potential probe Rhodamine 123 in the presence of FCCP, which revealed significant differences between control and PD hiA lines (p鈥?鈥?.0365); (Fig. 3e鈥揻). Taken together, these finding confirm that hiA, with differences between healthy and PD astrocytes, express functional receptors for ATP, typical astrocytic Ca2+ signalling machinery; the PD-derived astrocytes also demonstrated signs of mitochondrial malfunction.Fig. 3: Cytosolic Ca2+ responses to ATP and FCCP in human astrocytes.a鈥?b>b Time-courses show basal cytosolic Ca2+ responses in control and PD hiA loaded with fura-2 (n鈥?鈥墄-y cells). c Ca2+ responses evoked by ATP (100鈥壩糓) in hiA (n鈥?鈥墄-y cells). d Comparison of the area under the curve (AUC) calculated for each experimental condition (n鈥?鈥? cultures). e鈥?b>f Measurement of mitochondrial membrane potential in hiA. *p鈥?鈥?.0365. One-way ANOVA followed by Newman鈥揔euls tests. Ctrls, control. PDs Parkinson麓s disease. Data are presented as mean values鈥壜扁€塖EM with n鈥?鈥?.Full size imageMitochondrial impairment in PD astrocytesIt is well known that LRRK2 protein interacts with mitochondrial membranes and affects mitochondrial respiration13,28. We, thus, analysed mitochondrial metabolism in healthy and in PD astrocytes using Seahorse technology. We first measured the mitochondrial oxygen consumption rate (OCRs) of hiA in a live-cell metabolic assay (Fig. 4a). PD astrocytes showed lower OCRs in both basal (Fig. 4b) and maximal (Fig. 4c) respiration paradigms, when compared to healthy cells. PD astrocytes also produced less ATP (Fig. 4d). We did not, however, observe significant differences between healthy and PDs astrocytes in terms of spare respiratory capacity or proton leak (Fig. 4e鈥揻). Consistent with a mitochondrial respiration deficit, PD astrocytes displayed increased glycolytic capacity as determined by changes in the extracellular acidification rate (ECAR) (Fig. 5a), both in basal (Fig. 5b) and in compensatory glycolysis (Fig. 5c). Similarly, basal proton efflux rate (PER, the measure of extracellular acidification, Fig. 5d), but not the PER derived from glycolysis (Fig. 5e), was increased in PD astrocytes. Collectively these results indicate that astrocytes in PD switch from oxidative phosphorylation to the aerobic glycolytic respiration.Fig. 4: Mitochondrial metabolism and respiration.a Oxygen consumption rates (OCRs) of Ctrl and PD astrocytes. Oligomycin, FCCP and rotenone (Rot) were added, respectively, after 20, 40, and 60鈥塵in as respiratory chain blockers. OCRs are expressed in b鈥?b>f as pmol por minute after cell viability normalisation with calcein staining. b Basal respiration, c maximal respiration and d ATP production are reduced in PD astrocytes compared to the controls. e Spare respiratory capacity and f H+ Leak do not show statistically significant changes (n鈥?鈥?). Statistical analysis was performed using one-way ANOVA. Data are presented as mean values鈥壜扁€塖EM.Full size imageFig. 5: Glycolytic activity.a Extracellular acidification rate (ECAR) of Ctrl and PD astrocytes. b Basal glycolysis, expressed as pmol/min of glycolytic proton efflux rate (PER), c Compensatory glycolysis and d Basal PER, expressed as pmol/min of PER, are increased in PD astrocytes compared to the controls. e PER from glycolysis is used as an internal control and it is similar in the four lines (n鈥?鈥?). Statistical analysis was performed using one-way ANOVA. Data are presented as mean values鈥壜扁€塖EM.Full size imageMitochondrial malfunction is frequently associated with aberrant morphology29 and, therefore, we compared intracellular distribution and the ultrastructure of mitochondria in healthy and PD astrocytes. Mitochondrial distribution and gross morphology was visualised by staining with Rhodamin 123. In healthy astrocytes, mitochondria were elongated and interconnected, forming a homogenous network distributed throughout the entire cytoplasm, being present in the soma and in the principal processes (Fig. 6a). In contrast, PD astrocytes had fewer mitochondria, which were apparently more fragmented and mainly concentrated in the perinuclear region; in addition mitochondria were absent from short processes (Fig. 6a). The very same distribution pattern was observed after staining with Mitotracker (Supplementary Fig. 5), which demonstrates evident perinuclear concentration of mitochondria in PD astrocytes. Ultrastructural analysis of mitochondria (Fig. 6b), revealed further differences between the healthy and PD astrocytes. The measurement of the circularity, usually used as an index of ROS production, demonstrated that mitochondria in PD astrocytes were more rounded than in the control cells (Fig. 6c). Accordingly, the Aspect Ratio (the major axis divided the minor axis of the mitochondria) was higher in healthy astrocytes indicating the presence of more elongated mitochondria.Fig. 6: Analysis of mitochondrial morphology.a Mitochondrial staining with Rhodamin 123 of hiA cultures from healthy (Ctrl1-2) and patients (PD1-2) donors . Images were taken with the confocal microscope Leica TCS STED CW SP8. Squared inlets represent a higher magnification of the field. Scale bar 20鈥壩糾. b Representative images of mitochondrial ultrastructure in Ctrl and PD astrocytes. c Circularity is measured considering 1 as the perfect circle and d aspect ratio (ratio of circularity vs. elongation) reveal a more rounded shape in PD astrocyte mitochondria compared to the control. More than 100 mitochondria were analysed for each line. Statistical analysis was performed using one-way ANOVA. Data are presented as mean values鈥壜扁€塖EM.Full size imageAccording to the hypothesis by which fragmented mitochondria are associated with higher levels of ROS29,30, we investigated astrocytic metabolic profile. Using Oxyblot analysis, we measured the carbonyl groups of total proteins extracted from healthy and diseased lines as a readout of the oxidative status of the proteins. We found higher amount of oxidised proteins (32%) in PD astrocytes when compared to the controls (Fig. 7), suggesting a basal oxidative status of astrocytes in PD higher than in healthy astrocytes. We may conclude, therefore, that LRRK2G2019S mutation corresponds to a general mitochondrial dysfunction in astrocytes, with impaired mitochondrial respiration, cellular localisation and mitochondrial ultrastructure.Fig. 7: Detection of oxidised proteins in total astrocyte protein.Total proteins from hiA cultures were extracted after 60 days of in vitro maturation. Oxidised proteins are visualised after Western blot analysis as the conversion of the 2,4-dinitrophenol (DNP) to 2,4-dinitrophenylhydrazine (DNPH). Each sample is loaded as a negative control (Neg) with non-derivatised procedure. DNPH levels were normalised with total proteins stained with Red Ponceau.Full size imageDiscussionTo study human diseases, the 鈥渉umanised鈥?experimental preparations are essential; even the most sophisticated animal models of human pathologies are not faithful replicas31,32. In this paper, we analysed morphological characteristics and metabolic profile of astrocytes derived from iPSCs generated from PD patients bearing the LRRK2G2019S mutation. Using different combinations of several laminins coating, we obtained almost homogeneous cultures of human astrocytes (95%鈥?8%). The purity of cultures was confirmed by citofluorimetry analysis (Supplementary Fig. 3). We simulated the physiological conditions occurring during the embryonic development by mixing laminins LN521 and LN511. Both these laminins are expressed in the inner cell mass and support survival and self-renewal of the pluripotent stem cells through the interaction with 伪6尾1 integrin and PI3/Akt activation33,34. In contrast, mature astrocytes express LN111 and LN21135,36; activation of these two laminins supports cell differentiation and specialisation, such as, for example, the maintenance of the blood-brain barrier integrity36.The role of astrocytes in pathological progress of PD is yet to be fully characterised. Recent works conducted on some inflammatory experimental paradigms have suggested two subtypes of astrocytes, A1 and A2 with neurotoxic and neuroprotective profiles37. The A1/A2 dichotomy has been based on correlative analysis of limited number of genes detected for specific conditions in the in vitro settings. This binary polarisation has not been confirmed38,39,40,41,42,43 and, similarly to once popular, but now discarded, M1/M2 microglial polarisation concept, has been repudiated by neuroglial community44.Nonetheless, early in vitro experiments have clearly demonstrated that astrocytes protect and support survival of dopaminergic neurones45. Subsequent studies revealed that functional exhaustion and loss of astroglial homoeostatic support are dominant glial contribution to the PD, and the special definition of 鈥渄ysfunctional鈥?astrocytes has been introduced22,46. Furthermore, analysis of post-mortem samples of susbtantia nigra obtained from PD patients demonstrated significant decrease in expression of astroglial markers compared to healthy controls47,48; these findings being in general agreement with our concept of astroglial atrophy linked to the disease. Astroglial asthenia, atrophy and loss of homoeostatic and neuroprotective capacities were noted in aging40 and in various neurodegenerative and neuropsychiatric diseases23,49; astroglial atrophy thus represents a defined class of astrogliopathies50.Obtaining an almost pure population of iPSC-derived astrocytes allowed us to study human astrocytes bearing pathophysiological signature. We have found a prominent aberrant morphology of astrocytes derived from PD LRRK2G2019S patients. Differentiated astrocytic cultures, obtained from both healthy and PD subjects, expressed classical astrocyte markers (GFAP, S100B, CD49f and EAAT2). The PD astrocytes, however, were characterised by substantially smaller area and perimeter; they also show diminished complexity of primary and secondary processes as evidenced by high-content screening and Sholl analysis. These morphological changes do not represent culture artefact because this atrophy was observed only in fully differentiated astrocytes and not at preceding derivation stages. Previously published study of iPSCs-derived astrocytes with LRRK2G2019S mutation51 did not found conspicuous morphological changes, although astrocytic appearance was not analysed in detail. We assume that the use of specific feeder layers (laminins) and smaller number of cell passages in our protocol (differentiation to astrocyte proceeds with weekly passages) diminishes cell reactivity thus reliably revealing cell morphology. Our findings of pronounced morphological atrophy in human iPSCs parallel recent demonstration of similar morphological atrophy in iPSC-derived astrocytes generated from early familial and late sporadic AD patients52. Morphological atrophy of astrocytes is arguably associated with neuronal damage (due to failed homoeostatic support) and aberrant synaptic connectivity manifest in neurodegenerative and psychiatric diseases (for a review see ref. 50). In many cases, astrocytic atrophy precedes cell death and neuronal degeneration. For example, in acute excitotoxic neurodegeneration and ALS, morphological aberrations are accompanied with the down-regulation of glutamate transporters and increased excitotoxicity18. Morphological atrophy in AD has been described in animal models54,55,56, in human iPSC-derived astrocytes from patients53, in deprenil-based brain imaging in patients57, and in post-mortem brain at late stages of the disease (Rodriguez and Verkhratsky, unpublished results). In our culture conditions atrophic astrocytes from PD patients showed normal viability, as demonstrated by expression of classical markers (Fig. 1) and by physiological [Ca2+]i dynamics (Fig. 3). At the same time PD astrocytes demonstrate reduced mitochondrial functionality (Figs 4鈥?a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"/articles/s41531-021-00175-w#Fig6\">6). Mitochondrial aberrations and morphological atrophy may explain why astrocytes in PD with LRRK2G2019S mutation fail to support and protect neurones. This loss of function became even more evident in specific brain regions, specifically for substantia nigra pars compacta and striatum, where astrocytic density is lower compared to other regions20. Furthermore, astrocytes from substantia nigra seem to be unusually vulnerable to ischaemic attack58 and oxidative stress59. Loss of astroglial support may act as an exacerbating factor in neurodegenerative process; chronically malfunctional astroglia was suggested to contribute to death of dopaminergic neurones60. It is early to conclude that astrocytic atrophy is the hallmark of PD until the same astrocytic atrophy is characterised in situ in patient鈥檚 brain tissues or in astrocytes derived from other mutations. Detailed analysis of astrocyte morphology should be performed in other regions of the brain that are related with dopaminergic degeneration (e.g., subthalamic nucleus or globus pallidum) and not only in degenerating regions where astrocytes are pathologically remodelled.Mutation in the LRRK2 gene may be specifically responsible for both aberrant morphology and mitochondrial dysfunction observed in LRRK2G2019S hiA. Abnormalities in neurites outgrowth and branching are among the earliest pathological phenotypes observed in LRRK2 mutations61,62. It has been initially proposed that the origin of these morphological changes could be related to an apoptotic process62; however, further studies provided evidence for an association of LRRK2 with tubulin/actin, thus suggesting that morphological changes may be consequences of LRRK2-modulation of cytoskeletal dynamics63. Several lines of evidence suggest the relationship of LRRK2 protein with the cytoskeleton: (i) The GTPase domain of LRRK2 protein can pull-down 伪/尾 tubulin from cell lysates of mouse fibroblasts and human embryonic kidney64; (ii) LRRK2 co-precipitates with 尾 tubulin from wild-type mouse brain and (iii) Recombinant LRRK2 can phosphorylate 尾 tubulin in vitro65. High-throughput screening of LRRK2 interactome revealed proteins of the actin family and of the actin-regulatory network as interactors of LRRK2 in actin polymerisation in vitro16. We presume therefore, that the atrophy observed in PD LRRK2G2019S astrocytes could be a consequence of the mutated LRRK2 protein breakdown that becomes unable to properly modulate cytoskeletal dynamics. Similarly, LRRK2 mutation can be responsible for mitochondrial dysfunction and fragmentation, as already observed in fibroblasts, neural stem cells or neuroblastoma cell lines13,66,67,68. Multiple studies demonstrated that LRRK2 loss of function, associated with G2019S and R1441G mutations impair mitochondrial oxidative state increasing the neuronal susceptibility to oxidative stress damage69,70,71. One possible explanation might be a mitochondrial DNA damage induced by the LRRK2 mutations, which was observed in midbrain cultures and PD patient-derived lymphoblastoid cell lines72.The observations that LRRK2 mutation may be responsible for morphological atrophy and mitochondrial malfunction, indicate possible mechanism associated with reduced neuroprotection in this mutation carriers. A recent observation demonstrated that G2019S mutation in hiA alters the astrocyte-to-neurone communication mediated by extracellular vesicles73. In this work, the LRRK2 mutation in astrocytes was claimed to affect morphology and the content of extracellular vesicles and multivesicular bodies (MVB). The authors found that neurones incorporated astrocyte MVB with an abnormal accumulation of key PD-related proteins such as LRRK2 and phospho-S129 伪-Syn. Dopaminergic neurones incorporating the dysfunctional MVB released by the LRRK2G2019S astrocytes showed an aberrant morphology73.In this study, we propose an hallmark for PD with LRRK2G2019S mutation. Our hypothesis postulates that astrocytes with this mutation fail to support neurones because of loss of homoeostatic support resulting from substantial morphological atrophy and loss of complexity; in addition, astrocytes demonstrated mitochondrial dysfunction that also affects their neuroprotective capabilities.MethodsHuman samplesHuman fibroblasts were obtained from two healthy donors (Ctrl1 was purchased from AXOL and Ctrl 2 from the Coriell stem cell bank) and two Parkinson麓s disease patients with LRRK2G2019S mutation (PD1 from the Coriell stem cell bank and PD 2 provided by the BioDonostia Hospital, San Sebastian, Spain) (Supplementary Table 1). Control patients who matched PD donors in age and gender did not show any neurological symptoms. All procedures with human cells were approved by the National and local ethical committees (with code M30_2018_030) according to the National law 14/2007 on Biomedical research.Generation of human induced astrocytes (hiA)Fibroblasts were grown in DMEM F12 (Gibco/ThermoFisher, Spain) and infected with the CytoTune iPS 2.0 Sendai Reprogramming Kit (Thermofisher, Spain) as described in Supplementary Fig. 1. The commercial Sendai virus expressed the key genetic factors necessary for reprogramming somatic cells into iPSCs (Klf4/Oct3-4/Sox2-KOS, hc-Myc, Klf4). Infection efficiency was evaluated by co-infection with a EmGFP fluorescent reporter plasmid provided by the kit. Seven days later, transduced fibroblasts were seeded in Geltrex (ThermoFisher, Spain) in Essential 8 Flex medium (E8, Gibco/ThermoFisher, Spain). E8 medium was changed every day for 21 days until we observed the formation of iPSC colonies. Colonies were manually isolated using a 27G Braun Sterican Needle and replated in laminin-521 (LN521-Biolamina, Sundbyberg Sweden) with E8 medium and ROCK inhibitor (Y-27632; Millipore, Madrid, Spain). The day after, ROCK inhibitor was removed and replaced with fresh medium. Colonies were sequentially isolated and re-suspended at single cell level. Embryoid bodies (EB) were generated (Supplementary Fig. 2) after re-suspending the iPSC colonies in Essential 6 medium (E6, Gibco) for 2鈥? days in the AggrewellTM800 plates (StemCell, Grenoble, France). Half-medium in the microwells was replaced daily with fresh medium. EBs were then seeded in a LN521/LN211 mix (50% each) (Biolamina) and the differentiation to neural precursor cells (NPC) as neural rosettes was promoted using the STEMdiff Neural Induction Medium (Stemcell). After 7 days, neural rosettes were selected and detached using the STEMdiff Neural Rosette Selection Reagent (Stemcell). Cells were incubated for 2鈥塰 with this reagent at 37鈥壜癈 with 5% CO2 and then, mechanically re-suspended at single cell level and seeded in LN211/LN111 (50% each) (Biolamina). Differentiation of NPC to progenitor astrocytes was triggered using the astrocyte differentiation medium (STEMdiff astrocyte differentiation #100-0013, StemCell). To maintain the appropriate cell density (70% of confluence) cells were passed every week in the same coating mix during 21 days. Maturation. Finally, astrocytes progenitor cells were maturated in the Astrocyte Maturation Medium (STEMdiff astrocyte maturation #100-0016, StemCell) for 60 to 75 days. During the whole protocol, the correct state of the cells in each step was evaluated using the EVOS FL microscope (Life Technologies, AME4300). See Supplementary Fig. 1 for an overview of simplified protocol steps.Cytofluorimetry assayCells (500.000) were detached with TryPLE (Sigma, Spain) and fixed as a single cell suspension with PFA 4% for 10鈥塵in. Cells were washed in phosphate-buffered saline (PBS) at 400鈥墄鈥?i>g for 5鈥塵in and re-suspended in blocking solution (0.5鈥塯 BSA in PBS with 0.01% Triton (PBS-T) with agitation overnight at 4鈥壜癈. The following day cells were washed and incubated with the primary antibody goat anti-GFAP (Abcam, 53554) for 2鈥塰 at 4鈥壜癈. After further wash for 5鈥塵in in PBS-T 0.01% cell suspension was incubated with the secondary conjugated antibody Alexa fluor 488 donkey anti-goat for 1鈥塰 at 4鈥壜癈. After a further wash with PBS-T 0.01%, cells were finally re-suspended in PBS 1x. Cells were analysed in the BD FACSJazz (USB, inFlux Compact) analyser using the Blue 488 laser. Unstained cells were gated and used as a negative control.Calcium imagingCytosolic calcium (Ca2+) levels were estimated by the 340/380 ratiometric microfluorimetry as described previously74. Astrocytes were loaded with fura-2 AM (5鈥壩糓; ThermoFisher/Invitrogen) for 20 at 37鈥壜癈 and subsequently washed in the recording solution containing 137鈥塵M NaCl, 5.3鈥塵M KCl, 0.4鈥塵M KH2PO4, 0.35鈥塵M Na2HPO4, 20鈥塵M HEPES, 4鈥塵M NaHCO3, 10鈥塵M glucose, 1鈥塵M MgCl2, 2鈥塵M CaCl2 (pH 7.4) to allow de-esterification. In experiments with FCCP, Ca2+ was omitted from the recording solution. Experiments were performed in a coverslip chamber continuously perfused with buffer at 1鈥塵l/min by exposing the cells to ATP (100鈥壩糓) or FCCP (1鈥壩糓). The perfusion chamber was mounted on the stage of a Zeiss (Oberkochen, Germany) inverted epifluorescence microscope (Axiovert 35), equipped with a 150鈥塛 xenon lamp Polychrome IV (T.I.L.L. Photonics, Martinsried, Germany), and a Plan Neofluar 403 oil immersion objective (Zeiss). Cells were visualised with a high-resolution digital black/white CCD camera (ORCA, Hamamatsu Photonics Iberica, Barcelona, Spain) and images were acquired every 5鈥塻. Image acquisition and data analysis were performed using the AquaCosmos software programme (Hamamatsu Photonics Iberica). Intracellular Ca2+ measurements were expressed as the ratio of F340/F380 and normalised to baseline values. Results for statistical comparison were calculated as area under the curve (AUC) of the response for each cell from the start of each compound application.ImmunofluorescenceCell cultures were fixed in 4% para-formaldehyde (Merck/Sigma), permeabilised with 0.1% Triton (Sigma) and non-specific epitopes were blocked with 5% normal goat serum in PBS. Primary antibodies (Supplementary Table 2) were incubated overnight and then washed three times with 0.1% Triton in PBS. Secondary conjugated antibodies Alexa 488, Alexa 594, Alexa 647 or Alexa 405 (Invitrogen, 1:500), were incubated for 1鈥塰 in the dark at room temperature. After three washes with 0.1% Triton in PBS, cell nuclei were counter-stained for 1鈥塵in with DAPI (ThermoFisher). Finally, coverslips were mounted with Glycergel (Dako, Barcelona, Spain) and analysed using the confocal microscope Leica TCS STED CW SP8.Morphological analysis by high-content screening Cells were seeded in glass bottom Cellvis 24-well plates (Cellvis, Bilbao, Spain) coated with LN111/LN211 (Biolamina). After fixation with 4% PFA for 8鈥塵in, cells were immunostained for GFAP expression (Goat anti-GFAP, Abcam 53554). Alexa fluor Donkey anti-goat was used as a secondary antibody. Images were taken with the CellInsight CX7 high-content screening system (Thermo Scientific) using a 10x objective. Morphological parameters for area (defined as the number of microns squared of the object), perimeter (length of the boundary of the object) and ShapeP2A (measure of the ratio of the perimeter squared of the object to four times) were calculated with High-Content Analysis platform. More than 100 cells per cell line were analysed.Morphological assessmentSholl analysis was performed with the public software Fiji75, to measure the complexity of GFAP-positive human astrocytes. A transparent grid with concentric circles (every 5鈥壩糾 from the centre of the cell soma across the whole radius) were superimposed onto the cells after immunofluorescence with GFAP antiserum. Sholl measurements were obtained by quantifying the number of intersections with each concentric circle.Electron microscopyCells were fixed in 4% PFA for 10鈥塵in and post-fixed in 3% glutaraldehyde for 30鈥塵in. After a wash in phosphate buffer (PB) samples were osmicated (1% OsO4 in 0.1鈥塎 PB; pH 7.4) for 30鈥塵in. After 3鈥墄鈥?0鈥塵in washes in 0.1鈥塎 PB, samples were dehydrated in graded ethanol concentrations (50% to100%) to propylene oxide and embedded in epoxy resin (Sigma-Aldrich) by immersion in decreasing concentration of propylene oxide (1:3 for 30鈥塵in, 1:1 for 1 h and 3:1 for 2鈥塰). Samples were then embedded in fresh resin overnight and allowed to polymerise at 60鈥壜癈 for 2 days. Following visualisation at the light microscope, selected portions were trimmed and glued onto epoxy resin capsules. Semi-thin sections (500鈥塶m-thick were cut from epoxy blocks using a Power Tome ultramicrotome (RMC Boeckeler, Tucson, AZ, USA and stained with 1% toluidine blue. Ultrathin (50鈥?0鈥塶m thick) sections were then cut with diamond knife (Diatome, Hatfield PA, USA), collected on nickel mesh grids and stained with 4% uranyl acetate for 30鈥塵in and 2.5% lead citrate for electron microscope visualisation. For Image Acquisition and analysis, electron microscopy images of mitochondria were taken from randomly selected fields with a Jeol JEM 1400 Plus electron microscope at the Service of Analytical and High-Resolution Microscopy in Biomedicine of University of the Basque Country UPV/EHU. Images were taken at a magnification of 12,000X. Circularity and aspect ratio (ratio of circularity vs. elongation) were measured with Fiji-Software using a self-made plug-in. More than 100 mitochondria were analysed for each line.Mitochondrial membrane potential (螖唯m) measurementMitochondrial membrane potential (螖唯m) of human astrocytes was assessed by the Rhodamine 123 (Rh123) staining. Briefly, cells were seeded in 35鈥塵m glass bottom plates (Ibidi GmbH, Germany) at a mean density confluence of 50鈥?0% and loaded with 10鈥壩糓 Rh123 at 37鈥壜癈 and 5% CO2. After 15鈥塵in cells were washed with 900鈥壩糽 Hanks鈥?balanced salt solution and analysed by time lapse every 15鈥塻 for 5鈥塵in using the confocal microscope Leica TCS STED CW SP8. To establish the basal line, cells were stimulated with 1鈥壩糓 FCCP after the first 60鈥塻. Fluorescence intensity after FCCP treatment was measured with the Leica LASX Software and data were analysed with GraphPad Prism 5 (San Diego, CA, USA).Measurement of mitochondrial function and glycolytic activityThe oxygen consumption rate (OCR), as an indicator of mitochondrial respiration, the extracellular acidification rate (ECAR), as indicator of glycolytic activity, and the proton efflux rate (PER), which correlates with lactate production, were measured with the Seahorse XF96 extracellular flux analyser. For the analysis of mitochondrial respiration, human astrocytes (30,000 cells/ mm2) were seeded in LN211/LN111 (Biolamina) precoated wells. The day of the experiment, cell medium was changed to Mito XF Medium (XF basal medium with phenol red, 0.001鈥塎 piruvic acid, 0.002鈥塎 glutamine, glucose 0.01鈥塎, pH 7.4). The OCRs were obtained after the sequential treatment with oligomycin (2鈥壩糓), FCCP (1鈥壩糓), and rotenone combined with antimycin A (0.5鈥壩糓). To measure the glycolytic activity, we used the same protocol with the following modifications. The day of the experiment, cell medium was changed to Glico XF Medium without phenol red (DMEM Base Medium without Phenol Red with 5鈥塵M HEPES, 10鈥塵M glucose, 1鈥塵M sodium pyruvate, 2鈥塵M glutamine, pH 7.4 at 37鈥壜癈). The ECAR and PER were obtained after the sequential treatment with rotenone combined with antimycin A (0.5鈥壩糓) and 2DG (50鈥塵M), respectively. Four replicates were performed for each condition or cell type for every experiments (n鈥?鈥?). Data was analysed with the Wave 2.4.0 software.Western blot for detection of oxidatively modified proteins (oxyblot)Astrocytes (30.000/well) were maturated for 60 days in 24-well plates coated with LN211/LN111 (Biolamina) and solubilised for 20鈥塵in with equal volume of 2x Extraction Buffer. Samples were prepared according to manufacturer鈥檚 instruction with all the reagents provided in Oxidised protein western blot detection kit (ab178020; Abcam). Briefly, the carbonyl groups in the protein side chains were derivatised to 2,4-dinitrophenylhydrazone (DNP-Hydrazone) by reaction with 2,4-dinitrophenylhydrazine (DNPH). Two aliquots of each sample were prepared to be analysed simultaneously. One aliquot was treated with 鈥渄erivatisation reaction鈥?(DNPH Solution) and the other control aliquot was treated with 鈥渄erivatisation control reaction鈥? Protein concentration was quantified with a detergent-compatible assay reagent (Pierce BCA Protein Assay Kit) according to the manufacturer鈥檚 instructions (ThermoFisher Scientific). Proteins loading was normalised after Red Ponceau staining. All blots derive from the same experiment and they are processed in parallel.Statistical analysisResults are expressed as mean鈥壜扁€塻tandard error of the mean (S.E.M) with n corresponding to the number of cells or cultures tested. Data were analysed with Excel (Microsoft, Seattle, WA, USA) and GraphPad Prism software. Statistical significance between datasets was tested using one-way analysis of variance (ANOVA) followed by Newman鈥揔euls multiple comparison test, with a significance threshold of p鈥?lt;鈥?.05.Reporting summaryFurther information on research design is available in the Nature Research Reporting Summary linked to this article. The data that support the findings of this study are available from the corresponding author upon reasonable request. References1.Parkinson, J. An essay on the Shaking Palsy. Arch. Neurol. 20, 441鈥?45 (1969). Google Scholar聽 2.Reeve, A., Simcox, E. Turnbull, D. Ageing and Parkinson鈥檚 disease: why is advancing age the biggest risk factor? Ageing Res. Rev. 14, 19鈥?0 (2014).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 3.Spillantini, M. G. et al. 伪-synuclein in Lewy bodies. Nature 388, 839鈥?40 (1997).CAS聽Google Scholar聽 4.Ross, C. A. Poirier, M. A. Protein aggregation and neurodegenerative disease. Nat. Med. 10, S10鈥揝17 (2004).PubMed聽Google Scholar聽 5.Desplats, P. et al. Inclusion formation and neuronal cell death through neuron-to-neuron transmission of 伪-synuclein. Proc. Natl Acad. Sci. 106, 13010鈥?3015 (2009).CAS聽 PubMed聽Google Scholar聽 6.Blauwendraat, C., Nalls, M. A. Singleton, A. B. The genetic architecture of Parkinson鈥檚 disease. Lancet Neurol. 19, 170鈥?78 (2019).PubMed聽Google Scholar聽 7.De Wit, T., Baekelandt, V. Lobbestael, E. LRRK2 phosphorylation: behind the scenes. Neuroscientist 24, 486鈥?00 (2018).PubMed聽Google Scholar聽 8.Singleton, A. B., Farrer, M. J. Bonifati, V. The genetics of P arkinson鈥檚 disease: progress and therapeutic implications. Mov. Disord. 28, 14鈥?3 (2013).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 9.West, A. B. et al. Parkinson鈥檚 disease-associated mutations in LRRK2 link enhanced GTP-binding and kinase activities to neuronal toxicity. Hum. Mol. Genet. 16, 223鈥?32 (2007).CAS聽 PubMed聽Google Scholar聽 10.Ju谩rez-Flores, D. L. et al. Exhaustion of mitochondrial and autophagic reserve may contribute to the development of LRRK2 G2019S-Parkinson鈥檚 disease. J. Transl. Med. 16, 1鈥?3 (2018). Google Scholar聽 11.H盲big, K. et al. LRRK2 guides the actin cytoskeleton at growth cones together with ARHGEF7 and Tropomyosin 4. Biochim. Biophys. Acta (BBA)-Mol. Basis Dis. 1832, 2352鈥?367 (2013). Google Scholar聽 12.Mortiboys, H., Johansen, K. K., Aasly, J. O. Bandmann, O. Mitochondrial impairment in patients with Parkinson disease with the G2019S mutation in LRRK2. Neurology 75, 2017鈥?020 (2010).CAS聽 PubMed聽Google Scholar聽 13.Wang, X. et al. LRRK2 regulates mitochondrial dynamics and function through direct interaction with DLP1. Hum. Mol. Genet. 21, 1931鈥?944 (2012).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 14.Papkovskaia, T. D. et al. G2019S leucine-rich repeat kinase 2 causes uncoupling protein-mediated mitochondrial depolarization. Hum. Mol. Genet. 21, 4201鈥?213 (2012).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 15.Hsieh, C. H. et al. Functional impairment in miro degradation and mitophagy is a shared feature in familial and sporadic Parkinson鈥檚 disease. Cell Stem Cell 19, 709鈥?24 (2016).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 16.Meixner, A. et al. A QUICK screen for Lrrk2 interaction partners鈥搇eucine-rich repeat kinase 2 is involved in actin cytoskeleton dynamics. Mol. Cell. Proteomics 10 M110.001172 (2011)17.Verkhratsky, A. Nedergaard, M. Physiology of astroglia. Physiological Rev. 98, 239鈥?89 (2018).CAS聽Google Scholar聽 18.Pekny, M. et al. Astrocytes: a central element in neurological diseases. Acta Neuropathologica 131, 323鈥?45 (2016).CAS聽 PubMed聽Google Scholar聽 19.Verkhratsky, A., Steardo, L., Parpura, V. Montana, V. Translational potential of astrocytes in brain disorders. Prog. Neurobiol. 144, 188鈥?05 (2016).CAS聽 PubMed聽Google Scholar聽 20.von Bartheld, C. S., Bahney, J. Herculano-Houzel, S. The search for true numbers of neurons and glial cells in the human brain: a review of 150 years of cell counting. J. Comp. Neurol. 524, 3865鈥?895 (2016). Google Scholar聽 21.Song, Y. J. C. et al. Degeneration in different parkinsonian syndromes relates to astrocyte type and astrocyte protein expression. J. Neuropathol. Exp. Neurol. 68, 1073鈥?083 (2009).CAS聽 PubMed聽Google Scholar聽 22.Booth, H. D., Hirst, W. D. Wade-Martins, R. The role of astrocyte dysfunction in Parkinson鈥檚 disease pathogenesis. Trends Neurosci. 40, 358鈥?70 (2017).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 23.Verkhratsky, A., Marutle, A., Rodriguez-Arellano, J. J. Nordberg, A. Glial asthenia and functional paralysis: a new perspective on neurodegeneration and Alzheimer鈥檚 disease. Neuroscientist 21, 552鈥?68 (2015).CAS聽 PubMed聽Google Scholar聽 24.Lee, J. H. et al. Parkinson鈥檚 disease-associated LRRK2-G2019S mutant acts through regulation of SERCA activity to control ER stress in astrocytes. Acta Neuropathologica Commun. 7, 1鈥?9 (2019). Google Scholar聽 25.Shigetomi, E., Saito, K., Sano, F. Koizumi, S. Aberrant calcium signals in reactive astrocytes: a key process in neurological disorders. Int. J. Mol. Sci. 20, 996 (2019).CAS聽 PubMed Central聽Google Scholar聽 26.Grolla, A. A. et al. Amyloid-尾 and Alzheimer鈥檚 disease type pathology differentially affects the calcium signalling toolkit in astrocytes from different brain regions. Cell Death Dis. 4, e623 (2013).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 27.Kuchibhotla, K. V., Lattarulo, C. R., Hyman, B. T. Bacskai, B. J. Synchronous hyperactivity and intercellular calcium waves in astrocytes in Alzheimer mice. Science 323, 1211鈥?215 (2009).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 28.Stafa, K. et al. Functional interaction of Parkinson鈥檚 disease-associated LRRK2 with members of the dynamin GTPase superfamily. Hum. Mol. Genet. 23, 2055鈥?077 (2014).CAS聽 PubMed聽Google Scholar聽 29.Picard, M., Shirihai, O. S., Gentil, B. J. Burelle, Y. Mitochondrial morphology transitions and functions: implications for retrograde signaling? Am. J. Physiol. Regul. Integr. Comp. Physiol. 304, R393鈥揜406 (2013).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 30.Je啪ek, J., Cooper, K. F. Strich, R. Reactive oxygen species and mitochondrial dynamics: the yin and yang of mitochondrial dysfunction and cancer progression. Antioxidants 7, 13 (2018).PubMed Central聽Google Scholar聽 31.Hartung, T. Thoughts on limitations of animal models. Parkinsonism Relat. Disord. 14, S81鈥揝83 (2008).PubMed聽Google Scholar聽 32.Ransohoff, R. M. All (animal) models (of neurodegeneration) are wrong. Are they also useful? J. Exp. Med. 215, 2955 (2018).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 33.Domogatskaya, A., Rodin, S. Tryggvason, K. Functional diversity of laminins. Annu. Rev. Cell Dev. Biol. 28, 523鈥?53 (2012).CAS聽 PubMed聽Google Scholar聽 34.Rodin, S., Antonsson, L., Hovatta, O. Tryggvason, K. Monolayer culturing and cloning of human pluripotent stem cells on laminin-521鈥揵ased matrices under xeno-free and chemically defined conditions. Nat. Protoc. 9, 2354 (2014).CAS聽 PubMed聽Google Scholar聽 35.Liddelow, S. A. et al. Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541, 481鈥?87 (2017).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 36.Al-Dalahmah, O. et al. Single-nucleus RNA-seq identifies Huntington disease astrocyte states. Acta Neuropathol. Commun. 8, 19 (2020).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 37.Amini-Bavil-Olyaee, S. et al. Genotype characterization and phylogenetic analysis of hepatitis B virus isolates from Iranian patients. J. Med. Virol. 75, 227鈥?34 (2005).CAS聽 PubMed聽Google Scholar聽 38.Das, S., Li, Z., Noori, A., Hyman, B. T. Serrano-Pozo, A. Meta-analysis of mouse transcriptomic studies supports a context-dependent astrocyte reaction in acute CNS injury versus neurodegeneration. J. Neuroinflammation 17, 227 (2020).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 39.Diaz-Castro, B., Gangwani, M. R., Yu, X., Coppola, G. Khakh, B. S. Astrocyte molecular signatures in Huntington鈥檚 disease. Sci. Transl. Med. 11, eaaw8546 (2019).CAS聽 PubMed聽Google Scholar聽 40.Grubman, A. et al. A single-cell atlas of entorhinal cortex from individuals with Alzheimer鈥檚 disease reveals cell-type-specific gene expression regulation. Nat. Neurosci. 22, 2087鈥?097 (2019).CAS聽 PubMed聽Google Scholar聽 41.Zhou, Y. et al. Human and mouse single-nucleus transcriptomics reveal TREM2-dependent and TREM2-independent cellular responses in Alzheimer鈥檚 disease. Nat. Med. 26, 131鈥?42 (2020).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 42.Sixt, M. et al. Endothelial cell laminin isoforms, laminins 8 and 10, play decisive roles in T cell recruitment across the blood鈥揵rain barrier in experimental autoimmune encephalomyelitis. J. Cell Biol. 153, 933鈥?46 (2001).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 43.Yao, Y., Chen, Z. L., Norris, E. H. Strickland, S. Astrocytic laminin regulates pericyte differentiation and maintains blood brain barrier integrity. Nat. Commun. 5, 1鈥?2 (2014).CAS聽Google Scholar聽 44.Escartin, A. C. et al. Consensus paper: Reactive astrocyte nomenclature, definitions, and future directions. Nat. Neurosci. 24, 312鈥?25 (2021).CAS聽 PubMed聽Google Scholar聽 45.Mena, M. A., Casarejos, M. J., Carazo, A., Paino, C. L. de Y茅benes, J. G. Glia conditioned medium protects fetal rat midbrain neurones in culture from L-DOPA toxicity. Neuroreport 7, 441鈥?45 (1996).CAS聽 PubMed聽Google Scholar聽 46.Mena, M. A. Garcia De Yebenes, J. Glial cells as players in parkinsonism: the 鈥済ood,鈥?the 鈥渂ad,鈥?and the 鈥渕ysterious鈥?glia. Neuroscientist 14, 544鈥?60 (2008).CAS聽 PubMed聽Google Scholar聽 47.Tong, J. et al. Low levels of astroglial markers in Parkinson鈥檚 disease: relationship to alpha-synuclein accumulation. Neurobiol. Dis. 82, 243鈥?53 (2015).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 48.Kano, M. et al. Reduced astrocytic reactivity in human brains and midbrain organoids with PRKN mutations. NPJ Parkinson鈥檚 Dis. 6, 33 (2020).CAS聽Google Scholar聽 49.Verkhratsky, A. et al. Astroglial asthenia and loss of function, rather than reactivity, contribute to the ageing of the brain. Pflugers Arch-Eur. J. Physiol. E-pub ahead of print, https://doi.org/10.1007/s00424-020-02465-3 (2020).50.Verkhratsky, A., Rodrigues, J. J., Pivoriunas, A., Zorec, R. Semyanov, A. Astroglial atrophy in Alzheimer鈥檚 disease. Pfl眉gers Arch.-Eur. J. Physiol. 471, 1247鈥?261 (2019).CAS聽Google Scholar聽 51.Verkhratsky, A., Zorec, R. Parpura, V. Stratification of astrocytes in healthy and diseased brain. Brain Pathol. 27, 629鈥?44 (2017).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 52.di Domenico, A. et al. Patient-specific iPSC-derived astrocytes contribute to non-cell-autonomous neurodegeneration in Parkinson鈥檚 disease. Stem Cell Rep. 12, 213鈥?29 (2019). Google Scholar聽 53.Jones, V. C., Atkinson-Dell, R., Verkhratsky, A. Mohamet, L. Aberrant iPSC-derived human astrocytes in Alzheimer鈥檚 disease. Cell Death Dis. 8, e2696鈥揺2696 (2017).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 54.Olabarria, M., Noristani, H. N., Verkhratsky, A. Rodr铆guez, J. J. Concomitant astroglial atrophy and astrogliosis in a triple transgenic animal model of Alzheimer鈥檚 disease. Glia 58, 831鈥?38 (2010).PubMed聽Google Scholar聽 55.Beauquis, J. et al. Environmental enrichment prevents astroglial pathological changes in the hippocampus of APP transgenic mice, model of Alzheimer鈥檚 disease. Exp. Neurol. 239, 28鈥?7 (2013).CAS聽 PubMed聽Google Scholar聽 56.Polis, B., Srikanth, K. D., Elliott, E., Gil-Henn, H. Samson, A. O. L-Norvaline reverses cognitive decline and synaptic loss in a murine model of Alzheimer鈥檚 disease. Neurotherapeutics 15, 1036鈥?054 (2018).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 57.Rodriguez-Vieitez, E. et al. Diverging longitudinal changes in astrocytosis and amyloid PET in autosomal dominant Alzheimer鈥檚 disease. Brain 139, 922鈥?36 (2016).PubMed聽 PubMed Central聽Google Scholar聽 58.Karunasinghe, R. N., Dean, J. M. Lipski, J. Acute sensitivity of astrocytes in the Substantia Nigra to oxygen and glucose deprivation (OGD) compared with hippocampal astrocytes in brain slices. Neurosci. Lett. 685, 137鈥?43 (2018).CAS聽 PubMed聽Google Scholar聽 59.Elgayar, S. A., Abdel-Hafez, A. A., Gomaa, A. M. Elsherif, R. Vulnerability of glia and vessels of rat substantia nigra in rotenone Parkinson model. Ultrastructural Pathol. 42, 181鈥?92 (2018). Google Scholar聽 60.Kuter, K., Olech, 艁. G艂owacka, U. Prolonged dysfunction of astrocytes and activation of microglia accelerate degeneration of dopaminergic neurons in the rat substantia nigra and block compensation of early motor dysfunction induced by 6-OHDA. Mol. Neurobiol. 55, 3049鈥?066 (2018).CAS聽 PubMed聽Google Scholar聽 61.Tsika, E. et al. Conditional expression of Parkinson鈥檚 disease-related R1441C LRRK2 in midbrain dopaminergic neurons of mice causes nuclear abnormalities without neurodegeneration. Neurobiol. Dis. 71, 345鈥?58 (2014).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 62.MacLeod, D. et al. The familial Parkinsonism gene LRRK2 regulates neurite process morphology. Neuron 52, 587鈥?93 (2006).CAS聽 PubMed聽Google Scholar聽 63.Wallings, R., Manzoni, C. Bandopadhyay, R. Cellular processes associated with LRRK 2 function and dysfunction. FEBS J. 282, 2806鈥?826 (2015).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 64.Gandhi, P. N., Wang, X., Zhu, X., Chen, S. G. Wilson-Delfosse, A. L. The Roc domain of leucine-rich repeat kinase 2 (LRRK2) is sufficient for interaction with microtubules. J. Neurosci. Res. 87, 1711鈥?720 (2008). Google Scholar聽 65.Gillardon, F. Leucine-rich repeat kinase 2 phosphorylates brain tubulin-尾 isoforms and modulates microtubule stability鈥揳 point of convergence in Parkinsonian neurodegeneration? J. Neurochem. 110, 1514鈥?522 (2009).CAS聽 PubMed聽Google Scholar聽 66.Gr眉newald, A. et al. Does uncoupling protein 2 expression qualify as marker of disease status in LRRK2-associated Parkinson鈥檚 disease?. 20, Antioxid. Redox Signal. 1955鈥?960 (2014)67.Su, Y. C. Qi, X. Inhibition of excessive mitochondrial fission reduced aberrant autophagy and neuronal damage caused by LRRK2 G2019S mutation. Hum. Mol. Genet. 22, 4545鈥?561 (2013).CAS聽 PubMed聽Google Scholar聽 68.Smith, G. A. et al. Fibroblast biomarkers of sporadic Parkinson鈥檚 disease and LRRK2 kinase inhibition. Mol. Neurobiol. 53, 5161鈥?177 (2016).CAS聽 PubMed聽Google Scholar聽 69.Reinhardt, P. et al. Genetic correction of a LRRK2 mutation in human iPSCs links parkinsonian neurodegeneration to ERK-dependent changes in gene expression. Cell Stem Cell 12, 354鈥?67 (2013).CAS聽 PubMed聽Google Scholar聽 70.Cooper, O. et al. Pharmacological rescue of mitochondrial deficits in iPSC-derived neural cells from patients with familial Parkinson鈥檚 disease. Sci. Transl. Med. 4, 141ra90鈥?41ra90 (2012).PubMed聽 PubMed Central聽Google Scholar聽 71.Nguyen, H. N. et al. LRRK2 mutant iPSC-derived DA neurons demonstrate increased susceptibility to oxidative stress. Cell Stem Cell 8, 267鈥?80 (2011).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 72.Howlett, E. H. et al. LRRK2 G2019S-induced mitochondrial DNA damage is LRRK2 kinase dependent and inhibition restores mtDNA integrity in Parkinson鈥檚 disease. Hum. Mol. Genet. 26, 4340鈥?351 (2017).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 73.de Rus Jacquet, A. et al. The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson鈥檚 disease. Preprint at bioRxiv https://doi.org/10.1101/2020.07.02.178574 (2020).74.Mato, S., S谩nchez-G贸mez, M. V., Bernal-Chico, A. Matute, C. Cytosolic zinc accumulation contributes to excitotoxic oligodendroglial death. Glia 61, 750鈥?64 (2013).PubMed聽Google Scholar聽 75.Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676鈥?82 (2012).CAS聽Google Scholar聽 Download referencesAcknowledgementsThis work was supported by BIOEF (BIO17/ND/008 to FC), Euskampus, CIBERNED (CB06/0005/0076 to C.M.), the Ministry of Economy and Competitiveness, Government of Spain (SAF2016-75292-R to C.M. and PID2019-109724RB-I00 to C.M.), FEDER and ISCIII (AES 2018-PI18/00513 to S.M.) and the Basque Government (IT1203-19 to C.M.; PIBA19-0059 to S.M.). P.R.G. was supported by a fellowship from the Basque Government. This study used fibroblast samples from the NINDS Repository, as well as personal but anonymous data. NINDS Repository sample numbers corresponding to the sample used are 38530A (healthy control) and PD33879. We deeply thank Dr. L. Escobar for her valuable contribution with cytofluorimetry assays.Author informationAffiliationsDepartment of Neurosciences, University of the Basque Country UPV/EHU, Leioa, SpainPaula Ramos-Gonzalez,聽Susana Mato,聽Juan Carlos Chara,聽Carlos Matute聽 聽Fabio CavaliereAchucarro Basque Center for Neuroscience, Leioa, SpainPaula Ramos-Gonzalez,聽Susana Mato,聽Juan Carlos Chara,聽Alexei Verkhratsky,聽Carlos Matute聽 聽Fabio CavaliereCentro de Investigaci贸n Biom茅dica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, SpainSusana Mato,聽Juan Carlos Chara,聽Carlos Matute聽 聽Fabio CavaliereBiocruces, Bizkaia, Barakaldo, SpainSusana MatoFaculty of Biology, Medicine and Health, The University of Manchester, Manchester, M13 9PT, UKAlexei VerkhratskySechenov First Moscow State Medical University, Moscow, RussiaAlexei VerkhratskyAuthorsPaula Ramos-GonzalezView author publicationsYou can also search for this author in PubMed聽Google ScholarSusana MatoView author publicationsYou can also search for this author in PubMed聽Google ScholarJuan Carlos CharaView author publicationsYou can also search for this author in PubMed聽Google ScholarAlexei VerkhratskyView author publicationsYou can also search for this author in PubMed聽Google ScholarCarlos MatuteView author publicationsYou can also search for this author in PubMed聽Google ScholarFabio CavaliereView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsP.R.G., design, performing the experiments and writing; S.M., performing experiments and comment on the final version of the paper; J.C.C., preparation of samples and experiments with electronic microscope; A.V., writing and critical revision of the manuscript; C.M., design of the experiments and conceptualisation of the study; F.C., principal designer of the experiments, writing and conceptualisation of the experiments.Corresponding authorCorrespondence to Fabio Cavaliere.Ethics declarations Competing interests The authors declare no competing interests. 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