Age-dependent Progression from Clearance to Vulnerability in the Early Response of Periventricular Microglia to α-synuclein Toxic Species

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Molecular Neurodegeneration (2025) 20:26 https://doi.org/10.1186/s13024-025-00816-1 Molecular Neurodegeneration †Mª Salomé Sirerol-Piquer, Ana Perez-Villalba and Pere Duart-Abadia contributed equally to the work. *Correspondence: Mª Salomé Sirerol-Piquer M.Salome.P[email protected] Isabel Fariñas [email protected] 1Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain 2Departamento de Biología Celular, Biología Funcional y Antropología Física, Universidad de Valencia, Burjassot, Spain 3Instituto de Biotecnología y Biomedicina (BioTecMed), Universidad de Valencia, Burjassot, Spain 4Laboratory of Neurobiology, Institute of Neurosciences, Hospital Clínico San Carlos Health Research Institute, Universidad Complutense de Madrid, Madrid, Spain 5Instituto de Biomedicina de Sevilla (IBiS), Universidad de Sevilla, Seville, Spain 6Departamento Bioquímica y Biología Molecular, Universidad de Sevilla, Seville, Spain 7Univ. Bordeaux, CNRS, IMN, UMR 5293, Bordeaux F-33000, France 8Neurodegenerative Diseases Research Group, Vall d´Hebron Research Institute, Autonomous University of Barcelona, Barcelona, Spain 9Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain 10Present address: L.A.B.P. (Laboratory of Animal Behavior Phenotype), Facultad de Psicología. UCV, Valencia, Spain Abstract Cytoplasmic alpha-synuclein (αSyn) aggregates are a typical feature of Parkinson’s disease (PD). Extracellular insoluble αSyn can induce pathology in healthy neurons suggesting that PD neurodegeneration may spread through cell-to-cell transfer of αSyn proteopathic seeds. Early pro-homeostatic reaction of microglia to toxic forms of αSyn remains elusive, which is especially relevant considering the recently uncovered microglial molecular diversity. Here, we show that periventricular microglia of the subependymal neurogenic niche monitor the cerebrospinal fluid and can rapidly phagocytize and degrade different aggregated forms of αSyn delivered into the lateral ventricle. However, this clearing ability worsens with age, leading to an increase in microglia with aggregates in aged treated mice, an accumulation also observed in human PD samples. We also show that exposure of aged microglia to aggregated αSyn isolated from human PD samples results in the phosphorylation of the endogenous protein and the generation of αSyn seeds that can transmit the pathology to healthy neurons. Our data indicate that while microglial phagocytosis rapidly clears toxic αSyn, aged microglia can contribute to synucleinopathy spreading. Keywords Alpha-synuclein, Microglia, Aging, Parkinson’s disease, Lewy bodies, PFFs, CSF Age-dependent progression from clearance to vulnerability in the early response of periventricular microglia to α-synuclein toxic species Mª SaloméSirerol-Piquer1,2,3*†, AnaPerez-Villalba1,2,3,10†, PereDuart-Abadia1,2,3†, GermánBelenguer1,2,3, UlisesGómez-Pinedo4, LauraBlasco-Chamarro1,2,3, PauCarrillo-Barberà1,2,3, AzucenaPérez-Cañamás2,3, VictoriaNavarro-Garrido5,6, BenjaminDehay7, MiquelVila1,8,9, JavierVitorica1,5,6, FranciscoPérez-Sánchez1,2,3 and IsabelFariñas1,2,3* Page 2 of 20Sirerol-Piquer et al. Molecular Neurodegeneration (2025) 20:26 Introduction Parkinson’s disease (PD) is characterized by abnormal intraneuronal aggregates of the synaptic protein alphasynuclein (αSyn) known as Lewy bodies (LB) [1–3]. Mounting evidence sustains the idea that aggregated pathological αSyn may reach the extracellular milieu and induce the trans-cellular spreading of the pathology. αSyn deposits spanning progressively larger brain areas in postmortem PD samples can be directly associated with the course of the disease, and embryonic mesencephalic neurons grafted in the striatum of PD patients can develop LB pathology [4–7]. Injection of LB fractions into the brain parenchyma of mice and primates results in the long-term pathological modification and aggregation of endogenous αSyn into LB-like lesions [8–11]. In vitro-aggregated forms of recombinant monomeric αSyn, consisting of oligomers, ribbons, or pre-formed fibrils (PFFs), can also induce the seeding of αSyn pathology in neuronal cultures. Furthermore, toxic αSyn oligomercontaining exosomes from affected neurons reportedly mediate the spreading of the pathology to other neurons when injected in vivo [12–23]. Administration of these different αSyn toxic forms has, therefore, emerged as a promising modeling strategy for the study of pathological αSyn spreading, as it enables spatiotemporal studies of the induction of the disease and its progression [8, 20, 24–26]. Although cell-to-cell transmission of pathologic αSyn between interconnected brain regions has suggested prion-like effects, the spread pattern does not necessarily adhere to neural connectivity [24, 27]. In this context, the cerebrospinal fluid (CSF) has recently emerged as a potential brain reservoir of misfolded αSyn [28, 29]. Indeed, pathogenic aggregated αSyn found in exosomes isolated from the CSF of PD patients can initiate the oligomerization of soluble αSyn in cells [23, 30, 31]. The mammalian brain ventricles, where the CSF that bathes the central nervous system is generated, are lined up by a monolayer of ependymocytes that act as a physical barrier between the fluid and the brain parenchyma [32]. At the level of the lateral ventricles (LV), the lining wall includes the germinal niche known as the subependymal zone (SEZ), where glial fibrillary acidic protein (GFAP)-positive neural stem cells (NSCs) continuously produce new neurons for olfactory circuits. The SEZ includes the NSCs and their progeny and other cellular elements, such as niche astrocytes, vascular elements, axonal projections, and microglial cells [32]. Microglia are highly dynamic brain-resident innate immune cells that continuously survey their surrounding microenvironment to maintain homeostasis, mainly through the phagocytosis of pathogens, dead cells, and debris [33]. Across most adult brain regions, Tmem119-positive homeostatic microglial cells appear remarkably homogeneous at the molecular level. In contrast, developmental Tmem119-negative microglia are much more heterogeneous. These cells are found in different areas during fetal and postnatal development, but in the adult brain, they become restricted to regions of persistent neurogenesis, i.e., the SEZ [34, 35]. Intriguingly, these proliferative-region-associated microglia (PAM) exhibit gene expression signatures previously associated with the degenerative disease-associated microglia (DAM) found associated with Aβ-plaques and other abnormal proteinaceous deposits [36]. Although brain microglial cells are generally thought to participate in the phagocytosis and clearance of αSyn [37], the potential response of periventricular non-homeostatic microglia to aggregated CSF-derived αSyn remains elusive. Furthermore, the SEZ location provides an ideal scenario to test the early responses of cells to toxic proteins delivered into the CSF without the confounding tissue injury reaction that characterizes intraparenchymal injections. Here, we show that subependymal periventricular microglia actively survey the CSF by extending projections that cross the ependymal layer and are highly efficient in clearing both αSyn PFFs and LB-enriched fractions containing αSyn, but their phagocytic/degrading function to remove these toxic forms of αSyn is reduced with age. Compromised clearance of αSyn LBfractions with age results in the spreading of the pathology to other microglial cells and neurons. Our results indicate that periventricular microglia constitute a firstline defense to avoid the spreading of toxic forms of αSyn from the CSF, but can contribute to the disease progression in the aging brain. Results Periventricular microglia survey the lateral ventricles and phagocytize aggregated αSyn from the CSF We first addressed the possibility that neuron-derived αSyn could be naturally present in the CSF of mouse brains. Detection of αSyn by Western blot in CSF samples extracted from the cisterna magna of 2-month-old wildtype and TH-hαSyn transgenic mice expressing human αSyn only in catecholaminergic neurons [38] revealed an αSyn specific band that could not be seen in CSF obtained from Snca null (Snca−/−) mice (Fig.1a; striatal homogenates of the same mice were used as controls). To obtain a quantitative assessment of the CSF levels of αSyn produced by catecholaminergic neurons, we performed an ELISA using antibodies specific to the human protein. CSF and striatal homogenates pooled from three TH-hαSyn mice revealed concentrations of the transgenic αSyn of 0.24 ng/ml (within the range of human CSF concentrations [39]) and 17 ng/ml (see also [40, 41]), respectively. These results confirmed the natural presence of αSyn derived from monoaminergic neurons in Page 3 of 20Sirerol-Piquer et al. Molecular Neurodegeneration (2025) 20:26 the CSF, suggesting that this fluid can be a natural vehicle for the dissemination of neuronal αSyn. The SEZ neurogenic niche is located in the LV wall immediately adjacent to the lining ependymal layer, and whole-mount preparations of the LV wall allow the visualization of the 3D relationships among its different cell types and with the CSF [42] (Fig.1b). Confocal 3D microscopy analysis after immunofluorescent detection for cell-specific antigens has revealed that GFAP-positive NSCs intercalate their apical cytoplasmic process perpendicularly among the ependymocytes and access the CSF [42] (see Fig.1c for an example). Microglia is Fig. 1 Periventricular microglia have access to αSyn from the CSF. (a) Western blot showing the presence of αSyn in the CSF of Snca+/+ and TH-hSnca, but not Snca−/− mice; detection in the striatum is shown as a control. (b) Schematic representation illustrating the SEZ location in a mouse brain, a wholemount-en-face preparation and a coronal section containing the SEZ. Cx: cortex, Str: striatum, LV: lateral ventricle. (c) Confocal image of a whole-mounten-face preparation of a dissected SEZ immunostained for microglia (IBA1, green), GFAP (red) and β-catenin (blue). The dashed white line delineates the rosette of ependymocytes surrounding a few GFAP+ NSC cell apical processes. (d) Left panel: Confocal image of the ventricular surface of a SEZ whole-mount-en-face preparation stained for acetylated-tubulin (green), a marker of ependymal cilia, and IBA1 (red) showing cytoplasmic processes of microglial cells contacting the LV through the ependymal layer (pointed by arrowheads). Right panel: Z-stack of confocal images from the ependymal surface (0–6μm) showing the cell bodies of microglial cells that are the origin of the processes shown in the left panel. (e) Confocal images of coronal sections displaying microglia (IBA1, red) in the striatum (left panel) and the SEZ (middle panel). Right panel: confocal images of microglia (red) contacting the LV through the ependymal layer stained for S100β (green). (f) Light microscopy picture of a toluidine blue-stained 1-µm-thick section and EM micrographs showing direct contact of IBA1+ DAB-reacted microglia with the LV. Bottom: reconstruction of the microglia contacting the ventricle. Scale bars: c-e, 20μm; f, 10μm (light microscopy), 4μm (EM, low magnification), and 1μm (EM, high magnification) Page 4 of 20Sirerol-Piquer et al. Molecular Neurodegeneration (2025) 20:26 another conspicuous element in the SEZ and their labeling in whole-mount preparations with antibodies to ionized calcium-binding adaptor molecule 1 (IBA1) revealed IBA1-positive filopodia-like protrusions embedded among ependymal cells and contacting the ventricular space, as seen in confocal 3D projections (Fig.1c, d). Our analysis in static images indicated 7.44 ± 1.02 × 10− 4 microglial projections per µm2 contacting the LV. The intercalating protrusions could also be observed in conventional sections and were corroborated by correlative light-electron microscopy (EM) analysis in IBA1-immunoperoxidase reacted samples. Phagocytic protrusions were distinctly observed extending into the LV lumen (Fig. 1e, f). SEZ microglia could also be labeled with antibodies to the purinergic receptor P2RY12 or visualized in heterozygous mice of the Cx3r1eGFP knock-in reporter strain carrying an enhanced GFP-coding allele in the endogenous Cx3cr1 locus [43] (Suppl. Figure1a-c). In addition, some SEZ microglia exhibited specific PAM and DAM traits, such as increased levels of CLEC7A [36] and a characteristic semi-amoeboid shape compared to the ramified one of other brain areas, such as the adjacent striatum (Suppl. Figure1a-c), that is in line with their apparently higher state of reactivity compared to microglia in non-neurogenic regions [33, 44]. The results indicated that subependymal microglia can play a role in CSF surveillance, likely acting as one of the first lines of defense against toxic components in the CSF. Based on our observations, we decided to test the interaction of periventricular microglia with toxic forms of αSyn delivered into the CSF. αSyn pre-formed fibrils (PFFs) were generated in vitro from endotoxin-free recombinant murine αSyn following the standard procedures of the Michael J. Fox Foundation and tagged with an Alexa Fluor 555 fluorophore [24]. The resulting fibrillary assemblies were tested for size and morphology by sedimentation and EM after negative staining (Fig.2a-c). Subsequently, PFFs were sonicated to obtain assemblies of about 50nm average size (Fig.2c) that are reportedly optimal to reproduce αSyn pathology in culture or after injection [24]. Primary hippocampal neurons obtained from Snca wild-type and null E18 fetuses were seeded at a density of 50,000 cells per cm2, treated with 1µg/ml PFFs or PBS for 5 days, and analyzed 16 days later (Fig.2d). Analysis by Western blot of the cell soluble and insoluble protein fractions isolated from the treated cultures revealed that the treatment with PFFs resulted in the formation of aggregates of endogenous αSyn in wild-type, that were not detected in Snca null cultures. Furthermore, aggregated αSyn carried the pSer129 post-translational modification reportedly associated with toxicity (Fig.2e). Analysis by immunocytochemistry revealed that most neurons had internalized PFFs (83.68 ± 2.17%; n = 3) and were strongly positive for aggregated pSer129-αSyn (Fig. 2f). As a result, overall neuronal viability was compromised in the PFF treated cultures (Fig.2g). The results indicated that the PFFs generated in vitro could act as seeds and induce endogenous αSyn pathology in neurons, as reported for these types of assemblies [45]. We next performed stereotaxic injections of 2µl fluorescently tagged PFFs at 0.1 mg/ml into the LV of 2 month-old mice (Fig.3a). In contrast to intraparenchymal delivery, injections into the LV do not physically harm the SEZ allowing the evaluation of the short-term response of periventricular microglia without the confounding effects of direct tissue injury. Indeed, we determined the degree of microglial activation 15 days after the injection by measuring the number of IBA1-positive somas and the area occupied by them and found that intracerebroventricular injection of αSyn assemblies did not alter microglial morphology in the SEZ (Suppl. Figure2a). At this time, PFFs were never found inside S100β-positive ependymocytes that line up the LV and only 3.53 ± 0.73% (n = 3) of GFAP-positive astrocytes/ NSCs were labeled with PFFs (Fig.3b, c). In contrast, we could readily detect IBA1-positive, P2RY12-positive, and CLEC7A-positive periventricular microglial cells with PFFs inside (Fig.3d-f), suggesting the specific involvement of microglia in the clearance from the CSF. We could observe 48.37 ± 2.46% (n = 5) of IBA1+ and 30.92 ± 4.94% (n = 3) of CLEC7A+ microglial cells with PFFs inside. The specificity of the PFF uptake was studied in animals injected with PBS or monomeric αSyn as a control, in which we never observed any signal (Fig.3d). Interestingly, microglial cells with PFFs inside could be observed as early as 2h after the injection (Suppl. Figure2b, c) suggesting a very fast phagocytic response. We also used a cytometry-based strategy to quantitate the PFF engulfment by CLEC7A-positive microglia. To do so, we specifically isolated the CLEC7A-positive fraction within the CD11b+CD45low microglial population (Suppl. Figure3a, b) and the cells were incubated with PFFs in suspension for 5h, washed, and evaluated by flow cytometry. We found that CLEC7A-positive microglia efficiently and rapidly engulfed PFFs (Suppl. Figure3c). Together, the data indicated that periventricular microglia physiologically survey the CSF and are specifically involved in the clearance of toxic αSyn from the LV. To confirm the selective uptake of aggregated αSyn by microglia vs. other cells without the spatial constraint of the ependymal barrier, we obtained primary mixed glial cell cultures from the SEZ containing astrocytes and microglial cells and treated them with fluorescent PFFs or monomeric αSyn at 1µg/ml for 24h. In agreement with our in vivo analyses, microglial cells were heavily loaded with PFFs. In contrast, GFAP-positive astrocytes did not internalize any αSyn form despite direct exposure Page 5 of 20Sirerol-Piquer et al. Molecular Neurodegeneration (2025) 20:26 (Fig.3g). In turn, uptake by microglia was not dependent on the presence of astrocytes in the culture, as we could find efficient phagocytosis of PFFs also in pure cultures of brain primary microglia (Fig.3h). Because other authors have previously observed PFF uptake by astrocytes in vitro [46, 47], these results suggest that the highly efficient phagocytic activity of microglia takes over at young ages. The post-translational pSer129 phosphorylation targets αSyn for degradation by the lysosome [48–50]. We found that PFFs became phosphorylated in S129 after 24h inside microglial cells in pure cultures that were obtained from Snca null animals to eliminate the potentially confounding effect of endogenous αSyn phosphorylation (Fig.3i; Suppl. Figure4a). PFF and LysoTracker detection in real-time confocal microscopy revealed that PFF uptake follows the endocytic pathway (Suppl. Video 1). In addition, a Manders’ overlap coefficient M1 = 0.973 indicated that virtually all PFFs were in lysosomes 24h after treatment (Fig.3j). Fig. 2 αSyn PFFs production and validation. (a) Schematic representation of the protocol for generating αSyn PFFs from monomers (see Material and Methods). (b) Fibrillation assessment by EM and Western blot before sonication. (c) EM images and size distribution of the αSyn PFFs after sonication. (d) Schematic depicting the treatment of E18 hippocampal neurons with PFFs and analysis. (e) Detection of endogenous αSyn by Western blot in soluble (left) and insoluble (right) extracts from hippocampal neuron cultures of Snca+/+ and Snca−/− mice. (f) Confocal images of cultures of hippocampal neurons treated with PBS or PFFs (red) and immunostained for MAP2 (blue) and pSer129-αSyn (white). (g) Quantification of the number of neurons per field in hippocampal neuron cultures treated with PFFs. Scale bars: b and c, left image 200nm and right image 100nm; f, 20μm Page 6 of 20Sirerol-Piquer et al. Molecular Neurodegeneration (2025) 20:26 Fig. 3 (See legend on next page.) Page 7 of 20Sirerol-Piquer et al. Molecular Neurodegeneration (2025) 20:26 Age-related declines in periventricular microglia capacity reduce PFF clearance from the CSF Because aging remains the most significant risk factor for developing idiopathic PD, we next decided to evaluate the effect of age on the capacity of periventricular microglia to engulf and degrade αSyn PFFs. In aging mice, microglia reportedly undergo morphological changes, i.e., less ramified cell morphology, reduced process length, increased soma volume, and functional alterations, including impaired phagocytic and lysosomal dysfunction [51–55]. Indeed, immunostainings for the indicator of endosomal/lysosomal activity CD68 in 12vs. 3-month-old mice revealed an abnormal distribution of the staining in SEZ microglia of elderly mice, suggesting an age-dependent endolysosomal impairment (Suppl. Figure5a). To functionally evaluate the in vivo phagocytic capacity of SEZ microglia over time, we injected red fluorescent microspheres (FluoSpheres™) into the right LV of 2and 12-month-old mice (Fig.4a). Three days after the injection, the ipsilateral SEZs were disaggregated for flow cytometry analysis and the contralateral SEZs were fixed for microscopy analysis. Examination of the whole-mount-en-face preparation of the LV wall by confocal microscopy revealed an apparently reduced proportion of IBA1-positive cells with fluorescent spheres inside and fewer microspheres within each microglial cell in 12-month-old mice (Fig.4b). In good agreement with these observations, flow cytometry analysis showed that, although the CD11b+CD45low microglial fraction recovered from the SEZ of 2-month and 12-month old mice was similar (1.60 ± 0.39 and 1.86 ± 0.4% of all SEZ cells, respectively, n = 7), the proportion of fluorescent beadcontaining microglial cells was significantly reduced in the samples from aged mice (Fig.4c). We next set out to quantitatively test the PFF-specific short-term phagocytic capacity of microglia employing flow cytometry. CD11b+CD45low microglia from the brains of 2and 12-month-old mice were incubated with PFFs in suspension for 5h, washed, and assayed by flow cytometry. The percentage of phagocytic microglial cells from 12-month-old mice was significantly reduced and they displayed a reduced phagocytic index (Fig.4d). These data indicated that the phagocytic capacity of periventricular microglia declines over time. We next used an in vitro model to specifically analyze PFF degradation. We performed this experiment in mixed astroglial-microglial cell cultures, as pure microglial cultures from aged mice are extremely difficult to maintain. Cultures obtained from 12-month-old mice and incubated with 1 µg/ml PFFs for 3h were subsequently washed and fixed to set the initial uptake or washed and maintained alive for 8 days to evaluate clearance. Qualitatively, after 8 days, CD45+ microglial cells from 2-month-old mice presented an apparent reduction of PFF content in their cytoplasm, with only small aggregates surrounding the nucleus. In contrast, PFFs were more abundant and appeared more dispersed in the cytoplasm of microglial cells obtained from 12-month-old mice (Fig. 4e). Quantitative analysis showed a significant reduction in the degradative capacity from 82.53 ± 3.82 to 10.54 ± 2.88% (p < 0.001) in 2-month and 12-month old mice, respectively. Our in vitro and ex vivo data indicated that microglia lose phagocytic, but also degradative capacity over time. To evaluate whether the age-related functional decline in microglia capacities could affect the in vivo surveillance of CSF αSyn by microglia, we analyzed the microglial uptake of PFFs injected into the LV of 2and 12-monthold mice 15 days after infusion (Fig.4a). The percentage of microglia containing PFFs was significantly increased at 12 months and we could also observe an apparent increase in the PFF load per cell (Fig.4f, g) in line with the reduced degradation capacity observed in vitro. The immunostaining for pSer129 was significantly reduced in 12-month-old mice (Fig.4h, i), suggesting that a less effective degradation of αSyn assemblies could potentially be due to a less efficient phosphorylation at this age. We next decided to evaluate in vivo whether a deficient handling at 12 months could result in effects in other cells. As αSyn accumulations are found in astrocytes of autopsy PD samples [56], we paid attention to SEZ GFAP+ cells. Interestingly, we found a significantly higher proportion of astrocytes that had up-taken PFFs in 12-month vs. 2 month-old-mice (Suppl. Figure 6a, b). To test the possibility that reduced phagocytosis by microglia could indeed be responsible for the engagement of astrocytes in PFF uptake, we decided to evaluate PFF phagocytosis in knock-in Cx3cr1eGFP/eGFP young mice. It has been reported that Cx3cr1-deficient microglia from Cx3cr1eGFP/eGFP young mice exhibit a transcriptome consistent with that of aged Cx3cr1-sufficient animals, suggesting a premature aging transcriptomic signature [57]. In agreement with this, microglial cells (See figure on previous page.) Fig. 3 Microglia selectively phagocytize αSyn PFFs from the CSF and culture media. a. Schematic representation of the in vivo experiment. b-f. Confocal images of the SEZ showing αSyn PFFs (red) 15 days after their infusion into the LV. Absence of PFF uptake in astrocytes (GFAP, green) (b) and ependymocytes (S100β, green) (c). PFF uptake by microglial cells (green) immunostained for IBA1 (d), P2RY12 (e), and CLEC7A (f). Arrowheads point at PFFs within microglial cells. g. Top: schematic representation of the in vitro experiment using mixed glial cell cultures. At the bottom: Detection of αSyn monomer or PFFs (red) in mixed glial cell cultures composed of astrocytes (GFAP, cyan) and microglia (CD45, green). h. Top: schematic representation of the in vitro experiment using pure microglia cultures. Bottom: Detection of αSyn monomer or PFFs (red) in pure microglia cultures (CD45, green). i. Detection of PFFs (red) and αSyn pSer129 (green) in Snca−/− mixed glial cultures. j. Co-localization of PFFs (red) with lysosomes using LysoTracker (blue). DAPI: blue. Scale bars: b-e 20μm; g, 40μm; h, 20μm; i, 20μm; j, 30μm Page 8 of 20Sirerol-Piquer et al. Molecular Neurodegeneration (2025) 20:26 Fig. 4 Aging impairs phagocytic and degrading microglial capacity. (a) Schematic representation of the injection of red fluorescent microspheres (FluoSpheres, red) or αSyn PFFs (blue) into the right LV of 2and 12-month-old mice. The animals were euthanized 3 days after the FluoSpheres infusion or 15 days after the infusion of PFFs. (b) Whole-mount-en-face preparations of the contralateral dissected SEZ immunostained for γ-tubulin (blue; a marker of cilia centrosomes to show the ependymal surface) and microglia (IBA1, green). (c) Percentage of microglia isolated from the ipsilateral SEZ containing FluoSpheres (n = 5 mice). Data are presented as mean values ± SEM; Student’s t-test p < 0.05. (d) Ex vivo phagocytosis assay with αSyn PFFs in microglia isolated from 2and 12-month-old mice. Percentage of phagocytic microglia and phagocytic index in 2- (n = 3) and 12-month-old (n = 4) mice. Data are presented as mean values ± SEM; Student’s t-test p < 0.001. (e) In vitro degradation experiment. Mixed glial cultures obtained from young and aged mice were incubated for 3h with PFFs (red) and either fixed immediately (3h) or washed and maintained for 8 days in fresh media (8 days) before fixation and immunostaining for microglia (CD45, green). (f) Confocal images of the SEZ of 2and 12-month-old mice showing PFFs (red) and IBA1+ microglia (green) 15 days after infusion. DAPI: grey. (g) Quantification of the percentage of microglia containing αSyn PFFs in 2- (n = 4) and 12-month-old (n = 3) mice. Data are presented as mean values ± SEM; p < 0.01, Student’s t-test. (h) Confocal images of the SEZ of young and aged mice showing PFFs (red), pSer129 (blue) and IBA1+ microglia (green) 15 days after infusion. DAPI: grey. (i) Quantification of the percentage of microglia containing phosphorylated αSyn PFFs in 2- (n = 3) and 12-month-old (n = 3) mice. Data are presented as mean values ± SEM; Student’s t-test, p < 0.05. Scale bars: b, 50μm; e, 20μm; f, h 20μm Page 9 of 20Sirerol-Piquer et al. Molecular Neurodegeneration (2025) 20:26 isolated from young Cx3cr1eGFP/eGFP mice and incubated with PFFs in suspension for 5h evidenced a significantly reduced phagocytic activity, compared to Cx3cr1+/eGFP or Cx3cr1+/+, as assayed by flow cytometry (Suppl. Figure6c). In line with this and with the previous results, we observed increased proportions of astrocytes with PFFs inside in SEZ sections of young mice lacking CX3CR1 (Suppl. Figure6d, e). All these data together indicated that periventricular microglia phagocytize and degrade the PFFs less efficiently at 12 months and, furthermore, that this functional decline leads to the involvement of astrocytes/NSCs in the uptake of PFFs. Reduced clearance of LB-derived aggregated αSyn from the LV results in modification of the endogenous protein Because our data indicated that periventricular microglia can act as a first line of defense at the ependyma-CSF interface, we next decided to explore SEZ microglial cells in postmortem human samples by immunostaining PD and non-affected control autopsy brain samples ranging from 65 to 84 years (Fig.5a, b and Suppl. Table 1) with αSyn and pSer129 αSyn antibodies. We could readily detect pSer129/αSyn-positive aggregates in MAP2-positive neurons in the PD samples (Fig.5c), but also in periventricular microglia, with similar aggregates that were significantly more numerous in PD samples (Fig.5d, e). Although these results could be attributed to aggregation of pathological αSyn in microglia, they could also be in line with human periventricular microglia playing an active role in αSyn clearance from the CSF. We decided to explore these possibilities in mice using human LB fractions. There is an increasing appreciation that PFFs only reproduce specific aspects of LB pathology [58], fostering the usage of aggregated αSyn derived from neurons or αSyn-containing LB fractions. We had previously reported that inoculation of LB-enriched fractions of pathological αSyn purified by sucrose gradient from postmortem PD brains into the substantia nigra of young mice initiates a slowly progressive nigrostriatal degeneration that is not detected after inoculation of non-LB (NLB) fractions [9, 10]. We, therefore, decided to explore the short-term effects of the LV infusion of similar LB fractions, containing pathological αSyn, and NLB control fractions isolated from postmortem samples (Fig.6a). We next infused the fractions into the LV of young mice and fifteen days later, we immunostained SEZ-containing sections of the infused mice with antibodies specific to human αSyn (LB509 antibody) and to pSer129-αSyn. We found detectable uptake of the LB-derived human αSyn aggregates by periventricular IBA1-positive microglia (Fig. 6c). In line with our previous observations using PFFs, the percentage of microglia containing LBs was significantly higher in 12vs. 2-month-old mice (Fig.6c). To ensure the specificity of the LB509 and pSer129 antibodies employed in the analysis we also performed the infusions in Snca null mice. At 15 days after the infusion, labelings for LBs and pSer129 were only found in animals injected with LB, but not NLB fractions, highlighting the specificity of these antibodies (Fig. 6d, e). As with PFFs, we compared the infusions in mice at 2-months and 12-months of age and found LB material within more astrocytes in the latter (Suppl. Figure7a, b). The data indicated that periventricular microglia can also phagocytize LB-enriched fractions from the CSF and further sustain the idea that, with advancing age, astrocytes react more actively to toxic αSyn, likely due to microglial reduced efficiency. We next focused on the potential differences in the response of periventricular microglia to PFFs vs. LB fractions. In contrast to PFF infusions, and specifically at 12-months of age, we noticed some microglial cells with LB509+/pSer129+ aggregates inside that also displayed LB509-negative/pSer129+ aggregates, suggesting a potential seeding effect on microglia (Fig.6f). In addition, we observed pSer129+ cells close to LB-phagocytic microglia suggesting a potential spreading effect from microglia at 12-months (Fig.6g). To delve into this potential prionoid effect, we next analyzed the proportions of SEZ microglial cells and nearby MAP2-positive neurons that were positive for pSer129-αSyn, but negative for LB509 and we observed increased proportions of what appeared to be phosphorylated endogenous αSyn in both cell types in 12-month vs. 2-month-old mice (Fig.6h, i). Our results suggested a phosphorylation of the endogenous murine αSyn in microglia and in neurons of 12-month-old animals exposed to LB material and a potential spreading among periventricular microglia, in addition to adjacent neurons, at very short times. To assess the potential spread to more distant regions, analyses at durations beyond those examined in this study would be necessary. αSyn prion-like effects have been widely studied in neurons, but similar effects in microglial cells remain elusive. Previous reports have indicated the presence of αSyn in mouse microglial cells [59, 60], and we could readily detect αSyn protein in the intact murine SEZ (Fig.7a) and other brain regions such as cortex or dentate gyrus (Suppl. Figure8a, b) using immunofluorescent detection with specific antibodies. To obtain a more direct evidence that LB uptake by microglia could have non-autonomous effects on other cells, we turned to an in vitro system. Expression analysis by RT-PCR and Western blot analysis in dissociates of magnetically isolated brain microglia (CD11b-positive fraction), astrocyte-enriched cultures, and neuronal hippocampal cultures confirmed that microglia have levels of αSyn that are similar to those of neurons and much higher than those of astrocytes (Fig.7b). We, therefore, established mixed cell cultures Page 16 of 20Sirerol-Piquer et al. Molecular Neurodegeneration (2025) 20:26 ab5076), chicken antibodies to GFAP (1:800, Millipore, AB5541) or MAP2 (1:500, Abcam, ab5392) and rat antibodies to dectin 1 (CLEC7A) (1:50, InvivoGen, 6114-4301), alone or in different combinations for 24–48h at 4°C. After several washes, the sections were incubated for 1h at room temperature with appropriate fluorescentlylabeled secondary antibodies: Alexa Fluor® 488 Donkey Anti-mouse, Alexa Fluor® 488 Donkey Anti-rabbit, Alexa Fluor® 647 Donkey Anti-mouse, Alexa Fluor® 647 Donkey Anti-rabbit, Alexa Fluor® 647 Donkey Anti-goat (1:800 Molecular Probes), Alexa Fluor® 488 Donkey Antichicken, Cy3 Donkey Anti-rabbit, Cy3 Donkey Anti-goat (1:800 Jackson ImmunoResearch Laboratories). DAPI (1µg/ml, 4min, Sigma-Aldrich) was used for counterstaining. Images were acquired and processed using an Olympus Fluoview FV10i confocal microscope and the FV10-ASW 2.1 viewer software. Confocal images at 100x magnification were collected every 1μm covering all the open LV ventricular wall. Every region of interest in microscope images was 1,250μm length x 50μm width x 15μm thick (on average). The SEZ was fully included in 50μm from the LV and only cell nuclei included in this distance were considered in quantifications. Immunostained sections (at least 5 slices per experiment per mouse) were photographed using an Olympus FV-10i confocal microscope. Separate images were taken with restrictive excitation filters in each fluorescent channel every 1 μm. Subsequently, individual cells were identified across the tissue and quantitative analyses were performed on high-resolution image stacks. At least 10 sections were analyzed in every brain slice. In this way, the entire ventricular surface was scrutinized in detail. Quantification of microglial activation was performed by measuring the number of individual IBA1+ microglial cells and the area occupied by all of them with ImageJ [79] (version 2.1.0/1.53c) software using a single automated macro-script to quantitate areas. The IBA1+ area was normalized to the total tissue area using the DAPI signal. Electron microscopy Mice were transcardially perfused with saline followed by 4% PFA and 0.1% glutaraldehyde (EM grade, Electron Microscope Science) in PB. The brain was removed, post-fixed overnight in 4% PFA, and coronally sectioned with a vibratome at 50μm. Sections were pre-incubated in blocking solution (10% FBS and 0.1% Triton X-100 in PB 0.1M) and then incubated in primary antibody goat anti-IBA1 (1:500, Wako, 019-19741) prepared in blocking solution, on an orbital shaker for 24h at 4 ºC. After several washes in PB, tissue sections were incubated in biotinylated rabbit anti-goat IgGs (1:1,000, Vector Laboratories, BA5000) for 1h at RT. After washing, sections were incubated with an avidin-biotin-peroxidase complex (ABC, Elite Vector Laboratories), washed and revealed with 0.05% diaminobenzidine (DAB) and 0.01% hydrogen peroxide (Sigma) in PB. The DAB-stained sections were further processed for electron microscopy. Briefly, tissue was osmicated (1% OsO4 in PB, 20min), dehydrated in graded alcohols to propylene oxide, and plastic-embedded flat in Durcupan (Sigma). To study selected microglial cells in contact with the ventricle, serial 1.5-μm sections were cut with a diamond knife and stained with 1% toluidine blue. Subsequently, the area of interest was trimmed, and ultrathin Sects.(50–70nm) were obtained from this material, stained with lead citrate, and examined in a JEM 1010 (Jeol) electron microscope. Primary cell cultures and treatments For mixed cell cultures (astrocytes and microglia) and pure microglia cultures, cortices, or SEZs from 2and 12-month-old C57/Bl6 mice were dissected, minced, and enzymatically digested using the Neural Tissue Dissociation kit (P) (130092-628, Miltenyi Biotec) in a gentleMACS Octo Dissociator with heaters (Miltenyi Biotec; 37 ºC_ABDK_01 program). Digestion was diluted with 3ml of washing solution (0.6% glucose, 0.1% NaHCO3, 5 mM HEPES, 2 mM L-glutamine, 0.4% BSA, 1X antibiotic/antimicotic in DMEM/F-12) (all from Invitrogen) and digested pieces were mechanically dissociated by pipetting up and down 20 times through a plastic Pasteur pipette. Cell suspension was filtered through a 40μm nylon filter and then centrifuged. To prepare primary mixed glial cell cultures, the cell pellet was resuspended in complete medium (DMEM supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100mg/ml streptomycin) and seeded on polyD-lysine coated coverslips. Alternatively, the single-cell suspension was subjected to immunomagnetic CD11b positive selection using CD11b microbeads (130-049-601, Miltenyi Biotech) in combination with an OctoMACS following manufacturer´s instructions to obtain a microglia-rich fraction (CD11b+ fraction) and an astrocyte rich fraction (CD11b− fraction). Purified CD11b+ microglial cells were seeded in poly-D-lysine (100 µg/ml)-treated plates and cultured in TIC medium (DMEM/F12 containing 100 units/ml penicillin/streptomycin, 2 mM L-glutamine, 5µg/ml N-acetyl cysteine, 5µg/ml insulin, 100µg/ml apo-transferrin, 100 ng/ml sodium selenite, 2 ng/ml TGF-β2, 100 ng/ml murine IL-34, 1µg/ml heparan sulfate). Cells were maintained at 37 ºC and 5% CO2, changing the media every 2 days. For primary neuronal cultures, hippocampal neurons were prepared from E18 wild-type and Snca null mice. Hippocampi were isolated stereoscopically and enzymatically digested with 12 U/ml papain solution (Worthington) for 15min at 37 ºC. Dissociated hippocampal neurons were plated on coverslips or tissue culture plates coated with poly-D-lysine (Sigma) at a 50,000 cells/cm2 density and cultured in Neurobasal Page 17 of 20Sirerol-Piquer et al. Molecular Neurodegeneration (2025) 20:26 medium containing B27 supplement, L-glutamine and penicillin/streptomycin (Life technologies). Neurons were maintained by changing half of the media every 2 days. Confirmation of PFFs seeding capacity and toxicity Neurons cultured for 5 days were treated with 1µg/ml PFFs (or PBS as control) for only 24h or for 10 to 14 days. At the end of the treatment, neurons were scraped and collected in 1% Triton X-100 in Tris-buffered saline (TBS) (50 mM Tris HCl, 150 mM NaCl, pH 7.4) and protease and phosphatase inhibitor cocktail at 4°C. Lysates were sonicated and centrifuged at 100,000g for 30min. The supernatant (soluble fraction) and the pellet (insoluble fraction) were collected. The pellet was washed and resuspended in 2% SDS in TBS. Both fractions were analyzed by immunoblotting. On the other hand, neurons were fixed with 2% PFA for 20min at RT, blocked in 10% FBS in 0.1% Triton X-100 PBS for 1h at RT and then incubated with rabbit anti-MAP2 (1:800, Santa Cruz, SC-20172), mouse anti-NeuN (1:400, Millipore, MAB377) or mouse anti-pSer129 (1:500, BioLegend, 825702) overnight. After several washes, cells were incubated for 1h at RT with the appropriate secondary antibodies (1:800, Molecular Probes). DAPI (1µg/ml, 5min, Sigma) was used for counterstaining. The number of neurons per field was estimated in 30 different fields. Immunoblotting For Western blotting, cells were lysed in ice-cold RIPA buffer (50 mM Tris HCl, 150 mM NaCl, 1 mM MgCl2, 1.0% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 1 mM EDTA, 0.1% (w/v) SDS, pH 7.4) supplemented with phosphatase and protease inhibitors and total protein concentration was determined using a BCA kit (BCA Protein Assay-Kit, ThermoScientific, Sweden). Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad) using the TransBlot Turbo system from Bio-Rad. For dot blot, 5µl of serial diluted LB and NLB fractions were spotted directly onto a nitrocellulose membrane. Membranes were blocked for 1h with skim milk at 3% (w/v) in TBS-T, followed by incubation with mouse primary antibodies to humanαSyn LB509 (1:500, Abcam, ab27766), αSyn (1:500, BD, 610786), pS129 αSyn (1:500, Abcam, ab59264), α-tubulin (1:100, Sigma, T9026) and GAPDH (1:500, Millipore, mAb 374) or rabbit primary antibody to pS129 αSyn (1:500, Abcam, ab59264) overnight. Then, membranes were washed, incubated with appropriated secondary peroxidase-conjugated antibodies (1:1,000 of goat antimouse-HRP or mouse anti-rabbit-HRP from Dako and Santa Cruz, respectively) for 1h and reacted by chemiluminiscence (SuperSignal, Thermo Fisher Scientific). Flow cytometry analyses For in vivo phagocytic assay with red fluorescent microspheres, SEZ single cell suspensions obtained from young and aged mice 3 days after red fluorescent microsphere infusion were pelleted (300g, 10min), resuspended in 100µl blocking buffer (HBSS without calcium and magnesium, 10 mM HEPES, 2 mM EDTA, 0.1% glucose, 0.5% BSA) and incubated with primary antibodies CD45-BUV395 (1:200, BD, 565967), CD11b-APC (1:100, BD, 553312), DAPI 50µg/ml (1:500, Sigma, D9542) for 30min at 4 ºC. After washing with 1ml blocking buffer, labeled samples were centrifuged (300g, 10min, at 4 ºC) and resuspended in 0.5ml blocking buffer. Cells were analyzed using a LSR-Fortessa (BD) with 355, 561 and 640nm lasers. For ex vivo phagocytosis assays with PFFs, brain single-cell suspensions from young and aged mice were pelleted (300g, 10min), resuspended in 100µl complete medium containing 1µg/ml PFFs and incubated at 37 ºC for 5h. Cells were washed with blocking buffer to remove residual PFFs and then incubated with antibodies as before. After washing with 1ml of blocking buffer, labelled samples were centrifuged (300g, 10min, at 4 ºC) and resuspended in 0.5ml blocking buffer. Cells were analyzed using a LSR-Fortessa (BD) with 355, 561, and 640nm lasers. The phagocytic index was calculated by dividing the median fluorescence intensity (MFI) of each sample by the MFI of the fluorescence minus one (FMO) of its respective group (young or aged). Afterwards, a fold change analysis was performed to determine the magnitude of the observed alteration. In vitro PFFs uptake assay To analyze PFF uptake by microglia and astrocytes, confluent mixed cell cultures or pure microglia cells grown for 7 days were treated with either monomeric αSyn or PFFs at a concentration of 1µg/ml for 24h and then fixed with 2% PFA. Cells were blocked in 10% FBS and 0.01% Triton X-100PB and then incubated with chicken anti-GFAP (1:800, Millipore, ab55414), rat anti-CD45 (1:400, BD, 5530076) and mouse anti-pSer129 (1:500, BioLegend, 825702) antibodies. After several washes, cells were incubated for 1h at RT with Alexa 488-donkey anti-rat, Alexa 488-donkey anti-mouse, Alexa 647-donkey and anti-chicken (1:800, Molecular Probes) secondary antibodies. Finally, cells were counterstained with DAPI. PFF endocytosis in living cells was imaged by incubating pure microglial cell cultures with 40 µM LysoTracker Deep Red (L12492, Invitrogen) for 30min, followed by 1µg/ml PFFs and immediately visualized under an Olympus Fv10i confocal inverted microscope. Serial images were obtained every 5min for 30min. Alternatively, cells were treated with PFFs for 24h, preincubated with LysoTracker Deep Red, and then analyzed. Colocalization with LysoTracker was quantified using JACoP and Manders overlap coefficient. In vitro degradation assay Primary mixed glial cultures were treated with 1µg/ml PFFs for 3h at 37 ⁰C and then Page 18 of 20Sirerol-Piquer et al. Molecular Neurodegeneration (2025) 20:26 rinsed twice with PBS. A set of samples was fixed with 2% PFA (initial PFF uptake) for 20min at RT, while another set of samples was maintained for 8 days to allow PFF degradation and subsequently fixed. Finally, cells were immunostained for the microglial cell marker CD45 as mentioned before. Images were acquired under an Olympus Fv10i confocal inverted microscope. 3D image stacks were preprocessed with a Fiji [80] macro to generate 2D images through maximum projection. Then, CD45-positive cells were then annotated in QuPath (version 0.5.1) [81] for PFF quantification, and the PFF mask associated with each microglial cell was determined using the QuPath-integrated ImageJ’s Moments thresholding algorithm [79]. The final readout for each cell was the PFF area, measured in squared pixels. At least 30 cells per condition were analyzed. Prion-like effect in microglial cells Confluent primary mixed glial cultures were treated with LBs at 0.04 ng/ ml or vehicle for 5 days, rinsed twice with PBS and then, fresh media was added and replaced every 2 days for 21 days. Media from the last two changes was collected and concentrated 22.5x using 10kDa MW-cut-off Microcon filters (MRCPRT010; Millipore) to test spreading in hippocampal neuron cultures. To analyze seeding in microglia, cultures were fixed after 21 days and stained with antibodies to pSer129 αSyn (BioLegend) and CD45 (BD), as mentioned above. To analyze spreading from microglia, concentrated media obtained above was diluted 1:40 in neuronal medium and added to hippocampal neuron cultures for seeding capacity. After 10 days, hippocampal neuron cultures were fixed and immunostained for pSer129 αSyn (BioLegend) and MAP2, as described previously. Gene expression analysis Immunomagnetically purified CD11b+ microglial cells from young and aged mice were lysed with RLT plus buffer and the DNA extracted with a RNeasy Plus Mini Kit (QIAGEN) following the instructions of the manufacturer. The RNA obtained was quantified using the Qubit® RNA HS Assay Kit (Thermo Fisher) in a Qubit Fluorimeter (Thermo Fisher). For quantitative RT-PCR experiments, a total amount of 50–100 ng of RNA was retro-transcribed to cDNA using the PrimeScriptTM RT-PCR Kit (Clontech) according to the manufacturer instructions. Snca expression analysis was assessed using 5–10 ng of cDNA, specific Taqman probes (Applied Biosystems) and the Premix Ex Taq™ (Probe qPCR) Kit (Clontech). RT-qPCR was performed in a Step One Plus PCR device (Applied Biosystems). The expression level was obtained by relative quantification (2(– ΔΔCt)) using constitutive expression of Gapdh and 18S genes as housekeeping endogenous controls. Statistical analyses All statistical tests were performed using GraphPad Prism Software, version 5.00 for Windows. Analyses of significant differences between means were assessed using the unpaired or paired two-tailed Student’s t-test or one-way ANOVA with Tukey post-hoc test when appropriate. When comparisons were carried out with relative values (normalized values and percentages), data were first normalized by using a log or arcsin transformation, respectively. All p-values lower than 0.05 were considered statistically different and referred to as *p < 0.05, **p < 0.01, and ***p < 0.001. Data are always presented as the mean ± standard error of the mean (SEM). The number of experiments carried out with independent cultures/animals (n) is either shown as dots in the graphs or listed in Figure Legends. Supplementary Information The online version contains supplementary material available at h t t p s : / / d o i . o r g / 1 0 . 1 1 8 6 / s 1 3 0 2 4 - 0 2 5 - 0 0 8 1 6 - 1. Supplementary Material 1 Supplementary Material 2 Acknowledgements We thank M. J. Palop for help with the mouse colonies and technical assistance and acknowledge the support of the Servicio Central de Soporte a la Investigación Experimental (SCSIE-UVEG). This work was supported by grants PID2020-117937GB-I00, RED2018-102723-T, and CB06/05/0086 (CIBERNED) from Ministerio de Ciencia e Innovación (MICINN) and Prometeo 2021/028 from Generalitat Valenciana to I.F. P.D-A. is a recipient of a MICINN’s FPI predoctoral contract, and L.B.-C. was a recipient of an FPU predoctoral contract from Ministerio de Universidades. This study received financial support from the French government in the framework of the University of Bordeaux’s IdEx “Investments for the Future” program/GPR BRAIN_2030. Author contributions Conceptualization, A.P.-V., M.S.S.-P., M.V., I.F.; Methodology, B.D., G.B., U.G.P., P.D.-A., V.M., J.V., A.P.-V.; Formal Analysis, P.C.-B., A.P.-V., M.S.S.-P., P.D.-A.; Investigation, G.B., U.G.-P., P.D.-A., L.B.-C., A.P.-V., M.S.S.-P; Resources, B.D., U.G.P.; Data Curation, A.P.-V., M.S.S.-P., P.D.-A.; Writing – Original Draft, I.F., M.S.S.-P; Writing – Review & Editing, P.D.-A, G.B., L.B.-C., M.V., B.D., F.P.-S., A.P.-C., A.P.-V., M.S.S.-P., I.F.; Visualization, A.P.-V., M.S.S.-P., P.D.-A.; Supervision, I.F.; Project Administration, I.F.; Funding Acquisition: B.D, I.F. Data availability The authors declare that the data supporting the findings of this study are available within the paper and its supplementary information files. Declarations Competing interest The authors declare no competing financial interests. Received: 18 May 2024 / Accepted: 19 February 2025 References 1. Balestrino R, Schapira AHV. Parkinson disease. 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