Astroglia proliferate upon biogenesis of tunneling nanotubes via α-synuclein dependent transient nuclear translocation of focal adhesion kinase

Astroglia proliferate upon biogenesis of tunneling nanotubes via α-synuclein dependent transient nuclear translocation of focal adhesion kinase | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Astroglia proliferate upon biogenesis of tunneling nanotubes via α-synuclein dependent transient nuclear translocation of focal adhesion kinase Sangeeta Nath, Abinaya Raghavan, Rachana Kashyap, Sreedevi P, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3747717/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Astroglia play crucial neuroprotective roles by internalizing pathogenic aggregates and facilitating its degradation. Here, we show, that α-SYN protofibril-induced organelle toxicities and reactive oxygen species (ROS) cause premature cellular senescence in astrocytes and astrocytes origin cancer cells, resulting in a transient increase in biogenesis of tunneling nanotubes (TNTs). TNT-biogenesis and TNT-mediated cell-to-cell transfer lead to clearance of α-SYN-induced organelle toxicities, reduction in cellular ROS levels, and reversal of cellular senescence. Enhanced cell proliferation is seen in the post-recovered cells after relieving from α-SYN-induced organelle toxicities. Further, we show, that α-SYN-induced senescence promotes transient localization of focal adhesion kinase (FAK) in the nucleus. FAK-mediated regulation of Rho-associated kinases plays a significant role in the biogenesis of TNTs, and successively proliferation. Our study emphasizes that TNT biogenesis has a potential role in the clearance of α-SYN-induced cellular toxicities and reversal of stress-induced cellular senescence, consequences of which cause enhanced proliferation in the post-recovered astroglia cells. Biological sciences/Neuroscience/Glial biology/Astrocyte Biological sciences/Cell biology/Cell adhesion/Focal adhesion cell-cell transfer α- synuclein tunneling nanotubes reactive oxygen species (ROS) mitochondria cellular senescence focal adhesion kinase cell proliferation. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Highlights α-SYN protofibrils treated astroglia cells proliferate upon transient biogenesis of TNTs. Transient TNT biogenesis precedes clearance of α-SYN toxicities and reversal of senescence. Stress-induced senescence results in nuclear localization of FAK and ROCK-mediated TNT biogenesis. The rescued cells enhance proliferation through ROCK-mediated ERK1/2 and NFκB signalling cascades. Introduction Parkinson’s disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra (SN) due to cytoplasmic accumulation of Lewy bodies or Lewy neurites which mainly consists of α-synuclein (α-SYN) protein (Forno, 1996 , Kalia & Lang, 2015 ). Along with the accumulated cytoplasmic α-SYN, extracellular α-SYN also plays a significant role in neurodegeneration, progressive intercellular spreading of pathology, and neuroinflammation (Lee, Bae et al., 2014 , Yamada & Iwatsubo, 2018 ). Studies have shown that elevation of neuronal activity and various stress conditions increase extracellular release of α-SYN (Fortin, Nemani et al., 2005 , Yamada & Iwatsubo, 2018 ). Pathogenic aggregates of α-SYN can be released from degenerating neurons, which can be taken up by surrounding neurons and glial cells. Glial cells normally express low levels of α-SYN, however, at the advanced stage of PD, α-SYN aggregates in astrocytes and glial synucleinopathies are often detected (Krejciova, Carlson et al., 2019 ). Additionally, gliosis is a typical pathological feature of neurodegenerative diseases. Sustained activation and fibrous proliferation of glial cells, mainly astrocytes and microglia, are central features of dopaminergic neurodegeneration in PD (MacMahon Copas, McComish et al., 2021 ). Neuronal activity modulates cellular crosstalk and intercellular communication between neurons and the surrounding neuroglial cells (Gibson, Purger et al., 2014 , Venkatesh, Johung et al., 2015 , Venkatesh, Morishita et al., 2019 ). Recent studies have shown that mode of intercellular transfer of neurodegenerative proteins between glial cells facilitate the clearing of neurodegenerative aggregates, such as α-SYN and amyloid-β (Aβ) (Rostami, Holmqvist et al., 2017 , Rostami, Mothes et al., 2021 , Scheiblich, Dansokho et al., 2021 ). The spreading of neurodegenerative proteins through an intercellular mode of transfer and its role in pathology progression have widely been studied in several model systems (Neupane, De Cecco et al., 2022 , Victoria & Zurzolo, 2017 ). Exosomes, unconventional secretion, and direct cell-to-cell transfer via membrane nanotubes or tunneling nanotubes (TNTs) have been demonstrated by several studies as modes of intercellular transfer (Neupane et al., 2022 ). The discoveries of TNTs have opened up the possibility of direct long-range cell-to-cell communication (Rustom, Saffrich et al., 2004 ). TNTs have been shown as open-ended, and thin (diameter around 50–700 nm) intercellular membrane-actin continuity (long up to 300 µm) between distant cells. TNTs are reported to be a conduit for the direct transfer of organelles (Rustom et al., 2004 ), neurodegeneration-associated protein aggregates (Ramirez-Jarquin, Sharma et al., 2022 , Victoria & Zurzolo, 2017 ), viruses (Jansens, Tishchenko et al., 2020 ) and RNA. (Haimovich, Ecker et al., 2017 ) between cells. Neurodegenerative aggregate-induced endo-lysosomal toxicities and mitochondrial stress promote biogenesis of TNTs (Raghavan, Rao et al., 2021 , Victoria & Zurzolo, 2017 ). Propagation of pathogenic aggregates via TNTs has also been shown to aid in the progression of neurodegeneration (Dilna, Deepak et al., 2021 , Rostami et al., 2017 , Victoria & Zurzolo, 2017 ). In this context, several studies have shown that α-SYN aggregates in lysosomes transfer from neuron to neuron via TNTs in PD (Abounit, Bousset et al., 2016 , Dilsizoglu Senol, Samarani et al., 2021 , Victoria & Zurzolo, 2017 ). On the other hand, TNTs mediate crosstalk between astrocytes, and microglia to degrade toxic α-SYN through cell-to-cell transfer of aggregates and help to reduce ROS accumulation and oxidative stress (Rostami et al., 2017 , Scheiblich et al., 2021 ). Oxidative stress and ROS promote formation of TNTs and cell-to-cell transfer (Desir, Dickson et al., 2016 , Wang, Cui et al., 2011 ). ROS induced by the accumulation of neurotoxic proteins in neurons can induce cell senescence and that can aggravate neurodegeneration (Yoon, You et al., 2022 , Zizhen Si, 2021 ). Cellular senescence is a biologically homeostatic phenomenon that is defined by a persistent degree of cell cycle inhibition and cellular aging. Cell cycle arrest in senescence cells is believed to be irreversible, however, studies have shown that stress-induced premature senescence that is induced by DNA damage-mediated amplification of the p53/p21 signalling pathway can be reversed by modulating the levels of p53 and p21 expressions (Beausejour, Krtolica et al., 2003 , Macip, Igarashi et al., 2002 ). Senescent cells are more prevalent in brain tissues and exert roles in brain aging and neurodegeneration (Martinez-Cue & Rueda, 2020 ). Brain cells including astrocytes, microglia, oligodendrocytes, and epithelial cells undergo cellular senescence under oxidative stress, which aggravates neurodegeneration (Schousboe, Bak et al., 2013 ). On the contrary, glial cells, especially astrocytes, and microglia, play a critical role in protecting neurons by clearing toxic accumulations of neurodegenerative proteins from neurons and brains (Rostami et al., 2017 , Scheiblich et al., 2021 ). α-SYN induced ROS generation can produce cellular senescence in response to DNA damage (Yoon et al., 2022 ). Thus, the investigation into the mechanisms of TNT biogenesis and the role of TNTs in facilitating clearance of toxic α-SYN protofibrils from the brain to improve cell survival is needed. In this study, we have shown that transient localization of FAK/pFAK to the nucleus upon treatment with α-SYN protofibrils caused transient biogenesis of TNTs in the astroglia cells. Transient biogenesis of TNTs precedes clearance of α-SYN-induced organelle toxicities and reversal of ROS-induced premature senescence leading to enhanced cell proliferation in the post-recovered astroglia cells. This study emphasizes the potential role of TNTs in facilitating cellular clearance in pathological stress conditions which maintains the survival of astroglia cells, by enhancing cell proliferation. Materials and Methods Cell culture maintenance U-87 MG and U251 cell lines (astrocytoma-glioblastoma origin cancer cell lines) were kind gifts from Prof. Kumaravel Somasundaram of the Indian Institute of Science, Bangalore, India. Cells were cultured and maintained in DMEM (Gibco #2120395) media supplemented with 10% FBS (fetal bovine serum; Gibco #1600004, US Origin), along with 1% PSN (Penicillin-Streptomycin-Neomycin Mixture; Thermo Fisher Scientific #15640055) incubated at 37°C, 5% CO 2 . Neuro 2a (N2a) neuroblastoma cell line was procured from NCCS, India. The cells were cultured and maintained in DMEM media with 10% FBS and 1X Glutamax (Gibco #35050). N2a cells were differentiated with 10 µM Retinoic acid (RA; Sigma-Aldrich #R2625) in the presence of 2% FBS-supplemented media for 2–3 days. Differentiation was established from the neurites like morphology. Primary astrocytes culture Five-week-old mice (C57BL/6) were used for primary astrocyte culture. The mice were sacrificed using CO 2 , and the cerebral cortices were dissected from the mice's brains and observed using an Olympus SZ51 stereomicroscope. Pieces of cortical tissue were trypsinized using 0.25% trypsin-EDTA solution (Gibco Canada origin #25200-072) for 5 min. The tissue was washed twice in warm HBSS and transferred to Minimum Essential Media (Gibco 61100-087) with 10% FBS (Gibco #1600004, US Origin) containing HEPES (Sigma Aldrich H0887) and D-glucose (Sigma Aldrich G7021). The tissue was triturated using a fire-polished pipette and counted using Trypan Blue in an automated cell counter. Dissociated cells were plated in MEM with 10% FBS on Corning® 100mm TC-Treated Culture Dish at a density of 10 million cells per dish. The media was replaced with fresh MEM with 10% FBS on the next day and maintained the same for 1 week. After 1 week, cells were trypsinized and re-plated for experiments. Purification and Labelling of α-SYN protein The human α-SYN wild-type (Addgene ID #36046) and α-SYN (wt)-141C (Addgene ID #108866) with a cysteine residue at C-terminus, constructs were purchased and overexpressed in E. coli . Then purification was done from periplasmic fraction as described in (Huang, Ren et al., 2005 , Jos, Gogoi et al., 2021 ). Samples were aliquoted and flash frozen in liquid nitrogen and stored immediately at -80°C. The purified α-SYN (wt)-141C protein was thawed and pre-incubated with reducing agent TCEP (tris(2-carboxyethyl) phosphine-hydrochloride); Sigma Aldrich-C4706; 20mM) to reduce/dissociate any pre-existing cysteine bonds. Then the protein was labelled adding two-fold molar excess of TetramethylRhodamine-5-maleimide (TMR-maleimide; Sigma Aldrich #94506) in 20 mM HEPES buffer (pH 7.4). The protein solution was vortexed and the mixture was incubated for 1h at 20°C. The excess label was removed by using PD-10 columns (Himedia #TKC287) (Nath, Meuvis et al., 2010 ). Preparation and characterisation of α-SYN protofibrils Protofibrils of α-SYN (labelled and unlabelled) was prepared with 1 µM of unlabelled α-SYN wild-type and labelled α-SYN-141C by incubating with 0.65% 4-Hydroxynonenal (10mg/ml stock) (Sigma-Aldrich #393204-1MG) at 37°C for 7 days with moderate shaking (Sackmann, Sinha et al., 2019 ). The α-SYN protofibrils formed at the end of 7 days were then characterized by transmission electron microscopy (TEM). The unlabelled and labelled α-SYN protofibrils were resuspended in 1X PBS, coated on a carbon grid with uranyl acetate and dried in a desiccator. The grids were visualized using FEI Tecnai T20 at 200 KV. The α-SYN protofibrils were then lyophilized and stored at -20°C and resuspended in 1X TBS (Tris-buffered saline) buffer before experiments. The toxicity of the protofibrils was assessed by treating the differentiated neuronal cells seeded at a density of 30,000 cells / well in 24 well plates. Cell viability assay was performed using different concentrations of α-SYN protofibrils (0.5µM, 1µM, 2µM, 3µM) over different periods by MTT assay. Treatment conditions of astroglia cells by α-SYN protofibrils All the experiments were carried out by treatment of primary astrocytes, U-87 MG, and U251 cells with 1µM α-SYN protofibrils- unlabelled or TMR-labelled for 3h, 6h, 12h, and 24h at 37°C, 5% CO 2 unless mentioned otherwise. The cells were seeded at the same time for all the treated time points and controls. Once cells were adhered properly and appeared healthy after 24h of seeding, then treatments were done by adding (α-SYN 1µM) protofibrils in a reverse order of the time points. For example, 24h time points were treated first and then sequentially 12 h, 6 h, and 3 h points. Then, experiments were performed for all the time points and controls at a time. Therefore, the condition of the untreated control cells corresponds to all the treatment points. MTT assay and cell numbers counting MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay is a colorimetric assay used to measure cellular metabolic activity. Astrocytes, U-87 MG, and U251 cells were seeded at a density of 4000 cells per well in a 96-well plate and incubated overnight at 37°C, 5% CO 2 . Different concentrations of α-SYN protofibrils (0.5µM, 1µM, 2µM, 3µM) were added to the wells over different time periods (3h, 6h, 12h, 24h, and 48h) and treated with MTT reagent (Himedia # TC191-1G) for 2h. The insoluble formazan crystals were dissolved by adding dimethyl sulfoxide (DMSO; Himedia #TC185) which was later quantified by measuring absorbance at 570 nm using the PerkinElmer-Multimode plate reader spectrophotometer. Cell numbers were counted from the phase contrast microscopy images or DIC images using the multi-point option in Fiji (a Java-based program developed at the National Institutes of Health, USA). Immunocytochemistry (ICC) After treatment with the above-mentioned concentrations and time points adding α-SYN protofibrils, the cells were washed with 1X PBS and fixed with 4% PFA. The cells were then incubated with incubation buffer (1mg/ml Saponin and 5%FBS) for 20 min at RT. Respective primary antibodies (as mentioned below) were added and incubated overnight at 4°C, the cells were then washed 3 times with 1X PBS. All the secondary antibody (1:700 dilution) incubations were carried out in the dark for 2 hrs at RT, and 1X PBS washes were repeated. The coverslips were mounted on glass slides with ProLong Gold antifade reagent with DAPI, (Invitrogen P36941). Alternately, glass bottom 35mm dishes (Cellvis, D35-14-1.5-N) were used in a few experiments. Primary antibodies Ki67 anti-rabbit (Millipore B9260) as cell proliferation marker; FAK Rabbit (CST 3285T) and phospho-FAK ((Tyr397) pAb; Invitrogen 44-624G) as total and active FAK marker; Cathepsin D anti-rabbit (GeneTex #42368), LAMP-1 anti-mouse (BD Biosciences # 611042), LAMP-2 anti-mouse (Invitrogen #MA1-205) as lysosomal markers; βIII-tubulin anti-rabbit (Cloud-Clone #PAE711Hu01), Connexin43 Rabbit Ab (CST #3512S), SNCa rabbit pAb (Cloud-Clone #PAB222Hu01), GFAP rabbit pAb (Cloud-Clone #PAA068Mu01) and Phalloidin conjugated with iFlour555 (Abcam #176756) were used to stain TNTs and TMs. Secondary antibodies Alexa- 488 goat anti-rabbit (#A-11070; Invitrogen), Alexa- 555 anti-mouse (#A-1413312; Invitrogen), and Alexa- 488 anti-mouse IgG (H + L) (#A-11059; Invitrogen) were used respectively following the above-mentioned protocol. All primary antibodies were used at a dilution of 1:300, secondary antibodies at 1:500, and phalloidin at 1:700. ICC-stained cells were imaged with a confocal microscope (Zeiss LSM880, Carl Zeiss, Germany) or fluorescence microscope (IX73-Olympus). Western Blot U-87 MG cells were seeded at a density of 1 million per well in a 6-well plate and were treated with 1µM α-SYN protofibrils from 3h-24h. Post-treatment media was removed and the cells were washed with 1X PBS. RIPA buffer was added and the cells were scrapped and collected in 1.5 ml tubes. The tubes were incubated on ice with intermittent vortexing. Later the suspension was spun at 12,000 rpm for 10 min and the supernatant was collected and stored at -20 0 C. After normalizing the protein concentration, western blot assay was performed with primary antibodies GAPDH Mouse (Cloud-Clone # CAB932Hu22 dilution 1:1000), ROCK1 (CST # 4035T dilution 1:1000), ROCK2 (CST # 8236S dilution 1:1000), NF-kB p65/RelA Mouse mAb (Abclonal # A10609 dilution 1:1000), phospho ERK1-T202 + ERK2- T18 (Abclonal # AP0485 dilution 1:1000), CDK1 Rabbit pAb (Abclonal #A0220 dilution 1:1000), p21 Waf1/Cip1 (12D1) Rabbit mAb (CST #2947 dilution 1:1000) and secondary antibodies, Goat anti-mouse (H + L) (Invitrogen #32430 dilution 1:1500) and Goat Anti-Rabbit (H + L) (Invitrogen #32460 1:1500). The blot was developed with ECL solution (SuperSignal West Femto Trial kit Invitrogen #34094) and were quantified by densitometry using gel analyzer plugin of Fiji software. α-SYN internalization U-87 MG cells were seeded in a 24-well plate at a density of 0.1 million/well, pre-treated with 0.5 µM cytochalasin D and 5 µM Y-27632 (ROCK inhibitor), then treated (before 30min) with TMR labelled 1µM α-SYN protofibrils. Post-treatment U-87 MG cells were trypsinized (0.25% trypsin-EDTA solution; Gibco Canada origin #25200-072). The pellet was collected and resuspended in 1X PBS and twice washed with 1X PBS. α-SYN-TMR levels were quantified by measuring the fluorescence intensity of TMR at excitation/emission 555/585nm by using a flow cytometer (BD LSR II) and analysis was done using analysis software BD LSR-II analysis software. Live cell imaging using mitotracker and lysotracker U-87 MG cells (80,000 cells/well) were seeded on a 35 mm glass bottom dish (Cellvis, D35-14-1.5-N) and treated with 1µM TMR labelled α-SYN for 0h, 3h, 6h, 12h, and 24 h. The 0h cells were immediately washed before staining with lysotracker and mitotracker. Cells were stained with mitotracker green (Invitrogen #M7514) and lysotracker deep red (Invitrogen #L12492) and were incubated at 37°C for 15 min. Post incubation, the cells were washed with 10% DMEM and time-lapse and z-stack images were taken using confocal microscopy (Zeiss LSM880, Carl Zeiss, Germany). This was done to comprehend the localization of α-SYN and the transfer of organelles through TNTs. Live Cell Imaging using DiD®vybrant membrane dye U-87 MG cells (80,000 cells/well) were seeded on a 35 mm dish and treated with 1µM α-SYN as mentioned above. Cells were stained with cell-labelling dye DiD®Vybrant (Invitrogen #V22887) to label cell membranes. The dye was diluted with 10% DMEM (phenol red free) in the ratio 1:200 and incubated at 37°C for 20 min. Post incubation, the cells were washed with DMEM for 10 min, and images of live cells were taken to visualise the number of thin membrane tubes like TNTs using a fluorescence microscope (IX73-Olympus). Estimation of cytosolic ROS 2′-7′-Dichlorodifluorescein diacetate (DCFDA) is used to detect ROS production in the cell. U-87 MG cells were seeded in a 24-well plate at a density of 0.1 million/well and treated with 1µM α-SYN protofibrils as mentioned above. Post-treatment U-87 MG cells were trypsinized (0.25% trypsin-EDTA solution; Gibco Canada origin #25200-072). The pellet was collected and resuspended in DMEM with 10% FBS and 20µM DCFDA. Cells were incubated for 30 min at 37°C then washed with 1X PBS to remove the extra dye. ROS levels were quantified by measuring the fluorescence of DCFDA stained cells at excitation/emission 488/520nm by using a flow cytometer (BD LSR II) and analysis was done using analysis software BD LSR-II analysis software. Mitochondrial membrane potential using JC-1 dye JC-1 (5,5,6,6’-tetrachloro-1,1’,3,3’ tetraethylbenzimi-dazolylcarbocyanine iodide) is the indicator of mitochondrial membrane potential. JC-1 is a cationic, lipophilic dye. Normal healthy cells show negative mitochondrial membrane potential where JC-1 dye can enter into the mitochondria and form J-aggregates. The aggregates exhibit excitation/emission maxima at 485/590 nm (red spectrum). Unhealthy cells show lower mitochondrial membrane potential as it loses the balance of electrochemical potential. At this condition, a lesser amount of JC-1 dye enters into the mitochondria and retains its monomeric form. These monomers exhibit excitation/emission maxima at 514/529 nm (green spectrum). To know the effect of α-synuclein aggregates on mitochondrial membrane potential, JC-1 assay (BD Biosciences #551302) was done. Astrocytes and U-87 MG cells were seeded at a density of 15,000 cells / well in the glass bottom area of the 35 mm imaging dishes (Cellvis, D35-14-1.5-N). Cells were treated with 1µM α-SYN for 3h-24h. Confocal images were also taken at an excitation of 490 nm and emission at 527 nm (green) and 590 nm (red). The ratio of green fluorescence versus red fluorescence per cell was analysed using Fiji image analysis software to quantify mitochondrial membrane potential. Co-culture model and mitochondria transfer One population of U-87 MG cells was transiently transfected with pLV-mitoDsRed (Addgene #44386; was a gift from Dr. Pantelis Tsoulfas's lab) plasmid and another population with mEGFP-lifeact-7 (Addgene #58470; was a gift from Michael Davidson) plasmid using lipofectamine 3000 (Invitrogen #44386) transfection reagent. An equal number of transfected cells from both populations were seeded together at a total density of 60,000 cells per glass bottom 35mm dish (Cellvis, #D35-14-1.5-N) and treated with α-SYN protofibrils. After treatment, the cells were fixed with 4% PFA and images were taken using confocal microscopy (Zeiss LSM880). β- galactosidase activity assay U-87 MG cells were seeded at the density of 10,000 cells per well in a 24-well plate. The cells were treated with 1µM α-SYN for 3h and 24h, these time points were chosen according to the data from other experiments. Post-treatment the β-galactosidase activity was measured by using the β-galactosidase staining kit (AKR- 100 Cell Biolabs Inc). Brightfield images of the stained cells were taken using a colour camera. Microscopy Fluorescence images were taken using Zeiss LSM880 confocal laser scanning microscope (Carl Zeiss, Germany) or fluorescence microscope (IX73-Olympus). The confocal images were taken using objectives Plan-Apochromat 40x/1.40 or 63x/1.40 Oil Dic M27, with the fluorescence filter sets DAPI, FITC, and TRITC (Carl Zeiss, Germany). Sequential images of the different fluorescence channels were taken with 405 nm, 488 nm, and 561 nm lasers. The images were captured with a pixel dwell of 1.02 µs and each xy-pixel of 220 nm 2 . For all the experiments at least 5–10 images per condition were taken from randomly selected areas. DIC (differential interference contrast) images were captured along with fluorescence channels to understand the morphology and cell boundary for both fixed and live imaging experiments. Time-lapse and z-stacks (6–12 stacks of z-scaling ~ 415 nm) were captured from the bottom to the top of the cells using a confocal microscope to identify TNTs and TMs. Trafficking of organelles through TNTs and TMs was tracked from time-lapse images. Wide-field fluorescence microscope (IX73-Olympus) was used to capture images for a few experiments using 20X/0.4 NA, and 40X/1.3 NA plan-apochromatic objectives. Image analysis a) TNT characterization Confocal images were analysed using Fiji a Java-based image processing software developed at the National Institutes of Health (NIH) and the Laboratory for Optical and Computational Instrumentation (LOCI). TNTs were characterized by their unique nature to hover and not attach to the substratum, images were taken with z-stacks using confocal microscopy as mentioned above. TNTs (less than 1µM in diameter) were found in the middle z-stacks while TMs are found in the initial z-stacks since they are attached to the surface. Images of z-stacks were reconstructed using the 3D volume view plugin of Fiji as described earlier (Dilna et al., 2021 ). TNTs were manually counted and plotted as the ratio of the number of TNTs to the number of cells per field (Valappil, Raghavan et al., 2022 ). b) Tracking of organelles Movements of lysotracker and mitotracker positive vesicles through TNTs and TMs were tracked using the Trackmate (manual) plugin in Fiji. Analysis was done from time-lapse videos taken for a minimum of 30 frames at an interval of 36 seconds for 18–20 mins. By setting each organelle as an object we used the semi-automatic tracking method of the trackmate plugin. The speed of the organelles was determined from the average displacement between each consecutive image. c) Size analysis The area of protofibrils, size of aggregates, and size of lysosomes and mitochondria were analysed per cell from the randomly taken images of 200–300 cells per condition. The analysis was performed by setting a threshold to separate the particles as individual points and then by using the ‘Analyze particle’ option of Fiji. d) Intensity analysis Similarly, the expression of Ki67, FAK, pFAK, α-SYN aggregates, lysosomes, and the ratio of green versus red fluorescence of the JC-1 experiment was analysed by quantifying the intensities of the labelled proteins per cell from the images of 200–300 cells for each condition. Intensities were analysed by drawing and selecting regions of interest using the ROI-plugin in Fiji. e) Mitochondria morphology analysis The quantification of mitochondria branching and branch length were determined using the MiNA plugin in the ImageJ software. We downloaded the MiNA analysis plug-in from the Git Hub repository of Stuart Lab. We cropped each cell stained with mitochondria and ran the MiNA plugin to determine the mean branch length of mitochondria. Statistics To validate the significance of the analysed data, one-way and two-way ANOVA tests were performed as per the experimental parameters as mentioned in the figure legends. For Fig. 5 C, we performed a student t-test since we had only two conditions. The statistics were calculated from the three biological repeats. Results α-SYN protofibrils induce biogenesis of transient TNTs The contributions of extracellular α-SYN protofibrils in the pathological spreading of PD in neuronal cell culture models are well-studied (Dieriks, Park et al., 2017 ). Recent studies have shown that aggregates of α-SYN can modulate cellular crosstalk between neurons and the surrounding neuroglial cells (Chakraborty, Nonaka et al., 2023 ), which plays an important role in getting rid of toxic loads of aggregates (Rostami et al., 2017 , Scheiblich et al., 2021 ). However, the mechanism of establishment of intercellular communications and their fate are yet to be understood. Therefore, we looked into the primary astrocytes and astrocyte-origin cancer cell lines (U-87 MG and U251) to understand the possible fate of the astroglia cells over time on α-SYN protofibril treatment. To understand this, purified α-SYN was converted into protofibrils that were either unlabelled or fluorescently (TMR) labelled and characterized using transmission electron microscopy (Fig. 1 A). Interestingly, after treatment with 1µM α-SYN protofibrils at 3h, 6h, 12h, and 24h, we observed formation of TNT-like membrane networks between primary astrocytes (Fig. 1 B), U-87 MG (Fig. 1 C, Supplementary Fig. 1A) and U251 cells (Supplementary Fig. 1B) for a transient period at early hours. Time points were chosen to quantify optimum biogenesis of membrane nanotubes after treatment with 1µM α-SYN protofibrils over 24 h (Supplementary Fig. 1C). Quantification of TNT-like structures was performed on confocal images of phalloidin-stained astrocytes (Fig. 1 B). DiD dye-stained membranes were imaged using a fluorescence microscope to detect the abundance of stained TNT-like membrane conduits in live cells (Fig. 1 C). DIC (Differential Interference Contrast) images of U-87 MG (Supplementary Fig. 1A) and U251 (Fig Supplementary Fig. 1B) cells were also taken to detect TNT-like structures. Quantification showed an increase in several hovering thin TNT-like structures per cell compared to controls in the primary astrocytes (Fig. 1 D). DiD images of U-87 MG also revealed transient increase in the biogenesis of thin (diameters < 1 µm), cell-to-cell membrane connections at early time points (3h and 6h) (Fig. 1 E-F). Similarly, quantification of DIC images of U-87 MG cells (Supplementary Fig. 1D) and U251 (Supplementary Fig. 1E) showed enhanced transient biogenesis of TNTs at early time points compared to their respective controls and later time points. α-SYN protofibrils induce open-ended TNTs and cell-to-cell transfer TNTs are actin-rich, nano-sized in diameter, and primarily defined as open-ended membrane tubes (Abounit, Delage et al., 2015 ). TNTs are distinguished from filopodia and neurites by their distinct characteristic to hover between two distant cells (Valappil et al., 2022 ). Actin-binding phalloidin and GFAP-stained, 3D-reconstructed confocal images show actin-positive thin TNTs that hover between two cells in primary astrocytes (Fig. 2 A). Astrocyte-origin cancer cell lines (U-87 MG and U251) are known to form tumour microtubes (TMs), close-ended thicker membrane networks (diameters > 1 µm) between cells and electrically coupled via gap junctions (Osswald, Jung et al., 2015 ). To distinguish TNTs and TMs, confocal z-stack images were obtained after immunocytochemical staining of cells using β-tubulin antibody and phalloidin dye. Thin actin-positive, hovering TNTs (Fig. 2 B) and thicker TMs (Fig. 2 C) are identified in U-87 MG cells. TMs are positive for both actin and β-tubulin. Studies have indicated that astrocytoma/glioblastoma cells express lower levels of neuronal marker β-tubulin and are stained faintly (Abbassi, Recasens et al., 2019 ). Despite that, β-tubulin staining is distinctly detected in thicker TMs and absent in TNTs (Supplementary Fig. 2A and B). DIC images clearly showed the nano-sized diameters of TNTs and thicker TMs (Fig. 2 A-C-upper panels). Reconstructed 3D volume images of confocal z-stacks show actin-positive, thin TNTs hover between two cells and are not attached to the substratum, whereas the tubulin-positive thicker TMs are present on the substratum (Fig. 2 A-C-lower panels). Several studies have shown that TNTs are open-ended membrane channels, and can transfer organelles directly from one cell to another (Abounit et al., 2016 , Rustom et al., 2004 ). However, few studies have referred close ended membrane continuities that are not involved in cell-to-cell transfer of organelles and electrically coupled via gap junctions at the endpoint as TNTs (Sowinski, Jolly et al., 2008 , Wang, Veruki et al., 2010 ). Connexin43 is the essential protein of the GAP junction. Therefore, TNTs were immunostained using gap junction protein connexin43, to identify open and close-ended TNTs with GAP junction at the junctional end. Images captured populations of both connexin43 negative (Fig. 2 D) and positive (Fig. 2 E) at the endpoint of TNTs. Non-hovering thicker TMs are connexin43 positive structures (Fig. 2 F). We further functionally characterized TNTs based on their ability to transfer organelles like lysosomes and mitochondria. Time-lapse and z-stack images were taken using confocal microscopy (using both fluorescence channels and DIC channel) in live U-87 MG cells, stained with lysotracker and mitotracker, post-treatment with 1µM α-SYN protofibrils (3h, 6h, 12h, and 24h). Time-lapse videos in DIC microscopy images revealed transient increase in the biogenesis of thin cell-to-cell membrane connections and unidirectional movements of organelles via the membrane nanotubes at early time points (3h and 6h) (Movies 1–2). Time-lapse videos captured cell-to-cell movement of α-SYN-TMR accumulated lysosomes and mitochondria through long (20 µm to 120 µm), thin TNTs after 3h and 6 h of treatments respectively (Movies 3 and 4). We validated from z-stack images, that the thin nano-sized membrane tubes or TNTs are hovering structures connecting between two distant cells. The DIC images show thin membrane nanotubes (red arrows) between cells that are clearly visible at a higher plane of z = 4. On the surface, at z = 0, filopodia-like membrane extensions (violet arrows) are visible (Fig. 2 G) which are unlike the TNTs. We tracked the movements of lysotracker (Fig. 2 H) and mitotracker-labelled (Fig. 2 I) organelles along the TNTs (identified from z-stacks) using the TrackMate plug-in available in FIJI and we observed that both the organelles travel unidirectionally from one cell towards another, with an average speed of 0.062 ± 0.003 µm/sec. Further, we captured time-lapse videos to detect direct cell-to-cell transfer of α-SYN-TMR accumulated lysotracker and mitochondria via open-ended TNTs between U-87 MG cells after 3h of treatments (Movies 5 and 6). Our results reveal the formation of open-ended functional TNTs and cell-to-cell transfer through them at 3h and 6h post-treatment with α-SYN. We observed a transient increase in the biogenesis of thin (diameters < 1 µm), hovering TNTs upon 1µM α-SYN protofibrils treatment at early hours (3h and 6h) by quantifying from the 3D reconstruction of confocal z-stack images (Fig. 2 J). Though control cells contain a higher percentage of TMs, we observed a transient increase of both open and close-ended TNTs, based on connexin43 staining after 3h and 6h post-treatment with α-SYN (Fig. 2 K). Length and diameters of TNTs were measured using the confocal z-stack images in all the cell types, primary astrocytes, U-87 MG, and U251 cells. The careful measurements of z-stack images detected that the diameter of TNTs ranges between 1–3 pixels, that is around 220 nm to 660 nm and length varies between 20 µm – 100 µm across all the cell types (Fig. 2 L and M). Transient lysosomal toxicities in astroglia cells upon treatment with α-SYN protofibrils Internalization of extracellularly applied α-SYN protofibrils into endo-lysosomal pathways induces transient accumulations and toxicities (3h and 6h) in primary astrocytes (Fig. 3 A) and U-87 MG cells (Fig. 3 B and C). α-SYN and LAMP1 were co-immunostained in primary astrocytes (Fig. 3 A) and U-87 MG cells treated with TMR-labelled α-SYN were stained with lysotracker (Fig. 3 B). Accumulations of α-SYN protofibrils in endo-lysosomes and resulting toxicities were studied following enlarged and distorted morphology of α-SYN accumulated endo-lysosomal vesicles at 3h and 6h (Fig. 3 A and B). The extent of cathepsin leakage from LAMP2-positive vesicles was also observed to follow lysosomal toxicities (Fig. 3 C). α-SYN protofibrils were internalized in primary astrocytes and colocalized in substantial amounts to LAMP1-immunostained positive vesicles (analysed using Coloc 2 plug-in in FIJI). Maximum colocalizations were observed at 3h of treatment which over time gradually decreased (Fig. 3 D). Internalization of protofibrils to lysosomal pathway caused transient accumulation and lysosomal toxicities, resulting in larger-sized LAMP1 positive vesicles that were transiently detectable at 3h and 6h after α-SYN protofibrils treatment in astrocytes (Fig. 3 E). Total areas of red puncta of α-SYN-TMR oligomers per cell were quantified measuring the size of each punctum. The results show that endo-lysosomal machinery in U-87 MG cells gradually clears up toxic protofibrils (Fig. 3 F). The analysis of cathepsin leakage in LAMP2-positive lysosomal vesicles indicated no significant increase of cathepsin-D to the cytosol (Fig. 3 C). However, we observed the larger cathepsin-D and LAMP2-positive vesicles after 3h of protofibril treatments (Fig. 3 G). Morphology of LAMP2-positive lysosomes at 3h is elongated and rough in comparison to control, 6h, 12h, and 24h treated cells (magnified images highlighted in the upper-left corner of Fig. 3 C). The time of transient lysosomal toxicity coincides with the transient increase of TNT biogenesis (Fig. 1 B-F). Altogether the results suggest that transfer of organelles via TNTs probably aids the cells to cope with protofibril-induced lysosomal toxicities. Transient mitochondrial toxicities in astroglia cells upon treatment with α-SYN protofibrils Similar to transient lysosomal toxicities and biogenesis of TNTs, mitochondrial toxicities were observed through morphological changes in the early time points (3h and 6h) using MitoProbe JC-1 dye which measures mitochondrial membrane potential (ΔΨ M ). Lipophilic, cationic JC-1 dye can enter healthy mitochondria and aggregate (emission @590 nm in the red channel) inside, whereas, in unhealthy mitochondria with decreased membrane potential, the dye crosses in and out through the relatively open membrane pores and stays as monomers (emission @527 nm in the green channel). Adding to our previous observations, we noticed a decrease of ΔΨ M transiently in the early time points (3h and 6h) in primary astrocytes, whereas at the later time points (12h and 24h) mitochondria looked healthier and were rescued from protofibrils-induced mitochondrial toxicities (Fig. 4 A and B). The quantification shows JC-1 labelled red coloured healthier, larger mitochondria in the control and recovered healthier cells after 24h (Fig. 4 C). The toxic mitochondria in early time points (3h and 6h) were observed to possess shorter branch lengths compared to the control and the recovered cells at later times (12h and 24h) (Fig. 4 D). Mitochondrial toxicities were also observed in U-87 MG cells through morphological changes using mitotracker staining (Fig. 4 E, upper panel) and mitochondrial membrane potential (ΔΨ M ) using MitoProbe JC-1 (Fig. 4 E, lower panel). Similar to our results in primary astrocytes, images of U-87 MG cells showed that at the early time points (3h and 6h) mitochondria were smaller sized, with fragmented morphology (Fig. 4 E, upper panel) and quantifications showed increased JC-1 stained greener mitochondria or mitochondria with decreased ΔΨ M (Fig. 4 F). Thus, it is evident that primary astrocytes and U-87 MG cells adapt and are rescued from α-SYN protofibril-induced transient mitochondrial toxicities at later times. Cell-to-cell transfer of mitochondria in α-SYN protofibril treated astroglia cells The transfer of mitochondria between α-SYN protofibrils-treated U-87 MG cells was further examined by co-culturing these cells (Fig. 5 A). MitoDsRed (red) and EGFP-lifeact (green) transfected U-87 MG cells were co-cultured and treated with α-SYN protofibrils for 3h, 6h, 12h, and 24h. Results demonstrate mitoDsRed labelled red mitochondria in the green EGFP-lifeact stained cells upon treatment with α-SYN protofibrils (Fig. 5 A). The transferred red mitochondria in the green cells were observed maximum at early time points (3h and 6h) when cells show enhanced mitochondrial toxicities and increased numbers of TNTs (Fig. 5 B). When conditional media from the mitoDsRed transfected cells were given to the green cells transfected with EGFP-lifeact, we did not observe any significant transfer of mitochondria (Supplementary Fig. 3). The result excludes the probability of transfer of mitochondria through exosomes. The morphology of the transferred mitochondria in the green cells mostly appears round in shape rather than elongated and healthy. Quantification shows that transferred mitochondria are smaller in size and round-shaped, compared to the mitochondria of the control cells ( Fig. 5 C ) . Therefore, we further studied the fate of cell-to-cell transfer of toxic mitochondria, following ROS levels in the cells upon protofibril treatment at different time intervals. We also observed increased total cellular ROS at an early time point (3h) and a gradual decrease in ROS levels at later time points (6h, 12h, and 24h) in U-87 MG cells (Fig. 5 D and E). The fluorescence intensity of DCFDA was analysed using flow cytometer to quantify the cellular ROS levels (Fig. 5 E). The quantification of DCFDA flow cytometer data show similar results in primary astrocytes with a significant increase in cellular ROS levels at 3h compared to the control cells which then decrease at later time points (6h, 12h, and 24h) (Fig. 5 F and G). α-SYN protofibril-induced ROS mediated cellular senescence and its correlation with transient biogenesis of TNTs Increased levels of cellular ROS play a significant role in inducing cellular senescence and reduction of ROS accumulation can reverse p-21 mediated cellular senescence (Macip et al., 2002 ). Studies have also shown the critical role of α-SYN-induced ROS in cellular senescence-associated neurodegeneration (Miller, Campbell et al., 2022 , Verma, Seo et al., 2021 ). The increased ROS levels and its elimination in correlation to TNT biogenesis, motivated us to look into the senescence states in astroglia cells (Macip et al., 2002 ). We have observed a transient increase in p-21 levels with α-SYN protofibrils treatment in western blot analysis at an earlier time point (3h) in U-87 MG, then p-21 levels decrease gradually over time (6h, 12h, and 24h) (Fig. 5 H). Further, we have observed irregular nuclei, invaginations, and fragmented nuclei during the transient time window 3h and 6h, which corresponds to the α-SYN protofibrils induced transient organelle toxicities, ROS production and biogenesis of TNTs in primary astrocytes (Fig. 5 I-J) and U251 cells (Supplementary Fig. 4A and B). We established cells undergo transient cellular senescence at an early time point (3h) by measuring β-galactosidase activity as the senescence marker (Supplementary Fig. 4C). It is known that DNA damage-related nuclear size irregularities are often associated with hyperproduction of cellular ROS and mediated senescence. The transient cellular senescence corresponds to the transient biogenesis of TNTs at early hours in α-SYN treated cells. Eventually, the reduction of ROS accumulation caused a reversal of p-21-dependent cellular senescence at later times. Clearance of α-SYN induced organelle toxicities and ROS in cell survival and proliferation It is a well-established fact that extracellularly applied α-SYN protofibrils are toxic to neurons and cause gradual neuronal death (Li, Yuan et al., 2021 ). Conversely, we observed astrocytes and astrocytes origin cancer cells survive by alleviating α-SYN protofibrils induced organelle toxicities and cellular ROS levels. We also observed that biogenesis of TNTs and cell-to-cell transfer precedes clearance of α-SYN protofibrils induced organelle toxicities and cellular ROS levels. Recent studies have shown that cell-to-cell transfer of mitochondria in astrocytes via TNTs facilitates cell survival (Valdebenito, Malik et al., 2021 ). Therefore, we investigated cell viability using α-SYN protofibrils in primary astrocytes, U-87 MG, and U251 astroglia cells. MTT assay measures cell metabolic activity and is used as an indicator for cell viability. The results pertaining to this assay show a concentration-dependent increase of cell viability upon treatment of toxic α-SYN protofibrils at later time points (12h and 24h) in astrocytes (Fig. 6 A), compared to the respective controls and early time points (3h and 6h). α-SYN protofibrils induced organelle toxicities at earlier times also resulted in decreased metabolic activity (Fig. 6 A). α-SYN protofibrils treated U-87 MG (Fig. 6 B) and U251 ( Fig. 6 D) cells show proliferation at later times (12h and 24h) compared to their respective controls. U-87 MG cells also show concentration-dependent proliferation at later times (24h) ( Fig. 6 C ) . Further, we manually counted the cells to reconfirm that the increasing concentrations of toxic protofibrils caused an increase in cell numbers or cell proliferations, after 24 and 48 h of treatments in astroglia cells ( Supplementary Fig. 5A and B). Overall, the results showed that, instead of developing progressive toxicities or cell death overtime α-SYN protofibrils treatment, the astroglia cells adapt to overcome the stress and proliferate during post-recovery time. We have verified the toxic effects of the protofibrils in the neurons derived from the differentiation of neuroblastoma (N2a) cells. The results show time- and concentration-dependent cell death in the treated cells (Supplementary Fig. 5C and D). Since we have observed a significant increase in cell numbers and cell viability upon α-SYN protofibrils treatment, we checked for cell proliferation using Ki67 as the marker. Astrocytes and U-87 MG cells immunostained with Ki67 antibody and nuclear stain DAPI (Fig. 6 E) show a significant increase in Ki67 overexpression in the nucleus at 12h and 24h in comparison to control and early time points (3h and 6h) (Fig. 6 F and G). Overall, the results indicate toxic α-SYN-induced initial stress (till 6 h) promotes biogenesis of TNTs and cell-to-cell transfer of organelles, consequences of which aid in rescuing the cells from organelle toxicities or ROS-induced cellular stresses. Further, to deal with recovery, cells probably facilitate proliferation. TNT biogenesis pathways in astroglia proliferation TNTs are structurally open-ended membrane actin conduits. Thereby, it is obvious that modulation of membrane and cytoskeleton will play a major role in their biogenesis. However, the exact mechanism of TNT biogenesis is not known. Studies on screening of inhibitors in the actin signalling pathways could unfold molecular events behind the α-SYN toxicity-induced biogenesis of TNTs. Therefore, we studied different actin inhibitors (Fig. 6 H ) to see their effects on biogenesis of TNTs at an early time (3h) (Fig. 6 I) and cell proliferation at a later time (24h) in U-87 MG cells (Fig. 6 J). We observed that Cytochalasin D (inhibits actin polymerization and interaction of G-actin-cofilin) and IPA-3 (PAK1/2 inhibitor) inhibited α-SYN protofibril-induced TNTs formation, whereas, CK-666 (Arp2/3 inhibitor), Blebbistatin (myosin-II-specific ATPase inhibitor), and Y-27632 (ROCK inhibitor) promote biogenesis of TNTs (Fig. 6 K). Similar to the reports of earlier studies (Henderson, Ljubojevic et al., 2022 ), we have also observed that Arp2/3 inhibitor CK-666 caused formation of significantly longer TNTs (Fig. 6 I and Supplementary Fig. 6). To understand the role of TNTs in cell proliferation, cell viability was assayed using MTT (Fig. 6 L), and cell numbers were determined by manual counting (Fig. 6 M). TNT-numbers (Fig. 6 K) and cell proliferation (Fig. 6 L and M) data showed that the actin inhibitors, Cytochalasin D, and IPA-3, which prevent biogenesis of TNTs, inhibit α-SYN protofibrils induced cell proliferation as well (Fig. 6 L and M). The actin inhibitors, CK-666 and Blebbistatin which facilitate biogenesis of TNTs, did not alter α-SYN protofibrils induced proliferation. However, the inhibitors alone (CK-666, and Blebbistatin) did not show a significant effect on cell proliferation. We observed the ROCK inhibitor Y-27632 in the presence and absence of α-SYN protofibrils significantly increased proliferation (Fig. 6 L and M). ` Biogenesis of α-SYN induced TNTs through modulation of ROCK pathway, resulting in increased cell survival and proliferation A recent study has shown that the ROCK inhibitor (Y-27632) is an intriguing compound that boosts biogenesis of TNTs via Myosin II-mediated actin modulation (Scheiblich et al., 2021 ). However, it is not yet known how α-SYN dominates modulation of ROCK inhibition signalling mediated TNT biogenesis, over the cofilin-G-actin interaction (Cytochalasin-D inhibited pathway) mediated actin remodulation. To unfold the mechanism, we followed the internalization of α-SYN-TMR protofibrils in the presence of Cytochalasin-D and ROCK inhibitor (Y-27632) using flow cytometry quantification (Fig. 7 A). We observed that Cytochalasin-D inhibited internalization of α-SYN protofibrils, however, Y-27632 did not show any effect (Fig. 7 B). α-SYN-induced cellular senescence results in translocation of FAK in the nucleus Our results showed α-SYN protofibrils induced nuclear deformity and cellular senescence in astroglia cells at early time points (3h and 6h) (Fig. 5 H-J). Several studies have shown that focal adhesion kinase (FAK) inhibition could induce the DNA damage and nucleus deformity that accompanies cellular senescence (Chuang, Wang et al., 2019 , Zhou, Yi et al., 2019 ). Loss of integrin-dependent cell adhesion in cellular stress modulates FAK and facilitates its translocation from the plasma membrane to enter to nucleus (Lietha, Cai et al., 2007 , Lim, Chen et al., 2008 , Lim, Miller et al., 2012 ). We observed de-adhesion of astroglia cells (U-87 MG) with α-SYN protofibrils treatments at early time points (3h and 6h) when cells show lysosomal-mitochondrial toxicities and transient biogenesis of TNTs, compared to the control and cells at later times (12h and 24h) (Supplementary Fig. 7A). Further, we noticed treatments with α-SYN protofibrils cause nuclear translocation of FAK in the astrocytes that are connected by TNTs at early time points (3h and 6h) (Fig. 7 C and D). The presence of nuclear FAK was verified by 3D images at xz and yz planes (Supplementary Fig. 7B). We also observed treatments with α-SYN protofibrils cause nuclear translocation of FAK to the nucleus in the U-87 MG (Fig. 7 E and F) and U251 (Supplementary Fig. 7C and D) cells at early time points (3h and 6h), whereas, at later timepoints in the post-recovered cells, FAK relocates back to PM and cytosol. We have also observed nuclear translocation of activated phospho-FAK (Tyr 397) transiently at early time points (3h and 6h) in astrocytes ( Fig. 7 G and H). These results clearly demonstrate that there is a transient translocation of FAK in the nucleus at the early and late time points on α-SYN treatment. Nuclear translocation of FAK modulates the ROCK pathway to induce TNT biogenesis and cell proliferation Inhibition of integrin-mediated FAK activation prevents ROCK activation and inhibits cell adhesion. Moreover, when active/inactive FAK is displaced from cell-adhesion sites in non-adherent cells, Rho-mediated activation of ROCK kinases maintains cytoskeleton tension by regulating actin re-modulation (Pirone, Liu et al., 2006 ). To understand the FAK-mediated regulation of ROCK kinases, we have performed western blots and quantified expressions of ROCK1 and ROCK2 in the α-SYN treated cells (Fig. 7 I and J). Results show ROCK2 inhibition was observed in the early hour (3 h) after α-SYN treatment, later expressions increased gradually (Fig. 7 I and J). However, ROCK1 expression with α-SYN treatment showed gradual inhibition over time. ROCK activation pathways involve in cell proliferation as well (Pirone et al., 2006 ). Our western blots showed increased levels of ROCK-mediated cell proliferation markers ERK1/2, NF-κB, and cdk1 (Fig. 7 K and L). We observed higher ROCK2 expression in the rescued cells (Fig. 7 I and J). Probably, ROCK2 activation at the later hours, results in increased cell proliferation through ERK1/2 and NF-κB signalling cascades. Overall, the results delineate that α-SYN protofibrils treatment caused transient localization of FAK/pFAK to the nucleus of astroglia cells, which could modulate ROCK inhibitory pathways to promote biogenesis of TNTs in the astroglia cells for a transient time at early hours after the treatment. The rescued cells post α-SYN treatment and transient TNT biogenesis, eventually may re-activate ROCK signalling to restore cytoskeleton tension, and promote enhanced cell proliferation (Fig. 8 ). Discussion PD pathology progresses with the spreading of misfolded α-SYN aggregates in the brain (Kalia & Lang, 2015 ). Even though cytoplasmic inclusion of α-SYN in the neurons is the central hallmark of neuropathology development in PD, evidence suggests release of α-SYN aggregates from degenerated neurons in the extracellular brain plays a significant role in PD pathology progression. The role of extracellular α-SYN in cell-to-cell transfer and PD pathology progression has widely been studied in several model systems (Neupane et al., 2022 ). Recent studies have shown that extracellular α-SYN aggregates, taken up by astrocytes and microglia, promote biogenesis of TNTs. The TNT-mediated glial crosstalk and cell-to-cell transfer facilitate degradation of the aggregates and clearance of toxic organelles (Rostami et al., 2017 , Scheiblich et al., 2021 ). Previously, the study by Loria et al., has shown that astrocytes facilitate degradation of α-SYN protofibrils by promoting TNT-mediated transfer (Loria, Vargas et al., 2017 ). The crosstalk between astrocytes and microglia via TNTs facilitates the degradation of α-SYN protofibrils even faster (Rostami et al., 2021 ). A recent study has reported, an on-demand formation of TNTs to clear toxic α-SYN protofibrils and alleviate ROS levels in microglia (Scheiblich et al., 2021 ). The study showed, the rescue of toxic burden of α-SYN via TNT-mediated borrowing of healthier mitochondria from the healthier neighbours and transfer of toxic aggregates to them (Scheiblich et al., 2021 ). Recently, a study demonstrated that neuronal cells transfer toxic mitochondria to microglia via TNTs, whereas microglia protect neurodegeneration by transferring healthier mitochondria to neurons (Chakraborty et al., 2023 ). However, the molecular players involved in the biogenesis of transient TNTs, the fates of TNT-mediated cell-to-cell transfer, and their dynamic interplay in the clearance of toxic neurodegenerative aggregates are not well explored. Our study has shown for the first time, that transient translocation of FAK/pFAK to the nucleus upon α-SYN protofibrils treatment causes transient biogenesis of TNTs, which eventually contributes to enhanced cell proliferation in the astroglia cells. Our study has also established a strong correlation between transient biogenesis of TNTs with α-SYN-induced toxic burden at early hours (3h and 6h). Cell-to-cell transfer through transient TNTs corresponds to cellular toxicities, like lysosomal, and mitochondrial toxicities, and increased levels of cellular ROS and associated cellular senescence. However, the astroglia cells recover from these cellular toxicities at later hours (12h and 24 h). The post-recovered astroglia cells proliferate with increasing concentrations of α-SYN protofibril treatments. Understanding of molecular players involved in the proliferation of astrocytes in response to α-SYN aggregates is highly important since astrogliosis and microgliosis act as essential mediators in maintaining cellular homeostasis in the PD degenerative brain (MacMahon Copas et al., 2021 ). Several studies have reported cell-to-cell transfer of mitochondria through TNTs in several pathophysiology conditions, where the transfer of healthy mitochondria results in rescue of cells from apoptosis or toxicities (Han & Wang, 2021 , Lou, Fujisawa et al., 2012 , Spees, Olson et al., 2006 ). However, it is not the case that TNTs transfer only healthy mitochondria to neighbouring cells. Microglia and astrocytes probably facilitate dilution of its toxic burden by transferring toxic α-SYN aggregates and toxic mitochondria by sharing with its neighbours through TNTs (Loria et al., 2017 , Scheiblich et al., 2021 ). We have observed α-SYN protofibrils induced increased ROS production at early time points (3h and 6h), causing reduced mitochondrial membrane potential, resulting in generation of fragmented mitochondria and disintegration of mitochondrial network. In the co-culture experiment, we observed only unidirectional transfer of toxic fragmented mitochondria to their neighbouring cells. Several studies have shown that accumulated ROS with transferred toxic mitochondria could facilitate cell proliferation (Heinke, 2022 , Kidwell, Casalini et al., 2023 ). The observations suggest that astrocytes and astroglia cells share their toxic organelles (lysosomes and mitochondria) with surrounding neighbours to dilute the toxic burden, and the transferred toxic mitochondria may contribute to facilitating proliferation. We have observed that α-SYN protofibrils induced increased ROS levels directly affect mitochondrial dysfunction, and DNA damage, which play key roles in inducing cellular senescence in reactive astrocytes (Davalli, Mitic et al., 2016 , Gallage & Gil, 2016 , Verma et al., 2021 ). We have observed α-SYN protofibrils induced mitochondrial abnormality, fragmented irregular nucleus, increased β-galactosidase activity, and p21-pathway-dependent premature cellular senescence for a transitory period. Astroglia cells recover from senescence-related toxicities by alleviating ROS levels, after transient biogenesis of TNTs and TNT-mediated cell-to-cell transfer. ROS-induced DNA damage and mitochondrial toxicity do not always lead to apoptosis (Borges, Linden et al., 2008 ). Cells activate repair mechanism/s in case of delayed apoptosis. Moreover, reduction of ROS accumulation can reverse p21-mediated stress-induced premature senescence or vice versa (Macip et al., 2002 ). Cell-to-cell transfer of neurodegenerative aggregates drive spreading of aggregates, which act as seeds for further propagation in neurons (Nath, Agholme et al., 2012 ), whereas, astrocytes and astroglia cells rescue cellular toxicities (Loria et al., 2017 , Nath et al., 2012 , Rostami et al., 2017 ). The role of the inherent conventional clearance efficiency of astrocytes cannot completely be ruled out. Unlike neurons, astrocytes lack endogenous α-SYN and the capacity of continuous seeding to form higher-order toxic aggregates is limited. This is to emphasize here that TNTs are predominantly observed in the cell types that possess inherent anti-apoptotic properties, like primary neurons, neuronal cells, neuroglial cells, and cancer cells (Gousset, Schiff et al., 2009 , Rustom et al., 2004 ). Neurons possess inherent anti-apoptotic properties like other brain cells, however, unlike glial cells they are mitotically incompetent (Sharma & Subramaniam, 2019 , Tardivel, Begard et al., 2016 ). We observed α-SYN induced TNT-mediated cell-to-cell transfer eventually leads to cell death in mitotically incompetent neurons, whereas astrocytes and astroglia cells clear toxic burden and enhance proliferation and cell survival. TNT-mediated cellular clearance and proliferation in astrocytes could play a significant role in PD. Therefore, the important question that needs to be understood is how biogenesis of TNTs and clearance of cellular toxicities are associated with proliferation in astrocytes. Several studies have indicated that biogenesis of TNTs may induce cell proliferation, to protect the cells from cell death under pathogenic conditions and chemo- or radio- therapy related stress (Han & Wang, 2021 , Osswald et al., 2015 , Saha, Dash et al., 2022 , Wang, Chen et al., 2021 ). The studies show mostly limited or indirect correlation between TNTs and cell proliferation. Our results on small molecule actin modulators indicate ROCK inhibitory pathway mediated biogenesis mechanism/s of TNTs and its correlation with enhanced proliferation. Small molecules actin modulators (Cytochalasin D, and IPA-3) inhibit biogenesis of TNTs and prevent α-SYN protofibril-induced cell proliferation. On the other hand, the actin modulators CK-666 and Blebbistatin, which facilitate biogenesis of TNTs, did not show a significant effect on the enhancement of cell proliferation. Even though these actin inhibitors (CK-666 and Blebbistatin) show better cell survival compared to the molecules that inhibit TNTs. Only ROCK inhibitor Y-27632 mediated biogenesis of TNTs significantly promotes proliferation, similar to proliferation induced by α-SYN protofibrils. ROCK inhibitor Y-27632 inhibits LIMK-dependent cofilin-G-actin interaction and inhibits actin polymerization whereas, IPA-3 and Cytochalasin-D could also inhibit actin polymerization through the LIMK pathway (Scheiblich et al., 2021 ). However, we observed ROCK inhibition promotes biogenesis of TNTs, whereas, IPA-3 and Cytochalasin-D inhibit TNTs. Our result suggests, that inhibition of ROCK kinase pathways may modulate TNT biogenesis by regulating actin cytoskeleton through myosin-II-specific downstream signalling molecules, which dominates over cofilin-mediated actin modulation. Similarly, a recent study (Scheiblich et al., 2021 ) has shown that α-SYN protofibrils promote biogenesis of TNTs in microglia cells via regulating ROCK inhibitory pathway by phosphorylation of myosin light chain phosphatase (MLCP). Thus, we tried to unfold how α-SYN protofibrils induced actin-remodulation may modulate ROCK inhibitory signalling cascades to induce TNT biogenesis. We have found that α-SYN-induced transient senescence-related toxicities caused nuclear localization of FAK and pFAK for the transitory period, which corresponds to TNT biogenesis at early time points (3h and 6h). Organization of FAK at focal adhesion sites of PM regulates activation of Rho-mediated ROCK signalling, and its de-localization from focal adhesion inhibits the ROCK pathway (Schober, Raghavan et al., 2007 ). On the other hand, in low adhesion conditions displacement of inactive/active FAK/pFAK from focal adhesion sites leads to Rho kinase-mediated activation of ROCK signalling, probably to restore the cytoskeletal tension (Pirone et al., 2006 ). Then, restoration of cytoskeleton tension may cause re-translocation of FAK/pFAK to PM, which could reinstate ROCK activation. In this line, we have detected α-SYN toxicities induced FAK translocation dynamics, which could modulate transient inhibition of ROCK2 signalling to induce biogenesis of TNTs at early times and later may activate to restore cytoskeleton tension. We have also seen the proliferation of astroglia cells via activation of ROCK signalling mediated ERK1/2 and NFκB proteins, in the post-recovered astroglia cells. Thus, our results suggest that biogenesis of TNTs is not the root cause of cell proliferation, rather modulation of α-SYN protofibrils induced ROCK-mediated actin-regulatory signalling pathway related to biogenesis of TNTs triggers cell proliferation. In conclusion, our study reveals α-SYN protofibrils induced biogenesis of TNTs aids in enhancing the clearance of toxic burden as a cellular survival strategy to rescue the astroglia cells from ROS-induced cell death/cellular senescence. α-SYN protofibrils regulate FAK-mediated modulation of ROCK signalling cascades to rescue the cells from toxic burden by promoting TNT biogenesis. The rescued cells, eventually re-activate ROCK2 signalling probably to restore cytoskeleton tension and enhance cell proliferation via ERK1/2 and NFκB signalling. α-SYN inclusions in the astroglia cells and glial inclusion or fibrous gliosis in the areas of neurodegeneration are widespread in PD (MacMahon Copas et al., 2021 ). Thus, the study is important for understanding the relevance of TNT-mediated crosstalk in the clearance of α-SYN protofibrils by astrocytes, and its implication in astroglia cell proliferation. Thus, the study will open up new strategies to design therapeutics targeting astrocytes in the PD brain. Declarations Author contributions S.N conceived and conducted the research; A.R, R.K, S.P (JNCASR), R.M (IISc), R.M (JNCASR), S.P (NIMHANS) and S.N designed and interpreted data; A.R, R.K, S.P (JNCASR), S.J, S.C, A.A, M.N.D and M.G performed experiments; A.R, R.K, S.P (JNCASR), S.C, and A.A analysed the data; A.R, R.M (JNCASR), and S.N wrote the paper taking valuable inputs from all the authors. Acknowledgment We thank Mrs. Suma (JNCASR) for the confocal microscopy and Mrs. Usha (JNCASR) for TEM imaging. We thank Dr. Anujith Kumar (MIRM-MAHE) for sharing his resources and Ms. Smitha Bhaskar (MIRM-MAHE) for helping with transfection using the electroporation technique. We thank Prof. Dipankar Nandi, Indian Institute of Science, for the DCFDA reagent. We thank Dr Lakshmi Balasubramanian (C-CAMP, Bangalore) and Mr. Vedam Pruthvi (MIRM-MAHE) for giving suggestions to develop an image analysis flow diagram. Funding A.R, and S.C thank Manipal Academy of higher education for Dr. TMA pai fellowship; A.R thanks ICMR SRF- Direct fellowship, R.K. thanks the Indian Council of Medical Research of India (#5/4-5/Ad-hoc/Neuro/216/2020-NCD-I) for her JRF fellowship; S.N thanks the Science and Engineering Research Board of India for the SERB-SRG (#SRG/2021/001315) grant; the Indian Council of Medical Research of India (#5/4-5/Ad-hoc/Neuro/216/2020-NCD-I) and the Intramural fund of Manipal Academy of Higher Education, Manipal, India (#MAHE/CDS/PHD/MIFR/2019) for financial support; The financial support from the DBT-RA program in Biotechnology and Life Sciences and DST-SERB NPDF to SP (JNCASR) is gratefully acknowledged; The financial support from Department of Biotechnology (DBT) grant in Life Science Research, Education and Training at JNCASR (BT/INF/22/SP27679/2018), S. Ramachandran-National Bioscience Award for Career Development (NBACD)-2020-21 (SAN No. 102/IFD/SAN/990/2021-22) and JNCASR intramural funds to RM is acknowledged. S.P (NIMHANS) thanks the Science and Engineering Research Board, Government of India for the funding support (ECR/2018/002219). References Abbassi RH, Recasens A, Indurthi DC, Johns TG, Stringer BW, Day BW, Munoz L (2019) Lower Tubulin Expression in Glioblastoma Stem Cells Attenuates Efficacy of Microtubule-Targeting Agents. ACS Pharmacol Transl Sci 2: 402-413 Abounit S, Bousset L, Loria F, Zhu S, de Chaumont F, Pieri L, Olivo-Marin JC, Melki R, Zurzolo C (2016) Tunneling nanotubes spread fibrillar alpha-synuclein by intercellular trafficking of lysosomes. EMBO J 35: 2120-2138 Abounit S, Delage E, Zurzolo C (2015) Identification and Characterization of Tunneling Nanotubes for Intercellular Trafficking. 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Biomedicine & Pharmacotherapy Additional Declarations There is no duality of interest Supplementary Files Movie1.mp4 Movie 1 Movie2.mp4 Movie 2 Movie3.mp4 Movie 3 Movie4.mp4 Movie 4 Movie5.mp4 Movie 5 Movie6.mp4 Movie 6 RaghavanetalCellDeathDiffsupplementaryMaterial.docx SUPPLEMENTAL MATERIAL Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3747717","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":262410553,"identity":"323b9129-3fca-4832-b563-e2415097029f","order_by":0,"name":"Sangeeta 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1","display":"","copyAsset":false,"role":"figure","size":3785915,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCharacterization of α-SYN protofibrils and their effect on TNTs formation in astroglia cells. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eA) TEM (transmission electron microscope) images of α-SYN protofibrils unlabelled and TMR fluorescent dye labelled respectively. Magnified structures of protofibrils are shown in the zoomed panel. B) Primary astrocytes treated with 1µM α-SYN protofibrils for 3h, 6h, 12h, and 24h, stained for actin (red) and GFAP (green), white arrows indicate formation of shorter and longer TNTs at 3h and 6h. C) DiD (membrane dye) stained U-87 MG cells showing a transient increase of TNT-like structures (red arrows) in the cells treated with α-SYN protofibrils (1 μM) for 3 -6 h, compared to the respective controls and the cells treated for 12 h and 24 h. Blue arrows indicate relatively thicker tumour microtubes (TMs) like connections. D) The percentage of TNTs from primary astrocytes was quantified by counting the numbers normalized with the number of cells. E, F) Quantification of the percentage of TNT-like structures and pairs of cells connected by them upon α-SYN protofibrils treatment in U-87 MG cells live stained with DiD. Quantifications are done from 10-15 image frames of a set and each image frame has 10-20 cells. Scale bars are denoted on the images. Data are expressed as mean ± SD, *** p ≤ 0.001. Statistics were analysed using two-way ANOVA. n=3.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3747717/v1/764bc5df3d095186a9e1806f.jpg"},{"id":49974664,"identity":"0497ad90-6a1e-4216-816d-859fca6181e3","added_by":"auto","created_at":"2024-01-22 14:32:25","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2407245,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCharacterization of TNTs formed upon treatment with α-SYN protofibrils in astroglia cells\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eA) The red arrow in the DIC image (a’) indicates TNT formed between two astrocytes on α-SYN protofibrils treatment. The bottom panel shows a 3D volume view of Phalloidin and GFAP-positive astrocytes connected by both long (a’’) and short TNTs (b). B, C) DIC images of U-87 MG cells show TNTs (B) and TMs (C). The cells were stained with phalloidin and β-tubulin, both bottom panels show 3D volume views of TNT as an actin-positive hovering structure (B) and tubulin-positive TM at the surface (C). Red arrows indicate actin-positive TNTs and blue arrows indicate β-tubulin-positive tumour microtubes (TM) respectively. D, E) Characterization of open (D) and close (E) ended TNTs based on Phalloidin and connexin43 staining at the tip of the TNT in U-87 MG cells. Pink arrows indicate close-ended TNT. The bottom panel shows 3D volume views of the same. F) Closed-ended TM characterized by the presence of connexin43 staining, indicated by pink arrows. The bottom panel shows 3D volume views of the TM at the surface. G) TNT-like structures (red arrows) are detectable in DIC images as focused structures at z=4. At z=0 filopodia-like extensions (magenta arrows) on the substratum are at focus. H, I) U87MG cells treated with 1µM α-SYN protofibrils for 3h and 6h, stained with lysotracker and mitotracker respectively. Red arrows indicate lysotracker and mitotracker through TNTs at 3h and 6h. Movement of lysotracker (H) and mitotracker (I) positive vesicles through TNTs were tracked using the trackmate plug-in of Fiji. \u0026nbsp;J) Quantification of the percentage of TNTs and K) percentage of open-ended TNTs, close-ended TNTs, and TMs from the confocal Z-stack images of U-87 MG cells. Quantification of length (L) and diameter (M) of TNTs formed by U-87 MG cells, U251 cells, and astrocytes. Quantifications are done from 10-15 image frames of a set and each image frame has 10-20 cells. Scale bars are denoted on the images. Data are expressed as mean ± SD, *** p ≤ 0.001. Statistics were analysed using two-way ANOVA (2J and 2K) and one-way ANOVA (2L and 2M). n=3.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3747717/v1/76a94dd33f341a594dfccc7a.jpg"},{"id":49974313,"identity":"60d00826-8090-4e6b-a867-e0278b6d5cdb","added_by":"auto","created_at":"2024-01-22 14:24:25","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2106692,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEffect of α-SYN protofibrils on lysosomes in U-87 MG cells and astrocytes\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eA) Mouse primary astrocytes treated with 1µM α-SYN protofibrils for 3h, 6h, 12h, and 24h. α-SYN protofibrils (green) in lamp1 positive vesicles (red). At 3h and 6h, larger sizes of lamp1 positive vesicles colocalized with α-SYN (yellow) are visible. B) U-87 MG cells treated with α-SYN-TMR protofibrils (red) in lysotracker-positive vesicles (green). C) U-87 MG cells treated with α-SYN protofibrils (1µM) were stained using Cathepsin D (red) and Lamp2 (green). At 3h larger sizes of lamp2 positive vesicles colocalized with cathepsin-D were observed, compared to the other time points (magnified images highlighted at upper-left corner). D) Quantification of co-localization of α-SYN protofibrils in lamp1 positive lysosomes and (E) Size of α-SYN accumulated in lysosomes in astrocytes. F) Quantification of α-SYN accumulation per cell and G) Lysosome size in U-87 MG cells. Quantifications are done by analysing randomly selected 10-15 cells from different image frames of an experimental set. Scale bars are denoted on the images. Data are expressed as mean ± SD, *** p ≤ 0.001. Statistics were analysed using two-way ANOVA. n=3.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3747717/v1/878e596e098030c56b7f411d.jpg"},{"id":49973771,"identity":"45b284f5-10ad-41d7-a377-dfbe741ae918","added_by":"auto","created_at":"2024-01-22 14:16:25","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1922255,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEffect of α-SYN protofibrils on mitochondria in astrocytes and U-87 MG cells\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePrimary astrocytes and U-87 MG were treated with 1µM α-SYN protofibrils for 3h, 6h, 12h, and 24h, and mitochondrial membrane potentials were detected using JC-1 staining (emission ratio of 527/590 nm). A) Primary astrocytes stained with JC-1. B) Quantification of green vs red intensities, C) Size of green/red mitochondria, and D) mitochondria branch length by MiNA analysis were quantified from JC-1 astrocyte images. E) U87MG cells treated with 1µM α-SYN protofibrils for 3-24 h, were stained with mitotracker (top panel -cyan) and JC-1 (bottom panel) to visualize mitochondrial morphology. F) Quantification of green vs red intensities from JC-1 U-87 MG images. Quantification of green vs red intensities. Quantifications are done from 10-15 image frames of a set and analysing 10-20 cells from each image. Scale bars are denoted on the images. Data are expressed as mean ± SD, *** p ≤ 0.001. Statistics were analysed using two-way ANOVA. n=3.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3747717/v1/cc9486cfbcbd057104935c60.jpg"},{"id":49973774,"identity":"5aa4ffac-10b4-49fc-b9b2-60560e26f6f8","added_by":"auto","created_at":"2024-01-22 14:16:25","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1963839,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCell-cell transfer of mitochondria and α-SYN-induced ROS-mediated cellular senescence.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eA) Transfer of mitochondria was detected in a co-culture experiment. U87MG cells were co-cultured using transiently transfected with Mito-ds-Red (red) and \u003c/em\u003eEGFP-lifeact\u003cem\u003e (green) respectively and treated with 1µM α-SYN protofibrils for 3h-24h. Confocal microscopy images show the transfer of Mito-ds-Red labelled mitochondria to \u003c/em\u003eEGFP-lifeact\u003cem\u003e labelled green cell population. B) Quantification of the percentage of acceptor cells with transferred mitochondria. C) quantification of the size of the transferred mitochondria vs the control mitochondria. Quantifications are done from 5 image frames of a set and each image frame has 10-15 cells. D, F) Estimation of total cellular ROS by DCFDA assay in the cells treated with 1µM α-SYN protofibrils for 3h-24h by flow cytometry in U-87 MG and astrocytes respectively. E, G) Quantification of the percentage of gating population of the DCFDA positive cells were obtained from the same. H) Western blot analysis to estimate the levels of senescence marker p21 in the cells treated with 1µM α-SYN protofibrils. Full-length western blots of four repeats were represented in Supplementary Figure 8. I) In astrocyte images white arrows indicate DAPI-stained fragmented nuclei while the red arrows indicate the formation of TNTs from those cells. J) Quantification of the percentage of fragmented nucleus on α-SYN protofibrils treatment. Quantifications are done from around 10 image frames of a set and each image frame has 10-20 cells. Scale bars are denoted on the images. Data are expressed as mean ± SD, *** p ≤ 0.001. Statistics were analysed using two-way ANOVA except graph C which was analysed by student t-test. n=3.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3747717/v1/e3c65526aa52b5222619a96d.jpg"},{"id":49973776,"identity":"46e9c9d8-7eae-494d-8b83-2a0a68f82248","added_by":"auto","created_at":"2024-01-22 14:16:25","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2014033,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eTNT biogenesis pathways in cell proliferation\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eA) Absorbance measured at 570 nm after MTT assay performed in astrocytes treated with varying concentrations (0.5µM, 1µM, 2µM, and 3µM) of α-SYN protofibrils at 3h, 6h, 12h and 24h. B) Cell number quantification of U-87 MG cells treated with 1µM α-SYN protofibrils after 3h, 6h, 12h, 18h and 24h. C) MTT absorbance estimated at 24h on treatment with varying concentrations (0.5µM, 1µM, 2µM, and 3µM) of α-SYN protofibrils in U-87 MG cells. D) Estimation of MTT absorbance at 12h and 24h after treatment with 1µM α-SYN protofibrils in U251 cells. E) Fluorescence images of astrocytes and U87MG cells treated with 1µM α-SYN protofibrils for 3h-24h were stained with proliferation marker Ki67 along with nuclear stain. F, G) Quantification of the intensity of Ki67 per cell in astrocytes and U-87 MG respectively. Quantifications are done from 15 image frames of a set and each image frame has 20-25 cells. H) Flow chart depicting the mode of action of actin inhibitors. I) DiD (membrane dye) stained, control and 1µM α-SYN protofibrils treated U-87 MG cells pre-treated (before 30min) with 3 µM IPA3, 0.5 µM cytochalasin D, 50 µM CK-666 (Arp2/3 inhibitor), 75 µM Blebbistatin and 5 µM Y-27632 (ROCK inhibitor). Red arrows indicate the formation of TNT-like structures and K) shows quantification of TNT numbers. J) representative images showing cell numbers with the above-mentioned treatment and L, M) quantification of MTT absorbance and cell numbers of the same respectively. Quantifications are done from 5 image frames of a set and each image frame has 15-20 cells. Scale bars are denoted on the images. Data are expressed as mean ± SD, *** p ≤ 0.001. Statistics were analysed using two-way ANOVA, and only graph 6B was analysed using one-way ANOVA. n=3.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3747717/v1/cb055529aad700edefd0217b.jpg"},{"id":49973775,"identity":"fa789372-d069-4dea-9aa1-4e1b9c744d3a","added_by":"auto","created_at":"2024-01-22 14:16:25","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2872427,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eFAK translocation, ROCK remodulation, and activation of proliferation pathway\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eA) Estimation of uptake of α-SYN-TMR protofibrils by U251 cells treated with 1µM α-SYN protofibrils and pre-treated (before 30min) with 0.5 µM cytochalasin D and 5 µM Y-27632 (ROCK inhibitor) by flow cytometry. B) Quantification of percentage gated fluorescence intensity of α-SYN-TMR protofibrils in the above experiment. C) Fluorescence images of maximum intensity projected confocal z-stacks in astrocytes stained with Phalloidin (green), and FAK (red). The upper panel with nucleus stained with DAPI and the lower panel without DAPI. White arrows indicate nuclear colocalization of FAK, red arrows indicate TNTs and pink arrows indicate FAK at focal adhesion. D) Quantification of FAK intensity in the nucleus of the cells with and without TNTs. E, G) Fluorescence images of maximum intensity projected confocal z-stacks in U-87 MG cells stained with FAK (red) and pFAK (red) with DAPI respectively. F, H) Quantification of FAK and pFAK fluorescence intensity per cell in the nucleus. Quantifications are done from 15 image frames of a set and each image frame has 15-20 cells. I) Western blot images showing the change in ROCK1 and ROCK2 expressions with time on 1µM α-SYN protofibrils treatment in U-87 MG cells. J) Quantification of ROCK1 and ROCK2 western blots. Full-length western blots of three repeats were represented in Supplementary Figure 9. K) Western blot images showing increased intensity of pERK1/pERK2, NF-κB, and cdk1 with time on 1µM α-SYN protofibrils treatment in U-87 MG cells. Full-length western blots of four repeats were represented in Supplementary Figure 10. L) Quantification of pERK1/pERK2, NF-κB and cdk1 western blots. Scale bars are denoted on the images. Data are expressed as mean ± SD, *** p ≤ 0.001. Statistics were analysed using two-way ANOVA. n=3.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3747717/v1/87a2dc7563294e201f9187c4.jpg"},{"id":49974315,"identity":"266992db-3646-4f74-82b5-77a15adab5a1","added_by":"auto","created_at":"2024-01-22 14:24:25","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1575168,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSummary Figure\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eα-SYN protofibrils-induced biogenesis of tunneling nanotubes (TNTs) aids in enhancing the cellular clearance of toxic burden as a cellular survival strategy. α-SYN protofibrils treated toxic senescence cells regulate FAK-mediated modulation of ROCK signalling cascades to promote TNT biogenesis and rescue the cellular toxicities. The rescued cells eventually enhance cell proliferation.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3747717/v1/63de8386662b81a99d52a3d0.jpg"},{"id":49975140,"identity":"b9643d23-aae8-42ce-a0fa-af4a29de46dc","added_by":"auto","created_at":"2024-01-22 14:40:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2398843,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3747717/v1/1c61b7a3-7aae-474b-8b65-a0cee3c789e2.pdf"},{"id":49973783,"identity":"e2f22771-945e-4f48-a190-e0b8dc90cd1e","added_by":"auto","created_at":"2024-01-22 14:16:25","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4032148,"visible":true,"origin":"","legend":"Movie 1","description":"","filename":"Movie1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3747717/v1/13634859b02a27bebb181d87.mp4"},{"id":49973780,"identity":"675f3149-55b2-4878-90b8-462b4efbd7e5","added_by":"auto","created_at":"2024-01-22 14:16:25","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3381563,"visible":true,"origin":"","legend":"Movie 2","description":"","filename":"Movie2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3747717/v1/480c934c27005aaab70a2c7b.mp4"},{"id":49974317,"identity":"ff06672c-632d-4166-bb85-5f43e2db7977","added_by":"auto","created_at":"2024-01-22 14:24:25","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4227311,"visible":true,"origin":"","legend":"Movie 3","description":"","filename":"Movie3.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3747717/v1/2b0bce252310b2e71d09d96f.mp4"},{"id":49974316,"identity":"647a5ef6-1ec5-4f02-aee6-b457c2519860","added_by":"auto","created_at":"2024-01-22 14:24:25","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1820219,"visible":true,"origin":"","legend":"Movie 4","description":"","filename":"Movie4.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3747717/v1/fbc3bfd838aa7b223da163b9.mp4"},{"id":49973784,"identity":"8f3bfc98-480f-49ff-9e67-dd675c8983d1","added_by":"auto","created_at":"2024-01-22 14:16:25","extension":"mp4","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":3232871,"visible":true,"origin":"","legend":"Movie 5","description":"","filename":"Movie5.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3747717/v1/23059cbbb61e4fb3c62f0107.mp4"},{"id":49973778,"identity":"44f432b4-11f2-42ac-9df0-eb44b9ef2b5d","added_by":"auto","created_at":"2024-01-22 14:16:25","extension":"mp4","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":3645630,"visible":true,"origin":"","legend":"Movie 6","description":"","filename":"Movie6.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3747717/v1/6fdb3630523833c2bab3515c.mp4"},{"id":49973785,"identity":"b326d4fc-bd9c-49c2-ba88-7cacb6765852","added_by":"auto","created_at":"2024-01-22 14:16:25","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":7267547,"visible":true,"origin":"","legend":"SUPPLEMENTAL MATERIAL","description":"","filename":"RaghavanetalCellDeathDiffsupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-3747717/v1/52d306b6c7f15b269fe13609.docx"}],"financialInterests":"There is no duality of interest","formattedTitle":"Astroglia proliferate upon biogenesis of tunneling nanotubes via α-synuclein dependent transient nuclear translocation of focal adhesion kinase","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003e\u0026alpha;-SYN protofibrils treated astroglia cells proliferate upon transient biogenesis of TNTs.\u003c/li\u003e\n \u003cli\u003eTransient TNT biogenesis precedes clearance of \u0026alpha;-SYN toxicities and reversal of senescence.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eStress-induced senescence results in nuclear localization of FAK and ROCK-mediated TNT biogenesis.\u003c/li\u003e\n \u003cli\u003eThe rescued cells enhance proliferation through ROCK-mediated ERK1/2 and NF\u0026kappa;B signalling cascades.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eParkinson\u0026rsquo;s disease (PD) is characterized by the loss of dopaminergic neurons in the \u003cem\u003esubstantia nigra\u003c/em\u003e (SN) due to cytoplasmic accumulation of Lewy bodies or Lewy neurites which mainly consists of α-synuclein (α-SYN) protein (Forno, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, Kalia \u0026amp; Lang, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Along with the accumulated cytoplasmic α-SYN, extracellular α-SYN also plays a significant role in neurodegeneration, progressive intercellular spreading of pathology, and neuroinflammation (Lee, Bae et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Yamada \u0026amp; Iwatsubo, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Studies have shown that elevation of neuronal activity and various stress conditions increase extracellular release of α-SYN (Fortin, Nemani et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Yamada \u0026amp; Iwatsubo, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Pathogenic aggregates of α-SYN can be released from degenerating neurons, which can be taken up by surrounding neurons and glial cells. Glial cells normally express low levels of α-SYN, however, at the advanced stage of PD, α-SYN aggregates in astrocytes and glial synucleinopathies are often detected (Krejciova, Carlson et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Additionally, gliosis is a typical pathological feature of neurodegenerative diseases. Sustained activation and fibrous proliferation of glial cells, mainly astrocytes and microglia, are central features of dopaminergic neurodegeneration in PD (MacMahon Copas, McComish et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Neuronal activity modulates cellular crosstalk and intercellular communication between neurons and the surrounding neuroglial cells (Gibson, Purger et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Venkatesh, Johung et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Venkatesh, Morishita et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecent studies have shown that mode of intercellular transfer of neurodegenerative proteins between glial cells facilitate the clearing of neurodegenerative aggregates, such as α-SYN and amyloid-β (Aβ) (Rostami, Holmqvist et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Rostami, Mothes et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Scheiblich, Dansokho et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The spreading of neurodegenerative proteins through an intercellular mode of transfer and its role in pathology progression have widely been studied in several model systems (Neupane, De Cecco et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Victoria \u0026amp; Zurzolo, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Exosomes, unconventional secretion, and direct cell-to-cell transfer \u003cem\u003evia\u003c/em\u003e membrane nanotubes or tunneling nanotubes (TNTs) have been demonstrated by several studies as modes of intercellular transfer (Neupane et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe discoveries of TNTs have opened up the possibility of direct long-range cell-to-cell communication (Rustom, Saffrich et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). TNTs have been shown as open-ended, and thin (diameter around 50\u0026ndash;700 nm) intercellular membrane-actin continuity (long up to 300 \u0026micro;m) between distant cells. TNTs are reported to be a conduit for the direct transfer of organelles (Rustom et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), neurodegeneration-associated protein aggregates (Ramirez-Jarquin, Sharma et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Victoria \u0026amp; Zurzolo, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), viruses (Jansens, Tishchenko et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and RNA. (Haimovich, Ecker et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) between cells. Neurodegenerative aggregate-induced endo-lysosomal toxicities and mitochondrial stress promote biogenesis of TNTs (Raghavan, Rao et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Victoria \u0026amp; Zurzolo, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Propagation of pathogenic aggregates \u003cem\u003evia\u003c/em\u003e TNTs has also been shown to aid in the progression of neurodegeneration (Dilna, Deepak et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Rostami et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Victoria \u0026amp; Zurzolo, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In this context, several studies have shown that α-SYN aggregates in lysosomes transfer from neuron to neuron \u003cem\u003evia\u003c/em\u003e TNTs in PD (Abounit, Bousset et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Dilsizoglu Senol, Samarani et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Victoria \u0026amp; Zurzolo, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). On the other hand, TNTs mediate crosstalk between astrocytes, and microglia to degrade toxic α-SYN through cell-to-cell transfer of aggregates and help to reduce ROS accumulation and oxidative stress (Rostami et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Scheiblich et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Oxidative stress and ROS promote formation of TNTs and cell-to-cell transfer (Desir, Dickson et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Wang, Cui et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eROS induced by the accumulation of neurotoxic proteins in neurons can induce cell senescence and that can aggravate neurodegeneration (Yoon, You et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Zizhen Si, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Cellular senescence is a biologically homeostatic phenomenon that is defined by a persistent degree of cell cycle inhibition and cellular aging. Cell cycle arrest in senescence cells is believed to be irreversible, however, studies have shown that stress-induced premature senescence that is induced by DNA damage-mediated amplification of the p53/p21 signalling pathway can be reversed by modulating the levels of p53 and p21 expressions (Beausejour, Krtolica et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, Macip, Igarashi et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSenescent cells are more prevalent in brain tissues and exert roles in brain aging and neurodegeneration (Martinez-Cue \u0026amp; Rueda, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Brain cells including astrocytes, microglia, oligodendrocytes, and epithelial cells undergo cellular senescence under oxidative stress, which aggravates neurodegeneration (Schousboe, Bak et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). On the contrary, glial cells, especially astrocytes, and microglia, play a critical role in protecting neurons by clearing toxic accumulations of neurodegenerative proteins from neurons and brains (Rostami et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Scheiblich et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). α-SYN induced ROS generation can produce cellular senescence in response to DNA damage (Yoon et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Thus, the investigation into the mechanisms of TNT biogenesis and the role of TNTs in facilitating clearance of toxic α-SYN protofibrils from the brain to improve cell survival is needed.\u003c/p\u003e \u003cp\u003eIn this study, we have shown that transient localization of FAK/pFAK to the nucleus upon treatment with α-SYN protofibrils caused transient biogenesis of TNTs in the astroglia cells. Transient biogenesis of TNTs precedes clearance of α-SYN-induced organelle toxicities and reversal of ROS-induced premature senescence leading to enhanced cell proliferation in the post-recovered astroglia cells. This study emphasizes the potential role of TNTs in facilitating cellular clearance in pathological stress conditions which maintains the survival of astroglia cells, by enhancing cell proliferation.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture maintenance\u003c/h2\u003e \u003cp\u003eU-87 MG and U251 cell lines (astrocytoma-glioblastoma origin cancer cell lines) were kind gifts from Prof. Kumaravel Somasundaram of the Indian Institute of Science, Bangalore, India. Cells were cultured and maintained in DMEM (Gibco #2120395) media supplemented with 10% FBS (fetal bovine serum; Gibco #1600004, US Origin), along with 1% PSN (Penicillin-Streptomycin-Neomycin Mixture; Thermo Fisher Scientific #15640055) incubated at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eNeuro 2a (N2a) neuroblastoma cell line was procured from NCCS, India. The cells were cultured and maintained in DMEM media with 10% FBS and 1X Glutamax (Gibco #35050). N2a cells were differentiated with 10 \u0026micro;M Retinoic acid (RA; Sigma-Aldrich #R2625) in the presence of 2% FBS-supplemented media for 2\u0026ndash;3 days. Differentiation was established from the neurites like morphology.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePrimary astrocytes culture\u003c/h2\u003e \u003cp\u003eFive-week-old mice (C57BL/6) were used for primary astrocyte culture. The mice were sacrificed using CO\u003csub\u003e2\u003c/sub\u003e, and the cerebral cortices were dissected from the mice's brains and observed using an Olympus SZ51 stereomicroscope. Pieces of cortical tissue were trypsinized using 0.25% trypsin-EDTA solution (Gibco Canada origin #25200-072) for 5 min. The tissue was washed twice in warm HBSS and transferred to Minimum Essential Media (Gibco 61100-087) with 10% FBS (Gibco #1600004, US Origin) containing HEPES (Sigma Aldrich H0887) and D-glucose (Sigma Aldrich G7021). The tissue was triturated using a fire-polished pipette and counted using Trypan Blue in an automated cell counter. Dissociated cells were plated in MEM with 10% FBS on Corning\u0026reg; 100mm TC-Treated Culture Dish at a density of 10\u0026nbsp;million cells per dish. The media was replaced with fresh MEM with 10% FBS on the next day and maintained the same for 1 week. After 1 week, cells were trypsinized and re-plated for experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePurification and Labelling of α-SYN protein\u003c/h2\u003e \u003cp\u003eThe human α-SYN wild-type (Addgene ID #36046) and α-SYN (wt)-141C (Addgene ID #108866) with a cysteine residue at C-terminus, constructs were purchased and overexpressed in \u003cem\u003eE. coli\u003c/em\u003e. Then purification was done from periplasmic fraction as described in (Huang, Ren et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Jos, Gogoi et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Samples were aliquoted and flash frozen in liquid nitrogen and stored immediately at -80\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe purified α-SYN (wt)-141C protein was thawed and pre-incubated with reducing agent TCEP (tris(2-carboxyethyl) phosphine-hydrochloride); Sigma Aldrich-C4706; 20mM) to reduce/dissociate any pre-existing cysteine bonds. Then the protein was labelled adding two-fold molar excess of TetramethylRhodamine-5-maleimide (TMR-maleimide; Sigma Aldrich #94506) in 20 mM HEPES buffer (pH 7.4). The protein solution was vortexed and the mixture was incubated for 1h at 20\u0026deg;C. The excess label was removed by using PD-10 columns (Himedia #TKC287) (Nath, Meuvis et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003ePreparation and characterisation of α-SYN protofibrils\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eProtofibrils of α-SYN (labelled and unlabelled) was prepared with 1 \u0026micro;M of unlabelled α-SYN wild-type and labelled α-SYN-141C by incubating with 0.65% 4-Hydroxynonenal (10mg/ml stock) (Sigma-Aldrich #393204-1MG) at 37\u0026deg;C for 7 days with moderate shaking (Sackmann, Sinha et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe α-SYN protofibrils formed at the end of 7 days were then characterized by transmission electron microscopy (TEM). The unlabelled and labelled α-SYN protofibrils were resuspended in 1X PBS, coated on a carbon grid with uranyl acetate and dried in a desiccator. The grids were visualized using FEI Tecnai T20 at 200 KV. The α-SYN protofibrils were then lyophilized and stored at -20\u0026deg;C and resuspended in 1X TBS (Tris-buffered saline) buffer before experiments.\u003c/p\u003e \u003cp\u003eThe toxicity of the protofibrils was assessed by treating the differentiated neuronal cells seeded at a density of 30,000 cells / well in 24 well plates. Cell viability assay was performed using different concentrations of α-SYN protofibrils (0.5\u0026micro;M, 1\u0026micro;M, 2\u0026micro;M, 3\u0026micro;M) over different periods by MTT assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTreatment conditions of astroglia cells by α-SYN protofibrils\u003c/h2\u003e \u003cp\u003eAll the experiments were carried out by treatment of primary astrocytes, U-87 MG, and U251 cells with 1\u0026micro;M α-SYN protofibrils- unlabelled or TMR-labelled for 3h, 6h, 12h, and 24h at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e unless mentioned otherwise. The cells were seeded at the same time for all the treated time points and controls. Once cells were adhered properly and appeared healthy after 24h of seeding, then treatments were done by adding (α-SYN 1\u0026micro;M) protofibrils in a reverse order of the time points. For example, 24h time points were treated first and then sequentially 12 h, 6 h, and 3 h points. Then, experiments were performed for all the time points and controls at a time. Therefore, the condition of the untreated control cells corresponds to all the treatment points.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMTT assay and cell numbers counting\u003c/h2\u003e \u003cp\u003eMTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay is a colorimetric assay used to measure cellular metabolic activity. Astrocytes, U-87 MG, and U251 cells were seeded at a density of 4000 cells per well in a 96-well plate and incubated overnight at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e. Different concentrations of α-SYN protofibrils (0.5\u0026micro;M, 1\u0026micro;M, 2\u0026micro;M, 3\u0026micro;M) were added to the wells over different time periods (3h, 6h, 12h, 24h, and 48h) and treated with MTT reagent (Himedia # TC191-1G) for 2h. The insoluble formazan crystals were dissolved by adding dimethyl sulfoxide (DMSO; Himedia #TC185) which was later quantified by measuring absorbance at 570 nm using the PerkinElmer-Multimode plate reader spectrophotometer. Cell numbers were counted from the phase contrast microscopy images or DIC images using the multi-point option in Fiji (a Java-based program developed at the National Institutes of Health, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eImmunocytochemistry (ICC)\u003c/h2\u003e \u003cp\u003eAfter treatment with the above-mentioned concentrations and time points adding α-SYN protofibrils, the cells were washed with 1X PBS and fixed with 4% PFA. The cells were then incubated with incubation buffer (1mg/ml Saponin and 5%FBS) for 20 min at RT. Respective primary antibodies (as mentioned below) were added and incubated overnight at 4\u0026deg;C, the cells were then washed 3 times with 1X PBS. All the secondary antibody (1:700 dilution) incubations were carried out in the dark for 2 hrs at RT, and 1X PBS washes were repeated. The coverslips were mounted on glass slides with ProLong Gold antifade reagent with DAPI, (Invitrogen P36941). Alternately, glass bottom 35mm dishes (Cellvis, D35-14-1.5-N) were used in a few experiments.\u003c/p\u003e \u003cp\u003ePrimary antibodies Ki67 anti-rabbit (Millipore B9260) as cell proliferation marker; FAK Rabbit (CST 3285T) and phospho-FAK ((Tyr397) pAb; Invitrogen 44-624G) as total and active FAK marker; Cathepsin D anti-rabbit (GeneTex #42368), LAMP-1 anti-mouse (BD Biosciences # 611042), LAMP-2 anti-mouse (Invitrogen #MA1-205) as lysosomal markers; βIII-tubulin anti-rabbit (Cloud-Clone #PAE711Hu01), Connexin43 Rabbit Ab (CST #3512S), SNCa rabbit pAb (Cloud-Clone #PAB222Hu01), GFAP rabbit pAb (Cloud-Clone #PAA068Mu01) and Phalloidin conjugated with iFlour555 (Abcam #176756) were used to stain TNTs and TMs. Secondary antibodies Alexa- 488 goat anti-rabbit (#A-11070; Invitrogen), Alexa- 555 anti-mouse (#A-1413312; Invitrogen), and Alexa- 488 anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) (#A-11059; Invitrogen) were used respectively following the above-mentioned protocol. All primary antibodies were used at a dilution of 1:300, secondary antibodies at 1:500, and phalloidin at 1:700. ICC-stained cells were imaged with a confocal microscope (Zeiss LSM880, Carl Zeiss, Germany) or fluorescence microscope (IX73-Olympus).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot\u003c/h2\u003e \u003cp\u003eU-87 MG cells were seeded at a density of 1\u0026nbsp;million per well in a 6-well plate and were treated with 1\u0026micro;M α-SYN protofibrils from 3h-24h. Post-treatment media was removed and the cells were washed with 1X PBS. RIPA buffer was added and the cells were scrapped and collected in 1.5 ml tubes. The tubes were incubated on ice with intermittent vortexing. Later the suspension was spun at 12,000 rpm for 10 min and the supernatant was collected and stored at -20\u003csup\u003e0\u003c/sup\u003eC. After normalizing the protein concentration, western blot assay was performed with primary antibodies GAPDH Mouse (Cloud-Clone # CAB932Hu22 dilution 1:1000), ROCK1 (CST # 4035T dilution 1:1000), ROCK2 (CST # 8236S dilution 1:1000), NF-kB p65/RelA Mouse mAb (Abclonal # A10609 dilution 1:1000), phospho ERK1-T202\u0026thinsp;+\u0026thinsp;ERK2- T18 (Abclonal # AP0485 dilution 1:1000), CDK1 Rabbit pAb (Abclonal #A0220 dilution 1:1000), p21 Waf1/Cip1 (12D1) Rabbit mAb (CST #2947 dilution 1:1000) and secondary antibodies, Goat anti-mouse (H\u0026thinsp;+\u0026thinsp;L) (Invitrogen #32430 dilution 1:1500) and Goat Anti-Rabbit (H\u0026thinsp;+\u0026thinsp;L) (Invitrogen #32460 1:1500). The blot was developed with ECL solution (SuperSignal West Femto Trial kit Invitrogen #34094) and were quantified by densitometry using gel analyzer plugin of Fiji software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eα-SYN internalization\u003c/h2\u003e \u003cp\u003eU-87 MG cells were seeded in a 24-well plate at a density of 0.1\u0026nbsp;million/well, pre-treated with 0.5 \u0026micro;M cytochalasin D and 5 \u0026micro;M Y-27632 (ROCK inhibitor), then treated (before 30min) with TMR labelled 1\u0026micro;M α-SYN protofibrils. Post-treatment U-87 MG cells were trypsinized (0.25% trypsin-EDTA solution; Gibco Canada origin #25200-072). The pellet was collected and resuspended in 1X PBS and twice washed with 1X PBS. α-SYN-TMR levels were quantified by measuring the fluorescence intensity of TMR at excitation/emission 555/585nm by using a flow cytometer (BD LSR II) and analysis was done using analysis software BD LSR-II analysis software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLive cell imaging using mitotracker and lysotracker\u003c/h2\u003e \u003cp\u003eU-87 MG cells (80,000 cells/well) were seeded on a 35 mm glass bottom dish (Cellvis, D35-14-1.5-N) and treated with 1\u0026micro;M TMR labelled α-SYN for 0h, 3h, 6h, 12h, and 24 h. The 0h cells were immediately washed before staining with lysotracker and mitotracker. Cells were stained with mitotracker green (Invitrogen #M7514) and lysotracker deep red (Invitrogen #L12492) and were incubated at 37\u0026deg;C for 15 min. Post incubation, the cells were washed with 10% DMEM and time-lapse and z-stack images were taken using confocal microscopy (Zeiss LSM880, Carl Zeiss, Germany). This was done to comprehend the localization of α-SYN and the transfer of organelles through TNTs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLive Cell Imaging using\u003c/b\u003e \u003cb\u003eDiD\u0026reg;vybrant membrane dye\u003c/b\u003e\u003c/p\u003e \u003cp\u003eU-87 MG cells (80,000 cells/well) were seeded on a 35 mm dish and treated with 1\u0026micro;M α-SYN as mentioned above. Cells were stained with cell-labelling dye DiD\u0026reg;Vybrant (Invitrogen #V22887) to label cell membranes. The dye was diluted with 10% DMEM (phenol red free) in the ratio 1:200 and incubated at 37\u0026deg;C for 20 min. Post incubation, the cells were washed with DMEM for 10 min, and images of live cells were taken to visualise the number of thin membrane tubes like TNTs using a fluorescence microscope (IX73-Olympus).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of cytosolic ROS\u003c/h2\u003e \u003cp\u003e2\u0026prime;-7\u0026prime;-Dichlorodifluorescein diacetate (DCFDA) is used to detect ROS production in the cell. U-87 MG cells were seeded in a 24-well plate at a density of 0.1\u0026nbsp;million/well and treated with 1\u0026micro;M α-SYN protofibrils as mentioned above. Post-treatment U-87 MG cells were trypsinized (0.25% trypsin-EDTA solution; Gibco Canada origin #25200-072). The pellet was collected and resuspended in DMEM with 10% FBS and 20\u0026micro;M DCFDA. Cells were incubated for 30 min at 37\u0026deg;C then washed with 1X PBS to remove the extra dye. ROS levels were quantified by measuring the fluorescence of DCFDA stained cells at excitation/emission 488/520nm by using a flow cytometer (BD LSR II) and analysis was done using analysis software BD LSR-II analysis software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial membrane potential using JC-1 dye\u003c/h2\u003e \u003cp\u003eJC-1 (5,5,6,6\u0026rsquo;-tetrachloro-1,1\u0026rsquo;,3,3\u0026rsquo; tetraethylbenzimi-dazolylcarbocyanine iodide) is the indicator of mitochondrial membrane potential. JC-1 is a cationic, lipophilic dye. Normal healthy cells show negative mitochondrial membrane potential where JC-1 dye can enter into the mitochondria and form J-aggregates. The aggregates exhibit excitation/emission maxima at 485/590 nm (red spectrum). Unhealthy cells show lower mitochondrial membrane potential as it loses the balance of electrochemical potential. At this condition, a lesser amount of JC-1 dye enters into the mitochondria and retains its monomeric form. These monomers exhibit excitation/emission maxima at 514/529 nm (green spectrum). To know the effect of α-synuclein aggregates on mitochondrial membrane potential, JC-1 assay (BD Biosciences #551302) was done. Astrocytes and U-87 MG cells were seeded at a density of 15,000 cells / well in the glass bottom area of the 35 mm imaging dishes (Cellvis, D35-14-1.5-N). Cells were treated with 1\u0026micro;M α-SYN for 3h-24h. Confocal images were also taken at an excitation of 490 nm and emission at 527 nm (green) and 590 nm (red). The ratio of green fluorescence versus red fluorescence per cell was analysed using Fiji image analysis software to quantify mitochondrial membrane potential.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCo-culture model and mitochondria transfer\u003c/h2\u003e \u003cp\u003eOne population of U-87 MG cells was transiently transfected with pLV-mitoDsRed (Addgene #44386; was a gift from Dr. Pantelis Tsoulfas's lab) plasmid and another population with mEGFP-lifeact-7 (Addgene #58470; was a gift from Michael Davidson) plasmid using lipofectamine 3000 (Invitrogen #44386) transfection reagent. An equal number of transfected cells from both populations were seeded together at a total density of 60,000 cells per glass bottom 35mm dish (Cellvis, #D35-14-1.5-N) and treated with α-SYN protofibrils. After treatment, the cells were fixed with 4% PFA and images were taken using confocal microscopy (Zeiss LSM880).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eβ- galactosidase activity assay\u003c/h2\u003e \u003cp\u003eU-87 MG cells were seeded at the density of 10,000 cells per well in a 24-well plate. The cells were treated with 1\u0026micro;M α-SYN for 3h and 24h, these time points were chosen according to the data from other experiments. Post-treatment the β-galactosidase activity was measured by using the β-galactosidase staining kit (AKR- 100 Cell Biolabs Inc). Brightfield images of the stained cells were taken using a colour camera.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMicroscopy\u003c/h2\u003e \u003cp\u003eFluorescence images were taken using Zeiss LSM880 confocal laser scanning microscope (Carl Zeiss, Germany) or fluorescence microscope (IX73-Olympus). The confocal images were taken using objectives Plan-Apochromat 40x/1.40 or 63x/1.40 Oil Dic M27, with the fluorescence filter sets DAPI, FITC, and TRITC (Carl Zeiss, Germany). Sequential images of the different fluorescence channels were taken with 405 nm, 488 nm, and 561 nm lasers. The images were captured with a pixel dwell of 1.02 \u0026micro;s and each xy-pixel of 220 nm\u003csup\u003e2\u003c/sup\u003e. For all the experiments at least 5\u0026ndash;10 images per condition were taken from randomly selected areas. DIC (differential interference contrast) images were captured along with fluorescence channels to understand the morphology and cell boundary for both fixed and live imaging experiments. Time-lapse and z-stacks (6\u0026ndash;12 stacks of z-scaling\u0026thinsp;~\u0026thinsp;415 nm) were captured from the bottom to the top of the cells using a confocal microscope to identify TNTs and TMs. Trafficking of organelles through TNTs and TMs was tracked from time-lapse images. Wide-field fluorescence microscope (IX73-Olympus) was used to capture images for a few experiments using 20X/0.4 NA, and 40X/1.3 NA plan-apochromatic objectives.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImage analysis\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ea) TNT characterization\u003c/h2\u003e \u003cp\u003eConfocal images were analysed using Fiji a Java-based image processing software developed at the National Institutes of Health (NIH) and the Laboratory for Optical and Computational Instrumentation (LOCI). TNTs were characterized by their unique nature to hover and not attach to the substratum, images were taken with z-stacks using confocal microscopy as mentioned above. TNTs (less than 1\u0026micro;M in diameter) were found in the middle z-stacks while TMs are found in the initial z-stacks since they are attached to the surface. Images of z-stacks were reconstructed using the 3D volume view plugin of Fiji as described earlier (Dilna et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). TNTs were manually counted and plotted as the ratio of the number of TNTs to the number of cells per field (Valappil, Raghavan et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eb) Tracking of organelles\u003c/h2\u003e \u003cp\u003eMovements of lysotracker and mitotracker positive vesicles through TNTs and TMs were tracked using the Trackmate (manual) plugin in Fiji. Analysis was done from time-lapse videos taken for a minimum of 30 frames at an interval of 36 seconds for 18\u0026ndash;20 mins. By setting each organelle as an object we used the semi-automatic tracking method of the trackmate plugin. The speed of the organelles was determined from the average displacement between each consecutive image.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003ec) Size analysis\u003c/h2\u003e \u003cp\u003eThe area of protofibrils, size of aggregates, and size of lysosomes and mitochondria were analysed per cell from the randomly taken images of 200\u0026ndash;300 cells per condition. The analysis was performed by setting a threshold to separate the particles as individual points and then by using the \u0026lsquo;Analyze particle\u0026rsquo; option of Fiji.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003ed) Intensity analysis\u003c/h2\u003e \u003cp\u003eSimilarly, the expression of Ki67, FAK, pFAK, α-SYN aggregates, lysosomes, and the ratio of green versus red fluorescence of the JC-1 experiment was analysed by quantifying the intensities of the labelled proteins per cell from the images of 200\u0026ndash;300 cells for each condition. Intensities were analysed by drawing and selecting regions of interest using the ROI-plugin in Fiji.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003ee) Mitochondria morphology analysis\u003c/h2\u003e \u003cp\u003eThe quantification of mitochondria branching and branch length were determined using the MiNA plugin in the ImageJ software. We downloaded the MiNA analysis plug-in from the Git Hub repository of Stuart Lab. We cropped each cell stained with mitochondria and ran the MiNA plugin to determine the mean branch length of mitochondria.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eTo validate the significance of the analysed data, one-way and two-way ANOVA tests were performed as per the experimental parameters as mentioned in the figure legends. For Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, we performed a student t-test since we had only two conditions. The statistics were calculated from the three biological repeats.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003eα-SYN protofibrils induce biogenesis of transient TNTs\u003c/h2\u003e \u003cp\u003eThe contributions of extracellular α-SYN protofibrils in the pathological spreading of PD in neuronal cell culture models are well-studied (Dieriks, Park et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Recent studies have shown that aggregates of α-SYN can modulate cellular crosstalk between neurons and the surrounding neuroglial cells (Chakraborty, Nonaka et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), which plays an important role in getting rid of toxic loads of aggregates (Rostami et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Scheiblich et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the mechanism of establishment of intercellular communications and their fate are yet to be understood. Therefore, we looked into the primary astrocytes and astrocyte-origin cancer cell lines (U-87 MG and U251) to understand the possible fate of the astroglia cells over time on α-SYN protofibril treatment.\u003c/p\u003e \u003cp\u003eTo understand this, purified α-SYN was converted into protofibrils that were either unlabelled or fluorescently (TMR) labelled and characterized using transmission electron microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Interestingly, after treatment with 1\u0026micro;M α-SYN protofibrils at 3h, 6h, 12h, and 24h, we observed formation of TNT-like membrane networks between primary astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), U-87 MG (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, Supplementary Fig.\u0026nbsp;1A) and U251 cells (Supplementary Fig.\u0026nbsp;1B) for a transient period at early hours. Time points were chosen to quantify optimum biogenesis of membrane nanotubes after treatment with 1\u0026micro;M α-SYN protofibrils over 24 h (Supplementary Fig.\u0026nbsp;1C). Quantification of TNT-like structures was performed on confocal images of phalloidin-stained astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). DiD dye-stained membranes were imaged using a fluorescence microscope to detect the abundance of stained TNT-like membrane conduits in live cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). DIC (Differential Interference Contrast) images of U-87 MG (Supplementary Fig.\u0026nbsp;1A) and U251 (Fig Supplementary Fig.\u0026nbsp;1B) cells were also taken to detect TNT-like structures. Quantification showed an increase in several hovering thin TNT-like structures per cell compared to controls in the primary astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). DiD images of U-87 MG also revealed transient increase in the biogenesis of thin (diameters\u0026thinsp;\u0026lt;\u0026thinsp;1 \u0026micro;m), cell-to-cell membrane connections at early time points (3h and 6h) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-F). Similarly, quantification of DIC images of U-87 MG cells (Supplementary Fig.\u0026nbsp;1D) and U251 (Supplementary Fig.\u0026nbsp;1E) showed enhanced transient biogenesis of TNTs at early time points compared to their respective controls and later time points.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eα-SYN protofibrils induce open-ended TNTs and cell-to-cell transfer\u003c/h2\u003e \u003cp\u003eTNTs are actin-rich, nano-sized in diameter, and primarily defined as open-ended membrane tubes (Abounit, Delage et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). TNTs are distinguished from filopodia and neurites by their distinct characteristic to hover between two distant cells (Valappil et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Actin-binding phalloidin and GFAP-stained, 3D-reconstructed confocal images show actin-positive thin TNTs that hover between two cells in primary astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Astrocyte-origin cancer cell lines (U-87 MG and U251) are known to form tumour microtubes (TMs), close-ended thicker membrane networks (diameters\u0026thinsp;\u0026gt;\u0026thinsp;1 \u0026micro;m) between cells and electrically coupled \u003cem\u003evia\u003c/em\u003e gap junctions (Osswald, Jung et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). To distinguish TNTs and TMs, confocal z-stack images were obtained after immunocytochemical staining of cells using β-tubulin antibody and phalloidin dye. Thin actin-positive, hovering TNTs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and thicker TMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) are identified in U-87 MG cells. TMs are positive for both actin and β-tubulin. Studies have indicated that astrocytoma/glioblastoma cells express lower levels of neuronal marker β-tubulin and are stained faintly (Abbassi, Recasens et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Despite that, β-tubulin staining is distinctly detected in thicker TMs and absent in TNTs (Supplementary Fig.\u0026nbsp;2A and B). DIC images clearly showed the nano-sized diameters of TNTs and thicker TMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C-upper panels). Reconstructed 3D volume images of confocal z-stacks show actin-positive, thin TNTs hover between two cells and are not attached to the substratum, whereas the tubulin-positive thicker TMs are present on the substratum (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C-lower panels).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSeveral studies have shown that TNTs are open-ended membrane channels, and can transfer organelles directly from one cell to another (Abounit et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Rustom et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). However, few studies have referred close ended membrane continuities that are not involved in cell-to-cell transfer of organelles and electrically coupled \u003cem\u003evia\u003c/em\u003e gap junctions at the endpoint as TNTs (Sowinski, Jolly et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Wang, Veruki et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Connexin43 is the essential protein of the GAP junction. Therefore, TNTs were immunostained using gap junction protein connexin43, to identify open and close-ended TNTs with GAP junction at the junctional end. Images captured populations of both connexin43 negative (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) and positive (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) at the endpoint of TNTs. Non-hovering thicker TMs are connexin43 positive structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eWe further functionally characterized TNTs based on their ability to transfer organelles like lysosomes and mitochondria. Time-lapse and z-stack images were taken using confocal microscopy (using both fluorescence channels and DIC channel) in live U-87 MG cells, stained with lysotracker and mitotracker, post-treatment with 1\u0026micro;M α-SYN protofibrils (3h, 6h, 12h, and 24h). Time-lapse videos in DIC microscopy images revealed transient increase in the biogenesis of thin cell-to-cell membrane connections and unidirectional movements of organelles \u003cem\u003evia\u003c/em\u003e the membrane nanotubes at early time points (3h and 6h) (Movies 1\u0026ndash;2). Time-lapse videos captured cell-to-cell movement of α-SYN-TMR accumulated lysosomes and mitochondria through long (20 \u0026micro;m to 120 \u0026micro;m), thin TNTs after 3h and 6 h of treatments respectively (Movies 3 and 4). We validated from z-stack images, that the thin nano-sized membrane tubes or TNTs are hovering structures connecting between two distant cells. The DIC images show thin membrane nanotubes (red arrows) between cells that are clearly visible at a higher plane of z\u0026thinsp;=\u0026thinsp;4. On the surface, at z\u0026thinsp;=\u0026thinsp;0, filopodia-like membrane extensions (violet arrows) are visible (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eG) which are unlike the TNTs. We tracked the movements of lysotracker (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eH) and mitotracker-labelled (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eI) organelles along the TNTs (identified from z-stacks) using the TrackMate plug-in available in FIJI and we observed that both the organelles travel unidirectionally from one cell towards another, with an average speed of 0.062\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 \u0026micro;m/sec. Further, we captured time-lapse videos to detect direct cell-to-cell transfer of α-SYN-TMR accumulated lysotracker and mitochondria \u003cem\u003evia\u003c/em\u003e open-ended TNTs between U-87 MG cells after 3h of treatments (Movies 5 and 6). Our results reveal the formation of open-ended functional TNTs and cell-to-cell transfer through them at 3h and 6h post-treatment with α-SYN.\u003c/p\u003e \u003cp\u003eWe observed a transient increase in the biogenesis of thin (diameters\u0026thinsp;\u0026lt;\u0026thinsp;1 \u0026micro;m), hovering TNTs upon 1\u0026micro;M α-SYN protofibrils treatment at early hours (3h and 6h) by quantifying from the 3D reconstruction of confocal z-stack images (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). Though control cells contain a higher percentage of TMs, we observed a transient increase of both open and close-ended TNTs, based on connexin43 staining after 3h and 6h post-treatment with α-SYN (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eK). Length and diameters of TNTs were measured using the confocal z-stack images in all the cell types, primary astrocytes, U-87 MG, and U251 cells. The careful measurements of z-stack images detected that the diameter of TNTs ranges between 1\u0026ndash;3 pixels, that is around 220 nm to 660 nm and length varies between 20 \u0026micro;m \u0026ndash; 100 \u0026micro;m across all the cell types (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eL and M).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eTransient lysosomal toxicities in astroglia cells upon treatment with α-SYN protofibrils\u003c/h2\u003e \u003cp\u003eInternalization of extracellularly applied α-SYN protofibrils into endo-lysosomal pathways induces transient accumulations and toxicities (3h and 6h) in primary astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and U-87 MG cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and C). α-SYN and LAMP1 were co-immunostained in primary astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and U-87 MG cells treated with TMR-labelled α-SYN were stained with lysotracker (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Accumulations of α-SYN protofibrils in endo-lysosomes and resulting toxicities were studied following enlarged and distorted morphology of α-SYN accumulated endo-lysosomal vesicles at 3h and 6h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). The extent of cathepsin leakage from LAMP2-positive vesicles was also observed to follow lysosomal toxicities (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). α-SYN protofibrils were internalized in primary astrocytes and colocalized in substantial amounts to LAMP1-immunostained positive vesicles (analysed using Coloc 2 plug-in in FIJI). Maximum colocalizations were observed at 3h of treatment which over time gradually decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Internalization of protofibrils to lysosomal pathway caused transient accumulation and lysosomal toxicities, resulting in larger-sized LAMP1 positive vesicles that were transiently detectable at 3h and 6h after α-SYN protofibrils treatment in astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Total areas of red puncta of α-SYN-TMR oligomers per cell were quantified measuring the size of each punctum. The results show that endo-lysosomal machinery in U-87 MG cells gradually clears up toxic protofibrils (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). The analysis of cathepsin leakage in LAMP2-positive lysosomal vesicles indicated no significant increase of cathepsin-D to the cytosol (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). However, we observed the larger cathepsin-D and LAMP2-positive vesicles after 3h of protofibril treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Morphology of LAMP2-positive lysosomes at 3h is elongated and rough in comparison to control, 6h, 12h, and 24h treated cells (magnified images highlighted in the upper-left corner of Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The time of transient lysosomal toxicity coincides with the transient increase of TNT biogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-F). Altogether the results suggest that transfer of organelles \u003cem\u003evia\u003c/em\u003e TNTs probably aids the cells to cope with protofibril-induced lysosomal toxicities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eTransient mitochondrial toxicities in astroglia cells upon treatment with α-SYN protofibrils\u003c/h2\u003e \u003cp\u003eSimilar to transient lysosomal toxicities and biogenesis of TNTs, mitochondrial toxicities were observed through morphological changes in the early time points (3h and 6h) using MitoProbe JC-1 dye which measures mitochondrial membrane potential (ΔΨ\u003csub\u003eM\u003c/sub\u003e). Lipophilic, cationic JC-1 dye can enter healthy mitochondria and aggregate (emission @590 nm in the red channel) inside, whereas, in unhealthy mitochondria with decreased membrane potential, the dye crosses in and out through the relatively open membrane pores and stays as monomers (emission @527 nm in the green channel). Adding to our previous observations, we noticed a decrease of ΔΨ\u003csub\u003eM\u003c/sub\u003e transiently in the early time points (3h and 6h) in primary astrocytes, whereas at the later time points (12h and 24h) mitochondria looked healthier and were rescued from protofibrils-induced mitochondrial toxicities (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B). The quantification shows JC-1 labelled red coloured healthier, larger mitochondria in the control and recovered healthier cells after 24h (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The toxic mitochondria in early time points (3h and 6h) were observed to possess shorter branch lengths compared to the control and the recovered cells at later times (12h and 24h) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Mitochondrial toxicities were also observed in U-87 MG cells through morphological changes using mitotracker staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, upper panel) and mitochondrial membrane potential (ΔΨ\u003csub\u003eM\u003c/sub\u003e) using MitoProbe JC-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, lower panel). Similar to our results in primary astrocytes, images of U-87 MG cells showed that at the early time points (3h and 6h) mitochondria were smaller sized, with fragmented morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, upper panel) and quantifications showed increased JC-1 stained greener mitochondria or mitochondria with decreased ΔΨ\u003csub\u003eM\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Thus, it is evident that primary astrocytes and U-87 MG cells adapt and are rescued from α-SYN protofibril-induced transient mitochondrial toxicities at later times.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eCell-to-cell transfer of mitochondria in α-SYN protofibril treated astroglia cells\u003c/h2\u003e \u003cp\u003eThe transfer of mitochondria between α-SYN protofibrils-treated U-87 MG cells was further examined by co-culturing these cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). MitoDsRed (red) and EGFP-lifeact (green) transfected U-87 MG cells were co-cultured and treated with α-SYN protofibrils for 3h, 6h, 12h, and 24h. Results demonstrate mitoDsRed labelled red mitochondria in the green EGFP-lifeact stained cells upon treatment with α-SYN protofibrils (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The transferred red mitochondria in the green cells were observed maximum at early time points (3h and 6h) when cells show enhanced mitochondrial toxicities and increased numbers of TNTs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). When conditional media from the mitoDsRed transfected cells were given to the green cells transfected with EGFP-lifeact, we did not observe any significant transfer of mitochondria (Supplementary Fig.\u0026nbsp;3). The result excludes the probability of transfer of mitochondria through exosomes. The morphology of the transferred mitochondria in the green cells mostly appears round in shape rather than elongated and healthy. Quantification shows that transferred mitochondria are smaller in size and round-shaped, compared to the mitochondria of the control cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Therefore, we further studied the fate of cell-to-cell transfer of toxic mitochondria, following ROS levels in the cells upon protofibril treatment at different time intervals. We also observed increased total cellular ROS at an early time point (3h) and a gradual decrease in ROS levels at later time points (6h, 12h, and 24h) in U-87 MG cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and E). The fluorescence intensity of DCFDA was analysed using flow cytometer to quantify the cellular ROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). The quantification of DCFDA flow cytometer data show similar results in primary astrocytes with a significant increase in cellular ROS levels at 3h compared to the control cells which then decrease at later time points (6h, 12h, and 24h) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eF and G).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eα-SYN protofibril-induced ROS mediated cellular senescence and its correlation with transient biogenesis of TNTs\u003c/h3\u003e\n\u003cp\u003eIncreased levels of cellular ROS play a significant role in inducing cellular senescence and reduction of ROS accumulation can reverse p-21 mediated cellular senescence (Macip et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Studies have also shown the critical role of α-SYN-induced ROS in cellular senescence-associated neurodegeneration (Miller, Campbell et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Verma, Seo et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The increased ROS levels and its elimination in correlation to TNT biogenesis, motivated us to look into the senescence states in astroglia cells (Macip et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). We have observed a transient increase in p-21 levels with α-SYN protofibrils treatment in western blot analysis at an earlier time point (3h) in U-87 MG, then p-21 levels decrease gradually over time (6h, 12h, and 24h) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Further, we have observed irregular nuclei, invaginations, and fragmented nuclei during the transient time window 3h and 6h, which corresponds to the α-SYN protofibrils induced transient organelle toxicities, ROS production and biogenesis of TNTs in primary astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eI-J) and U251 cells (Supplementary Fig.\u0026nbsp;4A and B). We established cells undergo transient cellular senescence at an early time point (3h) by measuring β-galactosidase activity as the senescence marker (Supplementary Fig.\u0026nbsp;4C). It is known that DNA damage-related nuclear size irregularities are often associated with hyperproduction of cellular ROS and mediated senescence. The transient cellular senescence corresponds to the transient biogenesis of TNTs at early hours in α-SYN treated cells. Eventually, the reduction of ROS accumulation caused a reversal of p-21-dependent cellular senescence at later times.\u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eClearance of α-SYN induced organelle toxicities and ROS in cell survival and proliferation\u003c/h2\u003e \u003cp\u003eIt is a well-established fact that extracellularly applied α-SYN protofibrils are toxic to neurons and cause gradual neuronal death (Li, Yuan et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Conversely, we observed astrocytes and astrocytes origin cancer cells survive by alleviating α-SYN protofibrils induced organelle toxicities and cellular ROS levels. We also observed that biogenesis of TNTs and cell-to-cell transfer precedes clearance of α-SYN protofibrils induced organelle toxicities and cellular ROS levels. Recent studies have shown that cell-to-cell transfer of mitochondria in astrocytes \u003cem\u003evia\u003c/em\u003e TNTs facilitates cell survival (Valdebenito, Malik et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, we investigated cell viability using α-SYN protofibrils in primary astrocytes, U-87 MG, and U251 astroglia cells. MTT assay measures cell metabolic activity and is used as an indicator for cell viability. The results pertaining to this assay show a concentration-dependent increase of cell viability upon treatment of toxic α-SYN protofibrils at later time points (12h and 24h) in astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), compared to the respective controls and early time points (3h and 6h). α-SYN protofibrils induced organelle toxicities at earlier times also resulted in decreased metabolic activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). α-SYN protofibrils treated U-87 MG (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) and U251 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD) cells show proliferation at later times (12h and 24h) compared to their respective controls. U-87 MG cells also show concentration-dependent proliferation at later times (24h) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Further, we manually counted the cells to reconfirm that the increasing concentrations of toxic protofibrils caused an increase in cell numbers or cell proliferations, after 24 and 48 h of treatments in astroglia cells ( Supplementary Fig.\u0026nbsp;5A and B). Overall, the results showed that, instead of developing progressive toxicities or cell death overtime α-SYN protofibrils treatment, the astroglia cells adapt to overcome the stress and proliferate during post-recovery time. We have verified the toxic effects of the protofibrils in the neurons derived from the differentiation of neuroblastoma (N2a) cells. The results show time- and concentration-dependent cell death in the treated cells (Supplementary Fig.\u0026nbsp;5C and D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince we have observed a significant increase in cell numbers and cell viability upon α-SYN protofibrils treatment, we checked for cell proliferation using Ki67 as the marker. Astrocytes and U-87 MG cells immunostained with Ki67 antibody and nuclear stain DAPI (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE) show a significant increase in Ki67 overexpression in the nucleus at 12h and 24h in comparison to control and early time points (3h and 6h) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF and G). Overall, the results indicate toxic α-SYN-induced initial stress (till 6 h) promotes biogenesis of TNTs and cell-to-cell transfer of organelles, consequences of which aid in rescuing the cells from organelle toxicities or ROS-induced cellular stresses. Further, to deal with recovery, cells probably facilitate proliferation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eTNT biogenesis pathways in astroglia proliferation\u003c/h2\u003e \u003cp\u003eTNTs are structurally open-ended membrane actin conduits. Thereby, it is obvious that modulation of membrane and cytoskeleton will play a major role in their biogenesis. However, the exact mechanism of TNT biogenesis is not known. Studies on screening of inhibitors in the actin signalling pathways could unfold molecular events behind the α-SYN toxicity-induced biogenesis of TNTs. Therefore, we studied different actin inhibitors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH\u003cb\u003e)\u003c/b\u003e to see their effects on biogenesis of TNTs at an early time (3h) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI) and cell proliferation at a later time (24h) in U-87 MG cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). We observed that Cytochalasin D (inhibits actin polymerization and interaction of G-actin-cofilin) and IPA-3 (PAK1/2 inhibitor) inhibited α-SYN protofibril-induced TNTs formation, whereas, CK-666 (Arp2/3 inhibitor), Blebbistatin (myosin-II-specific ATPase inhibitor), and Y-27632 (ROCK inhibitor) promote biogenesis of TNTs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK). Similar to the reports of earlier studies (Henderson, Ljubojevic et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), we have also observed that Arp2/3 inhibitor CK-666 caused formation of significantly longer TNTs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI and Supplementary Fig.\u0026nbsp;6).\u003c/p\u003e \u003cp\u003eTo understand the role of TNTs in cell proliferation, cell viability was assayed using MTT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL), and cell numbers were determined by manual counting (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM). TNT-numbers (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK) and cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL and M) data showed that the actin inhibitors, Cytochalasin D, and IPA-3, which prevent biogenesis of TNTs, inhibit α-SYN protofibrils induced cell proliferation as well (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL and M). The actin inhibitors, CK-666 and Blebbistatin which facilitate biogenesis of TNTs, did not alter α-SYN protofibrils induced proliferation. However, the inhibitors alone (CK-666, and Blebbistatin) did not show a significant effect on cell proliferation. We observed the ROCK inhibitor Y-27632 in the presence and absence of α-SYN protofibrils significantly increased proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL and M).\u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003e`\u003c/h2\u003e \u003cp\u003e \u003cb\u003eBiogenesis of α-SYN induced TNTs through modulation of ROCK pathway, resulting in increased cell survival and proliferation\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA recent study has shown that the ROCK inhibitor (Y-27632) is an intriguing compound that boosts biogenesis of TNTs \u003cem\u003evia\u003c/em\u003e Myosin II-mediated actin modulation (Scheiblich et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, it is not yet known how α-SYN dominates modulation of ROCK inhibition signalling mediated TNT biogenesis, over the cofilin-G-actin interaction (Cytochalasin-D inhibited pathway) mediated actin remodulation. To unfold the mechanism, we followed the internalization of α-SYN-TMR protofibrils in the presence of Cytochalasin-D and ROCK inhibitor (Y-27632) using flow cytometry quantification (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). We observed that Cytochalasin-D inhibited internalization of α-SYN protofibrils, however, Y-27632 did not show any effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003eα-SYN-induced cellular senescence results in translocation of FAK in the nucleus\u003c/h2\u003e \u003cp\u003eOur results showed α-SYN protofibrils induced nuclear deformity and cellular senescence in astroglia cells at early time points (3h and 6h) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eH-J). Several studies have shown that focal adhesion kinase (FAK) inhibition could induce the DNA damage and nucleus deformity that accompanies cellular senescence (Chuang, Wang et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Zhou, Yi et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Loss of integrin-dependent cell adhesion in cellular stress modulates FAK and facilitates its translocation from the plasma membrane to enter to nucleus (Lietha, Cai et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, Lim, Chen et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Lim, Miller et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). We observed de-adhesion of astroglia cells (U-87 MG) with α-SYN protofibrils treatments at early time points (3h and 6h) when cells show lysosomal-mitochondrial toxicities and transient biogenesis of TNTs, compared to the control and cells at later times (12h and 24h) (Supplementary Fig.\u0026nbsp;7A). Further, we noticed treatments with α-SYN protofibrils cause nuclear translocation of FAK in the astrocytes that are connected by TNTs at early time points (3h and 6h) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC and D). The presence of nuclear FAK was verified by 3D images at xz and yz planes (Supplementary Fig.\u0026nbsp;7B). We also observed treatments with α-SYN protofibrils cause nuclear translocation of FAK to the nucleus in the U-87 MG (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE and F) and U251 (Supplementary Fig.\u0026nbsp;7C and D) cells at early time points (3h and 6h), whereas, at later timepoints in the post-recovered cells, FAK relocates back to PM and cytosol. We have also observed nuclear translocation of activated phospho-FAK (Tyr 397) transiently at early time points (3h and 6h) in astrocytes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG and H). These results clearly demonstrate that there is a transient translocation of FAK in the nucleus at the early and late time points on α-SYN treatment.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eNuclear translocation of FAK modulates the ROCK pathway to induce TNT biogenesis and cell proliferation\u003c/h3\u003e\n\u003cp\u003eInhibition of integrin-mediated FAK activation prevents ROCK activation and inhibits cell adhesion. Moreover, when active/inactive FAK is displaced from cell-adhesion sites in non-adherent cells, Rho-mediated activation of ROCK kinases maintains cytoskeleton tension by regulating actin re-modulation (Pirone, Liu et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). To understand the FAK-mediated regulation of ROCK kinases, we have performed western blots and quantified expressions of ROCK1 and ROCK2 in the α-SYN treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI and J). Results show ROCK2 inhibition was observed in the early hour (3 h) after α-SYN treatment, later expressions increased gradually (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI and J). However, ROCK1 expression with α-SYN treatment showed gradual inhibition over time.\u003c/p\u003e \u003cp\u003eROCK activation pathways involve in cell proliferation as well (Pirone et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Our western blots showed increased levels of ROCK-mediated cell proliferation markers ERK1/2, NF-κB, and cdk1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eK and L). We observed higher ROCK2 expression in the rescued cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI and J). Probably, ROCK2 activation at the later hours, results in increased cell proliferation through ERK1/2 and NF-κB signalling cascades. Overall, the results delineate that α-SYN protofibrils treatment caused transient localization of FAK/pFAK to the nucleus of astroglia cells, which could modulate ROCK inhibitory pathways to promote biogenesis of TNTs in the astroglia cells for a transient time at early hours after the treatment. The rescued cells post α-SYN treatment and transient TNT biogenesis, eventually may re-activate ROCK signalling to restore cytoskeleton tension, and promote enhanced cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePD pathology progresses with the spreading of misfolded α-SYN aggregates in the brain (Kalia \u0026amp; Lang, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Even though cytoplasmic inclusion of α-SYN in the neurons is the central hallmark of neuropathology development in PD, evidence suggests release of α-SYN aggregates from degenerated neurons in the extracellular brain plays a significant role in PD pathology progression. The role of extracellular α-SYN in cell-to-cell transfer and PD pathology progression has widely been studied in several model systems (Neupane et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Recent studies have shown that extracellular α-SYN aggregates, taken up by astrocytes and microglia, promote biogenesis of TNTs. The TNT-mediated glial crosstalk and cell-to-cell transfer facilitate degradation of the aggregates and clearance of toxic organelles (Rostami et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Scheiblich et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Previously, the study by Loria et al., has shown that astrocytes facilitate degradation of α-SYN protofibrils by promoting TNT-mediated transfer (Loria, Vargas et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The crosstalk between astrocytes and microglia \u003cem\u003evia\u003c/em\u003e TNTs facilitates the degradation of α-SYN protofibrils even faster (Rostami et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). A recent study has reported, an on-demand formation of TNTs to clear toxic α-SYN protofibrils and alleviate ROS levels in microglia (Scheiblich et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The study showed, the rescue of toxic burden of α-SYN \u003cem\u003evia\u003c/em\u003e TNT-mediated borrowing of healthier mitochondria from the healthier neighbours and transfer of toxic aggregates to them (Scheiblich et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Recently, a study demonstrated that neuronal cells transfer toxic mitochondria to microglia \u003cem\u003evia\u003c/em\u003e TNTs, whereas microglia protect neurodegeneration by transferring healthier mitochondria to neurons (Chakraborty et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, the molecular players involved in the biogenesis of transient TNTs, the fates of TNT-mediated cell-to-cell transfer, and their dynamic interplay in the clearance of toxic neurodegenerative aggregates are not well explored.\u003c/p\u003e \u003cp\u003eOur study has shown for the first time, that transient translocation of FAK/pFAK to the nucleus upon α-SYN protofibrils treatment causes transient biogenesis of TNTs, which eventually contributes to enhanced cell proliferation in the astroglia cells. Our study has also established a strong correlation between transient biogenesis of TNTs with α-SYN-induced toxic burden at early hours (3h and 6h). Cell-to-cell transfer through transient TNTs corresponds to cellular toxicities, like lysosomal, and mitochondrial toxicities, and increased levels of cellular ROS and associated cellular senescence. However, the astroglia cells recover from these cellular toxicities at later hours (12h and 24 h). The post-recovered astroglia cells proliferate with increasing concentrations of α-SYN protofibril treatments. Understanding of molecular players involved in the proliferation of astrocytes in response to α-SYN aggregates is highly important since astrogliosis and microgliosis act as essential mediators in maintaining cellular homeostasis in the PD degenerative brain (MacMahon Copas et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral studies have reported cell-to-cell transfer of mitochondria through TNTs in several pathophysiology conditions, where the transfer of healthy mitochondria results in rescue of cells from apoptosis or toxicities (Han \u0026amp; Wang, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Lou, Fujisawa et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Spees, Olson et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). However, it is not the case that TNTs transfer only healthy mitochondria to neighbouring cells. Microglia and astrocytes probably facilitate dilution of its toxic burden by transferring toxic α-SYN aggregates and toxic mitochondria by sharing with its neighbours through TNTs (Loria et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Scheiblich et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We have observed α-SYN protofibrils induced increased ROS production at early time points (3h and 6h), causing reduced mitochondrial membrane potential, resulting in generation of fragmented mitochondria and disintegration of mitochondrial network. In the co-culture experiment, we observed only unidirectional transfer of toxic fragmented mitochondria to their neighbouring cells. Several studies have shown that accumulated ROS with transferred toxic mitochondria could facilitate cell proliferation (Heinke, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Kidwell, Casalini et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The observations suggest that astrocytes and astroglia cells share their toxic organelles (lysosomes and mitochondria) with surrounding neighbours to dilute the toxic burden, and the transferred toxic mitochondria may contribute to facilitating proliferation.\u003c/p\u003e \u003cp\u003eWe have observed that α-SYN protofibrils induced increased ROS levels directly affect mitochondrial dysfunction, and DNA damage, which play key roles in inducing cellular senescence in reactive astrocytes (Davalli, Mitic et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Gallage \u0026amp; Gil, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Verma et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We have observed α-SYN protofibrils induced mitochondrial abnormality, fragmented irregular nucleus, increased β-galactosidase activity, and p21-pathway-dependent premature cellular senescence for a transitory period. Astroglia cells recover from senescence-related toxicities by alleviating ROS levels, after transient biogenesis of TNTs and TNT-mediated cell-to-cell transfer. ROS-induced DNA damage and mitochondrial toxicity do not always lead to apoptosis (Borges, Linden et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Cells activate repair mechanism/s in case of delayed apoptosis. Moreover, reduction of ROS accumulation can reverse p21-mediated stress-induced premature senescence or \u003cem\u003evice versa\u003c/em\u003e (Macip et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCell-to-cell transfer of neurodegenerative aggregates drive spreading of aggregates, which act as seeds for further propagation in neurons (Nath, Agholme et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), whereas, astrocytes and astroglia cells rescue cellular toxicities (Loria et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Nath et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Rostami et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The role of the inherent conventional clearance efficiency of astrocytes cannot completely be ruled out. Unlike neurons, astrocytes lack endogenous α-SYN and the capacity of continuous seeding to form higher-order toxic aggregates is limited. This is to emphasize here that TNTs are predominantly observed in the cell types that possess inherent anti-apoptotic properties, like primary neurons, neuronal cells, neuroglial cells, and cancer cells (Gousset, Schiff et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Rustom et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Neurons possess inherent anti-apoptotic properties like other brain cells, however, unlike glial cells they are mitotically incompetent (Sharma \u0026amp; Subramaniam, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Tardivel, Begard et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). We observed α-SYN induced TNT-mediated cell-to-cell transfer eventually leads to cell death in mitotically incompetent neurons, whereas astrocytes and astroglia cells clear toxic burden and enhance proliferation and cell survival. TNT-mediated cellular clearance and proliferation in astrocytes could play a significant role in PD.\u003c/p\u003e \u003cp\u003eTherefore, the important question that needs to be understood is how biogenesis of TNTs and clearance of cellular toxicities are associated with proliferation in astrocytes. Several studies have indicated that biogenesis of TNTs may induce cell proliferation, to protect the cells from cell death under pathogenic conditions and chemo- or radio- therapy related stress (Han \u0026amp; Wang, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Osswald et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Saha, Dash et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Wang, Chen et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The studies show mostly limited or indirect correlation between TNTs and cell proliferation. Our results on small molecule actin modulators indicate ROCK inhibitory pathway mediated biogenesis mechanism/s of TNTs and its correlation with enhanced proliferation. Small molecules actin modulators (Cytochalasin D, and IPA-3) inhibit biogenesis of TNTs and prevent α-SYN protofibril-induced cell proliferation. On the other hand, the actin modulators CK-666 and Blebbistatin, which facilitate biogenesis of TNTs, did not show a significant effect on the enhancement of cell proliferation. Even though these actin inhibitors (CK-666 and Blebbistatin) show better cell survival compared to the molecules that inhibit TNTs. Only ROCK inhibitor Y-27632 mediated biogenesis of TNTs significantly promotes proliferation, similar to proliferation induced by α-SYN protofibrils. ROCK inhibitor Y-27632 inhibits LIMK-dependent cofilin-G-actin interaction and inhibits actin polymerization whereas, IPA-3 and Cytochalasin-D could also inhibit actin polymerization through the LIMK pathway (Scheiblich et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, we observed ROCK inhibition promotes biogenesis of TNTs, whereas, IPA-3 and Cytochalasin-D inhibit TNTs. Our result suggests, that inhibition of ROCK kinase pathways may modulate TNT biogenesis by regulating actin cytoskeleton through myosin-II-specific downstream signalling molecules, which dominates over cofilin-mediated actin modulation. Similarly, a recent study (Scheiblich et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) has shown that α-SYN protofibrils promote biogenesis of TNTs in microglia cells \u003cem\u003evia\u003c/em\u003e regulating ROCK inhibitory pathway by phosphorylation of myosin light chain phosphatase (MLCP). Thus, we tried to unfold how α-SYN protofibrils induced actin-remodulation may modulate ROCK inhibitory signalling cascades to induce TNT biogenesis.\u003c/p\u003e \u003cp\u003eWe have found that α-SYN-induced transient senescence-related toxicities caused nuclear localization of FAK and pFAK for the transitory period, which corresponds to TNT biogenesis at early time points (3h and 6h). Organization of FAK at focal adhesion sites of PM regulates activation of Rho-mediated ROCK signalling, and its de-localization from focal adhesion inhibits the ROCK pathway (Schober, Raghavan et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). On the other hand, in low adhesion conditions displacement of inactive/active FAK/pFAK from focal adhesion sites leads to Rho kinase-mediated activation of ROCK signalling, probably to restore the cytoskeletal tension (Pirone et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Then, restoration of cytoskeleton tension may cause re-translocation of FAK/pFAK to PM, which could reinstate ROCK activation. In this line, we have detected α-SYN toxicities induced FAK translocation dynamics, which could modulate transient inhibition of ROCK2 signalling to induce biogenesis of TNTs at early times and later may activate to restore cytoskeleton tension. We have also seen the proliferation of astroglia cells \u003cem\u003evia\u003c/em\u003e activation of ROCK signalling mediated ERK1/2 and NFκB proteins, in the post-recovered astroglia cells. Thus, our results suggest that biogenesis of TNTs is not the root cause of cell proliferation, rather modulation of α-SYN protofibrils induced ROCK-mediated actin-regulatory signalling pathway related to biogenesis of TNTs triggers cell proliferation.\u003c/p\u003e \u003cp\u003eIn conclusion, our study reveals α-SYN protofibrils induced biogenesis of TNTs aids in enhancing the clearance of toxic burden as a cellular survival strategy to rescue the astroglia cells from ROS-induced cell death/cellular senescence. α-SYN protofibrils regulate FAK-mediated modulation of ROCK signalling cascades to rescue the cells from toxic burden by promoting TNT biogenesis. The rescued cells, eventually re-activate ROCK2 signalling probably to restore cytoskeleton tension and enhance cell proliferation \u003cem\u003evia\u003c/em\u003e ERK1/2 and NFκB signalling. α-SYN inclusions in the astroglia cells and glial inclusion or fibrous gliosis in the areas of neurodegeneration are widespread in PD (MacMahon Copas et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Thus, the study is important for understanding the relevance of TNT-mediated crosstalk in the clearance of α-SYN protofibrils by astrocytes, and its implication in astroglia cell proliferation. Thus, the study will open up new strategies to design therapeutics targeting astrocytes in the PD brain.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eS.N conceived and conducted the research; A.R, R.K, S.P (JNCASR), R.M (IISc), R.M (JNCASR), S.P (NIMHANS) and S.N designed and interpreted data; A.R, R.K, S.P (JNCASR), S.J, S.C, A.A, M.N.D and M.G performed experiments; A.R, R.K, S.P (JNCASR), S.C, and A.A analysed the data; A.R, R.M (JNCASR), and S.N wrote the paper taking valuable inputs from all the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Mrs. Suma (JNCASR) for the confocal microscopy and Mrs. Usha (JNCASR) for TEM imaging. We thank Dr. Anujith Kumar (MIRM-MAHE) for sharing his resources and Ms. Smitha Bhaskar (MIRM-MAHE) for helping with transfection using the electroporation technique. We thank Prof. Dipankar Nandi, Indian Institute of Science, for the DCFDA reagent. We thank Dr Lakshmi Balasubramanian (C-CAMP, Bangalore) and Mr. Vedam Pruthvi (MIRM-MAHE) for giving suggestions to develop an image analysis flow diagram.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eA.R, and S.C thank Manipal Academy of higher education for Dr. TMA pai fellowship; A.R thanks ICMR SRF- Direct fellowship, R.K. thanks the Indian Council of Medical Research of India (#5/4-5/Ad-hoc/Neuro/216/2020-NCD-I) for her JRF fellowship; S.N thanks the Science and Engineering Research Board of India for the SERB-SRG (#SRG/2021/001315) grant; the Indian Council of Medical Research of India (#5/4-5/Ad-hoc/Neuro/216/2020-NCD-I) and the Intramural fund of Manipal Academy of Higher Education, Manipal, India (#MAHE/CDS/PHD/MIFR/2019) for financial support; The financial support from the DBT-RA program in Biotechnology and Life Sciences and DST-SERB NPDF to SP (JNCASR) is gratefully acknowledged; The financial support from Department of Biotechnology (DBT) grant in Life Science Research, Education and Training at JNCASR (BT/INF/22/SP27679/2018), S. Ramachandran-National Bioscience Award for Career Development (NBACD)-2020-21 (SAN No. 102/IFD/SAN/990/2021-22) and JNCASR intramural funds to RM is acknowledged. S.P (NIMHANS) thanks the Science and Engineering Research Board, Government of India for the funding support (ECR/2018/002219).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbbassi RH, Recasens A, Indurthi DC, Johns TG, Stringer BW, Day BW, Munoz L (2019) Lower Tubulin Expression in Glioblastoma Stem Cells Attenuates Efficacy of Microtubule-Targeting Agents. \u003cem\u003eACS Pharmacol Transl Sci\u003c/em\u003e 2: 402-413\u003c/li\u003e\n\u003cli\u003eAbounit S, Bousset L, Loria F, Zhu S, de Chaumont F, Pieri L, Olivo-Marin JC, Melki R, Zurzolo C (2016) Tunneling nanotubes spread fibrillar alpha-synuclein by intercellular trafficking of lysosomes. \u003cem\u003eEMBO J\u003c/em\u003e 35: 2120-2138\u003c/li\u003e\n\u003cli\u003eAbounit S, Delage E, Zurzolo C (2015) Identification and Characterization of Tunneling Nanotubes for Intercellular Trafficking. \u003cem\u003eCurr Protoc Cell Biol\u003c/em\u003e 67: 12 10 1-12 10 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Biomedicine \u0026amp; Pharmacotherapy \u003c/em\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"cell-cell transfer, α- synuclein, tunneling nanotubes, reactive oxygen species (ROS), mitochondria, cellular senescence, focal adhesion kinase, cell proliferation. ","lastPublishedDoi":"10.21203/rs.3.rs-3747717/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3747717/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAstroglia play crucial neuroprotective roles by internalizing pathogenic aggregates and facilitating its degradation. Here, we show, that α-SYN protofibril-induced organelle toxicities and reactive oxygen species (ROS) cause premature cellular senescence in astrocytes and astrocytes origin cancer cells, resulting in a transient increase in biogenesis of tunneling nanotubes (TNTs). TNT-biogenesis and TNT-mediated cell-to-cell transfer lead to clearance of α-SYN-induced organelle toxicities, reduction in cellular ROS levels, and reversal of cellular senescence. Enhanced cell proliferation is seen in the post-recovered cells after relieving from α-SYN-induced organelle toxicities. Further, we show, that α-SYN-induced senescence promotes transient localization of focal adhesion kinase (FAK) in the nucleus. FAK-mediated regulation of Rho-associated kinases plays a significant role in the biogenesis of TNTs, and successively proliferation. Our study emphasizes that TNT biogenesis has a potential role in the clearance of α-SYN-induced cellular toxicities and reversal of stress-induced cellular senescence, consequences of which cause enhanced proliferation in the post-recovered astroglia cells.\u003c/p\u003e","manuscriptTitle":"Astroglia proliferate upon biogenesis of tunneling nanotubes via α-synuclein dependent transient nuclear translocation of focal adhesion kinase","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-22 14:16:20","doi":"10.21203/rs.3.rs-3747717/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"615115bc-9a5c-46f3-ac6a-14f527be1657","owner":[],"postedDate":"January 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":27702933,"name":"Biological sciences/Neuroscience/Glial biology/Astrocyte"},{"id":27702934,"name":"Biological sciences/Cell biology/Cell adhesion/Focal adhesion"}],"tags":[],"updatedAt":"2024-01-22T14:16:20+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-22 14:16:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3747717","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3747717","identity":"rs-3747717","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}