α- Linoleanic acid modulates phagocytosis of extracellular Tau and induces microglial migration by actin-remodeling

Background Seeding effect of extracellular Tau species is an emerging aspect to study the Tauopathies in Alzheimer’s disease. Tau seeds enhance the propagation of disease along with its contribution to microglia-mediated inammation. Omega-3 fatty acids are known to exert the anti-inammatory property to microglia by modulating cell membrane compositions. The immunomodulatory function of omega-3 fatty acids exerts anti-inammatory property to microglia. Owing to the imparted anti-inammatory nature enhance phagocytosis and increased migration property has been observed in microglia. The dietary omega-3 fatty acids are found to change the lipid composition of the cell membrane that predominated many signaling cascade and by modulating specic receptor response. Thus the omega-3 fatty acids inuence microglial response in Tauopathy. Method N9 microglia cells were exposed to extracellular full-length Tau monomer and aggregates along with ALA (α- Linolenic acid) to study the internalization of exposed Tau. The degradation of internalized Tau studied with the endosomal markers Rab5 and Rab7. The nal degradation step in phagocytosis has been studied with LAMP-2A as lysosomal markers. The changes in the rate of migration of microglia were assessed by wound-scratch assay along with Microtubule organizing center (MTOC) reorientation were studied after exposure of Tau and ALA as the property of highly migratory microglia. The role of actin in phagocytosis and migration was observed with the study of actin structures lamellipodia, lopodia, and membrane ruing. The formation of extensive actin branching in lamellipodia and membrane ruing was studied with the help of ARP2/3 complex for nucleating actin network. Results The increased phagocytosis of extracellular Tau monomer and aggregates has been observed upon ALA exposure to microglia cells. The intracellular degradation of internalized Tau species was targeted by early and late endosomal markers Rab5 and Rab7. The increase levels of LAMP-2A and colocalization with internalized Tau indicated the degradation via lysosome. These results indicate the degradation of internalized Tau species in the presence of ALA instead of getting accumulated D) 100% 50 degrees for 2 The microscopic observation of ALA vesicles was done by Transmission electron microscopy for the morphological analysis. The enlarge area showing zoomed images of vesicles; scale bar is 100nm E) Kinetics of ThS uorescence assay, to observe the aggregation propensity of hTau40 on different time points in presence of heparin in vitro. F) SDS PAGE analysis of hTau40 aggregates. The the marking of 250kDa shows higher order bands corresponding to aggregates. G) TEM analysis of hTau40 aggregates after 120 hours. H) CD analysis to study the conformation changes of Tau on aggregation from random coiled to β-sheet structure. The spectra was analysed between 250-190 nm range. A) internalized Tau follows the nally lysosome-mediated In the pathway the maturation of phagocytic vesicle take place that can be marked with the early endosomal marker Rab 5 and late endosomal marker Rab 7. We observe the colocalization of internalized Tau with endosomal markers to trace the degradation of internalized Tau. B) The uorescence microscopy images indicates the levels of endosomal markers and their colocalization with internalized Tau, the zoomed area indicates the colocalized positions inside the cell, the white arrow marks indicates colocalization. C) The intensity analysis of endosomal markers was carried out and plotted as intensity per unit sq. area; signicance is P<0.05. D) Expression analysis of early endosomal marker was observed by western blot after various treatments of hTau40 monomer, aggregates and ALA after 24 hours. omega-3 fatty anti-inammatory phenotype. A) ALA observed to enhance the phagocytosis microglia that is also assisted by migration of microglia. The effect of ALA on migration under the inuence of hTau40 monomer and aggregates has been studied. B) The migration of microglia was studied by wound scratch assay. Migration of cells into the scratch was studied with different time intervals 0, 6, 12, 24 hours after the scratch observed with optical microscope. Scale bar is 100nm. C) In each treatment groups random ve elds were chosen and number of cells migrated into wound was counted. The comparison for each time point was carried out with its respective time point control (untreated) group; signicance is P<0.001.

In the central nervous system (CNS), embryonic mesoderm-derived microglia are the major group of resident immune cells, which consists of 20% of the total glial population. In a normal physiological condition, microglia displays rami ed morphology having long branched cellular processes, which senses the tissue damage, pathogenic invasions, etc [1]. The surveillant stage of microglia is maintained by neuronal and astrocytes-derived factors [2]. On external stimuli, microglia become activated and the rami ed morphology changes to amoeboid morphology. Microglia are either classically activated to give a proin ammatory response or follow alternative activation to show anti-in ammatory response. In Alzheimer's disease (AD), which is a progressive neurodegenerative disease shows a predominance of in ammatory microglia. Gliosis in AD pathology indicates abnormal morphology, excessive activation of microglia and astrocytes. Neuroin ammation acts as a key triggering process in AD where Amyloid-β and neuro brillary tangles of Tau are found to surrounded by microglia [3]. The presence of excessive proin ammatory cytokines IL-1β, TNF-α, IFN-γ and accumulation of aggregated protein's proin ammatory condition of microglia favors and hampers the phagocytic nature of microglia [4]. The phagocytic state of microglia is regulated by the expression of receptors on the cell surface, membrane uidity, downstream signaling, and rearrangement of the actin network. Environmental factors including dietary lipids contributes to the phagocytosis by microglia. The phagocytic property of microglia is regulated by environmental factors as well, including dietary lipids [5]. Dietary lipids affect the brain extensively since fatty acids are building blocks of the brain. Dietary fatty acids importantly polyunsaturated fatty acids (PUFAs) including omega-3 fatty acids Docosahexaenoic acid (DHA-22 n3:6), Eicosapentaenoic acid (EPA-20 n3:5) and α-Linolenic acid (ALA 18 n3:3) have bene cial effects on the brain. Omega-3 fatty acids enhance uidity of cell membrane by incorporating long-chain fatty acids into phospholipids of cell membrane. The increased uidity of the cell membrane holds the extent of receptor expression on the cell surface and their downstream signaling [6]. DHA and EPA are either taken up by dietary lipids or synthesized by α-Linolenic acid in the body. DHA and EPA are main regulators of lipid mediators that drive the resolution phase by suppressing the in ammatory response and helps to restore the homeostasis [6,7].
Microtubule-associated protein-Tau, which forms neuro brillary tangles (NFTs) in the neurons is considered one of the major consequences of AD along with extracellular amyloid-β plaques [8]. Glial activation acts as a major cause to drive the pathology-associated with AD. The establishment of Tau as a factor of neurotoxicity and neuroin ammation is still a matter of debate, but recently accepted the concept of Tau as a prion-like protein that supports this hypothesis [9,10]. The spreading of Tau and its ability to cause template-dependent deformation in the healthy neuron can be targeted [11,12]. Omega-3 fatty acids are found to implement the suppression of neuroin ammation and triggers polarization of microglia [13]. Omega-3 fatty acids elevate the resolution phase and mediate tissue repair, healing, and clearing of debris and maintain homeostasis by microglia. Enhanced phagocytic nature of microglial cells due to exposure of Omega-3 fatty acids could act as a therapeutic strategy to minimize the spreading of Tau in Tauopathies to reduce the propagation of disease [11,14]. In the process of phagocytosis Rab proteins especially Rab5, 7 plays an important role in intracellular vesicle tra cking and mediates the endocytic pathways. Rab5 is associated with early endosomal marker whereas; Rab7 identi es late endosomes in the phagocytosis process. Hence to study the internalization and the subsequent degradation, Rab 5, Rab 7 and their transition can help to provide the insights of the process [15,16]. The nal step of phagocytosis involves fusion of late endosome with lysosome to form phagolysosome as a microcidal compartment. The fusion of late-endosome involves LAMP-lysosome associated membrane proteins and other lumial proteases [17,18]. The high acidic pH (pH-4.5) along with other hydrolytic enzymes (cathepsins, proteases, lysozymes) ensures elimination and degradation of microorganisms [19,20]. Microglia activation leads to polarization and migrates in a particular direction, depending upon the directional clues. Cytokine and chemokine are response to play an important role in migration and polarization of microglia such as CX3CL1-CX3CR1, IL-4, CCR5, CCR3, and CCR1 mediated signaling [21]. For the 2-dimensional migratory motion of microglia, the cells have been supported by protruding fan-shaped actin network (lamellipodia) and on leading end and F-actin-rich thin extensions ( lopodia) on rear end [22]. In this migratory state microglia also shows presence of podosmes which are actin rich structures that mediates adhesion to substratum, migration and ECM degradation for invasion [23]. In phagocytosis after detecting target, the signaling cascades reulating actin cytoskeleton intitates. The membrane protrusion around targets, membrane ru ing to increase surface area intiates major actin remodeling [24]. The polarized state of microglia is maintained by the cytoskeletal network where actin provides directional sensing and microtubule dynamics for the mechanical strength to move cell forward [25]. The coordinated polymerization of actin laments provides a protrusive force for the cell to move forward. ARP2/3 complex along with other actin-binding proteins plays important role in nucleating thr branching of actin lament. The membrane protrusions and ru ing is initiated with ARP2/3 signaling cascade [26]. In the process of membrane ru ing, phagocytic cup formation along with actin network Iba-1(ionized calcium adapted molecule-1) protein of microglia plays an important role. Iba-1 is also reported to have a key role in the function of activated microglia [27,28].
In this study we have examined α-Linolenic acid as a precursor for the DHA and EPA, to increase the phagocytic capacity of microglia by enhancing the phagocytosis of extracellular propagating Tau; eventually reducing their spreading. The downstream degradation of internalized Tau was assessed by endosomal transition with Rab5, Rab7 endosomal markers and LAMP-2A as a lysosomal marker to track degradation of internalized Tau. The migration of microglia has been studied as one of the key properties of alternative anti-in ammatory phenotype of microglia. The migration pro le of microglia was studied with wound scratch assay, reorientation of microtubule-organizing center along with nuclear centrosomal (NC) axis and actin rearrangement as morphological hallmarks of activated microglia. We have also focused on the role of cytoskeletal protein actin in microglial migration, its role in phagocytosis and membrane ru ing along with the microglial activated protein Iba-1. The excessive branching of actin cytoskeleton in phagocytosis was studies with the help of ARP2/3 complex which intiates branching of parental actin laments. We have analyzed the enhancement of phagocytosis after ALA exposure by actin mediated membrane ru ing and the degradation was con rmed with the localization with Rab 5,7 and LAMP-2A. The ALA also improves the migration pro le of microglia that aids to the phagocytosis.

Protein expression and puri cation
Full-length wild type Tau protein (hTau40 wt ) was expressed in BL21* cells with 100 µg/ml of ampicillin antibiotic selection and puri ed with two-step chromatography methods, cation-exchange chromatography and size-exclusion chromatography (Gorantla, MiMB, 2018). Cells were grown at 37°C, scaled up and harvested after induction with 0.5 mM IPTG for 4 hours. Cells were subjected to homogenization to produce cell lysate at 15000-psi pressure. The cell lysate was subjected to 90°C heating in presence of 0.5 M NaCl and 5mM DTT for 20 min to denature structured proteins. The supernatant was collected after centrifugation at 40000 rpm for 45 minutes then put through dialysis overnight in 20 mM MES buffer supplemented with 50 mM NaCl. The supernatant was obtained again after centrifugation at 40000 rpm for 45 min passed through cation-exchange chromatography.
Sepharose fast-ow column was used for chromatography, using 20 mM MES buffer and 50 mM NaCl (Buffer A). Elution was done with 20 mM MES buffer and 1 M NaCl (Buffer B). A fractions containing Tau proteins were collected after cation exchange chromatography, it was then concentrated and subjected to size-exclusion chromatography. Size-exclusion chromatography was carried out in the Superdex 75 Hiload 16/600 column in 1X PBS supplemented with 2 mM DTT. A fractions containing Tau were collected, pooled, concentrated and the concentration of protein was determined with BCA (Bicinchoninic acid assay) assay.

Aggregation assay
Tau protein undergoes aggregation in presence of poly-anionic reagent such as heparin, arachidonic acid, etc., it is observed by the transition of random coiled structure to the β-sheet formation in protein [29]. In this study Tau aggregation was induced by heparin (MW-17500 Da) in the ratio of 1:4 heparin to Tau along with other additives 20 mM BES buffer, 25 mM NaCl, 1 mM DTT, 0.01% NaN 3, PIC. The effect of ALA on Tau aggregation was measured by Thio avin S (ThS) uorescence assay. ThS is a homogeneous mixture of methylation product of dehydrothiotoluidine in sulfonic acid, which can bind to β-sheet structure. Aggregation kinetics of Tau was studied with 2 µM of Tau and ThS in 1:4 ratios. The excitation wavelength for ThS is 440 nm and the emission wavelength is 521 nm, further analysis of data was done using Sigmaplot 10.0.

Transmission electron microscopy
Morphological analysis of Tau brils and ALA vesicles were studied by transmission electron microscopy (TEM). 2 µM Tau sample was incubated on 400 mesh, carbon-coated copper grid and stained with 2% uranyl acetate. For ALA vesicles working concentration of 40µM was taken for grid preparation. The images were taken with TECNAI T20 120 KV.

CD spectroscopy
Conformational changes in Tau from random coiled structure to β-sheet conformation on aggregation of protein was studied using CD spectroscopy, the spectra was collected as previously mentioned in UV region [30]. The measurement was done in Jasco J-815 spectrometer, cuvette path length was 1 mm , measurement was done in range of 250 to 190 nm, and with a data pitch of 1.0 nm, and scanning speed was kept 100 nm/min . For measurement 3 μM sample concentration was taken in phosphate buffer pH 6.8 all the spectra were taken at 25°C.
Cell culture N9 (microglia) cells were grown in RPMI media in T25 ask or 60mm dish supplemented with 10% heatinactivated serum, 1% penicillin-streptomycin antibiotic solution and glutamine for maintain the culture. Cells were passaged on 90% con uence using 0.25% trypsin-EDTA solution after washing with PBS. For western blotting experiment cells were seeded in 6 well plate. For α-Linolenic acid preparation, previously published protocol was followed [14]. Brie y, ALA was dissolved in 100% molecular biology grade ethanol and solubilized at 50°C in the stock concentration of 20 mM. The fatty acid solution was prepared freshly before every experiment. According to the previous studies 40 μM was the working concentration of ALA for carrying further experiments. The nal concentration of ethanol in cell culture media was maintained below 0.5%.

Tau internalization
To study the effect of ALA on microglial phagocytosis, N9 cels were treated with extracellular 1 µM monomer and aggregates along with 40 µM ALA. For the immuno uorescence experiemnt (25,000 cells/well),N9 cells were seeded on 12 mm glass coverslip in 24 well-plate. The cells were then incubated with 1 µM Tau monomer and aggregates along with 40 µM ALA for 24 hours. To compare the internalization ability the controls of 1 μM Tau monomer and aggregates alone for the comparative studies with ALA treatment, 40 μM ALA alone to check morphological changes in N9 cells and cell control (without treatment) were kept. The coverslips were then xed and stained for immuno uorescence analysis with antibodies Tau T-46 (1:400) and Iba-1 (1:500). The mounting of coverslips were done with mounting media (80% glycerol in 1X PBS). The intracellular intensity of microglia were calculated from uorescence images to quantify the internalization. The representation of intracellular intensity was done as an intensity/μm area.
To study the degradation of intracellular Tau after phagocytosis we have targeted early and late endosomal markers and lysosme marker for nal degradation process. The treatment was done as previously mentioned, after 24 hours of exposure cells were xed and stained for immuno uorescence analysis. The analysis of the process of degradation was done co-localization of internalized Tau  were studied as intensity/ μm area to understand the expression of proteins on ALA exposure. The colocalization of internalized Tau was studied with 3-D and orthogonal analysis of immuno uorescence images.

Wound scratch assay
To study the migration of microglia wound-scatch assay was performed. For the assay, (5,00,000 cells/well) N9 cells were seeded in a 6-well plate and maintained in RPMI media for 24 hours till the con uency reached to 80%. Scratch was created with sterile 200 μl pipette tip, followed by treatment with groups as mentioned previously. Cells were incubated further for 24 hours to study the migration of N9 cells into the wound. A number of cells migrated into the wound were calculated for 5 different areas of culture and the average was calculated to quantify the migration.

MTOC reorientation analysis
To study immuno uorecence experiment (25,000 cells/well) N9 cells were seeded on 12 mm coverslips in 24-well plate. The desired treatment of Tau monomer, aggregates and ALA was given to cells for 24 hours and xed for immuno uorescence staining. The MTOC positions were analyzed by β-tubulin Immuno uorescence analysis N9 cells were passaged in RPMI media supplemented with 10% FBS and 1% penicillin-streptomycin. For immuno uorescence studies, 25,000 cells were seeded on 12 mm coverslip (Bluestar) in 24 well plate. Supplemented with 0.5% serum-deprived RPMI media for the desired treatment. The treatment was given for 24 hours. Cells were then xed with chilled absolute distilled methanol for 20 minutes at -20°C then washed with 1X PBS thrice. Permeabilisation was carried out using 0.2% Triton X-100 for 15 Minutes, washed three times with 1X PBS followed by blocking with 2% serum in 1X PBS for 1 hour at room temperature. Primary antibody treatment was given to cells overnight at 4°C in 2% serum in 1X PBS in a moist chamber. The next day, cells were washed with PBS thrice. Then incubated in the desired secondary antibody in 2% serum at 37°C for 1 hour. Further cells were washed with 1X PBS 3 times and counterstained with DAPI (300 nM). Mounting of coverslip was done in mounting media (80% glycerol). Images were observed under a 63x oil immersion lens in Axio observer 7.0 Apotome 2.0 Zeiss microscope.

Confocal-Super-resolution microscopy analysis
To study the actin structures associated with migration, phagocytosis in presence of ALA Zeiss LSM 980 with Airy scan 2 in super-resolution mode was used. The immuno uorescence staining for the previously described conditions were carried out with β-Actin (1:500) and Iba-1 (1:500) proteins to study the microglia activation and actin structures. The super-resolution mode helped to resolved and understand the minute cell structures such as lamellipodia, lopodia, membrane ru ing and polarization state of microglia. The image processing was carried out with Zeiss ZEN 2.3 software.

Western blot
For detection of protein levels in cells (3,00,000 cells/well) N9 Cells were seeded in 6 well plate and after the desired treatment for 24 hours. Treatment exposure followed by washing with 1X PBS. Cell lysis was carried out using radioimmunoprecipitation (RIPA) assay buffer containing protease inhibitors for 20 min at 4°C. The cell lysate was centrifuged at 12000 rpm for 20 minutes. Protein concentration was checked by using Bradford's assay and equal amount of 75 µg total proteins for all the treatment groups were loaded on polyacrylamide gel electrophoresis of range 4-20% and the gel is electrophoretically transferred to polyvinylidene di uoride membrane and kept for primary antibody Rab5, Rab7, LAMP-2A, Iba-1 (1:1000)binding for overnight at 4°C. After the incubation washing of blot was carried out three times with 1X PBST (0.1% Tween-20). The secondary antibody were incubated for 1 hour at RT. Then the membrane was developed using chemiluminescence detection system. The relative quanti cation of protein was carried out with loading control β-Actin (1:5000) in each treatment group.

Statistical analysis
All the experiments have performed 3 times. The data is analyzed using SigmaPlot 10.0 and the statistical signi cance was calculated by student's t-test (ns-non-signi cant, * indicates P≤0.05, ** indicates P≤ 0.01, *** indicates P≤0.001). The quanti cation of levels of intracellular proteins in immuno uorescence experiments was carried out by measuring the absolute intensity of protein and the corresponding area of microglia with Zeiss ZEN 2.3 software for image processing.

Results
Tau aggregation in the presence of α-Linolenic acid (ALA) α-Linolenic acid (ALA) is an essential omega-3 fatty acid, which is a precursor of Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA) [31]. The role of Omega-3 fatty acids in cardiovascular diseases is well-studied but its role in neuroprotection is needs to be studied [32,33]. In this study, we aim to understand the role of ALA on the function of microglia and its effect on extracellular Tau in Alzheimer's disease. Tau, a natively unfolded protein stabilizes microtubules in neuron and other CNS cells. The longest isoform of Tau has 441 amino acids with two inserts, proline-rich domain and four imperfect repeat regions (Fig 1A). The positive charge of the repeat region of Tau facilitates the binding of anionic free fatty acids [34]. The bene cial effect of ALA as a potent anti-in ammatory agent and a precursor of other omega-3 fatty acids DHA, EPA provides therapeutic strategy in AD. In this study, we explored the neuroprotective anti-in ammatory role of ALA on exposure to microglia and its effect on phagocytosis of extracellular Tau (Fig. 1B). ALA is a polyunsaturated omega-3 fatty acid (18:3) having three double bonds in its structure and it is a precursor of DHA and EPA. For the preparation of ALA was dissolved in 100% ethanol and then solubilized at 50 degrees, they produce vesicles-like structure, which has been shown with transmission microscopy (TEM) images ( Fig. 1 C, D). Due to high hydrophilic nature, high net positive charge and lack of hydrophobic residues accounts for the natively unfolded nature of Tau. This exible structure of Tau due to unfolded nature aids for microtubule-binding and stability. The highly soluble form of Tau can be induced to aggregate in the presence of polyanionic agents such as heparin, which neutralize net positive charge in vitro. Free fatty acids such as arachidonic acid induce spontaneous self-assembly of Tau protein to form aggregates in dose-dependent manner [34]. In vitro aggregation of hTau40 Tau in presence of heparin was con rmed with ThS uorescence for time period of 120 hours, SDS PAGE analysis and TEM for visualization of aggregated Tau brils (Fig 1 E, F and G). The con rmation of for the aggregates formation in presence of Tau was carried out with the circular dichroism spectroscopy (CD). The native random coil nature of Tau changes to β-sheet conformation on formation of aggregates which can be detect with the shift in absorbance in CD data ( Fig. 1 H).

Internalization of extracellular Tau in presence of ALA in microglia
Long chain polyunsaturated Omega-3 fatty acids are integral part of membrane phospholipid [35]. Incorporation of long chain fatty acid in microglia cell membrane increases the uidity of membrane and hence enhances anti-in ammatory phenotype [5]. The intrinsic phagocytosis property of microglia enhances as exposed to long chain polyunsaturated fatty acids (PUFAs) since in case of microglia PUFAs exerts anti-in ammation properties and suppresses pro-in ammatory properties. We expose microglia cells (N9) with 40 µM ALA for 24 hours and checked for the internalization of extracellular Tau monomer and aggregates ( Fig. 2A). N9 cells were treated with 40 µM ALA as a control, 1 µM Tau monomer, aggregates and their respective treatment with ALA. Immuno uorescence staining was performed to study the internalization of Tau (red) in Iba-1 (green) positive microglia cells since Iba-1 is marker for microglia and involves in membrane-ru ing and phagocytosis [27]. Phagocytosis of extracellular Tau has increased in both Tau monomer and aggregates in presence of ALA (Fig. 2B). The internalization of extracellular aggregates observed to be increased as compared to extracellular monomer, thus indicates that ALA enhances phagocytosis ability of N9 cells. 3-D view of immuno uorescence images indicates the internalized Tau. The insight representation of single cell of 3-D immuno uorescence images indicated the internalized Tau shown with the white arrow marks. . Intracellular intensity of internalized Tau was quanti ed in uorescence images, signi cant increase in internalization quanti ed as an intensity/µm 2 area was observed in cells treated with ALA as compared to control (no treatment) cells (P<0.001) (Fig. 2C). ALA exposure increased the intrinsic phagocytic capacity of microglia in monomer and aggregates by 68 and 75% with P< 0.05, 0.01 respectively (Fig 2 D). Supplementary gure 1A(S1 A) incorporates the individual panel for all the lters given in the merge images for better understanding of morphology and immuno uorescence staining as Tau (red), Iba-1 (green), DAPI (blue) and DIC (Differential interference contrast). Supplementary gure 1B ( g. S1 B) shows the orthogonal view indicating x and y axis for the better understanding of localization of Tau.
Effect of ALA on endosomal tra cking of internalized Tau and its degradation pathway The phagosomes after internalization is subjected to to lysosome-mediated degradation via endosomal maturation process in phagocytosis [36,37] The phagosomes after internalization fuses with endosomal compartments-mediated by endosomal markers Rab5, Rab7, where maturation of endosome occurs and nally it fuses with lysosomes for degradation of internalized microorganisms in immune cells. We expect the colocalization of internalized Tau with endosome compartment since the endosomal maturation is followed to degradation pathway. We studied downstream early and late endosomal markers Rab5, Rab7 and LAMP-2A respectively for the colocalization with internalized Tau (Fig. 3A). The immuno uorescence images of Tau and endosomal, lysosomal markers after 24 hours of exposure with extracellular Tau monomer, aggregates and ALA showed the levels of endosomal and lysosomal markers in the cell and the colocalization with internalized Tau represented with white arrow marks in images. (Fig. 3B, 4A). The zoomed images shows the area of colocalization of internalized Tau with Rab5 and Rab7 in microglia (Fig. 3B, 4A). The intracellular intensity per unit area of Rab5 and Rab7 was estimated. The expression of Rab5 was found to increased signi cantly in case of aggregates with ALA treated cells (p<0.001), whereas Rab7 showed increased levels of protein with ALA treatment in both monomer and aggregates treated cells as compare to other control groups (P<0.001) (Fig. 3C, 4B). Expression of Rab5 and 7 was also checked with western blot, aggregates treated groups found to increase both Rab5 and 7protein levels (Fig. 3D, 4C). The increased levels of Rab 5 and 7 in case of ALA treated cells in both monomer and aggregates shows cells are undergoing more of phagocytosis and the internalizing Tau is channelizing towards degradation pathway. The mature late-endosome containg target then fuses with the lysosome. The high pH in lysosome compartment and other hyderolytic enzymes containg proteases, lipases, lysozymes, cathepsins induce degradation of internalized targets [38].
Effect of ALA on lysosome-mediated degradation of internalized Tau Further to understand lysosome-mediated degradation, N9 cells were treated with extracellular Tau monoer, aggregates and ALA and the cells were stained for immuno uorescence analysis with Tau (red) and LAMP-2A(green) post treatment. The levels of LAMP-2A and its colocalization internalized Tau indicated through immuno uorescence analysis (Fig. 5A). The 3-D representation of immuno uorescence images indicated colocalization of Tau and LAMP-2A, zoom image panel indicates colocalization spotted with white arrow marks (Fig. 5A). The intracellular intensity of LAMP-2A was calculated, which does not show changes in intensity (Fig. 5B). the levels of LAMP-2A by western blot was analysed, the ALA treated N9 cells showed increase in levels in both Tau monomer and aggregates exposed cells (Fig. 5C, D). For the better understanding of internalized Tau orthogonal view of immuno uorescence images was provided, the x and y axis of the images shows localization of Tau in cell (Fig. S2).

ALA enhances migration of microglia
Omega-3 fatty acid induces alternative anti-in ammatory phenotype of microglia; anti-in ammatory phenotype depicts increased migration of microglia. The alternative activation observed with IL-4 treatment to microglia induces excessive migration [22]. We hypothesize that exposure of ALA will modulate the cell membrane, and increased its uidity, which showed increased the migration of N9 cells (Fig. 6A). In this study we checked for migration ability of microglia by wound scratch assay. Timedependent migration of microglia was studied in presence of ALA for 0, 6, 12, and 24 hours (Fig. 6B). We quanti ed the number of cells into the wound after every time point by optical microscopy images. ALA found to increase the migration of microglia as compared to cell control. At 24 hours' time point monomer showed higher migration rate than aggregates, however with their respective exposure with ALA enhanced the migration to greater extent. The migration pro le at 24 hours found to be highest in aggregates with ALA condition (Fig. 6C). These results suggest that ALA supports microglia to increase the migration, which is one of the key properties of anti-in ammatory phenotype of microglia.

ALA polarize nuclear-centrosomal axis in microglia
Increased migration is also associated with the repolarization of MTOC along the nucleus centrosome (NC) axis in the cell. In migratory cells the microtubule network reorient to migratory leading end for the forward motion and hence the MTOC positions are found predominately in the anterior region of nucleus. In case of highly migratory cells the repolarization of MTOC are observed to be present on different positions such as anterior, posterior and lateral positions to nucleus. The microtubule network found to be dense at the nucleus, spreaded towards lamellum and bundled down to uropod at rear end (Fig 7A). In case of ALA treated cells in both monomers and aggregates showed MTOC orientation in all the different positions, whereas in other treatment groups anterior position of MTOC prevails (Fig 7B). In case of aggregates with ALA treatment the percentage occurrence of different positions of MTOC around the nucleus is near to equal (Fig 7B). The picture representation suggests that ALA treatment to microglia reorient the MTOC to different positions anterior, posterior and lateral to nucleus unlike unipolar control cells (Fig 7C). All the panel of immuno uorescence images with DIC is shown indicating exact positions of MTOC (Fig. S3).

Effect of ALA on actin dynamics and microglial activation
Actin cytoskeleton involves in membrane-remodeling, downstream signal transduction and provides mechanical framework for phagosomes after internalization. Actin cytoskeleton furnishes platform for receptors and other signaling cascades. Co-ordinated cycles of actin polymerization and depolymerization occurs at the phagosomes for its internalization and maturation [39]. Membraneru ing along with the Iba-1 and phagocytic cup formation is being assisted by actin cytoskeleton. Iba-1 enhances membrane-ru ing and crosslinking of actin, which up regulated the internalization events [27]. In phagocytosis after interaction of target with receptors actin cytoskeleton changes. The neccessay remodeling with the membrane associated actin and introduction of more membrane protrusion enhance the phagocytosis [38]. We studied the role of actin and Iba-1 in phagocytosis in N9 cells on ALA exposure by immuno uorescence staining. In activated migratory microglia the polymerization of actin at leading ends is signi es with the presence of fan shaped lamellipodia and thin lamentous lopodia-like structure whereas long extension is observed at rear end (Fig. 8A). For the migratory cells such as immune cells the front end protrusion of lammelipodia formed by the forces of actin polymerization indicates the migratory state of microglia since they sence the extracellular Tau species. Whereas the nger-like protrusion called as lopodia would cause the slow migration in absence of lammelipodia [40]. The immuno uorescence staining indicates enhanced colocalization between Iba-1 and actin at leading end of activates microglia cells in ALA treated cells (Fig. 8A). These results indicates involvement of actin and Iba-1 in phagocytic cup formation and membrane-ru ing [27]. Intracellular intensity of Iba-1 was quanti ed and it was found to increase signi cantly in both monomer and aggregates in presence of ALA (P<0.001) (Fig. 8B). Iba-1 levels were also quanti ed by western blot, with respect to cell control (without treatment) Iba-1 levels have not been increased signi cantly. The western blot quanti cation also suggests that no signi cant changes were observed as compared to cell control ( Fig. 8C and D). The microscopic images showing all the panel is represented in supplementary gure (Fig. S4) In this study we observe that, after exposure of ALA and Tau species to microglia triggers the microglial activation and increases the phagocytosis. In this process actin and Iba-1 plays major role in microglial migration and activation, which assists the phagocytosis process ( Fig 8E). After detection of target immune cells increase the membrane protrusion and membrane ru ing to increase the area in contact with the target to initiates the internalization process. Membrane ru ing enhanced by the actin polymerization and involvement of Iba-1 was shown with the super-resolution confocal microscopy images. The presence of ALA found to enhance the membrane ru ing in both monomer and aggregates trated cells indicating increased phagocytosis (Fig. 9). The overall view of the zoom images with separate panel is shown in supplementary gure (Fig. S5) ALA enhace actin structure and dynamics for phagocytosis Immune cells are charachterized with excessive motility to survey environment to nd and destroy target pathogens. Actin polymerization provide necessary protrusive for the cells to move forward, which can be observed with the lammelipodia and lopodia to sense distant targets [26]. We observed the role of actin structures lamellipodia and lopodia in migration and phagocytosis. The immuno uorescence staining of actin in microglia cells helped to study the minute structures (Fig. 10A). The percentage of lamellipodia and lopodia positive cells have increased with the ALA exposure in both monomer and aggregates treated cells (P<0.001) (Fig. 10B, C). The number of lopodia per cell has increased signi cantly with ALA exposure in monomer and aggregates treatment and the numbers of lopodia per cell is maximum in monomer with ALA treatment (P<0.001) (Fig. 10D). Actin plays major role in phagocytosis; migration and this can be observed with enhanced actin structures lamellipodia, lopodia (Fig. 10E). ALA enhace actin branching network via ARP2/3 complex.
In case of lamellipodia formation and protrusions to move forward the continuous branching of mother actin laments is necessary. The force required to move the plasma membrane forward is provided by actin lament polymerization one of the actin bingding protein ARP2/3 complex enhance the branching process. We have studied the presence of ARP2/3 complex in microglia after treatment of Tau monomer, aggregates and ALA. The 3D representation of images showing ARP2/3 (red), phalloidin (green) and DAPI indicates the presence of ARP2/3 at the leading ends of cells inducing more branching of actin laments for the protrusive force (Fig. 11A). The 2D representation of the immuno uorescence images with all the panel are shown for the better understanding (Fig. S6).

Discussion
The extracellular Tau species after recognition by immune cells induces immune response. Damping of response given by immune cells could be achieved with omega-3 fatty acids. Dietary omega-3 fatty acids involves microglia into anti-in ammatory immune response, which would enhance clearance of extracellular pathological Tau species. The insoluble pathological aggregated form of Tau prepared in vitro was con rmed by SDS PAGE, TEM, ThS uorescence and CD analysis.In present study, N9 microglia cells were exposed to ALA being an omega-3 fatty acid and observed for its bene cial effects. The antiin ammtory property of microglia was observed with the enhanced phagocytosis of extracellular Tau species. The effect of ALA on migration and actin dynamics has been studied on microglia cells as they assist the phagocytosis process. The enhanced phagocytosis in presence of ALA should also channel the internalized antigen towards lysosome-mediated degradation for desired clearance of extracellular antigens. The degradation of internalized Tau was denoted with the endosomal markers and their colocalization with internalized Tau. The reported results suggest the bene cial effects of ALA in brain.
In previous studies, it has been proven the seeding nature of Tau as it causes template-dependent aggregation on uptake by healthy neurons [11]. The aggregated extracellular Tau species secreted by various mechanisms have tendency to propagate the disease [41][42][43]. The use of other omega-3 fatty acids DHA, EPA has been studied for the uptake of extracellular Aβ-plaques and their clearance [14,44]. To study the bene cial role of ALA for the uptake of extracellular Tau, we had incubated Tau and ALA with N9 microglia cells for 24 hours. The increased phagocytosis of extracellular Tau has been observed with ALA treatment conditions [45]. The omega-3 fatty acids exerts ant-in ammatory properties to cells due to their ability to produce SPM (specialized pro-resolving molecules), which on attending certain concentration shows their effects [46]. DHA and EPA are the main omega-3 fatty acids increase the microglial activation and act as a main precursor of SPM, they also found to activate PPAR-γ to mediate anti-in ammatory response [13,47]. The clearance of extracellular Targeting species in case of AD could be objecti ed with dietary omega-3 fatty acids. Alzheimer's disease is also being characterized by the endo-lysosomal abnormalities and accumulation of Rab5 positive enlarged endosomes followed by detectable Aβ-plaques [16,48]. The accumulation also impairs the fusion of autophagosomes with lateendosomes and lysosomal degradation. The transition of phagosomes Rab5 to Rab7 is one of the important events, which speci es the degradation of internalized antigens [49]. In our results the levels of Rab5 and Rab7 found to increase in case of ALA treatment and there is also signi cant colocalization of internalized Tau with Rab5 and 7 was observed indicating that internalized Tau is undergoing degradation pathway instead of accumulating inside the cell. The nal step of phagosome maturation ends up with the fusion with lysosome. The formation of Phagolysosome regulated by Lysosomal associated membrane proteins. Double knock out of LAMP-2A found to impairs the maturation of phagosome by halting the process prior to acquisition of Rab7 and affects lysosome desity in cell [38,50]. LAMP-2A might be better target to study the lysosomal degradation of internalized Tau. We have observed from the results that ALA enhances LAMP-2A levels in cell and its colocalization with internalized Tau indicates the active phagocytosis.
Activation stage of microglia on stimulation is observed with increased migration, phagocytosis, proliferation and cell shape changes which are assisted by actin cytoskeleton [51]. The cell migration pro le for N9 cells treated with different groups was studied by wound scratch assay. Excessive migration was seen with the ALA treatment as compared to control groups. The protrusive and contractile force needed for the migration is supported by actin rearrangements. Hence excessive membrane ru es, lammellum, lopodia justi es the migration pattern. The polarization of microglia is supported by both actin and microtubule cytoskeleton. In migratory polarized microglia well assisted reorientation of NC axis is observed, in many migratory cells anterior NC axis is observed where MTOC, endoplasmic reticulum and Golgi apparatus are in front of nucleus stabilized front end. However in highly migratory ALA treated cells lacks the preference of NC axis and other positions such as posterior, lateral were observed [22]. This is also observed in highly migratory immune cells such as neutrophils and Tlymphocytes. The assembly of actin laments into membrane ru es, lopodia, and lamellipodia supports these processes.
Coordinated polymerization of branched network of actin laments is a key phenomenon to generated pushing force for the migratory cells. Extensive motility of immune cells is necessary to locate a target to destroy. The identi cation of targets via receptors intiated the signaling cascade involving actin polymerization to produce membrane protrusions around the target to engulf. After engulfment the target is enclosed in endocytic vesicle and pinched out from cell membrane. In this process actin remodeling plays a major role to induce a changes in plasma membrane, membrane ru ing and protrusions that speed up phagocytosis [26,52]. Membrane ru ing shows involvement of Iba-1 alond with the actin, Iba-1 mutants hampers membrane ru ing and phagocytic cup formation [27]. The role of Iba-1 as an actin binding protein has been studied. The activated microglia after 24 hours of treatment of ALA and Tau showed high colocalization between actin and Iba-1 and enhanced membrane ru ing, lamellipodia and lopodia. The ALA found to increased membrane ru es to the great extent, which was observed to be effective for increased phagocytosis. Previous studies have been reported the physical binding of Iba-1 to F-actin and their involvement in membrane ru ing supports the increased colocalization of Iba-1 and Actin in ALA treated cells. After CNS injury microglia has tendency to migrate towards site and migration is supported by rearrangement of actin cytoskeleton [39]. The actin structures and dynamics supports migration, where lamellipodia structure produce forward protrusive forces and thin protrusion of lopodia enspect distant targets. The ALA treated cells showed greater polarized morphology with presence of dense lammellum at leading ends and uropods at rear ends. The lammellum and lopodia extensions were high in ALA treated cells suggests the high directional migration and also supports protrusive and contractile migration. In the previous studies with the alternative activation of microglia by IL-4 treatment observed to have lammellum with more membrane ru es similar morphology has been observed with ALA treated cells. For the extensive branching required for the lamellipodium formation ARP2/3 complex plays important role. ARP2/3 introduce branching of mother actin laments to produce dense branching network of the actin. The density of ARP2/3 in migratory cells is higher at leading end to produce dense actin network and fade out in other cell body [26,52]. ALA found to enhace ARP2/3 at the leading ends since it enhaces lamellipodia formation. This depicts that ALA has tensdency to remodulate actin network (Fig. 12).  Availability of data and materials

Conclusions
All data generated or analyzed during this study are included in this published article and its supplementary information les.

Competing interests
The authors declare that they have no competing interests.

Additional information
Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional a liations.  Percentage increase in phagocytosis of hTau40 in microglia after ALA exposure to cells; percentage increase in aggregates to aggregates with ALA exposed groups and monomer to monomer with ALA groups calculated from the intracellular intensity of Tau amongst the groups, signi cance is P=0.02, 0.007 respectively.

Figure 3
Degradation of internalized Tau in microglia via endosome-lysosome pathway. A microglia cell were exposed to hTau40 monomer and aggregates in presence and absence of ALA and observed for the levels of Rab 5(green), and Tau (red) by uorescence microscopy. The degradation of internalized Tau was studied with the early endosomal marker and late endosomal markers. A) The internalized Tau follows the phagocytosis pathway and nally degrades via lysosome-mediated degradation. In the pathway the maturation of phagocytic vesicle take place that can be marked with the early endosomal marker Rab 5 and late endosomal marker Rab 7. We observe the colocalization of internalized Tau with endosomal markers to trace the degradation of internalized Tau   Migration analysis of microglia in presence of ALA. Increased migration microglia is a key property of anti-in ammatory phenotype. We desired to observe the effect of ALA on migration of microglia since omega-3 fatty acids enhance anti-in ammatory phenotype. A) ALA observed to enhance the phagocytosis of microglia that is also assisted by migration of microglia. The effect of ALA on migration under the in uence of hTau40 monomer and aggregates has been studied. B) The migration of microglia was studied by wound scratch assay. Migration of cells into the scratch was studied with different time intervals 0, 6, 12, 24 hours after the scratch observed with optical microscope. Scale bar is 100nm. C) In each treatment groups random ve elds were chosen and number of cells migrated into wound was counted. The comparison for each time point was carried out with its respective time point control (untreated) group; signi cance is P<0.001.
aggregates, ALA for 24 hours and observed for MTOC positioning with respect to nucleus in N9 cells.    Quanti cation of number of lopodia extensions present per cell per 10 elds of different groups, signi cance is p<0.001. E) Migration of microglia is important aspect in phagocytosis process; the ALA in presence absence of hTau40 monomer and aggregates enhances the process by promoting actin rich structure lamellipodia and lopodia, which helps the phagocytosis.

Figure 11
Enhancement of lamellipodia and ARP2/3 complex in presence of ALA in microglia. ARP2/3 complex plays important role in formation of lamellipodia and provide pushing force for cell to move forward.
After exposure hTAu40 monoer, aggregates and ALA the abundance of ARP2/3 in microglia was studied immuno uorescence images and plotted as intensity per sq. area. Signi cance is P< 0.05 for ALA treated groups.

Figure 12
Study of ALA as a anti-in ammatory dietary fatty acid to reduce Tau seeding. The picture reperesentation of experimental proceeding. ALA after exposure to N9 cells enhances extracellular Tau phagocytosis and its lysosome-mediated degradation. Thus reduces accumulation internalized Tau in microglia. ALA on the other hand enhances phagocytosis of microglia with increased migration, actin dynamics. The enhance actin dynamics, membrane ru ing supports the process of phagocytosis.

Supplementary Files
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