Chromogranin A deficiency attenuates tauopathy by altering epinephrine–alpha-adrenergic receptor signaling in PS19 mice

To quantify the difference in the level of seeding-competent Tau seeds between PS19 and CgA-KO/PS19 cortex, we employed the well-established Förster resonance energy transfer (FRET) assay in HEK 293 T cells expressing Tau-RD fused to CFP/YFP⁵⁰. Lower FRET signal was found in crude brain lysates of CgA-KO/PS19 mice compared to PS19 mice, indicating lower Tau seeding capacity (Fig. 2G, H). We further investigated CgA’s impact on Tau seeding and spreading by stereotaxically injecting synthetic K18 Tau fibrils into the dentate gyrus (DG) of three-month-old PS19 and CgA-KO/PS19 mice⁴⁷ (Fig. 2I). Fibril-induced Tau pathology spread to the CA3 region of the contralateral hippocampus was evident in PS19 mice 6 weeks post-injection, but minimal in CgA-KO/PS19 mice, suggesting that CgA promotes Tau spread (Fig. 2J, K, S-Fig. 3M, N). Our control experiment with PBS injection showed no MC1+ aggregates at this age (S-Fig. 3O–P). Furthermore, IHC staining with Myc and MC1 showed that K18 fibril induced Tau spreading pathology is from endogenous Tau aggregation rather than the injected K18 fibrils themselves (S-Fig. 3Q–R).

We performed ultrastructural analysis via transmission electron microscopy (TEM) and found amplified synapse numbers (marked by arrows) in the hippocampus of CgA-KO/PS19 mice compared to PS19 mice (S-Fig. 4A, B), consistent with increased PSD95 levels in the cortical and hippocampal lysates (Fig. 2C; S-Fig. 3F, G, J). Notably, PS19 mice had fewer synaptic vesicles (SVs) in their presynaptic neurons compared to WT and CgA-KO/PS19 mice (S-Fig. 4C, D). Collectively, these findings suggest that CgA promotes neurodegeneration in Tau-induced pathogenesis.

We next determined the lifespan of PS19 and CgA-KO/PS19 mice. The median lifespan of CgA-KO/PS19 mice extended to 13.22 months, compared to 10.12 months in PS19 mice (Fig. 3I), indicating a remarkable ~30% increase in lifespan upon CgA depletion. Notably, 57% (n = 8) of CgA-KO/PS19 mice exhibited relatively healthy lives until 12 months of age without discernible disease symptoms. Moreover, 23.6% (n = 5) of CgA-KO/PS19 mice survived until 14.5 months, in contrast with the PS19 mice, of which none had survived by this age. Collectively, these findings underscore the benefit of CgA depletion in the PS19 mouse model in improving their cognitive function and mitigating premature mortality.

Verification of protein levels for Adra1b and Adra2b in the cortex aligned with transcript levels. WB analysis demonstrated significantly higher Adra1b and lower Adra2b levels in the cortical extracts of PS19 mice compared to WT and CgA-KO/PS19 mice, while Adrb2 levels remained unchanged (Fig. 5H–J). IHC confirmed higher Adra1b protein levels in the DG and CA3 (mossy fibers) of PS19 mice compared to WT and CgA-KO/PS19 (Fig. 5K, L). Notably, the increase in the area fraction of Adra1b is negatively correlated with the hippocampal volume (Fig. 5M). Collectively, these findings suggest that upregulated Adra1 and compromised Adra2 signaling underlies CgA’s detrimental effect in Tauopathy brain.

To investigate the spatial relationship between CgA, Adra1b and Tau, we performed IHC with CgA, Adra1b, and MC1 antibodies (S-Fig. 8). Adra1b was predominantly localized in axons, where it was associated MC1+ misfolded Tau species (S-Fig. 8B–D). In contrast, CgA was observed as puncta in the soma of CA2 and CA3 regions of the hippocampus (S-Fig. 8C, D), where it partially colocalized with MC1+ misfolded Tau species (S-Fig. 8C, D, white arrows). Notably, there is minimal colocalization of CgA with Adra1b, suggesting that the effect of CgA on Adra1b is likely indirect.

Along with the altered expression of Adra1, we also observed a downregulation of phosphodiesterases (PDEs), including PDE1, PDE3, and PDE9, in PS19 mice compared to WT and CgA-KO/PS19 mice (S-Fig. 9A). Since PDEs degrades cAMP - a key second messenger in the adrenergic system, we measured cAMP levels in WT, PS19 and CgA-KO/PS19 mice. As anticipated, PS19 mice exhibited significantly higher cortical cAMP levels compared to WT and CgA-KO/PS19 mice (S-Fig. 9B). Consistently, we observed elevated cortical cAMP levels in AD Braak stage VI compared to Braak stage 0–II samples (S-Fig. 9C). These findings suggest that the upregulation of Adra1 and concurrent downregulation of Adra2 causes an imbalance signaling cascade with sustain elevated cAMP levels, potentially contributing to exacerbated Tau toxicity.

Since PR can cross the blood brain barrier (BBB)⁵⁵, we assessed its potential to reduce Tau pathology in PS19 mice. Six-month-old PS19 mice were treated with PR via weekly (two times a week) intraperitoneal injection for 12 weeks, followed by WB (Fig. 6E–G) and IHC analysis (Fig. 6H). Remarkably, PR treatment significantly reduced levels of pTau (Fig. 6E–G) and misfolded Tau species (Fig. 6H, S-Fig. 10J) in the hippocampus. Collectively, these results strongly suggest that Adra1 activation drives Tau pathology.

To test the effect of EPI on Tau pathology, we performed EPI treatment on WT OTSC expressing P301S human Tau and seeded with K18 fibrils. WB (Fig. 7F–H) and IHC (Fig. 7I, J) analysis showed that EPI significantly induced Tau phosphorylation (Fig. 7F–H) and aggregation (Fig. 7I, J). To further determine if EPI-induced Tau pathology is dependent on CgA, we treated CgA-KO OTSC with EPI and found that EPI failed to induce Tau phosphorylation (S-Fig. 11H–J) or Tau aggregate formation (S-Fig. 11K, L). These observations underscore the potential contribution of elevated EPI levels to the development of Tau pathology, which is regulated by CgA.

To establish the CgA-EPI-Adra1 signaling axis in regulation of Tau pathogenesis, we previously treated CgA-KO OTSC with EPI (S-Fig. 11H–L) and found no significant increase in Tau pathogenic burden. To further confirm CgA-EPI-Adra1 signaling axis in regulation of Tau pathogenesis, we performed co-treatment of EPI and PR in OTSC (Fig. 7K, L). Remarkably, EPI + PR co-treatment significantly reduced EPI-induced increase in Tau aggregation (Fig. 7L, M) and Tau phosphorylation (S-Fig. 11M–O), indicating that EPI increases Tau pathology via activation of Adra1 signaling.

All studies performed on animals were approved by the UCSD and Veteran Affairs San Diego Institutional Animal Care and Use Committee (IACUC) and conform to relevant National Institutes of Health guidelines. Chromogranin A knockout (CgA-KO) mice on a mixed background (50% 129/SvJ; 50% C57BL/6) were backcrossed to C57BL/6 J mice for 7 generations to get CgA-KO mice in C57BL/6 background²². Cre-loxP gene-targeting strategy was used to generate congenital and whole body CgA-KO mice. CgA is a unique pro-protein having counter-regulatory domains such as PST and CST. The above CgA-KO mice were backcrossed to B6C3F1/J mice for 4 generations to generate CgA-KO mice in B6C3F1/J background (Mahata Lab). These mice were crossed with PS19 (Jackson Lab) heterozygote mice (in B6C3F1/J background) to generate CgA-KO/PS19 mice. Animals were kept in a 12-hour light/12-hour dark cycle. Mice have access to food and water ad libitum. Mice were fed a regular chow diet (NCD: 14% calorie from fat; LabDiet 5P00).

Mice were euthanized using inhalational isoflurane in compliance with institutional IACUC protocols and the AVMA Guidelines for the Euthanasia of Animals (2020). Animals were placed in an induction chamber pre-filled with 3–5% isoflurane in oxygen (flow rate: 1–2 L/min) and continuously monitored until loss of the righting reflex and absence of response to toe pinch, confirming a surgical plane of anesthesia. Under deep anesthesia, tissues were collected, and euthanasia was completed by exsanguination. All animal procedures were approved by the Institutional Animal Care and Use Committee and conducted in accordance with the ARRIVE guidelines.

Mice were ear-tagged, and tail-snips were collected. Genomic DNA from tail-snips were isolated using the Accustart Genotyping kit and amplified by PCR with Accustart Geltrack PCR supermix. Primers used for PS19 genotyping are Forward (WT and mutant): 5′ TTG AAG TTG GGT TAT CAA TTT GG 3′, Reverse (WT): 5′ TTC TTG GAA CAC AAA CCA TTT C 3′, Reverse (Mutant): 5′ AAA TTC CTC AGC AAC TGT GGT 3′. Primers used for Chga genotyping are Forward: 5′GTA GCA TGG CCA CTA CCC AG 3′ and Reverse: 5′ ATC CTT CAG AGC CCC TCC TT 3′.

Organotypic hippocampal slice cultures (OTSC) were performed with hippocampal slices obtained from postnatal day 7-8 pups of WT or CgA-KO mice as described previously (Croft, Noble, 2018). Pups were culled by decapitation, and the hippocampus was quickly dissected in oxygenated artificial cerebrospinal fluid (ACSF). Approximately 18-24 400 μm thick slices were cut using a tissue chopper, and these were cultured in Millicell culture inserts (Millipore) in 6 well plates (6-8 slices per insert). Culture media was changed every 2-3 days, and the slices were harvested after 21 or 28 days. The composition of ACSF was 125 mM NaCl, 2.4 mM KCl, 1.2 mM NaH₂PO₄, 1 mM CaCl₂, 2 mM MgCl₂, 25 mM NaHCO₃, and 25 mM Glucose. The composition of OTSC culture media was 25 mM HEPES, 133 mM NaCl, 31 mM NaHCO₃, 45 mM D-Glucose, 5 mM KCl, 3.3 mM MgSO₄, 4.5 mM CaCl₂, 1 mM Na₂HPO₄, 0.5 mM Ascorbic Acid, 1.25 ml Pen-Strep, 2.5 ml Glut-Max, 62.5 ml Heat Inactivated Horse Serum and 82.5μl Insulin. To evaluate tau spreading, organotypic hippocampal slices were fixed in 4% paraformaldehyde (PFA) at room temperature for 4 hours. Following fixation, slices were permeabilized overnight in 1% Triton X-100 in PBS. Antigen retrieval was performed using citrate buffer (pH 6.0) under low pressure for 15 minutes. Slices were then blocked in 20% bovine serum albumin (BSA) with 0.4% Triton X-100 for 3 hours at room temperature. Slices were incubated with primary antibodies for 48 hours at 4°C. Afterward, they were washed six times for 10 minutes each in PBS, followed by incubation with fluorophore-conjugated secondary antibodies and DAPI for 2 hours at room temperature. Finally, slices were washed an additional six times for 10 minutes in PBS, mounted together with their membrane inserts onto glass slides, and coverslipped using Fluoromount-G.

Hippocampal slices were transduced with AAV viral vector expressing P301S human Tau (AAV2-smCBA-human_P301S_Tau-WPRE; 2×10¹⁰/mL) for 24 h. AAV was removed from culture media and K18 (preformed Tau fibrils; 1.5 μg/mL) were added before incubation for 72 h. Subsequently, fresh media without or with Phenylephrine:10 nM, Prazosin:0.5μM, or Epinephrine: 10 nM, was applied as indicated. Slices were harvested on day 21 for Western blot analysis and immunohistochemistry.

Mice were anesthetized with inhalation of 2% isoflurane for the duration of surgery and secured on a stereotaxic frame (Kopf Instruments). 3-month-old PS19 and CgA-KO/PS19 mice were injected stereotaxically at a rate of 0.5 μl/min, with 2 μL of 5 mg/ml K18 PFF into the CA1 region of the left hippocampus. The coordinates for injection were anterior-posterior − 2.5, medial-lateral + 2.0, dorsal-ventral −1.8. Mice were sacrificed 6 weeks after injection and transcardially perfused with PBS for immunohistochemistry analysis of Tau aggregation (MC1 + ) in the ipsilateral and contralateral sides.

The repeat domains (K18) of P301L mutant Tau with a Myc Tag was expressed in E. coli BL21 (DE3) strain. NaCl (500 mM) and betaine (10 mM), a small molecule chaperone, were added before induction with IPTG (200 μM) at 30 °C for 3.5 h. Cells were resuspended in lysis buffer (20 mM MES, pH 6.8, 1 mM EGTA, 0.2 mM MgCl₂, 1 mM PMSF, 5 mM DTT), and passed through a microfluidizer for lysis. After lysis, 5 M NaCl was added to a final concentration of 500 mM followed by heating at 90 °C for 20 min. was clarified by centrifugation and was dialyzed overnight (Buffer A: 50 mM NaCl, 1 mM MgCl₂, 0.1 mM PMSF, 2 mM DTT, 1 mM EGTA, 20 mM MES pH 6,8) to obtain the Tau-containing supernatant. Subsequently, cation exchange (HiTrap SP HP, 5 ml column from Cytiva) was performed to purify K18. The degradation products and other impurities were removed by running 15% elution buffer (50 mM NaCl, 1 mM MgCl₂, 0.1 mM PMSF, 2 mM DTT, 1 mM EGTA, 20 mM MES pH 6,8, 1 M NaCl). Subsequently, 15-60% gradient (NaCl gradient from 50 mM to 1 M) of NaCl was used to obtain pure K18, which was concentrated and further dialyzed against assay buffer (PBS pH 7.4, 1 mM DTT and 2 mM MgCl₂). A 10μM concentration of Tau was incubated in assay buffer for 36 h at 37 °C) upon adding 44μg/ml Heparin. The aggregates formed were snap frozen at −80 °C till further use.

Deeply anesthetized mice were flushed with a pre-warmed (37 °C) Hank’s balanced salt solution (HBSS), with calcium and magnesium by cardiac perfusion with freshly prepared pre-warmed (37 °C) fixative containing 2.5% glutaraldehyde and 2% paraformaldehyde in 0.15 M cacodylate buffer using a peristaltic pump. The hippocampus and cortex were dissected and postfixed in 1% OsO₄ in 0.1 M cacodylate buffer. The tissues were stained en bloc with 2-3% uranyl acetate and dehydrated in graded ethanol series. Sections of 50-60 nm thickness were cut, stained with 2% uranyl acetate and Sato’s lead stain. Grids were imaged with a JEOL JEM1400-plus TEM (JEOL, Peabody, MA) attached to a Gatan OneView digital camera with 4k x 4k resolution (Gatan, Pleasanton, CA).

Cortical and plasma catecholamines were measured upon separation by an Atlantis dC18 column (100 A, 3 µm, 3 mm×100 mm) on an ACQUITY UPLC H-Class System attached to an electrochemical detector (ECD model 2465; Waters Corp, Milford, MA) as described previously^18,79. The mobile phase (isocratic: 0.3 ml/min) contained 95:5 (vol/vol) of phosphate-citrate buffer and acetonitrile. An internal standard 3.4-dihydroxybenzylamine (DHBA 400 ng) was added to the cortical homogenate in 0.1 N HCl. The ECD was set at 500 pA for determination of brain catecholamines. For determination of CSF catecholamines, DHBA (2 ng) was added to 150 µl CSF and adsorbed with ~15 mg of activated aluminum oxide for 10 min in a rotating shaker. After washing with 1 ml water, adsorbed catecholamines were eluted with 100 µl of 0.1 N HCl. The ECD was set at 500 pA for determination of CSF catecholamines. The data were analyzed using Empower software (Waters Corp, Milford, MA). Catecholamine levels were assessed using the internal standard. Catecholamine levels were provided in nM (CSF) or ng/mg protein. For determination of plasma catecholamines, DHBA (2 ng) was added to 150 μl plasma and adsorbed with ~15 mg of activated aluminum oxide for 10 min in a rotating shaker. After washing with 1 ml water adsorbed catecholamines were eluted with 100 μl of 0.1 N HCl. The ECD was set at 500 pA for determination of plasma catecholamines. The data were analyzed using Empower software (Waters Corp, Milford, MA). Catecholamine levels were normalized with the recovery of the internal standard. Tissue and plasma catecholamines were expressed as ng/mg and ng/ml, respectively. Patient sample details are mentioned in Supplementary Table 1 and Supplementary Table 2.

Cortices from PS19, CgA-KO/PS19, WT and CgA-KO were homogenized in PBS followed by centrifugation at 12500 RPM for 30 min at 4 °C. The supernatant was collected and used in ELISA. Cytokines were measured using U-PLEX mouse cytokine assay kit (Meso Scale Diagnostics, Rockville, MD) following the manufacturer’s protocol in a MESO SECTOR S 600MM Ultra-Sensitive Plate Imager. The levels were presented in pg/mg protein. Plasma (25 µl) cytokines were measured using U-PLEX mouse cytokine assay kit (Meso Scale Diagnostics, Rockville, MD).

Behavioral studies including, Morris Water Maze, Novel Object Recognition and Rotarod test for all four cohorts of mice were performed in UCSD behavior core facility.

Human Tau RD P301S FRET biosensor expressing HEK 293 cells (ATCC CRL-3275) were plated on Millipore EZ chambered slide at a confluency of 1 ×103 Cells / well in DMEM Complete media (DMEM with 10%FBS + 100 μg penicillin and streptomycin). After 16 h, the media is replaced with OptiMEM and cells were treated with 2 μg lysate from CgA-KO/PS19 and PS19 cortex prepared in PBS and 4 μl Lipofectamine-3000. Post 24 h of treatment the media is replaced with complete DMEM. After 24 h the media was removed, and cells were fixed with 4% Paraformaldehyde and stained with DAPI. Cells were mounted and images were taken using Keyence Fluorescence Microscope under 40X lens. Four biological replicates per group were taken and 4 images each group were analyzed by ImageJ for quantification.

Isoflurane was used to anesthetize the mice followed by trans-cardial perfusion with PBS. The brain was dissected out and kept in Zn-Formalin for 48 hr. After that, Zn-Formalin is replaced with 30% sucrose incubated for 72 hr at 4 °C. The brains underwent coronal sectioning of 30 μm thick by sliding freezing microtome (Epredia). The sections were kept at −20 °C in cryoprotectant. Approximately 7-8 sections were taken for each animal covering from anterior to posterior region of hippocampus. The sections were washed thoroughly (6×10 min wash) with 1X PBS and then incubated with primary antibody in PBS containing 0.4% Triton-X for 24 hr at 4 °C. Following which the primary antibody is removed and washed with 1X PBS for 3X 15 min. Subsequently, the secondary antibody and DAPI (1:2000) were added and incubated for 1 hr at room temperature followed by 3X 15 min PBS wash. Next, the sections were mounted on glass slides with Fluoromount G. The stained slides were imaged under Keyence Fluorescence Microscope. Antibodies used are as follows: AT8 (1:500, Thermo Fisher Science Cat# MN1020), MC1 (1:500, Gift from P. Davies), CD68 (1:700, BIO-RAD Cat# MCA1957GA), GFAP (1:500, Cell Signaling Technology Cat# 3670), ADRA1B (1:500, Abcam, Cat# AB 169523), CgA (1:400, Invitrogen Cat# MA5-13093), NeuN (1:500, Proteintech 66836-1-Ig), Anti-mouse Alexa Fluor 568 (1:300, Invitrogen Cat# A11031), Anti-rabbit Alexa Fluor 488 (1:300, Invitrogen Cat# A10042), Strep-Alexa Fluor 568 (1:500, Thermo Fisher Science Cat#S11226↗), Strep-Alexa Fluor 647 (1:500, Thermo Fisher Science Cat#S32357↗).

Paraffin-embedded brain slices pre-mounted on slides from ADRC were de-paraffinized in xylene for 10 mins, followed by rehydration with sequential incubation in 100%, 90%, 70% EtOH, 5 mins each. Sections were placed in Citrate buffer (pH 6) solution in a pressure cooker at low pressure for 10 minutes for antigen retrieval. The slides were blocked (2.5% Normal Goat Serum for 1 hr at room temperature) and incubated with primary antibody for overnight at 4  °C, followed by 3X PBS wash for 15 min each. Next, a secondary antibody and DAPI (1:2000, Thermo Fisher Cat #D3571) were added and incubated for an hour at room temperature. After this, slides were dried and mounted with Fluoromount G. Images were taken with the Keyence Fluorescence microscope.

Investigators were blinded to the genotypes or treatment of the mice. For quantification of hippocampal volume, mice hemibrains were cut at 30 μm coronally, and all hippocampi, including sections, were collected. Brain sections were mounted on microscope slides (Fisher Scientific) in an anterior-to-posterior order, starting from the section where the hippocampal structure first becomes visible (first section) to the section where hippocampal structure just disappears (last section). Mice with missing sections were excluded from the analyses, a pre-established criterion. Mounted brain sections were dried at room temperature for 24 h and stained with cresyl violet (Nissl staining). After rehydrating with a quick wash in distilled water, sections were stained in FD Cresyl Violet Solution for 3 min. Next, sections were dehydrated in increasing ethanol concentrations and differentiated in 95% ethanol with 0.1% glacial acetic acid. After the final dehydration in 100% ethanol, sections were cleared in xylene and mounted with DePeX mounting media (VWR). Images were acquired with a Keyence BZ-9000 microscope. Hippocampal volume was estimated using ImageJ (NIH) Volumest plugin (http://lepo.it.da.ut.ee/~markkom/volumest/↗). To measure the thickness of CA1 pyramidal cell layer and dentate gyrus granule cells layer, a straight line perpendicular to the length of cell layers at fixed locations was drawn and measured using ImageJ.10–12 hippocampal-containing sections were typically used for each analysis.

15 mg tissue was incubated in RIPA buffer (150 mM NaCl, 1% Triton-X, 12 mM Na-deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0, 25 mM EDTA, 1 mM PMSF, 5% Glycerol, 50 mM NaF, 1 mM Na₃VO₄, 1 mM Na₄P₂O₇, 25 mM β-Glycerophosphate, 50 mM DTT and PIC) for 10 min. Homogenization was done by handheld motor homogenizer, followed by centrifugation at 12500 rpm for 30 min at 4 °C. The supernatant was collected, and protein concentration was measured using Bradford Protein Assay Reagent. 15μg protein was loaded in each lane of 12% Tris-Glycine SDS Gel and post-running protein was transferred to PVDF membrane followed by blocking with 5% BSA for 2hrs. Primary antibody incubation was done at 4 °C. Antibodies and dilution used as follows: AT8 (1:3000, Thermo Fisher Science Cat# MN1020), PHF1 (1:4000, Gift from P. Davies), CP13 (1:2000, Gift from P. Davies), HT7 (1:8000, Thermo Fisher Science Cat# MN1000), Actin (1:8000, Bio-Bharati, Cat# BB-AB0024), ADRA1B (1:2000, Abcam, Cat# AB 169523), ADRA2B (1:2000, Proteintech, Cat# 19778-1-AP), ADRB2 (1:2000, Cell Signaling Technology, Cat# 8513S), Alpha-Tubulin (1:6000, Cell Signaling Technology, Cat# 3873), CgA (1:2000, Invitrogen Cat# MA5-13093), Anti-Mouse-HRP (1:10000, Sigma, Cat# A0168), Anti-Rabbit-HRP (1:10000, Sigma, Cat# A0545), Strep-HRP (1:6000, Thermo Fisher Science Cat#N100).

The cortex tissue samples from mice or human were lysed in PBS along with PDE inhibitor (IBMX) and PIC (SIGMA) with homogenizer followed by centrifugation at 12500 rpm for 20 min at 4 °C. The cAMP level in the supernatant was analyzed using Perkin Elmer cAMP measurement kit as described in the manual. Briefly, the tissue lysate was incubated with anti-cAMP acceptor beads in corning dark 384 well plate and incubated for 30 min at room temperature in dark. Subsequently, biotinylated cAMP/streptavidin beads were added, and incubated at room temperature in dark for 1 h. Excitation was performed at 680 nm wave-length light and emission was measured at 550 nM to determine the cAMP concentration based on the standard curve.

The prazosin treatment was administered with utmost care in 6-month-old PS19 mice at a 2 mg/kg body weight dose⁸⁰. A stock concentration of Prazosin was made at 10 mg/ml in DMSO and stored at −20 °C. At the time of injection, the solution was warmed up to 37 °C and diluted in saline containing 10% DMSO. Saline containing 10% DMSO was used for the vehicle-treated experimental set. The mice received treatment twice a week for 12 consecutive weeks, after which they were sacrificed. Half of their brain was frozen for biochemical assays (western blotting) and the other half was fixed with Zn-formalin for immunohistochemistry.

Primary neuronal cultures were established from cortices of mouse pups on postnatal day 0 or 1. The cerebral cortices of 3-4 pups were dissected on ice, meninges removed and placed in the cold dissection medium (DM) consisting of 6 mM MgCl₂, 0.25 mM CaCl₂, 10 mM HEPEs (100X), 0.9% Glucose, 20 mM D-AP5 (Cayman, NC1368401), and 5 mM NBQX (Tocris Bioscience, 10-441-0). The tissues were digested with papain (Worthington, LK003176↗) in 37 C water bath for 20 min, followed by low OVO and DNase I incubation to stop the digestion for 5 min. The digested tissues were triturated and filtered through 70 mm cell strainer. The cell suspension was centrifuged at 1000 rpm for 10 min, and the cell pellet was gently resuspended in 20 mL B27/NBM/High glucose media and centrifuged again at 850 rpm for 5 min. The resulting cell pellet was resuspended in B27/NBM (1 mL/mouse brain). The cells were counted and plated onto the coverslips at 250,000 in 24-well plates for immunofluorescent imaging. Antibodies used: MAP2 (1:400, Millipore-Sigma Cat#AB-5622), MC1 (1:500, Gift from P. Davies).

The in vitro fibril-induced Tau spreading model was adapted and modified from previously described⁴⁸. Briefly, primary neurons cultured on coverslips were infected with AAV-P301S PS19 (Virovek, Inc.) on DIV3, and synthetic Tau fibrils (K18/P301L, 100 nM) were added to the medium on DIV5. Neurons without AAV infection (AAV-) and neurons treated with PBS instead of fibrils were included as negative controls. Neurons were incubated with the fibrils for 7–10 days. To test the effect of CgA treatment, recombinant CgA (5 ng/ml; R, D 10422-CH) were added to the culture medium together with the fibrils and replenished once after 3 days. Immunofluorescence staining using the MC1 antibody (1:500) was performed to detect misfolded Tau species. Briefly, neurons grown on coverslips were washed with fresh medium containing 0.01% trypsin, and fixed in fresh 4% PFA with 0.1% Triton-X, followed by regular immunofluorescence staining procedure.

RNA was isolated using TRIzol reagent and extracted further with phenol-chloroform method. RNA concentration was measured using Thermo Nanodrop and cDNA was synthesized from 500 ng of total RNA using Maxima RT cDNA synthesis kit. SYBR green containing NEB Luna qPCR mix was used to set up the qPCR in Bio-Rad qPCR machine (Primer details mentioned in Supplementary Table). 3

Total RNA was isolated from prefrontal cortex of 8-month-old mouse using RNeasy kit (Qiagen). RNA amount was quantified by ‘Nanodrop’ spectrophotometer and integrity was assessed by TapeStation (Agilent). Complementary DNA libraries were prepared from 500 ng of total RNA using the mRNA HyperPrep Kit (KAPA) according to manufacturer’s recommendations with Unique Dual-Indexed adapters (KAPA). Libraries were PCR-amplified for 10 cycles and quality was assessed by Tapestation. Libraries were then quantified by Qubit 2.0 fluorometer (Thermo), pooled, and analyzed by paired-end 100 sequencing on the NovaSeq 6000 platform (Illumina) at UCSD IGM Core.

RNA samples were first assessed for Sequencing quality by FASTQC and libraries with high quality reads were trimmed using Trimmomatic (v0.39). Reads were mapped to the mm10 mouse reference genome using STAR (v2.7.10b) and transcripts were quantified by StringTie (v1.3.6). Transcript counts were imported to R using prepDE.py3 for normalization and differential expression was analyzed by DESeq2 (v1.42.0). Gene clusters among differentially expressed genes were identified as common genes in comparisons of WT vs PS19 and CgA-KO/PS19 vs PS19 with log2 fold-change > +/− 0.5 and p value < 0.01. For gene ontology analysis, the enrichGO function from clusterProfiler (v4.10.0) (Yu et al., 2012) with the org.Mm.eg.db (v3.18.0) was used. Results were annotated along ontology of biological processes with the following parameters: p-value Cutoff = 0.05, q-value Cutoff = 0.05, and pAdjustMethod = ‘BH’ (Benjamani and Hochberg).

For human transcript analysis, normalized data of gene expression in parahippocampal gyrus (Brodmann area 36) was obtained from Mount Sinai Brain Bank (MSBB) through the Synapse platform (syn16795937). Total number of samples for each Braak stage considered for analysis is mentioned in Supplementary Table. 4

In figure legends ‘n’ represents the biological replicates for the group. In all in vivo experiments the male and female ratio of 50% was maintained. Normality was tested by Shapiro-Wilk test using GraphPad Prism version 10.4.1. Data were analyzed using unpaired two-tailed T-test, One-way or Two-way ANOVA as mentioned in the figure legends of each figure. Exact p-values are mentioned in the figures. Data are presented as mean values +/− SEM. In all figures except S-Fig4, n represents the number of biological replicates in the experiment.

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