(2024) 21:130
Ortiz et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03117-4

Journal of Neuroinflammation

Open Access

RESEARCH

Overexpression of pathogenic tau
in astrocytes causes a reduction in AQP4and GLT1, an immunosuppressed phenotype
and unique transcriptional responses
to repetitive mild TBI without appreciablechanges in tauopathy
Camila Ortiz1,2*, Andrew Pearson1,2, Robyn McCartan1, Shawn Roche1, Nolan Carothers1, Mackenzie Browning1,
Sylvia Perez3, Bin He3, Stephen D. Ginsberg4,5, Michael Mullan1, Elliott J. Mufson3, Fiona Crawford1,2,6 andJoseph Ojo1,2Abstract
Epidemiological studies have unveiled a robust link between exposure to repetitive mild traumatic brain injury(r-mTBI) and elevated susceptibility to develop neurodegenerative disorders, notably chronic traumatic encephalopathy (CTE). The pathogenic lesion in CTE cases is characterized by the accumulation of hyperphosphorylated tau
in neurons around small cerebral blood vessels which can be accompanied by astrocytes that contain phosphorylated tau, the latter termed tau astrogliopathy. However, the contribution of tau astrogliopathy to the pathobiology
and functional consequences of r-mTBI/CTE or whether it is merely a consequence of aging remains unclear. We
addressed these pivotal questions by utilizing a mouse model harboring tau-bearing astrocytes, G­ FAPP301L mice,subjected to our r-mTBI paradigm. Despite the fact that r-mTBI did not exacerbate tau astrogliopathy or generaltauopathy, it increased phosphorylated tau in the area underneath the impact site. Additionally, gene ontology
analysis of tau-bearing astrocytes following r-mTBI revealed profound alterations in key biological processes including immunological and mitochondrial bioenergetics. Moreover, gene array analysis of microdissected astrocytes
accrued from stage IV CTE human brains revealed an immunosuppressed astroglial phenotype similar to tau-bearing
astrocytes in the ­GFAPP301L model. Additionally, hippocampal reduction of proteins involved in water transport (AQP4)
and glutamate homeostasis (GLT1) was found in the mouse model of tau astrogliopathy. Collectively, these findings reveal the importance of understanding tau astrogliopathy and its role in astroglial pathobiology under normal
circumstances and following r-mTBI. The identified mechanisms using this ­GFAPP301L model may suggest targets
for therapeutic interventions in r-mTBI pathogenesis in the context of CTE.
Keywords Astrocytes, Tau astrogliopathy, Traumatic brain injury, Neuroinflammation, Chronic traumatic
encephalopathy*Correspondence:
Camila Ortiz
cortiz@roskampinstitute.org
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
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regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Ortiz et al. Journal of Neuroinflammation(2024) 21:130

Background
Repetitive mild traumatic brain injury (r-mTBI) is associated with an increased risk of neurodegenerative diseases such as Chronic Traumatic Encephalopathy (CTE).
Pathognomonic lesions in CTE are characterized by theaccumulation of phosphorylated tau in neurons, with orwithout tau-bearing astrocytes (i.e., 4R-tau astrogliopathy) within the depths of cortical sulci [1]. Tau astrogliopathy may play a key role in dysfunction of the central
nervous system (CNS) homeostasis, blood–brain barrier (BBB) integrity, and trophic and metabolic support
for neurons. In vitro studies have indicated that overexpression of 4R-tau in astrocytes renders them more vulnerable to oxidative stress and with an inability to clear
synaptic glutamate, which increases the vulnerability ofneurons to hyperexcitability and death [2, 3]. Transgenic
mouse models of astroglial tauopathy manifest a significant reduction of glial glutamate transporters, GLAST
and GLT1, as early as 5 months, in regions where astrocytic tau accumulation is most robust (e.g., brainstemand spinal cord) [4, 5].
To date, the effect of r-mTBI-induced tau astrogliopathy
upon astroglial homeostasis and pathobiology remains to
be determined. The present study interrogated the histopathological, biochemical and transcriptional effectsof tau astrogliopathy on astroglial pathobiology undernormal circumstances and after r-mTBI. Additionally,
we were also interested in comparing r-mTBI-induced
astroglial responses to endogenous overexpression ofmutant tau ­(GFAPP301L model) versus exogenous neuronal tau overproduction. We, therefore, used a mousemodel of neuronal tauopathy, the CaMKIIαP301L model,
also referred to as rTg4510 mice. We also assessed nonTBI related pathological effects of astroglial tauopathy
by using the r-sham groups (i.e., G­ FAPP301L versus WT
P301L
and CaMKIIα
versus WT). Finally, we compared
the genetic expression of tau-bearing astrocytes in thetransgenic GFAP tau mouse to astrocytes extracted fromhuman autopsy cases of CTE.
We hypothesized that overexpression of human pathogenic tau leads to an increased astroglial and microglial
response, dysregulation of astroglial homeostatic markers and transcriptional dysregulation compared to t­ auWT
astrocytes (i.e., astrocytes from WT and CaMKIIαP301Lmice). In addition, we posit early exposure to r-mTBI(pre-onset of tau astrogliopathy) will exacerbate tau
accumulation in astrocytes and brain-wide tauopathy, increase astroglial and microglial responses, cause
more pronounced dysregulation of astroglial homeostatic markers and transcriptional changes in mice harboring tau-bearing astrocytes compared to WT and
CaMKIIαP301L models. We further expect that tau-bearing astrocytes manifest changes in astroglial biology

Page 2 of 24

faithfully recapitulating pathological changes of astrocytes extracted from postmortem CTE cases.

Methods and materials
Animals

This study investigated three-month-old male andfemale C57BL/6 mice (n = 12), ­GFAPP301L (n = 12), and
CaMKIIαP301L (n = 12). Because ­GFAPP301L mice wereno longer available from Dr. Trojanowski [4, 5], we
generated a ­GFAPP301L transgenic mouse (Fig. 1A–C)
by crossing GFAP-tTa mice (a generous gift from Dr.
Brian Popko, Northwestern University, IL) with tetOMAPT*P301L (cat#015815; The Jackson Laboratory, ME,
USA). We favored the utilization of human P301L mutant
tau over human WT tau due to its preferential aggregation of 4R tau [6] which is particularly relevant in various
primary tauopathies such as CTE, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), andage-related tau astrogliopathy (ARTAG) [7]. ­GFAPP301L
and CaMKIIαP301L (Tg4510) were genetically engineered
to express the P301L MAPT mutation (4R/0N-human)
under the GFAP or CamMKIα promoter leading to tau
expression in astrocytes or neurons, respectively. Bothgenetically engineered models were on an FVB-C57BL/6
background. Mice were housed in a 12 h light/dark cyclewith food and water ad libitum. All experiments were
performed in accordance with Office of Laboratory Welfare and National Institutes of Health guidelines with
Roskamp Institute Institutional Animal Care and Use
Committee approval.
Traumatic brain injury administration

Our well-characterized closed-head mild TBI [8–11]
was used in a chronic repetitive injury paradigm involving 20 mTBI over the course of four weeks, as described
previously [12]. Briefly, mice subjected to r-mTBI wereanesthetized with 1.5 L/min and 3% isoflurane for 3 min
and the injury site was shaved. Mice were then positioned in a stereotaxic frame (Stereotaxic Instrument,
Stoelting, Wood Dale, Illinois) attached to an impactor (Impact One Stereotaxic Motorized Impactor, Richmond, Illinois). Mice were placed on a heating pad tomaintain body temperature at 37 °C during head impacts,
which were administered to the area of the shaved head
above the mid-sagittal suture in the central area of theskull. Head impact was performed using a 5 mm blunt
metal impactor tip with 1mm strike depth. The impact
was delivered at 5 m/s with a force of 72N. To prevent
hypothermia post-injury mice were placed on a heating pad set at 37 °C until they gained consciousness.
Mice were injured every weekday for 5 days a week for 4
weeks resulting in 20 hits in a month. Sham counterparts
were exposed to isoflurane with the same frequency andOrtiz et al. Journal of Neuroinflammation(2024) 21:130

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Fig. 1 Development of an inducible/reversible conditional mouse model of tau astrogliopathy. A Schematic drawing depicting the generationof GFAP-tTA(±)/tet0-MAPT*P301L(±) mice (referred to as ­GFAPP301L mice) by crossing tetO-MAPT*P301L transgenic mice (i.e., FVB-Fgf14Tg(tet0MAPT*P301L)4510kha/JlwsJ) with B6.Cg-Tg(GFAP-tTA)/110Pop/J mice that express a tetracycline-controlled transactivator protein (tTA) driven
by the human glial fibrillary acidic protein (GFAP) promoter. This bitransgenic mouse allows Tet-Off/Tet-On expression of a P301L mutant variantof human four-repeat microtubule-associated protein tau (4R0N tauP301L) under control of GFAP promoter, specifically in astrocytes. B Qualitative
micrographs of astrocyte colocalization with phosphorylated tau using S100 β/pTau T231 immunofluorescence (upper panel) and GFAP/pTau
T231 (lower panel). C Quantitative immunoblot of total tau protein (DA9) levels in astrocytes of GFAP-P301L vs non-carrier (control) mice. DA9
was normalized to house-keeper—β actin

duration. Mice were sacrificed 3 months post-last injury
for histopathological, biochemical, or transcriptomic
analyses (Fig. 2).
Intracerebral injection of AAV vector

Ten-month-old TauKI naïve mice were anesthetized with
4%isoflurane with 1.5 L/min oxygen and then placed on

the stereotaxic platform. A single injection of ketoprofen(5 mg/kg; Patterson Veterinary, USA) was administeredsubcutaneously before surgery for pre-operative analgesia. The head was shaved, and the exposed area wassterilized using betadine solution 5% povidone-iodine.
Using a scalpel, a midline incision was made in the skin

Ortiz et al. Journal of Neuroinflammation(2024) 21:130

Page 4 of 24

Fig. 2 Timeline of experiments and features of mouse models. A Three-month-old wild-type (WT), G­ FAPP301L, and CamkIIαP301L were subjected
to mild TBI every weekday for 5 days a week for four weeks resulting in 20 hits in a month. Three months post-last injury brain mouse tissue
was collected for histopathological, biochemical and transcriptional analyses. B mutant tau expression in the three mouse models utilized for thisstudy. − none; + some; +  +  + abundant

to expose the skull. Mice received unilateral intracranial
injections into the cortex (AP: − 1; ML: − 1 DV: − 1.3
from Bregma) through a small burr hole in the skull. Four
microliters of virus vector (AAV5-GFAP-htauP301L-GFP;
3.37 × ­1013 vg/mL) were injected using a 10 μl Hamilton syringe with a 30G needle and a rate of 0.2 μl/min.
The needle was kept in place for 10 min before withdrawal to avoid backflow. The incision was closed using
webglue (Pivetal, USA). Mice were monitored until
they recovered consciousness, at which point they wereadministered Ethiqa (3.25 mg/kg; Covetrus, USA) as a
post-operative analgesic (with 72 h duration). Mice weredaily monitored for 72 h after the injection. Transfection was allowed to occur for 6 weeks after the injection.
Mice were then subjected to the 20-hit r-mTBI paradigm described above. Six months after the last injury
brains were collected in 4% paraformaldehyde (PFA) in
phosphatase-buffered saline (PBS) and stored at 4 °C for
48 h. Brains were transferred to 20% sucrose PBS solution and stored at 4 °C until they were sectioned using
a vibratome Leica VT1200S (Leica, USA). Coronal brain
section (25 μm) were collected and immunostained with
AT8 in well-plates. Subsequently sections were mounted
on glass slides and imaged using ZEISS LSM 800 (ZEISS,
Germany).

Immunohistochemistry

Mice were deeply anesthetized with isoflurane and perfused transcardially with 4 °C cold phosphate-buffer
saline (PBS), decapitated and the right hemibrain wasplaced in 4% PFA in PBS solution for 24-48 h, then were
dehydrated and paraffin-embedded using the TissueTek® VIP and Tissue-Tek® TEC (Sakura, USA), respectively. Brains were sectioned at 6 μm using a Leica
RM2235 microtome (Leica, USA) and mounted on glass
slides. Sections were deparaffinized using ­HistoClear®
(National Diagnostics, USA) and rehydrated in a decreasing gradient of ethanol prior to immunohistochemistry.
Immunofluorescence: Following rehydration, slides were
submerged in PBS for 5 min to wash off the excess ethanol. For better detection of GFAP/RZ3 (Thr 231)/S100β
antigen retrieval was performed using an acidic buffer(pH 6). The antigen retrieval solution was warmed using apressure cooker, the antigen solution was prepared, slides
immersed and heated for 7 min in a microwave. Following antigen retrieval, sections were blocked with 5% normal donkey serum (NDS) dissolved in 0.1% TritonX-100
phosphate-buffered saline (PBST) at room temperature(RT) for 1 h. Sections were immunostained using primary antibodies (supplementary Table 1) in a PBST
solution containing 1% NDS. After overnight incubation

Ortiz et al. Journal of Neuroinflammation(2024) 21:130

at 4 °C, sections were rinsed with PBS and transferred
to a solution containing the appropriate fluorophoreconjugated secondary antibody (Alexa Fluor antibodies
648 nm, 555 nm, 488 nm 1:500, Thermofisher, MA, USA)at RT for 1 h. Autofluorescence was quenched with anautofluorescence removal reagent (Millipore Sigma, MA,
USA). Sections were mounted using anti-fade mounting
media with DAPI (Abcam, MA, USA). Z-stack images of
6 μm-thick sections were collected using a ZEISS LSM
800 (ZEISS, Germany) confocal microscope.
Immunohistochemistry: Following rehydration, slides
were submerged in PBS for 5 min to remove excess
ethanol. Slides were immersed in hydrogen peroxidefor 15 min to deactivate endogenous peroxidases. Antigen retrieval was performed using a basic buffer (pH 8).
Sections were incubated with 2.5% normal goat serum(VectorLabs, CA, USA) at RT for 1 h and subsequently
incubated with primary antibody at 4 °C overnight. Slides
were rinsed in PBS and incubated with horse radish peroxidase (HRP)-labeled secondary antibody (VectorLabs,
CA, USA) at RT for 1 h. Immunohistochemical signal was developed using 3,3’-Diaminobenzidine (DAB)(VectorLabs, CA, USA). After light counterstaining withhematoxylin, tissue was dehydrated in an increasing
series of ethanol (80%, 95%, and 100%). Next, slides werecover-slipped using toluene (Milipore Sigma, MA, USA).
Images were collected using an Olympus DP72 microscope (Olympus, PA, USA).
Microscopy and image analysis

Confocal microscopy: Multiple regions of interest in thecortex beneath the impact area were analyzed for eachmarker. A minimum of 20 microscopic fields (5 images
per sagittal section, 4 sections per mouse brain) wereimaged using a 20 × objective lens (total magnification
200×). Images from individual channels (wavelengths
648 nm, 555 nm, 488 nm, and 450 nm) were used independently for subsequent percentage area analysis. The
immunoreactivity of cell markers was measured by quantitative image analysis performed blinded by the investigator using ImageJ software. Quantification of G
­ FAP+ve/+ve
RZ3
astrocytes was performed throughout the entire
cortex of the 4 sagittal sections.
Bright-field microscopy: Multiple regions of interest in
the cortex (ventral to the impact site) were analyzed for
Iba1. A minimum of 20 microscopic fields (5 images per
sagittal section, 4 sections per mouse brain) were imaged
using a 40 × objective lens. Iba1 immunoreactivity wasmeasured blinded by the investigator using ImageJ software. Before quantitative analysis, color deconvolution
was applied on bright-field images to separate the DAB
reaction from the hematoxylin staining. Percentage area

Page 5 of 24

analysis was then performed using the “DAB” set ofimages.
Western blotting

Hippocampal samples were sonicated in 250 μl of mammalian protein extraction reagent, M-PER, (plus proteaseand phosphatase inhibitors, Thermofisher, MA, USA) toensure maximal protein extraction. Samples were centrifuged at 14,000 rpm for 20 min at 4 °C to pellet debris.
Total amount of protein was determined using the Bicinchoninic acid (BCA) assay (Thermofisher, MA, USA).
A total of 30 μg of tissue homogenate were mixed witha denaturing buffer containing 10% β-mercaptoethanoland boiled at 95 °C for 10 min. Samples were then loaded
on 4–15% SDS-PAGE gels for protein separation andthen transferred onto PVDF membranes overnight at 4
°C. Transferred membranes were blocked with 5% nonfat milk in 0.05% Tween 20 Tris-buffered Saline (TBST)
for 1 h at RT. Membranes were incubated with the primary antibodies (see Suppl. Table 1) overnight at 4 °C.
Membranes were washed in TBST 3 times (5 min each)prior to exposure of HRP-conjugated secondary antibodies (Cell Signaling, MA, USA) for 1 h at RT. Membranes
were washed in TBST and deionized water and developedby chemiluminescent (ECL or FEMTO) (Thermofisher,
MA, USA). Imaging was performed using a ChemoDoc
MP imager (BioRad, CA, USA) and densitometry analysis was performed with Image Lab software (Version 6.1,
BioRad, CA, USA). Because standardized housekeeper
proteins (e.g., β-actin and GAPDH) displayed genotypedependent variability, total protein obtained by PonceauS (Millipore, MA, USA) staining was used to normalize
the data.
Statistical analyses for histopathological and biochemical
analysesStatistical analyses were performed using GraphPad
Prism (Version 9, Dotmatics, CA, USA). Data were
tested for normality using the Shapiro–Wilk test. Statistical analysis was obtained using either t-test or twoway ANOVA accompanied by the Benjamini, Krieger,
and Yekuteli test to correct multiple comparisons. Whendatasets were not normally distributed, the datasets were
log-transformed. If after transformation, datasets maintained a non-normal distribution, the Mann–Whitney
test or Kruskal–Wallis non-parametric test were performed. Data are presented as mean ± SEM. A p value(p < 0.05) was considered statistically significant following
post-hoc testing. Asterisks in the Results represent different p value: *p < 0.05; **p < 0.01, and ***p < 0.001.

Ortiz et al. Journal of Neuroinflammation(2024) 21:130

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Human brain tissue

Hippocampi of 11 male athletes who participated in
American football with a history of repetitive mild TBI
were provided to an author (EJM) by Boston University
School of Medicine [13, 14] (see Table 1). The human
CTE cases (n = 11 with a mean age of 84.3) were diagnosed as stage-IV, the most severe category, according to
the neuropathological McKee and colleagues’ criteria forthis disorder [15]. In addition, we evaluated nine elderly
age-matched controls (2 males and 7 females) with amean age of 77.3 (see Table 1) lacking a history of brain
trauma.
Immunofluorescence of human tissue

Double stain was used to evaluate whether astrocytes
display tau pathology using antibodies to the astrocytic
marker GFAP and phosphorylated tau at S396/404, PHF1, in the hippocampus of CTE stage IV cases [16]. One
section per case was incubated overnight at room temperature with both GFAP (1:1000, DAKO Denmark)and PHF-1 (1:1000, gift from Peter Davies) after 10 min
citric acid pH 6 antigen retrieval. Then, the appropriate
secondary antibodies were applied for 1 h at room temperature as followed: first Cy5-conjugated donkey antimouse IgG for PHF-1 (1:400, Jackson Immuno-research,West Grove, PA) and secondly, after several washes,Cy2-donkey anti-rabbit IgG for GFAP (1:400, Jackson
Immuno-research). DAPI, a nuclear fluorescence stain,
was employed at 1:2000 at room temperature for 10 min.
Auto-fluorescence was blocked with Auto-fluorescence
Eliminator Reagent (Millipore, MA, USA) according
to manufacturer’s instructions and cover-slipped with
Table 1 Demographics of human CTE and healthy control(HC) cases used for microdissection and gene array analysis ofhippocampal astrocytes
HC

CTE IVN

911Sex (M/F)

2/7

11/0

Race (C/AA)

0/9

9/2

Sport Played (Soc/Rug/Hoc/Foot)

N/A

0/0/0/11

Sport Level (am/co/semipro/pro)

N/A

0/3/0/8

Military Service (Y/N)

N/A

2/9

Age begun

N/A

11.3 (±2.24)

Years of play

N/A

11.3 (±2.24)

Age of symptom onset

N/A

59.9 (±14.58)

Years retirement to symptom onset

N/A

31.9 (±16.28)

Years symptom onset to death

N/A

17.4 (±10.96)

Age at death84.3 (±12.25)

77.3 (±8.26)

HC healthy controls, M male, F female, C Caucasian, AA Afro-American, Soc
soccer, Rug rugby, Hoc hockey, Foot American football, am amateur, co college,
semipro semi-professional, pro professional, Y yes, N no, N/A not applicableaqueous mounting media (Thermo Scientific). Dualimmunofluorescence was visualized with the aid of a
Revolve Fluorescent Microscope (Echo laboratories, CA,
USA) with excitation filters for DAPI (pseudocoloredgreen), Cy2 (emission green; pseudocolored red) and Cy5(pseudocolored blue) as described previously [17].
Astroglial isolation

Following transcardial perfusion, left hemibrains were
removed and placed into a petri dish on wet ice. Enzymatic tissue digestion was performed using the Adult
Brain Dissociation Kit (Miltenyi Biotec, CA, USA). Brains
were minced using a sterile scalpel blade and transferred
to a 15 mL conical tube containing 1950 μl of enzyme
mix 1 (enzyme P and buffer Z) and 30 μl of enzyme mix
2 (enzyme A and buffer Y), incubated in the enzyme mix
for 30 min and then further dissociated using repeatedpipetting. Samples were briefly centrifuged and filteredthrough a 70 µm cell strainer to achieve a single-cellsuspension. Single cells were resuspended in 900 µl of
Debris removal solution mixed with 3.1 mL of PBS containing 0.5% fetal bovine serum (FBS) (PBS buffer) andthen transferred to a fresh 15 mL falcon tube, overlayedwith 4 mL of PBS buffer and centrifuged at 3000×g for
10 min. Cells were rinsed in PBS to remove any remaining debris and centrifuged for 10 min. The supernatant
was aspirated, and cells were labeled with FcR blocking
Reagent (Miltenyi Biotec, CA, USA) for 10 min at 4 °C toavoid non-specific binding of the astrocyte-specific magnetic bead-conjugated ASCA2 antibody (Miltenyi Biotec,
CA, USA). Samples were subsequently incubated with
ASCA2 antibody for 15 min at 4 °C, then loaded onto a
pre-conditioned LS separation column and rinsed threetimes to remove unlabeled cells. To elute A­ CSA2+ve cells,
the LS column was removed from the magnet and PBS
was used to elute the sample. Enriched ­ACSA2+ve cells
were centrifuged at 1000g for 5 min and stored at − 80
°C in Trizol until RNA extraction. Two left hemibrainsfrom two different mice that belonged to the same groupwere combined to collect enough material for one RNA
sample. In total we had an n = 3 RNA samples per groupper genotype for this analysis. These hemibrains weredepleted of hippocampi.
RNA isolation and sequencing

RNA isolation: RNA was extracted from astrocytes usinga Trizol-based protocol (Chomczynski and Mackey,
1995). Briefly, 500 µl of Trizol was added to cell isolates
obtained from the astrocyte isolation procedure. Samples were sonicated and allowed to dissociate at RT for 5
min. Samples were centrifugated at 14000rpm for 15 min.
Post-centrifugation, three layers were formed: the topmost layer was the aqueous phase and the one containingOrtiz et al. Journal of Neuroinflammation(2024) 21:130

RNA. The aqueous layer was transferred to a centrifuge
tube for further processing. Additionally, 1 µl of glycogen, per sample, was added to enhance RNA extraction. Serial centrifugation steps accompanied by ethanol
washes yielded the RNA pellet that was used for RNA
sequencing.
RNA sequencing: RNA samples were sent to GENEWIZ
LLC (South Plainfield, NJ, USA) for integrity checks(average RIN values = 5), library preparation and astrocyte-specific bulk sequencing. RNA sequencing libraries
were prepared from 100 ng of RNA using NEB Next Ultra
RNA Library Prep Kit for Illumina. Messenger RNA was
first enriched with Oligod(T) beads and fragmented, withthe 1st and 2nd strand cDNA subsequently synthesized.
cDNA fragments were end-repaired and adenylated,
and universal adaptor ligated to fragments, followed byindex addition and library enrichment with limited cycle
PCR. The sequencing library was validated on the Agilent
TapeStation and quantified using Qubit 20 Fluometer aswell as qPCR. The sequencing libraries were clustered on
one lane of a flowcell. After clustering, the flowcell was
loaded on the Illumina HiSeq4000 according to the manufacturer’s instructions. The samples were sequenced
using a 2 × 150 Paired End configuration. Raw sequence
data from Illumina HiSeq was converted into Fastq files
and de-multiplexed.
Bioinformatic analyses of mouse astrocytes

Bioinformatic analyses were obtained using the publicplatform usegalaxy.org. RNA sequences received from
GENEWIZ are uploaded to the platform where raw
data undergoes a quality check (FastQC on individual
files followed by MultiQC to obtain a quality report of
all files), and then the sequence reads are trimmed toremove adapter sequences, poly-N-containing reads andlow quality reads (Phred score <25) (TRimmomatic followed by MultiQC), and mapped to the Mus musculus
GRCm38 reference genome (HISAT2). Output files were
subjected to labeling of duplicated molecules (MarkDuplicates) for its posterior removal when measuring thegene expression in FPKM (fragments per kilobase million) (featureCounts). Data was inspected for outlying
samples using unsupervised hierarchical clustering andprincipal component analysis. Combat batch correction
was applied to combine the datasets and reduce systematic sources of variability. Differential gene expression analysis was conducted to determine relationships
between gene expression levels in the injury group versus
their sham counterparts (DESeq2). The IDs of the genes
were obtained using annotateMyIDs. The covariates were
included in all models to adjust for any potential confounding influence on gene expression between maingroup effects. This was conducted using the Wald test (inPage 7 of 24

DESeq2Genes with FDR adj p < 0.05 classified as differentially expressed genes (DEG). Values with an adjusted-p value of < 0.05 were processed for pathway enrichment analysis using Ingenuity Pathway Analysis (IPA,
QIAGEN). In rare instances where DEGs exceeded 5000
in number, we added another cut-off parameter for the
expression log2 fold change ± 1.2.
Single population gene expression analysis on humanhippocampal CTE astrocytes

Single GFAP-immunolabeled human hippocampal
astrocytes (a total of 50–100 astrocytes per CTE casepooled/per assay) from CTE Stage IV cases (n = 11) weremicroaspirated by laser capture microdissection (PALM
MicroBeam C IP, Carl Zeiss MicroImaging Inc., Thornwood, NY) [13, 18].
Microdissected astrocytes were homogenized in Trizol
solution and RNAs were extracted for reverse transcription in the presence of the poly d (T) primer (100 ng/mL) and TC primer (100 ng/mL) in 1 × first strand buffer,
500 μM deoxyribonucleotide triphosphate (dNTP)s,
5 mM dithiothreitol (DTT), 20 U of SuperRNase Inhibitor, and 200 U of reverse transcriptase. Single-stranded
complementary DNAs (cDNAs) were digested with
RNase H and re-annealed with the primers in a thermal
cycler: RNase H digestion step at 37 °C, 30 min; denaturation step at 95°C, 3 min; and primer reannealing step at
60 °C, 5 min. This step generated cDNAs with doublestranded regions at the primer interface [19–22]. Samples were then purified by column filtration (Montage
PCR filters; Millipore, MA, USA). RNAs hybridization
probes were synthesized by in vitro transcription withthe use of P incorporation in 40 mmol/L Tris (pH 7.5);
6 mmol/L MgCl2; 10 mmol/L NaCl; 2 mmol/L spermidine; 2.5 mmol/L DTT; 125 μmol/L adenosine triphosphate (ATP), guanosine triphosphate (GTP), and cytidine
triphosphate (CTP); 2.5 μmol/L cold uridine triphosphate(UTP); 20 U of RNase inhibitor; 2 kU of T7 RNA polymerase (Epicentre, Madison, WI); and 60 μCi of 33P-UTP(PerkinElmer, Waltham, MA). The labeling reaction was
performed at 37 °C for 4 h. Radiolabeled terminal continuation RNA probes were hybridized to custom-designed
microarrays without further purification [19–22]. Gene
array expression was run in triplicate for each case.
Custom-designed microarray platforms and data analysis: Platforms consist of 1 μg of linearized cDNA purified from plasmid preparations adhered to high-density
nitrocellulose (Hybond XL, GE Healthcare, Piscataway, NJ). cDNAs were verified by sequence analysis andrestriction digestion. Approximately 864 cDNAs wereused on our custom array platform [13, 18]. Arrays were
hybridized for 24 h in a solution consisting of 6 × salinesodium phosphate-ethylenediaminetetraacetic acid,Ortiz et al. Journal of Neuroinflammation(2024) 21:130

5 × Denhardt’s solution, 50% formamide, 0.1% sodium
dodecyl sulfate (SDS), and denatured salmon sperm
DNA (200 μg/mL) at 42°C in a rotisserie oven [19]. Following the hybridization protocol, arrays were washedsequentially in 2 × saline sodium citrate (SSC)/0.1% SDS,
1 × SSC/0.1% SDS, and 0.5 × SSC/0.1% SDS for 15 mineach at 37 °C. Arrays were placed in a phosphor screen
for 24 h and developed on a phosphor imager (Storm 840,
GE Healthcare, Piscataway, NJ).
Hybridization signal intensity was determined utilizing
ImageQuant software (GE Healthcare). Briefly, each array
was compared with negative control arrays utilizing therespective protocols without input RNA [19–21]. Expression of TC amplified RNA bound to each target minus
background was expressed as a ratio of the total hybridization signal intensity of the array (a global normalization approach) [19–21]. Global normalization effectively
minimizes variation because of differences in the specific activity of the synthesized probe and the absolute
quantity of the probe. Data analyzed in this manner
does not allow the absolute quantification of mRNAlevels generated [19]. However, an expression profile ofrelative changes in mRNA levels was generated. Relative changes in total hybridization signal intensity andindividual mRNAs were analyzed by one-way ANOVA
with post hoc Neumann-Keuls test analysis [19–21].
The level of significance was set at p < 0.01 for individualcomparisons.

Results
Three-month-old WT, ­GFAPP301L, and CaMKIIαP301L
mice were subjected to our 20-hit model of r-mTBI (orr-sham), and 3 months after the last injury (at the age of 7months), we performed histopathological and biochemical analyses on brain tissue, and transcriptomic analyses on isolated ex-vivo primary astrocytes to investigate
the effects of tau astrogliopathy on brain levels of phosphorylated and total, neuroinflammation (astrocyte andmicroglial reactivity), astrocytic homeostasis and astrocyte-specific pathological response under normal (noTBI) circumstances and three months post-last injury.
For the present study, normal circumstances refer to datafrom the sham cohorts of each genotype at 3 months
post-last isoflurane exposure.
Effects of astrocytic versus neuronal tau on tau
astrogliopathy and brain levels of phosphorylated
and total tau in the cortex and hippocampus under normal
circumstances and following r‑mTBI

To analyze brain levels of tau pathology in the regionsof interest we selected antigens against several epitopesof tau hyperphosphorylation and aggregation including
tau phosphorylated at Threonine 231 (RZ3), Serine 202

Page 8 of 24(CP13) and Serine 396/Serine 404 (PHF1) [23, 24]. First,
we examined the effects of astroglial vs neuronal mutant
human tau (­htauP301L) on promoting tau phosphorylation in cortical astrocytes under normal circumstances.
We quantified the number of astrocytes double labeledfor ­GFAP+/RZ3+ in 4 sagittal sections of the cortex per
mouse from each of the three mouse models examinedin this study. ­GFAP+/RZ3+ astrocytes were not seen in
WT and CaMKIIαP301L mice. In the ­GFAPP301L mice, anaverage of 6 ± 1.4 ­GFAP+/RZ3+ astrocytes were found in
the cortex. Three months post-last TBI, the number of­GFAP+/RZ3+ astrocytes remained unchanged comparedto their shams (Fig. 3A, B). Next, we assessed Threonine
231 (RZ3) immunoreactivity in the cortex beneath theimpact size of each mouse model. Under normal circumstances, the immunoreactivity of this tau epitope
was unchanged in the ­GFAPP301L mice compared to theWT mice (Fig. 3C, D). However, there was a significant ~ 12-fold increase in CaMKIIαP301L mice compared
to both WT and ­GFAPP301L (Fig. 3D). Three months postlast injury (3mpi), there were no changes in RZ3 immunoreactivity in the WT cohort; however, the presenceof astroglial pathogenic tau ­(GFAPP301L) and neuronalpathogenic tau (CaMKIIαP301L) mice resulted in a significant increase in p-tau (RZ3) immunoreactivity aroundthe impact site compared to their respective sham counterparts (Fig. 3D) (two-way ANOVA: genotype effect[F(2,22) = 62.53, p ≤ 0.001]; injury effect [F(1,22) = 4.87,
p = 0.038],
injury*age
interaction[F(2,22) = 1.27,
p = 0.301]). Because tau astrogliopathy in the entire cortex did not change, the observed changes in tau phosphorylation underneath the impact site may occur inneurons.
Next, we used immunoblotting to evaluate brain levelsof tauopathy by assessing tau phosphorylation markers(RZ3, CP13 and PHF1) and total tau (DA9) in the hippocampus. Because tau abundance in the CaMKIIαP301L
model was disproportionately increased compared to theother models, we performed a separate two-way analysis using WT and G­ FAPP301L data only, for all markers ofinterest.
In the hippocampus, under normal circumstances,
total tau levels displayed a significant 68-fold increasein CaMKIIαP301L compared to WT (two-way ANOVA:
genotype effect [F(2,26) = 101.2, p ≤ 0.001]) (Fig. 4G)
and a significant 18-fold increase in G
­ FAPP301L compared to WT mice (two-way ANOVA: genotype effect[F(1,19) = 28.19, p ≤ 0.001]) (Fig. 4H). CaMKIIαP301L
mice have around 4 times more levels of tau compared to­GFAPP301L.
Three months post-last injury, in the hippocampus,
r-mTBI does not cause changes in tau phosphorylation levels at any epitope in WT and G­ FAPP301L mice.

Ortiz et al. Journal of Neuroinflammation(2024) 21:130

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Fig. 3 RZ3 immunoreactivity in the cortex of WT, ­GFAPP301L and CaMKIIαP301L mice at 3 months post-last injury. Qualitative images (A)and quantification of RZ3/GFAP + cells in the cortex of ­GFAPP301L mice from 4 serial sagittal sections per mouse (n = 5–6 per group) 3 months
post-last injury (B). Qualitative images of phosphorylated tau (RZ3, red) in the cortex underneath the impact site of WT, ­GFAPP301L, and CaMKIIαP301L(C). RZ3 immunoreactive percent area in the cortex underneath the impact site 3 months post-last injury (n = 4–6 per group per genotype) (D).
Data were analyzed by Two-Way ANOVA followed by the Benjamini, Krieger, and Yekuteli test. Table under the graph details injury and genotype
effects and their interaction after Two-way ANOVA. Asterisks denote: *p < 0.05; **p < 0.01 and ***p < 0.001 for post-hoc analyses

In contrast, in CaMKIIαP301L mice, r-mTBI causes
an increase of tau phosphorylation levels at specificepitopes, but not total tau compared to their shamcontrols (Fig. 4A–H) (two-way ANOVA: RZ3 injury
effect [F(1,26) = 5.76, p = 0.024]; genotype*injury[F(2,26) = 5.36, p = 0.011]; CP13 [F(1,26) = 11.65,
p = 0.002];
genotype*injury[F(2,26) = 19.74,
p = 0 < 0.001];
PHF1[F(1,26) = 10.44
p = 0.003];
genotype*injury [F(2,26) = 8.97, p ≤ 0.001]; DA9[F(1,26) = 0.24, p = 0.623]). The lack of a TBI-mediatedeffect on tau phosphorylation in the hippocampus may

indicate dilution of the signal on regions distal to thesite of impact.
Lastly, we wanted to overcome any developmental
issues related to astrocytic tau production from birth inthe ­GFAPP301L model, by utilizing astrocyte-targeted AAV
transfection of the mutant tau. Six weeks after transfection
we confirmed mutant tau expression within astrocytes, andmice were subjected to our 20hit r-mTBI model (Fig. 5A).
Then we evaluated tau phosphorylation levels in the ipsilateral cortex across 7 coronal sections at a more chronic time
post last injury (6mpi) to possibly capture the progressive
nature and of tau phosphorylation spread. We observed

Ortiz et al. Journal of Neuroinflammation(2024) 21:130

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Fig. 4 Changes in Tau species (phosphorylated and total tau) in the hippocampus of WT, ­GFAPP301L and CaMKIIαP301L mice at 3 months
post-last injury. Levels of RZ3 (A, B), CP13 (C, D), PHF1 (E, F) and DA9 (G, H) in the hippocampus (HIPPO) at 3 months post-last injury (n = 5–6
per group per genotype). Data were analyzed by Two-Way ANOVA followed by the Benjamini, Krieger, and Yekuteli test. Table under the graphdetails injury and genotype effects and their interaction after Two-way ANOVA. Asterisks denote: *p < 0.05; **p < 0.01 and ***p < 0.001 for post-hoc
analyses. Graphs from B, D, F and H are from WT and GFAP-P301L cohorts alone. Representative immunoblots from the hippocampus are depictedon the left of the graphs

Ortiz et al. Journal of Neuroinflammation(2024) 21:130

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Fig. 5 Adeno-associated viral (AAV) mediated transfection of astrocytes with mutant P301L tau prior to r-mTBI/sham injuries in human Tau Knock
In (TauKI) mice. Schematic representation of AAV study design involving transfection of astrocytes under GFAP promoter with an intracerebral
injection of AAV-GFAP-eGFP-P301L-Tau-FlagTau vector, six weeks prior to exposure to r-mTBI/sham injuries in 10-month-old TauKI mice (A).
Qualitative micrographs of phosphorylated tau (AT8) marker by immunofluorescence 6 months post-last injury (B). AT8 immunoreactivity
in the cortex of injected naïve mice (n = 4–6 per group) (C). AT8 + astrocyte-like cells in the cortex of injected mice (n = 4–6 per group) (D). t-test
analysis yielded no significant changes

that tau phosphorylation across the cortex and the numberof cortical astrocyte-like cells that were positive for phosphorylated tau ­(AT8+) remained unchanged in sham and
TBI mice after transfection (Fig. 5B–D). Thus, we corroborated in mice, independent of the time of astrocytic tau
overexpression, TBI does not cause cortical-wide neuronaltauopathy or exacerbate tau astrogliopathy in the cortex,
even by 6 months post-injury.

Ortiz et al. Journal of Neuroinflammation(2024) 21:130

Page 12 of 24(See figure on next page.)
Fig. 6 Astrocyte reactivity (GFAP) and microglial reactivity (Iba1) in the cortex (CTX) of WT, ­GFAPP301L and CaMKIIαP301L mice 3-months after r-mTBI/
sham injury. Top right image is the overview of the region of interest (yellow box) where the images were collected from (red dot indicatesthe impact site). Qualitative images of GFAP and Iba1 in the cortex (A and B, respectively) of WT mice (top-two panels), ­GFAPP301L mice (middle-two
panels) and CaMKIIαP301L mice (bottom-two panels) 3-months after r-mTBI/sham injury. GFAP images were captured at × 20 magnification and
IBA1 images at x40 magnification. Percentage area of GFAP (C, D) and Iba1 (E, F) in the cortex tissue (n = 4–6 per group per genotype). Data wereanalyzed by Two-Way ANOVA followed by the Benjamini, Krieger, and Yekuteli test. Table under the graph details injury and genotype effects
and their interaction after Two-way ANOVA. Asterisks denote: *p < 0.05; **p < 0.01 and ***p < 0.001 for post-hoc analyses. Graphs from D and F arefrom WT and GFAP-P301L cohorts alone

Effects of astrocytic versus neuronal tau on astrocyte
and microglial reactivity in cortex under normal
circumstances and following r‑mTBI

To investigate the effect of astrocyte-derived and neuronal-derived tau on astrocyte and microglial morphological phenotype at baseline and following r-mTBI, wequantified the percent area of GFAP and Iba1 immunoreactivity to evaluate astrocyte and microglial reactivity, respectively, in the cortex of each mouse model(Fig. 6A–F). Under normal circumstances, the presenceof tau within astrocytes in the ­GFAPP301L model did
not impact GFAP and Iba1 immunoreactivity levels in
the cortex (Fig. 6C–F) compared to WT. The presenceof neuronal mutant tau in the CaMKIIαP301L mice led
to a significant tenfold increase in GFAP immunoreactivity in the cortex (two-way ANOVA: genotype effect[F(2,23) = 40.54, p ≤ 0.001]), compared to both WT and­GFAPP301L mice (Fig. 6E, I). Iba1 immunoreactivity inthe cortex was significantly upregulated compared tothe other two genotypes in the CaMKIIαP301L mice,
represented by a twofold increase (two-way ANOVA:
genotype effect [F(2,23) = 28.55, p ≤ 0.001]) (Fig. 6E, F).
Three months post-last injury, there was a significant
1.5-fold increase in GFAP immunoreactivity in the cortexof the CaMKIIαP301L r-mTBI group compared to shamcontrols (two-way ANOVA: injury effect [F(1,23) = 3.88,
p = 0.061]; genotype*injury [F(2,23) = 0.86, p = 0.435])(Fig. 6E). Because astroyte reactivity in the CaMKIIαP301L
model (both r-sham and r-mTBI mice cohorts) was disproportionately exacerbated compared to the other models, we performed a separate two-way analysis using only
the r-mTBI/sham WT and ­GFAPP301L data (see Fig. 6D).
Our analysis revealed that r-mTBI induced a significant
twofold increase in GFAP immunoreactivity in both
WT and G­ FAPP301L cohorts compared to sham counterparts (two-way ANOVA: injury effect [F(1,15) = 15.62,
p = 0.001]; genotype*injury [F(1,15) = 0.63, p = 0.437]).
Unlike exposure to exogenous tau (i.e., CaMKIIαP301L
model), the presence of tau within astrocytes (i.e., the­GFAPP301L model) was not sufficient to evoke an exacerbated TBI response compared to WT counterparts.

Furthermore, no synergistic TBI effect was found in the­GFAPP301L cohort compared to WT counterparts.
Iba1 immunoreactivity demonstrated no significant
changes in the CaMKIIαP301L cohort in the TBI groupcompared to shams (two-way ANOVA: injury effect[F(1,23) = 5.14, p = 0.033]; genotype*injury [F(2,23) = 0.47,
p = 6.27]) (Fig. 6E). Microglial reactivity was significantly increased in the TBI groups of both models
compared to sham counterparts (two-way ANOVA:
injury effect [F(1,15) = 11.98, p = 0.003]; genotype*injury[F(2,15) = 0.432, p = 0.521]) (Fig. 6F). The effect of r-mTBI
exposure was not worsened in the ­GFAPP301L cohort
compared to WT.
These data indicate presence of pathological tau within
astrocytes does not exacerbate the effect of r-mTBI on
chronic astrocyte and microglial reactivity at 3 months
post-injury. However, r-mTBI elicited an augmented glial
response in CaMKIIαP301L mice at 3 months post-last
injury.
Effects of astrocytic versus neuronal tau on astrocyte
homeostatic markers in the hippocampus under normal
circumstances and following r‑mTBI

Under normal circumstances, a significant reduction in AQP4 and GLT1 protein levels was observed in
the hippocampus of both tau models (­GFAPP301L and
CaMKIIαP301L) compared to WT (Fig. 7A–C) (two-way
ANOVA: genotype effect [F(2,24) = 24.43, p ≤ 0.001];
genotype effect [F(2,25) = 49.14, p ≤ 0.001], respectively).
GLAST levels in the hippocampus remained unchanged
across genotypes (Fig. 7D) (two-way ANOVA: genotype
effect [F(2,27) = 2.25, p = 0.125]).
Three months post-last injury, analyses revealed anabsence of TBI-dependent changes in the levels of AQP4,
GLT1, and GLAST in both regions of interest across thethree genotypes (Fig. 7) (two-way ANOVA: injury effect[F(1,24) = 0.668, p = 0.422]; injury effect [F(1,25) = 0.10,
p = 0.752]; injury effect [F(1,27) = 6.42, p = 0.017], respectively, none of them had genotype injury interaction).

Ortiz et al. Journal of Neuroinflammation(2024) 21:130

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Fig. 6 (See legend on previous page.)

Astrocyte‑specific transcriptional changes in response
to astroglial vs neuronal tau under normal circumstances

Through cell-specific bulk RNA sequencing of astrocytes

harvested from our three mouse models we were able toevaluate unbiased changes in gene expression and dysregulation of cellular pathways. First, we verified that

Ortiz et al. Journal of Neuroinflammation(2024) 21:130

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Fig. 7 Changes in astrocyte homeostatic protein markers in WT, ­GFAPP301L and CaMKIIαP301L mice at 3 months post-last injury. Qualitative (A)and quantitative immunoblotting levels of aquaporin 4 (AQP4) (B) and glutamate transporters GLT1 and GLAST (C, D) in the hippocampus (HIPPO)(n = 4–6 per group per genotype). Data were analyzed by Two-Way ANOVA followed by the Benjamini, Krieger, and Yekuteli test. Table
under the graph details injury and genotype effects and their interaction after Two-way ANOVA. Asterisks denote: *p < 0.05; **p < 0.01
and ***p < 0.001 for post-hoc analyses

our samples contained astrocytes by assessing the transcriptional expression of several published astrocytespecific markers [25]. A clear enrichment of protein andtranscriptomic astrocytic markers compared to thosefor microglia, endothelial cells, oligodendrocytes andneurons was found. Interrogating significantly alteredgenes (adjusted p-value < 0.05) in the ­GFAPP301L model,
the presence of tau within astrocytes dysregulated theexpression of 11,401 genes (5799 upregulated DEGs and5602 downregulated DEGs) compared to WT mice. In
comparison, the presence of neuronal tau in CaMKIIαP301L
astrocytes dysregulated 7900 genes (4298 upregulated DEGs and 3602 downregulated DEGs) compared toWT astrocytes (Fig. 8A).

Pathway enrichment analysis was performed on
DEGs (adjusted p-value < 0.05 and IPA fold change cutoff =  ± 1.2). Under normal conditions, interestingly, two
opposing astroglial profiles were observed. Tau-bearing
astrocytes show an immunosuppressed phenotype, characterized by the downregulation of interleukin signaling(IL-8, IL-15, IL-1), NFκB and STAT3 signaling, as well as
production of nitric oxide (NO), and reactive oxygen species (ROS) (compared to WT astrocytes (Fig. 8B). On thecontrary, CaMKIIαP301L astrocytes displayed upregulation of the above-mentioned pathways compared toWT astrocytes (Fig. 8C). Key pathways identified in­GFAPP301L astrocytes compared to WT include upregulation of calcium signaling, and antioxidant action ofVitamin C (ascorbic acid) compared to WT astrocytes.

Ortiz et al. Journal of Neuroinflammation(2024) 21:130

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Fig. 8 Astrocyte specific pathways that are dysregulated in ­GFAPP301L and CaMKIIαP301L mice compared to Wild-type mice at 7-month-old. Venn
diagram of differentially expressed genes (DEGs) of primary astrocytes isolated using MACS ACSA2 + beads from our mouse models are shown in A.
Histogram in B and C depicts results of IPA pathway analyses after analyzing DEGs between ­GFAPP301L vs WT and CaMKIIαP301L vs WT, respectively.
Upregulated and downregulated pathways in B–C are depicted in red and blue, respectively. Heat-bar in B–C represents –log 10 of the p value(yellow—Topmost significant; purple—least significant). Threshold for obtaining the DEGs: adj. p-value ≥ 0.05 with its respective –log value ≥ 1.3.
N = 3 per group per genotype

CaMKIIαP301L astrocytes also exhibited a downregulationof neurovascular coupling and antioxidant action transcripts. Together these findings suggest that G­ FAPP301L
astrocytes have an immunosuppressed phenotype withan enhanced antioxidant role and dysregulated calcium
signaling, while CaMKIIαP301L astrocytes have a proinflammatory phenotype and an impairment in antioxidant
defense.
To identify DEGs common to astrocytes expressing
human pathogenic tau ­(GFAPP301L) and those exposed
to neuronal-derived h­ tauP301L (CaMKIIαP301L), we queried DEGs obtained from G­ FAPP301L versus WT and
P301L
CaMKIIα
versus WT mice. This comparison found
2,138 convergently dysregulated genes (862 upregulated DEGs and 1,276 downregulated DEGs) (Fig. 8A).
Enriched pathway analysis identified that those convergent DEGs were associated with the upregulation ofchemokine signaling, synaptogenesis, neuroinflammation,and mitochondrial dysfunction; and the downregulationof neurovascular coupling and detoxification function(glutathione-mediated).
Astroglial transcriptome changes in response to r‑mTBI
three months post‑last hit

Three months post-last injury (3mpi), astrocyte-specificbulk RNA analysis showed that r-mTBI caused dysregulation of 57 genes (51 downregulated DEGs and 6 upregulated DEGs) in WT, 175 (63 downregulated DEGs and
112 upregulated DEGs) in CaMKIIαP301L, and 441 genes(157 downregulated DEGs and 284 upregulated DEGs)
in ­GFAPP301L mice (Fig. 9A–D). The presence of endogenous ­htauP301L in astrocytes results in greater transcriptomic dysregulation in response to r-mTBI compared tothe exposure of astrocytes to exogenous neuronal tau in
the CaMKIIαP301L or the absence of pathogenic humantau in the WT mice. No single gene was dysregulated
across cohorts in response to TBI (Fig. 9A) highlighting
the context-dependent nature of astroglial response.

Ortiz et al. Journal of Neuroinflammation(2024) 21:130

IPA analysis interrogating the 57 DEGs in the WT
mice revealed significant downregulation in 5 pathways: phagosome formation, insulin secretion, c-AMP
signaling, synaptogenesis and dendritic cell maturation(Fig. 9E). In the G­ FAPP301L cohort, 441 DEGs mapped
to 52 significant pathways included the downregulation of oxidative phosphorylation, synaptic long-term
potentiation, and opioid signaling; and upregulationof neuroinflammation, complement system signaling,
and ERK5-mediated cell activation (Fig. 9F). In the
CaMKIIαP301L cohort, 175 DEGs mapped to 17 significant pathways including downregulation of phagosome
formation and neuroprotective erythropoietin signaling, and upregulation of nitric oxide and reactive oxygen species production, and senescence (Fig. 9G). Theseobservations revealed that r-mTBI affects ­GFAPP301L
astrocytes by compromising mitochondrial function
and neuronal support mechanisms, which favors a proinflammatory state compared to WT and CaMKIIαP301L
mice.
Transcriptome changes in astroglia obtained from human
CTE tissue

Prior to performing gene array analysis, we used dual
immunofluorescence to determine whether GFAPstained hippocampal astrocytes colocalized with PHF-1
tau in individuals with a postmortem neuropathological diagnosis of stage IV CTE (CTE-IV). Qualitative
analysis showed that hippocampal CTE-IV astrocytes
did not contain PHF-1 phosphorylated tau (Fig. 10A–
F). Then, we evaluated changes in gene expression in
hippocampal astrocytes from CTE-IV brains comparedto hippocampal astrocytes from non-demented healthy
controls (HC). G­ FAP+ve astrocytes microaspirated from
CTE-IV brains revealed 156 DEGs (153 downregulated
and 3 upregulated) (Fig. 10G). Pathway enrichment
analysis by IPA on DEGs revealed CTE-IV astrocytes
had 100 dysregulated pathways including upregulation of thrombin signaling, and sphingosine signaling,
and downregulation of neuroinflammation, interleukin
signaling (IL-6, 8, 17), endothelin 1 signaling, calcium
signaling, synaptogenesis, and insulin-like growth factor

Page 16 of 24

signaling. These data indicate that CTE-IV astrocytes
exhibit a loss of neurorestorative function(s) accompanied by an immunosuppressed phenotype comparedto healthy controls. Interestingly, despite that CTE-IV
hippocampal astrocytes do not display phosphorylatedtau (PHF1) in their soma, CTE-IV astrocytes showedsignificant downregulation of genes associated withimmunological response including IL-8 signaling andNO and ROS production similar to tau bearing murine
astrocytes ­(GFAPP301L).

Discussion
Tau astrogliopathy is a pathological feature of primarytauopathies including CTE. However, the role that tau
astrogliopathy plays in the onset and/or progression of
TBI/CTE pathophysiology remains underinvestigated.
Using the G­ FAPP301L mouse model harboring tau-bearing astrocytes, we investigated the effects of endogenous­htauP301L accumulation, r-mTBI and how their interaction might affect astroglial pathological response. Ourfindings reveal that astroglial overexpression of h
­ tauP301L
does not alter markers of gliosis but elicits a significant
reduction in hippocampal homeostatic astrocyte-specificmarkers (AQP4 and GLT1) that regulate water transportand glutamate homeostasis, respectively. Additionally,
astrocytes overexpressing tau display an immunosuppressed phenotype similar to the phenotype observed instage IV CTE-derived astrocytes. Moreover, r-mTBI does
not augment or accelerate tau astrogliopathy or gliosis
but causes an increase in neuronal phosphorylated tau
in the cortical area underneath the impact site and astroglial mitochondrial dysfunction.
Pathological analyses of human CTE cases show significant increases in gliosis at the interface between
white and grey matter [26]. The present findings indicate that compared to astrocytic tau overexpression,
neuronal tau elicits significantly more astrogliosis andmicrogliosis in the cortex grey matter compared to WT
mice and in the white matter of the corpus callosum(supplementary Fig. 1). At the age of 7 months (miceage at 3mpi), CaMKIIαP301L mice already exhibit substantial tau pathology [27] while increased tau pathology in ­GFAPP301L mice does not occur until 24 months(See figure on next page.)
Fig. 9 Astrocyte specific pathways that are dysregulated in WT, ­GFAPP301L and CaMKIIαP301L mice at 3-month post-last injury. Venn diagram of injury
dependent differentially expressed genes (DEGs) primary astrocytes isolated using MACS ACSA2 + beads from our mouse models are shown
in A (i.e., entire DEGs, overlapping DEGs and unique DEGs). Volcano plot of injury dependent DEGs are shown in B (WT), C ­(GFAPP301L) and D(CaMKIIαP301L). Top 10 DEGs are highlighted on the volcano plots. Upregulated DEGs are in red, Downregulated DEGs are in blue. Histogram in E
(WT), F ­(GFAPP301L) and G (CaMKIIαP301L) depicts results of IPA pathway analyses after analyzing the entire DEG list between r-mTBI vs sham groups
for each of the 3 different genotypes. Upregulated and downregulated pathways in E–G are depicted in red and blue, respectively. Heat-bar in E–G
represents -log 10 of the p value (yellow—Topmost significant; purple—least significant). Threshold for obtaining the DEGs: adj. p-value ≥ 0.05
with its respective –log value ≥ 1.3. N = 3 per group per genotype

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Fig. 9 (See legend on previous page.)[5]. Foreman and colleagues reported that in regions of
the brain where tau astrogliopathy was robust, astrogliosis was also exacerbated. Together with the present

findings, the appearance of tau astrogliopathy at 7mo of
age is not sufficient to increase gliosis. Since our preliminary data (supplementary Fig. 2) and findings from otherOrtiz et al. Journal of Neuroinflammation(2024) 21:130

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Fig. 10 Absence of tau astrogliopathy in CTE-IV hippocampal astrocytes and astrocyte-specific pathways that are dysregulated in human
CTE (stage IV) cases versus healthy controls after laser microdissection of GFAP + astrocytes and gene array analyses. Immunofluorescent label
demonstrating a lack of colocalization between tau-bearing neurofibrillary tangles and ­GFAPve astrocytes in the CA1 region of the hippocampus
of a male Caucasian American football player that played for 25 years had an age of onset of symptoms at 66 years and died in his 70s. Postmortem
neuropathologic diagnosis revealed CTE stage IV. Low power Immunofluorescence images showing single labeled GFAP astrocytes (red) and PHF-1(tau phosphorylated at S396/404) positive NFTs (blue) and a merged image combined with staining for cell nuclei (green) in the hippocampusof the CTE stage IV case (A, B). High-power images of the GFAP astrocyte (upper left panel A, white arrow) and the NFT (D–F). Scale bar = 25 μm
in A-C; scale bar = 10 μm in D–F. Histogram depicts the results of IPA pathway analyses after analyzing the entire DEG list between CTE stage IV(n = 11) vs HC (n = 9) (G). Upregulated and downregulated pathways are depicted in red and blue, respectively. Heat-bar represents -log 10 of the Pvalue (yellow—Topmost significant; dark blue—least significant). The threshold for obtaining the DEGs: adj. p-value ≥ 0.05 with its respective –log
value ≥ 1.3. Approximately 50–100 astrocytes were micro-dissected and subjected to customized gene array analyses to interrogate > 850 genes
with > 20 gene ontology groupsOrtiz et al. Journal of Neuroinflammation(2024) 21:130

models of tauopathy [4, 5] suggest that G­ FAPP301L mice
do not exhibit robust astroglial tauopathy earlier than 24
months of age. Therefore, we combined r-mTBI (20-hit
paradigm) with the astroglial tau model to evaluate theonset and/or progression of tau astrogliopathy. Repetitive mTBI evoked a region-specific (i.e., underneath theimpact site) increase in cortical levels of tau phosphorylation at 3mpi but it did not affect cortical-wide tau
phosphorylation at 3 and 6mpi. Furthermore, the number of astrocytes containing phosphorylated tau (i.e.,
tau astrogliopathy) remained unchanged between shamand TBI at chronic timepoints, 3 and 6mpi. In the hippocampi of CTE-IV brains, we did not observe PHF-1
reactive astrocytes stained with GFAP, which suggeststhat tau astrogliopathy in the hippocampus is inconspicuous compared to neuronal tau [28, 29]. CTE pathology
in the dorsal lateral frontal cortex from this brain bankhas previously been shown to be primarily neuronal [28].
Further analysis with other markers of tau phosphorylation, such as AT8 or CP13, will be needed to confirm theextent of tau astrogliopathy in the hippocampus of CTE
samples used for this study. Additionally, since all astrocytes do not stain with GFAP, other homeostatic markers—e.g., S100β—may be also required. Three months
post-last brain injury, we detected increased astrogliosis
and microgliosis in the cortex and CC beneath the impact
of GFAP P301L mice (TBI vs sham) (Supplementary Fig. 1),
however, TBI-induced gliosis was not exacerbated compared to WT TBI counterparts.
These results suggest that overexpression of tau P301L inastrocytes in G
­ FAPP301L mice is not a necessary precondition to cause significant gliosis compared to WT mice.
Although r-mTBI did not increase tau astrogliopathy, it
initiated an increase in tau phosphorylation in the cortex beneath the impact. This increase in tau phosphorylation is not robust enough to exacerbate TBI-mediatedgliosis compared to the WT cohort. In the case of the
CaMKIIαP301L mice, the substantial presence of phosphorylated tau might underlie the greater glial response
seen in this transgenic mouse than in WT mice. We
suggest that TBI-dependent gliosis in humans might beorchestrated by an increase in neuronal tauopathy rather
than tau astrogliopathy. Further investigation on aged
­GFAPP301L mice could potentially aid in understanding the contribution of robust tau astrogliopathy to glial
responses.
Biochemical analysis of homeostatic astrocyte markers in our mouse models of tau astrogliopathy and neuronal tauopathy revealed a region-specific reductionof AQP4 and GLT1 in the hippocampus of shams compared to WT shams. AQP4 is a water channel locatedon astrocytic perivascular end-feet that is essential forwater flux and waste clearance termed the glymphatic

Page 19 of 24

system [30]. AQP4 is also present in perineuronal astrocytic processes where it is involved in maintaining water
volume and ion/neurotransmitter buffering in response
to synaptic transmission [31]. Skucas and colleagues(2011) reported that AQP4-/- mice have impaired memory consolidation linked to impairments in long-term
potentiation and long-term depression (LTP/LTD) [32].
Based on hippocampal AQP4 downregulation in bothtauopathy models, we expect ­GFAPP301L have impaired
memory. Interestingly, CamkIIαP301L mice exhibit behavioral changes at the age of 5 months [33], which likely arelinked to the substantial accumulation of pathogenic tau.
Moreover, r-mTBI did not elicit changes in AQP4 levelsin the mouse models confirming other studies that havereported no differences in AQP4 abundance after TBI[34, 35]. Glutamate transporter 1 (GLT1) an astroglialspecific marker was also affected by the presence of tau
overexpression in astrocytes or neurons only in the hippocampus. GLT1 is responsible for preventing excitotoxicity by recycling 85% of glutamate excess at the synaptic
cleft after synaptic transmission [36]. Thus, a significant
reduction of GLT1 expression would lead to a significant
increase in extracellular glutamate concentration which
ultimately can cause neuronal loss [2]. Previous studies
on CaMKIIαP301L mice have shown a ~ 55% loss of hippocampal neurons by the age of 6 months [37, 38], which
may suggest a link between a reduction of GLT1 and neuronal loss due to excitotoxicity. However, we observed
no genotype or injury-related changes in markers of synaptic integrity such as synaptophysin and PSD95 in ourmodels (data not shown) which suggests that the significant reduction in GLT1 levels might affect neurons at afunctional level. Further experiments assessing glutamate
concentration and electrophysiological properties of neurons in these models are needed to confirm this relationship. Moreover, r-mTBI did not cause changes in GLT1
or GLAST at 3mpi compared to controls. A previous
study showed a significant reduction of GLT1 in the cortical astrocytes after TBI [39]. The authors also noted that
the GLT1 downregulation was observed in astrocytes
directly below the impact site. Additional experiments
are essential to assess the mechanism and the functional
effects of alteration in glutamate transporter and AQP4
abundance.
Pathway analysis of DEGs shows that, under shamconditions, astrocytic tau overexpression compared toneuronal tau overexpression resulted in greater transcriptional astrocyte change. Tau-bearing astrocytes­(GFAPP301L astrocytes) showed an immunosuppressedphenotype. On the contrary, CaMKIIαP301L astrocytes
exposed to neuronal tau and robust microgliosis showeda pro-inflammatory phenotype and lost their neurovascular and antioxidant function. Under injury conditions,Ortiz et al. Journal of Neuroinflammation(2024) 21:130­ FAPP301L astrocytes showed greater transcriptionalGdysregulation compared to WT and CaMKIIαP301L astrocytes 3mpi.
Downregulation of neuroinflammatory pathways
including IL-12 and IL-15 in ­GFAPP301L sham astrocytes
might cause suppression of astrocyte-microglia bidirectional crosstalk since microglia respond to both interleukins by mounting immune responses [40, 41]. Thesefindings may explain the failed genotype effect observedin microgliosis and astrogliosis between G­ FAPP301L andWT sham groups. Interestingly, CTE-IV hippocampal
astrocytes (tau-negative) also manifest an immunosuppressed phenotype characterized by the downregulation of IL-8 signaling. Since IL-8 signaling is involved in
microglial activation [42], its downregulation may alter
the ability of astrocyte-microglia interaction needed tomount an immune response. This similarity might indicate that hippocampal human astrocytes and murine
astrocytes share a “loss of function” trait irrespective of
astrocytic tau accumulation. In contrast, Chancellor andcolleagues showed that CTE white matter astrocytes
exhibit downregulation of normal functioning, and dysfunctional mitochondrial metabolism accompanied byincreased neuroinflammation resulting in a “neurotoxic
profile” [43]. The observed discrepancies in transcriptional changes might be related to the heterogenicity
of astroglial response depending on the region they arefound respective to the impact site (hippocampus vswhite matter). Moreover, astrocytes harvested from individuals with other neurodegenerative conditions, namely,
AD, PD, MS, and HD show an upregulation of inflammatory responses [44] which contrasts with what we arereporting in the present studies. However, analogous towhat is seen in neurodegenerative diseases, murine astrocytes may display a senescent phenotype following TBI
characterized by the dysregulation of astrocyte-secreted
molecules important for cell-to-cell communication [46].
Additionally, it has also been reported that a sub-population of astrocytes in neurodegenerative conditions candisplay downregulation of transcripts involved in synaptogenesis and neuronal function [44, 45] similar to thephenotype of hippocampal CTE-IV astrocytes reported
here. This suggests that astrocytes might have a common
pathobiological response in neurodegenerative conditions characterized by the loss of their neuronal support
functions and mechanisms.
Altogether, these findings suggest that tau astrogliopathy in CTE might result in a “loss of function” phenotype
where hippocampal astrocytes fail to mount an immune
response, communicate to microglia, support synaptogenesis and maintain neurovascular coupling which
may be involved in the neurodegenerative nature of CTE.

Page 20 of 24

The prominent pathways identified in the present study
warrant further investigation to understand and determine if the molecular changes dictating their dysregulation might represent therapeutic targets. Given thattau-bearing astrocytes from the sham ­GFAPP301L mice (vsWT) also showed a similar immunosuppressed state wesuggest that the ­GFAPP301L model is a relevant platform
in which to study tau astrogliopathy in tauopathies.
Limitations of the study

Data presented here provides insights into the effectsof overexpression of pathogenic tau in astrocytes undernormal conditions and after r-mTBI, however, this study
has several limitations. The expression of pathogenic
tau in astrocytes in the G­ FAPP301L model occurs fromembryonic day 14 [46] and persists throughout the animals’ lifespan, leading to an age-dependent increase in
tau astrogliopathy. In humans, it has been suggested thatthe accumulation of tau within astrocytes is more likely
to progressively appear after an encounter with a triggering event (e.g., cerebrovascular accidents, gene predisposition, head trauma) that either increases the expression
and accumulation of astrocytic endogenous tau, or theinternalization and aggregation of secreted neuronal tau[29].
Tau expression in our model is controlled by the GFAP
promoter whose expression is region-dependent andknown to increase in response to TBI [47]. This leads todifferential expression/accumulation of tau within astrocytes throughout the brain which might contribute toregional astroglial response under normal (sham) conditions and r-mTBI. An increase in GFAP expression in
response to TBI is likely to confound the effects of TBI
alone on tau pathology in this model. In the future, wewill consider the utilization of an astrocyte-specific promoter that is known to not change in response to TBI
such as Aldehyde dehydrogenase 1 family, member L1(ALDH1) or Connexin 30 (Cx30) promoter [48].
It has been reported that the integration of the tetOMAPT*P301L transgene (~ 70 copies) into chromosome 14 results in a deletion of 244kbp in the Fgf14(Fibroblast growing factor 14). This is particularly relevant because functional knockout of FGF14 may contribute to accelerated neuronal loss and brain atrophy
in tetO-MAPT*P301L-Fgf14 models [49], and thus it
cannot be assumed that mutant tau, alone, drives thereported changes in neurodegeneration. Additionally,
the trans activator (tTA) transgene insertion, that drives
the expression of mutant tau either in astrocytes orneurons, disrupts another five genes which, collectively,
have been shown to be involved in synaptic transmission, plasticity, and neurogenesis [50, 51] which mayOrtiz et al. Journal of Neuroinflammation(2024) 21:130

indirectly confound our findings. However, relevantto the outcomes on this paper, we observed that hippocampal levels of AQP4 did not change while GLT1
levels seem to suffer a slight decrease (28% decrease) in
tetO-MAPT*P301L compared to WT (data not shown).
Thus the tetO*MAPT*P301L transgene insertion affects
hippocampal levels of GLT1 but the overproductionof mutant tau in astrocytes or neurons is what further
decreases GLT1 (93% decrease) in the ­GFAPP301L mice.
Further investigation is warranted to know the impactof these transgene insertions on other markers of normal physiology of astrocytes.
Astrocyte tau pathology in CTE has been shown to be
predominantly of 4R tau isoform, therefore parallelingthe phenotype in our G­ FAPP301L model. However, theP301L
CaMKIIα
model displays 4R-neuronal tauopathy
while neuronal pathology in CTE cases is mixed (3R/4R)[52]. Although the effects of 3R and 4R tau on pathology
are largely unclear, they may manifest different pathogenicity individually or synergistically. More studies are
therefore needed to delineate the isoform-specific effects
at the cell type level.
Human ­GFAP+ve astrocytes were microdissectedfrom the hippocampus of CTE-IV cases. As shown in
Fig. 10A–F, ­GFAP+ve astrocytes do not contain phosphorylated tau (PHF1), thus, it is possible that the harvested
human astrocytes do not contain tau. PHF1 antibodies
recognize both 3R and 4R pathogenic isoforms of tau andwere selected because it is widely used for the identification of both astroglial (predominantly 4R) and neuronal(both 3R/4R) tauopathies in CTE [53–55]. As mentioned
before, further investigation is warranted to know if othertau epitopes might be preferentially phosphorylated(AT8, CP13, or 4R-tau) in ­GFAP+ve astrocytes, therefore,
we cannot discard or prove that the analyzed astrocyteslack accumulation of tau. Nonetheless, we acknowledge
that astrocyte pathophysiology may differ in the presenceor absence of astrocytic tau accumulation. Furthermore,
previous reports have shown that astrocytes exhibit not
only region-dependent transcriptional changes but also
differential responses to stimuli [56–59]. Human RNA
data was obtained from hippocampal tau-negative astrocytes ­(GFAP+ve/PHF1−ve) while mouse data was obtained
from tau-overexpressing astrocytes harvested from theentire brain excluding the hippocampi. Therefore, directcomparisons of our human and mouse data and/or generalization of astrocytic changes based on the hippocampal
astrocyte population, here reported, should be avoided.
Additionally, we acknowledge that the age-matchedcohort has a sex disparity (2 males and 7 females). Thus,
we compared the transcriptome profile between females
and males (Supplementary Fig. 3) which did not reveal
obvious differences which allowed us to move forward

Page 21 of 24

with the CTE vs control comparisons. Lastly, the consensus RIN value for RNA samples is ≥ 7 [60], therefore
we acknowledge that the reduced RIN value (RIN ≥ 5) of
our samples might have resulted in under- or overrepresentation of transcripts in the library. However, Ilumina
HiSeq library used for the analysis of our samples has
been extensively used for the analysis of samples with a
RIN = 2 and still provides high-quality RNA sequencing
results [61].

Summary
We demonstrate r-mTBI did not significantly exacerbate
tau astrogliopathy, but increased tau phosphorylationin the cortical neurons beneath the impact site. Overexpression of h
­ tauP301L within astrocytes did not increase
astrogliosis and microgliosis compared to WT mice.
Moreover, r-mTBI did not exacerbate gliosis compared
to WT cohorts. Interestingly, tau-bearing astrocytes
undergo region-specific changes that alter homeostatic
markers related to optimal neuronal transmission andwater transport independent of TBI. Tau-bearing astrocytes have significantly more DEGs compared to WT
astrocytes and astrocytes exposed to robust neuronaltau pathology and microgliosis under normal circumstances. Tau-bearing murine astrocytes exhibit an immunosuppressed phenotype that might modify bidirectional
molecular communication with microglia limiting theability to mount immune responses. At three months
post-last injury, tau-bearing astrocytes display a greatertranscriptional dysregulation compared to WT and
CaMKIIαP301L astrocytes showing a compromised bioenergetic system which might interrupt their physiologicalroles. Finally, CTE-IV astrocytes display an immunosuppressed phenotype similar to the effects of tau overexpression seen in astrocytes from the G­ FAPP301L mousemodel.
Collectively, the findings of this study underscore thesignificance of unraveling mechanisms driving tau astrogliopathy and the pathobiological consequences on thenormal, injured, and tauopathic brain. Our unbiased
results imply a driving role in the astroglial immune
state and propagating microglial-mediated neuroinflammation observed after r-mTBI. These mechanisms may
suggest targeted interventions to address the pathobiology of r-mTBI that are both tau-dependent andtau-independent.
Abbreviations
AD	Alzheimer’s disease
ALDH1	Aldehyde dehydrogenase 1 family, member L1
ANOVA	Analysis of variance
AQP4	Aquaporin 4
ARTAG​	Age-related tau astrogliopathy
BBB	Blood–brain barrier
CaMKIIα	Calcium/calmodulin-dependent protein kinase II alphaOrtiz et al. Journal of Neuroinflammation(2024) 21:130

Page 22 of 24

CBD	Cortico-basal degeneration
CC	Corpus callosum
CNS	Central Nervous System
CTE	Chronic Traumatic Encephalopathy
CTX	Cortex
Cx30	Connexin 30
DAB	3,3’-Diaminobenzidine tetra-hydrochloride
DEGs	Differentially expressed genes
ERK	Extracellular signal-regulated kinase
FDR	False rate discovery
GFAP	Glial fibrillary acidic protein
GLAST	Glutamate transporter GLAST
GLT1	Glutamate transporter 1
HIPPO	Hippocampus
Iba1	Ionized calcium binding adapter molecule 1
IL-12	Interleukin 12
IL-15	Interleukin 15
IL-8	Interleukin 8
LDP/LTP	Long-term potentiation and long-term depression
mpi	Months post-last injury
HC	Healthy controlsHD	Huntington’s diseaseMS	Multiple sclerosis
NDS	Normal donkey serum
NFκB	Nuclear factor kappa beta
PBS	Phosphate-buffered saline
PD	Parkinson’s diseasePSP	Progressive supranuclear palsy
r-mTBI	Repetitive mild traumatic brain injury
RT	Room temperature
STAT3	Signal transducer and activator of transcription 3
TBI	Traumatic brain injury
TBST	Tris-base saline Tween 20
WT	Wild type

Acknowledgements
We would like to thank Boston University Brain Bank for providing human CTE
hippocampal tissue and the University of Kentucky Brain Bank for providing
healthy control hippocampal tissue to EJM. We would like to also thank Dr.
Brian Popko at Northwestern University, Evanston, IL, for providing us with
GFAP-tTa mice. Lastly, we would like to also thank Dr. Peter Davies for kindly
sharing tau antibodies (RZ3/CP13/PHF1/DA9) with us.

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12974-​024-​03117-4.
Suuplemenatry Material 1: Figure 1: Astrocyte reactivity (GFAP) andmicroglial reactivity (Iba1) in the corpus callosum (CC) of WT, ­GFAPP301L
and CaMKIIαP301L mice 3-months after r-mTBI/sham injury. Top right imageis the overview of the region of interest (yellow box) where the images
were collected from (red dot indicates the impact site). Qualitative images
of GFAP and Iba1 in the CC (A and B, respectively) of WT mice (top-two
panels), ­GFAPP301L mice (middle-two panels) and CaMKIIαP301L mice(bottom-two panels) 3-months after r-mTBI/sham injury. Images were
captured at x20 magnification. Percentage area of GFAP (C) and Iba1 (D) in
the CC (n=5-6 per group per genotype). Data were analyzed by Two-Way
ANOVA followed by the Benjamini, Krieger, and Yekuteli test. Table under
the graph details injury and genotype effects and their interaction afterTwo-way ANOVA. Asterisks denote: *P<0.05; **P<0.01 and ***P<0.001 forpost-hoc analyses.
Suuplementary Material 2: Figure 2: Tau astrogliopathy in the cortex of­GFAPP301L mice at 7 and 10 months of age. RZ3/GFAP+ cells in the cortex
from 4 serial sagittal sections at 7 and 10 months of age (n=5-6 pergroup). t-test analysis yielded significant changes p<0.001***
Supplementary Material 3: Figure 3: Heat map revealing expression levelsof all genes in the microarray of all healthy control (HC) cases and briefclinical demographics of CTE and HC cohorts. Heat map depicts relative
intensity score (i.e., expression levels) of all genes in the microarray from all
healthy control (HC) cases (n=9). Upregulated and downregulated genes
are depicted in red and blue, respectively.
Supplementary Material 4: Table 1. Antibodies used. Abbreviations: GFAP,
Glial Fibrillary Acidic Protein; Iba1, Ionized calcium-binding adaptor molecule; AQP4, aquaporin 4; GLAST, Glutamate transporter; GLT1, Glutamate
transporter 1. IF, Immunofluorescence; IHC, Immunohistochemistry; WB,
Western blotting.

Author contributions
CO, MM, FC, and JO designed the experiments; RM, SR, MB and NC contributed to histopathological staining and imaging acquisition. CO performed
animal experiments and data analysis and statical analysis. CO and AP isolated
astrocytes for RNA extraction and sequencing. BH and SP performed laser capture, RNA labeling and gene expression analysis in human astrocytes. CO andJO participated in writing the original draft. FC, EJM, SDG reviewed, and edited
the manuscript. MM, FC and JO were the supervisors and also participated infunding acquisition. All authors read and approved the final manuscript.
Funding
This research was supported by the U.S Department of Defense Peer Reviewed
Alzheimer’s Research Program (PRARP) and Congressionally Directed Medical
Research Programs (CDMRP) under award W81XWH-19-1-0535 to JO and
FC. FC is a Research Career Scientist at the James A. Haley Veterans Hospital,
Tampa, FL. U.S Department of Defense Grant 13685123 to EJM. National
Institutes of Health Funding AG014449, AG107617, AG072599, AG074004, andAG077103 to SDG.
Availability of data and materials
Data used and analyzed in the current study are available upon request fromthe corresponding author.

Declarations
Ethics approval and consent to participate
All experiments in mice were performed in accordance with Office ofLaboratory Welfare and National Institutes of Health guidelines with Roskamp
Institute Institutional Animal Care and Use Committee approval (protocol
R114). Institutional review board approval for brain donation was obtained
through the Boston University Alzheimer’s Disease Center CTE program andthe Bedford Veterans’ Affairs hospital. Institutional review board approval
for post-mortem review of clinical records, family member interviews andneuropathological analysis was obtained through Boston University Schoolof Medicine. Consent for tissue donations was attained from the subject ora family member and is part of a larger brain bank collaboration (VA-BU-CLF
Brain Bank, http://​www.​bu.​edu/​cte/​our-​resea​rch/​brain-​bank/).
Competing interests
The authors declare they have no competing interests.
Author details
1
The Roskamp Institute, Sarasota, FL, USA. 2 The Open University, Milton
Keynes, UK. 3 Barrow Neurological Institute, Phoenix, AZ, USA. 4 Center
for Dementia Research, Nathan Kline Institute, Orangeburg, NY, USA. 5 Departments of Psychiatry, Neuroscience and Physiology, and NYU Neuroscience
Institute, New York University Grossman School of Medicine, New York, NY,
USA. 6 James A. Haley Veterans Hospital, Tampa, FL, USA.
Received: 22 February 2024 Accepted: 30 April 2024

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