Abstract
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, GFAP^P301L mice, subjected to our r-mTBI paradigm. Despite
the fact that r-mTBI did not exacerbate tau astrogliopathy or general
tauopathy, 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 GFAP^P301L 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 GFAP^P301L model may suggest targets for therapeutic interventions
in r-mTBI pathogenesis in the context of CTE.
Supplementary Information
The online version contains supplementary material available at
10.1186/s12974-024-03117-4.
Keywords: Astrocytes, Tau astrogliopathy, Traumatic brain injury,
Neuroinflammation, Chronic traumatic encephalopathy
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
the accumulation of phosphorylated tau in neurons, with or without
tau-bearing astrocytes (i.e., 4R-tau astrogliopathy) within the depths
of cortical sulci [[49]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 of neurons to hyperexcitability and death [[50]2, [51]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., brainstem and spinal cord) [[52]4, [53]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 effects of tau astrogliopathy on astroglial
pathobiology under normal circumstances and after r-mTBI. Additionally,
we were also interested in comparing r-mTBI-induced astroglial
responses to endogenous overexpression of mutant tau (GFAP^P301L model)
versus exogenous neuronal tau overproduction. We, therefore, used a
mouse model of neuronal tauopathy, the CaMKIIα^P301L model, also
referred to as rTg4510 mice. We also assessed non-TBI related
pathological effects of astroglial tauopathy by using the r-sham groups
(i.e., GFAP^P301L versus WT and CaMKIIα^P301L versus WT). Finally, we
compared the genetic expression of tau-bearing astrocytes in the
transgenic GFAP tau mouse to astrocytes extracted from human 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 tau^WT astrocytes (i.e., astrocytes from WT and
CaMKIIα^P301L mice). 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 CaMKIIα^P301L models. We
further expect that tau-bearing astrocytes manifest changes in
astroglial biology faithfully recapitulating pathological changes of
astrocytes extracted from postmortem CTE cases.
Methods and materials
Animals
This study investigated three-month-old male and female C57BL/6 mice
(n = 12), GFAP^P301L (n = 12), and CaMKIIα^P301L (n = 12). Because
GFAP^P301L mice were no longer available from Dr. Trojanowski [[54]4,
[55]5], we generated a GFAP^P301L transgenic mouse (Fig. [56]1A–C) by
crossing GFAP-tTa mice (a generous gift from Dr. Brian Popko,
Northwestern University, IL) with tetO-MAPT*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 [[57]6] which is particularly relevant in various primary
tauopathies such as CTE, progressive supranuclear palsy (PSP),
corticobasal degeneration (CBD), and age-related tau astrogliopathy
(ARTAG) [[58]7]. GFAP^P301L and CaMKIIα^P301L (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. Both genetically engineered models were on an
FVB-C57BL/6 background. Mice were housed in a 12 h light/dark cycle
with 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.
Fig. 1.
[59]Fig. 1
[60]Open in a new tab
Development of an inducible/reversible conditional mouse model of tau
astrogliopathy. A Schematic drawing depicting the generation of
GFAP-tTA(±)/tet0-MAPT*P301L(±) mice (referred to as GFAP^P301L mice) by
crossing tetO-MAPT*P301L transgenic mice (i.e.,
FVB-Fgf14Tg(tet0-MAPT*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 variant of 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
[MATH: β :MATH]
/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—
[MATH: β :MATH]
actin
Traumatic brain injury administration
Our well-characterized closed-head mild TBI [[61]8–[62]11] was used in
a chronic repetitive injury paradigm involving 20 mTBI over the course
of four weeks, as described previously [[63]12]. Briefly, mice
subjected to r-mTBI were anesthetized 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 to
maintain 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 the skull. 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 duration. Mice were
sacrificed 3 months post-last injury for histopathological,
biochemical, or transcriptomic analyses (Fig. [64]2).
Fig. 2.
[65]Fig. 2
[66]Open in a new tab
Timeline of experiments and features of mouse models. A Three-month-old
wild-type (WT), GFAP^P301L, and CamkIIα^P301L 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 this study. − none; + some; + + + abundant
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
administered subcutaneously before surgery for pre-operative analgesia.
The head was shaved, and the exposed area was sterilized using betadine
solution 5% povidone-iodine. Using a scalpel, a midline incision was
made in the skin 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-htau^P301L-GFP; 3.37 × 10^13 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 were administered Ethiqa (3.25 mg/kg; Covetrus, USA) as a
post-operative analgesic (with 72 h duration). Mice were daily
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 was placed in 4% PFA in PBS
solution for 24-48 h, then were dehydrated and paraffin-embedded using
the Tissue-Tek^® 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 a
pressure 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 at 4 °C, sections were rinsed with PBS and
transferred to a solution containing the appropriate
fluorophore-conjugated 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 an autofluorescence 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 peroxide for 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 with hematoxylin, tissue was dehydrated in an
increasing series of ethanol (80%, 95%, and 100%). Next, slides were
cover-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 the cortex beneath
the impact area were analyzed for each marker. A minimum of 20
microscopic fields (5 images per sagittal section, 4 sections per mouse
brain) were imaged 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 GFAP^+ve/RZ3^+ve 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
was measured 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 analysis was then performed using the “DAB” set of
images.
Western blotting
Hippocampal samples were sonicated in 250 μl of mammalian protein
extraction reagent, M-PER, (plus protease and phosphatase inhibitors,
Thermofisher, MA, USA) to ensure 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 with a denaturing buffer containing 10%
β-mercaptoethanol and boiled at 95 °C for 10 min. Samples were then
loaded on 4–15% SDS-PAGE gels for protein separation and then
transferred onto PVDF membranes overnight at 4 °C. Transferred
membranes were blocked with 5% non-fat 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 developed
by 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 genotype-dependent variability, total protein obtained
by Ponceau-S (Millipore, MA, USA) staining was used to normalize the
data.
Statistical analyses for histopathological and biochemical analyses
Statistical 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 two-way ANOVA accompanied by the Benjamini, Krieger, and
Yekuteli test to correct multiple comparisons. When datasets 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.
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 [[67]13, [68]14] (see
Table [69]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 for this disorder
[[70]15]. In addition, we evaluated nine elderly age-matched controls
(2 males and 7 females) with a mean age of 77.3 (see Table [71]1)
lacking a history of brain trauma.
Table 1.
Demographics of human CTE and healthy control (HC) cases used for
microdissection and gene array analysis of hippocampal astrocytes
HC CTE IV
N 9 11
Sex (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 death 84.3 (±12.25) 77.3 (±8.26)
[72]Open in a new tab
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 applicable
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, PHF-1, in the hippocampus of CTE stage
IV cases [[73]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
anti-mouse 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 aqueous mounting media (Thermo Scientific). Dual
immunofluorescence was visualized with the aid of a Revolve Fluorescent
Microscope (Echo laboratories, CA, USA) with excitation filters for
DAPI (pseudocolored green), Cy2 (emission green; pseudocolored red) and
Cy5 (pseudocolored blue) as described previously [[74]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 repeated pipetting. Samples were briefly centrifuged and filtered
through a 70 µm cell strainer to achieve a single-cell suspension.
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) and then transferred to a fresh 15 mL falcon tube, overlayed
with 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 to avoid
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 three times to remove
unlabeled cells. To elute ACSA2^+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 hemibrains from two
different mice that belonged to the same group were combined to collect
enough material for one RNA sample. In total we had an n = 3 RNA
samples per group per genotype for this analysis. These hemibrains were
depleted of hippocampi.
RNA isolation and sequencing
RNA isolation: RNA was extracted from astrocytes using a 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 top-most layer was the aqueous phase and
the one containing 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, with the 1st and 2nd strand cDNA
subsequently synthesized. cDNA fragments were end-repaired and
adenylated, and universal adaptor ligated to fragments, followed by
index addition and library enrichment with limited cycle PCR. The
sequencing library was validated on the Agilent TapeStation and
quantified using Qubit 20 Fluometer as well 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 public platform
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 to remove adapter sequences,
poly-N-containing reads and low 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 the gene expression in FPKM (fragments per
kilobase million) (featureCounts). Data was inspected for outlying
samples using unsupervised hierarchical clustering and principal
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 main
group effects. This was conducted using the Wald test (in 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 human hippocampal CTE
astrocytes
Single GFAP-immunolabeled human hippocampal astrocytes (a total of
50–100 astrocytes per CTE case pooled/per assay) from CTE Stage IV
cases (n = 11) were microaspirated by laser capture microdissection
(PALM MicroBeam C IP, Carl Zeiss MicroImaging Inc., Thornwood, NY)
[[75]13, [76]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 double-stranded regions at the primer interface
[[77]19–[78]22]. Samples were then purified by column filtration
(Montage PCR filters; Millipore, MA, USA). RNAs hybridization probes
were synthesized by in vitro transcription with the 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 [[79]19–[80]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 and
restriction digestion. Approximately 864 cDNAs were used on our custom
array platform [[81]13, [82]18]. Arrays were hybridized for 24 h in a
solution consisting of 6 × saline-sodium
phosphate-ethylenediaminetetraacetic acid, 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 [[83]19]. Following
the hybridization protocol, arrays were washed sequentially in
2 × saline sodium citrate (SSC)/0.1% SDS, 1 × SSC/0.1% SDS, and
0.5 × SSC/0.1% SDS for 15 min each 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 the respective protocols without
input RNA [[84]19–[85]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) [[86]19–[87]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 mRNA
levels generated [[88]19]. However, an expression profile of relative
changes in mRNA levels was generated. Relative changes in total
hybridization signal intensity and individual mRNAs were analyzed by
one-way ANOVA with post hoc Neumann-Keuls test analysis
[[89]19–[90]21]. The level of significance was set at p < 0.01 for
individual comparisons.
Results
Three-month-old WT, GFAP^P301L, and CaMKIIα^P301L mice were subjected
to our 20-hit model of r-mTBI (or r-sham), and 3 months after the last
injury (at the age of 7 months), 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 and microglial reactivity), astrocytic
homeostasis and astrocyte-specific pathological response under normal
(no TBI) circumstances and three months post-last injury. For the
present study, normal circumstances refer to data from 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 regions of interest we
selected antigens against several epitopes of tau hyperphosphorylation
and aggregation including tau phosphorylated at Threonine 231 (RZ3),
Serine 202 (CP13) and Serine 396/Serine 404 (PHF1) [[91]23, [92]24].
First, we examined the effects of astroglial vs neuronal mutant human
tau (htau^P301L) on promoting tau phosphorylation in cortical
astrocytes under normal circumstances. We quantified the number of
astrocytes double labeled for GFAP^+/RZ3^+ in 4 sagittal sections of
the cortex per mouse from each of the three mouse models examined in
this study. GFAP^+/RZ3^+ astrocytes were not seen in WT and
CaMKIIα^P301L mice. In the GFAP^P301L mice, an average of 6 ± 1.4
GFAP^+/RZ3^+ astrocytes were found in the cortex. Three months
post-last TBI, the number of GFAP^+/RZ3^+ astrocytes remained unchanged
compared to their shams (Fig. [93]3A, [94]B). Next, we assessed
Threonine 231 (RZ3) immunoreactivity in the cortex beneath the impact
size of each mouse model. Under normal circumstances, the
immunoreactivity of this tau epitope was unchanged in the GFAP^P301L
mice compared to the WT mice (Fig. [95]3C, [96]D). However, there was a
significant ~ 12-fold increase in CaMKIIα^P301L mice compared to both
WT and GFAP^P301L (Fig. [97]3D). Three months post-last injury (3mpi),
there were no changes in RZ3 immunoreactivity in the WT cohort;
however, the presence of astroglial pathogenic tau (GFAP^P301L) and
neuronal pathogenic tau (CaMKIIα^P301L) mice resulted in a significant
increase in p-tau (RZ3) immunoreactivity around the impact site
compared to their respective sham counterparts (Fig. [98]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 in neurons.
Fig. 3.
[99]Fig. 3
[100]Open in a new tab
RZ3 immunoreactivity in the cortex of WT, GFAP^P301L and CaMKIIα^P301L
mice at 3 months post-last injury. Qualitative images (A) and
quantification of RZ3/GFAP + cells in the cortex of GFAP^P301L 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, GFAP^P301L, and
CaMKIIα^P301L (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
Next, we used immunoblotting to evaluate brain levels of tauopathy by
assessing tau phosphorylation markers (RZ3, CP13 and PHF1) and total
tau (DA9) in the hippocampus. Because tau abundance in the
CaMKIIα^P301L model was disproportionately increased compared to the
other models, we performed a separate two-way analysis using WT and
GFAP^P301L data only, for all markers of interest.
In the hippocampus, under normal circumstances, total tau levels
displayed a significant 68-fold increase in CaMKIIα^P301L compared to
WT (two-way ANOVA: genotype effect [F(2,26) = 101.2, p ≤ 0.001])
(Fig. [101]4G) and a significant 18-fold increase in GFAP^P301L
compared to WT mice (two-way ANOVA: genotype effect [F(1,19) = 28.19,
p ≤ 0.001]) (Fig. [102]4H). CaMKIIα^P301L mice have around 4 times more
levels of tau compared to GFAP^P301L.
Fig. 4.
[103]Fig. 4
[104]Open in a new tab
Changes in Tau species (phosphorylated and total tau) in the
hippocampus of WT, GFAP^P301L and CaMKIIα^P301L 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
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 B, D, F and H are from WT and
GFAP-P301L cohorts alone. Representative immunoblots from the
hippocampus are depicted on the left of the graphs
Three months post-last injury, in the hippocampus, r-mTBI does not
cause changes in tau phosphorylation levels at any epitope in WT and
GFAP^P301L mice. In contrast, in CaMKIIα^P301L mice, r-mTBI causes an
increase of tau phosphorylation levels at specific epitopes, but not
total tau compared to their sham controls (Fig. [105]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-mediated effect on tau
phosphorylation in the hippocampus may indicate dilution of the signal
on regions distal to the site of impact.
Lastly, we wanted to overcome any developmental issues related to
astrocytic tau production from birth in the GFAP^P301L model, by
utilizing astrocyte-targeted AAV transfection of the mutant tau. Six
weeks after transfection we confirmed mutant tau expression within
astrocytes, and mice were subjected to our 20hit r-mTBI model
(Fig. [106]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 that tau phosphorylation
across the cortex and the number of cortical astrocyte-like cells that
were positive for phosphorylated tau (AT8^+) remained unchanged in sham
and TBI mice after transfection (Fig. [107]5B–D). Thus, we corroborated
in mice, independent of the time of astrocytic tau overexpression, TBI
does not cause cortical-wide neuronal tauopathy or exacerbate tau
astrogliopathy in the cortex, even by 6 months post-injury.
Fig. 5.
[108]Fig. 5
[109]Open in a new tab
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
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, we quantified the percent area of GFAP and Iba1
immunoreactivity to evaluate astrocyte and microglial reactivity,
respectively, in the cortex of each mouse model (Fig. [110]6A–F). Under
normal circumstances, the presence of tau within astrocytes in the
GFAP^P301L model did not impact GFAP and Iba1 immunoreactivity levels
in the cortex (Fig. [111]6C–F) compared to WT. The presence of neuronal
mutant tau in the CaMKIIα^P301L 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 and
GFAP^P301L mice (Fig. [112]6E, I). Iba1 immunoreactivity in the cortex
was significantly upregulated compared to the other two genotypes in
the CaMKIIα^P301L mice, represented by a twofold increase (two-way
ANOVA: genotype effect [F(2,23) = 28.55, p ≤ 0.001]) (Fig. [113]6E, F).
Fig. 6.
[114]Fig. 6
[115]Open in a new tab
Astrocyte reactivity (GFAP) and microglial reactivity (Iba1) in the
cortex (CTX) of WT, GFAP^P301L and CaMKIIα^P301L 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
indicates the impact site). Qualitative images of GFAP and Iba1 in the
cortex (A and B, respectively) of WT mice (top-two panels), GFAP^P301L
mice (middle-two panels) and CaMKIIα^P301L 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 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. Graphs from D and F are from WT and GFAP-P301L cohorts alone
Three months post-last injury, there was a significant 1.5-fold
increase in GFAP immunoreactivity in the cortex of the CaMKIIα^P301L
r-mTBI group compared to sham controls (two-way ANOVA: injury effect
[F(1,23) = 3.88, p = 0.061]; genotype*injury [F(2,23) = 0.86,
p = 0.435]) (Fig. [116]6E). Because astroyte reactivity in the
CaMKIIα^P301L 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
GFAP^P301L data (see Fig. [117]6D). Our analysis revealed that r-mTBI
induced a significant twofold increase in GFAP immunoreactivity in both
WT and GFAP^P301L 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.,
CaMKIIα^P301L model), the presence of tau within astrocytes (i.e., the
GFAP^P301L model) was not sufficient to evoke an exacerbated TBI
response compared to WT counterparts. Furthermore, no synergistic TBI
effect was found in the GFAP^P301L cohort compared to WT counterparts.
Iba1 immunoreactivity demonstrated no significant changes in the
CaMKIIα^P301L cohort in the TBI group compared 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. [118]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. [119]6F). The effect of r-mTBI exposure was not worsened in the
GFAP^P301L 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 CaMKIIα^P301L 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
(GFAP^P301L and CaMKIIα^P301L) compared to WT (Fig. [120]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. [121]7D) (two-way
ANOVA: genotype effect [F(2,27) = 2.25, p = 0.125]).
Fig. 7.
[122]Fig. 7
[123]Open in a new tab
Changes in astrocyte homeostatic protein markers in WT, GFAP^P301L and
CaMKIIα^P301L 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
Three months post-last injury, analyses revealed an absence of
TBI-dependent changes in the levels of AQP4, GLT1, and GLAST in both
regions of interest across the three genotypes (Fig. [124]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).
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 to evaluate unbiased changes in
gene expression and dysregulation of cellular pathways. First, we
verified that our samples contained astrocytes by assessing the
transcriptional expression of several published astrocyte-specific
markers [[125]25]. A clear enrichment of protein and transcriptomic
astrocytic markers compared to those for microglia, endothelial cells,
oligodendrocytes and neurons was found. Interrogating significantly
altered genes (adjusted p-value < 0.05) in the GFAP^P301L model, the
presence of tau within astrocytes dysregulated the expression of 11,401
genes (5799 upregulated DEGs and 5602 downregulated DEGs) compared to
WT mice. In comparison, the presence of neuronal tau in CaMKIIα ^P301L
astrocytes dysregulated 7900 genes (4298 upregulated DEGs and 3602
downregulated DEGs) compared to WT astrocytes (Fig. [126]8A).
Fig. 8.
[127]Fig. 8
[128]Open in a new tab
Astrocyte specific pathways that are dysregulated in GFAP^P301L and
CaMKIIα^P301L 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 GFAP^P301L vs WT and CaMKIIα^P301L 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
Pathway enrichment analysis was performed on DEGs (adjusted
p-value < 0.05 and IPA fold change cut-off = ± 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. [129]8B). On the contrary, CaMKIIα^P301L astrocytes
displayed upregulation of the above-mentioned pathways compared to WT
astrocytes (Fig. [130]8C). Key pathways identified in GFAP^P301L
astrocytes compared to WT include upregulation of calcium signaling,
and antioxidant action of Vitamin C (ascorbic acid) compared to WT
astrocytes. CaMKIIα^P301L astrocytes also exhibited a downregulation of
neurovascular coupling and antioxidant action transcripts. Together
these findings suggest that GFAP^P301L astrocytes have an
immunosuppressed phenotype with an enhanced antioxidant role and
dysregulated calcium signaling, while CaMKIIα^P301L astrocytes have a
proinflammatory phenotype and an impairment in antioxidant defense.
To identify DEGs common to astrocytes expressing human pathogenic tau
(GFAP^P301L) and those exposed to neuronal-derived htau^P301L
(CaMKIIα^P301L), we queried DEGs obtained from GFAP^P301L versus WT and
CaMKIIα^P301L versus WT mice. This comparison found 2,138 convergently
dysregulated genes (862 upregulated DEGs and 1,276 downregulated DEGs)
(Fig. [131]8A). Enriched pathway analysis identified that those
convergent DEGs were associated with the upregulation of chemokine
signaling, synaptogenesis, neuroinflammation, and mitochondrial
dysfunction; and the downregulation of 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-specific bulk 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 CaMKIIα^P301L, and 441 genes (157
downregulated DEGs and 284 upregulated DEGs) in GFAP^P301L mice
(Fig. [132]9A–D). The presence of endogenous htau^P301L in astrocytes
results in greater transcriptomic dysregulation in response to r-mTBI
compared to the exposure of astrocytes to exogenous neuronal tau in the
CaMKIIα^P301L or the absence of pathogenic human tau in the WT mice. No
single gene was dysregulated across cohorts in response to TBI
(Fig. [133]9A) highlighting the context-dependent nature of astroglial
response.
Fig. 9.
[134]Fig. 9
[135]Open in a new tab
Astrocyte specific pathways that are dysregulated in WT, GFAP^P301L and
CaMKIIα^P301L 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 (GFAP^P301L) and D
(CaMKIIα^P301L). Top 10 DEGs are highlighted on the volcano plots.
Upregulated DEGs are in red, Downregulated DEGs are in blue. Histogram
in E (WT), F (GFAP^P301L) and G (CaMKIIα^P301L) 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
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. [136]9E). In the GFAP^P301L cohort, 441 DEGs mapped to
52 significant pathways included the downregulation of oxidative
phosphorylation, synaptic long-term potentiation, and opioid signaling;
and upregulation of neuroinflammation, complement system signaling, and
ERK5-mediated cell activation (Fig. [137]9F). In the CaMKIIα^P301L
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. [138]9G). These
observations revealed that r-mTBI affects GFAP^P301L astrocytes by
compromising mitochondrial function and neuronal support mechanisms,
which favors a pro-inflammatory state compared to WT and CaMKIIα^P301L
mice.
Transcriptome changes in astroglia obtained from human CTE tissue
Prior to performing gene array analysis, we used dual
immunofluorescence to determine whether GFAP-stained 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. [139]10A–F). Then, we evaluated changes
in gene expression in hippocampal astrocytes from CTE-IV brains
compared to hippocampal astrocytes from non-demented healthy controls
(HC). GFAP^+ve astrocytes microaspirated from CTE-IV brains revealed
156 DEGs (153 downregulated and 3 upregulated) (Fig. [140]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 signaling.
These data indicate that CTE-IV astrocytes exhibit a loss of
neurorestorative function(s) accompanied by an immunosuppressed
phenotype compared to healthy controls. Interestingly, despite that
CTE-IV hippocampal astrocytes do not display phosphorylated tau (PHF1)
in their soma, CTE-IV astrocytes showed significant downregulation of
genes associated with immunological response including IL-8 signaling
and NO and ROS production similar to tau bearing murine astrocytes
(GFAP^P301L).
Fig. 10.
[141]Fig. 10
[142]Open in a new tab
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 GFAP^ve 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 hippocampus of 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 P value
(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
Discussion
Tau astrogliopathy is a pathological feature of primary tauopathies
including CTE. However, the role that tau astrogliopathy plays in the
onset and/or progression of TBI/CTE pathophysiology remains
underinvestigated. Using the GFAP^P301L mouse model harboring
tau-bearing astrocytes, we investigated the effects of endogenous
htau^P301L accumulation, r-mTBI and how their interaction might affect
astroglial pathological response. Our findings reveal that astroglial
overexpression of htau^P301L does not alter markers of gliosis but
elicits a significant reduction in hippocampal homeostatic
astrocyte-specific markers (AQP4 and GLT1) that regulate water
transport and glutamate homeostasis, respectively. Additionally,
astrocytes overexpressing tau display an immunosuppressed phenotype
similar to the phenotype observed in stage 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 [[143]26]. The
present findings indicate that compared to astrocytic tau
overexpression, neuronal tau elicits significantly more astrogliosis
and microgliosis 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 (mice age at 3mpi), CaMKIIα^P301L mice already exhibit
substantial tau pathology [[144]27] while increased tau pathology in
GFAP^P301L mice does not occur until 24 months [[145]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 models of tauopathy
[[146]4, [147]5] suggest that GFAP^P301L 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 the onset and/or progression of tau astrogliopathy. Repetitive
mTBI evoked a region-specific (i.e., underneath the impact 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 sham and 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
suggests that tau astrogliopathy in the hippocampus is inconspicuous
compared to neuronal tau [[148]28, [149]29]. CTE pathology in the
dorsal lateral frontal cortex from this brain bank has previously been
shown to be primarily neuronal [[150]28]. Further analysis with other
markers of tau phosphorylation, such as AT8 or CP13, will be needed to
confirm the extent 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 in astrocytes
in GFAP^P301L 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-mediated gliosis compared to the WT
cohort. In the case of the CaMKIIα^P301L 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 be orchestrated by an increase in neuronal
tauopathy rather than tau astrogliopathy. Further investigation on aged
GFAP^P301L 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 reduction of AQP4 and GLT1 in the hippocampus of shams
compared to WT shams. AQP4 is a water channel located on astrocytic
perivascular end-feet that is essential for water flux and waste
clearance termed the glymphatic system [[151]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 [[152]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) [[153]32]. Based on hippocampal AQP4 downregulation in both
tauopathy models, we expect GFAP^P301L have impaired memory.
Interestingly, CamkIIα^P301L mice exhibit behavioral changes at the age
of 5 months [[154]33], which likely are linked to the substantial
accumulation of pathogenic tau. Moreover, r-mTBI did not elicit changes
in AQP4 levels in the mouse models confirming other studies that have
reported no differences in AQP4 abundance after TBI [[155]34, [156]35].
Glutamate transporter 1 (GLT1) an astroglial-specific 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 [[157]36]. Thus, a significant
reduction of GLT1 expression would lead to a significant increase in
extracellular glutamate concentration which ultimately can cause
neuronal loss [[158]2]. Previous studies on CaMKIIα^P301L mice have
shown a ~ 55% loss of hippocampal neurons by the age of 6 months
[[159]37, [160]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 our models (data not shown) which
suggests that the significant reduction in GLT1 levels might affect
neurons at a functional 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 [[161]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 sham conditions, astrocytic
tau overexpression compared to neuronal tau overexpression resulted in
greater transcriptional astrocyte change. Tau-bearing astrocytes
(GFAP^P301L astrocytes) showed an immunosuppressed phenotype. On the
contrary, CaMKIIα^P301L astrocytes exposed to neuronal tau and robust
microgliosis showed a pro-inflammatory phenotype and lost their
neurovascular and antioxidant function. Under injury conditions,
GFAP^P301L astrocytes showed greater transcriptional dysregulation
compared to WT and CaMKIIα^P301L astrocytes 3mpi.
Downregulation of neuroinflammatory pathways including IL-12 and IL-15
in GFAP^P301L sham astrocytes might cause suppression of
astrocyte-microglia bidirectional crosstalk since microglia respond to
both interleukins by mounting immune responses [[162]40, [163]41].
These findings may explain the failed genotype effect observed in
microgliosis and astrogliosis between GFAP^P301L and WT 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 [[164]42], its downregulation may alter the
ability of astrocyte-microglia interaction needed to mount 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
and colleagues showed that CTE white matter astrocytes exhibit
downregulation of normal functioning, and dysfunctional mitochondrial
metabolism accompanied by increased neuroinflammation resulting in a
“neurotoxic profile” [[165]43]. The observed discrepancies in
transcriptional changes might be related to the heterogenicity of
astroglial response depending on the region they are found respective
to the impact site (hippocampus vs white matter). Moreover, astrocytes
harvested from individuals with other neurodegenerative conditions,
namely, AD, PD, MS, and HD show an upregulation of inflammatory
responses [[166]44] which contrasts with what we are reporting in the
present studies. However, analogous to what 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
[[167]46]. Additionally, it has also been reported that a
sub-population of astrocytes in neurodegenerative conditions can
display downregulation of transcripts involved in synaptogenesis and
neuronal function [[168]44, [169]45] similar to the phenotype 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. 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 that
tau-bearing astrocytes from the sham GFAP^P301L mice (vs WT) also
showed a similar immunosuppressed state we suggest that the GFAP^P301L
model is a relevant platform in which to study tau astrogliopathy in
tauopathies.
Limitations of the study
Data presented here provides insights into the effects of
overexpression of pathogenic tau in astrocytes under normal conditions
and after r-mTBI, however, this study has several limitations. The
expression of pathogenic tau in astrocytes in the GFAP^P301L model
occurs from embryonic day 14 [[170]46] and persists throughout the
animals’ lifespan, leading to an age-dependent increase in tau
astrogliopathy. In humans, it has been suggested that the 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 the internalization
and aggregation of secreted neuronal tau [[171]29].
Tau expression in our model is controlled by the GFAP promoter whose
expression is region-dependent and known to increase in response to TBI
[[172]47]. This leads to differential expression/accumulation of tau
within astrocytes throughout the brain which might contribute to
regional 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,
we will 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 [[173]48].
It has been reported that the integration of the tetO-MAPT*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 [[174]49], and thus it cannot be assumed
that mutant tau, alone, drives the reported changes in
neurodegeneration. Additionally, the trans activator (tTA) transgene
insertion, that drives the expression of mutant tau either in
astrocytes or neurons, disrupts another five genes which, collectively,
have been shown to be involved in synaptic transmission, plasticity,
and neurogenesis [[175]50, [176]51] which may indirectly confound our
findings. However, relevant to 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 overproduction of mutant tau
in astrocytes or neurons is what further decreases GLT1 (93% decrease)
in the GFAP^P301L mice. Further investigation is warranted to know the
impact of 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 paralleling the phenotype in our GFAP^P301L
model. However, the CaMKIIα^P301L model displays 4R-neuronal tauopathy
while neuronal pathology in CTE cases is mixed (3R/4R) [[177]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 microdissected from the hippocampus of
CTE-IV cases. As shown in Fig. [178]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 and were selected
because it is widely used for the identification of both astroglial
(predominantly 4R) and neuronal (both 3R/4R) tauopathies in CTE
[[179]53–[180]55]. As mentioned before, further investigation is
warranted to know if other tau epitopes might be preferentially
phosphorylated (AT8, CP13, or 4R-tau) in GFAP^+ve astrocytes,
therefore, we cannot discard or prove that the analyzed astrocytes lack
accumulation of tau. Nonetheless, we acknowledge that astrocyte
pathophysiology may differ in the presence or 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 [[181]56–[182]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 the entire brain excluding the hippocampi. Therefore,
direct comparisons 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-matched cohort 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 with the CTE vs control comparisons. Lastly,
the consensus RIN value for RNA samples is ≥ 7 [[183]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
[[184]61].
Summary
We demonstrate r-mTBI did not significantly exacerbate tau
astrogliopathy, but increased tau phosphorylation in the cortical
neurons beneath the impact site. Overexpression of htau^P301L 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 and water transport independent of TBI. Tau-bearing
astrocytes have significantly more DEGs compared to WT astrocytes and
astrocytes exposed to robust neuronal tau pathology and microgliosis
under normal circumstances. Tau-bearing murine astrocytes exhibit an
immunosuppressed phenotype that might modify bidirectional molecular
communication with microglia limiting the ability to mount immune
responses. At three months post-last injury, tau-bearing astrocytes
display a greater transcriptional dysregulation compared to WT and
CaMKIIα^P301L astrocytes showing a compromised bioenergetic system
which might interrupt their physiological roles. Finally, CTE-IV
astrocytes display an immunosuppressed phenotype similar to the effects
of tau overexpression seen in astrocytes from the GFAP^P301L mouse
model.
Collectively, the findings of this study underscore the significance of
unraveling mechanisms driving tau astrogliopathy and the
pathobiological consequences on the normal, 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 and tau-independent.
Supplementary Information
[185]12974_2024_3117_MOESM1_ESM.png^ (17.2MB, png)
Suuplemenatry Material 1: Figure 1: Astrocyte reactivity (GFAP) and
microglial reactivity (Iba1) in the corpus callosum (CC) of WT,
GFAP^P301L and CaMKIIα^P301L 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 indicates the impact
site). Qualitative images of GFAP and Iba1 in the CC (A and B,
respectively) of WT mice (top-two panels), GFAP^P301L mice (middle-two
panels) and CaMKIIα^P301L 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 after Two-way ANOVA.
Asterisks denote: *P<0.05; **P<0.01 and ***P<0.001 for post-hoc
analyses.
[186]12974_2024_3117_MOESM2_ESM.png^ (119.3KB, png)
Suuplementary Material 2: Figure 2: Tau astrogliopathy in the cortex of
GFAP^P301L 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
per group). t-test analysis yielded significant changes p<0.001***
[187]12974_2024_3117_MOESM3_ESM.png^ (314.8KB, png)
Supplementary Material 3: Figure 3: Heat map revealing expression
levels of all genes in the microarray of all healthy control (HC) cases
and brief clinical 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.
[188]12974_2024_3117_MOESM4_ESM.docx^ (15.3KB, docx)
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.
Acknowledgements