Do Carmo et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03184-7(2024) 21:185

Journal of Neuroinflammation

Open Access

RESEARCH

Differential effect of an evolving amyloid
and tau pathology on brain phospholipids
and bioactive lipid mediators in rat modelsof Alzheimer-like pathology
Sonia Do Carmo1*, Marie-Audrey I. Kautzmann2, Surjyadipta Bhattacharjee2, Bokkyoo Jun2, Carolyn Steinberg1,
Joshua T. Emmerson1, Janice C. Malcolm3, Quentin Bonomo4, Nicolas G. Bazan1,2* and A. Claudio Cuello1,3,4,5*Abstract
Background Brain inflammation contributes significantly to the pathophysiology of Alzheimer’s disease, and it ismanifested by glial cell activation, increased production of cytokines/chemokines, and a shift in lipid mediators froma pro-homeostatic to a pro-inflammatory profile. However, whether the production of bioactive lipid mediators
is affected at earlier stages, prior to the deposition of Aβ plaques and tau hyperphosphorylation, is unknown. The
differential contribution of an evolving amyloid and tau pathology on the composition and abundance of membrane
phospholipids and bioactive lipid mediators also remains unresolved.
Methods In this study, we examined the cortical levels of DHA- and AA-derived bioactive lipid mediators and of
membrane phospholipids by liquid chromatography with tandem mass spectrometry in transgenic rat models of the
Alzheimer’s-like amyloid and tau pathologies at early and advanced pathological stages.
Results Our findings revealed a complex balance between pro-inflammatory and pro-resolving processes inwhich tau pathology has a more pronounced effect compared to amyloid pathology. At stages preceding tau
misfolding and aggregation, there was an increase in pro-resolving lipid mediators (RVD6 and NPD1), DHAcontaining phospholipids and IFN-γ levels. However, in advanced tau pathology displaying NFT-like inclusions,
neuronal death, glial activation and cognitive deficits, there was an increase in cytokine and PGD2, PGE2, and PGF2α
generation accompanied by a drop in IFN-γ levels. This pathology also resulted in a marked increase in AA-containing
phospholipids. In comparison, pre-plaque amyloid pathology already presented high levels of cytokines andAA-containing phospholipids together with elevated RVD6 and NPD1 levels. Finally, Aβ plaque deposition was
accompanied by a modest increase in prostaglandins, increased AA-containing phospholipids and reduced DHAcontaining phospholipids.

*Correspondence:
Sonia Do Carmo
sonia.docarmo@mcgill.ca
Nicolas G. Bazan
NBazan@lsuhsc.edu
A. Claudio Cuelloclaudio.cuello@mcgill.ca
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-NonCommercial-NoDerivatives 4.0
International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you
give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified thelicensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it.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 to thematerial. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation
or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http://
creativecommons.org/licenses/by-nc-nd/4.0/.

Do Carmo et al. Journal of Neuroinflammation(2024) 21:185

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Conclusions Our findings suggest a dynamic trajectory of inflammatory and lipid mediators in the evolving amyloid
and tau pathologies and support their differing roles on membrane properties and, consequentially, on signaltransduction.
Keywords Alzheimer’s disease, Amyloid pathology, Tauopathy, Transgenic rat, Lipidomics, Brain phospholipids,
Bioactive lipid mediators, Neuroinflammation

Background
Alzheimer’s disease (AD), the most common form ofdementia, is characterized by a progressive and irreversible loss of cognitive functions, resulting in the inability to carry out daily activities. AD pathology develops
for decades before its clinical presentation [1], at which
point currently available therapies are ineffective. In
addition to extracellular amyloid beta (Aβ) plaques andintraneuronal neurofibrillary tangles (NFTs) composedof abnormally phosphorylated and aggregated tau, neuroinflammation is increasingly considered as the third
core pathological feature of AD. This concept is supported by genome-wide association studies indicatingthat immune-related genes are significant risk factors forAD [2–7]. The trigger, function, and trajectory of ADrelated brain inflammation remain controversial. It isevident that neuroinflammation contributes significantly
to the development and progression of AD, aggravating
both Aβ and tau pathologies [4, 8, 9]. Evidence gatheredfrom human and animal studies also suggests that inflammation is a dynamic process differing at the early and latestages of AD [10] (reviewed in [11]). Neuroinflammatory molecules also accumulate in the brain in healthy
aging [12, 13]. Furthermore, epidemiological studies haveshown that cognitively intact individuals receiving sustained nonsteroidal anti-inflammatory drugs (NSAIDs)
had approximately 50% lower incidence of clinical AD
than the general population [14]. This finding promoted
AD anti-inflammatory therapy, which has proved ineffective after clinical presentation [14–19]. This highlights
the need for a better understanding of the driving forces
behind the constantly evolving inflammatory process.
As it is well-established, transient, self-limiting neuroinflammation represents the brain’s natural response toinvading pathogens and neuronal injury, and it is usually neuroprotective by contributing to the clearance ofdebris, sustaining synaptic plasticity, and promoting neurogenesis [20, 21]. However, when the active resolution
mechanisms fail, there is a self-perpetuating activation of
inflammatory processes, leading to chronic neuroinflammation and ultimately progressing into a neurodegenerative state. The resolution of inflammation is, in part,
controlled by a switch in the production of lipid mediators from a pro-inflammatory to a pro-resolving class[22]. Lipid mediators are synthesized from cell membrane
phospholipids through the action of several key enzymes,
including phospholipase A2 (PLA2). In response toinflammatory stimuli, PLA2 releases omega-6 (linoleic
acid (LA) and arachidonic acid (AA)) and omega-3(eicosapentaenoic acid (EPA) and docosahexaenoic acid
(DHA)) polyunsaturated fatty acids (PUFAs) from themembrane phospholipids into the cytosol where they
serve as substrates for the biosynthesis of bioactive lipid
mediators through the cyclooxygenase (COX), lipoxygenase (5-LOX/15-LOX) and cytochrome P450 (CYP) enzymatic pathways [23].
Reductions in the levels of pro-resolving lipid mediators and their receptors, as well as increases in proinflammatory lipid mediators, have been evidenced in
post-mortem brain tissue and in CSF from mild cognitive impairment (MCI) and AD patients, as well as in
AD experimental models [22, 24–27]. Importantly, in
CSF, specific pro-inflammatory and pro-resolving lipid
mediators showed significant correlations with cognitive measures and with classical biomarkers of AD (Aβ42,
tau, p-tau) as early as at subjective cognitive impairment(SCI) stages [22, 26]. However, whether the productionof bioactive lipid mediators is affected at earlier stages,
prior to the deposition of Aβ plaques and tau hyperphosphorylation, is unknown. The differential contribution ofan evolving amyloid and tau pathology on the composition and abundance of membrane phospholipids and bioactive lipid mediators also remains unresolved. To shedsome light on these questions, in this study, we examined the cortical levels of DHA- and AA-derived lipid
mediators and of phospholipids in transgenic rat modelsof the AD-like amyloid and tau pathologies at early andadvanced pathological stages.

Materials and methods
Animals

The McGill-R962-hTau (high expressor) and McGillR955-hTau (low expressor) transgenic rat lines overexpress the coding region of the 2N4R isoform of humantau (MAPT) bearing the P301S mutation causative of
FTDP-17 [28], under the control of the mouse CAMKIIαpromoter cassette, which directs expression to neuronsof the forebrain, as described previously [29–31]. Bothlines are derived from the founders obtained following
the same pronuclear injection. Heterozygous R955-hTau
rats possess a milder tau pathology phenotype compared to R962-hTau (summarized in Fig. 1), in accordance with their transgene copy number (estimated to 4
and 30 insertions, respectively). The McGill-R-Thy1-APP

Do Carmo et al. Journal of Neuroinflammation(2024) 21:185

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Fig. 1 Schematic representation of the evolution of the amyloid and tau pathologies in the 3 lines of transgenic rats applied in this study. Note the accelerated tau pathology in R962- compared to the R955-hTau line. The time-points studied are indicated with red arrows in McGill-APP+/+ rats (6 and 16
months) and with red font in hTau rats (10 and 20 months)transgenic rat line overexpresses the human APP751
isoform with the Swedish and Indiana mutations under
the control of the murine Thy1 promoter [32]. Rats werehoused in pairs in temperature and humidity regulatedrooms under a 12-hour light/dark cycle and were given
standard chow and water ad libitum. All animal procedures were carried out under strict adherence to theguidelines set down by the Canadian Council of Animal
Care and were approved by the Animal Care Committeeof McGill University.

cold physiological saline prior to harvesting the brains.
One hemisphere was post-fixed in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer for 24 h followedby saturation in 30% sucrose in 0.1 M phosphate buffer.
Coronal Sect. (40 μm-thick) were cut using a freezing
microtome (Leica SM 2000R, Germany) and stored at-20 °C in cryoprotectant solution (1.1 M sucrose, 37.5%
ethylene glycol in phosphate buffer saline (PBS)). The
other hemisphere was macrodissected, and the cerebral
cortex was snap-frozen on dry ice and stored at -80 °C.

Experimental design

Immunohistochemistry

We examined (sex-balanced) cohorts of heterozygous
male and female R955-hTau and R962-hTau transgenic
rats and their corresponding wild-type (wt) littermates at
10 and 20 months of age, representing progressive stagesof tau pathology as described in the results section and
in Fig. 1 (n = 3–5 rats per genotype at each age). We also
analyzed homozygous male and female McGill-APP rats
and their wt littermates at 6 and 16 months of age, representing pre- and post-plaque amyloid pathology stages,
respectively (n = 3–5 rats per genotype at each age).

To examine total human tau distributions, we applied
immunoenzymatic procedures as previously described[29–31]. Brain sections were quenched in 3% hydrogen
peroxide and 10% methanol in tris-buffered saline (TBS)for 30 min. Tissue was then blocked in 10% normal goat
serum (NGS) in TBS containing 0.1% Triton (TBS-T) for
1 h at room temperature. After blocking non-specificantibody binding, sections were incubated with the primary antibody anti-human Tau (HT7, 1:1200, ThermoFisher, USA) in 10% NGS in TBS-T overnight at 4°C.
After washing, sections were incubated with a goat antimouse-IgG (1:100, MP Biomedicals, USA) in 10% NGS in
TBS-T for 1 h at room temperature. Signal amplification
was performed with anti-horseradish peroxidase (HRP)

Tissue collection

Rats were deeply anesthetized with 1% pentobarbital (Equithesin) and then perfused transcardially with

Do Carmo et al. Journal of Neuroinflammation(2024) 21:185

monoclonal antibody (1:30) pre-incubated with 5 ug/
mL HRP (MAP kit; MediMabs, Canada). Stainings weredeveloped with 0.06% 3,3’-diaminobenzidine. Sections
were mounted onto gelatin-coated slides, dried overnight, and then dehydrated in increasing concentrationsof ethanol, delipidated in xylene and coverslipped with
Entellan mounting media (EM Science, USA).
A similar protocol was applied to examine levels of
intracellular amyloid beta (Aβ) peptides, as previously
described [33]. However, PBS was used as a buffer insteadof TBS at all steps. After quenching and blocking in 10%
NGS in PBS-T, sections were incubated with the primary
antibody anti-Aβ (McSA1, 1:4000; Medimabs, Canada)(Grant et al. 2000) in 5% NGS overnight at 4 °C. Then,
sections were incubated with a goat anti-mouse-IgG(1:100, MP Biomedicals, USA) in 5% NGS in PBS-T for
1 h at room temperature. The following steps were performed as described above.
Images were acquired using an Axioplan 2 imaging
microscope (Carl Zeiss, Germany) equipped with an
Axiocam 506 color digital camera (Zeiss) and running
Zen Blue software (Zeiss).
Immunofluorescence

When available, additional animals were included toinclude the statistical power of the analyses. Briefly,
40-µm-thick free floating brain Sect. (2 per animal) wereincubated 30 min at 80 °C in 10 mM citrate buffer (pH6.0) for antigen retrieval, then cooled for 20 min at room
temperature. Sections were then washed with PBS andincubated with 50% ethanol to permeabilize cell membrane. Hereafter PBS-T was used for all washes, andincubations were all performed at room temperature, if
not otherwise specified. Sections were washed and nonspecific binding sites were blocked by incubating 1 hwith 10% NGS in PBS-T. Sections were then incubatedovernight at 4 °C with rabbit polyclonal COX-2 antibody(1:100, Invitrogen) and mouse monoclonal AT8 antibody (1:200, Invitrogen) in 5% NGS-PBS-T. The following day, sections were washed and incubated with goatanti-rabbit Alexa 488 and goat anti-mouse Alexa 568
secondary antibody in 5% NGS-PBS-T for 2 h. Sections
were then washed and incubated with 0.3% Sudan Black
in 70% ethanol for 5 min to reduce background autofluorescence. After washing with PBS-T then PBS, sections
were mounted on gelatin-coated slides, coverslipped with
Aqua-Poly/Mount (Polysciences) and kept at 4 °C in thedark. Negative control experiments including application of secondary antibody alone (no primary) and primary alone (no secondary) were performed. Omissionof primary antibodies resulted in no detectable fluorescent staining. Images were acquired as described above.
Images from 2 sections per animal were acquired for thelamina I-III of the parietal cortex (two image regions)Page 4 of 21

and the CA1 (two image regions). To allow quantitative
comparisons, images were acquired with the same microscope settings, adjusted specifically for each marker
assessed. For image analyses, custom, automated ImageJ
macros were created for each target investigated. We
calculated COX-2 immunoreactivity (green) as the total
mean grey value (optical density).
Western blotting of cortical homogenates

Cortical tissue (20 mg) was homogenized by sonicationin 8 volumes of cell lysis buffer (20 mM Tris-HCL pH 7.5,
150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton,
2.5 mM sodium pyrophosphate, 1 mM Na3VO4, 1 µg/ml
of leupeptin, 1 mM β-glycerophosphate; Cell Signaling
Technologies) containing protease inhibitors (cOmplete™
Protease Inhibitor Cocktail, Roche Applied Sciences).
The homogenates were centrifuged at 13,000 rpm for
45 min at 4 °C and the supernatants were kept at -80 °C.
Protein concentration was measured using a modified
Lowry assay (DC Assay, Bio-Rad laboratories Inc). Equal
amounts of sample (20 µg) were diluted in loading buffer (10% glycerol, 0.08 M SDS, 5% β-mercaptoethanoland 0.05 M Tris pH 6.8), heated at 90 °C for 5 min, andrun on a 12% polyacrylamide gel at 100 V for 2 h. Proteins were transferred onto a methanol-activated polyvinylidene difluoride membrane at 0.3 A for 1 h. After
blocking in 5% non-fat milk in TBS-T for 1 h, membranes
were incubated with primary antibodies directed against
Tau (Tau5) (MAB361, Millipore; 1:750), NfL (ab7255,
Abcam, 1:5000) or βIII-tubulin (G712A, Promega;
1:1000), in 5% non-fat milk in TBS-T overnight at 4 °C.
Membranes were washed and incubated with a speciesspecific secondary antibody for 1 h at room temperature.
Immunoreactive bands were revealed using enhanced
chemiluminescence substrate (PerkinElmer, Inc.) in an
Amersham Imager 600. Optical density of each band wasdetermined using the ImageLab software. All values werenormalized by GAPDH and expressed as relative values
compared to wt. Each experiment was repeated a minimum of two times.
Quantification of Aβ42 and cytokines

Cortical tissue (40 mg) was homogenized by sonication in 8 volumes of Tris-buffered saline buffer (150 mM
NaCl, 50 mM Tris HCl, 5 mM EDTA, pH 7.6) containing protease inhibitors (cOmplete Mini Protease Inhibitor Cocktail, Roche). Homogenates were ultracentrifuged
at 100,000 g for 1 h (soluble fraction). The pellets were
resuspended in 5 volumes of guanidine buffer (5 M guanidine HCl, 50 mM Tris HCl, pH 8.0), sonicated, andincubated for 3 h. The supernatants (insoluble fraction)
were collected after ultracentrifugation at 100,000 g for
1 h.

Do Carmo et al. Journal of Neuroinflammation(2024) 21:185

Levels of human Aβ42 were assayed in soluble andinsoluble fractions in duplicate using the MesoScale
Discovery V-plex Aβ Peptide Panel 1 (6E10) kit and the
SECTOR Imager 6000 (MesoScale Discovery, Rockville,
MD). Soluble cortical fractions were pre-diluted to 4 ug
protein/ul and assayed at a 1:2 dilution in Diluent 35.
Insoluble cortical fractions were pre-diluted to 10 ug protein/ul and assayed at a 1:50 dilution. Analyte concentrations were calculated in reference to calibrators for eachindividual analyte using the MSD Discovery Workbench
software v. 4.0 (Meso Scale Discovery). Values were normalized to total protein input and expressed as pg Aβ
peptide/mg protein. The dynamic range for Aβ38 was
8.12–12,200 pg/ml, for Aβ40 4.89–15,300 pg/ml, and forAβ42 0.328–1710 pg/ml.
Levels of cytokines and chemokines were determinedin soluble fractions pre-diluted to 4 µg protein/ul andassayed at a 1:2 dilution in Diluent 42, in duplicate. Levels of 9 cytokines (IFN-γ, IL-1β, IL-4, IL-5, IL-6, KC/
GRO, IL-10, IL-13, TNF-α) were measured using the
MesoScale Discovery Proinflammatory Panel 2 (rat) kit.
Cytokine concentrations were calculated from a standard
curve generated by the MSD Discovery Workbench software v. 4.0. Values were normalized to total protein input
and expressed as pg cytokine/mg protein.
Lipid extraction

Each cortical sample was homogenized with 3 ml MeOH
and added with 9 ml CHCl3. An internal standard mixture of deuterium-labeled lipids (AA-d8, PGD2-d4, EPAd5, 15HETE-d8, and LTB4-d4) was added to each sample.
The samples were sonicated in a water bath for 30 min,and then stored at -80 °C. The next day, the samples were
centrifuged at 4200 × g for 30 min, and the supernatantscollected in a new tube. The pellets were washed with
CHCl3/MeOH (2:1) and centrifuged. The supernatantsfrom both centrifugations were combined. Two ml of distilled water, pH 3.5, were added to the combined supernatants, vortexed, and centrifuged, revealing two layers.
The lower phase was dried under N2 gas stream, resuspended in 200 µl MeOH, and transferred to MS vials. The
sample was resuspended with 200ul MeOH and transferred to MS vial. Samples were dried under N2 and thenresuspended with 30ul MeOH: H2O = 1:1 solvent.
LC-MS/MS

Xevo TQ-S equipped with Acquity I Class UPLC (Waters
Corporation, Milford, MA) was used for lipidomics.
Analysis of fatty acids and their derivatives was performed using a CORTECS 2.7 μm 4.6 × 100 mm C18
Column (Waters Corporation, Milford, MA). 45% ofsolvent A (H2O + 0.01% acetic acid) and 55% of solvent
B (MeOH + 0.01% acetic acid) with 0.4 ml/min flow
was used initially and gradient to 15% of A for the first

Page 5 of 21

10 min, then gradient to 2% of solvent A at 18 min. 2% ofsolvent A ran till 25 min, and gradient back to 45% of A
for re-equilibration till 30 min. The capillary voltage was− 2.5 kV, desolvation temperature at 600 C, desolvation
gas flow at 1100 L/Hr, cone gas at 150 L/Hr, and nebulizer pressure at 7.0 Bar with the source temperature at
150oC. MassLynx 4.1 software was used for the operation
and recording of the data. Lipid standards (Cayman, Ann
Arbor, MI, USA) were used for tuning and optimization,
as well as to create calibration curves for each compound.
MS data were analyzed and calculated with Excel.
Analysis of phospholipids was performed using an
Acquity UPLC BEH HILIC 1.7 μm 2.1 × 100 mm Column(Waters Corporation, Milford, MA) with solvent A (acetonitrile: water = 1:1, 10mM ammonium acetate pH 8.3)
and solvent B (acetonitrile: water = 95:5, 10mM ammonium acetate pH 8.3) as mobile phase. The solvent B(100%) ran for the first 5 min isocratically, then gradient
to 20% solvent A at 13 min and 65% of A at 13.5 min. For
16.5 min, it ran isocratically at 65% of A. It went back to
100% of B to 20 min for equilibration. The capillary voltage was 2.5 kV, desolvation temperature at 550 C, desolvation gas flow at 800 L/Hr, cone gas at 150 L/Hr, andnebulizer pressure at 7.0 Bar with the source temperature
at 120 C. Phospholipid molecular species were calculated
as % of the total amount in each sample.
Statistical analysis

Statistical analyses were performed using the software GraphPad Prism version 10 (La Jolla, USA). The
D’Agostino and Pearson omnibus normality test was used
to assess normal distribution of the data. Outliers were
excluded using the ROUT method (Q = 1%). Grapheddata is presented as mean values ± SEM. Given the small
sample size, two-group comparisons were performed
with the Mann-Whitney U test. Kruskal-Wallis with
Dunn’s post hoc tests was used for multiple comparisons.
Spearman’s correlation test was used to examine associations between analytes. Significance was set to p < 0.05.

Results
Transgenic rat models expressing mutant human APP and
MAPT exhibit progressive neuropathology

To assess the differential impact of cortical tau and amyloid pathology on bioactive lipid mediators, we examined cohorts of homozygous McGill-R-Thy1-APP rats
[32] and their wt littermates at 6 and 16 months of age,as well as cohorts of heterozygous McGill-R962-hTau[31] and McGill-R955-hTau [30] transgenic rats and theircorresponding wt littermates at 10 and 20 months of age(n = 3–5 rats per genotype at each age). A summary of thephenotypes of the three transgenic lines is provided inFig. 1.

Do Carmo et al. Journal of Neuroinflammation(2024) 21:185

McGill-R-Thy1-APP transgenic rats (hereafter McGillAPP rats) develop progressive AD-like amyloid pathology in the absence of tau pathology. The phenotypeof McGill-APP transgenic rats has been extensively
reported [34–45]. Six-month-old McGill-APP rats display intraneuronal amyloid beta (Aβ) throughout brain
regions including cortical and hippocampal areas but
are devoid of amyloid plaques, while extensive Aβ plaquepathology is present at 16 months, mainly in hippocampal but also in cortical areas as shown with the humanAβ-specific McSA1 antibody [46] (Fig. 2A). The Aβplaque pathology is reflected by higher levels of soluble
and foremost insoluble Aβ42, as measured by electrochemiluminescence immunoassays, in 16-month-old
McGill-APP rats compared to younger rats (Fig. 2B).
McGill-R955-hTau and R962-hTau lines display an
age-dependent accumulation of mutated human tau
in their brain. However, heterozygous R955-hTau rats

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possess a milder tau pathology phenotype compared toR962-hTau (summarized in Fig. 1). Thus, as previously
reported [30], 10-month-old McGill-R955-hTau+/- rats
display accumulation of total human tau as shown by
HT7 immunoreactivity in the subiculum and CA1, CA2
and CA3 regions of the hippocampus as well as in layers II, III of the cerebral cortices. The distribution ofhuman tau HT7 immunoreactivity was similar but more
intense in hippocampal and cortical areas at 20 monthsof age (Fig. 2C). In comparison, neurons of 10-monthold McGill-R962-hTau+/- rats are more heavily burdenedwith HT7 immunoreactivity. However, in the brain of
20-month-old McGill-R962-hTau+/- rats, in accordancewith a neurodegenerative phenotype, HT7 immunoreactivity becomes less intense while a shrinkage of the hippocampal and cortical areas as well as an enlargement of
the ventricles is evident (Fig. 2D). In line with the immunohistochemistry results, Western blot analyses applying

Fig. 2 Transgenic models expressing mutant human APP and MAPT exhibit progressive neuropathology. A. In 6-month-old McGill-R-Thy1-APP transgenic rats, human Aβ (McSA1) immunoreactivity is restricted to the intraneuronal compartment (left). By 16 months of age, human Aβ immunoreactivity
has transitioned to the extracellular compartment and extensive Aβ plaque pathology is present (right). B. Levels of TBS-soluble and insoluble (formicacid soluble) Aβ42 peptides were measured by ECLIA. C-D Both McGill-R955-hTau (C) and McGill-R962-hTau (D) transgenic rats display progressive
tauopathy as assessed by HT-7 immunoreactivity. Note the hippocampal shrinkage and ventricular dilation in 20-month-old R962-hTau rats (right). Scalebar = 1000 μm. E-F. Levels of total tau (applying the pan-species antibody Tau5) and levels of NfL were assessed by Western blot analysis of cortical homogenates. (F) Quantification of the intensity of Tau5 and NfL immunoreactive bands. Values are expressed as means ± SEM (n = 4–5/group). Two-groupcomparisons were performed with the Mann-Whitney U test. Kruskal-Wallis with Dunn’s post hoc tests was used for multiple comparisons. # p < 0.05, ##p < 0.01 compared to age-matched wt rats; * p < 0.05, ** p < 0.01 compared to other groups

Do Carmo et al. Journal of Neuroinflammation(2024) 21:185

the pan-species antibody Tau5 recognizing phosphorylated and non-phosphorylated isoforms of tau revealed
that by 10 months of age, R955-hTau rats exhibit 3-foldhigher levels of cortical total tau compared to wt rats andthese levels rise modestly with time, reaching levels oftau ~ 4-fold higher than wt rats by 20 months (Fig. 2E-F).
In comparison, the R962-hTau line produced about 3-foldhigher amounts of cortical tau at 10 months compared toR955-hTau. However, in 20-months-old R962-hTau rats
the levels of cortical tau decreased substantially to reachlevels similar to age-matched wt rats. The decreased taulevels in 20-month-old R962-hTau rats are likely causedby the extensive brain degeneration and atrophy occurring at this age [31], as evidenced by the changes inbrain structures and HT7 immunoreactivity (Fig. 2D) as
well as by a decrease in the levels of neurofilament lightchain (NfL) (Fig. 2E-F). Defects in the total amount ofneurofilaments, the predominant structural component of the adult neuronal cytoskeleton [47, 48], would
indicate a loss of neuronal integrity. Together with theline’s characteristics illustrated in Fig. 1, this highlights

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that R962-hTau+/- rats display a stronger tau pathology
phenotype than R955-hTau+/- rats and hence that tau
pathology is stronger in 10-month-old R962-hTau+/- rats
than in 20-month-old R955-hTau+/- rats.
Progressive tau and amyloid pathologies are accompanied
by increased production of cytokines/chemokines

We next investigated whether the evolution of tau andamyloid pathologies coincided with significant changes inneuroinflammatory signals. In our rat models of tauopathy, an elevation in inflammatory molecules was mainly
seen at late tau pathology stages. Thus, levels of IL-1β,
TNF-α, and KC/GRO raised progressively with advancing pathology and were found significantly elevated in
20-month-old R962-hTau rats compared to age-matched
wt rats. One exception was IFN-γ, which was elevated in
20-month-old R955-hTau rats but not in R962-hTau rats
(Fig. 3A).
In contrast to the inflammatory profile in the tau
pathology, in line with our previous publications [33,49], we found that in McGill-APP rats, the corticalFig. 3 Cortical production of cytokines and chemokines in progressive amyloid and tau pathologies. Cortical production of IL-1β, IL-4, IL-6, IL-10, TNFα,KC/GRO and IFNγ were measured by ECLIA in McGill-R-hTau rats (A) and in McGill-R-Thy1-APP rats (B). Values are expressed as means ± SEM (n = 4–5/
group). Two-group comparisons were performed with the Mann-Whitney U test. Kruskal-Wallis with Dunn’s post hoc tests was used for multiple comparisons. # p < 0.05 compared to age-matched wt rats. * p < 0.05 compared to other groups

Do Carmo et al. Journal of Neuroinflammation(2024) 21:185

production of the pro-inflammatory cytokines IL-1β
and IL-6 was already significantly elevated (while TNF-α
trended towards an elevation) compared to wt rats at
pre-plaque stages and remained elevated at post-plaquestages. However, levels of the anti-inflammatory cytokines IL-4 and IL-10 and of IFN-γ were only found elevated at post-plaque stages (Fig. 3B).
Differential effect of amyloid and tau pathology on
pro‑inflammatory and pro‑resolving lipid mediators

Given that the progressive amyloid and tau pathologies
were accompanied by elevations in cytokines/chemokines, we then investigated the levels of pro‑inflammatory and pro‑resolving lipid mediators by LC-MS/
MS. Our analyses revealed that in our models, the AAderived prostaglandins D2 (PGD2), E2 (PGE2), and F2α(PGF2α) are more affected by tau pathology than by amyloid pathology (Fig. 4). Indeed, the levels of PGD2, PGE2,
and PGF2α increased with the extent of tau pathology(Fig. 4B). R955-hTau+/- rats, which display modest levelsof tau pathology, showed levels of prostaglandins similar to age-matched controls. In contrast, R962-hTau rats
showed age- and pathology-dependent increases in thelevels of these prostaglandins. By 20 months of age, representing late stages of tauopathy, the levels of these prostaglandins were considerably heightened and reached up
to 7-fold higher levels compared to age-matched wt andR955-hTau rats. Interestingly, the levels of PGD2, PGE2,
and PGF2α were negatively correlated with NfL (Fig. 4D)
but did not correlate with Tau levels, suggesting that thelevels of these lipid mediators are modulated in response
to a secondary Tau-driven pathology rather than to thelevels of Tau protein per se.
However, in response to amyloid pathology, thechanges in AA-derived lipid mediators were more modest than in Tau transgenic rats (Fig. 4C). As such, postplaque amyloid pathology coincided with a 2-foldincrease in PGE2 levels in 16-month-old APP rats compared to age-matched wt rats while PGF2α trended
towards an increase. Still, these levels are 10-fold lower
than those detected in 20-month-old R962-hTau rats.
Intriguingly, this rise seemed to be caused by an agedriven decrease in PGE2 and PGF2α levels in 16-monthold wt rats, given that the levels of PGE2 and PGF2α in
16-month-old McGill-APP rats are similar to 6-monthold wt and McGill-APP rats. Therefore, in McGill-APP
transgenic rats, the presence of amyloid pathology counteracted the decline in prostaglandins due to aging.
Surprisingly, neither amyloid nor tau pathology
affected the levels of the mono-hydroxy AA metabolites
5-HETE, 12-HETE and 15-HETE (hydroxyeicosatetraenoic acids), nor the levels of leukotriene B4 (LTB4) andlipoxin A4 (LXA4) (Supplemental Fig. 1). This suggests
that, in our models, amyloid and tau pathologies have

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a greater impact on eicosanoids produced through the
COX pathway than on those produced through the lipoxygenase (LO) pathway.
Likewise, our analyses revealed that the DHA-derived
pro-resolving mediators resolvin D6 (RVD6) and
10,17 S-docosatriene (neuroprotectin D1, NPD1) are
impacted differentially by tau and amyloid pathology in
our models. However, the mono-hydroxy DHA metabolites 7-HDHA, 14 S-HDHA, 17 S-HDHA, and 20-HDHA
were affected neither by amyloid nor tau pathology (Supplemental Fig. 2). Therefore, RVD6 isoforms RVD6_8.29,
RVD6_8.46, and RVD6_8.56 show a stepwise decreasein their levels from the initial to the advanced stages oftauopathy (with significantly higher levels in 10-monthold R955-hTau rats compared to 20-month-old R962hTau rats), but with a slower rate of decay than the effectof aging alone (Fig. 5B). As a result, in Tau transgenic
rats, these RVD6 isoforms are positively correlated with
NfL, representative of Tau-driven brain degeneration, but
not with the levels of Tau (Fig. 5D). In addition, similar to
RVD6_8.29, RVD6_8.46, and RVD6_8.56, NPD1 showeda stepwise decrease in its levels from the initial to theadvanced stages of tauopathy, which is more pronounced
than the effect of aging alone (Fig. 5B).
In comparison, pre-plaque amyloid pathology had amore pronounced effect on levels of DHA-derived mediators. In pre-plaque McGill-APP rats (6 months), levels
of RVD6_8.29 and RVD6_8.46 isoforms were significantly
heightened compared to age-matched wt rats. Levels of
RVD6 isoforms decreased with amyloid pathology progression and by 16 months of age, levels of most RVD6
isoforms were similar to those seen in wt rats (Fig. 5C).
Similarly, NPD1 was increased at pre-plaque stages of theevolving amyloid pathology but decreased at post-plaquestages to reach levels similar to age-matched wt rats.
The special case of RVD6_8.65 is worth mentioning. Levels of RVD6_8.65, the most abundant isoform,
increased dramatically with age and the extent of tau
pathology. Thus, RVD6_8.65 levels were significantly
elevated in 20-month-old R955-hTau (4-fold) and R962hTau rats (38-fold) compared to wt rats (Fig. 5B). Surprisingly, however, RVD6_8.65 did not correlate with
NfL or Tau levels in transgenic rats (Fig. 5D). In contrast,
RVD6_8.65 levels were higher in younger McGill-APP
rats compared to age-matched wt rats (Fig. 5C).
As prostaglandin production depends on COX-2 levels
and activity, we then examined whether COX-2 expression would be differentially expressed in cortical neurons
in relation to tau pathology using immunofluorescenceapproaches and the AT8 antibody as well as an antibody recognizing COX-2. Contrary to our hypothesis,
although COX-2 immunofluorescence levels trended
towards an increase in relation to advancing tau pathology, this elevation did not reach statistical significance.

Do Carmo et al. Journal of Neuroinflammation(2024) 21:185

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Fig. 4 Arachidonic acid-derived prostaglandins are highly increased at advanced stages of tau pathology. A. Arachidonic acid metabolism and production of eicosanoid lipid mediators including prostaglandins, leukotrienes and hydroxyeicosatetraenoic acids (HETEs). B-C. Levels of PGD2, PGE2, PGF2α
in McGill-R-hTau rats (B) and in McGill-R-Thy1-APP rats (C). Two-group comparisons were performed with the Mann-Whitney U test. Kruskal-Wallis with
Dunn’s post hoc tests was used for multiple comparisons. # p < 0.05 compared to age-matched wt rats. * p < 0.05, ** p < 0.01,*** p < 0.001 compared toother groups. D. Levels of prostaglandins and NfL are inversely correlated as assessed by Spearman’s correlation test

However, as for PGD2, PGE2 and PGF2α, COX-2 immunofluorescence levels were correlated with NfL but notwith Tau5 immunoblotting signal intensity in transgenic
rats (Supplemental Fig. 3). Additionally, within neurons,
COX-2 and AT8 immunofluorescence intensities wereweakly associated. This signifies that a cell burdened with
pTau does not necessarily display higher COX-2 levels
compared to other cells.

Given the differential effects of amyloid and tau pathologies on AA- and DHA-derived lipid mediators, we thenexamined their effect on the abundance of free very
long-chain (> C24) polyunsaturated fatty acids (VLCPUFAs) which are produced from long-chain PUFAs(LC-PUFAs; C20–C22; e.g., DHA, AA, and EPA) by theelongation of very-long-chain fatty acids-4 (ELOVL4)
enzyme. Free FA24:6 and 26:6, the intermediate products

Do Carmo et al. Journal of Neuroinflammation(2024) 21:185

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Fig. 5 Docosahexaenoic acid-derived bioactive lipid mediators are affected by amyloid and tau pathology. A. Docosahexaenoic acid metabolism andproduction of DHA-derived pro-resolving lipid mediators including Resolvin RVD6, protectin (NPD1) and polyunsaturated fatty acids. B-C. Levels of RVD6
isoforms and NPD1 in McGill-hTau rats (B) and in McGill-APP rats (C). Two-group comparisons were performed with the Mann-Whitney U test. Kruskal-Wallis with Dunn’s post hoc tests was used for multiple comparisons. # p < 0.05, ### p < 0.001 compared to age-matched wt rats. * p < 0.05 compared to othergroups. D. Levels of RVD6_8.29, RVD6_8.46, and RVD6_8.65 are directly correlated with NfL in McGill-hTau rats as assessed by a Spearman’s correlation testof the ELOVL4 pathway, and free FA32:6 n3 and FA34:6
n3, precursors of elovanoids, were elevated in 10-monthold R955-hTau rats when compared to their wt littermates, but not in 20-month-old R955-hTau rats nor
in R962-hTau rats, suggesting that these VLC-PUFAs
only respond at the very early stages of tau pathology(Fig. 6A). In contrast, amyloid pathology had no effect on
the levels of these VLC-PUFAs (Fig. 6B).
Differential effect of amyloid and tau pathology on AAand DHA-containing phospholipids

Since DHA and AA are implicated in physiological andpathological processes both as bioactive lipid mediators
and as components of membrane phospholipids (PLs)[50–52], the later affecting membrane-based cellular processes [53, 54], we then examined the impact of the amyloid and tau pathologies on the abundance of DHA- andAA-containing PLs (Fig. 7).
We found alterations in the fatty acyl chain composition of PLs in both tau and amyloid transgenic rat models. As expected, these alterations coincided better withthe extent of pathology than with aging alone. Thus, few

changes in PLs abundance were seen in 10-month-old
R955-hTau rats. Interestingly, in 20-month-old R955hTau heterozygous rats a moderate level of tau pathology,
as represented by an elevation in pTau (Ser202, Thr205;
AT8), but in the absence of modifications at other tau
epitopes and neuronal loss, as previously reported [29]and summarized in Fig. 1, caused alterations in 15 PLs,
including a significant increase of DHA-containing PLs
and, to a lesser extent, a decrease in AA-containing PLs
compared to age-matched wt littermates (Supplemental Tables 1 and Fig. 7A). The PLs affected were mainly
phosphatidylcholines (PC) and phosphatidylethanolamines (PE). In 10-month-old R962-hTau rats displaying
increased tau phosphorylation, conformational changes,
and aggregation in the absence of neuronal loss, there
were alterations in 11 PLs equally distributed between
DHA- and AA-containing species, mostly PCs and PEs.
However, the direction of the changes in PLs contrasted
with that observed in 20-month-old R955-hTau rats.
There was an increase in AA-containing species and adecrease in DHA-containing species. This increase in
AA-containing species further progressed in advanced

Do Carmo et al. Journal of Neuroinflammation(2024) 21:185

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Fig. 6 Abundance of free very long-chain (> C24) polyunsaturated fatty acids (VLC-PUFA) is heightened in early-stage tau pathology. A. Levels of FA24:6,
FA26:6, FA32:6, FA34:6 are increased in 10-month-old McGill-R955-hTau rats but not at an older age nor in McGill-R962-hTau rats. Levels of these PUFAs are
not affected by amyloid pathology (B). Two-group comparisons were performed with the Mann-Whitney U test. Kruskal-Wallis with Dunn’s post hoc tests
was used for multiple comparisons. # p < 0.05 compared to age-matched wt rats. * p < 0.05 compared to other groups

tau pathology. As expected, the brain of 20-month-old
R962-hTau rats, which display extensive tau pathology,
neurodegeneration, and brain atrophy, showed the highest number of changes in PLs abundance. Notably, there
was an elevation predominantly in AA-containing PLs
but also in some DHA-containing PLs distributed equally
between PCs, PEs, phosphatidylserines (PS), and sphingomyelins (SM).
Accordingly, at the individual level, in response toprogressing tau pathology, most DHA-containing PLs
showed a stepwise decrease in their levels that was independent of aging (Fig. 7A-B). Consequently, 20-monthold R955-hTau rats displayed significantly higher levelsof those DHA-containing PLs than age-matched wt and20-month-old R962-hTau rats. Conversely, some DHAcontaining PLs showed a stepwise increase in their levels (PC34:7, PC42:10, PE40:8, PS48:10). In contrast, theincrease in AA-containing PLs occurred mostly at stages
with significant tau pathology in 10- and 20-month-old
R962-hTau rats. Remarkably, while most PLs were elevated only at 20 months of age, some PLs were elevated at
10 months to stabilize (PC38:4, PC40:4, PE38:4, PE40:4)
or even decrease (PS44:5) afterward (Fig. 7A-B).
The pattern of alterations in PLs levels in response toamyloid pathology in McGill-APP transgenic rats wasdifferent from that of tau transgenic rats (Supplemental Tables 1 and Fig. 7A). At early pre-plaque stages (6months), only 5 PLs were impacted: most of them were
AA-containing PLs and showed increased levels. At postplaque stages (16 months), McGill-APP rats showed amarked decrease in DHA-containing PLs and an increasein AA-containing PLs, which was modest in comparison
to the rise seen in response to late tau pathology.

Discussion
In the present study, we explored the effect of an evolving amyloid and tau pathology, separately, on the phospholipid composition of cell membranes as well as on
bioactive lipid mediators of inflammation synthesizedfrom phospholipids through the action of phospholipase
A. This study provides a much-needed landscape of thechanges in lipid mediators provoked by tau pathology
as represented in transgenic rat models recapitulating
the staging of human-like tauopathy [30, 31]. Furthermore, our findings show that tau pathology has a more
pronounced effect on the levels of phospholipids andbioactive lipid mediators than amyloid pathology. These
aspects are of significance given that tau pathology is ultimately the main cause of AD dementia. Cerebral amyloid
deposition in AD pathology is poorly associated with theseverity of cognitive symptoms [55–57]. In contrast, tau
pathology emerges much closer to cognitive symptomatology [58–65], and it is associated with grey matter loss
[66]. The close relationship between tauopathy and cognition is also present in the healthy aging brain [67, 68].
As summarized in Fig. 8, we found that in tau transgenic rats, the initial tau pathology preceding increased
tau phosphorylation at Ser202-Thr205 (AT8 immunoreactivity) [29], as represented in 10-month-old R955-hTau+/- rats, is accompanied by elevations in intermediates
of VLC-PUFA synthesis and in IFN-γ. As these elevations are specific to this early pathology stage and return

Do Carmo et al. Journal of Neuroinflammation(2024) 21:185

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Fig. 7 Altered abundance of DHA- and AA-containing phospholipids in response to amyloid and tau pathology. A. Heat map analysis of PCs, PEs, PSs,
and SMs of 10- and 20-month-old McGill-R955-hTau, McGill-R962-hTau and age-matched wt rats (left) and of 6- and 16-month-old McGill-R-Thy1-APP
and age-matched wt rats (right). Rows represent mean values of phospholipids and columns represent different ages and genotypes as indicated. B-C.
Scatter plots of selected DHA- and AA-containing phospholipids show a decrease in DHA-containing phospholipids and an increase in AA-containing
phospholipids in both APP and hTau rats. Two-group comparisons were performed with the Mann-Whitney U test. Kruskal-Wallis with Dunn’s post hoc
tests was used for multiple comparisons. # p < 0.05, ### p < 0.001 compared to age-matched wt rats. * p < 0.05, ** p < 0.01 compared to other groups

Do Carmo et al. Journal of Neuroinflammation(2024) 21:185

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Fig. 8 Summary of the changes observed in this study. A. Changes in R955-hTau and R962-hTau rats. B. Changes in McGill-APP rats

to baseline levels after tau aggregation, we may speculate that a sudden rise in their levels would help predict aforthcoming increment in tau pathology in combination
with classical markers of tau pathology. This aspect calls
for further investigations. Similarly, we found that an elevation in pTau Ser202-Thr205 (AT8 immunoreactivity),prior to tau misfolding and aggregation, as represented
in 20-month-old R955-hTau +/- rats, is accompanied byelevations in DHA-containing PLs, IFN-γ, RVD6, and
NPD1 as well as a decrease in AA-containing PLs. Thesechanges are also specific to this stage and wane after tau
aggregation. Furthermore, the establishment of NFT-likeinclusions prior to neuronal death and cognitive decline,
as revealed in 10-month-old R962-hTau +/- rats, isaccompanied by a modest reduction in DHA content andmoderate elevations in AA-containing PLs as well as in
PGD2, PGE2, and PGF2α. Finally, the presence of a fullblown tauopathy, as displayed in 20-month-old R962hTau +/- rats, is characterized by signification elevations
in cytokines, prostaglandins, and AA-containing PLs.

In contrast, in McGill-APP transgenic rats, the accumulation of iAβ before the deposition of plaques(6-month-old) is accompanied by elevations in cytokine
expression, RVD6 and NPD1 levels, and AA-containing
PLs (PE and SM). After plaque deposition (16-monthold), cytokine levels remain elevated while there is a drop
in RVD6, NPD1, and DHA-containing PLs. In addition,
there is an increase in AA-containing PLs (PC and PE) as
well as a stabilization in PGD2, PGE2, and PGF2α levels,
which do not decrease with aging as they do in wt rats.
A striking difference between the two pathologies is
the timing of production of cytokines in amyloid andtau pathology, which represents an early and late pathological feature, respectively. Therefore, elevations in
IL-1β, TNFα, and KC/GRO could only be detected atadvanced stages of tau pathology, as recapitulated in
20-month-old R962-hTau transgenic rats. It is worth noting that this cohort is the only one displaying overt glial
activation. Only minor changes in microglia morphology are observed in 10-month-old R962-hTau, while

Do Carmo et al. Journal of Neuroinflammation(2024) 21:185

glial changes are absent in R955-hTau rats at both timepoints. Therefore, the production of those cytokines
seems to be associated with glial activation. Elevations inthese cytokines have been reported in post-mortem brain
tissue from individuals with AD and other tauopathies,
including frontotemporal dementia and parkinsonism
linked to chromosome 17 (FTDP-17) and in mouse models carrying tau mutations [69–72]. In contrast, IFN-γ
was only elevated in early tau pathology, as represented
in 20-month-old R955-hTau transgenic rats, prior to tau’sconformational changes and aggregation, glial activationand overt cognitive deficits [30]. This observation is inline with studies showing that high IFN-γ levels are only
detected in mild, but not in severe, stages of AD [73].
On the other hand, an increased cytokine production(IL-1β, IL-6,) was detected from early pre-plaque stagesof the amyloid pathology (6-month-old McGill-APPrats) and remained elevated at post-plaque stages whileother cytokines (most of them displaying anti-inflammatory properties) came about at later stages (IL-4,
IL-10, IFNγ). This scenario would agree with our previous reports of a pro-inflammatory process occurring at
pre-plaque stages of amyloid pathology including elevations in COX-2, IL-1β, TNFα, IL-6, CCL-2, CCL-3 and
CX3CL1 [33, 49, 74, 75]. This inflammatory process is
initiated in Aβ-burdened hippocampal neurons and coincides with microglia activation and recruitment towards
those neurons [33, 49, 74, 75]. These findings reinforce
the notion that neurons are the first inflammatory agentsin response to amyloid but not to tau pathology, and theyhighlight differences in the neurochemical pathways activated in response to the two central AD pathologies.
Alternatively, and importantly, this may also suggest that oligomerization of amyloid and tau is a prerequisite for the initiation of inflammatory responses.
Indeed, oligomeric Aβ, as revealed by neuronal NU-1
immunoreactivity [76], could already be detected inside
neurons of the subiculum, CA1, CA3, and cortical layer
V in 5-month-old McGill-APP rats presenting intraneuronal production of cytokines [33]. On the other hand,in R955-hTau heterozygous rats, the occurrence of tau
oligomerization as detected using the tau oligomer-specific antibody T22 was only revealed at 24–26 months ofage (months after the last time-point examined in thisstudy) and coincided with neuronal loss [30]. Although
not demonstrated here, one can safely speculate thatoligomeric tau species are present in 20-month-old
R962-hTau heterozygous rats displaying advanced tau
pathology and neuronal loss.
This dichotomy in inflammatory responses to amyloid
vs. tau pathologies was reflected in the production oflipid mediators derived from AA and DHA. Our findings
showed that while amyloid pathology had a modest effecton the production of AA-derived pro-inflammatory

Page 14 of 21

prostaglandins [77], the advanced tau pathology, as
reflected in heterozygous R962-hTau rats, induced significant increases in PGD2, PGE2, and PGF2α. Despite
these differences, in both pathologies, increased prostaglandin production was a late pathological event, occurring after the aggregation of Aβ and tau into neuritic
plaques or NFT-like inclusions.
In addition to their role in neuroinflammation [78–80],
prostaglandins, in particular PGD2 and PGE2, play key
roles at the synapse and the neurovascular unit where
they regulate synaptic transmission and cerebral blood
flow. PGE2 has also been shown to mediate the effects
of IL-1β on learning impairments in rats [81, 82], to suppress myeloid cell bioenergetics in aging mice [83] andto regulate neuronal apoptosis and necrosis [84–87].
Importantly, in the context of an inflammatory response,
PGE2 induces lipid mediator class-switching by inhibiting 5-LOX translocation from the cytoplasm to the
nucleus, thereby abrogating leukotriene synthesis andfavoring lipoxin synthesis, which stimulates inflammation resolution [88].
The relationship between prostaglandins and amyloid
pathology in AD has been extensively studied [89–97].
PGE synthase has been found increased in AD brains,and PGD synthase was found in amyloid plaques in AD
brains and Tg2576 mice [98, 99]. Additionally, deletion of
the PGE2 EP3 receptor reduced pro-inflammatory cytokine production, oxidative stress, and production of Aβ
peptides in mouse models of amyloid pathology [94, 100,101]. Considering the above, the small effect of amyloid
pathology on prostaglandin levels in McGill-APP rats
was somewhat surprising. However, our findings do recapitulate those from APP NL-G-F Knock-in mice where
PGD2, PGE2, and PGF2α were only found elevated in
18-month-old mice, i.e., more than 15 months after the
initiation of plaque deposition [102]. Similarly, they alignwith findings reported in J20 mice where in cortical tissue, only minor elevations in LTB4 were observed, while
AA, AA-derived metabolites, and COX metabolites
remained unchanged. However, significant alterations in
AA, AA-derived metabolites, and metabolites of LOX,
COX, and CYP were found in hippocampal homogenates[103, 104].
In comparison, information on the interaction of prostaglandins and tau is scarce. PGF2α was reported tomediate the effects of TNF-α and Zn2 + in stimulating
tau phosphorylation [105]. It was also demonstrated invitro that PGJ2 induces caspase-mediated cleavage of tau,generating the aggregation-prone Δtau [106]. In addition,
pharmacological blockade of COX-1/2 and 5-LOX withflavocoxid blunted both Aβ and tau pathology in 3xTgAD mice [107], suggesting that prostaglandins play a rolein both pathologies.

Do Carmo et al. Journal of Neuroinflammation(2024) 21:185

Therefore, in addition to revealing a relationshipbetween tau pathology and prostaglandins, the present
findings would suggest that tau misfolding and aggregation are required to induce a burst of PGD2, PGE2, and
PGF2α production coinciding with microglia phenotypicchanges [31]. The production of prostaglandins is further
amplified at late tau pathology stages, presenting overt
microglia activation, cytokine production, and neuronal cell death where it correlates with NfL levels rather
than simply tau levels. This novel observation is of potential significance for our understanding of the pathology,
especially as it does not extend to LOX metabolites.
Our findings may also suggest that in AD, the increases
in prostaglandins are mostly driven by tau pathology,
although a contribution of late-stage Aβ plaque pathology cannot be ruled out.
However, the mechanisms underlying such prostaglandin elevation remain obscure. One possible explanation
could be the stimulation of COX-2 levels and activity by
tau pathology. COX-2 plays a central role in prostaglandin production and acts as a neuronal modulator [108]. It
has been shown that in P301S mice, tau-immunoreactive
nerve cells in the brainstem and spinal cord were strongly
immunoreactive for IL-1β and COX-2 [109]. It has also
been shown that in the brains of AD patients and individuals with Down syndrome with AD pathology, COX-2
induction notably occurs in neurons bearing NFTs and indamaged axons [110]. However, using immunofluorescence approaches we could not demonstrate unequivocally that COX-2 levels are affected by tau pathology
rather than by aging alone and AT8-immunoreactive
neurons do not display increased COX-2 levels comparedto other cortical cells. In addition, COX-2 levels, as prostaglandins, do not correlate with tau levels as measured
by western blotting (Tau5 immunoreactivity) but rather
with NfL diminution. This observation aligns with thework of Oka and collaborators showing increased COX-2
levels in damaged axons of AD and DS individuals [110].
Axons are likely damaged in our hTau rats given thedecreased NfL levels coinciding with tau pathology [110].
COX-2 is also known to be induced by inflammatory
stimuli, including pro-inflammatory cytokines such as
IL-1β, mediated through protein kinase C and mitogenactivated protein kinases [111, 112]. Interestingly, IL-1β
levels are elevated at late pathology stages in R962-hTaurats. However, previous research from our group has
shown that elevated IL-1β and COX-2 levels are alsopresent in McGill-APP rats [49], which do not exhibit
increased prostaglandin levels. This suggests that theincrease in PGD2, PGE2 and PGF2α in the brain of rats
reproducing advanced tau pathology potentially involves
also other mechanisms yet to be identified .The elevatedlevels of PGD2, PGE2, and PGF2α observed in the brainsof rats exhibiting advanced tau pathology could also

Page 15 of 21

potentially be linked to increased COX-2 enzymatic
activity facilitated by tau-induced extrasynaptic NMDAR
activation rather than its levels. Tau pathology has beenshown to promote extrasynaptic NMDAR activation,
which is associated with neurodegeneration [113]. This
form of NMDAR activation has also been found to notably elevate COX-2 enzymatic activity by increasing AA
release. In contrast, synaptic NMDAR activation primarily induces COX-2 expression with relatively lower prostaglandin (PG) production [114].
These investigations also revealed a contrasting effectof amyloid and tau on the production of DHA-derived
pro-resolving lipid mediators. As such, RVD6 and NPD1
were significantly elevated in pre-plaque amyloid pathology. However, RVD6 and NPD1 were little affected by
the evolving tau pathology and were only increased in
20-month-old R955-hTau +/- rats, which display an elevation in pTau Ser202-Thr205 (AT8 immunoreactivity)[29]. Despite these differences, in both pathologies, RVD6
and NPD1 elevations seemed limited to early diseasestages prior to amyloid and tau misfolding and aggregation. The cause and significance of these transient elevations in RVD6 and NPD1 remain unknown. The notionthat they might represent an attempt at counteracting thedeleterious actions of the initial disease-aggravating cytokine production in pre-plaque McGill-APP rats could be
advanced [33, 49, 74, 75]. However, their marginal elevation in 20-month-old R955-hTau +/- rats remains unresolved, as the only evidence of an ongoing inflammatory
process is the increased IFN-γ levels.
In addition to their effects on inflammation, proresolving lipid mediators improve neuronal survival
and increase Aβ phagocytosis [25, 115–118]. Decreased
levels of RvD1, LXA4, and NPD1 have been reported in
the brain and CSF of AD patients and in AD models [24,25, 27, 116, 117]. Administration of RVE1 and LXA4 in
AD models reduced Aβ plaque deposition, reversed theinflammatory process, and ameliorated cognition [119].
NPD1, which is also triggered by oxidative stress andactivation of neurotrophins [120], was shown to enhanceneuron survival and alleviate amyloidogenic processingof APP [27, 115, 121–123]. There are no reports on RVD6
levels and function in AD. However, RVD6 was reported
as decreasing inflammation and increasing nerve regeneration in the damaged cornea [124].
Moreover, this study provided evidence that the early
tau pathology (prior to tau phosphorylation at Ser202,
Thr205 (AT8 immunoreactivity)) as represented in
10-month-old R955-hTau rats [29], could be signaled
by a sudden and transient increase in free FA24:6 and26:6, the intermediate products of the ELOVL4 pathway, and free FA32:6 n3 and FA34:6 n3, which are precursors of elovanoids [125–129]. Such elevation was notpresent in rats with more pronounced tau pathology or

Do Carmo et al. Journal of Neuroinflammation(2024) 21:185

in McGill-APP rats. This may be suggestive of an initial
reparative effect of VCL-PUFAS against tau pathology.
However, the mechanisms and function underlying thiselevation remain to be determined.
VLC-PUFAs are highly enriched in the brain and modulate neuronal function and health [130, 131], including
the release of neurotransmitters [132, 133]. Oxidative
stress and oligomeric Aβ peptides are known to trigger
the release of VLC-PUFAs. Changes in the levels of VLCPUFAs can modify the structure, fluidity, and function ofcellular membranes [134–139] and lead to cellular dysfunction and death [132, 133, 140, 141]. Still, there is very
limited information regarding alterations in VLC-PUFAs
contained in phospholipid molecular species in the brain
affected by AD and other tauopathies. Accumulation of
VLC-PUFAs due to peroxisomal dysfunction [142] wasreported in the cortex of AD cases with Braak stage V-VI
pathology [143], and their levels correlated with cognitive
deficits. In contrast, a deficiency of VLC-PUFAs in PCs
was reported in the retina of 5xFAD mice displaying an
accelerated amyloid pathology [144].
As VLC-PUFAs are found mainly in the phospholipids
PC, PE, PS, and SM, we examined levels of phospholipids
as precursors to produce bioactive lipid mediators. Onceagain, tau pathology induced more pronounced changes
in the distribution of phospholipids than amyloid. The
progressive tau pathology first induced an increase in
DHA-containing PLs coinciding with p-Tau at pSer202pThr205 (AT8 immunoreactivity), which was followed bya shift towards increased AA-containing PLs when tau
conformational changes and NFT-like inclusions developed. This suggests the existence of a tilting point in thebalance between DHA and AA-containing PLs, which
would depend on tau oligomerization and conformational changes. It may also suggest that AA-containing
PLs are more toxic to cells.
In contrast, progressing amyloid pathology was dominated by an elevation in AA-containing PLs, which wereaccompanied by decreases in DHA-containing PLs at
post-plaque stages. The present findings would align withthose reported in APP NL-G-F Knock-in mice, showing
a marked increase in AA-containing PLs and a decreasein DHA-containing PLs at late amyloid pathology stages[102]. They also corroborate the described elevations in
3 AA-containing PLs in cortical tissue from FTD cases,
which were strongly correlated with ELOVL4 levels andincreased with disease severity and neurodegeneration[145]. Decreased brain phospholipid levels and alterations in brain phospholipid metabolism have also been
noted in both the white and grey matter [146–149] of
the AD brain. These alterations evolve with AD progression and would play different roles in the white and graymatter. Changes in the grey matter are minor comparedto those in the white matter but are associated with

Page 16 of 21

vulnerability to oxidative stress while the changes in thewhite matter would be associated with a protective profile [149]. In the present study, there was no evident lossof phospholipids in cortical tissue. Rather, there was analteration in phospholipid species with a drastic accumulation of AA-containing PLs in 20-month-old R962-hTau
rats, which coincided with myelin loss and overall brain
atrophy [31].
The initial rise in DHA-containing PLs, as well as themore dramatic changes at advanced tauopathy stages,
suggest that amyloid and tau have a different effect on thecontent in PLs and, therefore, on the properties, structure, and function of cellular membranes. These alterations may explain, at least partially, the differences foundhere in lipid mediators. How amyloid and tau specificallyaffect the content in PLs of cellular membranes remains
unknown. Tau interacts with different components ofcellular membranes, including lipids, proteoglycans, andmembrane proteins, as eloquently reviewed by Bok andcolleagues 2021 [150]. This interaction modulates theaggregation and toxic properties of tau. Of interest to
this study, two hexapeptide motifs of the microtubulebinding-domain of tau are critical for the formation of
the tau-phospholipid complexes, which promote folding
and fibril formation in acidic pH and are toxic to primaryhippocampal neurons [151–154]. Likewise, Aβ binds
directly to membrane lipids and modifies the lipid bilayer
structure [155–157].

Conclusions
This study provides a landscape of changes in lipid
mediators in the evolving amyloid and tau pathologies,
separately. Our findings reveal that tau has a more pronounced effect on lipid mediators than amyloid pathology, especially regarding prostaglandins, VLC-PUFAs
and phospholipids. Still, both pathologies display similarfeatures in the timing of the production of lipid mediators. Therefore, both pathologies showed increased
production of pro-resolving lipid mediators NPD1 andRVD6 at pathology stages preceding Aβ and tau misfolding and aggregation. They also illustrate an increased
PGD2, PGE2, and PGF2α production as well as elevatedAA-containing PLs and reduced DHA-containing PLs atlate pathological stages when Aβ and tau misfolding andaggregation are established. These findings reveal a complex balance between pro-inflammatory and pro-resolving processes, which, in combination with classical AD
markers, may help identify disease stages as well as differentiate AD from other tauopathies at stages preceding
overt neuronal loss and, therefore, stages more prone tointervention.

Do Carmo et al. Journal of Neuroinflammation(2024) 21:185

Limitations and considerations

This study is exploratory in nature, distinguished by its
utilization of LC-MS/MS to examine the pro-inflammatory and pro-resolving lipidome in samples from two
distinct transgenic models of AD-like pathology. However, additional research involving larger cohorts for eachgroup is warranted, particularly to expand the number ofage-matched animals. This is especially important given
the varied profile of changes observed with aging andmodel.
Additional limitations of this study include the lackof distinction between the sexes of the animals for eachcohort in the sample analysis. This could influence experimental outcomes, hinder the complete understanding ofcertain physiological events, contribute to biased interpretations, and limit the generalizability of findings.
Abbreviations
AA	Arachidonic acidAD	Alzheimer’s disease
APP	Amyloid precursor protein
Aβ	Amyloid beta
CAMKIIα	Calcium/calmodulin-dependent protein kinase type II subunit
alphaCOX	Cyclooxygenase
CSF	Cerebrospinal fluid
CYP	Cytochrome P450
DHA	Docosahexaenoic acid
ELOVL4	Elongation of very-long-chain fatty acids-4
EPA	Eicosapentaenoic acid
FTDP	17-Frontotemporal dementia and parkinsonism linked tochromosome 17
GFAP	Glial fibrillary acidic protein
HDHA	Hydroxy docosahexaenoic acid
HETE	Hydroxyeicosatetraenoic acid
HRP	Horseradish peroxidase
IFN	γ-Interferon gammaIL	Interleukin
KC/GRO	Keratinocyte chemoattractant (KC)/human growth-regulated
oncogene (GRO)
LA	Linoleic acidLC	MS/MS-Liquid Chromatography with tandem mass spectrometry
LOX	Lipoxygenase
LTB4	Leukotriene B4
LXA4	Lipoxin A4
MAPT	Microtubule associated protein tau
MCI	Mild cognitive impairment
NFT	Neurofibrillary tangle
NGS	Normal goat serum
NPD1	Neuroprotectin D1; 10,17 S-docosatriene
NSAIDs	Nonsteroidal anti-inflammatory drugs
PBS	Phosphate buffered saline
PC	Phosphatidylcholines
PE	Phosphatidylethanolamines
PGD2	Prostaglandin D2
PGE2	Prostaglandin E2
PGF2α	Prostaglandin F2α
PLA2	Phospholipase A2
PLs	Phospholipids
PS	Phosphatidylserines
PUFAs	Polyunsaturated fatty acids
RVD6	Resolvin D6
SCI	Subjective cognitive impairment
SM	Sphingomyelins
TNF	α-Tumour necrosis factor alpha
VLC	PUFAs-Very long chain polyunsaturated fatty acids

Page 17 of 21

Wt	Wild type

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03184-7.
Supplementary Material 1
Supplementary Material 2
Supplementary Material 3
Supplementary Material 4
Supplementary Material 5
Supplementary Material 6
Acknowledgements
We thank and acknowledge Dr. Alfredo Ribeiro-da-Silva for training, technicalassistance and access to the Axioplan 2 imaging microscope. We are gratefulto Dr. Jörg Hermann Fritz and Ms. Nailya Ismailova for access and technicalassistance with the MesoScale Discovery SECTOR Imager 6000 and software.
Author contributions
SDC, NGB, and ACC conceptualized and designed the study and edited
the final manuscript. SDC, JTE, and JCM collected brain tissue samples andperformed IHC procedures. SDC carried out ECLIA analyses. CS and SDC
performed Western blot analyses. QB performed IF procedures and analyses.
NGB, M-AIK, SB, and BJ prepared samples, performed LC-MS/MS lipidomics,
and analyzed data. SDC performed data analyses and wrote the manuscript.
Funding
This study was supported by the Canadian Institute of Health Research(CIHR-PJT-186111), the Healthy Brains Healthy Lives and BrainsCAN McGill- Western Collaboration Grant (MWCG), the Morris and Rosalind Goodman
Family Foundation and the Sylvester and Pauline Chuang Foundation to ACC’sLaboratory. This study was also supported by the EENT Foundation of New
Orleans (NGB). ACC is the holder of the Charles E. Frosst/Merck-endowed
Chair in Pharmacology and a member of the Canadian Consortium on
Neurodegeneration in Aging. SDC is the holder of the Charles E. Frosst/Merck
Research Associate position.
Data availability
No datasets were generated or analysed during the current study.

Declarations
Ethics approval and consent to participate
All animal work was carried out under strict adherence to the guidelines set
down by the Canadian Council of Animal Care and was approved by the
Animal Care Committee of McGill University.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1
Department of Pharmacology & Therapeutics, McGill University, 3655
Promenade Sir William Osler, Room 1210, Montreal H3G 1Y6, Canada
2
Neuroscience Center of Excellence, School of Medicine, Louisiana State
University Health New Orleans, 2020 Gravier Street, Suite D, New Orleans,
LA 70112, USA
3
Department of Cell Anatomy and Cell Biology, McGill University,
Montreal H3A 0C7, Canada
4
Department of Neurology and Neurosurgery, McGill University,
Montreal H3G 1Y6, Canada
5
Department of Pharmacology, Oxford University, Oxford OX1 3QT, UK

Do Carmo et al. Journal of Neuroinflammation(2024) 21:185

Received: 19 April 2024 / Accepted: 23 July 2024

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