A murine model of amyloid‐enhanced tauopathy: Comparison of wild‐type and P301L‐tau

Abstract

INTRODUCTION

To optimize a model of amyloid‐enhanced tauopathy we compared wild‐type (WT) tau with P301L‐tau in a mouse with mature amyloid.

METHODS

Mice transgenic for amyloid precursor protein and presenilin‐1 (APP+PS1, A/P) were injected intravenously with adeno‐associated virus with capsid B10 (AAV.CAP‐B10) expressing either WT or P301L human tau. Mice were behaviorally assessed 5 and 8 months and tissue collected 9 months after injection.

RESULTS

A/P+WT and A/P+P301L groups were equivalently impaired in spatial learning and memory. A/P+WT mice had significantly greater tau hyperphosphorylation than A/P+P301L mice. However, A/P+P301L mice had greater deposition of phospho‐tau and formation of Gallyas‐positive neurofibrillary tangles (NFTs).

DISCUSSION

These data demonstrate that WT‐tau is more highly phosphorylated than P301L‐tau in the presence of amyloid in mouse brain. Furthermore, despite the lack of NFT pathology in A/P+WT mice, WT‐tau still caused cognitive impairment. The use of WT‐tau in this model may make it more translatable for pre‐clinical assessments of potential therapeutics for AD.

Highlights

• P301L and non‐mutated (wild‐type [WT]) tau produce equivalent and severe cognitive deficits when introduced into mice with mature amyloid deposits.

• P301L‐tau deposits more but WT‐tau is more prone to phosphorylation.

• P301L‐tau results in formation of Gallyas‐positive neurofibrillary tangles, whereas WT‐tau does not.

• Enhanced translation to Alzheimer's disease will be observed with this model of amyloid‐enhanced tauopathy using WT‐tau.

1. BACKGROUND

Alzheimer's disease (AD) is a progressive neurodegenerative disease with the hallmark neuropathologies of extracellular amyloid beta (Aβ) plaque deposition and intracellular neurofibrillary tangles (NFTs) of tau (microtubule associated protein tau; MAPT). The amyloid cascade hypothesis stipulates that Aβ deposition initiates tau pathology, including hyperphosphorylation and deposition of tau. ¹ , ² Although Aβ deposition occurs first, it is the tau pathology the correlates best with neuronal loss and cognitive decline in patients with AD. ³ , ⁴

Animal models have been used extensively to investigate the underlying mechanisms of neuronal loss and cognitive decline in AD. However, many of these models have either Aβ pathology or tau pathology, but not both. ⁵ , ⁶ Furthermore, models of tauopathy often include a mutation associated with frontotemporal dementia (or other tauopathies) within the tau protein. Mouse models with tau mutations at the P301 position have been the most commonly used for treatment‐evaluation studies. ⁷ Prior work has highlighted the biochemical differences between mutant and wild‐type (WT) tau. WT‐tau has greater affinity for microtubules than P301L mutant tau. ⁸ Furthermore, P301L‐tau has an increased propensity to aggregate than WT‐tau, requiring only 4–6 moles of phosphate per mole of P301L‐tau whereas WT‐tau requires 8–10 moles of phosphate per mole of WT‐tau. ⁹ , ¹⁰ , ¹¹

Although few studies have compared WT‐tau and P301L‐tau directly in vivo, some differences have been reported. In tau transgenic mice, Gamache et al. ¹² reported that WT‐tau is neurotoxic during post‐natal development and induces cognitive deficits, whereas they did not observe neurotoxicity or cognitive deficits in transgenic mice expressing P301L‐tau. Using a gene therapy approach in middle‐aged nontransgenic (NonTg) mice, Tetlow et al. ¹³ injected adeno‐associated virus (AAV) intracranially expressing mutant or WT‐tau. The authors observed more severe neurotoxicity and increased phosphorylation, but reduced insoluble tau deposition with WT‐tau compared to P301L‐tau. In another study using AAV injection into NonTg mice, Koller et al. ¹⁴ injected neonatal (P0) pups with AAV‐tau and observed phospho‐tau after 3 months with both WT‐tau and P301L‐tau. Cognitive deficits and paired helical filament (PHF1)‐positive tau were observed at 6 months post‐injection, but neither WT‐tau nor P301L‐tau formed insoluble NFTs. Similarly, multiple markers of neurodegeneration were observed 6 weeks after P0 injection of AAV‐WT‐tau. ¹⁵ In a study of tau variants overexpressed by intracranial AAV injections into the CRND8 mouse model of amyloidosis, Koller et al. ¹⁶ reported that P301L‐tau, but not WT‐tau, formed neurofibrillary tangles, but only in the presence of amyloid plaques. Another study comparing P301S‐tau to WT‐tau yielded similar results in a transgenic amyloidosis model. ¹⁷ Additional work is needed to further refine a more translatable mouse model of AD with both hallmark pathologies using AD‐relevant wild‐type tau.

RESEARCH IN CONTEXT

1. Systematic review: The authors searched PubMed for articles that combined amyloid and tau pathologies with tau exposure initiated after amyloidosis was established, as in Alzheimer's disease (AD). None of these prior models used systemic administration of tau vectors. Only one compared different tau variants, including wild‐type (WT) tau.

2. Interpretation: We found that WT tau, the variant associated with AD, caused behavioral impairments that were equivalent to those observed with P301L‐tau, responsible for some frontal cortical dementias. We further identified that P301L‐tau was more prone to deposition. Unexpectedly, we found that WT tau was more prone to hyperphosphorylation.

3. Future directions: We believe that models using WT tau will have more relevance for AD. Other variants, such as PAD‐12 tau may be even more relevant, since it forms the same “fold” as found in tau aggregates from AD brain.

We have previously published on the characterization of a model of amyloid‐enhanced tauopathy in which we overexpressed P301L‐tau, using intravenous AAV, in transgenic amyloid precursor protein + presenilin‐1 (APP + PS1 [A/P]) mice with mature amyloid pathology. ¹⁸ These injections expose most brain regions to a relatively uniform and low level of tau overexpression. We observed significant enhancement of tauopathy in these A/P mice compared with NonTg littermate mice injected with AAV‐P301L‐tau. In order to increase the potential for translation to human AD, we wished to examine if WT‐tau would yield similar cognitive deficits and tauopathy as P301L‐tau when overexpressed in the presence of mature amyloid pathology. In the present study, 10‐month‐old APP+PS1 mice were intravenously injected with AAV.CAP‐B10 overexpressing either WT‐tau (A/P+WT) or P301L‐tau (A/P+P301L) and the resulting pathology was assessed after 9 months of survival time.

2. METHODS

2.1. Animals

The transgenic mouse line Tg2576, carrying mutated human APP K595N/M596L, was crossed with PS1 line 5.2, carrying mutated PS1 M146L, to generate A/P mice and NonTg littermates. ¹⁹ The genotype of experimental mice was determined by ear punch biopsy at weaning and confirmed after euthanasia by Transnetyx, Inc (Cordova, TN, USA). The animals were maintained on a 12‐hour light/12‐hour dark cycle and given food and water ad libitum. Mice were group housed until enrollment into the experiment, when possible. All mice were singly housed just prior to viral injection. Each treatment group consisted of approximately 50% male and 50% female mice. The P301L‐injected mice in this experiment are also used in our companion paper for comparison with an earlier timepoint of P301L‐injected mice. ²⁰ Some of the results from these mice are duplicated within the two papers. All animal experiments were conducted in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals. ²¹

2.2. Cloning and adeno‐associated virus (AAV) production

Human tau (4R2N), both WT and with the P301L mutation, was cloned into the pTR MCSW vector as reported previously. ¹⁸ Clones were confirmed by sequencing. The vectors contained AAV2 terminal repeats, the ubiquitous CAG promoter, human tau, the woodchuck hepatitis virus posttranscriptional regulatory element, and the bovine growth hormone poly‐A signal for transcription termination. Expression of these vectors was confirmed by transfection into 293T cells. The construct encoding AAV.CAP‐B10, pUCmini‐iCAP‐AAV.CAP‐B10, was a generous gift from Viviana Gradinaru (Addgene plasmid # 175004; http://n2t.net/addgene:175004; RRID:Addgene_175004). ²² Recombinant AAV.CAP‐B10 particles were generated using the triple transfection method as described previously and quantified using the dot‐blot method with a nonradioactive biotinylated probe for human tau generated by polymerase chain reaction (PCR). ²³

2.3. AAV injection

One cohort of 10‐month‐old A/P mice was injected into the retro‐orbital sinus with 100 µL of 9.45 × 10¹² vg/mL of AAV.CAP‐B10 hTau P301L (n = 10, five females and five males) or AAV.CAP‐B10 hTau WT (n = 9, four females and six males). These mice were behaviorally assessed on the radial‐arm water maze (RAWM) task 4 months after injection. A second cohort of 10‐month‐old A/P mice were injected with 100 µL of 9.45 × 10¹² vg/mL of AAV.CAP‐B10 hTau P301L (n = 13, five females and eight males) or AAV.CAP‐B10 hTau WT (n = 9, four females and five males). This second cohort was behaviorally assessed in the open field, elevated plus maze, and nest building 8 months post‐injection (Figure 1). The  mice in the A/P+P301L group collected at 19 months (Cohort 2) are also used for the comparison with A/P+P301L mice collected at 15 months of age in our companion paper describing the time course of A/P+P301L tau phenotype. ²⁰ Some of the results for these mice are duplicated in the two papers.

FIGURE 1.

Schematic of experimental design.

FIGURE 2.

Mutant P301L‐tau results in greater protein expression than wild type (WT)‐tau. (A) Graph of viral genomes per mouse genome measured by quantitative PCR (qPCR). Mice injected with P301L‐tau (A/P+P301L) had greater transduction efficiency than those injected with WT‐tau (A/P+WT). (B) Graph of human tau mRNA (MAPT) expression, relative to glyceraldehyde‐3‐phosphate dehydrogenase (Gapdh), measured by qPCR. Mice injected with P301L‐tau had greater MAPT expression than mice injected with WT‐tau. (C) Graph of MAPT mRNA expression normalized to viral genomes. (D) Graph of soluble total tau, measured by ELISA, normalized to MAPT mRNA expression, measured by qPCR. Mutant P301L‐tau results in greater relative translation than WT‐tau. Data from A/P+P301L mice also presented in. ²⁰ Data presented as mean ± standard error of the mean (SEM), with each data point representing the value of one animal (n = 9–14). Data analyzed by two‐sided Student's t‐test, * denotes p < 0.05, ** denotes p < 0.01, **** denotes p < 0.0001.

2.4. Behavioral assessment

Uninjected NonTg mice (n = 10, five females and five males) were used as indicators of normative performance on behavioral tasks. The experimenter was blinded to condition and mice were run in a group balanced order on each task. Dry mazes were cleaned with 70% ethanol between each mouse. To assess general activity levels, animals were recorded for 15 min in a 40 cm by 40 cm open field box with video‐tracking software. To assess anxiety‐like behaviors, animals were recorded for 5 min on an elevated plus maze with 35 cm by 5 cm arms. Video recordings were analyzed using ANY‐maze software, version 7.15 (Stoelting, Wood Dale, IL). A nesting score, which is sensitive to hippocampal lesions, was determined as described previously. ²⁴ Mice were given a single cotton nestlet and allowed to build a nest for 24 h before the nest was scored. The radial arm water maze (RAWM) task was performed as described in detail previously. ²⁵ Briefly, mice were trained to find an escape platform in a six‐armed water maze, using extra maze visual cues on Day 1. Each of the 15 trials lasted 60 s and the location of the platform (alternating between visible and hidden) remained the same for each mouse while the start arm changed for each trial. On Day 2, the mice were tested using only a hidden platform in the same location as on Day 1. On the third day, a reversal trial was performed with the platform hidden in the arm opposite from the goal arm on Days 1 and 2. Entry into an incorrect arm of the maze was counted as an error. Failure to enter an arm for 15 s was also recorded as an error. The errors of blocks of three trials were averaged for data analysis.

2.5. Euthanasia and tissue collection

Nine months after AAV injection, mice were weighed and injected with a pentobarbital (100 mg/kg) and phenytoin (12.5 mg/kg) solution. Deeply anesthetized mice were transcardially perfused with 25 mL of 0.9% saline. The right hemibrain was dissected into anterior cortex, posterior cortex, hippocampus, and cerebellum, which were snap frozen on dry ice and stored at –80°C. Left hemibrains were fixed in fresh 4% paraformaldehyde for 24 h at 4°C before being cryoprotected in sucrose by successive 24‐h incubations in 10%, 20%, and 30% sucrose solutions. Hemibrains were frozen on a cold stage of a sliding microtome and sectioned horizontally into 25‐µm thick sections. Sections were stored in phosphate‐buffered saline (PBS) with 10 mM sodium azide at 4°C.

2.6. Tissue homogenization and Enzyme‐Linked Immunosorbent Assay (ELISA)

Frozen hippocampi were weighed and homogenized in radioimmunoprecipitation assay (RIPA) buffer (10 µL per mg of tissue mass) with protease inhibitor cocktail (Sigma Aldrich, Cat. No. P8340) and phosphatase inhibitor cocktails II and III (Sigma Aldrich, Cat. No. P5726 & P0044, respectively). The crude homogenate was sonicated and centrifuged at 50,000 x g for 1 h at 4°C. The resulting supernatant, the RIPA‐soluble fraction, was transferred to a new tube. The RIPA‐insoluble pellet was resuspended in 70% formic acid (2 µL per mg tissue mass), sonicated, incubated for 1 h at room temperature, and neutralized in 167 mM Tris. Invitrogen enzyme‐linked immunosorbent assay (ELISA) kits were used to analyze human total tau (ThermoFisher, Cat. No. KHB0041), tau phosphorylated at serine residue 199 (pSer199) (ThermoFisher, Cat. No. KHB7041), tau phosporylated at serine residue (pSer396) (ThermoFisher, Cat. No. KHB7031), Aβ42 (ThermoFisher, Cat. No. KHB3441), Aβ40 (ThermoFisher, Cat. No. KHB3481), and Aβ aggregate (ThermoFisher, Cat. No. KHB3491). Prior to ELISA analysis, supernatant and formic acid samples were assayed for total protein (ThermoFisher, Cat. No. 23227), and then diluted in PBS and sample buffer to be within standard curve range of each respective ELISA. Kits were performed per manufacturer's instructions. Values were converted to mass of target per mass of total protein of that fraction. For tau species, a deposition ratio was calculated mathematically by dividing insoluble tau by soluble tau. Similarly, a phosphorylation ratio was calculated by dividing phospho‐tau by total tau within each fraction.

2.7. Nucleic acid isolation and quantitative PCR (qPCR)

Genomic DNA and RNA were isolated simultaneously from frozen posterior cortical samples using AllPrep kits per the manufacturer's instructions (Qiagen, Cat. No. 80004). RNA and genomic DNA were quantified by nanodrop. RNA was reverse transcribed to complementary DNA (cDNA) using Bio‐Rad iScript RT and the expression of human tau (microtubule‐associated protein tau [MAPT]) and mouse glyceraldehyde‐3‐phosphate dehydrogenase (Gapdh), a housekeeping gene, was assessed by qPCR using the relative standard curve method. Primer/probe sets were from ThermoFisher: MAPT assay number Hs00213941_m1 and Gapdh assay number Mm99999915_g1. The genomic DNA was assayed by PCR for AAV genomes, using the MAPT probe, and a single copy mouse gene (Gapdh) using the same method. AAV transduction efficiency was calculated for each mouse as the number of viral genomes per mouse genome. Transcription efficiency was mathematically determined by dividing, on a per‐animal basis, the MAPT messenger RNA (mRNA) expression by the transduction efficiency. Similarly, translation efficiency was mathematically determined by dividing the soluble total tau ELISA values by the MAPT mRNA expression (not corrected for differences in transduction).

2.8. Immunohistochemistry

Six, evenly spaced horizontal sections spanning the entire brain were chosen for analysis. Immunohistochemistry experiments were performed as described previously. ²⁶ The commercially available antibodies for total tau (HT7) and phospho‐tau (AT8) were purchased with biotin conjugation from ThermoFisher (HT7 Cat. No. MN1000B; AT8 Cat. No. MN1020B). Briefly, floating sections were placed into a multi‐sample staining tray. Endogenous peroxidases were blocked (10% methanol, 3% hydrogen peroxide in PBS) and tissue was permeabilized (0.2% lysine, 0.1% Triton X‐100, and 4% goat serum in PBS). Sections were incubated with primary antibody in 4% goat serum in PBS overnight at room temperature. The next day, sections were washed three times in PBS and incubated with Vectastain Elite ABC kit for enzyme conjugation for one hour at room temperature. Sections were washed again and stained with 0.05% diaminobenzidine with nickel enhancement and 0.03% hydrogen peroxide for 5 min at room temperature. Each assay omitted some sections from primary antibody incubation to evaluate nonspecific staining. Sections were mounted onto slides, allowed to dry, dehydrated in ethanol solutions, delipidated in Histoclear, and cover slipped.

For the Congo red and Gallyas stains, sections were first mounted onto slides and allowed to dry. Congo red and Gallyas silver stain were performed as described previously. ²⁷ , ²⁸ For Congo red, sections were briefly rehydrated in distilled water, and then incubated for 20 min in alkaline‐saturated sodium chloride solution. The slides were then incubated in filtered 0.2% Congo Red in alkaline saturated sodium chloride solution for 30 min. Slides were then dehydrated, delipidated, and cover slips applied. For Gallyas stain, slides were briefly rehydrated in distilled water. Slides were incubated in 5% periodic acid for 5 min, washed briefly with water, and incubated in silver iodide solution (1 min) before being placed in 0.5% acetic acid for 10 min. Sections were then placed in freshly made developer solution (2.5% sodium carbonate, 0.1% ammonium nitrate, 0.1% silver nitrate, 1% tungstosilicic acid, 0.7% formaldehyde) and incubated until color developed, after which they were incubated in 0.5% acetic acid solution for 3 min to stop the reaction. Slides were incubated in 0.1% gold chloride for 5 min, washed with water, and incubated in 1% sodium thiosulphate solution for 5 min. Finally, slides were washed in water, dehydrated in ethanol solutions, delipidated in Histoclear, and cover slipped.

Slides were digitized using the Zeiss AxioScan.Z1 scanning microscope using a 20x objective. Images were analyzed using Neurocyte IAE software (created by Andrew Lesniak) using hue, saturation, and intensity to segment the images. The lightest and darkest sections were used to determine threshold values for these settings. Once identified, these values were held constant for analysis of every section in that stain. The percent‐area staining was determined mathematically by dividing the number of positive pixels by the total number of pixels within the user‐defined region of interest (ROI). Hippocampal volume was estimated using the method of Cavalieri and reported as mean ± standard error of the mean (SEM).

2.9. Statistical analysis

Statistical analysis was performed using GraphPad Prism Statistics Software version 10.6.1 (Sand Diego, CA, USA). A one‐way or repeated‐measures analysis of variance (ANOVA) was used to analyze behavioral data (which included the NonTg mice), whereas a two‐tailed Student's t‐test was used to analyze tau pathology data (excluding NonTg mice lacking AAV‐tau). The threshold for significance was set at p = 0.05.

3. RESULTS

3.1. Increased translation of P301L mutant tau compared to WT‐tau

We first assessed transduction efficiency and the expression of both WT and P301L mutant tau. We observed that our P301L‐tau virus had greater transduction efficiency than the WT‐tau virus and, similarly, there was significantly more mRNA expression of the P301L‐tau than the WT‐tau (Figure 2A, B). However, the same amount of mRNA expression per viral genome was found for both tau constructs (transcription efficiency; Figure 2C). However, when comparing the amount of soluble total tau, we observed greater translation efficiency of P301L‐tau than WT‐tau, calculated by normalizing total soluble tau (measured by ELISA) to tau mRNA expression for each mouse (Figure 2D).

3.2. Both P301L mutant tau and WT‐tau impair cognitive function

Spatial learning and memory were assessed 4 months post‐injection by the RAWM behavioral task. Untreated NonTg control mice learned the location of the escape platform, making one or fewer errors per block by the end of the testing day. Both A/P+P301L and A/P+WT mice failed to learn the platform location, making significantly more errors than untreated NonTg control mice on the testing day (Figure 3). Figure 3 also contains data from the companion study that included A/P mice without AAV tau injections ²⁰ (solid black line). The performance of both AAV‐tau injected groups was worse on Day 2 than we observed in uninjected A/P mice. In the open field, A/P+P301L and A/P+WT mice both demonstrated hyperactivity, traveling a greater distance than the NonTg mice (Figure 4A). Similarly, both AAV‐tau injected groups demonstrated increased impulsivity, with more time spent in the open arms of the elevated plus maze than NonTg mice (Figure 4B). In a murine variant of “activities of daily living,” both A/P‐tau groups exhibited deficits in nest building compared to NonTg mice (Figure 4C).

FIGURE 3.

Both wild type (WT)‐tau and P301L‐tau worsen performance of amyloid precursor protein + presenilin (A/P) mice on the radial arm water maze. Graph of average errors on each block of trials. A/P+WT and A/P+P301L mice make significantly more errors than nontransgenic (NonTg) mice. The dotted line indicates criterion performance of ≤1 error per block of trials. Data presented as mean ± standard error of the mean (SEM; n = 9–14). Data analyzed by two‐way repeated‐measures ANOVA with Tukey's post hoc test. * denotes p < 0.05, A/P+P301L compared to NonTg; # denotes p < 0.05, A/P+WT compared to NonTg.

FIGURE 4.

Amyloid precursor protein + presenilin‐1 (A/P) mice Injected with tau virus are hyperactive, disinhibited, and build poor nests. (A) Graph of distance traveled in open field. A/P mice injected with either tau virus travel further than NonTg mice. (B) Graph of time spent in the open arms of the elevated plus maze. A/P+P301L and A/P+WT tau mice spend more time exploring the open arms of the elevated plus maze than NonTg mice. (C) Graph of nesting score after 24 h with a nestlet. A/P+P301L and A/P+WT tau mice build less complex nests than NonTg mice. Data from A/P+P301L mice also presented in. ²⁰ Data presented as mean ± standard error of the mean (SEM), with each data point representing the value of one animal (n = 9–14). Data analyzed by one‐way ANOVA with Tukey's post hoc test (A & B) or Kruskal–Wallis with Dunn's post hoc (C), * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001, **** denotes p < 0.0001.

3.3. Greater deposition of P301L‐tau but more extensive phosphorylation of WT‐tau

Neurochemical estimates of tauopathy were assessed by ELISA in RIPA‐soluble and RIPA‐insoluble fractions of hippocampal homogenates. Mice injected with P301L‐tau had significantly greater soluble total tau than mice injected with WT‐tau. However, there was no difference in soluble pS199 tau and, of interest, A/P+WT mice had significantly more soluble pS396 tau than A/P+P301L mice (Figure 5A–C). Similarly, A/P+P301L mice had significantly more insoluble total tau than A/P+WT mice. There were no significant differences in insoluble phospho‐tau between A/P+P301L and A/P+WT mice (Figure 5D–F). A deposition ratio was calculated by dividing insoluble tau by soluble tau for each tau species measured. We observed that A/P+P301L mice had significantly greater deposition ratio of pS396 tau than A/P+WT mice, with a trend for increased deposition ratio of pS199 tau (Figure 5G–I).

FIGURE 5.

Mutant P301L‐tau deposits more but wild type (WT‐tau is more prone to phosphorylation. (A) Graph of soluble total tau. Mice injected with P301L‐tau have greater soluble total tau than mice injected with WT‐tau as measured by enzyme‐linked immunosorbent assay (ELISA). (B) Graph of soluble tau phosphorylated at serine residue 199 (pSer199). (C) Graph of soluble tau phosphorylated at serine residue 396 (pSer396). Mice injected with WT‐tau have greater pS396 tau levels than mice injected with P301L‐tau as measured by ELISA. (D) Graph of total insoluble tau. Mice injected with P301L‐tau have more insoluble total tau than mice injected with WT‐tau as measured by ELISA. (E) Graph of insoluble pS199 tau. (F) Graph of insoluble pS396 tau. (G) Graph of ratio of total insoluble tau to total soluble tau. (H) Graph of ratio of insoluble pS199 tau to soluble pS199 tau. (I) Graph of insoluble pS396 tau to soluble pS396 tau. Mice injected with P301L‐tau have greater ratio of insoluble pS396 tau than mice injected with WT‐tau. Data from A/P+P301L mice also presented in. ²⁰ Data are ELISA values normalized to total protein in respective fraction and presented as mean ± standard error of the mean (SEM), with each data point representing the value of one animal (n = 9–14). Data analyzed by two‐sided Student's t‐test, * denotes p < 0.05, ** denotes p < 0.01, **** denotes p < 0.0001.

To quantify the fraction of soluble or insoluble tau that was phosphorylated, we calculated a phosphorylation ratio by dividing phospho‐tau by total tau within each fraction. Surprisingly, we observed that a significantly greater fraction of tau was phosphorylated at pS199 and pS396 in both soluble and insoluble fractions of A/P+WT mice than A/P+P301L mice (Figure 6).

FIGURE 6.

Enhanced phosphorylation of wild type (WT‐tau compared to P301L‐tau. (A) Graph of soluble tau phosphorylated at serine residue 199 (pSer199) relative to total soluble tau. (B) Graph of soluble tau phosphorylated at serine residue 396 (pSer396) relative to total soluble tau. Mice injected with WT‐tau have significantly more soluble phospho‐tau, relative to total soluble tau levels, than mice injected with P301L‐tau. (C) Graph of insoluble pS199 tau relative to total insoluble tau. (D) Graph of insoluble pS396 tau relative to total insoluble tau. Mice injected with WT‐tau have significantly more insoluble phospho‐tau, relative to total insoluble tau levels, than mice injected with P301L‐tau. Ratios mathematically derived from enzyme‐linked immunosorbent assay (ELISA) values. Data from A/P+P301L mice also presented in. ²⁰ Data presented as mean ± standard error of the mean (SEM), with each data point representing the value of one animal (n = 9–14). Data analyzed by two‐sided Student's t‐test, **** denotes p < 0.0001.

3.4. P301L‐tau deposits into argyrophilic tangles and exacerbates gliosis

Tau pathology was also assessed histologically. In general, both tau variants stained cells with neuronal morphology and in the hippocampal neuronal laminae. In the cortex there are staining voids, which in other work we found were also Congo red positive. ¹⁸ In addition, we observed a similar pattern of staining as we reported in Finneran et al. (2024) ¹⁸ with widespread staining of total human tau, but phospho‐tau staining was restricted to brain regions with amyloid burden (Figure S1). A/P+P301L mice had significantly greater total human tau area staining in the hippocampus than A/P+WT mice, with a similar trend in the cortex (Figure 7C). This is consistent with the ELISA measures of total tau presented earlier. However, A/P+WT mice had significantly greater AT8 phospho‐tau area staining in both the cortex and hippocampus than A/P+P301L mice (Figure 7D), concordant with the ELISA measures of phosphorylated tau. Gallyas silver stain was performed to assess insoluble tau deposits that are consistent with NFT pathology. We observed significantly greater area staining of reaction product in the cortex of A/P+P301L mice compared to A/P+WT mice (Figure 7F), paralleling the increased deposition ratio measured by ELISA. What little Gallyas labeling there was in A/P+WT mice appeared morphologically to be blood vessels rather than intraneuronal deposits. We did not observe Gallyas staining within the hippocampus of either group of A/P‐tau mice. In order to assess atrophy, an estimate of hippocampal volume was calculated from stained sections. Neither A/P+WT mice (11.65 ± 0.50mm³) nor A/P+P301L (11.03 ± 0.42 mm³) mice had significantly altered hippocampal volume compared to nontransgenic control mice (11.54 ± 0.40 mm³).

FIGURE 7.

Greater total tau area staining and argyrophilic deposits in mice injected with P301L‐tau. (A) Representative images of HT7 staining in the cortex and hippocampus of mice injected with P301L‐tau and wild type (WT)‐tau. Scale bar in cortex = 100 µm; scale bar in hippocampus = 200 µm; scale bar of insets = 50 µm. (B) Representative images of AT8 staining in the cortex and hippocampus of mice injected with P301L‐tau and WT‐tau. Scale bar in cortex = 100 µm; scale bar in hippocampus = 200 µm; scale bar in insets = 50 µm. (C) Graph of area staining of HT7 reaction product. Mice injected with P301L‐tau had significantly more area staining in hippocampus than mice injected with WT‐tau. A similar trend was observed in cortex (p = 0.08). (D) Graph of area staining of AT8 reaction product. Mice injected with WT‐tau had greater AT8 area staining in both cortex and hippocampus than mice injected with P301L‐tau. (E) Representative images of Gallyas silver stain in anterior cortex of A/P+P301L and A/P‐WT mice. Scale bar = 100 µm. (F) Graph of area staining of Gallyas silver staining. Mice injected with P301L‐tau develop mature, silver‐positive tau deposits, which are undetectable in mice injected with WT‐tau. Data from A/P+P301L mice also presented in. ²⁰ Data presented as mean ± standard error of the mean (SEM) with each data point representing the value of one animal (n = 9–14). Data analyzed by two‐sided Student's t‐test, * denotes p < 0.05, ** denotes p < 0.01.

Furthermore, we examined microgliosis and astrogliosis immunohistochemically. We observed a significant increase in area staining of ionized calcium‐binding adapter molecule 1 (Iba‐1) in the cortex of A/P+P301L mice compared to A/P+WT mice. We observed a similar trend in the hippocampus that did not reach statistical significance (p = 0.13; Figure 8A, B). Similarly, we observed significantly greater area staining of glial fibrillary acidic protein (GFAP) in both the cortex and hippocampus of A/P+P301L mice compared to A/P+WT mice (Figure 8C, D).

FIGURE 8.

Mice injected with P301L‐tau have greater gliosis than mice injected with wild type (WT)‐tau. (A) Representative images of ionized calcium‐binding adapter molecule 1 (Iba‐1) immunostaining in the cortex and hippocampus of amyloid precursor protein + presenilin‐1 (A/P)+P301L and A/P+WT mice. Scale bar in cortex = 100 µm; scale bar in hippocampus = 200 µm; scale bar of insets = 50 µm. (B) Graph of percent area staining of Iba‐1 reaction product. Analyzed by two‐tailed Student's t‐test. (C) Representative images of glial fibrillary acidic protein (GFAP) immunostaining in the cortex and hippocampus of A/P+P301L and A/P+WT mice. Scale bar in cortex = 100 µm; scale bar in hippocampus = 200 µm; scale bar of insets = 50 µm. (D) Graph of percent area staining of GFAP reaction product. Analyzed by Mann–Whitney test. Data from A/P+P301L mice also presented in. ²⁰ Data presented as mean ± standard error of the mean (SEM), with each data point representing the value of one animal (n = 8–13). * denotes p < 0.05; n.s. denotes not significant.

To rule out group differences in amyloid pathology as a possible explanation for the differences in tauopathy observed, Aβ was assayed by ELISA and histology. No significant differences in soluble Aβ40 or Aβ42 were detected between A/P+P301L and A/P+WT mice. Similarly, there were no significant differences in insoluble Aβ40 or Aβ42. Soluble Aβ oligomers were also assayed and no significant differences were detected. The histological stain Congo red indicated no significant differences in plaque burden in either the cortex or hippocampus comparing A/P+P301L and A/P+WT mice (Figure S2).

4. DISCUSSION

In this study, we examined how the presence of mature amyloidosis impacts the emergence of tauopathy using two tau variants: WT 4R2N tau and the frontotemporal dementia (FTD) associated P301L mutant tau. Most in vivo investigations of tau pathology and evaluations of interventions overexpress mutant forms of tau in the absence of amyloidosis. However, mouse models expressing mutant human tau may be better predictors of treatment effects in frontotemporal dementia or other primary tauopathies that lack amyloidosis than predictors of interventions benefiting AD. In addition, it is known that the structure of the tau fold in AD is distinct from that in other tauopathies, such as those associated with the tau mutations. ²⁹ We are seeking to establish a potentially more translatable model of AD with sequential development of both amyloid pathology and tau pathology with WT‐tau. We observed significant differences in the tauopathy resulting from overexpression of these two tau variants in A/P mice.

We, and others, have established that overexpressing tau in amyloid‐depositing mice exacerbates cognitive impairments. ¹⁷ , ¹⁸ In the present experiment, we observed that A/P+WT and A/P+P301L mice have a similar degree of cognitive impairment. Both groups of mice are hyperactive in the open field, disinhibited in the elevated plus maze, and make poor nests. Furthermore, both A/P+WT and A/P+P301L mice are severely impaired on a spatial learning and memory task, the RAWM, performing at chance levels (similar to blind mice ²⁵ ) and not exhibiting any improvement over 30 trials in the task. These data are consistent with prior reports that cognitive impairment in tauopathy models can occur prior to or in the absence of frank NFT pathology. ¹² , ¹⁴ , ³⁰

We did observe a significant difference in transduction efficiency between A/P+WT and A/P+P301L. However, the resulting amount of tau expressed, when normalized to the transduction efficiency, was the same for both WT‐tau and P301L‐tau. Surprisingly, we observed more soluble total tau translated per message of P301L‐tau than WT‐tau. To our knowledge, other studies have not measured this translation efficiency directly.

Recent studies have highlighted the physiological role of tau beyond microtubule binding. ³¹ , ³² , ³³ The tau has been reported to bind ribosomal proteins in influence translation of mRNA in neurons. This binding is altered by phosphorylation of tau as well as by FTD‐associated mutations. ³¹ The outcome of tau's association with ribosomes, however, is the subject of conflicting reports. Radford et al. ³⁴ report a reduction in total protein synthesis in rTg4510, but only after the onset of neurodegeneration at 6 months of age. In a Drosophila model of tauopathy, Zuniga et al. ³³ report an increase in the rate of mRNA translated globally during early stages of the disease. Similar results have been reported in SH‐SY5Y cells. ³⁵ It is possible that the A/P+P301L mice are in an early stage of disease and thus have enhanced translation of tau mRNA. This apparent increase in translation efficiency could also reflect differences in protein stability or might be a consequence of overall impairment of proteostasis caused by the increase in deposited tau. Postmortem studies to examine similar relationships in human mutation carriers would likely require tissues obtained prior to the onset of neurodegeneration, which would be expected to reduce brain mRNA levels contributed by neurons.

In concurrence with the increased mRNA and translation efficiency, we observed significantly greater levels of total tau in A/P+P301L mice than A/P+WT mice both by ELISA and immunohistochemistry. Of interest, it appears that WT‐tau is prone to greater phosphorylation than P301L‐tau. We observed significantly more soluble pS396 phospho‐tau by ELISA, and more AT8 tau by immunohistochemistry, in A/P+WT mice than A/P+P301L mice. Due to the significant differences in total tau, we sought to highlight this enhanced phosphorylation of WT‐tau by normalizing phospho‐tau by total tau ELISA values. Corrected for this difference in total tau, we observed significantly more phospho‐tau levels, by at least a factor of two, in both soluble and insoluble fractions in A/P+WT mice compared to A/P+P301L mice. Similar results have been reported in vitro ³⁶ as well as in vivo. ³⁰ This may be due to the increased propensity of P301L‐tau to aggregate, compared to WT‐tau. ⁸ Separately, using an in vitro assay, studies have suggested that it requires fewer moles of phosphate for P301L‐tau to begin to aggregate than WT‐tau. ¹⁰ , ¹¹

In order to assess the propensity of each tau variant to form insoluble deposits, we normalized the ELISA values of the insoluble fraction to the ELISA values of the soluble fraction and termed this the deposition ratio. Surprisingly we did not observe a significant difference in total tau deposition ratio. However, we did observe a significant increase, an approximate doubling, in the deposition ratio of pS396 tau in A/P+P301L mice compared to A/P+WT mice. There was a trend for a significant increase in the deposition ratio of pS199 tau in A/P+P301L mice compared to A/P+WT mice. Similarly, we observed Gallyas silver positive NFTs in the anterior cortex, but not hippocampus, of A/P+P301L mice that were absent in A/P+WT mice. Measures of gliosis followed this pattern, being increased in A/P+P301L mice but not A/P+WT mice, suggesting a role for insoluble tau rather than phospho‐tau in activating glia in AD. Argyrophilic inclusions have been observed in viral tauopathy models using P301L‐tau in both NonTg mice ¹³ and in a model with amyloidosis ¹⁶ as well as tau transgenic mice. ³⁷ , ³⁸ The lack of these tangle‐like structures in the A/P+WT group may be due to the much less overexpression of tau using intravenous compared to intracranial AAV‐tau injections. We find that the average expression of AAV tau is roughly equal to murine tau at the RNA level using intravenous administration, ¹⁸ whereas there is large gradient of expression radiating from the injection site with intracranial administration. However, it is generally recognized that mutant forms of tau tend to aggregate more readily than WT‐tau both in vitro and in vivo. ⁸ , ¹⁶

We suggest that these models may be useful depending on the goal of the study in which they are employed. The use of WT‐tau is more directly translatable to AD in humans but may not be most appropriate for the study of agents that are hypothesized to affect tau deposition. Notably, tau mutations do not associate with increased risk of AD. In addition, we believe that this model offers advantages over others in that it faithfully recapitulates the staging of pathologies observed in AD patients rather than congenital expression that occurs in transgenic mice. Furthermore, the presence of amyloid enhances the tau pathology, which allows for the study of mechanisms by which this occurs, and the degree of human tau expression is comparable to that of endogenous mouse tau.

Limitations of this study include the cross‐sectional design. It is possible that WT‐tau would have formed NFTs had the survival time been extended or the dose increased. An additional confound, which we have tried to address mathematically through normalization, is the difference in viral genomes per mouse genome between the two groups. We delivered the same volume of virus at the same concentration of viral genomes per milliliter and the two viral genomes differ by only a single nucleotide, resulting in the P301L mutation. However, the two AAV preparations compared here were prepared at different times. There may be lot‐to‐lot variations in the viral preparations used for each virus type as well as different storage times between virus preparation and injection into mice. Although AAV has been shown to be stable at 4°C for extended periods, ³⁹ it is possible that a longer storage time resulted in reduced activity of the WT‐tau virus compared to the P301L‐tau virus. We do not believe that the differences in transduction efficiency are due to the effects of mutant versus WT‐tau, but technical differences in the AAV production or storage.

We conclude that there are differences in the tau pathology produced by WT‐tau versus mutant tau pathology, even in the presence of amyloidosis. WT‐tau appears more prone to phosphorylation, presumably leading to reduced binding to microtubules and their destabilization. Mutant tau appears more prone to aggregation and subsequent deposition. Nonetheless, both variants would appear to form intermediate‐sized aggregates and can result in cognitive and functional impairments consistent with dementia in mouse models of amyloidosis. Given these slight differences in phenotype, it seems plausible that use of WT‐tau in models of amyloid‐enhanced tauopathy is potentially more translatable than similar models using mutant forms of tau.

CONSENT STATEMENT

No human subjects took part in this research, so no consent is necessary.

CONFLICT OF INTEREST STATEMENT

M.N.G. is a member of the Alzheimer's Association International Research Grant Program (IGRP) Council, for which she receives complementary membership in the Alzheimer's Association International Society to Advance Alzheimer's Research and Treatment (ISTAART) and registration for the Alzheimer's Association International Conference (AAIC). D.M. serves on the external advisory boards for SynapsDx, MindImmune, and InMed Pharmaceuticals, and receives project support from Bright Minds and Avanex. None of these relationships are related to the topic of this manuscript. The remaining authors have nothing to disclose. Author disclosures are available in the Supporting Information.

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Supporting Information

Supporting Information

Supporting Information