P301S Tg mice exhibited sex-specific differences in behavior
To determine the applicability of behavioral tests for male and female P301S Tg mice, we analyzed sex- and age-related changes in behavior, including body weight, grip force test, accelerating rotarod test, stride length, ledge test, hind limb clasping, gait grade, kyphosis grade, nesting and Morris water maze.
Weight change and survival
We first assessed body weights in P301S Tg mice and WT littermates at different ages and sexes. Two cohorts of Tg mice with age and sex-matched WT littermates were used for long term behavioral testing at 3.5-, 5-, 6-, 7-, 8-, 9-, 10, 11- and 12-month-old time points. There was a significant difference in weight between P301S mice and WT mice across all ages (Fig. 1A; Additional file 1: Figure S1A; Additional file 2: Table S2). P301S Tg mice reached maximum weight at 8-months-old, while WT mice continued to gain weight with age. Both male and female WT mice exhibited a similar weight growth curve from the age of 3.5 months to 12 months. However, the weights of male WT mice were significantly higher than those of female WT mice at all disease stages (Fig. 1B; Additional file 2: Table S3). We also observed that P301S Tg mice exhibited sex-specific differences in weight changes. Male Tg mice gained weight slowly, similar to female Tg mice before 8 months of age, but this declined sharply thereafter (Fig. 1B; Additional file 1: Figure S1B). Conversely, the weights of female P301S Tg mice showed small changes from the age of 8 months to 12 months. Taken together, P301S Tg mice exhibited sex-specific differences in weight changes, male Tg mice showed a more noticeable decrease in weight than that of female mice at the late stage of disease progression.
Considering the significant differences in weight changes between P301S mice and WT littermates, we also assessed weight changes of different organs including the heart, liver, lung, kidney and spleen. The weights of the lung and spleen in P301S mice showed a similar change tendency to that of WT mice (Fig. 1C, 1D), without abnormal pathology in HE slides (Additional file 1: Figure S2, Figure S3). Consistent with the changes in body weight, liver weights in tau Tg mice reached maximum at the age of 6 months, and subsequently dropped dramatically until 12 months of age, revealing impaired liver function in P301S Tg mice due to the development of tau pathology (Fig.1E). However, we did not observe any significant pathological changes in both Tg and WT mice by HE staining, except slight nucleolar enlargement in the livers of 6 to 12-month-old Tg mice (Additional file 1: Figure S4). Weights of the heart and kidney in both male and female P301S mice changed minimally with age, while those in WT mice increased in an age-dependent manner (Fig. 1F, Fig. 1G). HE staining of heart tissue revealed myoneme atrophy and widened gaps between myofilaments in Tg mice across all periods, but normal histology was observed in WT littermates (Additional file 1: Figure S5). No obvious pathological changes were observed in the kidney (Additional file 1: Figure S6). These changes in the organs of P301S Tg mice suggested metabolic abnormalities in Tg mice.
We also analyzed the survival rate of different mouse cohorts. As shown in the survival analysis in Fig. 1H, all WT mice remained alive for more than 1 year. For P301S Tg mice, female Tg mice begin to die at 10 months old, and 90% of animals were still alive after 1-year observation. In contrast, male P301S Tg mice started to die at 4 months of age and final survival rate dropped to 32% at 12 months of age. Thus, female P301S Tg mice have a significant survival advantage over male Tg mice, probably due to fewer comorbidities.
P301S mice exhibited sex differences in coordination score system
P301S mice present with muscular atrophy and motor dysfunction as the disease develops . We therefore applied tests for grip strength, accelerating rotarod, stride length and coordination score system to determine the impact of sex differences on motor dysfunction in P301S Tg mice.
In the grip strength test, the strength of P301S Tg mice was significantly reduced at the late disease stage compared to that of WT mice (Fig. 2A; Additional file 2: Table S2). Male but not female Tg mice exhibited decreased grip strength from the age of 10 to 12 months when compared with sex-matched WT littermates (Fig. 2B; Additional file 2: Table S3). Hence, the difference in grip strength between WT and P301S Tg mice was mainly due to sex.
We employed the accelerating rotarod test to evaluate motor performance and balance. Compared with the WT group, P301S Tg mice demonstrated 10% prolonged latency (Fig. 2C; Additional file 2: Table S2). There was no significant latency difference between male and female Tg mice (Fig. 2D; Additional file 2: Table S3).
We performed the stride length test to compare muscular function of hind limbs between P301S Tg and WT mice. There were no significant differences between these two groups (Additional file 2: Table S2). However, we observed that male Tg mice showed a gradually shortening stride length with age. In contrast, female P301S Tg mice maintained a steady curve of stride length with ageing (Fig. 2E; Additional file 2: Table S3). Thus, male P301S Tg mice showed more serious damage in motor ability, and male Tg mice were more sensitive than female mice in the stride length test.
The coordination score system consisted of four experiments including ledge test, gait, hind limb clasping and kyphosis, which individually or in combination characterized equilibrium and muscular atrophy of mice. When we first assessed P301S Tg and WT mice in the ledge test, both groups exhibited no significant differences in coordination (Additional file 1: Figure S7B; Additional file 2: Table S2). Similarly, P301S Tg and WT mice performed similarly in the gait study (Additional file 1: Figure S7D). However, when comparing the behavior of P301S Tg and WT mice in the hind limb clasping and kyphosis test, Tg mice presented with a state of tauopathy and performed worse than WT mice did (Additional file 1: Figure S7C, Figure S7E). Furthermore, when taking sex into account, we observed that female Tg mice displayed similar results to those of female WT mice in all four measurements (Fig. 2G-2J; Additional file 2: Table S3). In contrast, male P301S Tg mice exhibited a progressive tauopathy phenotype that was significantly different from that of WT male littermates beginning at 5 months, indicating a more notable impairment of coordination and muscle function in male P301S Tg mice but not in female mice.
When we combined the ledge walking, gait, clasping and kyphosis assessments into a composite phenotype score for each individual mouse, we observed that male P301S Tg mice exhibited an increasing composite phenotype score that was significantly different from male WT littermates beginning at 4 months, consistent with the progressive nature of AD (Fig. 2F). Nevertheless, female P301S Tg mice showed a similar composite phenotype score as female WT mice did, suggesting that female Tg mice were not sensitive in this composite phenotype assessment (Additional file 2: Table S3).
Male and female P301S mice differed in cognitive impairments
To determine if there was sex-dependent memory and cognitive impairments in P301S Tg mice, we assessed spatial learning and memory capacity of Tg mice and WT control mice at different ages using the MWM. Four cohorts of Tg mice and their littermates were utilized for MWM at the age of 3-, 6-, 9- and 12-months old. Before 9 months of age, Tg mice did not differ from WTs in latency to find the hidden platform during the acquisition phase (Fig. 3A-C; Additional file 1: Figure S9; Additional file 2: Table S4, Table S5), number of crossings over the target platform, or time spent in the target quadrant in the probe trial (Fig. 3E-G; Additional file 1:Figure S8A-L; Additional file 2: Table S4, Table S5), indicating that cognitive capacity was not impaired in 9-month-old Tau Tg mice. We noticed that 12-month-old female P301S Tg mice showed striking longer escape latencies than those of age-matched WT mice during acquisition and slightly decreased number of crossings over the target platform on the probe trial, although these differences were not significant between Tg mice and WTs (Fig. 3D, Fig. 3H). In contrast, no significant differences in performance in the MWM were observed between male Tg mice and controls, potentially due to rapid dyskinesia in male tau P301S Tg mice. This highlighted later memory decline of female Tg mice which began from 12-months-old, and a sex-specific response to memory tests among Tau P301S Tg mice.
We also used the OFT to analyze the influence of sex on exploratory behavior and general activity of P301S Tg mice. Our results demonstrated an age dependent decrease in exploratory activity in both P301S mice and non-Tg mice, and Tg mice typically exhibited longer travel distances and higher movement velocity than that of WTs. This diversity was amplified in females (Fig. 3I; Additional file 1: Figure S10A-B). Compared to female WT mice, female P301S mice displayed enhanced levels of locomotor activity, significantly increased freezing time (Additional file 1: Figure S10E), and more frequent appearances in the central area despite moving (Fig. 3J，Fig. 3L, Fig. 3N) or being in a silent state (Additional file 1: Figure S10F). This was especially evident for freezing time in the corner area (Fig. 3K, Fig. 3M, Fig. 3O). However, we also observed that the differences between female Tg mice and sex-matched WTs decreased over time. In addition, similar hyperactivity state made it difficult to distinguish male P301S mice and WT littermates. Rearing and grooming behavior (Additional file 1: Figure S10G-I) as well as autonomic nervous system (Additional file 1: Figure S10J) activity did not differ among Tg and WT mice. Thus, female P301S Tg mice were more suitable for this measurement at younger ages.
Non-maternal nest-building performance is sensitive to hippocampal damage and is used in murine models of psychiatric disorders [20–23]. We therefore assessed the nest-building capacity of P301S Tg mice of different sexes and ages. Compared to WT littermates, P301S Tg mice showed significantly decreased nest-building capacity throughout disease progression (Additional file 1: Figure S11; Additional file 2: Table S2). Furthermore, male P301S Tg mice exhibited a gradual decrease in nest-building ability from 6 to 12-months-old, which was substantially different from the steady building capacity of male WT control mice (Fig. 3P-Q; Additional file 2: Table S3). In contrast, female Tg mice performed similarly to WT mice in the nest-building test throughout disease progression. Hence, nest-building performance is a valid behavior measurement for TauP301S Tg mice, and male but not female Tg mice are applicable for this assessment. Further, the 24h-detection time point is most sensitive.
Pathological Tau development in P301S transgenic mice of different ages and sex
NFTs accumulated by hyperphosphorylated Tau proteins are the vital features of AD and related tauopathy. We measured the levels of NFT deposition and tau phosphorylation in the hippocampus by immunohistochemistry (IHC) and total brain homogenates by western blot (WB) in P301S Tg mice and non-Tg littermates at different ages and sexes, respectively. Human tau in P301S Tg mice began to accumulate at 3-month-old, and kept increasing until 12 months of age. Female Tg mice exhibited stronger expression than that of males in both soluble and insoluble fractions in total brain (Fig. 4J-L, Additional file 3). Immunoreactivity with AT8 antibody raised against tau phosphorylated at serine 202 and threonine 205 (pTauS202/T205) was only observed in the hippocampus of P301S mice at 6-month-old in both sexes but not in WT controls (Fig. 5B). In male P301S Tg mice, tau phosphorylation levels at S202/T205 were enriched at 9 months of age and showed a slight decline at 12 months in the aqueous fraction. Instead, we detected a decline in 12-month-old mice in soluble RAB fraction (Fig. 4A), and an increase in both RIPA fraction (Fig. 4B) and insoluble urea fraction (Fig. 4C), indicating that tau proteins containing phosphorylation at Ser202/Thr205 formed insoluble NFTs. We noticed that the enrichment time of pTau(S202/T205) in IHC was earlier than that in WB, suggesting that this form of phosphorylated tau protein may develop earlier in the hippocampus than in other brain regions. We also noticed that the concentration of pTau(S202/T205) in male P301S mice was higher than that in female Tg mice, indicating the more severe dementia in male P301S Tg mice (Fig. 4A-C). Similar changes were found in tau phosphorylation levels at the S396 (pTauS396) and S404 (pTauS404). IHC staining results showed an age-dependent increase at the S396 and S404 phosphorylated points across the observation period (Fig. 5C-D) and more substantial increase in female Tg mice than in males. Western blot with the PHF13 antibody and polyclonal pTau(S404) antibody revealed that the level of phosphorylated tau at S396 and S404 increased to nearly the highest concentration in both RAB and RIPA soluble fraction of brain homogenates at the age of 6 months and 3 months, respectively (Fig. 4D-F, Fig. 4G-I). Furthermore, female Tg mice showed a faster decrease than that of males in both the level of pTau(S396) and pTau(S404) in brain homogenates just after 6 months in aqueous fractions, suggesting that more hyperphosphorylated tau proteins with pTau(S396) and pTau(S404) transferred into insoluble fractions in female P301S Tg mice (Fig. 4D-F, Fig. 4G-I). We also observed that the enrichment time of pTau(S404) in WB was earlier than that in IHC, indicating that this form of phosphorylated Tau protein may be extensively expressed in various brain regions. Consistent with the variations in phosphorylated tau protein in Tg mice of different sexes, the amount of neurons exhibited a significant decline from the age of 6 months in male P301S mice, but only a small decrease in females was observed until 12-month-old, demonstrating a more toxic effect of pTau(S202/T205) on neurons (Fig. 4O). In sum, phosphorylated tau protein showed different degrees of abundance in male and female P301S Tg mice. pTau(S202/T205) may play a key role in NFT formation in male mice, while pTau(S404) is likely to exert a dominant function in NFT formation in female mice.
Microglia and astrocytes maintain homeostasis in the central nervous system by engulfment and degradation of extracellular material via phagocytosis . We assessed the activation of microglia and astrocytes in the brains of P301S mice and WT littermates at different ages and sex. In the hippocampus of both male and female P301S mice, the amount of microglia kept increasing from the age of 3 months to 12 months but was maintained at a low level in WT littermates (Fig. 5E). Nevertheless, active microglia in the brain homogenates of WT mice appeared higher than that in P301S mice, which only showed a slight increase with aging (Fig. 4M). This implied that the microglia in the whole brain of P301S mice were in a quiescent or inhibited condition before typical tau pathology, and accumulated and converted to an active state in the hippocampal region induced by tau hyper-phosphorylation.
In addition, both IHC and WB for GFAP, a marker for astrocytes, revealed that GFAP was already activated in the hippocampus of 3-month-old P301S mice of both sexes (Fig. 5F, Fig. 4N). However, the astrocyte burden sharply declined at the age of 6 months, and then gradually increased, suggesting a significant induction of astrogliosis in P301S mice after tau phosphorylation. We noticed that the increase in astrocyte burden in the brains of male P301S mice was significantly higher than that in female Tg mice, possibly due to the higher level of hyper-phosphorylated tau in the brains of male Tg mice.
Absence of MIP-3α and reduced sex-specific cytokines in P301S mouse plasma
We examined whether sex differences influenced specific plasma and brain proteins in P301S Tg mice. We first checked the level of human tau in the plasma of different cohorts of mice. No human tau protein was detected in the plasma of WT mice. In the plasma of male and female P301S mice, plasma tau exhibited a tendency to increase between the age of 3 months to 8 months and was maintained at a stable and high level after 9 months, indicating early cerebrovascular lesions before pathologic changes of Tau in the brain, and potential leakage of anomalous tau protein may have engaged changes in the peripheral nervous system and immune system (Fig. 6A).
We then analyzed the concentration of inflammatory cytokines and chemokines in the plasma and soluble fractions of brain homogenates of P301S mice and WT littermates at different ages and sexes. As shown in the heat map Fig. 6J, no MIP-3α (also termed CCL20) was detected in the plasma of both male and female P301S mice entire life , while this protein showed an increasing tendency in WT mice across all ages (Fig. 6B). However, there was no significant difference in the concentration of brain homogenate MIP-3α between Tg and WTs (Additional file 1: Figure S12). The concentration of plasma IFN-γ, IL-5 and IL-6 in P301S mice were higher than that in sex- or age-matched WT littermates during the ages of 6 months to 10 months (Fig. 6C-E), which showed a synchronous accumulation with hyper-phosphorylated tau protein in the brain. Hence, systematic inflammation in the peripheral circulation such as the increase in IFN-γ, IL-5 and IL-6 may be a signal of abnormal phosphorylation of tau protein in P301S mice, and may be a subsequent event during pathological progression of tau. In addition, the continuous increase in the concentration of MIG (monokine induced by IFN-γ, also termed CXCL9) only occurred in male P301S male mice, whereas the concentration of MIG in female P301S mice maintained a low and steady tendency similar to that of WT mice (Fig. 6F). We also detected a higher level of TNF-α in the plasma of 7-month-old male P301S mice (Fig. 6G). Analogous abnormal levels were also observed with IL-10 and IL-13, which were only expressed in male P301S mice (Fig. 6H-I). In the soluble brain homogenates of these mice cohorts, no obvious differences in the concentration of these factors were found except IL-6, which exhibited a lower level in P301S mice and implied astrocytic malfunction (Additional file 1: Figure S12).
Correlation analysis among pathological, ethological and inflammatory factors
Based on the above findings, we aimed figure to elucidate a potential ethological index and specific disease-related plasma inflammatory factors to track disease progression during preclinical treatment of P301S mice. We included the average of all behavioral, pathological and serological data of each subgroup of mice with the same sex and genotype during the observation period, and then calculated the Pearson correlations of all parameters (Fig. 7A). Some factors of all the detected mice were selected to calculate the Pearson correlation one-on-one (Fig. 7B).
Levels of tau phosphorylation are widely used as the golden standard for the identification of tau pathological progression, so we first analyzed the correlations between tau phosphorylation and other factors. The load of various phosphorylated tau protein in the hippocampus showed high correlations with each other, with Pearson correlation coefficients (r) of 0.938, 0.875, and 0.828 among pTau(S202/T205)-pTau(S396), pTau(S202/T205)-pTau(S404), and pTau(S404)-pTau(S396), respectively. Furthermore, the relevance of these phosphorylated tau proteins were not different between males and females. The level of activated microglia (detected by Iba1 antibody) in the hippocampus was more relevant to pTau(S202/T205) (r=0.828) and pTau(S396) (r=0.831) and less relevant to pTau(S404)(r=0.728), but the level of astrocytes (detected by anti-GFAP antibody) in the hippocampus were more relevant to pTau(S404) (r=0.844) than to pTau(S202/T205) (r=0.651) and pTau(S396) (r=0.517). When we calculated the Pearson correlation individually, a weaker relationship between microglia and pTau(S404), or astrocytes and pTau(S202/T205) was observed, possibly due to sex-induced differences.
The level of human tau proteins in plasma is widely use as biomarker to reflect the progression of tau pathology. In our experiment, it indeed showed a high correlation with phosphorylated Tau in the hippocampus detected by AT8 (r=0.843) and PHF13 (r=0.931), but had a low correlation with pTau(S404) (r=0.614), which also varied with sex. A similar tendency was observed between human tau proteins in plasma and the level of microglia (r=0.77) or astrocytes (r=0.304) in the hippocampus, and male mice showed better responses to these factors.
The level of pTau(S202/T205) and pTau(S396) was also related to kyphosis score (r=0.824 and r=0.784, respectively) and score of nesting test (r=-0.656 and r=-0.522, respectively). They both exhibited a strong relationship only in male mice with all detected phosphorylation points of tau protein. However, for other behavioral experiments including grip strength test, stride length test or accelerating rotarod test, they all exhibited low correlations with tau hyper-phosphorylation, which may be the reason for which those behavioral tests are not applicable for the assessment of tau P301S transgenic mice. In addition, the concentration of pTau(S202/T205), pTau(S396) and pTau(S404) were also strongly related to the level of brain IP-10 (r=0.918, r=0.824 and r=0.945, respectively), MIP-1α (r=0.802, r=0.772 and r=0.760, respectively), MIP-1β (r=0.887, r=0.829 and r=0.879, respectively), and BLC (r=0.894, r=0.852 and r=0.751, respectively), suggesting that IP-10, MIP-1α, MIP-1β and LIX may participate in pathological progression of tau and impair neuronal function. Furthermore, we observed that the concentration of brain IP-10, MIP-1α, MIP-1β and LIX were highly relevant to kyphosis score (r=0.806, r=0.920, r=0.928 and r=0.913, respectively), score of nesting test adjusted at the 24th hour (r=-0.663, r=-0.846, r=-0.835 and r=-0.801, respectively), and astrocytes (r=0.843, r=0.803, r=0.802 and 0.705).