Exercise suppresses mouse systemic AApoAII amyloidosis through enhancement of the p38 MAPK signaling pathway

ABSTRACT Exercise interventions are beneficial for reducing the risk of age-related diseases, including amyloidosis, but the underlying molecular links remain unclear. Here, we investigated the protective role of interval exercise training in a mouse model of age-related systemic apolipoprotein A-II amyloidosis (AApoAII) and identified potential mechanisms. Mice subjected to 16 weeks of exercise showed improved whole-body physiologic functions and exhibited substantial inhibition of amyloidosis, particularly in the liver and spleen. Exercise activated the hepatic p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway and the downstream transcription factor tumor suppressor p53. This activation resulted in elevated expression and phosphorylation of heat shock protein beta-1 (HSPB1), a chaperone that defends against protein aggregation. In amyloidosis-induced mice, the hepatic p38 MAPK-related adaptive responses were additively enhanced by exercise. We observed that with exercise, greater amounts of phosphorylated HSPB1 accumulated at amyloid deposition areas, which we suspect inhibits amyloid fibril formation. Collectively, our findings demonstrate the exercise-activated specific chaperone prevention of amyloidosis, and suggest that exercise may amplify intracellular stress-related protective adaptation pathways against age-associated disorders, such as amyloidosis.


Introduction
Amyloidosis is a group of diseases characterized by misfolded amyloid precursor proteins accumulating and forming amyloid fibrils that have abundant cross-β conformation. The intervals that are equivalent to >70% and ~40% of individual peak aerobic capacity (VO 2peak ), respectively. These repetitions of muscle contraction and relaxation at the required intensity, like traditional resistance exercise, lead to increases in thigh muscle mass and strength and VO 2peak in older humans, suggesting that IWT combines resistance and aerobic training (Nemoto et al., 2007). In this study, we developed an interval training (IT) protocol for mice that mimics the human IWT and used it in a unique mouse model of age-related systemic AApoAII amyloidosis. Our current data show that exercise effectively delays the progression of systemic amyloidosis, especially in the liver and spleen. Further mechanistic analyses suggested that the stress-sensitive p38 MAPK signaling pathway upregulated by exercise is further activated by the unfolded protein response with amyloid deposition, leading to elevated expression and phosphorylation of the molecular chaperone HSPB1.

IT improved physiological characteristics
To investigate the effects of exercise on physical function and AApoAII amyloidosis, female R1.P1-Apoa2 c mice underwent IT or training volume-matched moderate-intensity continuous exercise training (CT) for 16 weeks (Fig. 1). The body weights of mice among groups were not different across the 16-week training period (Fig. S1, Fig. S2A). After training, the weight of white adipose tissue (WAT) in IT groups tended to be reduced compared to sedentary mice, but serum lipid profiles (triglyceride, total-cholesterol, HDL-cholesterol) were not different among groups (Fig. S2B, C). Blood pressure increased with age in sedentary mice, but it improved after training (Fig. S2D). Glucose tolerance tests (IGTT) and the areas under the curve (AUC) showed that in vehicle groups, age-related deterioration of glucose tolerance was observed for sedentary mice (VS), but exercises prevented this deterioration (VI and VC) ( Fig. S2E, Fig. 2A). Additionally, the AUC in amyloidosis-induced groups regardless of exercises was lower compared with vehicle groups in the post-check (Fig. S2E, Fig. 2A). The 15-h fasting temperature decreased with age in amyloidosis-induced sedentary mice (FS), but exercises rescued this decline and reduced the difference between feeding and fasting temperatures (FI and FC), indicating exercise better maintained constant temperature after fasting (Fig. 2B). Maximal running speed (V max ) and 24-h voluntary activities tended to increase in exercised groups particularly in amyloidosis-induced IT mice (FI) (Fig. 2C, D). Of note, both IT and CT mice exhibited higher quadriceps muscle mass relative to sedentary mice after training (Fig. 2E).
Muscle is the organ most directly affected by exercise. Thus, we determined the molecular responses. Interleukin-6 (IL6) is a myokine involved in muscle-liver and muscle-systemic crosstalk, and it has a role in glucose uptake in muscle cells (Pedersen and Febbraio et al., 2012). IL6 mRNA levels in quadriceps were dramatically upregulated more than 5-fold by IT compared to sedentary mice regardless of influence by AApoAII amyloidosis, whereas IL6 mRNA levels in the CT groups were elevated around 2-fold (Fig. 2F). Quadriceps from the IT mice had higher mRNA levels of the mitochondria regulator gene peroxisome-proliferator-activated receptor γ coactivator 1α (Ppargc1a), the glucose uptake regulating gene glucose transporter 4 (Glut4) and the fatty acid oxidation biomarker gene pyruvate dehydrogenase kinase 4 (Pdk4) (Fig. 2F). Together, these results indicated a healthier physiological profile in exercised mice compared with sedentary mice.

IT and CT suppressed AApoAII amyloid deposition
To further investigate the effects of exercise on AApoAII amyloidosis, mouse organs were obtained within 24 h of completing the 16-week training. AApoAII amyloid deposition was analyzed by apple-green birefringence in Congo Red-stained tissue sections and immunohistochemical staining (IHC) with anti-ApoA-II antiserum. Vehicle groups without amyloidosis induction showed no amyloid deposition (data not shown). The amyloid index (AI), a semi-quantitative parameter for evaluating the degree of systemic AApoAII deposition, was significantly lower in both FI and FC groups compared with the FS group (Fig. 3A).
Livers and spleens from mice in the amyloidosis-induced groups that underwent IT and CT showed significant and similar alleviations of AApoAII amyloid deposition (Fig. 3B, C). To quantify amyloid deposition in the liver and spleen, the ratios of areas positively stained with anti-ApoA-II antiserum to the whole section area were calculated. This quantification confirmed that AApoAII amyloid deposition was indeed dramatically suppressed in the liver and spleen following both exercise regimens (Fig. 3D). Additionally, amyloid deposition in the other examined organs tended to decrease in exercised mice, particularly in the FI group, but this difference did not attain significance (Fig. S3). These results suggested that both exercise regimens induced organ-dependent suppression of amyloidosis.
The level of amyloid precursor protein is an important risk factor that has a positive precursor protein ApoA-II are mainly found in serum HDL, and account for about 75% and 20%, respectively, of apolipoproteins in HDL. Exercises did not affect either the serum levels of ApoA-II in vehicle groups or the ApoA-II mRNA levels in liver (Fig. S4). We observed lower ApoA-II serum levels and higher ApoA-I serum levels as well as lower ApoA-II/ApoA-I ratios in the amyloidosis-induced groups (Fig. S4), indicating that serum ApoA-II deposited into AApoAII amyloid fibrils.

Transcriptome analysis revealed a significant increase of p38 MAPK and Hspb1 in response to IT and amyloidosis
To identify the potential signaling pathways or effector molecules for IT-mediated prevention of amyloidosis, we performed RNA sequencing analysis to investigate transcriptome changes in response to IT and amyloidosis in the liver. Analysis of differentially expressed genes (DEGs) (> 2-fold-change and corrected P value < 0.05) showed that IT induced 247 DEGs in vehicle groups (VI vs. VS) and 370 DEGs in fibril groups (FI vs. FS) (Fig. 4A, Table S2). The overlapping 76 DEGs between the vehicle groups and fibril groups showed common profiles in response to IT regardless of amyloidosis (Fig. 4B, showed significantly higher phosphorylated p38 MAPK and total HSPB1 protein levels in the IT groups (Fig. 5A). Notably, levels of both phosphorylated p38 MAPK and total HSPB1 were additively enhanced in amyloidosis-induced IT mice (FI) compared to the VS group (Fig.   5A). Thus, the levels of total HSPB1 protein were coordinately upregulated with the change in phosphorylated p38 MAPK (Fig. 5A). In contrast, higher phosphorylation of HSPB1 only occurred with IT.
We observed accumulation of p-HSPB1 in amyloid deposition areas using IHC with specific p-HSPB1 antibody and Congo Red staining of paraffin-embedded liver sections (Fig. 5B). IT induced greater amounts of p-HSPB1 indicated by a brown stain that reacted with anti-ApoA-II antiserum in hepatocytes cytoplasm and at amyloid deposition sites (Fig. 5B).
This observation was in line with the Western blot analysis. Co-localization of p-HSPB1 and extracellular AApoAII amyloid deposition may suggest the interaction between p-HSPB1 and extracellular AApoAII amyloid deposition.
We subsequently asked whether Hspb1 played a protective role against amyloid deposition in the spleen (Fig. 3B) and lungs (Fig. S5A), where IT reduced (spleen) or did not reduce (lungs) amyloid deposition. Real-time qPCR of Hspb1 mRNA expression and Western blot analysis of total and phosphorylated HSPB1 levels in the spleen showed similar results as that for the liver (Fig. S5B). Similarly, we observed large amounts of p-HSPB1 accumulated at amyloid deposition sites in the FI group in the spleen (Fig. S5C). On the other hand, mRNA expression and total-protein levels of Hsbp1 remained unchanged by either IT or amyloid deposition in the lungs (Fig. S5D). Therefore, we considered the induction of p-HSPB1 as one possible mechanism for IT-mediated prevention of AApoAII amyloidosis.

P53 signaling pathway upregulated Hspb1 levels in mice with IT
The tumor suppressor protein p53 is a well-defined downstream transcription factor of p38  6A). Mice with amyloidosis also exhibited higher TP53 levels compared with vehicle groups.
Importantly, IT and amyloid deposition (FI) additively enhanced TP53 levels ( Fig. 6A), data consistent with the change of phosphorylated p38 MAPK and total HSPB1 protein levels ( We found that the levels of HSPB1 and TP53 proteins induced by IT and amyloidosis were similar ( Fig. 5A and Fig. 6A). Pearson Analysis of those protein levels in the liver showed a strong positive correlation (P < 0.001, Fig. 6C). Predictive analyses of the putative binding site for TP53 in the transcriptional regulatory region of Hspb1 revealed a homology higher than 80% with the 18-mer TP53-motif sequence. That finding suggested that the Hspb1 gene may be regulated by TP53 (Fig. 6D). These data suggested that IT-mediated activation of p38 MAPK might induce TP53-dependent transcriptional regulation of Hspb1.

Amyloid deposition induced the unfolded protein response and activated p38 MAPK
We sought to identify gene clusters that were related to amyloid deposition and were also associated with the activation of p38 MAPK by amyloid deposition. Thus, we performed propose that the anchoring of active p38 MAPK to its target gene Hspb1 may be mediated by the transcription factor p53 (Fig. 6).
Our current data demonstrated that p-HSPB1 was upregulated in livers and spleens in which amyloid deposition was mitigated, and it accumulated at sites of amyloid deposition. Thus, we suggest that the induction of p-HSPB1 could be a novel mechanism by which exercise reduces amyloidosis. Hspb1 can be elevated in the brain and accumulate in senile plaques in We previously found that the mRNA levels of UPR/ER stress-related genes were elevated in the liver of mice with AApoAII amyloidosis (Luo et al., 2015). Here, we found that UPR was significantly upregulated in the liver of sedentary mice with amyloidosis (Fig. 7).  Here, we utilized two exercise regimens. IT for mice mimics the human IWT, an exercise program at submaximal intensity that combines aerobic and resistance training. The molecular mechanisms underlying this regimen have not been elucidated because there were no appropriate animal models. Another exercise regimen, CT, uses aerobic/endurance training at moderate intensity. We observed that both IT and CT increased muscle mass, improved glucose intolerance and several physiological functions. We also observed that both IT and CT upregulated myokine IL6 expression in the muscle after exercise, but the effect of IT was more significant. Interestingly, both exercise regimens had similar positive effects on systemic amyloidosis. We assume that the adaptive response to a CT regimen could be sufficient to suppress the progression of AApoAII amyloidosis. In other age-related diseases, such as type 2 diabetes, four-month resistance training or IWT training is more effective in In conclusion, to our knowledge, this is the first demonstration of one possible mechanism in which exercise additively enhances the expression of HSPB1 in the presence of amyloid deposition, and activates the anti-amyloid activity of HSPB1 (p-HSPB1) which prevents amyloidosis in vivo. Although further investigation is needed to better characterize the participating molecular pathways, we suspect that exercise can regulate signaling programs that enhance the expression of appropriate adaptive molecules in the presence of harmful extra-or intracellular conditions (such as amyloidosis). Our findings suggest a biochemical basis that explains how exercise reduces the risk of age-related disorders. New therapeutic strategies should build upon these findings, further improving treatment strategies. In terms of experimental design (Fig.1) avoidance behaviors more than three consecutive times (i.e., less than ten min at each exercise interval) for mice in the IT and CT groups; and iii) unexpected death (Table S1 and

3) Moderate-intensity continuous training (CT) protocol
CT training was performed 4 days per week on a treadmill based on a protocol that was slightly modified from a previous study (He et al., 2012). For CT, the abovementioned warm-up was used and then the mice undertook continuous running for 30 minutes at a speed that produced 50% of the pre-check V max . After 8 weeks of training, the V max was adjusted as described above.

Induction of AApoAII amyloidosis
AApoAII amyloid fibrils were isolated from the livers of R1.P1-Apoa2 c mice with severe amyloid deposition using a modified Pras method as described previously (Pras et al., 1969).
Mice in the amyloidosis-induced groups (FS, FI and FC) were injected intravenously at 10 weeks of age with 1 μg AApoAII fibrils in 100 μL PBS to induce AApoAII amyloidosis.
AApoAII fibrils were sonicated on ice according to our previous method (Xing et al., 2001) before injection.

Detection of amyloid deposition in mice
The main organs were fixed in 10% neutral buffered formalin, then embedded in paraffin and cut into 4-μm sections using standard procedures. Amyloid deposition was identified using polarizing light microscopy (LM) (Axioskop 2, Carl Zeiss Japan, Tokyo, Japan) to observe apple-green birefringence in tissue sections stained with Congo Red (Sawashita et al., 2009).
The degree of amyloid deposition formation in examined organs was scored from 0 to 4 (amyloid score) according to a previously described scoring principle (Higuchi et al., 1998).
The systemic degree of amyloid deposition in each mouse was determined semi-quantitatively using an amyloid index (AI) that represents the average of amyloid scores for seven organs (heart, liver, spleen, tongue, stomach, small intestine and skin) (Higuchi et al., 1998).

Measurements of physiological indices and serum lipid parameters
We determined physiological indices, including 24-h voluntary activity, body temperature, 15-h fasting body temperature, heart rate, blood pressure and IGTT for each mouse at two time points: before training (when mice were 8 weeks old) termed the pre-check, and 2 weeks before the end of the 16-week training (when mice were 24 weeks old) termed the post-check.

Immunohistochemical and immunofluorescent analysis
AApoAII deposition and phospho-HSPB1 were evaluated by immunohistochemistry using the horseradish peroxidase-labeled streptavidin-biotin method with specific antibodies. Rabbit Signaling Technology) was then applied as the secondary antibody and incubated with the membranes for 1 h at room temperature. Protein bands were detected by enhanced chemiluminescence (ECL), and the target proteins were analyzed using NIH ImageJ software.

Liver RNA sequence analysis
Although both IT and CT effectively suppressed AApoAII amyloid deposits in the liver to similar degrees, we selected liver samples from the IT groups for RNA sequence analysis because IT stimuli offered more metabolic benefits in skeletal muscle at a transcriptional level. Briefly, 10 mg liver samples taken from each mouse and stored at -80 ̊ C were homogenized in TRIzol RNA isolation reagent (Invitrogen-Thermo Fisher, Tokyo Japan) and then pooled in 4 sample tubes (VS, VI, FS and FI groups, N = 4). The sample tubes were sent to Novogene (Nagoya, Japan) for analysis. Total RNA was extracted, and RNA purity and integrity were confirmed by Novogene. Sequencing analysis of expressed RNAs was performed using an Illumina next generation sequencing platform. Sequencing count data were analyzed using edgeR R package (

Gene expression analysis with quantitative real time PCR
Analysis of mRNA expression in the liver, skeletal muscle, spleen, and lung was performed as Quantitative real-time qPCR analysis was carried out using a sequence detection system (ABI PRISM 7500, Applied Biosystems, Foster City CA, USA) with SYBR Green (TaKaRa Bio, Tokyo, Japan). Gene expression was normalized relative to 18S ribosomal RNA. The forward and reverse primer sequences are listed in Table S3.

TUNEL assay for apoptosis detection
Formalin-fixed, paraffin-embedded tissue blocks were cut into 4-mm sections and processed for use in TUNEL assays (Luo et al., 2015). Sections were stained with an In Situ Apoptosis Detection Kit (TaKaRa, MK500, Japan), according to the manufacturer's instructions.
TUNEL-positive cells in tissues were counted in three fields per section at 200-fold and 400-fold magnifications, using light microscopy.

Fig. S7. ER stress responses in the livers of amyloidosis-induced mice
Heat-map analysis of gene-set in ER stress identified by GSEA.