Age-related metabolic and neurodegenerative changes in SAMP8 mice

Results

Behavioral experiments

Behavioral experiments were performed to monitor several aspects of the behavior of SAMP8 mice compared to their SAMR1 controls at the age of 3, 6 and 9 months; activity of mice was examined in the open field, anxiety was evaluated in the open field and elevated plus maze, or spatial working memory was assessed in the Y-maze.

Increased activity and decreased anxiety of SAMP8 mice

During the 10 minutes that mice spent in the open field, SAMP8 mice from the age of 3 months ran a significantly longer total distance than their SAMR1 controls, as shown in Figure 1A, and covered a significantly larger area of the ground (Figure 1B); the representative tracks of the SAMR1 and SAMP8 mice at the age of 6 months are shown in Figure 1D, 1E, respectively. Wall distance was significantly decreased only in 3-month-old SAMP8 mice, which indicates higher anxiety compared to SAMR1 mice (Figure 1C). To further examine anxiety-like behavior, the elevated plus maze was performed at the age of 6 and 9 months. Compared to the open arms, both SAMR1 and SAMP8 mice at both ages spent more time in the closed arms (Figure 1F, 1G). However, at the age of 6 months, SAMP8 mice spent significantly more time in the open arms than SAMR1 controls; at the age of 9 months, only a tendency toward increased time was observed. At both ages, SAMP8 mice spent less time in the closed arms than SAMR1 controls.

Figure 1. Increased activity measured by open field (A–E) and decreased anxiety measured by elevated plus maze (F, G) of SAMP8 mice. Mice spent 10 minutes in open field where the: (A) total distance (B) covered area (C) wall distance were measured. The representative tracks of 6 months old (D) SAMR1 mice (E) SAMP8 mice. Mice spent 10 minutes in elevated plus maze: (F) 6 months old mice (G) 9 months old mice. Data are mean ± SEM, analyzed by 2-way ANOVA, repeated measures, with Bonferroni post test. Significance is *P < 0.05, **P < 0.01 and ***P < 0.001. *SAMP8 mice compared to SAMR1, + age-dependent changes in SAMR1, # age-dependent changes in SAMP8, and ^† compared closed arm to open arm. n = 12 (3 months old), 10 (6 months old) or 5 (9 months old) mice per group.

Impaired spatial working memory of the SAMP8 mice in Y maze

To evaluate spatial working memory, the Y maze test was performed at the age of 3, 6, and 9 months (Figure 2A–2C, respectively). From the age of 6 months, SAMP8 mice spent significantly less time in the new arm of the maze, as well as increased time in the known entry arm, indicating decreased spatial working memory compared to SAMR1 mice.

Figure 2. Impaired spatial working memory of SAMP8 mice in Y maze (A) 3 months old mice (B) 6 months old mice (C) 9 months old mice. Mice spent 5 minutes in the entry and open arm, then 5 minutes in free access to new, open and entry arms. Data are mean ± SEM, analyzed by 2-way ANOVA, repeated measures, with Bonferroni post test. Significance is **P < 0.01 and ***P < 0.001. * SAMP8 compared to SAMR1 mice, and ^† open and entry arm compared to the new arm. n = 12 (3 months old), 10 (6 months old) or 5 (9 months old) mice per group.

Lower body weight in SAMP8 mice

As shown in Figure 3, from the 2^nd month of age, the SAMP8 mice were significantly leaner than their SAMR1 controls; this phenomenon was enhanced during the ageing.

Figure 3. Lower body weight SAMP8 mice from 2 months of age. Body weight was measured every month. Data are mean ± SEM, analyzed by 2-way ANOVA with Bonferroni post test. n=14-15 (2-3 months), 10 (4-6 months), 5 (7-9 months).

In concordance with lower body weight, a lower weight of white adipose tissue (WAT, subcutaneous and epididymal) was observed at the ages of 6 and 9 months in SAMP8 mice than in SAMR1 controls (Table 1) and a significantly decreased level of the adipokine leptin was detected at the age of 9 months. On the other hand, no differences were observed in the plasma levels of triacylglycerols (TAGs), or free fatty acids (FFAs). The concentration of plasma cholesterol was significantly lower in 6- and 9-month-old SAMP8 mice than in SAMR1 mice. There were no significant differences in the concentration of blood glucose between SAMR1 and SAMP8 mice. The level of plasma insulin did not differ between SAMP8 mice and their controls.

Table 1. Metabolic parameters of SAMR1 and SAMP8 mice.

		WAT	Leptin	FFAs	TAGs	Cholesterol	Glucose	Insulin
		[g]	[ng/ml]	[mmol/l]	[mmol/l]	[mmol/l]	[mmol/l]	[ng/ml]
3 M	SAMR1	0.27 ± 0.06	0.19 ± 0.04	0.27 ± 0.05	0.52 ± 0.08	2.57 ± 0.10	9.3 ± 0.6	0.54 ± 0.18
	SAMP8	0.39 ± 0.07	0.48 ± 0.19	0.21 ± 0.03	0.74 ± 0.07	2.01 ± 0.04	11.4 ± 1.7	0.37 ± 0.11
6 M	SAMR1	0.78 ± 0.10	0.35 ± 0.17	0.37 ± 0.03	0.65 ± 0.05	2.79 ± 0.30	8.9 ± 1.8	0.05 ± 0.02
	SAMP8	0.42 ± 0.17	0.94 ± 0.69	0.51 ± 0.12	0.81 ± 0.12	1.76 ± 0.17 **	6.5 ± 1.1	0.10 ± 0.02
9 M	SAMR1	1.47 ± 0.17	2.94 ± 0.68	0.45 ± 0.04	0.54 ± 0.07	3.09 ± 0.08	10.0 ± 0.7	0.19 ± 0.09
	SAMP8	0.46 ± 0.13 ***	0.85 ± 0.11 ***	0.34 ± 0.04	0.48 ± 0.14	1.86 ± 0.27 **	8.8 ± 0.6	0.16 ± 0.02
Data are mean ± SEM analyzed by 2-way ANOVA, Bonferroni post-hoc test. *P < 0.05, **P < 0.01 and ***P < 0.001 (*SAMP8 compared to SAMR1 at the same age), n = 4-5 mice per group.

Increased rectal temperature and increased cytokine levels in SAMP8 mice

SAMP8 mice had significantly increased rectal temperature compared to age-matched SAMR1 mice (shown in Table 2). In brown adipose tissue (BAT), mRNA expression of uncoupling protein 1 (UCP-1), a marker of increased thermogenesis, was significantly higher in SAMP8 mice than in SAMR1 mice only at the age of 3 months, increasing significantly between the 3^rd and 6^th months of age in both SAMP8 mice and their respective controls, as shown in Figure 4. Between the 6^th and 9^th months, decreased expression of UCP-1 was observed in SAMP8 mice.

Table 2. Rectal temperature, and level of CRP and cytokines in blood plasma of SAMR1 and SAMP8 mice.

		Rectal temperature	CRP	TNFα	IL-6
		[° C]	[μg/ml]	[pg/ml]	[pg/ml]
3 M	SAMR1	37.0 ± 0.2	4.50 ± 0.30	2.51 ± 0.52	2.16 ± 0.70
	SAMP8	37.9 ± 0.2*	3.20 ± 0.69*	2.48 ± 0.92	4.09 ± 1.29
6 M	SAMR1	37.0 ± 0.1	4.16 ± 0.86	3.09 ± 1.56	4.03 ± 0.39
	SAMP8	38.3 ± 0.3***	6.02 ± 0.44**	4.64 ± 1.01	20.64 ± 12.04
9 M	SAMR1	36.4 ± 0.2	4.30 ± 0.76	4.15 ± 0.69	18.21 ± 4.39
	SAMP8	37.4 ± 0.3*	4.61 ± 0.40	6.66 ± 3.20	36.22 ± 8.10*
Data are mean ± SEM analyzed by 2-way ANOVA, Bonferroni post-hoc test. *P < 0.05, **P < 0.01 and ***P < 0.001 (* SAMP8 compared to SAMR1 at the same age), n = 5 mice per group.

Figure 4. Increased mRNA expression of UCP-1 in brown adipose tissue of 3 months old SAMP8 mice. Data are mean ± SEM, analyzed by 2-way ANOVA with Bonferroni post test. Significance is *P < 0.05, and ***P < 0.001; *SAMP8 mice compared to SAMR1, +age-dependent changes in SAMR1, and # age-dependent changes in SAMP8. n = 4-5 mice per group.

At the age of 3 and 6 months, level of c-reactive protein (CRP) was significantly increased in SAMP8 mice compared to their controls. Concentration of proinflammatory cytokines TNFα and IL-6 tended to increase in SAMP8 mice from the 6^th month. Significantly increased concentration of IL-6 was observed in blood plasma of 9-month-old SAMP8 mice compared to SAMR1 (as shown in Table 2).

Worsened neuroinflammation with ageing in the brains of SAMP8 mice

Six- and 9-month-old SAMP8 mice showed significantly increased glial fibrillary acidic protein (GFAP) reactivity in the cortex compared to the age-matched controls; a similar trend was also observed in the 3-month-old mice. Figure 5A–5H illustrates the visible clusters of GFAP-stained reactive astrocytes and the quantification of the immunohistochemical staining (IHC).

Figure 5. Increased neuroinflammation in the brains of SAMP8 mice: (A–G) marker of reactive astrocytes GFAP in the cortex, and (H) its quantification, and (I, J) marker of microglia Iba1 in the brainstem and (K) its quantification. Data are mean ± SEM, analyzed by 2-way ANOVA with Bonferroni post test (GFAP) or t-test (Iba1). Significance is *P < 0.05 and **P < 0.01. * SAMP8 compared to SAMR1, + age-dependent changes in SAMR1, and # age-dependent changes in SAMP8. n = 4-5 mice per group, 8-10 sections per brain.

Staining with ionized calcium-binding adapter molecule 1 (Iba1), a marker expressed by both resting and activated microglia and macrophages, revealed clusters of activated microglia in the brainstems of 9-month-old SAMP8 mice in contrast to the age-matched controls which did not develop any microglial activation (Figure 5I, 5J); the percentage of the area stained with Iba1 was significantly increased in the SAMP8 mice compared to the SAMR1 controls (Figure 5K). However, in the hippocampus and cortex, there were no visible clusters in SAMR1 or SAMP8 mice of any age, and no significant difference in the intensity of Iba1 staining was found between the strains (Supplementary Figure 1A–1C).

Decreased number of neurons and neurogenesis in the brains of SAMP8 mice

Three- and 6-month-old SAMP8 mice showed significantly decreased number of neurons (marked by NeuN, Figure 6) in both the cortex and hippocampus compared to the age-matched controls (SAMR1 mice). At the age of 9 months, there was no significant difference between strains; however, the number of neurons was reduced in the SAMR1 controls compared to those 3 months old.

Figure 6. Decreased total number of neurons in the cortex and hippocampus of SAMP8 immunohistochemically stained for NeuN: representative photomicrographs of the brains of SAMR1 (A–C) and SAMP8 (D–F) mice. Black-framed inserts in right down corners show a magnified area of hippocampus, and (G, H) the quantification. Total number of stained particles was counted in cortex, percentage of the stained area expressed as a % of a respective control group was used in hippocampus. Data are mean ± SEM, analyzed by 2-way ANOVA with Bonferroni post test. Significance is *P < 0.05 and **P < 0.01. * SAMP8 compared to SAMR1,+ age-dependent changes in SAMR1, and # age-dependent changes in SAMP8. n = 4-5 mice per group, 8-10 sections per brain.

Similarly, adult neurogenesis, measured as the number of doublecortin (DCX)-positive cells, was significantly reduced in 3-month-old SAMP8 mice compared to the age-matched control strain. The same trend was observed in 6-month-old mice, but the difference did not reach significance. The number of newly generated neurons dropped sharply with increasing age in both strains, and at the age of 9 months, there was no apparent difference between strains (Figure 7).

Figure 7. Decreased neurogenesis in the hippocampi of SAMP8 mice immunohistochemically stained for doublecortin (DCX): representative photomicrographs of the brains of SAMR1 (A–C) and SAMP8 (D–F) mice, (G) a detail of DCX-stained neurons in the subgranular zone of the hippocampus, and (H) the quantification. Data are mean ± SEM, analyzed by 2-way ANOVA with Bonferroni post test. Significance is ***P < 0.001. * SAMP8 compared to SAMR1, + age-dependent changes in SAMR1, and # age-dependent changes in SAMP8. n = 4-5 mice per group, 8-10 sections per brain.

Decreased synaptic plasticity in the hippocampi of SAMP8 mice

At all examined ages, significantly decreased levels of the presynaptic marker synaptophysin were observed in SAMP8 mice compared to SAMR1 controls (Figure 8). The level of postsynaptic density protein 95 (PSD95) did not differ at any age between SAMP8 and SAMR1 mice. The level of the most abundant postsynaptic density protein, calcium/calmodulin-dependent protein kinase II α (CaMKIIα), did not differ between strains and with ageing; however, its activating phosphorylation at Thr286 was significantly lower in SAMP8 mice than in SAMR1 mice at 3 months of age. CaMKIIα is a kinase of c-AMP-response element binding (CREB), and analogously, significantly decreased activating phosphorylation at Ser133 was observed in the hippocampi of SAMP8 mice with ageing.

Figure 8. Decreased markers of synaptic plasticity in the hippocampi of SAMP8 (A) western blots and (B) their quantification. Data are mean ± SEM, analyzed by 2-way ANOVA with Bonferroni post test. Significance is *P <0.05, **P < 0.01 and ***P < 0.001. * SAMP8 compared to SAMR1, + age-dependent changes in SAMR1, and # age-dependent changes in SAMP8. n = 4-5 mice per group.

Impaired activation of the insulin signaling cascade in the hippocampi of SAMP8 mice

In the hippocampi of SAMP8 mice, insulin resistance was observed (Figure 9). First, SAMP8 mice showed decreased hippocampal insulin receptor β at the age of 6 months compared to SAMR1 controls. Insulin receptor substrate 1 (IRS-1) decreased significantly with age in SAMR1 mice; a nonsignificant decrease was observed at 3-month-old SAMP8 mice in comparison with SAMR1 mice. Significantly increased phosphorylation of IRS-1 at Ser612 was observed in 3-month-old SAMP8 mice. Decreasing levels of IRS-2 was observed with ageing in SAMP8 mice; however, they did not differ from those in age-matched SAMR1 mice. Additionally, kinases implicated in the insulin signaling cascade, such as phosphoinositide 3-kinases (PI3K) and phosphoinositide-dependent kinase-1 (PDK-1), were reduced in SAMP8 mice compared to SAMR1 controls. Phosphorylation of Akt at Thr308 increased in SAMR1 mice with ageing; on the other hand, in SAMP8 mice, phosphorylation was less pronounced and was significantly lower at 9 months of age in SAMP8 mice than in SAMR1 controls. Phosphorylation of Akt at Ser473 decreased with ageing in both SAMR1 and SAMP8 mice; no differences were observed between SAMR1 and SAMP8 mice.

Figure 9. Impaired activation of the insulin signaling cascade in the hippocampi of SAMP8 mice (A) western blots and (B) their quantification. Data are mean ± SEM, analyzed by 2-way ANOVA with Bonferroni post test. Significance is *P < 0.05, **P < 0.01 and ***P < 0.001; * SAMP8 compared to SAMR1, + age-dependent changes in SAMR1, and # age-dependent changes in SAMP8. n = 4-5 mice per group.

Decreased activity of tau phosphatase PP2A in the hippocampi of SAMP8 mice

The level of total glycogen synthase kinase 3β (GSK-3β), one of the main tau kinases that is also implicated in the insulin signaling cascade, did not differ during ageing or between the SAMR1 and SAMP8 mice at any age (Figure 10). However, phosphorylation at the inhibitory epitope Ser9 significantly decreased during the ageing of both strains. Total level of main tau phosphatase called protein phosphatase 2A (PP2A) was significantly lower in SAMP8 mice compared to SAMR1, at the age of 6 and 9 months; in concordance, the level of PP2A methylated at Leu9, which is the marker of its activation, also significantly decreased at 6-month-old SAMP8 mice and nonsignificantly at the age of 9 months.

Figure 10. Increased activation of tau kinase GSK-3β and decreased activation of protein phosphatase 2A sub C in the hippocampi of SAMP8 (A) western blots and (B) their quantification. Data are mean ± SEM, analyzed by 2-way ANOVA with Bonferroni post test. Significance is *P < 0.05, **P < 0.01 and ***P < 0.001; * SAMP8 compared to SAMR1, + age-dependent changes in SAMR1, and # age-dependent changes in SAMP8. n = 4-5 mice per group.

Hyperphosphorylation of tau protein at different epitopes in the hippocampi of SAMP8 mice

In SAMR1 mice, increased levels of total tau protein with ageing were observed in the hippocampi (Figure 11). SAMP8 mice had increased phosphorylation of tau protein at different epitopes, such as Ser396, Thr231 and Ser214 in comparison with age-matched SAMR1 controls at the age of 6 or 9 months. Increased tau phosphorylation at Ser396 was confirmed by immunofluorescence; a more intense fluorescence signal was observed in the CA1 area of the hippocampus of SAMP8 mice than in SAMR1 mice at the ages of 6 and 9 months.

Figure 11. Hyperphosphorylation of tau protein in the hippocampi of SAMP8 mice (A) western blots, (B) their quantification, and (C) immunofluorescence of p-tau [Ser396] at CA1 area of the hippocampus. Data are mean ± SEM, analyzed by 2-way ANOVA with Bonferroni post test. Significance is *P < 0.05, **P < 0.01 and ***P < 0.001; * SAMP8 compared to SAMR1, + age-dependent changes in SAMR1, and # age-dependent changes in SAMP8. n = 4-5 mice per group.

In addition to tau hyperphosphorylation, AD is characterized by the accumulation of Aβ plaques. However, in the brains of SAMP8 mice, no Aβ plaques were detected using IHC (Supplementary Figure 2).

Increased peripheral insulin resistance in the skeletal muscle of SAMP8 mice

In skeletal muscle (Figure 12), SAMP8 mice showed significantly decreased glucose transporter 4 (GLUT 4) from the 6^th month and increased levels of forkhead box protein O1 (FoxO1) at the age of 9 months compared to controls. The level of total AMP-activated protein kinase (AMPK) did not differ between SAMR1 and SAMP8 mice; however, the level increased with ageing in both strains. SAMP8 mice had significantly increased levels of AMPK phosphorylated at Thr172 at the 3^rd month. A significantly increased level of total Akt was observed in SAMP8 mice from the 6^th month; on the other hand, significantly decreased Akt activation manifested by significantly decreased phosphorylation at Thr308 and Ser473 was observed in 9-month-old SAMP8 mice and at Ser473 in 6-month-old SAMP8 mice.

Figure 12. Increased insulin resistance in skeletal muscle of SAMP8 (A) western blots and (B) their quantification. Data are mean ± SEM, analyzed by 2-way ANOVA with Bonferroni post test. Significance is *P <0.05, **P < 0.01 and ***P < 0.001; * SAMP8 compared to SAMR1, + age-dependent changes in SAMR1, and # age-dependent changes in SAMP8. n = 4-5 mice per group.

NMR metabolomics revealed significant differences in urine composition between SAMP8 and SAMR1 mice at the age of 3 months

The differences between urinary metabolic profiles of both strains were evaluated by both multivariate and univariate approaches. PCA did not detect any outliers in the SAMP8 or SAMR1 groups. Then, a PLS-DA model was built; leave-one-out cross-validation showed satisfactory values (as shown in Supplementary Figure 3).

The highest VIP score of the PLS-DA model identified bins, corresponding to the signals of metabolites responsible for group separation. The difference between SAMP8 mice and their SAMR1 controls at 3 months of age is caused mainly by the combination of increased levels of trimethylamine, dimethylamine, citrate, and N-carbamoyl-β-alanine together with decreased concentrations of creatine, methanol, n-hexanoylglycine, butyrylglycine and vinylacetylglycine. As the multivariate approach is based on the simultaneous evaluation of all metabolite levels and the model is characterized as the unique combination of their changes, univariate statistics were used to describe the significance of changes in individual metabolite concentrations.

Within a set of sixty signals quantified in urine spectra, twenty metabolites were evaluated as significantly changed in SAMP8 mice compared to SAMR1 controls and are summarized in Table 3.

Table 3. Significantly changed metabolites in 3-month-old SAMP8 mice.

Metabolite	NMR signal used for the quantitation	SAMP8 vs. SAMR1 ∆ %
1-Methylnicotinamide	8.90 (m)	29.4 **
3-Indoxylsulfate	7.71 (m)	52.2 *
Acetate	1.92 (s)	-59.1 *
Butyrylglycine	1.61 (m)	-23.9 **
Carnitine	3.23 (s)	50.6 **
Creatine (phosphate)	3.93 (s)	-53.9 **
Creatinine	4.06 (s)	23.7 **
Dimethylamine	2.72 (s)	76.5 ***
Glycine	3.55 (s)	71.7 **
n-Hexanoylglycine	0.88 (t)	-17.9 *
Methanol	3.37 (s)	-51.5 *
Methylamine	2.61 (s)	32.3 *
N-Carbamoyl β-alanine	3.31 (m)	58.3 **
Niacinamide	8.72 (m)	-15.0 *
Nicotinamide N-oxide	8.75 (m)	36.4 *
Orotate	6.20 (s)	49.2 **
Putrescine	1.85 (m)	-38.1 ***
Tartrate	4.34 (s)	137.3 *
Trimethylamine	2.88 (s)	155.9 **
Vinylacetylglycine	1.88 (dd)	-36.1 **
Unknown triplet	3.17 (t)	65.0 ***
The results are expressed as the percentage change of concentration in SAMP8 vs. SAMR1 analyzed by Wilcoxon-Mann-Whitney test. *P < 0.05, **P < 0.01 and ***P < 0.001. Signal multiplicity is marked as follows: (s)-singlet, (t)-triplet, (dd)-doublet od doublets, (m)-multiplet.

Author Contributions

A P, M H, L M and J K designed the experiments; A P, M H, L H, V S, B N and H P performed the experiments; A P, M H and H P analyzed the data and wrote the manuscript; M K, B Ž, J K, and L M edited the manuscript; A P, M H, B Ž, J K and L M approved the final manuscript.

Acknowledgments

We would like greatly acknowledge Hedvika Vysušilová for her excellent technical assistance and great animal care.

Conflicts of Interest

Authors disclose no conflicts of interest.

Ethical Statement

All animal experiments followed the ethical guidelines for animal experiments and the Czech Republic Act No. 246/1992 and were approved by the Committee for Experiments with Laboratory Animals of the CAS.

Funding

The study was supported by Czech Science Foundation: grant 20-00546S and Czech Academy of Sciences RVO: 61388963, RVO: 67985823 and RVO: 61388971.

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