Exogenous Hsp70 delays senescence and improves cognitive function in aging mice

Abstract

Molecular chaperone Heat Shock Protein 70 (Hsp70) plays an important protective role in various neurodegenerative disorders often associated with aging, but its activity and availability in neuronal tissue decrease with age. Here we explored the effects of intranasal administration of exogenous recombinant human Hsp70 (eHsp70) on lifespan and neurological parameters in middle-aged and old mice. Long-term administration of eHsp70 significantly enhanced the lifespan of animals of different age groups. Behavioral assessment after 5 and 9 mo of chronic eHsp70 administration demonstrated improved learning and memory in old mice. Likewise, the investigation of locomotor and exploratory activities after eHsp70 treatment demonstrated a significant therapeutic effect of this chaperone. Measurements of synaptophysin show that eHsp70 treatment in old mice resulted in larger synaptophysin-immunopositive areas and higher neuron density compared with control animals. Furthermore, eHsp70 treatment decreased accumulation of lipofuscin, an aging-related marker, in the brain and enhanced proteasome activity. The potential of eHsp70 intranasal treatment to protect synaptic machinery in old animals offers a unique pharmacological approach for various neurodegenerative disorders associated with human aging.

Keywords: Hsp70; aging; memory; proteasome; therapy.

Fig. 1.

Effect of chronic eHsp70 treatment on longevity. (A) Longevity of old animals (experimental and control mice; n = 33 and 34, respectively) when eHsp70 treatment was started at 17 mo of age and lasted until animals’ death. (B) Longevity of middle-aged animals (experimental + control mice n = 25 + 27) when eHsp70 treatment was started at 12 mo of age and lasted until animals’ death. Experimental results were statistically processed with the use of the Statistica.10 software (Statsoft). The choice of the most appropriate model was carried out with the use of the weighted least-squares method. Parameters of the Gompertz model were estimated by using the maximum-likelihood ratio. Kaplan and Meier product-limit method was used to compare untreated (control) and eHsp70-treated samples of mice. Experimental data are designated with color circles. Cox's F test: F(66, 68) = 1.661273; P = 0.01959 for old mice; Cox's F-Test: F(50, 54) = 1.927759; P = 0.00949 for middle-aged mice. Confidence intervals for the parameters were obtained by using the bootstrap method. Treatment with eHsp70 improved longevity from 29 to 31 mo in both age groups, but more importantly, eHsp70 improved survival rates; at 22 mo, 55.5% of animals in the experimental middle-aged group were alive, compared with 22.2% in the control group. After 22 mo, in the untreated old-age group, survival was just over 35.2%, whereas in the experimental group, 54.5% were still alive.

Fig. 2.

Effect of eHsp70 treatment on learning in old and middle-aged mice. Learning was assessed in old (n = 9; A) and middle-aged (n = 9; B) animals after 5 mo of eHsp70 treatment and again in old (n = 8; C) and middle-aged (n = 9; D) animals after 9 mo of eHsp70 treatment. Treatment with eHsp70 decreased latency to finding the platform in both old and middle-aged mice at both time points investigated. *P < 0.05.

Fig. 3.

The effects of chronic intranasal administration of eHsp70 for 5 or 9 mo on spatial memory in old and middle-aged animals. Animals were subjected to probe trial after 5 d of training trials in the Morris water maze. (A and B) The number of entries into each the four sectors of the Morris water maze was assessed in old (n = 13; A) and middle-aged (n = 14; B) animals. (C and D) Time spent in each sector of the Morris water maze was also analyzed for old (n = 13; C) and middle-aged (n = 14; D) animals. The target sector is shaded. In middle-aged animals, treatment with eHsp70 for 5 or 9 mo did not induce changing in recognition of target sector in comparison with age-matched control mice. In contrast, old animals only improved their spatial memory after 9 mo of treatment with eHsp70. *P < 0.05; **P < 0.01; ***P < 0.001 compared with age-matched control groups.

Fig. 4.

Effect of chronic intranasal administration of eHsp70 for 5 or 9 mo on locomotor activity in an OFT. We assessed locomotor activity in middle-aged (n = 26; B and D) and old (n = 27; A and C) mice during each minute of the test. In old animals treated with eHsp70 for 5 mo, locomotor activity increased compared with age-matched control (n = 7; A). This effect was not apparent after 9 mo of treatment. Furthermore, eHsp70 treatment had no effect in middle-aged animals. **P < 0.01; ***P < 0.001.

Fig. S1.

Effect of chronic administration of eHsp70 on the behavioral profile of middle-aged and old mice in the OFT. We measured the number of rearings (A and B), indicating exploratory behavior, and the number of central zone crossings (C and D), an indicator of fear and anxiety. There was no significant effect of eHsp70 treatment in middle-aged animals (n = 14), but eHsp70 did increase exploratory behavior in old animals (n = 13) after 5 and 9 mo of treatment and also decreased anxiety in old animals after 5 mo of treatment (n = 7). This effect was gone after 9 mo of treatment. *P < 0.05; **P < 0.01; ***P < 0.001. Error bars indicate SEM.

Fig. S2.

The effect of eHsp70 administration on neuron density. We assessed neuronal density in the temporal cortex (A) and hippocampal area CA1–CA2 (B) of young and old mice. The x axis represents the experimental groups: 2.5-mo mice (n = 6); untreated old mice (n = 5; age 26.2 ± 2.61 mo); and old mice treated with eHsp70 for 9 mo (n = 5; age 28.2 ± 3.12). All data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5.

Effect of eHsp70 administration for 5 mo on molecular markers of aging. (A and B) Levels of synaptophysin (A) or lipofuscin (B) were measured in the brains of eHsp70-treated old mice (n = 9) and age-matched controls (n = 8). Error bars represent mean SEM. Treatment with eHsp70 increased synaptophysin immunostaining in the cortex and hippocampus and decreased lipofuscin autofluorescence in the hippocampus. (C) Proteolytic activities of proteasomes in lysates from the cerebral cortex of experimental old mice. Average relative activity levels in 1 µL of tissue lysate of eHsp70-treated (n = 8) and control (n = 5) groups are shown. Treatment with eHsp70 increased chymotrypsin-like activity, but had no effect on caspase-like activity. (D) Expression of proteasome subunits in cortex lysates. Treatment with eHsp70 increased expression of the Rpt6, β6, and β5i subunits. Error bars represent SD of the mean. Significance was tested by using the Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001, calculated by two-tailed Student’s t test.

Fig. S3.

The protective effect of eHsp70 treatment on levels of lipofuscin immunostaining in the hippocampus and cortex is evident. Representative photomicrograph of lipofuscin in the hippocampus (A) and temporal cortex (B) of eHsp70-treated and control old mice. (A, i) General view of hippocampus in HSP70-treated animal. (A, ii) The same as A, i at higher magnification. (A, iii) General view of hippocampus in control animal. (A, iv) The same as A, iii at higher magnification. (B, i) General view of temporal cortex in control animal. (B, ii) The same as B, ii at higher magnification. (B, iii) General view of temporal cortex in control animal. (B, iv) The same as B, iiii, at higher magnification. [Scale bars: 20 μm (general views of cortex and hippocampus); 5 μm (photos with higher magnification).]

Fig. S4.

Accumulation of proteasomal subunits in the cerebral cortex lysates of control and experimental mice. Proteins were detected by Western blot with specific antibodies to 20S proteasome α1,2,3,5,6,7 subunits; 19S regulator subunit Rpt6; PA28 alpha subunit; beta subunits β6, β5, β1i, β5i, and to IFN-γ. After development, the membranes were stripped and stained with anti–β-actin antibodies for signal normalization. Western blots were repeated three times.

Fig. S5.

eHsp70 and BSA stained with Roti Blue quick. A total of 1.45 μg of eHsp70 protein was used. Gradient was 4–20% (wt/vol) SDS/PAGE.