Centella asiatica attenuates hippocampal mitochondrial dysfunction and improves memory and executive function in β-amyloid overexpressing mice

Abstract

Centella asiatica is a medicinal plant used to enhance memory. We have previously shown that a water extract of Centella asiatica (CAW) attenuates β-amyloid (Aβ)-induced spatial memory deficits in mice and improves neuronal health. Yet the effect of CAW on other cognitive domains remains unexplored as does its in vivo mechanism of improving Aβ-related cognitive impairment. This study investigates the effects of CAW on learning, memory and executive function as well as mitochondrial function and antioxidant response in the 5xFAD model of Aβ accumulation. Seven month old 5xFAD female mice were treated with CAW (2 mg/mL) in their drinking water for two weeks prior to behavioral testing. Learning, memory and executive function were assessed using the object location memory task (OLM), conditioned fear response (CFR) and odor discrimination reversal learning (ODRL) test. Mitochondrial function was profiled using the Seahorse XF platform in hippocampal mitochondria isolated from these animals and tissue was harvested for assessment of mitochondrial, antioxidant and synaptic proteins. CAW improved performance in all behavioral tests in the 5xFAD but had no effect on WT animals. Hippocampal mitochondrial function was improved and hippocampal and cortical expression of mitochondrial genes was increased in CAW-treated 5xFAD mice. Gene expression of the transcription factor NRF2, as well as its antioxidant target enzymes, was also increased with CAW treatment in both WT and 5xFAD mice. CAW treatment also decreased Aβ-plaque burden in the hippocampus of treated 5xFAD mice but had no effect on plaques in the cortex. These data show that CAW can improve many facets of Aβ-related cognitive impairment in 5xFAD mice. Oral treatment with CAW also attenuates hippocampal mitochondrial dysfunction in these animals. Because mitochondrial dysfunction and oxidative stress accompany cognitive impairment in many pathological conditions beyond Alzheimer's disease, this suggests potentially broad therapeutic utility of CAW.

Keywords: Antioxidant; Beta amyloid; Executive function; Mitochondrial function.

Figure 1:. Timeline of CAW treatment and behavioral assessment.

Mice were treated with CAW (2g/L) two weeks prior to the beginning of behavioral testing and treatment continued throughout the experiment. After testing, animals were sacrificed and tissue was harvested. CAW treatment lasted a total of 5 weeks.

Figure 2:. CAW treatment improves object location memory retention in 5×FAD mice.

A) Schematic of the Object Location Memory task (OLM) set up. B) At the 2h retention time point 5×FAD mice showed significantly reduced preference for the novel location. CAW-treatment attenuated this impairment in 5×FAD mice. n=5-7 mice in each group, *p<0.05

Figure 3:. CAW treatment improves contextual memory in 5×FAD mice.

A) Schematic of the conditioned fear response (CFR) testing set up. B) 5×FAD mice froze significantly less in the testing phase than WT mice. CAW restored freezing behavior in 5×FAD mice to WT levels but had no effect on WT mice. Data is represented as the difference in freezing time between the test phase and the habituation phase, n=5-7 mice in each group, *p<0.05, **p<0.01.

Figure 4:. CAW treatment improves executive function in 5×FAD mice.

In the acquisition phase of the ODRL the number of trials to reach criteria was significantly increased in 5×FAD mice. The same trend was evident in the shift phase. CAW treatment reduced the number of trials to reach criteria in both phases of the ODRL although this reduction only reached statistical significance in the shift phase. n=5–7 mice in each group, *p<0.05.

Figure 5:. CAW reduces Aβ accumulation in the hippocampus but not the cortex of 5×FAD mice.

A) CAW significantly reduced the Aβ plaque burden in the hippocampus of 5×FAD mice. B) There was no effect of CAW treatment on Aβ accumulation in the cortex. n=10-14 mice each group, *p<0.05

Figure 6:. CAW increases mitochondrial, antioxidant and synaptic proteins in the hippocampus and cortex of 5×FAD mice.

Synaptic gene expression was reduced in 5×FAD mice in the hippocampus (A) and frontal cortex (B) but this was attenuated by CAW treatment. CAW treatment also increased expression of NRF2 and its ARE-containing target genes in the hippocampus (C) and frontal cortex (D) of both 5×FAD and WT animals. 5×FAD mice had a reduction in expression of ETC genes in the hippocampus (E) and frontal cortex (F) and CAW treatment increased this expression in both brain regions. n=8-10, *p<0.05, **p<0.01, ***p<0.001.

Figure 7:. CAW increases mitochondrial respiration in the hippocampus of 5×FAD mice.

A) The bioenergetic profile of 5×FAD mice, as determined by Seahorse XF analyzer, was impaired relative to WT mice and CAW treatment partially attenuated this impairment. B) CAW reduced the deficit in both basal and maximal respiration in hippocampal mitochondria isolated from 5×FAD mice but had no effect on RCR. n=4-5 per group, *p<0.05, **p<0.01, ***p<0.001.