INTRODUCTION

Alzheimer’s disease (AD) is the most common neurodegenerative disorder associated with gradual deterioration in memory and cognitive function¹. Age and gender are considered as major factors to influence the progression of AD, and the number of women suffering from AD is always higher than that of males in respective age groups from 60 years to 90. It may be attributed to the higher longevity of women at higher age groups, but at the age of 60 to 80 difference in mortality of males and females is not significant to account for the differences in the disease severity². The 3xTg-AD mouse is an advanced model of AD which exhibit both Aβ plaques and neurofibrillary tangles³. The perturbation in neurometabolite homeostasis and regional glucose hypometabolism has been reported in the AD condition^4,5. The objective of the current study is to assess the severity of AD in males and females by monitoring behavioral phenotype and neurometabolism using 3xTg-AD mice.

MATERIALS AND METHODS

All animal experiments were performed under approved protocols by the Institutional Animal Ethics Committee of CCMB. For behavioral assessment, 12 months old 3xTg-AD and B6129SF2/J (Control) mice were divided into four groups: (A) Control Female, n=8; (B) 3xTgAD Female, n=5; (C) Control Male, n=12; (D) 3xTg-AD Male, n=7. Morris Water Maze was performed to assess the learning and memory in mice⁶. For measurement of metabolic activity, animals were fasted 3 hrs prior to the study. Mice were infused with [1,6-¹³C₂]glucose via tail vein. Blood was collected from retro orbital sinus followed by euthanization using focused beam microwave irradiation (4KW, 1 s) at the end of 7 minutes after infusion⁷. Different brain regions were dissected, and metabolites were extracted from cortical tissue. The concentration and ¹³C labeling of cortical amino acids were measured in ¹H-[¹³C]-NMR spectra recorded at 600 MHz Bruker Avance HD III NMR spectrometer⁸. The cerebral metabolic rates of glucose oxidation by glutamatergic and GABAergic neurons were determined from the ¹³C labeling of brain amino acids from [1,6-¹³C₂]glucose⁹. One way Annova was carried out to find the statistical significance of difference among different groups. The post hoc Tuckey honest test was carried out to further identify the difference between groups. The values are presnted as mean± SEM.

RESULTS AND DISCUSSIONS

Learning of 3xTg-AD males and females was compromised when compared with the respective controls (Figure 1A). The memory assessment carried out on 7^th day shows that escape latency of 3xTg-AD males (47.7±4.7 s vs 23.7±2.8 s, p=0.008) and females (55.1±4.8 s vs 27.5±6.6 s, p=0.013) was almost doubled as comapred with the respective control. Moreover, the retention of memory analysis suggested that time spent in quadrant 4 by male (8.8±1.1 s vs 15.4±1.0 s, p=0.001) and female (9.5±0.9 s vs 16.4±0.8 s, p=0.002) 3xTg-AD mice is significantly less than controls (Figure 1B). These results suggests learning and memory impairment in 3xTg-AD. The level of glutamate was decreased (Cont 12.9±0.3 μmol/g; 3xTg-AD 11.7±0.3 μmol/g; p=0.008), and choline was increased (Cont 0.05±0.01 μmol/g, 3xTg-AD 0.09±0.02 μmol/g, p=0.029) in male 3xTg-AD mice. Moreover, the concentrations of ¹³C lebeled GluC4 (p=0.001), GlnC4 (p=0.020), AspC3 (p=0.003) and GluC3 (p=0.012) were decreased significantly in male 3xTg-AD mice (Figure 2, Table 1) when compared with controls. There was no significant change in the level of neurometabolites and ¹³C labeling in female 3xTg-AD mice. The reduction in concentration of ¹³C labeled amino acids resulted into decreased metabolic activity of glutamatergic (Cont 0.40±0.01 μmol/g/min; 3xTg-AD 0.31±0.02 μmol/g/min; p=0.002) and GABAergic neurons (Cont 0.10±0.004 μmol/g/min; 3xTg-AD 0.07±0.004 μmol/g/min; p=0.004) in male 3xTg-AD mice when compared with controls (Figure 3). The neurometabolism was unpertubed in transgenic female mice. Although, there was a loss in learning and memory in case of female 3xTg-AD mice which is important behavioral phenotype in AD condition, there was no perturbation in neurometabolic activity. In contrast, 3xTg-AD male mice exhibited stronger behavioral phenotype and show pertubation in neurometabolite homeostasis and imapiment in neurometabolic activity.The data presented in the study provide an experimental evidence for increased severity of AD condition in 3xTg-AD male mice, and existence of some neuroprotective mechanism in female during their reproductive age. The severity of AD beyond the reproductive age needs further investigations.

Acknowledgements

The study was supported by funding from CSIR-CCMB.