(M1130-05-27) Brain Mitochondrial Metabolic Alterations In VCD-Induced Menopause Model

Purpose: Loss of estrogen is implicated in age-related cognitive decline and increased risk of age-related dementias including Alzheimer’s disease [1], contributing to higher prevalence in females.[2] Brain dementia such as Alzheimer’s disease (AD) disproportionately affects women, with nearly two-thirds of patients being postmenopausal females.[3] The menopausal transition (MT) is associated with declining levels of ovarian hormones, leading to a loss of neuroprotective support and an increase in brain metabolic vulnerability.[4] In this context, metabolic vulnerabilities in mid-life’s brain contributes as a risk factor for the development of brain dementia. [5-7], Metabolic shifts within the cortex, hippocampus and hypothalamus during this period are thought to contribute to the development of cognitive decline and dementia highlighting the importance of early metabolic detection as a strategy for identifying individuals at risk. Previous studies have shown that perimenopausal women experience reduced brain glucose metabolism associating with bioenergetic crisis and mitochondrial dysfunction, which are observable via FDG-PET imaging.[7] These alterations in brain energy metabolism have been shown to precede the onset of Alzheimer’s disease symptoms, underscoring the potential of early metabolic detection for identifying individuals at risk.[8] The objective of this study is to characterize menopause-associated alterations in brain metabolism, structure, and mitochondrial function in a VCD-induced mouse model. Specifically, we investigated how ovarian hormone loss affects brain metabolism, structure and mitochondrial function, particularly in the hippocampus, a critical brain region involved in memory consolidation and learning, using a preclinical model of menopause.

Methods: We induced menopause in female C57BL/6 mice using 4-vinylcyclohexene diepoxide (VCD) to model the gradual loss of ovarian hormones observed in humans. VCD (160mg/kg, 100ul/mouse of VCD in sesame oil) was administered to mice to induce ovarian failure. Three mice groups: menopause (12months, VCD), perimenopause (12 months) and Young (6 months) were used. Menopausal status was confirmed by evaluating vaginal cytology using Wright-Giemsa-stained smears. Blood samples were analyzed for estrogen and follicle-stimulating hormone (FSH) levels using mouse-specific ELISA kits for estrogen (R&D Systems, Bio-Techne) and FSH (Invitrogen, Thermo Fisher Scientific), respectively. T2-weighted (T2WI) magnetic resonance imaging (MRI) was performed on each group. MRI were conducted on a cryogen-free MRI magnet at 7T (MRS*DRYMAG7107) equipped with a 17-cm bore magnet with 600 mT/m RF gradients. T2WI were acquired using the following parameters: FSE sequence, FOV of 12 mm, matrix size of 256 × 256, TR 5000 ms, TE 45 ms, 32 slices with slice thickness of 0.50. Image post-processing was conducted using VivoQuant 2022 software and multi-Atlas segmentation tool was used to analyze substructure regional brain volume measurement in each group. Mitochondrial metabolism in the CA3 region of the hippocampus was assessed using the Seahorse XFe24 Analyzer (Agilent Technologies, CA) with the Cell Mito Stress Test. Parameters including Oxygen consumption rate, basal respiration, ATP-linked respiration, and maximal respiration were measured. One-way analysis of variance (ANOVA) revealed significant group differences. P-value < 0.05 will be considered as statistically significant.

Results: Vaginal smear analysis confirmed (Fig 1A). The young mice had a relatively regular estrous cycle, perimenopause showed an irregular cycle, and the menopause group had persistent acyclicity (Fig 1 B-D.), consistent with the characteristic phenotypes of each reproductive stage. Further validated with serum estrogen and FSH levels. Menopause group had a significantly lower estrogen (42.296 ± 7.123 pg/mL) (Fig 1E.) with higher FSH (7.530 ± 1.052 ng/mL) (Fig 1F.), compared to perimenopause (estrogen, (55.094 ± 3.057 pg/mL) and FSH (4.344 ± 0.124 ng/mL)),   and young mice groups (estrogen, (76.079 ± 5.209 pg/mL) and FSH (3.477 ± 0.389 ng/mL)).  The menopause group had significantly lower estrogen levels (P < 0.0009) and higher FSH levels compared to the young group (P < 0.0004. (p values varied from *p < 0.05, **p < 0.01, ***p < 0.001). One-way ANOVA followed by Tukey’s multiple comparison. The results are shown as the mean ± SD. Comparatively the CA3 region of menopause mice had significantly lower basal respiratory rate compared to perimenopause mice (60 ± 37.373 pmol/min vs 108.624 ± 54.173 pmol/min, P< 0.0392) due to a decreased glucose metabolic phenotype (Fig 2B). In addition, ATP-linked respiration was significantly reduced in menopause mice (6.534 ± 6.965 pmol/min) compared to perimenopause mice (19.845 ± 15.216 pmol/min), P < 0.02. (Fig 2F) The MRI showed a comparable hippocampus volume across groups (Fig 3.)

Conclusion: This study highlights the early metabolic dysfunction in the hippocampal mitochondria after ovarian hormone loss, particularly within the CA3 region, despite no detectable structural changes. This lack of detectable regional brain atrophy may reflect the resolution limitations of MRI in capturing subtle cellular changes, which may precede overt atrophy. The findings suggest that assessment of mitochondrial function in the hippocampus may be useful as a biomarker of menopause related cognitive decline and a potential target for early intervention in age-related neurodegenerative diseases.

References: 1. Scheyer, O., et al., Female Sex and Alzheimer's Risk: The Menopause Connection. The Journal of Prevention of Alzheimer's Disease, 2018. 5(4): p. 225-230.
2. Itoh, N., et al., Estrogen receptor beta in astrocytes modulates cognitive function in mid-age female mice. Nat Commun, 2023. 14(1): p. 6044.
3. 2023 Alzheimer's disease facts and figures. Alzheimers Dement, 2023. 19(4): p. 1598-1695.
4. Abi-Ghanem, C., et al., Effects of menopause and high fat diet on metabolic outcomes in a mouse model of Alzheimer’s disease. bioRxiv, 2023: p. 2023.11.21.568069.
5. Onaolapo, A.Y., et al., Diabetes Mellitus and Energy Dysmetabolism in Alzheimer's Disease: Understanding the Relationships and Potential Therapeutic Targets. Curr Diabetes Rev, 2023. 19(8): p. e020123212333.
6. Kim, Y.J., et al., Associations between metabolic syndrome and type of dementia: analysis based on the National Health Insurance Service database of Gangwon province in South Korea. Diabetol Metab Syndr, 2021. 13(1): p. 4.
7. Mosconi, L., et al., Increased Alzheimer's risk during the menopause transition: A 3-year longitudinal brain imaging study. PLoS One, 2018. 13(12): p. e0207885.
8. Marcus, C., E. Mena, and R.M. Subramaniam, Brain PET in the diagnosis of Alzheimer's disease. Clin Nucl Med, 2014. 39(10): p. e413-22; quiz e423-6.

Acknowledgements:

Funding: Pharmacology and Neuroscience Department Pilot Grant, UNT Health at Fort Worth

FIGURE 1

FIGURE 2

FIGURE 3