Ginkgo biloba extract EGb 761® ameliorates cognitive impairment and alleviates TNFα response in 5xFAD Alzheimer's disease model mice

Background: Ginkgo biloba leaf extract EGb 761 (R) has shown clinical efficacy in patients with mild cognitive impairment and dementia. However, the pharmacological action of EGb 761 (R) in Alzheimer's disease (AD) remains unclear and molecular mechanisms targeted in the brain are not completely understood. Hypothesis/Purpose: We aimed to investigate 1) the potential sex-dependent effects of oral administration of EGb 761 (R) in 5xFAD mice, an AD mouse model, and 2) the underlying microglial subtype responsible for the observed anti-inflammatory effects in the brain. Methods: Eight-week old 5xFAD and wild type mice received EGb 761 (R)-supplemented diet or control diet for eight weeks. The study investigated changes in cognitive function as well as amyloid plaque load, expression of AD-related genes, and anti-inflammatory effects. Moreover, we used organotypic brain slices for confirmation and assessment of concentration-dependency of the observed EGb 761 (R) effects and performed single cell RNA sequencing on the prefrontal cortex of male mice with focus on microglia. Results: We demonstrate that EGb 761 (R) treatment improves cognitive function in 5xFAD mice in several behavioral tests. Analysis of the brain tissue from these animals indicated a reduction in amyloid plaque load in the prefrontal cortex (PFC). This brain area was further investigated to assess the molecular changes that occurred following EGb 761 (R) intake. Alterations in the expression of genes related to AD were highly sex-specific with effects on the cholinergic system, the gamma-secretase complex, and neuroinflammation. Anti-inflammatory effects of EGb 761 (R) with a particularly pronounced reduction of the TNF alpha-response could be shown for the PFC but also peripherally in the serum of 5xFAD mice of both sexes. Single-cell RNA sequencing revealed that EGb761 (R) mainly affected disease-associated microglia stage 2 (DAM2), which are thought to have a detrimental role in AD. Conclusions: EGb 761 (R) shows efficacy in the treatment of cognitive deficits in the 5xFAD mouse model via multimodal activity, including sex-specific and sex-unrelated mechanisms including the normalization of neuroinflammatory parameters.