(M1130-04-24) Excitatory Amino Acid Transporter 2 (EAAT2): A Novel Therapeutic Target for Treatment of Alzheimer’s Disease (AD)

Purpose: AD is the most common form of dementia and is expected to exceed 150 million cases worldwide by 2050¹. At present, there are few FDA approved treatments that can limit the progression of AD. Primary therapeutic targets that have been interrogated for AD include protein aggregates comprised of amyloid beta (Ab) protein and hyperphosphorylated tau². Nonetheless, selection of these proteins as primary targets for AD therapeutics remains controversial. This is highlighted by the recent clinical experience with aducanumab, an Ab-targeted antibody that failed to produce significant therapeutic benefits over placebo in the ENGAGE trial which exhibited serious safety concerns including an increased risk of cerebral edema and intracranial hemorrhage^3,4. This suggests a need to reconsider AD drug development strategies and focus on alternative targets that can modulate both AD pathology and improve functional cognitive performance. Studies by our collaborative and multidisciplinary team have shown that AD involves complex pathological processes via interactive networks of molecular pathways in glial cells (i.e., astrocytes, microglia). One such target that has been identified and validated by our group is EAAT2 (also known as solute carrier (SLC) 1A2). EAAT2 is known to be highly expressed on astrocytes where it plays an essential role in homeostasis of the cerebral glutamate-glutamine cycle⁵. More recently, our group has shown that EAAT2 is functionally expressed on microglia, a primary regulator of phagocytosis, which can promote both neural repair and blood-brain barrier (BBB) protection⁶. Using a combination of computational biology approaches and cell-based assays, we have identified 13 small molecule compounds that target EAAT2. As such, we hypothesize that these small molecules include compounds that can stimulate EAAT2 activity in microglia, thereby attenuating brain expression of biomarkers associated with AD pathogenesis and improving cognitive performance in a rodent AD model.

Methods: Using a focused in vitro screen, we identified a potent small molecule EAAT2 activator (designated as C41) that exhibited both rescue of human neuronal cell viability following exposure to Ab(1-42) (i.e., the most abundant Ab species found in neurite plaques) and the ability to enhance phagocytosis of Ab(1-42) in primary cultures of human microglia. This compound was selected for evaluation in all subsequent experiments. Protein expression of EAAT2 in primary cultures of human microglia was confirmed by western blot analysis. Stimulation of EAAT2-mediated transport by C41 (10 mM) was evaluated in primary cultures of human microglia using [³H] glutamic acid (1.0 mCi/ml) as a probe substrate. Assessment of phagocytosis stimulation by C41 was performed in primary cultures of human microglia exposed to fluorescein isothiocyanate (FITC) labeled Ab(1-42) for 24 h. Selectivity for EAAT2-mediated transport was determined using WAY-213613 (10 mM), a competitive EAAT2 inhibitor. In vivo experiments were conducted in 6-month-old male and female 3xTg mice, which display measurable deficits in working memory and intraneuronal Ab deposition in both the hippocampus and amygdala. Animals were administered vehicle (10% DMSO, 90% saline, i.p.), C41 (0.25 mg/kg, i.p.) or riluzole (4.0 mg/kg, i.p.) once daily over 28 days. Working memory performance was evaluated using a modified conditioned place preference (CPP) paradigm that was developed in our laboratory for AD research. Brain concentrations of Ab proteins were measured in whole brain homogenates using an enzyme-linked immunosorbent assay (ELISA). In all experiments, the FDA-approved EAAT2 agonist drug riluzole was used as a positive control.

Results: Western blot analysis detected a single band at 62 kDa (i.e., the known molecular weight of EAAT2) in primary cultures of microglia. Both C41 and riluzole increased time-dependent uptake of [³H] glutamic acid, suggesting a stimulatory effect on EAAT2-mediated transport. Transport specificity was confirmed using WAY-213613, which reduced uptake of [³H] glutamic acid in primary cultures of human microglia treated with C41 or riluzole. Interestingly, both drugs enhanced phagocytosis of Ab(1-42) in primary cultures of human microglia, an effect that was attenuated in the presence of WAY-213613. In 3xTg mice treated for 28 days with C41 or riluzole, working memory was significantly improved relative to vehicle-treated mice. This effect existed in both males and females, which implies that our novel EAAT2 activating drug can improve cognitive performance in early-stage AD for both sexes. Interestingly, C41 demonstrated higher therapeutic effects than riluzole in female 3xTg mice. Additionally, C41 and riluzole reduced brain concentrations of two major amyloid beta proteins (i.e., Ab(1-40) and Ab(1-42)) associated with AD pathology in both male and female 3xTg mice.

Conclusion: Overall, our work demonstrates that EAAT2 can be targeted for discovery and development of novel compounds for treatment of AD. Additionally, our translational studies have resulted in a strong preclinical data set that supports the continued development of small molecule EAAT2 activators such as C41 towards future clinical evaluation.

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Acknowledgements: This work was supported by a grant from the National Institutes on Aging (R41-AG080957) to RC, WW, and PTR.