ASTROCYTE RESPONSE TO HYPERHOMOCYSTEINEMIA (HHCY)

Abstract

Homocysteine (Hcy) is a non-protein forming amino acid involved in the production of both methionine and cysteine. Elevated Hcy can result from mutations in methylenetetrahydrofolate reductase (MTHFR) or cystathionine beta synthase (CBS) genes, as well as low levels of vitamins B6, B9, and B12. Elevated Hcy is termed hyperhomocysteinemia (HHcy). HHcy is a risk factor for vascular cognitive impairment and dementia (VCID), as well as Alzheimer's disease. The mechanism by which HHcy promotes VCID or AD remains unknown. Astrocytes play a crucial role in both potassium buffering and neurovascular coupling. The astrocytic end-feet essentially ensheath intraparenchymal blood vessels in the brain and express a variety of channels and markers indicative of their specialized functions. Channels enriched at the astrocytic end-feet include the aquaporin 4 water channel (AQP4), the inward rectifying potassium channel Kir4.1 and the calcium-dependent potassium channel BK. These channels are anchored at the astrocytic end-foot by dystrophin-1 (Dp71), as well as other anchoring proteins. To determine the cell specific effects of Hcy, C8-D1A astrocytes were treated with 50mM of Hcy for 24, 48, 72 and 96 hours. Gene expression for several astrocyte specific markers was then analyzed. To determine the in vivo effects of HHcy, wildtype mice were subject to a HHcy-inducing diet for a period of 6, 10 or 14 weeks. Tissue was then histologically examined for the astrocytic end-foot markers AQP4, Kir4.1, BK and dystrophin-1 (Dp71). Astrocytes treated with homocysteine exhibit an increased GFAP expression, suggesting homocysteine to be an inflammatory stimulus for astrocytes. While GFAP is increased, AQP4, Kir4.1, and BK expression levels were decreased. Our HHcy model displays significant astrocyctic end-foot disruptions. While AQP4 becomes dislocalized from the end-feet, Kir4.1 and BK protein expression decreases. In addition, the anchoring protein Dp71, also decreases. We examined mice subjected to the HHcy-inducing diet for 6, 10, and 14 weeks and find that end-disruption becomes progressively more severe as the mice continue the diet. This corresponds with progressively worse cognitive performance in the radial-arm water maze. HHcy results in the disruption of the astrocytic end-foot connection, which we hypothesize contributes to the cognitive decline observed in our HHcy mouse model.