Astrocytic abnormalities in brain-specific Cacna1c-deficient mice: Implications for BBB impairment in neuropsychiatric diseases associated with CACNA1C mutations

Abstract

Intronic genetic variants within the CACNA1C gene, which encodes the pore-forming alpha 1c subunit of the Ca_v1.2 L-type calcium channel, are significant risk factors for a multitude of neuropsychiatric disorders. In most cases, these intronic SNPs have been associated with reduced CACNA1C expression. Here, we demonstrate that targeted genetic deletion of Cacna1c in mouse brain leads to increased astrocyte reactivity, increased expression of aquaporin 4 (AQP4) in astrocytes adjacent to the blood-brain barrier (BBB), and neuroinflammation, including changes in the levels of brain chemokines and inflammatory cytokines. Astrocytes are vital for maintaining BBB integrity, with AQP4 predominantly expressed in astrocytic endfeet where it regulates water balance in the brain. This function is critical to brain health, and deterioration of the BBB is a major feature of virtually all forms of neuropsychiatric disease. Our results highlight a previously unrecognized role for CACNA1C in astrocytes at the BBB, which could be a major factor in how intronic CACNA1C SNPs broadly increase the risk of multiple forms of major neuropsychiatric disease.

Keywords: AQP4; CACNA1C; Calcium; Cav1.2; aquaporin 4; neuropsychiatric disease.

Figure 1.

Elimination of Cacna1c in the brain leads to abnormal expression of AQP4 in the brain. a-b. Dual ISH data of Cacna1c (green, left) and nes (red, middle) mRNA show significant reduction of Cacna1c mRNA in nes-positive cells (right) in the cerebral cortex of Nes-Cacna1c^KO mice (three males and two females), compared to Nes-Cacna1c^WT littermates (two males and three females) (unpaired t-test). c-d. AQP4 (water channel localized to astrocytic endfeet, green) and CD31 (blood vessel, red) staining reveals significantly increased AQP4 expression around the blood vessels in the cerebral cortex of Nes-Cacna1c^KO mice (two males and one female), compared to Nes-Cacna1c^WT littermates (two males and two females) (unpaired t-test). e-f. Western blot (left) and its quantification (right) of AQP4 show elevated levels in the cerebral cortex of Nes-Cacna1c^KO mice (five females), relative to Nes-Cacna1c^WT littermate controls (five females) (unpaired t-test). In all graphs, datapoints indicate values of individual mice.

Figure 2.

Elimination of Cacna1c in the brain increases neuroinflammation and oxidative stress. a-b. Western blot (left) and its quantification (right) of GFAP shows that GFAP protein expression is up-regulated in the cerebral cortex of Nes-Cacna1c^KO mice compared to Nes-Cacna1c^WT controls (female, unpaired t-test). c. Cytokine screening from brain lysates reveals brain cytokine levels that are altered in the cerebral cortex of Nes-Cacna1c^KO mice, compared to Nes-Cacna1c^WT controls (* p < 0.05, ** p < 0.01, female, unpaired t-test). d. Immunohistochemistry of 3-NT shows that oxidative stress is increased in the cerebral cortex of Nes-Cacna1c^KO mice compared to Nes-Cacna1c^WT controls (male and female, unpaired t-test). In all graphs, datapoints indicate values of individual mice.