Mitochondrial mechanism of neuroprotection by CART

Abstract

We previously demonstrated that the neuropeptide cocaine- and amphetamine-regulated transcript (CART) is protective against focal cerebral ischemia in vivo and against neuronal cell death in culture induced by oxygen-glucose deprivation (OGD). The mechanism of neuroprotection by CART is unknown, in part due to lack of knowledge regarding its putative receptor. Using a yeast two-hybrid system with CART's carboxy-terminal to screen a mouse brain cDNA library, we uncovered a potential direct interaction between CART and subunit B of the mitochondrial enzyme succinate dehydrogenase (SDHB). We confirmed CART/SDHB binding using in vitro pull-down assay, and tested the effects of CART peptide on SDH activity, Complex II (CII) activity and ATP production in primary cultured cortical neurons under basal conditions and after OGD. At concentrations between 0.2 and 4 nM, CART significantly increased SDH function, CII activity and ATP generation in purified mitochondria and intact neurons under baseline conditions. Furthermore, pretreatment with CART enhanced mitochondrial mechanisms of neuronal survival and prevented the decline in SDH and CII activities and ATP production after OGD. The findings suggest that CART's neuroprotective mechanism of action may be linked to preservation of mitochondrial function and prevention of energy failure after ischemia-reperfusion injury.

Fig. 1

Identification of cocaine- and amphetamine-regulated transcript (CART)-interacting partner using yeast two-hybrid system and β-Gal filter assay. Yeast cells transformed with clone #35 containing the sequence for SDHB (prey) and CART (bait), but not empty vector plasmid (lower panel), induce reporter gene (β-gal) expression (upper panel).

Fig. 2

In vitro pull-down assay. Cell lysates were incubated with purified enhanced green fluorescent protein cocaine- and amphetamine-regulated transcript (EGFPCART) proteins (lanes 1 and 3) or EGFP proteins (2 and 4), pulled down by protein G beads linked to anti-CII (SDHB) antibody, and then probed with either anti-EGFP (A) or anti-CART (B) antibodies (same membrane stripped and reprobed). Lysates in lanes 1 and 2 were prepared from PC3 cells, 3 and 4 from HEK293 cells using CelLytic Reagent. Lane 5 is input loaded with approximately 10% of the EGFPCART protein used in the binding reaction. HC, heavy chain of Ig; LC, light chain of Ig.

Fig. 3

Cocaine- and amphetamine-regulated transcript (CART) stimulates basal succinate dehydrogenase (SDH) and CII activities in mitochondrial extracts from primary cortical neurons. (A) CART increases SDH activity determined spectrophotometrically at 650 nm by the reduction of 2,6-dichloroindiphenol (DCIP) after addition of the SDH substrate succinate at time 0. (B) CART increases CII activity in mitochondrial extract measured spectrophotometrically at 595 nm by DCIP reduction after the addition of Coenzyme Q at 3 min *P < 0.05 compared with control.

Fig. 4

Cocaine- and amphetamine-regulated transcript (CART) prevents the decline in CII activities after oxygen-glucose deprivation (OGD) in mitochondrial extracts from primary cortical neurons. CART was added at 0.2 nM concentration 30 min prior to 2 h OGD, and mitochondria extracted at 24 h after OGD. *P < 0.05 compared with OGD, ^#P < 0.05 compared with OGD plus vehicle (VEH). DCIP, 2,6-dichloroindiphenol.

Fig. 5

Cocaine- and amphetamine-regulated transcript (CART) increases basal ATP production and prevents the decline in ATP after oxygen-glucose deprivation (OGD). (A) CART increases ATP production in mitochondria extracted from normal cultured cortex neuronal cells. ATP levels were measured using a luciferase-based bioluminescence detection assay (n = 4 per group). The CII inhibitor 3-nitropropionic acid (3-NPA, 50 mM) was added to demonstrate specificity. *P < 0.05 compared with control. (B) CART preserves mitochondrial ATP production capacity after OGD and reperfusion (n = 4 per group). CART was added at 0.2 nM 30 min prior to 2 h OGD, and mitochondrial extract isolated at 24 h of reoxygenation after OGD. *P < 0.05 compared with control; ^#P < 0.05 compared with OGD alone.

Fig. 6

Cocaine- and amphetamine-regulated transcript (CART) improves mitochondrial dysfunction and neuronal viability after oxygen-glucose deprivation (OGD). (A) CART improves neuronal survival as determined by the reduction of MTT by mitochondrial dehydrogenases (n = 7 per group, *P < 0.05 compared with control; ^#P < 0.05 compared with OGD alone). (B) CART decreases OGD-induced cell death as determined by PI/calcein staining at 24 h after 2 h OGD (n = 6 per group, *P < 0.05 compared with control; ^#P < 0.05 compared with OGD alone).