G protein-coupled receptors (GPCRs) are classically characterized as cell-surface receptors transmitting extracellular signals into cells. Here we show that central components of a GPCR signaling system comprised of the melatonin type 1 receptor (MT), its associated G protein, and β-arrestins are on and within neuronal mitochondria. We discovered that the ligand melatonin is exclusively synthesized in the mitochondrial matrix and released by the organelle activating the mitochondrial MT signal-transduction pathway inhibiting stress-mediated cytochrome release and caspase activation. These findings coupled with our observation that mitochondrial MT overexpression reduces ischemic brain injury in mice delineate a mitochondrial GPCR mechanism contributing to the neuroprotective action of melatonin. We propose a new term, "automitocrine," analogous to "autocrine" when a similar phenomenon occurs at the cellular level, to describe this unexpected intracellular organelle ligand-receptor pathway that opens a new research avenue investigating mitochondrial GPCR biology.

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The age-related decline in plasticity of the brain may be one factor underlying poor functional recovery after stroke. In the present work we tested the hypothesis that the attenuation of neural plasticity in old age could be the result of an altered temporal relationship between factors promoting brain plasticity [microtubule-associated protein 1B (MAP1B)] and neurotoxic factors such as C-terminal betaAPP. Focal cerebral ischemia was produced by reversible occlusion of the right middle cerebral artery in 3- and 20-month-old male Sprague-Dawley rats. The functional outcome was assessed in neurobehavioral tests at 3, 7, 14 and 28 days after surgery. At the indicated timepoints, brains were removed and immunostained for C- and N-terminal betaAPP and MAP1B. At 2 weeks poststroke, we found an age-related increase in the amount of the C-terminal fragment of betaAPP in the peri-infarcted area and the infarct core as well as an early, vigorous incorporation of N-terminal betaAPP into the developing astroglial scar. The recovery of the plasticity-associated protein MAP1B following stroke was delayed in both age groups and became prominent between days 14 and 28. As aged rats showed diminished functional recovery compared with young rats, these results suggest that the accumulation of C-terminal betaAPP, together with the early incorporation of N-terminal betaAPP into the glial scar, may over-ride the beneficial role of plasticity factors such as MAP1B.

MMP-9 deficiency protected against photochemical thrombosis induced brain hemorrhagic transformation (HT), but it did not protect against tissue plasminogen activator induced brain hemorrhage. The roles of MMP-2 and/or MMP-9 knockout (KO) in mechanical reperfusion induced HT after ischemia have not been investigated. Here we assessed the effects of MMP-2 KO, MMP-9 KO and MMP-2/9 double KO (dKO) in protecting against mechanical reperfusion induced HT and other brain injuries after the early stages of cerebral ischemia in mice of the same genetic background. Middle cerebral artery occlusion (MCAO) was performed in mice. Reperfusion was started at 1 or 1.5 hours after onset of MCAO. All mice were sacrificed 8 hours after MCAO. We found that both pro- and active MMP-2 and MMP-9 levels were significantly elevated in the early ischemic brain. After the early stages of ischemia and reperfusion, the hemorrhagic incidence was reduced in the cortex of MMP-2 KO mice (p < 0.05 vs. WT). The hemorrhagic volume was significantly decreased in the cortexes of MMP-2 and/or -9 knockout mice (MMP-9 KO vs. WT: p < 0.01, MMP-2 KO and dKO vs. WT: p < 0.001). In the basal ganglia, MMP-2 KO and MMP-2/9 dKO mice displayed a remarkable decrease in hemorrhagic volume (p < 0.01 or 0.05 vs. WT), but MMP-9 KOs did not protect against hemorrhage. MMP-2 and/or -9 knockout mice displayed significantly decreased infarction volume in both the cortex and striatum, in addition to improved neurological function (p < 0.001 vs. WT). The results suggested that MMP-2 deficiency and MMP-2 and MMP-9 double deficiency were more protective than MMP-9 deficiency against HT after the early stages of ischemia and reperfusion. These studies increase our understanding of MMP-2 and MMP-9 in HT development and will help to selectively target MMPs to protect the post-ischemic brain from injury and HT.