Biosynthesis of neuroprotective melatonin is dysregulated in Huntington's disease.

Huntington's disease (HD) is a progressive neurodegenerative brain disorder associated with uncontrolled body movements, cognitive decline, and reduced circulating melatonin levels. Melatonin is a potent antioxidant and exogenous melatonin treatment is neuroprotective in experimental HD models. In neurons, melatonin is exclusively synthesized in the mitochondrial matrix. Thus, we investigated the integrity of melatonin biosynthesis pathways in pineal and extrapineal brain areas in human HD brain samples, in the R6/2 mouse model of HD and in full-length mutant huntingtin knock-in cells. Aralkylamine N-acetyltransferase (AANAT) is the rate-limiting step enzyme in the melatonin biosynthetic pathway. We found that AANAT expression is significantly decreased in the pineal gland and the striatum of HD patients compared to normal controls. In the R6/2 mouse forebrain, AANAT protein expression was decreased in synaptosomal, but not nonsynaptosomal, mitochondria and was associated with decreased synaptosomal melatonin levels compared to wild type mice. We also demonstrate sequestration of AANAT in mutant-huntingtin protein aggregates likely resulting in decreased AANAT bioavailability. Paradoxically, AANAT mRNA expression is increased in tissues where AANAT protein expression is decreased, suggesting a potential feedback loop that is, ultimately unsuccessful. In conclusion, we demonstrate that pineal, extrapineal, and synaptosomal melatonin levels are compromised in the brains of HD patients and R6/2 mice due, at least in part, to protein aggregation.

Neuronal melatonin type 1 receptor overexpression promotes M2 microglia polarization in cerebral ischemia/reperfusion-induced injury.

Microglial activation is readily detected following cerebral ischemia/reperfusion-induced injury. Activated microglia polarize into either classic pro-inflammatory M1 or protective M2 microglia following ischemia/reperfusion-induced injury. Melatonin is protective immediately after ischemia/reperfusion-induced brain injury. However, the ability of melatonin to affect longer-term recovery from ischemic/reperfusion-induced injury as well as its ability to modulate microglia/macrophage polarization are unknown. The goal of this study is to understand the impact of melatonin on mice 14 days after injury, as well as to understand how melatonin affects microglial polarization of neuronal MT1 activation following cerebral ischemia/reperfusion. We utilized NSEMT1-GFP transgenic mice which overexpress MT1 (melatonin type 1 receptor) in neurons. Melatonin-treated or vehicle treated wild type and NSEMT1-GFP mice underwent middle cerebral artery occlusion (MCAO)/reperfusion and followed for 14 days. Neuronal MT1 overexpression significantly reduced infarct volumes, improved motor function, and ameliorated weight loss. Additionally, melatonin treatment reduced infarct volume in NSEMT1-GFP mice as compared to untreated wild type, melatonin treated wild type, and untreated NSEMT1-GFP mice. Melatonin improved neurological function and prevented weight loss in NSEMT1-GFP mice compared with melatonin treated wild type mice. Finally, melatonin treatment in combination with MT1 overexpression reduced the numbers of Iba1+/CD16+ M1 microglia and increased the numbers of Iba1+/ CD206+ M2 microglia after ischemic injury. In conclusion, neuronal MT1 mediates melatonin-induced long-term recovery after cerebral ischemia, at least in part, by shifting microglial polarization toward the neuroprotective M2 phenotype.

Melatonin inhibits cytosolic mitochondrial DNA-induced neuroinflammatory signaling in accelerated aging and neurodegeneration.

Chronic inflammation is a pathologic feature of neurodegeneration and aging; however, the mechanism regulating this process is not understood. Melatonin, an endogenous free radical scavenger synthesized by neuronal mitochondria, decreases with aging and neurodegeneration. We proposed that insufficient melatonin levels impair mitochondrial homeostasis, resulting in mitochondrial DNA (mtDNA) release and activation of cytosolic DNA-mediated inflammatory response in neurons. We found increased mitochondrial oxidative stress and decreased mitochondrial membrane potential, with higher mtDNA release in brain and primary cerebro-cortical neurons of melatonin-deficient aralkylamine N-acetyltransferase (AANAT) knockout mice. Cytosolic mtDNA activated the cGAS/STING/IRF3 pathway, stimulating inflammatory cytokine generation. We found that Huntington's disease mice had increased mtDNA release, cGAS activation, and inflammation, all inhibited by exogenous melatonin. Thus, we demonstrated that cytosolic mtDNA activated the inflammatory response in aging and neurodegeneration, a process modulated by melatonin. Furthermore, our data suggest that AANAT knockout mice are a model of accelerated aging.

Mir-155 knockout protects against ischemia/reperfusion-induced brain injury and hemorrhagic transformation.

MiR-155 negatively regulates translation of mRNA targets to proteins involved in processes that modulate ischemic brain injury including neuroinflammation, blood-brain barrier (BBB) permeability, and apoptosis. However, reports of the effect of cerebral miR-155 expression changes after ischemic brain injury are equivocal and miR-155 modulates molecular pathways with opposing effects on these processes. The role of miR-155 in postischemic cerebral hemorrhagic transformation remains unknown. To understand the net effect of complete inactivation of miR-155, miR-155 knockout mice were studied in a cerebral ischemia/reperfusion (I/R) model of infarction and hemorrhagic transformation as compared with those of wild type mice. Wild type and miR-155 knockout mice underwent one hour of middle cerebral artery occlusion (MCAO) followed by up to 71 hours of reperfusion. The effects of miR-155 knockout on cerebral infarct size, incidence and extent of hemorrhagic transformation, and neurological outcome were determined. We found that miR-155 was significantly upregulated after cerebral I/R in wild type mice, and miR-155 knockout mice had comparably smaller cerebral infarct size and improved neurological deficits. Similarly, wild type mice had significant hemorrhagic burden after cerebral I/R, the incidence and volume of which was reduced in miR-155 knockout mice. Although miR-155 can have opposite effects on cerebral I/R-injury-related processes, the net effect of miR-155 knockout is neuroprotective. Thus, the increase in miR-155 expression observed after cerebral I/R may be considered deleterious and inhibition of this expression and its effects a potential therapeutic target.

Increased Serotonin Transporter Expression in Huntington's Disease Patients Is Not Consistently Replicated in Murine Models.

BACKGROUND: Selective serotonin reuptake inhibitors (SSRIs) target the serotonin transporter (SERT) and are commonly prescribed for depression in Huntington's disease (HD) patients. However, SERT expression in HD has not been carefully evaluated in patients or mouse models. OBJECTIVE: In this study, we investigated SERT levels in HD patients and HD mouse models. METHODS: We obtained HD patient brain striatal samples and matched controls, as well as brain tissue from CAG140 and R6/2 mice. SERT mRNA and protein levels were analyzed using quantitative RT-PCR and immunoblotting. RESULTS AND CONCLUSIONS: We found that SERT protein, but not mRNA is markedly increased in grade 4 HD patient striatal tissue. These findings suggest posttranscriptional or translational SERT dysregulation as a possible etiologic factor modulating psychopathology in HD. Interestingly, SERT expression is variable in mouse models of the disease. Increased SERT levels are demonstrated in the brain of CAG140 mice, a full-length knock-in mouse model of the disease, but not in the striatum of the R6/2 fragment murine model of the disease. Based on this parameter, the CAG140 huntingtin knock-in mouse model is more suitable than the R6/2 model for the study of serotonergic pathway pathology in Huntington's disease.

Mitochondria modulate programmed neuritic retraction.

Neuritic retraction in the absence of overt neuronal death is a shared feature of normal aging and neurodegenerative disorders, but the intracellular mechanisms modulating this process are not understood. We propose that cumulative distal mitochondrial protein damage results in impaired protein import, leading to mitochondrial dysfunction and focal activation of the canonical apoptosis pathway in neurites. This is a controlled process that may not lead to neuronal death and, thus, we term this phenomenon "neuritosis." Consistent with our hypothesis, we show that in primary cerebrocortical neurons, mitochondrial distance from the soma correlates with increased mitochondrial protein damage, PINK1 accumulation, reactive oxygen species production, and decreased mitochondrial membrane potential and depolarization threshold. Furthermore, we demonstrate that the distance-dependent mitochondrial membrane potential gradient exists in vivo in mice. We demonstrate that impaired distal mitochondria have a lower threshold for focal/nonlethal neuritic caspase-3 activation in normal neurons that is exacerbated in aging, stress, and neurodegenerative conditions, thus delineating a fundamental mechanistic underpinning for synaptic vulnerability.

Dual role of mitochondria in producing melatonin and driving GPCR signaling to block cytochrome c release.

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 (MT1), its associated G protein, and ß-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 MT1 signal-transduction pathway inhibiting stress-mediated cytochrome c release and caspase activation. These findings coupled with our observation that mitochondrial MT1 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.

Systemic antimiR-337-3p delivery inhibits cerebral ischemia-mediated injury.

Modulation of miRNA expression has been shown to be beneficial in the context of multiple diseases. The purpose of this study was to determine if an inhibitor of miR-337-3p is neuroprotective for hypoxic injury after tail vein injection. We evaluated miR-337-3p expression levels and in brain tissue in vivo before and after permanent middle cerebral artery occlusion (pMCAO) in mice. Subsequently, a custom locked nucleic acid (LNA) antimir-337-3p oligonucleotide was developed and tested in vitro after induction of oxygen glucose-deprivation (OGD) and in vivo by injection into the mouse tail vein for 3 consecutive days before pMCAO. Ischemic lesion volume was measured by TTC staining. We show that systemically administered LNA antimir-337-3p crosses the blood brain-brain-barrier (BBB), penetrates into neurosn, downregulates endogenous miR-337-3p expression and reduces ischemic brain injury. The findings support the use of similar antimir-LNA constructs as novel therapies in neurological disease.

Peroxynitrite decomposition catalyst prevents matrix metalloproteinase activation and neurovascular injury after prolonged cerebral ischemia in rats.

Matrix metalloproteinases (MMPs) play an important role in reperfusion-induced brain injury following ischemia. To define the effects of peroxynitrite decomposition catalyst on MMP activation and neurovascular reperfusion injury, 5,10,15,20-tetrakis (2,4,6-trimethyl-3,5-disulfonatophenyl)-porphyrin iron (III) (FeTMPyP) was administered intravenously 30 min prior to reperfusion following a middle cerebral artery occlusion. Activation of MMP was assessed by in situ and gel zymography. Neurovascular injury was assessed using endothelial barrier antigen, collagen IV immunohistochemistry and Cresyl violet staining. Results were compared with sham and ischemia alone groups. We found that administration of FeTMPyP just before reperfusion after ischemia inhibited MMP-9 activation and total MMP-2 increases in the cortex and decreased active MMP-9 along with the total amounts of active MMP-9 and active MMP-2 in the striatum. Reperfusion-induced injury to the basal lamina of collagen IV-immunopositive microvasculature and neural cells in cortex and striatum was ameliorated by FeTMPyP. Losses of blood vessel endothelium produced by ischemia or reperfusion were also decreased in the cortex. These results suggest that administration of FeTMPy prior to reperfusion decreases MMP activation and neurovascular injury after prolonged cerebral ischemia. This strategy may be useful for future therapies targeted at preventing breakdown of the blood-brain barrier and hemorrhagic transformation.

Proteomic identification of an upregulated isoform of annexin A3 in the rat brain following reversible cerebral ischemia.

We used proteomics to identify regulated proteins following cerebral ischemia in a rat model. Young rats were subjected to reversible middle cerebral artery (MCA) occlusion and proteins were extracted from the peri-infarcted and the corresponding contralateral area at days 3 and 14 postischemia. Proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometry. We report for the first time that an isoform of annexin A3 (ANXA3) was among the upregulated proteins in the postischemic rat brain. The results were confirmed by real-time PCR and by western blotting. Double- and triple-immunostaining with neuronal and microglia/macrophagic markers demonstrated that ANXA3 is produced by resting microglia in control tissue and by activated microglial/macrophage cells in the infarcted area. 3D-images of the infarcted area suggest that ANXA3 is associated with a phagocytic phenotype. Our study identifies ANXA3 as a novel marker of brain microglia, which should be of substantial value in future studies of microglial cells and its role in the postischemic brain.

Proteomic identification of the involvement of the mitochondrial rieske protein in epilepsy.

PURPOSE: Kindled seizures are widely used to model epileptogenesis, but the molecular mechanisms underlying the attainment of kindling status are largely unknown. Recently we showed that achievement of kindling status in the Sprague-Dawley rat is associated with a critical developmental interval of 25 +/- 1 days; the identification of this long, well-defined developmental interval for inducing kindling status makes possible a dissection of the cellular and genetic events underlying this phenomenon and its relation to normal and pathologic brain function. METHODS: By using proteomics on cerebral tissue from our new rat kindling model, we undertook a global analysis of protein expression in kindled animals. Some of the identified proteins were further investigated by using immunohistochemistry. RESULTS: We report the identification of a modified variant of the Rieske iron-sulfur protein, a component of the mitochondrial cytochrome bc1 complex, whose isoelectric point is shifted toward more alkaline values in the hippocampus of kindled rats. By immunohistochemistry, the Rieske protein is well expressed in the hippocampus, except in the CA1 subfield, an area of selective vulnerability to seizures in humans and animal models. We also noted an asymmetric, selective expression of the Rieske protein in the subgranular neurons of the dorsal dentate gyrus, a region implicated in neurogenesis. CONCLUSIONS: These results indicate that the Rieske protein may play a role in the response of neurons to seizure activity and could give important new insights into the molecular pathogenesis of epilepsy.