Activation of testosterone-androgen receptor mediates cerebrovascular protection by photobiomodulation treatment in photothrombosis-induced stroke rats.

RATIONALE: Numerous epidemiological studies have reported a link between low testosterone levels and an increased risk of cerebrovascular disease in men. However, there is ongoing controversy surrounding testosterone replacement therapy due to potential side effects. PBMT has been demonstrated to improve cerebrovascular function and promote testosterone synthesis in peripheral tissues. Despite this, the molecular mechanisms that could connect PBMT with testosterone and vascular function in the brain of photothrombosis (PT)-induced stroke rats remain largely unknown. METHODS: We measured behavioral performance, cerebral blood flow (CBF), vascular permeability, and the expression of vascular-associated and apoptotic proteins in PT-induced stroke rats treated with flutamide and seven consecutive days of PBM treatment (350 mW, 808 nM, 2 min/day). To gain further insights into the mechanism of PBM on testosterone synthesis, we used testosterone synthesis inhibitors to study their effects on bEND.3 cells. RESULTS: We showed that PT stroke caused a decrease in cerebrovascular testosterone concentration, which was significantly increased by 7-day PBMT (808 nm, 350 mW/cm2 , 42 J/cm2 ). Furthermore, PBMT significantly increased cerebral blood flow (CBF) and the expression of vascular-associated proteins, while inhibiting vascular permeability and reducing endothelial cell apoptosis. This ultimately mitigated behavioral deficits in PT stroke rats. Notably, treatment with the androgen receptor antagonist flutamide reversed the beneficial effects of PBMT. Cellular experiments confirmed that PBMT inhibited cell apoptosis and increased vascular-associated protein expression in brain endothelial cell line (bEnd.3) subjected to oxygen-glucose deprivation (OGD). However, these effects were inhibited by flutamide. Moreover, mechanistic studies revealed that PBMT-induced testosterone synthesis in bEnd.3 cells was partly mediated by 17ß-hydroxysteroid dehydrogenase 5 (17ß-HSD5). CONCLUSIONS: Our study provides evidence that PBMT attenuates cerebrovascular injury and behavioral deficits associated with testosterone/AR following ischemic stroke. Our findings suggest that PBMT may be a promising alternative approach for managing cerebrovascular diseases.

The role of serum/glucocorticoid-regulated kinase 1 in brain function following cerebral ischemia.

Cardiopulmonary arrest (CA) is a major cause of death/disability in the U.S. with poor prognosis and survival rates. Current therapeutic challenges are physiologically complex because they involve hypoperfusion (decreased cerebral blood flow), neuroinflammation, and mitochondrial dysfunction. We previously discovered novel serum/glucocorticoid-regulated kinase 1 (SGK1) is highly expressed in brain of neurons that are susceptible to ischemia (hippocampus and cortex). We inhibited SGK1 and utilized pharmacological (specific inhibitor, GSK650394) and neuron-specific genetic approaches (shRNA) in rodent models of CA to determine if SGK1 is responsible for hypoperfusion, neuroinflammation, mitochondrial dysfunctional, and neurological deficits after CA. Inhibition of SGK1 alleviated cortical hypoperfusion and neuroinflammation (via Iba1, GFAP, and cytokine array). Treatment with GSK650394 enhanced mitochondrial function (via Seahorse respirometry) in the hippocampus 3 and 7 days after CA. Neuronal injury (via MAP2, dMBP, and Golgi staining) in the hippocampus and cortex was observed 7 days after CA but ameliorated with SGK1-shRNA. Moreover, SGK1 mediated neuronal injury by regulating the Ndrg1-SOX10 axis. Finally, animals subjected to CA exhibited learning/memory, motor, and anxiety deficits after CA, whereas SGK1 inhibition via SGK1-shRNA improved neurocognitive function. The present study suggests the fundamental roles of SGK1 in brain circulation and neuronal survival/death in cerebral ischemia-related diseases.

PRMT7 can prevent neurovascular uncoupling, blood-brain barrier permeability, and mitochondrial dysfunction in repetitive and mild traumatic brain injury.

Mild traumatic brain injury (TBI) comprises the largest percentage of TBI-related injuries, with pathophysiological and functional deficits that persist in a subset of TBI patients. In our three-hit paradigm of repetitive and mild traumatic brain injury (rmTBI), we observed neurovascular uncoupling via decreased red blood cell velocity, microvessel diameter, and leukocyte rolling velocity 3 days post-rmTBI via intra-vital two-photon laser scanning microscopy. Furthermore, our data suggest increased blood-brain barrier (BBB) permeability (leakage), with corresponding decrease in junctional protein expression post-rmTBI. Mitochondrial oxygen consumption rates (measured via Seahorse XFe24) were also altered 3 days post-rmTBI, along with disrupted mitochondrial dynamics of fission and fusion. Overall, these pathophysiological findings correlated with decreased protein arginine methyltransferase 7 (PRMT7) protein levels and activity post-rmTBI. Here, we increased PRMT7 levels in vivo to assess the role of the neurovasculature and mitochondria post-rmTBI. In vivo overexpression of PRMT7 using a neuronal specific AAV vector led to restoration of neurovascular coupling, prevented BBB leakage, and promoted mitochondrial respiration, altogether to suggest a protective and functional role of PRMT7 in rmTBI.

A role for protein arginine methyltransferase 7 in repetitive and mild traumatic brain injury.

Mild traumatic brain injury affects the largest proportion of individuals in the United States and world-wide. Pre-clinical studies of repetitive and mild traumatic brain injury (rmTBI) have been limited in their ability to recapitulate human pathology (i.e. diffuse rotational injury). We used the closed-head impact model of engineered rotation acceleration (CHIMERA) to simulate rotational injury observed in patients and to study the pathological outcomes post-rmTBI using C57BL/6J mice. Enhanced cytokine production was observed in both the cortex and hippocampus to suggest neuroinflammation. Furthermore, microglia were assessed via enhanced iba1 protein levels and morphological changes using immunofluorescence. In addition, LC/MS analyses revealed excess glutamate production, as well as diffuse axonal injury via Bielschowsky's silver stain kit. Moreover, the heterogeneous nature of rmTBI has made it challenging to identify drug therapies that address rmTBI, therefore we sought to identify novel targets in the concurrent rmTBI pathology. The pathophysiological findings correlated with a time-dependent decrease in protein arginine methyltransferase 7 (PRMT7) protein expression and activity post-rmTBI along with dysregulation of PRMT upstream mediators s-adenosylmethionine and methionine adenosyltransferase 2 (MAT2) in vivo. In addition, inhibition of the upstream mediator MAT2A using the HT22 hippocampal neuronal cell line suggest a mechanistic role for PRMT7 via MAT2A in vitro. Collectively, we have identified PRMT7 as a novel target in rmTBI pathology in vivo and a mechanistic link between PRMT7 and upstream mediator MAT2A in vitro.

Protein arginine methyltransferase 4 modulates nitric oxide synthase uncoupling and cerebral blood flow in Alzheimer's disease.

Alzheimer's disease (AD) is the leading cause of mortality, disability, and long-term care burden in the United States, with women comprising the majority of AD diagnoses. While AD-related dementia is associated with tau and amyloid beta accumulation, concurrent derangements in cerebral blood flow have been observed alongside these proteinopathies in humans and rodent models. The homeostatic production of nitric oxide synthases (NOS) becomes uncoupled in AD which leads to decreased NO-mediated vasodilation and oxidative stress via the production of peroxynitrite (ONOO-∙) superoxide species. Here, we investigate the role of the novel protein arginine methyltransferase 4 (PRMT4) enzyme function and its downstream product asymmetric dimethyl arginine (ADMA) as it relates to NOS dysregulation and cerebral blood flow in AD. ADMA (type-1 PRMT product) has been shown to bind NOS as a noncanonic ligand causing enzymatic dysfunction. Our results from RT-qPCR and protein analyses suggest that aged (9-12 months) female mice bearing tau- and amyloid beta-producing transgenic mutations (3xTg-AD) express higher levels of PRMT4 in the hippocampus when compared to age- and sex-matched C57BL6/J mice. In addition, we performed studies to quantify the expression and activity of different NOS isoforms. Furthermore, laser speckle contrast imaging analysis was indicative that 3xTg-AD mice have dysfunctional NOS activity, resulting in reduced production of NO metabolites, enhanced production of free-radical ONOO-, and decreased cerebral blood flow. Notably, the aforementioned phenomena can be reversed via pharmacologic PRMT4 inhibition. Together, these findings implicate the potential importance of PRMT4 signaling in the pathogenesis of Alzheimer's-related cerebrovascular derangement.

Long-term exercise pre-training attenuates Alzheimer's disease-related pathology in a transgenic rat model of Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia. Despite enormous efforts around the world, there remains no effective cure for AD. This study was performed to investigate the effects of long-term exercise pretreatment on the typical pathology of AD in a novel transgenic AD rat model. Male 2-month-old animals were divided into the following groups: wild-type (WT) rats, AD rats, and AD rats with treadmill exercise pretreatment (AD-Exe). After exercise pretreatment, the Barnes maze task, passive avoidance task, and cued fear conditioning test were performed to test learning and memory function. The elevated plus maze, open field test, sucrose preference test, and forced swim test were conducted to measure anxious-depressive-like behavior. Immunofluorescence staining, Golgi staining, transmission electron microscopy, Western blot analysis, F-Jade C staining, TUNEL staining, and related assay kits were conducted to measure Aß plaques, tau hyperphosphorylation, neuronal damage, neuronal degeneration, dendritic spine density, synapses, synaptic vesicles, mitochondrial morphology, mitochondrial dynamic, oxidative stress, and neuroinflammation. Behavioral tests revealed that long-term exercise pretreatment significantly alleviated learning and memory dysfunction and anxious-depressive-like behaviors in AD animals. In addition, exercise pretreatment attenuated amyloid-ß deposition and tau hyperphosphorylation and preserved spine density, synapses, and presynaptic vesicles. Exercise also inhibited neuronal damage, neuronal apoptosis, and neuronal degeneration. Additional studies revealed the imbalance of mitochondrial dynamics was significantly inhibited by exercise pretreatment accompanied by a remarkable suppression of oxidative stress and neuroinflammation. Our findings suggest that long-term exercise pretreatment alleviated behavioral deficits and typical pathologies of the AD rat model, supporting long-term exercise pretreatment as a potential approach to delay the progression of AD.

Activation of Neuropeptide Y2 Receptor Can Inhibit Global Cerebral Ischemia-Induced Brain Injury.

Cardiopulmonary arrest (CA) can greatly impact a patient's life, causing long-term disability and death. Although multi-faceted treatment strategies against CA have improved survival rates, the prognosis of CA remains poor. We previously reported asphyxial cardiac arrest (ACA) can cause excessive activation of the sympathetic nervous system (SNS) in the brain, which contributes to cerebral blood flow (CBF) derangements such as hypoperfusion and, consequently, neurological deficits. Here, we report excessive activation of the SNS can cause enhanced neuropeptide Y levels. In fact, mRNA and protein levels of neuropeptide Y (NPY, a 36-amino acid neuropeptide) in the hippocampus were elevated after ACA-induced SNS activation, resulting in a reduced blood supply to the brain. Post-treatment with peptide YY3-36 (PYY3-36), a pre-synaptic NPY2 receptor agonist, after ACA inhibited NPY release and restored brain circulation. Moreover, PYY3-36 decreased neuroinflammatory cytokines, alleviated mitochondrial dysfunction, and improved neuronal survival and neurological outcomes. Overall, NPY is detrimental during/after ACA, but attenuation of NPY release via PYY3-36 affords neuroprotection. The consequences of PYY3-36 inhibit ACA-induced 1) hypoperfusion, 2) neuroinflammation, 3) mitochondrial dysfunction, 4) neuronal cell death, and 5) neurological deficits. The present study provides novel insights to further our understanding of NPY's role in ischemic brain injury.

Palmitic acid methyl ester inhibits cardiac arrest-induced neuroinflammation and mitochondrial dysfunction.

We previously discovered that palmitic acid methyl ester (PAME) is a potent vasodilator released from the sympathetic ganglion with vasoactive properties. Post-treatment with PAME can enhance cortical cerebral blood flow and functional learning and memory, while inhibiting neuronal cell death in the CA1 region of the hippocampus under pathological conditions (i.e. cerebral ischemia). Since mechanisms underlying PAME-mediated neuroprotection remain unclear, we investigated the possible neuroprotective mechanisms of PAME after 6 min of asphyxial cardiac arrest (ACA, an animal model of global cerebral ischemia). Our results from capillary-based immunoassay (for the detection of proteins) and cytokine array suggest that PAME (0.02 mg/kg) can decrease neuroinflammatory markers, such as ionized calcium binding adaptor molecule 1 (Iba1, a specific marker for microglia/macrophage activation) and inflammatory cytokines after cardiopulmonary resuscitation. Additionally, the mitochondrial oxygen consumption rate (OCR) and respiratory function in the hippocampal slices were restored following ACA (via Seahorse XF24 Extracellular Flux Analyzer) suggesting that PAME can ameliorate mitochondrial dysfunction. Finally, hippocampal protein arginine methyltransferase 1 (PRMT1) and PRMT8 are enhanced in the presence of PAME to suggest a possible pathway of methylated fatty acids to modulate arginine-based enzymatic methylation. Altogether, our findings suggest that PAME can provide neuroprotection in the presence of ACA to alleviate neuroinflammation and ameliorate mitochondrial dysfunction.

Upregulation of serum and glucocorticoid-regulated kinase 1 exacerbates brain injury and neurological deficits after cardiac arrest.

Cardiopulmonary arrest (CA) is the leading cause of death and disability in the United States. CA-induced brain injury is influenced by multifactorial processes, including reduced cerebral blood flow (hypoperfusion) and neuroinflammation, which can lead to neuronal cell death and functional deficits. We have identified serum and glucocorticoid-regulated kinase-1 (SGK1) as a new target in brain ischemia previously described in the heart, liver, and kidneys (i.e., diabetes and hypertension). Our data suggest brain SGK1 mRNA and protein expression (i.e., hippocampus), presented with hypoperfusion (low cerebral blood flow) and neuroinflammation, leading to further studies of the potential role of SGK1 in CA-induced brain injury. We used a 6-min asphyxia cardiac arrest (ACA) rat model to induce global cerebral ischemia. Modulation of SGK1 was implemented via GSK650394, a SGK1-specific inhibitor (1.2 µg/kg icv). Accordingly, treatment with GSK650394 attenuated cortical hypoperfusion and neuroinflammation (via Iba1 expression) after ACA, whereas neuronal survival was enhanced in the CA1 region of the hippocampus. Learning/memory deficits were observed 3 days after ACA but ameliorated with GSK650394. In conclusion, SGK1 is a major contributor to ACA-induced brain injury and neurological deficits, while inhibition of SGK1 with GSK650394 provided neuroprotection against CA-induced hypoperfusion, neuroinflammation, neuronal cell death, and learning/memory deficits. Our studies could lead to a novel, therapeutic target for alleviating brain injury following cerebral ischemia.NEW & NOTEWORTHY Upregulation of SGK1 exacerbates brain injury during cerebral ischemia. Inhibition of SGK1 affords neuroprotection against cardiac arrest-induced hypoperfusion, neuroinflammation, neuronal cell death, and neurological deficits.

Stearic acid methyl ester affords neuroprotection and improves functional outcomes after cardiac arrest.

Cardiac arrest causes neuronal damage and functional impairments that can result in learning/memory dysfunction after ischemia. We previously identified a saturated fatty acid (stearic acid methyl ester, SAME) that was released from the superior cervical ganglion (sympathetic ganglion). The function of stearic acid methyl ester is currently unknown. Here, we show that SAME can inhibit the detrimental effects of global cerebral ischemia (i.e. cardiac arrest). Treatment with SAME in the presence of asphyxial cardiac arrest (ACA) revived learning and working memory deficits. Similarly, SAME-treated hippocampal slices after oxygen-glucose deprivation inhibited neuronal cell death. Moreover, SAME afforded neuroprotection against ACA in the CA1 region of the hippocampus, reduced ionized calcium-binding adapter molecule 1 expression and inflammatory cytokines/chemokines, with restoration in mitochondria respiration. Altogether, we describe a unique and uncharted role of saturated fatty acids in the brain that may have important implications against cerebral ischemia.

Protein Arginine Methyltransferases in Cardiovascular and Neuronal Function.

The methylation of arginine residues by protein arginine methyltransferases (PRMTs) is a type of post-translational modification which is important for numerous cellular processes, including mRNA splicing, DNA repair, signal transduction, protein interaction, and transport. PRMTs have been extensively associated with various pathologies, including cancer, inflammation, and immunity response. However, the role of PRMTs has not been well described in vascular and neurological function. Aberrant expression of PRMTs can alter its metabolic products, asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA). Increased ADMA levels are recognized as an independent risk factor for cardiovascular disease and mortality. Recent studies have provided considerable advances in the development of small-molecule inhibitors of PRMTs to study their function under normal and pathological states. In this review, we aim to elucidate the particular roles of PRMTs in vascular and neuronal function as a potential target for cardiovascular and neurological diseases.

Palmitic acid methyl ester is a novel neuroprotective agent against cardiac arrest.

We previously discovered that palmitic acid methyl ester (PAME) is a potent vasodilator first identified and released from the superior cervical ganglion and remain understudied. Thus, we investigated PAME's role in modulating cerebral blood flow (CBF) and neuroprotection after 6 min of cardiac arrest (model of global cerebral ischemia). Our results suggest that PAME can enhance CBF under normal physiological conditions, while administration of PAME (0.02 mg/kg) immediately after cardiopulmonary resuscitation can also enhance CBF in vivo. Additionally, functional learning and spatial memory assessments (via T-maze) 3 days after asphyxial cardiac arrest (ACA) suggest that PAME-treated rats have improved learning and memory recovery versus ACA alone. Furthermore, improved neuronal survival in the CA1 region of the hippocampus were observed in PAME-treated, ACA-induced rats. Altogether, our findings suggest that PAME can enhance CBF, alleviate neuronal cell death, and promote functional outcomes in the presence of ACA.

Memantine inhibits α3ß2-nAChRs-mediated nitrergic neurogenic vasodilation in porcine basilar arteries.

Memantine, an NMDA receptor antagonist used for treatment of Alzheimer's disease (AD), is known to block the nicotinic acetylcholine receptors (nAChRs) in the central nervous system (CNS). In the present study, we examined by wire myography if memantine inhibited α3ß2-nAChRs located on cerebral perivascular sympathetic nerve terminals originating in the superior cervical ganglion (SCG), thus, leading to inhibition of nicotine-induced nitrergic neurogenic dilation of isolated porcine basilar arteries. Memantine concentration-dependently blocked nicotine-induced neurogenic dilation of endothelium-denuded basilar arteries without affecting that induced by transmural nerve stimulation, sodium nitroprusside, or isoproterenol. Furthermore, memantine significantly inhibited nicotine-elicited inward currents in Xenopous oocytes expressing α3ß2-, α7- or α4ß2-nAChR, and nicotine-induced calcium influx in cultured rat SCG neurons. These results suggest that memantine is a non-specific antagonist for nAChR. By directly inhibiting α3ß2-nAChRs located on the sympathetic nerve terminals, memantine blocks nicotine-induced neurogenic vasodilation of the porcine basilar arteries. This effect of memantine is expected to reduce the blood supply to the brain stem and possibly other brain regions, thus, decreasing its clinical efficacy in the treatment of Alzheimer's disease.

Sympathetic α3ß2-nAChRs mediate cerebral neurogenic nitrergic vasodilation in the swine.

The α(7)-nicotinic ACh receptor (α(7)-nAChR) on sympathetic neurons innervating basilar arteries of pigs crossed bred between Landrace and Yorkshire (LY) is known to mediate nicotine-induced, ß-amyloid (Aß)-sensitive nitrergic neurogenic vasodilation. Preliminary studies, however, demonstrated that nicotine-induced cerebral vasodilation in pigs crossbred among Landrace, Yorkshire, and Duroc (LYD) was insensitive to Aß and α-bungarotoxin (α-BGTX). We investigated nAChR subtype on sympathetic neurons innervating LYD basilar arteries. Nicotine-induced relaxation of porcine isolated basilar arteries was examined by tissue bath myography, inward currents on nAChR-expressing oocytes by two-electrode voltage recording, and mRNA and protein expression in the superior cervical ganglion (SCG) and middle cervical ganglion (MCG) by reverse transcription PCR and Western blotting. Nicotine-induced basilar arterial relaxation was not affected by Aß, α-BGTX, and α-conotoxin IMI (α(7)-nAChR antagonists), or α-conotoxin AuIB (α(3)ß(4)-nAChR antagonist) but was inhibited by tropinone and tropane (α(3)-containing nAChR antagonists) and α-conotoxin MII (selective α(3)ß(2)-nAChR antagonist). Nicotine-induced inward currents in α(3)ß(2)-nAChR-expressing oocytes were inhibited by α-conotoxin MII but not by α-BGTX, Aß, or α-conotoxin AuIB. mRNAs of α(3)-, α(7)-, ß(2)-, and ß(4)-subunits were expressed in both SCGs and MCGs with significantly higher mRNAs of α(3)-, ß(2)-, and ß(4)-subunits than that of α(7)-subunit. The Aß-insensitive sympathetic α(3)ß(2)-nAChR mediates nicotine-induced cerebral nitrergic neurogenic vasodilation in LYD pigs. The different finding from Aß-sensitive α(7)-nAChR in basilar arteries of LY pigs may offer a partial explanation for different sensitivities of individuals to Aß in causing diminished cerebral nitrergic vasodilation in diseases involving Aß.