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.

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.

EphA2 signaling within integrin adhesions regulates fibrillar adhesion elongation and fibronectin deposition.

The multifunctional glycoprotein fibronectin influences several crucial cellular processes and contributes to multiple pathologies. While a link exists between fibronectin-associated pathologies and the receptor tyrosine kinase EphA2, the mechanism by which EphA2 promotes fibronectin matrix remodeling remains unknown. We previously demonstrated that EphA2 deletion reduces smooth muscle fibronectin deposition and blunts fibronectin deposition in atherosclerosis without influencing fibronectin expression. We now show that EphA2 expression is required for contractility-dependent elongation of tensin- and α5ß1 integrin-rich fibrillar adhesions that drive fibronectin fibrillogenesis. Mechanistically, EphA2 localizes to integrin adhesions where focal adhesion kinase mediates ligand-independent Y772 phosphorylation, and mutation of this site significantly blunts fibrillar adhesion length. EphA2 deficiency decreases smooth muscle cell contractility by enhancing p190RhoGAP activation and reducing RhoA activity, whereas stimulating RhoA signaling in EphA2 deficient cells rescues fibrillar adhesion elongation. Together, these data identify EphA2 as a novel regulator of fibrillar adhesion elongation and provide the first data identifying a role for EphA2 signaling in integrin adhesions.

Protein arginine methyltransferase 8 modulates mitochondrial bioenergetics and neuroinflammation after hypoxic stress.

Protein arginine methyltransferases (PRMTs) are a family of enzymes involved in gene regulation and protein/histone modifications. PRMT8 is primarily expressed in the central nervous system, specifically within the cellular membrane and synaptic vesicles. Recently, PRMT8 has been described to play key roles in neuronal signaling such as a regulator of dendritic arborization, synaptic function and maturation, and neuronal differentiation and plasticity. Here, we examined the role of PRMT8 in response to hypoxia-induced stress in brain metabolism. Our results from liquid chromatography mass spectrometry, mitochondrial oxygen consumption rate, and protein analyses indicate that PRMT8(-/-) knockout mice presented with altered membrane phospholipid composition, decreased mitochondrial stress capacity, and increased neuroinflammatory markers, such as tumor necrosis factor alpha and ionized calcium binding adaptor molecule 1 (Iba1, a specific marker for microglia/macrophage activation) after hypoxic stress. Furthermore, adenovirus-based overexpression of PRMT8 reversed the changes in membrane phospholipid composition, mitochondrial stress capacity, and neuroinflammatory markers. Together, our findings establish PRMT8 as an important regulatory component of membrane phospholipid composition, short-term memory function, mitochondrial function, and neuroinflammation in response to hypoxic stress.

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.

Selective role of Nck1 in atherogenic inflammation and plaque formation.

Although the Canakinumab Anti-Inflammatory Thrombosis Outcomes Study (CANTOS) established the role of treating inflammation in atherosclerosis, our understanding of endothelial activation at atherosclerosis-prone sites remains limited. Disturbed flow at atheroprone regions primes plaque inflammation by enhancing endothelial NF-κB signaling. Herein, we demonstrate a role for the Nck adaptor proteins in disturbed flow-induced endothelial activation. Although highly similar, only Nck1 deletion, but not Nck2 deletion, limited flow-induced NF-κB activation and proinflammatory gene expression. Nck1-knockout mice showed reduced endothelial activation and inflammation in both models, disturbed flow- and high fat diet-induced atherosclerosis, whereas Nck2 deletion did not. Bone marrow chimeras confirmed that vascular Nck1, but not hematopoietic Nck1, mediated this effect. Domain-swap experiments and point mutations identified the Nck1 SH2 domain and the first SH3 domain as critical for flow-induced endothelial activation. We further characterized Nck1's proinflammatory role by identifying interleukin 1 type I receptor kinase-1 (IRAK-1) as a Nck1-selective binding partner, demonstrating that IRAK-1 activation by disturbed flow required Nck1 in vitro and in vivo, showing endothelial Nck1 and IRAK-1 staining in early human atherosclerosis, and demonstrating that disturbed flow-induced endothelial activation required IRAK-1. Taken together, our data reveal a hitherto unknown link between Nck1 and IRAK-1 in atherogenic inflammation.