Multiubiquitination of TRPV4 reduces channel activity independent of surface localization.

Ubiquitin (Ub)-mediated regulation of plasmalemmal ion channel activity canonically occurs via stimulation of endocytosis. Whether ubiquitination can modulate channel activity by alternative mechanisms remains unknown. Here, we show that the transient receptor potential vanilloid 4 (TRPV4) cation channel is multiubiquitinated within its cytosolic N-terminal and C-terminal intrinsically disordered regions (IDRs). Mutagenizing select lysine residues to block ubiquitination of the N-terminal but not C-terminal IDR resulted in a marked elevation of TRPV4-mediated intracellular calcium influx, without increasing cell surface expression levels. Conversely, enhancing TRPV4 ubiquitination via expression of an E3 Ub ligase reduced TRPV4 channel activity but did not decrease plasma membrane abundance. These results demonstrate Ub-dependent regulation of TRPV4 channel function independent of effects on plasma membrane localization. Consistent with ubiquitination playing a key negative modulatory role of the channel, gain-of-function neuropathy-causing mutations in the TRPV4 gene led to reduced channel ubiquitination in both cellular and Drosophila models of TRPV4 neuropathy, whereas increasing mutant TRPV4 ubiquitination partially suppressed channel overactivity. Together, these data reveal a novel mechanism via which ubiquitination of an intracellular flexible IDR domain modulates ion channel function independently of endocytic trafficking and identify a contributory role for this pathway in the dysregulation of TRPV4 channel activity by neuropathy-causing mutations.

Inflammatory profile in a canine model of hypothermic circulatory arrest.

BACKGROUND: Hypothermic circulatory arrest (HCA) is a technique used for complex repair of the aorta, but it can be associated with neurologic morbidity. To better understand the molecular changes that underlie ischemic brain injury, we assessed gene expression and cytokine/chemokine polypeptide concentration in brain tissue and cerebrospinal fluid (CSF) of canines that underwent two hours of HCA. MATERIALS AND METHODS: Adult male canines were cannulated peripherally for cardiopulmonary bypass, cooled to 18°C, and arrested for two hours. Animals were euthanized two, eight, or 24 hours post-HCA (n = 8 per group), and their brains were compared to brains from eight normal canines, using gene expression microarray analysis, cytokine assay, and histopathology. RESULTS: Two to eight hours after HCA, pro-inflammatory cytokine mRNAs increased markedly, and gene expression was enriched within signaling pathways related to neuroinflammation or ischemic injury. Concentrations of pro-inflammatory cytokine polypeptides IL-6, IL-8, IL-1ß, and CCL2 were very low in normal canine brain, whereas anti-inflammatory IL-10 and TGF-ß1 were expressed at moderate levels. Pro-inflammatory cytokine concentrations rose robustly in cerebral tissue and CSF after HCA. IL-6 and IL-8 peaked at eight hours and declined at 24 hours, while IL-1ß and CCL2 remained elevated. Concentrations of anti-inflammatory IL-10 and TGF-ß1 were maintained after HCA, with a significant increase in TGF-ß1 at 24 hours. CONCLUSIONS: These cytokines represent potential diagnostic markers for ischemic neurologic injury that could be used to assess neurologic injury in patients undergoing HCA. The cellular mechanisms underlying this pro-inflammatory, ischemic-induced injury represent potential targets for neuroprotection in the future.

Early Detection of Hypothermic Neuroprotection Using T2-Weighted Magnetic Resonance Imaging in a Mouse Model of Hypoxic Ischemic Encephalopathy.

Perinatal hypoxic-ischemic encephalopathy (HIE) can lead to neurodevelopmental disorders, including cerebral palsy. Standard care for neonatal HIE includes therapeutic hypothermia, which provides partial neuroprotection; magnetic resonance imaging (MRI) is often used to assess injury and predict outcome after HIE. Immature rodent models of HIE are used to evaluate mechanisms of injury and to examine the efficacy and mechanisms of neuroprotective interventions such as hypothermia. In this study, we first confirmed that, in the CD1 mouse model of perinatal HIE used for our research, MRI obtained 3 h after hypoxic ischemia (HI) could reliably assess initial brain injury and predict histopathological outcome. Mice were subjected to HI (unilateral carotid ligation followed by exposure to hypoxia) on postnatal day 7 and were imaged with T2-weighted MRI and diffusion-weighted MRI (DWI), 3 h after HI. Clearly defined regions of increased signal were comparable in T2 MRI and DWI, and we found a strong correlation between T2 MRI injury scores 3 h after HI and histopathological brain injury 7 days after HI, validating this method for evaluating initial injury in this model of HIE. The more efficient, higher resolution T2 MRI was used to score initial brain injury in subsequent studies. In mice treated with hypothermia, we found a significant reduction in T2 MRI injury scores 3 h after HI, compared to normothermic littermates. Early hypothermic neuroprotection was maintained 7 days after HI, in both T2 MRI injury scores and histopathology. In the normothermic group, T2 MRI injury scores 3 h after HI were comparable to those obtained 7 days after HI. However, in the hypothermic group, brain injury was significantly less 7 days after HI than at 3 h. Thus, early neuroprotective effects of hypothermia were enhanced by 7 days, which may reflect the additional 3 h of hypothermia after imaging or effects on later mechanisms of injury, such as delayed cell death and inflammation. Our results demonstrate that hypothermia has early neuroprotective effects in this model. These findings suggest that hypothermia has an impact on early mechanisms of excitotoxic injury and support initiation of hypothermic intervention as soon as possible after diagnosis of HIE.

Uptake of dendrimer-drug by different cell types in the hippocampus after hypoxic-ischemic insult in neonatal mice: Effects of injury, microglial activation and hypothermia.

Perinatal hypoxic-ischemic encephalopathy (HIE) can result in neurodevelopmental disability, including cerebral palsy. The only treatment, hypothermia, provides incomplete neuroprotection. Hydroxyl polyamidoamine (PAMAM) dendrimers are being explored for targeted delivery of therapy for HIE. Understanding the biodistribution of dendrimer-conjugated drugs into microglia, neurons and astrocytes after brain injury is essential for optimizing drug delivery. We conjugated N-acetyl-L-cysteine to Cy5-labeled PAMAM dendrimer (Cy5-D-NAC) and used a mouse model of perinatal HIE to study effects of timing of administration, hypothermia, brain injury, and microglial activation on uptake. Dendrimer conjugation delivered therapy most effectively to activated microglia but also targeted some astrocytes and injured neurons. Cy5-D-NAC uptake was correlated with brain injury in all cell types and with activated morphology in microglia. Uptake was not inhibited by hypothermia, except in CD68+ microglia. Thus, dendrimer-conjugated drug delivery can target microglia, astrocytes and neurons and can be used in combination with hypothermia for treatment of HIE.