Gulnaz Begum

Docosahexaenoic Acid Reduces ER Stress and Abnormal Protein Accumulation and Improves Neuronal Function Following Traumatic Brain Injury

In this study, we investigated the development of endoplasmic reticulum (ER) stress after traumatic brain injury (TBI) and the efficacy of post-TBI administration of docosahexaenoic acid (DHA) in reducing ER stress. TBI was induced by cortical contusion injury in Sprague-Dawley rats. Either DHA (16 mg/kg in DMSO) or vehicle DMSO (1 ml/kg) was administered intraperitoneally at 5 min after TBI, followed by a daily dose for 3–21 d. TBI triggered sustained expression of the ER stress marker proteins including phosphorylated eukaryotic initiation factor-2α, activating transcription factor 4, inositol requiring kinase 1, and C/EBP homologous protein in the ipsilateral cortex at 3–21 d after TBI. The prolonged ER stress was accompanied with an accumulation of abnormal ubiquitin aggregates and increased expression of amyloid precursor protein (APP) and phosphorylated tau (p-Tau) in the frontal cortex after TBI. The ER stress marker proteins were colocalized with APP accumulation in the soma. Interestingly, administration of DHA attenuated all ER stress marker proteins and reduced the accumulation of both ubiquitinated proteins and APP/p-Tau proteins. In addition, the DHA-treated animals exhibited early recovery of their sensorimotor function after TBI. In summary, our study demonstrated that TBI induces a prolonged ER stress, which is positively correlated with abnormal APP accumulation. The sustained ER stress may play a role in chronic neuronal damage after TBI. Our findings illustrate that post-TBI administration of DHA has therapeutic potentials in reducing ER stress, abnormal protein accumulation, and neurological deficits.

Inhibition of WNK3 Kinase Signaling Reduces Brain Damage and Accelerates Neurological Recovery After Stroke

Background and Purpose— WNK kinases, including WNK3, and the associated downstream Ste20/SPS1-related proline-alanine–rich protein kinase (SPAK) and oxidative stress responsive 1 (OSR1) kinases, comprise an important signaling cascade that regulates the cation-chloride cotransporters. Ischemia-induced stimulation of the bumetanide-sensitive Na+-K+-Cl− cotransporter (NKCC1) plays an important role in the pathophysiology of experimental stroke, but the mechanism of its regulation in this context is unknown. Here, we investigated the WNK3-SPAK/OSR1 pathway as a regulator of NKCC1 stimulation and their collective role in ischemic brain damage. Method— Wild-type WNK3 and WNK3 knockout mice were subjected to ischemic stroke via transient middle cerebral artery occlusion. Infarct volume, brain edema, blood brain barrier damage, white matter demyelination, and neurological deficits were assessed. Total and phosphorylated forms of WNK3 and SPAK/OSR1 were assayed by immunoblotting and immunostaining. In vitro ischemia studies in cultured neurons and immature oligodendrocytes were conducted using the oxygen-glucose deprivation/reoxygenation method. Results— WNK3 knockout mice exhibited significantly decreased infarct volume and axonal demyelination, less cerebral edema, and accelerated neurobehavioral recovery compared with WNK3 wild-type mice subjected to middle cerebral artery occlusion. The neuroprotective phenotypes conferred by WNK3 knockout were associated with a decrease in stimulatory hyperphosphorylations of the SPAK/OSR1 catalytic T-loop and of NKCC1 stimulatory sites Thr203/Thr207/Thr212, as well as with decreased cell surface expression of NKCC1. Genetic inhibition of WNK3 or small interfering RNA knockdown of SPAK/OSR1 increased the tolerance of cultured primary neurons and oligodendrocytes to in vitro ischemia. Conclusions— These data identify a novel role for the WNK3-SPAK/OSR1-NKCC1 signaling pathway in ischemic neuroglial injury and suggest the WNK3-SPAK/OSR1 kinase pathway as a therapeutic target for neuroprotection after ischemic stroke.

Administration of DHA Reduces Endoplasmic Reticulum Stress-Associated Inflammation and Alters Microglial or Macrophage Activation in Traumatic Brain Injury

We investigated the effects of the administration of docosahexaenoic acid (DHA) post-traumatic brain injury (TBI) on reducing neuroinflammation. TBI was induced by cortical contusion injury in Sprague Dawley rats. Either DHA (16 mg/kg in dimethyl sulfoxide) or vehicle dimethyl sulfoxide (1 ml/kg) was administered intraperitonially at 5 min after TBI, followed by a daily dose for 3 to 21 days. TBI triggered activation of microglia or macrophages, detected by an increase of Iba1 positively stained microglia or macrophages in peri-lesion cortical tissues at 3, 7, and 21 days post-TBI. The inflammatory response was further characterized by expression of the proinflammatory marker CD16/32 and the anti-inflammatory marker CD206 in Iba1+ microglia or macrophages. DHA-treated brains showed significantly fewer CD16/32+ microglia or macrophages, but an increased CD206+ phagocytic microglial or macrophage population. Additionally, DHA treatment revealed a shift in microglial or macrophage morphology from the activated, amoeboid-like state into the more permissive, surveillant state. Furthermore, activated Iba1+ microglial or macrophages were associated with neurons expressing the endoplasmic reticulum (ER) stress marker CHOP at 3 days post-TBI, and the administration of DHA post-TBI concurrently reduced ER stress and the associated activation of Iba1+ microglial or macrophages. There was a decrease in nuclear translocation of activated nuclear factor kappa-light-chain-enhancer of activated B cells protein at 3 days in DHA-treated tissue and reduced neuronal degeneration in DHA-treated brains at 3, 7, and 21 days after TBI. In summary, our study demonstrated that TBI mediated inflammatory responses are associated with increased neuronal ER stress and subsequent activation of microglia or macrophages. DHA administration reduced neuronal ER stress and subsequent association with microglial or macrophage polarization after TBI, demonstrating its therapeutic potential to ameliorate TBI-induced cellular pathology.

WNK1-OSR1 kinase-mediated phospho-activation of Na+-K+-2Cl- cotransporter facilitates glioma migration.

BackgroundThe bumetanide (BMT)-sensitive Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) maintains cell volume homeostasis by increasing intracellular K+ and Cl- content via regulatory volume increase (RVI). Expression levels of NKCC1 positively correlate with the histological grade and severity of gliomas, the most common primary adult brain tumors, and up-regulated NKCC1 activity facilitates glioma cell migration and apoptotic resistance to the chemotherapeutic drug temozolomide (TMZ). However, the cellular mechanisms underlying NKCC1 functional up-regulation in glioma and in response to TMZ administration remain unknown.MethodsExpression of NKCC1 and its upstream kinases With-No-K (Lysine) kinase 1 (WNK1) and oxidative stress-responsive kinase-1 (OSR1) in different human glioma cell lines and glioma specimens were detected by western blotting and immunostaining. Live cell imaging and microchemotaxis assay were applied to record glioma cell movements under different treatment conditions. Fluorescence indicators were utilized to measure cell volume, intracellular K+ and Cl- content to reflect the activity of NKCC1 on ion transportation. Small interfering RNA (siRNA)-mediated knockdown of WNK1 or OSR1 was used to explore their roles in regulation of NKCC1 activity in glioma cells. Results of different treatment groups were compared by one-way ANOVA using the Bonferroni post-hoc test in the case of multiple comparisons.ResultsWe show that compared to human neural stem cells and astrocytes, human glioma cells exhibit robust increases in the activation and phosphorylation of NKCC1 and its two upstream regulatory kinases, WNK1 and OSR1. siRNA-mediated knockdown of WNK1 or OSR1 reduces intracellular K+ and Cl- content and RVI in glioma cells by abolishing NKCC1 regulatory phospho-activation. Unexpectedly, TMZ activates the WNK1/OSR1/NKCC1 signaling pathway and enhances glioma migration. Pharmacological inhibition of NKCC1 with its potent inhibitor BMT or siRNA knockdown of WNK1 or OSR1 significantly decreases glioma cell migration after TMZ treatment.ConclusionTogether, our data show a novel role for the WNK1/OSR1/NKCC1 pathway in basal and TMZ-induced glioma migration, and suggest that glioma treatment with TMZ might be improved by drugs that inhibit elements of the WNK1/OSR1/NKCC1 signaling pathway.

ER Stress and Effects of DHA as an ER Stress Inhibitor

The endoplasmic reticulum (ER) functions in the synthesis, folding, modification, and transport of newly synthesized transmembrane and secretory proteins. The ER also has important roles in the storage of intracellular Ca2+ and regulation of Ca2+ homeostasis. The integrity of the Ca2+ homeostasis in the ER lumen is vital for proper folding of proteins. Dysregulation of ER Ca2+ could result in an increase in unfolded or misfolded proteins and ER stress. ER stress triggers activation of the unfolded protein response (UPR), which is a fundamentally adaptive cell response and functions as a cytoprotective mechanism by over-expression of relevant chaperones and the global shutdown of protein synthesis. UPR activation occurs when three key ER membrane-sensor proteins detect an accumulation of aberrant proteins. The UPR acts to alleviate ER stress, but if the stress is too severe or prolonged, apoptosis will be triggered. In this review, we focused on ER stress and the effects of docosahexaenoic acid (DHA) on ER stress. DHA and its bioactive compounds, such as protectins and resolvins, provide neuroprotection against oxidative stress and apoptosis and have the ability to resolve inflammation in neurological diseases. New studies reveal that DHA blocks inositol trisphosphate receptor (IP3R)-mediated ER Ca2+ depletion and ER stress. The administration of DHA post-traumatic brain injury (TBI) reduces ER stress, aberrant protein accumulation, and neurological deficits. Therefore, DHA presents therapeutic potentials for TBI via its pleiotropic effects including inhibition of ER stress.

DHA inhibits ER Ca2+ release and ER stress in astrocytes following in vitro ischemia

J. Neurochem. (2012) 120, 622–630.

Generation of WNK1 knockout cell lines by CRISPR/Cas-mediated genome editing

Sodium-coupled SLC12 cation chloride cotransporters play important roles in cell volume and chloride homeostasis, epithelial fluid secretion, and renal tubular salt reabsorption. These cotransporters are phosphorylated and activated indirectly by With-No-Lysine (WNK) kinases through their downstream effector kinases, Ste20- and SPS1-related proline alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1). Multiple WNK kinases can coexist within a single cell type, although their relative contributions to SPAK/OSR1 activation and salt transport remain incompletely understood. Deletion of specific WNKs from cells that natively express a functional WNK-SPAK/OSR1 network will help resolve these knowledge gaps. Here, we outline a simple method to selectively knock out full-length WNK1 expression from mammalian cells using RNA-guided clustered regularly interspaced short palindromic repeats/Cas9 endonucleases. Two clonal cell lines were generated by using a single-guide RNA (sgRNA) targeting exon 1 of the WNK1 gene, which produced indels that abolished WNK1 protein expression. Both cell lines exhibited reduced endogenous WNK4 protein abundance, indicating that WNK1 is required for WNK4 stability. Consistent with an on-target effect, the reduced WNK4 abundance was associated with increased expression of the KLHL3/cullin-3 E3 ubiquitin ligase complex and was rescued by exogenous WNK1 overexpression. Although the morphology of the knockout cells was indistinguishable from control, they exhibited low baseline SPAK/OSR1 activity and failed to trigger regulatory volume increase after hypertonic stress, confirming an essential role for WNK1 in cell volume regulation. Collectively, our data show how this new, powerful, and accessible gene-editing technology can be used to dissect and analyze WNK signaling networks.

Glial Na+‐dependent ion transporters in pathophysiological conditions

Sodium dynamics are essential for regulating functional processes in glial cells. Indeed, glial Na+ signaling influences and regulates important glial activities, and plays a role in neuron‐glia interaction under physiological conditions or in response to injury of the central nervous system (CNS). Emerging studies indicate that Na+ pumps and Na+‐dependent ion transporters in astrocytes, microglia, and oligodendrocytes regulate Na+ homeostasis and play a fundamental role in modulating glial activities in neurological diseases. In this review, we first briefly introduced the emerging roles of each glial cell type in the pathophysiology of cerebral ischemia, Alzheimers disease, epilepsy, Parkinsons disease, Amyotrophic Lateral Sclerosis, and myelin diseases. Then, we discussed the current knowledge on the main roles played by the different glial Na+‐dependent ion transporters, including Na+/K+ ATPase, Na+/Ca2+ exchangers, Na+/H+ exchangers, Na+‐K+‐Cl− cotransporters, and Na+‐ HCO3− cotransporter in the pathophysiology of the diverse CNS diseases. We highlighted their contributions in cell survival, synaptic pathology, gliotransmission, pH homeostasis, and their role in glial activation, migration, gliosis, inflammation, and tissue repair processes. Therefore, this review summarizes the foundation work for targeting Na+‐dependent ion transporters in glia as a novel strategy to control important glial activities associated with Na+ dynamics in different neurological disorders. GLIA 2016;64:1677–1697

Endoplasmic reticulum Ca2+ signaling and mitochondrial Cyt c release in astrocytes following oxygen and glucose deprivation.

J. Neurochem. (2010) 114, 1436–1446.

Peripheral motor neuropathy is associated with defective kinase regulation of the KCC3 cotransporter

Evaluation of a patient with peripheral motor weakness reveals a key role for phosphorylation-dependent regulation of the transporter KCC3 in the peripheral nervous system. Neuropathic KCC3 activity Clinical presentation of disease by patients can lead to profoundly important discoveries about basic biology. In this case, a child with progressive, early-onset, motor neuropathy resulting in profound disability revealed a key role for the phosphorylation-mediated regulation of the K+-Cl− transporter KCC3 in the peripheral nervous system. Kahle et al. discovered a point mutation in the gene encoding KCC3 in the patient that prevented the transporter from being inhibited through phosphorylation and resulted in its constitutive activation. Mice expressing KCC3 with the same mutation had increased transporter activity and impaired locomotor function, suggesting that the normal function of peripheral neurons depends on the regulation of KCC3 function. Using exome sequencing, we identified a de novo mutation (c.2971A>G; T991A) in SLC12A6, the gene encoding the K+-Cl− cotransporter KCC3, in a patient with an early-onset, progressive, and severe peripheral neuropathy primarily affecting motor neurons. Normally, the WNK kinase–dependent phosphorylation of T991 tonically inhibits KCC3; however, cell swelling triggers Thr991 dephosphorylation to activate the transporter and restore cell volume. KCC3 T991A mutation in patient cells abolished Thr991 phosphorylation, resulted in constitutive KCC3 activity, and compromised cell volume homeostasis. KCC3T991A/T991A mutant mice exhibited constitutive KCC3 activity and recapitulated aspects of the clinical, electrophysiological, and histopathological findings of the patient. These results suggest that the function of the peripheral nervous system depends on finely tuned, kinase-regulated KCC3 activity and implicate abnormal cell volume homeostasis as a previously unreported mechanism of axonal degeneration.