WNK Kinase Signaling in Ion Homeostasis and Human Disease

This is the author accepted manuscript. The final version is available from Elsevier via the DOI in this record.WNK kinases, along with their upstream regulators (CUL3/KLHL3) and downstream targets (the SPAK/OSR1 kinases and the cation-Cl- cotransporters [CCCs]), comprise a signaling cascade essential for ion homeostasis in the kidney and nervous system. Recent work has furthered our understanding of the WNKs in epithelial transport, cell volume homeostasis, and GABA signaling, and uncovered novel roles for this pathway in immune cell function and cell proliferation.This work was supported by a NIHNRCDP grant (K.T.K.), Simons Foundation grant #400947 (K.T.K.), March of Dimes Basil O’Connor Award (K.T.K.), and NIH grant DK93501 to E.D

Autocrine Netrin Function Inhibits Glioma Cell Motility and Promotes Focal Adhesion Formation

Deregulation of mechanisms that control cell motility plays a key role in tumor progression by promoting tumor cell dissemination. Secreted netrins and their receptors, Deleted in Colorectal Cancer (DCC), neogenin, and the UNC5 homologues, regulate cell and axon migration, cell adhesion, and tissue morphogenesis. Netrin and netrin receptor expression have previously been shown to be disrupted in invasive tumors, including glioblastoma. We determined that the human glioblastoma cell lines U87, U343, and U373 all express neogenin, UNC5 homologues, and netrin-1 or netrin-3, but only U87 cells express DCC. Using transfilter migration assays, we demonstrate DCC-dependent chemoattractant migration of U87 cells up a gradient of netrin-1. In contrast, U343 and U373 cells, which do not express DCC, were neither attracted nor repelled. Ectopic expression of DCC by U343 and U373 cells resulted in these cells becoming competent to respond to a gradient of netrin-1 as a chemoattractant, and also slowed their rate of spontaneous migration. Here, in addition to netrins' well-characterized chemotropic activity, we demonstrate an autocrine function for netrin-1 and netrin-3 in U87 and U373 cells that slows migration. We provide evidence that netrins promote the maturation of focal complexes, structures associated with cell movement, into focal adhesions. Consistent with this, netrin, DCC, and UNC5 homologues were associated with focal adhesions, but not focal complexes. Disrupting netrin or DCC function did not alter cell proliferation or survival. Our findings provide evidence that DCC can slow cell migration, and that neogenin and UNC5 homologues are not sufficient to substitute for DCC function in these cells. Furthermore, we identify a role for netrins as autocrine inhibitors of cell motility that promote focal adhesion formation. These findings suggest that disruption of netrin signalling may disable a mechanism that normally restrains inappropriate cell migration

Loss of neuronal potassium/chloride cotransporter 3 (KCC3) is responsible for the degenerative phenotype in a conditional mouse model of hereditary motor and sensory neuropathy associated with agenesis of the corpus callosum

Disruption of the potassium/chloride cotransporter 3 (KCC3), encoded by the SLC12A6 gene, causes hereditary motor and sensory neuropathy associated with agenesis of the corpus callosum (HMSN/ACC), a neurodevelopmental and neurodegenerative disorder affecting both the peripheral nervous system and CNS. However, the precise role of KCC3 in the maintenance of ion homeostasis in the nervous system and the pathogenic mechanisms leading to HMSN/ACC remain unclear. We established two Slc12a6 Cre/LoxP transgenic mouse lines expressing C-terminal truncated KCC3 in either a neuron-specific or ubiquitous fashion. Our results suggest that neuronal KCC3 expression is crucial for axon volume control. We also demonstrate that the neuropathic features of HMSN/ACC are predominantly due to a neuronal KCC3 deficit, while the auditory impairment is due to loss of non-neuronal KCC3 expression. Furthermore, we demonstrate that KCC3 plays an essential role in inflammatory pain pathways. Finally, we observed hypoplasia of the corpus callosum in both mouse mutants and a marked decrease in axonal tracts serving the auditory cortex in only the general deletion mutant. Together, these results establish KCC3 as an important player in both central and peripheral nervous system maintenance

Potential Application of Novel DNA Editing Techniques in Translational Neuroscience

No abstract is provided

Netrin-1 signaling : cellular consequences and molecular mechanisms

During the embryonic development of the nervous System, groups of multipotent cells proliferate, migrate, and differentiate to become neurons. The new born neurons need to make precise connections with their targets. These may be a great distance away from their soma. To make these connections, neurons send their axon through a biochemically complex environment and their growth cones may make many guidance decisions on the ways to their targets. The existence of axon guidance molecules was inferred by the early classical neuroanatomists. We now know of multiple examples of secreted or membrane-bound proteins that either attract or repel axonal growth cônes by causing the growth cône to collapse or extend. The molecular mechanisms that regulate growth cone collapse or extension are closely linked to reorganization of the cytoskeleton. The small Rho GTPases, Cdc42, Rac, and Rho play key roles in neuronal growth cônes by regulating the organization of the cytoskeleton. Netrins are a small family of secreted guidance eues that are implicated in the attraction and repulsion of axons during the development of the nervous System. The netrin-1 receptor deleted in colorectal cancer (DCC) is highly expressed by commissural neurons in the developing spinal cord. The findings described here show that DCC is present at the tips of filopodia and the edges of lamellipodia of HEK293T, NG108-15 neuroblastoma-glioma cells, and the growth cones of embryonic rat spinal commissural neurons. Furthermore, netrin-1 protein causes an increase in filopodia number and surface area of embryonic rat commissural neuron growth cônes and the cell fines when transfected to express DCC. Further experiments indicated that netrin-1 activates the small GTPases Cdc42 and Racl in the cell lines and the embryonic rat commissural spinal cord neurons. The activation of Cdc42 and Racl by netrin-1 requires DCC. Furthermore, netrin-1 causes increased phosphorylation of Pakl, an effector for both Cdc42 and Racl. Expression of a dominant negative form of N-WASP, an effector for Cdc42, blocks the netrin-1 induced morphological changes in the growth cones of commissural neurons. Evidence is presented that netrin-1 induces the formation of a complex of proteins interacting with the intra-cellular domain of DCC that includes Nckl, Cdc42, Racl, Pakl, and N-WASP. The findings described lead to a model whereby netrin-1 binding to DCC triggers the activation of Cdc42 and Racl, which leads to actin based membrane extension and changes in growth cone morphology

Gene regulation of amyloid precursor protein during differentiation of neuroblastoma cells

Expression of APP (amyloid precursor protein) during brain development, specific expression of APP 695 isoform in neurons, and the degeneration of cholinergic neurons in Alzheimer's disease point to the important physiological function of APP in nervous system development and metabolism. We studied the regulation of APP mRNA expression in NG108-15 neuroblastoma x glioma cells which had been induced to differentiate in the presence of dibutyryl cAMP. APP mRNA levels gradually increased concomitant with the appearance of differentiated features. In the present studies, Northern blot analysis showed a 3.1 fold increase in APP expression at day 6 of dibutyryl cAMP treatment along with increase in expression of the neuronal markers, GAP-43 and NF-L. Run-on assay experiments confirmed a 2-fold increase in the rate of APP mRNA transcription. Although the half-life of APP mRNA was long, differentiated and nondifferentiated cells showed the same half-life of about 21h. The presence of APP mRNA binding proteins was also investigated. We found that RNA binding proteins were present in both differentiated and non-differentiated cells. These experiments strongly suggest that APP mRNA induction is due to increase in transcription rate. Studies of mRNA decay in the presence of actinomycin D, an inhibitor of transcription, in primary cultures revealed large differences in APP mRNA half-life between fetal neurons and fetal astrocytes without any qualitative changes in APP mRNA binding proteins

The effect of obesity, macronutrients, fasting and nutritional status on drug-metabolizing cytochrome P450s : a systematic review of current evidence on human studies

Background: Cytochrome P450s (CYPs) are a class of hemoproteins involved in drug metabolism. It has been reported that body composition, proportion of dietary macronutrients, fasting and nutritional status can interfere with the activity of drug-metabolizing CYPs. Objectives: The present systematic review was conducted to summarize the effect of obesity, weight reduction, macronutrients, fasting and malnutrition on the CYP-mediated drug metabolism. Methods: PubMed (Medline), Scopus, Embase and Cochrane Library databases and Google Scholar were searched up to June 2020 to obtain relevant studies. The PRISMA guidelines were employed during all steps. Two reviewers independently extracted the information from the included studies. Studies investigating CYPs activity directly or indirectly through pharmacokinetics of probe drugs, were included. Increase in clearance (CL) or decrease in elimination half-life (t½) and area under the curve (AUC) of probe drugs were considered as increase in CYPs activity. Results: A total of 6545 articles were obtained through searching databases among which 69 studies with 126 datasets fully met the inclusion criteria. The results indicated that obesity might decrease the activity of CYP3A4/5, CYP1A2 and CYP2C9 and increase the activity of CYP2E1. The effect of obesity on CYP2D6 is controversial. Also, weight loss increased CYP3A4 activity. Moreover, CYP1A2 activity was decreased by high carbohydrate diet, increased by high protein diet and fasting and unchanged by malnutrition. The activity of CYP2C19 was less susceptible to alterations compared to other CYPs. Conclusion: The activity of drug-metabolizing CYPs are altered by body composition, dietary intake and nutritional status. This relationship might contribute to drug toxicity or reduce treatment efficacy and influence cost-effectiveness of medical care

Dietary green-synthesized curcumin-mediated zinc oxide nanoparticles promote growth performance, haemato-biochemical profile, antioxidant status, immunity, and carcass quality in Nile tilapia (Oreochromis niloticus)

This study evaluated the effect of dietary curcumin-assisted green synthesized zinc oxide nanoparticles (CUR-ZnONPs) on growth performance, haemato-biochemical profile, antioxidant capacity, immune response, carcass composition, and bioaccumulation of Zn in Nile tilapia. The fish (‎16.20 ± 1.02 g‎) were fed with diets enriched with 0 (CZP1), 5 (CZP2), 7.5 (CZP3), and 10 (CZP4) mg/kg CUR-ZnONPs for 56 days and then challenged with Aeromonas hydrophila. The highest growth performance (final weight, weight gain, and specific growth rate) and the lowest feed conversion ratio were recorded in CZP4 (P  0.05). The mortality rate of the fish infected with A. hydrophila was significantly lower in the group that received CZP4 compared to the other experimental groups (P < 0.05). The principal component analysis demonstrated that the fish fed with CUR-ZnONPs have significantly higher growth performance, immune responses, and antioxidant status than the fish fed with the control diet. Overall, it is suggested to use dietary CUR-ZnONPs, especially at 10 mg/kg, as a beneficial feed supplement to improve growth performance, antioxidant status, and immune parameters in Nile tilapia