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

Analysis workflow to assess de novo genetic variants from human whole-exome sequencing

Here, we present a protocol to analyz

DNA Damage Response and Repair: Insights into Strategies for Radiation Sensitization

The incorporation of radiotherapy into multimodality treatment plans has led to significant improvements in glioma patient survival. However, local recurrence from glioma resistance to ionizing radiation remains a therapeutic challenge. The tumoricidal effect of radiation therapy is largely attributed to the induction of dsDNA breaks (DSBs). In the past decade, there have been tremendous strides in understanding the molecular mechanisms underlying DSB repair. The identification of gene products required for DSB repair has provided novel therapeutic targets. Recent studies revealed that many US FDA-approved cancer agents inhibit DSB repair by interacting with repair proteins. This article will aim to provide discussion of DSB repair mechanisms to provide molecular targets for radiation sensitization of gliomas and a discussion of FDA-approved cancer therapies that modulate DSB repair to highlight opportunities for combination therapy with radiotherapy for glioma therapy

Molecular diagnostic yield of exome sequencing in patients with congenital hydrocephalus: A systematic review and meta-analysis

IMPORTANCE: Exome sequencing (ES) has been established as the preferred first line of diagnostic testing for certain neurodevelopmental disorders, such as global developmental delay and autism spectrum disorder; however, current recommendations are not specific to or inclusive of congenital hydrocephalus (CH). OBJECTIVE: To determine the diagnostic yield of ES in CH and whether ES should be considered as a first line diagnostic test for CH. DATA SOURCES: PubMed, Cochrane Library, and Google Scholar were used to identify studies published in English between January 1, 2010, and April 10, 2023. The following search terms were used to identify studies: congenital hydrocephalus, ventriculomegaly, cerebral ventriculomegaly, primary ventriculomegaly, fetal ventriculomegaly, prenatal ventriculomegaly, molecular analysis, genetic cause, genetic etiology, genetic testing, exome sequencing, whole exome sequencing, genome sequencing, microarray, microarray analysis, and copy number variants. STUDY SELECTION: Eligible studies included those with at least 10 probands with the defining feature of CH and/or severe cerebral ventriculomegaly that had undergone ES. Studies with fewer than 10 probands, studies of mild or moderate ventriculomegaly, and studies using genetic tests other than ES were excluded. A full-text review of 68 studies was conducted by 2 reviewers. Discrepancies were resolved by consensus. DATA EXTRACTION AND SYNTHESIS: Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines and Meta-Analysis of Observational Studies in Epidemiology guidelines were used by 2 reviewers to extract data. Data were synthesized using a random-effects model of single proportions. Data analysis occurred in April 2023. MAIN OUTCOMES AND MEASURES: The primary outcome was pooled diagnostic yield. Additional diagnostic yields were estimated for specific subgroups on the basis of clinical features, syndromic presentation, and parental consanguinity. For each outcome, a 95% CI and estimate of interstudy heterogeneity (I2 statistic) was reported. RESULTS: From 498 deduplicated and screened records, 9 studies with a total of 538 CH probands were selected for final inclusion. The overall diagnostic yield was 37.9% (95% CI, 20.0%-57.4%; I2 = 90.1). The yield was lower for isolated and/or nonsyndromic cases (21.3%; 95% CI, 12.8%-31.0%; I2 = 55.7). The yield was higher for probands with reported consanguinity (76.3%; 95% CI, 65.1%-86.1%; I2 = 0) than those without (16.2%; 95% CI, 12.2%-20.5%; I2 = 0). CONCLUSIONS AND RELEVANCE: In this systematic review and meta-analysis of the diagnostic yield of ES in CH, the diagnostic yield was concordant with that of previous recommendations for other neurodevelopmental disorders, suggesting that ES should also be recommended as a routine diagnostic adjunct for patients with CH

Glioblastoma Mimicking an Arteriovenous Malformation

Abnormal cerebral vasculature can be a manifestation of a vascular malformation or a neoplastic process. We report the case of a patient with angiography-negative subarachnoid hemorrhage (SAH) who re-presented 3 years later with a large intraparenchymal hemorrhage. Although imaging following the intraparenchymal hemorrhage was suggestive of arteriovenous malformation, the patient was ultimately found to have an extensive glioblastoma associated with abnormal tumor vasculature. The case emphasizes the need for magnetic resonance imaging to investigate angiography-negative SAH in suspicious cases to rule out occult etiologies, such as neoplasm. We also discuss diagnostic pitfalls when brain tumors are associated with hemorrhage and abnormal vasculature

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

Background: The 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. Methods: Expression 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. Results: We 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. Conclusion: Together, 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

Current view on the functional regulation of the neuronal K+-Cl- cotransporter KCC2

Peer reviewe

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

This is the author accepted manuscript. The final version is available from American Association for the Advancement of Science via the DOI in this record.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 T(991) tonically inhibits KCC3; however, cell swelling triggers Thr(991) dephosphorylation to activate the transporter and restore cell volume. KCC3 T991A mutation in patient cells abolished Thr(991) phosphorylation, resulted in constitutive KCC3 activity, and compromised cell volume homeostasis. KCC3(T991A/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 in humans.This work was supported by NIH research grant GM74771 (E.D.). K.T.K. was supported by a Harvard-MIT Neuroscience Grant, the Manton Center for Orphan Disease Research at Harvard Medical School, and the March of Dimes. C.G.B. is supported by intramural funds of the NINDS. B.F. and D.B.G. received support from NIH grants 2T32MH064913-11A1 and T32-AR056993, respectively