Protein kinase D1 variant associated with human epilepsy and peripheral nerve hypermyelination.

We report the case of a patient with severe progressive epilepsy and peripheral neuropathy and a novel de novo inactivating variant (p.E79X) in Protein Kinase D1 (PKD1). Using CRISPR/Cas9, we engineered the homologous variant in mice and showed that in the homozygote mouse, it recapitulated the patient peripheral nerve hypermyelination pathology. The lethality of the homozygote mouse prevented us from performing an assessment of locomotor behavior. The mutant heterozygote mouse; however, exhibited a significant increase in kainate-induced seizure activity over wild-type mice, supporting the hypothesis that the PKD1 variant is a candidate for the cause of the patient epilepsy. Because PKD1 was previously identified in a kinomic screen as an interacting partner of the K-Cl cotransporter 3 (KCC3), and since KCC3 is involved in peripheral nerve disease and brain hyperexcitability, one possible mechanism of action of PKD1 in disease is through KCC3. We show that catalytically inactive PKD1 stimulates KCC3 activity, consistent with tonic relief of inhibitory phosphorylation. Our findings implicate a novel role for PKD1 in the human nervous system, and uncover a mechanism that could serve as a potential target to promote nervous system myelination.

A mutation in the Na-K-2Cl cotransporter-1 leads to changes in cellular metabolism.

The Na-K-Cl cotransporter-1 (NKCC1), by mediating the electroneutral transport of Na+ , K+ , and Cl- plays an important role in cell volume regulation, epithelial transport, and the control of neuronal excitability. Recently, we reported the first known human mutation in SLC12A2, the gene encoding NKCC1. The 17-year old patient suffers from multiorgan failure. Laboratory tests conducted on muscle and liver biopsies of the patient showed abnormal increase in mitochondrial DNA copy number and increased glycogen levels, indicating the possibility that the transporter may play a role in energy metabolism. Here, we show that fibroblasts isolated from the patient demonstrate a significant increase in mitochondrial respiration, compared to fibroblasts isolated from healthy individuals. Similarly, Madin Darby canine kidney (MDCK) cells transfected with enhanced green fluorescent protein (EGFP)-tagged mutant NKCC1 DNA demonstrated increased mitochondrial respiration when compared to MDCK cells expressing EGFP-tagged wild-type (WT) cotransporter. Direct inhibition of the cotransporter through addition of bumetanide did not change the rate of basal respiration, but led to increased maximal mitochondrial respiration. Fibroblasts extracted from NKCC1WT/DFX and NKCC1DFX/DFX mice also demonstrated a significant elevation in mitochondrial respiration, compared to fibroblasts isolated from their WT littermates. Expression of the mutant protein was associated with an increase in hydrogen peroxide and peroxidase activity and a decrease in messenger RNA transcript levels for protein involved in the unfolded protein response. These data reveal that cells expressing the mutant cotransporter demonstrate increased mitochondrial respiration and behave like they are experiencing a state of starvation.

A dileucine motif in the COOH-terminal domain of NKCC1 targets the cotransporter to the plasma membrane.

Na+-K+-2Cl- cotransporter-1 (NKCC1) mediates the electroneutral transport of Na+, K+, and Cl- and is normally localized to the basolateral membrane of polarized epithelial cells. We recently reported the first known solute carrier family 12 member 2 ( SLC12A2) mutation (we call NKCC1-DFX) that causes epithelial dysfunction in an undiagnosed disease program case. The heterozygous mutation leads to truncation of the COOH-terminal tail of the cotransporter, resulting in both mutant and wild-type cotransporters being mistrafficked to the apical membrane of polarized epithelial cells. Here we demonstrate by using consecutive truncations and site-directed mutagenesis of the COOH-terminal domain of NKCC1 that truncation of NKCC1 COOH domain uncouples the cotransporter from the lateral membrane. We identify a dileucine motif that, when mutated, leads to cotransporter accumulation in the cytoplasm and mistrafficking to the apical/subapical region of epithelial cells, thereby recapitulating the phenotype observed with the patient mutation. We show that truncation deletion and LL substitution mutants are trafficked out of the endoplasmic reticulum and trans-Golgi network but accumulate in early and late endosomes where they are degraded.

Mistargeting of a truncated Na-K-2Cl cotransporter in epithelial cells.

We recently reported the case of a young patient with multisystem failure carrying a de novo mutation in SLC12A2, the gene encoding the Na-K-2Cl cotransporter-1 (NKCC1). Heterologous expression studies in nonepithelial cells failed to demonstrate dominant-negative effects. In this study, we examined expression of the mutant cotransporter in epithelial cells. Using Madin-Darby canine kidney (MDCK) cells grown on glass coverslips, permeabilized support, and Matrigel, we show that the fluorescently tagged mutant cotransporter is expressed in cytoplasm and at the apical membrane and affects epithelium integrity. Expression of the mutant transporter at the apical membrane also results in the mislocalization of some of the wild-type transporter to the apical membrane. This mistargeting is specific to NKCC1 as the Na+-K+-ATPase remains localized on the basolateral membrane. To assess transporter localization in vivo, we created a mouse model using CRISPR/cas9 that reproduces the 11 bp deletion in exon 22 of Slc12a2. Although the mice do not display an overt phenotype, we show that the colon and salivary gland expresses wild-type NKCC1 abundantly at the apical pole, confirming the data obtained in cultured epithelial cells. Enough cotransporter must remain, however, on the basolateral membrane to participate in saliva secretion, as no significant decrease in saliva production was observed in the mutant mice.

Novel Human NKCC1 Mutations Cause Defects in Goblet Cell Mucus Secretion and Chronic Inflammation.

BACKGROUND & AIMS: Infections resulting from intestinal yeast and bacteria affect a large number of patients with deficits in absorptive or secretory epithelial transport mechanisms. The basolateral Na+-K+-2Cl- cotransporter (NKCC1) has been implicated in intestinal epithelial fluid secretion. Two patients with deleterious heterozygous (NKCC1-DFX, DFX for Asp-Phe-stop codon) or homozygous (Kilquist) mutations in SLC12A2 (NKCC1) suffered from gastrointestinal deficits. Because of chronic infections, the colon and the small intestine of the NKCC1-DFX patient were resected surgically. METHODS: To investigate how NKCC1 affects the integrity and function of the gut epithelia, we used a mouse model recapitulating the NKCC1-DFX patient mutation. Electron microscopy and immunostaining were used to analyze the integrity of the colonic mucus layers and immune cell infiltration. Fluorescence in situ hybridization was performed on the distal colon sections to measure bacteria translocation to the mucosa and submucosa. Citrobacter rodentium was used to measure mouse ability to clear enteric infection. A multiplex cytokine assay was used to analyze mouse inflammatory response to infection. RESULTS: We show that NKCC1-DFX expression causes defective goblet cell mucus granule exocytosis, leading to secretion of intact granules into the lumen of the large intestine. In addition, NKCC1-DFX colon submucosal glands secrete mucus that remained attached to the epithelium. Importantly, expression of the mutant NKCC1 or complete loss of NKCC1 function leads to aggravated inflammatory response to C rodentium infection. Compared with wild-type, NKCC1-DFX mice showed decreased expression of claudin-2, a tight junction protein involved in paracellular Na+ and water transport and enteric infection clearance. CONCLUSIONS: Our data indicate that NKCC1-DFX impairs gut barrier function by affecting mucus secretion and immune properties.

A patient with multisystem dysfunction carries a truncation mutation in human SLC12A2, the gene encoding the Na-K-2Cl cotransporter, NKCC1.

This study describes a 13-yr-old girl with orthostatic intolerance, respiratory weakness, multiple endocrine abnormalities, pancreatic insufficiency, and multiorgan failure involving the gut and bladder. Exome sequencing revealed a de novo, loss-of-function allele in SLC12A2, the gene encoding the Na-K-2Cl cotransporter-1. The 11-bp deletion in exon 22 results in frameshift (p.Val1026Phefs*2) and truncation of the carboxy-terminal tail of the cotransporter. Preliminary studies in heterologous expression systems demonstrate that the mutation leads to a nonfunctional transporter, which is expressed and trafficked to the plasma membrane alongside wild-type NKCC1. The truncated protein, visible at higher molecular sizes, indicates either enhanced dimerization or misfolded aggregate. No significant dominant-negative effect was observed. K+ transport experiments performed in fibroblasts from the patient showed reduced total and NKCC1-mediated K+ influx. The absence of a bumetanide effect on K+ influx in patient fibroblasts only under hypertonic conditions suggests a deficit in NKCC1 regulation. We propose that disruption in NKCC1 function might affect sensory afferents and/or smooth muscle cells, as their functions depend on NKCC1 creating a Cl- gradient across the plasma membrane. This Cl- gradient allows the γ-aminobutyric acid (GABA) receptor or other Cl- channels to depolarize the membrane affecting processes such as neurotransmission or cell contraction. Under this hypothesis, disrupted sensory and smooth muscle function in a diverse set of tissues could explain the patient's phenotype.