Muriel Auberson

Leukoencephalopathy upon Disruption of the Chloride Channel ClC-2

ClC-2 is a broadly expressed plasma membrane chloride channel that is modulated by voltage, cell swelling, and pH. A human mutation leading to a heterozygous loss of ClC-2 has previously been reported to be associated with epilepsy, whereas the disruption of Clcn2 in mice led to testicular and retinal degeneration. We now show that the white matter of the brain and spinal cord of ClC-2 knock-out mice developed widespread vacuolation that progressed with age. Fluid-filled spaces appeared between myelin sheaths of the central but not the peripheral nervous system. Neuronal morphology, in contrast, seemed normal. Except for the previously reported blindness, neurological deficits were mild and included a decreased conduction velocity in neurons of the central auditory pathway. The heterozygous loss of ClC-2 had no detectable functional or morphological consequences. Neither heterozygous nor homozygous ClC-2 knock-out mice had lowered seizure thresholds. Sequencing of a large collection of human DNA and electrophysiological analysis showed that several ClC-2 sequence abnormalities previously found in patients with epilepsy most likely represent innocuous polymorphisms.

Disrupting MLC1 and GlialCAM and ClC-2 interactions in leukodystrophy entails glial chloride channel dysfunction

Defects in the astrocytic membrane protein MLC1, the adhesion molecule GlialCAM or the chloride channel ClC-2 underlie human leukoencephalopathies. Whereas GlialCAM binds ClC-2 and MLC1, and modifies ClC-2 currents in vitro, no functional connections between MLC1 and ClC-2 are known. Here we investigate this by generating loss-of-function Glialcam and Mlc1 mouse models manifesting myelin vacuolization. We find that ClC-2 is unnecessary for MLC1 and GlialCAM localization in brain, whereas GlialCAM is important for targeting MLC1 and ClC-2 to specialized glial domains in vivo and for modifying ClC-2s biophysical properties specifically in oligodendrocytes (OLs), the cells chiefly affected by vacuolization. Unexpectedly, MLC1 is crucial for proper localization of GlialCAM and ClC-2, and for changing ClC-2 currents. Our data unmask an unforeseen functional relationship between MLC1 and ClC-2 in vivo, which is probably mediated by GlialCAM, and suggest that ClC-2 participates in the pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts.

No evidence for a role of CLCN2 variants in idiopathic generalized epilepsy

To the Editor: We note the retraction of a paper published in Nature Genetics in 2003, which had reported that mutations in CLCN2 (NCBI Reference Sequence NC_000003.11), the gene encoding the chloride channel ClC-2, were associated with several subtypes of idiopathic generalized epilepsy1. Despite the retraction, Kleefuß-Lie et al.2 recently asserted that “other major aspects of the work remain unaltered” and that their electrophysiological studies are supported by further work published subsequently2. We believe that their logical argument is flawed and that, in addition, the assertion misrepresents the work of others, including our own. First, the authors maintain that they “still believe that the reported genetic variations may contribute to the epileptic phenotypes”2. Without the link between the reported genetic variations and epilepsy, there is no rational basis for such a belief. Second, concerning the functional consequences of the mutations, Kleefuß-Lie et al.2 state that “studies in other laboratories... supported some of the functional changes that were originally reported.” This statement is untrue. The two papers cited as “studies in other laboratories” come from our respective groups3,4. The first of these papers (Niemeyer et al.3) in fact contradicts every one of the functional findings of Haug et al.1. The first mutant, 3792_3793insG (M200fsX231), corresponding to family 1 in the retracted publication, predicts a truncated protein lacking 13 out of 18 expected membrane helices including most putative pore-forming regions. The second consists of an 11-bp deletion (2776_2788del11) in intron 2 close to the splice acceptor site, which was suggested to lead preferentially to an alternatively spliced mRNA and a protein, V74_Q117del, lacking most of transmembrane α-helix B, the largest α-helix predicted to lie at the interface between the ClC-2 channel and the membrane. Our results showed that, in contrast to the claims in the retracted paper, these altered proteins did not reach the plasma membrane and did not exert any dominant negative effect on the function of normal ClC-2 (ref. 3). Also, in regard to the 2776_2788del11 mutation, using a minigene approach, we could find no difference in the proportion of exon-skipped to normally spliced mRNA as a consequence of the mutation and, on this basis, predicted no alteration in ClC-2–channel expression in affected individuals. A third mutation, G8794A, produces an amino acid replacement (G715E) purportedly associated with a gain of function1, allowing the channel to be conductive at reduced intracellular Cl– concentration. We could not reproduce this result of the retracted paper either3. The contrast between our results and those in the retracted paper was reflected in the first paragraph of our Discussion section, which reads: “Our results are in marked contrast to those reported previously by Haug et al. and suggest that the pathophysiological mechanisms proposed by these authors to account for the phenotype need to be revised” 3. The other paper cited as supporting the functional results of the retracted paper is that by Blanz et al.4, in which we in fact confirm the failure to reproduce the dominant negative effect of mutation 3792_3793insG (M200fsX231) reported by Haug et al.1. We concluded that “our electrophysiological analysis of CLCN2 sequence abnormalities described in patients with epilepsy (Haug et al.) did not provide evidence for them being epileptogenic”4. In the same paper4, we showed that other CLCN2 sequence variants identified more recently in patients with epilepsy5 did not alter the biophysical properties of ClC-2 and were also found in humans not displaying epilepsy. We have also reported that the ClC-2–null genotype in mice failed to induce spontaneous seizures or to alter the seizure threshold for the response of the animals to proconvulsants4,6. We discussed these points in recent reviews7,8 and had concluded before the retraction of the paper by Haug et al.1 that “the sum of these observations... warrants skepticism toward the proposed causative role of ClC-2 in epilepsy”7. These observations both suggest that even the functional results of the retracted paper cannot be relied upon, and they also support the view that there is no basis to claim that CLCN2 plays a role in epilepsy.

Renal Fanconi Syndrome and Hypophosphatemic Rickets in the Absence of Xenotropic and Polytropic Retroviral Receptor in the Nephron

Tight control of extracellular and intracellular inorganic phosphate (Pi) levels is critical to most biochemical and physiologic processes. Urinary Pi is freely filtered at the kidney glomerulus and is reabsorbed in the renal tubule by the action of the apical sodium-dependent phosphate transporters, NaPi-IIa/NaPi-IIc/Pit2. However, the molecular identity of the protein(s) participating in the basolateral Pi efflux remains unknown. Evidence has suggested that xenotropic and polytropic retroviral receptor 1 (XPR1) might be involved in this process. Here, we show that conditional inactivation of Xpr1 in the renal tubule in mice resulted in impaired renal Pi reabsorption. Analysis of Pi transport in primary cultures of proximal tubular cells or in freshly isolated renal tubules revealed that this Xpr1 deficiency significantly affected Pi efflux. Further, mice with conditional inactivation of Xpr1 in the renal tubule exhibited generalized proximal tubular dysfunction indicative of Fanconi syndrome, characterized by glycosuria, aminoaciduria, calciuria, and albuminuria. Dramatic alterations in the renal transcriptome, including a significant reduction in NaPi-IIa/NaPi-IIc expression, accompanied these functional changes. Additionally, Xpr1-deficient mice developed hypophosphatemic rickets secondary to renal dysfunction. These results identify XPR1 as a major regulator of Pi homeostasis and as a potential therapeutic target in bone and kidney disorders.

Altered Prostasin (CAP1/Prss8) Expression Favors Inflammation and Tissue Remodeling in DSS-induced Colitis.

Background:Inflammatory bowel diseases (IBD) including ulcerative colitis and Crohns disease are diseases with impaired epithelial barrier function. We aimed to investigate whether mutated prostasin and thus, reduced colonic epithelial sodium channel activity predisposes to develop an experimentally dextran sodium sulfate (DSS)–induced colitis. Methods:Wildtype, heterozygous (frCR/+), and homozygous (frCR/frCR) prostasin-mutant rats were treated 7 days with DSS followed by 7 days of recovery and analyzed with respect to histology, clinicopathological parameters, inflammatory marker mRNA transcript expression, and sodium transporter protein expression. Results:In this study, a more detailed analysis on rat frCR/frCR colons revealed reduced numbers of crypt and goblet cells, and local angiodysplasia, as compared with heterozygous (frCR/+) and wildtype littermates. Following 2% DSS treatment for 7 days followed by 7 days recovery, frCR/frCR animals lost body weight, and reached maximal diarrhea score and highest disease activity after only 3 days, and strongly increased cytokine levels. The histology score significantly increased in all groups, but frCR/frCR colons further displayed pronounced histological alterations with near absence of goblet cells, rearrangement of the lamina propria, and presence of neutrophils, eosinophils, and macrophages. Additionally, frCR/frCR colons showed ulcerations and edemas that were absent in frCR/+ and wildtype littermates. Following recovery, frCR/frCR rats reached, although significantly delayed, near-normal diarrhea score and disease activity, but exhibited severe architectural remodeling, despite unchanged sodium transporter protein expression. Conclusions:In summary, our results demonstrate a protective role of colonic prostasin expression against experimental colitis, and thus represent a susceptibility gene in the development of inflammatory bowel disease.

Increased bone resorption by osteoclast-specific deletion of the sodium/calcium exchanger isoform 1 (NCX1)

Calcium is a key component of the bone mineral hydroxyapatite. During osteoclast-mediated bone resorption, hydroxyapatite is dissolved and significant quantities of calcium are released. Several calcium transport systems have previously been identified in osteoclasts, including members of the sodium/calcium exchanger (NCX) family. Expression pattern and physiological role of NCX isoforms in osteoclasts, however, remain largely unknown at the moment. Our data indicate that all three NCX isoforms (NCX1, NCX2, and NCX3) are present in murine osteoclasts. RANKL-induced differentiation of murine osteoclast precursors into mature osteoclasts significantly attenuated the expression of NCX1, while NCX2 and NCX3 expressions were largely unaffected. To study the role of NCX1 during osteoclast differentiation and bone resorption, we crossed mice with exon 11 of the NCX1 gene flanked by loxP sites with cathepsin K-Cre transgenic mice. Mature osteoclasts derived from transgenic mice exhibited an 80–90% reduction of NCX1 protein. In vitro studies indicate that NCX1 is dispensable for osteoclast differentiation, but NCX1-deficient osteoclasts exhibited increased resorptive activity. In line with these in vitro findings, mice with an osteoclast-targeted deletion of the NCX1 gene locus displayed an age-dependent loss of bone mass. Thus, in summary, our data reveal NCX1 as a regulator of osteoclast-mediated bone resorption.