Cellular basis of ClC-2 Cl− channel–related brain and testis pathologies
2020; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1074/jbc.ra120.016031
ISSN1083-351X
AutoresCorinna Göppner, Audrey H. Soria, Maja B. Hoegg-Beiler, Thomas J. Jentsch,
Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoThe ClC-2 chloride channel is expressed in the plasma membrane of almost all mammalian cells. Mutations that cause the loss of ClC-2 function lead to retinal and testicular degeneration and leukodystrophy, whereas gain-of-function mutations cause hyperaldosteronism. Leukodystrophy is also observed with a loss of GlialCAM, a cell adhesion molecule that binds to ClC-2 in glia. GlialCAM changes the localization of ClC-2 and opens the channel by altering its gating. We now used cell type–specific deletion of ClC-2 in mice to show that retinal and testicular degeneration depend on a loss of ClC-2 in retinal pigment epithelial cells and Sertoli cells, respectively, whereas leukodystrophy was fully developed only when ClC-2 was disrupted in both astrocytes and oligodendrocytes. The leukodystrophy of Glialcam−/− mice could not be rescued by crosses with Clcn2op/op mice in which a mutation mimics the "opening" of ClC-2 by GlialCAM. These data indicate that GlialCAM-induced changes in biophysical properties of ClC-2 are irrelevant for GLIALCAM-related leukodystrophy. Taken together, our findings suggest that the pathology caused by Clcn2 disruption results from disturbed extracellular ion homeostasis and identifies the cells involved in this process. The ClC-2 chloride channel is expressed in the plasma membrane of almost all mammalian cells. Mutations that cause the loss of ClC-2 function lead to retinal and testicular degeneration and leukodystrophy, whereas gain-of-function mutations cause hyperaldosteronism. Leukodystrophy is also observed with a loss of GlialCAM, a cell adhesion molecule that binds to ClC-2 in glia. GlialCAM changes the localization of ClC-2 and opens the channel by altering its gating. We now used cell type–specific deletion of ClC-2 in mice to show that retinal and testicular degeneration depend on a loss of ClC-2 in retinal pigment epithelial cells and Sertoli cells, respectively, whereas leukodystrophy was fully developed only when ClC-2 was disrupted in both astrocytes and oligodendrocytes. The leukodystrophy of Glialcam−/− mice could not be rescued by crosses with Clcn2op/op mice in which a mutation mimics the "opening" of ClC-2 by GlialCAM. These data indicate that GlialCAM-induced changes in biophysical properties of ClC-2 are irrelevant for GLIALCAM-related leukodystrophy. Taken together, our findings suggest that the pathology caused by Clcn2 disruption results from disturbed extracellular ion homeostasis and identifies the cells involved in this process. Chloride channels are molecularly very diverse. They can reside in the plasma membrane or intracellular organelles and perform a plethora of functions. These include transepithelial transport, the modulation of cellular excitability, or the regulation of both intracellular and extracellular ion concentrations and of cell volume (1Jentsch T.J. Stein V. Weinreich F. Zdebik A.A. Molecular structure and physiological function of chloride channels.Physiol. Rev. 2002; 82: 503-568Crossref PubMed Scopus (1061) Google Scholar, 2Jentsch T.J. Pusch M. CLC chloride channels and transporters: structure, function, physiology, and disease.Physiol. Rev. 2018; 98: 1493-1590Crossref PubMed Scopus (195) Google Scholar, 3Jentsch T.J. VRACs and other ion channels and transporters in the regulation of cell volume and beyond.Nat. Rev. Mol. Cell Biol. 2016; 17: 293-307Crossref PubMed Scopus (183) Google Scholar). Accordingly, both loss- and gain-of-function mutations in diverse chloride channel genes, both in humans and animal models, result in a large spectrum of disease phenotypes. These in turn allow conclusions on the physiological roles of individual channels. ClC-2 (4Thiemann A. Gründer S. Pusch M. Jentsch T.J. A chloride channel widely expressed in epithelial and non-epithelial cells.Nature. 1992; 356: 57-60Crossref PubMed Scopus (505) Google Scholar) is a widely expressed member of the CLC gene family of Cl− channels and 2Cl−/H+ transporters (2Jentsch T.J. Pusch M. CLC chloride channels and transporters: structure, function, physiology, and disease.Physiol. Rev. 2018; 98: 1493-1590Crossref PubMed Scopus (195) Google Scholar) whose first member, ClC-0, was cloned from electric fish (5Jentsch T.J. Steinmeyer K. Schwarz G. Primary structure of Torpedo marmorata chloride channel isolated by expression cloning in Xenopus oocytes.Nature. 1990; 348: 510-514Crossref PubMed Scopus (421) Google Scholar). ClC-2 is expressed at the plasma membrane where it mediates inwardly rectifying Cl− currents that are slowly activated by membrane hyperpolarization (4Thiemann A. Gründer S. Pusch M. Jentsch T.J. A chloride channel widely expressed in epithelial and non-epithelial cells.Nature. 1992; 356: 57-60Crossref PubMed Scopus (505) Google Scholar, 6Gründer S. Thiemann A. Pusch M. Jentsch T.J. Regions involved in the opening of CIC-2 chloride channel by voltage and cell volume.Nature. 1992; 360: 759-762Crossref PubMed Scopus (361) Google Scholar). ClC-2 can also be activated by cell swelling (6Gründer S. Thiemann A. Pusch M. Jentsch T.J. Regions involved in the opening of CIC-2 chloride channel by voltage and cell volume.Nature. 1992; 360: 759-762Crossref PubMed Scopus (361) Google Scholar), but, in contrast to volume-regulated LRRC8/VRAC anion channels (3Jentsch T.J. VRACs and other ion channels and transporters in the regulation of cell volume and beyond.Nat. Rev. Mol. Cell Biol. 2016; 17: 293-307Crossref PubMed Scopus (183) Google Scholar, 7Voss F.K. Ullrich F. Münch J. Lazarow K. Lutter D. Mah N. Andrade-Navarro M.A. von Kries J.P. Stauber T. Jentsch T.J. Identification of LRRC8 heteromers as an essential component of the volume-regulated anion channel VRAC.Science. 2014; 344: 634-638Crossref PubMed Scopus (399) Google Scholar, 8Qiu Z. Dubin A.E. Mathur J. Tu B. Reddy K. Miraglia L.J. Reinhardt J. Orth A.P. Patapoutian A. SWELL1, a plasma membrane protein, is an essential component of volume-regulated anion channel.Cell. 2014; 157: 447-458Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar), appears to lack a prominent role in cell volume regulation (2Jentsch T.J. Pusch M. CLC chloride channels and transporters: structure, function, physiology, and disease.Physiol. Rev. 2018; 98: 1493-1590Crossref PubMed Scopus (195) Google Scholar), although Bergmann glia from Clcn2−/− mice appeared swollen (9Hoegg-Beiler M.B. Sirisi S. Orozco I.J. Ferrer I. Hohensee S. Auberson M. Gödde K. Vilches C. de Heredia M.L. Nunes V. Estévez R. Jentsch T.J. Disrupting MLC1 and GlialCAM and ClC-2 interactions in leukodystrophy entails glial chloride channel dysfunction.Nat. Commun. 2014; 5: 3475Crossref PubMed Scopus (72) Google Scholar). Regions and residues important for the slow opening of ClC-2 by hyperpolarization or cell swelling have been mapped to the amino terminus of ClC-2 (6Gründer S. Thiemann A. Pusch M. Jentsch T.J. Regions involved in the opening of CIC-2 chloride channel by voltage and cell volume.Nature. 1992; 360: 759-762Crossref PubMed Scopus (361) Google Scholar) and an intracellular loop (10Jordt S.E. Jentsch T.J. Molecular dissection of gating in the ClC-2 chloride channel.EMBO J. 1997; 16: 1582-1592Crossref PubMed Scopus (204) Google Scholar). Mutations in these regions virtually abolish gating and result in large Cl− currents with an almost ohmic behavior. Interestingly, coexpression with the cell adhesion protein GlialCAM (also known as HepaCAM), which can bind ClC-2 and localize it to cell–cell contacts, similarly "opens" ClC-2 by changing its gating (9Hoegg-Beiler M.B. Sirisi S. Orozco I.J. Ferrer I. Hohensee S. Auberson M. Gödde K. Vilches C. de Heredia M.L. Nunes V. Estévez R. Jentsch T.J. Disrupting MLC1 and GlialCAM and ClC-2 interactions in leukodystrophy entails glial chloride channel dysfunction.Nat. Commun. 2014; 5: 3475Crossref PubMed Scopus (72) Google Scholar, 11Jeworutzki E. Lagostena L. Elorza-Vidal X. López-Hernández T. Estévez R. Pusch M. GlialCAM, a CLC-2 Cl− channel subunit, activates the slow gate of CLC chloride channels.Biophys. J. 2014; 107: 1105-1116Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 12Jeworutzki E. López-Hernández T. Capdevila-Nortes X. Sirisi S. Bengtsson L. Montolio M. Zifarelli G. Arnedo T. Müller C.S. Schulte U. Nunes V. Martínez A. Jentsch T.J. Gasull X. Pusch M. et al.GlialCAM, a protein defective in a leukodystrophy, serves as a ClC-2 Cl− channel auxiliary subunit.Neuron. 2012; 73: 951-961Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 13Maduke M.C. Reimer R.J. Biochemistry to the rescue: a ClC-2 auxiliary subunit provides a tangible link to leukodystrophy.Neuron. 2012; 73: 855-857Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar). However, the physiological role of this effect remains unclear. To elucidate the physiological roles of ClC-2 we previously generated constitutive ClC-2 KO mice (Clcn2−/− mice) (14Bösl M.R. Stein V. Hübner C. Zdebik A.A. Jordt S.E. Mukhophadhyay A.K. Davidoff M.S. Holstein A.F. Jentsch T.J. Male germ cells and photoreceptors, both depending on close cell-cell interactions, degenerate upon ClC-2 Cl−-channel disruption.EMBO J. 2001; 20: 1289-1299Crossref PubMed Scopus (260) Google Scholar). These mice display male infertility and blindness due to early postnatal degeneration of the testes and retina, respectively (14Bösl M.R. Stein V. Hübner C. Zdebik A.A. Jordt S.E. Mukhophadhyay A.K. Davidoff M.S. Holstein A.F. Jentsch T.J. Male germ cells and photoreceptors, both depending on close cell-cell interactions, degenerate upon ClC-2 Cl−-channel disruption.EMBO J. 2001; 20: 1289-1299Crossref PubMed Scopus (260) Google Scholar). They also slowly develop leukodystrophy (15Blanz J. Schweizer M. Auberson M. Maier H. Muenscher A. Hübner C.A. Jentsch T.J. Leukoencephalopathy upon disruption of the chloride channel ClC-2.J. Neurosci. 2007; 27: 6581-6589Crossref PubMed Scopus (131) Google Scholar) in which vacuoles appear in the white matter. These symptoms were reproduced in independently generated Clcn2−/− mice (16Cortez M.A. Li C. Whitehead S.N. Dhani S.U. D'Antonio C. Huan L.J. Bennett S.A. Snead 3rd, O.C. Bear C.E. Disruption of ClC-2 expression is associated with progressive neurodegeneration in aging mice.Neuroscience. 2010; 167: 154-162Crossref PubMed Scopus (14) Google Scholar, 17Nehrke K. Arreola J. Nguyen H.V. Pilato J. Richardson L. Okunade G. Baggs R. Shull G.E. Melvin J.E. Loss of hyperpolarization-activated Cl− current in salivary acinar cells from Clcn2 knockout mice.J. Biol. Chem. 2002; 277: 23604-23611Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) and in Clcn2 mutant mice obtained in a chemical mutagenesis screen (18Edwards M.M. Marin de Evsikova C. Collin G.B. Gifford E. Wu J. Hicks W.L. Whiting C. Varvel N.H. Maphis N. Lamb B.T. Naggert J.K. Nishina P.M. Peachey N.S. Photoreceptor degeneration, azoospermia, leukoencephalopathy, and abnormal RPE cell function in mice expressing an early stop mutation in CLCN2.Invest. Ophthalmol. Vis. Sci. 2010; 51: 3264-3272Crossref PubMed Scopus (24) Google Scholar). Several years later, homozygous CLCN2 loss-of-function mutations were identified in patients with leukodystrophy, in some cases accompanied by visual problems (19Depienne C. Bugiani M. Dupuits C. Galanaud D. Touitou V. Postma N. van Berkel C. Polder E. Tollard E. Darios F. Brice A. de Die-Smulders C.E. Vles J.S. Vanderver A. Uziel G. et al.Brain white matter oedema due to ClC-2 chloride channel deficiency: an observational analytical study.Lancet Neurol. 2013; 12: 659-668Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 20Giorgio E. Vaula G. Benna P. Lo Buono N. Eandi C.M. Dino D. Mancini C. Cavalieri S. Di Gregorio E. Pozzi E. Ferrero M. Giordana M.T. Depienne C. Brusco A. A novel homozygous change of CLCN2 (p.His590Pro) is associated with a subclinical form of leukoencephalopathy with ataxia (LKPAT).J. Neurol. Neurosurg. Psychiatry. 2017; 88: 894-896Crossref PubMed Scopus (11) Google Scholar, 21Hanagasi H.A. Bilgic B. Abbink T.E. Hanagasi F. Tufekcioglu Z. Gurvit H. Basak N. van der Knaap M.S. Emre M. Secondary paroxysmal kinesigenic dyskinesia associated with CLCN2 gene mutation.Parkinsonism Relat. Disord. 2015; 21: 544-546Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 22Guo Z. Lu T. Peng L. Cheng H. Peng F. Li J. Lu Z. Chen S. Qiu W. CLCN2-related leukoencephalopathy: a case report and review of the literature.BMC Neurol. 2019; 19: 156Crossref PubMed Scopus (15) Google Scholar) or azoospermia-related infertility (23Di Bella D. Pareyson D. Savoiardo M. Farina L. Ciano C. Caldarazzo S. Sagnelli A. Bonato S. Nava S. Bresolin N. Tedeschi G. Taroni F. Salsano E. Subclinical leukodystrophy and infertility in a man with a novel homozygous CLCN2 mutation.Neurology. 2014; 83: 1217-1218Crossref PubMed Scopus (34) Google Scholar). Of note, mutations in GLIALCAM cause a related form of leukodystrophy (megalencephalic leukoencephalopathy with subcortical cysts, MLC) (24López-Hernández T. Ridder M.C. Montolio M. Capdevila-Nortes X. Polder E. Sirisi S. Duarri A. Schulte U. Fakler B. Nunes V. Scheper G.C. Martínez A. Estévez R. van der Knaap M.S. Mutant GlialCAM causes megalencephalic leukoencephalopathy with subcortical cysts, benign familial macrocephaly, and macrocephaly with retardation and autism.Am. J. Hum. Genet. 2011; 88: 422-432Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). MLC can also be caused by mutations in MLC1, which encodes a membrane protein of unknown function (25Leegwater P.A. Yuan B.Q. van der Steen J. Mulders J. Konst A.A. Boor P.K. Mejaski-Bosnjak V. van der Maarel S.M. Frants R.R. Oudejans C.B. Schutgens R.B. Pronk J.C. van der Knaap M.S. Mutations of MLC1 (KIAA0027), encoding a putative membrane protein, cause megalencephalic leukoencephalopathy with subcortical cysts.Am. J. Hum. Genet. 2001; 68: 831-838Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). MLC1, GlialCAM, and ClC-2 apparently form ternary complexes in glial membranes. Loss of either GlialCAM or MLC1 altered localization and decreased ClC-2 expression levels in glia (9Hoegg-Beiler M.B. Sirisi S. Orozco I.J. Ferrer I. Hohensee S. Auberson M. Gödde K. Vilches C. de Heredia M.L. Nunes V. Estévez R. Jentsch T.J. Disrupting MLC1 and GlialCAM and ClC-2 interactions in leukodystrophy entails glial chloride channel dysfunction.Nat. Commun. 2014; 5: 3475Crossref PubMed Scopus (72) Google Scholar), suggesting a loss of ClC-2 function as a common factor in MLC, but it had remained unclear whether the loss of ClC-2 "opening" by GlialCAM contributes to the pathology. To explain the degenerative phenotypes in testis, retina, and brain, we have speculated that ClC-2 regulates the extracellular ion homeostasis in the clefts between cells (9Hoegg-Beiler M.B. Sirisi S. Orozco I.J. Ferrer I. Hohensee S. Auberson M. Gödde K. Vilches C. de Heredia M.L. Nunes V. Estévez R. Jentsch T.J. Disrupting MLC1 and GlialCAM and ClC-2 interactions in leukodystrophy entails glial chloride channel dysfunction.Nat. Commun. 2014; 5: 3475Crossref PubMed Scopus (72) Google Scholar, 14Bösl M.R. Stein V. Hübner C. Zdebik A.A. Jordt S.E. Mukhophadhyay A.K. Davidoff M.S. Holstein A.F. Jentsch T.J. Male germ cells and photoreceptors, both depending on close cell-cell interactions, degenerate upon ClC-2 Cl−-channel disruption.EMBO J. 2001; 20: 1289-1299Crossref PubMed Scopus (260) Google Scholar, 15Blanz J. Schweizer M. Auberson M. Maier H. Muenscher A. Hübner C.A. Jentsch T.J. Leukoencephalopathy upon disruption of the chloride channel ClC-2.J. Neurosci. 2007; 27: 6581-6589Crossref PubMed Scopus (131) Google Scholar). Genetic analysis of patients with hyperaldosteronism (26Scholl U.I. Stolting G. Schewe J. Thiel A. Tan H. Nelson-Williams C. Vichot A.A. Jin S.C. Loring E. Untiet V. Yoo T. Choi J. Xu S. Wu A. Kirchner M. et al.CLCN2 chloride channel mutations in familial hyperaldosteronism type II.Nat. Genet. 2018; 50: 349-354Crossref PubMed Scopus (130) Google Scholar, 27Fernandes-Rosa F.L. Daniil G. Orozco I.J. Göppner C. El Zein R. Jain V. Boulkroun S. Jeunemaitre X. Amar L. Lefebvre H. Schwarzmayr T. Strom T.M. Jentsch T.J. Zennaro M.C. A gain-of-function mutation in the CLCN2 chloride channel gene causes primary aldosteronism.Nat. Genet. 2018; 50: 355-361Crossref PubMed Scopus (110) Google Scholar) recently revealed CLCN2 missense mutations in regions known to affect ClC-2 gating (6Gründer S. Thiemann A. Pusch M. Jentsch T.J. Regions involved in the opening of CIC-2 chloride channel by voltage and cell volume.Nature. 1992; 360: 759-762Crossref PubMed Scopus (361) Google Scholar, 10Jordt S.E. Jentsch T.J. Molecular dissection of gating in the ClC-2 chloride channel.EMBO J. 1997; 16: 1582-1592Crossref PubMed Scopus (204) Google Scholar). By opening the ClC-2 "gate", these human mutations drastically increase ClC-2 Cl− currents (27Fernandes-Rosa F.L. Daniil G. Orozco I.J. Göppner C. El Zein R. Jain V. Boulkroun S. Jeunemaitre X. Amar L. Lefebvre H. Schwarzmayr T. Strom T.M. Jentsch T.J. Zennaro M.C. A gain-of-function mutation in the CLCN2 chloride channel gene causes primary aldosteronism.Nat. Genet. 2018; 50: 355-361Crossref PubMed Scopus (110) Google Scholar, 28Göppner C. Orozco I.J. Hoegg-Beiler M.B. Soria A.H. Hübner C.A. Fernandes-Rosa F.L. Boulkroun S. Zennaro M.C. Jentsch T.J. Pathogenesis of hypertension in a mouse model for human CLCN2 related hyperaldosteronism.Nat. Commun. 2019; 10: 4678Crossref PubMed Scopus (19) Google Scholar). We generated knockin mice (Clcn2op/op) in which an N-terminal deletion, based on our previous structure-function analysis (6Gründer S. Thiemann A. Pusch M. Jentsch T.J. Regions involved in the opening of CIC-2 chloride channel by voltage and cell volume.Nature. 1992; 360: 759-762Crossref PubMed Scopus (361) Google Scholar), opens ClC-2 to a similar degree as aldosteronism-associated human mutations (28Göppner C. Orozco I.J. Hoegg-Beiler M.B. Soria A.H. Hübner C.A. Fernandes-Rosa F.L. Boulkroun S. Zennaro M.C. Jentsch T.J. Pathogenesis of hypertension in a mouse model for human CLCN2 related hyperaldosteronism.Nat. Commun. 2019; 10: 4678Crossref PubMed Scopus (19) Google Scholar). Adrenal zona glomerulosa cells of Clcn2op/op mice are strongly depolarized by large Cl− currents, resulting in increased Ca2+-influx and stimulation of aldosterone synthesis (28Göppner C. Orozco I.J. Hoegg-Beiler M.B. Soria A.H. Hübner C.A. Fernandes-Rosa F.L. Boulkroun S. Zennaro M.C. Jentsch T.J. Pathogenesis of hypertension in a mouse model for human CLCN2 related hyperaldosteronism.Nat. Commun. 2019; 10: 4678Crossref PubMed Scopus (19) Google Scholar). These Clcn2op/op mice can now be used to explore effects of markedly increased Cl− currents in other cells and tissues. To better understand the mechanisms underlying ClC-2-related disease, we used novel mouse models to genetically identify the cell types that are critically involved in the pathogenesis. Targeted deletion of ClC-2 in Sertoli or retinal pigment epithelial cells revealed that lack of ClC-2 in those cells is responsible for the loss of germ cells and photoreceptors, respectively. Specific Clcn2 disruption in astrocytes or oligodendrocytes produced no or only mild leukodystrophy, respectively, whereas the full extent of Clcn2-related leukodystrophy was reproduced by combined disruption in both cell types. Finally, the leukodystrophy of Glialcam−/− mice could not be rescued by crosses with Clcn2op/op mice, showing that the lack of opening of ClC-2 by GlialCAM plays no significant role in the pathology of Glialcam−/− mice. Our work bolsters the notion that ClC-2 is crucial for extracellular ion homeostasis in various tissues and identifies the cell types responsible for that regulation. Conditional Clcn2lox/lox mice were generated by homologous recombination in embryonic stem cells. Exons 2 and 3 of the mouse Clcn2 gene were flanked with loxP sites (Fig. S1, A and B). Excision of these exons by the Cre recombinase is expected to result in a frameshift and termination of protein synthesis by an early stop codon that occurs before the first transmembrane domain. Western blots of brain lysates revealed that Clcn2lox/lox mice expressed normal amounts of ClC-2 protein (Fig. S1C). When crossed with deleter mice that express the Cre-recombinase in all tissues (29Schwenk F. Baron U. Rajewsky K. A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells.Nucleic Acids Res. 1995; 23: 5080-5081Crossref PubMed Scopus (995) Google Scholar), the brain had lost detectable ClC-2 immunoreactivity just like brain from Clcn2−/− mice (Fig. S1C). After this validation, Clcn2lox/lox mice were used with various cell type–specific Cre lines to identify the cell types causally related to the various degenerative phenotypes of Clcn2−/− mice (14Bösl M.R. Stein V. Hübner C. Zdebik A.A. Jordt S.E. Mukhophadhyay A.K. Davidoff M.S. Holstein A.F. Jentsch T.J. Male germ cells and photoreceptors, both depending on close cell-cell interactions, degenerate upon ClC-2 Cl−-channel disruption.EMBO J. 2001; 20: 1289-1299Crossref PubMed Scopus (260) Google Scholar). Male Clcn2−/− mice are infertile owing to early postnatal testicular degeneration and the ensuing azoospermia (14Bösl M.R. Stein V. Hübner C. Zdebik A.A. Jordt S.E. Mukhophadhyay A.K. Davidoff M.S. Holstein A.F. Jentsch T.J. Male germ cells and photoreceptors, both depending on close cell-cell interactions, degenerate upon ClC-2 Cl−-channel disruption.EMBO J. 2001; 20: 1289-1299Crossref PubMed Scopus (260) Google Scholar). Infertility associated with azoospermia has also been found in a male patient with subclinical CLCN2-related leukodystrophy (23Di Bella D. Pareyson D. Savoiardo M. Farina L. Ciano C. Caldarazzo S. Sagnelli A. Bonato S. Nava S. Bresolin N. Tedeschi G. Taroni F. Salsano E. Subclinical leukodystrophy and infertility in a man with a novel homozygous CLCN2 mutation.Neurology. 2014; 83: 1217-1218Crossref PubMed Scopus (34) Google Scholar). In Clcn2−/− mice, seminiferous tubules fail to develop normal lumina and germ cells do not complete meiosis. Eventually, germ cells of all stages are lost, resulting in a Sertoli cell–only phenotype (14Bösl M.R. Stein V. Hübner C. Zdebik A.A. Jordt S.E. Mukhophadhyay A.K. Davidoff M.S. Holstein A.F. Jentsch T.J. Male germ cells and photoreceptors, both depending on close cell-cell interactions, degenerate upon ClC-2 Cl−-channel disruption.EMBO J. 2001; 20: 1289-1299Crossref PubMed Scopus (260) Google Scholar). Based on the immunohistochemical detection of ClC-2 in Sertoli cells, and their abnormal morphology in Clcn2−/− mice, we hypothesized that the azoospermia was due to a loss of ClC-2 in Sertoli cells that normally provide vital support to germ cells (14Bösl M.R. Stein V. Hübner C. Zdebik A.A. Jordt S.E. Mukhophadhyay A.K. Davidoff M.S. Holstein A.F. Jentsch T.J. Male germ cells and photoreceptors, both depending on close cell-cell interactions, degenerate upon ClC-2 Cl−-channel disruption.EMBO J. 2001; 20: 1289-1299Crossref PubMed Scopus (260) Google Scholar). Considering the nearly ubiquitous expression pattern of ClC-2, other possibilities could not be excluded, however. We now crossed Clcn2lox/lox mice with AMH-Cre mice (30Lécureuil C. Fontaine I. Crepieux P. Guillou F. Sertoli and granulosa cell-specific Cre recombinase activity in transgenic mice.Genesis. 2002; 33: 114-118Crossref PubMed Scopus (176) Google Scholar) to create Sertoli cell–specific Clcn2 KO mice (named SC-ΔC2 mice), and with Stra8-Cre (31Sadate-Ngatchou P.I. Payne C.J. Dearth A.T. Braun R.E. Cre recombinase activity specific to postnatal, premeiotic male germ cells in transgenic mice.Genesis. 2008; 46: 738-742Crossref PubMed Scopus (198) Google Scholar) to generate germ cell–specific GC-ΔC2 KO mice. In Western blot analysis of membrane proteins from whole testes ClC-2 protein levels appeared somewhat more reduced in GC-ΔC2 than in SC-ΔC2 testes (Fig. 1A, Fig. S2A). The apparent discrepancy to immunofluorescence, which detects ClC-2 in Sertoli, but not germ cells (14Bösl M.R. Stein V. Hübner C. Zdebik A.A. Jordt S.E. Mukhophadhyay A.K. Davidoff M.S. Holstein A.F. Jentsch T.J. Male germ cells and photoreceptors, both depending on close cell-cell interactions, degenerate upon ClC-2 Cl−-channel disruption.EMBO J. 2001; 20: 1289-1299Crossref PubMed Scopus (260) Google Scholar) (Fig. 1B), might be explained by the easily detectable, patchy expression of ClC-2 in Sertoli cells. Confirming the Sertoli cell–specific deletion of Clcn2, these ClC-2-positive patches were no longer visible in SC-ΔC2 testis, whereas ClC-2 could still be detected in cells outside the tubules (Fig. 1B). In adult SC-ΔC2 mice, testes and epididymis were significantly smaller than in the WT (Fig. 1C), closely resembling the corresponding phenotype of Clcn2−/− mice (14Bösl M.R. Stein V. Hübner C. Zdebik A.A. Jordt S.E. Mukhophadhyay A.K. Davidoff M.S. Holstein A.F. Jentsch T.J. Male germ cells and photoreceptors, both depending on close cell-cell interactions, degenerate upon ClC-2 Cl−-channel disruption.EMBO J. 2001; 20: 1289-1299Crossref PubMed Scopus (260) Google Scholar). Accordingly, the weight of both tissues was markedly reduced (Fig. 1D). The reduced weight of SC-ΔC2 epididymis might result from the absence of germ cells in adult SC-ΔC2 mice (Fig. 1E). The time course of testicular degeneration was examined by hematoxylin and eosin staining (Fig. 1E). Similar to Clcn2−/− mice, testicular degeneration of SC-ΔC2 mice started at an age of 2 weeks: cells of the tubular lumen were disorganized, with many cells found in the center of the seminiferous tubule rather than being close to its inner walls as in control Clcn2lox/lox mice. In 3-week-old SC-ΔC2 mice, clusters of degenerating cells could be observed in some tubules, which were, however, mainly filled with Sertoli cells. Such clusters of degenerating cells had almost disappeared in SC-ΔC2 mice at 4 weeks of age, a time point at which the tubules of control mice were filled with germ cells of different developmental stages. Eventually, adult SC-ΔC2 mice displayed a Sertoli cell–only syndrome. They were unable to produce offspring during their entire lifespan. By contrast, GC-ΔC2 mice showed no signs of testicular degeneration (Fig. 1E) and were fertile. Hence, ClC-2 appears to be dispensable in germ cells but is essential for the role of Sertoli cells in maintaining normal spermatogenesis. Clcn2−/− mice are blind because of an early loss of photoreceptors, which is already visible at postnatal day 14 (P14) (14Bösl M.R. Stein V. Hübner C. Zdebik A.A. Jordt S.E. Mukhophadhyay A.K. Davidoff M.S. Holstein A.F. Jentsch T.J. Male germ cells and photoreceptors, both depending on close cell-cell interactions, degenerate upon ClC-2 Cl−-channel disruption.EMBO J. 2001; 20: 1289-1299Crossref PubMed Scopus (260) Google Scholar, 18Edwards M.M. Marin de Evsikova C. Collin G.B. Gifford E. Wu J. Hicks W.L. Whiting C. Varvel N.H. Maphis N. Lamb B.T. Naggert J.K. Nishina P.M. Peachey N.S. Photoreceptor degeneration, azoospermia, leukoencephalopathy, and abnormal RPE cell function in mice expressing an early stop mutation in CLCN2.Invest. Ophthalmol. Vis. Sci. 2010; 51: 3264-3272Crossref PubMed Scopus (24) Google Scholar). Although Clcn2 is expressed both in the retinal pigment epithelium (RPE) and in all layers of the neuronal retina, we speculated that photoreceptors degenerate because of a lack of support from the RPE (14Bösl M.R. Stein V. Hübner C. Zdebik A.A. Jordt S.E. Mukhophadhyay A.K. Davidoff M.S. Holstein A.F. Jentsch T.J. Male germ cells and photoreceptors, both depending on close cell-cell interactions, degenerate upon ClC-2 Cl−-channel disruption.EMBO J. 2001; 20: 1289-1299Crossref PubMed Scopus (260) Google Scholar). To test this hypothesis, we now crossed Clcn2lox/lox mice with Trp1-Cre mice (32Mori M. Metzger D. Garnier J.M. Chambon P. Mark M. Site-specific somatic mutagenesis in the retinal pigment epithelium.Invest. Ophthalmol. Vis. Sci. 2002; 43: 1384-1388PubMed Google Scholar) to generate RPE-ΔC2 mice that lack ClC-2 specifically in the RPE. These mice displayed retinal degeneration that closely resembled that of Clcn2−/− mice (Fig. 2). In 2-week-old mice, the outer nuclear layer and the photoreceptor layer (Fig. 2) appeared disorganized in both RPE-ΔC2 and Clcn2−/− mice. At 4 weeks of age, all retinal layers were thinner in either mouse model. However, at this age photoreceptors of Clcn2−/− mice were completely lost, whereas residual parts of the photoreceptor layer could still be detected in RPE-ΔC2 mice. This difference might be due to a mosaic-like expression pattern of the Cre-recombinase in Trp1-Cre mice (33Thanos A. Morizane Y. Murakami Y. Giani A. Mantopoulos D. Kayama M. Roh M.I. Michaud N. Pawlyk B. Sandberg M. Young L.H. Miller J.W. Vavvas D.G. Evidence for baseline retinal pigment epithelium pathology in the Trp1-Cre mouse.Am. J. Pathol. 2012; 180: 1917-1927Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), which presumably spared some RPE cells from Clcn2 ablation. In adult mice, the photoreceptor layer was almost completely lost in either mouse model. These results buttress the hypothesis (14Bösl M.R. Stein V. Hübner C. Zdebik A.A. Jordt S.E. Mukhophadhyay A.K. Davidoff M.S. Holstein A.F. Jentsch T.J. Male germ cells and photoreceptors, both depending on close cell-cell interactions, degenerate upon ClC-2 Cl−-channel disruption.EMBO J. 2001; 20: 1289-1299Crossref PubMed Scopus (260) Google Scholar) that the loss of photoreceptors in Clcn2−/− mice is secondary to a malfunction of nurturing RPE cells. Loss of ClC-2 leads to leukodystrophy in both mice (15Blanz J. Schweizer M. Auberson M. Maier H. Muenscher A. Hübner C.A. Jentsch T.J. Leukoencephalopathy upon disrupti
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