Michaela Schweizer

Loss of the ClC-7 Chloride Channel Leads to Osteopetrosis in Mice and Man

Chloride channels play important roles in the plasma membrane and in intracellular organelles. Mice deficient for the ubiquitously expressed ClC-7 Cl(-) channel show severe osteopetrosis and retinal degeneration. Although osteoclasts are present in normal numbers, they fail to resorb bone because they cannot acidify the extracellular resorption lacuna. ClC-7 resides in late endosomal and lysosomal compartments. In osteoclasts, it is highly expressed in the ruffled membrane, formed by the fusion of H(+)-ATPase-containing vesicles, that secretes protons into the lacuna. We also identified CLCN7 mutations in a patient with human infantile malignant osteopetrosis. We conclude that ClC-7 provides the chloride conductance required for an efficient proton pumping by the H(+)-ATPase of the osteoclast ruffled membrane.

Disruption of ClC-3, a Chloride Channel Expressed on Synaptic Vesicles, Leads to a Loss of the Hippocampus

Several plasma membrane chloride channels are well characterized, but much less is known about the molecular identity and function of intracellular Cl- channels. ClC-3 is thought to mediate swelling-activated plasma membrane currents, but we now show that this broadly expressed chloride channel is present in endosomal compartments and synaptic vesicles of neurons. While swelling-activated currents are unchanged in mice with disrupted ClC-3, acidification of synaptic vesicles is impaired and there is severe postnatal degeneration of the retina and the hippocampus. Electrophysiological analysis of juvenile hippocampal slices revealed no major functional abnormalities despite slightly increased amplitudes of miniature excitatory postsynaptic currents. Mice almost lacking the hippocampus survive and show several behavioral abnormalities but are still able to acquire motor skills.

Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration

ClC‐7 is a chloride channel of late endosomes and lysosomes. In osteoclasts, it may cooperate with H+‐ATPases in acidifying the resorption lacuna. In mice and man, loss of ClC‐7 or the H+‐ATPase a3 subunit causes osteopetrosis, a disease characterized by defective bone resorption. We show that ClC‐7 knockout mice additionally display neurodegeneration and severe lysosomal storage disease despite unchanged lysosomal pH in cultured neurons. Rescuing their bone phenotype by transgenic expression of ClC‐7 in osteoclasts moderately increased their lifespan and revealed a further progression of the central nervous system pathology. Histological analysis demonstrated an accumulation of electron‐dense material in neurons, autofluorescent structures, microglial activation and astrogliosis. Like in human neuronal ceroid lipofuscinosis, there was a strong accumulation of subunit c of the mitochondrial ATP synthase and increased amounts of lysosomal enzymes. Such alterations were minor or absent in ClC‐3 knockout mice, despite a massive neurodegeneration. Osteopetrotic oc/oc mice, lacking a functional H+‐ATPase a3 subunit, showed no comparable retinal or neuronal degeneration. There are important medical implications as defects in the H+‐ATPase and ClC‐7 can underlie human osteopetrosis.

Age-Dependent Neurodegeneration and Alzheimer-Amyloid Plaque Formation in Transgenic Drosophila

β-Amyloid peptides that are cleaved from the amyloid precursor protein (APP) play a critical role in Alzheimers disease (AD) pathophysiology. Here, we show that in Drosophila, the targeted expression of the key genes of AD, APP, theβ-site APP-cleaving enzyme BACE, and the presenilins led to the generation of β-amyloid plaques and age-dependent neurodegeneration as well as to semilethality, a shortened life span, and defects in wing vein development. Genetic manipulations or pharmacological treatments with secretase inhibitors influenced the activity of the APP-processing proteases and modulated the severity of the phenotypes. This invertebrate model of amyloid plaque pathology demonstrates Aβ-induced neurodegeneration as a basic biological principle and may allow additional genetic analyses of the underlying molecular pathways.

Loss of K-Cl co-transporter KCC3 causes deafness, neurodegeneration and reduced seizure threshold

K‐Cl co‐transporters are encoded by four homologous genes and may have roles in transepithelial transport and in the regulation of cell volume and cytoplasmic chloride. KCC3, an isoform mutated in the human Anderman syndrome, is expressed in brain, epithelia and other tissues. To investigate the physiological functions of KCC3, we disrupted its gene in mice. This severely impaired cell volume regulation as assessed in renal tubules and neurons, and moderately raised intraneuronal Cl− concentration. Kcc3−/− mice showed severe motor abnormalities correlating with a progressive neurodegeneration in the peripheral and CNS. Although no spontaneous seizures were observed, Kcc3−/− mice displayed reduced seizure threshold and spike‐wave complexes on electrocorticograms. These resembled EEG abnormalities in patients with Anderman syndrome. Kcc3−/− mice also displayed arterial hypertension and a slowly progressive deafness. KCC3 was expressed in many, but not all cells of the inner ear K+ recycling pathway. These cells slowly degenerated, as did sensory hair cells. The present mouse model has revealed important cellular and systemic functions of KCC3 and is highly relevant for Anderman syndrome.

Oligomerization of KCC2 correlates with development of inhibitory neurotransmission

The neuron-specific K+–Cl− cotransporter KCC2 extrudes Cl− and renders GABA and glycine action hyperpolarizing. Thus, it plays a pivotal role in neuronal inhibition. Development-dependent KCC2 activation is regulated at the transcriptional level and by unknown posttranslational mechanisms. Here, we analyzed KCC2 activation at the protein level in the developing rat lateral superior olive (LSO), a prominent auditory brainstem structure. Electrophysiology demonstrated ineffective KCC2-mediated Cl− extrusion in LSO neurons at postnatal day 3 (P3). Immunohistochemical analyses by confocal and electron microscopy revealed KCC2 signals at the plasma membrane in the somata and dendrites of both immature and mature neurons. Biochemical analysis demonstrated mature glycosylation pattern of KCC2 at both stages. Immunoblot analysis of the immature brainstem demonstrated mainly monomeric KCC2. In contrast, three KCC2 oligomers with molecular masses of ∼270, ∼400, and ∼500 kDa were identified in the mature brainstem. These oligomers were sensitive to sulfhydryl-reducing agents and resistant to SDS, contrary to the situation seen in the related Na+–(K+)–Cl− cotransporter. In HEK-293 cells, coexpressed hemagglutinin-tagged KCC2 assembled with histidine-tagged KCC2, demonstrating formation of homomers. Based on these findings, we conclude that the oligomers represent KCC2 dimers, trimers, and tetramers. Finally, immunoblot analysis identified a development-dependent increase in the oligomer/monomer ratio from embryonic day 18 to P30 throughout the brain that correlates with KCC2 activation. Together, our data indicate that the developmental shift from depolarization to hyperpolarization can be determined by both increased gene expression and KCC2 oligomerization.

Mice with altered KCNQ4 K + channels implicate sensory outer hair cells in human progressive deafness

KCNQ4 is an M‐type K+ channel expressed in sensory hair cells of the inner ear and in the central auditory pathway. KCNQ4 mutations underlie human DFNA2 dominant progressive hearing loss. We now generated mice in which the KCNQ4 gene was disrupted or carried a dominant negative DFNA2 mutation. Although KCNQ4 is strongly expressed in vestibular hair cells, vestibular function appeared normal. Auditory function was only slightly impaired initially. It then declined over several weeks in Kcnq4−/− mice and over several months in mice carrying the dominant negative allele. This progressive hearing loss was paralleled by a selective degeneration of outer hair cells (OHCs). KCNQ4 disruption abolished the IK,n current of OHCs. The ensuing depolarization of OHCs impaired sound amplification. Inner hair cells and their afferent synapses remained mostly intact. These cells were only slightly depolarized and showed near‐normal presynaptic function. We conclude that the hearing loss in DFNA2 is predominantly caused by a slow degeneration of OHCs resulting from chronic depolarization.

Conditional Ablation of the Neural Cell Adhesion Molecule Reduces Precision of Spatial Learning, Long-Term Potentiation, and Depression in the CA1 Subfield of Mouse Hippocampus

NCAM, a neural cell adhesion molecule of the immunoglobulin superfamily, is involved in neuronal migration and differentiation, axon outgrowth and fasciculation, and synaptic plasticity. To dissociate the functional roles of NCAM in the adult brain from developmental abnormalities, we generated a mutant in which the NCAM gene is inactivated by cre-recombinase under the control of the calcium-calmodulin-dependent kinase II promoter, resulting in reduction of NCAM expression predominantly in the hippocampus. This mutant (NCAMff+) did not show the overt morphological and behavioral abnormalities previously observed in constitutive NCAM-deficient (NCAM-/-) mice. However, similar to the NCAM-/- mouse, a reduction in long-term potentiation (LTP) in the CA1 region of the hippocampus was revealed. Long-term depression was also abolished in NCAMff+ mice. The deficit in LTP could be rescued by elevation of extracellular Ca2+ concentrations from 1.5 or 2.0 to 2.5 mm, suggesting an involvement of NCAM in regulation of Ca2+-dependent signaling during LTP. Contrary to the NCAM-/- mouse, LTP in the CA3 region was normal, consistent with normal mossy fiber lamination in NCAMff+ as opposed to abnormal lamination in NCAM-/- mice. NCAMff+ mutants did not show general deficits in short- and long-term memory in global landmark navigation in the water maze but were delayed in the acquisition of precise spatial orientation, a deficit that could be overcome by training. Thus, mice conditionally deficient in hippocampal NCAM expression in the adult share certain abnormalities characteristic of NCAM-/- mice, highlighting the role of NCAM in the regulation of synaptic plasticity in the CA1 region.

Lysosomal storage disease upon disruption of the neuronal chloride transport protein ClC-6

Mammalian CLC proteins function as Cl− channels or as electrogenic Cl−/H+ exchangers and are present in the plasma membrane and intracellular vesicles. We now show that the ClC-6 protein is almost exclusively expressed in neurons of the central and peripheral nervous systems, with a particularly high expression in dorsal root ganglia. ClC-6 colocalized with markers for late endosomes in neuronal cell bodies. The disruption of ClC-6 in mice reduced their pain sensitivity and caused moderate behavioral abnormalities. Neuronal tissues showed autofluorescence at initial axon segments. At these sites, electron microscopy revealed electron-dense storage material that caused a pathological enlargement of proximal axons. These deposits were positive for several lysosomal proteins and other marker proteins typical for neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. However, the lysosomal pH of Clcn6−/− neurons appeared normal. CLCN6 is a candidate gene for mild forms of human NCL. Analysis of 75 NCL patients identified ClC-6 amino acid exchanges in two patients but failed to prove a causative role of CLCN6 in that disease.

Lysosomal Pathology and Osteopetrosis upon Loss of H+-Driven Lysosomal Cl– Accumulation

Chloride Balancing Act The ionic composition of the cytosol and intracellular organelles must be regulated in the face of ongoing membrane traffic into and out of the cell. Now, two papers address the consequences of a change in the transport phenotype of an intracellular Cl− transport protein from a coupled exchanger to a passive Cl− conductor (see the Perspective by Smith and Schwappach). Novarino et al. (p. 1398, published online 29 April) investigated the consequence of a knock-in of the uncoupled ClC-5 transporter into mouse. The knock-out mouse of this endosomal kidney transporter has a severe endocytic phenotype believed to be due to a defect in vesicular acidification. The current study shows a similarly impaired endocytic phenotype for the uncoupled mutant, but the acidification of endosomes was unaffected. Weinert et al. (p. 1401, published online 29 April) used a similar strategy to investigate the consequence of the equivalent mutation in the lysosomal transporter ClC-7, which is highly expressed in the resorption lacuna of osteoclasts and whose knock-out in mice produces lysosomal storage disease and severe osteopetrosis. A similar (though less severe) phenotype was observed in the knock-in mice containing the uncoupled ClC-7, indicating that coupled transport plays a critical role in lysosomes. Chloride conductance and chloride-proton exchange have distinct effects on endolysosomal physiology in mice. During lysosomal acidification, proton-pump currents are thought to be shunted by a chloride ion (Cl–) channel, tentatively identified as ClC-7. Surprisingly, recent data suggest that ClC-7 instead mediates Cl–/proton (H+) exchange. We generated mice carrying a point mutation converting ClC-7 into an uncoupled (unc) Cl– conductor. Despite maintaining lysosomal conductance and normal lysosomal pH, these Clcn7unc/unc mice showed lysosomal storage disease like mice lacking ClC-7. However, their osteopetrosis was milder, and they lacked a coat color phenotype. Thus, only some roles of ClC-7 Cl–/H+ exchange can be taken over by a Cl– conductance. This conductance was even deleterious in Clcn7+/unc mice. Clcn7–/– and Clcn7unc/unc mice accumulated less Cl– in lysosomes than did wild-type mice. Thus, lowered lysosomal chloride may underlie their common phenotypes.