Communicated by: Shoichiro Tsukita
CLC-3 deficiency leads to phenotypes similar to human neuronal ceroid lipofuscinosis
Article first published online: 6 JUN 2002
Genes to Cells
Volume 7, Issue 6, pages 597–605, June 2002
How to Cite
Yoshikawa, M., Uchida, S., Ezaki, J., Rai, T., Hayama, A., Kobayashi, K., Kida, Y., Noda, M., Koike, M., Uchiyama, Y., Marumo, F., Kominami, E. and Sasaki, S. (2002), CLC-3 deficiency leads to phenotypes similar to human neuronal ceroid lipofuscinosis. Genes to Cells, 7: 597–605. doi: 10.1046/j.1365-2443.2002.00539.x
- Issue published online: 6 JUN 2002
- Article first published online: 6 JUN 2002
- Received: 20 February 2002 Accepted: 20 March 2002
Background: CLC-3 is a member of the CLC chloride channel family and is widely expressed in mammalian tissues. To determine the physiological role of CLC-3, we generated CLC-3-deficient mice (Clcn3–/–) by targeted gene disruption.
Results: Together with developmental retardation and higher mortality, the Clcn3–/– mice showed neurological manifestations such as blindness, motor coordination deficit, and spontaneous hyperlocomotion. In histological analysis, the Clcn3–/– mice showed a pattern of progressive degeneration of the retina, hippocampus and ileal mucosa, which resembled the phenotype observed in cathepsin D knockout mice. The defect of cathepsin D results in a lysosomal accumulation of ceroid lipofuscin containing the mitochondrial F1F0 ATPase subunit c. In immunohistochemistry and Western blot analysis, we found that the subunit c was heavily accumulated in the lysosome of Clcn3–/– mice. Furthermore, we detected an elevation in the endosomal pH of the Clcn3–/– mice.
Conclusions: These results indicated that the neurodegeneration observed in the Clcn3–/– mice was caused by an abnormality in the machinery which degrades the cellular protein and was associated with the phenotype of neuronal ceroid lipofuscinosis (NCL). The elevated endosomal pH could be an important factor in the pathogenesis of NCL.
In mammals, the CLC chloride channel family consists of nine known members. The detection of inherited diseases that results from mutations in these genes and analyses of knockout mouse models are beginning to highlight the critical importance of the CLCs in biological processes. Mutations in the CLCN1 gene lead to myotonia congenita, a disease characterized by disturbed muscle relaxation (Koch et al. 1992). Mutations in the CLCNKB gene cause Bartter’s syndrome, a disease of renal salt wasting (Simon et al. 1997). Disruption of the Clcnk1 gene in mice (the homologue of human CLCNKA) leads to nephrogenic diabetes insipidus, a disease caused by impairment of the countercurrent system (Matsumura et al. 1999). These phenotypes suggest that these chloride channels reside in the plasma membrane and mediate transepithelial chloride transport. On the other hand, mutations of CLC-5 lead to Dent’s disease, a disorder characterized by the urinary loss of low-molecular-weight proteins, phosphate and calcium, and often lead to kidney stones (Günther et al. 1998). CLC-5 has been identified as an intracellular chloride channel co-localized with H+-ATPase in endosomes of the proximal tubules in the kidney (Günther et al. 1998; Sakamoto et al. 1999) and it has been shown to play a role in endocytosis (Piwon et al. 2000). While these findings suggest that CLC-5 provides a chloride shunt pathway for the efficient acidification of endosomes, this has not been confirmed by direct evidence. Considering the structural similarity between CLC-3 and CLC-5, the former may play a similar role to the latter in other tissues and cells. Recently, Stobrawa and colleagues reported that the disruption of Clcn3 leads to a severe postnatal degeneration of the retina and hippocampus (Stobrawa et al. 2001). However, it is still unclear how the disruption of Clcn3 leads to neurodegeneration.
To gain more insight into this pathogenesis, we generated and analysed the Clcn3–/– mice. We found that the neuronal abnormalities in the Clcn3–/– mice closely matched those in cathepsin D knockout mice (Saftig et al. 1995; Koike et al. 2000) and met the criteria for neuronal ceroid lipofuscinosis (NCL).
Targeted disruption of the mouse Clcn3 gene
We disrupted the Clcn3 locus in mice by homologous recombination using a replacement vector with positive (neo) and negative (TK) selection markers (Fig. 1A). This deleted exons 5 and 6 encoding transmembrane segments D2–D4 of the CLC-3 protein, an important region for ion permeation (Fahlke et al. 1997). Two recombinant ES cells were isolated and confirmed to be Clcn3+/–. These two clones generated five chimeras, four of which transmitted the mutant allele to their offspring. We subsequently intercrossed these heterozygous mice and yielded wild-type, heterozygous and homozygous progeny as determined by Southern blot analysis (Fig. 1B).
CLC-3 protein analysis
The absence of the CLC-3 protein was verified by Western blot analysis of protein fractions from brain tissues. We used a commercially available anti-CLC-3 antibody, which was recently shown not to cross-react with the closely related CLC-4 and CLC-5 proteins (Schmieder et al. 2001). In our Western blot analysis, this antibody recognized a ≈ 110 kDa smeary band in the synaptosomes (P2 in Fig. 1C) and in the synaptic vesicles (P3 in Fig. 1C) of the Clcn3+/+ mice, but no such band was found in the Clcn3–/– mice. These results proved the absence of the CLC-3 protein in the Clcn3–/– mice.
Skeletal abnormality, developmental retardation and higher mortality
The Clcn3–/– mice were smaller than their normal littermates soon after birth. At 6 weeks the average body weight of Clcn3−/– male mice was 18.1 ± 1.1 g (n = 7), or ≈ 25% lower than that of their normal littermates (24.0 ± 0.9 g, n= 9). In an evaluation of their skeletal system by X-ray, they showed kyphoscoliosis (data not shown). The Clcn3–/– mice also showed a higher mortality during the 3–4 weeks after weaning (33/116 in Clcn3–/– and 1/198 in Clcn3+/+). The mice that died were especially small, and necrosis of the whole small intestine was observed in all of their autopsies (Fig. 2A).
Since CLC-3 was abundantly expressed in almost all regions in the brain, we performed a systemic analysis of gross neurological functions. The visible version of the water maze test suggested that the Clcn3−/– mice were blind. While their muscle power was equal to that of weight-matched wild-type mice, we observed a performance deficit on the hanging wire test (Sango et al. 1996) (57.7 ± 2.0 s in Clcn3+/+, n = 12; 28.4 ± 7.4 s, Clcn3–/–, n= 10; P < 0.01, anova). In the rotarod test, the Clcn3–/– mice performed significantly worse than their littermate controls (29.5 ± 3.5 s in Clcn3+/+, n = 12; 7.2 ± 1.6 s in Clcn3−/–, n = 10, P < 0.001, anova). We analysed the locomotor behaviour of the Clcn3−/– mice in an open field and found that they spontaneously entered the hyperlocomotion phase at night (data not shown).
Selective degeneration of ileal mucosa, retina and hippocampus
We compared the gross morphology and histology of major organs between Clcn3+/+ and Clcn3–/– mice at two months of age. Sagittal sections of brain revealed that the lateral ventricular system was enlarged to fill the void left by the atrophic hippocampus. At higher magnifications, we found a reduction in the density of pyramidal cells in the hippocampus in the Clcn3–/– mice (Fig. 2B). The Ileal mucosa was focally lost, even in the Clcn3–/– mice (Fig. 2B) that were not dying. In the retina, the outer and inner segments and the outer nuclear layer were completely absent (Fig. 2B), indicating photoreceptor degeneration.
Lysosomal accumulation of subunit c of mitochondrial F1F0ATPase
During our morphological examination of the Clcn3–/– mice, we noticed that our findings resembled the phenotype observed in cathepsin D knockout mice (Saftig et al. 1995; Koike et al. 2000). Recently, cathepsin D knockout mice were shown to have the characteristics of NCL (Koike et al. 2000), which showed massive lysosomal storage with autofluorescent lipopigment. To test whether the neuronal degeneration in the Clcn3–/– mice resulted from a similar mechanism, we examined the morphological, immunohistochemical and biochemical features of the Clcn3–/– mice. Electron microscopy showed abundant lysosome-like structures in the perikarya of pyramidal neurones in the hippocampus (Fig. 3a). These structures resembled lipofuscin granules and exhibited a similar irregular profile and heterogeneous electron-density. In the astrocytes, we observed autolysosome- or autophagosome-like bodies encircled by a single layer of a limiting membrane and containing numerous membranous structures (Fig. 3b). In many types of NCLs and cathepsin D knockout mice, subunit c of mitochondrial F1F0 ATPase is stored in lysosomes. An immunohistochemical analysis performed on Clcn3−/– mice at the age of 6 weeks, just before the hippocampus was completely degenerated, showed a clear positive staining of subunit c in neuronal cells in the olfactory bulb, hippocampus, cerebellum and cerebral cortex (Fig. 4), i.e. in all of the organs where CLC-3 mRNA was abundantly expressed (Kawasaki et al. 1994).
Next, we biochemically examined the lysosomal localization of stored subunit c in brains and livers. Figure 5 summarizes the reactivities of the antibodies against various mitochondrial proteins (subunit c, β of ATP synthase, and subunit IV of cytochrome oxidase) and lysosomal proteinases (cathepsin D and TPP-I). A significant amount of subunit c was detected in the lysosomal fractions in the Clcn3–/– mice, whereas subunit c was found almost exclusively in the mitochondrial fractions in the Clcn3+/+ mice. At the age of 27 weeks, the lysosomal accumulation of subunit c was slightly increased in the liver, but somewhat decreased in the brain, reflecting a loss of damaged cells. The β subunit of ATP synthase and cytochrome oxidase subunit IV were only detected in mitochondrial fractions of Clcn3–/– and Clcn3+/+ mice. Finally, we examined whether the levels of TPP-I and cathepsin D, two lysosomal proteases which are known to be involved in the lysosomal degradation of subunit c (Ezaki et al. 1999; Koike et al. 2000), are processed into the mature active forms in Clcn3–/– mice. As shown in Fig. 5, the mature form of TPP-I (46 kDa) and the single-chain mature form of cathepsin D were observed in lysosomal fractions of the brain and liver of Clcn3–/– mice, whereas the TPP-I precursor form (67 kDa) and procathepsin D (53 kDa) were undetectable. These data suggested that the precursor forms of TPP-I and cathepsin D were effectively processed into the enzymatically active mature forms in the brains and livers of both the Clcn3–/– mice and control mice.
Impaired acidification of endosomal compartments
To gain more insight into the pathogenesis of NCL in the Clcn3–/– mice, we measured the intraluminal pH in the CLC-3-containing intracellular vesicles in the liver, since the NCL phenotype was observed in the liver (Fig. 5) and intravesicular pH measurement in the brain cells was not successful. In a Western blot analysis, CLC-3 was found to be concentrated in the P3 fraction (100 000 g pellet) containing endosomes, and not in the P2 fraction (10 000 g pellet) containing lysosomes and mitochondria (data not shown). We prepared a FITC-dextran-loaded P3 fraction from the wild-type and Clcn3–/– mice, and compared their steady-state intravesicular pH values in the presence of ATP. The mean steady-state pH in the vesicles was 6.37 ± 0.04 for the Clcn3+/+ mice (n = 4) and 6.76 ± 0.09 for the Clcn3–/– mice (n = 3). The difference between groups was statistically significant (P < 0.01, unpaired t-test), confirming that CLC-3 is involved in the regulation of intravesicular pH homeostasis.
While we were preparing this manuscript, Stobrawa et al. published a separate report on the generation of Clcn3–/– mice (Stobrawa et al. 2001). Growth defects, higher mortality and hyperlocomotion were common findings. They also demonstrated a postnatal degeneration of the retina and hippocampus, but they did not mention a degeneration of the ileal mucosa and they did not describe the pathogenesis of neuronal degeneration. A morphological examination of the Clcn3–/– mice showed that they resembled cathepsin D knockout mice in terms of retinal atrophy, neuronal degeneration, and focal, sometimes total, atrophy of the ileal mucosa (Saftig et al. 1995; Koike et al. 2000). Recently, Koike et al. reported that cathepsin D knockout mice had characteristics of NCL (Koike et al. 2000).
NCLs are a group of inherited lysosomal storage diseases characterized by progressive blindness, psychomotor retardation and premature death (Goebel 1995; Goebel & Sharp 1998; Dawson & Cho 2000). Pathologically, autofluorescent lipopigment accumulates in lysosome-derived organelles in neurones and cells in other organs. Recent studies have shown that the various forms of NCLs result from mutations in at least eight genes. These include the soluble lysosomal enzymes palmitoyl protein thioesterase 1 (CLN1) (Vesa et al. 1995), TPP-I (CLN2) (Sleat et al. 1997), and cathepsin D (Tyynela et al. 2000), and three membrane proteins of unknown function [CLN3 (International Batten Disease Consortium 1995), CLN5 (Savukoski et al. 1998), and CLN8 (Ranta et al. 1999)]. Based on the substrate specificity of the above-mentioned three lysosomal enzymes and the lysosomal localization of the CLN3 protein (Jarvela et al. 1999), the NCLs seem to result from defects in a specific pathway through which membrane-associated hydrophobic proteins are normally degraded in lysosomes.
To investigate whether the neuronal degeneration in the Clcn3–/– mice had characteristics of NCL, we began a study by electron microscopy. As shown in Fig. 3, we confirmed the presence of lipofuscin-containing lysosomes in neurones and glias in the Clcn3–/– mice, although these deposits were not as conspicuous as those in the cathepsin D knockout mice (Koike et al. 2000). This may be related to the severity of NCL, since cathepsin D knockout mice die at postnatal day 26 ± 1 (Saftig et al. 1995) whereas some Clcn3–/– mice survive for longer. Subunit c of mitochondrial F1F0 ATPase, a very hydrophobic peptide, is known to be specifically accumulated in cells in many forms of NCL except in the infantile form (Hall et al. 1991; Kominami et al. 1992). Accordingly, we performed immunohistochemistry for subunit c in the brain and found that neuronal cell bodies in various regions of the brain in the Clcn3–/– mice were immunopositive (Fig. 4). This is consistent with the additional finding of cerebral atrophy in the aged Clcn3–/– mice, although drastic atrophy was predominant in the hippocampus. Western blotting of proteins after subcellular fractionation confirmed the accumulation of subunit c in the lysosomes in both the brain and liver. While small amounts of subunit c accumulation were found in neuronal cells, even in some types of lysosomal stroage disorders other than NCL, there was no evidence of a significant lysosomal accumulation of subunit c in non-neuronal cells (Elleder et al. 1997). These data strongly suggested that the lysosomal accumulation of subunit c in the Clcn3–/– mice resulted from the dysfunction of a specific degradation pathway rather than a nonspecific one. This notion was also supported by the evidence that other mitochondrial proteins such as subunit β of ATP synthase and subunit IV of cytochrome oxidase were not accumulated in the lysosome in the Clcn3–/– mice (Fig. 5).
How could the CLC-3 deficiency impair the specific cellular degradation pathway? We confirmed that cathepsin D and TPP-I, enzymes with mutations that result in the accumulation of subunit c in lysosomes, were not impaired in maturation or protein abundance in lysosomes (Fig. 5). The molecular mechanism of accumulation of ceroid lipofuscin have not been clearly explained in most NCLs, especially in those whose responsible genes have been identified by reverse genetics (CLN3, CLN5, CLN8), such as those in this study. This study showed that the CLC-3 gene is important for the degradation of cellular proteins. Moreover, we were clearly able to show that the CLC-3 deficiency led to an elevation of pH within endosomes. As a chloride shunt pathway has been established to be necessary for efficient acidification within intracellular organelles, particularly in endosomes (Sonawane et al. 2002), CLC-3 could be a channel responsible for this pathway in endosomes. Elevated lysosomal pH was recently reported in several forms of the NCLs (Holopainen et al. 2001), supporting the idea that the elevated pH in lysosomes may affect the catalytic activity of lysosomal enzymes, thus leading to the accumulation of ceroid-lipofuscin-like materials. However, the pattern of pH elevation in our NCL model, i.e. pH elevation not in the lysosome but in endosomes, could postulate that the elevated intravesicular pH directly influences not only the catalytic activity of lysosomal enzymes in situ, but also other biological processes such as the maturation of endosomes/autophagosomes, the binding of late endosomes to lysosomes, and the enhancement of the translocation or dislocation of proteins across the endosomes/autophagosomes to mature lysosomes. Thus, this study might shed new light on the pathogenesis of the NCLs. Similarly, Lonka et al. recently demonstrated that the CLN8 gene product is a protein not in lysosomes but in the endoplasmic reticulum. They speculated that CLN8 might act as a regulator of intracellular membrane transport (Lonka et al. 2000). At present, there is no unifying hypothesis which can explain the molecular mechanisms leading from defects of CLN proteins and CLC-3 to a relative uniform cellular phenotype. However, this study further supports the idea that pH homeostasis in intracellular organelles other than lysosomes may also be one of the major factors for proper proteolysis machinery.
In summary, we generated Clcn3–/– mice by targeted gene disruption and found that the CLC-3 deficiency in mice led to elevated intraendosomal pH, influenced the cellular protein degradation cascade, and caused phenotypes similar to human NCL.
Clcn3 gene targeting
We isolated two overlapping λ phage clones from a 129/Sv strain mouse genomic library (Stratagene) using mouse Clcn3 cDNA. An approximately 7.2-kb BamHI fragment and a 1.9-kb XbaI fragment were isolated as the long arm and short arm, respectively, and used to construct a targeting vector in pLNTK. To generate the Clcn3−/– mice, we electroporated and selected 129/Sv-derived E14 ES cells according to standard procedures. To screen homologous recombinant ES clones, we used a Southern blot analysis of EcoRI-digested DNA with the probe described in Fig. 1A. The expected sizes of the EcoRI bands for wild-type and mutant alleles are 5.8 kb and 3.7 kb, respectively. ES cells heterozygous for the targeted mutation were injected into C57BL/6 blastocysts to generate chimeras, and the resulting chimeras were then mated with C57BL/6 mice. The germ-line transmission of injected ES cells was confirmed by the inheritance of agouti coat colour in the F1 animals, and all agouti offspring were tested for the presence of the mutated Clcn3 allele by Southern blot analysis.
Brain tissues were immediately frozen in liquid nitrogen and carefully broken down to a fine powder. Next, we suspended the powder in ice-cold 320 mm sucrose and homogenized the suspension with a glass-Teflon homogenizer (8 strokes, 1000 r.p.m). The homogenate was twice centrifuged for 10 min at 47 000 g, and the resulting supernatant (S1) was combined and centrifuged for 40 min at 120 000 g to obtain S2 and P2. The S2 supernatant was further fractionated on a 700 mm sucrose cushion for 2 h at 260 000 g to obtain P3. We performed a Western analysis of each fraction using anti-rat CLC-3 antibody (1 : 200 dilution) (Alomone Laboratories, Jerusalem, Israel) and a Western Blue detection system (Promega).
The hanging wire test (Sango et al. 1996) was performed by placing the mouse on the wire cage lid and turning the lid upside down. The investigator quantified the latency with which the mice fell from the wire lid. In the rotarod test, each mouse was given three consecutive trials at 5 r.p.m. In both tests a 60 s cut-off time was used for the standard test session. Locomotor activity was measured in the Multi-Digital 32-Port Counter System (Neuroscience Inc., Tokyo, Japan).
Whole eyes, brains and internal viscera were fixed in 10% buffered neutral formalin. Routine processing, paraffin embedding and haematoxylin and eosin staining (H&E) were performed.
Electron microscopic analysis
Mice were fixed by cardiac perfusion with 2% paraformaldehyde-2% glutaraldehyde buffered with 0.1 m phosphate buffer (pH 7.2). Brains were cut into small pieces, and further immersed in the same fixative overnight at 4 °C. After washing thoroughly with the same buffer containing 7.5% sucrose, the samples were post-fixed with 1% OsO4 with the same buffer containing 7.5% sucrose at 4 °C for 2 h. The brains were then block-stained with a 2% aqueous solution of uranyl acetate for 1 h, dehydrated with a series of ethanol, and embedded in Epon 812. Silver sections were cut with an ultramicrotome, stained with lead citrate and uranyl acetate, and observed with a Hitachi H-7100 electron microscope.
Mouse brains were perfused with 4% paraformaldehyde/PBS, immersed overnight, and snap-frozen in OCT compound in liquid nitrogen. Cryostat sections were prepared and fixed again in 4% paraformaldehyde/PBS for 15 min at room temperature. After three washes with Tris-buffered saline with 1 mm calcium chloride (TBS-Ca), the sections were immersed in cold methanol for 10 min at −20 °C, and nonspecific binding was blocked in 5% skimmed milk in TBS-Ca for 30 min at room temperature. Anti-F1F0 ATPase subunit c antibody diluted at 1 : 200 in 10% sheep serum in TBS-Ca was applied to each section and incubated overnight. After one wash with TBS-Ca, we applied Cy3-conjugated secondary antibody (Sigma) in 5% skimmed milk in TBS-Ca and incubated for 1 h at room temperature. Immunofluorescence was examined with an LSM-510 laser-scanning microscope (Carl Zeiss). A Vectastain ABC kit (Vector Laboratories, Burlingame, CA) was also used for staining subunit c in the paraffin sections.
Percoll density fractionation of brain and liver
Brains and livers were fractionated in Percoll density gradients into mitochondrial and lysosomal fractions as previously described (Ohshita & Kido 1995; Ohshita & Hiroi 1998). Equal amounts of proteins from control and Clcn3–/– mice were separated on 16.5% Tricine-SDS-PAGE for subunit c of ATP synthase and subunit IV of cytochrome oxidase, or on 12.5% SDS-PAGE for β-subunit of ATP synthase, cathepsin D, and tripeptidylpeptidase I (TPP-I). The gels were analysed by immunoblotting.
Fluorescence pH measurement of liver endosomes
Endosomal pH measurement was performed as previously described (Van Dyke 1993). Briefly, after an intraperitoneal injection of 10 mg 70 000 molecular weight FITC-dextran, the livers were homogenated and centrifuged at 1000 g for 10 min to remove nuclei and unbroken cells. The supernatant (S1) was removed and centrifuged at 10 000 g for 20 min, and then the supernatant (S2) was removed and centrifuged at 100 000 g for 60 min to obtain a microsomal pellet containing FITC-dextran-loaded endosomes (P3). P3 was suspended in a small volume of incubation medium (100 mm KCl/10 mm MgCl2/20 mm HEPES-KOH, pH 7.0/1 mm ATP), and the fluorescence was measured in a Spectrafluor fluorescence spectrophotometer (Wako). The vesicle pH was determined from the ratio of fluorescence intensity at two excitation wavelengths (492 and 450 nm excitation, 535 nm emission). In the range of pH 5.0–7.0, the ratio of fluorescence intensity was linearly related to the pH (data not shown). The addition of Triton X-100 disrupted the vesicles and exposed the incubation medium (pH 5.0 and pH 7.0) to FITC-dextran, thereby providing the calibration line.
This research was supported by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Culture of Japan, and the Salt Science Research Foundation.
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