Human Molecular Genetics Advance Access originally published online on December 12, 2006
Human Molecular Genetics 2007 16(2):115-128; doi:10.1093/hmg/ddl446
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A Gne knockout mouse expressing human V572L mutation develops features similar to distal myopathy with rimmed vacuoles or hereditary inclusion body myopathy
Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi-cho, Kodaira, Tokyo 187-8502, Japan
* To whom correspondence should be addressed. Tel: +81 423461712; Fax: +81 423461742; Email: noguchi{at}ncnp.go.jp
Received October 3, 2006; Revised November 2, 2006; Accepted November 21, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Distal myopathy with rimmed vacuoles (DMRV) or hereditary inclusion myopathy (h-IBM) is an early adult-onset distal myopathy caused by mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene which encodes for a bifunctional enzyme involved in sialic acid biosynthesis. It is pathologically characterized by the presence of rimmed vacuoles especially in atrophic fibers, which also occasionally contain congophilic materials that are immunoreactive to ß-amyloid, lysosomal proteins, ubiquitin and tau proteins. To elucidate the pathomechanism of this myopathy and to explore the treatment options, we generated a mouse model of DMRV/h-IBM. We knocked out the Gne gene in the mouse, but this resulted in embryonic lethality. We therefore generated a transgenic mouse that expressed the human GNEV572L mutation, which is the most prevalent among Japanese DMRV patients, and crossed this with Gne(+/–) mouse to obtain Gne(–/–)hGNEV572L-Tg. Interestingly, these mice exhibit marked hyposialylation in serum, muscle and other organs. Reduction in motor performance in these mice can only be seen from 30 weeks of age. A compelling finding is the development of ß-amyloid deposition in myofibers by 32 weeks, which clearly precedes rimmed vacuole formation at 42 weeks. These results show that the Gne(–/–) hGNEV572L-Tg mouse mimics the clinical, histopathological and biochemical features of DMRV/h-IBM, making it useful for understanding the pathomechanism of this myopathy and for employing different strategies for therapy. Our findings underscore the notion that hyposialylation plays an important role in the pathomechanism of DMRV/h-IBM.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Distal myopathy with rimmed vacuoles (DMRV) is an autosomal recessive myopathy which was originally reported by Nonaka et al., (1) thus it is also known as Nonaka myopathy. It is the same entity with hereditary inclusion body myopathy (h-IBM), which was initially reported among Iranian Jews (2). DMRV/h-IBM usually starts affecting adults from ages 15 to 40 years with an average onset of 26 years and with an initial symptom of altered gait (1). It is gradually progressive, and patients become wheelchair-bound between 26 and 57 years of age, or about an average of 12 years after the onset of symptoms (3).
DMRV/h-IBM is characterized by preferential involvement of the distal muscles of lower extremities especially the tibialis anterior muscles, with relative sparing of the quadriceps, hence the term ‘quadriceps-sparing’ rimmed vacuolar myopathy (2). Other muscles are involved as well, especially late in the course of the disease (3). Serum creatine kinase (CK) level is normal or mildly elevated. The characteristic finding on muscle biopsy is the presence of rimmed vacuoles (RVs), which are actually empty spaces surrounded by aggregation of autophagic vacuoles. These RVs occasionally contain congophilic materials that are immunoreactive to various proteins, including amyloid ß, phosphorylated tau, ubiquitin and 
-synuclein. Necrotic and regenerating fibers and areas of inflammation are uncommon but can be seen. Ultrastructurally, filamentous inclusions measuring 18–20 nm in diameter are seen in both the cytoplasm and nucleus (3), in addition to the presence of autophagy and various inclusions.
DMRV/h-IBM was mapped to chromosome 9 (4,5), and was shown to be associated with mutations in the GNE gene (6,7), which encodes for a bifunctional enzyme that catalyzes the rate-limiting step in sialic acid biosynthesis (8). All patients acquire the disease by autosomal recessive pattern, and have at least one missense mutation in one allele, including the most common mutations V572L and M712T among Japanese and Iranian Jews, respectively. No patient with homozygous null mutation was identified. Genetically confirmed DMRV/h-IBM diseases, initially recognized among Japanese and Iranian Jews (6,9–11), appear to afflict patients with diverse nationalities and cultural backgrounds (12–16).
The mechanism by which mutations in the GNE lead to the phenotype in DMRV/h-IBM has remained unclear. We previously demonstrated that mutations in the GNE led to the reduction in either the UDP-GlcNAc 2-epimerase or ManNAc kinase activity (17); moreover, we have shown that myotubes from DMRV patients are hyposialylated, and this phenomenon can be corrected by the addition of free sialic acid and/or its precursor. Other groups have shown similar results regarding GNE activity, but in contrast, they suggested that only the cells derived from a patient carrying a homozygous epimerase mutation had a significant reduction in the overall membrane-bound sialic acid (18), and that GNE mutations may not contribute to alteration in sialylation in h-IBM myoblasts (19). To address these issues, we developed a mouse model for the disease. In this article, we present the first DMRV/h-IBM mouse model that expressed only the mutated human GNE and show that this mouse evidently displays features of DMRV/h-IBM seen in human patients.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Production of Gne(–/–)hGNEV572L-Tg
In Figure 1, the genomic configuration of the Gne gene (A) and the targeting construct (B) are shown. The inserted Neo cassette replaced the 1.4 kb upstream of exon 3, exon 3 and 1.4 kb downstream of exon 3. Only WT and Gne(+/–) mice were generated; no Gne(–/–) mouse was produced (data not shown), in concurrence with a previous report (20). We then proceeded to generate a transgenic mouse (hGNEV572L-Tg) that expressed the human mutated GNE with V572L, the most common GNE mutation in Japan, the structure of which is shown in Figure 1D. Of the resulting litters, nine mice were found to incorporate the hGNEV572L by PCR analysis of genomic DNA isolated from tail snips, but only four lines were able to generate offspring. Using quantitative RT-PCR, we quantified mRNA expression of hGNEV572L and endogenous Gne in muscle and other organs of these transgenic mice. Transgene expression was highest in the skeletal muscle, followed by heart, kidney, brain, spleen and liver (Fig. 2), while endogenous Gne expression was barely detected. We also determined the copy number using quantitative PCR by comparing the amplification of hGNEV572L with endogenous Gne. We calculated the copy numbers for lines 3, 6, 7 and 9 as 2, 4, 3 and 5, respectively. Consequently, we used this transgenic line 9 for producing the model mouse.
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We crossed the hGNEV572L transgenic mouse with a Gne(+/–) to obtain a Gne(+/–)hGNEV572L-Tg. Further, we crossed this Gne(+/–)hGNEV572L-Tg with a Gne(+/–) to generate our model mouse, a transgenic mouse on a Gne knockout background, Gne(–/–)hGNEV572L-Tg. Analysis of 823 newborn mice from independent heterozygous crosses indicated that the numbers of mice with the five genotypes, Gne(–/–)hGNEV572L-Tg (+), Gne(+/–)hGNEV572L-Tg(+), Gne(+/–)hGNEV572L-Tg(–), Gne(+/+)hGNEV572L-Tg(+) and Gne(+/+)hGNEV572L-Tg(–) were 72 (9%), 225 (28%), 193 (24%), 177 (22%) and 136 (17%), respectively, almost approximating the expected ratio of Mendelian inheritance. Mice of the latter four genotypes did not demonstrate unusual phenotype, and thus were considered as control littermates. Images for routine PCR for checking Gne genotype and the incorporation of the human GNEV572L are shown in Figure 1E, with the corresponding PCR fragments illustrated in A, C and D.
Hyposialylation is evident in the Gne(–/–)hGNEV572L-Tg
Understandably, mutations in the GNE can affect sialylation of glycoconjugates because of the gene's role in sialic acid synthesis. We therefore measured the sialic acid levels in the Gne(–/–)hGNEV572L-Tg mice using HPLC with fluorometric detection. In wild-type (WT) mice, sialic acid levels are highest in the brain, followed by the liver, spleen and kidney (Fig. 3B, open boxes). In both skeletal and cardiac muscles, sialic acid levels are evidently lower than in other tissues. As we have expected, the total sialic acid in the Gne(–/–)hGNEV572L-Tg mice is remarkably lower than WT. This hyposialylation is most remarkable in the serum (Fig. 3A). A significant reduction in total sialic acid level is seen in various tissues examined (Fig. 3B, closed boxes). We also measured sialic acid level in the hGNEV572L-Tg and noted that sialic acid levels are comparable with WT mice (Fig. 3B, gray boxes), although the transgenic expression was extremely higher than endogenous GNE.
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Gne(–/–)hGNEV572L-Tg has lower median of survival than littermate
The Gne(–/–)hGNEV572L-Tg mice were indistinguishable from their littermates at birth and seemed healthy (Fig. 4A and B). After 30 weeks of age, these mice weighed less than their littermates (Fig. 4). Significant difference in weight is more pronounced and earlier in female Gne(–/–)hGNEV572L-Tg (Fig. 4C) mice when compared with male (Fig. 4D). To investigate plausible explanations for this difference in weight, we performed gross inspection of the muscles, and found out that some muscles, especially the gastrocnemius, were atrophic in the Gne(–/–)hGNEV572L-Tg when compared with control (Fig. 4E and F), and this finding was more remarkable among females (Fig. 4E).
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Surprisingly, the median survival rate were lower in the Gne(–/–)hGNEV572L-Tg as seen in Figure 4B. The cause of death could not be ascertained, but upon necropsy, no external gross abnormalities were seen and the internal organs appeared normal. On pathological examination, five out of 12 (41%) mice that died had RVs in the skeletal muscle; among these five mice, only one was before 40 weeks of age. Twenty five percent had fibrosis and a few RVs in the diaphragm. Thirty-three percent had fibrosis in the cardiac muscles.
Gne(–/–)hGNEV572l-Tg shows clinical phenotype
Using a tool for assessing general muscle strength, these mice notably performed worse than their littermates (Fig. 5A). Interestingly, significant change in muscle power is noted after 30 weeks of age. We then proceeded to measure serum CK activity in the mice and found out that CK was significantly elevated in the Gne(–/–)hGNEV572L-Tg mice when compared with their littermates (Fig. 5B), albeit the observation that these values are much lower when compared with muscular dystrophy models like Largemyd and Sgcb(–/–) mice (data not shown). Because the appearance of phenotype seemed to be related to age, we measured CK activity according to different age groups. From Figure 5C, we note that elevation of CK activity starts at 30 weeks of age. Using gel electrophoresis, we verified that CK-MM isozyme was primarily increased (data not shown).
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Gne(–/–)hGNEV572l-Tg shows characteristic pathological features of DMRV
We checked if the Gne(–/–)hGNEV572L-Tg mice showed muscle phenotype not only by the analysis of muscle power but also by evaluating biopsy samples in five different age groups (10, 20, 30, 40 and 50 weeks). Morphometric analysis of the fibers in different age groups showed that the variation in fiber size becomes more marked with age, preferentially affecting the gastrocnemius and quadriceps muscles (data not shown); for this reason, we used the gastrocnemius muscle in further experiments. We found that at young age, they developed neither clinical nor pathological phenotype, as they were comparable with WT. Figure 6A, D and G shows representative sections from the Gne(–/–)hGNEV572L-Tg mice per age group. Histopathological analysis revealed almost normal findings in the muscle sections before 30 weeks of age (data not shown). In general, necrotic and regenerating processes are not observed in the young mice, although a few necrotic fibers are observed as they grow older. No endomysial or perimysial inflammation is seen.
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Scattered small angular fibers are noted by 30 weeks of age (Fig. 6A), which is not seen in the littermates (Fig. 6B and C). The variation in fiber size becomes more noticeable as the mice grow older. In addition, fibers appear atrophic by 40 weeks of age, in support with the observation that the gastrocnemius muscle is relatively atrophic by gross inspection. Remarkably after 40 weeks, RVs are seen in scattered fibers (arrows in Fig. 6D and G). Occasionally, inclusion bodies are found in the fibers with or without RVs (arrowhead in Fig. 7A and B). Like in humans, these RVs are intensely stained with acid phosphatase, giving the impression that autophagic process is activated (Fig. 7C). We confirmed this by checking the expression of lysosomal-associated proteins (LAMPs) 1 and 2, and LC3 in muscle sections, all of which are upregulated. LAMP-1 is predominantly expressed within the vicinity of RVs (Fig. 7E). LAMP-2, on the other hand, noticeably is also expressed in the subsarcolemmal areas aside from its localization in the area of RVs (Fig. 7F). LC3 immunoreactivity is almost similar to LAMP-2, except that the perinuclear region is also highlighted (Fig. 7G).
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Various proteins are expressed in the Gne(–/–)hGNEV572L-Tg muscles
One of the defining hallmark features of DMRV/h-IBM is the presence of inclusion bodies that are presumed to have a role in muscle degeneration. These deposits have been shown to be immunoreactive to several proteins. Similar to human cases of DMRV, muscle cross sections obtained from the Gne(–/–)hGNEV572L-Tg mice reveal positive Congo red staining (Fig. 7D), which is not observed in the myofibers of control mice (data not shown). Intense, demarcated signals are seen within the area of RVs and more frequently co-localizing with inclusion bodies which are often seen in DMRV/h-IBM. As congophilia denotes deposition of proteins assuming a beta-pleated structure, we used the well-characterized 6E10, Aß1–42, Aß1–40 and A11 (amyloid ß-oligomer), and ß-site amyloid precursor protein cleaving enzyme (BACE2) antibodies to check for intracellular accumulation of amyloid. Amyloid depositions occur within the myofibers, and are seen to be occasionally associated with vacuolated fibers, as
62% of RVs are positive for amyloid expression (data not shown). These amyloid inclusions are also noted in non-vacuolated fibers, including those which appear normal. Amyloid ß precursor protein (AßPP), which is recognized by 6E10 antibody (Fig. 7I) has intense, large, fairly demarcated immunoreactive signals within the RVs, similar to the staining pattern of the fibrillar forms of amyloid ß or amyloid ß peptides 1–42 and 1–40 (Fig. 7J and K). In good agreement with finding amyloid deposits in the myofibers, BACE2, which purportedly represents ß-secretase activity, is upregulated in these myofibers and are seen as granular staining in the cytoplasm and intense immunoreactivity at subsarcolemmal areas (Fig. 7H). Interestingly, the oligomer form of amyloid ß, which is recognized by A11, is also expressed in the myofibers; positive signals are seen as aggregates around the RVs which are localized in areas distinct from fibrillar forms of amyloid (Fig. 7L). We then analyzed skeletal muscles of mice from different age groups to see whether these amyloid accumulations are related to or can be considered as a function of age. We found out that these accumulations start to occur from 32 to 34 weeks of age, a period when virtually no RV is seen in the myofibers, and muscle pathology is characterized mainly by mild variation in fiber size (Fig. 8A and C). Both AßPP (Fig. 8B) and amyloid ß 1–42 peptide (Fig. 8D) show positive immunoreactivity within the myofibers.
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The microtubule-associated protein tau, a cytoskeletal protein, has been shown to be abnormally phosphorylated and accumulated in DMRV and other muscle disorders (21–23). Similarly, in these mice, these deposits are evident as squiggly inclusions which are occasionally seen in vacuolated fibers (Fig. 7M).
SM-31, an antibody which detects neurofilaments, has been well-characterized in DMRV/h-IBM (21,24). In muscle sections, positive staining is seen within the vicinity of RVs (Fig. 7N); not all RVs, however, show immunoreactivity with this antibody. SM-310, on the other hand, only stains the intramuscular nerve bundles (Fig. 7O).
Because of the accumulation of several proteins in the myofibers, ER stress and the unfolded protein response (UPR) have been implicated in the pathogenesis of DMRV/h-IBM. Using an antibody which recognizes one of the ER chaperones, we show that the UPR activation occurs in the Gne(–/–)hGNEV572L-Tg mice. Intracellular Grp-94 immunoreactivity is seen exclusively in vacuolated fibers (Fig. 7P). In the myofibers of the mice, strong reactivity to ubiquitin antibody in vacuolated and non-vacuolated fibers are seen (Fig. 7Q), suggesting that the ubiquitin–proteosome system may as well be involved in the degradation of abnormal protein accumulations in the muscle, and that misfolded proteins are ubiquitinated but not degraded.
Sarcolemmal proteins are also accumulated in DMRV/h-IBM myofibers. Within the vicinity of the RVs, positive -dystroglycan (Fig. 7R), ß-dystroglycan (Fig. 7S) and -sarcoglycan (Fig. 7T) signals are observed.
In the myofibers of the control mice, no protein depositions were appreciated (data not shown).
Electron microscopic studies show evidence of autophagy and inclusions in the Gne(–/–)hGNEV572L-Tg muscles
Ultrastructural studies confirm the activation of autophagy in Gne(–/–)hGNEV572L-Tg muscles (Fig. 9). We obtained samples from a 42-week-old female mouse which had RVs as seen in light microscopy. In these samples, disorganization of myofibrils was seen in the vicinity of RVs. In about 500 myofibers examined, 10% showed ultrastructural evidence of autophagy. Collections of lysosomal autophagosomes containing undigested intracellular debris were seen, usually enclosed by a limiting membrane (Fig. 9A, arrow). The debris are often composed of light or electron-dense amorphous materials, and appeared like myelin whorls. Multiple small double membrane-bound autophagic vacuoles were often contained within a larger autophagic vesicle (AV), suggesting that autophagy in these myofibers involves a continual process of AV consolidation (Fig. 9A, arrowhead). Multilamellar bodies are also observed (Fig. 9A, double arrows). Probable amyloid deposits are seen as amorphous and granular material (Fig. 9B, magnified from A). Interestingly, ovoid and densely granular deposits, which may also be amyloid-like structures, are noted not only in the areas of autophagy (Fig. 9A, asterisk), but also in areas where myofibrillar architecture is well preserved (Fig. 9C). Occasionally, autophagic vacuoles are seen within the substance of these deposits (Fig. 9C, arrow).
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Gne(–/–)hGNEV572L-Tg shows pathological changes in the diaphragm and cardiac muscles
It has been a well-accepted fact that DMRV/h-IBM primarily involved the skeletal muscles, and that respiratory muscles are assumed to be spared as there had been no reports implying the involvement of the respiratory system. Interestingly, in the Gne(–/–)hGNEV572L-Tg mice, we found that even diaphragm muscles are involved, although the findings range from almost normal findings to the presence of marked fibrosis and RVs in the myofibers (Fig. 10A). Likewise, we have observed inclusion bodies which are seen in both vacuolated and non-vacuolated fibers (data not shown).
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It is now being recognized that some patients manifest with a variety of cardiac abnormalities, from the seemingly benign right bundle branch block to fatal arrhythmias. This led us to carefully check the status of cardiac muscles in the mice. We found out that few mice (around 20%) develop fibrosis in the cardiac tissue after the age of 30 weeks, and some show marked endomysial fibrosis (Fig. 10B). Moreover, amyloid deposition (Fig. 10C) and, occasionally, RVs (Fig. 10D) are also observed in cardiomyocytes. We also tried to functionally evaluate the heart using 2D echocardiography and electrocardiogram, but we did not observe any abnormality pointing to definite cardiomyopathy or conduction defects (data not shown), although we only tested a limited number of mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Sialylation of oligosaccharide chains is a common and physiologically important event, and sialic acids are probably the most biologically important monosaccharide units of glycoconjugates. These negatively charged sugars at the terminal ends of glycoconjugates have very important biological roles in mammalian development, and this is underscored by the embryonic lethality resulting from attempts to knock-out Gne in the mice (20), and further supported by the absence of homozygous null mutations in humans. Making a transgenic GNE mouse on a Gne knockout background thus allowed us to rescue the phenotype in Gne knockout. Clearly, the Gne(–/–)hGNEV572L-Tg resembles the phenotype in human DMRV/h-IBM patients.
It is conceivable that a mutation in the GNE, a gene responsible for catalyzing the rate-limiting step in sialic acid biosynthesis, can lead to hyposialylation. Most, if not all, of the mutations causing DMRV caused partial reduction of the enzymatic activity of either UDP-GlcNAc 2-epimerase or ManNAc kinase of the GNE (17,19). As we have predicted, our results show that there is a marked reduction in sialic acid level, which can reflect the enzymatic activity of GNE, in the serum and other tissues of the Gne(–/–)hGNEV572L-Tg mice. With regards to the expression of GNE in various tissues, it has been shown that expression in the muscle is very low (25). Our results show, on the other hand, that mRNA expression of hGNEV572L-Tg is highest in the muscle, and we attribute this to the promoter that we used in the transgene construct. Previously, we have shown that CAG promoter efficiently promotes expression of a gene into adult skeletal muscles (26).
Skeletal muscle is mainly affected in DMRV/h-IBM, although it is reasonable to expect multi-organ involvement because of the ubiquitous expression of GNE. In our mice, the skeletal muscle is clearly affected despite the data that hyposialylation is not that remarkable when compared with other organs. Our results suggest that even a slight reduction in sialic acid level can cause symptoms in skeletal muscles; however, the selectivity of skeletal muscle may not be explained by the Gne expression levels and sialic acid levels in each organ.
It is notable that some of the Gne(–/–)hGNEV572L-Tg mice die sooner than their littermates, but the precise reason for this is not known at present. It is, however, evident that a significant number of the autopsied mice showed pathological findings in the diaphragm and the heart. In humans, there was a report on two siblings with the homozygous V572L mutation who died from arrhythmia (27), but there had been no reports on respiratory involvement among patients.
The onset of symptoms among DMRV patients has been reported to be from the second to the third decade (3), although there were anecdotal reports of earlier onset (28). Interestingly, in the Gne(–/–)hGNEV572L-Tg mice, the onset of clinical phenotype is noted around 30 weeks of age, which can be considered to be similar to that in humans, using lifespan and ability to reproduce for points of comparison. It is peculiar that gastrocnemius and quadriceps muscles are preferentially involved in mice, while in humans, the tibialis anterior is remarkably involved while the quadriceps are affected relatively late in the course of the disease. In our recent data on the clinical presentation of DMRV, however, it is clear that the gastrocnemius can be affected more severely in some cases (28).
We tried to check fiber type involvement in these muscles, and found out that both slow and fast fibers are affected in human and mice, in terms of the presence of RVs, but fast type fibers are predominantly involved (data not shown). Sporadic IBM has some pathological similarities with DMRV; recently, it has been shown that the presence of inclusions on routine histochemistry and the pathogenic accumulation of ß-amyloid protein occur in fast twitch muscles, both in a transgenic model of IBM and in IBM patients (29), implying that fast type fibers are more vulnerable to pathological changes. Further analysis is needed on this aspect to derive a more conclusive data.
CK levels are reported to be mildly or moderately elevated in patients, although there were isolated cases where the CK activity was above 1000 IU/L (11). CK elevation has always been correlated with the presence of necrotic and regenerating processes in the skeletal muscle, but which are only occasionally found in DMRV/h-IBM. Elevation of serum CK is also seen in the Gne(–/–)hGNEV572L-Tg mice, although necrotic and regenerating process is barely detectable. Our data suggest that there might be other mechanisms which trigger CK release into the circulation, aside from myonecrosis. It has not been clarified if CK release into the blood stream may be induced by deglycosylation of membrane proteins, although some studies suggested that removal of sialic acids by neuraminidase treatment may influence sarcolemmal permeability (30). Further tests are clearly needed to shed some insight on the CK elevation in DMRV/h-IBM and Gne(–/–)hGNEV572L-Tg mice.
A subject of poignant interest is whether RV formation, one of the hallmarks of DMRV/h-IBM, is the primary event that induces muscle fiber atrophy and loss, notwithstanding the fact that RVs are non-specific and could be seen in a multitude of myopathies. In the Gne(–/–)hGNEV572L-Tg mice, weakness is clearly noted before the occurrence of RVs, implying that other factors should be responsible for the earlier onset of weakness. Consistently, we have documented that serum and other tissues are hyposialylated, and this phenomenon is not at all correlated with age, strongly suggesting that hyposialylation may play a role in the development of clinical manifestations exhibited by patients. Previous studies have implicated that sialic acid directly contributes to the negative surface potential of cells, because desialylation of rat skeletal muscle sodium channel leads to reduced sensitivity of these channels to the effects of external calcium (31). This would mean that voltage gating parameters are shifted to the point that channels required a larger depolarization in order to gate, which may suggest that the mechanism of weakness may be due to the reduced excitability of the muscle membrane as a result of sodium channel desialylation.
The hallmarks of DMRV/h-IBM include RVs that are autophagic in nature (32) and cytoplasmic inclusions in vacuolated and non-vacuolated fibers, both of which are seen in muscle sections from the Gne(–/–)hGNEV572L-Tg mice. Several proteins have been shown to accumulate in DMRV myofibers (33,34), and most of which have been demonstrated to be mainly associated with amyloid because of the positive reactivity to crystal violet and Congo red, suggesting that they assume the beta-pleated sheet configuration. In general, more than 20 unrelated proteins, including ß-amyloid (34), prion, tau (21) and transthyretin, can abnormally unfold and self-aggregate to form beta-pleated sheet amyloid (35). The association of these proteins with DMRV/h-IBM pathomechanism has largely been enigmatic up to this time, but unfolding and misfolding of proteins most probably play a role. Previous reports have alluded to the role of sialic acid in proper folding of proteins (35–37). The ultimate fate of aggregated, misfolded glycoproteins is degradation, hence the activation of UPR is expected, which could explain the presence of ubiquitin signals in the myofibers of the Gne(–/–)hGNEV572L-Tg mice and upregulation of ubiquitin and proteasome in DMRV/h-IBM myofibers (38).
The implication of amyloid deposition in the formation of RVs in both DMRV/h-IBM and s-IBM (39) is supported by our finding that the occurrence of amyloid inclusions in the myofibers preceded RV formation. Amyloid itself has been shown in vitro to block the degradation of ubiquitinated proteins by inhibiting proteasome activity (40), hence its accumulation may not only lead to cytotoxicity, but also may further aggravate protein misfolding. In addition, it has been clarified that overproduction of amyloid can induce tau hyperphosphorylation and decrease its solubility (41). Sialylation and glycosylation of amyloid precursor protein, which contains both O- and N-glycans, appear to be important for its proteolytic processing, secretion and metabolism (42–45). Interference with the formation of N-linked glycans resulted in a decrease in secreted AßPP and an increase in the level of the cellular form of the protein, which has a higher propensity to form amyloid ß peptide (42,46). Although amyloid fibrils were the structure previously considered to be cytotoxic, there is current experimental evidence that pre-amyloid oligomeric complexes or aggregates, either diffuse or in a protofibril stage, can be very cytotoxic (47). The presence of dense deposits in areas with relatively preserved myofibrillar architecture on electron microscopy strongly suggest that deposition of amyloid and amyloid-like structures pre-date RV formation.
Because DMRV/h-IBM patients do not present, in general, with symptoms reflecting involvement of the respiratory system, it is assumed that the diaphragm is relatively spared in this myopathy. In the Gne(–/–)hGNEV572L-Tg mice, it is clear that the diaphragm can be involved, despite the absence of overt respiratory difficulties. The presence of pathological findings in the sacrificed mice, and not only in the ones that died suddenly, may suggest that the presence of RVs per se, may not correlate with severity in phenotype, with respect to involvement of diaphragm. A more sensitive method of assessing the respiratory status of these mice, vis-à-vis a plain observation, might be helpful in clarifying the extent to which respiratory system is involved. Our results suggest that careful evaluation of respiratory and cardiovascular functions is logical and warranted in human patients.
In the Gne(–/–)hGNEV572L-Tg mice, we have seen RVs in the cardiac muscles obtained from a couple of mice, clearly supporting the presence of cardiac involvement in DMRV/h-IBM. It has always been reported that DMRV involves primarily skeletal muscles but recently, however, it is being recognized that other organs may likewise be involved. For example, cardiac involvement is not very rare as it is seen in 18% of patients, with a spectrum of manifestations ranging from an incomplete right bundle-branch block to a fatal arrhythmia which led to sudden death (25,26). Sialic acid was shown to be an important component on the surface of heart muscle cells, because its removal reduced the cell surface negative charge by 25% (48) and produced a large increase in cardiac myocyte Ca2+, followed by marked cell contracture (49), emphasizing the importance of negatively charged sialic acid-containing gangliosides in the maintenance of cardiac cell physiological Ca2+ permeability. More importantly, it has been demonstrated that in myocardial cells, desialylation of cells by neuraminidase treatment causes aberrant electrical activity (50), and may lead to arrhythmia (51).
In conclusion, we have generated the first mouse model of DMRV/h-IBM, which resembles the clinical, pathological and biochemical features of the disease in humans. The Gne(–/–)hGNEV572L-Tg mouse is a concrete evidence that mutations in the GNE are causative of DMRV/h-IBM. Indeed, these DMRV/h-IBM mice will be a valuable tool to search for further clues in unraveling the pathomechanism of this myopathy. As we have clearly documented in these mice, hyposialylation plays a key role in the pathogenesis of DMRV/h-IBM, and is of paramount importance in considering therapeutic trials.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation of Gne knockout mice
The Gne knockout mice [Gne(–/–)] was produced in inGenious Targeting Laboratory (New York, NY, USA). The 17 kb mouse genomic DNA fragment, containing exons 3–5, was cloned from the mouse 129Sv/Ev lambda genomic library. The Neo cassette that was inserted replaced the 1.4 kb upstream of exon 3, exon 3 and 124 bp downstream of exon 3 (Fig. 1). The resulting targeting vector was linearized by NotI, purified and then transfected by electroporation into ES cells. Positive clones after neomycin selection were identified using PCR (primer sequences available upon request).
Generation of hGNEV572L-Tg
The cDNA for GNE mutant was obtained by reverse transcribed-PCR from skeletal muscle RNA of a DMRV patient with the V572L mutation and cloned into pCR-Blunt vector (Invitrogen, Carlsbad, CA, USA), as described previously (17). Cloned cDNA was sequenced by ABI cycle-sequencing procedures using an ABI 3100 (Applied Biosystems, Foster City, CA, USA). The XhoI fragment containing GNE mutant cDNA was excised and inserted into pCAGGS vector in which gene expression is driven by a CAG promoter (52). loxP sequences were introduced to flank the cDNA insert. SalI fragment was purified and injected into C57BL/6 oocytes and subsequently transplanted into recipient mice. Founders were bred to WT C57BL/6 females to check for germline transmission, which was confirmed by PCR analyses on genomic DNA.
Production of Gne(–/–)hGNEV572L-Tg
To maintain the same copy number of transgene, stringent measures were taken in generating mice. The hGNEV572L-Tg mouse was crossbred to Gne heterozygous mouse [Gne(+/–)] to create a Gne(+/–) mouse that carried the human GNE [Gne(+/–)hGNEV572L-Tg]. The latter was then mated with a Gne(+/–) mouse, to obtain a mouse that harbors the human V572L mutated GNE in a Gne knockout background.
For genotyping, DNA was isolated from mouse tails. Gne mice genotyping was carried out using PCR analysis on tail genomic DNA with the following primers: Neo, WT3 and S2 (primer sequences available upon request). Further, DNA was digested with BamHI, subjected to Southern blotting and then analyzed by hybridization to a 500 bp probe.
For transgenic mice, the following oligonucleotides were used to amplify a 318 bp segment found specifically in human GNE: 1186F, CTTCAAGAGCCACTGCAAA; 1504R, CAATTCCTTCCCGAGGATT.
mRNA expression and determination of copy number
Mouse skeletal muscles, heart, brain, spleen and liver were dissected and rapidly frozen in liquid-nitrogen. Total RNA was extracted from cryostat sections of tissues with TRIzol (Invitrogen) following the manufacturer's protocol. First-strand cDNA was synthesized from RNA by reverse transcription using the Superscript RNase H– Reverse Transcriptase (Invitrogen) and random hexamers. Gene expression was measured by quantitative real-time PCR in i-Cycler IQ system (Bio-Rad Laboratories, Hercules, CA, USA). Primers (1186F and 1504R) were used to span exon–intron junctions to prevent amplification of genomic DNA. Relative quantification of gene expression was determined by comparison of threshold values as suggested by the manufacturer. All results were normalized with respect to Gapdh expression.
Transgene copy number was determined by the i-Cycler IQ system using the SYBR Green reagent kit according to the manufacturer's instructions. Triplicate samples of tail DNA from transgenic mice of each line were analyzed concurrently against a standard curve of scaled concentrations of an external standard. Primers were designed to amplify the transgene hGNEV572L and endogenous Gne; twice the ratio of the hGNEV572L/Gne amplicons was interpreted as copy number.
Sialic acid measurement
The bound sialic acids from the serum and pieces of different tissues were released using 20 mM sulfuric acid hydrolysis for 1 h at 80°C. Free sialic acids were then derivitized with 1, 2-diamino-4, 5-methylenedioxybenzene and analyzed by reverse-phase HPLC fluorescence detection as described previously (53). The eluant was monitored by fluorescence and measured by comparison with Neu5Ac and Neu5Gc standards (from 0.05 nmol/µl to 5 nmol/µl). Total protein from tissues was measured using the Bio-Rad Protein Assay (Bio-Rad Laboratories) according to the manufacturer's protocol.
General assessment for motor strength and fatigability
Whole-animal strength and fatigability were measured according to a test procedure (here referred to as rod-climbing test) previously reported (54). In brief, this test required the mice to pull themselves on top of a suspended rod (3 mM in diameter). The measurement of muscle weakness was based on the mean percentage of passes over 15 trials of the test in a 3-min period. Fatigability was assessed as the average pass rate over time for each group of mice. The test was repeated at least three times after a 2-week period.
Histopathological and histochemical analyses
Fresh specimens from individual skeletal and cardiac muscles were snap-frozen in liquid-nitrogen-cooled isopentane and stored at – 80°C until further processing. We stained frozen sections (6 µm) of transversal skeletal and cardiac muscles with a battery of histochemical stains including hematoxylin and eosin, modified Gomori trichrome and acid phosphatase. Sections were analyzed by light microscopy. We performed Congo red staining in 10 µm cryosections following the Putchler's modification, and viewed sections under light microscope and conventional fluorescence microscope using Texas-red filters (39). For immunohistochemical analysis, tissue sections were fixed either in acetone or paraformaldehyde, depending on the primary antibody used, and blocked with 5% normal serum and 2% bovine serum albumin in phosphate-buffered saline. The primary antibodies used are listed in Table 1. We used several antibodies which recognize amyloidß. 6E10, which is a human-specific antibody, but also reacts to murine tissue when the amyloid burden is high, primarily recognizes AßPP (residues 1–16) after -secretase cleavage. It also recognizes, in addition, C99 fragment and amyloid ß peptides (1–40 and 1–42) which have been shown to be prone to aggregation. The anti-oligomeric antibody (A11) is specific to the oligomeric structure of ß amyloid peptides. The following secondary antibodies were used appropriately: anti-goat IgG F (ab')-2-fragment, FITC conjugated (EY Laboratories, San Mateo, CA, USA); anti-rabbit IgG (H + L), Alexa Fluor conjugated (Molecular Probes, Eugene, OR, USA); anti-mouse IgG1, FITC conjugated (Sanbio/Monosan, Uden, The Netherlands). Images were collected and analyzed with a laser scanning microscope (Olympus, Tokyo, Japan) with its appropriate software.
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Morphometric analysis of fibers
Muscle cross sections were stained with rabbit polyclonal antibody against caveolin-3 followed by a fluorescent secondary antibody. Digital images from fluorescence signals were observed under a confocal microscope and the widest diameter was recorded for 600 or more fibers using Image-J software from the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). Results were analyzed using Statistics Software for Social Sciences (SPSS for Windows, Rel. 11.0.0. 2001, SPSS Inc., Chicago) software.
Electron microscopy
The muscle specimens were immediately fixed for 2 h in 2.5% cold glutaraldehyde with 0.1 M cacodylate buffer, pH 7.3. After washing in cacodylate buffer, the specimens were post-fixed in 1% osmium tetroxide in the same buffer, dehydrated with graded series of ethanol and embedded in Epon. Semi-thin sections (0.5 µm) were stained with toluidine blue alkaline. Ultrathin sections were stained with uranyl acetate, citrated and observed with a H-600 electron microscope (Hitachi, Tokyo, Japan) at 75 kV.
Serum CK
Blood samples were obtained either by inferior vena cava aspiration, or careful collection from mouse tail. Total CK activity was measured by a spectrophotometric assay employing a commercial kit (CPK-L Determiner, Kyowa MEDEX, Tokyo, Japan). For confirmation, CK isoforms were electrophoretically analyzed using Titan Gel CK Isozyme kit (Helena Laboratories, Beaumont, TX, USA) following the manufacturer's protocol.
Statistical analysis
Data were entered in SPSS version 11.0 and were analyzed by computation of the frequency and the mean ± SD and/or percentage. The data were then subjected to a univariate analysis (Fisher's exact test), Student's t-test, Wilcoxon paired test, ANOVA or Mann–Whitney U test, log-rank test or multiple regression analysis, whichever was appropriate. P-values less than 0.05 were considered to be statistically significant.
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ACKNOWLEDGEMENTS |
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The authors thank the following persons for their invaluable support and assistance: Yoko Keira, Genri Kawahara, Mari Okada and Kumiko Murayama. This study is supported by the ‘Research on Psychiatric and Neurological Diseases and Mental Health’ from Health and Labour Sciences Research Grants; the ‘Research on Health Sciences focusing on Drug Innovation’ from the Japanese Health Sciences Foundation; the ‘Research Grant (16B-2, 17A-10) for Nervous and Mental Disorders’ from the Ministry of Health, Labour and Welfare; and the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO).
Conflict of Interest statement. None declared.
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