Human Molecular Genetics, 2001, Vol. 10, No. 18 1963-1970
© 2001 Oxford University Press
Loss of Uch-L1 and Uch-L3 leads to neurodegeneration, posterior paralysis and dysphagia
Howard Hughes Medical Institute and Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA, 1Department of Animal Models for Human Disease and 2Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo 187-8502, Japan
Received May 8, 2001; Revised and Accepted June 27, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Altered function of the ubiquitin pathway has been implicated in the etiology of neurodegeneration. For example, gracile axonal dystrophy (gad) mutant mice, which harbor a deletion within the gene encoding ubiquitin C-terminal hydrolase L1 (Uch-L1), display sensory ataxia followed by posterior paralysis and lethality. We previously showed that mice homozygous for a targeted deletion of the related Uch-L3 gene are indistinguishable from wild-type. To assess whether the two hydrolases have redundant function, we generated mice homozygous for both Uch-L1gad and Uch-L3
3–7. The double homozygotes weigh 30% less than single homozygotes and display an earlier onset of lethality, possibly due to dysphagia, a progressive loss in the ability to swallow food. This is consistent with histological analysis that revealed axonal degeneration of the nucleus tractus solitarius (NTS) and area postrema (AP) of the medulla. The NTS is essential for central nervous system control of swallowing. The double homozygotes also display a more severe axonal degeneration of the gracile tract of the medulla and spinal cord than had been observed in Uch-L1gad single homozygotes. In addition, degeneration of dorsal root ganglia cell bodies was detected in both the double homozygotes and Uch-L33–7 single homozygotes. Given that both Uch-L1gad and Uch-L33–7 single homozygotes display distinct degenerative defects that are exacerbated in the double homozygotes, we conclude that Uch-L1 and Uch-L3 have both separate and overlapping functions in the maintenance of neurons of the gracile tract, NTS and AP. This study is the first to successfully document dysphagia in the mouse and is a potentially valuable resource for understanding human neurodegenerative disorders that cause swallowing defects. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The ubiquitin pathway is responsible for the turnover of both short-lived regulatory proteins and damaged proteins within the cell. This pathway consists of enzymes that ubiquitinate a target protein and the proteasome that is required for subsequent degradation (1,2). Although this pathway is constitutive and essential for viability in organisms from yeast to humans, mutations in various ubiquitin pathway enzymes lead to specific defects due to either substrate specificity or restricted spatial or temporal expression. For example, maternal inheritance of mutations in the imprinted ubiquitin ligase UBE3A leads to Angelman syndrome, a human disorder characterized by motor dysfunction and mental retardation (3). The specific defects may arise from central nervous system (CNS)-specific imprinting of UBE3A during neuronal development, which silences the paternal allele since UBE3A is expressed from both parental alleles in unaffected fetal tissues (4).
The ubiquitin pathway has been implicated in the etiology of neurodegenerative disease (5). Under conditions that induce protein misfolding such as oxidative stress or neurotoxin exposure, misfolded proteins are normally degraded. However, if the ubiquitin pathway becomes overloaded or disabled, misfolded proteins aggregate and form inclusion bodies characteristic of most neurodegenerative disorders. Recently, Bence et al. (6) found that protein aggregation directly impairs the function of the ubiquitin pathway. In addition, ubiquitin immunoreactivity has been observed in inclusion bodies for the majority of neurodegenerative disorders analyzed such as Huntington’s and Alzheimer’s disease (7,8). Also, specific mutations within ubiquitin pathway enzymes and substrates lead to neurodegeneration. For example, mutation of the Parkin ubiquitin ligase causes one form of autosomal recessive Parkinson’s disease (9). Mutations that extend the half-life of the ubiquitin pathway substrate 
-synuclein cause one form of autosomal dominant Parkinson’s disease (10).
Analysis of the gracile axonal dystrophy (gad) mutant mouse uncovered another example of a ubiquitin pathway enzyme involved in neurodegeneration. First isolated as a spontaneous mutation in 1984, Saigoh et al. (11) positionally cloned the corresponding lesion and identified a deletion within Uch-L1 [the gene encoding ubiquitin C-terminal hydrolase (UCH) L1] that removed residues critical for enzymatic activity. The primary defect in Uch-L1gad homozygotes is axonal degeneration of the gracile tract, which results in sensory ataxia (12). The gracile tract consists of thoracic, lumbar and sacral dorsal root ganglion (DRG) axons that travel up the dorsal column of the spinal cord within the gracile fascicle, where they terminate in the medulla within the gracile nucleus. These axons carry afferent sensory information from the hindlimbs and trunk required for proprioception. Uch-L1gad homozygotes display dying-back type axonal degeneration within this tract which causes a block in delivery of sensory information leading to ataxia (13,14). Over time, the axonal degeneration in Uch-L1gad homozygotes spreads to motor tracts and higher centers of the CNS leading to paralysis and eventually death between 150 and 180 days of age (15,16).
Uch-L1 is closely related to a second UCH, Uch-L3. Four genes of the UCH class of ubiquitin hydrolases have been identified that share homology surrounding residues critical for enzymatic activity (17). Both Uch-L1 and Uch-L3 encode proteins of similar size that display 52% amino acid identity, whereas UCH37 and BAP1 are more divergent (18–21). The UCH family differs from the large and highly diverse UBP family of de-ubiquitinating enzymes, although UCH function may be contributed by one or more of the numerous UBP enzymes (17,22). Based on in vitro substrate specificity studies, UCH-L1 and UCH-L3 are thought to be involved in ubiquitin recycling to maintain pools of monomeric ubiquitin necessary for proteolysis (17,23). Uch-L1 is expressed highly in all neurons as well as testis (18,24), whereas Uch-L3 mRNA was detected in all tissues analyzed, including brain and testis (19). Initially, we proposed that Uch-L3 may be required during embryonic development based on its location within a deletion locus conferring embryonic lethality as well as its embryonic expression pattern (L.J.Kurihara, E.Semenova, R.S.Ingram, X.-J.Guan and S.M.Tilghman, submitted). However, we found that mice homozygous for a null allele of Uch-L3 were indistinguishable from wild-type, despite the fact that its amino acid sequence is 97% conserved between mouse and human (19). In this manuscript we set out to determine whether Uch-L1 and Uch-L3 perform a redundant function, particularly in the CNS and testis where the two genes are co-expressed.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation of Uch-L1gad/Uch-L3
3–7 miceTo generate Uch-L1gad/Uch-L3
3–7 double homozygotes, we crossed Uch-L1gad heterozygous mice to Uch-L33–7 homozygous mice. Fifty percent of the resulting progeny were double heterozygotes for both Uch-L1gad and Uch-L33–7. These mice were crossed to generate double homozygotes, wild-type and all heterozygous combinations of the four alleles within a single cross, the latter of which served as appropriate strain-matched controls. All genotypes were obtained at the expected frequency at weaning; double homozygotes represented 4.9% (12/247) of the total progeny, an insignificant deviation from the expected 6.25%. The single and double homozygotes were indistinguishable from their wild-type and heterozygous littermates at weaning. Contrary to our expectation that spermatogenesis might be affected in the double homozygotes (since Uch-L1 and Uch-L3 are co-expressed in testis) both male and female double homozygous mice were fertile. In addition, standard histopathology indicated that testis and ovary as well as liver, lung, kidney, adrenal, spleen, thymus, heart, stomach and intestine were normal in double homozygous mice (data not shown).
Neural degeneration in Uch-L1gad/Uch-L33–7 mice
The Uch-L1gad/Uch-L33–7 double homozygotes developed sensory ataxia with 100% penetrance starting at 
80 days of age, similar to the Uch-L1gad single homozygotes. We analyzed the severity of sensory ataxia by footpad usage when monitored by ink test after 80 days. Wild-type mice produced an ink pattern indicative of their hindlimb digits making contact with the surface while walking. However, Uch-L1gad and Uch-L1gad/Uch-L33–7 homozygous mice produced an ink pattern indicative of both their hindlimb digits and footpads making contact. This ‘flat-footed’ hindlimb gait was more severe in the double homozygotes compared with Uch-L1gad single homozygotes but was difficult to quantitate (data not shown). Instead, we relied on quantitative pathology to determine the severity of axonal degeneration underlying the sensory ataxia within the gracile tract.
We compared wild-type, single and double homozygous mice at 90 days of age for axonal degeneration within the gracile tract by quantitating the occurrence of eosinophilic spheroids that are indicative of dystrophic axons. As shown in Figure 1, sections of the gracile nucleus from both Uch-L1gad single homozygotes and Uch-L1gad/Uch-L33–7 double homozygotes showed the presence of dystrophic axons or spheroids whereas wild-type and Uch-L33–7 single homozygotes did not. The Uch-L1gad/Uch-L33–7 double homozygotes had a significant increase in the number of spheroids when compared with Uch-L1gad single homozygotes (Fig. 2). This increase occurred within the gracile nucleus of the medulla (P < 10–6), as well as the gracile fascicle of the spinal cord at the cervical (P < 0.004) and thoracic (P < 0.009) levels. This pathology is the likely explanation for the more severe sensory ataxia we observed in the double homozygotes.
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In addition, we found that the DRG cell bodies from which the gracile axons emanate also showed signs of increased degeneration in the double homozygotes as indicated by smaller cell diameter and more basophilic cytoplasm; this cell morphology increased 4.1-fold in double homozygotes compared with wild-type (Fig. 3 and Table 1). This correlates with previous data indicating that larger neurons in the lumbar DRG are the most vulnerable and severely affected by dying-back axonopathy (25). Interestingly, the Uch-L3
3–7 single homozygotes also demonstrated a significant degeneration of the DRG cell bodies [3.6-fold increase (P = 3 x 10–5) in small, basophilic cells] in contrast to the Uch-L1gad single homozygotes which displayed a 2.2-fold increase (P = 0.02). This is the first difference that we have detected between wild-type and Uch-L33–7 homozygotes. The single homozygous phenotypes suggest that there is a greater requirement for Uch-L3 function in maintaining DRG cell bodies whereas Uch-L1 function is more important in maintaining the integrity of the axon.
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We next analyzed other regions of the CNS in Uch-L1gad/Uch-L3
3–7 double homozygotes for potentially novel defects not observed in Uch-L1gad single homozygotes. Although dystrophic axons in double homozygotes were occasionally observed in the dorsal horn, motor nucleus and white matter of the spinal cord, and in the cerebellar white matter, their low frequency was indistinguishable from Uch-L1gad single homozygotes. However, we found that the occurrence of dystrophic axons had extended into two nuclei neighboring the gracile nucleus: the nucleus tractus solitarius (NTS) and area postrema (AP) (Fig. 3). When we quantitated spheroid number in these nuclei (Fig. 2), there was a highly significant increase in spheroid formation within Uch-L1gad/Uch-L33–7 double homozygotes when compared with Uch-L1gad single homozygotes (P < 0.0006 and P < 0.01, for the NTS and AP, respectively). A low level of axonal dystrophy was observed in Uch-L1gad homozygotes that had not been reported previously. As in wild-type, spheroids were not found in Uch-L33–7 single homozygotes. The NTS is the primary region where the peripheral cardiovascular, respiratory, gustatory and general visceral inputs are processed (26–28). The NTS receives neurons from cranial nerves IX and X that carry afferent sensory information from the tongue, palate, pharynx, larynx and gut that are required for taste and swallowing. Multiple neural and vascular interactions exist between the NTS and AP (26). We assumed that similar to the gracile tract, a dying-back type axonal degeneration within the NTS and AP would block this sensory information path and we investigated the phenotype that might result from these lesions.
Dysphagia in Uch-L1gad/Uch-L33–7 mice
Although the mice were indistinguishable at weaning, by 80 days of age the Uch-L1gad/Uch-L33–7 double homozygotes weighed 45% less than wild-type (P < 10–6) and 30% less than single homozygotes (Fig. 4). This reflects the fact that both single homozygotes were 20% smaller than wild-type, an observation that had previously gone undetected for Uch-L33–7 (19). Quantitation of weight differences among the various genotypes indicates that although Uch-L1 and Uch-L3 display functional redundancy, they clearly also retain separate functions, based on a comparison among genotypes with a UCH gene dosage of 2. Double heterozygotes depicted as ‘h/h’ possess one copy of each gene and are equivalent to wild-type in weight. However, if the mice instead have two copies of only Uch-L3 (depicted as –/+) or two copies of only Uch-L1 (depicted as +/–), even though the UCH gene dosage is still 2, the mice show a moderate (20%) but significant (P = 10–6) decrease in weight when compared with wild-type. This shows that the functions of Uch-L1 and Uch-L3 are not equivalent. These data also indicate the lack of a haplo-insufficient phenotype given that there are no size differences between Uch-L1 and Uch-L3 single homozygous genotypes with a UCH gene dosage of 2 versus 1 (Fig. 4); the –/+ and –/h mice weigh the same as well as +/– and h/– mice. Therefore, a single copy of either Uch-L1 or Uch-L3 is sufficient.
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10 mice per genotypeNext we wanted to determine the underlying cause of the decreased weight of the double homozygotes. In Uch-L1gad single homozygotes, paralysis interferes with their ability to efficiently obtain food and eventually leads to death. Therefore it was possible that the decreased weight of the double homozygotes was due solely to the more severe axonal degeneration of the gracile tract. Alternatively, hyperkinesia or altered metabolism with no change in food intake could underlie the decreased weight. However, given the novel pathology we observed in the NTS and AP, we performed a food intake assay since this region of the medulla is important for control of swallowing (29,30).
We utilized precision pelleted food and cage bedding that allowed easy retrieval of partially consumed food pellets. We found that the double homozygotes consumed less food than wild-type or single homozygotes. However, this difference was proportional to their progressive decrease in body weight (data not shown). More striking, the cage floor housing double homozygotes showed an accumulation of chewed but uningested food. An example is shown in Figure 5 where a Uch-L1gad/Uch-L33–7 double homozygote generated ‘crumbs’ but an Uch-L33–7 single homozygote given an equal number of food pellets and time did not. We do not believe the excess food crumbs were generated as a result of an increase in activity or chewing; in fact, dysphagic mice were less active and did not shred their cage bedding as did the non-dysphagic mice. Thus the double homozygotes retained an appetite for food but were not able to ingest it efficiently, suggesting that the dysphagia probably results from a swallowing defect. To our knowledge such a phenotype has not been previously documented in a mutant mouse line.
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The penetrance of this dramatic phenotype was high; 11 of 15 double homozygotes displayed dysphagia compared with only two of 14 Uch-L1gad single homozygotes and none of eight Uch-L3
3–7 single homozygotes. On average, dysphagic mice generated 1–2 g of crumbs while ingesting a similar amount of food per day. The dysphagia was associated with terminal stages and the duration ranged from several days to more than several weeks prior to death. Ingestion of food often stopped entirely several days prior to death. Although dysphagia is the most likely explanation for the decreased weight, we cannot exclude other possibilities, especially given that Uch-L33–7 single homozygotes do not display overt dysphagia or NTS/AP degeneration. Most likely as a result of the exacerbated and novel defects, the double homozygotes displayed earlier onset lethality relative to Uch-L1gad single homozygotes (Fig. 6). Consistent with previous reports, single homozygotes survived an average of 187 ± 42 days with a terminal phenotype of complete paralysis rendering them incapable of obtaining food and water. There was no significant difference in lifespan between Uch-L1 single homozygotes that were either wild-type (194 ± 48) or heterozygous (183 ± 39) for Uch-L3, once again indicating that a single copy of Uch-L3 is sufficient. In contrast, the double homozygotes survived only an average of 118 ± 37 days, 69 days less than Uch-L1gad single homozygotes. At this stage, the double homozygotes displayed only moderate posterior paralysis similar to age matched single homozygotes, and were still fully capable of obtaining food and water. However necropsy performed on dysphagic double homozygous mice indicated that the stomach and gut content was very low. This leads us to believe that the double homozygotes die from starvation caused by dysphagia, a novel cause of death.
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Spatial expression of Uch-L1 and Uch-L3 mRNA
One puzzle that arose from the identification of Uch-L1 as the gene responsible for the gad mutant phenotype was how loss of a protein expressed highly in all neurons would initially lead to axonal degeneration within a single tract (11). A similar question can be raised about Uch-L3 and its role in the defects associated with loss of Uch-L1 and Uch-L3; do these specific defects result from restricted expression of Uch-L3 in the CNS? To resolve this issue, we performed northern analysis on dissected regions of the CNS from wild-type mice (Fig. 7). Uch-L3 mRNA was detected in all five regions of the CNS examined at levels that appeared to be lower than that of Uch-L1. Therefore, we conclude that the double homozygous phenotype is not restricted to the medulla and spinal cord due to limited expression of Uch-L3. In addition, we performed northern analysis of Uch-L3
3–7 and Uch-L1gad single homozygous CNS tissues to determine if either gene was up-regulated in the absence of its redundant partner (Fig. 7). This was not the case as shown by examples of CNS tissues affected (spinal cord) and unaffected (olfactory bulb) in the double homozygotes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Uch-L1gad/Uch-L3
3–7 double homozygotes displayed a more severe axonal and cell body degeneration of the gracile tract relative to Uch-L1gad single homozygotes. Furthermore, we uncovered axonal degeneration of the NTS and AP of the medulla that was more severe in double homozgyotes compared with Uch-L1gad single homozgyotes. As the double homozygotes progressed in age, their failure to thrive became more and more pronounced, marked by continued weight loss, a shift from an ataxic gait to moderate posterior paralysis and dysphagia. These defects were manifested at an earlier age with greater severity in the Uch-L1gad/Uch-L33–7 double homozygotes and contributed to an earlier onset lethality. We also found that Uch-L33–7 single homozygotes undergo DRG cell body degeneration and weigh 20% less than wild-type. Given that each single homozygote displays unique neurodegenerative defects that are exacerbated in the double homozygotes, we conclude that Uch-L1 and Uch-L3 perform both separate and overlapping functions in the maintenance of neurons within the gracile tract, NTS and AP. It is striking that the double homozygous phenotype is region-specific within the CNS given that the pattern of expression for both genes is not. There are several possible explanations for these findings. Upon discovery that a deletion within Uch-L1 was the underlying cause of the gad mutant phenotype, Saigoh et al. (11) proposed that DRGs may be the most sensitive to loss of Uch-L1 because of their extremely long axons. However, given that the NTS and AP have short axons relative to the DRGs, such an argument does not apply to the region-specific pathogenesis observed in the double homozygotes. Alternatively, Saigoh et al. (11) proposed there may be Uch-L1 substrates with restricted expression that cause the region-specific pathogenesis. Although this does not seem likely given that Uch-L1 and Uch-L3 hydrolyze bonds between small adducts and ubiquitin to generate free monomeric ubiquitin (17,23), perhaps substrates specific to the gracile tract, NTS and AP are particularly sensitive to a compromised ubiquitin pathway. Such a model does not require Uch-L1 and Uch-L3 to possess substrate specificity for unknown substrates whose improper processing underlie the resulting pathology.
The single homozygous phenotypes indicate a greater requirement for Uch-L3 function in DRG cell bodies whereas Uch-L1 function is required more in the axon. Perhaps this is caused by differences in subcellular localization of substrates that prefer either Uch-L1 or Uch-L3. Alternatively, differences in the subcellular localization or concentration of the UCH enzymes themselves could be the cause. Our results do not address Uch-L1 and Uch-L3 substrate specificity since overlapping function in neuronal maintenance could occur by regulation/turnover of either common or distinct substrates.
Another issue concerning Uch-L1 and Uch-L3 function is whether they possess an important role in the processing of ubiquitin pathway substrates known to induce neurodegeneration. This is especially relevant given that a missense mutation in human Uch-L1 has been linked to Parkinson’s disease in a German family (31). Furthermore, Uch-L1 immunoreactivity has also been reported for Lewy bodies found in Parkinson’s disease (32). In addition, previous analysis of gad mutant mice indicated an accumulation of aggregates possessing ubiquitin immunoreactivity, similar to human neurodegenerative disorders (16). Finally, in a screen for enhancers and suppressors of a mutant eye phenotype induced by expression of human ataxin-1 (spinocerebellar ataxia type 1) in Drosophila, a UCH mutation was identified as an enhancer along with other components of the ubiquitin pathway (33). Therefore, we propose that altered Uch-L1 and Uch-L3 function is likely to contribute to the pathogenesis of other neurodegenerative diseases. To test this, we are crossing mice carrying transgenes that cause Huntington’s (34) and spinocerebellar ataxia-like (35) symptoms to Uch-L3 null mice to determine whether loss of Uch-L3 exacerbates the effects of these transgenes.
The dysphagia was the most significant defect we observed in the double homozygotes and we propose that the novel axonal degeneration of the NTS and AP underlies this abnormal behavior. Nearly three-quarters of the double homozygotes displayed dysphagia and had the most severe NTS and AP degeneration. Only 15% of Uch-L1gad single homozygotes displayed dysphagia and had only a mild degeneration of the NTS and AP. Evidence for a correlation between NTS degeneration and dysphagia comes from functional studies (29,30). The NTS receives sensory input that triggers a sequential excitation of motor nuclei to elicit the events that evoke swallowing. This model is based on anatomical studies of neuronal connections and functional analysis from electrical lesions and electrical/chemical stimulation experiments (36). The NTS is a likely site for peripheral afferent and AP interaction since the NTS receives a large percentage of the efferents of the AP and is also the primary terminal for peripheral afferents (26). Although the NTS is also important for gustatory control (37–39), altered taste function is usually manifested by changes in food preference, not in food consumption. Therefore, a defect in swallowing is the most likely explanation for the dysphagia we observed in the double homozygotes, particularly since the mice retained their appetite and did not appear to have difficulty in chewing their food. Although the NTS innervates structures required for both the oral and pharyngeal phases of swallowing, either phase could be affected in the double homozygous mice. It seems that the double homozygous mice experience difficulty in delivering a bolus of food to the throat, suggesting at least an oral phase defect. Further studies will be required to pinpoint the exact physiological cause of the dysphagia.
This study is the first to successfully document dysphagia in the mouse, and will be a potentially valuable resource for understanding a number of human neurodegenerative disorders that cause swallowing defects (40). For example, bulbar amyotrophic lateral sclerosis (or Lou Gehrig’s disease) causes a progressive loss in the ability to swallow, speak and eventually breathe. Fifty percent of Parkinson’s disease patients experience dysphagia, a problem that leads to other complications, particularly aspiration pneumonia which kills 50 000 people annually in the USA. Less frequent neurodegenerative disorders such as Kennedy’s disease also cause swallowing defects. Finally, stroke victims with lateral medullary infarctions such as those with Wallenberg’s syndrome also experience dysphagia; this pathology most closely resembles that of the double homozygous mice. While these diseases are manifested by multiple complex motor and sensory defects, study of the double homozygote will provide new insights into the molecular pathogenesis of neurodegeneration and associated neurological defects.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mice. To generate Uch-L1gad/Uch-L3
3–7 double homozygous mice, we crossed Uch-L1gad heterozygotes maintained on a mixed CBA/Nga and RFM/Nga strain background to Uch-L33–7 homozygotes maintained on a mixed 129 Sv/Ev Tac and C57BL/6J strain background. To generate appropriate mixed strain matched controls the double heterozygotes resulting from the first cross were used to generate double homozygotes, wild-type and all heterozygous combinations of the four segregating alleles within a single cross. Throughout our analyses, we did not observe significant phenotypic variation within a given genotype, suggesting that mixing strain backgrounds did not uncover any modifier loci. PCR genotyping was performed following DNA extraction from tail biopsy using previously described primers to detect Uch-L1, Uch-L1gad, Uch-L3 and Uch-L33–7 (11,19). Reaction conditions were 35 cycles at 94°C for 1 min, 55°C for 1 min and 72°C for 1 min in standard buffer (Perkin Elmer) plus 12% sucrose, 0.02 mM cresol red. PCR reactions to detect Uch-L1 and Uch-L1gad also required the addition of Perfect Match (Stratagene). Pathology. Mice were anesthetized by CO2 asphyxiation and fixed by transcardiac perfusion with 150 ml of Zamboni’s solution (2% paraformaldehyde with 0.2% picric acid in 0.1 M PBS adjusted to pH 7.3). Mice were post-fixed in the same solution for 3–4 days at 4°C. Brain and spinal cord were excised and embedded in paraffin. According to the Atlas of the Mouse Brain and Spinal Cord (41), coronal sections were made (6 µm) of the brain at the levels of 220, 300, 407 and 535, and of cervical (C3), thoracic (T8) and lumber (L2) spinal cord segments. Saggital sections of cerebellum were also prepared. These sections were stained with hematoxylin and eosin (H&E), luxol fast blue and cresyl violet (KB) and Bodian’s methods. The DRGs were excised at the level of lumbar segments and embedded in Epon812 after fixation with Zamboni’s solution. Pathological analysis was made in the gracile nucleus, solitary nucleus and AP in a section at level 535 and in 1 µm sections of the DRGs which were stained with toluidin blue. Quantitation of eosinophilic spheroid number was performed on H&E-stained sections by determining the total number of spheroids within these regions from a single section as specified above. The total number of spheroids in the NTS was the sum of numbers from the dorsomedial, medial, intermediate and commissural subnuclear regions according to Zittel et al. (42). The total number of DRG neurons was expressed as a sum of one to three DRGs from four mice per genotype. The area of neurons was measured by the Imagin system KS-100 (Kontron Elektronik, Germany). Statistical values were calculated and graphed using Microsoft Excel for mean, SD and P-value.
Food intake assay. To determine whether food consumption was affected in Uch-L1gad/Uch-L33–7 double homozygotes, animals were fed dustless precision pelleted rodent formula (Bio-Serv) provided ad libitum and housed in iso-PAD (Omni BioResources) cage bedding which allowed for efficient recovery of partially consumed food pellets. Body and food weights were taken twice weekly and consumption was normalized to body weight. When dysphagia was observed, food crumbs were collected and weighed separately from intact pelleted food. Because the dysphagia was associated with terminal stages, this assay was continued until death.
Northern analysis. Total RNA was extracted from dissected regions of wild-type and mutant mouse brain and spinal cord with Trizol (Gibco BRL). Fifteen micrograms of RNA was separated in 1% agarose gels containing (MOPS)-formaldehyde and transferred to Hybond N+ membranes (Amersham). Blots were hybridized in Church Buffer (43) at 65°C and washed in 0.1x SSC, 0.1% SDS at 23°C and 65°C. Radiolabeled probes were synthesized from cDNA fragments corresponding to Uch-L1 (AA060272), Uch-L3 (kocDNA) and ß-actin (Clontech).
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ACKNOWLEDGEMENTS |
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We thank John Levorse for performing perfusions and dissections, Ms Hisae Kikuchi for technical assistance with histopathology and statistical analysis and Mrs Masako Shikama for breeding and care of gad mice. We also thank Robert M. Miller of the University of Washington Medical Center and Hitoshi Osaka and Yu-Lai Wang of the NCNP for helpful discussions. Histological analysis of tissues (excluding CNS) was performed at the University of California Davis Histo-Pathology Laboratory. S.M.T. is an investigator of the Howard Hughes Medical Institute. This study was also supported in part by the Human Health Science Foundation (T.K.) and Brain Science research grants (K.W.) from the Ministry of Health, Labour and Welfare in Japan.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 609 258 2900; Fax: +1 609 258 3345; Email: stilghman@molbio.princeton.edu
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REFERENCES |
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