Assessment of dietary therapies in a canine model of Batten disease.

The neuronal ceroid lipofuscinoses (NCLs) are inherited neurodegenerative diseases that occur in a number of animal species, including dogs. A study was conducted to determine whether the resupply of nutrients lost in NCL English Setter dogs would modify the course of the disease. Carnitine and polyunsaturated fatty acids have been reported to be reduced in NCL English Setters. Therefore, the normal laboratory diets of NCL dogs were supplemented with carnitine, fish oil and corn oil and the disease progression was compared with that of an untreated litter mate. The following specific prognostic indicators of NCL were monitored: cognitive function, brain atrophy, brain glucose metabolism and lifespan. Carnitine, with or without lipid supplements, dramatically delayed the progression of cognitive decline in NCL dogs. When fish oil and corn oil only were supplied, brain atrophy was reduced. A combination of all three supplements preserved cognitive function and increased lifespan by 10%. However, brain glucose hypometabolism and cerebral atrophy were not reduced. The results in this study indicated that the effectiveness of therapeutic interventions can be assessed by non-invasive methods at a relatively early stage of the disease process. Our study suggests that dietary supplementation with carnitine is a promising new approach for delaying or preventing the cognitive decline in dogs, and perhaps, with human NCL patients.

Studies of renal injury III: lipid-induced nephropathy in type II diabetes.

UNLABELLED: Studies of renal injury III: Lipid-induced nephropathy in type II diabetes. BACKGROUND: Nephrotoxicity from elevated circulating lipids occurs in experimental and clinical situations. We tested the hypothesis that lipid-induced nephropathy causes advanced renal failure in rats with type II diabetes and dyslipidemia. METHODS: First generation (F1) hybrid rats derived from the spontaneous hypertensive heart failure rat (SHHF/Gmi-fa) and the LA/NIH-corpulent rat (LA/N-fa) were studied for 41 weeks while being on specific diets. Group 1 (14 rats) ingested 11.5% protein, 47.9% fat, and 40.6% carbohydrate. Group 2 (8 rats) ingested 26.9% protein, 16.7% animal fat, and 56.4% carbohydrate, and group 3 (20 rats) ingested 20.2% protein, 40.4% soy and coconut oil, and 39.4% carbohydrate. RESULTS: Hyperglycemia was more severe in rat groups 1 and 2 than in group 3. In contrast, circulating cholesterol and hydroperoxide levels were highest in group 3, intermediate in group 2, and lowest in group 1. Group 3 had severe renal failure secondary to glomerulosclerosis and tubulointerstitial disease, with striking deposition of the lipid peroxidation stress biomarker 4-hydroxynonenal in glomeruli and renal microvessels. Moreover, in group 3, increased arterial wall thickness also connoted vascular injury. In contrast, the glycoxidation stress biomarkers pentosidine and carboxymethyl-lysine were preferentially localized to renal tubules of hyperglycemic rats in groups 1 and 2 and did not segregate with the most severe renal injury. Glomerular and interstitial fibrosis was accompanied by proportional increases in renal transforming growth factor-beta1 levels, which were threefold higher in the hypercholesterolemic rats of group 3 than in the hyperglycemic rats of group 1. CONCLUSIONS: Acquisition of non-nodular glomerular sclerosis and tubulointerstitial disease is dependent on lipoxidation stress in rats with type II diabetes. On the other hand, in the absence of hypercholesterolemia, prolonged glycoxidation stress does not appear to be uniquely nephrotoxic.

Mutational analysis of the defective protease in classic late-infantile neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disorder.

The late-infantile form of neuronal ceroid lipofuscinosis (LINCL) is a progressive and ultimately fatal neurodegenerative disease of childhood. The defective gene in this hereditary disorder, CLN2, encodes a recently identified lysosomal pepstatin-insensitive acid protease. To better understand the molecular pathology of LINCL, we conducted a genetic survey of CLN2 in 74 LINCL families. In 14 patients, CLN2 protease activities were normal and no mutations were identified, suggesting other forms of NCL. Both pathogenic alleles were identified in 57 of the other 60 LINCL families studied. In total, 24 mutations were associated with LINCL, comprising six splice-junction mutations, 11 missense mutations, 3 nonsense mutations, 3 small deletions, and 1 single-nucleotide insertion. Two mutations were particularly common: an intronic G-->C transversion in the invariant AG of a 3' splice junction, found in 38 of 115 alleles, and a C-->T transition in 32 of 115 alleles, which prematurely terminates translation at amino acid 208 of 563. An Arg-->His substitution was identified, which was associated with a late age at onset and protracted clinical phenotype, in a number of other patients originally diagnosed with juvenile NCL.

Altered mitochondrial function in canine ceroid-lipofuscinosis.

The neuronal ceroid-lipofuscinoses (NCL) are a group of autosomal recessively inherited neurodegenerative disorders characterized by progressive dementia, neuronal atrophy, and premature death. The late infantile and juvenile types of NCL show massive accumulation of mitochondrial ATP synthase subunit c protein in both mitochondria and lysosomes. The specific accumulation of this mitochondrial protein suggests that mitochondrial function may be impaired in the NCL diseases. Therefore, a study was conducted to determine whether oxidative phosphorylation is altered in liver mitochondria from English setters with NCL, an animal model in which there is also massive accumulation of the subunit c protein. The ADP/O ratios were significantly depressed in affected and carrier dogs, suggesting that the disease mutation led to a partial uncoupling of oxidative phosphorylation. On the other hand, ADP-stimulated respiration rates were higher than normal in both carriers and affected dogs. The increased respiration rates were highest in the carriers, and may reflect a compensatory response to the reduced efficiency of oxidative phosphorylation. Accompanying the increased respiration rates were elevations in mitochondrial ADP content with the elevation being greater in the carriers than in the affected dogs. This suggests that the increased respiration rates may be due, at least in part, to enhanced ADP uptake by the mitochondria. In the carriers, the enhanced respiration rate may be sufficient to offset the reduced efficiency of oxidative phosphorylation. In the affected animals, which had lower respiration rates than the carriers, the enhanced respiration rates may not be sufficient to offset the reduced efficiency of oxidative phosphorylation. Impaired mitochondrial function may therefore contribute to the disease pathology.

Molecular genetics of palmitoyl-protein thioesterase deficiency in the U.S.

Mutations in a newly described lysosomal enzyme, palmitoyl-protein thioesterase (PPT), were recently shown to be responsible for an autosomal recessive neurological disorder prevalent in Finland, infantile neuronal ceroid lipofuscinosis. The disease results in blindness, motor and cognitive deterioration, and seizures. Characteristic inclusion bodies (granular osmiophilic deposits [GROD]) are found in the brain and other tissues. The vast majority of Finnish cases are homozygous for a missense mutation (R122W) that severely affects PPT enzyme activity, and the clinical course in Finnish children is uniformly rapidly progressive and fatal. To define the clinical, biochemical, and molecular genetic characteristics of subjects with PPT deficiency in a broader population, we collected blood samples from U.S. and Canadian subjects representing 32 unrelated families with neuronal ceroid lipofuscinosis who had GROD documented morphologically. We measured PPT activity and screened the coding region of the PPT gene for mutations. In 29 of the families, PPT deficiency was found to be responsible for the neurodegenerative disorder, and mutations were identified in 57 out of 58 PPT alleles. One nonsense mutation (R151X) accounted for 40% of the alleles and was associated with severe disease in the homozygous state. A second mutation (T75P) accounted for 13% of the alleles and was associated with a late onset and protracted clinical course. A total of 19 different mutations were found, resulting in a broader spectrum of clinical presentations than previously seen in the Finnish population. Symptoms first appeared at ages ranging from 3 mo to 9 yr, and about half of the subjects have survived into the second or even third decades of life.

Coding sequence and exon/intron organization of the canine CLN3 (Batten disease) gene and its exclusion as the locus for ceroid-lipofuscinosis in English setter dogs.

Hereditary ceroid-lipofuscinosis in English setters has been proposed to be the canine equivalent of human juvenile ceroid-lipofuscinosis, which results from defects in the CLN3 gene. Analyses were performed to determine whether the disease in English setters is also the consequence of a CLN3 gene mutation. Canine CLN3 cDNA was found to contain a 1,314-bp open reading frame predicting a derived amino acid sequence which is 89%, 85%, and 84% identical to the predicted amino acid sequences for the human, mouse, and rabbit CLN3 proteins, respectively. The canine gene has sixteen exons. No differences were detected when cDNA nucleotide sequences from an English setter with ceroid-lipofuscinosis and from a normal dog were compared. Moreover, alleles of the canine CLN3 gene distinguished by an intragenic marker segregated independently from the disease in an English setter family, eliminating CLN3 as the locus for the canine disease. A ceroid-lipofuscinosis-affected Tibetan terrier was homozygous for a Gly70Glu CLN3 variant; however, this allele is common in dog breeds considered free of ceroid-lipofuscinosis.

Palmitoyl-protein thioesterase deficiency in a novel granular variant of LINCL.

Typically, late infantile neuronal ceroid-lipofuscinosis (LINCL) patients present between the ages of 2 and 4 years with progressive dementia, blindness, seizures, and motor dysfunction. Curvilinear profiles are seen on electron microscopic examination of tissues derived from those patients. Data were collected on 122 LINCL cases, representing 81 independent families, diagnosed on the basis of age of onset, clinical symptomatology, and pathologic findings. Careful analysis of our data has revealed that 20% of these cases (24 of 122) show either an atypical clinical course or atypical pathologic findings and may represent variants of LINCL. Recent progress in the biochemistry and molecular genetics of NCL has led us to reevaluate these atypical cases. Five atypical LINCL cases (representing three independent families) manifested granular inclusions when examined by electron microscopy, a finding normally associated with the infantile form of NCL. In addition, these five cases did not show elevated subunit c levels in urine (typically seen in LINCL). In these five cases, palmitoyl-protein thioesterase activity was found to be deficient (less than 10% normal activity), suggesting that these cases represent INCL, presenting at a later age of onset. These findings suggest that palmitoyl-protein thioesterase deficiency is not restricted to infantile onset cases, and they raise the possibility that milder forms of INCL may result from less deleterious mutations.

Acetaldehyde-modified and 4-hydroxynonenal-modified proteins in the livers of rats with alcoholic liver disease.

Liver proteins form adducts with acetaldehyde and are modified by products of lipid peroxidation in alcohol-fed animals. It has been hypothesized that the formation of these modified liver proteins may contribute to liver injury in alcoholic liver disease. The present work was performed to determine the extent of protein modification in rats with experimental alcoholic liver disease. Rats were fed ethanol intragastrically with medium chain triglycerides (MCTs), palm oil, corn oil, or fish oil. The group fed MCTs and ethanol showed no liver injury, rats fed palm oil and ethanol showed only fatty liver, rats fed corn oil and ethanol showed fatty liver with moderate necrosis and inflammation, and rats fed fish oil and ethanol showed fatty liver with severe necrosis and inflammation. Antibodies were raised by using keyhole limpet hemocyanin modified in vitro by 4-hydroxynonenal (4-HNE) or acetaldehyde as immunogens. When liver extracts were examined by Western blot analysis, the intensities of the acetaldehyde-modified protein band (37 kd) in the alcohol-fed animals were significantly different among the ethanol-treated groups and correlated with plasma acetaldehyde concentrations. It was strongest in rats fed fish oil and ethanol, followed by rats fed palm oil and ethanol and rats fed corn oil and ethanol, whereas rats fed MCTs and ethanol showed the weakest intensity. The 37-kd protein-adetaldehyde adduct was located mainly in the pericentral region of the liver. No acetaldehyde adduct was detected in the control rats that were pair-fed with isocaloric amounts of dextrose. Western blot analysis using the anti-4-HNE antibody showed four distinctive bands (48, 45, 40, and 38 kd) in the liver extracts of alcohol-fed rats. Control animals showed only a weak 38-kd band. Although the intensities of the 48-, 40-, and 38-kd bands were similar among the different ethanol-treated groups, the intensity of the 45-kd band decreased from MCTs and ethanol > palm oil and ethanol > or = corn oil and ethanol > fish oil and ethanol. The data indicate that the degree of liver protein modification by acetaldehyde correlates well with the severity of liver injury in ethanol-fed rats, whereas modification by the lipid peroxidation product 4-HNE shows no correlation with the severity of liver injury.

Late-infantile ceroid-lipofuscinosis: lysine methylation of mitochondrial ATP synthase subunit c from lysosomal storage bodies.

Late-infantile ceroid-lipofuscinosis is a fatal autosomal recessively inherited disease characterized by massive accumulations of lysosomal storage bodies in many tissues. A major constituent of the storage bodies is the subunit c protein of mitochondrial ATP synthase. Juvenile ceroid-lipofuscinosis, a disease that is similar to but genetically distinct from the late-infantile disorder, also involves lysosomal accumulation of the subunit c protein. In the juvenile disease, the stored form of the protein contains an epsilon-N-trimethyllysine (TML) residue at position 43. Analyses were performed to determine whether subunit c protein stored in the late-infantile disease is also trimethylated at lysine residue 43. Amino acid composition analysis of the subunit c protein stored in brains from subjects with the late-infantile disease indicated that one of the two lysine residues in the protein is trimethylated. Data from molecular mass analysis of the protein was consistent with the presence of three methyl groups not present in the unmodified protein. The TML in the storage body subunit c protein was found by amino acid sequence analysis to occur exclusively at residue 43. The lysine at this position in the stored protein was completely methylated. Recent studies suggest that the subunit c protein from normal mitochondria may also have the same amino acid modification. Thus, it appears that specific methylation of lysine residue 43 of mitochondrial ATP synthase subunit c is probably a normal post-translational modification, and that the lysosomal storage of this protein in late-infantile, as well as in juvenile ceroid-lipofuscinosis, does not result from a defect in its methylation.

Low molecular weight storage material in infantile ceroid lipofuscinosis (CLN1).

The identification of the genetic defect in CLN1 as a palmitoyl-protein thioesterase deficiency initiated a search for the lysosomal storage material. Pulse-chase labelling of fibroblasts and lymphoblastoid cell lines with [35S]cysteine revealed the presence of lipid [35S]cysteine material in CLN1 fibroblasts and not in controls, CLN2 or CLN3 patients or other patients with lipidosis. A single band comigrated with the acylcysteine standard and labelling with [3H]palmitate showed a band of material which eluted from the silicic acid column with the phospholipid fraction and which co-migrated with the lipid-[35S]cysteine band. The storage material is tentatively identified as palmitoylcysteine.

Mitochondrial abnormalities in CLN2 and CLN3 forms of Batten disease.

The storage of subunit c of mitochondrial ATP synthase, other hydrophobic peptides, and autofluorescent pigment in both late infantile (CLN2) and juvenile (CLN3) neuronal ceroid lipofuscinosis, but not in infantile (CLN1), has raised the question of abnormal mitochondrial function. We now report a partial deficiency in three types of fatty acid oxidation in intact skin fibroblasts from CLN2 and CLN3 patients, but not CLN1. We observed a statistically significant 33% reduction in palmitate (beta-oxidation; mainly mitochondrial) and lignocerate (beta-oxidation; mainly peroxisomal), and a 50% reduction in phytanic acid (alpha-oxidation; mainly peroxisomal) in the absence of exogenous carnitine. In contrast, when we measured fatty acid beta-oxidation (lignoceric acid and palmitic acid), in the same human skin fibroblasts, following lysis in the presence of carnitine, we found no difference in enzyme activity among normal, CLN1, CLN2, and CLN3. However, we did observe a 40% reduction in peroxisomal particulate (bound) catalase activity in CLN1 and CLN2 fibroblasts, which typically results from organellar lipid accumulation or a membrane abnormality. However, total catalase levels were normal, and Western blot analysis of this and three other major oxidant protective enzymes (Mn-dependent superoxide dismutase [MnSOD], CuZn-dependent superoxide dismutase [CuZnSOD], and glutathione peroxidase) were normal in CLN1, CLN2, and CLN3, as well as in liver from an animal (English Setter dog) model for CLN, which shows similar pathology and subunit c storage. Our data showing differences between CLN1 and forms CLN2 and CLN3 suggest some type of mitochondrial membrane abnormality as the source of the pathology in CLN2 and CLN3.

Low-molecular-weight, calcium-dependent phospholipase A2 genes are linked and map to homologous chromosome regions in mouse and human.

The Group IIA phospholipase gene (PLA2G2A) protein coding regions exhibit significant homology with recently described Group IIC (PLA2G2C) and Group V (PLA2GV) genes. All three genes are present in many mammalian species and are expressed in a tissue-specific pattern. Here, we demonstrate in human that they are tightly linked and map to chromosome 1p34-p36.1. We also show that the homologous mouse loci are tightly linked (no observed recombination) on the distal part of chromosome 4, a region exhibiting synteny with human 1p34-p36. Unlike its rodent counterpart, human PLA2G2C appears to be a nonfunctional pseudogene.

Diagnoses of neuronal ceroid-lipofuscinosis by immunochemical methods.

The neuronal ceroid-lipofuscinoses (NCL), also known as Batten disease, are a not uncommon group of disorders affecting infants, children, and young adults. The abnormal ultrastructural profiles seen in NCL are used for standard diagnosis; however, they can be missed, and are also found in other neurodegenerative conditions. Furthermore, there is an overlap between the types of inclusion profiles among the different forms of NCL. Therefore, a more specific and biochemically-based marker is necessary to confirm the diagnosis of NCL. Antibodies raised against the storage material from the ovine form of NCL (mitochondrial ATP synthase subunit c) were utilized to determine whether NCL could be distinguished from other metabolic-neurodegenerative disorders. By immunoblotting and immunohistochemistry, several brain samples of well-evaluated NCL cases confirmed increased accumulations in all NCL cases except in the brain of an infantile-onset NCL patient. The immunoblot studies of skin fibroblasts and brain were sensitive but not highly specific to NCL, due to the recognition of this material in normal controls as well as in other neurogenetic diseases. Immunocytochemistry of skin fibroblasts clearly distinguished LINCL and JNCL cases from controls, and with further refinement has the potential for becoming a diagnostic tool.

Early detection of canine ceroid-lipofuscinosis (CCL): an ultrastructural study.

English setters from the Koppang line have been used as a model for the juvenile type of Batten disease in human patients. This disorder, canine ceroid-lipofuscinosis (CCL), has been shown by Koppang [1973: Mech Ageing Dev 2:421-445] to be an autosomal recessive disorder. Homozygous animals show the typical storage granules by autofluorescence; however, the autofluorescence material cannot easily be detected in affected dogs before age 3-6 months. At the present time the early stages of CCL can be studied only in litters from homozygous matings. Electron microscopy was used to study the reliability of diagnosing affected animals in heterozygous-heterozygous (HET-HET) and heterozygous-homozygous (HET-HOM) litters at age 2-3 weeks. The electron micrographs of brain biopsies were examined and the number of all dense bodies (d.b.), including linear and fingerprint patterns typical of CCL, were counted per neuron. The results indicated that affected animals have higher numbers of dense bodies than nonaffected carriers. Four-6 months later confirmatory diagnoses were made on a second contralateral biopsy taken and examined as a frozen section by autofluorescence. The diagnoses made by electron microscopy at age 2-3 weeks appeared identical to the results obtained by autofluorescence at 4-6 months of age.

Canine hereditary ceroid-lipofuscinosis: evidence for a defect in the carnitine biosynthetic pathway.

The ceroid-lipofuscinoses are a group of autosomal-recessive hereditary lysosomal storage diseases that have been characterized in humans and other mammalian species. In a canine model for the juvenile form of the human disease, a major constituent of the storage bodies is the subunit c protein of mitochondrial ATP synthase that contains an epsilon-N-trimethyllysine (TML) residue. TML is a precursor in carnitine biosynthesis. To determine whether accumulation of the TML-containing protein could result from a defect in the carnitine biosynthetic pathway, plasma carnitine and trimethyllysine levels were measured in homozygous affected, heterozygous carriers, and in normal dogs. When compared top normal animals, mean carnitine levels were reduced by 67% in affected and 50% in carrier dogs. Mean plasma TML levels were elevated almost 50% above control levels in the carriers, but were decreased by approximately 25% in the affected animals. The changes in plasma carnitine and TML levels in the carriers are consistent with the possibility that the disease involves a defect in the carnitine biosynthetic pathway. Secondary effects of the disease process may account for the apparently contradictory decrease in plasma TML levels in affected animals.

Biosynthesis and metabolism of 4-hydroxynonenal in canine ceroid-lipofuscinosis.

Canine ceroid-lipofuscinosis (CCL) is a model of the juvenile type of Batten disease in human patients. Abnormalities have been reported previously in 4-hydroxynonenal (HNE) levels in English setters with CCL. The purpose of this study was to examine the sources of HNE in neutrophil membranes and plasma of CCL dogs. The fatty acid composition of neutrophil phospholipids, i.e., phosphatidyl ethanolamine and phosphatidyl serine, was determined by gas-liquid-chromatography (GLC) since some polyunsaturated fatty acids (PUFA) are precursors of HNE. The copper catalyzed peroxidation of low density lipoprotein (LDL) was examined to determine the susceptibility of LDL from CCL dogs to peroxidation. The results indicated that a number of PUFA precursors of HNE decreased in affected an carrier neutrophil phospholipids, indicating that this source of HNE may be disease specific. The Cu++ catalyzed formation of HNE from LDL demonstrated that carrier and normal LDL produced large amounts of HNE, while LDL from affected dogs required much higher concentrations of Cu++ for maximal HNE production. These results provide additional support for the role of HNE in the pathogenetic events in NCL and support the view that lipid peroxidation may be an important contributor to the complex pathogenesis of the NCL.

Quantitative measurement of 4-hydroxyalkenals in oxidized low-density lipoprotein by gas chromatography-mass spectrometry.

Previously, we reported on the determination of 4-hydroxyalkenals as pentafluorobenzyl oxime derivatives by gas chromatography-mass spectrometry. In this study, an improved method is presented which allows quantitative detection of 4-hydroxyalkenals in tissues by using two stable isotope-labeled internal standards, 9D3-4-hydroxynonenal and 9D3-4-hydroxyhexenal. This assay was used to quantitate 4-hydroxyalkenals in copper-oxidized human low-density lipoprotein, and we found more 4-hydroxynonenal than 4-hydroxyhexenal. This is consistent with the fact that there are more omega-6 than omega-3 fatty acids in human low-density lipoprotein samples, which are the sources of 4-hydroxynonenal and 4-hydroxyhexenal, respectively.

Lysine methylation of mitochondrial ATP synthase subunit c stored in tissues of dogs with hereditary ceroid lipofuscinosis.

Certain forms of ceroid lipofuscinosis, a hereditary degenerative disease, are characterized by accumulation of large amounts of subunit c of mitochondrial ATP synthase in lysosomal storage bodies of numerous tissues. The subunit c protein appears to constitute a major fraction of the total storage body protein. In previous studies it was demonstrated that hydrolysates of total storage body protein from affected humans and sheep contain significant amounts of epsilon-N-trimethyllysine (TML). This finding suggested that one or both of the two lysine residues of subunit c might be methylated in the stored form of the protein. The normal subunit c protein from mitochondria does not appear to be methylated. Using a putative canine model for the juvenile form of ceroid lipofuscinosis, analyses were conducted to determine whether lysosomal storage of subunit c was accompanied by lysine methylation of this protein. In affected dogs, as in humans and sheep with hereditary ceroid lipofuscinosis, the storage bodies were found to contain large amounts of subunit c protein, as indicated by polyacrylamide gel electrophoresis and partial amino acid sequence analysis. The subunit c protein partially purified from isolated storage bodies was found to contain lysine and TML in an almost equimolar ratio. Normal subunit c contains 2 lysine residues, one at position 7 and the other at position 43. Removal of the first 7 residues of the partially purified protein through sequential Edman degradation resulted in a dramatic increase in the TML to lysine ratio in the residual protein. This suggests that lysine residue 43 is methylated. Confirmation that residue 43 of the stored protein is TML was obtained by amino acid sequence analysis after cleavage of the protein with trypsin. This finding strongly suggests that specific methylation of lysine residue 43 of mitochondrial ATP synthase plays a central role in the lysosomal storage of this protein.

The A-1 antigen: a novel marker in experimental peripheral nerve injury.

Expression of the Schwann cell phenotype is regulated by signals from the adjoining axon. After axotomy, the Schwann cell ceases the production and maintenance of the myelin sheath and assumes phagocytic properties necessary to digest its own myelin. The molecular mechanisms responsible for this behavior remain unclear. A monoclonal antibody termed BIKS was produced after the immunization of mice with guinea pig lymphoid tissue. This antibody recognizes a cytoplasmic vesicle-associated molecule (A-1 antigen) which is abundant in all tissue macrophages but is also expressed in small amounts in normal Schwann cells. Following axotomy, the A-1 antigen appears to be translocated from a perinuclear site to accumulate in large quantities around myelin ovoids in Schwann cells, as well at the nodes of Ranvier-sites where Wallerian degeneration is known to commence. The level of the antigen remains high when axons are prevented from regeneration. During repair of crush injury, however, the level of antigen drops concomitant with the ingrowth of regenerating axons, suggesting axonal control of A-1 antigen expression.