Aristotle N. Siakotos

The Morphogenesis and Biochemical Characteristics of Ceroid Isolated from Cases of Neuronal Ceroid-Lipofuscinosis

Two distinctly different groups of disorders have emerged from the conditions generically classified as amaurotic familial idiocies. One is characterized by grossly abnormal profiles for cerebral sphingolipids, for example the GM1 and GM2 gangliosides. The other group is composed of patients with normal sphingolipid profiles, but with neuronal accumulation of lipopigments of the ceroid-lipofuscin type. The sphingolipidoses have been shown by a number of investigators to meet the classic concept of Hers (1965) for lysosomal diseases. This view has been repeatedly reinforced by continuing studies which show the lack or reduction of specific hydrolases, resulting in the accumulation of biochemical compounds which cannot be degraded to metabolically utilizable substances. The concept of lysosomal diseases has led many investigators to search for a single accumulating lipid or a deficient hydrolytic enzyme unique to neuronal ceroid-lipofuscinoses. Since many of the lysosomal disorders became better understood by the elucidation of the chemical properties of a specific lipid, present in unphysiologically large quantities, the pronouncement by Donahue et al. (1966) before this group, that the “chemical analysis of cytoplasmic lipopigment granules (from the brains of patients with Batten’s disease) will contribute little towards an understanding of this disease” was decidedly unwise. Although it is correct, that autofluorescent lipopigments lack chemical specificity and result from a great variety of pathogenetic situations (Porta and Hartroft, 1969), Donahue et al. (1966) failed to recognize the distinct possibility, that certain repetitive patterns in the chemical composition and ultrastructure of these residual bodies may indicate a specific formative pathogenesis. On the strength of this argument, we developed methods to isolate lipopigments in pure preparations and the first results have proved already the soundness of this concept. As it turned out, the previously held concept of a close relationship between lipofuscin and ceroid had to be abandoned and replaced by the theses that these classes of lipopigment are distinctly different entities, albeit both represent residual bodies and both contain polymeric substances (Siakotos et al., 1970).

Early lesion of Pelizaeus-Merzbacher disease: electron microscopic and biochemical study.

Ultrastructural and biochemical studies on the cerebral biopsy of a 3-month-old male with a clinical diagnosis of Pelizaeus-Merzbacher disease revealed normal myelin sheaths but characteristic alterations in the oligodendrocytes. They displayed unique vacuoles with concentric lamellae in the perikarya and numerous myelin balls in the terminal processes. Very few macrophages containing non-specific sudanophilic lipids were encountered. Cerebroside concentration and several acid hydrolases were normal. The above findings differed distinctly from those obtained on a previous unrelated case of Pelizaeus-Merzbacher disease (35). It remains unknown whether these differences are an expression of genetic heterogeneity.

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.

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.

Improved Synthesis of Various Isotope Labeled 4-Hydroxyalkenals and Peroxidation Intermediates

Abstract A concise and isotope specific synthesis of perfluorobenzyl 5,5,6,6-tetradeutero-9-oxononanoate and an abbreviated general multiisotope synthesis of 4-hydroxyalkenals is reported.

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. J. Neurosci. Res. 52:268–275, 1998.

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.

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.

Elevated levels of neutrophil 4-hydroxynonenal in canine neuronal ceroid-lipofuscinosis and human immortalized lymphocytes of NCL patients

The hallmark of the neuronal ceroid lipofuscinoses (NCL), or Batten disorders, is the massive accumulation of ceroid/lipofuscin or autofluorescent lipopigments in tissues, especially in brain and the eye (Zeman 1976). Zeman (1976) hypothesized that the NCL were a result of the increased formation or diminished breakdown of lipid hydroperoxides from membrane polyunsaturated fatty acids (PUFA). Decreases in PUFA levels in brain samples and blood cells of NCL patients have been observed (Hagberg et al 1968; Pullarkat et al 1978, 1982). Hypotheses to explain these observations have included deficiencies of peroxidases (Armstrong et al 1974a,b) or reduced levels of antioxidants (Zeman 1976). While both of these hypotheses have largely been refuted (Rider et al 1992), abnormalities in the metabolism or production of secondary products of lipid peroxidation have not been ruled out in the NCL. Our laboratory reported significantly higher levels of 4-hydroxynonenal (HNE), a secondary product of lipid peroxidation, in blood cells and tissues in English setters with NCL and in carriers (Siakotos et al 1988). In this study we present new data on the mechanism of HNE production in canine NCL and preliminary data showing elevated HNE levels in immortalized human lymphocytes from NCL patients.

Reduced phospholipase activity, peptide storage and the pathogenesis of canine neuronal ceroid-lipofuscinosis

The spectrum of neuronal ceroid lipofuscinosis (CNL) encompasses the infantile (gene assigned to the short arm of chromosome 1), late-infantile (unassigned but not 1 or 16), juvenile (assigned to chromsome 16) through adult forms, with many variants (Rider et al 1992). The common biochemical thread is the deposition of autofluorescent pigment of granular, curvilinear or fingerprint nature, the storage of PAS-positive dolichol mannosides (Hall and Patrick 1988), hydrophobic peptides (Palmer et al 1989, 1992), evidence of peroxidative lipid damage in the form of conjugated 4-hydroxynonenals (Siakotos et al 1988), altered polyunsaturated fatty acids (Dawson et al 1990) and reduced lysosomal phospholipase A1 activity, especially in neurons. Much attention in recent years has focused on the accumulation of the hydrophobic (DCCD-binding) subunit c of mitochondrial ATP synthetase in the late-infantile form (CLN2) and, to a lesser extent, in the juvenile form (CNL3) and the sheep model of CNL3, but this has not led to elucidation of the gene defect. Thus the genes for the c-peptide are on chromosomes 12 and 17, and the highly conserved approximately 40 amino acid sequence is completely normal in sheep and human CNL3. CNL1 patients appear to store a different hydrophobic peptide and Jolly (1993) has suggested a pattern of hydrophobic peptide storage in certain disease types. For example, the autofluorescent age pigment in the thyroid consists largely of dolichols and a hydrophobic peptide derived from thyroglobulin. This has led to a working hypothesis that the gene defect results in impaired disposal of a hydrophobic protein, which might be diseaseand tissue-specific. At present very little is known of the mechanisms by which organelles such as mitochondria are disposed of and the peptide or carbohydrate motifs that might be involved in this trafficking. Katz and Rodrigues (1991) have observed the presence of S-methylmethionine in CNL2 and methylhistidine in CNL3 storage bodies, suggesting that methylation could be blocking normal disposal signals analogous to the ubiquitin pathway (Hershko 1988). However, others (Palmer et al 1993) have failed to find evidence for the covalent linkage of such amino acid derivatives to the storage material in CNL2 or CNL3. By analogy with the retinal degenerative diseases, which largely result from inherited point mutations in rhodopsin and the