Disruption of PPT1 or PPT2 causes neuronal ceroid lipofuscinosis in knockout mice.

PPT1 and PPT2 encode two lysosomal thioesterases that catalyze the hydrolysis of long chain fatty acyl CoAs. In addition to this function, PPT1 (palmitoyl-protein thioesterase 1) hydrolyzes fatty acids from modified cysteine residues in proteins that are undergoing degradation in the lysosome. PPT1 deficiency in humans causes a neurodegenerative disorder, infantile neuronal ceroid lipofuscinosis (also known as infantile Batten disease). In the current work, we engineered disruptions in the PPT1 and PPT2 genes to create "knockout" mice that were deficient in either enzyme. Both lines of mice were viable and fertile. However, both lines developed spasticity (a "clasping" phenotype) at a median age of 21 wk and 29 wk, respectively. Motor abnormalities progressed in the PPT1 knockout mice, leading to death by 10 mo of age. In contrast, the majority of PPT2 mice were alive at 12 mo. Myoclonic jerking and seizures were prominent in the PPT1 mice. Autofluorescent storage material was striking throughout the brains of both strains of mice. Neuronal loss and apoptosis were particularly prominent in PPT1-deficient brains. These studies provide a mouse model for infantile neuronal ceroid lipofuscinosis and further suggest that PPT2 serves a role in the brain that is not carried out by PPT1.

Infantile neuronal ceroid lipofuscinosis: no longer just a 'Finnish' disease.

The neuronal ceroid lipofuscinoses (NCLs) are a group of enigmatic neurodegenerative disorders of children that have in common the storage of autofluorescent lipofuscin, or aging pigment, in the brain. With the identification of the three major genes involved in the disorder, the NCLs are now appreciated to represent true lysosomal storage disorders. The most severe (infantile) form of NCL is caused by mutations in a lysosomal thioesterase that removes fatty acids from modified cysteine residues in proteins. Although the disorder was first described in Finland (and the identification of the underlying gene (CLN1) made in this population) defects in CLN1 and the underlying deficiency have been widely reported outside of Scandinavia. In this report, we summarize the relationship of genotype to phenotype in the disorder and evaluate known mutations in light of the recently solved crystal structure of defective enzyme, palmitoyl-protein thioesterase (PPT). We also discuss progress in identifying the fatty acyl cysteine thioesters that accumulate in PPT deficiency and in working toward animal models of NCL. Recent progress in these areas holds promise for the eventual treatment of the disorder.

Tripeptidyl-peptidase I in neuronal ceroid lipofuscinoses and other lysosomal storage disorders.

The classic late infantile form of neuronal ceroid lipofuscinosis (CLN2, cLINCL) is associated with mutations in the gene encoding tripeptidyl-peptidase I (TPP-I), a lysosomal aminopeptidase that cleaves off tripeptides from the free N-termini of oligopeptides. To date over 30 different mutations and 14 polymorphisms associated with CLN2 disease process have been identified. In the present study, we analysed the molecular basis of 15 different mutations of TPP-I by using immunocytochemistry, immunofluorescence, Western blotting, enzymatic assay and subcellular fractionation. In addition, we studied the expression of TPP-I in other lysosomal storage disorders such as CLN1, CLN3, muccopolysaccharidoses and GM1 and GM2 gangliosidoses. Our study shows that TPP-I is absent or appears in very small amounts not only in cLINCL subjects with mutations producing severely truncated protein, but also in individuals with missense point mutations, which correlates with loss of TPP-I activity. Of interest, small amounts of TPP-I were detected in lysosomal fraction from fibroblasts from cLINCL subject with protracted form. This observation suggests that the presence of small amounts of TPP-I in lysosomes is able to delay significantly CLN2 disease process. We also show that TPP-I immunoreactivity is increased in the brain tissue of CLN1 and CLN3 subjects, stronger in glial cells and macrophages than neurons. Less prominent increase of TPP-I staining was found in muccopolysaccharidoses and GM1 and GM2 gangliosidoses. These data suggest that TPP-I participates in lysosomal turnover of proteins in pathological conditions associated with cell/tissue injury.

CLN3 disease process: missense point mutations and protein depletion in vitro.

Although the CLN3 gene associated with the disease process in subjects with the juvenile form of neuronal ceroid lipofuscinosis was discovered in 1995, our knowledge of the physiological function of its gene product, CLN3 protein, is still incomplete. To gain more insight into the structural properties and function of CLN3 protein we studied at present: i) how the naturally occurring point mutations Arg334Cys and Leu101Pro affect the biological properties of CLN3 protein, and ii) whether depletion of CLN3 protein synthesis by using an antisense approach induces a distinct phenotype in cells of neuronal origin in vitro. Here we report that although both CLN3 mutant proteins are targeted to lysosomes, thus similar to wild-type CLN3 protein, they are devoid of the biological activity of wild-type CLN3 protein such as its effect on lysosomal pH or intracellular processing of amyloid-beta protein precursor and cathepsin D in vitro. The Leu101Pro mutation affected significantly the maturation and stability of CLN3 protein. The Arg334Cys mutation influenced mildly the maturation and turnover of CLN3 protein, but at the same time abolished the function of CLN3 protein in vitro, which suggests that the Arg334 may constitute a part of the active site of CLN3 protein. In addition, we show that depletion of CLN3 protein synthesis in human neuroblastoma cells in vitro induces outgrowth of long cellular processes and formation of cellular aggregates and affects the viability of these cells. This finding suggests that CLN3 protein is implicated in biological processes associated with the differentiation of cells of neuronal origin.

Impaired temporo-occipital blood flow in an atypical CLN1 case with late infantile onset and granular osmiophilic deposits.

A 5-year-old boy presented with frequent absences. Speech began to regress. He became ataxic, barely able to walk. Studies with Xe-133 and hexamethylpropylene amine oxime single-photon emission computed tomography revealed sharply decreased cerebral blood flow, especially in the occipital area. Landau-Kleffner syndrome was suspected but a sleep electroencephalogram showed few abnormalities. He was started on clorazepate and diltiazem. A skin biopsy to rule out possible CLN2 revealed, instead of the predicted curvilinear profiles, granular osmiophilic deposits, consistent with infantile neuronal ceroid lipofuscinosis (CLN1). The family reported increased seizure frequency and consulted with a colleague, who advised them to resume valproate and discontinue diltiazem. The boy died shortly thereafter. Decreased cerebral blood flow is a new finding in CLN1 with delayed onset. Calcium-channel blockers improve cerebral blood flow and perhaps delay clinical regression.

Pheno/genotypic correlations of neuronal ceroid lipofuscinoses.

The neuronal ceroid lipofuscinoses (NCL) are a large group of autosomal recessive lysosomal storage disorders with both enzymatic deficiency and structural protein dysfunction. Previously, diagnosis of NCL was based on age at onset and clinicopathologic (C-P) findings, classified as 1) infantile (INCL), 2) late infantile (LINCL), 3) juvenile (JNCL), and 4) adult (ANCL). Most patients with NCL have progressive ocular and cerebral dysfunction, including cognitive/motor dysfunction and uncontrolled seizures. After reviewing 319 patients with NCL, the authors found that 64 (20%) did not fit into this classification of NCL. With research progress, four additional forms have been recognized: 5) Finnish, 6) Gypsy/Indian, and 7) Turkish variants of LINCL and 8) northern epilepsy, also known as progressive epilepsy with mental retardation. These eight NCL forms resulted from 100 different mutations on genes CLN1to CLN8 causing different phenotypes (http://www.ucl.ac.uk/ncl). The genes CLN1 and CLN2 encode lysosomal palmitoyl protein thioesterase and tripeptidyl peptidase 1. The function of CLN3, CLN5, and CLN8 gene-encoded products is unknown, although their predicted amino acid sequences suggest they have a transmembrane topology. The diagnosis of NCL is based on C-P findings, enzymatic assay, and molecular genetic testing. Before biochemical and genetic tests are conducted, ultrastructural studies (i.e., blood [buffy coat] or punch biopsies [skin, conjunctiva]) must be performed to confirm the presence and nature of lysosomal storage material (fingerprint or curvilinear profiles or granular osmiophilic deposits). The recognition of variable onset from infancy to middle age supersedes the traditional emphasis on age-related NCL forms.

Neuronal ceroid lipofuscinoses: classification and diagnosis.

The neuronal ceroid lipofuscinoses (NCLs) are neurodegenerative disorders characterized by accumulation of ceroid lipopigment in lysosomes in various tissues and organs. The childhood forms of the NCLs represent the most common neurogenetic disorders of childhood and are inherited in an autosomal-recessive mode. The adult form of NCL is rare and shows either an autosomal-recessive or autosomal dominant mode of inheritance. Currently, five genes associated with various childhood forms of NCLs, designated CLN1, CLN2, CLN3, CLN5, and CLN8, have been isolated and characterized. Two of these genes, CLN1 and CLN2, encode lysosomal enzymes: palmitoyl protein thioesterase 1 (PPT1) and tripetidyl peptidase 1 (TPP1), respectively. CLN3, CLN5, and CLN8 encode proteins of predicted transmembrane topology, whose function has not been characterized yet. Two other genes, CLN6 and CLN7, have been assigned recently to small chromosomal regions. Gene(s) associated with the adult form of NCLs (CLN4) are at present unknown. This study summarizes the current classification and new diagnostic criteria of NCLs based on clinicopathological, biochemical, and molecular genetic data. Material includes 159 probands with NCL (37 CLNI, 72 classical CLN2, 10 variant LINCL, and 40 CLN3) collected at the New York State Institute for Basic Research in Developmental Disabilities (IBR) as well as a comprehensive review of the literature. The results of our study indicate that although only biochemical and molecular genetic studies allow for definitive diagnosis, ultrastructural studies of the biopsy material are still very useful. Thus, although treatments for NCLs are not available at present, the diagnosis has become better defined.

Outlook for future treatment.

Currently, no treatment is available for neuronal ceroid lipofuscinoses. The progress of human genome project will stimulate molecular cloning of unidentified genes underlying the NCLs, which will lead eventually clinical management and therapies for NCL. Characterizing the native substrate(s) for the palmitoyl-protein thioesterase-1 (PPT1) and tripeptidyl peptidase 1 (TPP1), understanding the protein functions encoded by CLN genes, and uncovering the pathological metabolic mechanism for the NCLs are the bases of designing rational treatments for the NCLs. Testing potential therapeutic agents, replacing deficient enzymes, and developing gene therapy will be the major tasks for NCL researchers.

Cellular pathology and pathogenic aspects of neuronal ceroid lipofuscinoses.

Lysosomal accumulation of autofluorescent, ceroid lipopigment material in various tissues and organs is a common feature of the neuronal ceroid lipofuscinoses (NCLs). However, recent clinicopathologic and genetic studies have evidenced that NCLs encompass a group of highly heterogeneous disorders. In five of the eight NCL variants distinguished at present, genes associated with the disease process have been isolated and characterized (CLN1, CLN2, CLN3, CLN5, CLN8). Only products of two of these genes, CLN 1 and CLN2, have structural and functional properties of lysosomal enzymes. Nevertheless, according to the nature of the material accumulated in the lysosomes, NCLs in humans as well as natural animal models of these disorders can be divided into two major groups: those characterized by the prominent storage of saposins A and D, and those showing the predominance of subunit c of mitochondrial ATP synthase accumulation. Thus, taking into account the chemical character of the major component of the storage material, NCLs can be classified currently as proteinoses. Of importance, although lysosomal storage material accumulates in NCL subjects in various organs, only brain tissue shows severe dysfunction and cell death, another common feature of the NCL disease process. However, the relation between the genetic defects associated with the NCL forms, the accumulation of storage material, and tissue damage is still unknown. This chapter introduces the reader to the complex pathogenesis of NCLs and summarizes our current knowledge of the potential consequences of the genetic defects of NCL-associated proteins on the biology of the cell.

Lysosomal ceroid depletion by drugs: therapeutic implications for a hereditary neurodegenerative disease of childhood.

Neuronal ceroid lipofuscinoses (NCLs) are the most common hereditary neurodegenerative diseases of childhood. The infantile form, INCL, is caused by lysosomal palmitoyl-protein thioesterase (PPT) deficiency, which impairs the cleavage of thioester linkages in palmitoylated proteins, preventing their hydrolysis by lysosomal proteinases. Consequent accumulation of these lipid-modified proteins (constituents of ceroid) in lysosomes leads to INCL. Because thioester linkages are susceptible to nucleophilic attack, drugs with this property may have therapeutic potential for INCL. We report here that two such drugs, phosphocysteamine and N-acetylcysteine, disrupt thioester linkages in a model thioester compound, [14C]palmitoyl approximately CoA. Most importantly, in lymphoblasts derived from INCL patients, phosphocysteamine, a known lysosomotrophic drug, mediates the depletion of lysosomal ceroids, prevents their re-accumulation and inhibits apoptosis. Our results define a novel pharmacological approach to lysosomal ceroid depletion and raise the possibility that nucleophilic drugs such as phosphocysteamine hold therapeutic potential for INCL.

Distribution of tripeptidyl peptidase I in human tissues under normal and pathological conditions.

Tripeptidyl peptidase I (TPP I) is a lysosomal exopeptidase that cleaves tripeptides from the free N-termini of oligopeptides. Mutations in this enzyme are associated with the classic late-infantile form of neuronal ceroid lipofuscinosis (CLN2), an autosomal recessive disorder leading to severe brain damage. To gain more insight into CLN2 pathogenesis and the role of TPP I in human tissues in general, we analyzed the temporal and spatial distribution of TPP I in the brain and its localization in internal organs under normal and pathological conditions. We report that TPP I immunoreactivity appears in neurons late in gestation and increases gradually in the postnatal period, matching significantly the final differentiation and maturation of neural tissue. Endothelial cells, choroid plexus, microglial cells, and ependyma showed TPP I immunostaining distinctly earlier than neurons. Acquisition of the adult pattern of TPP I distribution in the brain at around the age of 2 years correlates with the onset of clinical signs in CLN2 subjects. In adults, TPP I was found in all types of cells in the brain and internal organs we studied, although the intensity of TPP I labeling varied among several types of cells and showed a noticeable predilection for cells and/or organs associated with peptide hormone and neuropeptide production. In addition, TPP I immunoreactivity was increased in aging brain, neurodegenerative and lysosomal storage disorders, and some differentiated neoplasms and was reduced in ischemic/anoxic areas and undifferentiated tumors. These findings suggest that TPP I is involved in general protein turnover and that its expression may be controlled by various regulatory mechanisms, which highlights the importance of this enzyme for normal function of cells and organs in humans.

Neuronal ceroid lipofuscinoses: research update.

This study describes the phenotype/genotype analysis of 159 probands with neuronal ceroid lipofuscinosis (37 CLN1, 72 classic CLN2, 10 variant LINCL, and 40 CLN3) collected at the New York State Institute for Basic Research in Developmental Disabilities (IBR). Phenotype/genotype comparison showed that mutations in the CLN1 gene were associated with different phenotypes: infantile, late infantile, and juvenile. Two common mutations (223A-->C and 451C-->T) were found in 26 of 37 CLN1 subjects (64% of alleles examined). A nonsense point mutation, 451C-->T, was the most common in CLN1 subjects with infantile onset at 0-2 years, accounting for 50% of alleles studied. A missense point mutation, 223A-->C, was the most common among CLN1 subjects with juvenile onset older than 4 years, accounting for 45% of alleles examined. Twenty-one other CLN1 mutations were identified in 4 of 37 subjects with infantile onset, 6 of 37 with late-infantile onset, and 6 of 37 with juvenile onset. All CLN1 probands were palmitoyl-protein thioesterase (PPT)-deficient and showed granular osmiophilic deposits (GROD) at the electron microscopic (EM) level. In the group of classic CLN2 (72 probands), two common mutations were found: an intronic 3556G-->C transversion in the invariant AG of 3' splice junction in 55% of probands, and a nonsense mutation 3670C-->T in 30% of probands. Classic late-infantile onset (2-4 years) was found in 68 of 72 (95%) cases, whereas juvenile onset (> 4 years) occurred only in 4 of 72 (5%) cases. All probands had deficiency of tripeptidyl-peptidase I (TPP1) activity and, at the EM level, curvilinear profiles. Ten probands with late-infantile onset did not show mutations in the CLN2 gene, had normal TPP1 activity, and at the EM level had mixed profiles. Further studies are in progress to identify genetic defect(s) in these subjects. The CLN3 group (40 probands) was divided into two categories: classic or typical presentation, and delayed classic or atypical presentation. All CLN3 patients had onset of symptoms after 4 years of age. In 40 probands, the 1.02-kb common deletion was found in one or two alleles of the CLN3 gene. Homozygotes for the common CLN3 deletion showed the classic phenotype. The phenotype in compound heterozygotes was either the classic or the delayed classic or atypical form. Thus, our study indicates that some mutations in the CLN1 and CLN2 genes may be associated with juvenile onset of the disease process and a more benign clinical course. Interfamilial and intrafamilial variations also were found, especially in the speed of becoming blind and neurologically disabled.

CLN-encoded proteins do not interact with each other.

The lysosomal storage of lipofuscins is the common pathological feature that characterizes the infantile, late-infantile, juvenile (Batten's disease), and Finnish-variant neuronal ceroid lipofuscinosis (INCL, LINCL, JNCL and FNCL), which are due to mutations in the genes CLN1, CLN2, CLN3, and CLN5, respectively. The CLN1 and CLN2 genes encode lysosomal enzymes, but the CLN3 and CLN5 genes encode membrane-spanning proteins. Why deficiencies of lysosomal enzymes and membrane-spanning proteins produce similar clinical phenotypes and pathological changes is still unanswered. We hypothesize that CLN-encoded proteins may comprise a functional pathogenic pathway, in which protein associations may play important roles. To test this hypothesis, we studied protein-protein interactions among the CLN1-, CLN2-, and CLN3-encoded proteins using a yeast two-hybrid system. Our results provided no evidence that CLN-encoded proteins interact with each other. This suggests there may be unidentified components in NCL pathogenesis.

CLN3 protein regulates lysosomal pH and alters intracellular processing of Alzheimer's amyloid-beta protein precursor and cathepsin D in human cells.

Maintenance of the appropriate pH in the intracellular vacuolar compartments is essential for normal cell function. Here, we report that CLN3 protein, which is associated with the juvenile form of neuronal ceroid lipofuscinosis (JNCL), participates in lysosomal pH homeostasis in human cells. We show that CLN3 protein increases lysosomal pH in cultured human embryonal kidney cells, whereas inhibition of CLN3 protein synthesis by antisense approach acidifies lysosomal compartments. These changes in lysosomal pH are sufficient to exert a significant biological effect and modify intracellular processing of amyloid-beta protein precursor and cathepsin D, model proteins whose metabolism is influenced by the pH of acidic organelles. Mutant CLN3 protein (R334C) that is associated with the classical JNCL phenotype was devoid of biological activities of wild-type CLN3 protein. These data suggest that the pathogenesis of juvenile neuronal ceroid lipofuscinosis is associated with altered acidification of lysosomal compartments. Furthermore, our study indicates that CLN3 protein affects metabolism of proteins essential for cell functions, such as amyloid-beta protein precursor, implicated in Alzheimer's disease pathogenesis.

Temporal accrual of complement proteins in amyloid plaques in Down's syndrome with Alzheimer's disease.

The complement system constitutes a series of enzymatic steps involved in the inflammatory response and is activated in Alzheimer's disease (AD). Using Down's syndrome (DS) brains as a temporal model for the progression of AD, we examined components of the complement cascade and their relationship to other principal events in AD pathology: Abeta42 deposition, neuritic changes, neurofibrillary tangles (NFTs), and gliosis (reactive astrocytes, activated microglia). Adjacent sections of frontal cortex from 24 DS subjects ranging in age from 12 to 73 years were immunohistochemically examined for immunoreactivity (IR) of classical complement proteins (Clq and C3), markers indicating activation of complement (C4d and C5b-9), the complement inhibitor apolipoprotein J (apo J), and markers of AD neuropathology. Abeta42-labeled diffuse plaques were first detected in a 12-year-old DS subject and were not labeled by any of the complement antibodies. Colocalization of Abeta42 with Clq, C3, C4d, and/or apo J was first detected in compacted plaques in the brain of a 15-year-old DS patient with features of mature AD pathology, such as reactive astrocytes, activated microglia, dystrophic neurites, and a few NFTs. IR for C4d and C5b-9 (membrane attack complex, MAC) was observed in small numbers of plaque-associated dystrophic neurites and in focal regions of pyramidal neurons in this 15-year-old. The only other young (

Identification of three novel mutations of the palmitoyl-protein thioesterase-1 (PPT1) gene in children with neuronal ceroid-lipofuscinosis.

Eight unrelated children with progressive neurological deterioration and granular osmiophilic deposits (GROD) due to an underlying palmitoyl-protein thioesterase deficiency were analyzed for mutations in the PPT1 gene. Three novel mutations (G118D, Q291X and F84del) were identified. The novel Q291X mutation was observed in an African-American child. The G118D and Q291X mutations occurred in infantile-onset subjects. These two mutations would be predicted to have severe effects on enzyme activity. The novel F84del mutation involves an invariant phenylalanine residue. A missense mutation, Q177E, occurred in three subjects from two families with late-infantile NCL, confirming an association of the Q177E mutation with a late-infantile phenotype. Other previously described mutations were R151X (5/16 alleles), T75P (3/16 alleles), R164X (1/16 alleles), and V181M (1/16 alleles). The current study expands the spectrum of mutations in PPT1 deficiency and further confirms the broad range of age of onset of symptoms resulting from an enzyme deficiency previously associated only with infantile NCL.

Molecular diagnosis of and carrier screening for the neuronal ceroid lipofuscinoses.

The neuronal ceroid lipofuscinoses (NCLs) are a large group of autosomal recessive lysosomal storage disorders with both enzymatic deficiency and structural protein dysfunction. Three typical forms, the infantile (INCL), late-infantile (LINCL), and juvenile (JNCL), are among the most common childhood-onset neurodegenerative disorders. They result from mutations on genes CLN1, CLN2, and CLN3, respectively. We determined that the mutations 223A --> G and 451C --> T in CLN1, T523-1G --> C, and 636 C --> T in CLN2, and deletion of a 1.02-kb genomic fragment in CLN3 are the five common mutations for NCL. To offer clinical genetic testing for the NCLs, we have developed simple and quick PCR-based molecular tests for detecting INCL-, LINCL-, and JNCL-affected individuals from 180 NCL families (27 INCL, 76 LINCL, and 77 JNCL). The sensitivity of testing to detect NCL patients among clinically suspected individuals was determined to be 78% (21/27) for INCL, 66% (54/76) for LINCL, and 75% (58/77) for JNCL. When molecular screening for carriers was conducted among the normal siblings or parents of the probands, we identified two carriers out of three individuals tested for INCL, 20/56 (35.7%) carriers for LINCL, and 48/106 (45.3%) carriers for JNCL families. In addition, 5% (9/180) of NCL patients revealed genetic heterogeneity and were reclassified. Seven patients previously diagnosed as having JNCL were now found to carry mutations of CLN2 (5/7) or CLN1 (2/7) and 2 with late-infantile onsets were identified as carrying mutations of CLN1. Our data demonstrate the importance of DNA testing to detect accurately both affected individuals and carriers in NCL families.

Heterogeneity of late-infantile neuronal ceroid lipofuscinosis.

PURPOSE: Late-infantile neuronal ceroid lipofuscinosis (LINCL), an autosomal recessively inherited lysosomal storage disorder characterized by autofluorescent inclusions and rapid progression of neurodegeneration, is due to CLN2 gene mutations. However, CLN2 mutation analysis has failed to identify some clinically diagnosed "late-infantile" NCL cases. This study was conducted to further characterize genetic heterogeneity in families affected by LINCL. METHODS: DNA mutations in the CLN1, CLN2, and CLN3 genes that underlie INCL (infantile NCL), LINCL, and JNCL (juvenile NCL), respectively, were studied with molecular analyses. RESULTS: A total of 252 families affected by childhood NCL were studied. Of 109 families clinically diagnosed as having LINCL, 3 were determined to have either INCL or JNCL by identification of mutation(s) in CLN1 or CLN3. Six families diagnosed initially as having JNCL were found to have LINCL based on the finding of mutations in the CLN2 gene. In addition, several novel mutations were identified. CONCLUSIONS: Clinical and genetic heterogeneity of LINCL was demonstrated in nine LINCL families studied.