👤 T M Wisniewski

Nilotinib, a tyrosine kinase inhibitor that targets the Abelson tyrosine kinase (c-Abl) signaling pathway, is FDA-approved to treat chronic myeloid leukemia. Nilotinib has properties indicative of a possible utility in neuroprotection that have prompted exploration of repurposing the drug for the treatment of Alzheimer's disease (AD) and Parkinson's disease (PD). AD is a progressive age-related neurodegenerative disorder characterized by the deposition of extracellular amyloid-β plaques and intracellular neurofibrillary tangles. It is incurable and affects approximately 50 million patients worldwide. Nilotinib reduces c-Abl phosphorylation, amyloid-β levels, and dopaminergic neuron degeneration in preclinical AD models. This study explores the effects of nilotinib on amyloid processing and mitochondrial functioning in the SH-SY5Y human neuroblastoma cell line. SH-SY5Y cells were exposed to nilotinib (1, 5, and 10 µM). Real-time PCR and immunoblot analysis were performed to quantify the expression of genes pertaining to amyloid-β processing and neuronal health. Nilotinib did not significantly change APP, BACE1, or ADAM10 mRNA levels. However, BACE1 protein was significantly increased at 1 µM, and ADAM10 was increased at 10 µM nilotinib without affecting APP protein expression. Further, nilotinib treatment did not affect the expression of genes associated with neuronal health and mitochondrial functioning. Taken together, our findings do not support the efficacy of nilotinib treatment for neuroprotection. Show less

Dietary lipids induce apolipoprotein A4 (APOA4) production and brown adipose tissue (BAT) thermogenesis. Administration of exogenous APOA4 elevates BAT thermogenesis in chow-fed mice, but not high-fat diet (HFD)-fed mice. Chronic feeding of HFD attenuates plasma APOA4 production and BAT thermogenesis in wildtype (WT) mice. In light of these observations, we sought to determine whether steady production of APOA4 could keep BAT thermogenesis elevated, even in the presence of HFD consumption, with an aim toward eventual reduction of body weight, fat mass and plasma lipid levels. Transgenic mice with overexpression of mouse APOA4 in the small intestine (APOA4-Tg mice) produce greater plasma APOA4 than their WT controls, even when fed an atherogenic diet. Thus, we used these mice to investigate the correlation of levels of APOA4 and BAT thermogenesis during HFD consumption. The hypothesis of this study was that overexpression of mouse APOA4 in the small intestine and increased plasma APOA4 production would increase BAT thermogenesis and consequently reduce fat mass and plasma lipids of HFD-fed obese mice. To test this hypothesis, BAT thermogenic proteins, body weight, fat mass, caloric intake, and plasma lipids in male APOA4-Tg mice and WT mice fed either a chow diet or a HFD were measured. When fed a chow diet, APOA4 levels were elevated, plasma triglyceride (TG) levels were reduced, and BAT levels of UCP1 trended upward, while body weight, fat mass, caloric intake, and plasma lipids were comparable between APOA4-Tg and WT mice. After a four-week feeding of HFD, APOA4-Tg mice maintained elevated plasma APOA4 and reduced plasma TG, but UCP1 levels in BAT were significantly elevated in comparison to WT controls; body weight, fat mass and caloric intake were still comparable. After 10-week consumption of HFD, however, while APOA4-Tg mice still exhibited increased plasma APOA4, UCP1 levels and reduced TG levels, a reduction in body weight, fat mass and levels of plasma lipids and leptin were finally observed in comparison to their WT controls and independent of caloric intake. Additionally, APOA4-Tg mice exhibited increased energy expenditure at several time points when measured during the 10-week HFD feeding. Thus, overexpression of APOA4 in the small intestine and maintenance of elevated levels of plasma APOA4 appear to correlate with elevation of UCP1-dependent BAT thermogenesis and subsequent protection against HFD-induced obesity in mice. Show less

How host and microbial factors combine to structure gut microbial communities remains incompletely understood. Redox potential is an important environmental feature affected by both host and microbial actions. We assessed how antibiotics, which can impact host and microbial function, change redox state and how this contributes to post-antibiotic succession. We showed gut redox potential increased within hours of an antibiotic dose in mice. Host and microbial functioning changed under treatment, but shifts in redox potentials could be attributed specifically to bacterial suppression in a host-free ex vivo human gut microbiota model. Redox dynamics were linked to blooms of the bacterial family Enterobacteriaceae. Ecological succession to pre-treatment composition was associated with recovery of gut redox, but also required dispersal from unaffected gut communities. As bacterial competition for electron acceptors can be a key ecological factor structuring gut communities, these results support the potential for manipulating gut microbiota through managing bacterial respiration. Show less

Genotype-phenotype associations were studied in 517 subjects clinically affected by classical neuronal ceroid lipofuscinosis (NCL). Genetic loci CLN1-3 were analyzed in regard to age of onset, initial neurological symptoms, and electron microscope (EM) profiles. The most common initial symptom leading to a clinical evaluation was developmental delay (30%) in NCL1, seizures (42.4%) in NCL2, and vision problems (53.5%) in NCL3. Eighty-two percent of NCL1 cases had granular osmiophilic deposits (GRODs) or mixed-GROD-containing EM profiles; 94% of NCL2 cases had curvilinear (CV) or mixed-CV-containing profiles; and 91% of NCL3 had fingerprint (FP) or mixed-FP-containing profiles. The mixed-type EM profile was found in approximately one-third of the NCL cases. DNA mutations within a specific CLN gene were further correlated with NCL phenotypes. Seizures were noticed to associate with common mutations 523G>A and 636C>T of CLN2 in NCL2 but not with common mutations 223G>A and 451C>T of CLN1 in NCL1. Vision loss was the initial symptom in all types of mutations in NCL3. Surprisingly, our data showed that the age of onset was atypical in 51.3% of NCL1 (infantile form) cases, 19.7% of NCL2 (late-infantile form) cases, and 42.8% of NCL3 (juvenile form) cases. Our data provide an overall picture regarding the clinical recognition of classical childhood NCLs. This may assist in the prediction and genetic identification of NCL1-3 via their characteristic clinical features. Show less

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. Show less

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. Show less

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. Show less

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. Show less

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. Show less

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. 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. 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. Clinical and genetic heterogeneity of LINCL was demonstrated in nine LINCL families studied. Show less

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. Show less

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. Show less

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. Show less

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. Show less

The gene for Batten disease, the CLN3 gene, encodes a novel, highly hydrophobic, multitransmembrane protein, predicted to consist of 438 amino acid residues. We have expressed a full-length CLN3 protein in fusion with green fluorescent protein in various cell lines to provide its initial biochemical characterization and subcellular localization. By using Western blotting, Percoll density gradient fractionation, and Triton X-114 extraction, we demonstrate that the product of the CLN3 gene, which we call battenin, in mammalian expression system studied is a highly glycosylated protein of lysosomal membrane. In addition our data suggest that CLN3 protein is processed proteolytically in acidic compartments of the cell. Thus, battenin represents the novel constituent of a growing family of lysosomal membrane proteins. Show less

The CLN3 gene associated with Batten disease and encoding a novel protein of a predicted 438 amino acids was cloned in 1995 by the International Batten Disease Consortium. The function of CLN3 protein remains unknown. Computer-based analysis predicted that CLN3 may contain several posttranslational modifications. Thus, to study the posttranslational modification of CLN3 protein, we have expressed a full-length CLN3 protein as a C-terminal fusion with green fluorescent protein of the jellyfish Aequerea victoria in a Chinese hamster ovary cell line. Previously, we have shown that CLN3 is a glycosylated protein from lysosomal compartment, and now, by using in vivo labeling with 32P, detection with anti-phosphoamino acid antibodies, and phosphoamino acid analysis, we demonstrate that CLN3 is a phosphorylated protein. We demonstrate that CLN3 protein does not undergo mannose 6-phosphate modification and that it is a membrane protein. Furthermore, we show that the level of CLN3 protein phosphorylation may be modulated by several protein kinases and phosphatases activators or inhibitors. Show less

CLN3 gene, associated with juvenile neuronal ceroid lipofuscinosis, encodes a novel protein of a predicted 438 amino acid residues. We have expressed a full-length CLN3 protein and fragments thereof in fusion with green fluorescent protein in Chinese hamster ovary and human neuroblastoma cell lines to study its subcellular localization and intracellular trafficking pattern. By using laser scanning confocal microscopy, we demonstrate that the full-length CLN3 fusion protein is targeted to lysosomal compartments. Tunicamycin treatment did not alter the lysosomal targeting of the CLN3 protein, which indicates that extensive N-glycosylation of the full-length CLN3 fusion protein is not engaged in its lysosomal sorting. Monensin produced retention of CLN3 fusion protein in vesicular structure of the Golgi apparatus in the perinuclear space, suggesting that CLN3 fusion protein is transported to the lysosomal compartments through the trans-Golgi cisternae. Neither of the truncated CLN3 fusion proteins encompassing its 1-138, 1-322, and 138-438 amino acid residues was disclosed in lysosomal compartments. However, CLN3 fusion protein showing double-point mutations at amino acid residues 425 and 426, thus at its putative dileucine lysosomal signaling motif, was still targeted to lysosomes, suggesting that a dileucine motif alone is not sufficient for lysosomal sorting of the CLN3 fusion protein. Show less

The product of the CLN3 gene is a novel protein of unknown function. Simulations using amphiphacy algorithms have shown that structurally CLN3 may be another candidate for the family of membranous proteins. Signals controlling intracellular targeting of many membrane proteins are present as short sequences within their cytoplasmic domains. In fact, the sequence of CLN3 protein contains several such signaling sequences, which are conserved among mammals. First, at the N-terminus, potential N-myristoylation motif is present. Second, the C-terminal part of CLN3 protein contains both the dileucine motif, which is a potential lysosomal targeting signal, and the prenylation motif. There is scanty evidence of lysosomal and/or mitochondrial localization of CLN3 protein. However, the question of where the functional site of the cln3 protein exists in vivo remains unanswered. From theoretical calculations, we hypothesized that CLN3 should be an integral part of the membranous micro-environment. First, to test this hypothesis, we initiated detergent-partitioning experiments, localizing CLN3 predominantly in a pool of membranous protein. Further studies have shown that CLN3 protein integrates spontaneously with cellular membranes. Second, based on the prenylation results of CLN3 protein in vitro, we discussed the possible topological consequences of C-terminal fragment of CLN3 protein. Show less

This study describes the phenotype/genotype analyses of 56 probands with a juvenile onset, some of which had atypical features of neuronal ceroid lipofuscinosis, collected at the New York State Institute for Basic Research (IBR). In this group, we found probands with abundant curvilinear profiles in lysosomal storage material, deficiency of pepstatin-insensitive peptidase, and mutations in the CLN2 gene, as well as patients with a predominance of granular osmiophilic deposits in the lysosomal storage material, deficiency of palmitoyl-protein thioesterase, and mutations in the CLN1 gene. We have divided the probands into two categories: typical (or classic) and atypical. Most of the typical and atypical probands had onset of symptoms about or after 4 years of age. Interfamiliar and intrafamiliar variations were found, especially in the speed of becoming practically blind. Thus, our study indicates that some mutations in the CLN1, CLN2, and CLN3 genes may be associated with late onset of the disease process, may have a more benign clinical course, and clinic overlap with other forms of neuronal ceroid lipofuscinosis. Show less

The infantile form of neuronal ceroid lipofuscinosis (NCL) has been well studied in Finland, where there is a high carrier frequency (1:70) for a single mutation in the causative gene, CLN1, or PPT. We have recently studied a group of 29 NCL subjects in the United States with palmitoyl-protein thioesterase (PPT) deficiency and described 19 different CLN1/PPT mutations in our population. In this report, we present a review of our previous findings, including a more detailed analysis of phenotype-genotype correlations, and present previously unpublished data concerning the clinical manifestations of the disorder in children of families with multiple affected members. Our studies indicate that about half of PPT-deficient patients in the United States are very similar to Finnish infants with INCL, but that a different mutation (R151X) accounts for 40% of U.S. alleles. The Finnish mutation (R122W) is rare in the United States. The other half of U.S. PPT-deficient patients develop symptoms after the age of 2 years, much later than Finnish patients. One common mutation (the "Scottish" allele, T75P) accounts for 13% of alleles and results in a juvenile-onset phenotype that is clinically indistinguishable from JNCL with CLN3 mutations. Other rare mutations were also associated with JNCL phenotypes, such as D79G and G250V. A preliminary expression study of two of these mutant enzymes supports the conclusion that juvenile-onset NCL (JNCL with GROD) is caused by missense mutations in the PPT gene that result in mutated enzymes with residual PPT enzyme activity. Show less

Recently, the CLN3 gene associated with Batten disease (juvenile neuronal ceroid lipofuscinosis, JNCL), a recessively inherited, progressive, neurodegenerative disorder of childhood, has been identified. The CLN3 gene encodes a novel protein (battenin) of a predicted 438 amino acids containing several potential posttranslational modifications. We have expressed a full-length CLN3 protein as a C-terminal fusion with green fluorescent protein (GFP) to evaluate whether CLN3 protein is phosphorylated. By using in vivo labeling with 32P, detection with anti-phosphoamino acid antibodies, and phosphoamino acid analysis, we demonstrate that the CLN3 protein is phosphorylated on both serine and threonine residues. We also demonstrate that CLN3 protein is not modified by mannose 6-phosphate. Furthermore, we show that phosphorylation of CLN3 protein is carried out by protein kinase A (cAMP-dependent protein kinase, PKA), protein kinase G (cGMP-dependent protein kinase, PKG), and casein kinase II and that it is enhanced by inhibition of protein phosphatase 1 (PP 1) or protein phosphatase 2A (PP 2A). Show less

In the United States, juvenile neuronal ceroid-lipofuscinosis (JNCL) is the most common form of NCL. This study analyzed 191 cases, diagnosed on the basis of age-at-onset, clinical symptomatology, and pathologic findings. Twenty percent (40/191) of these cases from 24/120 families manifested atypical clinical symptomatology and/or pathologic findings (typical revealed fingerprints and atypical revealed mixed inclusions, or only curvilinear or granular profiles) and, therefore, represent variant forms of JNCL. Those patients in the study with typical JNCL were a uniform group of cases, whereas the atypical were heterogenous and were divided into 8 subgroups based on the clinicopathologic findings. Forty-three families were analyzed (27 typical, 16 atypical) for the common 1.02 kb deletion and several pedigrees for novel mutations. In typical JNCL the common 1.02 kb deletion in both alleles (homozygous) were observed in 23/27, and only 1 allele (heterozygous) was exhibited in 4/27 families. In atypical JNCL families, 5/16 were heterozygous for the common 1.02 kb deletion. None of the remaining 11/16 families had the common 1.02 kb deletion in either allele, but in 9/11 cases the palmitoyl-protein thioesterase (PPT) levels were deficient. In cases where the mutation in CLN3 gene has not been identified, several possibilities may exist. The phenotype may be caused by a yet undefined mutation in CLN3 or may be due to overlapping with other forms of NCL. Show less

Batten disease, the juvenile form of neuronal ceroid lipofuscinosis, is a prevalent neuron degenerative disorder of childhood. A 1.02-kb genomic deletion in the Batten disease gene CLN3 has been determined to be a common mutation. We developed a PCR method to screen for this deletion and tested 43 Batten disease probands. We found 36% (31/86) of Batten disease chromosomes did not carry the 1.02-kb deletion. Of the three heterozygotes for the 1.02-kb deletion, a novel G-to-A missense mutation at nucleotide 1020 of the CLN3 cDNA sequence was found on two of the non-1.02-kb deletion chromosomes. The missense mutation resulted in a substitution of glutamic acid (E) by lysine (K) at position 295 (E295 K). The E295 K mutation causes a change in predicted local protein conformation. This glutamic acid is a highly conserved acidic amino acid, being present in human, mouse, dog and yeast, which suggests it may play an important role in the function of the Batten disease protein. Show less

We present a clinicopathological study and the first molecular genetic analysis of a family with 2 siblings affected by a rare, protracted form of juvenile neuronal ceroid lipofuscinosis (JNCL). Molecular genetic studies showed that both siblings, in addition to being heterozygous for the 1.02-kb CLN3 deletion, a common mutation in JNCL, also had a G-to-A missense mutation at nucleotide 1,020 of the CLN3 cDNA sequence on the non-1.02-kb deletion chromosomes. This point mutation resulted in a substitution of glutamic acid by lysine at position 295 of the CLN3 protein. Thus, a single point mutation at residue 295 of the CLN3 protein in protracted JNCL may underlie the phenotype in this form, which differs from that in classic JNCL. Show less

Expression of the gene for Batten disease (CLN3) was studied in Escherichia coli and in a cell-free rabbit reticulocyte expression systems. A full-length recombinant fusion CLN3 protein was not produced in the bacterial systems used. However, both N-terminal fragment encompassing 246 amino acids and short C-terminal fragment containing 428-438 amino acids of the CLN3 protein were successfully overexpressed in bacteria. Further studies showed that the C-terminal sequence of the CLN3 protein corresponding to the 356-438 amino acid residues was responsible for inhibition of protein synthesis in bacteria. The full-length CLN3 gene product was readily synthesized in vitro in the cell-free rabbit reticulocyte expression system. The product obtained, corresponding to core CLN3 protein, showed an approximate molecular weight of 43 kDa. Immunoprecipitation of this product with pAb to 4-19 amino acids of the CLN3 protein allows us to suggest that CLN3 protein translation starts at Met-1. Show less

The neuronal ceroid-lipofuscinoses (NCL) are a group of different genetic diseases. The major types of NCL are expressed by six forms which represent different clinicopathologic and genetic forms. These are CLN-1, Infantile; CLN-2, Late Infantile; CLN-3, Juvenile; CLN-4, Adult-Recessive; CLN-5, Adult-Dominant; and CLN-6, Early Juvenile. The distinction between CLN-4 and CLN-5 is still disputatious. CLN-6 has been called CLN-5. A seventh classification of NCL represents from 12 to 20% of those afflicted. This group consists of an extensive array of atypical types of ceroid-lipofuscin accumulation in the secondary lysosomes of neurons and cells of other tissues (e.g., skin, conjunctiva, and lymphocytes) or by presumed clinical and genetic relationships. The authors have identified 15 atypical subtypes of NCL. These as a group are here described as a seventh form. Further biochemical, molecular, and genetic studies will identify more precisely the phenotypic and genotypic expression of these "minor" forms of NCL. Show less