Juvenile Neuronal Ceroid Lipofuscinosis Research Articles

CLN3 Defines a Novel Antiapoptotic Pathway Operative in Neurodegeneration and Mediated by Ceramide

Juvenile neuronal ceroid lipofuscinosis or Batten disease (JNCL) is a neurodegenerative disorder characterized by blindness, seizures, cognitive decline and early death. Brain atrophy and retinitis pigmentosa ensue because of neuronal and photoreceptor apoptosis. The CLN3 gene defective in JNCL encodes a novel 438 amino acid protein. Most affected genes harbor a deletion resulting in a truncated protein. CLN3 overexpression in NT2 cells enhances growth, reverses growth inhibition induced by serum starvation and protects from apoptosis induced by vincristine, staurosporine, and etoposide but not from death caused by ceramide. CLN3 modulates endogenous and vincristine-activated ceramide, and therefore suppresses apoptosis by impacting generation of ceramide.

Tissue Expression and Subcellular Localization of CLN3, the Batten Disease Protein

Juvenile neuronal ceroid lipofuscinosis (Batten disease) is a progressive neurologic disorder which results from mutations in the CLN3 gene, which normally produces a 48-kDa polypeptide of unknown function. To help characterize the CLN3 protein, we have studied its tissue distribution and subcellular localization in human tissues using three epitope-specific polyclonal antibodies to human CLN3 by immunoblot, immunocytochemical, and immunoelectron microscopic analysis. The most abundant CLN3 protein expression was in the gray matter of the brain, where it was localized to astrocytes, capillary endothelium, and neurons. CLN3 was also evident in peripheral nerve, in pancreatic islet cells, and within the seminiferous tubules in the testis. Staining was generally diffuse within the cytoplasm with some nuclear reactivity. Subcellular localization identified the CLN3 protein within the nucleus and along cell membranes. These results were contrasted with the cellular distribution of palmitoyl-protein thioesterase (PPT), the enzyme whose deficiency is responsible for infantile neuronal ceroid lipofuscinosis (CLN1). PPT was most abundant in brain and visceral macrophages where it displayed a coarse granular staining pattern typical of lysosomal distribution. Immunoelectron microscopy confirmed that PPT immunoreactivity was limited to lysosomes.

Intracellular Trafficking of the JNCL Protein CLN3

Juvenile neuronal ceroid lipofuscinosis is a lysosomal storage disease that causes visual impairment, progressive mental deterioration, and eventually death. A predominant 1.02-kb deletion as well as other mutations have been described in the CLN3 gene. Lacking significant identity with proteins of known function and no overt targeting signals within the primary amino acid sequence, accurate predictions of the intracellular location and function could not be made. Further, recent conflicting reports identified CLN3 as either a lysosomal or a mitochondrial protein. Transfection experiments using native and epitope-tagged fusion proteins were evaluated to help delineate CLN3 localization. We confirmed by immunohistochemistry and brefeldin A treatment that NH2-terminal green fluorescence protein (GFP)–CLN3 fusion proteins were retained in the Golgi apparatus, with no colocalization with mitochondrial markers. Anti-CLN3 antibodies directed against amino acids 67–90 of CLN3 were generated and shown to be specific for a 50-kDa protein in HEK 293 cells and GFP–CLN3 in transfected cells. However, cells transfected with nontagged CLN3 or carboxyl-terminal-tagged CLN3 were not immunoreactive with anti-CLN3 antibodies, suggesting that normally, the amino terminus interacts with other molecules. Thus, tags on the NH2-terminus probably inhibited these interactions and movement of CLN3 from the Golgi to more distal compartments. Also, CLN3 tagged at the COOH-terminus with either GFP or FLAG epitopes were retained in the ER, indicating a role for the COOH-terminus in trafficking. Taken together, these data confirm that CLN3 traffics through the ER and Golgi.

Analysis of Intracellular Distribution and Trafficking of the CLN3 Protein in Fusion with the Green Fluorescent Proteinin Vitro

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 thetrans-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.

Clinical and Molecular Analysis of Japanese Patients with Neuronal Ceroid Lipofuscinosis

Neuronal ceroid lipofuscinosis (NCL) is one of the most common inherited neurological diseases in childhood. It occurs every 12,500 births in northern-European populations. Mental retardation, visual impairment, and seizures are common symptoms. The prevalence of NCL is variable depending upon the races or countries. Although a wealth information is available in Caucasian populations, there is little information about NCL in Asian people. Because a nationwide survey in Japanese patients with NCL has never been performed, we pursued an epidemiological survey. We identified 36 NCL patients in Japan. Patients with infantile, late infantile, juvenile, and adult type accounted for 2, 15, 15, and 4 cases, respectively. Seizures were a major initial symptom in the late infantile type. In the juvenile type, visual failure was present in 73% at onset. Recently, the juvenile NCL (Batten disease) gene has been isolated. Studies of the mutations in this gene demonstrated that a 1.02-kb deletion was the most prevalent mutation among Caucasian patients, accounting for 81% of total alleles. To determine the prevalence of this 1.02-kb deletion in Japanese patients, we performed a rapid allele-specific polymerase chain reaction test. No 1.02-kb major deletion was detected in 5 Japanese juvenile NCL cases. These data suggest that the distribution of NCL and clinical findings are similar to those of Caucasian subjects; however, prevalence of mutations in Japanese patients with NCL would be distinct from that observed in Caucasians.

Progress in Neuropathology of the Neuronal Ceroid Lipofuscinoses

Since the last, 6th, International Congress on Neuronal Ceroid-Lipofuscinoses, neuropathological advances in neuronal ceroid lipofuscinoses (NCL) have been made in several areas: (1) In adult NCL (ANCL) lipopigments have now been repeatedly confirmed to contain subunit c of mitochondrial ATP synthase and even sphingolipid activators (saposins). ANCL lipopigments have also been confirmed in extracerebral tissues including skin, skeletal muscle, and spleen, but not yet lymphocytes (2). Among circulating blood cells not only B cells and subclasses of T lymphocytes, i.e., CD4+, CD8+, and CD56 cells, but also monocytes have been found to contain NCL lipopigments, indicating that this precursor cell in the digesting macrophage system also has an impaired metabolic catabolism for lipopigments (3). Immunohistochemical studies indicate that microglial reaction in NCL brain is limited to resident microglia without contribution by circulating monocytes (4). The granular osmiophilic deposit (GROD) type of NCL has now been established not only in infantile, but also in late-infantile, juvenile, and protracted-juvenile NCL (5). A European Tissue Registry established within the framework of a European Concerted Action on Neuronal Ceroid-Lipofuscinosis may form the basis for additional collaborative studies on NCL, including both biopsy and autopsy tissues.

Genetic Heterogeneity of Neuronal Ceroid Lipofuscinosis in the Netherlands

An overview of patients in the Netherlands who are known to us with neuronal ceroid lipofuscinosis (NCL) is presented. Several CLN genes involved in NCL have been isolated or mapped. We have analyzed families with different types of NCL with polymorphic markers linked to CLN loci to investigate the genetic heterogeneity of NCL in the Netherlands. Haplotype analysis suggests that in addition to the CLN2 and CLN6 genes another gene is involved in at least one family with late infantile NCL in the Netherlands. The CLN2 and CLN6 loci have also been excluded in a family with protracted juvenile NCL.

The Batten disease gene product (CLN3p) is a Golgi integral membrane protein.

Batten disease (juvenile neuronal ceroid lipofuscinosis) is a recessive neurodegenerative disorder of childhood. The gene, CLN3, was recently identified and found to encode a novel 438 amino acid protein of unknown function. In order to gain insight into the function of the Batten disease protein (CLN3p), we investigated its subcellular localization. Protein constructs incorporating CLN3p fused to the green fluorescence protein or an eight amino acid peptide tag were transiently expressed in fibroblasts, HeLa and COS-7 cells. A juxtanuclear, asymmetric localization pattern was observed that correlated with the Golgi apparatus in all three cell types. However, a proportion of transiently transfected cells exhibited a punctate vesicular distribution throughout the cytoplasm in addition to or without the Golgi localization. In order to account for localization patterns arising from intracellular protein transport disruption due to exaggerated overexpression in transiently transfected cells, we isolated a stably transfected cell line expressing only one copy of the CLN3 -GFP DNA construct. Fluorescence and biochemical analyses using this cell line demonstrated that CLN3p is an integral membrane protein that localizes primarily in the Golgi apparatus. The functional implications of this finding are discussed.

Phenotypes of Juvenile Batten Disease

The phenotypes of 10 Finnish juvenile neuronal ceroid lipofuscinosis (JNCL; late-onset Batten disease) patients were correlated with the genotypes in a study at Helsinki University, Finland; and the Rayne Institute, University College, London, UK.

Delayed classic and protracted phenotypes of compound heterozygous juvenile neuronal ceroid lipofuscinosis.

To correlate the phenotypes with the genotypes of 10 Finnish juvenile neuronal ceroid lipofuscinosis (JNCL; late-onset Batten disease) patients who all are compound heterozygotes for the major 1.02-kb deletion in the CLN3 gene. The mutations on the non-1.02-kb deletion chromosomes were screened in 6 patients; in the other 4 patients the mutations were known (one affecting a splice site, two missense mutations, and one deletion of exons 10 through 13). Clinical features were examined, and MRI, MRS, somatosensory evoked magnetic field (SEF), and overnight polysomnography (PSG) studies were performed. A novel deletion of exons 10 through 13 was found in 6 patients belonging to three families. In the patients carrying the deletions of exons 10 through 13 the clinical course of the disease was fairly similar. Variation was greatest in the time course to blindness. In these patients the mental and motor decline was slower than in classic JNCL, but more severe than in the two patients with missense mutations in exons 11 and 13. MRI showed brain atrophy in 4 patients. One patient had hyperintense periventricular white matter, otherwise brain signal intensities were normal. SEFs were enhanced in patients older than 14 years, whereas in PSG all but the youngest 6-year-old patient showed epileptiform activity in slow-wave sleep. JNCL can manifest as at least three different phenotypes: classic, delayed classic, and protracted JNCL with predominantly ocular symptoms. Finnish compound heterozygotes have the delayed classic or the protracted form of JNCL.

039 Hormonal changes in girls with juvenile neuronal ceroid lipofuscinosis

039 Hormonal changes in girls with juvenile neuronal ceroid lipofuscinosis

A disrupted homologue of the human CLN3 or juvenile neuronal ceroid lipofuscinosis gene in Saccharomyces cerevisiae: a model to study Batten disease.

1. In order to investigate the biological function of the human CLN3 gene that is defective in Batten disease, we created a yeast strain by PCR-targeted disruption of the yeast gene (YHC3), which is a homologue of the human CLN3 gene. 2. The phenotypic characterization revealed that the yhc3 delta mutants are more sensitive to combined heat and alkaline stress than the wild-type strains as determined by inhibition of cell proliferation. 3. This suggests that the yhc3 delta mutant is a good model to investigate the biological function of human CLN3 gene in mammalian cells and to understand the pathophysiology of juvenile Batten disease.

Evidence for Phosphorylation of CLN3 Protein Associated with Batten Disease

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 usingin vivolabeling with32P, 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).

First African‐American child with juvenile neuronal ceroid lipofuscinosis

The neuronal ceroid lipofuscinoses are among the most common forms of progressive neurodegenerative disease of childhood. They appear to be panethnic, but there is a special predilection of the infantile subtype in Finland. In the United States, the Batten disease registry of 731 cases shows that juvenile neuronal ceroid lipofuscinosis (JNCL) is the most common form. Here, we report on the first known African-American child with JNCL. Genetic study showed the 1.02-kb deletion typically seen in JNCL cases. Am. J. Med. Genet. 79:335–336, 1998. © 1998 Wiley-Liss, Inc.

Mutations in the palmitoyl-protein thioesterase gene (PPT; CLN1) causing juvenile neuronal ceroid lipofuscinosis with granular osmiophilic deposits.

A subtype of neuronal ceroid lipofuscinosis (NCL) is well recognized which has a clinical course consistent with juvenile NCL (JNCL) but the ultrastructural characteristics of infantile NCL (INCL): granular osmiophilic deposits (GROD). Evidence supporting linkage of this phenotype, designated vJNCL/GROD, to the INCL region of chromosome 1p32 was demonstrated (pairwise lod score with D1S211 , Z max = 2.63, straight theta = 0.00). The INCL gene, palmitoyl-protein thioesterase (PPT ; CLN1), was therefore screened for mutations in 11 vJNCL/GROD families. Five mutations in the PPT gene were identified: three missense mutations, Thr75Pro, Asp79Gly, Leu219Gln, and two nonsense mutations, Leu10STOP and Arg151STOP. The missense mutation Thr75Pro accounted for nine of the 22 disease chromosomes analysed and the nonsense mutation Arg151STOP for seven. Nine out of 11 patients were shown to combine a missense mutation on one disease chromosome with a nonsense mutation on the other. Mutations previously identified in INCL were not observed in vJNCL/GROD families. Thioesterase activity in peripheral blood lymphoblast cells was found to be markedly reduced in vJNCL/GROD patients compared with controls. These results demonstrate that this subtype of JNCL is allelic to INCL and further emphasize the correlation which exists between genetic basis and ultrastructural changes in the NCLs.

Studies of atypical JNCL suggest overlapping with other NCL forms

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.

The neuronal ceroid-lipofuscinoses. Recent advances.

The neuronal ceroid lipofuscinoses (NCLs) represent a group of neurodegenerative disorders characterised by progressive visual failure, neurodegeneration, epilepsy and the accumulation of an autofluorescent lipopigment in neurons and other cells. The main childhood subtypes are infantile (INCL;CLN1), classical late infantile (LINCL;CLN2) and juvenile NCL (JNCL;CLN3), distinguished on the basis of age of onset, clinical course and ultrastructural morphology, and recently genetic analysis. In addition several variant forms of the disease complex have been described as well as a rare adult onset form. Advances in both genetics and biochemistry have led to the identification of the genes for the three main subtypes of childhood NCL and their corresponding protein products and to mapping of two additional genes for two variant forms. The disease causing genes in both INCL and classical LINCL have been shown to encode lysosomal enzymes whilst the JNCL gene codes for a protein whose function is as yet unknown.

Rapid detection of the major deletion in the Batten disease gene CLN3 by allele specific PCR.

The recent isolation of the CLN3 gene involved in Batten disease (juvenile neuronal ceroid lipofuscinosis) creates possibilities for direct detection of mutations which can confirm or indicate the clinical diagnosis...

Somatosensory evoked magnetic fields from primary sensorimotor cortex in juvenile neuronal ceroid lipofuscinosis.

The present study evaluated neurophysiologic function of the primary sensorimotor cortex in juvenile neuronal ceroid lipofuscinosis. A 122-channel magnetometer, which allowed studies of the somatosensory system in millimeter and millisecond precision, was used to record somatosensory evoked magnetic fields to median nerve stimulation from 10 patients and their matched control subjects. In both patients and controls, the somatosensory evoked magnetic fields from primary sensorimotor area typically consisted of N20m, P35m, and P60m deflections. In the patients, N20m was significantly delayed, whereas P35m peaked earlier than in the control subjects. The source strengths for N20m and P35m were greater in the patients than in the controls. Both deflections showed a significant positive correlation with the disease duration: the sources were stronger in the older patients than in the younger ones. P60m deflections were normal or reduced in the patients. The results indicated increased thalamocortical excitability in the sensorimotor cortex in juvenile neuronal ceroid lipofuscinosis.

18F]fluorodopa PET shows striatal dopaminergic dysfunction in juvenile neuronal ceroid lipofuscinosis.

OBJECTIVES: To investigate whether nigrostriatal dopaminergic hypofunction is related to the extrapyramidal symptoms in patients with juvenile neuronal ceroid lipofuscinosis (JNCL). METHODS: Nine patients with JNCL and seven healthy controls...