Therapeutic approaches to the challenge of neuronal ceroid lipofuscinoses.

The Neuronal Ceroid Lipofuscinoses (NCLs) are lysosomal storage diseases (LSDs) affecting the central nervous system (CNS), with generally recessive inheritance. They are characterized by pathological lipofuscin-like material accumulating in cells. The clinical phenotypes at all onset ages show progressive loss of vision, decreasing cognitive and motor skills, epileptic seizures and premature death, with dementia without visual loss prominent in the rarer adult forms. Eight causal genes, CLN10/CTSD, CLN1/PPT1, CLN2/TPP1, CLN3, CLN5, CLN6, CLN7/MFSD8, CLN8, with more than 265 mutations and 38 polymorphisms (http://www.ucl.ac.uk/ncl) have been described. Other NCL genes are hypothesized, including CLN4 and CLN9; CLCN6, CLCN7 and possibly SGSH are under study. Some therapeutic strategies applied to other LSDs with significant systemic involvement would not be effective in NCLs due to the necessity of passing the blood brain barrier to prevent the neurodegeneration, repair or restore the CNS functionality. There are therapies for the NCLs currently at preclinical stages and under phase 1 trials to establish safety in affected children. These approaches involve enzyme replacement, gene therapy, neural stem cell replacement, immune therapy and other pharmacological approaches. In the next decade, progress in the understanding of the natural history and the biochemical and molecular cascade of events relevant to the pathogenesis of these diseases in humans and animal models will be required to achieve significant therapeutic advances.

CLN5 mutations are frequent in juvenile and late-onset non-Finnish patients with NCL.

OBJECTIVES: To explore a potential expansion of the phenotypic and genotypic characteristics of Finnish variant late-infantile neuronal ceroid lipofuscinosis (NCL), we screened a collection of 47 patients with clinically diagnosed NCL in whom no molecular diagnosis had been made. METHODS: We used PCR amplification of genomic DNA, followed by fluorescent-labeled dideoxy-nucleotide chain termination sequencing and multiplex ligation-dependent probe amplification, to screen our cohort of patients for mutations in CLN5. We collected ethnic background, clinical, and pathologic information, as available, to clarify the breadth of CLN5 disease expression and to explore possible genotype-phenotype correlations. RESULTS: We identified 10 patients with pathogenic CLN5 mutations, including 11 mutations not previously described: 4 missense, 5 out-of-frame insertion/deletion mutations, and 2 large intragenic deletions. We also documented 3 previously reported CLN5 mutations. The age at disease onset in this cohort is predominantly juvenile rather than late infantile. Importantly, we have identified 2 adult-onset patients who share a common pathogenic allele. The majority of patients presented with motor and visual impairments and not seizures. In those patients with available longitudinal data, most had progressed to global neurodevelopmental and visual failure with seizures within 1 to 4 years. CONCLUSIONS: Our study suggests that CLN5 mutations 1) are more common in patients with neuronal ceroid lipofuscinosis (NCL) than previously reported, 2) are found in non-Finnish NCL patients of broad ethnic diversity, and 3) can be identified in NCL patients with disease onset in adult and juvenile epochs. CLN5 genetic testing is warranted in a wider population with clinical and pathologic features suggestive of an NCL disorder.

A CLN5 mutation causing an atypical neuronal ceroid lipofuscinosis of juvenile onset.

Three related patients from Colombia presented with a juvenile-onset neuronal ceroid lipofuscinosis. Electron microscopy of one case showed condensed fingerprint profiles, and genetic analyses identified a novel missense mutation in CLN5. The authors demonstrate the existence of pathogenic CLN5 mutations outside northern Europe and that mutations in this gene can lead to an atypical late-onset neuronal ceroid lipofuscinosis disease, in addition to the late infantile form first described in Finland.

Neurodegenerative disease: the neuronal ceroid lipofuscinoses (Batten disease).

In the past decade there have been significant advances in our understanding of the molecular genetic basis of the neuronal ceroid lipofuscinoses, a clinically and genetically heterogeneous group of childhood neurodegenerative storage disorders. Recent research progress is reviewed here, to summarize new disease gene identification, diagnostics, treatment, protein functional studies and investigations into the underlying molecular pathogenesis of these devastating disorders.

Identification of a transactivation motif in the CLN3 protein.

A transactivation motif has been identified in the neurodegenerative disease protein, CLN3. The C-terminal domain (residues 394-438) of CLN3 can function as a transcriptional activator when fused to the DNA binding domain, LexA. A series of deletion and substitution constructs have been generated to identify the essential region for transactivation. A similar motif is also present in the POU domain transcription factor, nubbin. However, this domain alone does not activate transcription, allowing further localisation of the critical residues in CLN3 required for activity.

New mutations in the neuronal ceroid lipofuscinosis genes.

Thirty-eight mutations and seven polymorphisms have recently been reported in the genes underlying the neuronal ceroid lipofuscinoses (NCLs) including 11 new mutations described here. A total of 114 mutations and 28 polymorphisms have now been described in the five human genes identified which cause NCL. Thirty-eight mutations are recorded for CLN1/PPT; 40 for CLN2/TTP-1, 31 for CLN3, four for CLN5, one for CLN8. Two mutations have been described in animal genes (cln8/mnd, CTSD). All mutations in NCL genes are contained in the NCL Mutation Database (http://www.ucl.ac.uk/NCL).

Analysis of CLN3-protein interactions using the yeast two-hybrid system.

Juvenile neuronal ceroid lipofuscinosis (Batten disease) is a childhood neurodegenerative disease that is caused by mutations in the CLN3 gene. The protein encoded by CLN3 has no homology with any proteins of known function and its cellular role remains elusive. In order to investigate the role played by the CLN3 protein we aimed to identify interacting proteins. Here, we describe the yeast two-hybrid system as the approach taken to investigate such protein-protein interactions. CLN3 was expressed as a fusion protein with a DNA-binding domain and used to screen a library of human fetal brain cDNAs fused to a transcriptional activation domain. Owing to low level expression of the full length CLN3 fusion protein, truncated regions corresponding to the predicted hydrophilic regions were also tested. No proteins that interact with CLN3 were detected, nor was there any evidence for CLN3-CLN3 interactions. Potential interaction of CLN3 with subunit c of mitochondrial ATP synthase, the major component of the storage material that accumulates in Batten disease patients, was also tested. No interaction was detected suggesting that the accumulation of subunit c does not result from loss of a process that requires a direct interaction with CLN3. We conclude that either CLN3 does not interact with other proteins or such interactions cannot be detected using the two-hybrid system.

Genomic structure of three CLN3-like genes in Caenorhabditis elegans.

The genome of Caenorhabditis elegans is predicted to carry three genes similar to CLN3, the gene underlying juvenile neuronal ceroid lipofuscinosis. All three genes are transcribed and the genomic structure has been determined. The number and position of exons for two of the genes differ from that predicted from the genomic sequence, but no discrepancies with the genomic nucleotide sequence were found. Gene F07B10.1 (cln-3.1) is predicted to have 7 exons and to encode a protein of 424 amino acids. Gene C01G8.2 (cln-3.2) has 9 exons and encodes a protein of 435 amino acids. Gene ZC190.1 (cln-3.3) is predicted to have 9 exons and to encode a protein of 416 amino acids.

Turkish variant late infantile neuronal ceroid lipofuscinosis (CLN7) may be allelic to CLN8.

One variant form of late infantile neuronal ceroid lipofuscinosis (LINCL) is found predominantly within the Turkish population (CLN7). Exclusion mapping showed that CLN7 was not an allelic variant of known NCL loci (CLN1, CLN2, CLN3, CLN5 or CLN6). Using the method of homozygosity mapping, a genome-wide search was undertaken and a total of 358 microsatellite markers were typed at an average distance of about 10 cM. A region of shared homozygosity was identified on chromosome 8p23. This telomeric region contained the recently identified CLN8 gene. A missense mutation in CLN8 causes progressive epilepsy with mental retardation (EPMR) or Northern epilepsy, which has so far been reported only from Finland and is now classified as an NCL. The mouse model mnd has been shown to carry a 1 bp insertion in the orthologous Cln8 gene. Statistically significant evidence for linkage was obtained in this region, with LOD scores > 3, assuming either homogeneity or heterogeneity. Flanking recombinants defined a critical region of 14 cM between D8S504 and D8S1458 which encompasses CLN8. This suggests that Turkish variant LINCL, despite having an earlier onset and more severe phenotype, may be an allelic variant of Northern epilepsy. However mutation analysis has not so far identified a disease causing mutation within the coding or non-coding exons of CLN8 in the families. The Turkish variant LINCL disease-causing mutation remains to be delineated.

Analysis of candidate genes in the CLN6 critical region using in silico cloning.

CLN6, the gene for variant late infantile neuronal ceroid lipofuscinosis, was mapped to a 4 cM region on chromosome 15q22-23. Subsequently the critical region was narrowed to less than 1 cM between microsatellite markers D15S988 and D15S1000 by additional marker typing in an expanded family resource. A physical map was constructed across this region using YAC and PAC clones and sequence was generated from two PAC clones. This sequence was analysed together with overlapping sequence generated by the Human Genome Project to identify genes within the region using an in silico cloning approach. In all, 29 genes have been identified and 18 have been analysed for mutations by direct sequencing. This powerful new approach will lead to the identification of CLN6.

Full-field ERG in patients with Batten/Spielmeyer-Vogt disease caused by mutations in the CLN3 gene.

PURPOSE: To investigate, using full-field ERG, the retinal function in patients with Batten/Spielmeyer-Vogt disease caused by mutations in the CLN(3) gene. METHODS: Batten disease status of five patients was confirmed by the presence of vacuolated lymphocytes in peripheral blood and the identification of mutations in the Batten disease gene (CLN(3)). Visual acuity, fundus appearance, and full-field ERG were examined in all patients (age 4-19 years). The examination was repeated in one patient after 16 months. RESULTS: Three unrelated patients were homozygous for the most common mutation in CLN(3), the 1.02 kb deletion; two patients (sisters) were heterozygous for the 1.02 kb deletion and an as yet unidentified mutation in the CLN(3) gene. Full-field ERG recordings in all five patients demonstrated no rod responses and only small remaining cone responses, which could be detected with 30 Hz-flicker stimulation. Re-examination of a six-year-old girl after 16 months revealed a fast progression of the retinal degeneration. CONCLUSION: Full-field ERG recordings in Batten disease patients, both homozygous and heterozygous for the 1.02 kb deletion in the CLN( 3) gene, confirm retinal degeneration to be severe, widespread, and with a rapid progression early in the disease course. The onset of visual failure may be delayed when compared to the classic disease course, particularly in patients who are not homozygous for the most common CLN(3) mutation, a 1.02 kb deletion. In that case, the disease progression in terms of other symptoms may also be further delayed.

Molecular basis of the neuronal ceroid lipofuscinoses: mutations in CLN1, CLN2, CLN3, and CLN5.

The neuronal ceroid lipofuscinoses (NCLs), also referred to as Batten disease, are a group of neurodegenerative disorders characterised by the accumulation of an autofluorescent lipopigment in many cell types. Different NCL types are distinguished according to age of onset, clinical phenotype, ultrastructural characterisation of the storage material, and chromosomal location of the disease gene. At least eight genes underlie the NCLs, of which four have been isolated and mutations characterised: CLN1, CLN2, CLN3, CLN5. Two of these genes encode lysosomal enzymes, and two encode transmembrane proteins, at least one of which is likely to be in the lysosomal membrane. The basic defect in the NCLs appears to be associated with lysosomal function.

The molecular basis of GROD-storing neuronal ceroid lipofuscinoses in Scotland.

Two distinct clinical subtypes of neuronal ceroid lipofuscinosis caused by mutations in the PPT gene, INCL and vJNCL/GROD, occur at a high frequency in the central region of Scotland. In this paper we summarize the clinical details and the molecular basis underlying the disease in the Scottish patients. Comparison of the combination of mutations in the different clinical types reveals a clear genotype-phenotype correlation.

A murine model for juvenile NCL: gene targeting of mouse Cln3.

JNCL is a neurodegenerative disease of childhood caused by mutations in the CLN3 gene. A mouse model for JNCL was created by disrupting exons 1-6 of Cln3, resulting in a null allele. Cln3 null mice appear clinically normal at 5 months of age; however, like JNCL patients, they exhibit intracellular accumulation of autofluorescent material. A second approach will generate mice in which exons 7 and 8 of Cln3 are deleted, mimicking the common mutation in JNCL patients.

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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Epitope mapping of antibodies recognising the N-terminal domain of simian virus large tumour antigen.

The large tumour antigen (TAg) of simian virus 40 is the main regulatory protein in viral replication. We have investigated possible functional roles for the N-terminal domain of TAg while defining the epitopes of antibodies binding within that region. The epitopes recognised by eight monoclonal anti-TAg antibodies were identified using synthetic peptides in an enzyme-linked immunosorbent assay. For antibodies PAb 223, 281, 211, 219 and 224, epitopes varied in length between ten and fifteen amino acids. Two antibodies, PAb 210 and PAb 419, could not be mapped any further using this technique. Antibody PAb 416 recognised a pentapeptide WEQWW that could be involved in topoisomerase I binding and possibly in the interaction between topoisomerase I and simian virus reverse transcriptase.

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.