Characterization of candidate genes for neuronal ceroid lipofuscinosis in dog

The neuronal ceroid lipofuscinoses (NCL) are a heterogenous group of monogenic autosomal recessive inherited progressive neurodegenerative diseases characterized by brain and retinal atrophy and the intracellular accumulation of autofluorescent lysosomal storage bodies resembling lipofuscin in neurons and other cells. Until today, eight forms of NCL have been classified in humans by clinical criteria, which result from mutations in at least six different genes (TPP1, CLN2, PPT1, CLN5, CLN6, and CLN8). NCL has also been reported in various domestic animal species including cattle, goat, sheep, cat, and certain dog breeds. In this report, the experimental analysis of canine PPT1, CLN5, CLN6, and CLN8 full-length cDNA sequences is described, and the current whole genome sequence assembly was used for gene structure analyses. Characterization of the four canine genes revealed a conserved organization with respect to the human orthologs. In general the gene size in dog is smaller compared to the human sequence due to shorter intron length. Using four individuals of Tibetan terrier with NCL, and a single affected Polish Owczarek Nizinny (PON) dog, we excluded the complete coding region of canine PPT1 and CLN8 and three of four exons of CLN5 and six of seven exons of CLN6 harboring disease-causing mutations.     

Genetic heterogeneity in neuronal ceroid lipofuscinosis (NCL): Evidence that the late-infantile subtype (Jansky-Bielschowsky disease; CLN2) is not an allelic form of the juvenile or infantile subtypes

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative disorders characterized by the accumulation of autofluorescent lipopigment in neurons and other cell types. Inheritance is autosomal recessive. Three main childhood subtypes are recognized: infantile (Haltia-Santavuori disease; MIM 256743), late infantile (Jansky-Bielschowsky disease; MIM 204500), and juvenile (Spielmeyer-Sjögren-Vogt, or Batten, disease; MIM 204200). The gene loci for the juvenile (CLN3) and infantile (CLN1) types have been mapped to human chromosomes 16p and 1p, respectively, by linkage analysis. Linkage analysis of 25 families segregating for late-infantile NCL has excluded these regions as the site of this disease locus (CLN2). The three childhood subtypes of NCL therefore arise from mutations at distinct loci.     

Biochemical Characterization of a Lysosomal Protease Deficient in Classical Late Infantile Neuronal Ceroid Lipofuscinosis (LINCL) and Development of an Enzyme-Based Assay for Diagnosis and Exclusion of LINCL in HUman Specimens and Animal Models

Classical late-infantile neuronal ceroid lipofuscinosis (LINCL), a progressive and fatal neurodegenerative disease of childhood, results from mutations in a gene (CLN2) that encodes a protein with significant sequence similarity to prokaryotic pepstatin-insensitive acid proteases. We have developed a sensitive protease activity assay that allows biochemical characterization of the CLN2 gene product in various human biological samples, including solid tissues (brain and chorionic villi), blood (buffy coat leukocytes, platelets, granulocytes, and mononuclear cells), and cultured cells (lymphoblasts, fibroblasts, and amniocytes). The enzyme has a pH optimum of 3.5 and is rapidly inactivated at neutral pH. A survey of fibroblasts and lymphoblasts demonstrated that lack of activity was associated with LINCL arising from mutations in the CLN2 gene but not other neuronal ceroid lipofuscinoses (NCLs), including the CLN6 variant LINCL, classical infantile NCL, classical juvenile NCL, and adult NCL (Kufs' disease). A study conducted using blood samples collected from classical LINCL families whose affliction was confirmed by genetic analysis indicates that the assay can distinguish homozygotes, heterozygotes, and normal controls and thus is useful for diagnosis and carrier testing. Analysis of archival specimens indicates that several specimens previously classified as LINCL have enzyme activity and thus disease is unlikely to arise from mutations in CLN2. Conversely, a specimen previously classified as juvenile NCL lacks pepinase activity and is associated with mutations in CLN2. In addition, several animals with NCL-like neurodegenerative symptoms [mutant strains of mice (nclf and mnd), English setter, border collie, and Tibetan terrier dogs, sheep, and cattle] were found to contain enzyme activity and are thus unlikely to represent models for classical LINCL. Subcellular fractionation experiments indicate that the CLN2 protein is located in lysosomes, which is consistent with its acidic pH optimum for activity and the presence of mannose 6-phosphate. Taken together, these findings indicate that LINCL represents a lysosomal storage disorder that is characterized by the absence of a specific protease activity.     

Involvement of the mitochondrial compartment in human NCL fibroblasts

Neuronal ceroid lipofuscinosis (NCL) are a group of progressive neurodegenerative disorders of childhood, characterized by the endo-lysosomal storage of autofluorescent material. Impaired mitochondrial function is often associated with neurodegeneration, possibly related to the apoptotic cascade. In this study we investigated the possible effects of lysosomal accumulation on the mitochondrial compartment in the fibroblasts of two NCL forms, CLN1 and CLN6. Fragmented mitochondrial reticulum was observed in all cells by using the intravital fluorescent marker Mitotracker, mainly in the perinuclear region. This was also associated with intense signal from the lysosomal markers Lysotracker and LAMP2. Likewise, mitochondria appeared to be reduced in number and shifted to the cell periphery by electron microscopy; moreover the mitochondrial markers VDCA and COX IV were reduced following quantitative Western blot analysis. Whilst there was no evidence of increased cell death under basal condition, we observed a significant increase in apoptotic nuclei following Staurosporine treatment in CLN1 cells only. In conclusion, the mitochondrial compartment is affected in NCL fibroblasts invitro, and CLN1 cells seem to be more vulnerable to the negative effects of stressed mitochondrial membrane than CLN6 cells.     

Neuronal ceroid lipofuscinosis in Devon cattle is caused by a single base duplication (c.662dupG) in the bovine CLN5 gene

The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are recessively inherited neurodegenerative disorders that affect humans and other animals, characterised by brain atrophy and the accumulation of lysosome derived fluorescent storage bodies in neurons and most other cells. Common clinical signs include blindness, ataxia, dementia, seizures and premature death. The associated genes for six different human forms have been identified (CLN1, CLN2, CLN3, CLN5, CLN6 and CLN8), and three other human forms suggested (CLNs 4, 7 and 9). A form of NCL in Australian Devon cattle is caused by a single base duplication (c.662dupG) in bovine CLN5. This mutation causes a frame-shift and premature termination (p.Arg221GlyfsX6) which is predicted to result in a severely truncated protein, analogous to disease causing mutations in human Finnish late infantile variant NCL (CLN5), and a simple genetic diagnostic test has been developed. The symptoms and disease course in cattle also matches CLN5. Only one initiation site was found in the bovine gene, equivalent to the third of four possible initiation sites in the human gene. As cattle are anatomically and physiologically similar to humans with a human-like central nervous system and easy to maintain and breed, they provide a valuable alternative model for CLN5 studies.     

Neuronal ceroid lipofuscinoses are connected at molecular level: interaction of CLN5 protein with CLN2 and CLN3

Neuronal ceroid lipofuscinoses (NCLs) are neurodegenerative storage diseases characterized by mental retardation, visual failure, and brain atrophy as well as accumulation of storage material in multiple cell types. The diseases are caused by mutations in the ubiquitously expressed genes, of which six are known. Herein, we report that three NCL disease forms with similar tissue pathology are connected at the molecular level: CLN5 polypeptides directly interact with the CLN2 and CLN3 proteins based on coimmunoprecipitation and in vitro binding assays. Furthermore, disease mutations in CLN5 abolished interaction with CLN2, while not affecting association with CLN3. The molecular characterization of CLN5 revealed that it was synthesized as four precursor forms, due to usage of alternative initiator methionines in translation. All forms were targeted to lysosomes and the longest form, translated from the first potential methionine, was associated with membranes. Interactions between CLN polypeptides were shown to occur with this longest, membrane-bound form of CLN5. Both intracellular targeting and posttranslational glycosylation of the polypeptides carrying human disease mutations were similar to wild-type CLN5.     

Early changes in gene expression in two models of Batten disease

Infantile and juvenile neuronal ceroid lipofuscinosis (NCLs) are progressive neurodegenerative disorders of childhood with distinct ages of clinical onset, but with a similar pathological outcome. Infantile and juvenile NCL are inherited in an autosomal recessive manner due to mutations in the CLN1 and CLN3 genes, respectively. Recently developed Cln1- and Cln3-knockout mouse models share similarities in pathology with the respective human disease. Using oligonucleotide arrays we identified reproducible changes in gene expression in the brains of both 10-week-old Cln1- and Cln3-knockout mice as compared to wild-type controls, and confirmed changes in levels of several of the cognate proteins by immunoblotting. Despite the similarities in pathology, the two mutations affect the expression of different, non-overlapping sets of genes. The possible significance of these changes and the pathological mechanisms underlying NCL diseases are discussed.     

Progress towards understanding disease mechanisms in small vertebrate models of neuronal ceroid lipofuscinosis

Model systems provide an invaluable tool for investigating the molecular mechanisms underlying the NCLs, devastating neurodegenerative disorders that affect the relatively inaccessible tissues of the central nervous system. These models have enabled the assessment of behavioural, pathological, cellular, and molecular abnormalities, and also allow for development and evaluation of novel therapies. This review highlights the relative advantages of the two available small vertebrate species, the mouse and zebrafish, in modelling NCL disease, summarising how these have been useful in NCL research and their potential for the development and testing of prospective disease treatments. A panel of mouse mutants is available representing all the cloned NCL gene disorders (Cathepsin D, CLN1, CLN2, CLN3, CLN5, CLN6, CLN8). These NCL mice all have progressive neurodegenerative phenotypes that closely resemble the pathology of human NCL. The analysis of these models has highlighted several novel aspects underlying NCL pathogenesis including the selective nature of neurodegeneration, evidence for glial responses that precede neuronal loss and identification of the thalamus as an important pathological target early in disease progression. Studies in mice have also highlighted an unexpected heterogeneity underlying NCL phenotypes, and novel potential NCL-like mouse models have been described including mice with mutations in cathepsins, CLC chloride channels, and other lysosome-related genes. These new models are likely to provide significant new information on the spectrum of NCL disease. Information on NCL mice is available in the NCL Mouse Model Database (). There are homologs of most of the NCL genes in zebrafish, and NCL zebrafish models are currently in development. This model system provides additional advantages to those provided by NCL mouse models including high-throughput mutational, pharmacogenetic and therapeutic technique analyses. Mouse and zebrafish models are an important shared resource for NCL research, offering a unique possibility to dissect disease mechanisms and to develop therapeutic approaches.     

Elevated lysosomal pH in neuronal ceroid lipofuscinoses (NCLs).

We report here the intracellular (pHi) and lysosomal pH in fibroblasts of six forms of neuronal ceroid lipofuscinoses (NCLs). Acid extrusion rate and pH(i) values were measured by the membrane-permeant acetoxymethyl ester of the indicator dye, 2',7'-bis(carboxyethyl)-5-(and-6)-carboxy-fluorescein (BCECF) and lysosomal pH by a spectrofluorometric assay utilizing a novel acidotropic probe, Lysosensor yellow/blue. Intracellular pH was normal in all NCLs. Elevated lysosomal pH was detected in all NCL forms except CLN2 and CLN8. Elevated pH most probably disturbs the catalytic activity of lysosomes and is one important factor in explaining accumulation of ceroid and lipofuscin-like autofluorescent lipopigments characteristic of NCLs. Using the novel spectrofluorometric assay introduced in this study provides a fast and repeatable technique to measure intralysosomal pH from cell suspensions.     

Variant late infantile ceroid lipofuscinoses associated with novel mutations in CLN6

The neuronal ceroid lipofuscinoses (NCL) are heterogeneous neurodegenerative disorders with typical autofluorescence material stored in tissues. Ten clinical NCL forms and eight causative genes are known. Mutations in CLN6 have been reported in roughly 30 patients, mostly in association with the variant late-infantile NCL (v-LINCL) phenotype. We screened CLN6 in 30 children from a cohort of 53 v-LINCL cases and revised their clinical and ultrastructural features. We detected 11 mutations, eight of which are novel, all predicting a direct impairing of the putative gene function. No clear-cut genotype-phenotype correlations were observed, with inter- and intra-familial variability evident for few recurrent mutations. Ultrastructural findings were suggestive of an impaired regulation of the autophagic vacuoles turnover. While expanding the array of CLN6 mutations, we showed that more than half of our v-LINCL cases lack a DNA confirmation and further molecular etiologies are to be searched.     

A missense mutation (c.184C>T) in ovine CLN6 causes neuronal ceroid lipofuscinosis in Merino sheep whereas affected South Hampshire sheep have reduced levels of CLN6 mRNA

The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are a group of fatal recessively inherited neurodegenerative diseases of humans and animals characterised by common clinical signs and pathology. These include blindness, ataxia, dementia, behavioural changes, seizures, brain and retinal atrophy and accumulation of fluorescent lysosome derived organelles in most cells. A number of different variants have been suggested and seven different causative genes identified in humans (CLN1, CLN2, CLN3, CLN5, CLN6, CLN8 and CTSD). Animal models have played a central role in the investigation of this group of diseases and are extremely valuable for developing a better understanding of the disease mechanisms and possible therapeutic approaches. Ovine models include flocks of affected New Zealand South Hampshires and Borderdales and Australian Merinos. The ovine CLN6 gene has been sequenced in a representative selection of these sheep. These investigations unveiled the mutation responsible for the disease in Merino sheep (c.184C>T; p.Arg62Cys) and three common ovine allelic variants (c.56A>G, c.822G>A and c.933_934insCT). Linkage analysis established that CLN6 is the gene most likely to cause NCL in affected South Hampshire sheep, which do not have the c.184C>T mutation but show reduced expression of CLN6 mRNA in a range of tissues as determined by real-time PCR. Lack of linkage precludes CLN6 as a candidate for NCL in Borderdale sheep.     

Analysis of NCL Proteins from an Evolutionary Standpoint

The Neuronal Ceroid Lipofuscinoses (NCLs) are the most common group of neurodegenerative disorders of childhood. While mutations in eight different genes have been shown to be responsible for these clinically distinct types of NCL, the NCLs share many clinical and pathological similarities. We have conducted an exhaustive Basic Local Alignment Search Tool (BLAST) analysis of the human protein sequences for each of the eight known NCL proteins- CLN1, CLN2, CLN3, CLN5, CLN6, CLN7, CLN8 and CLN10. The number of homologous species per CLN-protein identified by BLAST searches varies depending on the parameters set for the BLAST search. For example, a lower threshold is able to pull up more homologous sequences whereas a higher threshold decreases this number. Nevertheless, the clade confines are consistent despite this variation in BLAST searching parameters. Further phylogenetic analyses on the appearance of NCL proteins through evolution reveals a different time line for the appearance of the CLN-proteins. Moreover, divergence of each protein shows a different pattern, providing important clues on the evolving role of these proteins. We present and review in-depth bioinformatic analysis of the NCL proteins and classify the CLN-proteins into families based on their structures and evolutionary relationships, respectively. Based on these analyses, we have grouped the CLN-proteins into common clades indicating a common evolving pathway within the evolutionary tree of life. CLN2 is grouped in Eubacteria, CLN1 and CLN10 in Viridiplantae, CLN3 in Fungi/ Metazoa, CLN7 in Bilateria and CLN5, CLN6 and CLN8 in Euteleostomi.     

Exome Sequencing Is an Efficient Tool for Variant Late-Infantile Neuronal Ceroid Lipofuscinosis Molecular Diagnosis

The neuronal ceroid-lipofuscinoses (NCL) is a group of neurodegenerative disorders characterized by epilepsy, visual failure, progressive mental and motor deterioration, myoclonus, dementia and reduced life expectancy. Classically, NCL-affected individuals have been classified into six categories, which have been mainly defined regarding the clinical onset of symptoms. However, some patients cannot be easily included in a specific group because of significant variation in the age of onset and disease progression. Molecular genetics has emerged in recent years as a useful tool for enhancing NCL subtype classification. Fourteen NCL genetic forms (CLN1 to CLN14) have been described to date. The variant late-infantile form of the disease has been linked to CLN5, CLN6, CLN7 (MFSD8) and CLN8 mutations. Despite advances in the diagnosis of neurodegenerative disorders mutations in these genes may cause similar phenotypes, which rends difficult accurate candidate gene selection for direct sequencing. Three siblings who were affected by variant late-infantile NCL are reported in the present study. We used whole-exome sequencing, direct sequencing and in silico approaches to identify the molecular basis of the disease. We identified the novel c.1219T>C (p.Trp407Arg) and c.1361T>C (p.Met454Thr) MFSD8 pathogenic mutations. Our results highlighted next generation sequencing as a novel and powerful methodological approach for the rapid determination of the molecular diagnosis of NCL. They also provide information regarding the phenotypic and molecular spectrum of CLN7 disease.     

Novel interactions of CLN5 support molecular networking between Neuronal Ceroid Lipofuscinosis proteins

Background  

Neuronal ceroid lipofuscinoses (NCLs) comprise at least eight genetically characterized neurodegenerative disorders of childhood. Despite of genetic heterogeneity, the high similarity of clinical symptoms and pathology of different NCL disorders suggest cooperation between different NCL proteins and common mechanisms of pathogenesis. Here, we have studied molecular interactions between NCL proteins, concentrating specifically on the interactions of CLN5, the protein underlying the Finnish variant late infantile form of NCL (vLINCL_Fin).     

The Gene Mutated in Variant Late-Infantile Neuronal Ceroid Lipofuscinosis (CLN6) and in nclf Mutant Mice Encodes a Novel Predicted Transmembrane Protein

The neuronal ceroid lipofuscinoses (NCLs) are a group of autosomal recessive neurodegenerative diseases characterized by the accumulation of autofluorescent lipopigment in various tissues and by progressive cell death in the brain and retina. The gene for variant late-infantile NCL (vLINCL), CLN6, was previously mapped to chromosome 15q21-23 and is predicted to be orthologous to the genes underlying NCL in nclf mice and in South Hampshire and Merino sheep. The gene underlying this disease has been identified with six different mutations found in affected patients and with a 1-bp insertion in the orthologous Cln6 gene in the nclf mouse. CLN6 encodes a novel 311-amino acid protein with seven predicted transmembrane domains, is conserved across vertebrates and has no homologies with proteins of known function. One vLINCL mutation, affecting a conserved amino acid residue within the predicted third hydrophilic loop of the protein, has been identified, suggesting that this domain may play an important functional role.     

CLN6, which is associated with a lysosomal storage disease, is an endoplasmic reticulum protein

The neuronal ceroid lipofuscinoses (NCLs) are severe inherited neurodegenerative disorders affecting children. In this disease, lysosomes accumulate autofluorescent storage material and there is death of neurons. Five types of NCL are caused by mutations in lysosomal proteins (CTSD, CLN1/PPT1, CLN2/TTPI, CLN3 and CLN5), and one type is caused by mutations in a protein that recycles between the ER and ERGIC (CLN8). The CLN6 gene underlying a variant of late infantile NCL (vLINCL) was recently identified. It encodes a novel 311 amino acid transmembrane protein. Antisera raised against CLN6 peptides detected a protein of 30 kDa by Western blotting of human cells, which was missing in cells from some CLN6 deficient patients. Using immunofluorescence microscopy, CLN6 was shown to reside in the endoplasmic reticulum (ER). CLN6 protein tagged with GFP at the C-terminus and expressed in HEK293 cells was also found within the ER. Investigation of the effect of five CLN6 disease mutations that affect single amino acids showed that the mutant proteins were retained in the ER. These data suggest that CLN6 is an ER resident protein, the activity of which, despite this location, must contribute to lysosomal function.     

Linkage disequilibrium between the juvenile neuronal ceroid lipofuscinosis gene and marker loci on chromosome 16p 12.1.

The neuronal ceroid lipofuscinoses (NCL; Batten disease) are a collection of autosomal recessive disorders characterized by the accumulation of autofluorescent lipopigments in the neurons and other cell types. Clinically, these disorders are characterized by progressive encephalopathy, loss of vision, and seizures. CLN3, the gene responsible for juvenile NCL, has been mapped to a 15-cM region flanked by the marker loci D16S148 and D16S150 on human chromosome 16. CLN2, the gene causing the late-infantile form of NCL (LNCL), is not yet mapped. We have used highly informative dinucleotide repeat markers mapping between D16S148 and D16S150 to refine the localization of CLN3 and to test for linkage to CLN2. We find significant linkage disequilibrium between CLN3 and the dinucleotide repeat marker loci D16S288 (chi 2(7) = 46.5, P < .005), D16S298 (chi 2(6) = 36.6, P < .005), and D16S299 (chi 2(7) = 73.8, P < .005), and also a novel RFLP marker at the D16S272 locus (chi 2(1) = 5.7, P = .02). These markers all map to 16p12.1. The D16S298/D16S299 haplotype "5/4" is highly overrepresented, accounting for 54% of CLN3 chromosomes as compared with 8% of control chromosomes (chi 2 = 117, df = 1, P < .001). Examination of the haplotypes suggests that the CLN3 locus can be narrowed to the region immediately surrounding these markers in 16p12.1. Analysis of D16S299 in our LNCL pedigrees supports our previous finding that CLN3 and CLN2 are different genetic loci. This study also indicates that dinucleotide repeat markers play a valuable role in disequilibrium studies.     

Experimental gene therapies for the NCLs

The neuronal ceroid lipofuscinoses (NCLs), also known as Batten disease, are a group of rare monogenic neurodegenerative diseases predominantly affecting children. All NCLs are lethal and incurable and only one has an approved treatment available. To date, 13 NCL subtypes (CLN1-8, CLN10-14) have been identified, based on the particular disease-causing defective gene. The exact functions of NCL proteins and the pathological mechanisms underlying the diseases are still unclear. However, gene therapy has emerged as an attractive therapeutic strategy for this group of conditions. Here we provide a short review discussing updates on the current gene therapy studies for the NCLs.     

Palmitoyl-protein thioesterase 1 deficiency in Drosophila melanogaster causes accumulation of abnormal storage material and reduced life span

Human neuronal ceroid lipofuscinoses (NCLs) are a group of genetic neurodegenerative diseases characterized by progressive death of neurons in the central nervous system (CNS) and accumulation of abnormal lysosomal storage material. Infantile NCL (INCL), the most severe form of NCL, is caused by mutations in the Ppt1 gene, which encodes the lysosomal enzyme palmitoyl-protein thioesterase 1 (Ppt1). We generated mutations in the Ppt1 ortholog of Drosophila melanogaster to characterize phenotypes caused by Ppt1 deficiency in flies. Ppt1-deficient flies accumulate abnormal autofluorescent storage material predominantly in the adult CNS and have a life span 30% shorter than wild type, phenotypes that generally recapitulate disease-associated phenotypes common to all forms of NCL. In contrast, some phenotypes of Ppt1-deficient flies differed from those observed in human INCL. Storage material in flies appeared as highly laminar spherical deposits in cells of the brain and as curvilinear profiles in cells of the thoracic ganglion. This contrasts with the granular deposits characteristic of human INCL. In addition, the reduced life span of Ppt1-deficient flies is not caused by progressive death of CNS neurons. No changes in brain morphology or increases in apoptotic cell death of CNS neurons were detected in Ppt1-deficient flies, even at advanced ages. Thus, Ppt1-deficient flies accumulate abnormal storage material and have a shortened life span without evidence of concomitant neurodegeneration.