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.     

Bioinformatic perspectives in the neuronal ceroid lipofuscinoses

The neuronal ceroid lipofuscinoses (NCLs) are a group of rare genetic diseases characterised clinically by the progressive deterioration of mental, motor and visual functions and histopathologically by the intracellular accumulation of autofluorescent lipopigment – ceroid – in affected tissues. The NCLs are clinically and genetically heterogeneous and more than 14 genetically distinct NCL subtypes have been described to date (CLN1–CLN14) (Haltia and Goebel, 2012 [1]). In this review we will chronologically summarise work which has led over the years to identification of NCL genes, and outline the potential of novel genomic techniques and related bioinformatic approaches for further genetic dissection and diagnosis of NCLs. This article is part of a Special Issue entitled: The Neuronal Ceroid Lipofuscinoses or Batten Disease.     

Functional biology of the neuronal ceroid lipofuscinoses (NCL) proteins

Neuronal ceroid lipofucinoses (NCLs) are a group of severe neurodegenerative disorders characterized by accumulation of autofluorescent ceroid lipopigment in patients' cells. The different forms of NCL share many similar pathological features but result from mutations in different genes. The genes affected in NCLs encode both soluble and transmembrane proteins and are localized to ER or to the endosomes/lysosomes. Due to selective vulnerability of the central nervous system in the NCL disorders, the corresponding proteins are proposed to have important, tissue specific roles in the brain. The pathological similarities of the different NCLs have led not only to the grouping of these disorders but also to suggestion that the NCL proteins function in the same biological pathway. Despite extensive research, including the development of several model organisms for NCLs and establishment of high-throughput techniques, the precise biological function of many of the NCL proteins has remained elusive. The aim of this review is to summarize the current knowledge of the functions, or proposed functions, of the different NCL proteins.     

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.     

Molecular diagnosis of and carrier screening for the neuronal ceroid lipofuscinoses

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

A two-dimensional protein fragmentation-proteomic study of neuronal ceroid lipofuscinoses: identification and characterization of differentially expressed proteins

The neuronal ceroid lipofuscinoses (NCLs) are a group of neuronal degenerative diseases that primarily affect children. Previously we hypothesized that the similarity of the phenotypes among the variant subtypes of NCL suggests that the NCLs share a common metabolic functional pathway. To test our hypothesis, we have studied several candidate proteins identified using a proteomic approach. We analyzed their differential expression and cataloged their functions and involved pathways. Forty protein peaks, differentially expressed in NCLs, were selected from two-dimensional protein fragmentation (PF2D) maps and twenty-four proteins were identified by MALDI-TOF-MS or LC-ESI-MS/MS. Six proteins were verified by further Western blotting. Our results showed that annexin A1, annexin A2, and vimentin were significantly down-regulated in NCL1, NCL2, NCL3, and NCL8 cells; galectin-1 was down-regulated in NCL1, NCL3, and NCL8 but up-regulated in NCL2 cells; and isoform 5 of caldesmon was up-regulated in all NCL cell types. The histone 2B was down-regulated in NCL3. Functional analysis showed that the differentially expressed proteins identified by PF2D could be grouped into categories of intermediate filaments, cell motility, apoptosis, cytoskeleton, membrane trafficking, calcium binding, nucleosome assembly, pigment granule and cell development. Immunocytochemistry revealed nuclear translocalization of annexin A1 in CLN2-deficient fibroblasts and abnormal distribution of L-caldesmon in cultured CLN1, CLN2, CLN3 and CLN8-deficient fibroblasts. Finding differentially expressed proteins in variant NCLs, which showed disturbances of cytoskeleton, RAGE-dependent cellular pathways and decreased glycolysis provides evidence supporting our hypothesis. These findings may contribute to the discovery of molecular biomarkers and may help further elucidate the pathogenic mechanisms underlying the NCLs.     

Moving towards a new era of genomics in the neuronal ceroid lipofuscinoses

The neuronal ceroid lipofuscinoses (NCL) are a group of disorders defined by shared clinical and pathological features, including seizures and progressive decline in vision, neurocognition, and motor functioning, as well as accumulation of autofluorescent lysosomal storage material, or ‘ceroid lipofuscin’. Research has revealed thirteen distinct genetic subtypes. Precisely how the gene mutations lead to the clinical phenotype is still incompletely understood, but recent research progress is starting to shed light on disease mechanisms, in both gene-specific and shared pathways. As the application of new sequencing technologies to genetic disease diagnosis has grown, so too has the spectrum of clinical phenotypes caused by mutations in the NCL genes. Most genes causing NCL have probably been identified, underscoring the need for a shift towards applying genomics approaches to achieve a deeper understanding of the molecular basis of the NCLs and related disorders. Here, we summarize the current understanding of the thirteen identified NCL genes and the proteins they encode, touching upon the spectrum of clinical manifestations linked to each of the genes, and we highlight recent progress leading to a broader understanding of key pathways involved in NCL disease pathogenesis and commonalities with other neurodegenerative diseases.     

Tripeptidyl peptidase I, the late infantile neuronal ceroid lipofuscinosis gene product, initiates the lysosomal degradation of subunit c of ATP synthase

The specific accumulation of a hydrophobic protein, subunit c of ATP synthase, in lysosomes from the cells of patients with the late infantile form of NCL (LINCL) is caused by a defect in the CLN2 gene product, tripeptidyl peptidase I (TPP-I). The data here show that TPP-I is involved in the initial degradation of subunit c in lysosomes and suggest that its absence leads directly to the lysosomal accumulation of subunit c. The inclusion of a specific inhibitor of TPP-I, Ala-Ala-Phe-chloromethylketone (AAF-CMK), in the culture medium of normal fibroblasts induced the lysosomal accumulation of subunit c. In an in vitro incubation experiment the addition of AAF-CMK to mitochondrial-lysosomal fractions from normal cells inhibited the proteolysis of subunit c, but not the b-subunit of ATP synthase. The use of two antibodies that recognize the aminoterminal and the middle portion of subunit c revealed that the subunit underwent aminoterminal proteolysis, when TPP-I, purified from rat spleen, was added to the mitochondrial fractions. The addition of both purified TPP-I and the soluble lysosomal fractions, which contain various proteinases, to the mitochondrial fractions resulted in rapid degradation of the entire molecule of subunit c, whereas the degradation of subunit c was markedly delayed through the specific inhibition of TPP-I in lysosomal extracts by AAF-CMK. The stable subunit c in the mitochondrial-lysosomal fractions from cells of a patient with LINCL was degraded on incubation with purified TPP-I. The presence of TPP-I led to the sequential cleavage of tripeptides from the N-terminus of the peptide corresponding to the amino terminal sequence of subunit c.     

Neuronal ceroid lipofuscinoses caused by defects in soluble lysosomal enzymes (CLN1 and CLN2)

Infantile and classical late infantile neuronal ceroid lipofuscinoses (NCL) are two recent additions to the expanding spectrum of lysosomal storage disorders caused by deficiencies in lysosomal hydrolases. They are latecomers to the lysosomal storage disorders, probably because of the heterogeneous nature of the storage material, which precluded meaningful biochemical analysis. Infantile NCL is caused by deficiency in palmitoyl-protein thioesterase, an enzyme that hydrolyzes fatty acids from cysteine residues in lipid-modified proteins. Classical late-infantile NCL is caused by a deficiency in tripeptidyl amino peptidase-I, a lysosomal peptidase that removes three amino acids from the free amino terminus of peptides or small proteins. Late-onset forms of these disorders have been described. The clinical, biochemical, and molecular genetic aspects of these two latest lysosomal storage disorders are discussed in this review. In addition, approaches to treatment and future directions for research are examined.     

RAGE signaling contributes to neuroinflammation in infantile neuronal ceroid lipofuscinosis

Palmitoyl-protein thioesterase-1 (PPT1) deficiency causes infantile neuronal ceroid lipofuscinosis (INCL), a devastating childhood neurodegenerative storage disorder. We previously reported that neuronal apoptosis in INCL is mediated by endoplasmic reticulum-stress. ER-stress disrupts Ca²⁺-homeostasis and stimulates the expression of Ca²⁺-binding proteins. We report here that in the PPT1-deficient human and mouse brain the levels of S100B, a Ca²⁺-binding protein, and its receptor, RAGE (receptor for advanced glycation end-products) are elevated. We further demonstrate that activation of RAGE signaling in astroglial cells mediates pro-inflammatory cytokine production, which is inhibited by SiRNA-mediated suppression of RAGE expression. We propose that RAGE signaling contributes to neuroinflammation in INCL.     

Cardiac pathology in neuronal ceroid lipofuscinoses (NCL): More than a mere co-morbidity

The neuronal ceroid lipofuscinoses (NCLs) are mostly seen as diseases affecting the central nervous system, but there is accumulating evidence that they have co-morbidities outside the brain. One of these co-morbidities is a decline in cardiac function. This is becoming increasingly recognised in teenagers and adolescents with juvenile CLN3, but it may also occur in individuals with other NCLs. The purpose of this review is to summarise the current knowledge of the structural and functional changes found in the hearts of animal models and people diagnosed with NCL. In addition, we present evidence of structural changes that were observed in a systematic comparison of the cardiomyocytes from CLN3^Δex7/8 mice.     

Infantile neuronal ceroid lipofuscinosis (CLN1): linkage disequilibrium in the Finnish population and evidence that variant late infantile form (variant CLN2) represents a nonallelic locus

Two forms of neuronal ceroid lipofuscinosis (CLN) are enriched in the Finnish population: the infantile form (CLN1), which is the most common progressive encephalopathy of small children, and the variant late infantile form (variant CLN2), which is a rare, atypical form of neuronal ceroid lipofuscinosis. We recently established the linkage of the infantile form (CLN1) to the short arm of chromosome 1 close to the anchor marker D1S7. Here we demonstrate a linkage disequilibrium of CLN1 chromosomes using the two closest markers, DIS62 and L-MYC at the short arm of chromosome 1 (P less than 0.0025). The results of linkage analyses in Finnish variant CLN2 families using the markers linked to CLN1 revealed an exclusion; i.e., this form of CLN is caused by a locus different from that of CLN1. This finding was confirmed with the result of the M-test for heterogeneity. The genealogical data collected further support the molecular genetic findings and provide evidence that the mutation causing CLN1 in Finland is very old, whereas the mutation causing the variant CLN2 could be a result of a younger, i.e., more recent founder effect.     

Pathogenesis and therapies for infantile neuronal ceroid lipofuscinosis (infantile CLN1 disease)

The neuronal ceroid lipofuscinoses (NCL, Batten disease) are a group of inherited neurodegenerative diseases. Infantile neuronal ceroid lipofuscinosis (INCL, infantile Batten disease, or infantile CLN1 disease) is caused by a deficiency in the soluble lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1) and has the earliest onset and fastest progression of all the NCLs. Several therapeutic strategies including enzyme replacement, gene therapy, stem cell-mediated therapy, and small molecule drugs have resulted in minimal to modest improvements in the murine model of PPT1-deficiency. However, more recent studies using various combinations of these approaches have shown more promising results; in some instances more than doubling the lifespan of PPT1-deficient mice. These combination therapies that target different pathogenic mechanisms may offer the hope of treating this profoundly neurodegenerative disorder. Similar approaches may be useful when treating other forms of NCL caused by deficiencies in soluble lysosomal proteins. Different therapeutic targets will need to be identified and novel strategies developed in order to effectively treat forms of NCL caused by deficiencies in integral membrane proteins such as juvenile neuronal ceroid lipofuscinosis. Finally, the challenge with all of the NCLs will lie in early diagnosis, improving the efficacy of the treatments, and effectively translating them into the clinic. This article is part of a Special Issue entitled: The Neuronal Ceroid Lipofuscinoses or Batten Disease.     

Human palmitoyl protein thioesterase: evidence for lysosomal targeting of the enzyme and disturbed cellular routing in infantile neuronal ceroid lipofuscinosis.

Palmitoyl protein thioesterase (PPT) is an enzyme that removes palmitate residues from various S-acylated proteins in vitro. We recently identified mutations in the human PPT gene in patients suffering from a neurodegenerative disease in childhood, infantile neuronal ceroid lipofuscinosis (INCL), with dramatic manifestations limited to the neurons of neocortical origin. Here we have expressed the human PPT cDNA in COS-1 cells and demonstrate the lysosomal targeting of the enzyme via the mannose 6-phosphate receptor-mediated pathway. The enzyme was also secreted into the growth medium and could be endocytosed by recipient cells. We further demonstrate the disturbed intracellular routing of PPT carrying the worldwide most common INCL mutation, Arg122Trp, to lysosomes. The results provide evidence that INCL represents a novel lysosomal enzyme deficiency. Further, the defect in the PPT gene causing a neurodegenerative disorder suggests that depalmitoylation of the still uncharacterized substrate(s) for PPT is critical for postnatal development or maintenance of cortical neurons.     

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.     

Systemic administration of tripeptidyl peptidase I in a mouse model of late infantile neuronal ceroid lipofuscinosis: effect of glycan modification

Late-infantile neuronal ceroid lipofuscinosis (LINCL) is a recessive genetic disease of childhood caused by deficiencies in the lysosomal protease tripeptidyl peptidase I (TPP1). Disease is characterized by progressive and extensive neuronal death. One hurdle towards development of enzyme replacement therapy is delivery of TPP1 to the brain. In this study, we evaluated the effect of modifying N-linked glycans on recombinant human TPP1 on its pharmacokinetic properties after administration via tail vein injection to a mouse model of LINCL. Unmodified TPP1 exhibited a dose-dependent serum half-life of 12 min (0.12 mg) to 45 min (2 mg). Deglycosylation or modification using sodium metaperiodate oxidation and reduction with sodium borohydride increased the circulatory half-life but did not improve targeting to the brain compared to unmodified TPP1. Analysis of liver, brain, spleen, kidney and lung demonstrated that for all preparations, >95% of the recovered activity was in the liver. Interestingly, administration of a single 2 mg dose (80 mg/kg) of unmodified TPP1 resulted in ～10% of wild-type activity in brain. This suggests that systemic administration of unmodified recombinant enzyme merits further exploration as a potential therapy for LINCL.     

Characterization of endopeptidase activity of tripeptidyl peptidase-I/CLN2 protein which is deficient in classical late infantile neuronal ceroid lipofuscinosis

Endopeptidase activities of the CLN2 gene product (Cln2p)/tripeptidyl peptidase I (TPP-I), purified from rat spleen, were studied using the synthetic fluorogenic substrates. We designed and constructed decapeptides, based on the known sequence cleavage specificities of bacterial pepstatin-insensitive carboxyl proteases (BPICP). MOCAc-Gly-Lys-Pro-Ile-Pro-Phe-Phe-Arg-Leu-Lys(Dnp)r-NH(2) is readily hydrolyzed by Cln2p/TPP-I (K(cat)/K(m) = 7.8 s(-1) mM(-1)). The enzyme had a maximal activity at pH 3.0 for an endopeptidase substrate, but at pH 4.5 with respect to tripeptidyl peptidase activity. Both endopeptidase and tripeptidyl peptidase activities were strongly inhibited by Ala-Ala-Phe-CH(2)Cl, but not inhibited by tyrostatin, an inhibitor of bacterial pepstatin-insensitive carboxyl proteases, pepstatin, or inhibitors of serine proteases. Fibroblasts from classical late infantile neuronal ceroid lipofuscinosis patients have less than 5% of the normal tripeptidyl peptidase activity and pepstatin-insensitive endopeptidase activity. Cln2p/TPP-I is a unique enzyme with both tripeptidyl peptidase and endopeptidase activities for certain substrate specificity.