Human Neuronal Ceroid Lipofuscinoses Research Articles

A mixed breed dog with neuronal ceroid lipofuscinosis is homozygous for a CLN5 nonsense mutation previously identified in Border Collies and Australian Cattle Dogs

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative disorders characterized by progressive declines in neurological functions following normal development. The NCLs are distinguished from similar disorders by the accumulation of autofluorescent lysosomal storage bodies in neurons and many other cell types, and are classified as lysosomal storage diseases. At least 13 genes contain pathogenic sequence variants that underlie different forms of NCL. Naturally occurring canine NCLs can serve as models to develop better understanding of the disease pathologies and for preclinical evaluation of therapeutic interventions for these disorders. To date 14 sequence variants in 8 canine orthologs of human NCL genes have been found to cause progressive neurological disorders similar to human NCLs in 12 different dog breeds. A mixed breed dog with parents of uncertain breed background developed progressive neurological signs consistent with NCL starting at approximately 11 to 12 months of age, and when evaluated with magnetic resonance imaging at 21 months of age exhibited diffuse brain atrophy. Due to the severity of neurological decline the dog was euthanized at 23 months of age. Cerebellar and cerebral cortical neurons contained massive accumulations of autofluorescent storage bodies the contents of which had the appearance of tightly packed membranes. A whole genome sequence, generated with DNA from the affected dog contained a homozygous C-to-T transition at position 30,574,637 on chromosome 22 which is reflected in the mature CLN5 transcript (CLN5: c.619C > T) and converts a glutamine codon to a termination codon (p.Gln207Ter). The identical nonsense mutation has been previously associated with NCL in Border Collies, Australian Cattle Dogs, and a German Shepherd-Australian Cattle Dog mix. The current whole genome sequence and a previously generated whole genome sequence for an Australian Cattle Dog with NCL share a rare homozygous haplotype that extends for 87 kb surrounding 22: 30, 574, 637 and includes 21 polymorphic sites. When genotyped at 7 of these polymorphic sites, DNA samples from the German Shepherd-Australian Cattle Dog mix and from 5 Border Collies with NCL that were homozygous for the CLN5: c.619 T allele also shared this homozygous haplotype, suggesting that the NCL in all of these dogs stems from the same founding mutation event that may have predated the establishment of the modern dog breeds. If so, the CLN5 nonsence allele is probably segregating in other, as yet unidentified, breeds. Thus, dogs exhibiting similar NCL-like signs should be screened for this CLN5 nonsense allele regardless of breed.

ATP13A2 missense variant in Australian Cattle Dogs with late onset neuronal ceroid lipofuscinosis

The neuronal ceroid lipofuscinoses (NCLs) are lysosomal storage disorders characterized by progressive neurodegeneration and declines in neurological functions. Pathogenic sequence variants in at least 13 genes underlie different forms of NCL, almost all of which are recessively inherited. To date 13 sequence variants in 8 canine orthologs of human NCL genes have been found to occur in 11 dog breeds in which they result in progressive neurological disorders similar to human NCLs. Canine NCLs can serve as models for preclinical evaluation of therapeutic interventions for these disorders. In most NCLs, the onset of neurological signs occurs in childhood, but some forms have adult onsets. Among these is CLN12 disease, also known as Kufor-Rakeb syndrome, PARK9, and spastic paraplegia78. These disorders result from variants in ATP13A2 which encodes a putative transmembrane ion transporter important for lysosomal function. Three Australian Cattle Dogs (a female and two of her offspring) were identified with a progressive neurological disorder with an onset of clinical signs at approximately 6 years of age. The affected dogs exhibited clinical courses and histopathology characteristic of the NCLs. Whole genome sequence analysis of one of these dogs revealed a homozygous c.1118C > T variant in ATP13A2 that predicts a nonconservative p.(Thr373Ile) amino acid substitution. All 3 affected dogs were homozygous for this variant, which was heterozygous in 42 of 394 unaffected Australian Cattle Dogs, the remainder of which were homozygous for the c.1118C allele. The high frequency of the mutant allele in this breed suggests that further screening for this variant should identify additional homozygous dogs and indicates that it would be advisable to perform such screening prior to breeding Australian Cattle Dogs.

Comparative transcriptomics reveals mechanisms underlying cln3-deficiency phenotypes in Dictyostelium

Mutations in CLN3 cause a juvenile form of neuronal ceroid lipofuscinosis (NCL). This devastating neurological disorder, commonly known as Batten disease, is currently untreatable due to a lack of understanding of the physiological role of the protein. Recently, work in the social amoeba Dictyostelium discoideum has provided valuable new insight into the function of CLN3 in the cell. More specifically, research has linked the Dictyostelium homolog (gene: cln3, protein: Cln3) to protein secretion, adhesion, and aggregation during starvation, which initiates multicellular development. In this study, we used comparative transcriptomics to explore the mechanisms underlying the aberrant response of cln3− cells to starvation. During starvation, 1153 genes were differentially expressed in cln3− cells compared to WT. Among the differentially expressed genes were homologs of other human NCL genes including TPP1/CLN2, CLN5, CTSD/CLN10, PGRN/CLN11, and CTSF/CLN13. STRING and GO term analyses revealed an enrichment of genes linked to metabolic, biosynthetic, and catalytic processes. We then coupled the findings from the RNA-seq analysis to biochemical assays, specifically showing that loss of cln3 affects the expression and activity of lysosomal enzymes, increases endo-lysosomal pH, and alters nitric oxide homeostasis. Finally, we show that cln3− cells accumulate autofluorescent storage bodies during starvation and provide evidence linking the function of Cln3 to Tpp1 and CtsD activity. In total, this study enhances our knowledge of the molecular mechanisms underlying Cln3 function in Dictyostelium.

Flupirtine derivatives as potential treatment for the neuronal ceroid lipofuscinoses.

ObjectiveNeuronal Ceroid Lipofuscinoses (NCL) are fatal inherited neurodegenerative diseases with established neuronal cell death and increased ceramide levels in brain, hence, a need for disease‐modifying drug candidates, with potential to enhance growth, reduce apoptosis and lower ceramide in neuronal precursor PC12 cells and human NCL cell lines using enhanced flupirtine aromatic carbamate derivatives in vitro.MethodsAromatic carbamate derivatives were tested by establishing growth curves under pro‐apoptotic conditions and activity evaluated by trypan blue and JC‐1 staining, as well as a drop in pro‐apoptotic ceramide in neuronal precursor PC12 cells following siRNA knockdown of the CLN3 gene, and CLN1‐/CLN2‐/CLN3‐/CLN6‐/CLN8 patient‐derived lymphoblasts. Ceramide levels were determined in CLN1‐/CLN2‐/CLN3‐/CLN6‐/CLN8 patient‐derived lymphoblasts before and after treatment. Expression of BCL‐2, ceramide synthesis enzymes (CERS2/CERS6/SMPD1/DEGS2) and Caspases 3/8/9 levels were compared in treated versus untreated CLN3‐deficient PC12 cells by qRT‐PCR.ResultsRetigabine, the benzyl‐derivatized carbamate and an allyl carbamate derivative were neuroprotective in CLN3‐defective PC12 cells and rescued CLN1‐/CLN2‐/CLN3‐/CLN6‐/CLN8 patient‐derived lymphoblasts from diminished growth and accelerated apoptosis. All drugs decreased ceramide in CLN1‐/CLN2‐/CLN3‐/CLN6‐/CLN8 patient‐derived lymphoblasts. Increased BCL‐2 and decreased ceramide synthesis enzyme expression were established in CLN3‐derived PC12 cells treated with the benzyl and allyl carbamate derivatives. They down‐regulated Caspase 3/Caspase 8 expression. Caspase 9 expression was reduced by the benzyl‐derivatized carbamate.InterpretationThese findings establish that compounds analogous to flupirtine demonstrate anti‐apoptotic activity with potential for treatment of NCL disease and use of ceramide as a marker for these diseases.

Using the social amoeba Dictyostelium to study the functions of proteins linked to neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinosis (NCL), also known as Batten disease, is a debilitating neurological disorder that affects both children and adults. Thirteen genetically distinct genes have been identified that when mutated, result in abnormal lysosomal function and an excessive accumulation of ceroid lipofuscin in neurons, as well as other cell types outside of the central nervous system. The NCL family of proteins is comprised of lysosomal enzymes (PPT1/CLN1, TPP1/CLN2, CTSD/CLN10, CTSF/CLN13), proteins that peripherally associate with membranes (DNAJC5/CLN4, KCTD7/CLN14), a soluble lysosomal protein (CLN5), a protein present in the secretory pathway (PGRN/CLN11), and several proteins that display different subcellular localizations (CLN3, CLN6, MFSD8/CLN7, CLN8, ATP13A2/CLN12). Unfortunately, the precise functions of many of the NCL proteins are still unclear, which has made targeted therapy development challenging. The social amoeba Dictyostelium discoideum has emerged as an excellent model system for studying the normal functions of proteins linked to human neurological disorders. Intriguingly, the genome of this eukaryotic soil microbe encodes homologs of 11 of the 13 known genes linked to NCL. The genetic tractability of the organism, combined with its unique life cycle, makes Dictyostelium an attractive model system for studying the functions of NCL proteins. Moreover, the ability of human NCL proteins to rescue gene-deficiency phenotypes in Dictyostelium suggests that the biological pathways regulating NCL protein function are likely conserved from Dictyostelium to human. In this review, I will discuss each of the NCL homologs in Dictyostelium in turn and describe how future studies can exploit the advantages of the system by testing new hypotheses that may ultimately lead to effective therapy options for this devastating and currently untreatable neurological disorder.

Mutational analysis of the feline CLN3 gene and an ultrastructural evaluation of lysosomal storage materials in a cat with neuronal ceroid lipofuscinosis: An investigation into the molecular basis of the disease

Neuronal ceroid lipofuscinosis (NCL) is a neurodegenerative disease caused by a number of different genes. A mutational analysis of the feline CLN3 gene was performed in a cat with NCL that had vacuolated lymphocytes, which is a feature of human NCL caused by defects of the CLN3 gene. To determine the candidate gene(s) responsible for this case, NCL-specific ultrastructures of storage materials were analysed. A sequence analysis indicated that the CLN3 gene was not likely to be responsible for this case of feline NCL because no deleterious mutation was detected. An ultrastructural analysis did not reveal any candidate gene because of inconsistency with any pattern found in human NCL. These findings suggest that the diagnostic criteria for human NCL are not directly applicable to feline NCL.

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 in vitro, and CLN1 cells seem to be more vulnerable to the negative effects of stressed mitochondrial membrane than CLN6 cells.

Deficient mitochondrial Ca 2+ buffering in the Cln8mnd mouse model of neuronal ceroid lipofuscinosis

Neuronal ceroid lipofuscinoses (NCLs) are a group of genetic childhood-onset progressive brain diseases characterized by a decline in mental and motor capacities, epilepsy, visual loss and premature death. Using patch clamp, fluorescence imaging and caged Ca 2+ photolysis, we evaluated the mechanisms of neuronal Ca 2+ clearance in Cln8 mnd mice, a model of the human NCL caused by mutations in the CLN8 gene. In Cln8 mnd hippocampal slices, Ca 2+ clearance efficiency in interneurons and, to some extent, principal neurons declined with age. In cultured Cln8 mnd hippocampal neurons, clearance of large Ca 2+ loads was inefficient due to impaired mitochondrial Ca 2+ uptake. In contrast, neither Ca 2+ uptake by sarco/endoplasmic reticulum Ca 2+ ATPase, nor Ca 2+ extrusion through plasma membrane was affected by the Cln8 mutation. Excitotoxic glutamate challenge caused Ca 2+ deregulation more readily in Cln8 mnd than in wt neurons. We propose that neurodegeneration in human CLN8 disorders is primarily caused by reduced mitochondrial Ca 2+ buffering capacity.

A truncating mutation in ATP13A2 is responsible for adult-onset neuronal ceroid lipofuscinosis in Tibetan terriers

A recessive, adult-onset neuronal ceroid-lipofuscinosis (NCL) occurs in Tibetan terriers. A genome-wide association study restricted this NCL locus to a 1.3Mb region of canine chromosome 2 which contains canine ATP13A2. NCL-affected dogs were homozygous for a single-base deletion in ATP13A2, predicted to produce a frameshift and premature termination codon. Homozygous truncating mutations in human ATP13A2 have been shown by others to cause Kufor-Rakeb syndrome (KRS), a rare neurodegenerative disease. These findings suggest that KRS is also an NCL, although analysis of KRS brain tissue will be needed to confirm this prediction. Generalized brain atrophy, behavioral changes, and cognitive decline occur in both people and dogs with ATP13A2 mutations; however, other clinical features differ between the species. For example, Tibetan terriers with NCL develop cerebellar ataxia not reported in KRS patients and KRS patients exhibit parkinsonism and pyramidal dysfunction not observed in affected Tibetan terriers. To see if ATP13A2 mutations could be responsible for some cases of human adult-onset NCL (Kufs disease), we resequenced ATP13A2 from 28 Kufs disease patients. None of these patients had ATP13A2 sequence variants likely to be causal for their disease, suggesting that mutations in this gene are not common causes of Kufs disease.

A Missense Mutation in Canine CLN6 in an Australian Shepherd with Neuronal Ceroid Lipofuscinosis

The childhood neuronal ceroid lipofuscinoses (NCLs) are inherited neurodegenerative diseases that are progressive and ultimately fatal. An Australian Shepherd that exhibited a progressive neurological disorder with signs similar to human NCL was evaluated. The cerebral cortex, cerebellum, and retina were found to contain massive accumulations of autofluorescent inclusions characteristic of the NCLs. Nucleotide sequence analysis of DNA from the affected dog identified a T to C variant (c.829T>C) in exon 7 of CLN6. Mutations in the human ortholog underlie a late-infantile form of NCL in humans. The T-to-C transition results in a tryptophan to arginine amino acid change in the predicted protein sequence. Tryptophans occur at homologous positions in the CLN6 proteins from all 13 other vertebrates evaluated. The c.829T>C transition is a strong candidate for the causative mutation in this NCL-affected dog. Dogs with this mutation could serve as a model for the analogous human disorder.

Retinal Pathology in a Canine Model of Late Infantile Neuronal Ceroid Lipofuscinosis

Late infantile neuronal ceroid lipofuscinosis (NCL) is an inherited disorder characterized by progressive vision loss. The disease results from mutations in the TPP1 (CLN2) gene. Studies were undertaken to characterize the effects of a TPP1 frameshift mutation on the retina in Dachshunds. A litter of four puppies consisting of one homozygous affected dog, two heterozygotes, and one homozygous normal dog were monitored for neurologic and retinal changes through 10 months of age. The affected and homozygous normal dogs, as well as one of the heterozygotes, were then euthanatized, and the retinas were examined morphologically. The affected dog exhibited normal visual behavior and retinal function at 3 months of age, but vision was clearly impaired by 7 months, with markedly reduced ERG b-wave amplitudes. Beyond 7 months of age, the affected dog was functionally blind, and pupillary light reflexes and ERG response amplitudes continued to decline through 10 months of age. Both rod and cone system functions were severely impaired. The retina exhibited accumulation of autofluorescent storage bodies with distinctive curvilinear contents. Substantial cell loss occurred in the inner nuclear layer, with a smaller reduction in photoreceptor cell density. The canine TPP1 mutation results in progressive vision loss and retinal degeneration similar to that which occurs in human late infantile NCL. With the canine model, the natural history of disease progression in the retina provides a better understanding of the pathologic course of the disease and provides objective markers that can be used to assess the efficacy of therapeutic interventions.

Accumulation of glial fibrillary acidic protein and histone H4 in brain storage bodies of Tibetan terriers with hereditary neuronal ceroid lipofuscinosis

The neuronal ceroid lipofuscinoses (NCLs) are inherited neurodegenerative diseases characterized by massive accumulation of autofluorescent storage bodies in neurons and other cells. A late-onset form of NCL occurs in Tibetan terrier dogs. Gel electrophoretic analyses of isolated storage body proteins from brains of affected dogs indicated that a protein of approximately 50 kDa was consistently prominent and a 16 kDa component was present in some brain storage body preparations. Mass spectral analysis identified the 50 kDa protein as glial fibrillary acidic protein (GFAP), isoform 2. GFAP identification was supported by immunoblot and immunohistochemical analyses. Histone H4 was the major protein in the 16 kDa component. Specific accumulation of GFAP and histone H4 in storage bodies has not been previously reported for any of the NCLs. Tibetan terrier NCL may be the canine correlate of one of the human adult-onset NCLs for which the genetic bases and storage body compositions have not yet been determined.

Lipofuscinose ceróide neuronal: achados clínicos e neurorradiológicos

The neuronal ceroid lipofuscinoses (NCL) are a group of neurodegenerative disorders, characterized by abnormal storage of an autofluorescent substance of lipopigments, resembling ceroid and lipofuscin, within lysosomes of neurons and other types of cells. The main phenotypic subtypes have been established on the basis of age of onset, clinical course, and ultra structural morphology, and classified as infantile, late infantile, juvenile and adult forms. Six genes have been associated with human NCL and approximately 150 mutations have been described. The aim of this study is to report the clinical, neuroradiological, and morphological characteristics of seven patients evaluated at Sarah Network of Hospitals for Reabilitation-Fortaleza-Ceará-Brazil. Five cases were histopathologically confirmed with skin biopsy and two were siblings of confirmed patients. An early diagnosis of NCL, an autosomal recessive disease, is mandatory for genetic counseling and to avoid further cases in the family. Imaging findings can contribute to the differential diagnosis.

Lysosomal storage disease upon disruption of the neuronal chloride transport protein ClC-6

Mammalian CLC proteins function as Cl(-) channels or as electrogenic Cl(-)/H(+) exchangers and are present in the plasma membrane and intracellular vesicles. We now show that the ClC-6 protein is almost exclusively expressed in neurons of the central and peripheral nervous systems, with a particularly high expression in dorsal root ganglia. ClC-6 colocalized with markers for late endosomes in neuronal cell bodies. The disruption of ClC-6 in mice reduced their pain sensitivity and caused moderate behavioral abnormalities. Neuronal tissues showed autofluorescence at initial axon segments. At these sites, electron microscopy revealed electron-dense storage material that caused a pathological enlargement of proximal axons. These deposits were positive for several lysosomal proteins and other marker proteins typical for neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. However, the lysosomal pH of Clcn6(-/-) neurons appeared normal. CLCN6 is a candidate gene for mild forms of human NCL. Analysis of 75 NCL patients identified ClC-6 amino acid exchanges in two patients but failed to prove a causative role of CLCN6 in that disease.

A frame shift mutation in canine TPP1 (the ortholog of human CLN2) in a juvenile Dachshund with neuronal ceroid lipofuscinosis

The neuronal ceroid lipofuscinoses (NCLs) are inherited lysosomal storage diseases characterized by progressive neuropathy and the accumulation of autofluorescent cytoplasmic granules. Clinical signs of a new canine NCL began in a 9-month-old male Dachshund with vomiting, mental dullness, and loss of previously learned commands and rapidly progressed to include disorientation, ataxia, visual deficits, generalized myoclonic seizures, and death at 12 months of age. Neurons throughout the CNS contained autofluorescent storage granules that stained with periodic acid-Schiff and Luxol fast blue stains. Electron microscopy revealed that the storage granule contents consisted of curvilinear-appearing material characteristic of human late infantile NCL caused by CLN2 mutations. Nucleotide sequence analysis of canine TPP1, the ortholog of human CLN2, revealed a single nucleotide deletion in exon 4 which predicted a frame shift with a premature stop codon. Brain tissue from the affected dog lacked detectable activity of the tripeptidyl-peptidase enzyme encoded by TPP1, whereas the specific activities of 15 other lysosomal enzymes were higher than those in the brains of three control dogs. The affected Dachshund was homozygous for the mutant c.325delC allele, his sire and dam were heterozygotes, and 181 unrelated dogs, including 77 Dachshunds, were all homozygous for the wild-type allele. A DNA assay that detects the mutant allele will help Dachshund breeders avoid producing affected puppies in future generations. Furthermore, this Dachshund NCL may prove to be a useful model for studying the pathogenesis of neurodegeneration in human late infantile NCL and for evaluating novel therapeutic interventions for this disease.

Another disorder finds its gene

Siintola et al . describe for the first time three patients with a deficiency of the lysosomal aspartyl proteinase cathepsin D (CTSD). In one of these patients, the authors found a mutational inactivation of the cathepsin D gene ( CTSD ) that encodes CTSD. All the patients described had severe neurological abnormalities at birth including intractable seizures, spasticity, apnoea and microcephaly. This deficiency is proposed as the underlying cause of congenital neuronal ceroid lipofuscinosis (CNCL) (Humphreys et al ., 1985; Garborg et al ., 1987; Barohn et al ., 1992). Originally described in a sheep model (Tyynela et al ., 2000, 2001) and further corroborated by findings in a canine model (Awano et al ., 2006), defects in CTSD were considered to be part of the neuronal ceroid lipofuscinoses (NCLs). The ovine model proved to be a feasible tool to study the disease. The affected sheep have a mutation in the active site of CTSD and newborn lambs suffer similar pathological and neurological consequences as seen in the human NCLs. The NCLs encompass a group of lysosomal storage diseases sharing similar clinical features (Goebel et al ., 1999; Herva et al ., 2000; Santavuori et al ., 2000; Weimer et al ., 2002; Mole, 2004). Described in the 19th and early 20th century as one disease with a variable age of onset, these diseases are now known to be caused by mutations in six different genes ( CLN1 – CLN3 , CLN5 , CLN6 and CLN8 ) located on different chromosomes (for a review see Mole, 2004). Adult NCL is expected to be caused by mutations in a gene not yet identified ( CLN4 ). The worldwide incidence of the NCLs is 1 : 30 000 live births (Santavuori et al ., 2000). The NCLs present with …

Characterization of the canine CLCN3 gene and evaluation as candidate for late-onset NCL

BackgroundThe neuronal ceroid lipofuscinoses (NCL) are a heterogenous group of inherited progressive neurodegenerative diseases in different mammalian species. Tibetan Terrier and Polish Owczarek Nizinny (PON) dogs show rare late-onset NCL variants with autosomal recessive inheritance, which can not be explained by mutations of known human NCL genes. These dog breeds represent animal models for human late-onset NCL. In mice the chloride channel 3 gene (Clcn3) encoding an intracellular chloride channel was described to cause a phenotype similar to NCL.ResultsTwo full-length cDNA splice variants of the canine CLCN3 gene are reported. The current canine whole genome sequence assembly was used for gene structure analyses and revealed 13 coding CLCN3 exons in 52 kb of genomic sequence. Sequence analysis of the coding exons and flanking intron regions of CLCN3 using six NCL-affected Tibetan terrier dogs and an NCL-affected Polish Owczarek Nizinny (PON) dog, as well as eight healthy Tibetan terrier dogs revealed 13 SNPs. No consistent CLCN3 haplotype was associated with NCL.ConclusionFor the examined animals we excluded the complete coding region and adjacent intronic regions of canine CLCN3 to harbor disease-causing mutations. Therefore it seems to be unlikely that a mutation in this gene is responsible for the late-onset NCL phenotype in these two dog breeds.

A mutation in the cathepsin D gene ( CTSD) in American Bulldogs with neuronal ceroid lipofuscinosis

We obtained DNA, brains, and eyes from American Bulldogs with neurodegeneration due to neuronal ceroid lipofuscinosis (NCL). The diagnosis of NCL was confirmed by detection of autofluorescent cytoplasmic inclusions within neurons throughout the brains, in retinal ganglion cells, and along outer limiting membranes of the retinas. Electron microscopy revealed that the inclusions had coarsely granular matrices surrounding well-delineated spherical structures and that the inclusions near the retinal outer limiting membranes were within photoreceptor cells, mostly cones. Affected American Bulldogs were homozygous for the A allele of a G to A transition in the cathepsin D gene ( CTSD), which predicts the conversion of methionine-199 to an isoleucine. Only the G allele was detected in DNA samples from 131 randomly selected dogs representing 108 breeds other than American Bulldog; however, the A allele had a frequency of 0.28 among 123 genotyped American Bulldogs. Transmission analysis in a 99 dog pedigree of American Bulldogs indicated a probability of less than 10 −7 that alleles from any mutation unlinked to CTSD would be concordant with the pedigree and phenotypes of the dogs. Brain samples from affected dogs had 36% of the cathepsin D-specific enzymatic activity found in control dog brains; whereas, specific enzymatic activities of 15 other lysosomal enzymes were unchanged or increased. Compared to previously described NCLs in mice and sheep that completely lack cathepsin D activity, the clinical course of NCL in the American Bulldogs was less severe and more closely resembled that of many human NCLs.

Cathepsin Deficiency as a Model for Neuronal Ceroid Lipofuscinoses

Lysosomal storage disorders arise from genetic alterations in lysosomal function and result in the aberrant accumulation of undigested lipoproteins.1 Neuronal ceroid lipofuscinoses (NCL), collectively known as Batten disease, are autosomal recessive and classical lysosomal storage disorders and are the most common human pediatric neurodegenerative diseases, occurring in 4 to 8 of every 100,000 children.2 NCL is a progressive and fatal neurodegenerative disease currently lacking any effective treatment or cure, thus underscoring the need for a greater understanding of its pathophysiology. In this issue of The American Journal of Pathology, Koike and colleagues3 elegantly describe how the cathepsin D (CD)-deficient mouse and the combined cathepsins B and L (CB/CL)-deficient mouse share neuropathological features common to NCL. Although mutations and/or deficiencies in cathepsins have not been described clinically in NCL, striking similarities exist in the neuropathology of these mice compared to established mouse models of NCL and to human NCL, suggesting involvement of similar neurodegenerative pathways. Previous reports of cell death in NCL have focused on apoptotic cell death as its principal death pathway. However, other death pathways also appear to be involved. Cell death has been defined previously by morphological criteria as either type I apoptotic or type II autophagic.4 Apoptotic death is regulated by pro- and anti-apoptotic members of the Bcl-2 family5 and culminates in the activation of caspases, which are responsible for causing the nuclear condensation, DNA fragmentation, and cell shrinkage that characterize apoptotic morphology.4 Autophagy is a normal cellular process whereby cytoplasmic material and organelles are shuttled to the lysosomes by a complex, regulated series of vesicle fusion events for degradation and ultimate recycling of lysosomal contents.6 Autophagic death is defined morphologically by the aberrant accumulation of autophagic vacuoles in a degenerating cell that in addition often displays morphological features of apoptosis.4 In agreement with such mechanisms, reports from Koike and colleagues,3,7 and Nakanishi and colleagues8 indicate the contribution of both apoptotic and autophagic neuron death, suggesting an interrelationship between these two pathways in neuron death and neurodegeneration in NCL.

A mutation in canine CLN5 causes neuronal ceroid lipofuscinosis in Border collie dogs

Neuronal ceroid lipofuscinosis (NCL) is a neurodegenerative disease found in Border collie dogs, humans, and other animals. Disease gene studies in humans and animals provided candidates for the NCL gene in Border collies. A combination of linkage analysis and comparative genomics localized the gene to CFA22 in an area syntenic to HSA13q that contains the CLN5 gene responsible for the Finnish variant of human late infantile NCL. Sequencing of CLN5 revealed a nonsense mutation (Q206X) within exon 4 that correlated with NCL in Border collies. This truncation mutation should result in a protein product of a size similar to that of some mutations identified in human CLN5 and therefore the Border collie may make a good model for human NCL. A simple test was developed to enable screening of the Border collie population for carriers so the disease can be eliminated as a problem in the breed.