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.     

Application of neuronal cell culture in human degenerative brain disorders

Neuronal cell culture system has been used for the study of pathochemical evaluations in human degenerative brain disorders, particularly for Krabbe's disease and neuronal ceroid lipofuscinosis. To understand the pathochemistry of Krabbe's disease, we added psychosine into neuronal cell cultures and psychosine treated cells showed the destruction of cytoskeleton and pathy intracellular changes. Electron microscopic finding showed the swelling of the mitochondria. Oligodendrocytes and Schwann cells were isolated from the brains and sciatic nerve of twitcher mouse as an authentic murine model of globoid cell leukodystrophy. Oligodendroglial cells cultured for 22 days were stained by anti-galactocerebroside antibodies. In twitcher oligodendrocyte processes were wirelike and progressively degenerated and there were few membranous expansion. Schwann cells from twitcher could not elongated their processes. These data suggest that psychosine might be important factor to result in these pathological conditions. Furthermore, we studied the effect of protease inhibitors, E-64 on dissociated primary cultures from fetal rat brain. After treated with E-64 in a concentration from 0.1-50 micrograms/ml, numerous cytoplasmic accumulations appeared in neuronal cells. These morphological pictures resemble with those of neuronal ceroid lipofuscinosis, Batten disease. We will discuss the relationship between the deficiency of catepsin H in Batten disease and inclusion bodies found in E-64.     

Autophagy is disrupted in a knock-in mouse model of juvenile neuronal ceroid lipofuscinosis

Juvenile neuronal ceroid lipofuscinosis is caused by mutation of a novel, endosomal/lysosomal membrane protein encoded by CLN3. The observation that the mitochondrial ATPase subunit c protein accumulates in this disease suggests that autophagy, a pathway that regulates mitochondrial turnover, may be disrupted. To test this hypothesis, we examined the autophagic pathway in Cln3(Deltaex7/8) knock-in mice and CbCln3(Deltaex7/8) cerebellar cells, accurate genetic models of juvenile neuronal ceroid lipofuscinosis. In homozygous knock-in mice, we found that the autophagy marker LC3-II was increased, and mammalian target of rapamycin was down-regulated. Moreover, isolated autophagic vacuoles and lysosomes from homozygous knock-in mice were less mature in their ultrastructural morphology than the wild-type organelles, and subunit c accumulated in autophagic vacuoles. Intriguingly, we also observed subunit c accumulation in autophagic vacuoles in normal aging mice. Upon further investigation of the autophagic pathway in homozygous knock-in cerebellar cells, we found that LC3-positive vesicles were altered and overlap of endocytic and lysosomal dyes was reduced when autophagy was stimulated, compared with wildtype cells. Surprisingly, however, stimulation of autophagy did not significantly impact cell survival, but inhibition of autophagy led to cell death. Together these observations suggest that autophagy is disrupted in juvenile neuronal ceroid lipofuscinosis, likely at the level of autophagic vacuolar maturation, and that activation of autophagy may be a prosurvival feedback response in the disease process.     

A metabolomic comparison of mouse models of the Neuronal Ceroid Lipofuscinoses

The Neuronal Ceroid Lipofuscinoses (NCL) are a group of fatal inherited neurodegenerative diseases in humans distinguished by a common clinical pathology, characterized by the accumulation of storage body material in cells and gross brain atrophy. In this study, metabolic changes in three NCL mouse models were examined looking for pathways correlated with neurodegeneration. Two mouse models; motor neuron degeneration (mnd) mouse and a variant model of late infantile NCL, termed the neuronal ceroid lipofuscinosis (nclf) mouse were investigated experimentally. Both models exhibit a characteristic accumulation of autofluorescent lipopigment in neuronal and non neuronal cells. The NMR profiles derived from extracts of the cortex and cerebellum from mnd and nclf mice were distinguished according to disease/wildtype status. In particular, a perturbation in glutamine and glutamate metabolism, and a decrease in γ-amino butyric acid (GABA) in the cerebellum and cortices of mnd (adolescent mice) and nclf mice relative to wildtype at all ages were detected. Our results were compared to the Cln3 mouse model of NCL. The metabolism of mnd mice resembled older (6?month) Cln3 mice, where the disease is relatively advanced, while the metabolism of nclf mice was more akin to younger (1-2?months) Cln3 mice, where the disease is in its early stages of progression. Overall, our results allowed the identification of metabolic traits common to all NCL subtypes for the three animal models.     

Mutations in DNAJC5, encoding cysteine-string protein alpha, cause autosomal-dominant adult-onset neuronal ceroid lipofuscinosis

Autosomal-dominant adult-onset neuronal ceroid lipofuscinosis (ANCL) is characterized by accumulation of autofluorescent storage material in neural tissues and neurodegeneration and has an age of onset in the third decade of life or later. The genetic and molecular basis of the disease has remained unknown for many years. We carried out linkage mapping, gene-expression analysis, exome sequencing, and candidate-gene sequencing in affected individuals from 20 families and/or individuals with simplex cases; we identified in five individuals one of two disease-causing mutations, c.346_348delCTC and c.344T>G, in DNAJC5 encoding cysteine-string protein alpha (CSPα). These mutations-causing a deletion, p.Leu116del, and an amino acid exchange, p.Leu115Arg, respectively-are located within the cysteine-string domain of the protein and affect both palmitoylation-dependent sorting and the amount of CSPα in neuronal cells. The resulting depletion of functional CSPα might cause in parallel the presynaptic dysfunction and the progressive neurodegeneration observed in affected individuals and lysosomal accumulation of misfolded and proteolysis-resistant proteins in the form of characteristic ceroid deposits in neurons. Our work represents an important step in the genetic dissection of a genetically heterogeneous group of ANCLs. It also confirms a neuroprotective role for CSPα in humans and demonstrates the need for detailed investigation of CSPα in the neuronal ceroid lipofuscinoses and other neurodegenerative diseases presenting with neuronal protein aggregation.     

A Murine Model of Variant Late Infantile Ceroid Lipofuscinosis Recapitulates Behavioral and Pathological Phenotypes of Human Disease

Neuronal ceroid lipofuscinoses (NCLs; also known collectively as Batten Disease) are a family of autosomal recessive lysosomal storage disorders. Mutations in as many as 13 genes give rise to ∼10 variants of NCL, all with overlapping clinical symptomatology including visual impairment, motor and cognitive dysfunction, seizures, and premature death. Mutations in CLN6 result in both a variant late infantile onset neuronal ceroid lipofuscinosis (vLINCL) as well as an adult-onset form of the disease called Type A Kufs. CLN6 is a non-glycosylated membrane protein of unknown function localized to the endoplasmic reticulum (ER). In this study, we perform a detailed characterization of a naturally occurring Cln6 mutant (Cln6^nclf) mouse line to validate its utility for translational research. We demonstrate that this Cln6^nclf mutation leads to deficits in motor coordination, vision, memory, and learning. Pathologically, we demonstrate loss of neurons within specific subregions and lamina of the cortex that correlate to behavioral phenotypes. As in other NCL models, this model displays selective loss of GABAergic interneuron sub-populations in the cortex and the hippocampus with profound, early-onset glial activation. Finally, we demonstrate a novel deficit in memory and learning, including a dramatic reduction in dendritic spine density in the cerebral cortex, which suggests a reduction in synaptic strength following disruption in CLN6. Together, these findings highlight the behavioral and pathological similarities between the Cln6^nclf mouse model and human NCL patients, validating this model as a reliable format for screening potential therapeutics.     

Juvenile neuronal ceroid lipofuscinosis and education

Juvenile neuronal ceroid lipofuscinosis (JNCL) is characterized by severe visual impairment with onset around age 4–8 years, and a developmental course that includes blindness, epilepsy, speech problems, dementia, motor coordination problems, and emotional reactions. There is presently no cure and the disease leads to premature death. There have been few studies of non-medical intervention for individuals with JNCL, probably because of the negative prognosis. The present chapter discusses the education of children and adolescents with JNCL on the basis of current knowledge about the variation in perceptual, cognitive and language abilities through the course of the disease, and the possibilities that exist for supporting coping and learning within and outside the classroom. Adapted and special needs education may contribute significantly to improved learning conditions, better maintenance of skills and less frustration for individuals with JNCL. This article is part of a Special Issue entitled: The Neuronal Ceroid Lipofuscinoses or Batten Disease.     

Ovine ceroid lipofuscinosis. The major lipopigment protein and the lipid-binding subunit of mitochondrial ATP synthase have the same NH2-terminal sequence

Previous studies on lipopigment isolated from sheep affected with ceroid lipofuscinosis (Batten's disease) showed that the disease is a lysosomal proteinosis, involving specific storage of peptide(s) that migrate in dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr of 3500. This band is the dominant contributor to the lipopigment mass. When purified total lipopigment proteins were loaded onto a protein sequencer, a dominant sequence was found, identical to the NH2 terminus of the lipid-binding subunit of protein translocating mitochondrial ATP synthase. This sequence was determined to 40 residues and a minimum estimate of 40% made for its contribution to the lipopigment protein mass. The full lipid-binding subunit has physical and chemical properties similar to those of the specifically stored low Mr peptide, which may be the full protein or a large NH2-terminal fragment of it. Lipopigments in the human ceroid lipofuscinoses also contain a major component with similar physical and chemical properties. These and previous results indicate that the genetic lesion in ovine ceroid lipofuscinosis causes an abnormal accumulation of this peptide in lysosomes, i.e. the disease is a proteolipid proteinosis, specifically a lysosomal mitochondrial ATP synthase lipid-binding subunit proteinosis. The analogous human diseases are likely to reflect storage of the same or similar peptides.     

Neurodevelopmental delay in the Cln3Δex7/8 mouse model for Batten disease

Juvenile neuronal ceroid lipofuscinosis (JNCL), also known as Batten disease, is a fatal inherited neurodegenerative disorder. The major clinical features of this disease are vision loss, seizures and progressive cognitive and motor decline starting in childhood. Mutations in CLN3 are known to cause the disease, allowing the generation of mouse models that are powerful tools for JNCL research. In this study, we applied behavioural phenotyping protocols to test for early behavioural alterations in Cln3 ^Δex7/8 knock-in mice, a genetic model that harbours the most common disease-causing CLN3 mutation. We found delayed acquisition of developmental milestones, including negative geotaxis, grasping, wire suspension time and postural reflex in both homozygous and heterozygous Cln3 ^Δex7/8 preweaning pups. To further investigate the consequences of this neurodevelopmental delay, we studied the behaviour of juvenile mice and found that homozygous and heterozygous Cln3 ^Δex7/8 knock-in mice also exhibit deficits in exploratory activity. Moreover, when analysing motor behaviour, we observed severe motor deficits in Cln3 ^Δex7/8 homozygous mice, but only a mild impairment in motor co-ordination and ambulatory gait in Cln3 ^Δex7/8 heterozygous animals. This study reveals previously overlooked behaviour deficits in neonate and young adult Cln3^Δex7/8 mice indicating neurodevelopmental delay as a putative novel component of JNCL.     

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.     

Drosophila as an In Vivo Model for Human Neurodegenerative Disease

With the increase in the ageing population, neurodegenerative disease is devastating to families and poses a huge burden on society. The brain and spinal cord are extraordinarily complex: they consist of a highly organized network of neuronal and support cells that communicate in a highly specialized manner. One approach to tackling problems of such complexity is to address the scientific questions in simpler, yet analogous, systems. The fruit fly, Drosophila melanogaster, has been proven tremendously valuable as a model organism, enabling many major discoveries in neuroscientific disease research. The plethora of genetic tools available in Drosophila allows for exquisite targeted manipulation of the genome. Due to its relatively short lifespan, complex questions of brain function can be addressed more rapidly than in other model organisms, such as the mouse. Here we discuss features of the fly as a model for human neurodegenerative disease. There are many distinct fly models for a range of neurodegenerative diseases; we focus on select studies from models of polyglutamine disease and amyotrophic lateral sclerosis that illustrate the type and range of insights that can be gleaned. In discussion of these models, we underscore strengths of the fly in providing understanding into mechanisms and pathways, as a foundation for translational and therapeutic research.     

Palmitoylation-induced Aggregation of Cysteine-string Protein Mutants That Cause Neuronal Ceroid Lipofuscinosis

Recently, mutations in the DNAJC5 gene encoding cysteine-string protein α (CSPα) were identified to cause the neurodegenerative disorder adult-onset neuronal ceroid lipofuscinosis. The disease-causing mutations (L115R or ΔL116) occur within the cysteine-string domain, a region of the protein that is post-translationally modified by extensive palmitoylation. Here we demonstrate that L115R and ΔL116 mutant proteins are mistargeted in neuroendocrine cells and form SDS-resistant aggregates, concordant with the properties of other mutant proteins linked to neurodegenerative disorders. The mutant aggregates are membrane-associated and incorporate palmitate. Indeed, co-expression of palmitoyltransferase enzymes promoted the aggregation of the CSPα mutants, and chemical depalmitoylation solubilized the aggregates, demonstrating that aggregation is induced and maintained by palmitoylation. In agreement with these observations, SDS-resistant CSPα aggregates were present in brain samples from patients carrying the L115R mutation and were depleted by chemical depalmitoylation. In summary, this study identifies a novel interplay between genetic mutations and palmitoylation in driving aggregation of CSPα mutant proteins. We propose that this palmitoylation-induced aggregation of mutant CSPα proteins may underlie the development of adult-onset neuronal ceroid lipofuscinosis in affected families.     

Genetic basis and phenotypic correlations of the neuronal ceroid lipofusinoses

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative disorders that mainly affect children and are grouped together by similar clinical features and the accumulation of autofluorescent storage material. More than a dozen genes containing nearly 400 mutations underlying human NCLs have been identified. Most of the mutations in these genes are associated with a typical disease phenotype, but some result in variable disease onset, severity and progression. There are still disease subgroups with unknown molecular genetic backgrounds. This article is part of a Special Issue entitled: The Neuronal Ceroid Lipofuscinoses or Batten Disease.     

A model of tripeptidyl-peptidase I (CLN2), a ubiquitous and highly conserved member of the sedolisin family of serine-carboxyl peptidases

Background  

Tripeptidyl-peptidase I, also known as CLN2, is a member of the family of sedolisins (serine-carboxyl peptidases). In humans, defects in expression of this enzyme lead to a fatal neurodegenerative disease, classical late-infantile neuronal ceroid lipofuscinosis. Similar enzymes have been found in the genomic sequences of several species, but neither systematic analyses of their distribution nor modeling of their structures have been previously attempted.     

Complex translocation t(9;21)(9;22)(q12p13)(q12q11) in the family of a child with 9p trisomy syndrome

Summary Leukocyte peroxidase activity was estimated in 5 patients with the juvenile form of neuronal ceroid lipofuscinosis (Spielmeyer-Vogt's disease) and in 15 healthy controls. In contradiction to recent reports normal activity of p-phenylene diamine mediated peroxidase was found in the patients. The possible role of contamination of the white cell preparation with hemoglobin is discussed.     

Dipeptidyl-peptidase I does not functionally compensate for the loss of tripeptidyl-peptidase I in the neurodegenerative disease late-infantile neuronal ceroid lipofuscinosis

LINCL (late-infantile neuronal ceroid lipofuscinosis) is a fatal neurodegenerative disease resulting from mutations in the gene encoding the lysosomal protease TPPI (tripeptidyl-peptidase I). TPPI is expressed ubiquitously throughout the body but disease appears restricted to the brain. One explanation for the absence of peripheral pathology is that in tissues other than brain, other proteases may compensate for the loss of TPPI. One such candidate is another lysosomal aminopeptidase, DPPI (dipeptidyl-peptidase I), which appears to have overlapping substrate specificity with TPPI and is expressed at relatively low levels in brain. Compensation for the loss of TPPI by DPPI may have therapeutic implications for LINCL and, in the present study, we have investigated this possibility using mouse genetic models. Our rationale was that if DPPI could compensate for the loss of TPPI in peripheral tissues, then its absence should exacerbate disease in an LINCL mouse model but, conversely, increased CNS (central nervous system) expression of DPPI should ameliorate disease. By comparing TPPI and DPPI single mutants with a double mutant lacking both proteases, we found that the loss of DPPI had no effect on accumulation of storage material, disease severity or lifespan of the LINCL mouse. Transgenic expression of DPPI resulted in a approximately 2-fold increase in DPPI activity in the brain, but this had no significant effect on survival of the LINCL mouse. These results together indicate that DPPI cannot functionally compensate for the loss of TPPI. Therapeutic approaches to increase neuronal expression of DPPI are therefore unlikely to be effective for treatment of LINCL.     

Ceroid lipofuscinosis in sheep. II. The major component of the lipopigment in liver, kidney, pancreas, and brain is low molecular weight protein

Previous studies on the lipopigment from the livers of sheep affected with ceroid lipofuscinosis showed that the disease does not involve a defect in lipid metabolism or abnormal lipid peroxidation and that most of the lipopigment was proteinaceous. In this study, lipopigment was isolated from liver, kidney, pancreas, and brain of affected sheep without the use of proteolytic enzymes. Lipopigment from all tissues was two-thirds protein. Modified silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a major band of Mr = 14,800, heterogeneous material between Mr = 5,000 and 9,000, and a major band of Mr = 3,500. These compounds did not stain for RNA or carbohydrate and were digested by a nuclease-free protease as expected for protein. They are not normal lysosomal proteins. Lipopigment levels of dolichol, ubiquinone, and cholesterol were consistent with the lipopigment being protein-enriched lysosome-derived cytosomes. The presence of the Mr = 3,500 proteins in whole affected tissue homogenates distinguished them from homogenates of normal tissues. It was concluded that low Mr proteins are specifically stored in ovine ceroid lipofuscinosis and that the ceroid lipofuscinoses may result from inherited defects in lysosomal protein catabolism.