Lysosomal Storage Bodies Research Articles

Neuronal Ceroid Lipofuscinosis in a Mixed-Breed Dog with a Splice Site Variant in CLN6.

A 23-month-old neutered male dog of unknown ancestry presented with a history of progressive neurological signs that included anxiety, cognitive impairment, tremors, seizure activity, ataxia, and pronounced visual impairment. The clinical signs were accompanied by global brain atrophy. Due to progression in the severity of disease signs, the dog was euthanized at 26 months of age. An examination of the tissues collected at necropsy revealed dramatic intracellular accumulations of autofluorescent inclusions in the brain, retina, and cardiac muscle. The inclusions were immunopositive for subunit c of mitochondrial ATP synthase, and their ultrastructural appearances were similar to those of lysosomal storage bodies that accumulate in some neuronal ceroid lipofuscinosis (NCL) diseases. The dog also exhibited widespread neuroinflammation. Based on these findings, the dog was deemed likely to have suffered from a form of NCL. A whole genome sequence analysis of the proband's DNA revealed a homozygous C to T substitution that altered the intron 3-exon 4 splice site of CLN6. Other mutations in CLN6 cause NCL diseases in humans and animals, including dogs. The CLN6 protein was undetectable with immunolabeling in the tissues of the proband. Based on the clinical history, fluorescence and electron-microscopy, immunohistochemistry, and molecular genetic findings, the disorder in this dog was classified as an NCL resulting from the absence of the CLN6 protein. Screening the dog's genome for a panel of breed-specific polymorphisms indicated that its ancestry included numerous breeds, with no single breed predominating. This suggests that the CLN6 disease variant is likely to be present in other mixed-breed dogs and at least some ancestral breeds, although it is likely to be rare since other cases have not been reported to date.

Homozygous CNP Mutation and Neurodegeneration in Weimaraners: Myelin Abnormalities and Accumulation of Lipofuscin-like Inclusions.

A progressive neurological disorder was observed in a male neutered Weimaraner. Clinical signs included fecal incontinence, lethargy, moderate paraparesis, proprioceptive pelvic limb ataxia, falling, cognitive decline, incoordination, decreased interest in food, changes in posture, and episodes of trance-like behavior. Neurologic signs were first observed at approximately 4 years, 10 months of age and progressed slowly. Magnetic resonance imaging showed generalized brain atrophy with areas of white matter pathology. Humane euthanasia was elected at 6 years, 7 months of age due to increasing severity of the neurological signs. Autofluorescent intracellular granules were observed in the cerebral and cerebellar cortexes, optic nerve, and cardiac muscle of the affected dog. These abnormal inclusions in the cerebral cortex and cardiac muscle immunolabeled with antibodies to mitochondrial ATP synthase subunit c protein, like that observed in the neuronal ceroid lipofuscinosis group of lysosomal storage diseases. Immunolabeling also demonstrated pronounced neuroinflammation in brain tissues. The ultrastructural appearances of the disease-related inclusion bodies in the brain and optic nerve were quite variable. The ultrastructure and locations of many of the inclusions in the nervous tissues suggested that they were derived, at least in part, from the myelin surrounding axons. The storage bodies in the cardiac muscle were located in mitochondria-rich regions and consisted of parallel arrays of membrane-like components interspersed with electron-dense flocculent material. The disease was characterized by pronounced abnormalities in the myelin of the brain and optic nerve consisting of distinctive areas of ballooning between the layers of myelin. The whole genome sequence generated from the affected dog contained a homozygous G-to-A missense mutation in CNP, which encodes proteins with CNPase enzyme activity and a structural role in myelin. The mutation predicts a Thr42Met amino acid sequence substitution. Genotyping of archived Weimaraner DNA samples identified an additional G > A variant homozygote with a clinical history and brain lesions similar to those of the proband. Of 304 Weimaraners and over 4000 other dogs of various breeds, the proband and the other Weimaraner that exhibited similar signs were the only two that were homozygous for the CNP missense variant. CNPase immunolabeling was widespread in brain tissues from normal dogs but was undetectable in the same tissues from the proband. Based on the clinical history, fluorescence and electron-microscopy, immunohistochemistry, and molecular genetic findings, the late-onset Weimaraner disorder likely results from the missense mutation that results in CNPase deficiency, leading to myelin abnormalities, accumulation of lysosomal storage bodies, and brain atrophy. Similar disorders have been associated with different CNP variants in Dalmatians and in human subjects.

Visual system pathology in a canine model of CLN5 neuronal ceroid lipofuscinosis

CLN5 neuronal ceroid lipofuscinosis is a hereditary neurodegenerative disease characterized by progressive neurological decline, vision loss and seizures. Visual impairment in children with CLN5 disease is attributed to a progressive decline in retinal function accompanied by retinal degeneration as well as impaired central nervous system function associated with global brain atrophy. We studied visual system pathology in five Golden Retriever littermates homozygous for the CLN5 disease allele previously identified in the breed. The dogs exhibited signs of pronounced visual impairment by 21–22 months of age. Electroretinogram recordings showed a progressive decline in retinal function primarily affecting cone neural pathways. Altered visual evoked potential recordings indicated that disease progression affected visual signal processing in the brain. Aside from several small retinal detachment lesions, no gross retinal abnormalities were observed with in vivo ocular imaging and histologically the retinas did not exhibit apparent abnormalities by 23 months of age. However, there was extensive accumulation of autofluorescent membrane-bound lysosomal storage bodies in almost all retinal layers, as well as in the occipital cortex, by 20 months of age. In the retina, storage was particularly pronounced in retinal ganglion cells, the retinal pigment epithelium and in photoreceptor cells just interior to the outer limiting membrane. The visual system pathology of CLN5-affected Golden Retrievers is similar to that seen early in the human disease. It was not possible to follow the dogs to an advanced stage of disease progression due to the severity of behavioral and motor disease signs by 23 months of age. The findings reported here indicate that canine CLN5 disease will be a useful model of visual system disease in CLN5 neuronal ceroid lipofuscinosis. The baseline data obtained in this investigation will be useful in future therapeutic intervention studies. The findings indicate that there is a fairly broad time frame after disease onset within which treatments could be effective in preserving vision.

Fundus autofluorescence imaging.

Fundus autofluorescence (FAF) imaging is an in vivo imaging method that allows for topographic mapping of naturally or pathologically occurring intrinsic fluorophores of the ocular fundus. The dominant sources are fluorophores accumulating as lipofuscin in lysosomal storage bodies in postmitotic retinal pigment epithelium cells as well as other fluorophores that may occur with disease in the outer retina and subretinal space. Photopigments of the photoreceptor outer segments as well as macular pigment and melanin at the fovea and parafovea may act as filters of the excitation light. FAF imaging has been shown to be useful with regard to understanding of pathophysiological mechanisms, diagnostics, phenotype-genotype correlation, identification of prognostic markers for disease progression, and novel outcome parameters to assess efficacy of interventional strategies in chorio-retinal diseases. More recently, the spectrum of FAF imaging has been expanded with increasing use of green in addition to blue FAF, introduction of spectrally-resolved FAF, near-infrared FAF, quantitative FAF imaging and fluorescence life time imaging (FLIO). This article gives an overview of basic principles, FAF findings in various retinal diseases and an update on recent developments.

Differential accumulation of storage bodies with aging defines discrete subsets of microglia in the healthy brain.

To date, microglia subsets in the healthy CNS have not been identified. Utilizing autofluorescence (AF) as a discriminating parameter, we identified two novel microglia subsets in both mice and non-human primates, termed autofluorescence-positive (AF+) and negative (AF-). While their proportion remained constant throughout most adult life, the AF signal linearly and specifically increased in AF+ microglia with age and correlated with a commensurate increase in size and complexity of lysosomal storage bodies, as detected by transmission electron microscopy and LAMP1 levels. Post-depletion repopulation kinetics revealed AF- cells as likely precursors of AF+ microglia. At the molecular level, the proteome of AF+ microglia showed overrepresentation of endolysosomal, autophagic, catabolic, and mTOR-related proteins. Mimicking the effect of advanced aging, genetic disruption of lysosomal function accelerated the accumulation of storage bodies in AF+ cells and led to impaired microglia physiology and cell death, suggestive of a mechanistic convergence between aging and lysosomal storage disorders.

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.

Extraneuronal pathology in a canine model of CLN2 neuronal ceroid lipofuscinosis after intracerebroventricular gene therapy that delays neurological disease progression

CLN2 neuronal ceroid lipofuscinosis is a hereditary lysosomal storage disease with primarily neurological signs that results from mutations in TPP1, which encodes the lysosomal enzyme tripeptidyl peptidase-1 (TPP1). Studies using a canine model for this disorder demonstrated that delivery of TPP1 enzyme to the cerebrospinal fluid (CSF) by intracerebroventricular administration of an AAV-TPP1 vector resulted in substantial delays in the onset and progression of neurological signs and prolongation of life span. We hypothesized that the treatment may not deliver therapeutic levels of this protein to tissues outside the central nervous system that also require TPP1 for normal lysosomal function. To test this hypothesis, dogs treated with CSF administration of AAV-TPP1 were evaluated for the development of non-neuronal pathology. Affected treated dogs exhibited progressive cardiac pathology reflected by elevated plasma cardiac troponin-1, impaired cardiac function and development of histopathological myocardial lesions. Progressive increases in the plasma activity levels of alanine aminotransferase and creatine kinase indicated development of pathology in the liver and muscles. The treatment also did not prevent disease-related accumulation of lysosomal storage bodies in the heart or liver. These studies indicate that optimal treatment outcomes for CLN2 disease may require delivery of TPP1 systemically as well as directly to the central nervous system.

Neuronal ceroid lipofuscinosis associated with an MFSD8 mutation in Chihuahuas.

The neuronal ceroid lipofuscinoses (NCLs) are hereditary neurodegenerative disorders characterized by progressive declines in neurological functions, seizures, and premature death. NCLs result from mutations in at least 13 different genes. Canine versions of the NCLs can serve as important models in developing effective therapeutic interventions for these diseases. NCLs have been described in a number of dog breeds, including Chihuahuas. Studies were undertaken to further characterize the pathology of Chihuahua NCL and to verify its molecular genetic basis. Four unrelated client owned Chihuahuas from Japan, Italy and England that exhibited progressive neurological signs consistent with a diagnosis of NCL underwent neurological examinations. Brain and in some cases also retinal and heart tissues were examined postmortem for the presence of lysosomal storage bodies characteristic of NCL. The affected dogs exhibited massive accumulation of autofluorescent lysosomal storage bodies in the brain, retina and heart accompanied by brain atrophy and retinal degeneration. The dogs were screened for known canine NCL mutations previously reported in a variety of dog breeds. All 4 dogs were homozygous for the MFSD8 single base pair deletion (MFSD8:c.843delT) previously associated with NCL in a Chinese Crested dog and in 2 affected littermate Chihuahuas from Scotland. The dogs were all homozygous for the normal alleles at the other genetic loci known to cause different forms of canine NCL. The MFSD8:c.843delT mutation was not present in 57 Chihuahuas that were either clinically normal or suffered from unrelated diseases or in 1761 unaffected dogs representing 186 other breeds. Based on these data it is almost certain that the MFSD8:c.843delT mutation is the cause of NCL in Chihuahuas. Because the disorder occurred in widely separated geographic locations or in unrelated dogs from the same country, it is likely that the mutant allele is widespread among Chihuahuas. Genetic testing for this mutation in other Chihuahuas is therefore likely to identify intact dogs with the mutant allele that could be used to establish a research colony that could be used to test potential therapeutic interventions for the corresponding human disease.

Intracerebroventricular gene therapy that delays neurological disease progression is associated with selective preservation of retinal ganglion cells in a canine model of CLN2 disease

CLN2 disease is one of a group of lysosomal storage disorders called the neuronal ceroid lipofuscinoses (NCLs). The disease results from mutations in the TPP1 gene that cause an insufficiency or complete lack of the soluble lysosomal enzyme tripeptidyl peptidase-1 (TPP1). TPP1 is involved in lysosomal protein degradation, and lack of this enzyme results in the accumulation of protein-rich autofluorescent lysosomal storage bodies in numerous cell types including neurons throughout the central nervous system and the retina. CLN2 disease is characterized primarily by progressive loss of neurological functions and vision as well as generalized neurodegeneration and retinal degeneration. In children the progressive loss of neurological functions typically results in death by the early teenage years. A Dachshund model of CLN2 disease with a null mutation in TPP1 closely recapitulates the human disorder with a progression from disease onset at approximately 4 months of age to end-stage at 10–11 months. Delivery of functional TPP1 to the cerebrospinal fluid (CSF), either by periodic infusion of the recombinant protein or by a single administration of a TPP1 gene therapy vector to the CSF, significantly delays the onset and progression of neurological signs and prolongs life span but does not prevent the loss of vision or modest retinal degeneration that occurs by 11 months of age. In this study we found that in dogs that received the CSF gene therapy treatment, the degeneration of the retina and loss of retinal function continued to progress during the prolonged life spans of the treated dogs. Eventually the normal cell layers of the retina almost completely disappeared. An exception was the ganglion cell layer. In affected dogs that received TPP1 gene therapy to the CSF and survived an average of 80 weeks, ganglion cell axons were present in numbers comparable to those of normal Dachshunds of similar age. The selective preservation of the retinal ganglion cells suggests that while TPP1 protein delivered via the CSF may protect these cells, preservation of the remainder of the retina will require delivery of normal TPP1 more directly to the retina, probably via the vitreous body.

A CLN8 nonsense mutation in the whole genome sequence of a mixed breed dog with neuronal ceroid lipofuscinosis and Australian Shepherd ancestry

The neuronal ceroid lipofuscinoses (NCLs) are hereditary neurodegenerative diseases characterized by seizures and progressive cognitive decline, motor impairment, and vision loss accompanied by accumulation of autofluorescent lysosomal storage bodies in the central nervous system and elsewhere in the body. Mutations in at least 14 genes underlie the various forms of NCL. One of these genes, CLN8, encodes an intrinsic membrane protein of unknown function that appears to be localized primarily to the endoplasmic reticulum. Most CLN8 mutations in people result in a form of NCL with a late infantile onset and relatively rapid progression. A mixed breed dog with Australian Shepherd and Blue Heeler ancestry developed neurological signs characteristic of NCL starting at about 8months of age. The signs became progressively worse and the dog was euthanized at 21months of age due to seizures of increasing frequency and severity. Postmortem examination of the brain and retinas identified massive accumulations of intracellular autofluorescent inclusions characteristic of the NCLs. Whole genome sequencing of DNA from this dog identified a CLN8:c.585G>A transition that predicts a CLN8:p.Trp195* nonsense mutation. This mutation appears to be rare in both ancestral breeds. All of our 133 archived DNA samples from Blue Heelers, and 1481 of our 1488 archived Australian Shepherd DNA samples tested homozygous for the reference CLN8:c.585G allele. Four of the Australian Shepherd samples tested heterozygous and 3 tested homozygous for the mutant CLN8:c.585A allele. All 3 dogs homozygous for the A allele exhibited clinical signs of NCL and in 2 of them NCL was confirmed by postmortem evaluation of brain tissue. The occurrence of confirmed NCL in 3 of 4 CLN8:c.585A homozygous dogs, plus the occurrence of clinical signs consistent with NCL in the fourth homozygote strongly suggests that this rare truncating mutation causes NCL. Identification of this NCL-causing mutation provides the opportunity for identifying dogs that can be used to establish a canine model for the CLN8 disease (also known as late infantile variant or late infantile CLN8 disease).

A One Base Pair Deletion in the Canine ATP13A2 Gene Causes Exon Skipping and Late-Onset Neuronal Ceroid Lipofuscinosis in the Tibetan Terrier

Neuronal ceroid lipofuscinosis (NCL) is a progressive neurodegenerative disease characterized by brain and retinal atrophy and the intracellular accumulation of autofluorescent lysosomal storage bodies resembling lipofuscin in neurons and other cells. Tibetan terriers show a late-onset lethal form of NCL manifesting first visible signs at 5–7 years of age. Genome-wide association analyses for 12 Tibetan-terrier-NCL-cases and 7 Tibetan-terrier controls using the 127K canine Affymetrix SNP chip and mixed model analysis mapped NCL to dog chromosome (CFA) 2 at 83.71–84.72 Mb. Multipoint linkage and association analyses in 376 Tibetan terriers confirmed this genomic region on CFA2. A mutation analysis for 14 positional candidate genes in two NCL-cases and one control revealed a strongly associated single nucleotide polymorphism (SNP) in the MAPK PM20/PM21 gene and a perfectly with NCL associated single base pair deletion (c.1620delG) within exon 16 of the ATP13A2 gene. The c.1620delG mutation in ATP13A2 causes skipping of exon 16 presumably due to a broken exonic splicing enhancer motif. As a result of this mutation, ATP13A2 lacks 69 amino acids. All known 24 NCL cases were homozygous for this deletion and all obligate 35 NCL-carriers were heterozygous. In a sample of 144 dogs from eleven other breeds, the c.1620delG mutation could not be found. Knowledge of the causative mutation for late-onset NCL in Tibetan terrier allows genetic testing of these dogs to avoid matings of carrier animals. ATP13A2 mutations have been described in familial Parkinson syndrome (PARK9). Tibetan terriers with these mutations provide a valuable model for a PARK9-linked disease and possibly for manganese toxicity in synucleinopathies.

Mice deficient in Neu4 sialidase exhibit abnormal ganglioside catabolism and lysosomal storage

Mammalian sialidase Neu4, ubiquitously expressed in human tissues, is located in the lysosomal and mitochondrial lumen and has broad substrate specificity against sialylated glycoconjugates. To investigate whether Neu4 is involved in ganglioside catabolism, we transfected beta-hexosaminidase-deficient neuroglia cells from a Tay-Sachs patient with a Neu4-expressing plasmid and demonstrated the correction of storage due to the clearance of accumulated GM2 ganglioside. To further clarify the biological role of Neu4, we have generated a stable loss-of-function phenotype in cultured HeLa cells and in mice with targeted disruption of the Neu4 gene. The silenced HeLa cells showed reduced activity against gangliosides and had large heterogeneous lysosomes containing lamellar structures. Neu4(-/-) mice were viable, fertile and lacked gross morphological abnormalities, but showed a marked vacuolization and lysosomal storage in lung and spleen cells. Lysosomal storage bodies were also present in cultured macrophages preloaded with gangliosides. Thin-layer chromatography showed increased relative level of GD1a ganglioside and a markedly decreased level of GM1 ganglioside in brain of Neu4(-/-) mice suggesting that Neu4 may be important for desialylation of brain gangliosides and consistent with the in situ hybridization data. Increased levels of cholesterol, ceramide and polyunsaturated fatty acids were also detected in the lungs and spleen of Neu4(-/-) mice by high-resolution NMR spectroscopy. Together, our data suggest that Neu4 is a functional component of the ganglioside-metabolizing system, contributing to the postnatal development of the brain and other vital organs.

Phenotypic characterization of a mouse model of juvenile neuronal ceroid lipofuscinosis

Juvenile neuronal ceroid lipofuscinosis (JNCL) is an autosomal recessively inherited neurodegenerative disorder that results from mutations in the CLN3 gene. JNCL is characterized by accumulation of autofluorescent lysosomal storage bodies, vision loss, seizures, progressive cognitive and motor decline, and premature death. Studies were undertaken to characterize the neuronal ceroid lipofuscinosis phenotype in a Cln3 knockout mouse model. Progressive accumulation of autofluorescent storage material was observed in brain and retina of affected mice. The Cln3 −/− mice exhibited progressively impaired inner retinal function, altered pupillary light reflexes, losses of inner retinal neurons, and reduced brain mass. Behavioral changes included reduced spontaneous activity levels and impaired learning and memory. In addition, Cln3 −/− mice had significantly shortened life spans. These phenotypic features indicate that the mouse model will be useful for investigating the mechanisms underlying the disease pathology in JNCL and provide quantitative markers of disease pathology that can be used for evaluating the efficacies of therapeutic interventions.

Pathogenic mechanisms in lysosomal disease: a reappraisal of the role of the lysosome

The view that lysosomes simply represent end organelles in the serial degradation of polymeric molecules derived from the cell surface and its interior has led to major misconceptions about the nature of lysosomal storage diseases and the pathogenic cascades that characterize them. Accordingly, lysosomal storage bodies are often considered 'inert', inducing cell dysfunction and death primarily through mechanical overcrowding of normal organelles or by other non-specific means leading to generalized cytotoxicity. However, modern studies of lysosomes and their component proteins provide evidence to support a far greater role for these organelles in cell metabolism. In intimate association with endosomal, autophagosomal and related vesicular systems, the greater lysosomal system can be conceptualized as a vital recycling centre that serves as a central metabolic coordinator, influencing literally every aspect of the cell, from signal transduction to regulation of gene expression. This broader view of the role of lysosomes in cells not only provides insight into how single gene defects impacting on lysosomal function can result in the plethora of complex cellular transformations characteristic of these diseases, but also suggests new and innovative therapies that may hold considerable promise for ameliorating disease progression.

Ocular phenotype in a mouse gene knockout model for infantile neuronal ceroid lipofuscinosis

Mutations in the human protein palmitoyl thioesterase-1 (PPT-1) gene result in an autosomal recessive neurodegenerative disorder designated neuronal ceroid lipofuscinosis (NCL), type CLN1, or infantile NCL. Among the symptoms of the CLN1 disease are accumulation of autofluorescent lysosomal storage bodies in neurons and other cell types, seizures, motor and cognitive decline, blindness, and premature death. Development of an effective therapy for this disorder will be greatly assisted by the availability of suitable animal models. A mouse PPT-1 gene knockout model has recently been generated. Studies were performed to determine whether the mouse model exhibits ocular features of the human CLN1 disorder. A progressive accumulation of autofluorescent storage material in all layers of the retina was observed in the PPT-1 knockout mice. Accompanying the storage body accumulation was a modest loss of cells with nuclei in the outer and inner nuclear layers. As indicated by electroretinogram (ERG) responses, retinal function was only mildly impaired at 4 months of age but was severely impaired by 8 months, despite only modest changes in retinal morphology. The pupillary light reflex (PLR), on the other hand, was exaggerated in the knockout mice. The apparent anomaly between the ERG and the PLR findings suggests that disease-related PLR changes may be due to changes in extraocular signal processing. The pronounced ocular phenotype in the PPT-1 knockout mice makes these animals a good model for testing therapeutic interventions for treatment of the human CLN1 disorder.

Embryonic Stem Cell-Derived Neural Progenitors Incorporate into Degenerating Retina and Enhance Survival of Host Photoreceptors

Embryonic stem (ES) cells differentiate into all cell types of the body during development, including those of the central nervous system (CNS). After transplantation, stem cells have the potential to replace host cells lost due to injury or disease or to supply host tissues with therapeutic factors and thus provide a functional benefit. In the current study, we assessed whether mouse neuralized ES cells can incorporate into retinal tissue and prevent retinal degeneration in mnd mice. These mice have an inherited lysosomal storage disease characterized by retinal and CNS degeneration. Sixteen weeks after intravitreal transplantation into adult mice, donor cells had incorporated into most layers of the retina, where they resembled retinal neurons in terms of morphology, location in the retina, and expression of cell type-specific marker proteins. Presence of these donor cells was correlated with a reduction in the sizes and numbers of lysosomal storage bodies in host retinal cells. The presence of transplanted donor cells was also accompanied by enhanced survival of host retinal neurons, particularly photoreceptors. These results demonstrate that neuralized ES cells protect host neurons from degeneration and appear to replace at least some types of lost 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.

RPE65 gene mutation prevents development of autofluorescence in retinal pigment epithelial phagosomes

During senescence, autofluorescent lysosomal storage bodies known as lipofusin or age pigment accumulate in many post-mitotic types of cells. Among these cell types is the retinal pigment epithelium (RPE) of the mammalian eye. The mechanisms of lipofuscin formation and accumulation have been studied more extensively in the RPE than in any other cell type. Substantial evidence indicates that Vitamin A derivatives (retinoids) are required for RPE lipofuscin formation. The RPE and adjacent retina contain retinoids in the forms of retinol, retinyl esters, and retinaldehyde. Previous research has demonstrated that retinaldehydes are directly involved in the formation of one RPE lipofuscin fluorophore. However, RPE lipofuscin contains many other fluorophores. It has not been determined which retinoids are involved in the formation of these fluorescent compounds. Mice with a mutation in the Rpe65 gene contain substantial levels of retinol and retinyl esters in the RPE, but little if any retinaldehydes in either the RPE or retina. Therefore, these mice could be used to determine whether retinaldehydes are required for formation of all of the RPE lipofuscin fluorophores. Normal mice were given intraocular injections of a protease inhibitor, which resulted in the rapid accumulation in the RPE of lipofuscin-like inclusions. These inclusions exhibited fluorescence properties typical of RPE lipofuscin. Rpe65 −/− mice treated with the protease inhibitor also accumulated inclusions similar to those observed in the normal mice. However, these inclusions did not fluoresce under the conditions used to visualize lipofuscin fluorescence. These findings indicate that the aldehyde form of Vitamin A is required for the formation of not only one, but all of the RPE lipofuscin fluorophores.

Mutation in the gene encoding lysosomal acid phosphatase (Acp2) causes cerebellum and skin malformation in mouse

We report a novel spontaneous mutation named nax in mice, which exhibit delayed hair appearance and ataxia in a homozygote state. Histological analyses of nax brain revealed an overall impairment of the cerebellar cortex. The classical cortical cytoarchitecture was disrupted, the inner granule cell layer was not obvious, the Purkinje cells were not aligned as a Purkinje cell layer, and Bergmann glias did not span the molecular layer. Furthermore, histological analyses of skin showed that the hair follicles were also abnormal. We mapped the nax locus between marker D2Mit158 and D2Mit100 within a region of 800 kb in the middle of chromosome 2 and identified a missense mutation (Gly244Glu) in Acp2, a lysosomal monoesterase. The Glu244 mutation does not affect the stability of the Acp2 transcript, however it renders the enzyme inactive. Ultrastructural analysis of nax cerebellum showed lysosomal storage bodies in nucleated cells, suggesting progressive degeneration as the underlying mechanism. Identification of Acp2 as the gene mutated in nax mice provides a valuable model system for studying the role of Acp2 in cerebellum and skin homeostasis.

Lysine 43 Is Trimethylated in Subunit c from Bovine Mitochondrial ATP Synthase and in Storage Bodies Associated with Batten Disease

The hydrophobic membrane protein, subunit c, has been isolated from ATP synthase purified from bovine heart mitochondria. It has also been obtained from lysosomal storage bodies associated with ceroid lipofuscinosis from ovine liver and from human brain tissue of a victim of Batten disease. It is likely that the lysosomal protein has originated from the mitochondrion. These samples have been characterized by mass spectrometric methods. Irrespective of its source, subunit c has an intact molecular mass of 7650 Da, 42 Da greater than the value calculated from the amino acid sequence, and the protein has been modified post-translationally. In all three samples, the modification is associated with lysine 43, which lies in a polar loop region linking the two transmembrane alpha-helices of the protein. This residue is conserved throughout vertebrate sequences. The additional mass arises from trimethylation and not acetylation at the epsilon-N-position of the residue. These experiments show that the post-translational modification of subunit c is not, as has been suggested, an abnormal phenomenon associated with the etiology of Batten disease and ceroid lipofucinoses. Evidently, it occurs either before or during import of the protein into mitochondria or at a mitochondrial location after completion of the import process. The function of the trimethyllysine residue in the assembled ATP synthase complex is obscure. The residue and the modification are not conserved in all ATP synthases, and their role in the assembly and (or) functioning of the enzyme appear to be confined to higher organisms.