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A Murine Model for Juvenile NCL: Gene Targeting of MouseCln3
Abstract
JNCL is a neurodegenerative disease of childhood caused by mutations in the CLN3 gene. A mouse model for JNCL was created by disrupting exons 1-6 of Cln3, resulting in a null allele. Cln3 null mice appear clinically normal at 5 months of age; however, like JNCL patients, they exhibit intracellular accumulation of autofluorescent material. A second approach will generate mice in which exons 7 and 8 of Cln3 are deleted, mimicking the common mutation in JNCL patients.
... The Cln3-targeting vector was constructed as outlined previously (Greene et al., 1999). The insert from mouse Cln3 cDNA clone pMNCL (Taschner et al., 1997) was tested by Southern blot hybridization to verify it was single copy sequence. ...
... To inactivate Cln3, exons 2-6 and most of exon 1, including the start codon, were deleted in TC1 embryonic stem cells (Figs. 1A and 1B) by replacement with a neomycin resistance gene transcribed in reverse orientation from a mouse PGK promoter, as previously described (Greene et al., 1999). Chimeras established from these cells were used to generate both 129/Sv inbred and 129/Sv ϫ Black Swiss outbred lines; the outbred line was used for subsequent analysis (Fig. 1B). ...
... 2A-2D). Storage levels at 5 months of age have also previously been described (Greene et al., 1999). Stored material fluoresced over a wide range of excitatory and barrier filter combinations. ...
Batten disease, a degenerative neurological disorder with juvenile onset, is the most common form of the neuronal ceroid lipofuscinoses. Mutations in the CLN3 gene cause Batten disease. To facilitate studies of Batten disease pathogenesis and treatment, a murine model was created by targeted disruption of the Cln3 gene. Mice homozygous for the disrupted Cln3 allele had a neuronal storage disorder resembling that seen in Batten disease patients: there was widespread and progressive intracellular accumulation of autofluorescent material that by EM displayed a multilamellar rectilinear/fingerprint appearance. Inclusions contained subunit c of mitochondrial ATP synthase. Mutant animals also showed neuropathological abnormalities with loss of certain cortical interneurons and hypertrophy of many interneuron populations in the hippocampus. Finally, as is true in Batten disease patients, there was increased activity in the brain of the lysosomal protease Cln2/TPP-1. Our findings are evidence that the Cln3-deficient mouse provides a valuable model for studying Batten disease.
... In knock-in models, exons 7 and 8 are replaced with a PGKneo cassette . Both types of mutated murine models exhibit a similar autofluorescent storage material accumulation to human JNCL, and that is SCMAS Greene et al., 1999). CLN3 mice show a progressive but delayed onset, deficit pattern of neurological symptoms, including motor dysfunction, visual and learning deficits, etc., that correlate with neuron loss in the thalamus, cerebellum, substantia nigra, cortex, striatum, and retina (Cao et al., 2011;Greene et al., 1999;Mitchison et al., 1999;Pontikis et al., 2005;Seigel et al., 2002). ...
... Both types of mutated murine models exhibit a similar autofluorescent storage material accumulation to human JNCL, and that is SCMAS Greene et al., 1999). CLN3 mice show a progressive but delayed onset, deficit pattern of neurological symptoms, including motor dysfunction, visual and learning deficits, etc., that correlate with neuron loss in the thalamus, cerebellum, substantia nigra, cortex, striatum, and retina (Cao et al., 2011;Greene et al., 1999;Mitchison et al., 1999;Pontikis et al., 2005;Seigel et al., 2002). It has been found that CLN3 localizes in endosomes and lysosomes as an integral membrane glycoprotein. ...
Batten disease consists of a family of primarily autosomal recessive, progressive neuropediatric disorders, also known as neuronal ceroid lipofuscinoses (NCLs). These pathologies are characterized by seizures and visual, cognitive and motor decline, and premature death. The pathophysiology of this rare disease is still unclear despite the years of trials and financial aids. This paper has reviewed advantages and limits of in vivo and in vitro models of Batten disease from murine and larger animal models to primitive unicellular models, until the most recently developed patient-derived induced pluripotent stem cells. For each model advantages, limits and applications were analyzed. The first prototypes investigated were murine models that due to their limits were replaced by larger animals. In vitro models gradually replaced animal models for practical, cost, and ethical reasons. Using induced pluripotent stem cells to study neurodegeneration is a new way of studying the disease, since they can be distinguished into differentiating elements like neurons, which are susceptible to neurodegeneration. In vivo and in vitro models have contributed to clarifying to some extent the pathophysiology of the disease. The collection and sharing of suitable human bio samples likely through biobanks can contribute to a better understanding, prevention, and to identify possible treatment strategies of Batten disease.
... Transgenic animals have been generated for NCL genes CLN2, CLN3 and cathepsin D (Greene, et al. 1999, Katz, et al. 1999a, Katz, et al. 1999b, Saftig, et al. 1995. Two separate CLN3 transgenic models have been generated using gene targeted disruption of the CLN3 gene, through homologous recombination, to create a null allele. ...
... Successive rounds of mating produced mice homozygous for the null allele (Greene, et al. 1999, Katz, et al. 1999b. By one year old the CLN3 knockout mice (-/-) did not display features of a clinical phenotype similar to human JNCL, although examination of the neuronal cells of CLN3-/mice revealed the accumulation of autofluorescent storage material and characteristic fingerprint morphology. ...
The neuronal ceroid lipofuscinoses (NCL; Batten disease) are a group of recessively inherited childhood neurodegenerative disorders characterised by the accumulation of autofluorescent material in many cell types. To date, at least six genetically distinct NCL loci have been mapped and five genes have been cloned. Mutations in three genes cause onset in late infancy. Classical late infantile NCL (LINCL) presents between the ages of 2 - 4 years with seizures followed by ataxia. Children lose the ability to walk by age six years, and progressive visual failure from age three eventually results in blindness by six years. Death usually occurs by 15 years. Variant forms of LINCL have a similar clinical course although the age of onset may be slightly later. A variant form of late infantile NCL (LINCL) is found predominantly within the Turkish population (CLN7). Exclusion mapping showed that CLN7 was not an allelic variant of known NCL loci (CLN1, CLN2, CLN3, CLN5 or CLN6). Using the method of homozygosity mapping, a genome wide search was undertaken and a total of 358 microsatellite markers were typed at an average distance of 10 cM. A region of shared homozygosity was identified in chromosome 8p23. This telomeric region contained the recently identified CLN8 gene. A missense mutation in CLN8 causes the disease Northern epilepsy or 'Progressive epilepsy with mental retardation' (EPMR), which is now classified as an NCL. The mouse model mnd was also shown to carry a 1 bp insertion in the orthologous gene. Five Turkish families were shown to be consistent with linkage across a 700 kb region in 8p23 that contained CLN8 and is defined by the microsatellite markers 140CA and 159CA. This suggests that Turkish variant LINCL, despite an earlier onset, may be an allelic variant of the Finnish disease EPMR. Mutation analysis of the Turkish variant LINCL families examined the coding or non-coding exons of CLN8 by direct sequencing of both genomic and cDNA sequences. A C>T transition at position 509 was identified in family 360. This substitution encodes the amino acid methionine and not the predicted threonine. Computer predictions suggest that the substitution would alter the hydrophobicity of the protein resulting in the introduction of two additional transmembrane domains and presumably disrupt the normal CLN8 protein function. The identification of a putative disease-causing mutation in family 360 suggests that the Turkish variant LINCL family may be an allelic variant of CLN8. No mutations were identified in the remaining Turkish families, and the disease-causing mutation(s) remains to be delineated in these families.
... Mammalian models exist in the form of sheep (CLN6), 15 cow (CLN5), 42 dog (CLN2 and CLN6), 8,28 and mouse (CTSD, CLN1, CLN2, CLN3, CLN5, CLN6 and CLN8). 14,19,30,36,38,45,47,48,51,54,58,77,[88][89][90] We will focus on mouse models of JNCL (CLN3) and summarize those studies that pertain to the visual system. ...
... Accumulation of autofluorescent material, carbohydrate storage material, and apoptotic cell death were detected in these mice. 36,59 MRI studies have revealed global changes in the brains. 37 These mice do not manifest retinal degeneration, suggesting that the presence of autofluorescent materials alone may not be sufficient to affect retinal function. ...
Juvenile neuronal ceroid lipofuscinoses, or Batten disease, is the most common type of NCL in the United States and Europe. This devastating disorder presents with vision failure and progresses to include seizures, motor dysfunction, and dementia. Death usually occurs in the third decade, but some patients die before age twenty. Though the mechanism of visual failure remains poorly understood, recent advances in molecular genetics have improved diagnostic testing and suggested possible therapeutic strategies. The ophthalmologist plays a crucial role in both early diagnosis and documentation of progression of juvenile neuronal ceroid lipofuscinoses. We update Batten disease research, particularly as it relates to the eye, and present various theories on the pathophysiology of retinal degeneration.
... To further our understanding of the function of CLN3 and to gain an insight into the disease process, several laboratories have developed murine models of disease. The original, and most widely utilized model, known as the Cln3 Dex1-6 mouse was created by Mitchison and colleagues and was intended to provide a complete null mutation of Cln3 by replacing the start codon and first six exons with a neo cassette (7,8). The Cln3 Dex7/8 Katz mouse was created at around the same time using a different strategy that replaced the majority of exon 7 and all of exon 8 with a neo cassette in an attempt to create a genetically similar mutation to the 1 kb deletion mutation found in the majority of human patients (9). ...
... The Cln3 Dex1-6 knockout mouse (7,8) is the most widely utilized model animal for JNCL research, containing a null mutation designed to ablate Cln3 function by the replacement of the majority of exon 1 (including the AUG start condon) through exon 6 of the genomic sequence with a Neo cassette in the reverse orientation. Homozygous Cln3 Dex1-6 mice were previously shown to express Neo but not Cln3 exons 1 and 2 demonstrating the fidelity of the deletion (7). ...
Juvenile neuronal ceroid lipofuscinoses (JNCL), commonly known as Batten disease, is a progressive neurodegenerative disorder of childhood characterized by blindness, seizures, motor and cognitive decline, leading to death in early adulthood. Mutations within the CLN3 gene, which encodes a putative lysosomal protein of unknown function, are the underlying cause of JNCL. Over 85% of JNCL patients harbor a 1 kb deletion that is predicted to result in a truncated CLN3 protein and is presumed to be a null mutation. A recent study by Kitzmuller et al. (1) suggested that the 1 kb deletion-associated truncated protein may have partial function, and proposed that JNCL is a mutation-specific disease. In addition, the validity of the original and most widely utilized JNCL mouse model, the Cln3(Deltaex1-6) mouse, as a true null mutant was questioned. We report a substantial decrease in the transcript level of the truncated CLN3 gene product in cells from 1 kb deletion patients. We contend that the truncated CLN3 protein is unlikely to be expressed in JNCL patients since cellular quality control mechanisms at the RNA and protein levels are likely to degrade the mutant transcript and polypeptides. Moreover, we present analysis identifying the expressed transcripts present in Cln3(Deltaex1-6) mouse brain. From the analysis of expressed Cln3(Deltaex1-6) mouse transcripts, combined with in silico prediction of the expected consequences of the Cln3(Deltaex1-6) mutation on these transcripts, we argue that aberrant Cln3 proteins are unlikely to be expressed in this disease model. Taken together our results indicate that the most common mutation associated with JNCL results in a loss of functional CLN3, that the Cln3(Deltaex1-6) mouse harbors a null Cln3 allele, and that it therefore represents a valid model for this disease.
... Genotyping of Tpp1 À/À and Cln3 À/À mice was conducted as described. 19,28 Experimental cohorts contained $equal numbers of males and female animals and were analyzed at $120 days of age. For biochemical analyses, animals were deeply anesthetized with sodium pentobarbital/ phenytoin (a 1:4 dilution of Euthasol; Delmarva Laboratories, Midlothian, VA) and euthanized by transcardial perfusion with 0.9% saline. ...
Late-infantile neuronal ceroid lipofuscinosis (LINCL) and juvenile neuronal ceroid lipofuscinosis (JNCL) are inherited neurodegenerative diseases caused by mutations in the genes encoding lysosomal proteins tripeptidyl peptidase 1 (TPP1) and CLN3 protein, respectively. TPP1 is well-understood and, aided by animal models that accurately recapitulate the human disease, enzyme replacement therapy has been approved and other promising therapies are emerging. In contrast, there are no effective treatments for JNCL, partly because the function of the CLN3 protein remains unknown but also because animal models have attenuated disease and lack robust survival phenotypes. Mouse models for LINCL and JNCL, with mutations in Tpp1 and Cln3 respectively, have been thoroughly characterized but the phenotype of a double Cln3/Tpp1 mutant remains unknown. We created this double mutant and find that its phenotype is essentially indistinguishable from the single Tpp1-/- mutant in terms of survival and brain pathology. Analysis of brain proteomic changes in the single Tpp1-/- and double Cln3-/- ;Tpp1-/- mutants indicates largely overlapping sets of altered proteins and reinforces earlier studies that highlight GPNMB, LYZ2 and SERPINA3 as promising biomarker candidates in LINCL while several lysosomal proteins including SMPD1 and NPC1 appear to be altered in the Cln3-/- animals. An unexpected finding was that Tpp1 heterozygosity significantly decreased lifespan of the Cln3-/- mouse. The truncated survival of this mouse model makes it potentially useful in developing therapies for JNCL using survival as an endpoint. In addition, this model may also provide insights into CLN3 protein function and its potential functional interactions with TPP1. This article is protected by copyright. All rights reserved.
... These models contribute to therapeutic development through facilitating investigation of disease pathophysiology, aiding in identification of therapeutic targets, and facilitating initial assessment of efficacy of therapeutic approaches. To date, murine models have been identified for CLN1 (14)(15)(16)(17), CLN2 (18), CLN3 (17,(19)(20)(21)(22)(23)(24)(25)(26)(27)(28), CLN5 (29), CLN6 (30), CLN8 (31)(32)(33)(34)(35), and CLN10 (36,37). Reports of retinopathy characterization in murine models of NCL predominantly describe findings in CLN3 (19,28), CNL5 (38), CNL6 (39), and CLN8 (32)(33)(34)(35), though the available descriptions of ocular disease associated with CLN8 are limited to homozygous motor neuron degeneration (mnd) murine models. ...
Background:
Ceroid lipofuscinosis type 8 belongs to a heterogenous group of vision and life-threatening neurodegenerative diseases, neuronal ceroid lipofuscinosis (NCL). Effective therapy is limited to a single drug for treatment of ceroid lipofuscinosis type 2, necessitating animal disease models to facilitate further therapeutic development. Murine models are advantageous for therapeutic development due to easy genetic manipulation and rapid breeding, however appropriate genetic models need to be identified and characterized before being used for therapy testing. To date, murine models of ocular disease associated with ceroid lipofuscinosis type 8 have only been characterized in motor neuron degeneration mice.
Methods:
Cln8-/- mice were produced by CRISPR/Cas9 genome editing through the International Mouse Phenotyping Consortium. Ophthalmic examination, optical coherence tomography, electroretinography, and ocular histology was performed on Cln8-/- mice and controls at 16 weeks of age. Quantification of all retinal layers, retinal pigmented epithelium, and the choriocapillaris was performed using images acquired with ocular coherence tomography and planimetry of histologic sections. Necropsy was performed to investigate concurrent systemic abnormalities. Clinical correlation with human patients with CLN8-associated retinopathy is provided.
Results:
Retinal degeneration characterized by retinal pigment epithelium mottling, scattered drusen, and retinal vascular attenuation was noted in all Cln8-/- mice. Loss of inner and outer photoreceptor segment demarcation was noted on optical coherence tomography, with significant thinning of the whole retina (P=1e-9), outer nuclear layer (P=1e-9), and combined photoreceptor segments (P=1e-9). A global reduction in scotopic and photopic electroretinographic waveforms was noted in all Cln8-/- mice. Slight thickening of the inner plexiform layer (P=0.02) and inner nuclear layer (P=0.004), with significant thinning of the whole retina (P=0.03), outer nuclear layer (P=0.01), and outer photoreceptor segments (P=0.001) was appreciated on histologic sections. Scattered lipid vacuoles were noted in splenic red pulp of all Cln8-/- mice, though no gross systemic abnormalities were detected on necropsy. Retinal findings are consistent with those seen in patients with ceroid lipofuscinosis type 8.
Conclusions:
This study provides detailed clinical characterization of retinopathy in adult Cln8-/- mice. Findings suggest that Cln8-/- mice may provide a useful murine model for development of novel therapeutics needed for treating ocular disease in patients with ceroid lipofuscinosis type 8.
... Cln3 knockout mouse lines and two Cln3 knock-in lines [68][69][70][71], allowing for 3 evaluation of potential therapeutic approaches using AAV vectors. To date, AAV gene 4 therapy has only been studied with the Cln3 Δex7/8 knock-in mice developed by Cotman 5 et al [68] (Table 3). ...
The neuronal ceroid lipofuscinoses (NCLs), also known as Batten disease, are a group of rare monogenic neurodegenerative diseases predominantly affecting children. All NCLs are lethal and incurable and only one has an approved treatment available. To date, 13 NCL subtypes (CLN1-8, CLN10-14) have been identified, based on the particular disease-causing defective gene. The exact functions of NCL proteins and the pathological mechanisms underlying the diseases are still unclear. However, gene therapy has emerged as an attractive therapeutic strategy for this group of conditions. Here we provide a short review discussing updates on the current gene therapy studies for the NCLs.
... In the first mouse model, exons 1-6 of Cln3 were disrupted, creating a null allele (Cln3 −/− ; generated in 129/SvEvTac (129S6) TC1 ES cells). 66,67 On a 129/ SvEvTac background, there was significant accumulation of intracellular autofluorescent inclusions in the Cln −/− brain as well as in the retinal ganglion cells and inner nuclear layer as early as 2.5-3 months old when no obvious visual impairment was observed, 67,68 suggesting that this autofluorescent material may not be critical in the progression of the ensuing retinal degeneration. At 15 months old, the fundus autofluorescence pattern was enhanced in the retinal ganglion cells, inner plexiform layer, and photoreceptor-RPE zone. ...
Juvenile neuronal ceroid lipofuscinosis (JNCL; also known as CLN3 disease) is a devastating neurodegenerative lysosomal storage disorder and the most common form of Batten disease. Progressive visual and neurological symptoms lead to mortality in patients by the third decade. Although ceroid-lipofuscinosis, neuronal 3 (CLN3) has been identified as the sole disease gene, the biochemical and cellular bases of JNCL and the functions of CLN3 are yet to be fully understood. As severe ocular pathologies manifest early in disease progression, the retina is an ideal tissue to study in the efforts to unravel disease etiology and design therapeutics. There are significant discrepancies in the ocular phenotypes between human JNCL and existing murine models, impeding investigations on the sequence of events occurring during the progression of vision impairment. This review focuses on current understanding of vision loss in JNCL and discusses future research directions toward molecular dissection of the pathogenesis of the disease and associated vision problems in order to ultimately improve the quality of patient life and cure the disease.
... Cln3Ϫ/Ϫ mice were created on a 129ev/ TAC background (also known as 129S6), as previously described. 14,20,21 Briefly, a PGK neomycin cassette was inserted into a plasmid designed to delete and replace exons 1 to 6 of the cln3 gene. with 4% paraformaldehydeϩ2.5% glutaraldehyde in phosphate buffer (pH 7.4). ...
To investigate optic nerve degeneration associated with CLN3 deficiency in a murine model of juvenile neuronal ceroid lipofuscinosis (Batten disease).
Using light and electron microscopy, the density and diameter of axons and the thickness of myelin in optic nerve were compared between age-matched cln3 knock-out (cln3-/-) and wild-type (129ev/TAC) mice. Western blot analysis was used to assay expression of Cln3 in mouse and primate retina and optic nerve.
Morphologically identified mast cells were present in the meningeal sheaths surrounding the cln3-/- nerve and in the nerve itself. The cln3-/- optic nerve exhibited an overall loss of uniformity and integrity. Axon density in cln3-/- optic nerve was only 64% of that in wild-type optic nerve (P < 0.01). Accounting for differences in axon density, the diameter of axons in cln3-/- optic nerve was 1.2 times greater than in wild-type optic nerve (P < 0.01). Electron micrographs revealed large spaces between axons and 32% thinner myelin surrounding axons in cln3-/- mice than in wild type (P < 0.01). Western blot analysis demonstrated that Cln3 was expressed in retinas and optic nerves of mouse and primate.
The presence of apparent mast cells in cln3-/- optic nerve suggests compromise of the blood-brain barrier. The absence of Cln3 causes loss of axons, axonal hypertrophy, and a reduction in myelination of retinal ganglion cells. Furthermore, expression of CLN3 in mouse and primate optic nerve links degeneration to loss of Cln3.
... To date, no animal model has been described for cLINCL, although there are models for other forms of human NCLs. For infantile NCL and juvenile NCL, mouse models have been generated by targeting genes CLN1 (Gupta et al., 2001) and CLN3 (Greene et al., 1999;Katz et al., 1999), respectively. Spontaneous mouse mutants nclf and mnd have defects in CLN6 (Gao et al., 2002;Wheeler et al., 2002) and CLN8 , respectively, providing models for a variant late-infantile neuronal cer-oid lipofuscinosis and epilepsy, progressive, with mental retardation. ...
Mutations in the CLN2 gene, which encodes a lysosomal serine protease, tripeptidyl-peptidase I (TPP I), result in an autosomal recessive neurodegenerative disease of children, classical late-infantile neuronal ceroid lipofuscinosis (cLINCL). cLINCL is inevitably fatal, and there currently exists no cure or effective treatment. In this report, we provide the characterization of the first CLN2-targeted mouse model for cLINCL. CLN2-targeted mice were fertile and apparently healthy at birth despite an absence of detectable TPP I activity. At approximately 7 weeks of age, neurological deficiencies became evident with the onset of a tremor that became progressively more severe and was eventually accompanied by ataxia. Lifespan of the affected mice was greatly reduced (median survival, 138 d), and extensive neuronal pathology was observed including a prominent accumulation of cytoplasmic storage material within the lysosomal-endosomal compartment, a loss of cerebellar Purkinje cells, and widespread axonal degeneration. The CLN2-targeted mouse therefore recapitulates much of the pathology and clinical features of cLINCL and represents an animal model that should provide clues to the normal cellular function of TPP I and the pathogenic processes that underlie neuronal death in its absence. In addition, the CLN2-targeted mouse also represents a valuable model for the evaluation of different therapeutic strategies.
... Efforts to develop therapeutic interventions for the human NCLs will be greatly enhanced by the availability of appropriate animal models. Mouse models for most of the NCLs have been developed since the characterization of the human NCL genes [7][8][9][10][11][12]. However, the utility of these mouse models is limited because, in some cases, affected mice do not develop phenotypic features of the diseases seen in the corresponding human disorders [13]. ...
A heritable neurodegenerative disease of English Setters has long been studied as a model of human neuronal ceroid-lipofuscinosis (NCL). Megablast searches of the first build of the canine genome for potential causative genes located the CLN8 gene near the q telomere of canine chromosome 37, close to a marker previously linked to English Setter NCL. Sequence analysis of the coding region from affected dogs revealed a T-to-C transition in the CLN8 gene that predicts a p.L164P missense mutation. Leucine 164 is conserved in four other mammalian species. The C allele co-segregated with the disease phenotype in a two-generation English Setter family in a pattern consistent with autosomal recessive inheritance. All four NCL-affected family members were C/C homozygotes and all four obligate carriers were C/T heterozygotes; whereas, 103 unrelated dogs were all T/T homozygotes. These findings indicate that the CLN8 T-to-C transition is the likely cause of English Setter NCL.
Juvenile neuronal ceroid lipofuscinosis (JNCL) is a fatal inherited neurodegenerative disease of children that occurs due to defective function of the lysosomal membrane glycoprotein CLN3. JNCL features glial activation and accumulation of autofluorescent storage material containing subunit c of mitochondrial ATP synthase (SCMAS), ultimately resulting into neuronal loss. Until now, no effective therapy is available for JNCL. This study underlines the possible therapeutic importance of gemfibrozil, an activator of peroxisome proliferator-activated receptor α (PPARα) and food and drug administration (FDA)-approved lipid-lowering drug, in an animal model of JNCL. Oral gemfibrozil treatment reduced microglial and astroglial activation, attenuated neuroinflammation, restored the level of TFEB (the master regulator of lysosomal biogenesis), and decreased the accumulation of storage material SCMAS in somatosensory barrel field (SBF) cortex of Cln3 Δex7/8 (Cln3ΔJNCL) mice of both sexes. Accordingly, gemfibrozil treatment also improved locomotor activities of Cln3ΔJNCL mice. While investigating the mechanism, we found marked loss of PPARα in the SBF cortex of Cln3ΔJNCL mice, which increased after gemfibrozil treatment. Oral gemfibrozil also stimulated the recruitment of PPARα to the Tfeb gene promoter in vivo in the SBF cortex of Cln3ΔJNCL mice, indicating increased transcription of Tfeb in the CNS by gemfibrozil treatment via PPARα. Moreover, disease pathologies aggravated in Cln3ΔJNCL mice lacking PPARα (Cln3ΔJNCL ΔPPARα ) and gemfibrozil remained unable to decrease SCMAS accumulation, reduce glial activation and improve locomotor performance of Cln3ΔJNCL ΔPPARα mice. These results suggest that activation of PPARα may be beneficial for JNCL and that gemfibrozil may be repurposed for the treatment of this incurable disease.
SIGNIFICANCE STATEMENT:
Despite intense investigations, no effective therapy is available for JNCL, an incurable inherited lysosomal storage disorder. Here, we delineate that oral administration of gemfibrozil, a lipid-lowering drug, decreases glial inflammation, normalizes and/or upregulates TFEB and reduces accumulation of autofluorescent storage material in SBF cortex to improve locomotor activities in Cln3 Δex7/8 (Cln3ΔJNCL) mice. Aggravation of disease pathology in Cln3ΔJNCL mice lacking PPARα (Cln3ΔJNCL ΔPPARα ) and inability of gemfibrozil to decrease SCMAS accumulation, reduce glial activation and improve locomotor performance of Cln3ΔJNCL ΔPPARα mice delineates an important role of PPARα in this process. These studies highlight a new property of gemfibrozil and indicate its possible therapeutic use in JNCL patients.
The NCLs (neuronal ceroid lipofuscinosis) are forms of neurodegenerative disease that affect people of all ages and ethnicities but are most prevalent in children. Commonly known as Batten disease, this debilitating neurological disorder is comprised of 13 different subtypes that are categorized based on the particular gene that is mutated (CLN1-8, CLN10-14). The pathological mechanisms underlying the NCLs are not well understood due to our poor understanding of the functions of NCL proteins. Only one specific treatment (enzyme replacement therapy) is approved, which is for the treating the brain in CLN2 disease. Hence there remains a desperate need for further research into disease-modifying treatments. In this review, we present and evaluate the genes, proteins and studies performed in the social amoeba, nematode, fruit fly, zebrafish, mouse and large animals pertinent to NCL. In particular, we highlight the use of multicellular model organisms to study NCL protein function, pathology and pathomechanisms. Their use in testing novel therapeutic approaches is also presented. With this information, we highlight how future research in these systems may be able to provide new insight into NCL protein functions in human cells and aid in the development of new therapies.
Treatments are emerging for the neuronal ceroid lipofuscinoses (NCLs), a group of similar but genetically distinct lysosomal storage diseases. Clinical ratings scales measure long-term disease progression and response to treatment but clinically useful biomarkers have yet to be identified in these diseases. We have conducted proteomic analyses of brain and cerebrospinal fluid (CSF) from mouse models of the most frequently diagnosed NCL diseases: CLN1 (infantile NCL), CLN2 (classical late infantile NCL) and CLN3 (juvenile NCL). Samples were obtained at different stages of disease progression and proteins quantified using isobaric labeling. In total, 8303 and 4905 proteins were identified from brain and CSF, respectively. We also conduced label-free analyses of brain proteins that contained the mannose 6-phosphate lysosomal targeting modification. In general, we detect few changes at presymptomatic timepoints but later in disease, we detect multiple proteins whose expression is significantly altered in both brain and CSF of CLN1 and CLN2 animals. Many of these proteins are lysosomal in origin or are markers of neuroinflammation, potentially providing clues to underlying pathogenesis and providing promising candidates for further validation.
Over the past 20 years, there has been tremendous progress in retinal gene therapy. The safety and efficacy results in one early-onset severe blinding disease may lead to the first gene therapy drug approval in the United States. Here, we review how far the field has come over the past two decades and speculate on the directions that the field will take in the future. There has been tremendous progress in retinal gene therapy over the past two decades. This progress and the current status of retinal gene therapy are reviewed. Furthermore, there is speculation as to the future directions of this field.
Lysosomal storage diseases are inherited metabolic disorders characterized by severe pathology, typically involving the brain. Although individually rare, they collectively represent a significant group of diseases that primarily present in early infancy or childhood. In recent years, considerable progress has been made in understanding the molecular mechanisms that lead to disordered function of the lysosomal system and to lysosomal storage. Unravelling the basis for these diseases is providing unique insight into the normal biology of cells and pointing the way to the development of therapeutic strategies for their treatment. This book details recent advances in the molecular and cellular pathologies of these diseases and in the development of effective therapies. After an overview of the biology of the endosomal-lysosomal system and the types of diseases resulting from defects in this system, the book describes in detail the molecular mechanisms of storage, model systems and pathophysiological mechanisms, and finally, new advances toward treatment.
As with many human maladies, the search for effective treatments or cures for the various ceroid lipofuscinoses would be greatly facilitated if appropriate animal models were available for studies on the mechanisms underlying the disease pathologies and for screening potential therapeutic interventions. This is particularly true for relatively rare diseases for which, it would be difficult to amass a sufficiently large pool of human subjects for controlled clinical intervention trials. The hereditary neuronal ceroid lipofuscinoses (NCLs) are autosomal recessively inherited neurodegenerative diseases that have a number of clinical and pathological features in common. Despite their similarities, however, the NCLs are actually a group of distinct disorders that result from defects in different genes. The most common diseases that are classified as NCLs result from defects in the CLN1, CLN2, and CLN3 genes associated with what are commonly referred to as infantile, late-infantile, and juvenile NCL, respectively. NCL-like disorders have been reported in a number of mammalian species. These include dogs, cats, cattle, sheep, and mice. Many of the disorders in animals were reported as isolated cases in veterinary patients, and only a small number of the animal diseases have been evaluated as potential models for the human disorders. The animal NCLs that are most extensively studied are described briefly. The chapter discusses the animal models created through molecular genetic manipulation, namely, mouse gene knockout models of juvenile NCL (CLN3 ) and mouse gene knockout model of late-infantile NCL. None of the animal models identified or developed to date replicate the human NCL disorders perfectly. Nonetheless, they have been and will continue to be valuable tools in studies to determine the mechanisms that underlie the human disease pathologies.
Juvenile neuronal ceroid lipofuscinosis (CLN3, JNCL, and Batten or Spielmeyer-Vogt-Sjogren disease, MIM304200) is the most common neurodegenerative disorder of childhood. Inheritance of the disease is autosomal recessive. There is no cure for JNCL. Treatment is largely symptomatic. Eiberg first mapped the JNCL locus CLN3 to chromosome 16. It is discovered a previously unreported subunit 9 gene, P3, and demonstrated the mapping of this gene to chromosome 2, also excluding it as the JNCL gene. Refined localization of CLN3 only became possible with the advent of highly polymorphic microsatellite markers. In parallel to efforts to refine the genetic locus of CLN3, a physical map consisting of yeast artificial chromosomes (YACs) and cosmid contigs is generated to span the candidate region. Cosmid clone NL11A, the most likely site of the JNCL gene CLN3, is selected for exon trapping for the isolation of candidate JNCL gene transcripts. The common mutation in JNCL is a 1-kb genomic deletion. The finding of additional mutations of the gene encoding cDNA2-3 in JNCL patients supported the conclusion that this gene is the JNCL gene CLN3. The tissue expression of CLN3 reflects the expression pattern of the other NCL genes so far identified, and offers no clue toward understanding the neuronal specificity of this group of disorders. No homology has been found at either the nucleotide or the protein level with any known gene, indicating that CLN3 encodes a novel protein of unknown function. The chapter discusses the animal models of JNCL. These animal models are critical for studies of the pathogenesis underlying the disease and for the evaluation of therapies.
PurposeTo describe the pathophysiologic features of retinal degeneration in Batten disease (juvenile neuronal ceroid lipofuscinosis [JNCL]) caused by mutations in the CLN3 gene.
The neuronal ceroid lipofuscinoses (NCL) are the most common childhood neurodegenerative disorders with a worldwide incidence of up to 1 in 12, 500 live births. Various subtypes have been described on a clinical and genetic level, with mutations in one of at least eight genes, termed CLN1-8, forming the molecular basis of the disease. Since mutations in distinct genes result in similar pathologies, suggesting a common biological pathway, it is important to not only elucidate the function of the proteins they encode but also to examine the possible consequences of protein dysfunction in cellular processes. Development of animal models of NCL disease and advancements in genomic and proteomic technologies provide valuable tools in the search for the underlying basis of disease. Application of DNA microarray analysis has revealed alterations in the expression of genes involved in a number of cellular processes including inflammation, neuronal function, oxidative stress, energy metabolism and proteolytic processing. Comparison of DNA microarray data from various NCL animal models has revealed not only alterations in a number of common pathological pathways characteristic of neurodegenerative disorders, but also some unique changes that may provide an insight into the function of the mutated proteins that underlie these diseases. Here, we review and discuss how such studies have furthered our current understanding of protein function and related pathological processes.
Neuronal ceroid lipofuscinosis (Batten disease) encompasses a group of 8 or more inherited lysosomal storage diseases, with an overall frequency of 1 in 12,500 births. All are characterized by progressive blindness and dementia and were initially classified on the basis of age of onset, clinical phenotype and ultrastructural characterization of the storage material as granular osmiophilic deposits, curvilinear bodies or fingerprint bodies. Recent research has shown that the various forms of Batten disease result from mutations in at least 8 genes which code for proteins involved in different aspects of lysosomal protein catabolism. These include palmitoyl:protein thioesterase 1 (CLN1), tripeptidylpeptidase 1 (CLN2), cathepsin D (CLN8), and two membrane proteins of unknown function (CLN3 and CLN5). Biochemically, Batten disease is characterized by the accumulation in neurons and other cells of an autofluorescent pigment which has resisted many attempts at analysis. In this review we attempt to relate our current understanding of the nature of the storage material in Batten disease with this genetic information. We conclude that the 8 genes probably code for proteins which facilitate the degradation of post-translationally modified proteins in lysosomes, suggesting that the turnover of these proteins is highest in cortical neurons. J. Neurosci. Res. 60:133–140, 2000 © 2000 Wiley-Liss, Inc.
The neuronal ceroid lipofuscinoses (NCL, also known as Batten disease) is a devastating neurodegenerative diseases caused by mutations in either soluble enzymes or membrane-associated structural proteins that result in lysosome dysfunction. Different forms of NCL were defined initially by age of onset, affected population and/or type of storage material but collectively represent the most prevalent pediatric hereditary neurovisceral storage disorder. Specific gene mutations are now known for each subclass of NCL in humans that now largely define the disease: cathepsin D (CTSD) for congenital (CLN10 form); palmitoyl protein thioesterase 1 (PPT1) for infantile (CLN1 form); tripeptidyl peptidase 1 (TPP1) for classic late infantile (CLN2 form); variant late infantile-CLN5, CLN6 or CLN8 for variant late infantile forms; and CLN3 for juvenile (CLN3 form). Several mouse models of NCL have been developed, or in some cases exist sporadically, that exhibit mutations producing a progressive neurodegenerative phenotype similar to that observed in human NCL. The study of these mouse models of NCL has dramatically advanced our knowledge of NCL pathophysiology and in some cases has helped delineate the function of proteins mutated in human NCL. In addition, NCL mutant mice have been tested for several different therapeutic approaches and as such they have become important pre-clinical models for validating treatment options. In this review we will assess the current state of mouse models of NCL with regards to their unique pathophysiology and how these mice have helped investigators achieve a better understanding of human NCL disease and therapy.
A critical analysis of the literature of mitochondrial disorders reveals that genetic diseases of oxidative phosphorylation are often associated with impaired beta-oxidation, and vice versa, and preferentially affect brain, retina, heart and skeletal muscle, tissues which depend on docosahexaenoic (22:6n-3)-containing phospholipids for functionality. Evidence suggests that an increased NADH/NAD(+) ratio generated by reduced flux through the respiratory chain inhibits beta-oxidation, producing secondary carnitine deficiency while increasing reactive oxygen species and depleting alpha-tocopherol (alpha-TOC). These events result in impairment of the recently elucidated mitochondrial pathway for synthesis of 22:6n-3-containing phospholipids, since carnitine and alpha-TOC are involved in their biosynthesis. Therapeutic supplementation with 22:6n-3 and alpha-TOC is suggested.
Neuronal ceroid lipofuscinosis (Batten disease) encompasses a group of 8 or more inherited lysosomal storage diseases, with an overall frequency of 1 in 12,500 births. All are characterized by progressive blindness and dementia and were initially classified on the basis of age of onset, clinical phenotype and ultrastructural characterization of the storage material as granular osmiophilic deposits, curvilinear bodies or fingerprint bodies. Recent research has shown that the various forms of Batten disease result from mutations in at least 8 genes which code for proteins involved in different aspects of lysosomal protein catabolism. These include palmitoyl:protein thioesterase 1 (CLN1), tripeptidylpeptidase 1 (CLN2), cathepsin D (CLN8), and two membrane proteins of unknown function (CLN3 and CLN5). Biochemically, Batten disease is characterized by the accumulation in neurons and other cells of an autofluorescent pigment which has resisted many attempts at analysis. In this review we attempt to relate our current understanding of the nature of the storage material in Batten disease with this genetic information. We conclude that the 8 genes probably code for proteins which facilitate the degradation of post-translationally modified proteins in lysosomes, suggesting that the turnover of these proteins is highest in cortical neurons.
The neuronal ceroid lipofuscinoses (NCLs) are an intriguing group of inherited neurodegenerative disorders characterized by blindness, progressive psychomotor deterioration and death of neocortical neurons. Clinically, four major NCL groups have been identified: infantile, late infantile, juvenile and adult. In recent years, our understanding of the molecular basis of different NCLs has advanced significantly. The accumulation of autofluorescent material in patients' tissues has been shown to be caused by defects in either lysosomal enzymes or in novel membrane proteins of unknown function. Although the accumulated material is biochemically well defined and some of the causative mutations are known, a unifying hypothesis for the molecular basis of the NCLs remains elusive. Further work will be required to characterize the interactiving molecules and metabolic pathways involved in the pathogenesis of NCLs.
To describe the pathophysiologic features of retinal degeneration in Batten disease (juvenile neuronal ceroid lipofuscinosis [JNCL]) caused by mutations in the CLN3 gene.
Comparative human tissue study.
The retina and other ocular tissues of a 22-year-old man with JNCL were compared with the same tissues of a healthy 30-year-old man. DNA from whole blood and RNA from retina were used for genotype analysis.
The retinas, corneas, conjunctiva, and ciliary body were processed for histopathologic and immunofluorescence analysis. Genomic DNA was subjected to polymerase chain reaction (PCR) and nucleotide sequence analyses. Reverse transcriptase/PCR and sequence analysis were performed on retinal RNA.
The JNCL donor was heterozygous for a approximately 1 kb deletion in CLN3, as found in most JNCL patients. The other allele had a single base pair deletion in exon 6 that resulted in a frame shift. Gross pathology of the JNCL retina resembled that in retinitis pigmentosa, including deposits of bone spicule pigment. Histopathologic studies revealed loss of neurons from all retinal layers. Immunofluorescence labeling with antibodies to rhodopsin, recoverin, and cone opsin demonstrated degenerate rods and cones with short outer segments in the far periphery. Autofluorescent lipopigment granules were prominent in ganglion cells and some cells of the inner nuclear layer, but not in the photoreceptors. The retinal pigment epithelium (RPE) had fewer lipofuscin granules than the control specimen. Increased numbers of lipofuscin granules were found in the epithelia of the ciliary body and conjunctiva, but not in the cornea of the JNCL eye.
Immunofluorescence studies revealed degenerate rods and cones in the far periphery. Lipofuscin granules were decreased in the RPE, consistent with loss of photoreceptor outer segments. The novel finding that degenerate photoreceptors did not contain autofluorescent inclusions suggests that granule accumulation may not precede photoreceptor degeneration in JNCL. The presence of normal photoreceptor proteins in the degenerate rods and cones suggests that these cells may be capable of functional regeneration if a therapy for Batten disease is developed.
Batten disease, the juvenile-onset form of neuronal ceroid lipofuscinosis (NCL), is a progressive neurodegenerative disorder of childhood with an age of onset of 5-10 years of age. JNCL is caused by mutations in the CLN3 gene which encodes a membrane protein of unknown function. Magnetic resonance imaging of the brain of juvenile NCL patients has revealed changes in signal intensity and tissue atrophy, predominantly in the cortex and cerebellum. A mouse model for Batten disease was created by targeted disruption of the murine Cln3 gene in order to further understanding of the pathophysiology of Batten disease and to evaluate potential therapeutic approaches. Several features of the disease are displayed by Cln3 mice including accumulation of characteristic storage material in neurons. The aim of this work was to investigate neurodegeneration in the Cln3 mouse model using high resolution magnetic resonance imaging to measure signal intensity ratios in selected regions of interest. Global changes were observed in the brains of 12-month-old mutant mice that mirror those seen in juvenile NCL patients. There is a decrease in signal intensity ratio in grey matter regions including cortex, hippocampus and cerebellum, tissues where neuronal storage accumulation and cell loss have been seen in the mouse model. The alterations seen in Cln3 mutant mice support the validity of further imaging studies and suggest that this method will have application in assessment of therapeutic approaches in the study of mutant mouse models of NCL including the Cln3 mouse.
Batten disease or JNCL, is the juvenile form of Neuronal Ceroid Lipofuscinosis (NCL) an autosomal recessive neurodegenerative disorder. Since retinal degeneration is an early consequence of Batten disease, we examined the eyes of Cln3 knockout mice (1-20 months of age), along with heterozygotes and appropriate controls, to determine whether or not the Cln3 defect would lead to characteristic retinal degeneration and visual loss. Accumulation of autofluorescent material and intracellular inclusions were markedly increased in Cln3 knockout retinal ganglion cells, as well as most other nuclear layers. Nerve fiber density was also significantly decreased in Cln3 knockout retinae. Apoptosis was observed in the photoreceptor layer of Cln3 knockout. However, the degree of retinal degeneration up to age 20 months was not extensive. Fundus examinations of Cln3 knockout mice showed no significant abnormalities, while electroretinograms remained robust through 11 months of age. In summary, it appears that accumulation of autofluorescent material, carbohydrate storage material, as well as apoptotic cell death are retinal manifestations of the Cln3 defect that do not appear to extinguish retinal function in this mouse model of Batten disease.
Positional cloning efforts of genes mutated in Batten disease and in the Finnish type of variant late infantile neuronal ceroid lipofuscinosis resulted in the identification of two novel genes, CLN3 and CLN5, and corresponding gene products that proved to be residents of lysosomes. Although the clinical phenotype of these NCL subtypes differs in the age of onset, average life span and EEG findings, the major component of material accumulating in patients' lysosomes is subunit c of mitochondrial ATPase in both these diseases. The CLN3 and CLN5 genes show ubiquitous expression patterns and are targeted to lysosomes in vitro, but the observed synaptosomal localization of the CLN3 protein in neurons would suggest some cell specificity in targeting and function of these proteins. So far, 31 different mutations of the CLN3 gene have been described in Batten patients, with one deletion of 1.02 kb accounting for 75% of disease alleles worldwide. Four CLN5 mutations are known, with one premature stop representing the major founder mutation in the isolated Finnish population. Functional studies of the yeast homolog of CLN3 and increased pH in patients' lysosomes would suggest an involvement of this protein in lysosomal pH homeostasis. Knock-out mouse models for CLN3 have been produced and the histopathology bears a close resemblance to human counterparts with characteristic lysosomal accumulations. Both CLN3 and CLN5 mouse models will provide experimental tools to resolve the pathological cascade in these neurodegenerative diseases.
The neuronal ceroid-lipofuscinoses (NCLs) collectively constitute the most common group of neurodegenerative diseases in childhood and usually show an autosomal recessive mode of inheritance. Despite varying ages of onset and clinical course characterized in most instances by progressive mental and motor deterioration, blindness, epileptic seizures, and premature death, all forms of NCL show unifying histopathological features. There is accumulation of autofluorescent, periodic acid-Schiff-, and Sudan black B-positive granules that are resistant to lipid solvents in the cytoplasm of most nerve cells and. to a lesser degree, of many other cell types. The storage process is associated with progressive and selective neuronal loss and gliosis with secondary white matter lesions. The ultrastructure of the storage deposits varies between different forms of NCL and, along with the age of onset, has provided the basis for the traditional classification of NCLs. Recent molecular genetic findings have established that defects in at least 7 different genes underlie the various forms of NCL. The purpose of this paper is to provide an overview of the NCLs, review recent molecular genetic and biochemical findings, and discuss their impact on our views on the classification and pathogenesis of these devastating brain disorders.
There are more than 40 different forms of inherited lysosomal storage diseases (LSDs) known to occur in humans and the aggregate incidence has been estimated to approach 1 in 7000 live births. Most LSDs are associated with high morbidity and mortality and represent a significant burden on patients, their families, and health care providers. Except for symptomatic therapies, many LSDs remain untreatable, and gene therapy is among the only viable treatment options potentially available. Therapies for some LSDs do exist, or are under evaluation, including heterologous bone marrow transplantation (BMT), enzyme replacement therapy (ERT), and substrate reduction therapy (SRT), but these treatment options are associated with significant concerns, including high morbidity and mortality (BMT), limited positive outcomes (BMT), incomplete response to therapy (BMT, ERT, and SRT), life-long therapy (ERT, SRT), and cost (BMT, ERT, SRT). Gene therapy represents a potential alternative therapy, albeit a therapy with its own attendant concerns. Animal models of LSDs play a critical role in evaluating the efficacy and safety of therapy for many of these conditions. Naturally occurring animal homologs of LSDs have been described in the mouse, rat, dog, cat, guinea pig, emu, quail, goat, cattle, sheep, and pig. In this review we discuss those animal models that have been used in gene therapy experiments and those with promise for future evaluations.
Juvenile Neuronal Ceroid Lipofuscinosis (JNCL), or Batten disease, is a childhood neurodegenerative disease that is characterized clinically by progressive visual loss, seizures, dementia, and motor incoordination. Children affected with this disease tend to develop normally for the first 5 years of life. However, once disease onset occurs, they decline rapidly and die in their late 20s to early 30s. Though this represents the typical disease course, the onset and severity of disease symptoms can vary. This variability is presumed to be the result of both differences in the causative genetic mutation in the CLN3 gene as well as environmental influences. Most cases of JNCL are caused by a 1 kb deletion in the CLN3 gene, resulting in a frameshift mutation predicted to leave the first 153 amino acids of the CLN3 protein intact, followed by the addition of 28 novel amino acids. Here we report the discovery of a novel mutation identified as a G to T transversion at nucleotide 49 (G49T) in exon 2 of CLN3, introducing a premature stop codon (E17X) near the N-terminus. This mutation represents the most 5' mutation described to date. The patient examined in this study was heterozygous for the common 1 kb deletion and E17X. She had classical disease progression, suggesting that this mutation in CLN3 mimics the more prevalent 1 kb deletion and that progression of JNCL is predominantly the result of loss of CLN3 function.
We describe a transgenic mouse line carrying the cre transgene under the control of the adenovirus EIIa promoter that targets expression of the Cre recombinase to the early mouse embryo. To assess the ability of this recombinase to excise loxP-flanked DNA sequences at early stages of development, we bred EIIa-cre transgenic mice to two different mouse lines carrying loxP-flanked target sequences: (i) a strain with a single gene-targeted neomycin resistance gene flanked by 1oxP sites and (ii) a transgenic line carrying multiple transgene copies with internal loxP sites. Mating either of these loxP-carrying mouse lines to EIIa-cre mice resulted in first generation progeny in which the loxP-flanked sequences had been efficiently deleted from all tissues tested, including the germ cells. Interbreeding of these first generation progeny resulted in efficient germ-line transmission of the deletion to subsequent generations. These results demonstrate a method by which loxP-flanked DNA sequences can be efficiently deleted in the early mouse embryo. Potential applications of this approach are discussed, including reduction of multicopy transgene loci to produce single-copy transgenic lines and introduction of a variety of subtle mutations into the line.
The neuronal ceroid lipofuscinoses (NCLs) comprise a set of at least 6 distinct human and an unknown number of animal diseases characterized by storage of proteolipids in lysosomes of many cell types. By unknown mechanisms, this accumulation leads to or is associated with severe neuronal and retinal degeneration. The genes for 3 human NCLs, infantile, late infantile, and juvenile, have been cloned. The first murine form of NCL, the motor neuron degeneration (mnd) mouse, has been described and mapped to proximal Chromosome 8. Here we describe a second genetic variant of NCL in the mouse, neuronal ceroid lipofuscinosis, nclf. These mice exhibited a phenotype that was almost exactly the same as that observed in mnd/mnd mice. Homozygous nclf mice developed progressive retinal atrophy early in life and become paralyzed at around 9 months of age. They accumulated luxol fast blue staining material in cytoplasm of neurons and many other cell types. Ultrastructurally, affected lysosomes had a “finger print pattern” with membranous material arranged in “pentalaminar” patterns. Affected mice developed severe cerebral gliosis in late stages of their disease. They also had severe Wallerian degeneration of long tracts in spinal cord and brain stem, lesions that accounted for the distinctive upper motor neuron signs displayed by both nclf/nclf and mnd/mnd mice. By crossing nclf/nclf mice with CAST/Ei mice, linkage analysis of nclf with respect to SSLP markers was performed, showing that nclf is located on Chromosome 9 between D9Mit164 and D9Mit165, in a region that is homologous with human Ch 15q21, where the gene for one variant of late infantile NCL, CLN6, recently has been mapped. The genes for two proteolipids known to be stored in lysosomes of animals and people with NCL were also mapped in this study and found not to map to the mnd or nclf loci nor to any mouse locus homologous to any known human NCL disease locus. Am. J. Med. Genet. 77:289–297, 1998. © 1998 Wiley‐Liss, Inc.
Batten disease (also known as juvenile neuronal ceroid lipofuscinosis) is a recessively inherited neurodegenerative disorder of childhood characterized by progressiveloss of vision, seizures, and psychomotor disturbances. The Batten disease gene, CLN3, maps to chromosome 16p12.1. The so-called 56 chromosome haplotype defined by alleles at the D16S299 and D16S298 loci is shared by 73% of Batten disease chromosomes. Exon amplification of a cosmid containing D16S298 has yielded a candidate gene that is disrupted by a 1 kb genomic deletion in all patients carrying the 56 chromosome. Two separate deletions and a point mutation altering a splice site in three unrelated families have confirmed the candidate as the CLN3 gene. The disease gene encodes a novel 438 amino acid protein of unknown function.
The motor neuron degeneration mutation (Mnd) causes a late-onset, progressive degeneration of upper and lower motor neurons in mice. After establishing genetic and environmental conditions that distinguish the phenotypes of Mnd/Mnd from +/Mnd mice, Mnd was mapped to proximal Chr 8, using endogenous retroviruses as markers. The map location was confirmed with additional linked polymorphic markers. The outcross/intercross matings to the strain AKR/J, which were used to follow the segregation of the retroviral markers with respect to Mnd, also revealed the existence of a timing effect. Approximately one-fourth of the affected Mnd/Mnd F2 progeny showed accelerated disease. The Mnd mouse model should allow study of mechanisms affecting onset and progression of specific neuronal degeneration in both animal and human neurological disease.
Progressive epilepsy with mental retardation (EPMR) is an autosomal recessive central nervous system disorder characterized by childhood onset epilepsy and subsequent mental retardation. The locus for EPMR has been mapped to human chromosome 8p23. We recently reported the construction of a YAC contig across the 4 centimorgan minimum genetic region that harbors the disease locus. We now report further delineation of the critical region to <700 kb. Our mapping strategy relied on the identification of nine novel microsatellite markers and the construction of a complete BAC contig across the critical region. Several partial gene sequences have been identified from the region and are being analyzed as candidate genes for EPMR.
[The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AF009188 – AF009214 .]
The c-abl proto-oncogene, which encodes a cytoplasmic protein-tyrosine kinase, is expressed throughout murine gestation and ubiquitously in adult mouse tissues. However, its levels are highest in thymus, spleen, and testes. To examine the in vivo role of c-abl, the gene was disrupted in embryonic stem cells, and the resulting genetically modified cells were used to establish a mouse strain carrying the mutation. Most mice homozygous for the c-abl mutation became runted and died 1 to 2 weeks after birth. In addition, many showed thymic and splenic atrophy and a T and B cell lymphopenia.
A late-onset neurological disease has been identified in a substrain of C57Bl/6 mice. The disorder is characterized by hindlimb weakness and ataxia starting at 5-11 months of age, progressing to severe spastic paralysis of all limbs, with premature death. Histopathology reveals degeneration of upper and lower motoneurons. Both sexes are affected; the mice are fertile, although breeding efficiency is reduced. In outcrosses to wild-type, symptoms have been observed in all obligate heterozygotes, with a similar age range for onset to that of homozygotes. We have designated this autosomal dominant disorder Motor neuron degeneration (Mnd).
Multiple forms of ceroid-lipofuscinosis occur in human beings and animals. They are characterized by brain and retinal atrophy associated with selective necrosis of neurons. This neurodegenerative disease appears associated with the disease process rather than storage of fluorescent lipopigment per se, and there is now growing evidence that pathogenesis may involve mitochondria rather than a primary defect of lysosomal catabolism. Of the forms of ceroid-lipofuscinosis studied, most but not all reflect accumulation of subunit c of mitochondrial ATP synthase. If there is a common denominator between all forms other than the presence of fluorescent lipopigment, then it may be the accumulation of hydrophobic protein. Analogous diseases in animals can be expected to reflect the same spectrum of biochemical changes, and they warrant in-depth study to help understand the pathogenesis and heterogeneity of the group.
Pathological studies of mice homozygous for the motor neuron degeneration (Mnd) mutation show abnormalities similar to those of the human neuronal ceroid lipofuscinoses: sudanophilic, autofluorescent intraneuronal inclusions that are immunoreactive with antibodies to subunit c of mitochondrial ATP synthase. Ultrastructurally, the inclusions have the pentalaminar structure characteristic of some form of human neuronal ceroid lipofuscinosis and of canine and ovine models of neuronal ceroid lipofuscinosis. Similar inclusions are observed in many somatic organs and in the retina, which develops photoreceptor degeneration. This mutation, previously considered a model of amyotrophic lateral sclerosis, may be a useful model for molecular and genetic studies of human neuronal ceroid lipofuscinosis because mice have been well characterized genetically. Since they are inexpensive to breed and maintain, they can also be used to test therapeutic interventions.
Endochondral ossification is a major mode of bone that occurs as chondrocytes undergo proliferation, hypertrophy, cell death, and osteoblastic replacement. We have identified a role for fibroblast growth factor receptor 3 (FGFR-3) in this process by disrupting the murine Fgfr-3 gene to produce severe and progressive bone dysplasia with enhanced and prolonged endochondral bone growth. This growth is accompanied by expansion of proliferating and hypertrophic chondrocytes within the cartilaginous growth plate. Thus, FGFR-3 appears to regulate endochondral ossification by an essentially negative mechanism, limiting rather than promoting osteogenesis. In light of these mouse results, certain human disorders, such as achondroplasia, can be interpreted as gain-of-function mutations that activate the fundamentally negative growth control exerted by the FGFR-3 kinase.
The neuronal ceroid lipofuscinoses (NCLs) are a group of clinically and genetically heterogenenous neurodegenerative disorders characterised by the accumulation of autofluorescent storage material in many cell types. Most display autosomal recessive inheritance. More than 400 mutations have been described in 13 genes: PPT1 , TPP1 , CLN3 , CLN5 , CLN6 , MFSD8 , CLN8 , CTSD, GRN, ATP13A2, KCTD7, CTSF and DNAJC5 , and none show obvious mutation hotspots. The functions of most of these genes remain elusive. The majority of the genetic defects identified are private, but there are geographically widespread or localised common founder mutations. The NCLs exemplify both phenotypic convergence and divergence, and there can be very wide disease severity caused by mutations in the same gene. Some recently identified mutations are in genes that are associated with other diseases. The recent expansion of knowledge in the genetic understanding of the NCLs is leading to improved diagnostic approaches, and the recent adoption of a new nomenclature.
Key Concepts
More than 400 mutations are known across 13 genes causing disease in families diagnosed with NCL.
Most NCLs are autosomal recessive.
NCLs are a heterogenous group of disorders with common features of progressive degeneration of the brain, and often the retina, and intracellular storage of material similar to ceroid and lipofuscin.
NCLs are monogenic disorders.
One type of NCL is autosomal dominant.
There remain families diagnosed with NCL in which the genetic cause is not yet determined.
We describe the isolation and chromosomal mapping of a mouse homolog of the Batten disease gene, CLN3. Like its human counterpart, the mouse cDNA contains an open reading frame of 1314 bp encoding a predicted protein product of 438 amino acids. The mouse and human coding regions are 82 and 85% identical at the nucleic acid and amino acid levels, respectively. The mouse gene maps to distal Chromosome 7, in a region containing genes whose homologs are on human chromosome 16p12, where CLN3 maps. Isolation of a mouse CLN3 homolog will facilitate the creation of a mouse model of Batten disease.
The childhood neuronal ceroid lipofuscinoses (NCLs) are a group of autosomal recessive neurodegenerative disorders characterised by progressive visual failure, neurodegeneration, epilepsy and the accumulation of an autofluorescent lipopigment in neurones and other cells. Three main subtypes have been identified according to age of onset, clinical features and ultrastructural morphology. These are infantile NCL (INCL; CLN1), classical late infantile NCL (LINCL; CLN2) and juvenile NCL (JNCL; CLN3). Several atypical forms of late infantile NCL (LINCL) have also been described including a Finnish variant LINCL (CLN5). The CLN2 gene has been excluded from the CLN1, CLN3 and CLN5 loci. A genome search was initiated using a homozygosity mapping strategy in five classical LINCL and two variant LINCL consanguineous families. A common region of homozygosity was identified on chromosome 11p15 in two of the classical families. Analysis of a further 33 classical LINCL families supported linkage in this region (Zmax = 3.07 at theta = 0.06 at D11S1338). A common region of homozygosity was also observed on chromosome 15q21-23 in the two variant LINCL families. Extension of the analysis to include a further seven families of identical ultrastructural phenotype established linkage to this region (Zmax = 6.00 at theta = 0.00 at D15S1020).
Batten disease (juvenile-onset neuronal ceroid lipofuscinosis [JNCL]) is an autosomal recessive condition characterized by accumulation of lipopigments (lipofuscin and ceroid) in neurons and other cell types. The Batten disease gene, CLN3, was recently isolated, and four disease-causing mutations were identified, including a 1.02-kb deletion that is present in the majority of patients (The International Batten Disease Consortium 1995). One hundred eighty-eight unrelated patients with JNCL were screened in this study to determine how many disease chromosomes carried the 1.02-kb deletion and how many carried other mutations in CLN3. One hundred thirty-nine patients (74%) were found to have the 1.02-kb deletion on both chromosomes, whereas 49 patients (41 heterozygous for the 1.02-kb deletion) had mutations other than the 1.02-kb deletion. SSCP analysis and direct sequencing were used to screen for new mutations in these individuals. Nineteen novel mutations were found: six missense mutations, five nonsense mutations, three small deletions, three small insertions, one intronic mutation, and one splice-site mutation. This report brings the total number of disease-associated mutations in CLN3 to 23. All patients homozygous for mutations predicted to give rise to truncated proteins were found to have classical JNCL. However, a proportion of the patients (n = 4) who were compound heterozygotes for a missense mutation and the 1.02-kb deletion were found to display an atypical phenotype that was dominated by visual failure rather than by severe neurodegeneration. All missense mutations were found to affect residues conserved between the human protein and homologues in diverse species.
The neuronal ceroid lipofuscinoses (NCLs) comprise a set of at least 6 distinct human and an unknown number of animal diseases characterized by storage of proteolipids in lysosomes of many cell types. By unknown mechanisms, this accumulation leads to or is associated with severe neuronal and retinal degeneration. The genes for 3 human NCLs, infantile, late infantile, and juvenile, have been cloned. The first murine form of NCL, the motor neuron degeneration (mnd) mouse, has been described and mapped to proximal Chromosome 8. Here we describe a second genetic variant of NCL in the mouse, neuronal ceroid lipofuscinosis, nclf. These mice exhibited a phenotype that was almost exactly the same as that observed in mnd/mnd mice. Homozygous nclf mice developed progressive retinal atrophy early in life and become paralyzed at around 9 months of age. They accumulated luxol fast blue staining material in cytoplasm of neurons and many other cell types. Ultrastructurally, affected lysosomes had a "finger print pattern" with membranous material arranged in "pentalaminar" patterns. Affected mice developed severe cerebral gliosis in late stages of their disease. They also had severe Wallerian degeneration of long tracts in spinal cord and brain stem, lesions that accounted for the distinctive upper motor neuron signs displayed by both nclf/nclf and mnd/mnd mice. By crossing nclf/nclf mice with CAST/Ei mice, linkage analysis of nclf with respect to SSLP markers was performed, showing that nclf is located on Chromosome 9 between D9Mit164 and D9Mit165, in a region that is homologous with human Ch 15q21, where the gene for one variant of late infantile NCL, CLN6, recently has been mapped. The genes for two proteolipids known to be stored in lysosomes of animals and people with NCL were also mapped in this study and found not to map to the mnd or nclf loci nor to any mouse locus homologous to any known human NCL disease locus.
Isolation of a novel gene underlying Batten disease
- International Batten
- Consortium
International Batten Disease Consortium. Isolation of a novel gene underlying Batten disease, CLN3. Cell 82:949 – 957, 1995.
Mapping of the motor neuron degeneration (Mnd) gene, a ics 18
- A Messer
- J Plummer
- P Maskin
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