Cln3-knockout Mice Research Articles

Defective synaptic transmission causes disease signs in a mouse model of juvenile neuronal ceroid lipofuscinosis.

Juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease) caused by mutations in the CLN3 gene is the most prevalent inherited neurodegenerative disease in childhood resulting in widespread central nervous system dysfunction and premature death. The consequences of CLN3 mutation on the progression of the disease, on neuronal transmission, and on central nervous network dysfunction are poorly understood. We used Cln3 knockout (Cln3Δex1-6) mice and found increased anxiety-related behavior and impaired aversive learning as well as markedly affected motor function including disordered coordination. Patch-clamp and loose-patch recordings revealed severely affected inhibitory and excitatory synaptic transmission in the amygdala, hippocampus, and cerebellar networks. Changes in presynaptic release properties may result from dysfunction of CLN3 protein. Furthermore, loss of calbindin, neuropeptide Y, parvalbumin, and GAD65-positive interneurons in central networks collectively support the hypothesis that degeneration of GABAergic interneurons may be the cause of supraspinal GABAergic disinhibition.

Age-dependent therapeutic effect of memantine in a mouse model of juvenile Batten disease

Currently there is no treatment for juvenile Batten disease, a fatal childhood neurodegenerative disorder caused by mutations in the CLN3 gene. The Cln3-knockout (Cln3Δex1-6) mouse model recapitulates several features of the human disorder. Cln3Δex1-6 mice, similarly to juvenile Batten disease patients, have a motor coordination deficit detectable as early as postnatal day 14. Previous studies demonstrated that acute attenuation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptor activity by the non-competitive AMPA antagonist, EGIS-8332, in both 1- and 6–7-month-old Cln3Δex1-6 mice results in improvement in motor coordination. Here we show that acute inhibition of N-methyl-d-aspartate (NMDA)-type glutamate receptors by memantine (1 and 5 mg/kg i.p.) had no effect on the impaired motor coordination of one-month-old Cln3Δex1-6 mice. At a later stage of the disease, in 6–7-month-old Cln3Δex1-6 mice, memantine induced a delayed but extended (8 days) improvement of motor skills similarly to that observed previously with EGIS-8332 treatment. An age-dependent therapeutic effect of memantine implies that the pathomechanism in juvenile Batten disease changes during disease progression. In contrast to acute treatment, repeated administration of memantine or EGIS-8332 (1 mg/kg, once a week for 4 weeks) to 6-month-old Cln3Δex1-6 mice had no beneficial effect on motor coordination. Moreover, repeated treatments did not impact microglial activation or the survival of vulnerable neuron populations. Memantine did not affect astrocytosis in the cortex. EGIS-8332, however, decreased astrocytic activation in the somatosensory barrelfield cortex.Acute inhibition of NMDA receptors can induce a prolonged therapeutic effect, identifying NMDA receptors as a new therapeutic target for juvenile Batten disease.

Temporary inhibition of AMPA receptors induces a prolonged improvement of motor performance in a mouse model of juvenile Batten disease

Mutations in the CLN3 gene cause juvenile Batten disease, a fatal pediatric neurodegenerative disorder. The Cln3-knockout (Cln3Δex1–6) mouse model of the disease displays many pathological characteristics of the human disorder including a deficit in motor coordination. We have previously found that attenuation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptor activity in one-month-old Cln3Δex1–6 mice resulted in an immediate improvement of their motor skills. Here we show that at a later stage of the disease, in 6-7-month-old Cln3Δex1–6 mice, acute inhibition of AMPA receptors by a single intraperitoneal injection (1mg/kg) of the non-competitive AMPA antagonist, EGIS-8332, does not have an immediate effect. Instead, it induces a delayed but prolonged improvement of motor skills. Four days after the injection of the AMPA antagonist, Cln3Δex1–6 mice reached the same motor skill level as their wild type (WT) counterparts, an improvement that persisted for an additional four days. EGIS-8332 was rapidly eliminated from the brain as measured by HPLC-MS/MS. Histological analysis performed 8 days after the drug administration revealed that EGIS-8332 did not have any impact upon glial activation or the survival of vulnerable neuron populations in 7-month-old Cln3Δex1–6 mice.We propose that temporary inhibition of AMPA receptors can induce a prolonged correction of the pre-existing abnormal glutamatergic neurotransmission in vivo for juvenile Batten disease.

INTERMITTENT PREDNISOLONE AND AUTOANTIBODIES TO GAD65 IN JUVENILE NEURONAL CEROID LIPOFUSCINOSIS

Juvenile neuronal ceroid lipofuscinosis (JNCL) is a recessively inherited, progressive neurologic disease caused by mutations in the CLN3 gene, coding for a transmembrane protein with unknown function.1 The symptoms include visual failure, noticed around the age of 4 to 7 years, mental decline, epilepsy, parkinsonism, psychiatric symptoms, and sleeping difficulties. Thus far, no curative treatment is available and the disease leads to an early death at the age of 15 to 30 years. Autoantibodies to glutamic acid decarboxylase 65 (GAD65) were detected in the sera of patients with JNCL and in CLN3 knock-out mice.2 As a biologic sign of these antibodies, decreased GAD65 activity and increased levels of glutamate were observed in the mice brains. Together these findings suggest that an immune-mediated reaction to GAD65 may be involved in the pathogenesis of JNCL. Our aim was to test the effect of immunosuppressive therapy on the GAD65-antibodies, symptoms, and disease progression in patients with JNCL. ### Methods. Four girls and four boys with JNCL (table) were included in this 1-year study, approved by the Ethics Committee of the Hospital for Children and Adolescents, University of Helsinki. View this table: Table Patients, their GAD65-autoantibodies, verbal IQ, and weight for height ratio in percentage at the beginning (0 mo) and end …

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.

Neuronal vulnerability of CLN3 deletion to calcium-induced cytotoxicity is mediated by calsenilin

Calsenilin/DREAM/KChIP3, a neuronal Ca(2+)-binding protein, has multifunctions in nucleus and cytosol. Here, we identified CLN3 as a calsenilin-binding partner whose mutation or deletion is observed in Batten disease. In vitro binding and immunoprecipitation assays show that calsenilin interacts with the C-terminal region of CLN3 and the increase of Ca(2+) concentration in vitro and in cells causes significant dissociation of calsenilin from CLN3. Ectopic expression of CLN3 or its deletion mutant containing only the C-terminus (153-438) and capable of binding to calsenilin suppresses thapsigargin or A23187-induced death of neuronal cells. In contrast, CLN3 deletion mutant containing the N-terminus (1-153) or (1-263), which is frequently found in Batten disease, induces the perturbation of Ca(2+) transient and fails to inhibit the cell death. In addition, the expression of calsenilin is increased in the brain tissues of CLN3 knock-out mice and SH-SY5Y/CLN3 knock-down cells. Down-regulation of CLN3 expression sensitizes SH-SY5Y cells to thapsigargin or A23187. However, additional decrease of calsenilin expression rescues the sensitivity of SH-SY5Y/CLN3 knock-down cells to Ca(2+)-mediated cell death. These results suggest that the vulnerability of CLN3 knock-out or CLN3 deletion (1-153)-expressing neuronal cells to Ca(2+)-induced cell death may be mediated by calsenilin.

Selectively increased sensitivity of cerebellar granule cells to AMPA receptor-mediated excitotoxicity in a mouse model of Batten disease

Batten disease, a lysosomal storage disorder, is caused by mutations in the CLN3 gene. The Cln3-knockout (Cln3-/-) mouse model of the disease exhibits many characteristic pathological features of the human disorder. Here, we show that Cln3-/- mice, similarly to Batten disease patients, have a deficit in cerebellar motor coordination. To explore the possible cellular cause of this functional impairment, we compared the vulnerability of wild type (WT) and Cln3-/- cerebellar granule cell cultures to different toxic insults. We have found that cultured Cln3-/- cerebellar granule cells are selectively more vulnerable to AMPA-type glutamate receptor-mediated toxicity than their WT counterparts. This selective sensitivity was also observed in organotypic cerebellar slice cultures. Our results suggest that lack of the CLN3 protein has a significant influence on the function of AMPA receptors in cerebellar granule neurons, and that AMPA receptor dysregulation may be a major contributor to the cerebellar dysfunction in Batten disease.

High Resolution 1H NMR-based Metabolomics Indicates a Neurotransmitter Cycling Deficit in Cerebral Tissue from a Mouse Model of Batten Disease

The neuronal ceroid lipofuscinoses (NCLs) constitute a range of progressive neurological disorders primarily affecting children. Although six of the causative genes have been characterized, the underlying disease pathogenesis for this family of disorders is unknown. Using a metabolomics approach based on high resolution 1H NMR spectroscopy of the cortex, cerebellum, and remaining regions of the brain in conjunction with statistical pattern recognition, we report metabolic deficits associated with juvenile NCL in a Cln3 knock-out mouse model. Tissue from Cln3 null mutant mice aged 1-6 months was characterized by an increased glutamate concentration and a decrease in -amino butyric acid (GABA) concentration in aqueous extracts from the three regions of the brain. These changes are consistent with the reported altered expression of genes involved in glutamate metabolism in older mice and imply a change in neurotransmitter cycling between glutamate/glutamine and the production of GABA. Further variations in myo-inositol, creatine, and N-acetyl-aspartate were also identified. These metabolic changes were distinct from the normal aging/developmental process. Together, these changes represent the first documented pre-symptomatic symptoms of the Cln3 mouse at 1 month of age and demonstrate the versatility of 1H NMR spectroscopy as a tool for phenotyping mouse models of disease.

Elevation of Hook1 in a disease model of Batten disease does not affect a novel interaction between Ankyrin G and Hook1

Hook1 is a member of a family of microtubule-binding proteins. Studies on the Drosophila homolog of Hook1 have suggested a role in the maturation and trafficking of internalized proteins to the late endosome. A weak interaction between Hook1 and the lysosomal/late endosomal protein, CLN3, was recently reported. Mutations in CLN3 result in the neurological disorder Batten disease. Here we show a novel interaction between Hook1 and Ankyrin G, an adaptor protein that binds the spectrin–actin cytoskeleton and targets proteins to the peripheral membrane. Although we demonstrate co-localization of Hook1 and Ankyrin G, Hook1 also localizes to additional regions of the cell devoid of Ankyrin G where it likely interacts with other proteins. There is no disruption of the Hook1–Ankyrin G interaction or localization in tissue derived from a Cln3-knockout mouse despite a nearly threefold increase in the expression of Hook1. However, mutation of CLN3 could lead to alterations in the functioning and positioning of organelles and membrane proteins through this Hook1–Ankyrin G interaction.

Altered gene expression in the eye of a mouse model for batten disease.

Juvenile neuronal ceroid lipofuscinosis (JNCL or Batten Disease) is one of the most common progressive neurodegenerative disorders of childhood, resulting from autosomal recessive inheritance of mutations in the CLN3 gene. Pathologically, Batten disease is characterized by lysosomal storage of autofluorescent material in all tissue types. Although characterized by seizures, mental retardation, and loss of motor skills, the first presenting symptom of Batten disease is vision loss. High-density oligonucleotide arrays were used to profile approximately 19,000 mRNAs in the eye of 10-week-old Cln3-knockout and normal mice, and the data were compared with that for the cerebellum in the same model as a means to identify gene expression changes that are specific to the eye. A detailed list was compiled of 285 functionally categorized genes that have altered expression in the eye of Cln3-knockout mice before the appearance of the characteristic lysosomal storage material. Furthermore, 18 genes were identified and 6 validated by semiquantitative RT-PCR that have altered expression in the eye, but not in the cerebellum of Cln3-knockout mice. The genes that have altered expression specific to the eye of the Cln3-knockout mouse may be of importance in understanding the function of CLN3 in different tissues. Downregulation of genes associated with energy production in the mitochondria appears to be specific to the eye. The CLN3 defect may result in altered mitochondrial function in eye but not other tissue. More detailed experimentation is needed to understand the contribution of these changes in expression to disease state, and whether these changes are specific for certain cell types within the eye.

Optic nerve degeneration in a murine model of juvenile ceroid lipofuscinosis.

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.

Early changes in gene expression in two models of Batten disease

Infantile and juvenile neuronal ceroid lipofuscinosis (NCLs) are progressive neurodegenerative disorders of childhood with distinct ages of clinical onset, but with a similar pathological outcome. Infantile and juvenile NCL are inherited in an autosomal recessive manner due to mutations in the CLN1 and CLN3 genes, respectively. Recently developed Cln1- and Cln3-knockout mouse models share similarities in pathology with the respective human disease. Using oligonucleotide arrays we identified reproducible changes in gene expression in the brains of both 10-week-old Cln1- and Cln3-knockout mice as compared to wild-type controls, and confirmed changes in levels of several of the cognate proteins by immunoblotting. Despite the similarities in pathology, the two mutations affect the expression of different, non-overlapping sets of genes. The possible significance of these changes and the pathological mechanisms underlying NCL diseases are discussed.

Functional categorization of gene expression changes in the cerebellum of a Cln3-knockout mouse model for Batten disease

Juvenile neuronal ceroid lipofuscinosis (JNCL or Batten Disease) is the most common progressive neurodegenerative disorder of childhood. The disease is inherited in an autosomal recessive manner and is the result of mutations in the CLN3 gene. One brain region severely affected in Batten disease is the cerebellum. Using a mouse model for Batten disease which shares pathological similarities to the disease in humans we have used oligonucleotide arrays to profile approximately 19,000 mRNAs in the cerebellum. We have identified reproducible changes of twofold or more in the expression of 756 gene products in the cerebellum of 10-week-old Cln3-knockout mice as compared to wild-type controls. We have subsequently divided these genes with altered expression into 14 functional categories. We report a significant alteration in expression of genes associated with neurotransmission, neuronal cell structure and development, immune response and inflammation, and lipid metabolism. An apparent shift in metabolism toward gluconeogenesis is also evident in Cln3-knockout mice. Further experimentation will be necessary to understand the contribution of these changes in expression to a disease state. Detailed analysis of the functional consequences of altered expression of genes in the cerebellum of the Cln3-knockout mice may provide valuable clues in understanding the molecular basis of the pathological mechanisms underlying Batten disease.

An autoantibody to GAD65 in sera of patients with juvenile neuronal ceroid lipofuscinoses.

Juvenile neuronal ceroid lipofuscinoses (Batten disease) is a neurodegenerative disease that presents in early childhood. The main features of this disease are failure of psychomotor development, impaired vision, and seizures. Death usually occurs in late teens to early twenties. Batten disease is an autosomal-recessive disorder, resulting from mutations in the CLN3 gene product.1 Genetic testing can identify the most common mutation in CLN3 , a 1.02-kb deletion. However this accounts for only about 74% of the mutations identified thus far in CLN3 ,1,2⇓ and because identification of rarer point mutations in CLN3 is often difficult, another diagnostic tool would be useful. We have previously shown that a mouse model for Batten disease, i.e., cln3-knockout mice, and eight individuals with Batten disease have an autoimmune response to GAD65.3 In the same study we reported that a total of 20 individuals with Batten disease tested positive for the presence of autoantibodies to glutamate decarboxylase, GAD65. Furthermore, studies in the mouse revealed that the autoantibody to GAD65 is associated with astrocytic hypertrophy in the brain. Moreover, the cln3-knockout mice that have the autoimmune response to GAD65 have decreased activity of glutamic acid decarboxylase and consequently elevated levels of presynaptic glutamate in the brain.3 It has been suggested that a preferential loss of GABAergic neurons is a major factor in the progression of Batten disease.4 It is therefore plausible to predict that the GAD65 autoantibody and its resultant effects on the brain are possible contributors to the pathology associated …

An autoantibody inhibitory to glutamic acid decarboxylase in the neurodegenerative disorder Batten disease.

Mutations in the CLN3 gene are responsible for the neurodegenerative disorder Batten disease; however, the molecular basis of this disease remains unknown. In studying a mouse model for Batten disease, we report the presence of an autoantibody to glutamic acid decarboxylase (GAD65) in cln3-knockout mice serum that associates with brain tissue but is not present in sera or brain of normal mice. The autoantibody to GAD65 has the ability to inhibit the activity of glutamic acid decarboxylase. Furthermore, brains from cln3-knockout mice have decreased activity of glutamic acid decarboxylase as a result of the inhibition of this enzyme by the autoantibody, resulting in brain samples from cln3-knockout mice having elevated levels of glutamate as compared with normal. This elevated glutamate in the brain of cln3-knockout mice co-localizes with presynaptic markers. The decreased activity of GAD65 and increased levels of glutamate may have a causative role in astrocytic hypertrophy evident in cln3-knockout mice, and in altered expression of genes involved in the synthesis and utilization of glutamate that underlie a shift from synthesis to utilization of glutamate. An autoantibody to GAD65 is also present in sera of 20 out of 20 individuals tested who have Batten disease. Postmortem tissue shows decreased reactivity to an anti-GAD65 antibody that may be due to loss of GAD65-positive neurons or due to the reactive epitope being blocked by the presence of the autoantibody. We propose that an autoimmune response to GAD65 may contribute to a preferential loss of GABAergic neurons associated with Batten disease.

Retinal Pathology and Function in a Cln3 Knockout Mouse Model of Juvenile Neuronal Ceroid Lipofuscinosis (Batten Disease)

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.

A mouse gene knockout model for juvenile ceroid-lipofuscinosis (batten disease)

The human hereditary ceroid-lipofuscinoses are a group of autosomal recessively inherited diseases characterized by massive accumulations of autofluorescent lysosomal storage bodies in the cells of many tissues and by neuronal degeneration throughout the central nervous system. There are a number of clinically and genetically distinct forms of ceroid-lipofuscinosis, the most common of which is the juvenile type, also known as Batten disease and CLN3. To study the mechanisms that lead to pathology in CLN3 and to evaluate potential therapies, a mouse model has been generated by targeted disruption of the mouse ortholog of the CLN3 gene (Cln3). As in affected humans, mice homozygous for the disrupted Cln3 allele show accumulation of autofluorescent storage material in neurons and other cell types. The storage material consists of membrane-bounded intracellular inclusions with ultrastructural features typical of the ceroid-lipofuscinoses. The accumulation of this storage material validates the Cln3 knockout mice as a model for the human disorder.