Juvenile Form Of Neuronal Ceroid Lipofuscinosis Research Articles

Synapse alterations precede neuronal damage and storage pathology in a human cerebral organoid model of CLN3-juvenile neuronal ceroid lipofuscinosis

The juvenile form of neuronal ceroid Lipofuscinosis (JNCL) is the most common form within this group of rare lysosomal storage disorders, causing pediatric neurodegeneration. The genetic disorder, which is caused by recessive mutations affecting the CLN3 gene, features progressive vision loss, cognitive and motor decline and other psychiatric conditions, seizure episodes, leading to premature death. Animal models have traditionally aid the understanding of the disease mechanisms and pathology and are very relevant for biomarker research and therapeutic testing. Nevertheless, there is a need for establishing reliable and predictive human cellular models to study the disease. Since patient material, particularly from children, is scarce and difficult to obtain, we generated an engineered a CLN3-mutant isogenic human induced pluripotent stem cell (hiPSC) line carrying the c.1054C → T pathologic variant, using state of the art CRISPR/Cas9 technology. To prove the suitability of the isogenic pair to model JNCL, we screened for disease-specific phenotypes in non-neuronal two-dimensional cell culture models as well as in cerebral brain organoids. Our data demonstrates that the sole introduction of the pathogenic variant gives rise to classical hallmarks of JNCL in vitro. Additionally, we discovered an alteration of the splicing caused by this particular mutation. Next, we derived cerebral organoids and used them as a neurodevelopmental model to study the particular effects of the CLN3Q352X mutation during brain formation in the disease context. About half of the mutation -carrying cerebral organoids completely failed to develop normally. The other half, which escaped this severe defect were used for the analysis of more subtle alterations. In these escapers, whole-transcriptome analysis demonstrated early disease signatures, affecting pathways related to development, corticogenesis and synapses. Complementary metabolomics analysis confirmed decreased levels of cerebral tissue metabolites, some particularly relevant for synapse formation and neurotransmission, such as gamma-amino butyric acid (GABA). Our data suggests that a mutation in CLN3 severely affects brain development. Furthermore, before disease onset, disease -associated neurodevelopmental changes, particular concerning synapse formation and function, occur.

Screening for calcium channel modulators in CLN3 siRNA knock down SH-SY5Y neuroblastoma cells reveals a significant decrease of intracellular calcium levels by selected L-type calcium channel blockers

Background Defects of the CLN3 gene on chromosome 16p12.1 lead to the juvenile form of neuronal ceroid-lipofuscinosis (JNCL, Batten Disease), the most common recessive inherited neurodegenerative disorder in children. Dysregulation of intracellular calcium homeostasis in the absence of a functional CLN3 protein (CLN3P, Battenin) has been linked to synaptic dysfunction and accelerated apoptosis in vulnerable neuronal cells. Prolonged increase of intracellular calcium concentration is considered to be a significant trigger for neuronal apoptosis and cellular loss in JNCL. Methods We examined the potential effect of 41 different calcium channel modulators on intracellular calcium concentration in CLN3 siRNA knock down SH-SY5Y neuroblastoma cells. Results Six drugs belonging to the group of voltage dependent L-type channel blockers show significant lowering of the increased intracellular calcium levels in CLN3 siRNA knock down cells. Conclusions Our studies provide important new data suggesting possible beneficial effects of the tested drugs on calcium flux regulated pathways in neuronal cell death. Therapeutic intervention in this untreatable disease will likely require drugs that cross the blood–brain barrier as did all of the positively screened drugs in this study. General significance Better comprehension of the mechanism of neurodegeneration in rare recessive disorders, such as neuronal ceroid-lipofuscinoses, is likely to help to better understand mechanisms involved in more complex genetic neurodegenerative conditions, such as those associated with aging.

Nitric oxide signaling is disrupted in the yeast model for Batten disease.

The juvenile form of neuronal ceroid lipofuscinoses (JNCLs), or Batten disease, results from mutations in the CLN3 gene, and it is characterized by the accumulation of lipopigments in the lysosomes of several cell types and by extensive neuronal death. We report that the yeast model for JNCL (btn1-Delta) that lacks BTN1, the homologue to human CLN3, has increased resistance to menadione-generated oxidative stress. Expression of human CLN3 complemented the btn1-Delta phenotype, and equivalent Btn1p/Cln3 mutations correlated with JNCL severity. We show that the previously reported decreased levels of L-arginine in btn1-Delta limit the synthesis of nitric oxide (.NO) in both physiological and oxidative stress conditions. This defect in .NO synthesis seems to suppress the signaling required for yeast menadione-induced apoptosis, thus explaining btn1-Delta phenotype of increased resistance. We propose that in JNCL, a limited capacity to synthesize .NO directly caused by the absence of Cln3 function may contribute to the pathology of the disease.

IgG entry and deposition are components of the neuroimmune response in Batten disease

Patients and a mouse model of Batten disease, the juvenile form of neuronal ceroid lipofuscinosis (JNCL), raise autoantibodies against GAD65 and other brain-directed antigens. Here we investigate the adaptive component of the neuroimmune response. Cln3(-/-) mice have autoantibodies to GAD65 in their cerebrospinal fluid and elevated levels of brain bound immunoglobulin G (IgG). IgG deposition was found within human JNCL autopsy material, a feature that became more evident with increased age in Cln3(-/-) mice. The lymphocyte infiltration present in human and murine JNCL occurred late in disease progression, and was not capable of central/intrathecal IgG production. In contrast, we found evidence for an early systemic immune dysregulation in Cln3(-/-) mice. In addition evidence for a size-selective breach in the blood-brain barrier integrity in these mice suggests that systemically produced autoantibodies can access the JNCL central nervous system and contribute to a progressive inflammatory response.

Protracted juvenile neuronal ceroid lipofuscinosis—An autopsy report and immunohistochemical analysis

The juvenile form of neuronal ceroid lipofuscinosis (JNCL) is caused by mutations in the CLN3 gene, and is characterized by progressive loss of vision and development of motor deficits. A few patients exhibit a more protracted clinical course and are diagnosed with protracted JNCL (PJNCL). Here, we report the autopsy in a case of PJNCL in a 55-year-old male and immunohistochemical examination of the involvement of oxidative stress and glutamate excitotoxicity in neurodegeneration. The patient was born to consanguineous parents (I assume this means that the parents were related. If not, then the sentence will need to be changed again.) and had brothers with similar neurological disease. He showed mental retardation and visual impairment in the first decade which gradually developed along with motor dysfunction for over 40 years. At autopsy, the cerebral pyramidal neurons revealed deposition of lipopigments, which demonstrated ‘finger print’ and curvilinear profiles on electron microscopy. He also exhibited cerebellar cortical atrophy, fibrillary gliosis in the white matter, and rarefication in the globus pallidus. Immunohistochemically, the number of neurons immunoreactive for advanced glycation end product was elevated in the cerebellar cortex and midbrain. Immunoreactivity for excitatory amino acid transporter 1 was reduced in the cerebellar dentate and inferior olivary nuclei. These findings suggest that oxidative damage to proteins and disturbed glutamate transport can be involved in PJNCL.

Mechanisms of neurodegeneration in neuronal ceroid-lipofuscinoses

Neuronal ceroid-lipofuscinoses (NCL) are a group of neurodegenerative diseases and autosomal recessive lysosomal storage disorders. We examined the involvement of cell death, oxidative stress, and glutamate excitotoxicity using immunohistochemistry against Bcl-2, Bcl-x, oxidative products to proteins, lipids and DNA, calcium-binding proteins (calbindin-D28K, parvalbumin, calretinin), and glial glutamate transporters (excitatory amino acid transporters 1 and 2), in addition to terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) in the brains from three cases of late infantile form of NCL (LINCL) and one case of juvenile form of NCL (JNCL) to investigate the neurodegenerative mechanisms. In the cerebral and cerebellar cortex, all of three LINCL cases demonstrated neurons with TUNEL-immunoreactive nuclei, whereas the JNCL case did not show TUNEL-immunoreactive nuclei. The coexistence of the nuclear TUNEL-immunoreactivity nuclei and cytoplasmic deposition of 4-hydroxy-2-nonenal-modified protein in the frontal cortex and hypoglossal nucleus may suggest a possible interrelationship between DNA fragmentation and lipid oxidation in LINCL. Additionally, glycoxidation of protein and oxidative stress to DNA seemed to be involved in the cerebellar and cerebral degeneration, respectively. Interneurons immunoreactive for calbindin-D28K and parvalbumin were severely reduced in the cerebral cortex, whereas those for calretinin were comparatively well preserved in LINCL, indicating the possibility of altered GABAergic system. The disturbance of expression of glial glutamate transporters seemed to be heterogeneous and mild. These findings suggest the possibility of new treatments for neurodegeneration in LINCL using antioxidative agents and/or GABAergic medications.

CLN3 Protein Regulates Lysosomal pH and Alters Intracellular Processing of Alzheimer's Amyloid-β Protein Precursor and Cathepsin D in Human Cells

Maintenance of the appropriate pH in the intracellular vacuolar compartments is essential for normal cell function. Here, we report that CLN3 protein, which is associated with the juvenile form of neuronal ceroid lipofuscinosis (JNCL), participates in lysosomal pH homeostasis in human cells. We show that CLN3 protein increases lysosomal pH in cultured human embryonal kidney cells, whereas inhibition of CLN3 protein synthesis by antisense approach acidifies lysosomal compartments. These changes in lysosomal pH are sufficient to exert a significant biological effect and modify intracellular processing of amyloid-β protein precursor and cathepsin D, model proteins whose metabolism is influenced by the pH of acidic organelles. Mutant CLN3 protein (R334C) that is associated with the classical JNCL phenotype was devoid of biological activities of wild-type CLN3 protein. These data suggest that the pathogenesis of juvenile neuronal ceroid lipofuscinosis is associated with altered acidification of lysosomal compartments. Furthermore, our study indicates that CLN3 protein affects metabolism of proteins essential for cell functions, such as amyloid-β protein precursor, implicated in Alzheimer's disease pathogenesis.

Polyunsaturated fatty acids reverse the lysosomal storage and accumulation of subunit 9 of mitochondrial F1F0-ATP synthase in cultured lymphoblasts from patients with Batten disease.

Batten disease (McKusick 204200), the juvenile form of neuronal ceroid lipofuscinosis (JNCL) is a severe autosomal recessive neurodegenerative disease characterized by progressive loss of vision, seizures, degenerating motor function and ultimately death, usually in the third decade of life (Kohlschutter et al 1993). The gene CLN3, for JNCL has recently been isolated. The protein encoded by the CLN3 gene is a highly lipophilic protein of 438 amino acid residues and molecular mass 48kDa (Lerner et al 1995). The function of the protein is unknown. The characteristic storage material has been shown to be proteolipid in nature, with subunit 9 of mitochondrial F 1 F 0 -ATP synthase being a major component (Hall et al 1991; Palmer et al 1992). We recently reported upon the reversibility of induced lysosomal storage in JNCL lymphoblast cell lines when incubated with gentamycin (an agent known to damage mitochondria) and a defined mixture of polyunsaturated fatty acids (Boriack et al 1995). In the experiments reported here we demonstrate that ATP synthase subunit 9 storage can be induced in this in vitro model and that this can be reversed by polyunsaturated fatty acids. This provides a good system for testing therapeutic regimens for this disease.

Lectin histochemistry in brains with juvenile form of neuronal ceroid-lipofuscinosis (Batten disease)

Defective utilization of dolichols in the synthesis of glycoprotein leads to an accumulation of the storage, pigment "ceroid" lipofuscin, containing high-mannose-type glycoconjugates, in brains affected by neuronal ceroid-lipofuscinoses (NCL). We have employed lectin histochemistry to study the distribution of such compounds and the composition of other glycoconjugates in brains of patients with a juvenile form of the disease (JNCL). Concanavalin A detected the high-mannose glycoconjugates in all neurons of brains with JNCL, in lipofuscin-containing neurons of aging brains and in some neurons of age-matched control brains. Three other lectins (soybean agglutinin, Peanut agglutinin and Ulex europaeus agglutinin-I) recognized sugar moieties in neurons containing lipofuscin in patients only with JNCL and not in age-matched or aging brains. The results led to the conclusion, that the binding pattern of these three lectins may differentiate between storage materials of NCL and aging brains.

β-Protein immunoreactivity in brains of patients with neuronal ceroid lipofuscinosis: Ultrastructural and biochemical demonstration

The storage pigment in neuronal ceroid lipofuscinoses (NCL) has a close similarity to age pigment lipofuscin. We studied immunoreactivity of isolated neuronal pigments from the juvenile form of NCL and aging control, using monoclonal antibodies (mAbs) against amyloid β-protein. Ultrastructural localization of the immunoreactivity demonstrated that in NCL the epitopes are distributed mainly in curvilinear multilamellar arrays of the storage pigments and less in fingerprint profiles, while in aging control they are more homogeneously distributed on age pigment lipofuscin. The different distribution of the epitopes may reflect some catabolic as well as morphologic differences in lysosomes. A unique 31-kDa polypeptide detected on Western blots in NCL possibly derives from the same precursor, amyloid β-protein precursor (ABPP). ABPP processing may be aberrant in NCL brains, and this can be detected as a 31-kDa polypeptide reactive with the mAbs.

Topographic heterogeneity of amyloid B-protein epitopes in brains with various forms of neuronal ceroid lipofuscinoses suggesting defective processing of amyloid precursor protein

To verify our hypothesis of defective protease inhibitor domains that are encoded by abnormal processing of amyloid precursor protein (APP) in brains of patients with neuronal ceroid lipofuscinoses (NCL), immunohistochemical and cytochemical studies were performed with monoclonal antibodies (mAbs) directed against various domains of APP. For the studies, 22 autopsy brains were used: 12 with different forms of NCL, and 10 control brains. The staining procedure for the avidin-biotin complex (ABC) technique and the postembedding gold-labelled procedure for electron microscopy (EM) were employed. Of all mAbs used for the study, only mAbs generated against amyloid B-protein bound to neural tissue were affected with NCL. The strongest immunostaining of neurons and of some reactive glial cells was found in brains with the juvenile form of NCL. Only in the infantile form of the disease were some neurons overloaded with storage material weakly immunoreactive. In brains of patients with the adult form of NCL, immunoreactivity was found in affected neurons and in extracellularly deposited material of senile plaques. The results of EM study showed that the immunoreactivity was restricted to lysosomal cytosomes in neural tissue with any form of NCL selectively localized on the curvilinear and fingerprint proteinaceous component of ceroid lipofuscin. Studies performed on control aging brains and Alzheimer's disease (AD) brains confirmed previous observations of immunoreactivity being found diffusely in the protein component of some neurons containing lipopigment.(ABSTRACT TRUNCATED AT 250 WORDS)

Kufs' disease: a critical reappraisal.

A review of 118 cases published as Kufs' disease revealed only 50 cases, including 2 patients described herein, that fulfilled our criteria for this diagnosis. Of the other 68 cases, 16 had inadequate data for analysis, 21 had evidence of a storage disease other than Kufs' disease, 10 did not have clear evidence of any neuronal storage, and 21 had atypical clinical features considered outside the spectrum of Kufs' disease. The 50 cases accepted as Kufs' disease comprised two clinical phenotypes; progressive myoclonus epilepsy (Type A) and dementia with motor disturbances (Type B). Marked photosensitivity was a striking feature of some Type A cases, and facial dyskinesias were common amongst Type B patients. Onset was typically at around the age of 30 years. A few cases began in adolescence; these differ from the protracted juvenile form of neuronal ceroid-lipofuscinosis by the absence of visual failure. Demonstration of fingerprint profiles or granular osmiophilic deposits by electron microscopy is mandatory for definitive diagnosis. Urinary sediment dolichol levels were markedly elevated in our 2 cases. This biochemical finding confirms the relationship of Kufs' disease to the early forms of neuronal ceroid-lipofuscinosis and is consistent with our hypothesis that these diseases are due to defects in the intracellular processing of lysosomal and related membranes.

Urinary sediment dolichols in the diagnosis of neuronal ceroid-lipofuscinosis.

Long-chain polyisoprenol alcohol (dolichols) levels are significantly increased in the urinary sediment of patients with infantile, late-infantile, and juvenile forms of neuronal ceroid-lipofuscinosis (NCL). The values in obligate heterozygotes for these diseases are similar to those in patients with other neurological diseases and in healthy controls. Antioxidant treatment of patients with juvenile NCL has no effect on dolichol values. The rate of false-negative results is 13.9% in infantile, 7.5% in late-infantile, and 15.0% in juvenile NCL. False-positive results were found in 8.2 to 14.3% of patients with other neurological diseases and in 15.4% of healthy controls. The test is of considerable value in the diagnosis of NCL and in decisions on whether to perform a biopsy. It is not useful in the screening of random samples, however.

Leukocyte docosahexaenoic acid in juvenile form of ceroid-lipofuscinosis.

Leukocyte glycerolipid fatty acid compositions were determined in four patients of juvenile form of neuronal ceroid-lipofuscinosis (Battens disease), their parents and age-matched controls. A 54 to 93% reduction in docosahexaenoic acid (22:6, n-3) was noted in patient leukocytes. The parents also showed reduction in docosahexaenoic acid content, but to a lesser degree. The levels of linolenic family (n-6) polyunsaturated fatty acids were unchanged suggesting that the metabolic defect in this disease is specific to the linolenic family (n-3) of fatty acids.

Human leukocyte peroxidase: Activity of a soluble and membrane-bound enzyme form in normal persons and patients with neuronal ceroid-lipofuscinosis

Human leukocytes contain a peroxidase fraction soluble in 0.1 M phosphate buffer and an insoluble peroxidase component with 10–15 times higher specific activity which can be extracted by 0.1 M phosphate buffer + 0.2% Triton X-100 + 0.2% sodium taurocholate and sonication. Both enzyme components have been estimated spectrophotometrically with the substrate hydrogen peroxide (final concentration 1 m M) and the hydrogen donor p-phenylenediamine (final concentration 28–55 m M) within the first 60 sec. The pH-optimum of the soluble and membrane-bound leukocyte peroxidase is at pH 7.0 with a second smaller peak at pH 5.5. Using 0.2 M boric acid 0.05 M sodium borate buffer (pH 7.6) instead of phosphate buffer a 40%–50% increase of enzyme activity can be achieved. In two patients with the juvenile form of neuronal ceroid-lipofuscinosis (type Spielmeyer-Vogt) the activity of soluble leukocyte peroxidase was considerably reduced, in one patient with the late infantile form (type Jansky-Bielschowsky) the activity was just below the normal range, and in two patients with the adult form (type Kuf) activity was normal. In all patients the activity of membrane-bound leukocyte peroxidase was not significantly altered. Only one of four heterozygotes for the juvenile type had deficient values of the soluble enzyme. The variability of the peroxidase findings in patients and carriers with neuronal ceroid-lipofuscinosis makes it uncertain whether this represents the primary enzymic defect.

Human saliva peroxidase: microanalytical isoelectric fractionation and properties in normal persons and in cases with neuronal ceroid-lipofuscinosis

Human saliva contains a high peroxidase activity that can be estimated spectrophotometrically with the hydrogen donor p-phenylenediamine and the substrate hydrogen peroxide from 20 mul of material. The pH optimum of the enzyme with citrate-phosphate buffer is 5.5. After microanalytical isoelectric fractionation 3 main isoenzyme components at pI 8.6, 6.5 and 4.3, and a number of isoenzyme subfractions at pI 9.5, 7.3 and 3.8 are detectable. In 3 patients with the juvenile form of neuronal ceroid-lipofuscinosis (type Spielmeyer-Vogt), in which a deficiency of leukocyte peroxidase had been reported by other authors, both the total activity of saliva peroxidase and the activity of individual isoenzymes were found to be within normal limits. These findings are not consistent with a generalized peroxidase deficiency in this disease.