Juvenile NCL Research Articles

Combined Anti-inflammatory and Neuroprotective Treatments Have the Potential to Impact Disease Phenotypes in Cln3 -/- Mice.

Batten disease, or juvenile NCL, is a fatal neurodegenerative disorder that occurs due to mutations in the CLN3 gene. Because the function of CLN3 remains unclear, experimental therapies for JNCL have largely concentrated upon the targeting of downstream pathomechanisms. Neuron loss is preceded by localized glial activation, and in this proof-of-concept study we have investigated whether targeting this innate immune response with ibuprofen in combination with the neuroprotective agent lamotrigine improves the previously documented beneficial effects of immunosuppressants alone. Drugs were administered daily to symptomatic Cln3−/− mice over a 3 month period, starting at 6 months of age, and their impact was assessed using both behavioral and neuropathological outcome measures. During the treatment period, the combination of ibuprofen and lamotrigine significantly improved the performance of Cln3−/− mice on the vertical pole test, slowing the disease-associated decline, but had less of an impact upon their rotarod performance. There were also moderate and regionally dependent effects upon astrocyte activation that were most pronounced for ibuprofen alone, but there was no overt effect upon microglial activation. Administering such treatments for longer periods will enable testing for any impact upon the neuron loss that occurs later in disease progression. Given the partial efficacy of these treatments, it will be important to test further drugs of this type in order to find more effective combinations.

Longitudinal MRI brain volumetry in CLN3 disease: Introduction of a new sensitive and objective marker for the measurement of clinical disease outcome

Objective: A precise description of disease progression in CLN3 disease is urgently needed in order to establish an evaluation tool for future experimental treatments. This is especially challenging as CLN3 disease may present with variable clinical disease progression despite most patients harboring the same 1 kb deletion in the CLN3 gene. Gray matter atrophy due to neuronal loss is a striking feature of patients with juvenile CLN3 disease. Therefore, to develop a quantitative marker to measure brain volume outcome, we analyzed the longitudinal volumetric development of gray matter (GM), white matter (WM) and lateral ventricles and correlated those with the clinical course and with patient age. Methods: 122 MRIs of 35 patients (14 male; 21 female; age 15.3±4.8 years) with genetically confirmed CLN3 disease were performed on 1.5T scanners. A 3D T1-weighted MP- RAGE sequence was acquired. Volumetric segmentation of the brain was performed using Freesurfer Software suite. The clinical severity was assessed by the Hamburg juvenile NCL score, a disease-specific scoring system. Results: The volumes of cortical GM, cerebellar GM and WM, basal ganglia + thalami and hippocampus significantly (all P<0.0001) decreased with age, while the lateral ventricle volume increased (P<0.0001). Supratentorial WM volume correlated much poorer with age than the other observed regions (R=0.21, P=0.0094). Decrease of cortical gray matter volume showed the most uniform decrease with the strongest correlation with age (R=0.814, P<0.0001). CLN3 patients lost on average 4.6% (± 0.2 %) of cortical GM per year. In addition, a strong correlation with the Hamburg JNCL Score existed for the cortical GM (R=0.823, P=<0.0001). Conclusion: Cortical GM volume decline seems to be the most sensitive parameter for assessment of disease progression and represents a potential objective and sensitive outcome measure for evaluation of future therapies.

Clinicopathological study of neuronal ceroid lipofuscinosis in Egyptian patients, role of gingival biopsy

Neuronal ceroid lipofuscinosis (Batten disease, NCL) represents a group of common childhood neurodegenerative diseases with a shared feature of deposition of abnormal metabolic products in neurons and other tissues, including peripheral blood lymphocytes. Clinical findings and pathological features of 12 Egyptian patients affected with NCL are reviewed. The patient group included three children affected with the late-infantile form of NCL, five patients affected with the juvenile form NCL, and four infantile cases. Ultrastructural examination of gingival biopsies revealed the characteristic deposits in fibroblasts and the endothelial cells. In spite of recent advances in immunohistochemical identification of biochemical markers, the ultrastructural identification of lipofuscinic pigments in an easily accessible tissue like the gingiva or skin remains the gold standard to identify NCL, together with clinical aspects.

The neuronal ceroid‐lipofuscinoses

The neuronal ceroid-lipofuscinoses (NCL's, Batten disease) represent a group of severe neurodegenerative diseases, which mostly present in childhood. The phenotypes are similar and include visual loss, seizures, loss of motor and cognitive function, and early death. At autopsy, there is massive neuronal loss with characteristic storage in remaining neurons. Neurons appear to die because of increased rates of apoptosis and altered autophagy. Ten genes have been identified so far that result in an NCL (CLN1-10). The most common forms are CLN1, CLN2, and CLN3, which were previously known as Infantile, Late-Infantile, and Juvenile NCL's, respectively. CLN1 and CLN2 result from mutations in soluble lysosomal enzymes palmitoyl-protein thioesterase (PPT) and tripeptidyl peptidase 1 (TPP1), which can be measured in white blood cells for clinical diagnosis. Molecular diagnostic testing is routinely available for CLN1, CLN2, and CLN3. Sequencing of other NCL genes may be required to establish a diagnosis when the common forms are ruled out. The pathogenesis of NCL neuronal loss resulting from loss of function of any of the NCL gene products remains unknown and no treatment options are presently available.

A phase II, randomized, controlled trial of Mycophenolate Mofetil in children with juvenile NCL (JNCL)

A phase II, randomized, controlled trial of Mycophenolate Mofetil in children with juvenile NCL (JNCL)

Moving towards therapies for Juvenile Batten disease?

The neuronal ceroid lipofuscinoses (NCLs or Batten disease) are a group of at least nine autosomal recessively inherited monogenetic storage disorders. Because there are no effective therapies available, all forms of NCL invariably prove fatal after a prolonged period of disability. Indeed, for the forms of NCL that are the result of mutations in transmembrane proteins, the therapeutic outlook remains uniformly bleak. This includes juvenile NCL (JNCL); the most prevalent form of Batten disease that is the result of mutations in the CLN3 gene. Characterizing Cln3 deficient mice is now revealing important clues about the pathogenesis of JNCL. This includes evidence for elevated levels of glutamate within the JNCL CNS and cell type selective sensitivity to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type glutamate receptor overactivation. These findings raise the possibility that AMPA-receptor blockade may potentially be beneficial in JNCL. This possibility has now begun to be tested in Cln3 mutant mice using a single intraperitoneal injection of the non-competitive AMPA antagonist EGIS-8332 by Kovács and Pearce [Kovács, A.D., Pearce, D.A., 2008. Attenuation of AMPA receptor activity improves motor skills in a mouse model of juvenile Batten disease. Exp. Neurol. 209, 288–291.]. Although a positive effect of upon motor coordination deficits in this mouse model of JNCL is reported in this acute study, it remains unclear whether EGIS-8332 provides any lasting benefit or effects upon other aspects of their disease phenotype. Although supplying the first evidence for any form of improvement in a disease-relevant phenotype in Cln3 mutant mice, more detailed studies will be needed to determine whether these preliminary findings will translate into a successful therapy for either murine or human JNCL.

Thalami and corona radiata in juvenile NCL (CLN3): a voxel‐based morphometric study

Juvenile neuronal ceroid lipofuscinosis (CLN3) is characterized by progressive cerebral atrophy. The purpose of this study was to re-evaluate the three-dimensional magnetic resonance (3D-MR) images of patients with CLN3 using voxel-based morphometry (VBM) to achieve a detailed understanding of the affected brain regions. T1-weighted 3D-MR images of 15 patients with CLN3 (age range: 12-25 years, mean age 17.6 years) and 15 age- and sex-matched controls were analyzed using VBM. VBM showed strikingly focal alterations in the brains of CLN3 patients: the gray matter volume was significantly decreased in the dorsomedial part of the thalami of CLN3 patients. In addition, the volume of the white matter was significantly decreased in the corona radiata, containing cortical efferents and afferents in the transition between the internal capsule and the subcortical white matter. These data suggest that the dorsomedial part of the thalamus and the corona radiata may have a central, previously unrecognized role in the pathogenesis of CLN3.

Progressive myoclonic epilepsy: A clinical, electrophysiological and pathological study from South India

Progressive myoclonic epilepsy (PME) is a syndrome complex encompassing different diagnostic entities and often cause problems in diagnosis. We describe the clinical, electrophysiological and pathological features of 97 patients with the diagnosis of PME evaluated over 25 years. Case records of confirmed patients of Neuronal ceroid lipofuscinosis (NCL = 40), Lafora body disease (LBD = 38), Myoclonic epilepsy with ragged red fibers (MERRF = 10), and probable Unverricht–Lundberg disease (ULD = 9) were reviewed. The mean age at onset in patients with NCL ( n = 40) was 5.9 ± 9.1 years (M:F:: 28:12). Subtypes of NCL were: late infantile ( n = 19), infantile ( n = 8), juvenile ( n = 11) and adult ( n = 2) NCL. EEG ( n = 37) showed varying degree of diffuse slowing of background activity in 94.6% and epileptiform discharges in 81.1% of patients. Slow frequency photic stimulation evoked photo-convulsive response in 5 patients only. Giant SSEP was demonstrated in 7 and VEP study revealed a prolonged P100 (2) and absent waveform (7). Electrophysiological features of neuropathy were present in 3 patients. Presence of PAS and Luxol Fast Blue (LFB) positive, auto fluorescent (AF) ceroid material in brain tissue ( n = 12) and electron microscopy of brain ( n = 5), skin ( n = 28) and muscle ( n = 1) samples showing curvilinear and lamellar bodies established the diagnosis. Patients of LBD (mean age of onset at 14.4 ± 3.9 years, M:F:: 24:14) with triad of PME symptoms were evaluated. EEG ( n = 37) showed variable slowing of background activity in 94.6% and epileptiform discharges in 97.4%. Photosensitivity with fast frequency was observed only in 5 patients. CT ( n = 32) and MRI ( n = 4) revealed diffuse cortical atrophy. Giant SSEP was demonstrated in 24 patients of LBD while VEP study revealed a prolonged P100 (4) and absent waveform (8). Electrophysiological features of neuropathy were present in one patient. Diagnosis was established by the presence of PAS positive diastase resistant, Lugol's Iodine labeled inclusions in sweat glands of axillary skin ( n = 35), brain ( n = 2) and liver ( n = 1). Ten patients with MERRF (mean age at onset: 14.6 ± 5.8 years; M: F:: 3:2) had triad of PME symptoms. Muscle biopsy revealed oxidative reaction product and classical ragged red fibers. In nine patients of PME without cognitive decline, probable diagnosis of ULD (mean age at onset: 13.8 ± 9.5 years) was considered after biopsy of skin and/or muscle excluded other forms of PMEs. Neuronal ceroid lipofuscinosis and Lafora body diseases were the common causes of PME in the series from south India. This is one of the largest series from the Indian subcontinent to the best of our knowledge. Photosensitivity is notably less common in LBD/NCL in this series distinctly different from those reported in the literature. Further exploration is required to determine whether different genotype is responsible. Morphological changes were helpful in diagnosis and could be confirmed by biopsy of peripheral tissues like skin and muscle in majority (60%). Electron microscopy was helpful in the diagnosis NCL and MERRF.

Heterogeneity of late-infantile neuronal ceroid lipofuscinosis

Late-infantile neuronal ceroid lipofuscinosis (LINCL), an autosomal recessively inherited lysosomal storage disorder characterized by autofluorescent inclusions and rapid progression of neurodegeneration, is due to CLN2 gene mutations. However, CLN2 mutation analysis has failed to identify some clinically diagnosed "late-infantile" NCL cases. This study was conducted to further characterize genetic heterogeneity in families affected by LINCL. DNA mutations in the CLN1, CLN2, and CLN3 genes that underlie INCL (infantile NCL), LINCL, and JNCL (juvenile NCL), respectively, were studied with molecular analyses. A total of 252 families affected by childhood NCL were studied. Of 109 families clinically diagnosed as having LINCL, 3 were determined to have either INCL or JNCL by identification of mutation(s) in CLN1 or CLN3. Six families diagnosed initially as having JNCL were found to have LINCL based on the finding of mutations in the CLN2 gene. In addition, several novel mutations were identified. Clinical and genetic heterogeneity of LINCL was demonstrated in nine LINCL families studied.

Mutational Analysis of the Defective Protease in Classic Late-Infantile Neuronal Ceroid Lipofuscinosis, a Neurodegenerative Lysosomal Storage Disorder

The late-infantile form of neuronal ceroid lipofuscinosis (LINCL) is a progressive and ultimately fatal neurodegenerative disease of childhood. The defective gene in this hereditary disorder, CLN2, encodes a recently identified lysosomal pepstatin-insensitive acid protease. To better understand the molecular pathology of LINCL, we conducted a genetic survey of CLN2 in 74 LINCL families. In 14 patients, CLN2 protease activities were normal and no mutations were identified, suggesting other forms of NCL. Both pathogenic alleles were identified in 57 of the other 60 LINCL families studied. In total, 24 mutations were associated with LINCL, comprising six splice-junction mutations, 11 missense mutations, 3 nonsense mutations, 3 small deletions, and 1 single-nucleotide insertion. Two mutations were particularly common: an intronic G-->C transversion in the invariant AG of a 3' splice junction, found in 38 of 115 alleles, and a C-->T transition in 32 of 115 alleles, which prematurely terminates translation at amino acid 208 of 563. An Arg-->His substitution was identified, which was associated with a late age at onset and protracted clinical phenotype, in a number of other patients originally diagnosed with juvenile NCL.

A Murine Model for Juvenile NCL: Gene Targeting of MouseCln3

JNCL is a neurodegenerative disease of childhood caused by mutations in theCLN3gene. A mouse model for JNCL was created by disrupting exons 1–6 ofCln3,resulting in a null allele.Cln3null 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 ofCln3are deleted, mimicking the common mutation in JNCL patients.

A Rapid Fluorogenic Palmitoyl-Protein Thioesterase Assay: Pre- and Postnatal Diagnosis of INCL

A deficiency of palmitoyl-protein thioesterase (PPT) was recently shown to be the primary defect in infantile neuronal ceroid lipofuscinosis (INCL). The available enzyme assays are complicated and impractical for diagnostic use. We have recently developed a new, fluorometric assay for PPT based on the sensitive fluorochrome 4-methylumbelliferone, requiring an overnight incubation to measure PPT. Now we have synthesized an analogue of this substrate which allows PPT determinations in 1 h. This improved PPT assay is simple, sensitive, and robust and will facilitate the definition of the full clinical spectrum associated with a deficiency of PPT. PPT activity was readily detectable in fibroblasts, leukocytes, amniotic fluid cells, chorionic villi, plasma, and cerebrospinal fluid from controls. PPT activity was profoundly deficient in these tissues and fluids from INCL patients. Similarly, a deficiency of PPT activity was demonstrated in patients with the variant juvenile NCL with GROD. These results show the feasibility of rapid pre- and postnatal diagnosis of INCL and its variants.

Translocation 10;18 in a patient with juvenile neuronal ceroid‐lipofuscinosis (Batten disease)

We report the first observation of a chromosome abnormality in a patient with typical juvenile ceroid-lipofuscinosis (NCL), who was found to have an apparently balanced translocation between chromosomes 10 and 18 [t(10;18)(q22.1;q21.1)]. Since juvenile NCL was previously mapped to 16p12, this report raises the possibility of heterogeneity in this form of NCL.

Somatosensory evoked potentials with high cortical amplitudes: clinical data in 31 children.

Between 1989 and 1993, somatosensory evoked potentials (SEP) were recorded as part of the diagnostic work-up in 282 children with different neurologic disorders. In thirty-one children with N20/P25/N35 amplitudes were enhanced compared to our control group (highest amplitude 14.1 microV). Four children had amplitudes > 40 microV ("giant"), fifteen between 20-39.9 microV ("elevated") and twelve between 14-19.9 microV ("borderline"). Enhanced cortical SEPs were seen in all patients with neuronal ceroid lipofuscinosis (5 late-infantile NCL > 20 microV, 1 juvenile NCL 14.7 microV). In addition, five of six NCL children showed bilaterally prolonged cervico-cortical conduction times, otherwise only seen in a 4-month-old child following hypoxia. "Borderline" and "elevated" SEPs occurred in patients with heterogeneous neurologic disorders. Follow-up recordings showed inconsistent results: seven children had amplitudes > 14 microV in all recordings, six only at the first examination, and six only at follow-up. In six children with hemiparesis enhanced SEPs were recorded over both (n = 2) or only over the unaffected hemisphere (n = 4). Myoclonic seizures were observed only in five children with NCL. Similar to other SEP parameters, enhanced amplitudes are an unspecific indicator of an ongoing neurologic disorder. However, in neuronal ceroid lipofuscinosis, enhanced SEP amplitudes may be a useful diagnostic criterium.

59 LOW PLASMA MEMBRANE FLUIDITY IN JUVENILE NEURONAL CEROID LIPOFUSCINOSIS (NCL) LYMPHOCYTES

NCL is a group of genetic lipid storage disorders of unknown etiology which share the intracellular accumulation of a lipoperoxide containing material, possibly membrane breakdown products. The storage could be due to a yet undefined degrading enzyme deficiency or to an alteration of the lipid substrate. We investigated the plasma membranes of isolated intact lymphocytes from 7 patients with juvenile NCL and from 30 control children with steady-state fluorescence polarization. The membrane fluidity was determined by the anisotropy measured. Fluorescent probes used were diphenylhexatriene (DPH), a cationic analogue (TMA-DPH), and a set of n-(9-anthroyloxy) fatty acids (nAS; n = fatty acid carbon numbers 6, 7, 9, 12, and 16, respectively). Membrane fluidity was significantly decreased in NCL vs. control lymphocytes when measured with the labels TMA-DPH (P<.005), 7AS (p<.01) and 9AS (p<.01). Fluidity was less significantly decreased when measured with the other probes. The results indicate a decreased fluidity of the outer membrane leaflet in NCL lymphocytes. Explanations of these changes include lower fatty acid unsaturation or raised levels of cholesterol, sphingomyelin, or protein. Further studies should focus on the composition of intact NCL membranes.

Ultrastructural studies on the retinal pigment epithelium in the neuronal ceroid-lipofuscinoses

In human neuronal ceroid-lipofuscinosis, the retina is affected twofold: (1) There is ubiquitous accumulation of lipopigments in various cell types of the retina that are similar to lysosomal accretion in many other lysosomal disorders. (2) There is loss of photoreceptors followed by atrophy of the remaining inner part of the retina. The retinal pigment epithelium may also be affected by showing atrophy and proliferation and lack of phagocytosed photoreceptor outer segments.Curvilinear profiles, characteristic of late-infantile NCL, may accumulate in RPE cells, either within lipopigments or combined with melanin pigment, while fingerprint profiles, typical of juvenile NCL are difficult to identify in Rpe cells of juvenile NCL. Canine NCL is an established spontaneous model for juvenile human NCL but only statement 1 not statement 2 as mentioned above, is correct for diseased English setters. The RPE in canine NCL contains phagocytosed photoreceptor cells but not disease specific lipopigments, contrary to ...

Fingerprint profiles in lymphocytic vacuoles of mucopolysaccharidoses I-H, II, III-A, and III-B.

Fingerprint (FP) profiles in vacuolated lymphocytes of mucopolysaccharidoses I-H, II, III-A, and III-B are a numerically rare, but possibly consistent finding as they have not been seen in vacuolated lymphocytes of other non-neuronal lipofuscinosis (NCL) lysosomal diseases. Their nosologic significance is not clear, but they may be as non-specific as tubular inclusions in lymphocytes and they are identical to those FP profiles seen in juvenile NCL.