Lack of Cathepsin D in the Central Nervous System Results in Microglia and Astrocyte Activation and the Accumulation of Proteinopathy-related Proteins

Neuronal ceroid lipofuscinosis is one of many neurodegenerative storage diseases characterized by excessive accumulation of lipofuscins. CLN10 disease, an early infantile neuronal ceroid lipofuscinosis, is associated with a gene that encodes cathepsin D (CtsD), one of the major lysosomal proteases. Whole body CtsD-knockout mice show neurodegenerative phenotypes with the accumulation of lipofuscins in the brain and also show defects in other tissues including intestinal necrosis. To clarify the precise role of CtsD in the central nervous system (CNS), we generated a CNS-specific CtsD-knockout mouse (CtsD-CKO). CtsD-CKO mice were born normally but developed seizures and their growth stunted at around postnatal day 23±1. CtsD-CKO did not exhibit apparent intestinal symptoms as those observed in whole body knockout. Histologically, autofluorescent materials were detected in several areas of the CtsD-CKO mouse’s brain, including: thalamus, cerebral cortex, hippocampus, and cerebellum. Expression of ubiquitin and autophagy-associated proteins was also increased, suggesting that the autophagy-lysosome system was impaired. Microglia and astrocytes were activated in the CtsD-CKO thalamus, and Inducible nitric oxide synthase (iNOS), an inflammation marker, was increased in the microglia. Interestingly, deposits of proteinopathy-related proteins, phosphorylated α-synuclein and Tau protein, were also increased in the thalamus of CtsD-CKO infant mice. Considering these results, it is likely that the CtsD-CKO mouse is a useful mouse model to investigate the contribution of cathepsin D to the early phases of neurodegenerative diseases in relation to lipofuscins, proteinopathy-related proteins and activation of microglia and astrocytes.

Experience in Diagnosing Neuronal Ceroid Lipofuscinosis Type-2

Introduction: Developmental regression is always an alarming symptom in children as it is an early sign of some genetic disorders, one of which is neuronal ceroid lipofuscinosis (NCL). NCL is a group of rare neurodegenerative disorder caused by accumulation of intracellular ceroid lipofuscin. Since 2017 an enzyme replacement therapy (ERT) has been approved by Food and Drug Administration (FDA) for this disease. The symptoms of NCL could be managed by ERT if detected early, and the child could live normally.Case: We present a case of a 6-year-and-5-month-old boy with developmental regression, speech delay, recurrent seizure, and visual impairment, who was diagnosed with NCL type 2 after genetic testing. Compound heterozygous mutations in tripeptidyl-peptidase 1 (TPP1) gene was revealed, consistent with very low level of TPP1 enzyme in this patient.Discussion: NCL is a fatal disease which is often misdiagnosed in early stage. Diagnostic delay of NCL often occurs due to lack of awareness which often leads to premature death.Conclusion: Knowledge regarding the disease is important for early detection and to slow down the disease progression.

Towards Splicing Therapy for Lysosomal Storage Disorders: Methylxanthines and Luteolin Ameliorate Splicing Defects in Aspartylglucosaminuria and Classic Late Infantile Neuronal Ceroid Lipofuscinosis

Splicing defects caused by mutations in the consensus sequences at the borders of introns and exons are common in human diseases. Such defects frequently result in a complete loss of function of the protein in question. Therapy approaches based on antisense oligonucleotides for specific gene mutations have been developed in the past, but they are very expensive and require invasive, life-long administration. Thus, modulation of splicing by means of small molecules is of great interest for the therapy of genetic diseases resulting from splice-site mutations. Using minigene approaches and patient cells, we here show that methylxanthine derivatives and the food-derived flavonoid luteolin are able to enhance the correct splicing of the AGA mRNA with a splice-site mutation c.128-2A>G in aspartylglucosaminuria, and result in increased AGA enzyme activity in patient cells. Furthermore, we also show that one of the most common disease causing TPP1 gene variants in classic late infantile neuronal ceroid lipofuscinosis may also be amenable to splicing modulation using similar substances. Therefore, our data suggest that splice-modulation with small molecules may be a valid therapy option for lysosomal storage disorders.

Adult-Onset Neuronal Ceroid Lipofuscinosis in a Shikoku Inu

A two-year-and-eleven-month-old male Shikoku Inu was referred for evaluation of progressive gait abnormality that had begun three months prior. Neurological examination revealed ventral flexion of the neck, a wide-based stance in the hindlimb, wide excursions of the head from side to side, tremor in all four limbs, hypermetria in all four limbs, proprioceptive deficits in all four limbs, reduced patellar reflex in both hindlimbs, and postural vertical nystagmus. Later, behavioral and cognitive dysfunction, ataxia, and visual deficits slowly progressed. Magnetic resonance imaging revealed symmetrical progressive atrophy of the whole brain and cervical spinal cord. Bilateral retinal degeneration was observed, and both flush and flicker electroretinograms were bilaterally non-recordable at the age of five years and eight months, and the dog was euthanized. Histopathologically, faint-to-moderate deposition of light-brown pigments was frequently observed in the cytoplasm of neurons throughout the cerebrum, cerebellum, and nuclei of the brainstem. The pigments were positive for Luxol fast blue, periodic acid–Schiff, and Sudan black B, and exhibited autofluorescence. Electron microscopic examination revealed the accumulation of membranous material deposition in the neuronal cytoplasm. Small foci of pigment-containing macrophages were frequently observed around the capillary vessels. Based on these clinical and pathological findings, the animal was diagnosed with adult-onset neuronal ceroid lipofuscinosis.

Next-generation sequencing in childhood-onset epilepsies: Diagnostic yield and impact on neuronal ceroid lipofuscinosis type 2 (CLN2) disease diagnosis

Epilepsy is one of the most common childhood-onset neurological conditions with a genetic etiology. Genetic diagnosis provides potential for etiologically-based management and treatment. Existing research has focused on early-onset (<24 months) epilepsies; data regarding later-onset epilepsies is limited. The goal of this study was to determine the diagnostic yield of a clinically available epilepsy panel in a selected pediatric epilepsy cohort with epilepsy onset between 24–60 months of life and evaluate whether this approach decreases the age of diagnosis of neuronal ceroid lipofuscinosis type 2 (CLN2). Next-generation sequencing (NGS)-based epilepsy panels, including genes associated with epileptic encephalopathies and inborn errors of metabolism (IEMs) that present with epilepsy, were used. Copy-number variant (CNV) detection from NGS data was included. Variant interpretation was performed per American College of Medical Genetics and Genomics (ACMG) guidelines. Results are reported from 211 consecutive patients with the following inclusion criteria: 24–60 months of age at the time of enrollment, first unprovoked seizure at/after 24 months, and at least one additional finding such as EEG/MRI abnormalities, speech delay, or motor symptoms. Median age was 42 months at testing and 30 months at first seizure onset; the mean delay from first seizure to comprehensive genetic testing was 10.3 months. A genetic diagnosis was established in 43 patients (20.4%). CNVs were reported in 25.6% diagnosed patients; 27.3% of CNVs identified were intragenic. Within the diagnosed cohort, 11 (25.6%) patients were diagnosed with an IEM. The predominant molecular diagnosis was CLN2 (14% of diagnosed patients). For these patients, diagnosis was achieved 12–24 months earlier than reported by natural history of the disease. This study supports comprehensive genetic testing for patients whose first seizure occurs ≥ 24 months of age. It also supports early application of testing in this age group, as the identified diagnoses can have significant impact on patient management and outcome.