Childhood-onset Neurodegenerative Disorder Research Articles

Exploration of dietary interventions to treat mitochondrial fatty acid disorders in a mouse model

Mitochondrial fatty acids synthesis (mtFAS) is a conserved metabolic pathway essential for mitochondrial respiration. The best characterized mtFAS product is the medium-chain fatty acid octanoate (C8) used as a substrate in the synthesis of lipoic acid (LA), a cofactor required by several mitochondrial enzyme complexes. In humans, mutations in the mtFAS component enoyl reductase MECR cause childhood-onset neurodegenerative disorder MEPAN. A complete deletion of Mecr in mice is embryonically lethal, while selective deletion of Mecr in cerebellar Purkinje cells causes neurodegeneration in these cells. A fundamental question in the research of mtFAS deficiency is if the defect is amenable to treatment by supplementation with known mtFAS products. Here we used the Purkinje-cell specific mtFAS deficiency neurodegeneration model mice to study if feeding the mice with a medium-chain triacylglycerol-rich formula supplemented with LA could slow down or prevent the neurodegeneration in Purkinje cell-specific Mecr KO mice. Feeding started at the age of 4 weeks and continued until the age of 9 months. The neurological status on the mice was assessed at the age of 3, 6, and 9 months with behavioral tests and the state of the Purkinje cell deterioration in the cerebellum was studied histologically. We showed that feeding the mice with medium chain triacylglycerols and LA affected fatty acid profiles in the cerebellum and plasma but did not prevent the development of neurodegeneration in these mice. Our results indicate that dietary supplementation with medium chain fatty acids and LA alone is not an efficient way to treat mtFAS disorders.

Expanding the Clinical Spectrum of UBTF-Related Neurodevelopmental Disorder.

UBTF1 gene encodes for Upstream Binding Transcription Factor, an essential protein for RNA metabolism. A recurrent de novo variant (c.628G>A; p.Glu210Lys) has recently been associated with a childhood-onset neurodegenerative disorder characterized by motor and language regression, ataxia, dystonia, and acquired microcephaly. In this study, we report the clinical, metabolic, molecular genetics and neuroimaging findings and histologic, histochemical, and electron microscopy studies in muscle samples of 2 patients from unrelated families with a neurodevelopmental disorder. Data were retrospectively analyzed by medical charts revision. Patient 1, a 16-year-old boy, presented a childhood-onset slowly progressive neurodegenerative disorder mainly affecting language skills, behavior, and motor coordination. Patient 2, a 22-year-old woman, presented with a severe and rapidly progressive disease with dystonic tetra paresis, acquired microcephaly, and severe cognitive deficit complicated by pseudobulbar syndrome characterized by involuntary and uncontrollable outbursts of laughing, dysphagia requiring tube feeding, and nocturnal hypoventilation treated with noninvasive ventilation. Both patients carried the recurrent previously described UBTF1 de novo variant and had signs of mitochondrial dysfunction at muscle biopsy. The metabolic profile of patient 2 also revealed a decrease in CSF biopterin. These case reports add new insights to the UBTF1 disease spectrum instrumental to improving the diagnostic rate in neurodevelopmental disorders.

Whole exome screening of neurodevelopmental regression disorders in a cohort of Egyptian patients

Developmental regression describes a child who begins to lose his previously acquired milestones skills after he has reached a certain developmental stage and though affects his childhood development. It is associated with neurodegenerative diseases including leukodystrophy and neuronal ceroid lipofuscinosis diseases (NCLs), one of the most frequent childhood-onset neurodegenerative disorders. The current study focused on screening causative genes of developmental regression diseases comprising neurodegenerative disorders in Egyptian patients using next-generation sequencing (NGS)-based analyses as well as developing checklist to support clinicians who are not familiar with these diseases. A total of 763 Egyptian children (1 to 11 years), mainly diagnosed with developmental regression, seizures, or visual impairment, were studied using whole exome sequencing (WES). Among 763 Egyptian children, 726 cases were early clinically and molecularly diagnosed, including 482 cases that had pediatric stroke, congenital infection, and hepatic encephalopathy; meanwhile, 192 had clearly dysmorphic features, 31 showed central nervous system (CNS) malformation, 17 were diagnosed by leukodystrophy, 2 had ataxia telangiectasia, and 2 were diagnosed with tuberous sclerosis. The remained 37 out of 763 candidates were suspected with NCLs symptoms; however, 28 were confirmed to be NCLs patients, 1 was Kaya-Barakat-Masson syndrome, 1 was diagnosed as infantile neuroaxonal dystrophy, and 7 cases required further molecular diagnosis. This study provided an NGS-based approach of the genetic causes of developmental regression and neurodegenerative diseases as it comprised different variants and de novo mutations with complex phenotypes of these diseases which in turn help in early diagnoses and counseling for affected families.

Loss of IRF2BPL impairs neuronal maintenance through excess Wnt signaling.

De novo truncations in Interferon Regulatory Factor 2 Binding Protein Like (IRF2BPL) lead to severe childhood-onset neurodegenerative disorders. To determine how loss of IRF2BPL causes neural dysfunction, we examined its function in Drosophila and zebrafish. Overexpression of either IRF2BPL or Pits, the Drosophila ortholog, represses Wnt transcription in flies. In contrast, neuronal depletion of Pits leads to increased wingless (wg) levels in the brain and is associated with axonal loss, whereas inhibition of Wg signaling is neuroprotective. Moreover, increased neuronal expression of wg in flies is sufficient to cause age-dependent axonal loss, similar to reduction of Pits. Loss of irf2bpl in zebrafish also causes neurological defects with an associated increase in wnt1 transcription and downstream signaling. WNT1 is also increased in patient-derived astrocytes, and pharmacological inhibition of Wnt suppresses the neurological phenotypes. Last, IRF2BPL and the Wnt antagonist, CKIα, physically and genetically interact, showing that IRF2BPL and CkIα antagonize Wnt transcription and signaling.

Progressive spastic tetraplegia and axial hypotonia (STAHP) due to SOD1 deficiency: is it really a new entity?

BackgroundAmyotrophic Lateral Sclerosis (ALS) is a rare, progressive, and fatal neurodegenerative disease due to upper and lower motor neuron involvement with symptoms classically occurring in adulthood with an increasing recognition of juvenile presentations and childhood neurodegenerative disorders caused by genetic variants in genes related to Amyotrophic Lateral Sclerosis. The main objective of this study is detail clinical, radiological, neurophysiological, and genetic findings of a Brazilian cohort of patients with a recent described condition known as Spastic Tetraplegia and Axial Hypotonia (STAHP) due to SOD1 deficiency and compare with other cases described in the literature and discuss whether the clinical picture related to SOD1 protein deficiency is a new entity or may be represent a very early-onset form of Amyotrophic Lateral Sclerosis.MethodsWe conducted a case series report which included retrospective data from five Brazilian patients with SOD1 protein deficiency of a Brazilian reference center for Neuromuscular Disorders. Clinical data were obtained from a review of the medical records and descriptive statistics and variables were summarized using counts and percentages of the total population.ResultsAll 5 patients presented with a childhood-onset neurodegenerative disorders characterized by spastic tetraplegia with axial hypotonia in all cases, with gestational history showing polyhydramnios in 4/5 and intrauterine growth restriction in 3/5 patients, with most patients initially presenting a normal motor development until the six month of life or during the first year followed by a rapidly progressive motor decline with severe dysphagia and respiratory insufficiency in all patients accompanied by cognitive impairment in 3/5 patients. All patients were homozygous for the c.335dupG (p.Cys112Trpfs*11) mutation in the SOD1 gene with completely decreased enzyme activity.ConclusionsThis case series is the biggest data collection of the new recent clinical entity described as Spastic Tetraplegia and Axial Hypotonia (STAHP) due to SOD1 deficiency.

Mitochondrial Abnormalities in Induced Pluripotent Stem Cells-Derived Motor Neurons from Patients with Riboflavin Transporter Deficiency.

Riboflavin transporter deficiency (RTD) is a childhood-onset neurodegenerative disorder characterized by sensorineural deafness and motor neuron degeneration. Since riboflavin plays key functions in biological oxidation-reduction reactions, energy metabolism pathways involving flavoproteins are affected in RTD. We recently generated induced pluripotent stem cell (iPSC) lines from affected individuals as an in vitro model of the disease and documented mitochondrial impairment in these cells, dramatically impacting cell redox status. This work extends our study to motor neurons (MNs), i.e., the cell type most affected in patients with RTD. Altered intracellular distribution of mitochondria was detected by confocal microscopic analysis (following immunofluorescence for superoxide dismutase 2 (SOD2), as a dual mitochondrial and antioxidant marker), and βIII-Tubulin, as a neuronal marker. We demonstrate significantly lower SOD2 levels in RTD MNs, as compared to their healthy counterparts. Mitochondrial ultrastructural abnormalities were also assessed by focused ion beam/scanning electron microscopy. Moreover, we investigated the effects of combination treatment using riboflavin and N-acetylcysteine, which is a widely employed antioxidant. Overall, our findings further support the potential of patient-specific RTD models and provide evidence of mitochondrial alterations in RTD-related iPSC-derived MNs—emphasizing oxidative stress involvement in this rare disease. We also provide new clues for possible therapeutic strategies aimed at correcting mitochondrial defects, based on the use of antioxidants.

Current Insights in Elucidation of Possible Molecular Mechanisms of the Juvenile Form of Batten Disease.

The neuronal ceroid lipofuscinoses (NCLs) collectively constitute one of the most common forms of inherited childhood-onset neurodegenerative disorders. They form a heterogeneous group of incurable lysosomal storage diseases that lead to blindness, motor deterioration, epilepsy, and dementia. Traditionally the NCL diseases were classified according to the age of disease onset (infantile, late-infantile, juvenile, and adult forms), with at least 13 different NCL varieties having been described at present. The current review focuses on classic juvenile NCL (JNCL) or the so-called Batten (Batten-Spielmeyer-Vogt; Spielmeyer-Sjogren) disease, which represents the most common and the most studied form of NCL, and is caused by mutations in the CLN3 gene located on human chromosome 16. Most JNCL patients carry the same 1.02-kb deletion in this gene, encoding an unusual transmembrane protein, CLN3, or battenin. Accordingly, the names CLN3-related neuronal ceroid lipofuscinosis or CLN3-disease sometimes have been used for this malady. Despite excessive in vitro and in vivo studies, the precise functions of the CLN3 protein and the JNCL disease mechanisms remain elusive and are the main subject of this review. Although the CLN3 gene is highly conserved in evolution of all mammalian species, detailed analysis of recent genomic and transcriptomic data indicates the presence of human-specific features of its expression, which are also under discussion. The main recorded to date changes in cell metabolism, to some extent contributing to the emergence and progression of JNCL disease, and human-specific molecular features of CLN3 gene expression are summarized and critically discussed with an emphasis on the possible molecular mechanisms of the malady appearance and progression.

HEREDITARY NEUROPATHIES & ALS: P.107 Review of ophthalmologic findings following intrathecal gene transfer for giant axonal neuropathy

Giant Axonal Neuropathy (GAN) is an ultra-rare childhood onset neurodegenerative disorder of the peripheral and central nervous system. Recessive GAN mutations cause loss of function of gigaxonin, a cytoskeletal regulatory protein, leading to progressive sensorimotor and optic neuropathy, CNS involvement and respiratory failure. We report on the ophthalmologic findings of a single site, phase I, non-randomized, open label dose escalation gene transfer study for GAN (NCT02362438), a first-in-human intrathecal (IT) AAV9 mediated gene transfer. Data from 11 patients with GAN dosed at 3 dose levels; 3.5 × 1013 (1X), 1.2 × 1014 (3.3X), and 1.8 × 1014 (5X) with scAAV9-JeT-GAN with up to 3 years of follow up is analyzed. Ophthalmic evaluations include best-corrected visual acuity, color discrimination thresholds, fundus imaging, and measurement of retinal nerve fiber later (RNFL) thickness through optical coherence tomography. Photopic electroretinography including a photopic negative response and visual evoked potentials (VEP) were also recorded. GAN natural history study data is used for comparison of visual acuity and RNFL thickness as well as correlation of RNFL thickness with validated outcome measures of motor function. Cross sectional natural history analysis shows significant correlation between RNFL thickness and the MFM32 total score. Interim analysis reveals a trend towards stabilization and slowing of the expected decline in RNFL thickness and visual acuity compared to pre-trial lead in data and natural history. VEP results were variable with no consistent trends across natural history and clinical trial cohorts. The interim analysis of ophthalmologic assessments highlights several key aspects: 1) RNFL thickness, a structural assessment of optic nerve atrophy and axonal nerve degeneration at the level of the CNS is a biomarker strongly correlated to overall disease severity. In GAN patients, the decline in RNFL precedes change in visual acuity, and changes can be detected longitudinally. 2) The trend towards stabilization of RNFL thickness post gene transfer suggests that RNFL may be an objective and responsive biomarker of visual system involvement and overall nervous system degeneration in GAN. 3) Assessment of RNFL thickness post gene transfer may be used as an exploratory marker of feasibility of CNS transduction following IT gene transfer.

Mitochondrial and Peroxisomal Alterations Contribute to Energy Dysmetabolism in Riboflavin Transporter Deficiency.

Riboflavin transporter deficiency (RTD) is a childhood-onset neurodegenerative disorder characterized by progressive pontobulbar palsy, sensory and motor neuron degeneration, sensorineural hearing loss, and optic atrophy. As riboflavin (RF) is the precursor of FAD and FMN, we hypothesize that both mitochondrial and peroxisomal energy metabolism pathways involving flavoproteins could be directly affected in RTD, thus impacting cellular redox status. In the present work, we used induced pluripotent stem cells (iPSCs) from RTD patients to investigate morphofunctional features, focusing on mitochondrial and peroxisomal compartments. Using this model, we document the following RTD-associated alterations: (i) abnormal colony-forming ability and loss of cell-cell contacts, revealed by light, electron, and confocal microscopy, using tight junction marker ZO-1; (ii) mitochondrial ultrastructural abnormalities, involving shape, number, and intracellular distribution of the organelles, as assessed by focused ion beam/scanning electron microscopy (FIB/SEM); (iii) redox imbalance, with high levels of superoxide anion, as assessed by MitoSOX assay accompanied by abnormal mitochondrial polarization state, evaluated by JC-1 staining; (iv) altered immunofluorescence expression of antioxidant systems, namely, glutathione, superoxide dismutase 1 and 2, and catalase, as assessed by quantitatively evaluated confocal microscopy; and (v) peroxisomal downregulation, as demonstrated by levels and distribution of fatty acyl β-oxidation enzymes. RF supplementation results in amelioration of cell phenotype and rescue of redox status, which was associated to improved ultrastructural features of mitochondria, thus strongly supporting patient treatment with RF, to restore mitochondrial- and peroxisomal-related aspects of energy dysmetabolism and oxidative stress in RTD syndrome.

CMT AND NEUROGENIC DISEASE: P.337Giant axonal neuropathy presenting as CMT2: results from the NIH natural history study

Giant axonal neuropathy (GAN) is a rare childhood onset neurodegenerative disorder of the peripheral and central nervous system caused by recessive mutations in the GAN gene resulting in loss of function of gigaxonin, a cytoskeletal regulatory protein. The true incidence of GAN is unknown but it has been reported in approximately 100 patients worldwide since the identification of the causative gene in 2000. In its classical form GAN displays a consistent phenotype with 'kinky' hair, early gait abnormality from sensory ataxia followed by a progressive length dependent decline in strength with uniform sensory more than motor axonal polyneuropathy on electrophysiology. Patients later develop progressive cerebellar dysfunction, optic neuropathy, respiratory insufficiency and seizures. Death from respiratory complications usually ensues by the 3rd decade. With the advent of whole exome sequencing patients with milder disease, often initially clinically diagnosed with hereditary axonal sensory motor polyneuropathy (CMT2), have been recognized. Here we conduct a careful clinical and genetic characterization of 6 individuals age 8-21 years with genetically confirmed GAN with a milder phenotype who participated in the single site NIH GAN natural history study evaluating 35 patients age 5-21 years. In addition to comparatively better motor function they displayed curly rather than kinky hair, minimal to no brain imaging abnormalities, normal intellect and pulmonary function and no optic neuropathy. No definitive genotype-phenotype correlations emerged. Overall GAN of milder clinical severity can present with a CMT2-like phenotype. Accurate and early diagnosis can be achieved by the inclusion of GAN on CMT NGS panels to facilitate timely initiation of disease modifying therapy, to improve understanding of the natural history of this disorder, and to further elucidate the pathogenic mechanism underlying a milder disease course.

NEW THERAPEUTIC APPROACHES AND THEIR READOUT: O.19First-in-human intrathecal gene transfer study for giant axonal neuropathy: review of safety, immunologic responses and interim analysis of efficacy

Giant axonal neuropathy (GAN) is a rare childhood onset neurodegenerative disorder of the peripheral and central nervous system. Recessive GAN mutations cause loss of function of gigaxonin, a cytoskeletal regulatory protein, leading to progressive sensorimotor and optic neuropathy, CNS involvement and respiratory failure. We report on a single site, phase I, non-randomized, open label dose escalation gene transfer study for GAN (NCT02362438), a first-in-human intrathecal (IT) AAV9 mediated gene transfer trial for any indication. 9 GAN patients have been dosed at three dose levels (ranging from 3.5 × 1013vg to 1.8 × 1014vg) with scAAV9-JeT-GAN, with up to 2 year post-gene transfer follow up. We review safety and immunologic response to the gene vector and transgene (serum and CSF AAV9 neutralizing antibody titers, interferon-γ ELISpot analysis to GAN and AAV9 epitopes, CSF and peripheral cytokine analysis and white blood cell flow cytometry), and present an interim efficacy analysis. GAN natural history study data is used for comparison of outcome measures which include: Motor function measure 32 (MFM32), Neuropathy impairment score (NIS), Friedreich's Ataxia rating scale (FARS), myometry, grip and pinch strength, timed testing, electrophysiology, and neuroimaging. We highlight the overall safety and feasibility of an intrathecal route of AAV9 based gene transfer. The efficacy interim analysis highlights several key aspects: Feasibility for adequately targeting the nervous system, preferred IT dosing regimens needed for effective transduction, relevance of baseline neurologic impairment and disease progression in patient selection and stratification. This data also provides insight into the immunological implications of gene transfer with concomitant immunosuppressive regimens and their impact on AAV9 gene transfer safety and efficacy. This study is a proof of concept for IT gene transfer as a strategy for gene replacement targeting the central nervous system.

Do Perineuronal Net Elements Contribute to Pathophysiology of Spinal Muscular Atrophy? In Vitro and Transcriptomics Insights.

Spinal muscular atrophy (SMA) is one of the most common childhood onset neurodegenerative disorders in global health whereby novel biomarkers and therapeutic targets are sorely needed. SMA is an autosomal recessive genetic disorder resulting in degeneration of α-motor neurons in the brain stem and spinal cord that leads to mortality in infants worldwide. In majority of the patients, SMA is caused by homozygous deletion of the SMN1 gene. The clinical spectrum of the SMA displays, however, large person-to-person variations where the underlying mechanisms are poorly understood. We report in this study transcriptomics insights gleaned from patients with the severe type I (GM03813 and GM09677) and the mild type III. Pathway enrichment and functional analysis showed that especially extracellular matrix (ECM), synapse organization, and ECM receptor interaction pathways were affected. Among the neural ECM components, hyaluronan and proteoglycan link protein (HAPLN1), which is a key triggering molecule of the perineuronal net (PNN), was significantly downregulated in type I fibroblasts compared to type III. PNN is a specialized form of neural ECM around the neuronal cell bodies and dendrites in the central nervous system. In addition, we evaluated the PNN expression in vitro in a model established by SMN silencing in the PC12 rat pheochromocytoma cell line which can be differentiated into neurons with nerve growth factor treatment. In this neuronal in vitro model, we found that HAPLN1 showed a significant 50% decrease. Our results describe the association between PNN elements, especially HAPLN1, and SMA pathophysiology for the first time. These observations collectively inform future translational research on SMA for discovery of novel molecular targets for diagnostics and precision medicine innovation.

Cathepsin D Polymorphism C224T in Childhood-Onset Neurodegenerative Disorders: No Impact for Childhood Dementia

Compromised lysosomal functioning has been identified as a major risk factor for neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Furthermore, the association between a defined cathepsin D ( CTSD ) polymorphism and a higher risk of sporadic Alzheimer's disease has been established for particular populations. Here, we analyzed 189 children with rare neurodegenerative disease for carrying the T-allele by polymerase chain reaction-restriction fragment length polymorphism. We found no statistical differences in genotype and allele frequencies between the neurodegenerative group and European descent participants of genetic studies using the Cochran-Armitage's trend test. In contrast to adult-onset neurodegenerative diseases, analysis of clinical datasets of children carrying the T-allele did not demonstrate differences to the general disease group.

Hospitalizations of Children with Huntington's Disease in the United States.

Juvenile Huntington's disease (JHD) is a childhood-onset neurodegenerative disorder. Although it is caused by the same pathologic expansion of CGA repeats as adult-onset Huntington's disease, JHD has distinct clinical features. Most clinical research in HD focuses in the adult-onset disease; therefore, little is known about acute care outcomes for patients with JHD. The Kids' Inpatient Database (KID) was used to examine hospitalizations of children with JHD and to determine the diagnoses and procedures associated with inpatient care for JHD. Regression models were built to examine acute care outcomes, including death, length of stay, and disposition at discharge in patients with JHD compared with patients in the general KID data. The proportion of JHD cases among hospitalized children was 1.23 per 100,000 KID inpatient stays. Seizures/convulsions (58.5%) and psychiatric conditions (26.1%) were the most common primary or secondary diagnoses among JHD patient hospitalizations. The most common procedure was percutaneous endoscopic gastrostomy tube placement (8.6%). Compared with hospitalizations of the general population, hospitalizations of patients with JHD had a lower odds of discharge to home (adjusted odds ratio [AOR], 0.23; 95% confidence interval [CI], 0.14-0.37) and an increased likelihood of death (AOR, 8.03; 95% CI, 2.98-21.60) or discharge to a short-term care facility (AOR, 4.44; 95% CI, 2.59-7.61). A diagnosis of JHD was associated with increased length of stay (7.04 vs. 3.75 days; P < 0.01). Children with JHD have unique acute care patterns. Future studies are needed to determine the extent to which coordinated care may impact inpatient and disposition needs.

Brain inflammation is accompanied by peripheral inflammation in Cstb -/- mice, a model for progressive myoclonus epilepsy.

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessively inherited childhood-onset neurodegenerative disorder, characterized by myoclonus, seizures, and ataxia. Mutations in the cystatin B gene (CSTB) underlie EPM1. The CSTB-deficient (Cstb −/−) mouse model recapitulates key features of EPM1, including myoclonic seizures. The mice show early microglial activation that precedes seizure onset and neuronal loss and leads to neuroinflammation. We here characterized the inflammatory phenotype of Cstb −/− mice in more detail. We found higher concentrations of chemokines and pro-inflammatory cytokines in the serum of Cstb −/− mice and higher CXCL13 expression in activated microglia in Cstb −/− compared to control mouse brains. The elevated chemokine levels were not accompanied by blood-brain barrier disruption, despite increased brain vascularization. Macrophages in the spleen and brain of Cstb −/− mice were predominantly pro-inflammatory. Taken together, these data show that CXCL13 expression is a hallmark of microglial activation in Cstb −/− mice and that the brain inflammation is linked to peripheral inflammatory changes, which might contribute to the disease pathology of EPM1.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0764-7) contains supplementary material, which is available to authorized users.

Low-dose, continual enzyme delivery ameliorates some aspects of established brain disease in a mouse model of a childhood-onset neurodegenerative disorder

To determine the capacity of continual low-dose lysosomal enzyme infusion into the cerebrospinal fluid of mucopolysaccharidosis type IIIA (MPS IIIA) mice to reverse established neurodegenerative disease. The rationale behind the study is that there is only limited animal model-derived evidence supporting treatment of symptomatic patients, principally because few studies have been designed to examine disease reversibility. Twelve-week old MPS IIIA mice were implanted with indwelling unilateral intra-ventricular cannulae. These were connected to subcutaneous mini-osmotic pumps infusing recombinant human sulphamidase. Pump replacement was carried out in some mice at 16-weeks of age, enabling treatment to continue for a further month. Control affected/unaffected mice received vehicle via the same method. Behavioural, neuropathological and biochemical parameters of disease were assessed. Improvement in some, but not all, behavioural parameters occurred. Sulphamidase infusion mediated a statistically significant reduction in primary (heparan sulphate) and secondary (gangliosides GM2, GM3) substrate accumulation in the brain, with small reductions in micro- but not astro-gliosis. There was no change in axonal spheroid number. All mice developed a humoural response, however the antibodies were non-neutralising and no adverse clinical effects were observed. Continual infusion of replacement enzyme partially ameliorates clinical, histological and biochemical aspects of MPS IIIA mice, when treatment begins at an early symptomatic stage.

Chapter 14 - Ataxia telangiectasia

Ataxia telangiectasia (AT) is an autosomal recessive multisystem genetic disorder caused by a mutation in the ATM gene encoding for the ATM protein. AT systemic manifestations include cutaneous telangiectasias, radiosensitivity, immune deficiency with recurrent sinopulmonary infections, and a tendency to develop lymphoid malignancies. These complications are explained by the major role played by ATM in DNA repair. AT is also the second most common childhood onset neurodegenerative disorder of the cerebellum, presenting with progressive ataxia and oculomotor apraxia and often accompanied by extrapyramidal movement disorders. Ataxia typically begins around the time children start to walk at about 1 year of age and leads to wheelchair dependence by the second decade of life. Cerebellar atrophy is evident on imaging after 2 years of life and is progressive. Abnormal DNA repair mechanisms do not entirely explain the pathophysiology in nondividing neurons. The nervous system involvement is better explained by the role ATM plays in antioxidative defense, mitochondrial homeostasis, and DNA chromatin packing. A better understanding of the underlying pathophysiologic mechanisms of this devastating disease may enable disease-modifying treatments in the future. Meanwhile, treatment is mainly supportive and does not change the poor prognosis of the disease although it improves the patient's quality of life.

Parkin-mediated K63-polyubiquitination targets ubiquitin C-terminal hydrolase L1 for degradation by the autophagy-lysosome system.

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a key neuronal deubiquitinating enzyme which is mutated in Parkinson disease (PD) and in childhood-onset neurodegenerative disorder with optic atrophy. Furthermore, reduced UCH-L1 protein levels are associated with a number of neurodegenerative diseases, whereas up-regulation of UCH-L1 protein expression is found in multiple types of cancer. However, very little is known about how UCH-L1 protein level is regulated in cells. Here, we report that UCH-L1 is a novel interactor and substrate of PD-linked E3 ubiquitin-protein ligase parkin. We find that parkin mediates K63-linked polyubiquitination of UCH-L1 in cooperation with the Ubc13/Uev1a E2 ubiquitin-conjugating enzyme complex and promotes UCH-L1 degradation by the autophagy-lysosome pathway. Targeted disruption of parkin gene expression in mice causes a significant decrease in UCH-L1 ubiquitination with a concomitant increase in UCH-L1 protein level in brain, supporting an in vivo role of parkin in regulating UCH-L1 ubiquitination and degradation. Our findings reveal a direct link between parkin-mediated ubiquitin signaling and UCH-L1 regulation, and they have important implications for understanding the roles of these two proteins in health and disease.

CLN3 Loss Disturbs Membrane Microdomain Properties and Protein Transport in Brain Endothelial Cells

Juvenile neuronal ceroid lipofuscinosis (JNCL) is a fatal childhood-onset neurodegenerative disorder caused by mutations in ceroid lipofuscinosis neuronal-3 (CLN3), a hydrophobic transmembrane protein of unresolved function. Previous studies indicate blood-brain barrier (BBB) defects in JNCL, and our earlier report showed prominent Cln3 expression in mouse brain endothelium. Here we find that CLN3 is necessary for normal trafficking of the microdomain-associated proteins caveolin-1, syntaxin-6, and multidrug resistance protein 1 (MDR1) in brain endothelial cells. Correspondingly, CLN3-null cells have reduced caveolae, and impaired caveolae- and MDR1-related functions including endocytosis, drug efflux, and cell volume regulation. We also detected an abnormal blood-brain barrier response to osmotic stress in vivo. Evaluation of the plasma membrane with fluorescent sphingolipid probes suggests microdomain destabilization and enhanced fluidity in CLN3-null cells. In further work we found that application of the glycosphingolipid lactosylceramide to CLN3-deficient cells rescues protein transport and caveolar endocytosis. Last, we show that CLN3 localizes to the trans-Golgi network (TGN) and partitions with buoyant microdomain fractions. We propose that CLN3 facilitates TGN-to-plasma membrane transport of microdomain-associated proteins. Insult to this pathway may underlie BBB dysfunction and contribute to JNCL pathogenesis.

Npc1 Acting in Neurons and Glia Is Essential for the Formation and Maintenance of CNS Myelin

Cholesterol availability is rate-limiting for myelination, and prior studies have established the importance of cholesterol synthesis by oligodendrocytes for normal CNS myelination. However, the contribution of cholesterol uptake through the endocytic pathway has not been fully explored. To address this question, we used mice with a conditional null allele of the Npc1 gene, which encodes a transmembrane protein critical for mobilizing cholesterol from the endolysosomal system. Loss of function mutations in the human NPC1 gene cause Niemann-Pick type C disease, a childhood-onset neurodegenerative disorder in which intracellular lipid accumulation, abnormally swollen axons, and neuron loss underlie the occurrence of early death. Both NPC patients and Npc1 null mice exhibit myelin defects indicative of dysmyelination, although the mechanisms underlying this defect are incompletely understood. Here we use temporal and cell-type-specific gene deletion in order to define effects on CNS myelination. Our results unexpectedly show that deletion of Npc1 in neurons alone leads to an arrest of oligodendrocyte maturation and to subsequent failure of myelin formation. This defect is associated with decreased activation of Fyn kinase, an integrator of axon-glial signals that normally promotes myelination. Furthermore, we show that deletion of Npc1 in oligodendrocytes results in delayed myelination at early postnatal days. Aged, oligodendocyte-specific null mutants also exhibit late stage loss of myelin proteins, followed by secondary Purkinje neuron degeneration. These data demonstrate that lipid uptake and intracellular transport by neurons and oligodendrocytes through an Npc1-dependent pathway is required for both the formation and maintenance of CNS myelin.