Infantile Neuronal Ceroid Lipofuscinosis Research Articles

Decreased sensitivity of palmitoyl protein thioesterase 1-deficient neurons to chemical anoxia.

Infantile CLN1 disease, also known as infantile neuronal ceroid lipofuscinosis, is a fatal childhood neurodegenerative disorder caused by mutations in the CLN1 gene. CLN1 encodes a soluble lysosomal enzyme, palmitoyl protein thioesterase 1 (PPT1), and it is still unclear why neurons are selectively vulnerable to the loss of PPT1 enzyme activity in infantile CLN1 disease. To examine the effects of PPT1 deficiency on several well-defined neuronal signaling and cell death pathways, different toxic insults were applied in cerebellar granule neuron cultures prepared from wild type (WT) and palmitoyl protein thioesterase 1-deficient (Ppt1 -/- ) mice, a model of infantile CLN1 disease. Glutamate uptake inhibition by t-PDC (L-trans-pyrrolidine-2,4-dicarboxylic acid) or Zn2+-induced general mitochondrial dysfunction caused similar toxicity in WT and Ppt1 -/- cultures. Ppt1 -/- neurons, however, were more sensitive to mitochondrial complex I inhibition by MPP+ (1-methyl-4-phenylpyridinium), and had significantly decreased sensitivity to chemical anoxia induced by the mitochondrial complex IV inhibitor, sodium azide. Our results indicate that PPT1 deficiency causes alterations in the mitochondrial respiratory chain.

The paediatric rheumatologist and orphan disease - a story without happy ending.

Orphan diseases are not a common challenge in the everyday practice of the rheumatologist. Despite their extremely rare occurrence one of the patients under our care developed one of them – neuronal ceroid lipofuscinosis, the most frequent neurodegenerative disease observed in the paediatric population. We report a case of 2-year-old girl diagnosed with oligoarticular form of juvenile idiopathic arthritis treated in our Department with steroids and methotrexate and staying in the stage of disease remission. During routine checkups at Outpatient Clinic we observed progressive deterioration of girls neurological condition resulting in ataxia, gait disturbances with no rheumatological cause behind and speech impairment. The appearance of the symptoms was accompanied by frequent episodes of epileptic seizures, with little clinical improvement on combined antiepileptic treatment. Magnetic resonance imaging that we performed showed a picture highly suggestive of neuronal ceroid lipofuscinosis – atrophy of the patients cerebrum and cerebellum. Genetic testing conducted resulted in the diagnosis of late infantile neuronal ceroid lipofuscinosis (LINCL).

27. Therapeutic Efficacy of Intracranial and Intrathecal AAV2/9-PPT1 in Infantile Batten Disease

Background The neuronal ceroid lipofuscinoses (NCLs) are a group of the most common pediatric neurodegenerative lysosomal storage disorders. Infantile NCL (INCL), caused by a deficiency in the lysosomal enzyme palmitoyl-protein thioesterase-1 (PPT1), is characterized clinically by progressive cognitive and motor decline, profound neurodegeneration and neuroinflammation, and accumulation of autofluorescent storage material (AFSM). Infantile NCL murine model recapitulates the human disease. AAV2/5-PPT1 intracranial (IC) delivery delayed the onset of INCL histopathological markers in the forebrain and cerebellum and improved preclinical outcome measures. However, overall disease progression was only partially corrected suggesting peripheral nervous system involvement. In collaboration with Dr. Jon Cooper (King's College, London), we discovered substantial progressive pathology in the spinal cord: neuronal loss and axon density, significant microgliosis and astrocytosis, and AFSM (Nelvagal H etal, manuscript in prep). These data suggest that the spinal cord could be an important therapeutic target. We hypothesize that IC and intrathecal (IT) gene therapy in combination will significantly improve the lifespan, preclinical outcome measures, and histopathological markers as compared to either therapy alone. Methods We generated five groups (n=10): PPT1-/-, wild type, and PPT1-/- injected with IC, IT, or the combination IC/IT AAV2/9-PPT1. For IC injections, 3-2µl bilateral intracranial injections were performed. For IT injections, one 15µl bolus injection into the lumbar subarachnoid space was performed. The AAV2/9-PPT1 virus was diluted to 1×1012 viral particles/ml. To date, we have collected 3, 5, and 7-month time points for all groups, and have generated a 9-month time point. Samples will be analyzed for PPT1 enzyme activity, AFSM, neuroinflammation and neurohistopathology, spinal cord pathology, and a histochemical stain for PPT1. Lifespan, behavior, and brain weight (gross measure of atrophy) will be analyzed. Significance was determined using a 2-way ANOVA test. Results PPT1-/- mice have a median lifespan of 35.8 weeks and rapid decline in rotarod performance beginning at 5 months. There is a progressive decline in PPT1-/- brain weight beginning at 3 months. IT AAV2/9-PPT1 mice have a median lifespan of 48.4 weeks and have a steady decline in rotarod performance beginning at 7 months. There is a progressive decline in the IT mice brain weight compared to wild-type, reaching significance at 7 months (p<. 001); however, it had significantly less atrophy than PPT1-/- brains until 7 months (p<0.05). IC AAV2/9-PPT1 mice have a median lifespan of 58.5 weeks and a rapid decline in rotarod performance beginning at 9 months. IC AAV2/9-PPT1 mice brain weight are not significantly different than wild-type. To date, at 66 weeks, all IC/IT AAV2/9-PPT1 mice are alive. There is a significant decline in IC/IT mice rotarod performance at 15 months. The IC/IT mice brain weight is not significantly different than wild-type. Data for the enzyme activity, neuroinflammatory markers, histopathology, and histochemical stain will be complete by April 2016. Conclusions To date, these data confirm that targeting the entire CNS will provide a significant step for INCL therapy. The combination therapy significantly increases the lifespan beyond that of an additive benefit. As expected, modifying the gene therapy vector from IC-AAV2/5 to IC-AAV2/9 significantly improved preclinical outcome measures. Lastly, the IT AAV2/9-PPT1 injections suggest that spinal cord disease plays an important role in INCL pathogenesis. These findings could form the basis for an effective therapeutic strategy that incorporates targeting multiple facets of INCL disease.

362. Translatable Gene Therapy for Infantile Neuronal Lipofuscinosis

Infantile Neuronal Ceroid Lipofuscinosis (INCL) is a rare lysosomal storage disease caused by mutations in the CLN1 gene, which encodes the protein palmitoyl-protein thioesterase-1 (PPT1). In the absence of the PPT1 enzyme, osmiophilic granules accumulate in cells, eventually leading to cell dysfunction and neurodegeneration. In the classic form in human patients, the damage from the storage material and resulting cell death causes visual failure, speech and motor deterioration, and seizures to appear between the ages of 6 and 24 months, with premature death, usually between 3 to 5 years of age. INCL mice (CLN1 knock-out) accurately model the disease symptoms and pathogenesis, leading to premature death at approximately 8 months of age. Previous results from Mark Sands’ laboratory have shown that intracranial administration of AAV5 vectors in newborn INCL mice results in a significant extension of lifespan of approximately 50% along with improvements in motor function. While encouraging, the translation of this approach in humans is complicated by the reduced relative biodistribution of the vector after intracranial administration when moving from a rodent to human brain, and because a newborn mouse corresponds to a prenatal time period in humans. With the ability of AAV9 to cross the blood-brain barrier, intravenous (IV) administration of the vector can achieve widespread distribution of the transgene in the Central Nervous System (CNS), while intrathecal (IT) administration of AAV9 vectors into the cerebrospinal fluid (CSF) can achieve a similar CNS distribution at a considerably lower dose more amenable to human translation. Currently, self-complementary AAV9 vectors are being used in ongoing Phase I human clinical trials for Spinal Muscular Atrophy, administered IV (NCT02122952), and for Giant Axonal Neuropathy, administered IT (NCT02122952). We have pursued two global and translationally-relevant gene transfer approaches for INCL: IT delivery of scAAV9/CLN1 into the CSF of the lumbar cistern at a dose of 7×1010 vg per mouse, and IV delivery of scAAV9.47/CLN1, a liver-detargeted vector, at a dose of 1×1012 vg per mouse. Both approaches were evaluated at different ages: 1 week and 4 weeks (pre-symptomatic), 20 weeks (early-symptomatic) and 26 weeks (post-symptomatic). While we failed to rescue mice injected at 20 weeks or later, mice treated at 1 and 4 weeks of age increased their survival to approximately twice their expected lifespan (ongoing), concurrent with improvement of their quality of life. These results will be compared to a cohort of mice injected as neonates, which are alive at 1 year and being monitored for long-term benefits. Our results indicate that, at the current doses, either treatment approach is effective early in the disease course, providing a dramatic survival benefit and improved quality of life, but it is wholly ineffective once significant symptoms have emerged. The IT route offers a comparable rescue at < 1/10 the dose, making this approach potentially more favorable for human translation. We propose this as a realistic and readily translatable approach to treat INCL.

Brain Region-Specific Degeneration with Disease Progression in Late Infantile Neuronal Ceroid Lipofuscinosis (CLN2 Disease).

Late infantile neuronal ceroid lipofuscinosis (CLN2 disease) is a uniformly fatal lysosomal storage disease resulting from mutations in the CLN2 gene. Our hypothesis was that regional analysis of cortical brain degeneration may identify brain regions that are affected earliest and most severely by the disease. Fifty-two high-resolution 3T MR imaging datasets were prospectively acquired on 38 subjects with CLN2. A retrospective cohort of 52 disease-free children served as a control population. The FreeSurfer software suite was used for calculation of cortical thickness. An increased rate of global cortical thinning in CLN2 versus control subjects was the primary finding in this study. Three distinct patterns were observed across brain regions. In the first, subjects with CLN2 exhibited differing rates of cortical thinning versus age. This was true in 22 and 26 of 34 regions in the left and right hemispheres, respectively, and was also clearly discernable when considering brain lobes as a whole and Brodmann regions. The second pattern exhibited a difference in thickness from healthy controls but with no discernable change with age (9 left hemispheres, 5 right hemispheres). In the third pattern, there was no difference in either the rate of cortical thinning or the mean cortical thickness between groups (3 left hemispheres, 3 right hemispheres). This study demonstrates that CLN2 causes differential rates of degeneration across the brain. Anatomic and functional regions that degenerate sooner and more severely than others compared with those in healthy controls may offer targets for directed therapies. The information gained may also provide neurobiologic insights regarding the mechanisms underlying disease progression.

Tissue-specific variation in nonsense mutant transcript level and drug-induced read-through efficiency in the Cln1(R151X) mouse model of INCL.

About 10% of inherited diseases are caused by nonsense mutations [Trends Mol Med 18 (2012) 688], and nonsense suppression drug therapy promoting translation through premature stop codons is an emerging therapeutic approach. Infantile neuronal ceroid lipofuscinosis (INCL), a childhood neurodegenerative disease, results from mutations in the CLN1 gene encoding the lysosomal enzyme, palmitoyl-protein thioesterase 1 (PPT1) [Biochim Biophys Acta 1832 (2013) 1806, Hum Mutat (2012) 63, Biochim Biophys Acta 1832 (2013) 1881]. The nonsense mutation p.R151X is the most common disease-causing CLN1 mutation Hum Mutat (2012) 63. In the novel Cln1(R151X) mouse model of INCL, we found large, tissue-specific variations in Cln1(R151X) mRNA level and PPT1 residual enzyme activity. These tissue-specific differences strongly influenced the read-through efficiency of ataluren (PTC124), a well-known nonsense suppression drug. A two-day treatment with ataluren (10 mg/kg) increased PPT1 enzyme activity in the liver and muscle, but not in any other tissue examined. Our study identifies a new challenge/hurdle for read-through drug therapy: variable efficiency of read-through therapy in the different tissues/organs because of tissue-specific variations in nonsense mutant transcript levels.

Histochemical localization of palmitoyl protein thioesterase-1 activity.

Infantile neuronal ceroid lipofuscinosis (INCL, Infantile Batten disease) is an invariably fatal neurodegenerative pediatric disorder caused by an inherited mutation in the PPT1 gene. Patients with INCL lack the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1, EC 3.1.2.22), resulting in intracellular accumulation of autofluorescent storage material and subsequent neuropathology. The Ppt1(-/-) mouse is deficient in PPT1 activity and represents a useful animal model of INCL that recapitulates most of the clinical and pathological aspects of the disease. Preclinical therapeutic experiments performed in the INCL mouse include CNS-directed gene therapy and recombinant enzyme replacement therapy; both seek to re-establish therapeutic levels of the deficient enzyme. We present a novel method for the histochemical localization of PPT1 activity in the Ppt1(-/-) mouse. By utilizing the substrate CUS-9235, tissues known to be positive for PPT1 activity turn varying intensities of blue. Presented here are histochemistry data showing the staining pattern in Ppt1(-/-), wild type, and Ppt1(-/-) mice treated with enzyme replacement therapy or AAV2/9-PPT1-mediated gene therapy. Results are paired with quantitative biochemistry data that confirm the ability of CUS-9235 to detect and localize PPT1 activity. This new method complements the current tools for the study of INCL and evaluation of effective therapies.

Suppression of agrin-22 production and synaptic dysfunction in Cln1 (-/-) mice.

Oxidative stress in the brain is highly prevalent in many neurodegenerative disorders including lysosomal storage disorders, in which neurodegeneration is a devastating manifestation. Despite intense studies, a precise mechanism linking oxidative stress to neuropathology in specific neurodegenerative diseases remains largely unclear. Infantile neuronal ceroid lipofuscinosis (INCL) is a devastating neurodegenerative lysosomal storage disease caused by mutations in the ceroid lipofuscinosis neuronal-1 (CLN1) gene encoding palmitoyl-protein thioesterase-1. Previously, we reported that in the brain of Cln1 (-/-) mice, which mimic INCL, and in postmortem brain tissues from INCL patients, increased oxidative stress is readily detectable. We used molecular, biochemical, immunohistological, and electrophysiological analyses of brain tissues of Cln1 (-/-) mice to study the role(s) of oxidative stress in mediating neuropathology. Our results show that in Cln1 (-/-) mice oxidative stress in the brain via upregulation of the transcription factor, CCAAT/enhancer-binding protein-δ, stimulated expression of serpina1, which is an inhibitor of a serine protease, neurotrypsin. Moreover, in the Cln1 (-/-) mice, suppression of neurotrypsin activity by serpina1 inhibited the cleavage of agrin (a large proteoglycan), which substantially reduced the production of agrin-22, essential for synaptic homeostasis. Direct whole-cell recordings at the nerve terminals of Cln1 (-/-) mice showed inhibition of Ca(2+) currents attesting to synaptic dysfunction. Treatment of these mice with a thioesterase-mimetic small molecule, N-tert (Butyl) hydroxylamine (NtBuHA), increased agrin-22 levels. Our findings provide insight into a novel pathway linking oxidative stress with synaptic pathology in Cln1 (-/-) mice and suggest that NtBuHA, which increased agrin-22 levels, may ameliorate synaptic dysfunction in this devastating neurodegenerative disease.

Comprehensive functional characterization of murine infantile Batten disease including Parkinson-like behavior and dopaminergic markers.

Infantile neuronal ceroid lipofuscinosis (INCL, Infantile Batten disease) is a neurodegenerative lysosomal storage disease caused by a deficiency in palmitoyl protein thioesterase-1 (PPT1). The PPT1-deficient mouse (Cln1−/−) is a useful phenocopy of human INCL. Cln1−/− mice display retinal dysfunction, seizures, motor deficits, and die at ~8 months of age. However, little is known about the cognitive and behavioral functions of Cln1−/− mice during disease progression. In the present study, younger (~1–2 months of age) Cln1−/− mice showed minor deficits in motor/sensorimotor functions while older (~5–6 months of age) Cln1−/− mice exhibited more severe impairments, including decreased locomotor activity, inferior cued water maze performance, decreased running wheel ability, and altered auditory cue conditioning. Unexpectedly, certain cognitive functions such as some learning and memory capabilities seemed intact in older Cln1−/− mice. Younger and older Cln1−/− mice presented with walking initiation defects, gait abnormalities, and slowed movements, which are analogous to some symptoms reported in INCL and parkinsonism. However, there was no evidence of alterations in dopaminergic markers in Cln1−/− mice. Results from this study demonstrate quantifiable changes in behavioral functions during progression of murine INCL and suggest that Parkinson-like motor/sensorimotor deficits in Cln1−/− mice are not mediated by dopamine deficiency.

MRI and proton spectroscopy in children with late infantile neuronal ceroid lipofuscinosis: Preliminary results

Aim of the study was to characterize findings on magnetic resonance imaging (MRI) and proton magnetic resonance spectroscopy ( 1 H-MRS) in children with late infantile neuronal ceroid lipofuscinosis (NCL) and the relationship between these results and duration of the disorder. Three children with late infantile NCL and six age-matched controls were examined with MRI and by localized 1 H-MRS. Voxel regions studied were periventricular frontal white matter, thalami and cerebellar deep white matter. N-acetylaspartate/creatine (NAA/Cr) and choline/creatine (Cho/Cr) ratio was calculated. MRI showed various degrees of cortical atrophy and increased periventricular white matter signal intensity on T2- and -fluid attenuated inversion recovery-weighted images. There was no significant volume loss in the brainstem. Metabolic ratio (NAA/Cr and Cho/Cr) showed progressive decrease with longer disease duration in thalamic and cerebellar voxels and exhibited a negative relationship in frontal voxels. In conclusion, the most significant changes on neuroimaging in late infantile NCL are progressive cortical and cerebellar atrophy, which are more pronounced in the cerebellum. Decreased signal intensity in the thalami may also be observed as in other forms of NCL. 1 H-MRS is the most sensitive method for measuring metabolic changes. The decreased level of NAA although

Cln1gene disruption in mice reveals a common pathogenic link between two of the most lethal childhood neurodegenerative lysosomal storage disorders

Neurodegeneration is a devastating manifestation in the majority of >50 lysosomal storage disorders (LSDs). Neuronal ceroid lipofuscinoses (NCLs) are the most common childhood neurodegenerative LSDs. Mutations in 13 different genes (called CLNs) underlie various types of NCLs, of which the infantile NCL (INCL) and congenital NCL (CNCL) are the most lethal. Although inactivating mutations in the CLN1 gene encoding palmitoyl-protein thioesterase-1 (PPT1) cause INCL, those in the CLN10 gene encoding cathepsin D (CD) underlie CNCL. PPT1 is a lysosomal thioesterase that cleaves the thioester linkage in S-acylated proteins required for their degradation by lysosomal hydrolases like CD. Thus, PPT1 deficiency causes lysosomal accumulation of these lipidated proteins (major constituents of ceroid) leading to INCL. We sought to determine whether there is a common pathogenic link between INCL and CNCL. Using biochemical, histological and confocal microscopic analyses of brain tissues and cells from Cln1(-/-) mice that mimic INCL, we uncovered that Cln10/CD is overexpressed. Although synthesized in the endoplasmic reticulum, the CD-precursor protein (pro-CD) is transported through endosome to the lysosome where it is proteolytically processed to enzymatically active-CD. We found that despite Cln10 overexpression, the maturation of pro-CD to enzymatically active-CD in lysosome was disrupted. This defect impaired lysosomal degradative function causing accumulation of undegraded cargo in lysosome leading to INCL. Notably, treatment of intact Cln1(-/-) mice as well as cultured brain cells derived from these animals with a thioesterase-mimetic small molecule, N-tert-butyl-hydroxylamine, ameliorated the CD-processing defect. Our findings are significant in that they define a pathway in which Cln1 mutations disrupt the maturation of a major degradative enzyme in lysosome contributing to neuropathology in INCL and suggest that lysosomal CD deficiency is a common pathogenic link between INCL and CNCL.

Evaluation of disease progression in INCL by MR spectroscopy.

ObjectiveInfantile neuronal ceroid lipofuscinosis (INCL) is a devastating neurodegenerative storage disease caused by palmitoyl-protein thioesterase-1 deficiency, which impairs degradation of palmitoylated proteins (constituents of ceroid) by lysosomal hydrolases. Consequent lysosomal ceroid accumulation leads to neuronal injury. As part of a pilot study to evaluate treatment benefits of cysteamine bitartrate and N-acetylcysteine, we quantitatively measured brain metabolite levels using magnetic resonance spectroscopy (MRS).MethodsA subset of two patients from a larger treatment and follow-up study underwent serial quantitative single-voxel MRS examinations of five anatomical sites. Three echo times were acquired in order to estimate metabolite T2. Measured metabolite levels included correction for partial volume of cerebrospinal fluid. Comparison of INCL patients was made to a reference group composed of asymptomatic and minimally symptomatic Niemann-Pick disease type C patients.ResultsIn INCL patients, N-acetylaspartate (NAA) was abnormally low at all locations upon initial measurement, and further declined throughout the follow-up period. In the cerebrum (affected early in the disease course), choline and myo-inositol were initially elevated and fell during the follow-up period, whereas in the cerebellum and brainstem (affected later), choline and myo-inositol were initially normal and rose subsequently.InterpretationCholine and myo-inositol levels in our patients are consistent with patterns of neuroinflammation observed in two INCL mouse models. Low, persistently declining NAA was expected based on the progressive, irreversible nature of the disease. Progression of metabolite levels in INCL has not been previously quantified; therefore the results of this study serve as a reference for quantitative evaluation of future therapeutic interventions.

Selective N-Hydroxyhydantoin Carbamate Inhibitors of Mammalian Serine Hydrolases

Serine hydrolase inhibitors, which facilitate enzyme function assignment and are used to treat a range of human disorders, often act by an irreversible mechanism that involves covalent modification of the serine hydrolase catalytic nucleophile. The portion of mammalian serine hydrolases for which selective inhibitors have been developed, however, remains small. Here, we show that N-hydroxyhydantoin (NHH) carbamates are a versatile class of irreversible serine hydrolase inhibitors that can be modified on both the staying (carbamylating) and leaving (NHH) groups to optimize potency and selectivity. Synthesis of a small library of NHH carbamates and screening by competitive activity-based protein profiling furnished selective, in vivo-active inhibitors and tailored activity-based probes for multiple mammalian serine hydrolases, including palmitoyl protein thioesterase 1, mutations of which cause the human disease infantile neuronal ceroid lipofuscinosis.

Quantitative analysis of PPT1 interactome in human neuroblastoma cells.

Mutations in the CLN1 gene that encodes Palmitoyl protein thioesterase 1 (PPT1) or CLN1, cause Infantile NCL (INCL, MIM#256730). PPT1 removes long fatty acid chains such as palmitate from modified cysteine residues of proteins. The data shown here result from isolated protein complexes from PPT1-expressing SH-SY5Y stable cells that were subjected to single step affinity purification coupled to mass spectrometry (AP-MS). Prior to the MS analysis, we utilised a modified filter-aided sample preparation (FASP) protocol. Based on label free quantitative analysis of the data by SAINT, 23 PPT1 interacting partners (IP) were identified. A dense connectivity in PPT1 network was further revealed by functional coupling and extended network analyses, linking it to mitochondrial ATP synthesis coupled protein transport and thioester biosynthetic process. Moreover, the terms: inhibition of organismal death, movement disorders and concentration of lipid were predicted to be altered in the PPT1 network. Data presented here are related to Scifo et al. (J. Proteomics, 123 (2015) 42–53).

Protracted late infantile ceroid lipofuscinosis due to TPP1 mutations: Clinical, molecular and biochemical characterization in three sibs

ObjectiveThis work investigated the molecular cause responsible for a late-onset parkinsonism–dystonia phenotype in three Italian siblings, and clinically characterize this condition. MethodsExtensive neurophysiological and neuroradiological exams were performed on the three sibs. Most frequent late-onset metabolic diseases were ruled out through laboratory and biochemical analyses. A whole exome sequencing (WES) approach was used to identify the molecular cause underlying this condition. Results and conclusionsPeculiar neurologic phenotype was characterized by dystonia–parkinsonism, cognitive impairment, gait ataxia and apraxia, pyramidal signs. WES analysis allowed the identification of a compound heterozygosity for two nucleotide substitutions (c.1340G>A, p.R447H; c.790C>T, p.Q264X) affecting the TPP1 gene in the three affected siblings. Biochemical analyses demonstrated abrogated TPP1 catalytic activity in primary skin fibroblasts, but revealed residual activity in leukocytes. Our findings document that late infantile neuronal ceroid lipofuscinosis (CLN2), which is caused by TPP1 gene mutations, should be considered in the differential diagnosis of autosomal recessive dystonia–parkinsonism syndromes. The availability of enzyme replacement therapy and other therapeutic approaches for ceroid lipofuscinoses emphasizes the value of reaching an early diagnosis in patients with atypical and milder presentation of these disorders.

Sustained Neural Stem Cell-Based Intraocular Delivery of CNTF Attenuates Photoreceptor Loss in the nclf Mouse Model of Neuronal Ceroid Lipofuscinosis.

A sustained intraocular administration of neurotrophic factors is among the strategies aimed at establishing treatments for currently untreatable degenerative retinal disorders. In the present study we have analyzed the neuroprotective effects of a continuous neural stem (NS) cell-based intraocular delivery of ciliary neurotrophic factor (CNTF) on photoreceptor cells in the nclf mouse, an animal model of the neurodegenerative lysosomal storage disorder variant late infantile neuronal ceroid lipofuscinosis (vLINCL). To this aim, we genetically modified adherently cultivated NS cells with a polycistronic lentiviral vector encoding a secretable variant of CNTF together with a Venus reporter gene (CNTF-NS cells). NS cells for control experiments (control-NS cells) were modified with a vector encoding the reporter gene tdTomato. Clonal CNTF-NS and control-NS cell lines were established using fluorescent activated cell sorting and intravitreally grafted into 14 days old nclf mice at the onset of retinal degeneration. The grafted cells preferentially differentiated into astrocytes that were attached to the posterior side of the lenses and the vitreal side of the retinas and stably expressed the transgenes for at least six weeks, the latest post-transplantation time point analyzed. Integration of donor cells into host retinas, ongoing proliferation of grafted cells or adverse effects of the donor cells on the morphology of the host eyes were not observed. Quantitative analyses of host retinas two, four and six weeks after cell transplantation revealed the presence of significantly more photoreceptor cells in eyes with grafted CNTF-NS cells than in eyes with grafted control-NS cells. This is the first demonstration that a continuous intraocular administration of a neurotrophic factor attenuates retinal degeneration in an animal model of neuronal ceroid lipofuscinosis.

Intrathecal enzyme replacement therapy improves motor function and survival in a preclinical mouse model of infantile neuronal ceroid lipofuscinosis.

The neuronal ceroid lipofuscinoses (NCLs) are a group of related hereditary lysosomal storage disorders characterized by progressive loss of neurons in the central nervous system resulting in dementia, loss of motor skills, seizures and blindness. A characteristic intralysosomal accumulation of autofluorescent storage material occurs in the brain and other tissues. Three major forms and nearly a dozen minor forms of NCL are recognized. Infantile-onset NCL (CLN1 disease) is caused by severe deficiency in a soluble lysosomal enzyme, palmitoyl-protein thioesterase-1 (PPT1) and no therapy beyond supportive care is available. Homozygous Ppt1 knockout mice reproduce the known features of the disease, developing signs of motor dysfunction at 5 months of age and death around 8 months. Direct delivery of lysosomal enzymes to the cerebrospinal fluid is an approach that has gained traction in small and large animal models of several other neuropathic lysosomal storage diseases, and has advanced to clinical trials. In the current study, Ppt1 knockout mice were treated with purified recombinant human PPT1 enzyme delivered to the lumbar intrathecal space on each of three consecutive days at 6 weeks of age. Untreated PPT1 knockout mice and wild-type mice served as additional controls. Four enzyme concentration levels (0, 2.6, 5.3 and 10.6 mg/ml of specific activity 20 U/mg) were administered in a volume of 80 μl infused over 8 min. Each group consisted of 16-20 mice. The treatment was well tolerated. Disease-specific survival was 233, 267, 272, and 284days for each of the four treatment groups, respectively, and the effect of treatment was highly significant (p<0.0001). The timing of motor deterioration was also delayed. Neuropathology was improved as evidenced by decreased autofluorescent storage material in the spinal cord and a decrease in CD68 staining in the cortex and spinal cord. The improvements in motor function and survival are similar to results reported for preclinical studies involving other lysosomal storage disorders, such as CLN2/TPP1 deficiency, for which intraventricular ERT is being offered in clinical trials. If ERT delivery to the CSF proves to be efficacious in these disorders, PPT1 deficiency may also be amenable to this approach.

501. Radioiodinated Adeno-Associated Virus: A Promising New Approach for Monitoring Gene Therapy

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a form of Batten disease that is caused by mutations in the CLN2 gene. These defects cause widespread neurodegeneration resulting in death by the age of 10-12 years. One treatment for LINCL that has shown promise in animal and clinical studies is gene therapy using adeno-associated virus (AAV) as a vehicle to deliver the CLN2 gene throughout the brain. This is currently accomplished by direct infusion, but there is no way to measure the spatial distribution of administered vector. Iodine-124 labeling of the viral capsid may offer a means for non-invasive determination of spatial distribution using MicroPET imaging. The production of AAVrh.10CLN2 under GMP conditions met endotoxin, mycoplasma, sterility and transgene expression release criteria. Purified AAVrh.10CLN2 was concentrated to 1013 gene copies/ml. Labeling with Na124I was carried out at 2-5 0C under mild oxidizing conditions in pH 7.5 buffer. Following radiolabeling, the product mixture was purified using an anion exchange cartridge and centrifugal filtration. Purified I124-AAVrh.10CLN2 was formulated in a pH 7.4 PBS buffer. The sterile formulation was injected (2 ml at 2.5 μCi/ml) intraparenchymally to the striatum in the murine brain and imaged on a Siemens Inveon MicroPET scanner. Thirty minute PET scans were acquired for each mouse (n=3). For the control group, we performed the same procedure using free Na 124I alone (n=3). The radiolabeling efficiency of AAV was in the range of 12-14%. PET/CT imaging clearly demonstrated the spatial distribution of vector over a ten day period, with minimal 124I uptake in the unblocked thyroid. In contrast, free iodide was rapidly cleared from the brain within 2 days (Figure 1). In conclusion, adeno-associated virus was successfully labeled with 124I and its distribution in the mouse brain was observed. This radiolabeling approach has the potential for wide application in gene therapy trials. View Large Image | Download PowerPoint Slide

PP03.7 – 2754: KCTD7 mutation causing infantile neuronal ceroid lipofucinosis

Objective Neuronal ceroid lipofuscinosis (NCL) is one of the progressive myoclonic epilepsies (PME) of childhood with 14 types currently described. We present a case of NCL with a novel potassium channel tetramerization containing 7 (KCTD7) gene mutations. Case A 10 year old previously well boy presented at 23 months old with rapid onset global regression. Within 6 months he was non-ambulant and had developed generalised dystonia, non-epileptic myoclonus and epilepsy (tonic clonic seizures). Family history was unremarkable. The EEG at 29 months demonstrated a diffuse slow background and frequent independent spike and sharp waves bilaterally. MRI brain (with spectroscopy) was initially normal but at 5 and 7 years showed signal changes in both cerebellar hemispheres with mild global atrophy. Extensive neurometabolic investigations were normal including ultrastructural examination of lymphocytes, white cells and cultured fibroblasts lysosomal enzyme activity, muscle histology and respiratory chain enzymes, electroretinogram and visual evoked potentials were normal. Brain biopsy at 8 years demonstrated membrane-bound lysosomal accumulation of electron dense material in numerous neurons but did not contain curvilinear inclusion material characteristic of NCL. Repeat ultrastructural examination of lymphocytes and white cell enzyme activity was again normal. Results Genetic testing by next-generation sequencing, through DDD study, revealed autosomal recessive compound heterozygote mutations in KCTD7 gene exon 2 (c.190A>G, p. Thr64Ala) and 4 (c.631C>T, p.Arg211X). This was confirmed by DNA sequence analysis. The KCTD7 gene has recently been associated with infantile-onset NCL subtype CLN14 and PME type 3 (EPM3). Conclusion KCTD7 mutation testing should be considered in children with suspected infantile NCL when first line diagnostic tests are normal.

A lysosomal targeting defect of V0a1 suppresses V‐ATPase activity elevating lysosomal pH in Ppt1‐/‐ mice: amelioration by NtBuHA

Lysosomal pH regulation is impaired in virtually all lysosomal storage disorders (LSDs). However, the mechanism of this defect remains unexplained. The vacuolar H+‐transporting ATPase (v‐ATPase), a multi‐subunit protein‐complex, regulates endosomal/lysosomal pH. The reversible assembly of its membrane‐bound V0 sector and cytosolic V1 sector regulates acidification of the lysosomes. Infantile Neuronal Ceroid Lipofuscinoses (INCL) is a neurodegenerative LSD caused by mutations in the CLN1 gene encoding palmitoyl‐protein thioesterase‐1(PPT1), a depalmitoylating enzyme. Dynamic palmitoylation (palmitoylation‐depalmitoylation) is critical for regulating the functions of many proteins, which requires palmitoyl‐acyltransferases (PATs) for palmitoylation and palmitoyl‐protein thioesterases (PPTs) for depalmitoylation.Using Ppt1‐/‐ mice that mimic INCL, we uncovered that subunit a1 of V0‐ATPase (V0a1) requires palmitoylation on Cys‐25 for localization to endosomal/lysosomal membrane. V0a1 is transported via clathrin/AP2 pathway from plasma membrane to early endosomes where PPT1‐deficiency suppressed depalmitoylation impairing clathrin/AP2/V0a1 dissociation. This defect prevented interaction of V0a1 with AP3, essential for trafficking to lysosomes and clathrin/AP2/V0a1 was recycled back to the plasma membrane. Consequently, disruption of V0/V1 assembly on lysosomal membrane suppressed V‐ATPase activity, essential for pH regulation. Importantly, a PPT1‐mimetic, NtBuHA, ameliorated these defects. Our findings uncover a novel mechanism underlying increased lysosomal pH in INCL and suggest that varying factors impairing V‐ATPase activity may disrupt lysosomal pH regulation in other LSDs.