RESUMEN
Niemann-Pick disease type C1 (NPC1) is a lysosomal disorder due to impaired intracellular cholesterol transport out of the endolysosomal compartment.. Marked heterogeneity has been observed in individuals with the same NPC1 genotype, thus suggesting a significant effect of modifier genes. Prior work demonstrated that decreased SOAT1 activity decreased disease severity in an NPC1 mouse model. Thus, we hypothesized that a polymorphism associated with decreased SOAT1 expression might influence the NPC1 phenotype. Phenotyping and genomic sequencing of 117 individuals with NPC1 was performed as part of a Natural History trial. Phenotyping included determination of disease severity and disease burden. Significant clinical heterogeneity is present in individuals homozygous for the NPC1I1061T variant and in siblings. Analysis of the SOAT1 polymorphism, rs1044925 (A>C), showed a significant association of the C-allele with earlier age of neurological onset. The C-allele may be associated with a higher Annualized Severity Index Score as well as increased frequency of liver disease and seizures. A polymorphism associated with decreased expression of SOAT1 appears to be a genetic modifier of the NPC1 phenotype. This finding is consistent with prior data showing decreased phenotypic severity in Npc1-/-:Soat1-/- mice and supports efforts to investigate the potential of SOAT1 inhibitors as a potential therapy for NPC1.
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Enfermedad de Niemann-Pick Tipo C , Esterol O-Aciltransferasa , Enfermedad de Niemann-Pick Tipo C/genética , Enfermedad de Niemann-Pick Tipo C/metabolismo , Humanos , Masculino , Femenino , Esterol O-Aciltransferasa/genética , Esterol O-Aciltransferasa/metabolismo , Proteína Niemann-Pick C1 , Niño , Polimorfismo de Nucleótido Simple , Animales , Ratones , Fenotipo , Adolescente , Preescolar , Genes Modificadores , Adulto , Alelos , Índice de Severidad de la Enfermedad , Genotipo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Adulto JovenRESUMEN
[This corrects the article DOI: 10.1016/j.ymgmr.2023.101001.].
RESUMEN
Background: Creatine transporter deficiency (CTD) is a rare X-linked disorder of creatine transport caused by pathogenic variants in SLC6A8 (Xq28). The disorder is marked by developmental delay, especially speech delay. The biomarkers Aß40, Aß42 and total tau are abnormal in Alzheimer disease (AD), a common neurodegenerative disorder pathologically characterized by Aß peptide containing amyloid plaques and tau neurofibrillary tangles. Although CTD results in neuronal energy deficiency, the pathological processes underlying the CTD phenotype are not fully characterized. Methods: Cerebral spinal fluid (CSF) was collected as an optional part of a natural history study of CTD. Aß40, Aß42 and total tau levels were quantified in CSF from individuals with CTD and from age-appropriate comparison samples. Neuro3-Plex enzyme-linked immunoassay was performed on a Quanterix SR-X instrument. The Vineland Adaptive Behavior Scale, 3rd Edition was used to determine an overall Adaptive Behavior Composite (ABC) standard score. Results: CSF from 12 individuals with CTD and 23 age appropriate non-CTD comparison samples were analyzed. We found that levels of total tau [t(32) = 4.05, p = 0.0003], Aß40 [t(31) = 6.11, p < 0.0001], and Aß42 [t(32) = 3.20, p = 0.003] were elevated in the participants with CTD relative to the comparison group. Additionally, except for one individual that we considered an outlier, all three biomarkers correlated inversely with the adaptive behavior score (total tau: ρ = -0.60 [-0.88, 0.005]; Aß40: ρ = -0.67 [-0.91, -0.12]; Aß42: ρ = -0.62 [-0.89, -0.02]). Conclusion: We describe here the novel finding of elevated protein biomarkers in the CSF of individuals with CTD. Aß40, Aß42 and total tau are markedly elevated in individuals with CTD compared to comparison samples, and increased levels of these biomarkers inversely correlated with ABC scores. We hypothesize that elevated CSF levels of Aß40 and Aß42 are due to cellular energy deficiency. Elevated CSF total tau levels may indicate ongoing neuronal damage. The observed inverse correlation of Vineland ABC scores with increased biomarker levels needs to be confirmed in a larger CTD cohort; however, our observation of increased Aß40, Aß42 and total tau levels in CSF from individuals with CTD may provide insight into pathological mechanisms contributing to the CTD phenotype and may prove useful as supportive data in future therapeutic trials.
RESUMEN
BACKGROUND: Niemann-Pick disease, type C1 (NPC1) is an ultrarare, recessive disorder due to pathological variants of NPC1. The NPC1 phenotype is characterized by progressive cerebellar ataxia and cognitive impairment. Although classically a childhood/adolescent disease, NPC1 is heterogeneous with respect to the age of onset of neurological signs and symptoms. While miglustat has shown to be clinically effective, there are currently no FDA approved drugs to treat NPC1. Identification and characterization of biomarkers may provide tools to facilitate therapeutic trials. Ubiquitin C-terminal hydrolase-L1 (UCHL1) is a protein which is highly expressed by neurons and is a biomarker of neuronal damage. We thus measured cerebrospinal fluid (CSF) levels of UCHL1 in individuals with NPC1. METHODS: CSF levels of UCHL1 were measured using a Quanterix Neuroplex 4 assay in 94 individuals with NPC1 and 35 age-appropriate comparison samples. Cross-sectional and longitudinal CSF UCHL1 levels were then evaluated for correlation with phenotypic measures and treatment status. RESULTS: CSF UCHL1 levels were markedly elevated (3.3-fold) in individuals with NPC1 relative to comparison samples. The CSF UCHL1 levels showed statistically significant (adj p < 0.0001), moderate, positive correlations with both the 17- and 5-domain NPC Neurological Severity Scores and the Annual Severity Increment Scores. Miglustat treatment significantly decreased (adj p < 0.0001) CSF UCHL1 levels by 30% (95% CI 17-40%). CONCLUSIONS: CSF UCHL1 levels are elevated in NPC1, increase with increasing clinical severity and decrease in response to therapy with miglustat. Based on these data, UCHL1 may be a useful biomarker to monitor disease progression and therapeutic response in individuals with NPC1.
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Enfermedad de Niemann-Pick Tipo C , Adolescente , Niño , Humanos , Biomarcadores/metabolismo , Estudios Transversales , Enfermedad de Niemann-Pick Tipo C/tratamiento farmacológico , Enfermedad de Niemann-Pick Tipo C/genética , Enfermedad de Niemann-Pick Tipo C/metabolismo , Fenotipo , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/uso terapéuticoRESUMEN
BACKGROUND: Niemann-Pick disease, type C1 (NPC1) is an ultrarare, recessive, lethal, lysosomal disease characterized by progressive cerebellar ataxia and cognitive impairment. Although the NPC1 phenotype is heterogeneous with variable age of onset, classical NPC1 is a pediatric disorder. Currently there are no therapies approved by the FDA and therapeutics trials for NPC1 are complicated by disease rarity, heterogeneity, and the relatively slow rate of neurological decline. Thus, identification of disease relevant biomarkers is necessary to provide tools that can support drug development efforts for this devastating neurological disease. METHODS: Proximal extension assays (O-link® Explore 1536) were used to compare cerebrospinal fluid (CSF) samples from individuals with NPC1 enrolled in a natural history study and non-NPC1 comparison samples. Relative expression levels of 1467 proteins were determined, and candidate protein biomarkers were identified by evaluating fold-change and adjusted Kruskal-Wallis test p-values. Selected proteins were orthogonally confirmed using ELISA. To gain insight into disease progression and severity we evaluated the altered protein expression with respect to clinically relevant phenotypic aspects: NPC Neurological Severity Score (NPC1 NSS), Annual Severity Increment Score (ASIS) and age of neurological onset. RESULTS: This study identified multiple proteins with altered levels in CSF from individuals with NPC1 compared to non-NPC1 samples. These included proteins previously shown to be elevated in NPC1 (NEFL, MAPT, CHIT1, CALB1) and additional proteins confirmed by orthogonal assays (PARK7, CALB2/calretinin, CHI3L1/YKL-40, MIF, CCL18 and ENO2). Correlations with clinically relevant phenotypic parameters demonstrated moderate negative (p = 0.0210, r = -0.41) and possible moderate positive (p = 0.0631, r = 0.33) correlation of CSF CALB2 levels with age of neurological onset and ASIS, respectively. CSF CHI3L1 levels showed a moderate positive (p = 0.0183, r = 0.40) correlation with the concurrent NPC1 NSS. A strong negative correlation (p = 0.0016, r = -0.648) was observed between CSF CCL18 and age of neurological onset for childhood/adolescent cases. CSF CCL18 levels also showed a strong positive correlation (p = 0.0017, r = 0.61) with ASIS. CONCLUSION: Our study identified and validated multiple proteins in CSF from individuals with NPC1 that are candidates for further investigation in a larger cohort. These analytes may prove to be useful as supportive data in therapeutic trials. TRIAL REGISTRATIONS: NCT00344331, NCT00001721, NCT02931682.
RESUMEN
Complex asparagine-linked glycosylation plays key roles in cellular functions, including cellular signaling, protein stability, and immune response. Previously, we characterized the appearance of a complex asparagine-linked glycosylated form of lysosome-associated membrane protein 1 (LAMP1) in the cerebellum of Npc1-/- mice. This LAMP1 form was found on activated microglia, and its appearance correlated both spatially and temporally with cerebellar Purkinje neuron loss. To test the importance of complex asparagine-linked glycosylation in NPC1 pathology, we generated NPC1 knock-out mice deficient in MGAT5, a key Golgi-resident glycosyl transferase involved in complex asparagine-linked glycosylation. Our results show that Mgat5-/-:Npc1-/- mice were smaller than Mgat5+/+:Npc1-/- mice, and exhibited earlier NPC1 disease onset and reduced lifespan. Western blot and lectin binding analyses of cerebellar extracts confirmed the reduction in complex asparagine-linked glycosylation, and the absence of the hyper-glycosylated LAMP1 previously observed. Western blot analysis of cerebellar extracts demonstrated reduced calbindin staining in Mgat5-/-:Npc1-/- mice compared to Mgat5+/+:Npc1-/- mutant mice, and immunofluorescent staining of cerebellar sections indicated decreased levels of Purkinje neurons and increased astrogliosis in Mgat5-/-:Npc1-/- mice. Our results suggest that reduced asparagine-linked glycosylation increases NPC1 disease severity in mice, and leads to the hypothesis that mutations in genes involved in asparagine-linked glycosylation may contribute to disease severity progression in individuals with NPC1. To examine this with respect to MGAT5, we analyzed 111 NPC1 patients for two MGAT5 SNPs associated with multiple sclerosis; however, we did not identify an association with NPC1 phenotypic severity.
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N-Acetilglucosaminiltransferasas , Enfermedad de Niemann-Pick Tipo C , Animales , Asparagina/metabolismo , Asparagina/farmacología , Glicosilación , Humanos , Ratones , Ratones Endogámicos BALB C , N-Acetilglucosaminiltransferasas/metabolismo , Enfermedad de Niemann-Pick Tipo C/metabolismo , Enfermedad de Niemann-Pick Tipo C/patologíaRESUMEN
Identifying meaningful predictors of therapeutic efficacy from preclinical studies is challenging. However, clinical manifestations occurring in both patients and mammalian models offer significant translational value. Many neurological disorders, including inherited, metabolic Niemann-Pick disease, type C (NPC), exhibit ataxia. Both individuals with NPC and murine models manifest ataxia, and investigational therapies impacting this phenotype in mice have been reported to slow disease progression in patients (e.g. miglustat, intrathecal 2-hydroxypropyl-beta-cyclodextrin, and acetyl-L-leucine). Reproducible phenotypic scoring of animal models can facilitate comparisons between genotypes, sexes, disease course, and therapies. Previously, other groups have developed a composite phenotypic scoring system (CPSS), which was subsequently used to distinguish strain-dependent phenotypes and, with modifications, to evaluate potential therapies. However, high inter-rater reliability is paramount to widespread use. We have created a comprehensive, easy-to-follow phenotypic assessment based on the CPSS and have verified its reproducibility using murine models of NPC disease. Application of this scoring system is not limited to NPC disease and may be applicable to other models of neurodegeneration exhibiting motor incoordination, thereby increasing its utility in translational studies.
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Enfermedad de Niemann-Pick Tipo C , Animales , Ataxia/diagnóstico , Ataxia/etiología , Modelos Animales de Enfermedad , Humanos , Mamíferos , Ratones , Enfermedad de Niemann-Pick Tipo C/diagnóstico , Enfermedad de Niemann-Pick Tipo C/tratamiento farmacológico , Enfermedad de Niemann-Pick Tipo C/genética , Fenotipo , Reproducibilidad de los ResultadosRESUMEN
Niemann-Pick disease, type C1 is a progressive, lethal, neurodegenerative disorder due to endolysosomal storage of unesterified cholesterol. Cerebellar ataxia, as a result of progressive loss of cerebellar Purkinje neurons, is a major symptom of Nieman-Pick disease, type C1. Comparing single cell RNAseq data from control (Npc1+/+) and mutant (Npc1-/-) mice, we observed significantly decreased expression of Slc1a3 in Npc1-/- astrocytes. Slc1a3 encodes a glutamate transporter (GLAST, EAAT1) which functions to decrease glutamate concentrations in the post synaptic space after neuronal firing. Glutamate is an excitatory neurotransmitter and elevated extracellular levels of glutamate can be neurotoxic. Impaired EAAT1 function underlies type-6 episodic ataxia, a rare disorder with progressive cerebellar dysfunction, thus suggesting that impaired glutamate uptake in Niemann-Pick disease, type C1 could contribute to disease progression. We now show that decreased expression of Slc1a3 in Npc1-/- mice has functional consequences that include decreased surface protein expression and decreased glutamate uptake by Npc1-/- astrocytes. To test whether glutamate neurotoxicity plays a role in Niemann-Pick disease, type C1 progression, we treated NPC1 deficient mice with ceftriaxone and riluzole. Ceftriaxone is a ß-lactam antibiotic that is known to upregulate the expression of Slc1a2, an alternative glial glutamate transporter. Although ceftriaxone increased Slc1a2 expression, we did not observe a treatment effect in NPC1 mutant mice. Riluzole is a glutamate receptor antagonist that inhibits postsynaptic glutamate receptor signaling and reduces the release of glutamate. We found that treatment with riluzole increased median survival in Npc1-/- by 12%. Given that riluzole is an approved drug for the treatment of amyotrophic lateral sclerosis, repurposing of this drug may provide a novel therapeutic approach to decrease disease progression in Niemann-Pick disease type, C1 patients.
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Ceftriaxona/uso terapéutico , Ácido Glutámico/toxicidad , Enfermedad de Niemann-Pick Tipo C/tratamiento farmacológico , Riluzol/uso terapéutico , Animales , Astrocitos/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Transportador 1 de Aminoácidos Excitadores/fisiología , Femenino , Ácido Glutámico/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Proteína Niemann-Pick C1/fisiologíaRESUMEN
PURPOSE: Creatine transporter deficiency (CTD) is a rare X-linked disorder of creatine transport caused by pathogenic variants in SLC6A8 (Xq28). CTD features include developmental delay, seizures, and autism spectrum disorder. This study was designed to investigate CTD cardiac phenotype and sudden death risk. METHODS: We performed a cross-sectional analysis of CTD males between 2017 and 2020. Subjects underwent evaluation with electrocardiogram (ECG), echocardiography, and ambulatory ECG with comparable analysis in creatine transporter deficient mice (Slc6a8-/y) using ECG, echocardiography, exercise testing, and indirect calorimetry. RESULTS: Eighteen subjects with CTD (18 males, age 7.4 [3.8] years) were evaluated: seven subjects (39%) had QTc ≥ 470 milliseconds: 510.3 ± 29.0 vs. 448.3 ± 15.9, P < 0.0001. The QTc ≥ 470 milliseconds cohort had increased left ventricular internal dimension (diastole) ([LVIDd] Z-score: 0.22 ± 0.74, n = 7 vs. -0.93 ± 1.0, n = 11, P = 0.0059), and diminished left ventricular posterior wall dimension (diastole) ([LVPWDd, in mm]: 5.0 ± 0.6, n = 7 vs. 5.7 ± 0.8, n = 11, P = 0.0183), when compared to subjects with normal or borderline QTc prolongation. Similar ECG and echocardiographic abnormalities were seen in Slc6a8-/y mice. Additionally, Slc6a8-/y mice had diminished survival (65%). CONCLUSION: Prolonged QTc and abnormal echocardiographic parameters consistent with developing cardiomyopathy are seen in some male subjects with CTD. Slc6a8-/y mice recapitulated these cardiac abnormalities. Male CTD subjects may be at increased risk for cardiac dysfunction and sudden death.
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Trastorno del Espectro Autista , Creatina , Animales , Encefalopatías Metabólicas Innatas , Creatina/deficiencia , Estudios Transversales , Muerte Súbita , Humanos , Masculino , Discapacidad Intelectual Ligada al Cromosoma X , Ratones , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/deficienciaRESUMEN
Niemann-Pick type C1 (NPC1) is a rare neurodegenerative disease. In NPC1 mouse cerebella, the antibacterial enzyme, lysozyme (Lyz2), is significantly increased in multiple cell types. Due to its possible role in toxic fibril deposition, we confirmed Lyz2 overexpression in culture in different control and NPC1 cell types including human NPC1 fibroblasts. Lyz2 expression is induced by Toll-like receptors potentially in response to lipid storage but does not play a functional role in NPC disease pathology.
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Péptidos y Proteínas de Señalización Intracelular/genética , Muramidasa/genética , Enfermedad de Niemann-Pick Tipo C/genética , Receptores Toll-Like/genética , Animales , Astrocitos/metabolismo , Fibroblastos , Expresión Génica/genética , Humanos , Ratones , Ratones Noqueados , Microglía/metabolismo , Proteína Niemann-Pick C1 , Enfermedad de Niemann-Pick Tipo C/patologíaRESUMEN
Niemann-Pick disease, type C1 (NPC1) is a lysosomal disease characterized by endolysosomal storage of unesterified cholesterol and decreased cellular cholesterol bioavailability. A cardinal symptom of NPC1 is cerebellar ataxia due to Purkinje neuron loss. To gain an understanding of the cerebellar neuropathology we obtained single cell transcriptome data from control (Npc1+/+) and both three-week-old presymptomatic and seven-week-old symptomatic mutant (Npc1-/-) mice. In seven-week-old Npc1-/- mice, differential expression data was obtained for neuronal, glial, vascular, and myeloid cells. As anticipated, we observed microglial activation and increased expression of innate immunity genes. We also observed increased expression of innate immunity genes by other cerebellar cell types, including Purkinje neurons. Whereas neuroinflammation mediated by microglia may have both neuroprotective and neurotoxic components, the contribution of increased expression of these genes by non-immune cells to NPC1 pathology is not known. It is possible that dysregulated expression of innate immunity genes by non-immune cells is neurotoxic. We did not anticipate a general lack of transcriptomic changes in cells other than microglia from presymptomatic three-week-old Npc1-/- mice. This observation suggests that microglia activation precedes neuronal dysfunction. The data presented in this paper will be useful for generating testable hypotheses related to disease progression and Purkinje neurons loss as well as providing insight into potential novel therapeutic interventions.
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Cerebelo , Perfilación de la Expresión Génica , Microglía , Enfermedad de Niemann-Pick Tipo C , Células de Purkinje , Análisis de la Célula Individual , Animales , Cerebelo/metabolismo , Cerebelo/patología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Microglía/metabolismo , Microglía/patología , Enfermedad de Niemann-Pick Tipo C/genética , Enfermedad de Niemann-Pick Tipo C/metabolismo , Enfermedad de Niemann-Pick Tipo C/patología , Células de Purkinje/metabolismo , Células de Purkinje/patologíaRESUMEN
Niemann-Pick disease, type C1, is a cholesterol storage disease where unesterified cholesterol accumulates intracellularly. In the cerebellum this causes neurodegeneration of the Purkinje neurons that die in an anterior-to-posterior and time-dependent manner. This results in cerebellar ataxia as one of the major outcomes of the disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a significant role in the regulation of serum cholesterol levels by modulating LDL receptor levels on peripheral tissues. In the central nervous system, PCSK9 may have a similar effect on the closely related VLDL and ApoE2 receptors to regulate brain cholesterol. In addition, regulation of VLDLR and ApoER2 by PCSK9 may contribute to neuronal apoptotic pathways through Reelin, the primary ligand of VLDLR and ApoER2. Defects in reelin signaling results in cerebellar dysfunction leading to ataxia as seen in the Reeler mouse. Our recent findings that Pcsk9 is expressed ~8-fold higher in the anterior lobules of the cerebellum compared to the posterior lobule X, which is resistant to neurodegeneration, prompted us to ask whether PCSK9 could play a role in NPC1 disease progression. We addressed this question genetically, by characterizing NPC1 disease in the presence or absence of PCSK9. Analysis of double mutant Pcsk9-/-/Npc1-/- mice by disease severity scoring, motor assessments, lifespan, and cerebellar Purkinje cell staining, showed no obvious difference in NPC1 disease progression with that of Npc1-/- mice. This suggests that PCSK9 does not play an apparent role in NPC1 disease progression.
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Colesterol/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Enfermedad de Niemann-Pick Tipo C/genética , Enfermedad de Niemann-Pick Tipo C/metabolismo , Proproteína Convertasa 9/genética , Proproteína Convertasa 9/metabolismo , Animales , Apolipoproteína E2 , Cerebelo/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Enfermedades Neurodegenerativas , Proteína Niemann-Pick C1 , Células de Purkinje/metabolismo , Receptores de LDL/metabolismo , Proteína ReelinaRESUMEN
A hallmark of Niemann-Pick disease, type C (NPC) is the progressive degeneration of Purkinje neurons in the cerebellum caused by the accumulation of free cholesterol and glycosphingolipids in the lysosome. Recent studies suggest that the state of glycosylation of lysosomal membrane proteins may play a role in disease progression. Our study has identified the presence of a highly glycosylated form of Lysosome Associated Membrane Protein 1 (LAMP1) that correlated spatiotemporally with Purkinje neuron loss. This form of LAMP1 was predominantly localized to activated microglia; showing a ~5-fold increase in surface labeling by FACS analysis. This suggests a potential role for LAMP1 in the neuro-inflammatory process in these mice during disease progression. Analysis of other mouse models of neurodegeneration that exhibit neuro-inflammation showed little or no presence of this glycosylated form of LAMP1, suggesting this observation for LAMP1 is specific to NPC disease. Furthermore, early treatment of Npc1-/- mice with 2-hydroxypropyl-ß-cyclodextrin (HPßCD), significantly prevented the appearance of the glycosylated LAMP1 in the cerebellum of Npc1-/- mice at 7 weeks, consistent with the prevention of neuro-inflammation in mice treated with this drug. Treatment of Npc1-/- mice with HPßCD at 7 weeks, after disease onset, did not reverse or prevent further appearance of the hyperglycosylated LAMP1, demonstrating that once this aspect of neuro-inflammation began, it continued despite the HPßCD treatment. Analysis of LAMP1 in cerebellar tissue of NPC1 patients showed a small level of hyperglycosylated LAMP1 in the tissue, however, this was not seen in the CSF of patients.
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Proteínas de Membrana de los Lisosomas/metabolismo , Enfermedad de Niemann-Pick Tipo C/patología , Animales , Cerebelo/metabolismo , Cerebelo/patología , Modelos Animales de Enfermedad , Femenino , Glicosilación , Humanos , Proteínas de Membrana de los Lisosomas/análisis , Masculino , Ratones Endogámicos BALB C , Enfermedad de Niemann-Pick Tipo C/metabolismo , Células de Purkinje/metabolismo , Células de Purkinje/patologíaRESUMEN
BACKGROUND: Lysosomal storage diseases (LSD) are a large family of inherited disorders characterized by abnormal endolysosomal accumulation of cellular material due to catabolic enzyme and transporter deficiencies. Depending on the affected metabolic pathway, LSD manifest with somatic or central nervous system (CNS) signs and symptoms. Neuroinflammation is a hallmark feature of LSD with CNS involvement such as mucolipidosis type IV, but not of others like Fabry disease. METHODS: We investigated the properties of microglia from LSD with and without major CNS involvement in 2-month-old mucolipidosis type IV (Mcoln1-/-) and Fabry disease (Glay/-) mice, respectively, by using a combination of flow cytometric, RNA sequencing, biochemical, in vitro and immunofluorescence analyses. RESULTS: We characterized microglia activation and transcriptome from mucolipidosis type IV and Fabry disease mice to determine if impaired lysosomal function is sufficient to prime these brain-resident immune cells. Consistent with the neurological pathology observed in mucolipidosis type IV, Mcoln1-/- microglia demonstrated an activation profile with a mixed neuroprotective/neurotoxic expression pattern similar to the one we previously observed in Niemann-Pick disease, type C1, another LSD with significant CNS involvement. In contrast, the Fabry disease microglia transcriptome revealed minimal alterations, consistent with the relative lack of CNS symptoms in this disease. The changes observed in Mcoln1-/- microglia showed significant overlap with alterations previously reported for other common neuroinflammatory disorders including Alzheimer's, Parkinson's, and Huntington's diseases. Indeed, our comparison of microglia transcriptomes from Alzheimer's disease, amyotrophic lateral sclerosis, Niemann-Pick disease, type C1 and mucolipidosis type IV mouse models showed an enrichment in "disease-associated microglia" pattern among these diseases. CONCLUSIONS: The similarities in microglial transcriptomes and features of neuroinflammation and microglial activation in rare monogenic disorders where the primary metabolic disturbance is known may provide novel insights into the immunopathogenesis of other more common neuroinflammatory disorders. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01067742, registered on February 12, 2010.
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Microglía/metabolismo , Mucolipidosis/genética , Mucolipidosis/patología , Transcriptoma , Animales , Enfermedad de Fabry/genética , Enfermedad de Fabry/metabolismo , Enfermedad de Fabry/patología , Humanos , Ratones , Ratones Transgénicos , Microglía/patología , Mucolipidosis/metabolismoRESUMEN
: Niemann-Pick disease, type C1 (NPC1) is a lysosomal disease characterized by progressive cerebellar ataxia. In NPC1, a defect in cholesterol transport leads to endolysosomal storage of cholesterol and decreased cholesterol bioavailability. Purkinje neurons are sensitive to the loss of NPC1 function. However, degeneration of Purkinje neurons is not uniform. They are typically lost in an anterior-to-posterior gradient with neurons in lobule X being resistant to neurodegeneration. To gain mechanistic insight into factors that protect or potentiate Purkinje neuron loss, we compared RNA expression in cerebellar lobules III, VI, and X from control and mutant mice. An unexpected finding was that the gene expression differences between lobules III/VI and X were more pronounced than those observed between mutant and control mice. Functional analysis of genes with anterior to posterior gene expression differences revealed an enrichment of genes related to neuronal cell survival within the posterior cerebellum. This finding is consistent with the observation, in multiple diseases, that posterior Purkinje neurons are, in general, resistant to neurodegeneration. To our knowledge, this is the first study to evaluate anterior to posterior transcriptome-wide changes in gene expression in the cerebellum. Our data can be used to not only explore potential pathological mechanisms in NPC1, but also to further understand cerebellar biology.
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Cerebelo , Regulación de la Expresión Génica , Enfermedad de Niemann-Pick Tipo C/metabolismo , Células de Purkinje , Animales , Cerebelo/metabolismo , Cerebelo/patología , Modelos Animales de Enfermedad , Ratones , Ratones Noqueados , Enfermedad de Niemann-Pick Tipo C/genética , Enfermedad de Niemann-Pick Tipo C/patología , Células de Purkinje/metabolismo , Células de Purkinje/patologíaRESUMEN
Niemann-Pick disease, type C1 (NPC1) is a neurodegenerative disorder with limited treatment options. NPC1 is associated with neuroinflammation; however, attempts to therapeutically target neuroinflammation in NPC1 have had mixed success. We show here that NPC1 neuroinflammation is characterized by an atypical microglia activation phenotype. Specifically, Npc1-/- microglia demonstrated altered morphology, reduced levels of lineage markers and a shift toward glycolytic metabolism. Treatment with 2-hydroxypropyl-ß-cyclodextrin (HPßCD), a drug currently being studied in a phase 2b/3 clinical trial, reversed all microglia-associated defects in Npc1-/- animals. In addition, impairing microglia mediated neuroinflammation by genetic deletion of IRF8 led to decreased symptoms and increased lifespan. We identified CD22 as a marker of dysregulated microglia in Npc1 mutant mice and subsequently demonstrated that elevated cerebrospinal fluid levels of CD22 in NPC1 patients responds to HPßCD administration. Collectively, these data provide the first in-depth analysis of microglia function in NPC1 and suggest possible new therapeutic approaches.
Asunto(s)
Inflamación/tratamiento farmacológico , Proteína Niemann-Pick C1/genética , Enfermedad de Niemann-Pick Tipo C/tratamiento farmacológico , Lectina 2 Similar a Ig de Unión al Ácido Siálico/genética , 2-Hidroxipropil-beta-Ciclodextrina/administración & dosificación , Adolescente , Adulto , Animales , Cerebelo/efectos de los fármacos , Cerebelo/patología , Niño , Preescolar , Modelos Animales de Enfermedad , Femenino , Humanos , Lactante , Inflamación/genética , Inflamación/patología , Factores Reguladores del Interferón , Masculino , Ratones , Microglía/efectos de los fármacos , Microglía/patología , Enfermedad de Niemann-Pick Tipo C/líquido cefalorraquídeo , Enfermedad de Niemann-Pick Tipo C/genética , Enfermedad de Niemann-Pick Tipo C/patología , Lectina 2 Similar a Ig de Unión al Ácido Siálico/líquido cefalorraquídeoRESUMEN
BACKGROUND: Niemann-Pick disease, type C (NPC) is a rare lysosomal storage disorder characterized by progressive neurodegeneration, splenomegaly, hepatomegaly, and early death. NPC is caused by mutations in either the NPC1 or NPC2 gene. Impaired NPC function leads to defective intracellular transport of unesterified cholesterol and its accumulation in late endosomes and lysosomes. A high frequency of Crohn disease has been reported in NPC1 patients, suggesting that gastrointestinal tract pathology may become a more prominent clinical issue if effective therapies are developed to slow the neurodegeneration. The Npc1 nih mouse model on a BALB/c background replicates the hepatic and neurological disease observed in NPC1 patients. Thus, we sought to characterize the gastrointestinal tract pathology in this model to determine whether it can serve as a model of Crohn disease in NPC1. METHODS: We analyzed the gastrointestinal tract and isolated macrophages of BALB/cJ cNctr-Npc1m1N/J (Npc1-/-) mouse model to determine whether there was any Crohn-like pathology or inflammatory cell activation. We also evaluated temporal changes in the microbiota by 16S rRNA sequencing of fecal samples to determine whether there were changes consistent with Crohn disease. RESULTS: Relative to controls, Npc1 mutant mice demonstrate increased inflammation and crypt abscesses in the gastrointestinal tract; however, the observed pathological changes are significantly less than those observed in other Crohn disease mouse models. Analysis of Npc1 mutant macrophages demonstrated an increased response to lipopolysaccharides and delayed bactericidal activity; both of which are pathological features of Crohn disease. Analysis of the bacterial microbiota does not mimic what is reported in Crohn disease in either human or mouse models. We did observe significant increases in cyanobacteria and epsilon-proteobacteria. The increase in epsilon-proteobacteria may be related to altered cholesterol homeostasis since cholesterol is known to promote growth of this bacterial subgroup. CONCLUSIONS: Macrophage dysfunction in the BALB/c Npc1-/- mouse is similar to that observed in other Crohn disease models. However, neither the degree of pathology nor the microbiota changes are typical of Crohn disease. Thus, this mouse model is not a good model system for Crohn disease pathology reported in NPC1 patients.
Asunto(s)
Enfermedad de Crohn/etiología , Enfermedad de Crohn/patología , Tracto Gastrointestinal/patología , Enfermedad de Niemann-Pick Tipo C/patología , Animales , Modelos Animales de Enfermedad , Tracto Gastrointestinal/microbiología , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos BALB C , Enfermedad de Niemann-Pick Tipo C/microbiologíaRESUMEN
Report of a human with a homozygous truncating null mutation of the Carboxypeptidase E (CPE) gene with endocrinological and neurological deficits prompted us to search for other mutations in the human CPE gene that might be linked to disease. We searched an EST database and identified from a small population of patients, a novel T to C single nucleotide polymorphism (SNP) in the CPE gene at bp980 of exon 4, herein called TC-CPE. This introduces a tryptophan to arginine (W235R) mutation in the catalytic domain of human CPE protein. Over-expression of TC-CPE in N2A cells, a neuroendocrine cell line, showed that it was synthesized, but was found in lesser amounts compared to over-expressed WT-CPE in these cells. Furthermore, TC-CPE was secreted poorly from these N2A cells. The levels of TC-CPE were significantly increased after the N2A cells were treated with MG132 (a proteasome inhibitor), suggesting that TC-CPE was targeted to proteasomes for degradation in N2A cells. In addition, TC-CPE induced ER stress as demonstrated by the increased expression of CHOP in N2A cells. Double labeling of CPE and calnexin (and ER marker) suggested the accumulation of TC-CPE in the ER, and the accumulation appears to be enhanced by the treatment of MG132 in the cells. Moreover, the secreted levels of TC-CPE were not affected by the treatment of MG132 in the cells. Over-expression studies revealed that while N2A cells transfected with WT-CPE showed reduced cytotoxicity when challenged with H2O2 compared to cells expressing an empty vector, cells transfected with TC-CPE had no effect. Furthermore, WT-CPE condition medium showed protective effect against oxidative stress, but not TC-CPE condition medium. Although co-expression of WT-CPE and TC-CPE in N2A cells resulted in the reduction in secretion of WT-CPE, co-expression of WT-CPE and TC-CPE did not significantly affect the protective effect of WT-CPE. Taken together, we have identified a novel SNP in the CPE gene which results in the loss of its neuroprotective function in cells and may confer neurological disorders in humans.
Asunto(s)
Carboxipeptidasa H/genética , Sistema Nervioso/patología , Polimorfismo de Nucleótido Simple , Línea Celular , Humanos , Sistemas de Lectura AbiertaRESUMEN
Embryonic neurodevelopment involves inhibition of proliferation of multipotent neural stem cells (NSCs) followed by differentiation into neurons, astrocytes and oligodendrocytes to form the brain. We have identified a new neurotrophic factor, NF-α1, which inhibits proliferation and promotes differentiation of NSC/progenitors derived from E13.5 mouse cortex. Inhibition of proliferation of these cells was mediated through negatively regulating the Wnt pathway and decreasing ß-catenin. NF-α1 induced differentiation of NSCs to astrocytes by enhancing Glial Fibrillary Acidic Protein (GFAP) expression through activating the ERK1/2-Sox9 signaling pathway. Cultured E13.5 cortical stem cells from NF-α1-knockout mice showed decreased astrocyte numbers compared to wild-type mice, which was rescued by treatment with NF-α1. In vivo, immunocytochemistry of brain sections and Western blot analysis of neocortex of mice showed a gradual increase of NF-α1 expression from E14.5 to P1 and a surge of GFAP expression at P1, the time of increase in astrogenesis. Importantly, NF-α1-Knockout mice showed â¼49% fewer GFAP positive astrocytes in the neocortex compared to WT mice at P1. Thus, NF-α1 is critical for regulating antiproliferation and cell fate determination, through differentiating embryonic stem cells to GFAP-positive astrocytes for normal neurodevelopment. Stem Cells 2017;35:557-571.