RESUMO
Unstable proteins are prone to form non-native interactions with other proteins and thereby may become toxic. To mitigate this, destabilized proteins are targeted by the protein quality control network. Here we present systematic studies of the cytosolic aspartoacylase, ASPA, where variants are linked to Canavan disease, a lethal neurological disorder. We determine the abundance of 6152 of the 6260 ( ~ 98%) possible single amino acid substitutions and nonsense ASPA variants in human cells. Most low abundance variants are degraded through the ubiquitin-proteasome pathway and become toxic upon prolonged expression. The data correlates with predicted changes in thermodynamic stability, evolutionary conservation, and separate disease-linked variants from benign variants. Mapping of degradation signals (degrons) shows that these are often buried and the C-terminal region functions as a degron. The data can be used to interpret Canavan disease variants and provide insight into the relationship between protein stability, degradation and cell fitness.
Assuntos
Amidoidrolases , Doença de Canavan , Proteólise , Humanos , Amidoidrolases/genética , Amidoidrolases/metabolismo , Doença de Canavan/genética , Doença de Canavan/metabolismo , Células HEK293 , Substituição de Aminoácidos , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Estabilidade Proteica , Ubiquitina/metabolismo , TermodinâmicaRESUMO
Canavan disease (CD, OMIM# 271900) is an autosomal recessive neurodegenerative disorder caused by homozygous or compound heterozygous mutations in ASPA gene, which result in catalytic deficiency of the aspartoacylase enzyme and the accumulation of N-acetylaspartic acid (NAA). Clinical presentation varies according to the age of disease onset. Here, we generated a human induced pluripotent stem cell line (hiPSCs) SDQLCHi064-A from a 5-month old boy with CD carrying two novel frame shift mutations c.556_559dupGTTC (p.L187Rfs*5) and c.919delA (p.S307Vfs*24) of the ASPA gene, in order for us to better understanding the disease.
Assuntos
Doença de Canavan , Células-Tronco Pluripotentes Induzidas , Masculino , Humanos , Lactente , Doença de Canavan/genética , Doença de Canavan/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação/genética , Homozigoto , Amidoidrolases/genética , Amidoidrolases/metabolismoRESUMO
Canavan disease (CD) is a leukodystrophy caused by mutations in the N-acetylaspartate (NAA) hydrolase aspartoacylase (ASPA). Inability to degrade NAA and its accumulation in the brain results in spongiform myelin degeneration. NAA is mainly synthesized by neurons, where it is also a precursor of the neuropeptide N-acetylaspartylglutamate (NAAG). Hydrolysis of this peptide by glutamate carboxypeptidases is an additional source of extracellular NAA besides the instant neuronal release of NAA. This study examines to what extent NAA released from NAAG contributes to NAA accumulation and pathogenesis in the brain of Aspanur7/nur7 mutant mice, an established model of CD. Towards this aim, Aspanur7/nur7 mice with additional deficiencies in NAAG synthetase genes Rimklb and/or Rimkla were generated. Loss of myelin in Aspanur7/nur7 mice was not significantly affected by Rimkla and Rimklb deficiency and there was also no obvious change in the extent of brain vacuolation. Astrogliosis was slightly reduced in the forebrain of Rimkla and Rimklb double deficient Aspanur7/nur7 mice. However, only minor improvements at the behavioral level were found. The brain NAA accumulation in CD mice was, however, not significantly reduced in the absence of NAAG synthesis. In summary, there was only a weak tendency towards reduced pathogenic symptoms in Aspanur7/nur7 mice deficient in NAAG synthesis. Therefore, we conclude that NAAG metabolism has little influence on NAA accumulation in Aspanur7/nur7 mice and development of pathological symptoms in CD.
Assuntos
Doença de Canavan , Camundongos , Animais , Doença de Canavan/genética , Doença de Canavan/metabolismo , Doença de Canavan/patologia , Encéfalo/patologia , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Neurônios/metabolismo , Amidoidrolases/genética , Amidoidrolases/metabolismo , Modelos Animais de Doenças , Ácido Aspártico/metabolismo , Ligases/metabolismoRESUMO
N-acetyl-L-aspartic acid (NAA) is a prominent amino acid derivative primarily associated with vertebrate brain metabolism. This review delineates the critical role of NAA across various cell types and its significance in pathophysiological contexts, including Canavan disease and cancer metabolism. Although traditionally linked with myelination and aspartoacylase-driven carbon donation, its significance as a carbon source for myelination remains debated. Evidence suggests that intact NAA might substantially impact cellular signaling, particularly processes such as histone acetylation. Beyond the brain, NAA metabolism's relevance is evident in diverse tissues, such as adipocytes, immune cells, and notably, cancer cells. In several cancer types, there is an observed upregulation of NAA synthesis accompanied by a simultaneous downregulation of its degradation. This pattern highlights the potential signaling role of intact NAA in disease.
Assuntos
Doença de Canavan , Neoplasias , Humanos , Ácido Aspártico/metabolismo , Encéfalo/metabolismo , Doença de Canavan/metabolismo , Carbono/metabolismo , Neoplasias/metabolismoRESUMO
Canavan disease (CD) is a recessively inherited pediatric leukodystrophy resulting from inactivating mutations to the oligodendroglial enzyme aspartoacylase (ASPA). ASPA is responsible for hydrolyzing the amino acid derivative N-acetyl-L-aspartate (NAA), and without it, brain NAA concentrations increase by 50% or more. Infants and children with CD present with progressive cognitive and motor delays, cytotoxic edema, astroglial vacuolation, and prominent spongiform brain degeneration. ASPA-deficient CD mice (Aspanur7/nur7 ) present similarly with elevated NAA, widespread astroglial dysfunction, ataxia, and Purkinje cell (PC) dendritic atrophy. Bergmann glia (BG), radial astrocytes essential for cerebellar development, are intimately intertwined with PCs, where they regulate synapse stability, functionality, and plasticity. BG damage is common to many neurodegenerative conditions and frequently associated with PC dysfunction and ataxia. Here, we report that, in CD mice, BG exhibit significant morphological alterations, decreased structural associations with PCs, loss of synaptic support proteins, and altered calcium dynamics. We also find that BG dysfunction predates cerebellar vacuolation and PC damage in CD mice. Previously, we developed an antisense oligonucleotide (ASO) therapy targeting Nat8l (N-acetyltransferase-8-like, "Nat8l ASO") that inhibits the production of NAA and reverses ataxia and PC atrophy in CD mice. Here, we show that Nat8l ASO administration in adult CD mice also leads to BG repair. Furthermore, blocking astroglial uptake of NAA is neuroprotective in astroglia-neuron cocultures exposed to elevated NAA. Our findings suggest that restoration of BG structural and functional integrity could be a mechanism for PC regeneration and improved motor function.
Assuntos
Doença de Canavan , Doenças Neurodegenerativas , Humanos , Criança , Lactente , Camundongos , Animais , Doença de Canavan/genética , Doença de Canavan/metabolismo , Doença de Canavan/patologia , Cálcio , Ataxia/patologia , Oligodendroglia/metabolismo , Doenças Neurodegenerativas/patologia , Ácido Aspártico , Atrofia/complicações , Atrofia/patologiaRESUMO
Demyelinating disorders are among the most common and debilitating diseases in neurology. Canavan disease (CD) is a lethal demyelinating disease caused by mutation of the aspartoacylase (ASPA) gene, which leads to the accumulation of its substrate N-acetyl-l-aspartate (NAA), and consequently demyelination and vacuolation in the brain. In this study, hypoimmunogenic human induced pluripotent stem cell (iPSC)-derived oligodendrocyte progenitor cells (OPC) are developed from a healthy donor as an "off-the-shelf" cell therapy. Hypoimmunogenic iPSCs are generated through CRISPR/Cas9 editing of the human leukocyte antigen (HLA) molecules in healthy donor-derived iPSCs and differentiated into OPCs. The OPCs are engrafted into the brains of CD (nur7) mice and exhibit widespread distribution in the brain. The engrafted OPCs mature into oligodendrocytes that express the endogenous wildtype ASPA gene. Consequently, the transplanted mice exhibit elevated human ASPA expression and enzymatic activity and reduced NAA level in the brain. The transplanted OPCs are able to rescue major pathological features of CD, including defective myelination, extensive vacuolation, and motor function deficits. Moreover, the hypoimmunogenic OPCs exhibit low immunogenicity both in vitro and in vivo. The hypoimmunogenic OPCs can be used as "off-the-shelf" universal donor cells to treat various CD patients and many other demyelinating disorders, especially autoimmune demyelinating diseases, such as multiple sclerosis.
Assuntos
Doença de Canavan , Células-Tronco Pluripotentes Induzidas , Esclerose Múltipla , Células Precursoras de Oligodendrócitos , Humanos , Camundongos , Animais , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Células Precursoras de Oligodendrócitos/patologia , Oligodendroglia/metabolismo , Doença de Canavan/genética , Doença de Canavan/metabolismo , Doença de Canavan/patologiaRESUMO
Myelinating oligodendrocytes are essential for neuronal communication and homeostasis of the central nervous system (CNS). One of the most abundant molecules in the mammalian CNS is N-acetylaspartate (NAA), which is catabolized into L-aspartate and acetate by the enzyme aspartoacylase (ASPA) in oligodendrocytes. The resulting acetate moiety is thought to contribute to myelin lipid synthesis. In addition, affected NAA metabolism has been implicated in several neurological disorders, including leukodystrophies and demyelinating diseases such as multiple sclerosis. Genetic disruption of ASPA function causes Canavan disease, which is hallmarked by increased NAA levels, myelin and neuronal loss, large vacuole formation in the CNS, and early death in childhood. Although NAA's direct role in the CNS is inconclusive, in peripheral adipose tissue, NAA-derived acetate has been found to modify histones, a mechanism known to be involved in epigenetic regulation of cell differentiation. We hypothesize that a lack of cellular differentiation in the brain contributes to the disruption of myelination and neurodegeneration in diseases with altered NAA metabolism, such as Canavan disease. Our study demonstrates that loss of functional Aspa in mice disrupts myelination and shifts the transcriptional expression of neuronal and oligodendrocyte markers towards less differentiated stages in a spatiotemporal manner. Upon re-expression of ASPA, these oligodendrocyte and neuronal lineage markers are either improved or normalized, suggesting that NAA breakdown by Aspa plays an essential role in the maturation of neurons and oligodendrocytes. Also, this effect of ASPA re-expression is blunted in old mice, potentially due to limited ability of neuronal, rather than oligodendrocyte, recovery.
Assuntos
Doença de Canavan , Camundongos , Animais , Doença de Canavan/genética , Doença de Canavan/metabolismo , Linhagem da Célula , Epigênese Genética , Sistema Nervoso Central/metabolismo , Oligodendroglia , Bainha de Mielina/metabolismo , MamíferosRESUMO
Canavan disease is caused by ASPA mutations that diminish brain aspartoacylase activity, and it is characterized by excessive brain storage of the aspartoacylase substrate, N-acetyl-l-aspartate (NAA), and by astroglial and intramyelinic vacuolation. Astroglia and the arachnoid mater express sodium-dependent dicarboxylate transporter (NaDC3), encoded by SLC13A3, a sodium-coupled transporter for NAA and other dicarboxylates. Constitutive Slc13a3 deletion in aspartoacylase-deficient Canavan disease mice prevents brain NAA overaccumulation, ataxia, and brain vacuolation. ANN NEUROL 2021;90:845-850.
Assuntos
Encéfalo/efeitos dos fármacos , Doença de Canavan/metabolismo , Transportadores de Ácidos Dicarboxílicos/metabolismo , Simportadores/genética , Animais , Astrócitos/metabolismo , Encéfalo/metabolismo , Transportadores de Ácidos Dicarboxílicos/genética , Modelos Animais de Doenças , Camundongos Transgênicos , Doenças Neurodegenerativas/genética , Simportadores/metabolismoAssuntos
Doença de Canavan , Substância Branca , Amidoidrolases/genética , Doença de Canavan/diagnóstico por imagem , Doença de Canavan/genética , Doença de Canavan/metabolismo , Doença de Canavan/patologia , Pré-Escolar , Humanos , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Masculino , Substância Branca/diagnóstico por imagem , Substância Branca/patologiaRESUMO
Canavan disease, a leukodystrophy caused by loss-of-function ASPA mutations, is characterized by brain dysmyelination, vacuolation, and astrogliosis ("spongiform leukodystrophy"). ASPA encodes aspartoacylase, an oligodendroglial enzyme that cleaves the abundant brain amino acid N-acetyl-L-aspartate (NAA) to L-aspartate and acetate. Aspartoacylase deficiency results in a 50% or greater elevation in brain NAA concentration ([NAAB]). Prior studies showed that homozygous constitutive knockout of Nat8l, the gene encoding the neuronal NAA synthesizing enzyme N-acetyltransferase 8-like, prevents aspartoacylase-deficient mice from developing spongiform leukodystrophy. We now report that brain Nat8l knockdown elicited by intracerebroventricular/intracisternal administration of an adeno-associated viral vector carrying a short hairpin Nat8l inhibitory RNA to neonatal aspartoacylase-deficient AspaNur7/Nur7 mice lowers [NAAB] and suppresses development of spongiform leukodystrophy.
Assuntos
Acetiltransferases/genética , Amidoidrolases/deficiência , Doença de Canavan/genética , Doença de Canavan/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Doença de Canavan/patologia , Doença de Canavan/fisiopatologia , Dependovirus/genética , Modelos Animais de Doenças , Expressão Gênica , Técnicas de Silenciamento de Genes , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Camundongos , Camundongos Knockout , Atividade Motora , Neurônios/metabolismo , RNA Mensageiro/genética , Transdução GenéticaRESUMO
N-Acetylaspartate (NAA) is the second most abundant organic metabolite in the brain, but its physiological significance remains enigmatic. Toxic NAA accumulation appears to be the key factor for neurological decline in Canavan disease-a fatal neurometabolic disorder caused by deficiency in the NAA-degrading enzyme aspartoacylase. To date clinical outcome of gene replacement therapy for this spongiform leukodystrophy has not met expectations. To identify the target tissue and cells for maximum anticipated treatment benefit, we employed comprehensive phenotyping of novel mouse models to assess cell type-specific consequences of NAA depletion or elevation. We show that NAA-deficiency causes neurological deficits affecting unconscious defensive reactions aimed at protecting the body from external threat. This finding suggests, while NAA reduction is pivotal to treat Canavan disease, abrogating NAA synthesis should be avoided. At the other end of the spectrum, while predicting pathological severity in Canavan disease mice, increased brain NAA levels are not neurotoxic per se. In fact, in transgenic mice overexpressing the NAA synthesising enzyme Nat8l in neurons, supra-physiological NAA levels were uncoupled from neurological deficits. In contrast, elimination of aspartoacylase expression exclusively in oligodendrocytes elicited Canavan disease like pathology. Although conditional aspartoacylase deletion in oligodendrocytes abolished expression in the entire CNS, the remaining aspartoacylase in peripheral organs was sufficient to lower NAA levels, delay disease onset and ameliorate histopathology. However, comparable endpoints of the conditional and complete aspartoacylase knockout indicate that optimal Canavan disease gene replacement therapies should restore aspartoacylase expression in oligodendrocytes. On the basis of these findings we executed an ASPA gene replacement therapy targeting oligodendrocytes in Canavan disease mice resulting in reversal of pre-existing CNS pathology and lasting neurological benefits. This finding signifies the first successful post-symptomatic treatment of a white matter disorder using an adeno-associated virus vector tailored towards oligodendroglial-restricted transgene expression.
Assuntos
Ácido Aspártico/análogos & derivados , Encéfalo/metabolismo , Encéfalo/patologia , Doença de Canavan/metabolismo , Doença de Canavan/terapia , Acetiltransferases/metabolismo , Amidoidrolases/administração & dosagem , Amidoidrolases/genética , Amidoidrolases/metabolismo , Animais , Ácido Aspártico/metabolismo , Encéfalo/diagnóstico por imagem , Doença de Canavan/patologia , Modelos Animais de Doenças , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Potenciais Evocados Visuais/fisiologia , Feminino , Terapia Genética , Humanos , Masculino , Camundongos Transgênicos , Neurônios/metabolismo , Neurônios/patologia , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Fenótipo , RNA Mensageiro/metabolismoRESUMO
Aspartoacylase (ASPA) is a zinc-dependent abundant enzyme in the brain, which catalyzes the conversion of N-acetyl aspartate (NAA) into acetate and aspartate. Mutations in the ASPA gene are associated with the development of Canavan disease (CD), leading to the deficiency of ASPA activity. Patients with CD were characterized by degeneration of the white matter of the brain. We reported earlier on two patients with severe form of CD that both had two novel missense mutations in the ASPA: c.427 A > G; p. I143V and c.557 T > A; p. V186D (Zaki et al. 2017a), patient 1 harbored both mutations (p.I143V and p.V186D) in a heterozygous form together with four other mutations, and patient 2 had both mutations in homozygous form. Wijayasinghe et al. (2014) crystallized the 3D structures of four different ASPA mutants (p.K213E, p.Y231C, p.E285A, and p.F295S). In this study, we used in silico prediction methods and molecular dynamics simulation (MDS) to understand the structural impact of all these mutations. Moreover, we used molecular docking (MD) to investigate the binding patterns of the NAA substrate to the native and mutant proteins. Among the mutations, p.E285A (crystallized mutant) was predicted to be the most deleterious for the protein function and the least deleteriousness mutant was the p.I143V (novel mutant). Among the novel mutations, p.V186D was observed to be disruptive for both the zinc binding and NAA binding than the p.I143V. This study provides practical insights on the effect of these mutations on the ASPA function and might serve as a platform for drug design for CD treatment.
Assuntos
Amidoidrolases/genética , Encéfalo/metabolismo , Doença de Canavan/genética , Amidoidrolases/metabolismo , Doença de Canavan/metabolismo , Bases de Dados Genéticas , Humanos , Modelos Moleculares , Simulação de Acoplamento MolecularRESUMO
Canavan disease (CD) is a debilitating and lethal leukodystrophy caused by mutations in the aspartoacylase (ASPA) gene and the resulting defect in N-acetylaspartate (NAA) metabolism in the CNS and peripheral tissues. Recombinant adeno-associated virus (rAAV) has the ability to cross the blood-brain barrier and widely transduce the CNS. We developed a rAAV-based and optimized gene replacement therapy, which achieves early, complete, and sustained rescue of the lethal disease phenotype in CD mice. Our treatment results in a super-mouse phenotype, increasing motor performance of treated CD mice beyond that of WT control mice. We demonstrate that this rescue is oligodendrocyte independent, and that gene correction in astrocytes is sufficient, suggesting that the establishment of an astrocyte-based alternative metabolic sink for NAA is a key mechanism for efficacious disease rescue and the super-mouse phenotype. Importantly, the use of clinically translatable high-field imaging tools enables the noninvasive monitoring and prediction of therapeutic outcomes for CD and might enable further investigation of NAA-related cognitive function.
Assuntos
Amidoidrolases/genética , Doença de Canavan/terapia , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Amidoidrolases/metabolismo , Animais , Ácido Aspártico/análogos & derivados , Barreira Hematoencefálica/metabolismo , Doença de Canavan/genética , Doença de Canavan/metabolismo , Sistema Nervoso Central , Dependovirus/genética , Modelos Animais de Doenças , Humanos , Camundongos , Oligodendroglia/metabolismoRESUMO
Canavan disease is a leukodystrophy caused by aspartoacylase (ASPA) deficiency. The lack of functional ASPA, an enzyme enriched in oligodendroglia that cleaves N-acetyl-l-aspartate (NAA) to acetate and l-aspartic acid, elevates brain NAA and causes "spongiform" vacuolation of superficial brain white matter and neighboring gray matter. In children with Canavan disease, neuroimaging shows early-onset dysmyelination and progressive brain atrophy. Neuron loss has been documented at autopsy in some cases. Prior studies have shown that mice homozygous for the Aspa nonsense mutation Nur7 also develop brain vacuolation. We now report that numbers of cerebral cortical and cerebellar neurons are decreased and that cerebral cortex progressively thins in AspaNur7/Nur7 mice. This neuronal pathology is prevented by constitutive disruption of Nat8l, which encodes the neuronal NAA-synthetic enzyme N-acetyltransferase-8-like. SIGNIFICANCE STATEMENT: This is the first demonstration of cortical and cerebellar neuron depletion and progressive cerebral cortical thinning in an animal model of Canavan disease. Genetic suppression of N-acetyl-l-aspartate (NAA) synthesis, previously shown to block brain vacuolation in aspartoacylase-deficient mice, also prevents neuron loss and cerebral cortical atrophy in these mice. These results suggest that lowering the concentration of NAA in the brains of children with Canavan disease would prevent or slow progression of neurological deficits.
Assuntos
Ácido Aspártico/análogos & derivados , Doença de Canavan/metabolismo , Modelos Animais de Doenças , Neurônios/metabolismo , Animais , Ácido Aspártico/biossíntese , Ácido Aspártico/deficiência , Ácido Aspártico/genética , Doença de Canavan/genética , Doença de Canavan/patologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/patologiaRESUMO
Canavan disease (CD) is a rare fatal childhood neurological autosomal recessive genetic disease caused by mutations in the ASPA gene, which lead to catalytic deficiency of the ASPA enzyme, which catalyzes the hydrolysis of N-acetyl-L-aspartate (NAA) into aspartate and acetate. CD occurs frequently among Ashkenazi Jewish population, however it has been reported in many other ethnic groups with significantly lower frequency. Here, we report on two Egyptian patients diagnosed with CD, the first patient harbors five missense mutations (c.427 A > G; p. I143V, c.502C > T; p. R168C, c.530 T > C; p. I177T, c.557 T > C; p. V186D c.548C > T; p. P183L) and a silent mutation (c.693 C > T; p. Y231Y). The second patient was found to be homozygous for two missense mutations (c.427 A > G; p. I143V and c.557 T > A; p. V186D). Furthermore, molecular modeling of the novel mutation p. P183L provides an instructive explanation of the mutational impact on the protein structure that can affect the function of the ASPA. Here, the clinical, radiological, and biochemical profile of the two patients are reviewed in details.
Assuntos
Encéfalo/diagnóstico por imagem , Doença de Canavan/metabolismo , Modelos Moleculares , Mutação , Doença de Canavan/diagnóstico por imagem , Doença de Canavan/genética , Humanos , Lactente , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Masculino , Mutação de Sentido Incorreto , Conformação ProteicaRESUMO
Canavan disease is a fatal neurological disorder caused by defects in the metabolism of N-acetyl-l-aspartate (NAA). Recent work has shown that the devastating symptoms of this disorder are correlated with the elevated levels of NAA observed in these patients, caused as a consequence of the inability of mutated forms of aspartoacylase to adequately catalyze its breakdown. The membrane-associated enzyme responsible for the synthesis of NAA, aspartate N-acetyltransferase (ANAT), has recently been purified and examined (Wang et al., Prot Expr Purif. 2016;119:11). With the availability, for the first time, of a stable and soluble form of ANAT we can now report the identification of initial inhibitors against this biosynthetic enzyme, obtained from the screening of several focused compound libraries. Two core structures of these moderate binding compounds have subsequently been optimized, with the most potent inhibitors in these series possessing sub-micromolar inhibition constants (Ki values) against ANAT. Slowing the production of NAA via the inhibition of ANAT will lower the elevated levels of this metabolite and can potentially serve as a treatment option to moderate the symptoms of Canavan disease.
Assuntos
Acetiltransferases/antagonistas & inibidores , Doença de Canavan/tratamento farmacológico , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Acetiltransferases/metabolismo , Doença de Canavan/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Aspartoacylase (ASPA) is an abundant enzyme in the brain, which catalyzes the conversion of N-acetylaspartate into acetate and aspartate, deficiency in its activity leads to degeneration of the white matter of the brain and is a recognized cause of Canavan disease (CD), which affect children. Although genotype-phenotype correlation have been reported for Canavan disease patients, this relationships is still not straightforward. In this communication, we use molecular modeling to address the structural consequences resulting from the missense variant p.V31F in the ASPA enzyme, which we previously reported in a homozygous form in an Egyptian patient with infantile CD. This modeling suggests that this variant brings significant changes to the catalytic core by introducing structural flexibility through neighbouring key residues. In particular, it provides a molecular explanation for the pathogenic effect of this variant and provides a meaningful genotype-phonotype relationships. The mutational impact appears to have an influence on the function of the protein and initiates molecular event for the mechanism of the disease.
Assuntos
Amidoidrolases/genética , Doença de Canavan/genética , Mutação , Amidoidrolases/metabolismo , Doença de Canavan/metabolismo , Genótipo , Humanos , Modelos MolecularesRESUMO
Canavan disease is caused by inactivating ASPA (aspartoacylase) mutations that prevent cleavage of N-acetyl-L-aspartate (NAA), resulting in marked elevations in central nervous system (CNS) NAA and progressively worsening leukodystrophy. We now report that ablating NAA synthesis by constitutive genetic disruption of Nat8l (N-acetyltransferase-8 like) permits normal CNS myelination and prevents leukodystrophy in a murine Canavan disease model.
Assuntos
Ácido Aspártico/análogos & derivados , Doença de Canavan/metabolismo , Doença de Canavan/prevenção & controle , Modelos Animais de Doenças , Animais , Ácido Aspártico/deficiência , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Doença de Canavan/genética , Feminino , Masculino , Camundongos , Camundongos KnockoutRESUMO
The primary purpose of this work was to assess long-term in vitro reproducibility of metabolite levels measured using 1H MRS (proton magnetic resonance spectroscopy). The secondary purpose was to use the in vitro results for interpretation of 1H MRS in vivo spectra acquired from patients diagnosed with Canavan disease. 1H MRS measurements were performed in the period from April 2006 to September 2010. 118 short and 116 long echo spectra were acquired from a stable phantom during this period. Change-point analysis of the in vitro N-acetylaspartate levels was exploited in the computation of fT factor (ratio of the actual to the reference N-acetylaspartate level normalized by the reciprocity principle). This coefficient was utilized in the interpretation of in vivo spectra analyzed using absolute reference technique. The monitored time period was divided into six time intervals based on short echo in vitro data (seven time intervals based on long echo in vitro data) characterized by fT coefficient ranging from 0.97 to 1.09 (based on short echo data) and from 1.0 to 1.11 (based on long echo data). Application of this coefficient to interpretation of in vivo spectra confirmed increased N-acetylaspartate level in Canavan disease. Long-term monitoring of an MRS system reproducibility, allowing for absolute referencing of metabolite levels, facilitates interpretation of metabolic changes in white matter disorders.
Assuntos
Ácido Aspártico/análogos & derivados , Encéfalo/metabolismo , Doença de Canavan/diagnóstico , Doença de Canavan/metabolismo , Diagnóstico por Computador/métodos , Espectroscopia de Prótons por Ressonância Magnética/instrumentação , Espectroscopia de Prótons por Ressonância Magnética/métodos , Adolescente , Adulto , Algoritmos , Ácido Aspártico/metabolismo , Biomarcadores/metabolismo , Criança , Pré-Escolar , Feminino , Humanos , Técnicas In Vitro , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Pesquisa Translacional Biomédica , Adulto JovemRESUMO
Canavan Disease (CD) is a leukodystrophy caused by homozygous null mutations in the gene encoding aspartoacylase (ASPA). ASPA-deficiency is characterized by severe psychomotor retardation, and excessive levels of the ASPA substrate N-acetylaspartate (NAA). ASPA is an oligodendrocyte marker and it is believed that CD has a central etiology. However, ASPA is also expressed by Schwann cells and ASPA-deficiency in the periphery might therefore contribute to the complex CD pathology. In this study, we assessed peripheral and central auditory function in the AspalacZ/lacZ rodent model of CD using auditory brainstem response (ABR). Increased ABR thresholds and the virtual loss of waveform peaks 4 and 5 from AspalacZ/lacZ mice, indicated altered central auditory processing in mutant mice compared with Aspawt/wt controls and altered central auditory processing. Analysis of ABR latencies recorded from AspalacZ/lacZ mice revealed that the speed of nerve conduction was unchanged in the peripheral part of the auditory pathway, and impaired in the CNS. Histological analyses confirmed that ASPA was expressed in oligodendrocytes and Schwann cells of the auditory system. In keeping with our physiological results, the cellular organization of the cochlea, including the organ of Corti, was preserved and the spiral ganglion nerve fibres were normal in ASPA-deficient mice. In contrast, we detected substantial hypomyelination in the central auditory system of AspalacZ/lacZ mice. In summary, our data suggest that the lack of ASPA in the CNS is responsible for the observed hearing deficits, while ASPA-deficiency in the cochlear nerve fibres is tolerated both morphologically and functionally.