CANAVAN DISEASE: SUPPRESSING THE PHENOTYPE BY INHIBITING NAA SYNTHESIS

 描述(申请人提供)：Canavan病是一种由天冬氨酸氨基转移酶(ASPA)失活突变引起的隐性遗传性疾病，其特征是前脑、小脑和脑干空泡变性、星形胶质细胞增生症和髓鞘萎缩(海绵状脑白质营养不良)。目前还没有治疗加那文病的方法被证明是有效的。天冬氨酸氨基转移酶是一种寡突胶质细胞酶，能裂解N-乙酰-L-天冬氨酸，释放乙酸酯和L-天冬氨酸。NAA是由神经元酶N-乙酰转移酶-8样(NAT8L)合成的，在正常的CNS中维持在约10 mM，但在Canavan病中，CNS NAA远远高于这一水平。一种流行的假说是，醋酸盐从NAA中释放出来，并被少突胶质细胞乙酰辅酶A合成酶转化为乙酰辅酶A，这是维持少突胶质细胞产脂乙酰辅酶A池所必需的，而Canavan病中的海绵状脑白质营养不良是由于少突胶质细胞乙酰辅酶A“饥饿”所致。但我们的初步数据挑战了NAA衍生的乙酸酯对中枢神经系统髓鞘脂类合成的必要性，因为我们的数据表明，在纯合子结构性Nat8L基因敲除(Nat8LKO/KO)小鼠的中枢神经系统中，乙酰辅酶A含量和髓鞘形成是正常的，其中NAA是1H-磁共振波谱无法检测到的。特定目标1将评估另一种假说，即Canavan病中的海绵状脑白质营养不良是中枢神经系统NAA浓度持续升高的结果。我们将使用一个特征良好的小鼠Canavan模型，该模型是由ASPA无意义突变(ASPAnur7)的纯合引起的。通过与携带ASPAnur7和Nat8LKO等位基因的小鼠杂交，将产生携带2、1或0个Nat8LKO等位基因的ASPAnur7/Nur7小鼠，并将用于测试抑制ASPAnur7/Nur7小鼠NAA合成将防止海绵状白质营养不良的预测。在特定的目标2中，我们将他莫昔芬应用于有症状的年轻成年ASPAnur7/Nur7小鼠，这些小鼠携带有广泛表达的他莫昔芬诱导的cre转基因基因(Nat8Lflx)，这些小鼠是条件Nat8L等位基因纯合的，以测试延迟抑制NAA合成是否会逆转它们先前存在的海绵状脑白质营养不良。如果是这样的话，预防Canavan病儿童中枢神经系统NAA水平升高的干预措施可能具有治疗价值。

 DESCRIPTION (provided by applicant): Canavan disease is a recessively inherited disorder caused by inactivating aspartoacylase (ASPA) mutations and characterized by forebrain, cerebellar, and brainstem vacuolar degeneration, astrogliosis, and dysmyelination ("spongiform leukodystrophy"). No treatments for children with Canavan disease have yet proven effective. ASPA is an oligodendroglial enzyme that cleaves N-acetyl-L-aspartate (NAA), releasing acetate and L-aspartate. NAA is synthesized by the neuronal enzyme N-acetyltransferase-8 like (NAT8L), and is maintained at approximately 10mM in the normal CNS, but in Canavan disease, CNS NAA climbs far above that level. A popular hypothesis is that acetate released from NAA and converted to acetyl-CoA by oligodendroglial acetyl-CoA synthetase is essential to maintain the oligodendroglial lipogenic acetyl-CoA pool, and that spongiform leukodystrophy in Canavan disease is attributable to oligodendroglial acetyl-CoA "starvation". But our preliminary data challenge the necessity for NAA-derived acetate for CNS myelin lipid synthesis by showing that, in the CNS of homozygous constitutive NAT8L knockout (NAT8LKO/KO) mice, in which NAA is undetectable by 1H-magnetic resonance spectroscopy, acetyl-CoA content and myelination are normal. Specific Aim 1 will evaluate an alternative hypothesis, that spongiform leukodystrophy in Canavan disease is a consequence of persistently elevated CNS concentrations of NAA. We will employ a well characterized mouse Canavan model caused by homozygosity for a nonsense ASPA mutation (ASPAnur7). By crossing mice carrying ASPAnur7 and NAT8LKO alleles, ASPAnur7/nur7 mice carrying 2, 1, or 0 NAT8LKO alleles will be generated, and will be used to test the prediction that suppressing NAA synthesis in ASPAnur7/nur7 mice will prevent spongiform leukodystrophy. In Specific Aim 2, we will administer tamoxifen to symptomatic young adult ASPAnur7/nur7 mice that are homozygous for a conditional NAT8L allele (NAT8Lflox) and carrying a widely expressed tamoxifen-inducible cre transgene to test whether delayed suppression of NAA synthesis will reverse their pre-existing spongiform leukodystrophy. If so, interventions to prevent elevated CNS NAA levels in children with Canavan disease may be of therapeutic value.