CASK-related neurodevelopmental disorders are untreatable. Affected children show variable severity, with microcephaly, intellectual disability (ID), and short stature as common features. X-linked human CASK shows dosage sensitivity with haploinsufficiency in females. CASK protein has multiple domains, binding partners, and proposed functions at synapses and in the nucleus. Human and Drosophila CASK show high amino-acid-sequence similarity in all functional domains. Flies homozygous for a hypomorphic CASK mutation (∆18) have motor and cognitive deficits. A Drosophila genetic model of CASK-related disorders could have great scientific and translational value. We assessed the effects of CASK loss of function on morphological phenotypes in Drosophila using established genetic, histological, and primary neuronal culture approaches. NeuronMetrics software was used to quantify neurite-arbor morphology. Standard nonparametric statistics methods were supplemented by linear mixed effects modeling in some cases. Microfluidic devices of varied dimensions were fabricated and numerous fluid-flow parameters were used to induce oscillatory stress fields on CNS tissue. Dissociation into viable neurons and neurite outgrowth in vitro were assessed. We demonstrated that ∆18 homozygous flies have small brains, small heads, and short bodies. When neurons from developing CASK-mutant CNS were cultured in vitro, they grew small neurite arbors with a distinctive, quantifiable “bushy” morphology that was significantly rescued by transgenic CASK+. As in humans, the bushy phenotype showed dosage-sensitive severity. To overcome the limitations of manual tissue trituration for neuronal culture, we optimized the design and operation of a microfluidic system for standardized, automated dissociation of CNS tissue into individual viable neurons. Neurons from CASK-mutant CNS dissociated in the microfluidic system recapitulate the bushy morphology. Moreover, for any given genotype, device-dissociated neurons grew larger arbors than did manually dissociated neurons. This automated dissociation method is also effective for rodent CNS. These biological and engineering advances set the stage for drug discovery using the Drosophila model of CASK-related disorders. The bushy phenotype provides a cell-based assay for compound screening. Nearly a dozen genes encoding CASK-binding proteins or transcriptional targets also have brain-development mutant phenotypes, including ID. Hence, drugs that improve CASK phenotypes might also benefit children with disorders due to mutant CASK partners.

中文翻译：

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果蝇 CASK 调节大脑大小和神经元形态发生，提供适合药物发现的出生后小头畸形遗传模型

CASK 相关的神经发育障碍是无法治疗的。受影响的儿童表现出不同的严重程度，以小头畸形、智力障碍 (ID) 和身材矮小为常见特征。X 连锁的人类 CASK 在女性中表现出单倍体不足的剂量敏感性。CASK 蛋白具有多个结构域、结合伙伴以及在突触和细胞核中的拟议功能。人类和果蝇 CASK 在所有功能域中都显示出高度的氨基酸序列相似性。具有亚等位性 CASK 突变 (Δ18) 的纯合果蝇具有运动和认知缺陷。CASK 相关疾病的果蝇遗传模型可能具有巨大的科学和转化价值。我们使用已建立的遗传、组织学和原代神经元培养方法评估了 CASK 功能丧失对果蝇形态表型的影响。NeuronMetrics 软件用于量化神经突-乔木形态。在某些情况下，标准非参数统计方法由线性混合效应模型进行补充。制造了不同尺寸的微流体装置，并使用大量流体流动参数在中枢神经系统组织上诱导振荡应力场。评估体外解离成活神经元和神经突生长。我们证明 Δ18 纯合果蝇的大脑较小，头部较小，身体较短。当来自发育中的 CASK 突变中枢神经系统的神经元在体外培养时，它们会长出小的神经突乔木，具有独特的、可量化的“浓密”形态，而转基因 CASK+ 显着挽救了这种形态。与人类一样，浓密的表型表现出剂量敏感的严重程度。为了克服神经元培养的手动组织研磨的局限性，我们优化了微流体系统的设计和操作，以将中枢神经系统组织标准化、自动分离成单个可行的神经元。来自微流体系统中解离的 CASK 突变中枢神经系统的神经元再现了浓密的形态。此外，对于任何给定的基因型，装置分离的神经元比手动分离的神经元长出更大的乔木。这种自动解离方法对啮齿动物中枢神经系统也有效。这些生物和工程进展为利用 CASK 相关疾病的果蝇模型发现药物奠定了基础。浓密的表型为化合物筛选提供了基于细胞的测定方法。近十几个编码 CASK 结合蛋白或转录靶标的基因也具有大脑发育突变表型，包括 ID。因此，改善 CASK 表型的药物也可能使因 CASK 伴侣突变而患有疾病的儿童受益。