Sexual Divergence in Microtubule Function: The Novel Intranasal Microtubule Targeting SKIP Normalizes Axonal Transport and Enhances Memory

Activity-dependent neuroprotective protein (ADNP), essential for brain formation, is a frequent autism spectrum disorder (ASD)-mutated gene. ADNP associates with microtubule end binding proteins (EBs) through its SxIP motif, to regulate dendritic spine formation and brain plasticity. Here, we reveal SKIP, a novel 4 amino acid peptide representing an EB-binding site, as a replacement therapy in an outbred Adnp-deficient mouse model. We discovered, for the first time, axonal transport deficits in Adnp+/- mice (measured by manganese-enhanced magnetic resonance imaging), with significant male-female differences. Furthermore, the Adnp+/- mice exhibited impaired hippocampal expression of key ASD-linked genes including the serotonin transporter (Slc6a4), the calcium channel (VDCC) and the autophagy regulator, BECN1 (Beclin1), in a sex-dependent manner. RNA-seq evaluations corroborated, in part, immunohistochemical and functional results. Intranasal SKIP treatment normalized social memory in 8-9-month-old Adnp+/--treated mice to placebo-control levels, while protecting axonal transport and ameliorating changes in ASD-like gene expression. SKIP presents a novel lead compound for ASD drug development, a prevalent unmet medical need. 8 different samples were used, all in biological triplicates except for one sample with duplicates. Samples include 1 month and 5 months mice hippocampus of males and females, wild-type and Adnp+-.活性依赖神经保护蛋白（Activity-dependent Neuroprotective Protein, ADNP）是大脑形成所必需的蛋白质，同时也是自闭症谱系障碍（Autism Spectrum Disorder, ASD）的高频突变基因。ADNP可通过其SxIP基序（SxIP motif）与微管末端结合蛋白（Microtubule End Binding Proteins, EBs）结合，进而调控树突棘形成与大脑可塑性。本研究首次报道，SKIP——一种新型的、可结合EB的4氨基酸肽——可作为替代治疗手段，应用于远交系Adnp缺陷小鼠模型。本研究同时首次在Adnp杂合缺陷（Adnp+/-）小鼠中检测到轴突运输缺陷，该缺陷通过锰增强磁共振成像（Manganese-Enhanced Magnetic Resonance Imaging）检测，且存在显著的性别差异。此外，Adnp+/-小鼠的海马体中，与ASD相关的关键基因表达出现异常，包括5-羟色胺转运体（Serotonin Transporter, Slc6a4）、钙离子通道（VDCC）以及自噬调节因子BECN1（Beclin1），且该表达异常呈现性别依赖性。RNA测序（RNA-seq）结果在一定程度上验证了免疫组化与功能实验的结论。鼻腔给予SKIP可使8~9月龄的Adnp+/-模型小鼠的社交记忆恢复至安慰剂对照组水平，同时能够保护轴突运输功能，并改善ASD样基因的表达异常。SKIP有望成为ASD药物开发的新型先导化合物，而ASD作为一类高发疾病，目前仍存在未被满足的临床医疗需求。本研究共纳入8组样本：除1组样本采用生物学重复两次外，其余各组均为生物学重复三次。样本涵盖雌雄两性1月龄与5月龄小鼠的海马组织，包含野生型与Adnp+/-两种基因型。