Isolated loss of the AUTS2 long isoform, brain-wide or targeted to Calbindin-lineage cells, generates a specific suite of brain, behavioral and molecular pathologies

Rearrangements within the AUTS2 region are associated with a rare syndromic disorder with intellectual disability, developmental delay and behavioral abnormalities as core features. In addition, smaller regional variants are linked to wide range of neuropsychiatric disorders, underscoring the gene's essential role in brain development. Like many essential neurodevelopmental genes, AUTS2 is large and complex, generating distinct long (AUTS2-l) and short (AUTS2-s) protein isoforms from alternative promoters. Although evidence suggests unique isoform functions, the contributions of each isoform to specific AUTS2-linked phenotypes have not been clearly resolved. Furthermore, Auts2 is widely expressed across the developing brain, but cell populations most central to disease presentation have not been determined. In this study, we focused on the specific roles of AUTS2-l in brain development, behavior, and postnatal brain gene expression, showing that brain-wide AUTS2-l ablation leads to specific subsets of the recessive pathologies associated with C-terminal mutations that disrupt both isoforms. We identify downstream genes that could explain expressed phenotypes including hundreds of putative direct AUTS2-l target genes. Furthermore, in contrast to C-terminal Auts2 mutations which lead to dominant hypoactivity, AUTS2-l loss-of-function is associated with dominant hyperactivity, a phenotype exhibited by many human patients. Finally, we show that AUTS2-l ablation in Calbindin 1-expressing cell lineages is sufficient to yield learning/memory deficits and hyperactivity with abnormal dentate gyrus granule cell maturation, but not other phenotypic effects. These data provide new clues to in vivo AUTS2-l functions and novel information relevant to genotype-phenotype correlations in the human AUTS2 region. Overall design: RNA Sequencing was performed on RNA samples collected from individual forebrain of P0 or hippocampus of P14 mice. Two mouse strains were sampled in biological triplicate, Cmv-cre, Auts2-ex7-/-, and Nes-cre, Auts2-ex7-/-. Paired end RNA Libraries were prepared and sequenced on a Hiseq 4000 sequencer with a read length of 2x150bp.

AUTS2区域内的染色体重排与一种以智力障碍（intellectual disability）、发育迟缓（developmental delay）及行为异常（behavioral abnormalities）为核心特征的罕见综合征性疾病相关。此外，更小范围的区域变异与多种神经精神疾病（neuropsychiatric disorders）存在关联，凸显了该基因在脑发育过程中的关键作用。与诸多关键神经发育基因类似，AUTS2基因体量庞大且结构复杂，可通过可变启动子（alternative promoters）产生长型（AUTS2-l）与短型（AUTS2-s）两种不同的蛋白异构体（protein isoforms）。尽管已有研究提示两种异构体功能存在差异，但二者各自对AUTS2相关特定表型的贡献尚未得到明确解析。此外，Auts2在发育中的大脑中广泛表达，但与疾病表型最相关的细胞群体仍未确定。本研究聚焦AUTS2-l在脑发育、行为调控及产后脑基因表达中的特定功能，证实全脑AUTS2-l敲除可引发仅与同时破坏两种异构体的C末端突变相关的隐性病理特征中的特定子集。研究鉴定了可解释观测到的表型的下游基因，包括数百个推定的AUTS2-l直接靶基因。进一步而言，与引发显性活动减退的C末端Auts2突变不同，AUTS2-l功能缺失与显性活动亢进相关——这一表型亦是诸多人类患者所表现出的症状。最后，本研究表明，在表达钙结合蛋白1（Calbindin 1）的细胞谱系中敲除AUTS2-l，即可独立引发学习/记忆缺陷与活动亢进，并伴随齿状回颗粒细胞成熟异常，但不会产生其他表型效应。上述研究结果为体内AUTS2-l的功能提供了新的研究线索，也为人类AUTS2区域的基因型-表型关联研究提供了新颖的参考信息。整体实验设计：对P0代小鼠全前脑或P14代小鼠海马体提取的RNA样本进行RNA测序（RNA Sequencing）。以生物学重复三次的方式采集两种小鼠品系的样本：Cmv-cre、Auts2-ex7-/-，以及Nes-cre、Auts2-ex7-/-。构建双端RNA文库（paired end RNA Libraries），并在Hiseq 4000测序仪（Hiseq 4000 sequencer）上以2×150bp的读长完成测序。