Choroid plexus mis-splicing and altered cerebrospinal fluid composition in myotonic dystrophy type 1 [mouse]

Myotonic dystrophy type 1 is a dominantly inherited multisystemic disease caused by CTG tandem repeat expansions in the DMPK 3' untranslated region. These expanded repeats are transcribed and produce toxic CUG RNAs that sequester and inhibit activities of the MBNL family of developmental RNA processing factors. Although myotonic dystrophy is classified as a muscular dystrophy, the brain is also severely affected by an unusual cohort of symptoms, including hypersomnia, executive dysfunction, as well as early onsets of tau/MAPT pathology and cerebral atrophy. To address the molecular and cellular events that lead to these pathological outcomes, we recently generated a mouse Dmpk CTG expansion knockin model and identified choroid plexus epithelial cells as particularly affected by the expression of toxic CUG expansion RNAs. To determine if toxic CUG RNAs perturb choroid plexus functions, alternative splicing analysis was performed on lateral and hindbrain choroid plexi from Dmpk CTG knockin mice. Choroid plexus transcriptome-wide changes were evaluated in Mbnl2 knockout mice, a developmental-onset model of myotonic dystrophy brain dysfunction. To determine if transcriptome changes also occurred in the human disease, we obtained post-mortem choroid plexus for RNA-seq from donors without neurologically unaffected (two females, three males; ages 50-70) and myotonic dystrophy type 1 donors (one female, three males; ages 50-70). To test that choroid plexus transcriptome alterations resulted in altered CSF composition, we obtained CSF via lumbar puncture from patients with myotonic dystrophy type 1 (five females, five males; ages 35-55) and non-myotonic dystrophy patients (three females, four males; ages 26-51) and Western blot and osmolarity analyses were used to test CSF alterations predicted by choroid plexus transcriptome analysis. We determined that CUG RNA induced toxicity was more robust in the lateral choroid plexus of Dmpk CTG knockin mice due to comparatively higher Dmpk and lower Mbnl RNA levels. Impaired transitions to adult splicing patterns during choroid plexus development were identified in Mbnl2 knockout mice, including mis-splicing previously found in Dmpk CTG knockin mice. Whole transcriptome analysis of myotonic dystrophy type 1 choroid plexus revealed disease-associated RNA expression and mis-splicing events. Based on these RNA changes, predicted alterations in ion homeostasis, secretory output, and CSF composition were confirmed by analysis of myotonic dystrophy type 1 CSF. Our results implicate choroid plexus spliceopathy and concomitant alterations in CSF homeostasis as an unappreciated contributor to myotonic dystrophy type 1 CNS pathogenesis. We collected and performed RNA-seq on Choroid plexus tissue from different mouse developmental stages either wild type or knockout of splicing regulator Mbnl2. Transcriptomic analysis was performed to identify alternative splicing and gene expression changes during development and upon loss of Mbnl21型肌强直性营养不良（Myotonic dystrophy type 1）是一种由DMPK基因3'非翻译区（3' untranslated region）的CTG串联重复扩增引发的显性遗传性多系统疾病。这类扩增的重复序列会被转录，产生毒性CUG RNA，后者会结合并抑制发育性RNA加工因子MBNL家族（MBNL family）的活性。尽管肌强直性营养不良被归类为肌营养不良症，但其脑部也会受到一系列异常症状的严重累及，包括过度嗜睡、执行功能障碍，以及tau/MAPT病理和大脑萎缩的早发。 为阐明介导上述病理结局的分子与细胞事件，我们近期构建了Dmpk CTG扩增敲入小鼠模型，并鉴定到脉络丛上皮细胞（choroid plexus epithelial cells）受毒性CUG扩增RNA的表达影响尤为显著。为探究毒性CUG RNA是否会扰乱脉络丛功能，我们对Dmpk CTG敲入小鼠的侧脑室脉络丛（lateral choroid plexi）和后脑脉络丛（hindbrain choroid plexi）开展了可变剪接分析（alternative splicing analysis）。我们还在Mbnl2敲除小鼠（Mbnl2 knockout mice，一种肌强直性营养不良脑功能障碍的发育模型）中评估了脉络丛的全转录组（transcriptome-wide）变化。 为确定转录组变化是否也存在于人类疾病中，我们获取了无神经系统受累的对照供者（2名女性、3名男性；年龄50~70岁）及1型肌强直性营养不良供者（1名女性、3名男性；年龄50~70岁）的死后脉络丛组织，进行RNA测序（RNA-seq）。 为验证脉络丛转录组改变是否会导致脑脊液（cerebrospinal fluid, CSF）成分异常，我们通过腰椎穿刺（lumbar puncture）采集了1型肌强直性营养不良患者（5名女性、5名男性；年龄35~55岁）与非肌强直性营养不良患者（3名女性、4名男性；年龄26~51岁）的脑脊液，并采用蛋白质印迹法（Western blot）与渗透压分析（osmolarity analyses）验证了由脉络丛转录组分析预测的脑脊液改变。 我们发现，由于Dmpk表达水平相对更高、Mbnl RNA水平相对更低，毒性CUG RNA诱导的毒性在Dmpk CTG敲入小鼠的侧脑室脉络丛中更为显著。在Mbnl2敲除小鼠的脉络丛发育过程中，我们鉴定到向成年剪接模式的转化受损，包括此前在Dmpk CTG敲入小鼠中发现的异常剪接事件。对1型肌强直性营养不良脉络丛的全转录组分析揭示了疾病相关的RNA表达与异常剪接事件。基于这些RNA变化，我们通过分析1型肌强直性营养不良患者的脑脊液，证实了离子稳态、分泌输出与脑脊液成分的预测性改变。本研究结果表明，脉络丛剪接异常及伴随的脑脊液稳态改变是1型肌强直性营养不良中枢神经系统（central nervous system, CNS）发病机制中未被重视的致病因素。 我们收集了不同发育阶段的野生型或剪接调控因子（splicing regulator）Mbnl2敲除小鼠的脉络丛组织，并开展了RNA测序。我们进行了转录组分析，以鉴定发育过程中及Mbnl2缺失时的可变剪接与基因表达变化。