Loss of the APP regulator RHBDL4 preserves memory in an Alzheimer's disease mouse model.

Characteristic cerebral pathological changes of Alzheimer's disease (AD) such as glucose hypometabolism or the accumulation of cleavage products of the amyloid precursor protein (APP), known as Aß peptides, lead to sustained endoplasmic reticulum (ER) stress and neurodegeneration. To preserve ER homeostasis, cells activate their unfolded protein response (UPR). The rhomboid-like-protease 4 (RHBDL4) is an enzyme that participates in the UPR by targeting proteins for proteasomal degradation. We demonstrated previously that RHBLD4 cleaves APP in HEK293T cells, leading to decreased total APP and Aß. More recently, we showed that RHBDL4 processes APP in mouse primary mixed cortical cultures as well. Here, we aim to examine the physiological relevance of RHBDL4 in the brain. We first found that brain samples from AD patients and an AD mouse model (APPtg) showed increased RHBDL4 mRNA and protein expression. To determine the effects of RHBDL4's absence on APP physiology in vivo, we crossed APPtg mice to a RHBDL4 knockout (R4-/-) model. RHBDL4 deficiency in APPtg mice led to increased total cerebral APP and amyloidogenic processing when compared to APPtg controls. Contrary to expectations, as assessed by cognitive tests, RHBDL4 absence rescued cognition in 5-month-old female APPtg mice. Informed by unbiased RNAseq data, we demonstrated in vitro and in vivo that RHBDL4 absence leads to greater levels of active ß-catenin due to decreased proteasomal clearance. Decreased ß-catenin activity is known to underlie cognitive defects in APPtg mice and AD. Our work suggests that RHBDL4's increased expression in AD, in addition to regulating APP levels, leads to aberrant degradation of ß-catenin, contributing to cognitive impairment. Overall design: RNAseq profiling of wild-type and RHBDL4-knockout mouse ebryonic fibroblasts at standard culturing conditions after 48 h of seeding.

阿尔茨海默病（Alzheimer's disease, AD）的特征性脑病理改变，如葡萄糖代谢减低或淀粉样前体蛋白（amyloid precursor protein, APP）的裂解产物β淀粉样肽（Aβ）的沉积，可引发持续性内质网（endoplasmic reticulum, ER）应激与神经退行性变。为维持内质网稳态，细胞会激活未折叠蛋白反应（unfolded protein response, UPR）。类菱形蛋白酶4（rhomboid-like-protease 4, RHBDL4）是一类可通过靶向蛋白进行蛋白酶体降解，参与未折叠蛋白反应的酶。我们此前已证实，RHBDL4可在HEK293T细胞中裂解淀粉样前体蛋白APP，使总APP与Aβ水平降低。近期我们进一步发现，RHBDL4同样可在小鼠原代混合皮层培养物中加工处理APP。

本研究旨在探究RHBDL4在大脑中的生理学相关功能。我们首先检测发现，AD患者与AD小鼠模型（APPtg）的脑组织样本中，RHBDL4的mRNA与蛋白表达水平均显著升高。为明确体内敲除RHBDL4对APP生理过程的影响，我们将APPtg小鼠与RHBDL4基因敲除（RHBDL4 knockout, R4-/-）模型小鼠进行杂交。与APPtg对照组小鼠相比，APPtg小鼠中RHBDL4缺失会导致脑内总APP水平升高以及淀粉样蛋白生成通路活化增强。

与预期结果相悖的是，通过认知功能评估发现，RHBDL4缺失可改善5月龄雌性APPtg小鼠的认知功能。基于无偏倚RNA测序（RNAseq）数据，我们在体外与体内实验中证实，RHBDL4缺失会因蛋白酶体清除能力降低，使活性β-连环蛋白（β-catenin）的水平升高。已知β-连环蛋白活性降低是APPtg小鼠与AD患者认知缺陷的潜在分子机制。我们的研究表明，AD患者脑内RHBDL4表达升高，除调控APP水平外，还会异常降解β-连环蛋白，进而参与认知损伤的发生发展。

本研究的总体实验设计为：对标准培养条件下接种48小时后的野生型与RHBDL4基因敲除小鼠胚胎成纤维细胞进行RNA测序分析。