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Abstract：
Rationale: Tripeptidyl peptidase II (TPP2) has been proven to be related to human immune and neurological diseases. It is generally considered as a cytosolic protein which forms the largest known protease complex in eukaryotic cells to operate mostly downstream of proteasomes for degradation of longer peptides. However, this canonical function of TPP2 cannot explain its role in a wide variety of biological and pathogenic processes. The mechanistic interrelationships and hierarchical order of these processes have yet to be clarified. Methods: Animals, cells, plasmids, and viruses established and/or used in this study include: TPP2 knockout mouse line, TPP2 conditional knockout mouse lines (different neural cell type oriented), TRE-TPP2 knockin mouse line on the C57BL/6 background; 293T cells with depletion of TPP2, ATF6, IRE1, PERK, SYVN1, UCHL1, ATG5, CEPT1, or CCTα, respectively; 293T cells stably expressing TPP2, TPP2 S449A, TPP2 S449T, or CCTα-KDEL proteins on the TPP2-depleted background; Plasmids for eukaryotic transient expression of rat CYP19A1-Flag, CYP19A1 S118A-Flag, CYP19A1 S118D-Flag, Sac I ML GFP Strand 11 Long, OMMGFP 1-10, G-CEPIA1er, GCAMP2, CEPIA3mt, ACC-GFP, or SERCA1-GFP; AAV2 carrying the expression cassette of mouse CYP19A1-3 X Flag-T2A-ZsGreen. Techniques used in this study include: Flow cytometry, Immunofluorescence (IF) staining, Immunohistochemical (IHC) staining, Luxol fast blue (LFB) staining, β-galactosidase staining, Lipid droplet (LD) staining, Calcium (Ca2+) staining, Stimulated emission depletion (STED) imaging, Transmission electron microscopic imaging, Two-photon imaging, Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end Labeling (TUNEL) assay, Bromodeoxyuridine (BrdU) assay, Enzymatic activity assay, Proximity ligation assay (PLA), In vivo electrophysiological recording, Long-term potentiation (LTP) recording, Split-GFP-based mitochondria-associated membrane (MAM) detection, Immunoprecipitation (IP), Cellular fractionation, In situ hybridization, Semi-quantitative RT-PCR, Immunoblot, Mass spectrometry-based lipidomics, metabolomics, proteomics, Primary hippocampal neuron culture and Morris water maze (MWM) test. Results: We found that TPP2, independent of its enzymatic activity, plays a crucial role in maintaining the homeostasis of intracellular Ca2+ and phosphatidylcholine (PC) in the central nervous system (CNS) of mice. In consistence with the critical importance of Ca2+ and PC in the CNS, TPP2 gene ablation causes presenile dementia in female mice, which is closely associated with Ca2+/PC dysregulation-induced endoplasmic reticulum (ER) stress, abnormal autophagic degradation of CYP19A1 (aromatase), and estrogen depletion. This work therefore uncovers a new role of TPP2 in lipogenesis and neurosteroidogenesis which is tightly related to cognitive function of adult female mice. Conclusion: Our study reveals a crucial role of TPP2 in controlling homeostasis of Ca2+ and lipids in CNS, and its deficiency causes sexual dimorphism in dementia. Thus, this study is not only of great significance for elucidating the pathogenesis of dementia and its futural treatment, but also for interpreting the role of TPP2 in other systems and their related disorders.
摘要：
原理：三肽基肽酶II（TPP2）已被证明与人类免疫和神经系统疾病有关。它通常被认为是在真核细胞中形成最大的已知蛋白酶复合物的细胞溶质蛋白,主要在蛋白酶体的下游操作以降解较长的肽。然而,TPP2的这种典型功能不能解释其在多种生物学和致病过程中的作用。这些过程的机械相互关系和分层顺序尚未阐明。方法：动物，细胞,质粒,本研究中建立和/或使用的病毒包括：TPP2敲除小鼠系，TPP2条件性敲除小鼠系（不同神经细胞类型定向），TRE-TPP2敲入C57BL/6背景上的小鼠系；293T细胞耗尽TPP2，ATF6，IRE1，PERK，SYVN1、UCHL1、ATG5、CEPT1或CCTα，分别稳定表达TPP2，TPP2S449A，TPP2S449T,或TPP2耗尽背景上的CCTα-KDEL蛋白；用于真核瞬时表达大鼠CYP19A1-Flag的质粒，CYP19A1S118A-标志,CYP19A1S118D-标志,SacIMLGFPStand11Long,OMMGFP1-10，G-CEPIA1er，GCAMP2，CEPIA3mt，ACC-GFP,或SERCA1-GFP;AAV2携带小鼠CYP19A1-3XFlag-T2A-ZsGreen的表达盒。本研究中使用的技术包括：流式细胞术，免疫荧光（IF）染色，免疫组化（IHC）染色，Luxol快速蓝（LFB）染色，β-半乳糖苷酶染色,脂滴（LD）染色，钙（Ca2+）染色，受激发射损耗（STED）成像，透射电子显微镜成像，双光子成像,末端脱氧核苷酸转移酶（TdT）dUTP缺口末端标记（TUNEL）测定，溴脱氧尿苷（BrdU）测定，酶活性测定，邻近连接测定(PLA)，体内电生理记录，长期增强（LTP）记录，基于分裂GFP的线粒体相关膜（MAM）检测，免疫沉淀(IP),细胞分馏,原位杂交,半定量RT-PCR,免疫印迹,基于质谱的脂质组学，代谢组学，蛋白质组学，原代海马神经元培养和Morris水迷宫(MWM)试验。结果：我们发现TPP2与其酶活性无关，在维持小鼠中枢神经系统（CNS）中细胞内Ca2和磷脂酰胆碱（PC）的稳态中起着至关重要的作用。与Ca2+和PC在中枢神经系统中的关键重要性一致，TPP2基因消融导致雌性小鼠发生早老性痴呆,与Ca2+/PC失调诱导的内质网（ER）应激密切相关，CYP19A1（芳香化酶）的异常自噬降解，和雌激素消耗。因此，这项工作揭示了TPP2在脂肪生成和神经类固醇生成中的新作用，这与成年雌性小鼠的认知功能密切相关。结论：我们的研究揭示了TPP2在控制中枢神经系统Ca2+和脂质稳态中的关键作用。它的缺乏会导致痴呆症的性二态性。因此，这项研究不仅对阐明痴呆的发病机制及其未来治疗具有重要意义，还用于解释TPP2在其他系统及其相关疾病中的作用。
