AbstractIt has been shown recently, without an explanation of the possible molecular mechanisms involved, that 4-(2-hydroxyethyl)-1-piperazinepropanesulphonic (EPPS) acid effectively protects from the neurotoxicity induced by oligomers and plaques formed by the protein amyloid-β protein. Here we report the same protective effect, obtained in vitro (HT22-diff cell line) and ex vivo (hippocampal slices) models, against amyloid neurotoxicity induced by oligomers of salmon Calcitonin (sCT), which has been shown to be a good model for the study of neurodegenerative diseases.

摘要最近有研究表明，在没有解释可能的分子机制的情况下，4-（2-羟乙基）-1-哌嗪丙烷磺酸（EPPS）可以有效地保护蛋白质淀粉样蛋白-β蛋白形成的寡聚体和斑块引起的神经毒性。在这里，我们报告了体外（HT22-diff细胞系）和离体（海马切片）模型获得的相同保护作用，对抗鲑鱼降钙素（sCT）寡聚体诱导的淀粉样蛋白神经毒性，这已被证明是一个很好的模型。神经退行性疾病的研究。

Based on biophysical studies focusing on the protein aggregation kinetic and the interaction of the aggregates with model membranes, we propose a possible molecular mechanism underlying the protective effects. Taken together, our results indicate that EPPS is able to counteract the direct association (primary aggregation) of harmless low-molecular weight aggregates (dimers and trimers) or their aggregation catalysed by surfaces present in the solution (secondary aggregation).

基于关注蛋白质聚集动力学和聚集体与模型膜相互作用的生物物理研究，我们提出了保护作用的可能分子机制。综上所述，我们的研究结果表明，EPPS能够抵消无害的低分子量聚集体（二聚体和三聚体）的直接缔合（一次聚集）或溶液中存在的表面催化的聚集（二次聚集）。

Thus, EPPS stabilizes harmless aggregates and hinders the formation of toxic and metastable prefibrillar oligomers. Overall, our data demonstrate that EPPS is an excellent drug candidate for the treatment of neurodegeneration due to misfolded proteins, such as Alzheimer’s or Parkinson’s disease..

因此，EPPS稳定了无害的聚集体，并阻碍了有毒和亚稳的原纤低聚物的形成。总的来说，我们的数据表明，EPPS是治疗蛋白质错误折叠（如阿尔茨海默病或帕金森病）引起的神经退行性疾病的优秀候选药物。。

IntroductionNeurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and other less frequent such Huntington’s (HD), Prion (Pr) and dementia with Lewy bodies diseases1,2, are a heterogeneous class of severe and incurable disorders3 characterized by the progressive loss of structure and function of neurons4.

引言神经退行性疾病，如阿尔茨海默病（AD），帕金森病（PD）和其他不太常见的亨廷顿病（HD），朊病毒（Pr）和路易体病痴呆症1,2，是一类异质性的严重和不可治愈的疾病3，其特征是神经元结构和功能的逐渐丧失4。

Among these, some are particularly frequent and include AD and PD (AD today affects approximately 5% of people over 60 years old and in Italy there are an estimated 500 thousand sufferers), other are rare diseases such as Prion’s Protein disease and the large family of polyglutamine diseases (United Europe defines “rare disease” when its prevalence is below 0.05% ofthe population).Despite the great and many differences in clinical manifestation and incidence, neurodegenerative diseases share several characteristics such as increasing occurence with age and chronic and progressive nature2,5,6,7.It’s now generally accepted that many disorders are related to the misfolding and the following aggregation of the specific proteins involved, called amyloid proteins.

其中，一些特别常见，包括AD和PD（今天AD影响大约5%的60岁以上人群，意大利估计有50万患者），其他是罕见疾病，如朊病毒蛋白病和聚谷氨酰胺大家族疾病（当其患病率低于人口的0.05%时，联合欧洲定义为“罕见疾病”）。尽管在临床表现和发病率方面存在巨大差异，但神经退行性疾病具有几个特征，例如随着年龄的增长和慢性和进行性疾病的发生率增加2,5,6,7。现在普遍认为，许多疾病与所涉及的特定蛋白质（称为淀粉样蛋白）的错误折叠和随后的聚集有关。

Seven of the 37 proteins associated with amyloid disease form deposits in the CNS, giving rise to severe neurodegenerative conditions such as AD and PD6. The Central Nervous System (CNS) seems a suitable environment and this could be due to neurons that, compared to other cell types, are especially susceptible.

。中枢神经系统（CNS）似乎是一个合适的环境，这可能是由于与其他细胞类型相比，神经元特别容易受到影响。

Infact, due to their long lifetime, when damaged they are not readily replenished through cell division.Normally, misfolded proteins are degraded inside the cell in proteasomes or outside the cell by macrophages8. In amyloidosis, a condition associated with various hereditary and inflammatory diseases9, these control mechanisms are less efficient and, therefore, cause misfolde.

事实上，由于它们的寿命长，当它们受损时，不容易通过细胞分裂补充。通常，错误折叠的蛋白质在蛋白酶体的细胞内或巨噬细胞的细胞外降解8。在淀粉样变性（一种与各种遗传性和炎性疾病相关的疾病）中，这些控制机制效率较低，因此会导致错误折叠。

(i)

（一）

EPPS, maintained for 60 min up to the induction of the tetanic stimulus (minute 77, third arrow) (orange trace);

EPPS，维持60分钟直至诱导强直刺激（第77分钟，第三个箭头）（橙色迹线）；

(ii)

（二）

EPPS, maintained for 40 min up to the administration of the mixture EPPS + PFOs (minute 57, second arrow), which was maintained up to the induction of the tetanic stimulus (minute 77, third arrow) (blue trace);

EPPS，维持40分钟直至给予混合物EPPS+ PFOs（第57分钟，第二个箭头），其维持至强直刺激的诱导（第77分钟，第三个箭头）（蓝色迹线）；

(iii)

（三）

plain aCSF solution, maintained for 40 min up to the administration of the sCT-NA solution (minute 57, second arrow), which was maintained for 20 min up to the induction of the tetanic stimulus (minute 77, third arrow) (green trace);

普通aCSF溶液，维持40分钟直至施用sCT-NA溶液（第57分钟，第二箭头），维持20分钟直至诱导强直刺激（第77分钟，第三箭头）（绿色迹线）；

(iv)

（四）

plain aCSF solution, maintained for 40 min up to the administration of the PFOs solution (minute 57, second arrow), which was maintained for 20 min up to the induction of the tetanic stimulus (minute 77, third arrow) (red trace).

普通aCSF溶液，在施用全氟辛烷磺酸溶液（第57分钟，第二个箭头）之前保持40分钟，在诱导强直刺激（第77分钟，第三个箭头）（红色迹线）之前保持20分钟。

Fig. 3Synaptic plasticity in CA1 subfield of mouse hippocampal slices. Spectra relative to LTP induced in hippocampal brain slices by PFOs (red) (n = 6) and NA solutions (green) (n = 6), EPPS solution (orange) (n = 10) and EPPS + PFOs solution (blue) (n = 6), compared to untreated samples (CTRL in black) (n = 6).

图3小鼠海马切片CA1亚区的突触可塑性。与未处理的样品（CTRL为黑色）（n=6）相比，PFOs（红色）（n=6）和NA溶液（绿色）（n=6），EPPS溶液（橙色）（n=10）和EPPS+PFOs溶液（蓝色）（n=6）在海马脑切片中诱导LTP的光谱。

The first arrow from the left represents the starting point of the EPPS treatments, when performed (EPPS and EPPS + PFOs). The second arrow represents the administration of NA, PFOs and PFOs + EPPS solutions, which were maintained (20 min.) up to the tetanic stimulations (third arrow). The insert (top, left) shows in detail the BST.Full size imagePS amplitude values, for each group of hippocampal slices, were recorded from the beginning up to 170 min and are reported in Table 1.Table 1 Percentage of PS amplitude values of BST and LTP recorded in the CA1 region of hippocampal slices from the control group and groups treated with different substances at different times.Full size tableAs previously reported by our group, sCT-PFOs resulted to be toxic32.

。第二个箭头表示NA，PFOs和PFOs+ EPPS溶液的施用，其维持（20分钟）直至强直刺激（第三个箭头）。插入物（顶部，左侧）详细显示了BST。每组海马切片的全尺寸imagePS振幅值从开始到170分钟记录，并在表1中报告。表1在对照组和在不同时间用不同物质处理的组的海马切片的CA1区记录的BST和LTP的PS振幅值的百分比。全尺寸表正如我们小组先前报道的那样，sCT PFOs具有毒性32。

In fact, the treatment with plain sCT-PFOs (red) induced a strong reduction in LTP with PS values that reached values similar to those of BST 170 min after the high-frequency stimulation (HFS) (Fig. 3; Table 1: row 6). However, in this case we didn’t observe any significant alteration in the BST with respect to the CTRL (inset of Fig. 3; Table 1: row 3).The treatment with sCT-NA solutions (green) didn’t alter either BST or LTP (Fig. 3; Table 1: row 3 and 6).

事实上，用普通sCT全氟辛烷磺酸（红色）治疗引起LTP的强烈降低，PS值在高频刺激（HFS）后170分钟达到与BST相似的值（图3；表1：第6行）。然而，在这种情况下，我们没有观察到BST相对于CTRL的任何显着变化（图3的插图；表1：第3行）。用sCT-NA溶液（绿色）处理不会改变BST或LTP（图3；表1：第3行和第6行）。

This confirmed that the toxic aggregates (pentamers, hexamers and heptamers) were not abundant in this solution.Concerning the effects of EPPS, we can say that its presence induced an increase in the BST, without alterations of LTP. In particular, we observed (.

这证实了有毒聚集体（五聚体，六聚体和七聚体）在该溶液中不丰富。关于EPP的影响，我们可以说它的存在诱导了BST的增加，而没有改变LTP。特别是，我们观察到（。

Data availability

数据可用性

Data availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

数据可用性当前研究中使用和/或分析的数据集可根据合理要求从通讯作者处获得。

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Download referencesAcknowledgementsHT22 cell line was kindly gifted from David Schubert of “The Salk Institute”, La Jolla, CA (USA). C.G., C.B., M.D. are grateful to the Consiglio Nazionale delle Ricerche (CNR, Rome - Italy) and Universidad de Antioquia (Medellín - Colombia) for the support given to the project n.2024-68170 entitled:” The study of the protective effect exerted by 4-(2-hydroxyethyl)-1-piperazinepropanesulphonic acid (EPPS) on neurodegeneration induced by prefibrillar salmon Calcitonin (sCT) amyloid oligomers” stipulated between the Institute for Biological System of Consiglio Nazionale delle Ricerche (ISB-CNR) and Universidad de Antioquia, within the cooperation framework agreement (MOU) n° 005-2022.FundingThis work was supported by the Italian “Ministero della Salute” with the “Progetto Ordinario di Ricerca Finalizzata (RF-2013-02355682)” entitled: “Calcitonin oligomers as a model to study the amyloid neurotoxicity: the focal role played by lipid rafts in the prevention and cure”.Author informationAuthor notesBeatrice Zarrilli and Roberto Bonanni, Marco Diociaiuti and Giovanna D’Arcangelo contributed equally to this work.Authors and AffiliationsDepartment of Systems Medicine, “Tor vergata” University of Rome, Via Montpellier 1, 00133, Rome, ItalyBeatrice Zarrilli, Ida Cariati, Virginia Tancredi & Giovanna D’ArcangeloLaboratory of Experimental Neurology, IRCCS Fondazione Santa Lucia, Rome, ItalyBeatrice ZarrilliDepartment of Biomedicine and Prevention, “Tor vergata” University of Rome, Via Montpellier 1, 00133, Rome, ItalyRoberto BonanniNational Centre for Drug Research and Evaluation, Istituto Superiore di Sanità, Rome, ItalyMarcello Belfiore, Mariagrazia Severino & Giacomo CappellaDepartment of Infectious Diseases, Istituto Superiore di.

下载参考文献致谢HT22细胞系由美国加利福尼亚州拉霍亚“索尔克研究所”的戴维·舒伯特（DavidSchubert）赠送。C、 G.，C.B.，M.D.感谢意大利罗马国家理工大学（CNR）和哥伦比亚麦德林大学（Medellín）对国家理工大学生物系统研究所（ISB-CNR）和哥伦比亚大学（Universidad de Ricerche）之间规定的题为“4-（2-羟乙基）-1-哌嗪丙磺酸（EPPS）对由原发性鲑鱼降钙素（sCT）淀粉样寡聚体诱导的神经变性的保护作用研究”的项目2024-68170的支持Antioquia，在合作框架协议（MOU）第005-2022号范围内。资助这项工作得到了意大利“La Salute部长”和“Progetto Ordinario di Ricerca Finalizata（RF-2013-02355682）”的支持，题为“降钙素寡聚体作为研究淀粉样神经毒性的模型：脂筏在预防和治疗中发挥的重要作用”。作者信息作者注意到，比阿特丽斯·扎里利（Beatrice Zarrilli）和罗伯托·博南尼（Roberto Bonanni），马尔科·迪奥·迪亚乌蒂（Marco Diociaiuti）和乔瓦纳·达坎杰洛（Giovanna D'Arcangelo）对这项工作做出了同样的贡献。作者和附属机构系统医学系，“Tor vergata”罗马大学，Via Montpellier 100133，Rome，ItalyBeatrice Zarrilli，Ida Cariati，Virginia Tancredi＆Giovanna D'Arcangelo实验神经病学实验室，IRCCS Fondazione Santa Lucia，Rome，ItalyBeatrice Zarrillide生物医学与预防系，“Tor vergata”罗马大学，Via Mon意大利博南尼国家药物研究与评估中心，罗马高级卫生研究所，意大利马切洛·贝尔菲奥雷，马里亚格拉齐亚·塞韦里诺和贾科莫·卡佩拉传染病研究所。

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PubMed Google ScholarContributionsConceptualization B.Z., C.G., C.F., V.T., M.D., G.D. Designed the experiments B.Z., R.B., I.C., S.S., C.B., M.D., G.D Performed citotoxicity experiments R.B., M.B., M.G.S. Performed plasticity experiments B.Z Performed protein sample preparation and characterization experiments M.B., M.G.S., R.F., G.C. Biophysical experiments S.S., C.B., M.D., G.C. Writing-Original Draft preparation, B.Z., M.D.

PubMed谷歌学术贡献概念化B.Z.，C.G.，C.F.，V.T.，M.D.，G.D.设计了实验B.Z.，R.B.，I.C.，S.S.，C.B.，M.D.，G.D.进行了城市毒性实验R.B.，M.B.，M.G.S.进行了可塑性实验B.Z进行了蛋白质样品制备和表征实验M.B.，M.G.S.，R.F.，G.C.生物物理实验S.S.，C.B.，M.D.，G.C.撰写原始草案准备，B.Z.，M.D。

Writing-Review and Editing, B.Z., C.F., V.T., G.D., M.D. Super-vision, M.D., G.D. All authors approved the final manuscript.Corresponding authorCorrespondence to.

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Reprints and permissionsAbout this articleCite this articleZarrilli, B., Bonanni, R., Belfiore, M. et al. Molecular mechanisms at the basis of the protective effect exerted by EPPS on neurodegeneration induced by prefibrillar amyloid oligomers.

转载和许可本文引用本文Zarrilli，B.，Bonanni，R.，Belfiore，M。等人。基于EPPS对纤维前淀粉样蛋白寡聚体诱导的神经变性的保护作用的分子机制。

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KeywordsneurodegenerationAlzheimer’s disease (AD)salmon calcitonin (sCT)4-(2-hydroxyethyl)-1-piperazinepropanesulphonic acid (EPPS)Prefibrillar Oligomers (PFOs)

关键词神经退行性疾病（AD）鲑鱼降钙素（sCT）4-（2-羟乙基）-1-哌嗪丙磺酸（EPPS）纤维前低聚物（PFOs）

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Biological physicsDrug discoveryIntrinsically disordered proteinsLearning and memoryMembrane biophysicsMolecular biophysicsMolecular medicineMolecular neuroscienceSynaptic plasticity

生物物理药物发现本质上无序的蛋白质学习和记忆膜生物物理分子生物物理分子医学分子神经科学突触可塑性