ABSTRACT

Impairment of the macroautophagy/autophagy-lysosomal pathway (ALP) can lead to amyloid plaque accumulation in Alzheimer disease (AD); however, the underlying mechanism remains unresolved. This study revealed a mechanism of ALP impairment mediated by gain-of-function of lysosomal TPCN2/TPC2 (two pore segment channel 2) and suggests a molecular target for AD intervention. Using mutant PSEN1/PS1 (presenilin 1)-expressing human neuroblastoma SH-SY5Y and familial AD fibroblasts collected from human patients, we showed lysosomal pH was increased in AD cells due to exaggerated TPCN2-mediated calcium (Ca²⁺) release which increases lysosomal pH and compromises ALP degradation. Genetic knockdown or pharmacological inhibition of the TPCN2 channel restored lysosomal Ca²⁺ homeostasis, acidity of the lysosome and ALP function. Furthermore, AD mice (5xFAD) that had received treatment with the TPCN2 inhibitor tetrandrine for 6 months or injection of AAV-shTpcn2 to knock down Tpcn2 showed reduction in amyloid plaques in cortical and hippocampal regions. These treatments also improved spine morphology and density and corrected cognitive deficits in 5xFAD mice assayed by immunohistochemistry, behavioral tests, and electrophysiological measurements, respectively. Taken together, these findings reveal a previously under-appreciated role of lysosomal TPCN2 in ALP impairment of AD. They also suggest targeting the lysosomal TPCN2 can serve as a therapeutic strategy for AD treatment in future drug development.

Abbreviations

Aβ: β-amyloid; AD: Alzheimer disease; AIF1/IBA1: allograft inflammatory factor 1; ALP: autophagy-lysosomal pathway; APP: amyloid beta precursor protein; ATP6V1B1/V-ATPase V1b1: ATPase H+ transporting V1 subunit B1; AVs: autophagy vacuoles; BAF: bafilomycin A₁; CFC: contextual/cued fear conditioning assay; CHX: Ca²⁺/H⁺ exchanger; CTF-β: carboxy-terminal fragment derived from β-secretase; CTSD: cathepsin D; fAD: familial Alzheimer disease; GFAP: glial fibrillary acidic protein; LAMP1: lysosomal associated membrane protein 1; LTP: long‐term potentiation; MCOLN1/TRPML1: mucolipin 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAPT: microtubule associated protein tau; MWM: Morris water maze; NFT: neurofibrillary tangles; PFC: prefrontal cortex; PSEN1: presenilin 1; SQSTM1/p62: sequestosome 1; TBS: theta burst stimulation; TEM: transmission electronic microscopy; TPCN2/TPC2: two pore segment channel 2; WT: wild-type; V-ATPase: vacuolar type H⁺-ATPase

中文翻译：

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溶酶体 TPCN（两个孔段通道）抑制可改善 β-淀粉样蛋白病理学并减轻阿尔茨海默病的记忆障碍

摘要

巨自噬/自噬-溶酶体途径 (ALP) 的损害可导致阿尔茨海默病 (AD) 中的淀粉样蛋白斑块积聚；然而，潜在的机制仍未解决。该研究揭示了溶酶体 TPCN2/TPC2（两个孔段通道 2）功能获得介导的 ALP 损伤机制，并提出了 AD 干预的分子靶点。使用表达突变体 PSEN1/PS1（早老素 1）的人类神经母细胞瘤 SH-SY5Y 和从人类患者收集的家族性 AD 成纤维细胞，我们发现由于 TPCN2 介导的钙（Ca ²⁺）释放过度增加溶酶体pH 值和损害 ALP 降解。TPCN2 通道的基因敲除或药理抑制可恢复溶酶体 Ca ²⁺稳态、溶酶体的酸度和 ALP 功能。此外，接受 TPCN2 抑制剂粉防己治疗 6 个月或注射 AAV-sh Tpcn2以敲低Tpcn2的 AD 小鼠 (5xFAD)显示皮质和海马区域的淀粉样蛋白斑减少。这些治疗还分别通过免疫组织化学、行为测试和电生理测量分析，改善了 5xFAD 小鼠的脊柱形态和密度，并纠正了认知缺陷。总之，这些发现揭示了溶酶体 TPCN2 在 AD 的 ALP 损伤中的先前被低估的作用。他们还建议靶向溶酶体 TPCN2 可以作为未来药物开发中 AD 治疗的治疗策略。

缩写

Aβ：β-淀粉样蛋白；AD：阿尔茨海默病；AIF1/IBA1：同种异体炎症因子 1；ALP：自噬-溶酶体途径；APP：β淀粉样蛋白前体蛋白；ATP6V1B1/V-ATPase V1b1：ATPase H+ 转运 V1 亚基 B1；AVs：自噬泡；BAF：巴弗洛霉素 A ₁；CFC：上下文/提示恐惧条件分析；CHX：Ca ²⁺ /H ⁺交换器；CTF-β：衍生自 β-分泌酶的羧基末端片段；CTSD：组织蛋白酶 D；fAD：家族性阿尔茨海默病；GFAP：胶质纤维酸性蛋白；LAMP1：溶酶体相关膜蛋白 1；LTP：长时程增强；MCOLN1/TRPML1：粘磷脂1；MAP1LC3B/LC3B：微管相关蛋白1轻链3β；MAPT：微管相关蛋白 tau；MWM：莫里斯水迷宫；NFT：神经原纤维缠结；PFC：前额叶皮层；PSEN1：早老素 1；SQSTM1/p62：隔离体 1；TBS：θ爆发刺激；TEM：透射电子显微镜；TPCN2/TPC2：两个孔段通道2；WT：野生型；V-ATP酶：液泡型H ⁺ -ATP酶