Alzheimer's Disease Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital/Harvard Medical School, 114, 16th Street, Charlestown, MA 02129, USA.
Sensors (Basel). 2023 Feb 28;23(5):2651. doi: 10.3390/s23052651.
Our unique multiplexed imaging assays employing FRET biosensors have previously detected that γ-secretase processes APP C99 primarily in late endosomes and lysosomes in live/intact neurons. Moreover we have shown that Aβ peptides are enriched in the same subcellular loci. Given that γ-secretase is integrated into the membrane bilayer and functionally links to lipid membrane properties in vitro, it is presumable that γ-secretase function correlates with endosome and lysosome membrane properties in live/intact cells. In the present study, we show using unique live-cell imaging and biochemical assays that the endo-lysosomal membrane in primary neurons is more disordered and, as a result, more permeable than in CHO cells. Interestingly, γ-secretase processivity is decreased in primary neurons, resulting in the predominant production of long Aβ42 instead of short Aβ38. In contrast, CHO cells favor Aβ38 over the Aβ42 generation. Our findings are consistent with the previous in vitro studies, demonstrating the functional interaction between lipid membrane properties and γ-secretase and provide further evidence that γ-secretase acts in late endosomes and lysosomes in live/intact cells.
我们先前使用 FRET 生物传感器的独特多重成像检测方法发现 γ-分泌酶主要在活体内完整神经元的晚期内体和溶酶体中加工 APP C99。此外，我们还发现 Aβ 肽在相同的亚细胞位置富集。鉴于 γ-分泌酶整合到膜双层中，并在体外与脂膜特性功能相关，因此可以推测 γ-分泌酶的功能与活体内完整细胞的内体和溶酶体膜特性相关。在本研究中，我们使用独特的活细胞成像和生化检测方法表明，原代神经元中的内体 - 溶酶体膜比 CHO 细胞更无序，因此更具通透性。有趣的是，γ-分泌酶的连续性在原代神经元中降低，导致主要产生长 Aβ42 而不是短 Aβ38。相比之下，CHO 细胞更倾向于产生 Aβ38 而不是 Aβ42。我们的研究结果与之前的体外研究一致，证明了脂膜特性与 γ-分泌酶之间的功能相互作用，并进一步证明 γ-分泌酶在活体内完整细胞的晚期内体和溶酶体中发挥作用。
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