Institute of Neuropathology, University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany.
Department of Neurology, Experimental Research in Stroke and Inflammation (ERSI), University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany.
J Extracell Vesicles. 2024 Nov;13(11):e70011. doi: 10.1002/jev2.70011.
Extracellular vesicles (EVs) have gained significant attention as pathology mediators and potential diagnostic tools for neurodegenerative diseases. However, isolation of brain-derived EVs (BDEVs) from tissue remains challenging, often involving enzymatic digestion steps that may compromise the integrity of EV proteins and overall functionality. Here, we describe that collagenase digestion, commonly used for BDEV isolation, produces undesired protein cleavage of EV-associated proteins in brain tissue homogenates and cell-derived EVs. In order to avoid this effect, we studied the possibility of isolating BDEVs with a reduced amount of collagenase or without any protease. Characterization of the isolated BDEVs from mouse and human samples (both female and male) revealed their characteristic morphology and size distribution with both approaches. However, we show that even minor enzymatic digestion induces 'artificial' proteolytic processing in key BDEV markers, such as Flotillin-1, CD81, and the cellular prion protein (PrP), whereas avoiding enzymatic treatment completely preserves their integrity. We found no major differences in mRNA and protein content between non-enzymatically and enzymatically isolated BDEVs, suggesting that the same BDEV populations are purified with both approaches. Intriguingly, the lack of Golgi marker GM130 signal, often referred to as contamination indicator (or negative marker) in EV preparations, seems to result from enzymatic digestion rather than from its actual absence in BDEV samples. Overall, we show that non-enzymatic isolation of EVs from brain tissue is possible and avoids artificial pruning of proteins while achieving an overall high BDEV yield and purity. This protocol will help to understand the functions of BDEV and their associated proteins in a near-physiological setting, thus opening new research approaches.
细胞外囊泡 (EVs) 作为病理学介质和神经退行性疾病的潜在诊断工具引起了广泛关注。然而，从组织中分离脑源性 EVs (BDEVs) 仍然具有挑战性，通常涉及酶消化步骤，这可能会破坏 EV 蛋白的完整性和整体功能。在这里，我们描述了胶原酶消化，通常用于 BDEV 分离，会在脑组织匀浆和细胞衍生的 EV 中产生 EV 相关蛋白的不期望的蛋白切割。为了避免这种影响，我们研究了用较少量的胶原酶或不使用任何蛋白酶来分离 BDEVs 的可能性。对来自小鼠和人类样本（雌性和雄性）的分离的 BDEVs 的特征描述揭示了它们的特征形态和大小分布，这两种方法都可以实现。然而，我们表明，即使是轻微的酶消化也会诱导关键 BDEV 标志物（如 Flotillin-1、CD81 和细胞朊病毒蛋白 (PrP)）的“人为”蛋白水解加工，而完全避免酶处理则完全保留其完整性。我们发现非酶和酶分离的 BDEVs 之间在 mRNA 和蛋白质含量上没有重大差异，这表明这两种方法都可以纯化相同的 BDEV 群体。有趣的是，缺少高尔基体标志物 GM130 信号，通常在 EV 制剂中被称为污染指标（或阴性标志物），似乎是由于酶消化引起的，而不是由于 BDEV 样本中实际上不存在。总体而言，我们表明从脑组织中非酶分离 EVs 是可能的，可以避免蛋白质的人为修剪，同时实现高的 BDEV 产量和纯度。该方案将有助于在近生理条件下理解 BDEVs 及其相关蛋白的功能，从而开辟新的研究方法。
