Department of Medicine (Royal Melbourne Hospital), The University of Melbourne, Parkville, Victoria, Australia.
Florey Institute of Neuroscience and Mental Health, Parkville, Victoria, Australia.
PLoS Pathog. 2019 Apr 10;15(4):e1007712. doi: 10.1371/journal.ppat.1007712. eCollection 2019 Apr.
Although considerable evidence supports that misfolded prion protein (PrPSc) is the principal component of "prions", underpinning both transmissibility and neurotoxicity, clear consensus around a number of fundamental aspects of pathogenesis has not been achieved, including the time of appearance of neurotoxic species during disease evolution. Utilizing a recently reported electrophysiology paradigm, we assessed the acute synaptotoxicity of ex vivo PrPSc prepared as crude homogenates from brains of M1000 infected wild-type mice (cM1000) harvested at time-points representing 30%, 50%, 70% and 100% of the terminal stage of disease (TSD). Acute synaptotoxicity was assessed by measuring the capacity of cM1000 to impair hippocampal CA1 region long-term potentiation (LTP) and post-tetanic potentiation (PTP) in explant slices. Of particular note, cM1000 from 30% of the TSD was able to cause significant impairment of LTP and PTP, with the induced failure of LTP increasing over subsequent time-points while the capacity of cM1000 to induce PTP failure appeared maximal even at this early stage of disease progression. Evidence that the synaptotoxicity directly related to PrP species was demonstrated by the significant rescue of LTP dysfunction at each time-point through immuno-depletion of >50% of total PrP species from cM1000 preparations. Moreover, similar to our previous observations at the terminal stage of M1000 prion disease, size fractionation chromatography revealed that capacity for acute synpatotoxicity correlated with predominance of oligomeric PrP species in infected brains across all time points, with the profile appearing maximised by 50% of the TSD. Using enhanced sensitivity western blotting, modestly proteinase K (PK)-resistant PrPSc was detectable at very low levels in cM1000 at 30% of the TSD, becoming robustly detectable by 70% of the TSD at which time substantial levels of highly PK-resistant PrPSc was also evident. Further illustrating the biochemical evolution of acutely synaptotoxic species the synaptotoxicity of cM1000 from 30%, 50% and 70% of the TSD, but not at 100% TSD, was abolished by digestion of immuno-captured PrP species with mild PK treatment (5μg/ml for an hour at 37°C), demonstrating that the predominant synaptotoxic PrPSc species up to and including 70% of the TSD were proteinase-sensitive. Overall, these findings in combination with our previous assessments of transmitting prions support that synaptotoxic and infectious M1000 PrPSc species co-exist from at least 30% of the TSD, simultaneously increasing thereafter, albeit with eventual plateauing of transmitting conformers.
尽管有大量证据表明错误折叠的朊病毒蛋白 (PrPSc) 是“朊病毒”的主要成分，支持传染性和神经毒性，但在发病机制的一些基本方面尚未达成明确共识，包括在疾病演变过程中神经毒性物质出现的时间。利用最近报道的电生理学范例，我们评估了从感染 M1000 野生型小鼠大脑中制备的粗匀浆（cM1000）的急性突触毒性，这些匀浆取自代表疾病终末期（TSD）的 30%、50%、70%和 100%的时间点。通过测量 cM1000 对海马 CA1 区长时程增强（LTP）和强直后增强（PTP）的损伤能力来评估急性突触毒性。值得注意的是，cM1000 从 TSD 的 30%就能够引起 LTP 和 PTP 的显著损伤，随着时间的推移，诱导的 LTP 失败增加，而 cM1000 诱导 PTP 失败的能力即使在疾病进展的早期阶段似乎也是最大的。通过免疫耗竭 cM1000 制剂中超过 50%的总 PrP 物种，显著挽救了每个时间点的 LTP 功能障碍，证明了这种突触毒性直接与 PrP 物种有关。此外，类似于我们在 M1000 朊病毒疾病终末期的先前观察结果，大小分级色谱显示，在所有时间点，急性突触毒性的能力与感染大脑中寡聚 PrP 物种的优势相关，在 TSD 的 50%时达到最大值。使用增强的灵敏度 Western blot 检测，在 TSD 的 30%时，可以在 cM1000 中检测到非常低水平的适度蛋白酶 K（PK）抗性 PrPSc，到 TSD 的 70%时可以可靠地检测到，此时还可以检测到大量高度 PK 抗性 PrPSc。进一步说明了急性突触毒性物质的生化演变，cM1000 从 TSD 的 30%、50%和 70%，但不是 100%TSD，的突触毒性通过用温和的 PK 处理（37°C 下 1 小时 5μg/ml）消化免疫捕获的 PrP 物种而被消除，证明在 TSD 的 70%及之前，主要的突触毒性 PrPSc 物种是蛋白酶敏感的。总的来说，这些发现与我们之前对传播朊病毒的评估相结合，支持从 TSD 的至少 30%开始就存在突触毒性和感染性 M1000 PrPSc 物种，此后同时增加，尽管随后传播构象达到平台期。
