Cytosolic prion protein (PrP) is not toxic in N2a cells and primary neurons expressing pathogenic PrP mutations
Cytosolic prion protein (PrP) is not toxic in N2a cells and primary neurons expressing pathogenic PrP mutations
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DOI：
10.1074/jbc.m412441200
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发表时间：
2005-03-25
影响因子：
4.8
通讯作者：
Chiesa, R
中科院分区：
文献类型：
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作者：
Fioriti, L;Dossena, S;Chiesa, R
遗传性朊病毒疾病与朊病毒蛋白（PrP）基因的突变有关，这种突变有利于将PrP转化为构象改变的致病性同种异构体。这一过程导致神经功能障碍的细胞机制尚不清楚。有研究认为，由于从内质网逆转录的错误折叠的PrP分子的蛋白酶体降解受损，胞浆中PrP的积累可能引发神经元死亡（Ma， J., Wollmann， R., and Lindquist， S. (2002) Science 298, 1781-1785）。为了测试这种神经毒性机制是否在遗传性朊病毒疾病中起作用，我们评估了蛋白酶体抑制剂对转染的N2a细胞和表达D178N和9个八肽突变的小鼠PrP同源物的原代神经元活力的影响。我们发现，这些抑制剂仅在表达巨细胞病毒启动子PrP的转染N2a中引起非糖基化、聚集形式的PrP的积累。这种形式含有一个未裂解的信号肽，表明它代表了在合成过程中未能转运到内质网腔的多肽链，而不是逆行转运的PrP。在蛋白酶体抑制剂存在的情况下，对N2a活力的定量分析表明，这种形式的积累是无毒的。在表达内源性启动子的野生型或突变型PrP的转基因小鼠的小脑颗粒神经元中没有发现胞质PrP的证据，这些神经元也不比PrP敲除小鼠的神经元更容易受到蛋白酶体抑制剂的毒性影响。我们的分析未能证实之前的观察结果，即PrP在细胞质中的错位具有神经毒性，并反驳了通过蛋白酶体途径扰乱PrP代谢在朊病毒疾病中起致病作用的假设。
Inherited prion diseases are linked to mutations in the prion protein (PrP) gene, which favor conversion of PrP into a conformationally altered, pathogenic isoform. The cellular mechanism by which this process causes neurological dysfunction is unknown. It has been proposed that neuronal death can be triggered by accumulation of PrP in the cytosol because of impairment of proteasomal degradation of misfolded PrP molecules retrotranslocated from the endoplasmic reticulum (Ma, J., Wollmann, R., and Lindquist, S. (2002) Science 298, 1781-1785). To test whether this neurotoxic mechanism is operative in inherited prion diseases, we evaluated the effect of proteasome inhibitors on the viability of transfected N2a cells and primary neurons expressing mouse PrP homologues of the D178N and nine octapeptide mutations. We found that the inhibitors caused accumulation of an unglycosylated, aggregated form of PrP exclusively in transfected N2a expressing PrP from the cytomegalovirus promoter. This form contained an uncleaved signal peptide, indicating that it represented polypeptide chains that had failed to translocate into the ER lumen during synthesis, rather than retrogradely translocated PrP. Quantification of N2a viability in the presence of proteasome inhibitors demonstrated that accumulation of this form was not toxic. No evidence of cytosolic PrP was found in cerebellar granule neurons from transgenic mice expressing wild-type or mutant PrPs from the endogenous promoter, nor were these neurons more susceptible to proteasome inhibitor toxicity than neurons from PrP knock-out mice. Our analysis fails to confirm the previous observation that mislocation of PrP in the cytosol is neurotoxic, and argues against the hypothesis that perturbation of PrP metabolism through the proteasomal pathway plays a pathogenic role in prion diseases.