TDP-43在哺乳動物脊髓運動神經元的生理功能和病理功能缺失中所扮演的角色

Abstract

TDP-43〈TAR去氧核糖核酸結合酶-43〉為Tardbp基因所轉譯的多功能RNA與DNA結合蛋白，近幾年在許多具有泛素沉澱物堆積的神經退化性疾病中，發現主要的疾病蛋白為TDP-43。目前至少九個基因突變與TDP-43泛素沉澱物堆積的運動神經元退化性疾病(俗稱漸凍人，Amyotrophic Lateral Sclerosis, ALS)相關，包含Tardbp基因本身，在ALS病人中，約有三十幾個胺基酸突變被發現在Tardbp基因上，然而其致病機轉仍然未知。於小鼠運動神經元細胞株中，表現野生型〈wild type〉或突變型〈mutant〉人類TDP-43蛋白造成顯著的細胞凋亡，此現象並沒有在其他神經元細胞株或纖維母細胞株中發現；此外，表現相同濃度的野生型及突變型人類TDP-43質體DNA，突變型TDP-43在老鼠運動神經元細胞中造成較高的細胞死亡率，由實驗結果得知此現象與突變型TDP-43蛋白本身相對於野生型TDP-43蛋白擁有較高的穩定度相關。為更進一步了解TDP-43蛋白的生理功能，我們利用基因標靶技術剃除小鼠體內TDP-43蛋白的表現，缺少TDP-43蛋白表現的小鼠死於胚胎著床時期，約胚胎期三點五至六點五天，觀察基因剔除小鼠囊胚〈blastocysts〉顯示外觀正常但無法於體外培養的情形下正常成長成內細胞團〈Inner cell mass〉，為了研究TDP-43代謝異常在ALS疾病中扮演的角色，我們利用條件性基因標靶技術於小鼠運動神經元細胞中專一性剃除小鼠TDP-43基因表現，此小鼠具有類ALS的症狀，例如運動神經元退化症的駝背、運動失能、肌肉萎縮、運動神經元缺失及神經膠細胞增生等等，泛素蛋白更顯著地堆積在脊髓運動神經元內。根據細胞實驗和TDP-43蛋白過度表現之小鼠模式所得到的結果，我們推測TDP-43基因突變會導致TDP-43蛋白穩定性增加，進而使突變型TDP-43蛋白量增加，運動神經元細胞株中對於TDP-43蛋白量具有較低的耐受性，因而在ALS疾病裡造成運動神經退化及病變；全身性剃除TDP-43蛋白表現的小鼠模式中，顯示TDP-43蛋白為體外培養內細胞團生長所必須，脊髓運動神經元內專一性剔除TDP-43表現的小鼠模式中，顯示TDP-43在哺乳類脊髓運動神經元長期生存扮演重要的角色，TDP-43蛋白缺失可能為造成具有TDP-43蛋白病變的ALS疾病中神經退化的主要原因。

TDP-43, a multi-functional DNA/ RNA-binding protein encoded by the TARDBP gene, has emerged as a major patho-signature factor of the ubiquitinated intracellular inclusions (UBIs) in the diseased cells of a range of neurodegenerative diseases. Mutations in at least 9 different genes including TARDBP have been identified in ALS with TDP-43 (+)-UBIs. Thus far, the pathogenic role(s) of the more than 30 ALS-associated mutations in the TARDBP gene has not been well defined. By transient DNA transfection studies, we show that exogenously expressed human TDP-43 (hTDP-43), either wild type (WT) or 2 different ALS mutant (MT) forms, could cause significantly higher apoptotic death rate of a mouse spinal motor neuron-like cell line (NSC34) than other types of cells, e.g. mouse neuronal Neuro2a and human fibroblast HEK293T cells. Furthermore, at the same plasmid DNA dose(s) used for transfection, the percentages of NSC34 cell death caused by the 2 exogenously expressed hTDP-43 mutants are all higher than that caused by the WT hTDP-43. Significantly, the above observations are correlated with higher steady-state levels of the mutant hTDP-43 proteins as well as their stabilities than the WT. To understand the physiological functions of TDP-43 in vivo, we have used gene targeting approach to disrupt the expression of TDP-43 in mouse. Loss of the TDP-43 expression results in peri-implantation lethality of mice between embryonic day (E) 3 3.5 to 6.5. Blastocysts of the homozygous Tardbp null mutants are morphologically normal but exhibit defective outgrowth of the inner cell mass (ICM) cells in vitro. Further emphasized the role of the mismetabolism of TDP-43 in the pathogenesis of ALS is addressed by the conditional mouse gene targeting approach, we show that mice with inactivation of the Tardbp gene in the spinal cord motor neurons (HB9:Cre-Tardbplx/-) exhibit progressive and male-dominant development of ALS-related phenotypes including kyphosis, motor dysfunctions, muscle weakness/ atrophy, motor neuron loss, and astrocytosis in the spinal cord. Significantly, ubiquitinated proteins accumulate in the TDP-43-depleted motor neurons of the spinal cords of HB9:Cre–Tardbplx/– mice with the ALS phenotypes. Based on the data of cell culture experiments and HB9-hTDP-43 transgenic mice, we suggest that one major common consequence of the different ALS-associated TDP-43 mutations is the stabilization of the hTDP-43 polypeptide. The resulting elevation of the steady state level of hTDP-43 in combination with the relatively low tolerance of the spinal motor neurons to the increased amount of hTDP-43 lead to the neurodegeneration and pathogenesis of ALS, and of diseases with TDP-43 proteinopathies in general. The EIIa:Cre–Tardbplx/lx Mice provides genetic evidence for the in vivo role of TDP-43 as an essential gene for the viability and expansion of ICM cells of the implanting blastocysts. The study of HB9:Cre–Tardbplx/–mice not only establishes an important role of TDP-43 in the long term survival and functioning of the mammalian spinal cord motor neurons, but it also establishes that loss-of-TDP-43 function could be one major cause for neurodegeneration in ALS with TDP-43 proteinopathies.