Mishra Suman, Maurya Shashank Kumar, Srivastava Khushboo, Shukla Sachin, Mishra Rajnikant
Biochemistry and Molecular Biology Lab, Department of Zoology, Banaras Hindu University, Varanasi- 221005, INDIA.
Ann Neurosci. 2015 Oct;22(4):226-31. doi: 10.5214/ans.0972.7531.220407.
Pax6, a highly conserved multifunctional transcription factor, has been critical for neurogenesis and neuronal plasticity. It is presumed that if level of Pax6 approaches either low or null, critical genes responsible for maintaining functional status of neurons or glia would be modulated.
Therefore, it has been intended to explore possibility of either direct or indirect influence of Pax6 in neurodegeneration.
The cell lines having origin of murine embryonic fibroblast (Pax6-non expressing, NIH3T3-cell line), murine neuroblastoma (Pax6-expressing brain-derived, Neuro-2a-cell line), and human glioblastoma-astrocytoma (U87MG) were cultured and maintained in a CO2 incubator at 37°C and 5% CO2 in DMEM containing 10% fetal bovine serum. The knockdown of endogenous Pax6 in Neuro-2a cells was achieved through siRNA based gene knock-down approach. The efficiency and validation of knock-down was done by real time PCR. The knock-down of Pax6 was successfully achieved.
The levels of expression of transcripts of some of the proposed putative markers of neurodegeneration like Pax6, S100β, GFAP, BDNF, NGN2, p73α, p73δ, LDH, SOD, and Catalase were analyzed in Pax6 knockdown condition for analysis of role of Pax6 in neurodegeneration. Since the Pax6 has been proposed to bind to promoter sequences of catalase, and catalase suppresses TGFβ, relative lower levels of catalase in Neuro-2a and U-87MG as compared to NIH-3T3 indicates a possible progressive dominant negative impact of Pax6. However, presence of SOD and LDH indicates alternative protective mechanism.
Presence of BDNF and TGFβ indicates association between them in glioblastoma-astrocytoma. Therefore, Pax6 seems to be involved directly with p53 and TGFβ mediated pathways and indirectly with redox-sensitive pathway regulation. The neurodegenerative markers S100β, GFAP, BDNF, NGN2, p73α, p73δ, observed downregulated in Pax6 knockdown condition suggest Pax6-mediated regulation of these markers. Observations enlighten Pax6-mediated influences on cascades of genes involved in growth, differentiation and maturation of neurons and glia.
Pax6是一种高度保守的多功能转录因子，对神经发生和神经元可塑性至关重要。据推测，如果Pax6水平接近低水平或无水平，负责维持神经元或神经胶质细胞功能状态的关键基因将受到调节。
因此，旨在探索Pax6在神经退行性变中直接或间接影响的可能性。
培养具有小鼠胚胎成纤维细胞起源（不表达Pax6，NIH3T3细胞系）、小鼠神经母细胞瘤（表达Pax6，脑源性，Neuro-2a细胞系）和人胶质母细胞瘤-星形细胞瘤（U87MG）的细胞系，并在含有10%胎牛血清的DMEM中于37°C和5% CO₂的二氧化碳培养箱中进行培养和维持。通过基于小干扰RNA（siRNA）的基因敲除方法实现Neuro-2a细胞中内源性Pax6的敲除。通过实时定量聚合酶链反应（PCR）对敲除效率和验证进行检测。成功实现了Pax6的敲除。
在Pax6敲除条件下，分析了一些拟议的神经退行性变推定标志物如Pax6、S100β、胶质纤维酸性蛋白（GFAP）、脑源性神经营养因子（BDNF）、神经生成素2（NGN2）、p73α、p73δ、乳酸脱氢酶（LDH）、超氧化物歧化酶（SOD）和过氧化氢酶的转录本表达水平，以分析Pax6在神经退行性变中的作用。由于已提出Pax6与过氧化氢酶的启动子序列结合，且过氧化氢酶抑制转化生长因子β（TGFβ），与NIH-3T3相比，Neuro-2a和U-87MG中过氧化氢酶水平相对较低表明Pax6可能存在渐进性显性负性影响。然而，SOD和LDH的存在表明存在替代保护机制。
BDNF和TGFβ的存在表明它们在胶质母细胞瘤-星形细胞瘤中存在关联。因此，Pax6似乎直接参与p53和TGFβ介导的途径，并间接参与氧化还原敏感途径的调节。在Pax6敲除条件下观察到神经退行性变标志物S100β、GFAP、BDNF、NGN2、p73α、p73δ下调，提示Pax6对这些标志物的介导调节作用。这些观察结果揭示了Pax6对参与神经元和神经胶质细胞生长、分化和成熟的基因级联反应的介导影响。
