Results

ATG12-ATG5 and LC3-II Conjugation after Traumatic Brain Injury

The biochemical hallmark of autophagic initiation is the consecutive formation of two key ATG conjugates:

first ATG12-ATG5, followed by LC3-II (LC3-II is a mammalian homologue of yeast ATG8-PE). After

conjugation, ATG12-ATG5 and LC3-II became components of double-membraned cisterns and/or AP

and hence were distributed into membrane fractions. We therefore prepared brain tissue homogenate

(H) as well as standard P1, P2, P3, and S3subcellular fractions from neocortical brain tissues subjected either to sham surgery or TBI followed by 4 h, and 1, 3, 5, and 15 days of recovery. To ensure equal loading of protein samples on immunoblots, b-actin immunoreactivity was measured by Western blotting and used as an endogenous control. As shown in Figure 3A, b-actin immunoreactivity on immunoblots was not significantly altered in brain tissue homogenate and any subcellular fraction after TBI. ATG5 antibody was primarily labeled the ATG12-ATG5 conjugated form (B53 kDa) in all subcellular fractions (Figure 3B), whereas the unconjugated (free form) ATG5 (B32 kDa) was barely detected in the homogenate (H) and S3 fraction (Figure 3B, H and S3, arrowhead). Relative to shamoperated controls, the ATG12-ATG5 conjugate was not significantly changed in tissue homogenate (H) and P1 fraction, but was significantly upregulated in the P2 fraction at 3 days of reperfusion after TBI (Figures 3B and 3C). In comparison with P1 and P2 fractions, ATG12-ATG5 in the pre-AP-containing P3 fraction was drastically downregulated at 4 h of recovery and then rebounded significantly above the control level during 5 to 15 days of recovery after

TBI (Figures 3B and 3C). ATG12-ATG5 in S3 fraction tended to decrease, but the difference did not reach

statistical significance during 1 to 15 days of recovery after TBI (Figures 3B and 3C). In addition, the ATG12-ATG5 level was the highest in S3 fraction, followed by P3 fraction, and much lower in P1 and P2 fractions (Figure 3B). Microtubule-associated protein light chain 3 is synthesized as a proform that is cleaved by ATG4 protease to become LC3-I (Tanida et al, 2004). Microtubule-associated protein light chain 3-I conjugates covalently with a neutral lipid PE via its C-terminal glycine residue to form lipidated LC3-II,

which has a faster mobility on immunoblots. Thus, the N-terminal LC3 antibody was able to recognize

both the upper LC3-I band and the lower LC3-IIband in homogenate (H) as well as in P1, P2, and P3

fractions, and also pro-LC3 and LC3-I in the cytosolic (S3) fraction (Figure 4A), consistent with previous studies (Tanida et al, 2004). After conjugation, LC3-II became a component of the AP and then the AL membranes and hence was distributed mainly to P1 and P2 fractions and to a much lesser degree also to P3 fraction after TBI (Figure 4A). The LC3-I level tended to decrease in H, P1, P2, and P3 fractions, although the changes did not reach statistical significance, probably owing to its large residual contents in these fractions (Figure 4A, LC3-I and Figure 4B, white bars). The LC3-II protein level, however, was significantly and persistently increased in the pellet fractions, particularly in P1 and P2 fractions during 1 to 15 days of recovery after TBI (Figure 4A, LC3-II and Figure 4B, black bars). Pro-LC3 was detected only in S3 fraction and its level seemed to be not obviously altered after TBI (Figure 4A, S3, pro-LC3). Only LC3-I, but not LC3-II, was detected in S3 fraction (Figure 4A), consistent with previous reports (Tanida et al, 2004).

外伤性脑损伤后自噬基因ATG12-ATG5复合物和LC3-II的结合

自噬开始的生化特征是连续形成两种关键的自噬相关基因轭合物：首先是ATG12-ATG5结合物，接着是LC3-II.( LC3-II是哺乳动物中酵母ATG8基因和PE结合的同源物)

结合后，ATG12-ATG5 和 LC3-II 变成双膜泡和/或自噬小体的一部分，从而分布到膜部分里。因此，我们分别准备了从假手术对照组到TBI后4小时和手术后恢复1，3，5和15天的脑组织匀浆(H)，及制备的标准的P1, P2, P3, 和S3的亚细胞组分。

为确定在免疫印迹时载入等量样品蛋白，先用蛋白印迹测量了β―actin免疫活性并用做一个内部标准控制。如图3A所示，在TBI后脑组织匀浆与所有亚细胞部分的上β―actin免疫活性在免疫印迹都没有显著性改变。ATG5抗体标志到了最初所有亚细胞部分的ATG12-ATG5共轭体形态（图3，B53kDa），然而，这游离的非结合体ATG5(B32kDa)仅在组织匀浆及S3部分发现(Figure 3B, H and S3, arrow head)。

 与对照组相比， ATG12-ATG5 轭合物在组织匀浆及P1部分无显著变化，但是在TBI后再灌注3天的P2部分有显著上调。（图3B与3C）

比较P1与P2部分，包含前自噬小体的P3部分的ATG12-ATG5在恢复4小时明显下降，然后在TBI后恢复第5天到15天重新回弹超过对照组以上水平(Figures 3B and 3C)。

TBI后的恢复过程中，在S3部分的ATG12-ATG5表现出下降趋势，但其差异未达到统计显著性程度。(Figures 3B and 3C)。另外，ATG12-ATG5水平在S3部分最高，其次是P3，而P1与P2则小得多。(Figure 3B)

微管相关蛋白轻链 LC3被合成为ATG4蛋白酶裂解形成的LC3-I的形式(Tanida et al, 2004)。

微管相关蛋白轻链LC3-I与一个中性脂类磷脂酰乙醇胺PE通过其C端甘胺酸残端共价联接，形成脂质的LC3-II,它在免疫印迹中,LC3-II的迁移速率要快于LC3-I，因此，N端LC3抗体能识别匀浆中，P1，P2，P3部分的上端LC3-1带和低端LC3-II带。也能识别S3中的LC3前体和LC3-I (Figure 4A)，这与以前的研究相一致，Tanida et al, 2004)。

结合后，LC3-II变成AP的一部分，然后是AL膜，因此它主要分布在TBI后的P1与P2部分，在P3部分里则少得多。(Figure 4A)

组织匀浆H, P1, P2, and P3 部分中的LC3-I水平倾向于下降，虽然这变化没达到统计学显著意义。可能的原因是这些部分中存有大量的残留物。(Figure 4A, LC3-I and Figure 4B, white bars)。

但是,小球部分的LC3-II蛋白水平，特别是在TBI后恢复1到15天的P1与P2部分，呈现显著与持续的增加。(Figure 4A, LC3-II and Figure 4B, black bars)。

LC3前体只在S3部分检测到,而且它的水平似乎在TBI后没有明显改变(Figure 4A, S3, pro-LC3)

只有LC3-I, 但LC3-II没有在S3部分中被检测到(Figure 4A)，与以前的报导相一致(Tanida et al, 2004).