Discussion

This study shows that the autophagy pathway is markedly changed in vulnerable brain regions after TBI. Under EM, both APs and ALs accumulate markedly in neurons after TBI.Western blot analysis consistently clearly shows upregulation in two key autophagic markers, ATG12-ATG5 and LC3-II, in pre-AP- and AP-containing subcellular fractions during the post-TBI phase. Microtubule-associated protein light chain 3 immunoreactivity is located mainly in living neurons under confocal microscopy. The ultrastructural and biochemical results clearly show that the autophagy pathway is significantly activated in neurons after TBI. The autophagy pathway is the chief route for bulk degradation of damaged cell membranes, neuronal processes, and organelles after TBI. Therefore,activation of the autophagy pathway may play a key role in removing damaged cellular componentsafter TBI.

Ultrastructural Features of Autophagy after Traumatic Brain Injury

All these ultrastructural features of autophagy are clearly observed by transmission EM in TBI neurons (see Figures 1 and 2). The ultrastructural hallmarks for induction of autophagy are the manifestation of (i) double-membraned cistern structures (see Figures 1 and 2); (ii) APs containing cytoplasmic materials or aberrant organelles (APs, see Figures 1 and 2); and (iii) ALs that contain partially digestedheterogeneous dense (dark) materials at an early stage and ultimately digested homogeneous dense material (ALs, see Figures 1 and 2). These results provide solid ultrastructural evidence showing that the autophagy pathway is activated after TBI.

Biochemical Changes in Autophagy after Traumatic Brain Injury

Biochemical hallmarks of autophagy initiation are the formation of two conjugates: (i) ATG12-ATG5 and (ii) LC3-II. ATG12-ATG5 is required for transformation from cup-shaped double-membraned cisterns to APs. Immediately before or after AP formation is completed, ATG12-ATG5 detaches from the membrane and is then recycled by ATG4 protease for the next round of AP formation (Mizushima et al, 2001). It remains unclear whether ATG12-ATG5 is also degraded during this process. This study shows that the ATG5 antibody predominantly labels the ATG12-ATG5 conjugated form (B53 kDa) mainly in S3 and P3 fractions but to a much lesser degree in P1 and P2 fractions (see Figure 3). The free or unconjugated ATG5 is barely detected in homogenate and cytosolic fractions (see Figure 3B, H and S3, arrowhead). The result is in

line with previous reports showing that almost all ATG5 exists in the ATG12-ATG5 conjugated form (Mizushima et al, 2001). The predominant cytosolic pool of ATG12-ATG5 suggests that ATG12-ATG5 conjugate is ready to initiate AP formation in response to physiologic and pathologic changes. This is consistent with the fact that autophagy is a nonstop renewal process even under physiologic conditions. In addition, this study shows that ATG12-ATG5 in P3 fraction is significantly reduced at 4 h and is then drastically upregulated during the late periods of recovery after TBI. The changes in ATG12-ATG5 level in P3 fraction are likely because of its redistribution among P2, P3, and S3 fractions after TBI, because ATG12-ATG5 in homogenate is not significantly changed (see Figure 3). The late increases in ATG12-ATG5 in P3 fraction indicate that AP formation is upregulated during the late period of recovery after TBI. These results are consistent with the increases in APs and ALs observed by EM (see Figures 1 and 2). Microtubule-associated protein light chain 3-II, as one of the mammalian homologues of ATG8-PE conjugate, is also recruited into double-membraned cisterns in an ATG12-ATG5-dependent manner (Mizushima et al, 2001). However, unlike ATG12-ATG5 conjugate that issociates from the membraneimmediately after AP formation, LC3-II conjugate remains on the AP membrane even after AP merges with lysosome (Kabeya et al, 2000). Hence, the LC3-II protein level has been used as a molecular marker to assess cellular AP numbers, and it has proved to be both more sensitive and specific than the less quantitative EM method (Koike et al, 2005). This study clearly shows that LC3-II is drastically and persistently upregulated in neurons from 1 day onward after TBI, thus providing solid quantitative evidence for dynamic upregulation of APs after TBI. The result is also complementary to the less quantitative EM observation of increases in APs after TBI (see Figures 1 and 2). In addition, upregulation of LC3-II is found mainly in P1 and P2 fractions but to a much lesser degree in the P3 fraction after TBI. This is consistent with the fact that LC3-II is located mainly in AP and also to a lesser degree in AL membranes (Kabeya et al, 2000; Tanida et al, 2004). In comparison with LC3-II, changes in ATG12-ATG5 conjugate occur mainly in S3 and P3 fractions after TBI. Taken together, these results support the fact that ATG12-ATG5 is associated

with double-membrane cisterns (pre-APs) located in microsomal P3 fraction, whereas LC3-II in AP and AL membranes is distributed mainly into P1 and P2 fractions from brain tissues. Confocal microscopic immunolabeling of LC3 in brain sections indicates that LC3 is mainly located in living neurons. However, the punctate LC3 immunostaining of APs seems not obvious after TBI. This may be due to a large non-AP LC3-I pool in neurons, as indicated by  mmunoblotting shown in Figure 4. There are a few studies recently showing LC3 changes after brain hypoxia–ischemia (HI). Zhu et al (2005, 2006) reported that the LC3-II protein level was upregulated mainly in adult rather thanneonatal brains after HI. Adhami et al (2006, 2007)

found a reduction of LC3-I after HI but did not detect upregulation of LC3-II on immunoblot in mature HI mouse model. This study suggests that, relative to mild changes in LC3-II after HI in the previous reports, alterations in LC3-II after TBI appear more robust. During the revision of this paper, a study of autophagy after TBI in the ahead of print status by Lai et al (2007) also shows an increase in LC3-II in a mouse TBI model. The reason for the difference in the LC3-II level between TBI and HI is unknown, but it is probably because the pathophysiology incurred by TBI is far from identical to that of HI (Siesjo et al,1995). Traumatic brain injury produces shear forces that primarily damage cell bodies and processes, whereas HI leads to metabolic failure (Bramlett and Dietrich, 2004; Zhu et al, 2006; Chu, 2006).

讨论

    本研究显示，自噬途径在外伤性脑损伤的后的脑受伤区域内有明显的变化。电镜下观察，TBI之后的神经元内存在明显的自噬小体与自噬溶酶体积聚。蛋白印迹分析一直清楚地显示，包含亚细胞碎片的两个关键自噬标记前自噬小体和自噬小体中的ATG12-ATG5结合物和 LC3-II在外伤性脑损伤后明显上调。在激光共焦显微镜下,微管相关蛋白轻链LC3的免疫反应主要位于存活的神经元内。超微结构与生化结果清楚的显示, TBI之后,自噬路径在神经元内被显著激活。自噬路径是TBI之后，大量受损细胞膜和细胞器官降解的主要途径。因此，自噬途径的激活可能会在TBI之后清除受损细胞扮演着关键角色。

外伤性脑损伤后自噬的超微结构特征

    在透射电镜下(图1和2)，TBI神经元的自噬超微特征可以清楚地观察到,导致自噬的超微结构的标志主要表现在：一，双层膜结构(图1和2)；二,包含胞质的物质或异常细胞器的自噬小体(图1和2)；三,包含部分降解异常物质和完全降解同质密度材料的自噬溶酶体(图1和2)。这些结果提供了可靠的超微结构证据，显示TBI之后，自噬途径被激活。

外伤性脑损伤后自噬的生化变化

    自噬开始的生化标志物是形成二个轭合物：一，ATG12-ATG5结合物,二,LC3-II。在自噬小体形成的前后，ATG12-ATG5结合物从膜上被分离，然后由ATG4蛋白酶再分配回收用于下一轮自噬小体的形成。在这个过程中，ATG12-ATG5结合物是否也被降解，现在不清楚。本研究显示：主要标记ATG12-ATG5共轭形态的ATG5抗体，在S3，P3部分里有很多，但在P1，P2部分里就少得多了(图3)。游离的ATG5仅仅在组织匀浆和细胞溶质部分中被找到。(图3B,H和S3箭头)。以前文献显示，几乎所有的ATG5都以ATG12-ATG5共轭形式存在。细胞质里大量的ATG12-ATG5存在显示ATG12-ATG5共轭体是准备启动自噬小体的生成以应对生理与病理变化。这与下面的事实相符合：自噬是一个持续更新的过程，即使在生理条件下也是这样。另外，本研究显示，P3部分的ATG12-ATG5在TBI 4小时后显著减少，然后在后面的恢复过程中明显上调。P3部分中ATG12-ATG5 水平的变化似乎是由于TBI之后它在P2, P3和 S3 部分之间的再分配。因为在组织匀浆中ATG12-ATG5没有显著变化。P3部分里ATG12-ATG5在以后的增加说明，自噬小体的生成在TBI后恢复期明显上调。这个结果也符合电镜下观察的自噬小体和自噬溶酶体增多是一致的。

    微管相关蛋白轻链（LC3）是一种哺乳动物中ATG8-PE共轭体的同源物，也被补充到ATG12-ATG5双膜泡的方式。但是，不象ATG12-ATG5 共轭体在自噬小体形成后从膜上脱离开来，LC3-II 共轭体则一直保持在AP膜上，甚至自噬小体与自噬溶酶体结合之后。因此，LC3-II 蛋白水平被当作评价细胞中自噬小体数量的分子标记。这个研究清楚的显示，LC3-II在TBI之后一天起持久上调。因而提供了TBI之后自噬小体的动态上调的有力定量证据。这个结果也是对TBI后自噬小体的低定量性的电镜观察的一个补充。另外，在TBI后LC3-II的上调在P1 和 P2 部分也被发现占主要作用，但比P3低得多。这也符合下述事实：LC3-II主要定位于自噬小体，并且更少程度的存在于自噬溶酶体膜上。与LC3-II相比，ATG12-ATG5共轭体的变化主要发生在TBI之后S3 和P3部分。总之，这些结果支持的事实是：ATG12-ATG5与位于线粒体P3部分的双膜泡 (前自噬小体)有关联，而自噬小体与自噬溶酶体膜上的LC3-II则主要分布于脑组织P1 和 P2 部分。

    脑切片在共焦显微镜免疫印迹下显示LC3主定位于活的神经元上。但是在TBI之后的LC3 免疫染色似乎并没有明显观察到自噬小体。这可能是由于在神经元里的一个大的非AP LC3-I 汇聚，如图4的免疫印迹所示。

    最近有一些对低氧性脑缺血（HI）后LC3变化的研究。在HI后大幅上调，这主要表现在成人而不是新生儿。Adhami 等人发现, 成年鼠HI模型在HI后LC3-I减少，但免疫印迹显示LC3-II没有上调。此研究提示，相对于以前所报导的HI之后LC3-II的轻缓变化，TBI之后的LC3-II的变化大得多。在本论文修订期间，另一篇Lai et al (2007)出版的文章也显示鼠TBI模型中的LC3-II增加。导致LC3-II水平在TBI与HI之间不同的原因不明，目前认为这可能是由于TBI导致的病理生理变化远不同于脑缺血缺氧损伤。外伤性脑损伤产生剪切力，主要损害细胞体和形成过程，而HI导致的是代谢的障碍。