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
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
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
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).