Department of Pharmacology, Physiology and Neuroscience, Rutgers-New Jersey Medical School, Newark, NJ 07103, USA.
Federated Department of Biological Sciences, New Jersey Institute of Technology, Rutgers University, Newark, NJ 07102, USA.
Cells. 2021 Apr 14;10(4):898. doi: 10.3390/cells10040898.
Recent studies of cerebral hypoxia-ischemia (HI) have highlighted slowly progressive neurodegeneration whose mechanisms remain elusive, but if blocked, could considerably improve long-term neurological function. We previously established that the cytokine transforming growth factor (TGF)β1 is highly elevated following HI and that delivering an antagonist for TGFβ receptor activin-like kinase 5 (ALK5)-SB505124-three days after injury in a rat model of moderate pre-term HI significantly preserved the structural integrity of the thalamus and hippocampus as well as neurological functions associated with those brain structures. To elucidate the mechanism whereby ALK5 inhibition reduces cell death, we assessed levels of autophagy markers in neurons and found that SB505124 increased numbers of autophagosomes and levels of lipidated light chain 3 (LC3), a key protein known to mediate autophagy. However, those studies did not determine whether (1) SB was acting directly on the CNS and (2) whether directly inducing autophagy could decrease cell death and improve outcome. Here we show that administering an ALK5 antagonist three days after HI reduced actively apoptotic cells by ~90% when assessed one week after injury. Ex vivo studies using the lysosomal inhibitor chloroquine confirmed that SB505124 enhanced autophagy flux in the injured hemisphere, with a significant accumulation of the autophagic proteins LC3 and p62 in SB505124 + chloroquine treated brain slices. We independently activated autophagy using the stimulatory peptide Tat-Beclin1 to determine if enhanced autophagy is directly responsible for improved outcomes. Administering Tat-Beclin1 starting three days after injury preserved the structural integrity of the hippocampus and thalamus with improved sensorimotor function. These data support the conclusion that intervening at this phase of injury represents a window of opportunity where stimulating autophagy is beneficial.
近期关于脑缺氧缺血（HI）的研究强调了缓慢进行性神经退行性变，其机制仍不清楚，但如果加以阻断，可能会显著改善长期神经功能。我们之前的研究表明，细胞因子转化生长因子（TGF）β1 在 HI 后高度升高，并且在中度早产 HI 大鼠模型中，在损伤后三天给予 TGFβ 受体激活素样激酶 5 (ALK5) 的拮抗剂 SB505124 可显著保留丘脑和海马的结构完整性以及与这些脑结构相关的神经功能。为了阐明 ALK5 抑制减少细胞死亡的机制，我们评估了神经元中自噬标记物的水平，发现 SB505124 增加了自噬体的数量和脂化轻链 3（LC3）的水平，LC3 是一种已知介导自噬的关键蛋白。然而，这些研究并未确定（1）SB 是否直接作用于中枢神经系统，以及（2）直接诱导自噬是否可以减少细胞死亡并改善预后。在这里，我们表明，在 HI 后三天给予 ALK5 拮抗剂可在损伤后一周评估时将活跃的凋亡细胞减少约 90%。使用溶酶体抑制剂氯喹进行的离体研究证实，SB505124 增强了损伤半球中的自噬流，在 SB505124 +氯喹处理的脑片中，自噬蛋白 LC3 和 p62 明显积累。我们使用刺激肽 Tat-Beclin1 独立激活自噬，以确定增强自噬是否直接导致改善结果。在损伤后三天开始给予 Tat-Beclin1 可保持海马体和丘脑的结构完整性，并改善感觉运动功能。这些数据支持这样的结论，即在损伤的这个阶段进行干预代表了一个机会窗口，刺激自噬是有益的。
