Uchiyama Yasuo, Koike Masato, Shibata Masahiro, Sasaki Mitsuho
Department of Cell Biology and Neuroscience, Juntendo University Graduate School of Medicine, Tokyo, Japan.
Methods Enzymol. 2009;453:33-51. doi: 10.1016/S0076-6879(08)04003-2.
Neurons of the central nervous system (CNS) tissue are terminally differentiated cells and have large volumes, unlike cells of peripheral tissues. Such neurons possess abundant lysosomes in which damaged and unneeded intracellular constituents are degraded. A cellular process to bring the unneeded constituents to lysosomes is referred to as macroautophagy (autophagy), which is essential for the maintenance of cellular metabolism under physiological conditions. In fact, mice deficient in Atg7 or Atg5 specifically in CNS tissue have ubiquitin aggregates in neurons and massive loss of cerebral and cerebellar cortical neurons, resulting in neurodegeneration and short life span. In addition, acceleration of autophagy induced by the loss of lysosomal proteinases such as cathepsin D or cathepsins B and L, or by hypoxic/ischemic (H/I) brain injury, causes neurodegeneration. Moreover, lysosomes with undigested materials due to loss of proteinases are enwrapped by double membranes to produce autophagosomes, resulting in the further accumulation of autolysosomes. H/I brain injury at birth that is an important cause of cerebral palsy, mental retardation, and epilepsy causes energy failure, oxidative stress, and unbalanced ion fluxes, leading to a high induction of autophagy in brain neurons. Since mice that are unable to execute autophagy (due to brain-specific deletion of Atg7 or Atg5) die as a result of massive loss of cerebral and cerebellar neurons with accumulation of ubiquitin aggregates, induction of neuronal autophagy after H/I injury is generally considered neuroprotective, as it maintains cellular homeostasis. However, our data showing that H/I injury-induced pyramidal neuron death in the neonatal hippocampus is largely prevented by Atg7 deficiency indicate the presence of autophagic neuron death. In this section, we introduce various methods for the detection of autophagic neuron death in addition to other death modes of CNS neurons.
与外周组织的细胞不同，中枢神经系统（CNS）组织中的神经元是终末分化细胞，体积较大。这类神经元拥有丰富的溶酶体，受损及不需要的细胞内成分在其中被降解。将不需要的成分转运至溶酶体的细胞过程称为巨自噬（自噬），这在生理条件下对维持细胞代谢至关重要。事实上，中枢神经系统组织中特异性缺失Atg7或Atg5的小鼠，神经元中会出现泛素聚集体，大脑和小脑皮质神经元大量丢失，导致神经退行性变和寿命缩短。此外，诸如组织蛋白酶D或组织蛋白酶B和L等溶酶体蛋白酶缺失，或缺氧/缺血（H/I）性脑损伤诱导的自噬加速，会导致神经退行性变。而且，由于蛋白酶缺失而含有未消化物质的溶酶体被双膜包裹形成自噬体，导致自噬溶酶体进一步积累。出生时的H/I性脑损伤是脑瘫、智力发育迟缓及癫痫的重要病因，可导致能量衰竭、氧化应激和离子通量失衡，从而使脑神经元中的自噬大量诱导。由于无法进行自噬的小鼠（因脑特异性缺失Atg7或Atg5）会因大脑和小脑神经元大量丢失及泛素聚集体积累而死亡，H/I损伤后神经元自噬的诱导通常被认为具有神经保护作用，因为它能维持细胞稳态。然而，我们的数据表明，Atg7缺陷可在很大程度上预防H/I损伤诱导的新生海马锥体神经元死亡，这表明存在自噬性神经元死亡。在本节中，除了中枢神经系统神经元的其他死亡模式外，我们还介绍了检测自噬性神经元死亡的各种方法。
