Filamentous Aggregation of Sequestosome-1/p62 in Brain Neurons and Neuroepithelial Cells upon Tyr-Cre-Mediated Deletion of the Autophagy Gene Atg7

Abstract

Defects in autophagy and the resulting deposition of protein aggregates have been implicated in aging and neurodegenerative diseases. While gene targeting in the mouse has facilitated the characterization of these processes in different types of neurons, potential roles of autophagy and accumulation of protein substrates in neuroepithelial cells have remained elusive. Here we report that Atg7f/f Tyr-Cre mice, in which autophagy-related 7 (Atg7) is conditionally deleted under the control of the tyrosinase promoter, are a model for accumulations of the autophagy adapter and substrate sequestosome-1/p62 in both neuronal and neuroepithelial cells. In the brain of Atg7f/f Tyr-Cre but not of fully autophagy competent control mice, p62 aggregates were present in sporadic neurons in the cortex and other brain regions as well in epithelial cells of the choroid plexus and the ependyma. Western blot analysis confirmed a dramatic increase of p62 abundance and formation of high-molecular weight species of p62 in the brain of Atg7f/f Tyr-Cre mice relative to Atg7f/f controls. Immuno-electron microscopy showed that p62 formed filamentous aggregates in neurons and ependymal cells. p62 aggregates were also highly abundant in the ciliary body in the eye. Atg7f/f Tyr-Cre mice reached an age of more than 2 years although neurological defects manifesting in abnormal hindlimb clasping reflexes were evident in old mice. These results show that p62 filaments form in response to impaired autophagy in vivo and suggest that Atg7f/f Tyr-Cre mice are a model useful to study the long-term effects of autophagy deficiency on the homeostasis of different neuroectoderm-derived cells.Electronic supplementary materialThe online version of this article (10.1007/s12035-018-0996-x) contains supplementary material, which is available to authorized users.

Structural basis of p62/SQSTM1 helical filaments and their role in cellular cargo uptake

“…The principal dimension of the observed structures is consistent in width and length with previous measurements in vitro 8 . The structures are compatible with recently observed aggregates of p62 in brain neurons and neuroepithelial cells 20 . Due to the limited length and flexibility, p62 filaments pack loosely into a spheroid-shaped, meshwork-like superstructure.…”

“…Due to p62's involvement in protein homeostasis, the impairment of autophagy or oxidative stress results in aggregation or upregulation of p62, including increased body formation 19,20 . Recently, we and others independently found that p62 reconstituted with other components of the autophagy pathway, such as ubiquitinated model cargo, and the selective autophagy receptor NBR1, spontaneously coalesces into p62 bodies in vitro 21 and shows the characteristics of liquid-liquid-phase separation in vivo 22 .…”

Protection effect of gastrodin on learning and memory ability in vascular dementia by promoting autophagy flux via Ca2+/CaMKII signal pathway

“…p62 protein is located at the site of autophagosome formation and can bind to autophagosome localization protein LC3 and ubiquitin protein. Therefore, p62 is a recognition receptor for ubiquitin protein and organelle degradation [17,36]. The decrease of p62 level leads to neuropathological changes, including excessive accumulation of tau and Aβ proteins, and even neuronal apoptosis [56].…”

“…Expressions of LC3Ⅱ was significantly increased in a dose-dependent manner ( Fig. 3d), suggesting an increase in the number of autophagic vacuoles, which may be due to elevation of autophagosome formation or suppression of autophagy degradation.In addition to LC3, the level of p62, as an autophagy substrate whichcan be attached to LC3 and ubiquitinated substrates, and then integrated into autophagosomes and degraded in autophagolysosomes [17,35], are significantly up-regulated ( Fig. 3d-e).…”

“…As a shuttle protein transporting lysosome and proteasome-degrading ubiquitinated proteins, p62 has been highlighted on a variety of diseases such as PD, HD, and AD [16]. When autophagy deficiency occurs in the brain, neurons in almost all regions are accompanied with polyubiquitin and p62 accumulation, suggesting that p62 plays a key role in neurons [17]. In the early stage of neurodegenerative diseases, the activation of autophagy accelerates the removal of the denatured protein and delay the development of the disease [18].…”