Sequential formation of different layers of dystrophic neurites in Alzheimer's brains

Alzheimer's disease (AD) is characterized by the presence of neuritic plaques in which dystrophic neurites (DNs) are typical constituents. We recently showed that DNs labeled by antibodies to the tubular endoplasmic reticulum (ER) protein reticulon-3 (RTN3) are enriched with clustered tubular ER. However, multi-vesicle bodies are also found in DNs, suggesting that different populations of DNs exist in brains of AD patients. To understand how different DNs evolve to surround core amyloid plaques, we monitored the growth of DNs in AD mouse brains (5xFAD and APP/PS1 Delta E9 mice) by multiple approaches, including two-dimensional and three-dimensional (3D) electron microscopy (EM). We discovered that a pre-autophagosome protein ATG9A was enriched in DNs when a plaque was just beginning to develop. ATG9A-positive DNs were often closer to the core amyloid plaque, whereas RTN3 immunoreactive DNs were mostly located in the outer layers of ATG9A-positive DNs. Proteins such as RAB7 and LC3 appeared in DNs at later stages during plaque growth, likely accumulated as a part of large autophagy vesicles, and were distributed relatively furthest from the core amyloid plaque. Reconstructing the 3D structure of different morphologies of DNs revealed that DNs in AD mouse brains were constituted in three layers that are distinct by enriching different types of vesicles, as validated by immune-EM methods. Collectively, our results provide the first evidence that DNs evolve from dysfunctions of pre-autophagosomes, tubular ER, mature autophagosomes, and the ubiquitin proteasome system during plaque growth.