What are the first changes in the brain as Alzheimer’s pathology takes hold? According to a postmortem proteomics study published in Alzheimer’s and Dementia on April 19, it is proteins involved in vesicular functions, including endocytosis, secretion, and synaptic vesicle transport. Researchers led by Dietmar Thal at KU Leuven in Belgium identified 53 proteins whose abundance changed at different stages of AD, including seven proteins that had not been tied to AD before. The scientists reported that, as Aβ plaques and tau tangles overtake the brain and cognitive symptoms surface, other biological pathways show signs of change, and some proteins become harder to solubilize. In all, the findings implicate endosomal and synaptic malfunctions as foundational to the disease process.
- Proteins changed in abundance in response to amyloid plaques.
- Endocytosis and synaptic vesicle function is hit in preclinical stage.
- Some proteins become harder to solubilize as disease progresses.
“This proteomic analysis provides one more piece of the puzzle that indicates that endosomal/secretory defects are an essential mechanism early in AD,” commented Claudia Almeida of the University of Lisbon, Portugal. “Moreover, it supports that synaptic dysfunction, namely of the synaptic vesicle cycle, is an early target in AD.”
Numerous proteomic studies have graced the field in recent years, with some taking stock of proteins in thousands of postmortem brain samples to peg proteomic signatures of AD (Jul 2017 conference news; Mendonça et al., 2019; May 2020 news). Thal and colleagues opted to perform a smaller, more in-depth study with strict standards for distinguishing controls from people in different stages of AD. They also analyzed proteins based on their solubility using an ad hoc extraction method.
Stage Fractions. Neocortical homogenates prepared from postmortem brain samples of controls, people with preclinical AD (p-pre-AD), and people with symptomatic AD (left) were fractionated based on solubility (middle). Proteomic analysis of each fraction compared abundance and solubility of proteins between disease groups (right). [Courtesy of Li et al., Alzheimer’s & Dementia, 2021.]
First author Xiaohang Li and colleagues tracked not only the abundance of proteins, but how their solubility shifted from one disease stage to the next. They analyzed postmortem neocortical samples from, coincidentally, 53 people, including 18 non-AD controls who had died without any signs of AD pathology in the brain; 18 people who had been cognitively normal but harbored Aβ pathology; and 17 people who had had symptomatic AD. They also sampled the occipital cortex, a region that accumulates amyloid plaques early in disease, but stays free of tau tangles or neurodegeneration until the latest stages. For two non-AD cases, only frontal cortex samples were available, but the researchers later established that these frontal proteomes did not differ markedly from occipital proteomes of other controls.
Using a fractionation method they had previously developed, the researchers split each brain sample into four: a soluble fraction; a dispersible fraction containing insoluble protein oligomers and small vesicles such as synaptosomes and endosomes; a detergent-soluble fraction containing larger organelles and membrane–associated proteins; and a formic acid fraction containing highly insoluble, aggregated proteins such as those associated with plaques (Upadhaya et al., 2014). Then they measured the proteins in each fraction via mass spectrometry.
All told, the researchers detected 1,449 unique proteins across all fractions and samples. Of those, 53 differed in abundance between control, preclinical, and symptomatic AD groups. Levels of four proteins differed between controls and preclinical AD; the scientists called them “early responding proteins.” Another 26, called “late responding proteins,” changed between the preclinical and symptomatic stages, while another 20 proteins changed gradually in abundance from control through symptomatic stages.
Five proteins shifted fractions—moving from the dispersible fraction in controls or preclinical samples to the detergent fraction in symptomatic AD. The researchers reported that levels of early responding proteins correlated strongly with the presence of Aβ plaques. By contrast, the gradually changing and late-responding proteins correlated more tightly with measures of disease progression, including Aβ load, Braak neurofibrillary tangle stage, and neuritic plaque score.
In the earliest stage of disease, when Aβ plaques were present but cognitive symptoms had not yet surfaced, levels of calmodulin-1, endophilin-A1, adaptor-related protein complex 1-b (AP1B1), and ribosomal protein L31 had changed relative to controls. Calmodulin-1 increased, but the other three—found within the vesicle-laden, dispersible fraction—all decreased in preclinical AD. Both endophilin-A1 and AP1B1 facilitate clathrin-mediated endocytosis. Endophilin-A is crucial for the endocytosis of synaptic vesicles and the maintenance of dendritic spines. L31 is part of the large ribosomal subunit.
Endocytosis, secretory pathways, and/or synaptic vesicles also featured prominently among the gradually changing and late-responding proteins. In addition, pathway analysis placed the gradually and late-responding proteins in caspase-mediated cleavage and MHC Class II antigen presentation pathways. Notably, APP/Aβ (mass spec does not distinguish between the two) ramped up in the formic acid fraction, while tau increased in the dispersible, i.e., oligomeric, fraction as disease progressed.
Two of the fraction-shifting proteins—plectin and UCH-L1—not only were less soluble in samples from more advanced disease, but were also less abundant in people with symptomatic AD.
Using a protein-protein interaction network database called STRING, the researchers next asked how the 53 AD-related proteins fit into a wider picture of protein function. The analysis identified two functional networks—APP metabolism and endocytosis. When the researchers wove in AD risk genes identified in genome-wide association studies, three functional networks emerged: APP/Aβ, tau-related, and synaptic vesicle/endocytosis/secretory pathways.
In all, the findings suggest that endocytic and synaptic vesicle functions become impaired in the earliest stage of AD, at a time when a person has Aβ plaques but tau tangles have not yet spread extensively in his or her cortex. This suggests that these early neocortical changes are caused by Aβ-related processes, rather than by tau accumulation. This is especially true in the sampled region—the occipital lobe—which is inundated by Aβ pathology early in disease but does not bear tau tangles until the latest stages. An alternative explanation, Thal said, is that tau pathology in subcortical regions such as the nucleus basalis of Meynert, the raphe nuclei, and the locus coeruleus, which arises early in disease, may affect neurons in connected regions of the brain, including the occipital lobe.
Thal hesitated to speculate about the role of any one protein in AD, noting that proteomic studies are best suited for assessing the entire forest, rather than individual trees. Future studies will focus on how specific proteins and cellular processes influence disease progression in detail, he said.—Jessica Shugart
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