Best known for its role in churning out Aβ peptides as the catalytic subunit of γ-secretase, presenilin-1 may also promote digestion of those Aβ peptides within microglia. That is the upshot of a study published August 13 in Molecular Psychiatry. Led by the late Paul Greengard of Rockefeller University in New York, the study found that in mice expressing a form of PS1 that cannot be phosphorylated on serine-367, microglia became overloaded with autophagic vacuoles and lysosomes that failed to acidify. These microglia migrated sluggishly in response to brain injury. In a mouse model of amyloidosis, the phosphorylation-deficient PS1-S367A mutant stymied microglial digestion of Aβ and exacerbated its accumulation. Together with previous findings from the lab, the study attempts to build a case that presenilin-1 influences amyloidosis via multiple mechanisms and within different cell types.
- PS1-S367A knock-in mice cannot phosphorylate PS1 on serine-367.
- Their microglia migrate slowly and have lysosomal defects.
- In 5xFAD mice, PS1- S367A exacerbates Aβ accumulation.
“This study reminds us that PS1 harbors multiple functions other than the proteolysis,” wrote Taisuke Tomita of the University of Tokyo. “Further analysis of this regulatory mechanism would provide a novel understanding of PS1 biology.” Tomita added that it remains unclear whether this microglial PS1 function is relevant to the pathogenesis of AD. He wondered how familial AD mutations in PS1 might affect its microglial-specific functions.
Previously, a pair of studies from Greengard’s lab cast PS1 as a driver of autophagy in neurons (Jun 2017 news). The researchers reported that when phosphorylated at serine-367, PS1 teamed up with Annexin 2 and other vesicular proteins to facilitate the fusion of autophagosomes to lysosomes. This promoted the autophagic digestion of APP β-C-terminal fragments (CTF), thus keeping Aβ production in check. When the researchers swapped endogenous mouse PS1 for the S367A mutant, amyloidosis skyrocketed in J20 mice. Thwarting phosphorylation of PS1 at this residue did not appear to affect the processing of APP by γ-secretase, suggesting the modification is not essential for proteolysis.
In the new study, first author Jose Ledo and colleagues expanded their investigation to microglia, since these cells use autophagic digestion to clear extracellular debris, including Aβ. The researchers started by assessing fundamental characteristics of microglia—migration and ramification—in PS1-S367A knock-in mice. Using cranial windows to watch the cells in action, the researchers found that they migrated more slowly to the site of a laser-induced injury than did microglia in wild-type mice. Microglia in the knock-ins also extended fewer processes, with less elaborate branching patterns than those expressing normal PS1. Phospho-deficient PS1 did not appear to change how many microglia were there.
How did it alter microglial function? The researchers gleaned some inkling from gene-expression analyses, which uncovered 121 differentially expressed genes when compared with microglia from wild-type mice. Genes involved in phagosome maturation and autophagy were among those most affected. Microglial expression of ATP6v0a1, a vacuolar-ATPase that acidifies the lysosome, was reduced by half in the knock-ins.
Microglial Indigestion. Electron microscopy indicates an overabundance of autophagic vacuoles (arrows) in microglia sorted from phospho-deficient PS1-S367A mice (right) compared with wild-type mice (left). Vacuoles are quantified on right. [Courtesy of Ledo et al., Molecular Psychiatry, 2020.]
In keeping with the gene-expression data, microglia isolated from PS1-S367A mice had a glut of autophagic vacuoles chock-full of undigested material. Their lysosomes had a higher pH, suggesting acidification was not working properly. In agreement with the defect, primary microglia from the knock-in mice internalized Aβ oligomers normally, but did not go on to digest them. The researchers could correct lysosomal defects by overexpressing TFEB, a master transcriptional regulator of autophagy. Ramping up TFEB in primary microglia not only restored levels of ATP6v0a1, but normalized lysosomal pH.
Ralph Nixon, Nathan Kline Institute, New York, is unconvinced that reductions in vacuolar ATPase explain the lysosomal deficits. He has previously reported that APP CTFs raise lysosomal pH. “It is puzzling why this demonstrated action of APP-CTF on lysosomal pH, given the group’s previous report of APP-βCTF elevations in presumably the same lysosomal compartment, was not considered as a possible mechanism for the phospho-deficient mutant, but instead a highly speculative alternative was favored,” he wrote (see comment below).
Does this microglial indigestion influence amyloidosis? To investigate, the researchers crossed the PS1 phospho-deficient mice with 5xFAD mice. Confocal microscopy of brain sections from 3-month-old offspring revealed microglia stuffed with Aβ. Congo Red staining followed by iDISCO, a technique that renders brain tissue transparent, revealed Aβ plaques throughout the brain. Compared with 5xFAD mice, those expressing the phospho-deficient PS1 had a higher plaque load in 35 of about 180 brain regions analyzed.
Aβ Plaque Disco. Tissue clearing reveals substantially more Aβ plaques (Congo Red, gold) in brain hemispheres from PS1-S367A mice. [Courtesy of Ledo et al., Molecular Psychiatry, 2020.]
By mixing APP with microglial extracts, the researchers again found that γ-secretase processing of its C-terminal fragments were unaffected by the S367A mutation in PS1. However, the same was not true for another γ-secretase substrate, Notch. Its processing was enhanced in microglial extracts from the knock-in mice compared with those from wild-type mice. This suggested that PS1 S367 phosphorylation affects γ-secretase activity toward some substrates and not others.
Hui Zheng of Baylor College of Medicine in Houston noted that it is difficult to comprehend how S367A phosphorylation would mediate distinct effects on APP and Notch, and the phenomenon requires further study. “Further, since the S367A mutation is not found in PSEN1 FAD cohorts, the relevance of the microglial phenotypes observed in the KI mice to AD is not evident,” Zheng added.
Ledo told Alzforum that he sees this study as a start to investigating the function of PS1 in microglia. Most studies have focused on the role of the protein in neurons. Previous studies from Greengard’s lab suggested that in neurons, PS1 drives autophagy by promoting the fusion of autophagosomes to lysosomes. However, in microglia, phosphorylation of the protein appears to influence acidification of lysosomes. It is possible that PS1 function is regulated differently depending on cell type, he said. Investigation of PS1 function in microglia is an ongoing focus in the lab, he said, including the generation of conditional knock-in mice that express phospho-deficient PS1, or PS1 with familial AD mutations, only in microglia.
It remains to be seen if these lysosomal functions of PS1 are relevant for the human brain. Ledo said that he has detected phosphorylation of S367 in human fibroblasts, and phosphorylation of the residue has been documented for numerous other human cell types (see PhosphoSitePlus). Rapid changes in phosphorylation complicate postmortem analyses of PS1 phosphorylation in the human brain, though Ledo plans to work with brain banks to minimize this problem. The researchers are also investigating PS1 function in human iPSC-derived neurons and microglia. One hint that PS1 phosphorylation might be affected in the AD brain comes from a methylation study, which found that the gene encoding CK1g2—the kinase that phosphorylates serine-367—is hypermethylated in the brains of people with sporadic AD and poorly expressed (Semick et al., 2019). Reduced expression of this kinase would theoretically lead to less phosphorylation of PS1 and greater Aβ accumulation, Ledo noted.—Jessica Shugart
Research Models Citations
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- Matz A, Halamoda-Kenzaoui B, Hamelin R, Mosser S, Alattia JR, Dimitrov M, Moniatte M, Fraering PC. Identification of new Presenilin-1 phosphosites: implication for γ-secretase activity and Aβ production. J Neurochem. 2015 May;133(3):409-21. Epub 2015 Feb 24 PubMed.
- Ledo JH, Liebmann T, Zhang R, Chang JC, Azevedo EP, Wong E, Silva HM, Troyanskaya OG, Bustos V, Greengard P. Presenilin 1 phosphorylation regulates amyloid-β degradation by microglia. Mol Psychiatry. 2020 Aug 13; PubMed.