A new APP knock-in mouse has hit the scene. Researchers led by Pascal Sanchez, Denali Therapeutics, San Francisco, California, described the details in a paper posted to bioRxiv on January 20. They humanized the Aβ sequence in the mouse APP gene and knocked in three familial Alzheimer’s disease mutations: Swedish, Arctic, and Austrian. TREM2 expression and cytokine concentrations spiked in brain tissue, microglia flocked to plaques, neurites swelled, and tau and neurofilament light collected in their cerebrospinal fluid. Notably, microglia surrounding plaques seemed distressed—the cells filled with lipids and revved up transcription of genes previously linked to a subset of microglia isolated from AD brain tissue. Denali, in collaboration with the Jackson Laboratory, will make this model open access for academics and companies alike.
- Mice have humanized APP and Swedish, Arctic, Austrian mutations.
- Microglial transcriptome resembles human AD profile.
- Stressed microglia accumulate Aβ and lipids.
In 2014, researchers led by Takaomi Saido, RIKEN Brain Science Institute, Wako, Japan, had already created two APP knock-in mice: NL-F mice carrying the Swedish (KM670/671NL) and Iberian (I716F) mutations, and NL-G-F animals with the additional Arctic (E693G) mutation. Saido’s knock-ins make more Aβ than do wild-type mice, accumulate plaques in the brain, have obvious gliosis, and perform poorly in cognitive tests (April 2014 webinar; Saito et al., 2014).
Model Strategy. While healthy microglia protect neurons (bottom, left), those in SAA mice (top) become phagocytic (bottom, middle). Plaque-associated microglia swell with Aβ plaques and lipids, unable to cope with the high phagocytic load (bottom, right). [Courtesy of Pascal Sanchez, 2021.]
Alas, obtaining those mice for study proved difficult for many labs outside of Japan, so Denali scientists decided to make their own. Co-first authors Dan Xia, Steve Lianoglou, and colleagues used homologous recombination to humanize the Aβ sequence of the mouse APP gene, incorporating three FAD APP mutations, Swedish, Arctic, and Austrian (T714I), rather than the Iberian mutation used in the NL-G-F model. “This new knock-in model is conceptually identical to our NL-G-F,” Saido wrote to Alzforum. “The Swedish mutations increase the total amount of Aβ; the Arctic mutation makes Aβ prone to oligomerization and resistant to degradation; and the Austrian or Iberian mutation increases the ratio of Aβ42/40.” (Full comment below.)
Denali, in collaboration with the Jackson Laboratory, Bar Harbor, Maine, promises to allow unrestricted use of the SAA mice by academic and industry researchers alike. Mike Sasner from Jax noted the struggle company researchers especially often face to obtain models. “While the Jackson Lab has a lot of APP models, legal restrictions imposed by donating investigators prevent us from distributing them to companies,” he explained. “To get around this, companies tend to make their own mouse models, but keep them in house without sharing them.”
Christian Haass, German Center for Neurodegenerative Diseases (DZNE), Munich, Germany, agreed. “Model availability is a big issue, considering that many experiments were blocked or slowed due to legal issues,” he wrote to Alzforum (see comment below). “Making this mouse model freely available is a great sign for our research community.”
As expected, in the new homozygous SAA mice, the Aβ42/40 ratio in brain tissue extracts, CSF, and plasma rose higher than in wild-type controls in animals as young as 2 months of age, two months before plaques were detectable. Beginning when mice were 4 months old, plaques spread, starting in the cortex/hippocampus, moving to the entorhinal region, then spilling into the amygdala, thalamus, and striatum among other regions, similar to the phases of Aβ deposition see in the human brain (Thal et al., 2002). Plaques packed the mouse cortex and hippocampus and Aβ deposited in blood vessel walls of the meninges (see image below).
Amyloid on the Brain. Whole-brain heatmaps light up plaque distribution in 8-month-old SAA knock-in mice (left). Immunostaining revealed Aβ deposition in blood vessels of the meninges (arrows, right). [Courtesy of Xia et al., bioRxiv, 2021.]
By 8 months, the brain was packed with plaques, and neurites swelled with phosphorylated tau detected by the AT-8 antibody. Neurofilament light (NfL), and the lysosomal marker LAMP1 ticked up—all characteristic of AD. These changes correlated with increased total tau and NfL in the CSF. TREM2 expression and cytokine concentrations spiked in brain tissue. Microglia also flocked to plaques.
Were these microglia different from those in wild-type mice? Expression of more than 600 genes was different in microglia from SAA mice compared to wild-type. Transcripts related to cholesterol metabolism, glycolysis, and phagocytic/lysosomal function ticked up in the knock-ins. Disease-associated microglia (DAM) genes that were previously linked to pathology in amyloid models were particularly upregulated (Jun 2017 news).
What about microglia that swarmed around plaques? The researchers labeled microglia with methoxy-X04, a fluorescent dye that binds to Aβ fibrils, then sorted fibril-positive from fibril-negative cells. Those that had swallowed Aβ fibrils altered expression of more than 800 genes, including those of the DAM and plaque-induced gene (PIG) variety. PIGs were found when researchers in Bart De Strooper’s lab in KU Leuven, Belgium, carried out a spatial transcriptomics study in brain tissue from NL-G-F mice and in postmortem tissue taken from people who had had AD. They found a suite of genes that were up- or down-regulated in microglia adjacent to plaques (July 2020 news).
Some of the differentially expressed genes in the SAA phagocytic microglia are orthologs of those in a sub-cluster of microglial transcripts found in people with AD (May 2019 news). In SAA mice, expression of neurotrophic genes, such as those involved in neuronal development, axon guidance, and spine morphogenesis, was reduced (see image below). Overall, the microglia surrounding plaques in SAA mice had a transcriptional signature that tracked with those in other mouse models and in the human brain.
Plaque-associated microglia also highly expressed genes involved in lipid clearance and metabolism. What did this have to do with their function? Despite the ramp-up, lipids, such as ganglioside GM3, filled the cells, hinting at lysosomal dysfunction. They also accumulated spermine, a molecular distress signal that cells release when they are overworked. “Highly phagocytic, distressed microglia had difficulty coping with getting rid of so much debris, which is reflected in their mishandled lipid metabolism,” suggested Sanchez. “We are now investigating whether this metabolic dysregulation will lead to microglia dysfunction.”
Sasner wants to cross these SAA mice with different genetic backgrounds to create new combinations of alleles, such as with APOE4 and humanized tau. “Because the SAA model does not have tau pathology, we are hoping that crossing it with a humanized tau model will give us both phenotypes,” he told Alzforum.
Initial mouse distribution is estimated for August 2021. Interested researchers can pre-order on the Jackson Laboratory website.—Chelsea Weidman Burke
Research Models Citations
- Hot DAM: Specific Microglia Engulf Plaques
- Paper Alert: Those PIGs! Spatial Transcriptomics Add Human Data
- When It Comes to Alzheimer’s Disease, Do Human Microglia Even Give a DAM?
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