Single-cell gene studies at Duke-NUS Medical School in Singapore and Monash University in Melbourne, Australia, have shown that gene expression signatures underlie the microglial phagocytosis of beta-amyloid (Abeta) plaque in patients with Alzheimer's disease (AD), the authors reported in the May 20, 2021, edition of Nature Communications.
"This is the first study to directly elucidate the gene expression signatures underlying the microglial phagocytosis of Abeta plaques in an AD mouse model," said study co-senior author Enrico Petretto, an associate professor with the cardiovascular and metabolic disease program at Duke-NUS Medical School. Such single-cell genetic studies are important, as "they help us to understand the role of different cell types such as microglia in AD," said Petretto.
"This provides greater resolution for studying gene transcriptional signatures at the single-cell level, providing an opportunity for cell-specific therapies," he told BioWorld Science.
The most common cause of dementia worldwide, AD, despite decades of intensive research, has remained essentially incurable.
This is primarily due to the "genetic and cellular complexity of the disease, with different drivers of the disease possibly being active at different disease stages," said co-first author Alexandra Grubman, an adjunct reserch fellow at the Monash BioMedicine Discovery Institute.
In addition, said Grubman, "there is a lack of translatability from mouse models to humans, while the role of non-neuronal cells such as microglia in AD, is not well understood."
The immune cells of the brain, microglia are responsible for eliminating foreign proteins and maintaining brain homeostasis by clearing toxic waste including Abeta, which accumulates in AD.
However, the exact role of microglia in AD and the relationship to Abeta plaque accumulation remain unclear, but could offer a promising new target for interventions addressing underlying AD mechanisms.
To investigate differences between healthy brains and those of AD patients at the single cell level, the scientists had previously studied gene expression changes in specific human brain cell types associated with AD progression.
Those findings, published in Nature Neuroscience in 2019, prompted the team to focus on microglia and molecular differences between microglia actively phagocytosing Abeta plaque in AD and those that were not.
"We found that transcriptional signatures underlying microglia in AD were different from those underlying healthy microglia, suggesting that this difference might be partly attributed to plaque phagocytosis in AD," said Petretto.
This was done using methoxy-XO4, which specifically stains microglia that have engulfed Abeta plaques. The researchers used the stain in preclinical AD models and examined gene expression in stained microglia.
"Staining experiments were done in healthy and 5 x FAD mice, in which neurons express five familial AD mutations resulting in rapid Abeta buildup," said Grubman.
"Others have described an intermediate signature called DAM1, which is a step toward becoming disease associated microglia (DAM)," she noted.
"Our study showed that microglia that had not phagocytosed Abeta plaque were functionally deregulated, set on a trajectory of accelerated aging, and not a transcriptional intermediate with respect to the phagocytosis trajectory as are DAM1."
The researchers then investigated differences in gene expression underlying microglial ability to ingest particles such as Abeta plaque and identified associated regulatory molecules.
"Understanding this mechanism has importantly identified several new targets, which may open a new front against this devastating disease," said study co-senior author Jose Polo, a professor at the Monash Biomedicine Discovery Institute.
The gene expression patterns of microglia that had not taken up Abeta plaque were most similar to those of aged microglia, which are known to be dysfunctional and are important in AD pathogenesis.
Hif1alpha was reduced either using RNA interference or the mTOR pathway inhibitor rapamycin. This is important because it shows that phagocytosis can be modulated by Hif1a. In addition, it suggests that drugs that affect Hif1a activity might also affect microglia phagocytosis of synaptosomes.
"We showed that increasing Hif1a expression could increase synaptosome phagocytosis by 1.5 times in vitro, whereas Abeta phagocytosis was only increased by 10% in vitro by Hif1a overexpression," said Petretto.
In addition, after engulfing Abeta, the microglia developed a characteristic gene expression signature, which was shown to be induced, in part, by the Hif1a gene.
This altered gene expression was shown to increase the ability of microglia to take up proteins such as Abeta, while reducing Hif1a achieved the reverse, stressing the importance of Hif1a in controlling microglia function.
This regulatory role of Hif1a might also apply to the microglial function of removing damaged synapses and other unwanted debris.
This process is initially protective, with the microglia effectively pruning damaged synapses located near plaques, but it may malfunction as AD progresses.
The researchers also used computational models to predict networks of molecules involved in microglia uptake of proteins and identified potential targets to study for drug development.
For example, the widely used immunosuppressant drug rapamycin was found to block Hif1a from triggering microglia to engulf amyloid plaques.
"This relationship between Hif1a and cognitive decline in AD has yet to be comprehensively revealed," said study co-first author Gabriel Chew.
"Future work could focus on using the gene editing tool CRISPR to test the impact of manipulating Hif1a on symptom severity and disease progression," suggested Chew, a PhD student at Monash.
"Our findings suggest that the future development of AD drugs could focus on microglia-specific therapeutics by modulating Hif1a," Petretto said.
"In future," he added, "research may include investigating the mechanism behind Hif1a's modulation in phagocytosis by using knockout mice models, and the effect of this on disease progression at different times throughout the disease course, which could assist translational studies in humans."