Summary: Deletion of the neurodegenerative disease associated microglial gene CX3CR1 aggravated the disease state and increased the accumulation of plaques in the brains of mouse models of Alzheimer’s disease. Deficiencies of the gene also impaired the movement of microglia toward the plaques.
Source: Indiana University
Indiana University School of Medicine researchers are investigating how the deficiency of a gene in immune cells can shape the progression of Alzheimer’s disease.
The study, published in Molecular Neurodegeneration, found that deleting CX3CR1, a microglial gene associated with neurodegenerative diseases, in Alzheimer’s disease animal models resulted in an aggravated disease state and accumulation of plaques in the brain. The deficiency of the gene also impaired the movement of microglia—the brain’s immune cells—toward the plaques.
“This investigation shows that microglia in Alzheimer’s disease become dysfunctional earlier in the disease course in the absence of CX3CR1, and this dysfunction results in the cascade of neurotoxic events in the brain,” said Shweta Puntambekar, MS, Ph.D., assistant research professor of medical and molecular genetics.
“For the larger research community, this research pinpoints how we can target this cell type early in the disease in order to modulate how the disease progresses in the brain and ultimately modulate cognitive outcomes in Alzheimer’s disease.”
CX3CR1 has been shown in both past human and animal studies to be downregulated in neurodegenerative diseases when microglia are activated. The CX3CR1-V249I, a loss-of-function gene variant, was first identified and associated with macular degeneration and was later shown to relate to neurodegeneration in Alzheimer’s disease and ALS.
Puntambekar, first author of the journal article, said the study also looked at the connection between amyloid beta and tau in the brain—hallmark proteins commonly associated with neurodegenerative diseases. Amyloid beta proteins clump together and form plaques, which destroy nerve cell connections. Tau then can later form in the brain after amyloid plaques.
“The study has made a connection not just between amyloid and tau, but how microglia can shape the entire disease process,” Puntambekar said.
In the absence of this gene, the microglia—which act as the first line of defense against viruses, toxic materials and damaged neurons—cannot move closer to plaques to clear up proteins. This occurs early in the disease and leads to more neurotoxic events, such as accumulations of other toxic species of amyloid beta and aggravated tau in later disease stages.
Some of those species of amyloid beta aren’t deposited in the brain as “insoluble” plaques, Puntambekar said, but rather accumulate in the brain as soluble plaques and have been shown to also be associated with cognitive decline. These species were increased in the absence of CX3CR1, she added.
Most therapies that target amyloid beta proteins in the brain focus on insoluble plaques, but drugs for years have been proven ineffective in clinical trials.
“With this new data set, we can now start asking if the limited clinical efficiencies of Alzheimer’s disease therapies are due to not targeting the correct species of amyloid beta and whether we should start targeting other soluble species to get better cognitive outcomes,” Puntambekar said.
CX3CR1 deficiency aggravates amyloid driven neuronal pathology and cognitive decline in Alzheimer’s disease
Despite its identification as a key checkpoint regulator of microglial activation in Alzheimer’s disease, the overarching role of CX3CR1 signaling in modulating mechanisms of Aβ driven neurodegeneration, including accumulation of hyperphosphorylated tau is not well understood.
Accumulation of soluble and insoluble Aβ species, microglial activation, synaptic dysregulation, and neurodegeneration is investigated in 4- and 6-month old 5xFAD;Cx3cr1+/+ and 5xFAD;Cx3cr1−/− mice using immunohistochemistry, western blotting, transcriptomic and quantitative real time PCR analyses of purified microglia. Flow cytometry based, in-vivo Aβ uptake assays are used for characterization of the effects of CX3CR1-signaling on microglial phagocytosis and lysosomal acidification as indicators of clearance of methoxy-X-04+ fibrillar Aβ. Lastly, we use Y-maze testing to analyze the effects of Cx3cr1 deficiency on working memory.
Disease progression in 5xFAD;Cx3cr1−/− mice is characterized by increased deposition of filamentous plaques that display defective microglial plaque engagement. Microglial Aβ phagocytosis and lysosomal acidification in 5xFAD;Cx3cr1−/− mice is impaired in-vivo. Interestingly, Cx3cr1 deficiency results in heighted accumulation of neurotoxic, oligomeric Aβ, along with severe neuritic dystrophy, preferential loss of post-synaptic densities, exacerbated tau pathology, neuronal loss and cognitive impairment. Transcriptomic analyses using cortical RNA, coupled with qRT-PCR using purified microglia from 6 month-old mice indicate dysregulated TGFβ-signaling and heightened ROS metabolism in 5xFAD;Cx3cr1−/− mice. Lastly, microglia in 6 month-old 5xFAD;Cx3cr1−/− mice express a ‘degenerative’ phenotype characterized by increased levels of Ccl2, Ccl5, Il-1β, Pten and Cybb along with reduced Tnf, Il-6 and Tgfβ1 mRNA.
Cx3cr1 deficiency impairs microglial uptake and degradation of fibrillar Aβ, thereby triggering increased accumulation of neurotoxic Aβ species. Furthermore, loss of Cx3cr1 results in microglial dysfunction typified by dampened TGFβ-signaling, increased oxidative stress responses and dysregulated pro-inflammatory activation. Our results indicate that Aβ-driven microglial dysfunction in Cx3cr1−/− mice aggravates tau hyperphosphorylation, neurodegeneration, synaptic dysregulation and impairs working memory.