As the resident innate immune cells of the brain, microglia are emerging as key drivers of neurological diseases, but as yet there is no systematic way of exploring their potential as drug targets.
That is about to change, with the development of a screening platform for evaluating the functional consequences of genetic perturbation in microglia derived from human induced pluripotent stem cells (iPSCs). The screen has been used to pinpoint genes involved in microglial survival, activation and phagocytosis, including genes that are associated with neurodegeneration. The induced microglia adopt a spectrum of states, mirroring those observed in human brains and making it possible to identify regulators of these states.
In addition, researchers led by Martin Kampmann at the Sandler Neurosciences Center, University of California, San Francisco, have demonstrated the screen picks up gene targets that can be inhibited by drug compounds, to influence the state of microglia.
"We are now in a position to systematically dissect the changes microglia undergo in diseases such as Alzheimer's disease and to uncover druggable targets to manipulate microglia for therapeutic benefit," Kampmann told BioWorld Science.
The work underpinning development of the platform and research carried out to demonstrate its validity is described in a paper published in Nature Neuroscience, on August 11. This is not the first time iPSCs have been used to generate microglia, but Kampmann and colleagues have overcome two significant barriers to deploying these cells at scale in drug screening.
First, the current protocols for differentiating microglia from iPSCs are cumbersome and lengthy, requiring either lentiviral gene transfer of transcription factors to drive differentiation, or differentiation using small molecules, recombinant proteins and growth factors, a process requiring significant know-how and technical skill.
Kampmann has speeded up and simplified the differentiation process by engineering iPSCs to inducibly express all the transcription factors required for them to become microglia. It takes 8 days for this transformation to complete and the microglia generated maintain viability for 8 days. These cells, which the researchers term induced-transcription factor microglia-like cells (iTF-microglia), resemble other iPSC-derived counterparts in their gene expression profiles, response to inflammatory stimuli, phagocytic capabilities and capacity to be co-cultured with iPSC-derived neurons.
Microglia adopt a large number of functional states in health and disease. While other researchers are actively mapping these, it is not possible to study how these distinct microglial states contribute to brain function or disease using gene knockouts.
In a second advance over current approaches, Kampmann has addressed this by using CRISPR interference and CRISPR activation (CRISPR i/a) gene editing to enable changes to expression levels of endogenous genes, mimicking what happens when cells regulate genes up and down. "CRISPR knockout screens are more limited. They enable inactivation of genes, but not overexpression or partial knockdown, which can be an issue for example when studying essential genes," Kampmann said.
Scalable modelling of changes in gene expression will make it possible to discover regulatory mechanisms in healthy microglia and in those derived from patients with neurological diseases. "Our platform is the first to enable large-scale CRISPR-based screens in human microglia without the use of robotic/high-throughput equipment," said Kampmann. "We believe it will accelerate microglia research: both insights into microglia functions in health and disease, and the discovery of therapeutic targets in microglia."
Kampmann said the protocol with be freely available for others to use. He previously published a protocol using CRISPR i/a in iPSC-derived neurons which he said has been widely adopted.
Exemplifying the microglia screening platform
The first test of the platform's validity was to identify modifiers of microglial survival and proliferation in a library of 2,325 genes encoding kinases, phosphatases and other classes of druggable proteins. The researchers uncovered several microglial-specific modifiers.
The researchers also used the screen to look for modifiers of inflammatory activation of microglia. They identified several genes that regulated cell surface levels of CD38, a known controller of microglial activation.
Dysfunctional or dysregulated phagocytosis of synaptosomes by microglia is implicated in neurodegenerative and psychiatric diseases, and the screen was able to identify known and novel phagocytosis modulators in microglia.
Several genes associated with neurodegenerative diseases, including the actin binding protein PFN1, defects of which cause amyotrophic lateral sclerosis, and inositol polyphosphate-5-phosphatase D, an Alzheimer's disease risk factor, were shown to be modulators of phagocytosis in microglia. That points to a possible cellular mechanism by which variants in these genes contribute to disease.
In addition to target discovery and studying microglial biology, the researchers say their iTF-microglia could be introduced into brain organoids to investigate their interaction with other types of brain cells. Similarly, transplanting iTF-microglia into immune-deficient humanized mice could be a way to investigate factors controlling microglia in models of neurological diseases."Microglia are highly sensitive to their environment. Placing them in a more brain-like environment can likely yield important insights," said Kampmann.