An optimized integrated functional genomics toolkit identifies novel role of SORL1 in microglia
Falak Sher- Psychiatry and Mental health
- Cellular and Molecular Neuroscience
- Geriatrics and Gerontology
- Neurology (clinical)
- Developmental Neuroscience
- Health Policy
- Epidemiology
Abstract
Background
Recent genome wide association studies implicated microglia in the pathophysiology of neurodegeneration; however, the translation of these genetic discoveries is hindered by the current lack of a robust human microglia in vitro model system and standardized protocols for functional genomics assessment. Though microglia differentiated from human induced pluripotent stem cells (iMG) could be a promising cellular model, the available differentiation protocols are variable, lengthy and give inefficient, mixed yields, limiting our ability to utilize modern gene editing tools (e.g., CRISPR‐Cas9) in this model for elucidating the functional consequences of disease relevant genes and variants.
Methods
In order to streamline microglia functional genomics, we optimized a highly efficient protocol to differentiate human induced pluripotent stem cells into iMG in two easy steps, offering a significant improvement in purity and time requirement for iMG production. We validated this iMG model system in a robust way through a comprehensive single cell RNA‐sequencing (scRNA‐Seq) study, mass spectrometry‐based proteomics and microglial functional assays. In addition, the iMG differentiation protocol includes sequential iMG harvesting and cell freezing steps, that will allow the researchers to scale up the production of iMG for high‐throughput assays. Furthermore, the toolkit also includes a drug inducible CRISPR‐ON/OFF approach in our newly established microglia model system that enables the investigation of gene function in a temporal fashion. All the data generated as part of the comprehensive validation is publicly available in the form of a searchable online platform (
Results
We used this newly developed integrated microglia functional genomics toolkit, along with human primary microglia freshly purified from dorsolateral prefrontal cortex (DLPFC), to investigate the thus far unidentified role of SORL1, an AD associated gene, in microglia. By combining state‐of‐the art (e.g. single cell RNA‐Seq, proteomics and focused CRISPR‐Cas9 screen) and standard (immunohistochemistry on postmortem human aged brain tissues) approaches we established the molecular mechanisms by which SORL1 shapes the functional phenotypes of human microglia. Most interestingly, we have found divergent roles for SORL1 in microglia when compared to neurons.
Conclusions
Our findings highlight the importance of cell type specific functional genomics of susceptibility genes in AD.