The discovery by Dr. Rudy Tanzi and his lab that certain variants of the CD33 gene increased Alzheimer’s disease (AD) risk was a highlight of the early years of the CureAlz Alzheimer’s Genome ProjectÔ because it was pivotal in establishing that immune response is central to AD risk and progression and to launching the study of microglial dysfunction and neuroinflammation in response to AD pathology. CD33 is a cell surface membrane receptor primarily expressed by microglia that, when activated, suppresses the ability of these immune cells to engulf and clear harmful substances from the brain. Subsequent investigations identified protective CD33 gene variants, which prompted researchers to investigate how each of the different CD33 protein isoforms alters microglial response to amyloid and tau pathology.
This project will add an additional layer of investigation to these studies. New technologies have allowed biomedical science to discover that the translation of the DNA blueprint into protein can vary, including in health versus disease. One type of variation is driven by alterations in RNA splicing. RNA splicing is a process where non-coding segments (introns) are removed from a gene’s initial RNA transcript, while coding sequences (exons) are joined together to create the final messenger RNA, which is translated into a protein. Under normal conditions, alternative splicing that includes, excludes, or repeats different exons allows a single gene to generate multiple useful protein variants with different functional impacts. However, when this process becomes dysregulated and the pattern of splicing changes, splicing removes or retains a different mix of exons and may produce entirely different protein variants and/or a different mix of variants. The resulting yield of protein variants will have an altered functional impact, which may have either harmful or beneficial consequences for the molecular pathways in which they act.
Dr. Tilgner, in collaboration with the lab of RLG member Dr. Li Gan, previously identified specific patterns of splicing associated with either higher risk of or resilience to AD. The Tilgner lab now seeks to understand the relationship between changes in splicing and amyloid beta and tau pathology, as well as the role of nearby microglia carrying CD33 variants. They hypothesize that different CD33 variants induce distinct splicing patterns and that amyloid and tau pathologies may also directly associate with specific splicing patterns in certain cell types. Using spatial analysis technologies, the team will test these relationships by examining whether splicing variations increase with closer proximity to amyloid beta, tau pathology, or specific CD33 isoforms. Since nearby cells communicate through chemical signals, such proximity patterns would suggest causal relationships between them. Dr. Tilgner will investigate the role and consequence of splicing alterations by asking three questions: Do these splicing changes happen mainly in or near brain cells that contain tau tangles? Are the splicing problems worse in areas close to amyloid plaques? And do these splicing issues occur more frequently near microglia, and do they differ according to the CD33 isoform present in those microglia?
To answer these questions, the Tilgner lab has developed an innovative combinatorial methodology to first detect, quantify, and locate individual RNA isoforms, AD pathology, and CD33 isoforms, and then determine the spatial relationships among them. The lab will examine brain samples from individuals across the AD spectrum to assess how splicing differs at different pathological and clinical stages. They will first detect and quantify specific RNA isoforms at single-cell and single-nucleus resolution in brain samples sliced to be sufficiently thin to enable these light-driven technologies. These slices are so thin that they are virtually two-dimensional, meaning that nearby and relevant structures and cells that might be affecting what is captured in a slice can be missed if they are slightly out of plane from it. To combat this, the Tilgner lab will then use computational modeling to reconstruct the 3D architecture from consecutive slices. Since this enables precise visualization and colocalization in all three spatial dimensions of Alzheimer’s pathologies, microglia cells containing specific CD33 isoforms, and the protein products of the altered splicing patterns, the actual distances between them can be measured. The team will assess splicing alterations within specific cell types, as well as across donor demographics, to determine if sex or age plays a role in the frequency or type of these alterations.
This project connects amyloid, tau, and neuroinflammation by investigating how splicing dysregulation is associated with AD risk and progression of different pathologies. As a cell surface membrane protein, CD33 is a relatively accessible potential target for small-molecule or other therapeutic intervention. The identification of how altered RNA splicing connects to AD pathology will generate new molecular processes and pathways for future therapeutic targeting.
