Strong evidence implicates microglia cells—agents of the brain’s innate immune response—in the onset and progression of Alzheimer’s disease (AD). Many of the ~75 genetic variants identified through genome-wide association studies (GWAS) for their impact on classic AD risk are primarily expressed by microglia. However, in many cases, the function of the proteins encoded by these genes remains poorly understood. It is hypothesized that optimizing the immune response to amyloid beta and tau AD pathology—ensuring that microglia neither over- nor under-respond—could prevent the neurodegeneration associated with disease progression and clinical syndromes. Thus, the Cruchaga lab and the field seek to understand how these genetic risk factors influence microglia to reveal new insights into the disease and help identify potential drug targets.
TREM2 (Triggering Receptor Expressed on Myeloid Cells 2) is one of the most well-studied of the genetic risk factors for late-onset AD because some of its variants have a strong impact on AD risk, and the protein it encodes has a well-established role in microglial activation. The TREM2 protein sits on the surface of microglia, where it helps detect signals from outside the cell and trigger an appropriate microglia response, such as clearing amyloid plaques. TREM2 can also be cleaved at the cell surface, producing an extracellular fragment called soluble TREM2 (sTREM2), which may play a role in inflammation and microglia survival. Along with RLG member Dr. Christian Haass, Dr. Cruchaga and others discovered that sTREM2 is present in cerebrospinal fluid (CSF), that its levels are higher in AD patients, and that sTREM2 levels correlate with tau pathology biomarkers.
Using a novel GWAS approach, the Cruchaga team then identified gene variants within specific regions (loci) of the genome associated with elevated sTREM2 levels in the CSF. Notably, these loci include genes such as MS4A4A and APOE—both already well-known AD risk genes—as well as TGFBR2, which is reduced in human AD. This suggests that these genes may interact within the same pathway as TREM2. In their current study, the Cruchaga are investigating whether and how genetic variations in TREM2, TGFBR2, and the genetic locus that contains APOE and another gene named NECTIN2 influence TREM2 function. The team also plans to identify and describe TREM2’s many effects by comparing how different TREM2 genetic variants influence microglia signaling pathways in AD.
Dr. Cruchaga’s proposal has three aims. In the first aim, the Cruchaga lab is identifying which specific cellular pathways and molecules differ in carriers of TREM2 risk variants compared to non-carriers. They have already generated—or have access to—data from 3,000 patient samples. They are using cutting-edge proteomic and metabolomic methods to measure the levels of 7,000 proteins and related metabolites in CSF and blood samples. The lab also has access to a smaller subset of brain tissue samples collected from healthy controls, patients with Alzheimer’s disease, and TREM2 variant carriers at the Washington University Knight ADRC and by the Alzheimer’s Disease Neuroimaging Initiative (ADNI). As part of their second and third aims, the team is investigating how variants in a gene called TGFBR2 affect TREM2 cleavage and its release as sTREM2. To explore this in Aim 2, they are using CRISPR to modify TGFBR2 in human microglia derived from stem cells. By testing how different variants of TGFBR2 impact sTREM2 levels and microglia functions—and by testing the effects of completely silencing TGFBR2—they hope to uncover its role in AD and brain immune responses. In the third aim, the team is studying two genes located in the same genomic region: APOE and NECTIN2. The Cruchaga lab has not yet pinpointed (or fine-mapped) whether the genetic variants linked to sTREM2 levels identified by GWAS are associated with one or both genes. To investigate this, they are using CRISPR to increase or decrease the expression of APOE and NECTIN2 and measuring the impact on sTREM2 levels and signaling. After this, they will conduct similar experiments as outlined in the second aim.
At the end of the first year, the team has made significant progress on Aims 1 and 2. For Aim 1, they identified ~700 proteins and metabolites in CSF that differ between carriers and non-carriers of different TREM2 variants, providing insights into the metabolic differences in disease progression. Next, they plan to expand their comparisons to include blood samples. In Aim 2, they found that reducing TGFBR2 levels also decreased sTREM2 levels, which highlights TGFBR2’s role in regulating TREM2 biology. These data may indicate that TGFBR2 contributes to microglial maintenance or activation. They are now conducting experiments to uncover exactly how TGBFR2 modifies TREM2 biology. In year two, the team will focus on Aim 3, studying how APOE and NECTIN2 variants impact sTREM2 levels and signaling.
