There is mounting evidence of a link between pathological tau (p-tau) and neuroinflammation in neurodegenerative diseases including Alzheimer’s Disease (AD). In previous studies, we found that p-tau can induce neuroinflammation through activating inflammasomes, a multi-protein complex that recognizes danger signals including bacterial toxins lipopolysaccharide (LPS), nigericin and endogenous danger signals including extracellular ATP and p-tau. Activation of inflammasomes leads to maturation of pro-inflammatory cytokines interleukin-1 (IL)-1b and IL-18, causing neuroinflammation. We aim to obtain quantitative measurements of activated inflammasomes, specifically the levels of assembled ASC (apoptosis-associated speck-like protein containing a CARD), a key adaptor protein of inflammasomes, in the Cerebrospinal Fluid (CSF) samples of AD patients to test if this serves as a surrogate marker of brain inflammasome activation.
Immortalized mouse macrophages that constitutively express ASC conjugated to the fluorescent protein cerulean (ASC-cerulean cells) were treated with LPS and nigericin to induce inflammasome assembly and secretion into extracellular space. Extracellular ASC specks were collected, labelled with ASC antibody, followed by the Alexa Fluor 647-conjugated secondary antibody, then analyzed with Amnis ImageStream® imaging flow cytometry (Amnis). CSF samples from healthy control and AD patients were labelled and analyzed as above.
ASC-cerulean cells treated with LPS and nigericin released 1.5 to 2.6 times more ASC specks positive for both cerulean and Alexa Fluor 647 compared to untreated samples. Furthermore, CSF samples from AD patients had a significantly higher number of ASC specks compared to healthy controls.
Our results indicate Amnis can detect extracellular ASC specks, and there are more ASC specks in AD CSF samples compared to healthy controls. These results suggest that differential levels of ASC specks in extracellular environments including CSF may correlate with disease severity and potentially serve as a biomarker for AD and related dementia, which needs further investigation.
Dong, Qi "Phoebe"; Shanya Jiang; Kathryn Sanchez; Gary Rosenberg; and Kiran Bhaskar. "Extracellular ASC Level as a Potential Biomarker for Alzheimer’s Disease." (2021). https://digitalrepository.unm.edu/hsc-bbhrd/25