The STING pathway plays a central role in innate immune activation by recognizing and responding to foreign or damaged DNA. It has been linked to neurodegenerative diseases, including ALS, for which global STING inhibition improves disease progression. This improvement has been thought to result from STING deletion in immune cells; however, because neuronal accumulation of DNA damage is a key feature of ALS, we hypothesized that STING signaling can initiate an innate immune response within neurons. Our findings demonstrate STING pathway integrity in neurons and selective activation in ALS-vulnerable neurons in addition to immune cells. The presence of STING activation in both microglia and neuron in ALS highlights the need to understand how STING signaling affects these cell types. This proposal aims to define the specific contributions of STING signaling activation in disease-relevant cell types in ALS using iPSC models. We will generate spinal motor neurons and microglia across different ALS variants and control lines. We will determine the extent and changes in STING activation and downstream pathways in both cell types and lines, in both single and co-culture conditions. By combining unbiased bioinformatic analysis of published datasets with validation in human postmortem tissue and CRISPR-mediated gene editing technology, we will identify candidate cell-type-specific genes and pathways involved in non-cell autonomous interactions in ALS between neurons and microglia.