PROJECT SUMMARY Alzheimer’s disease (AD) is the most common form of dementia and affects over 6 million Americans. There is no cure for AD, and many potential treatments have failed, creating a critical need for the development of additional disease models for novel therapeutic discovery and testing. The hallmark pathology of AD includes amyloid-β (Aβ) plaques and hyperphosphorylated tau (ptau)-containing neurofibrillary tangles. This pathology is associated with memory loss, which is diagnostic for AD, but is now known to be preceded by earlier prodromal symptoms including mood and sleep disorders. Dysregulation of the neurotransmitter norepinephrine (NE) is associated with AD symptoms, as its release in the forebrain regulates attention, arousal, stress response, and learning and memory. Importantly, the locus coeruleus (LC), the main noradrenergic nucleus in the brain and source of NE in the forebrain, is the first brain region to develop ptau inclusions, may transmit pathogenic tau to the forebrain, and undergoes catastrophic degeneration in later stages of the disease. Recent evidence indicates that that early ptau-mediated LC dysfunction contributes to the prodromal symptoms of AD, while LC neuronal loss later in the disease exacerbates cognitive impairment. However, nearly all information about the LC in AD has been derived from rodent models and postmortem or human imaging studies. It is not known how the early ptau accumulation in the human LC affects neuronal function or NE transmission. Organoids, which are 3D human cell culture models derived from human induced pluripotent stem cells, emulate the architecture and function of human tissue. The goal of this proposal is to develop the first human LC organoid model to address questions related to the earliest stages of AD. In Aim 1, I will validate our LC organoids by comparing their structure and function to human and rodent LC neurons. I will then combine LC organoids with cortical organoids to define assembloid connectivity by tracking NE innervation of the cortical organoids. In Aim 2, I will derive LC organoids from AD patients and compare their structure, function, and AD pathology to that of LC organoids derived from healthy subjects. Following LC-cortical assembloid creation, I will assess the consequences of NE AD pathology transmission from AD LC organoids to control cortical organoids. Together, the proposed experiments will provide new insights on AD pathology and normal and AD LC-cortex connectivity in living human neurons. This information would greatly impact AD research and provides a novel model for the development of new treatment strategies for patients in the earliest stages of AD.