Regulation of intercellular communication by Alzheimer's disease genetic risk factors

Abstract

Intercellular communication between glial cells, neurons, and the vasculature drives the progression of neurodegenerative diseases like Alzheimer’s disease (AD). The accumulation of misfolded proteins initiates neuroinflammation, activating microglia to release proinflammatory signals that, in turn, drive astrocyte reactivity. This feedback loop sustains and amplifies inflammation, disrupts vascular function, and accelerates the progression of synapse loss and neurodegeneration. Although hallmark features of AD—tau tangles, Aβ deposits, and synapse loss—emphasize neuronal dysfunction, genetic studies underscore the pivotal role of glial-specific genes, including TREM2, APOE, and CLU, in late-onset AD (LOAD). In contrast, familial AD (fAD) is driven by mutations in APP and PSEN1/2, which are known to induce altered Aβ production within neurons. Understanding how glial-driven LOAD risk factors contribute to neuronal dysfunction and how fAD-linked mutations disrupt glial communication is crucial for unraveling the interconnected mechanisms of AD pathogenesis. Advancements in single-nucleus RNA sequencing (snRNAseq) have uncovered diverse cellular states implicated in neurodegenerative diseases, including disease-associated microglia (DAM), which are closely tied to neuronal function. However, testing hypotheses regarding glial-neuronal intercellular communication derived from these datasets requires a reproducible human model system capable of capturing the complexity of these interactions. To address this, we developed a robust human iPSC-derived triple-culture platform incorporating astrocytes, neurons, and microglia. Analyses of each cell type in mono- and co-culture uncovered distinct transcriptional signatures uniquely shaped by co-culture interactions. For example, astrocyte co-culture strongly induced the upregulation of DAM-associated proteins, including TREM2, SPP1, APOE, and GPNMB. Strikingly, exposure to fAD neurons initially suppressed astrocyte-mediated DAM induction while activating NfB-dependent inflammatory responses. These findings validate our platform's ability to model glial-neuronal interactions and provide insights into how fAD mutations disrupt intercellular signaling. We then leveraged this platform to investigate the intercellular mechanisms underlying the AD risk gene Clusterin (CLU). Genetic studies implicate CLU in AD pathogenesis, and CLU levels are elevated in the brains of individuals with AD. Despite nearly three decades of research, the role of CLU remains enigmatic: it is unclear whether CLU upregulation is neuroprotective, contributes to pathology, or serves merely as a biomarker. Based on multi-omic analyses of postmortem human brain tissue, we hypothesized that sufficient astrocytic CLU upregulation in response to neuropathology preserves cognitive function, while reduced CLU expression, as seen in individuals carrying CLU risk alleles, increases disease susceptibility. Using human iPSC-based models, we explored the molecular and functional consequences of CLU deficiency. Unbiased proteomic profiling and functional validation revealed that CLU deficiency activates NFκB-dependent signaling, leading to elevated secretion of complement component C3 and proinflammatory cytokines. By establishing co-cultures of astrocytes with neurons, microglia, or both, we demonstrate an intricate network of intercellular signaling, leading to microglia-dependent tau phosphorylation, increased microglia phagocytosis, and reduced synapse density in CLU deficient conditions. Remarkedly, longitudinal analysis of human plasma samples revealed that individuals with CLU protective alleles showed an increase in CLU levels over time without changes in inflammatory markers, while those with risk alleles exhibited stable CLU levels alongside an upregulation in inflammatory markers. By integrating mouse and human cellular models, we demonstrate that CLU risk alleles recapitulate CLU-loss-of-function phenotypes under neuropathological burden. In vivo, mice carrying a humanized CLU risk allele showed reduced CLU protein levels and increased expression of phagocytosis- and complement-related genes. In vitro, we used genetically diverse iPSC-derived astrocytes to demonstrate that CLU risk alleles led to reduced CLU and APOE levels and increased complement protein and phosphorylated tau levels in co-cultures with microglia and neurons. Taken together, our findings establish a mechanistic link between AD genetic risk factors, astrocyte reactivity, and microglia-mediated effects on synaptic integrity, underscoring CLU as a pivotal neuroprotective factor in AD pathogenesis and brain health. Our triple-culture model effectively captures critical signaling dynamics among microglia, neurons, and astrocytes but lacks vascular components, such as brain endothelial cells and pericytes, which are integral to the blood-brain barrier (BBB). The BBB, a cornerstone of the neurovascular unit (NVU), preserves brain homeostasis, and its dysfunction contributes to neurodegenerative processes. To extend our platform, we developed an all-human BBB model that incorporates endothelial cells, pericytes, astrocytes, neurons, and microglia. We demonstrate the utility of this model by profiling the molecular responses of BBB cells exposed to fAD neurons, revealing dysregulated pathways across multiple cell types, including extracellular matrix (ECM) degradation, complement activation, and TNF signaling via NFκB. Furthermore, endothelial cells exhibited upregulation of matrisome proteins (e.g., SMOC1, SPOCK3, MDK) and increased matrix metalloproteinase activity, recapitulating vascular changes observed in the AD brain. Our findings provide a powerful resource for investigating cell-type-specific responses to pathogenic Aβ and provide a platform for exploring therapeutic interventions targeting the NVU. Collectively, these studies illuminate the critical role of glial-neuronal and vasculature interactions in AD pathogenesis, revealing how glial-driven risk factors affect neuronal homeostasis and how fAD-associated mutations disrupt intercellular communication. Using our co-culture systems, we found that acute exposure to fAD neurons suppresses astrocyte-induced DAM states, triggers inflammatory responses, and upregulates matrisome proteins in BBB cell types, reflecting AD-related vascular changes. Moreover, we demonstrate that CLU protects neuronal synapses by mitigating complement and inflammatory signaling between microglia and astrocytes. These findings underscore the utility of co-culture models in uncovering mechanisms by which glial-neuronal and vasculature interactions preserve brain homeostasis and how their disruption by genetic risk factors contributes to neurodegeneration.