Neurodegenerative diseases (NDs), often described as a major health challenge of the 21st century, are closely associated with brain inflammation. The Calvaria project was motivated by the recent discovery of skull–meninges connections (SMCs), suggesting that skull bone marrow may directly communicate with the meninges and contribute to neuroimmune processes. This raised the possibility that the calvaria represents a previously unrecognized and accessible immune compartment involved in brain pathology, with potential relevance for both diagnostics and therapeutic intervention. However, the structural, molecular, and functional properties of this system, and its relationship to neurological disease, remained largely unknown.
To address this gap, the project combined tissue clearing, advanced imaging, spatial omics, and computational analysis to systematically investigate the calvaria–meninges–brain axis. The main objectives were to: (1) characterize the molecular, cellular, and structural features of calvaria bone marrow under physiological conditions; (2) determine how this system responds to neurological disorders, including stroke and neurodegeneration; and (3) explore its potential for diagnostic imaging and therapeutic targeting.
Over the course of the project, we demonstrated that skull bone marrow exhibits distinct molecular and functional properties compared to peripheral bones. Using intravital imaging and spatial transcriptomics, we showed that skull immune cells are dynamically activated following stroke and display a specific transcriptional program characterized by enrichment of regulatory pathways such as Nr4a1 (Nur77) and CXCR2 signaling, together with differential regulation of inflammatory mediators, including TREM1. These findings indicate that skull marrow represents a specialized neuroimmune niche with distinct activation dynamics. The functional relevance of this system was further supported by TSPO-PET imaging in humans, which revealed disease-specific skull activation patterns across neurological conditions, including stroke and Alzheimer’s disease. These signals correlated with brain inflammation and clinical severity and were validated ex vivo, supporting the concept that the skull can serve as an accessible proxy for neuroinflammatory processes.
Beyond local skull–brain interactions, the project extended to systemic and disease contexts. Using the MouseMapper platform, a deep learning–based framework for whole-body segmentation and quantitative analysis of nervous and immune system architecture, we identified coordinated immune and neural remodeling across the whole body in obesity, including structural and functional alterations in trigeminal sensory pathways. In parallel, analysis of aging using the VesselPro pipeline, an integrated approach combining perfusion-based labeling, tissue clearing, 3D imaging, and spatial proteomics, revealed that cerebrovascular aging follows two distinct trajectories, including a previously unrecognized hypervascular state associated with blood–brain barrier disruption and cognitive decline, which could be reversed by Tie2 pathway activation. Finally, in the context of SARS-CoV-2 infection, we identified persistent accumulation of viral spike protein within the skull–meninges–brain axis in both humans and mice, accompanied by sustained inflammatory and neurodegeneration-associated changes. Functional experiments demonstrated that the spike protein alone is sufficient to induce neuroinflammation and worsen neurological outcomes.
Together, these findings establish the calvaria–meninges–brain axis as a functionally relevant and dynamically regulated neuroimmune interface, integrating local and systemic signals, and provide a foundation for future diagnostic and therapeutic strategies targeting neuroinflammation.
