Periodic Reporting for period 4 - CALVARIA (Translational aspects of the discovery of skull marrow – meninges connections)
Período documentado: 2025-07-01 hasta 2025-12-31
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.
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.
Over the course of the project, we combined advanced imaging, tissue clearing, spatial omics, and computational analysis to investigate the calvaria–meninges–brain axis across multiple scales and disease contexts. This included the generation of comprehensive datasets using single-cell transcriptomics, proteomics, intravital two-photon imaging, and whole-organ and whole-body 3D imaging in both mouse models and human samples. In parallel, we developed computational frameworks enabling quantitative analysis of large-scale imaging and molecular datasets. These efforts led to several key findings. We demonstrated that skull bone marrow exhibits distinct molecular and cellular characteristics compared to peripheral bones and responds dynamically to brain pathology, including stroke. Transcriptomic and proteomic analyses identified specific regulatory pathways, including Nr4a1 (Nur77), CXCR2 signaling, and differential TREM1 regulation, supporting a specialized role of skull-derived immune cells in neuroinflammation. In humans, TSPO-PET imaging revealed disease-specific activation patterns in the skull that correlate with brain inflammation and clinical outcomes.
We further extended our analysis to systemic and disease contexts. Using deep learning–based whole-body analysis, we identified coordinated neural and immune remodeling in obesity. In parallel, vascular mapping approaches revealed distinct aging trajectories associated with blood–brain barrier disruption and cognitive decline, which could be modulated through Tie2 pathway activation. In the context of SARS-CoV-2 infection, we observed persistent accumulation of spike protein within the skull–meninges–brain axis, associated with sustained inflammatory and neurodegenerative changes and functional impairment in experimental models.
The results of the project have been disseminated through high-impact publications and presentations at international conferences. In addition, the project generated methodological outputs, including whole-body analysis tools and spatial omics workflows, which are being further developed and applied in ongoing studies. The translational potential of these findings is reflected in follow-up funding applications, including an ERC Proof of Concept proposal that received a Seal of Excellence.
We further extended our analysis to systemic and disease contexts. Using deep learning–based whole-body analysis, we identified coordinated neural and immune remodeling in obesity. In parallel, vascular mapping approaches revealed distinct aging trajectories associated with blood–brain barrier disruption and cognitive decline, which could be modulated through Tie2 pathway activation. In the context of SARS-CoV-2 infection, we observed persistent accumulation of spike protein within the skull–meninges–brain axis, associated with sustained inflammatory and neurodegenerative changes and functional impairment in experimental models.
The results of the project have been disseminated through high-impact publications and presentations at international conferences. In addition, the project generated methodological outputs, including whole-body analysis tools and spatial omics workflows, which are being further developed and applied in ongoing studies. The translational potential of these findings is reflected in follow-up funding applications, including an ERC Proof of Concept proposal that received a Seal of Excellence.
This project advances the understanding of neuroimmune interactions by demonstrating that skull bone marrow is an active and specialized immune compartment with distinct molecular identity and dynamic responses to brain pathology. The identification of skull-specific transcriptional programs and signaling pathways extends current models of neuroinflammation beyond the brain parenchyma to include its anatomical interfaces. A major advance is the development of integrated methodological frameworks enabling anatomically unbiased analysis of intact systems. These include whole-body imaging combined with deep learning–based quantification (MouseMapper), spatial transcriptomics in cleared tissues (DISCO-seq), and large-scale vascular and molecular mapping (VesselPro). Together, these approaches enable the integration of structural, molecular, and functional data across organs and scales, overcoming limitations of traditional section-based analyses.
Importantly, the project demonstrates that neuroimmune and vascular processes must be understood in a systemic context. The identification of coordinated immune and neural remodeling in obesity, as well as distinct vascular aging trajectories linked to cognitive outcomes, reveals previously unrecognized heterogeneity in disease mechanisms. The finding that hypervascular brain aging represents a maladaptive but reversible state provides a new framework for understanding vascular contributions to neurodegeneration. The project also provides translational advances, including the use of TSPO-PET imaging of the skull as a non-invasive readout of brain inflammation and the identification of persistent viral components at the brain borders in COVID-19. These findings support new approaches for monitoring and potentially modulating neuroinflammatory processes.
Building on these advances, the project establishes a foundation for future work aimed at patient stratification based on neuroimmune and vascular profiles, as well as for developing targeted intervention strategies in stroke, vascular dementia, and post-viral neurological conditions.
Importantly, the project demonstrates that neuroimmune and vascular processes must be understood in a systemic context. The identification of coordinated immune and neural remodeling in obesity, as well as distinct vascular aging trajectories linked to cognitive outcomes, reveals previously unrecognized heterogeneity in disease mechanisms. The finding that hypervascular brain aging represents a maladaptive but reversible state provides a new framework for understanding vascular contributions to neurodegeneration. The project also provides translational advances, including the use of TSPO-PET imaging of the skull as a non-invasive readout of brain inflammation and the identification of persistent viral components at the brain borders in COVID-19. These findings support new approaches for monitoring and potentially modulating neuroinflammatory processes.
Building on these advances, the project establishes a foundation for future work aimed at patient stratification based on neuroimmune and vascular profiles, as well as for developing targeted intervention strategies in stroke, vascular dementia, and post-viral neurological conditions.