Probes for INS

The molecular mediators of cell death and inflammation in Alzheimer's disease (AD) have yet to be fully elucidated. Caspase-8 is a critical regulator of several cell death and inflammatory pathways; however, its role in AD pathogenesis has not yet been examined in detail. In the absence of Caspase-8, mice are embryonic lethal due to excessive RIPK3-dependent necroptosis. Compound RIPK3 and Caspase-8 mutants rescue embryonic lethality, which we leveraged to examine the roles of these pathways in an amyloid beta (Aβ)-mediated mouse model of AD. We find that combined deletion of Caspase-8 and RIPK3, but not RIPK3 alone, leads to diminished Aβ deposition and microgliosis in the 5xFAD mouse model of AD. Despite its well-known role in cell death, Caspase-8 does not appear to impact cell loss in the 5xFAD model. In contrast, we found that Caspase-8 is a critical regulator of Aβ-driven inflammasome gene expression and IL-1β release. Interestingly, loss of RIPK3 had only a modest effect on disease progression suggesting that inhibition of necroptosis or RIPK3-mediated cytokine pathways are not critical during mid stages of Aβ amyloidosis. These findings suggest that therapeutics targeting Caspase-8 may represent a novel strategy to limit Aꞵ amyloidosis and neuroinflammation in AD.

Research in recent years firmly established that microglial cells play an important role in the pathogenesis of Alzheimer's disease (AD). In parallel, a series of studies showed that, under both homeostatic and pathological conditions, microglia are a heterogeneous cell population. In AD, amyloid-β (Aβ) plaque-associated microglia (PAM) display a clearly distinct phenotype compared to plaque-distant microglia (PCM), suggesting that these two microglia subtypes likely differently contribute to disease progression. So far, molecular characterization of PAM was performed indirectly using single cell RNA sequencing (scRNA-seq) approaches or based on markers that are supposedly up-regulated in this microglia subpopulation.In this study based on a well-characterized AD mouse model, we combined cell-specific laser capture microdissection and RNA-seq analysis to i) identify, without preconceived notions of the molecular and/or functional changes that would affect these cells, the genes and gene networks that are dysregulated in PAM or PCM at three critical stages of the disease, and ii) to investigate the potential contribution of both plaque-associated and plaque-distant microglia.First, we established that our approach allows selective isolation of microglia, while preserving spatial information and preventing transcriptome changes induced by classical purification approaches. Then, we identified, in PAM and PCM subpopulations, networks of co-deregulated genes and analyzed their potential functional roles in AD. Finally, we investigated the dynamics of microglia transcriptomic remodeling at early, intermediate and late stages of the disease and validated select findings in postmortem human AD brain.Our comprehensive study provides useful transcriptomic information regarding the respective contribution of PAM and PCM across the Aβ pathology progression. It highlights specific pathways that would require further study to decipher their roles across disease progression. It demonstrates that the proximity of microglia to Aβ-plaques dramatically alters the microglial transcriptome and reveals that these changes can have both positive and negative impacts on the surrounding cells. These opposing effects may be driven by local microglia heterogeneity also demonstrated by this study. Our approach leads to molecularly define the less well studied plaque-distant microglia. We show that plaque-distant microglia are not bystanders of the disease, although the transcriptomic changes are far less striking compared to what is observed in plaque-associated microglia. In particular, our results suggest they may be involved in Aβ oligomer detection and in Aβ-plaque initiation, with increased contribution as the disease progresses.

Traumatic brain injury (TBI) is a leading cause of death and disability that lacks neuroprotective therapies. Following a TBI, secondary injury response pathways are activated and contribute to ongoing neurodegeneration. Microglia and astrocytes are critical neuroimmune modulators with early and persistent reactivity following a TBI. Although histologic glial reactivity is well established, a precise understanding of microglia and astrocyte function following trauma remains unknown.Adult male C57BL/6J mice underwent either fluid percussion or sham injury. RNA sequencing of concurrently isolated microglia and astrocytes was conducted 7 days post-injury to evaluate cell-type-specific transcriptional responses to TBI. Dual in situ hybridization and immunofluorescence were used to validate the TBI-induced gene expression changes in microglia and astrocytes and to identify spatial orientation of cells expressing these genes. Comparative analysis was performed between our glial transcriptomes and those from prior reports in mild TBI and other neurologic diseases to determine if severe TBI induces unique states of microglial and astrocyte activation.Our findings revealed sustained, lineage-specific transcriptional changes in both microglia and astrocytes, with microglia showing a greater transcriptional response than astrocytes at this subacute time point. Microglia and astrocytes showed overlapping enrichment for genes related to type I interferon signaling and MHC class I antigen presentation. The microglia and astrocyte transcriptional response to severe TBI was distinct from prior reports in mild TBI and other neurodegenerative and neuroinflammatory diseases.Concurrent lineage-specific analysis revealed novel TBI-specific transcriptional changes; these findings highlight the importance of cell-type-specific analysis of glial reactivity following TBI and may assist with the identification of novel, targeted therapies.