Microglia are the resident myeloid cells in the central nervous system (CNS). The majority of microglia rely on CSF1R signaling for survival. However, a small subset of microglia in mouse brains can survive without CSF1R signaling and reestablish the microglial homeostatic population after CSF1R signaling returns. Using single-cell transcriptomic analysis, we characterized the heterogeneous microglial populations under CSF1R inhibition, including microglia with reduced homeostatic markers and elevated markers of inflammatory chemokines and proliferation. Importantly, MAC2/Lgals3 was upregulated under CSF1R inhibition, and shared striking similarities with microglial progenitors in the yolk sac and immature microglia in early embryos. Lineage-tracing studies revealed that these MAC2+ cells were of microglial origin. MAC2+ microglia were also present in non-treated adult mouse brains and exhibited immature transcriptomic signatures indistinguishable from those that survived CSF1R inhibition, supporting the notion that MAC2+ progenitor-like cells are present among adult microglia.
Pathological hallmarks of Alzheimer’s disease (AD) precede clinical symptoms by years, indicating a period of cognitive resilience before the onset of dementia. Here, we report that activation of cyclic GMP–AMP synthase (cGAS) diminishes cognitive resilience by decreasing the neuronal transcriptional network of myocyte enhancer factor 2c (MEF2C) through type I interferon (IFN-I) signaling. Pathogenic tau activates cGAS and IFN-I responses in microglia, in part mediated by cytosolic leakage of mitochondrial DNA. Genetic ablation of Cgas in mice with tauopathy diminished the microglial IFN-I response, preserved synapse integrity and plasticity and protected against cognitive impairment without affecting the pathogenic tau load. cGAS ablation increased, while activation of IFN-I decreased, the neuronal MEF2C expression network linked to cognitive resilience in AD. Pharmacological inhibition of cGAS in mice with tauopathy enhanced the neuronal MEF2C transcriptional network and restored synaptic integrity, plasticity and memory, supporting the therapeutic potential of targeting the cGAS–IFN–MEF2C axis to improve resilience against AD-related pathological insults.
Ferroptosis is a regulated, iron-dependent form of necrosis that is triggered by the accumulation of oxidatively damaged phospholipids [1][2][3] . Glutathione peroxidase 4 (GPX4) prevents ferroptosis by converting phospholipid hydroperoxides into non-toxic lipid alcohols 4,5 . Ferroptosis has been implicated in the pathology of several degenerative conditions and inhibiting GPX4 activity has emerged as a therapeutic strategy to induce cancer cell death 1,2 . However, many cancer cell lines are resistant to GPX4 inhibition 6 , and the mechanisms that regulate GPX4 activity and ferroptosis resistance remain incompletely understood. Here, employing a synthetic lethal CRISPR-Cas9 screen in a triple negative breast cancer (TNBC) cell line, we identify LRP8 (also known as ApoER2) as a ferroptosis resistance factor. LRP8 is upregulated in cancer, and we find that it promotes ferroptosis resistance in cancer cells in both 2-dimensional (2-D) cell culture and 3-dimensional (3-D) spheroid models. Mechanistically, loss of LRP8 decreases cellular selenium levels, resulting in the reduced expression of a subset of selenoproteins, including GPX4. Remarkably, the reduction in GPX4 is not due to the classic hierarchical selenoprotein regulatory program 7,8 . Instead, our findings demonstrate that the translation of GPX4 is severely impaired in the selenium-deficient LRP8 knockout (KO) cells due to extensive ribosome stalling at the inefficiently decoded GPX4 selenocysteine (SEC) UGA codon, which results in ribosome collisions and early translation termination. Thus, our findings reveal ribosome stalling and collisions during GPX4 translation as targetable ferroptosis vulnerabilities in cancer cells. MAINTwo primary cellular mechanisms that prevent ferroptosis are the conversion of phospholipid hydroperoxides into non-toxic lipid alcohols by GPX4 4 and the generation of radical trapping antioxidants to block the propagation of lipid peroxidation by enzymes such as ferroptosis suppressor protein 1 (FSP1) 9,10 , dihydroorotate dehydrogenase (DHODH) 11 , and GTP cyclohydrolase-1 (GCH1) 12,13 . Targeting the pathways that mediate ferroptosis resistance has emerged as a promising cancer therapeutic strategy, but how known protective pathways are regulated and whether additional mechanisms of ferroptosis resistance exist remain outstanding questions. LRP8 is a candidate ferroptosis regulator that is upregulated in cancerHigh FSP1 expression is strongly correlated with resistance to GPX4 inhibitors 9,10 , consistent with the role of FSP1 in preventing ferroptosis. To identify additional ferroptosis resistance mechanisms, we searched for cell lines displaying FSP1-independent resistance. Analysis of cancer cell line sensitivities to ferroptosis inducers reported in the Cancer TherapeuticsResponse Portal (CTRP) 14,15 identified a subset of cancer cell lines, including the MDA-MB-453 TNBC cell line, that are resistant to GPX4 inhibitors (RSL3, ML162, and ML210) despite low amounts of FSP1 mRNA (Fig. 1a and Extended Data Fig. 1a,b). Consist...
The heterogeneous pathophysiology of traumatic brain injury (TBI) is a barrier to advancing diagnostics and therapeutics, including targeted drug delivery. We used a unique discovery pipeline to identify novel targeting motifs that recognize specific temporal phases of TBI pathology. This pipeline combined in vivo biopanning with domain antibody (dAb) phage display, next-generation sequencing analysis, and peptide synthesis. We identified targeting motifs based on the complementarity-determining region 3 structure of dAbs for acute (1 day post-injury) and subacute (7 days post-injury) post-injury time points in a preclinical TBI model (controlled cortical impact). Bioreactivity and temporal sensitivity of the targeting motifs were validated via immunohistochemistry. Immunoprecipitation–mass spectrometry indicated that the acute TBI targeting motif recognized targets associated with metabolic and mitochondrial dysfunction, whereas the subacute TBI motif was largely associated with neurodegenerative processes. This pipeline successfully discovered temporally specific TBI targeting motif/epitope pairs that will serve as the foundation for the next-generation targeted TBI therapeutics and diagnostics.
The heterogeneous injury pathophysiology of traumatic brain injury (TBI) is a barrier to developing highly sensitive and specific diagnostic tools. Embracing neural injury complexity is critical for the development and advancement of diagnostics and therapeutics. The current study employs a unique discovery pipeline to identify targeting motifs that recognize specific phases of TBI pathology. This pipeline entails in vivo biopanning with a domain antibody (dAb) phage display library, next generation sequencing (NGS) analysis, and peptide synthesis. Here, we identify targeting motifs based on the HCDR3 structure of dAbs for acute (1 day) and subacute (7 days) postinjury timepoints using a mouse controlled cortical impact model. Their bioreactivity was validated using immunohistochemistry and candidate target epitopes were identified via immunoprecipitation-mass spectrometry. The acute targeting motif recognizes targets associated with metabolic and mitochondrial dysfunction whereas the subacute motif was largely associated with neurodegenerative processes. This phage display biomarker discovery pipeline for TBI successfully achieved discovery of temporally specific TBI targeting motif/epitope pairs that will advance the TBI diagnostics and therapeutics.
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