Defining blood-induced microglia functions in neurodegeneration through multiomic profiling.

Blood protein extravasation through a disrupted blood-brain barrier and innate immune activation are hallmarks of neurological diseases and emerging therapeutic targets. However, how blood proteins polarize innate immune cells remains largely unknown. Here, we established an unbiased blood-innate immunity multiomic and genetic loss-of-function pipeline to define the transcriptome and global phosphoproteome of blood-induced innate immune polarization and its role in microglia neurotoxicity. Blood induced widespread microglial transcriptional changes, including changes involving oxidative stress and neurodegenerative genes. Comparative functional multiomics showed that blood proteins induce distinct receptor-mediated transcriptional programs in microglia and macrophages, such as redox, type I interferon and lymphocyte recruitment. Deletion of the blood coagulation factor fibrinogen largely reversed blood-induced microglia neurodegenerative signatures. Genetic elimination of the fibrinogen-binding motif to CD11b in Alzheimer's disease mice reduced microglial lipid metabolism and neurodegenerative signatures that were shared with autoimmune-driven neuroinflammation in multiple sclerosis mice. Our data provide an interactive resource for investigation of the immunology of blood proteins that could support therapeutic targeting of microglia activation by immune and vascular signals.

Triaging of α-helical proteins to the mitochondrial outer membrane by distinct chaperone machinery based on substrate topology.

Mitochondrial outer membrane ⍺-helical proteins play critical roles in mitochondrial-cytoplasmic communication, but the rules governing the targeting and insertion of these biophysically diverse proteins remain unknown. Here, we first defined the complement of required mammalian biogenesis machinery through genome-wide CRISPRi screens using topologically distinct membrane proteins. Systematic analysis of nine identified factors across 21 diverse ⍺-helical substrates reveals that these components are organized into distinct targeting pathways that act on substrates based on their topology. NAC is required for the efficient targeting of polytopic proteins, whereas signal-anchored proteins require TTC1, a cytosolic chaperone that physically engages substrates. Biochemical and mutational studies reveal that TTC1 employs a conserved TPR domain and a hydrophobic groove in its C-terminal domain to support substrate solubilization and insertion into mitochondria. Thus, the targeting of diverse mitochondrial membrane proteins is achieved through topological triaging in the cytosol using principles with similarities to ER membrane protein biogenesis systems.

Triaging of α-helical proteins to the mitochondrial outer membrane by distinct chaperone machinery based on substrate topology.

Mitochondrial outer membrane α-helical proteins play critical roles in mitochondrial-cytoplasmic communication, but the rules governing the targeting and insertion of these biophysically diverse substrates remain unknown. Here, we first defined the complement of required mammalian biogenesis machinery through genome-wide CRISPRi screens using topologically distinct membrane proteins. Systematic analysis of nine identified factors across 21 diverse α-helical substrates reveals that these components are organized into distinct targeting pathways which act on substrates based on their topology. NAC is required for efficient targeting of polytopic proteins whereas signal-anchored proteins require TTC1, a novel cytosolic chaperone which physically engages substrates. Biochemical and mutational studies reveal that TTC1 employs a conserved TPR domain and a hydrophobic groove in its C-terminal domain to support substrate solubilization and insertion into mitochondria. Thus, targeting of diverse mitochondrial membrane proteins is achieved through topological triaging in the cytosol using principles with similarities to ER membrane protein biogenesis systems.

Dietary restriction ameliorates TBI-induced phenotypes in Drosophila melanogaster.

Traumatic brain injury (TBI) affects millions annually and is associated with long-term health decline. TBI also shares molecular and cellular hallmarks with neurodegenerative diseases (NDs), typically increasing in prevalence with age, and is a major risk factor for developing neurodegeneration later in life. While our understanding of genes and pathways that underlie neurotoxicity in specific NDs has advanced, we still lack a complete understanding of early molecular and physiological changes that drive neurodegeneration, particularly as an individual ages following a TBI. Recently Drosophila has been introduced as a model organism for studying closed-head TBI. In this paper, we deliver a TBI to flies early in adult life, and then measure molecular and physiological phenotypes at short-, mid-, and long-term timepoints following the injury. We aim to identify the timing of changes that contribute to neurodegeneration. Here we confirm prior work demonstrating a TBI-induced decline in lifespan, and present evidence of a progressive decline in locomotor function, robust acute and modest chronic neuroinflammation, and a late-onset increase in protein aggregation. We also present evidence of metabolic dysfunction, in the form of starvation sensitivity and decreased lipids, that persists beyond the immediate injury response, but does not differ long-term. An intervention of dietary restriction (DR) partially ameliorates some TBI-induced phenotypes, including lifespan and locomotor function, though it does not alter the pattern of starvation sensitivity of injured flies. In the future, molecular pathways identified as altered following TBI-particularly in the short-, or mid-term-could present potential therapeutic targets.

MTCH2 is a mitochondrial outer membrane protein insertase.

In the mitochondrial outer membrane, α-helical transmembrane proteins play critical roles in cytoplasmic-mitochondrial communication. Using genome-wide CRISPR screens, we identified mitochondrial carrier homolog 2 (MTCH2), and its paralog MTCH1, and showed that it is required for insertion of biophysically diverse tail-anchored (TA), signal-anchored, and multipass proteins, but not outer membrane ß-barrel proteins. Purified MTCH2 was sufficient to mediate insertion into reconstituted proteoliposomes. Functional and mutational studies suggested that MTCH2 has evolved from a solute carrier transporter. MTCH2 uses membrane-embedded hydrophilic residues to function as a gatekeeper for the outer membrane, controlling mislocalization of TAs into the endoplasmic reticulum and modulating the sensitivity of leukemia cells to apoptosis. Our identification of MTCH2 as an insertase provides a mechanistic explanation for the diverse phenotypes and disease states associated with MTCH2 dysfunction.

Fibrinogen Induces Microglia-Mediated Spine Elimination and Cognitive Impairment in an Alzheimer's Disease Model.

Cerebrovascular alterations are a key feature of Alzheimer's disease (AD) pathogenesis. However, whether vascular damage contributes to synaptic dysfunction and how it synergizes with amyloid pathology to cause neuroinflammation and cognitive decline remain poorly understood. Here, we show that the blood protein fibrinogen induces spine elimination and promotes cognitive deficits mediated by CD11b-CD18 microglia activation. 3D molecular labeling in cleared mouse and human AD brains combined with repetitive in vivo two-photon imaging showed focal fibrinogen deposits associated with loss of dendritic spines independent of amyloid plaques. Fibrinogen-induced spine elimination was prevented by inhibiting reactive oxygen species (ROS) generation or genetic ablation of CD11b. Genetic elimination of the fibrinogen binding motif to CD11b reduced neuroinflammation, synaptic deficits, and cognitive decline in the 5XFAD mouse model of AD. Thus, fibrinogen-induced spine elimination and cognitive decline via CD11b link cerebrovascular damage with immune-mediated neurodegeneration and may have important implications in AD and related conditions.