Cx3cr1-deficient microglia exhibit a premature aging transcriptome.

CX3CR1, one of the highest expressed genes in microglia in mice and humans, is implicated in numerous microglial functions. However, the molecular mechanisms underlying Cx3cr1 signaling are not well understood. Here, we analyzed transcriptomes of Cx3cr1-deficient microglia under varying conditions by RNA-sequencing (RNA-seq). In 2-mo-old mice, Cx3cr1 deletion resulted in the down-regulation of a subset of immune-related genes, without substantial epigenetic changes in markers of active chromatin. Surprisingly, Cx3cr1-deficient microglia from young mice exhibited a transcriptome consistent with that of aged Cx3cr1-sufficient animals, suggesting a premature aging transcriptomic signature. Immunohistochemical analysis of microglia in young and aged mice revealed that loss of Cx3cr1 modulates microglial morphology in a comparable fashion. Our results suggest that CX3CR1 may regulate microglial function in part by modulating the expression levels of a subset of inflammatory genes during chronological aging, making Cx3cr1-deficient mice useful for studying aged microglia

[PSI+] Prion Induction within the Yeast Saccharomyces Genera

Prions are self-perpetuating protein aggregates that cause neurodegenerative diseases in mammals and carry heritable traits in yeast. Yeast [PSI+] prion is the prion isoform of Sup35 protein, an essential translation termination factor. The [PSI+] prion state can be induced in [psi-] cells by the overexpression of the full-length Sup35 protein, or only a certain portion of the protein. However, this induction requires the presence of a second yeast prion known as [PIN+]. While [PSI+] is present in lab cultures of S. cerevisiae, it has not been reported in any other Saccharomyces species. In this study, we attempted to induce [PSI+] in S. paradoxus and S. bayanus, two close relatives of S. cerevisiae. As non-cerevisiae species of Saccharomyces lack other known prions, we employed a new induction approach based on the overproduction of a chimeric protein composed of portions of Sup35 and Human Progesterone Receptor 6.6 (HPR6.6). This construct can induce [PSI+] in the absence of other endogenous prions. We next engineered S. paradoxus and S. bayanus strains with markers that allowed for prion studies in these species. The novel Sup35-HPR6.6 fusion constructs led to [PSI+] induction in S. paradoxus, the sister species of S. cerevisiae, but not in the more distantly related S. bayanus. We also showed that the prion isoform of Sup35 from S. paradoxus, previously known to produce only unstable prions in the heterologous S. cerevisiae system, can generate mitotically stable prions in the homologous S. paradoxus system. Finally, we propose a model which explains the possible role of the Sup35 and HPR6.6 fusion constructs in [PIN+]-independent [PSI+] induction.Committee Member/Second Reader: Storici, Francesca; Faculty Mentor: Chernoff, Yur

Systemic Inflammation Regulates Microglial Responses to Tissue Damage in Vivo

Microglia, the resident immune cells of the central nervous system, exist in either a “resting” state associated with physiological tissue surveillance or an “activated” state in neuroinflammation. We recently showed that ATP is the primary chemoattractor to tissue damage in vivo and elicits opposite effects on the motility of activated microglia in vitro through activation of adenosine A2A receptors. However, whether systemic inflammation affects microglial responses to tissue damage in vivo remains largely unknown. Using in vivo two-photon imaging of mice, we show that injection of lipopolysaccharide (LPS) at levels that can produce both clear neuroinflammation and some features of sepsis significantly reduced the rate of microglial response to laser-induced ablation injury in vivo. Under proinflammatory conditions, microglial processes initially retracted from the ablation site, but subsequently moved toward and engulfed the damaged area. Analyzing the process dynamics in 3D cultures of primary microglia indicated that only A2A, but not A1 or A3 receptors, mediate process retraction in LPS-activated microglia. The A2A receptor antagonists caffeine and preladenant reduced adenosine-mediated process retraction in activated microglia in vitro. Finally, administration of preladenant before induction of laser ablation in vivo accelerated the microglial response to injury following systemic inflammation. The regulation of rapid microglial responses to sites of injury by A2A receptors could have implications for their ability to respond to the neuronal death occurring under conditions of neuroinflammation in neurodegenerative disorders. GLIA 2014;62:1345–136

Design, Synthesis, and Structure–Activity Relationship of a Novel Series of GluN2C-Selective Potentiators

NMDA receptors are tetrameric complexes composed of GluN1 and GluN2A–D subunits that mediate a slow Ca²⁺<b>-</b>permeable component of excitatory synaptic transmission. NMDA receptors have been implicated in a wide range of neurological diseases and thus represent an important therapeutic target. We herein describe a novel series of pyrrolidinones that selectively potentiate only NMDA receptors that contain the GluN2C subunit. The most active analogues tested were over 100-fold selective for recombinant GluN2C-containing receptors over GluN2A/B/D-containing NMDA receptors as well as AMPA and kainate receptors. This series represents the first class of allosteric potentiators that are selective for diheteromeric GluN2C-containing NMDA receptors

CCR2 deficiency alters activation of microglia subsets in traumatic brain injury.

In traumatic brain injury (TBI), a diversity of brain resident and peripherally derived myeloid cells have the&nbsp;potential to worsen damage and/or to assist in healing. We define the heterogeneity of microglia and macrophage phenotypes during TBI in wild-type (WT) mice and Ccr2-/- mice, which lack macrophage influx following TBI and are resistant to brain damage. We use unbiased single-cell RNA sequencing methods to uncover 25 microglia, monocyte/macrophage, and dendritic cell subsets in acute TBI and normal brains. We find alterations in transcriptional profiles of microglia subsets in Ccr2-/- TBI mice compared to WT TBI mice indicating that infiltrating monocytes/macrophages influence microglia activation to promote a type I IFN response. Preclinical pharmacological blockade of hCCR2 after injury reduces expression of IFN-responsive gene, Irf7, and improves outcomes. These data extend our understanding of myeloid cell diversity and crosstalk in brain trauma and identify therapeutic targets in myeloid subsets