Excessive poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) activation kills cells via a cell-death process designated "parthanatos" in which PAR induces the mitochondrial release and nuclear translocation of apoptosis-inducing factor to initiate chromatinolysis and cell death. Accompanying the formation of PAR are the reduction of cellular NAD + and energetic collapse, which have been thought to be caused by the consumption of cellular NAD + by PARP-1. Here we show that the bioenergetic collapse following PARP-1 activation is not dependent on NAD + depletion. Instead PARP-1 activation initiates glycolytic defects via PAR-dependent inhibition of hexokinase, which precedes the NAD + depletion in N-methyl-N-nitroso-N-nitroguanidine (MNNG)-treated cortical neurons. Mitochondrial defects are observed shortly after PARP-1 activation and are mediated largely through defective glycolysis, because supplementation of the mitochondrial substrates pyruvate and glutamine reverse the PARP-1-mediated mitochondrial dysfunction. Depleting neurons of NAD + with FK866, a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase, does not alter glycolysis or mitochondrial function. Hexokinase, the first regulatory enzyme to initiate glycolysis by converting glucose to glucose-6-phosphate, contains a strong PAR-binding motif. PAR binds to hexokinase and inhibits hexokinase activity in MNNG-treated cortical neurons. Preventing PAR formation with PAR glycohydrolase prevents the PAR-dependent inhibition of hexokinase. These results indicate that bioenergetic collapse induced by overactivation of PARP-1 is caused by PAR-dependent inhibition of glycolysis through inhibition of hexokinase.

Inhibition or genetic deletion of poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) is protective against toxic insults in many organ systems. The molecular mechanisms underlying PARP-1–dependent cell death involve release of mitochondrial apoptosis-inducing factor (AIF) and its translocation to the nucleus, which results in chromatinolysis. We identified macrophage migration inhibitory factor (MIF) as a PARP-1–dependent AIF-associated nuclease (PAAN). AIF was required for recruitment of MIF to the nucleus, where MIF cleaves genomic DNA into large fragments. Depletion of MIF, disruption of the AIF-MIF interaction, or mutation of glutamic acid at position 22 in the catalytic nuclease domain blocked MIF nuclease activity and inhibited chromatinolysis, cell death induced by glutamate excitotoxicity, and focal stroke. Inhibition of MIF's nuclease activity is a potential therapeutic target for diseases caused by excessive PARP-1 activation.

T cell therapies require the removal and culture of T cells ex vivo to expand several thousandfold. However, these cells often lose the phenotype and cytotoxic functionality necessary to mediate an effective therapeutic response. The extracellular matrix has been used to preserve and augment cell phenotype; however, it has not been applied to cellular immunotherapies.Here we engineered a hyaluronic acid (HA)-based hydrogel to present the two stimulatory signals required for T-cell activation-termed an artificial T cell stimulating matrix (aTM).We found that biophysical properties of the aTM-stimulatory ligand density, stiffness, and extracellular matrix (ECM) proteins-potentiate T cell signaling and skew phenotype of both murine and human T cells. Importantly, it was only the combination of the ECM environment and mechanically sensitive TCR signaling from the aTM that produced nearly 4-times the number of rare, antigen-specific CD8+ T cells. Adoptive transfer of these tumor-specific cells significantly suppressed tumor growth and improved animal survival as compared with T cells stimulated by traditional methods. Beyond immediate immunotherapeutic applications, demonstrating that the environment influences the cellular therapeutic product delineates the importance of the ECM and provides a case study of how to engineer ECM-mimetic materials for therapeutic immune stimulation in the future.

Injury and loss of oligodendrocytes can cause demyelinating diseases such as multiple sclerosis. To improve our understanding of human oligodendrocyte development, which could facilitate development of remyelination-based treatment strategies, here we describe time-course single-cell-transcriptomic analysis of developing human stem cell-derived oligodendrocyte-lineage-cells (hOLLCs). The study includes hOLLCs derived from both genome engineered embryonic stem cell (ESC) reporter cells containing an Identification-and-Purification tag driven by the endogenous PDGFRα promoter and from unmodified induced pluripotent (iPS) cells. Our analysis uncovers substantial transcriptional heterogeneity of PDGFRα-lineage hOLLCs. We discover sub-populations of human oligodendrocyte progenitor cells (hOPCs) including a potential cytokine-responsive hOPC subset, and identify candidate regulatory genes/networks that define the identity of these sub-populations. Pseudotime trajectory analysis defines developmental pathways of oligodendrocytes vs astrocytes from PDGFRα-expressing hOPCs and predicts differentially expressed genes between the two lineages. In addition, pathway enrichment analysis followed by pharmacological intervention of these pathways confirm that mTOR and cholesterol biosynthesis signaling pathways are involved in maturation of oligodendrocytes from hOPCs.