Ziv Porat

Early activation of microglia as antigen-presenting cells correlates with T cell-mediated protection and repair of the injured central nervous system

After an injury to the central nervous system (CNS), activated microglia have been shown to contribute to the ongoing destructive processes leading to secondary neuronal degeneration. They can, however, also express neuroprotective activity. Studies from our laboratory point to the existence of a physiological T cell-mediated neuroprotective mechanism (adaptive immunity) that is amenable to boosting. We postulate that the beneficial or destructive outcome of the local microglial (innate) response is determined by a well-controlled dialog between the innate and the adaptive immune players. Here, we show that spontaneous or exogenously boosted T cell-mediated neuroprotection is correlated with early activation of microglia as antigen-presenting cells. We suggest that such microglial activity, if well controlled, is a crucial step in determining the fate of the neurons in a hostile environment.

Monocytes-macrophages that express α-smooth muscle actin preserve primitive hematopoietic cells in the bone marrow

Hematopoietic stem and progenitor cells (HSPCs) are regulated by various bone marrow stromal cell types. Here we identified rare activated bone marrow monocytes and macrophages with high expression of α-smooth muscle actin (α-SMA) and the cyclooxygenase COX-2 that were adjacent to primitive HSPCs. These myeloid cells resisted radiation-induced cell death and further upregulated COX-2 expression under stress conditions. COX-2-derived prostaglandin E2 (PGE2) prevented HSPC exhaustion by limiting the production of reactive oxygen species (ROS) via inhibition of the kinase Akt and higher stromal-cell expression of the chemokine CXCL12, which is essential for stem-cell quiescence. Our study identifies a previously unknown subset of α-SMA+ activated monocytes and macrophages that maintain HSPCs and protect them from exhaustion during alarm situations.

An MTCH2 pathway repressing mitochondria metabolism regulates haematopoietic stem cell fate

The metabolic state of stem cells is emerging as an important determinant of their fate. In the bone marrow, haematopoietic stem cell (HSC) entry into cycle, triggered by an increase in intracellular reactive oxygen species (ROS), corresponds to a critical metabolic switch from glycolysis to mitochondrial oxidative phosphorylation (OXPHOS). Here we show that loss of mitochondrial carrier homologue 2 (MTCH2) increases mitochondrial OXPHOS, triggering HSC and progenitor entry into cycle. Elevated OXPHOS is accompanied by an increase in mitochondrial size, increase in ATP and ROS levels, and protection from irradiation-induced apoptosis. In contrast, a phosphorylation-deficient mutant of BID, MTCH2s ligand, induces a similar increase in OXPHOS, but with higher ROS and reduced ATP levels, and is associated with hypersensitivity to irradiation. Thus, our results demonstrate that MTCH2 is a negative regulator of mitochondrial OXPHOS downstream of BID, indispensible in maintaining HSC homeostasis.

Overexpression of antizyme-inhibitor in NIH3T3 fibroblasts provides growth advantage through neutralization of antizyme functions

Antizyme inhibitor (AzI) is a homolog of ornithine decarboxylase (ODC), a key enzyme of polyamine synthesis. Antizyme inhibitor retains no enzymatic activity, but exhibits high affinity to antizyme (Az), a negative regulator of polyamine homeostasis. As polyamines are involved in maintaining cellular proliferation, and since AzI may negate Az functions, we have investigated the role of AzI in regulating cell growth. We show here that overexpression of AzI in NIH3T3 cells increased growth rate, enabled growth in low serum, and permitted anchorage-independent growth in soft agar, while reduction of AzI levels by AzI siRNA reduced cellular proliferation. Moreover, AzI overproducing cells gave rise to tumors when injected into nude mice. AzI overexpression resulted in elevation of ODC activity and of polyamine uptake. These effects of AzI are a result of its ability to neutralize Az, as overexpression of an AzI mutant with reduced Az binding failed to alter cellular polyamine metabolism and growth properties. We also demonstrate upregulation of AzI in Ras transformed cells, suggesting its relevance to some naturally occurring transformations. Finally, increased uptake activity rendered AzI overproducing and Ras-transformed cells more sensitive to toxic polyamine analogs. Our results therefore imply that AzI has a central and meaningful role in modulation of polyamine homeostasis, and in regulating cellular proliferation and transformation properties.

Inhibition of triple-negative breast cancer models by combinations of antibodies to EGFR

Breast tumors lacking expression of human epidermal growth factor receptor 2 (HER2) and the estrogen and the progesterone receptors (triple negative; TNBC) are more aggressive than other disease subtypes, and no molecular targeted agents are currently available for their treatment. Because TNBC commonly displays EGF receptor (EGFR) expression, and combinations of monoclonal antibodies to EGFR effectively inhibit other tumor models, we addressed the relevance of this strategy to treatment of TNBC. Unlike a combination of the clinically approved monoclonal antibodies, cetuximab and panitumumab, which displaced each other and displayed no cooperative effects, several other combinations resulted in enhanced inhibition of TNBC’s cell growth both in vitro and in animals. The ability of certain antibody mixtures to remove EGFR from the cell surface and to promote its intracellular degradation correlated with the inhibitory potential. However, unlike EGF-induced sorting of EGFR to lysosomal degradation, the antibody-induced pathway displayed independence from the intrinsic kinase activity and dimer formation ability of EGFR, and it largely avoided the recycling route. In conclusion, although TNBC clinical trials testing EGFR inhibitors reported lack of benefit, our results offer an alternative strategy that combines noncompetitive antibodies to achieve robust degradation of EGFR and tumor inhibition.

PAR1 signaling regulates the retention and recruitment of EPCR-expressing bone marrow hematopoietic stem cells

Retention of long-term repopulating hematopoietic stem cells (LT-HSCs) in the bone marrow is essential for hematopoiesis and for protection from myelotoxic injury. We report that signaling cascades that are traditionally viewed as coagulation related also control retention of endothelial protein C receptor–positive (EPCR+) LT-HSCs in the bone marrow and their recruitment to the blood via two pathways mediated by protease activated receptor 1 (PAR1). Thrombin-PAR1 signaling induces nitric oxide (NO) production, leading to EPCR shedding mediated by tumor necrosis factor-α–converting enzyme (TACE), enhanced CXCL12-CXCR4–induced motility and rapid stem and progenitor cell mobilization. Conversely, bone marrow blood vessels provide a microenvironment enriched with activated protein C (aPC) that retains EPCR+ LT-HSCs by limiting NO generation, reducing Cdc42 activity and enhancing integrin VLA4 affinity and adhesion. Inhibition of NO production by aPC-EPCR-PAR1 signaling reduces progenitor cell egress from the bone marrow, increases retention of bone marrow NOlow EPCR+ LT-HSCs and protects mice from chemotherapy-induced hematological failure and death. Our study reveals new roles for PAR1 and EPCR in controlling NO production to balance maintenance and recruitment of bone marrow EPCR+ LT-HSCs, with potential clinical relevance for stem cell transplantation.

Dynamic Response Diversity of NFAT Isoforms in Individual Living Cells

Processing of external information by mammalian cells often involves seemingly redundant isoforms of signaling molecules and transcription factors. Understanding the functional relevance of coexpressed isoforms that respond to the same signal and control a shared set of genes is still limited. Here we show, using imaging of individual living mammalian cells, that the closely related transcription factors NFAT1 and NFAT4 possess distinct nuclear localization dynamics in response to cell stimulation. NFAT4 shows a fast response, with rapid stochastic bursts of nuclear localization. Burst frequency grows with signal level, while response amplitude is fixed. In contrast, NFAT1 has a slow, continuous response, and its amplitude increases with signal level. These diverse dynamical features observed for single cells are translated into different impulse response strategies at the cell population level. We suggest that dynamic response diversity of seemingly redundant genes can provide cells with enhanced capabilities of temporal information processing.

miR-142 orchestrates a network of actin cytoskeleton regulators during megakaryopoiesis

Genome-encoded microRNAs (miRNAs) provide a posttranscriptional regulatory layer that controls the differentiation and function of various cellular systems, including hematopoietic cells. miR-142 is one of the most prevalently expressed miRNAs within the hematopoietic lineage. To address the in vivo functions of miR-142, we utilized a novel reporter and a loss-of-function mouse allele that we have recently generated. In this study, we show that miR-142 is broadly expressed in the adult hematopoietic system. Our data further reveal that miR-142 is critical for megakaryopoiesis. Genetic ablation of miR-142 caused impaired megakaryocyte maturation, inhibition of polyploidization, abnormal proplatelet formation, and thrombocytopenia. Finally, we characterized a network of miR-142-3p targets which collectively control actin filament homeostasis, thereby ensuring proper execution of actin-dependent proplatelet formation. Our study reveals a pivotal role for miR-142 activity in megakaryocyte maturation and function, and demonstrates a critical contribution of a single miRNA in orchestrating cytoskeletal dynamics and normal hemostasis. DOI: http://dx.doi.org/10.7554/eLife.01964.001

Cerebral nitric oxide represses choroid plexus NFκB-dependent gateway activity for leukocyte trafficking.

Chronic neuroinflammation is evident in brain aging and neurodegenerative disorders and is often associated with excessive nitric oxide (NO) production within the central nervous system (CNS). Under such conditions, increased NO levels are observed at the choroid plexus (CP), an epithelial layer that forms the blood–cerebrospinal fluid barrier (BCSFB) and serves as a selective gateway for leukocyte entry to the CNS in homeostasis and following injury. Here, we hypothesized that elevated cerebral NO levels interfere with CP gateway activity. We found that induction of leukocyte trafficking determinants by the CP and sequential leukocyte entry to the CSF are dependent on the CP epithelial NFκB/p65 signaling pathway, which was inhibited upon exposure to NO. Examining the CP in 5XFAD transgenic mouse model of Alzheimers disease (AD‐Tg) revealed impaired ability to mount an NFκB/p65‐dependent response. Systemic administration of an NO scavenger in AD‐Tg mice alleviated NFκB/p65 suppression at the CP and augmented its gateway activity. Together, our findings identify cerebral NO as a negative regulator of CP gateway activity for immune cell trafficking to the CNS.

Promoter activity dynamics in the lag phase of Escherichia coli

BackgroundLag phase is a period of time with no growth that occurs when stationary phase bacteria are transferred to a fresh medium. Bacteria in lag phase seem inert: their biomass does not increase. The low number of cells and low metabolic activity make it difficult to study this phase. As a consequence, it has not been studied as thoroughly as other bacterial growth phases. However, lag phase has important implications for bacterial infections and food safety. We asked which, if any, genes are expressed in the lag phase of Escherichia coli, and what is their dynamic expression pattern.ResultsWe developed an assay based on imaging flow cytometry of fluorescent reporter cells that overcomes the challenges inherent in studying lag phase. We distinguish between lag1 phase- in which there is no biomass growth, and lag2 phase- in which there is biomass growth but no cell division. We find that in lag1 phase, most promoters are not active, except for the enzymes that utilize the specific carbon source in the medium. These genes show promoter activities that increase exponentially with time, despite the fact that the cells do not measurably increase in size. An oxidative stress promoter, katG, is also active. When cells enter lag2 and begin to grow in size, they switch to a full growth program of promoter activity including ribosomal and metabolic genes.ConclusionsThe observed exponential increase in enzymes for the specific carbon source followed by an abrupt switch to production of general growth genes is a solution of an optimal control model, known as bang-bang control. The present approach contributes to the understanding of lag phase, the least studied of bacterial growth phases.