Hematopoietic stem cell (HSC) aging has become a concern in chemotherapy of older patients. Humoral and paracrine signals from the bone marrow (BM) hematopoietic microenvironment (HM) control HSC activity during regenerative hematopoiesis. Connexin-43 (Cx43), a connexin constituent of gap junctions (GJs) is expressed in HSCs, down-regulated during differentiation, and postulated to be a self-renewal gene. Our studies, however, reveal that hematopoietic-specific Cx43 deficiency does not result in significant long-term competitive repopulation deficiency. Instead, hematopoietic Cx43 (H-Cx43) deficiency delays hematopoietic recovery after myeloablation with 5-fluorouracil (5-FU). 5-FU-treated H-Cx43-deficient HSC and progenitors (HSC/P) cells display decreased survival and fail to enter the cell cycle to proliferate. Cell cycle quiescence is associated with down-regulation of cyclin D1, up-regulation of the cyclin-dependent kinase inhibitors, p21 cip1. and p16 INK4a , and Forkhead transcriptional factor 1 (Foxo1), and activation of p38 mitogen-activated protein kinase (MAPK), indicating that H-Cx43-deficient HSCs are prone to senescence. The mechanism of increased senescence in H-Cx43-deficient HSC/P cells depends on their inability to transfer reactive oxygen species (ROS) to the HM, leading to accumulation of ROS within HSCs. In vivo antioxidant administration prevents the defective hematopoietic regeneration, as well as exogenous expression of Cx43 in HSC/P cells. Furthermore, ROS transfer from HSC/P cells to BM stromal cells is also rescued by reexpression of Cx43 in HSC/P. Finally, the deficiency of Cx43 in the HM phenocopies the hematopoietic defect in vivo. These results indicate that Cx43 exerts a protective role and regulates the HSC/P ROS content through ROS transfer to the HM, resulting in HSC protection during stress hematopoietic regeneration.

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Neurotransmission through electrical synapses plays an important role in the spike synchrony among neurons and oscillation of neuronal networks. Indeed, electrical transmission has been implicated in the hypersynchronous electrical activity of epilepsy. We have investigated the influence of intracellular pH on the strength of electrical coupling mediated by connexin36 (Cx36), the principal gap junction protein in the electrical synapses of vertebrates. In striking contrast to other connexin isoforms, the activity of Cx36 channels decreases following alkalosis rather than acidosis when it is expressed in Xenopus oocytes and N2A cells. This uncoupling of Cx36 channels upon alkalinization occurred in the vertebrate orthologues analyzed (human, mouse, chicken, perch, and skate). While intracellular acidification caused a mild or moderate increase in the junctional conductance of virtually all these channels, the coupling of the skate Cx35 channel was partially blocked by acidosis. The mutational analysis suggests that the Cx36 channels may contain two gating mechanisms operating with opposing sensitivity to pH. One gate, the dominant mechanism, closes for alkalosis and it probably involves an interaction between the Cand N-terminal domains, while a secondary acid sensing gate only causes minor, albeit saturating, changes in coupling following acidosis and alkalosis. Thus, we conclude that neuronal Cx36 channels undergo unique regulation by pH i since their activity is inhibited by alkalosis rather than acidosis. These data provide a novel basis to define the relevance and consequences of the pH-dependent modulation of Cx36 synapses under physiological and pathological conditions. alkalosis ͉ electrical synapses ͉ intracellular pH ͉ pH gating ͉ gap junction

Connexin-43 (Cx43), a gap junction protein involved in control of cell proliferation, differentiation and migration, has been suggested to have a role in hematopoiesis. Cx43 is highly expressed in osteoblasts and osteogenic progenitors (OB/P). To elucidate the biologic function of Cx43 in the hematopoietic microenvironment (HM) and its influence in hematopoietic stem cell (HSC) activity, we studied the hematopoietic function in an IntroductionIn the adult bone marrow (BM), the hematopoietic function is supported by the proliferation and differentiation of a finite number of transplantable hematopoietic stem cells and progenitors (HSCs/Ps) that have self-renewal and multipotential differentiation capacity. Endosteal and neighboring vascular/perivascular niches have been proposed as anatomic sites that control HSC/P activity, providing signals that regulate the stem cell pool size, survival, and migration. [1][2][3][4] The definition of the cell components of the BM hematopoietic microenvironment (HM) is an area of debate. Endothelial cells, 2 mesenchymal stem cells (MSCs), 5 and osteoblasts and osteogenic progenitors (OB/Ps) 1,4,6 represent major cell components of the BM HM.A poorly studied mechanism of intercellular communication (IC) in the HM is mediated by gap junction (GJ) channels. GJ channels are formed by a large family of proteins called connexins (Cxs). Each neighboring cell contributes cell-to-cell channels with 1 hemichannel that dock head-to-head. GJ channels have the ability to transfer ions and low-molecular weight secondary messengers from one cell to another depending on concentration gradients and Cx-dependent selective permeability. 7 Connexin-43 (Cx43), one member of this protein family, is highly expressed by adult BM stromal cells, 8 osteoblasts, 9 endothelial cells, 2 and MSCs, 5 and is also expressed by HSCs. 10 The presence of a functional Cx43-dependence of GJIC between osteoblasts and stromal cells, as well as between stromal cells and HSCs, has been confirmed in vitro. 11 After HSC transplantation, HSCs must first home to the BM, and then localize and anchor in suitable microenvironments within the BM, a process known as lodgment. 12 This process involves the migration of HSC/Ps through different layers of cell components in the HM, including endothelial cells and mesenchymal-origin cells, which act as extrinsic HSC/P migration regulators, 13 and is directed by chemokine gradients. Myeloablation through irradiation has been postulated to modify the functional ability of the BM HM to allow HSC homing and retention through clearance of HSC niches. 14 The effect of irradiation on the BM HM is not well known, but diverse studies have shown that it is not innocuous 15 and includes the up-regulation of expression and secretion of Cxcl12 16 and the up-regulation of the expression of Cx43 in the BM. 17 It has been proposed that Cx43 hemichannels are important in reinforcing cell-to-cell migration and that Cx43-dependent GJ are needed in the process of neuronal migration during neocortex form...

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Mutations in the potassium channel gene KCNQ4 underlie DFNA2, a subtype of autosomal dominant progressive, high-frequency hearing loss. Based on a phenotype-guided mutational screening we have identified a novel mutation c.886G>A, leading to the p.G296S substitution in the pore region of KCNQ4 channel. The possible impact of this mutation on total KCNQ4 protein expression, relative surface expression and channel function was investigated. When the G296S mutant was expressed in Xenopus oocytes, electrophysiological recordings did not show voltage-activated K(+) currents. The p.G296S mutation impaired KCNQ4 channel activity in two manners. It greatly reduced surface expression and, secondarily, abolished channel function. The deficient expression at the cell surface membrane was further confirmed in non-permeabilized NIH-3T3 cells transfected with the mutant KCNQ4 tagged with the hemagglutinin epitope in the extracellular S1-S2 linker. Co-expression of mutant and wild type KCNQ4 in oocytes was performed to mimic the heterozygous condition of the p.G296S mutation in the patients. The results showed that the G296S mutant exerts a strong dominant-negative effect on potassium currents by reducing the wild type KCNQ4 channel expression at the cell surface. This is the first study to identify a trafficking-dependent dominant mechanism for the loss of KCNQ4 channel function in DFNA2.

In the bone marrow (BM), hematopoietic progenitors (HP) reside in specific anatomical niches near osteoblasts (Ob), macrophages (MΦ) and other cells forming the BM microenvironment. A connection between immunosurveillance and traffic of HP has been demonstrated but the regulatory signals that instruct immune regulation on HP circulation are unknown. We discovered that the BM microenvironment deficiency of p62, an autophagy regulator and signal organizer, results in loss of autophagic repression of macrophage contact-dependent activation of Ob NF-κB signaling. Consequently, Ob p62-deficient mice lose bone, Ob Ccl4 expression and HP chemotaxis towards Cxcl12 resulting in egress of short-term hematopoietic stem cells and myeloid progenitors. Finally, Ccl4 expression and myeloid progenitor egress are reversed by the deficiency of the p62 PB1 binding partner Nbr1. A functional ‘MΦ-Ob niche’ is required for myeloid progenitor/short-term stem cell retention, in which Ob p62 is required to maintain NF-κB signaling repression, osteogenesis and BM progenitor retention.

We herein report the synthesis, antioxidant power and neuroprotective properties of nine homo-bisnitrones HBNs 1-9 as alpha-phenyl-N-tert-butylnitrone (PBN) analogues for stroke therapy. In vitro neuroprotection studies of HBNs 1-9 against Oligomycin A/Rotenone and in an oxygen-glucosedeprivation model of ischemia in human neuroblastoma cell cultures, indicate that (1Z,1′Z)-1,1′-(1,3phenylene)bis(N-benzylmethanimine oxide) (HBN6) is a potent neuroprotective agent that prevents the decrease in neuronal metabolic activity (EC 50 = 1.24 ± 0.39 μM) as well as necrotic and apoptotic cell death. HBN6 shows strong hydroxyl radical scavenger power (81%), and capacity to decrease superoxide production in human neuroblastoma cell cultures (maximal activity = 95.8 ± 3.6%), values significantly superior to the neuroprotective and antioxidant properties of the parent PBN. The higher neuroprotective ability of HBN6 has been rationalized by means of Density Functional Theory calculations. Calculated physicochemical and ADME properties confirmed HBN6 as a hit-agent showing suitable drug-like properties. Finally, the contribution of HBN6 to brain damage prevention was confirmed in a permanent MCAO setting by assessing infarct volume outcome 48 h after stroke in drug administered experimental animals, which provides evidence of a significant reduction of the brain lesion size and strongly suggests that HBN6 is a potential neuroprotective agent against stroke. Bis-nitrones are well-known antioxidant and neuroprotective agents showing high clinical potential. For instance, bis-nitrone W-AZN (Fig. 1), an azulenyl spin trap possessing neuroprotective effects in an animal model of