Long-term combination therapy with metformin and oxymetholone in a Fanconi anemia mouse model.

Fanconi anemia (FA) is a disease caused by defective deoxyribonucleic acid (DNA) repair that manifests as bone marrow failure, cancer predisposition, and developmental defects. We previously reported that monotherapy with either metformin (MET) or oxymetholone (OXM) improved peripheral blood (PB) counts and the number and functionality of bone marrow hematopoietic stem progenitor cells (HSPCs) number in Fancd2-/- mice. To evaluate whether the combination treatment of these drugs has a synergistic effect to prevent bone marrow failure in FA, we treated cohorts of Fancd2-/- mice and wildtype controls with either MET alone, OXM alone, MET+OXM, or placebo diet from age 3 weeks to 18 months. The OXM treated animals showed modest improvements in blood parameters including platelet count (p = .01) and hemoglobin levels (p < .05). In addition, the percentage of quiescent hematopoietic stem cell (HSC) (LSK [Lin-Sca+c-Kit+]) was significantly increased (p = .001) by long-term treatment with MET alone. The combination of metformin and oxymetholone did not result in a significant synergistic effect in any hematopoietic parameter. Gene expression analysis of liver tissue from these animals showed that some of the expression changes caused by Fancd2 deletion were partially normalized by metformin treatment. Importantly, no adverse effects of the individual or combination therapies were observed, despite the long-term administration. We conclude that androgen therapy is not a contraindication to concurrent metformin administration in clinical trials. HIGHLIGHTS: Long-term coadministration of metformin in combination with oxymetholone is well tolerated by Fancd2-/- mice. Hematopoietic stem cell quiescence in mutant mice was enhanced by treatment with metformin alone. Metformin treatment caused a partial normalization of gene expression in the livers of mutant mice.

Abcb10 regulates murine hematopoietic stem cell potential and erythroid differentiation.

Erythropoiesis in the adult bone marrow relies on mitochondrial membrane transporters to facilitate heme and hemoglobin production. Erythrocytes in the bone marrow are produced although the differentiation of erythroid progenitor cells that originate from hematopoietic stem cells (HSCs). Whether and how mitochondria transporters potentiate HSCs and affect their differentiation toward erythroid lineage remains unclear. Here, we show that the ATP-binding cassette (ABC) transporter 10 (Abcb10), located on the inner mitochondrial membrane, is essential for HSC maintenance and erythroid-lineage differentiation. Induced deletion of Abcb10 in adult mice significantly increased erythroid progenitor cell and decreased HSC number within the bone marrow (BM). Functionally, Abcb10-deficient HSCs exhibited significant decreases in stem cell potential but with a skew toward erythroid-lineage differentiation. Mechanistically, deletion of Abcb10 rendered HSCs with excess mitochondrial iron accumulation and oxidative stress yet without alteration in mitochondrial bioenergetic function. However, impaired hematopoiesis could not be rescued through the in vivo administration of a mitochondrial iron chelator or antioxidant to Abcb10-deficient mice. Abcb10-mediated mitochondrial iron transfer is thus pivotal for the regulation of physiologic HSC potential and erythroid-lineage differentiation.

Deregulated protein homeostasis constrains fetal hematopoietic stem cell pool expansion in Fanconi anemia.

Demand-adjusted and cell type specific rates of protein synthesis represent an important safeguard for fate and function of long-term hematopoietic stem cells. Here, we identify increased protein synthesis rates in the fetal hematopoietic stem cell pool at the onset of hematopoietic failure in Fanconi Anemia, a prototypical DNA repair disorder that manifests with bone marrow failure. Mechanistically, the accumulation of misfolded proteins in Fancd2-/- fetal liver hematopoietic stem cells converges on endoplasmic reticulum stress, which in turn constrains midgestational expansion. Restoration of protein folding by the chemical chaperone tauroursodeoxycholic acid, a hydrophilic bile salt, prevents accumulation of unfolded proteins and rescues Fancd2-/- fetal liver long-term hematopoietic stem cell numbers. We find that proteostasis deregulation itself is driven by excess sterile inflammatory activity in hematopoietic and stromal cells within the fetal liver, and dampened Type I interferon signaling similarly restores fetal Fancd2-/- long-term hematopoietic stem cells to wild type-equivalent numbers. Our study reveals the origin and pathophysiological trigger that gives rise to Fanconi anemia hematopoietic stem cell pool deficits. More broadly, we show that fetal protein homeostasis serves as a physiological rheostat for hematopoietic stem cell fate and function.

Thermal gradient ring for analysis of temperature-dependent behaviors involving TRP channels in mice.

There are a lot of temperature-sensitive proteins including transient receptor potential (TRP) channels. Some TRP channels are temperature receptors having specific activation temperatures in vitro that are within the physiological temperature range. Mice deficient in specific TRP channels show abnormal thermal behaviors, but the role of TRP channels in these behaviors is not fully understood. The Thermal Gradient Ring is a new apparatus that allows mice to freely move around the ring floor and not stay in a corner. The system can analyze various factors (e.g., 'Spent time', 'Travel distance', 'Moving speed', 'Acceleration') associated with temperature-dependent behaviors of TRP-deficient mice. For example, the Ring system clearly discriminated differences in temperature-dependent phenotypes between mice with diabetic peripheral neuropathy and TRPV1-/- mice, and demonstrated the importance of TRPV3 in temperature detection in skin. Studies using the Thermal Gradient Ring system can increase understanding of the molecular basis of thermal behaviors in mice and in turn help develop strategies to affect responses to different temperature conditions in humans.

Cell death induction and protection by activation of ubiquitously expressed anion/cation channels. Part 3: the roles and properties of TRPM2 and TRPM7.

Cell volume regulation (CVR) is a prerequisite for animal cells to survive and fulfill their functions. CVR dysfunction is essentially involved in the induction of cell death. In fact, sustained normotonic cell swelling and shrinkage are associated with necrosis and apoptosis, and thus called the necrotic volume increase (NVI) and the apoptotic volume decrease (AVD), respectively. Since a number of ubiquitously expressed ion channels are involved in the CVR processes, these volume-regulatory ion channels are also implicated in the NVI and AVD events. In Part 1 and Part 2 of this series of review articles, we described the roles of swelling-activated anion channels called VSOR or VRAC and acid-activated anion channels called ASOR or PAC in CVR and cell death processes. Here, Part 3 focuses on therein roles of Ca2+-permeable non-selective TRPM2 and TRPM7 cation channels activated by stress. First, we summarize their phenotypic properties and molecular structure. Second, we describe their roles in CVR. Since cell death induction is tightly coupled to dysfunction of CVR, third, we focus on their participation in the induction of or protection against cell death under oxidative, acidotoxic, excitotoxic, and ischemic conditions. In this regard, we pay attention to the sensitivity of TRPM2 and TRPM7 to a variety of stress as well as to their capability to physicall and functionally interact with other volume-related channels and membrane enzymes. Also, we summarize a large number of reports hitherto published in which TRPM2 and TRPM7 channels are shown to be involved in cell death associated with a variety of diseases or disorders, in some cases as double-edged swords. Lastly, we attempt to describe how TRPM2 and TRPM7 are organized in the ionic mechanisms leading to cell death induction and protection.

Long-term combination therapy with Metformin and Oxymetholone in a Fanconi Anemia mouse model.

Fanconi Anemia (FA) is a disease caused by defective DNA repair which manifests as bone marrow failure, cancer predisposition, and developmental defects. Mice containing inactivating mutations in one or more genes in the FA pathway partially mimic the human disease. We previously reported that monotherapy with either metformin (MET) or oxymetholone (OXM) improved peripheral blood (PB) counts and the number and functionality of bone marrow (BM) hematopoietic stem progenitor cells (HSPCs) number in Fancd2-/- mice. To evaluate whether the combination treatment of these drugs has a synergistic effect to prevent bone marrow failure in FA, we treated cohorts of Fancd2-/- mice and wild-type controls with either MET alone, OXM alone, MET+OXM or placebo diet. Both male and female mice were treated from age 3 weeks to 18 months. The OXM treated animals showed modest improvements in blood parameters including platelet count (p=0.01) and hemoglobin levels (p<0.05). In addition, the percentage of quiescent HSC (LSK) was significantly increased (p=0.001) by long-term treatment with MET alone. However, the absolute number of progenitors, measured by LSK frequency or CFU-S, was not significantly altered by MET therapy. The combination of metformin and oxymetholone did not result in a significant synergistic effect on any parameter. Male animals on MET+OXM or MET alone were significantly leaner than controls at 18 months, regardless of genotype. Gene expression analysis of liver tissue from these animals showed that some of the expression changes caused by Fancd2 deletion were partially normalized by metformin treatment. Importantly, no adverse effects of the individual or combination therapies were observed, despite the long-term administration.

Replication stress increases mitochondrial metabolism and mitophagy in FANCD2 deficient fetal liver hematopoietic stem cells.

Fanconi Anemia (FA) is an inherited bone marrow (BM) failure disorder commonly diagnosed during school age. However, in murine models, disrupted function of FA genes leads to a much earlier decline in fetal liver hematopoietic stem cell (FL HSC) number that is associated with increased replication stress (RS). Recent reports have shown mitochondrial metabolism and clearance are essential for long-term BM HSC function. Intriguingly, impaired mitophagy has been reported in FA cells. We hypothesized that RS in FL HSC impacts mitochondrial metabolism to investigate fetal FA pathophysiology. Results show that experimentally induced RS in adult murine BM HSCs evoked a significant increase in mitochondrial metabolism and mitophagy. Reflecting the physiological RS during development in FA, increase mitochondria metabolism and mitophagy were observed in FANCD2-deficient FL HSCs, whereas BM HSCs from adult FANCD2-deficient mice exhibited a significant decrease in mitophagy. These data suggest that RS activates mitochondrial metabolism and mitophagy in HSC.

Protein kinase C-mediated phosphorylation of transient receptor potential melastatin type 2 Thr738 counteracts the effect of cytosolic Ca2+ and elevates the temperature threshold.

The transient receptor potential melastatin type 2 (TRPM2) channel is a non-selective cation channel that has high Ca2+ permeability. TRPM2 is sensitive to warm temperatures and is expressed in cells and tissues that are maintained at core body temperature. TRPM2 activity is also regulated by endogenous factors including redox signalling, cytosolic Ca2+ and adenosine diphosphate ribose. As a result of its wide expression and function at core body temperature, these endogenous factors could regulate TRPM2 activity at body temperature under physiological and pathophysiological conditions. We previously reported that cellular redox signalling can lower TRPM2 temperature thresholds, although the mechanism that regulates these thresholds is unclear. Here, we used biochemical and electrophysiological techniques to explore another regulatory mechanism for TRPM2 temperature thresholds that is mediated by TRPM2 phosphorylation. Our results show that: (1) the temperature threshold for TRPM2 activation is lowered by cytosolic Ca2+ ; (2) protein kinase C-mediated phosphorylation of TRPM2 counteracts the effect of cytosolic Ca2+ ; and (3) Thr738 in mouse TRPM2 that lies near the Ca2+ binding site in the cytosolic cleft of the transmembrane domain is a potential phosphorylation site that may be involved in phosphorylation-mediated elevation of TRPM2 thresholds. These findings provide structure-based evidence to understand how temperature thresholds of thermo-sensitive TRP channels (thermo-TRPs) are determined and regulated. KEY POINTS: The transient receptor potential melastatin type 2 (TRPM2) ion channel is temperature-sensitive and Ca2+ -permeable. Endogenous factors and pathways such as redox signalling can regulate TRPM2 activity at body temperature under physiological and pathophysiological conditions. In the present study, we report the novel finding that cytosolic Ca2+ lowers the temperature threshold for TRPM2 activation in a concentration-dependent manner. Protein kinase C-mediated phosphorylation of TRPM2 at amino acid Thr782 elevates the temperature threshold for activation by counteracting the effects of cytosolic Ca2+ . These findings provide structure-based evidence to understand how temperature thresholds of thermo-sensitive TRP channels are determined and regulated.

Single amino acids set apparent temperature thresholds for heat-evoked activation of mosquito transient receptor potential channel TRPA1.

Animals detect heat using thermosensitive transient receptor potential (TRP) channels. In insects, these include TRP ankyrin 1 (TRPA1), which in mosquitoes is crucial for noxious heat avoidance and thus is an appealing pest control target. However, the molecular basis for heat-evoked activation has not been fully elucidated, impeding both studies of the molecular evolution of temperature sensitivity and rational design of inhibitors. In TRPA1 and other thermosensitive TRPs, the N-terminal cytoplasmic ankyrin repeat (AR) domain has been suggested to participate in heat-evoked activation, but the lack of a structure containing the full AR domain has hindered our mechanistic understanding of its role. Here, we focused on elucidating the structural basis of apparent temperature threshold determination by taking advantage of two closely related mosquito TRPA1s from Aedes aegypti and Culex pipiens pallens with 86.9% protein sequence identity but a 10 °C difference in apparent temperature threshold. We identified two positions in the N-terminal cytoplasmic AR domain of these proteins, E417 (A. aegypti)/Q414 (C. pipiens) and R459 (A. aegypti)/Q456 (C. pipiens), at which a single exchange of amino acid identity was sufficient to change apparent thresholds by 5 to 7 °C. We further found that the role of these positions is conserved in TRPA1 of a third related species, Anopheles stephensi. Our results suggest a structural basis for temperature threshold determination as well as for the evolutionary adaptation of mosquito TRPA1 to the wide range of climates inhabited by mosquitoes.

TRP channels in thermosensation.

The ability to sense external temperature is assumed by somatosensory neurons, in which temperature information is converted to neural activity by afferent input to the central nervous system. Somatosensory neurons consist of various populations with specialized gene expression, including thermosensitive transient receptor potential ion channels (thermo-TRPs). Thermo-TRPs are responsible for thermal transduction at the peripheral ends of somatosensory neurons and over a wide range of temperatures. In this review, we focus on several thermo-TRPs expressed in sensory neurons: TRPV1, TRPV4, TRPM2, TRPM3, TRPM8, TRPC5, and TRPA1. TRPV3, TRPV4, and TRPC5 expressed in non-neuronal cells that are also involved in somatosensation are also discussed, whereas TRPM2 and TRPM8 are involved in thermosensation in the brain.

Thermal gradient ring reveals different temperature-dependent behaviors in mice lacking thermosensitive TRP channels.

Transient receptor potential (TRP) channels are known as temperature receptors. Each channel has an activation temperature in vitro within the physiological temperature range. Mice deficient in specific TRP channels show abnormal thermal behaviors. However, the role of TRP channels in mouse thermal behavior is not fully understood. We measured thermal behavior using a new type of thermal gradient system, where mice can freely move around the ring floor, thereby avoiding the stereotypical habit that mice have of staying in a corner, as occurs in a rectangular system. With this system, we can also analyze various factors, such as "Spent time," "Travel distance," "Moving speed," and "Acceleration," to provide more accurate information about mouse behaviors. Further analysis using this system would lead to a better understanding of the molecular basis of thermal behaviors in mice, which could help us develop ways of making humans comfortable in different temperature conditions.

[Mechanisms Underlying Thermosensation].

The ability to sense ambient temperatures is essential for human survival. Sensory nerve endings contain ion channels that are activated by temperature stimuli, which lead to cation influx and depolarization with consequent action potential generation via activation of voltage-gated Na+ channels. Thermosensitive transient receptor potential channels play a key role in cation channels. In addition to sensory neurons, the skin is known to detect ambient temperatures. Furthermore, hypothalamic neurons directly detect brain temperature.

Thermosensitive TRPV4 channels mediate temperature-dependent microglia movement.

Microglia maintain central nervous system homeostasis by monitoring changes in their environment (resting state) and by taking protective actions to equilibrate such changes (activated state). These surveillance and protective roles both require constant movement of microglia. Interestingly, induced hypothermia can reduce microglia migration caused by ischemia, suggesting that microglia movement can be modulated by temperature. Although several ion channels and transporters are known to support microglia movement, the precise molecular mechanism that regulates temperature-dependent movement of microglia remains unclear. Some members of the transient receptor potential (TRP) channel superfamily exhibit thermosensitivity and thus are strong candidates for mediation of this phenomenon. Here, we demonstrate that mouse microglia exhibit temperature-dependent movement in vitro and in vivo that is mediated by TRPV4 channels within the physiological range of body temperature. Our findings may provide a basis for future research into the potential clinical application of temperature regulation to preserve cell function via manipulation of ion channel activity.

Mitochondria Turnover and Lysosomal Function in Hematopoietic Stem Cell Metabolism.

Hematopoietic stem cells (HSCs) reside in a hypoxic microenvironment that enables glycolysis-fueled metabolism and reduces oxidative stress. Nonetheless, metabolic regulation in organelles such as the mitochondria and lysosomes as well as autophagic processes have been implicated as essential for the determination of HSC cell fate. This review encompasses the current understanding of anaerobic metabolism in HSCs as well as the emerging roles of mitochondrial metabolism and lysosomal regulation for hematopoietic homeostasis.

Prolonged maintenance of hematopoietic stem cells that escape from thrombopoietin deprivation.

Hematopoietic stem cells (HSC) rarely divide, rest in quiescence, and proliferate only upon stress hematopoiesis. The cytokine thrombopoietin (Thpo) has been perplexingly described to induce quiescence and promote self-renewal divisions in HSCs. To clarify the contradictory effect of Thpo, we conducted a detailed analysis on conventional (Thpo-/-) and liver-specific (Thpofl/fl;AlbCre+/-) Thpo-deletion models. Thpo-/- HSCs exhibited profound loss of quiescence, impaired cell cycle progression, and increased apoptosis. Thpo-/- HSCs also exhibited diminished mitochondrial mass and impaired mitochondrial bioenergetics. Abnormal HSC phenotypes in Thpo-/- mice were reversible after HSC transplantation into wild-type recipients. Moreover, Thpo-/- HSCs acquired quiescence with extended administration of a Thpo receptor agonist, romiplostim, and were prone to subsequent stem cell exhaustion during competitive bone marrow transplantation. Thpofl/fl;AlbCre+/- HSCs exhibited similar stem cell phenotypes but to a lesser degree compared with Thpo-/- HSCs. HSCs that survive Thpo deficiency acquire quiescence in a dose-dependent manner through the modification of their metabolic state.

Thermosensation involving thermo-TRPs.

The transient receptor potential (TRP) channels constitute a superfamily of large ion channels that are activated by a wide range of chemical, mechanical and thermal stimuli. TRP channels with temperature sensitivity are called thermo-TRPs. They are involved in diverse physiological functions through their detection of external environmental temperature and internal body temperature. Each thermo-TRP has its own characteristic temperature threshold for activation. As a group, they cover temperatures ranging from cold to nociceptive high temperatures. Recently, many studies have identified the functions of thermo-TRPs residing in deep organs where they are exposed to body temperature. Importantly, temperature thresholds of thermo-TRPs can be regulated by physiological factors enabling their function at relatively constant body temperature. Moreover, several thermo-TRPs are reportedly engaged in body temperature regulation. This review will summarize the current understanding of thermo-TRPs, including their roles in thermosensation and functional regulation of physiological responses at body temperature and the regulation of body temperature.

Involvement of TRPM2 and TRPM8 in temperature-dependent masking behavior.

Masking is a direct behavioral response to environmental changes and plays an important role in the temporal distribution of activity. However, the mechanisms responsible for masking remain unclear. Here we identify thermosensors and a possible neural circuit regulating temperature-dependent masking behavior in mice. Analysis of mice lacking thermosensitive transient receptor potential (TRP) channels (Trpv1/3/4 and Trpm2/8) reveals that temperature-dependent masking is impaired in Trpm2- and Trpm8-null mice. Several brain regions are activated during temperature-dependent masking, including the preoptic area (POA), known as the thermoregulatory center, the suprachiasmatic nucleus (SCN), which is the primary circadian pacemaker, the paraventricular nucleus of the thalamus (PVT), and the nucleus accumbens (NAc). The POA, SCN, PVT are interconnected, and the PVT sends dense projections to the NAc, a key brain region involved in wheel-running activity. Partial chemical lesion of the PVT attenuates masking, suggesting the involvement of the PVT in temperature-dependent masking behavior.

AAV-Mediated Gene Delivery to Taste Cells of the Tongue.

Taste sensation is initiated in sensory cells within the taste buds (taste cells), in which the cooperation of many signaling molecules leads to the coding and transmission of information on the quality and intensity of taste to the afferent gustatory nerves. Here, we describe our method for inducing foreign gene expression in taste cells of fungiform papillae in a living mouse using a recombinant adeno-associated virus (AAV) vector, enabling us to study and control the function of a gene product in vivo. Among the serotypes tested to date, only AAV-DJ, a synthetic serotype, can transduce taste cells in vivo. We also describe how to validate intragemmal foreign gene expression in fungiform taste buds using an immunohistochemical approach.