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Indiana University-Purdue University Indianapolis (IUPUI) / Hematopoietic stem cell (HSC) transplantation is a lifesaving therapy for a number of hematological disorders. However, to be effective, transplanted HSCs must efficiently “home” to supportive niches within the bone marrow. Limited HSC number and poor function are complications of transplant in some circumstances, and can lead to delayed engraftment and immune reconstitution, or in some cases, bone marrow failure. Enhancing HSC homing is a strategy to improve stem cell transplantation efficiency. We have previously shown that ex vivo treatment of mouse or human HSCs with 16-16 dimethyl PGE2 (dmPGE2) increases their bone marrow homing efficiency and engraftment, resulting in part from upregulation of surface CXCR4 expression. We now show that pulse-treatment of mouse or human HSPCs with dmPGE2 stabilizes HIF1α in HSPCs, and that similar treatment with the hypoxia mimetic DMOG produces analogous effects to dmPGE2 on HSPC CXCR4 expression and homing. This suggests that HIF1α is responsible for PGE2’s enhancing effects on HSPCs. Pharmacological inhibition of HIF1α stabilization in vitro with Sodium Nitroprusside (SNP), confirms the requirement of HIF1α for dmPGE2-enhanced migration and CXCR4 upregulation. Additionally, we confirm the requirement for HIF1α in dmPGE2-enhanced in vivo homing using a conditional knockout mouse model of HIF1α gene deletion. Finally, we validate that the hypoxia response element located 1.3kb from the transcriptional start site within the CXCR4 promoter is required for enhanced CXCR4 expression after PGE2 treatment. Interestingly, we also observe an increase in the small GTPase Rac1 after dmPGE2 treatment, as well as a defect in PGE2-enhanced migration and CXCR4 expression in Rac1 knockout HSPCs. Using state-of-the-art imaging technology we, confirm an increase in Rac1 and CXCR4 colocalization after dmPGE2 treatment that likely explains enhanced sensitivity of PGE2-treated HSPCs to SDF-1. Taken together, these results define a precise mechanism through which ex vivo pulse treatment of HSPC with dmPGE2 enhances HSPC function through alterations in cell motility and homing, and describe a role for hypoxia and HIF1α in enhancement of hematopoietic transplantation.

Indiana University-Purdue University Indianapolis (IUPUI) / The hematopoietic system is highly proliferative, making hematopoietic stem and progenitor cells (HSPC) sensitive to radiation damage. Total body irradiation and chemotherapy, as well as the risk of radiation accident, create a need for countermeasures that promote recovery of hematopoiesis. Substantive damage to the bone marrow from radiation exposure results in the hematopoietic syndrome of the acute radiation syndrome (HS-ARS), which includes life-threatening neutropenia, lymphocytopenia, thrombocytopenia, and possible death due to infection and/or hemorrhage. Given adequate time to recover, expand, and appropriately differentiate, bone marrow HSPC may overcome HS-ARS and restore homeostasis of the hematopoietic system. Prostaglandin E2 (PGE2) is known to have pleiotropic effects on hematopoiesis, inhibiting apoptosis and promoting self-renewal of hematopoietic stem cells (HSC), while inhibiting hematopoietic progenitor cell (HPC) proliferation. We assessed the radiomitigation potential of modulating PGE2 signaling in a mouse model of HS-ARS. Treatment with the PGE2 analog 16,16 dimethyl PGE2 (dmPGE2) at 24 hours post-irradiation resulted in increased survival of irradiated mice compared to vehicle control, with greater recovery in HPC number and colony-forming potential measured at 30 days post-irradiation. In a sublethal mouse model of irradiation, dmPGE2-treatment at 24 hours post-irradiation is associated with enhanced recovery of HSPC populations compared to vehicle-treated mice. Furthermore, dmPGE2-treatment may also act to promote recovery of the HSC niche through enhancement of osteoblast-supporting megakaryocyte (MK) migration to the endosteal surface of bone. A 2-fold increase in MKs within 40 um of the endosteum of cortical bone was seen at 48 hours post-irradiation in mice treated with dmPGE2 compared to mice treated with vehicle control. Treatment with the non-steroidal anti-inflammatory drug (NSAID) meloxicam abrogated this effect, suggesting an important role for PGE2 signaling in MK migration. In vitro assays support this data, showing that treatment with dmPGE2 increases MK expression of the chemokine receptor CXCR4 and enhances migration to its ligand SDF-1, which is produced by osteoblasts. Our results demonstrate the ability of dmPGE2 to act as an effective radiomitigative agent, promoting recovery of HSPC number and enhancing migration of MKs to the endosteum where they play a valuable role in niche restoration.

Indiana University-Purdue University Indianapolis (IUPUI) / Chronic inflammatory disorders are characterized by an increase in excitability of small diameter sensory neurons located in dorsal root ganglia (DRGs). This sensitization of neurons is a mechanism for chronic inflammatory pain and available therapies have poor efficacy and severe adverse effects when used chronically. Prostaglandin E₂ (PGE₂) is an inflammatory mediator that plays an important role in sensitization by activating G-protein coupled receptors (GPCRs) known as E-series prostaglandin receptors (EPs) coupled to the protein kinase A (PKA) pathway. EPs are known to downregulate upon prolonged exposure to PGE₂ or in chronic inflammation, however, sensitization persists and the mechanism for this is unknown. I hypothesized that persistence of PGE₂-induced hypersensitivity is associated with a switch in signaling caused by prolonged exposure to PGE₂ or the neurotrophin nerve growth factor (NGF), also a crucial inflammatory mediator. DRG cultures grown in the presence or absence of either PGE₂ or NGF were used to study whether re-exposure to the eicosanoid is able to cause sensitization and activate PKA. When cultures were grown in the presence of NGF, PGE₂-induced sensitization was not attenuated by inhibitors of PKA. Activation of PKA by PGE₂ was similar in DRG cultures grown in the presence or absence of NGF when phosphatase inhibitors were added to the lysis and assay buffers, but significantly less in cultures grown in the presence of NGF when phosphatase inhibitors were not added. In DRG cultures exposed to PGE₂ for 12 hours-5 days, sensitization after re-exposure to PGE₂ is maintained and resistant to PKA inhibition. Prolonged exposure to the eicosanoid caused complete loss of PKA activation after PGE₂ re-exposure. This desensitization was homologous, time dependent, reversible, and insurmountable by a higher concentration of PGE₂. Desensitization was attenuated by reduction of expression of G-protein receptor kinase 2 and was not mediated by PKA or protein kinase C. The presented work provides evidence for persistence of sensitization by PGE₂ as well as switch from the signaling pathway mediating this sensitization after long-term exposure to NFG or PGE₂.

Heterocyclic amino acids can serve as tools in structure-activity relationship (SAR) studies. They can also act as peptide secondary structure initiators. Incorporation of heterocyclic amino acid units into peptides can limit flexibility, improve receptor binding affinity, enhance selectivity, and augment potency. Among heterocyclic amino acids, substituted prolines, alpha-amino-delta-lactams, and indolizidine-2-one amino acid derivatives have shown significant utility; however, their syntheses remain challenging. Various unsaturated amino acids have previously been synthesized using copper catalyzed SN2’ reactions of the zincate from beta-iodoalanine derivatives onto allylic halides, such as (Z)-1,4-dichlorobut-2-ene, 3-chloro-2-(chloromethyl)prop-1-ene, and (E)-1,3-dichloroprop-1-ene. Employing the resulting olefins as building block, a variety of substituted heterocyclic amino acids have now been prepared by routes featuring halide displacements and olefin oxidation. For example, 2-N-(Boc)amino-4-(chloromethyl)hexenoate was employed in halide displacements to synthesize 4-vinylproline (4-Vyp), 4-vinylornithine (4-Von) and gamma-vinyl-alpha-amino-delta-lactams. Moreover, unsaturated diamino azelates were employed to synthesize 6-hydroxymethyl and 5- and 7-hydroxy indolizidine-2-one amino acid (I2aa) derivatives by routes featuring olefin oxidation. X-ray studies have demonstrated that 6-hydroxymethyl and 7-hydroxy I2aa residues can mimic the backbone geometry of the central residues of ideal type II’ beta-turns. Replacement of the I2aa residue of a potent prostaglandin-F2α (PGF2alpha) receptor (FP) modulator by the 5-, 6- and 7-substituted counterparts was performed to study the influences of substituents and backbone geometry on inhibitory effects on myometrial contractility in mouse models. A promising gateway for preparing different heterocyclic amino acids has been opened by employing unsaturated amino acid building blocks. In these routes, the olefin has served as a means for adding functional group diversity onto the ring systems. Access to a variety of substituted ring systems has expanded the toolbox for studying peptide structures using substituted heterocyclic amino acids. / Les acides aminés hétérocycliques peuvent servir d'outils dans les études des relations structure-activité (RSA). Ils peuvent également jouer le rôle d'initiateurs de structure secondaire peptidique. L'incorporation d'unités d'acides aminés hétérocycliques dans des peptides peut limiter la flexibilité, améliorer l'affinité de liaison au récepteur, améliorer la sélectivité et augmenter l’activité. Parmi les acides aminés hétérocycliques, les prolines substituées, les alpha-amino-delta-lactames et les dérivés d'acides aminés indolizidine-2-one ont montré une utilité significative. Cependant, leurs synthèses restent difficiles. Divers acides aminés insaturés ont déjà été synthétisés à l'aide de réactions SN2' catalysées par le cuivre de zincate à partir de dérivés de beta-iodoalanine sur des halogénures allyliques, tels que le (Z)-1,4-dichlorobut-2-ène, le 3-chloro-2-(chlorométhyl)prop-1-ène et (E)-1,3-dichloroprop-1-ène. En utilisant les oléfines résultantes comme synthons, une variété d'acides aminés hétérocycliques substitués ont maintenant été préparés via les déplacements d'halogénures et oxydations d’oléfines. Par exemple, le 2-N-(Boc)amino-4-(chlorométhyl)hexénoate a été utilisé dans les déplacements d'halogénures pour synthétiser la 4-vinylproline (4-Vyp), la 4-vinylornithine (4-Von) et le gamma-vinyl-alpha-amino-delta-lactamines. De plus, des azélates de diamines insaturées ont été utilisés pour synthétiser des dérivés d'acides aminés 6-hydroxyméthyle et 5- et 7-hydroxy indolizidine-2-one (I2aa) par des voies comportant une oxydation des oléfines. Des études par rayons X ont démontré que les résidus 6-hydroxyméthyle et 7-hydroxy I2aa peuvent imiter la géométrie du squelette des résidus centraux des tours beta de type II idéal. Le remplacement du résidu I2aa d'un puissant modulateur de la prostaglandine-F2α (récepteur PGF2alpha (FP) par les homologues substitués en position 5, 6 et 7 a été effectué pour étudier les influences des substituants et la géométrie du squelette sur les effets inhibiteurs sur la contractilité myométriale chez modèles de souris. Une passerelle prometteuse pour la préparation de différents acides aminés hétérocycliques a été ouverte en utilisant des acides aminés insaturés comme synthons de départ. Dans ces voies, l'oléfine a servi comme moyen d’élargir la diversité des groupes fonctionnels sur les systèmes cycliques. L'accès à une variété de systèmes cycliques substitués a élargi la boîte à outils pour étudier les structures peptidiques à l'aide d'acides aminés hétérocycliques substitués.

Balding causes widespread psychological distress but is poorly controlled. The commonest treatment, minoxidil, was originally an antihypertensive drug that promoted unwanted hair. We hypothesized that another serendipitous discovery, increased eyelash growth side-effects of prostamide F(2alpha)-related eyedrops for glaucoma, may be relevant for scalp alopecias. Eyelash hairs and follicles are highly specialized and remain unaffected by androgens that inhibit scalp follicles and stimulate many others. Therefore, we investigated whether non-eyelash follicles could respond to bimatoprost, a prostamide F(2alpha) analog recently licensed for eyelash hypotrichosis. Bimatoprost, at pharmacologically selective concentrations, increased hair synthesis in scalp follicle organ culture and advanced mouse pelage hair regrowth in vivo compared to vehicle alone. A prostamide receptor antagonist blocked isolated follicle growth, confirming a direct, receptor-mediated mechanism within follicles; RT-PCR analysis identified 3 relevant receptor genes in scalp follicles in vivo. Receptors were located in the key follicle regulator, the dermal papilla, by analyzing individual follicular structures and immunohistochemistry. Thus, bimatoprost stimulates human scalp follicles in culture and rodent pelage follicles in vivo, mirroring eyelash behavior, and scalp follicles contain bimatoprost-sensitive prostamide receptors in vivo. This highlights a new follicular signaling system and confirms that bimatoprost offers a novel, low-risk therapeutic approach for scalp alopecias.

Various molecular pathways in the brain shape our understanding of good and bad, as well as our motivation to seek and avoid such stimuli. This work evolves around how systemic inflammation causes aversion; and why general unpleasant states such as sickness, stress, pain and nausea are encoded by our brain as undesirable; and contrary to these questions, how drugs of abuse can subjugate the motivational neurocircuitry of the brain. A common feature of these various disease states is involvement of the motivational neurocircuitry - from mesolimbic to striatonigral pathways. Having an intact motivational system is what helps us evade negative outcomes and approach natural positive reinforcers, which is essential for our survival. During disease-states the motivational neurocircuitry may be overthrown by the molecular mechanisms that originally were meant to aid us. In study I, to investigate how inflammation is perceived as aversive, we used a behavioral test based on Pavlovian place conditioning with the aversive inflammatory stimulus E. coli lipopolysaccharide (LPS). Using a combination of cell-type specific gene deletions, pharmacology, and chemogenetics, we uncovered that systemic inflammation triggered aversion by MyD88-dependent activation of the brain endothelium followed by COX1-mediated cerebral prostaglandin E2 (PGE2) synthesis. Moreover, we showed that inflammation-induced PGE2 targeted EP1 receptors on striatal dopamine D1 receptor–expressing neurons and that this signaling sequence induced aversion through GABA-mediated inhibition of dopaminergic cells. Finally, inflammation-induced aversion was not an indirect consequence of fever or anorexia but constituted an independent inflammatory symptom triggered by a unique molecular mechanism. Collectively, these findings demonstrate that PGE2-mediated modulation of the dopaminergic circuitry is a key mechanism underlying inflammation-induced aversion. In study II, we investigate the role of peripheral IFN-γ in LPS induced conditioned place aversion by employing a strategy based on global and cell-type specific gene deletions, combined with measures of gene-expression. LPS induced IFN-ɣ expression in the blood, and deletion of IFN-ɣ or its receptor prevented conditioned place aversion (CPA) to LPS. LPS increased the expression of chemokine Cxcl10 in the striatum of normal mice. This induction was absent in mice lacking IFN-ɣ receptors or Myd88 in blood brain barrier endothelial cells. Furthermore, inflammation-induced aversion was blocked in mice lacking Cxcl10 or its receptor Cxcr3. Finally, mice with a selective deletion of the IFN-ɣ receptor in brain endothelial cells did not develop inflammation-induced aversion. Collectively, these findings demonstrate that circulating IFN-ɣ binding to receptors on brain endothelial cells which induces Cxcl10, is a central link in the signaling chain eliciting inflammation-induced aversion. In study III, we explored the role of melanocortin 4 receptors (MC4Rs) in aversive processing using genetically modified mice in CPA to various stimuli. In normal mice, robust aversions were induced by systemic inflammation, nausea, pain and kappa opioid receptor-induced dysphoria. In sharp contrast, mice lacking MC4Rs displayed preference towards most of the aversive stimuli, but were indifferent to pain. The unusual flip from aversion to reward in mice lacking MC4Rs was dopamine-dependent and associated with a change from decreased to increased activity of the dopamine system. The responses to aversive stimuli were normalized when MC4Rs were re-expressed on dopamine D1 receptor-expressing cells or in the striatum of mice otherwise lacking MC4Rs. Furthermore, activation of arcuate nucleus proopiomelanocortin neurons projecting to the ventral striatum increased the activity of striatal neurons in a MC4R-dependent manner and elicited aversion. Our findings demonstrate that melanocortin signaling through striatal MC4Rs is critical for assigning negative motivational valence to harmful stimuli. The neurotransmitter acetylcholine has been implied in reward learning and drug addiction. However, the role of cholinergic receptor subtypes in such processes remains elusive. In study IV we investigated the function of muscarinic M4Rs on dopamine D1R expressing neurons and acetylcholinergic neurons, using transgenic mice in various reward-enforced behaviors and in a “waiting”-impulsivity test. Mice lacking M4-receptors from D1-receptor expressing neurons exhibited an escalated reward seeking phenotype towards cocaine and natural reward, in Pavlovian conditioning and an operant self-administration task, respectively. In addition, the M4-D1RCre mice showed impaired waiting impulsivity in the 5-choice-serial-reaction-time-task. On the contrary, mice without M4Rs in acetylcholinergic neurons were unable to learn positive reinforcement to natural reward and cocaine, in an operant runway paradigm and in Pavlovian conditioning.  Immediate early gene expression mirrored the behavioral findings arising from M4R-D1R knockout, as cocaine induced cFos and FosB was significantly increased in the forebrain of M4-D1RCre mice, whereas it remained normal in the M4R-ChatCre mice. Our study illustrates that muscarinic M4Rs on specific neural populations, either cholinergic or D1R-expressing, are pivotal for learning processes related to both natural reward and drugs of abuse, with opposing functionality.