Purmorphamine Research Articles

Modulating sonic hedgehog (SHH) pathway to create a rapid CNS-TB model: Facilitating drug discovery

Tuberculous meningitis, a severe complication of Mycobacterium tuberculosis (M. tb) infection, involves the dissemination of bacilli in the brain. This study explored the role of the sonic hedgehog (SHH) signaling pathway in regulating blood-brain barrier (BBB) integrity, M. tb invasion into the central nervous system (CNS), and disease progression of Central Nervous System Tuberculosis (CNS-TB) in a Balb/c mouse model. The modulation of the SHH pathway using agonist Purmorphamine (PUR) and antagonist Cyclopamine (CYC) revealed that CYC treatment led to a rapid and extensive invasion of M. tb in the brain, with bacterial loads increasing by 99 % compared to the untreated-infected group. In contrast, PUR reduced M. tb loads by 50 % and delayed disease progression. Histopathological analysis showed that CYC exacerbated inflammation and immune cell infiltration, while PUR mitigated these responses. Immunohistochemistry demonstrated that CYC caused severe BBB breakdown and reactive gliosis, while PUR partially attenuated this response. Further analysis revealed that CYC upregulated Matrix Metalloproteinase-9 (MMP-9) secretion, a key contributor to BBB disruption. These findings highlight the critical role of the SHH pathway in maintaining BBB integrity and regulating the immunopathological response during CNS-TB, opening up future scope for drug discovery. This Cyclopamine-induced model of rapid M. tb invasion and chronic inflammation provides a new tool for studying CNS-TB pathogenesis and evaluating potential therapeutic interventions targeting the SHH signaling axis. Significance statementUnderstanding how tuberculosis (TB) infection can spread to the brain is crucial, as this “central nervous system TB” (CNS-TB) is a serious and potentially life-threatening health complication. However, studying CNS-TB in humans is very difficult. Animal models are needed to better understand how TB gets into the brain and the resulting damage. This study in mice showed that blocking a signaling pathway called Sonic Hedgehog (SHH) allowed TB to rapidly spread to the brain, damaging the blood-brain barrier and causing severe inflammation. In contrast, activating the SHH pathway helped protect the brain from TB invasion. These findings provide important insights that could lead to new ways to prevent or treat this dangerous form of TB.

Resveratrol Induces Apoptosis, Suppresses Migration, and Invasion of Cervical Cancer Cells by Inhibiting the Hedgehog Signaling Pathway.

To investigate the effect and mechanism of resveratrol on the biological behavior of cervical cancer cells (HeLa cells), the apoptosis, migration, and invasion of HeLa cells were detected by flow cytometry, wound healing, and transwell assays. The expression levels of Hedgehog signal pathway proteins (smoothened (SMO), zinc finger transcription factors (Gli1), and sonic hedgehog homolog (Shh)) were detected by quantitative real-time PCR (qPCR) and western blotting. Compared with that control group, resveratrol (RES) significantly induced apoptosis, inhibited the migration and invasion of the HeLa cells. The expression of SMO, Gli1, and Shh were downregulated in the HeLa cells treated with RES. The Hedgehog agonist purmorphamine (PUR) reversed the RES-induced increase of apoptosis and reduction of migration and invasion in the HeLa cells. In conclusion, RES induced the apoptosis and suppressed the migration and invasion of HeLa cells by inhibiting Hedgehog signal pathway.

Magnesium Oxide/Poly(l‐lactide‐co‐ε‐caprolactone) Scaffolds Loaded with Neural Morphogens Promote Spinal Cord Repair through Targeting the Calcium Influx and Neuronal Differentiation of Neural Stem Cells (Adv. Healthcare Mater. 15/2022)

Spinal Cord Injury In article number 2200386, by Bin Li, Saijilafu, and co-workers, a novel purmorphamine (PUR)/retinoic acid (RA)-loaded MgO/PLCL scaffold for repairing spinal cord injury is developed. The magnisum ions released from MgO nanoparticles protect the neurons and the PUR/RA promotes the recruitment and neuronal differentiation of endogenous neural stem cells.

Phenotypic, metabolic, and biogenesis properties of human stem cell-derived cerebellar spheroids

Human cerebellum consists of high density and complexity of neurons. Thus, it is challenging to differentiate cerebellar-like organoids with similar cellular markers and function to the human brain. Our previous study showed that the combination of retinoic acid (RA), Wingless/integrated (Wnt) activator, and Sonic Hedgehog (SHH) activator promotes cerebellar differentiation from human induced pluripotent stem cells (hiPSCs). This study examined phenotypic, metabolic, and biogenesis in early cerebellar development. Cerebellum spheroids were differentiated from human iPSK3 cells. During day 7–14, RA and Wnt activator CHIR99021 were used and SHH activator purmorphamine (PMR) was added later to promote ventralization. Gene expression for early cerebellar layer markers, metabolism, and extracellular vesicle (EV) biogenesis were characterized. Zinc-induced neurotoxicity was investigated as a proof-of-concept of neurotoxicity study. Flow cytometry results showed that there was no significant difference in NEPH3, PTF1A, OLIG2, and MATH1 protein expression between RCP (RA-CHIR-PMR) versus the control condition. However, the expression of cerebellar genes for the molecular layer (BHLE22), the granule cell layer (GABRB2, PAX6, TMEM266, KCNIP4), the Bergmann glial cells (QK1, DAO), and the Purkinje cell layer (ARHGEF33, KIT, MX1, MYH10, PPP1R17, SCGN) was significantly higher in the RCP condition than the control. The shift in metabolic pathways toward glycolysis was observed for RCP condition. The EV biogenesis marker expression was retained. Mild zinc-induced neurotoxicity may exist when zinc exposure exceeds 1.0 µM. RCP treatment can promote specific cerebellar-like differentiation from hiPSCs indicated by gene expression of early cerebellar markers and regionally enriched genes. The higher cerebellar marker expression is accompanied by the elevated glycolysis with the retained EV biogenesis. This study should advance the understanding of biomarkers during early cerebellar development for cerebellum organoid engineering and neurotoxicity study.

Smo-Shh signaling activator purmorphamine ameliorates neurobehavioral, molecular, and morphological alterations in an intracerebroventricular propionic acid-induced experimental model of autism.

Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disease characterized by cognitive and sensorimotor impairment. Numerous research findings have consistently shown that alteration of Smo-Shh (smoothened-sonic hedgehog) signaling during the developmental process plays a significant role in ASD and triggers neuronal changes by promoting neuroinflammation and apoptotic markers. Purmorphamine (PUR), a small purine-derived agonist of the Smo-Shh pathway, shows resistance to hippocampal neuronal cell oxidation and decreases neuronal cell death. The goal of this study was to investigate the neuroprotective potential of PUR in brain intoxication induced by intracerebroventricular-propionic acid (ICV-PPA) in rats, with a focus on its effect on Smo-Shh regulation in the brain of rats. In addition, we analyze the impact of PUR on myelin basic protein (MBP) and apoptotic markers such as Caspase-3, Bax (pro-apoptotic), and Bcl-2 (anti-apoptotic) in rat brain homogenates. Chronic ICV-PPA infusion was administered consecutively for 11 days to induce autism in rats. In order to investigate behavioral alterations, rats were tested for spatial learning in the Morris Water Maze (MWM), locomotive alterations using actophotometer, and beam crossing task, while Forced Swimming Test (FST) for depressive behavior. PUR treatment with 5 mg/kg and 10 mg/kg (i.p.) was administered from day 12 to 44. Besides cellular, molecular and neuroinflammatory analyses, neurotransmitter levels and oxidative markers have also been studied in brain homogenates. The results of this study have shown that PUR increases the level of Smo-Shh and restores the neurochemical levels, and potentially prevents morphological changes, including demyelination.

Cerebellar Differentiation from Human Stem Cells Through Retinoid, Wnt, and Sonic Hedgehog Pathways.

Differentiating cerebellar organoids can be challenging due to complex cell organization and structure in the cerebellum. Different approaches were investigated to recapitulate differentiation process of the cerebellum from human-induced pluripotent stem cells (hiPSCs) without high efficiency. This study was carried out to test the hypothesis that the combination of different signaling factors including retinoic acid (RA), Wnt activator, and sonic hedgehog (SHH) activator promotes the cerebellar differentiation of hiPSCs. Wnt, RA, and SHH pathways were activated by CHIR99021 (CHIR), RA, and purmorphamine (PMR), respectively. Different combinations of the morphogens (RA/CHIR, RA/PMR, CHIR/PMR, and RA/CHIR/PMR) were utilized, and the spheroids (day 35) were characterized for the markers of three cerebellum layers (the molecular layer, the Purkinje cell layer, and the granule cell layer). Of all the combinations tested, RA/CHIR/PMR promoted both the Purkinje cell layer and the granule cell layer differentiation. The cells also exhibited electrophysiological characteristics using whole-cell patch clamp recording, especially demonstrating Purkinje cell electrophysiology. This study should advance the understanding of different signaling pathways during cerebellar development to engineer cerebellum organoids for drug screening and disease modeling. Impact statement This study investigated the synergistic effects of retinoic acid, Wnt activator, and sonic hedgehog activator on cerebellar patterning of human-induced pluripotent stem cell (hiPSC) spheroids and organoids. The results indicate that the combination promotes the differentiation of the Purkinje cell layer and the granule cell layer. The cells also exhibit electrophysiological characteristics using whole-cell patch clamp recording, especially demonstrating Purkinje cell electrophysiology. The findings are significant for understanding the biochemical signaling of three-dimensional microenvironment on neural patterning of hiPSCs for applications in organoid engineering, disease modeling, and drug screening.

Purmorphamine Attenuates Neuro-Inflammation and Synaptic Impairments After Hypoxic-Ischemic Injury in Neonatal Mice via Shh Signaling.

Purmorphamine (PUR), an agonist of the Smoothened (Smo) receptor, has been shown to function as a neuroprotectant in acute experimental ischemic stroke. Its role in hypoxic-ischemic (HI) brain injury in neonatal mice remains unknown. Here we show that PUR attenuated acute brain injury, with a decrease in Bax/Bcl-2 ratio as well as inhibition of caspase-3 activation. These beneficial effects of PUR were associated with suppressing neuro-inflammation and oxidative stress. PUR exerted long-term protective effects upon tissue loss and improved neurobehavioral outcomes as determined at 14 and 28 days post-HI insult. Moreover, PUR increased synaptophysin (Syn) and postsynaptic density (PSD) protein 95 expression in HI-treated mice and attenuated synaptic loss. PUR upregulated the expression of Shh pathway mediators, while suppression of the Shh signaling pathway with cyclopamine (Cyc) reversed these beneficial effects of PUR on HI insult. Our study suggests a therapeutic potential for short-term PUR administration in HI-induced injury as a result of its capacity to exert multiple protective actions upon acute brain injury, long-term memory deficits, and impaired synapses. Moreover, we provide evidence indicating that one of the mechanisms underlying these beneficial effects of PUR involves activation of the Shh signaling pathway.

Genetic and pharmacological activation of Hedgehog signaling inhibits osteoclastogenesis and attenuates titanium particle-induced osteolysis partly through suppressing the JNK/c-Fos-NFATc1 cascade.

Rationale: Wear particle-induced periprosthetic osteolysis (PPO) is a common long-term complication of total joint arthroplasty, and represents the major cause of aseptic loosening and subsequent implant failure. Previous studies have identified the central role of osteoclast-mediated bone resorption in the pathogenesis of PPO. Thus, therapeutic approaches of inhibiting osteoclast formation and activity are considered to be of great potential to prevent and treat this osteolytic disease. Hedgehog (Hh) signaling has been shown to play an important role in promoting osteoblast differentiation and bone formation. While Hh signaling is also implicated in regulating osteoclastogenesis, whether it can directly inhibit osteoclast differentiation and bone resorption remains controversial. Moreover, its potential therapeutic effects on PPO have never been assessed. In this study, we explored the cell-autonomous role of Hh signaling in regulating osteoclastogenesis and its therapeutic potential in preventing wear particle-induced osteolysis.Methods: Hh signaling was activated in macrophages by genetically ablating Sufu in these cells using LysM-Cre or by treating them with purmorphamine (PM), a pharmacological activator of Smoothened (Smo). In vitro cell-autonomous effects of Hh pathway activation on RANKL-induced osteoclast differentiation and activity were evaluated by TRAP staining, phalloidin staining, qPCR analyses, and bone resorption assays. In vivo evaluation of its therapeutic efficacy against PPO was performed in a murine calvarial model of titanium particle-induced osteolysis by μCT and histological analyses. Mechanistic details were explored in RANKL-treated macrophages through Western blot analyses.Results: We found that Sufu deletion or PM treatment potently activated Hh signaling in macrophages, and strongly inhibited RANKL-induced TRAP+ osteoclast production, F-actin ring formation, osteoclast-specific gene expression, and osteoclast activity in vitro. Furthermore, we found that Sufu deletion or PM administration significantly attenuated titanium particle-induced osteoclast formation and bone loss in vivo. Our mechanistic study revealed that activation of Hh signaling suppressed RANKL-induced activation of JNK pathway and downregulated protein levels of two key osteoclastic transcriptional factors, c-Fos and its downstream target NFATc1.Conclusions: Both genetic and pharmacological activation of Hh signaling can cell-autonomously inhibit RANKL-induced osteoclast differentiation and activity in vitro and protect against titanium particle-induced osteolysis in vivo. Mechanistically, Hh signaling hinders osteoclastogenesis partly through suppressing the JNK/c-Fos-NFATc1 cascade. Thus, Hh signaling may serve as a promising therapeutic target for the prevention and treatment of PPO and other osteolytic diseases.

Neuroprotective effects of a Smoothened receptor agonist against postoperative cognitive dysfunction by promoting autophagy in the dentate gyrus of aged rats

ABSTRACTObjectives: To investigate the effect of purmorphamine (PUR), a Shh co-receptor Smoothened (Smo) agonist, on postoperative cognitive dysfunction (POCD) rat models.Methods: Eighteen-month-old male Sprague–Dawley rats were subjected to intramedullary fixation of a tibial fracture with 7% chloral hydrate anesthesia to mimic human clinical surgery. PUR was administered via an intraperitoneal injection at a dose of 15mg/kg/day for 3 consecutive days at 6 h after surgery. The aged rats were sacrificed after performing a Morris water maze test 1, 3, and 7 days postoperatively to evaluate the expression of related proteins at the appointed time.Results: Compared to the POCD + vehicle group and sham + PUR group, the POCD + PUR group restored neurological deficit (P = 0.01). PUR administration induced upregulation of Shh expression on postoperative day 1 (P = 0.02), which continued on the third day (P = 0.008) but dropped by the 7th day (P = 0.03). Immunofluorescent analysis, similar to western blot analysis, showed a significant increase in the autophagy-marker LC3 (P = 0.006) as well as p62 degradation (P = 0.000) in the dentate gyrus of the aged rats (P = 0.000) after PUR treatment. Importantly, LC3 was mainly found in the presynaptic and postsynaptic membranes of the hippocampus.Conclusions: These results indicate a link between Shh and autophagy in the rat model of POCD, providing new insights into Shh signaling pathway-mediated mechanisms of neuroprotection and cognitive repair after POCD. It also provides a potential entry point for the development of clinical drugs.

Myocardial Preconditioning Potential of Hedgehog Activator Purmorphamine (Smoothened Receptor Agonist) against Ischemia-Reperfusion in Deoxycortisone Acetate Salt-Induced Hypertensive Rat Hearts

Aim: To investigate the myocardial preconditioning potential of hedgehog activator smoothened (SMO) receptor agonist purmorphamine (PUR) against ischemia-reperfusion (I/R) injury in deoxycortisone acetate (DOCA) salt-induced hypertensive rat heart. Methods: Hypertension in rats was produced by surgical removal of the kidney and DOCA salt administration for 6 weeks. Isolated rat heart was subjected to 30 min ischemia followed by 120 min of I/R. The heart was subjected to pharmacological preconditioning with the hedgehog (Hh) activator PUR (1 μM), PUR (2 μM), and atractyloside (4 μM), an mPTP opener, in the last episode of reperfusion before I/R. Elevated blood pressure was evaluated through tail-cuff method. Myocardial infarction was assessed in terms of the increase in lactate dehydrogenase, creatinine kinase-muscle/brain, and infarct size through triphenyltetrazolium chloride staining. Further, there were a decrease in the release of nitrite and coronary flow rate. Immunohistochemistry was performed for the assessment of tumor necrosis factor-α level in cardiac tissue. Results: Pharmacological preconditioning with PUR significantly attenuated I/R-induced myocardial infarction. However, atractyloside limits the ameliorative preconditioning potential of PUR and confirmed the role of Hh pathway in ischemic preconditioning. Conclusion: It may be concluded that the Hh activator PUR (SMO receptor agonist) prevents I/R injury in DOCA salt-induced hypertensive rat heart.

Activation of the Gi protein-RHOA axis by non-canonical Hedgehog signaling is independent of primary cilia.

Primary cilia are solitary organelles that emanate from the plasma membrane during growth arrest in almost all mammalian cells. The canonical Hedgehog (HH) pathway requires trafficking of the G protein-coupled receptor SMOOTHENED (SMO) and the GLI transcription factors to the primary cilium upon binding of a HH ligand to PATCHED1. However, it is unknown if activation of the small GTPase RHOA by SMO coupling to heterotrimeric Gi proteins, a form of non-canonical HH signaling, requires localization of SMO in the primary cilium. In this study, we compared RHOA and Gi protein stimulation by activation of SMO or sphingosine 1-phosphate receptor (S1P) receptors in WT and KIF3A-deficient mouse embryonic fibroblasts that lack primary cilia. We found that activation of SMO in response to Sonic HH (SHH) or purmorphamine (PUR), a small molecule agonist of SMO, stimulates Gi proteins and RHOA independently of the presence of primary cilia, similar to the effects of S1P. However, while S1P induced a fast activation of AKT that is sensitive to the Gi inhibitor pertussis toxin, HH pathway activators did not significantly activate AKT, suggesting that RHOA activation is not downstream of AKT. Our findings demonstrate that early events in some forms of non-canonical HH signaling occur in extraciliary membranes, which might be particularly relevant for actively-cycling cells, for some cancers characterized by loss of primary cilia, and in ciliopathies.

The research of differentiation of skin-derived precursors into motor neurons induced by retinoic acid combined with Purmorphamine

Objective To explore the differentiation of skin-derived precursors (SKPs) into motor neurons (MNs) induced by retinoic acid (RA) combined with Purmorphamine (PM), and provide the evidence for motor neuron transplantation in the treatment of denervated muscle atrophy. Methods SKPs were isolated from the skin of neonatal rats, and cultured and proliferated in vitro. The expression of SKPs markers Nestin and Fibronectin was detected by immunocytochemistry. RA and PM were used as inducers to induce the differentiation of the third generation SKPs to MNs, and the growth factors such as GDNF and BDNF were added to promote the axon elongation and MNs maturity. The expression of MNs marker HB9 and choline acetyltransferase (ChAT) was detected by immunocytochemistry to identify whether the differentiated cells were MNs or not. The positive rate of ChAT and HB9 was calculated. Results The differentiated cells from SKPs induced by RA and PM had the morphology of MNs, and express the MNs specific markers HB9 and ChAT. The positive rate was (72.61±2.25)% for ChAT and (75.16±3.62)% for HB9. Conclusion The use of RA and PM can induce SKPs to differentiate into MNs. Key words: Motor neurons; Cell differentiation; Skin-derived precursors; Retinoic acid

Klf10 silencing inhibited mechanical force induced osteogenic differentiation of human periodontal ligament cells

To investigate the effect of Klf10 silence on human periodontal ligament cells (HPDLCs) under mechanical force. HPDLCs were isolated and transfected with Klf10 siRNA, and then exposed to centrifugal force for 6 h at 631 r/min. Purmorphamine, an hedgehog signaling pathway agonist, was used for intervention. The activity of alkaline phosphatase (ALP) was detected by ELISA. RT-PCR and Western blot were performed to detect the mRNA and protein expression of Klf10, Runt related transcription factor 2 (Runx2), osteopontin (OPN) and osteocalcin (OCN). The protein expression of glioma associated oncogene homolog 1 (Gli1) and patched-1 (PTCH1) was detected by Western blot. SPSS 20.0 software package was used to analyze the data. Mechanical force increased mRNA and protein level of Klf10, Runx2, OPN and OCN, and elevated ALP activity significantly (P<0.05). Mechanical force also upregulated the protein expression of GLI1 and Ptch1 significantly (P<0.05). Compared with the control group, Klf10 siRNA transfection significantly decreased mRNA and protein level of Klf10 (P<0.05). Klf10 siRNA significantly inhibited the activity of ALP, and downregulated mRNA and protein expression of Runx2, OPN and OCN (P<0.05). Moreover, Klf10 siRNA significantly inhibited protein expression of GLI1 and Ptch1 (P<0.05), and purmorphamine obviously inhibited the effect of Klf10 siRNA (P<0.05). Klf10 silencing could inhibit bone differentiation of human periodontal ligament cells under mechanical force, which may be through regulation of hedgehog signaling pathway.

Neuroprotective Effects of a Smoothened Receptor Agonist against Early Brain Injury after Experimental Subarachnoid Hemorrhage in Rats

The sonic hedgehog (Shh) signaling pathway plays a fundamental role in the central nervous system (CNS) development, but its effects on neural cell survival and brain repair after subarachnoid hemorrhage (SAH) has not been well-investigated. The present study was undertaken to evaluate the influence of an agonist of the Shh co-receptor Smoothened (Smo), purmorphamine (PUR), on early brain injury (EBI) as well as the underlying mechanisms after SAH. PUR was administered via an intraperitoneal injection with a dose of 0.5, 1, and 5 mg/kg at 2, 6, 24, and 46 h after SAH in rat model. The results showed that PUR treatment significantly ameliorated brain edema, improved neurobehavioral function, and attenuated neuronal cell death in the prefrontal cortex (PFC), associated with a decrease in Bax/Bcl-2 ratio and suppression of caspase-3 activation at 48 h after SAH. PUR also promoted phospho-ERK levels. Additionally, PUR treatment markedly decreased MDA concentration accompanied with the elevation in the expression of nuclear factor erythroid 2-related factor 2 and heme oxygenase-1 in PFC. Notably, PUR treatment significantly reversed the changes of Shh pathway mediators containing Patched, Gli1, and Shh by SAH insult, and the neuroprotection of PUR on SAH was blocked by Smo antagonist cyclopamine. These results indicated that PUR exerts neuroprotection against SAH-evoked injury in rats, mediated in part by anti-apoptotic and anti-oxidant mechanism, up-regulating phospho-ERK levels, mediating Shh signaling molecules in the PFC.

Purmorphamine as a Shh Signaling Activator Small Molecule Promotes Motor Neuron Differentiation of Mesenchymal Stem Cells Cultured on Nanofibrous PCL Scaffold.

There is variety of stem cell sources but problems in ethical issues, contamination, and normal karyotype cause many limitations in obtaining and using these cells. The cells in Wharton's jelly region of umbilical cord are abundant and available stem cells with low immunological incompatibility, which could be considered for cell replacement therapy. Small molecules have been presented as less expensive biologically active compounds that can regulate different developmental process. Purmorphamine (PMA) is a small molecule that, according to some studies, possesses certain differentiation effects. In this study, we investigated the effect of the PMA on Wharton's jelly mesenchymal stem cell (WJ-MSC) differentiation into motor neuronal lineages instead of sonic hedgehog (Shh) on PCL scaffold. After exposing to induction media for 15days, the cells were characterized for expression of motor neuron markers including PAX6, NF-H, Islet1, HB9, and choline acetyl transferase (ChAT) by quantitative reverse transcription (PCR) and immunocytochemistry. Our results demonstrated that induced WJ-MSCs with PMA could significantly express motor neuron markers in RNA and protein levels 15days post induction. These results suggested that WJ-MSCs can differentiate to motor neuron-like cells with PMA on PCL scaffold and might provide a potential source in cell therapy for nervous system.

Empowering sonic hedgehog to rescue brain cells after ischemic stroke.

Ischemic stroke occurs when blood supply to the brain is interrupted. This can cause irreversible injury to the central nervous system (CNS) tissue. Each year in the United States almost 800,000 people experience a new or recurrent stroke. 15% of stroke patients die shortly after insult and only 10% recover completely, leaving the majority of surviving stroke patients with disabilities. Tissue-type plasminogen activator (tPA) is the only available therapy for stroke but its clinical use is limited because of associated danger of intracranial hemorrhage. Therefore, there is an emergent need for stroke therapeutics that are safe and effective when administered at a later time point after insult.

A Smoothened receptor agonist is neuroprotective and promotes regeneration after ischemic brain injury.

Ischemic stroke occurs as a result of blood supply interruption to the brain causing tissue degeneration, patient disabilities or death. Currently, treatment of ischemic stroke is limited to thrombolytic therapy with a narrow time window of administration. The sonic hedgehog (Shh) signaling pathway has a fundamental role in the central nervous system development, but its impact on neural cell survival and tissue regeneration/repair after ischemic stroke has not been well investigated. Here we report the neuroprotective properties of a small-molecule agonist of the Shh co-receptor Smoothened, purmorphamine (PUR), in the middle cerebral artery occlusion model of ischemic stroke. We found that intravenous administration of PUR at 6 h after injury was neuroprotective and restored neurological deficit after stroke. PUR promoted a transient upregulation of tissue-type plasminogen activator in injured neurons, which was associated with a reduction of apoptotic cell death in the ischemic cortex. We also observed a decrease in blood–brain barrier permeability after PUR treatment. At 14 d postinjury, attenuation of inflammation and reactive astrogliosis was found in PUR-treated animals. PUR increased the number of newly generated neurons in the peri-infarct and infarct area and promoted neovascularization in the ischemic zone. Notably, PUR treatment did not significantly alter the ischemia-induced level of Gli1, a Shh target gene of tumorigenic potential. Thus our study reports a novel pharmacological approach for postischemic treatment using a small-molecule Shh agonist, providing new insights into hedgehog signaling-mediated mechanisms of neuroprotection and regeneration after stroke.

Reagents for developmental regulation of Hedgehog signaling

We have examined a number of reagents for their ability to modulate activity of the Hh signaling pathway during embryonic development of Xenopus. In particular we have focused on regulation of events occurring during tailbud stages and later. Two inducible protein reagents based on the Gli1 and Gli3 transcription factors were generated and the activity of these proteins was compared to the Hh signaling pathway inhibitor, cyclopamine, and the activators, Smoothened agonist (SAG) and purmorphamine (PMA). Effectiveness of reagents was assayed using both molecular biological techniques and biological readouts. We found that the small molecule modulators of the Hh pathway were highly specific and effective and produced results generally superior to the more conventional protein reagents for examination of later stage developmental processes.

Efficient derivation of NPCs, spinal motor neurons and midbrain dopaminergic neurons from hESCs at 3% oxygen.

This protocol has been designed to generate neural precursor cells (NPCs) from human embryonic stem cells (hESCs) using a physiological oxygen (O(2)) level of 3% (previously termed hypoxia) and chemically defined conditions. The first stage involves suspension culture of hESC colonies at 3% O(2), where they acquire a neuroepithelial identity over a period of 2 weeks. This timescale is comparable to that observed at 20% O(2), but survival is enhanced. Sequential application of retinoic acid and purmorphamine (PM), from day 14 to day 28, directs differentiation toward spinal motor neurons. Alternatively, addition of fibroblast growth factor-8 and PM generates midbrain dopaminergic neurons. OLIG2 (encoding oligodendrocyte lineage transcription factor 2) induction in motor neuron precursors is twofold greater than that at 20% O(2), whereas EN1 (encoding engrailed homeobox 1) expression is enhanced fivefold. NPCs (at 3% O(2)) can be differentiated into all three neural lineages, and such cultures can be maintained long term in the absence of neurotrophins. The ability to generate defined cell types at 3% O(2) should represent a significant advancement for in vitro disease modeling and potentially for cell-based therapies.

Hedgehog signaling and osteogenic differentiation in multipotent bone marrow stromal cells are inhibited by oxidative stress

Oxidative stress may play a major role in age-related osteoporosis in part by inhibiting osteoblast generation from osteoprogenitors cells. In the present study, we hypothesized that oxidative stress may inhibit the osteogenic differentiation of bone marrow stromal cells (MSC) in part by inhibiting the Hedgehog (Hh) signaling pathway, which is essential for bone development and maintenance and induces osteogenic differentiation of osteoprogenitor cells. To test this hypothesis, we examined the effects of oxidative stress on Sonic Hh (Shh)-induced osteogenic differentiation and signaling in M2-10B4 (M2) MSC, C3H10T1/2 embryonic fibroblasts, and mouse primary MSC. Treatment of cells with H(2)O(2) inhibited Shh-induced osteogenic differentiation determined by the inhibition of Shh-induced expression of osteogenic differentiation markers alkaline phosphatase (ALP), osterix (OSX), and bone sialoprotein (BSP). Similar effects were found when oxidative stress was induced by xanthine/xanthine oxidase (XXO) or minimally oxidized LDL (MM-LDL). H(2)O(2) , XXO, and MM-LDL treatment inhibited Shh-induced expression of the Hh target genes Gli1 and Patched1 as well as Gli-dependent transcriptional activity in M2 cells. H(2)O(2) treatment also inhibited Hh signaling induced by the direct activation of Smoothened by purmorphamine (PM), but not by Gli1 overexpression. This suggests that oxidative stress may inhibit Hh signaling upstream of Gli activation and Gli-induced gene expression. These findings demonstrate for the first time that oxidative stress inhibits Hh signaling associated with osteogenic differentiation. Inhibition of Hh signaling-mediated osteogenic differentiation of osteoprogenitor cells may in part explain the inhibitory effects of oxidative stress on osteoblast development, differentiation, and maintenance in aging.