Second-generation non-hematopoietic erythropoietin-derived peptide for neuroprotection.

Erythropoietin (EPO) is a well-known erythropoietic cytokine having a tissue-protective effect in various tissues against hypoxic stress, including the brain. Thus, its recombinants may function as neuroprotective compounds. However, despite considerable neuroprotective effects, the EPO-based therapeutic approach has side effects, including hyper-erythropoietic and tumorigenic effects. Therefore, some modified forms and derivatives of EPO have been proposed to minimize the side effects. In this study, we generated divergently modified new peptide analogs derived from helix C of EPO, with several amino acid replacements that interact with erythropoietin receptors (EPORs). This modification resulted in unique binding potency to EPOR. Unlike recombinant EPO, among the peptides, ML1-h3 exhibited a potent neuroprotective effect against oxidative stress without additional induction of cell-proliferation, owing to a differential activating mode of EPOR signaling. Furthermore, it inhibited neuronal death and brain injury under hypoxic stress in vitro and in an in vivo ischemic brain injury model. Therefore, the divergent modification of EPO-derivatives for affinity to EPOR could provide a basis for a more advanced and optimal neuroprotective strategy.

Visualizing in deceased COVID-19 patients how SARS-CoV-2 attacks the respiratory and olfactory mucosae but spares the olfactory bulb.

Anosmia, the loss of smell, is a common and often the sole symptom of COVID-19. The onset of the sequence of pathobiological events leading to olfactory dysfunction remains obscure. Here, we have developed a postmortem bedside surgical procedure to harvest endoscopically samples of respiratory and olfactory mucosae and whole olfactory bulbs. Our cohort of 85 cases included COVID-19 patients who died a few days after infection with SARS-CoV-2, enabling us to catch the virus while it was still replicating. We found that sustentacular cells are the major target cell type in the olfactory mucosa. We failed to find evidence for infection of olfactory sensory neurons, and the parenchyma of the olfactory bulb is spared as well. Thus, SARS-CoV-2 does not appear to be a neurotropic virus. We postulate that transient insufficient support from sustentacular cells triggers transient olfactory dysfunction in COVID-19. Olfactory sensory neurons would become affected without getting infected.

The erythropoietin-derived peptide MK-X and erythropoietin have neuroprotective effects against ischemic brain damage.

Erythropoietin (EPO) has been well known as a hematopoietic cytokine over the past decades. However, recent reports have demonstrated that EPO plays a neuroprotective role in the central nervous system, and EPO has been considered as a therapeutic target in neurodegenerative diseases such as ischemic stroke. Despite the neuroprotective effect of EPO, clinical trials have shown its unexpected side effects, including undesirable proliferative effects such as erythropoiesis and tumor growth. Therefore, the development of EPO analogs that would confer neuroprotection without adverse effects has been attempted. In this study, we examined the potential of a novel EPO-based short peptide, MK-X, as a novel drug for stroke treatment in comparison with EPO. We found that MK-X administration with reperfusion dramatically reduced brain injury in an in vivo mouse model of ischemic stroke induced by middle cerebral artery occlusion, whereas EPO had little effect. Similar to EPO, MK-X efficiently ameliorated mitochondrial dysfunction followed by neuronal death caused by glutamate-induced oxidative stress in cultured neurons. Consistent with this effect, MK-X significantly decreased caspase-3 cleavage and nuclear translocation of apoptosis-inducing factor induced by glutamate. MK-X completely mimicked the effect of EPO on multiple activation of JAK2 and its downstream PI3K/AKT and ERK1/2 signaling pathways, and this signaling process was involved in the neuroprotective effect of MK-X. Furthermore, MK-X and EPO induced similar changes in the gene expression patterns under glutamate-induced excitotoxicity. Interestingly, the most significant difference between MK-X and EPO was that MK-X better penetrated into the brain across the brain-blood barrier than did EPO. In conclusion, we suggest that MK-X might be used as a novel drug for protection from brain injury caused by ischemic stroke, which penetrates into the brain faster in comparison with EPO, even though MK-X and EPO have similar protective effects against excitotoxicity.

Neuroprotective Effects of an Erythropoietin-Derived Peptide in PC1 2 Cells under Oxidative Stress.

Erythropoietin (EPO) has been shown to be a key cytokine in the production of erythrocytes from erythroblasts. Recently, attempts have been made to adopt EPO as a drug target for neuroprotection in selected neurological pathologies. In the current study, a novel EPO-derived peptide which mimics the weak binding site of EPO to its receptor (MK-X) was generated. Experimental results demonstrated that MK-X was able to ameliorate neuronal death due to reactive oxygen species and conditions of oxidative stress similar to EPO. In addition, MK-X induced long-lasting Extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and Akt activation. Furthermore, treatment with inhibitors of ERK1/2 and Akt abolished the neuroprotective effect of MK-X. Unlike EPO, however, MK-X did not induce cellular proliferation. Collectively, the results of the current study suggested that MK-X may be useful as a novel neuroprotective reagent.

Nano-patterned SU-8 surface using nanosphere-lithography for enhanced neuronal cell growth.

Mimicking the nanoscale surface texture of the extracellular matrix can affect the regulation of cellular behavior, including adhesion, differentiation, and neurite outgrowth. In this study, SU-8-based polymer surfaces with well-ordered nanowell arrays were fabricated using nanosphere lithography with polystyrene nanoparticles. We show that the SU-8 surface with nanowells resulted in similar neuronal development of rat pheochromocytoma (PC12) cells compared with an unpatterned poly-L-lysine (PLL)-coated SU-8 surface. Additionally, even after soaking the substrate in cell culture medium for two weeks, cells on the nanowell SU-8 surface showed long-term neurite outgrowth compared to cells on the PLL-coated SU-8 surface. The topographical surface modification of the nanowell array demonstrates potential as a replacement for cell adhesive material coatings such as PLL, for applications requiring long-term use of polymer-based implantable devices.

Odorant Stimulation Promotes Survival of Rodent Olfactory Receptor Neurons via PI3K/Akt Activation and Bcl-2 Expression.

Olfactory stimulation activates multiple signaling cascades in order to mediate activity-driven changes in gene expression that promote neuronal survival. To date, the mechanisms involved in activity-dependent olfactory neuronal survival have yet to be fully elucidated. In the current study, we observed that olfactory sensory stimulation, which caused neuronal activation, promoted activation of the phosphatidylinositol 3'-kinase (PI3K)/Akt pathway and the expression of Bcl-2, which were responsible for olfactory receptor neuron (ORN) survival. We demonstrated that Bcl-2 expression increased after odorant stimulation both in vivo and in vitro. We also showed that odorant stimulation activated Akt, and that Akt activation was completely blocked by incubation with both a PI3K inhibitor (LY294002) and Akt1 small interfering RNA. Moreover, blocking the PI3K/Akt pathway diminished the odorant-induced Bcl-2 expression, as well as the effects on odorant-induced ORN survival. A temporal difference was noted between the activation of Akt1 and the expression of Bcl-2 following odorant stimulation. Blocking the PI3K/Akt pathway did not affect ORN survival in the time range prior to the increase in Bcl-2 expression, implying that these two events, activation of the PI3K pathway and Bcl-2 induction, were tightly connected to promote post-translational ORN survival. Collectively, our results indicated that olfactory activity activated PI3K/Akt, induced Bcl-2, and promoted long term ORN survival as a result.

Phosphoinositide and Erk signaling pathways mediate activity-driven rodent olfactory sensory neuronal survival and stress mitigation.

Olfactory sensory neurons (OSNs) are the initial site for olfactory signal transduction. Therefore, their survival is essential to olfactory function. In the current study, we demonstrated that while odorant stimulation promoted rodent OSN survival, it induced generation of reactive oxygen species in a dose- and time-dependent manner as well as loss of membrane potential and fragmentation of mitochondria. The MEK-Erk pathway played a critical role in mediating these events, as its inhibition decreased odorant stimulation-dependent OSN survival and exacerbated intracellular stress measured by reactive oxygen species generation and heat-shock protein 70 expression. The phosphoinositide pathway, rather than the cyclic AMP pathway, mediated the odorant-induced activation of the MEK-Erk pathway. These findings provide important insights into the mechanisms of activity-driven OSN survival, the role of the phosphoinositide pathway in odorant signaling, and demonstrate that odorant detection and odorant stimulation-mediated survival proceed via independent signaling pathways. This mechanism, which permits independent regulation of odorant detection from survival signaling, may be advantageous if not diminished by repeated or prolonged odor exposure. We investigated the role of odorant stimulation in generating cellular stress and the molecular mechanisms mitigating such stress and promoting neuronal survival. Odorant stimulation promoted olfactory sensory neuron (OSN) survival and also induced intracellular oxidative stress, which was exacerbated when MEK/Erks pathway was inhibited. Sensory stimulation simultaneously activated at least two parallel pathways, the AC/cAMP cascade responsible for odorant detection, and phosphoinositide hydrolysis to promote odorant stimulation-dependent neuronal survival odorants may activate parallel signaling cascades to mediate sensory detection and sensory stimulation-dependent survival. AC, adenylyl cyclase; cAMP, cyclic adenosine monophosphate; Erk, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase.

Formation of perfect ohmic contact at indium tin oxide/N,N'-di(naphthalene-1-yl)-N,N'-diphenyl-benzidine interface using ReO3.

A perfect ohmic contact is formed at the interface of indium tin oxide (ITO) and N,N'-di(naphthalene-1-yl)-N,N'-diphenyl-benzidine (NPB) using ReO3 as the interfacial layer. The hole injection efficiency is close to 100% at the interface, which is much higher than those for interfacial layers of 1,4,5,8,9,11-hexaazatripheylene hexacarbonitrile (HAT-CN) and MoO3. Interestingly, the ReO3 and MoO3 interfacial layers result in the same hole injection barrier, ≈0.4 eV, to NPB, indicating that the Fermi level is pinned to the NPB polaron energy level. However, a significant difference is observed in the generated charge density in the NPB layer near the interfacial layer/NPB interface, indicating that charge generation at the interface plays an important role in forming the ohmic contact.

Induction of neural stem cell-like cells (NSCLCs) from mouse astrocytes by Bmi1.

Recently, Bmi1 was shown to control the proliferation and self-renewal of neural stem cells (NSCs). In this study, we demonstrated the induction of NSC-like cells (NSCLCs) from mouse astrocytes by Bmi1 under NSC culture conditions. These NSCLCs exhibited the morphology and growth properties of NSCs, and expressed NSC marker genes, including nestin, CD133, and Sox2. In vitro differentiation of NSCLCs resulted in differentiated cell populations containing astrocytes, neurons, and oligodendrocytes. Following treatment with histone deacetylase inhibitors (trichostatin A and valproic acid), the potential of NSCLCs for proliferation, dedifferentiation, and self-renewal was significantly inhibited. Our data indicate that multipotent NSCLCs can be generated directly from astrocytes by the addition of Bmi1.

Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment.

Cell replacement therapy is a promising approach for the treatment of cardiac diseases. It is, however, challenged by a limited supply of appropriate cells. Therefore, we have investigated whether functional cardiomyocytes can be efficiently generated from human embryonic stem cells (hESCs). In this study, we developed an efficient protocol for the generation of functional cardiomyocytes from hESCs by combining hanging drop culture and 5-azacytidine, a well-known demethylating agent, and then evaluated the expression of cardiac-specific markers. hESCs were cultured both in the medium without or with 0.1, 1, or 10 microM of 5-azacytidine under a hanging drop culture. The expression of several cardiac-specific markers was determined by real-time PCR, RT-PCR, immunofluorescence, and confocal microscopy. To verify the structural and functional properties of hESC-derived cardiomyocytes, we performed electron microscopy and electrophysiological recording. The efficiency of beating cell generation was significantly improved in the hanging drop culture compared with that in suspension culture. Treatment of hESCs with 0.1 microM of 5-azacytidine for 1-3 days significantly increased the number of beating cells and simultaneously enhanced the expression of cardiac-specific markers. Transmission electron microscopy and electrophysiological recording showed that hESC-derived cardiomyocytes acquired structural and functional properties of cardiomyocytes. In conclusion, these results suggest that differentiation of hESCs into cardiomyocytes can be enhanced by the combination of hanging drop culture and 5-azacytidine treatment. Also the methylation status of genes related to cardiomyocyte development may play an important role in the differentiation of hESCs into cardiomyocytes.

Contribution of the PI3K/Akt/PKB signal pathway to maintenance of self-renewal in human embryonic stem cells.

Although basic fibroblast growth factor (FGF2) is generally included in the media for maintenance of human embryonic stem cells (hESCs), the action of FGF2 in these cells has not been well defined. Here, we determined the roles of FGF2 in maintaining hESC self-renewal. Withdrawal of FGF2 from the media led to acquisition of typical differentiated characteristics in hESCs. In the presence of FGF2, which is normally required for proliferation in an undifferentiated state, inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt/PKB signal stimulated differentiation and attenuated the expression of extracellular matrix (ECM) molecules. We suggest that FGF2 maintains hESC self-renewal by supporting stable expression of ECM molecules through activation of the PI3K/Akt/PKB pathway.