Neurobiochemical characteristics of arginine-rich peptides explain their potential therapeutic efficacy in neurodegenerative diseases.

Neurodegenerative diseases, including Alzheimer̕ s disease (AD), Parkinson̕ s disease (PD), Huntington̕ s disease (HD), and Amyotrophic Lateral Sclerosis (ALS) require special attention to find new potential treatment methods. This review aims to summarize the current knowledge of the relationship between the biochemical properties of arginine-rich peptides (ARPs) and their neuroprotective effects to deal with the harmful effects of risk factors. It seems that ARPs have portrayed a promising and fantastic landscape for treating neurodegeneration-associated disorders. With multimodal mechanisms of action, ARPs play various unprecedented roles, including as the novel delivery platforms for entering the central nervous system (CNS), the potent antagonists for calcium influx, the invader molecules for targeting mitochondria, and the protein stabilizers. Interestingly, these peptides inhibit the proteolytic enzymes and block protein aggregation to induce pro-survival signaling pathways. ARPs also serve as the scavengers of toxic molecules and the reducers of oxidative stress agents. They also have anti-inflammatory, antimicrobial, and anti-cancer properties. Moreover, by providing an efficient nucleic acid delivery system, ARPs can play an essential role in developing various fields, including gene vaccines, gene therapy, gene editing, and imaging. ARP agents and ARP/cargo therapeutics can be raised as an emergent class of neurotherapeutics for neurodegeneration. Part of the aim of this review is to present recent advances in treating neurodegenerative diseases using ARPs as an emerging and powerful therapeutic tool. The applications and progress of ARPs-based nucleic acid delivery systems have also been discussed to highlight their usefulness as a broad-acting class of drugs.

Novel therapeutic approach to slow down the inflammatory cascade in acute/subacute spinal cord injury: Early immune therapy with lipopolysaccharide enhanced neuroprotective effect of combinational therapy of granulocyte colony-stimulating factor and bone-marrow mesenchymal stem cell in spinal cord injury.

Bone-marrow mesenchymal stem cells (BM-MSCs) have not yet proven any significant therapeutic efficacy in spinal cord injury (SCI) clinical trials, due to the hostile microenvironment of the injured spinal cord at the acute phase. This study aims to modulate the inflammatory milieu by lipopolysaccharide (LPS) and granulocyte colony-stimulating factor (G-CSF) to improve the BM-MSCs therapy. For this purpose, we determined the optimum injection time and sub-toxic dosage of LPS following a T10 contusion injury. Medium-dose LPS administration may result in a local anti-inflammatory beneficial role. This regulatory role is associated with an increase in NF-200-positive cells, significant tissue sparing, and improvement in functional recovery compared to the SCI control group. The second aim was to examine the potential ability of LPS and LPS + G-CSF combination therapy to modulate the lesion site before BM-MSC (1 × 105 cells) intra-spinal injection. Our results demonstrated combination therapy increased potency to enhance the anti-inflammatory response (IL-10 and Arg-1) and decrease inflammatory markers (TNF-α and CD86) and caspase-3 compared to BM-MSC monotherapy. Histological analysis revealed that combination groups displayed better structural remodeling than BM-MSC monotherapy. In addition, Basso-Beattie-Bresnahan (BBB) scores show an increase in motor recovery in all treatment groups. Moreover, drug therapy shows faster recovery than BM-MSC monotherapy. Our results suggest that a sub-toxic dose of LPS provides neuroprotection to SCI and can promote the beneficial effect of BM-MSC in SCI. These findings suggest that a combination of LPS or LPS + G-CSF prior BM-MSC transplantation is a promising approach for optimizing BM-MSC-based strategies to treat SCI. However, because of the lack of some methodological limitations to examine the survival rate and ultimate fate of transplanted BM-MSCs followed by LPS administration in this study, further research needs to be done in this area. The presence of only one-time point for evaluating the inflammatory response (1 week) after SCI can be considered as one of the limitations of this study. We believed that the inclusion of additional time points would provide more information about the effect of our combination therapy on the microglia/macrophage polarization dynamic at the injured spinal cord.

Long-term administration of bumetanide improve functional recovery after spinal cord injury in rats.

Ion disturbances are among the most remarkable deficits in spinal cord injury (SCI). GABA is an integral part of neural interaction. Action of the GABAA receptor depends on the amount of intracellular chloride. Homeostasis of chloride is controlled by two co-transporters, NKCC1 and KCC2. Previous studies revealed that NKCC1 are disturbed in SCI. In this study, NKCC1 is highly expressed in the epicenter of the lesioned spinal cord at 3 hours after induction of the lesion and reached the peak around 6 hours after SCI. Bumetanide (2 and 4 mg/day), as a specific NKCC1 inhibitor, was used at 3 hours post SCI for 28 days. The functional recovery outcomes were measured by the Basso-Beattie-Bresnahan (BBB) locomotor rating scale, ladder walking test, and hot plate test. The rats that received bumetanide 4 mg/day exhibited improved recovery of locomotor function, reduction of NKCC1 gene expression, and upregulation of GAP protein levels 28 days post SCI. Histological tissue evaluations confirmed bumetanide's neuroprotective and regenerative effects. This study provides novel evidence for the benefits of bumetanide in early administration after SCI.

NKCC1 to KCC2 mRNA Ratio in Schizophrenia and Its Psychopathology: a Case-Control Study.

Schizophrenia (SCZ) is a debilitating, destructive, and chronic mental disorder and affects approximately one percent of the human population. Diagnosis in psychiatry is based on the patient's descriptions of his/her symptoms, interviewer's observations, history of disorder over time, and response to treatment. All of these data measure phenotype-based functions. But it appears that accurate diagnosis of such a complex disorder must be based on valid and reliable factors. In the present study, gene selection was based on the possible role of γ-aminobutyric acid (GABA) in psychopathology of SCZ and expression in blood. We evaluated the association of Na+-K+-Cl- co-transporter 1 (NKCC1) and K+-Cl- co-transporter 2 (KCC2) genes' messenger ribonucleic acid (mRNA) levels, and also the NKCC1/KCC2 ratio with positive and negative syndrome scale (PANSS) and brief psychiatric rating scale (BPRS) scores in an SCZ group. By using real-time PCR (RT-PCR), the present study is the first attempt to explore levels of NKCC1 and KCC2 expression at mRNA level and their relative expression in human peripheral blood of patients with SCZ. Our results showed that the NKCC1 to KCC2 mRNA ratio is significantly increased (but based on the delta cycle of threshold [∆Ct] is significantly lower) in the total sample of cases rather than controls (p = 0.045) and also higher in male sample cases rather than male controls (p = 0.016). In female samples, we found a trend toward a significant effect between the case and control participants (p = 0.075). We also found statistically significant association between mRNA of NKCC1 and KCC2 genes and NKCC1/KCC2 mRNA ratio with the positive and negative syndrome scale (PANSS) and brief psychiatric rating scale (BPRS) scores.

Ac-SDKP peptide improves functional recovery following spinal cord injury in a preclinical model.

Damage to the spinal cord triggers a local complex inflammatory reaction that results in irreversible impairments or complete loss of motor function. The evidence suggested that inhibiting the pro-inflammatory macrophage/microglia (M1 subsets) and stimulating the anti-inflammatory macrophage/microglia (M2 subsets) are potential strategies for the treatment of neuroinflammation-related diseases. We evaluated the potentially protective effect of Ac-SDKP as an endogenous tetrapeptide on rat spinal cord injury (SCI). Wistar rats were subjected to a weight-drop contusion model and were treated with Ac-SDKP (0.8 mg/kg) given subcutaneously once a day for 7 days starting at two clinically relevant times, at 2 h or 6 h post-injury. The effect of Ac-SDKP was assessed by motor functional analysis, real-time PCR (CD86 and CD206 mRNA), western blot (caspase-3), ELISA (TNF-a, IL-10), and histological analysis (toluidine blue staining). Ac-SDKP improved locomotor recovery and rescue motor neuron loss after SCI. Moreover, a decreased in TNF-a level as well as caspase 3 protein levels occurred in the lesion epicenter of the spinal cord following treatment. In addition, CD206 mRNA expression level increased significantly in Ac-SDKP treated rats compared with SCI. Together these data suggest that Ac-SDKP might be a novel immunomodulatory drug. It may be beneficial for the treatment of SCI with regards to increasing CD206 gene expression and suppress inflammatory cytokine to improve motor function and reducing histopathological lesion.

ß2-Adrenergic Receptor Stimulation Upregulates Cx43 Expression on Glioblastoma Multiforme and Olfactory Ensheathing Cells.

Glioblastoma multiforme (GBM) is described as an invasive astrocytic tumor in adults. Despite current standard treatment approaches, the outcome of GBM remains unfavorable. The downregulation of connexin 43 (Cx43) expression is one of the molecular transformations in GBM cells. The Cx43 levels and subsequently gap junctional intercellular communication (GJIC) have an important role in the efficient transfer of cytotoxic drugs to whole tumor cells. As shown in our previous study, the stimulation of the ß2-adrenergic receptor (ß2-AR) leads to the modulation of Cx43 expression level in the GBM cell line. Here we further examine the effect of clenbuterol hydrochloride as a selective ß2-AR agonist on the Cx43 expression in human GBM-derived astrocyte cells and human olfactory ensheathing cells (OECs) as a potent vector for future gene therapy. In this experiment, first we established a primary culture of astrocytes from GBM samples and verified the purity using immunocytofluorescent staining. Western blot analysis was performed to evaluate the Cx43 protein level. Our western blot findings reveal that clenbuterol hydrochloride upregulates the Cx43 protein level in both primary human astrocyte cells and human OECs. Conversely, ICI 118551 as a ß2-AR antagonist inhibits these effects. Moreover, clenbuterol hydrochloride increases the Cx43 expression in primary human astrocyte cells and OECs co-culture systems, and ICI 118551 reverses these effects. To confirm the western blot results, immunocytofluorescent staining was performed to evaluate the ß2-AR agonist effect on Cx43 expression. Our immunocytofluorescent results supported western blot analysis in primary human astrocyte cells and the OECs co-culture system. The results of this study suggest that the activation of ß2-AR with regard to Cx43 protein levels enhancement in GBM cells and OECs might be a promising approach for GBM treatment in the future.

Involvement of central opioid receptors in protective effects of methadone on experimental colitis in rats.

PURPOSE: There are several lines of evidence on the protective roles of opioids in gastrointestinal inflammatory conditions. This study aims to distinguish the central and peripheral roles of methadone, a non-selective opioid receptor agonist, in an acute model of ulcerative colitis in male rats. METHODS: Ulcerative colitis was induced by intrarectal administration of acetic acid 4%. Methadone was injected subcutaneously (s.c.), 5 and 10 mg/kg, and intracerebroventricular (i.c.v.), 50 and 300 ng/rat. Opioid antagonists were employed. Methylnaltrexone (MNTX; 5 mg/kg, i.p.), a peripherally acting opioid receptor antagonist, and naltrexone (NTX; 5 mg/kg, i.p. and 10 ng/rat, i.c.v.), a peripherally and centrally acting opioid receptor antagonist were injected before methadone (10 mg/kg, s.c. and or 300 ng/rat, i.c.v.) administration. NTX (5 mg/kg, i.p. and 10 ng/rat, i.c.v.) were administered 30 min prior to administration of methadone (10 mg/kg, s.c. and 300 ng/rat, i.c.v.), respectively. MNTX (5 mg/kg, i.p.) was injected 30 min prior to methadone (10 mg/kg, s.c.). Seventy-two hours following colitis induction, macroscopic and microscopic mucosal lesions, and the colonic levels of tumor necrosis factor-alpha (TNF-α) and interleukin-1ß (IL-1ß) were determined. RESULTS: Methadone (300 ng/rat, i.c.v.) and Methadone (5 and 10 mg/kg, s.c.) improved the macroscopic and microscopic scores through opioid receptors. Also, a significant reduction in TNF-α and IL-1ß was observed. Peripherally and centrally injected NTX significantly reversed methadone 10 mg/kg s.c. anti-inflammatory effects while MNTX could not completely reverse this effect. Moreover, centrally administered methadone (300 ng/rat) showed the anti-inflammatory effect which was reversed by central administration of NTX (10 ng/rat). CONCLUSIONS: The opioid receptors mainly the central opioid receptors may mediate the protective actions of methadone on the experimental model of inflammatory bowel disease in rat.

In Vitro Differentiation of Neural-Like Cells from Human Embryonic Stem Cells by A Combination of Dorsomorphin, XAV939, and A8301.

OBJECTIVES: Motor neuron differentiation from human embryonic stem cells (hESCs) is a goal of regenerative medicine to provide cell therapy as treatments for diseases that damage motor neurons. Most protocols lack adequate efficiency in generating functional motor neurons. However, small molecules present a new approach to overcome this challenge. The aim of this research is to replace morphogen factors with a cocktail of efficient, affordable small molecules for effective, low cost motor neuron differentiation. MATERIALS AND METHODS: In this experimental study, hESCs were differentiated into motor neuron by the application of a small molecule cocktail that consisted of dorsomorphin, A8301, and XAV939. During the differentiation protocol, we selected five stages and assessed expressions of neural markers by real-time polymerase chain reaction (PCR), immunofluorescence staining, and flow cytometry. Motor neuron ion currents were determined by whole cell patch clamp recording. RESULTS: Immunofluorescence staining and flow cytometry analysis of hESC-derived neural ectoderm (NE) indicated that they were positive for NESTIN (92.68%), PAX6 (64.40%), and SOX1 (82.11%) in a chemically defined adherent culture. The replated (hESC)-derived NE differentiated cells were positive for TUJ1, MAP2, HB9 and ISL1. We evaluated the gene expression levels with real-time reverse transcriptase-PCR at different stages of the differentiation protocol. Voltage gated channel currents of differentiated cells were examined by the whole-cell patch clamp technique. The hESC-derived motor neurons showed voltage gated delay rectifier K+, Na+ and Ca2+ inward currents. CONCLUSIONS: Our results indicated that hESC-derived neurons expressed the specific motor neuron markers specially HB9 and ISL1 but voltage clamp recording showed small ionic currents therefore it seems that voltage gated channel population were inadequate for firing action potentials.

A Universal and Robust Integrated Platform for the Scalable Production of Human Cardiomyocytes From Pluripotent Stem Cells.

UNLABELLED: Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs), in conjunction with the promising outcomes from preclinical and clinical studies, have raised new hopes for cardiac cell therapy. We report the development of a scalable, robust, and integrated differentiation platform for large-scale production of hPSC-CM aggregates in a stirred suspension bioreactor as a single-unit operation. Precise modulation of the differentiation process by small molecule activation of WNT signaling, followed by inactivation of transforming growth factor-ß and WNT signaling and activation of sonic hedgehog signaling in hPSCs as size-controlled aggregates led to the generation of approximately 100% beating CM spheroids containing virtually pure (∼90%) CMs in 10 days. Moreover, the developed differentiation strategy was universal, as demonstrated by testing multiple hPSC lines (5 human embryonic stem cell and 4 human inducible PSC lines) without cell sorting or selection. The produced hPSC-CMs successfully expressed canonical lineage-specific markers and showed high functionality, as demonstrated by microelectrode array and electrophysiology tests. This robust and universal platform could become a valuable tool for the mass production of functional hPSC-CMs as a prerequisite for realizing their promising potential for therapeutic and industrial applications, including drug discovery and toxicity assays. SIGNIFICANCE: Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) and the development of novel cell therapy strategies using hPSC-CMs (e.g., cardiac patches) in conjunction with promising preclinical and clinical studies, have raised new hopes for patients with end-stage cardiovascular disease, which remains the leading cause of morbidity and mortality globally. In this study, a simplified, scalable, robust, and integrated differentiation platform was developed to generate clinical grade hPSC-CMs as cell aggregates under chemically defined culture conditions. This approach resulted in approximately 100% beating CM spheroids with virtually pure (∼90%) functional cardiomyocytes in 10 days from multiple hPSC lines. This universal and robust bioprocessing platform can provide sufficient numbers of hPSC-CMs for companies developing regenerative medicine technologies to rescue, replace, and help repair damaged heart tissues and for pharmaceutical companies developing advanced biologics and drugs for regeneration of lost heart tissue using high-throughput technologies. It is believed that this technology can expedite clinical progress in these areas to achieve a meaningful impact on improving clinical outcomes, cost of care, and quality of life for those patients disabled and experiencing heart disease.

Induction of Neural Progenitor-Like Cells from Human Fibroblasts via a Genetic Material-Free Approach.

BACKGROUND: A number of studies generated induced neural progenitor cells (iNPCs) from human fibroblasts by viral delivering defined transcription factors. However, the potential risks associated with gene delivery systems have limited their clinical use. We propose it would be safer to induce neural progenitor-like cells from human adult fibroblasts via a direct non-genetic alternative approach. METHODOLOGY/PRINCIPAL FINDINGS: Here, we have reported that seven rounds of TAT-SOX2 protein transduction in a defined chemical cocktail under a 3D sphere culture gradually morphed fibroblasts into neuroepithelial-like colonies. We were able to expand these cells for up to 20 passages. These cells could give rise to cells that expressed neurons and glia cell markers both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: These results show that our approach is beneficial for the genetic material-free generation of iNPCs from human fibroblasts where small chemical molecules can provide a valuable, viable strategy to boost and improve induction in a 3D sphere culture.

Exogenous treatment with eicosapentaenoic acid supports maturation of cardiomyocytes derived from embryonic stem cells.

Embryonic stem cells offer multiple advantages over adult stem cells in terms of achieving acceptable number of functional cardiomyocytes to be exploited in cell therapy. However, differentiation efficacy is still a major issue to be solved before moving to regenerative medicine. Although a vast number of chemical compounds have been tested on efficiency of cardiac differentiation, the effect of fish oil components, such as eicosapentaenoic acid (EPA) on developmental bioenergetics, and hence cardiac differentiation, remained unstudied. EPA has been reported to have several cardioprotective effects, but there is no study addressing its role in cardiac differentiation. After mesoderm induction of embryoid bodies (EBs) derived from mouse embryonic stem cells (mESCs) in hanging drops initiated by ascorbic acid, they were treated with various concentrations of EPA. Gene and protein expression and functional properties of cardiomyocytes derived from ESCs were evaluated following treatment with various concentrations of EPA. Exposure to low concentrations of EPA (10 µM) increased percentage of beating colonies and beating area. This treatment also resulted in up to 3 fold increase in expression of NKX2-5, MEF2C, MYH6, TNNT2 and CX43. FACS analysis confirmed gene expression analysis with increased percentage of MYH6 positive cells in EPA-treated group compared to the control group. In contrast, the expression of genes coding for cardiac differentiation, remained constant or even declined with higher concentrations of EPA. In conclusion, we have demonstrated that treatment of mESCs undergoing cardiac differentiation with low concentration, but not high concentration of EPA up-regulate transcription of genes associated with cardiac development.

Basolateral amygdala CB1 cannabinoid receptors mediate nicotine-induced place preference.

In the present study, the effects of bilateral microinjections of cannabinoid CB1 receptor agonist and antagonist into the basolateral amygdala (intra-BLA) on nicotine-induced place preference were examined in rats. A conditioned place preference (CPP) apparatus was used for the assessment of rewarding effects of the drugs in adult male Wistar rats. Subcutaneous (s.c.) administration of nicotine (0.2mg/kg) induced a significant CPP, without any effect on the locomotor activity during the testing phase. Intra-BLA microinjection of a non-selective cannabinoid CB1/CB2 receptor agonist, WIN 55,212-2 (0.1-0.5 µg/rat) with an ineffective dose of nicotine (0.1mg/kg, s.c.) induced a significant place preference. On the other hand, intra-BLA administration of AM251 (20-60 ng/rat), a selective cannabinoid CB1 receptor antagonist inhibited the acquisition of nicotine-induced place preference. It should be considered that the microinjection of the same doses of WIN 55,212-2 or AM251 into the BLA, by itself had no effect on the CPP score. The administration of a higher dose of AM251 (60 ng/rat) during the acquisition decreased the locomotor activity of animals on the testing phase. Interestingly, the microinjection of AM251 (20 and 40 ng/rat), but not WIN55,212-2 (0.1-0.5 µg/rat), into the BLA inhibited the expression of nicotine-induced place preference without any effect on the locomotor activity. Taken together, these findings support the possible role of endogenous cannabinoid system of the BLA in the acquisition and the expression of nicotine-induced place preference. Furthermore, it seems that there is a functional interaction between the BLA cannabinoid receptors and nicotine in producing the rewarding effects.