Altered expression of miR-29a-3p and miR-34a-5p by specific inhibition of GSK3ß in the MPP+ treated SH-SY5Y Parkinson's model.

In the current study, the effects of 7-BIO as a specific GSK3ß inhibitor was examined on cell survival and expression of miR-29a-3p and miR-34a-5p in neurotoxin MPP+ treated SH-SY5Y cells. Our findings revealed that while co-treatment of the cells with 7-BIO and MPP+ did not alter the toxicity induced by MPP+, pretreatment with 3.5 µM 7-BIO for 6 h increased the survival of the 2 mM MPP+ treated cells. Also, qRT-PCR analysis of gene expression showed that while miR-29a-3p was unchanged in cells treated with either 2 mM MPP+ or 3.5 µM 7-BIO alone, miR-34a-5p was increased by MPP+ but decreased by 7-BIO. Pretreatment with 3.5 µM 7-BIO prior to MPP+ however, increased miR-29a-3p but decreased miR-34a-5p induced by MPP+. We therefore suggest that 7-BIO inhibition of GSK3ß alleviates the MPP+ induced neurotoxicity by regulating miR-29a-3p and miR-34a-5p expressions in Parkinson's disease model SH-SY5Y cells.

Transcriptional drug repositioning and cheminformatics approach for differentiation therapy of leukaemia cells.

Differentiation therapy is attracting increasing interest in cancer as it can be more specific than conventional chemotherapy approaches, and it has offered new treatment options for some cancer types, such as treating acute promyelocytic leukaemia (APL) by retinoic acid. However, there is a pressing need to identify additional molecules which act in this way, both in leukaemia and other cancer types. In this work, we hence developed a novel transcriptional drug repositioning approach, based on both bioinformatics and cheminformatics components, that enables selecting such compounds in a more informed manner. We have validated the approach for leukaemia cells, and retrospectively retinoic acid was successfully identified using our method. Prospectively, the anti-parasitic compound fenbendazole was tested in leukaemia cells, and we were able to show that it can induce the differentiation of leukaemia cells to granulocytes in low concentrations of 0.1 µM and within as short a time period as 3 days. This work hence provides a systematic and validated approach for identifying small molecules for differentiation therapy in cancer.

Lithium chloride improves the efficiency of induced pluripotent stem cell-derived neurospheres.

Induced pluripotent stem cell (iPSC)-derived neurospheres, which consist mainly of neural progenitors, are considered to be a good source of neural cells for transplantation in regenerative medicine. In this study, we have used lithium chloride, which is known to be a neuroprotective agent, in an iPSC-derived neurosphere model, and examined both the formation rate and size of the neurospheres as well as the proliferative and apoptotic status of their contents. Our results showed that lithium enhanced the formation and the sizes of the iPSC-derived neurospheres, increased the number of Ki67-positive proliferating cells, but reduced the number of the TUNEL-positive apoptotic cells. This increased number of Ki67 proliferating cells was secondary to the decreased apoptosis and not to the stimulation of cell cycle entry, as the expression of the proliferation marker cyclin D1 mRNA did not change after lithium treatment. Altogether, we suggest that lithium enhances the survival of neural progenitors and thus the quality of the iPSC-derived neurospheres, which may strengthen the prospect of using lithium-treated pluripotent cells and their derivatives in a clinical setting.

Activation of TGFß1 signaling enhances early dopaminergic differentiation in unrestricted somatic stem cells.

So far there is increasing evidence for the involvement of transforming growth factor beta TGFß (transforming growth factor) in differentiation and maintenance of midbrain dopaminergic neurons. Considering that USSCs (unrestricted somatic stem cells) have the potentials to differentiate into neuron-like cells and even dopaminergic neurons and that no evidence available on the role of TGFß signaling in dopaminergic differentiation of these cells, we investigated the presence of TGFß signaling components in USSCs and their involvement on USSCs differentiation into early dopaminergic neurons. Our results showed that components of TGFß signaling were present and functional in undifferentiated USSCs, after which the neurally induced USSCs treated with TGFß1 for 3 days resulted in increased expression of ß-tubulin III (a general neuronal marker) and Nurr-1 (an early dopaminergic marker) at both mRNA and protein levels. Consistently, TGFß inhibition in culture medium by using SB431542 in the presence or absence of TGFß1, significantly decreased the expression of both neural markers. We therefore suggest that activation of TGFß signaling-pathway in neurally induced USSCs enhances neural differentiation with an early dopaminergic phenotype which points at the positive role of the TGFß signaling pathway toward dopaminergic fate.

Lithium induces follicular atresia in rat ovary through a GSK-3ß/ß-catenin dependent mechanism.

Lithium chloride (LiCl) is a drug used to treat bipolar disorder, but has side effects in the female reproductive system. Although lithium is known to decrease folliculogenesis and induce follicular atresia in rodent ovaries, its cellular and molecular effects in the ovary have not yet been addressed. To investigate these effects, 23-day-old immature female rats were injected with 10 IU pregnant mare serum gonadotropin (PMSG), followed by injections of 250 mg/kg LiCl every 12 hr for four doses. Ovaries were removed 40 and 48 hr after PMSG administration and prepared for histology, immunohistochemistry, Western blotting, and DNA laddering analysis. Our results showed that in the ovaries of LiCl-treated rats, few antral but more atretic follicles were present compared to those of the control rats. The induction of atresia by LiCl was further confirmed by the presence of DNA fragmentation, accompanied by a reduced level of 17ß-estradiol in the serum. At the cellular level, lithium significantly decreased the number of proliferating cell nuclear antigen (PCNA)-positive cells and conversely increased the number of TUNEL-positive cells in the granulosa layer of the antral follicles. At the molecular level, lithium increased the level of phosphorylated glycogen synthase kinase-3ß, and unexpectedly decreased the expression of active (stabilized) ß-catenin. Altogether, our results indicate that lithium disrupts the balance between proliferation and apoptosis in granulosa cells, leading to follicular atresia possibly through the reduction in both the stabilized ß-catenin and 17ß-estradiol synthesis.

Inhibition of GSK-3ß enhances neural differentiation in unrestricted somatic stem cells.

GSK-3ß is a key molecule in several signalling pathways, including the Wnt/ß-catenin signalling pathway. There is increasing evidence suggesting Wnt/ß-catenin signalling is involved in the neural differentiation of embryonic, somatic and neural stem cells. However, a large body of evidence indicates that this pathway maintains stem cells in a proliferative state. To address this controversy, we have investigated whether the Wnt/ß-catenin pathway is present and involved in the neural differentiation of newly introduced USSCs (unrestricted somatic stem cells). Our results indicate that the components of Wnt/ß-catenin signalling are present in undifferentiated USSCs. We also show that the treatment of neurally induced USSCs with BIO (6-bromoindirubin-3'-oxime), a specific GSK-3ß inhibitor and Wnt activator, for 5 and 10 days results in increased expression of a general neuronal marker (ß-tubulin III). Moreover, the expression of pGSK-3ß and stabilized ß-catenin increased by BIO in neurally induced USSCs, indicates that the Wnt pathway is activated and functional in these cells. Thus, inhibition of GSK-3ß in USSCs enhances their neural differentiation, which suggests a positive role of the Wnt/ß-catenin signalling pathway towards neural fate.

Microinjection of l-arginine into corpus callosum cause reduction in myelin concentration and neuroinflammation.

Role of nitric oxide (NO) in inflammationary diseases such as multiple sclerosis (MS) has been proposed previously. We sought to examine if NO plays centrally a key role in MS related phenomena; demyelination or neuroinflammation. Female Wistar rats (weighing 200-250 g) were mounted in a stereotaxic apparatus and received injections of l-arginine aimed at corpus callosum (AP: 1.2, L: ±1.8, V: 3.2). The drug (50-200 µg/rat) was microinjected intra-corpus callosum repeatedly (3-5 times/each per day). Control groups solely received saline (1 µg/rat) into the corpus callosum. The animals were tested for the novelty seeking behavior using the conditioning task. Memory impairment was examined using the shuttle box and Y-maze. l-NAME was pre-injected to l-arginine to involve the NO. All animals' brains were also processed for histological evaluation. l-arginine produced significant changes in the novelty seeking behavior but not in the memory formation, evidenced by passive avoidance and alternation behaviors. Pre-injection of l-NAME reversed the response to l-arginine. Present study further revealed a prominent inflammation as well as myelin elimination in the l-arginine treated rats' brains. These data suggest that the NO infusion in the myelin rich areas such as corpus callosum may lead to MS signs centrally.

An Iranian herbal-marine medicine, MS14, ameliorates experimental allergic encephalomyelitis.

Multiple sclerosis is an inflammatory and demyelinating disease of the central nervous system which mainly affects young adults. To overcome wide spectrum troublesome symptoms of multiple sclerosis which affects the quality of life both in patients and their families, new drugs and remedies have been examined and offered. The preclinical beneficial effects of different medicines have mostly been examined in an animal model of multiple sclerosis called experimental allergic encephalomyelitis (EAE). In this study we have tested a traditionally used natural (herbal-marine) product called MS(14) in EAE mice. EAE mice were fed with MS(14) containing diet (30%) on the immunization day and monitored for 20 days. The results show that while clinical scores and therefore severity of the disease was progressive in normal-fed EAE mice, the disease was slowed down in MS(14)-fed EAE mice. Moreover, while there were moderate to severe neuropathological changes in normal fed mice, milder changes were seen in MS(14) fed mice.

Induction of experimental allergic encephalomyelitis in C57/BL6 Mice: an animal model for multiple sclerosis.

Basic research on the autoimmune disease multiple sclerosis has been performed mainly on its animal model namely experimental allergic encephalomyelitis. There are many different approaches established to get this model. Despite the existence of many references in literature in this regard, we have been faced with many difficulties generating the model suitable for studying different therapies. After a long time of challenging to get a reliable and replicable method, we came up with the following major points: First, the key element for getting a maximum number of sick animals at a defined time is to consider the most appropriate animal body weight (19-20 gr). Even though the age of immunized animals (6-8 week old) is highlighted in literature, we found out that body weight is of a greater importance. Secondly, because the only available susceptible mice strain in Iran is C57/BL6, the choice of peptide for immunization would be myelin oligodendrocyte glycoprotein (35-55 sequence of this peptide 200 mg/animal). Finally, pertussis toxin which is a costly reagent plays a key role in stimulating the immune response. Altogether, we recommend that considering the above mentioned tricks and tracks, one would definitely be able to generate a chronic progressive type of model, for basic research on therapies of multiple sclerosis.