Comparing The Effects of Small Molecules BIX-01294, Bay K8644, RG-108 and Valproic Acid, and Their Different Combinations on Induction of Pluripotency Marker-Genes by Oct4 in The Mouse Brain

Objective: Every cell type is characterized by a specific transcriptional profile together with a unique epigenetic landscape. Reprogramming factors such as Oct4, Klf4, Sox2 and c-Myc enable somatic cells to change their transcriptional profile and convert them to pluripotent cells. Small molecules such as BIX-01294, Bay K8644, RG-108 and valproic acid (VPA) are reported as effective molecules for enhancing induction of pluripotency in vitro, however, their effects during in vivo reprogramming are addressed in this experimental study. Materials and Methods: In this experimental study, Oct4 expressing lentiviral particles and small molecules BIX-01294, Bay K8644 and RG-108 were injected into the right ventricle of mice brain and VPA was systematically administered as oral gavages. Animals treated with different combinations of small molecules for 7 or 14 days in concomitant with Oct4 exogenous expression were compared for expression of pluripotency markers. Total RNA was isolated from the rims of the injected ventricle and quantitative polymerase chain reaction (PCR) was performed to evaluate the expression of endogenous Oct4, Nanog, c-Myc, klf4 and Sox2 as pluripotency markers, and Pax6 and Sox1 as neural stem cell (NSC) markers. Results: Results showed that Oct4 exogenous expression for 7 days induced pluripotency slightly as it was detected by significant enhancement in expression of Nanog (p<0.05). Combinatorial administration of Oct4 expressing vector and BIX-01294, Bay K8644 and RG-108 did not affect the expression of pluripotency and NSC markers, but VPA treatment along with Oct4 exogenous expression induced Nanog, Klf4 and c-Myc (p<0.001). VPA treatment before the induction of exogenous Oct4 was more effective and significantly increased the expression of endogenous Oct4, Nanog, Klf4, c-Myc (p<0.01), Pax6 and Sox1 (p<0.001). Conclusion: These results suggest VPA as the best enhancer of pluripotency among the chemicals tested, especially when applied prior to pluripotency induction by Oct4

The gut-brain axis affecting TLR4 in Parkinson’s diseases: Gut-brain axis in Parkinson's disease

Parkinson’s disease (PD) is a chronic neurodegenerative illness, which is increasing in developing countries and creating a burden on these economies. Multiple motor and non-motor symptoms have been connected to PD, and patients are diagnosed using clinical observations. Unfortunately, clinical symptoms are found in the late stages of the disease, when preventing is no longer an option. The gastrointestinal system, and more specifically the gut microbiota have an important role in the bidirectional communication taking place between the gut and brain. As a result, the gut microbiota dysbiosis and its effect on the brain-gut axis are among important factors to be considered in PD pathology. In addition, the role of Toll-like receptors (TLRs) in recognizing pathogenic molecules and creating immune responses can affect PD pathogenesis. In this review, we have tried to better understand the effect of TLR4 on the gut microbiota and their dysbiosis, creating a bidirectional feedback loop. Even though the effect of blocking TLR4 signaling on PD is still not well known, it affects PD, and brings hope of using microbial based medications to control this chronic neurodegenerative disease

Comparison of Rat Primary Midbrain Neurons Cultured in DMEM/F12 and Neurobasal Mediums

Introduction: Midbrain dopaminergic neurons are involved in various brain functions, including motor behavior, reinforcement, motivation, learning, and cognition. Primary dopaminergic neurons and also several lines of these cells are extensively used in cell culture studies. Primary dopaminergic neurons prepared from rodents have been cultured in both DMEM/F12 and neurobasal mediums in several studies. However, there is no document reporting the comparison of these two mediums. So in this study, we evaluated the neurons and astroglial cells in primary midbrain neurons from rat embryos cultured in DMEM/F12 and neurobasal mediums. Methods: Primary mesencephalon cells were prepared from the E14.5 rat embryo. Then they were seeded in two different mediums ( Dulbeccochr('39')s Modified Eagle Medium/Nutrient Mixture F-12 [DMEM/F12] and neurobasal). On day 3 and day 5, half of the medium was replaced with a fresh medium. On day 7, β3-tubulin-, GFAP (Glial fibrillary acidic protein)- and Tyrosine Hydroxylase TH-positive cells were characterized as neurons, astrocytes, and dopaminergic neurons, respectively, using immunohistochemistry. Furthermore, the morphology of the cells in both mediums was observed under light microscopy on days 1, 3, and 5. Results: The cells cultured in both mediums were similar under light microscopy regarding the cell number, but in a neurobasal medium, the cells have aggregated and formed clustering structures. Although GFAP-immunoreactive cells were lower in neurobasal compared to DMEM/F12, the number of β3-tubulin- and TH-positive cells in both cultures was the same. Conclusion: This study’s findings demonstrated that primary midbrain cells from the E14.5 rat embryo could grow in both DMEM/F12 and neurobasal mediums. Therefore, considering the high price of a neurobasal medium, it can be replaced with DMEM/F12 for culturing primary dopaminergic neurons

The Effect of Intrahippocampal Insulin Injection on Scopolamine-induced Spatial Memory Impairment and Extracellular Signal-regulated Kinases Alteration

Introduction: It is well documented that insulin has neuroprotective and neuromodulator effects and can protect against different models of memory loss. Furthermore, cholinergic activity plays a significant role in memory, and scopolamine-induced memory loss is widely used as an experimental model of dementia. The current study aimed at investigating the possible effects of insulin against scopolamine-induced memory impairment in Wistar rat and its underlying molecular mechanisms. Methods: Accordingly, animals were bilaterally cannulated in CA1, hippocampus. Intrahippocampal administration of insulin 6 MU and 12 MU in CA1 per day was performed during first 6 days after surgery. During next four days, the animal’s spatial learning and memory were assessed in Morris water maze test (three days of learning and one day of retention test). The animals received scopolamine (1 mg/kg) Intraperitoneally (IP) 30 minutes before the onset of behavioral tests in each day. In the last day, the hippocampi were dissected and the levels of MAPK (mitogen-activated protein kinases) and caspase-3 activation were analyzed by Western blot technique.  Results: The behavioral results showed that scopolamine impaired spatial learning and memory without activating casapase-3, P38, and JNK, but chronic pretreatment by both doses of insulin was unable to restore this spatial memory impairment. In addition, scopolamine significantly reduced Extracellular signal-Regulated Kinases (ERKs) activity and insulin was unable to restore this reduction. Results revealed that scopolamine-mediated memory loss was not associated with hippocampal damage. Conclusion: Insulin as a neuroprotective agent cannot restore memory when there is no hippocampal damage. In addition, the neuromodulator effect of insulin is not potent enough to overwhelm scopolamine-mediated disruptions of synaptic neurotransmission

Investigating the role of P38, JNK and ERK in LPS induced hippocampal insulin resistance and spatial memory impairment

Despite the consensus that neuro-inflammation and insulin resistance (IR) are two hallmarks of Alzheimer disease (AD), the molecular mechanisms responsible for the development of IR remain uncharacterized. MAPKs are signaling molecules that are implicated in the pathology of AD and have a role in IR development. Given that inflammatory mediators are shown to interfere with insulin signaling pathway in different cell types, the present work aimed to investigate whether neuro-inflammation induced memory loss is associated with hippocampal IR and whether insulin treatment protects against this IR. Subsequently, possible roles of MAPKs in this situation were investigated. Male Wistar rats were cannulated, and LPS (15 μg, day 0), insulin (3 mU) or saline (vehicle) were administered intra-cerebroventricularly (ICV) (days 1-6). Spatial memory performance was assessed during days 7-10 by Morris Water Maze test. Consequently, analysis of the amount of hippocampal phosphorylated forms of P38, JNK, ERK, IRS1 (ser307) and Akt (ser473) were done by Western blot. The outcomes indicated that while LPS induced memory loss and hippocampal IR (shown by elevated IRS1 and decreased Akt phosphorylation),insulin treatment nullified these effects. Molecular results also showed that LPS mediated IR and memory loss are associated with P38 but not JNK and ERK activation; this P38 activation was reversed by insulin treatment. These observations implied that one of the ways by which neuro-inflammation participates in AD is via induction of IR. It seems that this IR is mainly mediated by P38. Therefore, P38 could be considered as a molecular target for preventing IR development