Exosomal miR-132-3p from Mesenchymal Stromal Cells Improve Synaptic Dysfunction and Cognitive Decline in Vascular Dementia

Background/Aims: Vascular dementia (VD) results in cognition and memory deficit. Exosomes and their carried microRNAs (miRs) contribute to the neuroprotective effects of mesenchymal stromal cells, and miR-132-3p plays a key role in neuron plasticity. Here we investigated the role and underlying mechanism of MSC EX and their miR-132-3p cargo in rescuing cognition and memory deficit in VD mice. Methods: Bilateral carotid artery occlusion was used to generate a VD mouse model. MiR-132-3p and MSC EX levels in the hippocampus and cortex were measured. At 24 h post-VD induction, mice were administered with MSC EX infected with control lentivirus (EXCon), pre-miR-132-3p-expressing lentivirus (EXmiR−132−3p), or miR-132-3p antago lentivirus (EXantagomiR−132−3p) intravenously. Behavioral and cognitive tests were performed and the mice were sacrificed in 21 days after VD. The effects of MSC EX on neuron number, synaptic plasticity, dendritic spine density, and Aβ and p-Tau levels in the hippocampus and cortex were determined. The effects of MSC EX on oxygen-glucose deprivation (OGD)-injured neurons with respect to apoptosis, and neurite elongation and branching were determined. Finally, the expression levels of Ras, phosphorylation of Akt, GSK-3β, and Tau were also measured. Results: Compared with normal mice, VD mice exhibited significantly decreased miR-132-3p and MSC EX levels in the cortex and hippocampus. Compared with EXCon treatment, the infusion of EXmiR−132−3p was more effective at improving cognitive function and increasing miR-132-3p level, neuron number, synaptic plasticity, and dendritic spine density, while decreasing Aβ and p-Tau levels in the cortex and hippocampus of VD mice. Conversely, EXantagomiR−132−3p treatment significantly decreased miR-132-3p expression in cortex and hippocampus, as well as attenuated EXmiR−132−3p treatment-induced functional improvement. In vitro, EXmiR−132−3p treatment inhibited RASA1 protein expression, but increased Ras and the phosphorylation of Akt and GSK-3β, and decreased p-Tau levels in primary neurons by delivering miR-132-3p, which resulted in reduced apoptosis, and increased neurite elongation and branching in OGD-injured neurons. Conclusions: Our studies suggest that miR-132-3p cluster-enriched MSC EX promotes the recovery of cognitive function by improving neuronal and synaptic dysfunction through activation of the Ras/Akt/GSK-3β pathway induced by downregulation of RASA1.

In Vitro and In Vivo Effects of Nobiletin on DRG Neurite Elongation and Axon Growth after Sciatic Nerve Injury

Sciatic nerve injury (SNI) leads to sensory and motor dysfunctions. Nobiletin is a major component of polymethoxylated flavonoid extracted from citrus fruits. The role of nobiletin on sciatic nerve regeneration is still unclear. Thus, the purpose of this study was to investigate whether nobiletin increases DRG neurite elongation and regeneration-related protein expression after SNI. Cytotoxicity of nobiletin was measured in a concentration–dependent manner using the MTT assay. For an in vitro primary cell culture, the sciatic nerve on the middle thigh was crushed by holding twice with forceps. Dorsal root ganglion (DRG) and Schwann cells were cultured 3 days after SNI and harvested 36 h later and 3 days later, respectively. In order to evaluate specific regeneration-related markers and axon growth in the injured sciatic nerve, we applied immunofluorescence staining and Western blot techniques. Nobiletin increased cell viability in human neuroblastoma cells and inhibited cytotoxicity induced by exposure to H2O2. Mean neurite length of DRG neurons was significantly increased in the nobiletin group at a dose of 50 and 100 μM compared to those at other concentrations. GAP-43, a specific marker for axonal regeneration, was enhanced in injury preconditioned Schwann cells with nobiletin treatment and nobiletin significantly upregulated it in injured sciatic nerve at only 3 days post crush (dpc). In addition, nobiletin dramatically facilitated axonal regrowth via activation of the BDNF-ERK1/2 and AKT pathways. These results should provide evidence to distinguish more accurately the biochemical mechanisms regarding nobiletin-activated sciatic nerve regeneration.

Glutathione S-transferase Pi (Gstp) Proteins Regulate Neuritogenesis in the Developing Cerebral Cortex

GSTP proteins are metabolic enzymes involved in removal of oxidative stress and intracellular signaling and also have inhibitory effects on JNK activity. However, the functions of Gstp proteins in the developing brain are unknown. In mice, there are three Gstp proteins, Gstp1, 2 and 3, while there is only one GSTP in humans. By RT-PCR analysis, we found that Gstp1 was expressed beginning at E15.5 in the cortex, but Gstp2 and 3 started expressing at E18.5. Gstp 1 and 2 knockdown caused decreased neurite number in cortical neurons, implicating them in neurite initiation. Using in utero electroporation to knockdown Gstp1 and 2 in layer 2/3 pyramidal neurons in vivo, we found abnormal swelling of the apical dendrite at P3 and reduced neurite number at P15. Using time-lapse live imaging, we found that the apical dendrite orientation was skewed compared to the control, but these defects were ameliorated. Overexpression of Gstp 1 or 2 resulted in changes in neurite length, suggesting a role in neurite elongation. We explored the molecular mechanism and found that JNK inhibition rescued reduced neurite number caused by Gstp knockdown, indicating that Gstp regulates neurite formation through JNK signaling. Thus, we found novel functions of Gstp proteins in neurite initiation during cortical development. Furthermore, the overexpression experiments suggest different functions of Gstp1 and 2 in neurite elongation. Since previous studies have shown the potential implication of Gstp in Autism Spectrum Disorder, our findings will attract more clinical interests in Gstp proteins in neurodevelopmental disorders.SignificanceNeurite formation, including neurite initiation and elongation, is the first step of generating polarized neuronal morphology in developing neurons, and thus is essential for establishing a neuronal network. Therefore, it is crucial to understand the mechanisms of neurite formation. Limited studies have been performed to clarify the mechanisms of neurite formation, especially neurite initiation. In this present study, we report a novel, essential role of Gstp in neurite initiation in mouse cortical neurons in vitro and in vivo. We also found that Gstp regulates neurite formation via JNK signaling pathways. These findings not only provide novel functions of Gstp proteins in neuritogenesis during cortical development but also help us to understand the complexity of neurite formation.

Madecassic Acid Reduces Fast Transient Potassium Channels and Promotes Neurite Elongation in Hippocampal CA1 Neurons

Background and Objective: Madecassic Acid (MA) is well known to induce neurite elongation. However, its correlation with the expression of fast transient potassium (AKv) channels during neuronal development has not been well studied. Therefore, the present study was designed to investigate the effects of MA on the modulation of AKv channels during neurite outgrowth. Methods: Neurite outgrowth was measured with morphometry software, and Kv4 currents were recorded by using the patch clamp technique. Results: The ability of MA to promote neurite outgrowth is dose-dependent and was blocked by using the mitogen/extracellular signal-regulated kinase (MEK) inhibitor U0126. MA reduced the peak current density and surface expression of the AKv channel Kv4.2 with or without the presence of NaN3. The surface expression of Kv4.2 channels was also reduced after MA treatment of growing neurons. Ethylene glycol tetraacetic acid (EGTA) and an N-methyl-D-aspartate (NMDA) receptor blocker, MK801 along with MA prevented the effect of MA on neurite length, indicating that calcium entry through NMDA receptors is necessary for MA-induced neurite outgrowth. Conclusion: The data demonstrated that MA increased neurite outgrowth by internalizing AKv channels in neurons. Any alterations in the precise density of ion channels can lead to deleterious consequences on health because it changes the electrical and mechanical function of a neuron or a cell. Modulating ion channel’s density is exciting research in order to develop novel drugs for the therapeutic treatment of various diseases of CNS.

Quercetin is a Useful Medicinal Compound Showing Various Actions Including Control of Blood Pressure, Neurite Elongation and Epithelial Ion Transport

Quercetin has multiple potential to control various cell function keeping our body condition healthy. In this review article, we describe the molecular mechanism on how quercetin exerts its action on blood pressure, neurite elongation and epithelial ion transport based from a viewpoint of cytosolic Cl- environments, which is recently recognized as an important signaling factor in various types of cells. Recent studies show various roles of cytosolic Cl- in regulation of blood pressure and neurite elongation, and prevention from bacterial and viral infection. We have found the stimulatory action of quercetin on Cl- transporter, Na+-K+-2Cl- cotransporter 1 (NKCC1; an isoform of NKCC), which has been recognized as one of the most interesting, fundamental actions of quercetin. In this review article, based on this stimulatory action of quercetin on NKCC1, we introduce the molecular mechanism of quercetin on: 1) blood pressure, 2) neurite elongation, and 3) epithelial Cl- secretion including tight junction forming in epithelial tissues. 1) Quercetin induces elevation of the cytosolic Cl- concentration via activation of NKCC1, leading to anti-hypertensive action by diminishing expression of epithelial Na+ channel (ENaC), a key ion channel involved in renal Na+ reabsorption, while quercetin has no effects on the blood pressure with normal salt intake. 2) Quercetin also has stimulatory effects on neurite elongation by elevating the cytosolic Cl- concentration via activation of NKCC1 due to tubulin polymerization facilitated through Cl--induced inhibition of GTPase. 3) Further, in lung airway epithelia quercetin stimulates Cl- secretion by increasing the driving force for Cl- secretion via elevation of the cytosolic Cl- concentration: this leads to water secretion, participating in prevention of our body from bacterial and viral infection. In addition to transcellular ion transport, quercetin regulates tight junction function via enhancement of tight junction integrity by modulating expression and assembling tight junction-forming proteins. Based on these observations, it is concluded that quercetin is a useful medicinal compound keeping our body to be in healthy condition.