Heat-induced antigen retrieval utilizing modified Tris-EDTA buffer for reprobing of Western blots on nitrocellulose paper.

The development of heat-induced antigen retrieval technologies with Tris-EDTA buffer has dramatically improved immunostaining of specific antigens for routine immunohistochemical detection (Krenacs et al., 2010) [1]. However, little evidence exists on whether heat-Induced antigen retrieval utilizing Tris-EDTA buffer can strip western blot (WB) membranes and allow sequential reprobing. Here, we serendipitously discover that ∼95 °C Tris-EDTA buffer with 0.01% Tween 20 could repeatedly strip the Nitrocellulose membranes (NC). After electroblotting, NC blots were soaked into Tris-EDTA stripping buffer (∼95 °C, 10-25min) and we could perform at least five rounds (the following antibodies used: Vinculin, Atg7, Caspase-3, UBA5, JNK and ERK1/2) stripping in sequential chemiluminescent detections. The NC membranes also show clear western signals and background without losing transferred proteins during the reprobing process of WB. Hence, this study report additional new roles of the heat-Induced antigen retrieval Tris-EDTA buffer with 0.01% Tween 20. The method is simpler, more affordable and harmless for the nitrocellulose paper, which will be helpful for effective reprobing in western blotting applications.

Inactivation of horseradish peroxidase with crotonic acid for reprobing of western blotting.

Inactivation of horseradish peroxidase (HRP) treatment is a conventional preference to stripping for sequential detections of different proteins of chemiluminescent western blotting (WB). However, little evidence exists on whether other chemical substances treatment can affects the biological activity of HRP during stripping and re-probing of WB blots. Here, we successfully develop 20% crotonic acid (CA) as an alternative to stripping to inhibit HRP used for sequential chemiluminescent WB on polyvinylidene difluoride (PVDF) and Nitrocellulose (NC) membrane. Moreover, NC blots incubation in CA (40 °C, 30min) allow us to perform three round HRP inhibition in sequential detections without losing transferred proteins and damaging membrane. Hence, the method will help us save time and valuable samples without the need to rerun gels.

Total protein staining with Congo red as an alternative loading control for western blot analysis.

For western blot analysis, a housekeeping protein, such as ß-actin or glyceraldehyde-3-phosphate dehydrogenase, is used as loading control with the assumption that these proteins are stable. In practice, these internal loading control proteins vary with different cell states and tissue types. These internal standards are not appropriate for use with serum, extracellular secretion, cerebrospinal fluid analysis or for protein purification. We investigated total protein measurement using Congo red staining and found it to be a superior alternative to routine loading controls. Advantages include lower cost, technical simplicity and improved linear regression. We propose using Congo red staining for total protein immunoblotting to evaluate protein loading in western blots.

Short-term high-fat diet favors the appearances of apoptosis and gliosis by activation of ERK1/2/p38MAPK pathways in brain.

High-fat diet (HFD) has been associated with neuroinflammation and apoptosis in distinct brain regions. To explore the effect of short-term (7, 14 and 21 days) high-fat overfeeding on apoptosis, inflammatory signaling proteins, APP changes and glial cell activities in cerebral cortex and cerebellum. Mice were fed with HFD for different lengths (up to 21 days) and after each time body weights of mice was tested, then the apoptotic proteins, IL-1ß, APP, BACE1and MAPKs, Akt and NF-κB signaling activity were evaluated by western blots. Results demonstrate that short period of high-fat overnutrition significantly promotes apoptosis, APP expression at day 21 of cerebral cortex and at day 7 of cerebellum compared to chow diet. In addition, increased GFAP+astrocytes, Iba-1+microglia and IL-1ß 30 were observed in cerebral cortex after 21 days HFD, but no changes for 7 days overfeeding of cerebellum. Serendipitously, ERK1/2 pathway was activated both in cerebral cortex and cerebellum for different time course of HFD. Furthermore, increased phospho-p38 MAPK level was observed in cerebellum only. In consistent with in vivo results, SH-SY5Y cells treatment with cholesterol (50 µM, 100 µM) for 48 h culture in vitro demonstrated that pro-apoptotic proteins were enhanced as well. In brief, short-term HFD consumption increases sensitivity to apoptosis, APP and IL-1ß production as well as gliosis in cerebral cortex and cerebellum, which may be related to enhancement of ERK1/2 and p38 MAPK activation.

Nogo-A-Δ20/EphA4 interaction antagonizes apoptosis of neural stem cells by integrating p38 and JNK MAPK signaling.

Nogo-A protein consists of two main extracellular domains: Nogo-66 (rat amino acid [aa] 1019-1083) and Nogo-A-Δ20 (extracellular, active 180 amino acid Nogo-A region), which serve as strong inhibitors of axon regeneration in the adult CNS (Central Nervous System). Although receptors S1PR2 and HSPGs have been identified as Nogo-A-Δ20 binding proteins, it remains at present elusive whether other receptors directly interacting with Nogo-A-Δ20 exist, and decrease cell death. On the other hand, the key roles of EphA4 in the regulation of glioblastoma, axon regeneration and NSCs (Neural Stem Cells) proliferation or differentiation are well understood, but little is known the relationship between EphA4 and Nogo-A-Δ20 in NSCs apoptosis. Thus, we aim to determine whether Nogo-A-Δ20 can bind to EphA4 and affect survival of NSCs. Here, we discover that EphA4, belonging to a member of erythropoietin-producing hepatocellular (Eph) receptors family, could be acting as a high affinity ligand for Nogo-A-Δ20. Trans-membrane protein of EphA4 is needed for Nogo-A-Δ20-triggered inhibition of NSCs apoptosis, which are mediated by balancing p38 inactivation and JNK MAPK pathway activation. Finally, we predict at the atomic level that essential residues Lys-205, Ile-190, Pro-194 in Nogo-A-Δ20 and EphA4 residues Gln-390, Asn-425, Pro-426 might play critical roles in Nogo-A-Δ20/EphA4 binding via molecular docking.

A sensitive and reversible staining of proteins on blot membranes.

A modified, sensitive and reversible method for protein staining on nitrocellulose (NC) and polyvinylidine fluoride (PVDF) membranes was developed in Western blotting. The method employed Congo red staining to visualize proteins on different blot membranes. Staining of proteins with Congo red dye is more faster procedures. According to the experimental results, approximate 20 ng proteins could be detected in 3 min in room temperature. The staining on the proteins is easily reversible with Congo red destaining solution for NC and PVDF membranes, so that the blot membranes can be reused for Western blotting. In addition, we confirmed that the staining method is fully compatible with Western blot detection. NC and PVDF membranes treatment with Congo red staining does not interfere with conventional chemiluminescent substrates of peroxidase. As compared to MemCode reversible protein stain kits from Pirece Biotechnology, the staining technique is more sensitive, lower of cost, convenient and not adversely affecting subsequent Western blotting results. On the other hand, the stain is more sensitive than the Ponceau S staining. Therefore, Congo red staining is a promising and ideal alternative for current protein stain. Besides, the binding modes of Congo red or Ponceau S stain were investigated using various 2D and 3D molecular docking and demonstrated potential molecular basis for sensitivity of Congo red staining are higher than Ponceau S.

Astrocytes autophagy in aging and neurodegenerative disorders.

Astrocytes can serve multiple functions in maintaining cellular homeostasis of the central nervous system (CNS), and normal functions for autophagy in astrocytes is considered to have very vital roles in the pathogenesis of aging and neurodegenerative diseases. Autophagy is a major intracellular lysosomal (or its yeast analog, vacuolar) clearance pathways involved in the degradation and recycling of long-lived proteins, oxidatively damaged proteins and dysfunctional organelles by lysosomes. Current evidence has shown that autophagy might influence inflammation, oxidative stress, aging and function of astrocytes. Although the interrelation between autophagy and inflammation, oxidative stress, aging or neurological disorders have been addressed in detail, the influence of astrocytes mediated-autophagy in aging and neurodegenerative disorders has yet to be fully reviewed. In this review, we will summarize the most up-to-date findings and highlight the role of autophagy in astrocytes and link autophagy of astrocytes to aging and neurodegenerative diseases. Due to the prominent roles of astrocytic autophagy in age-related neurodegenerative diseases, we believe that we can provide new suggestions for the treatment of these disorders.

Tph2 Genetic Ablation Contributes to Senile Plaque Load and Astrogliosis in APP/PS1 Mice.

BACKGROUND: Amyloid-ß (Aß) accumulation plays a critical role in the pathogenesis of Alzheimer's disease (AD) lesions. Deficiency of Serotonin signaling recently has been linked to the increased Aß level in transgenic mice and humans. In addition, tryptophan hydroxylase-2 (Tph2), a second tryptophan hydroxylase isoform, controls brain serotonin synthesis. However, it remains to be determined that whether Tph2 deficient APP/PS1mice affect the formation of Aß plaques in vivo. METHODS: Both quantitative and qualitative immunochemistry methods, as well as Congo red staining were used to evaluate the Aß load and astrogliosis in these animals. RESULTS: we studied alterations of cortex and hippocampus in astrocytes and senile plaques by Tph2 conditional knockout (Tph2 CKO) AD mice from 6-10 months of age. Using Congo red staining and immunostained with Aß antibody, we showed that plaques load or plaques numbers significantly increased in Tph2 CKO experimental groups at 8 to 10 months old, compared to wild type (WT) group, respectively. Using GFAP+ astrocytes immunofluorescence method, we found that the density of GFAP+ astrocytes markedly enhanced in Tph2 CKO at 10 months. We showed Aß plaques co-localized autophagic markers LC3 and p62. Nevertheless, we did not observe any co-localization between GFAP+ astrocytes and autophagic markers, but detected the co-localization between ßIII-tubulin+ neurons and autophagic markers. CONCLUSION: Overall, our work provides the preliminary evidence in vivo that Tph2 plays a role in amyloid plaques generation.

The effect of curcumin on the differentiation, apoptosis and cell cycle of neural stem cells is mediated through inhibiting autophagy by the modulation of Atg7 and p62.

Curcumin is an orange-yellow colored, lipophilic polyphenol substance derived from the rhizome of Curcuma longa that is widely used in many countries. Curcumin has many reported functions, including antioxidant and anti­inflammatory effects. Autophagy removes damaged organelles and protein aggregates in the cell. However, whether curcumin mediates its effects on neural stem cell (NSC) differentiation, cell cycle and apoptosis through autophagy is unknown. In the present study, the effects of curcumin and 3­methyladenine (3MA; an autophagy inhibitor, as a positive control) on the autophagy, differentiation, cell cycle progression and apoptosis of NSCs in different culture states were examined. In order to confirm the role of autophagy in these processes of NSC behavioral change, the protein expression level changes of markers of autophagy, such as autophagy­related protein 7 (Atg7), light chain (LC)3 and p62, were assessed. When NSCs were in an adherent state, 10 µM curcumin inhibited their differentiation into GFAP+ astrocytes or DCX+ immature neurons, while Atg7 and p62 protein expression were also reduced compared with the untreated control group. When NSCs were in a suspended state, 10 µM curcumin inhibited the cell cycle progression and apoptosis of NSCs as determined by western blotting, which was associated with a decreased autophagic flux and Atg7 expression. In addition, the curcumin­treated group trended in a similar direction to the 3MA­treated group. Thus, the data suggest that curcumin can inhibit differentiation, promote cell survival and inhibit cell cycle progression from G1 to S in NSCs, and that these effects are mediated through the regulation of Atg7 and p62.

The ROCK/GGTase Pathway Are Essential to the Proliferation and Differentiation of Neural Stem Cells Mediated by Simvastatin.

Simvastatin, a lipophilic and fermentation-derived natural statin, is reported to treat neurological disorders, such as traumatic brain injury, Parkinson's disease (PD), Alzheimer disease (AD), etc. Recently, research also indicated that simvastatin could promote regeneration in the dentate gyrus of adult mice by Wnt/ß-catenin signaling (Robin et al. in Stem Cell Reports 2:9-17, 2014). However, the effect and mechanisms by which simvastatin may affect the neural stem cells (NSCs; from the embryonic day 14.5 (E14.5) SD rat brain) are not fully understood. Here, we investigated the effects of different doses of simvastatin on the survival, proliferation, differentiation, migration, and cell cycle of NSCs as well as underlying intracellular signaling pathways. The results showed that simvastatin not only inhibits the proliferation of NSCs but also enhances the ßIII-tubulin+ neuron differentiation rate. Additionally, we find that simvastatin could also promote NSC migration and induce cell cycle arrest at M2 phrase. All these effects of simvastatin on NSCs were mimicked with an inhibitor of Rho kinase (ROCK) and a specific inhibitor of geranylgeranyl transferase (GGTase). In conclusion, these data indicate that simvastatin could promote neurogenesis of neural stem cells, and these effects were mediated through the ROCK/GGTase pathway.

Autophagy mediates oral submucous fibrosis.

Oral submucous fibrosis (OSF) is a chronic insidious disease of the oral mucosa, well-recognized as a premalignant condition and commonly found in Southern China. It is primarily caused by the habit of areca nut or gutkha chewing. OSF is believed to be a homeostatic disorder of the extracellular matrix and fibroblast proliferation. The present study demonstrated a novel link between autophagy and OSF. Tissue samples from human OSF showed an overexpression of the autophagy marker microtubule-associated protein 1 light chain 3 using immunohistochemistry and quantitative polymerase chain reaction. With regard to the crucial role of transforming growth factor (TGF)-ß in OSF disease, western blot analysis demonstrated that TGF-ß signaling was shown to contribute to the activation of autophagy in fibroblasts in vitro; however, a cell apoptosis and MTS assay demonstrated that the suppression of autophagy ameliorated the fibrosis induced by active TGF-ß receptor type I signaling, as well as promoted fibroblast apoptosis and suppressed proliferation. Therefore, the present results suggest that autophagy serves a crucial function in OSF.

The Emerging Therapeutic Role of NGF in Alzheimer's Disease.

Alzheimer's disease (AD) is the most common type of neurodegenerative dementia that affects the elderly population. Nerve growth factor (NGF) contributes to the survival, regeneration and death of neurons during aging and in neurodegenerative diseases. Recently, research has shown that NGF is related to the pathology, mechanisms and symptoms of AD. Therefore, there is a need to summarize the new advancements in NGF research and its potential therapeutic implications in AD. In this review, we will focus on NGF distribution, production, and function; the interaction of Aß and NGF; and the effect of different therapy methods on AD. In summary, we hope to describe the experimental and clinical data demonstrating the important roles of NGF for AD treatment.

Ninety day toxicity and toxicokinetics of fluorochloridone after oral administration in rats.

The ninety day toxicity and toxicokinetics of fluorochloridone (FLC) were accessed in Wistar rats. Animals were gavaged with FLC at doses of 31.25 mg/kg, 125 mg/kg and 500 mg/kg for ninety days, followed by thirty days for recovery. On the 1st, 60th, 75th and 90th days of the dosing phase, plasma of ten animals of all groups treated with FLC was collected for toxicokinetic analysis of FLC by an UPLC-MS/MS method. Numerous changes in body weight, hematology, serum chemistry, and organ weight ratios were observed by the 45th and 90th dosing day. Most changes in groups treated with FLC were absent on the last recovery day. Testis and epididymis lesions were consistently seen in histopathological observations on the 45th, 90th dosing day and the last recovery day. Repeated administration of FLC increased the level of testosterone in serum in male rats on the 90th dosing day. FLC plasma concentrations could be detected in all animal drug-treated groups during the dosing phase, and a dose proportional relationship was seen between FLC dose and AUC or Cmax. This study will support future studies on the mechanism of FLC-induced toxicity.

The Neural Stem Cell Microenvironment: Focusing on Axon Guidance Molecules and Myelin-Associated Factors.

Neural stem cells (NSCs) could produce various cell phenotypes in the subventricular zone (SVZ) and dentate gyrus of the hippocampus in the central nervous system (CNS), where neurogenesis has been determined to occur. The extracellular microenvironment also influences the behaviors of NSCs during development and at CNS injury sites. Our previous study indicates that myelin, a component of the CNS, could regulate the differentiation of NSCs in vitro. Recent reports have implicated three myelin-derived inhibitors, NogoA, myelin-associated glycoprotein (MAG), and oligodendrocyte-myelin glycoprotein (OMgp), as well as several axon guidance molecules as regulators of NSC survival, proliferation, migration, and differentiation. However, the molecular mechanisms underlying the behavior of NSCs are not fully understood. In this study, we summarize the current literature on the effects of different extrinsic factors on NSCs and discuss possible mechanisms, as well as future possible clinical applications.

Evaluation of blood-brain barrier permeability in tryptophan hydroxylase 2-knockout mice.

The blood-brain barrier (BBB) is critical to the health of the central nervous system (CNS). The possibility that 5-hydroxytryptamine (5-HT) participates in the alteration of the BBB has been previously demonstrated. Tryptophan hydroxylase 2 (TPH2) is a unique genetic enzyme isoform that catalyzes the rate-limiting step in the biosynthesis of 5-HT in the CNS; however, its role in the permeability changes of the BBB remains unclear. In the present study, TPH2-knockout mice were utilized in the assessment of BBB disruption, as measured by the Evans Blue (EB) extravasation or fluorescein isothiocyanate-albumin leakage assay in the brain. EB was not found to be retained in the brain in the TPH2-knockout mice or the wild-type controls. The results of the study demonstrate that TPH2 knockout has no effect on BBB permeability, indicating that TPH2 and the 5-HT system in the CNS are not sufficient to influence the BBB leakage.

[Transplantation of neural stem cells and bone marrow mesenchymal stem cells in treatment of spinal cord injury].

OBJECTIVE: To review the progress in the treatment of spinal cord injury (SCI) by graft of neural stem cells (NSCs) or bone marrow mesenchymal stem cells (BMSCs) as well as immune characteristics of two stem cells. METHODS: Different kinds of documents were widely collected, and then immunologic characteristics of NSCs and BMSCs were summarized. The therapy of SCI by stem cell transplantation was reviewed. Additionally, some problems in treatment were analyzed. RESULTS: Experimental study showed that graft of NSCs and BMSCs can promote the functional recovery of the injured spinal cord in animals. Due to immunologic properties of two stem cells, rejection reaction of transplantation could produce a harmful effect on SCI treatment. CONCLUSION: Transplantation of NSCs or BMSCs might be an effective measure for SCI treatment, but immunologic rejection reaction must be considered.

Effect of central myelin on the proliferation and differentiation into O4(+) oligodendrocytes of GFP-NSCs.

Myelin is a biologically active mutilamellar that is formed by oligodendrocytes (OLs) in the central nervous system (CNS) and ensheathes axons (Ishii et al., Proc Natl Acad Sci USA 106:14605-14610, 2009). Myelin damage is related to neurological trauma such as spinal cord injury (SCI). In this article, we investigated whether myelin derived from rat spinal cord can influence the proliferation of neural stem cells (NSCs) and NSCs differentiation into oligodendrocytes in vitro. After extracting myelin, we verified that myelin preparation was successful by western blot analysis. Then, we explored the effects of different myelin concentrations on the NSCs proliferation by MTT assays. Our results showed that 2 µg/ml myelin can promote the proliferation of NSCs, while NgR antibody can antagonize the effect. In addition, myelin can inhibit the differentiation of NSCs into O4(+) oligodendrocytes impeding them maturation. In conclusion, these results suggested that central myelin can affect the proliferation and differentiation of NSCs, thus promoting us to understand further the complex roles of myelin in NSCs after CNS injury.

Long-term fate of allogeneic neural stem cells following transplantation into injured spinal cord.

To characterize the fate of allogeneic neural stem cells (NSCs) following transplantation into injured spinal cord, green fluorescent protein (GFP)-NSCs isolated from GFP transgenic Sprague-Dawley rat embryos were transplanted into contused spinal cords of Wistar rats. The GFP-NSCs survived for at least 6 months in injured spinal cord; most of them differentiated rapidly into astrocytes, and a few were able to undergo proliferation. After transplantation, the GFP-NSCs remained in the transplantation site at the early stage, and then migrated along white-matter, and gathered around the injured cavity. At 6 months post-transplantation, CD8 T-lymphocytes infiltrated the spinal cord, and mixed lymphocyte culture from host and donor showed that lymphocytes from the host spleen were primed by allogeneic GFP-NSCs. At 12 months post-transplantation, most GFP cells in the spinal cord lost their morphology and disintegrated. The Basso, Beattie and Bresnahan score and footprint analysis indicated that the improvement of locomotor function in transplanted rats appeared only at the early stage, and was not seen even at 6 months after transplantation All these results suggest that the allogeneic NSCs, after transplantation into injured spinal cord, activate the host immune system. Therefore, if immunosuppressive agents are not used, the grafted allogeneic NSCs, although they can survive for a long time, are subjected to host immune rejection, and the effect of NSCs on functional recovery is limited.