Insights of Extracellular Vesicles of Mesenchymal Stem Cells: a Prospective Cell-Free Regenerative Medicine for Neurodegenerative Disorders.

Mesenchymal stem cells (MSCs) are multipotent, adult stem cells which are found in numerous tissues like the umbilical cord, Wharton's jelly, bone marrow, and adipose tissue. They possess the capacity of self-renewal by dividing and differentiating into various cellular lineages. Their characteristic therapeutic potential exploited so far has made them a desirable candidate in regenerative medicine. Neurodegenerative diseases (NDs) like Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and ischemic stroke have been treated with MSCs and MSC-derived products. Over the past few decades, we have witnessed significant contributions in discovering the etiology of various NDs and their possible therapeutic solutions. One of the MSC-based therapeutics is extracellular vesicles (EVs), which contain multiple biologically active molecules like nucleic acids and proteins. The contents of EVs are ferried between cells for intercellular communication which then leads to regulation of the homeostasis of recipient cells. EVs serve as a considerable means of cell-free therapies like for tissue repair or regeneration as EVs can maintain therapeutically effective cargo of parent cells and are free of various ethical issues in cell-based therapies. Due to paucity of standard protocols in extraction procedures of EVs and their pharmacological properties and mechanisms, the development of new EV dependent therapies is challenging. With this review, an attempt has been made to annotate these mechanisms, which can help advance the novel therapeutic approaches towards the treat and define a more narrowed down approach for each ND to devise effective MSC-based therapies to cure and avert these diseases.

Secretome of Differentiated PC12 Cells Restores the Monocrotophos-Induced Damages in Human Mesenchymal Stem Cells and SHSY-5Y Cells: Role of Autophagy and Mitochondrial Dynamics.

A perturbed cellular homeostasis is a key factor associated with xenobiotic exposure resulting in various ailments. The local cellular microenvironment enriched with secretory components aids in cell-cell communication that restores this homeostasis. Deciphering the underlying mechanism behind this restorative potential of secretome could serve as a possible solution to many health hazards. We, therefore, explored the protective efficacy of the secretome of differentiated PC12 cells with emphasis on induction of autophagy and mitochondrial biogenesis. Monocrotophos (MCP), a widely used neurotoxic organophosphate, was used as the test compound at sublethal concentration. The conditioned medium (CM) of differentiated PC12 cells comprising of their secretome restored the cell viability, oxidative stress and apoptotic cell death in MCP-challenged human mesenchymal stem cells and SHSY-5Y, a human neuroblastoma cell line. Delving further to identify the underlying mechanism of this restorative effect we observed a marked increase in the expression of autophagy markers LC3, Beclin-1, Atg5 and Atg7. Exposure to autophagy inhibitor, 3-methyladenine, led to a reduced expression of these markers with a concomitant increase in the expression of pro-apoptotic caspase-3. Besides that, the increased mitochondrial fission in MCP-exposed cells was balanced with increased fusion in the presence of CM facilitated by AMPK/SIRT1/PGC-1α signaling cascade. Mitochondrial dysfunctions are strongly associated with autophagy activation and as per our findings, cellular secretome too induces autophagy. Therefore, connecting these three potential apices can be a major breakthrough in repair and rescue of xenobiotic-damaged tissues and cells.

Secretome of Differentiated PC12 Cells Enhances Neuronal Differentiation in Human Mesenchymal Stem Cells Via NGF-Like Mechanism.

The secretome-mediated responses over cellular physiology are well documented. Stem cells have been ruling the field of secretomics and its role in regenerative medicine since the past few years. However, the mechanistic aspects of secretome-mediated responses and the role of other cells in this area remain somewhat elusive. Here, we investigate the effects of secretome-enriched conditioned medium (CM) of neuronally differentiated PC12 cells on the neuronal differentiation of human mesenchymal stem cells (hMSCs). The exposure to CM at a ratio of 1:1 (CM: conditioned medium of PC12 cells) led to neuronal induction in hMSCs. This neuronal induction was compared with a parallel group of cells exposed to nerve growth factor (NGF). There was a marked increase in neurite length and expression of neuronal markers (ß-III tubulin, neurofilament-M (NF-M), synaptophysin, NeuN in exposed hMSCs). Experimental group co-exposed to NGF and CM showed an additive response via MAPK signaling and directed the cells particularly towards cholinergic lineage. The ability of CM to enhance the neuronal properties of stem cells could aid in their rapid differentiation into neuronal subtypes in case of stem cell transplantation for neuronal injuries, thus broadening the scope of non-stem cell-based applications in the area of secretomics.

Resveratrol Prevents the Cellular Damages Induced by Monocrotophos via PI3K Signaling Pathway in Human Cord Blood Mesenchymal Stem Cells.

The role of resveratrol (RV) as a neuroprotectant is well recognized, and cellular molecules involved in imparting the physiological effect have been well illustrated. However, some ambiguity still prevails as the specific receptor, and downstream signaling molecules are not yet clearly stated. So, we investigated the signaling pathway(s) involved in its cellular protection in the human umbilical cord blood mesenchymal stem cell (hUCB-MSC) derived neuronal cells. The mesenchymal stem cells were exposed to various concentrations (10, 100, 1000 µM) of monocrotophos (MCP), a known developmental neurotoxic organophosphate pesticide, for a period of 24 h. The MAPK signaling pathways (JNK, p38, and ERK) known to be associated with MCP-induced damages were also taken into consideration to identify the potential connection. The biological safe dose of RV (10 µM) shows a significant restoration in the MCP-induced alterations. Under the specific growth conditions, RV exposure was found to promote neuronal differentiation in the hUCB-MSCs. The exposure of cells to a specific pharmacological inhibitor (LY294002) of PI3K confirms the significant involvement of PI3K-mediated pathway in the ameliorative responses of RV against MCP exposure. Our data identifies the substantial role of RV in the restoration of MCP-induced cellular damages, thus proving to have a therapeutic potential against organophosphate pesticide-induced neurodegeneration.

PKA-GSK3ß and ß-Catenin Signaling Play a Critical Role in Trans-Resveratrol Mediated Neuronal Differentiation in Human Cord Blood Stem Cells.

The role of resveratrol (RV), a natural polyphenol, is well documented, although its role on neurogenesis is still controversial and poorly understood. Therefore, to decipher the cellular insights of RV on neurogenesis, we investigated the potential effects of the compound on the survival, proliferation, and neuronal differentiation of human cord blood-derived mesenchymal stem cells (hCBMSCs). For neuronal differentiation, purified and characterized hCBMSCs were exposed to biological safe doses of RV (10 µM) alone and in combination with nerve growth factor (NGF-50 ng). The cells exposed only to NGF (50 ng/mL) served as positive control for neuronal differentiation. The genes showing significant involvement in the process of neuronal differentiation were further funneled down at transcriptional and translational level. It was observed that RV promotes PKA-mediated neuronal differentiation in hCBMSCs by inducing canonical pathway. The studies with pharmacological inhibitors also confirmed that PKA significantly induces ß-catenin expression via GSK3ß induction and stimulates CREB phosphorylation and pERK1/2 induction. Besides that, the studies also revealed that RV additionally possesses the binding sites for molecules other than PKA and GSK3ß, with which it interacts. The present study therefore highlights the positive impact of RV over the survival, proliferation, and neuronal differentiation in hCBMSCs via PKA-mediated induction of GSK3ß, ß catenin, CREB, and ERK1/2.

A Case of Non-Traumatic Avascular Necrosis of Femur in Case of Transfusion-Dependent Thalassemia.

INTRODUCTION: Avascular necrosis of the head of femur (AVNF) has frequently been reported with sickle cell anemia but is not commonly associated with beta thalassemia. CASE REPORT: We report a case of 14-year-old male with transfusion-dependent thalassemia (TDT) and hepatitis C, who developed bilateral atraumatic AVNF requiring surgical correction. The likely etiopathogenesis and the review of literature for this uncommon finding are discussed. CONCLUSION: AVNF should be considered as a possibility in a patient with TDT presenting with hip pain. Multiple disease and treatment related factors are likely to play a key role in its causation.

Adoptive Autophagy Activation: a Much-Needed Remedy Against Chemical Induced Neurotoxicity/Developmental Neurotoxicity.

The profound significance of autophagy as a cell survival mechanism under conditions of metabolic stress is a well-proven fact. Nearly a decade-long research in this area has led scientists to unearth various roles played by autophagy other than just being an auto cell death mechanism. It is implicated as a vital cell survival pathway for clearance of all the aberrant cellular materials in case of cellular injury, metastasis, disease states, cellular stress, neurodegeneration and so on. In this review, we emphasise the critical role of autophagy in the environmental stressors-induced neurotoxicity and its therapeutic implications for the same. We also attempt to shed some light on the possible protective role of autophagy in developmental neurotoxicity (DNT) which is a rapidly growing health issue of the human population at large and hence a point of rising concern amongst researchers. The intimate association between DNT and neurodegenerative disorders strongly indicates towards adopting autophagy activation as a much-needed remedy for DNT.

Stem Cells in Neurotoxicology/Developmental Neurotoxicology: Current Scenario and Future Prospects.

Stem cell biology has played a pivotal role in the field of disease modeling, regenerative medicine, and tissue engineering. The scope of stem cell research has been further extended to address the issues associated with toxicity and biosafety. However, its role in the field of neurotoxicity (NT) and the emerging field of developmental neurotoxicity (DNT) is somewhat underrepresented and needs thorough investigation. Several challenges have hindered the progress of NT and DNT studies, and there is a dire need for human-specific high-throughput in vitro system(s) as a tool with better predictivity, reliability, and reproducibility. The unique proliferation and pluripotency of stem cells makes them a tremendous resource for human material, allowing the prediction of drug toxicity and metabolic effects of chemicals. Recognizing the growing importance of NT and DNT and the application of stem cell biology, in this review article, we provide the diversified approaches of stem cell research which can be effectively applied to the NT and DNT studies and provide an update of the recent progress made so far. We further provide a futuristic approach towards novel stem cell-based strategies for NT and DNT testing. We have further discussed the current technologies, role of induced pluripotent stem cells, the application of three-dimensional (3D) cultures and role of stem cell-derived organs in the NT and DNT studies.

Lead Intoxication Synergies of the Ethanol-Induced Toxic Responses in Neuronal Cells--PC12.

Lead (Pb)-induced neurodegeneration and its link with widespread neurobehavioral changes are well documented. Experimental evidences suggest that ethanol could enhance the absorption of metals in the body, and alcohol consumption may increase the susceptibility to metal intoxication in the brain. However, the underlying mechanism of ethanol action in affecting metal toxicity in brain cells is poorly understood. Thus, an attempt was made to investigate the modulatory effect of ethanol on Pb intoxication in PC12 cells, a rat pheochromocytoma. Cells were co-exposed to biological safe doses of Pb (10 µM) and ethanol (200 mM), and data were compared to the response of cells which received independent exposure to these chemicals at similar doses. Ethanol (200 mM) exposure significantly aggravated the Pb-induced alterations in the end points associated with oxidative stress and apoptosis. The finding confirms the involvement of reactive oxygen species (ROS)-mediated oxidative stress, and impairment of mitochondrial membrane potential, which subsequently facilitate the translocation of triggering proteins between cytoplasm and mitochondria. We further confirmed the apoptotic changes due to induction of mitochondria-mediated caspase cascade. These cellular changes were found to recover significantly, if the cells are exposed to N-acetyl cysteine (NAC), a known antioxidant. Our data suggest that ethanol may potentiate Pb-induced cellular damage in brain cells, but such damaging effects could be recovered by inhibition of ROS generation. These results open up further possibilities for the design of new therapeutics based on antioxidants to prevent neurodegeneration and associated health problems.

Emergence of ceftriaxone resistant Shigella.

Shigellosis is endemic in many resource-poor countries due to feco-oral transmission, resulting in considerable morbidity and mortality. There is rapid emergence of multi-drug resistant (MDR) Shigella spp. resulting in poor reliability of first line antibiotics like quinolones, co-trimoxazole and ampicillin. Ceftriaxone has been used as a reserved antibiotic for treatment of MDR Shigella spp. The authors report a case of ceftriaxone resistant Shigella flexneri successfully managed with meropenem. As occurrence of ceftriaxone resistant Shigella is still rare, the objective of reporting this case is to highlight the possible failure of ceftriaxone in treating shigellosis which if not detected timely can result in mortality.

Short-term exposure of 4-hydroxynonenal induces mitochondria-mediated apoptosis in PC12 cells.

4-Hydroxynonenal (4-HNE) is one of the most reactive aldehydic by-products of lipid peroxidation. The role of 4-HNE in the etiology of various neurodegenerative disorders including cerebral ischemia/reperfusion, Alzheimer's disease, Parkinson's disease, etc. has been documented. We and others have reported that long-term toxic insults of 4-HNE triggers apoptotic signals and oxidative stress in various cells. However, the status of apoptosis following short-term exposure and underlying mechanisms has not been explored so far. We studied the apoptotic changes in PC12 cells receiving short-term exposure of 4-HNE. A significant dose-dependent induction in reactive oxygen species (ROS) and early response markers (c-Fos, c-Jun, and GAP-43) were observed in cells exposed to 4-HNE (10, 25, and 50 µM) for 1h. Following the exposure of PC12 cells to 4-HNE, the levels of protein and messenger RNA expressions of P(53), Bax, and caspase 3 were significantly upregulated, whereas the levels of Bcl(2) was downregulated. We could record the apoptotic signals and ROS generation in PC12 cells receiving 4-HNE exposure for such a short period of time. Induction in the expression and activity of caspase 3 has also indicated the mitochondrial mediation in the apoptosis induction.

Protective potential of 17ß-estradiol against co-exposure of 4-hydroxynonenal and 6-hydroxydopamine in PC12 cells.

4-hydroxynonenal (4-HNE) and 6-hydroxydopamine (6-OHDA)-mediated damage in dopaminergic neurons is well documented. Protective potential of steroidal hormone (17ß-estradiol) has also been suggested. However, therapeutic potential of such promising hormone is hampered due to complex brain anatomy and physiology. Thus, the present investigations were studied to suggest the applicability of dopamine expressing PC12 cells as in vitro tool to screen the pharmacological potential of 17ß-estradiol against 4-HNE and 6-OHDA. MTT assay was conducted for cytotoxicity assessment of both 4-HNE (1 µM to 50 µM) and 6-OHDA (10(-4) to 10(-7) M). Non-cytotoxic concentrations, that is, 4-HNE (1 µM) and 6-OHDA (10(-6) M) were selected to study the synergetic/additive responses. PC12 cells were found to be more vulnerable towards co-exposure of individual exposure of 4-HNE and 6-OHDA, even at non-cytotoxic concentrations. Then, cells were subjected to pre-treatment (24 hours) of 17ß-estradiol (1 µM), followed by a permutation of combinations of both 4-HNE and 6-OHDA. Pretreatment of 17ß-estradiol was found to be significantly effective against the cytotoxic responses of 4-HNE and 6-OHDA, when the damage was at lower level. However, 17ß-estradiol was found to be ineffective against higher concentrations. Physiological-specific responses of PC12 cells against 4-HNE/6-OHDA and 17ß-estradiol suggest its applicability as first tier of screening tool.

Differential protection of pre-, co- and post-treatment of curcumin against hydrogen peroxide in PC12 cells.

Pharmacological potential of curcumin was assessed in PC12 cells against hydrogen peroxide (H(2) O(2)) exposure. In MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and lactate dehydrogenase (LDH) assays, 24-hour exposure of H(2)O(2) (0.5 mM and above) was found to be cytotoxic. A significant (p < 0.001) increase in percentage cell viability was recorded in PC12 cells pretreated with curcumin (25, 50 and 100 µg/mL) for 24 hours prior to H(2)O(2) (0.5 and 1 mM) exposure for 24 hours. Co-exposure to H(2)O(2) and curcumin was also found effective. However, a therapeutic treatment of curcumin for 24 hours after H(2)O(2) exposure to the cells was found ineffective. Differential response of PC12-H(2)O(2) model to curcumin in MTT and LDH assays suggests the utility of these endpoints to sort the drug candidates to study their antioxidant potential.

Caspase cascade regulated mitochondria mediated apoptosis in monocrotophos exposed PC12 cells.

Monocrotophos (MCP) is a commonly used organophosphorus (OP) pesticide. We studied apoptotic changes in PC12 cells exposed to MCP. A significant induction in reactive oxygen species (ROS), lipid peroxide (LPO), and the ratio of glutathione disulfide (GSSG)/reduced glutathione (GSH) was observed in cells exposed to selected doses of MCP. Following the exposure of PC12 cells to MCP, the levels of protein and mRNA expressions of Caspase-3, Caspase-9, Bax, p53, P(21), Puma, and cytochrome-c were significantly upregulated, whereas the levels of Bcl(2), Bcl(w), and Mcl1 were downregulated. TUNEL assay, DNA laddering, and micronuclei induction show that long-term exposure of PC12 cells to MCP at higher concentration (10(-5) M) decreases the number of apoptotic events due to an increase in the number of necrotic cells. MCP-induced translocation of Bax and cytochrome-c proteins between the cytoplasm and mitochondria confirmed the role of p53 and Puma in mitochondrial membrane permeability. Mitochondria mediated apoptosis induction was confirmed by the increased activity of caspase cascade. We believe that this is the first report showing MCP-induced apoptosis in PC12 cells, which is mitochondria mediated and regulated through the caspase cascade. Our data demonstrates that MCP induced the apoptotic cell death in neuronal cells and identifies the possible cellular and molecular mechanisms of organophosphate pesticide-induced apoptosis in neuronal cells.

Association of dopamine DA-D2 receptor in rotenone-induced cytotoxicity in PC12 cells.

The investigations were aimed to study the possible association of dopamine DA-D(2) receptor in rotenone-induced cytotoxicity in PC12 cells, one among the most studied cell line in neurotoxicity studies. PC12 cells were subjected to receive an exposure of rotenone (10(-6) to 10(-4) M) for 24 and 48 hours. Cytotoxicity studies were carried out using standard end points including, (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT), lactate dehydrogenase (LDH) release and neutral red uptake (NRU). Cells were found to be vulnerable to rotenone in dose-dependent manner. In general, 10(-4) and 10(-5) M concentrations were found to be cytotoxic, whereas 10(-6) M and lower concentrations used have shown nonsignificant effect on cell viability. Further, studies were extended to study the rotenone-induced alterations in cellular glutathione (GSH) level and dopamine DA-D(2) receptor expression. Significant (p < 0.001) chronological depletion in GSH levels were recorded following rotenone exposure. Expression of dopamine DA-D(2) receptor was also found to be effected significantly (p < 0.001) at 24 hours of rotenone exposure (10(-4) and 10(-5)). However, no further depletion in the expression of dopamine DA-D(2) receptor could be recorded with extended exposure period, that is, 48 hours. Rotenone at 10(-6) M and lower concentrations was found to be ineffective in PC12 cells. Data suggest the vulnerability of PC12 cells against experimental exposure of rotenone, which possibly routed through dopamine DA-D(2) receptor and oxidative stress machinery.

NGF induced differentiated PC12 cells as in vitro tool to study 4-hydroxynonenal induced cellular damage.

Investigations were carried out to examine the suitability of PC12 cells as an in vitro tool to examine 4-hydroxynonenal (4-HNE)-induced toxicity in nervous tissue. On day 8 of differentiation, markers of neural effects and oxidative stress were measured following exposure of PC12 cells to 1-50 microM 4-HNE for 1-8h. Endpoints included dopamine DA-D(2) receptor and glutathione S-transferase (GSTP1-1) protein levels, 4-HNE-protein binding, glutathione (GSH) concentrations and intracellular calcium levels. GSH levels were maximally depleted after 4h. 4-HNE also induced depletion of GSTP1-1 and increased intracellular Ca(++), with the latter seen as early as 1h after exposure. Responses at 8h were not greater than responses at earlier times. The experiments suggest that PC12 cells could be an in vitro tool for understanding toxicant-cell interactions, especially those that result in oxidative stress.

Oxygen glucose deprivation model of cerebral stroke in PC-12 cells: glucose as a limiting factor.

Optimum time points for oxygen-glucose deprivation (OGD) and re-oxygenation have been identified to suggest the suitability of PC-12 cells as rapid and sensitive in vitro model of cerebral stroke. Further, the precise role of glucose as one of the limiting factors was ascertained. PC-12 cells were subjected to receive OGD of 1-8 h followed by re-oxygenation for 6 to 96 h in medium having glucose 0-10 mg/ml. Loss of cell viability was assessed using trypan blue dye exclusion and MTT assays. The significant (p < 0.05) reduction in percent viable cell count was started at 2 h of OGD (80.7 +/- 2.0) and continued in further OGD periods (3, 4, 5, 6, 7, and 8 h), i.e. 65.7 +/- 3.5, 59.7 +/- 4.6, 54.3 +/- 3.2, 44.7 +/- 2.9, 20.3 +/- 4.3, 5.7 +/- 2.0 of counted cells, respectively. Cells growing in glucose-free medium have shown a gradual (p < 0.001) decrease in cell viability throughout the re-oxygenation. Re-oxygenation of 24 h was found to be first statistically significant time point for all the glucose concentrations. Glucose concentration during re-oxygenation was found to be one of the key factors involved in the growth and proliferation in PC-12 cells. The OGD of 6 h followed by a re-oxygenation period of 24 h with 4-6 mg/ml glucose concentration could be recorded as optimum conditions under our experimental conditions.

Platelet dopamine: D2 receptor binding in patients with migraine.

Clinical, genetic and pharmacological evidences suggest an abnormality of the dopaminergic system in the pathogenesis of migraine. Direct evidence of an abnormal metabolism of dopamine in migraine, however, is lacking. Platelets are a useful model to understand brain dopaminergic mechanisms. The present study has been undertaken to study the status of platelet dopamine receptor binding by carrying out radioligand receptor binding assay. Binding of (3)H-spiperone to platelet membranes, known to label dopamine (DA)-D2 receptors, was conducted in 20 patients with migraine and an equal number of healthy controls. The equilibrium dissociation constant (Kd) in patients with migraine (1.71 +/- 0.19 nM) was found to be significantly lower (P < 0.001) as compared with controls (3.14 +/- 0.33 nM). However, no significant change was observed in the maximal number of binding sites (Bmax) in patients with migraine. No relationship of Kd with type of migraine, presence of vomiting, family history, frequency of attack, duration of illness and menstrual migraine was observed. The findings of the present study provide support for the involvement of the dopaminergic system in migraine.