BDNF-modified human umbilical cord mesenchymal stem cells-derived dopaminergic-like neurons improve rotation behavior of Parkinson's disease rats through neuroprotection and anti-neuroinflammation.

Parkinson's disease (PD) is a neurodegenerative disease still without any cure. Brain-derived neurotrophic factor (BDNF) has shown therapeutic potential in PD, which is limited by its short half-life and inability to penetrate the blood-brain barrier. Stem cells not only present migration, differentiation and neurotrophy characteristics, but also can be used as delivery vectors for BDNF. This study aimed to investigate the therapeutic effects and possible mechanisms of BDNF-modified human umbilical cord mesenchymal stem cells (hUC-MSCs)-derived dopaminergic (DAergic)-like neurons in the PD rats. Results showed that transplantation of BDNF-modified hUC-MSCs-derived DAergic-like neurons improved the apomorphine induced rotation behavior of PD rats, increased the dopamine concentration and the expression of glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule-1 (Iba-1) in the striatum, promoted the expression of tyrosine hydroxylase (TH), nuclear receptor-related factor 1 (Nurr1), pituitary homeobox 3 (Pitx3), BDNF, tyrosine kinase B (TrkB), phosphatidylinositol-3-hydroxykinase (PI3K), phosphorylated protein kinase B (p-Akt), heat shock protein 60 (Hsp60), toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) and inhibited the neural apoptosis in the substantia nigra (SN) and striatum. Results suggest that BDNF-modified hUC-MSCs-derived DAergic-like neurons improve the rotation of PD rats might through neuroprotection and anti-neuroinflammation by regulating the BDNF-TrkB-PI3K/Akt and Hsp60-TLR4/MyD88 signaling pathways, respectively.

BDNF-overexpressing human umbilical cord mesenchymal stem cell-derived motor neurons improve motor function and prolong survival in amyotrophic lateral sclerosis mice.

OBJECTIVE: To investigate the beneficial effect of brain-derived neurotrophic factor (BDNF) -overexpressing human umbilical cord mesenchymal stem cell (hUC-MSC)-derived motor neurons in the human Cu, Zn-superoxide dismutase1 (hSOD1)G93A amyotrophic lateral sclerosis (ALS) mice. METHODS: The BDNF gene was transfected into hUC-MSC-derived motor neurons by the lentivirus-mediated method. hSOD1G93A mice were assigned to the ALS, ALS/MN, and ALS/MN-BDNF groups, and intrathecally administrated phosphate-buffered saline (PBS), motor neurons, or motor neurons overexpressing BDNF, respectively. The control group included non-transgenic wild-type littermates administrated PBS. One month after transplantation, the motor function of the mice was assessed by the rotarod test, and the lumbar enlargements were then isolated to detect the expression of hSOD1 and BDNF by western blotting, and the expression of choline acetyltransferase (ChAT), homeobox protein 9 (HB9), major histocompatibility complex I (MHCI) and microtubule-associated protein-2 (MAP-2) by immunofluorescence assay. RESULTS: After transplantation, mice in the ALS/MN-BDNF and ALS/MN groups both exhibited longer latency to fall and longer survival than those in the ALS group (P < 0.01 vs. P < 0.05), and the improvement was more significant in the former than in the latter. However, cell transplantation did not delay disease onset. In the lumbar enlargements of the ALS/MN-BDNF and ALS/MN groups, the expression of hSOD1 was slightly reduced without statistical significance (P > 0.05), but the expression of BDNF, ChAT and HB9, and the co-expression of MHCI and MAP-2 were significantly greater than in the ALS group (P < 0.01), with the differences also being more prominent in the former group than in the latter. CONCLUSIONS: Transplantation of BDNF-overexpressing hUC-MSC-derived motor neurons can improve motor performance and prolong the survival of hSOD1G93A mice. Combining stem cell-derived motor neurons with BDNF might provide a new therapeutic strategy for ALS.

Advances in treatment of neurodegenerative diseases: Perspectives for combination of stem cells with neurotrophic factors.

Neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis, are a group of incurable neurological disorders, characterized by the chronic progressive loss of different neuronal subtypes. However, despite its increasing prevalence among the ever-increasing aging population, little progress has been made in the coincident immense efforts towards development of therapeutic agents. Research interest has recently turned towards stem cells including stem cells-derived exosomes, neurotrophic factors, and their combination as potential therapeutic agents in neurodegenerative diseases. In this review, we summarize the progress in therapeutic strategies based on stem cells combined with neurotrophic factors and mesenchymal stem cells-derived exosomes for neurodegenerative diseases, with an emphasis on the combination therapy.

Brain-derived neurotrophic factor modified human umbilical cord mesenchymal stem cells-derived cholinergic-like neurons improve spatial learning and memory ability in Alzheimer's disease rats.

Alzheimer's disease (AD) is an age-related neurodegenerative disease and the most common type of dementia. Although it is still incurable, stem cell replacement therapy provides new hope for AD. Human umbilical cord mesenchymal stem cells (hUC-MSCs) have multiple differentiation potentials, which can differentiate into cholinergic-like neurons and promote the release of acetylcholine. Brain-derived neurotrophic factor (BDNF) can also promote neurogenesis and synaptic formation, reduce oxidative stress and cell death. Therefore, we investigated the therapeutic effects of BDNF modified hUC-MSCs-derived cholinergic-like neurons in AD rats in this study. To make AD models, 1 µl beta amyloid (Aß)1-42 was injected into the right hippocampus of the rats. After two weeks, the hUC-MSCs-derived cholinergic-like neurons null cells or overexpressing BDNF cells delivered by lentiviralvectors were slowly injected into the right hippocampus of the AD rats. After 8 weeks of transplantation, Morris water maze test, Western blotting, Immunohistochemistry, Immunofluorescence assay and TdT mediated dUTP Nick End Labeling (TUNEL) detection were performed. Transplantation of BDNF modified hUC-MSCs-derived cholinergic-like neurons significantly improved spatial learning and memory abilities in the AD rats, increased the release of acetylcholine and ChAT expression in the hippocampus, enhanced the activation of astrocytes and microglia, reduced the expression of Aß and recombinant human beta-site APP-cleaving enzyme1 (BACE1), inhibited neuronal apoptosis, and promoted neurogenesis. Our results demonstrate that BDNF modified hUC-MSCs-derived cholinergic-like neurons might be a promising therapeutic strategy for AD.

Rig-I is involved in inflammation through the IPS-1/TRAF6 pathway in astrocytes under chemical hypoxia.

The retinoic acid-inducible gene I (RIG-I) is a crucial cytoplasmic pathogen recognition receptor involved in neuroinflammation in degenerative diseases. In the present study, in vitro human astrocytes were subjected to a chemical hypoxia model using cobalt chloride pretreatment. Chemical hypoxia induces the up-regulation of RIG-I in astrocytes and results in the expression of inflammatory cytokines IL-1ß, IL-6, and TNF-α in an NF-κB dependent manner. Elevated RIG-I modulates the interaction of interferon-ß promoter stimulator-1 (IPS-1) and TNF receptor-associated factor 6 (TRAF6) following chemical hypoxia. Inhibition of IPS-1 or TRAF6 suppresses RIG-I-induced NF-κB activation and inflammatory cytokines in response to chemical hypoxia. These data suggest that chemical hypoxia leads to RIG-I activation and the expression of inflammatory cytokines through the NF-κB pathway. Blocking IPS-1/TRAF6 pathway relieves RIG-I-induced neuroinflammation in astrocytes subjected to hypoxia.

Association between FGF20 rs12720208 gene polymorphism and Parkinson's disease: a meta-analysis.

Previous studies have claimed the association of rs12720208 polymorphism in the fibroblast growth factor 20 (FGF20) gene with the increased risk of Parkinson's disease (PD), but results from the published data were controversial. The aim of our present meta-analysis was to estimate the overall association between FGF20 rs12720208 polymorphism and the risk of PD. Case-control studies with sufficient data evaluating the association between rs12720208 C/T polymorphism and PD susceptibility were systematically identified in PubMed, OVID, SinoMed, Chinese National Knowledge Infrastructure (CNKI) up to July 10, 2015. A total of 3402 PD patients and 3739 controls from seven case-control studies were collected for this meta-analysis. The pooled odds ratio (OR) with its 95 % confidence interval (CI) was calculated to assess the genetic association between FGF20 rs12720208 polymorphism and the risk of PD. In this study, no enough proof was found to prove the association in any genetic models with random-effects model (CT+TT vs. CC: OR = 1.147, 95 % CI: 0.883-1.489, P = 0.304; TT vs. CC+CT: OR = 1.754, 95 % CI: 0.878-3.505, P = 0.112; T vs. C: OR = 1.169, 95 % CI = 0.919-1.487, P = 0.204; TT+CC vs. CT: OR = 0.906, 95 % CI = 0.694-1.182, P = 0.466). Our results suggest that there is no sufficient evidence to support the association between rs12720208 polymorphism and PD risk. Studies with larger sample size across diverse populations and subgroup analyses are necessary in the future.

Heat shock protein 60 affects behavioral improvement in a rat model of Parkinson's disease grafted with human umbilical cord mesenchymal stem cell-derived dopaminergic-like neurons.

Parkinson's disease (PD) is a neurodegenerative disorder that is caused by a loss of dopaminergic (DAergic) neurons in mesencephalic substantia nigra (SN). Human umbilical cord mesenchymal stem cells (hUC-MSCs) are capable of self-renewal and differentiation into multiple cell lineages, including DAergic neurons. Thus, hUC-MSCs could be a promising alternative to compensate for the loss of DAergic neurons in PD. In the current study, hUC-MSCs and hUC-MSCs-derived DAergic-like neurons were transplanted into the striatum and SN of a rat model of PD that is induced by 6-hydroxydopamine (6-OHDA). We evaluated their therapeutic effects on improving rotation behavior in the rat and on modulating the level of heat shock protein 60 (Hsp60) expression in the brain. After transplantation, an amelioration of rotation behavior was observed in rats that underwent cell grafting, and hUC-MSCs-derived DAergic-like neurons were superior to hUC-MSCs at inducing behavioral improvement. Western blot and immunohistochemistry analysis indicated significantly elevated levels of Hsp60 in cell-grafted rats compared to 6-OHDA-lesioned (PD) rats. These results demonstrate that hUC-MSCs-based cell transplantation is potential therapeutic treatment for PD, and hUC-MSCs-derived DAergic-like neurons appear to be favorable candidates for cell replacement therapy in PD. Finally, Hsp60 could be involved in a mechanism of behavioral recovery.

Variants in neuronal nitric oxide synthase gene may contribute to increased ischemic stroke susceptibility in a Han Chinese population.

Variants in neuronal NOS (nNOS) gene were associated with atherosclerosis and stroke susceptibility. We aimed to investigate the association between nNOS gene polymorphism and risk of ischemic stroke caused by small-artery occlusion (SAO) and large-artery atherosclerosis (LAA) in a Chinese population. We conducted a case-control study involving 381 ischemic stroke patients and 366 healthy subjects. Selected SNPs (rs1483757, rs2293050, and rs2139733) were genotyped and assessed; the association with the risk of ischemic stroke was analyzed. Furthermore, gender- and etiologic subtype-stratified analyses were also carried out to evaluate the association between nNOS polymorphisms and risk of ischemic stroke. No significant difference was observed between selected nNOS loci and risk of ischemic stroke in alleles or any genetic models in total study population, males or females, adjusted with age, drinking and smoking status. Rs2293050 and rs2139733 genotypes were associated with total cholesterol (rs2293050, P = 0.026; rs2139733, P = 0.040) and LDL (rs2293050, P = 0.031; rs2139733, P = 0.046) in females. A significant difference in allele distribution of rs2293050 (P = 0.040) and a marginally significant difference of rs2139733 (P = 0.061) in LAA-caused ischemic stroke cases and controls were observed in total population. No association between rs1483757 and ischemic stroke was found in this study. T allele of rs2293050 and A allele of rs2139733 in nNOS gene may contribute to increased susceptibility of LAA-caused ischemic stroke in Han Chinese.

WITHDRAWN: Increase in phosphorylation of PDK1 and cell survival after acute spinal cord injury.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, doi:10.1016/j.jns.2012.06.003. The duplicate article has therefore been withdrawn.

Increase in phosphorylation of PDK1 and cell survival after acute spinal cord injury.

3-Phosphoinositidedependent protein kinase-1 (PDK1), which phosphorylates and activates a group of kinases, plays important roles in cellular metabolism, growth, proliferation and survival. However, the functions of PDK1 in central nervous system (CNS) injury remain an enigma. To elucidate the expressions and possible functions of PDK1 and its phosphorylation in CNS injury and repair, we performed an acute spinal cord injury (SCI) model in adult rats and detected the expression and localization of serine-241 phosphorylated PDK1 (p-PDK1s241). Western blot and immunohistochemistry showed that serine-241 phosphorylated PDK1 (p-PDK1s241) started increasing by 6h after damage and peaked at 12h, then declined to basal levels by 3days after injury. Immunohistochemical staining also revealed subcellular localization changes of p-PDK1s241 staining between nucleus and cytoplasm after injury including neurons and glial cells. Double immunofluorescence labeling suggested that p-PDK1s241 primarily localizes in neurons and oligodendrocytes. It might also be expressed in other glial cells of spinal cord tissues within 2mm from the epicenter at 12h post-injury. Moreover, double staining indicated that p-PDK1s241 and active caspase-3 showed different cellular distributions after SCI. Together with previous reports, we hypothesize that phosphorylation of PDK1 may be associated with cell survival and suggest PDK1 as a novel target for neuroprotection and functional repair in SCI.

DL-3-n-Butylphthalide protects rat bone marrow stem cells against hydrogen peroxide-induced cell death through antioxidation and activation of PI3K-Akt pathway.

Bone marrow stem cells (BMSCs) have been one of the most important cell sources for cell replacement therapy (CRT) in cerebral infarction. However, long-lasting oxidative stress during stroke, which plays an important role in neuron damage, deteriorates the microenvironment for cell survival, differentiation and removal. Thus the outcome of CRT in ischemic diseases was poor. DL-3-n-Butylphthalide (NBP) has protective effects on ischemic brain tissue through multiple mechanisms and has been used for stroke treatment in China for several years. In this study, hydrogen peroxide (H(2)O(2)) was used to induce oxidative stress injury to rat bone marrow stem cells (rBMSCs), imitating the microenvironment surrounding transplanted cells in the ischemic brain in vitro. The protective effects of NBP on rBMSCs against apoptosis induced by oxidative stress were investigated. Our results indicated that NBP could protect rBMSCs against apoptosis due to antioxidative properties and modulation of PI3K/Akt pathway. NBP could be used in combination with BMSCs for the treatment of cerebral infarction by improving the oxidative stress microenvironments and cell survival, however, further studies remain warranted.

Cocaine exposure at a sublethal concentration downregulates CREB functions in cultured neuroblastoma cells.

Previous studies showed that prenatal cocaine in an animal model decreased brain-derived neurotrophic factor (BDNF) activity in offspring's brain. Since BDNF is one of target genes of cAMP response element-binding protein (CREB), this study examined effects of cocaine on CREB activities in a human neuroblastoma (SK-N-AS) cell line. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazodium bromide) assay indicated that cocaine exposure at 5 microM for 24 h had no significant influences on cell viability. However, a 24-h exposure to cocaine at the same concentration significantly decreased the level of phosphorylated CREB, although no significant changes in total CREB proteins were observed. Consistent with reduced CREB phosphorylation, the electrophoretic mobility shift assay showed that exposure to 5 microM of cocaine for 24 h also inhibited CREB binding activity and significantly decreased BDNF mRNA expression. In addition, exposure to 5 microM cocaine for 24 h attenuated the glutamic acid-evoked increase in the intracellular Ca2+ concentration. Taken together, these findings suggest that cocaine exposure at the sublethal concentration downregulates CREB functions in the cultured SK-N-AS cell line, and that diminished intracellular Ca2+ responses may be associated in part with cocaine-induced downregulation of CREB activity.

Involvement of 5-HT1B receptors within the ventral tegmental area in ethanol-induced increases in mesolimbic dopaminergic transmission.

Evidence suggests that 5-hydroxytriptamine-1B (5-HT1B) receptors play a role in modifying ethanol's reinforcing effects and voluntary intake, and that 5-HT1B receptors within the ventral tegmental area (VTA) are involved in regulation of mesolimbic dopaminergic neuronal activity. Since increased mesolimbic dopaminergic transmission has been implicated in ethanol's reinforcing properties, this study was designed to assess the involvement of VTA 5-HT1B receptors in mediating the stimulatory effects of ethanol on VTA dopaminergic neurons. Dual-probe microdialysis was performed in freely moving adult Sprague-Dawley rats with one probe within the VTA and the other within the ipsilateral nucleus accumbens (NACC). Dopamine (DA) levels in dialysates from both areas, as the index of the activity of mesolimbic DA neurons, were measured simultaneously. The results showed that intraperitoneal injection of ethanol at the doses of 1 and 2 g/kg increased extracellular DA concentrations in both the VTA and the NACC, suggesting increased DA neuronal activity. These ethanol-induced increases of the DA release in the VTA and the NACC were significantly attenuated by intra-tegmental infusion of SB 216641 (a 5-HT(1B) receptor antagonist), but not BRL 15572 (a 5-HT(1D/1A) receptor antagonist) or WAY 100635 (a 5-HT1A receptor antagonist). Administration of ethanol at the same doses did not significantly alter extracellular levels of GABA in the VTA. The results also showed that intra-tegmental infusion of CP 94253, a 5-HT1B receptor agonist, significantly prolonged the effects of ethanol on NACC DA. The results suggest that blockade and activation of VTA 5-HT1B receptors attenuates and potentiates the neurochemical effects of ethanol, respectively, and support the suggestion that VTA 5-HT(1B) receptors may be involved in part in mediating the activating effects of ethanol on mesolimbic DA neurons.

Effects of prenatal alcohol exposure on brain-derived neurotrophic factor and its receptor tyrosine kinase B in offspring.

Prenatal alcohol exposure produces many developmental defects in the central nervous system. The underlying molecular mechanism, however, has not been fully understood. The present study was undertaken to examine the effects of prenatal alcohol exposure on brain-derived neurotrophic factor (BDNF) and its receptor tyrosine kinase B (TrkB) in offspring. The pregnant Sprague-Dawley rats received 1 or 3 g/kg of alcohol or an isocaloric solution by intragastric intubation once a day from gestational day (GD) 5 to GD 20. On postnatal day 7-8, pups were killed and the hippocampus, striatum, cortex, and cerebellum dissected out. Levels of BDNF mRNA and proteins, total TrkB proteins and receptor phosphorylation were measured. The results showed that prenatal alcohol exposure at the dose of 1 g/kg/day did not significantly affect BDNF protein levels in any region examined. However, administration of alcohol at the dose of 3 g/kg/day markedly reduced levels of BDNF protein and mRNA in the cortex and hippocampus of offspring. Western blotting showed that prenatal alcohol exposure at the dose of 3 g/kg/day also inhibited TrkB phosphorylation in the hippocampus although no changes in total TrkB protein levels were observed in any region examined. Our data suggest that prenatal alcohol exposure alters both presynaptic and postsynaptic BDNF function in certain brain areas of offspring. These alterations in BDNF function may contribute to the development of alcohol-related birth defects.

Different expression of brain-derived neurotrophic factor in the nucleus accumbens of alcohol-preferring (P) and -nonpreferring (NP) rats.

Recent studies suggest that the gene that encodes brain-derived neurotrophic factor (BDNF) might be linked with vulnerability to alcohol abuse. We have now compared BDNF protein levels in several brain regions between alcohol-naive alcohol-preferring (P) and -nonpreferring (NP) rats using the enzyme-linked immunosorbent assay (ELISA) procedure. The results showed that BDNF levels in the nucleus accumbens of the P rats were significantly lower than those of the NP rats, suggesting that this innate difference may contribute to the disparate alcohol drinking behavior of the P and NP rats.