Brain-derived Neurotrophic Factor Secretion Research Articles

The Brain-Derived Neurotrophic Factor Val66Met Polymorphism Can Protect Against Cognitive Impairment in Multiple Sclerosis.

Introduction: Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family, involved in neuronal survival and synaptic plasticity. The BDNF Val66Met polymorphism is known to reduce BDNF expression and secretion; its role in multiple sclerosis (MS) is poorly investigated.Objectives and Methods: In this multicenter, retrospective study, we assessed the role of BDNF Val66Met polymorphism on cognitive and motor disability in MS patients consecutively referred to the University of Florence and the Hospital of Barletta. All patients underwent a genetic analysis for the presence of Val66Met polymorphism and a comprehensive neuropsychological examination on the Rao's Brief Repeatable Battery and the Stroop Color Word Test. Possible predictors of the Expanded Disability Status Scale (EDSS) score and number of failed neuropsychological tests were assessed through linear multivariable regression models.Results: Ninety-eight patients were recruited. Patients with the BDNF Val66Met polymorphism (35.7%) were more frequently males (p = 0.020), more disabled (p = 0.026) and, marginally, older (p = 0.064). In the multivariable analysis, BDNF Val66Met polymorphism was associated with a better cognitive performance (B = −1.1 ± 0.5, p = 0.027). Higher EDSS score was associated with a progressive disease course (B = 3.4, p < 0.001) and, marginally, with the presence of the BDNF Val66Met polymorphism (B = 0.56, p = 0.066).Discussion: Our results preliminarily suggest a protective role of BDNF Val66Met polymorphism against cognitive impairment in MS patients, possibly related to a detrimental effect of increased BDNF concentration in a neuroinflammatory environment.

BDNF-Gene Transfected Schwann Cell-Assisted Axonal Extension and Sprouting on New PLA-PPy Microfiber Substrates.

The work here reported analyzes the effect of increased efficiency of brain-derived neurotrophic factor (BDNF) production by electroporated Schwann cells (SCs) on the axonal extension in a coculture system on a biomaterial platform that can be of interest for the treatment of injuries of the nervous system, both central and peripheral. Rat SCs are electrotransfected with a plasmid coding for the BDNF protein in order to achieve an increased expression and release of this protein into the culture medium of the cells, performing the best balance between the level of transfection and the number of living cells. Gene-transfected SCs show an about 100-fold increase in the release of BDNF into the culture medium, compared to nonelectroporated SCs. Cocultivation of electroporated SCs with rat dorsal root ganglia (DRG) is performed on highly aligned substrates of polylactic acid (PLA) microfibers coated with the electroconductive polymer polypyrrol (PPy). The coculture of DRG with electrotransfected SCs increase both the axonal extension and the axonal sprouting from DRG neurons compared to the coculture of DRG with nonelectroporated SCs. Therefore, the use of PLA-PPy highly aligned microfiber substrates preseeded with electrotransfected SCs with an increased BDNF secretion is capable of both guiding and accelerating axonal growth.

Peripheral BDNF correlated with miRNA in BD-II patients

ObjectivesWe have identified the association between peripheral levels of candidate miRNAs (miR-7-5p, miR-142-3p, miR-221-5p, and miR-370-3p) for BD-II in previous study. Most of these miRNAs are associated with regulation of expression of peripheral brain derived neurotrophic factor (BDNF) levels. In order to clarify the underlying mechanism of BDNF and miRNAs in the pathogenesis of BD-II, it is of interest to investigate the relation between the peripheral levels of miR-7-5p, miR-142-3p, miR-221-5p, miR-370-3p with BDNF levels. Because the BDNF Val66Met polymorphism influence the secretion of BDNF, we further stratified the above correlations by this polymorphism. MethodsWe have recruited 98 BD-II patients. Beside analyzing peripheral levels of miR-7-5p, miR-142-3p, miR-221-5p, miR-370-3p, and BDNF, the genetic distribution of the BDNF Val66Met polymorphism was also analyzed. ResultsWe found that the miR7-5p, miR221-5p, and miR370-3p significantly correlated with the BDNF levels for all patients. If stratified by the BDNF Val66Met polymorphism, the significant correlation between miR221-5p and miR370-3p with BDNF only remained in the Val/Met genotype. However, the correlation between miR7-5p and BDNF level is significant in all 3 genotypes. ConclusionOur result supported that these miRNAs may be involved in the pathomechanism of BD-II through relation with BDNF.

Sea urchin gangliosides exhibit neuritogenic effects in neuronal PC12 cells via TrkA- and TrkB-related pathways.

Gangliosides (GLSs) are ubiquitously distributed in all tissues but highly enriched in nervous system. Currently, it is unclear how exogenous GLSs regulate neuritogenesis, although neural functions of endogenous GLSs are widely studied. Herein, we evaluated the neuritogenic activities and mechanism of sea urchin gangliosides (SU-GLSs) in vitro. These different glycosylated SU-GLSs, including GM4(1S), GD4(1S), GD4(2A), and GD4(2G), promoted differentiation of NGF-induced PC12 cells in a dose-dependent and structure-selective manner. Sulfate-type and disialo-type GLSs exhibited stronger neuritogenic effects than monosialoganglioside GM1. Furthermore, SU-GLSs might act as neurotrophic factors possessing neuritogenic effects, via targeting tyrosine-kinase receptors (TrkA and TrkB) and activating MEK1/2-ERK1/2-CREB and PI3K-Akt-CREB pathways. This activation resulted in increased expression and secretion of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). These pathways were verified by specific inhibitors. Our results confirmed the neuritogenic functions of SU-GLS in vitro and indicated their potential roles as natural nutrition for neuritogenesis.

Association study of the BDNF gene polymorphism (G196A) with overweight/obesity among women from Northwest of Iran

BackgroundObesity is a health problem defined by surplus body fat accumulation and is one of the leading causes of morbidity and mortality. Earlier studies indicated the influence of brain-derived neurotrophic factor (BDNF) molecular alterations in the development of obesity. One of these variations is the G196A single nucleotide polymorphism (Val66Met; SNP rs6265), which impairs intracellular trafficking and reduces the secretion of BDNF. In this study, we evaluated the possible association of G196A polymorphism of the BDNF gene with body mass index (BMI) among women from the Iranian Azeri Turkish ethnic group. Four hundred eighty-four women including 343 women with obesity or overweight and 141 age-sex and ethnically matched healthy controls were genotyped for G196A SNP of BDNF gene by applying polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) method. The association of this polymorphism with BMI was evaluated using analysis of covariance (ANCOVA), and the comparison of alleles and genotypes frequencies between patients (obese and/or overweight participants) and healthy controls was carried out using logistic regression models.ResultsIndividuals carrying Met-Met genotype have a significantly lower mean of BMI in comparison to those carrying non-Met/Met polymorphisms (P = 0.0138).ConclusionsIn this study, the association of the Val66Met polymorphism of the BDNF gene with BMI as an obesity trait has been confirmed among the women from the Northwest of Iran.

Treatment of ischemic neuronal death by introducing brain-derived neurotrophic factor mRNA using polyplex nanomicelle

Ischemic neuronal death causes serious lifelong neurological deficits; however, there is no proven effective treatment that can prevent neuronal death after the ischemia. We investigated the feasibility of mRNA therapeutics for preventing the neuronal death in a rat model of transient global ischemia (TGI). By intraventricular administration of mRNA encoding brain-derived neurotrophic factor (BDNF) using a polymer-based carrier, polyplex nanomicelle, the mRNA significantly increased the survival rate of hippocampal neurons after TGI, with a rapid rise of BDNF in the hippocampus. Interestingly, mRNA administration on Day 2 after TGI provided significantly better survival rate than the administration immediately after TGI. Eventually, dosing twice on Day 2 and 5 exerted long-term therapeutic effects, which were confirmed by a Y-maze behavioral test demonstrating improved spatial memory compared with untreated rats on Day 20. Immunohistochemical analysis showed that astrocytes were chief targets of the BDNF mRNA-loaded nanomicelles, suggesting that the augmented BDNF secretion from astrocytes creates a supportive microenvironment for the neurons to tolerate changes caused by ischemic stresses, and terminate the process of progressive neuronal death after the ischemic attack. Overall, the unique mechanism of action of mRNA therapeutics provide a promising approach for preventing ischemic neuronal death.

Gut neurotoxin p-cresol induces brain-derived neurotrophic factor secretion and increases the expression of neurofilament subunits in PC-12 cells

Increased p-cresol levels reportedly alter brain dopamine metabolism and exacerbate neurological disorders in experimental animals. In contrast to toxic concentrations, low doses of p-cresol may have distinct effects on neuronal metabolism. However, the role of p-cresol in synapse remodeling, neurite outgrowth, and other anabolic processes in neurons remains elusive. We propose that low doses of p-cresol affect neuronal cell structural remodeling compared with the high concentration-mediated harmful effects. Thus, the effects of p-cresol on the secretion of brain-derived neurotrophic factor (BDNF) and neurofilament subunit expression were examined using rat pheochromocytoma cells (PC-12 cells). We observed that low doses of p-cresol potentiated nerve growth factor-induced differentiation via secretion of BDNF in cultured PC-12 cells. Opioidergic compounds modulated these p-cresol effects, which were reversed by oxytocin. We propose that this effect of p-cresol has an adaptive and compensatory character and can be attributed to the induction of oxidative stress. Accordingly, we hypothesize that low doses of p-cresol induce mild oxidative stress, stimulating BDNF release by activating redox-sensitive genes. Given that the intestinal microbiome is the primary source of endogenous p-cresol, the balance between gut microbiome strains (especially Clostridium species) and opioidergic compounds may directly influence neuroplasticity.

Role of Astrocytic Inwardly Rectifying Potassium (Kir) 4.1 Channels in Epileptogenesis.

Astrocytes regulate potassium and glutamate homeostasis via inwardly rectifying potassium (Kir) 4.1 channels in synapses, maintaining normal neural excitability. Numerous studies have shown that dysfunction of astrocytic Kir4.1 channels is involved in epileptogenesis in humans and animal models of epilepsy. Specifically, Kir4.1 channel inhibition by KCNJ10 gene mutation or expressional down-regulation increases the extracellular levels of potassium ions and glutamate in synapses and causes hyperexcitation of neurons. Moreover, recent investigations demonstrated that inhibition of Kir4.1 channels facilitates the expression of brain-derived neurotrophic factor (BDNF), an important modulator of epileptogenesis, in astrocytes. In this review, we summarize the current understanding on the role of astrocytic Kir4.1 channels in epileptogenesis, with a focus on functional and expressional changes in Kir4.1 channels and their regulation of BDNF secretion. We also discuss the potential of Kir4.1 channels as a therapeutic target for the prevention of epilepsy.

Brain-Derived Neurotrophic Factor Regulates Ishikawa Cell Proliferation through the TrkB-ERK1/2 Signaling Pathway.

(1) Background: Endometrial regulation is a necessary condition for maintaining normal uterine physiology, which is driven by many growth factors. Growth factors produced in the endometrium are thought to be related to the proliferation of endometrial cells induced by estradiol-17β (E2). In this study, we found that E2 can induce the secretion of brain-derived neurotrophic factor (BDNF) in Ishikawa cells (the cells of an endometrial cell line). Furthermore, Ishikawa cells were used in exploring the regulatory role of BDNF in endometrial cells and to clarify the potential mechanism. (2) Methods: Ishikawa cells were treated with different concentrations of BDNF (100, 200, 300, 400, and 500 ng/mL). The mRNA expression levels of various proliferation-related genes were detected through quantitative reverse transcription polymerase chain reaction, and the expression of various proliferation-related genes was detected by knocking out BDNF or inhibiting the binding of BDNF to its receptor TrkB. The expression levels of various proliferation-related genes were detected by performing Western blotting on the TrkB-ERK1/2 signaling pathway. (3) Results: Exogenous BDNF promoted the growth of the Ishikawa cells, but the knocking down of BDNF or the inhibition of TrkB reduced their growth. Meanwhile, BDNF enhanced cell viability and increased the expression of proliferation-related genes, including cyclin D1 and cyclin E2. More importantly, the BDNF-induced proliferation of the Ishikawa cells involved the ERK1/2 signaling pathway. (4) Conclusions: The stimulating effect of exogenous E2 on the expression of BDNF in the uterus and the action of BDNF promoted the proliferation of the Ishikawa cells through the TrkB-ERK1/2 signal pathway.

Nicotine induces P2X4 receptor, interleukin-1 beta, and brain-derived neurotrophic factor expression in BV2 microglia cells.

Upregulation of P2X4 receptor (P2X4R), brain-derived neurotrophic factor (BDNF), and interleukin-1 beta (IL-1β) in activated microglia is associated with hyperalgesia. This study investigated whether nicotine increases pain hypersensitivity by altering the expression of these molecules in microglia. We also examined the role of interferon regulatory factor 8 (IRF8) in this process. Experiments were performed in BV2 microglial cells. IRF8 was knocked down or overexpressed using lentiviruses harboring a short hairpin RNA targeting IRF8 or an IRF8 overexpression construct, respectively. P2X4R, BDNF, and IL-1β mRNA and protein levels were evaluated by real-time PCR and western blotting, respectively, and BDNF and IL-1β secretion was assessed by ELISA. Chronic nicotine exposure enhanced the expression of P2X4R, BDNF, and IL-1β in BV2 cells, and stimulated the release of BDNF and IL-1β in the presence of ATP. IRF8 was found to mediate the nicotine-induced increases in BDNF and IL-1β mRNA and P2X4R protein levels in BV2 cells. Nicotine may increase pain hypersensitivity by promoting the expression of P2X4R, BDNF, and IL-1β through modulation of IRF8 levels in microglial cells.

Co-transplantation of Schwann cells and neural stem cells in the laminin-chitosan-PLGA nerve conduit to repair the injured recurrent laryngeal nerve in SD rats.

The objective of this paper is to investigate the possibility and efficacy of recurrent laryngeal nerve repair by transplantation of co-cultured Schwann cells and neural stem cells (NSCs) in laminin-chitosan-poly-lactic-co-glycolic acid (laminin-chitosan-PLGA) nerve conduits in rats. A laminin-chitosan-PLGA conduit was used in a rat recurrent laryngeal nerve transection model. The rat recurrent laryngeal nerve was dissected to generate a 5 mm defect. Then, a laminin-chitosan-PLGA nerve conduit with or without Schwann cells and NSCs in the lumen was transplanted into the defect. A total of 96 female rats were randomised into six groups: co-culture of NSCs and Schwann cells in the nerve conduit group (CO), Schwann cells only in the nerve conduit group (SC), neural stem cells only in the nerve conduit group (NSC-only), nerve conduit group (null), autologous nerve graft group (autograft) and sham operation group (sham). Regenerated nerves were evaluated by histological and functional assessment at 8 and 12 weeks after surgery. The diameter and area of the regenerated myelin sheath, as well as the secretion of brain-derived neurotrophic factor and glial cell-derived neurotrophic factor in laryngeal muscle or regenerated nerve tissue in the CO group, were significantly better than they were in the SC, NSC-only and null groups (all P values < 0.05). Immunofluorescence showed that the CO group had significantly more neurofilament-200 immunoreactive and S-100 immunoreactive fibres than the SC, NSC-only and null groups (all P values < 0.05). The performance of the CO groups and autograft groups was found to be similar by laryngoscopy. Arytenoid cartilage motion recovery in these two groups was significantly better than it was in the other groups (all P values < 0.05). Our results indicated that co-culture of Schwann cells and NSCs in laminin-chitosan-PLGA conduits might promote injured nerve regeneration. This method might be a promising alternative for defective nerve repair.

Neural Regenerative Potential of Stem Cells Derived from the Tooth Apical Papilla.

The regenerative effects of stem cells derived from dental tissues have been previously investigated. This study assessed the potential of human tooth stem cells from apical papilla (SCAP) on nerve regeneration. The SCAP collected from nine individuals were characterized and polarized by exposure to interferon-γ (IFN-γ). IFN-γ increased kynurenine and interleukin-6 (IL-6) production by SCAP, without affecting the cell viability. IFN-γ-primed SCAP exhibited a decrease of brain-derived neurotrophic factor (BDNF) mRNA levels, followed by an upregulation of glial cell-derived neurotrophic factor mRNA. Ex vivo, the co-culture of SCAP with neurons isolated from the rat dorsal root ganglion induced neurite outgrowth, accompanied by increased BDNF secretion, irrespective of IFN-γ priming. In vivo, the local application of SCAP reduced the mechanical and thermal hypersensitivity in Wistar rats that had been submitted to sciatic chronic constriction injury. The SCAP also reduced the pain scores, according to the evaluation of the Grimace scale, partially restoring the myelin damage and BDNF immunopositivity secondary to nerve lesion. Altogether, our results provide novel evidence about the regenerative effects of human SCAP, indicating their potential to handle nerve injury-related complications.

BDNF secretion from C2C12 cells is enhanced by methionine restriction

Brain derived neurotrophic factor (BDNF) is produced in skeletal muscle as a myokine that plays a role in muscle metabolism. However, how metabolic changes affect skeletal muscle BDNF expression and release remains to be fully understood. Amino acid restrictions such as methionine restriction (MR) are considered as an alternative fasting approach. Here we reported that in C2C12 myotubes, MR enhanced BDNF release, which was measured using ELISA, RT-qPCR, cell immunostaining, and Western blot. Inhibition of protein transport pathway blocked the MR enhanced BDNF release, confirming that MR-induced BDNF release involved classic protein secretory pathway. MR increased l-lactate product in media, suggesting that MR promoted glycolysis. Treatment with 2-deoxy glucose (2-DG) attenuated lactate production as well as BDNF release, suggesting that glycolysis is involved in the enhanced BDNF release induced by MR. Moreover, treatment with l-Lactate, the end-product of glycolysis, enhanced BDNF gene expression and release in control cells in a dose dependent manner, suggesting lactate produced by glycolysis may mediate the enhanced BDNF release by MR. Overall, the results of this study suggest that MR promotes BDNF secretion from C2C12 myotubes at least partially via enhancing glycolysis and lactate production.

BMP2 increases the production of BDNF through the upregulation of proBDNF and furin expression in human granulosa-lutein cells.

Locally produced in human granulosa cells of the developing follicle, bone morphogenetic protein 2 (BMP2) plays a crucial role in the regulation of ovarian folliculogenesis and luteal formation. Brain-derived neurotrophic factor (BDNF) is an intraovarian neurotrophic factor that has been shown to promote oocyte maturation and subsequent fertilization competency. At present, little is known regarding the intracellular regulation, assembly and secretion of endogenous BDNF in human granulosa cells. The aim of this study was to explore the effect of BMP2 on the expression and production of BDNF in human granulosa cells and the molecular mechanisms underlying this effect. An immortalized human granulosa cell line (SVOG) and primary human granulosa-lutein (hGL) cells were utilized as in vitro study models. Our results showed that BMP2 significantly increased the mRNA and secreted levels of BDNF. Additionally, BMP2 upregulated the expression of furin at the transcriptional and translational levels. Knockdown of endogenous furin partially attenuated the BMP2-induced increase in BDNF production, indicating that furin is involved in the maturation process of BDNF. Using pharmacological (kinase receptor inhibitors) and siRNA-mediated inhibition approaches, we demonstrated that BMP2-induced upregulation of BDNF and furin expression is most likely mediated by the activin receptor-like kinase (ALK)2/ALK3-SMAD4 signaling pathway. Notably, analysis using clinical samples revealed that there was a positive correlation between follicular fluid concentrations of BMP2 and those of BDNF. These results indicate that BMP2 increases the production of mature BDNF by upregulating the precursor BDNF and promoting the proteolytic processing of mature BDNF. Finally, we also investigated the effects of BMP2 on ovarian steroidogenesis and the results showed that BMP2 treatment significantly increased the accumulated level of estradiol (by upregulating the expression of FSH receptor and cytochrome P450 aromatase), whereas it decreased the accumulated level of progesterone (by downregulating the expression of LH receptors and steroidogenic acute regulatory protein) in primary hGL cells. Our findings provide a novel paracrine mechanism underlying the regulation of an intraovarian growth factor in human granulosa cells.

Journey of brain-derived neurotrophic factor: from intracellular trafficking to secretion.

Brain-derived neurotrophic factor (BDNF) is known to control a wide variety of brain functions, ranging from memory formation to food intake. However, since the BDNF levels are extremely low in the nervous system, the dynamics in neurons from intracellular trafficking to secretion is absolutely complicated; the understanding is not fully promoted. We here review the findings of those critical mechanisms from intracellular trafficking to the secretion of BDNF. Furthermore, to solve this issue, technological advances for the detection, measurement, and imaging of this growth factor are essential. We believe that this review helps the study of these complex but critical mechanisms of BDNF.

Peripheral BDNF Response to Physical and Cognitive Exercise and Its Association With Cardiorespiratory Fitness in Healthy Older Adults.

Physical exercise (PE) has been shown to improve brain function via multiple neurobiological mechanisms promoting neuroplasticity. Cognitive exercise (CE) combined with PE may show an even greater effect on cognitive function. Brain-derived neurotrophic factor (BDNF) is important for neuroplastic signaling, may reduce with increasing age, and is confounded by fitness. The source and physiological role of human peripheral blood BDNF in plasma (pBDNF) is thought to differ from that in serum (sBDNF), and it is not yet known how pBDNF and sBDNF respond to PE and CE. A training intervention study in healthy older adults investigated the effects of acute (35 min) and prolonged (12 weeks, 30 sessions) CE and PE, both alone and in combination, on pBDNF and sBDNF. Cross-sectional associations between baseline pBDNF, sBDNF and cardiorespiratory fitness (CRF) were also investigated. Participants (65–75 years) were randomly assigned to four groups and prescribed either CE plus 35 min of rest (n = 21, 52% female); PE [performed on a cycle ergometer at moderate intensity (65–75% of individual maximal heart rate)] plus 35 min of rest (n = 27, 56% female); CE plus PE (n = 24, 46% female), or PE plus CE (n = 25, 52% female). Groups were tested for CRF using a maximal treadmill ergometer test (VO2peak); BDNF levels (collected 48 h after CRF) during baseline, after first exercise (PE or CE) and after second exercise (PE, CE or rest); and cognitive ability pre and post 12-week training. At both pre and post, pBDNF increased after CE and PE (up to 222%), and rest (∼67%), whereas sBDNF increased only after PE (up to 18%) and returned to baseline after rest. Acute but not prolonged PE increased both pBDNF and sBDNF. CE induced acute changes in pBDNF only. Baseline pBDNF was positively associated with baseline sBDNF (n = 93, r = 0.407, p < 0.001). No changes in CRF were found in any of the groups. Baseline CRF did not correlate with baseline BDNF. Even though baseline pBDNF and sBDNF were associated, patterns of changes in pBDNF and sBDNF in response to exercise were explicitly different. Further experimental scrutiny is needed to clarify the biological mechanisms of these results.

The rs6265 polymorphism might not affect the secretion of BDNF protein directedly

Tinnitus is an auditory disorder with ringing, buzzing, and hissing of the ears. Tinnitus occurs by perceiving sounds without attribution of external sources and has various risk factors such as loss of hearing, anxiety, depression, and hypertension. While there is a wide range of factors contributing to tinnitus, the precise molecular mechanism is poorly appreciated. Recent studies on tinnitus evoked potential evidence for the contribution of neurological factors in the progress of tinnitus. Here we used PCR-RFLP to assess a single nucleotide polymorphism in Brain-Derived Neurotrophic Factor (BDNF), which substitutes valine 66 to methionine amino acid (known as the rs6265). The BDNF functions in the development of the inner ear and has a crucial role in the central auditory pathway. The rs6265 causes a functional missense mutation that inhibits the secretion of BDNF from the synapses. Here we tested 127 Iranian subjects from the city of Yazd (90 normal subjects and 37 patients) to analyze the rs6265 polymorphism in the BDNF gene. Our analysis found no association between the investigated polymorphism of the BDNF gene and the presence of tinnitus in Iranian patients. Further, we proposed a hypothesis that SORT1 and TRAF6 might facilitate the BDNF secretion, and this process might be independent of the rs6265 polymorphism. Therefore, this study indicates that other genetic factors may be relevant to the development of tinnitus disease.

CaMKII controls neuromodulation via neuropeptide gene expression and axonal targeting of neuropeptide vesicles.

Ca2+/calmodulin-dependent kinase II (CaMKII) regulates synaptic plasticity in multiple ways, supposedly including the secretion of neuromodulators like brain-derived neurotrophic factor (BDNF). Here, we show that neuromodulator secretion is indeed reduced in mouse α- and βCaMKII-deficient (αβCaMKII double-knockout [DKO]) hippocampal neurons. However, this was not due to reduced secretion efficiency or neuromodulator vesicle transport but to 40% reduced neuromodulator levels at synapses and 50% reduced delivery of new neuromodulator vesicles to axons. αβCaMKII depletion drastically reduced neuromodulator expression. Blocking BDNF secretion or BDNF scavenging in wild-type neurons produced a similar reduction. Reduced neuromodulator expression in αβCaMKII DKO neurons was restored by active βCaMKII but not inactive βCaMKII or αCaMKII, and by CaMKII downstream effectors that promote cAMP-response element binding protein (CREB) phosphorylation. These data indicate that CaMKII regulates neuromodulation in a feedback loop coupling neuromodulator secretion to βCaMKII- and CREB-dependent neuromodulator expression and axonal targeting, but CaMKIIs are dispensable for the secretion process itself.

Engineered Perineural Vascular Plexus for Modeling Developmental Toxicity.

There is a vital need to develop in vitro models of the developing human brain to recapitulate the biological effects that toxic compounds have on the brain. To model perineural vascular plexus (PNVP) in vitro, which is a key stage in embryonic development, human embryonic stem cells (hESC)-derived endothelial cells (ECs), neural progenitor cells, and microglia (MG) with primary pericytes (PCs) in synthetic hydrogels in a custom-designed microfluidics device are cocultured. The formation of a vascular plexus that includes networks of ECs (CD31+, VE-cadherin+), MG (IBA1+), and PCs (PDGFRβ+), and an overlying neuronal layer that includes differentiated neuronal cells (βIII Tubulin+, GFAP+) and radial glia (Nestin+, Notch2NL+), are characterized. Increased brain-derived neurotrophic factor secretion and differential metabolite secretion by the vascular plexus and the neuronal cells over time are consistent with PNVP functionality. Multiple concentrations of developmental toxicants (teratogens, microglial disruptor, and vascular network disruptors) significantly reduce the migration of ECs and MG toward the neuronal layer, inhibit formation of the vascular network, and decrease vascular endothelial growth factor A (VEGFA) secretion. By quantifying 3D cell migration, metabolic activity, vascular network disruption, and cytotoxicity, the PNVP model may be a useful tool to make physiologically relevant predictions of developmental toxicity.

Effect of cytokine treatment on the expression and secretion of brain derived neurotrophic factor in the smooth muscle of the rat colon.

The production of pro-inflammatory cytokines and chemokines is increased during inflammatory bowel disease (IBD). Previously, it was demonstrated that brain derived neurotrophic factor (BDNF) expression is increased in experimental models of colitis. BDNF is partially responsible for the structural and functional changes that take place during IBD. However, the exact mechanisms underlying the upregulation of BDNF during gut inflammation are unknown. The aim of the present study was to determine the effects of direct treatment of smooth muscle cells with inflammatory cytokines on the synthesis and secretion of BDNF. BDNF expression and secretion levels were measured using ELISA kits on tissue lysates and on incubation media used to culture the rat colon smooth muscle tissues treated for 24 h with either tumor necrosis factor (TNF)-α or interleukin (IL)-1β. Compared with the control tissue samples, treatment with TNF-α and IL-1β resulted in a significant increase in the protein expression levels of BDNF in the incubated smooth muscle tissue. TNF-α and IL-1β also stimulated the secretion of BDNF. Chelation of intracellular Ca2+ with BABTA-AM prevented the TNF-α and IL-1β-induced increase in BDNF protein expression and secretion levels. Furthermore, inhibition of protein kinase A (PKA) significantly reduced BDNF expression levels when treated with cytokines but not secretion. In conclusion, proinflammatory cytokines that are upregulated during IBD, directly stimulated BDNF expression and secretion in a Ca2+ dependent manner. Considering the ability of BDNF to enhance smooth muscle contraction and pain sensation, this autocrine loop may partially explain the characteristic hypercontractility and hypersensitivity associated with IBD.