Primary Cultures Of Cortical Neurons Research Articles

Calcium Export from Neurons and Multi-Kinase Signaling Cascades Contribute to Ouabain Neuroprotection in Hyperhomocysteinemia.

Pathological homocysteine (HCY) accumulation in the human plasma, known as hyperhomocysteinemia, exacerbates neurodegenerative diseases because, in the brain, this amino acid acts as a persistent N-methyl-d-aspartate receptor agonist. We studied the effects of 0.1–1 nM ouabain on intracellular Ca2+ signaling, mitochondrial inner membrane voltage (φmit), and cell viability in primary cultures of rat cortical neurons in glutamate and HCY neurotoxic insults. In addition, apoptosis-related protein expression and the involvement of some kinases in ouabain-mediated effects were evaluated. In short insults, HCY was less potent than glutamate as a neurotoxic agent and induced a 20% loss of φmit, whereas glutamate caused a 70% decrease of this value. Subnanomolar ouabain exhibited immediate and postponed neuroprotective effects on neurons. (1) Ouabain rapidly reduced the Ca2+ overload of neurons and loss of φmit evoked by glutamate and HCY that rescued neurons in short insults. (2) In prolonged 24 h excitotoxic insults, ouabain prevented neuronal apoptosis, triggering proteinkinase A and proteinkinase C dependent intracellular neuroprotective cascades for HCY, but not for glutamate. We, therefore, demonstrated here the role of PKC and PKA involving pathways in neuronal survival caused by ouabain in hyperhomocysteinemia, which suggests existence of different appropriate pharmacological treatment for hyperhomocysteinemia and glutamate excitotoxicity.

Vexin is upregulated in cerebral cortical neurons by brain-derived neurotrophic factor.

AimChromosome 8 open reading frame 46 (C8orf46), a human protein‐coding gene, has recently been named Vexin. A recent study indicated that Vexin is involved in embryonic neurogenesis. Additionally, some transcriptomic studies detected changes in the mRNA levels of patients with psychiatric and neurological diseases. In our previous study, we sought for target genes of brain‐derived neurotrophic factor (BDNF) in cultured rat cortical neurons, finding that BDNF potentially leads to the upregulation of Vexin mRNA. However, its underlying mechanisms are unknown. In the present study, we assessed the regulatory mechanisms of the BDNF‐induced gene expression of Vexin in vitro.MethodsWe reanalyzed ChIP‐seq data in various human organs provided by the ENCODE project, evaluating acetylation levels of the 27th lysine residue of the histone H3 (H3K27ac) at the Vexin locus. The transcriptomic effects of BDNF on rat Vexin (RGD1561849) were evaluated by real‐time quantitative PCR (RT‐qPCR) in primary cultures of cerebral cortical neurons, in the presence or absence of inhibitors for signaling molecules activated by BDNF.ResultsThe Vexin locus and its promoter region in the brain angular gyrus show higher acetylation levels of the H3K27 than those in other organs. Stimulation of cultured rat cortical neurons, but not astrocyte, with BDNF, led to marked elevations in the mRNA levels of Vexin, which was inhibited in the presence of K252a and U0126.ConclusionThe upregulated H3K27ac in the brain may be associated with the enriched gene expression of Vexin in the brain. It is indicated that BDNF induces the gene expression of Vexin in the cortical neurons via the TrkB‐MEK signaling pathway.

Enhanced phosphorylation of PERK in primary cultured neurons as an autonomous neuronal response to prion infection.

Conversion of cellular prion protein (PrPC) into the pathogenic isoform of prion protein (PrPSc) in neurons is one of the key pathophysiological events in prion diseases. However, the molecular mechanism of neurodegeneration in prion diseases has yet to be fully elucidated because of a lack of suitable experimental models for analyzing neuron-autonomous responses to prion infection. In the present study, we used neuron-enriched primary cultures of cortical and thalamic mouse neurons to analyze autonomous neuronal responses to prion infection. PrPSc levels in neurons increased over the time after prion infection; however, no obvious neuronal losses or neurite alterations were observed. Interestingly, a finer analysis of individual neurons co-stained with PrPSc and phosphorylated protein kinase RNA-activated-like endoplasmic reticulum (ER) kinase (p-PERK), the early cellular response of the PERK-eukaryotic initiation factor 2 (eIF2α) pathway, demonstrated a positive correlation between the number of PrPSc granular stains and p-PERK granular stains, in cortical neurons at 21 dpi. Although the phosphorylation of PERK was enhanced in prion-infected cortical neurons, there was no sign of subsequent translational repression of synaptic protein synthesis or activations of downstream unfolded protein response (UPR) in the PERK-eIF2α pathway. These results suggest that PrPSc production in neurons induces ER stress in a neuron-autonomous manner; however, it does not fully activate UPR in prion-infected neurons. Our findings provide insights into the autonomous neuronal responses to prion propagation and the involvement of neuron-non-autonomous factor(s) in the mechanisms of neurodegeneration in prion diseases.

Partial reduction of amyloid \u03b2 production by \u03b2-secretase inhibitors does not decrease synaptic transmission

BackgroundAlzheimer’s disease (AD) is the most common form of age-related neurodegenerative diseases. Cerebral deposition of Aβ peptides, especially Aβ42, is considered the major neuropathological hallmark of AD and the putative cause of AD-related neurotoxicity. Aβ peptides are produced by sequential proteolytic processing of APP, with β-secretase (BACE) being the initiating enzyme. Therefore, BACE has been considered an attractive therapeutic target in AD research and several BACE inhibitors have been tested in clinical trials, but so far, all have had negative outcomes or even led to worsening of cognitive function. AD can be triggered by Aβ years before the first symptoms appear and one reason for the failures could be that the clinical trials were initiated too late in the disease process. Another possible explanation could be that BACE inhibition alters physiological APP processing in a manner that impairs synaptic function, causing cognitive deterioration.MethodsThe aim of this study was to investigate if partial BACE inhibition, mimicking the putative protective effect of the Icelandic mutation in the APP gene, could reduce Aβ generation without affecting synaptic transmission. To investigate this, we used an optical electrophysiology platform, in which effects of compounds on synaptic transmission in cultured neurons can be monitored. We employed this method on primary cortical rat neuronal cultures treated with three different BACE inhibitors (BACE inhibitor IV, LY2886721, and lanabecestat) and monitored Aβ secretion into the cell media.ResultsWe found that all three BACE inhibitors tested decreased synaptic transmission at concentrations leading to significantly reduced Aβ secretion. However, low-dose BACE inhibition, resulting in less than a 50% decrease in Aβ secretion, did not affect synaptic transmission for any of the inhibitors tested.ConclusionOur results indicate that Aβ production can be reduced by up to 50%, a level of reduction of relevance to the protective effect of the Icelandic mutation, without causing synaptic dysfunction. We therefore suggest that future clinical trials aimed at prevention of Aβ build-up in the brain should aim for a moderate CNS exposure of BACE inhibitors to avoid side effects on synaptic function.

Down-Regulation of miR-23a-3p Mediates Irradiation-Induced Neuronal Apoptosis

Radiation-induced central nervous system toxicity is a significant risk factor for patients receiving cancer radiotherapy. Surprisingly, the mechanisms responsible for the DNA damage-triggered neuronal cell death following irradiation have yet to be deciphered. Using primary cortical neuronal cultures in vitro, we demonstrated that X-ray exposure induces the mitochondrial pathway of intrinsic apoptosis and that miR-23a-3p plays a significant role in the regulation of this process. Primary cortical neurons exposed to irradiation show the activation of DNA-damage response pathways, including the sequential phosphorylation of ATM kinase, histone H2AX, and p53. This is followed by the p53-dependent up-regulation of the pro-apoptotic Bcl2 family molecules, including the BH3-only molecules PUMA, Noxa, and Bim, leading to mitochondrial outer membrane permeabilization (MOMP) and the release of cytochrome c, which activates caspase-dependent apoptosis. miR-23a-3p, a negative regulator of specific pro-apoptotic Bcl-2 family molecules, is rapidly decreased after neuronal irradiation. By increasing the degradation of PUMA and Noxa mRNAs in the RNA-induced silencing complex (RISC), the administration of the miR-23a-3p mimic inhibits the irradiation-induced up-regulation of Noxa and Puma. These changes result in an attenuation of apoptotic processes such as MOMP, the release of cytochrome c and caspases activation, and a reduction in neuronal cell death. The neuroprotective effects of miR-23a-3p administration may not only involve the direct inhibition of pro-apoptotic Bcl-2 molecules downstream of p53 but also include the attenuation of secondary DNA damage upstream of p53. Importantly, we demonstrated that brain irradiation in vivo results in the down-regulation of miR-23a-3p and the elevation of pro-apoptotic Bcl2-family molecules PUMA, Noxa, and Bax, not only broadly in the cortex and hippocampus, except for Bax, which was up-regulated only in the hippocampus but also selectively in isolated neuronal populations from the irradiated brain. Overall, our data suggest that miR-23a-3p down-regulation contributes to irradiation-induced intrinsic pathways of neuronal apoptosis. These regulated pathways of neurodegeneration may be the target of effective neuroprotective strategies using miR-23a-3p mimics to block their development and increase neuronal survival after irradiation.

Silver nanoparticles induce size-dependent and particle-specific neurotoxicity to primary cultures of rat cerebral cortical neurons.

Silver nanoparticles (AgNPs) are widely applied in many fields because of their excellent antibacterial activities. Toxicological studies have showed that AgNPs can cross the blood-brain barrier and exhibit high retention in the brain. Therefore, the potential neurotoxicity of AgNPs is raising serious concerns. This study investigated the neurotoxicological effects of AgNPs with two different sizes (20nm and 70nm, AgNPs-20 and AgNPs-70) using primary cultures of rat cerebral cortical neurons in mature and developing stages. The contribution of silver ion release was investigated by testing the effects of ionic silver in parallel. The results showed that both AgNPs-20 and AgNPs-70 significantly decreased neuronal cell viability, and AgNPs-20 had stronger toxicity compared with AgNPs-70. AgNP applications caused the granulated skeleton structure of the mature neurons with some broken synapses after a 24-h exposure, and inhibited neuronal growth during a 7-day exposure. Intracellular silver accumulation at non-cytotoxic exposure levels inhibited dopamine efflux, which was particle-specific and free of released silver ions. The findings herein can aid in guiding the proper applications of AgNPs in different areas, especially in medical use.

Lauric acid promotes neuronal maturation mediated by astrocytes in primary cortical cultures

Previous studies have suggested the potential efficacy of middle chain fatty acids (MCFAs) in the treatment of mood disorders and cognitive dysfunction. MCFAs are metabolized to ketone bodies in astrocytes; however, their effects on neuronal development including neurotrophic factor level are not well-understood. In the present study, we examined the effect of MCFAs on the mRNA expression of growth factors and cytokines in primary cultures of cortical astrocytes. The effect of MCFAs on neuron-astrocyte interaction in neuronal maturation was also determined using co-culture and astrocyte-conditioned medium. Lauric acid (LA) typically increased the mRNA expression of glial-derived neurotrophic factor (Gdnf), interleukin-6 (Il6), and C–C motif chemokine 2 (Ccl2) in astrocytes. LA-induced phosphorylation of extracellular signal-regulated kinase contributed to these changes. In primary cultures of cortical neurons containing astrocytes, LA enhanced the presynaptic protein levels. Astrocyte-conditioned medium after LA treatment also enhanced the presynaptic protein levels in the cortical neuron cultures. These results suggest that LA increase the mRNA expression of GDNF and cytokines in astrocytes, and thereby, enhances the presynaptic maturation.

Chitinase 3-like 1 is neurotoxic in primary cultured neurons

Chitinase 3-like 1 (CHI3L1) is known to play a role as prognostic biomarker in the early stages of multiple sclerosis (MS), and patients with high cerebrospinal fluid CHI3L1 levels have an increased risk for the development of neurological disability. Here, we investigated its potential neurotoxic effect by adding recombinant CHI3L1 in vitro to primary cultures of mouse cortical neurons and evaluating both neuronal functionality and survival by immunofluorescence. CHI3L1 induced a significant neurite length retraction after 24 and 48 hours of exposure and significantly reduced neuronal survival at 48 hours. The cytotoxic effect of CHI3L1 was neuron-specific and was not observed in mouse immune or other central nervous system cells. These results point to a selective neurotoxic effect of CHI3L1 in vitro and suggest a potential role of CHI3L1 as therapeutic target in MS patients.

Beneficial Effects of Inhibition of the Mitochondrial Pyruvate Carrier and the Mechanism of Action of Thiazolidinediones

We demonstrated a number of years ago that thiazolidinediones including pioglitazone (insulin sensitizers used in the treatment of type 2 diabetes) have a direct inhibitory effect on the mitochondrial pyruvate carrier (MPC). This metabolite transporter of the inner mitochondrial membrane is responsible for carrying pyruvate from the cytoplasm into mitochondrial matrix for further oxidation, and is key to determining the pattern of cellular substrate oxidation. Since then, multiple studies have described somewhat counter‐intuitive findings of potential benefit of loss of MPC activity through genetic or pharmacologic means in normal cells both in vitro and in vivo. For instance, we have provided evidence that loss of MPC activity in myocytes induces changes consistent with insulin sensitization, including enhanced fatty acid oxidation, elevation of glucose uptake, and phosphorylation of AMPK, similar to what is observed in the skeletal muscle of diabetic patients treated with pioglitazone. Perhaps the more surprising finding was in primary cultures of cortical neurons. Mapping the pattern of neuronal metabolism upon MPC inhibition using stable isotope tracing and metabolomics allowed us to determine the extent of metabolic flexibility in neurons, and determine whether adjustments in neuronal substrate oxidation could affect susceptibility to injury by excessive exposure to the neurotransmitter glutamate. We found that metabolism of cortical neurons in culture and hippocampal slices is characterized by metabolic plasticity, and oxidative metabolism can be maintained in spite of large reductions in mitochondrial pyruvate uptake because of a selective increase in glutamate oxidation as an energy substrate, even in the presence of ketone bodies. Increased glutamate oxidation decreased the glutamate available for packaging into synaptic vesicles and synaptic release and, in turn, minimized the positive feedback cascade of excitotoxic injury. Thus, reductions in mitochondrial pyruvate metabolism can be neuroprotective. These findings will be placed in the context of other studies designed to enhance healthy energy metabolism in neurological disease, as well as a re‐evaluation of the mechanism of action of thiazolidinediones.Support or Funding InformationNIH R01NS087611

‐The Function of CASK in Transcriptional Regulation in Neurons

CASK is a scaffold protein that functions in trafficking and targeting of synaptic proteins in the central nervous system. Emerging literature suggests that CASK localizes inside nucleus and might regulate transcription by interacting with TBR1, a transcription factor essential for brain development. However, these regulatory mechanisms are not well established. We hypothesize that CASK is involved in neuronal activity‐regulated transcription. To test our hypothesis, we used primary cultures of mouse cortical neurons stimulated with KCL depolarization buffer, which is known to activate a robust CREB‐dependent gene expression program and to induce the activation of distal enhancer elements. To measure gene expression genome‐wide, we performed RNAseq in unstimulated or KCL‐treated neurons after knock‐down of CASK expression using specific shRNA. Our results show that CASK knock‐down strongly reduces the number of genes that are regulated by depolarization. These results indicate that CASK is required for gene expression regulation in neuronal cells. To gain further insights in the mechanisms by which CASK regulate transcription, we used RNA in situ hybridization (RNAscope) to test the hypothesis that CASK is not only required for the transcription of coding genes, but also of non‐coding RNAs, termed ‘enhancer RNAs (eRNAs), in proximity of KCL‐regulated genes. We showed that RNAscope is a valid method to measure eRNAs induction after KCl treatment. Ongoing experiments in the lab are assessing the effect of CASK knock‐down on eRNA transcription. Interestingly, we observed by immunoprecipitation that nuclear CASK dimerizes, and that the stability of this homodimer is dependent on the presence of RNA. Finally, we used biochemical fractionation to study CASK localization in the nucleus in both mouse neuronal cultures and brain tissues. These results shed light on how CASK functions in neurons. This study is relevant to human disease because mutations in CASK gene are linked to microcephaly and X‐linked intellectual disability.Support or Funding InformationThis project has been supported by CABRI Undergraduate Research Fellowship

Chronic Activation of Gp1 mGluRs Leads to Distinct Refinement of Neural Network Activity through Non-Canonical p53 and Akt Signaling

Group 1 metabotropic glutamate receptors (Gp1 mGluRs), including mGluR1 and mGluR5, are critical regulators for neuronal and synaptic plasticity. Dysregulated Gp1 mGluR signaling is observed with various neurologic disorders, including Alzheimer’s disease, Parkinson’s disease, epilepsy, and autism spectrum disorders (ASDs). It is well established that acute activation of Gp1 mGluRs leads to elevation of neuronal intrinsic excitability and long-term synaptic depression. However, it remains unknown how chronic activation of Gp1 mGluRs can affect neural activity and what molecular mechanisms might be involved. In the current study, we employed a multielectrode array (MEA) recording system to evaluate neural network activity of primary mouse cortical neuron cultures. We demonstrated that chronic activation of Gp1 mGluRs leads to elevation of spontaneous spike frequency while burst activity and cross-electrode synchronization are maintained at the baseline. We further showed that these neural network properties are achieved through proteasomal degradation of Akt that is dependent on the tumor suppressor p53. Genetically knocking down p53 disrupts the elevation of spontaneous spike frequency and alters the burst activity and cross-electrode synchronization following chronic activation of Gp1 mGluRs. Importantly, these deficits can be restored by pharmacologically inhibiting Akt to mimic inactivation of Akt mediated by p53. Together, our findings reveal the effects of chronic activation of Gp1 mGluRs on neural network activity and identify a unique signaling pathway involving p53 and Akt for these effects. Our data can provide insights into constitutively active Gp1 mGluR signaling observed in many neurologic and psychiatric disorders.

A proteolytic C-terminal fragment of Nogo-A (reticulon-4A) is released in exosomes and potently inhibits axon regeneration

Glial signals are known to inhibit axonal regeneration and functional recovery after mammalian central nervous system trauma, including spinal cord injury. Such signals include membrane-associated proteins of the oligodendrocyte plasma membrane and astrocyte-derived, matrix-associated proteins. Here, using cell lines and primary cortical neuron cultures, recombinant protein expression, immunoprecipitation and immunoblot assays, transmission EM of exosomes, and axon regeneration assays, we explored the secretion and activity of the myelin-associated neurite outgrowth inhibitor Nogo-A and observed exosomal release of a 24-kDa C-terminal Nogo-A fragment from cultured cells. We found that the cleavage site in this 1192-amino-acid-long fragment is located between amino acids 961-971. We also detected a Nogo-66 receptor (NgR1)-interacting Nogo-66 domain on the exosome surface. Enzyme inhibitor treatment and siRNA knockdown revealed that β-secretase 1 (BACE1) is the protease responsible for Nogo-A cleavage. Functionally, exosomes with the Nogo-66 domain on their surface potently inhibited axonal regeneration of mechanically injured cerebral cortex neurons from mice. Production of this fragment was observed in the exosomal fraction from neuronal tissue lysates after spinal cord crush injury of mice. We also noted that, relative to the exosomal marker Alix, a Nogo-immunoreactive, 24-kDa protein is enriched in exosomes 2-fold after injury. We conclude that membrane-associated Nogo-A produced in oligodendrocytes is processed proteolytically by BACE1, is released via exosomes, and is a potent diffusible inhibitor of regenerative growth in NgR1-expressing axons.

Neuroprotective effects of leptin on cerebral ischemia through JAK2/STAT3/PGC-1-mediated mitochondrial function modulation

Neuroprotective effects of leptin have been shown in mouse model of cerebral ischemia/reperfusion injury and primary cortical neuronal culture with oxygen-glucose deprivation (OGD), while the underlying mechanisms are less understood. In the present study, we investigated whether leptin modulated mitochondrial function through JAK2/STAT3 in vivo mouse model of transient middle cerebral artery occlusion (MCAO) and in OGD-challenged primary neuronal cultures. JAK2/STAT3; mitochondrial biogenesis markers (PGC-1α); and apoptosis-associated proteins (caspase-3, BCL-2, BCL-XL, and cytochrome c) were detected by western blotting and reverse transcription-polymerase chain reaction at 1 h before and after ischemia/reperfusion. P-STAT3 and PGC-1α in neurons and astrocytes were detected. Moreover, mitochondrial morphology of the ischemic ipsilateral penumbra is examined using transmission electron microscopy. Primary cerebral cortical neurons were evaluated for viability, mitochondrial membrane potential (MMP), and apoptosis to assess whether dose-dependent neuroprotective effects of leptin during OGD were mitigated by the JAK2/STAT3 inhibitor AG490. Leptin activated JAK2/STAT3 signaling in neurons and astrocytes distributed in the ischemic ipsilateral penumbra, with peak p-STAT3 levels observed at 1 h after reperfusion. Leptin increased PGC-1α, BCL-2, and BCL-XL protein levels, cell viability, and MMP and decreased apoptosis both in vitro and in vivo; these effects were reversed by AG490 treatment. Our findings suggest that leptin-mediated neuroprotective effects in tMCAO may peak at 1 h to induce the transcription of its target gene PGC-1α, stabilization of MMP, inhibition of mitochondrial permeability transition pore opening, release of cytochrome c, and apoptosis.

CREB Family Transcription Factors Are Major Mediators of BDNF Transcriptional Autoregulation in Cortical Neurons.

BDNF signaling via its transmembrane receptor TrkB has an important role in neuronal survival, differentiation, and synaptic plasticity. Remarkably, BDNF is capable of modulating its own expression levels in neurons, forming a transcriptional positive feedback loop. In the current study, we have investigated this phenomenon in primary cultures of rat cortical neurons using overexpression of dominant-negative forms of several transcription factors, including CREB, ATF2, C/EBP, USF, and NFAT. We show that CREB family transcription factors, together with the coactivator CBP/p300, but not the CRTC family, are the main regulators of rat BDNF gene expression after TrkB signaling. CREB family transcription factors are required for the early induction of all the major BDNF transcripts, whereas CREB itself directly binds only to BDNF promoter IV, is phosphorylated in response to BDNF-TrkB signaling, and activates transcription from BDNF promoter IV by recruiting CBP. Our complementary reporter assays with BDNF promoter constructs indicate that the regulation of BDNF by CREB family after BDNF-TrkB signaling is generally conserved between rat and human. However, we demonstrate that a nonconserved functional cAMP-responsive element in BDNF promoter IXa in humans renders the human promoter responsive to BDNF-TrkB-CREB signaling, whereas the rat ortholog is unresponsive. Finally, we show that extensive BDNF transcriptional autoregulation, encompassing all major BDNF transcripts, occurs also in vivo in the adult rat hippocampus during BDNF-induced LTP. Collectively, these results improve the understanding of the intricate mechanism of BDNF transcriptional autoregulation.SIGNIFICANCE STATEMENT Deeper understanding of stimulus-specific regulation of BDNF gene expression is essential to precisely adjust BDNF levels that are dysregulated in various neurological disorders. Here, we have elucidated the molecular mechanisms behind TrkB signaling-dependent BDNF mRNA induction and show that CREB family transcription factors are the main regulators of BDNF gene expression after TrkB signaling. Our results suggest that BDNF-TrkB signaling may induce BDNF gene expression in a distinct manner compared with neuronal activity. Moreover, our data suggest the existence of a stimulus-specific distal enhancer modulating BDNF gene expression.

Human Milk Oligosaccharide 2'-Fucosyllactose Reduces Neurodegeneration in Stroke Brain.

2'-Fucosyllactose (2'-FL) is a major oligosaccharide in human milk and is present at trace levels in cow milk. 2'-FL reduces inflammation in the gastrointestinal tract. Its action in the central nervous system has not been well characterized. The purpose of this study is to determine 2'-FL-mediated neural protection and repair in culture and stroke brain. In rat primary cortical neuronal cultures, 2'-FL significantly antagonized N-methyl-D-aspartate (NMDA) or glutamate-mediated changes in ATP production, MAP2 immunoreactivity, and TUNEL. The influx of Ca++ (Ca++i) was examined in primary cortical neurons expressing GCaMP5, an endogenous calcium probe. NMDA increased Ca++i; 2'-FL significantly attenuated this reaction. In a rat middle cerebral artery occlusion model of stroke, we found that intracerebroventricular pretreatment or oral posttreatment with 2'-FL significantly reduced brain infarction, mitigated microglial activation, improved locomotor activity, and upregulated brain-derived neurotrophic factor (BDNF) expression. Post-stroke delivery of 2'-FL increased bromodeoxyuridine (BrdU) labeling in the perilesioned area. These BrdU cells co-expressed NeuN, or nestin, or GFAP. Using subventricular Matrigel cultures, we demonstrated that 2'-FL increased cell migration from subventricular zone explant. This response was reduced by anti-BDNF blocking antibody. In conclusion, our data suggest that 2'-FL has neuroprotective action through inhibition of Ca++i, inflammation, and apoptosis. Posttreatment with 2'-FL facilitates neural repair in stroke brain.

Effect of vitamin D treatment on VDR expression in primary cerebral cortical cells in induced oxidative stress

BACKGROUND: In addition to calcium and phosphate homeostasis in peripheral tissues; vitamin D performs a neuroprotection role in the nervous system. The neuroprotective actions of vitamin D include: increasing vitamin D receptor (VDR) expression, control glutathione synthesis and nitric oxide synthase activity and induce neurotrophins such as nerve growth factor (NGF). VDR mediates cellular actions, and biological responses of the vitamin D. OBJECTIVE: To study the effect of VDR and NGF expression levels by vitamin D3 treatment in induced oxidative stress in primary cortical neuronal cultures. METHOD: Primary neuronal cultures were set up from the cortex region of neonatal rat’s brain. They were cultured for up to 72 h in the presence of 0.25μg/ml vitamin D3. These cells were exposed to 0.5 mM H2O2 for two hours before collecting cell pellet and medium for biochemical assays. Control and H2O2 treated cells were cultured in the absence of vitamin D3 treatment. Sandwich ELISA was used to study NGF expression. Western blotting and immunofluorescence of cultured cells were used to estimate the expression of VDR. RESULTS: Vitamin D3 treatment increased more significantly (P < 0.001) NGF levels with and without induced oxidative stress. Protein expression studies confirmed the positive correlation between VDR expression and vitamin D3 treatment after 72 h in culture. Moreover, pre-treating the cells with vitamin D3 before H2O2 exposure significantly increase (P < 0.05) VDR expression in comparison with the cells exposed to H2O2 alone. CONCLUSION: The neuroprotective effect of vitamin D3 against oxidative stress could be through up-regulating VDR and NGF levels.

Therapeutic Potential of Vitamin D and Curcumin in an In Vitro Model of Alzheimer Disease.

Background:Alzheimer disease is a progressive neurodegenerative disease, affecting a very high proportion of the aging population. Several studies have demonstrated that one of the main contributors to this disease is oxidative stress (OS), which causes peroxidation of protein, lipids, and DNA resulting in the formation of advanced glycosylated end products (AGE) in the brain tissues. These AGE are usually associated with the amyloid β (Aβ), which could further aggravate its toxicity and its clearance. Antioxidants counteract the deterioration caused by OS.Objective:We aimed to evaluate the effect of vitamin D3 and curcumin on primary cortical neuronal cultures exposed to Aβ1-42 toxicity for different time periods.Methods:Primary cortical neuronal cultures were set up and exposed to Aβ1-42 for up to 72 hours. Cell viability was studied by 3[4,5-dimethylthiazole-2-yl]-2,5-dipheyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assay. Biochemical assays for OS such as lipid peroxidation, reduced Glutathione(GSH), Glutathione S-transferase (GST), catalase, and superoxide dismutase (SOD) were conducted. Sandwich enzyme-linked immunosorbent assay (ELISA) was used to study the neurotrophic growth factor (NGF) expression.Results:Treatments with Aβ1-42 caused an elevation in lipid peroxidation products, which were ameliorated in the presence of vitamin D3 and curcumin. Both enzymatic (GST, catalase, and SOD) and nonenzymatic antioxidants (reduced GSH) were raised significantly in the presence of vitamin D3 and curcumin, which resulted in the better recovery of neuronal cells from Aβ1-42 treatment. Treatment with vitamin D3 and curcumin also resulted in the upregulation of NGF levels.Conclusions:This study suggests that vitamin D3 and curcumin can be a promising natural therapy for the treatment of Alzheimer disease.

Chitosan-g-oligo(L,L-lactide) copolymer hydrogel for nervous tissue regeneration in glutamate excitotoxicity: in vitro feasibility evaluation

Over the last decade, a number of hydrogels attracted great attention in the area of brain tissue engineering. The hydrogels are composed of hydrophilic polymers forming 3D network in water. Their function is promoting structural and functional restoration of damaged brain tissues by providing mechanical support and navigating cell fate. This paper reports on the neurocompatibility of chitosan-g-oligo(L,L-lactide) copolymer hydrogel with primary rat cortical neuron culture. The hydrogel was produced by a molding technique on the base of photocurable composition consisting of chitosan-g-oligo(L,L-lactide) copolymer, poly(ethylene glycol) diacrylate and photosensitizer Irgacure 2959. The influence of the hydrogel on cell viability, phenotype and calcium homeostasis, mitochondrial potential and oxygen consumption rate in glutamate excitotoxicity was analyzed using primary neuron cultures obtained from a neonatal rat cortex. This study revealed that the hydrogel is non-cytotoxic. Dissociated neonatal rat cortical cells were actively attaching to the hydrogel surface and exhibited the phenotype, calcium homeostasis and mitochondrial function in both standard conditions and glutamate excitotoxicity (100 μM) similar to the control cells cultured without the hydrogel. To conclude, in this study we assessed the feasibility of the application of chitosan-g-oligo(L,L-lactide) copolymer hydrogel for tissue engineering therapy of brain injury in an in vitro model. The results support that the hydrogel is able to sustain realization of the functional metabolic activity of neonatal rat cortical cells in response to glutamate excitotoxicity.

Glucocerebrosidase Activity Modulates Neuronal Susceptibility to Pathological α-Synuclein Insult

Mutations in the GBA1 gene are the most common genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB). GBA1 encodes the lysosomal lipid hydrolase glucocerebrosidase (GCase), and its activity has been linked to accumulation of α-synuclein. The current study systematically examines the relationship between GCase activity and both pathogenic and non-pathogenic forms of α-synuclein in primary hippocampal, cortical, and midbrain neuron and astrocyte cultures, as well as in transgenic mice and a non-transgenic mouse model of PD. We find that reduced GCase activity does not result in aggregation of α-synuclein. However, in the context of extant misfolded α-synuclein, GCase activity modulates neuronal susceptibility to pathology. Furthermore, this modulation does not depend on neuron type but rather is driven by the level of pathological α-synuclein seeds. This study has implications for understanding how GBA1 mutations influence PD pathogenesis and provides a platform for testing novel therapeutics.

Quercetin Attenuates Decrease of Thioredoxin Expression Following Focal Cerebral Ischemia and Glutamate-induced Neuronal Cell Damage

Quercetin is a bioactive flavonoid which abundantly exists in vegetables and fruits. Quercetin exerts a neuroprotective effect against cerebral ischemia. Thioredoxin acts as antioxidant by regulating redox signaling. This study investigated whether quercetin regulates thioredoxin expression in focal cerebral ischemia and glutamate-induced neuronal cell death. Male Sprague Dawley rats (210–230 g) were intraperitoneally injected with vehicle or quercetin (10 mg/kg) 1 h prior to middle cerebral artery occlusion (MCAO). Cerebral cortex was collected 24 h after MCAO. MCAO led to neurological movement deficits, brain edema, and serious histopathological damages in cerebral cortex, and quercetin alleviated these damages following MCAO. We observed the change of thioredoxin expression in MCAO animals with quercetin using proteomic approach, reverse-transcription PCR, and Western blot analyses. Thioredoxin expression decreased in vehicle-treated MCAO animals, while quercetin attenuated this decrease. Moreover, quercetin treatment alleviated the decrease in the number of thioredoxin-positive cells in cerebral cortex of MCAO animals. Furthermore, immunoprecipitation analysis demonstrated that interaction of apoptosis signal-regulating kinase 1 (ASK1) and thioredoxin was decreased in MCAO animals with vehicle, while quercetin prevented MCAO-induced decrease in these binding. In addition, quercetin also alleviated the reduction of cell viability and the decrease in thioredoxin expression in glutamate-treated hippocampal cell line and primary cultures of cortical neurons. However in thioredoxin-silenced cortical neuron, anti-apoptotic effect of quercetin was decreased. Thus, changes of thioredoxin expression by quercetin may contribute to the neuroprotective effect of quercetin in focal cerebral ischemia. Our findings suggest that quercetin mediates its neuroprotective function by regulation of thioredoxin expression and maintenance of interaction between ASK1 and thioredoxin.