Zirconium dioxide nanoparticles induced cytotoxicity in rat cerebral cortical neurons and apoptosis in neuron-like N2a and PC12 cell lines.

During recent decades, the application of zirconium dioxide nanoparticles (ZrO2-NP) has been expanded in various fields ranging from medicine to industry. It has been shown that ZrO2-NP has the potential to cross the blood-brain barrier (BBB) and induce neurotoxicity. In the current study, we investigated the in vivo neurotoxicity, as well as, the cellular mechanism of ZrO2-NP toxicity on two neuronal-like cell lines, PC12 and N2a. PC12 and N2a cells were exposed to increasing concentrations of ZrO2-NP (0-2000 µg/ml) for 48 h. The apoptotic effect of ZrO2-NP was determined using annexin V/propidium iodide double staining (by flow cytometry), and western blot analysis of relative apoptotic proteins, including caspase-3, caspase-9, bax, and bcl2. Based on our results, ZrO2-NP at concentrations of 250-2000 µg/mL increased both early and late-stage apoptosis in a concentration-dependent manner. Moreover, the expressions of cleaved-caspase-3 and -9 proteins and the bax/bcl2 ratio were significantly increased. In addition, oral administration of ZrO2-NP (50 mg/kg) to male Wistar rats for 28 days led to the loss of neuronal cells in the cerebral cortex. Taken together, our findings highlighted the role of apoptosis on cytotoxicity induced by ZrO2-NP.

Characterization and Hydrolysis Studies of a Prodrug Obtained as Ester Conjugate of Geraniol and Ferulic Acid by Enzymatic Way.

Ferulic acid (Fer) and geraniol (Ger) are natural compounds whose antioxidant and anti-inflammatory activity confer beneficial properties, such as antibacterial, anticancer, and neuroprotective effects. However, the short half-lives of these compounds impair their therapeutic activities after conventional administration. We propose, therefore, a new prodrug (Fer-Ger) obtained by a bio-catalyzed ester conjugation of Fer and Ger to enhance the loading of solid lipid microparticles (SLMs) designed as Fer-Ger delivery and targeting systems. SLMs were obtained by hot emulsion techniques without organic solvents. HPLC-UV analysis evidenced that Fer-Ger is hydrolyzed in human or rat whole blood and rat liver homogenates, with half-lives of 193.64 ± 20.93, 20.15 ± 0.75, and 3.94 ± 0.33 min, respectively, but not in rat brain homogenates. Studies on neuronal-differentiated mouse neuroblastoma N2a cells incubated with the reactive oxygen species (ROS) inductor H2O2 evidenced the Fer-Ger ability to prevent oxidative injury, despite the fact that it appears ROS-promoting. The amounts of Fer-Ger encapsulated in tristearin SLMs, obtained in the absence or presence of glucose, were 1.5 ± 0.1%, allowing the control of the prodrug release (glucose absence) or to sensibly enhance its water dissolution rate (glucose presence). These new "green" carriers can potentially prolong the beneficial effects of Fer and Ger or induce neuroprotection as nasal formulations.

Neuroprotective Effect of Riboflavin Producing Lactic Acid Bacteria in Parkinsonian Models.

Oxidative stress and inflammatory processes might contribute to the cascade of events leading Parkinson disease (PD); and vitamins such as riboflavin can exert protection on vulnerable neurons in neurodegenerative conditions. Previously, it was demonstrated that a mixture of lactic acid bacteria (including a riboflavin-producing strain) improved motor skills in a parkinsonian model. The aim of the present work was to investigate the neuroprotective potential of Lactiplantibacillus (L.) plantarum CRL2130, a riboflavin-producing strain in PD models. In vitro, N2a differentiated neurons were exposed the neurotoxin 1-methyl-4-phenylpyridinium (MPP+) and treated with intracellular bacterial extracts or commercial riboflavin. In vivo, adult male C57BL/6 mice were injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and probenecid, and received orally L. plantarum CRL2130, L. plantarum CRL725 (parent strain that produces low levels of riboflavin) or commercial vitamin. Results showed that when N2a cells were incubated with intracellular extract from L. plantarum CRL2130 maintained the viability, and significantly decreased the release of IL-6 and the formation of reactive oxygen species (ROS), all affected by MPP+. In vivo, the administration of L. plantarum CRL2130 attenuated motor deficits and prevented dopaminergic neuronal death. Decrease of pro-inflammatory cytokines and increase of IL-10 in both serum and brain were observed in samples from mice that received L. plantarum CRL2130 compared to MPTP control group (without treatment). In addition, these beneficial effects were similar or improved when compared with animals that received commercial riboflavin. In conclusion, L. plantarum CRL2130 showed a neuroprotective effect in both PD models through anti-oxidant/anti-inflammatory mechanisms.

Estrogen-induced downregulation of TASK-1 expression through estrogen receptor ß in N2A cells.

BACKGROUND: Our previous data revealed that reduction of TASK-1 expression, as a consequence of exposure to 17ß-estradiol, could participate in neuroprotective effects in N2A cells. However, it is unclear which estrogen receptor underlies these effects of 17ß-estradiol. METHODS AND RESULTS: In this study, the knockdown experiments are carried out to clarify the estrogen receptor responsible for effects of estrogen on TASK-1 channels. Subsequently, data from QPCR measurements reveal that estrogen receptor ß (ERß), but not estrogen receptor α, serves as a binding target for 17ß-estradiol after a 48-h treatment. CONCLUSIONS: The current result suggests the implication of the ERß-dependent manner in the pro-proliferative action of estrogen via TASK-1 channels.

Structural and optical properties of dysprosium-doped hydroxyapatite nanoparticles and the use as a bioimaging probe in human cells.

In this work, the hydroxyapatite nanoparticles doped with trivalent dysprosium ions were synthesized by a co-precipitation method. The characterization techniques such as X-ray diffraction (XRD), transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDX) were carried out to determine the crystalline and structural properties. The Rietveld structural refinement of the XRD patterns confirmed the purity of the phase formation of the synthesized nanoparticles. The photoluminescence emission spectra exhibited intense emissions in the blue region at 450 nm and 476 nm along with less intense yellow emission at 573 nm which can be attributed to the magnetic dipole and electric dipole transitions of dysprosium respectively. In order to evaluate the colour tunability of the emitted light CIE chromaticity coordinate values were calculated. The intense blue emissions from the synthesized sample were found to be favourable for bioimaging. The images obtained from the fluorescence microscopy revealed that the dysprosium-doped hydroxyapatite nanoparticles are potential bioimaging probes in human cells.

Blockage of KHSRP-NLRP3 by MCC950 Can Reverse the Effect of Manganese-Induced Neuroinflammation in N2a Cells and Rat Brain.

Manganese neurotoxicity has been reported to cause a neurodegenerative disease known as parkinsonism. Previous reports have shown that the expression of the KH-type splicing regulatory protein (KHSRP), a nucleic acid-binding protein, and NLRP3 is increased upon Mn exposure. However, the relation between these two during Mn toxicity has not been fully deduced. The mouse neuroblastoma (N2a) and SD rats are treated with LPS and MnCl2 to evaluate the expression of KHSRP and NLRP3. Further, the effect of the NLRP3 inhibitor MCC950 is checked on the expression of NLRP3, KHSRP and pro-inflammatory markers (TNFα, IL-18 and IL-1ß) as well as the caspase-1 enzyme. Our results demonstrated an increment in NLRP3 and KHSRP expression post-MnCl2 exposure in N2a cells and rat brain, while on the other hand with LPS exposure only NLRP3 expression levels were elevated and KHSRP was found to be unaffected. An increased expression of KHSRP, NLRP3, pro-inflammatory markers and the caspase-1 enzyme was observed to be inhibited with MCC950 treatment in MnCl2-exposed cells and rats. Manganese exposure induces NLRP3 and KHSRP expression to induce neuroinflammation, suggesting a correlation between both which functions in toxicity-related pathways. Furthermore, MCC950 treatment reversed the role of KHSRP from anti-inflammatory to pro-inflammatory.

Cytotoxic and Antioxidant Activities of Imine Analogs of Trans-Resveratrol towards Murine Neuronal N2a Cells.

Trans-resveratrol is a natural polyphenol showing numerous biological properties, especially anti-tumoral and antioxidant activity. Among numerous resveratrol derivatives, aza-stilbenes, which bear an imine bound, show interesting biological activities. In the present study, we synthesized a series of imine analogs of trans-resveratrol (seven aza-stilbenes) following an easy and low-cost procedure of green chemistry. The toxicity of synthesized aza-stilbenes, which is currently unknown, was evaluated on murine neuronal N2a cells, comparatively to trans-resveratrol, by considering: cell density evaluated by staining with sulforhodamine 101; esterase activity, which is a criteria of cell viability, by staining with fluorescein diacetate; and transmembrane mitochondrial potential, which is known to decrease during cell death, by staining with DiOC6(3) using flow cytometry. In addition, the antioxidant activity was quantified with the KRL (Kit Radicaux Libres) assay, the DPPH (2,2'-diphenyl-1-picrylhydrazyl radical) assay and the FRAP (ferric reducing antioxidant power) assay. The PAOT (Pouvoir Antioxidant Total) score was also used. The aza-stilbenes provide different cytotoxic and antioxidant activities, which are either higher or lower than those of trans-resveratrol. Based on their cytotoxic and antioxidant characteristics, all synthesized aza-stilbenes are distinguished from trans-resveratrol.

Nanonization of a chemically synthesized flavone HMDF (3-hydroxy-3',4'-methylenedioxyflavone) by entrapping within calcium phosphate nanoparticles and exploring its antioxidant role on neural cellsin vitroand zebrafishin vivo.

The chemical synthesis of 3-hydroxy-3',4'-methylenedioxyflavone (HMDF) was reported to generate a modified flavone of potent antioxidant activity with significant neuropharmacological properties. In this study, HMDF was nanonized by entrapping within calcium phosphate nanoparticles (CPNPs). HMDF-CPNPs were of (i) size 25 nm, (ii) zeta potential (-) [22 ± 3] mV and (iii) entrapment efficiency 67%. HMDF-CPNPs, but not HMDF alone, inhibited thein vitroactivity of acetylcholinesterase enzymes to break down the major neurotransmitter compound acetylcholine. Moreover, nanonized HMDF had more antioxidant activity than bulk HMDF, as observed from its ability to protect mouse neural (N2A) cells from oxidative damage caused by H2O2exposure at the levels of cell viability, intracellular reactive oxygen species, mitochondrial membrane potential, cell cycle stages, nuclear integrity and neural connectivity. Anin vivostudy on zebrafish larvae (Denio rerio) also demonstrated that H2O2-mediated larval death was checked by HMDF-CPNP treatment. These results, therefore, suggest that HMDF-CPNPs may be developed as a potential antioxidant, particularly as a neuroprotectant.

JAK2/STAT3 involves oxidative stress-induced cell injury in N2a cells and a rat MCAO model.

Purpose: In this study, we sought to test the hypothesis that oxidative stress injury in ischemic brains and H2O2-treated mouse neuroblastoma Neuro-2a cells (N2a) was related to STAT3 activation.Materials and methods: Rat middle cerebral artery occlusion (MCAO) model and H2O2-treated mouse neuroblastoma Neuro-2a cells (N2a) were used to investigate the relationship between oxidative stress injury and STAT3 activation.Results: 8-Hydroxy-2'-deoxyguanosine (8-OHdG) content and STAT3 protein phosphorylation level were significantly increased after cerebral ischemia-reperfusion. H2O2 treatment inhibited the cell viability, induced the apoptosis, and further raised pSTAT3 protein level in N2a cells. Moreover, the addition of AG490, the protein inhibitor of JAK2, significantly alleviated cerebral ischemic damage in vivo and H2O2-induced injury in vitro, and JAK2 siRNA also alleviated H2O2-induced injury in N2a cell.Conclusions: JAK2/STAT3 pathway may play a crucial role in mediating reactive oxidative species (ROS)-induced cell injury in rat middle cerebral artery occlusion (MCAO) model and N2a cells. ROS scavenging and down-regulation of STAT3 activation might be a candidate design of therapeutic strategies against oxidative stress-related neurological diseases.

Green Tea Seed Isolated Theasaponin E1 Ameliorates AD Promoting Neurotoxic Pathogenesis by Attenuating Aß Peptide Levels in SweAPP N2a Cells.

Alzheimer's disease (AD) is the most frequent type of dementia affecting memory, thinking and behaviour. The major hallmark of the disease is pathological neurodegeneration due to abnormal aggregation of Amyloid beta (Aß) peptides generated by ß- and γ-secretases via amyloidogenic pathway. Purpose of the current study was to evaluate the effects of theasaponin E1 on the inhibition of Aß producing ß-, γ-secretases (BACE1, PS1 and NCT) and acetylcholinesterase and activation of the non-amyloidogenic APP processing α-secretase (ADAM10). Additionally, theasaponin E1 effects on Aß degrading and clearing proteins neprilysin and insulin degrading enzyme (IDE). The effect of theasaponin E1 on these crucial enzymes was investigated by RT-PCR, ELISA, western blotting and fluorometric assays using mouse neuroblastoma cells (SweAPP N2a). theasaponin E1 was extracted and purified from green tea seed extract via HPLC, and N2a cells were treated with different concentrations for 24 h. Gene and protein expression in the cells were measured to determine the effects of activation and/or inhibition of theasaponin E1 on ß- and γ-secretases, neprilysin and IDE. Results demonstrated that theasaponin E1 significantly reduced Aß concentration by activation of the α-secretase and neprilysin. The activities of ß- and γ-secretase were reduced in a dose-dependent manner due to downregulation of BACE1, presenilin, and nicastrin. Similarly, theasaponin E1 significantly reduced the activity of acetylcholinesterase. Overall, from the results it is concluded that green tea seed extracted saponin E1 possess therapeutic significance as a neuroprotective natural product recommended for the treatment of Alzheimer's disease.

Prevention of 7-Ketocholesterol-Induced Overproduction of Reactive Oxygen Species, Mitochondrial Dysfunction and Cell Death with Major Nutrients (Polyphenols, ω3 and ω9 Unsaturated Fatty Acids) of the Mediterranean Diet on N2a Neuronal Cells.

The brain, which is a cholesterol-rich organ, can be subject to oxidative stress in a variety of pathophysiological conditions, age-related diseases and some rare pathologies. This can lead to the formation of 7-ketocholesterol (7KC), a toxic derivative of cholesterol mainly produced by auto-oxidation. So, preventing the neuronal toxicity of 7KC is an important issue to avoid brain damage. As there are numerous data in favor of the prevention of neurodegeneration by the Mediterranean diet, this study aimed to evaluate the potential of a series of polyphenols (resveratrol, RSV; quercetin, QCT; and apigenin, API) as well as ω3 and ω9 unsaturated fatty acids (α-linolenic acid, ALA; eicosapentaenoic acid, EPA; docosahexaenoic acid, DHA, and oleic acid, OA) widely present in this diet, to prevent 7KC (50 µM)-induced dysfunction of N2a neuronal cells. When polyphenols and fatty acids were used at non-toxic concentrations (polyphenols: ≤6.25 µM; fatty acids: ≤25 µM) as defined by the fluorescein diacetate assay, they greatly reduce 7KC-induced toxicity. The cytoprotective effects observed with polyphenols and fatty acids were comparable to those of α-tocopherol (400 µM) used as a reference. These polyphenols and fatty acids attenuate the overproduction of reactive oxygen species and the 7KC-induced drop in mitochondrial transmembrane potential (ΔΨm) measured by flow cytometry after dihydroethidium and DiOC6(3) staining, respectively. Moreover, the studied polyphenols and fatty acids reduced plasma membrane permeability considered as a criterion for cell death measured by flow cytometry after propidium iodide staining. Our data show that polyphenols (RSV, QCT and API) as well as ω3 and ω9 unsaturated fatty acids (ALA, EPA, DHA and OA) are potent cytoprotective agents against 7KC-induced neurotoxicity in N2a cells. Their cytoprotective effects could partly explain the benefits of the Mediterranean diet on human health, particularly in the prevention of neurodegenerative diseases.

TLR4 and nucleolin influence cell injury, apoptosis and inflammatory factor expression in respiratory syncytial virus-infected N2a neuronal cells.

Respiratory syncytial virus (RSV) infection was recently reported to be associated with central nervous system (CNS) symptoms and neurological complications; however, related studies are very limited. Moreover, the molecular mechanism underlying RSV neuropathogenesis is still unclear. Our previous study revealed that toll-like receptor 4 (TLR4) and nucleolin (C23) could be modulated and that they played a role during RSV infection in mouse neuronal-2a (N2a) cells. In the present study, the effects of silencing of TLR4 and C23 on RSV propagation and N2a cellular responses were examined by using RNA interference technology. Four N2a cell treatment groups were established, namely, a normal control group, RSV control group, TLR4 siRNA + RSV group, and C23 siRNA + RSV group. Expression changes in NeuN protein and colocalization of C23 and TLR4 with RSV F protein were assessed using confocal microscopy. Changes in TLR4 and C23 mRNA expression, TLR4, C23, TLR3, TLR7, and p-NF-κB protein expression, and interleukin (IL)-8, IL-6, and tumor necrosis factor (TNF-α) cytokine secretion was measured using quantitative real-time reverse-transcription polymerase chain reaction, Western blot analysis, and enzyme-linked immunosorbent assay, respectively. RSV titers and the apoptotic status of N2a cells were monitored using plaque formation assays and flow cytometry, respectively. The results indicated that TLR4 and C23 gene knockdown decreased the amount of F protein in RSV-infected N2a cells, inhibited RSV propagation, attenuated N2a neuronal injury, diminished cell apoptosis levels, downregulated TLR3 and TLR7 protein expression, and reduced inflammatory protein expression. Therefore, TLR4 and C23 knockdown influences cell injury, apoptosis and inflammatory protein expression in RSV-infected N2a cells.

Octadecaneuropeptide (ODN) Induces N2a Cells Differentiation through a PKA/PLC/PKC/MEK/ERK-Dependent Pathway: Incidence on Peroxisome, Mitochondria, and Lipid Profiles.

Neurodegenerative diseases are characterized by oxidative stress, mitochondrial damage, and death of neuronal cells. To counteract such damage and to favor neurogenesis, neurotrophic factors could be used as therapeutic agents. Octadecaneuropeptide (ODN), produced by astrocytes, is a potent neuroprotective agent. In N2a cells, we studied the ability of ODN to promote neuronal differentiation. This parameter was evaluated by phase contrast microscopy, staining with crystal violet, cresyl blue, and Sulforhodamine 101. The effect of ODN on cell viability and mitochondrial activity was determined with fluorescein diacetate and DiOC6(3), respectively. The impact of ODN on the topography of mitochondria and peroxisomes, two tightly connected organelles involved in nerve cell functions and lipid metabolism, was evaluated by transmission electron microscopy and fluorescence microscopy: detection of mitochondria with MitoTracker Red, and peroxisome with an antibody directed against the ABCD3 peroxisomal transporter. The profiles in fatty acids, cholesterol, and cholesterol precursors were determined by gas chromatography, in some cases coupled with mass spectrometry. Treatment of N2a cells with ODN (10-14 M, 48 h) induces neurite outgrowth. ODN-induced neuronal differentiation was associated with modification of topographical distribution of mitochondria and peroxisomes throughout the neurites and did not affect cell viability and mitochondrial activity. The inhibition of ODN-induced N2a differentiation with H89, U73122, chelerythrine and U0126 supports the activation of a PKA/PLC/PKC/MEK/ERK-dependent signaling pathway. Although there is no difference in fatty acid profile between control and ODN-treated cells, the level of cholesterol and some of its precursors (lanosterol, desmosterol, lathosterol) was increased in ODN-treated cells. The ability of ODN to induce neuronal differentiation without cytotoxicity reinforces the interest for this neuropeptide with neurotrophic properties to overcome nerve cell damage in major neurodegenerative diseases.

Respiratory syncytial virus prolifically infects N2a neuronal cells, leading to TLR4 and nucleolin protein modulations and RSV F protein co-localization with TLR4 and nucleolin.

BACKGROUND: Respiratory syncytial virus (RSV) infects the central nervous system, resulting in neurological symptoms. However, the precise underlying pathogenic mechanisms have not been elucidated. In the present study, the infectivity of RSV on N2a neuronal cells and the possible roles of Toll-like receptor 4 (TLR4) and nucleolin (C23) during RSV infection were investigated. METHODS: We compared two experimental groups (infected and non-infected) and monitored the RSV viral titers in the culture supernatant by a viral plaque assay. We also inspected the morphology of the nucleus in infected N2a cells. We measured the level of RSV F protein and studied its co-localization with TLR4 and nucleolin using immunofluorescence assays and laser confocal microscopy. The potential interaction of RSV F protein with TLR4 and nucleolin was examined by coimmunoprecipitation. The expression changes of TLR4, nucleolin, TLR3 and TLR7 proteins in N2a cells and IL-6 and TNF-α in the culture supernatant were investigated by Western Blot analysis and ELISA assay. Changes in neuronal cell apoptosis status was examined by flow cytometry. RESULTS: The results demonstrated prolific RSV infection of N2a cells, which triggered a decrease of NeuN protein expression, coinciding with an increase of nuclear lesions, F protein expression, RSV viral titers, and late apoptotic levels of N2a cells. RSV infection induced co-localization of RSV F protein with TLR4 and nucleolin, which could potentially lead to a direct interaction. Furthermore, it was found that TLR4 and nucleolin levels increased early after infection and decreased subsequently, whereas TLR3 and TLR7 expression increased throughout RSV infection. CONCLUSION: The RSV Long strain can prolifically infect N2a neuronal cells, modulating the expression of TLR4 and nucleolin, as well as TLR3, TLR7 and their downstream inflammatory factors, and inducing the co-localization of the RSV F protein with TLR4 and nucleolin.

Ectonucleotide pyrophosphatase/phosphodiesterase activity in Neuro-2a neuroblastoma cells: changes in expression associated with neuronal differentiation.

Neuro-2a (N2a) neuroblastoma cells display an ectoenzymatic hydrolytic activity capable of degrading diadenosine polyphosphates. The Apn A-cleaving activity has been analysed with the use of the fluorogenic compound BODIPY FL guanosine 5'-O-(3-thiotriphosphate) thioester. Hydrolysis of this dinucleotide analogue showed a hyperbolic kinetic with a Km value of 4.9 ± 1.3 µM. Diadenosine pentaphosphate, diadenosine tetraphosphate, diadenosine triphosphate, and the nucleoside monophosphate AMP behaved as an inhibitor of BODIPY FL guanosine 5'-O-(3-thiotriphosphate) thioester extracellular degradation. Ectoenzymatic activity shared the typical characteristics of the ectonucleotide pyrophosphatase/phosphodiesterase family, as hydrolysis reached maximal activity at alkaline pH and was dependent on the presence of divalent cations, being strongly inhibited by EDTA and activated by Zn(2+) ions. Both NPP1 and NPP3 isozymes are expressed in N2a cells, their expression levels substantially changing when cells differentiate into a neuronal-like phenotype. In this sense, it is relevant to point the expression pattern of the NPP3 protein, whose levels were drastically reduced in the differentiated cells, being almost completely absent after 24 h of differentiation. Enzymatic activity assays carried out with differentiated N2a cells showed that NPP1 is the main isozyme involved in the extracellular degradation of dinucleotides in these cells, this enzyme reducing its activity and changing its subcellular location following neuronal differentiation. We described the presence of an ectoenzymatic activity able to hydrolyse diadenosine polyphosphates (ApnA) in N2a cells. This activity displays biochemical features that are typical of the ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP) family members, as demonstrated by the use of the fluorogenic compound BODIPY-FL-GTPγS. Both NPP1 and NPP3 ectoenzymes are expressed in N2a cells, their levels dramatically changing when cells differentiate into a neuronal-like phenotype. Activity assays in differentiated cells showed that the ApnA-hydrolytic activity largely depends on the NPP1 isozyme.

Negative Association of Cognitive Performance With Blood Serum Neurotoxicity and Its Modulation by Human Herpes Virus 5 (HHV5) Seropositivity in Healthy Women.

Identification of early influences on cognitive decline is of paramount importance in order to stem the impacts of decrements in cognitive functioning and to potentially intervene. Thus, here we focused on 132 healthy adult women (age range 26-98 years) to (a) determine whether factors circulating in serum may exert neurotoxic effects in vitro, (b) evaluate associations between serum neurotoxicity and cognitive performance, and (c) assess the influence of human herpes virus (HHV) seroprevalence and other factors on apoptosis and cognitive performance. The results documented that the addition of serum from healthy adult women to neural cell cultures resulted in apoptosis, indicating the presence of circulating neurotoxic factors in the serum. Furthermore, apoptosis increased with age, and was associated with decreased cognitive performance. Stepwise regression evaluating the influence of 6 HHVs on apoptosis and cognitive function revealed that only HHV5 (cytomegalovirus; CMV) seropositivity was significantly associated with apoptosis and cognitive decline, controlling for age. These findings document neurotoxic effects of serum from healthy women across the adult lifespan and suggest a unique detrimental influence associated with CMV seropositivity.

Isolation of Exosomes or Extracellular Vesicles from West Nile Virus-Infected N2a Cells, Primary Cortical Neurons, and Brain Tissues.

Several flaviviruses compromise the blood-brain barrier integrity, infect the central nervous system, and elicit neuroinvasion to successfully cause neuropathogenesis in the vertebrate host. Therefore, understanding the pathway(s) and mechanism(s) to block the transmission and/or dissemination of flaviviruses and perhaps other neuroinvasive viruses is considered as an important area of research. Moreover, studies that address mechanism(s) of neuroinvasion by flaviviruses are limited. In this chapter, we discuss detailed methods to isolate exosomes or extracellular vesicles (EVs) from mouse and human N2a cells, primary cultures of murine cortical neurons, and mouse brain tissue. Two different methods including differential ultracentrifugation and density gradient exosome (DG-Exo) isolation are described for the preparation of exosomes/EVs from N2a cells and cortical neurons. In addition, we discuss the detailed DG-Exo method for the isolation of exosomes from murine brain tissue. Studies on neuronal exosomes will perhaps enhance our understanding of the mechanism of neuroinvasion by these deadly viruses.

Non-prophylactic resveratrol-mediated protection of neurite integrity under chronic hypoxia is associated with reduction of Cav1.2 channel expression and calcium overloading.

Cellular hypoxia is a major cause of oxidative stress, culminating in neuronal damage in neurodegenerative diseases. Numerous ex vivo studies have implicated that hypoxia episodes leading to disruption of Ca2+ homeostasis and redox status contribute to the progression of various neuropathologies and cell death. Isolation and maintenance of primary cell culture being cost-intensive, the details of the time course relationship between Ca2+ overload, L-type Ca2+ channel function, and neurite retraction under chronic and long-term hypoxia remain undefined. In order to explore the effect of oxidative stress and Ca2+ overload on neurite length, first, we developed a 5-day-long neurite outgrowth model using N2a cell line. Second, we propose a chronic hypoxia model to investigate the modulation of the L-type Ca2+ channel (Cav1.2) and oxidative resistance gene (OXR1) expression level during the process of neurite retraction and neuronal damage over 32 h. Thirdly, we developed a framework for quantitative analysis of cytosolic Ca2+, superoxide formation, neurite length, and constriction formation in individual cells using live imaging that provides an understanding of molecular targets. Our findings suggest that an increase in cytosolic Ca2+ is a feature of an early phase of hypoxic stress. Further, we demonstrate that augmentation in the L-type channel leads to amplification in Ca2+ overload, ROS accumulation, and a reduction in neurite length during the late phase of hypoxic stress. Next, we demonstrated that non-prophylactic treatment of resveratrol leads to the reduction of calcium overloading under chronic hypoxia via lowering of L-type channel expression. Finally, we demonstrate that resveratrol-mediated reduction of Cav1.2 channel and STAT3 expression are associated with retention of neurite integrity. The proposed in vitro model assumes significance in the context of drug designing and testing that demands monitoring of neurite length and constriction formations by imaging before animal testing.

CRISPR-Mediated Activation of αV Integrin Subtypes Promotes Neuronal Differentiation of Neuroblastoma Neuro2a Cells.

Neuronal differentiation is a complex process whose dysfunction can lead to brain disorders. The development of new tools to target specific steps in the neuronal differentiation process is of paramount importance for a better understanding of the molecular mechanisms involved, and ultimately for developing effective therapeutic strategies for neurodevelopmental disorders. Through their interactions with extracellular matrix proteins, the cell adhesion molecules of the integrin family play essential roles in the formation of functional neuronal circuits by regulating cell migration, neurite outgrowth, dendritic spine formation and synaptic plasticity. However, how different integrin receptors contribute to the successive phases of neuronal differentiation remains to be elucidated. Here, we implemented a CRISPR activation system to enhance the endogenous expression of specific integrin subunits in an in vitro model of neuronal differentiation, the murine neuroblastoma Neuro2a cell line. By combining CRISPR activation with morphological and RT-qPCR analyses, we show that integrins of the αV family are powerful inducers of neuronal differentiation. Further, we identify a subtype-specific role for αV integrins in controlling neurite outgrowth. While αVß3 integrin initiates neuronal differentiation of Neuro2a cells under proliferative conditions, αVß5 integrin appears responsible for promoting a complex arborization in cells already committed to differentiation. Interestingly, primary neurons exhibit a complementary expression pattern for ß3 and ß5 integrin subunits during development. Our findings reveal the existence of a developmental switch between αV integrin subtypes during differentiation and suggest that a timely controlled modulation of the expression of αV integrins by CRISPRa provides a means to promote neuronal differentiation.

Potentilla anserine L. polysaccharide protects against cadmium-induced neurotoxicity.

Cadmium is a toxic metal that can damage the brain and other organs. This study aimed to explore the protective effects of Potentilla anserine L. polysaccharide (PAP) against CdCl2-induced neurotoxicity in N2a and SH-SY5Y cells and in the cerebral cortex of BALB/c mice. In addition, we aimed to identify the potential mechanisms underlying these protective effects. Relative to CdCl2 treatment alone, pretreatment with PAP prevented the reduction in cell viability evoked by CdCl2, decreased rates of apoptosis, promoted calcium homeostasis, decreased ROS accumulation, increased mitochondrial membrane potential, inhibited cytochrome C and AIF release, and prevented the cleavage of caspase-3 and PARP. In addition, PAP significantly decreased the CdCl2-induced phosphorylation of CaMKII, Akt, and mTOR. In conclusion, PAP represents a potential therapeutic agent for the treatment of Cd-induced neurotoxicity, functioning in part via attenuating the activation of the mitochondrial apoptosis pathway and the Ca2+-CaMKII-dependent Akt/mTOR pathway.