Neuronal Cultures Research Articles

Compartmentalized Primary Murine Neuron Culture Using Plastic Microfluidic Chips

Compartmentalized Primary Murine Neuron Culture Using Plastic Microfluidic Chips

Compartmentalized Primary Murine Neuron Culture Using Plastic Microfluidic Chips

Compartmentalized Primary Murine Neuron Culture Using Plastic Microfluidic Chips

Real-Time Analysis of Neuronal Cell Cultures for CNS Drug Discovery.

The ability to screen for agents that can promote the development and/or maintenance of neuronal networks creates opportunities for the discovery of novel agents for the treatment of central nervous system (CNS) disorders. Over the past 10 years, advances in robotics, artificial intelligence, and machine learning have paved the way for the improved implementation of live-cell imaging systems for drug discovery. These instruments have revolutionized our ability to quickly and accurately acquire large standardized datasets when studying complex cellular phenomena in real-time. This is particularly useful in the field of neuroscience because real-time analysis can allow efficient monitoring of the development, maturation, and conservation of neuronal networks by measuring neurite length. Unfortunately, due to the relative infancy of this type of analysis, standard practices for data acquisition and processing are lacking, and there is no standardized format for reporting the vast quantities of data generated by live-cell imaging systems. This paper reviews the current state of live-cell imaging instruments, with a focus on the most commonly used equipment (IncuCyte systems). We provide an in-depth analysis of the experimental conditions reported in publications utilizing these systems, particularly with regard to studying neurite outgrowth. This analysis sheds light on trends and patterns that will enhance the use of live-cell imaging instruments in CNS drug discovery.

Protection from oxygen–glucose deprivation by neurosteroid treatment in primary neurons and oligodendrocytes

Preterm birth results in an increased risk of neonatal brain injury and neurobehavioural disorders. Despite the seriousness of these adverse outcomes, there are currently no effective therapies to protect the vulnerable developing brain. We propose that neurosteroid replacement therapy may be a novel approach in reducing detrimental neurological outcomes following preterm birth. The use of guinea pig primary neuronal and oligodendrocyte cultures with relevance to late gestation allows insight into the mechanisms behind the effectiveness of these treatments. Primary neuronal and oligodendrocyte cultures were derived from fetal guinea pig frontal cortex brain tissue at gestational age 62 (GA62). Cell cultures were pre-treated with either etifoxine (5 µM) or zuranolone (1 µm) for 24 h prior to insult. Cells were then exposed to either oxygen–glucose deprivation (OGD; 0% O2 and no glucose DMEM; preterm birth insult) or sham (standard cell culture conditions; 25 mM DMEM) for 2 h. Lactate dehydrogenase assay (LDH) was performed following OGD as a measure of cytotoxicity. Relative mRNA expression of key neuronal and oligodendrocyte markers, as well as neuronal receptors and transporters, were quantified using high throughput (Fluidigm) RT-PCR. OGD significantly increased cellular cytotoxicity in both neurons and oligodendrocytes. Additionally, key neuronal marker mRNA expression was reduced following OGD, and oligodendrocytes displayed arrested mRNA expression of key markers of lineage progression. Treatment with etifoxine restored a number of parameters back to control levels, whereas treatment with zuranolone provided a robust improvement in all parameters examined. This study has demonstrated the neuroprotective potential of neurosteroid replacement therapy in a model of hypoxia related to preterm birth. Neuroprotection appears to be mediated through glutamate reduction and increased brain derived neurotrophic factor (BDNF). Future work is warranted in examining these treatments in vivo, with the overall aim to suppress preterm associated brain damage and reduce long term outcomes for affected offspring.

On the Feasibility of Using an Acedane-Based Fluorescent Probe to Monitor Hydrogen Sulfide in Primary Neuronal Cultures

Hydrogen sulfide (H₂S), which, under physiological conditions, exists in cells mainly in the form of anion HS–, is considered as a gaseous transmitter of inter- and intracellular signals along with nitrogen oxide and carbon monoxide. Analysis of the dynamics of H₂S content in living cells is impossible without creating sensitive and specific probes. Several acedan-based compounds have been synthesized in the group of K.H. Ahn (Singha et al., 2015. Anal. Chem. 87 (2), 1188–1195). In the presence of H₂S these probes attach to the sulfhydrilic group and form fluorescent carbocyclic compounds. The carbocyclic derivative of P3, compound csP3, was found to be optimal for fluorescence-microscopic studies in terms of spectral characteristics and response time to H₂S. In this work, we tested the suitability of csP3 to record H₂S changes in buffers mimicking the salt composition of the intracellular environment and in primary neuronal culture cells from rat cerebral cortex. It was found that reducing the polarity of the solution by adding dimethyl sulfoxide (30% by volume) caused a blue shift of the emission by ~10 nm and a twofold increase in fluorescence intensity. The csP3 fluorescence depends on the salt composition and increases in the presence of bicarbonate (NaHCO₃, 10 mM). Addition of P3 or csP3 to the neuronal culture caused a rapid increase in fluorescence, which was followed by a slow increase in fluorescence signal after 3–5 min. Glutamate (10 μM, in the presence of 10 μM glycine, 0 Mg2+) increased probe fluorescence, but only in those neurons in which delayed deregulation of calcium homeostasis did not occur. We conclude that the product of the reaction of P3 with H₂S is sensitive to a change in the salt composition of the intracellular medium and can be redistributed in cells between water and more hydrophobic environment. This means that an increase in P3 fluorescence in cells, especially after the addition of glutamate to neurons, does not necessarily indicate an increase in H₂S concentration. To confirm the feasibility of using P3 and structurally related probes as quantitative indicators of H₂S presence, additional studies of the properties of these compounds are needed.

Human tripartite cortical network model for temporal assessment of alpha-synuclein aggregation and propagation in Parkinson’s Disease

Previous studies have shown that aggregated alpha-synuclein (α-s) protein, a key pathological marker of Parkinson’s disease (PD), can propagate between cells, thus participating in disease progression. This prion-like propagation has been widely studied using in vivo and in vitro models, including rodent and human cell cultures. In this study, our focus was on temporal assessment of functional changes during α-s aggregation and propagation in human induced pluripotent stem cell (hiPSC)-derived neuronal cultures and in engineered networks. Here, we report an engineered circular tripartite human neuronal network model in a microfluidic chip integrated with microelectrode arrays (MEAs) as a platform to study functional markers during α-s aggregation and propagation. We observed progressive aggregation of α-s in conventional neuronal cultures and in the exposed (proximal) compartments of circular tripartite networks following exposure to preformed α-s fibrils (PFF). Furthermore, aggregated forms propagated to distal compartments of the circular tripartite networks through axonal transport. We observed impacts of α-s aggregation on both the structure and function of neuronal cells, such as in presynaptic proteins, mitochondrial motility, calcium oscillations and neuronal activity. The model enabled an assessment of the early, middle, and late phases of α-s aggregation and its propagation during a 13-day follow-up period. While our temporal analysis suggested a complex interplay of structural and functional changes during the in vitro propagation of α-s aggregates, further investigation is required to elucidate the underlying mechanisms. Taken together, this study demonstrates the technical potential of our introduced model for conducting in-depth analyses for revealing such mechanisms.

SARS-CoV-2 Rapidly Infects Peripheral Sensory and Autonomic Neurons, Contributing to Central Nervous System Neuroinvasion before Viremia.

Neurological symptoms associated with COVID-19, acute and long term, suggest SARS-CoV-2 affects both the peripheral and central nervous systems (PNS/CNS). Although studies have shown olfactory and hematogenous invasion into the CNS, coinciding with neuroinflammation, little attention has been paid to susceptibility of the PNS to infection or to its contribution to CNS invasion. Here we show that sensory and autonomic neurons in the PNS are susceptible to productive infection with SARS-CoV-2 and outline physiological and molecular mechanisms mediating neuroinvasion. Our infection of K18-hACE2 mice, wild-type mice, and golden Syrian hamsters, as well as primary peripheral sensory and autonomic neuronal cultures, show viral RNA, proteins, and infectious virus in PNS neurons, satellite glial cells, and functionally connected CNS tissues. Additionally, we demonstrate, in vitro, that neuropilin-1 facilitates SARS-CoV-2 neuronal entry. SARS-CoV-2 rapidly invades the PNS prior to viremia, establishes a productive infection in peripheral neurons, and results in sensory symptoms often reported by COVID-19 patients.

Biocompatibility and customizability: Expanding possibilities with 3D printed guide cannulas

BackgroundIntracerebral cannulation bypasses the blood-brain barrier, and is frequently used for targeted drug delivery to specific brain structures. Despite the availability of brain infusion kits and manual injections without cannulation, the traditional design of guide cannulas continues to be utilized in research. Several protocols describing guide cannula manufacture from stainless steel needles have been published previously. New methodWe describe a method for producing the first fully plastic guide cannula intended for intracerebroventricular injections in mice using Dental Sand A1-A2 resin and digital light processing 3D printing. ResultsThe lack of resin neurotoxicity for primary rat cortical neuron cultures was shown. Histological evaluations performed 6 weeks after guide cannula implantation to C57/black mice show that plastic cannula are biocompatible. Microglial and astroglial reactions to plastic cannulas are reduced compared to lab-made stainless steel cannulas. Plastic cannulas are less prone to obstruction, and remained unobstructed over the course of 3 weeks of daily injections, while 50 % of stainless steel cannula became impassable by the 2 week mark. Comparison with existing methodsThese are the first published cannulas intended for applications in mice which combine the presence of usable threads, allowing dummy cannula fixation, with a low profile and small footprint compared to commercially available cannulas. ConclusionsEditable parametric and stl files for reproducing the cannulas presented in this manuscript are included. The method described in this paper is accessible to most laboratories, enabling near-perfect standardization in length combined with a high level of customizability.

Role of the telomeric factor TRF2 in post-hypoxic brain damages

The neuronal excitotoxicity that follows reoxygenation after a hypoxic period may contribute to epilepsy, Alzheimer's disease, Parkinson's disease and various disorders that are related to inadequate supplement of oxygen in neurons. Therefore, counteracting the deleterious effects of post-hypoxic stress is an interesting strategy to treat a large spectrum of neurodegenerative diseases. Here, we show that the expression of the key telomere protecting protein Trf2 decreases in the brain of mice submitted to a post-hypoxic stress. Moreover, downregulating the expression of Terf2 in hippocampal neural cells of unchallenged mice triggers an excitotoxicity-like phenotype including glutamate overexpression and behavioral alterations while overexpressing Terf2 in hippocampal neural cells of mice subjected to a post-hypoxic treatment prevents brain damages. Moreover, Terf2 overexpression in culture neurons counteracts the oxidative stress triggered by glutamate. Finally, we provide evidence that the effect of Terf2 downregulation on excitotoxicity involves Sirt3 repression leading to mitochondrial dysfunction. We propose that increasing the level of Terf2 expression is a potential strategy to reduce post-hypoxic stress damages.

MC4R Localizes at Excitatory Postsynaptic and Peri-Postsynaptic Sites of Hypothalamic Neurons in Primary Culture.

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor (GPCR) that is expressed in several brain locations encompassing the hypothalamus and the brainstem, where the receptor controls several body functions, including metabolism. In a well-defined pathway to decrease appetite, hypothalamic proopiomelanocortin (POMC) neurons localized in the arcuate nucleus (Arc) project to MC4R neurons in the paraventricular nuclei (PVN) to release the natural MC4R agonist α-melanocyte-stimulating hormone (α-MSH). Arc neurons also project excitatory glutamatergic fibers to the MC4R neurons in the PVN for a fast synaptic transmission to regulate a satiety pathway potentiated by α-MSH. By using super-resolution microscopy, we found that in hypothalamic neurons in a primary culture, postsynaptic density protein 95 (PSD95) colocalizes with GluN1, a subunit of the ionotropic N-methyl-D-aspartate receptor (NMDAR). Thus, hypothalamic neurons form excitatory postsynaptic specializations. To study the MC4R distribution at these sites, tagged HA-MC4R under the synapsin promoter was expressed in neurons by adeno-associated virus (AAV) gene transduction. HA-MC4R immunofluorescence peaked at the center and in proximity to the PSD95- and NMDAR-expressing sites. These data provide morphological evidence that MC4R localizes together with glutamate receptors at postsynaptic and peri-postsynaptic sites.

Cholinergic stimulation stabilizes TRPM4 in the plasma membrane of cortical pyramidal neurons.

TRPM4 is a calcium activated non-selective cation channel, impermeable to Ca2+, in neurons it has been implicated in the regulation of the excitability and in the persistent firing. Cholinergic stimulation is also implicated in changes in excitability that leads neurons to an increased firing frequency, however it is not clear whether TRPM4 is involved in the cholinergic-induced increase in firing frequency. Here using a combination of patch clamp electrophysiology, Ca2+ imaging, immunofluorescence, fluorescence recovery after photobleaching (FRAP) and pharmacological approach, we demonstrate that carbachol (Cch) increases firing frequency, intracellular Ca2+ and that TRPM4 inhibition using 9-Ph and CBA reduces firing frequency and decreases the peak in intracellular Ca2+ induced by Cch in cortical pyramidal neurons in culture. Moreover, we determined that cholinergic stimulation reduces TRPM4 recycling and stabilizes TRPM4 in the plasma membrane. Together our results indicate that cholinergic stimulation increases firing in a TRPM4 dependent manner, and also increases the TRPM4 stability in the membrane, suggesting that TRPM4 is locked in microdomains in the membrane, possibly signaling or cytoskeleton proteins complexes.

Ca2+-permeable AMPA receptor-dependent silencing of neurons by KCa3.1 channels during epileptiform activity

Ca2+-activated KCa3.1 channels are known to contribute to slow afterhyperpolarization in pyramidal neurons of several brain areas, while Ca2+-permeable AMPA receptors (CP-AMPARs) may provide a subthreshold source of Ca2+ elevation in the cytoplasm. The functionality of these two types of channels has also been shown to be altered by epileptic disorders. However, the link between KCa3.1 channels and CP-AMPARs is poorly understood, and their potential interaction in epilepsy remains unclear. Here, we address this issue by overexpressing the KCNN4 gene, which encodes the KCa3.1 channel, using patch clamp, imaging, and channel blockers in an in vitro model of epilepsy in neuronal culture. We show that KCNN4 overexpression causes strong hyperpolarization and substantial silencing of neurons during epileptiform activity events, which also prevents KCNN4-positive neurons from firing action potentials (APs) during experimentally induced status epilepticus. Intracellular blocker application experiments showed that the amplitude of hyperpolarization was strongly dependent on CP-AMPARs, but not on NMDA receptors. Taken together, our data strongly suggest that subthreshold Ca2+ elevation produced by CP-AMPARs can trigger KCa3.1 channels to hyperpolarize neurons and protect them from seizures.

A comprehensive protocol for efficient differentiation of human NPCs into electrically competent neurons

BackgroundThe study of neurons is fundamental to unraveling the complexities of the nervous system. Primary neuronal cultures from rodents have long been a cornerstone of experimental studies, yet limitations related to their non-human nature and ethical concerns have prompted the development of alternatives. In recent years, the derivation of neurons from human-induced pluripotent stem cells (hiPSCs) has emerged as a powerful option, offering a scalable source of cells for diverse applications. Neural progenitor cells (NPCs) derived from hiPSCs can be efficiently differentiated into functional neurons, providing a platform to study human neural physiology and pathology in vitro. However, challenges persist in achieving consistent and reproducible outcomes across experimental settings. Comparison with existing methodsOur aim is to provide a step-by-step methodological protocol, augmenting existing procedures with additional instructions and parameters, to guide researchers in achieving reproducible results. Methods and resultsWe outline procedures for the differentiation of hiPSC-derived NPCs into electrically competent neurons, encompassing initial cell density, morphology, maintenance, and differentiation. We also describe the analysis of specific markers for assessing neuronal phenotype, along with electrophysiological analysis to evaluate biophysical properties of neuronal excitability. Additionally, we conduct a comparative analysis of three different chemical methods—KCl, N-methyl-D-aspartate (NMDA), and bicuculline—to induce neuronal depolarization and assess their effects on the induction of both fast and slow post-translational, transcriptional, and post-transcriptional responses. ConclusionOur protocol provides clear instructions for generating reliable human neuronal cultures with defined electrophysiological properties to investigate neuronal differentiation and model diseases in vitro.

Methadone directly impairs central nervous system cells in vitro

Methadone is a synthetic long-acting opioid that is increasingly used in the replacement therapy of opioid-addicted patients, including pregnant women. However, methadone therapy in this population poses challenges, as it induces cognitive and behavioral impairments in infants exposed to this opioid during prenatal development. In animal models, prenatal methadone exposure results in detrimental consequences to the central nervous system, such as: (i) increased neuronal apoptosis; (ii) disruption of oligodendrocyte maturation and increased apoptosis and (iii) increased microglia and astrocyte activation. However, it remains unclear whether these deleterious effects result from a direct effect of methadone on brain cells. Therefore, our goal was to uncover the impact of methadone on single brain cell types in vitro. Primary cultures of rat neurons, oligodendrocytes, microglia, and astrocytes were treated for three days with 10 µM methadone to emulate a chronic administration. Apoptotic neurons were identified by cleaved caspase-3 detection, and synaptic density was assessed by the juxtaposition of presynaptic and postsynaptic markers. Apoptosis of oligodendrocyte precursors was determined by cleaved caspase-3 detection. Oligodendrocyte myelination was assessed by immunofluorescence, while microglia and astrocyte proinflammatory activation were assessed by both immunofluorescence and RT-qPCR. Methadone treatment increased neuronal apoptosis and reduced synaptic density. Furthermore, it led to increased oligodendrocyte apoptosis and a reduction in the myelinating capacity of these cells, and promoted the proinflammatory activation of microglia and astrocytes. We showed that methadone, the most widely used drug in opioid replacement therapy for pregnant women with opioid addiction, directly impairs brain cells in vitro, highlighting the need for developing alternative therapies to address opioid addiction in this population.

Neuronal expression of herpes simplex virus-1 VP16 protein induces pseudorabies virus escape from silencing and reactivation.

Alpha herpesvirus (α-HV) particles enter their hosts from mucosal surfaces and efficiently maintain fast transport in peripheral nervous system (PNS) axons to establish infections in the peripheral ganglia. The path from axons to distant neuronal nuclei is challenging to dissect due to the difficulty of monitoring early events in a dispersed neuron culture model. We have established well-controlled, reproducible, and reactivateable latent infections in compartmented rodent neurons by infecting physically isolated axons with a small number of viral particles. This system not only recapitulates the physiological infection route but also facilitates independent treatment of isolated cell bodies or axons. Consequently, this system enables study not only of the stimuli that promote reactivation but also the factors that regulate the initial switch from productive to latent infection. Adeno-associated virus (AAV)-mediated expression of herpes simplex-1 (HSV-1) VP16 alone in neuronal cell bodies enabled the escape from silencing of incoming pseudorabies virus (PRV) genomes. Furthermore, the expression of HSV VP16 alone reactivated a latent PRV infection in this system. Surprisingly, the expression of PRV VP16 protein supported neither PRV escape from silencing nor reactivation. We compared transcription transactivation activity of both VP16 proteins in primary neurons by RNA sequencing and found that these homolog viral proteins produce different gene expression profiles. AAV-transduced HSV VP16 specifically induced the expression of proto-oncogenes including c-Jun and Pim2. In addition, HSV VP16 induces phosphorylation of c-Jun in neurons, and when this activity is inhibited, escape of PRV silencing is dramatically reduced.IMPORTANCEDuring latency, alpha herpesvirus genomes are silenced yet retain the capacity to reactivate. Currently, host and viral protein interactions that determine the establishment of latency, induce escape from genome silencing or reactivation are not completely understood. By using a compartmented neuronal culture model of latency, we investigated the effect of the viral transcriptional activator, VP16 on pseudorabies virus (PRV) escape from genome silencing. This model recapitulates the physiological infection route and enables the study of the stimuli that regulate the initial switch from a latent to productive infection. We investigated the neuronal transcriptional activation profiles of two homolog VP16 proteins (encoded by HSV-1 or PRV) and found distinct gene activation signatures leading to diverse infection outcomes. This study contributes to understanding of how alpha herpesvirus proteins modulate neuronal gene expression leading to the initiation of a productive or a latent infection.

Viscolin-mediated antiapoptotic and neuroprotective effects in cortical neurons exposed to oxygen-glucose deprivation and rats subjected to transient focal cerebral ischemia

ABSTRACT Objective Previously, we have successfully purified and synthesized viscolin, an agent derived from Viscum coloratum extract, which has shown significant potential in the treatment of stroke. Our study aimed to evaluate the neuroprotective effects of viscolin. Methods We first assessed the cytotoxicity of viscolin on primary neuronal cultures and determined its antioxidant and radical scavenging properties. Subsequently, we identified the optimal dose-response of viscolin in protecting against glutamate-induced neurotoxicity. Results Our results demonstrated that viscolin at a concentration of 10 μM effectively reduced neuronal cell death up to 6 hours after glutamate-induced neurotoxicity. Additionally, we investigated the therapeutic window of opportunity and the potential of viscolin in preventing necrotic and apoptotic damage in cultured neurons exposed to oxygen glucose deprivation-induced neurotoxicity. Our findings showed that viscolin treatment significantly reduced DNA breakage, prevented the release of cytochrome c from mitochondria to cytosol, increased the expression of anti-apoptotic protein Bcl-2, decreased the expression of pro-apoptotic protein Bax, and reduced the number of TUNEL-positive cells. Additionally, our in vivo investigation demonstrated a reduction in brain infarction following middle cerebral artery occlusion. Conclusion Viscolin has potential utility as a therapeutic agent in the treatment of stroke.

Two-layered microwell-array device for preparation of single-neuron culture samples

When a single neuron is cultured in isolation from other neurons, its axon connects with its own dendrites to form a simple, independent network with no synaptic inputs from other neurons. This culture system enables detailed analysis of synaptic function and morphology change in neurites at the single-neuron level, which is useful for elucidating the pathogenesis of neurological diseases and for evaluating the efficacy of therapeutic drugs for them. However, there was previously no device technology capable of simultaneously forming multiple single-neuron samples while allowing co-culture with astrocytes, which is essential for culture of a single neuron isolated from other neurons. In this study, we propose a novel microwell-array device for preparing single-neuron samples. The device consists of an upper layer for cell seeding and a lower layer for cell culture. Each layer has 16 × 16 microwells, and the bottom of each well is made of a 1 μm thick silicon nitride membrane. The membrane of the upper well has one microhole for seeding a single neuron, and the lower membrane has multiple microholes for interaction between a single neuron and astrocytes which are co-cultured back-to-back on both sides of the membrane. When neurons are seeded into the upper well, only one of them passes through the microhole in the upper membrane and falls onto the lower membrane. We evaluated a seeding efficiency of single neurons by changing seeding hole diameter and seeding density. The results showed that the yield of more than 20% was obtained regardless of the seeding density when the seeding hole diameter was 13 μm. We also confirmed that single neurons seeded in this manner and co-cultured with astrocytes developed neurites and formed synapses. These results demonstrated the usefulness of this device for the preparation of single-neuron culture samples.

Investigating the effect of pregabalin on neuronal development using ultrashort self-assembling peptides: Assessing 3D neuronal cultures with high throughput robotic 3D bioprinting

Pregabalin is a widely prescribed drug for various neurological disorders, yet its effects on embryonic cortical neuron development when given to pregnant women remain inadequately explored. In this study, we employed advanced three-dimensional (3D) culturing and in-house developed high-throughput robotic 3D bioprinting technologies to evaluate their potential in neuropharmacology applications, using pregabalin as a model compound. Using a robotic 3D bioprinter and tetrameric IIZK peptide hydrogel as bioink, we created constructs with pregabalin-treated and untreated primary mouse embryonic cortical neurons. This setup allowed us to study the drug’s effects on cell viability, expression of neuronal markers, and neuron development. Our comparative analysis between 2D and 3D peptide-based cell culture models revealed that at a therapeutic concentration of 10 μM, pregabalin does not affect neuronal viability or the morphogenesis of cortical neurons. However, it significantly alters adenosine triphosphate (ATP) release, suggesting potential disruptions in mitochondrial function. Moreover, gene expression analysis of key genes involved in the development of the forebrain and the differentiation and maturation of neurons revealed significant alterations, including the downregulation of Dlx2, Nhlh2, Otp, and Gad67. These findings, together with observed alterations in neuronal activity and oscillations, emphasize the complex impact of pregabalin on neuronal development and function. They highlight the necessity for comprehensive clinical evaluations of its use during pregnancy. Furthermore, our research demonstrates the feasibility and value of integrating 3D cultures with high-throughput 3D bioprinting in neuropharmacology, opening new avenues for investigating drug effects on neuronal development and function, and contributing to safer clinical practices.

Rasagiline Exerts Neuroprotection towards Oxygen-Glucose-Deprivation/Reoxygenation-Induced GAPDH-Mediated Cell Death by Activating Akt/Nrf2 Signaling.

Rasagiline (Azilect®) is a selective monoamine oxidase B (MAO-B) inhibitor that provides symptomatic benefits in Parkinson's disease (PD) treatment and has been found to exert preclinical neuroprotective effects. Here, we investigated the neuroprotective signaling pathways of acute rasagiline treatment for 22 h in PC12 neuronal cultures exposed to oxygen-glucose deprivation (OGD) for 4 h, followed by 18 h of reoxygenation (R), causing 40% aponecrotic cell death. In this study, 3-10 µM rasagiline induced dose-dependent neuroprotection of 20-80%, reduced the production of the neurotoxic reactive oxygen species by 15%, and reduced the nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by 75-90%. In addition, 10 µM rasagiline increased protein kinase B (Akt) phosphorylation by 50% and decreased the protein expression of the ischemia-induced α-synuclein protein by 50% in correlation with the neuroprotective effect. Treatment with 1-5 µM rasagiline induced nuclear shuttling of transcription factor Nrf2 by 40-90% and increased the mRNA levels of the antioxidant enzymes heme oxygenase-1, (NAD (P) H- quinone dehydrogenase, and catalase by 1.8-2.0-fold compared to OGD/R insult. These results indicate that rasagiline provides neuroprotection to the ischemic neuronal cultures through the inhibition of α-synuclein and GAPDH-mediated aponecrotic cell death, as well as via mitochondrial protection, by increasing mitochondria-specific antioxidant enzymes through a mechanism involving the Akt/Nrf2 redox-signaling pathway. These findings may be exploited for neuroprotective drug development in PD and stroke therapy.

Homeostatic Synaptic Plasticity of Miniature Excitatory Postsynaptic Currents in Mouse Cortical Cultures Requires Neuronal Rab3A.

Following prolonged activity blockade, amplitudes of miniature excitatory postsynaptic currents (mEPSCs) increase, a form of plasticity termed "homeostatic synaptic plasticity." We previously showed that a presynaptic protein, the small GTPase Rab3A, is required for full expression of the increase in miniature endplate current amplitudes following prolonged blockade of action potential activity at the mouse neuromuscular junction in vivo (Wang et al., 2011), but it is unknown whether this form of Rab3A-dependent homeostatic plasticity shares any characteristics with central synapses. We show here that homeostatic synaptic plasticity of mEPSCs is impaired in mouse cortical neuron cultures prepared from Rab3A-/- and mutant mice expressing a single point mutation of Rab3A, Rab3A Earlybird mice. To determine if Rab3A is involved in the well-established homeostatic increase in postsynaptic AMPA-type receptors (AMPARs), we performed a series of experiments in which electrophysiological recordings of mEPSCs and confocal imaging of synaptic AMPAR immunofluorescence were assessed within the same cultures. We found that Rab3A was required for the increase in synaptic AMPARs following prolonged activity blockade, but the increase in mEPSC amplitudes was not always accompanied by an increase in postsynaptic AMPAR levels, suggesting other factors may contribute. Finally, we demonstrate that Rab3A is acting in neurons because only selective loss of Rab3A in neurons, not glia, disrupted the homeostatic increase in mEPSC amplitudes. This is the first demonstration that neuronal Rab3A is required for homeostatic synaptic plasticity and that it does so partially through regulation of the surface expression of AMPA receptors.