Cultures Of Cerebellar Neurons Research Articles

Patients' IgLON5 autoantibodies interfere with IgLON5-protein interactions.

Anti-IgLON5 disease is a rare neurological disorder characterized by autoantibodies against IgLON5, and pathological evidence of neurodegeneration. IgLON5 is a cell adhesion molecule but its physiological function is unknown. Our aim was to investigate the IgLON5 interactome and to determine if IgLON5 antibodies (IgLON5-abs) affect these protein interactions. IgLON5 interactome was investigated by mass spectrometry sequencing of proteins immunoprecipitated by IgLON5 autoantibodies using cultures of rat cerebellar granular neurons (CGNs). Shedding of IgLON5 was explored using HEK cells transfected with human IgLON5 plasmid and in CGNs. Interactions of IgLON5 with identified binding partners and IgLON5-abs effects were confirmed by immunofluorescence in transfected HEK cells and rat hippocampal neurons. Patients' IgLON5 antibodies co-precipitated all members of the IgLON family and three 3 additional surface proteins. IgLON5 predominantly establishes homomeric and heteromeric cis (within the cell) and trans (between cells)-interactions with other IgLON family members and undergoes spontaneous ectodomain shedding. Antibodies from patients with anti-IgLON5 disease prevent trans-interactions in hippocampal neurons independently of the IgLON5 IgG subclass distribution. We show a potentially novel pathogenic mechanism of IgLON5-abs that consists in blocking IgLON5 interactions with its binding partners. These findings extend our knowledge about the physiological role of IgLON5 and pave the way to future understanding of the pathological mechanisms of anti-IgLON5 disease.

Raw Data of Immunofluorescence experiments related to the paper entitled "Potential Neurotoxic Effects of Glioblastoma-Derived Exosomes in Primary Cultures of Cerebellar Neurons via Oxidant Stress and Glutathione Depletion" (Data not published).

Raw Data of Immunofluorescence experiments related to the paper entitled "Potential Neurotoxic Effects of Glioblastoma-Derived Exosomes in Primary Cultures of Cerebellar Neurons via Oxidant Stress and Glutathione Depletion" (Data not published).

Meconic acid is a possible neuroprotector: justification on <i>in vitro </i>experiments and its physico-chemical properties

Main representatives of gamma pyronic acid are meconic, comenic, chelidonic and kojic acid. It was found that comenic acid exerts a neuroprotective effect, and chelidonic acid has a pronounced anti-inflammatory effect. It was not studied whether meconic acid exhibits neuroprotective effects. The aim of this work was to assess the neuroprotective potential of meconic acid, taking into accout its physicochemical properties, using an in vitro model of ischemic stroke. Primary neuroglial culture was obtained from the cerebellum of 7-8-day-old Wistar rat pups by mechanical tissue dissociation. The protective effect of meconic acid on the culture of cerebellar neurons was studied using the model of glutamate toxicity and oxygen-glucose deprivation. Quantum mechanical calculations were used and experiments in the model system citrate-phosphate-luminol were conducted by the method of chemiluminescent analysis to investigate the antioxidant activity of meconic acid. The chelating properties of meconic acid with respect to Fe3+ in solutions were studied using Job's method. Meconic acid has been found to have a protective effect in in vitro models of ischemia. Its action leads to a decrease in the level of intracellular calcium and the restoration of the membrane potential of mitochondria in a culture of cerebellar neurons under glutamate exposure, resulting in an increase in the percentage of living cells under oxygen-glucose deprivation. Meconic acid has a high calculated antioxidant potential, confirmed experimentally. With an increase in the pH of the medium, stepwise binding of meconic acid with Fe3+ occurs with the formation of complexes with different ligand/metal ratios. At physiological pH, the composition of the resulting complex is 1:3. The obtained antioxidant, chelating, and cytoprotective action of meconic acid provides a basis for further study of the possible neuroprotective properties of this compound in in vivo experiments, and the data obtained in the work on its physicochemical properties can be useful for the synthesis and study of new coordination compounds based on meconic acid.

Potential Neurotoxic Effects of Glioblastoma-Derived Exosomes in Primary Cultures of Cerebellar Neurons via Oxidant Stress and Glutathione Depletion

High-grade gliomas are the most fatal brain tumors. Grade 4 gliomas are called glioblastoma multiforme (GBM), which are associated with the poorest survival and a 5-year survival rate of less than 4%. Many patients with GBM developed concomitant cognitive dysfunctions and epilepsy. Although the cognitive decline is well defined in glioblastomas, the neurotoxic factors underlying this pathology are not well understood in GBM patients. In this study, we aimed to investigate whether GBM-derived exosomes play a role in neuronal toxicity. For this purpose, exosomes obtained from T98G and U373 GBM cells were applied to primary neuron culture at different concentrations. Subsequently, MTT, LDH, GSH, TAS, and TOS tests were performed. Both GBM-derived exosomes induced a dose-dependent and statistically significant increase of LDH release in cerebellar neurons. MTT assay revealed as both T98G and U373 GBM-derived exosomes induced dose-dependent neurotoxic effects in cerebellar neurons. To the best of our knowledge, this study is the first study demonstrating the toxic potential of GBM-derived exosomes to primary neurons, which may explain the peritumoral edema and cognitive decline in GBM patients.

β-micrustoxin (Mlx-9), a PLA2 from Micrurus lemniscatus snake venom: biochemical characterization and anti-proliferative effect mediated by p53.

Background Endogenous phospholipases A2 (PLA2) play a fundamental role in inflammation, neurodegenerative diseases, apoptosis and cellular senescence. Neurotoxins with PLA2 activity are found in snake venoms from the Elapidae and Viperidae families. The mechanism of action of these neurotoxins have been studied using hippocampal and cerebellar neuronal cultures showing [Ca2+]i increase, mitochondrial depolarization and cell death. Astrocytes are rarely used as a model, despite being modulators at the synapses and responsible for homeostasis and defense in the central nervous system. Preserving the cell division ability, they can be utilized to study the cell proliferation process. In the present work cultured astrocytes and glioblastoma cells were employed to characterize the action of β-micrustoxin (previously named Mlx-9), a PLA2 isolated from Micrurus lemniscatus snake venom. The β-micrustoxin structure was determined and the cell proliferation, cell cycle phases and the regulatory proteins p53, p21 and p27 were investigated.Methods β-micrustoxin was characterized biochemically by a proteomic approach. Astrocytes were obtained by dissociation of pineal glands from Wistar rats; glioblastoma tumor cells were purchased from ATCC and Sigma and cultured in DMEM medium. Cell viability was evaluated by MTT assay; cell proliferation and cell cycle phases were analyzed by flow cytometry; p53, p21 and p27 proteins were studied by western blotting and immunocytochemistry. Results Proteomic analysis revealed fragments on β-micrustoxin that aligned with a PLA2 from Micrurus lemniscatus lemniscatus previously identified as transcript ID DN112835_C3_g9_i1/m.9019. β-micrustoxin impaired the viability of astrocytes and glioblastoma tumor cells. There was a reduction in cell proliferation, an increase in G2/M phase and activation of p53, p21 and p27 proteins in astrocytes. Conclusion These findings indicate that β-micrustoxin from Micrurus lemniscatus venom could inhibit cell proliferation through p53, p21 and p27 activation thus imposing cell cycle arrest at the checkpoint G2/M.

Reduction of Deuterium Level Supports Resistance of Neurons to Glucose Deprivation and Hypoxia: Study in Cultures of Neurons and on Animals.

The effect of a reduced deuterium (D) content in the incubation medium on the survival of cultured neurons in vitro and under glucose deprivation was studied. In addition, we studied the effect of a decrease in the deuterium content in the rat brain on oxidative processes in the nervous tissue, its antioxidant protection, and training of rats in the T-shaped maze test under hypoxic conditions. For experiments with cultures of neurons, 7–8-day cultures of cerebellar neurons were used. Determination of the rate of neuronal death in cultures was carried out using propidium iodide. Acute hypoxia with hypercapnia was simulated in rats by placing them in sealed vessels with a capacity of 1 L. The effect on oxidative processes in brain tissues was assessed by changes in the level of free radical oxidation and malondialdehyde. The effect on the antioxidant system of the brain was assessed by the activity of catalase. The study in the T-maze was carried out in accordance with the generally accepted methodology, the skill of alternating right-sided and left-sided loops on positive reinforcement was developed. This work has shown that a decrease in the deuterium content in the incubation medium to a level of −357‰ has a neuroprotective effect, increasing the survival rate of cultured neurons under glucose deprivation. When exposed to hypoxia, a preliminary decrease in the deuterium content in the rat brain to −261‰ prevents the development of oxidative stress in their nervous tissue and preserves the learning ability of animals in the T-shaped maze test at the level of the control group. A similar protective effect during the modification of the 2H/1H internal environment of the body by the consumption of DDW can potentially be used for the prevention of pathological conditions associated with the development of oxidative stress with damage to the central nervous system.

Electrophysiological Activity and Survival Rate of Rats Nervous Tissue Cells Depends on D/H Isotopic Composition of Medium.

The deuterium content modification in an organism has a neuroprotective effect during the hypoxia model, affecting anxiety, memory and stress resistance. The aim of this work was to elucidate the possible mechanisms of the medium D/H composition modification on nerve cells. We studied the effect of an incubation medium with a 50 ppm deuterium content compared to a medium with 150 ppm on: (1) the activity of Wistar rats’ hippocampus CA1 field neurons, (2) the level of cultured cerebellar neuron death during glucose deprivation and temperature stress, (3) mitochondrial membrane potential (MMP) and the generation of reactive oxygen species in cultures of cerebellar neurons. The results of the analysis showed that the incubation of hippocampal sections in a medium with a 50 ppm deuterium reduced the amplitude of the pop-spike. The restoration of neuron activity was observed when sections were returned to the incubation medium with a 150 ppm deuterium content. An environment with a 50 ppm deuterium did not significantly affect the level of reactive oxygen species in neuron cultures, while MMP decreased by 16–20%. In experiments with glucose deprivation and temperature stress, the medium with 50 ppm increased the death of neurons. Thus, a short exposure of nerve cells in the medium with 50 ppm deuterium acts as an additional stressful factor, which is possibly associated with the violation of the cell energy balance. The decrease in the mitochondrial membrane potential, which is known to be associated with ATP synthesis, indicates that this effect may be associated with the cell energy imbalance. The decrease in the activity of the CA1 field hippocampal neurons may reflect reversible adaptive changes in the operation of fast-reacting ion channels.

PSA mimetic 5-nonyloxytryptamine protects cerebellar neurons against glutamate induced excitotoxicity: An in vitro perspective

PSA-NCAM is a molecule of therapeutic interest for its key role in promoting neuritogenesis and synaptic plasticity. The current study was aimed to investigate the neuroregenerative potential of 5-nonyloxytryptamine (5-NOT) as PSA mimetic compound against glutamate induced excitotoxicity. 2D and 3D cultures of cerebellar neurons challenged with glutamate were used to ascertain the effect of 5-NOT on neurite outgrowth, migration and expression of neuronal plasticity markers. Glutamate excitotoxicity is one of the major underlying pathological factor responsible for neurodegeneration in various neurological disorders such as trauma, stroke, ischemia, epilepsy and neurodegenerative diseases.5-NOT treatment was observed to promote axonal growth and defasiculation in glutamate challenged neurons as well as promoted the migration of cerebellar neurons in both wound scratched area and cerebellar explant cultures. Further, 5-NOT treatment upregulated the expression of synaptic plasticity and cell survival pathway proteins which showed reduced expression after glutamate induced excitotoxicity. Thus, this preliminary data reveals thatPSA-mimetic,5-NOT may prove to be a potential neuroprotective candidate for neurodegenerative diseases.

Effects of a human-based mixture of persistent organic pollutants on the in vivo exposed cerebellum and cerebellar neuronal cultures exposed in vitro

Exposure to persistent organic pollutants (POPs), encompassing chlorinated (Cl), brominated (Br) and perfluoroalkyl acid (PFAA) compounds is associated with adverse neurobehaviour in humans and animals, and is observed to cause adverse effects in nerve cell cultures. Most studies focus on single POPs, whereas studies on effects of complex mixtures are limited. We examined the effects of a mixture of 29 persistent compounds (Cl + Br + PFAA, named Total mixture), as well as 6 sub-mixtures on in vitro exposed rat cerebellar granule neurons (CGNs). Protein expression studies of cerebella from in vivo exposed mice offspring were also conducted. The selection of chemicals for the POP mixture was based on compounds being prominent in food, breast milk or blood from the Scandinavian human population. The Total mixture and sub-mixtures containing PFAAs caused greater toxicity in rat CGNs than the single or combined Cl/Br sub-mixtures, with significant impact on viability from 500x human blood levels. The potencies for these mixtures based on LC50 values were Br + PFAA mixture > Total mixture > Cl + PFAA mixture > PFAA mixture. These mixtures also accelerated induced lipid peroxidation. Protection by the competitive N-methyl-D-aspartate (NMDA) receptor antagonist 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) indicated involvement of the NMDA receptor in PFAA and Total mixture-, but not Cl mixture-induced toxicity. Gene-expression studies in rat CGNs using a sub-toxic and marginally toxic concentration ((0.4 nM-5.5 µM) 333x and (1 nM-8.2 µM) 500x human blood levels) of the mixtures, revealed differential expression of genes involved in apoptosis, oxidative stress, neurotransmission and cerebellar development, with more genes affected at the marginally toxic concentration. The two important neurodevelopmental markers Pax6 and Grin2b were downregulated at 500x human blood levels, accompanied by decreases in PAX6 and GluN2B protein levels, in cerebellum of offspring mice from mothers exposed to the Total mixture throughout pregnancy and lactation. In rat CGNs, the glutathione peroxidase gene Prdx6 and the regulatory transmembrane glycoprotein gene Sirpa were highly upregulated at both concentrations. In conclusion, our results support that early-life exposure to mixtures of POPs can cause adverse neurodevelopmental effects.

Synthetic Polymers Provide a Robust Substrate for Functional Neuron Culture.

Substrates for neuron culture and implantation are required to be both biocompatible and display surface compositions that support cell attachment, growth, differentiation, and neural activity. Laminin, a naturally occurring extracellular matrix protein is the most widely used substrate for neuron culture and fulfills some of these requirements, however, it is expensive, unstable (compared to synthetic materials), and prone to batch-to-batch variation. This study uses a high-throughput polymer screening approach to identify synthetic polymers that supports the in vitro culture of primary mouse cerebellar neurons. This allows the identification of materials that enable primary cell attachment with high viability even under "serum-free" conditions, with materials that support both primary cells and neural progenitor cell attachment with high levels of neuronal biomarker expression, while promoting progenitor cell maturation to neurons.

Comparative impact analysis of neuronal and glial progenitors conditioned medium on cerebellar neurons under glutamate exitotoxicity

One of the main causes of cell death in neurodegenerative diseases is excitotoxicity. Today the potential directions of treatment neurodegenerative diseases are including cell therapy, the purpose of which is to replace lost nerve tissue with donor cells. Transplanted cells along with replaced lost tissues have a paracrine effect, which requires careful study. The aim of this work was to study the effect of conditioned media, obtaining from neuronal and glial progenitor cells, on a primary culture of cerebellar neurons in a model of glutamate excitotoxicity. The cell viability, expression of marker genes for apoptosis and neuritogenesis, and the number of necrotic and apoptotic cells were determined in the culture of cerebellar neurons. The composition of the studied conditioned media was analyzed for the content of neurotrophins. A comparative analysis was revealed differences in the secretion of neurotrophins between the obtained cultures: the amount of brain-derived neurotrophic factor, nerve growth factor, ciliary neurotrophic factor and glial neurotrophic factor was higher in the secretion of glial progenitors. It was shown that the addition of conditioned media from neuronal cells does not significantly affect the viability of cerebellar neurons, whereas preincubation with media from glial progenitors has a neuroprotective effect by increasing the viability of cerebellar neurons, and during long-term cultivation promotes the growth of neurites by increasing the expression level of MAP2 and GAP43 genes.

Guanosine and GMP increase the number of granular cerebellar neurons in culture: dependence on adenosine A2A and ionotropic glutamate receptors.

The guanine-based purines (GBPs) have essential extracellular functions such as modulation of glutamatergic transmission and trophic effects on neurons and astrocytes. We previously showed that GBPs, such as guanosine-5'-monophosphate (GMP) or guanosine (GUO), promote the reorganization of extracellular matrix proteins in astrocytes, and increase the number of neurons in a neuron-astrocyte co-culture protocol. To delineate the molecular basis underlying these effects, we isolated cerebellar neurons in culture and treated them with a conditioned medium derived from astrocytes previously exposed to GUO or GMP (GBPs-ACM) or, directly, with GUO or GMP. Agreeing with the previous studies, there was an increase in the number of β-tubulin III-positive neurons in both conditions, compared with controls. Interestingly, the increase in the number of neurons in the neuronal cultures treated directly with GUO or GMP was more prominent, suggesting a direct interaction of GBPs on cerebellar neurons. To investigate this issue, we assessed the role of adenosine and glutamate receptors and related intracellular signaling pathways after GUO or GMP treatment. We found an involvement of A2A adenosine receptors, ionotropic glutamate N-methyl-D-aspartate (NMDA), and non-NMDA receptors in the increased number of cerebellar neurons. The signaling pathways extracellular-regulated kinase (ERK), calcium-calmodulin-dependent kinase-II (CaMKII), protein kinase C (PKC), phosphatidilinositol-3'-kinase (PI3-K), and protein kinase A (PKA) are also potentially involved with GMP and GUO effect. Such results suggest that GMP and GUO, and molecules released in GBPs-ACM promote the survival or maturation of primary cerebellar neurons or both via interaction with adenosine and glutamate receptors.

Identification and characterization of synthetic chondroitin-4-sulfate binding peptides in neuronal functions

Chondroitin sulfate proteoglycans (CSPGs), up-regulated in and around the glial scar after mammalian spinal cord injury, have been suggested to be key inhibitory molecules for functional recovery by impeding axonal regrowth/sprouting and synaptic rearrangements. CSPG-mediated inhibition is mainly associated with the glycosaminoglycan chains of CSPGs, and chondroitin-4-sulfate (C4S) is the predominant sulfated structure that regulates axonal guidance and growth in the adult nervous system. With the aim to find molecules that neutralize the inhibitory functions of C4S, we screened a phage display library for peptides binding to C4S. From the phage clones binding to C4S we selected three peptides for further analysis. We observed that these peptides bind to C4S, but not chondroitin-6-sulfate, heparin sulfate or dermatan sulfate, in a concentration-dependent and saturable manner, whereas the scrambled peptides showed highly reduced or no binding to C4S. The C4S-binding peptides, but not their scrambled counterparts, when added to cultures of mouse cerebellar neurons and human neuroblastoma cells, neutralized the inhibitory functions of the C4S- and CSPG-coated substrate on cell adhesion, neuronal migration and neurite outgrowth. These results indicate that the C4S-binding peptides neutralize several inhibitory functions of CSPGs, suggesting that they may be beneficial in repairing mammalian nervous system injuries.

Study of the Mechanism of the Neuron Sensitization to the Repeated Glutamate Challenge

Exposure of cultured neurons to high concentrations of Glu leads to a strong depolarization of mitochondria, which develops synchronously with the secondary rise in the intracellular Ca2+ concentration (delayed calcium deregulation, DCD). In this study, using the primary culture of rat cerebellar neurons, we investigated the mechanism of neuronal sensitization, which manifests itself in the reduction of latent periods of DCD during repeated exposures to Glu. It was shown that the most likely cause of sensitization is the inability of mitochondria to maintain a high transmembrane potential (ΔΨm) as a result of an increase in the proton conductivity of the internal mitochondrial membrane, but not the opening of the mitochondrial permeability transition pore in the inner mitochondrial membrane. Mitochondrial dysfunction reduces the production of ATP, leading to the inability of neurons to quickly restore the concentration of Na+, ATP, and NADH in the intervals between successive Glu administrations. One of the reasons that aggravate the dysfunction of mitochondria and contribute to the sensitization of neurons to the repeated action of Glu is Ca2+ accumulated in the mitochondria during the first glutamate impact.

Collapse of Neuronal Energy Balance As a Basis of L-Homocysteine Neurotoxicity

Using fluorescence detection methods, neurotoxic effects of L-homocysteine (HCY), L-glutamate (Glu), and N-methyl-D-aspartate (NMDA) on primary culture of rat cerebellar neurons were compared and the agonist-evoked intracellular Ca2+ responses and changes in mitochondrial membrane potential were studied. Long-term (5 h) action of HCY, Glu, or NMDA caused neuronal apoptosis and necrosis that was followed by a decrease of quantity of live cells to 40%. It was revealed using Fluo-3 that neurons differed by intracellular Ca2+ responses to 2-min applications of HCY. In response to all studied agonists, a brief peak or gradual increase of intracellular Ca2+ concentration was observed. Some neurons did not respond to HCY, but all responded to Glu and NMDA. A prolonged (60 min) treatment with agonists caused a rapid or delayed Ca2+ overload, while only a small portion of neurons were able to compensate the intracellular Ca2+ elevation. Six-minute applications of HCY or Glu to neurons induced similar changes of mitochondrial potential (φmit) measured by rhodamine123. In this protocol, the ability of the NMDA receptor agonists to cause the mitochondrial dysfunction could be arranged in the following order: NMDA > Glu = HCY. After a 60-min treatment the observed difference vanished because all of the agonists reduced φmit so that an uncoupling agent FCCP did not cause any additional changes in φmit. Thus, HCY-induced neurotoxicity in cerebellar neurons is comparable to that of Glu. In this feature cerebellar neurons differ from cortical neurons, in which HCY did not significantly change φmit during short-term application. This difference could be related with peculiarities of the HCY action on NMDA receptor subtypes expressed by cerebellar neurons.

Tinospora cordifolia as a potential neuroregenerative candidate against glutamate induced excitotoxicity: an in vitro perspective

BackgroundGlutamate, the major excitatory neurotransmitter of CNS acts as a neurotoxin at higher concentrations. Prolonged activation of glutamate receptors results in progressive neuronal damage by aggravating calcium influx, inducing mitochondrial damage and oxidative stress. Excitotoxic cell death is associated with the pathogenesis of various neurodegenerative disorders such as trauma, brain injury and neurodegenerative diseases. The current study was designed to investigate the neuroprotective and neuroregenerative potential of Tinospora cordifolia against glutamate-induced excitotoxicity using primary cerebellar neuronal cultures as a model system.MethodsMonosodium salt of glutamate was used to induce neurotoxic injury in primary cerebellar neurons. Four extracts including Hexane extract, Chloroform extract, Ethyl acetate, and Butanol extract were obtained from fractionation of previously reported aqueous ethanolic extract of T. cordifolia and tested for neuroprotective activity. Out of the four fractions, Butanol extract of T. cordifolia (B-TCE) exhibited neuroprotective potential by preventing degeneration of neurons induced by glutamate. Expression of different neuronal, apoptotic, inflammatory, cell cycle regulatory and plasticity markers was studied by immunostaining and Western blotting. Neurite outgrowth and migration were also studied using primary explant cultures, wound scratch and gelatin zymogram assay.ResultsAt molecular level, B-TCE pretreatment of glutamate-treated cultures normalized the stress-induced downregulation in the expression of neuronal markers (MAP-2, GAP-43, NF200) and anti-apoptotic marker (Bcl-xL). Further, cells exposed to glutamate showed enhanced expression of inflammatory (NF-κB, AP-1) and senescence markers (HSP70, Mortalin) as well as the extent of mitochondrial damage. However, B-TCE pretreatment prevented this increase and inhibited glutamate-induced onset of inflammation, stress and mitochondrial membrane damage. Furthermore, B-TCE was observed to promote regeneration, migration and plasticity of cerebellar neurons, which was otherwise significantly inhibited by glutamate treatment.ConclusionThese results suggest that B-TCE may have neuroprotective and neuroregenerative potential against catastrophic consequences of glutamate-mediated excitotoxicity and could be a potential therapeutic candidate for neurodegenerative diseases.

Effect of chondroitin sulfate proteoglycans on neuronal cell adhesion, spreading and neurite growth in culture.

As one major component of extracellular matrix (ECM) in the central nervous system, chondroitin sulfate proteoglycans (CSPGs) have long been known as inhibitors enriched in the glial scar that prevent axon regeneration after injury. Although many studies have shown that CSPGs inhibited neurite outgrowth in vitro using different types of neurons, the mechanism by which CSPGs inhibit axonal growth remains poorly understood. Using cerebellar granule neuron (CGN) culture, in this study, we evaluated the effects of different concentrations of both immobilized and soluble CSPGs on neuronal growth, including cell adhesion, spreading and neurite growth. Neurite length decreased while CSPGs concentration arised, meanwhile, a decrease in cell density accompanied by an increase in cell aggregates formation was observed. Soluble CSPGs also showed an inhibition on neurite outgrowth, but it required a higher concentration to induce cell aggregates formation than coated CSPGs. We also found that growth cone size was significantly reduced on CSPGs and neuronal cell spreading was restrained by CSPGs, attributing to an inhibition on lamellipodial extension. The effect of CSPGs on neuron adhesion was further evidenced by interference reflection microscopy (IRM) which directly demonstrated that both CGNs and cerebral cortical neurons were more loosely adherent to a CSPG substrate. These data demonstrate that CSPGs have an effect on cell adhesion and spreading in addition to neurite outgrowth.

Developmental Changes of Synaptic and Extrasynaptic NMDA Receptor Expression in Rat Cerebellar Neurons In Vitro.

Transient expression of different NMDA receptors (NMDARs) plays a role in development of the cerebellum. Whether similar processes undergo during neuronal differentiation in culture is not clearly understood. We studied NMDARs in cerebellar neurons in cultures of 7 and 21days in vitro (DIV) using immunocytochemical and electrophysiological approaches. Whereas at 7 DIV, the vast majority of neurons were immunopositive for GluN2 subunits, further synaptoginesis was accompanied by the time-dependent loss of NMDARs. In contrast to GluN2B- and GluN2C-containing NMDARs, which at 7 DIV exhibited homogenous distribution in extrasynaptic regions, GluN2A-containing receptors were aggregated in spots both in cell bodies and dendrites. Double staining for GluN2A subunits and synaptophysin, a widely used marker for presynaptic terminals, revealed their co-localization in about 75% of dendrite GluN2A fluorescent spots, suggesting postsynaptic origin of GluN2A subunits. In agreement, diheteromeric GluN2A-containing NMDARs contributed to postsynaptic currents recorded in neurons throughout the timescale under study. Diheteromeric GluN2B-containing NMDARs escaped postsynaptic regions during differentiation. Finally, the developmental switch favored the expression of triheteromeric NMDARs assembled of 2 GluN1/1 GluN2B/1 GluN2C or GluN2D subunits in extrasynaptic regions. At 21 DIV, these receptors represented over 60% of the NMDAR population. Thus, cerebellar neurons in primary culture undergo transformations with respect to the expression of di- and triheteromeric NMDARs that should be taken into account when studying cellular aspects of their pharmacology and functions.

Neuroligin-induced presynaptic differentiation through SLM2-mediated splicing modifications of neurexin in cerebellar cultures

Neurexins (NRXs) and neuroligins (NLs) play important roles in synapse specification. The alternatively spliced segment 4 (AS4) of NRX genes (Nrxn) is a critical element in selective trans-synaptic interactions. However, the role of splicing of NRXs and NLs in synapse specification is not fully understood. To investigate the exact role of splice-dependent NRX−NL interaction in the specification of glutamatergic and gamma-aminobutyric acid (GABA)-ergic synapses in the cerebellum, we evaluated the synaptogenic receptor activity of NL1/2/3 isoforms in a neuron-fibroblast co-culture system, in which the Nrxn AS4 segments are manipulated using SLM2, a selective and dominant regulator of AS4 splicing. We show that ectopic SLM2 expression (SLM2 E/E) causes marked skipping of exon 20 of AS4 in cerebellar neuron culture. Whereas NLs can induce VAMP2+ presynaptic contacts from mainly glutamatergic neurons in both uninfected (control) and SLM2 E/E co-cultures, they induce VGAT+ GABAergic contacts in the control culture, but not properly in the SLM2 E/E culture. Furthermore, Nrxn3 is responsible for the NL-induced assembly of GABAergic synapses in co-culture. Importantly, lentivirus-based expression of Nrxn3 containing exon 20 restores the reduced NL-induced GABAergic contacts in the SLM2 E/E co-culture. Therefore, our findings may provide further insights into NRX−NL mediated synapse specification.

Нейропротекторный эффект комената кальция при окислительном стрессе и глутаматной цитотоксичности на модели культивируемых нейронов мозжечка и спинальных ганглиев

Изучено влияние нового фармакологического соединения кальциевой соли коменовой кислоты (CaK) на генерацию активных форм кислорода в модельной системе ЦФЛ (цитрат фосфатный буфер с добавлением люминола), на токсичность глутамата в диссоциированных культурах нейронов мозжечка крыс, а также его влияние на ростовые процессы культивируемых нейронов спинальных ганглиев эмбрионов кур в условиях окислительного стресса в сравнении с коменовой кислотой (КК). Установлено, что CaK повышает устойчивость культивируемых нейронов мозжечка к глутаматной цитотоксичности, проявляет выраженный нейротрофический эффект — защищает нейроны спинальных ганглиев эмбрионов кур от окислительного воздействия перекиси водорода, значительно снижает содержание свободных радикалов в модельной системе ЦФЛ (на 64,6 %, p < 0,001). Антиокислительные свойства CaK практически не отличаются от КК, в тоже время максимальный нейропротекторный эффект CaK проявляется в более низких (1 и 10 мкМ), чем КК (1000 мкМ) концентрациях, а нейротрофическое действие, в отличие от КК, в более широком диапазоне концентраций: CaK — 0,01 и 0,001 мМ, КК — 0,01 мМ.