Cultured Rat Hippocampal Neurons Research Articles

The Rab5-Rab11 Endosomal Pathway is Required for BDNF-Induced CREB Transcriptional Regulation in Hippocampal Neurons.

Brain-derived neurotrophic factor (BDNF) is a key regulator of the morphology and connectivity of central neurons. We have previously shown that BDNF/TrkB signaling regulates the activity and mobility of the GTPases Rab5 and Rab11, which in turn determine the postendocytic sorting of signaling TrkB receptors. Moreover, decreased Rab5 or Rab11 activity inhibits BDNF-induced dendritic branching. Whether Rab5 or Rab11 activity is important for local events only or for regulating nuclear signaling and gene expression is unknown. Here, we investigated, in rat hippocampal neuronal cultures derived from embryos of unknown sex, whether BDNF-induced signaling cascades are altered when early and recycling endosomes are disrupted by the expression of dominant-negative mutants of Rab5 and Rab11. The activity of both Rab5 and Rab11 was required for sustained activity of Erk1/2 and nuclear CREB phosphorylation, and increased transcription of a BDNF-dependent program of gene expression containing CRE binding sites, which includes activity-regulated genes such as Arc, Dusp1, c-fos, Egr1, and Egr2, and growth and survival genes such as Atf3 and Gem Based on our results, we propose that early and recycling endosomes provide a platform for the integration of neurotrophic signaling from the plasma membrane to the nucleus in neurons, and that this mechanism is likely to regulate neuronal plasticity and survival.SIGNIFICANCE STATEMENT BDNF is a neurotrophic factor that regulates plastic changes in the brain, including dendritic growth. The cellular and molecular mechanisms underlying this process are not completely understood. Our results uncover the cellular requirements that central neurons possess to integrate the plasma membrane into nuclear signaling in neurons. Our results indicate that the endosomal pathway is required for the signaling cascade initiated by BDNF and its receptors at the plasma membrane to modulate BDNF-dependent gene expression and neuronal dendritic growth mediated by the CREB transcription factor. CREB is a key transcription factor regulating circuit development and learning and memory.

Inhibition of Mg2+ Extrusion Attenuates Glutamate Excitotoxicity in Cultured Rat Hippocampal Neurons.

Magnesium plays important roles in the nervous system. An increase in the Mg2+ concentration in cerebrospinal fluid enhances neural functions, while Mg2+ deficiency is implicated in neuronal diseases in the central nervous system. We have previously demonstrated that high concentrations of glutamate induce excitotoxicity and elicit a transient increase in the intracellular concentration of Mg2+ due to the release of Mg2+ from mitochondria, followed by a decrease to below steady-state levels. Since Mg2+ deficiency is involved in neuronal diseases, this decrease presumably affects neuronal survival under excitotoxic conditions. However, the mechanism of the Mg2+ decrease and its effect on the excitotoxicity process have not been elucidated. In this study, we demonstrated that inhibitors of Mg2+ extrusion, quinidine and amiloride, attenuated glutamate excitotoxicity in cultured rat hippocampal neurons. A toxic concentration of glutamate induced both Mg2+ release from mitochondria and Mg2+ extrusion from cytosol, and both quinidine and amiloride suppressed only the extrusion. This resulted in the maintenance of a higher Mg2+ concentration in the cytosol than under steady-state conditions during the ten-minute exposure to glutamate. These inhibitors also attenuated the glutamate-induced depression of cellular energy metabolism. Our data indicate the importance of Mg2+ regulation in neuronal survival under excitotoxicity.

Deuterated Glutamate-Mediated Neuronal Activity on Micro-Electrode Arrays.

The excitatory synaptic transmission is mediated by glutamate (GLU) in neuronal networks of the mammalian brain. In addition to the synaptic GLU, extra-synaptic GLU is known to modulate the neuronal activity. In neuronal networks, GLU uptake is an important role of neurons and glial cells for lowering the concentration of extracellular GLU and to avoid the excitotoxicity. Monitoring the spatial distribution of intracellular GLU is important to study the uptake of GLU, but the approach has been hampered by the absence of appropriate GLU analogs that report the localization of GLU. Deuterium-labeled glutamate (GLU-D) is a promising tracer for monitoring the intracellular concentration of glutamate, but physiological properties of GLU-D have not been studied. Here we study the effects of extracellular GLU-D for the neuronal activity by using primary cultured rat hippocampal neurons that form neuronal networks on microelectrode array. The frequency of firing in the spontaneous activity of neurons increased with the increasing concentration of extracellular GLU-D. The frequency of synchronized burst activity in neurons increased similarly as we observed in the spontaneous activity. These changes of the neuronal activity with extracellular GLU-D were suppressed by antagonists of glutamate receptors. These results suggest that GLU-D can be used as an analog of GLU with equivalent effects for facilitating the neuronal activity. We anticipate GLU-D developing as a promising analog of GLU for studying the dynamics of glutamate during neuronal activity.

Effects of IgLON5 Antibodies on Neuronal Cytoskeleton: A Link between Autoimmunity and Neurodegeneration.

Anti-IgLON5 disease is a neurological disorder characterized by autoantibodies against IgLON5 and pathological evidence of neuronal-specific tau accumulation. Here, we report that patients' IgLON5 IgG, but not other cell-surface antibodies, disrupt the cytoskeletal organization in cultured rat hippocampal neurons, resulting in dystrophic neurites and axonal swelling. Adsorption of IgLON5 IgG with HEK293 cells expressing IgLON5 abrogated the indicated cytoskeletal changes. These findings, along with an increase of levels of neurofilaments in patients' cerebrospinal fluid, suggest that IgLON5 IgG, unlike other cell-surface antibodies, disrupts neuronal cytoskeleton maintenance, providing a link between autoimmunity and neurodegeneration. ANN NEUROL 2020;88:1023-1027.

Lycopene antagonizes lead toxicity by reducing mitochondrial oxidative damage and mitochondria-mediated apoptosis in cultured hippocampal neurons.

Lead (Pb) exhibits serious adverse effects on the central nervous system, and the major pathogenic mechanism of Pb toxicity is oxidative stress. As one of the carotenoid family members with potent antioxidant properties, lycopene has shown its protections by inhibiting oxidative stress damage in numerous models of neurotoxicity. The current study was designed to explore the possible protective property in primary cultured rat hippocampal neurons challenged with Pb. We observed that 5 μM lycopene pretreatment for 4 h efficiently ameliorated Pb‐caused damage in cell viability, accumulation of reactive oxygen species (ROS), and apoptosis in a dose‐dependent manner. Moreover, lycopene (5 μM) attenuated the 50 μM Pb‐induced mitochondrial ROS production, improved the activities of mitochondrial respiratory chain enzymes and ATP production, and ameliorated the 50 μM Pb‐induced depolarization of mitochondrial membrane potential as well as opening of mitochondrial permeability transition pores. In addition, 5 μM lycopene restored the imbalance of Bax/Bcl‐2, inhibited translocation of cytochrome c, and reduced caspase‐3 activation. Taken together, these findings indicate that lycopene antagonizes against Pb‐induced neurotoxicity and the underlying mechanism probably involves reduction of mitochondrial oxidative damage and mitochondria‐mediated apoptosis.

Ginsenoside Rg1 reduces β‑amyloid levels by inhibiting CDΚ5‑induced PPARγ phosphorylation in a neuron model of Alzheimer's disease.

The accumulation of β-amyloid peptides (Aβ) in the brain is a hallmark of Alzheimer's disease (AD). Studies have indicated that ginsenoside Rg1, a primary component of ginseng (Panax ginseng), reduces brain Aβ levels in an AD model through peroxisome proliferator-activated receptor γ (PPARγ), thereby regulating the expression of insulin-degrading enzyme (Ide) and β-amyloid cleavage enzyme 1 (Bace1), which are PPARγ target genes. However, the effects of ginsenoside Rg1 on PPARγ remain unclear. Since cyclin-dependent kinase 5 (CDK5) mediates PPARγ phosphorylation in adipose tissue, this study aimed to investigate whether ginsenoside Rg1 regulates PPARγ target genes and reduces Aβ levels by inhibiting PPARγ phosphorylation through the CDK5 pathway. In the present study, a model of AD was established by treating primary cultured rat hippocampal neurons with Aβ1-42. The cells were pretreatment with ginsenoside Rg1 and roscovitine, a CDK5-inhibitor, prior to the treatment with Aβ1-42. Neuronal apoptosis was detected using TUNEL staining. PPARγ phosphorylation and protein expression levels of PPARγ, CDK5, IDE, BACE1, amyloid precursor protein (APP) and Aβ1-42 were measured by western blotting. The mRNA expression levels of PPARγ, CDK5, IDE, BACE1 and APP were assessed using reverse transcription-quantitative PCR. The results of the present study demonstrated that in an AD model induced by Aβ1-42, ginsenoside Rg1 significantly decreased CDK5 expression, inhibited PPARγ phosphorylation at serine 273, elevated IDE expression, downregulated BACE1 and APP expression, decreased Aβ1-42 levels and attenuated neuronal apoptosis. The CDK5 inhibitor, roscovitine, demonstrated similar effects. These results suggest that ginsenoside Rg1 has neuroprotective properties and has potential for use in the treatment of AD.

Alzheimer's disease risk gene BIN1 induces Tau-dependent network hyperexcitability.

Genome-wide association studies identified the BIN1 locus as a leading modulator of genetic risk in Alzheimer's disease (AD). One limitation in understanding BIN1's contribution to AD is its unknown function in the brain. AD-associated BIN1 variants are generally noncoding and likely change expression. Here, we determined the effects of increasing expression of the major neuronal isoform of human BIN1 in cultured rat hippocampal neurons. Higher BIN1 induced network hyperexcitability on multielectrode arrays, increased frequency of synaptic transmission, and elevated calcium transients, indicating that increasing BIN1 drives greater neuronal activity. In exploring the mechanism of these effects on neuronal physiology, we found that BIN1 interacted with L-type voltage-gated calcium channels (LVGCCs) and that BIN1-LVGCC interactions were modulated by Tau in rat hippocampal neurons and mouse brain. Finally, Tau reduction prevented BIN1-induced network hyperexcitability. These data shed light on BIN1's neuronal function and suggest that it may contribute to Tau-dependent hyperexcitability in AD.

Discovery of a novel small molecule PT109 with multi-targeted effects against Alzheimer's disease in vitro and in vivo

Alzheimer's disease (AD), which is characterized by impairment of cognitive functions, is a chronic neurodegenerative disease that mainly affects the elderly. Currently available anti-AD drugs can only offer limited symptom-relieving effects. “One-compound-Multitargeted Strategy” have been recognized as the promising way to win the war against AD. Herein we report a potential anti-AD agent PT109 with multi-functions. First, an 81-kinase screening was carried out and results showed that PT109 potently inhibited c-Jun N-terminal kinases and Serum and glucocorticoid-inducible kinase 1, which are the important signaling molecules involved in neurogenesis, neuroprotection and neuroinflammation and mildly inhibit glycogen synthase kinase-3β as well as protein kinase C gamma, both are involved in AD pathological processes. In addition, in vitro studies of immunofluorescent staining and Western blot showed that PT109 might promote the neurogenesis of C17.2 cells and induce synaptogenesis in primary cultured rat hippocampal neurons. We detected and confirmed the neuroprotective effect of PT109 in cultured HT22 cells by MTT assay, dehydrogenase assay, glutathione assay and reactive oxygen species assay. Furthermore, the results of Western blot, ELISA assay and immunofluorescent staining indicated that PT109 attenuated lipopolysaccharide-induced inflammation in BV2 cells and primary astrocytes. The results of Morris water maze and Step-through test indicated that PT109 improved the spatial learning ability in APP/PS1 mice. More importantly, the in vivo pharmacokinetic parameters indicated that PT109 had better medicinal properties. Taken together, our findings suggest that PT109 may be a promising candidate for treating AD through multiple targets although further studies are ought to be conducted.

NMDAR Antibodies Alter Dopamine Receptors and Cause Psychotic Behavior in Mice.

The aim was to demonstrate that antibodies from patients with anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis alter the levels of dopamine 1 receptor (D1R) and dopamine 2 receptor (D2R) and cause psychotic-like features in mice. Cultured rat hippocampal neurons were treated with cerebrospinal fluid (CSF) from patients with anti-NMDAR encephalitis or controls, and the effects on clusters of D1R and D2R were quantified. In vivo studies included 71 C57BL/6J mice that were chronically infused with CSF from patients or controls through ventricular catheters connected to subcutaneous osmotic pumps. Prepulse inhibition of the acoustic startling reflex (PPI; a marker of psychotic-like behavior), memory, locomotor activity, and the density of cell-surface and synaptic D1R, D2R, and NMDAR clusters were examined at different time points using reported techniques. In cultured neurons, CSF from patients, but not from controls, caused a significant decrease of cell-surface D1R and an increase of D2R clusters. In mice, CSF from patients caused a significant decrease of synaptic and total cell-surface D1R clusters and an increase of D2R clusters associated with a decrease of PPI. These effects were accompanied by memory impairment and a reduction of surface NMDARs, as reported in this model. The psychotic-like features, memory impairment, and changes in levels of D1R, D2R, and NMDAR progressively improved several days after the infusion of CSF from patients stopped. In addition to memory deficits and reduction of NMDARs, CSF antibodies from patients with anti-NMDAR encephalitis cause reversible psychotic-like features accompanied by changes (D1R decrease, D2R increase) in cell-surface dopamine receptor clusters. ANN NEUROL 2020 ANN NEUROL 2020;88:603-613.

Ouabain Modulates the Functional Interaction Between Na,K-ATPase and NMDA Receptor

The N-methyl-D-aspartate (NMDA) receptor plays an essential role in glutamatergic transmission and synaptic plasticity and researchers are seeking for different modulators of NMDA receptor function. One possible mechanism for its regulation could be through adjacent membrane proteins. NMDA receptors coprecipitate with Na,K-ATPase, indicating a potential interaction of these two proteins. Ouabain, a mammalian cardiotonic steroid that specifically binds to Na,K-ATPase and affects its conformation, can protect from some toxic effects of NMDA receptor activation. Here we have examined whether NMDA receptor activity and downstream effects can be modulated by physiological ouabain concentrations. The spatial colocalization between NMDA receptors and the Na,K-ATPase catalytic subunits on dendrites of cultured rat hippocampal neurons was analyzed with super-resolution dSTORM microscopy. The functional interaction was analyzed with calcium imaging of single hippocampal neurons exposed to 10 μM NMDA in presence and absence of ouabain and by determination of the ouabain effect on NMDA receptor–dependent long-term potentiation. We show that NMDA receptors and the Na,K-ATPase catalytic subunits alpha1 and alpha3 exist in same protein complex and that ouabain in nanomolar concentration consistently reduces the calcium response to NMDA. Downregulation of the NMDA response is not associated with internalization of the receptor or with alterations in its state of Src phosphorylation. Ouabain in nanomolar concentration elicits a long-term potentiation response. Our findings suggest that ouabain binding to a fraction of Na,K-ATPase molecules that cluster with the NMDA receptors will, via a conformational effect on the NMDA receptors, cause moderate but consistent reduction of NMDA receptor response at synaptic activation.

Neuroprotection of GLP-1/GIP receptor agonist via inhibition of mitochondrial stress by AKT/JNK pathway in a Parkinson's disease model

ObjectivesTo investigate the effect of glucagon-like peptide-1 (GLP-1) receptor and glucose dependent insulinotrophic polypeptide (GIP) receptor dual agonist DA-JC4 on alleviating Parkinson's disease (PD) and unveil related cellular mechanisms. MethodsRotenone was injected to generate a rat PD model, on which the effect of DA-JC4 on motor functions was evaluated by rotational behavioral assay and open field test. The survival of dopaminergic neurons was analyzed, in addition to assays for mitochondrial stress and quantification of neurotransmitter levels using high performance liquid chromatography (HPLC). In cultured hippocampal neurons, the effect of DA-JC4 on mitochondrial stress and related cellular mechanism was analyzed by Flow cytometry, western blotting and reactive oxygen species (ROS). ResultsDA-JC4 significantly improved motor functions in PD rats, and elevated levels of major neurotransmitters. By histological analysis, DA-JC4 protected dopaminergic neurons from rotenone-induced cell death, which was associated with reduced mitochondrial stress. Experiments in cultured rat hippocampal neurons validated the neuroprotective role of DA-JC4 against cell apoptosis and mitochondrial stress induced by rotenone. The protective effect of DA-JC4 was later found to be dependent on AKT/JNK signal pathway, as treatment using AKT inhibitor or JNK activator abolished such effects. ConclusionOur results showed that the dual agonist of GLP-1/GIP receptor could ameliorate motor dysfunctions of PD by protecting dopaminergic neurons which was mediated by relieved mitochondrial stress and apoptosis via AKT/JNK signal pathway.

Fabrication and validation of flexible 3D pillar electrodes for neural electrophysiological recording

One in fifty people in the United States suffer from paralysis or another neurological disorder. In order to restore functionality and quality of life to these patients, single electrodes and microelectrode arrays (MEAs) have been implanted to record and stimulate electrical signals in the brain. These treatments have shown promise to restore mobility to stroke and amputee patients and to restore quality of life for people suffering from Parkinson’s disease and severe depression. However, current silicon electrodes are 108 times stiffer than brain tissue and lead to chronic inflammation and scarring, eventually leading to the electrode becoming nonfunctional. Our lab has developed a novel MEA design with flexible 3D pillar electrodes made of polydimethylsiloxane (PDMS) and gold-coated nickel to better match the stiffness and elasticity of the brain than currently used stiff silicon electrodes. We describe the fabrication to achieve a high-aspect ratio flexible pillar electrode with a length up to ten times longer than width. As a first step, we have tested the functionality and biocompatibility of the array and pillars in vitro using cultured rat hippocampal neurons. Neurons cultured on our arrays were comparable in health to neurons grown on commercial MEAs, as demonstrated by morphology and electrophysiology during multiple weeks in culture. The pillar electrodes were sensitive enough to detect electrical signals with comparable signal-to-noise ratio, and signal activity to both custom and commercial flat electrodes. Based on this in vitro device validation, we believe this approach of making flexible, high-aspect ratio pillar electrode arrays may be translatable into an in vivo system for use as a neural prosthesis.

Reduced neurosteroid potentiation of GABAA receptors in epilepsy and depolarized hippocampal neurons.

ObjectiveNeurosteroids regulate neuronal excitability by potentiating γ‐aminobutyric acid type‐A receptors (GABARs). In animal models of temporal lobe epilepsy, the neurosteroid sensitivity of GABARs is diminished and GABAR subunit composition is altered. We tested whether similar changes occur in patients with epilepsy and if depolarization‐induced increases in neuronal activity can replicate this effect.MethodsWe determined GABAR α4 subunit expression in cortical tissue resected from pediatric epilepsy patients. Modulation of human GABARs by allopregnanolone and Ro15‐4513 was measured in Xenopus oocytes using whole‐cell patch clamp. To extend the findings obtained using tissue from epilepsy patients, we evaluated GABAR expression and modulation by allopregnanolone and Ro15‐4513 in cultured rat hippocampal neurons exposed to high extracellular potassium (HK) to increase neuronal activity.ResultsExpression of α4 subunits was increased in pediatric cortical epilepsy specimens encompassing multiple pathologies. The potentiation of GABA‐evoked currents by the neurosteroid allopregnanolone was decreased in Xenopus oocytes expressing GABARs isolated from epilepsy patients. Furthermore, receptors isolated from epilepsy but not control tissue were sensitive to potentiation by Ro15‐4513, indicating higher expression of α 4 β x γ 2 subunit‐containing receptors. Correspondingly, increasing the activity of cultured rat hippocampal neurons reduced allopregnanolone potentiation of miniature inhibitory postsynaptic currents (mIPSCs), increased modulation of tonic GABAR current by Ro15‐4513, upregulated the surface expression of α4 and γ2 subunits, and increased the colocalization of α4 and γ2 subunit immunoreactivity.InterpretationThese findings suggest that seizure activity‐induced upregulation of α 4 β x γ 2 subunit‐containing GABARs could affect the anticonvulsant actions of neurosteroids.

Interaction Between CRIPT and PSD-95 Is Required for Proper Dendritic Arborization in Hippocampal Neurons.

CRIPT, the cysteine-rich PDZ-binding protein, binds to the third PDZ domain of PSD-95 (postsynaptic density protein 95) family proteins and directly binds microtubules, linking PSD-95 family proteins to the neuronal cytoskeleton. Here, we show that overexpression of a full-length CRIPT leads to a modest decrease, and knockdown of CRIPT leads to an increase in dendritic branching in cultured rat hippocampal neurons. Overexpression of truncated CRIPT lacking the PDZ domain-binding motif, which does not bind to PSD-95, significantly decreases dendritic arborization. Conversely, overexpression of a full-length CRIPT significantly increases the number of immature and mature dendritic spines, and this effect is not observed when CRIPT∆PDZ is overexpressed. Competitive inhibition of CRIPT binding to the third PDZ domain of PSD-95 with PDZ3-binding peptides resulted in differential effects on dendritic arborization based on the origin of respective peptide sequence. These results highlight multifunctional roles of CRIPT during development and underscore the significance of the interaction between CRIPT and the third PDZ domain of PSD-95.

MicroRNA-183 regulates lipopolysaccharide-induced oxidative stress of hippocampal neurons by targeting the fibronectin 1 gene

Oxidative stress is implicated in the initiation and progression of human and animal diseases. MicroRNA (MiR) has been reported to be involved in the body’s regulation to oxidative stress. We investigated if miR-183 regulates lipopolysaccharide (LPS)-induced oxidative stress in the hippocampus of weaned piglets. LPS-treated piglets had lower expression of miR-183 and higher expression of the fibronectin(FN)1 gene in their hippocampus than control piglets. The expression profiles of miR-183 and the FN1 gene in primary cultured rat hippocampal neurons exposed to LPS were consistent with those in the hippocampus of LPS-treated piglets. The LPS-induced expression of FN1 was reversed in hippocampal neurons by transfection with an miR-183 mimic. A luciferase reporter assay further demonstrated that the FN1 gene is a direct target of miR-183. Taken together, our results demonstrated that miR-183 regulates LPS-induced oxidative stress at least in part by targeting FN1.

Toxicity of dibutyl phthalate in primary cultured rat hippocampal neurons and the toxicological mechanism

To investigate the neurotoxicity and toxicological mechanism of dibutyl phthalate (DBP) in primary cultured rat hippocampal neurons. Primary rat hippocampal neurons cultured for 4 days were exposed to 1 g/L DBP for 24, 48, or 96 h. Immunofluorescence assay and transmission electron microscopy (TEM) were used to observe the morphological changes of the axons and the ultrastructure of DBP-treated neurons. The action potential (AP) of the hippocampal neurons was measured with patch-clamp electrophysiology. CCK-8 assay was used to detect the viability of the hippocampal neurons, and Western blotting was performed to determine the mRNA and protein expressions of brain-derived neurotrophic factor (BDNF), neuropeptide Y (NPY) and estrogen receptor β (ERβ). High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS) was employed to detect the release of the neurotransmitter GABA. After exposure to DBP for 96 h, the cellular network of the hippocampal neurons became sparse, and the neurons showed significantly decreased axonal length (P < 0.01) and presented with round cell nuclei, chromatin aggregation and cytoplasmic vacuolization. Patch-clamp electrophysiology revealed depolarization drift and increased frequency of discharge in the exposed neurons (P < 0.01). The neurons with DBP exposure for 24, 48 and 96 h all showed significantly decreased cell viability (P < 0.01). DBP exposure for 48 and 96 h significantly lowered the protein expressions of ERβ, BDNF and NPY, and a 96-h exposure significantly reduced the release of the neurotransmitter GABA in the neurons (P < 0.05). DBP exposure causes morphological and functional damages of primary cultured rat hippocampal neurons. DBP-induced neurotoxicity is probably associated with GABA-mediated blockage of the ERβ-BDNF-NPY signaling communication.

Remodeling of Intracellular Ca2+ Homeostasis in Rat Hippocampal Neurons Aged In Vitro.

Aging is often associated with a cognitive decline and a susceptibility to neuronal damage. It is also the most important risk factor for neurodegenerative disorders, particularly Alzheimer’s disease (AD). AD is related to an excess of neurotoxic oligomers of amyloid β peptide (Aβo); however, the molecular mechanisms are still highly controversial. Intracellular Ca2+ homeostasis plays an important role in the control of neuronal activity, including neurotransmitter release, synaptic plasticity, and memory storage, as well as neuron cell death. Recent evidence indicates that long-term cultures of rat hippocampal neurons, resembling aged neurons, undergo cell death after treatment with Aβo, whereas short-term cultures, resembling young neurons, do not. These in vitro changes are associated with the remodeling of intracellular Ca2+ homeostasis with aging, thus providing a simplistic model for investigating Ca2+ remodeling in aging. In vitro aged neurons show increased resting cytosolic Ca2+ concentration, enhanced Ca2+ store content, and Ca2+ release from the endoplasmic reticulum (ER). Ca2+ transfer from the endoplasmic reticulum (ER) to mitochondria is also enhanced. Aged neurons also show decreased store-operated Ca2+ entry (SOCE), a Ca2+ entry pathway related to memory storage. At the molecular level, in vitro remodeling is associated with changes in the expression of Ca2+ channels resembling in vivo aging, including changes in N-methyl-D-aspartate NMDA receptor and inositol 1,4,5-trisphosphate (IP3) receptor isoforms, increased expression of the mitochondrial calcium uniporter (MCU), and decreased expression of Orai1/Stim1, the molecular players involved in SOCE. Additionally, Aβo treatment exacerbates most of the changes observed in aged neurons and enhances susceptibility to cell death. Conversely, the solely effect of Aβo in young neurons is to increase ER–mitochondria colocalization and enhance Ca2+ transfer from ER to mitochondria without inducing neuronal damage. We propose that cultured rat hippocampal neurons may be a useful model to investigate Ca2+ remodeling in aging and in age-related neurodegenerative disorders.

Astrocytes and their potential role in anti-NMDA receptor encephalitis

The N-methyl-d-aspartate receptor (NMDAR) encephalitis is the most common form of autoimmune encephalitis. Antibodies against the GluN1 subunit of the NMDAR showed in primary cultures of rat hippocampal neurons and in a mouse model pathogenic effects including cross-linking and internalization of the target receptors (NMDAR). Several studies demonstrated that not only neurons, but also astrocytes express functional NMDA receptors including GluN1 subunit. It is conceivable that the pathogenic antibodies against the NMDAR causing the anti-NMDAR encephalitis affect not only the neuronal receptors, but also the NMDAR on astrocytes. We hypothesize that antibodies against NMDAR can lead to cross-linking and internalization of the target receptors in astrocytes similar to neurons with disruption of the calcium release within the astrocytes and consequently blocking release of inhibitory gliotransmitters. Further, we assume influence on expression of aquaporin 4 channels and gap-junctional communication due to modification of the astrocytic NMDAR. The disruption of these interactions and dysbalance could result in impairment of CNS homeostasis and co-determine the severity of clinical disease manisfestation and recovery.

In-vitro neuroprotective effect and mechanism of 2β-hydroxy-δ-cadinol against amyloid β-induced neuronal apoptosis.

Amyloid beta (Aβ) neurotoxicity plays a causative role in the pathogenesis of Alzheimer's disease. Accumulating evidence demonstrates that Aβ neurotoxicity is mediated by glutamate excitotoxicity. In our previous study, a sesquiterpenoid compound 2β-hydroxy-δ-cadinol (HOC) which exhibited antiglutamate excitotoxicity effect was isolated from the fruits of Alpinia oxyphylla Miquel. Based on the antiglutamate excitotoxicity effect of HOC, in this study, we investigated the potential benefit of HOC in preventing Aβ(1-42)-induced neuronal apoptosis in cultured rat hippocampal neurons. The neuroprotective effect of HOC against Aβ(1-42)-induced neuronal apoptosis was assessed by Hoechst 33258 staining, reactive oxygen species (ROS) production, caspase-3 activation and caspase-3 activity. Results demonstrated that HOC treatment significantly prevented Aβ(1-42)-induced neuronal apoptosis. The underlying molecular mechanisms of HOC in preventing Aβ(1-42)-induced neuronal apoptosis may be via inhibiting Aβ(1-42)-induced ROS production, attenuating Aβ(1-42)-induced caspase-3 activation and inhibiting caspase-3 activity. This study suggests that HOC may be a potential agent for the prevention of Aβ neurotoxicity.

Spatial dynamics of the insulin receptor in living neurons.

Insulin signaling through the insulin receptor has long been studied in classic target organs, such as adipose tissue and skeletal muscle, where one of its effects is to increase glucose uptake. Insulin and insulin receptor are present in many areas of the brain, but the functions of brain insulin signaling outside feeding circuits are not well defined. It has been proposed that hippocampal insulin signaling is important for memory, that brain insulin signaling is deficient in Alzheimer's disease, and that intranasal insulin treatment improves cognition, but the mechanisms remain unclear and do not seem to involve increased glucose uptake by neurons. The molecular behavior of the insulin receptor itself is not well known in living neurons; therefore, we investigated the spatial dynamics of the insulin receptor on somatodendritic membranes of live rat hippocampal neurons in culture. Using single-molecule tracking of quantum dot-tagged insulin receptors and single-particle tracking photoactivation localization microscopy, we show that the insulin receptor is distributed over both dendritic shafts and spines. Using colocalization with synaptic markers, we also show that in contrast to the glutamate receptor subunit glutamate receptor subunit A1, the dynamics of the insulin receptor are not affected by association with excitatory synapses; however, the insulin receptor is immobilized by components of inhibitory synapses. The mobility of the insulin receptor is reduced both by low concentrations of the pro-inflammatory cytokine tumor necrosis factor α and by cholesterol depletion, suggesting an association with sphingolipid-rich membrane domains. On the other hand, the insulin receptor dynamics in hippocampal neurons are not affected by increased excitatory signaling. Finally, using real-time single-event quantification, we find evidence of strong insulin receptor exocytosis on dendritic shafts. Our results suggest an association of the neuronal insulin receptor with specific elements of the dendritic shaft, rather than excitatory synapses.