Neuronal Cell Cultures Research Articles

Gadolinium chelates inhibit intracellular calcium signaling in rat trigeminal ganglion neurons similar to rat dorsal root ganglion neurons, but less effective

Objectives: Gadolinium (Gd3+) based MR contrast agents (GBMCA) are manufactured with a chelation process, where organic ligand molecules form a stable complex around Gd3+. The aim of the present study was to investigate the possible impact of free gadolinium and GBMCA in ganglia (trigeminal and dorsal root) neuron cell cultures on intracellular calcium (Ca2+) since these agents could dissociate and lead to the release of toxic Gd3+ ions in metabolic conditions. Methods: Ganglia cells were studied for responses to agents and nonspecific depolarization by high KCl+ in Fura 2 AM loaded neurons by monitoring the changes in [Ca2+]i using a microscopic digital image analysis system. GBMCA and molecular gadolinium doses of 0.1 mmol/kg were prepared with the same pH and temperature and applied to the cells. Results: Application of Gd3+, gadobenate dimeglumine, gadobutrol and gadodiamide to rat ganglia neurons led to significant decreases in basal intracellular calcium levels, while gadoterate meglumine administration did not have a significant impact. As expected, Gd3+ was more effective when compared tı GBMCA. While the impact was almost full with gadodiamide, Gd3+ and other GBMCAs had irreversible effects at different levels. The impact of gadoterate meglumine was neglectable when compared to Gd3+ and other GBMCAs, although it exhibited slightly gadodiamide-like effects. Conclusions: In conclusion, gadolinium chelates released gadolinium and irreversibly reduced the intracellular calcium levels in identical TG and DRG cells at different levels.

Autophagy and protein aggregation as a mechanism of dopaminergic degeneration in a primary human dopaminergic neuronal model

The pathophysiology underlying the loss of dopaminergic neurons in Parkinson’s disease (PD) is unclear. A gap of knowledge in the molecular and cellular events leading to degeneration of the nigrostriatal DA system is a major barrier to the development of effective therapies for PD. 1-methyl-4-phenylpyridinium (MPP+) is used as a reliable in vitro model of PD in dopaminergic neurons; however, the molecular mechanisms that lead to cell death with this model are not fully understood. Additionally, there is a lack of translational in vitro models to fully understand progressive dopaminergic neurotoxicity. Here, we propose cultures of primary human dopaminergic neuronal precursor cells (HDNPCs) as a model to study progressive dopaminergic toxicity and neuronal damage in PD. We evaluated the concentration-response of MPP+ (0–10 mM) at 24 h, using cell viability and mitochondrial activity assays (LDH, XTT, Live/Dead staining, and MitoTracker). Based on concentration-response data, we chose two concentrations (1.0 and 2.5 mM) of MPP+ to evaluate markers of autophagy and dopaminergic status [tyrosine hydroxylase (TH)] after a 24-h exposure. Exposure to MPP+ induced cytotoxicity, reduced cell viability, and decreased mitochondrial activity. MPP+ at 1.0 and 2.5 mM also induced expression of lysosome-associated membrane protein 1 (LAMP-1) and increased the ratio of light chain 3 (LC3), LC3BII/LC3BI. The expression of TH also decreased. Furthermore, α-synuclein (α-SYN) and parkin were evaluated by immunofluorescence (IF) at 1.0 and 2.5 mM MPP+ after 24 h. A qualitative analysis revealed decreased parkin expression while α-SYN aggregation was observed in the cytoplasm and the nucleus. These data suggest that in HDNPCs MPP+ can cause cytotoxicity and neuronal damage. This damage may be mediated by autophagy, dopamine synthesis, and protein aggregation. The combination of HDNPCs and MPP+ may serve as valuable in vitro model of progressive dopaminergic neurotoxicity for research into potential treatments for PD.

In Vivo Imaging of Axonal Organelle Transport in the Mouse Brain.

Visualization and analysis of axonal organelle transport has been mostly conducted in vitro, using primary neuronal cell cultures, although more recently, intravital organelle imaging has been established in model organisms such as drosophila, zebrafish, and mouse. In this chapter, we describe a method to visualize axonal transport of cellular organelles such as dense core vesicles or mitochondria in the living mouse brain in order to study organelle transport in its native environment. We achieve this goal by injecting adeno-associated viruses expressing fluorescently tagged marker proteins into thalamic nuclei of mice, thereby transducing neurons that project to the surface of the brain. Axonal projections and trafficking of organelles can be imaged with a 2-photon microscope through a chronically implanted window in the mouse skull in anesthetized as well as awake mice.

Diverse approaches to neuronal cell culture from iPS cells: toward a model for neurodevelopmental disorders

The use of patient-derived iPS (induced-pluripotent stem) cells has become very meaningful for the development of pathological models and therapies for neuronal diseases. However, when modeling pathological conditions in vitro, it is very important to know which cell culture method to use. There are strong and weak points to each type of stem cell-based culture. We have been developing neuronal induction methods for pathological modeling of psychiatric and developmental disorders. In this study, we used both 2-D and 3-D cultures effectively. Directed neural differentiation by Dual SMAD inhibition and addition a morphogen allows us to observe neurogenesis-based tissue formation, which may reveal cellular phenotypes during development: this is achieved by 3D culture system. However, it is not always possible to obtain disease-causing cells under these conditions. In this case, it would be useful to use transient expression of transcription factors in iPS cells to directly differentiate into subtype-specific cells: this is achieved by 2D culture system. Each differentiation and culture technique is constantly evolving. It will be necessary to consider how to generate phenotypes more robustly. In this symposium, we introduce our research on modeling of psychiatric and developmental disorders based on customized neural differentiation system.

In vivo Evidence for Brain Region-Specific Molecular Interactions Between Cannabinoid and Orexin Receptors.

The endocannabinoid and orexin neuromodulatory systems serve key roles in many of the same biological functions such as sleep, appetite, pain processing, and emotional behaviors related to reward. The type 1 cannabinoid receptor (CB1R) and both subtypes of the orexin receptor, orexin receptor type 1 (OX1R) and orexin receptor type 2 (OX2R) are not only expressed in the same brain regions modulating these functions, but physically interact as heterodimers in recombinant and neuronal cell cultures. In the current study, male and female C57BL/6 mice were co-treated with the cannabinoid receptor agonist CP55,940 and either the OX2R antagonist TCS-OX2-29 or the dual orexin receptor antagonist (DORA) TCS-1102. Mice were then evaluated for catalepsy, body temperature, thermal anti-nociception, and locomotion, after which their brains were collected for receptor colocalization analysis. Combined treatment with the DORA TCS-1102 and CP55,940 potentiated catalepsy more than CP55,940 alone, but this effect was not observed for changes in body temperature, nociception, locomotion, or via selective OX2R antagonism. Co-treatment with CP55,940 and TCS-1102 also led to increased CB1R-OX1R colocalization in the ventral striatum. This was not seen following co-treatment with TCS-OX2-29, nor in CB1R-OX2R colocalization. The magnitude of effects following co-treatment with CP55,940 and either the DORA or OX2R-selective antagonist was greater in males than females. These data show that CB1R-OX1R colocalization in the ventral striatum underlies cataleptic additivity between CP55,940 and the DORA TCS-1102. Moreover, cannabinoid-orexin receptor interactions are sex-specific with regards to brain region and functionality. Physical or molecular interactions between these two systems may provide valuable insight into drug-drug interactions between cannabinoid and orexin drugs for the treatment of insomnia, pain, and other disorders.

Neuroprotective Effect of Clobenpropit against Lipopolysaccharide-Induced Cognitive Deficits via Attenuating Neuroinflammation and Enhancing Mitochondrial Functions in Mice.

Clobenpropit (CLO), an antagonist on histamine H3 receptors (HH3R), has been shown to protect NMDA-induced neuronal necrosis in cortical neuronal cell culture from rats. In this work, we explored its potential on lipopolysaccharide (LPS)-induced memory deficits, neuroinflammation, and mitochondrial dysfunction in mice. CLO (1 and 3 mg/kg, p.o.) was treated continually for 30 days, and neurotoxicity was induced by four doses of LPS (250 µg/kg, i.p.). The radial arm maze (RAM) was used to access memory behaviors. After the REM test, brain tissue was collected from each mouse to estimate pro-inflammatory cytokines (TNFα and IL6), anti-inflammatory cytokines (TGF-β1 and IL-10), cyclooxygenase-2 (COX 2), and mitochondrial respiratory chain complex (MRCC- I, II and IV) enzymes. CLO treatment reversed the LPS-induced behavioral deficits by a significant reduction in time taken to consume all five bites (TTB), working memory error (WME), and reference memory error (REM) in the REM test. Regarding neuroinflammation, it attenuated the release of COX, TNF-α, and IL-6, and augmented TGF-β1 and IL-10 levels in the brain. Reversal of LPS-induced brain MRCC (I, II, and IV) levels also resulted with CLO treatment. From these findings, CLO promises neuroprotection against LPS-induced cognitive deficits by ameliorating neuroinflammation and restoring the MRCC enzymes in mice.

Facile method to incorporate high-affinity ACAT/SOAT1 inhibitor F12511 into stealth liposome-based nanoparticle and demonstration of its efficacy in blocking cholesteryl ester biosynthesis without overt toxicity in neuronal cell culture

BackgroundAcyl-CoA:cholesterol acyltransferase (ACAT) inhibitors have been considered as potential therapeutic agents to treat several diseases, including Alzheimer’s disease, atherosclerosis, and cancer. While many ACAT inhibitors are readily available, methods to encapsulate them as nanoparticles have not been reported. New methodWe report a simple method to encapsulate ACAT inhibitors, using the potent hydrophobic ACAT inhibitor F12511 as an example. By mixing DSPE-PEG2000, egg phosphatidylcholine (PC), and F12511 in ethanol, followed by drying, resuspension and sonication in buffer, we show that F12511 can be encapsulated as stealth liposomes at high concentration. ResultsWe successfully incorporated F12511 into nanoparticles and found that increasing PC in the nanoparticles markedly increased the amount of F12511 incorporated in stealth liposomes. The nanoparticles containing F12511 (Nanoparticle F) exhibit average size of approximately 200 nm and are stable at 4 ºC for at least 6 months. Nanoparticle F is very effective at inhibiting ACAT in human and mouse neuronal and microglial cell lines. Toxicity tests using mouse primary neuronal cells show that F12511 alone or Nanoparticle F added at concentrations from 2 to 10 µM for 24-, 48-, and 72-hours produces minimal, if any, toxicity. Comparison with existing method(s)Unlike existing methods, the current method is simple, cost effective, and can be expanded to produce tagged liposomes to increase specificity of delivery. This also offers opportunity to embrace water soluble agent(s) within the aqueous compartment of the nanoparticles for potential combinatorial therapy. ConclusionsThis method shows promise for delivery of hydrophobic ACAT inhibitors at high concentration in vivo.

PrNP-induced effect over NMDA expression and signalling.

Etiology of Alzheimer's disease (AD) remain unclear. Different pathological processes have been described, such as phosphorylation of tau protein, amyloid plaques, oxidative stress or neuroinflammation. It has been described that over-activation of the NMDA receptor due to an increase in glutamate levels induces excitotoxicity in neurodegenerative disorders, on the other hand, these disorders course with a chronic neuroinflammation where PrNP prion protein could play an important role. We have also analyzed NMDA induced neuronal activity using Fluo4AM and MEAs for real time measurement of calcium-fluorescence. Different signaling pathways have been analyzed, such as MAPK phosphorylation (determined using AlphaScreen® SureFire® kit (Perkin Elmer) or Dynamic Mass Redistribution (analyzed by label-free-DMR technology). We have observed that the expression of PrNP protein in cortical and hippocampal primary cultures of microglial cells showed an important decrease in NMDA induced signaling. Moreover, expression of PrNP protein in cortical and hippocampal primary cultures of neuronal cells showed an important decrease in NMDA induced signaling. Finally, it has been observed that NMDA receptor functionality in the presence or in the absence of Tau protein (phosphorylated or not) and PrNP negatively affects NMDA induced activation of calcium release in neuronal primary cultures. NMDA receptor activation is negatively affected by the presence of PrNP prion protein by decreasing the receptor expression levels and the induced function.

Increased levels of phospho-tau217 in neuron cultures and CVN mice infected with Porphyromonas gingivalis.

The oral pathogen Porphyromonas gingivalis has been identified as a causative agent of Alzheimer's disease (AD) pathology and neurodegeneration. Previously, we have shown that P. gingivalis can act as a neurotrophic pathogen and that microtubule associated tau protein is a substrate for lysine-gingipain (Kgp), a major protease virulence factor of P. gingivalis. After infection of neuron cultures, we found tau protein to be hyperphosphorylated at S231, but other phosphorylation sites had not been investigated. Recently, an increased level of phospho-tau217 has been identified as a blood and CSF biomarker for AD. Here we aim to investigate the effect of P. gingivalis infection on tau phosphorylation at T217 in vitro and in vivo. We used IPSC-derived neuron cultures, neuron-astrocyte-microglia co-cultures and CVN mice (APPSwDI/NOS2 bigenic) as model systems for P. gingivalis-induced AD. Samples were analyzed for protein and RNA expression by ELISA, western blot and nanostring. In infected neuron cultures and co-cultures tau protein was susceptible to P. gingivalis-induced and gingipain dependent digestion in a dose- dependent manner. Tau degradation was completely inhibited by treatment with the specific Kgp inhibitor COR388 (atuzaginstat). At lower infection levels, tau protein persisted and an elevated p-tau217/total tau ratio was observed. In CVN mice chronically infected with P. gingivalis, the p-tau217/total tau ratio was elevated in the brain in infected compared to uninfected mice. Furthermore, after 5 weeks of treatment with the Kgp inhibitor COR588 the level of p-tau217 was similar to that seen in uninfected controls. P. gingivalis infected neuronal cell cultures and chronically infected CVN mice display elevated p-tau217. In vitro this was apparent at low infection levels that might reflect the physiological level of gingipain exposure. Phospho-tau217 is emerging as a specific biomarker for AD and these results further validate P. gingivalis-infected neuron cultures and mice as models for AD.

Poly(allylguanidine)-Coated Surfaces Regulate TGF-β in Glioblastoma Cells to Induce Apoptosis via NF-κB Pathway Activation.

Polycationic biomaterials are currently widely applied in neuronal cell cultures to promote cell adhesion and viability. However, polycations generally have cytotoxic properties that limit their application in the field of biomaterials. In this study, we examined the use of a novel polycation poly(allylguanidine) (PAG), which contains a guanidine group in the side chain and a structure similar to poly(allylamine hydrochloride) (PAH), an example of another commonly used polycation. Our findings showed that exposure to PAG induced apoptosis in glioblastoma (GBM) cells, while exposure to PAH induced necrosis. Compared to control groups, the PAG coating significantly limited the proliferation of GBM8901 in vitro and in vivo. Furthermore, GBM8901 cells exposed to the PAG coating exhibited increased levels of phospho-p65 and phosphor-IκB, implying that GBM8901 cells underwent apoptotic cell death via the NF-κB pathway by the regulation of TGF-β. This result was further confirmed to be consistent with the experimental results from western blot protein analysis and apoptosis/necrosis assays. These findings indicate that the polycation PAG has the potential to not only suppress the proliferation of GBM8901 cancer cells but also improve the neural viability and promote the differentiation of neural stem/precursor cells into mature neurons. In conclusion, biomaterials such as PAG act as extremely potent options for applications in the treatment of pathological conditions such as brain cancer.

Insulin-Like Growth Factor-1 Differentially Modulates Glutamate-Induced Toxicity and Stress in Cells of the Neurogliovascular Unit.

The age-related reduction in circulating levels of insulin-like growth factor-1 (IGF-1) is associated with increased risk of stroke and neurodegenerative diseases in advanced age. Numerous reports highlight behavioral and physiological deficits in blood-brain barrier function and neurovascular communication when IGF-1 levels are low. Administration of exogenous IGF-1 reduces the extent of tissue damage and sensorimotor deficits in animal models of ischemic stroke, highlighting the critical role of IGF-1 as a regulator of neurovascular health. The beneficial effects of IGF-1 in the nervous system are often attributed to direct actions on neurons; however, glial cells and the cerebrovasculature are also modulated by IGF-1, and systemic reductions in circulating IGF-1 likely influence the viability and function of the entire neuro-glio-vascular unit. We recently observed that reduced IGF-1 led to impaired glutamate handling in astrocytes. Considering glutamate excitotoxicity is one of the main drivers of neurodegeneration following ischemic stroke, the age-related loss of IGF-1 may also compromise neural function indirectly by altering the function of supporting glia and vasculature. In this study, we assess and compare the effects of IGF-1 signaling on glutamate-induced toxicity and reactive oxygen species (ROS)-produced oxidative stress in primary neuron, astrocyte, and brain microvascular endothelial cell cultures. Our findings verify that neurons are highly susceptible to excitotoxicity, in comparison to astrocytes or endothelial cells, and that a prolonged reduction in IGFR activation increases the extent of toxicity. Moreover, prolonged IGFR inhibition increased the susceptibility of astrocytes to glutamate-induced toxicity and lessened their ability to protect neurons from excitotoxicity. Thus, IGF-1 promotes neuronal survival by acting directly on neurons and indirectly on astrocytes. Despite increased resistance to excitotoxic death, both astrocytes and cerebrovascular endothelial cells exhibit acute increases in glutamate-induced ROS production and mitochondrial dysfunction when IGFR is inhibited at the time of glutamate stimulation. Together these data highlight that each cell type within the neuro-glio-vascular unit differentially responds to stress when IGF-1 signaling was impaired. Therefore, the reductions in circulating IGF-1 observed in advanced age are likely detrimental to the health and function of the entire neuro-glio-vascular unit.

Hypothalamic miR-1983 Targets Insulin Receptor β and the Insulin-mediated miR-1983 Increase Is Blocked by Metformin.

MicroRNAs (miRNAs) expressed in the hypothalamus are capable of regulating energy balance and peripheral metabolism by inhibiting translation of target messenger RNAs (mRNAs). Hypothalamic insulin resistance is known to precede that in the periphery, thus a critical unanswered question is whether central insulin resistance creates a specific hypothalamic miRNA signature that can be identified and targeted. Here we show that miR-1983, a unique miRNA, is upregulated in vitro in 2 insulin-resistant immortalized hypothalamic neuronal neuropeptide Y-expressing models, and in vivo in hyperinsulinemic mice, with a concomitant decrease of insulin receptor β subunit protein, a target of miR-1983. Importantly, we demonstrate that miR-1983 is detectable in human blood serum and that its levels significantly correlate with blood insulin and the homeostatic model assessment of insulin resistance. Levels of miR-1983 are normalized with metformin exposure in mouse hypothalamic neuronal cell culture. Our findings provide evidence for miR-1983 as a unique biomarker of cellular insulin resistance, and a potential therapeutic target for prevention of human metabolic disease.

Development of an Axon-Guiding Aligned Nanofiber-Integrated Compartmentalized Microfluidic Neuron Culture System

Microfluidic-based neuron cell culture systems have recently gained a lot of attention due to their efficiency in supporting the spatial and temporal control of cellular microenvironments. However, the lack of axon guidance is the key limitation in current culture systems. To combat this, we have developed electrospun aligned nanofiber-integrated compartmentalized microfluidic neuron culture systems (NIMSs), where the nanofibers have enabled axonal guidance and stability. The resulting platform significantly improved axon alignment, length, and stability for both rat primary embryonic motor neurons (MNs) and dorsal root ganglia (DRG) neurons compared to the conventional glass-based microfluidic systems (GMSs). The results showed that axonal growth covered more than two times the area on the axonal chamber of NIMSs compared to the area covered for GMSs. Overall, this platform can be used as a valuable tool for fundamental neuroscience research, drug screening, and biomaterial testing.

Real-Time Monitoring of Levetiracetam Effect on the Electrophysiology of an Heterogenous Human iPSC-Derived Neuronal Cell Culture Using Microelectrode Array Technology.

Levetiracetam (LEV) is a broad-spectrum and widely used antiepileptic drug that also has neuroprotective effects in different neurological conditions. Given its complex interaction with neuronal physiology, a better comprehension of LEV effects on neurons activity is needed. Microelectrode arrays (MEAs) represent an advanced technology for the non-invasive study of electrophysiological activity of neuronal cell cultures. In this study, we exploited the Maestro Edge MEA system, a platform that allows a deep analysis of the electrical network behavior, to study the electrophysiological effect of LEV on a mixed population of human neurons (glutamatergic, GABAergic and dopaminergic neurons, and astrocytes). We found that LEV significantly affected different variables such as spiking, single-electrode bursting, and network bursting activity, with a pronounced effect after 15 min. Moreover, neuronal cell culture completely rescued its baseline activity after 24 h without LEV. In summary, MEA technology confirmed its high sensitivity in detecting drug-induced electrophysiological modifications. Moreover, our results allow one to extend the knowledge on the electrophysiological effects of LEV on the complex neuronal population that resembles the human cortex.

In vitro transcriptome response to propolis in differentiated SH-SY5Y neurons.

Propolis is the extract of a resinous compound that protects plants from both cold and microorganism attack and has gained a strong and sticky property because it is transformed after being collected by honey bees. Up to date, many studies have shown that propolis exhibited various beneficial biological activities, such as antifungal, antibacterial, antiviral, antioxidant, antimutagenic, and antitumor effects. Recent reports propounded the in vitro and in vivo neuroprotective effect of propolis; however, the exact molecular genetic mechanisms are still unclear. Therefore, we aimed to investigate the toxicogenomic and beneficial properties, including cytotoxic, antioxidant, apoptotic/necrotic as well as genotoxic effects of propolis (1.56-200µg/ml) on differentiated SH-SY5Y neuronal cells. Additionally, microarray analysis was conducted on cell cultures following propolis application to explore gene differentiation. Differentially expressed genes were further analyzed using string software to characterize protein-protein interactions between gene pathways. Our results revealed that propolis applications could not have a prominent effect on cell viability even at concentrations up to 200µg/ml. The highest propolis concentration induced apoptotic rather than necrotic cell death. The alterations in gene expression profiles, including CYP26A1, DHRS2, DHRS3, DYNC1I1, IGF2, ITGA4, SVIL, TGFβ1, and TGM2 could participate in the neuroprotective effects of propolis. In conclusion, propolis supplementation exerted remarkable advantageous; thus, it may offer great potential as a natural component in the prevention and treatment of neurodegenerative disorders. Whole-genome gene expression pattern following propolis application was investigated for the first time in neuronal cell culture to fill a gap in the literature about propolis toxicogenomics. PRACTICAL APPLICATIONS: Propolis is a very rich product in terms of benefits. In addition to its antibacterial, antiviral, antifungal, and anti-inflammatory content, it is known to have preventive and therapeutic properties for many different ailments. On the other hand, molecular mechanisms of propolis on gene expression differentiations haven't been investigated until now. Moreover, gene expression pattern is vital for all living organisms to maintain homeostasis. Thus, we conduct an experiment series for analyzing gene expression differentiation effects on neuronal cells to understand beneficial properties of propolis. Hence, it could be possible to comment on the use of propolis as a nutritional factor and beneficial diet.

Rabies virus infection at different time intervals in neuronal cell culture

Rabies virus is a highly neurotropic virus that mainly infects all mammals. At present, it has been well recognized among neglected tropical diseases, pediatric diseases, and infectious diseases due to its zoonotic nature. More than 60,000 people including men, women, and children die of rabies every year. The present study has been designed to understand the distribution pattern of rabies virus (RABV) infection among cultured neuronal cells at different time intervals.

Decreased mitochondrial DNA copy number in nerve cells and the hippocampus during nicotine exposure is mediated by autophagy

Cigarette smoke is a harmful air pollutant and nicotine dependence is the essential cause of the tobacco epidemic. Since mitochondrial abnormalities are associated with substance addiction, in this work we used mitochondrial DNA (mtDNA) copy number as an indicator of mitochondrial function to investigate whether nicotine addicts also exhibit mitochondrial abnormalities. We found significantly lower mtDNA copy number in the peripheral blood of healthy nicotine addicts than in non-smokers, indicating that long-term nicotine exposure through smoking has detrimental effects on mitochondria. We also examined the effects of nicotine on mtDNA levels in a rat conditioned place preference (CPP) model of addiction and in cultured neuron cells, which revealed that the mtDNA copy number was significantly reduced in the hippocampus of CPP rats, in human neuroblastoma SH-SY5Y cells, and in rat pheochromocytoma PC12 cells, suggesting that significantly reduced mtDNA copy number is a potential biomarker of nicotine addiction. In SH-SY5Y cells, nicotine treatment induced several mitochondrial defects, such as increased mtDNA damage, increased reactive oxygen species (ROS) levels, decreased mitochondrial membrane potential (△Ψm), and stimulation of autophagic flux via transcriptional up-regulation of several autophagy-related genes and elevated marker protein accumulation, although genes controlling mtDNA replication were unaffected. In addition, pretreatment with the autophagy inhibitor Bafilomycin A1 led to accumulation of microtubule-associated protein 1 light chain 3b-II (LC3B-II) and counteracted the nicotine-induced decrease in mtDNA copy number. These results were recapitulated in PC12 cells, which also showed significant down-regulation of the marker SQSTM1/P62, suggesting that the decrease in mtDNA copy number is mediated by autophagy. This study shows that prolonged nicotine exposure, such as that in nicotine addicts, leads to a decrease of mtDNA copy number in neurons due to enhanced induction of autophagy. CapsuleIt was found that smoking or nicotine exposure decreased mtDNA copy number based on population, animal, and cell models, and these effects appear to be mediated by autophagy.

The CNS-penetrant soluble guanylate cyclase stimulator CYR119 attenuates markers of inflammation in the central nervous system

BackgroundInflammation in the central nervous system (CNS) is observed in many neurological disorders. Nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate (NO–sGC–cGMP) signaling plays an essential role in modulating neuroinflammation. CYR119 is a CNS-penetrant sGC stimulator that amplifies endogenous NO–sGC–cGMP signaling. We evaluated target engagement and the effects of CYR119 on markers of neuroinflammation in vitro in mouse microglial cells and in vivo in quinolinic acid (QA)-induced and high-fat diet-induced rodent neuroinflammation models.MethodsTarget engagement was verified in human embryonic kidney (HEK) cells, rat primary neurons, mouse SIM-A9 cells, and in rats by measuring changes in cGMP and downstream targets of sGC signaling [phosphorylated vasodilator-stimulated phosphoprotein (pVASP), phosphorylated cAMP-response element binding (pCREB)]. In SIM-A9 cells stimulated with lipopolysaccharides (LPS), markers of inflammation were measured when cells were treated with or without CYR119. In rats, microinjections of QA and vehicle were administered into the right and left hemispheres of striatum, respectively, and then rats were dosed daily with either CYR119 (10 mg/kg) or vehicle for 7 days. The activation of microglia [ionized calcium binding adaptor molecule 1 (Iba1)] and astrocytes [glial fibrillary acidic protein (GFAP)] was measured by immunohistochemistry. Diet-induced obese (DIO) mice were treated daily with CYR119 (10 mg/kg) for 6 weeks, after which inflammatory genetic markers were analyzed in the prefrontal cortex.ResultsIn vitro, CYR119 synergized with exogenous NO to increase the production of cGMP in HEK cells and in primary rat neuronal cell cultures. In primary neurons, CYR119 stimulated sGC, resulting in accumulation of cGMP and phosphorylation of CREB, likely through the activation of protein kinase G (PKG). CYR119 attenuated LPS-induced elevation of interleukin 6 (IL-6) and tumor necrosis factor (TNF) in mouse microglial cells. Following oral dosing in rats, CYR119 crossed the blood–brain barrier (BBB) and stimulated an increase in cGMP levels in the cerebral spinal fluid (CSF). In addition, levels of proinflammatory markers associated with QA administration or high-fat diet feeding were lower in rodents treated with CYR119 than in those treated with vehicle.ConclusionsThese data suggest that sGC stimulation could provide neuroprotective effects by attenuating inflammatory responses in nonclinical models of neuroinflammation.

A Compartmentalized Neuronal Cell-Culture Platform Compatible With Cryo-Fixation by High-Pressure Freezing for Ultrastructural Imaging.

The human brain contains a wide array of billions of neurons and interconnections, which are often simplified for analysis in vitro using compartmentalized microfluidic devices for neuronal cell culturing, to better understand neuronal development and disease. However, such devices are traditionally incompatible for high-pressure freezing and high-resolution nanoscale imaging and analysis of their sub-cellular processes by methods including electron microscopy. Here we develop a novel compartmentalized neuronal co-culture platform allowing reconstruction of neuronal networks with high variable spatial control, which is uniquely compatible for high-pressure freezing. This cryo-fixation method is well-established to enable high-fidelity preservation of the reconstructed neuronal networks and their sub-cellular processes in a near-native vitreous state without requiring chemical fixatives. To direct the outgrowth of neurites originating from two distinct groups of neurons growing in the two different compartments, polymer microstructures akin to microchannels are fabricated atop of sapphire disks. Two populations of neurons expressing either enhanced green fluorescent protein (EGFP) or mCherry were grown in either compartment, facilitating the analysis of the specific interactions between the two separate groups of cells. Neuronally differentiated PC12 cells, murine hippocampal and striatal neurons were successfully used in this context. The design of this device permits direct observation of entire neuritic processes within microchannels by optical microscopy with high spatial and temporal resolution, prior to processing for high-pressure freezing and electron microscopy. Following freeze substitution, we demonstrate that it is possible to process the neuronal networks for ultrastructural imaging by electron microscopy. Several key features of the embedded neuronal networks, including mitochondria, synaptic vesicles, axonal terminals, microtubules, with well-preserved ultrastructures were observed at high resolution using focused ion beam – scanning electron microscopy (FIB-SEM) and serial sectioning – transmission electron microscopy (TEM). These results demonstrate the compatibility of the platform with optical microscopy, high-pressure freezing and electron microscopy. The platform can be extended to neuronal models of brain disease or development in future studies, enabling the investigation of subcellular processes at the nanoscale within two distinct groups of neurons in a functional neuronal pathway, as well as pharmacological testing and drug screening.

Developmental dysregulation of excitatory-to-inhibitory GABA-polarity switch may underlie schizophrenia pathology: A monozygotic-twin discordant case analysis in human iPS cell-derived neurons

Schizophrenia is a major psychiatric disorder, but the molecular mechanisms leading to its initiation or progression remain unclear. To elucidate the pathophysiology of schizophrenia, we used an in vitro neuronal cell culture model involving human induced pluripotent stem cells (hiPSCs) derived from a monozygotic-twin discordant schizophrenia pair. The cultured neurons differentiated from hiPSCs were composed of a mixture of glutamatergic excitatory neurons and gamma aminobutyric acid (GABA)ergic inhibitory neurons. In the electrophysiological analysis, a different pattern of spontaneous neuronal activity was observed under the condition without any stimulants. The frequency of spontaneous excitatory post-synaptic currents (sEPSCs) was significantly higher in the hiPSC-derived neurons of the patient with schizophrenia than in the control sibling at day-in-vitro 30. However, the synaptic formation was not different between the patient with schizophrenia and the control sibling during the same culture period. To explain underlying mechanisms of higher excitability of presynaptic cells, we focused on the potassium-chloride co-transporter KCC2, which contributes to excitatory-to-inhibitory GABA polarity switch in developing neurons. We also revealed the altered expression pattern of KCC2 in hiPSC-derived neurons from the patient with schizophrenia, which could contribute to understanding the pathology of schizophrenia in the developing nervous system.