Search Results (11)

The ability to study mature neuronal cells ex vivo is complicated by their non-dividing nature and difficulty in obtaining large numbers of primary cells from organisms. Thus, numerous transformed progenitor models have been developed that can be routinely cultured, then scaled, and differentiated to mature neurons. In this paper, we present a new method for differentiating one such model, the Lund human mesencephalic (LUHMES) dopaminergic neurons. This method is two days faster than some established protocols, results in nearly five times greater numbers of mature neurons, and involves fewer handling steps that could introduce technical variability. Moreover, it overcomes the problem of cell aggregate formation that commonly impedes high-resolution imaging, cell dissociation, and downstream analysis. While recently established for herpes simplex virus type 1, we demonstrate that LUHMES neurons can facilitate studies of other herpesviruses, as well as RNA viruses associated with childhood encephalitis and hemorrhagic fever. This protocol provides an improvement in the generation of large-scale neuronal cultures, which may be readily applicable to other neuronal 2D cell culture models and provides a system for studying neurotrophic viruses. We named this method the Streamlined Protocol for Enhanced Expansion and Differentiation Yield, or SPEEDY, method. Full article

Thymoquinone (TQ), an active compound from Nigella sativa seeds, is often described as a pharmacologically relevant compound with antioxidative properties, while the synthesis of TQ in the plant via oxidations makes it inapplicable for scavenging radicals. Therefore, the present study was designed to reassess the radical scavenging properties of TQ and explore a potential mode of action. The effects of TQ were studied in models with mitochondrial impairment and oxidative stress induced by rotenone in N18TG2 neuroblastoma cells and rotenone/MPP⁺ in primary mesencephalic cells. Tyrosine hydroxylase staining revealed that TQ significantly protected dopaminergic neurons and preserved their morphology under oxidative stress conditions. Quantification of the formation of superoxide radicals via electron paramagnetic resonance showed an initial increase in the level of superoxide radicals in the cell by TQ. Measurements in both cell culture systems revealed that the mitochondrial membrane potential was tendentially lowered, while ATP production was mostly unaffected. Additionally, the total ROS levels were unaltered. In mesencephalic cell culture under oxidative stress conditions, caspase-3 activity was decreased when TQ was administered. On the contrary, TQ itself tremendously increased the caspase-3 activity in the neuroblastoma cell line. Evaluation of the glutathione level revealed an increased level of total glutathione in both cell culture systems. Therefore, the enhanced resistance against oxidative stress in primary cell culture might be a consequence of a lowered caspase-3 activity combined with an increased pool of reduced glutathione. The described anti-cancer ability of TQ might be a result of the pro-apoptotic condition in neuroblastoma cells. Our study provides evidence that TQ has no direct scavenging effect on superoxide radicals. Full article

This is the first study aiming to develop a method for the long-term visualization of living nigrostriatal dopaminergic neurons using 1-(2-(bis(4-fluorophenyl)methoxy)ethyl)-4-(3-phenylpropyl)piperazine-BODIPY (GBR-BP), the original fluorescent substance, which is a derivative of GBR-12909, a dopamine uptake inhibitor. This method is based on the authors’ hypothesis about the possibility of specifically internalizing into dopaminergic neurons substances with a high affinity for the dopamine transporter (DAT). Using a culture of mouse embryonic mesencephalic and LUHMES cells (human embryonic mesencephalic cells), as well as slices of the substantia nigra of adult mice, we have obtained evidence that GBR-BP is internalized specifically into dopaminergic neurons in association with DAT via a clathrin-dependent mechanism. Moreover, GBR-BP has been proven to be nontoxic. As we have shown in a primary culture of mouse metencephalon, GBR-BP is also specifically internalized into some noradrenergic and serotonergic neurons, but is not delivered to nonmonoaminergic neurons. Our data hold great promise for visualization of dopaminergic neurons in a mixed cell population to study their functioning, and can also be considered a new approach for the development of targeted drug delivery to dopaminergic neurons in pathology, including Parkinson’s disease. Full article

Background: The prevention of age-related neurodegenerative disorders is an important issue in an aging society. Microglia-mediated neuroinflammation resulting in dopaminergic neuron loss may lead to the pathogenesis of Parkinson’s disease (PD). Lipopolysaccharide (LPS), an endotoxin, induces neuroinflammatory microglial activation, contributing to dopaminergic neuron damage. Diosgenin is a phytosteroid sapogenin with a wide spectrum of pharmacological activities, e.g., anti-inflammatory activity. However, the preventive effect of diosgenin on neuroinflammation is not clear. Thus, in this study, we further investigated the neuroprotective effect of diosgenin on LPS-induced neural damage in vitro and in vivo. Methods: For in vitro experiments, primary mesencephalic neuron-glia cultures and primary microglia cultures isolated from Sprague–Dawley rats were used. Cells were pretreated with diosgenin and then stimulated with LPS. The expression of proinflammatory cytokines or tyrosine hydroxylase (TH) in the cells was analyzed. In vivo, rats were fed a diet containing 0.1% (w/w) diosgenin for 4 weeks before being administered a unilateral substantia nigra (SN) injection of LPS. Four weeks after the LPS injection, the rats were assessed for lesion severity using the amphetamine-induced rotation test and TH immunohistochemistry. Results: Diosgenin pretreatment prevented LPS-induced neurite shortening in TH-positive neurons in mesencephalic neuron-glia cultures. In addition, pretreatment of primary microglia with diosgenin significantly reduced tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) expression. Moreover, diosgenin pretreatment significantly suppressed LPS-induced extracellular signal-regulated kinase (ERK) activation. In vivo, the intranigral injection of LPS in rats fed a diosgenin-containing diet significantly improved motor dysfunction and reduced TH expression in SN. Conclusion: These results support the effectiveness of diosgenin in protecting dopaminergic neurons from LPS-induced neuroinflammation. Full article

Parkinson’s disease (PD) is a multifactorial neurodegenerative disease. In recent years, several studies demonstrated that the gastroenteric system and intestinal microbiome influence central nervous system function. The pathological mechanisms triggered thereby change neuronal function in neurodegenerative diseases including dopaminergic neurons in Parkinson´s disease. In this study, we employed a model system for PD of cultured primary mesencephalic cells and used the pesticide rotenone to model dopaminergic cell damage. We examined neuroprotective effects of the Rho kinase inhibitor Fasudil and the short chain fatty acid (SCFA) propionic acid on primary neurons in cell morphological assays, cell survival, gene and protein expression. Fasudil application resulted in significantly enhanced neuritic outgrowth and increased cell survival of dopaminergic cells. The application of propionic acid primarily promoted cell survival of dopaminergic cells against rotenone toxicity and increased neurite outgrowth to a moderate extent. Interestingly, Fasudil augmented gene expression of synaptophysin whereas gene expression levels of tyrosine hydroxylase (TH) were substantially increased by propionic acid. Concerning protein expression propionic acid treatment increased STAT3 levels but did not lead to an increased phosphorylation indicative of pathway activation. Our findings indicate that both Fasudil and propionic acid treatment show beneficial potential in rotenone-lesioned primary mesencephalic cells. Full article

Phytocannabinoids protect neurons against stressful conditions, possibly via the heme oxygenase (HO) system. In cultures of primary mesencephalic neurons and neuroblastoma cells, we determined the capability of cannabidiol (CBD) and tetrahydrocannabinol (THC) to counteract effects elicited by complex I-inhibitor rotenone by analyzing neuron viability, morphology, gene expression of IL6, CHOP, XBP1, HO-1 (stress response), and HO-2, and in vitro HO activity. Incubation with rotenone led to a moderate stress response but massive degeneration of dopaminergic neurons (DN) in primary mesencephalic cultures. Both phytocannabinoids inhibited in-vitro HO activity, with CBD being more potent. Inhibition of the enzyme reaction was not restricted to neuronal cells and occurred in a non-competitive manner. Although CBD itself decreased viability of the DNs (from 100 to 78%), in combination with rotenone, it moderately increased survival from 28.6 to 42.4%. When the heme degradation product bilirubin (BR) was added together with CBD, rotenone-mediated degeneration of DN was completely abolished, resulting in approximately the number of DN determined with CBD alone (77.5%). Using N18TG2 neuroblastoma cells, we explored the neuroprotective mechanism underlying the combined action of CBD and BR. CBD triggered the expression of HO-1 and other cell stress markers. Co-treatment with rotenone resulted in the super-induction of HO-1 and an increased in-vitro HO-activity. Co-application of BR completely mitigated the rotenone-induced stress response. Our findings indicate that CBD induces HO-1 and increases the cellular capacity to convert heme when stressful conditions are met. Our data further suggest that CBD via HO may confer full protection against (oxidative) stress when endogenous levels of BR are sufficiently high. Full article

Epidemiological studies have shown that coffee consumption decreases the risk of Parkinson’s disease (PD). Caffeic acid (CA) and chlorogenic acid (CGA) are coffee components that have antioxidative properties. Rotenone, a mitochondrial complex I inhibitor, has been used to develop parkinsonian models, because the toxin induces PD-like pathology. Here, we examined the neuroprotective effects of CA and CGA against the rotenone-induced degeneration of central dopaminergic and peripheral enteric neurons. Male C57BL/6J mice were chronically administered rotenone (2.5 mg/kg/day), subcutaneously for four weeks. The animals were orally administered CA or CGA daily for 1 week before rotenone exposure and during the four weeks of rotenone treatment. Administrations of CA or CGA prevented rotenone-induced neurodegeneration of both nigral dopaminergic and intestinal enteric neurons. CA and CGA upregulated the antioxidative molecules, metallothionein (MT)-1,2, in striatal astrocytes of rotenone-injected mice. Primary cultured mesencephalic or enteric cells were pretreated with CA or CGA for 24 h, and then further co-treated with a low dose of rotenone (1–5 nM) for 48 h. The neuroprotective effects and MT upregulation induced by CA and CGA in vivo were reproduced in cultured cells. Our data indicated that intake of coffee components, CA and CGA, enhanced the antioxidative properties of glial cells and prevents rotenone-induced neurodegeneration in both the brain and myenteric plexus. Full article

Cerebral dopamine neurotrophic factor (CDNF), previously known as the conserved dopamine neurotrophic factor, belongs to the evolutionarily conserved CDNF/mesencephalic astrocyte-derived neurotrophic factor MANF family of neurotrophic factors that demonstrate neurotrophic activities in dopaminergic neurons. The function of CDNF during brain ischemia is still not known. MANF is identified as an endoplasmic reticulum (ER) stress protein; however, the role of CDNF in ER stress remains to be fully elucidated. Here, we test the neuroprotective effect of CDNF on middle cerebral artery occlusion (MCAO) rats and neurons and astrocytes treated with oxygen–glucose depletion (OGD). We also investigate the expression of CDNF in cerebral ischemia and in primary neurons treated with ER stress-inducing agents. Our results show that CDNF can significantly reduce infarct volume, reduce apoptotic cells and improve motor function in MCAO rats, while CDNF can increase the cell viability of neurons and astrocytes treated by OGD. The expression of CDNF was upregulated in the peri-infarct tissue at 2 h of ischemia/24 h reperfusion. ER stress inducer can induce CDNF expression in primary cultured neurons. Our data indicate that CDNF has neuroprotective effects on cerebral ischemia and the OGD cell model and the protective mechanism of CDNF may occur through ER stress pathways. Full article

Lipopolysaccharide (LPS)-induced neuroinflammation triggers and accelerates the pathogenesis of Parkinson’s disease (PD). Carthamus tinctorius L., a traditional Chinese medicine, has been widely used for the treatment of cerebrovascular disease. Hydroxysafflor Yellow A (HSYA) is an active component of C. tinctorius. The purpose of this study was to investigate whether HSYA could attenuate LPS-induced neurotoxicity and neuroinflammation in primary mesencephalic cultures. Cell viability was measured by MTT and LDH assays. The number of tyrosine hydroxylase (TH) positive neuron was observed by immunohistochemistry. NF-κB p65 and iNOS expressions were evaluated with western blotting method. Pro-inflammatory cytokines including IL-1β and TNF-α were determined by ELISA kits. Nitric oxide (NO) content in the culture medium was assayed. The results showed that HSYA treatment significantly attenuated the LPS-induced dopaminergic neurons damage. HSYA partially inhibited the expressions of NF-κB p65 and iNOS. Furthermore, HSYA decreased the content of IL-1β, TNF-α and NO in the supernatants. Taken together, these results suggest that HSYA exerts protective effects on LPS-induced neurotoxicity in dopaminergic neurons and the mechanisms may be associated with the inhibition of inflammatory response. Full article

The neuroprotective effects of Licochalcone A (Lico.A), a flavonoid isolated from the herb licorice, in Parkinson’s disease (PD) have not been elucidated. The prominent pathological feature of PD is the loss of dopaminergic neurons. The crucial role of neuroinflammation induced by activated microglia in dopaminergic neurodegeneration has been validated. In this study, we explore the therapeutic effects of Lico.A in lipopolysaccharide (LPS)-induced PD models in vivo and in vitro. We find that Lico.A significantly inhibits LPS-stimulated production of pro-inflammatory mediators and microglial activation by blocking the phosphorylation of extracellular signal-regulated kinase (ERK1/2) and nuclear factor κB (NF-κB) p65 in BV-2 cells. In addition, through cultured primary mesencephalic neuron-glia cell experiments, we illustrate that Lico.A attenuates the decrease in [³H] dopamine (DA) uptake and the loss of tyrosine hydroxylase-immunoreactive (TH-ir) neurons in LPS-induced PD models in vitro. Furthermore, LPS intoxication in rats results in microglial activation, dopaminergic neurodegeneration and significant behavioral deficits in vivo. Lico.A treatment prevents microglial activation and reduction of dopaminergic neuron and ameliorates PD-like behavioral impairments. Thus, these results demonstrate for the first time that the neuroprotective effects of Lico.A are associated with microglia and anti-inflammatory effects in PD models. Full article

Interleukin (IL)-10, an anti-inflammatory cytokine, is expressed in the brain and can inhibit microglial activation. Herein, we utilized lipopolysaccharide (LPS)-induced inflammatory Parkinson’s disease (PD) cell model to determine whether microglia and astrocytes are necessary targets for IL-10 neuroprotection. Primary ventral mesencephalic (VM) cultures with different composition of neurons, microglia and astrocytes were prepared. The cells were exposed to IL-10 (15, 50 or 150 ng/mL) 1 h prior to LPS (50 ng/mL) treatment. LPS induced dopaminergic and non-dopaminergic neuronal loss in VM cultures, VM neuron-enriched cultures, and neuron-microglia co-cultures, but not in neuron-astrocyte co-cultures. IL-10 reduced LPS-induced neuronal loss particularly in single VM neuron cultures. Pro-inflammatory mediators (TNF-α, IL-1β, inducible nitric oxide synthase and cyclooxygenase-2) were upregulated in both neuron-microglia and neuron-astrocyte co-cultures by LPS. In contrast, neurotrophic factors (brain-derived neurotrophic factor, insulin-like growth factor-1 or glial cell-derived neurotrophic factor) were downregulated in neuron-microglia co-cultures, but upregulated in neuron-astrocyte co-cultures by LPS. IL-10 reduced both the increase in production of the pro-inflammatory mediators and the decrease in production of the neurotrophic factors induced by LPS. These results suggest that astrocytes can balance LPS neurotoxicity by releasing more neurotrophic factors and that IL-10 exerts neuroprotective property by an extensive action including direct on neurons and indirect via inhibiting microglial activation. Full article