Mesencephalic Cultures Research Articles

Rho-inhibition and neuroprotective effect on rotenone-treated dopaminergic neurons in vitro

Mesencephalic cell cultures are a good model to study the vulnerability of dopaminergic neurons and reproduce, in vitro, experimental models of Parkinson’s disease. Rotenone associated as an environmental neurotoxin related to PD, is able to provoke dopaminergic neuron degeneration by inhibiting complex I of the mitochondrial respiratory chain and by inducing accumulation of α-synuclein. Recently, rotenone has been described to activate RhoA, a GTPase protein.In the present study we evaluated a possible neuroprotective effect of Rho-inhibitor molecules on rotenone-damaged dopaminergic (DA) neurons obtained from mouse primary mesencephalic cell culture.Our results showed that Clostridium Botulinum C3 toxin (C3) and simvastatin, as RhoA inhibitors, were able to protect DA neurons from rotenone damages. In fact, pretreatment with C3 or simvastatin significantly prevented the reduction of [3H]dopamine uptake, neurites injury and the expression patterns of proteins like α-syn, actin and connexin 43.

Neurotoxic effects of acrylamide on dopaminergic neurons in primary mesencephalic cell culture

Exposure to acrylamide is increasing worldwide as a result of its heavy use in industry and formation in carbohydrate-rich food cooked at high temperature. Despite its neurotoxicity, no studies have shown its toxic effects on dopaminergic neurons yet. Therefore, the current study was carried out to show whether acrylamide adversely affects primary cultured dopaminergic neurons. Acrylamide (0.001, 0.01, 0.1, 1, 2 mM) was added to two different groups of primary mesencephalic cell cultures on the 9th day in vitro for 24 and 48 h, respectively. Moreover, a group of cultures was treated with lower concentrations of acrylamide (0.01, 0.05, 0.1, 0.5 mM) on the 6th day in vitro for 5 consecutive days to investigate its long-term effects on dopaminergic neurons. Following each treatment, culture media were obtained for measuring lactate dehydrogenase, and cultured cells were stained immunocytochemically against tyrosine hydroxylase and neuronal nuclear antigens. Treatment of cultures with acrylamide for 48 h significantly reduced the number of dopaminergic neurons, adversely altered the morphology of the surviving neurons and increased levels of lactate dehydrogenase in the culture media. Similar treatment of cultures with acrylamide also resulted in lower numbers of total neuronal cells as shown by a reduced expression of the neuronal nuclear antigen. Prolonged treatment of cultures with lower concentrations of acrylamide slightly reduced the survival of dopaminergic neurons but increased the release of lactate dehydrogenase into the culture media as well. The current study shows, for the first time, neurotoxicity of acrylamide on dopaminergic neurons in the primary mesencephalic cell culture.

The novel protective effects of loganin against 1-methyl-4-phenylpyridinium-induced neurotoxicity: Enhancement of neurotrophic signaling, activation of IGF-1R/GLP-1R, and inhibition of RhoA/ROCK pathway.

Loganin, a major iridoid glycoside obtained from fruits of Cornus officinalis, possesses anti-inflammatory, antitumor, antidiabetic, and osteoporosis prevention effects. Loganin has been linked to neuroprotection in several models of neurodegeneration, including Parkinson's disease (PD). However, mechanisms underlying the neuroprotective effects of loganin are still mostly unknown. Here, we demonstrated the protective effects of loganin against PD mimetic toxin 1-methyl-4-phenylpyridinium (MPP+ ) and the important roles of insulin-like growth factor 1 receptor (IGF-1R) and glucagon-like peptide 1 receptor (GLP-1R) in the neuroprotective mechanisms of loganin. In primary mesencephalic neuronal cultures treated with or without MPP+ , loganin up-regulated expressions of neurotrophic signals including IGF-1R, GLP-1R, p-Akt, BDNF, and tyrosine hydroxylase. Loganin protected against MPP+ -induced apoptosis by up-regulating antiapoptotic protein and down-regulating proapoptotic protein. Moreover, loganin attenuated MPP+ -induced neurite damage via up-regulation of GAP43 and down-regulation of membrane-RhoA/ROCK2/p-LIMK/p-cofilin. Loganin also attenuated MPP+ -induced reactive oxygen species (ROS) production. However, both AG1024, an IGF-1R antagonist, and exendin 9-39, a GLP-1R antagonist, attenuated the protective effects of loganin on MPP+ -induced cytotoxicity, apoptosis, neurite length decrease, and ROS production. Our results suggest that loganin attenuates MPP+ -induced apoptotic death, neurite damage, and oxidative stress through enhancement of neurotrophic signaling, activation of IGF-1R/GLP-1R, and inhibition of RhoA/ROCK pathway, providing the evidence that loganin possesses novel neuroprotective effects.

Nicotine-Induced Neuroprotection in Rotenone In Vivo and In Vitro Models of Parkinson's Disease: Evidences for the Involvement of the Labile Iron Pool Level as the Underlying Mechanism.

Parkinson's disease (PD) is characterized by the degeneration of the dopaminergic neurons in the substantia nigra pars compacta (SNpc). Clinical and experimental evidence suggest that the activation of the nicotinic acetylcholine receptor (nAChR) could be protective for PD. In this study, we investigated the neuroprotective capacity of nicotine in a rat PD model. Considering that iron metabolism has been implicated in PD pathophysiology and nicotine has been described to chelate this metal, we also studied the effect of nicotine on the cellular labile iron pool (LIP) levels. Rotenone (1μg) was unilaterally injected into the median forebrain bundle to induce the degeneration of the nigrostriatal pathway. Nicotine administration (1mg/K, s.c. daily injection, starting 5days before rotenone and continuing for 30days) attenuated the dopaminergic cell loss in the SNpc and the degeneration of the dopaminergic terminals provoked by rotenone, as assessed by immunohistochemistry. Furthermore, nicotine partially prevented the reduction on dopamine levels in the striatum and improved the motor deficits, as determined by HPLC-ED and the forelimb use asymmetry test, respectively. Studies in primary mesencephalic cultures showed that pretreatment with nicotine (50μM) improved the survival of tyrosine hydroxylase-positive neurons after rotenone (20nM) exposure. Besides, nicotine induced a reduction in the LIP levels assessed by the calcein dequenching method only at the neuroprotective dose. These effects were prevented by addition of the nAChRs antagonist mecamylamine (100μM). Overall, we demonstrate a neuroprotective effect of nicotine in a model of PD in rats and that a reduction in iron availability could be an underlying mechanism.

Long-term neurotoxic effects of domoic acid on primary dopaminergic neurons

Domoic acid, an excitatory neurotoxin produced by certain algae, reaches the food chain through accumulation in some sea organisms. To investigate its long-term neurotoxicity on dopaminergic neurons, prepared primary mesencephalic cell cultures were exposed to different concentrations of domoic acid (0.1, 1, 10, 100 μM) on the 8th day in vitro (DIV) for 4 days. On the 12th DIV, culture media were collected for measurement of lactate dehydrogenase and cultured cells were subjected to immunohistochemistry against tyrosine hydroxylase, neuronal nuclear antigen and glial fibrillary acidic protein, and fluorescence staining using H2DCFDA, JC-1 and Hoechst 33342 dyes. Moreover, roles of AMPA/KA and NMDA receptors in domoic acid neurotoxicity were also investigated. Domoic acid significantly decreased the number of dopaminergic neurons and adversely affected their morphology, and slightly reduced the expression of neuronal nuclear antigen and glial fibrillary acidic protein. Co-treatment of cultures with domoic acid and the AMPA/KA or NMDA receptor antagonists NBQX and MK-801 rescued significant number of dopaminergic neurons. Domoic acid significantly decreased red:green fluorescence ratio of JC-1 and did not affect production of reactive oxygen species and apoptotic cell death. In conclusions, the present study reveals that long-term treatment of primary mesencephalic cell culture with domoic acid significantly destroyed dopaminergic neurons. This effect appears to be attributed to activation of AMPA/KA and NMDA receptors and mitochondrial damage.

Hydroxysafflor Yellow A Attenuates Lipopolysaccharide-Induced Neurotoxicity and Neuroinflammation in Primary Mesencephalic Cultures.

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.

An Easy-to-Implement Protocol for Preparing Postnatal Ventral Mesencephalic Cultures.

Postnatally derived cultures of ventral mesencephalic neurons offer several crucial advantages over embryonic ventral mesencephalic cultures, including a higher content of TH-positive cells and the ability to derive cells from the substantia nigra, which contains the neurons most vulnerable to Parkinson’s disease. On the other hand, these cultures are more challenging to produce consistently. Here, we provide an easy-to-implement protocol for culturing postnatal ventral mesencephalic cells from the substantia nigra (SN) and the ventral tegmental area using commercially available media, dishes, and general lab equipment, avoiding extensive material and equipment purchases. The protocol can be completed in about 5 h and provides ventral midbrain neuron cultures on cortex glia feeder layers in three weeks’ time. The protocol uses an optimized protease digestion, tissue storage in Hibernate A during dissection and purification of neurons on an OptiPrep density gradient.

Neurotoxic effects of domoic acid on dopaminergic neurons in primary mesencephalic cell culture.

Domoic acid is a potent marine neurotoxin produced by certain species of the diatom genus Pseudonitzschia. To our knowledge, there are no studies that have investigated neurotoxic effects of domoic acid on dopaminergic neurons. Accordingly, the present study was carried out to investigate the potential neurotoxic effects of domoic acid on dopaminergic neurons in primary mesencephalic cell culture. Cultures prepared from embryonic mouse mesencephala (total of 250 embryos) were treated with different concentrations of domoic acid (0.1, 1, 10, 100 µM) on the 10th DIV for 48 h. On the 12th DIV, culture media were used for measurement of lactate dehydrogenase and cultured cells were subjected to immunostaining for tyrosine hydroxylase, neuronal nuclear antigen and glial fibrillary acidic protein, and fluorescence staining using H2DCFDA, JC-1 and DAPI stains. Moreover, roles of AMPA/KA and NMDA receptors in domoic acid neurotoxicity were also investigated. Domoic acid significantly decreased the number of dopaminergic neurons, decreased the expression of neuronal nuclear antigen and slightly affected astrocyte populations, and increased the release of lactate dehydrogenase into the culture media. AMPA/KA receptor antagonist NBQX but not NMDA receptor antagonist MK-801 significantly inhibited the neurotoxic effect of domoic acid on dopaminergic neurons. H<SUB>2</SUB>DCFDA, JC-1 and DAPI fluorescence staining, respectively, revealed that DomA slightly raised ROS production, and significantly decreased mitochondrial membrane potential and increased apoptotic cell death of cultured cells. The current study presents for the first time the neurotoxic effects of domoic acid on dopaminergic neurons and this effect appears to be attributed to activation of AMPA/KA receptors on dopaminergic neurons.

Culture of Rat Mesencephalic Dopaminergic Neurons and Application to Neurotoxic and Neuroprotective Agents.

Dopaminergic neuronal cell degeneration is the principal characteristic feature of the neuropathology of Parkinson disease. Cultures of mesencephalic neurons are widely used as a source of dopaminergic neurons for the study of mechanisms implicated in dopaminergic cell death and for the evaluation of potential dopaminergic neuroprotective agents, including neurotrophic factors. This chapter presents a detailed protocol for the preparation of rat mesencephalic cell cultures and their application to evaluating the effect of the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium and the neuroprotective action of brain-derived neurotrophic factor.

Neuroprotective effect of an Nrf2-ARE activator identified from a chemical library on dopaminergic neurons

The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway, which induces the production of antioxidant enzymes, is a possible therapeutic target for treating diseases related to oxidative stress. Nrf2 activators often exhibit cytotoxicity due to nonspecific electrophilic reactions with thiol groups. We screened a chemical library to explore Nrf2 activators with a wide safety margin. In at least in vitro experiments, TPNA10168, identified from the library, showed a higher efficacy in Nrf2 activation and a lower cytotoxicity than sulforaphane, a well-known Nrf2 activator. The present study demonstrated the protective effect of TPNA10168 against 6-hydroxydopamine-induced cytotoxicity. In PC12 cells, NAD(P)H:quinone oxidoreductase 1 was upregulated by TPNA10168 and participated in the protective effect. In primary mesencephalic cultures, heme oxygenase-1, upregulated by TPNA10168 in astrocytes, provided protection of dopaminergic neurons via a guanylate cyclase/protein kinase G signaling pathway via carbon monoxide. These results suggest that the compound identified from the chemical library may be suitable as a neuroprotective agent with the ability to induce antioxidant enzymes.

94 - Glutamate Excitotoxicity Induced by Nitric Oxide Mediated Dysfunction of the Mitochondrial 2-Oxoglutarate Dehydrogenase Complex

Increased levels of nitric oxide (NO) and peroxynitrite (ONOO‒), mitochondrial dysfunction at complex I, and excessive accumulation of glutamate have been suggested as the key pathophysiological events leading to brain damage upon traumatic brain injury (TBI). The aim of this study was to delineate the relationship between these three events and to begin to evaluate their potential pathophysiological relevance. In experiments with rat cortex homogenates we have shown that NO, at the concentrations previously shown to be present in the cortex upon TBI, substantially reduced 2-oxoglutarate dehydrogenase complex (OGDHC) activity, but did not affect mitochondrial respiration. Mitochondrial respiration was impaired by peroxynitrite (ONOO‒) and tert-butylperoxide (TBP) in a manner similar to that observed in the in vivo model of TBI. Incubation of neuronal cell line B35 with an NO donor did not induce cell necrosis (estimated by measurement of lactate dehydrogenase (LDH) release to the supernatant). Cell viability was also largely unaffected by either thiamine (TH) or by succinyl phosphonate (SP), an activator and an inhibitor of OGDHC, respectively. In contrast, incubation of the cells with glutamate increased extracellular LDH concentrations, which was inhibited by TH, with the effect reversed by SP. A similar protective effect of TH was also observed in primary murine mesencephalic cultures exposed to glutamate. Glutamate drastically increased the rate of cell death and this effect was ameliorated by TH. Taken together, these data suggest that NO generated from NOS upon neuro-inflammation inhibits OGDHC, causing reduced glutamate uptake by the tricarboxylic acid cycle (TCA) cycle, followed by the intracellular accumulation of glutamate, culminating in glutamate toxicity and neuronal death. Cell injury can be prevented by TH. Thus, the impairment of OGDHC plays a key role in the glutamate mediated neurotoxicity in neurons during TBI; pharmacological activation of OGDHC may thus be of neuroprotective potential.

Paracrine and Intracrine Angiotensin 1-7/Mas Receptor Axis in the Substantia Nigra of Rodents, Monkeys, and Humans.

In addition to the classical hormonal (tissue-to-tissue) renin-angiotensin system (RAS), there are a paracrine (cell-to-cell) and an intracrine (intracellular/nuclear) RAS. A local paracrine brain RAS has been associated with several brain disorders, including Parkinson’s disease (PD). Classically, angiotensin II (Ang II) is the main RAS effector peptide and acts through two major receptors: Ang II type 1 and 2 (AT1 and AT2) receptors. It has been shown that enhanced activation of the Ang II/AT1 axis exacerbates dopaminergic cell death. Several new components of the RAS have more recently been discovered. However, the role of new Ang 1-7/Mas receptor RAS component was not investigated in the brain and particularly in the dopaminergic system. In the present study, we observed Mas receptor labeling in dopaminergic neurons and glial cells in rat mesencephalic primary cultures; substantia nigra of rats, monkeys, and humans; and human induced pluripotent stem (iPS) cells derived from healthy controls and sporadic PD patients. The present data support a neuroprotective role of the Ang 1-7/Mas receptor axis in the dopaminergic system. We observed that this axis is downregulated with aging, which may contribute to the aging-related vulnerability to neurodegeneration. We have also identified an intracellular Ang 1-7/Mas axis that modulates mitochondrial and nuclear levels of superoxide. The present data suggest that nuclear RAS receptors regulate the adequate balance between the detrimental and the protective arms of the cell RAS. The results further support that the brain RAS should be taken into account for the design of new therapeutic strategies for PD.

Acylated and unacylated ghrelin confer neuroprotection to mesencephalic neurons

The polypeptide ghrelin is an endogenous ligand at the growth hormone secretagogue receptor 1a. To ghrelin multiple functions have been ascribed including promotion of gastrointestinal motility. Postprandial ghrelin levels have been reported to be reduced in patients suffering from Parkinson disease (PD). Experimental studies revealed neuroprotective effects of ghrelin in different PD models. The purpose of the present study was (i) to further elucidate the mechanism underlying the neuroprotective action of ghrelin and (ii) to determine whether these effects occur with both the acylated and the unacylated form. The study was conducted in primary mesencephalic cultures treated with mitochondrial complex I and complex II inhibitors. We show that protective effects of ghrelin against complex I inhibition with MPP+ were independent of the acylation status of ghrelin, although acylated ghrelin appeared to be more potent. Protection by both forms was also observed when neurons were exposed to the complex II inhibitor 3-NP. Both forms led to higher oxygen consumption rates upon electron transport chain uncoupling, indicating that the two peptides may exert uncoupling effects themselves. We demonstrate that the rescue provided by ghrelin required calcium influx through L-type voltage-gated calcium channels. Whereas the protective effects of acylated ghrelin required receptor binding, effects of the unacylated form remained unaffected by treatment with a ghrelin receptor antagonist. Importantly, inhibition of ghrelin O-acyltransferase failed to reduce the activity of unacylated ghrelin. Overall, our data suggest that both acylated and unacylated ghrelin afford protection to dopamine neurons but through mechanisms that only partially overlap.

Prostaglandin EP2 Receptors Mediate Mesenchymal Stromal Cell-Neuroprotective Effects on Dopaminergic Neurons.

Mesenchymal stromal cells (MSCs) have been shown to have useful properties for cell therapy and have been proposed for treatment of neurodegenerative diseases, including Parkinson's disease. However, the mechanisms involved in recovering dopaminergic neurons are not clear. The present study aims to evaluate the pathways and molecules involved in the neuroprotective effect of MSCs. We analyzed the viability of dopaminergic cells from different sources in response to conditioned medium derived from bone marrow MSC (MSC-CM). MSC-CM increased the viability of dopaminergic cells of rat and human origins, having both neuroprotective and neurorescue activities against effects of dopaminergic neurotoxin 6-hydroxydopamine. We found that lipid removal, inhibition of the prostaglandin E2 receptor 2 (EP2), and its signaling pathway were able to block the effects of MSC-CM on a pure population of dopaminergic neurons. Moreover, in primary mesencephalic cultures and hiPSC-derived neurons, inhibition of EP2 signaling caused a reduction in the number of dopaminergic neurons obtained in culture. Taken together, our results demonstrate for the first time the involvement of prostaglandin signaling from MSC in dopaminergic neuron survival through EP2 receptors, and suggest new approaches for treatment of Parkinson's disease.

Aminochrome induces microglia and astrocyte activation

Aminochrome has been suggested as a more physiological preclinical model capable of inducing five of the six mechanisms of Parkinson's Disease (PD). Until now, there is no evidence that aminochrome induces glial activation related to neuroinflammation, an important mechanism involved in the loss of dopaminergic neurons. In this study, the potential role of aminochrome on glial activation was studied in primary mesencephalic neuron-glia cultures and microglial primary culture from Wistar rats. We demonstrated that aminochrome induced a reduction in the number of viable cells on cultures exposed to concentration between 10 and 100μM. Moreover, aminochrome induces neuronal death determined by Fluoro-jade B. Furthermore, we demonstrated that aminochrome induced reduction in the number of TH-immunoreactive neurons and reactive gliosis, featured by morphological changes in GFAP+ and Iba1+ cells, increase in the number of OX-42+ cells and increase in the number of NF-κB p50 immunoreactive cells. These results demonstrate aminochrome neuroinflammatory ability and support the hypothesis that it may be a better PD preclinical model to find new pharmacological treatment that stop the development of this disease.

A Subpopulation of Dopaminergic Neurons Coexpresses Serotonin in Ventral Mesencephalic Cultures but not after Intrastriatal Transplantation in a Rat Model of Parkinson's Disease

Cell replacement therapy is a promising avenue into the investigation and treatment of Parkinson's disease (PD), and in some cases, significant long-term motor improvements have been demonstrated. The main source of donor tissue is the human fetal ventral mesencephalon (FVM), which consists of a mixed neuronal population, and its heterogeneity likely contributes to the inconsistent outcome observed in clinical trials. Therefore, detailed knowledge about the neuronal subpopulations in the VM seems essential for successful cell transplantation. Interestingly, it has been reported that some tyrosine hydroxylase-positive (TH+) neurons in the VM of adult rats and in cultured midbrain-derived neuroblasts coexpress additional neurotransmitters. Thus, the present study investigated, by means of colocalization analyses, the possible expression of GABA or serotonin in TH+ neurons. For that purpose, both fetal rat and human dissociated, organotypic and neurosphere FVM cultures as well as an animal model of PD were investigated. In dissociated rat FVM cultures, approximately 30% of the TH+ neurons coexpressed serotonin, while no colocalization with GABA was observed. Interestingly, coexpression of TH and serotonin was found to be dependent on the time in culture, the plating density, and the exposure to neurotrophic factors, that is, higher cell densities and treatment with brain-derived neurotrophic factor resulted in a significantly reduced coexpression rate. Notably, even though approximately 30% of the dopaminergic neurons in the donor tissue coexpressed serotonin, no colocalization could be detected in grafts 1 month after intrastriatal transplantation into hemiparkinsonian rats. In conclusion, a significant and susceptible subpopulation of dopaminergic neurons in FVM tissues coexpresses serotonin. This might have potential implications for the future selection and handling of cells prior to transplantation in PD.

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression.

In Parkinson's Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated "tagmentation" to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson's Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated "tagmentation" to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

Protective effect of Nrf2–ARE activator isolated from green perilla leaves on dopaminergic neuronal loss in a Parkinson's disease model

Parkinson's disease (PD) is a neurodegenerative disorder characterized by a selective loss of dopaminergic neurons in the substantia nigra (SN), and oxidative stress is thought to contribute to the pathogenesis. The nuclear factor erythroid 2-related factor 2 (Nrf2)–antioxidant response element (ARE) pathway, which is a cellular defense system against oxidative stress, is a promising target for therapeutics aimed at reducing neuronal death in PD. Previously, we have isolated 2′,3′-dihydroxy-4′,6′-dimethoxychalcone (DDC) from green perilla leaves as an activator of the Nrf2–ARE pathway. The present study showed the protective effect of DDC on PD models in vivo and in vitro. In a 6-hydroxydopamine (6-OHDA)-induced hemiparkinson's disease mouse model, intracerebral administration of DDC suppressed the dopaminergic neuronal loss and behavioral dysfunction. DDC upregulated the expression of heme oxygenase-1 (HO-1), one of the ARE-driven antioxidant enzymes, in astrocytes and microglia of the SN. In primary mesencephalic cultures, treatment with DDC also increased the HO-1 expression in astrocytes and microglia. DDC showed a protective effect against 6-OHDA-induced dopaminergic neuronal death, and the effect was suppressed by an HO-1 inhibitor. These results suggest that DDC prevents dopaminergic neurons from oxidative stress by upregulation of glial expression of HO-1.

Th17 Cells Induce Dopaminergic Neuronal Death via LFA-1/ICAM-1 Interaction in a Mouse Model of Parkinson's Disease.

T helper (Th)17 cells, a subset of CD4+ T lymphocytes, have strong pro-inflammatory property and appear to be essential in the pathogenesis of many inflammatory diseases. However, the involvement of Th17 cells in Parkinson's disease (PD) that is characterized by a progressive degeneration of dopaminergic (DAergic) neurons in the nigrostriatal system is unclear. Here, we aimed to demonstrate that Th17 cells infiltrate into the brain parenchyma and induce neuroinflammation and DAergic neuronal death in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- or 1-methyl-4-phenylpyridinium (MPP+)-induced PD models. Blood-brain barrier (BBB) disruption in the substantia nigra (SN) was assessed by the signal of FITC-labeled albumin that was injected into blood circulation via the ascending aorta. Live cell imaging system was used to observe a direct contact of Th17 cells with neurons by staining these cells using the two adhesion molecules, leukocyte function-associated antigen (LFA)-1 and intercellular adhesion molecule (ICAM)-1, respectively. Th17 cells invaded into the SN where BBB was disrupted in MPTP-induced PD mice. Th17 cells exacerbated DAergic neuronal loss and pro-inflammatory/neurotrophic factor disorders in MPP+-treated ventral mesencephalic (VM) cell cultures. A direct contact of LFA-1-stained Th17 cells with ICAM-1-stained VM neurons was dynamically captured. Either blocking LFA-1 in Th17 cells or blocking ICAM-1 in VM neurons with neutralizing antibodies abolished Th17-induced DAergic neuronal death. These results establish that Th17 cells infiltrate into the brain parenchyma of PD mice through lesioned BBB and exert neurotoxic property by promoting glial activation and importantly by a direct damage to neurons depending on LFA-1/ICAM-1 interaction.