Neuroprotective actions of a fatty acid nitroalkene in Parkinson's disease.

To date there are no therapeutic strategies that limit the progression of Parkinson's disease (PD). The mechanisms underlying PD-related nigrostriatal neurodegeneration remain incompletely understood, with multiple factors modulating the course of PD pathogenesis. This includes Nrf2-dependent gene expression, oxidative stress, α-synuclein pathology, mitochondrial dysfunction, and neuroinflammation. In vitro and sub-acute in vivo rotenone rat models of PD were used to evaluate the neuroprotective potential of a clinically-safe, multi-target metabolic and inflammatory modulator, the electrophilic fatty acid nitroalkene 10-nitro-oleic acid (10-NO2-OA). In N27-A dopaminergic cells and in the substantia nigra pars compacta of rats, 10-NO2-OA activated Nrf2-regulated gene expression and inhibited NOX2 and LRRK2 hyperactivation, oxidative stress, microglial activation, α-synuclein modification, and downstream mitochondrial import impairment. These data reveal broad neuroprotective actions of 10-NO2-OA in a sub-acute model of PD and motivate more chronic studies in rodents and primates.

The Parkinson's disease protein alpha-synuclein is a modulator of processing bodies and mRNA stability.

Alpha-synuclein (αS) is a conformationally plastic protein that reversibly binds to cellular membranes. It aggregates and is genetically linked to Parkinson's disease (PD). Here, we show that αS directly modulates processing bodies (P-bodies), membraneless organelles that function in mRNA turnover and storage. The N terminus of αS, but not other synucleins, dictates mutually exclusive binding either to cellular membranes or to P-bodies in the cytosol. αS associates with multiple decapping proteins in close proximity on the Edc4 scaffold. As αS pathologically accumulates, aberrant interaction with Edc4 occurs at the expense of physiologic decapping-module interactions. mRNA decay kinetics within PD-relevant pathways are correspondingly disrupted in PD patient neurons and brain. Genetic modulation of P-body components alters αS toxicity, and human genetic analysis lends support to the disease-relevance of these interactions. Beyond revealing an unexpected aspect of αS function and pathology, our data highlight the versatility of conformationally plastic proteins with high intrinsic disorder.

NADPH oxidase 2 activity in Parkinson's disease.

Mitochondrial dysfunction and oxidative stress are strongly implicated in Parkinson's disease (PD) pathogenesis and there is evidence that mitochondrially-generated superoxide can activate NADPH oxidase 2 (NOX2). Although NOX2 has been examined in the context of PD, most attention has focused on glial NOX2, and the role of neuronal NOX2 in PD remains to be defined. Additionally, pharmacological NOX2 inhibitors have typically lacked specificity. Here we devised and validated a proximity ligation assay for NOX2 activity and demonstrated that in human PD and two animal models thereof, both neuronal and microglial NOX2 are highly active in substantia nigra under chronic conditions. However, in acute and sub-acute PD models, we observed neuronal, but not microglial NOX2 activation, suggesting that neuronal NOX2 may play a primary role in the early stages of the disease. Aberrant NOX2 activity is responsible for the formation of oxidative stress-related post-translational modifications of α-synuclein, and impaired mitochondrial protein import in vitro in primary ventral midbrain neuronal cultures and in vivo in nigrostriatal neurons in rats. In a rat model, administration of a brain-penetrant, highly specific NOX2 inhibitor prevented NOX2 activation in nigrostriatal neurons and its downstream effects in vivo, such as activation of leucine-rich repeat kinase 2 (LRRK2). We conclude that NOX2 is an important enzyme that contributes to progressive oxidative damage which in turn can lead to α-synuclein accumulation, mitochondrial protein import impairment, and LRRK2 activation. In this context, NOX2 inhibitors hold potential as a disease-modifying therapy in PD.

LRRK2 and idiopathic Parkinson's disease.

The etiology of idiopathic Parkinson's disease (iPD) is multifactorial, and both genetics and environmental exposures are risk factors. While mutations in leucine-rich repeat kinase-2 (LRRK2) that are associated with increased kinase activity are the most common cause of autosomal dominant PD, the role of LRRK2 in iPD, independent of mutations, remains uncertain. In this review, we discuss how the architecture of LRRK2 influences kinase activation and how enhanced LRRK2 substrate phosphorylation might contribute to pathogenesis. We describe how oxidative stress and endolysosomal dysfunction, both of which occur in iPD, can activate non-mutated LRRK2 to a similar degree as pathogenic mutations. Similarly, environmental toxicants that are linked epidemiologically to iPD risk can also activate LRRK2. In aggregate, current evidence suggests an important role for LRRK2 in iPD.

Measurement of LRRK2 Kinase Activity by Proximity Ligation Assay.

Missense mutations in leucine rich-repeat kinase 2 (LRRK2) cause forms of familial Parkinson's disease and have been linked to 'idiopathic' Parkinson's disease. Assessment of LRRK2 kinase activity has been very challenging due to its size, complex structure, and relatively low abundance. A standard in the field to assess LRRK2 kinase activity is to measure the level of substrate phosphorylation (pThr73-Rab10) or autophosphorylation of serine 1292 (i.e., phosphoserine 1292; pS1292). The levels of pS1292 have typically been assessed by western blotting, which limits cellular and anatomical resolution. Here, we describe the method for a novel proximity ligation assay (PLA) that can detect endogenous LRRK2 kinase activity (PLA LRRK2) in situ at cellular and subcellular resolutions. PLA is a fluorescence- or chromogen-based assay that can be used to either (1) detect protein-protein interactions or (2) detect and amplify post-translational modifications on proteins. We used PLA for in situ detection and amplification of LRRK2 autophosphorylation levels at serine 1292. Our findings demonstrate that PLA LRRK2 is a highly sensitive and specific assay that can be used for assessing kinase activity in cultured cells and postmortem tissues.

Selective Fatty Acid Amide Hydrolase Inhibitors as Potential Novel Antiepileptic Agents.

Temporal lobe epilepsy is the most common form of epilepsy, and current antiepileptic drugs are ineffective in many patients. The endocannabinoid system has been associated with an on-demand protective response to seizures. Blocking endocannabinoid catabolism would elicit antiepileptic effects, devoid of psychotropic effects. We herein report the discovery of selective anandamide catabolic enzyme fatty acid amide hydrolase (FAAH) inhibitors with promising antiepileptic efficacy, starting from a further investigation of our prototypical inhibitor 2a. When tested in two rodent models of epilepsy, 2a reduced the severity of the pilocarpine-induced status epilepticus and the elongation of the hippocampal maximal dentate activation. Notably, 2a did not affect hippocampal dentate gyrus long-term synaptic plasticity. These data prompted our further endeavor aiming at discovering new antiepileptic agents, developing a new set of FAAH inhibitors (3a-m). Biological studies highlighted 3h and 3m as the best performing analogues to be further investigated. In cell-based studies, using a neuroblastoma cell line, 3h and 3m could reduce the oxinflammation state by decreasing DNA-binding activity of NF-kB p65, devoid of cytotoxic effect. Unwanted cardiac effects were excluded for 3h (Langendorff perfused rat heart). Finally, the new analogue 3h reduced the severity of the pilocarpine-induced status epilepticus as observed for 2a.

α-Synuclein amplifies cytoplasmic peroxide flux and oxidative stress provoked by mitochondrial inhibitors in CNS dopaminergic neurons in vivo.

Convergent evidence implicates impaired mitochondrial function and α-Synuclein accumulation as critical upstream events in the pathogenesis of Parkinson's disease, but comparatively little is known about how these factors interact to provoke neurodegeneration. We previously showed that α-Synuclein knockdown protected rat substantia nigra dopaminergic neurons from systemic exposure to the mitochondrial complex I inhibitor rotenone. Here we show that motor abnormalities prior to neuronal loss in this model are associated with extensive α-Synuclein-dependent cellular thiol oxidation. In order to elucidate the underlying events in vivo we constructed novel transgenic zebrafish that co-express, in dopaminergic neurons: (i) human α-Synuclein at levels insufficient to provoke neurodegeneration or neurobehavioral abnormalities; and (ii) genetically-encoded ratiometric fluorescent biosensors to detect cytoplasmic peroxide flux and glutathione oxidation. Live intravital imaging of the intact zebrafish CNS at cellular resolution showed unequivocally that α-Synuclein amplified dynamic cytoplasmic peroxide flux in dopaminergic neurons following exposure to the mitochondrial complex I inhibitors MPP+ or rotenone. This effect was robust and clearly evident following either acute or prolonged exposure to each inhibitor. In addition, disturbance of the resting glutathione redox potential following exogenous hydrogen peroxide challenge was augmented by α-Synuclein. Together these data show that α-Synuclein is a critical determinant of the redox consequences of mitochondrial dysfunction in dopaminergic neurons. The findings are important because the mechanisms underlying α-Synuclein-dependent reactive oxygen species fluxes and antioxidant suppression might provide a pharmacological target in Parkinson's disease to prevent progression from mitochondrial dysfunction and oxidative stress to cell death.

LRRK2 inhibition prevents endolysosomal deficits seen in human Parkinson's disease.

LRRK2 has been implicated in endolysosomal function and likely plays a central role in idiopathic Parkinson's disease (iPD). In iPD, dopaminergic neurons within the substantia nigra are characterized by increased LRRK2 kinase activity, endolysosomal deficits, and accumulation of autophagic vesicles with incompletely degraded substrates, including α-synuclein. Although LRRK2 has been implicated in endolysosomal and autophagic function, it remains unclear whether inhibition of LRRK2 kinase activity can prevent endolysosomal deficits or reduce dopaminergic neurodegeneration. In this study, we characterized the endolysosomal and autophagic defects in surviving dopaminergic neurons of iPD patient brain tissue. We next showed that these defects could be reproduced reliably in vivo using the rotenone model of iPD. Results suggested that there was impaired endosomal maturation, resulting in lysosomal dysfunction and deficits in protein degradation. A highly selective, brain-penetrant LRRK2 kinase inhibitor not only improved apparent endosomal maturation and lysosomal function, but also prevented rotenone-induced neurodegeneration in vivo. The fact that a LRRK2 kinase inhibitor was capable of preventing the neuropathological and endolysosomal abnormalities observed in human iPD suggests that LRRK2 inhibitors may have broad therapeutic utility in iPD, not only in those who carry a LRRK2 mutation.

WIN 55,212-2 Reverted Pilocarpine-Induced Status Epilepticus Early Changes of the Interaction among 5-HT2C/NMDA/CB1 Receptors in the Rat Hippocampus.

The molecular basis for temporal lobe epileptogenesis  remains poorly defined. Recent evidence shows that serotonin 2C receptors (5-HT2CRs), NR2A and NR2B subunit-containing N-methyl-d-aspartate receptors (NMDARs) and cannabinoid 1 receptors (CB1Rs) may be involved in the progression of the epileptic disorders. Moreover, CB1R activation has been reported to modulate the activity of 5-HT2C and NMDA receptors. Here, we treated Sprague-Dawley rats with the pro-convulsant agent pilocarpine (PILO) to induce status epilepticus (SE) in order to study the effect, with regards to receptor signaling and their interactions, of the preactivation of the CB1Rs on early changes that occur 24 h from the initial insult in the hippocampus. Pretreatment with the synthetic CB1/2R agonist WIN 55,212-2 (2 mg/kg, ip) counteracted PILO-induced 5-HT2CR downregulation. Moreover, WIN 55,212-2 uncoupled PILO-induced 5-HT2CR/NR2A and prevented NR2ATyr1325 phosphorylation indirectly since no 5-HT2CR/CB1R interactions were observed. WIN 55,212-2 treatment also prevented PILO-mediated impairment of CB1R/NR2B interactions and NR2B subunit internalization, suggesting a possible role of CB1R in NR2B-containing NMDAR turn over. All the effects observed in animals treated with WIN 55,212-2 were blocked by pretreatment with the selective CB1R antagonist AM251 (1 mg/kg, ip) given 45 min before PILO injection. These results, obtained in vivo in post-PILO-induced SE, provide new insights on the early cellular responses during epileptogenesis and identify new CB1R-mediated mechanisms by which cannabinoids may prevent the development of chronic epilepsy.

Synergistic action of CB1 and 5-HT2B receptors in preventing pilocarpine-induced status epilepticus in rats.

Endocannabinoids (eCBs) and serotonin (5-HT) play a neuromodulatory role in the central nervous system. Both eCBs and 5-HT regulate neuronal excitability and their pharmacological potentiation has been shown to control seizures in pre-clinical and human studies. Compelling evidence indicates that eCB and 5-HT systems interact to modulate several physiological and pathological brain functions, such as food intake, pain, drug addiction, depression, and anxiety. Nevertheless, there is no evidence of an eCB/5-HT interaction in experimental and human epilepsies, including status epilepticus (SE). Here, we performed video-EEG recording in behaving rats treated with the pro-convulsant agent pilocarpine (PILO), in order to study the effect of the activation of CB1/5-HT2 receptors and their interaction on SE. Synthetic cannabinoid agonist WIN55,212-2 (WIN) decreased behavioral seizure severity of PILO-induced SE at 2 mg/kg (but not at 1 and 5 mg/kg, i.p.), while 5-HT2B/2C receptor agonist RO60-0175 (RO; 1, 3, 10 mg/kg, i.p.) was devoid of any effect. RO 3 mg/kg was instead capable of potentiating the effect of WIN 2 mg/kg on the Racine scale score. Surprisingly, neither WIN 2 mg/kg nor RO 3 mg/kg had any effect on the incidence and the intensity of EEG seizures when administered alone. However, WIN+RO co-administration reduced the incidence and the severity of EEG SE and increased the latency to SE onset after PILO injection. WIN+RO effects were blocked by the selective CB1R antagonist AM251 and the 5-HT2BR antagonist RS127445, but not by the 5-HT2CR antagonist SB242084 or the 5-HT2AR antagonist MDL11,939. These data revealed a synergistic interaction between CB1R/5-HT2BR in the expression of PILO-induced SE.

LRRK2 activation in idiopathic Parkinson's disease.

Missense mutations in leucine-rich repeat kinase 2 (LRRK2) cause familial Parkinson's disease (PD). However, a potential role of wild-type LRRK2 in idiopathic PD (iPD) remains unclear. Here, we developed proximity ligation assays to assess Ser1292 phosphorylation of LRRK2 and, separately, the dissociation of 14-3-3 proteins from LRRK2. Using these proximity ligation assays, we show that wild-type LRRK2 kinase activity was selectively enhanced in substantia nigra dopamine neurons in postmortem brain tissue from patients with iPD and in two different rat models of the disease. We show that this occurred through an oxidative mechanism, resulting in phosphorylation of the LRRK2 substrate Rab10 and other downstream consequences including abnormalities in mitochondrial protein import and lysosomal function. Our study suggests that, independent of mutations, wild-type LRRK2 plays a role in iPD. LRRK2 kinase inhibitors may therefore be useful for treating patients with iPD who do not carry LRRK2 mutations.

Generation of three-dimensional human neuronal cultures: application to modeling CNS viral infections.

BACKGROUND: A variety of neurological disorders including neurodegenerative diseases and infection by neurotropic viruses can cause structural and functional changes in the central nervous system (CNS), resulting in long-term neurological sequelae. An improved understanding of the pathogenesis of these disorders is important for developing efficacious interventions. Human induced pluripotent stem cells (hiPSCs) offer an extraordinary window for modeling pathogen-CNS interactions, and other cellular interactions, in three-dimensional (3D) neuronal cultures that can recapitulate several aspects of in vivo brain tissue. METHODS: Herein, we describe a prototype of scaffold-free hiPSC-based adherent 3D (A-3D) human neuronal cultures in 96-well plates. To test their suitability for drug screening, A-3D neuronal cultures were infected with herpes simplex virus type 1 (HSV-1) with or without acyclovir. RESULTS: The half maximal inhibitory concentration (IC50) of acyclovir was 3.14 µM and 3.12 µM determined using flow cytometry and the CX7 High Content Screening platform, respectively. CONCLUSIONS: Our A-3D neuronal cultures provide an unprecedented opportunity for high-content drug screening programs to treat human CNS infections.

Preferential modulation of the lateral habenula activity by serotonin-2A rather than -2C receptors: Electrophysiological and neuroanatomical evidence.

AIMS: Serotonergic (5-HT) modulation of the lateral habenula (LHb) activity is central in normal and pathologic conditions such as mood disorders. Among the multiple 5-HT receptors (5-HTRs) involved, the 5-HT2C R seems to play a pivotal role. Yet, the role of 5-HT2A Rs in the control of the LHb neuronal activity is completely unknown. METHODS: Single-cell extracellular recording of the LHb neurons was used in rats to study the effect of the general activation and blockade of the 5-HT2C R and 5-HT2A R with Ro 60-0175 and SB242084, TCB-2 and MDL11939, respectively. The expression of both receptors in the LHb was confirmed using immunohistochemistry. RESULTS: Cumulative doses (5-640 µg/kg, iv) of Ro 60-0175 and TCB-2 affected the activity of 34% and 63% of the LHb recorded neurons, respectively. LHb neurons were either inhibited at low doses or excited at higher doses of the 5-HT2A/C R agonists. SB242084 or MDL11939 (both at 200 µg/kg, iv) did not modify neuronal firing when injected alone, but reverted the bidirectional effects of Ro 60-0175 or TCB-2, respectively. 5-HT2C Rs and 5-HT2A Rs are expressed in less than the 20% of the LHb neurons, and they neither colocalize nor make heterodimers. Strikingly, only 5-HT2A Rs are expressed by the majority of LHb astrocyte cells. CONCLUSIONS: Peripheral administration of 5-HT2A R agonist promotes a heterogeneous pattern of neuronal responses in the LHb, and these effects are more prominent than those induced by the 5-HT2C R activation.

α-Synuclein binds to TOM20 and inhibits mitochondrial protein import in Parkinson's disease.

α-Synuclein accumulation and mitochondrial dysfunction have both been strongly implicated in the pathogenesis of Parkinson's disease (PD), and the two appear to be related. Mitochondrial dysfunction leads to accumulation and oligomerization of α-synuclein, and increased levels of α-synuclein cause mitochondrial impairment, but the basis for this bidirectional interaction remains obscure. We now report that certain posttranslationally modified species of α-synuclein bind with high affinity to the TOM20 (translocase of the outer membrane 20) presequence receptor of the mitochondrial protein import machinery. This binding prevented the interaction of TOM20 with its co-receptor, TOM22, and impaired mitochondrial protein import. Consequently, there were deficient mitochondrial respiration, enhanced production of reactive oxygen species, and loss of mitochondrial membrane potential. Examination of postmortem brain tissue from PD patients revealed an aberrant α-synuclein-TOM20 interaction in nigrostriatal dopaminergic neurons that was associated with loss of imported mitochondrial proteins, thereby confirming this pathogenic process in the human disease. Modest knockdown of endogenous α-synuclein was sufficient to maintain mitochondrial protein import in an in vivo model of PD. Furthermore, in in vitro systems, overexpression of TOM20 or a mitochondrial targeting signal peptide had beneficial effects and preserved mitochondrial protein import. This study characterizes a pathogenic mechanism in PD, identifies toxic species of wild-type α-synuclein, and reveals potential new therapeutic strategies for neuroprotection.

Zn(2+) -induced Ca(2+) release via ryanodine receptors triggers calcineurin-dependent redistribution of cortical neuronal Kv2.1 K(+) channels.

KEY POINTS: Increases in intracellular Zn(2+) concentrations are an early, necessary signal for the modulation of Kv2.1 K(+) channel localization and physiological function. Intracellular Zn(2+) -mediated Kv2.1 channel modulation is dependent on calcineurin, a Ca(2+) -activated phosphatase. We show that intracellular Zn(2+) induces a significant increase in ryanodine receptor-dependent cytosolic Ca(2+) transients, which leads to a calcineurin-dependent redistribution of Kv2.1 channels from pre-existing membrane clusters to diffuse localization. As such, the link between Zn(2+) and Ca(2+) signalling in this Kv2.1 modulatory pathway is established. We observe that a sublethal ischaemic preconditioning insult also leads to Kv2.1 redistribution in a ryanodine receptor-dependent fashion. We suggest that Zn(2+) may be an early and ubiquitous signalling molecule mediating Ca(2+) release from the cortical endoplasmic reticulum via ryanodine receptor activation. ABSTRACT: Sublethal injurious stimuli in neurons induce transient increases in free intracellular Zn(2+) that are associated with regulating adaptive responses to subsequent lethal injury, including alterations in the function and localization of the delayed-rectifier potassium channel, Kv2.1. However, the link between intracellular Zn(2+) signalling and the observed changes in Kv2.1 remain undefined. In the present study, utilizing exogenous Zn(2+) treatment, along with a selective Zn(2+) ionophore, we show that transient elevations in intracellular Zn(2+) concentrations are sufficient to induce calcineurin-dependent Kv2.1 channel dispersal in rat cortical neurons in vitro, which is accompanied by a relatively small but significant hyperpolarizing shift in the voltage-gated activation kinetics of the channel. Critically, using a molecularly encoded calcium sensor, we found that the calcineurin-dependent changes in Kv2.1 probably occur as a result of Zn(2+) -induced cytosolic Ca(2+) release via activation of neuronal ryanodine receptors. Finally, we couple this mechanism with an established model for in vitro ischaemic preconditioning and show that Kv2.1 channel modulation in this process is also ryanodine receptor-sensitive. Our results strongly suggest that intracellular Zn(2+) -initiated signalling may represent an early and possibly widespread component of Ca(2+) -dependent processes in neurons.

Post-status epilepticus treatment with the cannabinoid agonist WIN 55,212-2 prevents chronic epileptic hippocampal damage in rats.

Repeated seizures are often associated with development of refractory chronic epilepsy, the most common form of which is temporal lobe epilepsy. G-protein-coupled cannabinoid receptors (CB1 and CB2 receptors) regulate neuronal excitability and have been shown to mediate acute anticonvulsant effects of cannabinoids in animal models. However, the potential of cannabinoids to prevent chronic neuronal damage and development of epilepsy remains unexplored. We hypothesized that treatment with a CB receptor agonist after an episode of status epilepticus--but before development of spontaneous recurrent seizures--might prevent the development of functional changes that lead to chronic epilepsy. Using the rat pilocarpine model, a therapeutic approach was simulated by administering the CB agonist, WIN 55,212-2 after an episode of status epilepticus. Epileptic behavior was monitored during development of spontaneous recurrent seizures for up to 6 months. Histology, neurochemistry, redox status and NMDA receptor subunit expression were assessed at 6 months after pilocarpine-induced seizures. Sub-acute treatment with WIN 55,212-2 (for 15 days starting 24h after PILO injection) dramatically attenuated the severity, duration and frequency of spontaneous recurrent seizures. Further, in contrast to vehicle-treated animals, hippocampi from WIN 55,212-2-treated animals showed: normal thiol redox state, normal NR2A and NR2B subunit expression, preservation of GABAergic neurons and prevention of abnormal proliferation of GABAergic progenitors. This study shows for the first time that, after a known inciting event, treatment with a compound targeting CB receptors has the potential to prevent the epileptogenic events that result in chronic epileptic damage.

Behavioral, neurochemical, and pathologic alterations in bacterial artificial chromosome transgenic G2019S leucine-rich repeated kinase 2 rats.

Mutations in leucine-rich repeated kinase 2 (LRRK2) cause autosomal dominant late-onset Parkinson's disease (PD), and the G2019S mutation in the kinase domain of LRRK2 is the most common genetic cause of familial PD. Enhanced kinase activity of G2019S LRRK2 is a suspected mechanism for carriers to develop PD but pathophysiological function of G2019S LRRK2 is not clear. The objective of the present study was to characterize a bacterial artificial chromosome rat expressing human G2019S LRRK2. Immunoblotting analysis showed that G2019S LRRK2 expression was approximately 5-8 times higher than wild-type rat LRRK2. At ages of 4, 8, and 12 months, our characterization showed that expression of G2019S LRRK2 induced oxidative stress in striatum and substantia nigra, increased inducible nitric oxide synthase expression in nigral dopamine neurons, and abnormal morphology of nigral dopaminergic neurons in transgenic rats compared with wild-type, without inducing overt neurodegeneration in nigrostriatal dopaminergic neurons. Thus, we conclude that although this model does not reproduce the key features of end-stage PD, important preclinical features of the disease are evident, which may be useful in studying the earliest stages of PD and for gene-environment interaction studies.

Transient muscarinic and glutamatergic stimulation of neural stem cells triggers acute and persistent changes in differentiation.

While aberrant cell proliferation and differentiation may contribute to epileptogenesis, the mechanisms linking an initial epileptic insult to subsequent changes in cell fate remain elusive. Using both mouse and human iPSC-derived neural progenitor/stem cells (NPSCs), we found that a combined transient muscarinic and mGluR1 stimulation inhibited overall neurogenesis but enhanced NPSC differentiation into immature GABAergic cells. If treated NPSCs were further passaged, they retained a nearly identical phenotype upon differentiation. A similar profusion of immature GABAergic cells was seen in rats with pilocarpine-induced chronic epilepsy. Furthermore, live cell imaging revealed abnormal de-synchrony of Ca(++) transients and altered gap junction intercellular communication following combined muscarinic/glutamatergic stimulation, which was associated with either acute site-specific dephosphorylation of connexin 43 or a long-term enhancement of its degradation. Therefore, epileptogenic stimuli can trigger acute and persistent changes in cell fate by altering distinct mechanisms that function to maintain appropriate intercellular communication between coupled NPSCs.

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation.

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases.