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1.
Brain ; 137(Pt 8): 2287-302, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24934288

RESUMO

Dopamine midbrain neurons within the substantia nigra are particularly prone to degeneration in Parkinson's disease. Their selective loss causes the major motor symptoms of Parkinson's disease, but the causes for the high vulnerability of SN DA neurons, compared to neighbouring, more resistant ventral tegmental area dopamine neurons, are still unclear. Consequently, there is still no cure available for Parkinson's disease. Current therapies compensate the progressive loss of dopamine by administering its precursor l-DOPA and/or dopamine D2-receptor agonists. D2-autoreceptors and Cav1.3-containing L-type Ca(2+) channels both contribute to Parkinson's disease pathology. L-type Ca(2+) channel blockers protect SN DA neurons from degeneration in Parkinson's disease and its mouse models, and they are in clinical trials for neuroprotective Parkinson's disease therapy. However, their physiological functions in SN DA neurons remain unclear. D2-autoreceptors tune firing rates and dopamine release of SN DA neurons in a negative feedback loop through activation of G-protein coupled potassium channels (GIRK2, or KCNJ6). Mature SN DA neurons display prominent, non-desensitizing somatodendritic D2-autoreceptor responses that show pronounced desensitization in PARK-gene Parkinson's disease mouse models. We analysed surviving human SN DA neurons from patients with Parkinson's disease and from controls, and detected elevated messenger RNA levels of D2-autoreceptors and GIRK2 in Parkinson's disease. By electrophysiological analysis of postnatal juvenile and adult mouse SN DA neurons in in vitro brain-slices, we observed that D2-autoreceptor desensitization is reduced with postnatal maturation. Furthermore, a transient high-dopamine state in vivo, caused by one injection of either l-DOPA or cocaine, induced adult-like, non-desensitizing D2-autoreceptor responses, selectively in juvenile SN DA neurons, but not ventral tegmental area dopamine neurons. With pharmacological and genetic tools, we identified that the expression of this sensitized D2-autoreceptor phenotype required Cav1.3 L-type Ca(2+) channel activity, internal Ca(2+), and the interaction of the neuronal calcium sensor NCS-1 with D2-autoreceptors. Thus, we identified a first physiological function of Cav1.3 L-type Ca(2+) channels in SN DA neurons for homeostatic modulation of their D2-autoreceptor responses. L-type Ca(2+) channel activity however, was not important for pacemaker activity of mouse SN DA neurons. Furthermore, we detected elevated substantia nigra dopamine messenger RNA levels of NCS-1 (but not Cav1.2 or Cav1.3) after cocaine in mice, as well as in remaining human SN DA neurons in Parkinson's disease. Thus, our findings provide a novel homeostatic functional link in SN DA neurons between Cav1.3- L-type-Ca(2+) channels and D2-autoreceptor activity, controlled by NCS-1, and indicate that this adaptive signalling network (Cav1.3/NCS-1/D2/GIRK2) is also active in human SN DA neurons, and contributes to Parkinson's disease pathology. As it is accessible to pharmacological modulation, it provides a novel promising target for tuning substantia nigra dopamine neuron activity, and their vulnerability to degeneration.


Assuntos
Autorreceptores/metabolismo , Canais de Cálcio Tipo L/fisiologia , Neurônios Dopaminérgicos/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Proteínas Sensoras de Cálcio Neuronal/fisiologia , Neuropeptídeos/fisiologia , Doença de Parkinson/metabolismo , Substância Negra/metabolismo , Área Tegmentar Ventral/metabolismo , Animais , Sinalização do Cálcio/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/patologia , Receptores de Dopamina D2/metabolismo , Substância Negra/citologia , Substância Negra/patologia , Área Tegmentar Ventral/citologia , Área Tegmentar Ventral/patologia
2.
J Pharmacol Toxicol Methods ; 128: 107529, 2024 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-38857637

RESUMO

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) have found utility for conducting in vitro drug screening and disease modelling to gain crucial insights into pharmacology or disease phenotype. However, diseases such as atrial fibrillation, affecting >33 M people worldwide, demonstrate the need for cardiac subtype-specific cells. Here, we sought to investigate the base characteristics and pharmacological differences between commercially available chamber-specific atrial or ventricular hiPSC-CMs seeded onto ultra-thin, flexible PDMS membranes to simultaneously measure contractility in a 96 multi-well format. We investigated the effects of GPCR agonists (acetylcholine and carbachol), a Ca2+ channel agonist (S-Bay K8644), an HCN channel antagonist (ivabradine) and K+ channel antagonists (4-AP and vernakalant). We observed differential effects between atrial and ventricular hiPSC-CMs on contractile properties including beat rate, beat duration, contractile force and evidence of arrhythmias at a range of concentrations. As an excerpt of the compound analysis, S-Bay K8644 treatment showed an induced concentration-dependent transient increase in beat duration of atrial hiPSC-CMs, whereas ventricular cells showed a physiological increase in beat rate over time. Carbachol treatment produced marked effects on atrial cells, such as increased beat duration alongside a decrease in beat rate over time, but only minimal effects on ventricular cardiomyocytes. In the context of this chamber-specific pharmacology, we not only add to contractile characterization of hiPSC-CMs but propose a multi-well platform for medium-throughput early compound screening. Overall, these insights illustrate the key pharmacological differences between chamber-specific cardiomyocytes and their application on a multi-well contractility platform to gain insights for in vitro cardiac liability studies and disease modelling.

3.
J Vis Exp ; (188)2022 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-36342136

RESUMO

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold promise to serve as a human-relevant model in preclinical phases of drug discovery and safety pharmacology, their maturity is still controversial in the scientific community and under constant development. We present a hybrid contractility and impedance/extracellular field potential (EFP) technology, adding significant pro-maturation features to an industry-standard 96-well platform. The impedance/EFP system monitors cellular functionality in real-time. Besides the beat rate of contractile cells, the electrical impedance spectroscopy readouts detect compound-induced morphological changes like cell density and integrity of the cellular monolayer. In the other component of the hybrid cell analysis system, the cells are cultured on bio-compliant membranes that mimic the mechanical environment of real heart tissue. This physiological environment supports the maturation of hiPSC-CMs in vitro, leading to more adult-like contractile responses including positive inotropic effects after treatment with isoproterenol, S-Bay K8644, or omecamtiv mecarbil. Parameters such as the amplitude of contraction force (mN/mm2) and beat duration also reveal downstream effects of compounds with influence on electrophysiological properties and calcium handling. The hybrid system provides the ideal tool for holistic cell analysis, allowing preclinical cardiac risk assessment beyond the current perspectives of human-relevant cell-based assays.


Assuntos
Células-Tronco Pluripotentes Induzidas , Adulto , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Contração Miocárdica , Fenômenos Eletrofisiológicos , Células Híbridas , Células Cultivadas
4.
J Pharmacol Toxicol Methods ; 105: 106892, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32629160

RESUMO

INDUCTION: Despite increasing acceptance of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in safety pharmacology, controversy remains about the physiological relevance of existing in vitro models for their mechanical testing. We hypothesize that existing signs of immaturity of the cell models result from an improper mechanical environment. With the presented study, we aimed at validating the newly developed FLEXcyte96 technology with respect to physiological responses of hiPSC-CMs to pharmacological compounds with known inotropic and/or cardiotoxic effects. METHODS: hiPSC-CMs were cultured in a 96-well format on hyperelastic silicone membranes imitating their native mechanical environment. Cardiomyocyte contractility was measured contact-free by application of capacitive displacement sensing of the cell-membrane biohybrids. Acute effects of positive inotropic compounds with distinct mechanisms of action were examined. Additionally, cardiotoxic effects of tyrosine kinase inhibitors and anthracyclines were repetitively examined during repeated exposure to drug concentrations for up to 5 days. RESULTS: hiPSC-CMs grown on biomimetic membranes displayed increased contractility responses to isoproterenol, S-Bay K8644 and omecamtiv mecarbil without the need for additional stimulation. Tyrosine kinase inhibitor erlotinib, vandetanib, nilotinib, gefitinib, A-674563 as well as anthracycline idarubicin showed the expected cardiotoxic effects, including negative inotropy and induction of proarrhythmic events. DISCUSSION: We conclude that the FLEXcyte 96 system is a reliable high throughput tool for invitro cardiac contractility research, providing the user with data obtained under physiological conditions which resemble the native environment of human heart tissue. We showed that the results obtained for both acute and sub-chronic compound administration are consistent with the respective physiological responses in humans.


Assuntos
Cardiotoxicidade/diagnóstico , Ensaios de Triagem em Larga Escala/métodos , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Antraciclinas/efeitos adversos , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Inibidores de Proteínas Quinases/efeitos adversos
5.
EBioMedicine ; 39: 401-408, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30503201

RESUMO

BACKGROUND: Small fiber neuropathy (SFN) is a severe and disabling chronic pain syndrome with no causal and limited symptomatic treatment options. Mechanistically based individual treatment is not available. We report an in-vitro predicted individualized treatment success in one therapy-refractory Caucasian patient suffering from SFN for over ten years. METHODS: Intrinsic excitability of human induced pluripotent stem cell (iPSC) derived nociceptors from this patient and respective controls were recorded on multi-electrode (MEA) arrays, in the presence and absence of lacosamide. The patient's pain ratings were assessed by a visual analogue scale (10: worst pain, 0: no pain) and treatment effect was objectified by microneurography recordings of the patient's single nerve C-fibers. FINDINGS: We identified patient-specific changes in iPSC-derived nociceptor excitability in MEA recordings, which were reverted by the FDA-approved compound lacosamide in vitro. Using this drug for individualized treatment of this patient, the patient's pain ratings decreased from 7.5 to 1.5. Consistent with the pain relief reported by the patient, microneurography recordings of the patient's single nerve fibers mirrored a reduced spontaneous nociceptor (C-fiber) activity in the patient during lacosamide treatment. Microneurography recordings yielded an objective measurement of altered peripheral nociceptor activity following treatment. INTERPRETATION: Thus, we are here presenting one example of successful patient specific precision medicine using iPSC technology and individualized therapeutic treatment based on patient-derived sensory neurons.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Lacosamida/administração & dosagem , Nociceptores/citologia , Neuropatia de Pequenas Fibras/tratamento farmacológico , Idoso , Células Cultivadas , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Lacosamida/farmacologia , Modelos Biológicos , Nociceptores/efeitos dos fármacos , Medição da Dor , Medicina de Precisão , Pesquisa Translacional Biomédica
6.
Nat Commun ; 10(1): 5094, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31704946

RESUMO

Degeneration of dopaminergic neurons in the substantia nigra causes the motor symptoms of Parkinson's disease. The mechanisms underlying this age-dependent and region-selective neurodegeneration remain unclear. Here we identify Cav2.3 channels as regulators of nigral neuronal viability. Cav2.3 transcripts were more abundant than other voltage-gated Ca2+ channels in mouse nigral neurons and upregulated during aging. Plasmalemmal Cav2.3 protein was higher than in dopaminergic neurons of the ventral tegmental area, which do not degenerate in Parkinson's disease. Cav2.3 knockout reduced activity-associated nigral somatic Ca2+ signals and Ca2+-dependent after-hyperpolarizations, and afforded full protection from degeneration in vivo in a neurotoxin Parkinson's mouse model. Cav2.3 deficiency upregulated transcripts for NCS-1, a Ca2+-binding protein implicated in neuroprotection. Conversely, NCS-1 knockout exacerbated nigral neurodegeneration and downregulated Cav2.3. Moreover, NCS-1 levels were reduced in a human iPSC-model of familial Parkinson's. Thus, Cav2.3 and NCS-1 may constitute potential therapeutic targets for combatting Ca2+-dependent neurodegeneration in Parkinson's disease.


Assuntos
Envelhecimento/genética , Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/genética , Sobrevivência Celular/genética , Neurônios Dopaminérgicos/metabolismo , Proteínas Sensoras de Cálcio Neuronal/genética , Neuropeptídeos/genética , Doença de Parkinson/genética , Envelhecimento/metabolismo , Animais , Canais de Cálcio Tipo R/metabolismo , Sinalização do Cálcio , Proteínas de Transporte de Cátions/metabolismo , Neurônios Dopaminérgicos/patologia , Humanos , Células-Tronco Pluripotentes Induzidas , Camundongos , Camundongos Knockout , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Neuropeptídeos/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Substância Negra/metabolismo , Substância Negra/patologia , Regulação para Cima , Área Tegmentar Ventral/metabolismo , Área Tegmentar Ventral/patologia
7.
Artigo em Inglês | MEDLINE | ID: mdl-29355722

RESUMO

Safety pharmacology studies that evaluate drug candidates for potential cardiovascular liabilities remain a critical component of drug development. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have recently emerged as a new and promising tool for preclinical hazard identification and risk assessment of drugs. Recently, Pluriomics organized its first User Meeting entitled 'Combining Pluricyte® Cardiomyocytes & MEA for Safety Pharmacology applications', consisting of scientific sessions and live demonstrations, which provided the opportunity to discuss the application of hiPSC-CMs (Pluricyte® Cardiomyocytes) in cardiac safety assessment to support early decision making in safety pharmacology. This report summarizes the outline and outcome of this Pluriomics User Meeting, which took place on November 24-25, 2016 in Leiden (The Netherlands). To reflect the content of the communications presented at this meeting we have cited key scientific articles and reviews.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Miócitos Cardíacos/efeitos dos fármacos , Cardiotoxicidade/prevenção & controle , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/instrumentação , Avaliação Pré-Clínica de Medicamentos/normas , Eletrodos , Guias como Assunto , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Técnicas de Patch-Clamp/instrumentação , Técnicas de Patch-Clamp/métodos
8.
Sci Rep ; 5: 13688, 2015 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-26381090

RESUMO

The preferential degeneration of Substantia nigra dopamine midbrain neurons (SN DA) causes the motor-symptoms of Parkinson's disease (PD). Voltage-gated L-type calcium channels (LTCCs), especially the Cav1.3-subtype, generate an activity-related oscillatory Ca(2+) burden in SN DA neurons, contributing to their degeneration and PD. While LTCC-blockers are already in clinical trials as PD-therapy, age-dependent functional roles of Cav1.3 LTCCs in SN DA neurons remain unclear. Thus, we analysed juvenile and adult Cav1.3-deficient mice with electrophysiological and molecular techniques. To unmask compensatory effects, we compared Cav1.3 KO mice with pharmacological LTCC-inhibition. LTCC-function was not necessary for SN DA pacemaker-activity at either age, but rather contributed to their pacemaker-precision. Moreover, juvenile Cav1.3 KO but not WT mice displayed adult wildtype-like, sensitised inhibitory dopamine-D2-autoreceptor (D2-AR) responses that depended upon both, interaction of the neuronal calcium sensor NCS-1 with D2-ARs, and on voltage-gated T-type calcium channel (TTCC) activity. This functional KO-phenotype was accompanied by cell-specific up-regulation of NCS-1 and Cav3.1-TTCC mRNA. Furthermore, in wildtype we identified an age-dependent switch of TTCC-function from contributing to SN DA pacemaker-precision in juveniles to pacemaker-frequency in adults. This novel interplay of Cav1.3 L-type and Cav3.1 T-type channels, and their modulation of SN DA activity-pattern and D2-AR-sensitisation, provide new insights into flexible age- and calcium-dependent activity-control of SN DA neurons and its pharmacological modulation.


Assuntos
Autorreceptores/metabolismo , Canais de Cálcio Tipo L/deficiência , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Neurônios Dopaminérgicos/metabolismo , Receptores de Dopamina D2/metabolismo , Substância Negra/metabolismo , Fatores Etários , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Regulação da Expressão Gênica , Masculino , Potenciais da Membrana , Camundongos , Camundongos Knockout , Proteínas Sensoras de Cálcio Neuronal/genética , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
9.
Neurobiol Aging ; 35(10): 2302-15, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24742361

RESUMO

Progressive loss of substantia nigra dopamine neurons (SN DA) is a hallmark of aging and of Parkinson's disease (PD). Mutations in PARK genes cause familial PD forms. Increased expression of alpha-synuclein (PARK4) is a disease-triggering event in familial PD and also observed in SN DA neurons in sporadic PD but related transcriptional changes are unknown. With optimized single-cell quantitative real-time polymerase chain reaction analysis, we compared messenger RNA and microRNA levels in SN DA neurons from sporadic PD patients and controls. Non-optimally matched donor ages and RNA integrities are common problems when analyzing human samples. We dissected the influence of distinct ages and RNA integrities of our samples by applying a specifically-optimized, linear-mixed-effects model to quantitative real-time polymerase chain reaction-data. We identified that elevated alpha-synuclein messenger RNA levels in SN DA neurons of human PD brains were positively correlated with corresponding elevated levels of mRNAs for functional compensation of progressive SN DA loss and for enhanced proteasomal (PARK5/UCHL1) and lysosomal (PARK9/ATPase13A2) function, possibly counteracting alpha-synuclein toxicity. In contrast, microRNA miR-133b levels, previously implicated in transcriptional dysregulation in PD, were not altered in SN DA neurons in PD.


Assuntos
Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Doença por Corpos de Lewy/genética , MicroRNAs/metabolismo , Doença de Parkinson/genética , RNA Mensageiro/metabolismo , alfa-Sinucleína/deficiência , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/genética , Neurônios Dopaminérgicos/patologia , Feminino , Humanos , Lisossomos/fisiologia , Masculino , Pessoa de Meia-Idade , Mutação , Doença de Parkinson/patologia , Complexo de Endopeptidases do Proteassoma/fisiologia , Substância Negra/citologia , alfa-Sinucleína/genética
10.
Neuron ; 59(3): 392-8, 2008 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-18701065

RESUMO

In the mammalian central nervous system, slow synaptic excitation involves the activation of metabotropic glutamate receptors (mGluRs). It has been proposed that C1-type transient receptor potential (TRPC1) channels underlie this synaptic excitation, but our analysis of TRPC1-deficient mice does not support this hypothesis. Here, we show unambiguously that it is TRPC3 that is needed for mGluR-dependent synaptic signaling in mouse cerebellar Purkinje cells. TRPC3 is the most abundantly expressed TRPC subunit in Purkinje cells. In mutant mice lacking TRPC3, both slow synaptic potentials and mGluR-mediated inward currents are completely absent, while the synaptically mediated Ca2+ release signals from intracellular stores are unchanged. Importantly, TRPC3 knockout mice exhibit an impaired walking behavior. Taken together, our results establish TRPC3 as a new type of postsynaptic channel that mediates mGluR-dependent synaptic transmission in cerebellar Purkinje cells and is crucial for motor coordination.


Assuntos
Desempenho Psicomotor/fisiologia , Transmissão Sináptica/fisiologia , Canais de Cátion TRPC/fisiologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Comportamento Animal/fisiologia , Cálcio/metabolismo , Cerebelo/citologia , Estimulação Elétrica/métodos , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos da radiação , Técnicas In Vitro , Metoxi-Hidroxifenilglicol/análogos & derivados , Metoxi-Hidroxifenilglicol/farmacologia , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Vias Neurais/fisiologia , Vias Neurais/efeitos da radiação , Técnicas de Patch-Clamp/métodos , Desempenho Psicomotor/efeitos dos fármacos , Células de Purkinje/fisiologia , Canais de Cátion TRPC/deficiência , Canais de Cátion TRPC/genética
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