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1.
Pain ; 164(8): 1718-1733, 2023 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-36727909

RESUMO

ABSTRACT: Induced pluripotent stem cells (iPSCs) have enabled the generation of various difficult-to-access cell types such as human nociceptors. A key challenge associated with human iPSC-derived nociceptors (hiPSCdNs) is their prolonged functional maturation. While numerous studies have addressed the expression of classic neuronal markers and ion channels in hiPSCdNs, the temporal development of key signaling cascades regulating nociceptor activity has remained largely unexplored. In this study, we used an immunocytochemical high-content imaging approach alongside electrophysiological staging to assess metabotropic and ionotropic signaling of large scale-generated hiPSCdNs across 70 days of in vitro differentiation. During this period, the resting membrane potential became more hyperpolarized, while rheobase, action potential peak amplitude, and membrane capacitance increased. After 70 days, hiPSCdNs exhibited robust physiological responses induced by GABA, pH shift, ATP, and capsaicin. Direct activation of protein kinase A type II (PKA-II) through adenylyl cyclase stimulation with forskolin resulted in PKA-II activation at all time points. Depolarization-induced activation of PKA-II emerged after 35 days of differentiation. However, effective inhibition of forskolin-induced PKA-II activation by opioid receptor agonists required 70 days of in vitro differentiation. Our results identify a pronounced time difference between early expression of functionally important ion channels and emergence of regulatory metabotropic sensitizing and desensitizing signaling only at advanced stages of in vitro cultivation, suggesting an independent regulation of ionotropic and metabotropic signaling. These data are relevant for devising future studies into the development and regulation of human nociceptor function and for defining time windows suitable for hiPSCdN-based drug discovery.


Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Analgésicos Opioides , Colforsina/farmacologia , Nociceptividade , Células Receptoras Sensoriais , Canais Iônicos
2.
Nat Neurosci ; 25(2): 168-179, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34931070

RESUMO

Bacterial products can act on neurons to alter signaling and function. In the present study, we found that dorsal root ganglion (DRG) sensory neurons are enriched for ANTXR2, the high-affinity receptor for anthrax toxins. Anthrax toxins are composed of protective antigen (PA), which binds to ANTXR2, and the protein cargoes edema factor (EF) and lethal factor (LF). Intrathecal administration of edema toxin (ET (PA + EF)) targeted DRG neurons and induced analgesia in mice. ET inhibited mechanical and thermal sensation, and pain caused by formalin, carrageenan or nerve injury. Analgesia depended on ANTXR2 expressed by Nav1.8+ or Advillin+ neurons. ET modulated protein kinase A signaling in mouse sensory and human induced pluripotent stem cell-derived sensory neurons, and attenuated spinal cord neurotransmission. We further engineered anthrax toxins to introduce exogenous protein cargoes, including botulinum toxin, into DRG neurons to silence pain. Our study highlights interactions between a bacterial toxin and nociceptors, which may lead to the development of new pain therapeutics.


Assuntos
Antraz , Bacillus anthracis , Toxinas Bacterianas , Células-Tronco Pluripotentes Induzidas , Animais , Antraz/microbiologia , Antraz/terapia , Bacillus anthracis/metabolismo , Toxinas Bacterianas/metabolismo , Gânglios Espinais/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Nociceptores/metabolismo , Dor , Receptores de Peptídeos/metabolismo
3.
Front Cell Neurosci ; 16: 1039957, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36733665

RESUMO

Human induced pluripotent stem cells (hiPSCs) are a promising approach to study neurological and neuropsychiatric diseases. Most methods to record the activity of these cells have major drawbacks as they are invasive or they do not allow single cell resolution. Genetically encoded voltage indicators (GEVIs) open the path to high throughput visualization of undisturbed neuronal activity. However, conventional GEVIs perturb membrane integrity through inserting multiple copies of transmembrane domains into the plasma membrane. To circumvent large add-ons to the plasma membrane, we used a minimally invasive novel hybrid dark quencher GEVI to record the physiological and pathological firing patterns of hiPSCs-derived sensory neurons from patients with inherited erythromelalgia, a chronic pain condition associated with recurrent attacks of redness and swelling in the distal extremities. We observed considerable differences in action potential firing patterns between patient and control neurons that were previously overlooked with other recording methods. Our system also performed well in hiPSC-derived forebrain neurons where it detected spontaneous synchronous bursting behavior, thus opening the path to future applications in other cell types and disease models including Parkinson's disease, Alzheimer's disease, epilepsy, and schizophrenia, conditions associated with disturbances of neuronal activity and synchrony.

4.
Oxid Med Cell Longev ; 2021: 6924251, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34691359

RESUMO

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease in humans and remains to have a fatal prognosis. Recent studies in animal models and human ALS patients indicate that increased reactive oxygen species (ROS) play an important role in the pathogenesis. Considering previous studies revealing the influence of ROS on mitochondrial physiology, our attention was focused on mitochondria in the murine ALS model, wobbler mouse. The aim of this study was to investigate morphological differences between wild-type and wobbler mitochondria with aid of superresolution structured illumination fluorescence microscopy, TEM, and TEM tomography. To get an insight into mitochondrial dynamics, expression studies of corresponding proteins were performed. Here, we found significantly smaller and degenerated mitochondria in wobbler motor neurons at a stable stage of the disease. Our data suggest a ROS-regulated, Ox-CaMKII-dependent Drp1 activation leading to disrupted fission-fusion balance, resulting in fragmented mitochondria. These changes are associated with numerous impairments, resulting in an overall self-reinforcing decline of motor neurons. In summary, our study provides common pathomechanisms with other ALS models and human ALS cases confirming mitochondria and related dysfunctions as a therapeutic target for the treatment of ALS.


Assuntos
Esclerose Lateral Amiotrófica/genética , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Camundongos
5.
Mol Autism ; 11(1): 99, 2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33308283

RESUMO

The controlled differentiation of pluripotent stem cells (PSCs) into neurons and glia offers a unique opportunity to study early stages of human central nervous system development under controlled conditions in vitro. With the advent of cell reprogramming and the possibility to generate induced pluripotent stem cells (iPSCs) from any individual in a scalable manner, these studies can be extended to a disease- and patient-specific level. Autism spectrum disorder (ASD) is considered a neurodevelopmental disorder, with substantial evidence pointing to early alterations in neurogenesis and network formation as key pathogenic drivers. For that reason, ASD represents an ideal candidate for stem cell-based disease modeling. Here, we provide a concise review on recent advances in the field of human iPSC-based modeling of syndromic and non-syndromic forms of ASD, with a particular focus on studies addressing neuronal dysfunction and altered connectivity. We further discuss recent efforts to translate stem cell-based disease modeling to 3D via brain organoid and cell transplantation approaches, which enable the investigation of disease mechanisms in a tissue-like context. Finally, we describe advanced tools facilitating the assessment of altered neuronal function, comment on the relevance of iPSC-based models for the assessment of pharmaceutical therapies and outline potential future routes in stem cell-based ASD research.


Assuntos
Transtorno do Espectro Autista/patologia , Transtorno do Espectro Autista/fisiopatologia , Células-Tronco Pluripotentes Induzidas/patologia , Modelos Biológicos , Neurônios/patologia , Animais , Reprogramação Celular/genética , Humanos , Organoides/patologia
6.
Mol Neurobiol ; 55(11): 8414-8424, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29549647

RESUMO

Amyotrophic lateral sclerosis is a devastating motor neuron disease and to this day not curable. While 5-10% of patients inherit the disease (familiar ALS), up to 95% of patients are diagnosed with the sporadic form (sALS). ALS is characterized by the degeneration of upper motor neurons in the cerebral cortex and of lower motor neurons in the brainstem and spinal cord. The wobbler mouse resembles almost all phenotypical hallmarks of human sALS patients and is therefore an excellent motor neuron disease model. The motor neuron disease of the wobbler mouse develops over a time course of around 40 days and can be divided into three phases: p0, presymptomatic; p20, early clinical; and p40, stable clinical phase. Recent findings suggest an essential implication of the NAD+-producing enzyme Nmnat2 in neurodegeneration as well as maintenance of healthy axons. Here, we were able to show a significant downregulation of both gene and protein expression of Nmnat2 in the spinal cord of the wobbler mice at the stable clinical phase. The product of the enzyme NAD+ is also significantly reduced, and the values of the reactive oxygen species are significantly increased in the spinal cord of the wobbler mouse at p40. Thus, the deregulated expression of Nmnat2 appears to have a great influence on the cellular stress in the spinal cord of wobbler mice.


Assuntos
Regulação para Baixo , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Espécies Reativas de Oxigênio/metabolismo , Medula Espinal/metabolismo , Animais , Substância Cinzenta/metabolismo , Substância Cinzenta/patologia , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Modelos Biológicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , NAD/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
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