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1.
J Nanobiotechnology ; 17(1): 46, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30935413

RESUMO

BACKGROUND: Silica-ε-polycaprolactone-nanoparticles (SiPCL-NPs) represent a promising tool for laser-tissue soldering in the brain. After release of the SiPCL-NPs in the brain, neuronal differentiation might be modulated. The present study was performed to determine effects of SiPCL-NP-exposure at different stages of neuronal differentiation in neuron-like SH-SY5Y cells. The resulting phenotypes were analyzed quantitatively and signaling pathways involved in neuronal differentiation and degeneration were studied. SH-SY5Y cells were differentiated with all-trans retinoic acid or staurosporine to obtain predominantly cholinergic or dopaminergic neurons. The resulting phenotype was analyzed at the end of differentiation with and without the SiPCL-NPs given at various times during differentiation. RESULTS: Exposure to SiPCL-NPs before and during differentiation led to a decreased cell viability of SH-SY5Y cells depending on the differentiation protocol used. SiPCL-NPs co-localized with the neuronal marker ß-3-tubulin but did not alter the morphology of these cells. A significant decrease in the number of tyrosine hydroxylase (TH) immunoreactive neurons was found in staurosporine-differentiated cells when SiPCL-NPs were added at the end of the differentiation. TH-protein expression was also significantly downregulated when SiPCL-NPs were applied in the middle of differentiation. Protein expression of the marker for the dopamine active transporter (DAT) was not affected by SiPCL-NPs. SiPCL-NP-exposure predominantly decreased the expression of the high-affinity choline transporter 1 (CHT1) when the NPs were given before the differentiation. Pathways involved in neuronal differentiation, namely Akt, MAP-K, MAP-2 and the neurodegeneration-related markers ß-catenin and GSK-3ß were not altered by NP-exposure. CONCLUSIONS: The decrease in the number of dopaminergic and cholinergic cells may implicate neuronal dysfunction, but the data do not provide evidence that pathways relevant for differentiation and related to neurodegeneration are impaired.


Assuntos
Neurônios Colinérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/efeitos dos fármacos , Nanopartículas/toxicidade , Poliésteres/toxicidade , Dióxido de Silício/toxicidade , Diferenciação Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neurônios Colinérgicos/citologia , Neurônios Colinérgicos/metabolismo , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Humanos , Nanopartículas/química , Fenótipo , Poliésteres/química , Transdução de Sinais , Dióxido de Silício/química , Estaurosporina/farmacologia , Tretinoína/farmacologia
2.
Nat Commun ; 10(1): 1944, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-31028266

RESUMO

Heart rate is under the precise control of the autonomic nervous system. However, the wiring of peripheral neural circuits that regulate heart rate is poorly understood. Here, we develop a clearing-imaging-analysis pipeline to visualize innervation of intact hearts in 3D and employed a multi-technique approach to map parasympathetic and sympathetic neural circuits that control heart rate in mice. We identify cholinergic neurons and noradrenergic neurons in an intrinsic cardiac ganglion and the stellate ganglia, respectively, that project to the sinoatrial node. We also report that the heart rate response to optogenetic versus electrical stimulation of the vagus nerve displays different temporal characteristics and that vagal afferents enhance parasympathetic and reduce sympathetic tone to the heart via central mechanisms. Our findings provide new insights into neural regulation of heart rate, and our methodology to study cardiac circuits can be readily used to interrogate neural control of other visceral organs.


Assuntos
Frequência Cardíaca/fisiologia , Neurônios Motores/fisiologia , Animais , Neurônios Colinérgicos/metabolismo , Neurônios Colinérgicos/fisiologia , Eletrofisiologia , Feminino , Masculino , Camundongos , Sistema Nervoso Periférico/metabolismo , Sistema Nervoso Periférico/fisiologia , Nervo Vago/metabolismo , Nervo Vago/fisiologia
3.
EBioMedicine ; 41: 649-658, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30819512

RESUMO

BACKGROUND: The most prevalent inherited form of generalized dystonia is caused by a mutation in torsinA (DYT1, ∆GAG) with incomplete penetrance. Rodent models with mutated torsinA do not develop dystonic symptoms, but previous ex vivo studies indicated abnormal excitation of cholinergic interneurons (ChI) and increased striatal acetylcholine. METHODS: We used in vivo optogenetics to exacerbate this endophenotype in order to determine its capacity to trigger dystonic symptoms in freely behaving mice. Tor1a+/Δgag DYT1 mice and wildtype littermates expressing channelrhodopsin2 under the Chat promotor were implanted bilaterally with optical LED cannulae and stimulated with blue light pulses of varied durations. FINDINGS: Six months old DYT1 KI mice but not wildtype controls responded with hyperactivity to blue light specifically at 25 ms pulse duration, 10 Hz frequency. Neuronal activity (c-Fos) in cholinergic interneurons was increased immediately after light stimulation and persisted only in DYT1 KI over 15 min. Substance P was increased specifically in striosome compartments in naïve DYT1 KI mice compared to wildtype. Under optogenetic stimulation substance P increased in wildtype to match levels in Dyt1 KI, and acetylcholinesterase was elevated in the striatum of stimulated DYT1 KI. No signs of dystonic movements were observed under stimulation of up to one hour in both genotypes and age groups, and the sensorimotor deficit previously observed in 6 months old DYT1 KI mice persisted under stimulation. INTERPRETATION: Overall this supports an endophenotype of dysregulated cholinergic activity in DYT1 dystonia, but depolarizing cholinergic interneurons was not sufficient to induce overt dystonia in DYT1 KI mice.


Assuntos
Neurônios Colinérgicos/metabolismo , Chaperonas Moleculares/genética , Optogenética , Acetilcolinesterase/genética , Acetilcolinesterase/metabolismo , Animais , Comportamento Animal/efeitos da radiação , Channelrhodopsins/metabolismo , Endofenótipos , Feminino , Técnicas de Introdução de Genes , Luz , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Substância P/genética , Substância P/metabolismo
4.
Proc Natl Acad Sci U S A ; 116(15): 7483-7492, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30923117

RESUMO

Three-dimensional cell cultures are leading the way to the fabrication of tissue-like constructs useful to developmental biology and pharmaceutical screenings. However, their reproducibility and translational potential have been limited by biomaterial and culture media compositions, as well as cellular sources. We developed a construct comprising synthetic multifunctionalized hydrogels, serum-free media, and densely seeded good manufacturing practice protocol-grade human neural stem cells (hNSC). We tracked hNSC proliferation, differentiation, and maturation into GABAergic, glutamatergic, and cholinergic neurons, showing entangled electrically active neural networks. The neuroregenerative potential of the "engineered tissue" was assessed in spinal cord injuries, where hNSC-derived progenitors and predifferentiated hNSC progeny, embedded in multifunctionalized hydrogels, were implanted. All implants decreased astrogliosis and lowered the immune response, but scaffolds with predifferentiated hNSCs showed higher percentages of neuronal markers, better hNSC engraftment, and improved behavioral recovery. Our hNSC-construct enables the formation of 3D functional neuronal networks in vitro, allowing novel strategies for hNSC therapies in vivo.


Assuntos
Diferenciação Celular , Proliferação de Células , Células Imobilizadas , Hidrogéis , Células-Tronco Neurais , Regeneração , Traumatismos da Medula Espinal , Animais , Células Imobilizadas/metabolismo , Células Imobilizadas/patologia , Células Imobilizadas/transplante , Neurônios Colinérgicos/metabolismo , Neurônios Colinérgicos/patologia , Modelos Animais de Doenças , Feminino , Xenoenxertos , Humanos , Hidrogéis/química , Hidrogéis/farmacologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Células-Tronco Neurais/transplante , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/terapia
5.
Neuron ; 101(4): 662-672.e5, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30638901

RESUMO

Cholinergic interneurons (ChIs) in the nucleus accumbens (NAc) have been implicated in drug addiction, reward, and mood disorders. However, the physiological role of ChIs in depression has not been characterized. Here, we show that the tonic firing rate of ChIs in NAc shell is reduced in chronic stress mouse models and in a genetic mouse model of depression. Chemogenetic inhibition of NAc ChIs renders naive mice susceptible to stress, whereas enhancement of ChI activity reverses depressive phenotypes. As a component of the molecular mechanism, we found that the expression and function of the hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) are decreased in ChIs of NAc shell in depressed mice. Overexpression of HCN2 channels in ChIs enhances cell activity and is sufficient to rescue depressive phenotypes. These data suggest that enhancement of HCN2 channel activity in NAc ChIs is a feasible approach for the development of a new class of antidepressants.


Assuntos
Neurônios Colinérgicos/metabolismo , Transtorno Depressivo/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Interneurônios/metabolismo , Núcleo Accumbens/metabolismo , Potenciais de Ação , Animais , Neurônios Colinérgicos/fisiologia , Transtorno Depressivo/fisiopatologia , Feminino , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Interneurônios/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Núcleo Accumbens/citologia , Núcleo Accumbens/fisiopatologia
6.
Biochem Biophys Res Commun ; 509(3): 763-766, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30612733

RESUMO

The neurotrophin Nerve growth factor (NGF) plays a critical role in the mature and developing nervous system. A point mutation (R100W) in the NGFB gene was found in patients with Hereditary Sensory and Autonomic Neuropathy type V (HSAN V), which leads to pain insensitivity. In a previous work it has been shown that the mutation provokes a reduced secretion of mature NGF. In this study we generated and analyzed homozygous NGFR100W/R100W mice to understand whether the reduced NGF bioavailability can contribute to the clinical phenotype of the homozygous condition. We found that the majority of NGFR100W/R100W mice were born normal but failed to reach the first month of age. This early lethality was rescued by daily treatment with wild type NGF. In addition, we found that the density of cholinergic neurons of homozygous mice was unaffected in the medial septum and in the nucleus basalis of Meynert, whereas, suprisingly, it was increased specifically in the striatum. Due to the known action of the striatal cholinergic tone in modulating pain, our findings support the hypothesis that a central mechanism, linked to the NGFR100W-dependent increase of the striatal cholinergic tone, can contribute to the pain insensitivity observed in HSAN V patients.


Assuntos
Neurônios Colinérgicos/efeitos dos fármacos , Corpo Estriado/efeitos dos fármacos , Neuropatias Hereditárias Sensoriais e Autônomas/terapia , Fator de Crescimento Neural/uso terapêutico , Animais , Disponibilidade Biológica , Neurônios Colinérgicos/citologia , Neurônios Colinérgicos/metabolismo , Corpo Estriado/citologia , Corpo Estriado/metabolismo , Neuropatias Hereditárias Sensoriais e Autônomas/genética , Homozigoto , Humanos , Camundongos , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/farmacocinética , Mutação Puntual
7.
Neuron ; 101(3): 444-458.e6, 2019 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-30658860

RESUMO

The motor symptoms of Parkinson's disease (PD) are thought to stem from an imbalance in the activity of striatal direct- and indirect-pathway spiny projection neurons (SPNs). Disease-induced alterations in the activity of networks controlling SPNs could contribute to this imbalance. One of these networks is anchored by the parafascicular nucleus (PFn) of the thalamus. To determine the role of the PFn in striatal PD pathophysiology, optogenetic, chemogenetic, and electrophysiological tools were used in ex vivo slices from transgenic mice with region-specific Cre recombinase expression. These studies revealed that in parkinsonian mice, the functional connectivity of PFn neurons with indirect pathway SPNs (iSPNs) was selectively enhanced by cholinergic interneurons acting through presynaptic nicotinic acetylcholine receptors (nAChRs) on PFn terminals. Attenuating this network adaptation by chemogenetic or genetic strategies alleviated motor-learning deficits in parkinsonian mice, pointing to a potential new therapeutic strategy for PD patients.


Assuntos
Neurônios Colinérgicos/fisiologia , Corpo Estriado/fisiopatologia , Potenciais Pós-Sinápticos Excitadores , Interneurônios/fisiologia , Doença de Parkinson/fisiopatologia , Tálamo/fisiopatologia , Animais , Neurônios Colinérgicos/metabolismo , Corpo Estriado/citologia , Ácido Glutâmico/metabolismo , Interneurônios/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doença de Parkinson/metabolismo , Receptores Nicotínicos/metabolismo , Tálamo/citologia
8.
Mol Brain ; 12(1): 4, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30626449

RESUMO

Traumatic spinal cord injury (SCI) causes serious disruption of neuronal circuits that leads to motor functional deficits. Regeneration of disrupted circuits back to their original target is necessary for the restoration of function after SCI, but the pathophysiological condition of the caudal spinal cord has not been sufficiently studied. Here we investigated the histological and biological changes in the distal part of the injured spinal cord, using a mice model of complete thoracic SCI in the chronic stage (3 months after injury). Atrophic changes were widely observed in the injured spinal cord both rostral and caudal to the lesion, but the decrease in area was mainly in the white matter in the rostral spinal cord while both the white and gray matter decreased in the caudal spinal cord. The number of the motor neurons was maintained in the chronic phase of injury, but the number of presynaptic boutons decreased in the lumbar motor neurons caudal to the lesion. Using laser microdissection, to investigate gene expressions in motor neurons caudal to the lesion, we observed a decrease in the expressions of neuronal activity markers. However, we found that the synaptogenic potential of postsynapse molecules was maintained in the motor neurons after SCI with the expression of acetylcholine-related molecules actually higher after SCI. Collectively, our results show that the potential of synaptogenesis is maintained in the motor neurons caudal to the lesion, even though presynaptic input is decreased. Although researches into SCI concentrate their effort on the lesion epicenter, our findings suggest that the area caudal to the lesion could be an original therapeutic target for the chronically injured spinal cord.


Assuntos
Neurônios Motores/patologia , Traumatismos da Medula Espinal/patologia , Acetilcolina/metabolismo , Animais , Atrofia , Orientação de Axônios , Neurônios Colinérgicos/metabolismo , Doença Crônica , Feminino , Regulação da Expressão Gênica , Microdissecção e Captura a Laser , Vértebras Lombares/patologia , Camundongos Endogâmicos C57BL , Neurotransmissores/metabolismo , Terminações Pré-Sinápticas/patologia , Medula Espinal/patologia , Sinapses/metabolismo
9.
Neuron ; 101(3): 486-499.e4, 2019 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-30594427

RESUMO

The ascending cholinergic system dynamically regulates sensory perception and cognitive function, but it remains unclear how this modulation is executed in neocortical circuits. Here, we demonstrate that the cholinergic system controls the integrative operations of neocortical principal neurons by modulating dendritic excitability. Direct dendritic recordings revealed that the optogenetic-evoked release of acetylcholine (ACh) transformed the pattern of dendritic integration in layer 5B pyramidal neurons, leading to the generation of dendritic plateau potentials which powerfully drove repetitive action potential output. In contrast, the synaptic release of ACh did not positively modulate axo-somatic excitability. Mechanistically, the transformation of dendritic integration was mediated by the muscarinic ACh receptor-dependent enhancement of dendritic R-type calcium channel activity, a compartment-dependent modulation which decisively controlled the associative computations executed by layer 5B pyramidal neurons. Our findings therefore reveal a biophysical mechanism by which the cholinergic system controls dendritic computations causally linked to perceptual detection.


Assuntos
Potenciais de Ação , Neurônios Colinérgicos/fisiologia , Dendritos/fisiologia , Potenciais Pós-Sinápticos Excitadores , Neocórtex/fisiologia , Células Piramidais/fisiologia , Acetilcolina/metabolismo , Animais , Canais de Cálcio Tipo R/metabolismo , Neurônios Colinérgicos/metabolismo , Dendritos/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neocórtex/citologia , Neocórtex/metabolismo , Células Piramidais/metabolismo , Ratos , Ratos Wistar , Receptores Muscarínicos/metabolismo
10.
Dev Biol ; 446(1): 80-93, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30529058

RESUMO

Insect mushroom bodies (MB) have an ensemble of synaptic connections well-studied for their role in experience-dependent learning and several higher cognitive functions. MB requires neurotransmission for an efficient flow of information across synapses with different flexibility to meet the demand of the dynamically changing environment of an insect. Neurotransmitter transporters coordinate appropriate changes for an efficient neurotransmission at the synapse. Till date, there is no transporter reported for any of the previously known neurotransmitters in the intrinsic neurons of MB. In this study, we report a highly enriched expression of Choline Transporter (ChT) in Drosophila MB. We demonstrate that knockdown of ChT in a sub-type of MB neurons called α/ß core (α/ßc) and ϒ neurons leads to eclosion failure, peristaltic defect in larvae, and altered NMJ phenotype. These defects were neither observed on knockdown of proteins of the cholinergic locus in α/ßc and ϒ neurons nor by knockdown of ChT in cholinergic neurons. Thus, our study provides insights into non-canonical roles of ChT in MB.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Corpos Pedunculados/metabolismo , Junção Neuromuscular/metabolismo , Neurônios/metabolismo , Animais , Animais Geneticamente Modificados , Neurônios Colinérgicos/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Proteínas de Membrana Transportadoras/genética , Corpos Pedunculados/citologia , Corpos Pedunculados/crescimento & desenvolvimento , Junção Neuromuscular/genética , Junção Neuromuscular/crescimento & desenvolvimento , Pupa/genética , Pupa/crescimento & desenvolvimento , Pupa/metabolismo , Interferência de RNA , Transmissão Sináptica/genética , Transmissão Sináptica/fisiologia
11.
Cell Mol Gastroenterol Hepatol ; 7(3): 655-678, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30594740

RESUMO

BACKGROUND & AIMS: RET, the receptor for the glial cell line-derived neurotrophic factor (GDNF) family ligands, is the most frequently mutated gene in congenital aganglionic megacolon or Hirschsprung's disease (HSCR). The leading cause of mortality in HSCR is HSCR-associated enterocolitis (HAEC), which is characterized by altered mucin composition, mucin retention, bacterial adhesion to enterocytes, and epithelial damage, although the order of these events is obscure. In mice, loss of GDNF signaling leads to a severely underdeveloped enteric nervous system and neonatally fatal kidney agenesis, thereby precluding the use of these mice for modeling postnatal HSCR and HAEC. Our aim was to generate a postnatally viable mouse model for HSCR/HAEC and analyze HAEC etiology. METHODS: GDNF family receptor alpha-1 (GFRa1) hypomorphic mice were generated by placing a selectable marker gene in the sixth intron of the Gfra1 locus using gene targeting in mouse embryonic stem cells. RESULTS: We report that 70%-80% reduction in GDNF co-receptor GFRa1 expression levels in mice results in HSCR and HAEC, leading to death within the first 25 postnatal days. These mice mirror the disease progression and histopathologic findings in children with untreated HSCR/HAEC. CONCLUSIONS: In GFRa1 hypomorphic mice, HAEC proceeds from goblet cell dysplasia, with abnormal mucin production and retention, to epithelial damage. Microbial enterocyte adherence and tissue invasion are late events and therefore unlikely to be the primary cause of HAEC. These results suggest that goblet cells may be a potential target for preventative treatment and that reduced expression of GFRa1 may contribute to HSCR susceptibility.


Assuntos
Enterocolite/complicações , Enterocolite/metabolismo , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Doença de Hirschsprung/complicações , Doença de Hirschsprung/metabolismo , Animais , Proteínas Sanguíneas/metabolismo , Neurônios Colinérgicos/metabolismo , Colo/inervação , Colo/patologia , Citocinas/genética , Citocinas/metabolismo , Sistema Nervoso Entérico/metabolismo , Sistema Nervoso Entérico/patologia , Enterocolite/sangue , Genótipo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Células Caliciformes/patologia , Doença de Hirschsprung/sangue , Homozigoto , Hipertrofia , Mucosa Intestinal/patologia , Camundongos Endogâmicos C57BL , Mucinas/metabolismo , Células-Tronco Neurais/metabolismo , Proteínas Proto-Oncogênicas c-ret , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
12.
Biomed Pharmacother ; 111: 496-502, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30594789

RESUMO

Certain biflavonoids have been proven to protect against cognitive dysfunction. A new biflavonoid, CGY-1, isolated from Cardiocrinum giganteum seeds, has not yet been reported to have any neuroprotective effect. In this study, a scopolamine-induced memory deficit model was used to explore the neuroprotective effect of CGY-1. Behavioral experiments, such as tests using the Morris water maze, the Y-maze and the fear conditioning test, were conducted. The results revealed that oral administration of CGY-1 (20 and 40 mg/kg) and donepezil shortened the escape latency, improved the percentage of spontaneous alternation, and increased the freezing times, respectively. CGY-1 decreased the levels of reactive oxygen species and malondialdehyde and increased the activities of superoxide dismutase and glutathione peroxidase in the hippocampus. In addition, CGY-1 decreased the activity of acetylcholinesterase and increased the activities of choline acetyltransferase and acetylcholine in the hippocampus. Furthermore, qPCR and western blot results revealed that the expressions of neurotrophic factors, brain-derived neurotrophic factor and nerve growth factor were upregulated in the hippocampus after CGY-1 treatment. In conclusion, CGY-1 could be a promising candidate for the treatment of cognitive dysfunction.


Assuntos
Biflavonoides/uso terapêutico , Neurônios Colinérgicos/efeitos dos fármacos , Medicamentos de Ervas Chinesas/uso terapêutico , Lilium , Transtornos da Memória/tratamento farmacológico , Escopolamina/toxicidade , Animais , Biflavonoides/isolamento & purificação , Biflavonoides/farmacologia , Antagonistas Colinérgicos/toxicidade , Neurônios Colinérgicos/metabolismo , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/farmacologia , Masculino , Transtornos da Memória/induzido quimicamente , Transtornos da Memória/metabolismo , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Sementes
13.
PLoS One ; 13(12): e0209363, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30571745

RESUMO

One of the pathological site effects in excitotoxic activation is Zn2+ overload to postsynaptic neurons. Such an effect is considered to be equivalent to the glutamate component of excitotoxicity. Excessive uptake of Zn2+ by active voltage-dependent transport systems in these neurons may lead to significant neurotoxicity. The aim of this study was to investigate whether and which antagonists of the voltage gated calcium channels (VGCC) might modify this Zn2+-induced neurotoxicity in neuronal cells. Our data demonstrates that depolarized SN56 neuronal cells may take up large amounts of Zn2+ and store these in cytoplasmic and mitochondrial sub-fractions. The mitochondrial Zn2+ excess suppressed pyruvate uptake and oxidation. Such suppression was caused by inhibition of pyruvate dehydrogenase complex, aconitase and NADP-isocitrate dehydrogenase activities, resulting in the yielding of acetyl-CoA and ATP shortages. Moreover, incoming Zn2+ increased both oxidized glutathione and malondialdehyde levels, known parameters of oxidative stress. In depolarized SN56 cells, nifedipine treatment (L-type VGCC antagonist) reduced Zn2+ uptake and oxidative stress. The treatment applied prevented the activities of PDHC, aconitase and NADP-IDH enzymes, and also yielded the maintenance of acetyl-CoA and ATP levels. Apart from suppression of oxidative stress, N- and P/Q-type VGCCs presented a similar, but weaker protective influence. In conclusion, our data shows that in the course of excitotoxity, impairment to calcium homeostasis is tightly linked with an excessive neuronal Zn2+ uptake. Hence, the VGCCs types L, N and P/Q share responsibility for neuronal Zn2+ overload followed by significant energy-dependent neurotoxicity. Moreover, Zn2+ affects the target tricarboxylic acid cycle enzymes, yields acetyl-CoA and energy deficits as well.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo N/metabolismo , Neurônios Colinérgicos/efeitos dos fármacos , Neurotoxinas/metabolismo , Zinco/metabolismo , Animais , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neurônios Colinérgicos/metabolismo , Metabolismo Energético/efeitos dos fármacos , Camundongos , Mitocôndrias/metabolismo , Neuroblastoma/patologia , Nifedipino/farmacologia
14.
Food Chem Toxicol ; 121: 297-308, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30213552

RESUMO

Cadmium, a neurotoxic environmental compound, produces cognitive disorders, although the mechanism remains unknown. Cadmium induces a more pronounced cell death on cholinergic neurons from basal forebrain (BF), mediated, in part, by increase in Aß and total and phosphorylated Tau protein levels, which may explain cadmium effects on learning and memory processes. Cadmium downregulates the expression of heat shock proteins (HSPs) HSP 90, HSP70 and HSP27, and of HSF1, the master regulator of the HSP pathway. HSPs proteins reduce the production of Aß and phosphorylated Tau proteins and avoid cell death pathways induction. Thus, we hypothesized that cadmium induced the production of Aß and Tau proteins by HSP pathway disruption through HSF1 expression alteration, leading to BF cholinergic neurons cell death. Our results show that cadmium downregulates HSF1, leading to HSP90, HSP70 and HSP27 gene expression downregulation in BF SN56 cholinergic neurons. In addition, cadmium induced Aß and total and phosphorylated Tau proteins generation, mediated partially by HSP90, HSP70 and HSP27 disruption, leading to cell death. These results provide new understanding of the mechanisms contributing to cadmium harmful effects on cholinergic neurons.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Cádmio/toxicidade , Morte Celular/efeitos dos fármacos , Neurônios Colinérgicos/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Proteínas tau/metabolismo , Animais , Caspase 3/genética , Caspase 3/metabolismo , Caspase 7/genética , Caspase 7/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neurônios Colinérgicos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Compostos de Fósforo , Reação em Cadeia da Polimerase em Tempo Real
15.
Nutrients ; 10(7)2018 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-30018265

RESUMO

Cholinergic dysfunction, impaired brain-derived neurotrophic factor and cAMP response element binding protein (BDNF-CREB) signaling are one of the major pathological hallmarks of cognitive impairment. Therefore, improving cholinergic neurotransmission, and regulating the BDNF-CREB pathway by downregulating apoptosis genes is one strategy for inhibiting the etiology of dementia. This study evaluates the potential effects of Stachys sieboldii MIQ (SS) extract against cognitive dysfunction and its underlying mechanisms. SS supplementation for 33 days improved scopolamine-induced memory impairment symptoms in Morris water maze test and Y-maze test. SS reduced the acetylcholineesterase activity and significantly increase acetylcholine and cholineacetyltransferase activity in the brain. In the subsequent mechanism study, SS regulated the mRNA expression level of neuronal plasticity molecules such as (nerve growth factor) NGF, BDNF, CREB, and its downstream molecules such as Bcl-2 and Egr-1 by downregulating the neuronal apoptosis targets in both hippocampus and frontal cortex. Additionally, inward currents caused by SS in hippocampal CA1 neurons was partially blocked by the GABA receptor antagonist picrotoxin (50 µM), suggesting that SS acts on synaptic/extrasynaptic GABAA receptors. These findings indicate that SS may function in a way that is similar to nootropic drugs by inhibiting cholinergic abnormalities, and neuronal apoptosis targets and ultimately increasing the expression of BDNF-CREB.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Suplementos Nutricionais , Transtornos da Memória/prevenção & controle , Nootrópicos/uso terapêutico , Extratos Vegetais/uso terapêutico , Stachys/química , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/agonistas , Fator Neurotrófico Derivado do Encéfalo/genética , Colina O-Acetiltransferase/química , Colina O-Acetiltransferase/metabolismo , Neurônios Colinérgicos/enzimologia , Neurônios Colinérgicos/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/agonistas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Etnofarmacologia , Lobo Frontal/enzimologia , Lobo Frontal/crescimento & desenvolvimento , Lobo Frontal/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hipocampo/enzimologia , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Masculino , Medicina Tradicional Coreana , Transtornos da Memória/enzimologia , Transtornos da Memória/metabolismo , Camundongos Endogâmicos ICR , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nootrópicos/administração & dosagem , Extratos Vegetais/administração & dosagem , Distribuição Aleatória , Ratos Sprague-Dawley , República da Coreia
16.
Nutrients ; 10(7)2018 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-29966363

RESUMO

In the recent years, interest in soybean as a neuroprotective nutrient in the management of Alzheimer’s disease (AD) has increased and soy isoflavones (SI), as kinds of soybean phytochemicals, are thought to be biologically active components that confer this beneficial effect against neurodegenerative diseases. However, the neuroprotective effect of SI is not well understood. Therefore, the present study (30 days) was conducted to investigate the neuroprotective effects of soy isoflavones (SI) on scopolamine (SCOP)-induced memory impairments in Institute of Cancer Research (ICR) mice (aged 4 weeks) and to elucidate its underlying mechanisms of action. SI (40 mg/kg) administration improved the cognitive performance of SCOP-treated mice in an object location recognition task and the Morris water maze test. SI (40 mg/kg) administration significantly enhanced cholinergic system function and suppressed oxidative stress levels in the hippocampus of SCOP-treated mice. Furthermore, SI (40 mg/kg) treatment markedly upregulated the phosphorylation levels of extracellular signal-regulated kinase (ERK), cAMP response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) expression levels in the hippocampus. Taken together, these results demonstrated that soy isoflavones exerted a significant neuroprotective effect on cognitive dysfunctions induced by scopolamine, suggesting that soy isoflavones could be a good candidate for possible treatment of neurodegenerative diseases, such as Alzheimer’s disease (AD).


Assuntos
Amnésia/prevenção & controle , Comportamento Animal/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Isoflavonas/farmacologia , Memória/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Escopolamina , Soja , Acetilcolina/metabolismo , Acetilcolinesterase/metabolismo , Amnésia/induzido quimicamente , Amnésia/metabolismo , Amnésia/psicologia , Animais , Antioxidantes/farmacologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Colina O-Acetiltransferase/metabolismo , Neurônios Colinérgicos/efeitos dos fármacos , Neurônios Colinérgicos/metabolismo , Cognição/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Ligadas por GPI/metabolismo , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Isoflavonas/isolamento & purificação , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos Endogâmicos ICR , Fármacos Neuroprotetores/isolamento & purificação , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , Soja/química , Fatores de Tempo
17.
Mol Neurodegener ; 13(1): 33, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29945658

RESUMO

BACKGROUND: Type 2 diabetes (T2D) is a recognized risk factor for the development of cognitive impairment (CI) and/or dementia, although the exact nature of the molecular pathology of T2D-associated CI remains obscure. One link between T2D and CI might involve decreased insulin signaling in brain and/or neurons in either animal or postmortem human brains as has been reported as a feature of Alzheimer's disease (AD). Here we asked if neuronal insulin resistance is a cell autonomous phenomenon in a familial form of AD. METHODS: We have applied a newly developed protocol for deriving human basal forebrain cholinergic neurons (BFCN) from skin fibroblasts via induced pluripotent stem cell (iPSC) technology. We generated wildtype and familial AD mutant PSEN2 N141I (presenilin 2) BFCNs and assessed if insulin signaling, insulin regulation of the major AD proteins Aß and/or tau, and/or calcium fluxes is altered by the PSEN2 N141I mutation. RESULTS: We report herein that wildtype, PSEN2 N141I and CRISPR/Cas9-corrected iPSC-derived BFCNs (and their precursors) show indistinguishable insulin signaling profiles as determined by the phosphorylation of canonical insulin signaling pathway molecules. Chronic insulin treatment of BFCNs of all genotypes led to a reduction in the Aß42/40 ratio. Unexpectedly, we found a CRISPR/Cas9-correctable effect of PSEN2 N141I on calcium flux, which could be prevented by chronic exposure of BFCNs to insulin. CONCLUSIONS: Our studies indicate that the familial AD mutation PSEN2 N141I does not induce neuronal insulin resistance in a cell autonomous fashion. The ability of insulin to correct calcium fluxes and to lower Aß42/40 ratio suggests that insulin acts to oppose an AD-pathophysiology. Hence, our results are consistent with a potential physiological role for insulin as a mediator of resilience by counteracting specific metabolic and molecular features of AD.


Assuntos
Doença de Alzheimer/metabolismo , Neurônios Colinérgicos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Insulina/metabolismo , Doença de Alzheimer/genética , Diabetes Mellitus Tipo 2/complicações , Feminino , Humanos , Resistência à Insulina/fisiologia , Masculino , Mutação , Presenilina-2/genética
18.
Toxicol Lett ; 294: 73-86, 2018 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-29775722

RESUMO

Bifenthrin (BF) is a synthetic pyrethroid pesticide widely used in several countries to manage insect pests on diverse agricultural crops. Growing evidence indicates that BF exposure is associated with an increased risk of developing neurodegenerative disorders. However, the mechanisms by which BF induces neurological and anxiety alterations in the frontal cortex and striatum are not well known. The present in vivo study was carried out to determine whether reactive oxygen species (ROS)-mediated oxidative stress (OS) and neuroinflammation are involved in such alterations. Thirty-six Wistar rats were thus randomly divided into three groups and were orally administered with BF (0.6 and 2.1 mg/kg body weight, respectively) or the vehicle (corn oil), on a daily basis for 60 days. Results revealed that BF exposure in rats enhanced anxiety-like behavior after 60 days of treatment, as assessed with the elevated plus-maze test by decreases in the percentage of time spent in open arms and frequency of entries into these arms. BF-treated rats also exhibited increased oxidation of lipids and carbonylated proteins in the frontal cortex and striatum, and decreased glutathione levels and antioxidant enzyme activities including superoxide dismutase, catalase and glutathione peroxidase. Treatment with BF also increased protein synthesis and mRNA expression of the inflammatory mediators cyclooxygenase-2 (COX-2), microsomal prostaglandin synthase-1 (mPGES-1) and nuclear factor-kappaBp65 (NF-kBp65), as well as the production of tumor necrosis factor-α (TNF-α) and ROS. Moreover, BF exposure significantly decreased protein synthesis and mRNA expression of nuclear factor erythroid-2 (Nrf2) and acetylcholinesterase (AChE), as well as gene expression of muscarinic-cholinergic receptors (mAchR) and choline acetyltransferase (ChAT) in the frontal cortex and striatum. These data suggest that BF induced neurological alterations in the frontal cortex and striatum of rats, and that this may be associated with neuroinflammation and oxidative stress via the activation of Nrf2/NF-kBp65 pathways, which might promote anxiety-like behavior.


Assuntos
Ansiedade/etiologia , Inseticidas/toxicidade , Neurite (Inflamação)/induzido quimicamente , Síndromes Neurotóxicas/fisiopatologia , Estresse Oxidativo/efeitos dos fármacos , Piretrinas/toxicidade , Tremor/etiologia , Animais , Comportamento Animal/efeitos dos fármacos , Biomarcadores/metabolismo , Neurônios Colinérgicos/efeitos dos fármacos , Neurônios Colinérgicos/imunologia , Neurônios Colinérgicos/metabolismo , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/imunologia , Corpo Estriado/metabolismo , Relação Dose-Resposta a Droga , Comportamento Exploratório/efeitos dos fármacos , Lobo Frontal/efeitos dos fármacos , Lobo Frontal/imunologia , Lobo Frontal/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Inseticidas/administração & dosagem , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurite (Inflamação)/imunologia , Neurite (Inflamação)/metabolismo , Neurite (Inflamação)/fisiopatologia , Síndromes Neurotóxicas/imunologia , Síndromes Neurotóxicas/metabolismo , Piretrinas/administração & dosagem , Distribuição Aleatória , Ratos Wistar
19.
Neurochem Res ; 43(7): 1392-1404, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29796737

RESUMO

Based on the scientific evidence supporting the neuroinflammatory response contributes the cognitive impairment associated with chronic alcoholism and the neuroprotective actions of mefenamic acid with reversal of memory loss and brain inflammation in mice, this study was designed to evaluate the effect of mefenamic acid against chronic alcohol induced cognitive impairment in zebrafish model. Zebrafish were grouped and subjected to normal behavioral analysis in light-dark chamber for 10 days. The preference to dark compartment was noted in zebrafish. Zebrafish were grouped and exposed to escalating doses of alcohol for 28 days with and without mefenamic acid exposure (100 and 200 µg/L) and subjected to a fear conditioning passive avoidance task from day 13 of 28. The cognitive evaluation was performed for 10 days and the brain tissue was isolated to estimate acetylcholinesterase activity. In cognitive evaluation study, the normal zebrafish retained the memory of the learned task and avoided the dark. The alcohol exposed zebrafish showed impairment in retaining the memory of learned task. Mefenamic acid exposed zebrafish showed a significant protection against cognitive impairment caused by alcohol and retained the memory of learned task with a significant decrease in AChE activity in brain homogenate compared to alcohol exposed zebrafish. The results of this study suggest that the memory enhancing activity of mefenamic acid might be due to activation of cholinergic transmission that has protected neuroinflammatory and neurodegenerative conditions caused by alcohol.


Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Neurônios Colinérgicos/efeitos dos fármacos , Disfunção Cognitiva/induzido quimicamente , Disfunção Cognitiva/tratamento farmacológico , Etanol/toxicidade , Ácido Mefenâmico/uso terapêutico , Acetilcolinesterase/metabolismo , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Aprendizagem da Esquiva/efeitos dos fármacos , Aprendizagem da Esquiva/fisiologia , Neurônios Colinérgicos/metabolismo , Disfunção Cognitiva/metabolismo , Etanol/administração & dosagem , Feminino , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Masculino , Ácido Mefenâmico/farmacologia , Peixe-Zebra
20.
J Mol Histol ; 49(4): 339-345, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29667149

RESUMO

Functional integrity of the regenerated tissues requires not only structural integrity but also vascularization and innervation. We previously demonstrated that the three-dimensional (3D) reconstructed eccrine sweat glands had similar structures as those of the native ones did, but whether the 3D reconstructed glands possessing vascularization and innervation was still unknown. In the study, Matrigel-embedded eccrine sweat gland cells were implanted under the inguinal skin. Ten weeks post-implantation, the vascularization, and innervation in the 10-week reconstructed eccrine sweat glands and native human eccrine sweat glands were detected by immunofluorescence staining. The results showed that the fluorescent signals of general neuronal marker protein gene product 9.5, adrenergic nerve fiber marker tyrosine hydroxylase, and cholinergic nerve fiber markers acetylcholinesterase and vasoactive intestinal peptide embraced the 3D reconstructed glands in circular patterns, as the signals appeared in native eccrine sweat glands. There were many CD31- and von Willebrand factor-positive vessels growing into the plugs. We demonstrated that the 3D reconstructed eccrine sweat glands were nourished by blood vessels, and we for the first time demonstrated that the engineering sweat glands were innervated by both cholinergic and adrenergic fibers. In conclusion, the 3D reconstructed eccrine sweat glands may have functions as the native ones do.


Assuntos
Neurônios Adrenérgicos/metabolismo , Neurônios Colinérgicos/metabolismo , Glândulas Écrinas/irrigação sanguínea , Glândulas Écrinas/inervação , Imagem Tridimensional , Fibras Adrenérgicas/metabolismo , Animais , Criança , Fibras Colinérgicas/metabolismo , Glândulas Écrinas/citologia , Humanos , Camundongos Endogâmicos BALB C
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