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1.
J Neuroinflammation ; 20(1): 129, 2023 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-37245024

RESUMO

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by accumulation of extracellular amyloid beta (Aß) and intracellular neurofibrillary tangles, leading to chronic activation of astrocytes and microglia and persistent neuroinflammation. Aß-linked activation of microglia and astrocytes leads to increased intracellular calcium and production of proinflammatory cytokines, impacting the progression of neurodegeneration. An N-terminal Aß fragment (Aß1-15) and a shorter hexapeptide core sequence within the N-Aß fragment (N-Aßcore: Aß10-15) have previously been shown to protect against Aß-induced mitochondrial dysfunction, oxidative stress and apoptosis in neurons and rescue synaptic and spatial memory deficits in an APP/PSEN1 mouse model. Here, we hypothesized that the N-Aß fragment and N-Aßcore are protective against Aß-induced gliotoxicity, promoting a neuroprotective environment and potentially alleviating the characteristically persistent neuroinflammation present in AD. METHODS: We treated ex vivo organotypic brain slice cultures from an aged familial AD mouse model, 5xFAD, with the N-Aßcore and used immunocytochemistry to assess the impact on astrogliosis and microgliosis and alterations in synaptophysin-positive puncta engulfed by microglia. Isolated neuron/glia cultures, mixed glial cultures or a microglial cell line were treated with oligomeric human Aß at concentrations mimicking the pathogenic concentrations (µM) observed in AD in the absence or presence of the non-toxic N-terminal Aß fragments. Resultant changes in synaptic density, gliosis, oxidative stress, mitochondrial dysfunction, apoptosis, and the expression and release of proinflammatory markers were then determined. RESULTS: We demonstrate that the N-terminal Aß fragments mitigated the phenotypic switch leading to astrogliosis and microgliosis induced by pathological concentrations of Aß in mixed glial cultures and organotypic brain slice cultures from the transgenic 5xFAD mouse model, while protecting against Aß-induced oxidative stress, mitochondrial dysfunction and apoptosis in isolated astrocytes and microglia. Moreover, the addition of the N-Aßcore attenuated the expression and release of proinflammatory mediators in microglial cells activated by Aß and rescued microglia-mediated loss of synaptic elements induced by pathological levels of Aß. CONCLUSIONS: Together, these findings indicate the protective functions of the N-terminal Aß fragments extend to reactive gliosis and gliotoxicity induced by Aß, by preventing or reversing glial reactive states indicative of neuroinflammation and synaptic loss central to AD pathogenesis.


Assuntos
Doença de Alzheimer , Camundongos , Humanos , Animais , Idoso , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/toxicidade , Peptídeos beta-Amiloides/metabolismo , Gliose/metabolismo , Doenças Neuroinflamatórias , Camundongos Transgênicos , Inflamação/metabolismo , Microglia/metabolismo , Modelos Animais de Doenças
2.
J Biol Chem ; 296: 100402, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33571523

RESUMO

Beta-amyloid (Aß) has been recognized as an early trigger in the pathogenesis of Alzheimer's disease (AD) leading to synaptic and cognitive impairments. Aß can alter neuronal signaling through interactions with nicotinic acetylcholine receptors (nAChRs), contributing to synaptic dysfunction in AD. The three major nAChR subtypes in the hippocampus are composed of α7-, α4ß2-, and α3ß4-nAChRs. Aß selectively affects α7- and α4ß2-nAChRs, but not α3ß4-nAChRs in hippocampal neurons, resulting in neuronal hyperexcitation. However, how nAChR subtype selectivity for Aß affects synaptic function in AD is not completely understood. Here, we showed that Aß associated with α7- and α4ß2-nAChRs but not α3ß4-nAChRs. Computational modeling suggested that two amino acids in α7-nAChRs, arginine 208 and glutamate 211, were important for the interaction between Aß and α7-containing nAChRs. These residues are conserved only in the α7 and α4 subunits. We therefore mutated these amino acids in α7-containing nAChRs to mimic the α3 subunit and found that mutant α7-containing receptors were unable to interact with Aß. In addition, mutant α3-containing nAChRs mimicking the α7 subunit interact with Aß. This provides direct molecular evidence for how Aß selectively interacted with α7- and α4ß2-nAChRs, but not α3ß4-nAChRs. Selective coactivation of α7- and α4ß2-nAChRs also sufficiently reversed Aß-induced AMPA receptor dysfunction, including Aß-induced reduction of AMPA receptor phosphorylation and surface expression in hippocampal neurons. Moreover, costimulation of α7- and α4ß2-nAChRs reversed the Aß-induced disruption of long-term potentiation. These findings support a novel mechanism for Aß's impact on synaptic function in AD, namely, the differential regulation of nAChR subtypes.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Receptores Nicotínicos/metabolismo , Potenciais Sinápticos/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/farmacologia , Experimentação Animal , Animais , Células Cultivadas , Células HEK293 , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Potenciação de Longa Duração , Camundongos , Neurônios/metabolismo , Neurônios/patologia
3.
J Neurosci ; 38(15): 3708-3728, 2018 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-29540552

RESUMO

The c-Jun N-terminal kinase (JNK) signal transduction pathway is implicated in learning and memory. Here, we examined the role of JNK activation mediated by the JNK-interacting protein 1 (JIP1) scaffold protein. We compared male wild-type mice with a mouse model harboring a point mutation in the Jip1 gene that selectively blocks JIP1-mediated JNK activation. These male mutant mice exhibited increased NMDAR currents, increased NMDAR-mediated gene expression, and a lower threshold for induction of hippocampal long-term potentiation. The JIP1 mutant mice also displayed improved hippocampus-dependent spatial memory and enhanced associative fear conditioning. These results were confirmed using a second JIP1 mutant mouse model that suppresses JNK activity. Together, these observations establish that JIP1-mediated JNK activation contributes to the regulation of hippocampus-dependent, NMDAR-mediated synaptic plasticity and learning.SIGNIFICANCE STATEMENT The results of this study demonstrate that c-Jun N-terminal kinase (JNK) activation induced by the JNK-interacting protein 1 (JIP1) scaffold protein negatively regulates the threshold for induction of long-term synaptic plasticity through the NMDA-type glutamate receptor. This change in plasticity threshold influences learning. Indeed, mice with defects in JIP1-mediated JNK activation display enhanced memory in hippocampus-dependent tasks, such as contextual fear conditioning and Morris water maze, indicating that JIP1-JNK constrains spatial memory. This study identifies JIP1-mediated JNK activation as a novel molecular pathway that negatively regulates NMDAR-dependent synaptic plasticity and memory.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Plasticidade Neuronal , Memória Espacial , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células Cultivadas , Condicionamento Clássico , Hipocampo/citologia , Hipocampo/metabolismo , Hipocampo/fisiologia , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Neurônios/fisiologia , Mutação Puntual , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo
4.
J Neurochem ; 144(2): 201-217, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29164616

RESUMO

High levels (µM) of beta amyloid (Aß) oligomers are known to trigger neurotoxic effects, leading to synaptic impairment, behavioral deficits, and apoptotic cell death. The hydrophobic C-terminal domain of Aß, together with sequences critical for oligomer formation, is essential for this neurotoxicity. However, Aß at low levels (pM-nM) has been shown to function as a positive neuromodulator and this activity resides in the hydrophilic N-terminal domain of Aß. An N-terminal Aß fragment (1-15/16), found in cerebrospinal fluid, was also shown to be a highly active neuromodulator and to reverse Aß-induced impairments of long-term potentiation. Here, we show the impact of this N-terminal Aß fragment and a shorter hexapeptide core sequence in the Aß fragment (Aßcore: 10-15) to protect or reverse Aß-induced neuronal toxicity, fear memory deficits and apoptotic death. The neuroprotective effects of the N-terminal Aß fragment and Aßcore on Aß-induced changes in mitochondrial function, oxidative stress, and apoptotic neuronal death were demonstrated via mitochondrial membrane potential, live reactive oxygen species, DNA fragmentation and cell survival assays using a model neuroblastoma cell line (differentiated NG108-15) and mouse hippocampal neuron cultures. The protective action of the N-terminal Aß fragment and Aßcore against spatial memory processing deficits in amyloid precursor protein/PSEN1 (5XFAD) mice was demonstrated in contextual fear conditioning. Stabilized derivatives of the N-terminal Aßcore were also shown to be fully protective against Aß-triggered oxidative stress. Together, these findings indicate an endogenous neuroprotective role for the N-terminal Aß fragment, while active stabilized N-terminal Aßcore derivatives offer the potential for therapeutic application.


Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/toxicidade , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/farmacologia , Peptídeos beta-Amiloides/química , Animais , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Condicionamento Operante/efeitos dos fármacos , Medo , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Oligopeptídeos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fragmentos de Peptídeos/química , Espécies Reativas de Oxigênio/metabolismo
5.
J Biol Chem ; 290(35): 21409-20, 2015 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-26139609

RESUMO

Among putative downstream synaptic targets of ß-amyloid (Aß) are signaling molecules involved in synaptic function, memory formation and cognition, such as the MAP kinases, MKPs, CaMKII, CREB, Fyn, and Tau. Here, we assessed the activation and interaction of signaling pathways upon prolonged exposure to Aß in model nerve cells expressing nicotinic acetylcholine receptors (nAChRs). Our goal was to characterize the steps underlying sensitization of the nerve cells to neurotoxicity when Aß-target receptors are present. Of particular focus was the connection of the activated signaling molecules to oxidative stress. Differentiated neuroblastoma cells expressing mouse α4ß2-nAChRs were exposed to Aß1-42 for intervals from 30 min to 3 days. The cells and cell-derived protein extracts were then probed for activation of signaling pathway molecules (ERK, JNK, CaMKII, CREB, MARCKS, Fyn, tau). Our results show substantial, progressive activation of ERK in response to nanomolar Aß exposure, starting at the earliest time point. Increased ERK activation was followed by JNK activation as well as an increased expression of PHF-tau, paralleled by increased levels of reactive oxygen species (ROS). The impact of prolonged Aß on the levels of pERK, pJNK, and ROS was attenuated by MEK-selective and JNK-selective inhibitors. In addition, the MEK inhibitor as well as a JNK inhibitor attenuated Aß-induced nuclear fragmentation, which followed the changes in ROS levels. These results demonstrate that the presence of nAChRs sensitizes neurons to the neurotoxic action of Aß through the timed activation of discrete intracellular signaling molecules, suggesting pathways involved in the early stages of Alzheimer disease.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Sistema de Sinalização das MAP Quinases , Neurônios/metabolismo , Neurônios/patologia , Fragmentos de Peptídeos/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Linhagem Celular Tumoral , Camundongos , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Proteínas tau/metabolismo
6.
J Neurosci ; 34(43): 14210-8, 2014 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-25339735

RESUMO

Soluble ß-amyloid has been shown to regulate presynaptic Ca(2+) and synaptic plasticity. In particular, picomolar ß-amyloid was found to have an agonist-like action on presynaptic nicotinic receptors and to augment long-term potentiation (LTP) in a manner dependent upon nicotinic receptors. Here, we report that a functional N-terminal domain exists within ß-amyloid for its agonist-like activity. This sequence corresponds to a N-terminal fragment generated by the combined action of α- and ß-secretases, and resident carboxypeptidase. The N-terminal ß-amyloid fragment is present in the brains and CSF of healthy adults as well as in Alzheimer's patients. Unlike full-length ß-amyloid, the N-terminal ß-amyloid fragment is monomeric and nontoxic. In Ca(2+) imaging studies using a model reconstituted rodent neuroblastoma cell line and isolated mouse nerve terminals, the N-terminal ß-amyloid fragment proved to be highly potent and more effective than full-length ß-amyloid in its agonist-like action on nicotinic receptors. In addition, the N-terminal ß-amyloid fragment augmented theta burst-induced post-tetanic potentiation and LTP in mouse hippocampal slices. The N-terminal fragment also rescued LTP inhibited by elevated levels of full-length ß-amyloid. Contextual fear conditioning was also strongly augmented following bilateral injection of N-terminal ß-amyloid fragment into the dorsal hippocampi of intact mice. The fragment-induced augmentation of fear conditioning was attenuated by coadministration of nicotinic antagonist. The activity of the N-terminal ß-amyloid fragment appears to reside largely in a sequence surrounding a putative metal binding site, YEVHHQ. These findings suggest that the N-terminal ß-amyloid fragment may serve as a potent and effective endogenous neuromodulator.


Assuntos
Peptídeos beta-Amiloides/farmacologia , Cálcio/fisiologia , Condicionamento Psicológico/fisiologia , Medo/fisiologia , Plasticidade Neuronal/fisiologia , Terminações Pré-Sinápticas/fisiologia , Sequência de Aminoácidos , Peptídeos beta-Amiloides/fisiologia , Animais , Linhagem Celular Tumoral , Condicionamento Psicológico/efeitos dos fármacos , Medo/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Plasticidade Neuronal/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Terminações Pré-Sinápticas/efeitos dos fármacos
7.
J Biol Chem ; 288(16): 11175-90, 2013 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-23479730

RESUMO

Although the interaction between ß-amyloid (Aß) and nicotinic acetylcholine receptors has been widely studied, the impact of prolonged exposure to Aß on nAChR expression and signaling is not known. In this study, we employed a neuronal culture model to better understand the impact of sustained exposure of Aß on the regulation of cellular and synaptic function. The differentiated rodent neuroblastoma cell line NG108-15 expressing exogenous high-affinity α4ß2 nAChRs was exposed to soluble oligomeric Aß for several days. Ca(2+) responses, expression levels of α4ß2 nAChRs, rate of mitochondrial movement, mitochondrial fission, levels of reactive oxygen species, and nuclear integrity were compared between Aß-treated and untreated cells, transfected or not (mock-transfected) with α4ß2 nAChRs. Sustained exposure of Aß(1-42) to α4ß2 nAChR-transfected cells for several days led to increased Ca(2+) responses on subsequent acute stimulation with Aß(1-42) or nicotine, paralleled by increased expression levels of α4ß2 nAChRs, likely the result of enhanced receptor recycling. The rate of mitochondrial movement was sharply reduced, whereas the mitochondrial fission protein pDrp-1 was increased in α4ß2 nAChR-transfected cells treated with Aß(1-42). In addition, the presence of α4ß2 nAChRs dramatically enhanced Aß(1-42)-mediated increases in reactive oxygen species and nuclear fragmentation, eventually leading to apoptosis. Our data thus show disturbed calcium homeostasis coupled with mitochondrial dysfunction and loss of neuronal integrity on prolonged exposure of Aß in cells transfected with α4ß2 nAChRs. Together, the results suggest that the presence of nAChRs sensitizes neurons to the toxic actions of soluble oligomeric Aß, perhaps contributing to the cholinergic deficit in Alzheimer disease.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Cálcio/metabolismo , Homeostase , Modelos Biológicos , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores Nicotínicos/metabolismo , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/genética , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Neurônios/patologia , Fragmentos de Peptídeos/genética , Espécies Reativas de Oxigênio/metabolismo , Receptores Nicotínicos/genética
8.
Br J Pharmacol ; 181(17): 3172-3191, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38720171

RESUMO

BACKGROUND AND PURPOSE: Oligomeric amyloid ß 1-42 (oAß1-42) exhibits agonist-like action at human α7- and α7ß2-containing nicotinic receptors. The N-terminal amyloid ß1-15 fragment (N-Aß fragment) modulates presynaptic calcium and enhances hippocampal-based synaptic plasticity via α7-containing nicotinic receptors. Further, the N-Aß fragment and its core sequence, the N-amyloid-beta core hexapeptide (N-Aßcore), protect against oAß1-42-associated synapto- and neurotoxicity. Here, we investigated how oAß1-42, the N-Aß fragment, and the N-Aßcore regulate the single-channel properties of α7- and α7ß2-nicotinic receptors. EXPERIMENTAL APPROACH: Single-channel recordings measured the impact of acetylcholine, oAß1-42, the N-Aß fragment, and the N-Aßcore on the unitary properties of human α7- and α7ß2-containing nicotinic receptors expressed in nicotinic-null SH-EP1 cells. Molecular dynamics simulations identified potential sites of interaction between the N-Aß fragment and orthosteric α7+/α7- and α7+/ß2- nicotinic receptor binding interfaces. KEY RESULTS: The N-Aß fragment and N-Aßcore induced α7- and α7ß2-nicotinic receptor single-channel openings. Relative to acetylcholine, oAß1-42 preferentially enhanced α7ß2-nicotinic receptor single-channel open probability and open-dwell times. Co-application with the N-Aßcore neutralized these effects. Further, administration of the N-Aß fragment alone, or in combination with acetylcholine or oAß1-42, selectively enhanced α7-nicotinic receptor open probability and open-dwell times (compared to acetylcholine or oAß1-42). CONCLUSIONS AND IMPLICATIONS: Amyloid-beta peptides demonstrate functional diversity in regulating α7- and α7ß2-nicotinic receptor function, with implications for a wide range of nicotinic receptor-mediated functions in Alzheimer's disease. The effects of these peptides on α7- and/or α7ß2-nicotinic receptors revealed complex interactions with these subtypes, providing novel insights into the neuroprotective actions of amyloid ß-derived fragments against the toxic effects of oAß1-42.


Assuntos
Peptídeos beta-Amiloides , Fragmentos de Peptídeos , Receptor Nicotínico de Acetilcolina alfa7 , Humanos , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/farmacologia , Peptídeos beta-Amiloides/toxicidade , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Fragmentos de Peptídeos/farmacologia , Fármacos Neuroprotetores/farmacologia , Simulação de Dinâmica Molecular , Acetilcolina/farmacologia , Receptores Nicotínicos/metabolismo
9.
J Neurosci ; 31(7): 2537-48, 2011 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-21325521

RESUMO

Diverse nicotinic acetylcholine receptor (nAChR) subtypes containing different subunit combinations can be placed on nerve terminals or soma/dendrites in the ventral tegmental area (VTA). nAChR α6 subunit message is abundant in the VTA, but α6*-nAChR cellular localization, function, pharmacology, and roles in cholinergic modulation of dopaminergic (DA) neurons within the VTA are not well understood. Here, we report evidence for α6ß2*-nAChR expression on GABA neuronal boutons terminating on VTA DA neurons. α-Conotoxin (α-Ctx) MII labeling coupled with immunocytochemical staining localizes putative α6*-nAChRs to presynaptic GABAergic boutons on acutely dissociated, rat VTA DA neurons. Functionally, acetylcholine (ACh) induces increases in the frequency of bicuculline-, picrotoxin-, and 4-aminopyridine-sensitive miniature IPSCs (mIPSCs) mediated by GABA(A) receptors. These increases are abolished by α6*-nAChR-selective α-Ctx MII or α-Ctx PIA (1 nm) but not by α7 (10 nm methyllycaconitine) or α4* (1 µm dihydro-ß-erythroidine)-nAChR-selective antagonists. ACh also fails to increase mIPSC frequency in VTA DA neurons prepared from nAChR ß2 knock-out mice. Moreover, ACh induces an α-Ctx PIA-sensitive elevation in intraterminal Ca(2+) in synaptosomes prepared from the rat VTA. Subchronic exposure to 500 nm nicotine reduces ACh-induced GABA release onto the VTA DA neurons, as does 10 d of systemic nicotine exposure. Collectively, these results indicate that α6ß2*-nAChRs are located on presynaptic GABAergic boutons within the VTA and modulate GABA release onto DA neurons. These presynaptic α6ß2*-nAChRs likely play important roles in nicotinic modulation of DA neuronal activity.


Assuntos
Dopamina/metabolismo , Neurônios/citologia , Terminações Pré-Sinápticas/fisiologia , Receptores Nicotínicos/metabolismo , Área Tegmentar Ventral/citologia , Ácido gama-Aminobutírico/metabolismo , 4-Aminopiridina/farmacologia , Acetilcolina/farmacologia , Aconitina/análogos & derivados , Aconitina/farmacologia , Animais , Animais Recém-Nascidos , Bungarotoxinas/farmacologia , Cálcio/metabolismo , Conotoxinas/metabolismo , Di-Hidro-beta-Eritroidina/farmacologia , Dopamina/farmacologia , Interações Medicamentosas , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Feminino , GABAérgicos/farmacologia , Glutamato Descarboxilase/metabolismo , Técnicas In Vitro , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Neurônios/efeitos dos fármacos , Nicotina/farmacologia , Antagonistas Nicotínicos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores Nicotínicos/deficiência , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo
10.
J Biol Chem ; 286(39): 34373-81, 2011 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-21828053

RESUMO

Soluble ß-amyloid (Aß) resides in certain regions of the brain at or near picomolar concentration, rising in level during the prodromic stage of Alzheimer disease. Recently, we identified the homomeric α7 nicotinic acetylcholine receptor (α7-nAChR) as one possible functional target for picomolar Aß. This study was aimed at addressing which residues in α7-nAChRs potentially interact with Aß to regulate the presynaptic function of this receptor. Site-directed mutagenesis was carried out to study the key aromatic residues in the mouse α7-nAChR agonist-binding pocket. Mutations of tyrosine188 resulted in a decrease in activation of presynaptic α7-nAChRs by ACh and Aß but with no change in response to nicotine, indicating the critical role of Tyr-188 in presynaptic regulation by Aß. Coimmunoprecipitation additionally revealed direct binding of Aß to α7-nAChRs and to the Tyr-188 mutant receptor. In contrast, mutations of Tyr-195 in α7-nAChR led to decreased activation by nicotine without apparent effects on ACh- or Aß-induced responses. Agonist-induced responses of Tyr-93 mutant α7-nAChRs indicated possible interactions of nicotine and Aß with its hydroxyl group, but there was no change in presynaptic responses after mutation of Trp-149. All of the mutants were shown to be expressed on the plasma membrane using cell surface labeling. Together, these results directly demonstrate an essential role for the aromatic residue Tyr-188 as a key component in the agonist binding domain for the activation of α7-nAChRs by Aß.


Assuntos
Aminoácidos Aromáticos/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Amiloide/metabolismo , Receptores Nicotínicos/metabolismo , Aminoácidos Aromáticos/genética , Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Linhagem Celular , Camundongos , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Mapeamento de Peptídeos , Estrutura Terciária de Proteína , Receptores Nicotínicos/genética , Receptor Nicotínico de Acetilcolina alfa7
11.
ACS Chem Neurosci ; 13(1): 43-52, 2022 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-34913695

RESUMO

Neuroinflammation contributes to the pathogenesis of several neurodegenerative disorders. Glycogen synthase kinase-3ß (GSK-3ß) regulates the release of proinflammatory cytokines and promotes inflammatory responses in immune cells. Microglia are the resident mononuclear immune cells of the central nervous system. Here, we investigated the anti-neuroinflammatory effects of (2S,3S,4R,5R,6S)-6-(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-6-yl)-3,4,5-trihydroxy-N-((S)-1,1,1-trifluoropropan-2-yl)tetrahydro-2H-pyran-2-carboxamide (TFGF-18), a semisynthetic GSK-3ß inhibitor, in lipopolysaccharide (LPS) activation of spontaneously immortalized SIM-A9 microglial cells and of mouse cortical microglia. TFGF-18 at 2.5 µM concentration inhibited LPS-induced production of nitric oxide by 56.3% and the proinflammatory cytokines TNF-α and IL-1ß by 28.3 and 59.2% in SIM-A9 cells, respectively, relative to the LPS treatment control group. Pretreatment of mouse primary microglial cells with TFGF-18 at 2.5 µM concentration led to a reduction of 58.7% in TNF-α+ microglial cells at 24 h post-LPS stimulation. The migration of LPS-activated SIM-A9 cells was also reduced by 26.7% with pretreatment of TFGF-18 in a scratch assay. Analyses of signaling pathways demonstrated that TFGF-18 led to the suppression of LPS-induced GSK-3ß activation and p65/NF-κB activity. Furthermore, the co-culture of SIM-A9 with SH-SY5Y neuroblastoma cells showed the suppression of TFGF-18 to microglia-mediated neurotoxicity in vitro. The findings indicate strong inhibitory effects of TFGF-18 on LPS-induced microglia activation via regulation of GSK-3ß and downstream p65/NF-κB signaling. The results suggest a potential role of TFGF-18 in neuroprotection via its anti-neuroinflammatory effect.


Assuntos
Lipopolissacarídeos , Microglia , Animais , Glicogênio Sintase Quinase 3 beta , Luteolina , Camundongos , Microglia/metabolismo , NF-kappa B/metabolismo , Doenças Neuroinflamatórias , Óxido Nítrico Sintase Tipo II/metabolismo
12.
Front Mol Neurosci ; 14: 576038, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33912008

RESUMO

Alzheimer's disease (AD) is the most common cause of dementia in the aging population. Evidence implicates elevated soluble oligomeric Aß as one of the primary triggers during the prodromic phase leading to AD, effected largely via hyperphosphorylation of the microtubule-associated protein tau. At low, physiological levels (pM-nM), however, oligomeric Aß has been found to regulate synaptic plasticity as a neuromodulator. Through mutational analysis, we found a core hexapeptide sequence within the N-terminal domain of Aß (N-Aßcore) accounting for its physiological activity, and subsequently found that the N-Aßcore peptide is neuroprotective. Here, we characterized the neuroprotective potential of the N-Aßcore against dysfunction of synaptic plasticity assessed in ex vivo hippocampal slices from 5xFAD APP/PS1 mice, specifically hippocampal long-term potentiation (LTP) and long-term depression (LTD). The N-Aßcore was shown to reverse impairment in synaptic plasticity in hippocampal slices from 5xFAD APP/PS1 model mice, both for LTP and LTD. The reversal by the N-Aßcore correlated with alleviation of downregulation of hippocampal AMPA-type glutamate receptors in preparations from 5xFAD mice. The action of the N-Aßcore depended upon a critical di-histidine sequence and involved the phosphoinositide-3 (PI3) kinase pathway via mTOR (mammalian target of rapamycin). Together, the present findings indicate that the non-toxic N-Aßcore hexapeptide is not only neuroprotective at the cellular level but is able to reverse synaptic dysfunction in AD-like models, specifically alterations in synaptic plasticity.

13.
Behav Brain Res ; 398: 112968, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33069740

RESUMO

ß-Amyloid (Aß) elevation, tau hyperphosphorylation, and neuroinflammation are major hallmarks of Alzheimer's disease (AD). Glycogen synthase kinase-3ß (GSK-3ß) is a key protein kinase implicated in the pathogenesis of AD. Blockade of GSK-3ß is an attractive therapeutic strategy for AD. Isoorientin, a 6-C-glycosylflavone, was previously shown to be a highly selective inhibitor of GSK-3ß, while exerting neuroprotective effects in neuronal models of AD. In the present study, we evaluated the in vivo effects of isoorientin on GSK-3ß, tau phosphorylation, Aß deposition, neuroinflammatory response, long-term potentiation, and spatial memory in amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice using biochemical, electrophysiological, and behavioral tests. Chronic oral administration of isoorientin to APP/PS1 mice at 8 months of age attenuated multiple AD pathogenic hallmarks in the brains, including GSK-3ß overactivation, tau hyperphosphorylation, Aß deposition, and neuroinflammation. For neuroinflammation, isoorientin treatment reduced the number of activated microglia associated with Aß-positive plaques, and in parallel reduced the levels of pro-inflammatory factors in the brains of APP/PS1 mice. Strikingly, isoorientin reversed deficits in synaptic long-term potentiation and spatial memory relevant to cognitive functions. Together, the findings suggest that isoorientin is a brain neuroprotector and may be a promising drug lead for treatment of AD and related neurodegenerative disorders.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Precursor de Proteína beta-Amiloide/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Inflamação/tratamento farmacológico , Luteolina/farmacologia , Transtornos da Memória/tratamento farmacológico , Microglia/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Presenilina-1/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Memória Espacial/efeitos dos fármacos , Proteínas tau/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Transgênicos , Fosforilação/efeitos dos fármacos
14.
Eur J Neurosci ; 31(5): 788-96, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20374280

RESUMO

Beta amyloid (Abeta) plays a central role in the pathogenesis of Alzheimer's disease. Abeta is the major constituent of senile plaques, but there is a significant presence of Abeta in the brain in soluble forms. The results of functional studies indicate that soluble Abeta interacts with the alpha7 nicotinic acetylcholine receptor (nAChR) complex with apparent high affinity. However, conflicting data exist as to the nature of the Abeta-alpha7 nAChR interaction, and whether it is the result of specific binding. Moreover, both agonist-like and antagonist-like effects have been reported. In particular, agonist-like effects have been observed for presynaptic nAChRs. Here, we demonstrate Abeta(1-42)-evoked stimulatory changes in presynaptic Ca(2+) level via exogenous alpha7 nAChRs expressed in the axonal varicosities of differentiated hybrid neuroblastoma NG108-15 cells as a model, presynaptic system. The Abeta(1-42)-evoked responses were concentration-dependent and were sensitive to the highly selective alpha7 nAChR antagonist alpha-bungarotoxin. Voltage-gated Ca(2+) channels and internal Ca(2+) stores were both involved in Abeta(1-42)-evoked increases in presynaptic Ca(2+) following activation of alpha7 nAChRs. In addition, disruption of lipid rafts by cholesterol depletion led to substantially attenuated responses to Abeta(1-42), whereas responses to nicotine were largely intact. These results directly implicate the nicotinic receptor complex as a target for the agonist-like action of pico- to nanomolar concentrations of soluble Abeta(1-42) on the presynaptic nerve terminal, including the possible involvement of receptor-associated lipid rafts. This interaction probably plays an important neuromodulatory role in synaptic dynamics.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Microdomínios da Membrana/metabolismo , Neurônios/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Potenciais Evocados/fisiologia , Imuno-Histoquímica , Camundongos , Microscopia Confocal , Ratos , Sinapses/metabolismo , Receptor Nicotínico de Acetilcolina alfa7
15.
Sci Rep ; 10(1): 5696, 2020 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-32231242

RESUMO

Understanding the specific gene changes underlying the prodromic stages of Alzheimer's disease pathogenesis will aid the development of new, targeted therapeutic strategies for this neurodegenerative disorder. Here, we employed RNA-sequencing to analyze global differential gene expression in a defined model nerve cell line expressing α4ß2 nicotinic receptors (nAChRs), high-affinity targets for beta amyloid (Aß). The nAChR-expressing neuronal cells were treated with nanomolar Aß1-42 to gain insights into the molecular mechanisms underlying Aß-induced neurotoxicity in the presence of this sensitizing target receptor. We identified 15 genes (out of 15,336) that were differentially expressed upon receptor-linked Aß treatment. Genes up-regulated with Aß treatment were associated with calcium signaling and axonal vesicle transport (including the α4 nAChR subunit, the calcineurin regulator RCAN3, and KIF1C of the kinesin family). Downregulated genes were associated with metabolic, apoptotic or DNA repair pathways (including APBA3, PARP1 and RAB11). Validation of the differential expression was performed via qRT-PCR and immunoblot analysis in the defined model nerve cell line and primary mouse neurons. Further verification was performed using immunocytochemistry. In conclusion, we identified apparent changes in gene expression on Aß treatment in the presence of the sensitizing nAChRs, linked to early-stage Aß-induced neurotoxicity, which may represent novel therapeutic targets.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Receptores Nicotínicos/metabolismo , Transcriptoma , Doença de Alzheimer/metabolismo , Animais , Linhagem Celular , Reparo do DNA , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos Transgênicos , Neurônios/metabolismo , Reação em Cadeia da Polimerase
16.
J Neurochem ; 109(5): 1452-8, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19457164

RESUMO

Disruption of neuronal signaling by soluble beta-amyloid has been implicated in deficits in short-term recall in the early stages of Alzheimer's disease. One potential target for beta-amyloid is the synapse, with evidence for differential interaction with both pre- and post-synaptic elements. Our previous work revealed an agonist-like action of soluble beta-amyloid (pM to nM) on isolated pre-synaptic terminals to increase [Ca(2+)]i, with apparent involvement of pre-synaptic nicotinic receptors. To directly establish the role of nicotinic receptors in pre-synaptic Ca(2+) regulation, we investigated the pre-synaptic action of beta-amyloid on terminals isolated from mice harboring either beta2 or alpha7 nicotinic receptor null mutants (knockouts). Average pre-synaptic responses to beta-amyloid in hippocampal terminals of alpha7 knockout mice were unchanged, whereas responses in hippocampal terminals from beta2 knockout mice were strongly attenuated. In contrast, pre-synaptic responses to soluble beta-amyloid were strongly attenuated in cortical terminals from alpha7 knockout mice but were moderately attenuated in cortical terminals from beta2 knockout mice. The latter responses, having distinct kinetics, were completely blocked by alpha-bungarotoxin. The use of receptor null mutants thus permitted direct demonstration of the involvement of specific nicotinic receptors in pre-synaptic Ca(2+) regulation by soluble beta-amyloid, and also indicated differential neuromodulation by beta-amyloid of synapses in hippocampus and cortex.


Assuntos
Peptídeos beta-Amiloides/farmacologia , Córtex Cerebral/citologia , Hipocampo/citologia , Fragmentos de Peptídeos/farmacologia , Receptores Nicotínicos/fisiologia , Sinaptossomos/fisiologia , Animais , Bungarotoxinas/farmacologia , Cálcio/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/deficiência , Sinaptossomos/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7
17.
Acta Pharmacol Sin ; 30(6): 788-94, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19498419

RESUMO

AIM: Substantial colocalization of functionally independent alpha4 nicotinic acetylcholine receptors and 5-HT(3) serotonin receptors on presynaptic terminals has been observed in brain. The present study was aimed at addressing whether nicotinic acetylcholine receptors and 5-HT(3) serotonin receptors interact on the same presynaptic terminal, suggesting a convergence of cholinergic and serotonergic regulation. METHODS: Ca(2+) responses in individual, isolated nerve endings purified from rat striatum were measured using confocal imaging. RESULTS: Application of 500 nmol/L nicotine following sustained stimulation with the highly selective 5-HT(3) receptor agonist m-chlorophenylbiguanide at 100 nmol/L resulted in markedly reduced Ca(2+) responses (28% of control) in only those striatal nerve endings that originally responded to m-chlorophenylbiguanide. The cross-regulation developed over several minutes. Presynaptic nerve endings that had not responded to m-chlorophenylbiguanide, indicating that 5-HT(3) receptors were not present, displayed typical responses to nicotine. Application of m-chlorophenylbiguanide following sustained stimulation with nicotine resulted in partially attenuated Ca(2+) responses (49% of control). Application of m-chlorophenylbiguanide following sustained stimulation with m-chlorophenylbiguanide also resulted in a strong attenuation of Ca(2+) responses (12% of control), whereas nicotine-induced Ca(2+) responses following sustained stimulation with nicotine were not significantly different from control. CONCLUSION: These results indicate that the presynaptic Ca(2+) increases evoked by either 5-HT(3) receptor or nicotinic acetylcholine receptor activation regulate subsequent responses to 5-HT(3) receptor activation, but that only 5-HT(3) receptors cross-regulate subsequent nicotinic acetylcholine receptor-mediated responses. The findings suggest a specific interaction between the two receptor systems in the same striatal nerve terminal, likely involving Ca(2+)-dependent intracellular pathways that regulate these signaling systems at one or more levels.


Assuntos
Cálcio/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores Nicotínicos/metabolismo , Receptores 5-HT3 de Serotonina/metabolismo , Animais , Biguanidas/farmacologia , Corpo Estriado/metabolismo , Masculino , Microscopia Confocal , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores Nicotínicos/efeitos dos fármacos , Receptores 5-HT3 de Serotonina/efeitos dos fármacos , Agonistas do Receptor de Serotonina/farmacologia , Transdução de Sinais
18.
Trends Mol Med ; 25(8): 685-695, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31248781

RESUMO

Alzheimer's disease (AD) is a relentlessly progressive neurodegenerative disease, currently incurable, which presents one of the largest unmet needs in medicine. AD is histologically characterized by the accumulation of extracellular amyloid-beta (Aß), evident as senile plaques and intracellular neurofibrillary tangles composed of hyperphosphorylated tau. However, the levels of diffusible extracellular Aß, a neuropeptide largely present in oligomeric form, rise by orders of magnitude many years before evident pathology and subsequent AD diagnosis. The long delay in neurotoxicity and synaptic dysfunction triggered by Aß and driven by abnormal tau indicates the presence of inherent neuroprotective systems in brain. Here, we propose that strategic approaches for the identification and implementation of neuroprotective agents could provide novel therapeutics for this devastating disease.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Descoberta de Drogas , Fármacos Neuroprotetores/farmacologia , Agregação Patológica de Proteínas/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Biomarcadores , Gerenciamento Clínico , Progressão da Doença , Suscetibilidade a Doenças , Descoberta de Drogas/métodos , Humanos , Terapia de Alvo Molecular , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Agregação Patológica de Proteínas/tratamento farmacológico , Transdução de Sinais , Sinapses/efeitos dos fármacos , Sinapses/metabolismo
19.
Trends Pharmacol Sci ; 39(4): 354-366, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29428175

RESUMO

The α7 nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel (LGIC) that plays an important role in cellular calcium signaling and contributes to several neurological diseases. Agonist binding to the α7 nAChR induces fast channel activation followed by inactivation and prolonged desensitization while triggering long-lasting calcium signaling. These activities foster neurotransmitter release, synaptic plasticity, and somatodendritic regulation in the brain. We discuss here the ability of α7 nAChRs to operate in ionotropic (α7i) and metabotropic (α7m) modes, leading to calcium-induced calcium release (CICR) and G protein-associated inositol trisphosphate (IP3)-induced calcium release (IICR), respectively. Metabotropic activity extends the spatial and temporal aspects of calcium signaling by the α7 channel beyond its ionotropic limits, persisting into the desensitized state. Delineation of the ionotropic and metabotropic properties of the α7 nAChR will provide definitive indicators of moment-to-moment receptor functional status that will, in turn, spearhead new drug development.


Assuntos
Cálcio/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Animais , Sinalização do Cálcio , Proteínas de Ligação ao GTP/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Plasticidade Neuronal , Transdução de Sinais , Transmissão Sináptica , Receptor Nicotínico de Acetilcolina alfa7/química
20.
Brain Res ; 1182: 82-9, 2007 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-17935702

RESUMO

The levels of soluble beta-amyloid (Abeta) are correlated with symptom severity in Alzheimer's disease. Soluble Abeta has been shown to disrupt synaptic function and it has been proposed that accumulation of soluble Abeta triggers synapse loss over the course of the disease. Numerous studies indicate that soluble Abeta has multiple targets, one of which appears to be the nicotinic acetylcholine receptor, particularly for Abeta concentrations of pM to nM. Moreover, pM to nM soluble Abeta was found to increase presynaptic Ca(2+) levels, suggesting that it may have an impact on neurotransmitter release. In the present study, soluble Abeta was perfused into mouse prefrontal cortex and the effect on the release of dopamine outflow via microdialysis was assessed. In the presence of tetrodotoxin, Abeta(1-42) at 100 nM evoked the release of dopamine to approximately 170% of basal levels. The Abeta(1-42)-evoked dopamine release was sensitive to antagonists of alpha7 nicotinic receptors and was absent in mice harboring a null mutation for the alpha7 nicotinic subunit, but was intact in mice harboring a null mutation for the beta2 nicotinic subunit. The control peptide Abeta(40-1) was without effect. In contrast, Abeta(1-42) at 1-10 pM caused a profound but slowly developing decrease in dopamine outflow. These results suggest that Abeta alters dopamine release in mouse prefrontal cortex, perhaps involving distinct targets as it accumulates during Alzheimer's disease and leading to disruption of synaptic signaling.


Assuntos
Peptídeos beta-Amiloides/farmacologia , Dopamina/metabolismo , Fragmentos de Peptídeos/farmacologia , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Receptores Nicotínicos/fisiologia , Análise de Variância , Animais , Bungarotoxinas/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microdiálise/métodos , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Receptor Nicotínico de Acetilcolina alfa7
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