RESUMEN
It has been recently established that GPR158, a class C orphan G protein-coupled receptor, serves as a metabotropic glycine receptor. GPR158 is highly expressed in the nucleus accumbens (NAc), a major input structure of the basal ganglia that integrates information from cortical and subcortical structures to mediate goal-directed behaviors. However, whether glycine modulates neuronal activity in the NAc through GPR158 activation has not been investigated yet. Using whole-cell patch-clamp recordings, we found that glycine-dependent activation of GPR158 increased the firing rate of NAc medium spiny neurons (MSNs) while it failed to significantly affect the excitability of cholinergic interneurons (CIN). In MSNs GPR158 activation reduced the latency to fire, increased the action potential half-width, and reduced action potential afterhyperpolarization, effects that are all consistent with negative modulation of potassium M-currents, that in the central nervous system are mainly carried out by Kv7/KCNQ-channels. Indeed, we found that the GPR158-induced increase in MSN excitability was associated with decreased M-current amplitude, and selective pharmacological inhibition of the M-current mimicked and occluded the effects of GPR158 activation. In addition, when the protein kinase A (PKA) or extracellular signal-regulated kinase (ERK) signaling was pharmacologically blocked, modulation of MSN excitability by GPR158 activation was suppressed. Moreover, GPR158 activation increased the phosphorylation of ERK and Kv7.2 serine residues. Collectively, our findings suggest that GPR158/PKA/ERK signaling controls MSN excitability via Kv7.2 modulation. Glycine-dependent activation of GPR158 may significantly affect MSN firing in vivo, thus potentially mediating specific aspects of goal-induced behaviors.
Asunto(s)
Potenciales de Acción , Glicina , Neuronas , Núcleo Accumbens , Receptores Acoplados a Proteínas G , Animales , Glicina/farmacología , Glicina/metabolismo , Núcleo Accumbens/metabolismo , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/citología , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Masculino , Potenciales de Acción/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Receptores de Glicina/metabolismo , Técnicas de Placa-Clamp , Fosforilación/efectos de los fármacos , Neuronas Espinosas MedianasRESUMEN
AIMS: Early dysfunction in Alzheimer's disease (AD) is characterised by alterations of synapse structure and function leading to dysmorphic neurites, decreased spine density, impaired synaptic plasticity and cognitive deficits. The class II member HDAC4, which recently emerged as a crucial factor in shaping synaptic plasticity and memory, was found to be altered in AD. We investigated how the modulation of HDAC4 may contribute to counteracting AD pathogenesis. METHODS: Using a cytoplasmic HDAC4 mutant (HDAC4SD ), we studied the recovery of synaptic function in hippocampal tissue and primary neurons from the triple-transgenic mouse model of AD (3×Tg-AD). RESULTS: Here, we report that in wild-type mice, HDAC4 is localised at synapses and interacts with postsynaptic proteins, whereas in the 3×Tg-AD, it undergoes nuclear import, reducing its interaction with synaptic proteins. Of note, HDAC4 delocalisation was induced by both amyloid-ß and tau accumulation. Overexpression of the HDAC4SD mutant in CA1 pyramidal neurons of organotypic hippocampal slices obtained from 3×Tg-AD mice increased dendritic length and promoted the enrichment of N-cadherin, GluA1, PSD95 and CaMKII proteins at the synaptic level compared with AD neurons transfected with the empty vector. Moreover, HDAC4 overexpression recovered the level of SUMO2/3ylation of PSD95 in AD hippocampal tissue, and in AD organotypic hippocampal slices, the HDAC4SD rescued spine density and synaptic transmission. CONCLUSIONS: These results highlight a new role of cytoplasmic HDAC4 in providing a structural and enzymatic regulation of postsynaptic proteins. Our findings suggest that controlling HDAC4 localisation may represent a promising strategy to rescue synaptic function in AD, potentially leading to memory improvement.
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Enfermedad de Alzheimer , Animales , Ratones , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Modelos Animales de Enfermedad , Hipocampo/patología , Ratones Transgénicos , Sinapsis/patología , Transmisión Sináptica/fisiología , Citoplasma/metabolismoRESUMEN
Histamine, a monoamine implicated in stress-related arousal states, is synthesized in neurons exclusively located in the hypothalamic tuberomammillary nucleus (TMN) from where they diffusely innervate striatal and mesolimbic networks including the nucleus accumbens (NAc), a vital node in the limbic loop. Since histamine-containing TMN neuron output increases during stress, we hypothesized that exposure of mice to acute restrain stress (ARS) recruits endogenous histamine type 2 receptor (H2R) signaling in the NAc, whose activation increases medium spiny neurons (MSNs) intrinsic excitability via downregulation of A-type K+ currents. We employed an ARS paradigm in which mice were restrained for 120 min, followed by a 20-min recovery period, after which brain slices were prepared for ex vivo electrophysiology. Using whole-cell patch-clamp recordings, we found that pharmacological activation of H2R failed to affect MSN excitability and A-type K+ currents in mice that underwent ARS. Interestingly, in mice treated with H2R-antagonist prior to ARS paradigm, H2R activation increased evoked firing and decreased A-type K+ currents similarly to what observed in control mice. Furthermore, H2R-antagonist treatment ameliorated anxiety-like behavior in ARS mice. Together, our findings indicate that ARS paradigm recruits endogenous H2R signaling in MSNs and suggest the involvement of H2R signaling in stress-related motivational states.
Asunto(s)
Histamina , Núcleo Accumbens , Ratones , Animales , Potenciales de Acción/fisiología , Neuronas Espinosas Medianas , Técnicas de Placa-ClampRESUMEN
Glycogen synthase kinase 3ß (GSK3) is a multifaceted serine/threonine (S/T) kinase expressed in all eukaryotic cells. GSK3ß is highly enriched in neurons in the central nervous system where it acts as a central hub for intracellular signaling downstream of receptors critical for neuronal function. Unlike other kinases, GSK3ß is constitutively active, and its modulation mainly involves inhibition via upstream regulatory pathways rather than increased activation. Through an intricate converging signaling system, a fine-tuned balance of active and inactive GSK3ß acts as a central point for the phosphorylation of numerous primed and unprimed substrates. Although the full range of molecular targets is still unknown, recent results show that voltage-gated ion channels are among the downstream targets of GSK3ß. Here, we discuss the direct and indirect mechanisms by which GSK3ß phosphorylates voltage-gated Na+ channels (Nav1.2 and Nav1.6) and voltage-gated K+ channels (Kv4 and Kv7) and their physiological effects on intrinsic excitability, neuronal plasticity, and behavior. We also present evidence for how unbalanced GSK3ß activity can lead to maladaptive plasticity that ultimately renders neuronal circuitry more vulnerable, increasing the risk for developing neuropsychiatric disorders. In conclusion, GSK3ß-dependent modulation of voltage-gated ion channels may serve as an important pharmacological target for neurotherapeutic development.
Asunto(s)
Glucógeno Sintasa Quinasa 3 , Neuronas , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Canales Iónicos/metabolismo , Neuronas/metabolismo , Fosforilación , Proteínas Serina-Treonina QuinasasRESUMEN
Histaminergic neurons are exclusively located in the hypothalamic tuberomammillary nucleus, from where they project to many brain areas including the nucleus accumbens (NAc), a brain area that integrates diverse monoaminergic inputs to coordinate motivated behaviours. While the NAc expresses various histamine receptor subtypes, the mechanisms by which histamine modulates NAc activity are still poorly understood. Using whole-cell patch-clamp recordings, we found that pharmacological activation of histamine 2 (H2) receptors elevates the excitability of NAc medium spiny neurons (MSNs), while activation of H1 receptors failed to significantly affect MSN excitability. The evoked firing of MSNs increased after seconds of local H2 agonist administration and remained elevated for minutes. H2 receptor (H2R) activation accelerated subthreshold depolarization in response to current injection, reduced the latency to fire, diminished action potential afterhyperpolarization and increased the action potential half-width. The increased excitability was protein kinase A-dependent and associated with decreased A-type K+ currents. In addition, selective pharmacological inhibition of the Kv4.2 channel, the main molecular determinant of A-type K+ currents in MSNs, mimicked and occluded the increased excitability induced by H2R activation. Our results indicate that histaminergic transmission in the NAc increases MSN intrinsic excitability through H2R-dependent modulation of Kv4.2 channels. Activation of H2R will significantly alter spike firing in MSNs in vivo, and this effect could be an important mechanism by which these receptors mediate certain aspects of goal-induced behaviours. KEY POINTS: Histamine is synthesized and released by hypothalamic neurons of the tuberomammillary nucleus and serves as a general modulator for whole-brain activity including the nucleus accumbens. Histamine receptors type 2 (HR2), which are expressed in the nucleus accumbens, couple to Gαs/off proteins which elevate cyclic adenosine monophosphate levels and activate protein kinase A. Whole-cell patch-clamp recordings revealed that H2R activation increased the evoked firing in medium spiny neurons of the nucleus accumbens via protein kinase A-dependent mechanisms. HR2 activation accelerated subthreshold depolarization in response to current injection, reduced the latency to fire, diminished action potential medium after-hyperpolarization and increased the action potential half-width. HR2 activation also reduced A-type potassium current. Selective pharmacological inhibition of the Kv4.2 channel mimicked and occluded the increased excitability induced by H2R activation.
Asunto(s)
Histamina , Núcleo Accumbens , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Histamina/farmacología , Neuronas/fisiología , Núcleo Accumbens/fisiología , Receptores Histamínicos H2RESUMEN
Glioblastoma is the most aggressive and lethal brain cancer. Current treatments involve surgical resection, radiotherapy and chemotherapy. However, the life expectancy of patients with this disease remains short and chemotherapy leads to severe adverse effects. Furthermore, the presence of the blood-brain barrier (BBB) makes it difficult for drugs to effectively reach the brain. A promising strategy lies in the use of graphene quantum dots (GQDs), which are light-responsive graphene nanoparticles that have shown the capability of crossing the BBB. Here we investigate the effect of GQDs on U87 human glioblastoma cells and primary cortical neurons. Non-functionalized GQDs (NF-GQDs) demonstrated high biocompatibility, while dimethylformamide-functionalized GQDs (DMF-GQDs) showed a toxic effect on both cell lines. The combination of GQDs and the chemotherapeutic agent doxorubicin (Dox) was tested. GQDs exerted a synergistic increase in the efficacy of chemotherapy treatment, specifically on U87 cells. The mechanism underlying this synergy was investigated, and it was found that GQDs can alter membrane permeability in a manner dependent on the surface chemistry, facilitating the uptake of Dox inside U87 cells, but not on cortical neurons. Therefore, experimental evidence indicates that GQDs could be used in a combined therapy against brain cancer, strongly increasing the efficacy of chemotherapy and, at the same time, reducing its dose requirement along with its side effects, thereby improving the life quality of patients.
RESUMEN
Recent evidence has shown that graphene quantum dots (GQDs) are capable of crossing the blood-brain barrier, the barrier that reduces cancer therapy efficacy. Here, we tested three alternative GQDs' surface chemistries on two neural lineages (glioblastoma cells and mouse cortical neurons). We showed that surface chemistry modulates GQDs' biocompatibility. When used in combination with the chemotherapeutic drug doxorubicin, GDQs exerted a synergistic effect on tumor cells, but not on neurons. This appears to be mediated by the modification of membrane permeability induced by the surface of GQDs. Our findings highlight that GQDs can be adopted as a suitable delivery and therapeutic strategy for the treatment of glioblastoma, by both directly destabilizing the cell membrane and indirectly increasing the efficacy of chemotherapeutic drugs.
Asunto(s)
Doxorrubicina/química , Doxorrubicina/farmacología , Embrión de Mamíferos/efectos de los fármacos , Glioblastoma/tratamiento farmacológico , Grafito/química , Neuronas/efectos de los fármacos , Puntos Cuánticos , Animales , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacología , Apoptosis , Proliferación Celular , Embrión de Mamíferos/citología , Glioblastoma/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Células Tumorales CultivadasRESUMEN
The voltage-gated sodium (Nav) channel complex is comprised of pore-forming α subunits (Nav1.1-1.9) and accessory regulatory proteins such as the intracellular fibroblast growth factor 14 (FGF14). The cytosolic Nav1.6 C-terminal tail binds directly to FGF14 and this interaction modifies Nav1.6-mediated currents with effects on intrinsic excitability in the brain. Previous studies have identified the FGF14V160 residue within the FGF14 core domain as a hotspot for the FGF14:Nav1.6 complex formation. Here, we used three short amino acid peptides around FGF14V160 to probe for the FGF14 interaction with the Nav1.6 C-terminal tail and to evaluate the activity of the peptide on Nav1.6-mediated currents. In silico docking predicts FLPK to bind to FGF14V160 with the expectation of interfering with the FGF14:Nav1.6 complex formation, a phenotype that was confirmed by the split-luciferase assay (LCA) and surface plasmon resonance (SPR), respectively. Whole-cell patch-clamp electrophysiology studies demonstrate that FLPK is able to prevent previously reported FGF14-dependent phenotypes of Nav1.6 currents, but that its activity requires the FGF14 N-terminal tail, a domain that has been shown to contribute to Nav1.6 inactivation independently from the FGF14 core domain. In medium spiny neurons in the nucleus accumbens, where both FGF14 and Nav1.6 are abundantly expressed, FLPK significantly increased firing frequency by a mechanism consistent with the ability of the tetrapeptide to interfere with Nav1.6 inactivation and potentiate persistent Na+ currents. Taken together, these results indicate that FLPK might serve as a probe for characterizing molecular determinants of neuronal excitability and a peptide scaffold to develop allosteric modulators of Nav channels.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Neuronas/metabolismo , Fragmentos de Péptidos/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Animales , Factores de Crecimiento de Fibroblastos/química , Factores de Crecimiento de Fibroblastos/genética , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Canal de Sodio Activado por Voltaje NAV1.6/química , Canal de Sodio Activado por Voltaje NAV1.6/genética , Neuronas/efectos de los fármacos , Fragmentos de Péptidos/química , Unión Proteica , Mapas de Interacción de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
Although major depressive disorder (MDD) is highly prevalent, its pathophysiology is poorly understood. Recent evidence suggests that glycogen-synthase kinase 3ß (GSK3ß) plays a key role in memory formation, yet its role in mood regulation remains controversial. Here, we investigated whether GSK3ß activity in the nucleus accumbens (NAc) is associated with depression-like behaviors and synaptic plasticity. We performed whole-cell patch-clamp recordings of medium spiny neurons (MSNs) in the NAc and determined the role of GSK3ß in spike timing-dependent long-term potentiation (tLTP) in the chronic unpredictable mild stress (CUMS) mouse model of depression. To assess the specific role of GSK3ß in tLTP, we used in vivo genetic silencing by an adeno-associated viral vector (AAV2) short hairpin RNA against GSK3ß. In addition, we examined the role of the voltage-gated potassium Kv4.2 subunit, a molecular determinant of A-type K+ currents, as a potential downstream target of GSK3ß. We found increased levels of active GSK3ß and augmented tLTP in CUMS mice, a phenotype that was prevented by selective GSK3ß knockdown. Furthermore, knockdown of GSK3ß in the NAc ameliorated depressive-like behavior in CUMS mice. Electrophysiological, immunohistochemical, biochemical, and pharmacological experiments revealed that inhibition of the Kv4.2 channel through direct phosphorylation at Ser-616 mediated the GSK3ß-dependent tLTP changes in CUMS mice. Our results identify GSK3ß regulation of Kv4.2 channels as a molecular mechanism of MSN maladaptive plasticity underlying depression-like behaviors and suggest that the GSK3ß-Kv4.2 axis may be an attractive therapeutic target for MDD.
Asunto(s)
Trastorno Depresivo Mayor/patología , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Plasticidad Neuronal , Núcleo Accumbens/patología , Canales de Potasio Shal/metabolismo , Potenciales de Acción , Animales , Conducta Animal , Trastorno Depresivo Mayor/etiología , Trastorno Depresivo Mayor/psicología , Modelos Animales de Enfermedad , Masculino , Ratones , Neuronas/patología , Núcleo Accumbens/citología , Técnicas de Placa-Clamp , Estrés Psicológico/complicaciones , Estrés Psicológico/psicología , Factores de TiempoRESUMEN
Impairment of adult hippocampal neurogenesis is an early event in Alzheimer's disease (AD), playing a crucial role in cognitive dysfunction associated with this pathology. However, the mechanisms underlying defective neurogenesis in AD are still unclear. Recently, the nucleoporin Nup153 has been described as a new epigenetic determinant of adult neural stem cell (NSC) maintenance and fate. Here we investigated whether Nup153 dysfunction could affect the plasticity of NSCs in AD. Nup153 expression was strongly reduced in AD-NSCs, as well as its interaction with the transcription factor Sox2, a master regulator of NSC stemness and their neuronal differentiation. Similar Nup153 reduction was also observed in WT-NSCs treated with amyloid-ß (Aß) or stimulated with a nitric oxide donor. Accordingly, AD-NSCs treated with either a γ-secretase inhibitor or antioxidant compounds showed higher Nup153 levels suggesting that both nitrosative stress and Aß accumulation affect Nup153 expression. Of note, restoration of Nup153 levels in AD-NSCs promoted their proliferation, as assessed by BrdU incorporation, neurosphere assay, and stemness gene expression analysis. Nup153 overexpression also recovered AD-NSC response to differentiation, increasing the expression of pro-neuronal genes, the percentage of cells positive for neuronal markers, and the acquisition of a more mature neuronal phenotype. Electrophysiological recordings revealed that neurons differentiated from Nup153-transfected AD-NSCs displayed higher Na+ current density, comparable to those deriving from WT-NSCs. Our data uncover a novel role for Nup153 in NSCs from animal model of AD and point to Nup153 as potential target to restore physiological NSC behavior and fate in neurodegenerative diseases.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Separación Celular , Células-Madre Neurales/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Ratones Transgénicos , Óxido Nítrico/metabolismo , Nitrosación , Factores de Transcripción SOXB1/metabolismo , Transducción de SeñalRESUMEN
The dopamine D3 receptor (D3R), in the nucleus accumbens (NAc), plays an important role in alcohol reward mechanisms. The major neuronal type within the NAc is the GABAergic medium spiny neuron (MSN), whose activity is regulated by dopaminergic inputs. We previously reported that genetic deletion or pharmacological blockade of D3R increases GABAA α6 subunit in the ventral striatum. Here we tested the hypothesis that D3R-dependent changes in GABAA α6 subunit in the NAc affect voluntary alcohol intake, by influencing the inhibitory transmission of MSNs. We performed in vivo and ex vivo experiments in D3R knockout (D3R -/-) mice and wild type littermates (D3R +/+). Ro 15-4513, a high affinity α6-GABAA ligand was used to study α6 activity. At baseline, NAc α6 expression was negligible in D3R+/+, whereas it was robust in D3R-/-; other relevant GABAA subunits were not changed. In situ hybridization and qPCR confirmed α6 subunit mRNA expression especially in the NAc. In the drinking-in-the-dark paradigm, systemic administration of Ro 15-4513 inhibited alcohol intake in D3R+/+, but increased it in D3R-/-; this was confirmed by intra-NAc administration of Ro 15-4513 and furosemide, a selective α6-GABAA antagonist. Whole-cell patch-clamp showed peak amplitudes of miniature inhibitory postsynaptic currents in NAc medium spiny neurons higher in D3R-/- compared to D3R+/+; Ro 15-4513 reduced the peak amplitude in the NAc of D3R-/-, but not in D3R+/+. We conclude that D3R-dependent enhanced expression of α6 GABAA subunit inhibits voluntary alcohol intake by increasing GABA inhibition in the NAc.
Asunto(s)
Consumo Excesivo de Bebidas Alcohólicas/genética , Neuronas GABAérgicas/patología , Receptores de Dopamina D3/genética , Receptores de GABA-A/genética , Animales , Consumo Excesivo de Bebidas Alcohólicas/patología , Neuronas GABAérgicas/metabolismo , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Noqueados , Núcleo Accumbens/metabolismo , Núcleo Accumbens/patología , Subunidades de Proteína/genética , ARN Mensajero/genéticaRESUMEN
Spike timing-dependent plasticity (STDP) is a form of activity-dependent remodeling of synaptic strength that underlies memory formation. Despite its key role in dictating learning rules in the brain circuits, the molecular mechanisms mediating STDP are still poorly understood. Here, we show that spike timing-dependent long-term depression (tLTD) and A-type K+ currents are modulated by pharmacological agents affecting the levels of active glycogen-synthase kinase 3 (GSK3) and by GSK3ß knockdown in layer 2/3 of the mouse somatosensory cortex. Moreover, the blockade of A-type K+ currents mimics the effects of GSK3 up-regulation on tLTD and occludes further changes in synaptic strength. Pharmacological, immunohistochemical and biochemical experiments revealed that GSK3ß influence over tLTD induction is mediated by direct phosphorylation at Ser-616 of the Kv4.2 subunit, a molecular determinant of A-type K+ currents. Collectively, these results identify the functional interaction between GSK3ß and Kv4.2 channel as a novel mechanism for tLTD modulation providing exciting insight into the understanding of GSK3ß role in synaptic plasticity.
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Glucógeno Sintasa Quinasa 3 beta/metabolismo , Depresión Sináptica a Largo Plazo/fisiología , Neuronas/fisiología , Canales de Potasio Shal/metabolismo , Corteza Somatosensorial/fisiología , Animales , Potenciales Postsinápticos Excitadores , Ratones Endogámicos C57BL , Neuronas/metabolismo , Fosforilación , Corteza Somatosensorial/metabolismoRESUMEN
Resilience and vulnerability to neuropsychiatric disorders are linked to molecular changes underlying excitability that are still poorly understood. Here, we identify glycogen-synthase kinase 3ß (GSK3ß) and voltage-gated Na+ channel Nav1.6 as regulators of neuroplasticity induced by environmentally enriched (EC) or isolated (IC) conditions-models for resilience and vulnerability. Transcriptomic studies in the nucleus accumbens from EC and IC rats predicted low levels of GSK3ß and SCN8A mRNA as a protective phenotype associated with reduced excitability in medium spiny neurons (MSNs). In vivo genetic manipulations demonstrate that GSK3ß and Nav1.6 are molecular determinants of MSN excitability and that silencing of GSK3ß prevents maladaptive plasticity of IC MSNs. In vitro studies reveal direct interaction of GSK3ß with Nav1.6 and phosphorylation at Nav1.6T1936 by GSK3ß. A GSK3ß-Nav1.6T1936 competing peptide reduces MSNs excitability in IC, but not EC rats. These results identify GSK3ß regulation of Nav1.6 as a biosignature of MSNs maladaptive plasticity.
Asunto(s)
Glucógeno Sintasa Quinasa 3 beta/metabolismo , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Condicionamiento Físico Animal , Aislamiento Social , Animales , Potenciales Evocados , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3 beta/genética , Células HEK293 , Humanos , Masculino , Canal de Sodio Activado por Voltaje NAV1.6/química , Canal de Sodio Activado por Voltaje NAV1.6/genética , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Técnicas de Placa-Clamp , Fosfopéptidos/análisis , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , TranscriptomaRESUMEN
High-fat diet (HFD) and metabolic diseases cause detrimental effects on hippocampal synaptic plasticity, learning, and memory through molecular mechanisms still poorly understood. Here, we demonstrate that HFD increases palmitic acid deposition in the hippocampus and induces hippocampal insulin resistance leading to FoxO3a-mediated overexpression of the palmitoyltransferase zDHHC3. The excess of palmitic acid along with higher zDHHC3 levels causes hyper-palmitoylation of AMPA glutamate receptor subunit GluA1, hindering its activity-dependent trafficking to the plasma membrane. Accordingly, AMPAR current amplitudes and, more importantly, their potentiation underlying synaptic plasticity were inhibited, as well as hippocampal-dependent memory. Hippocampus-specific silencing of Zdhhc3 and, interestingly enough, intranasal injection of the palmitoyltransferase inhibitor, 2-bromopalmitate, counteract GluA1 hyper-palmitoylation and restore synaptic plasticity and memory in HFD mice. Our data reveal a key role of FoxO3a/Zdhhc3/GluA1 axis in the HFD-dependent impairment of cognitive function and identify a novel mechanism underlying the cross talk between metabolic and cognitive disorders.
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Encéfalo/fisiología , Proteína Forkhead Box O3/metabolismo , Hipocampo/fisiología , Resistencia a la Insulina , Memoria/fisiología , Plasticidad Neuronal/fisiología , Receptores AMPA/metabolismo , Animales , Células Cultivadas , Cognición/fisiología , Dieta Alta en Grasa , Lipoilación , Potenciación a Largo Plazo/fisiología , Masculino , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Neuronas/fisiología , Interferencia de ARNRESUMEN
Hippocampal plasticity is triggered by a variety of stimuli including sensory inputs, neurotrophins and inflammation. Leptin, whose primary function is to regulate food intake and energy expenditure, has been recently shown to affect hippocampal neurogenesis and plasticity. Interestingly, mice fed a high-fat diet (HFD) exhibit impaired hippocampal function, but the underlying mechanisms are poorly understood. To address this issue, we compared leptin responsiveness of hippocampal neurons in control and HFD-fed mice by combining single-cell electrophysiology and biochemical assays. We found that leptin modulated spontaneous and evoked synaptic transmission in control, but not HFD, mice. This functional impairment was paralleled by blunted activation of STAT-3, one of the key signal transduction pathways controlled by the fully functional isoform of the leptin receptor, ObRb. In addition, SOCS-3 expression was non-responsive to leptin, indicating that modulation of negative feedback impinging on ObRb was also altered. Our results advance the understanding of leptin action on hippocampal plasticity and, more importantly, suggest that leptin resistance is a key determinant of hippocampal dysfunction associated with hypercaloric diet.
RESUMEN
Amyloid ß-protein (Aß) pathologies have been linked to dysfunction of excitability in neurons of the hippocampal circuit, but the molecular mechanisms underlying this process are still poorly understood. Here, we applied whole-cell patch-clamp electrophysiology to primary hippocampal neurons and show that intracellular Aß42 delivery leads to increased spike discharge and action potential broadening through downregulation of A-type K(+) currents. Pharmacologic studies showed that caspases and glycogen synthase kinase 3 (GSK-3) activation are required for these Aß42-induced effects. Extracellular perfusion and subsequent internalization of Aß42 increase spike discharge and promote GSK-3-dependent phosphorylation of the Kv4.2 α-subunit, a molecular determinant of A-type K(+) currents, at Ser-616. In acute hippocampal slices derived from an adult triple-transgenic Alzheimer's mouse model, characterized by endogenous intracellular accumulation of Aß42, CA1 pyramidal neurons exhibit hyperexcitability accompanied by increased phosphorylation of Kv4.2 at Ser-616. Collectively, these data suggest that intraneuronal Aß42 accumulation leads to an intracellular cascade culminating into caspases activation and GSK-3-dependent phosphorylation of Kv4.2 channels. These findings provide new insights into the toxic mechanisms triggered by intracellular Aß42 and offer potentially new therapeutic targets for Alzheimer's disease treatment.
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Enfermedad de Alzheimer/etiología , Péptidos beta-Amiloides/toxicidad , Caspasas/fisiología , Fenómenos Electrofisiológicos/genética , Glucógeno Sintasa Quinasa 3/fisiología , Hipocampo/fisiopatología , Neuronas/fisiología , Fragmentos de Péptidos/toxicidad , Canales de Potasio Shal/metabolismo , Potenciales de Acción/genética , Enfermedad de Alzheimer/terapia , Péptidos beta-Amiloides/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Hipocampo/citología , Hipocampo/metabolismo , Técnicas In Vitro , Ratones Endogámicos C57BL , Ratones Transgénicos , Terapia Molecular Dirigida , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , Fosforilación/genéticaRESUMEN
Intracellular accumulation of amyloid-ß (Aß) protein has been proposed as an early event in AD pathogenesis. In patients with mild cognitive impairment, intraneuronal Aß immunoreactivity was found especially in brain regions critically involved in the cognitive deficits of AD. Although a large body of evidence demonstrates that Aß42 accumulates intraneuronally ((in)Aß), the action and the role of Aß42 buildup on synaptic function have been poorly investigated. Here, we demonstrate that basal synaptic transmission and LTP were markedly depressed following Aß42 injection into the neuron through the patch pipette. Control experiments performed with the reverse peptide (Aß42-1) allowed us to exclude that the effects of (in)Aß depended on changes in oncotic pressure. To further investigate (in)Aß synaptotoxicity we used an Aß variant harboring oxidized methionine in position 35 that does not cross the neuronal plasma membrane and is not uploaded from the extracellular space. This Aß42 variant had no effects on synaptic transmission and plasticity when applied extracellularly, but induced synaptic depression and LTP inhibition after patch-pipette dialysis. Finally, the injection of an antibody raised against human Aß42 (6E10) in CA1 pyramidal neurons of mouse hippocampal brain slices and autaptic microcultures did not, per se, significantly affect LTP and basal synaptic transmission, but it protected against the toxic effects of extracellular Aß42. Collectively, these findings suggest that Aß42-induced impairment of glutamatergic synaptic function depends on its internalization and intracellular accumulation thus paving the way to a systemic proteomic analysis of intracellular targets/partners of Aß42.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/farmacología , Ácido Glutámico/fisiología , Hipocampo/efectos de los fármacos , Plasticidad Neuronal/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Transmisión Sináptica/efectos de los fármacos , Péptidos beta-Amiloides/administración & dosificación , Animales , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/fisiología , Espacio Intracelular/efectos de los fármacos , Potenciación a Largo Plazo/efectos de los fármacos , Potenciación a Largo Plazo/fisiología , Masculino , Ratones , Microinyecciones , Plasticidad Neuronal/fisiología , Fragmentos de Péptidos/administración & dosificación , Cultivo Primario de Células , Transmisión Sináptica/fisiologíaRESUMEN
Cocaine addiction is characterized by compulsive drug use despite adverse consequences and high rate of relapse during periods of abstinence. Increasing consensus suggests that addiction to drugs of abuse usurps learning and memory mechanisms normally related to natural rewards, ultimately producing long-lasting neuroadaptations in the mesocorticolimbic system. This system, formed in part by the ventral tegmental area and nucleus accumbens (NAc), has a central role in the development and expression of addictive behaviors. In addition to a broad spectrum of changes that affect morphology and function of NAc excitatory circuits in cocaine-treated animals, impaired N-methyl-D-aspartate receptor (NMDAR)-dependent synaptic plasticity is a typical feature. D-serine, a D-amino acid that has been found at high levels in mammalian brain, binds with high affinity the co-agonist site of NMDAR and mediates, along with glutamate, several important processes including synaptic plasticity. Here we review recent literature focusing on cocaine-induced impairment in synaptic plasticity mechanisms in the NAc and on the fundamental role of D-serine as co-agonist of NMDAR in functional and dysfunctional synaptic plasticity within this nucleus. The emerging picture is that reduced D-serine levels play a crucial role in synaptic plasticity relevant to cocaine addiction. This finding opens new perspectives for therapeutic approaches to treat this addictive state.
RESUMEN
Neural stem cells generate neurons in the hippocampal dentate gyrus in mammals, including humans, throughout adulthood. Adult hippocampal neurogenesis has been the focus of many studies due to its relevance in processes such as learning and memory and its documented impairment in some neurodegenerative diseases. However, we are still far from having a complete picture of the mechanism regulating this process. Our study focused on the possible role of cyclic nucleotide-gated (CNG) channels. These voltage-independent channels activated by cyclic nucleotides, first described in retinal and olfactory receptors, have been receiving increasing attention for their involvement in several brain functions. Here we show that the rod-type, CNGA1, and olfactory-type, CNGA2, subunits are expressed in hippocampal neural stem cells in culture and in situ in the hippocampal neurogenic niche of adult mice. Pharmacological blockade of CNG channels did not affect cultured neural stem cell proliferation but reduced their differentiation towards the neuronal phenotype. The membrane permeant cGMP analogue, 8-Br-cGMP, enhanced neural stem cell differentiation to neurons and this effect was prevented by CNG channel blockade. In addition, patch-clamp recording from neuron-like differentiating neural stem cells revealed cGMP-activated currents attributable to ion flow through CNG channels. The current work provides novel insights into the role of CNG channels in promoting hippocampal neurogenesis, which may prove to be relevant for stem cell-based treatment of cognitive impairment and brain damage.