RESUMEN
Excitatory neurotransmission meditated by glutamate receptors including N-methyl-D-aspartate receptors (NMDARs) is pivotal to brain development and function. NMDARs are heterotetramers composed of GluN1 and GluN2 subunits, which bind glycine and glutamate, respectively, to activate their ion channels. Despite importance in brain physiology, the precise mechanisms by which activation and inhibition occur via subunit-specific binding of agonists and antagonists remain largely unknown. Here, we show the detailed patterns of conformational changes and inter-subunit and -domain reorientation leading to agonist-gating and subunit-dependent competitive inhibition by providing multiple structures in distinct ligand states at 4 Å or better. The structures reveal that activation and competitive inhibition by both GluN1 and GluN2 antagonists occur by controlling the tension of the linker between the ligand-binding domain and the transmembrane ion channel of the GluN2 subunit. Our results provide detailed mechanistic insights into NMDAR pharmacology, activation, and inhibition, which are fundamental to the brain physiology.
Asunto(s)
Receptores de N-Metil-D-Aspartato/metabolismo , Sitios de Unión , Unión Competitiva , Microscopía por Crioelectrón , Cristalografía por Rayos X , Dimerización , Ácido Glutámico/química , Ácido Glutámico/metabolismo , Glicina/química , Glicina/metabolismo , Humanos , Ligandos , Simulación de Dinámica Molecular , Estructura Cuaternaria de Proteína , Subunidades de Proteína/agonistas , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Neurotransmission mediated by diverse subtypes of N-methyl-D-aspartate receptors (NMDARs) is fundamental for basic brain functions and development as well as neuropsychiatric diseases and disorders. NMDARs are glycine- and glutamate-gated ion channels that exist as heterotetramers composed of obligatory GluN1 and GluN2(A-D) and/or GluN3(A-B). The GluN2C and GluN2D subunits form ion channels with distinct properties and spatio-temporal expression patterns. Here, we provide the structures of the agonist-bound human GluN1-2C NMDAR in the presence and absence of the GluN2C-selective positive allosteric potentiator (PAM), PYD-106, the agonist-bound GluN1-2A-2C tri-heteromeric NMDAR, and agonist-bound GluN1-2D NMDARs by single-particle electron cryomicroscopy. Our analysis shows unique inter-subunit and domain arrangements of the GluN2C NMDARs, which contribute to functional regulation and formation of the PAM binding pocket and is distinct from GluN2D NMDARs. Our findings here provide the fundamental blueprint to study GluN2C- and GluN2D-containing NMDARs, which are uniquely involved in neuropsychiatric disorders.
Asunto(s)
Ácido Glutámico , Receptores de N-Metil-D-Aspartato , Humanos , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Ácido Glutámico/metabolismo , Glicina/metabolismo , Transmisión Sináptica , Subunidades de Proteína/metabolismoRESUMEN
N-methyl-D-aspartate receptors (NMDARs emerging from GRIN genes) are tetrameric receptors that form diverse channel compositions in neurons, typically consisting of two GluN1 subunits combined with two GluN2(A-D) subunits. During prenatal stages, the predominant channels are di-heteromers with two GluN1 and two GluN2B subunits due to the high abundance of GluN2B subunits. Postnatally, the expression of GluN2A subunits increases, giving rise to additional subtypes, including GluN2A-containing di-heteromers and tri-heteromers with GluN1, GluN2A, and GluN2B subunits. The latter emerge as the major receptor subtype at mature synapses in the hippocampus. Despite extensive research on purely di-heteromeric receptors containing two identical GRIN variants, the impact of a single variant on the function of other channel forms, notably tri-heteromers, is lagging. In this study, we systematically investigated the effects of two de novo GRIN2B variants (G689C and G689S) in pure, mixed di- and tri-heteromers. Our findings reveal that incorporating a single variant in mixed di-heteromers or tri-heteromers exerts a dominant negative effect on glutamate potency, although 'mixed' channels show improved potency compared to pure variant-containing di-heteromers. We show that a single variant within a receptor complex does not impair the response of all receptor subtypes to the positive allosteric modulator pregnenolone-sulfate (PS), whereas spermine completely fails to potentiate tri-heteromers containing GluN2A and -2B-subunits. We examined PS on primary cultured hippocampal neurons transfected with the variants, and observed a positive impact over current amplitudes and synaptic activity. Together, our study supports previous observations showing that mixed di-heteromers exhibit improved glutamate potency and extend these findings towards the exploration of the effect of Loss-of-Function variants over tri-heteromers. Notably, we provide an initial and crucial demonstration of the beneficial effects of GRIN2B-relevant potentiators on tri-heteromers. Our results underscore the significance of studying how different variants affect distinct receptor subtypes, as these effects cannot be inferred solely from observations made on pure di-heteromers. Overall, this study contributes to ongoing efforts to understand the pathophysiology of GRINopathies and provides insights into potential treatment strategies.
Asunto(s)
Pregnenolona , Receptores de N-Metil-D-Aspartato , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Pregnenolona/farmacología , Pregnenolona/metabolismo , Humanos , Animales , Células HEK293 , Hipocampo/metabolismo , Mutación con Pérdida de Función , Multimerización de Proteína , Neuronas/metabolismo , Subunidades de Proteína/metabolismo , Subunidades de Proteína/genéticaRESUMEN
ATB[Formula: see text] (SLC6A14) is a member of the amino acid transporter branch of the SLC6 family along with GlyT1 (SLC6A9) and GlyT2 (SLC6A5), two glycine-specific transporters coupled to 2:1 and 3:1 Na[Formula: see text]:Cl[Formula: see text], respectively. In contrast, ATB[Formula: see text] exhibits broad substrate specificity for all neutral and cationic amino acids, and its ionic coupling remains unsettled. Using the reversal potential slope method, we demonstrate a 3:1:1 Na[Formula: see text]:Cl[Formula: see text]:Gly stoichiometry for ATB[Formula: see text] that is consistent with its 2.1 e/Gly charge coupling. Like GlyT2, ATB[Formula: see text] behaves as a unidirectional transporter with virtually no glycine efflux at negative potentials after uptake, except by heteroexchange as remarkably shown by leucine activation of NMDARs in Xenopus oocytes coexpressing both membrane proteins. Analysis and computational modeling of the charge movement of ATB[Formula: see text] reveal a higher affinity for sodium in the absence of substrate than GlyT2 and a gating mechanism that locks Na[Formula: see text] into the apo-transporter at depolarized potentials. A 3:1 Na[Formula: see text]:Cl[Formula: see text] stoichiometry justifies the concentrative transport properties of ATB[Formula: see text] and explains its trophic role in tumor growth, while rationalizing its phylogenetic proximity to GlyT2 despite their extreme divergence in specificity.
Asunto(s)
Proteínas de Transporte de Glicina en la Membrana Plasmática , Sodio , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos , Glicina/metabolismo , Proteínas de Transporte de Glicina en la Membrana Plasmática/química , Iones/metabolismo , Leucina , Filogenia , Sodio/metabolismoRESUMEN
BACKGROUND: Mainly known as a transcription factor patterning the rostral brain and governing its histogenesis, FOXG1 has been also detected outside the nucleus; however, biological meaning of that has been only partially clarified. RESULTS: Prompted by FOXG1 expression in cytoplasm of pallial neurons, we investigated its implication in translational control. We documented the impact of FOXG1 on ribosomal recruitment of Grin1-mRNA, encoding for the main subunit of NMDA receptor. Next, we showed that FOXG1 increases GRIN1 protein level by enhancing the translation of its mRNA, while not increasing its stability. Molecular mechanisms underlying this activity included FOXG1 interaction with EIF4E and, possibly, Grin1-mRNA. Besides, we found that, within murine neocortical cultures, de novo synthesis of GRIN1 undergoes a prominent and reversible, homeostatic regulation and FOXG1 is instrumental to that. Finally, by integrated analysis of multiple omic data, we inferred that FOXG1 is implicated in translational control of hundreds of neuronal genes, modulating ribosome engagement and progression. In a few selected cases, we experimentally verified such inference. CONCLUSIONS: These findings point to FOXG1 as a key effector, potentially crucial to multi-scale temporal tuning of neocortical pyramid activity, an issue with profound physiological and neuropathological implications.
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Factores de Transcripción Forkhead , Neocórtex , Proteínas del Tejido Nervioso , Neuronas , Receptores de N-Metil-D-Aspartato , Animales , Femenino , Masculino , Ratones , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Neocórtex/metabolismo , Neocórtex/embriología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Biosíntesis de Proteínas/genética , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Dynamic reconfiguration of circuit function subserves the flexibility of innate behaviors tuned to physiological states. Internal energy stores adaptively regulate feeding-associated behaviors and integrate opposing hunger and satiety signals at the level of neural circuits. Across vertebrate lineages, the neuropeptides cocaine- and amphetamine-regulated transcript (CART) and neuropeptide Y (NPY) have potent anorexic and orexic functions, respectively, and show energy-state-dependent expression in interoceptive neurons. However, how the antagonistic activities of these peptides modulate circuit plasticity remains unclear. Using behavioral, neuroanatomical, and activity analysis in adult zebrafish of both sexes, along with pharmacological interventions, we show that CART and NPY activities converge on a population of neurons in the dorsomedial telencephalon (Dm). Although CART facilitates glutamatergic neurotransmission at the Dm, NPY dampens the response to glutamate. In energy-rich states, CART enhances NMDA receptor (NMDAR) function by protein kinase A/protein kinase C (PKA/PKC)-mediated phosphorylation of the NR1 subunit of the NMDAR complex. Conversely, starvation triggers NPY-mediated reduction in phosphorylated NR1 via calcineurin activation and inhibition of cAMP production leading to reduced responsiveness to glutamate. Our data identify convergent integration of CART and NPY inputs by the Dm neurons to generate nutritional state-dependent circuit plasticity that is correlated with the behavioral switch induced by the opposing actions of satiety and hunger signals.SIGNIFICANCE STATEMENT Internal energy needs reconfigure neuronal circuits to adaptively regulate feeding behavior. Energy-state-dependent neuropeptide release can signal energy status to feeding-associated circuits and modulate circuit function. CART and NPY are major anorexic and orexic factors, respectively, but the intracellular signaling pathways used by these peptides to alter circuit function remain uncharacterized. We show that CART and NPY-expressing neurons from energy-state interoceptive areas project to a novel telencephalic region, Dm, in adult zebrafish. CART increases the excitability of Dm neurons, whereas NPY opposes CART activity. Antagonistic signaling by CART and NPY converge onto NMDA-receptor function to modulate glutamatergic neurotransmission. Thus, opposing activities of anorexic CART and orexic NPY reconfigure circuit function to generate flexibility in feeding behavior.
Asunto(s)
Neuropéptido Y , Neuropéptidos , Masculino , Animales , Femenino , Neuropéptido Y/metabolismo , Pez Cebra/metabolismo , Neuropéptidos/metabolismo , Neuronas/metabolismo , Prosencéfalo/metabolismo , GlutamatosRESUMEN
Learning, memory, and cognition are thought to require synaptic plasticity, specifically including hippocampal long-term potentiation and depression (LTP and LTD). LTP versus LTD is induced by high-frequency stimulation versus low-frequency, but stimulating ß-adrenergic receptors (ßARs) enables LTP induction also by low-frequency stimulation (1 Hz) or theta frequencies (â¼5 Hz) that do not cause plasticity by themselves. In contrast to high-frequency stimulation-LTP, such ßAR-LTP requires Ca2+-flux through L-type voltage-gated Ca2+-channels, not N-methyl-D-aspartate-type glutamate receptors. Surprisingly, we found that ßAR-LTP still required a nonionotropic scaffolding function of the N-methyl-D-aspartate-type glutamate receptor: the stimulus-induced binding of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) to its GluN2B subunit that mediates CaMKII movement to excitatory synapses. In hippocampal neurons, ß-adrenergic stimulation with isoproterenol (Iso) transformed LTD-type CaMKII movement to LTP-type movement, resulting in CaMKII movement to excitatory instead of inhibitory synapses. Additionally, Iso enabled induction of a major cell-biological feature of LTP in response to LTD stimuli: increased surface expression of GluA1 fused with super-ecliptic pHluorein. Like for ßAR-LTP in hippocampal slices, the Iso effects on CaMKII movement and surface expression of GluA1 fused with super-ecliptic pHluorein involved L-type Ca2+-channels and specifically required ß2-ARs. Taken together, these results indicate that Iso transforms LTD stimuli to LTP signals by switching CaMKII movement and GluN2B binding to LTP mode.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Potenciación a Largo Plazo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Receptores Adrenérgicos beta/metabolismo , Ácido D-Aspártico/metabolismo , Ácido D-Aspártico/farmacología , Depresión Sináptica a Largo Plazo/fisiología , Hipocampo/metabolismo , Sinapsis/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
BACKGROUND: Early diagnosis of encephalitis involves identifying signs of neuroinflammation, including cerebrospinal fluid (CSF) pleocytosis. However, absence of CSF pleocytosis in encephalitis has been described, most notably in autoimmune encephalitis. We examined clinical characteristics and outcomes associated with the absence or presence of CSF white blood cell pleocytosis (≥ 5 cells/µL), to inform timely diagnosis and management of encephalitis. METHODS: This retrospective study compares initial CSF profiles in 597 adult patients with all-cause encephalitis. RESULTS: Of the 597 patients, 446 (74.7%) had CSF pleocytosis while 151 (25.3%) did not. CSF pleocytosis occurred more commonly in infectious cases (200/446, 44.8%), along with 59 (13.2%) autoimmune cases, comprised chiefly of anti-NMDAR encephalitis (37/59, 62.7%). Notably, the group without pleocytosis was comprised of similar proportions of infectious (47/151, 31.1%) and autoimmune (38/151, 25.92%; p>0.05) encephalitis. Among those with infectious encephalitis, 47/247 (19%) had absent pleocytosis, including 18/76 (23.7%) with HSV-1 encephalitis. The absence of pleocytosis was associated with a decreased rate of acyclovir administration (47.7% in patients without pleocytosis vs. 71.1% in patients with pleocytosis, p<0.001). Despite pleocytosis being associated with some measures of clinical severity at admission such as a Full Outline of UnResponsiveness (FOUR) score ≤14, it was not associated with mortality or prolonged hospitalization. CONCLUSION: CSF pleocytosis is an important criterion for encephalitis diagnosis, but 25.3% of patients with all-cause encephalitis and 23.7% of those with HSV-1 encephalitis exhibit absence of pleocytosis on initial LP. Acyclovir initiation should not be delayed in the absence of pleocytosis in patients with suspected encephalitis.
RESUMEN
Deciphering the molecular pathways associated with N-methyl-D-aspartate receptor (NMDAr) hypofunction and its interaction with antipsychotics is necessary to advance our understanding of the basis of schizophrenia, as well as our capacity to treat this disease. In this regard, the development of human brain-derived models that are amenable to studying the neurobiology of schizophrenia may contribute to filling the gaps left by the widely employed animal models. Here, we assessed the proteomic changes induced by the NMDA glutamate receptor antagonist MK-801 on human brain slice cultures obtained from adult donors submitted to respective neurosurgery. Initially, we demonstrated that MK-801 diminishes NMDA glutamate receptor signaling in human brain slices in culture. Next, using mass-spectrometry-based proteomics and systems biology in silico analyses, we found that MK-801 led to alterations in proteins related to several pathways previously associated with schizophrenia pathophysiology, including ephrin, opioid, melatonin, sirtuin signaling, interleukin 8, endocannabinoid, and synaptic vesicle cycle. We also evaluated the impact of both typical and atypical antipsychotics on MK-801-induced proteome changes. Interestingly, the atypical antipsychotic clozapine showed a more significant capacity to counteract the protein alterations induced by NMDAr hypofunction than haloperidol. Finally, using our dataset, we identified potential modulators of the MK-801-induced proteome changes, which may be considered promising targets to treat NMDAr hypofunction in schizophrenia. This dataset is publicly available and may be helpful in further studies aimed at evaluating the effects of MK-801 and antipsychotics in the human brain.
Asunto(s)
Antipsicóticos , Clozapina , Animales , Humanos , Clozapina/farmacología , Haloperidol/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Maleato de Dizocilpina/farmacología , Proteoma/metabolismo , N-Metilaspartato , Ácido Glutámico/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Proteómica , Antipsicóticos/farmacología , Encéfalo/metabolismoRESUMEN
Early life, or juvenility, stands out as the most pivotal phase in neurodevelopment due to its profound impact over the long-term cognition. During this period, significant changes are made in the brain's connections both within and between different areas, particularly in tandem with the development of more intricate behaviors. The hippocampus is among the brain regions that undergo significant postnatal remodeling, including dendritic arborization, synaptogenesis, the formation of complex spines and neuron proliferation. Given the crucial role of the hippocampus in spatial memory processing, it has been observed that spatial memory abilities continue to develop as the hippocampus matures, particularly before puberty. The N-methyl-d-aspartate (NMDA) type of glutamate receptor channel is crucial for the induction of activity-dependent synaptic plasticity and spatial memory formation in both rodents and humans. Although extensive evidence shows the role of NMDA receptors (NMDAr) in spatial memory and synaptic plasticity, the studies addressing the role of NMDAr in spatial memory of juveniles are sparse and mostly limited to adult males. In the present study, we, therefore, aimed to investigate the effects of systemic NMDAr blockade by the MK-801 on spatial memory (novel object location memory, OLM) and hippocampal plasticity in the form of long-term potentiation (LTP) of both male and female juvenile rats. Our results show the sex-dimorphic role of NMDAr in spatial memory and plasticity during juvenility, as systemic NMDAr blockade impairs the OLM and LTP in juvenile males without an effect on juvenile females. Taken together, our results demonstrate that spatial memory and hippocampal plasticity are NMDAr-dependent in juvenile males and NMDAr-independent in juvenile females. These sex-specific differences in the mechanisms of spatial memory and plasticity may imply gender-specific treatment for spatial memory disorders even in children.
RESUMEN
The transport of N-methyl-d-aspartate receptors (NMDARs) is crucial for neuronal plasticity and synapse formation. Here, we show that KIF3B, a member of the kinesin superfamily proteins (KIFs), supports the transport of vesicles simultaneously containing NMDAR subunit 2A (NR2A) and the adenomatous polyposis coli (APC) complex. Kif3b+/- neurons exhibited a reduction in dendritic levels of both NR2A and NR2B due to the impaired transport of NR2A and increased degradation of NR2B. In Kif3b+/- hippocampal slices, electrophysiological NMDAR response was found decreased and synaptic plasticity was disrupted, which corresponded to a common feature of schizophrenia (SCZ). The histological features of Kif3b+/- mouse brain also mimicked SCZ features, and Kif3b+/- mice exhibited behavioral defects in prepulse inhibition (PPI), social interest, and cognitive flexibility. Indeed, a mutation of KIF3B was specifically identified in human SCZ patients, which was revealed to be functionally defective in a rescue experiment. Therefore, we propose that KIF3B transports NR2A/APC complex and that its dysfunction is responsible for SCZ pathogenesis.
Asunto(s)
Cinesinas/genética , Cinesinas/fisiología , Mutación , Neuronas/patología , Receptores de N-Metil-D-Aspartato/metabolismo , Esquizofrenia/etiología , Sinapsis/patología , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Animales , Conducta Animal , Movimiento Celular , Humanos , Relaciones Interpersonales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Fenotipo , Subunidades de Proteína , Transporte de Proteínas , Esquizofrenia/metabolismo , Esquizofrenia/patología , Sinapsis/metabolismoRESUMEN
Nasopharyngeal carcinoma, a malignant tumor prevalent in southeast Asia and north Africa, still lacks effective treatment. Esketamine, an N-methyl-D-aspartatic acid (NMDA) receptor (NMDAR) antagonist, is widely used in clinical anesthesia. Emerging evidence suggests that esketamine plays an important role in inhibiting tumor cell activity. However, the underlying mechanisms of esketamine on nasopharyngeal carcinoma remain unknown. In this study, we found that esketamine inhibited the proliferation and migration of nasopharyngeal carcinoma cells. Mechanically, transcriptome sequencing and subsequent verification experiments revealed that esketamine promoted the apoptosis of nasopharyngeal carcinoma cells through endoplasmic reticulum stress PERK/ATF4/CHOP signaling pathway mediated by NMDAR. Additionally, when combined with esketamine, the inhibitory effect of cisplatin on the proliferation of nasopharyngeal carcinoma cells was significantly enhanced. These findings provide new insights into future anti-nasopharyngeal carcinoma clinical strategies via targeting the NMDAR/PERK/CHOP axis alone or in combination with cisplatin.
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Ketamina , Neoplasias Nasofaríngeas , eIF-2 Quinasa , Humanos , eIF-2 Quinasa/metabolismo , Cisplatino/farmacología , Carcinoma Nasofaríngeo/tratamiento farmacológico , Apoptosis , Neoplasias Nasofaríngeas/tratamiento farmacológico , Estrés del Retículo Endoplásmico , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , Factor de Transcripción Activador 4/metabolismoRESUMEN
The phenomenon of ischemic postconditioning (PostC) is known to be neuroprotective against ischemic reperfusion (I/R) injury. One of the key processes in PostC is the opening of the mitochondrial ATP-dependent potassium (mito-KATP) channel and depolarization of the mitochondrial membrane, triggering the release of calcium ions from mitochondria through low-conductance opening of the mitochondrial permeability transition pore. Mitochondrial calcium uniporter (MCU) is known as a highly sensitive transporter for the uptake of Ca2+ present on the inner mitochondrial membrane. The MCU has attracted attention as a new target for treatment in diseases, such as neurodegenerative diseases, cancer, and ischemic stroke. We considered that the MCU may be involved in PostC and trigger its mechanisms. This research used the whole-cell patch-clamp technique on hippocampal CA1 pyramidal cells from C57BL mice and measured changes in spontaneous excitatory post-synaptic currents (sEPSCs), intracellular Ca2+ concentration, mitochondrial membrane potential, and N-methyl-D-aspartate receptor (NMDAR) currents under inhibition of MCU by ruthenium red 265 (Ru265) in PostC. Inhibition of MCU increased the occurrence of sEPSCs (p = 0.014), NMDAR currents (p < 0.001), intracellular Ca2+ concentration (p < 0.001), and dead cells (p < 0.001) significantly after reperfusion, reflecting removal of the neuroprotective effects in PostC. Moreover, mitochondrial depolarization in PostC with Ru265 was weakened, compared to PostC (p = 0.004). These results suggest that MCU affects mitochondrial depolarization in PostC to suppress NMDAR over-activation and prevent elevation of intracellular Ca2+ concentrations against I/R injury.
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Lesiones Encefálicas , Canales de Calcio , Poscondicionamiento Isquémico , Compuestos de Rutenio , Animales , Ratones , Ratones Endogámicos C57BL , Receptores de N-Metil-D-Aspartato , Adenosina TrifosfatoRESUMEN
BACKGROUND: The pathway involving PTEN-induced putative kinase 1 (PINK1) and PARKIN plays a crucial role in mitophagy, a process activated by artesunate (ART). We propose that patients with anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis exhibit insufficient mitophagy, and ART enhances mitophagy via the PINK1/PARKIN pathway, thereby providing neuroprotection. METHODS: Adult female mice aged 8-10 weeks were selected to create a passive transfer model of anti-NMDAR encephalitis. We conducted behavioral tests on these mice within a set timeframe. Techniques such as immunohistochemistry, immunofluorescence, and western blotting were employed to assess markers including PINK1, PARKIN, LC3B, p62, caspase3, and cleaved caspase3. The TUNEL assay was utilized to detect neuronal apoptosis, while transmission electron microscopy (TEM) was used to examine mitochondrial autophagosomes. Primary hippocampal neurons were cultured, treated, and then analyzed through immunofluorescence for mtDNA, mtROS, TMRM. RESULTS: In comparison to the control group, mitophagy levels in the experimental group were not significantly altered, yet there was a notable increase in apoptotic neurons. Furthermore, markers indicative of mitochondrial leakage and damage were found to be elevated in the experimental group compared to the control group, but these markers showed improvement following ART treatment. ART was effective in activating the PINK1/PARKIN pathway, enhancing mitophagy, and diminishing neuronal apoptosis. Behavioral assessments revealed that ART ameliorated symptoms in mice with anti-NMDAR encephalitis in the passive transfer model (PTM). The knockdown of PINK1 led to a reduction in mitophagy levels, and subsequent ART intervention did not alleviate symptoms in the anti-NMDAR encephalitis PTM mice, indicating that ART's therapeutic efficacy is mediated through the activation of the PINK1/PARKIN pathway. CONCLUSIONS: At the onset of anti-NMDAR encephalitis, mitochondrial damage is observed; however, this damage is mitigated by the activation of mitophagy via the PINK1/PARKIN pathway. This regulatory feedback mechanism facilitates the removal of damaged mitochondria, prevents neuronal apoptosis, and consequently safeguards neural tissue. ART activates the PINK1/PARKIN pathway to enhance mitophagy, thereby exerting neuroprotective effects and may achieve therapeutic goals in treating anti-NMDAR encephalitis.
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Encefalitis Antirreceptor N-Metil-D-Aspartato , Artesunato , Modelos Animales de Enfermedad , Fármacos Neuroprotectores , Proteínas Quinasas , Animales , Artesunato/farmacología , Artesunato/uso terapéutico , Ratones , Femenino , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Encefalitis Antirreceptor N-Metil-D-Aspartato/patología , Encefalitis Antirreceptor N-Metil-D-Aspartato/tratamiento farmacológico , Proteínas Quinasas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/patología , Neuronas/metabolismo , Microscopía Electrónica de Transmisión , Mitofagia/efectos de los fármacos , Apoptosis/efectos de los fármacos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Hipocampo/patología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismoRESUMEN
OBJECTIVE: Genetic variations in proteins of the mechanistic target of rapamycin (mTOR) pathway cause a spectrum of neurodevelopmental disorders often associated with brain malformations and with intractable epilepsy. The mTORopathies are characterized by hyperactive mTOR pathway and comprise tuberous sclerosis complex (TSC) and focal cortical dysplasia (FCD) type II. How hyperactive mTOR translates into abnormal neuronal activity and hypersynchronous network remains to be better understood. Previously, the role of upregulated GluN2C-containing glutamate-gated N-methyl-D-aspartate receptors (NMDARs) has been demonstrated for germline defects in the TSC genes. Here, we questioned whether this mechanism would expand to other mTORopathies in the different context of a somatic genetic variation of the MTOR protein recurrently found in FCD type II. METHODS: We used a rat model of FCD created by in utero electroporation of neural progenitors of dorsal telencephalon with expression vectors encoding either the wild-type or the pathogenic MTOR variant (p.S2215F). In this mosaic configuration, patch-clamp whole-cell recordings of the electroporated, spiny stellate neurons and extracellular recordings of the electroporated areas were performed in neocortical slices. Selective inhibitors were used to target mTOR activity and GluN2C-mediated currents. RESULTS: Neurons expressing the mutant protein displayed an excessive activation of GluN2C NMDAR-mediated spontaneous excitatory postsynaptic currents. GluN2C-dependent increase in spontaneous spiking activity was detected in the area of electroporated neurons in the mutant condition and was restricted to a critical time window between postnatal days P9 and P20. SIGNIFICANCE: Somatic MTOR pathogenic variant recurrently found in FCD type II resulted in overactivation of GluN2C-mediated neuronal NMDARs in neocortices of rat pups. The related and time-restricted local hyperexcitability was sensitive to subunit GluN2C-specific blockade. Our study suggests that GluN2C-related pathomechanisms might be shared in common by mTOR-related brain disorders.
Asunto(s)
Neuronas , Receptores de N-Metil-D-Aspartato , Serina-Treonina Quinasas TOR , Animales , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Serina-Treonina Quinasas TOR/genética , Ratas , Neuronas/metabolismo , Femenino , Malformaciones del Desarrollo Cortical/genética , Malformaciones del Desarrollo Cortical/fisiopatología , Modelos Animales de Enfermedad , Ratas Sprague-Dawley , Técnicas de Placa-Clamp , Malformaciones del Desarrollo Cortical de Grupo I/genética , Displasia Cortical Focal , EpilepsiaRESUMEN
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-ß, leading to N-methyl-D-aspartate (NMDA) receptor-dependent synaptic depression, spine elimination, and memory deficits. Glycine transporter type 1 (GlyT1) modulates glutamatergic neurotransmission via NMDA receptors (NMDAR), presenting a potential alternative therapeutic approach for AD. This study investigates the neuroprotective potential of GlyT1 inhibition in an amyloid-ß-induced AD mouse model. C57BL/6 mice were treated with N-[3-([1,1-Biphenyl]-4-yloxy)-3-(4-fluorophenyl)propyl]-N-methylglycine (NFPS), a GlyT1 inhibitor, 24 h prior to intrahippocampal injection of amyloid-ß. NFPS pretreatment prevented amyloid-ß-induced cognitive deficits in short-term and long-term memory, evidenced by novel object recognition and spatial memory tasks. Moreover, NFPS pretreatment curbed microglial activation, astrocytic reactivity, and subsequent neuronal damage from amyloid-ß injection. An extensive label-free quantitative UPLC-MSE proteomic analysis was performed on the hippocampi of mice treated with NFPS. In proteomics, KEGG enrichment analysis revealed increased in dopaminergic synapse, purine-containing compound biosynthetic process and long-term potentiation, and a reduction in Glucose catabolic process and glycolytic process pathways. The western blot analysis confirmed that NFPS treatment elevated BDNF levels, correlating with enhanced TRKB phosphorylation and mTOR activation. Moreover, NFPS treatment reduced the GluN2B expression after 6 h, which was associated with an increase on CaMKIV and CREB phosphorylation. Collectively, these findings demonstrate that GlyT1 inhibition by NFPS activates diverse neuroprotective pathways, enhancing long-term potentiation signaling and countering amyloid-ß-induced hippocampal damage.
Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , Proteínas de Transporte de Glicina en la Membrana Plasmática , Hipocampo , Ratones Endogámicos C57BL , Fármacos Neuroprotectores , Animales , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/inducido químicamente , Enfermedad de Alzheimer/prevención & control , Péptidos beta-Amiloides/metabolismo , Masculino , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Ratones , Hipocampo/metabolismo , Hipocampo/efectos de los fármacos , Proteínas de Transporte de Glicina en la Membrana Plasmática/antagonistas & inhibidores , Proteínas de Transporte de Glicina en la Membrana Plasmática/metabolismo , Modelos Animales de Enfermedad , Sarcosina/análogos & derivados , Sarcosina/farmacología , Sarcosina/uso terapéutico , Neuroprotección/efectos de los fármacos , Neuroprotección/fisiologíaRESUMEN
4-Chlorokynurenine (4-Cl-KYN, AV-101) is a prodrug of a NMDA receptor antagonist and is in clinical development for potential CNS indications. We sought to further understand the distribution and metabolism of 4-Cl-KYN, as this information might provide a strategy to enhance the clinical development of this drug. We used excretion studies in rats, in vitro transporter assays, and pharmacogenetic analysis of clinical trial data to determine how 4-Cl-KYN and metabolites are distributed. Our data indicated that a novel acetylated metabolite (N-acetyl-4-Cl-KYN) did not affect the uptake of 4-Cl-KYN across the blood-brain barrier via LAT1. 4-Cl-KYN and its metabolites were found to be renally excreted in rodents. In addition, we found that N-acetyl-4-Cl-KYN inhibited renal and hepatic transporters involved in excretion. Thus, this metabolite has the potential to limit the excretion of a range of compounds. Our pharmacogenetic analysis found that a SNP in N-acetyltransferase 8 (NAT8, rs13538) was linked to levels of N-acetyl-4-Cl-KYN relative to 4-Cl-KYN found in the plasma and that a SNP in SLC7A5 (rs28582913) was associated with the plasma levels of the active metabolite, 7-Cl-KYNA. Thus, we have a pharmacogenetics-based association for plasma drug level that could aid in the drug development of 4-Cl-KYN and have investigated the interaction of a novel metabolite with drug transporters.
Asunto(s)
Ácido Quinurénico , Fármacos Neuroprotectores , Ratas , Animales , Quinurenina , Analgésicos , Fármacos Neuroprotectores/metabolismoRESUMEN
BACKGROUND: Persistent somatoform pain disorder (PSPD) is often the initial diagnosis in patients seeking treatment in psychiatric departments, making it challenging to consider organic nervous system diseases. However, autoimmune encephalitis can present with atypical initial symptoms, leading to misdiagnosis or missed diagnosis. Lumbar puncture, with antibody support, plays a crucial role in diagnosing autoimmune encephalitis. CASE PRESENTATION: This report describes a 40-year-old male adult patient who was initially diagnosed with persistent somatoform pain disorder in 2022. The patient reported a reduction in pain while resting on his back. There were no fever or relevant medical history. Despite 8 months of symptomatic treatment, the symptoms did not improve. Moreover, the patient developed confusion, gibberish speech, non-cooperation during questioning, and increased frequency and amplitude of upper limb convulsions. Lumbar puncture revealed elevated protein levels and protein-cell dissociation. The autoimmune encephalitis antibody NMDAR (+) was detected, leading to a diagnosis of autoimmune encephalitis (NMDAR). CONCLUSION: Autoimmune encephalitis (NMDAR), starting with persistent somatoform pain (PSPD), often presents with atypical symptoms and can be easily misdiagnosed. Therefore, it is important to consider the possibility of organic nervous system disease in time, and to test serum or cerebrospinal fluid antibodies to rule out organic nervous system disease after symptomatic treatment of mental disorders is ineffective. This approach facilitates the early diagnosis of autoimmune encephalitis and other underlying organic neurological disorders.
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Encefalitis Antirreceptor N-Metil-D-Aspartato , Humanos , Masculino , Adulto , Encefalitis Antirreceptor N-Metil-D-Aspartato/diagnóstico , Encefalitis Antirreceptor N-Metil-D-Aspartato/complicaciones , Dolor Musculoesquelético/diagnóstico , Dolor Musculoesquelético/etiología , Trastornos Somatomorfos/diagnósticoRESUMEN
Patients with herpes simplex virus (HSV) encephalitis (HSE) often develop neuronal autoantibody-associated encephalitis (AE) post-infection. Risk factors of AE are unknown. We tested the hypotheses that predisposition for AE post-HSE may be involved, including genetic variants at specific loci, human leucocyte (HLA) haplotypes, or the blood innate immune response against HSV, including type I interferon (IFN) immunity. Patients of all ages with HSE diagnosed between 1 January 2014 and 31 December 2021 were included in one of two cohorts depending on whether the recruitment was at HSE onset (Spanish Cohort A) or by the time of new neurological manifestations (international Cohort B). Patients were assessed for the type of neurological syndromes; HLA haplotypes; blood type I-IFN signature [RNA quantification of 6 or 28 IFN-response genes (IRG)] and toll-like receptor (TLR3)-type I IFN-related gene mutations. Overall, 190 patients (52% male) were recruited, 93 in Cohort A and 97 in Cohort B. Thirty-nine (42%) patients from Cohort A developed neuronal autoantibodies, and 21 (54%) of them developed AE. Three syndromes (choreoathetosis, anti-NMDAR-like encephalitis and behavioural-psychiatric) showed a high (≥95% cases) association with neuronal autoantibodies. Patients who developed AE post-HSE were less likely to carry the allele HLA-A*02 (4/21, 19%) than those who did not develop AE (42/65, 65%, P = 0.0003) or the Spanish general population (2005/4335, 46%, P = 0.0145). Blood IFN signatures using 6 or 28 IRG were positive in 19/21 (91%) and 18/21 (86%) patients at HSE onset, and rapidly decreased during follow-up. At Day 21 after HSE onset, patients who later developed AE had higher median IFN signature compared with those who did not develop AE [median Zs-6-IRG 1.4 (0.6; 2.0) versus 0.2 (-0.4; 0.8), P = 0.03]. However, a very high median Zs-6-IRG (>4) or persistently increased IFN signature associated with uncontrolled viral infection. Whole exome sequencing showed that the percentage of TLR3-IFN-related mutations in patients who developed AE was not different from those who did not develop AE [3/37 (8%) versus 2/57 (4%), P = 0.379]. Multivariate logistic regression showed that a moderate increase of the blood IFN signature at Day 21 (median Zs-6-IRG >1.5 but <4) was the most important predictor of AE post-HSE [odds ratio 34.8, interquartile ratio (1.7-691.9)]. Altogether, these findings show that most AE post-HSE manifest with three distinct syndromes, and HLA-A*02, but not TLR3-IFN-related mutations, confer protection from developing AE. In addition to neuronal autoantibodies, the blood IFN signature in the context of HSE may be potentially useful for the diagnosis and monitoring of HSE complications.
Asunto(s)
Encefalitis por Herpes Simple , Interferón Tipo I , Enfermedades del Sistema Nervioso , Humanos , Masculino , Femenino , Encefalitis por Herpes Simple/complicaciones , Encefalitis por Herpes Simple/genética , Receptor Toll-Like 3/genética , Autoanticuerpos , Antígenos HLA-ARESUMEN
Nuclear Ca2+ waves elicited by NMDAR and L-type voltage-gated Ca2+-channels as well as protein transport from synapse-to-nucleus are both instrumental in control of plasticity-related gene expression. At present it is not known whether fast [Ca2+]n transients converge in the nucleus with signaling of synapto-nuclear protein messenger. Jacob is a protein that translocate a signalosome from N-methyl-D-aspartate receptors (NMDAR) to the nucleus and that docks this signalosome to the transcription factor CREB. Here we show that the residing time of Jacob in the nucleoplasm strictly correlates with nuclear [Ca2+]n transients elicited by neuronal activity. A steep increase in [Ca2+]n induces instantaneous uncoupling of Jacob from LaminB1 at the nuclear lamina and promotes the association with the transcription factor cAMP-responsive element-binding protein (CREB) in hippocampal neurons. The size of the Jacob pool at the nuclear lamina is controlled by previous activity-dependent nuclear import, and thereby captures the previous history of NMDAR-induced nucleocytoplasmic shuttling. Moreover, the localization of Jacob at the nuclear lamina strongly correlates with synaptic activity and [Ca2+]n waves reflecting ongoing neuronal activity. In consequence, the resulting extension of the nuclear residing time of Jacob amplifies the capacity of the Jacob signalosome to regulate CREB-dependent gene expression and will, thereby, compensate for the relatively small number of molecules reaching the nucleus from individual synapses.