RESUMEN

Currently, stem cells technology is an effective tool in regenerative medicine. Cell therapy is based on the use of stem/progenitor cells to repair or replace damaged tissues or organs. This approach can be used to treat various diseases, such as cardiovascular, neurological diseases, and injuries of various origins. The mechanisms of cell therapy therapeutic action are based on the integration of the graft into the damaged tissue (replacement effect) and the ability of cells to secrete biologically active molecules such as cytokines, growth factors and other signaling molecules that promote regeneration (paracrine effect). However, cell transplantation has a number of limitations due to cell transportation complexity and immune rejection. A potentially more effective therapy is using only paracrine factors released by stem cells. Secreted factors can positively affect the damaged tissue: promote forming new blood vessels, stimulate cell proliferation, and reduce inflammation and apoptosis. In this work, we have studied the anti-inflammatory and neuroprotective effects of proteins with a molecular weight below 100 kDa secreted by glial progenitor cells obtained from human induced pluripotent stem cells. Proteins secreted by glial progenitor cells exerted anti-inflammatory effects in a primary glial culture model of LPS-induced inflammation by reducing nitric oxide (NO) production through inhibition of inducible NO synthase (iNOS). At the same time, added secreted proteins neutralized the effect of glutamate, increasing the number of viable neurons to control values. This effect is a result of decreased level of intracellular calcium, which, at elevated concentrations, triggers apoptotic death of neurons. In addition, secreted proteins reduce mitochondrial depolarization caused by glutamate excitotoxicity and help maintain higher NADH levels. This therapy can be successfully introduced into clinical practice after additional preclinical studies, increasing the effectiveness of rehabilitation of patients with neurological diseases.

RESUMEN

BACKGROUND: Ischemic stroke is a significant cause of death and disability with a limited treatment time window. The reduction of early glutamate excitotoxicity using neuroprotective agents targeting N-methyl-d-aspartic acid (NMDA) receptors have attracted recent research attention. SHPL-49, a structurally modified derivative of salidroside, was synthesized by our team. Previous studies have confirmed the neuroprotective efficacy of SHPL-49 in rats with ischemic stroke. However, the underlying mechanisms need to be clarified. METHODS: We conducted in vivo experiments using the permanent middle cerebral artery occlusion rat model to investigate the role of SHPL-49 in glutamate release at different time points and treatment durations. Glutamate transporters and receptor proteins and neural survival proteins in the brain were also examined at the same time points. In vitro, primary neurons and the coculture system of primary neurons-astrocytes were subjected to oxygen-glucose deprivation and glutamate injury. Proteomics and parallel reaction monitoring analyses were performed to identify potential therapeutic targets of SHPL-49, which were further confirmed through in vitro experiments on the inhibition and mutation of the target. RESULTS: SHPL-49 significantly reduced glutamate release caused by hypoxia-ischemia. One therapeutic pathway of SHPL-49 was promoting the expression of glutamate transporter-1 to increase glutamate reuptake and further reduce the occurrence of subsequent neurotoxicity. In addition, we explored the therapeutic targets of SHPL-49 and its regulatory effects on glutamate receptors for the first time. SHPL-49 enhanced neuroprotection by activating the NMDA subunit NR2A, which upregulated the cyclic-AMP response binding protein (CREB) neural survival pathway and Akt phosphorylation. Since calcium/calmodulin-dependent kinase IIα (CaMKIIα) is necessary for synaptic transmission of NMDA receptors, we explored the interaction between CaMKIIα and SHPL-49, which protected CaMKIIα from hypoxia-ischemia-induced autophosphorylation damage. CONCLUSION: Overall, SHPL-49 enhanced neuronal survival and attenuated acute ischemic stroke by promoting the NR2A-CAMKⅡα-Akt/CREB pathway. Our study provides the first evidence demonstrating that the neuroprotective effect of SHPL-49 is achieved by promoting the NR2A subunit to extend the treatment time window, making it a promising drug for ischemic stroke.

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Autism spectrum disorder (ASD) is a neurodevelopmental condition marked by restricted and repetitive behaviors as well as difficulties with social interaction. Numerous studies have revealed aberrant lipid mediators and autoimmunity as a recognized etiological cause of ASD that is amenable to therapeutic intervention. In this study, the relationship between the relative cyclooxygenase-2/prostaglandin E2 ratio (COX-2/PGE2) as a lipid mediator marker and anti-nucleosome autoantibodies as an autoimmunity marker of ASD was investigated using multiple regression and combined receiver operating characteristic (ROC) curve analyses. The study also sought to identify the linear combination of these variables that optimizes the partial area under the ROC curves. There were forty ASD children and forty-two age- and gender-matched controls included in the current study. Using combined ROC curve analysis, a notable increase in the area under the curve was seen in the patient group, using the control group as a reference group. Additionally, it was reported that the combined markers had improved specificity and sensitivity. This study demonstrates how the predictive value of particular biomarkers associated with lipid metabolism and autoimmunity in children with ASD can be measured using a ROC curve analysis. This technique should help us better understand the etiological mechanism of ASD and how it may adversely affect cellular homeostasis, which is essential to maintaining healthy metabolic pathways. Early diagnosis and intervention may be facilitated by this knowledge.

RESUMEN

There is a public health concern about the use of methylphenidate (MPH) since the higher prescription for young individuals and non-clinical purposes is addressed to the limited understanding of its neurochemical and psychiatric consequences. This study aimed to evaluate the impact of early and chronic MPH treatment on the striatum focusing on amino acid profile, glutamatergic excitotoxicity, redox status, neuroinflammation and glial cell responses. Male Wistar rats were treated with MPH (2.0 mg/kg) or saline solution from the 15th to the 44th postnatal day. Biochemical and histological analyses were conducted after the last administration. MPH altered the amino acid profile in the striatum, increasing glutamate and ornithine levels, while decreasing the levels of serine, phenylalanine, and branched-chain amino acids (leucine, valine, and isoleucine). Glutamate uptake and Na+,K+-ATPase activity were decreased in the striatum of MPH-treated rats as well as increased ATP levels, as indicator of glutamatergic excitotoxicity. Moreover, MPH caused lipid peroxidation and nitrative stress, increased TNF alpha expression, and induced high levels of astrocytes, and led to a decrease in BDNF levels. In summary, our results suggest that chronic early-age treatment with MPH induces parallel activation of damage-associated pathways in the striatum and increases its vulnerability during the juvenile period. In addition, data presented here contribute to shedding light on the mechanisms underlying MPH-induced striatal damage and its potential implications for neurodevelopmental disorders.

Asunto(s)

Aminoácidos , Astrocitos , Estimulantes del Sistema Nervioso Central , Cuerpo Estriado , Ácido Glutámico , Metilfenidato , Ratas Wistar , Animales , Masculino , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Metilfenidato/toxicidad , Metilfenidato/farmacología , Ácido Glutámico/metabolismo , Ratas , Estimulantes del Sistema Nervioso Central/toxicidad , Estimulantes del Sistema Nervioso Central/farmacología , Aminoácidos/metabolismo , Peroxidación de Lípido/efectos de los fármacos

RESUMEN

Despite the successes in the prevention and treatment of strokes, it is still necessary to search for effective cytoprotectors that can suppress the damaging factors of cerebral ischemia. Among the known neuroprotectors, there are a number of drugs with a protein nature. In the present study, we were able to obtain recombinant SELENOM, a resident of the endoplasmic reticulum that exhibits antioxidant properties in its structure and functions. The resulting SELENOM was tested in two brain injury (in vitro) models: under ischemia-like conditions (oxygen-glucose deprivation/reoxygenation, OGD/R) and glutamate excitotoxicity (GluTox). Using molecular biology methods, fluorescence microscopy, and immunocytochemistry, recombinant SELENOM was shown to dose-dependently suppress ROS production in cortical cells in toxic models, reduce the global increase in cytosolic calcium ([Ca2+]i), and suppress necrosis and late stages of apoptosis. Activation of SELENOM's cytoprotective properties occurs due to its penetration into cortical cells through actin-dependent transport and activation of the Ca2+ signaling system. The use of SELENOM resulted in increased antioxidant protection of cortical cells and suppression of the proinflammatory factors and cytokines expression.

RESUMEN

Cytotoxic neuronal swelling and glutamate excitotoxicity are two hallmarks of ischemic stroke. However, the underlying molecular mechanisms are not well understood. Here, it is reported that SWELL1, the essential subunit of the volume-regulated anion channel (VRAC), plays a dual role in ischemic injury by promoting neuronal swelling and glutamate excitotoxicity. SWELL1 expression is upregulated in neurons and astrocytes after experimental stroke in mice. The neuronal SWELL1 channel is activated by intracellular hypertonicity, leading to Cl- influx-dependent cytotoxic neuronal swelling and subsequent cell death. Additionally, the SWELL1 channel in astrocytes mediates pathological glutamate release, indicated by increases in neuronal slow inward current frequency and tonic NMDAR current. Pharmacologically, targeting VRAC with a new inhibitor, an FDA-approved drug Dicumarol, attenuated cytotoxic neuronal swelling and cell death, reduced astrocytic glutamate release, and provided significant neuroprotection in mice when administered either before or after ischemia. Therefore, these findings uncover the pleiotropic effects of the SWELL1 channel in neurons and astrocytes in the pathogenesis of ischemic stroke and provide proof of concept for therapeutically targeting it in this disease.

RESUMEN

Glutamate excitotoxicity is involved in retinal ganglion cell (RGC) death in various retinal degenerative diseases, including ischemia-reperfusion injury and glaucoma. Excitotoxic RGC death is caused by both direct damage to RGCs and indirect damage through neuroinflammation of retinal glial cells. Omidenepag (OMD), a novel E prostanoid receptor 2 (EP2) agonist, is a recently approved intraocular pressure-lowering drug. The second messenger of EP2 is cyclic adenosine monophosphate (cAMP), which activates protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). In this study, we investigated the neuroprotective effects of OMD on excitotoxic RGC death by focusing on differences in cAMP downstream signaling from the perspective of glia-neuron interactions. We established a glutamate excitotoxicity model in vitro and NMDA intravitreal injection model in vivo. In vitro, rat primary RGCs were used in an RGC survival rate assay. MG5 cells (mouse microglial cell line) and A1 cells (astrocyte cell line) were used for immunocytochemistry and Western blotting to evaluate the expressions of COX-1/2, PKA, Epac1/2, pCREB, cleaved caspase-3, inflammatory cytokines, and neurotrophic factors. Mouse retinal specimens underwent hematoxylin and eosin staining, flat-mounted retina examination, and immunohistochemistry. OMD significantly suppressed excitotoxic RGC death, cleaved caspase-3 expression, and activated glia both in vitro and in vivo. Moreover, it inhibited Epac1 and inflammatory cytokine expression and promoted COX-2, pCREB, and neurotrophic factor expression. OMD may have neuroprotective effects through inhibition of the Epac pathway and promotion of the COX-2-EP2-cAMP-PKA pathway by modulating glia-neuron interaction.

Asunto(s)

Proteínas Quinasas Dependientes de AMP Cíclico , AMP Cíclico , Ciclooxigenasa 2 , Neuroglía , Fármacos Neuroprotectores , Células Ganglionares de la Retina , Animales , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/metabolismo , Fármacos Neuroprotectores/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ciclooxigenasa 2/metabolismo , AMP Cíclico/metabolismo , Ratones , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/antagonistas & inhibidores , Subtipo EP2 de Receptores de Prostaglandina E/agonistas , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Ratas Sprague-Dawley , Ratas , Ácido Glutámico/metabolismo , Ácido Glutámico/toxicidad , Ratones Endogámicos C57BL , Masculino , N-Metilaspartato/farmacología , N-Metilaspartato/toxicidad , Neuronas/efectos de los fármacos , Neuronas/metabolismo

RESUMEN

Excessive levels of glutamate activity could potentially damage and kill neurons. Glutamate excitotoxicity is thought to play a critical role in many CNS and retinal diseases. Accordingly, glutamate excitotoxicity has been used as a model to study neuronal diseases. Immune proteins, such as major histocompatibility complex (MHC) class I molecules and their receptors, play important roles in many neuronal diseases, while T-cell receptors (TCR) are the primary receptors of MHCI. We previously showed that a critical component of TCR, CD3ζ, is expressed by mouse retinal ganglion cells (RGCs). The mutation of CD3ζ or MHCI molecules compromises the development of RGC structure and function. In this study, we investigated whether CD3ζ-mediated molecular signaling regulates RGC death in glutamate excitotoxicity. We show that mutation of CD3ζ significantly increased RGC survival in NMDA-induced excitotoxicity. In addition, we found that several downstream molecules of TCR, including Src (proto-oncogene tyrosine-protein kinase) family kinases (SFKs) and spleen tyrosine kinase (Syk), are expressed by RGCs. Selective inhibition of an SFK member, Hck, or Syk members, Syk or Zap70, significantly increased RGC survival in NMDA-induced excitotoxicity. These results provide direct evidence to reveal the underlying molecular mechanisms that control RGC death under disease conditions.

Asunto(s)

Complejo CD3 , Ácido Glutámico , Células Ganglionares de la Retina , Transducción de Señal , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/patología , Animales , Ácido Glutámico/metabolismo , Transducción de Señal/efectos de los fármacos , Complejo CD3/metabolismo , Ratones , Ratones Endogámicos C57BL , N-Metilaspartato/toxicidad , Supervivencia Celular/efectos de los fármacos , Retina/metabolismo , Retina/patología , Familia-src Quinasas/metabolismo , Quinasa Syk/metabolismo

RESUMEN

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairments in social interaction and restricted and repetitive behaviors. Oxidative stress may be a critical link between mitochondrial dysfunction and ASD as reactive oxygen species (ROS) generated from pro-oxidant environmental toxicants and activated immune cells can result in mitochondrial failure. Recently, mitochondrial dysfunction, autoimmunity, and abnormal lipid mediators have been identified in multiple investigations as an acknowledged etiological mechanism of ASD that can be targeted for therapeutic intervention. METHODS: The relationship between lipid mediator markers linked to inflammation induction, such as phospholipase A2/cyclooxygenase-2 (PLA2/Cox-2), and the mitochondrial dysfunction marker anti-mitochondrial antibodies (AMA-M2), and anti-histone autoantibodies in the etiology of ASD was investigated in this study using combined receiver operating characteristic (ROC) curve analyses. This study also sought to identify the linear combination for a given set of markers that optimizes the partial area under ROC curves. This study included 40 age- and sex-matched controls and 40 ASD youngsters. The plasma of both groups was tested for PLA2/COX-2, AMA-M2, and anti-histone autoantibodies' levels using ELISA kits. ROC curves and logistic regression models were used in the statistical analysis. RESULTS: Using the integrated ROC curve analysis, a notable rise in the area under the curve was noticed. Additionally, the combined markers had markedly improved specificity and sensitivity. CONCLUSIONS: The current study suggested that measuring the predictive value of selected biomarkers related to mitochondrial dysfunction, autoimmunity, and lipid metabolism in children with ASD using a ROC curve analysis could lead to a better understanding of the etiological mechanism of ASD as well as its relationship with metabolism.

RESUMEN

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disorder. While there are five FDA-approved drugs for treating this disease, each has only modest benefits. To design new and more effective therapies for ALS, particularly for sporadic ALS of unknown and diverse etiologies, we must identify key, convergent mechanisms of disease pathogenesis. This review focuses on the origin and effects of glutamate-mediated excitotoxicity in ALS (the cortical hyperexcitability hypothesis), in which increased glutamatergic signaling causes motor neurons to become hyperexcitable and eventually die. We characterize both primary and secondary contributions to excitotoxicity, referring to processes taking place at the synapse and within the cell, respectively. 'Primary pathways' include upregulation of calcium-permeable AMPA receptors, dysfunction of the EAAT2 astrocytic glutamate transporter, increased release of glutamate from the presynaptic terminal, and reduced inhibition by cortical interneurons-all of which have been observed in ALS patients and model systems. 'Secondary pathways' include changes to mitochondrial morphology and function, increased production of reactive oxygen species, and endoplasmic reticulum (ER) stress. By identifying key targets in the excitotoxicity cascade, we emphasize the importance of this pathway in the pathogenesis of ALS and suggest that intervening in this pathway could be effective for developing therapies for this disease.

Asunto(s)

Esclerosis Amiotrófica Lateral , Ácido Glutámico , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Humanos , Ácido Glutámico/metabolismo , Animales , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Envejecimiento/metabolismo , Receptores AMPA/metabolismo , Estrés del Retículo Endoplásmico , Mitocondrias/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Astrocitos/metabolismo , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

Huntington's disease (HD) is a neurodegenerative genetic disorder caused by an expansion in the CAG repeat tract of the huntingtin (HTT) gene resulting in behavioural, cognitive, and motor defects. Current knowledge of disease pathogenesis remains incomplete, and no disease course-modifying interventions are in clinical use. We have previously reported the development and characterisation of the OVT73 transgenic sheep model of HD. The 73 polyglutamine repeat is somatically stable and therefore likely captures a prodromal phase of the disease with an absence of motor symptomatology even at 5-years of age and no detectable striatal cell loss. To better understand the disease-initiating events we have undertaken a single nuclei transcriptome study of the striatum of an extensively studied cohort of 5-year-old OVT73 HD sheep and age matched wild-type controls. We have identified transcriptional upregulation of genes encoding N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors in medium spiny neurons, the cell type preferentially lost early in HD. Further, we observed an upregulation of astrocytic glutamate uptake transporters and medium spiny neuron GABAA receptors, which may maintain glutamate homeostasis. Taken together, these observations support the glutamate excitotoxicity hypothesis as an early neurodegeneration cascade-initiating process but the threshold of toxicity may be regulated by several protective mechanisms. Addressing this biochemical defect early may prevent neuronal loss and avoid the more complex secondary consequences precipitated by cell death.

Asunto(s)

Modelos Animales de Enfermedad , Ácido Glutámico , Enfermedad de Huntington , Neuronas , Receptores de N-Metil-D-Aspartato , Animales , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Ovinos , Neuronas/metabolismo , Neuronas/patología , Ácido Glutámico/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , RNA-Seq , Receptores AMPA/genética , Receptores AMPA/metabolismo , Muerte Celular/genética , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Animales Modificados Genéticamente , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Humanos , Transcriptoma/genética , Receptores de Ácido Kaínico/genética , Receptores de Ácido Kaínico/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/genética , Neuronas Espinosas Medianas

RESUMEN

The primary neural circuit affected in Amyotrophic Lateral Sclerosis (ALS) patients is the corticospinal motor circuit, originating in upper motor neurons (UMNs) in the cerebral motor cortex which descend to synapse with the lower motor neurons (LMNs) in the spinal cord to ultimately innervate the skeletal muscle. Perturbation of these neural circuits and consequent loss of both UMNs and LMNs, leading to muscle wastage and impaired movement, is the key pathophysiology observed. Despite decades of research, we are still lacking in ALS disease-modifying treatments. In this review, we document the current research from patient studies, rodent models, and human stem cell models in understanding the mechanisms of corticomotor circuit dysfunction and its implication in ALS. We summarize the current knowledge about cortical UMN dysfunction and degeneration, altered excitability in LMNs, neuromuscular junction degeneration, and the non-cell autonomous role of glial cells in motor circuit dysfunction in relation to ALS. We further highlight the advances in human stem cell technology to model the complex neural circuitry and how these can aid in future studies to better understand the mechanisms of neural circuit dysfunction underpinning ALS.

Asunto(s)

Esclerosis Amiotrófica Lateral , Neuronas Motoras , Esclerosis Amiotrófica Lateral/fisiopatología , Esclerosis Amiotrófica Lateral/patología , Humanos , Neuronas Motoras/patología , Neuronas Motoras/fisiología , Animales , Red Nerviosa/fisiopatología , Red Nerviosa/patología , Unión Neuromuscular/fisiopatología , Unión Neuromuscular/patología , Modelos Animales de Enfermedad , Corteza Motora/fisiopatología , Corteza Motora/patología

RESUMEN

Excitotoxicity is a common pathological process in neurological diseases caused by excess glutamate. The purpose of this study was to evaluate the effect of gypenoside XVII (GP-17), a gypenoside monomer, on the glutamatergic system. In vitro, in rat cortical nerve terminals (synaptosomes), GP-17 dose-dependently decreased glutamate release with an IC50 value of 16 µM. The removal of extracellular Ca2+ or blockade of N-and P/Q-type Ca2+ channels and protein kinase A (PKA) abolished the inhibitory effect of GP-17 on glutamate release from cortical synaptosomes. GP-17 also significantly reduced the phosphorylation of PKA, SNAP-25, and synapsin I in cortical synaptosomes. In an in vivo rat model of glutamate excitotoxicity induced by kainic acid (KA), GP-17 pretreatment significantly prevented seizures and rescued neuronal cell injury and glutamate elevation in the cortex. GP-17 pretreatment decreased the expression levels of sodium-coupled neutral amino acid transporter 1, glutamate synthesis enzyme glutaminase and vesicular glutamate transporter 1 but increased the expression level of glutamate metabolism enzyme glutamate dehydrogenase in the cortex of KA-treated rats. In addition, the KA-induced alterations in the N-methyl-D-aspartate receptor subunits GluN2A and GluN2B in the cortex were prevented by GP-17 pretreatment. GP-17 also prevented the KA-induced decrease in cerebral blood flow and arginase II expression. These results suggest that (i) GP-17, through the suppression of N- and P/Q-type Ca2+ channels and consequent PKA-mediated SNAP-25 and synapsin I phosphorylation, reduces glutamate exocytosis from cortical synaptosomes; and (ii) GP-17 has a neuroprotective effect on KA-induced glutamate excitotoxicity in rats through regulating synaptic glutamate release and cerebral blood flow.

Asunto(s)

Proteínas Quinasas Dependientes de AMP Cíclico , Ácido Glutámico , Gynostemma , Animales , Ácido Glutámico/metabolismo , Ratas , Masculino , Gynostemma/química , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ratas Sprague-Dawley , Sinaptosomas/metabolismo , Sinaptosomas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Ácido Kaínico/toxicidad , Convulsiones/inducido químicamente , Convulsiones/metabolismo , Convulsiones/tratamiento farmacológico , Convulsiones/prevención & control , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Proteína 25 Asociada a Sinaptosomas/metabolismo , Sinapsinas/metabolismo , Fosforilación/efectos de los fármacos , Calcio/metabolismo , Extractos Vegetales

RESUMEN

Due to the putative role of butyrylcholinesterase (BChE) in regulation of acetylcholine levels and functions in the late stages of the Alzheimer's disease (AD), the potential of selective inhibitors (BChEIs) has been envisaged as an alternative to administration of acetylcholinesterase inhibitors (AChEIs). Starting from our recent findings, herein the synthesis and in vitro evaluation of cholinesterase (ChE) inhibition of a novel series of some twenty 3,4,5,6-tetrahydroazepino[4,3-b]indol-1(2H)-one derivatives, bearing at the indole nitrogen diverse alkyl-bridged 4-arylalkylpiperazin-1-yl chains, are reported. The length of the spacers, as well as the type of arylalkyl group affected the enzyme inhibition potency and BChE/AChE selectivity. Two compounds, namely 14c (IC50 = 163 nM) and 14d (IC50 = 65 nM), bearing at the nitrogen atom in position 6 a n-pentyl- or n-heptyl-bridged 4-phenethylpiperazin-1-yl chains, respectively, proved to be highly potent mixed-type inhibitors of both equine and human BChE isoforms, showing more than two order magnitude of selectivity over AChE. The study of binding kinetics through surface plasmon resonance (SPR) highlighted differences in their BChE residence times (8 and 47 s for 14c and 14d, respectively). Moreover, 14c and 14d proved to hit other mechanisms known to trigger neurodegeneration underlying AD and other CNS disorders. Unlike 14c, compound 14d proved also capable of inhibiting by more than 60% the in vitro self-induced aggregation of neurotoxic amyloid-ß (Aß) peptide at 100 µM concentration. On the other hand, 14c was slightly better than 14d in counteracting, at 1 and 10 µM concentration, glutamate excitotoxicity, due to over-excitation of NMDA receptors, and hydrogen peroxide-induced oxidative stress assessed in neuroblastoma cell line SH-SY5Y. This paper is dedicated to Prof. Marcello Ferappi, former dean of the Faculty of Pharmacy of the University of Bari, in the occasion of his 90th birthday.

Asunto(s)

Enfermedad de Alzheimer , Neuroblastoma , Humanos , Animales , Caballos , Inhibidores de la Colinesterasa/química , Butirilcolinesterasa/metabolismo , Acetilcolinesterasa/metabolismo , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Línea Celular Tumoral , Nitrógeno , Relación Estructura-Actividad , Simulación del Acoplamiento Molecular

RESUMEN

Glutamate excitotoxicity accompanies numerous brain pathologies, including traumatic brain injury, ischemic stroke, and epilepsy. Disturbances of the ion homeostasis, mitochondria dysfunction, and further cell death are considered the main detrimental consequences of excitotoxicity. It is well known that neurons demonstrate different vulnerability to pathological exposures. In this regard, neurons containing calcium-permeable AMPA receptors (CP-AMPARs) may show higher susceptibility to excitotoxicity due to an additional pathway of Ca2+ influx. Here, we demonstrate that neurons containing CP-AMPARs are characterized by the higher amplitude of the glutamate-induced elevation of intracellular Ca2+ concentration ([Ca2+]i) and slower restoration of [Ca2+]i level compared to non-CP-AMPA neurons. Moreover, we have found that NASPM, an antagonist of CP-AMPARs, significantly decreases the amplitude of the [Ca2+]i elevation induced by glutamate or selective AMPARs agonist, 5-fluorowillardiine. In contrast, the antagonists of NMDARs or KARs affect insignificantly. We have also described some peculiarities of Na+, K+, and H+ intracellular dynamics in neurons containing CP-AMPARs. In particular, the amplitude of [Na+]i elevation was lower compared to non-CP-AMPA neurons, whereas the amplitude of [K+]i decrease was higher. We have shown the significant inverse correlation between [K+]i and [Ca2+]i and between intracellular pH and [Na+]i in CP-AMPARs-containing and non-CP-AMPA neurons upon glutamate excitotoxicity. Our data indicate that CP-AMPARs-mediated Ca2+ influx and slow removal of Ca2+ from the cytosol may underlie the vulnerability of the CP-AMPARs-containing neurons to glutamate excitotoxicity. Further studies of the mechanisms mediating the disturbances in ion homeostasis are crucial for developing new approaches for protecting these neurons at brain pathologies.

Asunto(s)

Calcio , Receptores AMPA , Receptores AMPA/fisiología , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/metabolismo , Calcio/metabolismo , Neuronas/metabolismo , Ácido Glutámico/metabolismo , Homeostasis

RESUMEN

Vitamin C (Vc) plays a pivotal role in a series of pathological processes, such as tumors, immune diseases, and neurological disorders. However, its therapeutic potential for tinnitus management remains unclear. In this study, we find that Vc relieves tinnitus in noise-exposed rats. In the 7-day therapy groups, spontaneous firing rate (SFR) increases from 1.17 ± 0.10 Hz to 1.77 ± 0.15 Hz after noise exposure. Vc effectively reduces the elevated SFR to 0.99 ± 0.07 and 0.55 ± 0.05 Hz at different doses. The glutamate level in auditory cortex of noise-exposed rats (3.78 ± 0.42 µM) increases relative to that in the control group (1.34 ± 0.22 µM). High doses of Vc (500 mg/kg/day) effectively reduce the elevated glutamate levels (1.49 ± 0.28 µM). Mechanistic studies show that the expression of glutamate transporter 1 (GLT-1) is impaired following noise exposure and that Vc treatment effectively restores GLT-1 expression in the auditory cortex. Meanwhile, the GLT-1 inhibitor, dl-threo-beta-benzyloxyaspartic acid (dl-TBOA), invalidates the protection role of Vc. Our finding shows that Vc substantially enhances glutamate clearance by upregulating GLT-1 and consequently alleviates noise-induced tinnitus. This study provides valuable insight into a novel biological target for the development of therapeutic interventions that may prevent the onset of tinnitus.

Asunto(s)

Corteza Auditiva , Acúfeno , Ratas , Animales , Corteza Auditiva/metabolismo , Ácido Ascórbico/farmacología , Ácido Ascórbico/metabolismo , Neuroprotección , Acúfeno/tratamiento farmacológico , Acúfeno/metabolismo , Ácido Glutámico/metabolismo , Modelos Animales de Enfermedad , Sistema de Transporte de Aminoácidos X-AG/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo

RESUMEN

Glutamate excitotoxicity and oxidative stress represent two major pathological mechanisms implicated in retinal disorders. In Diabetic Retinopathy (DR), oxidative stress is correlated to NADPH oxidase (NOX), a major source of Reactive Oxygen Species (ROS), and glutamate metabolism impairments. This study investigated the role of NOX2 and the novel NOX2 inhibitor, GLX7013170, in two models of a) retinal AMPA excitotoxicity [AMPA+GLX7013170 (10-4 M, intravitreally)] and b) early-stage DR paradigm (ESDR), GLX7013170: 14-day therapeutic treatment (topically, 20 µL/eye, 10 mg/mL (300 × 10-4 M), once daily) post-streptozotocin (STZ)-induced DR. Immunohistochemical studies for neuronal markers, nitrotyrosine, micro/macroglia, and real-time PCR, Western blot, and glutamate colorimetric assays were conducted. Diabetes increased NOX2 expression in the retina. NOX2 inhibition limited the loss of NOS-positive amacrine cells and the overactivation of micro/macroglia in both models. In the diabetic retina, GLX7013170 had no effect on retinal ganglion cell axons, but reduced oxidative damage, increased Bcl-2, reduced glutamate levels, and partially restored excitatory amino acid transporter (EAAT1) expression. These results suggest that NOX2 in diabetes is part of the triad, oxidative stress, NOX, and glutamate excitotoxicity, key players in the induction of DR. GLX7013170 is efficacious as a neuroprotective/anti-inflammatory agent and a potential therapeutic in retinal diseases, including ESDR.

RESUMEN

The disruption of the synaptic connection between the sensory inner hair cells (IHCs) and the auditory nerve fiber terminals of the type I spiral ganglion neurons (SGN) has been observed early in several auditory pathologies (e.g., noise-induced or ototoxic drug-induced or age-related hearing loss). It has been suggested that glutamate excitotoxicity may be an inciting element in the degenerative cascade observed in these pathological cochlear conditions. Moreover, oxidative damage induced by free hydroxyl radicals and nitric oxide may dramatically enhance cochlear damage induced by glutamate excitotoxicity. To investigate the underlying molecular mechanisms involved in cochlear excitotoxicity, we examined the molecular basis responsible for kainic acid (KA, a full agonist of AMPA/KA-preferring glutamate receptors)-induced IHC synapse loss and degeneration of the terminals of the type I spiral ganglion afferent neurons using a cochlear explant culture from P3 mouse pups. Our results demonstrated that disruption of the synaptic connection between IHCs and SGNs induced increased levels of oxidative stress, as well as altered both mitochondrial function and neurotrophin signaling pathways. Additionally, the application of exogenous antioxidants and neurotrophins (NT3, BDNF, and small molecule TrkB agonists) clearly increases synaptogenesis. These results suggest that understanding the molecular pathways involved in cochlear excitotoxicity is of crucial importance for the future clinical trials of drug interventions for auditory synaptopathies.

RESUMEN

This review advances an understanding of several dementias, based on four premises. One is that capillary hemorrhage is prominent in the pathogenesis of the dementias considered (dementia pugilistica, chronic traumatic encephalopathy, traumatic brain damage, Alzheimer's disease). The second premise is that hemorrhage introduces four neurotoxic factors into brain tissue: hypoxia of the tissue that has lost its blood supply, hemoglobin and its breakdown products, excitotoxic levels of glutamate, and opportunistic pathogens that can infect brain cells and induce a cytotoxic immune response. The third premise is that where organisms evolve molecules that are toxic to itself, like the neurotoxicity ascribed to hemoglobin, amyloid- (A), and glutamate, there must be some role for the molecule that gives the organism a selection advantage. The fourth is the known survival-advantage roles of hemoglobin (oxygen transport), of A (neurotrophic, synaptotrophic, detoxification of heme, protective against pathogens) and of glutamate (a major neurotransmitter). From these premises, we propose 1) that the brain has evolved a multi-factor response to intracerebral hemorrhage, which includes the expression of several protective molecules, including haptoglobin, hemopexin and A; and 2) that it is logical, given these premises, to posit that the four neurotoxic factors set out above, which are introduced into the brain by hemorrhage, drive the progression of the capillary-hemorrhage dementias. In this view, A expressed at the loci of neuronal death in these dementias functions not as a toxin but as a first responder, mitigating the toxicity of hemoglobin and the infection of the brain by opportunistic pathogens.

Asunto(s)

Enfermedad de Alzheimer , Humanos , Enfermedad de Alzheimer/patología , Hemorragia Cerebral/complicaciones , Encéfalo/patología , Hemoglobinas/metabolismo , Glutamatos

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a complex, neurodegenerative disorder in which alterations in structural, physiological, and metabolic parameters act synergistically. Over the last decade there has been a considerable focus on developing drugs to slow the progression of the disease. Despite this, only four disease-modifying therapies are approved in North America. Although additional research is required for a thorough understanding of ALS, we have accumulated a large amount of knowledge that could be better integrated into future clinical trials to accelerate drug development and provide patients with improved treatment options. It is likely that future, successful ALS treatments will take a multi-pronged therapeutic approach, targeting different pathways, akin to personalized medicine in oncology. In this review, we discuss the link between ALS pathophysiology and treatments, looking at the therapeutic failures as learning opportunities that can help us refine and optimize drug development.

Asunto(s)

Esclerosis Amiotrófica Lateral , Humanos , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/metabolismo