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BACKGROUND AND OBJECTIVES: To evaluate CSF inflammatory markers with accumulation of cortical damage as well as disease activity in patients with early relapsing-remitting MS (RRMS). METHODS: CSF levels of osteopontin (OPN) and 66 inflammatory markers were assessed using an immune-assay multiplex technique in 107 patients with RRMS (82 F/25 M, mean age 35.7 ± 11.8 years). All patients underwent regular clinical assessment and yearly 3T MRI scans for 2 years while 39 patients had a 4-year follow-up. White matter lesion number and volume, cortical lesions (CLs) and volume, and global cortical thickness (CTh) were evaluated together with the 'no evidence of disease activity' (NEDA-3) status, defined by no relapses, no disability worsening, and no MRI activity, including CLs. RESULTS: The random forest algorithm selected OPN, CXCL13, TWEAK, TNF, IL19, sCD30, sTNFR1, IL35, IL16, and sCD163 as significantly associated with changes in global CTh. OPN and CXCL13 were most related to accumulation of atrophy after 2 and 4 years. In a multivariate linear regression model on CSF markers, OPN (p < 0.001), CXCL13 (p = 0.001), and sTNFR1 (p = 0.024) were increased in those patients with accumulating atrophy (adjusted R-squared 0.615). The 10 markers were added in a model that included all clinical, demographic, and MRI variables: OPN (p = 0.002) and IL19 (p = 0.022) levels were confirmed to be significantly increased in patients developing more CTh change over the follow-up (adjusted R-squared 0.619). CXCL13 and OPN also revealed the best association with NEDA-3 after 2 years, with OPN significantly linked to disability accumulation (OR 2.468 [1.46-5.034], p = 0.004) at the multivariate logistic regression model. DISCUSSION: These data confirm and expand our knowledge on the prognostic role of the CSF inflammatory profile in predicting changes in cortical pathology and disease activity in early MS. The data emphasize a crucial role of OPN.
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Atrofia , Corteza Cerebral , Esclerosis Múltiple Recurrente-Remitente , Osteopontina , Humanos , Osteopontina/líquido cefalorraquídeo , Femenino , Masculino , Adulto , Esclerosis Múltiple Recurrente-Remitente/líquido cefalorraquídeo , Esclerosis Múltiple Recurrente-Remitente/patología , Esclerosis Múltiple Recurrente-Remitente/diagnóstico por imagen , Atrofia/patología , Persona de Mediana Edad , Corteza Cerebral/patología , Corteza Cerebral/diagnóstico por imagen , Imagen por Resonancia Magnética , Biomarcadores/líquido cefalorraquídeo , Estudios de Seguimiento , Adulto Joven , Progresión de la EnfermedadRESUMEN
Background: Cladribine has been introduced as a high-efficacy drug for treating relapsing-remitting multiple sclerosis (RRMS). Initial cohort studies showed early disease activity in the first year after drug initiation. Biomarkers that can predict early disease activity are needed. Aim: To estimate cerebrospinal fluid (CSF) markers of clinical and radiological responses after initiation of cladribine. Methods: Forty-two RRMS patients (30F/12M) treated with cladribine were included in a longitudinal prospective study. All patients underwent a CSF examination at treatment initiation, clinical follow-up including Expanded Disability Status Scale (EDSS) assessment, and a 3T MRI scan after 6,12 and 24 months, including the evaluation of white matter (WM) and cortical lesions (CLs). CSF levels of 67 inflammatory markers were assessed with immune-assay multiplex techniques. The 'no evidence of disease activity' (NEDA-3) status was assessed after two years and defined by no relapses, no disability worsening measured by EDSS and no MRI activity, including CLs. Results: Three patients were lost at follow-up. At the end of follow-up, 19 (48%) patients remained free from disease activity. IFNgamma, Chitinase3like1, IL32, Osteopontin, IL12(p40), IL34, IL28A, sTNFR2, IL20 and CCL2 showed the best association with disease activity. When added in a multivariate regression model including age, sex, and baseline EDSS, Chitinase 3 like1 (p = 0.049) significantly increased in those patients with disease activity. Finally, ROC analysis with Chitinase3like1 added to a model with EDSS, sex, age previous relapses, WM lesion number, CLs, number of Gad enhancing lesions and spinal cord lesions provided an AUC of 0.76 (95%CI 0.60-0.91). Conclusions: CSF Chitinase 3 like1 might provide prognostic information for predicting disease activity in the first years after initiation of cladribine. The drug's effect on chronic macrophage and microglia activation deserves further evaluation.
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Proteína 1 Similar a Quitinasa-3 , Cladribina , Esclerosis Múltiple Recurrente-Remitente , Humanos , Cladribina/uso terapéutico , Esclerosis Múltiple Recurrente-Remitente/diagnóstico por imagen , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Estudios Prospectivos , Proteína 1 Similar a Quitinasa-3/líquido cefalorraquídeoRESUMEN
Ketamine is clinically used fast-acting antidepressant. Its metabolite hydroxynorketamine (HNK) shows a robust antidepressant effect in animal studies. It is unclear, how these chemically distinct compounds converge on similar neuronal effects. While KET acts mostly as N-methyl-d-aspartate receptor (NMDAR) antagonist, the molecular target of HNK remains enigmatic. Here, we show that KET and HNK converge on rapid inhibition of glutamate release by reducing the release competence of synaptic vesicles and induce nuclear translocation of pCREB that controls expression of neuroplasticity genes connected to KET- and HNK-mediated antidepressant action. Ro25-6981, a selective antagonist of GluN2B, mimics effect of KET indicating that GluN2B-containing NMDAR might mediate the presynaptic effect of KET. Selective antagonist of α7 nicotinic acetylcholine receptors (α7nAChRs) or genetic deletion of Chrna7, its pore-forming subunit, fully abolishes HNK-induced synaptic and nuclear regulations, but leaves KET-dependent cellular effects unaffected. Thus, KET or HNK-induced modulation of synaptic transmission and nuclear translocation of pCREB can be mediated by selective signaling via NMDAR or α7nAChRs, respectively. Due to the rapid metabolism of KET to HNK, it is conceivable that subsequent modulation of glutamatergic and cholinergic neurotransmission affects circuits in a cell-type-specific manner and contributes to the therapeutic potency of KET. This finding promotes further exploration of new combined medications for mood disorders.
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Ketamina , Animales , Receptor Nicotínico de Acetilcolina alfa 7/genética , Antidepresivos/farmacología , Ácido Aspártico , Expresión Génica , Ketamina/análogos & derivados , Ketamina/farmacologíaRESUMEN
Ischemic stroke results in a loss of tissue homeostasis and integrity, the underlying pathobiology of which stems primarily from the depletion of cellular energy stores and perturbation of available metabolites 1 . Hibernation in thirteen-lined ground squirrels (TLGS), Ictidomys tridecemlineatus , provides a natural model of ischemic tolerance as these mammals undergo prolonged periods of critically low cerebral blood flow without evidence of central nervous system (CNS) damage 2 . Studying the complex interplay of genes and metabolites that unfolds during hibernation may provide novel insights into key regulators of cellular homeostasis during brain ischemia. Herein, we interrogated the molecular profiles of TLGS brains at different time points within the hibernation cycle via RNA sequencing coupled with untargeted metabolomics. We demonstrate that hibernation in TLGS leads to major changes in the expression of genes involved in oxidative phosphorylation and this is correlated with an accumulation of the tricarboxylic acid (TCA) cycle intermediates citrate, cis-aconitate, and α-ketoglutarate-αKG. Integration of the gene expression and metabolomics datasets led to the identification of succinate dehydrogenase (SDH) as the critical enzyme during hibernation, uncovering a break in the TCA cycle at that level. Accordingly, the SDH inhibitor dimethyl malonate (DMM) was able to rescue the effects of hypoxia on human neuronal cells in vitro and in mice subjected to permanent ischemic stroke in vivo . Our findings indicate that studying the regulation of the controlled metabolic depression that occurs in hibernating mammals may lead to novel therapeutic approaches capable of increasing ischemic tolerance in the CNS.
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The association between Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM) has been extensively demonstrated, but despite this, the pathophysiological mechanisms underlying it are still unknown. In previous work, we discovered a central role for the autophagy pathway in the common alterations observed between AD and T2DM. In this study, we further investigate the role of genes belonging to this pathway, measuring their mRNA expression and protein levels in 3xTg-AD transgenic mice, an animal model of AD. Moreover, primary mouse cortical neurons derived from this model and the human H4Swe cell line were used as cellular models of insulin resistance in AD brains. Hippocampal mRNA expression showed significantly different levels for Atg16L1, Atg16L2, GabarapL1, GabarapL2, and Sqstm1 genes at different ages of 3xTg-AD mice. Significantly elevated expression of Atg16L1, Atg16L2, and GabarapL1 was also observed in H4Swe cell cultures, in the presence of insulin resistance. Gene expression analysis confirmed that Atg16L1 was significantly increased in cultures from transgenic mice when insulin resistance was induced. Taken together, these results emphasise the association of the autophagy pathway in AD-T2DM co-morbidity, providing new evidence about the pathophysiology of both diseases and their mutual interaction.
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Enfermedad de Alzheimer , Diabetes Mellitus Tipo 2 , Resistencia a la Insulina , Ratones , Humanos , Animales , Enfermedad de Alzheimer/epidemiología , Diabetes Mellitus Tipo 2/epidemiología , Modelos Animales de Enfermedad , Comorbilidad , Ratones Transgénicos , Autofagia , ARN Mensajero , Proteínas PortadorasRESUMEN
Early stroke therapeutic approaches rely on limited options, further characterized by a narrow therapeutic time window. In this context, the application of transcranial direct current stimulation (tDCS) in the acute phases after brain ischemia is emerging as a promising non-invasive tool. Despite the wide clinical application of tDCS, the cellular mechanisms underlying its positive effects are still poorly understood. Here, we explored the effects of cathodal tDCS (C-tDCS) 6 h after focal forelimb M1 ischemia in Cx3CR1GFP/+ mice. C-tDCS improved motor functionality of the affected forelimb, as assessed by the cylinder and foot-fault tests at 48 h, though not changing the ischemic volume. In parallel, histological analysis showed that motor recovery is associated with decreased microglial cell density in the area surrounding the ischemic core, while astrocytes were not affected. Deeper analysis of microglia morphology within the perilesional area revealed a shift toward a more ramified healthier state, with increased processes' complexity and a less phagocytic anti-inflammatory activity. Taken together, our findings suggest a positive role for early C-tDCS after ischemia, which is able to modulate microglia phenotype and morphology in parallel to motor recovery.
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Isquemia Encefálica , Accidente Cerebrovascular , Estimulación Transcraneal de Corriente Directa , Animales , Isquemia Encefálica/patología , Modelos Animales de Enfermedad , Electrodos , Ratones , Microglía/patología , Accidente Cerebrovascular/patología , Estimulación Transcraneal de Corriente Directa/métodosRESUMEN
Amyloid beta (Aß) is linked to the pathology of Alzheimer's disease (AD). At physiological concentrations, Aß was proposed to enhance neuroplasticity and memory formation by increasing the neurotransmitter release from presynapse. However, the exact mechanisms underlying this presynaptic effect as well as specific contribution of endogenously occurring Aß isoforms remain unclear. Here, we demonstrate that Aß1-42 and Aß1-16, but not Aß17-42, increased size of the recycling pool of synaptic vesicles (SV). This presynaptic effect was driven by enhancement of endogenous cholinergic signalling via α7 nicotinic acetylcholine receptors, which led to activation of calcineurin, dephosphorylation of synapsin 1 and consequently resulted in reorganization of functional pools of SV increasing their availability for sustained neurotransmission. Our results identify synapsin 1 as a molecular target of Aß and reveal an effect of physiological concentrations of Aß on cholinergic modulation of glutamatergic neurotransmission. These findings provide new mechanistic insights in cholinergic dysfunction observed in AD.
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Péptidos beta-Amiloides/farmacología , Fragmentos de Péptidos/farmacología , Sinapsis/metabolismo , Sinapsinas/metabolismo , Vesículas Sinápticas/efectos de los fármacos , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Animales , Calcio/metabolismo , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Femenino , Humanos , Ratones , Ratones Noqueados , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neurotransmisores/metabolismo , Nicotina/farmacología , Fosforilación/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Vesículas Sinápticas/fisiología , Receptor Nicotínico de Acetilcolina alfa 7/deficiencia , Receptor Nicotínico de Acetilcolina alfa 7/genéticaRESUMEN
Proteasomes are protein complexes that mediate controlled degradation of damaged or unneeded cellular proteins. In neurons, proteasome regulates synaptic function and its dysfunction has been linked to neurodegeneration and neuronal cell death. However, endogenous mechanisms controlling proteasomal activity are insufficiently understood. Here, we describe a novel interaction between presynaptic scaffolding protein bassoon and PSMB4, a ß subunit of the 20S core proteasome. Expression of bassoon fragments that interact with PSMB4 in cell lines or in primary neurons attenuates all endopeptidase activities of cellular proteasome and induces accumulation of several classes of ubiquitinated and non-ubiquitinated substrates of the proteasome. Importantly, these effects are distinct from the previously reported impact of bassoon on ubiquitination and autophagy and might rely on a steric interference with the assembly of the 20S proteasome core. In line with a negative regulatory role of bassoon on endogenous proteasome we found increased proteasomal activity in the synaptic fractions prepared from brains of bassoon knock-out mice. Finally, increased activity of proteasome and lower expression levels of synaptic substrates of proteasome could be largely normalized upon expression of PSMB4-interacting fragments of bassoon in neurons derived from bassoon deficient mice. Collectively, we propose that bassoon interacts directly with proteasome to control its activity at presynapse and thereby it contributes to a compartment-specific regulation of neuronal protein homeostasis. These findings provide a mechanistic explanation for the recently described link of bassoon to human diseases associated with pathological protein aggregation. Presynaptic cytomatrix protein bassoon (Bsn) interacts with PSMB4, the ß7 subunit of 20S core proteasome, via three independent interaction interfaces. Bsn inhibits proteasomal proteolytic activity and degradation of different classes of proteasomal substrates presumably due to steric interference with the assembly of 20S core of proteasome. Upon Bsn deletion in neurons, presynaptic substrates of the proteasome are depleted, which can be reversed upon expression of PSMB4-interacting interfaces of Bsn. Taken together, bsn controls the degree of proteasome degradation within the presynaptic compartment and thus, contributes to the regulation of synaptic proteome.
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Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Animales , Humanos , Ratones , Ratones Noqueados , Agregado de Proteínas/genética , Agregación Patológica de Proteínas , Unión Proteica/genética , Mapas de Interacción de Proteínas/genética , Proteolisis , Sinapsis/genética , Sinapsis/metabolismo , Ubiquitina/genética , Ubiquitinación/genéticaRESUMEN
: The acid sphingomyelinase (ASM)/ceramide system exhibits a crucial role in the pathology of major depressive disorder (MDD). ASM hydrolyzes the abundant membrane lipid sphingomyelin to ceramide that regulates the clustering of membrane proteins via microdomain and lipid raft organization. Several commonly used antidepressants, such as fluoxetine, rely on the functional inhibition of ASM in terms of their antidepressive pharmacological effects. Transient receptor potential canonical 6 (TRPC6) ion channels are located in the plasma membrane of neurons and serve as receptors for hyperforin, a phytochemical constituent of the antidepressive herbal remedy St. John's wort. TRPC6 channels are involved in the regulation of neuronal plasticity, which likely contributes to their antidepressant effect. In this work, we investigated the impact of reduced ASM activity on the TRPC6 function in neurons. A lipidomic analysis of cortical brain tissue of ASM deficient mice revealed a decrease in ceramide/sphingomyelin molar ratio and an increase in sphingosine. In neurons with ASM deletion, hyperforin-mediated Ca2+-influx via TRPC6 was decreased. Consequently, downstream activation of nuclear phospho-cAMP response element-binding protein (pCREB) was changed, a transcriptional factor involved in neuronal plasticity. Our study underlines the importance of balanced ASM activity, as well as sphingolipidome composition for optimal TRPC6 function. A better understanding of the interaction of the ASM/ceramide and TRPC6 systems could help to draw conclusions about the pathology of MDD.
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Trastorno Depresivo Mayor , Neuronas , Esfingomielina Fosfodiesterasa , Canal Catiónico TRPC6 , Animales , Ratas , Trastorno Depresivo Mayor/sangre , Trastorno Depresivo Mayor/patología , Neuronas/metabolismo , Esfingomielina Fosfodiesterasa/efectos adversos , Canal Catiónico TRPC6/metabolismo , RatonesRESUMEN
Despite the central role of amyloid ß (Aß) peptide in the etiopathogenesis of Alzheimer's disease (AD), its physiological function in healthy brain is still debated. It is well established that elevated levels of Aß induce synaptic depression and dismantling, connected with neurotoxicity and neuronal loss. Growing evidence suggests a positive regulatory effect of Aß on synaptic function and cognition; however the exact cellular and molecular correlates are still unclear. In this work, we tested the effect of physiological concentrations of Aß species of endogenous origin on neurotransmitter release in rat cortical and hippocampal neurons grown in dissociated cultures. Modulation of production and degradation of the endogenous Aß species as well as applications of the synthetic rodent Aß40 and Aß42 affected efficacy of neurotransmitter release from individual presynapses. Low picomolar Aß40 and Aß42 increased, while Aß depletion or application of low micromolar concentration decreased synaptic vesicle recycling, showing a hormetic effect of Aß on neurotransmitter release. These Aß-mediated modulations required functional alpha7 acetylcholine receptors as well as extracellular and intracellular calcium, involved regulation of CDK5 and calcineurin signaling and increased recycling of synaptic vesicles. These data indicate that Aß regulates neurotransmitter release from presynapse and suggest that failure of the normal physiological function of Aß in the fine-tuning of SV cycling could disrupt synaptic function and homeostasis, which would, eventually, lead to cognitive decline and neurodegeneration.