RESUMEN
Kaempferol, a flavonoid present in many food products, has chemical and cellular antioxidant properties that are beneficial for protection against the oxidative stress caused by reactive oxygen and nitrogen species. Kaempferol administration to model experimental animals can provide extensive protection against brain damage of the striatum and proximal cortical areas induced by transient brain cerebral ischemic stroke and by 3-nitropropionic acid. This article is an updated review of the molecular and cellular mechanisms of protection by kaempferol administration against brain damage induced by these insults, integrated with an overview of the contributions of the work performed in our laboratories during the past years. Kaempferol administration at doses that prevent neurological dysfunctions inhibit the critical molecular events that underlie the initial and delayed brain damage induced by ischemic stroke and by 3-nitropropionic acid. It is highlighted that the protection afforded by kaempferol against the initial mitochondrial dysfunction can largely account for its protection against the reported delayed spreading of brain damage, which can develop from many hours to several days. This allows us to conclude that kaempferol administration can be beneficial not only in preventive treatments, but also in post-insult therapeutic treatments.
Asunto(s)
Lesiones Encefálicas , Accidente Cerebrovascular Isquémico , Fármacos Neuroprotectores , Nitrocompuestos , Propionatos , Accidente Cerebrovascular , Animales , Quempferoles/farmacología , Encéfalo , Estrés Oxidativo , Accidente Cerebrovascular/tratamiento farmacológico , Isquemia/tratamiento farmacológico , Lesiones Encefálicas/tratamiento farmacológico , Reperfusión , Accidente Cerebrovascular Isquémico/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéuticoRESUMEN
Highly neurotoxic A1-reactive astrocytes have been associated with several human neurodegenerative diseases. Complement protein C3 expression is strongly upregulated in A1 astrocytes, and this protein has been shown to be a specific biomarker of these astrocytes. Several cytokines released in neurodegenerative diseases have been shown to upregulate the production of amyloid ß protein precursor (APP) and neurotoxic amyloid ß (Aß) peptides in reactive astrocytes. Also, aberrant Ca2+ signals have been proposed as a hallmark of astrocyte functional remodeling in Alzheimer's disease mouse models. In this work, we induced the generation of A1-like reactive astrocytes after the co-treatment of U251 human astroglioma cells with a cocktail of the cytokines TNF-α, IL1-α and C1q. These A1-like astrocytes show increased production of APP and Aß peptides compared to untreated U251 cells. Additionally, A1-like astrocytes show a (75 ± 10)% decrease in the Ca2+ stored in the endoplasmic reticulum (ER), (85 ± 10)% attenuation of Ca2+ entry after complete Ca2+ depletion of the ER, and three-fold upregulation of plasma membrane calcium pump expression, with respect to non-treated Control astrocytes. These altered intracellular Ca2+ dynamics allow A1-like astrocytes to efficiently counterbalance the enhanced release of Ca2+ from the ER, preventing a rise in the resting cytosolic Ca2+ concentration.
Asunto(s)
Calcio , Enfermedades Neurodegenerativas , Ratones , Animales , Humanos , Calcio/metabolismo , Regulación hacia Arriba , Astrocitos/metabolismo , Péptidos beta-Amiloides/metabolismo , Señalización del Calcio , Precursor de Proteína beta-Amiloide/genética , Enfermedades Neurodegenerativas/metabolismo , Membrana Celular/metabolismoRESUMEN
Lipid membrane nanodomains or lipid rafts are 10-200 nm diameter size cholesterol- and sphingolipid-enriched domains of the plasma membrane, gathering many proteins with different roles. Isolation and characterization of plasma membrane proteins by differential centrifugation and proteomic studies have revealed a remarkable diversity of proteins in these domains. The limited size of the lipid membrane nanodomain challenges the simple possibility that all of them can coexist within the same lipid membrane domain. As caveolin-1, flotillin isoforms and gangliosides are currently used as neuronal lipid membrane nanodomain markers, we first analyzed the structural features of these components forming nanodomains at the plasma membrane since they are relevant for building supramolecular complexes constituted by these molecular signatures. Among the proteins associated with neuronal lipid membrane nanodomains, there are a large number of proteins that play major roles in calcium signaling, such as ionotropic and metabotropic receptors for neurotransmitters, calcium channels, and calcium pumps. This review highlights a large variation between the calcium signaling proteins that have been reported to be associated with isolated caveolin-1 and flotillin-lipid membrane nanodomains. Since these calcium signaling proteins are scattered in different locations of the neuronal plasma membrane, i.e., in presynapses, postsynapses, axonal or dendritic trees, or in the neuronal soma, our analysis suggests that different lipid membrane-domain subtypes should exist in neurons. Furthermore, we conclude that classification of lipid membrane domains by their content in calcium signaling proteins sheds light on the roles of these domains for neuronal activities that are dependent upon the intracellular calcium concentration. Some examples described in this review include the synaptic and metabolic activity, secretion of neurotransmitters and neuromodulators, neuronal excitability (long-term potentiation and long-term depression), axonal and dendritic growth but also neuronal cell survival and death.
Asunto(s)
Señalización del Calcio , Caveolina 1 , Caveolina 1/metabolismo , Calcio/metabolismo , Proteómica , Microdominios de Membrana/metabolismo , Neuronas/metabolismo , Gangliósidos , Neurotransmisores/metabolismoRESUMEN
Dysregulation in calcium signaling pathways plays a major role in the initiation of Alzheimer's disease (AD) pathogenesis. Accumulative experimental evidence obtained with cellular and animal models, as well as with AD brain samples, points out the high cytotoxicity of soluble small oligomeric forms of amyloid-ß peptides (Aß) in AD. In recent works, we have proposed that Aß-calmodulin (CaM) complexation may play a major role in neuronal Ca2+ signaling, mediated by CaM-binding proteins (CaMBPs). STIM1, a recognized CaMBP, plays a key role in store-operated calcium entry (SOCE), and it has been shown that the SOCE function is diminished in AD, resulting in the instability of dendric spines and enhanced amyloidogenesis. In this work, we show that 2 and 5 h of incubation with 2 µM Aß(1-42) oligomers of the immortalized mouse hippocampal cell line HT-22 leads to the internalization of 62 ± 11 nM and 135 ± 15 nM of Aß(1-42), respectively. Internalized Aß(1-42) oligomers colocalize with the endoplasmic reticulum (ER) and co-immunoprecipitated with STIM1, unveiling that this protein is a novel target of Aß. Fluorescence resonance energy transfer measurements between STIM1 tagged with a green fluorescent protein (GFP) and Aß(1-42)-HiLyte™-Fluor555 show that STIM1 can bind nanomolar concentrations of Aß(1-42) oligomers at a site located close to the CaM-binding site in STIM1. Internalized Aß(1-42) produced dysregulation of the SOCE in the HT-22 cells before a sustained alteration of cytosolic Ca2+ homeostasis can be detected, and is elicited by only 2 h of incubation with 2 µM Aß(1-42) oligomers. We conclude that Aß(1-42)-induced SOCE dysregulation in HT-22 cells is caused by the inhibitory modulation of STIM1, and the partial activation of ER Ca2+-leak channels.
Asunto(s)
Calcio , Calmodulina , Ratones , Animales , Calcio/metabolismo , Calmodulina/metabolismo , Canales de Calcio/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de la Membrana/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Señalización del Calcio , Proteína ORAI1/metabolismoRESUMEN
Amyloid ß1-42 (Aß(1-42)) oligomers have been linked to the pathogenesis of Alzheimer's disease (AD). Intracellular calcium (Ca2+) homeostasis dysregulation with subsequent alterations of neuronal excitability has been proposed to mediate Aß neurotoxicity in AD. The Ca2+ binding proteins calmodulin (CaM) and calbindin-D28k, whose expression levels are lowered in human AD brains, have relevant roles in neuronal survival and activity. In previous works, we have shown that CaM has a high affinity for Aß(1-42) oligomers and extensively binds internalized Aß(1-42) in neurons. In this work, we have designed a hydrophobic peptide of 10 amino acid residues: VFAFAMAFML (amidated-C-terminus amino acid) mimicking the interacting domain of CaM with Aß (1-42), using a combined strategy based on the experimental results obtained for Aß(1-42) binding to CaM and in silico docking analysis. The increase in the fluorescence intensity of Aß(1-42) HiLyteTM-Fluor555 has been used to monitor the kinetics of complex formation with CaM and with calbindin-D28k. The complexation between nanomolar concentrations of Aß(1-42) and calbindin-D28k is also a novel finding reported in this work. We found that the synthetic peptide VFAFAMAFML (amidated-C-terminus amino acid) is a potent inhibitor of the formation of Aß(1-42):CaM and of Aß(1-42):calbindin-D28k complexes.
Asunto(s)
Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/metabolismo , Calbindinas/metabolismo , Calmodulina/metabolismo , Enfermedad de Alzheimer/metabolismo , Aminoácidos/metabolismo , Calcio/metabolismo , Humanos , Neuronas/metabolismoRESUMEN
Intraneuronal amyloid ß (Aß) oligomer accumulation precedes the appearance of amyloid plaques or neurofibrillary tangles and is neurotoxic. In Alzheimer's disease (AD)-affected brains, intraneuronal Aß oligomers can derive from Aß peptide production within the neuron and, also, from vicinal neurons or reactive glial cells. Calcium homeostasis dysregulation and neuronal excitability alterations are widely accepted to play a key role in Aß neurotoxicity in AD. However, the identification of primary Aß-target proteins, in which functional impairment initiating cytosolic calcium homeostasis dysregulation and the critical point of no return are still pending issues. The micromolar concentration of calmodulin (CaM) in neurons and its high affinity for neurotoxic Aß peptides (dissociation constant ≈ 1 nM) highlight a novel function of CaM, i.e., the buffering of free Aß concentrations in the low nanomolar range. In turn, the concentration of Aß-CaM complexes within neurons will increase as a function of time after the induction of Aß production, and free Aß will rise sharply when accumulated Aß exceeds all available CaM. Thus, Aß-CaM complexation could also play a major role in neuronal calcium signaling mediated by calmodulin-binding proteins by Aß; a point that has been overlooked until now. In this review, we address the implications of Aß-CaM complexation in the formation of neurotoxic Aß oligomers, in the alteration of intracellular calcium homeostasis induced by Aß, and of dysregulation of the calcium-dependent neuronal activity and excitability induced by Aß.
Asunto(s)
Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Encéfalo/patología , Calmodulina/metabolismo , Degeneración Nerviosa/patología , Neuronas/metabolismo , Animales , HumanosRESUMEN
Lipid rafts are a primary target in studies of amyloid ß (Aß) cytotoxicity in neurons. Exogenous Aß peptides bind to lipid rafts, which in turn play a key role in Aß uptake, leading to the formation of neurotoxic intracellular Aß aggregates. On the other hand, dysregulation of intracellular calcium homeostasis in neurons has been observed in Alzheimer's disease (AD). In a previous work, we showed that Aß(1-42), the prevalent Aß peptide found in the amyloid plaques of AD patients, binds with high affinity to purified calmodulin (CaM), with a dissociation constant ≈1 nM. In this work, to experimentally assess the Aß(1-42) binding capacity to intracellular CaM, we used primary cultures of mature cerebellar granule neurons (CGN) as a neuronal model. Our results showed a large complexation of submicromolar concentrations of Aß(1-42) dimers by CaM in CGN, up to 120 ± 13 picomoles of Aß(1-42) /2.5 × 106 cells. Using fluorescence microscopy imaging, we showed an extensive co-localization of CaM and Aß(1-42) in lipid rafts in CGN stained with up to 100 picomoles of Aß(1-42)-HiLyteTM-Fluor555 monomers. Intracellular Aß(1-42) concentration in this range was achieved by 2 h incubation of CGN with 2 µM Aß(1-42), and this treatment lowered the resting cytosolic calcium of mature CGN in partially depolarizing 25 mM potassium medium. We conclude that the primary cause of the resting cytosolic calcium decrease is the inhibition of L-type calcium channels of CGN by Aß(1-42) dimers, whose activity is inhibited by CaM:Aß(1-42) complexes bound to lipid rafts.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Calcio/metabolismo , Calmodulina/metabolismo , Cerebelo/metabolismo , Citosol/metabolismo , Homeostasis , Microdominios de Membrana/metabolismo , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , Animales , Canales de Calcio Tipo L/metabolismo , Supervivencia Celular , Transferencia Resonante de Energía de Fluorescencia , Humanos , Unión Proteica , Ratas WistarRESUMEN
3-Nitropropionic acid (NPA) administration to rodents produces degeneration of the striatum, accompanied by neurological disturbances that mimic Huntington's disease (HD) motor neurological dysfunctions. It has been shown that inflammation mediates NPA-induced brain degeneration, and activated microglia secreting cytokines interleukin-1α (IL-1α) and tumor necrosis factor α (TNFα) can induce a specific type of reactive neurotoxic astrocytes, named A1, which have been detected in post-mortem brain samples of Huntington's, Alzheimer's, and Parkinson's diseases. In this work we used an experimental model based on the intraperitoneal (i.p.) administration of NPA to adult Wistar rats at doses that can elicit extensive brain degeneration, and brain samples were taken before and after extensive brain damage monitored using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Western blots and immunohistochemistry of brain slices show that i.p. NPA injections elicit significant increase in the expression levels of C3α subunit, a marker of generation of neurotoxic A1 astrocytes, and of cytokines IL-1α, TNFα, and C1q within the striatum, hippocampus, and cerebellum before the appearance of the HD-related neurological dysfunctions and neuronal death induced by NPA. Noteworthy, NPA administration primarily induces the generation of A1 astrocytes in the more recent phylogenetic area of the rat cerebellum. We conclude that the activation of complement C3 protein in the brain from Wistar rats is an early event in NPA-induced brain neurodegeneration.
Asunto(s)
Astrocitos/efectos de los fármacos , Encéfalo/efectos de los fármacos , Nitrocompuestos/toxicidad , Propionatos/toxicidad , Animales , Astrocitos/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Complemento C1q/metabolismo , Interleucina-1/metabolismo , Masculino , Ratas , Ratas Wistar , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Patients and family caregivers living with Congenital Disorders of Glycosylation (CDG) experience a heavy burden, which can impact their resiliency and quality of life. The study's purpose was to measure the resilience levels of patients and family caregivers living with CDG using the brief resilience coping scale. METHODS: We conducted an observational, cross-sectional study with 23 patients and 151 family caregivers living with CDG. Descriptive analyses were performed to characterize patients with CDG and family caregivers' samples. Additionally, we assessed correlations between resilience and specific variables (e.g., age, academic degree, time until diagnosis) and examined resilience differences between groups (e.g., sex, marital status, occupation, professional and social support). RESULTS: GNE myopathy was the most prevalent CDG among patients, while in family caregivers was PMM2-CDG. Both samples showed medium levels of resilience coping scores. Individuals with GNE myopathy had significantly higher scores of resilience compared to patients with other CDG. Resilience was positively correlated with educational degree in patients with CDG. Family caregivers had marginally significant higher scores of resilience coping if they received any kind of professional support or had contact with other families or people with the same or similar disease, compared with unsupported individuals. CONCLUSIONS: Despite the inherited difficulties of living with a life-threatening disease like CDG, patients and family caregivers showed medium resilient coping levels. Resilience scores changed significantly considering the CDG genotype, individual's academic degree and professional and social support. These exploratory findings can empower the healthcare system and private institutions by promoting the development of targeted interventions to enhance individuals` coping skills and improve the overall well-being and mental health of the CDG community.
Asunto(s)
Trastornos Congénitos de Glicosilación , Miopatías Distales , Resiliencia Psicológica , Humanos , Cuidadores , Estudios Transversales , Calidad de Vida , Habilidades de AfrontamientoRESUMEN
The Preyssler-type polyoxotungstate ({P5W30}) belongs to the family of polyanionic metal-oxides formed by group V and VI metal ions, such as V, Mo and W, commonly known as polyoxometalates (POMs). POMs have demonstrated inhibitory effect on a significant number of ATP-binding proteins in vitro. Purinergic P2 receptors, widely expressed in eukaryotic cells, contain extracellularly oriented ATP-binding sites and play many biological roles with health implications. In this work, we use the immortalized mouse hippocampal neuronal HT-22 cells in culture to study the effects of {P5W30} on the cytosolic Ca2+ concentration. Changes in cytosolic Ca2+ concentration were monitored using fluorescence microscopy of HT-22 cells loaded with the fluorescent Ca2+ indicator Fluo3. 31P-Nuclear magnetic resonance measurements of {P5W30} indicate its stability in the medium used for cytosolic Ca2+ measurements for over 30 min. The findings reveal that addition of {P5W30} to the extracellular medium induces a sustained increase of the cytosolic Ca2+ concentration within minutes. This Ca2+ increase is triggered by extracellular Ca2+ entry into the cells and is dose-dependent, with a half-of-effect concentration of 0.25 ± 0.05 µM {P5W30}. In addition, after the {P5W30}-induced cytosolic Ca2+ increase, the transient Ca2+ peak induced by extracellular ATP is reduced up to 100% with an apparent half-of-effect concentration of 0.15 ± 0.05 µM {P5W30}. Activation of metabotropic purinergic P2 receptors affords about 80% contribution to the increase of Fluo3 fluorescence elicited by {P5W30} in HT-22 cells, whereas ionotropic receptors contribute, at most, with 20%. These results suggest that {P5W30} could serve as a novel agonist of purinergic P2 receptors.
Asunto(s)
Calcio , Compuestos de Tungsteno , Animales , Ratones , Compuestos de Tungsteno/farmacología , Compuestos de Tungsteno/química , Calcio/metabolismo , Adenosina Trifosfato/metabolismo , Línea Celular , Hipocampo/metabolismo , Hipocampo/citología , Hipocampo/efectos de los fármacos , Agonistas del Receptor Purinérgico P2/farmacología , Citosol/metabolismoRESUMEN
Congenital disorders of glycosylation (CDG) are a complex and heterogeneous family of rare metabolic diseases. With a clinical history that dates back over 40 years, it was the recent multi-omics advances that mainly contributed to the fast-paced and encouraging developments in the field. However, much remains to be understood, with targeted therapies' discovery and approval being the most urgent unmet need. In this paper, we present the 2022 state of the art of CDG, including glycosylation pathways, phenotypes, genotypes, inheritance patterns, biomarkers, disease models, and treatments. In light of our current knowledge, it is not always clear whether a specific disease should be classified as a CDG. This can create ambiguity among professionals leading to confusion and misguidance, consequently affecting the patients and their families. This review aims to provide the CDG community with a comprehensive overview of the recent progress made in this field.
Asunto(s)
Trastornos Congénitos de Glicosilación , Humanos , Trastornos Congénitos de Glicosilación/genética , Trastornos Congénitos de Glicosilación/metabolismo , Glicosilación , Biomarcadores/metabolismo , Fenotipo , GenotipoRESUMEN
Kaempferol is a natural antioxidant present in vegetables and fruits used in human nutrition. In previous work, we showed that intraperitoneal (i.p.) kaempferol administration strongly protects against striatum neurodegeneration induced by i.p. injections of 3-nitropropionic acid (NPA), an animal model of Huntington's disease. Recently, we have shown that reactive A1 astrocytes generation is an early event in the neurodegeneration induced by NPA i.p. injections. In the present work, we have experimentally evaluated the hypothesis that kaempferol protects both against the activation of complement C3 protein and the generation of reactive A1 astrocytes in rat brain striatum and hippocampus. To this end, we have administered NPA and kaempferol i.p. injections to adult Wistar rats following the protocol described in previous work. Kaempferol administration prevents proteolytic activation of complement C3 protein and generation of reactive A1 astrocytes NPA-induced in the striatum and hippocampus. Also, it blocked the NPA-induced increase of NF-κB expression and enhanced secretion of cytokines IL-1α, TNFα, and C1q, which have been linked to the generation of reactive A1 astrocytes. In addition, kaempferol administration prevented the enhanced production of amyloid ß peptides in the striatum and hippocampus, a novel finding in NPA-induced brain degeneration found in this work.
Asunto(s)
Complemento C3 , Quempferoles , Péptidos beta-Amiloides/metabolismo , Animales , Astrocitos/metabolismo , Complemento C3/metabolismo , Cuerpo Estriado/metabolismo , Quempferoles/metabolismo , Quempferoles/farmacología , Nitrocompuestos/toxicidad , Propionatos/farmacología , Ratas , Ratas WistarRESUMEN
During the development of intranasal drug delivery systems for local/systemic effect or brain targeting, it is necessary to assess its cytotoxicity and drug transport through nasal epithelium. In order to avoid animal experiments or the use of excised tissues, in vitro cell models, such as RPMI 2650 cells, are being preferred during recent years. Nevertheless, the deposition of solid formulations into nasal cell layers with further transepithelial transport rate of drugs has been poorly studied or reported. Thus, the purpose of this work is to further investigate RPMI 2650 cell line as an effective alternative to animal tissues for solid drug-loaded formulations cytotoxicity and drug permeation studies in order to become an option as a tool for drug discovery. Furthermore, we wanted to determine the extent to which the administration of drugs in particulate forms would differ in relation to the permeability of the same compounds applied as solutions. RPMI 2650 cells were cultured in submersed or at air-liquid interface conditions and characterized regarding transepithelial electrical resistance (TEER) and production of mucus. Pure ketoprofen (used as model compound) and five formulations loaded with same drug, namely solid lipid particles (Gelucire 43/01™), structured lipid particles (Gelucire 43/01™:Glyceryl monooleate) and aerogel microparticles (Alginate, Alginate:Pectin, Alginate:Carrageenan), were evaluated with RPMI 2650 model in terms of cytotoxicity and permeability of drug (applied as solution, dispersion or powder+buffer). RPMI 2650 cells were capable to grow in monolayer and multilayer, showing the same permeability as excised human nasal mucosa for sodium fluorescein (paracellular marker), with analogous TEER values and production of mucus, as referred by other authors. None of the powders showed cytotoxicity when applied to RPMI 2650 cells. Regarding permeation of drug through cell layers, not only the form of application of powders but also their physical and chemical properties affected the final permeation of active pharmaceutical ingredient. Aerogel microparticles administered directly to the cell layer (powder+buffer) exhibited the highest permeation-enhancing effect compared to the pure drug, which can be attributed to the mucoadhesive properties of the materials composing the carriers, proving to be an attractive formulation for nasal drug delivery. According to these results, RPMI 2650 showed to be a promising alternative to ex vivo or in vivo nasal models for cytotoxicity and evaluation of drug permeability of nasal drug-loaded formulations.
Asunto(s)
Mucosa Nasal/metabolismo , Preparaciones Farmacéuticas/administración & dosificación , Administración Intranasal/métodos , Alginatos/química , Línea Celular , Química Farmacéutica/métodos , Sistemas de Liberación de Medicamentos/métodos , Fluoresceína/química , Ácido Glucurónico/química , Glicéridos/química , Ácidos Hexurónicos/química , Humanos , Modelos Biológicos , Permeabilidad , Preparaciones Farmacéuticas/química , Polvos/administración & dosificaciónRESUMEN
This study aims to synthesize, characterize and investigate the water solubility and cytotoxicity of branched anionic/cationic ß-cyclodextrins (bßCDs) obtained by reaction with epichlorohydrin and chloroacetic acid or choline chloride, respectively, by a single step polycondensation reaction. Obtained ionic bßCDs were investigated as an attempt to comparatively study anionic and cationic bßCDs. Water solubility of both ionic derivatives was similar (400 mg/mL) at neutral and basic pHs and remarkably higher than that of their neutral homologues. Additionally, a pH-dependent solubility of anionic bßCDs was observed. Cytotoxicity of ionic bßCDs was evaluated on Human colon carcinoma Caco-2 cells and high cell viability (>99%) was observed in the range of 0-100 mg/mL for anionic and cationic samples, in the same range of that of neutral and parent ß-CDs. Additionally, complexes formation capacity with triclosan, a poor water soluble antimicrobial agent, was confirmed by several techniques observing a complexation limit around 4 mg/mL for both systems and higher stability constant for anionic bßCDs than cationic derivatives.