RESUMEN

Hippocampal neurons exhibit activation of both the conventional transmembrane adenylyl cyclases (tmACs) and the non-canonical soluble adenylyl cyclase (sAC) as sources of cyclic AMP (cAMP). These two cAMP sources play crucial roles in mediating signaling pathways downstream of CRHR1 in neuronal and neuroendocrine contexts. In this study, we investigate the involvement of both cAMP sources in the molecular mechanisms triggered by CRHR2α. Here we provide evidence demonstrating that UCN1 and UCN3 exert a neuritogenic effect on HT22-CRHR2α cells, which is solely dependent on the cAMP pool generated by sAC and PKA activity but independent of ERK1/2 activation. Through the characterization of the effectors implicated in neurite elongation, we found that CREB phosphorylation and c-Fos induction rely on PKA activity and ERK1/2 phosphorylation, underscoring the critical role of signaling pathway regulation. These findings strengthen the concept that localized cAMP microdomains actively participate in the regulation of these signaling processes.

Asunto(s)

Adenilil Ciclasas , Proteínas Quinasas Dependientes de AMP Cíclico , AMP Cíclico , Receptores de Hormona Liberadora de Corticotropina , Transducción de Señal , AMP Cíclico/metabolismo , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Animales , Adenilil Ciclasas/metabolismo , Ratones , Fosforilación , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Urocortinas/metabolismo , Línea Celular , Neuritas/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Neuronas/metabolismo

RESUMEN

TRPM4 is a non-selective cation channel activated by intracellular Ca2+ but only permeable to monovalent cations, its activation regulates membrane potential and intracellular calcium. This channel participates in the migration and adhesion of non-excitable cells and forms an integral part of the focal adhesion complex. In neurons, TRPM4 expression starts before birth and its function at this stage is not clear, but it may function in processes such as neurite development. Here we investigate the role of TRPM4 in neuritogenesis. We found that neurons at DIV 0 express TRPM4, the inhibition of TRPM4 using 9-Ph reduces neurite number and slows the progression of neurite development, keeping neurons in stage 1. The genetic suppression of TRPM4 using an shRNA at later stages (DIV2) reduces neurite length. Conversely, at DIV 0, TRPM4 inhibition augments the Cch-induced Ca2 + i increase, altering the calcium homeostasis. Together, these results show that TRPM4 participates in progression of neurite development and suggest a critical role of the calcium modulation during this stage of neuronal development.

Asunto(s)

Calcio , Corteza Cerebral , Neuritas , Neurogénesis , Canales Catiónicos TRPM , Canales Catiónicos TRPM/metabolismo , Canales Catiónicos TRPM/antagonistas & inhibidores , Animales , Neuritas/metabolismo , Neuritas/efectos de los fármacos , Calcio/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Neuronas/metabolismo

RESUMEN

Neurodegenerative disorders, including traumatic injuries to the central nervous system (CNS) and neurodegenerative diseases, are characterized by early axonal damage, which does not regenerate in the adult mammalian CNS, leading to permanent neurological deficits. One of the primary causes of the loss of regenerative ability is thought to be a developmental decline in neurons' intrinsic capability for axon growth. Different molecules are involved in the developmental loss of the ability for axon regeneration, including many transcription factors. However, the function of microRNAs (miRNAs), which are also modulators of gene expression, in axon re-growth is still unclear. Among the various miRNAs recently identified with roles in the CNS, miR-17, which is highly expressed during early development, emerges as a promising target to promote axon regeneration. Here, we used adeno-associated viral (AAV) vectors to overexpress miR-17 (AAV.miR-17) in primary cortical neurons and evaluate its effects on neurite and axon regeneration in vitro. Although AAV.miR-17 had no significant effect on neurite outgrowth and arborization, it significantly enhances neurite regeneration after scratch lesion and axon regeneration after axotomy of neurons cultured in microfluidic chambers. Target prediction and functional annotation analyses suggest that miR-17 regulates gene expression associated with autophagy and cell metabolism. Our findings suggest that miR-17 promotes regenerative response and thus could mitigate neurodegenerative effects.

Asunto(s)

Axones , Dependovirus , MicroARNs , Regeneración Nerviosa , Neuritas , MicroARNs/genética , MicroARNs/metabolismo , Animales , Axones/metabolismo , Axones/fisiología , Regeneración Nerviosa/genética , Neuritas/metabolismo , Dependovirus/genética , Células Cultivadas , Vectores Genéticos/genética , Ratones , Neuronas/metabolismo

RESUMEN

Fluorescence recovery after photobleaching (FRAP) is a laser method of light microscopy to evaluate the rapid movement of fluorescent molecules. To have a more reliable approach to analyze data from FRAP, we designed Fraping, a free access R library to data analysis obtained from FRAP. Unlike other programs, Fraping has a new form of analyzing curves of FRAP using statistical analysis based on the average curve difference. To evaluate our library, we analyzed the differences of actin polymerization in real time between dendrites and secondary neurites of cultured neuron transfected with LifeAct to track F-actin changes of neurites. We found that Fraping provided greater sensitivity than the conventional model using mobile fraction analysis. Likewise, this approach allowed us to normalize the fluorescence to the size area of interest and adjust data curves choosing the best parametric model. In addition, this library was supplemented with data simulation to have a more significant enrichment for the analysis behavior. We concluded that Fraping is a method that reduces bias when analyzing two data groups as compared with the conventional methods. This method also allows the users to choose a more suitable analysis approach according to their requirements. RESEARCH HIGHLIGHTS: Fraping is a new programming tool to analyze FRAP data to normalize fluorescence recovery curves. The conventional method uses one-point analysis, and the new one compares all the points to define the similarity of the fluorescence recovery.

Asunto(s)

Actinas , Recuperación de Fluorescencia tras Fotoblanqueo , Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Actinas/análisis , Animales , Polimerizacion , Neuritas , Neuronas/metabolismo , Neuronas/química , Células Cultivadas , Dendritas/química , Dendritas/metabolismo

RESUMEN

Reactive oxygen species (ROS) function as signaling molecules in several physiologic and pathologic processes. In central nervous system, ROS are critical for differentiation, migration, polarization, and neurite growth. These actions are mediated by reversible oxidation of target proteins. On the other hand, PI3K/Akt signaling pathway is susceptible to be modulated by ROS and it has been implicated in neurite growth. In this study, we evaluated the participation of ROS in the neurite growth of cultured rat cerebellar granule neurons (CGN), as well as the possible regulation of the PI3K/Akt pathway by ROS during neurite outgrowth. For this purpose, CGN were treated with cellular or mitochondrial antioxidants, or an NOX inhibitor and neurite growth was evaluated. Moreover, to assess the participation Akt in this process, the p-Akt levels were measured in CGN treated with antioxidants or a NOX inhibitor. The effect of antioxidants on the neurite growth in the presence of a PI3K inhibitor was also measured. We found that cellular antioxidants and the NOX inhibitor decreased the neurite growth, but not the mitochondrial antioxidant. Interestingly, the antioxidants increased the p-Akt levels; however, the effect of antioxidants on neurite growth was no dependent on the Akt activity since the inhibitor of PI3K did not modify the antioxidant action on neurite growth. Our results show that the PI3K/Akt pathway participates in neurite growth and that ROS produced by NOX could function as signals in this process; however, this action is not mediated by a redox regulation of Akt activity.

Asunto(s)

Antioxidantes , Proteínas Proto-Oncogénicas c-akt , Ratas , Animales , Antioxidantes/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Neuritas , NADPH Oxidasas/metabolismo , NADPH Oxidasas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo

RESUMEN

Artepillin C is the most studied compound in Brazilian Green Propolis and, along with its acetylated derivative, displays neurotrophic activity on PC12 cells. Specific inhibitors of the trkA receptor (K252a), PI3K/Akt (LY294002), and MAPK/ERK (U0126) signaling pathways were used to investigate the neurotrophic mechanism. The expression of proteins involved in axonal and synaptic plasticity (GAP-43 and Synapsin I) was assessed by western blotting. Additionally, physicochemical properties, pharmacokinetics, and drug-likeness were evaluated by the SwissADME web tool. Both compounds induced neurite outgrowth by activating the NGF-signaling pathways but through different neuronal proteins. Furthermore, in silico analyses showed interesting physicochemical and pharmacokinetic properties of these compounds. Therefore, these compounds could play an important role in axonal and synaptic plasticity and should be further investigated.

Asunto(s)

Própolis , Ratas , Animales , Células PC12 , Própolis/farmacología , Própolis/metabolismo , Neuritas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Brasil , Transducción de Señal , Proyección Neuronal

RESUMEN

The Unfolded protein response (UPR), triggered by stress in the endoplasmic reticulum (ER), is a key driver of neurodegenerative diseases. GM2 gangliosidosis, which includes Tay-Sachs and Sandhoff disease, is caused by an accumulation of GM2, mainly in the brain, that leads to progressive neurodegeneration. Previously, we demonstrated in a cellular model of GM2 gangliosidosis that PERK, a UPR sensor, contributes to neuronal death. There is currently no approved treatment for these disorders. Chemical chaperones, such as ursodeoxycholic acid (UDCA), have been found to alleviate ER stress in cell and animal models. UDCA's ability to move across the blood-brain barrier makes it interesting as a therapeutic tool. Here, we found that UDCA significantly diminished the neurite atrophy induced by GM2 accumulation in primary neuron cultures. It also decreased the up-regulation of pro-apoptotic CHOP, a downstream PERK-signaling component. To explore its potential mechanisms of action, in vitro kinase assays and crosslinking experiments were performed with different variants of recombinant protein PERK, either in solution or in reconstituted liposomes. The results suggest a direct interaction between UDCA and the cytosolic domain of PERK, which promotes kinase phosphorylation and dimerization.

Asunto(s)

Gangliosidosis GM2 , Enfermedad de Sandhoff , Animales , Atrofia , Gangliosidosis GM2/metabolismo , Neuritas/metabolismo , Enfermedad de Sandhoff/terapia , Ácido Ursodesoxicólico/farmacología , eIF-2 Quinasa/metabolismo

RESUMEN

Down syndrome (DS) is characterized by the trisomy of chromosome 21 and by cognitive deficits that have been related to neuronal morphological alterations in humans, as well as in animal models. The gene encoding for amyloid precursor protein (APP) is present in autosome 21, and its overexpression in DS has been linked to neuronal dysfunction, cognitive deficit, and Alzheimer's disease-like dementia. In particular, the neuronal ability to extend processes and branching is affected. Current evidence suggests that APP could also regulate neurite growth through its role in the actin cytoskeleton, in part by influencing p21-activated kinase (PAK) activity. The latter effect is carried out by an increased abundance of the caspase cleavage-released carboxy-terminal C31 fragment. In this work, using a neuronal cell line named CTb, which derived from the cerebral cortex of a trisomy 16 mouse, an animal model of human DS, we observed an overexpression of APP, elevated caspase activity, augmented cleavage of the C-terminal fragment of APP, and increased PAK1 phosphorylation. Morphometric analyses showed that inhibition of PAK1 activity with FRAX486 increased the average length of the neurites, the number of crossings per Sholl ring, the formation of new processes, and stimulated the loss of processes. Considering our results, we propose that PAK hyperphosphorylation impairs neurite outgrowth and remodeling in the cellular model of DS, and therefore we suggest that PAK1 may be a potential pharmacological target.

Asunto(s)

Síndrome de Down , Ratones , Humanos , Animales , Síndrome de Down/tratamiento farmacológico , Síndrome de Down/genética , Trisomía , Neuronas/metabolismo , Neuritas/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Proyección Neuronal , Caspasas/metabolismo

RESUMEN

Increased collagen-derived advanced glycation end-products (AGEs) are consistently related to painful diseases, including osteoarthritis, diabetic neuropathy, and neurodegenerative disorders. We have recently developed a model combining a two-dimensional glycated extracellular matrix (ECM-GC) and primary dorsal root ganglion (DRG) that mimicked a pro-nociceptive microenvironment. However, culturing primary cells is still a challenge for large-scale screening studies. Here, we characterized a new model using ECM-GC as a stimulus for human sensory-like neurons differentiated from SH-SY5Y cell lines to screen for analgesic compounds. First, we confirmed that the differentiation process induces the expression of neuron markers (MAP2, RBFOX3 (NeuN), and TUBB3 (ß-III tubulin), as well as sensory neuron markers critical for pain sensation (TRPV1, SCN9A (Nav1.7), SCN10A (Nav1.8), and SCN11A (Nav1.9). Next, we showed that ECM-GC increased c-Fos expression in human sensory-like neurons, which is suggestive of neuronal activation. In addition, ECM-GC upregulated the expression of critical genes involved in pain, including SCN9A and TACR1. Of interest, ECM-GC induced substance P release, a neuropeptide widely involved in neuroinflammation and pain. Finally, morphine, the prototype opiate, decreased ECM-GC-induced substance P release. Together, our results suggest that we established a functional model that can be useful as a platform for screening candidates for the management of painful conditions.

Asunto(s)

Analgésicos/análisis , Analgésicos/farmacología , Colágeno/farmacología , Evaluación Preclínica de Medicamentos , Modelos Biológicos , Células Receptoras Sensoriales/citología , Animales , Antígenos de Neoplasias/metabolismo , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Matriz Extracelular/metabolismo , Galectina 3/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Glicosilación/efectos de los fármacos , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7/genética , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Receptores de Neuroquinina-1/genética , Receptores de Neuroquinina-1/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Sustancia P/metabolismo , betaendorfina/metabolismo

RESUMEN

Three dimensional (3D) in vitro neuronal cultures can better reproduce physiologically relevant phenotypes compared to 2D-cultures, because in vivo neurons reside in a 3D microenvironment. Interest in neuronal 3D cultures is emerging, with special attention to the mechanical forces that regulate axon elongation and sprouting in three dimensions. Type I collagen (Col-I) is a native substrate since it is present in the extracellular matrix and hence emulates an in vivo environment to study axon growth. The impact of its mechanical properties needs to be further investigated. Here, we generated Col-I 3D matrices of different mechanical stiffness and evaluated axon growth in three dimensions. Superior cervical ganglion (SCG) explants from neonatal rats were cultured in soft and stiff Col-I 3D matrices and neurite outgrowth was assessed by measuring: maximum neuritic extent; neuritic halo area and fasciculation. Axonal cytoskeletal proteins were examined. Axon elongation in stiff Col-I 3D matrices was reduced (31%) following 24 h in culture compared to soft matrices. In stiff matrices, neurites fasciculated and formed less dense halos. Consistently, almost no F-actin rich growth cones were recognized, and F-actin staining was strongly reduced in the axonal compartment. This study shows that stiffness negatively affects 3D neurite outgrowth and adds insights on the cytoskeletal responses upon mechanic interactions of axons with a 3D environment. Our data will serve to facilitate the development of model systems that are mechanically well-behaved but still mimic key physiologic properties observed in vivo.

Asunto(s)

Colágeno Tipo I , Conos de Crecimiento , Actinas , Animales , Axones/fisiología , Células Cultivadas , Matriz Extracelular , Neuritas , Ratas

RESUMEN

Cocaine use disorder (CUD) is characterized by a compulsive search for cocaine. Several studies have shown that cocaine users exhibit cognitive deficits, including lack of inhibition and decision-making as well as brain volume and diffusion-based white-matter alterations in a wide variety of brain regions. However, the non-specificity of standard volumetric and diffusion-tensor methods to detect structural micropathology may lead to wrong conclusions. To better understand microstructural pathology in CUD, we analyzed 60 CUD participants (3 female) and 43 non-CUD controls (HC; 2 female) retrospectively from our cross-sectional Mexican SUD neuroimaging dataset (SUDMEX-CONN), using multi-shell diffusion-weighted imaging and the neurite orientation dispersion and density imaging (NODDI) analysis, which aims to more accurately model microstructural pathology. We used Viso values of NODDI that employ a three-compartment model in white (WM) and gray-matter (GM). These values were also correlated with clinical measures, including psychiatric severity status, impulsive behavior and pattern of cocaine and tobacco use in the CUD group. We found higher whole-brain microstructural pathology in WM and GM in CUD patients than controls. ROI analysis revealed higher Viso-NODDI values in superior longitudinal fasciculus, cingulum, hippocampus cingulum, forceps minor and Uncinate fasciculus, as well as in frontal and parieto-temporal GM structures. We also found correlations between significant ROI and impulsivity, onset age of cocaine use and weekly dosage with Viso-NODDI. However, we did not find correlations with psychopathology measures. Overall, although their clinical relevance remains questionable, microstructural pathology seems to be present in CUD both in gray and white matter.

Asunto(s)

Trastornos Relacionados con Cocaína/patología , Cocaína/farmacología , Sustancia Gris/patología , Hipocampo/patología , Neuritas/patología , Sustancia Blanca/patología , Adulto , Encéfalo/patología , Estudios Transversales , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Conducta Impulsiva , Imagen por Resonancia Magnética , Masculino , México , Estudios Retrospectivos

RESUMEN

Leprosy, also known as Hansen's disease, continues to have a substantial impact on infectious diseases throughout the world. Leprosy is a chronic granulomatous infection caused by Mycobacterium leprae and shows a wide clinical and immunopathological spectrum related to the immune response of the host. This disease affects the skin and other internal organs with a predilection to infect Schwann cells, which play an active role during axonal degeneration, affecting peripheral nerves and promoting neurological damage. This chronic inflammation influences immune function, leading to neuroimmune disorders. Leprosy is also associated with neuroimmune reactions, including type 1 (reverse) and type 2 (erythema nodosum leprosum) reactions, which are immune-mediated inflammatory complications that can occur during the disease and appear to worsen dramatically; these complications are the main concerns of patients. The reactions may induce neuritis and neuropathic pain that progressively worsen with irreversible deformity and disabilities responsible for the immunopathological damage and glial/neuronal death. However, the neuronal damage is not always associated with the reactional episode. Also, the efficacy in the treatment of reactions remains low because of the nonexistence of a specific treatment and missing informations about the immunopathogenesis of the reactional episode. There is increasing evidence that peripheral neuron dysfunction strongly depends on the activity of neurotrophins. The most important neurotrophin in leprosy is nerve growth factor (NGF), which is decreased in the course of leprosy, as well as the presence of autoantibodies against NGF in all clinical forms of leprosy and neuroimmune reactions. The levels of autoantibodies against NGF are decreased by the immunomodulatory activity of cyclosporin A, which mainly controls pain and improves motor function and sensitivity. Therefore, the suppression of anti-NGF and the regulation of NGF levels can be attractive targets for immunomodulatory treatment and for controlling the neuroimmune reactions of leprosy, although further studies are needed to clarify this point.

Asunto(s)

Ciclosporina , Lepra , Humanos , Lepra/complicaciones , Lepra/tratamiento farmacológico , Mycobacterium leprae , Neuritas/patología , Células de Schwann/patología

RESUMEN

Neuroblastoma is the most common extracranial solid tumour in childhood, originated from cells of the neural crest during the development of the Sympathetic Nervous System. Retinoids are vitamin-A derived differentiating agents utilised to avoid disease resurgence in high-risk neuroblastoma treatment. Several studies indicate that hypoxia-a common feature of the tumoural environment-is a key player in cell differentiation and proliferation. Hypoxia leads to the accumulation of the hypoxia-inducible factor-1α (HIF-1α). This work aims to investigate the effects of the selective inhibition of HIF-1α on the differentiation induced by retinoic acid in human neuroblastoma cells from the SH-SY5Y lineage to clarify its role in cell differentiation. Our results indicate that HIF-1α inhibition impairs RA-induced differentiation by reducing neuron-like phenotype and diminished immunolabeling and expression of differentiation markers. HIF1A is involved in Retinoic Acid (RA) induced differentiation in SH-SY5Y neuroblastoma cells. siRNA HIF1A gene silencing leads to a weaker response to RA, demonstrated by changes in the neuro-like phenotype and diminished expression of differentiation markers.

Asunto(s)

Neuroblastoma , Tretinoina , Diferenciación Celular , Línea Celular Tumoral , Expresión Génica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Neuritas , Neuroblastoma/metabolismo , Tretinoina/farmacología

RESUMEN

BACKGROUND: Wnt signaling plays key roles in cellular and physiological processes, including cell proliferation, differentiation and migration during development and tissue homeostasis in adults. This pathway can be defined as Wnt/ß-catenin-dependent or ß-catenin-independent or "non-canonical", both signaling are involved in neurite and synapse development/maintenance. Porcupine (PORCN), an acylase that o-acylates Wnt ligands, a major modification in secretion and interaction with its receptors. We use Wnt-C59, a specific PORCN inhibitor, to block the secretion of endogenous Wnts in embryonic hippocampal neurons (DIV 4). Under these conditions, the activity of exogenous Wnt ligands on the complexity of the dendritic tree and axonal polarity were evaluated METHODS: Cultured primary embryonic hippocampal neurons obtained from Sprague-Dawley rat fetuses (E18), were cultured until day in vitro (DIV) 4 (according to Banker´s protocol) and treated with Wnt-C59 for 24 h, Wnt ligands were added to the cultures on DIV 3 for 24 h. Dendritic arbors and neurites were analysis by fluorescence microscopy. Transfection with Lipofectamine 2000 on DIV 2 of plasmid expressing eGFP and KIF5-Cherry was carried out to evaluate neuronal polarity. Immunostaining was performed with MAP1B and Tau protein. Immunoblot analysis was carried out with Wnt3a, ß-catenin and GSK-3ß (p-Ser9). Quantitative analysis of dendrite morphology was carried out with ImageJ (NIH) software with Neuron J Plugin. RESULTS: We report, here, that Wnt-C59 treatment changed the morphology of the dendritic arbors and neurites of embryonic hippocampal neurons, with decreases ß-catenin and Wnt3a and an apparent increase in GSK-3ß (p-Ser9) levels. No effect was observed on axonal polarity. In sister cultures, addition of exogenous Wnt3a, 5a and 7a ligands rescued the changes in neuronal morphology. Wnt3a restored the length of neurites to near that of the control, but Wnt7a increased the neurite length beyond that of the control. Wnt5a also restored the length of neurites relative to Wnt concentrations. CONCLUSIONS: Results indicated that Wnt ligands, added exogenously, restored dendritic arbor complexity in embryonic hippocampal neurons, previously treated with a high affinity specific Porcupine inhibitor. We proposed that PORCN is an emerging molecular target of interest in the search for preclinical options to study and treat Wnt-related diseases. Video Abstract.

Asunto(s)

Glucógeno Sintasa Quinasa 3 beta/genética , Neuronas/metabolismo , Proteína Wnt3A/genética , beta Catenina/genética , Animales , Axones/metabolismo , Bencenoacetamidas/farmacología , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Polaridad Celular/genética , Proliferación Celular/efectos de los fármacos , Feto , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/crecimiento & desarrollo , Ligandos , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Neuronas/efectos de los fármacos , Proteínas Proto-Oncogénicas/genética , Piridinas/farmacología , Ratas , Proteínas Wnt/genética , Proteína Wnt-5a/genética

RESUMEN

Neurodegenerative diseases are one of the major causes of death worldwide, characterized by neurite atrophy, neuron apoptosis, and synapse loss. No effective treatment has been indicated for such diseases so far, and the search for new drugs is being increased in the last years. Animal venoms' secretion/venom can be an alternative for the discovery of new molecules, which could be the prototype for a new treatment. Here, we present the biochemical characterization and activity of the extract from the box jellyfish Chiropsalmus quadrumanus (Cq) on neurites. The Cq methanolic extract was obtained and incubated to human SH-SY5Y neurons, and neurite parameters were evaluated. The extract was tested in other cell types to check its cytotoxicity and was submitted to biochemical analysis by mass spectrometry in order to check its composition. We could verify that the Cq extract increased neurite outgrowth length and branching junctions, amplifying the contact between SH-SY5Y neurons, without affecting cell body and viability. The extract action was selective for neurons, as it did not cause any effects on other cell types, such as tumor line, nontumor line, and red blood cells. Moreover, mass spectrometry analysis revealed that there are no proteins but several low molecular mass compounds and peptides. Three peptides, characterized as cryptides, and 14 low molecular mass compounds were found to be related to cytoskeleton reorganization, cell membrane expansion, and antioxidant/neuroprotective activity, which act together to increase neuritogenesis. After this evaluation, we conclude that the Cq extract is a promising tool for neuronal connection recovery, an essential condition for the treatment of neurodegenerative diseases.

Asunto(s)

Mezclas Complejas/farmacología , Cubomedusas/química , Neuritas/metabolismo , Fármacos Neuroprotectores/farmacología , Animales , Línea Celular Tumoral , Mezclas Complejas/química , Humanos , Fármacos Neuroprotectores/química

RESUMEN

Two billion people are chronically infected with Toxoplasma gondii worldwide with unknown consequences. Important neurological diseases have been associated to the brain infection, making essential to understand the neurophysiological changes associated with the neuronal encystment. T. gondii may subvert neuronal functions modifying neurotransmitter concentration in chronically infected mice but the molecular mechanisms involved are still unclear. Parasites were observed inside neuronal cells in cultures from 24-192 hs. The rate of infection increased with time. Neurite density decreased affecting network functionality. Neuronal survival was affected and we detected the presence of cysts inside neuronal bodies and dilated portions of neurites in association with a relative increase of TH-positive neuritic area without noticeable changes in DA immunofluorescence pattern. These results advance our knowledge of the interaction between T. gondii and the neuronal network of the host.

Asunto(s)

Neuritas/metabolismo , Neuritas/parasitología , Toxoplasma/patogenicidad , Tirosina 3-Monooxigenasa/metabolismo , Animales , Encéfalo/metabolismo , Ratones , Tubulina (Proteína)/metabolismo

RESUMEN

Neuropathic and inflammatory pain results from cellular and molecular changes in dorsal root ganglion (DRG) neurons. The type-2 receptor for Angiotensin-II (AT2R) has been involved in this type of pain. However, the underlying mechanisms are poorly understood, including the role of the type-1 receptor for Angiotensin-II (AT1R). Here, we used a combination of immunohistochemistry and immunocytochemistry, RT-PCR and in vitro and in vivo pharmacological manipulation to examine how cutaneous inflammation affected the expression of AT1R and AT2R in subpopulations of rat DRG neurons and studied their impact on inflammation-induced neuritogenesis. We demonstrated that AT2R-neurons express C- or A-neuron markers, primarily IB4, trkA, and substance-P. AT1R expression was highest in small neurons and co-localized significantly with AT2R. In vitro, an inflammatory soup caused significant elevation of AT2R mRNA, whereas AT1R mRNA levels remained unchanged. In vivo, we found a unique pattern of change in the expression of AT1R and AT2R after cutaneous inflammation. AT2R increased in small neurons at 1 day and in medium size neurons at 4 days. Interestingly, cutaneous inflammation increased AT1R levels only in large neurons at 4 days. We found that in vitro and in vivo AT1R and AT2R acted co-operatively to regulate DRG neurite outgrowth. In vivo, AT2R inhibition impacted more on non-peptidergic C-neurons neuritogenesis, whereas AT1R blockade affected primarily peptidergic nerve terminals. Thus, cutaneous-induced inflammation regulated AT1R and AT2R expression and function in different DRG neuronal subpopulations at different times. These findings must be considered when targeting AT1R and AT2R to treat chronic inflammatory pain. Cover Image for this issue: doi: 10.1111/jnc.14737.

Asunto(s)

Dermatitis/fisiopatología , Receptor de Angiotensina Tipo 1/fisiología , Receptor de Angiotensina Tipo 2/fisiología , Células Receptoras Sensoriales/fisiología , Animales , Células Cultivadas , Dermatitis/etiología , Femenino , Adyuvante de Freund/administración & dosificación , Ganglios Espinales/citología , Neuritas/fisiología , Dolor/fisiopatología , Ratas , Ratas Wistar , Receptor de Angiotensina Tipo 1/análisis , Receptor de Angiotensina Tipo 2/análisis , Células Receptoras Sensoriales/química , Piel/inervación

RESUMEN

Parkinson's disease (PD) is the second most common neurodegenerative condition, characterized by motor impairment due to the progressive degeneration of dopaminergic neurons in the substantia nigra and depletion of dopamine release in the striatum. Accumulating evidence suggest that degeneration of axons is an early event in the disease, involving destruction programs that are independent of the survival of the cell soma. Necroptosis, a programmed cell death process, is emerging as a mediator of neuronal loss in models of neurodegenerative diseases. Here, we demonstrate activation of necroptosis in postmortem brain tissue from PD patients and in a toxin-based mouse model of the disease. Inhibition of key components of the necroptotic pathway resulted in a significant delay of 6-hydroxydopamine-dependent axonal degeneration of dopaminergic and cortical neurons in vitro. Genetic ablation of necroptosis mediators MLKL and RIPK3, as well as pharmacological inhibition of RIPK1 in preclinical models of PD, decreased dopaminergic neuron degeneration, improving motor performance. Together, these findings suggest that axonal degeneration in PD is mediated by the necroptosis machinery, a process here referred to as necroaxoptosis, a druggable pathway to target dopaminergic neuronal loss.

Asunto(s)

Axones/patología , Necroptosis , Degeneración Nerviosa/patología , Enfermedad de Parkinson/patología , Animales , Biomarcadores/metabolismo , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora , Neuritas/patología , Oxidopamina , Enfermedad de Parkinson/fisiopatología , Fosforilación , Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Sustancia Negra/metabolismo , Sustancia Negra/patología

RESUMEN

During the progression of the neurodegenerative process, mitochondria participates in several intercellular signaling pathways. Voltage-dependent anion-selective channel 1 (VDAC1) is a mitochondrial porin involved in the cellular metabolism and apoptosis intrinsic pathway in many neuropathological processes. In spinal cord injury (SCI), after the primary cell death, a secondary response that comprises the release of pro-inflammatory molecules triggers apoptosis, inflammation, and demyelination, often leading to the loss of motor functions. Here, we investigated the functional role of VDAC1 in the neurodegeneration triggered by SCI. We first determined that in vitro targeted ablation of VDAC1 by specific morpholino antisense nucleotides (MOs) clearly promotes neurite retraction, whereas a pharmacological blocker of VDAC1 oligomerization (4, 4'-diisothiocyanatostilbene-2, 2'-disulfonic acid, DIDS), does not cause this effect. We next determined that, after SCI, VDAC1 undergoes conformational changes, including oligomerization and N-terminal exposition, which are important steps in the triggering of apoptotic signaling. Considering this, we investigated the effects of DIDS in vivo application after SCI. Interestingly, blockade of VDAC1 oligomerization decreases the number of apoptotic cells without interfering in the neuroinflammatory response. DIDS attenuates the massive oligodendrocyte cell death, subserving undisputable motor function recovery. Taken together, our results suggest that the prevention of VDAC1 oligomerization might be beneficial for the clinical treatment of SCI.

Asunto(s)

Neuritas/metabolismo , Traumatismos de la Médula Espinal/fisiopatología , Médula Espinal/metabolismo , Canal Aniónico 1 Dependiente del Voltaje/fisiología , Animales , Western Blotting , Células Cultivadas , Femenino , Técnica del Anticuerpo Fluorescente , Masculino , Microscopía Fluorescente , Ratas , Ratas Wistar , Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/metabolismo , Canal Aniónico 1 Dependiente del Voltaje/metabolismo

RESUMEN

Cholinergic transmission is critical to high-order brain functions such as memory, learning, and attention. Alzheimer's disease (AD) is characterized by cognitive decline associated with a specific degeneration of cholinergic neurons. No effective treatment to prevent or reverse the symptoms is known. Part of this might be due to the lack of in vitro models that effectively mimic the relevant features of AD. Here, we describe the characterization of an AD in vitro model using the SH-SY5Y cell line. Exponentially growing cells were maintained in DMEM/F12 medium and differentiation was triggered by the combination of retinoic acid (RA) and BDNF. Both acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) enzymatic activities and immunocontent were determined. For mimicking tau and amyloid-ß pathology, RA + BDNF-differentiated cells were challenged with okadaic acid (OA) or soluble oligomers of amyloid-ß (AßOs) and neurotoxicity was evaluated. RA + BDNF-induced differentiation resulted in remarkable neuronal morphology alterations characterized by increased neurite density. Enhanced expression and enzymatic activities of cholinergic markers were observed compared to RA-differentiation only. Combination of sublethal doses of AßOs and OA resulted in decreased neurite densities, an in vitro marker of synaptopathy. Challenging RA + BDNF-differentiated SH-SY5Y cells with the combination of sublethal doses of OA and AßO, without causing considerable decrease of cell viability, provides an in vitro model which mimics the early-stage pathophysiology of cholinergic neurons affected by AD.

Asunto(s)

Enfermedad de Alzheimer/patología , Diferenciación Celular , Neuronas Colinérgicas/patología , Modelos Biológicos , Neuroblastoma/patología , Enfermedad de Alzheimer/genética , Biomarcadores/metabolismo , Factor Neurotrófico Derivado del Encéfalo/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Neuroblastoma/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Tretinoina/farmacología