Short Daily Exposure to Environmental Enrichment, Fluoxetine, or Their Combination Reverses Deterioration of the Coat and Anhedonia Behaviors with Differential Effects on Hippocampal Neurogenesis in Chronically Stressed Mice.

Depression is a neuropsychiatric disorder with a high impact on the worldwide population. To overcome depression, antidepressant drugs are the first line of treatment. However, pre-clinical studies have pointed out that antidepressants are not entirely efficacious and that the quality of the living environment after stress cessation may play a relevant role in increasing their efficacy. As it is unknown whether a short daily exposure to environmental enrichment during chronic stress and antidepressant treatment will be more effective than just the pharmacological treatment, this study analyzed the effects of fluoxetine, environmental enrichment, and their combination on depressive-associated behavior. Additionally, we investigated hippocampal neurogenesis in mice exposed to chronic mild stress. Our results indicate that fluoxetine reversed anhedonia. Besides, fluoxetine reversed the decrement of some events of the hippocampal neurogenic process caused by chronic mild stress. Conversely, short daily exposure to environmental enrichment changed the deterioration of the coat and anhedonia. Although, this environmental intervention did not produce significant changes in the neurogenic process affected by chronic mild stress, fluoxetine plus environmental enrichment showed similar effects to those caused by environmental enrichment to reverse depressive-like behaviors. Like fluoxetine, the combination reversed the declining number of Ki67, doublecortin, calretinin cells and mature newborn neurons. Finally, this study suggests that short daily exposure to environmental enrichment improves the effects of fluoxetine to reverse the deterioration of the coat and anhedonia in chronically stressed mice. In addition, the combination of fluoxetine with environmental enrichment produces more significant effects than those caused by fluoxetine alone on some events of the neurogenic process. Thus, environmental enrichment improves the benefits of pharmacological treatment by mechanisms that need to be clarified.

Melatonin Reverses the Depression-associated Behaviour and Regulates Microglia, Fractalkine Expression and Neurogenesis in Adult Mice Exposed to Chronic Mild Stress.

Depression may be precipitated by the negative impact of chronic stress, which is considered to play a key role in this neuropsychiatric disorder. Interestingly, depressed patients show decreased levels of melatonin. This hormone acts pro-neurogenic and exhibits anti-depressant effects in rodent models of predictive antidepressant-like effects. However, the benefits of melatonin in reversing the deleterious effects of chronic mild stress on the alterations in behaviour and in the neurogenic niche of the hippocampus in male BALB/c mice are unknown. In this study, we compared the effects of melatonin (2.5 mg/kg) and citalopram (5 mg/kg), an antidepressant drug belonging to the selective serotonin reuptake inhibitors, in male BALB/c mice exposed to chronic mild stress (CMS). We also investigated the potential effects of melatonin and citalopram on microglial cells, hippocampal neurogenesis and peripheral cytokine profiles. Melatonin and citalopram induced similar antidepressant-like activities that occurred with some of the the following findings: (1) reversal of the morphological alterations in microglia; (2) reversal of the decreased immunoreactivity to CX3CL1 and CX3CR1 in the dentate gyrus; (3) positive regulation of cell proliferation, survival and complexity of the dendritic trees of doublecortin-cells; and (4) modifications of peripheral CX3CL1 expression. This outcome is consistent with the hypothesis about the antidepressant-like effect of melatonin and supports its relevance as a modulator of the niche in the dentate gyrus.

Melatonin Modulates Dendrite Maturation and Complexity in the Dorsal- and Ventral- Dentate Gyrus Concomitantly with Its Antidepressant-Like Effect in Male Balb/C Mice.

Adult neurogenesis occurs in the dentate gyrus (DG) of the hippocampus. New neurons help to counteract the effects of stress and several interventions including antidepressant drugs, environmental modifications and internal factors act pro-neurogenic with consequences in the dorsal and ventral DG. Melatonin, the main product synthesized by the pineal gland, induces antidepressant-like effects and modulates several events of the neurogenic process. However, the information related to the capability of melatonin to modulate dendrite maturation and complexity in the dorsal and ventral regions of the DG and their correlation with its antidepressant-like effect is absent. Thus, in this study, we analyzed the impact of melatonin (0, 0.5, 1, 2.5, 5 or 10 mg/kg) administered daily for fourteen days on the number, dendrite complexity and distribution of doublecortin (DCX)-cells in the dorsal-ventral regions of the DG in male Balb/C mice. Doublecortin is a microtubule-associated protein that is expressed during the course of dendritic maturation of newborn neurons. Also, we analyzed the impact of melatonin on despair-like behavior in the forced swim test. We first found a significant increase in the number and higher dendrite complexity, mainly with the doses of 2.5, 5 and 10 mg/kg of melatonin (81%, 122%, 78%). These cells showed more complex dendritic trees in the ventral- and the dorsal- DG. Concomitantly, the doses of 5 and 10 mg/kg of melatonin decreased depressant-like behavior (76%, 82%). Finally, the data corroborate the antidepressant-like effect of melatonin and the increasing number of doublecortin-associated cells. Besides, the data indicate that melatonin favors the number and dendrite complexity of DCX-cells in the dorsal- and ventral- region of the DG, which may explain part of the antidepressant-like effect of melatonin.

Newly Generated Cells in the Dentate Gyrus Differentially Respond to Brief Spatial Exploration and Forced Swim in Adult Female Balb/C Mice.

Neurogenesis in the hippocampus is influenced by several factors including external stimuli. In addition to their involvement in learning and memory processes, newborn neurons of the dentate gyrus (DG) buffer against the effects of stress. Although the response of these cells to environmental stimuli has been shown, the age of the cells that respond to a brief spatial exploration or a stressful situation produced by forced-swim stress in adult female Balb/C mice is still unknown. Here, we investigated the activation of newborn neurons after three (IdU) or six weeks (CldU) postlabelling with the expression of Arc in the same mice but exposed to different environmental stimuli. Mice housed in standard conditions showed an increase in the activation of CldU-labelled cells after two exposures to a brief spatial exploration but no increase in the activation of IdU-labelled cells compared with the control group. Additionally, we analysed neuronal activation in the DG of mice housed in standard conditions and further exposed to forced-swim stress. We found a decreased activation of IdU-labelled cells in mice exposed to forced-swim stress with increase number of CldU-labelled cells. Our results suggest that based on their time postlabelling, newly generated hippocampal neurons show a different response to several environmental stimuli.

Soluble Factors from Human Olfactory Neural Stem/Progenitor Cells Influence the Fate Decisions of Hippocampal Neural Precursor Cells.

Neurogenesis plays a significant role during adulthood, and the observation that neural stem cells reside in the central nervous system and the olfactory epithelium has attracted attention due to their importance in neuronal regeneration. In addition, soluble factors (SFs) release by neural stem cells may modulate the neurogenic process. Thus, in this study, we identified the SFs released by olfactory human neural stem/progenitor cells (hNS/PCs-OE). These cells express Ki67, nestin, and ßIII-tubulin, indicating their neural lineage. The hNS/PCs-OE also express PSD95 and tau proteins during proliferation, but increased levels are observed after differentiation. Thus, we evaluated the effects of SFs from hNS/PCs-OE on the viability, proliferation, and differentiation potential of adult murine hippocampal neural precursor cells (AHPCs). SFs from hNS/PCs-OE maintain cells in the precursor and proliferative stages and mainly promote the astrocytic differentiation of AHPCs. These effects involved the activation, as measured by phosphorylation, of several proteins (Erk1/2; Akt/PRAS40/GSK3ß and JAK/STAT) involved in key events of the neurogenic process. Moreover, according to the results from the antibody-based microarray approach, among the soluble factors, hNS/PCs-OE produce interleukin-6 (IL-6) and neurotrophin 4 (NT4). However, residual epidermal growth factor (EGF) was also detected. These proteins partially reproduced the effects of SFs from hNS/PCs-OE on AHPCs, and the mechanism underlying these effects is mediated by Src proteins, which have been implicated in EGF-induced transactivation of TrkB receptor. The results of the present study suggest the potential use of SFs from hNS/PCs-OE in controlling the differentiation potential of AHPCs. Thus, the potential clinical relevance of hNS/PCs-OE is worth pursuing.

Exposure to Patterned Auditory Stimuli during Acute Stress Prevents Despair-Like Behavior in Adult Mice That Were Previously Housed in an Enriched Environment in Combination with Auditory Stimuli.

Several interventions have been shown to counteract the effects of stress that may be related to improved neuroplasticity and neuronal activation. In this sense, environmental enrichment (ENR) protects against acute stress and increases neuroplasticity. It has been suggested that the use of patterned auditory stimuli (PAS) may be beneficial in increasing the effectiveness of ENR on disorders related to stress, such as depression and anxiety. Examples of PAS are classical music compositions that have interesting effects at both clinical and preclinical levels. Thus, we analyzed the effects of the exposure to PAS, represented in this study by Mozart's compositions, during ENR housing for 35 days in adult male Balb/C mice to evaluate depression-associated behavior using the forced-swim test (FST) paradigm with an additional short exposure to PAS. We found that the ENR mice that were exposed to PAS during both housing and behavioral task (ENR + PAS/FST + PAS) show decreased immobility and the number of despair episodes within a higher latency to show the first bout of immobility. Additionally, we found increased neuronal activation evaluated by the identification of activity-regulated cytoskeleton-associated protein- (Arc-) labeled cells in the prefrontal cortex (PFC) in mice exposed to PAS during housing and in the absence or presence of PAS during FST. Moreover, we found increased neuronal activation in the auditory cortex (AuCx) of mice exposed to PAS during FST. Our study suggests that the exposure to PAS during an emotional challenge decreases despair-like behavior in rodents that were previously housed in an enriched environment in combination with auditory stimuli. Thus, our data indicate that the role of the exposure to PAS as an intervention or in combination with positive environment to aid in treating neuropsychiatric disorders is worth pursuing.

Human neural stem/progenitor cells derived from the olfactory epithelium express the TrkB receptor and migrate in response to BDNF.

Neurogenesis constitutively occurs in the olfactory epithelium of mammals, including humans. The fact that new neurons in the adult olfactory epithelium derive from resident neural stem/progenitor cells suggests a potential use for these cells in studies of neural diseases, as well as in neuronal cell replacement therapies. In this regard, some studies have proposed that the human olfactory epithelium is a source of neural stem/progenitor cells for autologous transplantation. Although these potential applications are interesting, it is important to understand the cell biology and/or whether human neural stem/progenitor cells in the olfactory epithelium sense external signals, such as brain-derived neurotrophic factor (BDNF), that is also found in other pro-neurogenic microenvironments. BDNF plays a key role in several biological processes, including cell migration. Thus, we characterized human neural stem/progenitor cells derived from the olfactory epithelium (hNS/PCs-OE) and studied their in vitro migratory response to BDNF. In the present study, we determined that hNS/PCs-OE express the protein markers Nestin, Sox2, Ki67 and ßIII-tubulin. Moreover, the doubling time of hNS/PCs-OE was approximately 38h. Additionally, we found that hNS/PCs-OE express the BDNF receptor TrkB, and pharmacological approaches showed that the BDNF-induced (40ng/ml) migration of differentiated hNS/PCs-OE was affected by the compound K252a, which prevents TrkB activation. This observation was accompanied by changes in the number of vinculin adhesion contacts. Our results suggest that hNS/PCs-OE exhibit a migratory response to BDNF, accompanied by the turnover of adhesion contacts.

Brain-Derived Neurotrophic Factor Induces Cell Survival and the Migration of Murine Adult Hippocampal Precursor Cells During Differentiation In Vitro.

The generation of new neurons during adulthood involves local precursor cell migration and terminal differentiation in the dentate gyrus. These events are influenced by the hippocampal microenvironment. Brain-derived neurotrophic factor (BDNF) is relevant for hippocampal neuronal development and behavior. Interestingly, studies that have been performed in controlled in vitro systems that involve isolated precursor cells that were derived from the dentate gyrus (AHPCs) have shown that BDNF induces the activation of the TrkB receptor and, consequentially, might activate signaling pathways that favor survival and neuronal differentiation. Based on the fact that the cellular events of AHPCs that are induced by single factors can be studied in this controlled in vitro system, we investigated the ability of BDNF and the involvement of protein kinase C (PKC), as one of the TrkB-downstream activated signaling proteins, in the regulation of migration, here reflected by motility, of AHPCs. Precursor cells were cultured following a concentration-response curve (1-640 ng/ml) for 24 or 96 h. We found that BDNF favored cell survival without altering the viability under culture proliferative conditions of the AHPCs. Concomitantly, glial- and neuronal-differentiated precursor cells increased as a consequence of survival promoted by BDNF. Additionally, pharmacological approaches showed that BDNF (40 ng/ml)-induced migration of AHPCs was blocked with the compounds K252a and GF109203x, which prevent the activation of TrkB and PKC, respectively. The results indicate that in the in vitro migration of differentiated AHPCs it is involved the BDNF and TrkB cascade. Our results provide additional information about the mechanism by which BDNF impacts adult neurogenesis in the hippocampus.

The neurogenic effects of an enriched environment and its protection against the behavioral consequences of chronic mild stress persistent after enrichment cessation in six-month-old female Balb/C mice.

Because stress may underlie the presence of depressive episodes, strategies to produce protection against or to reverse the effects of stress on neuroplasticity and behavior are relevant. Preclinical studies showed that exposure to stimuli, such as physical activity and environmental enrichment (ENR), produce beneficial effects against stress causing antidepressant-like effects in rodents. Additionally, ENR induces positive effects on neuroplasticity, neurochemistry and behavior at any age of rodents tested. Here, we analyzed whether ENR exposure prevents the development of depressive-like behavior produced by unpredictable, chronic mild stress (CMS) exposure as well as changes in hippocampal neurogenesis in a six-month-old female Balb/C mice, strain that shows low baseline levels of hippocampal neurogenesis. Mice were assigned to one of four groups: (1) normal housing-normal housing (NH-NH), (2) NH-CMS, (3) ENR-NH, or (4) ENR-CMS. The animals were exposed over 46 days to ENR or NH and subsequently to NH or CMS for 4 weeks. ENR induces long-term effects protecting against CMS induction of anhedonia and hopelessness behaviors. Independent of housing conditions, ENR increased the number of proliferative cells (Ki67), and CMS decreased the number of proliferative cells. ENR increased the newborn cells (BrdU) and mature phenotypes of neurons; these effects were not changed by CMS exposure. Similarly, the number of doublecortin-positive cells was not affected by CMS in ENR mice, which showed more cells with complex dendrite arborizations. Our study suggests that ENR induces protection against the effects of CMS on behavior and neuroplasticity in six-month-old Balb/C mice.

Chronic administration of a melatonin membrane receptor antagonist, luzindole, affects hippocampal neurogenesis without changes in hopelessness-like behavior in adult mice.

Melatonin is involved in the regulation of hippocampal neuronal development during adulthood. Emerging evidence indicates that exogenous melatonin acts during different events of the neurogenic process and exerts antidepressant-like behavior in rodents. Thus, melatonin might act through different mechanism, including acting as an antioxidant, interacting with intracellular proteins and/or activating membrane receptors. The melatonin membrane receptors (MMRs; Mt1/Mt2) are distributed throughout the hippocampus with an interesting localization in the hippocampal neurogenic microenvironment (niche), suggesting the involvement of these receptors in the beneficial effects of melatonin on hippocampal neurogenesis and behavior. In this study, we analyzed the participation of MMRs in the baseline neurogenesis in C57BL/6 mice. To this end, we used a pharmacological approach, administering luzindole (10 mg/kg) for 14 days. We observed a decrease in the absolute number of doublecortin-positive cells (49%) without changes in either the dendrite complexity of mature doublecortin-cells or the number of apoptotic cells (TUNEL). However, after the chronic administration of luzindole, cell proliferation (Ki67) significantly decreased (36%) with increasing (>100%) number of neural stem cells (NSCs; GFAP(+)/Sox2(+)) in the subgranular zone of the dentate gyrus of the hippocampus. In addition, luzindole did not affect hopelessness-like behavior in the forced swim test (FST) or changes in the novelty suppressed feeding test (NST) after 14 days of treatment either neuronal activation in the dentate gyrus after FST. These results suggest that the MMRs are involved in the effects of endogenous melatonin to mediate the transition from NSCs and proliferative cells to the following developmental stages implicated in the hippocampal neurogenic process of adult female C57BL/6 mice.

Las zonas neurogénicas en el adulto y su relación con las enfermedades neuropsiquiátricas / Neurogenic regions in the adult: Relationship with the neuropsychiatric disorders

Neuropsychiatric diseases (NPD) are characterized by changes in brain plasticity involving alterations in the morphology and functionality of neurons. However, affectations of the neuronal development (neurogenesis) in the adult brain are also shown. The neurogenic process is widely regulated by different factors such as genes, microenvironment, hormones, neurotransmitters, environmental cues and, also, nutrition. Thus, alterations in these factors negatively impact the neuronal development. Several studies performed in humans have revealed alterations of neurogenesis in NPD. However, most of the knowledge derives from studies done in animal models of NPD. The evidences from animal models are controversial, thus the use of human-induced pluripotent stem cells as a model of NPD has marked a way to study alterations in the neuronal development. Recently, the use of another cellular model for studying NPD has been proposed. Multipotent stem cells derived from olfactory epithelium (MOESCs) are a good candidate. However, evidences are scarce and deeper studies are necessary to know if there is or not a correlation of alterations in neuronal development in the OE with the changes observed in the brain; or if the MOESCs can mimic alterations shown in NPD that could let to get more knowledge about the factors promoting these diseases. Thus, in this review we discuss basic information about adult neurogenesis under physiological and non-physiological conditions in the hippocampus, olfactory bulb and olfactory epithelium.

Las enfermedades neuropsiquiátricas (ENP) se caracterizan por cambios en la plasticidad cerebral que incluyen la pérdida neuronal en regiones específicas en el encéfalo, cambios en la transmisión sináptica originada por alteraciones en los contactos sinápticos y también por la expresión de genes. Además, otro proceso que forma parte de la plasticidad cerebral y que también se encuentra afectado en las ENP es la generación de nuevas neuronas (neurogénesis). El proceso neurogénico en el adulto es regulado de manera fina por diversos factores como los aspectos genéticos, celulares, el microambiente, los elementos neuroquímicos, los ambientales y los nutricionales. Las alteraciones de estos factores impactan en el desarrollo y en la función de las nuevas neuronas. Algunos estudios realizados en humanos han revelado las alteraciones en la neurogénesis en algunos ENP. Sin embargo los mayores avances logrados han utilizado modelos animales de ENP. En algunos casos estas evidencias son controvertidas y recientemente se han tratado de aclarar utilizando cultivos de células madre pluripotenciales-inducibles humanas como modelos de ENP. Otro modelo que se ha propuesto para estudiar las alteraciones en el desarrollo neuronal en las ENP son las células madre multipotenciales del epitelio olfatorio (CMPEO). Sin embargo las evidencias obtenidas con las CMPEO son escasas y resulta necesario demostrar si existe o no un correlato con las alteraciones que ocurren en el desarrollo neuronal a nivel central en las ENP, o bien si las CMPEO pueden mostrar las alteraciones observadas en las ENP que permitan obtener información acerca de los factores que promueven estas enfermedades. Por lo tanto en esta revisión se incluyen aspectos básicos de la neurogénesis e información relevante de las alteraciones de este proceso en las tres regiones neurogénicas en el adulto: el hipocampo, el bulbo olfatorio y el epitelio olfatorio.

Melatonin supplementation delays the decline of adult hippocampal neurogenesis during normal aging of mice.

Melatonin modulates adult hippocampal neurogenesis in adult mice. Also, plasma melatonin levels and new neuron formation decline during aging probably causing cognitive alterations. In this study, we analyzed the impact of exogenous supplementation with melatonin in three key events of hippocampal neurogenesis during normal aging of mice. The analysis was performed in rodents treated with melatonin during 3, 6, 9 or 12 months. We found an increase in cell proliferation in the dentate gyrus of the hippocampus after 3, 6 and 9 months of treatment (>90%). Additionally, exogenous melatonin promoted survival of new cells in the dentate gyrus (>50%). Moreover, melatonin increased the number of doublecortin-labeled cells after 6 and 9 months of treatment (>150%). In contrast, melatonin administered during 12 months did not induce changes in hippocampal neurogenesis. Our results indicate that melatonin also modulates the neurogenic process in the hippocampus during normal aging of mice. Together, the data support melatonin as one of the positive endogenous regulators of neurogenesis during aging.

Los fármacos antidepresivos como reguladores de la neurogénesis hipocámpica de roedores y humanos adultos / Regulation of rodent and human adult hippocampal neurogenesis by antidepressant drugs

New neuron formation in the adult brain extends our knowledge and incorporates a novel dimension about brain plasticity. Adult neurogenesis is a complex process regulated by different factors within the niche, where adult neural stem cells reside, proliferate and differentiate. Neural stem cell together with astrocytes and endothelial cells form the principle components of this complex niche. Other molecular factors that regulate adult neurogenesis are the neuro-transmitters (GABA, glutamate, serotonin, dopamine); hormones (prolactin, growth hormone, estrogens and melatonin); growth factors (FGF, EGF, VEGF) and neurotrophins (BDNF, NT3). All of them regulate different aspects of the neurogenic process. Behavioral regulators that influence new neuron formation in the adult brain include physical activity, complex stimulatory environment best known as enrichment environment, and social interaction. Voluntary physical activity with free access to the running wheel increases the number of proliferating cells, while the complex stimulatory environment provided by enriched environment preferentially influences survival of newborn cells. In addition, social interaction has a positive influence on the new neuron formation in the dentate gyrus (DG). Although adult hippocampal neurogenesis is positively regulated by the aforementioned factors, there are different conditions with negative influence on this process. Some of these conditions are stress exposure and sleep deprivation. Both conditions are present in neuropsychiatric diseases such as depression, anxiety and schizophrenia. Thus, stress and sleep deprivation impair adult hippocampal neurogenesis. Alteration of the neurogenic process following stress occurs due to the high levels of glucocorticoid receptors within the hippocampus and because exposure to stress causes the increase in glucocorticoid levels. Preclinical studies have shown that exposure to different classes of stressors affect hippocampal neurogenesis. Prolonged exposure to stressors (chronic mild stress), predatory odor, foot shock, acute force swimming and psychosocial stress not only affect mature neuronal plasticity but also hippocampal neurogenesis. Although there is information about the effects of stress on adult neurogenesis, the mechanism by which stress causes inhibition of hippocampal neurogenesis remains unclear. Recent work showed that exposure to stress increases the pro-inflammatory cytokine interleukin-1 β (IL-1 β) in several brain areas. Also, administration of IL-1β exerts stress-like effects including down-regulation of hippocampal brain derived neurotrophic factor (BDNF). Additionally, inhibition of the receptor for IL-1β prevents stress-like effects. Moreover, the suppression of cell proliferation is mediated by direct actions of IL-1 β on IL-1RI receptors localized on precursor cells. These findings support that IL-1 β is a critical mediator of the antineurogenic effect caused by acute and chronic stress. However, IL-1 β is not the unique mediator of stress that could be involved in the alteration of adult hippocampal neurogenesis. Recently it was reported that the decrease in cell proliferation concomitantly occurs with an increase of IL6 and TNFα levels. Preclinical studies have suggested that adult hippocampal neurogenesis is not a sole cause of depression or the sole mechanism of treatment efficacy, but it is likely an important contributor to this complex disorder. In order to revert the effects of stress on adult hippocampal neurogenesis, different therapies have been used, for example: electroconvulsive therapy (ECT), exercise, complex stimulatory environment and antidepressant drugs. Although the most rapid induction of neurogenesis is seen with ECT application, most studies have been done with antidepressant drugs. The effects of antidepressants are time-dependent as highest therapeutic effects are observed within the time course of weeks. Different types of antidepressants (serotonin and norepinephrine reuptake inhibitors, monoamine oxidase inhibitors and atypical antidepressants) have been used to study their influence on the neurogenic process. Despite that serotonin reuptake inhibitors are the most prescribed treatments for major depression and that the therapeutic effects of antidepressants require chronic treatment, the mechanisms by which these drugs exert their effects on hippocampal neurogenesis are still unknown. Although serotonin reuptake inhibitors are very fast in increasing serotonin levels, the antidepressant action is delayed possibly because of the induction of structural or functional changes that possibly need longer time (2-4 weeks). In this regard, one of the actions of antidepressants is the regulation of adult hippocampal neurogenesis, a process that is consistent with the delayed onset of therapeutic effects of antidepressants. Fluoxetine is one of the antidepressants more used to study its influence on adult neurogenesis. Fluoxetine targets amplifying neural progenitors by increasing the rate of symmetric divisions without altering the division of stem-like cells in the DG. Considering previous classification based on the temporal protein markers expression, the neural progenitors targeted by fluoxetine correspond to type 2a, 2b and type 3. In addition, the increase in new neurons caused by fluoxetine is due to the expansion of neural progenitors. In addition to cell proliferation, the neurogenic process also involves a maturation step, which is associated with the expression of doublecortin, a protein that binds to microtubules and that is expressed along the cytoplasm of the cell. Further maturation of immature neurons such as dendrite maturation, is controlled independently of the regulation of precursor cell proliferation. Thus, micro-regulatory events influence the course of adult hippocampal neurogenesis. Here, fluoxetine also affects dendrite maturation and functional integration of new neurons. Chronic fluoxetine treatment modifies dendrite morphology increasing dendrite arborisation and favors synaptic plasticity of newborn granule cells. Also, chronic administration of fluoxetine causes behavioral improvement, an effect that was blocked when neurogenesis was ablated by X-ray irradiation. Other important factor that influences the effect of antidepressants on adult neurogenesis is the genetic background. Then antidepressants induced behavioral improvement depending on the genetic background of the mouse strain used. Preclinical studies in mice have revealed different actions of antidepressants on adult hippocampal neurogenesis. However, studies in humans are scarce and deserve greater attention to discover the correlation between preclinical and clinical studies. Recent work in human brains shows contradictory evidences about the regulation of neuronal development by antidepressants. These evidences are in the same line as recent published work in which it was demonstrated that the effects of ADs are age-dependent. Altogether, multiple evidences indicate that antidepressants affect several aspects of the neurogenic process. Therefore, chronic treatment is necessary for the antidepressant-dependent regulation of adult hippocampal neurogenesis. In addition, it has been shown that antidepressants act through different pathways involving both neurogenesis-dependent and neurogenesis-independent actions. Although there is an important increase in the adult hippocampal neurogenesis field, it is necessary to increase the number of studies performed in human beings to correlate the preclinical findings with clinical studies to address the role of adult neurogenesis in neuropsychiatric disorders.

El hallazgo de la formación de nuevas neuronas en el giro dentado (GD) del hipocampo amplió el conocimiento acerca de la plasticidad del encéfalo. En este sentido, la neurogénesis es un proceso que involucra diferentes eventos celulares tales como: la división de las células madre, la proliferación de los neuroblastos, la migración y la sobrevivencia celular, así como la maduración dendrítica, la elongación axonal y la integración de las neuronas nuevas a los circuitos neuronales existentes. En conjunto, todas estas etapas causan cambios estructurales y funcionales en el cerebro. Por lo tanto, la formación de neuronas es un proceso regulado de manera fina por diferentes factores entre los que se incluyen: el nicho; algunos neurotransmisores como la serotonina, la dopamina, el glutamato y el GABA; factores de crecimiento como el factor de crecimiento de fibroblastos, el factor de crecimiento epidermal y el factor de crecimiento vascular endotelial (FGF, EGF y VEGF, por sus siglas en inglés); neurotrofinas como el factor neurotrópico derivado del cerebro y por la neurotrofina 3 (BDNF y NT3, por sus siglas en inglés). Aunado a la existencia de factores que favorecen la neurogénesis hipocámpica, también hay factores que influyen de manera negativa en la formación de neuronas. Entre éstos se encuentra el estrés, el cual se relaciona con algunas enfermedades neuropsiquiátricas como la depresión y la ansiedad. A este respecto, estudios preclínicos han revelado que la aplicación de diferentes tipos de estresores puede afectar la plasticidad neuronal al inducir alteraciones morfológicas y funcionales en el hipocampo, así como afectar el proceso neurogénico. Las alteraciones causadas por el estrés se han relacionado con un aumento considerable y sostenido de los niveles de glucocorticoides. Esto último afecta el proceso neurogénico debido a que el hipocampo es una estructura cerebral que expresa niveles altos de receptores para estas hormonas. Al ser activados de forma persistente, los receptores a glucocorticoides causan una alteración en la neuroplasticidad hipocámpica. De tal modo y considerando lo anterior, teorías recientes han asociado un fallo en la formación de neuronas en el hipocampo con algunos trastornos psiquiátricos como la demencia, la esquizofrenia y la depresión. No esta del todo elucidado el mecanismo a través del cual el estrés altera el proceso neurogénico. Sin embargo, trabajos recientes han revelado que la exposición a estrés causa un aumento en los niveles de ciertas citocinas proinflamatorias, tales como la interleucina-1 β (IL-1 β). El aumento en los niveles de esta citocina provoca un efecto tipo depresivo y una disminución en los niveles del BDNF, así como una alteración en la formación de nuevas neuronas. Estos hallazgos apoyan la idea de que la IL-1 β es un mediador crítico del efecto antineurogénico causado por el estrés crónico y agudo. Sin embargo, la IL-1 β no es la única citocina asociada con las alteraciones en el proceso neurogénico, ya que recientemente se reportó que la disminución en la proliferación celular causada por el estrés ocurre de manera paralela con el aumento en la expresión de los mensajeros de la IL-6 y del TNF-α. Una manera de contrarrestar los efectos del estrés sobre la plasticidad neuronal es a través de la administración de fármacos antidepresivos. Diversos trabajos han mostrado que el tratamiento crónico con este tipo de fármacos revierte las alteraciones en la neurogénesis hipocámpica y en la plasticidad neuronal causadas por el estrés. Finalmente, aun cuando existen evidencias del papel que desempeña la neurogénesis en modelos animales de algunas enfermedades neuropsiquiátricas y de la forma en que los fármacos antidepresivos favorecen la formación de neuronas, es importante contar con más estudios en humanos que permitan corroborar los hallazgos que se han obtenido en los estudios preclínicos. De algún modo todos los reportes apuntan a que los fármacos antidepresivos pueden actuar por mecanismos independientes o dependientes de la neurogénesis hipocámpica.

Chronic treatment with melatonin stimulates dendrite maturation and complexity in adult hippocampal neurogenesis of mice.

In the course of adult hippocampal neurogenesis, the postmitotic maturation and survival phase is associated with dendrite maturation. Melatonin modulates the survival of new neurons with relative specificity. During this phase, the new neurons express microtubule-associated protein doublecortin (DCX). Here, we show that the entire population of cells expressing DCX is increased after 14 days of treatment with melatonin. As melatonin also affects microtubule polymerization which is important for neuritogenesis and dendritogenesis, we studied the consequences of chronic melatonin administration on dendrite maturation of DCX-positive cells. Treatment with melatonin increased the number of DCX-positive immature neurons with more complex dendrites. Sholl analysis revealed that melatonin treatment lead to greater complexity of the dendritic tree. In addition, melatonin increased the total volume of the granular cell layer. Besides its survival-promoting effect, melatonin thus also increases dendritic maturation in adult neurogenesis. This might open the opportunity of using melatonin as an adjuvant in attempts to extrinsically stimulate adult hippocampal neurogenesis in neuropsychiatric disease, dementia or cognitive ageing.

ROCK-regulated cytoskeletal dynamics participate in the inhibitory effect of melatonin on cancer cell migration.

Cell movement is generated by a driving force provided by dynamic cytoskeletal organization. Two main cytoskeletal-dependent features, essential for migration, are the highly cell polarized structure and focal adhesion complexes. Cell migration and substrate anchorage are finely regulated by external signaling exerted by growth factors and hormones. In particular, the serine threonine kinase activated by the small GTPase Rho, the Rho-associated protein kinase (ROCK), participate in both processes through regulation of actin rearrangements in lamellipodia, filopodia, ruffles, and stress fibers. Melatonin, the main product secreted by the pineal gland has oncostatic properties. In MCF-7 cells, 1 nm melatonin reduces migration and invasiveness through increased expression of two cell surface adhesion proteins, E-cadherin and beta(1)-integrin. In this work, we studied the microfilament and microtubule rearrangements elicited by melatonin in migrating leader MCF-7 cells by a wound-healing assay. Additionally, cell anchorage was estimated by quantification of focal adhesions in MCF-7 cells cultured with melatonin. ROCK participation in the indole effects on anchorage and migration was explored by inhibition of the kinase activity with the specific inhibitor of ROCK, the Y-27632 compound. The results indicate that ROCK participates in the melatonin inhibitory effects on cell migration by changing cytoskeletal organization of leader MCF-7 cells. Also, they indicated that indole increased the number of focal contacts through ROCK. These results support the notion that melatonin inhibits cancer cell invasion and metastasis formation via ROCK-regulated microfilament and microtubule organization that converge in a migration/anchorage switch.

La melatonina: un coadyuvante potencial en el tratamiento de las demencias / Melatonin: A potential coadyuvant in dementia treatment

Alzheimer's disease is characterized by a progressive neuronal death and a lost of memory and cognition that unable the patient to perform daily tasks. Cytoskeleton alterations, identified as a major histopathologic hallmark of neurodegenerative diseases, occur in dementia. In this disease, neurons have pathologic inclusions containing fibrillar aggregates of hyperphosphorylated tau protein in absence of amyloid deposits. Abundant senile plaques and neurofibrillary tangles constitute the two major neuropathologic lesions present in hippocampal, neocortical, and forebrain cholinergic brain regions of Alzheimer's patients. Hyperphosphorylated tau and the subsequent formation of paired helical filaments loses the capabilities for maintaining highly asymmetrical neuronal polarity. Thus, in brains with a high content of hyperphosphorylated tau, microtubules are disassembled, the highly asymmetrical neural shape is lost and an impairment of axonal transport is produced together with a lost of dendrite arborizations. In addition, brain damage caused by free radicals occurs in Alzheimer's disease. This illness involves a reduction of the endogenous antioxidant enzyme system, increased senile-plaque formation, cytoskeletal collapse, and neuronal apoptosis induced by oxidative stress. Acetylcholinesterase inhibitors are the most commonly used compounds in the treatment of neurodegenerative diseases. However, despite their wide use in the treatment of Alzheimer's disease, these compounds have limited therapeutic effects and cause undesirable effects. Therefore it is necessary to investigate new alternatives in the Alzheimer's disease treatment. Considering that neurodegenerative diseases are cytoskeleton disorders, this cellular structure could be a drug target for therapeutic approaches by restoring normal cytoskeleton structure and by precluding damage caused by oxygen-reactive species. In this regard, melatonin, the indole secreted by the pineal gland during the dark phase of the photoperiod, has two important properties that may be useful for the treatment of mental disorders. One is that melatonin is a potent free-radical scavenger and the other is that this indole is a cytoskeletal modulator. A neuroprotective role for melatonin was initially suggested due to its free-radical scavenger properties. Melatonin detoxifies the highly toxic hydroxyl radical as well as the peroxyl radical, peroxynitrite anion, nitric oxide, and singlet oxygen, all of which can damage brain macromolecules. Moreover, melatonin stimulates the activity of antioxidative enzymes including superoxide dismutase, glutathione peroxidase, and glutathione reductase. Also, it is a lipophilic molecule able to cross the blood-brain barrier. All these properties make melatonin a highly effective pharmacologic agent against free-radical damage in the brain. Also, it is a useful neuroprotector in dementia because it synchronize the body rhythms with the photoperiod, which are altered in Alzheimer's disease and because normal circadian secretion of melatonin and sleep-wake cycle can be restored by the indolamine administration. Additionally, cytoskeletal modulation by melatonin is another relevant property of the indole for neurodegenerative diseases treatment. Direct assessment of melatonin effects on cytoskeletal organization in neuronal cells indicated that the indole promotes neuritogenesis in N1E-115 neuroblastoma cells at plasma melatonin concentration. Neurite formation is a complex process critical to establish synaptic connectivity that is lost in Alzheimer's disease. Neuritogenesis takes place by a dynamic cytoskeletal organization that involves microtubule enlargement, microfilament arrangement, and intermediate-filament reorganization. In particular, microtubule assembly participates in neurite formation elicited by melatonin through antagonism to calmodulin. Also, selective activation of protein kinase C (PKC) alpha by melatonin participates in vimentin intermediate filament rearrangements and actin dynamics for neurite outgrowth in neuroblastoma cells. In N1E-115 cells, melatonin at plasma and cerebrospinal fluid concentration caused an increase in microfilament arrays in stress fibers and their thickening, as well as increased growth cone formation, and augmented number of cells with microspikes. Recently, it was demonstrated that melatonin increased both the number of N1E-115 cells with filopodia and with long neurites through both PKC activation and Rho-associated kinase (ROCK) stimulation. The utility of melatonin to prevent damage in the cytoskeletal structure produced by neurodegenerative processes was demonstrated in N1E-115 neuroblastoma cells cultured with okadaic acid (OA), a specific inhibitor of the serine/threonine proteins phosphatases 1 and 2A that induces molecular and structural changes similar to those found in Alzheimer's disease. Melatonin prevented microtubule disruption followed by cell-shape changes and increased lipid peroxidation and apoptosis induced by OA. Melatonin effects on altered cytoskeletal organization induced by OA are dose-dependent and effects were observed at plasma -and cerebrospinal-fluid concentrations of the indole. These data support that melatonin can be useful in the treatment of neurodegenerative diseases by both its action on the cytoskeleton and by its free-radical scavenger properties.

La enfermedad de Alzheimer es una enfermedad neurodegenerativa progresiva que cursa con una deficiencia en las capacidades cognitivas, así como con la presencia de síntomas psiquiátricos y alteraciones conductuales. Las características histopatológicas más importantes en la enfermedad de Alzheimer son la formación de placas seniles, los ovillos neurofibrilares y un incremento en el estrés oxidativo. La polaridad estructural y la morfología neuronal se pierden en la enfermedad de Alzheimer. La proteína tau se encuentra anormalmente fosforilada, los microtúbulos se despolimerizan, se pierden la forma asimétrica de las neuronas y la conectividad sináptica, y se interrumpe el transporte axoplasmático. Asimismo, se ha sugerido que la inhibición o la pérdida en el balance de la formación de neuronas en el hipocampo puede participar en la fisiopatología de la enfermedad de Alzheimer debido a que el cerebro no puede reparar el daño neuronal y consecuentemente induce la pérdida de la cognición. Los agentes colinérgicos son los medicamentos más aceptados en el tratamiento de la enfermedad de Alzheimer en una etapa en que los síntomas se clasifican de medios a moderados. Sin embargo, el tratamiento de pacientes con enfermedad de Alzheimer grave es limitado. Por lo anterior se requiere la búsqueda de nuevas alternativas para el tratamiento de esta enfermedad. La melatonina es una indolamina que actúa como un potente antioxidante, como un modulador de la organización del citoesqueleto así como un factor de diferenciación celular. Diversos estudios han sugerido que la melatonina tiene un efecto neuroprotector por su capacidad de captar radicales libres. La melatonina disminuye la lipoperoxidación y la apoptosis producida por la administración de ácido ocadáico (AO) o peróxido de hidrógeno (H2O2). Se sabe que las especies reactivas de oxígeno producen alteraciones en la organización del citoesqueleto e influyen el estado de fosforilación de la proteína tau y que la melatonina previene la fosforilación de la proteína tau debido a su actividad antioxidante. Se ha descrito que la melatonina modula el arreglo de los microfilamentos de actina y la formación de fibras de tensión en las células Madin-Darby canine kidney (MDCK) por medio de una interacción concertada de la indolamina con la calmodulina y con la proteína cinasa C (PKC) y la participación de la proteína cinasa dependiente de Rho (ROCK). Asimismo, la melatonina participa en las etapas tempranas de la formación de neuritas en las células N1E-115 por medio de ROCK. Otros estudios han indicado que la melatonina previene el daño en el citoesqueleto producido por el AO en las células N1E-115. El AO se ha utilizado para reproducir en células en cultivo las alteraciones en el citoesqueleto y el incremento en el estrés oxidativo que ocurren en las neuronas de pacientes con enfermedad de Alzheimer. La melatonina en estas células previene la retracción del citoesqueleto, efecto del AO. La red del citoesqueleto se mantiene en el citoplasma y en las neuritas de las células N1E-115 cultivadas con melatonina, no obstante que sean tratadas con el AO posteriormente. Recientemente, se demostró que en las células de neuroblastoma N1E-115 incubadas con melatonina se previene la hiperfosforilación de la proteína tau causada por el AO. Aunado a lo anterior, se ha demostrado que la melatonina modula la formación de neuronas nuevas en un modelo in vitro utilizando células embrionarias y de corteza cerebral de ratón. La formación de neuronas inducida por la melatonina se corroboró utilizando células precursoras aisladas de animales adultos así como en animales adultos, y se encontró que la indolamina moduló la sobrevida de las células nuevas formadas, así como la diferenciación de éstas en neuronas nuevas. Las evidencias presentadas en esta revisión indican que la melatonina puede ser útil como un coadyuvante en el tratamiento de las demencias.

El neurocitoesqueleto: un nuevo blanco terapéutico para el tratamiento de la depresión

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Summary Postmortem and neuroimaging studies of Major Depressive Disorder patients have revealed changes in brain structure. In particular the reduction in prefrontal cortex and in hippocampus volume has been described. In addition, a variety of cytoarchitectural abnormalities have been described in limbic regions of major depressive patients. Decrease in neuronal density has been reported in the hippocampus, a structure involved in declarative, spatial and contextual memory. This structure undergoes atrophy in depressive illness along with impairment in cognitive function. Several studies suggest that reduction of hyppocampus volume is due to the decreased cell density and diminished axons and dendrites. These changes suggested a disturbance of normal neuronal polarity, established and maintained by elements of the neuronal cytoskeleton. In this review we describe evidence supporting that neuronal cytoskeleton is altered in depression. In addition, we present data indicating that the cytoskeleton can be a potential target in depression treatment. Neurons are structural polarized cells with a highly asymmetric shape. The cytoskeleton plays a key role in maintain the structural polarization in neurons which are differentiated in two structural domains: The somato-dendritic domain and the axonal domain. This differentiated asymmetric shape, depends of the cytoskeletal organization which support, transport and sorts various molecules and organelles in different compartments within the cell. Microtubules determine the asymmetrical shape and axonal structure of neurons and form the tracks for intracellular transport, of crucial importance in axonal flux. Actin microfilaments are involved in force generation during organization of neuronal shape in cellular internal and external movements and participate in growth cone formation. This important cytoskeletal organization preceed the formation of neurites that eventually will differentiated into axons or dendrites, a process that also comprises a dynamic assembly of the three cytoskeletal components. Intermediate filaments are known in neurons as neurofilaments spatially intercalated with microtubules in the axons and facilitate the radial axonal growth and the transport. Neurofilaments also act supporting other components of the cytoskeleton. All changes and movements of the cytoskeletal organization are coordinated by cytoskeletal associated proteins such as the protein tau and the microtubule associated proteins (MAPs). Also, specific interactions of microfilaments, microtubules and filaments which are regulated by extracellular signals take place in modulation of the cytoskeletal rearrangements. The polarized structure and the highly asymmetric shape of neurons are essentials for neuronal physiology and it appears to be lost in patients with a Major Depressive Disorder. Histopathological studies have shown that the hippocampus and frontal cortex of patients with major depressive disorder have diminished soma size, as well as, have decreased dendrites and cellular volume. Dendrite formation depends mainly in microfilaments organization as well as in polarization of the microtubule binding protein MAP2. In addition, there is a decreased synaptic connectivity and an increased oxidative stress, which originates abnormalities in the cytoskeletal structure. These neuronal changes originate alterations in the brain functionality such as decreased cognitive abilities and affective dis-regulations, usually encountered in patients with depression. Therefore, pathologic lesions implicating an altered cytoskeletal organization, may have an important role in decreased cognitive functions, observed in depression, as well as in changes in the brain volume, explained by a lost of neuronal processes such as axons, dendrite processes or dendritic spines, rather than by loss of neuronal or glial cell bodies. This explanation is supported by light immunomicroscopy of brain slices postmortem stained with specific antibodies. Psychological stress which causes oxidative stress has also been suggested to cause a decrease of neuronal volume in the prefrontal cortex, altering the synaptic connections established with the hippocampus. This conclusion was drawn from studies in animal models of psychological stress associated with molecular measurements where defects in the expression of MAP1 and sinaptophysin were found, suggesting that defects in cytoskeletal associated proteins could underlie some cytoarchitectural abnormalities described in depression. Together all the evidence accumulated indicates that major depression illness and bipolar depression are mental disorders that involve loss of axons and dendrites in neurons of the Central Nervous System, that in consequence cause disruption of synaptic connectivity. Thus is possible that depression can be considered as a cytoskeletal disorder, therefore this cellular structure could be a drug target for therapeutic approaches by restoring normal cytoskeleton structure and precluding damage caused by oxygen-reactive species. In this regard, melatonin, the hormone secreted by pineal gland during dark phase of the photoperiod, has two important properties that can be useful in treatment of mental disorders. First, the melatonin is a potent free-radical scavenger and second this hormone governs the assembly of the three main cytoskeletal components modulating the cytoskeletal organization. This notion is supported by direct action of melatonin effects on cytoskeletal organization in neuronal cells. In N1E-115 neuroblastoma cells, melatonin induced a two-fold increase in number of cells with neurites 1 day after plating; the effect lasting up to 4 days. Induction of neurite outgrowths is optimal at 1 nM melatonin and in presence of hormone the cells grew as clusters with long neurites forming a fine network to make contact with adjacent cells. Immunofluorescence of N1E-115 cells cultured under these conditions showed tubulin staining in long neurite processes connecting cells to each other. Neurite formation is a complex process that is critical to establish synaptic connectivity. Neuritogenesis takes place by a dynamic cytoskeletal organization that involves microtubule enlargement, microfilament arrangement, and intermediate- filament reorganization. In particular, it is known that vimentin intermediate filaments are reorganized during initial stages of neurite outgrowth in neuroblastoma cells and cultured hippocampal neurons. Evidence has been published indicating that increase in microtubule assembly participates in neurite formation elicited by melatonin antagonism to calmodulin. Moreover, recently it was reported that melatonin precludes cytoskeletal damage produced by high levels of free radicals produced by hydrogen peroxide, as well as, damage caused by higher doses of the antypsychotics haloperidol and clozapine. N1E-115 cells incubated with either 100 uM hydrogen peroxide, 100 uM haloperidol, or 100 uM clozapine undergo a complete cytoskeletal retraction around the nucleus. By contrast, NIE-115 cells incubated with hydrogen peroxide, clozapine, or haloperidol followed by the nocturnal cerebrospinal fluid concentration of melatonin (100 nM) showed a well preserved cytoskeleton and neuritogenesis. Thus melatonin is a neuroprotective compound, since protects the neurocytoskeletal organization against damage caused by high concentrations of antipsychotics and oxidative stress. As mentioned previously, polarity is intrinsic to neuronal function. In neurons, somatodendritic domain receives and decodes incoming information and axonal domain delivers information to target cells. Progressive loss of neuronal polarity is one of the histopathologic events in depression. Cytoskeletal collapse underlie the lost of structural polarity and it is known that precede neuronal death and disappearance of synaptic connectivity. Drugs that prevent the loss of polarity and cytoskeleton retraction intrinsic to these diseases, as well as damage in cytoskeletal structure produced by oxidative stress can be extremely useful in depression treatment. Melatonin is a potent free-radical scavenger that also acts as a cytoskeleton regulator; thus, we speculate that this hormone could be useful in prevention and alleviation of psychiatry diseases with synaptic connectivity disruption. Clinical trials show that melatonin administration is followed by alleviation of circadian disturbances and cognitive function in various neuropsychiatry diseases. Moreover, in depression, melatonin improves sleep. Thus, as suggestive as this information appears, controlled clinical trials will be necessary to investigate the beneficial effects of melatonin and other drugs in the depression treatment.

Melatonin induces neuritogenesis at early stages in N1E-115 cells through actin rearrangements via activation of protein kinase C and Rho-associated kinase.

Melatonin increases neurite formation in N1E-115 cells through microtubule enlargement elicited by calmodulin antagonism and vimentin intermediate filament reorganization caused by protein kinase C (PKC) activation. Microfilament rearrangement is also a necessary process in growth cone formation during neurite outgrowth. In this work, we studied the effect of melatonin on microfilament rearrangements present at early stages of neurite formation and the possible participation of PKC and the Rho-associated kinase (ROCK), which is a downstream kinase in the PKC signaling pathway. The results showed that 1 nm melatonin increased both the number of cells with filopodia and with long neurites. Similar results were obtained with the PKC activator phorbol 12-myristate 13-acetate (PMA). Both melatonin and PMA increased the quantity of filamentous actin. In contrast, the PKC inhibitor bisindolylmaleimide abolished microfilament organization elicited by either melatonin or PMA, while the Rho inhibitor C3, or the ROCK inhibitor Y27632, abolished the bipolar neurite morphology of N1E-115 cells. Instead, these inhibitors prompted neurite ramification. ROCK activity measured in whole cell extracts and in N1E-115 cells was increased in the presence of melatonin and PMA. The results indicate that melatonin increases the number of cells with immature neurites and suggest that these neurites can be susceptible to differentiation by incoming extracellular signals. Data also indicate that PKC and ROCK are involved at initial stages of neurite formation in the mechanism by which melatonin recruits cells for later differentiation.

Melatonin increases stress fibers and focal adhesions in MDCK cells: participation of Rho-associated kinase and protein kinase C.

Melatonin cyclically modifies water transport measured as dome formation in MDCK cells. An optimal increase in water transport, concomitant with elevated stress fiber (SF) formation, occurs at nocturnal plasma melatonin concentrations (1 nm) after 6 hr of incubation. Blockage in melatonin-elicited dome formation was observed with protein kinase C (PKC) inhibitors. Despite, this information on the precise mechanism by which melatonin increases SF formation involved in water transport is not known. Focal adhesion contacts (FAC) are cytoskeletal structures, which participate in MDCK membrane polarization. SF organization and vinculin phosphorylation are involved in FAC assembly and both processes are mediated by PKC, an enzyme stimulated by melatonin; in these processes also involved is Rho-associated kinase (ROCK). Thus, we studied FAC formation and the ROCK/PKC pathway as the mechanism by which melatonin increases SF formation and water transport. The results showed that 1 nM melatonin and the PKC agonist phorbol-12-miristate-13-acetate increased FAC. The PKC inhibitor GF109203x, and the ROCK inhibitor Y27632, blocked increased FAC caused by melatonin. ROCK and PKC activities, vinculin phosphorylation and FAC formation were increased with melatonin. The PKC inhibitor, GF109203x, abolished both melatonin stimulated FAC in whole cells and ROCK activity, indicating that ROCK is a downstream kinase in the melatonin-stimulated PKC pathway in MDCK cultured cells that causes an increase in SF and FAC formation. Data also document that melatonin modulates water transport through modifications of the cytoskeletal structure.