Fraping: A computational tool for detecting slight differences in fluorescence recovery after photobleaching (FRAP) data for actin polymerization analysis.

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.

Pharmacological Inhibition of p-21 Activated Kinase (PAK) Restores Impaired Neurite Outgrowth and Remodeling in a Cellular Model of Down Syndrome.

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.

SVCT2 Overexpression and Ascorbic Acid Uptake Increase Cortical Neuron Differentiation, Which Is Dependent on Vitamin C Recycling between Neurons and Astrocytes.

During brain development, sodium-vitamin C transporter (SVCT2) has been detected primarily in radial glial cells in situ, with low-to-absent expression in cerebral cortex neuroblasts. However, strong SVCT2 expression is observed during the first postnatal days, resulting in increased intracellular concentration of vitamin C. Hippocampal neurons isolated from SVCT2 knockout mice showed shorter neurites and low clustering of glutamate receptors. Other studies have shown that vitamin C-deprived guinea pigs have reduced spatial memory, suggesting that ascorbic acid (AA) and SVCT2 have important roles in postnatal neuronal differentiation and neurite formation. In this study, SVCT2 lentiviral overexpression induced branching and increased synaptic proteins expression in primary cultures of cortical neurons. Analysis in neuroblastoma 2a (Neuro2a) and human subventricular tumor C3 (HSVT-C3) cells showed similar branching results. SVCT2 was mainly observed in the cell membrane and endoplasmic reticulum; however, it was not detected in the mitochondria. Cellular branching in neuronal cells and in a previously standardized neurosphere assay is dependent on the recycling of vitamin C or reduction in dehydroascorbic acid (DHA, produced by neurons) by glial cells. The effect of WZB117, a selective glucose/DHA transporter 1 (GLUT1) inhibitor expressed in glial cells, was also studied. By inhibiting GLUT1 glial cells, a loss of branching is observed in vitro, which is reproduced in the cerebral cortex in situ. We concluded that vitamin C recycling between neurons and astrocyte-like cells is fundamental to maintain neuronal differentiation in vitro and in vivo. The recycling activity begins at the cerebral postnatal cortex when neurons increase SVCT2 expression and concomitantly, GLUT1 is expressed in glial cells.

Morphological neurite changes induced by porcupine inhibition are rescued by Wnt ligands.

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.

Vitamin C Recycling Regulates Neurite Growth in Neurospheres Differentiated In Vitro.

The reduced form of vitamin C, ascorbic acid (AA), has been related with gene expression and cell differentiation in the cerebral cortex. In neurons, AA is mainly oxidized to dehydroascorbic acid (DHA); however, DHA cannot accumulate intracellularly because it induces metabolic changes and cell death. In this context, it has been proposed that vitamin C recycling via neuron-astrocyte coupling maintains AA levels and prevents DHA parenchymal accumulation. To date, the role of this mechanism during the outgrowth of neurites is unknown. To stimulate neuronal differentiation, adhered neurospheres treated with AA and retinoic acid (RA) were used. Neuritic growth was analyzed by confocal microscopy, and the effect of vitamin C recycling (bystander effect) in vitro was studied using different cells. AA stimulates neuritic growth more efficiently than RA. However, AA is oxidized to DHA in long incubation periods, generating a loss in the formation of neurites. Surprisingly, neurite growth is maintained over time following co-incubation of neurospheres with cells that efficiently capture DHA. In this sense, astrocytes have high capacity to recycle DHA and stimulate the maintenance of neurites. We demonstrated that vitamin C recycling in vitro regulates the morphology of immature neurons during the differentiation and maturation processes.

Effects of Liposomes Contained in Thermosensitive Hydrogels as Biomaterials Useful in Neural Tissue Engineering.

Advances in the generation of suitable thermosensitive hydrogels for the delivery of cells in neural tissue engineering demonstrate a delicate relationship between physical properties and capabilities to promote cell proliferation and differentiation. To improve the properties of these materials, it is possible to add liposomes for the controlled release of bioactive elements, which in turn can affect the physical and biological properties of the hydrogels. In the present investigation, different hydrogels based on Pluronic F127 have been formulated with the incorporation of chitosan and two types of liposomes of two different sizes. The rheological and thermal properties and their relation with the neurite proliferation and growth of the PC12 cell line were evaluated. Our results show that the incorporation of liposomes modifies the properties of the hydrogels dependent on the concentration of chitosan and the lipid type in the liposomes, which directly affect the capabilities of the hydrogels to promote the viability and differentiation of PC12 cells.

Serotonin (5-HT) regulates neurite outgrowth through 5-HT1A and 5-HT7 receptors in cultured hippocampal neurons.

Serotonin (5-HT) production and expression of 5-HT receptors (5-HTRs) occur early during prenatal development. Recent evidence suggests that, in addition to its classical role as a neurotransmitter, 5-HT regulates neuronal connectivity during mammalian development by modulating cell migration and neuronal cytoarchitecture. Given the variety of 5-HTRs, researchers have had difficulty clarifying the specific role of each receptor subtype in brain development. Signalling mediated by the G-protein-coupled 5-HT1A R and 5-HT7 R, however, has been associated with neuronal plasticity. Thus, we hypothesized that 5-HT promotes neurite outgrowth through 5-HT1A R and 5-HT7 R. The involvement of 5-HT1A R and 5-HT7 R in the morphology of rat hippocampal neurons was evaluated by treating primary cultures at 2 days in vitro with 5-HT and specific antagonists for 5-HT1A R and 5-HT7 R (WAY-100635 and SB269970, respectively). The stimulation of hippocampal neurons with 100 nM 5-HT for 24 hr produced no effect on either the number or the length of primary neurites. Nonetheless, after 5HT7 R was blocked, the addition of 5-HT increased the number of primary neurites, suggesting that 5HT7 R could inhibit neuritogenesis. In contrast, 5-HT induced secondary neurite outgrowth, an effect inhibited by 1 µM WAY-100635 or SB269970. These results suggest that both serotonergic receptors participate in secondary neurite outgrowth. We conclude that 5-HT1A R and 5-HT7 R regulate neuronal morphology in primary hippocampal cultures by promoting secondary neurite outgrowth.

Avaliação clínico-epidemiológica em pacientes multibacilares em uma unidade de referência de hansenologia da amazônia / Clinical and epidemiologycal evaluation of multibacillary patients from a reference center for leprosy in the brazilian amazon

O mycobacterium leprae (bacilo de hansen) possui propriedades imunogênicas especiais, responsáveis pelo alto poder incapacitante da hanseníase. Objetivou-se estudar o perfil clínico-epidemiológico de pacientes hansenianos multibacilares, de acordo com a classificação de madri, correlacionar o índice baciloscópico com o número de troncos afetados pela neurite franca no início e término do tratamento, correlacionar o grau de incapacidade com a forma clínica, à entrada e à saída desses pacientes. Selecionaram-se 158 prontuários de pacientes diagnosticados com hanseníase multibacilar, avaliados pelo exame baciloscópico e neurológico. O estudo foi realizado no centro de referência em dermatologia sanitária dr. Marcelo cândia, em marituba, pará, brasil. Desses pacientes, 52% estavam na faixa etária de 15 a 54 anos, 80,4% eram do sexo masculino, 80% tiveram alta por cura e 84% eram casos novos. A forma clínica predominante foi a dimorfa, com 68% dos casos. A forma virchowiana (mhv) apresentou maior número de pacientes com grau de incapacidade ii. A presença de incapacidade grau zero foi estatisticamente significante na forma dimorfa (mhd), que possui aproximadamente 2,69 vezes maior probabilidade de evoluir para neurite que a mhv. Os nervos periféricos mais afetados foram: o tibial posterior, o ulnar, o fibular e o mediano. Conclui-se que a forma virchowiana tem maior potencial de produção de incapacidades tipo ii, enquanto que os portadores de mhd evoluem mais vezes para neurite; e que não há diferença no acometimento de troncos nervosos em relação ao índice baciloscópico.

Mycobacterium leprae (hansen's baccillus) displays special immunogenic properties responsible by the high incapacitating power of leprae. The aims of this study were to determine the clinical-epidemic profile of multibacillar leprosum patients according to madri's classification, correlate the baciloscopic index with the amount of nerve trunks affected by the classic neuritis prior and post-treatment, and correlate the inability degree of this patient with the clinical form at entrance and outcome. Medical records from 158 selected subjects with multibacilar leprosy from the center of reference in sanitary dermatology dr. Marcelo cândia, in marituba, pará, brazil were accessed to evaluate the baciloscopic and neurological exam. . Fifth two percent of the patients were in the range of 15 to 54 years, 80.4% Were male, 80% reach outcome for cure, and 84% were new cases. The predominant clinical form was dimorfa, comprising 68% of the cases. The virchowian form (mhv) was present in the majority of subjects with degree ii of incapacity, the presence of degree zero of incapacity was statistically significant in the dimorfa form (mhd), whose subjects displayed around 2.69 Fold more chance of evolving with neuritis symptoms than mhv. The most affected peripheric nerves at the moment and during the diagnosis were: tibia posterior, ulnar, fibula, and the median nerve. In conclusion, mhv is at greater potential to develop degree ii of incapacity, whereas mhd barriers more frequently evolve to neuritis, and there is no difference among the nervous trunks affected in regard to the bacterious index.

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.