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.

Vitamin C Recycling Regulates Neurite Growth in Neurospheres Differentiated in Vitro

Background: The reduced form of vitamin C, ascorbic acid (AA), has been related to antioxidant defense as well as 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.Methods: 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 (astrocytes, HL60 and U87). Reactive oxygen species (ROS) generation was also analyzed by flow cytometry and protein carbonylation / carboximetil-lysine production.Results: 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 (HL60, U87, or astrocytes) that efficiently capture DHA (Bystander effect). In this sense, astrocytes have high capacity to recycle DHA and stimulate the maintenance of neurites. Finally, our data indicate that DHA induces ROS generation, a condition that results in protein carbonylation and carboximetil-lysine production. Conclusions: We have demonstrated that vitamin C recycling in vitro regulates the morphology of immature neurons during the differentiation and maturation processes.

AutoNeuriteJ: An ImageJ plugin for measurement and classification of neuritic extensions

Morphometry characterization is an important procedure in describing neuronal cultures and identifying phenotypic differences. This task usually requires labor-intensive measurements and the classification of numerous neurites from large numbers of neurons in culture. To automate these measurements, we wrote AutoNeuriteJ, an imageJ/Fiji plugin that measures and classifies neurites from a very large number of neurons. We showed that AutoNeuriteJ is able to detect variations of neuritic growth induced by several compounds known to affect the neuronal growth. In these experiments measurement of more than 5000 mouse neurons per conditions was obtained within a few hours. Moreover, by analyzing mouse neurons deficient for the microtubule associated protein 6 (MAP6) and wild type neurons we illustrate that AutoNeuriteJ is capable to detect subtle phenotypic difference in axonal length. Overall the use of AutoNeuriteJ will provide rapid, unbiased and accurate measurement of neuron morphologies.

Inhibition of Biosynthesis and Biochemical Modulation of N-Acylneuraminic Acid (Biochemical Engineering of Sialoconjugates). A Review

The key enzyme of sialic acid biosynthesis is the bifunctional UDP-GlcNAc 2-epimerase/ ManNAc kinase. Novel inhibitors of this enzyme have been synthesized. TheN-acyl side chain of sialic acid can be biochemically engineered by incubating cells with non-naturalN-acylmannosamine analogues such asN-propionylmannosamine and related compounds. These modified sialic acids lead to various biological changes, such as stimulation of T-lymphocyte proliferation, inhibition of the uptake of influenza A virus, stimulation of neuritic growth, increased expression of sialyl-Lewisxand altered adhesion. A review with 41 references.

Modulators of axonal growth and guidance at the brain midline with special reference to glial heparan sulfate proteoglycans

Bilaterally symmetric organisms need to exchange information between the left and right sides of their bodies to integrate sensory input and to coordinate motor control. Thus, an important choice point for developing axons is the Central Nervous System (CNS) midline. Crossing of this choice point is influenced by highly conserved, soluble or membrane-bound molecules such as the L1 subfamily, laminin, netrins, slits, semaphorins, Eph-receptors and ephrins, etc. Furthermore, there is much circumstantial evidence for a role of proteoglycans (PGs) or their glycosaminoglycan (GAG) moieties on axonal growth and guidance, most of which was derived from simplified models. A model of intermediate complexity is that of cocultures of young neurons and astroglial carpets (confluent cultures) obtained from medial and lateral sectors of the embryonic rodent midbrain soon after formation of its commissures. Neurite production in these cocultures reveals that, irrespective of the previous location of neurons in the midbrain, medial astrocytes exerted an inhibitory or non-permissive effect on neuritic growth that was correlated to a higher content of both heparan and chondroitin sulfates (HS and CS). Treatment with GAG lyases shows minor effects of CS and discloses a major inhibitory or non-permissive role for HS. The results are discussed in terms of available knowledge on the binding of HSPGs to interative proteins and underscore the importance of understanding glial polysaccharide arrays in addition to its protein complement for a better understanding of neuron-glial interactions.