Fast and Sustained Axonal Growth by BDNF Released from Chitosan Microspheres.

Brain-derived neurotrophic factor (BDNF) regulates dendritic branching and dendritic spine morphology, as well as synaptic plasticity and long-term potentiation. Consequently, BDNF deficiency has been associated with some neurological disorders such as Alzheimer's, Parkinson's or Huntington's diseases. In contrast, elevated BDNF levels correlate with recovery after traumatic central nervous system (CNS) injuries. The utility of BDNF as a therapeutic agent is limited by its short half-life in a pathological microenvironment and its low efficacy caused by unwanted consumption of non-neuronal cells or inappropriate dosing. Here, we tested the activity of chitosan microsphere-encapsulated BDNF to prevent clearance and prolong the efficacy of this neurotrophin. Neuritic growth activity of BDNF release from chitosan microspheres was observed in the PC12 rat pheochromocytoma cell line, which is dependent on neurotrophins to differentiate via the neurotrophin receptor (NTR). We obtained a rapid and sustained increase in neuritic out-growth of cells treated with BDNF-loaded chitosan microspheres over control cells (p < 0.001). The average of neuritic out-growth velocity was three times higher in the BDNF-loaded chitosan microspheres than in the free BDNF. We conclude that the slow release of BDNF from chitosan microspheres enhances signaling through NTR and promotes axonal growth in neurons, which could constitute an important therapeutic agent in neurodegenerative diseases and CNS lesions.

Unraveling the Structural Landscape of Chitosan-Based Heparan Sulfate Mimics Binding to Growth Factors: Deciphering Structural Determinants for Optimal Activity.

Chitosan sulfates have demonstrated the ability to mimic heparan sulfate (HS) function. In this context, it is crucial to understand how the specific structural properties of HS domains determine their functionalities and biological activities. In this study, several HS-mimicking chitosans have been prepared to mimic the structure of HS domains that have proved to be functionally significant in cell processes. The results presented herein are in concordance with the hypothesis that sulfated chitosan-growth factor (GF) interactions are controlled by a combination of two effects: the electrostatic interactions and the conformational adaptation of the polysaccharide. Thus, we found that highly charged O-sulfated S-CS and S-DCS polysaccharides with a low degree of contraction interacted more strongly with GFs than N-sulfated N-DCS, with a higher degree of contraction and a low charge. Finally, the evidence gathered suggests that N-DCS would be able to bind to an allosteric zone and is likely to enhance GF signaling activity. This is because the bound protein remains able to bind to its cognate receptor, promoting an effect on cell proliferation as has been shown for PC12 cells. However, S-CS and S-DCS would sequester the protein, decreasing the GF signaling activity by depleting the protein or locally blocking its active site.

Inhibition of glial proliferation, promotion of axonal growth and myelin production by synthetic glycolipid: A new approach for spinal cord injury treatment.

PURPOSE: After spinal cord injury (SCI) a glial scar is generated in the area affected that forms a barrier for axon growth and myelination, preventing functional recovery. Recently, we have described a synthetic glycolipid (IG20) that inhibited proliferation of human glioma cells. We show now that IG20 inhibited the proliferation of astrocytes and microglial cells, the principal cellular components of the glial scar, and promoting axonal outgrowth and myelin production in vitro. METHODS: Glial cells were inhibited with IG20 (IC50≈10 µM) and studied by RT-PCR, Western Blotting, immunoprecipitation and fluorescence microscopy. Axonal outgrowth in dorsal root ganglia (DRG) and myelin production by oligodendrocytes were analyzed by immunocytochemistry. Adult rats were assayed in spinal cord contusion model and the recovery of treated animals (n = 6) and controls (n = 6) was followed. RESULTS: The IG20 was localized in the cytosol of glial cells, forming a complex with RhoGDIα, a regulator of RhoGTPases. Treatment of astroglial cultures with IG20 increase the expression of BDNF receptor genes (TrkBT1, TrkB Full). IG20 reduced the astroglial marker GFAP, while increasing production of myelin basic protein in oligodendrocytes and promoted axonal outgrowth from DRG neurons. Local injection of IG20, near a spinal cord contusion, promoted the recovery of lesioned animals analyzed by BBB test (P <  0.05). CONCLUSIONS: We propose that inhibition of astrocytes and microglia by IG20 could be diminished the glial scar formation, inducing the re-growth and myelination of axons, these elements constitute a new approach for SCI therapy.

Mass Spectrometry in Pharmacokinetic Studies of a Synthetic Compound for Spinal Cord Injury Treatment.

The studies of drugs that could constitute a palliative to spinal cord injury (SCI) are a continuous and increasing demand in biomedicine field from developed societies. Recently we described the chemical synthesis and antiglioma activity of synthetic glycosides. A synthetic sulfated glycolipid (here IG20) has shown chemical stability, solubility in polar solvents, and high inhibitory capacity over glioma growth. We have used mass spectrometry (MS) to monitor IG20 (m/z = 550.3) in cells and tissues of the central nervous system (CNS) that are involved in SCI recovery. IG20 was detected by MS in serum and homogenates from CNS tissue of rats, though in the latter a previous deproteinization step was required. The pharmacokinetic parameters of serum clearance at 24 h and half-life at 4 h were determined for synthetic glycoside in the adult rat using MS. A local administration of the drug near of spinal lesion site is proposed.

NFAT transcription factors regulate survival, proliferation, migration, and differentiation of neural precursor cells.

The study of factors that regulate the survival, proliferation, and differentiation of neural precursor cells (NPCs) is essential to understand neural development as well as brain regeneration. The Nuclear Factor of Activated T Cells (NFAT) is a family of transcription factors that can affect these processes besides playing key roles during development, such as stimulating axonal growth in neurons, maturation of immune system cells, heart valve formation, and differentiation of skeletal muscle and bone. Interestingly, NFAT signaling can also promote cell differentiation in adults, participating in tissue regeneration. The goal of the present study is to evaluate the expression of NFAT isoforms in NPCs, and to investigate its possible role in NPC survival, proliferation, migration, and differentiation. Our findings indicate that NFAT proteins are active not only in neurogenic brain regions such as hippocampus and subventricular zone (SVZ), but also in cultured NPCs. The inhibition of NFAT activation with the peptide VIVIT reduced neurosphere size and cell density in NPC cultures by decreasing proliferation and increasing cell death. VIVIT also decreased NPC migration and differentiation of astrocytes and neurons from NPCs. In addition, we identified NFATc3 as a predominant NFAT isoform in NPC cultures, finding that a constitutively-active form of NFATc3 expressed by adenoviral infection reduces NPC proliferation, stimulates migration, and is a potent inducer of NPC differentiation into astrocytes and neurons. In summary, our work uncovers active roles for NFAT signaling in NPC survival, proliferation and differentiation, and highlights its therapeutic potential for tissue regeneration.

Synthetic glycolipids for glioma growth inhibition developed from neurostatin and NF115 compound.

Neurostatin, a natural glycosphingolipid, and NF115, a synthetic glycolipid, are inhibitors of glioma growth. While neurostatin shows high inhibitory activity on gliomas its abundance is low in mammalian brain. On the contrary NF115 exhibits less inhibitory activity on gliomas, but could be prepared by chemical synthesis. In this study we describe synthetic compounds, structurally related to NF115, capable of inhibiting glioma growth at low micromolar range. We used DNA microarray technology to compare the genes inhibited in U373-MG human glioma cells after treatment with the natural or synthetic inhibitor. New synthetic compounds were developed to interact with the product of Rho GDP dissociation inhibitor alpha gene, which was repressed in both treatments. Compounds that were inhibitors of glioma cell growth in assays for [3H]-thymidine incorporation were then injected in C6 tumor bearing rats and the tumor size in each animal group were measured. The GC-17, GC-4 and IG-5 are new compounds derived from NF115 and showed high antiproliferative activity on tumor cell lines. The GC-17 compound inhibited U373-MG glioblastoma cells (3.2 µM), the effects was fifty times more potent than NF115, and caused a significant reduction of tumor volume (P<0.05) when tested in Wistar rats allotransplanted with C6 glioma cells.

Neural differentiation of transplanted neural stem cells in a rat model of striatal lacunar infarction: light and electron microscopic observations.

The increased risk and prevalence of lacunar stroke and Parkinson's disease (PD) makes the search for better experimental models an important requirement for translational research. In this study we assess ischemic damage of the nigrostriatal pathway in a model of lacunar stroke evoked by damaging the perforating arteries in the territory of the substantia nigra (SN) of the rat after stereotaxic administration of endothelin-1 (ET-1), a potent vasoconstrictor peptide. We hypothesized that transplantation of neural stem cells (NSCs) with the capacity of differentiating into diverse cell types such as neurons and glia, but with limited proliferation potential, would constitute an alternative and/or adjuvant therapy for lacunar stroke. These cells showed neuritogenic activity in vitro and a high potential for neural differentiation. Light and electron microscopy immunocytochemistry was used to characterize GFP-positive neurons derived from the transplants. 48 h after ET-1 injection, we characterized an area of selective degeneration of dopaminergic neurons within the nigrostriatal pathway characterized with tissue necrosis and glial scar formation, with subsequent behavioral signs of Parkinsonism. Light microscopy showed that grafted cells within the striatal infarction zone differentiated with a high yield into mature glial cells (GFAP-positive) and neuron types present in the normal striatum. Electron microscopy revealed that NSCs-derived neurons integrated into the host circuitry establishing synaptic contacts, mostly of the asymmetric type. Astrocytes were closely associated with normal small-sized blood vessels in the area of infarct, suggesting a possible role in the regulation of the blood brain barrier and angiogenesis. Our results encourage the use of NSCs as a cell-replacement therapy for the treatment of human vascular Parkinsonism.

Lipid and ganglioside alterations in tumor cells treated with antimitotic oleyl glycoside.

Oleyl 2-acetamido-2-deoxy-α-D-glucopyranoside (1) was previously shown to exhibit antimitotic activity on glioma (C6) and melanoma (A375) cell lines. Preliminary studies about its mechanism of action using (1)H MAS NMR suggested that 1 may be altering the metabolism of lipids. We have now studied the effect of 1 on the fatty acid, sphingolipid and ganglioside content in a line of carcinomic human alveolar epithelial cells (A549) using UPLC-MS. Oleic acid and NB-DNJ were used as positive controls for inhibition of fatty acid and ganglioside synthesis, respectively. Compound 1 (10 µM) was more efficient than oleic acid in reducing fatty acid levels of A549 cells, producing a decrease in the range of 40-15%, depending on the acyl chain length and the number of insaturations. In addition, glycoside 1 caused a reduction on ganglioside content of A549 tumor cell line and accumulation of lactosylceramide, the common metabolic precursor for ganglioside biosynthesis. Alteration of ganglioside metabolism was also observed with two galactosylated derivatives of 1, which caused a more pronounced increase in lactosylceramide levels. Compound 1 at higher concentrations (above 30 µM) produced drastic alterations in glycosphingolipid metabolism, leading to cell metabolic profiles very different from those obtained at 10 µM. These biochemical changes were ascribed to activation of endoplasmic reticulum stress pathways.

Detection of metabolite changes in C6 glioma cells cultured with antimitotic oleyl glycoside by 1H MAS NMR.

The synthetic glycoside, oleyl N-acetyl-alpha-D-glucosaminide (1), was previously shown to exhibit antimitotic activity on rat (C6) and human (U-373) glioma lines. To obtain information about its mechanism of action, metabolite changes in C6 glioma cells were analyzed after treatment with 1 using high-resolution magic angle spinning (1)H NMR. Compound 1 caused either a decrease or an increase in the intensity of the signal assigned to coenzyme A (CoA) metabolites depending on the concentration used. The data obtained from the (1)H NMR spectra of cells cultured with 1, combined with those obtained after treatment with oleic acid (an inhibitor of acetyl-CoA carboxylase) and phenyl butyrate (a known antineoplastic agent), suggest that 1 may be altering the metabolism of fatty acids and induce apoptosis of C6 glioma cells. These results point to NMR spectroscopy as an efficient technique for monitoring the response of the cells to therapeutic agents.

Design and synthesis of glycoside inhibitors of glioma and melanoma growth.

An N-acetylglucosaminide derivative with a pentaerythritol substituent at position C-6 was previously synthesized and shown to inhibit neural tumor growth. Now, we report the preparation of a series of new synthetic compounds introducing systematic changes in the nature, polarity, and size of the sugar substituents. The antimitotic activity of the new compounds was tested on cultured rat (C6) and human (U-373) glioma lines and on a human melanoma line (A-375). The antimitotic and antitumoral activity of the new compounds on glioma cell lines increased up to 2 orders of magnitude with respect to the parent compound or was abolished, permitting a detailed structure-function analysis of the new antitumorals. One of the glycosides inhibited melanoma division with an ID50 below the micromolar range.

Gelation under dynamic conditions: a strategy for in vitro cell ordering.

Ordered gelation under spin-coating conditions, as reported here, is a suitable method to order cells in biogels. Cell ordering is of great importance for functional repair of central nervous system (CNS) injuries, because therapies must include strategies to bridge chystic gaps and facilitate axon growth towards its target. Organized biocompatible and biodegradable substrates may be used for this purpose, to supply trophic support and provide directional cues for neuronal process outgrowth. Atomic force microscopy (AFM) and low temperature scanning electron microscopy (LTSEM), confirmed that fibrils in kappa-carrageenan/chitosan and fibrin hydrogels prepared under spin-coating conditions, were longitudinally arranged. The cell model was conveniently tested using rat C6 glioma cells. C6 cells were distributed regularly in fibrin gels formed under centrifugal force. The ability of ordered fibrin scaffolds to promote uniform distribution of transplanted cells, was confirmed by fluorescence microscopy.

Natural, synthetic and semisynthetic glycolipid inhibitors of glioma growth.

Neurostatin, a natural glycosphingolipid inhibitor of astroblast and astrocytoma division, present in mammalian brain, is the modified ganglioside O-acetylated GD1b. It is cytostatic for rat astroblasts, C6 glioma cells and various human astrocytoma lines grades III and IV, with median inhibitory dose values ranging 1 - 5 µM. Neurostatin was shown not to affect primary or transformed fibroblast division at concentrations of ≥ 10 µM. A synthetic neurostatin analogue, NF-115, consisting of an octyl N-acetylglucosaminide derivative with a pentaerythritol chain at position 6, loaded on a slow-delivery polymer disc, caused the destruction of cultured human astroblastoma, obtained after surgical biopsy, and destroyed human neuroectodermic tumours implanted in rats and human astrocytoma implanted in immunodeficient mice. Future antitumourals should combine the high activity of neurostatin with the ease of synthesis of NF-115.