Stem cells for amyotrophic lateral sclerosis modeling and therapy: myth or fact?

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease whose pathophysiology is poorly understood. Aiming to better understand the cause of motor neuron death, the use of experimental cell-based models increased significantly over the past years. In this scenario, much knowledge has been generated from the study of motor neurons derived from embryonic stem cells and induced pluripotent stem cells. These methods, however, have advantages and disadvantages, which must be balanced on experimental design. Preclinical studies provide valuable information, making it possible to combine diverse methods to build an expanded knowledge of ALS pathophysiology. In addition to using stem cells as experimental models for understanding disease mechanism, these cells had been quoted for therapy in ALS. Despite ethical issues involved in its use, cell therapy with neural stem cells stands out. A phase I clinical trial was recently completed and a phase II is on its way, attesting the method's safety. In another approach, mesenchymal stromal cells capable of releasing neuroregulatory and anti-inflammatory factors have also been listed as candidates for cell therapy for ALS, and have been admitted as safe in a phase I trial. Despite recent advances, application of stem cells as an actual therapy for ALS patients is still in debate. Here, we discuss how stem cells have been useful in modeling ALS and address critical topics concerning their therapeutic use, such as administration protocols, injection site, cell type to be administered, type of transplantation (autologous vs. allogeneic) among other issues with particular implications for ALS therapy.

Ultrastructural characterization of CD133+ stem cells bound to superparamagnetic nanoparticles: possible biotechnological applications.

CD133 antigen is an integral membrane glycoprotein that can bind with different cells. Originally, however, this cellular surface antigen was expressed in human stem cells and in various cellular progenitors of the haematopoietic system. Human cord blood has been described as an excellent source of CD133(+) haematopoietic progenitor cells with a large application potential. One of the main objectives of the present study is to describe for the first time the ultrastructural characteristics of CD133(+) stem cells using transmission electronic microscopy. Another objective of the manuscript is to demonstrate through transmission electronic microscopy the molecular image of magnetic nanoparticles connected to the stem cells of great biotechnological importance, as well as demonstrating the value of this finding for electronic paramagnetic resonance and its related nanobioscientific value. Ultrastructural results showed the monoclonal antibody anti-CD133 bound to the superparamagnetic nanoparticles by the presence of electrondense granules in cell membrane, as well as in the cytoplasm, revealing the ultrastructural characteristics of CD133(+) cells, exhibiting a round morphology with discrete cytoplasmic projections, having an active nucleus that follows this morphology. The cellular cytoplasm was filled up with mitochondrias, as well as microtubules and vesicles pinocitic, characterizing the process as being related to internalization of the magnetic nanoparticles that were endocyted by the cells in question. Electronic paramagnetic resonance analysis of the CD133(+) stem cells detected that the signal (spectrum) generated by the labelled cells comes from the superparamagnetic nanoparticles that are bound to them. These results strongly suggest that these CD133(+) cells can be used in nanobiotechnology applications, with benefits in different biomedical areas.

Changes in superoxide dismutase activity and photosynthetic pigment content during growth of marine phytoplankters in batch-cultures.

The ability of phytoplankton to cope with oxidative stress is one of the main factors that influence its survival in the marine environment, when senescence conditions prevail. In a first attempt to investigate the antioxidant strategies of different phytoplanktonic groups face to oxidative stress, the superoxide dismutase (SOD; EC 1.15.1.1) activity and photosynthetic pigment content along the growth curves of the dinoflagellate Lingulodinium polyedrum (Stein) Dodge, the prasinophycean Tetraselmis gracilis (Kylin) Butcher and the diatom Minutocellus polymorphus (Hargraves and Guillard) Hasle, von Stosch and Syvertsen were evaluated in batch-cultures. Total SOD activity was determined by an indirect method involving the inhibition of cytochrome c reduction. The contents of photosynthetic pigments were analysed by HPLC using a reverse phase column (RP-18), based on a ternary gradient. A peak of total SOD activity was detected at the beginning of the T. gracilis and M. polymorphus exponential growth. In L. polyedrum and M. polymorphus, SOD activity increased approximately three times by day 17 of growth, compared to the values obtained on day 3 (exponential phase) of the growth curve. All three species of microalgae had reduced SOD activity at the end of their growth. The levels of peridinin in L. polyedrum increased about 60% by day 17 of growth compared to the values obtained at exponential phase. Tetraselmis gracilis exhibited a remarkable increase (approximately 85%) in beta-carotene concentration after 10-14 days of growth whereas the beta-carotene levels in M. polymorphus decreased about 85% along its growth curve. These findings suggest that the antioxidant response during senescence in batch-cultures differ according to the species. Induction of SOD activity may occur either in the early exponential or stationary growth phases, which is important to prevent oxidative stress triggered by a number of factors that affects growth, such as nutrient and light availability.

Members of a dinoflagellate luciferase gene family differ in synonymous substitution rates.

Regulation and evolution of dinoflagellate luciferases are of particular interest since the enzyme is structurally unique and bioluminescence is under circadian control. In this study, three new members of the dinoflagellate luciferase gene family were identified and characterized from Pyrocystis lunula. These genes, lcfA, lcfB, and lcfC, also exhibit the unusual structure and organization previously reported for the luciferase gene of a related dinoflagellate, Lingulodinium polyedrum: three repeated domains, each encoding an active catalytic site, multiple gene copies, and tandem organization. The histidine residues involved in the pH regulation of L. polyedrum luciferase activity, and implicated in the regulation of flashing, are also fully conserved in P. lunula. The interspecific conservation between the individual luciferase domains of P. lunula and L. polyedrum is higher than among domains intramolecularly, indicating that this unique gene structure arose through duplication events that occurred prior to the divergence of these dinoflagellates. However, P. lunula luciferase genes differ from L. polyedrum in several respects, notably, the occurrence of an intron in one gene (lcfC), a 2.25-kb intergenic region connecting lcfA and lcfB, and, of particular interest, an invariant rate of synonymous (silent) substitutions along the repeat domains, in contrast to L. polyedrum luciferase, where the occurrence of synonymous substitutions is practically absent in the central region of the domains.

Different regulatory mechanisms modulate the expression of a dinoflagellate iron-superoxide dismutase.

Regulation of antioxidant enzymes is critical to control the levels of reactive oxygen species in cell compartments highly susceptible to oxidative stress. In this work, we studied the regulation of a chloroplastic iron superoxide dismutase (Fe-SOD) from Lingulodinium polyedrum (formerly Gonyaulax polyedra) under different physiological conditions. A cDNA-encoding Fe-SOD was isolated from this dinoflagellate, showing high sequence similarity to cyanobacterial, algal, and plant Fe-SODs. Under standard growth conditions, on a 12:12-h light-dark cycle, Lingulodinium polyedrum Fe-SOD exhibited a daily rhythm of activity and cellular abundance with the maximum occurring during the middle of the light phase. Northern analyses showed that this rhythmicity is not related to changes in Fe-SOD mRNA levels, indicative of translational regulation. By contrast, conditions of metal-induced oxidative stress resulted in higher levels of Fe-SOD transcripts, suggesting that transcriptional control is responsible for increased protein and activity levels. Daily (circadian) and metal-induced up-regulation of Fe-SOD expression in L. polyedrum are thus mediated by different regulatory pathways, allowing biochemically distinct changes appropriate to oxidative challenges.

Antioxidant modulation in response to metal-induced oxidative stress in algal chloroplasts.

To investigate adaptive responses to metal stress at the subcellular level, the oxidative balance in isolated chloroplasts was evaluated for the first time in the unicellular alga Gonyaulax polyedra exposed to the toxic metals Hg(2+), Cd(2+), Pb(2+), and Cu(2+). Different antioxidant responses were verified according to the metal and model of stress applied. Cells chronically exposed to metals exhibited high activity of the antioxidant enzymes superoxide dismutase and ascorbate peroxidase, high glutathione content, and decrease of peridinin levels, whereas no significant changes were detected for beta-carotene levels. In contrast, cells subjected to acute metal stress displayed twice as much beta-carotene but only a slight increase in superoxide dismutase and ascorbate peroxidase activities. The correlation of acute metal treatment and oxidative stress was inferred from the higher oxygen uptake and decreased reduced glutathione pool found in treated cells. In addition, increased oxidative damage to proteins and lipids occurred mainly in cells under acute stress. Pb(2+) was the most damaging toxicant, causing protein oxidation and lipid peroxidation even at chronic treatment. These results indicate that heavy metals are able to induce oxidative stress in chloroplasts of G. polyedra, particularly under acute conditions. Nevertheless, the maintenance of a high antioxidant capacity within chloroplasts seems to be an important strategy during acclimation of G. polyedra to chronic metal stress. By acting at the subcellular site, where oxidative stress is triggered, induction of such chloroplast antioxidants might be crucial for cell survival during exposure to heavy metals.

Acute and chronic effects of toxic metals on viability, encystment and bioluminescence in the dinoflagellate Gonyaulax polyedra.

Toxicity bioassays based on survival were carried out with cells of the marine dinoflagellate Gonyaulax polyedra exposed to mercury (Hg2+ ), cadmium (Cd2+), lead (Pb2+) and copper (Cu2+). The toxicity scale of these metals found was Hg2+ > Cu2+ > Cd2+ > Pb2+. Cells exposed to metals promptly underwent encystment, which is an important strategy for surviving metal exposure. Following 48 h exposure to Cu2+, complete excystment occurred within 96 h after reinoculation of cells in fresh metal-free media, and with Pb2+ partial recovery occurred in that time. Bioluminescence was affected by the metals in a dose-dependent manner primarily by increasing the frequency of flashing, but the glow emission was also altered with acute Cu2+ and Pb2+ treatments. Several physiological processes in G. polyedra are under circadian control. Chronic exposures to metals caused no substantial alterations in the circadian rhythm of bioluminescence glow, indicating that the biological clock of this dinoflagellate is not sensitive to these metals at the concentrations tested.

Response of superoxide dismutase to pollutant metal stress in the marine dinoflagellate Gonyaulax polyedra.

The response of superoxide dismutase (SOD) activity in the marine dinoflagellate Gonyaulax polyedra to chronic (5.0 ppb Hg, 0.5 ppm Cd, 2.0 ppm Pb and 0.1 ppm Cu, during 30 days) and acute (10.0 ppb Hg, 1.0 ppm Cd, 5.0 ppm Pb and 0.25 ppm Cu, during 48 hours) exposure to metals was investigated. Under chronic exposure to Hg, Cd, Pb, and Cu, total SOD activity of metal-treated cells increased during the first day of exposure to plateau levels of 134, 148, 127, and 139% of control values respectively. Under acute metal exposure, SOD activity increases were of similar magnitude but much more rapid (within several hours) and of shorter duration. In addition, assays for oxidative damage to lipids revealed high levels of lipid peroxidation in cells kept in either chronic or acute exposure to metals reaching values 2-fold greater than the control group. Changes in SOD activity were dependent on the metal, its concentration, and the time of exposure. Non-denaturing polyacrylamide gels revealed induction of Fe-SOD and Mn-SOD but not Cu-Zn-SOD isoforms in cells kept under acute exposure to metals. These results suggest that oxidative stress may be an important mediator of metal toxicity in algal systems, with SOD providing antioxidant protection.

The effect of light on the biosynthesis of beta-carotene and superoxide dismutase activity in the photosynthetic alga Gonyaulax polyedra.

Daily oscillations of both beta-carotene and superoxide dismutase (SOD) activity are related to the intracellular control of reactive oxygen species (ROS). It is well established that ROS are present in all aerobic cells. We studied the marine dinoflagellate Gonyaulax polyedra which has been extensively used as a model to understand the biological clock at the molecular level. beta-Carotene, besides suppressing singlet molecular oxygen (1O2), may act as a photoreceptor pigment in many photosynthetic cells. The levels of beta-carotene during the day phase were shown to be twice as high as during the night phase. The dose-response curve for light-induced carotenoid synthesis was linear for up to 45 min of light exposure, after which night phase cells contained the same levels of beta-carotene as day phase cells. Cells exposed to light pulses at different times during the dark period displayed the highest beta-carotene induction in the middle of the night. SOD activity of cell-free extracts of G. polyedra was three to four times higher during the day. This rhythm continued in cells kept in constant light, indicating that the regulation can be attributed to the cellular circadian clock. No-denaturing polyacrylamide gels revealed the presence of several SOD isoenzymes in G. polyedra, including CuZnSOD and MnSOD. Furthermore, G. polyedra SOD cross-reacts with a polyclonal antibody raised against SOD. In addition to being gene regulated by ROS concentration, G. polyedra SOD expression seems also to be under the control of the biological clock.

The effect of light on the biosynthesis of beta-carotene and superoxide dismutase activity in the photosynthetic alga Gonyaulax polyedra

Daily oscillations of both beta-carotene and superoxide dismutase (SOD) activity are related to the intracellular control of reactive oxygen species (ROS). It is well established that ROS are present in all aerobic cells. We studied the marine dinoflagellate Gonyaulax polyedra which has been extensively used as a model to understand the biological clock at the molecular level. beta-Carotene, besides suppressing singlet molecular oxygen (1O2), may act as a photoreceptor pigment in many photosynthetic cells. The levels of beta-carotene during the day phase were shown to be twice as high as during the night phase. The dose-response curve for light-induced carotenoid synthesis was linear for up to 45 min of light exposure, after which night phase cells contained the same levels of beta-carotene as day phase cells. Cells exposed to light pulses at different times during the dark period displayed the highest beta-carotene induction in the middle of the night. SOD activity of cell-free extracts of G. polyedra was three to four times higher during the day. This rhythm continued in cells kept in constant light, indicating that the regulation can be attributed to the cellular circadian clock. Non-denaturing polyacrylamide gels revealed the presence of several SOD isoenzymes in G. polyedra, including CuZnSOD and MnSOD. Furthermore, G. polyedra SOD cross-reacts with a polyclonal antibody raised against SOD. In addition to being gene regulated by ROS concentration, G. polyedra SOD expression seems also to be under the control of the biological clock.