International audienc

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron death. ALS can be induced by mutations in the superoxide dismutase 1 gene (SOD1). Evidence for the non-cell-autonomous nature of ALS emerged from the observation that wild-type glial cells extended the survival of SOD1 mutant motor neurons in chimeric mice. To uncover the contribution of astrocytes to human motor neuron degeneration, we cocultured hESC-derived motor neurons with human primary astrocytes expressing mutated SOD1. We detected a selective motor neuron toxicity that was correlated with increased inflammatory response in SOD1-mutated astrocytes. Furthermore, we present evidence that astrocytes can activate NOX2 to produce superoxide and that effect can be reversed by antioxidants. We show that NOX2 inhibitor, apocynin, can prevent the loss of motor neurons caused by SOD1-mutated astrocytes. These results provide an assay for drug screening using a human ALS in vitro astrocyte-based cell model.

A battery of monoclonal antibodies (mAbs) against brain cell nuclei has been generated by repeated immunizations. One of these, mAb A60, recognizes a vertebrate nervous system- and neuron-specific nuclear protein that we have named NeuN (Neuronal Nuclei). The expression of NeuN is observed in most neuronal cell types throughout the nervous system of adult mice. However, some major cell types appear devoid of immunoreactivity including cerebellar Purkinje cells, olfactory bulb mitral cells, and retinal photoreceptor cells. NeuN can also be detected in neurons in primary cerebellar cultures and in retinoic acid-stimulated P19 embryonal carcinoma cells. Immunohistochemically detectable NeuN protein first appears at developmental timepoints which correspond with the withdrawal of the neuron from the cell cycle and/or with the initiation of terminal differentiation of the neuron. NeuN is a soluble nuclear protein, appears as 3 bands (46-48 × 10(3) M(r)) on immunoblots, and binds to DNA in vitro. The mAb crossreacts immunohistochemically with nervous tissue from rats, chicks, humans, and salamanders. This mAb and the protein recognized by it serve as an excellent marker for neurons in the central and peripheral nervous systems in both the embryo and adult, and the protein may be important in the determination of neuronal phenotype.

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Alpha-modified minimum essential medium (MEM) has been found to cross-link a 1% gellan gum solution, resulting in the formation of a selfsupporting hydrogel in 1:1 and 5:1 ratios of polysaccharide: MEM. Rheological data from temperature sweeps confirm that in addition to orders of magnitude differences in G 0 between 1% gellan and 1% gellan with MEM, there is also a 20 C increase in the temperature at which the onset of gelation takes place when MEM is present. Frequency sweeps confirm the formation of a true gel; mechanical spectra for mixtures of gellan and MEM clearly demonstrate G 0 to be independent of frequency. It is possible to immobilize cells within a threedimensional (3D) gellan matrix that remain viable for up to 21 days in culture by adding a suspension of rat bone marrow cells (rBMC) in MEM to 1% gellan solution. This extremely simple approach to cell immobilization within 3D constructs, made possible by the fact that gellan solutions cross-link in the presence of millimolar concentrations of cations, poses a very low risk to a cell population immobilized within a gellan matrix and thus indicates the potential of gellan for use as a tissue engineering scaffold.