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STEM CELL Session Adult neural stem cells (ANSCs) from the subventricular zone (SVZ) of wobbler motor neuron disease mouse as a model to study the mechanisms leading to neurodegeneration. Botti Francesca1, Ottolina Arianna2, Mennini Tiziana3, Parati Eugenio2, Curti Daniela1. 1Dept. Cellular and Molecular Physiological and Pharmachological Sciences, University of Pavia, Pavia; 2Institute of Neurology “G Besta”, Dept. of Neuropharmacology, via Celoria, Milan; 3Lab. Receptor Pharmacology, “MarioNegri” Institute for Pharmacological Reasearch, via Eritrea 62, Milan, Italy. [email protected] The wobbler mouse bears a spontaneous recessive mutation of the gene encoding for the Vps54 membrane-trafficking protein (1). The homozygous mice develop progressive motor dysfunction with loss of motor neurons and astrogliosis, particularly in the cervical tract of the spinal cord. Astrogliosis is characterised by profound metabolic an morphological changes of astrocytes both in vivo and in primary cultures (2). We examined the adult neural stem cells (ANSCs), obtained from the subventricular zone (SVZ) of wobbler (wb) and disease free mice (ctr) of the same strain, for the ability to proliferate, the neurospheres forming capacity and their multipotentiality at low and high passages and in 4 different culturing conditions. Dissociated ctr and wb ANSCs, in medium containing growth factors, generated heterogeneous free-floating neurospheres. The proliferation rate of wb ANSCs was significantly lower after passage 4 (P4) than that of ctr ANSCs. Both at low (LP) and high passages (HP), the majority of differentiated ANSCs were astrocytes. However, in all cultural conditions used, at 5 days of differentiation, a higher number of neurons (tubulin III+ cells) were observed in the wb cultures in comparison with ctr ones. At LP the number of oligodendrocytes (O4+ cells) was similar whereas at HP it was higher in wb cultures respect to ctr cultures. Morphological differences between ctr and wb differentiated glial cells were seen at HP. A large fraction of wb astrocytes displayed an enlarged soma, short processes and strong immunoreactivity to glial fibrillar acidic protein (GFAP). The mitochondrial energy state of neurospheres and of GFAP+ cells differentiated from ctr and wb ANSCs was evaluated with the carbocyanine dye JC-1 and with laser scanning confocal microscopy (3). No difference was found between the mitochondrial membrane potential of ctr and wb cells in the neurospheres whereas, alterations were found in differentiated astrocytes both in basal conditions and in response to an uncoupler of oxidative phosphorylation (FCCP). In conclusion, the multipotentiality of ANSCs from the SVZ of wobbler motor neuron disease mice is preserved. However, the proliferative capacity is reduced, neurogenesis is increased and the astrocytes differentiated from the wobbler ANSCs display alterations resembling those observed in primary cultures of adult wobbler astrocytes. Our results suggest a primary glial defect in the wobbler pathology but we are currently studing the link between vps54 defects and motor neuron degeneration. (1) Schmitt-John T, Drepper C, Mussmann A, Hahn P, Kuhlmann M, Thiel C, Hafner M, Lengeling A, Heimann P, Jones JM, Meisler MH, Jockusch H. 2005. Mutation of Vps54 causes motor neuron disease and defective spermiogenesis in the wobbler mouse. Nat Genet 37:1213-1215. (2) Hantaz-Ambroise D, Aı¨t Ikhlef CJ, Parmentier C, Leclerc P, Cambier D, Zadigue G, Rieger F. 2001. Specific features of chronic astrocyte gliosis after experimental central nervous system (CNS) xenografting and in Wobbler neurological mutant CNS. Differentiation 69:100–107 (3) Bez A, Corsini E, Curti D, Biggiogera M, Colombo A, Nicosia RF, Pagano SF, Parati EA. 2003. Neurosphere and neurosphere-forming cells: morphological and ultrastructural characterization. Brain Res. 993:18-29.