Download Adult neural stem cells (ANSCs) from the subventricular zone (SVZ)

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.