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A secreted-factor based mechanism for olfactory ensheathing cell-mediated neurite outgrowth

University of British Columbia

The olfactory system continually turns over its neuronal population throughout life. As a consequence, newly generated olfactory receptor neurons (ORNs) grow axons centrally towards the olfactory bulb; thus the olfactory system continually accommodates axon growth and does so employing a unique glial cell type, olfactory ensheathing cells (OECs). Because of their involvement in providing a permissive environment for ORN axon growth, OECs have been employed in repair strategies in other parts of the nervous system, most prominently the injured spinal cord. However, little is known about how OECs promote regeneration. OECs reside in two compartments, the lamina propria and the nerve fibre layer of the olfactory bulb. LP-OECs were examined in vivo and in vitro and compared with what has been reported on olfactory bulb OECs (OB-OECs). LP-OECs are very similar to OB-OECs in vivo and in vitro with several subtle differences. LP-OECs express CD44 in vivo while OB-OECs do not. LP-OECs also proliferate robustly without exogenous mitogens added, suggesting a more immature phenotype. LP-OEC cultures can be purified to greater than 95% and express novel developmentally regulated markers such as p200 and NG2. Purified cultures of LP-OECs were used to elucidate mechanisms of OEC-mediated neurite outgrowth. Using an embryonic dorsal root ganglion (DRG) culture system, LPOECs promoted outgrowth in co-culture and also with LP-OEC conditioned media (LPOCM) alone. LP-OCM from passage 2 and passage 6 LP-OECs were assayed for biological activity using the outgrowth assay and passage 2 LP-OCM was found to have a more effective dose-response curve. Secreted factors underlying this difference in biological activity were identified using isotope coded affinity tags (ICAT) proteomics, which provides the identity and relative quantity. By correlating biological activity with relative quantity, SPARC (secreted protein acidic rich in cysteine) was identified as a candidate factor. Gain- and loss-offunction experiments confirmed the important role of SPARC in the outgrowth activity of LP-OCM. In summary, the work in this thesis characterizes a previously poorly understood cell type and uses it as a model system to prospectively elucidate mechanisms of OEC-mediated neurite outgrowth.

Text

2010-01-05

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http://hdl.handle.net/2429/17489

Neuroscience

University of British Columbia

UBCV

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