Lack of fibulin-3 alters regenerative tissue responses in the primary olfactory pathway
Introduction
The primary olfactory pathway is a unique part of the mammalian nervous system where a small proportion of olfactory sensory neurons (OSNs) is continuously being turned over from basal stem/progenitor cells in order to maintain a receptive field for the sense of smell (e.g. Graziadei et al., 1979, Harding et al., 1977, Leung et al., 2007; for review, see Schwob, 2002). Axons of adult-born, immature OSNs leave the epithelium through gaps in the basement membrane, after which they enter the underlying lamina propria and track through the olfactory nerve towards the olfactory bulb. Here, in the glomerular layer of the olfactory bulb, they can successfully (re-) establish appropriate connections with the dendrites of second-order neurons, i.e. mitral and tufted cells. A similar process, though on a much larger scale, occurs following experimentally-induced en mass OSN turnover (e.g. Cummings et al., 2000).
Olfactory ensheathing cells (OECs) are intimately associated with bundles of OSN axons, from their peripheral origin to the target structures in the central nervous system (e.g. Doucette, 1984, Field et al., 2003). These specialised glial cells are thought to support the growth of new sensory axons, coming from the olfactory epithelium (OE), in a way similar to Schwann cells during peripheral nerve growth and regeneration. OECs upregulate expression of low-affinity nerve growth factor receptor p75 after injury (Gong et al., 1994), and it has been suggested that the cells maintain open channels that provide a passage for de novo growing axons from new OSNs (Li et al., 2005, Williams et al., 2004a, Williams et al., 2004b). The extracellular matrix (ECM) constituents associated with OECs are likely to play an important role in olfactory neurite growth (Gong and Shipley, 1996, Tisay and Key, 1999, Treloar et al., 1996). In this context, we have recently reported that the glycoprotein fibulin-3 (also known as EFEMP-1, S1-5 or T16) is expressed along the primary olfactory pathway and produced by OECs in vitro (Vukovic et al., 2008).
Fibulin-3 belongs to a small family of glycoproteins that normally have widespread distribution in extracellular matrix structures such as basement membranes, microfibrils and elastic fibres (Argraves et al., 2003, Timpl et al., 2003, de Vega et al., 2009). A missense mutation in the fibulin-3 gene sequence has been linked to development of heritable macular degeneration in humans and mouse models (Stone et al., 1999, Fu et al., 2007, Marmorstein et al., 2007). Fibulin-3 deficiency, on the other hand, is hallmarked by reduced reproductivity, herniation, early ageing and reduced lifespan but not macular degeneration (McLaughlin et al., 2007). Additional in vitro work has shown that expression of fibulin-3 is significantly repressed in endothelial cells during capillary formation, i.e. vascular plasticity (Bell et al., 2001) while constitutive expression suppresses tumor angiogenesis (Albig et al., 2006). Based on its interaction with tissue inhibitor of metalloproteinases-3 (TIMP-3; Klenotic et al., 2004), a critical role for fibulin-3 in regulating matrix metalloproteinase (MMP) activity has been proposed (McLaughlin et al., 2007, Rahn et al., 2009). The latter molecules play an important role in matrix remodelling during periods of plasticity and growth.
To date, only three studies have mentioned fibulin-3 expression in context of the nervous system (Barkho et al., 2006; Thalmeier et al., 2008, Vukovic et al., 2008). Barkho et al. (2006) showed that fibulin-3 expression is downregulated in neurogenesis-promoting astrocytes. Our own earlier work has shown that manipulation of fibulin-3 expression in cultured OECs alters proliferation and migration (Vukovic et al., 2008). Specifically, knock-down of fibulin-3 resulted in reduced OEC proliferation whereas transgenic expression resulted in impaired migration of these glial cells. High levels of fibulin-3 also slowed down neurite growth. To further address a putative role for this glycoprotein in olfactory nerve growth and plasticity in vivo, the present study examined the role of fibulin-3 in olfactory nerve regeneration using comparative analysis between wild-type and fibulin-3 deficient (Efemp1−/−) mice.
Two different lesion models were employed: 1) Triton X-100 irrigation of the nasal cavity (Verhaagen et al., 1990) and 2) unilateral ablation of the olfactory bulb (Graziadei et al., 1979, Carr and Farbman, 1992). In both scenarios, rapid death of OSNs occurs in the first few days after injury and degenerating OSNs are phagocytised by infiltrating macrophages (Suzuki et al., 1995). This is then followed by a more prolonged wave of basal cell proliferation and differentiation. Reversible Triton X-100 lesion allows these adult-born OSNs to mature and re-establish connections with target glomeruli in the olfactory bulb, with complete recovery after 6 to 7 weeks (Cummings et al., 2000). In contrast, olfactory bulbectomy does not lead to recovery of the OE to a pre-injury state, there being a persistent increase in epithelial proliferation and cell death (Carr and Farbman, 1992, Verhaagen et al., 1990). Regenerative responses following these lesions were assessed by quantitative counts of olfactory marker protein (OMP) -positive OSNs in the OE at different time points (i.e. 3, 10, 42 days post surgery); OMP selectively stains mature OSNs (Monti-Graziadei et al., 1977). In addition, (re-)innervation of glomeruli following lesioning with Triton X-100 was quantified using densitometric analysis. Changes in vascular structure in the lamina propria underneath the OE in response to injury and fibulin-3 deficiency were also investigated.
Section snippets
Animals
Thirty-six BALB/c mice were used in this study (♂/♀, 8-week old), comprising 18 wild-type mice and 18 fibulin-3 deficient mice (Efemp1−/−; McLaughlin et al., 2007). Animals were housed under standard conditions and maintained on a 12 h light/dark cycle with free access to water and food. Experimental procedures were approved by the animal ethics committees of The University of Western Australia and The University of Arizona, and conformed to NHMRC and NIH guidelines.
Olfactory epithelium lesions
Transient deafferentation of
Fibulin-3 distribution in intact and injured olfactory system in adult mice
The overall distribution of members of the fibulin family of glycoproteins in the adult olfactory system has recently been described (Vukovic et al., 2008). Here the objective was to further characterize fibulin-3 distribution in the lamina propria, especially in relation to blood vessels and the lateral/medial aspects of the olfactory bulb, i.e. the olfactory nerve fibre layer and glomeruli. Fibulin-3 was detected mainly in the form of small microfibrils in close association with the basement
Discussion
We have recently reported that the matrix-associated glycoprotein fibulin-3 is produced by OECs, the glial cells of the olfactory nerve (Vukovic et al., 2008). The present study expands on these observations and is first to examine a putative role for this glycoprotein in vivo during regeneration of the olfactory system. Fibulin-3 protein was detected in association with basement membrane structures of blood vessels and nerve in the connective tissue (i.e. lamina propria) underneath the
Acknowledgments
This work was supported by the School of Anatomy and Human Biology and a Discovery Grant from the Australian Research Council (ARC DP0774113) to MJR. JV received additional support from the following sources: Australian Postgraduate Award, Woodside Neurotrauma Research PhD Excellence Award, Geoffrey Kennedy Postgraduate Research Travel Award and the University Graduate Research Candidate Travel Award (UWA). LYM is supported by NIH/NEI Grant EY13847 and a career development award from Research
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