Matrix metalloproteinase expression in the olfactory epithelium

Abstract

The olfactory epithelium contains neuronal progenitor cells capable of continuous neurogenesis and is a unique model for studying neural degeneration, regeneration, axon outgrowth and recovery from injury. Matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs), have been implicated in cell turnover, development, migration, and metastatic processes. We used Western blot and immunohistochemistry to determine whether MMP-2 and associated proteins TIMP-2 and membrane type 1 matrix metalloproteinase (MT1-MMP) are present in the olfactory epithelium of mice. We found MMP-2 expression localized to the olfactory basal cells and immature neurons. After injury-induced neural degeneration, MMP-2 and MT1-MMP levels decreased while TIMP-2 levels increased. However, following 35 days of neurogenesis and cell replacement TIMP-2 and MT1-MMP returned to control levels. The results show a correlation between MMP and TIMP levels and the stages of neural degeneration, regeneration and recovery of the olfactory epithelium following injury.

Fig.1

Western immunoblot analysis of protein expression in the olfactory epithelium. (a,b) Representative Western blot showing protein expression in control tissue (Ctrl), and changes during degeneration and recovery following bulbectomy. Both the pro form (heavy band) and active form (lighter band) of MMP-2 can be seen in control tissue. Growth-associated protein (GAP-43) and olfactory marker protein (OMP) were used to monitor changes in immature and mature olfactory neurons. (c,d) Plot of densitometric measurements for each protein shown in (a,b) normalization to cyclophyllin A (CPA) and expressed as a percentage of the maximum value. (c) Immediately following bulbectomy MMP-2 and MT1-MMP levels decreasedwhileTIMP-2 levels increased. Beginning at day 11, a gradual recovery in MMP-2 and MT1-MMP was observed and TIMP-2 began to decline. (d) During the first 7 days after bulbectomy, degeneration of the mature olfactory neurons was detected by a decrease in OMP. The increase inGAP-43 levels beginning at day 4 and peaking at day 15 indicates growth of the immature replacement neurons.

Fig.2

Expression and localization of MMP-2, GAP-43 and OMP in the olfactory epithelium of control and bulbectomizedmice. (Control) MMP-2 expression is seen among the basal cells and immature olfactory neurons (arrowheads). Basal cells (asterisks) located adjacent to the basement membrane (BM) did not stain with anti GAP-43 antibodies. Only the immature neurons, their dendrites and axon bundles were GAP-43-positive. OMP identified the population of mature olfactory neurons. (Day 7) After bulbectomy the thickness of the olfactory epithelium (OE) decreased and very few MMP-2 positive cells were observed (arrowhead). At this time point there were many GAP-43-positive cells and only an occasional OMP-positive neuron (Day 15).An increase in the number of MMP-2-positive cells was observed in the basal and immature cell layers (arrowheads) of the epithelium, and just below the basement membrane newly growing axons were observed (arrows).GAP-43-positive cells were present above the layer of MMP-2-positive cells and more OMP-positive cells were present in the epithelium. Tissue sections for each time point shown are near adjacent sections from the nasal septum. Brackets indicate thickness of the epithelium. Bar = 25 µm.

Fig.3

MMP-2 expression associated with axon outgrowth. (a,b) In control animals, MMP-2 expression was observed among basal cells and in the lamina propria (arrows). (b) High magnification of lamina propria shown in (a). (c,d) After recovery from bulbectomy (day 15), MMP-2 expression is observed among the basal cells and axon processes. A young developing neuron (arrowhead) and its axon process (arrows) can be seen passing through the basement membrane (BM). (d) Higher magnification of axon process shown in (c). Bar = 10 µm.