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. 2018 Aug 1;38(31):6806-6824.
doi: 10.1523/JNEUROSCI.3261-17.2018. Epub 2018 Jun 22.

The Neuroregenerative Capacity of Olfactory Stem Cells Is Not Limitless: Implications for Aging

Kevin M Child  1   2 , Daniel B Herrick  3   2 , James E Schwob  2 , Eric H Holbrook  4   2 , Woochan Jang  5
Affiliations

The Neuroregenerative Capacity of Olfactory Stem Cells Is Not Limitless: Implications for Aging

Kevin M Child et al. J Neurosci. .

Abstract

The olfactory epithelium (OE) of vertebrates is a highly regenerative neuroepithelium that is maintained under normal conditions by a population of stem and progenitor cells, globose basal cells (GBCs), which also contribute to epithelial reconstitution after injury. However, aging of the OE often leads to neurogenic exhaustion, the disappearance of both GBCs and olfactory sensory neurons (OSNs). Aneuronal tissue may remain as olfactory, with an uninterrupted sheet of apically arrayed microvillar-capped sustentacular cell, or may undergo respiratory metaplasia. We have generated a transgenic mouse model for neurogenic exhaustion using olfactory marker protein-driven Tet-off regulation of the A subunit of Diphtheria toxin such that the death of mature OSNs is accelerated. At as early as 2 months of age, the epithelium of transgenic mice, regardless of sex, recapitulates what is seen in the aged OE of humans and rodents. Areas of the epithelium completely lack neurons and GBCs; whereas the horizontal basal cells, a reserve stem cell population, show no evidence of activation. Surprisingly, other areas that were olfactory undergo respiratory metaplasia. The impact of accelerated neuronal death and reduced innervation on the olfactory bulb (OB) was also examined. Constant neuronal turnover leaves glomeruli shrunken and affects the dopaminergic interneurons in the periglomerular layer. Moreover, the acceleration of OSN death can be reversed in those areas where some GBCs persist. However, the projection onto the OB recovers incompletely and the reinnervated glomeruli are markedly altered. Therefore, the capacity for OE regeneration is tempered when GBCs disappear.SIGNIFICANCE STATEMENT A large percentage of humans lose or suffer a significant decline in olfactory function as they age. Therefore, quality of life suffers and safety and nutritional status are put at risk. With age, the OE apparently becomes incapable of fully maintaining the neuronal population of the epithelium despite its well known capacity for recovering from most forms of injury when younger. Efforts to identify the mechanism by which olfactory neurogenesis becomes exhausted with age require a powerful model for accelerating age-related tissue pathology. The current OMP-tTA;TetO-DTA transgenic mouse model, in which olfactory neurons die when they reach maturity and accelerated death can be aborted to assess the capacity for structural recovery, satisfies that need.

Keywords: aging; degeneration; neuroepithelium; olfactory; stem.

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Figures

Figure 1.
Figure 1.
Human OE becomes increasingly aneuronal with age. Autopsy sample taken from 74-year-old human shows that the OE contains prominent areas devoid of staining with neuronal markers. A, Whole mount of septum using an antibody against NST (visualized with DAB) to stain OSNs reveals regions of OE lacking any OSNs (indicated by gaps within the NST+ OE). Orientation of the tissue is indicated as dorsal (d), ventral (v), anterior (a), and posterior (p). B, Mosaic image of a section taken from the same sample shown in A showing the neuronal OE with OMP+ (green) and NST+ (magenta) OSNs (designated as “N”) and the RE with βIV-Tubulin+ (gold) columnar cells (designated as “R”); between the neurogenic OE and the RE is a patch of the aneuronal OE with neither of OMP+/NST+ OSNs or βIV-Tubulin+ columnar cells (designated as “A”). Arrows indicate borders between regions and arrowheads indicate basal lamina. Orientation of the tissue is indicated as dorsal (d) and ventral (v). C–F, High magnification of neurogenic regions of OE (C, D) and aneuronal OE (E, F). The neurogenic OE consists of OMP+ (green)/NST+ (magenta) OSNs (C) as well as Sox2+ (green) GBCs (open arrow), Sox2+ (green)/P63+ (magenta) HBCs (white arrows) above the basal lamina (arrowhead) in D and F and Sox2+-supporting cells apically (D). The aneuronal OE lacks OMP+/NST+ neurons (E) and Sox2+ GBCs; however, Sox2+ HBCs are present and remain P63+ indicated by white arrows (F). Scale bars: A, 3 mm; B, 300 μm; C, 20 μm (also applies to D–F). Dashed line indicates basal lamina in C and E.
Figure 2.
Figure 2.
The OE from aging mice contains aneuronal areas similar to the OE from aging humans. Sample taken from 2-year-old wild-type C57BL/6 mouse shows extensive areas devoid of neuronal markers. A, Low magnification of ectoturbinate II of the OE (black box in the diagram inset indicates the location within the OE) shows the lining of both normal and neuronal OE with OMP+ (green) mature and NST+ (magenta) immature OSNs and aneuronal OE lacking OSNs. The RE is identified as βIV-Tubulin+ (gold) at its apical surface. The white boxes depict the areas that were photographed in B–F. B–F, High magnification of neurogenic OE (B, C), aneuronal OE (D, E), and respiratory metaplasia (F) found in aging wild-type C57BL/6 mice. Similar to the human OE (Fig. 1), the normal, neuronal OE consists of OMP+ (green)/NST+ (magenta) OSNs (B) as well as Sox2+ (green) GBCs (open arrow) and Sox2+ (green)/P63+ (magenta) HBCs (white arrows) above the basal lamina (arrowhead) and Sox2+ supporting cells apically (C). The aneuronal OE contains neither OMP+/NST+ nor immature NST+ neurons (D) nor Sox2+ GBCs (E). As in human OE, Sox2+/P63+ HBCs (white arrows) and Sox2+ supporting cells are evident and evidently dormant (E). G, Areas of respiratory metaplasia found in K5-CreERT2;Rosa26-fl(stop)-TdTomato (KT) mice. Neither OMP+ or NST+ neurons are present, however, βIV-Tubulin+/TdTomato+ ciliated columnar cells can be identified. KT mice injected with tamoxifen at 6 weeks and killed at 18 months contain areas of metaplastic RE in which ciliated columnar cells are derived from TdT+ HBCs as a result of activation. Scale bars: A, 3150 μm; B, 20 μm (also applies to C–G). Dashed lines indicate basal lamina in B, C, F, and G; arrowheads indicate basal lamina in D and E.
Figure 3.
Figure 3.
Mouse model of accelerated aging via continuous DTA destruction of mature OSNs. A, Breeding strategy used to generate mice that are heterozygous for the OMP-tTA and TetO-DTA alleles (OMP-tTA;TetO-DTA mice). In this “Tet-Off” paradigm, transcription of DTA leads to the death of OSNs (“Die”) in the absence of doxycycline (−DOX). DTA is not expressed in the presence of doxycycline (+Dox) and OSNs remain alive (“Survive”). B, C, Coronal sections of the OE from a 4 month-old control mouse (+Dox; B) and a 4 month-old degeneration mouse (−DOX, DTA-on; C). Labeling with OMP (green) and CK19 (magenta) mark the mature OSNs in the OE and the apical lining of the RE, respectively. The OSN layer in the degeneration mouse is thinner, the axon bundles are smaller, and the CK19+ RE area expands (C). The higher-magnification inset highlights an example of a cyst commonly observed in degeneration mice. D–G, Confocal images of a cleared whole-mount of septal OE stained with OMP (green) and βIV-Tubulin (magenta) from a 6-month-old control mouse (D) with boxed area (E) at higher magnification and a 6-month-old-Dox OMP-tTA;TetO-DTA mouse (F) with boxed area (G) at higher magnification. The OE from −DOX OMP-tTA;TetO-DTA mouse (F, G) is interrupted with more expanded areas of RE (F and asterisk in G) and swaths of aneuronal OE. Scale bars: B, 600 μm (also applies to C); D, 1 mm (also applies to F). Scale in E and G is shown in Z stack 3-D view (B also applies to C). Orientation in D and E: dorsal (d), ventral (v), posterior (p), and anterior (a).
Figure 4.
Figure 4.
Epithelial grades of degeneration. The status of the OE in the Dox− OMP-tTA;TetO-DTA mice can be classified or graded on the basis of the abundance of mature and immature neurons, respiratory columnar cells, dividing GBCs, and upstream GBCs. Sections of the OE from a 6-month old Dox+ OMP-tTA;TetO-DTA control mouse (A–C) and a 6-month old Dox− OMP-tTA;TetO-DTA mouse (D–O) in which the OE is degenerating stained with multiple cell-specific markers demonstrate differences across Grades I–IV. As opposed to the normal-appearing OE in control mice (A), there is a progressive decrease in OMP+ (magenta)/NST+ (green) mature OSNs and OMP−/NST+ immature neurons in Grades I and II (D, G) with complete absence in Grades II and IV (J, M) and the presence of CK19+ (gold) RE columnar cells in Grade IV (M). As opposed to the occasional Sox2+ (green)/CK14− (gold)/Ki67+ (magenta) multipotent upstream GBCs (black arrow) in normal appearing control OE (B), Grade I displays an increase in upstream GBCs (black arrows in E). These proliferative, upstream Sox2+ GBCs decrease to normal levels in Grade II (H) and are essentially absent in Grade III and IV (K, N). Apically positioned Sox2+ Sus cells and basally positioned CK14+/Sox2+ HBCs are present throughout all grades and are rarely dividing. The presence of ND1+ (green)/BrdU+ (magenta) immediate neuronal precursor GBCs (black arrow) are also sporadic in normal appearing OE (C). These cells increase dramatically in Grade I (black arrows in F) and decrease again in Grade II (black arrows in I). As with dividing GBCs, the immediate neuronal precursors GBCs are absent in Grades III and IV OE (L, O). Nonactivated P63+ (gold) HBCs (white arrows in C–O) line the basal lamina (arrowheads) in all grades. Note the absence of cell bodies between the apical supporting cells and HBCs as indicated by the lack of nuclear staining (blue) in Grade III (J–L). Scale bar in A, 20 μm (also applies to B–O).
Figure 5.
Figure 5.
OE degeneration increases with time. A, Diagram renditions of coronal nasal sections through anterior and posterior OE areas after 2, 4, and 6 months of degeneration are labeled along the epithelium with colors indicating grades of degeneration: Grade I (blue), Grade II (yellow), Grade III (red), and Grade IV (black). Most severe degeneration can be seen in anterior sections in ectoturbinate 2 at 4 and 6 months. B, C, Averaged quantification of percentage total linear measurements of each grade at 2, 4, and 6 months for anterior (B) and posterior (C) sections demonstrate increases in more severe grades especially with anterior sections over time. Corresponding tables show p-values of t tests for comparisons after assigning numerical values of 1–4 for each grade for each measured length. Gray boxes represent significance of > 0.05. D, Western blots using antibodies against OMP, GAP43, and PGP9.5 were obtained from equal sized pieces of tissue removed from the septum of control and 6 month degeneration mice. E–G, Statistical analyses of the changes in the protein levels of OMP (E), GAP43 (F), and PGP9.5 (G). There is a significant decrease in OMP in degeneration mice compared with control at all ages and a decrease in PGP9.5 in degeneration mice compared with control at 4 and 6 months; however, GAP43 is not significantly changed. For E and F, *p < 0.05 (p-values are shown in the Results).
Figure 6.
Figure 6.
Numbers of GBCs across OE of degeneration mice vary with grade of degeneration, whereas HBCs remain stable. Cell counts of Sox2+ upstream GBCs per length of OE of ectoturbinate 2 in the anterior (A) and the posterior (B) regions were averaged for control mice and regions of Grade I–III of degeneration mice at 2, 4, and 6 months with representation in bar graphs. Overall, there is an increase in Sox2+ GBCs with Grade I degeneration compared with control and other grades. Similarly, ND1+ immediate neuronal precursor GBCs were counted in the anterior (C) and the posterior (D) Although the differences in the population of ND1+ cells do not reach statistical significance across various grades, the number of ND1+ GBCs appear to increase in Grade I OE. Tables in A–D provide p-values of ANOVA and t tests (gray boxes = p < 0.05). E, Immunohistochemical staining of the basal cell compartment (GBCs and HBCs) showing the differences between control tissue and each grade of degeneration (Grades I–III). ND1+ (green) and Sox2+ (gold) GBCs (arrowheads) are positioned above the P63+ (magenta) HBCs lining the basal lamina (dashed line) in a single row. F, Western blots of P63 protein levels in the septal OE from 6-month-old control and degeneration mice are not significantly different. Scale bar in E is 10 μm and applies to all images.
Figure 7.
Figure 7.
Macrophage recruitment and cell death is increased overall in Dox− OMP-tTA;TetO-DTA mice in which the OE is degenerating. A–C, Sections labeled for cleaved Caspase III+ (green) to mark dying cells demonstrate an increase in neuronal cell death in a 2 months DTA-on mouse (B) compared with control (A) cell counts were performed in ectoturbinate 2 including all grades (D). However, cleaved Caspase III+ cells are absent in aneuronal epithelium, as shown in the OE at 4 months of DTA-on (C). E–G, Macrophages are labeled with an antibody against Iba1 (green) in sections of OE from a control mouse at 2 months (E), degeneration mice at 2 months (F) and 4 months (G). CK19+ (magenta) cells are identified as HBCs (E–G). Macrophages appear clustered more densely in degeneration regions (F), but are infrequent in aneuronal epithelium as shown in the OE of a 4-month DTA-on mouse (G). The numbers of Iba1+ macrophages (H), CD3+ T-cells (I), CD19+ B-cells (J), and Ly6 g+ granulocytes (K) are increased in DTA-on mice compared with control without constraining for DTA status or age. A 4 × 2 × 2 ANOVA (cell type × DTA status × duration) was performed to assess the immune response to the lesion as a whole (L). Dashed line indicates the basal lamina. Arrowheads in A and B identify cleaved Caspase III+ dying neurons. Arrowheads identify individual macrophages in A–C). *p < 0.05. Scale bar in A, 20 μm (also applies to B, C, E–G).
Figure 8.
Figure 8.
Recovery of epithelium is substantial but incomplete. A, Diagram renditions of coronal nasal sections through anterior OE areas at 4 months of degeneration in a DTA-on mouse (image taken from Fig. 5A) and 4 months of degeneration (DTA-on) plus 2 months of recovery (DTA-off) are classified as to extent of the degeneration and recovery across the epithelium with colors indicating grades of degeneration: Grade 0 (equivalent to control, green), Grade I (blue), Grade II (yellow), Grade III (red), and Grade IV (black). Based on the extent of the damaged epithelium before and after recovery, the less severe Grades, for example, Grades I and II, appear to recover to normal (Grade 0), whereas OE that is aneuronal or metaplastic does not recover. B, Averaged extent of normal appearing OE (Grade 0) and each Grade of degeneration expressed as a percentage of the total length of OE for 2 months DTA-on, 2 months DTA-on plus 2 months DTA-off, 4 months DTA-on, and 4 months DTA-on plus 2 months DTA-off (n = 3 per conditions). The extent of normal (Grade 0) OE is markedly increased after the DTA-off recovery period in the 2 + 2 mice, whereas the extents of Grade I and II OE are significantly decreased after 2 months recovery (blue and yellow asterisks indicate that the decline in both Grade I and Grade II is significant at p < 0.05); however, severe degeneration (Grades III and IV and absent of GBCs) does not change. Similarly after 4 months of DTA-on degeneration, 2 months of DTA-off recovery increases normal appearing OE (Grade 0) and decreases Grade I degeneration (blue asterisks indicates p < 0.05), but regions of severe degeneration remain unchanged. C–F, Immunohistochemistry of sections from a 4-month DTA-on mouse (C, E) and a 4 month DTA-on + 2 months DTA-off recovery mouse (D, F) demonstrates a robust increase of OMP+ (green) mature OSNs and NST+ (magenta) immature neurons after recovery in neurogenic OE (D), whereas βIV-Tubulin− (gold) aneuronal regions (E, F) remain devoid of neurons. Arrowheads indicate basal lamina. Scale bar in C, is 20 μm (also applies to D–F).
Figure 9.
Figure 9.
Degeneration of the OE due to accelerated turnover alters the OB. A, B, Diagrams of coronal sections through the OB labeled with Vglut2 antibody reveal smaller glomeruli in the 6 months DTA-on mouse (B) compared with age-matched DTA-off control (A). C, D, In the DTA-off mice, individual glomeruli, labeled with OMP (magenta) and Vglut2 (green), are plump and normal in appearance as shown in this section from the medial surface of the OB (C), but glomeruli are markedly shrunken after 6 months of degeneration in the DTA-on mice (D). E, F, Likewise, periglomerular dopaminergic interneurons labeled with an antibody against TH (green) are prominent and surround OMP+ (magenta) glomeruli in the DTA-off control mice (E); in the DTA-on mice at 6 months of degeneration, TH+ are evident despite the substantial shrinkage of the glomeruli (F). G, H, Labeling of Tbx21+ (green) mitral cells appears unchanged comparing the OB of DTA-off control mice (G) versus the OB DTA-on mice at 4 months of degeneration (H). Total glomerular area was calculated from Vglut2 stained DTA-off coronal sections from anterior (I) and posterior (J) regions of the OB and from equivalent levels of the bulb from DTA-on mice after 2, 4, and 6 months of degeneration. There was a significant decrease in glomerular area compared with control (*p < 0.05) at all durations of degeneration points, but glomerular area is not further reduced by lengthening the period of degeneration to 4 and 6 months. K, Cell counts of TH+ interneurons from similar sections demonstrated no difference between control and degeneration mice. L, However, when TH protein levels were assessed using Western blots of OBs obtained from 2 and 4 month degeneration mice and normalized to Tbx21 protein concentration, there was a statistically significant decrease compared with age-matched controls (*p < 0.05). M, As with TH+ interneurons, counts of Tbx21+ mitral cells from OB sections did not change when comparing control and 2, 4, and 6 month degeneration mice. Scale bar in C, 50 μm (also applies to D–H).
Figure 10.
Figure 10.
The OB does not recover to normal after OE degeneration. A–D, Sections of OB from 4 month DTA-off (A, B) and 4 months of degeneration with 2 months of recovery (DTA-on to DTA-off) (C, D) mice labeled with OMP (magenta) to stain the axons of mature OSNs and with Vglut2+ (green) to mark axon terminals within glomeruli (A, C). Boxes in A and C indicate the locations of the higher-magnification images shown in G–J. Mosaics of OB sections stained for Vglut2 were acquired and then stained pixels were highlighted in ImageJ and rendered into schematics (B, D). The images were then split into medial (M, red) and lateral (L, black) compartments (B, D). E, F, Measures of total glomerular area were not statistically different when comparing 2 months DTA-off (control) versus 2 months DTA-on followed by 2 months DTA-off recovery (E) or 4 months control versus 4 months degeneration with 2 months recovery (F). G–J, Higher magnification of glomeruli labeled with antibodies against OMP (magenta) and Vglut2 (green); for purposes of the statistical comparison, a glomerulus is defined as an object composed of contiguous stained pixels that exceed background. The glomeruli in the degeneration–recovery mice are markedly smaller than in the DTA-off control bulb, but they are also more numerous (histograms below G–J). Mean glomerular size in square micrometers (red arrows) is much smaller in recovered mice than in control mice (graphs in G–J). K, L, The set of olfactory neurons that were labeled with antibodies against the MOR28 olfactory receptor (green) was mapped in control (K) and recovered (L) mice. OMP labeling (magenta) was used to mark all OSN axons in the glomerulus. K, L, Photographs documenting MOR28 staining on the medial side of the OB. In the control mice, each MOR28+ glomerulus was completely filled by that individual receptor type, whereas glomeruli in recovered mice are small in size and appeared to be more than one glomerulus on this side. M, N, Measurements of the size (M) and the complexity (N) of the glomeruli that are innervated by MOR28 neurons in both the lateral and medial OB from control and recovery mice. In the recovery mice, the size of the individual glomerular objects are smaller (M), but the complexity index (the complexity of the shape of objects relative to the complexity of perfect circle calculated as described in the Results) is higher (N) after recovery from degeneration compared with control, DTA-off mice on both sides of the OB. Scale bars: A, 300 μm (also applies to C); G, 100 μm (also applies to H–J), and K, 50 μm (also applies to L).
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