Inflammation-induced subventricular zone dysfunction leads to olfactory deficits in a targeted mouse model of multiple sclerosis

Abstract

Neural stem cells (NSCs) persist in defined brain niches, including the subventricular zone (SVZ), throughout adulthood and generate new neurons destined to support specific neurological functions. Whether brain diseases such as multiple sclerosis (MS) are associated with changes in adult NSCs and whether this might contribute to the development and/or persistence of neurological deficits remains poorly investigated. We examined SVZ function in mice in which we targeted an MS-like pathology to the forebrain. In these mice, which we refer to herein as targeted EAE (tEAE) mice, there was a reduction in the number of neuroblasts compared with control mice. Altered expression of the transcription factors Olig2 and Dlx2 in the tEAE SVZ niche was associated with amplification of pro-oligodendrogenic transit-amplifying cells and decreased neuroblast generation, which resulted in persistent reduction in olfactory bulb neurogenesis. Altered SVZ neurogenesis led to impaired long-term olfactory memory, mimicking the olfactory dysfunction observed in MS patients. Importantly, we also found that neurogenesis was reduced in the SVZ of MS patients compared with controls. Thus, our findings suggest that neuroinflammation induces functional alteration of adult NSCs that may contribute to olfactory dysfunction in MS patients.

Figure 1. Inflammation and demyelination in the forebrain of tEAE mice.

(A) Schematic illustration of sagittal mouse brain section indicating cytokine injection site used to achieve tEAE. (B) Cytokine injection in non-immunized mice induces restricted infiltration of CD45⁺ hematopoietic cells to injection site 3 days p.i. (C) Cytokine injection into MOG peptide–immunized mice induces widespread CD45⁺ cell infiltration at the injection site (arrow) and throughout the surrounding cortex and corpus callosum (arrowheads) during the first week p.i. (D) Co-immunolabeling for CD45 and MOG reveals inflammatory foci (arrowheads) in tEAE corpus callosum. (E) Toluidine blue–stained resin section of tEAE corpus callosum showing inflammatory cells surrounding a blood vessel (BV), 3 days p.i. (F) Bundles of demyelinated axons and (H) astrocytosis in the demyelinated area 2 weeks p.i. (G) Higher-power view of axons within the boxed area shown in F. (I–L) Electron micrographs of corpus callosum. (I).Oligodendrocytes (arrowheads) and myelinated axons in control mice. (J) Demyelination and axonal swelling in tEAE mice 2 weeks p.i. (K) Large astrocyte (A), active microglia (M), thin myelin sheets (arrowheads), and cell with young oligodendrocyte morphology (O) in tEAE tissue, 1 month p.i. (L) Higher-power view of the boxed area shown in K. Arrows indicate axons surrounded by thin myelin sheets as in K, while N labels an axon ensheathed by normal myelin. cc, corpus callosum; Ctx, cortex; St, striatum; V, lateral ventricle. Scale bars: 50 μm (B, D, and H), 100 μm (C), 25 μm (F), 10 μm (E, G, I–K), 2 μm (L).

Figure 2. SVZ inflammation correlates with modulation of proliferation in tEAE mice in a time-dependent manner.

(A–D) IHC for CD45 in the SVZ. (A) Absence of strongly labeled cells in the SVZ of control mice. (B) Cytokine injection into non-immunized mice leads to a moderate increase in hematopoietic cell numbers in the SVZ 3 days p.i. (cyt 3d). (C) Extensive increase in numbers of inflammatory cells during the first week p.i. occurs in MOG peptide–immunized mice (tEAE 3d). (D) Numbers of inflammatory cells decrease in tEAE mice sacrificed at 2 months p.i. (tEAE 2m) compared with early phases but do not reach control levels. (E) Quantification of CD45⁺ cells in the SVZ (n = 3–4 mice/group; *P = 0.04, ^†P = 0.01, ^‡P = 0.02, ^§P = 0.01, ^¶P = 0.04). (F–I) IHC for BrdU in the SVZ. (F) Control SVZ. (G) Decrease in BrdU⁺ cell numbers in tEAE SVZ 3 days p.i., while proliferation is upregulated in the neighboring parenchyma. (H) Increase in BrdU⁺ cell numbers in the SVZ at 7 days p.i. (I) Recovery of basal levels 2 months p.i. (J) Quantification of BrdU⁺ cells in the SVZ (n = 5–7 mice/group; **P = 0.0026, ^#P = 0.004 versus control). Error bars represent SEM. Scale bars: 50 μm.

Figure 3. Reduction in neuroblast chains in the tEAE SVZ.

(A) Numerous DCX⁺ cells are present in the control subependymal area. (B) SVZ areas free of DCX⁺ cells with chains emigrating into the neighboring striatum (arrowheads) along the blood vessels in tEAE tissue 3 days p.i. (C and D) SVZ in tEAE mice sacrificed 2 months p.i. (C) Areas containing few DCX⁺ cells alter with those containing bulks of DCX⁺ cells as shown in D. Arrowheads in D show individual DCX⁺ cells in the striatum near the SVZ. SVZ limits are indicated by white dashed lines. (E) Quantification of DCX immunoreactivity in the SVZ (n = 3–6 mice/group). Decreases compared with the controls are significant in tEAE mice at 3 days and 2 months p.i. (***P = 0.0002 and **P = 0.0087, respectively), but no significant changes in the amount of DCX were observed in non-immunized mice sacrificed 3 days following cytokine injection as compared with the controls. Error bars represent SEM. (f–h) RMS in control versus tEAE mice. (F) Organized chain migration toward OB in control RMS. (G) Accumulation of DCX⁺ cells within the RMS region proximal to the SVZ and emigration into the corpus callosum at 7 days p.i. (arrowheads). (H) Interruptions of the RMS and individual DCX⁺ cells in tEAE corpus callosum, 2 months p.i. (arrowheads). Scale bars: 50 μm.

Figure 4. Analysis of SVZ ultrastructure during tEAE.

(A–D) Schematic representation of SVZ organization, with each cell type shown in different color. Neuroblasts are shown in red. Reduction in red cells is obvious at 3 days p.i. in tEAE mice. Some recovery occurs at later time points, but control levels are not reached. Images are illustrations of the boxed areas within each schematic representation. (A) Chain of migrating neuroblasts (A) surrounded by astrocytes (B) in the control SVZ. (B) Macrophages (M) and pyknotic cell (P) next to blood vessels within tEAE SVZ 3 days p.i. (C) Upper panel: Large astrocytes (B) next to small neuroblast chain (A); lower panel: type C cells in tEAE SVZ 7 days p.i. (D) Astrocyte (B) processes and small myelinated axons (arrowheads) separate neuroblasts (A) and some C cells (C) from the ependyma in tEAE SVZ 1 month p.i. (E) Quantification of A cells in the SVZ of control and tEAE mice. **P = 0.002, *P = 0.01, ^†P = 0.005 versus control; n = 3–4 mice/group; error bars represent SEM. (F) SVZ composition represented as average percentages of each cell type within total SVZ cells (3–4 mice/group). While in the control, SVZ neuroblasts (red) predominate, their reduction is obvious during tEAE. The pathology, however, progressively increases type C cells (green). Astrocytes contacting the lateral ventricle are presented in white and non-identified cells in light blue.

Figure 5. Amplification of Olig2⁺Mash1⁺ cell pool and decrease in Dlx2 immunoreactivity in the tEAE SVZ.

(A) Quantification of Mash1⁺ SVZ cells (*P = 0.001, ^†P = 0.0001, ^‡P = 0.0003 versus control; n = 5–11 mice/group). (B) Quantification of Olig2⁺ cells in the SVZ (*P = 0.0007, ^†P = 0.0045 versus control; n = 5–11 mice/group). (C) Olig2⁺ fraction of the Mash1⁺ cell population in the SVZ increases during tEAE (*P = 0.0001, ^†P = 0.0182 versus control). (D–I) Representative images of Mash1 (green) and Olig2 (red) co-immunolabeling in the SVZ of control (D–F) and tEAE (G–I) mice at 2 months p.i. (J) Quantification of Dlx2⁺ cells in the SVZ (*P = 0.003, ^†P = 0.0091, ^‡P = 0.0133 versus control; n = 4–7 mice/group). (K) Dlx2⁺ fraction of the Mash1⁺ cell population in the SVZ diminishes during tEAE (*P = 0.003, ^†P = 0.0023, ^‡P = 0.0002 versus control). (L–Q) Representative images of Mash1 (green) and Dlx2 (red) co-localization in the SVZ in control (L–N) and tEAE (O–Q) mice. Error bars represent SEM. Scale bars: 20 μm.

Figure 6. OB neurogenesis decreases during tEAE.

Tracing of SVZ progeny was performed by two 2-hour BrdU pulses prior to pathology induction. (A–F) Representative images of granule cell layer in the OBs of sham-treated (A–C) and tEAE mice (D–F) sacrificed 2 weeks following pathology induction and BrdU injections. (I) BrdU labeling efficiency of SVZ cells is identical for sham-treated and tEAE mice immediately after the pulse. (J) Two weeks p.i., fewer (**P = 0.0037; n = 4–5 mice/group) BrdU⁺ cells are found in the OB of tEAE compared with sham-treated mice. (K) In the granule cell layer, a decrease (**P = 0.0006; n = 4–5 mice/group) in newly formed mature neurons (BrdU⁺NeuN⁺) is observed. (G, H, and L) Long-term evaluation of granule cell neurogenesis was performed by quantification of DCX⁺ immature neurons. (G and H) Representative images of coronal OB sections labeled with DCX antibody in sham-treated (G) and tEAE (H) mice sacrificed 3 months p.i. (L) Quantification of DCX immunoreactivity (**P = 0.0029; n = 6–7 mice/group). Error bars represent SEM. Scale bars: 20 μm (a–f), 50 μm (G and H).

Figure 7. Persistence of tEAE-induced neurogenesis impairment correlates with decreased p-SMAD signaling.

(A and B) IHC for p-SMAD1/5/8 on coronal sections of sham-treated (A) and tEAE (B) SVZ 3 months p.i. (C) Significant decrease in numbers of p-SMAD1/5/8⁺ SVZ cells in tEAE versus sham-treated mice (**P = 0.0013; n = 5 mice/group). (D) Schematic summary of tEAE-induced SVZ changes. In healthy SVZ, 3 type C cell subtypes are detected: the predominant is the Dlx2⁺ cell (green cells with yellow nucleus) in which Dlx suppresses Olig2 expression. In these cells, BMP signaling through p-SMAD is active, and migrating neuroblasts (violet unipolar cells) are produced. Neuroblasts migrate into the OB and generate new interneurons. The second type C cell subtype is the Olig2⁺ cell (green cells with red nucleus) in which Dlx2 expression is repressed. These cells are considered as an early source of oligodendrocyte progenitor cells (OPCs; red bipolar cells). A minor percentage of Mash1⁺ cells is negative for both Dlx2 and Olig2 (green cells with white nucleus). Inflammation induces some neuroblast apoptosis and emigration of others into the inflamed parenchyma. In addition, the pathology amplifies the Mash1⁺ SVZ population by increasing primarily numbers of Mash1⁺Olig2⁺ cells. Decreased p-SMAD signaling during tEAE may contribute to these changes. Moreover, neuroblasts generated still deviate from their default route, possibly due to structural changes within the neurogenic niche and/or attractive cues within the inflamed tissue. The final outcome of such changes is decreased neuronal OB supply. Scale bars: 20 μm.

Figure 8. Olfactory dysfunction in tEAE mice.

(A) Spontaneous olfactory discrimination. Histograms indicate mean time of odorant investigation within 2-minute exposure (rest intervals, 2 minutes). Eight sets of columns represent 4 habituations to heptanal (Hept), dishabituation (similar odorant, octanal [Oct]), 2 habituation recalls (Hept) and a final dishabituation (dissimilar odorant, geraniol [Ger]). ***P < 0.0001. (B) Reinforced olfactory discrimination between pairs of binary mixtures consisting of different linalool (L, rewarded odorant)/geraniol (no reward) ratios. Vertical axis represents correct response percentage for 10 blocks of 20 trials. Dashed line represents chance level (50%). (C and D) Short-term olfactory memory was tested by mint odorant presentation twice for 2 minutes (2-minute pause), followed by 30 minutes rest period and then 2-minute memory test (C). (D) Histograms indicate the mean investigation time. ***P < 0.0001. (E–I) Long-term olfactory memory. (E) Mice learned to discriminate (day 1) between 1% n-amyl acetate (rewarded odorant, S⁺) and 1% cineole (non-rewarded odorant, S^–), which was followed by a 4-day task consolidation (days 2–5) and then a 40-day rest period. Memory was tested in 1 block of 20 trials (10 S⁺, 10 S^–, random). (F) Mean percentages of correct responses in the training session 5 versus memory test. Chance level is represented by dashed line (50%); *P = 0.0132. (G) Mean error numbers for memory test: *P = 0.0151. (H) Mean error numbers in S⁺ memory trials: **P = 0.0067. (I) Mean probability of miss in S⁺ trials (error number in S⁺ trials/number of S⁺ trials): ***P = 0.0001. n = 7–9 mice/group. Error bars indicate SEM.

Figure 9. Neurogenesis is decreased in the SVZ of MS brain.

Lateral ventricular wall layers I–V were delimited by co-immunolabeling for GFAPδ (green) and MOG (red) in non-neurological control (A) and MS brains (B). The thickness of layer II is increased in MS compared with non-neurological controls. GFAPδ-expressing cells are mainly detected in layer III (astrocytic ribbon). DCX labeling in non-neurological controls (C) and MS SVZ (D). (E) The number of DCX⁺ neuroblasts (arrows in C and D) in the SVZ is significantly decreased in layers II, III, and IV. Error bars indicate SEM. *P ≤ 0.05. Scale bar: 50 μm.