Rejuvenation of regeneration in the aging central nervous system

Abstract

Remyelination is a regenerative process in the central nervous system (CNS) that produces new myelin sheaths from adult stem cells. The decline in remyelination that occurs with advancing age poses a significant barrier to therapy in the CNS, particularly for long-term demyelinating diseases such as multiple sclerosis (MS). Here we show that remyelination of experimentally induced demyelination is enhanced in old mice exposed to a youthful systemic milieu through heterochronic parabiosis. Restored remyelination in old animals involves recruitment to the repairing lesions of blood-derived monocytes from the young parabiotic partner, and preventing this recruitment partially inhibits rejuvenation of remyelination. These data suggest that enhanced remyelinating activity requires both youthful monocytes and other factors, and that remyelination-enhancing therapies targeting endogenous cells can be effective throughout life.

Figure 1. Heterochronic parabiosis stimulates OPC proliferation and angiogenesis

(A) Proliferating OPCs (Nkx2.2⁺/Ki67⁺), (B) CD34 blood vessels and (C) apoptotic OPCs (Nkx2.2⁺/TUNEL⁺) within lesions at 7 and 14dpl. Arrows indicate co-localisation. N ≥ 4 pairs were analyzed for each condition and timepoint. Quantified data are presented as means ± SEM. Data were analyzed by one-way ANOVA followed by Tukey’s post test. *denotes p < 0.05, **denotes p < 0.001. Scale bars: A,C 20 μm, B 125 μm. See also Fig. S1.

Figure 2. Heterochronic parabiosis stimulates OPC differentiation and remyelination

(A) Quantification of oligodendrocytes shows few CC1⁺/Olig2⁺ cells present in the lesion at 7dpl, independent of age. Higher oligodendrocyte densities at 14dpl indicates the generation of new oligodendrocytes, with significantly more differentiation occurring in heterochronic-old animals than in isochronic-old controls at 14 and 21dpl. Data are means ± SEM. (B) Oligodendrocytes in the lesion at 14dpl. Representative fields are shown for each parabiotic condition. (C) Ranking analysis of remyelination at 21dpl (rank 1 equals least remyelination). N ≥ 8 per group, horizontal line indicates median for each data set. Data analyzed by Kruskal-Wallis test followed by Dunn’s post test (D) Toluidine blue stained resin sections of lesions: representative images for each parabiotic condition at 21dpl. Widespread remyelination is evident in lesions in isochronic-young animals, contrasting with the relative paucity of remyelination in lesions of isochronic-old animals. Heterochronic-old animals showed increased remyelination compared to isochronic-old controls. Insets show magnification of boxed area. (E) Quantification of oligodendrocyte (Olig2⁺/CC1⁺) density in lesions of isochronic-young and heterochronic-young animals at 21dpl (n=4 animals per group). Data are means ± SEM and were analyzed with an unpaired two-tailed t test. No significant difference between the two groups was found. (F) Ranking analysis of remyelination at 21dpl (n=7 animals per group). Rank 1 equals least remyelination. The median is indicated by a horizontal line for each data set. Data were analyzed with a Mann-Whitney test, which did not detect a significant difference between the two groups. *denotes p < 0.05, **denotes p < 0.001, ns = not significant. Scale bars: B 50 μm, D 20μm (insets 2 μm)). See also Fig. S2.

Figure 3. Engrafted cells in the spinal cord lesions of parabionts are chiefly young-partner derived macrophages

(A) Parabiotic pairings in which cells of the young partner were transgenically labelled by GFP expression were established. In the old partner lesion from each GFP^young/WT^old pairing, GFP⁺ cells were confined to the lesion (B). (C) No GFP⁺ cells in old partner lesions were found to co-localize with Olig2⁺ nuclei. (D) Almost all GFP⁺ cells co-localized with the macrophage marker MAC1. (E) Characterization of the GFP⁺ inflammatory infiltrate at 5dpl in isochronic-young (iy) (n=3), isochronic-old (io) (n=2) and heterochronic-old (ho) animals (n=4). No statistically significant differences were found amongst the parabotic groups. The overwhelming majority of GFP⁺ cells were MAC1⁺ macrophages. Very few CD4⁺ T cells, CD8⁺ T cells, CD94⁺ natural killer cells, B220⁺ B cells and NIMP-R14⁺ neutrophils were present in the lesions. Data are means ± SEM. Scale bars: B 100 μm, C,D 20 μm.

Figure 4. Young macrophages play a central role in the rejuvenating effect of heterochronic parabiosis

(A) Experimental rationale: in CCR2^{−/− young}/WT^old parabionts, only old monocytes are recruited to the old partner lesion in response to MCP-1 signalling. Soluble factors retain access to the lesion by virtue of local disruption of the blood-spinal cord barrier (BBB). (B) Immunostaining for GFP and MAC1 in heterochronic WT^young/WT^old and CCR2^{−/− young}/WT^old pairs demonstrates the absence of donor-derived macrophages in CCR2^{−/− young}/WT^old lesions. (C) Comparison of the inflammatory infiltrate at 5 dpl shows that recruitment of donor-derived MAC1+ IB4+ macrophages predominates in old WT mice paired with young WT mice (n=3) and is abolished in old WT mice paired with young CCR2^−/− mice (n=4, white bars). There are small numbers of CD4⁺ T cells, CD8⁺ T cells, CD94⁺ natural killer cells, B220⁺ B cells and NIMP-R14⁺ neutrophils in the lesions of both pairings. (D) Oligodendrocyte (CC1⁺/Olig2⁺) density at 21 dpl in CCR2^{−/− young}/WT^old pairs was significantly reduced compared to heterochronic old WT pairs, but elevated compared to isochronic-old controls (n=4 pairs per condition). Data analyzed by one-way ANOVA followed by Dunnett’s post test (E,F) Oil Red O staining revealed significantly more lipid-rich myelin debris in the lesions of isochronic-old pairings than in isochronic-young pairings, consistent with the notion that myelin debris have an inhibitory effect on remyelination. Oil Red O staining in heterochronic old WT lesions was significantly lower than in isochronic old controls. This enhanced myelin debris clearance observed in heterochronic pairings was attenuated when the young partner was CCR2-deficient. Note that the staining appears less diffuse in the isochronic young and the heterochronic groups, suggesting that more myelin debris has been taken up by phagocytic cells. (G,H) Similar observations were made for intralesional degenerated myelin basic protein accumulation. *denotes p<0.05; ** denotes p<0.001. Data are means ± SEM. Scale bars: B 50 μm E,G 100 μm. See also Fig. S3 and Fig. S4.