Impact of heterochronic BMT on cognitive function

To evaluate the impact of hematopoietic age on cognitive function, we established a model of heterochronic BMT (Fig. 1a). Old (18-month) recipient mice were irradiated (without head-shielding) prior to injection of donor bone marrow cells from either young (4-month) or old (18-month) mice to achieve nearly complete (>90%) reconstitution with donor bone marrow (Supplementary Fig. 1a). Lymphocyte counts were initially slightly lower in old bone marrow recipients than young bone marrow recipients, but similar by 3 months post-transplantation (Supplementary Fig. 1b).

Fig. 1 Transplantation with young bone marrow preserved activity in old mice. a Timeline of bone marrow transplantation (BMT), hematopoietic reconstitution (0–3 months post-transplantation), behavioral analysis and tissue harvest for histology and molecular analyses (6 months post-transplantation). Old (18-month) mice received bone marrow from either young (4-month) or old (18-month) donors, and cognitive function was evaluated 6 months later. Non-transplanted old (24-month) and young (4-month) control mice were also evaluated. b–e Activity (b, c; includes horizontal locomotion and vertical rearing), distance covered (d), and speed (e) were assessed during four 5-min intervals over a 20-min period in an open field test, and the mean activity, distance and speed were calculated for each mouse. n = 32–51 mice per group, pooled from 3 independent experiments. Justification for pooling was confirmed by multi-ANOVA analysis. Similarly statistically significant differences were observed in all independent experiments. Box and whisker plots show median, 25th and 75th percentile, maximum and minimum values. ***p < 0.001 (ANOVA with Tukey-Kramer post-hoc test) Full size image

Behavioral testing was performed 6 months post-transplantation to assess activity and cognition (Fig. 1a). In an open field test, old control mice exploring a novel environment were less active than young control mice during the first 20 min of evaluation (Fig. 1b–e). Remarkably, however, young bone marrow recipients, but not old bone marrow recipients, were more active than old control mice (Fig. 1b–e). At the end of a 1-h assessment period, however, both transplanted groups and old control mice were similarly active (Supplementary Fig. 2a–c), suggesting that general wellness does not underlie the differences in exploratory behavior observed during the first 20 min of testing.

Exploratory behavior is thought to originate in the hippocampus17. We therefore evaluated hippocampus-dependent learning and memory. In the spontaneous alternation maze (Y-maze), old control mice performed worse than young control mice, but young bone marrow recipients completed more spontaneous alternations between the arms of the maze than both old bone marrow recipients and old control mice (Fig. 2a). This suggests that young BMT preserves spatial and working memory in old mice. The old control mice made fewer total arm entries than the young control mice, and transplantation with neither young nor old bone marrow reversed this (Supplementary Fig. 2d).

Fig. 2 Transplantation with young bone marrow prevented cognitive decline in old mice. a Spontaneous alternation between the arms of a Y-maze was assessed over an 8-min period. n = 44–46 mice per group, pooled from 3 independent experiments. Justification for pooling was confirmed by multi-ANOVA analysis. Similarly statistically significant differences or trends towards significance were observed in all independent experiments. b, c Memory of a prior electric shock was evaluated upon re-exposure to the same environment (context-dependent fear conditioning; b) or a tone that had preceded the shock (cue-dependent fear conditioning; c) by assessing freezing during a 5-min re-exposure period. n = 11 mice per group. d–f In a Barnes Maze test, the ability of mice to discover and then recall the location of an escape hole was evaluated during the learning phase (days 1–4; d), after a 2-day break (day 7; e), and following re-positioning of the escape hole (days 8–9; f). The number of errors made prior to successful location of the escape hole was recorded. n = 10–15 mice per group. Box and whisker plots show median, 25th and 75th percentile, maximum and minimum values. *p < 0.05, **p < 0.01, ***p < 0.001 (ANOVA with Tukey-Kramer post-hoc test) Full size image

A context-specific fear conditioning test was next used to evaluate hippocampus-dependent memory. When placed in the same environment in which they had previously received an electric shock, old control mice froze for shorter periods than young control mice (consistent with impaired memory), while young (but not old) bone marrow recipients froze for longer periods than old control mice (Fig. 2b). In contrast, a cue-specific fear conditioning test that assesses amygdala-dependent memory revealed no difference between any of the groups when mice were re-exposed to an audible cue that had preceded the electric shock (Fig. 2e). Collectively, these results confirm that a young hematopoietic system slows the aging-associated decline in hippocampal function.

Learning, spatial memory and memory recall were next assessed using a Barnes maze18. In comparison with old control mice and old bone marrow recipients, young control mice and young bone marrow recipients made only slightly fewer errors when locating an escape hole during the training phase (days 1–4; Fig. 2d). Following a 2-day break, however, young control mice and young bone marrow recipients made fewer errors than old control mice and old bone marrow recipients (day 7; Fig. 2e). The position of the escape hole was then changed and the number of errors made when discovering the new location was similar across all groups (day 8; Fig. 2f). However, the following day, young control mice and young bone marrow recipients made fewer errors compared to old control mice and old bone marrow recipients (day 9; Fig. 2f). In contrast, the time taken to successfully complete the test was similar across all groups on each day of testing (Supplementary Fig. 2c). Collectively, these data demonstrate that a young hematopoietic system preserves recall ability in old mice.

Impact of heterochronic BMT on neurons and glial cells

We next assessed neuron numbers and synapses in the hippocampus 6 months post-transplantation. Neuron numbers were reduced in the CA3 (but not the CA1) region of the hippocampus in old control mice (Fig. 3a, b and Supplementary Fig. 3a), consistent with previous studies19. Following young BMT, we observed a trend towards preservation of neuron numbers in the CA3 region, but it was not statistically significant (Fig. 3b). There was also an aging-associated reduction in the number of doublecortin (DCX)+ newly-born neurons in the dentate gyrus, and almost complete ablation of these cells in both young and old bone marrow recipients (Fig. 3c and Supplementary Fig. 3b), presumably due to the irradiation administered prior to transplantation15,16. We also assessed 5-bromo-2’-deoxyuridine (BrdU) incorporation in the dentate gyrus of bone marrow recipients at an earlier time point after transplantation (1 month post-transplant, when the mice were 19 months of age). We observed reduced BrdU incorporation in old control mice at this age compared to young control mice, and this was unchanged following transplantation with either old or young bone marrow (Supplementary Fig. 3c–d). Taken together these data indicate that preservation of cognitive function in the BMT model was independent of neurogenesis.

Fig. 3 Synapses were preserved in young bone marrow recipients. Neurons and synapses in the hippocampus were evaluated 6 months post-transplantation. a, b Neuron numbers in the CA1 (a) and CA3 (b) regions were assessed by NeuN staining. n = 6–8 mice per group, pooled from 2 independent experiments. c Newly-born neurons were assessed by counting doublecortin (DCX)+ cells. n = 6 mice per group. d Synapses were counted by evaluating co-localization of VGlut1 (pre-synaptic) and Homer1 (post-synaptic). n = 8 mice per group, pooled from 2 independent experiments. e Spine density was visualized and quantified on Golgi-impregnated slices. Scale bar: 2.5 μm. n = 5 mice per group. f Complement expression was assessed by RT-PCR (normalized to Gapdh mRNA). n = 5 mice per group, pooled from 2 independent experiments. g C3 deposition on synapses was evaluated by assessing C3 co-localization with Homer1. n = 8 mice per group, pooled from 2 independent experiments. The dotplots show mean plus standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (ANOVA with Tukey-Kramer post-hoc test) Full size image

Neuronal loss is normally preceded by synaptic loss in aging and aging-associated diseases20, and synaptic plasticity has been reported to improve in old mice following heterochronic parabiosis5. Thus, we postulated that synaptic connections may be maintained in young bone marrow recipients. Indeed, we observed an aging-associated decrease in co-localized VGlut1 (pre-synaptic) and Homer1 (post-synaptic) puncta in control mice, and preservation of co-localized puncta in recipients of young, but not old, bone marrow (Fig. 3d and Supplementary Fig. 3e). Moreover, Golgi impregnation of hippocampal neurons revealed a greater spine density in young, but not old, bone marrow recipients compared to old controls (Fig. 3e).

The complement factors C1qA and C3 are both over-expressed in the aging hippocampus and have been implicated in aging-associated cognitive decline19,21. C3 deposition on synapses labels them for detection by microglia, thereby inducing synaptic elimination19,22,23, which may be aberrantly over-active during aging20. We therefore assessed complement expression in the hippocampus, and found that C1qa and C3 mRNA levels were lower in young, but not old, bone marrow recipients (Fig. 3f). We also observed reduced C3 deposition on synapses (co-localized Homer1 and C3 puncta; Supplementary Fig. 3f) in young, but not old, bone marrow recipients compared to old control mice (Fig. 3g).

We next evaluated reactive astrocytes and microglia, which have also been shown to contribute to cognitive decline in aging23,24. We found no difference between the groups in the number of reactive glial fibrillary acidic protein (GFAP)+ astrocytes in the hippocampus (Fig. 4a and Supplementary Fig. 4a), but higher Gfap mRNA expression in old control mice and old bone marrow recipients compared to young controls (Fig. 4b). In contrast, Gfap mRNA levels in young bone marrow recipients were reduced to levels seen in young control mice (Fig. 4b). We also evaluated astrocyte hypertrophy by assessing the area and perimeter of GFAP+ cells, both of which were increased in old control mice (although only the area reached statistical significance; Fig. 4c). However, we did not observe a statistically significant reduction in astrocyte hypertrophy in the young bone marrow recipients, so preserved cognition could not be attributed to normalization of astrocyte morphology.

Fig. 4 Young BMT reduced the aging-associated activation of microglia. Activation of astrocytes and microglia was evaluated 6 months post-transplantation. a, b Astrogliosis was evaluated by counting GFAP+ cells in the hippocampus (a) and by assessing GFAP expression by RT-PCR (b). c Astrocyte hypertrophy was assessed by measuring the mean area and perimeter of GFAP+ cells. d–f Microglia were quantified by counting Iba1+ cells in the hippocampus (d, e) and by assessing Iba1 expression by RT-PCR (f). g–j The morphology of Iba1+ cells (g) was assessed by measuring their mean soma area (h), process complexity (i) and process length (j). k–m Microglial activation was also evaluated by assessing the proportion of Iba1+ cells with intense CD68 staining (k), Cd68 mRNA expression by RT-PCR (l), and proportion of Iba1+ cells with intense LAMP1 staining (m). Scale bars: 50 μm in d, 5 μm in g. RT-PCR data were normalized to Gapdh mRNA. For histological analyses, n = 5–8 mice per group, pooled from 2 independent experiments; for RT-PCR experiments, n = 5 mice per group. The dotplots show mean plus standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001 (ANOVA with Tukey-Kramer post-hoc test) Full size image

Microglia in the hippocampus are specifically vulnerable to aging25. While the number of Iba+ microglia was similar in all groups (Fig. 4d–f and Supplementary Fig. 4b), their activation state was very different (Fig. 4d, g–m and Supplementary Fig. 4b). Specifically, the microglia of old control mice exhibited a ‘reactive’ morphology26, with larger soma, as well as fewer and shorter processes than those of young control mice (Fig. 4g–j). This phenotype was reversed in recipients of young, but not old, bone marrow (Fig. 4g–j). In addition, quantification of reactive microglia by immunostaining for CD68 and LAMP1 revealed that more Iba1+ microglia co-stained with these lysosomal markers in old control mice than young control mice (Fig. 4d, k, m and Supplementary Fig. 4b), and Cd68 mRNA levels were also elevated (Fig. 4l). This phenotype was also completely reversed in recipients of young, but not old, bone marrow (Fig. 4d, k–m and Supplementary Fig. 4b). Thus, synapse preservation in the young bone marrow recipients may be a consequence of reduced phagocytic engulfment of synapses by microglia.

Impact of heterochronic BMT on CCL11 and β2-microglobulin

We next explored the mechanisms underlying the reduced activation of microglia. Previous studies have shown that donor-derived CD11b+ cells can be found in the choroid plexus and meninges of recipient mice following BMT, but rarely infiltrate the brain parenchyma in the absence of acute damage26. Similarly, we did not observe donor-derived cells in the brain 3 weeks after transplantation of young GFP-transgenic bone marrow into old mice (Supplementary Fig. 5a), but 3 months post-transplantation, GFP+ CD11b+ cells were seen in the choroid plexus and meninges (Supplementary Fig. 5a–c). This is consistent with previous reports that choroid plexus and meningeal macrophages are replaced throughout life26. However, donor-derived cells were not observed in the brain parenchyma (Supplementary Fig. 5a, d). Thus it is likely that the preservation of cognitive function is not due to direct effects of young hematopoietic cells in the hippocampus.

We therefore next investigated whether heterochronic BMT impacts the levels of circulating factors previously implicated in brain aging, focusing on β2-microglobulin and CCL11. Levels of β2-microglobulin and CCL11, which have been shown to suppress neurogenesis upon systemic injection into young mice, are elevated in the circulation and cerebrospinal fluid (CSF) of old mice and humans, and also increased in young parabionts following heterochronic parabiosis3,10,11. As expected, we observed higher plasma levels of both β2-microglobulin and CCL11 in old control mice than young control mice (Fig. 5a). Notably, however, plasma levels of CCL11, but not β2-microglobulin, were reduced in recipients of young (but not old) bone marrow (Fig. 5a). This suggests that non-hematopoietic cells are the main source of β2-microglobulin in old mice, while CCL11 is either derived from hematopoietic cells or produced by non-hematopoietic cells under the influence of hematopoietic cells. Moreover, it raises the possibility that reduced CCL11 production may underlie the preservation of cognitive function in old mice following young BMT.

Fig. 5 Circulating CCL11 levels were reduced in young bone marrow recipients and CCL11 injection into young mice mimicked hippocampal aging. a CCL11 and β2-microglobulin levels in the plasma of control mice and BMT recipients were quantified by ELISA. n = 12–20 mice per group, pooled from 2 independent experiments. b–i CCL11 was administered by intraperitoneal injection into young (4-month) mice (4 injections over 10 days), prior to histological and molecular analyses of the hippocampus. b Neuron numbers in the CA1 and CA3 regions were assessed by NeuN staining. n = 8–18 mice per group, pooled from 2 independent experiments. c Synapses were evaluated by co-localization of VGlut1 (pre-synaptic) and Homer1 (post-synaptic) markers. n = 14 mice per group, pooled from 2 independent experiments. d Spine density was assessed and quantified by Golgi impregnation of CA3 neurons. Scale bar: 2.5 μm. n = 5 mice per group. e C3 deposition on synapses was evaluated by C3 co-localization with Homer1. n = 16 mice per group, pooled from 2 independent experiments. f–i Microglial activation was assessed by measuring the percentage of Iba1+ cells with cytosolic co-staining of CD68 (f) or LAMP1 (g), as well as the complexity (h) and length (i) of microglial processes. n = 19–20 mice per group (CD68 and LAMP1 counts) or 10 mice per group (microglial morphology), pooled from 2 independent experiments. The dotplots show mean plus standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (a—ANOVA with Tukey-Kramer post-hoc test; b–i—Student’s t-test) Full size image

Effects of CCL11 injection into young mice

Microglia express higher levels of the CCL11 receptors CCR2, CCR3 and CCR5 than other brain cell types (including neurons and astrocytes)27. We therefore hypothesized that increased CCL11 production might be responsible for activation of microglia and aberrant synaptic pruning in old mice. To investigate this, we administered recombinant CCL11 to young mice via intraperitoneal injection, which achieves a physiologically relevant increase in circulating CCL11 levels3. Consistent with a previous study3, this resulted in reduced numbers of DCX+ newly-born neurons (Supplementary Fig. 6a). We also observed a reduction in CA3, but not CA1, region hippocampal neurons (Fig. 5b). The number of synapses (co-localized VGlut1 and Homer1 puncta) also decreased following CCL11 injection (Fig. 5c), as did the average spine density of Golgi-impregnated neurons (Fig. 5d). Moreover, quantification of co-localized Homer1 and C3 puncta showed that systemic CCL11 injection increased C3 deposition on synapses (Fig. 5e), which indicated that the reduction in synapses may be a consequence of pruning by microglia. Interestingly, astrocyte activation (GFAP+ cell number and Gfap mRNA expression) was unaffected by CCL11 administration (Supplementary Fig. 6b, c), and Iba1+ microglial numbers were unchanged (Supplementary Fig. 6d). In contrast, microglia in the hippocampus were more reactive, with increased CD68 and LAMP1 expression, as well as fewer and shorter processes (Fig. 5f–i and Supplementary Fig. 6e). Thus CCL11 injection reproduced the synaptic loss and microglial reactivity observed in old mice.