Adult neurogenesis occurs in local microenvironments, or neurogenic niches in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the hippocampus2,3
. Permissive cues within the neurogenic niche are thought to drive the production of new neurons and their subsequent integration into the neurocircuitry of the brain4,5
, directly contributing to cognitive processes including learning and memory6–8,9
. Importantly, the neurogenic niche is localized around blood vessels10,11
, allowing for potential communication with the systemic environment. Therefore, the possibility arises that diminished neurogenesis during aging may be modulated by the balance of two independent forces – intrinsic CNS-derived cues12–14
, and cues extrinsic to the CNS delivered by blood. Thus we hypothesized that age-related systemic molecular changes could cause a decline in neurogenesis and impair cognitive function during aging.
We first characterized cellular, electrophysiological and behavioral changes associated with the neurogenic niche in the dentate gyrus (DG) of the hippocampus in an aging cohort of mice. We observed cellular changes consistent with dramatically decreased adult neurogenesis1
and increased neuroinflammation with age15
(Supplementary Fig. 2a–e
). Additionally, we detected deficits in synaptic plasticity (Supplementary Fig. 3a–c
), and behavioral deficits in contextual fear conditioning (Supplementary Fig. 4a–c
) and radial arm water maze (RAWM; Supplementary Fig. 4d–f
) paradigms in old animals, consistent with decreased cognitive function during aging16
Next we investigated the contribution of peripheral systemic factors to the age-related decline in neurogenesis in the DG of the hippocampus in the setting of isochronic (young-young and old-old) and heterochronic (young-old) parabiosis (). Remarkably, the number of Doublecortin (Dcx)-positive newly born neurons (), BrdU-positive cells (), and Sox2-positive progenitors (Supplementary Fig. 5a,b
) decreased in young heterochronic parabionts. In contrast, we observed an increase in the number of Dcx-positive (), BrdU-positive () and Sox2-positive (Supplementary Fig. 5a,c
) cells in the old heterochronic parabionts. The number of Dcx-positive neurons between unpaired age-matched animals and isochronic animals showed no difference (Supplementary Fig. 6a,b
). As a control flow cytometry analysis confirmed a shared vasculature in a subset of parabiotic pairs, in which one parabiont was transgenic for green fluorescent protein (GFP, Supplementary Fig. 7a–d
). Together our findings suggest that global age-dependent systemic changes can modulate neurogenesis in both the young and aged neurogenic niche, potentially contributing to the decline in regenerative capacity observed in the normal aging brain.
Heterochronic parabiosis alters neurogenesis in an age-dependent fashion
As previously reported by others17
, we rarely detected peripherally derived GFP cells in the CNS of wild-type mice, and these numbers did not differ between isochronic and heterochronic pairings (Supplementary Fig. 7e
), suggesting the observed effects are mediated by soluble factors in plasma. To confirm that circulating factors within aged blood contribute to reduced neurogenesis with age, we intravenously injected plasma isolated from young or old mice into young adult animals (). The number of Dcx-positive cells in the DG decreased in animals receiving old plasma compared to animals receiving young plasma (), indicating that soluble factors present in old blood inhibit adult neurogenesis.
Factors from an old systemic environment decrease neurogenesis and impair learning and memory
To investigate the functional effect of the aging systemic milieu, extracellular electrophysiological recordings were done on hippocampal slices prepared from young isochronic and heterochronic parabionts (, Supplementary Fig. 6c
). A decrease in long-term potentiation (LTP) in the DG of heterochronic parabionts was detected (). These data indicate that age-related systemic changes can elicit deficits in synaptic plasticity. As LTP is considered a correlate of learning and memory18
, these finding suggest that age-related systemic changes may also impact cognitive functions during aging.
Subsequently, we tested hippocampal dependent learning and memory using contextual fear conditioning and RAWM paradigms in young adult mice intravenously injected with young or old plasma (). During fear conditioning training mice exhibited no differences in baseline freezing regardless of plasma injection treatment (Supplementary Fig. 8a
). However, mice receiving old plasma demonstrated decreased freezing in contextual (), but not cued (Supplementary Fig. 8b
), memory testing. During the training phase of the RAWM all mice showed similar swim speeds (Supplementary Fig. 8c
) and spatial learning capacity for the task (). However, during the testing phase animals administered with old plasma demonstrated impaired learning and memory for platform location (). As a control, we tested the RAWM paradigm in young adult mice with ablated hippocampal neurogenesis and observed corresponding behavioral deficits (Supplementary Fig. 9a–e
). Collectively, these data indicate that factors present in aging blood inhibit adult neurogenesis, and moreover functionally contribute to impairments in cognitive function.
Thus far circulating factors associated with aging and tissue degeneration, or tissue rejuvenation, have remained elusive in earlier studies of parabiosis19
. To identify such factors, we employed a proteomic approach in which relative levels of 66 cytokines, chemokines and other secreted signaling proteins were measured in the plasma of normal aging mice using standardized multiplex sandwich ELISAs (Luminex; Table S1
). Using multivariate analysis, we identified seventeen proteins whose levels increased and correlated with decreased neurogenesis during aging (, Supplementary Fig. 10a,b
). To identify systemic factors associated with heterochronic parabiosis, we analyzed plasma samples from young and old animals before and after pairings in an independent proteomic screen. Comparison of young isochronic and heterochronic cohorts identified fifteen factors that increased in heterochronic parabionts (, Supplementary Fig. 10c
), while comparison between old isochronic and heterochronic cohorts revealed four factors that decreased in isochronic parabionts (Supplementary Fig. 10c
). Interestingly, only six factors – CCL2, CCL11, CCL12, CCL19, Haptoglobin and β2-microglobulin – were elevated in old unpaired and young heterochronic cohorts (). CCL11, at the top of this list, is a chemokine involved in allergic responses and not previously linked to aging, neurogenesis, or cognition. Relative levels of CCL11 were increased in plasma of mice during normal aging () and within young mice during heterochronic parabiosis (). Furthermore, we detected an age-related increase in CCL11 in plasma (3d) and cerebrospinal fluid (CSF; ), from health human individuals between 20 and 90 years of age, suggesting that this age-related systemic increase is conserved across species.
Systemic chemokine levels increase during aging and heterochronic parabiosis and correlate with decreased neurogenesis
Having identified CCL11 as an age-related systemic factor associated with decreased neurogenesis, we tested its potential biological relevance in vivo. We administered CCL11 protein through intraperitoneal injections into young adult Doublecortin-luciferase reporter mice20
, and using a non-invasive bioluminescent imaging assay detected a significant decrease in neurogenesis (Supplementary Fig. 11b,c
). Using immunohistochemical analysis we investigated the effect of systemic CCL11 on adult hippocampal neurogenesis in young wild type adult mice. We administered CCL11 or vehicle alone, and in combination with either an anti-CCL11 neutralizing antibody or an isotype control antibody through intraperitoneal injections (). Systemic administration of CCL11 induced an increase in CCL11 plasma levels (Supplementary Fig. 11a
), and significantly decreased the number of Dcx-positive cells in the DG (). Importantly, this decrease in neurogenesis could be rescued by systemic neutralization of CCL11 (). Likewise, BrdU-positive cells also showed similar changes in cell number (Supplementary Fig. 11d,e
), and furthermore the percentage of cells expressing both BrdU and NeuN decreased (Supplementary Fig. 11f,g
). The percentage of cells expressing BrdU and GFAP did not significantly change (Supplementary Fig. 11f,h
). As a negative control we assayed neurogenesis after systemic administration of monocyte colony stimulating factor (MCSF), a measured protein not changing in plasma levels with age, and detected no change in Dcx-positive cells in the DG (Supplementary Fig. 12a–d
). Together, these data indicate that increasing the systemic level of the age-related factor CCL11 is sufficient to partially recapitulate some of the inhibitory effects observed with aging and heterochronic parabiosis.
Systemic exposure to CCL11 inhibits neurogenesis and impairs learning and memory
Additionally, we investigated the possibility that age-related blood-borne factors influence neural progenitor activity and neural differentiation in vitro. We assayed the number of neurospheres formed after exposure of primary NPCs to aged mouse serum and observed a 50% decrease when compared to young serum (Supplementary Fig. 13a
). We then tested the effect of CCL11 and observed that the number and size of neurospheres formed from primary NPCs exposed to CCL11 significantly decreased (Supplementary Fig. 13b–d
). Using a human derived NTERA cell line expressing eGFP under the Doublecortin promoter, we assayed neural differentiation and observed a significant decrease in eGFP expression after twelve days in culture with CCL11 (Supplementary Fig. 13e,f
). While these findings open the possibility of a direct interaction of systemic factors with progenitor cells in vivo during aging, they do not preclude the possibility of indirect actions by interactions with other neurogenic niche cell types.
To examine the effect of CCL11 on neurogenesis in the brain we stereotaxically injected CCL11 into the DG of young adult mice, and observed a decrease in the number of Dcx-positive cells compared with the contralateral DG receiving vehicle control (Supplementary Fig. 14a,b
). Furthermore, we examined whether the inhibitory effect of peripheral CCL11 could be restored locally within the hippocampus. We stereotaxically injected CCL11-specific neutralizing antibody and isotype control antibodies into the contralateral DG of young adult mice (). Following stereotaxic injection, we systemically administered CCL11 or vehicle control by intraperitoneal injections (). The decrease in Dcx-positive cell number observed in animals receiving systemic CCL11 administration could be rescued by neutralizing CCL11 within the DG (), suggesting the increase in systemic chemokine levels exerts a direct effect in the CNS.
Finally, to determine the physiological relevance of increased systemic CCL11 levels in mice we assessed hippocampal dependent learning and memory using contextual fear conditioning and RAWM paradigms (). Young adult mice received intraperitoneal injections of recombinant CCL11 or vehicle control. During fear conditioning training all mice, regardless of treatment, exhibited no differences in baseline freezing (Supplementary Fig. 15a
). However, mice receiving CCL11 demonstrated decreased freezing during contextual (), but not cued (Supplementary Fig. 15b
), memory testing. During the training phase of the RAWM task all mice regardless of treatment showed similar swim speeds (Supplementary Fig. 15c
) and learning capacity for the task (). However, by the end of the testing phase animals receiving CCL11 exhibited impaired learning and memory deficits (). Together, these functional data demonstrate that increasing the systemic level of CCL11 cannot only inhibit adult neurogenesis, but also impair learning and memory.
Cumulatively, our data link age-related molecular changes in the systemic milieu to the age-related decline in adult neurogenesis, and impairments in synaptic plasticity and cognitive function observed during aging (Supplementary Fig. 1
). While local immune signaling in the brain is emerging as a critical modulator of NPC function11,15,21,22
, we now identify systemic immune-related factors as potentially critical contributors to the susceptibility of the aging brain to cognitive impairments. Interestingly, members of the identified age-related chemokines (CCL2, CCL11 and CCL12) are localized to within 70kB on mouse chromosome 11, and within 40kB on human chromosome 17, implicating this genetic locus in normal brain aging and possibly aging in general. Indeed, work investigating cellular senescence, a known hallmark of aging, furthers the involvement of some of the individual systemic chemokines reported here (CCL2) in the aging process as components of the Senescence-Associated Secretory Phenotype23
. Lastly, while the proteomic platform we used here was sufficient to identify systemic inhibitory ‘aging’ factors it will be critical to develop and utilize broader proteomic screens to facilitate the discovery of systemic pro-neurogenic ‘rejuvenating’ factors with the potential to ameliorate age-related cognitive dysfunction.