Vascular niche contribution to age-associated neural stem cell dysfunction

Abstract

Neural stem cells (NSCs) persist throughout life in the dentate gyrus and the ventricular-subventricular zone, where they continuously provide new neurons and some glia. These cells are found in specialized niches that regulate quiescence, activation, differentiation, and cell fate choice. A key aspect of the regulatory niche is the vascular plexus, which modulates NSC behavior during tissue homeostasis and regeneration. During aging, NSCs become depleted and dysfunctional, resulting in reduced neurogenesis and poor brain repair. In this review, we discuss the emerging evidence that changes in the vascular niche both structurally and functionally contribute to reduced neurogenesis during aging and how this might contribute to reduced plasticity and repair in the aged brain.

the mammalian brain contains discrete regions of neural stem cells (NSCs) located in the ventricular-subventricular zone (V-SVZ), which lines the lateral ventricle, and the subgranular zone (SGZ) of the hippocampus (Fig. 1A) (1, 12, 14, 22). These NSCs undergo self-renewal throughout life and have the capacity to give rise to neurons, oligodendrocytes, and glia (12−14). In vivo, NSCs preferentially give rise to neurons, which are continuously turned over and thought to provide plasticity for learning and memory across the lifespan (7, 12, 15, 56). NSCs also proliferate in response to injuries, such as brain ischemia and traumatic brain injury, and, in the case of the V-SVZ, migrate out of their niche to the site of injury to give rise to a limited number of neurons and glia (28, 71–73). However, the capacity for neurogenesis and functional integration of neurons declines with age, leaving fewer proliferating NSCs and neuroblasts, which results in the geriatric brain having fewer options for plasticity and repair (8, 37, 38, 53). Extensive investigations are ongoing as to the cause of this decline in NSC function and number with age. Recent studies have pointed to the importance of the vasculature in the maintenance of NSCs, and it is currently thought that aging-related changes to the vasculature may mediate some of the deficits observed in these neurogenic regions. Maintenance of neurovascular integrity, especially in neurogenic regions, may be a viable therapeutic target to improve brain aging.

Fig. 1.

Aging results in reduced vascular density, proliferation, and neuroblast production. A: neural stem cells reside in the ventricular-subventricular zone (V-SVZ) and the subgranular zone (SGZ) in the hippocampus. V-SVZ neuroblasts migrate to the olfactory bulb (OB) via the rostral migratory stream (RMS), where they mature into neurons. B: a low-magnification image of the V-SVZ microdissected as a whole mount and immunostained for the endothelial marker CD31 showing the vascular plexus within the niche. Scale bars = 500 µm. C: doublecortin (DCX)-positive neuroblasts migrate in long chains through the young V-SVZ. Scale bar = 50 µm. D: the number of DCX-positive neuroblasts decreases with age in the V-SVZ, and the structure of the migrating neuroblast chains is compromised. Scale bar = 50 µm. E: proliferating neural stem cells (NSCs) labeled with 5-ethynyl-2′-deoxyuridine (EdU; green) are closely associated with the CD31-positive vasculature (red) in the young V-SVZ. Scale bars = 20 µm. F: the number of proliferating NSCs (EdU; green) declines in parallel with vascular density (CD31; red) in the aged V-SVZ. Scale bars = 20 µm.

The Vascular Niche Defined and Age-Associated Niche Changes

The “vascular niche” was first defined in the SGZ, where endothelial cells and NSCs were found to proliferate together near the vasculature. However, the role of the vasculature in NSC regulation has primarily been characterized in the young V-SVZ, and for the purpose of this review, we will mostly describe the molecular and structural properties of the vasculature in this region. In the V-SVZ, NSCs are highly organized into different compartments of the neurogenic niche (14). This arrangement is thought to be important in segregating signals for regulation of NSC quiescence, activation, proliferation, and migration (1, 20, 33, 42, 52, 54). V-SVZ NSCs, termed type B cells, exist in both a quiescent and activated state and are clustered near the ependymal layer lining the lateral ventricle. Type B NSCs extend an apical process into the lateral ventricle, making direct contact with cerebrospinal fluid, and a basal process toward a vast vascular plexus (Fig. 1B) that separates the niche from the underlying striatum (13, 42). This unique structure allows type B cells to receive signals from both the ependymal/ventricular compartment and the vascular compartment. Type B NSCs undergo asymmetric division to self-renew and give rise to a highly proliferative, transit amplifying cell (TAC), also termed a type C cell, which enlarges the progenitor pool and preferentially gives rise to fate-committed neuroblasts (type A cells) (12, 14). In the rodent, neuroblasts migrate out of the V-SVZ in chains (Fig. 1C) into the rostral migratory stream until they reach the olfactory bulb, where they use blood vessels as a physical substrate to migrate into their final position within the olfactory bulb (5, 6, 70). Both activated type B NSCs and TACs have been shown to preferentially divide near the vasculature (Fig. 1E) (52, 63). Interestingly, the blood-brain barrier in the V-SVZ is modified to have fewer astrocytic endfeet and less pericyte coverage, with proliferating NSCs and TACs making direct contact with endothelial cells. Furthermore, these modifications result in increased vascular permeability resulting in increased exposure of the V-SVZ to small molecules from the blood (52, 63). Thus, NSCs and TACs are uniquely poised to receive signals from both endothelial cells and the blood.

In the aging V-SVZ, structural and cell-intrinsic changes occur that contribute to diminished neurogenesis. The V-SVZ thins and the numbers of proliferating cells decline (Fig. 1F) (37, 40). The numbers of neuroblasts migrating to the olfactory bulb are reduced (Fig. 1D), resulting in functional deficits in olfactory memory (5, 18, 48). In the aged V-SVZ, the G₁ phase of the cell cycle is lengthened, leaving a larger portion of the remaining progenitor cells in a proliferative state (9, 10, 53), likely as an attempt to mitigate depletion of the NSC pool. Inflammatory markers are increased in the aged V-SVZ, with activated microglia and proinflammatory cytokine levels rising by mid-age, promoting an antineurogenic microenvironment (17, 43, 57). These changes to the niche and NSCs result in poor functionality of the neurogenic region and a reduced capacity for neurogenesis with age.

The Structural Integrity of the Vasculature Declines with Age

Cerebrovascular insufficiency is well documented in many pathological states as well as in the healthy aging brain (41, 58, 60). Density of the capillaries declines across the aged brain, including in the V-SVZ (5, 31, 58). The close proximity of NSCs to the vasculature is important for receiving a myriad of trophic and chemotactic signals from endothelial cells and blood that direct lineage progression and migration of NSCs (see Table 1). It is likely that age-related vascular rarefaction contributes to diminished neurogenesis in the aged brain due to decreased exposure to vascular-derived signals. In addition, V-SVZ blood vessels have been shown to undergo a 90° rotation in their orientation with age in the ventral V-SVZ, possibly due to stenosis of the lateral ventricle wall wherein the lateral and medial walls of the ventral V-SVZ become sealed together (38). Further evidence for remodeling of the vasculature within the V-SVZ with age is observed as a decrease in blood vessel volume, and subsequently diminished blood flow, in the aged V-SVZ compared with young V-SVZ as measured by magnetic resonance imaging (31). This suggests that the decrease in vascular density is likely contributing to poor perfusion of the tissue and reduced exposure to circulating factors that direct NSC activity. Together, these studies have shown age-related remodeling of the V-SVZ vasculature is robust and provide evidence of the vasculature as a critical component of the NSC niche (summarized in Fig. 2). The specific types of blood vessels that are affected in the aged V-SVZ, as well as the mechanisms by which vascular remodeling is achieved, have not yet been delineated. More detailed studies in the aging neurogenic niches are needed to address these important questions and begin to identify candidate molecules to prevent vascular remodeling and reduced neurogenesis in the aging brain.

Table 1.

Endothelial and circulating factors that affect neurogenesis

Circulating Factor	Change With Age	Effect On Neurogenesis When Circulating Factor Is Manipulated in the Aged Neurogenic Niche (References)
GDF11	↓	Neurogenesis rescued by exogenous GFD11 in aged animals (31, 65)
SDF1	↓	SDF1 antagonist impaired hippocampal learning and memory (47)
VEGF	↔	VEGF overexpression increases neurogenesis in adult hippocampus (35)
NT-3	↑	NT3 knockdown increases NSC proliferation (11)

Multiple endothelial and circulating factor levels increase or decrease with age and impact neurogenesis. Experiments in which these circulating factors have been restored to levels observed in young mice are summarized on the right. GDF11, growth differentiation factor 11; SDF1, stromal-derived factor 1; VEGF, vascular endothelial growth factor; NT3, neurotrophin-3; NSC, neural stem cell.

Fig. 2.

Cartoon depicting the age-related remodeling of the neurogenic niche. Left: young ventricular-subventricular zone (V-SVZ) neurogenic niche depicting abundant quiescent and activated type B cells (green), transit amplifying type C cells (blue), and migrating type A neuroblasts (yellow). The young V-SVZ has high vascular density (red). Age-related changes (middle) alter the proliferative and neurogenic capacity of the V-SVZ, resulting in fewer type B, type C, and type A cells (left). This is concomitant with a decrease in vascular density and structural changes, such as thinning and stenosis, in the aged V-SVZ.

Vascular Regulation of NSCs in the Young and Aged Brain

Early studies using cocultures of endothelial cells with NSCs showed that the vasculature secretes soluble factors that can promote self-renewal and proliferation of both fetal and adult NSCs. The endothelial secretome also increased the percentage of cells that differentiate into neurons as opposed to glia (51). Since then, studies have been undertaken in an attempt to identify the molecules secreted from endothelial cells that are important in NSC homeostasis and aging, although the exact nature and function of these signals are not yet fully characterized. More study will be needed to fully characterize the endothelium-derived molecules that support neurogenesis, and this is especially true in the context of aging, which remains almost completely unexplored. However, a few candidate molecules secreted by endothelial cells have been identified. For example, transplantation studies of NSCs into the V-SVZ niche of young mice revealed that activated type B NSCs and TACs migrate toward the vasculature by responding to the chemokine stromal-derived factor 1 (SDF1), which is secreted by the endothelial cells of blood vessels, creating a chemoattractive gradient (33). SDF1 recruits these activated NSCs to the vasculature by binding to its transmembrane receptor, chemokine (C-X-C motif) receptor-4 (CXCR4), which is highly expressed on V-SVZ progenitor cells. This suggests that endogenous NSCs also use this mechanism to home to the vascular compartment. Furthermore, exogenous treatment of cultured NSCs with SDF1 results in increased expression of the mitogen receptor epidermal growth factor receptor (EGFR), suggesting that SDF1 contributes to the proliferation of NSCs and TACs near the vasculature (33). It is unclear if SDF1/CXCR4 signaling is dysregulated during aging in the V-SVZ; however, this homing mechanism is compromised with age in multiple peripheral progenitor cell populations. In hematopoietic stem cells, SDF1/CXCR4 signaling decreases with age, resulting in poor engraftment of stem cells in the bone marrow compartment (23). Additionally, expression of CXCR4 is also decreased with age in progenitors isolated from bone marrow and the thymus, leading to dysfunctional chemotaxis and inefficient homing of CXCR4-expressing cells to their destined niche (23, 25, 50). In the brain, age-related decreases in SDF1/CXCR4 signaling have been described in a mouse model of Alzheimer’s disease, where the decline in SDF1 levels leads to deficits in hippocampal-dependent learning and memory (47). These deficits in SDF1/CXCR4 signaling in aged stem cell populations likely extend to the V-SVZ, which may contribute to the age-associated reduction in neurogenesis; however, studies are needed to directly test this hypothesis.

Vascular endothelial growth factor (VEGF) is a growth factor that stimulates angiogenesis by binding its receptor tyrosine kinase on endothelial cells, producing an outgrowth of blood vessels in both healthy tissue and in response to injury. In the young adult V-SVZ niche, VEGF is potent neurogenic factor that has been shown to increase the number of proliferating progenitors both in vivo and in vitro (29, 35). This dual role of VEGF as both a neurogenic and angiogenic growth factor in the V-SVZ is not unexpected given the close proximity of NSCs to the vasculature and is likely a critical response mechanism for the brain during ischemic injury, which occurs more frequently with age. VEGF preconditioning immediately after generation of new blood vessels in the hippocampus promotes long-term neurogenesis in aged mice (35). Cells in the V-SVZ niche become less responsive to VEGF with age, wherein both angiogenesis and neurogenesis are reduced, even when VEGF levels are “rescued” by overexpression in the aged brain (21). This finding suggests that an intrinsic change, likely at the VEGF receptor-2, occurs with age and diminishes the proliferative effects of VEGF (21).

Neurotrophin-3 (NT-3) is a neurotrophic factor that is spatially restricted to the heart and nervous system in adult mammals and is particularly active in neurogenic brain regions (3, 11). In the V-SVZ, NT-3 expression is restricted to choroid plexus and vascular endothelial cells (11). Knockdown of NT-3 results in increased proliferation of type B NSCs, suggesting that in addition to proliferation, endothelial cells can also contribute to the signals needed for quiescence (11). When NT-3 binds to its receptor, it rapidly results in phosphorylation and activation of the endothelial isoform of nitric oxide synthase in type B NSCs and promotes cytostasis by increasing nitric oxide production (11). By mid-age in NT-3-deficient mice, the stem cell pool is exhausted, resulting in fewer newborn neurons, indicating that NT-3 is necessary for preventing premature differentiation and loss of NSCs in the aging V-SVZ (11).

In addition to molecules secreted by endothelial cells, evidence suggests that molecules in the blood support NSC function in young mice, whereas blood from aged mice has either a reduced amount of proneurogenic molecules or molecules that directly inhibit neurogenesis. This evidence comes from heterochronic parabiosis experiments, where a young and aged rodent’s circulatory systems are surgically connected to one another such that the two animals now share a single circulatory system. In heterochronic parabiosis experiments, circulating factors from the young mouse restore the cerebrovascular architecture in the aged V-SVZ, increasing vascular density and restoring cerebral blood flow to levels observed in young mice (31). This restoration of the vasculature is accompanied by a partial restoration of V-SVZ neurogenesis (31). Heterochronic parabiosis experiments have also revealed that young circulating factors increase neurogenesis in the SGZ and result in improvements in hippocampal-dependent learning and memory in aged mice (65). Remarkably, this increase in hippocampal SGZ neurogenesis and cognitive function can also be achieved by isolating plasma from a young mouse and injecting it into an aged mouse via the tail vein (66), suggesting that plasma carries some of these proneurogenic molecules. In contrast, young mice saw reduced neurogenesis when exposed to old plasma (66). Many molecules in blood are likely to rejuvenate the aging niche. One candidate molecule for this effect is growth differentiation factor 11 (GDF11), which is reduced in the blood during aging. Injections of GDF11 into the bloodstream have been reported to mediate vascular remodeling and restore CD31-positive blood vessel volume in the V-SVZ of aged mice, although the specific blood vessels involved in the vascular remodeling have not been identified (31). This improvement in vascular function correlated with an increase in Sox2-positive cells, a marker for NSCs and TACs, in the aged V-SVZ (31). GDF11 has also been shown to improve the function of cardiac cells and muscle stem cells (36, 55). However, others have shown GDF11 to have the opposite or no effect (16, 68). These disparities in findings suggest further study is needed to clarify if GDF11 is a regenerative molecule.

Endogenous NSCs Respond Poorly to Ischemia in the Aged Brain

Brain ischemia or trauma stimulates NSC proliferation and maturation into neuroblasts, which migrate to the site of injury (4, 64). A limited number of these neuroblasts differentiate and integrate into the existing circuitry (2, 69), while others serve as neurotrophic factors that protect existing cells from injury and death (4). Blockade of neuroblast migration results in increased lesion volume and behavioral deficits (26, 64). Recent experiments have measured a marked increase in astrocytes postinjury and traced their lineage back to the V-SVZ progenitor cells (4, 19), however, whether these NSC-derived astrocytes are reparative or contribute to ischemic pathology is unclear. Despite the potential of NSCs to influence outcome after injury, brain repair is often limited, even in young mice. Elucidation of the molecular and cellular regulators of NSC proliferation will be important to increase the repair response following injury. However, these data illustrate the importance of cues from the microenvironment on NSC regulation, particularly after tissue damage. It is established that exogenous stem cell transplantation into areas of brain injury can decrease the infarct size and increase recovery after stroke (24). However, transplants often lead to unintended consequences, such as further tissue damage during implantation and immune rejection (1, 24). Mechanisms to enhance endogenous NSC involvement in the repair process after ischemic, traumatic, or neurodegenerative damage are less invasive and decrease stress on an already overburdened brain.

Angiogenesis is also altered in brain injury models. Along with neurogenesis, angiogenesis is upregulated in response to ischemia, with a substantial increase in blood vessel volume in the V-SVZ (24, 71) and in the adjacent striatum (64) in the weeks after induction of a middle cerebral artery occlusion (MCAO). New blood vessels combine with the existing vascular network to act as a scaffolding for NSC migration toward the site of injury (32, 49, 69). Angiogenesis appears to be necessary for NSC-mediated repair, as its inhibition substantially decreases the number of new neurons at the lesion, whereas enhancement of angiogenesis via endothelial progenitor cell transplants enhances neuronal migration (46, 61). When endothelial cells and neural progenitor cells are cotransplanted into the ischemic zone, proliferation, survival, and differentiation of neural progenitors are increased compared with that observed with transplantation of either cell type alone (44). Likewise, increases in VEGF levels under ischemic conditions led to increases in angiogenesis, neurogenesis, and neuronal migration toward the lesion (34, 59, 67, 74). Together, these studies provide evidence of the intricate and indispensable relationship between the vasculature and NSCs, particularly when faced with the challenge of brain repair.

The aged brain is highly susceptible to ischemic stroke, and the risk for an infarct increases dramatically with age (39). Given the distinct structural, functional, and metabolic changes experienced in the aging brain and vasculature, it is no surprise that cerebrovascular deficits are more difficult to address in geriatric patient populations (27, 30, 45). Much of the experimental rodent data to date has been derived primarily from work in young adult animals, which have not yet experienced the age-related changes that are common to most mammalian brains. However, several investigators have begun to examine the activity of NSCs in a more clinically relevant aged animal model of ischemic stroke. Tang et al. (62) examined the role of the aging microenvironment by transplanting NSCs from young rats into the striatum of young and aged rats using the MCAO model. Both young and aged animals showed a decreased striatal lesion volume and improved neurobehavioral scores, although the aged rats did not benefit as dramatically from the NSC transplant compared with young rats after MCAO (62). Curiously, NSC transplant promoted VEGF expression and endogenous angiogenesis and neurogenesis in aged rats (62), supporting the reciprocal relationship between VEGF expression and NSC response to brain damage. Other work has also found an age-dependent effect of ischemia on neurogenesis: transient MCAO produced an increase in 5-bromo-2′-deoxyuridine-labeled cells at 24 h after infarct in both young and aged rats, although again the number of new neurons in the young ischemic rat is nearly double the number observed in the aged rat (28).

Conclusions

Vascular challenges in the aging brain are notoriously difficult to address clinically, and even moderate dysfunction is likely to be detrimental to neurogenesis. Structurally, the vasculature of the neurogenic niche is optimized to support NSC activation, proliferation, differentiation, and migration by providing both chemical signals and physical structures that direct NSC function and activity. Numerous vascular-derived factors work in concert to balance the quiescent NSC population with activated NSCs undergoing lineage progression. This delicate balance is disrupted with age as the vasculature undergoes remodeling and rarefaction and develops functional deficits, thereby depriving NSCs of the specialized microenvironment needed to maintain function. This is observed in both studies of normal aging in the V-SVZ as well as in experimental ischemia and brain injury models. Preservation of the V-SVZ vasculature and reduction of endothelial impairment is a mechanism by which neurogenesis may be protected in the aging brain, resulting in a greater endogenous healing potential and improved clinical outcomes in geriatric patients. Much of what is currently known about vascular effects on neurogenesis has been determined from studies using young rodents, but to fully understand the delicate interactions between the aging microvasculature and NSCs, it is necessary to perform these studies in aged animals. By examining the vascular effects on the V-SVZ in the aged rodent brain, we may be able to identify, develop, and implement more effective interventions that support aging NSC function.

GRANTS

This work was supported by the Owen’s Medical Foundation and National Institute on Aging Grant T32-AG-021890.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

D.M.A. and E.K. drafted manuscript; E.K. edited and revised manuscript; E.K. approved final version of manuscript.