The peculiar plasticity of the CNS and of its NSCs manifests after CNS injury, when (i) proliferation and differentiation of NSCs is enhanced; (ii) striatal spiny interneurons and glutamatergic neurons are ectopically found in the injured cortex and in the striatum after stroke (Thored et al., 2007) while migrating neuroblasts become oligodendrocytes in area of demyelination after exiting the niche; (iii) ependymal cells behave as progenitors (Luo et al., 2008) and directly convert into neurons (Carlen et al., 2009); (iv) reactive astrocytes in ischemic brain injury exhibit self renewal capacity and multipotency (Buffo et al., 2008; Gabel et al., 2016).
The fact that non-neurogenic precursors can convert into neurogenic cells clearly highlights how exogenous factors can trigger plasticity within and outside of the niche (Carlen et al., 2009; Magnusson et al., 2014; Shetty and Hattiangady, 2016). Moreover, during pathology and steady-state, NSCs also exert trophic non-neurogenic functions which are crucial to maintain brain homeostasis.
In the following section we will explore how NSC niches function during CNS injury, with focus on stroke, multiple sclerosis and their animal models (Figures 1B,C, Table 1).
Effect of oxygen supply on neural stem cell plasticity, connection with stroke, and multiple sclerosis
In physiological conditions, neural stem cells are exposed to an oxygen concentration between 2.5 and 5.0%, which promotes NSC self-renewal via VEGF and erythropoietin (EPO) production induced by hypoxia inducible factor 1α, HIF1α (Pavlica et al., 2012; Li et al., 2014).
In the quiescent state, NSC mitochondria are quite immature with globular shape, do not depend much for energy on oxidative phosphorylation (OXPHOS), rather on glycolysis, with high lactate production (Zheng et al., 2016). Although glycolysis produces less ATP than mitochondrial OXPHOS, the pathway is very fast in NSCs (Ito and Suda, 2014). Their anaerobic metabolism is sustained mainly by mitochondria uncoupling protein 2 (UCP2), high level of hexokinase II and low pyruvate dehydrogenase to keep under control the production of reactive oxygen species (ROS) (Madhavan et al., 2006; Zheng et al., 2016). In this way, DNA and proteins of the cells are protected from ROS-dependent potential damage. Further, ROS, produced in limited amount in physiological conditions and normally neutralized, are also beneficial because they trigger self-renewal and neurogenesis (Le Belle et al., 2011). Conversely, differentiated cells present elongated, crystae-rich mitochondria, higher ratio of mitochondrial glucose oxidation (OXPHOS) over glycolysis as metabolic support (Zhang et al., 2014; Marcialis et al., 2016). Moreover, NSCs increase Krebs' cycle functionality and decrease lactate production, concurrent with increased number and total mitochondrial mass (Sola et al., 2013).
On the other side, the first consequence of the blood flow occlusion in brain ischemia is hypoxic injury that causes extensive neural cell death (Niquet et al., 2003). Nonetheless and surprisingly, hypoxia, while being detrimental in the acute phase of stroke, in a second step, contributes to promote neurogenesis (Arvidsson et al., 2002; Tonchev et al., 2003; Jin et al., 2006; Macas et al., 2006; Minger et al., 2007; Wang et al., 2011; Zhang and Chopp, 2016), to support replenishment of neurons in the RMS, migration in the region of ischemic brain injury, and growth of oligodendrocyte progenitors that disperse to the gray and white matter (Zhang et al., 2001, 2012; Parent et al., 2002; Minger et al., 2007; Li et al., 2010a; Zhang and Chopp, 2016).
Hypoxia has recently emerged as a concurrent complication of disease progression also in MS. Indeed, important lack of oxygen, occurring as a consequence of inflammation, has been measured in brain gray matter regions of MS patients, where it can per se be considered co-causative for neurodegeneration (Haider et al., 2014; Yang et al., 2015). Since hypoxia and inflammation are strictly linked, it is still difficult to tease apart their respective contributions in MS (Sun et al., 1998; Lassmann, 2003; Davies et al., 2013). It looks like hypoxia can occur in MS patients as a consequence of increased oxygen demand. When this request is not satisfied, hypoxia can be further detrimental for surrounding neural stem cells (Trapp and Stys, 2009).
Effect of inflammation on neural stem cell plasticity, connection with stroke, and multiple sclerosis
It has been well-documented that the brain cannot be simplistically considered an immune privileged site (Kleine and Benes, 2006) and actually immune system activation in the brain exerts both damaging and beneficial effects on CNS functions (Martino, 2004), depending on the onset of the inflammation, on the cell types involved in the process and on the chronicity of the response (Crutcher et al., 2006; Kyritsis et al., 2012).
NSCs share with the immune system an array of secreted mediators and receptors, which are all relevant for the maintenance of the neurogenic niche and, at the same time, represent the prerequisite for the interaction of NPCs with the microenvironment, especially during neuroinflammation (De Feo et al., 2012; Kokaia et al., 2012).
Indeed, inflammation, via its associated cues (i.e. cytokines, chemokines, chemical species….), strongly impacts structure and function of the stem cell niche, acts directly on NSCs and affects tissue restoration/regeneration via microglia and astrocyte activation (Ekdahl et al., 2003; Pluchino et al., 2008; Pourabdolhossein et al., 2017).
In homeostatic conditions, microglia are ramified in shape to maintain surveillance. Upon pathogen invasion or insults, they retract the protrusions, become amoeboid, increase their migratory behavior, and secrete cytokines. IL6, TNFα, IL1β, and complement 1 subunit q (C1q) are among the most potent microglia–derived cytokines, able to compromise the niche environment, inhibit neurogenesis (Vallieres et al., 2002; Monje et al., 2003), induce oligodendrogenesis (Valerio et al., 2002) and a subtype of reactive astrocytes (Liddelow et al., 2017). Further, while NSC-derived nitric oxide synthase promotes release of small amounts of nitric oxide (NO) with neurogenic properties (Carreira et al., 2010; Luo et al., 2010), abundant NO released by microglia or astrocytes in inflammatory conditions (i) inhibits proliferation of NSCs acting on the EGF receptor (Carreira et al., 2014) or on the transcription factor complex Neuron-Restrictive Silencer Factor /RE1-Silencing Transcription factor, NRSF/REST (Bergsland et al., 2014) and (ii) promotes gliogenesis (Bergsland et al., 2014).
IFNγ, a pleiotropic cytokine, has a central role in regulating NSCs proliferation and quiescence (Kulkarni et al., 2016). In inflammatory conditions, such as experimental autoimmune encephalomyelitis (EAE), the preclinical model of MS, IFNγ dramatically decreases progenitor proliferation (Pluchino et al., 2008; Pereira et al., 2015) and inhibits the recruitment of newborn neurons to the olfactory bulb (OB). IFNγ might also play a crucial role in regulating glioma-associated oncogene homolog-1 (Gli1), a Shh-induced trasncription factor that drives the oligodendroglial fate of NSCs (Wang et al., 2008; Li et al., 2010b; Samanta et al., 2015).
The complement system also inhibits NSC activity as demonstrated with complement receptor 2 (Cr2) knock out mice that have increased basal neurogenesis (Moriyama et al., 2011). Moreover, lipid modifiers present in inflammation, such as leukotriene, can negatively regulate NSC proliferation likely via TNFα and IL1β (Bonizzi et al., 1999; Wu et al., 2013). Of note, montelukast, a leukotriene receptor blocker protects from this event (Marschallinger et al., 2015).
IL6 is another crucial mediator of NSC function and its induction, e.g., during maternal infection and stroke, permanently perturbs NSC proliferation and neurogenesis (Gallagher et al., 2013; Chucair-Elliott et al., 2014; Meng et al., 2015).
Conversely, the anti-inflammatory interleukin-10 (IL10) in rodents keeps NSCs in an undifferentiated proliferation state, rather than promoting differentiation (Ben-Hur et al., 2003; Perez-Asensio et al., 2013; Wang et al., 2015).
Nevertheless, as anticipated, inflammation can be also beneficial for NSCs. First, the immune system in general and T cells in particular are involved in maintaining niche neurogenesis, as indeed genetic T-cell depleted mice present compromised cognitive functions (Ziv et al., 2006). Moreover, chemokines such as monocyte chemoattractant protein 1 (MCP1) or SDF1, released in inflammatory conditions, promote migration of NSCs to site of injury and local TNFα or IL1β trigger their proliferative capacity (Widera et al., 2006). Second, injection of moderately activated microglia can regulate brain homeostasis and neurogenesis (Butovsky et al., 2006; Bachstetter et al., 2011).
Further, NSCs directly produce inflammatory chemokines (Covacu et al., 2009), promoting a feed forward loop at sites of tissue damage (Imitola et al., 2004; Belmadani et al., 2006; Widera et al., 2006; Wang et al., 2007). NSCs can also directly change inflammatory responses through immunomodulatory factors (Pluchino et al., 2005; Ben-Hur, 2008; Yong and Rivest, 2009; Butti et al., 2012) or trophic factors (Huang et al., 2014). In particular, SVZ-NSCs can protect striatal neurons from the excitotoxic damage occurring in stroke and epilepsy, releasing endogenous endocannabinoids, likely upon inflammatory stimuli (Butti et al., 2012). Endogenous NSCs also secrete MCP1, VEGF, IL4, and interleukin-15 (IL15), the latter known to retain NK cells in the chronic phase of EAE in mice and of MS in humans. Indeed, in the SVZ, NK cells contribute to the dysfunction of the neurogenic niche observed in EAE by killing stem cells (Liu et al., 2016). Moreover, inflammatory cytokines lead to metabolic reprogramming of the arginase pathway in NSCs ultimately impacting on the NSC mediated anti-proliferative effect on T cells (Drago et al., 2016). Further, it has been reported that human NSCs hinder the differentiation of myeloid precursor cells into immature dendritic cells and the final maturation into functional antigen presenting cells (Pluchino et al., 2009).
In stroke, immune cell infiltration and inflammation take place as secondary events to hypoxic injury, which is responsible of the massive neuronal death. Upon brain injury, the blood-brain barrier (BBB) is disrupted, its permeability increases, migrating immature neurons associate with the angiogenic fenestrated endothelium, thus mimicking the neurogenic niche structure (Ohab et al., 2006; Ohab and Carmichael, 2008). Neutrophils and innate immune cells are the first players, although also T and B cells cross the damaged-BBB, inducing a rapid adaptive autoimmune response to neuronal antigens (Chamorro et al., 2012). As consequence of inflammation, neurogenesis is activated in stroke (Wang et al., 2011; Katajisto et al., 2015) supported by soluble factors such as bone morphogenic protein (Forni et al., 2013), retinoic acid (Plane et al., 2008), sonic hedgehog (Cheng et al., 2015), C-C motif chemokine Ligand 2 (CCL2) (Osman et al., 2016) along with SDF1 and angiopoietin-2 (Ang2) that home NSC progenitors to the site of injury (Thored et al., 2006), where they improve functional recovery (Guzman et al., 2008) and differentiate into neurons (Darsalia et al., 2007). Nevertheless, they present only limited reparative properties because matured neurons tend to die (Arvidsson et al., 2002; Marti-Fabregas et al., 2010; Kazanis et al., 2013). Moreover, given that NSCs themselves express and release respectively C-X-C motif chemokine receptor 4 (CXCR4) and SDF1, they might play a role in regulating axonal remodeling in ischemic brain. On the other hand, SVZ-derived astrocytes have a crucial role in ischemic stroke: photothrombotic cortical ischemia induces a strong gliogenic, Notch1-dependent response in the SVZ, which generates thrombospondin-4 (Thbs4)-positive astrocytes. Thbs4 astrocytes are essential components of the glial scar and their proliferation might be favored over neurogenesis after cortical injury (Benner et al., 2013). Conversely, it has also been reported that inflammation can promote neurogenesis in dormant neural progenitors in nonconventional neurogenic regions (Jiao and Chen, 2008).
Further, stroke increases generation of oligodendrocyte precursor cells (OPCs) from NSC in the ischemic brain, although it remains to be defined whether NSC-derived OPCs, beside oligodendrogenesis, help with brain repair, likely communicating with cerebrovasculature and other brain parenchyma cells (Itoh et al., 2015).
Overall, according to several evidences, inflammation in stroke can be beneficial for NSCs, nonetheless other reports, evaluating microglia function, claim that activated ED1+ microglia impair basal neurogenesis (Ekdahl et al., 2003) likely via TNFα/TNFR1 signaling (Iosif et al., 2008; Chen and Palmer, 2013; Gebara et al., 2013). In humans, a definitive robust evidence of clinical post-stroke neurogenesis remains a matter of investigation as analysis of autopsy tissue provided positive results (Ekonomou et al., 2012) which are instead lacking when labeling newly born neurons with 14C (Huttner et al., 2014).
In MS preclinical models, it is important to distinguish among experimental conditions since they influence the experimental evidence. Nonetheless, either chemically (LPC), focal (site injection of TNFα-IFNγ combined with subclinical immunization) or immune-mediated (EAE) demyelination causes reduced neuroblast proliferation in the olfactory bulb of mice, while neutralization of immune mediators, such as IFNγ and Galectin3 inflammatory cytokines, restores neurogenesis both in vitro (Pluchino et al., 2008; Tepavcevic et al., 2011) and in vivo (Monje et al., 2003; James et al., 2016). Indeed, in human samples, the SVZ of post-mortem MS brains is altered, with reduced number of neuroblasts in the SVZ of the lateral ventricle. Conversely, neuroinflammation increases neurotrophin levels (e.g., nerve growth factor-NGF) in the SVZ (Calza et al., 1998; Triaca et al., 2005), likely counteracting the inhibitory effect of proinflammatory molecules. Further, demyelination is able to boost proliferation of NSCs in the neurogenic niches promoting their differentiation (Pluchino et al., 2005; Menn et al., 2006). Only in conditions of acute cytokine exposure, either by focal intrathecal cytokine injection or as a consequence of strong microglia activation, the proliferative capacity of the niche is inhibited. In terms of gliogenic differentiation, inflammatory conditions may increase the percentage of Olig2+ cells originating from the SVZ via modulation of bone morphogenetic protein (BMP) signaling (Tepavcevic et al., 2011). The transcription factor Gli1 seems to be a key regulator of SVZ oligodendrogenesis and may be a promising target for reparative strategies in MS, as pharmacological inhibition of Gli1 during EAE promotes remyelination and improves clinical outcome (Samanta et al., 2015).
Leveraging the regenerative potential of NSCs and their immunomodulatory and trophic functions, NSC transplant has been proposed as therapeutic strategy for inflammatory diseases of the CNS. In stroke, transplanted NSCs directly modulate neuronal circuit plasticity (Zhang et al., 2009) through the expression of developmental molecules such as guidance molecules (i.e., slit, thrombospondin-1 and -2) and trophic factor such as VEGF (Andres et al., 2011). In fact, transplanted NSCs in stroke stimulate the proliferation of endogenous neural stem cells (Zhang et al., 2011; Hassani et al., 2012; Mine et al., 2013), increase endogenous angiogenesis after stroke (Jiang et al., 2005), scavenge the neurotoxic molecules (Emsley et al., 2004) and contribute both directly and indirectly (via astrocytes) to glutamate clearance (Bacigaluppi et al., 2009, 2016).
In EAE, transplanted NSCs directly inhibit both T cell and myeloid cell immune responses. In the CNS, they block inflammatory cell recruitment, T cell proliferation and promote the apoptosis of brain-reactive T cells (Einstein et al., 2003; Pluchino et al., 2005). In peripheral secondary lymphoid organs of EAE mice, antigen-specific proliferation of T cells, dendritic cell antigen presentation and chemotactic gradients are impaired by NSCs transplanted either subcutaneously or intravenously. As an example, NSC-secreted LIF, on one hand, stimulates endogenous remyelination in the CNS (Laterza et al., 2013), on the other, inhibits Th17 cell differentiation in the periphery (Cao et al., 2011).
Overall, the current literature shows that a controlled use of inflammation in CNS injury could be of help in regenerative approaches when NSC proliferation needs to be boosted and inflammatory tweaking can have beneficial outcomes. An option could consist in reducing NSC sensitivity to inflammatory mediators and, at the same time, increasing the therapeutic efficacy of NSCs when transplanting engineered cells.
Effect of aging on neural stem cell plasticity, connection with stroke, and multiple sclerosis
Aging is a common feature of both stroke and progressive multiple sclerosis and loss of niche integrity, depletion of the stem cell pool, cellular senescence, defect in cell-cell contact in the niche or metabolic changes are all shared characteristics that contribute to the demise of the aging neurogenic niche (Oh et al., 2014).
The most significant consequence of age in the SVZ consists in the alteration of the niche cytostructure and in reduction of the neuroblast population, with reduced proliferation and neurogenic capacity (Kerever et al., 2015). On the contrary, oligodendrogenesis is substantially not affected by aging, rather oligodendrocyte recruitment and differentiation are impaired (Sim et al., 2002; Franklin and Ffrench-Constant, 2008; Conover and Todd, 2016). The latter is indeed a causal pathogenic characteristic of chronically demyelinated MS lesions in humans (Kuhlmann et al., 2008).
Concurrently, given that senescence does not spare the vasculature (Farkas and Luiten, 2001), the environment of the niche may enrich for toxic factors which further sustain exhaustion, reduce neuroplasticity and cause cognitive decline (Villeda et al., 2011). Potential therapeutic strategies to counteract the decline of adult stem cells may involve the promotion of a rejuvenating environment (Katsimpardi et al., 2014) or the prevention of premature exhaustion of long-lived self-renewing NSC populations. Neurogenic decline in aged SVZ can occur because of the accumulation of damaged DNA and both nuclear and mitochondrial DNA are susceptible to age-related changes (Bailey et al., 2004). Upon sensing DNA damage, SVZ cells upregulate the inhibitor of cyclin kinase to post-pone cell cycle entry and reduce proliferation of NSCs (Molofsky et al., 2006).
NSC age-dependent cell plasticity is also influenced by metabolic changes (Rabie et al., 2011; Chaker et al., 2015). As far as nutrient sensing pathways, growth differentiation factor-11 (GDF11), has been identified as one of the extrinsic circulating youth signals that maintain neurogenesis (Katsimpardi et al., 2014), although those findings are currently matter of further validation (Egerman et al., 2015; Smith S. C. et al., 2015). It is still unknown whether the effect is direct onto NSCs or indirect via improved microvascular network. IGF1, another critical growth factor, per se stimulates proliferation and differentiation of NSCs (Aberg, 2010), but decreases with aging (Sonntag et al., 2005). Nonetheless, it is also life–long exposure to IGF1 that with age causes decline of neurogenesis (Chaker et al., 2015). Along the same line, NAD+ and AMP levels, which reflect cellular energy state, decline with age in the hippocampus, in parallel with reduction of differentiation and self-renewal capacity of NSCs, because their regulatory enzyme decreases (Liu et al., 2012; Stein and Imai, 2014). As far as oxygen-dependent effect, those are linked to failure in maintaining appropriate mitochondrial regulations. Indeed, accumulation of mutations in mitochondrial DNA (mtDNA) occurs during aging and comes along with abnormal accumulation of toxic by-products (Siqueira et al., 2005) because mtDNA lacks protective histones and is located close to the major source of ROS, the electron transport chain (Park and Larsson, 2011).
Inflammation is another comorbidity factor associated with aging. The immune molecules C-C motif chemokine-11 (CCL11) and β2-microglobulin (B2M), as well as CCL2, C-C motif chemokine-19 (CCL19) and haptoglobin, contribute to low-grade of inflammation in aging (inflammaging) (Franceschi and Campisi, 2014), and negatively affect neurogenesis (Villeda et al., 2011; Wyss-Coray, 2016). Indeed, heterochronic parabiosis between aged and young mice rejuvenates and reverses stem cell aging in numerous tissues (Conboy et al., 2005) or administration of plasma from young mice can ameliorate cognitive impairment in aged mice (Villeda et al., 2014).
Last, it is worth mentioning that also the cellular environment of the niche sustains aging features. Astrocytes of the niche, with age, reduce release of Wnt3 (Lie et al., 2005; Okamoto et al., 2011), that in turn down regulates Survivin expression and increases release of Dickkopf-related protein 1 (DKK1), a canonical Wnt signaling inhibitor. The final effect is induced quiescence on one side (Miranda et al., 2012) and negative regulation of NSC neurogenesis (Seib et al., 2013) on the other. This is paired with physical changes of the extracellular matrix, which also affect senescence of the niche (Gattazzo et al., 2014). Conversely, it is also possible that NSCs efficiently graft the SVZ and well differentiate in both young and aged hippocampus, suggesting that advanced age of the host at the time of grafting has no major adverse effects on engraftment, migration, and differentiation of SVZ-NSCs (Shetty and Hattiangady, 2016).
Age is an independent risk factor for brain ischemia (Marinigh et al., 2010; Roger et al., 2012) and stroke models using aged animals are clinically very relevant (Popa-Wagner et al., 2007). From work on animals, it has been reported a 50% decline in neurogenesis in the SVZ of elder compared with young–adult animals (Darsalia et al., 2005; Macas et al., 2006; Ahlenius et al., 2009). Unfortunately, although endogenous neurogenesis has been observed even in aged humans, it is still not clear to what extent newly formed neurons are functionally relevant for stroke recovery in human patients. Thus, cell therapies have been implemented to address this question. An open-label, single-site, dose-escalation study was performed on 13 aged patients after ischemic stroke, transplanting by stereotaxis the neural stem cell line CTX0E03 in the ipsilateral putamen. After 2 years, improvement in the disability score scale (NIHSS) ranging from 0 to 5 (median 2) points has been recorded (Borlongan, 2016; Kalladka et al., 2016). Similarly, studies in animal models examined whether exogenous NSC delivery might restore neurogenesis in the DG of old rodents. Both embryonic and fetal derived NSCs stimulated neurogenesis in young or old animal models. Likely the effect was exerted via suppression of microglia/macrophage activation (Jin et al., 2011). Of note, inhibition of microglial activation has also been reported in ischemic mice following systemic delivery of mouse NSCs (Bacigaluppi et al., 2009).
Those data suggest that the problem with NSCs in ischemic stroke is to some extent cell autonomous, because the environment of the aged brain does not preclude NSC engraftment.
In MS, loss of myelin, a specialized membrane produced by oligodendrocytes, essential for normal CNS function, is a characteristic feature. Aging impairs not only the neurogenic but also the oligodendrogenic properties of the brain germinal niches as a consequence of longer cell cycle length of NSCs and of their progeny, loss of growth factors and upregulation of inhibitors (Hamilton et al., 2013). Iron accumulation (Andersen et al., 2014), linked in cascade to oxidative alteration and to mitochondrial dysfunction (Mahad et al., 2015) are other recognized characteristics of neurodegeneration in progressive aged MS individuals. A direct impact also on neural stem cells has not been experimentally proven (Crabbe et al., 2010) but can reasonable be speculated.
In the context of demyelination, work by Crawford et al. demonstrated responsiveness of OPCs to demyelination, although remyelination and susceptibility to aging is regionally dependent: in chemically-induced demyelination of the corpus callosum in aged animals, dorsal SVZ-NSCs are efficiently recruited to lesions and their differentiation into OPCs is impaired, whereas ventral NSCs are recruited more slowly but differentiate rapidly into OPCs; similarly, while in aged animals the percentage of differentiated OPCs from dorsal NSCs is half of the one seen in young animals, the percentage of ventral NSCs differentiating into OPCs remained constant (Crawford et al., 2016). This evidence has significant implications for the progressive form of MS, for which age is the current best associated risk factor (Minden et al., 2004; Scalfari et al., 2011). As disease progresses with age, repair, remyelination and other physiological functions become less robust. Animal models have shown that while OPC recruitment toward lesions remains intact, differentiation into myelinating oligodendrocytes decreases with age with a slower and less effective remyelination process compared to young rats (Rist and Franklin, 2008; Ruckh et al., 2012). Similarly, elder human subjects showed reduced remyelinating capabilities. Multiple hypotheses can explain these findings, including either extrinsic factors or intrinsic characteristics such as epigenetic changes known to be modulated during aging and thus to impact OPC maturation (Rist and Franklin, 2008).