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Several neurological diseases are associated with, or are the cause of, movement impairments; among them, spinal cord injury, multiple sclerosis, and spinal muscular atrophy are examples with analogous effects on anti-gravity muscles. Similarly, it is well known that prolonged space missions and extended bed-rest induce functional alterations in many organs of the human body, including modifications of skeletal neuromuscular function (Kakurin et al., 1972) due to anti-gravity muscle reduced activity.

While the relationship between physical activity and cognitive ability has been known for centuries, recent studies demonstrate the significant impact that voluntary physical activity exerts on neurogenesis (van Praag et al., 1999; Adami and Bottai, 2016). Voluntary physical activity can both produce a significant increase in the levels of proliferative progenitor cells and restore neurogenesis artificially altered in rodent models of muscle disuse/inactivity (Farioli-Vecchioli et al., 2014). Voluntary physical activity has also been shown to overcome the age-dependent depletion of hippocampal neurogenesis (van Praag et al., 2005). In contrast, there is relatively little information available about the effect of prolonged muscle disuse on neurogenesis per se; previous studies describe in vivo changes with little focus on the differentiation process (Yasuhara et al., 2007). Thus we currently lack a detailed in vitro study of the influence of muscle reduced activity on neural stem cells (NSCs) characteristics.

Adult neurogenesis is restricted to few areas of the mammalian brain: the sub-ventricular zone of the lateral ventricles (SVZ), where it can be detected by evaluating the proliferation capability (for instance using marker associated to the cell cycle progression such as Ki67) (Shen et al., 2008; Liu and Crews, 2017), the sub-granular zone of the dentate gyrus of the hippocampus and the spinal cord (Bottai et al., 2003).

The synergistic action of extrinsic and intrinsic factors in the microenvironment of neurogenic areas controls the fate of the NSCs and is able to adjust the balance between undifferentiated progenitor cells and newly differentiated cells (Bottai et al., 2003).

The knowledge of the determinants affecting neurogenesis in individuals with movement restrictions is of pivotal interest in the attempt to develop new strategies to reduce the negative central and peripheral impact of motor deprivation in immobile patients and in astronauts. The effects of prolonged motor restraint on neurogenesis and the role of trophic determinants involved in this phenomenon can be studied using a recognized rodent model of severe motor deprivation: the so-called hindlimb unloading (HU) mouse model (Morey et al., 1979; Desaphy et al., 2005) which reproduces the absence of weight support on hindlimbs. In the literature, only a few studies have shown alterations in the levels of nerve growth factor (NGF) mRNA and of the brain-derived neurotrophic factor (BDNF) in the somatosensory cortex, supporting the hypothesis that disuse regulates neurotrophic factor expression (Dupont et al., 2005). A change has also been demonstrated in learning ability and memory in rats subjected to anti-gravity (Sun et al., 2009). The central effects of HU condition include a significant decrease in hindlimb representation on the motor cortex of the rat (Langlet et al., 2012). By contrast, physical exercise such as running leads to cell cycle shortening in some progenitors, and the S-phase shortening represents a major intrinsic regulator of the proneurogenic effect in the hippocampus exerted by running (Farioli-Vecchioli et al., 2014).

Low levels of exercise are thought to represent a major risk factor of developing metabolic alteration (Laaksonen et al., 2002) that could affect the central nervous system and in particular some neurogenic areas (Bottai and Adami, 2013; Adami and Bottai, 2016). L-lactate is a common metabolite in mammals, its production occurs in all cells including neurons and glia, and lactate is used actively by brain cells in culture (Medina and Tabernero, 2005). Pyruvate is formed during glycolysis and part of it is converted into L-lactate by lactate dehydrogenase (LDH). This prompted us to study lactate production as a marker of the metabolic activity of NSCs.

Our studies provide a new line of experimental investigation that can complement previous works on the role of exercise in neurogenesis. Overall, our analysis indicates the importance of the role of movement on NSCs properties in vitro.

A severe reduction of movement is present in many different pathologies such as spinal cord injury, spinal muscle atrophy, amyotrophic lateral sclerosis, and multiple sclerosis, and can be due to impairment of the skeleton muscular apparatus or of the central and peripheral nervous system. This condition is also common in bedrest patients (due to a wide range of pathologies) or in astronauts who experience low gravity for prolonged periods. Reduction of movement or gravity stimuli exerts an important effect on the human body, altering the activity of many organs including the brain. To study these effects, we took advantage of the hindlimb unloading protocol (Morey et al., 1979; Desaphy et al., 2005), which reproduces the reduction of movements or the absence of gravity in space.

Our previous experience with HU mice, which were used for the present and other studies (Brocca et al., 2010; Desaphy et al., 2010; Maffei et al., 2014; Cannavino et al., 2015), indicates that HU can produced significant stress in about 6% of cases and during the first 3 days of suspension (Kawano et al., 1994; Morey-Holton and Globus, 2002; Basco et al., 2010; Gaignier et al., 2014; Mysoet et al., 2014), after which the levels of stress, serum, and urine corticosterone measured, returned to the normal ones of the non-suspended animals. Indeed, it had already been demonstrated (Kawano et al., 1994) that in 5-week-old rats corticosterone urinary excretion in the control group was 87.4 ± 12.8 ng/day and did not change throughout the unloading time. A significant increase (more than 3-fold) in corticosterone excretion was found on days 1 and 3 during the suspension before returning to control levels, this time is little longer in older rats (Kawano et al., 1994). Meanwhile, the number of proliferating cells in rat neurogenic areas was not affected by adrenalectomy, the treatment of these animals with corticosteroids induced a significant increase in the hippocampus but not in the SVZ (Alonso, 2000). Other authors (Lee et al., 2016) demonstrated that chronic injection of corticosterone, for 28 days, can significantly reduce the cell proliferation and the differentiation in the SVZ. However, this treatment mimics a very long stress condition that is not analogous to our model. Indeed, even after 21 days, HU mice did not show any significant difference in corticosterone plasma level respect to control mice (Gaignier et al., 2014).

The thymus and the spleen weight (normalized by body weight) and the number of nucleated cells of the spleen were demonstrated not statistically different between CRT and HU (Gaignier et al., 2014). In our experience, we found no difference in the weight of the adrenal gland (normalized by body weight) between suspended and CTR mice (Bottai, Recchia, and Canepari unpublished results).

All these data indicate that the suspended animals, which can freely explore the cage without climbing, underwent little stress and most likely the changes in NSCs characteristics are due to the reduced overall amount of movements, by selective disuse of the hindlimb muscles. Although HU mice back limbs are free to make voluntary movements through the full range of motion, limb muscles are not exposed to the weight load and are lacking an active muscle force and strength development.

Many hypotheses can be drawn to explain the role of HU on NSCs properties. The reduction of movement of hindlimbs could induce an alteration in afferent signaling and feedback information from intramuscular receptors, to the cerebral cortex, due to a modification of the reflex organization in hindlimb muscle groups (D'Amelio et al., 1998). Similarly, a prolonged unilateral lower limb suspension, in humans, induces a variation of the nervous system plastic properties (Clark et al., 2006). On the site of neurotransmitters, it was demonstrated that 28 days of HU exposure in rats induced changes in the memory function and spatial learning that were related to protein expression changes (75 proteins were over-expressed and 72 down-regulated; Wang et al., 2017). In particular, the expression of Glutamate receptor (GluR) 1, GluR4, and the level of glutamate were up-regulated (Wang et al., 2017). On the other hand, the concentration of serotonin, dopamine, GABA, and epinephrine was reduced respect to control rats (Wang et al., 2017). Others studies indicated that HU-induced mRNA levels alterations of trophic factors such as NGF and BDNF in the somatosensory cortex (Dupont et al., 2005). In addition, many factors, such as cytokine and peptides, but also metabolites, exosomes, and miRNA, are released in the circulating system by the muscle during its activity allowing the possibility to influence other tissue (i.e., bone, liver, adipose tissue, cardiovascular system, brain) with autocrine, paracrine, and endocrine effects (Pedersen, 2011; Giudice and Taylor, 2017; Pourteymour et al., 2017; Safdar and Tarnopolsky, 2018; Whitham et al., 2018). Finally, other authors have previously demonstrated that the inhibition of bone formation, induced by unloading, is a consequence of the hindlimb elevation independently of the glucocorticoids since plasma corticosterone levels are not significantly different between normally loaded and unloaded rats and adrenalectomy had no protective effect on bone loss in unloaded animals (Halloran et al., 1988).

In our previous experience, where mice refused to eat during the first 2 days of suspension they were unfastened on the second day and discarded from the study. In the current study, none of the animals were discarded. Our previous observations indicated that the body weight of the animals that undergo suspension have a slight but significant decrease in their body mass (Desaphy et al., 2010), whereas other researchers even with prolonged suspension did not find differences compared to control group (Colaianni et al., 2017).

During the unloading experiment, mice were monitored daily for appropriate food and water intake, urination and defecation and normal grooming behavior, which were observed to be statistically similar between the two groups, indicating that they experienced very little stress. The weights of the antigravity muscles (gastrocnemius and soleus) were decreased significantly (respectively by 10 and 20%) after unloading, as already demonstrated in previous work of our group (Brocca et al., 2010) and from other groups (Hanson et al., 2013).

Our study adds more information for a better understanding of the role of movement reduction in NSCs features. It is known that physical inactivity is a risk factor for Alzheimer's disease (AD) (Hashimoto et al., 2017) since hippocampal atrophy was associated with the AD. In a recent meta-analysis, Guure and collaborators found that physical activity is more protective against AD than all the other forms of dementia (Guure et al., 2017).

Physical activity induces an increase in the hippocampal volume and ameliorates the neurogenesis (Bednarczyk et al., 2009) most likely via the augmentation of the blood flow (Cass, 2017). Consistently with this, the level of VEGF in the plasma of suspended rats decreases after 14 days of treatment, whereas it remains unaltered in the brain (Yasuhara et al., 2007) and in the soleus muscle (Wagatsuma, 2008). Long suspension (4 weeks) induced an inflammatory response in the common carotid artery of rats exposed to simulated microgravity, indicating that the inflammatory response may be a cellular mechanism that is responsible for the arterial remodeling during exposure to simulated microgravity (Liu et al., 2014). Prospective studies indicate that physical inactivity is one of the most frequent avoidable risk factors for developing AD. Moreover, elevated physical activity levels are associated with a lower risk of AD. The AD patient who undertook exercise training showed decreased neuropsychiatric symptoms, improvement in cognitive function, and a slower decline in the activities of daily life (Cass, 2017). After exercise AD patients regained some of their brain capabilities following appropriate and controlled physical training (Okonkwo et al., 2014).

We decided to study SVZ because its role in neurogenesis in the adult human might—albeit not comparable, in term of activity, with rodents—have a major impact on human brain health. It is already known that neuroblasts derived from SVZ migrate through the rostral migratory stream system into the olfactory bulb (Sanai et al., 2004; van den Berge et al., 2010). However, there is another major migratory pathway of immature neurons destined for the prefrontal cortex in infants (Sanai et al., 2011) and it was found, by measuring the levels of nuclear bomb test-derived ¹⁴C in genomic DNA (Bergmann et al., 2012), that there is insignificant neurogenesis in the human olfactory bulb. Surprisingly it was determined that there is a postnatal cell turnover in the striatum of adult humans (Ernst et al., 2014) that the authors suggested derives from the SVZ. Despite this evidence, there are limited hints about the role of exercise on neurogenesis in SVZ which impact remains debated (Brown et al., 2003; Bednarczyk et al., 2009; Blackmore et al., 2012; Chae et al., 2014). On the contrary, the positive role of exercise, especially in the case of previous brain impairment, is well assured (Lee et al., 2016; Mastrorilli et al., 2017). Finally, the impact on the hippocampus is more extensive and established (Lee et al., 2016; Mastrorilli et al., 2017; Snyder et al., 2017; Bouchard-Cannon et al., 2018; Firth et al., 2018; Masrour et al., 2018).

This range of evidence prompted us to carry out an ex vivo analysis demonstrating that, as already showed in the rat hippocampus model (Yasuhara et al., 2007), hindlimb unloading exerts a reduction in proliferating Ki67 (an endogenous marker of cell proliferation, expressed in the nucleus in all phases of the cell cycle except the resting phase, that is associated with, and necessary for, cellular proliferation) positive cells in the SVZ (Figure 1).

This first step allowed us to predict some alteration in NSCs attributes of the SVZ of the suspended animals; we then proceeded with the preparation of NSC cultures from this region.

We were able to produce NSC cultures from the SVZ of the HU and CTR mice; we found that HU NSCs neurospheres were more difficult to obtain since they emerged more slowly than those from the controls. The morphology of the neurospheres of the two culture groups was similar, with no difference in shape or adhesion to the plastic of the flask. However, we noticed before doing any counting that the size of the neurospheres was clearly smaller in the HU group. The proliferation analysis was performed measuring the cell number in the exponentially growing phase for many passages. This procedure showed that the proliferation capability of the HU derived SVZ NSCs was impaired, confirming the result obtained in ex vivo analysis (Figure 2). We analyzed the expression level of epitopes routinely detected in NSCs such as Nestin, GFAP, SOX2 (Kuhn et al., 2016), GLAST (Gubert et al., 2009), and Ki67, that is expressed in proliferating cells in all active phases of the cell cycle. The number of cells expressing Nestin, Ki67, and GFAP was significantly reduced in HU derived NSCs. The diminished number of Ki67 positive cells supports our results on NSCs proliferation (Figure 2). A decrease in Nestin and GFAP positive cells (Supplementary Figure S3) could be related to fewer committed glial cells that led to less glial differentiation as also shown in Figure 4. On the contrary, Sox2, that is considered a more trustworthy stem cell marker (Sarkar and Hochedlinger, 2013), did not show any changes in its expression. This result can be explained considering that we evaluated the number of positive cells, which is correlated with the change in cell cycle kinetics that we observed (Figure 3), rather than a decrease in the level of expression of these markers per cell. Further analysis will be necessary in order to understand these aspects.

The suspension induced an arrest of NSCs in the G0/G1 phase and a significant reduction in the G2/M phase (Figure 3), the G0/G1 arrest was also found in of rat bMSCs when exposed to simulated or real microgravity (Bradamante et al., 2014).

The effect of hindlimb unloading on the stereological parameters of the hippocampus and the motor cortex in male rats was recently assessed (see Supplementary discussion a).

Another interesting aspect is the potential reversibility of the alterations induced by adult NSCs in the SVZ, following a more or less long period of post-HU recovery. The recovery (after suspension) with or without exercise in rats were ineffective for the retrieval of proliferation and differentiation in SVZ (Yasuhara et al., 2007).

With regard to the metabolic capacity of the HU and CTR NSCs, it is recognized that intracellular pO₂ values of 0.5 Torr or less occur in O₂-limited oxidative phosphorylation and consequent lactate production and accumulation (Rogatzki et al., 2015). At low levels of oxygen, energy is provided by anaerobic metabolism, leading to the production of lactic acid (De Filippis and Delia, 2011). Stem cells, such as ESCs, HSCs, MSCs, and others, have immature mitochondrial morphology, a reduced oxidative capacity and enhanced anabolic levels in the glycolytic pathways (Folmes et al., 2012). Brain lactate is a major substrate for oxidative metabolism during development and it is selectively utilized as an anabolic reservoir for cell proliferation and differentiation. Furthermore, lactate is used actively by brain cells in culture (Medina and Tabernero, 2005). In this scenario, the results obtained from our work emphasize the involvement of metabolism alteration and NSCs properties during the reduction of motor activity.

The MTT assay has been widely used to assess cell viability; however, MTT effectively measures the metabolic activity of live cells, since it evaluates the capacity of cells to reduce the tetrazolium dye by functioning mitochondria. The levels of oxygen have been shown to affect NSCs characteristics during normal development, disease and culturing (Studer et al., 2000; Mohyeldin et al., 2010; Santilli et al., 2010). Since the NSCs have mostly a glycolytic metabolism, due to the low oxygen tension level (Zhang et al., 2015) the lower lactate level measured in HU NSCs indicates that these cells have a higher oxidative metabolism (Figure 6C,D), this was also demonstrated by other authors (Medina and Tabernero, 2005).

Contrary to what was expected, a gene, Cdk5 regulatory subunit-associated protein 1 (CDKrap1), was significantly reduced in HU samples relative to CTR (Figure 5). Cdk5rap1 was first found to be a negative Cdk5 regulator but in a subsequent study, Cdk5rap1 was found to be responsible for the post-synthetic conversion of the RNA modification N6-isopentenyladenosine (i⁶ A) into 2-methylthio-N6-isopentenyladenosine (ms² i⁶ A). (Cdk5rap1) is responsible for 2-methylthio modifications of mammalian mitochondria (mt)-tRNAs for Ser(UCN), Phe, Tyr, and Trp codons (Wang et al., 2015; Wei et al., 2015). In breast cancer cells MCF-7 Cdk5rap1 paucity induces cell cycle arrest (Wang et al., 2015). The Cdk5rap1-KO mice are sensitive to stress-induced mitochondrial remodeling (see Supplementary discussion b).

Another gene which expression was altered in HU NSCs, compared to CTR NSCs is Cdk6.

On the basis of our findings, we can speculate that the low expression level of Cdk6 in NSCs from CTR animals correlates with their higher proliferation capacity, so the elevated level of expression of this gene in HU derived NSC should be implicated in their altered capabilities (Figure 5). Cdk6, acting as cell cycle kinase, promotes the progression to S phase, chromatin status, cell death, cell survival, and DNA repair interacting with cyclin D; moreover, behaving as transcriptional regulators, Cdk6 interacts with other proteins such as RUNX1, NF-kB, STAT3, and AP-1 regulating respectively differentiation, inflammation, cell cycle arrest, stress hematopoiesis and angiogenesis (Tigan et al., 2016). Cdk6 and cyclin D are very important drivers of tumorigenesis. Cdk6 can have a role as tumor suppressor reducing proliferation in lymphoid malignancies (Kollmann et al., 2013), or restrain the proliferation of breast cancer cells (Lucas et al., 2004). Further analysis will be necessary to depict this aspect.

These data, to our knowledge, are the first evidence of a correlation between changes in NSCs attributes after movement restraint, metabolism modification, and gene expression changes. Interesting, the ability of HU cells to maintain their altered properties for more than 10 passages of culture suggests that an epigenetic modification might be involved. In these regards, the fact that suspended animals underwent a stress during the first three days of the unloading period could explain the gene expression alteration in terms of epigenetic changes. However, the gene expression analysis we performed on the cell cycle genes showed no changes in genes that are usually affected during stress induction namely Cyclin D1 and Cyclin-dependent kinase inhibitor 1A (P21) (Juszczak and Stankiewicz, 2018) but rather other genes were altered.

Epigenetic modifications affect not only DNA and proteins, but also coding and non-coding RNAs. RNAs can be modified in more than 100 ways including N6-methyladenosine (m6A), N7-methylguanosine (m7G), m5C, pseudouridine, and queuosine (Jia et al., 2013). Many such modifications have a basic function in controlling aspects of RNA metabolism, such as splicing, transport, translation, and degradation. These active RNA modifications represent another level of gene regulation, termed “epitranscriptomics.” The discovery that Cdk5rap1 (which acts as methylthiotransferase in mitochondria RNA) was altered in its expression after motor deprivation is very intriguing in this context, and opens a new link between neurogenesis metabolism and cell regulation.

RA and DB: designed research; RA, JP, MCo, NP, DR, MCa, and DB: performed research; RA, MCo, NP, RC, and DB: analyzed and interpreted data; RA and DB: wrote the paper; RA, JP, MCo, NP, DR, RC, RB, MCa, and DB: revised and approved the paper.