Dynamic Changes in the Neurogenic Potential in the Ventricular-Subventricular Zone of Common Marmoset during Postnatal Brain Development.

Even after birth, neuronal production continues in the ventricular-subventricular zone (V-SVZ) and hippocampus in many mammals. The immature new neurons ("neuroblasts") migrate and then mature at their final destination. In humans, neuroblast production and migration toward the neocortex and the olfactory bulb (OB) occur actively only for a few months after birth and then sharply decline with age. However, the precise spatiotemporal profiles and fates of postnatally born neurons remain unclear due to methodological limitations. We previously found that common marmosets, small nonhuman primates, share many features of V-SVZ organization with humans. Here, using marmosets injected with thymidine analogue(s) during various postnatal periods, we demonstrated spatiotemporal changes in neurogenesis during development. V-SVZ progenitor proliferation and neuroblast migration toward the OB and neocortex sharply decreased by 4 months, most strikingly in a V-SVZ subregion from which neuroblasts migrated toward the neocortex. Postnatally born neurons matured within a few months in the OB and hippocampus but remained immature until 6 months in the neocortex. While neurogenic activity was sustained for a month after birth, the distribution and/or differentiation diversity was more restricted in 1-month-born cells than in the neonatal-born population. These findings shed light on distinctive features of postnatal neurogenesis in primates.

Dynamic Changes in Ultrastructure of the Primary Cilium in Migrating Neuroblasts in the Postnatal Brain.

New neurons, referred to as neuroblasts, are continuously generated in the ventricular-subventricular zone of the brain throughout an animal's life. These neuroblasts are characterized by their unique potential for proliferation, formation of chain-like cell aggregates, and long-distance and high-speed migration through the rostral migratory stream (RMS) toward the olfactory bulb (OB), where they decelerate and differentiate into mature interneurons. The dynamic changes of ultrastructural features in postnatal-born neuroblasts during migration are not yet fully understood. Here we report the presence of a primary cilium, and its ultrastructural morphology and spatiotemporal dynamics, in migrating neuroblasts in the postnatal RMS and OB. The primary cilium was observed in migrating neuroblasts in the postnatal RMS and OB in male and female mice and zebrafish, and a male rhesus monkey. Inhibition of intraflagellar transport molecules in migrating neuroblasts impaired their ciliogenesis and rostral migration toward the OB. Serial section transmission electron microscopy revealed that each migrating neuroblast possesses either a pair of centrioles or a basal body with an immature or mature primary cilium. Using immunohistochemistry, live imaging, and serial block-face scanning electron microscopy, we demonstrate that the localization and orientation of the primary cilium are altered depending on the mitotic state, saltatory migration, and deceleration of neuroblasts. Together, our results highlight a close mutual relationship between spatiotemporal regulation of the primary cilium and efficient chain migration of neuroblasts in the postnatal brain.SIGNIFICANCE STATEMENT Immature neurons (neuroblasts) generated in the postnatal brain have a mitotic potential and migrate in chain-like cell aggregates toward the olfactory bulb. Here we report that migrating neuroblasts possess a tiny cellular protrusion called a primary cilium. Immunohistochemical studies with zebrafish, mouse, and monkey brains suggest that the presence of the primary cilium in migrating neuroblasts is evolutionarily conserved. Ciliogenesis in migrating neuroblasts in the rostral migratory stream is suppressed during mitosis and promoted after cell cycle exit. Moreover, live imaging and 3D electron microscopy revealed that ciliary localization and orientation change during saltatory movement of neuroblasts. Our results reveal highly organized dynamics in maturation and positioning of the primary cilium during neuroblast migration that underlie saltatory movement of postnatal-born neuroblasts.

Detachment of Chain-Forming Neuroblasts by Fyn-Mediated Control of cell-cell Adhesion in the Postnatal Brain.

In the rodent olfactory system, neuroblasts produced in the ventricular-subventricular zone of the postnatal brain migrate tangentially in chain-like cell aggregates toward the olfactory bulb (OB) through the rostral migratory stream (RMS). After reaching the OB, the chains are dissociated and the neuroblasts migrate individually and radially toward their final destination. The cellular and molecular mechanisms controlling cell-cell adhesion during this detachment remain unclear. Here we report that Fyn, a nonreceptor tyrosine kinase, regulates the detachment of neuroblasts from chains in the male and female mouse OB. By performing chemical screening and in vivo loss-of-function and gain-of-function experiments, we found that Fyn promotes somal disengagement from the chains and is involved in neuronal migration from the RMS into the granule cell layer of the OB. Fyn knockdown or Dab1 (disabled-1) deficiency caused p120-catenin to accumulate and adherens junction-like structures to be sustained at the contact sites between neuroblasts. Moreover, a Fyn and N-cadherin double-knockdown experiment indicated that Fyn regulates the N-cadherin-mediated cell adhesion between neuroblasts. These results suggest that the Fyn-mediated control of cell-cell adhesion is critical for the detachment of chain-forming neuroblasts in the postnatal OB.SIGNIFICANCE STATEMENT In the postnatal brain, newly born neurons (neuroblasts) migrate in chain-like cell aggregates toward their destination, where they are dissociated into individual cells and mature. The cellular and molecular mechanisms controlling the detachment of neuroblasts from chains are not understood. Here we show that Fyn, a nonreceptor tyrosine kinase, promotes the somal detachment of neuroblasts from chains, and that this regulation is critical for the efficient migration of neuroblasts to their destination. We further show that Fyn and Dab1 (disabled-1) decrease the cell-cell adhesion between chain-forming neuroblasts, which involves adherens junction-like structures. Our results suggest that Fyn-mediated regulation of the cell-cell adhesion of neuroblasts is critical for their detachment from chains in the postnatal brain.

Mechanisms of neuronal migration in the adult brain.

Adult neurogenesis was first observed nearly 60 years ago, and it has since grown into an important neurochemistry research field. Much recent research has focused on the treatment of brain diseases through neuronal regeneration with endogenously generated neurons. In the adult brain, immature neurons called neuroblasts are continuously generated in the ventricular-subventricular zone (V-SVZ). These neuroblasts migrate rapidly through the rostral migratory stream to the olfactory bulb, where they mature and are integrated into the neuronal circuitry. After brain insult, some of the neuroblasts in the V-SVZ migrate toward the lesion to repopulate the injured tissue. This notable migratory capacity of V-SVZ-derived neuroblasts is important for efficiently regenerating neurons in remote areas of the brain. As these neurons migrate for long distances through adult brain tissue, they are supported by various guidance cues and structures that act as scaffolds. Some of these mechanisms are unique to neuroblast migration in the adult brain, and are not involved in migration in the developing brain. Here, we review the latest findings on the mechanisms of neuroblast migration in the adult brain under physiological and pathological conditions, and discuss various issues that still need to be resolved. This article is part of the mini review series "60th Anniversary of the Japanese Society for Neurochemistry".

Identification of the growth cone as a probe and driver of neuronal migration in the injured brain.

Axonal growth cones mediate axonal guidance and growth regulation. We show that migrating neurons in mice possess a growth cone at the tip of their leading process, similar to that of axons, in terms of the cytoskeletal dynamics and functional responsivity through protein tyrosine phosphatase receptor type sigma (PTPσ). Migrating-neuron growth cones respond to chondroitin sulfate (CS) through PTPσ and collapse, which leads to inhibition of neuronal migration. In the presence of CS, the growth cones can revert to their extended morphology when their leading filopodia interact with heparan sulfate (HS), thus re-enabling neuronal migration. Implantation of an HS-containing biomaterial in the CS-rich injured cortex promotes the extension of the growth cone and improve the migration and regeneration of neurons, thereby enabling functional recovery. Thus, the growth cone of migrating neurons is responsive to extracellular environments and acts as a primary regulator of neuronal migration.

Human dental pulp-derived stem cells protect against hypoxic-ischemic brain injury in neonatal mice.

BACKGROUND AND PURPOSE: Perinatal hypoxia-ischemia (HI) has high rates of neurological deficits and mortality. So far, no effective treatment for HI brain injury has been developed. In this study, we investigated the therapeutic effects of stem cells from human exfoliated deciduous teeth (SHED) for the treatment of neonatal HI brain injury. METHODS: Unilateral HI was induced in postnatal day 5 (P5) mice. Twenty-four hours later, SHED, human skin fibroblasts, or serum-free conditioned medium derived from these cells was injected into the injured brain. The effects of cell transplantation or conditioned medium injection on the animals' neurological and pathophysiological recovery were evaluated. RESULTS: Transplanted SHED, but not fibroblasts, significantly reduced the HI-induced brain-tissue loss and improved neurological function. SHED also improved the survival of the HI mice. The engrafted SHED rarely differentiated into neural lineages; however, their transplantation inhibited the expression of proinflammatory cytokines, increased the expression of anti-inflammatory ones, and significantly reduced apoptosis. Notably, the intracerebral administration of SHED-conditioned medium also significantly improved the neurological outcome, inhibited apoptosis, and reduced tissue loss. CONCLUSIONS: SHED transplantation into the HI-injured brain resulted in remarkable neurological and pathophysiological recovery. Our findings indicate that paracrine factors derived from SHED support a neuroprotective microenvironment in the HI brain. SHED graft and SHED-conditioned medium may provide a novel neuroprotective therapy for HI.

Subventricular zone-derived oligodendrogenesis in injured neonatal white matter in mice enhanced by a nonerythropoietic erythropoietin derivative.

Perinatal hypoxia-ischemia (HI) frequently causes white-matter injury, leading to severe neurological deficits and mortality, and only limited therapeutic options exist. The white matter of animal models and human patients with HI-induced brain injury contains increased numbers of oligodendrocyte progenitor cells (OPCs). However, the origin and fates of these OPCs and their potential to repair injured white matter remain unclear. Here, using cell-type- and region-specific genetic labeling methods in a mouse HI model, we characterized the Olig2-expressing OPCs. We found that after HI, Olig2+ cells increased in the posterior part of the subventricular zone (pSVZ) and migrated into the injured white matter. However, their oligodendrocytic differentiation efficiency was severely compromised compared with the OPCs in normal tissue, indicating the need for an intervention to promote their differentiation. Erythropoietin (EPO) treatment is a promising candidate, but it has detrimental effects that preclude its clinical use for brain injury. We found that long-term postinjury treatment with a nonerythropoietic derivative of EPO, asialo-erythropoietin, promoted the maturation of pSVZ-derived OPCs and the recovery of neurological function, without affecting hematopoiesis. These results demonstrate the limitation and potential of endogenous OPCs in the pSVZ as a therapeutic target for treating neonatal white-matter injury.

Amphiphilic peptide-tagged N-cadherin forms radial glial-like fibers that enhance neuronal migration in injured brain and promote sensorimotor recovery.

The mammalian brain has very limited ability to regenerate lost neurons and recover function after injury. Promoting the migration of young neurons (neuroblasts) derived from endogenous neural stem cells using biomaterials is a new and promising approach to aid recovery of the brain after injury. However, the delivery of sufficient neuroblasts to distant injured sites is a major challenge because of the limited number of scaffold cells that are available to guide neuroblast migration. To address this issue, we have developed an amphiphilic peptide [(RADA)3-(RADG)] (mRADA)-tagged N-cadherin extracellular domain (Ncad-mRADA), which can remain in mRADA hydrogels and be injected into deep brain tissue to facilitate neuroblast migration. Migrating neuroblasts directly contacted the fiber-like Ncad-mRADA hydrogel and efficiently migrated toward an injured site in the striatum, a deep brain area. Furthermore, application of Ncad-mRADA to neonatal cortical brain injury efficiently promoted neuronal regeneration and functional recovery. These results demonstrate that self-assembling Ncad-mRADA peptides mimic both the function and structure of endogenous scaffold cells and provide a novel strategy for regenerative therapy.

Sensory input regulates spatial and subtype-specific patterns of neuronal turnover in the adult olfactory bulb.

Throughout life, new neurons are added and old ones eliminated in the adult mouse olfactory bulb. Previous studies suggested that olfactory experience controls the process by which new neurons are integrated into mature circuits. Here we report novel olfactory-experience-dependent mechanisms of neuronal turnover. Using two-photon laser-scanning microscopy and sensory manipulations in adult live mice, we found that the neuronal turnover was dynamically controlled by olfactory input in a neuronal subtype-specific manner. Olfactory input enhanced this turnover, which was characterized by the reiterated use of the same positions in the glomeruli by new neurons. Our results suggest that olfactory-experience-dependent modification of neuronal turnover confers structural plasticity and stability on the olfactory bulb.

Girdin is an intrinsic regulator of neuroblast chain migration in the rostral migratory stream of the postnatal brain.

In postnatally developing and adult brains, interneurons of the olfactory bulb (OB) are continuously generated at the subventricular zone of the forebrain. The newborn neuroblasts migrate tangentially to the OB through a well defined pathway, the rostral migratory stream (RMS), where the neuroblasts undergo collective migration termed "chain migration." The cell-intrinsic regulatory mechanism of neuroblast chain migration, however, has not been uncovered. Here we show that mice lacking the actin-binding Akt substrate Girdin (a protein that interacts with Disrupted-In-Schizophrenia 1 to regulate neurogenesis in the dentate gyrus) have profound defects in neuroblast chain migration along the RMS. Analysis of two gene knock-in mice harboring Girdin mutants identified unique amino acid residues in Girdin's C-terminal domain that are responsible for the regulation of neuroblast chain migration but revealed no apparent requirement of Girdin phosphorylation by Akt. Electron microscopic analyses demonstrated the involvement of Girdin in neuroblast cell-cell interactions. These findings suggest that Girdin is an important intrinsic factor that specifically governs neuroblast chain migration along the RMS.

Chitinase-like protein 3: A novel niche factor for mouse neural stem cells.

The concept of a perivascular niche has been proposed for neural stem cells (NSCs). This study examined endothelial colony-forming cell (ECFC)-secreted proteins as potential niche factors for NSCs. Intraventricle infusion with ECFC-secreted proteins increased the number of NSCs. ECFC-secreted proteins were more effective in promoting NSC self-renewal than marrow stromal cell (MSC)-secreted proteins. Differential proteomics analysis of MSC-secreted and ECFC-secreted proteins was performed, which revealed chitinase-like protein 3 (CHIL3; also called ECF-L or Ym1) as a candidate niche factor for NSCs. Experiments with recombinant CHIL3, small interfering RNA, and neutralizing antibodies demonstrated that CHIL3 stimulated NSC self-renewal with neurogenic propensity. CHIL3 was endogenously expressed in the neurogenic niche of the brain and retina as well as in the injured brain and retina. Transcriptome and phosphoproteome analyses revealed that CHIL3 activated various genes and proteins associated with NSC maintenance or neurogenesis. Thus, CHIL3 is a novel niche factor for NSCs.

Usefulness of pharmacist-assisted screening and psychiatric referral program for outpatients with cancer undergoing chemotherapy.

OBJECTIVE: Major depressive disorder (MDD) and adjustment disorder (AD) are common psychiatric disorders in cancer patients but are often overlooked in clinical oncology settings. We introduced a clinical screening program utilizing the Distress and Impact Thermometer (DIT) to identify MDD and AD in cancer outpatients receiving chemotherapy. This study assessed the usefulness of the screening program. METHODS: Pharmacists administered the DIT to consecutive patients undergoing chemotherapy at an outpatient clinic. Psychiatric treatment was recommended to all the patients with positive screening results. The proportion of patients referred to the Psychiatric Service during the program period was then compared with that during a usual care period. RESULTS: Of the 520 patients who started chemotherapy during the 6-month program period, 5.0% (26/520) were referred to the Psychiatric Service and 2.7% (15/520) were diagnosed as having MDD or AD. No statistically significant difference in the referral rates was observed between the two periods (2.7 vs 1.0%, p = 0.46). However, the period from the first chemotherapy treatment until the visit to the Psychiatric Service was significantly shorter during the program period than during the period of usual care (12.9±13.2 days vs 55.6±17.6 days, p<0.001). CONCLUSIONS: The proportion of patients referred to the Psychiatric Service for the treatment of MDD or AD during the program period was not different from that during the usual care period. However, the program was useful for introducing psychiatric treatment at an earlier stage. Further modifications to the program to improve the referral rate are necessary.

Neurogenesis and neuronal migration in the postnatal ventricular-subventricular zone: Similarities and dissimilarities between rodents and primates.

The ventricular-subventricular zone (V-SVZ) is located in the walls of the lateral ventricles and produces new neurons in the postnatal brain of mammals, including humans. Immature new neurons called "neuroblasts" generated by neural stem cells in the V-SVZ migrate toward their final destinations and contribute to brain development and plasticity. In this review, we describe recent progress in understanding the similarities and dissimilarities in postnatal neurogenesis and neuronal migration between rodents and primates. In rodents, most new V-SVZ-derived neurons migrate along the rostral migratory stream towards the olfactory bulb, where they differentiate into interneurons. In contrast, in humans, the extensive migration of new neurons towards the neocortex continues for several months after birth and might be involved in the development of the expanded neocortex. The mode of migration and the fate of neuroblasts seem to change depending on their environment, destination, and roles in the brain. A better understanding of these similarities and differences between rodents and primates will help translate important findings from animal models and may contribute to the development of clinical strategies for brain repair.

Efficient protein incorporation and release by a jigsaw-shaped self-assembling peptide hydrogel for injured brain regeneration.

During injured tissue regeneration, the extracellular matrix plays a key role in controlling and coordinating various cellular events by binding and releasing secreted proteins in addition to promoting cell adhesion. Herein, we develop a cell-adhesive fiber-forming peptide that mimics the jigsaw-shaped hydrophobic surface in the dovetail-packing motif of glycophorin A as an artificial extracellular matrix for regenerative therapy. We show that the jigsaw-shaped self-assembling peptide forms several-micrometer-long supramolecular nanofibers through a helix-to-strand transition to afford a hydrogel under physiological conditions and disperses homogeneously in the hydrogel. The molecular- and macro-scale supramolecular properties of the jigsaw-shaped self-assembling peptide hydrogel allow efficient incorporation and sustained release of vascular endothelial growth factor, and demonstrate cell transplantation-free regenerative therapeutic effects in a subacute-chronic phase mouse stroke model. This research highlights a therapeutic strategy for injured tissue regeneration using the jigsaw-shaped self-assembling peptide supramolecular hydrogel.

Metabolic fingerprints of fear memory consolidation during sleep.

Metabolites underlying brain function and pathology are not as well understood as genes. Here, we applied a novel metabolomics approach to further understand the mechanisms of memory processing in sleep. As hippocampal dentate gyrus neurons are known to consolidate contextual fear memory, we analyzed real-time changes in metabolites in the dentate gyrus in different sleep-wake states in mice. Throughout the study, we consistently detected more than > 200 metabolites. Metabolite profiles changed dramactically upon sleep-wake state transitions, leading to a clear separation of phenotypes between wakefulness and sleep. By contrast, contextual fear memory consolidation induced less obvious metabolite phenotypes. However, changes in purine metabolites were observed upon both sleep-wake state transitions and contextual fear memory consolidation. Dietary supplementation of certain purine metabolites impaired correlations between conditioned fear responses before and after memory consolidation. These results point toward the importance of purine metabolism in fear memory processing during sleep.

Phosphorylation of GAP-43 T172 is a molecular marker of growing axons in a wide range of mammals including primates.

GAP-43 is a vertebrate neuron-specific protein and that is strongly related to axon growth and regeneration; thus, this protein has been utilized as a classical molecular marker of these events and growth cones. Although GAP-43 was biochemically characterized more than a quarter century ago, how this protein is related to these events is still not clear. Recently, we identified many phosphorylation sites in the growth cone membrane proteins of rodent brains. Two phosphorylation sites of GAP-43, S96 and T172, were found within the top 10 hit sites among all proteins. S96 has already been characterized (Kawasaki et al., 2018), and here, phosphorylation of T172 was characterized. In vitro (cultured neurons) and in vivo, an antibody specific to phosphorylated T172 (pT172 antibody) specifically recognized cultured growth cones and growing axons in developing mouse neurons, respectively. Immunoblotting showed that pT172 antigens were more rapidly downregulated throughout development than those of pS96 antibody. From the primary structure, this phosphorylation site was predicted to be conserved in a wide range of animals including primates. In the developing marmoset brainstem and in differentiated neurons derived from human induced pluripotent stem cells, immunoreactivity with pT172 antibody revealed patterns similar to those in mice. pT172 antibody also labeled regenerating axons following sciatic nerve injury. Taken together, the T172 residue is widely conserved in a wide range of mammals including primates, and pT172 is a new candidate molecular marker for growing axons.

Dysfunction of the proteoglycan Tsukushi causes hydrocephalus through altered neurogenesis in the subventricular zone in mice.

The lateral ventricle (LV) is flanked by the subventricular zone (SVZ), a neural stem cell (NSC) niche rich in extrinsic growth factors regulating NSC maintenance, proliferation, and neuronal differentiation. Dysregulation of the SVZ niche causes LV expansion, a condition known as hydrocephalus; however, the underlying pathological mechanisms are unclear. We show that deficiency of the proteoglycan Tsukushi (TSK) in ependymal cells at the LV surface and in the cerebrospinal fluid results in hydrocephalus with neurodevelopmental disorder-like symptoms in mice. These symptoms are accompanied by altered differentiation and survival of the NSC lineage, disrupted ependymal structure, and dysregulated Wnt signaling. Multiple TSK variants found in patients with hydrocephalus exhibit reduced physiological activity in mice in vivo and in vitro. Administration of wild-type TSK protein or Wnt antagonists, but not of hydrocephalus-related TSK variants, in the LV of TSK knockout mice prevented hydrocephalus and preserved SVZ neurogenesis. These observations suggest that TSK plays a crucial role as a niche molecule modulating the fate of SVZ NSCs and point to TSK as a candidate for the diagnosis and therapy of hydrocephalus.

Effects of interferon-alpha on hippocampal neurogenesis and behavior in common marmosets.

In many mammalian species, the production of new neurons in the hippocampal dentate gyrus continues throughout life. Previous studies using rodents suggest that adult-born neurons are involved in memory and cognition tasks and mood regulation. Interferon-alpha (IFNα), a proinflammatory cytokine used for the treatment of chronic viral hepatitis and malignancies, frequently causes depressive symptoms in patients and animals, including non-human primates. We have previously demonstrated that chronic IFNα treatment decreases hippocampal neurogenesis in mice. Here, we investigated the effects of four-week human pegylated IFNα treatment on hippocampal neurogenesis and behavior in common marmosets. Continuous monitoring of voluntary activity levels using an actigraphy device suggested that adaptive ability is impaired in IFNα-treated animals. Analyses of BrdU-labeled cells expressing a marker for immature or mature neurons revealed a significant reduction in the number of new neurons in the hippocampus of IFNα-treated animals. These data indicate that chronic human IFNα treatment causes behavioral changes and a decrease in hippocampal neurogenesis in common marmosets.

Sparse Activity of Hippocampal Adult-Born Neurons during REM Sleep Is Necessary for Memory Consolidation.

The occurrence of dreaming during rapid eye movement (REM) sleep prompts interest in the role of REM sleep in hippocampal-dependent episodic memory. Within the mammalian hippocampus, the dentate gyrus (DG) has the unique characteristic of exhibiting neurogenesis persisting into adulthood. Despite their small numbers and sparse activity, adult-born neurons (ABNs) in the DG play critical roles in memory; however, their memory function during sleep is unknown. Here, we investigate whether young ABN activity contributes to memory consolidation during sleep using Ca2+ imaging in freely moving mice. We found that contextual fear learning recruits a population of young ABNs that are reactivated during subsequent REM sleep against a backdrop of overall reduced ABN activity. Optogenetic silencing of this sparse ABN activity during REM sleep alters the structural remodeling of spines on ABN dendrites and impairs memory consolidation. These findings provide a causal link between ABN activity during REM sleep and memory consolidation.