Neuraminidase inhibition promotes the collective migration of neurons and recovery of brain function.

In the injured brain, new neurons produced from endogenous neural stem cells form chains and migrate to injured areas and contribute to the regeneration of lost neurons. However, this endogenous regenerative capacity of the brain has not yet been leveraged for the treatment of brain injury. Here, we show that in healthy brain chains of migrating new neurons maintain unexpectedly large non-adherent areas between neighboring cells, allowing for efficient migration. In instances of brain injury, neuraminidase reduces polysialic acid levels, which negatively regulates adhesion, leading to increased cell-cell adhesion and reduced migration efficiency. The administration of zanamivir, a neuraminidase inhibitor used for influenza treatment, promotes neuronal migration toward damaged regions, fosters neuronal regeneration, and facilitates functional recovery. Together, these findings shed light on a new mechanism governing efficient neuronal migration in the adult brain under physiological conditions, pinpoint the disruption of this mechanism during brain injury, and propose a promising therapeutic avenue for brain injury through drug repositioning.

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.

Corrigendum: PlexinD1 signaling controls domain-specific dendritic development in newborn neurons in the postnatal olfactory bulb.

[This corrects the article DOI: 10.3389/fnins.2023.1143130.].

PlexinD1 signaling controls domain-specific dendritic development in newborn neurons in the postnatal olfactory bulb.

Newborn neurons show immature bipolar morphology and continue to migrate toward their destinations. After the termination of migration, newborn neurons undergo spatially controlled dendrite formation and change into a complex morphology. The mechanisms of dendritic development of newborn neurons have not been fully understood. Here, we show that in the postnatal olfactory bulb (OB), the Sema3E-PlexinD1 signaling, which maintains bipolar morphology of newborn neurons, also regulates their dendritic development after the termination of migration in a dendritic domain-specific manner. Genetic ablation of Sema3E or PlexinD1 enhanced dendritic branching in the proximal domain of the apical dendrites of OB newborn granule cells, whereas PlexinD1 overexpression suppressed it in a Rho binding domain (RBD)-dependent manner. Furthermore, RhoJ, a small GTPase that directly binds to PlexinD1RBD in vascular endothelial cells, is expressed in migrating and differentiating newborn granule cells in the OB and is also involved in the suppression of proximal branching of their apical dendrites. These results suggest that the Sema3E-PlexinD1-RhoJ axis regulates domain-specific dendrite formation of newborn neurons in the postnatal OB.

Seg2Link: an efficient and versatile solution for semi-automatic cell segmentation in 3D image stacks.

Recent advances in microscopy techniques, especially in electron microscopy, are transforming biomedical studies by acquiring large quantities of high-precision 3D cell image stacks. To examine cell morphology and connectivity in organs such as the brain, scientists need to conduct cell segmentation, which extracts individual cell regions of different shapes and sizes from a 3D image. This is challenging due to the indistinct images often encountered in real biomedical research: in many cases, automatic segmentation methods inevitably contain numerous mistakes in the segmentation results, even when using advanced deep learning methods. To analyze 3D cell images effectively, a semi-automated software solution is needed that combines powerful deep learning techniques with the ability to perform post-processing, generate accurate segmentations, and incorporate manual corrections. To address this gap, we developed Seg2Link, which takes deep learning predictions as inputs and use watershed 2D + cross-slice linking to generate more accurate automatic segmentations than previous methods. Additionally, it provides various manual correction tools essential for correcting mistakes in 3D segmentation results. Moreover, our software has been optimized for efficiently processing large 3D images in diverse organisms. Thus, Seg2Link offers an practical solution for scientists to study cell morphology and connectivity in 3D image stacks.

An Optochemical Oxygen Scavenger Enabling Spatiotemporal Control of Hypoxia.

We present an optochemical O2 scavenging system that enables precise spatiotemporal control of the level of hypoxia in living cells simply by adjusting the light intensity in the illuminated region. The system employs rhodamine containing a selenium or tellurium atom as an optochemical oxygen scavenger that rapidly consumes O2 by photochemical reaction with glutathione as a coreductant upon visible light irradiation (560-590 nm) and has a rapid response time, within a few minutes. The glutathione-consuming quantum yields of the system were calculated as about 5 %. The spatiotemporal O2 consuming in cultured cells was visualized with a hypoxia-responsive fluorescence probe, MAR. Phosphorescence lifetime imaging was applied to confirmed that different light intensities could generate different levels of hypoxia. To illustrate the potential utility of this system for hypoxia research, we show that it can spatiotemporally control calcium ion (Ca2+ ) influx into HEK293T cells expressing the hypoxia-responsive Ca2+ channel TRPA1.

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.

Habilitation Improves Mouse Gait Development Following Neonatal Brain Injury.

Objectives: Neonatal brain injury during gait development disrupts neural circuits and causes permanent gait dysfunction. Rehabilitation as an intervention to improve impaired gait function has been used in adults as a treatment for stroke and spinal cord injury. However, although neonates have greater neuroplasticity and regenerative capacity than adults, normal gait development and the effects of habilitation on gait function following neonatal brain injury are largely unknown. Methods: In this study, we generated cryogenic injury in mice at postnatal day 2 and subsequently performed habilitative training to promote autonomous limb movement for 4 weeks. We also quantitatively analyzed the gait acquisition process in developing mice using the Catwalk XT system. Results: Using quantitative gait analyses, we showed that during normal gait development in mice, stance phase function matures later than swing phase function. We also demonstrated that habilitation in which active limb movements were enhanced by suspending mice with a rubber band with no floor grounding promotes motor learning, including gait function, in mice with impaired acquisition of gait function resulting from neonatal brain injury. Conclusions: Our findings provide a basis for research on gait development in mice and suggest new habilitation strategies for patients with impaired gait development caused by perinatal brain diseases such as hypoxic-ischemic encephalopathy and periventricular leukomalacia.

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.

Synaptic pruning of murine adult-born neurons by microglia depends on phosphatidylserine.

New neurons, continuously added in the adult olfactory bulb (OB) and hippocampus, are involved in information processing in neural circuits. Here, we show that synaptic pruning of adult-born neurons by microglia depends on phosphatidylserine (PS), whose exposure on dendritic spines is inversely correlated with their input activity. To study the role of PS in spine pruning by microglia in vivo, we developed an inducible transgenic mouse line, in which the exposed PS is masked by a dominant-negative form of milk fat globule-EGF-factor 8 (MFG-E8), MFG-E8D89E. In this transgenic mouse, the spine pruning of adult-born neurons by microglia is impaired in the OB and hippocampus. Furthermore, the electrophysiological properties of these adult-born neurons are altered in MFG-E8D89E mice. These data suggest that PS is involved in the microglial spine pruning and the functional maturation of adult-born neurons. The MFG-E8D89E-based genetic approach shown in this study has broad applications for understanding the biology of PS-mediated phagocytosis in vivo.

Change in the central control of the bladder function of rats with focal cerebral infarction induced by photochemically-induced thrombosis.

The photochemically-induced thrombosis (photothrombosis) method can create focal cerebral infarcts anywhere in the relatively superficial layers of the cerebrum; it is easy to implement and minimally invasive. Taking advantage of this versatility, we aimed to establish a new rat model of urinary frequency with focal cerebral infarction, which was characterized by its simplicity, nonlethal nature, and high reproducibility. The prefrontal cortex and the anterior cingulate cortex, which are involved in lower urinary tract control, were targeted for focal cerebral infarction, and urinary parameters were measured by cystometrogram. Cystometric analysis indicated that micturition intervals significantly shortened in photothrombosis-treated rats compared with those in the sham operative group on Days 1 and 7 (P < 0.01), but prolonged after 14 days, with no difference between the two groups. Immunopathological evaluation showed an accumulation of activated microglia, followed by an increase in reactive astrocytes at the peri-infarct zone after photothrombotic stroke. Throughout this study, all postphotothrombosis rats showed cerebral infarction in the prefrontal cortex and anterior cingulate cortex; there were no cases of rats with fatal cerebral infarction. This model corresponded to the clinical presentation, in that the micturition status changed after stroke. In conclusion, this novel model combining nonlethality and high reproducibility may be a suitable model of urinary frequency after focal cerebral infarction.

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.

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.

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.

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.

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.

Postnatal neuronal migration in health and disease.

Postnatal neuronal migration modulates neuronal circuit formation and function throughout life and is conserved among species. Pathological conditions activate the generation of neuroblasts in the ventricular-subventricular zone (V-SVZ) and promote their migration towards a lesion. However, the neuroblasts generally terminate their migration before reaching the lesion site unless their intrinsic capacity is modified or the environment is improved. It is important to understand which factors impede neuronal migration for functional recovery of the brain. We highlight similarities and differences in the mechanisms of neuroblast migration under physiological and pathological conditions to provide novel insights into endogenous neuronal regeneration.

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.

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.