Nobuyuki Sakayori

The effects of Fabp7 and Fabp5 on postnatal hippocampal neurogenesis in the mouse.

New neurons are continually produced after birth from neural stem/progenitor cells (NSCs/NPCs) in the hippocampal dentate gyrus (DG). Recent studies have reported that fatty acid binding protein 7 (Fabp7/brain lipid binding protein (BLBP)) is required for the maintenance of embryonic NSCs/NPCs and have identified an association between the Fabp7 gene and behavioral paradigms that correlate with hippocampal functions. However, the specific roles of Fabps in postnatal neurogenesis remain unknown. Herein, we demonstrate the effects of Fabp7, and another Fabp, Fabp5, on postnatal neurogenesis. Fabp7 and Fabp5 were detected in the subgranular zone (SGZ) of the DG, and Fabp7+ cells were less differentiated than Fabp5+ cells. We analyzed the differentiation state of NSCs/NPCs in the SGZ of 4‐week‐old (4w) Fabp7 knockout (7KO), Fabp5 KO (5KO), and Fabp7/Fabp5 double KO (7/5KO) mice and found that the number of NSCs/NPCs was dramatically reduced compared with wild‐type mice. Although the uptake of BrdU 1 day after injection was decreased in all KO mice, the survival of BrdU+ cells 1 month after injection was increased in the 7/5KO mice compared to other three genotypes. We also observed an enhancement of neuronal differentiation in all Fabp KO mice. In addition, the proliferation and survival of NSCs/NPCs differed along the anterior‐posterior axis (A‐P axis). A greater number of newborn cells in the posterior region became extinct, but this tendency was not apparent in the Fabps KO mice. These data suggest that Fabp7 and Fabp5 have differential roles for proliferation and survival of the NSCs/NPCs during postnatal DG neurogenesis. STEM Cells2012;30:1532–1543

Distinctive effects of arachidonic acid and docosahexaenoic acid on neural stem /progenitor cells

Arachidonic acid (ARA) and docosahexaenoic acid (DHA), which are the dominant polyunsaturated fatty acids in the brain, have crucial roles in brain development and function. Recent studies have shown that ARA and DHA promote postnatal neurogenesis. However, the direct effects of ARA on neural stem/progenitor cells (NSPCs) and the effects of ARA and DHA on NSPCs at the neurogenic and subsequent gliogenic stages are still unknown. Here, we analyzed the effects of ARA and DHA on neurogenesis, specifically maintenance and differentiation, using neurosphere assays. We confirmed that primary neurospheres are neurogenic NSPCs and that tertiary neurospheres are gliogenic NSPCs. Regarding the effects of ARA and DHA on neurogenic NSPCs, ARA and DHA increased the number of neurospheres, whereas neither ARA nor DHA had a detectable effect on NSPCs in the differentiation condition. In gliogenic NSPCs, DHA increased the number of neurospheres, whereas ARA had no such effect. In contrast, ARA increased the number of astrocytes, whereas DHA increased the number of neurons in the differentiation condition. These results suggest that ARA promotes the maintenance of neurogenic NSPCs and might induce the glial differentiation of gliogenic NSPCs and that DHA promotes the maintenance of both neurogenic and gliogenic NSPCs and might lead to the neuronal differentiation of gliogenic NSPCs.

Maternal dietary imbalance between omega-6 and omega-3 polyunsaturated fatty acids impairs neocortical development via epoxy metabolites

Omega‐6 (n‐6) and omega‐3 (n‐3) polyunsaturated fatty acids (PUFAs) are essential nutrients. Although several studies have suggested that a balanced dietary n‐6:n‐3 ratio is essential for brain development, the underlying cellular and molecular mechanism is poorly understood. Here, we found that feeding pregnant mice an n‐6 excess/n‐3 deficient diet, which reflects modern human diets, impairsed neocortical neurogenesis in the offspring. This impaired neurodevelopment occurs through a precocious fate transition of neural stem cells from the neurogenic to gliogenic lineage. A comprehensive mediator lipidomics screen revealed key mediators, epoxy metabolites, which were confirmed functionally using a neurosphere assay. Importantly, although the offspring were raised on a well‐balanced n‐6:n‐3 diet, they exhibited increased anxiety‐related behavior in adulthood. These findings provide compelling evidence that excess maternal consumption of n‐6 PUFAs combined with insufficient intake of n‐3 PUFAs causes abnormal brain development that can have long‐lasting effects on the offsprings mental state. Stem Cells 2016;34:470–482

Molecular and Cellular Features of Murine Craniofacial and Trunk Neural Crest Cells as Stem Cell-Like Cells

The outstanding differentiation capacities and easier access from adult tissues, cells derived from neural crest cells (NCCs) have fascinated scientists in developmental biology and regenerative medicine. Differentiation potentials of NCCs are known to depend on their originating regions. Here, we report differential molecular features between craniofacial (cNCCs) and trunk (tNCCs) NCCs by analyzing transcription profiles and sphere forming assays of NCCs from P0-Cre/floxed-EGFP mouse embryos. We identified up-regulation of genes linked to carcinogenesis in cNCCs that were not previously reported to be related to NCCs, which was considered to be, an interesting feature in regard with carcinogenic potentials of NCCs such as melanoma and neuroblastoma. Wnt signal related genes were statistically up-regulated in cNCCs, also suggesting potential involvement of cNCCs in carcinogenesis. We also noticed intense expression of mesenchymal and neuronal markers in cNCCs and tNCCs, respectively. Consistent results were obtained from in vitro sphere-forming and differentiation assays. These results were in accordance with previous notion about differential potentials of cNCCs and tNCCs. We thus propose that sorting NCCs from P0-Cre/floxed-EGFP mice might be useful for the basic and translational research of NCCs. Furthermore, these newly-identified genes up-regulated in cNCC would provide helpful information on NC-originating tumors, developmental disorders in NCC derivatives, and potential applications of NCCs in regenerative medicine.

Mechanisms of DHA transport to the brain and potential therapy to neurodegenerative diseases.

Docosahexaenoic acid (DHA; 22:6 ω-3) is highly enriched in the brain and is required for proper brain development and function. Its deficiency has been shown to be linked with the emergence of neurological diseases. Dietary ω-3 fatty acid supplements including DHA have been suggested to improve neuronal development and enhance cognitive functions. However, mechanisms of DHA incorporation in the brain remain to be fully understood. Findings suggested that DHA is better incorporated when esterified within lysophospholipid rather than under its non-esterified form. Furthermore, DHA has the potential to be converted into diverse oxylipins with potential neuroprotective effects. Since DHA is poorly synthesized de novo, targeting the brain with specific carriers of DHA might provide novel therapeutic approaches to neurodegenerative diseases.

Ninein is essential for the maintenance of the cortical progenitor character by anchoring the centrosome to microtubules

Summary The mammalian cerebral cortex develops from proliferative apical progenitor cells (APs) that exhibit cell cycle-dependent nuclear movement (interkinetic nuclear migration; INM), which may be important for efficient and continuous production of neurons. The Pax6 transcription factor plays a major role in INM by regulating various downstream molecules. We have previously observed abnormal INM and unstable localization of the centrosome in APs of the Pax6 homozygous mutant rat embryo. To understand the mechanisms of INM, we focused on the centrosomes of APs. One of the centrosomal proteins, ninein, is specifically localized in the centrosome of APs. We observed a dramatic downregulation of ninein in APs of the Pax6 mutant. Moreover, knockdown of ninein by RNAi induced ectopic distribution of reduced numbers of BrdU-positive (S-phase) and PH3-positive (M-phase) cells. Furthermore, time-lapsed imaging demonstrated that knockdown of ninein in vivo induced abnormal INM. Finally, we observed impaired microtubule regrowth in neural progenitors taken from Pax6 homozygous mutant rat embryos, which was recovered by via ninein overexpression. We also found that ninein knockdown enlarged the surface size area of apical endfeet of the APs. Our results suggest that ninein plays a role in the molecular machinery essential for INM by connecting microtubules to the centrosome.

Impact of Lipid Nutrition on Neural Stem/Progenitor Cells

The neural system originates from neural stem/progenitor cells (NSPCs). Embryonic NSPCs first proliferate to increase their numbers and then produce neurons and glial cells that compose the complex neural circuits in the brain. New neurons are continually produced even after birth from adult NSPCs in the inner wall of the lateral ventricle and in the hippocampal dentate gyrus. These adult-born neurons are involved in various brain functions, including olfaction-related functions, learning and memory, pattern separation, and mood control. NSPCs are regulated by various intrinsic and extrinsic factors. Diet is one of such important extrinsic factors. Of dietary nutrients, lipids are important because they constitute the cell membrane, are a source of energy, and function as signaling molecules. Metabolites of some lipids can be strong lipid mediators that also regulate various biological activities. Recent findings have revealed that lipids are important regulators of both embryonic and adult NSPCs. We and other groups have shown that lipid signals including fat, fatty acids, their metabolites and intracellular carriers, cholesterol, and vitamins affect proliferation and differentiation of embryonic and adult NSPCs. A better understanding of the NSPCs regulation by lipids may provide important insight into the neural development and brain function.

Reduced proliferation and excess astrogenesis of Pax6 heterozygous neural stem/progenitor cells

Neural stem/progenitor cells (NSPCs) are generated in early embryonic brains and maintained to produce neurons and glial cells in the central nervous system throughout the lifespan. A transcription factor Pax6 is a pivotal player in various neurodevelopmental processes. Previously, we have shown that Pax6 heterozygous rodents have defects in hippocampal neurogenesis and production of olfactory bulb interneurons. However, characters of NSPCs derived from Pax6 heterozygous rodents have not been studied in vitro. Here we examined the maintenance/proliferation and differentiation of Pax6 heterozygous mutant (rSey(2)/+) rat NSPCs in the neurosphere culture system. We found that the proliferative activity of NSPCs derived from rSey(2)/+ rats was reduced after serial passages. We also observed an excess astrogenesis in serially passaged NSPCs from rSey(2)/+ rats. These results show that Pax6 is essential for maintaining NSPCs and determining their differentiation fates.

Maternal Nutritional Imbalance between Linoleic Acid and Alpha-Linolenic Acid Increases Offspring’s Anxious Behavior with a Sex-Dependent Manner in Mice

Omega-6 (n-6) and omega-3 (n-3) polyunsaturated fatty acids (PUFAs) are essential nutrients for normal brain development. The principal dietary n-6 and n-3 PUFAs are linoleic acid (LA) and α-linolenic acid (ALA), respectively, We have previously shown that maternal dietary imbalance between these PUFAs, i.e., rich in LA and poor in ALA, affected brain development and increased anxiety-related behavior in the mouse offspring. Here we further addressed sex difference in anxiety-related behavior in the offspring exposed to maternal LA:ALA imbalance. We fed pregnant mice a LA excess/ALA deficient (LA(ex)/ALA(def)) diet, and raised their offspring on a well-balanced LA:ALA diet from an early lactation period. When the offspring were grown to adulthood, they were subjected to behavioral and biochemical analyses. We found that both male and female offspring exposed to the LA(ex)/ALA(def) diet showed increased anxiety-related behavior compared to those exposed to the control diet, which was differently observed between the sexes. The female offspring also exhibited hyperactivity by maternal intake of the LA(ex)/ALA(def) diet. On the other hand, abnormal depressive behavior was undetected in both sexes. We also found that the ratio of n-6 to n-3 PUFAs in the brain was unaffected regardless of maternal diet or offsprings sex. Since the n-6/n-3 ratio is known to influence emotional behavior, it is reasonable to assume that LA:ALA imbalance exposed during brain development is the key for causing enhanced anxiety in adulthood. The present study indicates that maternal dietary imbalance between LA and ALA increases offsprings anxiety-related behavior with a sex-dependent manner.

Targeting the Brain with a Neuroprotective Omega-3 Fatty Acid to Enhance Neurogenesis in Hypoxic Condition in Culture

Docosahexaenoic acid (DHA, 22:6n-3) is an essential omega-3 polyunsaturated fatty acid (PUFA) that is required for proper brain development and cerebral functions. While DHA deficiency in the brain was shown to be linked to the emergence of cerebral diseases, a dietary intake of omega-3 PUFA could prevent or attenuate neurologic disturbances linked with aging or neurodegenerative diseases. In this context, targeting the brain with DHA might offer great promise in developing new therapeutics for neurodegenerative diseases. We previously synthesized a stabilized form of DHA-containing lysophosphatidylcholine a major vector of DHA transportation to the brain, which is 1-acetyl,2-docoshexaenoyl-glycerophosphocholine, named AceDoPC®. Injection of AceDoPC® or DHA after experimental ischemic stroke showed that both molecules had neuroprotective effects but AceDoPC® was the most potent. This study aims to investigate the beneficial effects of DHA either unesterified or esterified within AceDoPC® on a model of neurogenesis in vitro, under physiological or pathological conditions. The effect of protectin DX (PDX, a double lipoxygenase product of DHA) was also tested. We cultured neural stem progenitor cells (NSPCs) derived from the adult mouse brain under normal or hypoxigenic (ischemic) conditions in vitro. Neurogenesis study of cell cultures with AceDoPC® showed enhanced neurogenesis compared to addition of unesterified DHA, PDX, or vehicle control, especially under pathological conditions. Our studies of the potential mechanisms involved in neuroprotection hinted that AceDoPC® neuroprotective and regenerative effects might be due in part to its anti-oxidative effects. These results indicate the potential for novel therapeutics against stroke that target the brain.