N-WASP-Arp2/3 signaling controls multiple steps of dendrite maturation in Purkinje cells in vivo.

During neural development, the actin filament network must be precisely regulated to form elaborate neurite structures. N-WASP tightly controls actin polymerization dynamics by activating an actin nucleator Arp2/3. However, the importance of N-WASP-Arp2/3 signaling in the assembly of neurite architecture in vivo has not been clarified. Here, we demonstrate that N-WASP-Arp2/3 signaling plays a crucial role in the maturation of cerebellar Purkinje cell (PC) dendrites in vivo in mice. N-WASP was expressed and activated in developing PCs. Inhibition of Arp2/3 and N-WASP from the beginning of dendrite formation severely disrupted the establishment of a single stem dendrite, which is a characteristic basic structure of PC dendrites. Inhibition of Arp2/3 after stem dendrite formation resulted in hypoplasia of the PC dendritic tree. Cdc42, an upstream activator of N-WASP, is required for N-WASP-Arp2/3 signaling-mediated PC dendrite maturation. In addition, overactivation of N-WASP is also detrimental to dendrite formation in PCs. These findings reveal that proper activation of N-WASP-Arp2/3 signaling is crucial for multiple steps of PC dendrite maturation in vivo.

Molecular mechanisms regulating the spatial configuration of neurites.

Precise neural networks, composed of axons and dendrites, are the structural basis for information processing in the brain. Therefore, the correct formation of neurites is critical for accurate neural function. In particular, the three-dimensional structures of dendrites vary greatly among neuron types, and the unique shape of each dendrite is tightly linked to specific synaptic connections with innervating axons and is correlated with its information processing. Although many systems are involved in neurite formation, the developmental mechanisms that control the orientation, size, and arborization pattern of neurites definitively defines their three-dimensional structure in tissues. In this review, we summarize these regulatory mechanisms that establish proper spatial configurations of neurites, especially dendrites, in invertebrates and vertebrates.

Vascularization of human brain organoids.

Human brain organoids are three-dimensional tissues that are generated in vitro from pluripotent stem cells and recapitulate the early development of the human brain. Brain organoids consist mainly of neural lineage cells, such as neural stem/precursor cells, neurons, astrocytes, and oligodendrocytes. However, all human brain organoids lack vasculature, which plays indispensable roles not only in brain homeostasis but also in brain development. In addition to the delivery of oxygen and nutrition, accumulating evidence suggests that the vascular system of the brain regulates neural differentiation, migration, and circuit formation during development. Therefore, vascularization of human brain organoids is of great importance. Current trials to vascularize various organoids include the adjustment of cultivation protocols, the introduction of microfluidic devices, and the transplantation of organoids into immunodeficient mice. In this review, we summarize the efforts to accomplish vascularization and perfusion of brain organoids, and we discuss these attempts from a forward-looking perspective.

Challenges in Modeling Human Neural Circuit Formation via Brain Organoid Technology.

Human brain organoids are three-dimensional self-organizing tissues induced from pluripotent cells that recapitulate some aspects of early development and some of the early structure of the human brain in vitro. Brain organoids consist of neural lineage cells, such as neural stem/precursor cells, neurons, astrocytes and oligodendrocytes. Additionally, brain organoids contain fluid-filled ventricle-like structures surrounded by a ventricular/subventricular (VZ/SVZ) zone-like layer of neural stem cells (NSCs). These NSCs give rise to neurons, which form multiple outer layers. Since these structures resemble some aspects of structural arrangements in the developing human brain, organoid technology has attracted great interest in the research fields of human brain development and disease modeling. Developmental brain disorders have been intensely studied through the use of human brain organoids. Relatively early steps in human brain development, such as differentiation and migration, have also been studied. However, research on neural circuit formation with brain organoids has just recently began. In this review, we summarize the current challenges in studying neural circuit formation with organoids and discuss future perspectives.

The polymicrogyria-associated GPR56 promoter preferentially drives gene expression in developing GABAergic neurons in common marmosets.

GPR56, a member of the adhesion G protein-coupled receptor family, is abundantly expressed in cells of the developing cerebral cortex, including neural progenitor cells and developing neurons. The human GPR56 gene has multiple presumptive promoters that drive the expression of the GPR56 protein in distinct patterns. Similar to coding mutations of the human GPR56 gene that may cause GPR56 dysfunction, a 15-bp homozygous deletion in the cis-regulatory element upstream of the noncoding exon 1 of GPR56 (e1m) leads to the cerebral cortex malformation and epilepsy. To clarify the expression profile of the e1m promoter-driven GPR56 in primate brain, we generated a transgenic marmoset line in which EGFP is expressed under the control of the human minimal e1m promoter. In contrast to the endogenous GPR56 protein, which is highly enriched in the ventricular zone of the cerebral cortex, EGFP is mostly expressed in developing neurons in the transgenic fetal brain. Furthermore, EGFP is predominantly expressed in GABAergic neurons, whereas the total GPR56 protein is evenly expressed in both GABAergic and glutamatergic neurons, suggesting the GABAergic neuron-preferential activity of the minimal e1m promoter. These results indicate a possible pathogenic role for GABAergic neuron in the cerebral cortex of patients with GPR56 mutations.

Versatile Roles of LKB1 Kinase Signaling in Neural Development and Homeostasis.

Kinase signaling pathways orchestrate a majority of cellular structures and functions across species. Liver kinase B1 (LKB1, also known as STK11 or Par-4) is a ubiquitously expressed master serine/threonine kinase that plays crucial roles in numerous cellular events, such as polarity control, proliferation, differentiation and energy homeostasis, in many types of cells by activating downstream kinases of the AMP-activated protein kinase (AMPK) subfamily members. In contrast to the accumulating evidence for LKB1 functions in nonneuronal tissues, its functions in the nervous system have been relatively less understood until recently. In the brain, LKB1 initially emerged as a principal regulator of axon/dendrite polarity in forebrain neurons. Thereafter, recent investigations have rapidly uncovered diverse and essential functions of LKB1 in the developing and mature nervous system, such as migration, neurite morphogenesis, myelination and the maintenance of neural integrity, demonstrating that LKB1 is also a multifunctional master kinase in the nervous system. In this review article, we summarize the expanding knowledge about the functional aspects of LKB1 signaling in neural development and homeostasis.

The LKB1-SIK Pathway Controls Dendrite Self-Avoidance in Purkinje Cells.

Strictly controlled dendrite patterning underlies precise neural connection. Dendrite self-avoidance is a crucial system preventing self-crossing and clumping of dendrites. Although many cell-surface molecules that regulate self-avoidance have been identified, the signaling pathway that orchestrates it remains poorly understood, particularly in mammals. Here, we demonstrate that the LKB1-SIK kinase pathway plays a pivotal role in the self-avoidance of Purkinje cell (PC) dendrites by ensuring dendritic localization of Robo2, a regulator of self-avoidance. LKB1 is activated in developing PCs, and PC-specific deletion of LKB1 severely disrupts the self-avoidance of PC dendrites without affecting gross morphology. SIK1 and SIK2, downstream kinases of LKB1, mediate LKB1-dependent dendrite self-avoidance. Furthermore, loss of LKB1 leads to significantly decreased Robo2 levels in the dendrite but not in the cell body. Finally, restoration of dendritic Robo2 level via overexpression largely rescues the self-avoidance defect in LKB1-deficient PCs. These findings reveal an LKB1-pathway-mediated developmental program that establishes dendrite self-avoidance.

Elavl3 is essential for the maintenance of Purkinje neuron axons.

Neuronal Elav-like (nElavl or neuronal Hu) proteins are RNA-binding proteins that regulate RNA stability and alternative splicing, which are associated with axonal and synaptic structures. nElavl proteins promote the differentiation and maturation of neurons via their regulation of RNA. The functions of nElavl in mature neurons are not fully understood, although Elavl3 is highly expressed in the adult brain. Furthermore, possible associations between nElavl genes and several neurodegenerative diseases have been reported. We investigated the relationship between nElavl functions and neuronal degeneration using Elavl3-/- mice. Elavl3-/- mice exhibited slowly progressive motor deficits leading to severe cerebellar ataxia, and axons of Elavl3-/- Purkinje cells were swollen (spheroid formation), followed by the disruption of synaptic formation of axonal terminals. Deficit in axonal transport and abnormalities in neuronal polarity was observed in Elavl3-/- Purkinje cells. These results suggest that nElavl proteins are crucial for the maintenance of axonal homeostasis in mature neurons. Moreover, Elavl3-/- mice are unique animal models that constantly develop slowly progressive axonal degeneration. Therefore, studies of Elavl3-/- mice will provide new insight regarding axonal degenerative processes.

Cadherin-7 regulates mossy fiber connectivity in the cerebellum.

To establish highly precise patterns of neural connectivity, developing axons must stop growing at their appropriate destinations and specifically synapse with target cells. However, the molecular mechanisms governing these sequential steps remain poorly understood. Here, we demonstrate that cadherin-7 (Cdh7) plays a dual role in axonal growth termination and specific synapse formation during the development of the cerebellar mossy fiber circuit. Cdh7 is expressed in mossy fiber pontine nucleus (PN) neurons and their target cerebellar granule neurons during synaptogenesis and selectively mediates synapse formation between those neurons. Additionally, Cdh7 presented by mature granule neurons diminishes the growth potential of PN axons. Furthermore, knockdown of Cdh7 in PN neurons in vivo severely impairs the connectivity of PN axons in the developing cerebellum. These findings reveal a mechanism by which a single bifunctional cell-surface receptor orchestrates precise wiring by regulating axonal growth potential and synaptic specificity.

The long non-coding RNA nuclear-enriched abundant transcript 1_2 induces paraspeckle formation in the motor neuron during the early phase of amyotrophic lateral sclerosis.

BACKGROUND: A long non-coding RNA (lncRNA), nuclear-enriched abundant transcript 1_2 (NEAT1_2), constitutes nuclear bodies known as "paraspeckles". Mutations of RNA binding proteins, including TAR DNA-binding protein-43 (TDP-43) and fused in sarcoma/translocated in liposarcoma (FUS/TLS), have been described in amyotrophic lateral sclerosis (ALS). ALS is a devastating motor neuron disease, which progresses rapidly to a total loss of upper and lower motor neurons, with consciousness sustained. The aim of this study was to clarify the interaction of paraspeckles with ALS-associated RNA-binding proteins, and to identify increased occurrence of paraspeckles in the nucleus of ALS spinal motor neurons. RESULTS: In situ hybridization (ISH) and ultraviolet cross-linking and immunoprecipitation were carried out to investigate interactions of NEAT1_2 lncRNA with ALS-associated RNA-binding proteins, and to test if paraspeckles form in ALS spinal motor neurons. As the results, TDP-43 and FUS/TLS were enriched in paraspeckles and bound to NEAT1_2 lncRNA directly. The paraspeckles were localized apart from the Cajal bodies, which were also known to be related to RNA metabolism. Analyses of 633 human spinal motor neurons in six ALS cases showed NEAT1_2 lncRNA was upregulated during the early stage of ALS pathogenesis. In addition, localization of NEAT1_2 lncRNA was identified in detail by electron microscopic analysis combined with ISH for NEAT1_2 lncRNA. The observation indicating specific assembly of NEAT1_2 lncRNA around the interchromatin granule-associated zone in the nucleus of ALS spinal motor neurons verified characteristic paraspeckle formation. CONCLUSIONS: NEAT1_2 lncRNA may act as a scaffold of RNAs and RNA binding proteins in the nuclei of ALS motor neurons, thereby modulating the functions of ALS-associated RNA-binding proteins during the early phase of ALS. These findings provide the first evidence of a direct association between paraspeckle formation and a neurodegenerative disease, and may shed light on the development of novel therapeutic targets for the treatment of ALS.

Sox21 promotes hippocampal adult neurogenesis via the transcriptional repression of the Hes5 gene.

Despite the importance of the production of new neurons in the adult hippocampus, the transcription network governing this process remains poorly understood. The High Mobility Group (HMG)-box transcription factor, Sox2, and the cell surface activated transcriptional regulator, Notch, play important roles in CNS stem cells. Here, we demonstrate that another member of the SoxB (Sox1/Sox2/Sox3) transcription factor family, Sox21, is also a critical regulator of adult neurogenesis in mouse hippocampus. Loss of Sox21 impaired transition of progenitor cells from type 2a to type 2b, thereby reducing subsequent production of new neurons in the adult dentate gyrus. Analysis of the Sox21 binding sites in neural stem/progenitor cells indicated that the Notch-responsive gene, Hes5, was a target of Sox21. Sox21 repressed Hes5 gene expression at the transcriptional level. Simultaneous overexpression of Hes5 and Sox21 revealed that Hes5 was a downstream effector of Sox21 at the point where the Notch and Sox pathways intersect to control the number of neurons in the adult hippocampus. Therefore, Sox21 controls hippocampal adult neurogenesis via transcriptional repression of the Hes5 gene.

Neural RNA-binding protein Musashi1 controls midline crossing of precerebellar neurons through posttranscriptional regulation of Robo3/Rig-1 expression.

Precisely regulated spatiotemporal gene expression is essential for the establishment of neural circuits. In contrast to the increasing evidence for transcriptional regulation of axon guidance cues and receptors, the role of posttranscriptional regulation in axon guidance, especially in vivo, remains poorly characterized. Here, we demonstrate that the expression of Slit receptor Robo3/Rig-1, which plays crucial roles in axonal midline crossing, is regulated by a neural RNA-binding protein Musashi1 (Msi1). Msi1 binds to Robo3 mRNA through RNA recognition motifs and increases the protein level of Robo3 without affecting its mRNA level. In Msi1-deficient precerebellar neurons, Robo3 protein, but not its mRNA, is dramatically reduced. Moreover, similar to defects in Robo3-deficient mice, axonal midline crossing and neuronal migration of precerebellar neurons are severely impaired in Msi1-deficient mice. Together, these findings indicate that Msi1-mediated posttranscriptional regulation of Robo3 controls midline crossing of precerebellar neurons.

Disruption of the paternal necdin gene diminishes TrkA signaling for sensory neuron survival.

Necdin is a multifunctional signaling protein that stabilizes terminal differentiation of postmitotic neurons. The human necdin gene in chromosome 15q11-q12 is maternally imprinted, paternally transcribed, and not expressed in Prader-Willi syndrome, a human genomic imprinting-associated neurodevelopmental disorder. Although necdin-deficient mice display several abnormal phenotypes reminiscent of this syndrome, little is known about molecular mechanisms that lead to the neurodevelopmental defects. Here, we demonstrate that paternally expressed necdin is required for physiological development of nerve growth factor (NGF)-dependent sensory neurons. Mouse embryos defective in the paternal necdin allele displayed absent necdin expression in the dorsal root ganglia, in which the tropomyosin-related kinase A (TrkA) receptor tyrosine kinase and the p75 neurotrophin receptor were expressed in a normal manner. Necdin interacted with both TrkA and p75 to facilitate the association between these receptors. NGF-induced phosphorylation of TrkA and mitogen-activated protein kinase was significantly diminished in the necdin-null sensory ganglia. Furthermore, the mice lacking the paternal necdin allele displayed augmented apoptosis in the sensory ganglia in vivo and had a reduced population of substance P-containing neurons. These mutant mice showed significantly high tolerance to thermal pain, which is often seen in individuals with Prader-Willi syndrome. These results suggest that paternally expressed necdin facilitates TrkA signaling to promote the survival of NGF-dependent nociceptive neurons.

Necdin-related MAGE proteins differentially interact with the E2F1 transcription factor and the p75 neurotrophin receptor.

Necdin is a growth suppressor expressed predominantly in postmitotic neurons and implicated in their terminal differentiation. Necdin shows a moderate homology to the MAGE family proteins, the functional roles of which are largely unknown. Human genes encoding necdin, MAGEL2 (necdin-like 1), and MAGE-G1 (necdin-like 2) are located in proximal chromosome 15q, a region associated with neurodevelopmental disorders such as Prader-Willi syndrome, Angelman syndrome, and autistic disorder. The necdin and MAGEL2 genes are subjected to genomic imprinting and suggested to be involved in the etiology of Prader-Willi syndrome. In this study, we compared biochemical and functional characteristics of murine orthologs of these necdin-related MAGE proteins. The colony formation and bromodeoxyuridine incorporation analyses revealed that necdin and MAGE-G1, but not MAGEL2, induced growth arrest. Necdin and MAGE-G1 interacted with the transcription factor E2F1 via its transactivation domain, repressed E2F1-dependent transcription, and antagonized E2F1-induced apoptosis of N1E-115 neuroblastoma cells. In addition, necdin and MAGE-G1 interacted with the p75 neurotrophin receptor via its distinct intracellular domains. In contrast, MAGEL2 failed to bind to these necdin interactors, suggesting that MAGEL2 has no necdin-like function in developing brain. Overexpression of p75 translocated necdin and MAGE-G1 in the proximity of the plasma membrane and reduced their association with E2F1 to facilitate E2F1-induced death of neuroblastoma cells. These results suggest that necdin and MAGE-G1 target both E2F1 and p75 to regulate cell viability during brain development.

Upregulation and antiapoptotic role of endogenous Alzheimer amyloid precursor protein in dorsal root ganglion neurons.

The amyloid precursor protein (APP) is a transmembrane protein whose abnormal processing is associated with the pathogenesis of Alzheimer's disease. In this study, we examined the expression and role of cell-associated APP in primary dorsal root ganglion (DRG) neurons. When dissociated DRG cells prepared from mouse embryos were treated with nerve growth factor (NGF), neuronal APP levels were transiently elevated. DRG neurons treated with an antibody against cell surface APP failed to mature and underwent apoptosis. When NGF was withdrawn from the cultures after a 36-h NGF treatment, virtually all neurons underwent apoptosis by 48 h. During the course of apoptosis, some neurons with intact morphology contained increased levels of APP immunoreactivity, whereas the APP levels were greatly reduced in apoptotic neurons. Furthermore, affected neurons contained immunoreactivities for activated caspase-3, a caspase-cleaved APP fragment (APPDeltaC31), and Abeta. Downregulation of endogenous APP expression by treatment with an APP antisense oligodeoxynucleotide significantly increased the number of apoptotic neurons in NGF-deprived DRG cultures. Furthermore, overexpression of APP by adenovirus vector-mediated gene transfer reduced the number of apoptotic neurons deprived of NGF. These results suggest that endogenous APP is upregulated to exert an antiapoptotic effect on neurotrophin-deprived DRG neurons and subsequently undergoes caspase-dependent proteolysis.

Activation of calpain in cultured neurons overexpressing Alzheimer amyloid precursor protein.

We have previously reported that overexpression of wild-type amyloid precursor protein (APP) in postmitotic neurons induces cleavage-dependent activation of caspase-3 both in vivo and in vitro. In this study, we investigated the mechanism underlying APP-induced caspase-3 activation using adenovirus-mediated gene transfer into postmitotic neurons derived from human embryonal carcinoma NT2 cells. Overexpression of wild-type APP significantly increased intracellular (45)Ca(2+) content prior to the activation of caspase-3 in NT2-derived neurons. Chelation of intracellular Ca(2+) markedly suppressed APP-induced activation of caspase-3. Furthermore, calpain, a Ca(2+)-dependent cysteine protease, was activated in neurons overexpressing APP as assessed by increased levels of calpain-cleaved alpha-fodrin and autolytic mu-calpain fragments. Neither calpain nor caspase-3 was activated in neurons expressing an APP mutant defective in the Abeta(1-20) domain. Calpain inhibitors almost completely suppressed APP-induced activation of neuronal caspase-3. E64d, a membrane permeable inhibitor of calpain, significantly suppressed APP-induced neuronal death. These results suggest that overexpression of wild-type APP activates calpain that mediates caspase-3 activation in postmitotic neurons.