Zinc deficiency impairs axonal regeneration and functional recovery after spinal cord injury by modulating macrophage polarization via NF-κB pathway.

Background: Spinal cord injury (SCI) is a devastating disease that results in permanent paralysis. Currently, there is no effective treatment for SCI, and it is important to identify factors that can provide therapeutic intervention during the course of the disease. Zinc, an essential trace element, has attracted attention as a regulator of inflammatory responses. In this study, we investigated the effect of zinc status on the SCI pathology and whether or not zinc could be a potential therapeutic target. Methods: We created experimental mouse models with three different serum zinc concentration by changing the zinc content of the diet. After inducing contusion injury to the spinal cord of three mouse models, we assessed inflammation, apoptosis, demyelination, axonal regeneration, and the number of nuclear translocations of NF-κB in macrophages by using qPCR and immunostaining. In addition, macrophages in the injured spinal cord of these mouse models were isolated by flow cytometry, and their intracellular zinc concentration level and gene expression were examined. Functional recovery was assessed using the open field motor score, a foot print analysis, and a grid walk test. Statistical analysis was performed using Wilcoxon rank-sum test and ANOVA with the Tukey-Kramer test. Results: In macrophages after SCI, zinc deficiency promoted nuclear translocation of NF-κB, polarization to pro-inflammatory like phenotype and expression of pro-inflammatory cytokines. The inflammatory response exacerbated by zinc deficiency led to worsening motor function by inducing more apoptosis of oligodendrocytes and demyelination and inhibiting axonal regeneration in the lesion site compared to the normal zinc condition. Furthermore, zinc supplementation after SCI attenuated these zinc-deficiency-induced series of responses and improved motor function. Conclusion: We demonstrated that zinc affected axonal regeneration and motor functional recovery after SCI by negatively regulating NF-κB activity and the subsequent inflammatory response in macrophages. Our findings suggest that zinc supplementation after SCI may be a novel therapeutic strategy for SCI.

Reduced Neuroinflammation Via Astrocytes and Neutrophils Promotes Regeneration After Spinal Cord Injury in Neonatal Mice.

Neonatal spinal cord injury (SCI) shows better functional outcomes than adult SCI. Although the regenerative capability in the neonatal spinal cord may have cues in the treatment of adult SCI, the mechanism underlying neonatal spinal cord regeneration after SCI is unclear. We previously reported age-dependent variation in the pathogenesis of inflammation after SCI. Therefore, we explored differences in the pathogenesis of inflammation after SCI between neonatal and adult mice and their effect on axon regeneration and functional outcome. We established two-day-old spinal cord crush mice as a model of neonatal SCI. Immunohistochemistry of the spinal cord revealed that the nuclear translocation of NF-κB, which promotes the expression of chemokines, was significantly lower in the astrocytes of neonates than in those of adults. Flow cytometry revealed that neonatal astrocytes secrete low levels of chemokines to recruit circulating neutrophils (e.g., Cxcl1 and Cxcl2) after SCI in comparison with adults. We also found that the expression of a chemokine receptor (CXCR2) and an adhesion molecule (ß2 integrin) quantified by flow cytometry was lower in neonatal circulating neutrophils than in adult neutrophils. Strikingly, these neonate-specific cellular properties seemed to be associated with no neutrophil infiltration into the injured spinal cord, followed by significantly lower expression of inflammatory cytokines (Il-1ß, Il-6 and TNF-α) after SCI in the spinal cords of neonates than in those of adults. At the same time, significantly fewer apoptotic neurons and greater axonal regeneration were observed in neonates in comparison with adults, which led to a marked recovery of locomotor function. This neonate-specific mechanism of inflammation regulation may have potential therapeutic applications in controlling inflammation after adult SCI.

Macrophages play a leading role in determining the direction of astrocytic migration in spinal cord injury via ADP-P2Y1R axis.

After spinal cord injury (SCI), inflammatory cells such as macrophages infiltrate the injured area, and astrocytes migrate, forming a glial scar around macrophages. The glial scar inhibits axonal regeneration, resulting in significant permanent disability. However, the mechanism through which glial scar-forming astrocytes migrate to the injury site has not been clarified. Here we show that migrating macrophages attract reactive astrocytes toward the center of the lesion after SCI. Chimeric mice with bone marrow lacking IRF8, which controls macrophage centripetal migration after SCI, showed widely scattered macrophages in the injured spinal cord with the formation of a huge glial scar around the macrophages. To determine whether astrocytes or macrophages play a leading role in determining the directions of migration, we generated chimeric mice with reactive astrocyte-specific Socs3-/- mice, which showed enhanced astrocyte migration, and bone marrow from IRF8-/- mice. In this mouse model, macrophages were widely scattered, and a huge glial scar was formed around the macrophages as in wild-type mice that were transplanted with IRF8-/- bone marrow. In addition, we revealed that macrophage-secreted ATP-derived ADP attracts astrocytes via the P2Y1 receptor. Our findings revealed a mechanism through which migrating macrophages attract astrocytes and affect the pathophysiology and outcome after SCI.

Bone marrow-derived fibroblast migration via periostin causes irreversible arthrogenic contracture after joint immobilization.

Joint contracture causes distressing permanent mobility disorder due to trauma, arthritis, and aging, with no effective treatment available. A principal and irreversible cause of joint contracture has been regarded as the development of joint capsule fibrosis. However, the molecular mechanisms underlying contracture remain unclear. We established a mouse model of knee joint contracture, revealing that fibrosis in joint capsules causes irreversible contracture. RNA-sequencing of contracture capsules demonstrated a marked enrichment of the genes involved in the extracellular region, particularly periostin (Postn). Three-dimensional magnetic resonance imaging and immunohistological analysis of contracture patients revealed posterior joint capsule thickening with abundant type I collagen (Col1a2) and POSTN in humans. Col1a2-GFPTG ; Postn-/- mice and chimeric mice with Col1a2-GFPTG ; tdTomatoTG bone marrow showed fibrosis in joint capsules caused by bone marrow-derived fibroblasts, and POSTN promoted the migration of bone marrow-derived fibroblasts, contributing to fibrosis and contracture. Conversely, POSTN-neutralizing antibody attenuated contracture exacerbation. Our findings identified POSTN as a key inducer of fibroblast migration that exacerbates capsule fibrosis, providing a potential therapeutic strategy for joint contracture.

Macrophages play a leading role in determining the direction of astrocytic migration in spinal cord injury via ADP-P2Y1R axis.

After spinal cord injury (SCI), inflammatory cells such as macrophages infiltrate the injured area, and astrocytes migrate, forming a glial scar around macrophages. The glial scar inhibits axonal regeneration, resulting in significant permanent disability. However, the mechanism by which glial scar-forming astrocytes migrate to the injury site has not been clarified. Here we show that migrating macrophages attract reactive astrocytes toward the center of the lesion after SCI. Chimeric mice with bone marrow lacking IRF8, which controls macrophage centripetal migration after SCI, showed widely scattered macrophages in injured spinal cord with the formation of a huge glial scar around the macrophages. To determine whether astrocytes or macrophages play a leading role in determining the directions of migration, we generated chimeric mice with reactive astrocyte-specific Socs3 -/- mice, which showed enhanced astrocyte migration, and bone marrow from IRF8 -/- mice. In this mouse model, macrophages were widely scattered, and a huge glial scar was formed around the macrophages as in wild-type mice that were transplanted with IRF8 -/ bone marrow. In addition, we revealed that macrophage-secreted ATP-derived ADP attracts astrocytes via the P2Y1 receptor. Our findings revealed a mechanism in which migrating macrophages attracted astrocytes and affected the pathophysiology and outcome after SCI.

Glial scar survives until the chronic phase by recruiting scar-forming astrocytes after spinal cord injury.

Spinal cord injury (SCI) causes reactive astrogliosis, the sequential phenotypic change of astrocytes in which naïve astrocytes (NAs) transform into reactive astrocytes (RAs) and subsequently become scar-forming astrocytes (SAs), resulting in glial scar formation around the lesion site and thereby limiting axonal regeneration and motor/sensory functional recovery. Inhibiting the transformation of RAs into SAs in the acute phase attenuates the reactive astrogliosis and promotes regeneration. However, whether or not SAs once formed can revert to RAs or SAs is unclear. We performed selective isolation of astrocytes from glial scars at different time points for a gene expression analysis and found that the expression of Sox9, an important transcriptional factor for glial cell differentiation, was significantly increased in chronic phase astrocytes (CAs) compared to SAs in the sub-acute phase. Furthermore, CAs showed a significantly lower expression of chondroitin sulfate proteoglycan (CSPG)-related genes than SAs. These results indicated that SAs changed their phenotypes according to the surrounding environment of the injured spinal cord over time. Even though the integrin-N-cadherin pathway is critical for glial scar formation, collagen-I-grown scar-forming astrocytes (Col-I-SAs) did not change their phenotype after depleting the effect of integrin or N-cadherin. In addition, we found that Col-I-SAs transplanted into a naïve spinal cord formed glial scar again by maintaining a high expression of genes involved in the integrin-N-cadherin pathway and a low expression of CSPG-related genes. Interestingly, the transplanted Col-I-SAs changed NAs into SAs, and anti-ß1-integrin antibody blocked the recruitment of SAs while reducing the volume of glial scar in the chronic phase. Our findings indicate that while the characteristics of glial scars change over time after SCI, SAs have a cell-autonomous function to form and maintain a glial scar, highlighting the basic mechanism underlying the persistence of glial scars after central nervous system injury until the chronic phase, which may be a therapeutic target.

Zinc chelator treatment in crush syndrome model mice attenuates ischemia-reperfusion-induced muscle injury due to suppressing of neutrophil infiltration.

In crush syndrome, massive muscle breakdown resulting from ischemia-reperfusion muscle injury can be a life-threatening condition that requires urgent treatment. Blood reperfusion into the ischemic muscle triggers an immediate inflammatory response, and neutrophils are the first to infiltrate and exacerbate the muscle damage. Since free zinc ion play a critical role in the immune system and the function of neutrophils is impaired by zinc depletion, we hypothesized that the administration of a zinc chelator would be effective for suppressing the inflammatory reaction at the site of ischemia-reperfusion injury and for improving of the pathology of crush syndrome. A crush syndrome model was created by using a rubber tourniquet to compress the bilateral hind limbs of mice at 8 weeks. A zinc chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) was administered immediately after reperfusion in order to assess the anti-inflammatory effect of the chelator for neutrophils. Histopathological evaluation showed significantly less muscle breakdown and fewer neutrophil infiltration in TPEN administration group compared with control group. In addition, the expression levels of inflammatory cytokine and chemokine such as IL-6, TNFα, CXCL1, CXCL2, CXCR2, CCL2 in ischemia-reperfusion injured muscle were significantly suppressed with TPEN treatment. Less dilatation of renal tubules in histological evaluation in renal tissue and significantly better survival rate were demonstrated in TPEN treatment for ischemia-reperfusion injury in crush syndrome. The findings of our study suggest that zinc chelators contributed to the resolution of exacerbation of the inflammatory response and attenuation of muscle breakdown in the acute phase after crush syndrome. In addition, our strategy of attenuation of the acute inflammatory reaction by zinc chelators may provide a promising therapeutic strategy not only for crush syndrome, but also for other diseases driven by inflammatory reactions.

Microglial inflammation after chronic spinal cord injury is enhanced by reactive astrocytes via the fibronectin/ß1 integrin pathway.

BACKGROUND: After spinal cord injury (SCI), glial scarring is mainly formed around the lesion and inhibits axon regeneration. Recently, we reported that anti-ß1 integrin antibody (ß1Ab) had a therapeutic effect on astrocytes by preventing the induction of glial scar formation. However, the cellular components within the glial scar are not only astrocytes but also microglia, and whether or not ß1Ab treatment has any influence on microglia within the glial scar remains unclear. METHODS: To evaluate the effects of ß1Ab treatment on microglia within the glial scar after SCI, we applied thoracic contusion SCI to C57BL/6N mice, administered ß1Ab in the sub-acute phase, and analyzed the injured spinal cords with immunohistochemistry in the chronic phase. To examine the gene expression in microglia and glial scars, we selectively collected microglia with fluorescence-activated cell sorting and isolated the glial scars using laser-captured microdissection (LMD). To examine the interaction between microglia and astrocytes within the glial scar, we stimulated BV-2 microglia with conditioned medium of reactive astrocytes (RACM) in vitro, and the gene expression of TNFα (pro-inflammatory M1 marker) was analyzed via quantitative polymerase chain reaction. We also isolated both naïve astrocytes (NAs) and reactive astrocytes (RAs) with LMD and examined their expression of the ligands for ß1 integrin receptors. Statistical analyses were performed using Wilcoxon's rank-sum test. RESULTS: After performing ß1Ab treatment, the microglia were scattered within the glial scar and the expression of TNFα in both the microglia and the glial scar were significantly suppressed after SCI. This in vivo alteration was attributed to fibronectin, a ligand of ß1 integrin receptors. Furthermore, the microglial expression of TNFα was shown to be regulated by RACM as well as fibronectin in vitro. We also confirmed that fibronectin was secreted by RAs both in vitro and in vivo. These results highlighted the interaction mediated by fibronectin between RAs and microglia within the glial scar. CONCLUSION: Microglial inflammation was enhanced by RAs via the fibronectin/ß1 integrin pathway within the glial scar after SCI. Our results suggested that ß1Ab administration had therapeutic potential for ameliorating both glial scar formation and persistent neuroinflammation in the chronic phase after SCI.

Amyloidogenic processing of amyloid ß protein precursor (APP) is enhanced in the brains of alcadein α-deficient mice.

Alzheimer's disease (AD) is a very common neurodegenerative disorder, chiefly caused by increased production of neurotoxic ß-amyloid (Aß) peptide generated from proteolytic cleavage of ß-amyloid protein precursor (APP). Except for familial AD arising from mutations in the APP and presenilin (PSEN) genes, the molecular mechanisms regulating the amyloidogenic processing of APP are largely unclear. Alcadein α/calsyntenin1 (ALCα/CLSTN1) is a neuronal type I transmembrane protein that forms a complex with APP, mediated by the neuronal adaptor protein X11-like (X11L or MINT2). Formation of the ALCα-X11L-APP tripartite complex suppresses Aß generation in vitro, and X11L-deficient mice exhibit enhanced amyloidogenic processing of endogenous APP. However, the role of ALCα in APP metabolism in vivo remains unclear. Here, by generating ALCα-deficient mice and using immunohistochemistry, immunoblotting, and co-immunoprecipitation analyses, we verified the role of ALCα in the suppression of amyloidogenic processing of endogenous APP in vivo We observed that ALCα deficiency attenuates the association of X11L with APP, significantly enhances amyloidogenic ß-site cleavage of APP, especially in endosomes, and increases the generation of endogenous Aß in the brain. Furthermore, we noted amyloid plaque formation in the brains of human APP-transgenic mice in an ALCα-deficient background. These results unveil a potential role of ALCα in protecting cerebral neurons from Aß-dependent pathogenicity in AD.

Generation of the Rat Monoclonal Antibody Against the Extracellular Domain of Human CD63 by DNA Immunization.

Human cluster of differentiation 63 (hCD63) is one of the tetraspanin receptors that is abundant on the surface of exosomes. Exosomes are involved in cell-to-cell communication, including from cancer cells to normal cells. It is very important to detect exosomes as a marker for the diagnosis of various diseases. In this study, we report the generation and characterization of a monoclonal antibody (mAb) against the extracellular domain of hCD63 using DNA immunization. This mAb, clone 1C8-2B11, exhibits high performance for use in immunofluorescence and flow cytometry, and it has 10-fold higher affinity than the control antibody that is commercially available. mAb 1C8-2B11 has great potential to be a tool for research and clinical diagnosis.

Dmrt genes participate in the development of Cajal-Retzius cells derived from the cortical hem in the telencephalon.

BACKGROUND: During development, Cajal-Retzius (CR) cells are the first generated and essential pioneering neurons that control neuronal migration and arealization in the mammalian cortex. CR cells are derived from specific regions within the telencephalon, that is, the pallial septum in the rostromedial cortex, the pallial-subpallial boundary, and the cortical hem (CH) in the caudomedial cortex. However, the molecular mechanism underlying the generation of CR cell subtypes in distinct regions of origin is poorly understood. RESULTS: We found that double-sex and mab-3 related transcription factor (Dmrt) genes, that is, Dmrta1 and Dmrt3, were expressed in the progenitor domains that produce CR cells. The number of CH-derived CR cells was severely decreased in Dmrt3 mutants, especially in Dmrta1 and Dmrt3 double mutants. The reduced production of the CR cells was consistent with the developmental impairment of the CH structures in the medial telencephalon from which the CR cells are produced. CONCLUSION: Dmrta1 and Dmrt3 cooperatively regulate patterning of the CH structure and production of the CR cells from the CH during cortical development.

The beneficial aspects of spasticity in relation to ambulatory ability in mice with spinal cord injury.

STUDY DESIGN: Experimental study with mice. OBJECTIVES: Spasticity is a common complication after spinal cord injury (SCI) and has detrimental aspects, such as persistent pain and involuntary muscle spasms. This study aimed to assess the influence of antispastic therapy on locomotor function after SCI. SETTING: University-based laboratory in Fukuoka, Japan. METHODS: A mouse model of spasticity was developed by producing incomplete SCI at the 9th thoracic level. At 8 weeks after SCI, an antispastic drug, baclofen, was intraperitoneally administered to six injured and two sham-operated mice. The severity of spasticity was evaluated by the modified Ashworth scoring (MAS) system, and locomotor function was evaluated by the Basso-Beattie-Bresnahan (BBB) scale/Basso mouse score (BMS). RESULTS: The administration of baclofen significantly improved spasticity in the SCI mice and the mean MAS decreased to from 6.2 to 2.8. However, at the same time, it significantly exacerbated the locomotor dysfunction of the SCI mice and the mean BMS decreased from 4.7 to 2.3. The time-course of the changes in locomotor function coincided with the time-course of the spasticity score. We also confirmed that the administration of baclofen was not associated with any changes in either locomotor function or spasticity of the sham-operated control mice. CONCLUSIONS: Our results suggest that spasticity has a certain beneficial effect on ambulation ability. It is important to note that antispastic treatments may be associated with a risk of impairing the preserved function of chronic SCI patients.

Endfoot regeneration restricts radial glial state and prevents translocation into the outer subventricular zone in early mammalian brain development.

Neural stem cells, called radial glia, maintain epithelial structure during the early neocortical development. The prevailing view claims that when radial glia first proliferate, their symmetric divisions require strict spindle orientation; its perturbation causes precocious neurogenesis and apoptosis. Here, we show that despite this conventional view, radial glia at the proliferative stage undergo normal symmetric divisions by regenerating an apical endfoot even if it is lost by oblique divisions. We found that the Notch-R-Ras-integrin ß1 pathway promotes the regeneration of endfeet, whose leading edge bears ectopic adherens junctions and the Par-polarity complex. However, this regeneration ability gradually declines during the subsequent neurogenic stage and hence oblique divisions induce basal translocation of radial glia to form the outer subventricular zone, a hallmark of the development of the convoluted brain. Our study reveals that endfoot regeneration is a temporally changing cryptic property, which controls the radial glial state and its shift is essential for mammalian brain size expansion.

Dmrt factors determine the positional information of cerebral cortical progenitors via differential suppression of homeobox genes.

The spatiotemporal identity of neural progenitors and the regional control of neurogenesis are essential for the development of cerebral cortical architecture. Here, we report that mammalian DM domain factors (Dmrt) determine the identity of cerebral cortical progenitors. Among the Dmrt family genes expressed in the developing dorsal telencephalon, Dmrt3 and Dmrta2 show a medialhigh/laterallow expression gradient. Their simultaneous loss confers a ventral identity to dorsal progenitors, resulting in the ectopic expression of Gsx2 and massive production of GABAergic olfactory bulb interneurons in the dorsal telencephalon. Furthermore, double-mutant progenitors in the medial region exhibit upregulated Pax6 and more lateral characteristics. These ventral and lateral shifts in progenitor identity depend on Dmrt gene dosage. We also found that Dmrt factors bind to Gsx2 and Pax6 enhancers to suppress their expression. Our findings thus reveal that the graded expression of Dmrt factors provide positional information for progenitors by differentially repressing downstream genes in the developing cerebral cortex.

Tranexamic acid reduces heme cytotoxicity via the TLR4/TNF axis and ameliorates functional recovery after spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is a catastrophic trauma accompanied by intralesional bleeding and neuroinflammation. Recently, there is increasing interest in tranexamic acid (TXA), an anti-fibrinolytic drug, which can reduce the bleeding volume after physical trauma. However, the efficacy of TXA on the pathology of SCI remains unknown. METHODS: After producing a contusion SCI at the thoracic level of mice, TXA was intraperitoneally administered and the bleeding volume in the lesion area was quantified. Tissue damage was evaluated by immunohistochemical and gene expression analyses. Since heme is one of the degraded products of red blood cells (RBCs) and damage-associated molecular pattern molecules (DAMPs), we examined the influence of heme on the pathology of SCI. Functional recovery was assessed using the open field motor score, a foot print analysis, a grid walk test, and a novel kinematic analysis system. Statistical analyses were performed using Wilcoxon's rank-sum test, Dunnett's test, and an ANOVA with the Tukey-Kramer post-hoc test. RESULTS: After SCI, the intralesional bleeding volume was correlated with the heme content and the demyelinated area at the lesion site, which were significantly reduced by the administration of TXA. In the injured spinal cord, toll-like receptor 4 (TLR4), which is a DAMP receptor, was predominantly expressed in microglial cells. Heme stimulation increased TLR4 and tumor necrosis factor (TNF) expression levels in primary microglial cells in a dose-dependent manner. Similarly to the in vitro experiments, the injection of non-lysed RBCs had little pathological influence on the spinal cord, whereas the injection of lysed RBCs or heme solution significantly upregulated the TLR4 and TNF expression in microglial cells. In TXA-treated SCI mice, the decreased expressions of TLR4 and TNF were observed at the lesion sites, accompanied by a significant reduction in the number of apoptotic cells and better functional recovery in comparison to saline-treated control mice. CONCLUSION: The administration of TXA ameliorated the intralesional cytotoxicity both by reducing the intralesional bleeding volume and preventing heme induction of the TLR4/TNF axis in the SCI lesion. Our findings suggest that TXA treatment may be a therapeutic option for acute-phase SCI.

The acute phase serum zinc concentration is a reliable biomarker for predicting the functional outcome after spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is a devastating disorder for which the accurate prediction of the functional prognosis is urgently needed. Due to the lack of reliable prediction methods, the acute evaluation of SCI severity and therapeutic intervention efficacy is extremely difficult, presenting major obstacles to the development of acute SCI treatment. We herein report a novel method for accurately predicting the functional prognosis using the acute-phase serum zinc concentration after SCI. METHODS: We produced experimental animal SCI models with different prognoses and examined the relationship among the SCI severity, functional outcome, and acute-phase serum zinc concentration. We also examined whether we could predict the functional prognosis by evaluating the serum zinc concentration within 72 h after SCI in a human prospective study. FINDINGS: In a mouse model, the acute serum zinc concentrations decreased in proportion to SCI severity and the serum zinc concentrations at 12 h after SCI accurately predicted the functional prognosis. We clarified the mechanism underlying this serum zinc proportional decrease, showing that activated monocytes took up zinc from blood-serum and then infiltrated the lesion area in a severity-dependent manner. A non-linear regression analysis of 38 SCI patients showed that the serum zinc concentrations in the acute-phase accurately predicted the long-term functional outcome (R2 = 0·84) more accurately than any other previously reported acute-phase biomarkers. INTERPRETATION: The acute-phase serum zinc concentration could be a useful biomarker for predicting the functional prognosis. This simple method will allow for more objective clinical trials and the development of patient-tailored treatment for SCI.

Loss of the canonical spindle orientation function in the Pins/LGN homolog AGS3.

In many cell types, mitotic spindle orientation relies on the canonical "LGN complex" composed of Pins/LGN, Mud/NuMA, and Gαi subunits. Membrane localization of this complex recruits motor force generators that pull on astral microtubules to orient the spindle. Drosophila Pins shares highly conserved functional domains with its two vertebrate homologs LGN and AGS3. Whereas the role of Pins and LGN in oriented divisions is extensively documented, involvement of AGS3 remains controversial. Here, we show that AGS3 is not required for planar divisions of neural progenitors in the mouse neocortex. AGS3 is not recruited to the cell cortex and does not rescue LGN loss of function. Despite conserved interactions with NuMA and Gαiin vitro, comparison of LGN and AGS3 functional domains in vivo reveals unexpected differences in the ability of these interactions to mediate spindle orientation functions. Finally, we find that Drosophila Pins is unable to substitute for LGN loss of function in vertebrates, highlighting that species-specific modulations of the interactions between components of the Pins/LGN complex are crucial in vivo for spindle orientation.

Prdm16 is crucial for progression of the multipolar phase during neural differentiation of the developing neocortex.

The precise control of neuronal migration and morphological changes during differentiation is essential for neocortical development. We hypothesized that the transition of progenitors through progressive stages of differentiation involves dynamic changes in levels of mitochondrial reactive oxygen species (mtROS), depending on cell requirements. We found that progenitors had higher levels of mtROS, but that these levels were significantly decreased with differentiation. The Prdm16 gene was identified as a candidate modulator of mtROS using microarray analysis, and was specifically expressed by progenitors in the ventricular zone. However, Prdm16 expression declined during the transition into NeuroD1-positive multipolar cells. Subsequently, repression of Prdm16 expression by NeuroD1 on the periphery of ventricular zone was crucial for appropriate progression of the multipolar phase and was required for normal cellular development. Furthermore, time-lapse imaging experiments revealed abnormal migration and morphological changes in Prdm16-overexpressing and -knockdown cells. Reporter assays and mtROS determinations demonstrated that PGC1α is a major downstream effector of Prdm16 and NeuroD1, and is required for regulation of the multipolar phase and characteristic modes of migration. Taken together, these data suggest that Prdm16 plays an important role in dynamic cellular redox changes in developing neocortex during neural differentiation.

Cell-cycle-independent transitions in temporal identity of mammalian neural progenitor cells.

During cerebral development, many types of neurons are sequentially generated by self-renewing progenitor cells called apical progenitors (APs). Temporal changes in AP identity are thought to be responsible for neuronal diversity; however, the mechanisms underlying such changes remain largely unknown. Here we perform single-cell transcriptome analysis of individual progenitors at different developmental stages, and identify a subset of genes whose expression changes over time but is independent of differentiation status. Surprisingly, the pattern of changes in the expression of such temporal-axis genes in APs is unaffected by cell-cycle arrest. Consistent with this, transient cell-cycle arrest of APs in vivo does not prevent descendant neurons from acquiring their correct laminar fates. Analysis of cultured APs reveals that transitions in AP gene expression are driven by both cell-intrinsic and -extrinsic mechanisms. These results suggest that the timing mechanisms controlling AP temporal identity function independently of cell-cycle progression and Notch activation mode.