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Thermogenesis in brown and beige adipose tissue has important roles in maintaining body temperature and countering the development of metabolic disorders such as obesity and type 2 diabetes1,2. Although much is known about commitment and activation of brown and beige adipose tissue, its multiple and abundant immunological factors have not been well characterized3–6. Here we define a critical role of IL-27–IL-27Rα signalling in improving thermogenesis, protecting against diet-induced obesity and ameliorating insulin resistance. Mechanistic studies demonstrate that IL-27 directly targets adipocytes, activating p38 MAPK–PGC-1α signalling and stimulating the production of UCP1. Notably, therapeutic administration of IL-27 ameliorated metabolic morbidities in well-established mouse models of obesity. Consistently, individuals with obesity show significantly decreased levels of serum IL-27, which can be restored after bariatric surgery. Collectively, these findings show that IL-27 has an important role in orchestrating metabolic programs, and is a highly promising target for anti-obesity immunotherapy. Therapeutic administration of IL-27—serum levels of which are decreased in individuals with obesity—improves thermogenesis, protects against diet-induced obesity and ameliorates insulin resistance in mouse models of obesity.

Inhibitor of DNA binding 2 (Id2) can promote axonal regeneration after injury of the central nervous system. However, whether Id2 can promote axonal regeneration and functional recovery after peripheral nerve injury is currently unknown. In this study, we established a mouse model of bilateral sciatic nerve crush injury. Two weeks before injury, AAV9-Id2-3×Flag-GFP was injected stereotaxically into the bilateral ventral horn of lumbar spinal cord. Our results showed that Id2 was successfully delivered into spinal cord motor neurons projecting to the sciatic nerve, and the number of regenerated motor axons in the sciatic nerve distal to the crush site was increased at 2 weeks after injury, arriving at the tibial nerve and reinnervating a few endplates in the gastrocnemius muscle. By 1 month after injury, extensive neuromuscular reinnervation occurred. In addition, the amplitude of compound muscle action potentials of the gastrocnemius muscle was markedly recovered, and their latency was shortened. These findings suggest that Id2 can accelerate axonal regeneration, promote neuromuscular reinnervation, and enhance functional improvement following sciatic nerve injury. Therefore, elevating the level of Id2 in adult neurons may present a promising strategy for peripheral nerve repair following injury. The study was approved by the Experimental Animal Ethics Committee of Jinan University (approval No. 20160302003) on March 2, 2016.

The formation of a fluid-filled cystic cavity after spinal cord injury (SCI) is a major obstacle for neural regeneration. In this study, the post-SCI cavity was bridged by a functional self-assembling peptide (F-SAP) nanofiber hydrogel coupled with growth factor "cocktail". A sustained release of growth factors was achieved by carefully tailoring the physical hindrances and charge-induced interactions between the growth factors and the peptide nanofibers. Such an engineering microenvironment elicited axon regeneration, as determined by tracing of the descending pathway in the dorsal columns and immunochemical detection of regenerating axons beyond the lesion. Furthermore, the dynamic spatiotemporal activation line of endogenous NSCs (eNSCs) after severe SCI was thoroughly investigated. The results indicated that the growth factor-coupled F-SAP greatly facilitated eNSC proliferation, neuronal differentiation, maturation, myelination, and more importantly, the formation of interconnection with severed descending corticospinal tracts. The robust endogenous neurogenesis essentially led to the recovery of locomotion and electrophysiological properties. In conclusion, the growth factor-coupled F-SAP nanofiber hydrogel elucidated the therapeutic effect of eliciting endogenous neurogenesis by locally reassembling an extracellular matrix.

Spinal cord injury (SCI) is a devastating traumatic injury that causes persistent, severe motor and sensory dysfunction. Immune responses are involved in functional recovery after SCI. Mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) has been shown to regulate the survival and differentiation of immune cells and to play a critical role in many diseases, but its function in lesion recovery after SCI remains unclear. In this paper, we generated KI (knock in) mice with a point mutation (C472G) in the active center of MALT1 and found that the KI mice exhibited improved functional recovery after SCI. Fewer macrophages were recruited to the injury site in KI mice and these macrophages differentiated into anti-inflammatory macrophages. Moreover, macrophages from KI mice exhibited reduced phosphorylation of p65, which in turn resulted in decreased SOCS3 expression and increased pSTAT6 levels. Similar results were obtained upon inhibition of MALT1 paracaspase with the small molecule inhibitor “MI-2” or the more specific inhibitor “MLT-827”. In patients with SCI, peripheral blood mononuclear cells (PBMC) displayed increased MALT1 paracaspase. Human macrophages showed reduced pro-inflammatory and increased anti-inflammatory characteristics following the inhibition of MALT1 paracaspase. These findings suggest that inhibition of MALT1 paracaspase activity in the clinic may improve lesion recovery in subjects with SCI.

Neural progenitor proliferation, neuronal migration, areal organization, and pioneer axon wiring are critical events during early forebrain development, yet remain incompletely understood, especially in human. Here, we studied forebrain development in human embryos aged 5 to 8 postconceptional weeks (WPC5–8), stages that correspond to the neuroepithelium/early marginal zone (WPC5), telencephalic preplate (WPC6 & 7), and incipient cortical plate (WPC8). We show that early telencephalic neurons are formed at the neuroepithelial stage; the most precocious ones originate from local telencephalic neuroepithelium and possibly from the olfactory placode. At the preplate stage, forebrain organization is quite similar in human and mouse in terms of areal organization and of differentiation of Cajal-Retzius cells, pioneer neurons, and axons. Like in mice, axons from pioneer neurons in prethalamus, ventral telencephalon, and cortical preplate cross the diencephalon–telencephalon junction and the pallial–subpallial boundary, forming scaffolds that could guide thalamic and cortical axons at later stages. In accord with this model, at the early cortical plate stage, corticofugal axons run in ventral telencephalon in close contact with scaffold neurons, which express CELSR3 and FZD3, two molecules that regulates formation of similar scaffolds in mice.

Myelin-laden macrophages (mye-MΦ), resulting primarily from internalization of myelin debris by infiltrating bone marrow-derived macrophages in spinal cord injury (SCI), trigger inflammatory responses that largely contribute to secondary injury. Adiponectin, which is secreted from adipose tissue, is an important hormone that modulates macrophage inflammation. In the present study, we examined the role of adiponectin on macrophage-mediated neuroinflammation after SCI. We found that in vitro activation of adiponectin receptors (AdipoRs) by their agonist AdipoRon suppressed myelin lipid accumulation in mye-MΦ through APPL1/PPARγ/LXRα/ABCA1-mediated lipid efflux, subsequently inhibiting inflammation and restoring normal function to mye-MΦ. In vivo data further confirmed that intravenous administration of AdipoRon after SCI dampened recruitment of macrophages and reduced myelin lipid accumulation. Accordingly, AdipoRon treatment ameliorated post-SCI tissue damage and astrogliosis, resulting in improved motor function. Although there was no significant pathological exacerbation in adiponectin-null mice subjected to SCI, our work reveals a functional link between adiponectin and hematogenous macrophages in the context of SCI, suggesting that activation of adiponectin signaling is a promising therapeutic approach to mitigate mye-MΦ-mediated neuroinflammation in neurological disorders involving demyelination.

During development, cadherins Celsr2 and Celsr3 control axon navigation. Unlike Celsr3, Celsr2 remains expressed in the adult, suggesting unexplored roles in maintenance and repair. Here we show that Celsr2 knockdown promotes motor axon regeneration in mouse and human spinal cord explants and cultured motor neurons. Celsr2 downregulation is accompanied by increased levels of GTP-bound Rac1 and Cdc42, and of JNK and c-Jun proteins. Using a branchial plexus injury model, we show that forelimb functional recovery is improved in Celsr2 mutant versus control mice. Compared to controls, in mutant mice, reinnervated biceps muscles are less atrophic, contain more newly formed neuromuscular junctions, and generate larger electromyographic potentials, while motor neuron survival and axon regeneration are improved. GTP-bound Rac1 and Cdc42, JNK and c-Jun are upregulated in injured mutant versus control spinal cord. In conclusion, Celsr2 negatively regulates motor axon regeneration via Cdc42/Rac1/JNK/c-Jun signaling and is a target for neural repair.

The balance of excitatory and inhibitory neurons in the central nervous system is critical for maintaining brain function and sevoflurane, a general anesthetic and an GABA receptor modulator, may change the balance of excitatory and inhibitory neurons in the cortex during early brain development. Herein, we investigated whether prenatal sevoflurane exposure (PSE) disturbs cortical neuronal development and brain function. Pregnant rats at the gestational day 14.5 were subjected to sevoflurane exposure at 3.0% for 3 h and their offspring were studied thereafter. We found a significant increase of parvalbumin-positive neurons, vesicular GABA transporter (VGAT) and GAD67 expression, and GABA neurotransmitter, and a significant decrease of vesicular glutamate transporter 1 (VGLUT1) expression and glutamate in the medial prefrontal cortex (mPFC) of offspring. Pyramidal neurons showed atrophy with shorter dendrites, less branches and lower spine density visualized by Golgi stain and a decrease of excitability with the increased miniature inhibitory postsynaptic current (mIPSC) frequency and amplitude, the decreased miniature excitatory postsynaptic current (mEPSC) frequency and excitation/inhibition (E/I) ratio using whole-cell recording in offspring. There was a significant increase of inhibitory synapse in the mPFC detected by electron microscopy. Furthermore, PSE animals showed hypo-excitatory phenotype including depression-like behaviors and learning deficits. Thus, our studies provide novel evidence that PSE causes the persisted imbalance of excitatory and inhibitory neurons in the mPFC, and this is very likely the mechanisms of the sevoflurane-induced brain functional abnormalities.

Multiple types of stem cells have been proposed for the treatment of spinal cord injury, but their comparative information remains elusive. In this study, a rat model of T10 contusion spinal cord injury was established by the impactor method. Human umbilical cord-derived mesenchymal stem cells (UCMSCs) or human adipose tissue-derived mesenchymal stem cells (ADMSCs) (2.5 μL/injection site, 1 × 105 cells/μL) was injected on rostral and caudal of the injury segment on the ninth day after injury. Rats injected with mesenchymal stem cell culture medium were used as controls. Our results show that although transplanted UCMSCs and ADMSCs failed to differentiate into neurons or glial cells in vivo, both significantly improved motor and sensory function. After spinal cord injury, UCMSCs and ADMSCs similarly promoted spinal neuron survival and axonal regeneration, decreased glial scar and lesion cavity formation, and reduced numbers of active macrophages. Bio-Plex analysis of spinal samples showed a specific increase of interleukin-10 and decrease of tumor necrosis factor α in the ADMSC group, as well as a downregulation of macrophage inflammatory protein 3α in both UCMSC and ADMSC groups at 3 days after cell transplantation. Upregulation of interleukin-10 and interleukin-13 was observed in both UCMSC and ADMSC groups at 7 days after cell transplantation. Isobaric tagging for relative and absolute quantitation proteomics analyses showed that UCMSCs and ADMSCs induced changes of multiple genes related to axonal regeneration, neurotrophy, and cell apoptosis in common and specific manners. In conclusion, UCMSC and ADMSC transplants yielded quite similar contributions to motor and sensory recovery after spinal cord injury via anti-inflammation and improved axonal growth. However, there were some differences in cytokine and gene expression induced by these two types of transplanted cells. Animal experiments were approved by the Laboratory Animal Ethics Committee at Jinan University (approval No. 20180228026) on February 28, 2018, and the application of human stem cells was approved by the Medical Ethics Committee of Medical College of Jinan University of China (approval No. 2016041303) on April 13, 2016.

Spinal cord injury (SCI) is a worldwide problem and transplantation of neural progenitor cells (NPCs) represents a promising treatment strategy. Urine derived induced pluripotent stem cells (UiPSCs) which enable the generation of patient-specific NPCs, provide an invaluable source of autologous cells for future therapeutic applications after SCI. However, the fate and potential contribution of transplanted human UiPSCs-derived NPCs (UiPSC-NPCs) into injured spinal cords remain largely unknown. In this study, using a rat contusive SCI model, we evaluated the survival, migration and differentiation of UiPSC-NPCs after transplantation at subacute phase. Transplanted cells survived and migrated from the site of grafting towards the lesion epicenter. More than 25 % cells survived over 4 weeks post transplantation, with a few of them differentiated into neurons and astrocytes. Cytokine and chemokine levels within the injured spinal cord tissues were measured using multiplex immunoassays to evaluate the immune response. Pro-inflammatory factors and chemokines were significantly decreased at 3 days after UiPSC-NPCs transplantation. At 7 days post transplantation, a lower level of pro-inflammatory factor IFN-γ and a higher level of pro-inflammatory IL-2 were found in UiPSC-NPCs group than in the control. Transplantation of UiPSC-NPCs showed little effect on microglia activation at the lesion epicenter. However, the number of microglia cells at 4 mm rostral to the injury site was significantly decreased. The size of lesion cavity was reduced after transplantation of UiPSC-NPCs. In conclusions, the UiPSC-NPCs transplanted at the subacute phase of SCI showed a beneficial effect on tissue repairing.

The tangential migration of immature neurons in the postnatal brain involves consecutive migration cycles and depends on constant remodeling of the cell cytoskeleton, particularly in the leading process (LP). Despite the identification of several proteins with permissive and empowering functions, the mechanisms that specify the direction of migration remain largely unknown. Here, we report that planar cell polarity protein Celsr3 orients neuroblasts migration from the subventricular zone (SVZ) to olfactory bulb (OB). In Celsr3-forebrain conditional knockout mice, neuroblasts loose directionality and few can reach the OB. Celsr3-deficient neuroblasts exhibit aberrant branching of LP, de novo LP formation, and decreased growth rate of microtubules (MT). Mechanistically, we show that Celsr3 interacts physically with Kif2a, a MT depolymerizing protein and that conditional inactivation of Kif2a in the forebrain recapitulates the Celsr3 knockout phenotype. Our findings provide evidence that Celsr3 and Kif2a cooperatively specify the directionality of neuroblasts tangential migration in the postnatal brain.

The role of γδ T cells in spinal cord injury remains unknown. Sun et al. report that Vγ4 γδ T cells produce IFN-γ, promote inflammatory cytokine production of macrophages, and are detrimental for functional recovery after SCI., Immune responses and neuroinflammation are critically involved in spinal cord injury (SCI). γδ T cells, a small subset of T cells, regulate the inflammation process in many diseases, yet their function in SCI is still poorly understood. In this paper, we demonstrate that mice deficient in γδ T cells (TCRδ−/−) showed improved functional recovery after SCI. γδ T cells are detected at the lesion sites within 24 hours after injury and are predominantly of the Vγ4 subtype and express the inflammatory cytokine IFN-γ. Inactivating IFN-γ signaling in macrophages results in a significantly reduced production of proinflammatory cytokines in the cerebrospinal fluid (CSF) of mice with SCIs and improves functional recovery. Furthermore, treatment of SCI with anti-Vγ4 antibodies has a beneficial effect, similar to that obtained with anti–TNF-α. In SCI patients, γδ T cells are detected in the CSF, and most of them are IFN-γ positive. In conclusion, manipulation of γδ T cell functions may be a potential approach for future SCI treatment., Graphical Abstract

Summary Atypical cadherin Celsr3 is critical for brain embryonic development, and its role in the postnatal cerebellum remains unknown. Using Celsr3-GFP mice, Celsr3 shows high expression in postnatal Purkinje cells (PCs). Mice with conditional knockout (cKO) of Celsr3 in postnatal PCs exhibit deficit in motor coordination and learning, atrophic PC dendrites, and decreased synapses. Whole-PC recording in cerebellar slices discloses a reduction frequency of mEPSC and defective postsynaptic plasticity (LTP and LTD) in Celsr3 cKO mutants. Wnt5a perfusion enhances LTP formation, which could be occluded by cAMP agonist and diminished by cAMP antagonist in control, but not in Celsr3 cKO or Fzd3 cKO cerebellar slices. Celsr3 cKO resulted in the failure of mGluR1 agonist-induced LTD and paired stimulation-induced PKCα overexpression in PC dendrites, and downregulation of mGluR1 expression compvared to controls. In conclusion, Celsr3 is required for PCs maturation and regulates postsynaptic LTP and LTD through Wnt5a/cAMP and mGluR1/PKCα signaling respectively., Graphical abstract, Highlights • Celsr3 cKO in postnatal PCs impairs mouse motor coordination and learning • Celsr3 inactivation affects the maturation of PC dendrites and synapses • Celsr3 is required for the cerebellar LTP induction via the Wnt5a/cAMP signaling • Celsr3 regulates the cerebellar LTD induction through the mGluR1/PKCα pathway, Developmental neuroscience; Cognitive neuroscience

Dorsal root ganglion (DRG) neurons receive peripheral somatosensory information and send orderly projections to second-order relay nuclei in the spinal cord and in the brainstem. Atypical cadherin Celsr3 is known to play a critical role in wiring of several central and peripheral axons. Although Celsr3 mRNA is heavily expressed in DRG neurons, its role in the development of somatosensory projections remains unexplored. Here we assessed the role of Celsr3 in DRG using conditional gene inactivation in crosses with Wnt1-Cre mice. Using Celsr3-GFP transgenic mice, we found that Celsr3 was highly expressed in different DRG cells, such as Pavalbumin-, TrkB-, and calcitonin gene-related peptide (CGRP)-positive neurons. Wnt1-Cre;Celsr3f/- animals survived for a few weeks and looked smaller than littermate controls. DiI tracing showed that early DRG axons entered the spinal cord and reached spinal cord targets similarly in mutant and control mice. CGRP-positive fiber density was significantly decreased in lamina I in the mutant versus control spinal cord at postnatal day (P) 7 and P14. Furthermore, more Pavalbumin-positive fibers invaded the gray matter and made more contacts with spinal motor neurons in mutant than in control samples. Behavioral analysis showed that mutant animals were less sensitive to pain and more sensitive to mechanical stimulation than controls. In conclusion, Celsr3 is dispensable for the patterning of central DRG projections, but it regulates for the fine mapping of sensory fibers in the gray matter, which is important for somatosensory processing.

Background: POLR3-related leukodystrophy is an autosomal recessive neurodegenerative disorder characterized by onset time ranging from the neonatal period to late childhood, progressive motor decline that manifests as spasticity, ataxia, tremor, and cerebellar symptoms, as well as mild cognitive regression and hypodontia. POLR3-related leukodystrophy belongs to the family of RNA polymerase III-related leukodystrophy, which are caused by biallelic mutations in the POLR3A, POLR3B, POLRC1, or POLR3K genes. Case presentation: In this study, we report a female child with POLR3-related leukodystrophy manifesting as cognitive decline, moderate dysarthria, motor decline, cerebellar syndrome, short stature, dysphagia, hypodontia, and mild delayed myelination by brain imaging. Interestingly, polytrichia and bronchodysplasia were first observed in a POLR3-related leukodystrophy patient. Medical exome sequencing with high coverage depth was employed to identify potential genetic variants in the patient. Novel compound heterozygous mutations of the POLR3A gene, c.1771-6C>G and c.2611del (p.M871Cfs*8), were detected. One of them is an uncommon splice site mutation, and this is the first report of this mutation in a Chinese family. The father was determined to be a heterozygous carrier of the c.2611del (p.M871Cfs*8) mutation and the mother a heterozygous carrier of the c.1771-6C>G mutation. Conclusion: The patient’s newly emerged clinical features and mutations provide useful information for further exploration of genotype-phenotype correlations of POLR3-related leukodystrophy.

Our previous studies showed that mutant mice with congenital absence of the corticospinal tract (CST) undergo spontaneous remodeling of motor networks to partially compensate for absent CST function. Here, we asked whether voluntary wheel running could further improve locomotor plasticity in CST-deficient mice. Adult mutant mice were randomly allocated to a “runners” group with free access to a wheel, or a “non-runners” group with no access to a wheel. In comparison with non-runners, there was a significant motor improvement including fine movement, grip strength, decreased footslip errors in runners after 8-week training, which was supported by the elevated amplitude of electromyography recording and increased neuromuscular junctions in the biceps. In runners, terminal ramifications of monoaminergic and rubrospinal descending axons were significantly increased in spinal segments after 12 weeks of exercise compared to non-runners. 5-ethynyl-2′-deoxyuridine (EDU) labeling showed that proliferating cells, 90% of which were Olig2-positive oligodendrocyte progenitors, were 4.8-fold more abundant in runners than in non-runners. In 8-week runners, RNAseq analysis of spinal samples identified 404 genes up-regulated and 398 genes down-regulated, and 69 differently expressed genes involved in signal transduction, among which the NF-κB, PI3K-Akt and cyclic AMP (cAMP) signaling were three top pathways. Twelve-week training induced a significant elevation of postsynaptic density protein 95 (PSD95), synaptophysin 38 and myelin basic protein (MBP), but not of brain derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and insulin like growth factor-1 (IGF-1). Thus, locomotor training activates multiple signaling pathways, contributes to neural plasticity and functional improvement, and might palliate locomotor deficits in patients.

Spinal cord injury (SCI) can cause locomotor dysfunctions and sensory deficits. Evidence shows that functional nanodrugs can regulate macrophage polarization and promote anti-inflammatory cytokine expression, which is feasible in SCI immunotherapeutic treatments. Molybdenum disulfide (MoS2) nanomaterials have garnered great attention as potential carriers for therapeutic payload. Herein, we synthesize MoS2@PEG (MoS2 = molybdenum disulfide, PEG = poly (ethylene glycol)) nanoflowers as an effective carrier for loading etanercept (ET) to treat SCI. We characterize drug loading and release properties of MoS2@PEG in vitro and demonstrate that ET-loading MoS2@PEG obviously inhibits the expression of M1-related pro-inflammatory markers (TNF-α, CD86 and iNOS), while promoting M2-related anti-inflammatory markers (Agr1, CD206 and IL-10) levels. In vivo, the mouse model of SCI shows that long-circulating ET-MoS2@PEG nanodrugs can effectively extravasate into the injured spinal cord up to 96 h after SCI, and promote macrophages towards M2 type polarization. As a result, the ET-loading MoS2@PEG administration in mice can protect survival motor neurons, thus, reducing injured areas at central lesion sites, and significantly improving locomotor recovery. This study demonstrates the anti-inflammatory and neuroprotective activities of ET-MoS2@PEG and promising utility of MoS2 nanomaterial-mediated drug delivery.

Body-weight-supported locomotor training is an activity-based therapy used frequently to train individuals with spinal cord injury (SCI) for restoring walking ability. Locomotor training after SCI is developed on the basic scientific findings of activity-dependent neuroplasticity. Based on the research from animal SCI models, there exists a spinal neural networks for locomotion which can be reactivated by intense repetitive locomotor training. Notably, the effectiveness of locomotor training depends largely on the severity of injury and time after injury. Locomotor training, using body-weight-supported walking overground or on a treadmill, with assistance manually or robotically, with a variety of training intensity and training programs, has been shown to elicit improvements in locomotor function for incomplete SCI individuals. For chronic and motor complete SCI, other interventions with proven effectiveness such as epidural stimulation might be applied in addition to locomotor training to improve the chance of locomotor recovery. In this chapter, we review the factors that influence the functional outcomes of locomotor training. We also summarize the circuitry, cellular and molecular levels of mechanisms underlying the positive role of locomotor training in inducing neuroplasticity and functional recovery following SCI.

Celsr3 and Fzd3 regulate the development of reciprocal thalamocortical projections independently of their expression in cortical or thalamic neurons. To understand this cell non autonomous mechanism further, we tested whether Celsr3 and Fzd3 could act via Isl1-positive guidepost cells. Isl1-positive cells appear in the forebrain at embryonic day (E) 9.5-E10.5 and, from E12.5, they form 2 contingents in ventral telencephalon and prethalamus. In control mice, corticothalamic axons run in the ventral telencephalic corridor in close contact with Isl1-positive cells. When Celsr3 or Fzd3 is inactivated in Isl1-expressing cells, corticofugal fibers stall and loop in the ventral telencephalic corridor of high Isl1 expression, and thalamic axons fail to cross the diencephalon-telencephalon junction (DTJ). At E12.5, before thalamic and cortical axons emerge, pioneer projections from Isl1-positive cells cross the DTJ from both sides in control but not mutant embryos. These early projections appear to act like a bridge to guide later growing thalamic axons through the DTJ. Our data suggest that Celsr3 and Fzd3 orchestrate the formation of a scaffold of pioneer neurons and their axons. This scaffold extends from prethalamus to ventral telencephalon and subcortex, and steers reciprocal corticothalamic fibers.

SHANK2 is a scaffold protein that serves as a protein anchor at the postsynaptic density in neurons. Genetic variants of SHANK2 are strongly associated with synaptic dysfunction and the pathophysiology of autism spectrum disorder. Recent studies indicate that early neuronal developmental defects play a role in the pathogenesis of autism spectrum disorder, and that insulin-like growth factor 1 has a positive effect on neurite development. To investigate the effects of SHANK2 knockdown on early neuronal development, we generated a sparse culture system using human induced pluripotent stem cells, which then differentiated into neural progenitor cells after 3–14 days in culture, and which were dissociated into single neurons. Neurons in the experimental group were infected with shSHANK2 lentivirus carrying a red fluorescent protein reporter (shSHANK2 group). Control neurons were infected with scrambled shControl lentivirus carrying a red fluorescent protein reporter (shControl group). Neuronal somata and neurites were reconstructed based on the lentiviral red fluorescent protein signal. Developmental dendritic and motility changes in VGLUT1+ glutamatergic neurons and TH+ dopaminergic neurons were then evaluated in both groups. Compared with shControl VGLUT1+ neurons, the dendritic length and arborizations of shSHANK2 VGLUT1+ neurons were shorter and fewer, while cell soma speed was higher. Furthermore, dendritic length and arborization were significantly increased after insulin-like growth factor 1 treatment of shSHANK2 neurons, while cell soma speed remained unaffected. These results suggest that insulin-like growth factor 1 can rescue morphological defects, but not the change in neuronal motility. Collectively, our findings demonstrate that SHANK2 deficiency perturbs early neuronal development, and that IGF1 can partially rescue the neuronal defects caused by SHANK2 knockdown. All experimental procedures and protocols were approved by the Laboratory Animal Ethics Committee of Jinan University, China (approval No. 20170228010) on February 28, 2017.

Brachial plexus root avulsion (BPRA) results in the complete loss of motor function in the upper limb, mainly due to the death of spinal motoneurons (MNs). The survival of spinal MNs is the key to the recovery of motor function. Neuregulin-1 (Nrg1) plays fundamental roles in nervous system development and nerve repair. However, its functional role in BPRA remains unclear. On the basis of our findings that Nrg1 is down-regulated in the ventral horn in a mouse model of BPRA, Nrg1 may be associated with BPRA. Here, we investigated whether recombinant Nrg1β (rNrg1β) can enhance the survival of spinal MNs and improve functional recovery in mice following BPRA. In vitro studies on primary cultured mouse MNs showed that rNrg1β increased the survival rate in a dose-dependent manner, reaching a peak at 5 nM, which increased the survival rate and enhanced the pERK levels in MNs under H2O2-induced oxidative stress. In vivo studies revealed that rNrg1β improved the functional recovery of elbow flexion, promoted the survival of MNs, enhanced the re-innervation of biceps brachii, and decreased the muscle atrophy. These results suggest that Nrg1 may provide a potential therapeutic strategy for root avulsion.

After complete transection of the thoracic spinal segment, neonatal rats exhibit spontaneous locomotor recovery of hindlimbs, but this recovery is not found in adult rats after similar injury. The potential mechanism related to the difference in recovery of neonatal and adult rats remains unknown. In this study, 342 animals were analyzed. The vascular endothelial growth factor (VEGF) level in spinal segments below injury sites was significantly higher in postnatal day 1 rats (P1) compared with 28-day-old adult rats (P28) following a complete T9 transection. VEGF administration in P28 rats with T9 transection significantly improved the functional recovery; by contrast, treatment with VEGF receptor inhibitors in P1 rats with T9 transection slowed down the spontaneous functional recovery. Results showed more neurons reduced in the lumbar spinal cord and worse local neural network reorganization below injury sites in P28 rats than those in P1 rats. Transynaptic tracing with pseudorabies virus and double immunofluorescence analysis indicated that VEGF treatment in P28 rats alleviated the reduced number of neurons and improved their network reorganization. VEGF inhibition in neonates resulted in high neuronal death rate and deteriorated network reorganization. In in vivo studies, T9 transection induced less increase in the number of microglia in the spinal cord in P1 animals than P28 animals. VEGF treatment reduced the increase in microglial cells in P28 animals. VEGF administration in cultured spinal motoneurons prevented lipopolysaccharide (LPS)-induced neuronal death and facilitated neurite growth. Western blots of the samples of lumbar spinal cord after spinal transection and cultured spinal motoneurons showed a lower level of Erk1/2 phosphorylation after the injury or LPS induction compared with that in the control. The phosphorylation level increased after VEGF treatment. In conclusion, VEGF is a critical mediator involved in functional recovery after spinal transection and can be considered a potential target for clinical therapy.

Induced pluripotent stem cells (iPSCs) can differentiate into neural progenitor cells (NPC) under proper conditions. NPC can be used as a model and is a useful tool for disease mechanism exploration and drug screening. However, the characteristics of the cells in various stages from NPC to functional neurons have not been fully described. This study investigated the characteristics of iPSC-derived NPCs during differentiation. Morphological characteristics of the NPCs, including soma area, neurite length, and the number of neurite branches, were examined on selected differentiation days. Physiological functions were assessed by recordings of sodium current, spontaneous excitatory postsynaptic current (sEPSC), and spontaneous inhibitory postsynaptic current (sIPSC). Furthermore, gene expression patterns were assessed with RNA-seq. We found that NPCs derived from iPSCs can be differentiated into glutamatergic and gabaergic neurons. Cell growth peaked during differentiation day 7–12, as the soma area decreased after day 12, growth cone and the number of branches peaked at day 9 and decreased afterwards; whereas a functional synapse formed after day 23. RNA-seq analysis found that a differential expression pattern emerged by day 7. Overall, the study provides a framework for the differentiation process of hiPSC-derived NPCs.

Cognitive impairments in bipolar patients deteriorate as the disorder progresses. Little is known about whether genetic risks impact cognitive recovery during the course from depression to remission. In this six-week open-label trial, we shed light on the impacts of six single nucleotide polymorphisms (SNPs) in the calcium voltage-gated channel subunit alpha1 C (CACNA1C) gene on cognitive recovery in 192 bipolar patients suffering a major depressive episode (MDE). The primary outcome measures were changes in a battery of neuropsychological tests following 6-week treatment. Carriers with rs10466907 GT genotype did not significantly improve their executive function total scores on the Wisconsin Card Sorting Test after six weeks of treatment compared to the TT genotypes (β = −0.944, 95% Confidence Interval (CI) = −1.482–−0.405). Moreover, during a MDE carriers with rs58619945 GG and GA genotypes performed significantly worse than those with AA genotype on the categories completed (p = 0.013 and p = 0.001), total errors (p = 0.039 and p = 0.009), and random errors (p = 0.055 and p = 0.014, respectively). Our data suggest that the tested CACNA1C SNPs may have impacts on cognitive recovery from depression.

Commonly used anesthetic agents, e.g. ketamine, may be neurotoxic to the developing brain but there has been little attention to the neurobehavioral consequences for offspring when used for maternal anesthesia. We hypothesize that treatment of pregnant rats with ketamine during the second trimester would affect brain development of the offspring. Pregnant rats on gestational day 14, about equal to midtrimester pregnancy in humans, received a sedative dose of ketamine intravenously for 2 h. Brain hippocampal morphology of their pups at postnatal days 0 (P0) and P30 was examined by Nissl-staining and the characteristics of dendrites were determined using the Golgi-Cox staining, while cell proliferation in subventricular zone (SVZ) and dentate gyrus (DG) was labeled with bromodeoxyuridine (BrdU). Their neurobehavioral functions were tested at P25–30 after which the NR1 and NR2 subunits of N-methyl-d-aspartate (NMDA) receptor, brain-derived neurotrophic factor (BDNF) and postsynaptic density protein 95 (PSD-95) in the hippocampus were analyzed by western blot. When pregnant rats were exposed to ketamine, there was neuronal loss, pyramidal neuronal abnormality and reduced cell proliferation in the hippocampus of offspring. These morphological abnormalities were associated with depression- and anxiety-like behaviors, and impaired memory up to young adult age. The treatment further caused NR2A receptor subunit up-regulation and NR2B receptor subunit, BDNF and PSD-95 down-regulation. These data suggest that maternal anesthesia with ketamine during the fetal brain development period can cause fetal brain damage and subsequent neurobehavioral abnormality, which is likely associated with the imbalanced expression of NMDA receptor subunits.

The spinal cord plays a key role in motor behavior. It relays major sensory information, receives afferents from supraspinal centers and integrates movement in the central pattern generators. Spinal motor output is controlled via corticofugal pathways including corticospinal and cortico-subcortical projections. Spinal cord injury damages descending supraspinal as well as ascending sensory pathways. In adult rodent models, plasticity of the spinal cord is thought to contribute to functional recovery. How much spinal cord function depends on cortical input is not well known. Here, we address this question using Celsr3/Foxg1 mice, in which cortico-subcortical connections (including corticospinal tract (CST) and the terminal sensory pathway, the thalamocortical tract) are genetically ablated during early development. Although Celsr3/Foxg1 mice are able to eat, walk, climb on grids and swim, open-field tests showed them to be hyperactive. When compared with normal littermates, mutant animals had reduced number of spinal motor neurons, with atrophic dendritic trees. Furthermore, motor axon terminals were decreased in number, and this was confirmed by electromyography. The number of cholinergic, calbindin, and calretinin-positive interneurons was moderately increased in the mutant spinal cord, whereas that of reelin and parvalbumin-positive interneurons was unchanged. As far as we know, our study provides the first genetic evidence that the spinal motor network does not mature fully in the absence of corticofugal connections, and that some motor function is preserved despite congenital absence of the CST.

Purpose Frizzled3 (Fzd3), a member of the core planar cell polarity (PCP) family in mammals, contributes to visual development by guiding axonal projections of some retinal ganglion cells. However, its other functions in the maturation of the visual system, especially the retina, remain elusive. The present study explores the role of Fzd3 in retinal development by focusing on rod bipolar cells (RBCs). Methods Frizzled3 was conditionally removed from the retina of Isl1-Cre;Fzd3f/- mice using the Cre-loxP system. Electroretinograms (ERGs) were performed to measure the light response of retinas. Frizzled3 expression was monitored by β-galactosidase (β-gal) staining and anti-β-gal immunostaining. Immunofluorescence was used to examine cellular distribution and morphology during development, and electron microscopy was applied to visualize the dendritic invaginations of RBCs. Results Electroretinograms showed decreased b-wave amplitudes, and lower b- to a-wave ratios in Isl1-Cre;Fzd3f/- than in control (Isl1-Cre;Fzd3f/+) mice. In RBCs, where Fzd3 was expressed and inactivated, the planar organization, shape, and orientation of somas were disrupted. From P10, dendrites of these RBCs displayed reduced arborization with mistargeting. Furthermore, their dendritic invaginations into rod terminals were suppressed, and the density of rod ribbons in the OPL was reduced. Conclusions Frizzled3 is required to shape the pattern of RBC somas and dendrites, and the structural and functional connectivity between rods and RBCs. Our results highlight novel functions for Fzd3 in regulating retinal development.

Ependymal cells form the epithelial lining of cerebral ventricles. Their apical surface is covered by cilia that beat in a coordinated fashion to facilitate circulation of the cerebrospinal fluid (CSF). The genetic factors that govern the development and function of ependymal cilia remain poorly understood. We found that the planar cell polarity cadherins Celsr2 and Celsr3 control these processes. In Celsr2-deficient mice, the development and planar organization of ependymal cilia are compromised, leading to defective CSF dynamics and hydrocephalus. In Celsr2 and Celsr3 double mutant ependyma, ciliogenesis is markedly impaired, resulting in lethal hydrocephalus. The membrane distribution of Vangl2 and Fzd3, two key planar cell polarity proteins, was disturbed in Celsr2 mutants, and even more so in Celsr2 and Celsr3 double mutants. Our findings suggest that planar cell polarity signaling is involved in ependymal cilia development and in the pathophysiology of hydrocephalus, with possible implications in other ciliopathies.

As one major component of extracellular matrix (ECM) in the central nervous system, chondroitin sulfate proteoglycans (CSPGs) have long been known as inhibitors enriched in the glial scar that prevent axon regeneration after injury. Although many studies have shown that CSPGs inhibited neurite outgrowth in vitro using different types of neurons, the mechanism by which CSPGs inhibit axonal growth remains poorly understood. Using cerebellar granule neuron (CGN) culture, in this study, we evaluated the effects of different concentrations of both immobilized and soluble CSPGs on neuronal growth, including cell adhesion, spreading and neurite growth. Neurite length decreased while CSPGs concentration arised, meanwhile, a decrease in cell density accompanied by an increase in cell aggregates formation was observed. Soluble CSPGs also showed an inhibition on neurite outgrowth, but it required a higher concentration to induce cell aggregates formation than coated CSPGs. We also found that growth cone size was significantly reduced on CSPGs and neuronal cell spreading was restrained by CSPGs, attributing to an inhibition on lamellipodial extension. The effect of CSPGs on neuron adhesion was further evidenced by interference reflection microscopy (IRM) which directly demonstrated that both CGNs and cerebral cortical neurons were more loosely adherent to a CSPG substrate. These data demonstrate that CSPGs have an effect on cell adhesion and spreading in addition to neurite outgrowth.

Self-assembly peptide nanofibrous scaffold (SAPNS), such as RADA16-I, has been shown to reduce acute brain injury and enhance functional recovery in rat intracerebral hemorrhage (ICH) models. The acidic property of RADA16-I, however, limits its application in patients. In the present study, by using a modified neutral SAPNS (the RADA16mix) in collagenase IV induced ICH mice, we detected there were less microglial and apoptotic cells in mice injected with RADA16mix, meanwhile, more cells survived in this group. In addition, behavioral tests indicated that mice treated with RADA16mix showed better functional recovery than RADA16-I. Local delivery of RADA16mix reduces acute brain injury by lowering the number of apoptotic cells, decreasing glial reaction, reducing inflammatory response and, therefore promotes functional recovery. Moreover, new nerve fibers have grown into this new SAPNS, which indicates RADA16mix is able to serve as a bridge for nerve fibers to grow through. From the Clinical Editor Acute brain injury, such as intracerebral hemorrhage is a serious problem. In this work, self-assembly peptide nanofibrous scaffold (SAPNS) were tested in a rat model to aid functional recovery. Several items have been considered, such as histology, brain water content, hematoma volume, cell death and survival, inflammatory response, and nerve fiber growth. The positive data generated should pave the way towards better treatment options.

Ketamine is commonly used for anesthesia and as a recreational drug. In pregnant users, a potential neurotoxicity in offspring has been noted. Our previous work demonstrated that ketamine exposure of pregnant rats induces affective disorders and cognitive impairments in offspring. As the prefrontal cortex (PFC) is critically involved in emotional and cognitive processes, here we studied whether maternal ketamine exposure influences the development of the PFC in offspring. Pregnant rats on gestational day 14 were treated with ketamine at a sedative dose for 2 hrs, and pups were studied at postnatal day 0 (P0) or P30. We found that maternal ketamine exposure resulted in cell apoptosis and neuronal loss in fetal brain. Upon ketamine exposure in utero, PFC neurons at P30 showed more dendritic branching, while cultured neurons from P0 PFC extended shorter neurites than controls. In addition, maternal ketamine exposure postponed the switch of NR2B/2A expression, and perturbed pre- and postsynaptic protein expression in the PFC. These data suggest that prenatal ketamine exposure impairs neuronal development of the PFC, which may be associated with abnormal behavior in offsprings.

The arrival and refinement of corticospinal afferents are likely to influence the maturation of the spinal cord and sensory-motor networks. To understand this better, we studied the revision of monosynaptic muscle afferents, the expression of activity-related genes, neurotrophins and their receptors in the cervical spinal cord from postnatal day (P) 0 to 21. We compared control and Celsr3|Emx1 mice, in which corticospinal axons never develop. The corticospinal tract (CST), labeled by anti-protein kinase C gamma (PKCγ) antibody in the dorsal funiculus, increased gradually in the control, but was never visible in the mutant. Using anti-parvalbumin and choline acetyltransferase double immunostaining, close contacts between proprioceptive afferent fibers and spinal motor neurons appeared at P0 and were gradually eliminated thereafter, with no difference between control and mutant mice. In both genotypes, the number of parvalbumin-positive interneurons increased similarly from P7 to P21, and a comparable upregulation of c-Jun protein was seen at P7. Contrary to control samples, in which ciliary neurotrophic factor (CNTF) protein levels increased from P0 to P7 and gradually decreased after P14, CNTF concentrations were time-invariant in mutant samples. The dynamic profile of neurotrophin-3 (NT3) expression was also moderately affected in mutant mice. In control spinal cord, NT3 was increased at P7 and decreased at P14, but remained more stable in mutant samples. In contrast, expression profiles of brain-derived neurotrophic factor (BDNF), tropomyosin-related kinase (Trk) B, TrkC, p75 neurotrophin receptor (p75(NTR)) and glial cell-line-derived neurotrophic factor (GDNF) were similar in both genotypes. In conclusion, with the possible exception of CNTF and NT3 expression, most events that accompany maturation of the spinal cord appear largely independent of corticospinal inputs.

Celsr3 and Fzd3, members of "core planar cell polarity" (PCP) genes, were shown previously to control forebrain axon guidance and wiring by acting in axons and/or guidepost cells. Here, we show that Celsr2 acts redundantly with Celsr3, and that their combined mutation mimics that of Fzd3. The phenotypes generated upon inactivation of Fzd3 in different forebrain compartments are similar to those in conditional Celsr2-3 mutants, indicating that Fzd3 and Celsr2-3 act in the same population of cells. Inactivation of Celsr2-3 or Fzd3 in thalamus does not affect forebrain wiring, and joint inactivation in cortex and thalamus adds little to cortical inactivation alone in terms of thalamocortical projections. On the other hand, joint inactivation perturbs strongly the formation of the barrel field, which is unaffected upon single cortical or thalamic inactivation, indicating a role for interactions between thalamic axons and cortical neurons in cortical arealization. Unexpectedly, forebrain wiring is normal in mice defective in Vangl1 and Vangl2, showing that, contrary to epithelial PCP, axon guidance can be Vangl independent in some contexts. Our results suggest that Celsr2-3 and Fzd3 regulate axonal navigation in the forebrain by using mechanisms different from classical epithelial PCP, and require interacting partners other than Vangl1-2 that remain to be identified.

The aims of this study were to investigate the effect of chinonin in preventing 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurodegeneration in C57BL/6 mice and to examine the possible mechanisms. The neurotoxin MPTP was employed to create a subacute Parkinson's disease (PD)-like model in C57BL/6 mice. Chinonin (10, 20, 40 mg/kg body weight) was intraperitoneally administered 0.5 h after MPTP (30 mg/kg) injection for 7 d consecutively. Chinonin showed neuroprotective effects in the MPTP-treated mice PD model by ameliorating motor impairment in the catwalk and open-field tests. Consistently, chinonin reduced loss of dopaminergic neurons in the substantia nigra and prevented depletion of dopamine and its metabolites 3-methoxy-4-hydroxy-phenylacetic acid and homovanillic acid in the striatum of mice. Compared with the MPTP group, in the chinonin plus MPTP groups significant increases of superoxide dismutase activity and glutathione levels were observed as well as a distinct reduction of lipid peroxidation product malondialdehyde in the striatum. Taken together, we propose that chinonin exerts neuroprotective effects in C57BL/6 mouse model of PD and these effects may be due to chinonin's antioxidative property.

International audience; The cadherin Celsr3 regulates the directional growth and targeting of axons in the CNS, but whether it acts in collaboration with or in parallel to other guidance cues is unknown. Furthermore, the function of Celsr3 in the peripheral nervous system is still largely unexplored. Here we show that Celsr3 mediates pathfinding of motor axons innervating the hindlimb. In mice, Celsr3-deficient axons of the peroneal nerve segregate from those of the tibial nerve but fail to extend dorsally, and they stall near the branch point. Mutant axons respond to repulsive ephrinA-EphA forward signaling and glial cell-derived neurotrophic factor (GDNF). However, they are insensitive to attractive EphA-ephrinA reverse signaling. In transfected cells, Celsr3 immunoprecipitates with ephrinA2, ephrinA5, Ret, GDNF family receptor α1 (GFRα1) and Frizzled3 (Fzd3). The function of Celsr3 is Fzd3 dependent but Vangl2 independent. Our results provide evidence that the Celsr3-Fzd3 pathway interacts with EphA-ephrinA reverse signaling to guide motor axons in the hindlimb.

The andrographolide-lipoic acid conjugate AL-1 is a newly synthesized molecule by covalently linking andrographolide (Andro) with α-lipoic acid (LA). In the present work, the neuroprotective effect of AL-1 was investigated in vitro and in a mouse model of the Parkinson's disease (PD). We found that AL-1 significantly prevented 1-methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity in SH-SY5Y cells and primary cerebellar granule neurons. In a mouse model of Parkinson's disease, AL-1 rescued 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced loss of tyrosine hydroxylase (TH)-positive neurons, improved the behavioral impairment, and elevated the striatal levels of dopamine and its metabolites 3,4-dihydroxyphenylacetic acid. Furthermore, AL-1 remarkably lowered the nitric oxide and malondialdehyde levels while increased the superoxide dismutase level in the substantial nigra of MPTP-treated mice. The immunoblotting data showed that AL-1 significantly ameliorated the decreased expression of TH protein in the substantial nigra and inhibited the up-regulation of phosphorylated NF-κB p65 in vitro and in vivo. Taken together, AL-1 exerted neuroprotective effect in vitro and in animal model of PD through anti-oxidation and inhibition of NF-κB activation.

Atypical cadherin Celsr3, a regulator of planar cell polarity, is critical for the development of the axonal blueprint. We previously showed that expression of Celsr3 is necessary to establish forebrain connections such as the anterior commissure and thalamocortical and corticospinal tracts. The requirement for Celsr3 during hippocampal wiring and its action in the hippocampus remain largely unexplored. Here, we compared the connectivity and maturation of the hippocampal formation inCelsr3|Foxg1andCelsr3|Dlxmice.Celsr3is inactivated in the whole telencephalon, including the hippocampal primordium, inCelsr3|Foxg1mice, and in the early basal telencephalon, including ganglionic eminences and ventral diencephalon, inCelsr3|Dlxmice. Behavioral tests showed that both mutants were hyperactive and had impaired learning and memory. Abnormal cytoarchitecture of CA1, CA3, and dentate gyrus was found in theCelsr3|Foxg1mutant, in which afferent and efferent hippocampal pathways, as well as intrinsic connections, were dramatically disrupted. InCelsr3|Dlxmutant mice, hippocampal cytoarchitecture was mildly affected and extrinsic and intrinsic connectivity moderately disturbed. In both mutants, pyramidal neurons in CA1 harbored atrophic dendritic trees, with decreased synapse density and increased proportion of symmetric versus asymmetric synapses, and long-term potentiation was altered. In contrast, mutant hippocampal neurons extended neurites that were normal, even longer than those of control neurons, indicating that anomaliesin vivoare secondary to defective connections. Postnatal neurogenesis was preserved and mutant interneurons were able to migrate to the hippocampus. Thus, like in neocortex, Celsr3 is required for hippocampal development, connectivity and function, and for pyramidal cell maturation.

How much neocortical development depends on connections remains elusive. Here, we show that Celsr3, SCOPUS: ar.j, info:eu-repo/semantics/published

During hindbrain development, facial branchiomotor neurons (FBM neurons) migrate from medial rhombomere (r) 4 to lateral r6. In zebrafish, mutations in planar cell polarity genes celsr2 and frizzled3a block caudal migration of FBM neurons. Here, we investigated the role of cadherins Celsr1-3, and Fzd3 in FBM neuron migration in mice. In Celsr1 mutants (knock-out and Crash alleles), caudal migration was compromised and neurons often migrated rostrally into r2 and r3, as well as laterally. These phenotypes were not caused by defects in hindbrain patterning or neuronal specification. Celsr1 is expressed in FBM neuron precursors and the floor plate, but not in FBM neurons. Consistent with this, conditional inactivation showed that the function of Celsr1 in FBM neuron migration was non-cell autonomous. In Celsr2 mutants, FBM neurons initiated caudal migration but moved prematurely into lateral r4 and r5. This phenotype was enhanced by inactivation of Celsr3 in FBM neurons and mimicked by inactivation of Fzd3. Furthermore, Celsr2 was epistatic to Celsr1. These data indicate that Celsr1-3 differentially regulate FBM neuron migration. Celsr1 helps to specify the direction of FBM neuron migration, whereas Celsr2 and 3 control its ability to migrate.

The development of axonal tracts requires interactions between growth cones and the environment. Major bundles, particularly in the internal capsule, are completely defective in mice with constitutive mutation of Celsr3. In order to understand better how Celsr3 controls axonal tract formation, we generated a conditional allele that allowed inactivation of Celsr3 in different sectors of the forebrain. Effects of Celsr3 inactivation specifically in the telencephalon, in the ventral forebrain, or in the cortex, demonstrate essential roles for the gene, both in the neurons that project their axons to subcerebral targets such as the spinal cord, as well as in cells that guide projecting axons through the ventral forebrain. These observations provide unequivocal in vivo evidence that heterotypic interactions between axons and guidepost cells govern axonal path formation in mammals, and that Celsr3 plays a key role in this process. In absence of cortico-subcortical connections, mice can survive up to P20, allowing studies of behavior and cortical maturation. Mutant mice with defective corticospinal tracts survive normally and provide a model to evaluate in vivo the role of this tract in motor function in rodents.

Celsr3, the murine orthologue of Drosophila Flamingo/Starry night, is a brain-specific, atypical sevenpass cadherin that plays a key role during brain development. Celsr3 mutant mice die at birth of central hypoventilation. They have major anomalies of major tracts, particularly absence of anterior commissure and of all components of the internal capsule. In addition, the medial lemniscus and several longitudinal bundles in the brainstem and spinal cord are defective. This phenotype is similar to that generated by inactivation of Frizzled3 (Fzd3). As both Flamingo and Frizzled are two core planar cell polarity (PCP) genes in flies, our results indicate that Celsr3 and Fzd3 are part of a genetic network with similarity to the PCP network. We studied by in situ hybridization the expression patterns of other murine PCP genes Dyl1-3, Vangl1,2 and Prickle1,2. Together with data from the literature, results suggest a mechanism whereby Celsr1-3, Fzd3 and 6, and Vangl2 may interact to control neural tube closure and axonal development. In order to study this mechanism further, we generated a conditional Celsr3 mutant mouse that allows inactivation of Celsr3 in the forebrain and cerebral cortex, by crossing with mice that express Cre under the Foxg1 and Emx1 promoters, respectively.

Development of axonal tracts requires interactions between growth cones and the environment. Tracts such as the anterior commissure and internal capsule are defective in mice with null mutation of Celsr3 . We generated a conditional Celsr3 allele, allowing regional inactivation. Inactivation in telencephalon, ventral forebrain, or cortex demonstrated essential roles for Celsr3 in neurons that project axons to the anterior commissure and subcerebral targets, as well as in cells that guide axons through the internal capsule. When Celsr3 was inactivated in cortex, subcerebral projections failed to grow, yet corticothalamic axons developed normally, indicating that besides guidepost cells, additional Celsr3-independent cues can assist their progression. These observations provide in vivo evidence that Celsr3-mediated interactions between axons and guidepost cells govern axonal tract formation in mammals.

The cortex receives its major sensory input from the thalamus via thalamocortical axons, and cortical neurons are interconnected in complex networks by corticocortical and callosal axons. Our understanding of the mechanisms generating the circuitry that confers functional properties on cortical neurons and networks, although poor, has been advanced significantly by recent research on the molecular mechanisms of thalamocortical axonal guidance and ordering. Here we review recent advances in knowledge of how thalamocortical axons are guided and how they maintain order during that process. Several studies have shown the importance in this process of guidance molecules including Eph receptors and ephrins, members of the Wnt signalling pathway and members of a novel planar cell polarity pathway. Signalling molecules and transcription factors expressed with graded concentrations across the cortex are important in establishing cortical maps of the topography of sensory surfaces. Neural activity, both spontaneous and evoked, plays a role in refining thalamocortical connections but recent work has indicated that neural activity is less important than was previously thought for the development of some early maps. A strategy used widely in the development of corticocortical and callosal connections is the early overproduction of projections followed by selection after contact with the target structure. Here we discuss recent work in primates indicating that elimination of juvenile projections is not a major mechanism in the development of pathways feeding information forward to higher levels of cortical processing, although its use is common to developing feedback pathways.

Brachial plexus injury (BPI) and experimental spinal root avulsion result in loss of motor function in the affected segments. After root avulsion, significant motoneuron function is restored by re-implantation of the avulsed root. How much this functional recovery depends on corticospinal inputs is not known. Here, we studied that question using Celsr3|Emx1 mice, in which the corticospinal tract (CST) is genetically absent. In adult mice, we tore off right C5–C7 motor and sensory roots and re-implanted the right C6 roots. Behavioral studies showed impaired recovery of elbow flexion in Celsr3|Emx1 mice compared to controls. Five months after surgery, a reduced number of small axons, and higher G-ratio of inner to outer diameter of myelin sheaths were observed in mutant versus control mice. At early stages post-surgery, mutant mice displayed lower expression of GAP-43 in spinal cord and of myelin basic protein (MBP) in peripheral nerves than control animals. After five months, mutant animals had atrophy of the right biceps brachii, with less newly formed neuromuscular junctions (NMJs) and reduced peak-to-peak amplitudes in electromyogram (EMG), than controls. However, quite unexpectedly, a higher motoneuron survival rate was found in mutant than in control mice. Thus, following root avulsion/re-implantation, the absence of the CST is probably an important reason to hamper axonal regeneration and remyelination, as well as target re-innervation and formation of new NMJ, resulting in lower functional recovery, while fostering motoneuron survival. These results indicate that manipulation of corticospinal transmission may help improve functional recovery following BPI.

Despite the obvious clinical interest, our understanding of how developmental mechanisms are redeployed during degeneration and regeneration after brain and spinal cord injuries remains quite rudimentary. In animal models of spinal cord injury, although spontaneous regeneration of descending axons is limited, compensation by intact corticospinal axons, descending tracts from the brainstem, and local intrinsic spinal networks all contribute to the recovery of motor function. Here, we investigated spontaneous motor compensation and plasticity that occur in the absence of corticospinal tract, using Celsr3|Emx1 mice in which the corticospinal tract is completely and specifically absent as a consequence of Celsr3 inactivation in the cortex. Mutant mice had no paresis, but displayed hyperactivity in open-field, and a reduction in skilled movements in food pellet manipulation tests. The number of spinal motoneurons was reduced and their terminal arbors at neuromuscular junctions were atrophic, which was reflected in electromyography deficits. Rubrospinal projections, calretinin-positive propriospinal projections, afferent innervation of motoneurons by calretinin-positive segmental interneurons, and terminal ramifications of monoaminergic projections were significantly increased. Contrary to control animals, mutants also developed a severe and persistent disability of forelimb use following the section of the rubrospinal tract at the C4 spinal level. These observations demonstrate for the first time that the congenital absence of the corticospinal tract induces spontaneous plasticity, both at the level of the motor spinal cord and in descending monoaminergic and rubrospinal projections. Such compensatory mechanisms could be recruited in case of brain or spinal cord lesion or degeneration.

Spinal cord injury (SCI) is a devastating event that results in significant physical disabilities for affected individuals. Apart from local injury within the spinal cord, SCI patients develop a variety of complications characterized by multiple organ dysfunction or failure. These disorders, such as neurogenic pain, depression, lung injury, cardiovascular disease, liver damage, kidney dysfunction, urinary tract infection, and increased susceptibility to pathogen infection, are common in injured patients, hinder functional recovery, and can even be life threatening. Multiple lines of evidence point to pathological connections emanating from the injured spinal cord, post-injury systemic inflammation, and immune suppression as important multifactorial mechanisms underlying post-SCI complications. SCI triggers systemic inflammatory responses marked by increased circulation of immune cells and pro-inflammatory mediators, which result in the infiltration of inflammatory cells into secondary organs and persistence of an inflammatory microenvironment that contributes to organ dysfunction. SCI also induces immune deficiency through immune organ dysfunction, resulting in impaired responsiveness to pathogen infection. In this review, we summarize current evidence demonstrating the relevance of inflammatory conditions and immune suppression in several complications frequently seen following SCI. In addition, we highlight the potential pathways by which inflammatory and immune cues contribute to multiple organ failure and dysfunction and discuss current anti-inflammatory approaches used to alleviate post-SCI complications. A comprehensive review of this literature may provide new insights into therapeutic strategies against complications after SCI by targeting systemic inflammation.