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Cerebral creatine deficiency syndromes (CCDS) are rare inherited metabolic disorders caused by defective biosynthesis or transport of creatine. These conditions are characterized by reduced accumulation of creatine in the brain, mild to severe intellectual disability, global developmental delay, and speech-language disorders. The amount of brain creatine reduction needed to cause symptoms is not known. Here we report a new patient with creatine transporter deficiency (CTD) who presented at 15 months of age with seizures and global delays with no speech at 3 years of age. Brain MRI was normal, but brain MRS indicated creatine levels reduced to about 20 % of normal. He had normal levels of creatine and guanidinoacetate in plasma, but increased creatine/creatinine ratio in urine. DNA sequencing identified a hemizygous c.832C > T (p.Arg278Cys) variant in the creatine transporter gene SLC6A8. Fibroblasts from this patient had about 25 % of normal creatine transport activity, a value much higher than that measured in patients whose variants introduced premature stop codons in SLC6A8. The child was started on supplements of creatine, glycine, and arginine. His speech improved dramatically, and he had no more seizures, even during episodes of fever. Despite the clinical improvement, a repeat MRS demonstrated similar levels of brain creatine. This study suggests that a reduction in creatine transporter activity to 25 % or less is sufficient to cause symptoms of brain creatine deficiency and that functionally milder forms of CTD might respond to supplements aimed at replenishing brain creatine., Competing Interests: Declaration of competing interest The authors have no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Compulsive- and anxiety-like behaviour can be efficiently modelled in SAPAP3 knockout (KO) mice, a preclinical model of relevance to obsessive-compulsive disorder (OCD). Although there is emerging evidence in the clinical literature of gastrointestinal dysfunction in OCD, no previous studies have investigated gut function in preclinical models of relevance to OCD. Similarly, the effects of voluntary exercise (EX) or environmental enrichment (EE) have not yet been explored in this context., Method: We comprehensively phenotyped the SAPAP3 KO mouse model, including the assessment of grooming microstructure, anxiety- and depressive-like behaviour, and gastrointestinal function. Mice were exposed to either standard housing (SH), exercise (EX, provided by giving mice access to running wheels), or environmental enrichment (EE) for 4 weeks to investigate the effects of enriched housing conditions in this animal model relevant to OCD., Findings: Our study is the first to assess grooming microstructure, perseverative locomotor activity, and gastrointestinal function in SAPAP3 KO mice. We are also the first to report a sexually dimorphic effect of grooming in young-adult SAPAP3 KO mice; along with changes to grooming patterning and indicators of gut dysfunction, which occurred in the absence of gut dysbiosis in this model. Overall, we found no beneficial effects of voluntary exercise or environmental enrichment interventions in this mouse model; and unexpectedly, we revealed a deleterious effect of wheel-running exercise on grooming behaviour. We suspect that the detrimental effects of experimental housing in our study may be indicative of off-target effects of stress-a conclusion that warrants further investigation into the effects of chronic stress in this preclinical model of compulsive behaviour., Competing Interests: Declaration of competing interest The authors have nothing to disclose., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Social deficits represent a core symptom domain of autism spectrum disorder (ASD), which is often comorbid with sleep disturbances. In this issue of the JCI, Sun et al. explored a medial septum (MS) circuit linking these behaviors in a neuroligin 3 conditional knockout model of autism. They identified GABAergic neuron hyperactivity following neuroligin 3 deletion in the MS. This hyperactivity resulted in the inhibition of the downstream preoptic area (POA) and hippocampal CA2 region, resulting in sleep loss and social memory deficits, respectively. Inactivating the hyperactive MS GABA neurons or activating the POA or CA2 rescued the behavioral deficits. Together, these findings deepen our understanding of neural circuits underlying social and sleep deficits in ASD.

Abnormalities in gamma-aminobutyric acid (GABA)ergic neurotransmission play a role in the pathogenesis of autism, although the mechanisms responsible for alterations in specific brain regions remain unclear. Deficits in social motivation and interactions are core symptoms of autism, likely due to defects in dopaminergic neural pathways. Therefore, investigating the morphology and functional roles of GABAergic neurons within dopaminergic projection areas could elucidate the underlying etiology of autism. The aim of this study was to (1) compare the morphology and arborization of glutamate decarboxylase (GAD)-positive neurons from the midbrain tegmentum; (2) evaluate synaptic activity in primary neurons from the striatum; and (3) assess GABAergic postsynaptic puncta in the ventral striatum of wild-type (WT) and Shank3-deficient mice. We found a significant decrease in the number of short neurites in GAD positive primary neurons from the midbrain tegmentum in Shank3-deficient mice. The application of a specific blocker of GABA A receptors (GABA A R) revealed significantly increased frequency of spontaneous postsynaptic currents (sPSCs) in Shank3-deficient striatal neurons compared to their WT counterparts. The mean absolute amplitude of the events was significantly higher in striatal neurons from Shank3-deficient compared to WT mice. We also observed a significant reduction in gephyrin/GABA A R γ2 colocalization in the striatum of adult male Shank3-deficient mice. The gene expression of collybistin was significantly lower in the nucleus accumbens while gephyrin and GABA A R γ2 were lower in the ventral tegmental area (VTA) in male Shank3-deficient compared to WT mice. In conclusion, Shank3 deficiency leads to alterations in GABAergic neurons and impaired GABAergic function in dopaminergic brain areas. These changes may underlie autistic symptoms, and potential interventions modulating GABAergic activity in dopaminergic pathways may represent new treatment modality., (© 2024. The Author(s).)

Psilocybin is a serotonergic psychedelic compound which shows promise for treating compulsive behaviours. This is particularly pertinent as compulsive disorders require research into new pharmacological treatment options as the current frontline treatments such as selective serotonin reuptake inhibitors, require chronic administration, have significant side effects, and leave almost half of the clinical population refractory to treatment. In this study, we investigated psilocybin administration in male and female SAPAP3 knockout (KO) mice, a well-validated mouse model of obsessive compulsive and related disorders. We assessed the effects of acute psilocybin (1 mg/kg, intraperitoneal) administration on head twitch and locomotor behaviour as well as anxiety- and compulsive-like behaviours at multiple time-points (1, 3 and 8 days post-injection). While psilocybin did not have any effect on anxiety-like behaviours, we revealed that acute psilocybin administration led to enduring reductions in compulsive behaviour in male SAPAP3 KO mice and reduced grooming behaviour in female wild-type (WT) and SAPAP3 KO mice. We also found that psilocybin increased locomotion in WT littermates but not in SAPAP3 KO mice, suggesting in vivo serotonergic dysfunctions in KO animals. On the other hand, the typical head-twitch response following acute psilocybin (confirming its hallucinogenic-like effect at this dose) was observed in both genotypes. Our novel findings suggest that acute psilocybin may have potential to reduce compulsive-like behaviours (up to 1 week after a single injection). Our study can inform future research directions as well as supporting the utility of psilocybin as a novel treatment option for compulsive disorders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Pannexin 3 (Panx3) is a glycoprotein that forms mechanosensitive channels expressed in chondrocytes and annulus fibrosus cells of the intervertebral disc (IVD). Evidence suggests Panx3 plays contrasting roles in traumatic versus aging osteoarthritis (OA) and intervertebral disc degeneration (IDD). However, whether its deletion influences the response of joint tissue to forced use is unknown. The purpose of this study was to determine if Panx3 deletion in mice causes increased knee joint OA and IDD after forced treadmill running. Male and female wildtype (WT) and Panx3 knockout (KO) mice were randomized to either a no-exercise group (sedentary; SED) or daily forced treadmill running (forced exercise; FEX) from 24 to 30 weeks of age. Knee cartilage and IVD histopathology were evaluated by histology, while tibial secondary ossification centers were analyzed using microcomputed tomography (µCT). Both male and female Panx3 KO mice developed larger superficial defects of the tibial cartilage after forced treadmill running compared with SED WT mice. Additionally, Panx3 KO mice developed reduced bone volume, and female PANX3 KO mice had lengthening of the lateral tubercle at the intercondylar eminence. In the lower lumbar spine, both male and female Panx3 KO mice developed histopathological features of IDD after running compared to SED WT mice. These findings suggest that the combination of deleting Panx3 and forced treadmill running induces OA and causes histopathological changes associated with the degeneration of the IVDs in mice., (© 2024 The Authors. Journal of Orthopaedic Research ® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Patients with autism spectrum disorder (ASD) frequently experience sleep disturbance. Genetic mutations in the neuroligin 3 (NLG3) gene are highly correlative with ASD and sleep disturbance. However, the cellular and neural circuit bases of this correlation remain elusive. Here, we found that the conditional knockout of Nlg3 (Nlg3-CKO) in the medial septum (MS) impairs social memory and reduces sleep. Nlg3 CKO in the MS caused hyperactivity of MSGABA neurons during social avoidance and wakefulness. Activation of MSGABA neurons induced social memory deficits and sleep loss in C57BL/6J mice. In contrast, inactivation of these neurons ameliorated social memory deficits and sleep loss in Nlg3-CKO mice. Sleep deprivation led to social memory deficits, while social isolation caused sleep loss, both resulting in a reduction in NLG3 expression and an increase in activity of GABAergic neurons in the MS from C57BL/6J mice. Furthermore, MSGABA-innervated CA2 neurons specifically regulated social memory without impacting sleep, whereas MSGABA-innervating neurons in the preoptic area selectively controlled sleep without affecting social behavior. Together, these findings demonstrate that the hyperactive MSGABA neurons impair social memory and disrupt sleep resulting from Nlg3 CKO in the MS, and achieve the modality specificity through their divergent downstream targets.

Lowe Syndrome (LS) is a rare X-linked disorder characterized by renal dysfunction, cataracts, and several central nervous system (CNS) anomalies. The mechanisms underlying the neurological dysfunction in LS remain unclear, albeit they share some phenotypic characteristics similar to the deficiency or dysfunction of the Reelin signaling, a relevant pathway with roles in CNS development and neuronal functions. In this study, we investigated the role of OCRL1, an inositol polyphosphate 5-phosphatase encoded by the OCRL gene, mutated in LS, focusing on its impact on endosomal trafficking and receptor recycling in human neuronal cells. Specifically, we tested the effects of OCRL1 deficiency in the trafficking and signaling of ApoER2/LRP8, a receptor for the ligand Reelin. We found that loss of OCRL1 impairs ApoER2 intracellular trafficking, leading to reduced receptor expression and decreased levels at the plasma membrane. Additionally, human neurons deficient in OCRL1 showed impairments in ApoER2/Reelin-induced responses. Our findings highlight the critical role of OCRL1 in regulating ApoER2 endosomal recycling and its impact on the ApoER2/Reelin signaling pathway, providing insights into potential mechanisms underlying the neurological manifestations of LS.

Brain function is critically dependent on correct circuit assembly. Microglia are well-known for their important roles in immunological defense and neural plasticity, but whether they can also mediate experience-induced correction of miswired circuitry is unclear. Ten-m3 knockout (KO) mice display a pronounced and stereotyped visuotopic mismapping of ipsilateral retinal inputs in their visual thalamus, providing a useful model to probe circuit correction mechanisms. Environmental enrichment (EE) commenced around birth, but not later in life, can drive a partial correction of the most mismapped retinal inputs in Ten-m3 KO mice. Here, we assess whether enrichment unlocks the capacity for microglia to selectively engulf and remove miswired circuitry, and the timing of this effect. Expression of the microglial-associated lysosomal protein CD68 showed a clear enrichment-driven, spatially restricted change which had not commenced at postnatal day (P)18, was evident at P21, more robust at P25, and had ceased by P30. This was observed specifically at the corrective pruning site and was absent at a control site. An engulfment assay at the corrective pruning site in P25 mice showed EE-driven microglial-uptake of the mismapped axon terminals. This was temporally and spatially specific, as no enrichment-driven microglial engulfment was seen in P18 KO mice, nor the control locus. The timecourse of the EE-driven corrective pruning as determined anatomically, aligned with this pattern of microglia reactivity and engulfment. Collectively, these findings show experience can drive targeted microglial engulfment of miswired neural circuitry during a restricted postnatal window. This may have important therapeutic implications for neurodevelopmental conditions involving aberrant neural connectivity., (© 2024 The Authors. GLIA published by Wiley Periodicals LLC.)

Creatine transporter deficiency has been described with normal or uninformative levels of creatine and creatinine in plasma, while urine has been the preferred specimen type for biochemical diagnosis. We report a cohort of untreated patients with creatine transporter deficiency and abnormal plasma creatine panel results, characterized mainly by markedly decreased plasma creatinine. We conclude that plasma should be considered a viable specimen type for the biochemical diagnosis of this disorder, and abnormal results should be followed up with further confirmatory testing., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Creatine is an essential metabolite for the storage and rapid supply of energy in muscle and nerve cells. In humans, impaired metabolism, transport, and distribution of creatine throughout tissues can cause varying forms of mental disability, also known as creatine deficiency syndrome (CDS). So far, 80 mutations in the creatine transporter (SLC6A8) have been associated to CDS. To better understand the effect of human genetic variants on the physiology of SLC6A8 and their possible impact on CDS, we studied 30 missense variants including 15 variants of unknown significance, two of which are reported here for the first time. We expressed these variants in HEK293 cells and explored their subcellular localization and transport activity. We also applied computational methods to predict variant effect and estimate site-specific changes in thermodynamic stability. To explore variants that might have a differential effect on the transporter's conformers along the transport cycle, we constructed homology models of the inward facing, and outward facing conformations. In addition, we used mass-spectrometry to study proteins that interact with wild type SLC6A8 and five selected variants in HEK293 cells. In silico models of the protein complexes revealed how two variants impact the interaction interface of SLC6A8 with other proteins and how pathogenic variants lead to an enrichment of ER protein partners. Overall, our integrated analysis disambiguates the pathogenicity of 15 variants of unknown significance revealing diverse mechanisms of pathogenicity, including two previously unreported variants obtained from patients suffering from the creatine deficiency syndrome., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences GmbH. Published by Elsevier Ltd.. All rights reserved.)

Background/aim: We previously observed higher prevalence of high-grade pancreatic intraepithelial neoplasia (PanIN) in LSL-Kras G12D/+ ; Pdx1 Cre/+ (KC-Crmp4 wild ) mice than LSL-Kras G12D/+ ; Pdx1 Cre/+ ; Crmp4 -/- (KC-Crmp4 -/- ) mice. This study investigated the relationship between collapsin response mediator protein 4 (CRMP4) and immune cell infiltration in pancreatic cancer., Materials and Methods: PanIN was induced by intraperitoneal injection of caerulein into KC-Crmp4 wild and KC-Crmp4 -/- mice, and immune cells in PanIN lesions were compared. Subcutaneous tumors were created by injecting Pan02 cells, and tumor diameter was compared between Crmp4 wild and Crmp4 -/- mice every 7 days. Peritumoral immune cells were examined immunohisto chemically., Results: High-grade PanIN in KC mice showed statistically significantly high expression of CD163 (p=0.031) and CD11b (p=0.027). Following subcutaneous injection of Pan02 cells, tumor diameter was greater in Crmp4 wild mice than Crmp4 -/- mice. Crmp4 wild mice exhibited higher CD163 and CD11b expression than Crmp4 -/- mice in tumors (p<0.001)., Conclusion: CRMP4 might promote pancreatic cancer by up-regulating M2 macrophages and myeloid-derived suppressor cells., (Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Synaptic cell-adhesion molecules (CAMs) organize the architecture and properties of neural circuits. However, whether synaptic CAMs are involved in activity-dependent remodeling of specific neural circuits is incompletely understood. Leucine-rich repeat transmembrane protein 3 (LRRTM3) is required for the excitatory synapse development of hippocampal dentate gyrus (DG) granule neurons. Here, we report that Lrrtm3 -deficient mice exhibit selective reductions in excitatory synapse density and synaptic strength in projections involving the medial entorhinal cortex (MEC) and DG granule neurons, accompanied by increased neurotransmitter release and decreased excitability of granule neurons. LRRTM3 deletion significantly reduced excitatory synaptic innervation of hippocampal mossy fibers (Mf) of DG granule neurons onto thorny excrescences in hippocampal CA3 neurons. Moreover, LRRTM3 loss in DG neurons significantly decreased mossy fiber long-term potentiation (Mf-LTP). Remarkably, silencing MEC-DG circuits protected against the decrease in the excitatory synaptic inputs onto DG and CA3 neurons, excitability of DG granule neurons, and Mf-LTP in Lrrtm3 -deficient mice. These results suggest that LRRTM3 may be a critical factor in activity-dependent synchronization of the topography of MEC-DG-CA3 excitatory synaptic connections. Collectively, our data propose that LRRTM3 shapes the target-specific structural and functional properties of specific hippocampal circuits., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

It is very important to maintain normal levels of risk avoidance in daily life. We found that DISC1-N TM mice, which are a model for mental disorders, had a phenotype marked by a risk-avoidance impairment as measured in an open-field test (OFT). We used optogenetic methods to modulate glutamatergic neurons in the basolateral amygdala (BLA) in an attempt to rescue this risk-avoidance impairment. We found that photostimulation of BLA neurons at 20 Hz modified DISC1-N TM mouse behavior from low risk avoidance to high risk avoidance. We observed following photostimulation that, compared to controls, the number of entries to the center of the open field was lower and less time was spent in the central area. We also found that the time spent immobile was higher during photostimulation compared with WT mice. We also used a lower photostimulation frequency of 5 Hz, which activated BLA glutamatergic neurons and rescued the risk-avoidance impairment in DISC1-N TM mice. Our findings confirm that the BLA participates in diverse risk-avoidance behavior. Our results are also a reminder that differences in neuronal firing patterns within the same pathway may lead to different physiological functions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Inc.)

Prepulse inhibition (PPI) is a neurophysiological finding that is decreased in schizophrenia patients and has been used in pathophysiology studies of schizophrenia and the development of antipsychotic drugs. PPI is affected by several drugs including amphetamine, ketamine, and nicotinic agents, and it is reported that several brain regions and modulatory neurotransmitters are involved in PPI. Here we showed that mice with IRSp53 deletion in each dopaminergic, cholinergic, oxytocinergic, and serotoninergic modulatory neurons showed a decrease in PPI. Other than PPI, there were no other behavioral changes among IRSp53 deletion mice. Through this study, we could reconfirm that dysfunction of each modulatory neuron such as dopamine, acetylcholine, oxytocin, and serotonin can result in PPI impairment, and it should be considered that PPI could be broadly affected by changes in one of a certain kind of modulatory neurons., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The chemical synapse is one type of cell-adhesion system that transmits information from a neuron to another neuron in the complex neuronal network in the brain. Synaptic transmission is the rate-limiting step during the information processing in the neuronal network and its plasticity is involved in cognitive functions. Thus, morphological and electrophysiological analyses of synapses are of particular importance in neuroscience research. In the current study, we applied super-resolved three-dimensional stimulated emission depletion (3D-STED) microscopy for the morphological analyses of synapses. This approach allowed us to estimate the precise number of excitatory and inhibitory synapses in the mouse hippocampal tissue. We discovered a region-specific increase in excitatory synapses in a model mouse of autism spectrum disorder, Neuroligin-3 KO, with this method. This type of analysis will open a new field in developmental neuroscience in the future., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Ischemic stroke is an acute cerebrovascular disease characterized by high mortality, morbidity and disability rates. Ischemia/reperfusion is a critical pathophysiological basis of motor and cognitive dysfunction caused by ischemic stroke. Microglia, innate immune cells of the central nervous system, mediate the neuroinflammatory response to ischemia/reperfusion. PlexinA2 (PLXNA2) plays an important role in the regulation of neuronal axon guidance, the immune response and angiogenesis. However, it is not clear whether PLXNA2 regulates microglia polarization in ischemic stroke or the underlying mechanism. In the present study, we investigated the role of PLXNA2 in rats with middle cerebral artery occlusion/reperfusion (MCAO/R) and BV2 microglia cells with oxygen and glucose deprivation/reoxygenation (OGD/R). A battery of behavioral tests, including the beam balance test, forelimb placement test, foot fault test, cylinder test, CatWalk gait analysis and Morris water maze test were performed to evaluate sensorimotor function, locomotor activity and cognitive ability. The expression of M1/M2-specific markers in the ischemic penumbra and BV2 microglia cells was detected using immunofluorescence staining, quantitative real-time PCR analysis and Western blot analysis. Our study showed that PLXNA2 knockdown accelerated the recovery of motor function and cognitive ability after MCAO/R. In addition, PLXNA2 knockdown restrained proinflammatory cytokine release and promoted anti-inflammatory cytokine release, and the mammalian target of rapamycin (mTOR)/signal transducer and activator of transcription 3 (STAT3) pathway was involved in PLXNA2 regulated microglia polarization. Taken together, our results indicate that PLXNA2 knockdown reduces neuroinflammation by switching the microglia phenotype from M1 to M2 in the ischemic penumbra of MCAO/R-injured rats, which may be due to the inhibition of mTOR/STAT3 signaling. Treatments targeting PLXNA2 may be a promising therapeutic strategy for ischemic stroke., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Epilepsy Aphasia Syndromes (EAS) are a spectrum of childhood epileptic, cognitive, and language disorders of unknown etiology. CNKSR2 is a strong X-linked candidate gene implicated in EAS; however, there have been no studies of genetic models to dissect how its absence may lead to EAS. Here we develop a novel Cnksr2 KO mouse line and show that male mice exhibit increased neural activity and have spontaneous electrographic seizures. Cnksr2 KO mice also display significantly increased anxiety, impaired learning and memory, and a progressive and dramatic loss of ultrasonic vocalizations. We find that Cnksr2 is expressed in cortical, striatal, and cerebellar regions and is localized at both excitatory and inhibitory postsynapses. Proteomics analysis reveals Cnksr2 anchors key binding partners at synapses, and its loss results in significant alterations of the synaptic proteome, including proteins implicated in epilepsy disorders. Our results validate that loss of CNKSR2 leads to EAS and highlights the roles of Cnksr2 in synaptic organization and neuronal network activity. SIGNIFICANCE STATEMENT Epilepsy Aphasia Syndromes (EAS) are at the severe end of a spectrum of cognitive-behavioral symptoms seen in childhood epilepsies, and they remain an inadequately understood disorder. The prognosis of EAS is frequently poor, and patients have life-long language and cognitive disturbances. Here we describe a genetic mouse model of EAS, based on the KO of the EAS risk gene Cnksr2 We show that these mice exhibit electrophysiological and behavioral phenotypes similar to those of patients, providing an important new model for future studies of EAS. We also provide insights into the molecular disturbances downstream of Cnksr2 loss by using in vivo quantitative proteomics tools., (Copyright © 2021 the authors.)

Traumatic brain injury (TBI) can produce lasting cognitive, emotional, and somatic difficulties that can impact quality of life for patients living with an injury. Impaired hippocampal function and synaptic alterations have been implicated in contributing to cognitive difficulties in experimental TBI models. In the synapse, neuronal communication is facilitated by the regulated release of neurotransmitters from docking presynaptic vesicles. The synaptic vesicle glycoprotein 2 (SV2) isoforms SV2A and SV2B play central roles in the maintenance of the readily releasable pool of vesicles and the coupling of calcium to the N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex responsible for vesicle docking. Recently, we reported the findings of TBI-induced reductions in presynaptic vesicle density and SNARE complex formation; however, the effect of TBI on SV2 is unknown. To investigate this, rats were subjected to controlled cortical impact (CCI) or sham control surgery. Abundance of SV2A and SV2B were assessed at 1, 3, 7, and 14 days post-injury by immunoblot. SV2A and SV2B were reduced in the cortex at several time points and in the hippocampus at every time point assessed. Immunohistochemical staining and quantitative intensity measurements completed at 14 days post-injury revealed reduced SV2A immunoreactivity in all hippocampal subregions and reduced SV2B immunoreactivity in the molecular layer after CCI. Reductions in SV2A abundance and immunoreactivity occurred concomitantly with motor dysfunction and spatial learning and memory impairments in the 2 weeks post-injury. These findings provide novel evidence for the effect of TBI on SV2 with implications for impaired neurotransmission neurobehavioral dysfunction after TBI., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Spreading depolarization is a slowly propagating wave of massive cellular depolarization associated with acute brain injury and migraine aura. Genetic studies link depolarizing molecular defects in Ca2+ flux, Na+ current in interneurons, and glial Na+-K+ ATPase with spreading depolarization susceptibility, emphasizing the important roles of synaptic activity and extracellular ionic homeostasis in determining spreading depolarization threshold. In contrast, although gene mutations in voltage-gated potassium ion channels that shape intrinsic membrane excitability are frequently associated with epilepsy susceptibility, it is not known whether epileptogenic mutations that regulate membrane repolarization also modify spreading depolarization threshold and propagation. Here we report that the Kcnq2/Kv7.2 potassium channel subunit, frequently mutated in developmental epilepsy, is a spreading depolarization modulatory gene with significant control over the seizure-spreading depolarization transition threshold, bi-hemispheric cortical expression, and diurnal temporal susceptibility. Chronic DC-band cortical EEG recording from behaving conditional Kcnq2 deletion mice (Emx1cre/+::Kcnq2flox/flox) revealed spontaneous cortical seizures and spreading depolarization. In contrast to the related potassium channel deficient model, Kv1.1-KO mice, spontaneous cortical spreading depolarizations in Kcnq2 cKO mice are tightly coupled to the terminal phase of seizures, arise bilaterally, and are observed predominantly during the dark phase. Administration of the non-selective Kv7.2 inhibitor XE991 to Kv1.1-KO mice partly reproduced the Kcnq2 cKO-like spreading depolarization phenotype (tight seizure coupling and bilateral symmetry) in these mice, indicating that Kv7.2 currents can directly and actively modulate spreading depolarization properties. In vitro brain slice studies confirmed that Kcnq2/Kv7.2 depletion or pharmacological inhibition intrinsically lowers the cortical spreading depolarization threshold, whereas pharmacological Kv7.2 activators elevate the threshold to multiple depolarizing and hypometabolic spreading depolarization triggers. Together these results identify Kcnq2/Kv7.2 as a distinctive spreading depolarization regulatory gene, and point to spreading depolarization as a potentially significant pathophysiological component of KCNQ2-linked epileptic encephalopathy syndromes. Our results also implicate KCNQ2/Kv7.2 channel activation as a potential adjunctive therapeutic target to inhibit spreading depolarization incidence., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Numb, a stem cell fate determinant, acts as a tumor suppressor and is closely related to a wide variety of malignancies. Intrahepatic cholangiocarcinoma (iCCA) originates from hepatic progenitors (HPCs); however, the role of Numb in HPC malignant transformation and iCCA development is still unclear. A retrospective cohort study indicated that Numb was frequently decreased in tumor tissues and suggests poor prognosis in iCCA patients. Consistently, in a chemically induced iCCA mouse model, Numb was downregulated in tumor cells compared to normal cholangiocytes. In diet-induced chronic liver injury mouse models, Numb ablation significantly promoted histological impairment, HPC expansion, and tumorigenesis. Similarly, Numb silencing in cultured iCCA cells enhanced cell spheroid growth, invasion, metastasis, and the expression of stem cell markers. Mechanistically, Numb was found to bind to the Notch intracellular domain (NICD), and Numb ablation promoted Notch signaling; this effect was reversed when Notch signaling was blocked by γ-secretase inhibitor treatment. Our results suggested that loss of Numb plays an important role in promoting HPC expansion, HPC malignant transformation, and, ultimately, iCCA development in chronically injured livers. Therapies targeting suppressed Numb are promising for the treatment of iCCA., (© 2021. The Author(s).)

IQSEC2 is a guanine nucleotide exchange factor (GEF) for ADP-ribosylation factor 6 (Arf6), of which protein is exclusively localized to the postsynaptic density of the excitatory synapse. Human genome studies have revealed that the IQSEC2 gene is associated with X-linked neurodevelopmental disorders, such as intellectual disability (ID), epilepsy, and autism. In this study, we examined the behavior and synapse function in IQSEC2 knockout (KO) mice that we generated using CRIPSR/Cas9-mediated genome editing to solve the relevance between IQSEC2 deficiency and the pathophysiology of neurodevelopmental disorders. IQSEC2 KO mice exhibited autistic behaviors, such as overgrooming and social deficits. We identified that up-regulation of c-Fos expression in the medial prefrontal cortex (mPFC) induced by social stimulation was significantly attenuated in IQSEC2 KO mice. Whole cell electrophysiological recording identified that synaptic transmissions mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), N-methyl-D-aspartate receptor (NMDAR), and γ-aminobutyric acid receptor (GABAR) were significantly decreased in pyramidal neurons in layer 5 of the mPFC in IQSEC2 KO mice. Reexpression of IQSEC2 isoform 1 in the mPFC of IQSEC2 KO mice using adeno-associated virus (AAV) rescued both synaptic and social deficits, suggesting that impaired synaptic function in the mPFC is responsible for social deficits in IQSEC2 KO mice.

5-HT 7 R belongs to a family of G protein-coupled receptors and is associated with a variety of physiological processes in the central nervous system via the activation of the neurotransmitter serotonin (5-HT). To develop selective and biased 5-HT 7 R ligands, we designed and synthesized a series of pyrazolyl-diazepanes 2 and pyrazolyl-piperazines 3 , which were evaluated for binding affinities to 5-HTR subtypes and functional selectivity for G protein and β-arrestin signaling pathways of 5-HT 7 R. Among them, 1-(3-(3-chlorophenyl)-1 H -pyrazol-4-yl)-1,4-diazepane 2c showed the best binding affinity for 5-HT 7 R and selectivity over other 5-HTR subtypes. It was also revealed as a G protein-biased antagonist. The self-grooming behavior test was performed with 2c in vivo with Shank3 -/- transgenic (TG) mice, wherein 2c significantly reduced self-grooming duration time to the level of wild-type mice. The results suggest that 5-HT 7 R could be a potential therapeutic target for treating autism spectrum disorder stereotypy.

Autism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder characterized by impairments in social interaction, cognition, and communication, as well as the presence of repetitive or stereotyped behaviors and interests. ASD is most often studied as a neurodevelopmental disease, but it is a lifelong disorder. Adults with ASD experience more stressful life events and greater perceived stress, and frequently have comorbid mood disorders such as anxiety and depression. It remains unclear whether adult exposure to chronic stress can exacerbate the behavioral and neurodevelopmental phenotypes associated with ASD. To address this issue, we first investigated whether adult male and female Engrailed-2 deficient (En2-KO, En2-/-) mice, which display behavioral disturbances in avoidance tasks and dysregulated monoaminergic neurotransmitter levels, also display impairments in instrumental behaviors associated with motivation, such as the progressive ratio task. We then exposed adult En2-KO mice to chronic environmental stress (CSDS, chronic social defeat stress), to determine if stress exacerbated the behavioral and neuroanatomical effects of En2 deletion. En2-/- mice showed impaired instrumental acquisition and significantly lower breakpoints in a progressive ratio test, demonstrating En2 deficiency decreases motivation to exert effort for reward. Furthermore, adult CSDS exposure increased avoidance behaviors in En2-KO mice. Interestingly, adult CSDS exposure also exacerbated the deleterious effects of En2 deficiency on forebrain-projecting monoaminergic fibers. Our findings thus suggest that adult exposure to stress may exacerbate behavioral and neuroanatomical phenotypes associated with developmental effects of genetic En2 deficiency., (Copyright © 2021. Published by Elsevier B.V.)

Leukotoxin (LtxA) (Trade name, Leukothera) is a protein that is secreted from the oral bacterium Aggregatibacter actinomycetemcomitans, which targets and kills activated white blood cells (WBCs) by binding to lymphocyte function associated antigen-1 (LFA-1). Interaction between LtxA and Jurkat T-cells results in cell death and is characterized by increased intracellular Ca 2+ , activation of caspases, clustering of LtxA and LFA-1 within lipid rafts, and involvement of the Fas death receptor. Here, we show that LtxA can kill malignant lymphocytes via apoptotic and necrotic forms of cell death. We show that LtxA causes activation of caspases and PARP, cleavage of pannexin-1 (Panx1) channels, and expulsion of ATP, ultimately leading to cell death via apoptosis and necrosis. CRISPR-Cas9 mediated knockout (K/O) of Panx1 in Jurkat cells prevented ATP expulsion and resulted in resistance to LtxA for both apoptotic and necrotic forms of death. Resistance to necrosis could only be overcome when supplementing LtxA with endogenous ATP (bzATP). The combination of LtxA and bzATP promoted only necrosis, as no Panx1 K/O cells stained positive for phosphatidylserine (PS) exposure following the combined treatment. Inhibition of LtxA/bzATP-induced necrosis was possible when pretreating Jurkat cells with oATP, a P2X 7 R antagonist. Similarly, blockage of P2X 7 Rs with oATP prevented the intracellular mobilization of Ca 2+ , an important early step in LtxA induced cell death. We show that LtxA is able to kill malignant lymphocytes through an apoptotic death pathway which is potentially linked to a Panx1/P2X 7 R mediated necrotic form of death. Thus, inhibition of ATP release appears to significantly delay the onset of LtxA induced apoptosis while completely disabling the necrotic death pathway in T-lymphocytes, demonstrating the crucial role of ATP release in LtxA-mediated cell death., (© 2021. The Author(s).)

Microglia are brain-resident immune cells with a repertoire of functions in the brain. However, the extent of their interactions with the vasculature and potential regulation of vascular physiology has been insufficiently explored. Here, we document interactions between ramified CX3CR1  +  myeloid cell somata and brain capillaries. We confirm that these cells are bona fide microglia by molecular, morphological and ultrastructural approaches. Then, we give a detailed spatio-temporal characterization of these capillary-associated microglia (CAMs) comparing them with parenchymal microglia (PCMs) in their morphological activities including during microglial depletion and repopulation. Molecularly, we identify P2RY12 receptors as a regulator of CAM interactions under the control of released purines from pannexin 1 (PANX1) channels. Furthermore, microglial elimination triggered capillary dilation, blood flow increase, and impaired vasodilation that were recapitulated in P2RY12 -/- and PANX1 -/- mice suggesting purines released through PANX1 channels play important roles in activating microglial P2RY12 receptors to regulate neurovascular structure and function., (© 2021. The Author(s).)

Reelin is a large secreted glycoprotein that regulates neuronal migration, lamination and establishment of dendritic architecture in the embryonic brain. Reelin expression switches postnatally from Cajal-Retzius cells to interneurons. However, reelin function in interneuron development is still poorly understood. Here, we have investigated the role of reelin in interneuron development in the postnatal neocortex. To preclude early cortical migration defects caused by reelin deficiency, we employed a conditional reelin knockout (RelncKO) mouse to induce postnatal reelin deficiency. Induced reelin deficiency caused dendritic hypertrophy in distal dendritic segments of neuropeptide Y-positive (NPY+) and calretinin-positive (Calr+) interneurons, and in proximal dendritic segments of parvalbumin-positive (Parv+) interneurons. Chronic recombinant Reelin treatment rescued dendritic hypertrophy in Relncko interneurons. Moreover, we provide evidence that RelncKO interneuron hypertrophy is due to presynaptic GABABR dysfunction. Thus, GABABRs in RelncKO interneurons were unable to block N-type (Cav2.2) Ca2+ channels that control neurotransmitter release. Consequently, the excessive Ca2+ influx through AMPA receptors, but not NMDA receptors, caused interneuron dendritic hypertrophy. These findings suggest that reelin acts as a 'stop-growth-signal' for postnatal interneuron maturation., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Neuron migration is a hallmark of nervous system development that allows gathering of neurons from different origins for assembling of functional neuronal circuits. Cortical inhibitory interneurons arise in the ventral telencephalon and migrate tangentially forming three transient migratory streams in the cortex before reaching the final laminar destination. Although migration defects lead to the disruption of inhibitory circuits and are linked to aspects of psychiatric disorders such as autism and schizophrenia, the molecular mechanisms controlling cortical interneuron development and final layer positioning are incompletely understood. Here, we show that mouse embryos with a double deletion of FLRT2 and FLRT3 genes encoding cell adhesion molecules exhibit an abnormal distribution of interneurons within the streams during development, which in turn, affect the layering of somatostatin+ interneurons postnatally. Mechanistically, FLRT2 and FLRT3 proteins act in a noncell-autonomous manner, possibly through a repulsive mechanism. In support of such a conclusion, double knockouts deficient in the repulsive receptors for FLRTs, Unc5B and Unc5D, also display interneuron defects during development, similar to the FLRT2 / FLRT3 mutants. Moreover, FLRT proteins are chemorepellent ligands for developing interneurons in vitro , an effect that is in part dependent on FLRT-Unc5 interaction. Together, we propose that FLRTs act through Unc5 receptors to control cortical interneuron distribution in a mechanism that involves cell repulsion. SIGNIFICANCE STATEMENT Disruption of inhibitory cortical circuits is responsible for some aspects of psychiatric disorders such as schizophrenia or autism. These defects include interneuron migration during development. A crucial step during this process is the formation of three transient migratory streams within the developing cortex that determine the timing of interneuron final positioning and the formation of functional cortical circuits in the adult. We report that FLRT proteins are required for the proper distribution of interneurons within the cortical migratory streams and for the final laminar allocation in the postnatal cortex. These results expand the multifunctional role of FLRTs during nervous system development in addition to the role of FLRTs in axon guidance and the migration of excitatory cortical neurons., (Copyright © 2021 the authors.)

Background: Cardiac hypertrophy is an important prepathology of, and will ultimately lead to, heart failure. However, the mechanisms underlying pathological cardiac hypertrophy remain largely unknown. This study aims to elucidate the effects and mechanisms of HINT1 (histidine triad nucleotide-binding protein 1) in cardiac hypertrophy and heart failure., Methods: HINT1 was downregulated in human hypertrophic heart samples compared with nonhypertrophic samples by mass spectrometry analysis. Hint1 knockout mice were challenged with transverse aortic constriction surgery. Cardiac-specific overexpression of HINT1 mice by intravenous injection of adeno-associated virus 9 (AAV9)-encoding Hint1 under the cTnT (cardiac troponin T) promoter were subjected to transverse aortic construction. Unbiased transcriptional analyses were used to identify the downstream targets of HINT1. AAV9 bearing shRNA against Hoxa5 (homeobox A5) was administrated to investigate whether the effects of HINT1 on cardiac hypertrophy were HOXA5-dependent. RNA sequencing analysis was performed to recapitulate possible changes in transcriptome profile.Coimmunoprecipitation assays and cellular fractionation analyses were conducted to examine the mechanism by which HINT1 regulates the expression of HOXA5., Results: The reduction of HINT1 expression was observed in the hearts of hypertrophic patients and pressure overloaded-induced hypertrophic mice, respectively. In Hint1 -deficient mice, cardiac hypertrophy deteriorated after transverse aortic construction. Conversely, cardiac-specific overexpression of HINT1 alleviated cardiac hypertrophy and dysfunction. Unbiased profiler polymerase chain reaction array showed HOXA5 is 1 target for HINT1, and the cardioprotective role of HINT1 was abolished by HOXA5 knockdown in vivo. Hoxa5 was identified to affect hypertrophy through the TGF-β (transforming growth factor β) signal pathway. Mechanically, HINT1 inhibited PKCβ1 (protein kinase C β type 1) membrane translocation and phosphorylation via direct interaction, attenuating the MEK/ERK/YY1 (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/yin yang 1) signal pathway, downregulating HOXA5 expression, and eventually attenuating cardiac hypertrophy., Conclusions: HINT1 protects against cardiac hypertrophy through suppressing HOXA5 expression. These findings indicate that HINT1 may be a potential target for therapeutic interventions in cardiac hypertrophy and heart failure.

SLIT2 is a secreted polypeptide that guides migration of cells expressing Roundabout 1 and 2 (ROBO1 and ROBO2) receptors. Herein, we investigated SLIT2/ROBO signaling effects in gliomas. In patients with glioblastoma (GBM), SLIT2 expression increased with malignant progression and correlated with poor survival and immunosuppression. Knockdown of SLIT2 in mouse glioma cells and patient-derived GBM xenografts reduced tumor growth and rendered tumors sensitive to immunotherapy. Tumor cell SLIT2 knockdown inhibited macrophage invasion and promoted a cytotoxic gene expression profile, which improved tumor vessel function and enhanced efficacy of chemotherapy and immunotherapy. Mechanistically, SLIT2 promoted microglia/macrophage chemotaxis and tumor-supportive polarization via ROBO1- and ROBO2-mediated PI3K-γ activation. Macrophage Robo1 and Robo2 deletion and systemic SLIT2 trap delivery mimicked SLIT2 knockdown effects on tumor growth and the tumor microenvironment (TME), revealing SLIT2 signaling through macrophage ROBOs as a potentially novel regulator of the GBM microenvironment and immunotherapeutic target for brain tumors.

Identifying causative variants in cis-regulatory elements (CRE) in neurodevelopmental disorders has proven challenging. We have used in vivo functional analyses to categorize rigorously filtered CRE variants in a clinical cohort that is plausibly enriched for causative CRE mutations: 48 unrelated males with a family history consistent with X-linked intellectual disability (XLID) in whom no detectable cause could be identified in the coding regions of the X chromosome (chrX). Targeted sequencing of all chrX CRE identified six rare variants in five affected individuals that altered conserved bases in CRE targeting known XLID genes and segregated appropriately in families. Two of these variants, FMR1CRE and TENM1CRE, showed consistent site- and stage-specific differences of enhancer function in the developing zebrafish brain using dual-color fluorescent reporter assay. Mouse models were created for both variants. In male mice Fmr1CRE induced alterations in neurodevelopmental Fmr1 expression, olfactory behavior and neurophysiological indicators of FMRP function. The absence of another likely causative variant on whole genome sequencing further supported FMR1CRE as the likely basis of the XLID in this family. Tenm1CRE mice showed no phenotypic anomalies. Following the release of gnomAD 2.1, reanalysis showed that TENM1CRE exceeded the maximum plausible population frequency of a XLID causative allele. Assigning causative status to any ultra-rare CRE variant remains problematic and requires disease-relevant in vivo functional data from multiple sources. The sequential and bespoke nature of such analyses renders them time-consuming and challenging to scale for routine clinical use., Competing Interests: No authors have competing interests

The orphan G-protein-coupled receptor GPR139 is highly expressed in the habenula, a small brain nucleus that has been linked to depression, schizophrenia (SCZ), and substance-use disorder. High-throughput screening and a medicinal chemistry structure-activity relationship strategy identified a novel series of potent and selective benzotriazinone-based GPR139 agonists. Herein, we describe the chemistry optimization that led to the discovery and validation of multiple potent and selective in vivo GPR139 agonist tool compounds, including our clinical candidate TAK-041, also known as NBI-1065846 (compound 56 ). The pharmacological characterization of these GPR139 agonists in vivo demonstrated GPR139-agonist-dependent modulation of habenula cell activity and revealed consistent in vivo efficacy to rescue social interaction deficits in the BALB/c mouse strain. The clinical GPR139 agonist TAK-041 is being explored as a novel drug to treat negative symptoms in SCZ.

Supernumerary centrioles are frequently observed in diverse types of cancer cells. In this study, we investigated the mechanism underlying the generation of supernumerary centrioles during the M phase. We generated the TP53;PCNT;CEP215 triple knockout (KO) cells and determined the configurations of the centriole during the cell cycle. The triple KO cells exhibited a precocious separation of centrioles and unscheduled centriole assembly in the M phase. Supernumerary centrioles in the triple KO cells were present throughout the cell cycle; however, among all the centrioles, only two maintained an intact composition, including CEP135, CEP192, CEP295 and CEP152. Intact centrioles were formed during the S phase and the rest of the centrioles may be generated during the M phase. M-phase-assembled centrioles lacked the ability to organize microtubules in the interphase; however, a fraction of them may acquire pericentriolar material to organize microtubules during the M phase. Taken together, our work reveals the heterogeneity of the supernumerary centrioles in the triple KO cells.  .

Various neurodegenerative disorders are associated with human NTE/PNPLA6 dysfunction. Mechanisms of neuropathogenesis in these diseases are far from clearly elucidated. Hereditary spastic paraplegia belongs to a type of neurodegeneration associated with NTE/PNLPLA6 and is implicated in neuron death. In this study, we used Drosophila melanogaster to investigate the consequences of neuronal knockdown of swiss cheese ( sws )-the evolutionarily conserved ortholog of human NTE/PNPLA6- in vivo. Adult flies with the knockdown show longevity decline, locomotor and memory deficits, severe neurodegeneration progression in the brain, reactive oxygen species level acceleration, mitochondria abnormalities and lipid droplet accumulation. Our results suggest that SWS/NTE/PNPLA6 dysfunction in neurons induces oxidative stress and lipid metabolism alterations, involving mitochondria dynamics and lipid droplet turnover in neurodegeneration pathogenesis. We propose that there is a complex mechanism in neurological diseases such as hereditary spastic paraplegia, which includes a stress reaction, engaging mitochondria, lipid droplets and endoplasmic reticulum interplay.

PTX3 is a unique member of the long pentraxins family and plays an indispensable role in regulating the immune system. We previously showed that PTX3 deletion aggravates allergic inflammation via a Th17 -dominant phenotype and enhanced CD4 T cell survival using a murine model of ovalbumin (OVA) induced allergic inflammation. In this study, we identified that upon OVA exposure, increased infiltration of CD11c + CD11b + dendritic cells (DCs) was observed in the lungs of PTX3 -/- mice compared to wild type littermate. Further analysis showed that a short-term OVA exposure led to an increased number of bone marrow common myeloid progenitors (CMP) population concomitantly with increased Ly6C high CCR2 high monocytes and CD11c + CD11b + DCs in the lungs. Also, pulmonary CD11c + CD11b + DCs from OVA-exposed PTX3 -/- mice exhibited enhanced expression of maturation markers, chemokines receptors CCR2, and increased OVA uptake and processing compared to wild type controls. Taken together, our data suggest that PTX3 deficiency heightened lung CD11c + CD11b + DC numbers and function, hence exacerbating airway inflammatory response., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Balhara, Koussih, Mohammed, Shan, Lamkhioued and Gounni.)

Intact Disc1-L100P mice carrying a point mutation DISC1 Rgsc1390 in the second exon of the DISC1 gene (genetic model of schizophrenia) differ from the parental C57BL/6NCrl strain by higher content of CD3 + T cells and reduced number of CD19 + B cells in the peripheral blood and spleen. Analysis of T cell subpopulations revealed an increase in the number of CD3 + CD4 + T helpers in the blood of mutant mice and a decrease in the level of CD3 + CD8 + suppressor/cytotoxic T cells and CD3 + CD4 + CD25 + T-regulatory cells. The distribution pattern of inflammatory (IL-1β, IL-2, IL-6, IL-17, IFNγ, and TNFα) and anti-inflammatory (IL-4, IL-10) cytokines specific for Disc1-L100P mice was revealed in the brain structures involved in the pathogenesis of schizophrenia. A possible implication of immune mechanisms in the development of schizophrenia-like endophenotype of Disc1-L100P mice is discussed., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Endothelial cells (ECs) respond to blood shear stress by changing their morphology is important for maintaining vascular homeostasis. Studies have documented a relationship between endothelial cell shape and the stress flow, and however, the mechanism underlying this cytoskeletal rearrangement due to shear stress remains uncertain. In this paper, we demonstrate that laminar shear stress (LSS) significantly reduces latexin (LXN) expression in ECs. By using siRNA and cell imaging, we demonstrated that LXN knockdown results in the morphologic change and F-actin remodelling just like what LSS does in ECs. We further demonstrate that LXN interacts with Filamin A (FLNA) and regulates FLNA proteolytic cleavage and nuclei translocation. By constructing LXN -/- mice and ApoE -/- LXN -/- double knockout mice, we evaluated the effect of LXN knockout on aortic endothelium damage in mice. We found that LXN deficiency significantly improves vascular permeability, vasodilation and atherosclerosis in mice. Our findings provide confident evidence, for the first time, that LXN is a novel regulator for morphological maintenance of ECs, and LXN deficiency has a protective effect on vascular homeostasis. This provides new strategies and drug targets for the treatment of vascular diseases., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Macroautophagy dysregulation is implicated in multiple neurological disorders, such as Parkinson's disease. While autophagy pathways are heavily researched in heterologous cells and neurons, regulation of autophagy in the astrocyte, the most abundant cell type in the mammalian brain, is less well understood. Missense mutations in the Synj1 gene encoding Synaptojanin1 (Synj1), a neuron-enriched lipid phosphatase, have been linked to Parkinsonism with seizures. Our previous study showed that the Synj1 haploinsufficient (Synj1 +/- ) mouse exhibits age-dependent autophagy impairment in multiple brain regions. Here, we used cultured astrocytes from Synj1-deficient mice to investigate its role in astrocyte autophagy. We report that Synj1 is expressed in low levels in astrocytes and represses basal autophagosome formation. We demonstrate using cellular imaging that Synj1-deficient astrocytes exhibit hyperactive autophagosome formation, represented by an increase in the size and number of GFP-microtubule-associated protein 1A/1B-light chain 3 structures. Interestingly, Synj1 deficiency is also associated with an impairment in stress-induced autophagy clearance. We show, for the first time, that the Parkinsonism-associated R839C mutation impacts autophagy in astrocytes. The impact of this mutation on the phosphatase function of Synj1 resulted in elevated basal autophagosome formation that mimics Synj1 deletion. We found that the membrane expression of the astrocyte-specific glucose transporter GluT-1 was reduced in Synj1-deficient astrocytes. Consistently, AMP-activated protein kinase activity was elevated, suggesting altered glucose sensing in Synj1-deficient astrocytes. Expressing exogenous GluT-1 in Synj1-deficient astrocytes reversed the autophagy impairment, supporting a role for Synj1 in regulating astrocyte autophagy via disrupting glucose-sensing pathways. Thus, our work suggests a novel mechanism for Synj1-related Parkinsonism involving astrocyte dysfunction., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Centromeres are chromosomal loci where kinetochores assemble to ensure faithful chromosome segregation during mitosis. CENP-A defines the loci by serving as an epigenetic marker that recruits other centromere components for a functional structure. However, the mechanism that controls CENP-A regulation of centromeric chromatin integrity remains to be explored. Separate studies have shown that loss of CENP-A or the Cdk5 regulatory subunit associated protein 2 (Cdk5rap2), a key player in mitotic progression, triggers the occurrence of lagging chromosomes. This prompted us to investigate a potential link between CENP-A and Cdk5rap2 in the maintenance of centromeric chromatin integrity. Here, we demonstrate that loss of Cdk5rap2 causes reduced CENP-A expression while exogenous Cdk5rap2 expression in cells depleted of endogenous Cdk5rap2 restores CENP-A expression. Indeed, we show that Cdk5rap2 is a nuclear protein that acts as a positive transcriptional regulator of CENP-A. Cdk5rap2 interacts with the CENP-A promoter and upregulates CENP-A transcription. Accordingly, loss of Cdk5rap2 causes reduced level of centromeric CENP-A. Exogenous CENP-A expression partially inhibits the occurrence of lagging chromosomes in Cdk5rap2 knockdown cells, indicating that lagging chromosomes induced by loss of Cdk5rap2 is due, in part, to loss of CENP-A. Aside from manifesting lagging chromosomes, cells depleted of Cdk5rap2, and thus CENP-A, show increased micronuclei and chromatin bridge formation. Altogether, our findings indicate that Cdk5rap2 serves to maintain centromeric chromatin integrity partly through CENP-A., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Neurons have recently emerged as essential cellular constituents of the tumour microenvironment, and their activity has been shown to increase the growth of a diverse number of solid tumours 1 . Although the role of neurons in tumour progression has previously been demonstrated 2 , the importance of neuronal activity to tumour initiation is less clear-particularly in the setting of cancer predisposition syndromes. Fifteen per cent of individuals with the neurofibromatosis 1 (NF1) cancer predisposition syndrome (in which tumours arise in close association with nerves) develop low-grade neoplasms of the optic pathway (known as optic pathway gliomas (OPGs)) during early childhood 3,4 , raising  the possibility that postnatal light-induced activity of the optic nerve drives tumour initiation. Here we use an authenticated mouse model of OPG driven by mutations in the neurofibromatosis 1 tumour suppressor gene (Nf1) 5 to demonstrate that stimulation of optic nerve activity increases optic glioma growth, and that decreasing visual experience via light deprivation prevents tumour formation and maintenance. We show that the initiation of Nf1-driven OPGs (Nf1-OPGs) depends on visual experience during a developmental period in which Nf1-mutant mice are susceptible to tumorigenesis. Germline Nf1 mutation in retinal neurons results in aberrantly increased shedding of neuroligin 3 (NLGN3) within the optic nerve in response to retinal neuronal activity. Moreover, genetic Nlgn3 loss or pharmacological inhibition of NLGN3 shedding blocks the formation and progression of Nf1-OPGs. Collectively, our studies establish an obligate role for neuronal activity in the development of some types of brain tumours, elucidate a therapeutic strategy to reduce OPG incidence or mitigate tumour progression, and underscore the role of Nf1mutation-mediated dysregulation of neuronal signalling pathways in mouse models of the NF1 cancer predisposition syndrome.

One of the greatest barriers to curative treatment of neuroblastoma is its frequent metastatic outgrowth prior to diagnosis, especially in cases driven by amplification of the MYCN oncogene. However, only a limited number of regulatory proteins that contribute to this complex MYCN -mediated process have been elucidated. Here we show that the growth arrest-specific 7 ( GAS7 ) gene, located at chromosome band 17p13.1, is preferentially deleted in high-risk MYCN-driven neuroblastoma. GAS7 expression was also suppressed in MYCN -amplified neuroblastoma lacking 17p deletion. GAS7 deficiency led to accelerated metastasis in both zebrafish and mammalian models of neuroblastoma with overexpression or amplification of MYCN . Analysis of expression profiles and the ultrastructure of zebrafish neuroblastoma tumors with MYCN overexpression identified that GAS7 deficiency led to (i) downregulation of genes involved in cell-cell interaction, (ii) loss of contact among tumor cells as critical determinants of accelerated metastasis, and (iii) increased levels of MYCN protein. These results provide the first genetic evidence that GAS7 depletion is a critical early step in the cascade of events culminating in neuroblastoma metastasis in the context of MYCN overexpression. SIGNIFICANCE: Heterozygous deletion or MYCN -mediated repression of GAS7 in neuroblastoma releases an important brake on tumor cell dispersion and migration to distant sites, providing a novel mechanism underlying tumor metastasis in MYCN-driven neuroblastoma. See related commentary by Menard, p. 2815 ., (©2021 American Association for Cancer Research.)

Altered function of mitochondrial respiratory chain in brain cells is related to many neurodegenerative diseases. NADH Dehydrogenase (Ubiquinone) Fe-S protein 4 (Ndufs4) is one of the subunits of mitochondrial complex I and its mutation in human is associated with Leigh syndrome. However, the molecular biological role of Ndufs4 in neuronal function is poorly understood. In this study, upon Ndufs4 expression confirmation in NeuN-positive neurons, and GFAP-positive astrocytes in WT mouse hippocampus, we found significant decrease of mitochondrial respiration in Ndufs4-KO mouse hippocampus. Although there was no change in the number of NeuN positive neurons in Ndufs4-KO hippocampus, the expression of synaptophysin, a presynaptic protein, was significantly decreased. To investigate the detailed mechanism, we silenced Ndufs4 in Neuro-2a cells and we observed shorter neurite lengths with decreased expression of synaptophysin. Furthermore, western blot analysis for phosphorylated extracellular regulated kinase (pERK) revealed that Ndufs4 silencing decreases the activity of ERK signalling. These results suggest that Ndufs4-modulated mitochondrial activity may be involved in neuroplasticity via regulating synaptophysin expression.

Deletions of chromosome 1p36 are the most common telomeric deletions in humans and are associated with an increased risk of orofacial clefting. Deletion/phenotype mapping, combined with data from human and mouse studies, suggests the existence of multiple 1p36 genes associated with orofacial clefting including SKI, PRDM16, PAX7 and GRHL3. The arginine-glutamic acid dipeptide (RE) repeats gene (RERE) is located in the proximal critical region for 1p36 deletion syndrome and encodes a nuclear receptor co-regulator. Pathogenic RERE variants have been shown to cause neurodevelopmental disorder with or without anomalies of the brain, eye or heart (NEDBEH). Cleft lip has previously been described in one individual with NEDBEH. Here we report the first individual with NEDBEH to have a cleft palate. We confirm that RERE is broadly expressed in the palate during mouse embryonic development, and we demonstrate that the majority of RERE-deficient mouse embryos on C57BL/6 background have cleft palate. We go on to show that ablation of Rere in cranial neural crest (CNC) cells, mediated by a Wnt1-Cre, leads to delayed elevation of the palatal shelves and cleft palate and that proliferation of mesenchymal cells in the palatal shelves is significantly reduced in Rereflox/flox; Wnt1-Cre embryos. We conclude that loss of RERE function contributes to the development of orofacial clefts in individuals with proximal 1p36 deletions and NEDBEH and that RERE expression in CNC cells and their derivatives is required for normal palatal development., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Ganglioside-induced differentiation associated protein 1 (GDAP1) gene encodes a protein of the mitochondrial outer membrane and of the mitochondrial membrane contacts with the endoplasmic reticulum (MAMs) and lysosomes. Since mutations in GDAP1 cause Charcot-Marie-Tooth, an inherited motor and sensory neuropathy, its function is essential for peripheral nerve physiology. Our previous studies showed structural and functional defects in mitochondria and their contacts when GDAP1 is depleted. Nevertheless, the underlying axonal pathophysiological events remain unclear. Here, we have used embryonic motor neurons (eMNs) cultures from Gdap1 knockout (Gdap1 -/- ) mice to investigate in vivo mitochondria and calcium homeostasis in the axons. We imaged mitochondrial axonal transport and we found a defective pattern in the Gdap1 -/- eMNs. We also detected pathological and functional mitochondria membrane abnormalities with a drop in ATP production and a deteriorated bioenergetic status. Another consequence of the loss of GDAP1 in the soma and axons of eMNs was the in vivo increase calcium levels in both basal conditions and during recovery after neuronal stimulation with glutamate. Further, we found that glutamate-stimulation of respiration was lower in Gdap1 -/- eMNs showing that the basal bioenergetics failure jeopardizes a full respiratory response and prevents a rapid return of calcium to basal levels. Together, our results demonstrate that the loss of GDAP1 critically compromises the morphology and function of mitochondria and its relationship with calcium homeostasis in the soma and axons, offering important insight into the cellular mechanisms associated with axonal degeneration of GDAP1-related CMT neuropathies and the relevance that axon length may have., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Shadoo and PrP belongs to the same protein family, whose biological function remains poorly understood. Previous experiments reported potential functional redundancies or antagonisms between these two proteins, depending on the tissue analysed. While knockdown experiments suggested the requirement of Shadoo in the absence of PrP during early mouse embryogenesis, knockout ones, on the contrary, highlighted little impact, if any, of the double-knockout of these two loci. In the present study, we reinvestigated the phenotype associated with the concomitant knockout of these two genes using newly produced FVB/N Sprn knockout mice. In this genetic background, the combined two genes' knockout induces intra-uterine growth retardations, likely resulting from placental failures highlighted by transcriptomic analyses that revealed potential redundant or antagonist roles of these two proteins in different developmental-related pathways. It also induced an increased perinatal-lethality and ascertained the role of these two loci in the lactation process., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In schizophrenia (SCZ), neurons in the brain tend to undergo gross morphological changes, but the related molecular mechanism remains largely elusive. Using Kif3b +/- mice as a model with SCZ-like behaviors, we found that a high-betaine diet can significantly alleviate schizophrenic traits related to neuronal morphogenesis and behaviors. According to a deficiency in the transport of collapsin response mediator protein 2 (CRMP2) by the KIF3 motor, we identified a significant reduction in lamellipodial dynamics in developing Kif3b +/- neurons as a cause of neurite hyperbranching. Betaine administration significantly decreases CRMP2 carbonylation, which enhances the F-actin bundling needed for proper lamellipodial dynamics and microtubule exclusion and may thus functionally compensate for KIF3 deficiency. Because the KIF3 expression levels tend to be downregulated in the human prefrontal cortex of the postmortem brains of SCZ patients, this mechanism may partly participate in human SCZ pathogenesis, which we hypothesize could be alleviated by betaine administration., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Presynaptic neurotransmitter release is strictly regulated by SNARE proteins, Ca 2+ and a number of Ca 2+ sensors including synaptotagmins (Syts) and Double C 2 domain proteins (Doc2s). More than seventy years after the original description of spontaneous release, the mechanism that regulates this process is still poorly understood. Syt-1, Syt7 and Doc2 proteins contribute predominantly, but not exclusively, to synchronous, asynchronous and spontaneous phases of release. The proteins share a conserved tandem C 2 domain architecture, but are functionally diverse in their subcellular location, Ca 2+ -binding properties and protein interactions. In absence of Syt-1, Doc2a and -b, neurons still exhibit spontaneous vesicle fusion which remains Ca 2+ -sensitive, suggesting the existence of additional sensors. Here, we selected Doc2c, rabphilin-3a and Syt-7 as three potential Ca 2+ sensors for their sequence homology with Syt-1 and Doc2b. We genetically ablated each candidate gene in absence of Doc2a and -b and investigated spontaneous and evoked release in glutamatergic hippocampal neurons, cultured either in networks or on microglial islands (autapses). The removal of Doc2c had no effect on spontaneous or evoked release. Syt-7 removal also did not affect spontaneous release, although it altered short-term plasticity by accentuating short-term depression. The removal of rabphilin caused an increased spontaneous release frequency in network cultures, an effect that was not observed in autapses. Taken together, we conclude that Doc2c and Syt-7 do not affect spontaneous release of glutamate in hippocampal neurons, while our results suggest a possible regulatory role of rabphilin-3a in neuronal networks. These findings importantly narrow down the repertoire of synaptic Ca 2+ sensors that may be implicated in the spontaneous release of glutamate., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The core symptoms of many neurological disorders have traditionally been thought to be caused by genetic variants affecting brain development and function. However, the gut microbiome, another important source of variation, can also influence specific behaviors. Thus, it is critical to unravel the contributions of host genetic variation, the microbiome, and their interactions to complex behaviors. Unexpectedly, we discovered that different maladaptive behaviors are interdependently regulated by the microbiome and host genes in the Cntnap2 -/- model for neurodevelopmental disorders. The hyperactivity phenotype of Cntnap2 -/- mice is caused by host genetics, whereas the social-behavior phenotype is mediated by the gut microbiome. Interestingly, specific microbial intervention selectively rescued the social deficits in Cntnap2 -/- mice through upregulation of metabolites in the tetrahydrobiopterin synthesis pathway. Our findings that behavioral abnormalities could have distinct origins (host genetic versus microbial) may change the way we think about neurological disorders and how to treat them., Competing Interests: Declaration of interests A patent application related to the role of L. reuteri on social behavior has been filed by BCM. Findings regarding the manipulation of endogenous biotperin levels by the microbiome are the subject of a provisional patent application owned by BCM. M.C.-M. is a scientific co-founder of Mirkrovia. The authors declare no other competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Homozygous Dab1 yotari mutant mice, Dab1 yot (yot/yot) mice, have an autosomal recessive mutation of Dab1 and show reeler-like phenotype including histological abnormality of the cerebellum, hippocampus, and cerebral cortex. We here show abnormal hippocampal development of yot/yot mice where granule cells and pyramidal cells fail to form orderly rows but are dispersed diffusely in vague multiplicative layers. Possibly due to the positioning failure of granule cells and pyramidal cells and insufficient synaptogenesis, axons of the granule cells did not extend purposefully to connect with neighboring regions in yot/yot mice. We found that both hippocampal granule cells and pyramidal cells of yot/yot mice expressed proteins reactive with the anti-Dab1 antibody. We found that Y198- phosphorylated Dab1 of yot/yot mice was greatly decreased. Accordingly the downstream molecule, Akt was hardly phosphorylated. Especially, synapse formation was defective and the distribution of neurons was scattered in hippocampus of yot/yot mice. Some of neural cell adhesion molecules and hippocampus associated transcription factors of the neurons were expressed aberrantly, suggesting that the Reelin-Dab1 signaling pathway seemed to be importantly involved in not only neural migration as having been shown previously but also neural maturation and/or synaptogenesis of the mice. It is interesting to clarify whether the defective neural maturation is a direct consequence of the dysfunctional Dab1, or alternatively secondarily due to the Reelin-Dab1 intracellular signaling pathways., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Creatine transporter deficiency is an inborn error of metabolism caused by a deficiency in the creatine transporter protein encoded by the SLC6A8 gene. Previous treatment with creatine supplementation, either alone or in combination with creatine precursors (arginine or glycine), has been attempted; the efficacy of therapy, however, remains controversial., Methods and Results: To analyze the treatment efficacy of high-dose creatine supplementation on creatine transporter deficiency, we reported a child diagnosed with creatine transporter deficiency, who was treated with a conventional dose of creatine (400 mg/kg/d) for 1 month, then twice the dose (800 mg/kg/d) for 2 months, and finally 3 times the dose (1200 mg/kg/d) for 3 months. The patient tolerated the treatment well and showed improvements in muscle mass and strength when the creatine dose was gradually increased to 1200 mg/kg/d. However, when assessed by proton magnetic resonance spectroscopy (H-MRS), the brain creatine concentration did not increase, and there was no improvement in speech and neurodevelopmental symptoms., Conclusion: We conclude that high-dose creatine supplementation (1200 mg/kg/d) alone improved muscular symptoms, but did not improve cognitive symptoms and brain creatine concentration assessed using H-MRS. Therefore, new treatment strategies are required for the management of creatine transporter deficiency., (© 2021 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)