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BACKGROUND AND PURPOSE: All‐trans retinoic acid (ATRA) is a vitamin A metabolite, important in the developing and mature brain. Pre‐injury ATRA administration ameliorates ischaemic brain insults in rodents. This study examined the effects of post‐traumatic ATRA treatment in experimental traumatic brain injury (TBI). EXPERIMENTAL APPROACH: Male adult mice were subjected to the controlled cortical impact model of TBI or sham procedure and killed at 7 or 30 days post‐injury (dpi). ATRA (10 mg kg−1, i.p.) was given immediately after the injury and 1, 2 and 3 dpi. Neurological function and sensorimotor coordination were evaluated. Brains were processed for (immuno‐) histological, mRNA and protein analyses (qPCR and western blot). KEY RESULTS: ATRA treatment reduced brain lesion size, reactive astrogliosis and axonal injury at 7 dpi, and hippocampal granule cell layer (GCL) integrity was protected at 7 and 30 dpi, independent of cell proliferation in neurogenic niches and blood–brain barrier damage. Neurological and motor deficits over time and the brain tissue loss at 30 dpi were not affected by ATRA treatment. ATRA decreased gene expression of markers for damage‐associated molecular pattern (HMGB1), apoptosis (caspase‐3 and Bax), activated microglia (TSPO), and reactive astrogliosis (GFAP, SerpinA3N) at 7 dpi and a subset of markers at 30 dpi (TSPO, GFAP). CONCLUSION AND IMPLICATIONS: In experimental TBI, post‐traumatic ATRA administration exerted brain protective effects, including long‐term protection of GCL integrity, but did not affect neurological and motor deficits. Further investigations are required to optimize treatment regimens to enhance ATRA's brain protective effects and improve outcomes.

Background Recent emerging evidence suggests that extra-adrenal synthesis of aldosterone occurs (e.g., within the failing heart and in certain brain areas). In this study, the authors investigated evidence for a local endogenous aldosterone production through its key processing enzyme aldosterone synthase within peripheral nociceptive neurons. Methods In male Wistar rats (n = 5 to 8 per group) with Freund’s complete adjuvant hind paw inflammation, the authors examined aldosterone, aldosterone synthase, and mineralocorticoid receptor expression in peripheral sensory neurons using quantitative reverse transcriptase–polymerase chain reaction, Western blot, immunohistochemistry, and immunoprecipitation. Moreover, the authors explored the nociceptive behavioral changes after selective mineralocorticoid receptor antagonist, canrenoate-K, or specific aldosterone synthase inhibitor application. Results In rats with Freund’s complete adjuvant–induced hind paw inflammation subcutaneous and intrathecal application of mineralocorticoid receptor antagonist, canrenoate-K, rapidly and dose-dependently attenuated nociceptive behavior (94 and 48% reduction in mean paw pressure thresholds, respectively), suggesting a tonic activation of neuronal mineralocorticoid receptors by an endogenous ligand. Indeed, aldosterone immunoreactivity was abundant in peptidergic nociceptive neurons of dorsal root ganglia and colocalized predominantly with its processing enzyme aldosterone synthase and mineralocorticoid receptors. Moreover, aldosterone and its synthesizing enzyme were significantly upregulated in peripheral sensory neurons under inflammatory conditions. The membrane mineralocorticoid receptor consistently coimmunoprecipitated with endogenous aldosterone, confirming a functional link between mineralocorticoid receptors and its endogenous ligand. Importantly, inhibition of endogenous aldosterone production in peripheral sensory neurons by a specific aldosterone synthase inhibitor attenuated nociceptive behavior after hind paw inflammation (a 32% reduction in paw pressure thresholds; inflammation, 47 ± 2 [mean ± SD] vs. inflammation + aldosterone synthase inhibitor, 62 ± 2). Conclusions Local production of aldosterone by its processing enzyme aldosterone synthase within peripheral sensory neurons contributes to ongoing mechanical hypersensitivity during local inflammation via intrinsic activation of neuronal mineralocorticoid receptors. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

Gene transcription regulation is critical for the development of spinal microgliosis and neuropathic pain after peripheral nerve injury. Using a model of chronic constriction injury (CCI) of the sciatic nerve, this study characterized the role of SET domain containing lysine methyltransferase 7 (SETD7) which monomethylates histone H3 lysine 4 (H3K4me1), a marker for active gene transcription. SETD7 protein expression in the spinal dorsal horn ipsilateral to nerve lesion was increased from one day to 14 days after CCI, concomitantly with the expression of inflammatory genes, Ccl2, Il-6 and Il-1β. The CCI-induced SETD7 expression was predominantly localized to microglia, as demonstrated by immunohistochemistry and western blot from magnetic activated cell sorted spinal microglia. SETD7 knockdown by intrathecal lentivirus shRNA delivery prior to CCI prevented spinal microgliosis and neuropathic pain, whereas lentiviral SETD7 transduction exacerbated these symptoms. In addition, SETD7 regulated H3K4me1 level and expression of inflammatory mediators both in CCI rats and in the HAPI rat microglia cell line. Accordingly, PFI-2, a specific inhibitor of SETD7 monomethylation activity, suppressed the lipopolysaccharides-induced amoeboid morphology of primary microglia and the expression of inflammatory genes, Ccl2, Il-6 and Il-1β. Moreover, intrathecal administration of PFI-2 alleviated CCI-induced neuropathic pain. However, this effect was observed in male but not in female rats. These results demonstrate a critical role of SETD7 in the development of spinal microgliosis and neuropathic pain subsequently to peripheral nerve injury. The pharmacological approach further suggests that SETD7 is a new target for the treatment of neuropathic pain. The underlying mechanisms may involve H3K4me1-dependent regulation of inflammatory gene expression in microglia.

Objective Plasminogen activator inhibitor-1 (PAI-1) is the key endogenous inhibitor of fibrinolysis, and enhances clot formation after injury. In traumatic brain injury, dysregulation of fibrinolysis may lead to sustained microthrombosis and accelerated lesion expansion. In the present study, we hypothesized that PAI-1 mediates post-traumatic malfunction of coagulation, with inhibition or genetic depletion of PAI-1 attenuating clot formation and lesion expansion after brain trauma. Methods We evaluated PAI-1 as a possible new target in a mouse controlled cortical impact (CCI) model of traumatic brain injury. We performed the pharmacological inhibition of PAI-1 with PAI-039 and stimulation by tranexamic acid, and we confirmed our results in PAI-1-deficient animals. Results PAI-1 mRNA was time-dependently upregulated, with a 305-fold peak 12 hours after CCI, which effectively counteracted the 2- to 3-fold increase in cerebral tissue-type/urokinase plasminogen activator expression. PAI-039 reduced brain lesion volume by 26% at 24 hours and 43% at 5 days after insult. This treatment also attenuated neuronal apoptosis and improved neurofunctional outcome. Moreover, intravital microscopy demonstrated reduced post-traumatic thrombus formation in the pericontusional cortical microvasculature. In PAI-1-deficient mice, the therapeutic effect of PAI-039 was absent. These mice also displayed 13% reduced brain damage compared with wild type. In contrast, inhibition of fibrinolysis with tranexamic acid increased lesion volume by 25% compared with vehicle. Interpretation This study identifies impaired fibrinolysis as a critical process in post-traumatic secondary brain damage and suggests that PAI-1 may be a central endogenous inhibitor of the fibrinolytic pathway, promoting a procoagulatory state and clot formation in the cerebral microvasculature. Ann Neurol 2019;85:667-680.

L1 syndrome is a rare developmental disorder characterized by hydrocephalus of varying severity, intellectual deficits, spasticity of the legs, and adducted thumbs. Therapy is limited to symptomatic relief. Numerous gene mutations in the L1 cell adhesion molecule (L1CAM, hereafter abbreviated L1) were identified in L1 syndrome patients, and those affecting the extracellular domain of this transmembrane type 1 glycoprotein show the most severe phenotypes. Previously analyzed rodent models of the L1 syndrome focused on L1-deficient animals or mouse mutants with abrogated cell surface expression of L1, making it difficult to test L1 function-triggering mimetic compounds with potential therapeutic value. To overcome this impasse, we generated a novel L1 syndrome mouse with a mutation of aspartic acid at position 201 in the extracellular part of L1 (p.D201N, hereafter termed L1-201) that displays a cell surface-exposed L1 accessible to the L1 mimetics. Behavioral assessment revealed an increased neurological deficit score and increased locomotor activity in male L1-201 mice carrying the mutation on the X-chromosome. Histological analyses of L1-201 mice showed features of the L1 syndrome, including enlarged ventricles and reduced size of the corpus callosum. Expression levels of L1-201 protein as well as extent of cell surface biotinylation and immunofluorescence labelling of cultured cerebellar neurons were normal. Importantly, treatment of these cultures with the L1 mimetic compounds duloxetine, crotamiton, and trimebutine rescued impaired cell migration and survival as well as neuritogenesis. Altogether, the novel L1 syndrome mouse model provides a first experimental proof-of-principle for the potential therapeutic value of L1 mimetic compounds.

Progranulin (PGRN) is a neurotrophic and anti-inflammatory factor with protective effects in animal models of ischemic stroke, subarachnoid hemorrhage, and traumatic brain injury (TBI). Administration of recombinant (r) PGRN prevents exaggerated brain pathology after TBI in Grn-deficient mice, suggesting that local injection of recombinant progranulin (rPGRN) provides therapeutic benefit in the acute phase of TBI. To test this hypothesis, we subjected adult male C57Bl/6N mice to the controlled cortical impact model of TBI, administered a single dose of rPGRN intracerebroventricularly (ICV) shortly before the injury, and examined behavioral and biological effects up to 5 days post injury (dpi). The anti-inflammatory bioactivity of rPGRN was confirmed by its capability to inhibit the inflammation-induced hypertrophy of murine primary microglia and astrocytes in vitro. In C57Bl/6N mice, however, ICV administration of rPGRN failed to attenuate behavioral deficits over the 5-day observation period. (Immuno)histological gene and protein expression analyses at 5 dpi did not reveal a therapeutic benefit in terms of brain injury size, brain inflammation, glia activation, cell numbers in neurogenic niches, and neuronal damage. Instead, we observed a failure of TBI-induced mRNA upregulation of the tight junction protein occludin and increased extravasation of serum immunoglobulin G into the brain parenchyma at 5 dpi. In conclusion, single ICV administration of rPGRN had not the expected protective effects in the acute phase of murine TBI, but appeared to cause an aggravation of blood-brain barrier disruption. The data raise questions about putative PGRN-boosting approaches in other types of brain injuries and disease.

Background In naive rats, corticosteroids activate neuronal membrane–bound glucocorticoid and mineralocorticoid receptors in spinal cord and periphery to modulate nociceptive behavior by nongenomic mechanisms. Here we investigated inflammation-induced changes in neuronal versus glial glucocorticoid and mineralocorticoid receptors and their ligand-mediated nongenomic impact on mechanical nociception in rats. Methods In Wistar rats (n = 5 to 7/group) with Freund’s complete adjuvant hind paw inflammation, we examined glucocorticoid and mineralocorticoid receptor expression in spinal cord and peripheral sensory neurons versus glial using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot, immunohistochemistry, and radioligand binding. Moreover, we explored the expression of mineralocorticoid receptors protecting enzyme 11-betahydroxysteroid dehydrogenase type 2 as well as the nociceptive behavioral changes after glucocorticoid and mineralocorticoid receptors agonist or antagonist application. Results Hind paw inflammation resulted in significant upregulation of glucocorticoid receptors in nociceptive neurons of spinal cord (60%) and dorsal root ganglia (15%) as well as mineralocorticoid receptors, while corticosteroid plasma concentrations remained unchanged. Mineralocorticoid (83 ± 16 fmol/mg) but not glucocorticoid (104 ± 20 fmol/mg) membrane binding sites increased twofold in dorsal root ganglia concomitant with upregulated 11-betahydroxysteroid dehydrogenase type 2 (43%). Glucocorticoid and mineralocorticoid receptor expression in spinal microglia and astrocytes was small. Importantly, glucocorticoid receptor agonist dexamethasone or mineralocorticoid receptor antagonist canrenoate-K rapidly and dose-dependently attenuated nociceptive behavior. Isobolographic analysis of the combination of both drugs showed subadditive but not synergistic or additive effects. Conclusions The enhanced mechanical sensitivity of inflamed hind paws accompanied with corticosteroid receptor upregulation in spinal and peripheral sensory neurons was attenuated immediately after glucocorticoid receptor agonist and mineralocorticoid receptor antagonist administration, suggesting acute nongenomic effects consistent with detected membrane-bound corticosteroid receptors.

Affective and cognitive processing of nociception contributes to the development of chronic pain and vice versa, pain may precipitate psychopathologic symptoms. We hypothesized a higher risk for the latter with immanent neurologic diseases and studied this potential interrelationship in progranulin-deficient mice, which are a model for frontotemporal dementia, a disease dominated by behavioral abnormalities in humans. Young naïve progranulin deficient mice behaved normal in tests of short-term memory, anxiety, depression and nociception, but after peripheral nerve injury, they showed attention-deficit and depression-like behavior, over-activity, loss of shelter-seeking, reduced impulse control and compulsive feeding behavior, which did not occur in equally injured controls. Hence, only the interaction of 'pain x progranulin deficiency' resulted in the complex phenotype at young age, but neither pain nor progranulin deficiency alone. A deep proteome analysis of the prefrontal cortex and olfactory bulb revealed progranulin-dependent alterations of proteins involved in synaptic transport, including neurotransmitter transporters of the solute carrier superfamily. In particular, progranulin deficiency was associated with a deficiency of nuclear and synaptic zinc transporters (ZnT9/Slc30a9; ZnT3/Slc30a3) with low plasma zinc. Dietary zinc supplementation partly normalized the attention deficit of progranulin-deficient mice, which was in part reminiscent of autism-like and compulsive behavior of synaptic zinc transporter Znt3-knockout mice. Hence, the molecular studies point to defective zinc transport possibly contributing to progranulin-deficiency-associated psychopathology. Translated to humans, our data suggest that neuropathic pain may precipitate cognitive and psychopathological symptoms of an inherent, still silent neurodegenerative disease.

Traumatic brain injury (TBI) is a leading cause of disability and death and survivors often suffer from long-lasting motor impairment, cognitive deficits, anxiety disorders and epilepsy. Few experimental studies have investigated long-term sequelae after TBI and relations between behavioral changes and neural activity patterns remain elusive. We examined these issues in a murine model of TBI combining histology, behavioral analyses and single-photon emission computed tomography (SPECT) imaging of regional cerebral blood flow (CBF) as a proxy for neural activity. Adult C57Bl/6N mice were subjected to unilateral cortical impact injury and investigated at early (15-57 days after lesion, dal) and late (184-225 dal) post-traumatic time points. TBI caused pronounced tissue loss of the parietal cortex and subcortical structures and enduring neurological deficits. Marked perilesional astro- and microgliosis was found at 57 dal and declined at 225 dal. Motor and gait pattern deficits occurred at early time points after TBI and improved over the time. In contrast, impaired performance in the Morris water maze test and decreased anxiety-like behavior persisted together with an increased susceptibility to pentylenetetrazole-induced seizures suggesting alterations in neural activity patterns. Accordingly, SPECT imaging of CBF indicated asymmetric hemispheric baseline neural activity patterns. In the ipsilateral hemisphere, increased baseline neural activity was found in the amygdala. In the contralateral hemisphere, homotopic to the structural brain damage, the hippocampus and distinct cortex regions displayed increased baseline neural activity. Thus, regionally elevated CBF along with behavioral alterations indicate that increased neural activity is critically involved in the long-lasting consequences of TBI.

Background Traumatic brain injury (TBI) is a major cause of death and disability. T cells were shown to infiltrate the brain during the first days after injury and to exacerbate tissue damage. The objective of this study was to investigate the hitherto unresolved role of immunosuppressive, regulatory T cells (Tregs) in experimental TBI. Methods “Depletion of regulatory T cell” (DEREG) and wild type (WT) C57Bl/6 mice, treated with diphtheria toxin (DTx) to deplete Tregs or to serve as control, were subjected to the controlled cortical impact (CCI) model of TBI. Neurological and motor deficits were examined until 5 days post-injury (dpi). At the 5 dpi endpoint, (immuno-) histological, protein, and gene expression analyses were carried out to evaluate the consequences of Tregs depletion. Comparison of parametric or non-parametric data between two groups was done using Student’s t test or the Mann-Whitney U test. For multiple comparisons, p values were calculated by one-way or two-way ANOVA followed by specific post hoc tests. Results The overall neurological outcome at 5 dpi was not different between DEREG and WT mice but more severe motor deficits occurred transiently at 1 dpi in DEREG mice. DEREG and WT mice did not differ in the extent of brain damage, blood-brain barrier (BBB) disruption, or neuronal excitotoxicity, as examined by lesion volumetry, immunoglobulin G (IgG) extravasation, or calpain-generated αII-spectrin breakdown products (SBDPs), respectively. In contrast, increased protein levels of glial fibrillary acidic protein (GFAP) and GFAP+ astrocytes in the ipsilesional brain tissue indicated exaggerated reactive astrogliosis in DEREG mice. T cell counts following anti-CD3 immunohistochemistry and gene expression analyses of Cd247 (CD3 subunit zeta) and Cd8a (CD8a) further indicated an increased number of T cells infiltrating the brain injury sites of DEREG mice compared to WT. These changes coincided with increased gene expression of pro-inflammatory interferon-γ (Ifng) in DEREG mice compared to WT in the injured brain. Conclusions The results show that the depletion of Tregs attenuates T cell brain infiltration, reactive astrogliosis, interferon-γ gene expression, and transiently motor deficits in murine acute traumatic brain injury.

Peripheral nerve injury elicits spinal microgliosis, contributing to neuropathic pain. The aurora kinases A (AURKA), B (AURKB), and C (AURKC) are potential therapeutic targets in proliferating cells. However, their role has not been clarified in microglia. The aim of this study was to examine the regulation of aurora kinases and their roles and druggability in spinal microgliosis and neuropathic pain. Sprague-Dawley rats received chronic constriction injury (CCI). Gene expression of aurora kinases A-C was evaluated by quantitative RT-PCR and western blot, respectively, in spinal cords at 1, 3, 7, and 14 days after CCI. AURKB gene and protein expression was up-regulated concomitantly with the development of spinal microgliosis and neuropathic pain. Using lentiviral over-expression and adeno-associated viral knockdown approaches, the function of AURKB was further investigated by western blot, immunohistochemistry, RNA sequencing, and pain behavior tests. We found that AURKB over-expression in naive rats caused spinal microgliosis and pain hypersensitivity, whereas AURKB knockdown reduced microgliosis and alleviated CCI-induced neuropathic pain. Accordingly, RNA sequencing data revealed down-regulation of genes critically involved in signaling pathways associated with spinal microgliosis and neuropathic pain after AURKB knockdown in CCI rats. To examine its therapeutic potential for treatment of neuropathic pain, animals were treated intrathecally with the pharmacological AURKB inhibitor AZD1152-HQPA resulting in the alleviation of CCI-induced pain. Taken together, our findings indicated that AURKB plays a critical role in spinal microgliosis and neuropathic pain. Targeting AURKB may be an efficient method for treatment of neuropathic pain subsequent to peripheral nerve injury.

Neuronal growth regulator 1 (NEGR1) belongs to the immunoglobulin (IgLON) superfamily of cell adhesion molecules involved in cortical layering. Recent functional and genomic studies implicate the role of NEGR1 in a wide spectrum of psychiatric disorders, such as major depression, schizophrenia and autism. Here, we investigated the impact of Negr1 deficiency on brain morphology, neuronal properties and social behavior of mice. In situ hybridization shows Negr1 expression in the brain nuclei which are central modulators of cortical-subcortical connectivity such as the island of Calleja and the reticular nucleus of thalamus. Brain morphological analysis revealed neuroanatomical abnormalities in Negr1−/− mice, including enlargement of ventricles and decrease in the volume of the whole brain, corpus callosum, globus pallidus and hippocampus. Furthermore, decreased number of parvalbumin-positive inhibitory interneurons was evident in Negr1−/− hippocampi. Behaviorally, Negr1−/− mice displayed hyperactivity in social interactions and impairments in social hierarchy. Finally, Negr1 deficiency resulted in disrupted neurite sprouting during neuritogenesis. Our results provide evidence that NEGR1 is required for balancing the ratio of excitatory/inhibitory neurons and proper formation of brain structures, which is prerequisite for adaptive behavioral profiles. Therefore, Negr1−/− mice have a high potential to provide new insights into the neural mechanisms of neuropsychiatric disorders.

In response to traumatic brain injury (TBI) microglia/macrophages and astrocytes release inflammatory mediators with dual effects on secondary brain damage progression. The neurotrophic and anti-inflammatory glycoprotein progranulin (PGRN) attenuates neuronal damage and microglia/macrophage activation in brain injury but mechanisms are still elusive. Here, we studied histopathology, neurology and gene expression of inflammatory markers in PGRN-deficient mice (Grn-/- ) 24 h and 5 days after experimental TBI. Grn-/- mice displayed increased perilesional axonal injury even though the overall brain tissue loss and neurological consequences were similar to wild-type mice. Brain inflammation was elevated in Grn-/- mice as reflected by increased transcription of pro-inflammatory cytokines TNFα, IL-1β, IL-6, and decreased transcription of the anti-inflammatory cytokine IL-10. However, numbers of Iba1+ microglia/macrophages and immigrated CD45+ leukocytes were similar at perilesional sites while determination of IgG extravasation suggested stronger impairment of blood brain barrier integrity in Grn-/- compared to wild-type mice. Most strikingly, Grn-/- mice displayed exaggerated astrogliosis 5 days after TBI as demonstrated by anti-GFAP immunohistochemistry and immunoblot. GFAP+ astrocytes at perilesional sites were immunolabelled for iNOS and TNFα suggesting that pro-inflammatory activation of astrocytes was attenuated by PGRN. Accordingly, recombinant PGRN (rPGRN) attenuated LPS- and cytokine-evoked iNOS and TNFα mRNA expression in cultured astrocytes. Moreover, intracerebroventricular administration of rPGRN immediately before trauma reduced brain damage and neurological deficits, and restored normal levels of cytokine transcription, axonal injury and astrogliosis 5 days after TBI in Grn-/- mice. Our results show that endogenous and recombinant PGRN limit axonal injury and astrogliosis and suggest therapeutic potential of PGRN in TBI. GLIA 2017;65:278-292.

Motor neuron diseases such as amyotrophic lateral sclerosis (ALS) are now recognized as multi-system disorders also involving various non-motor neuronal cell types. The precise extent and mechanistic basis of non-motor neuron damage in human ALS and ALS animal models remain however unclear. To address this, we here studied progressive motor neuronopathy (pmn) mice carrying a missense loss-of-function mutation in tubulin binding cofactor E (TBCE). These mice manifest a particularly aggressive form of motor axon dying back and display a microtubule loss, similar to that induced by human ALS-linked TUBA4A mutations. Using whole nerve confocal imaging of pmn × thy1.2-YFP16 fluorescent reporter mice and electron microscopy, we demonstrate axonal discontinuities, bead-like spheroids and ovoids in pmn suralis nerves indicating prominent sensory neuropathy. The axonal alterations qualitatively resemble those in phrenic motor nerves but do not culminate in the loss of myelinated fibers. We further show that the pmn mutation decreases the level of TBCE, impedes microtubule polymerization in dorsal root ganglion (DRG) neurons and causes progressive loss of microtubules in large and small caliber suralis axons. Live imaging of axonal transport using GFP-tagged tetanus toxin C-fragment (GFP-TTC) demonstrates defects in microtubule-based transport in pmn DRG neurons, providing a potential explanation for the axonal alterations in sensory nerves. This study unravels sensory neuropathy as a pathological feature of mouse pmn, and discusses the potential contribution of cytoskeletal defects to sensory neuropathy in human motor neuron disease.

L1CAM gene mutations cause neurodevelopmental disorders collectively termed L1 syndrome. Insufficient information about L1CAM variants complicates clinical prognosis, genetic diagnosis and genetic counseling. We combined clinical data, in silico effect predictions and functional analysis of four L1CAM variants, p.I37N, p.T38M, p.M172I and p.D202Y, located to the two N-terminal Ig-like domains present in five families with symptoms of L1 syndrome. Software tools predicted destabilizing effects of p.I37N and p.D202Y but results for p.T38M and p.M172I were inconsistent. Cell surface expression of mutant proteins L1-T38M, L1-M172I and L1-D202Y was normal. Conversely, L1-I37N accumulated in the endoplasmic reticulum (ER) and showed temperature-sensitive protein maturation suggesting that p.I37N induces protein misfolding. L1CAM-mediated cell-cell aggregation was severely impaired by L1CAM variants p.I37N, p.M172I and p.D202Y but was preserved by the variant p.T38M. Our experimental data indicate that protein misfolding and accumulation in the ER affect function of the L1CAM variant p.I37N whereas the variants p.M172I and p.D202Y impair homophilic interaction at the cell surface.

Objectives:The gamma-aminobutyric acid modulator propofol induces neuronal cell death in healthy immature brains by unbalancing neurotrophin homeostasis via p75 neurotrophin receptor signaling. In adulthood, p75 neurotrophin receptor becomes down-regulated and propofol loses its neurotoxic effect. H

Clinical and animal studies have revealed sex-specific differences in histopathological and neurological outcome after traumatic brain injury (TBI). The impact of perioperative administration of sex steroid inhibitors on TBI is still elusive. Here, we subjected male and female C57Bl/6N mice to the controlled cortical impact (CCI) model of TBI and applied pharmacological inhibitors of steroid hormone synthesis, that is, letrozole (LET, inhibiting estradiol synthesis by aromatase) and finasteride (FIN, inhibiting dihydrotestosterone synthesis by 5α-reductase), respectively, starting 72 h prior CCI, and continuing for a further 48 h after CCI. Initial gene expression analyses showed that androgen (Ar) and estrogen receptors (Esr1) were sex-specifically altered 72 h after CCI. When examining brain lesion size, we found larger lesions in male than in female mice, but did not observe effects of FIN or LET treatment. However, LET treatment exacerbated neurological deficits 24 and 72 h after CCI. On the molecular level, FIN administration reduced calpain-dependent spectrin breakdown products, a proxy of excitotoxicity and disturbed Ca2+ homeostasis, specifically in males, whereas LET increased the reactive astrocyte marker glial fibrillary acid protein specifically in females. Examination of neurotrophins (brain-derived neurotrophic factor, neuronal growth factor, NT-3) and their receptors (p75NTR , TrkA, TrkB, TrkC) revealed CCI-induced down-regulation of TrkB and TrkC protein expression, which was reduced by LET in both sexes. Interestingly, FIN decreased neuronal growth factor mRNA expression and protein levels of its receptor TrkA only in males. Taken together, our data suggest a sex-specific impact on pathogenic processes in the injured brain after TBI. Sex hormones may thus modulate pathogenic processes in experimental TBI.

Neuronal growth regulator 1 (NEGR1), a member of the immunoglobulin superfamily cell adhesion molecule subgroup IgLON, has been implicated in neuronal growth and connectivity. In addition, genetic variants in or near the NEGR1 locus have been associated with obesity and more recently with learning difficulties, intellectual disability and psychiatric disorders. However, experimental evidence is lacking to support a possible link between NEGR1, neuronal growth and behavioral abnormalities. Initial expression analysis of NEGR1 mRNA in C57Bl/6 wildtype (WT) mice by in situ hybridization demonstrated marked expression in the entorhinal cortex (EC) and dentate granule cells. In co-cultures of cortical neurons and NSC-34 cells overexpressing NEGR1, neurite growth of cortical neurons was enhanced and distal axons occupied an increased area of cells overexpressing NEGR1. Conversely, in organotypic slice co-cultures, Negr1-knockout (KO) hippocampus was less permissive for axons grown from EC of β-actin-enhanced green fluorescent protein (EGFP) mice compared to WT hippocampus. Neuroanatomical analysis revealed abnormalities of EC axons in the hippocampal dentate gyrus (DG) of Negr1-KO mice including increased numbers of axonal projections to the hilus. Neurotransmitter receptor ligand binding densities, a proxy of functional neurotransmitter receptor abundance, did not show differences in the DG of Negr1-KO mice but altered ligand binding densities to NMDA receptor and muscarinic acetylcholine receptors M1 and M2 were found in CA1 and CA3. Activity behavior, anxiety-like behavior and sensorimotor gating were not different between genotypes. However, Negr1-KO mice exhibited impaired social behavior compared to WT littermates. Moreover, Negr1-KO mice showed reversal learning deficits in the Morris water maze and increased susceptibility to pentylenetetrazol (PTZ)-induced seizures. Thus, our results from neuronal growth assays, neuroanatomical analyses and behavioral assessments provide first evidence that deficiency of the psychiatric disease-associated Negr1 gene may affect neuronal growth and behavior. These findings might be relevant to further evaluate the role of NEGR1 in cognitive and psychiatric disorders.

See Contreras and Hippenmeyer (doi:10.1093/brain/awy218) for a scientific commentary on this article. Autism spectrum disorders (ASDs) are complex conditions with diverse aetiologies. Szczurkowska et al. demonstrate that two ASD-related molecules – FGFR2 and Negr1 – physically interact to act on the same downstream pathway, and regulate cortical development and ASD-relevant behaviours in mice. Identifying common mechanisms in ASDs may reveal targets for pharmacological intervention., Autism spectrum disorders are neurodevelopmental conditions with diverse aetiologies, all characterized by common core symptoms such as impaired social skills and communication, as well as repetitive behaviour. Cell adhesion molecules, receptor tyrosine kinases and associated downstream signalling have been strongly implicated in both neurodevelopment and autism spectrum disorders. We found that downregulation of the cell adhesion molecule NEGR1 or the receptor tyrosine kinase fibroblast growth factor receptor 2 (FGFR2) similarly affects neuronal migration and spine density during mouse cortical development in vivo and results in impaired core behaviours related to autism spectrum disorders. Mechanistically, NEGR1 physically interacts with FGFR2 and modulates FGFR2-dependent extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) signalling by decreasing FGFR2 degradation from the plasma membrane. Accordingly, FGFR2 overexpression rescues all defects due to Negr1 knockdown in vivo. Negr1 knockout mice present phenotypes similar to Negr1-downregulated animals. These data indicate that NEGR1 and FGFR2 cooperatively regulate cortical development and suggest a role for defective NEGR1-FGFR2 complex and convergent downstream ERK and AKT signalling in autism spectrum disorders.

After stroke, secondary brain damage is influenced by the extent of fibrin clot formation. This is counteracted by the endogenous fibrinolysis. Of major interest are the key players of the fibrinolytic plasminogen activator system including the urokinase plasminogen activator (uPA), the tissue-type plasminogen activator (tPA), and their endogenous inhibitors plasminogen activator inhibitor 1 (PAI-1) and PAI-2. The role of PAI-1 in brain injury is well established, whereas the importance of PAI-2 is unknown at present. The objectives of the present were twofold: first, to characterize the time-dependent cerebral mRNA expression of the plasminogen activator system (PAS) after brain ischemia and second, to investigate the impact of PAI-1 and PAI-2 on brain infarct volume using gene-deficient mice. Adult C57Bl/6J mice were subjected to unilateral transient middle cerebral artery occlusion (MCAO) followed by reperfusion for 3, 24, 72, or 120 h. Quantitative PCR revealed that brain mRNA expression levels of the PAS components, and particularly of PAI-1 (237-fold) and PAI-2 (19-fold), peaked at 24 h after stroke. Accordingly, PAI-1 plasma activity was strongly increased. Brain infarct volume in TTC (2,3,5-triphenyltetrazolium chloride)-stained brain sections was significantly smaller 24 h after MCAO in PAI-1-deficient mice (− 31%), but not in PAI-2-deficient mice (− 6%). Thus, endogenous upregulation of PAI-1, but not of PAI-2, might contribute to increased brain damage after acute ischemic stroke. The present study therefore shows that PAI-2 is induced by brain ischemia, but does not play an important or relevant role for secondary brain damage after brain injury.

Traumatic injury of the central nervous system is one of the leading causes of death and disability in young adults. Failure of regeneration is caused by autonomous neuronal obstacles and by formation of the glial scar, which is essential to seal the injury but also constitutes a barrier for regrowing axons. The scar center is highly inflammatory and populated by NG2+ glia, whereas astrocytes form the sealing border and trap regrowing axons, suggesting that the non-permissive environment of activated astrocytes and extracellular matrix components is one of the reasons for the regenerative failure. Particularly, secreted chondroitin-sulfate proteoglycans, CSPGs, of the lectican family hinder axonal regrowth. In contrast, the transmembrane CSPG, NG2/CSPG4, appears to be functionally closer related to axon growth permissive heparan sulfate proteoglycans, HSPGs, and synaptic adhesion molecules, which all regulate synaptic signaling and plasticity upon alpha-secretase mediated shedding. Consequently, knockout of NG2/CSPG4 aggravates tissue loss, inflammation and neurologic deficits after brain injury, a phenotype partly mimicked by deletion of HSPG-binding proteins such as the HSPG2/perlecan-interacting protein, progranulin that is also a functional ligand of Notch and Eph2a. Indeed, structural features or progranulin's targets and NG2 may point to direct reciprocal regulations that may act in concert to overcome injury-evoked inflammation and neuronal dystrophy. This review provides an overview of the pathophysiology of the glial scar after brain injury, with a specific focus on NG2/CSPG4, its functions before and after shedding and putative reciprocal influences with the glycoprotein progranulin.

In multiple sclerosis (MS), the immune cell attack leads to axonal injury as a major cause for neurological disability. Here, we report a novel role of the cell adhesion molecule L1 in the crosstalk between the immune and nervous systems. L1 was found to be expressed by CNS axons of MS patients and human T cells. In MOG35–55-induced murine experimental neuroinflammation, CD4+ T cells were associated with degenerating axons in the spinal cord, both expressing L1. However, neuronal L1 expression in the spinal cord was reduced, while levels of the transcriptional repressor REST (RE1-Silencing Transcription Factor) were up-regulated. In PLP139–151-induced relapsing–remitting neuroinflammation, L1 expression was low at the peak stage of disease, reached almost normal levels in the remission stage, but decreased again during disease relapse indicating adaptive expression regulation of L1. In vitro, activated CD4+ T cells caused contact-dependent down-regulation of L1, up-regulation of its repressor REST and axonal injury in co-cultured neurons. T cell adhesion to neurons and axonal injury were prevented by an antibody blocking L1 suggesting that down-regulation of L1 ameliorates neuroinflammation. In support of this hypothesis, antibody-mediated blocking of L1 in C57BL/6 mice as well as neuron-specific depletion of L1 in synapsinCre × L1fl/fl mice reduces disease severity and axonal pathology despite unchanged immune cell infiltration of the CNS. Our data suggest that down-regulation of neuronal L1 expression is an adaptive process of neuronal self-defense in response to pro-inflammatory T cells, thereby alleviating immune-mediated axonal injury.

International audience; Mutations in the gene encoding the neural cell adhesion molecule L1CAM cause several neurological disorders collectively referred to as L1 syndrome. We report here a family case of X-linked hydrocephalus in which an obligate female carrier has two exonic L1CAM missense mutations in trans substituting amino acids in the first (p.W635C) or second (p.V768I) fibronectin-type III domains. We performed various biochemical and cell biological in vitro assays to evaluate the pathogenicity of these variants. Mutant L1-W635C protein accumulates in the endoplasmic reticulum (ER), is not transported into axons, and fails to promote L1CAM-mediated cell-cell adhesion as well as neurite growth. Immunoprecipitation experiments show that L1-W635C associates with the molecular ER chaperone calnexin and is modified by poly-ubiquitination. The mutant L1-V768I protein localizes at the cell surface, is not retained in the ER, and promotes neurite growth similar to wild-type L1CAM. However, the p.V768I mutation impairs L1CAM-mediated cell-cell adhesion albeit less severe than L1-W635C. These data indicate that p.W635C is a novel loss-of-function L1 syndrome mutation. The p.V768I mutation may represent a non-pathogenic variant or a variant associated with low penetrance. The poly-ubiquitination of L1-W635C and its association with the ER chaperone calnexin provide further insights into the molecular mechanisms underlying defective cell surface trafficking of L1CAM in L1 syndrome.

Painful diabetic neuropathy is associated with impaired opioid analgesia; however, the precise mechanism in sensory neurons remains unclear. This study aimed to identify putative mechanisms involved in modified opioid responsiveness during early streptozotocin-induced diabetes in rats. In this study, we demonstrate that in diabetic animals, impaired peripheral opioid analgesia is associated with a reduction in functional mu-opioid receptor (MOR) G protein coupling. Mu-opioid receptor immunoreactive neurons colocalized with activated forms of protein kinase C (PKC) and with the receptor for advanced glycation end products (RAGE) during streptozotocin-induced diabetes. Moreover, MOR phosphorylation at Thr370 in sensory neurons of diabetic rats, and thus desensitization, was due to RAGE-dependent PKC activation. Importantly, blocking PKC activation using PKC selective inhibitor, silencing RAGE with intrathecal RAGE siRNA, or inhibiting advanced glycation end product (AGE) formation prevented sensory neuron MOR phosphorylation and, consequently, restored MOR G protein coupling and analgesic efficacy. Thus, our findings give the first in vivo evidence of a RAGE-dependent PKC-mediated heterologous MOR phosphorylation and desensitization in sensory neurons under pathological conditions such as diabetic neuropathy. This may unravel putative mechanisms and suggest possible prevention strategies of impaired opioid responsiveness.

To date, genome-wide association studies (GWAS) have identified at least 32 novel loci for obesity and body mass-related traits. However, the causal genetic variant and molecular mechanisms of specific susceptibility genes in relation to obesity are yet to be fully confirmed and characterised. Here, we examined whether the candidate gene NEGR1 encoding the neuronal growth regulator 1, also termed neurotractin or Kilon, accounts for the obesity association. To characterise the function of NEGR1 for body weight control in vivo, we generated two novel mutant mouse lines, including a constitutive NEGR1-deficient mouse line as well as an ENU-mutagenised line carrying a loss-of-function mutation (Negr1-I87N) and performed metabolic phenotypic analyses. Ablation of NEGR1 results in a small but steady reduction of body mass in both mutant lines, accompanied with a small reduction in body length in the Negr1-I87N mutants. Magnetic resonance scanning reveals that the reduction of body mass in Negr1-I87N mice is due to a reduced proportion of lean mass. Negr1-I87N mutants display reduced food intake and physical activity while normalised energy expenditure remains unchanged. Expression analyses confirmed the brain-specific distribution of NEGR1 including strong expression in the hypothalamus. In vitro assays show that NEGR1 promotes cell-cell adhesion and neurite growth of hypothalamic neurons. Our results indicate a role of NEGR1 in the control of body weight and food intake. This study provides evidence that supports the link of the GWAS candidate gene NEGR1 with body weight control.

International audience; OBJECTIVE: Recessive mutations in alsin, a guanine-nucleotide exchange factor for the GTPases Rab5 and Rac1, cause juvenile amyotrophic lateral sclerosis (ALS2) and related motoneuron disorders. Alsin function in motoneurons remained unclear because alsin knock-out mice do not develop overt signs of motoneuron degeneration. METHODS: To generate an alsin loss-of-function model in an ALS-relevant cell type, we developed a new small interfering RNA electroporation technique that allows efficient knock down of alsin in embryonic rat spinal motoneurons. RESULTS: After small interfering RNA-mediated alsin knockdown, cultured motoneurons displayed a reduced apparent size of EEA1-labeled early endosomes and an increased intracellular accumulation of transferrin and L1CAM. Alsin knockdown induced cell death in 32 to 48% of motoneurons and significantly inhibited axon growth in the surviving neurons. Both cellular phenotypes were mimicked by expression of a dominant-negative Rac1 mutant and were completely blocked by expression of a constitutively active Rac1 mutant. Expression of dominant-negative or constitutively active forms of Rab5 had no such effects. INTERPRETATION: Our data demonstrate that alsin controls the growth and survival of motoneurons in a Rac1-dependant manner. The strategy reported here illustrates how small interfering RNA electroporation can be used to generate cellular models of neurodegenerative disease involving a loss-of-function mechanism.

Tumour-specific splicing is known to contribute to cancer progression. In the case of the L1 cell adhesion molecule (L1CAM), which is expressed in many human tumours and often linked to bad prognosis, alternative splicing results in a full-length form (FL-L1CAM) and a splice variant lacking exons 2 and 27 (SV-L1CAM). It has not been elucidated so far whether SV-L1CAM, classically considered as tumour-associated, or whether FL-L1CAM is the metastasis-promoting isoform. Here, we show that both variants were expressed in human ovarian carcinoma and that exposure of tumour cells to pro-metastatic factors led to an exclusive increase of FL-L1CAM expression. Selective overexpression of one isoform in different tumour cells revealed that only FL-L1CAM promoted experimental lung and/or liver metastasis in mice. In addition, metastasis formation upon up-regulation of FL-L1CAM correlated with increased invasive potential and elevated Matrix metalloproteinase (MMP)-2 and -9 expression and activity in vitro as well as enhanced gelatinolytic activity in vivo. In conclusion, we identified FL-L1CAM as the metastasis-promoting isoform, thereby exemplifying that high expression of a so-called tumour-associated variant, here SV-L1CAM, is not per se equivalent to a decisive role of this isoform in tumour progression.