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The article presents scientific evidences that notes the deregulated expression of enhancer of zeste homolog 2 (EZH2) gene in cases of congenital brainstem disconnection (CBSD), an embryo-fetal defect focusing on the role of Polycomb Repressive Complex 2 (PRC2) as a promotor gene. Topics include interference with developing rhombencephalon, cerebral pathological analysis of patients with CBSD, and role of EZH2 in development of nervous system.

Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited peripheral neuropathy caused by a 1.5 Mb tandem duplication of chromosome 17 harbouring the PMP22 gene. This dose-dependent overexpression of PMP22 results in disrupted Schwann cell myelination of peripheral nerves. To obtain better insights into the underlying pathogenic mechanisms in CMT1A, we investigated the role of PMP22 duplication in cellular homeostasis in CMT1A mouse models and in patient-derived induced pluripotent stem cells differentiated into Schwann cell precursors (iPSC-SCPs). We performed lipidomic profiling and bulk RNA sequencing (RNA-seq) on sciatic nerves of two developing CMT1A mouse models and on CMT1A patient-derived iPSC-SCPs. For the sciatic nerves of the CMT1A mice, cholesterol and lipid metabolism was downregulated in a dose-dependent manner throughout development. For the CMT1A iPSC-SCPs, transcriptional analysis unveiled a strong suppression of genes related to autophagy and lipid metabolism. Gene ontology enrichment analysis identified disturbances in pathways related to plasma membrane components and cell receptor signalling. Lipidomic analysis confirmed the severe dysregulation in plasma membrane lipids, particularly sphingolipids, in CMT1A iPSC-SCPs. Furthermore, we identified reduced lipid raft dynamics, disturbed plasma membrane fluidity and impaired cholesterol incorporation and storage, all of which could result from altered lipid storage homeostasis in the patient-derived CMT1A iPSC-SCPs. Importantly, this phenotype could be rescued by stimulating autophagy and lipolysis. We conclude that PMP22 duplication disturbs intracellular lipid storage and leads to a more disordered plasma membrane owing to an alteration in the lipid composition, which might ultimately lead to impaired axo-glial interactions. Moreover, targeting lipid handling and metabolism could hold promise for the treatment of patients with CMT1A., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Introduction: Obesity has been linked to vascular dysfunction, cognitive impairment and neurodegenerative diseases. However, experimental models that recapitulate brain pathology in relation to obesity and vascular dysfunction are still lacking., Methods: In this study we performed the histological and histochemical characterization of brains from Ldlr-/-.Leiden mice, an established model for obesity and associated vascular disease. First, HFD-fed 18 week-old and 50 week-old Ldlr-/-.Leiden male mice were compared with age-matched C57BL/6J mice. We then assessed the effect of high-fat diet (HFD)-induced obesity on brain pathology in Ldlr-/-.Leiden mice and tested whether a treatment with an anti-complement component 5 antibody, a terminal complement pathway inhibitor recently shown to reduce vascular disease, can attenuate neurodegeneration and neuroinflammation. Histological analyses were complemented with Next Generation Sequencing (NGS) analyses of the hippocampus to unravel molecular pathways underlying brain histopathology., Results: We show that chow-fed Ldlr-/-.Leiden mice have more severe neurodegeneration and show an age-dependent astrogliosis that is not observed in age-matched C57BL/6J controls. This was substantiated by pathway enrichment analysis using the NGS data which showed that oxidative phosphorylation, EIF2 signaling and mitochondrial dysfunction pathways, all associated with neurodegeneration, were significantly altered in the hippocampus of Ldlr-/-.Leiden mice compared with C57BL/6J controls. Obesity-inducing HFD-feeding did not aggravate neurodegeneration and astrogliosis in Ldlr-/-.Leiden mice. However, brains from HFD-fed Ldlr-/-.Leiden mice showed reduced IBA-1 immunoreactivity and increased CD68 immunoreactivity compared with chow-fed Ldlr-/-.Leiden mice, indicating alteration of microglial immunophenotype by HFD feeding. The systemic administration of an anti-C5 treatment partially restored the HFD effect on microglial immunophenotype. In addition, NGS data of hippocampi from Ldlr-/-.Leiden mice showed that HFD feeding affected multiple molecular pathways relative to chow-fed controls: HFD notably inactivated synaptogenesis and activated neuroinflammation pathways. The anti-C5 treatment restored the HFD-induced effect on molecular pathways to a large extent., Conclusion: This study shows that the Ldlr-/-.Leiden mouse model is suitable to study brain histopathology and associated biological processes in a context of obesity and provides evidence of the potential therapeutic value of anti-complement therapy against obesity-induced neuroinflammation., 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 © 2023 Seidel, Fluiter, Kleemann, Worms, van Nieuwkoop, Caspers, Grigoriadis, Kiliaan, Baas, Michailidou and Morrison.)

Charcot-Marie-Tooth disease type 1A (CMT1A) is the most prevalent hereditary demyelinating neuropathy. This autosomal, dominantly inherited disease is caused by a duplication on chromosome 17p which includes the peripheral myelin protein 22 (PMP22) gene. There is clinical evidence that the disability in CMT1A is to a large extend due to axonal damage rather than demyelination. Over-expression of PMP22 is recently thought to impede cholesterol trafficking causing a total shutdown of local cholesterol and lipid synthesis in the Schwann cells, thus disturbing their ability to remyelinate. But there is a large variety in disease burden between CMT1A patients with the same genetic defect, indicating the presence of modifying factors that affect disease severity. One of these potential factors is the immune system. Several reports have described patients with co-occurrence of CMT1A with chronic inflammatory demyelinating disease or Guillain-Barré syndrome. We have previously shown in multiple animal models that the innate immune system and specifically the terminal complement system is a driver of inflammatory demyelination. To test the contribution of the terminal complement system to neuroinflammation and disease progression in CMT1A, we inhibited systemic complement C6 in two transgenic mouse models for CMT1A, the C3- PMP22 and C3- PMP22 c-JunP0Cre models. Both models over-express human PMP22 , and one (C3- PMP22 c-JunP0Cre) also has a Schwann cell-specific knockout of c-Jun, a crucial regulator of myelination controlling autophagy. We found that systemic inhibition of C6 using antisense oligonucleotides affects the neuroinflammation, Rho GTPase and ERK/MAPK signalling pathways in the CMT1A mouse models. The cholesterol synthesis pathway remained unaffected. Analysis of motor function during treatment with C6 antisense oligonucleotides did not reveal any significant improvement in the CMT1A mouse models. This study shows that the contribution of the terminal complement system to progressive loss of motor function in the CMT1A mouse models tested is limited., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Frank Baas reports a relationship with Complement Pharma BV that includes: board membership. Kees Fluiter reports a relationship with Complement Pharma BV that includes: consulting or advisory. Maarten Loos reports a relationship with Synaptologics BV that includes: employment. Bastijn Koopmans reports a relationship with Synaptologics BV that includes: employment. Nina Vlek reports a relationship with Synaptologics BV that includes: employment. Nina Straat reports a relationship with Synaptologics BV that includes: employment. Frank Baas has patent Complement inhibition for improved nerve regeneration issued to Regenesance BV. Frank Baas has patent COMPLEMENT ANTAGONISTS AND USES THEREOF issued to Regenesance BV. Kees Fluiter has patent COMPLEMENT ANTAGONISTS AND USES THEREOF issued to Regenesance BV. Frank Baas has patent #ANTIBODIES THAT BIND HUMAN C6 AND USES THEREOF issued to Regenesance BV., (© 2023 The Authors.)

Traumatic brain injury (TBI) is a leading cause of mortality, sensorimotor morbidity, and neurocognitive disability. Neuroinflammation is one of the key drivers causing secondary brain injury after TBI. Therefore, attenuation of the inflammatory response is a potential therapeutic goal. This review summarizes the most important neuroinflammatory pathophysiology resulting from TBI and the clinical trials performed to attenuate neuroinflammation. Studies show that non-selective attenuation of the inflammatory response, in the early phase after TBI, might be detrimental and that there is a gap in the literature regarding pharmacological trials targeting specific pathways. The complement system and its crosstalk with the coagulation system play an important role in the pathophysiology of secondary brain injury after TBI. Therefore, regaining control over the complement cascades by inhibiting overshooting activation might constitute useful therapy. Activation of the complement cascade is an early component of neuroinflammation, making it a potential target to mitigate neuroinflammation in TBI. Therefore, we have described pathophysiological aspects of complement inhibition and summarized animal studies targeting the complement system in TBI. We also present the first clinical trial aimed at inhibition of complement activation in the early days after brain injury to reduce the risk of morbidity and mortality following severe TBI., (© 2022. The Author(s).)

Damage and disease of nerves activates the complement system. We demonstrated that activation of the terminal pathway of the complement system leads to the formation of the membrane attack complex (MAC) and delays regeneration in the peripheral nervous system. Animals deficient in the complement component C6 showed improved recovery after neuronal trauma. Thus, inhibitors of the MAC might be of therapeutic use in neurological disease. Here, we describe the development, structure, mode of action, and properties of a novel therapeutic monoclonal antibody, CP010, against C6 that prevents formation of the MAC in vivo. The monoclonal antibody is humanized and specific for C6 and binds to an epitope in the FIM1-2 domain of human and primate C6 with sub-nanomolar affinity. Using biophysical and structural studies, we show that the anti-C6 antibody prevents the interaction between C6 and C5/C5b by blocking the C6 FIM1-2:C5 C345c axis. Systemic administration of the anti-C6 mAb caused complete depletion of free C6 in circulation in transgenic rats expressing human C6 and thereby inhibited MAC formation. The antibody prevented disease in experimental autoimmune myasthenia gravis and ameliorated relapse in chronic relapsing experimental autoimmune encephalomyelitis in human C6 transgenic rats. CP010 is a promising complement C6 inhibitor that prevents MAC formation. Systemic administration of this C6 monoclonal antibody has therapeutic potential in the treatment of neuronal disease., (© 2022 The Author(s). Published by S. Karger AG, Basel.)

Background: Chronic inflammation is an important driver in the progression of non-alcoholic steatohepatitis (NASH) and atherosclerosis. The complement system, one of the first lines of defense in innate immunity, has been implicated in both diseases. However, the potential therapeutic value of complement inhibition in the ongoing disease remains unclear., Methods: After 20 weeks of high-fat diet (HFD) feeding, obese Ldlr-/-.Leiden mice were treated twice a week with an established anti-C5 antibody (BB5.1) or vehicle control. A separate group of mice was kept on a chow diet as a healthy reference. After 12 weeks of treatment, NASH was analyzed histopathologically, and genome-wide hepatic gene expression was analyzed by next-generation sequencing and pathway analysis. Atherosclerotic lesion area and severity were quantified histopathologically in the aortic roots., Results: Anti-C5 treatment considerably reduced complement system activity in plasma and MAC deposition in the liver but did not affect NASH. Anti-C5 did, however, reduce the development of atherosclerosis, limiting the total lesion size and severity independently of an effect on plasma cholesterol but with reductions in oxidized LDL (oxLDL) and macrophage migration inhibitory factor (MIF)., Conclusion: We show, for the first time, that treatment with an anti-C5 antibody in advanced stages of NASH is not sufficient to reduce the disease, while therapeutic intervention against established atherosclerosis is beneficial to limit further progression.

Background: Traumatic brain injury (TBI) is a major cause of death and disability across all ages. After the primary impact, the pathophysiologic process of secondary brain injury consists of a neuroinflammation response that critically leads to irreversible brain damage in the first days after the trauma. A key catalyst in this inflammatory process is the complement system. Inhibiting the complement system could therefore be a therapeutic target in TBI., Objective: To study the safety and efficacy of C1-inhibitor (C1-INH) compared to placebo in patients with TBI. By temporarily blocking the complement system, we hypothesize a decrease in the posttraumatic neuroinflammatory response resulting in a less unfavorable clinical outcome for TBI patients., Methods: CIAO@TBI is a multicenter, randomized, blinded, phase II placebo-controlled trial. Adult TBI patients with GCS < 13 requiring intracranial pressure (ICP) monitoring will be randomized, using block randomization, within 12 h after trauma to one dose 6000 IU C1-INH or placebo. A total of 106 patients will be included, and follow-up will occur up to 12 months. The primary endpoints are (1) Therapy Intensity Level (TIL) Scale, (2) Glasgow Outcome Scale-Extended (GOSE) at 6 months, and (3) complication rate during hospitalization. Outcomes will be determined by a trial nurse blinded for the treatment allocation. Analyses will be conducted in an intention-to-treat analysis., Discussion: We expect that C1-INH administration will be safe and potentially effective to improve clinical outcomes by reducing neuroinflammation in TBI patients., Trial Registration: ClinicalTrials.gov NCT04489160. Registered on 27 July 2020. EudraCT 2020-000140-58., (© 2021. The Author(s).)

The complement system is a key driver of neuroinflammation. Activation of complement by all pathways, results in the formation of the anaphylatoxin C5a and the membrane attack complex (MAC). Both initiate pro-inflammatory responses which can contribute to neurological disease. In this study, we delineate the specific roles of C5a receptor signaling and MAC formation during the progression of experimental autoimmune encephalomyelitis (EAE)-mediated neuroinflammation. MAC inhibition was achieved by subcutaneous administration of an antisense oligonucleotide specifically targeting murine C6 mRNA (5 mg/kg). The C5a receptor 1 (C5aR1) was inhibited with the C5a receptor antagonist PMX205 (1.5 mg/kg). Both treatments were administered systemically and started after disease onset, at the symptomatic phase when lymphocytes are activated. We found that antisense-mediated knockdown of C6 expression outside the central nervous system prevented relapse of disease by impeding the activation of parenchymal neuroinflammatory responses, including the Nod-like receptor protein 3 (NLRP3) inflammasome. Furthermore, C6 antisense-mediated MAC inhibition protected from relapse-induced axonal and synaptic damage. In contrast, inhibition of C5aR1-mediated inflammation diminished expression of major pro-inflammatory mediators, but unlike C6 inhibition, it did not stop progression of neurological disability completely. Our study suggests that MAC is a key driver of neuroinflammation in this model, thereby MAC inhibition might be a relevant treatment for chronic neuroinflammatory diseases.

Background: MicroRNAs (miRNAs) are epigenetically involved in regulating gene expression. They may be of importance in the pathogenesis of inflammatory bowel disease (IBD). The aim of this study was to determine the role of miRNAs by their specific blocking in the CD4+CB45RBhi T-cell transfer model of chronic experimental colitis., Methods: Colitis caused by transfer of WT CD4+CD45RBhi T cells in severe combined immunodeficiency (SCID) mice shares many features with human IBD. Colonic miRNA expression levels were measured at three time points in colitic mice, where a time-dependent upregulation of multiple miRNAs was seen. To inhibit these miRNAs, specific locked-nucleic-acid-modified (LNA) oligonucleotides were administered in further experiments at the moment the mice demonstrated the first signs of colitis. As controls, PBS and a scrambled sequence of anti-miRNA were used. Genome-wide expression analyses were also performed in order to detect candidate target genes of miR-142-5p, of which inhibition resulted in most effective amelioration of colitis., Results: Anti-miR-142-5p reduced colitis and related wasting disease when administered in the T-cell transfer model, reflected in reduced weight loss and a lower disease activity index (DAI). In further validation experiments we also observed a higher survival rate and less colonic histological inflammation in the antagomir-treated mice. Moreover, by genome-wide expression analyses, we found downstream activation of the anti-inflammatory IL10RA pathway, including three genes also found in the top-20 candidate target genes of miR-142-5p., Conclusion: In conclusion, CD4+CD45RBhi-transfer colitis induces miR-142-5p. Blocking miR-142-5p reduced colitis and prevented wasting disease, possibly by activation of the IL10RA pathway.

Peripheral nerve damage is the hallmark of leprosy pathology but its etiology is unclear. We previously identified the membrane attack complex (MAC) of the complement system as a key determinant of post-traumatic nerve damage and demonstrated that its inhibition is neuroprotective. Here, we determined the contribution of the MAC to nerve damage caused by Mycobacterium leprae and its components in mouse. Furthermore, we studied the association between MAC and the key M. leprae component lipoarabinomannan (LAM) in nerve biopsies of leprosy patients. Intraneural injections of M. leprae sonicate induced MAC deposition and pathological changes in the mouse nerve, whereas MAC inhibition preserved myelin and axons. Complement activation occurred mainly via the lectin pathway and the principal activator was LAM. In leprosy nerves, the extent of LAM and MAC immunoreactivity was robust and significantly higher in multibacillary compared to paucibacillary donors (p = 0.01 and p = 0.001, respectively), with a highly significant association between LAM and MAC in the diseased samples (r = 0.9601, p = 0.0001). Further, MAC co-localized with LAM on axons, pointing to a role for this M. leprae antigen in complement activation and nerve damage in leprosy. Our findings demonstrate that MAC contributes to nerve damage in a model of M. leprae-induced nerve injury and its inhibition is neuroprotective. In addition, our data identified LAM as the key pathogen associated molecule that activates complement and causes nerve damage. Taken together our data imply an important role of complement in nerve damage in leprosy and may inform the development of novel therapeutics for patients.

Objective: Mesial temporal lobe epilepsy (MTLE) is one of the most common types of the intractable epilepsies and is most often associated with hippocampal sclerosis (HS), which is characterized by pronounced loss of hippocampal pyramidal neurons. microRNAs (miRNAs) have been shown to be dysregulated in epilepsy and neurodegenerative diseases, and we hypothesized that miRNAs could be involved in the pathogenesis of MTLE and HS., Methods: miRNA expression was quantified in hippocampal specimens from human patients using miRNA microarray and quantitative real-time polymerase chain reaction RT-PCR, and by RNA-seq on fetal brain specimens from domestic pigs. In situ hybridization was used to show the spatial distribution of miRNAs in the human hippocampus. The potential effect of miRNAs on targets genes was investigated using the dual luciferase reporter gene assay., Results: miRNA expression profiling showed that 25 miRNAs were up-regulated and 5 were down-regulated in hippocampus biopsies of MTLE/HS patients compared to controls. We showed that miR-204 and miR-218 were significantly down-regulated in MTLE and HS, and both were expressed in neurons in all subfields of normal hippocampus. Moreover, miR-204 and miR-218 showed strong changes in expression during fetal development of the hippocampus in pigs, and we identified four target genes, involved in axonal guidance and synaptic plasticity, ROBO1, GRM1, SLC1A2, and GNAI2, as bona fide targets of miR-218. GRM1 was also shown to be a direct target of miR-204., Significance: miR-204 and miR-218 are developmentally regulated in the hippocampus and may contribute to the molecular mechanisms underlying the pathogenesis of MTLE and HS., (Wiley Periodicals, Inc. © 2014 International League Against Epilepsy.)

Aims: The overloaded heart remodels by cardiomyocyte hypertrophy and interstitial fibrosis, which contributes to the development of heart failure. Signalling via the TGFβ-pathway is crucial for this remodelling. Here we tested the hypothesis that microRNAs in the overloaded heart regulate this remodelling process via inhibition of the TGFβ-pathway., Methods and Results: We show that the miRNA-15 family, which we found to be up-regulated in the overloaded heart in multiple species, inhibits the TGFβ-pathway by targeting of TGFBR1 and several other genes within this pathway directly or indirectly, including p38, SMAD3, SMAD7, and endoglin. Inhibition of miR-15b by subcutaneous injections of LNA-based antimiRs in C57BL/6 mice subjected to transverse aorta constriction aggravated fibrosis and to a lesser extent also hypertrophy., Conclusion: We identified the miR-15 family as a novel regulator of cardiac hypertrophy and fibrosis acting by inhibition of the TGFβ-pathway., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)