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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.)

Purpose: To describe a recessively inherited cerebral small vessel disease, caused by loss-of-function variants in Nitrilase1 (NIT1)., Methods: We performed exome sequencing, brain magnetic resonance imaging, neuropathology, electron microscopy, western blotting, and transcriptomic and metabolic analyses in 7 NIT1-small vessel disease patients from 5 unrelated pedigrees., Results: The first identified patients were 3 siblings, compound heterozygous for the NIT1 c.727C>T; (p.Arg243Trp) variant and the NIT1 c.198_199del; p.(Ala68∗) variant. The 4 additional patients were single cases from 4 unrelated pedigrees and were all homozygous for the NIT1 c.727C>T; p.(Arg243Trp) variant. Patients presented in mid-adulthood with movement disorders. All patients had striking abnormalities on brain magnetic resonance imaging, with numerous and massively dilated basal ganglia perivascular spaces. Three patients had non-lobar intracerebral hemorrhage between age 45 and 60, which was fatal in 2 cases. Western blotting on patient fibroblasts showed absence of NIT1 protein, and metabolic analysis in urine confirmed loss of NIT1 enzymatic function. Brain autopsy revealed large electron-dense deposits in the vessel walls of small and medium sized cerebral arteries., Conclusion: NIT1-small vessel disease is a novel, autosomal recessively inherited cerebral small vessel disease characterized by a triad of movement disorders, massively dilated basal ganglia perivascular spaces, and intracerebral hemorrhage., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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.)

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.)

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.

Changes in the expression and activation status of Ras proteins are thought to contribute to the pathological phenotype of stromal fibroblast-like synoviocytes (FLS) in rheumatoid arthritis, a prototypical immune-mediated inflammatory disease. Broad inhibition of Ras and related proteins has shown protective effects in animal models of arthritis, but each of the Ras family homologues (ie, H-, K-, and N-Ras) makes distinct contributions to cellular activation. We examined the expression of each Ras protein in synovial tissue and FLS obtained from patients with rheumatoid arthritis and other forms of inflammatory arthritis. Each Ras protein was expressed in synovial tissue and cultured FLS. Each homolog was also activated following FLS stimulation with tumor necrosis factor-α or interleukin (IL)-1β. Constitutively active mutants of each Ras protein enhanced IL-1β-induced FLS matrix metalloproteinase-3 production, while only active H-Ras enhanced IL-8 production. Gene silencing demonstrated that each Ras protein contributed to IL-1β-dependent IL-6 production, while H-Ras and N-Ras supported IL-1β-dependent matrix metalloproteinase-3 and IL-8 production, respectively. The overlap in contributions of Ras homologues to FLS activation suggests that broad targeting of Ras GTPases in vivo suppresses global inflammation and joint destruction in arthritis. Consistent with this, simultaneous silencing of H-Ras, K-Ras, and N-Ras expression significantly reduces inflammation and joint destruction in murine collagen-induced arthritis, while specific targeting of N-Ras alone is less effective in providing clinical benefits.

The clinical use of small interfering RNA (siRNA) is hampered by poor uptake by tissues and instability in circulation. In addition, off-target effects pose a significant additional problem for therapeutic use of siRNA. Chemical modifications of siRNA have been reported to increase stability and reduce off-target effects enabling possible therapeutic use of siRNA. Recently a large scale direct comparison of the impact of 21 different types of novel chemical modifications on siRNA efficiency and cell viability was published.1 It was found that several types of chemical modifications could enhance siRNA activity beyond that of an unmodified siRNA in vitro. In addition, a novel siRNA design, termed small internally segmented interfering RNA (sisiRNA), composed of an intact antisense strand and segmented guide strand stabilized using LNA was shown to be effective in cell based assays. In the present study we examined the in vivo efficacy of the LNA and UNA modified siRNA and sisiRNA in a mouse model bearing human tumor xenografts. We studied the biodistribution and efficacy of target knockdown in the mouse model. In addition we used whole genome profiling to assess the off-target effects in the liver of the mouse and the tumor xenografts. We report that LNA and UNA modified siRNA and sisiRNA improve the efficacy in target knockdown as compared with unmodified siRNA in the tumor xenografts without formulation. However, the level of off-target gene regulation in both the tumor and the liver correlated with the increase in efficacy in target knockdown, unless the seed region of the siRNA was modified.

Stability against nucleases, affinity for the targeted mRNA and the ability to recruit RNase H are prerequisites for antisense oligonucleotide (AON) applications where gene expression knockdown is required. Typically chimeric gapmer AON designs are used with a central continuous stretch of RNase H recruiting nucleotides (e.g. phosphorothioate DNA), flanked by affinity and stability-enhancing modified nucleotides. However, many types of nucleotide modifications in the central DNA gap can disturb RNase H function. Here we present studies into two different types of nucleotide modifications, a flexible acyclic RNA analog named unlocked nucleic acid (UNA) and 4'-C-hydroxymethyl-DNA in the gap of an LNA (locked nucleic acid) flanked gapmer. We compared the efficacy of mRNA degradation by the gap modified LNA antisense gapmers in cell-free assays and cultured cells. This study shows that both UNA and 4'-C-hydroxymethyl-DNA gap insertions are compatible with RNase H activity when used sparingly. However, multiple 4'-C-hydroxymethyl-DNA modifications are better tolerated by RNase H than multiple UNA modifications in the gap. Furthermore, this report shows that LNA gapmer AONs with multiple 4'-C-hydroxymethyl-DNA moieties in the gap can mediate target knockdown in vivo.

Allele-specific inhibition (ASI) is a new strategy to treat cancer through a vulnerability created by the loss of large segments of chromosomal material by loss of heterozygosity (LOH). Using antisense approaches, it is possible to target single nucleotide polymorphisms (SNP) in the remaining allele of an essential gene in the tumor, thus killing the tumor while the heterozygous patient survives at the expense of the other nontargeted allele lost by the tumor. In this study, the feasibility of using locked nucleic acid (LNA)-modified DNAzymes (LNAzymes) of the 10-23 motif as allele-specific drugs was investigated. We demonstrate that incorporation of LNA into 10-23 motif DNAzymes increases their efficacy in mRNA degradation and that, in a cell-free system, the 10-23 motif LNAzyme can adequately discriminate and recognize an SNP in the large subunit of RNA polymerase II (POLR2A), an essential gene frequently involved in LOH in cancer cells. However, the LNAzymes, optimized under in vitro conditions, are not always efficient in cleaving their RNA target in cell culture, and the efficiency of RNA cleavage in cell culture is cell type dependent. The cleavage rate of the LNAzyme is also much slower than RNase H-recruiting DNA phosphorothioate antisense oligonucleotides. Moreover, compared with DNA phosphorothioates, the ability of the LNAzymes to differentially knock down two POLR2A alleles in cultured cancer cells is limited.

Locked nucleic acid (beta-D-LNA) monomers are conformationally restricted nucleotides bearing a methylene 2'-O, 4'-C linkage that have an unprecedented high affinity for matching DNA or RNA. In this study, we compared the in vitro and in vivo properties of four different LNAs, beta-D-amino LNA (amino-LNA), beta-D-thio LNA (thio-LNA), beta-D-LNA (LNA), and its stereoisomer alpha-L-LNA in an antisense oligonucleotide (ODN). A well-known antisense ODN design against H-Ras was modified at the 5'- and 3'-ends with the different LNA analogues (LNA-DNA-LNA gapmer design). The resulting gapmers were tested in cancer-cell cultures and in a nude-mouse model bearing prostate tumor xenografts. The efficacy in target knockdown, the biodistribution, and the ability to inhibit tumor growth were measured. All anti H-Ras ODNs were very efficient in H-Ras mRNA knockdown in vitro, reaching maximum effect at concentrations below 5 nM. Moreover, the anti-H-Ras ODN containing alpha-L-LNA had clearly the highest efficacy in H-Ras knockdown. All LNA types displayed a great stability in serum. ODNs containing amino-LNA showed an increased uptake by heart, liver, and lungs as compared to the other LNA types. Both alpha-L-LNA and LNA gapmer ODNs had a high efficacy of tumor-growth inhibition and were nontoxic at the tested dosages. Remarkably, in vivo tumor-growth inhibition could be observed at dosages as low as 0.5 mg kg(-1) per day. These results indicate that alpha-L-LNA is a very promising member of the family of LNA analogues in antisense applications.

Among the diverse family of collagens, the widely expressed microfibrillar type VI collagen is believed to play a role in bridging cells with the extracellular matrix. Several observations imply substrate properties for cell attachment as well as association with major collagen fibers. Previously, we have established genetic linkage between the genes encoding the three constituent alpha-chains of type VI collagen and Bethlem myopathy. A distinctive feature of this autosomal dominant disorder consists of contractures of multiple joints in addition to generalized muscular weakness and wasting. Nine kindreds show genetic linkage to the COL6A1-COL6A2 cluster on chromosome 21q22.3 (refs 3,4; manuscript submitted) whereas one family shows linkage to markers on chromosome 2q37 close to COL6A3 (ref. 5). Sequence analysis in four families reveals a mutation in COL6A1 in one and a COL6A2 mutation in two other kindreds. Both mutations disrupt the Gly-X-Y motif of the triple helical domain by substitution of Gly for either Val or Ser. Analogous to the putative perturbation of the anchoring function of the dystrophin-associated complex in congenital muscular dystrophy with mutations in the alpha 2-subunit of laminin, our observations suggest a similar mechanism in Bethlem myopathy.