Search

Representatives of academia, patient organisations, industry and the United States Food and Drug Administration attended a workshop on dystrophin quantification methodology. The aims of the workshop were to provide an overview of methods used to quantify dystrophin levels in human skeletal muscle and their applicability to clinical trial samples, outline the gaps with regards to validating the methods for robust clinical applications prior to regulatory agency review, and to align future efforts towards further optimizing these methods. The workshop facilitated a constructive but also critical discussion on the potential and limitations of techniques currently used in the field of translational research (western blot and immunofluorescence analysis) and emerging techniques (mass spectrometry and capillary western immunoassay). Notably, all participants reported variation in dystrophin levels between muscle biopsies from different healthy individuals and agreed on the need for a common reference sample.

Nucleic acid‐based therapeutics that regulate gene expression have been developed towards clinical use at a steady pace for several decades, but in recent years the field has been accelerating. To date, there are 11 marketed products based on antisense oligonucleotides, aptamers and small interfering RNAs, and many others are in the pipeline for both academia and industry. A major technology trigger for this development has been progress in oligonucleotide chemistry to improve the drug properties and reduce cost of goods, but the main hurdle for the application to a wider range of disorders is delivery to target tissues. The adoption of delivery technologies, such as conjugates or nanoparticles, has been a game changer for many therapeutic indications, but many others are still awaiting their eureka moment. Here, we cover the variety of methods developed to deliver nucleic acid‐based therapeutics across biological barriers and the model systems used to test them. We discuss important safety considerations and regulatory requirements for synthetic oligonucleotide chemistries and the hurdles for translating laboratory breakthroughs to the clinic. Recent advances in the delivery of nucleic acid‐based therapeutics and in the development of model systems, as well as safety considerations and regulatory requirements for synthetic oligonucleotide chemistries are discussed in this review on oligonucleotide‐based therapeutics., Recent advances in the delivery of nucleic acid‐based therapeutics and in the development of model systems, as well as safety considerations and regulatory requirements for synthetic oligonucleotide chemistries are discussed in this review on oligonucleotide‐based therapeutics.

To establish the potential economic burden in caregivers to patients with DMD and the potential causative factors.Caregivers to patients with DMD were recruited through the DMD patients Register and questioned about several economic aspects using "ad-hoc" questionnaires.All families, apart from one (97.2% n = 36), incurred in monthly medical costs (44% of the families more than 50 euros/month). 97.2% of the households considered looking after a patient of DMD as financially burdensome, and 80.5% of households declared to have suffered work changes, especially the mothers (job timetable-related mainly). The presence of obsessive-compulsive disorders (OCD) in patients was significantly associated with caregivers' high financial burden as these were six times more likely to have this perception OR = 6468 IC 95% (1056-39,601), p = 0.043. Also, when patients had learning difficulties, caregivers had up to six times more chances to incur in monthly expenditure for formal care OR = 6089 IC 95% (1112-33,342), p = 0.037.Caregivers have relevant financial burden that might be conditioned by the clinical condition of the patient.Quantitative data about financial burden in DMD Spanish families providing informal care. Identification of the patient's main clinical issues associated with financial burden.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy characterized by dysfunction of the role of glial cells in controlling brain fluid and ion homeostasis. Patients affected by MLC present macrocephaly, cysts and white matter vacuolation, which lead to motor and cognitive impairments. To date, there is no treatment for MLC, only supportive care. MLC is caused by mutations in the MLC1 and GLIALCAM genes. MLC1 is a membrane protein with low identity to the Kv1.1 potassium channel and GlialCAM belongs to an adhesion molecule family. Both proteins form a complex with an as-yet-unknown function that is expressed mainly in the astrocytes surrounding the blood-brain barrier and in Bergmann glia. GlialCAM also acts as an auxiliary subunit of the chloride channel ClC-2, thus regulating its localization at cell-cell junctions and modifying its functional properties by affecting the common gate of ClC-2. Recent studies in Mlc1-, GlialCAM- and Clcn2-knockout mice or Mlc1-knockout zebrafish have provided fresh insight into the pathophysiology of MLC and further details about the molecular interactions between these three proteins. Additional studies have shown that GlialCAM/MLC1 also regulates other ion channels (TRPV4, VRAC) or transporters (Na+/K+-ATPase) in a not-understood manner. Furthermore, it has been shown that GlialCAM/MLC1 may influence signal transduction mechanisms, thereby affecting other proteins not related with transport such as the EGF receptor. Here, we offer a personal biochemical retrospective of the work that has been performed to gain knowledge of the pathophysiology of MLC, and we discuss future strategies that may be used to identify therapeutic solutions for MLC patients.

Aberrant activation of signaling pathways plays a pivotal role in central nervous system disorders, such as Alzheimer's disease (AD). Using a combination of virtual screening and experimental testing, novel small molecule inhibitors of tPA-mediated extracellular signal-regulated kinase (Erk)1/2 activation were identified that provide higher levels of neuroprotection from Aβ-induced apoptosis than Memantine, the most recently FDA-approved drug for AD treatment. Subsequent target deconvolution efforts revealed that they all share low micromolar affinity for the imidazoline I(2) receptor, while being devoid of any significant affinity to a list of AD-relevant targets, including the N-methyl-d-aspartate receptor (NMDAR), acetylcholinesterase (AChE), and monoamine oxidase B (MAO-B). Targeting the imidazoline I(2) receptor emerges as a new mechanism of action to inhibit tPA-induced signaling in neurons for the treatment of AD and other neurodegenerative diseases.

Cannabidiol (CBD) reduces seizures in childhood epilepsy syndromes including Dravet syndrome (DS). A formulation of CBD has obtained orphan drug designation for these syndromes and clinical trials are currently underway. The mechanism responsible for CBD effects is not known, although it could involve targets sensitive to CBD in other neurological disorders. We believe of interest to investigate whether these potential targets are altered in DS, in particular whether the endocannabinoid system is dysregulated. To this end, lymphocytes from patients and controls were used for analysis of gene expression of transmitter receptors and transporters, ion channels, and enzymes associated with CBD effects, as well as endocannabinoid genes. Plasma endocannabinoid levels were also analyzed. There were no differences between DS patients and controls in most of the CBD targets analyzed, except an increase in the voltage-dependent calcium channel alpha-1h subunit. We also found that cannabinoid type-2 (CB 2) receptor gene expression was elevated in DS patients, with no changes in other endocannabinoid-related receptors and enzymes, as well as in plasma levels of endocannabinoids. Such elevation was paralleled by an increase in CD70, a marker of lymphocyte activation, and certain trends in inflammation-related proteins (e.g., peroxisome proliferator-activated receptor-gamma receptors, cytokines). In conclusion, together with changes in the voltage-dependent calcium channel alpha-1h subunit, we found an upregulation of CB 2 receptors, associated with an activation of lymphocytes and changes in inflammation-related genes, in DS patients. Such changes were also reported in inflammatory disorders and may indirectly support the occurrence of a potential dysregulation of the endocannabinoid system in the brain.

A study on the preparation of N-alkylglycines (peptoids) that contain tertiary amino residues on the N-alkyl side chains is reported. The appropriate combination of the submonomer strategy with N-alkylglycine monomer couplings depending upon the structure of the N-alkyl side chain that must be incorporated into the peptoid is determinant for the efficiency of the synthetic pathway. The application of this strategy to the preparation of SICHI, an N-alkyglycine trimer containing tertiary amino residues in the three N-alkyl branches, and that has been identified as a potent Semaphorin 3A inhibitor, is presented.

SummaryIon fluxes mediated by glial cells are required for several physiological processes such as fluid homeostasis or the maintenance of low extracellular potassium during high neuronal activity. In mice, the disruption of the Cl− channel ClC-2 causes fluid accumulation leading to myelin vacuolation. A similar vacuolation phenotype is detected in humans affected with megalencephalic leukoencephalopathy with subcortical cysts (MLC), a leukodystrophy which is caused by mutations in MLC1 or GLIALCAM. We here identify GlialCAM as a ClC-2 binding partner. GlialCAM and ClC-2 colocalize in Bergmann glia, in astrocyte-astrocyte junctions at astrocytic endfeet around blood vessels, and in myelinated fiber tracts. GlialCAM targets ClC-2 to cell junctions, increases ClC-2 mediated currents, and changes its functional properties. Disease-causing GLIALCAM mutations abolish the targeting of the channel to cell junctions. This work describes the first auxiliary subunit of ClC-2 and suggests that ClC-2 may play a role in the pathology of MLC disease.Video Abstract

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy, in the majority of cases caused by mutations in the MLC1 gene. MRI from MLC patients shows diffuse cerebral white matter signal abnormality and swelling, with evidence of increased water content. Histopathology in a MLC patient shows vacuolation of myelin, which causes the cerebral white matter swelling. MLC1 protein is expressed in astrocytic processes that are part of blood- and cerebrospinal fluid-brain barriers. We aimed to create an astrocyte cell model of MLC disease. The characterization of rat astrocyte cultures revealed MLC1 localization in cell-cell contacts, which contains other proteins described typically in tight and adherent junctions. MLC1 localization in these contacts was demonstrated to depend on the actin cytoskeleton: it was not altered when disrupting the microtubule or the GFAP networks. In human tissues, MLC1 and the protein Zonula Occludens 1 (ZO-1), which is linked to the actin cytoskeleton, co-localized by EM immunostaining and were specifically co-immunoprecipitated. To create an MLC cell model, knockdown of MLC1 in primary astrocytes was performed. Reduction of MLC1 expression resulted in the appearance of intracellular vacuoles. This vacuolation was reversed by the co-expression of human MLC1. Re-examination of a human brain biopsy from an MLC patient revealed that vacuoles were also consistently present in astrocytic processes. Thus, vacuolation of astrocytes is also a hallmark of MLC disease. (C) 2011 Elsevier Inc. All rights reserved.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare leukodystrophy caused by mutations in MLC1 or GLIALCAM. The GLIALCAM gene product functions as an MLC1 beta-subunit. We aim to further clarify the molecular mechanisms of MLC caused by mutations in MLC1 or GLIALCAM. For this purpose, we analyzed a human post-mortem brain obtained from an MLC patient, who was homozygous for a missense mutation (S69L) in MLC1. We showed that this mutation affects the stability of MLC1 in vitro and reduces MLC1 protein levels in the brain to almost undetectable. However, the amount of GlialCAM and its localization were nearly unaffected, indicating that MLC1 is not necessary for GlialCAM expression or tar- geting. These findings were supported by experiments in primary astrocytes and in heterologous cells. In addition, we demonstrated that MLC1 and GlialCAM form homo- and hetero-complexes and that MLC-causing mutations in GLIALCAM mainly reduce the formation of GlialCAM homo-complexes, leading to a defect in the trafficking of GlialCAM alone to cell junctions. GLIALCAM mutations also affect the trafficking of its associ- ated molecule MLC1, explaining why GLIALCAM and MLC1 mutations lead to the same disease: MLC. © The Author 2011. Published by Oxford University Press.

We studied the changes in the distribution of a specific variant of Semaphorin Y/6C (Sema6C) in mouse forebrain after axotomy of the entorhino-hippocampal perforant pathway. We found this isoform to be widely expressed during development, remaining in the adult and showing variations in distribution when the perforant pathway was axotomized. These changes were detected in both the hippocampal and entorhinal cortices. Sema6C1 immunoreactivity (IR) was high in the stratum radiatum of the hippocampus proper and the inner molecular layer of the dentate gyrus; the entorhinal cortex showed Sema6C1 IR in both cell bodies and in fibers of the II/III and V/VI layers. In axotomized animals, the IR of the ipsilateral, but not the contralateral, hemisphere showed that IR had moved into the stratum lacunosum-moleculare, the medial molecular layer of the dentate gyrus and the fibers, but not the cell bodies, of the entorhinal cortex. These results were not reproduced after lateral axotomy of the fimbria fornix, indicating a specific role for Sema6C variants in the generation and/or stability of entorhino-hippocampal synapses. Growth cone collapse of entorhinal and pyramidal neurons, as well as activation of glycogen synthase kinase-3 (GSK-3) through depletion of the inactive pool, induced by diffusible Sema6C1 further supports this view.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a leukodystrophy characterized by early-onset macrocephaly and delayed-onset neurological deterioration. Recessive MLC1 mutations are observed in 75% of patients with MLC. Genetic-linkage studies failed to identify another gene. We recently showed that some patients without MLC1 mutations display the classical phenotype; others improve or become normal but retain macrocephaly. To find another MLC-related gene, we used quantitative proteomic analysis of affinity-purified MLC1 as an alternative approach and found that GlialCAM, an IgG-like cell adhesion molecule that is also called HepaCAM and is encoded by HEPACAM, is a direct MLC1-binding partner. Analysis of 40 MLC patients without MLC1 mutations revealed multiple different HEPACAM mutations. Ten patients with the classical, deteriorating phenotype had two mutations, and 18 patients with the improving phenotype had one mutation. Most parents with a single mutation had macrocephaly, indicating dominant inheritance. In some families with dominant HEPACAM mutations, the clinical picture and magnetic resonance imaging normalized, indicating that HEPACAM mutations can cause benign familial macrocephaly. In other families with dominant HEPACAM mutations, patients had macrocephaly and mental retardation with or without autism. Further experiments demonstrated that GlialCAM and MLC1 both localize in axons and colocalize in junctions between astrocytes. GlialCAM is additionally located in myelin. Mutant GlialCAM disrupts the localization of MLC1-GlialCAM complexes in astrocytic junctions in a manner reflecting the mode of inheritance. In conclusion, GlialCAM is required for proper localization of MLC1. HEPACAM is the second gene found to be mutated in MLC. Dominant HEPACAM mutations can cause either macrocephaly and mental retardation with or without autism or benign familial macrocephaly. © 2011 The American Society of Human Genetics. All rights reserved.