Targeting deregulated AMPK/mTORC1 pathways improves muscle function in myotonic dystrophy type I.

Myotonic dystrophy type I (DM1) is a disabling multisystemic disease that predominantly affects skeletal muscle. It is caused by expanded CTG repeats in the 3'-UTR of the dystrophia myotonica protein kinase (DMPK) gene. RNA hairpins formed by elongated DMPK transcripts sequester RNA-binding proteins, leading to mis-splicing of numerous pre-mRNAs. Here, we have investigated whether DM1-associated muscle pathology is related to deregulation of central metabolic pathways, which may identify potential therapeutic targets for the disease. In a well-characterized mouse model for DM1 (HSALR mice), activation of AMPK signaling in muscle was impaired under starved conditions, while mTORC1 signaling remained active. In parallel, autophagic flux was perturbed in HSALR muscle and in cultured human DM1 myotubes. Pharmacological approaches targeting AMPK/mTORC1 signaling greatly ameliorated muscle function in HSALR mice. AICAR, an AMPK activator, led to a strong reduction of myotonia, which was accompanied by partial correction of misregulated alternative splicing. Rapamycin, an mTORC1 inhibitor, improved muscle relaxation and increased muscle force in HSALR mice without affecting splicing. These findings highlight the involvement of AMPK/mTORC1 deregulation in DM1 muscle pathophysiology and may open potential avenues for the treatment of this disease.

Identification of Plant-derived Alkaloids with Therapeutic Potential for Myotonic Dystrophy Type I.

Myotonic dystrophy type I (DM1) is a disabling neuromuscular disease with no causal treatment available. This disease is caused by expanded CTG trinucleotide repeats in the 3' UTR of the dystrophia myotonica protein kinase gene. On the RNA level, expanded (CUG)n repeats form hairpin structures that sequester splicing factors such as muscleblind-like 1 (MBNL1). Lack of available MBNL1 leads to misregulated alternative splicing of many target pre-mRNAs, leading to the multisystemic symptoms in DM1. Many studies aiming to identify small molecules that target the (CUG)n-MBNL1 complex focused on synthetic molecules. In an effort to identify new small molecules that liberate sequestered MBNL1 from (CUG)n RNA, we focused specifically on small molecules of natural origin. Natural products remain an important source for drugs and play a significant role in providing novel leads and pharmacophores for medicinal chemistry. In a new DM1 mechanism-based biochemical assay, we screened a collection of isolated natural compounds and a library of over 2100 extracts from plants and fungal strains. HPLC-based activity profiling in combination with spectroscopic methods were used to identify the active principles in the extracts. The bioactivity of the identified compounds was investigated in a human cell model and in a mouse model of DM1. We identified several alkaloids, including the ß-carboline harmine and the isoquinoline berberine, that ameliorated certain aspects of the DM1 pathology in these models. Alkaloids as a compound class may have potential for drug discovery in other RNA-mediated diseases.

Improved Muscle Function in Duchenne Muscular Dystrophy through L-Arginine and Metformin: An Investigator-Initiated, Open-Label, Single-Center, Proof-Of-Concept-Study.

UNLABELLED: Altered neuronal nitric oxide synthase function in Duchenne muscular dystrophy leads to impaired mitochondrial function which is thought to be one cause of muscle damage in this disease. The study tested if increased intramuscular nitric oxide concentration can improve mitochondrial energy metabolism in Duchenne muscular dystrophy using a novel therapeutic approach through the combination of L-arginine with metformin. Five ambulatory, genetically confirmed Duchenne muscular dystrophy patients aged between 7­10 years were treated with L-arginine (3 x 2.5 g/d) and metformin (2 x 250 mg/d) for 16 weeks. Treatment effects were assessed using mitochondrial protein expression analysis in muscular biopsies, indirect calorimetry, Dual-Energy X-Ray Absorptiometry, quantitative thigh muscle MRI, and clinical scores of muscle performance. There were no serious side effects and no patient dropped out. Muscle biopsy results showed pre-treatment a significantly reduced mitochondrial protein expression and increased oxidative stress in Duchenne muscular dystrophy patients compared to controls. Post-treatment a significant elevation of proteins of the mitochondrial electron transport chain was observed as well as a reduction in oxidative stress. Treatment also decreased resting energy expenditure rates and energy substrate use shifted from carbohydrates to fatty acids. These changes were associated with improved clinical scores. In conclusion pharmacological stimulation of the nitric oxide pathway leads to improved mitochondria function and clinically a slowing of disease progression in Duchenne muscular dystrophy. This study shall lead to further development of this novel therapeutic approach into a real alternative for Duchenne muscular dystrophy patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT02516085.

Proteasome inhibitors increase missense mutated dysferlin in patients with muscular dystrophy.

No treatment is available for patients affected by the recessively inherited, progressive muscular dystrophies caused by a deficiency in the muscle membrane repair protein dysferlin. A marked reduction in dysferlin in patients harboring missense mutations in at least one of the two pathogenic DYSF alleles encoding dysferlin implies that dysferlin is degraded by the cell's quality control machinery. In vitro evidence suggests that missense mutated dysferlin might be functional if salvaged from degradation by the proteasome. We treated three patients with muscular dystrophy due to a homozygous Arg555Trp mutation in dysferlin with the proteasome inhibitor bortezomib and monitored dysferlin expression in monocytes and in skeletal muscle by repeated percutaneous muscle biopsy. Expression of missense mutated dysferlin in the skeletal muscle and monocytes of the three patients increased markedly, and dysferlin was correctly localized to the sarcolemma of muscle fibers on histological sections. Salvaged missense mutated dysferlin was functional in a membrane resealing assay in patient-derived muscle cells treated with three different proteasome inhibitors. We conclude that interference with the proteasomal system increases expression of missense mutated dysferlin, suggesting that this therapeutic strategy may benefit patients with dysferlinopathies and possibly other genetic diseases.

An essential role of MAG in mediating axon-myelin attachment in Charcot-Marie-Tooth 1A disease.

Charcot-Marie-Tooth disease type 1A (CMT1A) is a hereditary demyelinating peripheral neuropathy caused by the duplication of the PMP22 gene. Demyelination precedes the occurrence of clinical symptoms that correlate with axonal degeneration. It was postulated that a disturbed axon-glia interface contributes to altered myelination consequently leading to axonal degeneration. In this study, we examined the expression of MAG and Necl4, two critical adhesion molecules that are present at the axon-glia interface, in sural nerve biopsies of CMT1A patients and in peripheral nerves of mice overexpressing human PMP22, an animal model for CMT1A. We show an increase in the expression of MAG and a strong decrease of Necl4 in biopsies of CMT1A patients as well as in CMT1A mice. Expression analysis revealed that MAG is strongly upregulated during peripheral nerve maturation, whereas Necl4 expression remains very low. Ablating MAG in CMT1A mice results in separation of axons from their myelin sheath. Our data show that MAG is important for axon-glia contact in a model for CMT1A, and suggest that its increased expression in CMT1A disease has a compensatory role in the pathology of the disease. Thus, we demonstrate that MAG together with other adhesion molecules such as Necl4 is important in sustaining axonal integrity.

Global deprivation of brain-derived neurotrophic factor in the CNS reveals an area-specific requirement for dendritic growth.

Although brain-derived neurotrophic factor (BDNF) is linked with an increasing number of conditions causing brain dysfunction, its role in the postnatal CNS has remained difficult to assess. This is because the bdnf-null mutation causes the death of the animals before BDNF levels have reached adult levels. In addition, the anterograde axonal transport of BDNF complicates the interpretation of area-specific gene deletion. The present study describes the generation of a new conditional mouse mutant essentially lacking BDNF throughout the CNS. It shows that BDNF is not essential for prolonged postnatal survival, but that the behavior of such mutant animals is markedly altered. It also reveals that BDNF is not a major survival factor for most CNS neurons and for myelination of their axons. However, it is required for the postnatal growth of the striatum, and single-cell analyses revealed a marked decreased in dendritic complexity and spine density. In contrast, BDNF is dispensable for the growth of the hippocampus and only minimal changes were observed in the dendrites of CA1 pyramidal neurons in mutant animals. Spine density remained unchanged, whereas the proportion of the mushroom-type spine was moderately decreased. In line with these in vivo observations, we found that BDNF markedly promotes the growth of cultured striatal neurons and of their dendrites, but not of those of hippocampal neurons, suggesting that the differential responsiveness to BDNF is part of a neuron-intrinsic program.

Immunoglobulin M deposition in cutaneous nerves of anti-myelin-associated glycoprotein polyneuropathy patients correlates with axonal degeneration.

Anti-myelin-associated glycoprotein (MAG) neuropathy is an antibody-mediated polyneuropathy. We correlated clinical features, immunoglobulin (Ig) M blood levels, IgM deposition and axonal degeneration in skin biopsies of anti-MAG neuropathy patients. By confocal microscopy, IgM deposits were found exclusively within perineurium-enclosed nerves; they were not found on single, non-perineurium-ensheathed myelinated axons. There was a linear correlation between IgM accumulation in nerve fascicles with IgM blood levels but not with anti-MAG antibody titer or disease duration. Axons with specific IgM deposits had signs of axonal damage, including neurofilament disintegration. Nodal structures were intact even at sites where the axons showed pathologic changes. Ultrastructural analysis revealed degeneration of myelinating Schwann cells. Taken together, these findings suggest that in anti-MAG neuropathy patients, IgM deposits are entrapped within cutaneous perineurium-ensheathed nerve bundles where they accumulate in the endoneurial space. High local IgM levels in the endoneurium may be required for IgM deposition on myelin and subsequent axonal injury and degeneration. This study underlines the importance of early, effective anti-B-cell treatments for preventing progression of this neuropathy.

Allograft inflammatory factor-1: a pathogenetic factor for vasculitic neuropathy.

Allograft inflammatory factor-1 (AIF-1) is a cytokine that plays a major role in the immune response and proliferative vasculopathy that occur during chronic allograft rejection. The purpose of this study was to characterize the cellular expression pattern and pathogenetic role of AIF-1 in nerve biopsies from patients with vasculitic neuropathy. We performed immunohistochemistry in human nerve biopsies of 10 patients with vasculitic neuropathies (VASs), 6 with chronic inflammatory demyelinating polyneuropathies (CIDPs), 5 with noninflammatory axonal neuropathies (NIANs), and 3 control nerves (CNs). In the CIDP and VAS nerves, AIF-1 expression was higher than in CN and NIAN nerves (P < 0.05). AIF-1 was increased in the arterial walls of VAS compared with CIDP nerves (P < 0.05). Vascular smooth muscle cells in vasculitic nerve express AIF-1 at a higher level compared with CIDP and NIAN. AIF-1 plays a role in inflammatory nerve disease and vascular smooth muscle cell proliferation and may be a new molecular target for treatment.

Normal expression of myelin protein zero with frame-shift mutation correlates with mild phenotype.

Mutations in the gene encoding for myelin protein zero (MPZ) cause inherited demyelinating peripheral neuropathies of different severity. The molecular and cellular mechanisms by which the MPZ mutations cause neuropathy are incompletely understood. We investigated MPZ, myelin basic protein, and peripheral myelin protein 22 (PMP22) protein expression levels in a nerve biopsy of a Charcot-Marie-Tooth type 1B patient heterozygous for the Val 102 frame-shift mutation. We demonstrate by quantitative immunohistochemical as well as by Western blot analyses that MPZ expression levels were not reduced in myelin membranes, a finding that is in accordance with the mild phenotype of this patient. Our data show that heterozygous 'loss-of-function' of MPZ may not necessarily lead to reduced protein levels. In conclusion, we demonstrate that careful analysis of protein expression levels in peripheral nerve tissues provides important information with respect to the understanding of the molecular basis of these neuropathies.

Unraveling the differential expression of the two isoforms of myelin-associated glycoprotein in a mouse expressing GFP-tagged S-MAG specifically regulated and targeted into the different myelin compartments.

The two myelin-associated glycoprotein (MAG) isoforms are cell adhesion molecules that differ only in their cytoplasmic domains, but their specific roles are not well understood. In this study, we present a transgenic mouse line that specifically expresses GFP-tagged S-MAG correctly regulated and targeted into the myelin sheath allowing the specific discrimination of L- and S-MAG on the subcellular level. Here, we describe the differential expression pattern and spatial distribution of L- and S-MAG during development as well as in the adult central and peripheral nervous system. In peripheral nerves, where S-MAG is the sole isoform, we observed S-MAG concentrated in different ring-like structures such as periaxonal and abaxonal rings, and discs spanning through the compact myelin sheath perpendicular to the axon. In summary, our data provide new insight in the subcellular distribution of the two isoforms fundamental for the understanding of their specific functions in myelin formation and maintenance.

IgM deposits on skin nerves in anti-myelin-associated glycoprotein neuropathy.

Anti-myelin-associated glycoprotein (anti-MAG) neuropathy is a chronic demyelinating neuropathy with predominant involvement of large sensory fibers and deposits of IgM and complement on sural nerve myelinated fibers. We assessed the presence of IgM deposits on skin myelinated nerve fibers and the involvement of unmyelinated axons in anti-MAG neuropathy. Skin biopsies were performed in 14 patients with anti-MAG neuropathy, in 8 patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), and in 2 patients with IgM paraproteinemic neuropathy. Biopsies were taken at the proximal thigh in 20 patients, at the distal leg in 21 patients, at the proximal arm in 13 patients, and at the hand or fingertip in 10 patients. We found IgM deposits on dermal myelinated fibers in all anti-MAG neuropathy patients, with a greater prevalence at the distal site of the extremities. Deposits were located throughout the length of the fibers and at the paranodal loops. CIDP and IgM paraproteinemic neuropathies did not show any deposit of IgM. Anti-MAG neuropathy and CIPD patients showed a decrease in epidermal nerve fiber density reflecting an associated axonal loss. In anti-MAG neuropathy, both large- and small-diameter nerve fibers are affected, and specific deposits of IgM are found on skin myelinated nerve fibers.

The raft-associated protein MAL is required for maintenance of proper axon--glia interactions in the central nervous system.

The myelin and lymphocyte protein (MAL) is a tetraspan raft-associated proteolipid predominantly expressed by oligodendrocytes and Schwann cells. We show that genetic ablation of mal resulted in cytoplasmic inclusions within compact myelin, paranodal loops that are everted away from the axon, and disorganized transverse bands at the paranode--axon interface in the adult central nervous system. These structural changes were accompanied by a marked reduction of contactin-associated protein/paranodin, neurofascin 155 (NF155), and the potassium channel Kv1.2, whereas nodal clusters of sodium channels were unaltered. Initial formation of paranodal regions appeared normal, but abnormalities became detectable when MAL started to be expressed. Biochemical analysis revealed reduced myelin-associated glycoprotein, myelin basic protein, and NF155 protein levels in myelin and myelin-derived rafts. Our results demonstrate a critical role for MAL in the maintenance of central nervous system paranodes, likely by controlling the trafficking and/or sorting of NF155 and other membrane components in oligodendrocytes.

Microheterogeneity of anti-myelin-associated glycoprotein antibodies.

Antibodies to the myelin-associated glycoprotein (MAG) are implicated in the pathogenesis of an acquired demyelinating polyneuropathy. We studied IgM affinity to MAG in 18 patients with anti-MAG antibodies. Binding of sera was tested for anti-MAG immunoreactivity in central nervous system (CNS) by ELISA and in CNS and peripheral nervous system (PNS) by Western blot analysis. Furthermore, immunohistochemical characterization of IgM binding on sural nerve tissue was investigated using the indirect peroxidase method. Western blot analysis revealed that all sera detected MAG in central myelin, but only eight in peripheral myelin. Anti-MAG-IgM-ELISA-titers correlated significantly (p<0.0001) with PNS-Western blot results. By indirect immunoperoxidase immunohistochemistry, 12 sera stained myelin sheaths, while 6 sera showed no staining. These results demonstrate considerable variations in antibody binding strength to MAG between PNS myelin and CNS myelin. The relevance of these differences for the pathogenesis of the neuropathy and clinical impairment remains to be demonstrated.

Matrix metalloproteinases-9 and -2 in secondary vasculitic neuropathies.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endoproteinases that play an important role in inflammation and tissue degradation. MMP-9 and MMP-2 are gelatinases that have been implicated in the degradation of the blood-brain or blood-nerve barrier. We present an immunohistochemical study on 11 nerve biopsy samples of inflammatory and non-inflammatory polyneuropathies. Perineurium and endothelium were positive for MMP-2 in all tissue sections. In addition, there was a specific up-regulation of MMP-2 in stromal cells of chronic inflammatory demyelinating polyneuropathy (CIDP) and even more in vasculitic neuropathies. MMP-9-positive cells were detected in vessel walls, infiltrates, epineurium and endoneurium of vasculitic neuropathies. In CIDP, MMP-9-positive cells were prominent in vessel walls. Only a few MMP-9-positive cells were detected in noninflammatory controls in blood vessels and adhering to vessel walls. Double staining indicated that the infiltrating cells were T cells and macrophages. Our findings suggest that MMP-9 plays an important role in inflammatory peripheral neuropathy probably as means for inflammatory cell invasion.

Caspr3 and caspr4, two novel members of the caspr family are expressed in the nervous system and interact with PDZ domains.

The NCP family of cell-recognition molecules represents a distinct subgroup of the neurexins that includes Caspr and Caspr2, as well as Drosophila Neurexin-IV and axotactin. Here, we report the identification of Caspr3 and Caspr4, two new NCPs expressed in nervous system. Caspr3 was detected along axons in the corpus callosum, spinal cord, basket cells in the cerebellum and in peripheral nerves, as well as in oligodendrocytes. In contrast, expression of Caspr4 was more restricted to specific neuronal subpopulations in the olfactory bulb, hippocampus, deep cerebellar nuclei, and the substantia nigra. Similar to the neurexins, the cytoplasmic tails of Caspr3 and Caspr4 interacted differentially with PDZ domain-containing proteins of the CASK/Lin2-Veli/Lin7-Mint1/Lin10 complex. The structural organization and distinct cellular distribution of Caspr3 and Caspr4 suggest a potential role of these proteins in cell recognition within the nervous system.