Pathomechanisms of mutant proteins in Charcot-Marie-Tooth disease

We review the putative functions and malfunctions of proteins encoded by genes mutated in Charcot-Marie-Tooth disease (CMT; inherited motor and sensory neuropathies) in normal and affected peripheral nerves. Some proteins implicated in demyelinating CMT, peripheral myelin protein 22, protein zero (P0), and connexin32 (Cx32/GJB1) are crucial components of myelin. Periaxin is involved in connecting myelin to the surrounding basal lamina. Early growth response 2 (EGR2) and Sox10 are transcriptional regulators of myelin genes. Mutations in the small integral membrane protein of lysosome/late endosome, the myotubularin-related protein 2 (MTMR2), and MTMR13/set-binding factor 2 are involved in vesicle and membrane transport and the regulation of protein degradation. Pathomechanisms related to alterations of these processes are a widespread phenomenon in demyelinating neuropathies because mutations of myelin components may also affect protein biosynthesis, transport, and/or degradation. Related disease mechanisms are also involved inaxonal neuropathies although there is considerably more functional heterogeneity. Some mutations, most notably in P0, GJB1, ganglioside-induced differentiation-associated protein 1 (GDAP1), neurofilament light chain (NF-L), and dynamin 2 (DNM2), can result in demyelinating or axonal neuropathies introducing additional complexity in the pathogenesis. Often, this relates to the intinate connection between Schwann cells and neurons/axons leading to axonal damage even if the mutation-caused defect is Schwann-cell-autonomous. This mechanisms is likely for P0 and Cx32 mutations and provides the basis for the unifying hypothesis that also demyelinating neuropathies develop into functional axonopathies. In GDAP1 and DNM2 mutants, both Schwann cells and axons/neurons might be directly affected. NF-L mutants have a primary neuronal defect but also cause demyelination. The major challenge ahead lies in determining the individual contributions by neurons and Schwann cells to the pathology over time and to delineate the detailed molecular functions of the proteins associated with CMT in health and diseas

Ganglioside-induced differentiation associated protein 1 is a regulator of the mitochondrial network: new implications for Charcot-Marie-Tooth disease

Mutations in GDAP1 lead to severe forms of the peripheral motor and sensory neuropathy, Charcot-Marie-Tooth disease (CMT), which is characterized by heterogeneous phenotypes, including pronounced axonal damage and demyelination. We show that neurons and Schwann cells express ganglioside-induced differentiation associated protein 1 (GDAP1), which suggest that both cell types may contribute to the mixed features of the disease. GDAP1 is located in the mitochondrial outer membrane and regulates the mitochondrial network. Overexpression of GDAP1 induces fragmentation of mitochondria without inducing apoptosis, affecting overall mitochondrial activity, or interfering with mitochondrial fusion. The mitochondrial fusion proteins, mitofusin 1 and 2 and Drp1(K38A), can counterbalance the GDAP1-dependent fission. GDAP1-specific knockdown by RNA interference results in a tubular mitochondrial morphology. GDAP1 truncations that are found in patients who have CMT are not targeted to mitochondria and have lost mitochondrial fragmentation activity. The latter activity also is reduced strongly for disease-associated GDAP1 point mutations. Our data indicate that an exquisitely tight control of mitochondrial dynamics, regulated by GDAP1, is crucial for the proper function of myelinated peripheral nerves

Glutathione-conjugating and membrane-remodeling activity of GDAP1 relies on amphipathic C-terminal domain

Mutations in the ganglioside-induced differentiation associated protein 1 (GDAP1) cause severe peripheral motor and sensory neuropathies called Charcot-Marie-Tooth disease. GDAP1 expression induces fission of mitochondria and peroxisomes by a currently elusive mechanism, while disease causing mutations in GDAP1 impede the protein’s role in mitochondrial dynamics. In silico analysis reveals sequence similarities of GDAP1 to glutathione S-transferases (GSTs). However, a proof of GST activity and its possible impact on membrane dynamics are lacking to date. Using recombinant protein, we demonstrate for the first time theta-class-like GST activity for GDAP1, and it’s activity being regulated by the C-terminal hydrophobic domain 1 (HD1) of GDAP1 in an autoinhibitory manner. Moreover, we show that the HD1 amphipathic pattern is required to induce membrane dynamics by GDAP1. As both, fission and GST activities of GDAP1, are critically dependent on HD1, we propose that GDAP1 undergoes a molecular switch, turning from a pro-fission active to an auto-inhibited inactive conformation.ISSN:2045-232

Donor selection in a pediatric stem cell transplantation cohort using PIRCHE and HLA‐DPB1 typing

Background: New strategies to optimize donor selection for hematopoietic stem cell transplantation (HSCT) have mainly been evaluated in adults, but the disease spectrum requiring HSCT differs significantly in children and has consequences for the risk of complications, such as graft-versus-host disease (GvHD). Procedures: Here we evaluated whether HLA-DPB1 and Predicted Indirectly ReCognizable HLA-Epitope (PIRCHE) matching can improve donor selection and minimize risks specific for a pediatric cohort undergoing HSCT in Berlin between 2014 and 2016. Results: The percentage of HLA-DPB1–mismatched HSCT in the pediatric cohort was in line with the general distribution among matched unrelated donor HSCT. Nonpermissive HLA-DPB1 mismatches were not associated with a higher incidence of GvHD, but the incidence of relapse was higher in patients undergoing HSCT from HLA-DPB1–matched transplantations. High PIRCHE-I scores were associated with a significantly higher risk for developing GvHD in patients undergoing HSCT from nine of ten matched unrelated donors. This finding persisted after including HLA-DPB1 into the PIRCHE analysis. Conclusions: Implementing PIRCHE typing in the donor selection process for HSCT in children could particularly benefit children with nonmalignant diseases and support further validation of PIRCHE-based donor selection in a larger number of children treated at different sites

Dynamin 2 mutations in Charcot-Marie-Tooth neuropathy highlight the importance of clathrin-mediated endocytosis in myelination

Mutations in dynamin 2 (DNM2) lead to dominant intermediate Charcot-Marie-Tooth neuropathy type B, while a different set of DNM2 mutations cause autosomal dominant centronuclear myopathy. In this study, we aimed to elucidate the disease mechanisms in dominant intermediate Charcot-Marie-Tooth neuropathy type B and to find explanations for the tissue-specific defects that are associated with different DNM2 mutations in dominant intermediate Charcot-Marie-Tooth neuropathy type B versus autosomal dominant centronuclear myopathy. We used tissue derived from Dnm2-deficient mice to establish an appropriate peripheral nerve model and found that dominant intermediate Charcot-Marie-Tooth neuropathy type B-associated dynamin 2 mutants, but not autosomal dominant centronuclear myopathy mutants, impaired myelination. In contrast to autosomal dominant centronuclear myopathy mutants, Schwann cells and neurons from the peripheral nervous system expressing dominant intermediate Charcot-Marie-Tooth neuropathy mutants showed defects in clathrin-mediated endocytosis. We demonstrate that, as a consequence, protein surface levels are altered in Schwann cells. Furthermore, we discovered that myelination is strictly dependent on Dnm2 and clathrin-mediated endocytosis function. Thus, we propose that altered endocytosis is a major contributing factor to the disease mechanisms in dominant intermediate Charcot-Marie-Tooth neuropathy type

Silence on Shangri-La: attenuation of Huygens acoustic signals suggests surface volatiles

Objective: Characterize and understand acoustic instrument performance on the surface of Titan. Methods: The Huygens probe measured the speed of sound in Titan's atmosphere with a 1 MHz pulse time-of-flight transducer pair near the bottom of the vehicle. We examine the fraction of pulses correctly received as af unction of time. Results: This system returned good data from about 11 km altitude, where the atmosphere became thick enough to effectively transmit the sound, down to the surface just before landing: these data have been analyzed previously. After an initial transient at landing, the instrument operated nominally for about 10 min, recording pulses much as during descent. The fraction of pulses detected then declined and the transmitted sound ceased to be detected altogether, despite no indication of instrument or probe configuration changes. Conclusions: The most likely explanation appears to be absorption of the signal by polyatomic gases with relaxation losses at the instrument frequency, such as ethane, acetylene and carbon dioxide. These vapors, detected independently by the GCMS instrument, were evolved from the surface material by the warmth leaking from the probe, and confirm the nature of the surface materials as ‘damp’ with a cocktail of volatile compounds. Some suggestions for future missions are considered. Practice implications: None

Targeting and Function of the Mitochondrial Fission Factor GDAP1 Are Dependent on Its Tail-Anchor

Proteins controlling mitochondrial dynamics are often targeted to and anchored into the mitochondrial outer membrane (MOM) by their carboxyl-terminal tail-anchor domain (TA). However, it is not known whether the TA modulates protein function. GDAP1 is a mitochondrial fission factor with two neighboring hydrophobic domains each flanked by basic amino acids (aa). Here we define GDAP1 as TA MOM protein. GDAP1 carries a single transmembrane domain (TMD) that is, together with the adjacent basic aa, critical for MOM targeting. The flanking N-terminal region containing the other hydrophobic domain is located in the cytoplasm. TMD sequence, length, and high hydrophobicity do not influence GDAP1 fission function if MOM targeting is maintained. The basic aa bordering the TMD in the cytoplasm, however, are required for both targeting of GDAP1 as part of the TA and GDAP1-mediated fission. Thus, this GDAP1 region contains critical overlapping motifs defining intracellular targeting by the TA concomitant with functional aspects

The Gdap1 knockout mouse mechanistically links redox control to Charcot-Marie-Tooth disease

Mutations in the mitochondrial fission factor GDAP1 are associated with severe peripheral neuropathies, but why the CNS remains unaffected is unclear. Using a Gdap1−/− mouse, Niemann et al. demonstrate that a CNS-expressed Gdap1 paralogue changes its subcellular localisation under oxidative stress conditions to also act as a mitochondrial fission facto

Electrode positioning errors reduce current dose for focal tDCS set-ups: Evidence from individualized electric field mapping.

Electrode positioning errors contribute to variability of transcranial direct current stimulation (tDCS) effects. We investigated the impact of electrode positioning errors on current flow for tDCS set-ups with different focality. Deviations from planned electrode positions were determined using data acquired in an experimental study (N = 240 datasets) that administered conventional and focal tDCS during magnetic resonance imaging (MRI). Comparison of individualized electric field modeling for planned and empirically derived "actual" electrode positions was conducted to quantify the impact of positioning errors on the electric field dose in target regions for tDCS. Planned electrode positions resulted in higher current dose in the target regions for focal compared to conventional montages (7-12%). Deviations from planned positions significantly reduced current flow in the target regions, selectively for focal set-ups (26-30%). Dose reductions were significantly larger for focal compared to conventional set-ups (29-43%). Precise positioning is crucial when using focal tDCS set-ups to avoid significant reductions of current dose in the intended target regions. Our results highlight the urgent need to routinely implement methods for improving electrode positioning, minimization of electrode drift, verification of electrode positions before and/or after tDCS and also to consider positioning errors when investigating dose-response relationships, especially for focal set-ups

Real-Time Detection of Overloads on the Plasma-Facing Components of Wendelstein 7-X

Wendelstein 7-X (W7-X) is the leading experiment on the path of demonstrating that stellarators are a feasible concept for a future power plant. One of its major goals is to prove quasi-steady-state operation in a reactor-relevant parameter regime. The surveillance and protection of the water-cooled plasma-facing components (PFCs) against overheating is fundamental to guarantee a safe steady-state high-heat-flux operation. The system has to detect thermal events in real-time and timely interrupt operation if it detects a critical event. The fast reaction times required to prevent damage to the device make it imperative to automate fully the image analysis algorithms. During the past operational phases, W7-X was equipped with inertially cooled test divertor units and the system still required manual supervision. With the experience gained, we have designed a new real-time PFC protection system based on image processing techniques. It uses a precise registration of the entire field of view against the CAD model to determine the temperature limits and thermal properties of the different PFCs. Instead of reacting when the temperature limits are breached in certain regions of interest, the system predicts when an overload will occur based on a heat flux estimation, triggering the interlock system in advance to compensate for the system delay. To conclude, we present our research roadmap towards a feedback control system of thermal loads to prevent unnecessary plasma interruptions in long high-performance plasmas