H2A.Z is involved in premature aging and DSB repair initiation in muscle fibers.

Histone variants are key epigenetic players, but their functional and physiological roles remain poorly understood. Here, we show that depletion of the histone variant H2A.Z in mouse skeletal muscle causes oxidative stress, oxidation of proteins, accumulation of DNA damages, and both neuromuscular junction and mitochondria lesions that consequently lead to premature muscle aging and reduced life span. Investigation of the molecular mechanisms involved shows that H2A.Z is required to initiate DNA double strand break repair by recruiting Ku80 at DNA lesions. This is achieved via specific interactions of Ku80 vWA domain with H2A.Z. Taken as a whole, our data reveal that H2A.Z containing nucleosomes act as a molecular platform to bring together the proteins required to initiate and process DNA double strand break repair.

Molecular interactions of PCSK9 with an inhibitory nanobody, CAP1 and HLA-C: Functional regulation of LDLR levels.

OBJECTIVE: The liver-derived circulating PCSK9 enhances the degradation of the LDL receptor (LDLR) in endosomes/lysosomes. PCSK9 inhibition or silencing is presently used in clinics worldwide to reduce LDL-cholesterol, resulting in lower incidence of cardiovascular disease and possibly cancer/metastasis. The mechanism by which the PCSK9-LDLR complex is sorted to degradation compartments is not fully understood. We previously suggested that out of the three M1, M2 and M3 subdomains of the C-terminal Cys/His-rich-domain (CHRD) of PCSK9, only M2 is critical for the activity of extracellular of PCSK9 on cell surface LDLR. This likely implicates the binding of M2 to an unknown membrane-associated "protein X" that would escort the complex to endosomes/lysosomes for degradation. We reported that a nanobody P1.40 binds the M1 and M3 domains of the CHRD and inhibits the function of PCSK9. It was also reported that the cytosolic adenylyl cyclase-associated protein 1 (CAP1) could bind M1 and M3 subdomains and enhance the activity of PCSK9. In this study, we determined the 3-dimensional structure of the CHRD-P1.40 complex to understand the intricate interplay between P1.40, CAP1 and PCSK9 and how they regulate LDLR degradation. METHODS: X-ray diffraction of the CHRD-P1.40 complex was analyzed with a 2.2 Å resolution. The affinity and interaction of PCSK9 or CHRD with P1.40 or CAP1 was analyzed by atomic modeling, site-directed mutagenesis, bio-layer interferometry, expression in hepatic cell lines and immunocytochemistry to monitor LDLR degradation. The CHRD-P1.40 interaction was further analyzed by deep mutational scanning and binding assays to validate the role of predicted critical residues. Conformational changes and atomic models were obtained by small angle X-ray scattering (SAXS). RESULTS: We demonstrate that PCSK9 exists in a closed or open conformation and that P1.40 favors the latter by binding key residues in the M1 and M3 subdomains of the CHRD. Our data show that CAP1 is well secreted by hepatic cells and binds extracellular PCSK9 at distinct residues in the M1 and M3 modules and in the acidic prodomain. CAP1 stabilizes the closed conformation of PCSK9 and prevents P1.40 binding. However, CAP1 siRNA only partially inhibited PCSK9 activity on the LDLR. By modeling the previously reported interaction between M2 and an R-X-E motif in HLA-C, we identified Glu567 and Arg549 as critical M2 residues binding HLA-C. Amazingly, these two residues are also required for the PCSK9-induced LDLR degradation. CONCLUSIONS: The present study reveals that CAP1 enhances the function of PCSK9, likely by twisting the protein into a closed configuration that exposes the M2 subdomain needed for targeting the PCSK9-LDLR complex to degradation compartments. We hypothesize that "protein X", which is expected to guide the LDLR-PCSK9-CAP1 complex to these compartments after endocytosis into clathrin-coated vesicles, is HLA-C or a similar MHC-I family member. This conclusion is supported by the PCSK9 natural loss-of-function Q554E and gain-of-function H553R M2 variants, whose consequences are anticipated by our modeling.

Severe congenital myasthenic syndromes caused by agrin mutations affecting secretion by motoneurons.

Congenital myasthenic syndromes (CMS) are predominantly characterized by muscle weakness and fatigability and can be caused by a variety of mutations in genes required for neuromuscular junction formation and maintenance. Among them, AGRN encodes agrin, an essential synaptic protein secreted by motoneurons. We have identified severe CMS patients with uncharacterized p.R1671Q, p.R1698P and p.L1664P mutations in the LG2 domain of agrin. Overexpression in primary motoneurons cultures in vitro and in chick spinal motoneurons in vivo revealed that the mutations modified agrin trafficking, leading to its accumulation in the soma and/or in the axon. Expression of mutant agrins in cultured cells demonstrated accumulation of agrin in the endoplasmic reticulum associated with induction of unfolded protein response (UPR) and impaired secretion in the culture medium. Interestingly, evaluation of the specific activity of individual agrins on AChR cluster formation indicated that when secreted, mutant agrins retained a normal capacity to trigger the formation of AChR clusters. To confirm agrin accumulation and secretion defect, iPS cells were derived from a patient and differentiated into motoneurons. Patient iPS-derived motoneurons accumulated mutant agrin in the soma and increased XBP1 mRNA splicing, suggesting UPR activation. Moreover, co-cultures of patient iPS-derived motoneurons with myotubes confirmed the deficit in agrin secretion and revealed a reduction in motoneuron survival. Altogether, we report the first mutations in AGRN gene that specifically affect agrin secretion by motoneurons. Interestingly, the three patients carrying these mutations were initially suspected of spinal muscular atrophy (SMA). Therefore, in the presence of patients with a clinical presentation of SMA but without mutation in the SMN1 gene, it can be worth to look for mutations in AGRN.

Asialoglycoprotein receptor 1 is a novel PCSK9-independent ligand of liver LDLR cleaved by furin.

The hepatic carbohydrate-recognizing asialoglycoprotein receptor (ASGR1) mediates the endocytosis/lysosomal degradation of desialylated glycoproteins following binding to terminal galactose/N-acetylgalactosamine. Human heterozygote carriers of ASGR1 deletions exhibit ∼34% lower risk of coronary artery disease and ∼10% to 14% reduction of non-HDL cholesterol. Since the proprotein convertase PCSK9 is a major degrader of the low-density lipoprotein receptor (LDLR), we investigated the degradation and functionality of LDLR and/or PCSK9 by endogenous/overexpressed ASGR1 using Western blot and immunofluorescence in HepG2-naïve and HepG2-PCSK9-knockout cells. ASGR1, like PCSK9, targets LDLR, and both independently interact with/enhance the degradation of the receptor. This lack of cooperativity between PCSK9 and ASGR1 was confirmed in livers of wildtype (WT) and Pcsk9-/- mice. ASGR1 knockdown in HepG2-naïve cells significantly increased total (∼1.2-fold) and cell-surface (∼4-fold) LDLR protein. In HepG2-PCSK9-knockout cells, ASGR1 silencing led to ∼2-fold higher levels of LDLR protein and DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)-LDL uptake associated with ∼9-fold increased cell-surface LDLR. Overexpression of WT-ASGR1/2 primarily reduced levels of immature non-O-glycosylated LDLR (∼110 kDa), whereas the triple Ala-mutant of Gln240/Trp244/Glu253 (characterized by loss of carbohydrate binding) reduced expression of the mature form of LDLR (∼150 kDa), suggesting that ASGR1 binds the LDLR in both a sugar-dependent and -independent fashion. The protease furin cleaves ASGR1 at the RKMK103↓ motif into a secreted form, likely resulting in a loss of function on LDLR. Altogether, we demonstrate that LDLR is the first example of a liver-receptor ligand of ASGR1. We conclude that silencing of ASGR1 and PCSK9 may lead to higher LDL uptake by hepatocytes, thereby providing a novel approach to further reduce LDL cholesterol levels.

MACF1 controls skeletal muscle function through the microtubule-dependent localization of extra-synaptic myonuclei and mitochondria biogenesis.

Skeletal muscles are composed of hundreds of multinucleated muscle fibers (myofibers) whose myonuclei are regularly positioned all along the myofiber's periphery except the few ones clustered underneath the neuromuscular junction (NMJ) at the synaptic zone. This precise myonuclei organization is altered in different types of muscle disease, including centronuclear myopathies (CNMs). However, the molecular machinery regulating myonuclei position and organization in mature myofibers remains largely unknown. Conversely, it is also unclear how peripheral myonuclei positioning is lost in the related muscle diseases. Here, we describe the microtubule-associated protein, MACF1, as an essential and evolutionary conserved regulator of myonuclei positioning and maintenance, in cultured mammalian myotubes, in Drosophila muscle, and in adult mammalian muscle using a conditional muscle-specific knockout mouse model. In vitro, we show that MACF1 controls microtubules dynamics and contributes to microtubule stabilization during myofiber's maturation. In addition, we demonstrate that MACF1 regulates the microtubules density specifically around myonuclei, and, as a consequence, governs myonuclei motion. Our in vivo studies show that MACF1 deficiency is associated with alteration of extra-synaptic myonuclei positioning and microtubules network organization, both preceding NMJ fragmentation. Accordingly, MACF1 deficiency results in reduced muscle excitability and disorganized triads, leaving voltage-activated sarcoplasmic reticulum Ca2+ release and maximal muscle force unchanged. Finally, adult MACF1-KO mice present an improved resistance to fatigue correlated with a strong increase in mitochondria biogenesis.

Functional analysis of natural PCSK9 mutants in modern and archaic humans.

PCSK9 is the last member of the proprotein convertases (PCs) family and its gene is mutated in ~ 2% to 3% of individuals with familial hypercholesterolemia (FH). This protein enhances the degradation of the low-density lipoprotein receptor (LDLR) and hence increases the levels of circulating LDL-cholesterol (LDLc). Studies of the underlying mechanism(s) regulating the activity of different mutations in the PCSK9 gene are ongoing as they enhance our understanding of the biology and clinical relevance of PCSK9 and its partners. In an attempt to unravel the regulation of PCSK9 transcription and possibly identify mutation 'hot spot' regions with alterations in CpG methylation, we present for the first time the complete methylome profile of the PCSK9 gene in modern and archaic humanoids. Our data showed that the genomes of modern humans and archaic PCSK9 exhibit a similar methylation pattern. Next, we defined the mechanistic consequences of three PCSK9 natural mutations (PCSK9-R96L, -R105W, and -P174S) and one archaic Denisovan mutation (PCSK9-H449L) using various complementary cellular and in vitro binding assays. Our results showed that the PCSK9-H449L is a loss-of-function (LOF) mutation, likely due to its lower binding affinity to the LDLR. Similarly, PCSK9-R96L and -R105W are LOF mutations, even though they have been identified in FH patients. The PCSK9-R105W mutation leads to a significantly lower autocatalytic processing of proPCSK9. PCSK9-P174S resulted in a LOF in both extracellular and intracellular pathways. In conclusion, our extensive analyses revealed that all studied mutations result in PCSK9 LOF, via various mechanisms, leading to lower levels of LDLc.

Ser-Phosphorylation of PCSK9 (Proprotein Convertase Subtilisin-Kexin 9) by Fam20C (Family With Sequence Similarity 20, Member C) Kinase Enhances Its Ability to Degrade the LDLR (Low-Density Lipoprotein Receptor).

OBJECTIVE: PCSK9 (proprotein convertase subtilisin-kexin 9) enhances the degradation of the LDLR (low-density lipoprotein receptor) in endosomes/lysosomes. This study aimed to determine the sites of PCSK9 phosphorylation at Ser-residues and the consequences of such posttranslational modification on the secretion and activity of PCSK9 on the LDLR. Approach and Results: Fam20C (family with sequence similarity 20, member C) phosphorylates serines in secretory proteins containing the motif S-X-E/phospho-Ser, including the cholesterol-regulating PCSK9. In situ hybridization of Fam20C mRNA during development and in adult mice revealed a wide tissue distribution, including liver, but not small intestine. Here, we show that Fam20C phosphorylates PCSK9 at Serines 47, 666, 668, and 688. In hepatocytes, phosphorylation enhances PCSK9 secretion and maximizes its induced degradation of the LDLR via the extracellular and intracellular pathways. Replacing any of the 4 Ser by the phosphomimetic Glu or Asp enhanced PCSK9 activity only when the other sites are phosphorylated, whereas Ala substitutions reduced it, as evidenced by Western blotting, Elisa, and LDLR-immunolabeling. This newly uncovered PCSK9/LDLR regulation mechanism refines our understanding of the implication of global PCSK9 phosphorylation in the modulation of LDL-cholesterol and rationalizes the consequence of natural mutations, for example, S668R and E670G. Finally, the relationship of Ser-phosphorylation to the implication of PCSK9 in regulating LDL-cholesterol in the neurological Fragile X-syndrome disorder was investigated. CONCLUSIONS: Ser-phosphorylation of PCSK9 maximizes both its secretion and activity on the LDLR. Mass spectrometric approaches to measure such modifications were developed and applied to quantify the levels of bioactive PCSK9 in human plasma under normal and pathological conditions.

LSD1 Controls Timely MyoD Expression via MyoD Core Enhancer Transcription.

MyoD is a master regulator of myogenesis. Chromatin modifications required to trigger MyoD expression are still poorly described. Here, we demonstrate that the histone demethylase LSD1/KDM1a is recruited on the MyoD core enhancer upon muscle differentiation. Depletion of Lsd1 in myoblasts precludes the removal of H3K9 methylation and the recruitment of RNA polymerase II on the core enhancer, thereby preventing transcription of the non-coding enhancer RNA required for MyoD expression (CEeRNA). Consistently, Lsd1 conditional inactivation in muscle progenitor cells during embryogenesis prevented transcription of the CEeRNA and delayed MyoD expression. Our results demonstrate that LSD1 is required for the timely expression of MyoD in limb buds and identify a new biological function for LSD1 by showing that it can activate RNA polymerase II-dependent transcription of enhancers.

The Proprotein Convertase Subtilisin/Kexin Type 9-resistant R410S Low Density Lipoprotein Receptor Mutation: A NOVEL MECHANISM CAUSING FAMILIAL HYPERCHOLESTEROLEMIA.

Familial hypercholesterolemia (FH) is characterized by severely elevated low density lipoprotein (LDL) cholesterol. Herein, we identified an FH patient presenting novel compound heterozygote mutations R410S and G592E of the LDL receptor (LDLR). The patient responded modestly to maximum rosuvastatin plus ezetimibe therapy, even in combination with a PCSK9 monoclonal antibody injection. Using cell biology and molecular dynamics simulations, we aimed to define the underlying mechanism(s) by which these LDLR mutations affect LDL metabolism and lead to hypercholesterolemia. Our data showed that the LDLR-G592E is a class 2b mutant, because it mostly failed to exit the endoplasmic reticulum and was degraded. Even though LDLR-R410S and LDLR-WT were similar in levels of cell surface and total receptor and bound equally well to LDL or extracellular PCSK9, the LDLR-R410S was resistant to exogenous PCSK9-mediated degradation in endosomes/lysosomes and showed reduced LDL internalization and degradation relative to LDLR-WT. Evidence is provided for a tighter association of LDL with LDLR-R410S at acidic pH, a reduced LDL delivery to late endosomes/lysosomes, and an increased release in the medium of the bound/internalized LDL, as compared with LDLR-WT. These data suggested that LDLR-R410S recycles loaded with its LDL-cargo. Our findings demonstrate that LDLR-R410S represents an LDLR loss-of-function through a novel class 8 FH-causing mechanism, thereby rationalizing the observed phenotype.

MuSK frizzled-like domain is critical for mammalian neuromuscular junction formation and maintenance.

The muscle-specific kinase MuSK is one of the key molecules orchestrating neuromuscular junction (NMJ) formation. MuSK interacts with the Wnt morphogens, through its Frizzled-like domain (cysteine-rich domain [CRD]). Dysfunction of MuSK CRD in patients has been recently associated with the onset of myasthenia, common neuromuscular disorders mainly characterized by fatigable muscle weakness. However, the physiological role of Wnt-MuSK interaction in NMJ formation and function remains to be elucidated. Here, we demonstrate that the CRD deletion of MuSK in mice caused profound defects of both muscle prepatterning, the first step of NMJ formation, and synapse differentiation associated with a drastic deficit in AChR clusters and excessive growth of motor axons that bypass AChR clusters. Moreover, adult MuSKΔCRD mice developed signs of congenital myasthenia, including severe NMJs dismantlement, muscle weakness, and fatigability. We also report, for the first time, the beneficial effects of lithium chloride, a reversible inhibitor of the glycogen synthase kinase-3, that rescued NMJ defects in MuSKΔCRD mice and therefore constitutes a novel therapeutic reagent for the treatment of neuromuscular disorders linked to Wnt-MuSK signaling pathway deficiency. Together, our data reveal that MuSK CRD is critical for NMJ formation and plays an unsuspected role in NMJ maintenance in adulthood.

Schwann cells sense and control acetylcholine spillover at the neuromuscular junction by α7 nicotinic receptors and butyrylcholinesterase.

Terminal Schwann cells (TSCs) are key components of the mammalian neuromuscular junction (NMJ). How the TSCs sense the synaptic activity in physiological conditions remains unclear. We have taken advantage of the distinct localization of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) at the NMJ to bring out the function of different ACh receptors (AChRs). AChE is clustered by the collagen Q in the synaptic cleft and prevents the repetitive activation of muscle nicotinic AChRs. We found that BChE is anchored at the TSC by a proline-rich membrane anchor, the small transmembrane protein anchor of brain AChE. When BChE was specifically inhibited, ACh release was significant depressed through the activation of α7 nAChRs localized on the TSC and activated by the spillover of ACh. When both AChE and BChE were inhibited, the spillover increased and induced a dramatic reduction of ACh release that compromised the muscle twitch triggered by the nerve stimulation. α7 nAChRs at the TSC may act as a sensor for spillover of ACh adjusted by BChE and may represent an extrasynaptic sensor for homeostasis at the NMJ. In myasthenic rats, selective inhibition of AChE is more effective in rescuing muscle function than the simultaneous inhibition of AChE and BChE because the concomitant inhibition of BChE counteracts the positive action of AChE inhibition. These results show that inhibition of BChE should be avoided during the treatment of myasthenia and the pharmacological reversal of residual curarization after anesthesia.

An effective solution for capturing the single twitch of muscle: application to monitor muscle relaxation.

In this study, a fast algorithm was developed to capture of train of four and to filter extra contraction and noises. A low pass filter created to filter extra contraction and high frequency noises. Then, a TCA algorithm designed to capturing of the single twitch of muscle. The algorithm updated to remove embedded extra contraction and to derive boundary values in this location from cubic spline interpolation. Efficiency of TCA and effect of extra contraction tested in time and frequency domain.

Rab7 is functionally required for selective cargo sorting at the early endosome.

The small GTPases of the Rab family act as a molecular switch regulating various aspects of membrane trafficking through the selective recruitment of effector proteins. Whereas Rab7 has been classically involved in the regulation of transport within the endolysosomal network, persistent controversy remains as to whether Rab7 also plays a role in earlier steps of endosomal trafficking. In this study, we show that Rab7 depletion or inactivation results in enlargement of both early and late endosomes. Rab7 depletion led to the retention of a significant fraction of internalized low-density lipoproteins (LDL) mainly in enlarged early endosomes (EE). As a result, LDL processing and the transcriptional regulation of sterol-sensitive genes were impaired. We found that Rab7 activity was also required for the sorting of the mannose-6-phosphate receptor, the interferon alpha-receptor and the Shiga toxin B-subunit. In contrast, epidermal growth factor (EGF) sorting at the EE or the recycling of transferrin and LDL-R were not affected by Rab7 depletion. Our findings demonstrate that in addition to regulating late endosomes (LE) to lysosomes transport, Rab7 plays a functional role in the selective sorting of distinct cargos at the EE and that the Rab5 to Rab7 exchange occurs early in the endosomal maturation process.

Muscle histone deacetylase 4 upregulation in amyotrophic lateral sclerosis: potential role in reinnervation ability and disease progression.

Amyotrophic lateral sclerosis is a typically rapidly progressive neurodegenerative disorder affecting motor neurons leading to progressive muscle paralysis and death, usually from respiratory failure, in 3-5 years. Some patients have slow disease progression and prolonged survival, but the underlying mechanisms remain poorly understood. Riluzole, the only approved treatment, only modestly prolongs survival and has no effect on muscle function. In the early phase of the disease, motor neuron loss is initially compensated for by collateral reinnervation, but over time this compensation fails, leading to progressive muscle wasting. The crucial role of muscle histone deacetylase 4 and its regulator microRNA-206 in compensatory reinnervation and disease progression was recently suggested in a mouse model of amyotrophic lateral sclerosis (transgenic mice carrying human mutations in the superoxide dismutase gene). Here, we sought to investigate whether the microRNA-206-histone deacetylase 4 pathway plays a role in muscle compensatory reinnervation in patients with amyotrophic lateral sclerosis and thus contributes to disease outcome differences. We studied muscle reinnervation using high-resolution confocal imaging of neuromuscular junctions in muscle samples obtained from 11 patients with amyotrophic lateral sclerosis, including five long-term survivors. We showed that the proportion of reinnervated neuromuscular junctions was significantly higher in long-term survivors than in patients with rapidly progressive disease. We analysed the expression of muscle candidate genes involved in the reinnervation process and showed that histone deacetylase 4 upregulation was significantly greater in patients with rapidly progressive disease and was negatively correlated with the extent of muscle reinnervation and functional outcome. Conversely, the proposed regulator of histone deacetylase 4, microRNA-206, was upregulated in both patient groups, but did not correlate with disease progression or reinnervation. We conclude that muscle expression of histone deacetylase 4 may be a key factor for muscle reinnervation and disease progression in patients with amyotrophic lateral sclerosis. Specific histone deacetylase 4 inhibitors may then constitute a therapeutic approach to enhancing motor performance and slowing disease progression in amyotrophic lateral sclerosis.

Identification and functional characterization of a novel α-conotoxin (EIIA) from Conus ermineus.

Nicotinic acetylcholine receptors (nAChRs) are one of the most important families in the ligand-gated ion channel superfamily due to their involvement in primordial brain functions and in several neurodegenerative pathologies. The discovery of new ligands which can bind with high affinity and selectivity to nAChR subtypes is of prime interest in order to study these receptors and to potentially discover new drugs for treating various pathologies. Predatory cone snails of the genus Conus hunt their prey using venoms containing a large number of small, highly structured peptides called conotoxins. Conotoxins are classified in different structural families and target a large panel of receptors and ion channels. Interestingly, nAChRs represent the only subgroup for which Conus has developed seven distinct families of conotoxins. Conus venoms have thus received much attention as they could represent a potential source of selective ligands of nAChR subtypes. We describe the mass spectrometric-based approaches which led to the discovery of a novel α-conotoxin targeting muscular nAChR from the venom of Conus ermineus. The presence of several posttranslational modifications complicated the N-terminal sequencing. To discriminate between the different possible sequences, analogs with variable N-terminus were synthesized and fragmented by MS/MS. Understanding the fragmentation pathways in the low m/z range appeared crucial to determine the right sequence. The biological activity of this novel α-conotoxin (α-EIIA) that belongs to the unusual α4/4 subfamily was determined by binding experiments. The results revealed not only its selectivity for the muscular nAChR, but also a clear discrimination between the two binding sites described for this receptor.

Monoclonal antibodies to mouse butyrylcholinesterase.

Our immunization strategy introduced recombinant mouse butyrylcholinesterase (BChE) to naïve BChE knockout mice. An extraordinarily strong immune reaction gave rise to a whole spectrum of antibodies with different properties. Two selective and highly efficient monoclonal anti-mouse BChE antibodies 4H1 (IgG1) and 4 C9 (IgG2a), with Kd values in the nanomolar range were generated. ELISA detected BChE in as little as 20-50 nl of mouse plasma using 2 µg (4H1) or 4 µg (4C9). Both antibodies cross-reacted with BChE in dog plasma but only 4 H1 reacted with rat BChE, suggesting that the antibodies are targeted towards different epitopes. Surprisingly, neither recognized human BChE. The anti-mouse BChE antibodies were used in immunohistochemistry analysis of mouse muscle where they specifically stained the neuromuscular junction. The antibodies enable visualization of the BChE protein in the mouse tissue, thus complementing activity assays. They can be used to study a long-lasting question about the existence of mixed acetylcholinesterase/BChE oligomers in mouse tissues. Moreover, monoclonal anti-mouse BChE antibodies can provide a simple, fast and efficient way to purify mouse BChE from small amounts of starting material by using a single-step immunomagnetic bead-based protocol.

A new mouse model for the slow-channel congenital myasthenic syndrome induced by the AChR εL221F mutation.

We have generated a new mouse model for congenital myasthenic syndromes by inserting the missense mutation L221F into the ε subunit of the acetylcholine receptor by homologous recombination. This mutation has been identified in man to cause a mild form of slow-channel congenital myasthenic syndrome with variable penetrance. In our mouse model we observe as in human patients prolonged endplate currents. The summation of endplate potentials may account for a depolarization block at increasing stimulus frequencies, moderate reduced muscle strength and tetanic fade. Calcium and intracellular vesicle accumulation as well as junctional fold loss and organelle degeneration underlying a typical endplate myopathy, were identified. Moreover, a remodeling of neuromuscular junctions occurs in a muscle-dependent pattern expressing variable phenotypic effects. Altogether, this mouse model provides new insight into the pathophysiology of congenital myasthenia and serves as a new tool for deciphering signaling pathways induced by excitotoxicity at peripheral synapses.

The dynamin chemical inhibitor dynasore impairs cholesterol trafficking and sterol-sensitive genes transcription in human HeLa cells and macrophages.

Intracellular transport of cholesterol contributes to the regulation of cellular cholesterol homeostasis by mechanisms that are yet poorly defined. In this study, we characterized the impact of dynasore, a recently described drug that specifically inhibits the enzymatic activity of dynamin, a GTPase regulating receptor endocytosis and cholesterol trafficking. Dynasore strongly inhibited the uptake of low-density lipoprotein (LDL) in HeLa cells, and to a lower extent in human macrophages. In both cell types, dynasore treatment led to the abnormal accumulation of LDL and free cholesterol (FC) within the endolysosomal network. The measure of cholesterol esters (CE) further showed that the delivery of regulatory cholesterol to the endoplasmic reticulum (ER) was deficient. This resulted in the inhibition of the transcriptional control of the three major sterol-sensitive genes, sterol-regulatory element binding protein 2 (SREBP-2), 3-hydroxy-3-methyl-coenzymeA reductase (HMGCoAR), and low-density lipoprotein receptor (LDLR). The sequestration of cholesterol in the endolysosomal compartment impaired both the active and passive cholesterol efflux in HMDM. Our data further illustrate the importance of membrane trafficking in cholesterol homeostasis and validate dynasore as a new pharmacological tool to study the intracellular transport of cholesterol.

Distinct localization of collagen Q and PRiMA forms of acetylcholinesterase at the neuromuscular junction.

Acetylcholinesterase (AChE) terminates the action of acetylcholine at cholinergic synapses thereby preventing rebinding of acetylcholine to nicotinic postsynaptic receptors at the neuromuscular junction. Here we show that AChE is not localized close to these receptors on the postsynaptic surface, but is instead clustered along the presynaptic membrane and deep in the postsynaptic folds. Because AChE is anchored by ColQ in the basal lamina and is linked to the plasma membrane by a transmembrane subunit (PRiMA), we used a genetic approach to evaluate the respective contribution of each anchoring oligomer. By visualization and quantification of AChE in mouse strains devoid of ColQ, PRiMA or AChE, specifically in the muscle, we found that along the nerve terminus the vast majority of AChE is anchored by ColQ that is only produced by the muscle, whereas very minor amounts of AChE are anchored by PRiMA that is produced by motoneurons. In its synaptic location, AChE is therefore positioned to scavenge ACh that effluxes from the nerve by non-quantal release. AChE-PRiMA, produced by the muscle, is diffusely distributed along the muscle in extrajunctional regions.

The marine polyether gambierol enhances muscle contraction and blocks a transient K(+) current in skeletal muscle cells.

Gambierol is a complex marine toxin first isolated with ciguatoxins from cell cultures of the toxic dinoflagellate Gambierdiscus toxicus. Despite the chemical complexity of the polycyclic ether toxin, the total successful synthesis of gambierol has been achieved by different chemical strategies. In the present work the effects of synthetic gambierol on mouse and frog skeletal neuromuscular preparations and Xenopus skeletal myocytes have been studied. Gambierol (0.1-5 muM) significantly increased isometric twitch tension in neuromuscular preparations stimulated through the motor nerve. Less twitch augmentation was observed in directly stimulated muscles when comparing twitch tension-time integrals obtained by nerve stimulation. Also, gambierol induced small spontaneous muscle contraction originating from presynaptic activity that was completely inhibited by d-tubocurarine. Gambierol slowed the rate of muscle action potential repolarization, triggered spontaneous and/or repetitive action potentials, and neither affected action potential amplitude nor overshoot in skeletal muscle fibers. These results suggest that gambierol through an action on voltage-gated K(+) channels prolongs the duration of action potentials, enhances the extent and time course of Ca(2+) release from the sarcoplasmic reticulum, and increases twitch tension generation. Further evidence is provided that gambierol at sub-micromolar concentrations blocks a fast inactivating outward K(+) current that is responsible for action potential prolongation in Xenopus skeletal myocytes.