Gain-of-Function Dynamin-2 Mutations Linked to Centronuclear Myopathy Impair Ca2+-Induced Exocytosis in Human Myoblasts

Gain-of-function mutations of dynamin-2, a mechano-GTPase that remodels membrane and actin filaments, cause centronuclear myopathy (CNM), a congenital disease that mainly affects skeletal muscle tissue. Among these mutations, the variants p.A618T and p.S619L lead to a gain of function and cause a severe neonatal phenotype. By using total internal reflection fluorescence microscopy (TIRFM) in immortalized human myoblasts expressing the pH-sensitive fluorescent protein (pHluorin) fused to the insulin-responsive aminopeptidase IRAP as a reporter of the GLUT4 vesicle trafficking, we measured single pHluorin signals to investigate how p.A618T and p.S619L mutations influence exocytosis. We show here that both dynamin-2 mutations significantly reduced the number and durations of pHluorin signals induced by 10 μM ionomycin, indicating that in addition to impairing exocytosis, they also affect the fusion pore dynamics. These mutations also disrupt the formation of actin filaments, a process that reportedly favors exocytosis. This altered exocytosis might importantly disturb the plasmalemma expression of functional proteins such as the glucose transporter GLUT4 in skeletal muscle cells, impacting the physiology of the skeletal muscle tissue and contributing to the CNM disease.Fil: Bayonés, Lucas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Guerra Fernández, María José. Universidad de Valparaíso; ChileFil: Hinostroza, Fernando. Universidad Catolica de Maule; ChileFil: Báez Matus, Ximena. Universidad de Valparaíso; ChileFil: Vásquez Navarrete, Jacqueline. Universidad de Valparaíso; ChileFil: Gallo, Luciana Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Parra, Sergio. Universidad de Valparaíso; ChileFil: Martínez, Agustín D.. Universidad de Valparaíso; ChileFil: González Jamett, Arlek. Universidad de Valparaíso; ChileFil: Marengo, Fernando Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Cárdenas, Ana M.. Universidad de Valparaíso; Chil

Role of Tau Protein in Neuronal Damage in Alzheimer's Disease and Down Syndrome

Neurodegenerative disorders constitute a growing concern worldwide. Their incidence has increased steadily, in particular among the elderly, a high-risk population that is becoming an important segment of society. Neurodegenerative mechanisms underlie many ailments such as Parkinson’s disease, Huntington’s disease, Alzheimer’s disease (AD) and Down syndrome (DS, trisomy 21). Interestingly, there is increasing evidence suggesting that many such diseases share pathogenic mechanisms at the cellular and subcellular levels. These include altered protein misfolding, impaired autophagy, mitochondrial dysfunction, membrane damage, and altered axonal transport. Regarding AD and DS, the first common link comes from observations that DS patients undergo ADlike pathology early in adulthood. Also, the gene encoding for the amyloid precursor protein is present in human autosome 21 and in murine chromosome 16, an animal model of DS. Important functions related to preservation of normal neuronal architecture are impaired in both conditions. In particular, the stable assembly of microtubules, which is critical for the cytoskeleton, is impaired in AD and DS. In this process, tau protein plays a pivotal role in controlling microtubule stability. Abnormal tau expression and hyperphosphorylation are common features in both conditions, yet the mechanisms leading to these phenomena remain obscure. In the present report we review possible common mechanisms that may alter tau expression and function, in particular in relation to the effect of certain overexpressed DS-related genes, using cellular models of human DS. The latter contributes to the identification of possible therapeutic targets that could aid in the treatment of both AD and DS

Centronuclear myopathy-causing mutations in dynamin-2 impair actin-dependent trafficking in muscle cells

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N-Acetylcysteine Reduces Skeletal Muscles Oxidative Stress and Improves Grip Strength in Dysferlin-Deficient Bla/J Mice

Dysferlinopathy is an autosomal recessive muscular dystrophy resulting from mutations in the dysferlin gene. Absence of dysferlin in the sarcolemma and progressive muscle wasting are hallmarks of this disease. Signs of oxidative stress have been observed in skeletal muscles of dysferlinopathy patients, as well as in dysferlin-deficient mice. However, the contribution of the redox imbalance to this pathology and the efficacy of antioxidant therapy remain unclear. Here, we evaluated the effect of 10 weeks diet supplementation with the antioxidant agentN-acetylcysteine (NAC, 1%) on measurements of oxidative damage, antioxidant enzymes, grip strength and body mass in 6 months-old dysferlin-deficient Bla/J mice and wild-type (WT) C57 BL/6 mice. We found that quadriceps and gastrocnemius muscles of Bla/J mice exhibit high levels of lipid peroxidation, protein carbonyls and superoxide dismutase and catalase activities, which were significantly reduced by NAC supplementation. By using the Kondziela's inverted screen test, we further demonstrated that NAC improved grip strength in dysferlin deficient animals, as compared with non-treated Bla/J mice, without affecting body mass. Together, these results indicate that this antioxidant agent improves skeletal muscle oxidative balance, as well as muscle strength and/or resistance to fatigue in dysferlin-deficient animals.Comision Nacional de Investigacion Cientifica y Tecnologica (CONICYT) CONICYT FONDECYT 1160495 1151383 ICM-ANID, Chile P09-022-F Comision Nacional de Investigacion Cientifica y Tecnologica (CONICYT) FB0001 Millennium Scientific Initiative of the Ministerio de Economia, Fomento y Turism

Dynamin-2 Regulates Fusion Pore Expansion and Quantal Release through a Mechanism that Involves Actin Dynamics in Neuroendocrine Chromaffin Cells

<div><p>Over the past years, dynamin has been implicated in tuning the amount and nature of transmitter released during exocytosis. However, the mechanism involved remains poorly understood. Here, using bovine adrenal chromaffin cells, we investigated whether this mechanism rely on dynamin’s ability to remodel actin cytoskeleton. According to this idea, inhibition of dynamin GTPase activity suppressed the calcium-dependent <i>de novo</i> cortical actin and altered the cortical actin network. Similarly, expression of a small interfering RNA directed against dynamin-2, an isoform highly expressed in chromaffin cells, changed the cortical actin network pattern. Disruption of dynamin-2 function, as well as the pharmacological inhibition of actin polymerization with cytochalasine-D, slowed down fusion pore expansion and increased the quantal size of individual exocytotic events. The effects of cytochalasine-D and dynamin-2 disruption were not additive indicating that dynamin-2 and F-actin regulate the late steps of exocytosis by a common mechanism. Together our data support a model in which dynamin-2 directs actin polymerization at the exocytosis site where both, in concert, adjust the hormone quantal release to efficiently respond to physiological demands.</p></div

Amperometric parameters of exocytotic events induced by 10 µM DMPP in cells transfected with pEGFP, UnR-iRNA, iRNADyn2, Dyn2WT or Dyn2K44A.

<p>Data are means ± SEM of averages, where n is the number of cells. Imax corresponds to the spike amplitude.</p>*<p>p<0.05 compared with pEGFP-transfected cells;</p>†<p>p<0.05 compared with Dyn2 WT-transfected cells (Kruskal-Wallis test).</p

Calcium-dependent cortical actin polymerization in permeabilized chromaffin cells.

<p>Cultured chromaffin cells were permeabilized in buffer KGEP (mM: 139 K⁺-glutamate, 20 Pipes, 5 EGTA, 2 ATP-Mg and 0.01 free calcium, pH 6.6) during 6 minutes with 20 µM digitonin in the presence of 0.3 µM Alexa-Fluor488-G-actin conjugate (AF488-G-actin), fixed and visualized by confocal microscopy. <b>A:</b> Total F-actin was stained using 1 µM phalloidin-rodhamine B (red) and nuclei were stained with 5 µg/ml DAPI (blue). Note that newly synthesized actin was incorporated into pre-existing cortical filaments. <b>B–C:</b> The new formation of cortical actin filaments was assessed by quantifying AF488-G-actin staining mean intensity at the cell periphery in the presence of increasing free Ca²⁺ concentrations. Note that maximal cortical actin polymerization was observed at a range of 1–10 µM of free Ca^2+. Scale = 10 µm. Data are means of cortical actin fluorescence intensity from at least 12 cells per each Ca²⁺ concentration (12 cells for 0.01 µM Ca²⁺, 13 cells for 0.1 µM Ca²⁺, 15 cells for 1 µM Ca²⁺,and 18 cells for 10 µM Ca²⁺).</p

Impaired function or expression of dynamin-2 change F-actin organization pattern.

<p>Cells were transfected with Life-act-ruby (n = 11) or co-transfected with Life-act-ruby and pEGFP (n = 34), iRNA-UnR (n = 9) Dyn2WT (n = 21), Dyn2K44A (n = 31), iRNADyn2 (n = 38) or Eps15ED95/295 (n = 17) plasmids and visualized by TIRF microscopy 48 h later. To evaluate the effects of a pharmacological inhibition of dynamin, cells transfected with Life-act-ruby were treated with 100 µM dynasore (n = 28), or the vehicle DMSO (n = 25) during 1 hr at 37°C. The 81.8% of control cells exhibited a “normal” pattern with clear cortical actin fibers. This value was not significantly different in cells expressing pEGFP (73.6%), iRNA-UnR (88.9%) or Dyn2WT (85.7%) constructs. However, the expression of Dyn2K44A or iRNADyn2, as well as the treatment with dynasore, modified the cortical actin organization and 80.6%, 92.1% and 71.4% of the cells, respectively, exhibited a “punctuate” pattern. The treatment with 4 µM CytoD during 10 minutes at 37°C produced exactly the same effect: 84.6% of the cells displayed a “punctuate” pattern. Eps15ED95/295 expression did not alter actin organization (82.4% of cells exhibited a normal pattern), indicating that dynamin, but not of endocytosis disruption, modified the actin cytoskeleton pattern. Scale bar = 5 µm.</p

Dynamin-2 and actin polymerization regulate the fusion pore expansion and quantal size in BCC.

<p>Chromaffin cells were incubated with 4 µM CytoD during 10 minutes at 37°C. After that the exocytosis was evoked with 10 µM DMPP. <b>A–C:</b> Data show average values ± SEM of Q (A), t_1/2 (B) and foot duration (C) of amperometric spikes induced by 10 µM DMPP in cells transfected with pEGFP (n = 27), Dyn2K44A (n = 13) or iRNADyn2 (n = 16). All amperometric parameter values correspond to the median values of the events from individual cells, which were subsequently averaged per treatment group. Thus, n correspond to the number of cells in each treatment group. Note that the CytoD treatment (grey bars) significantly increased Q, t_1/2 and foot duration of the exocytotic events in cells transfected with pEGFP, without additional effects in cells transfected with Dyn2K44A or iRNADyn2. * p<0.05 compared with the untreated cells (Kruskal-Wallis test).</p