scholarly journals Caveolin 1 is required for axonal outgrowth of motor neurons and affects Xenopus neuromuscular development

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Marlen Breuer ◽  
Hanna Berger ◽  
Annette Borchers
Keyword(s):  
Motor Neurons ◽  
Muscle Development ◽  
Phosphorylation Site ◽  
Muscle Cells ◽  
Axonal Outgrowth ◽  
Loss Of Function ◽  
Caveolin 1 ◽  
Voltage Gated Sodium Channels ◽  
Resolution Imaging ◽  
Morpholino Oligonucleotides

Abstract Caveolins are essential structural proteins driving the formation of caveolae, specialized invaginations of the plasma membrane. Loss of Caveolin-1 (Cav1) function in mice causes distinct neurological phenotypes leading to impaired motor control, however, the underlying developmental mechanisms are largely unknown. In this study we find that loss-of-function of Xenopus Cav1 results in a striking swimming defect characterized by paralysis of the morphants. High-resolution imaging of muscle cells revealed aberrant sarcomeric structures with disorganized actin fibers. As cav1 is expressed in motor neurons, but not in muscle cells, the muscular abnormalities are likely a consequence of neuronal defects. Indeed, targeting cav1 Morpholino oligonucleotides to neural tissue, but not muscle tissue, disrupts axonal outgrowth of motor neurons and causes swimming defects. Furthermore, inhibition of voltage-gated sodium channels mimicked the Cav1 loss-of-function phenotype. In addition, analyzing axonal morphology we detect that Cav1 loss-of-function causes excessive filopodia and lamellipodia formation. Using rescue experiments, we show that the Cav1 Y14 phosphorylation site is essential and identify a role of RhoA, Rac1, and Cdc42 signaling in this process. Taken together, these results suggest a previously unrecognized function of Cav1 in muscle development by supporting axonal outgrowth of motor neurons.

scholarly journals The Link between VAPB Loss of Function and Amyotrophic Lateral Sclerosis

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1865
Author(s):  
Nica Borgese ◽  
Nicola Iacomino ◽  
Sara Francesca Colombo ◽  
Francesca Navone
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Motor Neurons ◽  
Adaptor Proteins ◽  
Loss Of Function ◽  
Membrane Contact Sites ◽  
Physiological Processes ◽  
Main Driver ◽  
Single Allele ◽  
Induced Pluripotent ◽  
Lateral Sclerosis

The VAP proteins are integral adaptor proteins of the endoplasmic reticulum (ER) membrane that recruit a myriad of interacting partners to the ER surface. Through these interactions, the VAPs mediate a large number of processes, notably the generation of membrane contact sites between the ER and essentially all other cellular membranes. In 2004, it was discovered that a mutation (p.P56S) in the VAPB paralogue causes a rare form of dominantly inherited familial amyotrophic lateral sclerosis (ALS8). The mutant protein is aggregation-prone, non-functional and unstable, and its expression from a single allele appears to be insufficient to support toxic gain-of-function effects within motor neurons. Instead, loss-of-function of the single wild-type allele is required for pathological effects, and VAPB haploinsufficiency may be the main driver of the disease. In this article, we review the studies on the effects of VAPB deficit in cellular and animal models. Several basic cell physiological processes are affected by downregulation or complete depletion of VAPB, impinging on phosphoinositide homeostasis, Ca2+ signalling, ion transport, neurite extension, and ER stress. In the future, the distinction between the roles of the two VAP paralogues (A and B), as well as studies on motor neurons generated from induced pluripotent stem cells (iPSC) of ALS8 patients will further elucidate the pathogenic basis of p.P56S familial ALS, as well as of other more common forms of the disease.

scholarly journals Effects of temperature on proliferation of myoblasts from donor piglets with different thermoregulatory maturities

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Katharina Metzger ◽  
Dirk Dannenberger ◽  
Armin Tuchscherer ◽  
Siriluck Ponsuksili ◽  
Claudia Kalbe
Keyword(s):  
Muscle Development ◽  
Muscle Growth ◽  
Extreme Temperature ◽  
Growth Inhibitor ◽  
Muscle Cells ◽  
The Body ◽  
Farm Animals ◽  
Neonatal Piglets ◽  
Protein Levels

Abstract Background Climate change and the associated risk for the occurrence of extreme temperature events or permanent changes in ambient temperature are important in the husbandry of farm animals. The aim of our study was to investigate the effects of permanent cultivation temperatures below (35 °C) and above (39 °C, 41 °C) the standard cultivation temperature (37 °C) on porcine muscle development. Therefore, we used our porcine primary muscle cell culture derived from satellite cells as an in vitro model. Neonatal piglets have limited thermoregulatory stability, and several days after birth are required to maintain their body temperature. To consider this developmental step, we used myoblasts originating from thermolabile (five days of age) and thermostable piglets (twenty days of age). Results The efficiency of myoblast proliferation using real-time monitoring via electrical impedance was comparable at all temperatures with no difference in the cell index, slope or doubling time. Both temperatures of 37 °C and 39 °C led to similar biochemical growth properties and cell viability. Only differences in the mRNA expression of myogenesis-associated genes were found at 39 °C compared to 37 °C with less MYF5, MYOD and MSTN and more MYH3 mRNA. Myoblasts grown at 35 °C are smaller, exhibit higher DNA synthesis and express higher amounts of the satellite cell marker PAX7, muscle growth inhibitor MSTN and metabolic coactivator PPARGC1A. Only permanent cultivation at 41 °C resulted in higher HSP expression at the mRNA and protein levels. Interactions between the temperature and donor age showed that MYOD, MYOG, MYH3 and SMPX mRNAs were temperature-dependently expressed in myoblasts of thermolabile but not thermostable piglets. Conclusions We conclude that 37 °C to 39 °C is the best physiological temperature range for adequate porcine myoblast development. Corresponding to the body temperatures of piglets, it is therefore possible to culture primary muscle cells at 39 °C. Only the highest temperature of 41 °C acts as a thermal stressor for myoblasts with increased HSP expression, but it also accelerates myogenic development. Cultivation at 35 °C, however, leads to less differentiated myoblasts with distinct thermogenetic activity. The adaptive behavior of derived primary muscle cells to different cultivation temperatures seems to be determined by the thermoregulatory stability of the donor piglets.

scholarly journals PDGFBB promotes proliferation and migration via regulating miR-1181/STAT3 axis in human pulmonary arterial smooth muscle cells

2018 ◽  
Vol 315 (6) ◽  
pp. L965-L976 ◽  
Author(s):  
Zhengjiang Qian ◽  
Yanjiao Li ◽  
Haiyang Yang ◽  
Jidong Chen ◽  
Xiang Li ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Causative Factor ◽  
Loss Of Function ◽  
Migration Flow ◽  
Pulmonary Arterial ◽  
And Migration ◽  
Proliferation And Migration

Platelet-derived growth factor (PDGF) can induce hyperproliferation of pulmonary artery smooth muscle cells (PASMCs), which is a key causative factor to the occurrence and progression of pulmonary arterial hypertension (PAH). We previously identified that miR-1181 is significantly downregulated by PDGFBB in human PASMCs. In this work, we further explore the function of miR-1181 and underlying regulatory mechanisms in PDGF-induced PASMCs. First, the expression pattern of miR-1181 was characterized under PDGFBB treatment, and PDGF receptor/PKCβ signaling was found to repress miR-1181 expression. Then, gain- and loss-of-function experiments were respectively conducted and revealed the prominent role of miR-1181 in inhibiting PASMC proliferation and migration. Flow cytometry analysis suggested that miR-1181 regulated the PASMC proliferation through influencing the cell cycle transition from G0/G1 to S phase. Moreover, we exhibited that miR-1181 targeting STAT3 formed a regulatory axis to modulate PASMC proliferation. Finally, serum miR-1181 expression was also observed to be reduced in adult and newborn patients with PAH. Overall, this study provides novel findings that the miR-1181/STAT3 axis mediated PDGFBB-induced dysfunction in human PASMCs, implying a potential use of miR-1181 as a therapeutic and diagnostic candidate for the vascular remodeling diseases.

scholarly journals Loss of TBK1 activity leads to TDP-43 proteinopathy through lysosomal dysfunction in human motor neurons

2021 ◽  
Author(s):  
Jin Hao ◽  
Michael F Wells ◽  
Gengle Niu ◽  
Irune Guerra San Juan ◽  
Francesco Limone ◽  
...  
Keyword(s):  
Motor Neuron ◽  
Neurodegenerative Disease ◽  
Motor Neurons ◽  
Inhibition Mechanism ◽  
Loss Of Function ◽  
Neurodegenerative Process ◽  
Lysosomal Dysfunction ◽  
Human Motor ◽  
Lateral Sclerosis ◽  
Lysosomal Acidification

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron loss accompanied by cytoplasmic localization of TDP-43 proteins and their insoluble accumulations. Haploinsufficiency of TBK1 has been found to associate with or cause ALS. However, the cell-autonomous mechanisms by which reduced TBK1 activity contributes to human motor neuron pathology remain elusive. Here, we generated a human cellular model harboring loss-of-function mutations of TBK1 by gene editing and found that TBK1 deficiency was sufficient to cause TDP-43 pathology in human motor neurons. In addition to its functions in autophagy, we found that TBK1 interacted with endosomes and was required for normal endosomal maturation and subsequent lysosomal acidification. Surprisingly, TDP-43 pathology resulted more from the dysfunctional endo-lysosomal pathway than the previously recognized autophagy inhibition mechanism. Restoring TBK1 levels ameliorated lysosomal dysfunction and TDP-43 pathology and maintained normal motor neuron homeostasis. Notably, using patient-derived motor neurons, we found that haploinsufficiency of TBK1 sensitized neurons to lysosomal stress, and chemical regulators of endosomal maturation rescued the neurodegenerative process. Together, our results revealed the mechanism of TBK1 in maintaining TDP-43 and motor neuron homeostasis and suggested that modulating endosomal maturation was able to rescue neurodegenerative disease phenotypes caused by TBK1 deficiency.

scholarly journals Inhibitory effect of caveolin-1 in vascular endothelial cells, pericytes and smooth muscle cells

Oncotarget ◽  
2017 ◽  
Vol 8 (44) ◽  
pp. 76165-76173 ◽  
Author(s):  
Hongping Xu ◽  
Liwei Zhang ◽  
Wei Chen ◽  
Jiazhou Xu ◽  
Ruting Zhang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Endothelial Cells ◽  
Muscle Cells ◽  
Inhibitory Effect ◽  
Vascular Endothelial ◽  
Caveolin 1

Urokinase-receptor-mediated phenotypic changes in vascular smooth muscle cells require the involvement of membrane rafts

2009 ◽  
Vol 423 (3) ◽  
pp. 343-351 ◽  
Author(s):  
Julia Kiyan ◽  
Graham Smith ◽  
Hermann Haller ◽  
Inna Dumler
Keyword(s):  
Smooth Muscle ◽  
Cell Migration ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Lipid Rafts ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Caveolin 1 ◽  
Phenotypic Changes ◽  
Dependent Cell

The cholesterol-enriched membrane microdomains lipid rafts play a key role in cell activation by recruiting and excluding specific signalling components of cell-surface receptors upon receptor engagement. Our previous studies have demonstrated that the GPI (glycosylphosphatidylinositol)-linked uPAR [uPA (urokinase-type plasminogen activator) receptor], which can be found in lipid rafts and in non-raft fractions, can mediate the differentiation of VSMCs (vascular smooth muscle cells) towards a pathophysiological de-differentiated phenotype. However, the mechanism by which uPAR and its ligand uPA regulate VSMC phenotypic changes is not known. In the present study, we provide evidence that the molecular machinery of uPAR-mediated VSMC differentiation employs lipid rafts. We show that the disruption of rafts in VSMCs by membrane cholesterol depletion using MCD (methyl-β-cyclodextrin) or filipin leads to the up-regulation of uPAR and cell de-differentiation. uPAR silencing by means of interfering RNA resulted in an increased expression of contractile proteins. Consequently, disruption of lipid rafts impaired the expression of these proteins and transcriptional activity of related genes. We provide evidence that this effect was mediated by uPAR. Similar effects were observed in VSMCs isolated from Cav1−/− (caveolin-1-deficient) mice. Despite the level of uPAR being significantly higher after the disruption of the rafts, uPA/uPAR-dependent cell migration was impaired. However, caveolin-1 deficiency impaired only uPAR-dependent cell proliferation, whereas cell migration was strongly up-regulated in these cells. Our results provide evidence that rafts are required in the regulation of uPAR-mediated VSMC phenotypic modulations. These findings suggest further that, in the context of uPA/uPAR-dependent processes, caveolae-associated and non-associated rafts represent different signalling membrane domains.

scholarly journals Regulation of the protein kinase PKR by the vaccinia virus pseudosubstrate inhibitor K3L is dependent on residues conserved between the K3L protein and the PKR substrate eIF2alpha.

1997 ◽  
Vol 17 (7) ◽  
pp. 4146-4158 ◽  
Author(s):  
M Kawagishi-Kobayashi ◽  
J B Silverman ◽  
T L Ung ◽  
T E Dever
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Vaccinia Virus ◽  
Defense Mechanism ◽  
Mutational Analysis ◽  
Substrate Recognition ◽  
Phosphorylation Site ◽  
Antiviral Defense ◽  
Loss Of Function ◽  
Yeast Saccharomyces Cerevisiae

The mammalian double-stranded RNA-activated protein kinase PKR is a component of the cellular antiviral defense mechanism and phosphorylates Ser-51 on the alpha subunit of the translation factor eIF2 to inhibit protein synthesis. To identify the molecular determinants that specify substrate recognition by PKR, we performed a mutational analysis on the vaccinia virus K3L protein, a pseudosubstrate inhibitor of PKR. High-level expression of PKR is lethal in the yeast Saccharomyces cerevisiae because PKR phosphorylates eIF2alpha and inhibits protein synthesis. We show that coexpression of vaccinia virus K3L can suppress the growth-inhibitory effects of PKR in yeast, and using this system, we identified both loss-of-function and hyperactivating mutations in K3L. Truncation of, or point mutations within, the C-terminal portion of the K3L protein, homologous to residues 79 to 83 in eIF2alpha, abolished PKR inhibitory activity, whereas the hyperactivating mutation, K3L-H47R, increased the homology between the K3L protein and eIF2alpha adjacent to the phosphorylation site at Ser-51. Biochemical and yeast two-hybrid analyses revealed that the suppressor phenotype of the K3L mutations correlated with the affinity of the K3L protein for PKR and was inversely related to the level of eIF2alpha phosphorylation in the cell. These results support the idea that residues conserved between the pseudosubstrate K3L protein and the authentic substrate eIF2alpha play an important role in substrate recognition, and they suggest that PKR utilizes sequences both near and over 30 residues from the site of phosphorylation for substrate recognition. Finally, by reconstituting part of the mammalian antiviral defense mechanism in yeast, we have established a genetically useful system to study viral regulators of PKR.

The C. elegans NeuroD homolog cnd-1 functions in multiple aspects of motor neuron fate specification

Development ◽  
2000 ◽  
Vol 127 (19) ◽  
pp. 4239-4252 ◽  
Author(s):  
S. Hallam ◽  
E. Singer ◽  
D. Waring ◽  
Y. Jin
Keyword(s):  
Motor Neuron ◽  
Motor Neurons ◽  
Expression Patterns ◽  
Loss Of Function ◽  
Variable Expression ◽  
C Elegans ◽  
Helix Loop Helix ◽  
Ventral Cord ◽  
Bhlh Proteins ◽  
Neuronal Type

The basic helix-loop-helix transcription factor NeuroD (Neurod1) has been implicated in neuronal fate determination, differentiation and survival. Here we report the expression and functional analysis of cnd-1, a C. elegans NeuroD homolog. cnd-1 expression was first detected in neuroblasts of the AB lineage in 14 cell embryos and maintained in many neuronal descendants of the AB lineage during embryogenesis, diminishing in most terminally differentiated neurons prior to hatching. Specifically, cnd-1 reporter genes were expressed in the precursors of the embryonic ventral cord motor neurons and their progeny. A loss-of-function mutant, cnd-1(ju29), exhibited multiple defects in the ventral cord motor neurons. First, the number of motor neurons was reduced, possibly caused by the premature withdrawal of the precursors from mitotic cycles. Second, the strict correlation between the fate of a motor neuron with respect to its lineage and position in the ventral cord was disrupted, as manifested by the variable expression pattern of motor neuron fate specific markers. Third, motor neurons also exhibited defects in terminal differentiation characteristics including axonal morphology and synaptic connectivity. Finally, the expression patterns of three neuronal type-specific transcription factors, unc-3, unc-4 and unc-30, were altered. Our data suggest that cnd-1 may specify the identity of ventral cord motor neurons both by maintaining the mitotic competence of their precursors and by modulating the expression of neuronal type-specific determination factors. cnd-1 appears to have combined the functions of several vertebrate neurogenic bHLH proteins and may represent an ancestral form of this protein family.

The role of the NK-homeobox gene slouch (S59) in somatic muscle patterning

Development ◽  
1999 ◽  
Vol 126 (20) ◽  
pp. 4525-4535 ◽  
Author(s):  
S. Knirr ◽  
N. Azpiazu ◽  
M. Frasch
Keyword(s):  
Muscle Development ◽  
Ectopic Expression ◽  
Homeobox Gene ◽  
Drosophila Embryo ◽  
Loss Of Function ◽  
Cell Fates ◽  
Somatic Muscle ◽  
Gene Encoding ◽  
Body Wall Muscles ◽  
Founder Cells

In the Drosophila embryo, a distinct class of myoblasts, designated as muscle founders, prefigures the mature pattern of somatic body wall muscles. Each founder cell appears to be instrumental in generating a single larval muscle with a defined identity. The NK homeobox gene S59 was the first of a growing number of proposed ‘identity genes’ that have been found to be expressed in stereotyped patterns in specific subsets of muscle founders and their progenitor cells and are thought to control their developmental fates. In the present study, we describe the effects of gain- and loss-of-function experiments with S59. We find that a null mutation in the gene encoding S59, which we have named slouch (slou), disrupts the development of all muscles that are derived from S59-expressing founder cells. The observed phenotypes upon mutation and ectopic expression of slouch include transformations of founder cell fates, thus confirming that slouch (S59) functions as an identity gene in muscle development. These fate transformations occur between sibling founder cells as well as between neighboring founders that are not lineage-related. In the latter case, we show that slouch (S59) activity is required cell-autonomously to repress the expression of ladybird (lb) homeobox genes, thereby preventing specification along the lb pathway. Together, these findings provide new insights into the regulatory interactions that establish the somatic muscle pattern.

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