scholarly journals The balance of mitochondrial fission and fusion in cortical axons depends on the kinases SadA and SadB

2021 ◽  
Author(s):  
Danila Di Meo ◽  
Priyadarshini Ravindran ◽  
Pratibha Dhumale ◽  
Andreas W Puschel
Keyword(s):  
Cortical Neurons ◽  
Actin Filaments ◽  
Hippocampal Neurons ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Fission ◽  
Metabolic Demand ◽  
Neuronal Homeostasis ◽  
Polarized Cells ◽  
Ankyrin B ◽  
High Metabolic Demand

Neurons are highly polarized cells that display characteristic differences in the organization of their organelles in axons and dendrites. Mitochondria are of particular importance for neuronal homeostasis due to their high metabolic demand. The kinases SadA and SadB (SadA/B) promote the formation of distinct axonal and dendritic extensions during the development of cortical and hippocampal neurons. Here, we show that SadA/B are required for the axon-specific dynamics of mitochondria. The interaction with Ankyrin B (AnkB) stimulates the activity of SadA/B that function as regulators of mitochondrial dynamics through the phosphorylation of Tau. Suppression of SadA/B or AnkB in cortical neurons induces the elongation of mitochondria by disrupting the balance of fission and fusion. The normal dynamics of axonal mitochondria could be restored by mild actin destabilization. Thus, the elongation after a loss of SadA/B results from an excessive stabilization of actin filaments and reduction of Drp1 recruitment to mitochondria.

scholarly journals Mitochondrial fission and mitophagy are independent mechanisms regulating ischemia/reperfusion injury in primary neurons

2021 ◽  
Vol 12 (5) ◽  
Author(s):  
Anthony R. Anzell ◽  
Garrett M. Fogo ◽  
Zoya Gurm ◽  
Sarita Raghunayakula ◽  
Joseph M. Wider ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Ischemia Reperfusion Injury ◽  
Mitochondrial Dynamics ◽  
Ischemia Reperfusion ◽  
Mitochondrial Fission ◽  
Conditional Knockout ◽  
Mitochondrial Morphology ◽  
Primary Neurons ◽  
Mitochondrial Fragmentation

AbstractMitochondrial dynamics and mitophagy are constitutive and complex systems that ensure a healthy mitochondrial network through the segregation and subsequent degradation of damaged mitochondria. Disruption of these systems can lead to mitochondrial dysfunction and has been established as a central mechanism of ischemia/reperfusion (I/R) injury. Emerging evidence suggests that mitochondrial dynamics and mitophagy are integrated systems; however, the role of this relationship in the context of I/R injury remains unclear. To investigate this concept, we utilized primary cortical neurons isolated from the novel dual-reporter mitochondrial quality control knockin mice (C57BL/6-Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl/J) with conditional knockout (KO) of Drp1 to investigate changes in mitochondrial dynamics and mitophagic flux during in vitro I/R injury. Mitochondrial dynamics was quantitatively measured in an unbiased manner using a machine learning mitochondrial morphology classification system, which consisted of four different classifications: network, unbranched, swollen, and punctate. Evaluation of mitochondrial morphology and mitophagic flux in primary neurons exposed to oxygen-glucose deprivation (OGD) and reoxygenation (OGD/R) revealed extensive mitochondrial fragmentation and swelling, together with a significant upregulation in mitophagic flux. Furthermore, the primary morphology of mitochondria undergoing mitophagy was classified as punctate. Colocalization using immunofluorescence as well as western blot analysis revealed that the PINK1/Parkin pathway of mitophagy was activated following OGD/R. Conditional KO of Drp1 prevented mitochondrial fragmentation and swelling following OGD/R but did not alter mitophagic flux. These data provide novel evidence that Drp1 plays a causal role in the progression of I/R injury, but mitophagy does not require Drp1-mediated mitochondrial fission.

scholarly journals Cofilin and Actin Dynamics: Multiple Modes of Regulation and Their Impacts in Neuronal Development and Degeneration

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2726
Author(s):  
James R. Bamburg ◽  
Laurie S. Minamide ◽  
O’Neil Wiggan ◽  
Lubna H. Tahtamouni ◽  
Thomas B. Kuhn
Keyword(s):  
Cortical Neurons ◽  
Hippocampal Neurons ◽  
Translational Regulation ◽  
Neuronal Development ◽  
Mitochondrial Fission ◽  
Amyloid Β ◽  
Cellular Prion Protein ◽  
Actin Dynamics ◽  
Specific Regulation ◽  
And Function

Proteins of the actin depolymerizing factor (ADF)/cofilin family are ubiquitous among eukaryotes and are essential regulators of actin dynamics and function. Mammalian neurons express cofilin-1 as the major isoform, but ADF and cofilin-2 are also expressed. All isoforms bind preferentially and cooperatively along ADP-subunits in F-actin, affecting the filament helical rotation, and when either alone or when enhanced by other proteins, promotes filament severing and subunit turnover. Although self-regulating cofilin-mediated actin dynamics can drive motility without post-translational regulation, cells utilize many mechanisms to locally control cofilin, including cooperation/competition with other proteins. Newly identified post-translational modifications function with or are independent from the well-established phosphorylation of serine 3 and provide unexplored avenues for isoform specific regulation. Cofilin modulates actin transport and function in the nucleus as well as actin organization associated with mitochondrial fission and mitophagy. Under neuronal stress conditions, cofilin-saturated F-actin fragments can undergo oxidative cross-linking and bundle together to form cofilin-actin rods. Rods form in abundance within neurons around brain ischemic lesions and can be rapidly induced in neurites of most hippocampal and cortical neurons through energy depletion or glutamate-induced excitotoxicity. In ~20% of rodent hippocampal neurons, rods form more slowly in a receptor-mediated process triggered by factors intimately connected to disease-related dementias, e.g., amyloid-β in Alzheimer’s disease. This rod-inducing pathway requires a cellular prion protein, NADPH oxidase, and G-protein coupled receptors, e.g., CXCR4 and CCR5. Here, we will review many aspects of cofilin regulation and its contribution to synaptic loss and pathology of neurodegenerative diseases.

scholarly journals Machine learning-based classification of mitochondrial morphology in primary neurons and brain

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Garrett M. Fogo ◽  
Anthony R. Anzell ◽  
Kathleen J. Maheras ◽  
Sarita Raghunayakula ◽  
Joseph M. Wider ◽  
...  
Keyword(s):  
Image Analysis ◽  
Cortical Neurons ◽  
Mitochondrial Dynamics ◽  
Automated Image Analysis ◽  
Mitochondrial Morphology ◽  
Analysis Pipeline ◽  
Genetic Ablation ◽  
Block Face

AbstractThe mitochondrial network continually undergoes events of fission and fusion. Under physiologic conditions, the network is in equilibrium and is characterized by the presence of both elongated and punctate mitochondria. However, this balanced, homeostatic mitochondrial profile can change morphologic distribution in response to various stressors. Therefore, it is imperative to develop a method that robustly measures mitochondrial morphology with high accuracy. Here, we developed a semi-automated image analysis pipeline for the quantitation of mitochondrial morphology for both in vitro and in vivo applications. The image analysis pipeline was generated and validated utilizing images of primary cortical neurons from transgenic mice, allowing genetic ablation of key components of mitochondrial dynamics. This analysis pipeline was further extended to evaluate mitochondrial morphology in vivo through immunolabeling of brain sections as well as serial block-face scanning electron microscopy. These data demonstrate a highly specific and sensitive method that accurately classifies distinct physiological and pathological mitochondrial morphologies. Furthermore, this workflow employs the use of readily available, free open-source software designed for high throughput image processing, segmentation, and analysis that is customizable to various biological models.

scholarly journals Syntaxin-17-Dependent Mitochondrial Dynamics is Essential for Protection Against Oxidative-Stress-Induced Apoptosis

Antioxidants ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 522 ◽  
Author(s):  
Wang ◽  
Xiao ◽  
Huang ◽  
Liu
Keyword(s):  
Oxidative Stress ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Fission ◽  
Autophagic Flux ◽  
Wild Type ◽  
Dual Roles ◽  
Defective Mutant ◽  
U2os Cells ◽  
Induced Apoptosis

In this study, cell death induced by the oxidant tert-butylhydroperoxide (tBH) was observed in U2OS cells; this phenotype was rescued by Syntaxin 17 (STX17) knockout (KO) but the mechanism is unknown. STX17 plays dual roles in autophagosome–lysosome fusion and mitochondrial fission. However, the contribution of the two functions of STX17 to apoptosis has not been extensively studied. Here, we sought to dissect the dual roles of STX17 in oxidative-stress-induced apoptosis by taking advantage of STX17 knockout cells and an autophagosome–lysosome fusion defective mutant of STX17. We generated STX17 knockout U2OS cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and the STX17 knockout cells were reconstituted with wild-type STX17 and its autophagosome–lysosome fusion defective mutant. Autophagy was assessed by autophagic flux assay, Monomer red fluorescent protein (mRFP)–GFP–LC3 assay and protease protection assay. Golgi, endoplasmic reticulum (ER)/ER–Golgi intermediate compartment (ERGIC) and mitochondrial dynamics were examined by staining the different indicator proteins. Apoptosis was evaluated by caspase cleavage assay. The general reactive oxygen species (ROS) were detected by flow cytometry. In STX17 complete knockout cells, sealed autophagosomes were efficiently formed but their fusion with lysosomes was less defective. The fusion defect was rescued by wild-type STX17 but not the autophagosome–lysosome fusion defective mutant. No obvious defects in Golgi, ERGIC or ER dynamics were observed. Mitochondria were significantly elongated, supporting a role of STX17 in mitochondria fission and the elongation caused by STX17 KO was reversed by the autophagosome–lysosome fusion defective mutant. The clearance of protein aggregation was compromised, correlating with the autophagy defect but not with mitochondrial dynamics. This study revealed a mixed role of STX17 in autophagy, mitochondrial dynamics and oxidative stress response. STX17 knockout cells were highly resistant to oxidative stress, largely due to the function of STX17 in mitochondrial fission rather than autophagy.

scholarly journals Non-alcoholic fatty liver disease in mice with hepatocyte-specific deletion of mitochondrial fission factor

Diabetologia ◽  
2021 ◽  
Author(s):  
Yukina Takeichi ◽  
Takashi Miyazawa ◽  
Shohei Sakamoto ◽  
Yuki Hanada ◽  
Lixiang Wang ◽  
...  
Keyword(s):  
Er Stress ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Fission ◽  
Primary Hepatocytes ◽  
Alcoholic Fatty Liver ◽  
Expression Of Genes ◽  
Specific Deletion

Abstract Aims/hypothesis Mitochondria are highly dynamic organelles continuously undergoing fission and fusion, referred to as mitochondrial dynamics, to adapt to nutritional demands. Evidence suggests that impaired mitochondrial dynamics leads to metabolic abnormalities such as non-alcoholic steatohepatitis (NASH) phenotypes. However, how mitochondrial dynamics are involved in the development of NASH is poorly understood. This study aimed to elucidate the role of mitochondrial fission factor (MFF) in the development of NASH. Methods We created mice with hepatocyte-specific deletion of MFF (MffLiKO). MffLiKO mice fed normal chow diet (NCD) or high-fat diet (HFD) were evaluated for metabolic variables and their livers were examined by histological analysis. To elucidate the mechanism of development of NASH, we examined the expression of genes related to endoplasmic reticulum (ER) stress and lipid metabolism, and the secretion of triacylglycerol (TG) using the liver and primary hepatocytes isolated from MffLiKO and control mice. Results MffLiKO mice showed aberrant mitochondrial morphologies with no obvious NASH phenotypes during NCD, while they developed full-blown NASH phenotypes in response to HFD. Expression of genes related to ER stress was markedly upregulated in the liver from MffLiKO mice. In addition, expression of genes related to hepatic TG secretion was downregulated, with reduced hepatic TG secretion in MffLiKO mice in vivo and in primary cultures of MFF-deficient hepatocytes in vitro. Furthermore, thapsigargin-induced ER stress suppressed TG secretion in primary hepatocytes isolated from control mice. Conclusions/interpretation We demonstrated that ablation of MFF in liver provoked ER stress and reduced hepatic TG secretion in vivo and in vitro. Moreover, MffLiKO mice were more susceptible to HFD-induced NASH phenotype than control mice, partly because of ER stress-induced apoptosis of hepatocytes and suppression of TG secretion from hepatocytes. This study provides evidence for the role of mitochondrial fission in the development of NASH. Graphical abstract

scholarly journals Reelin Signals through Phosphatidylinositol 3-Kinase and Akt To Control Cortical Development and through mTor To Regulate Dendritic Growth

2007 ◽  
Vol 27 (20) ◽  
pp. 7113-7124 ◽  
Author(s):  
Yves Jossin ◽  
André M. Goffinet
Keyword(s):  
Cortical Neurons ◽  
Hippocampal Neurons ◽  
Matrix Protein ◽  
Glycogen Synthase ◽  
Extracellular Matrix Protein ◽  
Cortical Plate ◽  
Target Cells ◽  
Neuronal Cultures ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

ABSTRACT Reelin is an extracellular matrix protein with various functions during development and in the mature brain. It activates different signaling cascades in target cells, one of which is the phosphatidylinositol 3-kinase (PI3K) pathway, which we investigated further using pathway inhibitors and in vitro brain slice and neuronal cultures. We show that the mTor (mammalian target of rapamycin)-S6K1 (S6 kinase 1) pathway is activated by Reelin and that this depends on Dab1 (Disabled-1) phosphorylation and activation of PI3K and Akt (protein kinase B). PI3K and Akt are required for the effects of Reelin on the organization of the cortical plate, but their downstream partners mTor and glycogen synthase kinase 3β (GSK3β) are not. On the other hand, mTor, but not GSK3β, mediates the effects of Reelin on the growth and branching of dendrites of hippocampal neurons. In addition, PI3K fosters radial migration of cortical neurons through the intermediate zone, an effect that is independent of Reelin and Akt.

scholarly journals High glucose induces Drp1-mediated mitochondrial fission via the Orai1 calcium channel to participate in diabetic cardiomyocyte hypertrophy

2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Qing-Rui Wu ◽  
Dan-Lin Zheng ◽  
Pei-Ming Liu ◽  
Hui Yang ◽  
Lu-An Li ◽  
...  
Keyword(s):  
Calcium Channel ◽  
Mitochondrial Dysfunction ◽  
High Glucose ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Fission ◽  
Calcium Handling ◽  
Cardiomyocyte Hypertrophy ◽  
Mitochondrial Morphology ◽  
Calmodulin Binding ◽  
Downstream Target

AbstractMitochondrial dysfunction and impaired Ca2+ handling are involved in the development of diabetic cardiomyopathy (DCM). Dynamic relative protein 1 (Drp1) regulates mitochondrial fission by changing its level of phosphorylation, and the Orai1 (Ca2+ release-activated calcium channel protein 1) calcium channel is important for the increase in Ca2+ entry into cardiomyocytes. We aimed to explore the mechanism of Drp1 and Orai1 in cardiomyocyte hypertrophy caused by high glucose (HG). We found that Zucker diabetic fat rats induced by administration of a high-fat diet develop cardiac hypertrophy and impaired cardiac function, accompanied by the activation of mitochondrial dynamics and calcium handling pathway-related proteins. Moreover, HG induces cardiomyocyte hypertrophy, accompanied by abnormal mitochondrial morphology and function, and increased Orai1-mediated Ca2+ influx. Mechanistically, the Drp1 inhibitor mitochondrial division inhibitor 1 (Mdivi-1) prevents cardiomyocyte hypertrophy induced by HG by reducing phosphorylation of Drp1 at serine 616 (S616) and increasing phosphorylation at S637. Inhibition of Orai1 with single guide RNA (sgOrai1) or an inhibitor (BTP2) not only suppressed Drp1 activity and calmodulin-binding catalytic subunit A (CnA) and phosphorylated-extracellular signal-regulated kinase (p-ERK1/2) expression but also alleviated mitochondrial dysfunction and cardiomyocyte hypertrophy caused by HG. In addition, the CnA inhibitor cyclosporin A and p-ERK1/2 inhibitor U0126 improved HG-induced cardiomyocyte hypertrophy by promoting and inhibiting phosphorylation of Drp1 at S637 and S616, respectively. In summary, we identified Drp1 as a downstream target of Orai1-mediated Ca2+ entry, via activation by p-ERK1/2-mediated phosphorylation at S616 or CnA-mediated dephosphorylation at S637 in DCM. Thus, the Orai1–Drp1 axis is a novel target for treating DCM.

scholarly journals Dynamin-related protein 1 deficiency accelerates lipopolysaccharide-induced acute liver injury and inflammation in mice

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Lixiang Wang ◽  
Xin Li ◽  
Yuki Hanada ◽  
Nao Hasuzawa ◽  
Yoshinori Moriyama ◽  
...  
Keyword(s):  
Liver Disease ◽  
Liver Injury ◽  
Er Stress ◽  
Disease Progression ◽  
Acute Liver Injury ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Fission ◽  
Mitochondrial Fusion ◽  
Related Protein ◽  
Liver Disease Progression

AbstractMitochondrial fusion and fission, which are strongly related to normal mitochondrial function, are referred to as mitochondrial dynamics. Mitochondrial fusion defects in the liver cause a non-alcoholic steatohepatitis-like phenotype and liver cancer. However, whether mitochondrial fission defect directly impair liver function and stimulate liver disease progression, too, is unclear. Dynamin-related protein 1 (DRP1) is a key factor controlling mitochondrial fission. We hypothesized that DRP1 defects are a causal factor directly involved in liver disease development and stimulate liver disease progression. Drp1 defects directly promoted endoplasmic reticulum (ER) stress, hepatocyte death, and subsequently induced infiltration of inflammatory macrophages. Drp1 deletion increased the expression of numerous genes involved in the immune response and DNA damage in Drp1LiKO mouse primary hepatocytes. We administered lipopolysaccharide (LPS) to liver-specific Drp1-knockout (Drp1LiKO) mice and observed an increased inflammatory cytokine expression in the liver and serum caused by exaggerated ER stress and enhanced inflammasome activation. This study indicates that Drp1 defect-induced mitochondrial dynamics dysfunction directly regulates the fate and function of hepatocytes and enhances LPS-induced acute liver injury in vivo.

scholarly journals Inhibition of Mitochondrial Dynamics Preferentially Targets Pancreatic Cancer Cells with Enhanced Tumorigenic and Invasive Potential

Cancers ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 698
Author(s):  
Sarah Courtois ◽  
Beatriz de Luxán-Delgado ◽  
Laure Penin-Peyta ◽  
Alba Royo-García ◽  
Beatriz Parejo-Alonso ◽  
...  
Keyword(s):  
Mitochondrial Dynamics ◽  
Mitochondrial Fission ◽  
Metastatic Potential ◽  
Ductal Adenocarcinoma ◽  
Expression Ratio ◽  
Cytotoxic Effects ◽  
Mitochondrial Homeostasis ◽  
Pancreatic Cancer Cells ◽  
Tumorigenic Potential ◽  
Dose Dependent

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest tumors, partly due to its intrinsic aggressiveness, metastatic potential, and chemoresistance of the contained cancer stem cells (CSCs). Pancreatic CSCs strongly rely on mitochondrial metabolism to maintain their stemness, therefore representing a putative target for their elimination. Since mitochondrial homeostasis depends on the tightly controlled balance between fusion and fission processes, namely mitochondrial dynamics, we aim to study this mechanism in the context of stemness. In human PDAC tissues, the mitochondrial fission gene DNM1L (DRP1) was overexpressed and positively correlated with the stemness signature. Moreover, we observe that primary human CSCs display smaller mitochondria and a higher DRP1/MFN2 expression ratio, indicating the activation of the mitochondrial fission. Interestingly, treatment with the DRP1 inhibitor mDivi-1 induced dose-dependent apoptosis, especially in CD133+ CSCs, due to the accumulation of dysfunctional mitochondria and the subsequent energy crisis in this subpopulation. Mechanistically, mDivi-1 inhibited stemness-related features, such as self-renewal, tumorigenicity, and invasiveness and chemosensitized the cells to the cytotoxic effects of Gemcitabine. In summary, mitochondrial fission is an essential process for pancreatic CSCs and represents an attractive target for designing novel multimodal treatments that will more efficiently eliminate cells with high tumorigenic potential.

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