The non-canonical Wnt/PKC pathway regulates mitochondrial dynamics through degradation of the ARM-like domain-containing protein Alex3

The regulation of mitochondrial dynamics is vital in complex cell types, such as neurons, that transport and localize mitochondria in high energy-demanding cell domains. The Armcx3 gene encodes a mitochondrial-targeted protein (Alex3) that contains several arm-like domains. In a previous study we showed that Alex3 protein regulates mitochondrial aggregation and trafficking. Here we studied the contribution of Wnt proteins to the mitochondrial aggregation and dynamics regulated by Alex3. Overexpression of Alex3 in HEK293 cells caused a marked aggregation of mitochondria, which was attenuated by treatment with several Wnts. We also found that this decrease was caused by Alex3 degradation induced by Wnts. While the Wnt canonical pathway did not alter the pattern of mitochondrial aggregation induced by Alex3, we observed that the Wnt/PKC non-canonical pathway regulated both mitochondrial aggregation and Alex3 protein levels, thereby rendering a mitochondrial phenotype and distribution similar to control patterns. Our data suggest that the Wnt pathway regulates mitochondrial distribution and dynamics through Alex3 protein degradation

The Armc10/SVH gene: Genome context, regulation of mitochondrial dynamics and protection against Aβ-induced mitochondrial fragmentation

Mitochondrial function and dynamics are essential for neurotransmission, neural function and neuronal viability. Recently, we showed that the eutherian-specific Armcx gene cluster (Armcx1-6 genes), located in the X chromosome, encodes for a new family of proteins that localise to mitochondria, regulating mitochondrial trafficking. The Armcx gene cluster evolved by retrotransposition of the Armc10 gene mRNA, which is present in all vertebrates and is considered to be the ancestor gene. Here we investigate the genomic organisation, mitochondrial functions and putative neuroprotective role of the Armc10 ancestor gene. The genomic context of the Armc10 locus shows considerable syntenic conservation among vertebrates, and sequence comparisons and CHIP-data suggest the presence of at least three conserved enhancers. We also show that the Armc10 protein localises to mitochondria and that it is highly expressed in the brain. Furthermore, we show that Armc10 levels regulate mitochondrial trafficking in neurons, but not mitochondrial aggregation, by controlling the number of moving mitochondria. We further demonstrate that the Armc10 protein interacts with the KIF5/Miro1-2/Trak2 trafficking complex. Finally, we show that overexpression of Armc10 in neurons prevents A beta-induced mitochondrial fission and neuronal death. Our data suggest both conserved and differential roles of the Armc10/Armcx gene family in regulating mitochondrial dynamics in neurons, and underscore a protective effect of the Armc10 gene against A beta-induced toxicity. Overall, our findings support a further degree of regulation of mitochondrial dynamics in the brain of more evolved mammals

The mixture of "ecstasy" and its metabolites impairs mitochondrial fusion/fission equilibrium and trafficking in hippocampal neurons, at in vivo relevant concentrations

3,4-Methylenedioxymethamphetamine (MDMA; "ecstasy") is a potentially neurotoxic recreational drug of abuse. Though the mechanisms involved are still not completely understood, formation of reactive metabolites and mitochondrial dysfunction contribute to MDMA-related neurotoxicity. Neuronal mitochondrial trafficking, and their targeting to synapses, is essential for proper neuronal function and survival, rendering neurons particularly vulnerable to mitochondrial dysfunction. Indeed, MDMAassociated disruption of Ca2+ homeostasis and ATP depletion have been described in neurons, thus suggesting possible MDMA interference on mitochondrial dynamics. In this study, we performed real-time functional experiments of mitochondrial trafficking to explore the role of in situ mitochondrial dysfunction in MDMA's neurotoxic actions. We show that the mixture of MDMA and six of its major in vivo metabolites, each compound at 10μM, impaired mitochondrial trafficking and increased the fragmentation of axonal mitochondria in cultured hippocampal neurons. Furthermore, the overexpression of mitofusin 2 (Mfn2) or dynamin-related protein 1 (Drp1) K38A constructs almost completely rescued the trafficking deficits caused by this mixture. Finally, in hippocampal neurons overexpressing a Mfn2 mutant, Mfn2 R94Q, with impaired fusion and transport properties, it was confirmed that a dysregulation of mitochondrial fission/fusion events greatly contributed to the reported trafficking phenotype. In conclusion, our study demonstrated, for the first time, that the mixture of MDMA and its metabolites, at concentrations relevant to the in vivo scenario, impaired mitochondrial trafficking and increasedmitochondrial fragmentation in hippocampal neurons, thus providing a new insight in the context of "ecstasy"-induced neuronal injury. © The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.Ministerio de Ciencia e Innovacion (MICINN), Spain (BFU2008-3980); Plan Nacional de Drogas, Spain; Fundação para a Ciência e a Tecnologia (Portugal) (FCT)

The Eutherian Armcx genes regulate mitochondrial trafficking in neurons and interact with Miro and Trak2

Producción CientíficaBrain function requires neuronal activity-dependent energy consumption. Neuronal energy supply is controlled by molecular mechanisms that regulate mitochondrial dynamics, including Kinesin motors and Mitofusins, Miro1-2 and Trak2 proteins. Here we show a new protein family that localizes to the mitochondria and controls mitochondrial dynamics. This family of proteins is encoded by an array of armadillo (Arm) repeat-containing genes located on the X chromosome. The Armcx cluster is unique to Eutherian mammals and evolved from a single ancestor gene (Armc10). We show that these genes are highly expressed in the developing and adult nervous system. Furthermore, we demonstrate that Armcx3 expression levels regulate mitochondrial dynamics and trafficking in neurons, and that Alex3 interacts with the Kinesin/Miro/Trak2 complex in a Ca2 + -dependent manner. Our data provide evidence of a new Eutherian-specific family of mitochondrial proteins that controls mitochondrial dynamics and indicate that this key process is differentially regulated in the brain of higher vertebrates.2015-03-3

Papel de Alex3 en la vía de señalización de Wnt y en la dinámica mitocondrial

La proteína Alex3 forma parte de la familia de genes exclusiva de los mamíferos euterios Armcx, caracterizada por presentar una alta expresión en el SNC, por encontrarse localizada en clúster en el cromosoma X y porque se originaron a partir de la retrotransposición del gen Armc10 y una rápida duplicación en tándem en una evolución temprana de los mamíferos euterios. Las proteínas Armcx/Armc10 poseen primariamente una localización subcelular bimodal, encontrándose asociadas a la membrana externa mitocondrial y en el núcleo celular, localización que concuerda con sus secuencias proteicas que poseen putativos dominios de localización en estos compartimentos. La sobreexpresión de las proteínas Armcx/Armc10 produce una profunda alteración de la red mitocondrial, demostrando que esta familia de proteínas juega un papel importante en la regulación de la dinámica y agregación mitocondrial y al menos, la sobreexpresión de la proteína Alex3, no induce cambios en los parámetros bio-energéticos mitocondriales, tales como el consumo de oxígeno, el potencial de membrana, el contenido de DNA mitocondrial, la actividad de la citocromo c oxidasa o la recaptación de Ca2+, ni alteran el balance de fisión/fusión mitocondrial. Tanto la sobreexpresión como el silenciamiento de las proteínas Alex3 y Armc10 en neuronas hipocampales se ha visto alteran la distribución y transporte mitocondrial. Las proteínas Alex3 y Armc10 interaccionan con el complejo Kinesina/Miro/Trak2, regulador del transporte mitocondrial, lo cual sugiere que esta familia de proteínas regularían el transporte y dinámica mitocondrial a través de este complejo de proteínas. La interacción de Alex3 con este complejo también se ha visto es dependiente de los niveles de Ca2+, reduciéndose la interacción de estas proteínas cuando los niveles de Ca2+ son elevados. Por otra parte, la vía de señalización asociada a proteínas Wnt se ha visto induce la degradación de la proteína Alex3 por un proceso independiente del proteosoma. Esta degradación no depende de los componentes de la vía canónica Dishevelled, GSK3-β y β-catenina ni de los componentes no canónicos JNK, CAMKII y calcineurina, habiéndose demostrado que la PKC y la CK2 juegan un papel principal en el control y degradación de los niveles de la proteína Alex3 de forma dependiente e independiente de las vías de señalización de Wnt. De manera similar, la depleción de los niveles intracelulares de Ca2+ también reproduce la degradación de Alex3. Además, la degradación de Alex3 a través de las vías de señalización asociadas a las proteínas Wnt revierte los fenotipos de agregación mitocondrial inducidos por la sobreexpresión de Alex3 y es evitado por la activación de la PKC, lo que sugiere que las proteínas Wnt podrían jugar un papel en el control de la dinámica mitocondrial mediante la regulación de las proteínas Armcx.Alex3 protein belongs to the eutherian specific family of genes Armcx, characterized by a high expression on the CNS, to be localized in a cluster on the X chromosome and to be originated by retrotransposition of Armc10 gene in a fast duplication in tandem. The Armcx/Armc10 proteins have a primary bimodal localization, both in nucleus and mitochondria as indicate their putative domains. Overexpression of Armcx/Armc10 proteins causes a profound alteration on the mitochondrial net showing that this family of proteins plays an important role in the regulation of the mitochondrial dynamics and at least, the overexpression of Alex3 protein neither change the bioenergetic parameters of mitochondria such as respiration, mitochondrial DNA content or calcium uptake nor alters the mitochondrial fusion/fission rate. Both the overexpression and knock-down of Alex3 and Armc10 proteins in hippocampal neurons alters the mitochondrial distribution and transport. Alex3 and Armc10 interact with the Kinesin/Miro/Trak2 mitochondrial transport regulator complex, suggesting that the Armcx protein family regulates mitochondrial dynamics through this complex. Moreover the interaction of Alex3 with this complex is dependent of calcium levels, diminishing the interaction when calcium levels are high. On the other hand, the Wnt signalling pathway induces the degradation of Alex3 protein in a proteosome independent process. This degradation is independent of the Wnt canonical and non-canonical members Dishevelled, GSK3β, β-catenin, JNK, calcineurin and CAMKII, but showing that the PKC and CKII members play a principal role in the control and degradation of Alex3 protein levels dependently and independently of Wnt pathways. Moreover, Alex3 degradation through Wnt signalling pathways, reverts the mitochondrial aggregation phenotypes and is avoided by PKC activation, suggesting that Wnt proteins can play a role in the control of mitochondrial dynamics through the regulation of Armcx proteins

ARMCX3 Mediates Susceptibility to Hepatic Tumorigenesis Promoted by Dietary Lipotoxicity

Lipotoxicitat; ObesitatLipotoxicity; ObesityLipotoxicidad; ObesidadARMCX3 is encoded by a member of the Armcx gene family and is known to be involved in nervous system development and function. We found that ARMCX3 is markedly upregulated in mouse liver in response to high lipid availability, and that hepatic ARMCX3 is upregulated in patients with NAFLD and hepatocellular carcinoma (HCC). Mice were subjected to ARMCX3 invalidation (inducible ARMCX3 knockout) and then exposed to a high-fat diet and diethylnitrosamine-induced hepatocarcinogenesis. The effects of experimental ARMCX3 knockdown or overexpression in HCC cell lines were also analyzed. ARMCX3 invalidation protected mice against high-fat-diet-induced NAFLD and chemically induced hepatocarcinogenesis. ARMCX3 invalidation promoted apoptotic cell death and macrophage infiltration in livers of diethylnitrosamine-treated mice maintained on a high-fat diet. ARMCX3 downregulation reduced the viability, clonality and migration of HCC cell lines, whereas ARMCX3 overexpression caused the reciprocal effects. SOX9 was found to mediate the effects of ARMCX3 in hepatic cells, with the SOX9 interaction required for the effects of ARMCX3 on hepatic cell proliferation. In conclusion, ARMCX3 is identified as a novel molecular actor in liver physiopathology and carcinogenesis. ARMCX3 downregulation appears to protect against hepatocarcinogenesis, especially under conditions of high dietary lipid-mediated hepatic insult.The study was supported by grant 201337-30-31-32 Fundació la Marató de TV3, grant SAF2017-85722R (Ministerio de Economia y Competitividad) to F.V., grant PID2019-106764RB-C21/ AEI/ 10.13039/501100011033 from the Spanish Ministry of Science and a collaborative grant from CIBERNED to E.S, grant PI18/00961 (Instituto de Salud Carlos III) to B.M, and the Hepacare Project grant from Fundación La Caixa to M.A.A. and C.B. F.V. and E.S. are ICREA researchers

The non-canonical Wnt/PKC pathway regulates mitochondrial dynamics through degradation of the ARM-like domain-containing protein Alex3

The regulation of mitochondrial dynamics is vital in complex cell types, such as neurons, that transport and localize mitochondria in high energy-demanding cell domains. The Armcx3 gene encodes a mitochondrial-targeted protein (Alex3) that contains several arm-like domains. In a previous study we showed that Alex3 protein regulates mitochondrial aggregation and trafficking. Here we studied the contribution of Wnt proteins to the mitochondrial aggregation and dynamics regulated by Alex3. Overexpression of Alex3 in HEK293 cells caused a marked aggregation of mitochondria, which was attenuated by treatment with several Wnts. We also found that this decrease was caused by Alex3 degradation induced by Wnts. While the Wnt canonical pathway did not alter the pattern of mitochondrial aggregation induced by Alex3, we observed that the Wnt/PKC non-canonical pathway regulated both mitochondrial aggregation and Alex3 protein levels, thereby rendering a mitochondrial phenotype and distribution similar to control patterns. Our data suggest that the Wnt pathway regulates mitochondrial distribution and dynamics through Alex3 protein degradation

The N-terminal domain of Alex3 is sufficient to induce mitochondrial aggregation.

<p>(<b>A–D</b>) Overexpression of Alex3-GFP (green) in HEK293T cells induces severe alterations of the mitochondrial network when compared with the expression of control GFP (<b>A</b>). (<b>B</b>) Illustrates an Alex3-transfected cell displaying normal mitochondrial morphology; (<b>C,D</b>) Alex3-overexpressing cells showing mild aggregating phenotypes (<b>C</b>) and severe aggregating mitochondrial phenotypes (<b>D</b>); Alex3 protein was visualized in green, mitochondria in red (MitDsRed), and nuclei were labeled with bisbenzimide (blue). (<b>E</b>) Quantification and graphical representation (mean ± standard deviation) of mitochondrial phenotypes in control (GFP) and Alex3-GFP-overexpressing cells. (<b>F</b>) Top: Scheme of the Alex3-GFP deletion constructs used for transfection. Bottom: Western Blot showing representative truncated Alex3-GFP constructs at the predicted protein sizes. (<b>G–J</b>) Photomicrographs illustrating that expression of the Alex3(1–200)-GFP (<b>G</b>), Alex3(1–106)-GFP (<b>H</b>) and Alex3(1–30)-GFP (<b>I</b>) constructs leads to mitochondrial aggregation; in contrast, deletion of the first N terminal 12 aa (GFP-Alex3ΔNt) targets Alex3 protein to the nucleus (<b>J</b>). Note that the 30 aa N-terminus deletion construct has a truncated outer mitochondrial membrane localization sequence, which may interfere with its mitochondrial targeting, thereby leading to nuclear localization. (<b>K</b>) Quantification and graphical representation (mean ± standard deviation) of mitochondrial phenotypes in HEK293T cells after transfection with several truncated Alex3-GFP constructs; the data show that all the constructs containing the N terminal region cause mitochondrial aggregation. Alex3 protein was visualized in green (GFP), mitochondria in red (MitDsRed) and nuclei in blue (bisbenzimide). Scale bar: 10 µm.</p

Alex3 degradation by Wnt1 is independent of the proteasome, JNK, CAMKII and Calcineurin pathways.

<p>(<b>A</b>) Proteasome inhibition with 10 µM MG-132 treatment blocks the normal turnover of Alex3 protein but not its Wnt1-induced degradation. (<b>B</b>) Numerous Alex3-overexpressing HEK293AD cells treated with the proteasomal inhibitor MG132 show the most severe mitochondrial aggregating phenotype. (<b>C</b>) Inhibition of JNK with 10 µM SP600125 (downstream effector of the Wnt/PCP pathway), CAMKII with 25 µM KN62 or Calcineurin with 10 µM Cypermetrin (downstream effectors of the Wnt/Ca²⁺ pathway) do not induce Alex3 protein degradation. Scale bar: 10 µm.</p

PKC and CKII phosphorylation protects against Wnt/Frizzled degradation of Alex3.

<p>(<b>A</b>) Inhibition of CKII (with 100 µM casein kinase II inhibitor I), downstream effector of the Wnt signaling pathway, is sufficient to trigger Alex3 degradation. (<b>B</b>) In contrast, PKC activation with 1 µM TPA protects against Wnt1-induced degradation of Alex3 protein. (<b>C</b>,<b>D</b>) Inhibition of PKC (with 1 µM Calphostin C) and treatment with 20 µM BAPTA/AM, an intracellular calcium chelator, also reproduces Wnt1 degradation. (<b>E</b>) Photomicrographs demonstrating that treatment with TPA prevents Alex3 degradation induced by Wnt1 and the reversion to normal mitochondrial phenotypes. (<b>F</b>) Quantification and graphical representation (mean ± standard deviation) of mitochondrial phenotypes in HEK293AD cells in the conditions shown in (<b>E</b>); note that incubation with TPA prevents the rescue of mitochondrial phenotypes induced by Wnt1. Scale bar: 10 µm. The quantification of Alex3 protein levels is shown at the bottom.</p