Function of Armcx3 and Armc10/svh genes in the regulation of progenitor proliferation and neural differentiation in the chicken spinal cord

The eutherian X-chromosome specific family of Armcx genes has been described asoriginating by retrotransposition from Armc10/SVH, a single Arm-containing somaticgene. Armcx3 and Armc10/SVH are characterized by high expression in the centralnervous system and they play an important role in the regulation of mitochondrialdistribution and transport in neurons. In addition, Armcx/Arm10 genes have severalArmadillo repeats in their sequence. In this study we address the potential role of thisgene family in neural development by using the chick neural tube as a model. Weshow that Armc10/SVH is expressed in the chicken spinal cord, and knocking-downArmc10/SVH by sh-RNAi electroporation in spinal cord reduces proliferation of neuralprecursor cells (NPCs). Moreover, we analyzed the effects of murine Armcx3 andArmc10 overexpression, showing that both proteins regulate progenitor proliferation,while Armcx3 overexpression also specifically controls neural maturation. We showthat the phenotypes found following Armcx3 overexpression require its mitochondriallocalization, suggesting a novel link between mitochondrial dynamics and regulation ofneural development. Furthermore, we found that both Armcx3 and Armc10 may act asinhibitors of Wnt-β-catenin signaling. Our results highlight both common and differentialfunctions of Armcx/Armc10 genes in neural development in the spinal cord

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

Blockade of the SNARE protein syntaxin 1 inhibits glioblastoma tumor growth

Glioblastoma (GBM) is the most prevalent adult brain tumor, with virtually no cure, and with a median overall survival of 15 months from diagnosis despite of the treatment. SNARE pro- teins mediate membrane fusion events in cells and are essential for many cellular process- es including exocytosis and neurotransmission, intracellular trafficking and cell migration. Here we show that the blockade of the SNARE protein Syntaxin 1 (Stx1) function impairs GBM cell proliferation. We show that Stx1 loss-of-function in GBM cells, through ShRNA lentiviral transduction, a Stx1 dominant negative and botulinum toxins, dramatically reduces the growth of GBM after grafting U373 cells into the brain of immune compromised mice. In- terestingly, Stx1 role on GBM progression may not be restricted just to cell proliferation since the blockade of Stx1 also reduces in vitro GBM cell invasiveness suggesting a role in several processes relevant for tumor progression. Altogether, our findings indicate that the blockade of SNARE proteins may represent a novel therapeutic tool against GBM

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

Reelin regulates the maturation of dendritic spines, synaptogenesis and glial ensheathment of newborn granule cells

The Reelin pathway is essential for both neural migration and for the development and maturation of synaptic connections. However, its role in adult synaptic formation and remodeling is still being investigated. Here, we investigated the impact of the Reelin/Dab1 pathway on the synaptogenesis of newborn granule cells (GCs) in the young-adult mouse hippocampus. We show that neither Reelin overexpression nor the inactivation of its intracellular adapter, Dab1, substantially alters dendritic spine numbers in these neurons. In contrast, 3D-electron microscopy (focused ion beam milling/scanning electron microscope) revealed that dysregulation of the Reelin/Dab1 pathway leads to both transient and permanent changes in the types and morphology of dendritic spines, mainly altering mushroom, filopodial, and branched GC spines. We also found that the Reelin/Dab1 pathway controls synaptic configuration of presynaptic boutons in the dentate gyrus, with its dysregulation leading to a substantial decrease in multi-synaptic bouton innervation. Lastly, we show that the Reelin/Dab1 pathway controls astroglial ensheathment of synapses. Thus, the Reelin pathway is a key regulator of adult-generated GC integration, by controlling dendritic spine types and shapes, their synaptic innervation patterns, and glial ensheathment. These findings may help to better understanding of hippocampal circuit alterations in neurological disorders in which the Reelin pathway is implicated. Significance Statement: The extracellular protein Reelin has an important role in neurological diseases, including epilepsy, Alzheimer's disease and psychiatric diseases, targeting hippocampal circuits. Here we address the role of Reelin in the development of synaptic contacts in adult-generated granule cells (GCs), a neuronal population that is crucial for learning and memory and implicated in neurological and psychiatric diseases. We found that the Reelin pathway controls the shapes, sizes, and types of dendritic spines, the complexity of multisynaptic innervations and the degree of the perisynaptic astroglial ensheathment that controls synaptic homeostasis. These findings show a pivotal role of Reelin in GC synaptogenesis and provide a foundation for structural circuit alterations caused by Reelin deregulation that may occur in neurological and psychiatric disorders

A conserved role for Syntaxin-1 in pre- and post-commissural midline axonal guidance in fly, chick, and mouse

Axonal growth and guidance rely on correct growth cone responses to guidance cues. Unlike the signaling cascades that link axonal growth to cytoskeletal dynamics, little is known about the crosstalk mechanisms between guidance and membrane dynamics and turnover. Recent studies indicate that whereas axonal attraction requires exocytosis, chemorepulsion relies on endocytosis. Indeed, our own studies have shown that Netrin-1/Deleted in Colorectal Cancer (DCC) signaling triggers exocytosis through the SNARE Syntaxin-1 (STX1). However, limited in vivo evidence is available about the role of SNARE proteins in axonal guidance. To address this issue, here we systematically deleted SNARE genes in three species. We show that loss-of-function of STX1 results in pre- and post-commissural axonal guidance defects in the midline of fly, chick, and mouse embryos. Inactivation of VAMP2, Ti-VAMP, and SNAP25 led to additional abnormalities in axonal guidance. We also confirmed that STX1 loss-of-function results in reduced sensitivity of commissural axons to Slit-2 and Netrin-1. Finally, genetic interaction studies in Drosophila show that STX1 interacts with both the Netrin-1/DCC and Robo/Slit pathways. Our data provide evidence of an evolutionarily conserved role of STX1 and SNARE proteins in midline axonal guidance in vivo, by regulating both pre- and post-commissural guidance mechanisms

Efectos del inhibidor de glicosilación benzil GaINAc sobre las rutas secretora y endocítica de células epiteliales

Consultable des del TDXTítol obtingut de la portada digitalitzadaEl benzil GalNAc fue descrito inicialmente como un inhibidor de la O-glicosilación de tipo mucina (Kuan et al., 1989). El tratamiento crónico de células mucosecretoras HT-29 M6 con este fármaco produjo una serie de efectos entre los que destacan: i) reducción de la proliferación, ii) inhibición de la secreción de mucinas, iii) acumulación de glicoproteínas apicales en vesículas citoplasmáticas electronlúcidas y, iv) reducción de la sialilación de glicoproteínas (Huet et al., 1998; (Hennebicq-Reig et al., 1998). El benzil GalNAc también inducía estos efectos sobre la población HT-29 parental y sobre otras poblaciones celulares derivadas de ésta, independientemente de su fenotipo. El benzil GalNAc es metabolizado a benzil GalNAc-Gal, el cual a su vez inhibe competitivamente la actividad ?2,3 sialiltransferasa de células HT-29, la actividad sialiltransferasa mayoritariamente expresada en estas células. Como resultado de ésto, se sintetizan compuestos sialilados como benzil GalNAc-Gal?2,3Neu5Ac (Huet et al., 1995; Delannoy et al., 1996). Por otro lado, el tratamiento con benzil GalNAc no afectaba sensiblemente a las células Caco-2, las cuales expresan fundamentalmente ?2,6 sialiltransferasas, enzimas que no son inhibidas por los metabolitos derivados del benzil GalNAc (Huet et al., 1998). El objetivo de este trabajo ha sido determinar el factor(es) responsable(s) del origen del fenotipo de células HT-29 tratadas crónicamente con benzil GalNAc. Más concretamente, nos interesó determinar cómo un inhibidor de glicosilación produce tantos efectos diferentes y si el efecto de este inhibidor estaba restringido o no a células derivadas de HT-29. La hipótesis que se manejó inicialmente proponía que los efectos globales del tratamiento con benzil GalNAc podrían estar causados por la hiposialilación de tardíos como, Rab7 y LBPA, pero no de endosomas primarios (Rab5, EEA1) y de Golgi (GRASP65, TGN46), colocalizaban con la ?1 integrina de las vesículas. El conjunto de los resultados obtenidos indica que las vesículas-BG corresponden a una población heterogénea de endosomas aberrantes, relacionados con endosomas tardíos. Las características fenotípicas de las células tratadas con benzil GalNAc reproducen parcialmente los defectos descritos en células de algunas enfermedades de depósito lisosomal (EDL), concretamente las que se producen por la acumulación de sacáridos en compartimentos degradativos. Así, la morfología de las vesículas-BG es similar a la de las que se originan en células tratadas con sacarosa (sacarosomas) y varias EDL como la sialidosis o la infantile sialic acid storage disease (ISSD). Análisis del procesamiento de la AAG en fibroblastos de pacientes con ISSD mostraron defectos similares a los encontrados en células HT-29 M6 tratadas con benzil GalNAc. Además, las células tratadas con benzil GalNAc también acumulan LBPA y colesterol, hallazgos característicos de algunas EDL. Confirmando la idea de que el benzil GalNAc induce un fenotipo de depósito lisosomal, el tratamiento con sacarosa en células IMIM-PC-1 produjo cambios sobre la morfología de las células y la acumulación intracelular de MUC1 y ?1 integrina, de manera semejante a lo observado en las células tratadas con benzil GalNAc. La sialilación de glicoproteínas en células IMIM-PC-1 tratadas con sacarosa no estaba alterada, resultado que indica que la hiposialilación no es un requisito para la acumulación intracelular de glicoproteínas de membrana. Dado que células tratadas con benzil GalNAc acumulan una gran cantidad de oligosacáridos derivados de esta droga, situación análoga a la que ocurre en células tratadas con sacarosa y algunas EDL, proponemos que la acumulación de estos metabolitos, y no la hiposialilación de glicoproteínas, es la que origina el fenotipo global de células HT-29 M6 e IMIM-PC-1 tratadas con este fármaco.Benzyl GalNAc was initially described as an inhibitor of mucin type O-glycosilation (Kuan et al., 1989). Long term exposure with this pharmac on mucosecretor HT-29 M6 colon cancer cells induced several effects: i) reduction of proliferation, ii) inhibition of the mucins secretion, iii) accumulation of apical glycoproteins into electron-lucid cytoplasmic vesicles (BG-vesicles) and iv) reduction of glycoprotein sialylation (Huet et al., 1998; Hennebicq-Reig et al., 1998). Moreover, these effects were induced in HT-29 parental and derived cells irrespectively of their phenotype. In HT-29 cells, benzyl GalNAc is metabolized to benzyl GalNAc-Gal, which in turn inhibits ?2,3 sialyltransferase activity, the most common in these cells. As a consequence of this, diverse sialylated compounds like benzyl GalNAc-Gal ?2,3 Neu5Ac are synthesised (Huet et al., 1995; Delannoy et al., 1996). On the other hand, benzyl GalNAc treatment did not affect Caco-2 colon cancer cells, which express fundamentally ?2,6 sialyltransferases, enzymes that are not inhibited by benzyl GalNAc (Huet et al., 1998). The aim of this work has been to determine the factor(s) responsible for the origin of the phenotype of HT-29 cells treated chronically with benzyl GalNAc. More in detail, we were interested in determine how a glycosylation inhibitor could produce a spectra of different effects and whether these effects were restricted only to HT-29 cells or not. Initially we worked with the hypothesis that the global effects of benzylGalNAc were derived from the glycoprotein hiposialylation. This hypothesis presumed a requirement of sialic acid for the apical glycoprotein transport/sorting. Thus, in the absence of sialic acid, this glycoproteins would be accumulated in TGN derived exocitic carriers, corresponding to the electron-lucid cytoplasmic vesicles (BG-vesicles). Consistent with this idea, we found through lectin analysis that ?2,3 linked sialic acid was distributed mainly in the apical membrane of epithelial cells both in culture and in tissues. Notoriously, ?2,6 linked sialic acid show a broad distribution and was located both in the apical membrane or in the basolateral membrane. The analysis of the treatment with benzyl GalNAc on a panel of cell lines indicated that its effects and severity were cell type depending but were not restricted only to HT-29 cells. Thus, benzyl GalNAc treated IMIM-PC-1 pancreas cancer cells shown a similar phenotype than benzyl GalNAc treated HT-29 cells. Notoriously, in these cells both apical (eg. MUC1) and basolateral (eg. ?1-integrin) glycoproteins were accumulated and colocalised into cytoplasmic vesicles. Benzyl GalNAc treatment affected the processing of apical and lysosomal glycoproteins in HT-29 M6 cells. We found that this pharmac induced a delay in maturation of cathepsin D and the blockage -in a post-TGN compartment- of the maturation of the lysosomal ?-glucosidasa. The endosomal route was affected in IMIM-PC-1 and HT-29 cells treated with benzyl GalNAc. Thus, benzyl GalNAc induced a reduction in the endocytosis rate and an accumulation of internalised material in IMIM-PC-1 cells. This material colocalised with vesicles positive for MUC1 or ?1 integrin. Late endosomes markers (Rab7, LBPA) but not early endosomes markers (Rab5, EEA1) or Golgi markers (GRASP65, TGN46) colocalised with vesicles accumulating ?1 integrin. Altogether, these results indicate that BG-vesicles correspond to an heterogenous population of aberrant endosomes related with late endosomes. The phenotype of benzyl GalNAc treated cells partially reproduces the defects seen in cells where saccharides are accumulated in the degradative compartiments. Thus, the morphology of the BG-vesicles is similar to those originated in sucrose treated cells (sucrosomes) and in cells from lysosome storage diseases (LSD) patients. The analysis of the processing of the lysosomal ?-glucosidase in fibroblasts of Infantile Sialic acid Storage Disease patients shown similar defects to those found in benzyl GalNAc HT-29 M6 treated cells. Moreover, benzyl GalNAc treated cells accumulate LBPA and cholesterol, commonly found in some LSD patients. Consistent with the idea that benzyl GalNAc induces a lysosomal storage defect, the sucrose treatment on IMIM-PC-1 cells induced the cytoplasmic accumulation of MUC1 and ?-1 integrin. In these cells, the glycoprotein sialylation was not affected, suggesting that in benzyl GalNAc treated cells the hiposialytion of membrane glycoproteins may not originate their intracelullar accumulation. In benzyl GalNAc treated cells a huge amount of benzyl GalNAc derived metabolites are accumulated, a scenario analogous to the sucrose treated or LSD cells. Therefore, we propose that is the metabolite accumulation, but not the glycoprotein hiposialylation, the responsible for the induction of the overall phenotype seen in benzyl GalNAc HT-29 and IMIM-PC-1 treated cells

Efectos del inhibidor de glicosilación benzil GalNAc sobre las rutas secretora y endocítica de células epiteliales

El benzil GalNAc fue descrito inicialmente como un inhibidor de la O-glicosilación de tipo mucina (Kuan et al., 1989). El tratamiento crónico de células mucosecretoras HT-29 M6 con este fármaco produjo una serie de efectos entre los que destacan: i) reducción de la proliferación, ii) inhibición de la secreción de mucinas, iii) acumulación de glicoproteínas apicales en vesículas citoplasmáticas electronlúcidas y, iv) reducción de la sialilación de glicoproteínas (Huet et al., 1998; (Hennebicq-Reig et al., 1998). El benzil GalNAc también inducía estos efectos sobre la población HT-29 parental y sobre otras poblaciones celulares derivadas de ésta, independientemente de su fenotipo. El benzil GalNAc es metabolizado a benzil GalNAc-Gal, el cual a su vez inhibe competitivamente la actividad ?2,3 sialiltransferasa de células HT-29, la actividad sialiltransferasa mayoritariamente expresada en estas células. Como resultado de ésto, se sintetizan compuestos sialilados como benzil GalNAc-Gal?2,3Neu5Ac (Huet et al., 1995; Delannoy et al., 1996). Por otro lado, el tratamiento con benzil GalNAc no afectaba sensiblemente a las células Caco-2, las cuales expresan fundamentalmente ?2,6 sialiltransferasas, enzimas que no son inhibidas por los metabolitos derivados del benzil GalNAc (Huet et al., 1998).El objetivo de este trabajo ha sido determinar el factor(es) responsable(s) del origen del fenotipo de células HT-29 tratadas crónicamente con benzil GalNAc. Más concretamente, nos interesó determinar cómo un inhibidor de glicosilación produce tantos efectos diferentes y si el efecto de este inhibidor estaba restringido o no a células derivadas de HT-29. La hipótesis que se manejó inicialmente proponía que los efectos globales del tratamiento con benzil GalNAc podrían estar causados por la hiposialilación de tardíos como, Rab7 y LBPA, pero no de endosomas primarios (Rab5, EEA1) y de Golgi (GRASP65, TGN46), colocalizaban con la ?1 integrina de las vesículas. El conjunto de los resultados obtenidos indica que las vesículas-BG corresponden a una población heterogénea de endosomas aberrantes, relacionados con endosomas tardíos.Las características fenotípicas de las células tratadas con benzil GalNAc reproducen parcialmente los defectos descritos en células de algunas enfermedades de depósito lisosomal (EDL), concretamente las que se producen por la acumulación de sacáridos en compartimentos degradativos. Así, la morfología de las vesículas-BG es similar a la de las que se originan en células tratadas con sacarosa (sacarosomas) y varias EDL como la sialidosis o la infantile sialic acid storage disease (ISSD). Análisis del procesamiento de la AAG en fibroblastos de pacientes con ISSD mostraron defectos similares a los encontrados en células HT-29 M6 tratadas con benzil GalNAc. Además, las células tratadas con benzil GalNAc también acumulan LBPA y colesterol, hallazgos característicos de algunas EDL. Confirmando la idea de que el benzil GalNAc induce un fenotipo de depósito lisosomal, el tratamiento con sacarosa en células IMIM-PC-1 produjo cambios sobre la morfología de las células y la acumulación intracelular de MUC1 y ?1 integrina, de manera semejante a lo observado en las células tratadas con benzil GalNAc. La sialilación de glicoproteínas en células IMIM-PC-1 tratadas con sacarosa no estaba alterada, resultado que indica que la hiposialilación no es un requisito para la acumulación intracelular de glicoproteínas de membrana.Dado que células tratadas con benzil GalNAc acumulan una gran cantidad de oligosacáridos derivados de esta droga, situación análoga a la que ocurre en células tratadas con sacarosa y algunas EDL, proponemos que la acumulación de estos metabolitos, y no la hiposialilación de glicoproteínas, es la que origina el fenotipo global de células HT-29 M6 e IMIM-PC-1 tratadas con este fármaco.Benzyl GalNAc was initially described as an inhibitor of mucin type O-glycosilation (Kuan et al., 1989). Long term exposure with this pharmac on mucosecretor HT-29 M6 colon cancer cells induced several effects: i) reduction of proliferation, ii) inhibition of the mucins secretion, iii) accumulation of apical glycoproteins into electron-lucid cytoplasmic vesicles (BG-vesicles) and iv) reduction of glycoprotein sialylation (Huet et al., 1998; Hennebicq-Reig et al., 1998). Moreover, these effects were induced in HT-29 parental and derived cells irrespectively of their phenotype. In HT-29 cells, benzyl GalNAc is metabolized to benzyl GalNAc-Gal, which in turn inhibits ?2,3 sialyltransferase activity, the most common in these cells. As a consequence of this, diverse sialylated compounds like benzyl GalNAc-Gal ?2,3 Neu5Ac are synthesised (Huet et al., 1995; Delannoy et al., 1996). On the other hand, benzyl GalNAc treatment did not affect Caco-2 colon cancer cells, which express fundamentally ?2,6 sialyltransferases, enzymes that are not inhibited by benzyl GalNAc (Huet et al., 1998).The aim of this work has been to determine the factor(s) responsible for the origin of the phenotype of HT-29 cells treated chronically with benzyl GalNAc. More in detail, we were interested in determine how a glycosylation inhibitor could produce a spectra of different effects and whether these effects were restricted only to HT-29 cells or not. Initially we worked with the hypothesis that the global effects of benzylGalNAc were derived from the glycoprotein hiposialylation. This hypothesis presumed a requirement of sialic acid for the apical glycoprotein transport/sorting. Thus, in the absence of sialic acid, this glycoproteins would be accumulated in TGN derived exocitic carriers, corresponding to the electron-lucid cytoplasmic vesicles (BG-vesicles).Consistent with this idea, we found through lectin analysis that ?2,3 linked sialic acid was distributed mainly in the apical membrane of epithelial cells both in culture and in tissues. Notoriously, ?2,6 linked sialic acid show a broad distribution and was located both in the apical membrane or in the basolateral membrane.The analysis of the treatment with benzyl GalNAc on a panel of cell lines indicated that its effects and severity were cell type depending but were not restricted only to HT-29 cells. Thus, benzyl GalNAc treated IMIM-PC-1 pancreas cancer cells shown a similar phenotype than benzyl GalNAc treated HT-29 cells. Notoriously, in these cells both apical (eg. MUC1) and basolateral (eg. ?1-integrin) glycoproteins were accumulated and colocalised into cytoplasmic vesicles. Benzyl GalNAc treatment affected the processing of apical and lysosomal glycoproteins in HT-29 M6 cells. We found that this pharmac induced a delay in maturation of cathepsin D and the blockage -in a post-TGN compartment- of the maturation of the lysosomal ?-glucosidasa.The endosomal route was affected in IMIM-PC-1 and HT-29 cells treated with benzyl GalNAc. Thus, benzyl GalNAc induced a reduction in the endocytosis rate and an accumulation of internalised material in IMIM-PC-1 cells. This material colocalised with vesicles positive for MUC1 or ?1 integrin. Late endosomes markers (Rab7, LBPA) but not early endosomes markers (Rab5, EEA1) or Golgi markers (GRASP65, TGN46) colocalised with vesicles accumulating ?1 integrin. Altogether, these results indicate that BG-vesicles correspond to an heterogenous population of aberrant endosomes related with late endosomes.The phenotype of benzyl GalNAc treated cells partially reproduces the defects seen in cells where saccharides are accumulated in the degradative compartiments. Thus, the morphology of the BG-vesicles is similar to those originated in sucrose treated cells (sucrosomes) and in cells from lysosome storage diseases (LSD) patients. The analysis of the processing of the lysosomal ?-glucosidase in fibroblasts of Infantile Sialic acid Storage Disease patients shown similar defects to those found in benzyl GalNAc HT-29 M6 treated cells. Moreover, benzyl GalNAc treated cells accumulate LBPA and cholesterol, commonly found in some LSD patients. Consistent with the idea that benzyl GalNAc induces a lysosomal storage defect, the sucrose treatment on IMIM-PC-1 cells induced the cytoplasmic accumulation of MUC1 and ?-1 integrin. In these cells, the glycoprotein sialylation was not affected, suggesting that in benzyl GalNAc treated cells the hiposialytion of membrane glycoproteins may not originate their intracelullar accumulation.In benzyl GalNAc treated cells a huge amount of benzyl GalNAc derived metabolites are accumulated, a scenario analogous to the sucrose treated or LSD cells. Therefore, we propose that is the metabolite accumulation, but not the glycoprotein hiposialylation, the responsible for the induction of the overall phenotype seen in benzyl GalNAc HT-29 and IMIM-PC-1 treated cells

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