Characterization of the Armcx/Almc10 gene family function in mitochondrial dynamics and neural development

Abstract

[spa]Los genes Armcx pertenecen a una misma familia localizada en el cromosoma X y caracterizada por la posesión de dominios armadillo en su secuencia proteica. En este trabajo se ha analizado la expresión de los genes Armcx/Armc10 durante el desarrollo embrionario, en los tejidos neurales en desarrollo y en el cerebro adulto. A continuación se ha analizado localización subcellular de las proteínas Armc10 y Alex3 (codificada por el gen Armcx3), que se encontraron mayoritariamente localizadas a núcleo y mitocondria. La sobreexpresión de Armc10 y Alex3 provoca la agregación y/o “tethering” mitocondrial en las neuronas, donde estos procesos sirven para anclar estas organelas en localizaciones específicas que requieren una alta demanda de energía y unos requerimientos de taponamiento de Ca2+. En la base de nuestros datos bioquímicos y de los estudios funcionales proponemos un modelo en el que las proteínas Alex3 y Armc10 son reguladores positivos del tráfico mitocondrial, interaccionando directamente con los complejos Miro/Trak2. Además, como se muestra para el complejo KIF5/Miro/Trak2, el incremento de la actividad neuronal que conlleva a incrementos en Ca2+ es probablemente la causa del desensamblaje del complejo y de la parada mitocondrial en los lugares de neurotransmisión activa, completando por lo tanto los requerimientos bioenergéticos de la transmisión neuronal. A continuaciòn utilizamos la médula espinal de pollo como modelo fisiológico in vivo para explorar la función de Alex3 y Armc10 en el desarrollo neural. Se encontró que Alex3 cumple un papel regulador por inhibición de la vía de Wnt/β-catenina. También demostramos que Alex3 está involucrado tanto en la regulación negativa del ciclo celular como en la inducción de la diferenciación de precursores neuronales. Por otro lado, Armc10 sólo se encontrò implicado en la regulación negativa del ciclo celular. Estas diferencias entre los efectos de la sobreexpresión de Alex3 y Armc10 en procesos llave del desarrollo de la médula espinal, evidencian las divergencias funcionales entre las dos proteínas, sugiriendo que Alex3 puede haber adquirido funciones adicionales respecto a Armc10, ancestro filogenético del cluster Armcx. Globalmente nuestros datos sugieren que la vía de señalización de Wnt puede estar regulando procesos llave del desarrollo a través de las proteínas mitocondriales Alex3 y Armc10.

[eng]The Armcx gene cluster arose by retrotransposition from a single Arm-containing gene (Armc10), and by subsequent short-range tandem duplications of a rapidly evolving region of the Eutherian X chromosome. In this work we analysed the expression of Armcx/Armc10 genes during embryonic development, describing their expression in the developing neural tissues. As yet shown for Alex3 protein, Armc10 also causes mitochondrial aggregation and/or tethering in neurons and HEK293 cells. In neurons, these processes are believed to serve to capture mitochondria at specific locations requiring high-energy and Ca2+ buffering conditions. The mechanism by which Alex3 and Armc10 cause mitochondrial aggregation may involve Mitofusins, as they both interact with Mfn1 and Mfn2. Nevertheless, we were unable to find evidence about the involvement of Alex3 protein in mitochondrial fusion. Alex3 and Armc10 interact with the KIF5/Miro/Trak2 complex (which controls mitochondrial dynamics in neurons), through a direct interaction with Miro1-2 and Trak2. Importantly, this interaction requires low Ca2+ concentrations. We suggest that the Ca2+-dependent conformational changes in Miro proteins are the essential mechanisms regulating the interaction between Alex3 and the Miro/Trak2complex. Thus, while low Ca2+ concentrations may favour the formation of KIF5/Miro/Trak2/Alex3 complex, increases in intracellular Ca2+ rapidly uncouple such complex (including Alex3), thereby arresting mitochondrial trafficking. The notion that Alex3 (and possibly also Armc10) interacts with the Miro/Trak2 complex when mitochondria are motile at low Ca2+ concentrations is further supported by our findings that knockdown of Alex3 (such as Armc10) results in a decrease in the percentages of motile mitochondria, similarly to what was observed in Miro/Trak2 loss-of-function. In conclusion, we described Alex3 and Armc10 as proteins with evolutionarily conserved functions in the regulation of mitochondrial dynamics and transport. However, gene-specific particularities are present, suggesting overlapping but differential levels and mechanism of regulation of mitochondrial dynamics and transport by Alex3 and Armc10 proteins and probably by the whole Armcx cluster. We next speculate that Alex3 (but also the whole Armcx cluster) could play a role in regulation of mitochondrial dynamics or function during neural development by regulating Wnt/β-catenin signaling pathway. Using the chicken spinal cord as physiological model, we found that Alex3 overexpression decreases TCF/LEF-transcriptional activity at basal condition and following Wnt3a or β-catenin induction, indicating that Alex3 substantially acts as an inhibitor of canonical Wnt/β-catenin pathway. Moreover, we showed that Alex3 is involved in both negative cell cycle regulation and induction of differentiation in chicken spinal cord, while Armc10 is only involved in negative cell cycle regulation. These differences between Alex3 and Armc10 overexpression effects on spinal cord developmental processes highlight functional divergences between the two proteins, suggesting that Alex3 may have acquired additional function respect to Armc10, phylogenetic ancestor of the whole Armcx gene cluster. The data collected in this study, describing Alex3 overexpression effects on spinal cord development, are coherent with the inhibitor function of Alex3 on Wnt/β-catenin pathway. However there is not sufficient evidence to sustain that these effects on progenitor cell cycle and neuronal differentiation are achieved by inhibition of Wnt/β-catenin pathway and further experiments are required to support this hypothesis. To shade light on the biological processes in which Alex3 can be involved (trough regulation of mitochondrial dynamics, regulation of Wnt/β-catenin pathway or new and different activity) we carried out a genome-wide analysis of changes in mRNA levels subjecting HEK293AD cells stably expressing Alex3GFP to microarray analysis. We found changes in global gene expression that involved key processes of cellular physiology, such as metabolic processes or cell cycle progression. However we are still far to identify the cellular and molecular mechanism by which Alex3 (and the whole Armcx/Armc10 cluster gene) acts in these processes. To achieve this goal it is essential to clarify Armcx/Armc10 function in nervous system development.