The multiple roles of TDP-43 in pre-mRNA processing and gene expression regulation.

Heterogeneous ribonucleoproteins (hnRNPs) are multifunctional RNA-binding proteins (RBPs) involved in many cellular processes. They participate in most gene expression pathways, from DNA replication and repair to mRNA translation. Among this class of proteins, TDP-43 (and more recently FUS/TLS) have received considerable attention due to their involvement in several neurodegenerative diseases. This finding has prompted many research groups to focus on the gene expression pathways that are regulated by these proteins. The results have uncovered a considerable complexity of TDP-43 and FUS/TLS functions due to the many independent mechanisms by which they may act to influence various cellular processes (such as DNA transcription, pre-mRNA splicing, mRNA export/import). The aim of this chapter will be to review especially some of the novel functions that have been uncovered, such as role in miRNA synthesis, regulation of transcript levels, and potential autoregulatory mechanisms in order to provide the basis for further investigations.

The splicing code.

This issue dedicated to the code of life tackles very challenging and open questions in Biology. The genetic code, brilliantly uncovered over 50 years ago is an example of a univocal biological code. In fact, except for very few and marginal variations, it is the same from bacteria to man, the RNA stretch: 5' GUGUUC 3' reads as the dipeptide: Val-Phe in bacteria, in yeast, in Arabidopsis, in zebra fish, in mouse and in human. A degree of ambiguity is possible if mutations are introduced in the tRNAs in a way that the anticodon reads one amino acid but the aminoacyl-transferase attaches a different one onto the tRNA. These were the very useful suppressor genes that aided greatly the study of bacterial genetics. Other biological codes however, are more akin to social codes and are less amenable to an unambiguous deciphering. Legal and ethical codes, weather we like it or not, are flexible and depend on the structure and history of the society that has produced them, as well as a specific point in time. The codes that govern RNA splicing have similar characteristics. In fact, the splicing code depends on a myriad of different factors that in part are influenced by the background in which they are read such as different cells, tissues or developmental stages. Given the complexity of the splicing process, the construction of an algorithm that can define exons or their fate with certainty has not yet been achieved. However a substantial amount of information towards the deciphering of the splicing code has been gathered and in this manuscript we summarize the point reached.

RhoGAPp190: A potential player in tbph-mediated neurodegeneration in Drosophila.

TDP-43 is an ubiquitous and highly conserved ribonucleoprotein involved in several cellular processes including pre-mRNA splicing, transcription, mRNA stability and transport. Notwithstanding the evidence of TDP-43 involvement in the pathogenesis of different neurodegenerative disorders (i.e. ALS and FTLD), the underlying mechanisms are still unclear. Given the high degree of functional similarity between the human and fly orthologs of TDP-43, Drosophila melanogaster is a simple and useful model to study the pathophysiological role of this protein in vivo. It has been demonstrated that the depletion of the TDP-43 fly ortholog (tbph) induces deficient locomotive behaviors and reduces life span and anatomical defects at the neuromuscular junction. In this study, using the known binding specificity of TDP-43/tbph for (UG) repeated sequences, we performed a bioinformatic screening for fly genes with at least 6 (TG) repeats in a row within the 3'-UTR regions in order to identify the genes that might be regulated by this factor. Among these genes, we were able to identify RhoGAPp190 as a potential target of the tbph-mediated neurodegeneration. RhoGAPp190 is a negative regulator of Drosophila RhoA, a GTPase protein implicated in the fine modulation of critical cellular processes including axon branch stability and motor axon defasciculation at muscle level and cognitive processes. We were able to demonstrate that the RhoGAPp190 expression is upregulated in a tbph-null fly model, providing evidence that this deregulation is associated to tbph silencing. Our results introduce RhoGAPp190 as a novel potential mediator in the complex scenario of events resulting from in vivo tbph loss-of-function.

Visión general y perspectivas futuras del estudio de las variaciones epigenéticas / Overview and future perspectives of the study of epigenetic variations / Visão geral e perspectivas futuras do estudo das variações epigenéticas / Reconocimiento a la trayectoria del Prof. Dr. Juan Miguel Castagnino†

Resumen Los cambios epigenéticos juegan en el organismo un papel importante en el control de la expresión génica, durante el desarrollo y a lo largo de toda la vida, sobre todo durante el envejecimiento. En los últimos años se han acumulado evidencias que avalan la participación de los procesos epigenéticos en el desarrollo y evolución de diversas enfermedades como procesos tumorales, enfermedades genéticas, cardiovasculares y neurodegenerativas. Además, los marcadores epigenéticos (metilación del ADN, modificaciones en las histonas y los ARN no codificantes) podrían indicar la predisposición del individuo a determinados procesos patológicos. La administración de fármacos epigenéticos ha demostrado ser eficiente en el tratamiento de enfermedades tales como la aterosclerosis, neoplasias, procesos neurodegenerativos, enfermedades hepáticas, etc. En este artículo se abordarán algunos ejemplos de la contribución que las modificaciones epigenéticas dan a la patogenia de las enfermedades neurodegenerativas y cardiovasculares. En el futuro, la bioquímica clínica será frecuentemente utilizada en los análisis epigenéticos y ayudará al diseño de fármacos y estrategias terapéuticas dirigidas a modificar el epigenoma.

Abstract In the organism, epigenetic changes play an important role in the control of gene expression, during its development and throughout life, especially during ageing. In recent years, evidence has accumulated that supports the participation of epigenetic processes in the development and evolution of various diseases such as tumor processes, genetic, cardiovascular and neurodegenerative diseases. In addition, epigenetic markers (DNA methylation, histone modifications and non-coding RNAs) could indicate the predisposition to certain pathological processes. The administration of epigenetic drugs has proven to be efficient in the treatment of diseases such as atherosclerosis, neoplasms, neurodegenerative processes, liver diseases, etc. In this article we will address some examples of the contribution that epigenetic modifications give to the pathogenesis of neurodegenerative and cardiovascular diseases. In the future, clinical biochemistry will be frequently used in epigenetic analyzes and will help design drugs and therapeutic strategies aimed to modify the epigenome.

Resumo As alterações epigenéticas têm no organismo um papel importante no controle da expressão gênica durante o desenvolvimento e ao longo de toda a vida, principalmente durante o envelhecimento. Nos últimos anos, foram acumuladas evidências que demonstram a participação dos processos epigenéticos no desenvolvimento e evolução de diversas doenças como, por exemplo, processos tumorais, doenças genéticas, cardiovasculares e neurodegenerativas. Além disso, os marcadores epigenéticos (metilação do DNA, modificações nas histonas e nos RNA não codificantes), poderiam indicar a predisposição do indivíduo a determinados processos patologicos. A administração de fármacos epigenéticos demonstrou ser eficiente no tratamento de doenças tais como a aterosclerose, neoplasias, processos neurodegenerativos, doenças hepáticas, etc. Neste estudo abordaremos alguns exemplos da contribuição que as alterações epigenéticas dão à patogenia das doenças neurodegenerativas e cardiovasculares. No futuro, a bioquímica clínica será frequentemente utilizada nas análises epigenéticas e ajudará ao desenho de medicamentos e estratégias terapêuticas dirigidas a modificar o epigenoma.

Impaired motor coordination in mice lacking the EDA exon of the fibronectin gene.

The extracellular matrix (ECM) plays an important role in the central nervous system (CNS) by modulating the migration of cells, axons and dendrites of neurons. Fibronectin (FN) is a major component of the ECM in the CNS and plays essential roles in development, cell adhesion and cell migration. Specific FN-isoforms, generated by alternative splicing at three conserved regions, the extra domain B (EDB), extra domain A (EDA) and type III homologies connecting segment (IIICS), have been shown to modulate these processes in vitro and in vivo. The inclusion of the EDA exon in the brain is highly regulated during development and aging, suggesting an important role of this exon in brain function. However, the direct role of FN-isoforms in brain function and behaviour is still obscure. Therefore, to directly assess the role of the FN-EDA exon in vivo, we have generated two mouse strains devoid of EDA exon regulated splicing in the FN gene that constitutively include (EDA(+/+)) or exclude (EDA(-/-)) the EDA exon in all tissues. Here, we show the behavioural consequences of the absence of regulated splicing of the EDA exon in the FN gene. Deletion of the EDA domain in the FN protein results in reduced motor-coordination abilities and vertical exploratory capacity, whereas mice that constitutively include the EDA domain displayed a decrease in locomotory activity in the open field (OF) test. These results strongly suggest that regulated splicing of the EDA exon is necessary for a normal function of the brain.

La agregación de TDP-43 como posible blanco terapéutico contra la Esclerosis Lateral Amiotrófica / TDP-43 aggregation as a possible therapeutic target for Amyotrophic Lateral Sclerosis / Agregação de TDP-43 como um possível alvo terapêutico para a Esclerose Lateral Amiotrófica

Los agregados de TDP-43 representan una de las característica histopatológicas más importantes de varias enfermedades neurodegenerativas, entre las que se incluye la Esclerosis Lateral Amiotrófica (ELA). TDP-43 está localizada principalmente en el núcleo. Sin embargo, los pacientes afectados por ELA presentan agregados de TDP-43 en el citoplasma de las neuronas comprometidas, con lo que se despoja al núcleo de TDP-43 funcional. Aún se desconoce si la degeneración causada por la agregación de TDP-43 es debida a una toxicidad intrínseca de los agregados o a la pérdida de función de TDP-43 como consecuencia del vaciamiento del núcleo. Varias investigaciones, incluidas las de estos autores, indican que la pérdida de función es el factor fundamental responsable de la neurodegeneración observada en presencia de inclusiones de TDP-43. Por otro lado, aún no existen tratamientos efectivos para la ELA. Por lo tanto, es de crucial importancia conocer las bases moleculares que conllevan al desarrollo de la enfermedad, con el objetivo de encontrar posibles estrategias terapéuticas. Para ello, estos autores han desarrollado un modelo celular capaz de imitar la agregación de TDP-43 y sus consecuencias. Finalmente, se ha utilizado este modelo para analizar el efecto de diferentes compuestos capaces de degradar los agregados de TDP-43 y se ha demostrado que esta podría ser una estrategia terapéutica válida para la ELA.

TDP-43 inclusions are important histopathological features of various neurodegenerative disorders, including Amyotrophic Lateral Sclerosis (ALS). TDP-43 is mainly a nuclear protein, but it shuffles from the nucleus to the cytoplasm. In patients’ brains, TDP-43 is retained in the cytoplasm of the affected motorneurons to form insoluble aggregates, which results in TDP-43 nuclear clearance. There is still no consensus whether TDP-43-mediated neurodegeneration results from a gain or loss of function of the protein or a combination of both. The work from several laboratories, including this, points towards a strong loss of function component. On the other hand, there is no effective treatment or cure for ALS. Thus, there is obviously a need to find new therapeutic strategies for ALS. In order to gain new insights into the molecular mechanism of the disease, and with the aim of looking for new methodologies that can revert it, a cellular model of TDP-43 aggregation that can mimic the phenotypic consequences found in ALS patients has been developed. Finally, this model was used to search for compounds that can dissolve these aggregates, and it was shown that the clearance of TDP-43 aggregates could be a therapeutic strategy for ALS.

Os agregados proteicos TDP-43 são características histopatológicas importantes de muitas doenças neurodegenerativas, incluindo a Esclerose Lateral Amiotrófica (ALS). A proteína TDP-43 se localiza principalmente no núcleo, porém nos cérebros de indivíduos afetados, a proteína TDP-43 fica retida no citoplasma dos neurônios motores, o que leva a formação de agregados insolúveis, resultando em deposição nuclear. Ainda não existe um consenso se a neurodegeneração mediada por TDP43 é causada por ganho ou perda da função da proteína ou uma combinação de ambos. O trabalho de muitos laboratórios, bem como este trabalho, apontam para uma forte perda da função da proteína. Por outro lado, não existe um tratamento efetivo ou cura para a ALS. Portanto, existe uma grande necessidade de identificar novos tratamentos para a ALS. Para entender o mecanismo molecular da doença, e com o objetivo de identificar novas metodologias para reverter a doença, desenvolvemos o modelo celular de agregados de TDP-43, o qual mimetiza as consequências fenotípicas encontradas em pacientes com ALS. Por fim, utilizamos esse modelo para identificar compostos que podem dissolver os agregados, e demonstramos que a liberação de inclusões de TDP-43 poderiam ser usados como tratamentos para a ALS.

Sam68 regulates EMT through alternative splicing-activated nonsense-mediated mRNA decay of the SF2/ASF proto-oncogene.

Epithelial-to-mesenchymal transition (EMT) and its reversal (MET) are crucial cell plasticity programs that act during development and tumor metastasis. We have previously shown that the splicing factor and proto-oncogene SF2/ASF impacts EMT/MET through production of a constitutively active splice variant of the Ron proto-oncogene. Using an in vitro model, we now show that SF2/ASF is also regulated during EMT/MET by alternative splicing associated with the nonsense-mediated mRNA decay pathway (AS-NMD). Overexpression and small interfering RNA experiments implicate the splicing regulator Sam68 in AS-NMD of SF2/ASF transcripts and in the choice between EMT/MET programs. Moreover, Sam68 modulation of SF2/ASF splicing appears to be controlled by epithelial cell-derived soluble factors that act through the ERK1/2 signaling pathway to regulate Sam68 phosphorylation. Collectively, our results reveal a hierarchy of splicing factors that integrate splicing decisions into EMT/MET programs in response to extracellular stimuli.