Line 1. Role of motor neuron survival factor (SMN) acetylation in the Cajal nuclear body assembly (Cajal body, CB): importance in spinal muscular atrophy (SMA). The core of Cajal (CB, Fig. 1) is the nuclear power plant that directs the final assembly and quality control of the snRNPs and snoRNPs (small nuclear nucleolar ribonucleoproteins) involved in the splicing of pre-mRNAs And in the maturation of pre-rRNAs. The mutation in the gene encoding one of its essential proteins, SMN (Survival Motor Neuron), is responsible for spinal muscular atrophy (SMA), which causes motor neuron degeneration and is the main cause of genetic-based mortality in the childhood. In addition to SMNs and snRNPs and snoRNPs, another essential component of CB is the coilin nucleating protein. The fundamental objective of this line is to analyze the mechanisms that regulate the molecular assembly of the CB and its importance in the SMA. We have recently shown that the SMN protein is a substrate of SUMO1 and that its conjugation with SUMO is another factor regulating the formation of CBs. We are currently studying the impact of another post-translational modification of SMN, acetylation, on the assembly of snRNPs and snoRNPs and the formation of CBs. Our preliminary experiments indicate that SMN is acetylated by acetyltranferase and CBP analysis with mass spectrometry demonstrates that acetylation modifies the interactions of the SMN with its target proteins, affecting the cellular localization of the protein and its ability to nuclear CBs .
Line 2. Importance of dysfunction of nuclear compartments of spinal motor neurons in the cellular and molecular pathophysiology of spinal muscular atrophy (SMA) in murine model SMNd7. As discussed above, mutation or deletion of the SMN1 gene in SMA produces a severe deficiency of the functional SMN protein, leading to degeneration and death of spinal neurons. In the pathophysiology of SMA, three essential mechanisms exist at the level of spinal motoneurons: splicing dysfunction of pre-mRNAs, axonal transport alterations and dysfunction of the neuromuscular synapse. All of them contribute to neurodegeneration and, consequently, to atrophy and muscular paralysis. Our research focuses on deepening the nuclear mechanisms affected by the disease, which results in an alteration of the nuclear metabolism of the RNAs in the motoneurons (Fig. 2). SMA has reported a deficit in the assembly of snRNPs for the spliceosome, the molecular machinery that governs the splicing of pre-mRNAs. The Cajal nuclear body (CB) is a nuclear power plant that governs the assembly and nuclear trafficking of snRNPs and snoRNPs to the spliceosome and nucleolus, respectively.
In this context, we consider that the loss of CBs and their interactions with the nucleolus, required for the transport of snoRNPs involved in nucleolar processing of rRNAs, are responsible for the severe disruption of splicing and biogenesis of ribosomes in the motoneurons of The SMA. To investigate these nuclear mechanisms we used the transgenic mouse SMNd7 as a model of SMA type I, the most severe that causes the degeneration of motor neurons and death of animals between postnatal days 13 and 16.
Line 3. Neuronal response to DNA damage: importance in neurodegeneration. There is growing evidence in the literature that defects in DNA repair and the consequent accumulation of DNA lesions play an important role in the molecular pathophysiology of multiple neurodegenerative processes. Our goal is to analyze the nuclear processing of DNA damage in normal, ganglionic and cerebral cortex neurons irradiated with X-rays (4 Gy) to induce DNA double-strand breaks. In this model we have observed that most of the neuronal DNA breaks are repaired in the first 24 hours, but there are persistent foci of unrepaired DNA that remain for months in a specific nuclear compartment. We have characterized the structural, molecular and spatial organization of this nuclear compartment as well as its transcriptional repression (Fig. 3). In addition we are characterizing with ChIP-seq techniques DNA sequences enriched in the "persistent foci" of cortical neurons. We have identified 17 sequences associated with genes essential for neuronal homeostasis whose dysfunction is related to pictures of human neuropathology. This line opens a new horizon that should allow to identify neural genes very vulnerable to DNA damage or difficult to repair, whose accumulation of lesions may be involved in neurodegenerative diseases