El splicing alternativo aberrante del mRNA-SMN2 induce en la SMA humana y murina (SMNΔ7) una miopatía de actina primaria: efecto corrector del tratamiento con el ASO Nusinersen

RESUMEN: La atrofia muscular espinal (SMA) es una enfermedad hereditaria, con patología neuromuscular, considerada la principal causa de mortalidad infantil de base genética. Está causada por la deleción o mutación del gen SMN1 que codifica el factor de supervivencia de las neuronas motoras (SMN). El déficit de SMN conduce a muerte de las motoneuronas espinales alfa y, consecuentemente, a la miopatía neurógénica. En humanos existe un segundo gen SMN2 con escasa eficiencia, sus transcritos experimentan un splicing alternativo aberrante con pérdida del exón 7 y producción de una proteína truncada no funcional (SMN∆7). La SMA no tiene tratamiento curativo pero la terapia génica con el oligonucleótido antisentido (ASO-Nusinersen) es prometedora en la clínica humana. No obstante, su eficiencia es limitada fuera del SNC, mejora la actividad motora pero solo parcialmente revierte las lesiones en las miofibras esqueléticas de la SMA. En este Tesis Doctoral se analiza la repercusión fisiopatológica que el déficit de SMN tiene a nivel muscular y la potencial eficiencia terapéutica del ASO-Nusinersen dirigida específicamente al músculo. Los resultados demuestran: i) que SMN es un componente esencial de la sarcómera humana y su déficit causa una miopatía primaria por acumulación de actina; ii) que el ratón SMN∆7 mimetiza la miopatía humana acompañada de disfunción en la secreción de mioquiinas, biogénesis mitocondrial y adquisición de miofibras tipo II; iii) que el tratamiento con el ASO-Nusinersen corrige el splicing aberrante del SMN2 mRNA en motoneuronas, mejorando la supervivencia y actividad motora en los ratones SMN∆7, pero solo revierte parcialmente la patología muscular; iv) que el sistema de liberación del ASO-Nusinersen, basado en nanopartículas biopoliméricas conjugadas con Nusinersen (Spinraza) y el Aptámero-AB01 específico del músculo, ofrece una potencial estrategia para la terapia correctora de la miopatía SMA.ABSTRACT: Spinal muscular atrophy (SMA) is an inherited disease with neuromuscular pathology, considered the leading cause of genetically based infant mortality. It is caused by deletion of the SMN1 gene encoding the survival motor neuron (SMN) factor. SMN deficiency leads to death of alpha spinal motor neurons and, consequently, to neurogenic myopathy. In humans, there is a second SMN2 gene with poor efficiency as its transcripts undergo aberrant alternative splicing with loss of exon 7 and production of a non-functional truncated protein (SMN∆7). SMA has no curative treatment but gene therapy with the antisense oligonucleotide (ASO-Nusinersen) is promising in the human clinic. However, its efficiency is limited outside the CNS as it improves motor activity but does not completely reverse the lesions in the skeletal myofibers of SMA. In this Doctoral Thesis we analyze the pathophysiological repercussions of SMN deficit at the muscular level and the potential therapeutic efficiency of ASO-Nusinersen specifically directed to this organ. The results demonstrate that (i) SMN is an essential component of the human sarcomere and its deficit causes a primary actin-accumulation myopathy; (ii) the mouse SMN∆7 mimics human SMA myopathy accompanied by dysfunction in myokine secretion, mitochondrial biogenesis and acquisition of type II (white and fast) myofibers; iii) the treatment with ASO-Nusinersen corrects aberrant SMN2 mRNA splicing, in motor neurons, enhancing survival and motor activity in SMN∆7 mice, but only partially ameliorates muscle pathology; iv) the delivery system based on biopolymeric nanoparticles conjugated with the ASO Nusinersen (Spinraza) and muscle-specific Aptamer-AB01 provides a potential therapeutic strategy for the SMA myopathy.Esta Tesis ha sido financiada con las siguientes ayudas: • Fundació La Marató de TV3 (Ref: 202005). “Preclinical analysis of new combinatorial treatments for spinal muscular atrophy (SMA): effects on motoneuron survival, synaptic integrity, and skeletal muscle preservtion”. • Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas. Red CIBERNED CB06/05/0037. • Instituto de Investigación Sanitaria Valdecilla (IDIVAL, Santander). Proyectos de investigación NVAL17/22 e INNVAL17/20

Neuroprotective Effect of Bexarotene in the SOD1(G93A) Mouse Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive weakness and muscle atrophy related to the loss of upper and lower motor neurons (MNs) without a curative treatment. There is experimental evidence suggesting that retinoids may be involved in ALS pathogenesis. Bexarotene (Bxt) is a retinoid-X receptor agonist used in the treatment of cutaneous lymphoma with a favorable safety profile whose effects have been recently investigated in other neurodegenerative diseases. In this study, we analyze the potential therapeutic effect of Bxt in the SOD1(G93A) mouse model of ALS. Mice were treated with Bxt or vehicle five times per week from day 60 onward. Survival, weight, and neuromuscular function studies together with histological and biochemical analyses were performed. Bxt significantly delayed motor function deterioration, ameliorated the loss of body weight, and extended mice survival up to 30% of the symptomatic period. Histological analyses of the lumbosacral spinal cord revealed that Bxt markedly delayed the early motor-neuron degeneration occurring at presymptomatic stages in ALS-transgenic mice. Bxt treatment contributed to preserve the MN homeostasis in the SOD1(G93A) mice. Particularly, it reduced the neuronal loss and the chromatolytic response, induced nucleolar hypertrophy, decreased the formation of ubiquitylated inclusions, and modulated the lysosomal response. As an agonist of the retinoic-X receptor (RXR) pathway, Bxt notably increased the nuclear expression of the RXRα throughout transcriptionally active euchromatin domains. Bxt also contributed to protect the MN environment by reducing reactive astrogliosis and preserving perisomatic synapsis. Overall, these neuroprotective effects suggest that treatment with Bxt could be useful in ALS, particularly in those cases related to SOD1 mutations

Satellite Glial Cells of the Dorsal Root Ganglion: A New ?Guest/Physiopathological Target? in ALS

Introduction: Amyotrophic lateral sclerosis (ALS) might not only be circumscribed to the motor system but also involves other neuronal systems including sensory abnormalities. In line with this notion, we aimed to assess the pathophysiology of sensory disturbances in the SOD1G93A mouse model of ALS, focusing on the satellite glial cells (SGCs) at the dorsal root ganglion (DRG) as a new potential target of the disease. MaterialandMethods: Thepresenceofsensorydisturbanceswasevaluatedusingvon Frey, hot plate, and hot water tail immersion tests at 75 days old, which represented the motor-pre-symptomatic stage. Cell biology analysis was performed at 75 and 95 days old and included conventional histology, immuno?uorescence, and electron microscopy of sensory neuron-SGC unit dissociates as a well as western blotting from DRG lysates. Results: At 75 days old, von Frey and hot plate tests demonstrated clear thermoalgesic disturbances in ALS transgenic mice. Histological studies of the SN-SGC units revealed abnormal SOD1 accumulation, which was associated with nitro-oxidative stress and biogenesis of lipid droplets in SGCs. Interestingly, these alterations led to a progressive lysosomal storage disorder and occasionally vacuolar degeneration in SGCs. Conclusions: SGCs emerge as a primary pathophysiological target in the SOD1 transgenic murine model of ALS, clearly reinforcing the pathogenic role of glial cells in motor neuron disease. Presymptomatic alterations of SGCs, might not only be responsibleofsensorydisturbancesinALS,butduetospinalcordsensory-motorcircuits could also contribute to anterior horn motor disturbance

A novel pathway of TEF regulation mediated by microRNA-125b contributes to the control of actin distribution and cell shape in fibroblasts

Background: Thyrotroph embryonic factor (TEF), a member of the PAR bZIP family of transcriptional regulators, has been involved in neurotransmitter homeostasis, amino acid metabolism, and regulation of apoptotic proteins. In spite of its relevance, nothing is known about the regulation of TEF. Principal findings: p53-dependent genotoxic agents have been shown to be much more harmful for PAR bZIP-deficient mice as compared to wild type animals. Here we demonstrate that TEF expression is controlled by p53 through upregulation of microRNA-125b, as determined by both regulating the activity of p53 and transfecting cells with microRNA-125b precursors. We also describe a novel role for TEF in controlling actin distribution and cell shape in mouse fibroblasts. Lack of TEF is accompanied by dramatic increase of cell area and decrease of elongation (bipolarity) and dispersion (multipolarity). Staining of actin cytoskeleton also showed that TEF (-/-) cells are characterized by appearance of circumferential actin bundles and disappearance of straight fibers. Interestingly, transfection of TEF (-/-) fibroblasts with TEF induced a wild type-like phenotype. Consistent with our previous findings, transfection of wild type fibroblasts with miR-125b promoted a TEF (-/-)-like phenotype, and a similar but weaker effect was observed following exogenous expression of p53. Conclusions/significance: These findings provide the first evidence of TEF regulation, through a miR-125b-mediated pathway, and describes a novel role of TEF in the maintenance of cell shape in fibroblasts

Neuronal accumulation of unrepaired DNA in a novel specific chromatin domain: structural, molecular and transcriptional characterization

There is growing evidence that defective DNA repair in neurons with accumulation of DNA lesions and loss of genome integrity underlies aging and many neurodegenerative disorders. An important challenge is to understand how neurons can tolerate the accumulation of persistent DNA lesions without triggering the apoptotic pathway. Here we study the impact of the accumulation of unrepaired DNA on the chromatin architecture, kinetics of the DNA damage response and transcriptional activity in rat sensory ganglion neurons exposed to 1-to-3 doses of ionizing radiation (IR). In particular, we have characterized the structural, molecular and transcriptional compartmentalization of unrepaired DNA in persistent DNA damaged foci (PDDF). IR induced the formation of numerous transient foci, which repaired DNA within the 24 h post-IR, and a 1-to-3 PDDF. The latter concentrate DNA damage signaling and repair factors, including ?H2AX, pATM, WRAP53 and 53BP1. The number and size of PDDF was dependent on the doses of IR administered. The proportion of neurons carrying PDDF decreased over time of post-IR, indicating that a slow DNA repair occurs in some foci. The fine structure of PDDF consisted of a loose network of unfolded 30 nm chromatin fiber intermediates, which may provide a structural scaffold accessible for DNA repair factors. Furthermore, the transcription assay demonstrated that PDDF are transcriptionally silent, although transcription occurred in flanking euchromatin. Therefore, the expression of ?H2AX can be used as a reliable marker of gene silencing in DNA damaged neurons. Moreover, PDDF were located in repressive nuclear environments, preferentially in the perinucleolar domain where they were frequently associated with Cajal bodies or heterochromatin clumps forming a structural triad. We propose that the sequestration of unrepaired DNA in discrete PDDF and the transcriptional silencing can be essential to preserve genome stability and prevent the synthesis of aberrant mRNA and protein products encoded by damaged genes

Oxidative-Stress-Associated Proteostasis Disturbances and Increased DNA Damage in the Hippocampal Granule Cells of the Ts65Dn Model of Down Syndrome

Oxidative stress (OS) is one of the neuropathological mechanisms responsible for the deficits in cognition and neuronal function in Down syndrome (DS). The Ts65Dn (TS) mouse replicates multiple DS phenotypes including hippocampal-dependent learning and memory deficits and similar brain oxidative status. To better understand the hippocampal oxidative profile in the adult TS mouse, we analyzed cellular OS-associated alterations in hippocampal granule cells (GCs), a neuronal population that plays an important role in memory formation and that is particularly affected in DS. For this purpose, we used biochemical, molecular, immunohistochemical, and electron microscopy techniques. Our results indicate that TS GCs show important OS-associated alterations in the systems essential for neuronal homeostasis: DNA damage response and proteostasis, particu larly of the proteasome and lysosomal system. Specifically, TS GCs showed: (i) increased DNA damage, (ii) reorganization of nuclear proteolytic factories accompanied by a decline in proteasome activity and cytoplasmic aggregation of ubiquitinated proteins, (iii) formation of lysosomal-related structures containing lipid droplets of cytotoxic peroxidation products, and (iv) mitochondrial ultrastructural defects. These alterations could be implicated in enhanced cellular senescence, accelerated aging and neurodegeneration, and the early development of Alzheimer?s disease neuropathology present in TS mice and the DS population.Funding: This work was supported by the following grants: “Instituto de Investigación Valdecilla” (IDIVAL; NVAL 19/23), Santander, Spain; “Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas” (CIBERNED; CB06/05/0037) Spain; and “Agencia Estatal de Investicación, MICIN” (grant number: PID2020-117601RB-I00). Acknowledgments: The authors would like to thank Raquel García-Ceballos and Eva García Iglesias for their technical assistance

Nucleolar disruption and cajal body disassembly are nuclear hallmarks of DNA damage-induced neurodegeneration in purkinje cells

The Purkinje cell (PC) degeneration (pcd) phenotype results from mutation in nna1 gene and is associated with the degeneration and death of PCs during the postnatal life. Although the pcd mutation is a model of the ataxic mouse, it shares clinical and pathological characteristics of inherited human spinocerebellar ataxias. PC degeneration in pcd mice provides a useful neuronal system to study nuclear mechanisms involved in DNA damage-dependent neurodegeneration, particularly the contribution of nucleoli and Cajal bodies (CBs). Both nuclear structures are engaged in housekeeping functions for neuronal survival, the biogenesis of ribosomes and the maturation of snRNPs and snoRNPs required for pre-mRNA and pre-rRNA processing, respectively. In this study, we use ultrastructural analysis, in situ transcription assay and molecular markers for DNA damage, nucleoli and CB components to demonstrate that PC degeneration involves the progressive accumulation of nuclear DNA damage associated with disruption of nucleoli and CBs, disassembly of polyribosomes into monoribosomes, ribophagy and shut down of nucleolar and extranucleolar transcription. Microarray analysis reveals that four genes encoding repressors of nucleolar rRNA synthesis (p53, Rb, PTEN and SNF2) are upregulated in the cerebellum of pcd mice. Collectively, these data support that nucleolar and CB alterations are hallmarks of DNA damage-induced neurodegeneration.ACKNOWLEDGMENTS: The authors wish to thank Raquel García-Ceballos and Saray Pereda for technical assistance. This work was supported by the following grants: Dirección General de Investigación (BFU2008- 00175); Instituto de Salud Carlos III (CIBERNED, CB06/05/ 0037), Ministerio de Ciencia y Tecnología (BFU2010-18284), Ministerio de Sanidad, Política Social e Igualdad (Plan Nacional Sobre Drogas), Instituto de Formación e Investigación Marqués de Valdecilla (IFIMAV, FMV/UC09-02), Junta de Castilla y León, Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León and Fundación Memoria D. Samuel Solórzano-Barruso, all of them from Spain

Persistent accumulation of unrepaired DNA damage in rat cortical neurons: nuclear organization and ChIP-seq analysis of damaged DNA

Neurons are highly vulnerable to DNA damage induced by genotoxic agents such as topoisomerase activity, oxidative stress, ionizing radiation (IR) and chemotherapeutic drugs. To avert the detrimental effects of DNA lesions in genome stability, transcription and apoptosis, neurons activate robust DNA repair mechanisms. However, defective DNA repair with accumulation of unrepaired DNA are at the basis of brain ageing and several neurodegenerative diseases. Understanding the mechanisms by which neurons tolerate DNA damage accumulation as well as defining the genomic regions that are more vulnerable to DNA damage or refractory to DNA repair and therefore constitute potential targets in neurodegenerative diseases are essential issues in the field. In this work we investigated the nuclear topography and organization together with the genome-wide distribution of unrepaired DNA in rat cortical neurons 15 days upon IR. About 5% of non-irradiated and 55% of irradiated cells accumulate unrepaired DNA within persistent DNA damage foci (PDDF) of chromatin. These PDDF are featured by persistent activation of DNA damage/repair signaling, lack of transcription and localization in repressive nuclear microenvironments. Interestingly, the chromatin insulator CTCF is concentrated at the PDDF boundaries, likely contributing to isolate unrepaired DNA from intact transcriptionally active chromatin. By confining damaged DNA, PDDF would help preserving genomic integrity and preventing the production of aberrant proteins encoded by damaged genes.ChIP-seq analysis of genome-wide ?H2AX distribution revealed a number of genomic regions enriched in ?H2AX signal in IR-treated cortical neurons. Some of these regions are in close proximity to genes encoding essential proteins for neuronal functions and human neurodegenerative disorders such as epm2a (Lafora disease), serpini1 (familial encephalopathy with neuroserpin inclusion bodies) and il1rpl1 (mental retardation, X-linked 21). Persistent ?H2AX signal close to those regions suggests that nearby genes could be either more vulnerable to DNA damage or more refractory to DNA repair.This work was supported by the following grants: “Dirección General de Investigación” (BFU2014–54754-P) and “Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas” (CIBERNED; CB06/05/0037) Spain

Nuclear Reorganization in Hippocampal Granule Cell Neurons from a Mouse Model of Down Syndrome: Changes in Chromatin Configuration, Nucleoli and Cajal Bodies

Down syndrome (DS) or trisomy of chromosome 21 (Hsa21) is characterized by impaired hippocampal-dependent learning and memory. These alterations are due to defective neurogenesis and to neuromorphological and functional anomalies of numerous neuronal populations, including hippocampal granular cells (GCs). It has been proposed that the additional gene dose in trisomic cells induces modifications in nuclear compartments and on the chromatin landscape, which could contribute to some DS phenotypes. The Ts65Dn (TS) mouse model of DS carries a triplication of 92 genes orthologous to those found in Hsa21, and shares many phenotypes with DS individuals, including cognitive and neuromorphological alterations. Considering its essential role in hippocampal memory formation, we investigated whether the triplication of this set of Hsa21 orthologous genes in TS mice modifies the nuclear architecture of their GCs. Our results show that the TS mouse presents alterations in the nuclear architecture of its GCs, affecting nuclear compartments involved in transcription and pre-rRNA and pre-mRNA processing. In particular, the GCs of the TS mouse show alterations in the nucleolar fusion pattern and the molecular assembly of Cajal bodies (CBs). Furthermore, hippocampal GCs of TS mice present an epigenetic dysregulation of chromatin that results in an increased heterochromatinization and reduced global transcriptional activity. These nuclear alterations could play an important role in the neuromorphological and/or functional alterations of the hippocampal GCs implicated in the cognitive dysfunction characteristic of TS mice

Hollow fiber membranes of PCL and PCL/graphene as scaffolds with potential to develop in vitro blood–brain barrier models

There is a huge interest in developing novel hollow fiber (HF) membranes able to modulate neural differentiation to produce in vitro blood–brain barrier (BBB) models for biomedical and pharmaceutical research, due to the low cell-inductive properties of the polymer HFs used in current BBB models. In this work, poly(ε-caprolactone) (PCL) and composite PCL/graphene (PCL/G) HF membranes were prepared by phase inversion and were characterized in terms of mechanical, electrical, morphological, chemical, and mass transport properties. The presence of graphene in PCL/G membranes enlarged the pore size and the water flux and presented significantly higher electrical conductivity than PCL HFs. A biocompatibility assay showed that PCL/G HFs significantly increased C6 cells adhesion and differentiation towards astrocytes, which may be attributed to their higher electrical conductivity in comparison to PCL HFs. On the other hand, PCL/G membranes produced a cytotoxic effect on the endothelial cell line HUVEC presumably related with a higher production of intracellular reactive oxygen species induced by the nanomaterial in this particular cell line. These results prove the potential of PCL HF membranes to grow endothelial cells and PCL/G HF membranes to differentiate astrocytes, the two characteristic cell types that could develop in vitro BBB models in future 3D co-culture systems.This research was funded by IDIVAL (INNVAL 17/20), MINECO/EIG-Concert Japan (X-MEM PCI2018-092929 project, International Joint Program 2018) and MINECO/Spain Feder (CTM-2016-75509-R project)