Secondary loss of miR-3607 reduced cortical progenitor amplification during rodent evolution

The evolutionary expansion and folding of the mammalian cerebral cortex resulted from amplification of progenitor cells during embryonic development. This process was reversed in the rodent lineage after splitting from primates, leading to smaller and smooth brains. Genetic mechanisms underlying this secondary loss in rodent evolution remain unknown. We show that microRNA miR-3607 is expressed embryonically in the large cortex of primates and ferret, distant from the primate-rodent lineage, but not in mouse. Experimental expression of miR-3607 in embryonic mouse cortex led to increased Wnt/β-catenin signaling, amplification of radial glia cells (RGCs), and expansion of the ventricular zone (VZ), via blocking the β-catenin inhibitor APC (adenomatous polyposis coli). Accordingly, loss of endogenous miR-3607 in ferret reduced RGC proliferation, while overexpression in human cerebral organoids promoted VZ expansion. Our results identify a gene selected for secondary loss during mammalian evolution to limit RGC amplification and, potentially, cortex size in rodents.This work was supported by Santiago Grisolía predoctoral fellowship (K.C.), Generalitat Valenciana I+D+i programs grant APOSTD/2019/059 (A.C.), Fundación Tatiana Pérez de Guzmán el Bueno predoctoral fellowship (A.P.-C.), Agencia Estatal de Investigación SVP-2014-068671 (A.V.), Spanish State Research Agency FPI contract (R.S.), Spanish State Research Agency grant RYC-2015-18056 (J.P.L.-A.), Spanish State Research Agency grant RTI2018-102260-B-100 (J.P.L.-A.), Spanish State Research Agency grant SAF2015-69168-R (V.B.), Spanish State Research Agency grant PGC2018-102172-B-I00 (V.B.), Spanish State Research Agency “Severo Ochoa” Programme for Centers of Excellence in R&D grant SEV-2017-0723 (V.B.), and European Research Council grant 309633 (V.B.).Peer reviewe

Epithelial to mesenchymal transition trajectories in developmental and disease

Resumen del trabajo presentado al 19th International Congress of Developmental Biology, celebrado en El Algarve (Portugal) del 16 al 20 de octubre de 2022.The Epithelial to Mesenchymal transition (EMT) triggers cell plasticity during embryonic development and tissue repair, but it can also promote tumor progression and organ degeneration. The reactivation of EMT in the adult promotes cell dedifferentiation and profound remodeling of the epithelial program, leading to multiple phenotypes, observed in response to injury, during organ fibrosis and cancer cell dissemination. Despite recent advances, identifying universal EMT molecular signatures and understanding how EMT can instructs different outcomes have remained elusive due to the intrinsic complexity and heterogeneity of the process. We have dissected how EMT transcription factors (EMT-TFs) orchestrate TGFBinduced EMT including phenotypic and behavioral states. Further, we have combined lineage tracing and single-cell transcriptomics in three EMT contexts, namely the neural crest, renal fibrosis, and breast cancer to reveal conserved EMT transcription factor codes and signaling pathways that discriminate different EMT states. After inferring cellular trajectories, we have reconstructed the evolution of EMT phenotypic and functional states in all these contexts. Finally, multiplex labeling allowed to spatially allocate distinct EMT programs in mouse and human tumor samples. Altogether, this work unveils distinct EMT trajectories in development and disease, which should also help propase improved therapeutic strategies for organ fibrosis and cancer.Peer reviewe

Two distinct epithelial to mesenchymal transition programmes. Control invasion and inflammation in segregated tumour cell populations

Resumen del trabajo presentado al 19th Christmas Meeting del Instituto de Neurociencias (CSIC-UMH) celebrado el 21 de diciembre de 2022.Epithelial plasticity is at the core of crucial processes including embryonic cell migration, cancer progression, organ tibrosis and tissue repair. The epithelial to mesenchymal transition (EMT) triggers cell plasticity in all these contexts, highlighting its pleiotropy and intrinsic complcxity. Seminal studies have classified EMT states in cancer celllines and animal modcls. This varicty ofEMT phenotypes necds further investigation, particularly those relevant to the progression ofprevalent and dcvastating diseases such as cancer. Our objcctive is to analyse at single-cell level how different EMT states are established in tumours and if different EMT states pcrform different functions during tumour progression.Peer reviewe

Microglia regulate learning and memory through NF-κB

Resumen del póster presentado al 19th Meeting Spanish Society of Neuroscience, celebrado en Lleida del 3 al 5 de noviembre de 2021.Microglia, the resident immune cells of the CNS, have been implicated in brain plasticity and function. However, the mechanisms remain largely unknown. Here, we show that Cre-dependent removal of the RelA subunit of the NF-κB transcription factor from adult microglia results in impaired learning and long-term potentiation. Depletion of RelA elicits changes in chromatin accessibility and transcriptome landscapes of microglia associated with specific gene regulatory programs driving the activation of specific microglia phenotypes. Our findings suggest that NF-κB gene products drive specific microglia phenotypes modulating neuronal circuits for learning and memory.Peer reviewe

Cell-specific vulnerability to metabolic failure: the crucial role of parvalbumin expressing neurons in creatine transporter deficiency

Mutations in the solute carrier family 6-member 8 (Slc6a8) gene, encoding the protein responsible for cellular creatine (Cr) uptake, cause Creatine Transporter Deficiency (CTD), an X-linked neurometabolic disorder presenting with intellectual disability, autistic-like features, and epilepsy. The pathological determinants of CTD are still poorly understood, hindering the development of therapies. In this study, we generated an extensive transcriptomic profile of CTD showing that Cr deficiency causes perturbations of gene expression in excitatory neurons, inhibitory cells, and oligodendrocytes which result in remodeling of circuit excitability and synaptic wiring. We also identified specific alterations of parvalbumin-expressing (PV+) interneurons, exhibiting a reduction in cellular and synaptic density, and a hypofunctional electrophysiological phenotype. Mice lacking Slc6a8 only in PV+ interneurons recapitulated numerous CTD features, including cognitive deterioration, impaired cortical processing and hyperexcitability of brain circuits, demonstrating that Cr deficit in PV+ interneurons is sufficient to determine the neurological phenotype of CTD. Moreover, a pharmacological treatment targeted to restore the efficiency of PV+ synapses significantly improved cortical activity in Slc6a8 knock-out animals. Altogether, these data demonstrate that Slc6a8 is critical for the normal function of PV+ interneurons and that impairment of these cells is central in the disease pathogenesis, suggesting a novel therapeutic venue for CTD.This work has been supported by grant GR-2017–02364378 funded by the Italian Ministry of Health and by Telethon grant GGP19177 to LB; Italian Ministry of Health, RC 2021; grant from Fondazione Cassa di Risparmio di Firenze “Human Brain Optical Mapping” to TP; grants from the Spanish Ministry of Science and Innovation (MICINN) co-financed by ERDF (grant no. RTI2018-102260-B-I00; Generalitat Valenciana, project no. PROMETEO/2020/007; and CSIC Interdisciplinary Thematic Platform (PTI +) NEURO-AGINGl + (PTI-NEURO-AGING +). C.M.N-I. was the recipient of a FPI fellowship from the MICINN. The Instituto de Neurociencias (UMH-CSIC) is a “Centre of Excellence Severo Ochoa” (grant no. SEV-2017–0723).Peer reviewe

Sublayer- and cell-type-specific neurodegenerative transcriptional trajectories in hippocampal sclerosis

Hippocampal sclerosis, the major neuropathological hallmark of temporal lobe epilepsy, is characterized by different patterns of neuronal loss. The mechanisms of cell-type-specific vulnerability and their progression and histopathological classification remain controversial. Using single-cell electrophysiology in vivo and immediate-early gene expression, we reveal that superficial CA1 pyramidal neurons are overactive in epileptic rodents. Bulk tissue and single-nucleus expression profiling disclose sublayer-specific transcriptomic signatures and robust microglial pro-inflammatory responses. Transcripts regulating neuronal processes such as voltage channels, synaptic signaling, and cell adhesion are deregulated differently by epilepsy across sublayers, whereas neurodegenerative signatures primarily involve superficial cells. Pseudotime analysis of gene expression in single nuclei and in situ validation reveal separated trajectories from health to epilepsy across cell types and identify a subset of superficial cells undergoing a later stage in neurodegeneration. Our findings indicate that sublayer- and cell-type-specific changes associated with selective CA1 neuronal damage contribute to progression of hippocampal sclerosis.This work was supported by grants from MICINN (RTI2018-098581-B-I00 to L.M.P.), Fundación Tatiana Pérez de Guzman el Bueno, and the SynCogDis Network (SAF2014-52624-REDT and SAF2017- 90664-REDT to L.M.P. and A. Bayes). Collaboration between L.M.d.l.P. and Y.H. was supported by Human Frontiers Science Program (HFSP) grant RGP0022/2013. J.P.L.-A. was supported by grants from MICIU co-financed by ERDF (RYC-2015-18056 and RTI2018-102260-B-I00) and Severo Ochoa grant SEV-2017-0723. R.R.-V. and A. Bayes were supported by MINECO BFU2015-69717-P and RTI2018-097037-B-100 and a Marie Curie career integration grant (ref. 304111). A.V.M. was supported by MICINN (SAF2017- 85717-R) and Fundación Alicia Koplowitz. A. Barco was supported by grants SAF2017-87928-R from MICINN co-financed by ERDF and RGP0039/2017 from the Human Frontiers Science Program Organization. The Instituto de Neurociencias is a ‘‘Centre of Excellence Severo Ochoa.’’ D.G.-D. and C.M.N. hold PhD fellowships from MICINN (BES-2013-064171 and BES2016-076281, respectively).Peer reviewe

A protocol to extract cell-type-specific signatures from differentially expressed genes in bulk-tissue RNA-seq

Bulk-tissue RNA-seq is widely used to dissect variation in gene expression levels across tissues and under different experimental conditions. Here, we introduce a protocol that leverages existing single-cell expression data to deconvolve patterns of cell-type-specific gene expression in differentially expressed gene lists from highly heterogeneous tissue. We apply this protocol to interrogate cell-type-specific gene expression and variation in cell type composition between the distinct sublayers of the hippocampal CA1 region of the brain in a rodent model of epilepsy. For complete details on the use and execution of this protocol, please refer to Cid et al. (2021).This work was supported by grants from MICINN (RTI2018-098581-B-I00 to L.M.P.), Fundación Tatiana Pérez de Guzman el Bueno to L.M.P., and the SynCogDis Network (SAF2014-52624-REDT and SAF2017-90664-REDT to L.M.P.). J.P.L.-A. was supported by grants from MICIU co-financed by ERDF (RYC-2015-18056 and RTI2018-102260-B-I00) and Severo Ochoa grant SEV-2017-0723. The Instituto de Neurociencias is a “Centre of Excellence Severo Ochoa.

Towards indentifying novel therapeutic approaches for melanoma brain metastases

Trabajo presentado a la EACR-AACR-ASPIC Basic and Translational Research Conference: Tumor Microenvironment, celebrada del 2 al 4 de marzo de 2020 en Lisboa (Portugal).Peer reviewe

Understanding the contribution of different macrophage populations to brain metastasis

Resumen del póster presentado al European Developmental Biology Congress (EDBC), celebrado en Alicante del 23 al 26 de octubre de 2019.Microglia, tissue-resident macrophages of the CNS, play key roles in brain homeostasis and disease. Inflammatory activation of microglia upon brain damage is considered a pathological hallmark and an important mechanism driving neurodegenerative diseases. Brain metastases can be conceptualized as a source of brain injury and as such, tumour cells promote microglial activation. In addition, recruited macrophages also contribute to the immune population of brain metastasis. Recent evidences suggest that the origins of the different macrophage populations influences their contribution to brain disease. While microglia derive from yolk-sac myeloid progenitors that colonize the brain during development, the major source of recruited macrophages is the bone marrow, however it has been classically difficult to distinguish both populations. Macrophages constitute critical regulators of tumour progression and responses to therapies, but their contribution to brain metastasis is poorly understood. To investigate this, we have established a brain colonization experimental model by injection of murine tumour cells into Cx3cr.1-CreERT2-YFP mice that can be used as a macrophage/microglia reporter model. Using this approach and recently described specific markers, we can observe that brain metastases are densely infiltrated by both microglia and recruited macrophages. We have isolated these specific populations by fluorescence activated cell sorting and perform RNA sequencing to identify differentially expressed genes and altered pathways that may regulate brain metastasis progression. In addition, we are performing single-cell RNA sequencing to help us understand the trajectory of cellular reprogramming of microglia and recruited macrophages in response to cancer cells.Peer reviewe

Understanding the contribution of brain macrophages and neuroinflammation to melanoma brain metastases

Resumen del trabajo presentado al EMBO / EMBL Symposium Defining and Defeating Metastasis, celebrado virtualmente del 19 al 22 de junio de 2022.Melanoma Brain Metastases (MBMs) are associated with very poor prognosis and constitute a very common clinical problem in melanoma patients. Systemic treatments including targeted therapy and immunotherapy can contribute to disease control in these patients but BMs very often behave differently to extracranial metastasis and this may be mostly due to the unique brain microenvironment. The majority of brain cells are glia, with key roles in homeostasis and in pathological conditions. In particular microglia, brain resident macrophages, as part of the innate immune system of this organ gets activated upon brain damage. However, sustained inflammatory activation of microglia is considerad a pathological hallmark and an important mechanism driving neurodegenerative diseases. Our analysis of preclinical models of MBMs indicates that microglia account for the majority of immune cells within MBMs. Although macrophages influence melanoma progression and constitute a source of resistance to therapies, very little is known about the contribution of brain macrophages and neuroinflammation to melanoma metastasis in the immuno-specialised microenvironment of the brain. We have established relevant MBMs preclinical models in glia-reporter transgenic mice that also allows gene silencing in specific populations to investigate the impact of neuroinflammation and the co-evolution of the immune system during MBMs progression and responses to therapies. Our aim is to identify novel targetable candidatas that will help to design better therapeutic strategies for patients with MBMs.Peer reviewe