Fernández-Sevilla, L. M., Belenguer, G., Martí-Prado, B., Ortiz-Sánchez, P., Ramírez, M., Varas, A., Fariñas, I., & Vicente, Á. (2022). Acute lymphoblastic leukemia cells are able to infiltrate the brain subventricular zone stem cell niche and impair neurogenesis. Haematologica, 107(4), 1004–1007.
Del Puerto, A., Pose-Utrilla, J., Simón-García, A., López-Menéndez, C., Jiménez, A. J., Porlan, E., Pajuelo, L., Cano-García, G., Martí-Prado, B., Sebastián-Serrano, Á., Sánchez-Carralero, M. P., Cesca, F., Schiavo, G., Ferrer, I., Fariñas, I., Campanero, M. R., & Iglesias, T. (2021). Kidins220 deficiency causes ventriculomegaly via SNX27-retromer-dependent AQP4 degradation. Molecular psychiatry, 26(11), 6411–6426.
Several psychiatric, neurologic and neurodegenerative disorders present increased brain ventricles volume, being hydrocephalus the disease with the major manifestation of ventriculomegaly caused by the accumulation of high amounts of cerebrospinal fluid (CSF). The molecules and pathomechanisms underlying cerebral ventricular enlargement are widely unknown. Kinase D interacting substrate of 220 kDa (KIDINS220) gene has been recently associated with schizophrenia and with a novel syndrome characterized by spastic paraplegia, intellectual disability, nystagmus and obesity (SINO syndrome), diseases frequently occurring with ventriculomegaly. Here we show that Kidins220, a transmembrane protein effector of various key neuronal signalling pathways, is a critical regulator of CSF homeostasis. We observe that both KIDINS220 and the water channel aquaporin-4 (AQP4) are markedly downregulated at the ventricular ependymal lining of idiopathic normal pressure hydrocephalus (iNPH) patients. We also find that Kidins220 deficient mice develop ventriculomegaly accompanied by water dyshomeostasis and loss of AQP4 in the brain ventricular ependymal layer and astrocytes. Kidins220 is a known cargo of the SNX27-retromer, a complex that redirects endocytosed plasma membrane proteins (cargos) back to the cell surface, thus avoiding their targeting to lysosomes for degradation. Mechanistically, we show that AQP4 is a novel cargo of the SNX27-retromer and that Kidins220 deficiency promotes a striking and unexpected downregulation of the SNX27-retromer that results in AQP4 lysosomal degradation. Accordingly, SNX27 silencing decreases AQP4 levels in wild-type astrocytes whereas SNX27 overexpression restores AQP4 content in Kidins220 deficient astrocytes. Together our data suggest that the KIDINS220-SNX27-retromer-AQP4 pathway is involved in human ventriculomegaly and open novel therapeutic perspectives.
Rojas-Vázquez, S., Blasco-Chamarro, L., López-Fabuel, I., Martínez-Máñez, R., & Fariñas, I. (2021). Vascular Senescence: A Potential Bridge Between Physiological Aging and Neurogenic Decline. Frontiers in neuroscience, 15, 666881.
The adult mammalian brain contains distinct neurogenic niches harboring populations of neural stem cells (NSCs) with the capacity to sustain the generation of specific subtypes of neurons during the lifetime. However, their ability to produce new progeny declines with age. The microenvironment of these specialized niches provides multiple cellular and molecular signals that condition NSC behavior and potential. Among the different niche components, vasculature has gained increasing interest over the years due to its undeniable role in NSC regulation and its therapeutic potential for neurogenesis enhancement. NSCs are uniquely positioned to receive both locally secreted factors and adhesion-mediated signals derived from vascular elements. Furthermore, studies of parabiosis indicate that NSCs are also exposed to blood-borne factors, sensing and responding to the systemic circulation. Both structural and functional alterations occur in vasculature with age at the cellular level that can affect the proper extrinsic regulation of NSCs. Additionally, blood exchange experiments in heterochronic parabionts have revealed that age-associated changes in blood composition also contribute to adult neurogenesis impairment in the elderly. Although the mechanisms of vascular- or blood-derived signaling in aging are still not fully understood, a general feature of organismal aging is the accumulation of senescent cells, which act as sources of inflammatory and other detrimental signals that can negatively impact on neighboring cells. This review focuses on the interactions between vascular senescence, circulating pro-senescence factors and the decrease in NSC potential during aging. Understanding the mechanisms of NSC dynamics in the aging brain could lead to new therapeutic approaches, potentially include senolysis, to target age-dependent brain decline.
Belenguer, G., Duart-Abadia, P., Domingo-Muelas, A., Morante-Redolat, J. M., & Fariñas, I. (2021). Cell population analysis of the adult murine subependymal neurogenic lineage by flow cytometry. STAR protocols, 2(2), 100425.
This protocol provides a flow-cytometry-based procedure to classify and isolate all cells of the adult rodent subependymal zone (SEZ) neurogenic lineage, without the need for reporter mice, into different cell populations, including three neural stem cell (NSC) fractions with molecular signatures that are coherent with single-cell transcriptomics. Additionally, their cycling behavior can be assessed by means of 5-ethynyl-2′-deoxyuridine (EdU) incorporation. Our method allows the isolation of different NSC fractions and the functional assay of their cycling heterogeneity and quiescence-activation transitions. For complete details on the use, execution, and outcomes of this protocol, please refer to Belenguer et al. (2021).
Bové, M., Monto, F., Guillem-Llobat, P., Ivorra, M. D., Noguera, M. A., Zambrano, A., Sirerol-Piquer, M. S., Requena, A. C., García-Alonso, M., Tejerina, T., Real, J. T., Fariñas, I., & D’Ocon, P. (2021). NT3/TrkC Pathway Modulates the Expression of UCP-1 and Adipocyte Size in Human and Rodent Adipose Tissue. Frontiers in endocrinology, 12, 630097.
Neurotrophin-3 (NT3), through activation of its tropomyosin-related kinase receptor C (TrkC), modulates neuronal survival and neural stem cell differentiation. It is widely distributed in peripheral tissues (especially vessels and pancreas) and this ubiquitous pattern suggests a role for NT3, outside the nervous system and related to metabolic functions. The presence of the NT3/TrkC pathway in the adipose tissue (AT) has never been investigated. Present work studies in human and murine adipose tissue (AT) the presence of elements of the NT3/TrkC pathway and its role on lipolysis and adipocyte differentiation. qRT-PCR and immunoblot indicate that NT3 (encoded by NTF3) was present in human retroperitoneal AT and decreases with age. NT3 was also present in rat isolated adipocytes and retroperitoneal, interscapular, perivascular, and perirenal AT. Histological analysis evidences that NT3 was mainly present in vessels irrigating AT close associated to sympathetic fibers. Similar mRNA levels of TrkC (encoded by NTRK3) and β-adrenoceptors were found in all ATs assayed and in isolated adipocytes. NT3, through TrkC activation, exert a mild effect in lipolysis. Addition of NT3 during the differentiation process of human pre-adipocytes resulted in smaller adipocytes and increased uncoupling protein-1 (UCP-1) without changes in β-adrenoceptors. Similarly, transgenic mice with reduced expression of NT3 (Ntf3 knock-in lacZ reporter mice) or lacking endothelial NT3 expression (Ntf3flox1/flox2;Tie2-Cre+/0) displayed enlarged white and brown adipocytes and lower UCP-1 expression.
Conclusions: NT3, mainly released by blood vessels, activates TrkC and regulates adipocyte differentiation and browning. Disruption of NT3/TrkC signaling conducts to hypertrophied white and brown adipocytes with reduced expression of the thermogenesis marker UCP-1.
Xie, X. P., Laks, D.R., Sun, D., Poran, A., Laughney, A. M., Wang, Z., Sam, J., Belenguer, G., Fariñas, I., Elemento, O., Zhou, X., & Parada, L. F. (2020). High-resolution mouse subventricular zone stem-cell niche transcriptome reveals features of lineage, anatomy, and aging. Proceedings of the National Academy of Sciences of the United States of America, 117(49), 31448-31458.
Adult neural stem cells (NSC) serve as a reservoir for brain plasticity and origin for certain gliomas. Lineage tracing and genomic approaches have portrayed complex underlying heterogeneity within the major anatomical location for NSC, the subventricular zone (SVZ). To gain a comprehensive profile of NSC heterogeneity, we utilized a well-validated stem/progenitor-specific reporter transgene in concert with single-cell RNA sequencing to achieve unbiased analysis of SVZ cells from infancy to advanced age. The magnitude and high specificity of the resulting transcriptional datasets allow precise identification of the varied cell types embedded in the SVZ including specialized parenchymal cells (neurons, glia, microglia) and noncentral nervous system cells (endothelial, immune). Initial mining of the data delineates four quiescent NSC and three progenitor-cell subpopulations formed in a linear progression. Further evidence indicates that distinct stem and progenitor populations reside in different regions of the SVZ. As stem/progenitor populations progress from neonatal to advanced age, they acquire a deficiency in transition from quiescence to proliferation. Further data mining identifies stage-specific biological processes, transcription factor networks, and cell-surface markers for investigation of cellular identities, lineage relationships, and key regulatory pathways in adult NSC maintenance and neurogenesis.
Belenguer, G., Duart-Abadia, P., Jordán-Pla, A., Domingo-Muelas, A., Blasco-Chamarro, L., Ferrón, S.R., Morante-Redolat, J.M., Fariñas, I. (2020). “Adult Neural Stem Cells Are Alerted by Systemic Inflammation through TNF-α Receptor Signaling”. Cell Stem Cell S1934-5909(20)30510-5.
Adult stem cells (SCs) transit between the cell cycle and a poorly defined quiescent state. Single neural SCs (NSCs) with quiescent, primed-for-activation, and activated cell transcriptomes have been obtained from the subependymal zone (SEZ), but the functional regulation of these states under homeostasis is not understood. Here, we develop a multilevel strategy to analyze these NSC states with the aim to uncover signals that regulate their level of quiescence/activation. We show that transitions between states occur in vivo and that activated and primed, but not quiescent, states can be captured and studied in culture. We also show that peripherally induced inflammation promotes a transient activation of primed NSCs (pNSCs) mediated by tumor necrosis factor α (TNF-α) acting through its receptor, TNF receptor 2 (TNFR2), and a return to quiescence in a TNF receptor 1 (TNFR1)-dependent manner. Our data identify a signaling pathway promoting NSC alertness and add to the emerging concept that SCs can respond to the systemic milieu.
Sirerol-Piquer, M. S., Belenguer, G., Morante-Redolat, J. M., Duart-Abadia, P., Perez-Villalba, A. and Farinas, I. (2019). «Physiological Interactions between Microglia and Neural Stem Cells in the Adult Subependymal Niche.» Neuroscience 405: 77-91.
Microglia are the prototypical innate immune cells of the central nervous system. They constitute a unique type of tissue-resident mononuclear phagocytes which act as glial cells. Elegant experiments in the last few years have revealed the origin, extraordinary molecular diversity, and phenotypic plasticity of these cells and how their potential relates to both immune and non-immune actions in the normal and diseased brain. Microglial cells originate in the yolk sac and colonize the brain during embryogenesis, playing a role in neural development and later in adult brain function. Neurogenesis continues after birth in discrete areas of the mammalian brain sustained by the postnatal persistence of neural stem cells in specific neurogenic niches. Recent data indicate that microglial cells are distinct cellular elements of these neurogenic niches where they regulate different aspects of stem cell biology. Interestingly, microglial and neural stem cells are specified very early in fetal development and persist as self-renewing populations throughout life, suggesting potential life-long interactions between them. We aim at reviewing these interactions in one neurogenic niche, the subependymal zone.
Garcia-Garrote, M., Perez-Villalba, A., Garrido-Gil, P., Belenguer, G., Parga, J. A., Perez-Sanchez, F., Labandeira-Garcia, J. L., Farinas, I. and Rodriguez-Pallares, J. (2019). «Interaction between Angiotensin Type 1, Type 2, and Mas Receptors to Regulate Adult Neurogenesis in the Brain Ventricular-Subventricular Zone.» Cells 8(12).
The renin-angiotensin system (RAS), and particularly its angiotensin type-2 receptors (AT2), have been classically involved in processes of cell proliferation and maturation during development. However, the potential role of RAS in adult neurogenesis in the ventricular-subventricular zone (V-SVZ) and its aging-related alterations have not been investigated. In the present study, we analyzed the role of major RAS receptors on neurogenesis in the V-SVZ of adult mice and rats. In mice, we showed that the increase in proliferation of cells in this neurogenic niche was induced by activation of AT2 receptors but depended partially on the AT2-dependent antagonism of AT1 receptor expression, which restricted proliferation. Furthermore, we observed a functional dependence of AT2 receptor actions on Mas receptors. In rats, where the levels of the AT1 relative to those of AT2 receptor are much lower, pharmacological inhibition of the AT1 receptor alone was sufficient in increasing AT2 receptor levels and proliferation in the V-SVZ. Our data revealed that interactions between RAS receptors play a major role in the regulation of V-SVZ neurogenesis, particularly in proliferation, generation of neuroblasts, and migration to the olfactory bulb, both in young and aged brains, and suggest potential beneficial effects of RAS modulators on neurogenesis.
Perez-Villalba, A., Sirerol-Piquer, M. S., Belenguer, G., Soriano-Canton, R., Munoz-Manchado, A. B., Villadiego, J., Alarcon-Aris, D., Soria, F. N., Dehay, B., Bezard, E., Vila, M., Bortolozzi, A., Toledo-Aral, J. J., Perez-Sanchez, F. and Farinas, I. (2018). «Synaptic Regulator alpha-Synuclein in Dopaminergic Fibers Is Essentially Required for the Maintenance of Subependymal Neural Stem Cells.» J Neurosci 38(4): 814-825.
Synaptic protein alpha-synuclein (alpha-SYN) modulates neurotransmission in a complex and poorly understood manner and aggregates in the cytoplasm of degenerating neurons in Parkinson’s disease. Here, we report that alpha-SYN present in dopaminergic nigral afferents is essential for the normal cycling and maintenance of neural stem cells (NSCs) in the brain subependymal zone of adult male and female mice. We also show that premature senescence of adult NSCs into non-neurogenic astrocytes in mice lacking alpha-SYN resembles the effects of dopaminergic fiber degeneration resulting from chronic exposure to 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine or intranigral inoculation of aggregated toxic alpha-SYN. Interestingly, NSC loss in alpha-SYN-deficient mice can be prevented by viral delivery of human alpha-SYN into their sustantia nigra or by treatment with l-DOPA, suggesting that alpha-SYN regulates dopamine availability to NSCs. Our data indicate that alpha-SYN, present in dopaminergic nerve terminals supplying the subependymal zone, acts as a niche component to sustain the neurogenic potential of adult NSCs and identify alpha-SYN and DA as potential targets to ameliorate neurogenic defects in the aging and diseased brain.SIGNIFICANCE STATEMENT We report an essential role for the protein alpha-synuclein present in dopaminergic nigral afferents in the regulation of adult neural stem cell maintenance, identifying the first synaptic regulator with an implication in stem cell niche biology. Although the exact role of alpha-synuclein in neural transmission is not completely clear, our results indicate that it is required for stemness and the preservation of neurogenic potential in concert with dopamine.
Porlan, E., Marti-Prado, B., Consiglio, A. and Farinas, I. (2016). «Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects.» J Vis Exp(108): 53282.
Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.
Belenguer, G., Domingo-Muelas, A., Ferron, S. R., Morante-Redolat, J. M. and Farinas, I. (2016). «Isolation, culture and analysis of adult subependymal neural stem cells.» Differentiation 91(4-5): 28-41.
Individual cells dissected from the subependymal neurogenic niche of the adult mouse brain proliferate in medium containing basic fibroblast growth factor (bFGF) and/or epidermal growth factor (EGF) as mitogens, to produce multipotent clonal aggregates called neurospheres. These cultures constitute a powerful tool for the study of neural stem cells (NSCs) provided that they allow the analysis of their features and potential capacity in a controlled environment that can be modulated and monitored more accurately than in vivo. Clonogenic and population analyses under mitogen addition or withdrawal allow the quantification of the self-renewing and multilineage potency of these cells and the identification of the mechanisms involved in these properties. Here, we describe a set of procedures developed and/or modified by our group including several experimental options that can be used either independently or in combination for the ex vivo assessment of cell properties of NSCs obtained from the adult subependymal niche.
Soriano-Canton, R., Perez-Villalba, A., Morante-Redolat, J. M., Marques-Torrejon, M. A., Pallas, M., Perez-Sanchez, F. and Farinas, I. (2015). «Regulation of the p19(Arf)/p53 pathway by histone acetylation underlies neural stem cell behavior in senescence-prone SAMP8 mice.» Aging Cell 14(3): 453-462.
Brain aging is associated with increased neurodegeneration and reduced neurogenesis. B1/neural stem cells (B1-NSCs) of the mouse subependymal zone (SEZ) support the ongoing production of olfactory bulb interneurons, but their neurogenic potential is progressively reduced as mice age. Although age-related changes in B1-NSCs may result from increased expression of tumor suppressor proteins, accumulation of DNA damage, metabolic alterations, and microenvironmental or systemic changes, the ultimate causes remain unclear. Senescence-accelerated-prone mice (SAMP8) relative to senescence-accelerated-resistant mice (SAMR1) exhibit signs of hastened senescence and can be used as a model for the study of aging. We have found that the B1-NSC compartment is transiently expanded in young SAMP8 relative to SAMR1 mice, resulting in disturbed cytoarchitecture of the SEZ, B1-NSC hyperproliferation, and higher yields of primary neurospheres. These unusual features are, however, accompanied by premature loss of B1-NSCs. Moreover, SAMP8 neurospheres lack self-renewal and enter p53-dependent senescence after only two passages. Interestingly, in vitro senescence of SAMP8 cells could be prevented by inhibition of histone acetyltransferases and mimicked in SAMR1 cells by inhibition of histone deacetylases (HDAC). Our data indicate that expression of the tumor suppressor p19, but not of p16, is increased in SAMP8 neurospheres, as well as in SAMR1 neurospheres upon HDAC inhibition, and suggest that the SAMP8 phenotype may, at least in part, be due to changes in chromatin status. Interestingly, acute HDAC inhibition in vivo resulted in changes in the SEZ of SAMR1 mice that resembled those found in young SAMP8 mice.
Recasens, A., Dehay, B., Bove, J., Carballo-Carbajal, I., Dovero, S., Perez-Villalba, A., Fernagut, P. O., Blesa, J., Parent, A., Perier, C., Farinas, I., Obeso, J. A., Bezard, E. and Vila, M. (2014). «Lewy body extracts from Parkinson disease brains trigger alpha-synuclein pathology and neurodegeneration in mice and monkeys.» Ann Neurol 75(3): 351-362.
OBJECTIVE: Mounting evidence suggests that alpha-synuclein, a major protein component of Lewy bodies (LB), may be responsible for initiating and spreading the pathological process in Parkinson disease (PD). Supporting this concept, intracerebral inoculation of synthetic recombinant alpha-synuclein fibrils can trigger alpha-synuclein pathology in mice. However, it remains uncertain whether the pathogenic effects of recombinant synthetic alpha-synuclein may apply to PD-linked pathological alpha-synuclein and occur in species closer to humans. METHODS: Nigral LB-enriched fractions containing pathological alpha-synuclein were purified from postmortem PD brains by sucrose gradient fractionation and subsequently inoculated into the substantia nigra or striatum of wild-type mice and macaque monkeys. Control animals received non-LB fractions containing soluble alpha-synuclein derived from the same nigral PD tissue. RESULTS: In both mice and monkeys, intranigral or intrastriatal inoculations of PD-derived LB extracts resulted in progressive nigrostriatal neurodegeneration starting at striatal dopaminergic terminals. No neurodegeneration was observed in animals receiving non-LB fractions from the same patients. In LB-injected animals, exogenous human alpha-synuclein was quickly internalized within host neurons and triggered the pathological conversion of endogenous alpha-synuclein. At the onset of LB-induced degeneration, host pathological alpha-synuclein diffusely accumulated within nigral neurons and anatomically interconnected regions, both anterogradely and retrogradely. LB-induced pathogenic effects required both human alpha-synuclein present in LB extracts and host expression of alpha-synuclein. INTERPRETATION: alpha-Synuclein species contained in PD-derived LB are pathogenic and have the capacity to initiate a PD-like pathological process, including intracellular and presynaptic accumulations of pathological alpha-synuclein in different brain areas and slowly progressive axon-initiated dopaminergic nigrostriatal neurodegeneration.
Porlan, E., Marti-Prado, B., Morante-Redolat, J. M., Consiglio, A., Delgado, A. C., Kypta, R., Lopez-Otin, C., Kirstein, M. and Farinas, I. (2014). «MT5-MMP regulates adult neural stem cell functional quiescence through the cleavage of N-cadherin.» Nat Cell Biol 16(7): 629-638.
The identification of mechanisms that maintain stem cell niche architecture and homeostasis is fundamental to our understanding of tissue renewal and repair. Cell adhesion is a well-characterized mechanism for developmental morphogenetic processes, but its contribution to the dynamic regulation of adult mammalian stem cell niches is still poorly defined. We show that N-cadherin-mediated anchorage of neural stem cells (NSCs) to ependymocytes in the adult murine subependymal zone modulates their quiescence. We further identify MT5-MMP as a membrane-type metalloproteinase responsible for the shedding of the N-cadherin ectodomain in this niche. MT5-MMP is co-expressed with N-cadherin in adult NSCs and ependymocytes and, whereas MT5-MMP-mediated cleavage of N-cadherin is dispensable for the regulation of NSC generation and identity, it is required for proper activation of NSCs under physiological and regenerative conditions. Our results indicate that the proliferative status of stem cells can be dynamically modulated by regulated cleavage of cell adhesion molecules.
Delgado, A. C., Ferron, S. R., Vicente, D., Porlan, E., Perez-Villalba, A., Trujillo, C. M., D’Ocon, P. and Farinas, I. (2014). «Endothelial NT-3 delivered by vasculature and CSF promotes quiescence of subependymal neural stem cells through nitric oxide induction.» Neuron 83(3): 572-585.
Interactions of adult neural stem cells (NSCs) with supportive vasculature appear critical for their maintenance and function, although the molecular details are still under investigation. Neurotrophin (NT)-3 belongs to the NT family of trophic factors, best known for their effects in promoting neuronal survival. Here we show that NT-3 produced and secreted by endothelial cells of brain and choroid plexus capillaries is required for the quiescence and long-term maintenance of NSCs in the mouse subependymal niche. Uptake of NT-3 from irrigating vasculature and cerebrospinal fluid (CSF) induces the rapid phosphorylation of endothelial nitric oxide (NO) synthase present in the NSCs, leading to the production of NO, which subsequently acts as a cytostatic factor. Our results identify a novel interaction between stem cells and vasculature/CSF compartments that is mediated by an unprecedented role of a neurotrophin and indicate that stem cells can regulate their own quiescence in response to endothelium-secreted molecules.
Porlan, E., Morante-Redolat, J. M., Marques-Torrejon, M. A., Andreu-Agullo, C., Carneiro, C., Gomez-Ibarlucea, E., Soto, A., Vidal, A., Ferron, S. R. and Farinas, I. (2013). «Transcriptional repression of Bmp2 by p21(Waf1/Cip1) links quiescence to neural stem cell maintenance.» Nat Neurosci 16(11): 1567-1575.
Relative quiescence and self renewal are defining features of adult stem cells, but their potential coordination remains unclear. Subependymal neural stem cells (NSCs) lacking cyclin-dependent kinase (CDK) inhibitor (CKI) 1a (p21) exhibit rapid expansion that is followed by their permanent loss later in life. Here we demonstrate that transcription of the gene encoding bone morphogenetic protein 2 (Bmp2) in NSCs is under the direct negative control of p21 through actions that are independent of CDK. Loss of p21 in NSCs results in increased levels of secreted BMP2, which induce premature terminal differentiation of multipotent NSCs into mature non-neurogenic astrocytes in an autocrine and/or paracrine manner. We also show that the cell-nonautonomous p21-null phenotype is modulated by the Noggin-rich environment of the subependymal niche. The dual function that we describe here provides a physiological example of combined cell-autonomous and cell-nonautonomous functions of p21 with implications in self renewal, linking the relative quiescence of adult stem cells to their longevity and potentiality.
Marques-Torrejon, M. A., Porlan, E., Banito, A., Gomez-Ibarlucea, E., Lopez-Contreras, A. J., Fernandez-Capetillo, O., Vidal, A., Gil, J., Torres, J. and Farinas, I. (2013). «Cyclin-dependent kinase inhibitor p21 controls adult neural stem cell expansion by regulating Sox2 gene expression.» Cell Stem Cell 12(1): 88-100.
In the adult brain, continual neurogenesis of olfactory neurons is sustained by the existence of neural stem cells (NSCs) in the subependymal niche. Elimination of the cyclin-dependent kinase inhibitor 1A (p21) leads to premature exhaustion of the subependymal NSC pool, suggesting a relationship between cell cycle control and long-term self-renewal, but the molecular mechanisms underlying NSC maintenance by p21 remain unexplored. Here we identify a function of p21 in the direct regulation of the expression of pluripotency factor Sox2, a key regulator of the specification and maintenance of neural progenitors. We observe that p21 directly binds a Sox2 enhancer and negatively regulates Sox2 expression in NSCs. Augmented levels of Sox2 in p21 null cells induce replicative stress and a DNA damage response that leads to cell growth arrest mediated by increased levels of p19(Arf) and p53. Our results show a regulation of NSC expansion driven by a p21/Sox2/p53 axis.
Eguren, M., Porlan, E., Manchado, E., Garcia-Higuera, I., Canamero, M., Farinas, I. and Malumbres, M. (2013). «The APC/C cofactor Cdh1 prevents replicative stress and p53-dependent cell death in neural progenitors.» Nat Commun 4: 2880.
The E3-ubiquitin ligase APC/C-Cdh1 is essential for endoreduplication but its relevance in the mammalian mitotic cell cycle is still unclear. Here we show that genetic ablation of Cdh1 in the developing nervous system results in hypoplastic brain and hydrocephalus. These defects correlate with enhanced levels of Cdh1 substrates and increased entry into the S phase in neural progenitors. However, cell division is prevented in the absence of Cdh1 due to hyperactivation of cyclin-dependent kinases, replicative stress, induction of p53, G2 arrest and apoptotic death of these progenitor cells. Concomitant ablation of p53 rescues apoptosis but not replicative stress, resulting in the presence of damaged neurons throughout the adult brain. These data indicate that the inactivation of Cdh1 in vivo results in replicative stress, cell cycle arrest and cell death, supporting recent therapeutic proposals aimed to inhibit the APC/C in tumours.
Ferron, S. R., Charalambous, M., Radford, E., McEwen, K., Wildner, H., Hind, E., Morante-Redolat, J. M., Laborda, J., Guillemot, F., Bauer, S. R., Farinas, I. and Ferguson-Smith, A. C. (2011). «Postnatal loss of Dlk1 imprinting in stem cells and niche astrocytes regulates neurogenesis.» Nature 475(7356): 381-385.
The gene for the atypical NOTCH ligand delta-like homologue 1 (Dlk1) encodes membrane-bound and secreted isoforms that function in several developmental processes in vitro and in vivo. Dlk1, a member of a cluster of imprinted genes, is expressed from the paternally inherited chromosome. Here we show that mice that are deficient in Dlk1 have defects in postnatal neurogenesis in the subventricular zone: a developmental continuum that results in depletion of mature neurons in the olfactory bulb. We show that DLK1 is secreted by niche astrocytes, whereas its membrane-bound isoform is present in neural stem cells (NSCs) and is required for the inductive effect of secreted DLK1 on self-renewal. Notably, we find that there is a requirement for Dlk1 to be expressed from both maternally and paternally inherited chromosomes. Selective absence of Dlk1 imprinting in both NSCs and niche astrocytes is associated with postnatal acquisition of DNA methylation at the germ-line-derived imprinting control region. The results emphasize molecular relationships between NSCs and the niche astrocyte cells of the microenvironment, identifying a signalling system encoded by a single gene that functions coordinately in both cell types. The modulation of genomic imprinting in a stem-cell environment adds a new level of epigenetic regulation to the establishment and maintenance of the niche, raising wider questions about the adaptability, function and evolution of imprinting in specific developmental contexts.
Mira, H., Andreu, Z., Suh, H., Lie, D. C., Jessberger, S., Consiglio, A., San Emeterio, J., Hortiguela, R., Marques-Torrejon, M. A., Nakashima, K., Colak, D., Gotz, M., Farinas, I. and Gage, F. H. (2010). «Signaling through BMPR-IA regulates quiescence and long-term activity of neural stem cells in the adult hippocampus.» Cell Stem Cell 7(1): 78-89.
Neural stem cells (NSCs) in the adult hippocampus divide infrequently, and the molecules that modulate their quiescence are largely unknown. Here, we show that bone morphogenetic protein (BMP) signaling is active in hippocampal NSCs, downstream of BMPR-IA. BMPs reversibly diminish proliferation of cultured NSCs while maintaining their undifferentiated state. In vivo, acute blockade of BMP signaling in the hippocampus by intracerebral infusion of Noggin first recruits quiescent NSCs into the cycle and increases neurogenesis; subsequently, it leads to decreased stem cell division and depletion of precursors and newborn neurons. Consistently, selective ablation of Bmpr1a in hippocampal NSCs, or inactivation of BMP canonical signaling in conditional Smad4 knockout mice, transiently enhances proliferation but later leads to a reduced number of precursors, thereby limiting neuronal birth. BMPs are therefore required to balance NSC quiescence/proliferation and to prevent loss of the stem cell activity that supports continuous neurogenesis in the mature hippocampus.
Ferron, S. R., Pozo, N., Laguna, A., Aranda, S., Porlan, E., Moreno, M., Fillat, C., de la Luna, S., Sanchez, P., Arbones, M. L. and Farinas, I. (2010). «Regulated segregation of kinase Dyrk1A during asymmetric neural stem cell division is critical for EGFR-mediated biased signaling.» Cell Stem Cell 7(3): 367-379.
Stem cell division can result in two sibling cells exhibiting differential mitogenic and self-renewing potential. Here, we present evidence that the dual-specificity kinase Dyrk1A is part of a molecular pathway involved in the regulation of biased epidermal growth factor receptor (EGFR) signaling in the progeny of dividing neural stem cells (NSC) of the adult subependymal zone (SEZ). We show that EGFR asymmetry requires regulated sorting and that a normal Dyrk1a dosage is required to sustain EGFR in the two daughters of a symmetrically dividing progenitor. Dyrk1A is symmetrically or asymmetrically distributed during mitosis, and biochemical analyses indicate that it prevents endocytosis-mediated degradation of EGFR by a mechanism that requires phosphorylation of the EGFR signaling modulator Sprouty2. Finally, Dyrk1a heterozygous NSCs exhibit defects in self-renewal, EGF-dependent cell-fate decisions, and long-term persistence in vivo, suggesting that symmetrical divisions play a role in the maintenance of the SEZ reservoir.
Wu, H. H., Bellmunt, E., Scheib, J. L., Venegas, V., Burkert, C., Reichardt, L. F., Zhou, Z., Farinas, I. and Carter, B. D. (2009). «Glial precursors clear sensory neuron corpses during development via Jedi-1, an engulfment receptor.» Nat Neurosci 12(12): 1534-1541.
During the development of peripheral ganglia, 50% of the neurons that are generated undergo apoptosis. How the massive numbers of corpses are removed is unknown. We found that satellite glial cell precursors are the primary phagocytic cells for apoptotic corpse removal in developing mouse dorsal root ganglia (DRG). Confocal and electron microscopic analysis revealed that glial precursors, rather than macrophages, were responsible for clearing most of the dead DRG neurons. Moreover, we identified Jedi-1, an engulfment receptor, and MEGF10, a purported engulfment receptor, as homologs of the invertebrate engulfment receptors Draper and CED-1 expressed in the glial precursor cells. Expression of Jedi-1 or MEGF10 in fibroblasts facilitated binding to dead neurons, and knocking down either protein in glial cells or overexpressing truncated forms lacking the intracellular domain inhibited engulfment of apoptotic neurons. Together, these results suggest a cellular and molecular mechanism by which neuronal corpses are culled during DRG development.
Folgueras, A. R., Valdes-Sanchez, T., Llano, E., Menendez, L., Baamonde, A., Denlinger, B. L., Belmonte, C., Juarez, L., Lastra, A., Garcia-Suarez, O., Astudillo, A., Kirstein, M., Pendas, A. M., Farinas, I. and Lopez-Otin, C. (2009). «Metalloproteinase MT5-MMP is an essential modulator of neuro-immune interactions in thermal pain stimulation.» Proc Natl Acad Sci U S A 106(38): 16451-16456.
Peripheral interactions between nociceptive fibers and mast cells contribute to inflammatory pain, but little is known about mechanisms mediating neuro-immune communication. Here we show that metalloproteinase MT5-MMP (MMP-24) is an essential mediator of peripheral thermal nociception and inflammatory hyperalgesia. We report that MT5-MMP is expressed by CGRP-containing peptidergic nociceptors in dorsal root ganglia and that Mmp24-deficient mice display enhanced sensitivity to noxious thermal stimuli under basal conditions. Consistently, mutant peptidergic sensory neurons hyperinnervate the skin, a phenotype that correlates with changes in the regulated cleavage of the cell-cell adhesion molecule N-cadherin. In contrast to basal nociception, Mmp24(-/-) mice do not develop thermal hyperalgesia during inflammation, a phenotype that appears associated with alterations in N-cadherin-mediated cell-cell interactions between mast cells and sensory fibers. Collectively, our findings demonstrate an essential role of MT5-MMP in the development of dermal neuro-immune synapses and suggest that this metalloproteinase may be a target for pain control.
Ferron, S. R., Marques-Torrejon, M. A., Mira, H., Flores, I., Taylor, K., Blasco, M. A. and Farinas, I. (2009). «Telomere shortening in neural stem cells disrupts neuronal differentiation and neuritogenesis.» J Neurosci 29(46): 14394-14407.
Proliferation in the subependymal zone (SEZ) and neurogenesis in the olfactory bulb decline in the forebrain of telomerase-deficient mice. The present work reveals additional effects of telomere shortening on neuronal differentiation, as adult multipotent progenitors with critically short telomeres yield reduced numbers of neurons that, furthermore, exhibit underdeveloped neuritic arbors. Genetic data indicate that the tumor suppressor protein p53 not only mediates the adverse effects of telomere attrition on proliferation and self-renewal but it is also involved in preventing normal neuronal differentiation of adult progenitors with dysfunctional telomeres. Interestingly, progenitor cells with short telomeres obtained from fetal brains do not exhibit any replicative defects but also fail to acquire a fully mature neuritic arbor, demonstrating cell cycle-independent effects of telomeres on neuronal differentiation. The negative effect of p53 on neuritogenesis is mechanistically linked to its cooperation with the Notch pathway in the upregulation of small GTPase RhoA kinases, Rock1 and Rock2, suggesting a potential link between DNA damage and the Notch signaling pathway in the control of neuritogenesis. We also show that telomerase expression is downregulated in the SEZ of aging mice leading to telomere length reductions in neurosphere-forming cells and deficient neurogenesis and neuritogenesis. Our results suggest that age-related deficits could be caused partly by dysfunctional telomeres and demonstrate that p53 is a central modulator of adult neurogenesis, regulating both the production and differentiation of postnatally generated olfactory neurons.
Andreu-Agullo, C., Morante-Redolat, J. M., Delgado, A. C. and Farinas, I. (2009). «Vascular niche factor PEDF modulates Notch-dependent stemness in the adult subependymal zone.» Nat Neurosci 12(12): 1514-1523.
We sought to address the fundamental question of how stem cell microenvironments can regulate self-renewal. We found that Notch was active in astroglia-like neural stem cells (NSCs), but not in transit-amplifying progenitors of the murine subependymal zone, and that the level of Notch transcriptional activity correlated with self-renewal and multipotency. Moreover, dividing NSCs appeared to balance renewal with commitment via controlled segregation of Notch activity, leading to biased expression of known (Hes1) and previously unknown (Egfr) Notch target genes in daughter cells. Pigment epithelium-derived factor (PEDF) enhanced Notch-dependent transcription in cells with low Notch signaling, thereby subverting the output of an asymmetrical division to the production of two highly self-renewing cells. Mechanistically, PEDF induced a non-canonical activation of the NF-kappaB pathway, leading to the dismissal of the transcriptional co-repressor N-CoR from specific Notch-responsive promoters. Our data provide a basis for stemness regulation in vascular niches and indicate that Notch and PEDF cooperate to regulate self-renewal.
Alcamo, E. A., Chirivella, L., Dautzenberg, M., Dobreva, G., Farinas, I., Grosschedl, R. and McConnell, S. K. (2008). «Satb2 regulates callosal projection neuron identity in the developing cerebral cortex.» Neuron 57(3): 364-377.
Satb2 is a DNA-binding protein that regulates chromatin organization and gene expression. In the developing brain, Satb2 is expressed in cortical neurons that extend axons across the corpus callosum. To assess the role of Satb2 in neurons, we analyzed mice in which the Satb2 locus was disrupted by insertion of a LacZ gene. In mutant mice, beta-galactosidase-labeled axons are absent from the corpus callosum and instead descend along the corticospinal tract. Satb2 mutant neurons acquire expression of Ctip2, a transcription factor that is necessary and sufficient for the extension of subcortical projections by cortical neurons. Conversely, ectopic expression of Satb2 in neural stem cells markedly decreases Ctip2 expression. Finally, we find that Satb2 binds directly to regulatory regions of Ctip2 and induces changes in chromatin structure. These data suggest that Satb2 functions as a repressor of Ctip2 and regulatory determinant of corticocortical connections in the developing cerebral cortex.
Ferron, S. R., Andreu-Agullo, C., Mira, H., Sanchez, P., Marques-Torrejon, M. A. and Farinas, I. (2007). «A combined ex/in vivo assay to detect effects of exogenously added factors in neural stem cells.» Nat Protoc 2(4): 849-859.
We describe a protocol developed/modified by our group for the ex vivo and in vivo assessment of the response to a soluble factor of murine neural stem cells from the adult sub-ventricular zone (SVZ). The procedure includes several experimental options that can be used either independently or in combination. Potential factor effects on self-renewal, survival and proliferation are assayed by means of neurosphere cultures, with the factor administered directly in vitro to the culture plates (Step 1) or infused in vivo immediately before tissue dissociation (Step 3). We also use bromodeoxiuridine (BrdU) retention to label slowly dividing cells in vivo and subsequently perform two different types of experiments. In one set of experiments, the factor is added to primary cultures of stem cells obtained from the BrdU-pulsed animals and effects are tested on label-retaining cells after immunocytochemistry (Step 2). In another set, prolonged intraventricular infusion of the factor in BrdU-pulsed animals is followed by immunohistochemical analysis of BrdU labeling in the intact SVZ (Step 4). The minimum estimated time for the full combined procedure is 45 d.
Ramirez-Castillejo, C., Sanchez-Sanchez, F., Andreu-Agullo, C., Ferron, S. R., Aroca-Aguilar, J. D., Sanchez, P., Mira, H., Escribano, J. and Farinas, I. (2006). «Pigment epithelium-derived factor is a niche signal for neural stem cell renewal.» Nat Neurosci 9(3): 331-339.
Adult stem cells are characterized by self-renewal and multilineage differentiation, and these properties seem to be regulated by signals from adjacent differentiated cell types and by extracellular matrix molecules, which collectively define the stem cell «niche.» Self-renewal is essential for the lifelong persistence of stem cells, but its regulation is poorly understood. In the mammalian brain, neurogenesis persists in two germinal areas, the subventricular zone (SVZ) and the hippocampus, where continuous postnatal neuronal production seems to be supported by neural stem cells (NSCs). Here we show that pigment epithelium-derived factor (PEDF) is secreted by components of the murine SVZ and promotes self-renewal of adult NSCs in vitro. In addition, intraventricular PEDF infusion activated slowly dividing stem cells, whereas a blockade of endogenous PEDF decreased their cycling. These data demonstrate that PEDF is a niche-derived regulator of adult NSCs and provide evidence for a role for PEDF protein in NSC maintenance.
Dobreva, G., Chahrour, M., Dautzenberg, M., Chirivella, L., Kanzler, B., Farinas, I., Karsenty, G. and Grosschedl, R. (2006). «SATB2 is a multifunctional determinant of craniofacial patterning and osteoblast differentiation.» Cell 125(5): 971-986.
Vertebrate skeletogenesis involves two processes, skeletal patterning and osteoblast differentiation. Here, we show that Satb2, encoding a nuclear matrix protein, is expressed in branchial arches and in cells of the osteoblast lineage. Satb2-/- mice exhibit both craniofacial abnormalities that resemble those observed in humans carrying a translocation in SATB2 and defects in osteoblast differentiation and function. Multiple osteoblast-specific genes were identified as targets positively regulated by SATB2. In addition, SATB2 was found to repress the expression of several Hox genes including Hoxa2, an inhibitor of bone formation and regulator of branchial arch patterning. Molecular analysis revealed that SATB2 directly interacts with and enhances the activity of both Runx2 and ATF4, transcription factors that regulate osteoblast differentiation. This synergy was genetically confirmed by bone formation defects in Satb2/Runx2 and Satb2/Atf4 double heterozygous mice. Thus, SATB2 acts as a molecular node in a transcriptional network regulating skeletal development and osteoblast differentiation.
Villadiego, J., Mendez-Ferrer, S., Valdes-Sanchez, T., Silos-Santiago, I., Farinas, I., Lopez-Barneo, J. and Toledo-Aral, J. J. (2005). «Selective glial cell line-derived neurotrophic factor production in adult dopaminergic carotid body cells in situ and after intrastriatal transplantation.» J Neurosci 25(16): 4091-4098.
Glial cell line-derived neurotrophic factor (GDNF) exerts a notable protective effect on dopaminergic neurons in rodent and primate models of Parkinson’s disease (PD). The clinical applicability of this therapy is, however, hampered by the need of a durable and stable GDNF source allowing the safe and continuous delivery of the trophic factor into the brain parenchyma. Intrastriatal carotid body (CB) autografting is a neuroprotective therapy potentially useful in PD. It induces long-term recovery of parkinsonian animals through a trophic effect on nigrostriatal neurons and causes amelioration of symptoms in some PD patients. Moreover, the adult rodent CB has been shown to express GDNF. Here we show, using heterozygous GDNF/lacZ knock-out mice, that unexpectedly CB dopaminergic glomus, or type I, cells are the source of CB GDNF. Among the neural or paraneural cells tested, glomus cells are those that synthesize and release the highest amount of GDNF in the adult rodent (as measured by standard and in situ ELISA). Furthermore, GDNF expression by glomus cells is maintained after intrastriatal grafting and in CB of aged and parkinsonian 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated animals. Thus, glomus cells appear to be prototypical abundant sources of GDNF, ideally suited to be used as biological pumps for the endogenous delivery of trophic factors in PD and other neurodegenerative diseases.
Ferron, S., Mira, H., Franco, S., Cano-Jaimez, M., Bellmunt, E., Ramirez, C., Farinas, I. and Blasco, M. A. (2004). «Telomere shortening and chromosomal instability abrogates proliferation of adult but not embryonic neural stem cells.» Development 131(16): 4059-4070.
Chromosome integrity is essential for cell viability and, therefore, highly proliferative cell types require active telomere elongation mechanisms to grow indefinitely. Consistently, deletion of telomerase activity in a genetically modified mouse strain results in growth impairments in all highly proliferative cell populations analyzed so far. We show that telomere attrition dramatically impairs the in vitro proliferation of adult neural stem cells (NSCs) isolated from the subventricular zone (SVZ) of telomerase-deficient adult mice. Reduced proliferation of postnatal neurogenic progenitors was also observed in vivo, in the absence of exogenous mitogenic stimulation. Strikingly, severe telomere erosion resulting in chromosomal abnormalities and nuclear accumulation of p53 did not affect the in vitro proliferative potential of embryonic NSCs. These results suggest that intrinsic differences exist between embryonic and adult neural progenitor cells in their response to telomere shortening, and that some populations of tissue-specific stem cells can bypass DNA damage check points.
Farinas, I., Jones, K. R., Tessarollo, L., Vigers, A. J., Huang, E., Kirstein, M., de Caprona, D. C., Coppola, V., Backus, C., Reichardt, L. F. and Fritzsch, B. (2001). «Spatial shaping of cochlear innervation by temporally regulated neurotrophin expression.» J Neurosci 21(16): 6170-6180.
Previous work suggested qualitatively different effects of neurotrophin 3 (NT-3) in cochlear innervation patterning in different null mutants. We now show that all NT-3 null mutants have a similar phenotype and lose all neurons in the basal turn of the cochlea. To understand these longitudinal deficits in neurotrophin mutants, we have compared the development of the deficit in the NT-3 mutant to the spatial-temporal expression patterns of brain-derived neurotrophic factor (BDNF) and NT-3, using lacZ reporters in each gene and with expression of the specific neurotrophin receptors, trkB and trkC. In the NT-3 mutant, almost normal numbers of spiral ganglion neurons form, but fiber outgrowth to the basal turn is eliminated by embryonic day (E) 13.5. Most neurons are lost between E13.5 and E15.5. During the period preceding apoptosis, NT-3 is expressed in supporting cells, whereas BDNF is expressed mainly in hair cells, which become postmitotic in an apical to basal temporal gradient. During the period of neuronal loss, BDNF is absent from the basal cochlea, accounting for the complete loss of basal turn neurons in the NT-3 mutant. The spatial gradients of neuronal loss in these two mutants appear attributable to spatial-temporal gradients of neurotrophin expression. Our immunocytochemical data show equal expression of their receptors, TrkB and TrkC, in spiral sensory neurons and thus do not relate to the basal turn loss. Mice in which NT-3 was replaced by BDNF show a qualitative normal pattern of innervation at E13.5. This suggests that the pattern of expression of neurotrophins rather than their receptors is essential for the spatial loss of spiral sensory neurons in NT-3 null mutants.
Abeliovich, A., Schmitz, Y., Farinas, I., Choi-Lundberg, D., Ho, W. H., Castillo, P. E., Shinsky, N., Verdugo, J. M., Armanini, M., Ryan, A., Hynes, M., Phillips, H., Sulzer, D. and Rosenthal, A. (2000). «Mice lacking alpha-synuclein display functional deficits in the nigrostriatal dopamine system.» Neuron 25(1): 239-252.
Alpha-Synuclein (alpha-Syn) is a 14 kDa protein of unknown function that has been implicated in the pathophysiology of Parkinson’s disease (PD). Here, we show that alpha-Syn-/- mice are viable and fertile, exhibit intact brain architecture, and possess a normal complement of dopaminergic cell bodies, fibers, and synapses. Nigrostriatal terminals of alpha-Syn-/- mice display a standard pattern of dopamine (DA) discharge and reuptake in response to simple electrical stimulation. However, they exhibit an increased release with paired stimuli that can be mimicked by elevated Ca2+. Concurrent with the altered DA release, alpha-Syn-/- mice display a reduction in striatal DA and an attenuation of DA-dependent locomotor response to amphetamine. These findings support the hypothesis that alpha-Syn is an essential presynaptic, activity-dependent negative regulator of DA neurotransmission.