Estudio de la neurogénesis adulta en un modelo murino de sobreexpresión de la glucógeno sintasa quinasa-3 Beta en precursores neuronales

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 17-01-2014GSK3 plays an important role in many physiological processes such as microtubule dynamics, cell cycle division, glucose metabolism, apoptosis, embryonic development, cell differentiation and adult neurogenesis. We have previously reported the effects on dentate gyrus (DG) neurogenesis of overexpress GSK3β in adult neurons under CamKIIα promoter. These mice present atrophied DG and less neurogenic niches which show a reduced proliferative activity leading to almost a total depletion with age. Here, by using GFAP promoter, we have generated a transgenic mice overexpressing GSK3β in glial cells. First, differences have been detected in GFAP/OE GSK-3β mice due to GSK3β overexpression such as an increase in brain weight, volume of dentate gyrus and number of mature granule neurons. Interestingly, main astrocytes overexpressing GSK3β are those present in the cerebellum as well as those present in the SGZ of the hippocampus. Behavioral tests suggest that GSK3β overexpression can be helpful as demonstrated by the Rotarod test or by the increasing freezing time observed in the fear conditioning test. Focusing our study in the SGZ, we have demonstrated that the increase in DG observed in this animal model could be due in part to alterations in neurogenesis. Thus, we have detected an increase in the number of neural progenitor cells in the overexpressing mice by using molecular markers such as BLBP, Sox2 and DCX. In good agreement, neural precursors isolated from GFAP/OE GSK3 mice, exhibit increased cell proliferation in culture. However, although that animal model has a higher number of neuronal progenitors we did not observed differences in parameters such as cell proliferation, survival of newly generated neurons or division cycle reentry. Interestingly, an increase in Dkk1 y sFRP3 mRNA, inhibitors of Wnt-frizzled complex, is observed. Moreover, some alterations occur because an increase in active microglial cells as well as changes in levels of some cytokine molecules is observed. This could be the explanation for changes in morphology of dendritic tree observed in mature newborn neurons of GFAP/OE GSK3 after switching off the transgene. In summary, these findings suggest that an increase in GSK3β activity in neural progenitors provokes an increase in these precursors at least in SGZ as well an increase in granular neurons in the DG

Novel connection between newborn granule neurons and the hippocampal CA2 field

© 2014 Elsevier Inc. Newborn neurons are continuously added to the hippocampal dentate gyrus (DG) throughout life. Mature and immature granule neurons are believed to send their axonal projections exclusively to the hippocampal CA3 field. However, recent data point to an alternative trisynaptic circuit, involving a direct axonal projection from mature granule neurons to the CA2 field. Whether this circuit takes place only in mature granule neurons or, on the contrary, whether immature granule neurons also contribute to this novel connection is unknown. We used various retroviral vectors to show that immature granule neurons send axonal processes to and establish synaptic contacts with CA2 pyramidal neurons and that axonal growth follows a similar time course to that described for CA3 innervation. In addition, we provide experimental evidence demonstrating that the pathway connecting newborn granule neurons and the CA2 field can be modulated by physiological and deleterious stimuli.Spanish Ministry of Health (SAF 2006-02424, BFU-2008-03980, BFU-2010-21507), the Comunidad de Madrid (SAL/0202/2006), the Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, ISCIII) (CB401), and the Fundación R. ArecesPeer Reviewe

Direct Evidence of Internalization of Tau by Microglia in Vitro and in Vivo

The microtubule-associated protein (MAP) tau plays a critical role in the pathogenesis of tauopathies. Excess tau can be released into the extracellular medium in a physiological or pathological manner to be internalized by surrounding neurons' a process that contributes to the spread of this protein throughout the brain. Such spreading may correlate with the progression of the abovementioned diseases. In addition to neurons, tau can be internalized into other cells. Here we demonstrate that microglia take up tau in vitro and in vivo. In this regard, microglia from primary cultures internalized soluble (human recombinant tau42) and insoluble (homogenates derived from human AD brain) tau in vitro. Furthermore, using stereotaxic injection of tau in mice in vivo, we show that murine microglia internalize human tau. In addition, we demonstrate, for the first time, that microglia colocalize with various forms of tau in postmortem brain tissue of patients with Alzheimer's disease and non-demented control subjects. Our data reveal a potential role of microglia in the internalization of tau that might be relevant for the design of strategies to enhance the clearance of extracellular tau in neurodegenerative diseases characterized by the accumulation of this protein.Spanish Ministry of Health, the Comunidad de Madrid, the Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, ISCIII), and the Alzheimer’s Association.Peer Reviewe

Excitotoxicity induced by kainic acid provokes glycogen synthase kinase-3 truncation in the hippocampus

© 2015 Elsevier B.V. In neuronal cultures, glycogen synthase kinase 3(GSK3) is truncated at the N-terminal end by calpain downstream of activated glutamate receptors. However, the in vivo biological significance of that truncation has not been explored. In an attempt to elucidate if GSK3 truncation has a pathophysiological relevance, we have used intraperitoneal injections of kainic acid (KA) in rats and intra-amygdala KA microinjections in mice as in vivo models of excitotoxicity. Spectrin cleavage analyzed by immunohistochemistry was observed in the CA1 hippocampal field in KA-intraperitoneal treated rats while the CA3 region was the hippocampal area affected after intra-amygdala KA microinjections. GSK3β immunofluorescence did not colocalize with truncated spectrin in both treatments using an antibody that recognize the N-terminal end of GSK3β. Thus, those neurons which are spectrin-positive do not show GSK3β immunolabelling. To study GSK3β truncation in vitro, we exposed organotypic hippocampal slices and cultured cortical neurons to KA leading to the truncation of GSK3 and we found that truncation was blocked by the calpain inhibitor calpeptin. These data suggest a relationship between N-terminal GSK3β truncation and excitotoxicity. Overall, our data reinforces the important relationship between glutamate receptors and GSK3 and their role in neurodegenerative processes in which excitotoxicity is involved.Ministerio de Educa ción y Ciencia (SAF2010-15525 and BUF2013-40664-P) and Comunidad de Madrid (S2010/BMD-2331). We also acknowl- edge institutional support from Fundación Ramón ArecesPeer Reviewe

Neuroprotective role of PKD1 against ischemic and kainic acid-induced brain injury

Trabajo presentado en el Second Spanish Molecular Imaging Network (SMIN) Meeting, celebrado en Madrid (España) el 26 de febrero de 2018

The Social Component of Environmental Enrichment Is a Pro-neurogenic Stimulus in Adult c57BL6 Female Mice

In rodents, the hippocampal dentate gyrus gives rise to newly generated dentate granule cells (DGCs) throughout life. This process, named adult hippocampal neurogenesis (AHN), converges in the functional integration of mature DGCs into the trisynaptic hippocampal circuit. Environmental enrichment (EE) is one of the most potent positive regulators of AHN. This paradigm includes the combination of three major stimulatory components, namely increased physical activity, constant cognitive stimulation, and higher social interaction. In this regard, the pro-neurogenic effects of physical activity and cognitive stimulation have been widely addressed in adult rodents. However, the pro-neurogenic potential of the social aspect of EE has been less explored to date. Here we tackled this question by specifically focusing on the effects of a prolonged period of social enrichment (SE) in adult female C57BL6 mice. To this end, 7-week-old mice were housed in groups of 12 per cage for 8 weeks. These mice were compared with others housed under control housing (2–3 mice per cage) or EE (12 mice per cage plus running wheels and toys) conditions during the same period. We analyzed the number and morphology of Doublecortin-expressing (DCX+) cells. Moreover, using RGB retroviruses that allowed the labeling of three populations of newborn DGCs of different ages in the same mouse, we performed morphometric, immunohistochemical, and behavioral determinations. Both SE and EE increased the number and maturation of DCX+ cells, and caused an increase in dendritic maturation in certain populations of newborn DGCs. Moreover, both manipulations increased exploratory behavior in the Social Interaction test. Unexpectedly, our data revealed the potent neurogenesis-stimulating potential of SE in the absence of any further cognitive stimulation or increase in physical activity. Given that an increase in physical activity is strongly discouraged under certain circumstances, our findings may be relevant in the context of enhancing AHN via physical activity-independent mechanisms

Adeno-associated viral vector serotype 9-based gene therapy for Niemann-Pick disease type A

Niemann-Pick disease type A (NPD-A) is a lysosomal storage disorder characterized by neurodegeneration and early death. It is caused by loss-of-function mutations in the gene encoding for acid sphingomyelinase (ASM), which hydrolyzes sphingomyelin into ceramide. Here, we evaluated the safety of cerebellomedullary (CM) cistern injection of adeno-associated viral vector serotype 9 encoding human ASM (AAV9-hASM) in nonhuman primates (NHP). We also evaluated its therapeutic benefit in a mouse model of the disease (ASM-KO mice). We found that CM injection in NHP resulted in widespread transgene expression within brain and spinal cord cells without signs of toxicity. CM injection in the ASM-KO mouse model resulted in hASM expression in cerebrospinal fluid and in different brain areas without triggering an inflammatory response. In contrast, direct cerebellar injection of AAV9-hASM triggered immune response. We also identified a minimally effective therapeutic dose for CM injection of AAV9-hASM in mice. Two months after administration, the treatment prevented motor and memory impairment, sphingomyelin (SM) accumulation, lysosomal enlargement, and neuronal death in ASM-KO mice. ASM activity was also detected in plasma from AAV9-hASM CM-injected ASM-KO mice, along with reduced SM amount and decreased inflammation in the liver. Our results support CM injection for future AAV9-based clinical trials in NPD-A as well as other lysosomal storage brain disorders.Nation Foundation and by grants from the Spanish Ministry of Economy and Competitivity (SAF-2014-57539-R and SAF2017-87698-R) to M.D.L. and from NIH-NINDS (R01NS073940) to K.S.B. A.P.-C. was a recipient of the FPU predoctoral fellowship from the Spanish Ministry of Economy and Competitivity and Fundación Ramón Areces to the Centro Biología Molecular Severo Ochoa

Excitotoxic inactivation of constitutive oxidative stress detoxification pathway in neurons can be rescued by PKD1

Excitotoxicity, a critical process in neurodegeneration, induces oxidative stress and neuronal death through mechanisms largely unknown. Since oxidative stress activates protein kinase D1 (PKD1) in tumor cells, we investigated the effect of excitotoxicity on neuronal PKD1 activity. Unexpectedly, we find that excitotoxicity provokes an early inactivation of PKD1 through a dephosphorylation-dependent mechanism mediated by protein phosphatase-1 (PP1) and dual specificity phosphatase-1 (DUSP1). This step turns off the IKK/NF-kappa B/SOD2 antioxidant pathway. Neuronal PKD1 inactivation by pharmacological inhibition or lentiviral silencing in vitro, or by genetic inactivation in neurons in vivo, strongly enhances excitotoxic neuronal death. In contrast, expression of an active dephosphorylation-resistant PKD1 mutant potentiates the IKK/NF-kappa B/SOD2 oxidative stress detoxification pathway and confers neuroprotection from in vitro and in vivo excitotoxicity. Our results indicate that PKD1 inactivation underlies excitotoxicity-induced neuronal death and suggest that PKD1 inactivation may be critical for the accumulation of oxidation-induced neuronal damage during aging and in neurodegenerative disorders

GSK-3β, a pivotal kinase in Alzheimer disease

Alzheimer disease (AD) is the most common form of age-related dementia. The etiology of AD is considered to be multifactorial as only a negligible percentage of cases have a familial or genetic origin. Glycogen synthase kinase-3 (GSK-3) is regarded as a critical molecular link between the two histopathological hallmarks of the disease, namely senile plaques and neurofibrillary tangles. In this review, we summarize current data regarding the involvement of this kinase in several aspects of AD development and progression, as well as key observations highlighting GSK-3 as one of the most relevant targets for AD treatment. © 2014 Llorens-Martín, Jurado, Hernández and Ávila.Peer Reviewe