David Díaz

NAPROC-13

MOTIVATION Although natural products represent a reservoir of molecular diversity, the process of isolating and identifying active compounds is a bottleneck in drug discovery programs. The rapid isolation and identification of the bioactive component(s) of natural product mixtures during the bioassay-guided fractionation have become crucial factors in the competition with chemical compound libraries and combinatorial synthetic efforts. In this respect, the use of spectral databases in identification processes is indispensable. RESULTS We have developed a database containing (13)C spectral information of over 6000 natural compounds, which allows for fast identifications of known compounds present in the crude extracts and provides insight into the structural elucidation of unknown compounds. AVAILABILITY http://c13.usal.es

Long-lasting changes in the anatomy of the olfactory bulb after ionizing irradiation and bone marrow transplantation.

The adult brain is considered to be a radioresistant organ since it is mainly composed of non-dividing cells. However, in adult animals there are a few neurogenic brain areas that are affected by ionizing radiation whose plasticity and capacity for recovery are still unclear. Here, mice were irradiated with a minimal lethal dose of radiation in order to determine its effects on the subventricular zone (SVZ), the rostral migratory stream (RMS), and the olfactory bulb (OB). These regions underwent a dramatic reduction in cell proliferation and ensuing morphological alterations, accompanied by a patent reactive gliosis. Bone marrow stem cell (BMSC) transplants were also performed after the radiation treatment to allow the mouse survival with a view to analyzing long-term effects. Normal proliferation rates were not recovered over time and although bone marrow-derived cells reached the brain, they were not incorporated into the SVZ-RMS-OB pathway in an attempt to rescue the damaged regions. Since neurogenesis produces new interneurones in the OB, thus feeding cell turnover, the volume and lamination of the OB were analyzed. The volume of the OB proved to be dramatically reduced at postnatal day 300 (P300), and this shrinkage affected the periependymal white matter, the granule cell layer, the external plexiform layer, and the glomerular layer. These results should be taken into account in cell therapies employing BMSC, since such cells reach the encephalon, although they cannot restore the damage produced in neurogenic areas. This study thus provides new insight into the long-term effects of ionizing radiation, widely employed in animal experimentation and even in clinical therapies for human beings.

Sexual dimorphic stages affect both proliferation and serotonergic innervation in the adult rostral migratory stream

One of the sexual dimorphic differences in adult rodents is neural proliferation. Here we demonstrate that physiological hormone stages can modulate this proliferation in the adult forebrain. Female mice, both pregnant and synchronized in oestrus, exhibited higher proliferating cell percentages than males in both the rostral migratory stream (RMS) and the olfactory bulb (OB). Moreover, although the hormonal component also influenced the subventricular zone (SVZ), no differences in proliferation were observed in this region. In addition, both groups of females had higher numbers of serotonergic fibres in these regions. Serotonin may therefore be related to the mechanism of action by which hormones can affect cell proliferation of this brain region. We also evaluated cell death in the SVZ in males and females, finding that this was higher in the former. Taken together, our results support the idea that in female rodents more neuroblasts are able to reach the RMS and then proliferate, apoptosis being an additional mechanism affecting the low proliferation of cells in the RMS and OB in males. Thus, proliferation in the RMS is influenced by sexual dimorphism.

Anosmin-1 over-expression increases adult neurogenesis in the subventricular zone and neuroblast migration to the olfactory bulb

New subventricular zone (SVZ)-derived neuroblasts that migrate via the rostral migratory stream are continuously added to the olfactory bulb (OB) of the adult rodent brain. Anosmin-1 (A1) is an extracellular matrix protein that binds to FGF receptor 1 (FGFR1) to exert its biological effects. When mutated as in Kallmann syndrome patients, A1 is associated with severe OB morphogenesis defects leading to anosmia and hypogonadotropic hypogonadism. Here, we show that A1 over-expression in adult mice strongly increases proliferation in the SVZ, mainly with symmetrical divisions, and produces substantial morphological changes in the normal SVZ architecture, where we also report the presence of FGFR1 in almost all SVZ cells. Interestingly, for the first time we show FGFR1 expression in the basal body of primary cilia in neural progenitor cells. Additionally, we have found that A1 over-expression also enhances neuroblast motility, mainly through FGFR1 activity. Together, these changes lead to a selective increase in several GABAergic interneuron populations in different OB layers. These specific alterations in the OB would be sufficient to disrupt the normal processing of sensory information and consequently alter olfactory memory. In summary, this work shows that FGFR1-mediated A1 activity plays a crucial role in the continuous remodelling of the adult OB

Bone Marrow Contributes Simultaneously to Different Neural Types in the Central Nervous System Through Different Mechanisms of Plasticity

Many studies have reported the contribution of bone marrow-derived cells (BMDC) to the CNS, raising the possibility of using them as a new source to repair damaged brain tissue or restore neuronal function. This process has mainly been investigated in the cerebellum, in which a degenerative microenvironment has been suggested to be responsible for its modulation. The present study further analyzes the contribution of BMDC to different neural types in other adult brain areas, under both physiological and neurodegenerative conditions, together with the mechanisms of plasticity involved. We grafted genetically marked green fluorescent protein/Cre bone marrow in irradiated recipients: a) the PCD (Purkinje Cell Degeneration) mutant mice, suffering a degeneration of specific neuronal populations at different ages, and b) their corresponding healthy controls. These mice carried the conditional lacZ reporter gene to allow the identification of cell fusion events. Our results demonstrate that BMDC mainly generate microglial cells, although to a lesser extent a clear formation of neuronal types also exists. This neuronal recruitment was not increased by the neurodegenerative processes occurring in PCD mice, where BMDC did not contribute to rescuing the degenerated neuronal populations either. However, an increase in the number of bone marrow-derived microglia was found along the life span in both experimental groups. Six weeks after transplantation more bone marrow-derived microglial cells were observed in the olfactory bulb of the PCD mice compared to the control animals, where the degeneration of mitral cells was in process. In contrast, this difference was not observed in the cerebellum, where Purkinje cell degeneration had been completed. These findings demonstrated that the degree of neurodegenerative environment can foster the recruitment of neural elements derived from bone marrow, but also provide the first evidence that BMDC can contribute simultaneously to different encephalic areas through different mechanisms of plasticity: cell fusion for Purkinje cells and differentiation for olfactory bulb interneurons.

Mild cerebellar neurodegeneration of aged heterozygous PCD mice increases cell fusion of Purkinje and bone marrow-derived cells.

Bone marrow-derived cells have different plastic properties, especially regarding cell fusion, which increases with time and is prompted by tissue injury. Several recessive mutations, including Purkinje Cell Degeneration, affect the number of Purkinje cells in homozygosis; heterozygous young animals have an apparently normal phenotype but they undergo Purkinje cell loss as they age. Our findings demonstrate that heterozygous pcd mice undergo Purkinje cell loss at postnatal day 300, this slow but steadily progressing cell death starting sooner than has been reported previously and without massive reactive gliosis or inflammation. Here, transplantation of bone marrow stem cells was performed to assess the arrival of bone marrow-derived cells in the cerebellum in these heterozygous mice. Our results reveal that a higher number of cell fusion events occurs in heterozygous animals than in the controls, on days 150 and 300 postnatally. In sum, this study indicates that mild cell death promotes the fusion of bone marrow-derived cells with surviving Purkinje neurons. This phenomenon suggests new therapies for long-lasting neurodegenerative disorders.

Bone Marrow Cell Transplantation Restores Olfaction in the Degenerated Olfactory Bulb

Bone marrow contains heterogeneous cell types including end-lineage cells, committed tissue progenitors, and multipotent stem/progenitor cells. The immense plasticity of bone marrow cells allows them to populate diverse tissues such as the encephalon, and give rise to a variety of cell types. This unique plasticity makes bone marrow-derived cells good candidates for cell therapy aiming at restoring impaired brain circuits. In the present study, bone marrow cells were transplanted into P20 mice that exhibit selective olfactory degeneration in adulthood between P60 and P150. These animals, the so-called Purkinje Cell Degeneration (PCD) mutant mice, suffer from a progressive and specific loss of a subpopulation of principal neurons of the olfactory bulb, the mitral cells (MCs), sparing the other principal neurons, the tufted cells. As such, PCD mice constitute an interesting model to evaluate the specific role of MCs in olfaction and to test the restorative function of transplanted bone marrow-derived cells. Using precision olfactometry, we revealed that mutant mice lacking MCs exhibited a deficit in odorant detection and discrimination. Remarkably, the transplantation of wild-type bone marrow-derived cells into irradiated PCD mutant mice generated a large population of microglial cells in the olfactory bulb and reduced the degenerative process. The alleviation of MC loss in transplanted mice was accompanied by functional recovery witnessed by significantly improved olfactory detection and enhanced odor discrimination. Together, these data suggest that: (1) bone marrow-derived cells represent an effective neuroprotective tool to restore degenerative brain circuits, and (2) MCs are necessary to encode odor concentration and odor identity in the mouse olfactory bulb.

Functional redundancy of Sos1 and Sos2 for lymphopoiesis and organismal homeostasis and survival.

ABSTRACT Sos1 and Sos2 are ubiquitously expressed, universal Ras guanine nucleotide exchange factors (Ras-GEFs) acting in multiple signal transduction pathways activated by upstream cellular kinases. The embryonic lethality of Sos1 null mutants has hampered ascertaining the specific in vivo contributions of Sos1 and Sos2 to processes controlling adult organism survival or development of hematopoietic and nonhematopoietic organs, tissues, and cell lineages. Here, we generated a tamoxifen-inducible Sos1-null mouse strain allowing analysis of the combined disruption of Sos1 and Sos2 (Sos1/2) during adulthood. Sos1/2 double-knockout (DKO) animals died precipitously, whereas individual Sos1 and Sos2 knockout (KO) mice were perfectly viable. A reduced percentage of total bone marrow precursors occurred in single-KO animals, but a dramatic depletion of B-cell progenitors was specifically detected in Sos1/2 DKO mice. We also confirmed a dominant role of Sos1 over Sos2 in early thymocyte maturation, with almost complete thymus disappearance and dramatically higher reduction of absolute thymocyte counts in Sos1/2 DKO animals. Absolute counts of mature B and T cells in spleen and peripheral blood were unchanged in single-KO mutants, while significantly reduced in Sos1/2 DKO mice. Our data demonstrate functional redundancy between Sos1 and Sos2 for homeostasis and survival of the full organism and for development and maturation of T and B lymphocytes.

The Olfactory System as a Puzzle: Playing With Its Pieces

The mammalian olfactory bulb (OB) has all the features of a whole mammalian brain but in a more reduced space: neuronal lamination, sensory inputs, afferences, or efferences to other centers of the central nervous system, or a contribution of new neural elements. Therefore, it is widely considered as “a brain inside the brain.” Although this rostral region has the same origin and general layering as the other cerebral cortices, some distinctive features make it very profitable in experimentation in neurobiology: the sensory inputs are driven directly on its surface, the main output can be accessed anatomically, and new elements appear in it throughout adult life. These three morphological characteristics have been manipulated to analyze further the response of the whole OB. The present review offers a general outlook into the consequences of such experimentation in the anatomy, connectivity and neurochemistry of the OB after (a) sensory deprivation, mainly by naris occlusion; (b) olfactory deinnervation by means of olfactory epithelium damage, olfactory nerve interruption, or even olfactory tract disruption; (c) the removal of the principal neurons of the OB; and (d) management of the arrival of newborn interneurons from the rostral migratory stream. These experiments were performed using surgical or chemical methods, but also by means of the analysis of genetic models, some of whose olfactory components are missing, colorless or mismatching within the wild‐type scenario of odor processing. Anat Rec, 296:1383‐1400, 2013.

Differential glial activation during the degeneration of Purkinje cells and mitral cells in the PCD mutant mice

Purkinje Cell Degeneration (PCD) mice harbor a nna1 gene mutation which leads to an early and rapid degeneration of Purkinje cells (PC) between the third and fourth week of age. This mutation also underlies the death of mitral cells (MC) in the olfactory bulb (OB), but this process is slower and longer than in PC. No clear interpretations supporting the marked differences in these neurodegenerative processes exist. Growing evidence suggests that either beneficial or detrimental effects of gliosis in damaged regions would underlie these divergences. Here, we examined the gliosis occurring during PC and MC death in the PCD mouse. Our results demonstrated different glial reactions in both affected regions. PC disappearance stimulated a severe gliosis characterized by strong morphological changes, enhanced glial proliferation, as well as the release of pro‐inflammatory mediators. By contrast, MC degeneration seems to promote a more attenuated glial response in the PCD OB compared with that of the cerebellum. Strikingly, cerebellar oligodendrocytes died by apoptosis in the PCD, whereas bulbar ones were not affected. Interestingly, the level of nna1 mRNA under normal conditions was higher in the cerebellum than in the OB, probably related to a faster neurodegeneration and stronger glial reaction in its absence. The glial responses may thus influence the neurodegenerative course in the cerebellum and OB of the mutant mouse brain, providing harmful and beneficial microenvironments, respectively.