Astrocytes play crucial roles in maintaining brain homeostasis and in orchestrating neural development, all through tightly coordinated steps that cooperate to maintain the balance needed for normal ...development. Here, we review the alterations in astrocyte functions that contribute to a variety of developmental neurometabolic disorders and provide additional data on the predominant role of astrocyte dysfunction in the neurometabolic neurodegenerative disease glutaric acidemia type I. Finally, we describe some of the therapeutical approaches directed to neurometabolic diseases and discuss if astrocytes can be possible therapeutic targets for treating these disorders.
•Three types of proliferating cells are distributed in neurogenic areas of fish brain.•Stem cells line the ventricular walls and represent around 10% of proliferating cells.•Proportion of ...proliferating cell types are maintained in all brain regions.•Migrating neurons were found farther from ventricular lumen.
Our previous studies demonstrated that Austrolebias charrua annual fish is an excellent model to study adult brain cell proliferation and neurogenesis due to the presence of active and fast neurogenesis in several regions during its short lifespan. Our main goal was to identify and localize the cells that compose the neurogenic areas throughout the Austrolebias brain. To do this, we used two thymidine halogenated analogs to detect cell proliferation at different survival times: 5-chloro-2′-deoxyuridine (CldU) at 1day and 5-iodo-2′-deoxyuridine (IdU) at 30days. Three types of proliferating cells were identified: I – transient amplifying or fast cycling cells that uptake CldU; II – stem cells or slow cycling cells, that were labeled with both CldU and IdU and did not migrate; and III – migrant cells that uptake IdU. Mapping and 3D-reconstruction of labeled nuclei showed that type I and type II cells were preferentially found close to ventricle walls. Type III cells appeared widespread and migrating in tangential and radial routes. Use of proliferation markers together with Vimentin or Nestin evidenced that type II cells are the putative stem cells that are located at the ventricular lumen. Double label cells with IdU+ and NeuN or HuC/D allowed us identify migrant neurons. Quantitation of labeled nuclei indicates that the proportion of putative stem cells is around 10% in all regions of the brain. This percentage of stem cells suggests the existence of a constant brain cell population in Austrolebias charrua that seems functional to the maintainance of adult neurogenesis.
Highlights • Olfactory bulb ventricular wall presents three different neurogenic niches. • The cellular composition is particular for each proliferative zone. • Elongated radial glial cells are ...present in the most proliferative ventricular regions. • Cells lining the ventricular wall are monociliated. • Migratory routes of neuroblast were found in transitional and ventral zones.
The annual killifish genus Austrolebias includes approximately 38 species distributed throughout the Paraná-Plata basin and Patos-Merín system. Within the Austrolebias adloffi species complex, the ...Uruguayan populations of Austrolebias charrua were considered as an intergradation between A. adloffi and Austrolebias viarius populations. Austrolebias charrua presents an intermediate phenotype between both taxa and high levels of morphological and chromatic variability. In the present study, we incorporate different methodological approaches (molecular, morphology, and gamete ultrastructure) to elucidate the pattern of differentiation among the parapatric taxa (A. charrua, Austrolebias reicherti, A. viarius) distributed in a Biosphere Reserve Site. Analyses of cytochrome b sequences show high values of DNA polymorphism, in particular for A. charrua. This is in accordance with both morphological and gametic variation. Using a statistical parsimony network based on these sequences and analysis of morphological data, past fragmentation and range expansion involving perhaps secondary contact between A. charrua and A. reicherti could be proposed. Coloration pattern and morphometric analyses showed an unexpected higher similarity between the most distantly-related taxa, A. viarius and A. charrua. This could be the result of retention of ancestral polymorphisms, especially in A. charrua populations from ponds of higher elevation, or to directional selection acting in similar ecological environments. Because these annual killifish species are considered endangered, our work reinforces the high priority need to include them in a conservation programme.
Introdução: A Doença de Behçet (DB) é uma vasculite multissistémica, de diagnóstico apenas clínico, e rara em idade pediátrica. Caso Clínico: Adolescente de 17 anos com úlceras aftosas orais de ...repetição desde há quatro anos, sem outros sinais ou sintomas associados. Hipóteses diagnósticas como infecções, hipovitaminoses, alergia alimentar e doença celíaca foram excluídas. O diagnóstico de DB foi também ponderado, tendo realizado teste de patergia, com resultado negativo. Aos 16 anos, a doente apresenta lesões papulopustulares mentonianas, indistinguíveis de acne vulgar, e aos 17 anos, um episódio de úlceras genitais. Foi referenciada a Reumatologia pediátrica e, perante um provável diagnóstico de DB, iniciou colchicina oral, com redução do número e da recorrência das úlceras aftosas orais. Discussão: A adolescente poderá apresentar critérios suficientes para se diagnosticar DB. Este caso exemplifica a importância do acompanhamento contínuo dos doentes, realça o carácter insidioso de algumas doenças reumáticas e a dificuldade de diagnosticar DB em idade pediátrica.
Chagas disease, endemic in 21 countries across Latin America, kills more people in the region each year than any other parasite-borne disease. Therapeutic options have problems ranging from toxicity, ...poor efficacy, drug resistance and high cost. Thus, cheaper and less toxic treatments are necessary. From our in-house chemical library of agents against Trypanosoma cruzi the most relevant N-oxide-containing heterocycles were selected for mode of action and type of death studies. Also included in these studies were two active nitrofuranes. Epimastigotes of T. cruzi were used as the biological model in this study. The metabolic profile was studied by 1H NMR in association with the MTT assay. Excreted catabolites data, using 1H NMR spectroscopy, showed that most of the studied N-oxides were capable of decreasing both the release of succinate and acetate shedding, the compounds therefore possibly acting on mitochondria. Only quinoxalines and the nitrofurane Nf1 showed significant mitochondrial dehydrogenase inhibitions, but with different dose-time profiles. In the particular case of quinoxaline Qx2 the glucose uptake study revealed that the integrity of some pathways into the glycosome could be affected. Optic, fluorescence (TUNEL and propidium iodide) and transmission electron microscopy (TEM) were employed for type of death studies. These studies were complemented with 1H NMR to visualize mobile lipids. At low concentrations none of the selected compounds showed a positive TUNEL assay. However, both quinoxalines, one furoxan and one benzofuroxan showed a necrotic effect at high concentrations. Curiously, one furoxan, Fx1, one benzofuroxan, Bfx1, and one nitrofurane, Nf1, caused a particular phenotype, with a big cytoplasmatic vacuole being observed while the parasite was still alive. Studies of TEM and employing a protease inhibitor (3-methyladenine) suggested an autophagic phenotype for Bfx1 and Nf1 and a 'BigEye' phenotype for Fx1.
LIM kinases 1 (LIMK1) and 2 (LIMK2) are major regulators of cytoskeletal dynamics in the cell. LIMK regulates actin dynamics by phosphorylating the actin-depolymerizing factor (ADF)/cofilin family of ...actin-binding proteins. Cofilin proteins bind preferentially and cooperatively to ADP-bound subunits in F-actin. This binding event changes the helical rotation of actin filaments, promoting actin filament severing. LIMKs promote actin filament stabilization by inactivating cofilin through phosphorylation of Ser3. Phosphorylation of cofilin at Ser3 deactivates cofilin severing activity by inhibition of cofilin binding to actin filaments. This cycling between actin depolymerization and polymerization impacts higher-order cellular processes, including motility, differentiation, and metastasis.In the past 25 years, LIMK and cofilin have been heavily studied, but important questions remain regarding kinase regulation. Current literature proposes a model of regulation in which the N-terminus, which contains two LIM domains and one PDZ domain, acts as a negative regulator of the C-terminal kinase domain. These N-terminal domains, known to mediate protein-protein interactions, remain understudied in the context of LIMK autoregulation. Previous studies have mainly focused on immunoprecipitation and pull-down assays of fragments of the N-terminal domains to the C-terminus kinase domain. However, no structure of the domains LIM and PDZ is published, nor details about the autoregulated complex is known. Thus, how these domains modulate the kinase activity of LIMK has yet to be revealed. The information in this dissertation aims to provide the molecular mechanism and structural details underlying the regulation of LIMK1 activity. I hypothesize that the N-terminus of LIMK1 negatively regulates its kinase activity via a direct "head-to-tail” interaction. I will test this hypothesis using biochemical, biophysical, cell-based, and structural biology techniques to understand molecular mechanisms underlying autoregulation.I will accomplish the goals of this dissertation by setting two aims. In Aim 1, I study the N-terminal domains of LIMK. I use biochemical and structural techniques to gain a molecular-level understanding of the PDZ domains. Specifically, I obtain the crystal structure of the hLIMK2 PDZ domain and map the conservation of this domain using both LIMK1 and LIMK2 sequence alignments. I find a surface in this domain that is conserved from mammals to insects. I use homology- and structure-driven mutations to validate structure-defined and functional mechanisms of PDZ domain regulation. To test the effect of these mutations, I reconstructed the human LIMK pathway in S. cerevisiae. Expression of human LIMK1 phosphorylates and inactivates endogenous yeast cofilin; thus, I observe alterations in LIMK activity by measuring yeast growth. Using this assay, I screened for LIMK1 PDZ mutants that may be involved in kinase autoregulation. I have successfully used radiolabel assays to test the impact of these mutations on kinase activity using cofilin as substrate in vitro. This combination of approaches allowed me to understand better the influence of the PDZ domain in kinase autoregulation.In Aim 2, I used biochemical, biophysical, and activity-based assays to elucidate how the N-terminus domains of LIMK are responsible for autoregulatory interactions with the kinase domain and if LIMK is regulated in cis or trans. I began by directly addressing whether, in addition to the PDZ, other domains in the N-terminus of LIMK are responsible for kinase autoregulation. I found that the LIMK2 LIM2-PDZ domain fragment reduces the kinase activity of LIMK2 catalytic domain (CAT) in vitro. Furthermore, I used SEC-MALS to study the molecular arrangement of the LIM2-PDZ domains in solution.Additionally, I explore the molecular arrangement of full-length LIMK. I purify human full-length LIMK2 protein and use negative staining electron microscopy to observe global conformational changes between the wild-type protein and kinase-inactive D451N mutant to differentiate between intra or intermolecular conformations. Negative staining electron microscopy suggests two different conformations where the full-length wild-type LIMK2 displays an elongated conformation, while the full-length catalytically inactive D451N mutant shows a more compact conformation. These discoveries lead me to propose that the N-terminal domains are responsible for the autoregulation of LIMKs and that the mode of regulation is intramolecular.These findings provide a foundation for studying N-terminal autoregulation of LIMK kinase activity. Here, I present studies of autoregulatory interaction in LIMK in purified systems as well as in a eukaryotic system. This work provides the first crystal of the human LIMK2 PDZ domain and an in-depth study of its fold and conservation. Mutagenesis studies of the PDZ domain reported here provide strong evidence for how this domain undergoes autoregulation. Likewise, I provide insight into the molecular arrangement of LIMK N-terminus domains and full-length protein and provide a low-resolution understanding of its oligomeric state using SAXS and negative stain electron microscopy. Together, I propose that the LIM2- PDZ region of the N-terminus autoregulates LIMK activity.
Glutaric acid (GA) is a neurotoxic metabolite that accumulates in the CNS of patients with glutaric acidemia-I (GA-I), a neurometabolic disease caused by deficient activity of glutaryl-CoA ...dehydrogenase. Most GA-I patients display characteristic CNS lesions, mainly in the gray and white matter of basal ganglia and cerebral cortex. Neurons and astrocytes are believed to be vulnerable to millimolar concentrations of GA. However, little is known about the effects of GA on oligodendrocytes (OL) and the myelination process in the postnatal brain. Here, we show that a single intracerebroventricular administration of GA to rat neonatal pups induced a selective and long-lasting myelination failure in the striatum but no deleterious effect in the myelination of the corpus callosum. At 45 days post-GA injection, the myelinated area of striatal axonal bundles was decreased by 35 %, and the expression of myelin basic protein and myelin-associated glycoprotein (MAG) reduced by 25 and 60 %, respectively. This was accompanied by long lasting cytopathology features in MAG and CC-1-expressing OLs, which was confirmed by transmission electron microscopy. Remarkably, GA did not induce acute loss of pre-OLs in the striatum as assessed by NG2 or PDGFRα immunohistochemistry, suggesting an indirect and progressive mechanism for OL damage. In accordance, GA-induced white matter injury was restricted to the striatum and associated to GA-induced astrocytosis and neuronal loss. In conclusion, the current evidence indicates a pathogenic mechanism by which GA can permanently affect myelin status.
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