Coronal brain atlas in stereotaxic coordinates of the African spiny mouse, Acomys cahirinus.

The African spiny mouse (Acomys cahirinus) is an emerging model of mammalian epimorphic regeneration that has aroused the interest of the scientific community in the last decade. To date, studies on brain repair have been hindered by the lack of knowledge on the neuroanatomy of this species. Here, we present a coronal brain atlas in stereotaxic coordinates, which allows for three-dimensional identification and localization of the brain structures of this species. The brain of 12-week-old spiny mice was mapped in stereotaxic coordinates using cresyl violet-stained brain sections obtained from coronal cryosectioning of the brain after transcardial perfusion with fixative. The atlas is presented in 42 plates representing sections spaced 240 µm apart. Stereotaxic coordinates were validated using both a model of Parkinsonian lesion of the striatum with 6-hydroxydopamine and labeling of the corticospinal tract in the spiny mouse spinal cord using AAV1/2-GFP intracortical injections. This work presents a new tool in A. cahirinus neurobiology and opens new avenues of research for the investigation of the regenerative ability of A. cahirinus in models of brain disorders.

Identification of new targets of S-nitrosylation in neural stem cells by thiol redox proteomics.

Nitric oxide (NO) is well established as a regulator of neurogenesis. NO increases the proliferation of neural stem cells (NSC), and is essential for hippocampal injury-induced neurogenesis following an excitotoxic lesion. One of the mechanisms underlying non-classical NO cell signaling is protein S-nitrosylation. This post-translational modification consists in the formation of a nitrosothiol group (R-SNO) in cysteine residues, which can promote formation of other oxidative modifications in those cysteine residues. S-nitrosylation can regulate many physiological processes, including neuronal plasticity and neurogenesis. In this work, we aimed to identify S-nitrosylation targets of NO that could participate in neurogenesis. In NSC, we identified a group of proteins oxidatively modified using complementary techniques of thiol redox proteomics. S-nitrosylation of some of these proteins was confirmed and validated in a seizure mouse model of hippocampal injury and in cultured hippocampal stem cells. The identified S-nitrosylated proteins are involved in the ERK/MAPK pathway and may be important targets of NO to enhance the proliferation of NSC.

S-Nitrosylation of Ras Mediates Nitric Oxide-Dependent Post-Injury Neurogenesis in a Seizure Model.

AIMS: Nitric oxide (NO) is involved in the upregulation of endogenous neurogenesis in the subventricular zone and in the hippocampus after injury. One of the main neurogenic pathways activated by NO is the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway, downstream of the epidermal growth factor receptor. However, the mechanism by which NO stimulates cell proliferation through activation of the ERK/MAPK pathway remains unknown, although p21Ras seems to be one of the earliest targets of NO. Here, we aimed at studying the possible neurogenic action of NO by post-translational modification of p21Ras as a relevant target for early neurogenic events promoted by NO in neural stem cells (NSCs). RESULTS: We show that NO caused S-nitrosylation (SNO) of p21Ras in Cys118, which triggered downstream activation of the ERK/MAPK pathway and proliferation of NSC. Moreover, in cells overexpressing a mutant Ras in which Cys118 was replaced by a serine-C118S-, cells were insensitive to NO, and no increase in SNO, in ERK phosphorylation, or in cell proliferation was observed. We also show that, after seizures, in the presence of NO derived from inducible nitric oxide synthase, there was an increase in p21Ras cysteine modification that was concomitant with the previously described stimulation of proliferation in the dentate gyrus. INNOVATION: Our work identifies p21Ras and its SNO as an early target of NO during signaling events that lead to NSC proliferation and neurogenesis. CONCLUSION: Our data highlight Ras SNO as an early event leading to NSC proliferation, and they may provide a target for NO-induced stimulation of neurogenesis with implications for brain repair. Antioxid. Redox Signal. 28, 15-30.

Calpastatin Overexpression Preserves Cognitive Function Following Seizures, While Maintaining Post-Injury Neurogenesis.

In the adult mammalian brain, new neurons continue to be produced throughout life in two main regions in the brain, the subgranular zone (SGZ) in the hippocampus and the subventricular zone in the walls of the lateral ventricles. Neural stem cells (NSCs) proliferate in these niches, and migrate as neuroblasts, to further differentiate in locations where new neurons are needed, either in normal or pathological conditions. However, the endogenous attempt of brain repair is not very efficient. Calpains are proteases known to be involved in neuronal damage and in cell proliferation, migration and differentiation of several cell types, though their effects on neurogenesis are not well known. Previous work by our group has shown that the absence of calpastatin (CAST), the endogenous inhibitor of calpains, impairs early stages of neurogenesis. Since the hippocampus is highly associated with learning and memory, we aimed to evaluate whether calpain inhibition would help improve cognitive recovery after lesion and efficiency of post-injury neurogenesis in this region. For that purpose, we used the kainic acid (KA) model of seizure-induced hippocampal lesion and mice overexpressing CAST. Selected cognitive tests were performed on the 3rd and 8th week after KA-induced lesion, and cell proliferation, migration and differentiation in the dentate gyrus (DG) of the hippocampus of adult mice were analyzed using specific markers. Cognitive recovery was evaluated by testing the animals for recognition, spatial and associative learning and memory. Cognitive function was preserved by CAST overexpression following seizures, while modulation of post-injury neurogenesis was similar to wild type (WT) mice. Calpain inhibition could still be potentially able to prevent the impairment in the formation of new neurons, given that the levels of calpain activity could be reduced under a certain threshold and other harmful effects from the pathological environment could also be controlled.

Perceção de autoeficácia dos pais para cuidar da criança com imobilização gessada no domicílio após a alta / Perceived self-efficacy of parents to care for the child with cast immobilization at home after discharge

O regresso a casa após hospitalização é atualmente um desafio, podendo ser influenciado pela perceção de autoeficácia dos pais ? a crença de cada um na sua capacidade para realizar com sucesso determinada atividade ? especialmente em casos como o tratamento da doença ortopédica. A perceção da capacidade para cuidar pode ser influenciada por uma boa preparação da alta, atendendo às necessidades e individualidades de cada família. O objetivo do estudo foi conhecer a perceção dos pais sobre a preparação do regresso a casa da criança com imobilização gessada e a sua autoeficácia percecionada para cuidar da criança no momento da alta e um mês após a mesma. Optou-se por um estudo quantitativo de cariz descritivo correlacional e uma vertente quase experimental. Utilizou-se, como instrumento de colheita de dados, um questionário composto por duas partes: a primeira parte, preenchida no momento da alta, constituída pelo questionário de caraterização da amostra, pelo ?Questionário sobre preparação para a alta?, pelo ?Questionário de perceção de autoeficácia para cuidar da criança/adolescente com gesso no momento da alta? e pela EAGP na versão portuguesa; a segunda parte, constituída pelo ?Questionário de perceção de autoeficácia para cuidar da criança/adolescente com gesso no domicílio após a alta?, preenchida e devolvida um mês após a alta. A colheita de dados decorreu entre novembro de 2015 e maio de 2016. A amostra foi constituída por um grupo de 51 pais. Os resultados evidenciam que a preparação para a alta foi entendida como positiva pelos pais. A autoeficácia percecionada pelos pais no momento da alta para cuidarem dos seus filhos com gesso mostrou-se elevada, mantendo-se igualmente elevada um mês após a alta. Verificou-se existir uma relação entre a autoeficácia e as variáveis nível socioeconómico e tipo de gesso da criança, não se tendo identificado correlação entre a autoeficácia e o tempo de internamento. Deste modo, organizar um plano seguro de preparação para a alta, atendendo à perceção de autoeficácia de cada família poderá ser um elemento essencial para evitar complicações e assegurar uma recuperação efetiva da criança no domicílio.

Differential contribution of the guanylyl cyclase-cyclic GMP-protein kinase G pathway to the proliferation of neural stem cells stimulated by nitric oxide.

Nitric oxide (NO) is an important inflammatory mediator involved in the initial boost in the proliferation of neural stem cells following brain injury. However, the mechanisms underlying the proliferative effect of NO are still unclear. The aim of this work was to investigate whether cyclic GMP (cGMP) and the cGMP-dependent kinase (PKG) are involved in the proliferative effect triggered by NO in neural stem cells. For this purpose, cultures of neural stem cells isolated from the mouse subventricular zone (SVZ) were used. We observed that long-term exposure to the NO donor (24 h), NOC-18, increased the proliferation of SVZ cells in a cGMP-dependent manner, since the guanylate cyclase inhibitor, ODQ, prevented cell proliferation. Similarly to NOC-18, the cGMP analogue, 8-Br-cGMP, also increased cell proliferation. Interestingly, shorter exposures to NO (6 h) increased cell proliferation in a cGMP-independent manner via the ERK/MAP kinase pathway. The selective inhibitor of PKG, KT5823, prevented the proliferative effect induced by NO at 24 h but not at 6 h. In conclusion, the proliferative effect of NO is initially mediated by the ERK/MAPK pathway, and at later stages by the GC/cGMP/PKG pathway. Thus, our work shows that NO induces neural stem cell proliferation by targeting these two pathways in a biphasic manner.

Comparative proteomic analysis of auxin-induced embryogenic and nonembryogenic tissues of the solanaceous tree Cyphomandra betacea (Tamarillo).

Cyphomandra betacea (tamarillo) is a tree that produces edible, highly nutritional fruits. In tamarillo, somatic embryogenesis (SE) is achieved through a two-step process starting with the formation of an embryogenic tissue on an auxin-rich medium and further development of embryos, following tissue transfer to an auxin-free medium. During the induction stage, both embryogenic (EC) and nonembryogenic calli (NEC) arise from the same explant (immature leaves or mature zygotic embryos) in the presence of either picloram or 2,4-dichlorophenoxyacetic acid. In an attempt to find somatic embryogenic-specific proteins, a comparative analysis of the proteome of tamarillo's EC and NEC was performed. Analysis of 2-DE gels revealed ca. 150 differentially expressed proteins, from which 22 have been identified by LC-MS/MS. Proteins exclusively or predominantly expressed in EC included metabolism-related proteins, such as enolases or treonine synthases, and also heat-shock and ribosomal proteins. Pathogenesis-related proteins were found mainly in NEC. A number of additional differentially expressed proteins involved in various functional categories were also identified. A quantitative real time PCR (qPCR) analysis revealed no significant differences at the mRNA level for 11 differentially expressed proteins, with exception of the pathogenesis-related proteins that were up-regulated in NEC. This seems to indicate that a posttranscriptional control might be responsible for the proteomic differences detected.