Vera Paschon

Blocking of Connexin-Mediated Communication Promotes Neuroprotection during Acute Degeneration Induced by Mechanical Trauma

Accruing evidence indicates that connexin (Cx) channels in the gap junctions (GJ) are involved in neurodegeneration after injury. However, studies using KO animal models endowed apparently contradictory results in relation to the role of coupling in neuroprotection. We analyzed the role of Cx-mediated communication in a focal lesion induced by mechanical trauma of the retina, a model that allows spatial and temporal definition of the lesion with high reproducibility, permitting visualization of the focus, penumbra and adjacent areas. Cx36 and Cx43 exhibited distinct gene expression and protein levels throughout the neurodegeneration progress. Cx36 was observed close to TUNEL-positive nuclei, revealing the presence of this protein surrounding apoptotic cells. The functional role of cell coupling was assessed employing GJ blockers and openers combined with lactate dehydrogenase (LDH) assay, a direct method for evaluating cell death/viability. Carbenoxolone (CBX), a broad-spectrum GJ blocker, reduced LDH release after 4 hours, whereas quinine, a Cx36-channel specific blocker, decreased LDH release as early as 1 hour after lesion. Furthermore, analysis of dying cell distribution confirmed that the use of GJ blockers reduced apoptosis spread. Accordingly, blockade of GJ communication during neurodegeneration with quinine, but not CBX, caused downregulation of initial and effector caspases. To summarize, we observed specific changes in Cx gene expression and protein distribution during the progress of retinal degeneration, indicating the participation of these elements in acute neurodegeneration processes. More importantly, our results revealed that direct control of GJ channels permeability may take part in reliable neuroprotection strategies aimed to rapid, fast treatment of mechanical trauma in the retina.

Interplay Between Exosomes, microRNAs and Toll-Like Receptors in Brain Disorders

Extracellular vesicles (EVs), including exosomes, microvesicles and apoptotic bodies, participate in intercellular communication, and particularly, in paracrine and endocrine signalling. The EVs and their specific contents have been considered hallmarks of different diseases. It has been recently discovered that EVs can co-transport nucleic acids such as DNAs, ribosomal RNAs, circular RNAs (circRNAs), long noncoding RNAs (lnRNAs) and microRNAs (miRNAs). miRNAs are important regulators of gene expression at the post-transcriptional level, although they may also play other roles. Recent evidence supports the hypothesis that miRNAs can activate Toll-like receptors (TLRs) under certain circumstances. TLRs belong to a multigene family of immune system receptors and have been recently described in the nervous system. In the immune system, TLRs are important for the recognition of the invading microorganisms, whereas in the nervous system, they recognise endogenous ligands released by undifferentiated or necrotic/injured cells. In the neuronal disease field, TLRs activity has been associated with amyotrophic lateral sclerosis (ALS), stroke, Alzheimer’s and Parkinson’s disease. Herein, we reviewed the current knowledge of the relationship between miRNA release by EVs and the inflammation signalling triggered by TLRs in neighbouring cells or during long-distance cell-to-cell communication. We highlight novel aspects of this communication mechanism, offering a valuable insight into such pathways in health and disease.

Connexin36, an essential element in the rod pathway, is highly expressed in the essentially rodless retina of Gallus gallus

Electrical coupling provided by connexins (Cx) in gap junctions (GJ) plays important roles in both the developing and the mature retina. In mammalian nocturnal species, Cx36 is an essential component in the rod pathway, the retinal circuit specialized for night, scotopic vision. Here, we report the expression of Cx36 in a species (Gallus gallus) that phylogenetic development endows with an essentially rodless retina. Cx36 gene is very highly expressed in comparison with other Cxs previously described in the adult retina, such as Cx43, Cx45, and Cx50. Moreover, real‐time PCR, Western blot, and immunofluorescence all revealed that Cx36 expression massively increased over time during development. We thoroughly examined Cx36 in the inner and outer plexiform layers, where this protein was particularly abundant. Cx36 was observed mainly in the off sublamina of the inner plexiform layer rather than in the on sublamina previously described in the mammalian retina. In addition, Cx36 colocalized with specific cell markers, revealing the expression of this protein in distinct amacrine cells. To investigate further the involvement of Cx36 in visual processing, we examined its functional regulation in retinas from dark‐adapted animals. Light deprivation markedly up‐regulates Cx36 gene expression in the retina, resulting in an increased accumulation of the protein within and between cone synaptic terminals. In summary, the developmental regulation of Cx36 expression results in particular circuitry‐related roles in the chick retina. Moreover, this study demonstrated that Cx36 onto‐ and phylogenesis in the vertebrate retina simultaneously exhibit similarities and particularities. J. Comp. Neurol. 512:651–663, 2009.

Lack of photoreceptor signaling alters the expression of specific synaptic proteins in the retina.

Synaptic modulation by activity-dependent changes constitutes a cellular mechanism for neuronal plasticity. However, it is not clear how the complete lack of neuronal signaling specifically affects elements involved in the communication between neurons. In the retina, it is now well established that both chemical and electrical synapses are essential to mediate the transmission of visual signaling triggered by the photoreceptors. In this study, we compared the expression of synaptic proteins in the retinas of wild-type (WT) vs. rd/rd mice, an animal model that displays inherited and specific ablation of photoreceptors caused by a mutation in the gene encoding the beta-subunit of rod cGMP-phosphodiesterase (Pde6brd1). We specifically examined the expression of connexins (Cx), the proteins that form the gap junction channels of electrical synapses, in addition to synaptophysin and synapsin I, which are involved in the release of neurotransmitters at chemical synapses. Our results revealed that Cx36 gene expression levels are lower in the retinas of rd/rd when compared with WT. Confocal analysis indicated that Cx36 immunolabeling almost disappeared in the outer plexiform layer without significant changes in protein distribution within the inner plexiform layer of rd/rd retinas. Likewise, synaptophysin expression remarkably decreased in the outer plexiform layer of rd/rd retinas, and this down-regulation was also associated with diminished transcript levels. Furthermore, we observed down-regulation of Cx57 gene expression in rd/rd retinas when compared with WT and also changes in protein distribution. Interestingly, Cx45 and synapsin I expression in rd/rd retinas showed no noticeable changes when compared with WT. Taken together, our results revealed that the loss of photoreceptors leads to decreased expression of some synaptic proteins. More importantly, this study provides evidence that neuronal activity regulates, but is not essential to maintain, the expression of synaptic elements.

Connexin-mediated communication controls cell proliferation and is essential in retinal histogenesis

Connexin (Cx) channels and hemichannels are involved in essential processes during nervous system development such as apoptosis, propagation of spontaneous activity and interkinetic nuclear movement. In the first part of this study, we extensively characterized Cx gene and protein expression during retinal histogenesis. We observed distinct spatio‐temporal patterns among studied Cx and an overriding, ubiquitous presence of Cx45 in progenitor cells. The role of Cx‐mediated communication was assessed by using broad‐spectrum (carbenoxolone, CBX) and Cx36/Cx50 channel‐specific (quinine) blockers. In vivo application of CBX, but not quinine, caused remarkable reduction in retinal thickness, suggesting changes in cell proliferation/apoptosis ratio. Indeed, we observed a decreased number of mitotic cells in CBX‐injected retinas, with no significant changes in the expression of PCNA, a marker for cells in proliferative state. Taken together, our results pointed a pivotal role of Cx45 in the developing retina. Moreover, this study revealed that Cx‐mediated communication is essential in retinal histogenesis, particularly in the control of cell proliferation.

Differential expression of connexins during histogenesis of the chick retina

Gap junction (GJ) channels couple adjacent cells, allowing transfer of second messengers, ions, and molecules up to 1 kDa. These channels are composed by a multigene family of integral membrane proteins called connexins (Cx). In the retina, besides being essential circuit element in the visual processing, GJ channels also play important roles during its development. Herein, we analyzed Cx43, Cx45, Cx50, and Cx56 expression during chick retinal histogenesis. Cx exhibited distinct expression profiles during retinal development, except for Cx56, whose expression was not detected. Cx43 immunolabeling was observed at early development, in the transition of ventricular zone and pigmented epithelium. Later, Cx43 was seen in the outer plexiform and ganglion cell layers, and afterwards also in the inner plexiform layer. We observed remarkable changes in the phosphorylation status of this protein, which indicated modifications in functional properties of this Cx during retinal histogenesis. By contrast, Cx45 showed stable gene expression levels throughout development and ubiquitous immunoreactivity in progenitor cells. From later embryonic development, Cx45 was mainly observed in the inner retina, and it was expressed by glial cells and neurons. In turn, Cx50 was virtually absent in the chick retina at initial embryonic phases. Combination of PCR, immunohistochemistry and Western blot indicated that this Cx was present in differentiated cells, arising in parallel with the formation of the visual circuitry. Characterization of Cx expression in the developing chick retina indicated particular roles for these proteins and revealed similarities and differences when compared to other species.

A New and Reliable Guide for Studies of Neuronal Loss Based on Focal Lesions and Combinations of In Vivo and In Vitro Approaches

In this study, we describe a simple and reliable method to study neuroprotective effects in living and organized neural tissue. This method, which was based on retinal explants for in vivo focal lesions, was conceived as a collection of modular procedures, which can be customized for particular demands. With this model, it is possible to combine immunohistochemistry with image data analysis to track the two- or three-dimensional redistribution of proteins as a time/space function of primary cell loss. At the same time, it is possible to finely control the exposure of the tissue to specific drugs and molecules. In order to illustrate the use of the proposed method, we tested the effects of two different nanotube compounds on retinal explant viability. Transcriptome analyses can be separately performed in the lesion focus and penumbra with laser capture microdissection followed by polymerase chain reaction analyses. In addition, other common experimental drawbacks, such as high individual variance, are eliminated. With intraocular injections, treatments can be verified in vivo, with one eye serving as the experimental tissue and the other serving as the control tissue. In summary, we describe a flexible and easy method, which can be useful in combination with a broad variety of recently developed neuroprotective strategies, to study neurodegeneration.

APAGAR A MEMÓRIA: Verdade ou Ficção?

APAGAR A MEMORIA: Verdade ou Ficcao?Luiz Fernando Rodrigues Gall, Vera Paschon, Alexandre Hiroaki KiharaLaboratorio de Neurogenetica / Nucleo de Cognicao e Sistemas Complexos / Centro de MatematicaComputacao e Cognicao / Universidade Federal do ABCVol. 1, N. 12, 03 de Junho de 2014DOI: http://dx.doi.org/10.15729/nanocellnews.2014.06.03.004Uma possibilidade abordada frequentemente na ficcao cientifica e o processo de apagar a memoria de umindividuo, seja por equipamentos sofisticados, drogas ou hipnose. Mas seria tal possibilidade apenas ficcao?Seria possivel de fato apagar algum evento da memoria de um individuo? Esse assunto tem recebido grandeatencao da comunidade cientifica e levanta discussoes sobre a natureza da memoria. Existem diversosparadigmas, ou modos como se entende o que e a memoria. O paradigma utilizado em um estudo conduzidopor pesquisadores britânicos divide o processo de memorizacao em tres etapas: a memoria sensorial, a de curtoprazo e a de longo prazo, cada qual com suas funcoes e caracteristicas distintas (Figura 1). De acordo comevidencias encontradas e de fato possivel apagar informacoes da memoria de curto prazo, porem ainda esta emdebate se o mesmo e possivel na memoria de longo prazo.Figura 1: Modelo de memoria de curto prazo: a informacao e processada inicialmente no nivel sensorial (oumemoria sensorial) e posteriormente pela memoria de curto prazo. Apos estas etapas, a informacao pode seresquecida ou consolidada na memoria de longo prazo.Estes pesquisadores realizaram um experimento em que individuos foram apresentados a um cenario virtual. Ocenario era uma oficina contendo diversas ferramentas (martelo, furadeira, etc.) que apareciam e desapareciam.Os individuos foram instruidos a sinalizar quando um objeto aparecesse, e manter a atencao fixa nele ate quedesaparecesse e aparecesse um novo objeto. Em cada teste, foram apresentados de 6 a 10 objetos pareados.Para ter certeza de que os individuos fixavam os objetos, seus olhos foram monitorados por uma câmera ocular.Ao final do experimento realizou-se um novo teste discriminatorio checando a memoria em relacao a algumobjeto presente durante o teste. Para indicar qual objeto apareceria, a apresentacao deste era seguida de umsom. Um som agudo indicava um possivel objeto a ser checado no final e um som grave um menos provavel.Quando o som agudo aparecia duas vezes surgia uma situacao em que o individuo passava a priorizar osegundo objeto e o primeiro era “apagado” da memoria. Os cientistas perceberam que a memoria dos objetos“apagados” era similar a memoria dos objetos que nao foram sinalizados, enquanto os mais sinalizados foram“mais lembrados” que os restantes. Assim, concluiu-se que quando um objeto era julgado irrelevante pelapessoa, este era descartado da memoria de curto prazo, mesmo que ja tivesse sido sinalizado como importanteanteriormente.

TERAPIA GÊNICA RECUPERA MEMÓRIA DE RATOS COM ALZHEIMER: um novo passo em direção à cura.

A doenca de Alzheimer foi descrita pelo psiquiatra alemao Alois Alzheimer. Em 1907, Alois publicou um artigo apresentando as caracteristicas clinicas de um caso bastante peculiar (1)(2). Apos cinco anos a “doenca descrita por Alzheimer” passou a ser usada para os casos de demencia que aconteciam antes dos 65 anos com sintomas como, perda de memoria, linguagem e habilidade de cuidar de si mesmo. O diagnostico para esta doenca foi proposto apenas por volta de 1984 e a partir de entao as pesquisas e atividades de atendimento clinico foram evoluindo (3).