Neurotrophins are important signaling molecules in neuronal survival and differentiation. The precursor forms of neurotrophins (proneurotrophins) are the dominant form of gene products in animals, which are cleaved to generate prodomain and mature neurotrophins, and are sorted to constitutive or regulated secretory pathway and released. Brain-derived neurotrophic factor (BDNF) plays a pivotal role in the brain development and in the pathogenesis of neurological diseases. In Huntington’s disease, the defective transport of BDNF in cortical and striatal neurons and the highly expressed polyQ mutant huntingtin (Htt) result in the degeneration of striatal neurons. The underlying mechanism of BDNF transport and release is remains to be investigated. Current studies were conducted to identify the mechanisms of how BDNF is transported in axons post Golgi trafficking. By using affinity purification and 2D-DIGE assay, we show Huntingtin-associated protein 1 (HAP1) interacts with the prodomain and mature BDNF. The GST pull-down assays have addressed that HAP1 directly binds to the prodomain but not to mature BDNF and this binding is decreased by PolyQ Htt. HAP1 immunoprecipitation shows that less proBDNF is associated with HAP1 in the brain homogenate of Huntington’s disease compared to the control. Co-transfections of HAP1 and BDNF plasmids in PC12 cells show HAP1 is colocalized with proBDNF and the prodomain, but not mature BDNF. ProBDNF was accumulated in the proximal and distal segments of crushed sciatic nerve in wild type mice but not in HAP1-/- mice. The activity-dependent release of the prodomain of BDNF is abolished in HAP1-/- mice. We conclude that HAP1 is the cargo-carrying molecule for proBDNF-containing vesicles and plays an essential role in the transport and release of BDNF in neuronal cells. 20-30% of people have a valine to methionine mutation at codon 66 (Val66Met) in the prodomain BDNF, which results in the retardation of transport and release of BDNF, but the mechanism is not known. Here, GST-pull down assays demonstrate that HAP1 binds Val66Met prodomain with less efficiency than the wild type and PolyQ Htt further reduced the binding, but the PC12 cells colocalization rate is almost the same between wt prodomain/HAP1 and Val66Met prodomain/HAP1, suggesting that the mutation in the prodomain may reduce the release by impairing the cargo-carrying efficiency of HAP1, but the mutation does not disrupt the sorting process. Recent studies have shown that proneurotrophins bind p75NTR and sortilin with high affinity, and trigger apoptosis of neurons in vitro. Here, we show that proBDNF plays a role in the death of axotomized sensory neurons. ProBDNF, p75NTR and sortilin are highly expressed in DRG neurons. The recombinant proBDNF induces the dose-dependent death of PC12 cells and the death activity is completely abolished in the presence of antibodies against the prodomain of BDNF. The exogenous proBDNF enhances the death of axotomized sensory neurons and the antibodies to the prodomain or exogenous sortilin-extracellular domain-Fc fusion molecule reduces the death of axotomized sensory neurons. We conclude that proBDNF induces the death of sensory neurons in neonatal rats and the suppression of endogenous proBDNF rescued the death of axotomized sensory neurons.

Submitted by Nathália Faria da Silva (nathaliafsilva.ufv@gmail.com) on 2017-06-14T14:43:21Z No. of bitstreams: 1 resumo.pdf: 19226 bytes, checksum: 3311f50c2162c5bb897dc91d378af6b2 (MD5) / Made available in DSpace on 2017-06-14T14:43:21Z (GMT). No. of bitstreams: 1 resumo.pdf: 19226 bytes, checksum: 3311f50c2162c5bb897dc91d378af6b2 (MD5) Previous issue date: 2005-05-15 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Na busca de novos resultados para elucidar o papel de HAP1 e ROX1, que codificam um ativador do metabolismo oxidativo e um repressor do metabolismo oxidorredutivo, respectivamente, na levedura Crabtree negativa Kluyveromyces lactis cuja versatilidade metabólica pode ser explorada em vários campos da biotecnologia, inicialmente, foi identificado um gene ortólogo ROX1 à S. cerevisiae na seqüência genômica de K.lactis. O gene KlROX1 possui 40% de identidade daquele presente em S. cerevisiae e um motif característico do domínio HMG (High Mobility Group). Com base nessa seqüência uma linhagem mutante com deleção de ROX1 foi construída e confirmada. O fenótipo URA + e rox1 - dos transformantes obtidos, K.lactis ∆rox1::URA3, foram 100% estáveis sob condição seletiva. O gene putativo ROX1 de K.lactis teve a sua função em resposta ao oxigênio confirmada em culturas de K. lactis sob regime contínuo e desrepressão por glicose, pois, a deleção de ROX1 induziu a um aumento no nível do transcrito do gene hipóxico HEM13. A análise dos produtos do metabolismo permitiu inferir que a deleção do gene ROX1 em K. lactis aumentou a capacidade fermentativa dessa levedura sob aerobiose e de desrepressão catabólica. A investigação em culturas K. lactis ∆hap1 submetidas ao cultivo contínuo aeróbico sob desrepressão por glicose revelou um fenótipo relacionado ao metabolismo oxidorredutivo, ou seja, K. lactis ∆hap1 é mais fermentativa levando a diversidade de metabólitos em torno do piruvato. A proposta da via de regulação parcial negativa controlando a expressão de HEM13 foi confirmada nas culturas K. lactis. / The objective of this study was to search for new results in order to elucidate the role of HAP1 and ROX1, which codify an oxidative metabolism activator and an oxidoreductive metabolism repressor respectively, in the Crabtree-negative yeast Kluyveromyces lactis, whose metabolic versatility can be exploited in several biotechnology fields. Initially, a ROX1 gene orthologous to S. cerevisiae was identified in the genomic sequence of K. lactis. The KlROX1 gene has 40% identity with the one present in S. cerevisiae and a characteristic motif of the HMG (High Mobility Group) domain. Based on this sequence, a mutant line with ROX1 deletion was built and confirmed. The obtained transformant URA + and rox1 - phenotypes, K. lactis ∆rox1::URA3, were 100% stable under selective condition. The putative K. lactis ROX1 gene had its function in response to oxygen confirmed in cultures of K. lactis under continuous regime and glucose derepression, since ROX1 deletion induced an increase in the level of the HEM13 hypoxic gene transcript. The analysis of metabolism products allowed inferring that the deletion of gene ROX1 in K. lactis increased the yeast fermentative capacity under aerobic and catabolic derepression. The investigation in K. lactis ∆hap1 cultures under continuous aerobic cultivation and glucose derepressiom revealed a phenotype related to oxidoreductive metabolism, in other words, K. lactis ∆hap1 is more fermentative, leading to metabolite diversity around the piruvate. The proposal of the partial negative regulation pathway controlling the HEM13 expression was confirmed in K. lactis cultures.

<p dir="ltr"><i>Candida glabrata</i> is a member of the gut microbiota that can become an opportunistic pathogen under certain conditions. It is known for its inherent resistance to azole antifungal drugs and its ability to rapidly develop resistance during treatment. However, the regulatory mechanisms that enable this commensal organism to survive in low-oxygen environments, such as the gut, and to develop antifungal resistance when it becomes pathogenic, are not fully understood. In this study, we demonstrate for the first time the roles of two zinc cluster transcription factors in <i>C. glabrata</i>, Hap1A and Hap1B, in contributing to azole drug resistance in both laboratory strains and drug-resistant clinical isolates, adaptation to hypoxia, and resistance to other antifungal drugs like polyenes and echinocandins under specific conditions.</p><p dir="ltr">Azole drugs, which target the Erg11 protein, are widely used to treat <i>Candida</i> infections. The regulation of azole-induced <i>ERG</i> gene expression and activation of drug efflux pumps in <i>C. glabrata</i> has primarily been linked to the zinc cluster transcription factors Upc2A and Pdr1. Here, we investigated the roles of <i>S. cerevisiae</i> Hap1 orthologs, Hap1A and Hap1B, in <i>C. glabrata</i> as direct regulators of <i>ERG</i> genes upon azole exposure.</p><p dir="ltr">Our research shows that deleting <i>HAP1</i> in the yeast model <i>S. cerevisiae</i> increases sensitivity to fluconazole due to the failure to induce <i>ERG11 </i>expression in the <i>hap1Δ</i> mutant compared to the wild-type strain. Although <i>C. glabrata</i> is closely related to <i>S. cerevisiae</i>, a whole genome duplication (WGD) event allowed <i>C. glabrata</i> to retain two HAP1 ohnologs, while <i>S. cerevisiae</i> lost one copy. Through phylogenetic and syntenic analyses, we identified Hap1A and Hap1B in <i>C. glabrata</i> as ohnologs of Hap1 in <i>S. cerevisiae</i>, which is known to regulate <i>ERG</i> gene expression under both aerobic and hypoxic conditions. Interestingly, deleting <i>HAP1B</i> in <i>C. glabrata</i> increased sensitivity to both triazole and imidazole drugs, similar to Hap1 in <i>S. cerevisiae</i>, while deleting <i>HAP1A </i>did not affect azole sensitivity.</p><p dir="ltr">Gene expression analysis revealed that the increased azole sensitivity in the <i>hap1BΔ </i>strain was due to reduced azole-induced <i>ERG</i> gene expression, leading to lower total endogenous ergosterol levels. Additionally, the loss of <i>HAP1B</i> in <i>C. glabrata</i> clinical isolates like SM1 and BG2, as well as in drug-resistant strains like SM3, also led to increased azole hypersusceptibility. While it was already known that losing <i>UPC2A</i> in <i>C. glabrata</i> increases azole sensitivity, our study is the first to demonstrate that the combined loss of both <i>HAP1B </i>and <i>UPC2A</i> makes <i>C. glabrata</i> strains even more sensitive to azoles than losing either gene alone. Additionally, we show that the loss of both <i>HAP1B </i>and the H3K4 histone methyltransferase <i>SET1</i> increases azole hypersensitivity more than the loss of either gene alone.</p><p dir="ltr">Interestingly, the Hap1A protein is barely detectable under aerobic conditions but is specifically induced under hypoxia, where it plays a crucial role in repressing <i>ERG</i> genes. In the absence of Hap1A, Hap1B compensates by acting as a transcriptional repressor. Our RNA sequencing analysis further showed that losing both <i>HAP1A</i> and <i>HAP1B</i> not only affects genes in the ergosterol biosynthesis pathway but also upregulates iron transport-related genes <i>FET3 </i>and <i>FTR1</i>. Moreover, we found that the hypoxic growth defect caused by the loss of both <i>HAP1A</i> and <i>HAP1B</i> is exacerbated when treated with the echinocandin caspofungin and the cell wall-damaging agent calcofluor white, indicating that these Hap1 ohnologs contribute to maintaining cell wall integrity under hypoxic conditions. Since <i>HAP1A</i> transcript levels remain stable under aerobic conditions, we suspect that Hap1A expression is regulated post-transcriptionally.</p><p dir="ltr">Furthermore, we discovered that the simultaneous loss of both HAP1A and HAP1B leads to increased hypersensitivity to the polyene antifungal drug amphotericin B, though the exact mechanism behind this phenotype remains unclear. Altogether, our study is the first to show that Hap1A and Hap1B have evolved distinct roles, enabling <i>C. glabrata</i> to adapt to specific host and environmental conditions.</p>