In S. cerevisiae, the accumulation of dysfunctional mitochondria activates a retrograde signal that is mediated through multiple cytosolic regulators. Central to activation is the cytosolic regulator Rtg2p which through its interaction with Mks1p, promotes the nuclear translocation of Rtg1p/3p. Nuclear localized Rtg1p/3p promotes transcription of target genes. Prior work has shown Rtg2p interaction with Mks1p is required for downstream signaling, however the Mks1p binding site within Rtg2p is unknown. To identify this motif, random mutations were generated in RTG2 and a red-white screening strategy was used to assess 14,001 clones. Sequence analysis identified four mutants with amino acid mutations in the carboxy-terminus of Rtg2p that gave rise to defects in CIT2 transcription and loss of Mks1p interaction. Relative to RTG2, all mutants had reduced Rtg2p protein half-lives. Together these results suggest that the carboxy-terminal domain of Rtg2p is essential for retrograde signaling as it may contain the Mks1p binding site.

Changes in gene expression in response to mitochondrial dysfunction are mediated by components of the retrograde signaling pathway. The mitochondrial signal is recognized and transferred to the nucleus by dynamic interactions between regulatory proteins Rtg2p, Mks1p and Bmh1p. Retrograde signaling genes have been well characterized in the budding yeast Saccharomyces cerevisiae but very little is known about the retrograde response of other fungi. To identify retrograde signaling proteins in other fungi, the protein sequence encoded by the S. cerevisiae RTG2 gene was used to search for fungal homologs using NCBI BlastP and the T-Coffee Multiple Sequence Alignment program. We selected four species having uncharacterized ORFs with more than 66% amino acid identity to Rtg2p for further analysis: Ashbya gossypii, Candida glabrata, Vanderwaltozyma polyspora and Kluyveromyces lactis. In S. cerevisiae, cells deleted for RTG2 are glutamate auxotrophs, and have reduced expression of Aco1p and Cit2p proteins. To determine whether the putative RTG2 genes we identified encode bonefide regulators of the retrograde response pathway, we used standard yeast genetic approaches and molecular biology tools to investigate their ability to complement the defects associated with the rtg2Ä mutant using our S. cerevisiae RTG2 shuffle strain. We investigated functional roles of Rtg2p homologs by comparing Cit2p and Aco1p protein levels, glutamate auxotrophy, as well as analyzing the interaction between Rtg2p homologs and Mks1p. We also analyzed sensitivity of mutant strains under various stress conditions to address possible signaling cross talk between the retrograde signaling pathway and the TOR pathway. Our data show that the fungal Rtg2p homologs from C. glabrata, V. polyspora and K. lactis are functional in mediating the mitochondrial signal through known components of the retrograde signaling cascade. Our immunoprecipitation data suggest that TOR and retrograde signaling may exhibit cross pathway activation under rapamycin treatment. We show that Mks1p, the negative regulator of retrograde signaling pathway is required for Cit2p expression under rapamycin treatment. Given that all Rtg2p homologs showed low affinity for Mks1p which was in turn paralleled by a higher affinity of Mks1p for Bmh1p suggests that Rtg2p may have an additional functional role in influencing the association of Mks1p with Bmh1p.

A sinalização retrógrada mitocondrial é uma via de comunicação entre a mitocôndria e o núcleo que regula a expressão de uma série de genes nucleares que codificam proteínas mitocondriais, em resposta a disfunções mitocondriais. Em Saccharomyces cerevisiae, a via depende de Rtg1p e Rtg3p, que juntos formam o fator de transcrição que regula a expressão gênica, e de Rtg2p, um ativador da via. Aqui, nós mostramos novos estudos direcionados à investigação do impacto da sinalização retrógrada RTG-dependente na fisiologia mitocondrial. Verificamos que mutantes incapazes de realizar sinalização retrógrada RTG-dependente apresentam consumo de oxigênio mais elevado e menor produção de peróxido de hidrogênio em fase estacionária quando comparados a células selvagens. Interessantemente, mutantes RTG são menos capazes de decompor peróxido de hidrogênio assim como manter-se viáveis quando desafiados com peróxido. Nossos resultados indicam que a sinalização por RTG está envolvida na indução hormética de defesas antioxidantes e de resistência a estresse, função ainda não descrita para este sistema. / Mitochondrial retrograde signaling is a communication pathway between the mitochondrion and the nucleus which regulates the expression of a subset of nuclear genes that codify mitochondrial proteins, mediating cell response to mitochondrial dysfunction. In Saccharomyces cerevisiae, the pathway depends on Rtg1p and Rtg3p, which together form the transcription factor that regulates gene expression, and Rtg2p, an activator of the pathway. Here, we provide novel studies aimed at assessing the functional impact of the lack of RTG-dependent signaling on mitochondrial activity. We show that mutants defective in RTG-dependent retrograde signaling present higher oxygen consumption and reduced hydrogen peroxide release in the stationary phase when compared to wild type cells. Interestingly, RTG mutants are less able to decompose hydrogen peroxide as well as maintain viability when challenged with hydrogen peroxide. Overall, our results indicate that RTG signaling is involved in the hormetic induction of antioxidant defenses and stress resistance, a function of this system not yet described.

Rtg2p é uma proteína que participa da sinalização retrógrada, uma via de comunicação da mitocôndria para o núcleo; também tem sido associada com a longevidade em S. cerevisiae. O objetivo deste trabalho foi identificar os determinantes estruturais de Rtg2p, envolvidos na sinalização retrógrada e no envelhecimento. Para isto foram produzidos treze mutantes pontuais a partir do desenho racional por decomposição de redes de correlação de aminoácidos (DRCN). Analisaram-se as cepas mutantes por ensaio de auxotrofia para glutamato, expressão do gene CIT2 e ensaio de longevidade replicativa. Em sua grande maioria as mutações realizadas causaram perturbações nas funções de Rtg2p, com destaque para as cepas E106A, R109E, E137A, T138A e D158A, que apresentaram longevidade igual à da cepa rtg2Δ, com apenas uma mutação pontual. Em conclusão, os resultados obtidos demonstram que o domínio N-terminal é muito importante para a função de Rtg2p, e indicam que existem determinantes estruturais que controlam a longevidade de forma dependente ou independente da resposta retrógrada. / Rtg2p is a protein involved in the retrograde signaling, a pathway of communcation from mitochondria to nucleus; also has been associated with longevity in S. cerevisiae. The goal of this study was to identify the structural determinants of Rtg2p, controlling the function of this protein in retrograde response and aging. For this purpose thirteen point mutants were produced by site-directed mutagenesis, using rational design by decomposition of residues correlation networks (DRCN). The strains was analyzed by glutamate auxotrophy, CIT2 gene expression and replicative life span assays. For the most of performed mutations, generated inactivation to Rtg2p functions, highlighting to R109E, E137A, T138A, and D158A showed longevity equal to rtg2Δ strain, even with a single amino acid change. In conclusion, our results demonstrate that the N-terminal domain is very important to the function of Rtg2p and also show there are structural determinants in Rtg2p that control longevity in both dependent or independent manner of the communication between mitochondria and nucleus.

The neuromuscular junction (nmj) is a commonly studied synapse, used often to investigate reciprocal signaling between a motor neuron and the appropriate target muscle. In Caenorhabditis elegans, ectopic nmjs can be created by eliminating selected embryonic muscle cells that act as guideposts for the migration of post-embryonic muscles. The ectopic muscles are required to induce sprouting from DD motor neurons, indicating the presence of a muscle derived signaling molecule that interacts with the neurons. A TGF-β homolog, unc-129, is reported to be transiently expressed in the dorsal body wall muscles. The timing of the expression of TGF-β coincides with the time that the DD motor neurons respecify their synapses. In this study, we show that TGF-β is expressed by the ectopic muscle and that in unc-129 mutant animals, the ectopic muscle is unable to induce sprouting from the DD motor neurons. Therefore, we conclude that TGF-β is necessary for ectopic nmj formation in C.elegans.

A sinalização retrógrada mitocondrial é uma via de comunicação entre a mitocôndria e o núcleo que regula a expressão de uma série de genes nucleares que codificam proteínas mitocondriais, em resposta a disfunções mitocondriais. Em Saccharomyces cerevisiae, a via depende de Rtg1p e Rtg3p, que juntos formam o fator de transcrição que regula a expressão gênica, e de Rtg2p, um ativador da via. Aqui, nós mostramos novos estudos direcionados à investigação do impacto da sinalização retrógrada RTG-dependente na fisiologia mitocondrial. Verificamos que mutantes incapazes de realizar sinalização retrógrada RTG-dependente apresentam consumo de oxigênio mais elevado e menor produção de peróxido de hidrogênio em fase estacionária quando comparados a células selvagens. Interessantemente, mutantes RTG são menos capazes de decompor peróxido de hidrogênio assim como manter-se viáveis quando desafiados com peróxido. Nossos resultados indicam que a sinalização por RTG está envolvida na indução hormética de defesas antioxidantes e de resistência a estresse, função ainda não descrita para este sistema. / Mitochondrial retrograde signaling is a communication pathway between the mitochondrion and the nucleus which regulates the expression of a subset of nuclear genes that codify mitochondrial proteins, mediating cell response to mitochondrial dysfunction. In Saccharomyces cerevisiae, the pathway depends on Rtg1p and Rtg3p, which together form the transcription factor that regulates gene expression, and Rtg2p, an activator of the pathway. Here, we provide novel studies aimed at assessing the functional impact of the lack of RTG-dependent signaling on mitochondrial activity. We show that mutants defective in RTG-dependent retrograde signaling present higher oxygen consumption and reduced hydrogen peroxide release in the stationary phase when compared to wild type cells. Interestingly, RTG mutants are less able to decompose hydrogen peroxide as well as maintain viability when challenged with hydrogen peroxide. Overall, our results indicate that RTG signaling is involved in the hormetic induction of antioxidant defenses and stress resistance, a function of this system not yet described.

Rtg2p é uma proteína que participa da sinalização retrógrada, uma via de comunicação da mitocôndria para o núcleo; também tem sido associada com a longevidade em S. cerevisiae. O objetivo deste trabalho foi identificar os determinantes estruturais de Rtg2p, envolvidos na sinalização retrógrada e no envelhecimento. Para isto foram produzidos treze mutantes pontuais a partir do desenho racional por decomposição de redes de correlação de aminoácidos (DRCN). Analisaram-se as cepas mutantes por ensaio de auxotrofia para glutamato, expressão do gene CIT2 e ensaio de longevidade replicativa. Em sua grande maioria as mutações realizadas causaram perturbações nas funções de Rtg2p, com destaque para as cepas E106A, R109E, E137A, T138A e D158A, que apresentaram longevidade igual à da cepa rtg2Δ, com apenas uma mutação pontual. Em conclusão, os resultados obtidos demonstram que o domínio N-terminal é muito importante para a função de Rtg2p, e indicam que existem determinantes estruturais que controlam a longevidade de forma dependente ou independente da resposta retrógrada. / Rtg2p is a protein involved in the retrograde signaling, a pathway of communcation from mitochondria to nucleus; also has been associated with longevity in S. cerevisiae. The goal of this study was to identify the structural determinants of Rtg2p, controlling the function of this protein in retrograde response and aging. For this purpose thirteen point mutants were produced by site-directed mutagenesis, using rational design by decomposition of residues correlation networks (DRCN). The strains was analyzed by glutamate auxotrophy, CIT2 gene expression and replicative life span assays. For the most of performed mutations, generated inactivation to Rtg2p functions, highlighting to R109E, E137A, T138A, and D158A showed longevity equal to rtg2Δ strain, even with a single amino acid change. In conclusion, our results demonstrate that the N-terminal domain is very important to the function of Rtg2p and also show there are structural determinants in Rtg2p that control longevity in both dependent or independent manner of the communication between mitochondria and nucleus.

No description available.

The survival of several neuron populations during development, including sympathetic neurons, is strictly regulated by neurotrophins such as nerve growth factor (NGF) released from innervation targets. NGF activates its receptor, TrkA, at axon terminals, to generate signals that are transmitted retrogradely to cell bodies to induce signaling cascades regulating survival. A general view of this process is that NGF generates retrograde survival signals that, when delivered to cell bodies, induce downstream survival signaling that prevents apoptosis. A retrograde survival signal proposed to be necessary for sympathetic neuron survival consists of endosomes containing NGF and phosphorylated TrkA. For this signal, phosphorylated TrkA arriving at cell bodies is required to initiate survival signaling. Studies have tested the necessity of TrkA phosphorylation in the cell bodies for survival: results from different studies contradict each other. Moreover, the Trk inhibitor, K252a, used in these studies, has reported non-specific effects. Using an alternate Trk inhibitor, Gö6976, data presented in this thesis demonstrates that NGF can promote survival by retrograde signaling that does not require TrkA phosphorylation in the cell bodies. These retrograde signals may be composed of signaling molecules activated downstream of TrkA in axons since pro-survival molecules downstream of TrkA, Akt and CREB, were found activated in the cell bodies/proximal axons. Data presented in this thesis also reveals a fundamentally different mechanism for how NGF promotes sympathetic neuron survival: a retrograde apoptotic signal that is suppressed by NGF. NGF withdrawal from axons induced the “axon apoptotic signal” that was retrogradely transmitted to cell bodies to activate a key pro-apoptotic molecule, c-jun. The axon apoptotic signal, which was blocked by the kinase inhibitors rottlerin and chelerythrine, was necessary for apoptosis in response to NGF deprivation. Evidence GSK3 is involved in generation or transmission of the axon apoptotic signal was provided by experiments with GSK3 inhibitors and siRNA. The axon apoptotic signal discovery refutes the previous view that NGF acting on axon terminals supports survival exclusively by generating retrograde survival signals. The axon apoptotic signal has broad implications for understanding nervous system development and other conditions where neuronal apoptosis occurs, such as neurotrauma and neurodegenerative diseases.

In murine and human brown adipose tissue (BAT), mitochondria are powerful generators of heat. Emerging evidence has suggested that the actions of mitochondria extend beyond this conventional biochemical role. In mouse BAT and cultured brown adipocytes, impaired mitochondrial respiratory capacity is accompanied by attenuated expression of Ucp1, a key thermogenic gene, implying a mitochondrial retrograde signaling. However, few have investigated this association in the context of mitochondria-nucleus communication. Using mice with adipose-specific ablation of LRPPRC, a regulator of respiratory capacity, we show that respiration-dependent retrograde signaling from mitochondria to nucleus contributes to transcriptional and metabolic reprogramming of BAT. Impaired respiratory capacity triggers down-regulation of thermogenic and oxidative genes, promoting a storage phenotype in BAT. This retrograde regulation functions by interfering with promoter-specific recruitment of PPARg. In addition, cytosolic calcium may mediate the retrograde signal from mitochondria to nucleus. These data are consistent with a model whereby BAT connects its respiratory capacity to thermogenic gene expression, which in turn contributes to determining its metabolic commitment. Additionally, we find that augmented respiratory capacity activates the thermogenic gene program in inguinal (subcutaneous) white adipose tissue (IWAT) from adipose-specific LRPPRC transgenic mice. When fed a high-fat diet at thermoneutrality, these mice exhibit metabolic improvements as shown by reduced fat mass and improved insulin sensitivity. Furthermore, there is increased recruitment of brown-like adipocytes in IWAT and thus energy expenditure is significantly increased, providing a potential explanation for protection from obesity. These data suggest that augmented respiratory capacity promotes ‘browning’ of IWAT, which has beneficial effects on obesity and diabetes.