The pharmacological regulation of cellular mitophagy

Small molecules are pharmacological tools of considerable value for dissecting complex biological processes and identifying potential therapeutic interventions. Recently, the cellular quality-control process of mitophagy has attracted considerable research interest; however, the limited availability of suitable chemical probes has restricted our understanding of the molecular mechanisms involved. Current approaches to initiate mitophagy include acute dissipation of the mitochondrial membrane potential (ΔΨm) by mitochondrial uncouplers (for example, FCCP/CCCP) and the use of antimycin A and oligomycin to impair respiration. Both approaches impair mitochondrial homeostasis and therefore limit the scope for dissection of subtle, bioenergy-related regulatory phenomena. Recently, novel mitophagy activators acting independently of the respiration collapse have been reported, offering new opportunities to understand the process and potential for therapeutic exploitation. We have summarized the current status of mitophagy modulators and analyzed the available chemical tools, commenting on their advantages, limitations and current applications

Hyperglycemia triggers HIPK2 protein degradation

Homeodomain interacting protein kinase-2 (HIPK2) is an evolutionary conserved kinase that modulates several key molecular pathways to restrain tumor growth and induce p53-depending apoptotic cell-death in response to anticancer therapies. HIPK2 silencing in cancer cells leads to chemoresistance and cancer progression, in part due to p53 inhibition. Recently, hyperglycemia has been shown to reduce p53 phosphorylation at serine 46 (Ser46), the target residue of HIPK2, thus impairing p53 apoptotic function. Here we asked whether hyperglycemia could, upstream of p53, target HIPK2. We focused on the effect of high glucose (HG) on HIPK2 protein stability and the underlying mechanisms. We found that HG reduced HIPK2 protein levels, therefore impairing HIPK2-induced p53 apoptotic activity. HG-triggered HIPK2 protein downregulation was rescued by both proteasome inhibitor MG132 and by protein phosphatase inhibitors Calyculin A (CL-A) and Okadaic Acid (OA). Looking for the phosphatase involved, we found that protein phosphatase 2A (PP2A) induced HIPK2 degradation, as evidenced by directly activating PP2A with FTY720 or by silencing PP2A with siRNA in HG condition. The effect of PP2A on HIPK2 protein degradation could be in part due to hypoxia-inducible factor-1 (HIF-1) activity which has been previously shown to induce HIPK2 proteasomal degradation through several ubiquitin ligases. Validation analysed performed with HIF-1α dominant negative or with silencing of Siah2 ubiquitin ligase clearly showed rescue of HG-induced HIPK2 degradation. These findings demonstrate how hyperglycemia, through a complex protein cascade, induced HIPK2 downregulation and consequently impaired p53 apoptotic activity, revealing a novel link between diabetes/obesity and tumor resistance to therapies

Impact of 2-bromopropane on mouse embryonic stem cells and related regulatory mechanisms

2-Bromopropane (2-BP), a cleaning agent, is used as an alternative to ozone-depleting solvents. Previously, 2-BP was shown to have cytotoxic effects on mouse blastocysts and is associated with defects in their subsequent development, both in vitro and in vivo. In addition, it was found that 2-BP also has cytotoxic effects on oocyte maturation and subsequent pre- and post implantation development in vitro and in vivo, and significantly reduces the rate of oocyte maturation, fertilization, and embryonic development in vitro. This study shows that 2-BP (5 to 10 μM) induces apoptotic processes in mouse embryonic stem cells (ESC-B5), but exerts no effects at treatment dosages below 5 μM. In ESC-B5 cells, 2-BP directly increased the content of reactive oxygen species (ROS), significantly increased the cytoplasmic free calcium and nitric oxide (NO) levels, triggered a loss of mitochondrial membrane potential (MMP), activated caspases-9 and -3, and induced cell death. Pre-treatment with NO scavengers suppressed the apoptotic biochemical changes induced by 10 μM 2-BP and promoted the gene expression levels of p53 and p21, which are involved in apoptotic signaling. These results demonstrate for the first time that 2-BP triggers apoptosis in mouse embryonic stem cells via ROS, NO and the activation of mitochondria-dependent cell death signaling.Keywords: 2-Bromopropane, apoptosis, oxidative stress, calcium, nitric oxideAfrican Journal of Biotechnology Vol. 12(20), pp. 3012-302

iASPP regulates neurite development by interacting with Spectrin proteins

IntroductionSince its discovery in 1999, a substantial body of research has shown that iASPP is highly expressed in various kinds of tumors, interacts with p53, and promotes cancer cell survival by antagonizing the apoptotic activity of p53. However, its role in neurodevelopment is still unknown.MethodsWe studied the role of iASPP in neuronal differentiation through different neuronal differentiation cellular models, combined with immunohistochemistry, RNA interference and gene overexpression, and studied the molecular mechanism involved in the regulation of neuronal development by iASPP through coimmunoprecipitation coupled with mass spectrometry (CoIP-MS) and coimmunoprecipitation (CoIP).ResultsIn this study, we found that the expression of iASPP gradually decreased during neuronal development. iASPP silencing promotes neuronal differentiation, while its overexpression inhibited neurite differentiation in a variety of neuronal differentiation cellular models. iASPP associated with the cytoskeleton-related protein Sptan1 and dephosphorylated the serine residues in the last spectrin repeat domain of Sptan1 by recruiting PP1. The non-phosphorylated and phosphomimetic mutant form of Sptbn1 inhibited and promoted neuronal cell development respectively.ConclusionOverall, we demonstrate that iASPP suppressed neurite development by inhibiting phosphorylation of Sptbn1

Identification of genes involved in ceramide-dependent neuronal apoptosis using cDNA arrays

BACKGROUND: Ceramide is important in many cell responses, such as proliferation, differentiation, growth arrest and apoptosis. Elevated ceramide levels have been shown to induce apoptosis in primary neuronal cultures and neuronally differentiated PC 12 cells. RESULTS: To investigate gene expression during ceramide-dependent apoptosis, we carried out a global study of gene expression in neuronally differentiated PC 12 cells treated with C(2)-ceramide using an array of 9,120 cDNA clones. Although the criteria adopted for differential hybridization were stringent, modulation of expression of 239 genes was identified during the effector phase of C(2)-ceramide-induced cell death. We have made an attempt at classifying these genes on the basis of their putative functions, first with respect to known effects of ceramide or ceramide-mediated transduction systems, and then with respect to regulation of cell growth and apoptosis. CONCLUSIONS: Our cell-culture model has enabled us to establish a profile of gene expression during the effector phase of ceramide-mediated cell death. Of the 239 genes that met the criteria for differential hybridization, 10 correspond to genes previously involved in C(2)-ceramide or TNF-α signaling pathways and 20 in neuronal disorders, oncogenesis or more broadly in the regulation of proliferation. The remaining 209 genes, with or without known functions, constitute a pool of genes potentially implicated in the regulation of neuronal cell death

Muscle wasting and the temporal gene expression pattern in a novel rat intensive care unit model

<p>Abstract</p> <p>Background</p> <p>Acute quadriplegic myopathy (AQM) or critical illness myopathy (CIM) is frequently observed in intensive care unit (ICU) patients. To elucidate duration-dependent effects of the ICU intervention on molecular and functional networks that control the muscle wasting and weakness associated with AQM, a gene expression profile was analyzed at time points varying from 6 hours to 14 days in a unique experimental rat model mimicking ICU conditions, i.e., post-synaptically paralyzed, mechanically ventilated and extensively monitored animals.</p> <p>Results</p> <p>During the observation period, 1583 genes were significantly up- or down-regulated by factors of two or greater. A significant temporal gene expression pattern was constructed at short (6 h-4 days), intermediate (5-8 days) and long (9-14 days) durations. A striking early and maintained up-regulation (6 h-14d) of muscle atrogenes (muscle ring-finger 1/tripartite motif-containing 63 and F-box protein 32/atrogin-1) was observed, followed by an up-regulation of the proteolytic systems at intermediate and long durations (5-14d). Oxidative stress response genes and genes that take part in amino acid catabolism, cell cycle arrest, apoptosis, muscle development, and protein synthesis together with myogenic factors were significantly up-regulated from 5 to 14 days. At 9-14 d, genes involved in immune response and the caspase cascade were up-regulated. At 5-14d, genes related to contractile (myosin heavy chain and myosin binding protein C), regulatory (troponin, tropomyosin), developmental, caveolin-3, extracellular matrix, glycolysis/gluconeogenesis, cytoskeleton/sarcomere regulation and mitochondrial proteins were down-regulated. An activation of genes related to muscle growth and new muscle fiber formation (increase of myogenic factors and JunB and down-regulation of myostatin) and up-regulation of genes that code protein synthesis and translation factors were found from 5 to 14 days.</p> <p>Conclusions</p> <p>Novel temporal patterns of gene expression have been uncovered, suggesting a unique, coordinated and highly complex mechanism underlying the muscle wasting associated with AQM in ICU patients and providing new target genes and avenues for intervention studies.</p

Microglia-derived TNFα induces apoptosis in neural precursor cells via transcriptional activation of the Bcl-2 family member Puma

Neuroinflammation is a common feature of acute neurological conditions such as stroke and spinal cord injury, as well as neurodegenerative conditions such as Parkinson’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis. Previous studies have demonstrated that acute neuroinflammation can adversely affect the survival of neural precursor cells (NPCs) and thereby limit the capacity for regeneration and repair. However, the mechanisms by which neuroinflammatory processes induce NPC death remain unclear. Microglia are key mediators of neuroinflammation and when activated to induce a pro-inflammatory state produce a number of factors that could affect NPC survival. Importantly, in the present study we demonstrate that tumor necrosis factor α (TNFα) produced by lipopolysaccharide-activated microglia is necessary and sufficient to trigger apoptosis in mouse NPCs in vitro. Furthermore, we demonstrate that microglia-derived TNFα induces NPC apoptosis via a mitochondrial pathway regulated by the Bcl-2 family protein Bax. BH3-only proteins are known to play a key role in regulating Bax activation and we demonstrate that microglia-derived TNFα induces the expression of the BH3-only family member Puma in NPCs via an NF-jB-dependent mechanism. Specifically, we show that NF-jB is activated in NPCs treated with conditioned media from activated microglia and that Puma induction and NPC apoptosis is blocked by the NF-jB inhibitor BAY-117082. Importantly, we have determined that NPC apoptosis induced by activated microglia-derived TNFα is attenuated in Puma-deficient NPCs, indicating that Puma induction is required for NPC death. Consistent with this, we demonstrate that Puma-deficient NPCs exhibit an B13-fold increase in survival as compared with wild-type NPCs following transplantation into the inflammatory environment of the injured spinal cord in vivo. In summary, we have identified a key signaling pathway that regulates neuroinflammation induced apoptosis in NPCs in vitro and in vivo that could be targeted to promote regeneration and repair in diverse neurological conditions

Biological evidence of the protective role of regucalcin in breast cancer

O cancro da mama é uma doença heterogénea que compreende uma grande variedade de alterações moleculares e diferentes tipos de resposta em termos clínicos. Esta diversidade reside nos múltiplos fatores que podem levar à transformação maligna das células, em consequência da desregulação de diferentes processos fisiológicos. A regucalcina (RGN) é uma proteína de ligação ao cálcio (Ca2+) e cuja principal função conhecida é regular a homeostase do Ca2+ intracelular, mas podendo também estar envolvida na regulação da proliferação celular, apoptose e metabolismo das células. A RGN também foi identificada como um gene regulado por hormonas, incluindo os esteroides sexuais como os androgénios. Para além disso, foi anteriormente associada a determinadas patologias e tendo mesmo sido identificada como uma proteína subexpressa em casos humanos de cancro da mama, próstata ou fígado. No fígado, a subexpressão da RGN foi detetada em lesões pré-neoplásicas, ou seja, antes da aquisição do fenótipo neoplásico, o que sugere que a sua diminuição pode estar ligada ao início do processo de transformação tumorigénico. Apesar destas evidências, os mecanismos moleculares subjacentes às funções da RGN na mama permanecem por identificar. Nesta tese, colocámos a hipótese de que a sobreexpressão da RGN poderá exercer uma ação protetora em relação à carcinogénese mamária. De modo a avaliar esta questão, o composto 7,12 dimetilbenz[α]antraceneno, o qual é conhecido por induzir carcinogénese mamária em rato, foi administrado a ratos transgénicos que sobreepressam a RGN (Tg-RGN) e aos respetivos controlos (Wt, do inglês wild-type). Os ratos Tg-RGN apresentaram, notavelmente, uma menor incidência de tumores (25.8 %) comparativamente aos animais controlo (100 %). A classificação histológica também demonstrou uma clara resistência dos ratos Tg-RGN à tumorigénese, ao serem bastante mais resistentes à progressão dos tumores para estadios mais agressivos. Verificou-se uma muito menor percentagem de tumores do tipo invasivo nos animais transgénicos (3.8 % vs 45.8 % nos Wt). Para além disso, foi observado um aumento da atividade proliferativa nos tumores não-invasivos nos Wt comparativamente aos animais TG-RGN, o que indica a menor capacidade invasiva. A avaliação metabólica dos tumores benignos da glândula demonstrou que os tumores de ratos Tg-RGN possuem uma menor expressão e atividade da lactato desidrogenase (LDH), característica que normalmente se encontra associada a uma restrição da progressão tumoral e a um decréscimo da agressividade. Contudo, em tecido mamário não-neoplásico de ratos Tg-RGN observou-se uma restrição do metabolismo glicolítico, o que é indicativo de uma redução dos níveis energéticos no tecido. Estes resultados podem ser de extrema importância para a diminuição da proliferação celular e constituir um mecanismo adicional, pelo qual a RGN previne o desenvolvimento tumoral. De facto, a sobreexpressão da RGN originou uma diminuição da expressão de genes envolvidos na regulação ciclo celular e de oncogenes na glândula mamária de ratos Tg-RGN. Mais ainda, a expressão do P53 e a atividade da caspase-3 também foi encontrada aumentadas concomitantemente com a sobreexpressão da RGN, o que sugere uma ação protetora da RGN no aparecimento do tumor, a qual pode ser mediada também pela regulação das vias apoptóticas. Em contrapartida, a expressão da RGN em células MCF-7 de cancro da mama diminui pela ação do androgénio não-aromatizável 5α-dihidrotestosterona, ao passo que a expressão do canal de Ca2+ do tipo L aumentou. Estes resultados sustentam a diminuição da viabilidade celular evidenciada nestas células e sugerem o envolvimento destes modeladores do Ca2+ no controlo da proliferação celular mamária, o que no caso do canal de Ca2+ do tipo L nunca antes tinha sido sugerido. Em conclusão, o trabalho apresentado nesta tese destaca a preponderância da RGN numa diversidade de mecanismos fisiológicos e fisiopatológicos na glândula mamária. As evidências biológicas aqui apresentadas confirmam o papel protetor da RGN na carcinogénese mamária, ao restringir processos biológicos reconhecidos como fundamentais para o desenvolvimento do cancro, nomeadamente, a proliferação celular e as alterações no metabolismo celular, ao mesmo tempo que aumenta a morte celular por apoptose.Breast cancer is a heterogeneous disease that comprises a wide variety of molecular alterations and divergent clinical behaviors. This diversity resides in a plethora of factors that can drive cell malignant transformation, by the deregulation of basic physiological pathways that are recognized as the cancer hallmarks. Regucalcin (RGN) is a calcium (Ca2+)-binding protein known to play an important role in intracellular Ca2+ homeostasis, but it is also involved in the regulation of multiple intracellular signaling pathways such as cell proliferation, apoptosis and metabolism. RGN also has been identified as a hormonally regulated gene, which includes the sex steroids androgens. Furthermore, RGN was previously associated with pathological conditions and described as a protein underexpressed in human breast, prostate and liver cancer cases. In the liver, it was demonstrated that RGN underexpression occurs in pre-neoplastic lesions before the acquisition of neoplastic phenotype, which thus implicates RGN loss in the tumorigenic transformation. In spite of this evidence, the molecular mechanisms underlying RGN actions in the breast remain to be identified. In the present thesis, we hypothesized that RGN overexpression may exert a protective action against mammary tumorigenesis. To address this issue, transgenic rats overexpressing RGN (Tg-RGN) and wild-type (Wt) controls were treated with 7,12-dimethylbenz[α]anthracene, a compound recognized to induce carcinogenesis of rat mammary gland. Tg-RGN rats displayed a remarkable lower incidence of tumors (25.8 %), comparatively with their Wt counterparts (100 %). Tumor histological classification also clearly showed that Tg-RGN rats are resistant to cancer progression into more aggressive stages, as indicated by the lower percentage of invasive tumors types (3.8 % vs 45.8 % in Wt). Moreover, higher proliferative activity was observed in non-invasive tumors of Wt comparatively with those of Tg-RGN animals. The metabolic evaluation of these tumors demonstrated a lower expression and activity of lactate dehydrogenase (LDH) in Tg-RGN rats, which is a feature associated with restricted tumor progression and lower aggressiveness. Notwithstanding, in the non-neoplastic mammary gland of Tg-RGN rats a restriction of the glycolytic metabolism was observed, which indicates a reduction of the energy levels in the tissue. These results may be quite relevant to slowdown cell proliferation, and may constitute an additional mechanism by which RGN prevent tumor development. Indeed, RGN overexpression suppressed the expression of cell cycle regulators and oncogenes in the mammary gland of Tg-RGN rats. Besides that, P53 expression and caspase-3 activity were also augmented in response to RGN overexpression, which suggests that the protective role of RGN against tumor onset may also be mediated by the modulation of apoptotic pathways. On the other hand, RGN and L-type Ca2+ channel were found to be down-regulated and up-regulated, respectively, by the non-aromatizable androgen 5α-didydrotestosterone in MCF-7 breast cancer cells. These results underpinned the decreased viability of MCF-7 cells and suggest the involvement of Ca2+ regulators, RGN and L-type Ca2+ channels, in the control of breast cell proliferation. In conclusion, the work presented in this thesis highlighted the influence of RGN in a plethora of physiologic and pathophysiologic mechanisms of the mammary gland. The biologic evidence presented herein confirmed the protective role of RGN in mammary carcinogenesis, by restraining biological processes recognized as the hallmarks of cancer, namely, cell proliferation and metabolism, or by enhancing apoptotic cell death