Die von der WHO zu den 17 wichtigsten NTDs gezählte Leishmaniose wird durch intrazelluläre Parasiten der Gattung Leishmania hervorgerufen. Der Lebenszyklus der Parasiten besteht aus zwei Phasen. Die länglichen und beweglichen Promastigoten kennzeichnen die Phase in der Sandmücke – der Vektor der Leishmaniose. Hingegen ist die Phase im Säugerwirt durch runde unbewegliche Amastigoten charakterisiert. Aufgrund des Mangels an potenten antileishmanialen Therapien wurde in der vorliegenden Arbeit die Interaktion zwischen L. m. Parasiten und der Hauptwirtszelle, der Makrophage, v. a. in Hinblick auf autophage Prozesse in den infizierten Makrophagen näher untersucht, um demgemäß neue Erkenntnisse zu gewinnen, welche bei der Herstellung zukünftiger anti-leishmanialer Medikamente helfen könnten. Bei der Autophagie handelt es sich um einen katabolen Prozess, wodurch Zellen bei Nahrungsmangel oder zellulärem Stress ihre Homöostase erhalten können. Durch diesen Prozess können überflüssige oder beschädigte Organellen recycelt werden, um die Funktionen der Zelle aufrechtzuerhalten. Daneben übernimmt Autophagie auch eine essenzielle Rolle bei der Abwehr von ins Zytosol eindringenden Pathogenen. Mittels des neu etablierten totalen Autophagiescore konnte festgestellt werden, dass Autophagie in L. m.-infizierten BMDM induziert wird. Die intrazellulären Amastigoten werden durch Autophagie in den BMDM verdaut. Die erhöhte autophage Aktivität konnte zudem durch Western-Blot-Analysen der autophagierelevanten Proteine ATG5, LC3B und UB bestätigt werden. Die molekulargenetischen Untersuchungen von L. m.-infizier-ten BMDM mithilfe von Affymetrix Microarrays führten zu einem Netzwerk aus autophagierelevanten und infektionsspezifischen Genen, welches als LISA bezeichnet worden ist. Hier hat sich ebenfalls eine starke Verknüpfung von autophagierelevanten Genen und den Genen der Glykolyse, einem zweiten katabolen Prozess, gezeigt. Zudem konnten zwei weitere autophagierelevante und infektionsspezifische Gene außerhalb von LISA identifiziert werden, nämlich Bnip3 und Ctse, welche im Anschluss genauer untersucht worden sind. Bei beiden Genen konnte auf Proteinebene gezeigt werden, dass sie in L. m.-infizierten BMDM signifikant erhöht sind. Durch siRNA-Analysen konnte überdies beobachtet werden, dass beide für die erfolgreiche Elimination der Amastigoten essenziell sind. Somit konnte mit den Proteinen BNIP3 und CTSE zwei potenzielle neue Ansatzpunkte für mögliche zukünftige antileishmaniale Therapien gefunden werden. Auch die in LISA enthaltenen Gene stellen prinzipiell vielversprechende Ziele für künftige Medikamente gegen Leishmaniose dar. Durch all diese Untersuchungen kommt man dem Ziel einer neuen, gezielten und nebenwirkungsärmeren Behandlung der Leishmaniose einen Schritt näher. / Leishmaniasis, listed by the WHO to be one of the 17 most important NTDs, is caused by intracellular parasites of the genus Leishmania. The life cycle of the parasites consists of two stages. The oblong and motile promastigotes characterize the stage in the sand fly, the vector of leishmaniasis. However, the stage in the vertebrate host is characterized by round immotile amastigotes. Due to a lack of capable antileishmanial therapies, the interaction between L. m. parasites and their main host cell, the macrophage, was investigated in the present work, huge focus on autophagic processes in infected macrophages. Our goal was to get new insights for the future production of antileishmanial drugs. Autophagy is a catabolic process whereby cells are able to maintain their homeostasis in times of starvation or cellular stress. During to this process, redundant or damaged organelles are recycled in order to sustain cellular viability. Furthermore, autophagy has an essential role in the defense of pathogens invading the cytosol. The newly established total autophagy score showed an autophagy induction in L. m.-infected BMDM. Intracellular amastigotes are digested by autophagy in BMDM. The increased autophagic activity could also be confirmed by western-blot analyses of the autophagy-relevant proteins ATG5, LC3B, and UB. Molecular genetic investigations of L. m.-infected BMDM by Affymetrix microarrays led to a network of autophagy-relevant and infection-specific genes, which was called LISA. Additionally, it showed a strong connection between autophagy-relevant genes and genes of the glycolysis, a second catabolic process. Moreover, we identified and further characterized two additional autophagy-relevant genes, Bnip3 and Ctse, which were not included in LISA. Both genes were significantly overexpressed on protein level in L. m.-infected BMDM. By siRNA analyses we also demonstrated their importance for successful elimination of amastigotes. Therefore, both proteins, BNIP3 and CTSE, could be new potential targets for possible future antileishmanial therapies. In addition, the genes included in LISA might be promising targets for future drugs against leishmaniasis. Due to all these investigations we are one step closer to our goal of a targeted and safe therapy of leishmaniasis.

Autophagy is a physiological process by which the cell eliminates damaged organelles, toxic agents, and long-lived proteins by degradation through lysosomal system. Mitophagy, the specific autophagic elimination of mitochondria, regulates mitochondrial number to match metabolic demand and is a core machinery of quality control to remove damaged mitochondria. A neuroprotective role of physiological autophagy/mitophagy has been discovered. However, recent studies suggested that highly accelerated autophagy/mitophagy might contribute to neuronal death in various pathological situations including cerebral ischemia. In this study, we aimed to investigate the activation of excessive autophagy, particularly, the more specific mitophagy, in neuronal tissues and its contribution to ischemia/hypoxia (I/H)-induced delayed neuronal death. I/H injury was induced by oxygen and glucose deprivation (OGD) followed by reperfusion (RP) on primary cortical neurons in vitro. Cerebral ischemia was induced by unilateral common carotid artery occlusion and hypoxia in neonatal mice in vivo. In order to determine the extent to which autophagy contributes to neuronal death in cerebral ischemia, we performed multiple methods and found that in both primary cortical neurons and SH-SY5Y cells exposed to OGD for 6 h and RP for 24, 48, and 72 h, respectively, an increase of autophagy was observed as determined by the increased ratio of LC3-II to LC3-I and Beclin 1 expression. Using Fluoro-Jade C and monodansylcadaverine double-staining, and electron microscopy we found the increment in autophagy after OGD/RP was accompanied by increased autophagic cell death, and this increased cell death was inhibited by the specific autophagy inhibitor, 3-methyladenine. The presence of large autolysosomes and numerous autophagosomes in cortical neurons were confirmed by electron microscopy. Autophagy activities were increased dramatically in the ischemic brains 3-7 days postinjury from a rat model of neonatal cerebral I/H as shown by increased punctate LC3 staining and Beclin-1 expression. We thus obtained the conclusion that excessive activation of autophagy contributes to neuronal death in cerebral ischemia. BNIP3 (Bcl-2/adenovirus E19 kD interacting protein 3), a member of a unique subfamily of death-inducing mitochondrial proteins, is highly associated with mitochondrial dysfunction and delayed neuronal death in stroke. It is known that BNIP3-induced neuronal death is caspase-independent and characterized by early mitochondrial damage. Recent evidence suggested that the BNIP3 family of proteins might be important regulators of mitophagy. Here, using both stroke models, we found that homodimer (60 kD) of BNIP3/NIX (BNIP3L) were highly expressed in a ‘delayed’ manner. Particularly, significant mitophagic activation was confirmed by electron microscopy. In contrast, both neonatal mitophagy and apoptosis were significantly inhibited in the BNIP3 knockout (KO) mice after I/H, which was also accompanied by a significantly increased autophagic response. In addition, the infarct volume in the BNIP3 KO mice was significantly reduced as compared to wild-type (WT) mice after 7 or 28 days recovery, showing a prominent neuroprotection of BNIP3 gene silencing. A protein-to-protein interaction of mitochondria-localized BNIP3 (60 kD) with the autophagosome marker, LC3, was confirmed by co-ip, immunocytochemistry and further quantified by ELISA, indicating BNIP3 was an effective LC3-binding target on damaged mitochondria. These data demonstrated a novel role of BNIP3 in regulating neuronal mitophagy and cell death during ischemic stroke.

Aims: This study was aimed to investigate the role of GSDME-mediated pyroptosis in cardiac injury induced by Doxorubicin (DOX), and to evaluate the role of BH3-only protein Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (Bnip3) in regulation of DOX-induced pyroptosis. Main methods: HL-1 cardiomyocytes and C57BL/6J mice were treated by DOX to establish DOX-induced cardiotoxicity in vitro and in vivo models, respectively. Cell transfection was applied to regulate the expression of caspase-3, GSDME and Bnip3. Western blot was used for measuring expression of protein level. LDH-cytotoxicity assay was used to detect the LDH release. The Flow cytometry analysis was used to detect the cell death. Echocardiography was used to determine the cardiac function. HE staining was used for observing pathological feature of heart tissues. Key findings: Our results showed that GSDME-mediated pyroptosis was involved in DOX-induced cardiotoxicity in vivo. We showed that HL-1 cardiomyocytes exposed to DOX exhibited morphological features of pyroptosis in vitro. We also showed that DOX induced activation of caspase-3 and eventually triggered GSDME-dependent pyroptosis, which was reduced by the silence or inhibitor of caspase-3. We further showed that knockdown of GSDME inhibited DOX-induced cardiomyocyte pyroptosis in vitro. Finally, DOX increased the expression of Bnip3, whereas silencing of Bnip3 blunted cardiomyocyte pyroptosis induced by DOX, which was regulated through caspase-3 activation and GSDME cleavage. Significance: Our findings revealed a novel pathway that cardiomyocyte pyroptosis is regulated through Bnip3-caspase-3-GSDME pathway following DOX treatment, suggesting that Bnip3-dependent pyroptosis may offer a novel therapeutic strategy to reduce cardiotoxicity induced by DOX.

No description available.

Prostate cancer is the second most common cancer among men in the United States. While treatments for prostate cancer exist, none are curative. As a solid tumor, prostate cancer can grow beyond the diffusion limits of oxygen, thereby resulting in a hypoxic environment. While hypoxia can cause death to a variety of cell types, tumor cells can develop resistance to hypoxia and survive under minimal oxygen conditions. Hypoxia in tumor cells has also been associated with poor prognosis, increased metastasis, and decreased efficacy of chemotherapy. BNIP3, a BH-3 only proapoptotic Bcl-2 family member, has been shown to play an important role in cell death under hypoxic conditions in a variety of cell types. In normoxia, BNIP3 shows little to no expression in both cardiomyocytes and many cancer cell types, but is then upregulated under hypoxic conditions. Previous work in our laboratory provides evidence that hypoxia alone, as well as the concomitant increase in BNIP3 expression, cannot cause death of rat neonatal cardiomyocytes. Instead, our studies found that hypoxia with concomitant intracellular acidosis is required. Further studies indicated that BNIP3 is also necessary for hypoxia-acidosis associated cell death in cardiomyocytes. Our results in rat neonatal cardiomyocytes led us to hypothesize that cell death could be induced in hypoxic prostate cancer cells if concomitant acidosis could be induced. Additionally, our intention was to determine if BNIP3 was required for any prostate cancer cell death that may occur under hypoxia-acidosis conditions.

Apoptotic cell death of cardiac myocytes plays an important pathological role after a myocardial infarction and during heart failure. Apoptotic myocytes are not regenerated because of the restricted ability of terminally differentiated cardiac myocytes to undergo cell division. Because ventricular function is directly related to the number of active muscle cells, the inappropriate loss or premature death of cardiac myocytes results in reduced cardiac performance. Bnip3 was previously identified by Dr. Lorrie Kirshenbaum’s laboratory as a critical mediator of hypoxia-induced apoptosis in the heart. Importantly, his lab established that the cytoprotective actions of NF-κB during hypoxia included the transcriptional repression of Bnip3. However, the mechanism by which NF-κB acted as a transcriptional repressor was undefined. The present work strongly supports the hypothesis that NF-κB-mediated inhibition of Bnip3 transcription is dependent on the recruitment of the corepressor protein HDAC1. Immunoprecipitation experiments revealed that HDAC1 and p65 NF-κB formed protein-protein interactions. ChIP assays demonstrated that HDAC1 and p65 NF-κB associated with the Bnip3 promoter. HDAC1-mediated repression of Bnip3 was lost in cells deficient for p65 NF-κB, and restored upon repletion of p65. A second avenue of investigation described in this work demonstrated that the cell cycle factor E2F-1 directly activated Bnip3 transcription. Earlier work by Dr. Kirshenbaum found that adenovirus-mediated overexpression of E2F-1 in ventricular myocytes induced apoptosis. Herein, it is shown that E2F-1-mediated cell death is largely Bnip3-dependent because functional loss of Bnip3 inhibited E2F-1-induced cell death. Concerning hypoxia, Bnip3 expression is dependent upon the loss of p65/HDAC1-mediated repression, and on the presence of transcriptionally active E2F-1. During hypoxia, overexpression of p65, HDAC1, or Rb, an endogenous inhibitor of E2F-1-dependent transcription, attenuated hypoxia-induced Bnip3 transcription. Based on these findings, future therapies may be designed to repress Bnip3 gene expression after a myocardial infarction, thereby averting cardiac cell death and preserving cardiac function post-infarction.

Apoptotic cell death of cardiac myocytes plays an important pathological role after a myocardial infarction and during heart failure. Apoptotic myocytes are not regenerated because of the restricted ability of terminally differentiated cardiac myocytes to undergo cell division. Because ventricular function is directly related to the number of active muscle cells, the inappropriate loss or premature death of cardiac myocytes results in reduced cardiac performance. Bnip3 was previously identified by Dr. Lorrie Kirshenbaum’s laboratory as a critical mediator of hypoxia-induced apoptosis in the heart. Importantly, his lab established that the cytoprotective actions of NF-κB during hypoxia included the transcriptional repression of Bnip3. However, the mechanism by which NF-κB acted as a transcriptional repressor was undefined. The present work strongly supports the hypothesis that NF-κB-mediated inhibition of Bnip3 transcription is dependent on the recruitment of the corepressor protein HDAC1. Immunoprecipitation experiments revealed that HDAC1 and p65 NF-κB formed protein-protein interactions. ChIP assays demonstrated that HDAC1 and p65 NF-κB associated with the Bnip3 promoter. HDAC1-mediated repression of Bnip3 was lost in cells deficient for p65 NF-κB, and restored upon repletion of p65. A second avenue of investigation described in this work demonstrated that the cell cycle factor E2F-1 directly activated Bnip3 transcription. Earlier work by Dr. Kirshenbaum found that adenovirus-mediated overexpression of E2F-1 in ventricular myocytes induced apoptosis. Herein, it is shown that E2F-1-mediated cell death is largely Bnip3-dependent because functional loss of Bnip3 inhibited E2F-1-induced cell death. Concerning hypoxia, Bnip3 expression is dependent upon the loss of p65/HDAC1-mediated repression, and on the presence of transcriptionally active E2F-1. During hypoxia, overexpression of p65, HDAC1, or Rb, an endogenous inhibitor of E2F-1-dependent transcription, attenuated hypoxia-induced Bnip3 transcription. Based on these findings, future therapies may be designed to repress Bnip3 gene expression after a myocardial infarction, thereby averting cardiac cell death and preserving cardiac function post-infarction.

No description available.

Bnip3 is a BH3-only member of the Bcl-2 family of apoptotic proteins. Our laboratory has previously shown that Bnip3 induces a unique pathway of cardiac myocyte cell death, characterized by mitochondrial dysfunction, cytochrome c release and DNA fragmentation. Bnip3 is induced by hypoxia and the death pathway is activated by concurrent acidosis. We have shown that hypoxia-acidosis creates an environment that is permissive to calpain but not caspase activation and is characterized by enhanced DNase(s) activity as evidenced by genomic DNA fragmentation. This dissertation describes the nuclear consequences of Bnip3 activation by hypoxia-acidosis. Chapter 3 presents my evidence that hypoxia with progressive acidosis in cardiac myocytes results in a biphasic activation of DNases. In phase 1, [pH]o 6.9-6.7, apoptosis-inducing factor (AIF) is released from the mitochondria and translocates to the nucleus. AIF release coincided with the loss of mitochondrial membrane potential and with the release of cytochrome c from the mitochondria. In Phase II, [pH]o 6.3-6.0, DNase II translocates from the cytoplasm to the nuclear compartment. Nuclear localization of DNase II was associated with the collapse of endosomal pH gradients, indicated by diffuse Lysotracker Red staining and with single strand DNA nicks. Both phases of DNase release were independent of Bnip3, the mPTP and calpains. Neither phase involved activation of caspase-dependent DNases. Chapter 4 describes a unique role for Bnip3 in the modulation of histone acetylation. I found that hypoxia with acidosis in cardiac myocytes but not hypoxia alone stimulated a global increase in the acetylation of histones H3 and H4. Acetylation was initiated at [pH]o ~ 6.8 and increased as the pH declined. Histone hyperacetylation was associated with an increase in histone acetyltransferase (HAT) activity but no change in deacetylase (HDAC) activity. Knockdown of Bnip3 protein expression with siRNA dramatically reduced both histone H3 and H4 acetylation levels and HAT activity indicating an essential role for Bnip3 in this process. Components of the hypoxia-acidosis death pathway including the mPTP and calpains are not required for Bnip3-mediated histone hyperacetylation. These results reveal a novel role for Bnip3 in regulating HAT activity and histone acetylation which may lead to altered cardiac gene expression.

As doenças cardiovasculares são a principal causa de morte no mundo, sendo a cardiomiopatia isquêmica a mais prevalente. Independente da sua etiologia, a via final comum da maioria das doenças cardiovasculares é a insuficiência cardíaca. Nos últimos anos, tem sido reportado que o acúmulo de proteínas mal enoveladas no retículo endoplasmático (estresse do RE) pode contribuir para redução da função cardíaca e instalação da insuficiência cardíaca. Apesar do mecanismo responsável pela disfunção contrátil induzida pelo estresse do RE ainda não ser conhecido, evidências sugerem que a inibição da via de sinalização PI3K/AKT pela proteína JNK pode estar envolvida nessa resposta. Na primeira parte desta tese, verificamos que a indução do estresse do RE em cardiomiócitos isolados ativou a JNK, mas não inibiu a via de sinalização PI3K/AKT. A inativação de JNK reverteu a disfunção contrátil e a redução da amplitude do transiente de Ca+2 de cardiomiócitos causados pelo estresse do RE. Pelo fato da via sinalização PI3K/AKT não estar envolvida na disfunção contrátil causada pelo estresse do RE, analisamos outro alvo de JNK, a proteína BNIP3, proteína pró-apoptótica e envolvida no controle de qualidade mitocondrial promovendo mitofagia quando ativada. O estresse do RE aumentou a expressão de BNIP3, a qual foi atenuada pela inibição de JNK. A depleção de BNIP3 impediu a disfunção contrátil dos cardiomiócitos e a redução da amplitude do transiente de Ca+2 induzidos pelo estresse do RE. Na segunda parte da tese, o objetivo foi avaliar se os efeitos observados em cardiomiócitos submetidos a estresse do RE poderiam ser observados em modelo experimental de doença cardiovascular. Nesse sentido, observamos que a disfunção cardíaca provocada pelo infarto do miocárdio em ratos foi acompanhada pelo quadro de estresse do RE e pela ativação da via de sinalização JNK/BNIP3. Entretanto, o treinamento físico aeróbico (TFA), uma das principais terapias não farmacológicas mais eficazes das doenças cardiovasculares, foi capaz de atenuar o estresse RE, a ativação da via de sinalização JNK/BNIP3 e a disfunção cardíaca de ratos infartados. Na terceira parte da tese, verificamos que o TFA aumentou a expressão proteica de DERLIN-1, uma proteína que atua retro-translocando proteínas mal enoveladas para o citosol, no miocárdio de ratos saudáveis. O aumento dos níveis proteicos de DERLIN-1 observado em ratos infartados foi atenuado pelo TFA. Apesar do aumento da proteína DERLIN-1, observamos que nos animais infartados as proteínas mal enoveladas acumulavam na forma de oligômeros e que o TFA atenuou essa resposta. Em conjunto, os resultados da presente tese sugerem que a ativação da via de sinalização JNK/BNIP3 pelo estresse do RE causa disfunção contrátil de cardiomiócitos e que o TFA é capaz de atenuar essa resposta no coração de ratos infartados, melhorando o controle de qualidade de proteína no músculo cardíaco / Cardiovascular diseases are currently the main cause of death worldwide, with the ischemic cardiomyopathy as the most prevalent ethiology. This is of particular interest, since ischemic cardiomyopathy advances to heart failure, a common endpoint of the most cardiovascular disease. In the last years, it has been showed that accumulation of unfolded protein in the endoplasmic reticulum (ER stress) may cause cardiac dysfunction and heart failure development. Despite the mechanisms behind this cardiac deterioration is still unknown, evidences suggest that ER stress-induced cardiomyocytes contractile dysfunction results from PI3K/AKT signaling pathway inhibition, which would be caused by JNK activation. In the first part of this thesis, we found that the ER stress activated JNK, but different from our hypothesis it was not accompanied by an inactivation of PI3K/AKT signaling pathway. The inhibition of JNK mitigated the reduction in cardiomyocytes shortening and amplitude of Ca+2 transient caused by ER stress. Once the PI3K/AKT signaling pathway was not involved in the ER stress-induced cardiomyocytes contractile dysfunction, we have analyzed protein expression of BNIP3, another JNK target involved in apoptosis and mitochondria quality control. We observed that the elevation in BNIP3 proteins levels after ER stress induction was prevented by inhibition of JNK. BNIP3 depletion attenuated the reduction in cardiomyocytes contractility and amplitude of Ca+2 transient induced by ER stress. In the second part of the thesis, we found that myocardial infarction-induced cardiac dysfunction in rats was accompanied by ER stress and activation of JNK/BNIP3 signaling pathway. However, the AET mitigated ER stress, activation of JNK/BNIP3 signaling pathway and cardiac dysfunction in infarcted rats. In third part of the thesis, we have identified that AET increased the protein expression of DERLIN-1 an ER membrane protein that retro-translocates unfolded proteins to cytosol in the myocardial of healthy rats. We observed that the increased DERLIN-1 protein levels in infarcted rats were mitigated by AET. Despite increased DERLIN-1 protein expression, we found high levels of oligomers in the myocardium of infarcted rat, which was reduced by AET. It suggests that unfolded protein degradation was reduced in infarcted hearts. Taken together, these results suggest that ER stress causes cardiomyocytes contractile dysfunction through JNK/BNIP3 signaling pathway activation and that AET mitigates the myocardial infarction-induced ER stress and activation of JNK/BNIP3 signaling pathway by restoring of ER-associated protein quality control in the cardiac muscle