Tauopathies are neurodegenerative diseases that affect millions of people around the world. Tauopathies include more than 20 neurodegenerative diseases. Some of the most common tauopathies are Alzheimer’s disease (AD), frontotemporal dementia (FTD), chronic traumatic encephalopathy (CTE), Pick’s disease, corticobasal degeneration, progressive supranuclear palsy (PSP), agyrophillic grain disease, and amyotrophic lateral sclerosis (ALS). These diseases can cause significant memory loss, behavioral changes, motor deficits and speech impairments. Tauopathies stem from accumulation of the microtubule associated protein tau (MAPT). Tau stabilizes microtubules and helps with axonal transport. In a disease state tau becomes hyperphosphorylated and truncated leading to its aggregation. More recently tau has been shown to propagate from cell to cell potentially acting as a signaling molecule that contributes to disease progression. In addition during disease, tau mislocalizes to dendrites leading to synaptic dysfunction. This mislocalization may also lead to subsequent neurodegeneration. Today, many strategies have been implemented to treat tauopathies. Some of these strategies include kinase inhibitors, immunotherapy, tau aggregation inhibitors, and microtubule-stabilizing compounds. However none these strategies have been effective in stopping tau pathology nor do they address tau degradation pathways. Therefore we hypothesized that utilizing small molecules that target degradation pathways such as autophagy or proteasomal degradation would improve clearance of aberrant tau. We previously showed that a natural product (+)-aR,11S-myricanol (1) from Myrica cerifica (bayberry/southern wax myrtle) root bark reduced levels of tau. In this study we discovered that 1 is composed of two enantiomers and two possible atropisomers. We found that one enantiomer (-)-aS,11R-myricanol (3) was responsible for the anti-tau activity of 1 in multiple models of tauopathy. We also found that 3 selectively targets and lowers specific tau species. To better understand how these tau species were being reduced we took a non-biased approach and subjected 3 treated samples to stable isotope labeling by amino acids in cell culture (SILAC) mass spectrometry (MS) proteomic analysis. We found that autophagy pathways were most affected by 3 and that 3 was predicted to mimic the drug rapamycin, a well-established macroautophagy activator. In addition we confirmed our MS findings by simultaneously giving 3 treated cells an autophagy inhibitor which blocked 3’s tau reductions. Moreover we created a tetralin derivative of 1, 13, that produced the same effects on tau as 3 but did not rely upon stereochemistry for its activity. This work supports targeting the autophagy degradation pathway as a viable approach to improving aberrant tau accumulation. In order to further support our hypothesis, we collected and screened several known heat shock protein 70 (Hsp70) inhibitors and tau aggregation inhibitors for cellular anti-tau activity. While it is known that Hsp70 inhibition facilitates tau clearance through proteasomal degradation, it is not known what role tau aggregation inhibition plays in the cellular degradation of tau. Moreover understanding which inhibitory activity contributes most to tau degradation would lead to the creation of better drug scaffolds. In this study, we found that several Hsp70 inhibitors from different scaffold backbones had varying effects on tau degradation. The rhodacyanine and phenothiazine compounds were most effective at lowering cellular tau while the adenosine analog, sulfonamide, dihyropyrimidine, piperidine-3-carboxamide, phenoxy-N-arylacetamide, and flavonol, were not as effective. We also examined the effects of several tau aggregation inhibitor scaffolds such as the carbocyanine, oleuropein, anthraquinone, aminothienopyridazine, hydroxytyrosol and rhodanine on tau expression reduction. We found that none had effective tau reductions except the carbocyanine. However when we performed a lactate dehydrogenase (LDH) assay, carbocyanine was shown to be extremely toxic. These results lead us to further investigate if the tau expression reducing Hsp70 inhibitors had anti-tau aggregation activity and if the tau aggregation inhibitors had any Hsp70 inhibitory activity. We discovered that many of the Hsp70 inhibitors also had anti-tau aggregation activity while none of the aggregation inhibitors had Hsp70 inhibitory activity. We found a positive correlation between tau expression reductions and anti-tau aggregation activity for the Hsp70 inhibitors. Our work demonstrates that both Hsp70 activity and tau aggregation in vitro best predicts anti-tau activity of small molecules. Also these dual acting Hsp70 inhibitors support our hypothesis that targeting the degradation pathways can improve tau clearance. Overall, this work indicates the importance of targeting degradation pathways to improve tau clearance. Utilizing small molecules that have dual activities against tau could prove beneficial as a novel therapeutic approach to treat tauopathies. In addition using small molecules that target different degradation pathways simultaneously could be another viable therapeutic strategy for treatment of tauopathies.

Le gène BAG3 a été identifié comme étant un nouveau gène responsable de cardiomyopathie dilatée (CMD), première cause d'insuffisance cardiaque (IC). La protéine BAG3 est une co-chaperonne qui participe au contrôle de l'homéostasie protéique via son rôle dans l'autophagie, protégeant ainsi les cellules contre la protéotoxicité induite par les protéines dégradées ou mal repliées. L'hypothèse qu'une inactivation de la voie autophagique contrôlée par BAG3 induirait une protéotoxicité cardiomyocytaire à l'origine de la CMD apparait particulièrement attractive et constitue l'hypothèse centrale de ce travail. Nos résultats indiquent que les mutations de BAG3 abolissent l'interaction avec la chaperonne HSP70, une protéine centrale du contrôle qualité des protéines. Nous avons observé une cytotoxicité des mutants BAG3, une altération de la fonction chaperonne HSP70-dépendante et une absence de réponse autophagique en condition de stress (jeun, choc thermique, expression d'une protéine pro-agrégante). In vivo (modèle poisson-zèbre) l'extinction de l'expression de BAG3 ou la surexpression des mutants conduisent à l'apparition d'un phénotype d'insuffisance cardiaque (¿dème péricardique) chez les embryons injectés. Par édition génomique, nous développons également un modèle de cardiomyocytes dérivés de cellules iPS porteurs ou non de la mutation afin d'explorer plus en avant la fonction contractile de ces cellules. Nos résultats confirment donc le rôle de BAG3 dans la CMD et indiquent que l'altération de la fonction protéostasique serait à l'origine de la maladie. Cette nouvelle voie physiopathologique dans la CMD pourrait s'avérer être, plus généralement, une voie centrale dans l'IC. / The BAG3 gene was identified as a novel gene responsible for dilated cardiomyopathy (DCM), a major cause of heart failure (HF). The BAG3 protein is a co-chaperone that participates in the control of protein homeostasis via its role in autophagy, protecting cells against the proteotoxicity induced by degraded or misfolded proteins. The hypothesis that inactivation of the autophagic pathway controlled by BAG3 would induce cardiomyocyte proteotoxicity behind the CMD appears particularly attractive and is the central hypothesis of this work. Our results indicate that BAG3 mutations abolish the interaction with the chaperone HSP70, a central actor of the protein quality control. We observed cytotoxicity of BAG3 mutants, an impaired HSP70-dependent chaperone function and absence of autophagic response under stress conditions (starvation, heat shock, expression of a pro-aggregating protein). In vivo (zebrafish model) the extinction of BAG3 expression or mutants overexpression lead to the occurrence of a heart failure phenotype (pericardial edema) in injected embryos. Through genomic edition, we also develop a model of iPS-derived cardiomyocytes carrying or not the mutation in order to further explore the contractile function of these cells. Our results confirm the role of BAG3 in DCM and indicate that the alteration of the proteostasis function is the cause of the disease. This new pathophysiological pathway in DCM may prove to be more generally, a central line in the IC.

Individual types of viability tests based on colorimetric changes of the solution are desribed in the theoretical part. Furthermore, HSP proteins are characterized, which are not connected only by heat shock, but also during other cell stresses such as exposure to UV, cold, extreme pH or heavy metals. They are important for the cell, because they help to reformulate proteins that have been damaged by cellular stress and also bind to new unpacked proteins and ensure their correct folding. Proteins that are affected by molecular chaperones are collectively called client proteins. Some HSPs also contribute to membrane transport or degradation. These proteins are co-operative with the cochaperones, which are important for heat shock proteins because they help them to pack protein, in particular by catalyzing the hydrolysis of ATP to ADP. Herein is also described cisplatin and its derivatives, including mechanism of action and adverse effects. This work was focused on detection cytotoxicity of cisplatin and its derivatives. Cells were exposed to stress condition induced by cytostatics and huge changes in heat shock proteins and cochaperon levels were observed. There was also observed colocalization of heat shock proteins and their client protein p53 by confocal microscopy in these stressing conditions.

OBJECTIVE - HSPA12B is the newest member of HSP70 family of proteins and is enriched in atherosclerotic lesions. This study focused on HSPA12B expression in mice and its involvement in angiogenesis. METHODS AND RESULTS - The expression of HSPA12B in mice and cultured cells was studied by: (1) Northern blot; (2) in situ hybridization; (3) immunostaining with HSPA12B-specific antibodies; and (4) expressing Enhanced-Green-Fluorescent-Protein under the control of the HSPA12B promoter in mice. The function of HSPA12B was probed by an in vitro angiogenesis assay (Matrigel) and a migration assay. Interacting proteins were identified through a yeast two-hybrid screening. HSPA12B is predominantly expressed in vascular endothelium and induced during angiogenesis. In vitro angiogenesis and migration are inhibited in human umbilical vein endothelial cells in the presence of HSPA12B-neutralizing antibodies. HSPA12B interacts with multiple proteins in yeast 2-hybrid system. CONCLUSIONS - We provide the first evidence to our knowledge that the HSPA12B is predominantly expressed in endothelial cells, required for angiogenesis, and interacts with known angiogenesis regulators. We postulate that HSPA12B provides a new mode of angiogenesis regulation and a novel therapeutic target for angiogenesis-related diseases.

No description available.

No description available.

No description available.

High temperatures are a significant biological stressor for mammals, which they may adapt to through behavioral changes, physiological plasticity, and via genetic adaptation. Savanna monkeys (genus Chlorocebus) have a wide climatic range in Africa south of the Sahara, making them a good model species for understanding adaptations to heat stress in primates. Savanna monkeys have been observed to behaviorally mitigate high temperatures, and genetic signs of selection in response to climate have also been found (specifically in relation to cold). In this study, I investigate whether there is genetic variation and evidence for selection related to function in a heat shock protein gene (HSPA1L) in 73 wild savanna monkeys ranging from equatorial Africa to the southern coast of South Africa. Given the important role of heat shock proteins in buffering heat stress, I hypothesized that genetic variation would be associated with maximum summer temperatures, as those are most likely to be warm enough to induce a heat shock response. I found 45 single nucleotide polymorphisms (SNPs) outside of Hardy-Weinberg Equilibrium, and 10 SNPs with significant integrated haplotype scores, only one of which was in a protein coding region (17:40210341; piHS = 2.20). Using phylogenetic least squares modeling I found that maximum temperature of the warmest month was strongly but not significantly associated with the frequency of a derived allele nested within a regulatory region for HSPA1L (17:40207386; piHS = 2.57; b = 0.044, p = 0.061) presumably experiencing selection. I discuss implications of these results for heat tolerance in primates and resilience to climate change.

Molecular chaperones play a key role in maintaining a healthy cellular proteome by performing protein quality control. Heat shock protein 70s (Hsp70s) are a diverse class of evolutionarily conserved chaperones that interact with short hydrophobic sequences presented in unfolded proteins, promoting productive folding, and preventing proteins from aggregation. Most of the extensive research on chaperone examines mechanism, substrate promiscuity, and engagement with many co-chaperones. Only recently were chaperones recognized to be frequent targets of post-translational modifications (PTMs). Despite the recent rise in PTMs identified, the impact of these modifications on chaperone function, whether singular or in concert with other modifications, remains elusive. To investigate the impact of PTMs on chaperone function, we chose to characterize two sites of phosphorylation on the linker of HspA1, the stress inducible human Hsp70. To mimic these phosphoserines, we used aspartate as a phosphomimetic substitution for all experiments. Interdomain allostery ties together chaperone structure and function. Therefore, the impact of phosphorylation on interdomain allostery is probed using biophysical and biochemical techniques. Altogether, data suggest that phosphorylation of the linker and SBD destabilizes the chaperone, while shifting the population towards the docked state. This result alludes to a previously described region of the protein that uncouples domain docking from conformational changes in the substrate-binding domain. The cross-communication between these phosphorylation sites reveals a novel, synergistic effect on chaperone structure and function.

L'ischémie-reperfusion (IR) rénale entraîne des lésions de nécrose tubulaire aigue, nécessitant parfois une épuration extra rénale transitoire voir définitive. La mitochondrie joue un rôle important dans la physiopathologie de ces lésions d'IR en entrainant la mort cellulaire. L'étude de l'IR dans la cellule cardiaque a permis de mettre en évidence le rôle central du pore de transition de perméabilité mitochondriale (mPTP) dans le déclenchement de cette mort cellulaire. La ciclosporine (CsA) a été proposée comme thérapeutique pour protéger la cellule des lésions d'IR en retardant l'ouverture de ce mPTP. Cependant la CsA a des effets rénaux vasoconstricteurs aigus, nécessitant une validation expérimentale de sa protection dans l'IR rénale. Au cours de ce travail de thèse, nous avons mis au point un modèle murin d'IR rénale. Ensuite nous avons montré que le post-conditionnement à la CsA, ainsi que le post-conditionnement ischémique, permettent d'améliorer la fonction rénale avec un retard à l'ouverture du mPTP. Dans un deuxième travail, nous montrons que le pré conditionnement à la CsA est dose et temps dépendant, et médié en partie par l'augmentation d'expression d'une protéine chaperonne, l'Heat Shock Protéine 70 (HSP70). L'injection en bolus de CsA permet également d'améliorer la fonction rénale dans ce modèle d'IR avec un retard à l'ouverture du mPTP. Nos résultats ouvrent de nouvelles perspectives dans la protection rénale, notamment dans la réduction des épisodes d'insuffisance rénale aigue après chirurgie aortique ou en transplantation rénale / Ischemia-reperfusion (IR) is a situation encountered in transplantation or during aortic surgery, which can result in renal damages, requiring sometimes transient or definitive dialysis. Mitochondria play a crucial role in the pathophysiology of IR causing cell death. Previous studies of cardiac IR highlighted the role of mitochondrial permeability transition pore (mPTP). Cyclosporin A (CsA) has been proposed as a treatment to protect the kidney from IR by the delay of the opening of the mPTP. However, CsA has acute renal hemodynamic effects and a long-term toxicity, requiring an experimental validation of its protection in the renal IR. In this work, we developed a mouse model of renal IR. In a first study, we showed that the post-conditioning with CsA and ischemic postconditioning improve renal function with a delay of the opening of the mPTP. In a second study, we showed that a high dose of CsA injected just before the ischemia improves renal function and leads to the delay of the opening of mPTP mediated by an increase of HSP70. Our results open new perspectives in renal protection, especially for reducing episodes of acute renal failure in aortic surgery or in renal transplantation