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Andrea C. Leblanc - One of the best experts on this subject based on the ideXlab platform.
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Methylene blue inhibits Caspase-6 activity, and reverses Caspase-6-induced cognitive impairment and neuroinflammation in aged mice
Acta neuropathologica communications, 2019Co-Authors: Libin Zhou, Joseph Flores, Anastasia Noël, Olivier Beauchet, P. Jesper Sjöström, Andrea C. LeblancAbstract:Activated Caspase-6 (Casp6) is associated with age-dependent cognitive impairment and Alzheimer disease (AD). Mice expressing human Caspase-6 in hippocampal CA1 neurons develop age-dependent cognitive deficits, neurodegeneration and neuroinflammation. This study assessed if methylene blue (MB), a phenothiazine that inhibits caspases, alters Caspase-6-induced neurodegeneration and cognitive impairment in mice. Aged cognitively impaired Casp6-overexpressing mice were treated with methylene blue in drinking water for 1 month. Methylene blue treatment did not alter Caspase-6 levels, assessed by RT-PCR, western blot and immunohistochemistry, but inhibited fluorescently-labelled Caspase-6 activity in acute brain slice intact neurons. Methylene blue treatment rescued Caspase-6-induced episodic and spatial memory deficits measured by novel object recognition and Barnes maze, respectively. Methylene blue improved synaptic function of hippocampal CA1 neurons since theta-burst long-term potentiation (LTP), measured by field excitatory postsynaptic potentials (fEPSPs) in acute brain slices, was successfully induced in the Schaffer collateral-CA1 pathway in methylene blue-treated, but not in vehicle-treated, Caspase-6 mice. Increased neuroinflammation, measured by ionized calcium binding adaptor molecule 1 (Iba1)-positive microglia numbers and subtypes, and glial fibrillary acidic protein (GFAP)-positive astrocytes, were decreased by methylene blue treatment. Therefore, methylene blue reverses Caspase-6-induced cognitive deficits by inhibiting Caspase-6, and Caspase-6-mediated neurodegeneration and neuroinflammation. Our results indicate that Caspase-6-mediated damage is reversible months after the onset of cognitive deficits and suggest that methylene blue could benefit Alzheimer disease patients by reversing Caspase-6-mediated cognitive decline.
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Rare human Caspase-6-R65W and Caspase-6-G66R variants identify a novel regulatory region of Caspase-6 activity.
Scientific Reports, 2018Co-Authors: Agne Tubeleviciute-aydin, Sergey N. Savinov, Jeanne A. Hardy, Gyanesh Sharma, Libin Zhou, Ishankumar V. Soni, Andrea C. LeblancAbstract:The cysteine protease Caspase-6 (Casp6) is a potential therapeutic target of Alzheimer Disease (AD) and age-dependent cognitive impairment. To assess if Casp6 is essential to human health, we investigated the effect of CASP6 variants sequenced from healthy humans on Casp6 activity. Here, we report the effects of two rare Casp6 amino acid polymorphisms, R65W and G66R, on the catalytic function and structure of Casp6. The G66R substitution eliminated and R65W substitution significantly reduced Casp6 catalytic activity through impaired substrate binding. In contrast to wild-type Casp6, both Casp6 variants were unstable and inactive in transfected mammalian cells. In addition, Casp6-G66R acted as a dominant negative inhibitor of wild-type Casp6. The R65W and G66R substitutions caused perturbations in substrate recognition and active site organization as revealed by molecular dynamics simulations. Our results suggest that full Casp6 activity may not be essential for healthy humans and support the use of Casp6 inhibitors against Casp6-dependent neurodegeneration in age-dependent cognitive impairment and AD. Furthermore, this work illustrates that studying natural single amino acid polymorphisms of enzyme drug targets is a promising approach to uncover previously uncharacterized regulatory sites important for enzyme activity.
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Caspase vinyl sulfone small molecule inhibitors prevent axonal degeneration in human neurons and reverse cognitive impairment in Caspase-6-overexpressing mice
Molecular Neurodegeneration, 2017Co-Authors: Prateep Pakavathkumar, Alain Noël, Clotilde Lecrux, Agne Tubeleviciute-aydin, Jan-eric Ahlfors, E. Hamel, Andrea C. LeblancAbstract:BackgroundThe activation of the aspartate-specific cysteinyl protease, Caspase-6, is proposed as an early pathogenic event of Alzheimer disease (AD) and Huntington’s disease. Caspase-6 inhibitors could be useful against these neurodegenerative diseases but most Caspase-6 inhibitors have been exclusively studied in vitro or show acute liver toxicity in humans. Here, we assessed vinyl sulfone small molecule peptide caspase inhibitors for potential use in vivo.MethodsThe IC50 of NWL vinyl sulfone small molecule caspase inhibitors were determined on Caspase-1 to 10, and Caspase-6-transfected human colon carcinoma HCT116 cells. Inhibition of Caspase-6-mediated axonal degeneration was assessed in serum-deprived or amyloid precursor protein-transfected primary human CNS neurons. Cellular toxicity was measured by phase contrast microscopy, mitochondrial and lactate dehydrogenase colorimetric activity assays, or flow cytometry. Caspase inhibition was measured by fluorogenic activity assays, fluorescence microscopy, and western blot analyses. The effect of inhibitors on age-dependent cognitive deficits in Caspase-6 transgenic mice was assessed by the novel object recognition task. Liquid chromatography coupled to tandem mass spectrometry assessed the blood-brain barrier permeability of inhibitors in Caspase-6 mice.ResultsVinyl sulfone NWL-117 caspase inhibitor has a higher selectivity against Caspase-6, −4, −8, −9, and −10 whereas NWL-154 has higher selectivity against Caspase-6, −8, and −10. The half-maximal inhibitory concentrations (IC50) of NWL-117 and NWL-154 is 192 nM and 100 nM against Caspase-6 in vitro, and 4.82 μM and 3.63 μM in Caspase-6-transfected HCT116 cells, respectively. NWL inhibitors are not toxic to HCT116 cells or to human primary neurons. NWL-117 and NWL-154 inhibit serum deprivation-induced Caspase-6 activity and prevent amyloid precursor protein-mediated neurite degeneration in human primary CNS neurons. NWL-117 crosses the blood brain barrier and reverses age-dependent episodic memory deficits in Caspase-6 mice.ConclusionsNWL peptidic vinyl methyl sulfone inhibitors are potent, non-toxic, blood-brain barrier permeable, and irreversible caspase inhibitors with neuroprotective effects in HCT116 cells, in primary human CNS neurons, and in Caspase-6 mice. These results highlight the therapeutic potential of vinyl sulfone inhibitors as caspase inhibitors against neurodegenerative diseases and sanction additional work to improve their selectivity against different caspases.
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Caspase-6 activity in the CA1 region of the hippocampus induces age-dependent memory impairment
Cell Death & Differentiation, 2014Co-Authors: Andrea C. Leblanc, Prateep Pakavathkumar, Jasmine Ramcharitar, Veronica M. Afonso, Edith Hamel, David A. Bennett, Steffen AlbrechtAbstract:Active Caspase-6 is abundant in the neuropil threads, neuritic plaques and neurofibrillary tangles of Alzheimer disease brains. However, its contribution to the pathophysiology of Alzheimer disease is unclear. Here, we show that higher levels of Caspase-6 activity in the CA1 region of aged human hippocampi correlate with lower cognitive performance. To determine whether Caspase-6 activity, in the absence of plaques and tangles, is sufficient to cause memory deficits, we generated a transgenic knock-in mouse that expresses a self-activated form of human Caspase-6 in the CA1. This Caspase-6 mouse develops age-dependent spatial and episodic memory impairment. Caspase-6 induces neuronal degeneration and inflammation. We conclude that Caspase-6 activation in mouse CA1 neurons is sufficient to induce neuronal degeneration and age-dependent memory impairment. These results indicate that Caspase-6 activity in CA1 could be responsible for the lower cognitive performance of aged humans. Consequently, preventing or inhibiting Caspase-6 activity in the aged may provide an efficient novel therapeutic approach against Alzheimer disease.
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Expression and activation of Caspase-6 in human fetal and adult tissues.
PLOS ONE, 2013Co-Authors: Nelly Godefroy, Cynthia G. Goodyer, Benedicte Foveau, Steffen Albrecht, Andrea C. LeblancAbstract:Caspase-6 is an effector caspase that has not been investigated thoroughly despite the fact that Caspase-6 is strongly activated in Alzheimer disease brains. To understand the full physiological impact of Caspase-6 in humans, we investigated Caspase-6 expression. We performed western blot analyses to detect the pro-Caspase-6 and its active p20 subunit in fetal and adult lung, kidney, brain, spleen, muscle, stomach, colon, heart, liver, skin, and adrenals tissues. The levels were semi-quantitated by densitometry. The results show a ubiquitous expression of Caspase-6 in most fetal tissues with the lowest levels in the brain and the highest levels in the gastrointestinal system. Caspase-6 active p20 subunits were only detected in fetal stomach. Immunohistochemical analysis of a human fetal embryo showed active Caspase-6 positive apoptotic cells in the dorsal root ganglion, liver, lung, kidney, ovary, skeletal muscle and the intestine. In the adult tissues, the levels of Caspase-6 were lower than in fetal tissues but remained high in the colon, stomach, lung, kidney and liver. Immunohistological analyses revealed that active Caspase-6 was abundant in goblet cells and epithelial cells sloughing off the intestinal lining of the adult colon. These results suggest that Caspase-6 is likely important in most tissues during early development but is less involved in adult tissues. The low levels of Caspase-6 in fetal and adult brain indicate that increased expression as observed in Alzheimer Disease is a pathological condition. Lastly, the high levels of Caspase-6 in the gastrointestinal system indicate a potential specific function of Caspase-6 in these tissues.
Anne-marie Courtot - One of the best experts on this subject based on the ideXlab platform.
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Lamin A/C, Caspase-6, and Chromatin Configuration During Meiosis Resumption in the Mouse Oocyte
Reproductive Sciences, 2010Co-Authors: Emilie Arnault, Mireille Doussau, Arlette Pesty, Brigitte Lefèvre, Anne-marie CourtotAbstract:After in vitro maturation (IVM), isolation of the healthiest oocytes is essential for successful in vitro fertilization. As germinal vesicle (GV) oocytes resume meiosis through healthy or apoptotic pathways without discernable morphological criteria, we checked for an apoptotic element acting at the nucleus level. We hypothesized that Caspase-6 with its corresponding substrate, lamin A/C, could be a potential target candidate, because Caspase-6 is the only functional caspase for lamin A/C. We used immunohistochemistry methods, Western blots, and a specific Caspase-6 inhibitor to determine the presence of lamin A/C and Caspase-6 during oogenesis and in isolated oocytes. Our results demonstrated that these proteins were always present and that their distributions were related to oocyte maturity, determined by chromatin configuration and oocyte diameter. Caspase-6 inhibition slowed meiosis resumption suggesting the involvement of Caspase-6 in the oocyte apoptotic pathway. Lamin A/C and Caspase-6 could be valuable tools in the knowledge of oocyte in vitro destiny.
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Review: Lamin A/C, Caspase-6, and chromatin configuration during meiosis resumption in the mouse oocyte.
Reproductive Sciences, 2010Co-Authors: Emilie Arnault, Mireille Doussau, Arlette Pesty, Brigitte Lefèvre, Anne-marie CourtotAbstract:After in vitro maturation (IVM), isolation of the healthiest oocytes is essential for successful in vitro fertilization. As germinal vesicle (GV) oocytes resume meiosis through healthy or apoptotic pathways without discernable morphological criteria, we checked for an apoptotic element acting at the nucleus level. We hypothesized that Caspase-6 with its corresponding substrate, lamin A/C, could be a potential target candidate, because Caspase-6 is the only functional caspase for lamin A/C. We used immunohistochemistry methods, Western blots, and a specific Caspase-6 inhibitor to determine the presence of lamin A/C and Caspase-6 during oogenesis and in isolated oocytes. Our results demonstrated that these proteins were always present and that their distributions were related to oocyte maturity, determined by chromatin configuration and oocyte diameter. Caspase-6 inhibition slowed meiosis resumption suggesting the involvement of Caspase-6 in the oocyte apoptotic pathway. Lamin A/C and Caspase-6 could be valuable tools in the knowledge of oocyte in vitro destiny.
Guy S. Salvesen - One of the best experts on this subject based on the ideXlab platform.
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An Optimized Activity-Based Probe for the Study of Caspase-6 Activation
Chemistry & Biology, 2012Co-Authors: Laura E. Edgington, Guy S. Salvesen, Bram J. Van Raam, Martijn Verdoes, Christoph Wierschem, Matthew BogyoAbstract:Summary Although significant efforts have been made to understand the mechanisms of caspase activation during apoptosis, many questions remain regarding how and when executioner caspases get activated. We describe the design and synthesis of an activity-based probe that labels caspase-3/-6/-7, allowing direct monitoring of all executioner caspases simultaneously. This probe has enhanced in vivo properties and reduced cross-reactivity compared to our previously reported probe, AB50. Using this probe, we find that Caspase-6 undergoes a conformational change and can bind substrates even in the absence of cleavage of the proenzyme. We also demonstrate that Caspase-6 activation does not require active caspase-3/-7, suggesting that it may autoactivate or be cleaved by other proteases. Together, our results suggest that Caspase-6 activation proceeds through a unique mechanism that may be important for its diverse biological functions.
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Engineering ML-IAP to produce an extraordinarily potent caspase 9 inhibitor: implications for Smac-dependent anti-apoptotic activity of ML-IAP.
The Biochemical journal, 2005Co-Authors: Dunja Vucic, Kanad Das, Hwain Shin, Linda O. Elliott, Saloumeh Kadkhodayan, Matthew C. Franklin, Heidi J. Wallweber, B P Eckelman, Kurt Deshayes, Guy S. SalvesenAbstract:ML-IAP (melanoma inhibitor of apoptosis) is a potent anti-apoptotic protein that is strongly up-regulated in melanoma and confers protection against a variety of pro-apoptotic stimuli. The mechanism by which ML-IAP regulates apoptosis is unclear, although weak inhibition of caspases 3 and 9 has been reported. Here, the binding to and inhibition of caspase 9 by the single BIR (baculovirus IAP repeat) domain of ML-IAP has been investigated and found to be significantly less potent than the ubiquitously expressed XIAP (X-linked IAP). Engineering of the ML-IAP-BIR domain, based on comparisons with the third BIR domain of XIAP, resulted in a chimeric BIR domain that binds to and inhibits caspase 9 significantly better than either ML-IAP-BIR or XIAP-BIR3. Mutational analysis of the ML-IAP-BIR domain demonstrated that similar enhancements in caspase 9 affinity can be achieved with only three amino acid substitutions. However, none of these modifications affected binding of the ML-IAP-BIR domain to the IAP antagonist Smac (second mitochondrial activator of caspases). ML-IAP-BIR was found to bind mature Smac with low nanomolar affinity, similar to that of XIAP-BIR2-BIR3. Correspondingly, increased expression of ML-IAP results in formation of a ML-IAP-Smac complex and disruption of the endogenous interaction between XIAP and mature Smac. These results suggest that ML-IAP might regulate apoptosis by sequestering Smac and preventing it from antagonizing XIAP-mediated inhibition of caspases, rather than by direct inhibition of caspases.
Emilie Arnault - One of the best experts on this subject based on the ideXlab platform.
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Lamin A/C, Caspase-6, and Chromatin Configuration During Meiosis Resumption in the Mouse Oocyte
Reproductive Sciences, 2010Co-Authors: Emilie Arnault, Mireille Doussau, Arlette Pesty, Brigitte Lefèvre, Anne-marie CourtotAbstract:After in vitro maturation (IVM), isolation of the healthiest oocytes is essential for successful in vitro fertilization. As germinal vesicle (GV) oocytes resume meiosis through healthy or apoptotic pathways without discernable morphological criteria, we checked for an apoptotic element acting at the nucleus level. We hypothesized that Caspase-6 with its corresponding substrate, lamin A/C, could be a potential target candidate, because Caspase-6 is the only functional caspase for lamin A/C. We used immunohistochemistry methods, Western blots, and a specific Caspase-6 inhibitor to determine the presence of lamin A/C and Caspase-6 during oogenesis and in isolated oocytes. Our results demonstrated that these proteins were always present and that their distributions were related to oocyte maturity, determined by chromatin configuration and oocyte diameter. Caspase-6 inhibition slowed meiosis resumption suggesting the involvement of Caspase-6 in the oocyte apoptotic pathway. Lamin A/C and Caspase-6 could be valuable tools in the knowledge of oocyte in vitro destiny.
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Review: Lamin A/C, Caspase-6, and chromatin configuration during meiosis resumption in the mouse oocyte.
Reproductive Sciences, 2010Co-Authors: Emilie Arnault, Mireille Doussau, Arlette Pesty, Brigitte Lefèvre, Anne-marie CourtotAbstract:After in vitro maturation (IVM), isolation of the healthiest oocytes is essential for successful in vitro fertilization. As germinal vesicle (GV) oocytes resume meiosis through healthy or apoptotic pathways without discernable morphological criteria, we checked for an apoptotic element acting at the nucleus level. We hypothesized that Caspase-6 with its corresponding substrate, lamin A/C, could be a potential target candidate, because Caspase-6 is the only functional caspase for lamin A/C. We used immunohistochemistry methods, Western blots, and a specific Caspase-6 inhibitor to determine the presence of lamin A/C and Caspase-6 during oogenesis and in isolated oocytes. Our results demonstrated that these proteins were always present and that their distributions were related to oocyte maturity, determined by chromatin configuration and oocyte diameter. Caspase-6 inhibition slowed meiosis resumption suggesting the involvement of Caspase-6 in the oocyte apoptotic pathway. Lamin A/C and Caspase-6 could be valuable tools in the knowledge of oocyte in vitro destiny.
Zhenfeng Ye - One of the best experts on this subject based on the ideXlab platform.
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MicroRNA-21 attenuates renal ischemia reperfusion injury via targeting caspase signaling in mice
American Journal of Nephrology, 2014Co-Authors: Honglin Hu, Cong Zou, Xiaoqing Xi, Wei Jiang, Zhenfeng YeAbstract:BACKGROUND: MicroRNAs (miR) have come into focus as powerful regulators of gene expression and potential diagnostic tools during renal ischemia reperfusion injury (IRI). The aim of this study was to investigate the molecular regulation and function of miR-21, and to analyze the relationship between caspases and miR-21 expression levels in an experimental model of renal IRI.\n\nMETHODS: IRI was induced by bilateral renal ischemia for 45 min followed by reperfusion. The male BALB/c mice were randomly assigned to the following groups: pre-miR-21 + IRI group, antagomiR-21 + IRI group, PBS + IRI group, pre-miR-21 + sham operation group, antagomiR-21 + sham operation group, PBS + sham operation group. The pre-miR-21 or antagomiR-21 was administered intraperitoneally (200 ng/kg weight) 24 and 6 h before induction of ischemia. Renal function, histological damage, renal cell apoptosis proteins were evaluated at 24 h after reperfusion.\n\nRESULTS: Mice upregulated miR-21 had lower plasma levels of blood urea nitrogen (BUN) and creatinine, lower histopathological scores and a decrease in programmed cell death 4 (PDCD4) mRNA and active caspase-3, caspase-8 proteins expressions.\n\nCONCLUSIONS: miR-21 is endowed with anti-apoptotic properties by suppressing the expression of PDCD4 gene and active caspase 3/8 fragments in the condition of renal IRI. miR-21 exerts significant functional protection in our renal murine model of IRI.