Decreased AdipoR1 signaling and its implications for obesity-induced male infertility.

Obesity is among the risk factors for male infertility. Although several mechanisms underlying obesity-induced male subfertility have been reported, the entire mechanism of obesity-induced male infertility still remains unclear. Here, we show that sperm count, sperm motility and sperm fertilizing ability were decreased in male mice fed a high-fat diet and that the expression of the AdipoR1 gene and protein was decreased, and the expression of pro-apoptotic genes and protein increased, in the testis from mice fed a high-fat diet. Moreover, we demonstrate that testes weight, sperm count, sperm motility and sperm fertilizing ability were significantly decreased in AdipoR1 knockout mice compared to those in wild-type mice; furthermore, the phosphorylation of AMPK was decreased, and the expression of pro-apoptotic genes and proteins, caspase-6 activity and pathologically apoptotic seminiferous tubules were increased, in the testis from AdipoR1 knockout mice. Furthermore, study findings show that orally administrated AdipoRon decreased caspase-6 activity and apoptotic seminiferous tubules in the testis, thus ameliorating sperm motility in male mice fed a high-fat diet. This was the first study to demonstrate that decreased AdipoR1/AMPK signaling led to increased caspase-6 activity/increased apoptosis in the testis thus likely accounting for male infertility.

Spinal Caspase-6 Contributes to Intrathecal Morphine-induced Acute Itch and Contact Dermatitis-induced Chronic Itch Through Regulating the Phosphorylation of Protein Kinase Mζ in Mice.

Patients receiving neuraxial treatment with morphine for pain relief often experience a distressing pruritus. Neuroinflammation-mediated plasticity of sensory synapses in the spinal cord is critical for the development of pain and itch. Caspase-6, as an intracellular cysteine protease, is capable of inducing central nociceptive sensitization through regulating synaptic transmission and plasticity. Given the tight interaction between protein kinase Mζ (PKMζ) and excitatory synaptic plasticity, this pre-clinical study investigates whether caspase-6 contributes to morphine-induced itch and chronic itch via PKMζ. Intrathecal morphine and contact dermatitis were used to cause pruritus in mice. Morphine antinociception, itch-induced scratching behaviors, spinal activity of caspase-6, and phosphorylation of PKMζ and ERK were examined. Caspase-6 inhibitor Z-VEID-FMK, exogenous caspase-6 and PKMζ inhibitor ZIP were utilized to reveal the mechanisms and prevention of itch. Herein, we report that morphine induces significant scratching behaviors, which is accompanied by an increase in spinal caspase-6 cleavage and PKMζ phosphorylation (but not expression). Intrathecal injection of Z-VEID-FMK drastically reduces morphine-induced scratch bouts and spinal phosphorylation of PKMζ, without abolishing morphine analgesia. Moreover, intrathecal strategies of ZIP dose-dependently reduce morphine-induced itch-like behaviors. Spinal phosphorylation of ERK following neuraxial morphine is down-regulated by ZIP therapy. Recombinant caspase-6 directly exhibits scratching behaviors and spinal phosphorylation of ERK, which is compensated by PKMζ inhibition. Also, spinal inhibition of caspase-6 and PKMζ reduces the generation and maintenance of dermatitis-induced chronic itch. Together, these findings demonstrate that spinal caspase-6 modulation of PKMζ phosphorylation is important in the development of morphine-induced itch and dermatitis-induced itch in mice.

Antisense oligonucleotide-mediated disruption of HTT caspase-6 cleavage site ameliorates the phenotype of YAC128 Huntington disease mice.

In Huntington disease, cellular toxicity is particularly caused by toxic protein fragments generated from the mutant huntingtin (HTT) protein. By modifying the HTT protein, we aim to reduce proteolytic cleavage and ameliorate the consequences of mutant HTT without lowering total HTT levels. To that end, we use an antisense oligonucleotide (AON) that targets HTT pre-mRNA and induces partial skipping of exon 12, which contains the critical caspase-6 cleavage site. Here, we show that AON-treatment can partially restore the phenotype of YAC128 mice, a mouse model expressing the full-length human HTT gene including 128 CAG-repeats. Wild-type and YAC128 mice were treated intracerebroventricularly with AON12.1, scrambled AON or vehicle starting at 6 months of age and followed up to 12 months of age, when MRI was performed and mice were sacrificed. AON12.1 treatment induced around 40% exon skip and protein modification. The phenotype on body weight and activity, but not rotarod, was restored by AON treatment. Genes differentially expressed in YAC128 striatum changed toward wild-type levels and striatal volume was preserved upon AON12.1 treatment. However, scrambled AON also showed a restorative effect on gene expression and appeared to generally increase brain volume.

Caspase 6 promotes innate immune activation by functional crosstalk between RIPK1-IκBα axis in liver inflammation.

BACKGROUND: Caspase 6 is an essential regulator in innate immunity, inflammasome activation and host defense. We aimed to characterize the causal mechanism of Caspase 6 in liver sterile inflammatory injury. METHODS: Human liver tissues were harvested from patients undergoing ischemia-related hepatectomy to evaluate Caspase 6 expression. Subsequently, we created Caspase 6-knockout (Caspase 6KO) mice to analyze roles and molecular mechanisms of macrophage Caspase 6 in murine models of liver ischemia/reperfusion (IR) injury. RESULTS: In human liver biopsies, Caspase 6 expression was positively correlated with more severe histopathological injury and higher serum ALT/AST level at one day postoperatively. Moreover, Caspase 6 was mainly elevated in macrophages but not hepatocytes in ischemic livers. Unlike in controls, the Caspase 6-deficient livers were protected against IR injury, as evidenced by inhibition of inflammation, oxidative stress and iron overload. Disruption of macrophage NF-κB essential modulator (NEMO) in Caspase 6-deficient livers deteriorated liver inflammation and ferroptosis. Mechanistically, Caspase 6 deficiency spurred NEMO-mediated IκBα phosphorylation in macrophage. Then phosphorylated-inhibitor of NF-κBα (p-IκBα) co-localized with receptor-interacting serine/ threonine-protein kinase 1 (RIPK1) in the cytoplasm to degradate RIPK1 under inflammatory conditions. The disruption of RIPK1-IκBα interaction preserved RIPK1 degradation, triggering downstream apoptosis signal-regulating kinase 1 (ASK1) phosphorylation and inciting NIMA-related kinase 7/NOD-like receptor family pyrin domain containing 3 (NEK7/NLRP3) activation in macrophages. Moreover, ablation of macrophage RIPK1 or ASK1 diminished NEK7/NLRP3-driven inflammatory response and dampened hepatocyte ferroptosis by reducing HMGB1 release from macrophages. CONCLUSIONS: Our findings underscore a novel mechanism of Caspase 6 mediated RIPK1-IκBα interaction in regulating macrophage NEK7/NLRP3 function and hepatocytes ferroptosis, which provides therapeutic targets for clinical liver IR injury. Video Abstract.

Role of cited-1 and caspase-6 expression in HELLP syndrome.

OBJECTIVE: In this study, we investigated the immunohistochemical staining of cited-1 and caspase-6 expression in the placentas of pregnant women with HELLP syndrome. PATIENTS AND METHODS: Placentas of 20 normotensive patients and 20 women with HELLP syndrome were processed for routine histological tissue processing. The biochemical and clinical parameters of patients were recorded. Placentas were stained with hematoxylin-eosin and cited-1 and caspase-6 immunostaining. RESULTS: Placentas of normotensive patients showed normal histology. Placentas of women with HELLP syndrome showed degenerated cells, hyalinization and vacuolization. Cited-1 expression was negative in normotensive group; however, it was increased in HELLP group, especially in decidual cells, endothelial cells and other placental cells. Caspase-6 expression was negative in placental structures of normotensive groups. However, it was intense in decidual cells, vacuolar and hyalinized areas, inflammatory cells and connective tissue cells in HELLP group. CONCLUSIONS: Cited-1 and caspase-6 are a marker in determining the severity of HELLP syndrome.

Royal jelly protects brain tissue against fluoride-induced damage by activating Bcl-2/NF-κB/caspase-3/caspase-6/Bax and Erk signaling pathways in rats.

This study is aimed at determining whether royal jelly (RJ) which has a powerful antioxidant property prevents fluoride-induced brain tissue damage and exploring whether Bcl-2/NF-κB/ and caspase-3/caspase-6/Bax/Erk pathways play a critical role in the neuroprotective effect of RJ. Wistar albino rats were chosen for the study, and they were randomly distributed into six groups: (i) control; (ii) royal jelly; (iii) fluoride-50; (iv) fluoride-100; (v) fluoride-50 + royal jelly; (vi) fluoride-100 + royal jelly. We established fluoride-induced brain tissue damage with 8-week-old male Wistar albino rats by administration of fluoride exposure (either 50 mg/kg or 100 mg/kg bw) through drinking water for 8 weeks. Then, the study duration is for 56 days where the rats were treated with or without RJ (100 mg/kg bw) through oral gavage. The effects of RJ on glutathione (GSH), catalase activity (CAT), and malondialdehyde (MDA) levels were determined via spectrophotometer. Western blot analysis was performed to investigate the effects of royal jelly on the protein expression levels of Bax, caspase-3, caspase-6, Bcl-2, NF-κB, COX-2, and Erk. It was also studied the effects of RJ on histopathological alterations in fluoride-induced damage to the rat brain. As a result, the Bcl-2, NF-κB, and COX-2 protein expression levels were increased in the fluoride-treated (50 and 100 mg/kg) groups but they were decreased significantly by RJ treatment in the brain tissue. Additionally, the protein expression of caspase-3, caspase-6, Bax, and Erk were decreased in fluoride-treated groups and they were significantly increased by RJ treatment compared to the un-treated rats. Our results suggested that RJ prevented fluoride-induced brain tissue damage through anti-antioxidant activities.

Caspase-6 is a key regulator of cross-talk signal way in PANoptosis in cancer.

Cysteinyl aspartate specific proteinase (caspase)-6 belongs to the caspase family and plays a vital role in mediating cell death. Under certain conditions, three pathways of programmed cell death (PCD), including apoptosis, necroptosis and pyroptosis (PANoptosis), transform one way into another, with enormous therapeutic potential. Initially, scholars reported that caspase-6 is a caspase executor that mediates apoptosis. With the ceaseless exploration of the PCD types, studies have demonstrated that caspase-6 mediates pyroptosis by regulating gasdermin D and mediates necroptosis by regulating mixed lineage kinase domain-like. By regulating PANoptosis, caspase-6 plays a crucial role in tumorigenesis in humans and mediates anti-tumour immunity. Therefore, a comprehensive understanding of caspase-6 function in cancer via PANoptosis is important for the prevention and therapy of tumours. This article summarized the function of caspase-6 in PANoptosis and its impact on cancer development, providing targets and strategies for tumour treatment.

Enzalutamide Induces Apoptotic Insults to Human Drug-Resistant and -Sensitive Glioblastoma Cells via an Intrinsic Bax-Mitochondrion-Cytochrome C Caspase Cascade Activation Pathway.

Glioblastoma multiforme (GBM) is the most common and malignant brain tumor. Temozolomide (TMZ) is the first-line chemotherapeutic drug for treating GBM. However, drug resistance is still a challenging issue in GBM therapy. Our preliminary results showed upregulation of androgen receptor (AR) gene expression in human GBM tissues. This study was designed to evaluate the effects of enzalutamide, a specific inhibitor of the AR, on killing drug-resistant and -sensitive glioblastoma cells and the possible mechanisms. Data mining from The Cancer Genome Atlas (TCGA) database revealed upregulation of AR messenger (m)RNA and protein expressions in human GBM tissues, especially in male patients, compared to normal human brains. In addition, expressions of AR mRNA and protein in human TMZ-sensitive U87 MG and -resistant U87 MG-R glioblastoma cells were elevated compared to normal human astrocytes. Exposure of human U87 MG and U87 MG-R cells to enzalutamide concentration- and time-dependently decreased cell viability. As to the mechanism, enzalutamide killed these two types of glioblastoma cells via an apoptotic mechanism. Specifically, exposure to enzalutamide augmented enzyme activities of caspase-9 rather than those of caspase-8. Moreover, enzalutamide successively triggered an elevation in levels of the proapoptotic Bax protein, a reduction in the mitochondrial membrane potential, release of cytochrome c, cascade activation of caspases-3 and -6, DNA fragmentation, and cell apoptosis in human TMZ-sensitive and -resistant glioblastoma cells. Pretreatment with Z-VEID-FMK, an inhibitor of caspase-6, caused significant attenuations in enzalutamide-induced morphological shrinkage, DNA damage, and apoptotic death. Taken together, this study showed that enzalutamide could significantly induce apoptotic insults to human drug-resistant and -sensitive glioblastoma cells via an intrinsic Bax-mitochondrion-cytochrome c-caspase cascade activation pathway. Enzalutamide has the potential to be a drug candidate for treating GBM by targeting the AR signaling axis.

A new approach on the regulation of NF-κB and Bax protein signaling pathway activation by royal jelly in fluoride-induced pancreas damage in rats.

Forty-two healthy adult male rats (Wistar albino, n = 42, 8 weeks old, starting weights 200-250 g) employed in this study were subdivided into six groups randomly with seven rats per group as follows: (i) Control group: received standard diet; (ii) RJ group: received standard diet supplemented with royal jelly; (iii) F50 group: received standard diet supplemented with fluoride (50 mg/kg BW); (iv) F100 group: received standard diet supplemented with fluoride (100 mg/kg BW); (v) F50 +RJ group: received standard diet supplemented with fluoride (50 mg/kg BW) and royal jelly; (iv) F100 +RJ group: received standard diet supplemented with fluoride (100 mg/kg BW) and royal jelly. The study continued for a total of eight weeks. Western blot analysis was conducted to determine the post-translational expression levels of NF-κB, Bax, Bcl-2, TNF-α, Caspase-3 and Caspase-6 proteins in pancreas tissue. The pancreatic tissue was subjected to histopathological evaluation. Furthermore, MDA, GSH and CAT activities were examined by spectrophotometric analyzes. Our findings demonstrate that, compared to the control and RJ groups, Bcl-2 protein expression was augmented and, conversely, Caspase-6, Caspase-3 and Bax protein levels were decreased upon fluoride treatment. A statistically significant increase in TNF-α and NF-κB protein expressions was observed in the groups with fluoride-induced damage compared to the control and RJ groups. The MDA levels were increased in all fluoride-treated rats compared to those in the control and RJ groups, whereas the CAT and GSH activities were reduced in all rats with fluoride- induced damage. Although there was not a great difference between the groups regarding histopathological findings, there was a tendency to decrease in the rate of damage upon royal jelly treatment.

Coronaviruses exploit a host cysteine-aspartic protease for replication.

Highly pathogenic coronaviruses, including severe acute respiratory syndrome coronavirus 2 (refs. 1,2) (SARS-CoV-2), Middle East respiratory syndrome coronavirus3 (MERS-CoV) and SARS-CoV-1 (ref. 4), vary in their transmissibility and pathogenicity. However, infection by all three viruses results in substantial apoptosis in cell culture5-7 and in patient tissues8-10, suggesting a potential link between apoptosis and pathogenesis of coronaviruses. Here we show that caspase-6, a cysteine-aspartic protease of the apoptosis cascade, serves as an important host factor for efficient coronavirus replication. We demonstrate that caspase-6 cleaves coronavirus nucleocapsid proteins, generating fragments that serve as interferon antagonists, thus facilitating virus replication. Inhibition of caspase-6 substantially attenuates lung pathology and body weight loss in golden Syrian hamsters infected with SARS-CoV-2 and improves the survival of mice expressing human DPP4 that are infected with mouse-adapted MERS-CoV. Our study reveals how coronaviruses exploit a component of the host apoptosis cascade to facilitate virus replication.

Protective effect of royal jelly on fluoride-induced nephrotoxicity in rats via the some protein biomarkers signalling pathways: a new approach for kidney damage.

INTRODUCTION: Protective effect of royal jelly (RJ) on fluoride-induced nephrotoxicity was investigated in this study. METHODS: 42 healthy male Wistar rats (n = 42, 8 weeks of age) were divided equally into 6 groups with 7 rats in each; (1) Group-1: Controls fed with standard diet; (2) Group-2: RJ [100 mg/kg] bw (body weight), by oral gavage; (3) Group-3: Fluoride [50 mg/kg] bw, in drinking water; (4) Group-4: Fluoride [100 mg/kg] bw, in drinking water; (5) Group-5: RJ [100 mg/kg] bw, by oral gavage + Fluoride [50 mg/kg] bw, in drinking water; (6) Group-6: RJ [100 mg/kg] bw, by oral gavage + Fluoride [100 mg/kg] bw, in drinking water. After 8 weeks, all rats were decapitated and their kidney tissues were removed for further analysis. The protein expression levels of caspase-3, caspase-6, caspase-9, Bcl-2, Bax, VEGF, GSK-3, BDNF, COX-2 and TNF-α proteins in kidney tissue were analysed by western blotting technique. RESULTS: RJ increased Bcl-2, COX-2, GSK-3, TNF-α and VEGF protein levels and a decreased caspase-3, caspase -6, caspase-9, Bax and BDNF protein levels in fluoride-treated rats. CONCLUSION: RJ application may have a promising therapeutical potential in the treatment of many diseases in the future by reducing kidney damage.

FGF4 protects the liver from nonalcoholic fatty liver disease by activating the AMP-activated protein kinase-Caspase 6 signal axis.

BACKGROUND AND AIMS: NAFLD represents an increasing health problem in association with obesity and diabetes with no effective pharmacotherapies. Growing evidence suggests that several FGFs play important roles in diverse aspects of liver pathophysiology. Here, we report a previously unappreciated role of FGF4 in the liver. APPROACH AND RESULTS: Expression of hepatic FGF4 is inversely associated with NAFLD pathological grades in both human patients and mouse models. Loss of hepatic Fgf4 aggravates hepatic steatosis and liver damage resulted from an obesogenic high-fat diet. By contrast, pharmacological administration of recombinant FGF4 mitigates hepatic steatosis, inflammation, liver damage, and fibrogenic markers in mouse livers induced to develop NAFLD and NASH under dietary challenges. Such beneficial effects of FGF4 are mediated predominantly by activating hepatic FGF receptor (FGFR) 4, which activates a downstream Ca2+ -Ca2+ /calmodulin-dependent protein kinase kinase beta-dependent AMP-activated protein kinase (AMPK)-Caspase 6 signal axis, leading to enhanced fatty acid oxidation, reduced hepatocellular apoptosis, and mitigation of liver damage. CONCLUSIONS: Our study identifies FGF4 as a stress-responsive regulator of liver pathophysiology that acts through an FGFR4-AMPK-Caspase 6 signal pathway, shedding light on strategies for treating NAFLD and associated liver pathologies.

Therapeutic potential of Nlrp1 inflammasome, Caspase-1, or Caspase-6 against Alzheimer disease cognitive impairment.

The sequential activation of Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing protein 1 (Nlrp1) inflammasome, Caspase-1 (Casp1), and Caspase-6 (Casp6) is implicated in primary human neuron cultures and Alzheimer Disease (AD) neurodegeneration. To validate the Nlrp1-Casp1-Casp6 pathway in vivo, the APPSwedish/Indiana J20 AD transgenic mouse model was generated on either a Nlrp1, Casp1 or Casp6 null genetic background and mice were studied at 4-5 months of age. Episodic memory deficits assessed with novel object recognition were normalized by genetic ablation of Nlrp1, Casp1, or Casp6 in J20 mice. Spatial learning deficits, assessed with the Barnes Maze, were normalized in genetically ablated J20, whereas memory recall was normalized in J20/Casp1-/- and J20/Casp6-/-, and improved in J20/Nlrp1-/- mice. Hippocampal CA1 dendritic spine density of the mushroom subtype was reduced in J20, and normalized in genetically ablated J20 mice. Reduced J20 hippocampal dentate gyrus and CA3 synaptophysin levels were normalized in genetically ablated J20. Increased Iba1+-microglia in the hippocampus and cortex of J20 brains were normalized by Casp1 and Casp6 ablation and reduced by Nlrp1 ablation. Increased pro-inflammatory cytokines, TNF-α and CXCL1, in the J20 hippocampus were normalized by Nlrp1 or Casp1 genetic ablation. CXCL1 was also normalized by Casp6 genetic ablation. IFN-γ was increased and total amyloid ß peptide was decreased in genetically ablated Nlrp1, Casp1 or Casp6 J20 hippocampi. We conclude that Nlrp1, Casp1, or Casp6 are implicated in AD-related cognitive impairment, inflammation, and amyloidogenesis. These results indicate that Nlrp1, Casp1, and Casp6 represent rational therapeutic targets against cognitive impairment and inflammation in AD.

Caspase-9 Activation of Procaspase-3 but Not Procaspase-6 Is Based on the Local Context of Cleavage Site Motifs and on Sequence.

Studying the interactions between a protease and its protein substrates at a molecular level is crucial for identifying the factors facilitating selection of particular proteolytic substrates and not others. These selection criteria include both the sequence and the local context of the substrate cleavage site where the active site of the protease initially binds and then performs proteolytic cleavage. Caspase-9, an initiator of the intrinsic apoptotic pathway, mediates activation of executioner procaspase-3 by cleavage of the intersubunit linker (ISL) at site 172IETD↓S. Although procaspase-6, another executioner, possesses two ISL cleavage sites (site 1, 176DVVD↓N; site 2, 190TEVD↓A), neither is directly cut by caspase-9. Thus, caspase-9 directly activates procaspase-3 but not procaspase-6. To elucidate this selectivity of caspase-9, we engineered constructs of procaspase-3 (e.g., swapping the ISL site, 172IETD↓S, with DVVDN and TEVDA) and procaspase-6 (e.g., swapping site 1, 176DVVD↓N, and site 2, 190TEVD↓A, with IETDS). Using the substrate digestion data of these constructs, we show here that the P4-P1' sequence of procaspase-6 ISL site 1 (DVVDN) can be accessed but not cleaved by caspase-9. We also found that caspase-9 can recognize the P4-P1' sequence of procaspase-6 ISL site 2 (TEVDA); however, the local context of this cleavage site is the critical factor that prevents proteolytic cleavage. Overall, our data have demonstrated that both the sequence and the local context of the ISL cleavage sites play a vital role in preventing the activation of procaspase-6 directly by caspase-9.

Rare CASP6N73T variant associated with hippocampal volume exhibits decreased proteolytic activity, synaptic transmission defect, and neurodegeneration.

Caspase-6 (Casp6) is implicated in Alzheimer disease (AD) cognitive impairment and pathology. Hippocampal atrophy is associated with cognitive impairment in AD. Here, a rare functional exonic missense CASP6 single nucleotide polymorphism (SNP), causing the substitution of asparagine with threonine at amino acid 73 in Casp6 (Casp6N73T), was associated with hippocampal subfield CA1 volume preservation. Compared to wild type Casp6 (Casp6WT), recombinant Casp6N73T altered Casp6 proteolysis of natural substrates Lamin A/C and α-Tubulin, but did not alter cleavage of the Ac-VEID-AFC Casp6 peptide substrate. Casp6N73T-transfected HEK293T cells showed elevated Casp6 mRNA levels similar to Casp6WT-transfected cells, but, in contrast to Casp6WT, did not accumulate active Casp6 subunits nor show increased Casp6 enzymatic activity. Electrophysiological and morphological assessments showed that Casp6N73T recombinant protein caused less neurofunctional damage and neurodegeneration in hippocampal CA1 pyramidal neurons than Casp6WT. Lastly, CASP6 mRNA levels were increased in several AD brain regions confirming the implication of Casp6 in AD. These studies suggest that the rare Casp6N73T variant may protect against hippocampal atrophy due to its altered catalysis of natural protein substrates and intracellular instability thus leading to less Casp6-mediated damage to neuronal structure and function.

Caspase-6-cleaved Tau fails to induce Tau hyperphosphorylation and aggregation, neurodegeneration, glial inflammation, and cognitive deficits.

Active Caspase-6 (Casp6) and Tau cleaved by Casp6 at amino acids 402 (Tau∆D402) and 421 (Tau∆D421) are present in early Alzheimer disease intraneuronal neurofibrillary tangles, which are made primarily of filamentous Tau aggregates. To assess whether Casp6 cleavage of Tau contributes to Tau pathology and Casp6-mediated age-dependent cognitive impairment, we generated transgenic knock-in mouse models that conditionally express full-length human Tau (hTau) 0N4R only (CTO) or together with human Casp6 (hCasp6) (CTC). Region-specific hippocampal and cortical hCasp6 and hTau expression were confirmed with western blot and immunohistochemistry in 2-25-month-old brains. Casp6 activity was confirmed with Tau∆D421 and Tubulin cleaved by Casp6 immunopositivity in 3-25-month-old CTC, but not in CTO, brains. Immunoprecipitated Tau∆D402 was detected in both CTC and CTO brains, but was more abundant in CTC brains. Intraneuronal hippocampal Tau hyperphosphorylation at S202/T205, S422, and T231, and Tau conformational change were absent in both CTC and CTO brains. A slight accumulation of Tau phosphorylated at S396/404 and S202 was observed in Cornu Ammonis 1 (CA1) hippocampal neuron soma of CTC compared to CTO brains. Eighteen-month-old CTC brains showed rare argentophilic deposits that increased by 25 months, whereas CTO brains only displayed them sparsely at 25 months. Tau microtubule binding was equivalent in CTC and CTO hippocampi. Episodic and spatial memory measured with novel object recognition and Barnes maze, respectively, remained normal in 3-25-month-old CTC and CTO mice, in contrast to previously observed impairments in ACL mice expressing equivalent levels of hCasp6 only. Consistently, the CTC and CTO hippocampal CA1 region displayed equivalent dendritic spine density and no glial inflammation. Together, these results reveal that active hCasp6 co-expression with hTau generates Tau cleavage and rare age-dependent argentophilic deposits but fails to induce cognitive deficits, neuroinflammation, and Tau pathology.

Analysis of the lamprey genotype provides insights into caspase evolution and functional divergence.

The cysteine-containing aspartate specific proteinase (caspase) family plays important roles in apoptosis and the maintenance of homeostasis in lampreys. We conducted genomic and functional comparisons of six distinct lamprey caspase groups with human counterparts to determine how these expanded molecules evolved to adapt to the changing caspase-mediated signaling pathways. Our results showed that lineage-specific duplication and rearrangement were responsible for expanding lamprey caspases 3 and 7, whereas caspases 1, 6, 8, and 9 maintained a relatively stable genome and protein structure. Lamprey caspase family molecules displayed various expression patterns and were involved in the innate immune response. Caspase 1 and 7 functioned as a pattern recognition receptor with a broad-spectrum of microbial recognition and bactericidal effect. Additionally, caspases 1 and 7 may induce cell apoptosis in a time- and dose-dependent manner; however, apoptosis was inhibited by caspase inhibitors. Thus, these molecules may reflect the original state of the vertebrates caspase family. Our phylogenetic and functional data provide insights into the evolutionary history of caspases and illustrate their functional characteristics in primitive vertebrates.

Remodeling hydrogen bond interactions results in relaxed specificity of Caspase-3.

Caspase (or cysteinyl-aspartate specific proteases) enzymes play important roles in apoptosis and inflammation, and the non-identical but overlapping specificity profiles (that is, cleavage recognition sequence) direct cells to different fates. Although all caspases prefer aspartate at the P1 position of the substrate, the caspase-6 subfamily shows preference for valine at the P4 position, while caspase-3 shows preference for aspartate. In comparison with human caspases, caspase-3a from zebrafish has relaxed specificity and demonstrates equal selection for either valine or aspartate at the P4 position. In the context of the caspase-3 conformational landscape, we show that changes in hydrogen bonding near the S3 subsite affect selection of the P4 amino acid. Swapping specificity with caspase-6 requires accessing new conformational space, where each landscape results in optimal binding of DxxD (caspase-3) or VxxD (caspase-6) substrate and simultaneously disfavors binding of the other substrate. Within the context of the caspase-3 conformational landscape, substitutions near the active site result in nearly equal activity against DxxD and VxxD by disrupting a hydrogen bonding network in the substrate binding pocket. The converse substitutions in zebrafish caspase-3a result in increased selection for P4 aspartate over valine. Overall, the data show that the shift in specificity that results in a dual function protease, as in zebrafish caspase-3a, requires fewer amino acid substitutions compared with those required to access new conformational space for swapping substrate specificity, such as between caspases-3 and -6.

Anticancer active trifluoromethylated fused triazinones are safe for early-life stages of zebrafish (Danio rerio) and reveal a proapoptotic action.

The main purpose of this investigation was to evaluate the effect of anticancer active compounds (I-VIII) on zebrafish development in order to select the safest molecules. Larval mortality, embryo hatchability and malformations were end-points used to assess the acute toxicity among embryos and larvae from compounds-/pemetrexed-treated and control groups. LC50 and MNLC (maximal non-lethal concentration) were determined. Lipophilicity-dependent structure-toxicity relationships were established. The results clearly indicated that the majority of test molecules are safe for zebrafish individuals and simultaneously are less toxic than an anticancer agent - pemetrexed. The subsequent aim of this study was to elucidate the molecular mechanism of antiproliferative activity of the most selective compounds. Substantially increased activation of caspase-6 and -8 in cancerous cell lines confirmed the proapoptotic action of molecules examined. Considering the safety for zebrafish individuals, the title compounds as inducers of apoptosis are promising drug candidates in the preclinical phase of drug development.