A Whole Cell Assay to Measure Caspase-6 Activity by Detecting Cleavage of Lamin A/C

Caspase-6 is a cysteinyl protease implicated in neurodegenerative conditions including Alzheimer's and Huntington's disease making it an attractive target for therapeutic intervention. A greater understanding of the role of caspase-6 in disease has been hampered by a lack of suitable cellular assays capable of specifically detecting caspase-6 activity in an intact cell environment. This is mainly due to the use of commercially available peptide substrates and inhibitors which lack the required specificity to facilitate development of this type of assay. We report here a 384-well whole-cell chemiluminescent ELISA assay that monitors the proteolytic degradation of endogenously expressed lamin A/C during the early stages of caspase-dependent apoptosis. The specificity of lamin A/C proteolysis by caspase-6 was demonstrated against recombinant caspase family members and further confirmed in genetic deletion studies. In the assay, plasma membrane integrity remained intact as assessed by release of lactate dehydrogenase from the intracellular environment and the exclusion of cell impermeable peptide inhibitors, despite the induction of an apoptotic state. The method described here is a robust tool to support drug discovery efforts targeting caspase-6 and is the first reported to specifically monitor endogenous caspase-6 activity in a cellular context

Specific induction of pp125 focal adhesion kinase in human breast cancer

The pp125 focal adhesion kinase (FAK) is involved in integrin-mediated cell signalling and overexpressed in a variety of solid tumours. Focal adhesion kinase expression has been correlated to invasion and metastasis, but the data on breast cancer are inconclusive. We analysed FAK mRNA, protein levels and expression patterns in primary breast cancer and normal breast tissue. FAK expression on the functional protein level and mRNA was determined in 55 matched pairs of breast cancer and corresponding normal tissue by Western blot, immunohistochemistry and RT–PCR. Using a score ranging from 0 to +5 for Western blots, we determined in normal breast tissue a score of 1.51±0.84 (mean±standard deviation), which was strongly induced to 2.91 (±1.22) in breast cancers (P<0.001). Overall, 45 out of 55 tissue pairs (81.8%) showed this upregulation of FAK protein in tumours in comparison to normal tissue. Immunohistochemistry confirmed these findings with a significant higher score for tumours vs physiological tissue (1.0±0.63 vs 2.27±0.91; P=0.001). Interestingly, no overall significant difference in the mRNA levels (P=0.359) was observed. In conclusion, expression levels of the FAK protein are specifically upregulated in breast cancer in comparison to matched normal breast tissue supporting its pivotal role in neoplastic signal transduction and representing a potential marker for malignant transformation

Familial amyloid precursor protein mutants cause caspase-6-dependent but amyloid β-peptide-independent neuronal degeneration in primary human neuron cultures.

Although familial Alzheimer disease (AD)-associated autosomal dominant mutants have been extensively studied, little is known about the underlying molecular mechanisms of neurodegeneration induced by these mutants in AD. Wild-type, Swedish or London amyloid precursor protein (APP) transfection in primary human neurons induced neuritic beading, in which several co-expressed proteins, such as enhanced green fluorescent protein, red fluorescent protein (RFP)-tau and RFP-ubiquitin, accumulated. APP-induced neuritic beading was dependent on caspase-6 (Casp6), because it was inhibited with 5 μM z-VEID-fmk or with dominant-negative Casp6. Neuritic beading was independent from APP-mediated amyloid β-peptide (Aβ) production, because the APPM596V (APPMV) mutant, which cannot generate Aβ, still induced Casp6-dependent neuritic beading. However, the beaded neurons underwent Casp6- and Aβ-dependent cell death. These results indicate that overexpression of wild-type or mutant APP causes Casp6-dependent but Aβ-independent neuritic degeneration in human neurons. Because Casp6 is activated early in AD and is involved in axonal degeneration, these results suggest that the inhibition of Casp6 may represent an efficient early intervention against familial forms of AD. Furthermore, these results indicate that removing Aβ without inhibiting Casp6 may have little effect in preventing the progressive dementia associated with sporadic or familial AD

Chemoattractant Receptor Homologous to the T Helper 2 Cell (CRTH2) Is Not Expressed in Human Amniocytes and Myocytes

BACKGROUND: 15-deoxy-Δ 12,14- Prostaglandin J2 (15dPGJ2) inhibits Nuclear factor kappa B (NF-κB) in human myocytes and amniocytes and delays inflammation induced preterm labour in the mouse. 15dPGJ2 is a ligand for the Chemoattractant Receptor Homologous to the T helper 2 cell (CRTH2), a G protein-coupled receptor, present on a subset of T helper 2 (Th2) cells, eosinophils and basophils. It is the second receptor for Prostaglandin D2, whose activation leads to chemotaxis and the production of Th2-type interleukins. The cellular distribution of CRTH2 in non-immune cells has not been extensively researched, and its identification at the protein level has been limited by the lack of specific antibodies. In this study we explored the possibility that CRTH2 plays a role in 15dPGJ2-mediated inhibition of NF-κB and would therefore represent a novel small molecule therapeutic target for the prevention of inflammation induced preterm labour. METHODS: The effect of a small molecule CRTH2 agonist on NF-κB activity in human cultured amniocytes and myocytes was assessed by detection of p65 and phospho-p65 by immunoblot. Endogenous CRTH2 expression in amniocytes, myocytes and peripheral blood mononuclear cells (PBMCs) was examined by PCR, western analysis and flow cytometry, with amniocytes and myocytes transfected with CRTH2 acting as a positive control in flow cytometry studies. RESULTS: The CRTH2 agonist had no effect on NF-κB activity in amniocytes and myocytes. Although CRTH2 mRNA was detected in amniocytes and myocytes, CRTH2 was not detectable at the protein level, as demonstrated by western analysis and flow cytometry. 15dPGJ2 inhibited phospho-65 in PBMC'S, however the CRTH2 antagonist was not able to attenuate this effect. In conclusion, CRTH2 is not expressed on human amniocytes or myocytes and plays no role in the mechanism of 15dPGJ2-mediated inhibition of NF-κB

Granulovacuolar Degenerations Appear in Relation to Hippocampal Phosphorylated Tau Accumulation in Various Neurodegenerative Disorders

BACKGROUND: Granulovacuolar degeneration (GVD) is one of the pathological hallmarks of Alzheimer's disease (AD), and it is defined as electron-dense granules within double membrane-bound cytoplasmic vacuoles. Several lines of evidence have suggested that GVDs appear within hippocampal pyramidal neurons in AD when phosphorylated tau begins to aggregate into early-stage neurofibrillary tangles. The aim of this study is to investigate the association of GVDs with phosphorylated tau pathology to determine whether GVDs and phosphorylated tau coexist among different non-AD neurodegenerative disorders. METHODS: An autopsied series of 28 patients with a variety of neurodegenerative disorders and 9 control patients were evaluated. Standard histological stains along with immunohistochemistry using protein markers for GVD and confocal microscopy were utilized. RESULTS: The number of neurons with GVDs significantly increased with the level of phosphorylated tau accumulation in the hippocampal regions in non-AD neurodegenerative disorders. At the cellular level, diffuse staining for phosphorylated tau was detected in neurons with GVDs. CONCLUSIONS: Our data suggest that GVDs appear in relation to hippocampal phosphorylated tau accumulation in various neurodegenerative disorders, while the presence of phosphorylated tau in GVD-harbouring neurons in non-AD neurodegenerative disorders was indistinguishable from age-related accumulation of phosphorylated tau. Although GVDs in non-AD neurodegenerative disorders have not been studied thoroughly, our results suggest that they are not incidental findings, but rather they appear in relation to phosphorylated tau accumulation, further highlighting the role of GVD in the process of phosphorylated tau accumulation

Metabolic State Determines Sensitivity to Cellular Stress in Huntington Disease: Normalization by Activation of PPARγ

Impairments in mitochondria and transcription are important factors in the pathogenesis of Huntington disease (HD), a neurodegenerative disease caused by a polyglutamine expansion in the huntingtin protein. This study investigated the effect of different metabolic states and peroxisome proliferator-activated receptor γ (PPARγ) activation on sensitivity to cellular stressors such as H2O2 or thapsigargin in HD. Striatal precursor cells expressing wild type (STHdhQ7) or mutant huntingtin (STHdhQ111) were prepared in different metabolic conditions (glucose vs. pyruvate). Due to the fact that STHdhQ111 cells exhibit mitochondrial deficits, we expected that in the pyruvate condition, where ATP is generated primarily by the mitochondria, there would be greater differences in cell death between the two cell types compared to the glucose condition. Intriguingly, it was the glucose condition that gave rise to greater differences in cell death. In the glucose condition, thapsigargin treatment resulted in a more rapid loss of mitochondrial membrane potential (ΔΨm), a greater activation of caspases (3, 8, and 9), and a significant increase in superoxide/reactive oxygen species (ROS) in STHdhQ111 compared to STHdhQ7, while both cell types showed similar kinetics of ΔΨm-loss and similar levels of superoxide/ROS in the pyruvate condition. This suggests that bioenergetic deficiencies are not the primary contributor to the enhanced sensitivity of STHdhQ111 cells to stressors compared to the STHdhQ7 cells. PPARγ activation significantly attenuated thapsigargin-induced cell death, concomitant with an inhibition of caspase activation, a delay in ΔΨm loss, and a reduction of superoxide/ROS generation in STHdhQ111 cells. Expression of mutant huntingtin in primary neurons induced superoxide/ROS, an effect that was significantly reduced by constitutively active PPARγ. These results provide significant insight into the bioenergetic disturbances in HD with PPARγ being a potential therapeutic target for HD

Formation and Toxicity of Soluble Polyglutamine Oligomers in Living Cells

Aggregation and cytotoxicity of mutant proteins containing an expanded number of polyglutamine (polyQ) repeats is a hallmark of several diseases, including Huntington's disease (HD). Within cells, mutant Huntingtin (mHtt) and other polyglutamine expansion mutant proteins exist as monomers, soluble oligomers, and insoluble inclusion bodies (IBs). Determining which of these forms constitute a toxic species has proven difficult. Recent studies support a role for IBs as a cellular coping mechanism to sequester levels of potentially toxic soluble monomeric and oligomeric species of mHtt.When fused to a fluorescent reporter (GFP) and expressed in cells, the soluble monomeric and oligomeric polyglutamine species are visually indistinguishable. Here, we describe two complementary biophysical fluorescence microscopy techniques to directly detect soluble polyglutamine oligomers (using Htt exon 1 or Htt(ex1)) and monitor their fates in live cells. Photobleaching analyses revealed a significant reduction in the mobilities of mHtt(ex1) variants consistent with their incorporation into soluble microcomplexes. Similarly, when fused to split-GFP constructs, both wildtype and mHtt(ex1) formed oligomers, as evidenced by the formation of a fluorescent reporter. Only the mHtt(ex1) split-GFP oligomers assembled into IBs. Both FRAP and split-GFP approaches confirmed the ability of mHtt(ex1) to bind and incorporate wildtype Htt into soluble oligomers. We exploited the irreversible binding of split-GFP fragments to forcibly increase levels of soluble oligomeric mHtt(ex1). A corresponding increase in the rate of IBs formation and the number formed was observed. Importantly, higher levels of soluble mHtt(ex1) oligomers significantly correlated with increased mutant cytotoxicity, independent of the presence of IBs.Our study describes powerful and sensitive tools for investigating soluble oligomeric forms of expanded polyglutamine proteins, and their impact on cell viability. Moreover, these methods should be applicable for the detection of soluble oligomers of a wide variety of aggregation prone proteins

PGH1, the Precursor for the Anti-Inflammatory Prostaglandins of the 1-series, Is a Potent Activator of the Pro-Inflammatory Receptor CRTH2/DP2

Prostaglandin H1 (PGH1) is the cyclo-oxygenase metabolite of dihomo-γ-linolenic acid (DGLA) and the precursor for the 1-series of prostaglandins which are often viewed as “anti-inflammatory”. Herein we present evidence that PGH1 is a potent activator of the pro-inflammatory PGD2 receptor CRTH2, an attractive therapeutic target to treat allergic diseases such as asthma and atopic dermatitis. Non-invasive, real time dynamic mass redistribution analysis of living human CRTH2 transfectants and Ca2+ flux studies reveal that PGH1 activates CRTH2 as PGH2, PGD2 or PGD1 do. The PGH1 precursor DGLA and the other PGH1 metabolites did not display such effect. PGH1 specifically internalizes CRTH2 in stable CRTH2 transfectants as assessed by antibody feeding assays. Physiological relevance of CRTH2 ligation by PGH1 is demonstrated in several primary human hematopoietic lineages, which endogenously express CRTH2: PGH1 mediates migration of and Ca2+ flux in Th2 lymphocytes, shape change of eosinophils, and their adhesion to human pulmonary microvascular endothelial cells under physiological flow conditions. All these effects are abrogated in the presence of the CRTH2 specific antagonist TM30089. Together, our results identify PGH1 as an important lipid intermediate and novel CRTH2 agonist which may trigger CRTH2 activation in vivo in the absence of functional prostaglandin D synthase

Neuronal Deletion of Caspase 8 Protects against Brain Injury in Mouse Models of Controlled Cortical Impact and Kainic Acid-Induced Excitotoxicity

system. mice demonstrated superior survival, reduced seizure severity, less apoptosis, and reduced caspase 3 processing. Uninjured aged knockout mice showed improved learning and memory, implicating a possible role for caspase 8 in cognitive decline with aging.Neuron-specific deletion of caspase 8 reduces brain damage and improves post-traumatic functional outcomes, suggesting an important role for this caspase in pathophysiology of acute brain trauma