An Ex Vivo Model for Studying Hepatic Schistosomiasis and the Effect of Released Protein from Dying Eggs

BACKGROUND: We report the use of an ex vivo precision cut liver slice (PCLS) mouse model for studying hepatic schistosomiasis. In this system, liver tissue is unfixed, unfrozen, and alive for maintenance in culture and subsequent molecular analysis.

METHODS AND FINDINGS: Using thick naive mouse liver tissue and sterile culture conditions, the addition of soluble egg antigen (SEA) derived from Schistosoma japonicum eggs, followed 4, 24 and 48 hrs time points. Tissue was collected for transcriptional analysis and supernatants collected to quantitate liver enzymes, cytokines and chemokines. No significant hepatotoxicity was demonstrated by supernatant liver enzymes due to the presence of SEA. A proinflammatory response was observed both at the transcriptional level and at the protein level by cytokine and chemokine bead assay. Key genes observed elevated transcription in response to the addition of SEA included: IL1-α and IL1-β, IL6, all associated with inflammation. The recruitment of antigen presenting cells was reflected in increases in transcription of CD40, CCL4 and CSF1. Indications of tissue remodeling were seen in elevated gene expression of various Matrix MetalloProteinases (MMP3, 9, 10, 13) and delayed increases in TIMP1. Collagen deposition was significantly reduced in the presence of SEA as shown in COL1A1 expression by qPCR after 24 hrs culture. Cytokine and chemokine analysis of the culture supernatants confirmed the elevation of proteins including IL6, CCL3, CCL4 and CXCL5.

CONCLUSIONS: This ex vivo model system for the synchronised delivery of parasite antigen to liver tissue provides an insight into the early phase of hepatic schistosomiasis, corresponding with the release of soluble proteins from dying schistosome eggs.

The role of intracellular calcium perturbations in muscle damage and dysfunction in mouse models of muscular dystrophy

Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by mutations in the dystrophin gene. DMD is clinically characterized by severe, progressive and irreversible loss of muscle function, in which most patients lose the ability to walk by their early teens and die by their early 20’s. Impaired intracellular calcium (Ca2+) regulation and activation of cell degradation pathways have been proposed as key contributors to DMD disease progression. This dissertation research consists of three studies investigating the role of intracellular Ca2+ in skeletal muscle dysfunction in different mouse models of DMD. Study one evaluated the role of Ca2+-activated enzymes (proteases) that activate protein degradation in excitation-contraction (E-C) coupling failure following repeated contractions in mdx and dystrophin-utrophin null (mdx/utr-/-) mice. Single muscle fibers from mdx/utr-/- mice had greater E-C coupling failure following repeated contractions compared to fibers from mdx mice. Moreover, protease inhibition during these contractions was sufficient to attenuate E-C coupling failure in muscle fibers from both mdx and mdx/utr-/- mice. Study two evaluated the effects of overexpressing the Ca2+ buffering protein sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1 (SERCA1) in skeletal muscles from mdx and mdx/utr-/- mice. Overall, SERCA1 overexpression decreased muscle damage and protected the muscle from contraction-induced injury in mdx and mdx/utr-/- mice. In study three, the cellular mechanisms underlying the beneficial effects of SERCA1 overexpression in mdx and mdx/utr-/- mice were investigated. SERCA1 overexpression attenuated calpain activation in mdx muscle only, while partially attenuating the degradation of the calpain target desmin in mdx/utr-/- mice. Additionally, SERCA1 overexpression decreased the SERCA-inhibitory protein sarcolipin in mdx muscle but did not alter levels of Ca2+ regulatory proteins (parvalbumin and calsequestrin) in either dystrophic model. Lastly, SERCA1 overexpression blunted the increase in endoplasmic reticulum stress markers Grp78/BiP in mdx mice and C/EBP homologous protein (CHOP) in mdx and mdx/utr-/- mice. Overall, findings from the studies presented in this dissertation provide new insight into the role of Ca2+ in muscle dysfunction and damage in different dystrophic mouse models. Further, these findings support the overall strategy for improving intracellular Ca2+ control for the development of novel therapies for DMD.

Increased CSF Aβ during the very early phase of cerebral Aβ deposition in mouse models

Abnormalities in brains of Alzheimer's disease (AD) patients are thought to start long before the first clinical symptoms emerge. The identification of affected individuals at this 'preclinical AD' stage relies on biomarkers such as decreased levels of the amyloid-β peptide (Aβ) in the cerebrospinal fluid (CSF) and positive amyloid positron emission tomography scans. However, there is little information on the longitudinal dynamics of CSF biomarkers, especially in the earliest disease stages when therapeutic interventions are likely most effective. To this end, we have studied CSF Aβ changes in three Aβ precursor protein transgenic mouse models, focusing our analysis on the initial Aβ deposition, which differs significantly among the models studied. Remarkably, while we confirmed the CSF Aβ decrease during the extended course of brain Aβ deposition, a 20-30% increase in CSF Aβ40 and Aβ42 was found around the time of the first Aβ plaque appearance in all models. The biphasic nature of this observed biomarker changes stresses the need for longitudinal biomarker studies in the clinical setting and the search for new 'preclinical AD' biomarkers at even earlier disease stages, by using both mice and human samples. Ultimately, our findings may open new perspectives in identifying subjects at risk for AD significantly earlier, and in improving the stratification of patients for preventive treatment strategies.

Microfluidics-based single-cell functional proteomics microchip for portraying protein signal transduction networks within the framework of physicochemical principles, with applications in fundamental and translational cancer research

Single-cell functional proteomics assays can connect genomic information to biological function through quantitative and multiplex protein measurements. Tools for single-cell proteomics have developed rapidly over the past 5 years and are providing unique opportunities. This thesis describes an emerging microfluidics-based toolkit for single cell functional proteomics, focusing on the development of the single cell barcode chips (SCBCs) with applications in fundamental and translational cancer research.

The microchip designed to simultaneously quantify a panel of secreted, cytoplasmic and membrane proteins from single cells will be discussed at the beginning, which is the prototype for subsequent proteomic microchips with more sophisticated design in preclinical cancer research or clinical applications. The SCBCs are a highly versatile and information rich tool for single-cell functional proteomics. They are based upon isolating individual cells, or defined number of cells, within microchambers, each of which is equipped with a large antibody microarray (the barcode), with between a few hundred to ten thousand microchambers included within a single microchip. Functional proteomics assays at single-cell resolution yield unique pieces of information that significantly shape the way of thinking on cancer research. An in-depth discussion about analysis and interpretation of the unique information such as functional protein fluctuations and protein-protein correlative interactions will follow.

The SCBC is a powerful tool to resolve the functional heterogeneity of cancer cells. It has the capacity to extract a comprehensive picture of the signal transduction network from single tumor cells and thus provides insight into the effect of targeted therapies on protein signaling networks. We will demonstrate this point through applying the SCBCs to investigate three isogenic cell lines of glioblastoma multiforme (GBM).

The cancer cell population is highly heterogeneous with high-amplitude fluctuation at the single cell level, which in turn grants the robustness of the entire population. The concept that a stable population existing in the presence of random fluctuations is reminiscent of many physical systems that are successfully understood using statistical physics. Thus, tools derived from that field can probably be applied to using fluctuations to determine the nature of signaling networks. In the second part of the thesis, we will focus on such a case to use thermodynamics-motivated principles to understand cancer cell hypoxia, where single cell proteomics assays coupled with a quantitative version of Le Chatelier's principle derived from statistical mechanics yield detailed and surprising predictions, which were found to be correct in both cell line and primary tumor model.

The third part of the thesis demonstrates the application of this technology in the preclinical cancer research to study the GBM cancer cell resistance to molecular targeted therapy. Physical approaches to anticipate therapy resistance and to identify effective therapy combinations will be discussed in detail. Our approach is based upon elucidating the signaling coordination within the phosphoprotein signaling pathways that are hyperactivated in human GBMs, and interrogating how that coordination responds to the perturbation of targeted inhibitor. Strongly coupled protein-protein interactions constitute most signaling cascades. A physical analogy of such a system is the strongly coupled atom-atom interactions in a crystal lattice. Similar to decomposing the atomic interactions into a series of independent normal vibrational modes, a simplified picture of signaling network coordination can also be achieved by diagonalizing protein-protein correlation or covariance matrices to decompose the pairwise correlative interactions into a set of distinct linear combinations of signaling proteins (i.e. independent signaling modes). By doing so, two independent signaling modes – one associated with mTOR signaling and a second associated with ERK/Src signaling have been resolved, which in turn allow us to anticipate resistance, and to design combination therapies that are effective, as well as identify those therapies and therapy combinations that will be ineffective. We validated our predictions in mouse tumor models and all predictions were borne out.

In the last part, some preliminary results about the clinical translation of single-cell proteomics chips will be presented. The successful demonstration of our work on human-derived xenografts provides the rationale to extend our current work into the clinic. It will enable us to interrogate GBM tumor samples in a way that could potentially yield a straightforward, rapid interpretation so that we can give therapeutic guidance to the attending physicians within a clinical relevant time scale. The technical challenges of the clinical translation will be presented and our solutions to address the challenges will be discussed as well. A clinical case study will then follow, where some preliminary data collected from a pediatric GBM patient bearing an EGFR amplified tumor will be presented to demonstrate the general protocol and the workflow of the proposed clinical studies.

Role of Ccbe1 during cardiac differentiation of mouse ESCs

Tese de Doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016

Regulation of electrical transmission by protein kinase C in Aplysia bag cell neurons

Electrical synapses are composed of gap junctions, made from paired hemi-channels that allow for the transfer of current from one neuron to another. Gap junctions mediate electrical transmission in neurons, where they synchronize spiking and promote rapid transmission, thereby influencing the coordination, pattern, and frequency of firing. In the marine snail, Aplysia calfornica, two clusters of neuroendocrine bag cell neurons use electrical synapses to synchronize a 30-min burst of action potentials, known as the afterdischarge, which releases egg-laying hormone and induces reproduction. In culture, paired bag cell neurons present a junctional conductance that is non-rectifying and largely voltage-independent. During the afterdischarge, PKC is activated, which is known to increase voltage-gated Ca2+ current; yet, little is understood as to how this pathway impacts electrical transmission. The transfer of presynaptic spike-like waveforms (generated in voltage-clamp) to the postsynaptic cell (measured in current-clamp) was monitored with or without PKC activation. It was found that pretreatment with the PKC activator, phorbol-12-myristate-13-acetate (PMA), enhanced junctional conductance between bag cell neurons. Furthermore, in control, presynaptic action potential waveforms mainly evoked postsynaptic electrotonic potentials at both -60 and -40 mV. However, with PKC activation the presynaptic stimulus consistently elicited postsynaptic action potentials from resting potentials of -40 mV, and would occasionally result in firing from repetitive input at -60 mV. Moreover, to assess whether this enhanced electrical transmission genuinely reflects a greater junctional conductance or a change in postsynaptic responsiveness, a fast-phase junctional-like current was applied to single bag cell neurons. Neurons in PMA always fired action potentials in response to current injection as opposed to control, which were less likely to spike. This outcome did not change when the junctional-like current was artificially enhanced in control conditions. Also, in response to fast- and slow-phase electrotonic potential (ETP) waveforms, Ca2+ current was markedly larger in single PMA-treated neurons. These findings suggest that PKC activation may contribute to afterdischarge fidelity by recruiting postsynaptic Ca2+ current to promote synchronous network firing. Finally, Aplysia gap junction genes (innexins) were transfected into mouse N2A cells and characterized. This revealed a biophysical and pharmacological profile similar to native gap junctions.

Salmonella Enterica serovar Typhimurium Infection in Mouse Pregnancy

Salmonella are Gram-negative, intracellular food-borne pathogens that cause pregnancy complications. In pregnant mice, Salmonella enterica serovar Typhimurium (S.Tm) infection results in placental bacterial replication, inflammation, necrosis, and fetal loss by unknown mechanisms. Necroptosis, or programmed necrosis mediated by RIPK3 (receptor-interacting protein kinase 3), an inflammatory cell death pathway, is implicated in the pathogenesis of S.Tm in non-pregnant mice. This goal of this thesis was to investigate the role of necroptosis in the pathogenesis of S.Tm infection during mouse pregnancy. I hypothesized that elimination of the key necroptotic cell death protein RIPK3 would decrease placental inflammation and trophoblast cell death, and increase conceptus survival compared to controls. Mice expressing a functional Slc11a1 (encodes the natural resistance-associated macrophage protein 1, NRAMP1) gene with or without RIPK3 function (Ripk3-/-Slc11a1+/+ compared to Slc11a1+/+) were infected with 103 S.Tm by tail vein injection on gestational day (GD) 12. Mice were euthanized on GD 14 (48h post-infection) or GD 15 (72h post-infection) and implantation sites (IS) and maternal serum were harvested for analyses. In nearly all challenged mice (except one outlier), S.Tm were detected in most IS within a litter but there was limited immune cell infiltration, placental damage or cell death in Slc11a1 competent mice regardless of Ripk3 gene deletion. Maternal serum cytokine analyses confirmed lack of maternal immune responses to S.Tm infection. IS amongst the litter of a single dam (Ripk3-/-Slc11a1+/+ at 72h postinfection) displayed heavy but not universal placental S.Tm infection of decidual tissues and spongiotrophoblast, associated with elevated maternal serum pro-inflammatory cytokines. S.Tm infection of the fetal yolk sac (YS) was observed in 54.5% of IS from this dam. YS infection was confirmed in archival samples in mice expressing Ripk3 with intact Slc11a1 and in mice lacking functional Slc11a1. In Slc11a1 incompetent mice, S.Tm were detected in placental labyrinthine trophoblast. Based on the available data, this thesis suggests that Ripk3 and necroptosis have no significant roles in either promotion or prevention of progressive Salmonella infection during mouse pregnancy. It also provides pilot data that NRAMP1 controls placental localization and lethality due to YS infection.

Chronic sustained hypoxia-induced redox remodeling causes contractile dysfunction in mouse sternohyoid muscle

Chronic sustained hypoxia (CH) induces structural and functional adaptations in respiratory muscles of animal models, however the underlying molecular mechanisms are unclear. This study explores the putative role of CH-induced redox remodeling in a translational mouse model, with a focus on the sternohyoid—a representative upper airway dilator muscle involved in the control of pharyngeal airway caliber. We hypothesized that exposure to CH induces redox disturbance in mouse sternohyoid muscle in a time-dependent manner affecting metabolic capacity and contractile performance. C57Bl6/J mice were exposed to normoxia or normobaric CH (FiO2 = 0.1) for 1, 3, or 6 weeks. A second cohort of animals was exposed to CH for 6 weeks with and without antioxidant supplementation (tempol or N-acetyl cysteine in the drinking water). Following CH exposure, we performed 2D redox proteomics with mass spectrometry, metabolic enzyme activity assays, and cell-signaling assays. Additionally, we assessed isotonic contractile and endurance properties ex vivo. Temporal changes in protein oxidation and glycolytic enzyme activities were observed. Redox modulation of sternohyoid muscle proteins key to contraction, metabolism and cellular homeostasis was identified. There was no change in redox-sensitive proteasome activity or HIF-1α content, but CH decreased phospho-JNK content independent of antioxidant supplementation. CH was detrimental to sternohyoid force- and power-generating capacity and this was prevented by chronic antioxidant supplementation. We conclude that CH causes upper airway dilator muscle dysfunction due to redox modulation of proteins key to function and homeostasis. Such changes could serve to further disrupt respiratory homeostasis in diseases characterized by CH such as chronic obstructive pulmonary disease. Antioxidants may have potential use as an adjunctive therapy in hypoxic respiratory disease.

Microglia as therapeutic targets in retinal degeneration: role of translocator protein (18 κDa) (TSPO) and minocycline.

Microglia are the resident immune cells of the central nervous system (CNS) and play an important role in innate immune defense as well as tissue homeostasis. Chronic microglial reactivity, microgliosis, is a general hallmark of inflammatory and degenerative diseases that affect the CNS, including the retina. There is increasing evidence that chronic microgliosis is more than just a bystander effect, but rather actively contributes to progression of degeneration through processes such as toxic nitric oxide (NO) production and even phagocytosis of stressed but viable photoreceptors. Therefore immunmodulation of microglia presents a possible therapeutic strategy for retinal degenerations. Notably, the expression of the mitochondrial translocator protein 18 (κDa) (TSPO) is highly elevated in reactive microglia as seen in several neuroinflammatory diseases such as Alzheimer’s disease, Parkinson’s disease and multiple sclerosis. Therefore it is used as a gliosis biomarker in the brain. Moreover TSPO ligands show potent effects in resolving neuroinflammatory brain disorders. However, TSPO expression in the eye had not been investigated before. Further, it was unknown whether TSPO ligands’ potent immunomodulatory effects could be used to treat retinal degenerations. To fill this gap, the study aimed to analyze whether TSPO is also a potential biomarker for degenerative processes in the retina. Moreover the thesis attempted to determine whether a specific TSPO ligand, XBD173, might modulate microglial reactivity and is a potent therapeutic, to treat retinal degenerative diseases. The findings revealed that TSPO is strongly upregulated in microglial cells of retinoschisin-deficient (RS1-/y) mice, a model of inherited retinal degeneration and in a murine light damage model. A co-localization of TSPO and microglia was furthermore detectable in human retinal sections, indicating a potential role for TSPO as a biomarker for retinal degenerations. In vitro assays showed that the TSPO ligand XBD173 effectively inhibited features of microglial activation such as morphological transformation into reactive phagocytes and enhanced expression of pro-inflammatory cytokines. XBD173 also reduced microglial migration and proliferation and reduced their neurotoxic potential on photoreceptor cells. In two independent mouse models of light-induced retinal degeneration, the treatment with XBD173 reduced accumulation of amoeboid, reactive microglia in the outer retina and attenuated degenerative processes, indicated by a nearly preserved photoreceptor layer. A further question addressed in this thesis was whether minocycline, an antibiotic with additional anti-inflammatory properties is able to reduce microglial neurotoxicity and to protect the retina from degeneration. Minocycline administration dampened microglial pro-inflammatory gene expression, NO production and neurotoxicity on photoreceptors. Interestingly, in addition to its immunomodulatory effect, minocycline also increased the viability of photoreceptors in a direct manner. In the light damage model, minocycline administration counter-acted microglial activation and blocked retinal degeneration. Taken together these results identified TSPO as a biomarker for microglial reactivity and as therapeutic target in the retina. Targeting TSPO with XBD173 was able to reverse microglial reactivity and could prevent degenerative processes in the retina. In addition, the study showed that the antibiotic minocycline effectively counter-regulates microgliosis and light-induced retinal degeneration. Considering that microgliosis is a major contributing factor for retinal degenerative disorders, this thesis supports the concept of a microglia-directed therapy to treat retinal degeneration.

Regulation of prostate cancer cell function by activators of AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is a key regulator of cell energy homeostasis. More recently, it has become apparent that AMPK regulates cell proliferation, migration and inflammation. Previous evidence has suggested that AMPK may influence proliferation and invasion by regulating the pro-proliferative mitogen-activated protein kinases (MAPKs). However, the mechanisms underlying this crosstalk between AMPK and MAPK signalling are not fully understood. As AMPK activation has been reported to have anti-proliferative effects, there has been increasing interest in AMPK activation as a therapeutic target for tumourigenesis. The aim of this study was to investigate whether AMPK activation influenced prostate cancer (PC) cell line proliferation, migration and signalling. Therefore, different PC cell lines were incubated with two structurally-unrelated molecules that activate AMPK by different mechanisms, AICAR and A769662. Both chemicals activated AMPK in a concentration- and time-dependent manner in PC3, DU145 and LNCaP cell lines. AMPK activity as assessed by AMPK activating phosphorylation as well as phosphorylation of the AMPK substrate ACC increased along with tumour severity in PC biopsies. Furthermore, both activators of AMPK decreased cell proliferation and migration in the androgen-independent PC cell lines PC3 and DU145. Inhibition of proliferation by A769662 was attenuated in AMPK α1-/- AMPK α2-/- knockout (KO) mouse embryonic fibroblasts (MEFs) compared to wild type (WT) MEFs, and the inhibitory effect on migration of AICAR lost significance in PC3 cells infected with adenoviruses expressing a dominant negative AMPK α mutant, indicating these effects are partially mediated by AMPK. Furthermore, long-term activation of AMPK was associated with inhibition of both the phosphatidylinositol 3’-kinase/protein kinase B (PI3K/Akt) signalling pathway in addition to the extracellular signal-regulated kinase 1/2 (ERK1/2) signalling pathway. Indeed, the actions of AMPK activators on PC cell line viability were mimicked by selective inhibitors of Akt and ERK1/2 pathways. In contrast to the effects of prolonged incubation with AMPK activators, short-term incubation with AMPK activators had no effect on epidermal growth factor (EGF)-stimulated ERK1/2 phosphorylation in PC cell lines. In addition, AMPK activation did not influence phosphorylation of the other MAPK family members p38 and JNK. Interestingly, both AICAR and A769662 decreased EGF-stimulated ERK5 phosphorylation in PC3, DU145 and LNCaP cells as assessed with an anti-phospho-ERK5 antibody. Further characterisation of this effect indicated that prior stimulation with the AMPK activators had no effect on ERK5 phosphorylation stimulated by transient transfection with a constitutively active ERK5 kinase (MEK5DD), which represents the only known canonical kinase for ERK5. Intriguingly, the pattern of EGF-stimulated ERK5 phosphorylation was distinct from that mediated by MEK5DD activation of ERK5. This finding indicates that AMPK activation inhibits EGF-stimulated ERK5 phosphorylation at a point at or above the level of MEK5, although why EGF and constitutively active MEK5 stimulate markedly different immunoreactive species recognised by the anti-phospho-ERK5 antibody requires further study. A769662 had a tendency to reduce EGF-stimulated ERK5 phosphorylation in WT MEFs, yet was without effect in MEFs lacking AMPK. These data indicate that AMPK may underlie the effect of A769662 to reduce EGF-stimulated ERK5 phosphorylation. Prolonged stimulation of PC cell lines with AICAR or A769662 inhibited EGF-stimulated Akt Ser473 phosphorylation, whereas only incubation with A769662 rapidly inhibited Akt phosphorylation. This difference in the actions of the different AMPK activators may suggest an AMPK-independent effect of A769662. Furthermore, AICAR increased phosphorylation of Akt in WT MEFs, an effect that was absent in MEFs lacking AMPK, indicating that this effect of AICAR may be AMPK-dependent. Taken together, the data presented in this study suggest that AMPK activators markedly inhibit proliferation and migration of PC cell lines, reduce EGF-stimulated ERK1/2 and Akt phosphorylation after prolonged incubation and rapidly inhibit ERK5 phosphorylation. Both AMPK activators exhibit a number of effects that are likely to be independent of AMPK in PC cell lines, although inhibition of ERK1/2, ERK5 and Akt may underlie the effects of AMPK activators on proliferation, viability and migration. Further studies are required to understand the crosstalk between those signalling pathways and their underlying significance in PC progression.

Modeling Alzheimer disease with iPSCs and mouse model for the identification of risk factors, new targets, and potential therapeutical strategies

Alzheimer's disease (AD) is the most common neurodegenerative disease in elderly. Donepezil is the first-line drug used for AD. In section one, the experimental activity was oriented to evaluate and characterize molecular and cellular mechanisms that contribute to neurodegeneration induced by the Aβ1-42 oligomers (Aβ1-42O) and potential neuroprotective effects of the hybrids feruloyl-donepezil compound called PQM130. The effects of PQM130 were compared to donepezil in a murine AD model, obtained by intracerebroventricular (i.c.v.) injection of Aβ1-42O. The intraperitoneal administration of PQM130 (0.5-1 mg/kg) after i.c.v. Aβ1-42O injection improved learning and memory, protecting mice against spatial cognition decline. Moreover, it reduced oxidative stress, neuroinflammation and neuronal apoptosis, induced cell survival and protein synthesis in mice hippocampus. PQM130 modulated different pathways than donepezil, and it is more effective in counteracting Aβ1-42O damage. The section two of the experimental activity was focused on studying a loss of function variants of ABCA7. GWA studies identified mutations in the ABCA7 gene as a risk factor for AD. The mechanism through which ABCA7 contributes to AD is not clear. ABCA7 regulates lipid metabolism and critically controls phagocytic function. To investigate ABCA7 functions, CRISPR/Cas9 technology was used to engineer human iPSCs and to carry the genetic variant Y622*, which results in a premature stop codon, causing ABCA7 loss-of-function. From iPSCs, astrocytes were generated. This study revealed the effects of ABCA7 loss in astrocytes. ABCA7 Y622* mutation induced dysfunctional endocytic trafficking, impairing Aβ clearance, lipid dysregulation and cell homeostasis disruption, alterations that could contribute to AD. Though further studies are needed to confirm the PQM130 neuroprotective role and ABCA7 function in AD, the provided results showed a better understanding of AD pathophysiology, a new therapeutic approach to treat AD, and illustrated an innovative methodology for studying the disease.

Cardiac functional and structural abnormalities in a mouse model of CDKL5 deficiency disorder

CDKL5 (cyclin-dependent kinase-like 5) deficiency disorder (CDD) is a rare and severe neurodevelopmental disease that mostly affects girls who are heterozygous for mutations in the X-linked CDKL5 gene. The lack of CDKL5 protein expression or function leads to the appearance of numerous clinical features, including early-onset seizures, marked hypotonia, autistic features, and severe neurodevelopmental impairment. Mouse models of CDD, Cdkl5 KO mice, exhibit several behavioral phenotypes that mimic CDD features, such as impaired learning and memory, social interaction, and motor coordination. CDD symptomatology, along with the high CDKL5 expression levels in the brain, underscores the critical role that CDKL5 plays in proper brain development and function. Nevertheless, the improvement of the clinical overview of CDD in the past few years has defined a more detailed phenotypic spectrum; this includes very common alterations in peripheral organ and tissue function, such as gastrointestinal problems, irregular breathing, hypotonia, and scoliosis, suggesting that CDKL5 deficiency compromises not only CNS function but also that of other organs/tissues. Here we report, for the first time, that a mouse model of CDD, the heterozygous Cdkl5 KO (Cdkl5 +/-) female mouse, exhibits cardiac functional and structural abnormalities. The mice also showed QTc prolongation and increased heart rate. These changes correlate with a marked decrease in parasympathetic activity to the heart and in the expression of the Scn5a and Hcn4 voltage-gated channels. Moreover, the Cdkl5 +/- heart shows typical signs of heart aging, including increased fibrosis, mitochondrial dysfunctions, and increased ROS production. Overall, our study not only contributes to the understanding of the role of CDKL5 in heart structure/function but also documents a novel preclinical phenotype for future therapeutic investigation.

Taurine Supplementation Increases K(atp) Channel Protein Content, Improving Ca2+ Handling And Insulin Secretion In Islets From Malnourished Mice Fed On A High-fat Diet.

Pancreatic β-cells are highly sensitive to suboptimal or excess nutrients, as occurs in protein-malnutrition and obesity. Taurine (Tau) improves insulin secretion in response to nutrients and depolarizing agents. Here, we assessed the expression and function of Cav and KATP channels in islets from malnourished mice fed on a high-fat diet (HFD) and supplemented with Tau. Weaned mice received a normal (C) or a low-protein diet (R) for 6 weeks. Half of each group were fed a HFD for 8 weeks without (CH, RH) or with 5% Tau since weaning (CHT, RHT). Isolated islets from R mice showed lower insulin release with glucose and depolarizing stimuli. In CH islets, insulin secretion was increased and this was associated with enhanced KATP inhibition and Cav activity. RH islets secreted less insulin at high K(+) concentration and showed enhanced KATP activity. Tau supplementation normalized K(+)-induced secretion and enhanced glucose-induced Ca(2+) influx in RHT islets. R islets presented lower Ca(2+) influx in response to tolbutamide, and higher protein content and activity of the Kir6.2 subunit of the KATP. Tau increased the protein content of the α1.2 subunit of the Cav channels and the SNARE proteins SNAP-25 and Synt-1 in CHT islets, whereas in RHT, Kir6.2 and Synt-1 proteins were increased. In conclusion, impaired islet function in R islets is related to higher content and activity of the KATP channels. Tau treatment enhanced RHT islet secretory capacity by improving the protein expression and inhibition of the KATP channels and enhancing Synt-1 islet content.

A Putative Otu Domain-containing Protein 1 Deubiquitinating Enzyme Is Differentially Expressed In Thyroid Cancer And Identifies Less-aggressive Tumours.

This study aimed to identify novel biomarkers for thyroid carcinoma diagnosis and prognosis. We have constructed a human single-chain variable fragment (scFv) antibody library that was selected against tumour thyroid cells using the BRASIL method (biopanning and rapid analysis of selective interactive ligands) and phage display technology. One highly reactive clone, scFv-C1, with specific binding to papillary thyroid tumour proteins was confirmed by ELISA, which was further tested against a tissue microarray that comprised of 229 thyroid tissues, including: 110 carcinomas (38 papillary thyroid carcinomas (PTCs), 42 follicular carcinomas, 30 follicular variants of PTC), 18 normal thyroid tissues, 49 nodular goitres (NG) and 52 follicular adenomas. The scFv-C1 was able to distinguish carcinomas from benign lesions (P=0.0001) and reacted preferentially against T1 and T2 tumour stages (P=0.0108). We have further identified an OTU domain-containing protein 1, DUBA-7 deubiquitinating enzyme as the scFv-binding antigen using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. The strategy of screening and identifying a cell-surface-binding antibody against thyroid tissues was highly effective and resulted in a useful biomarker that recognises malignancy among thyroid nodules and may help identify lower-risk cases that can benefit from less-aggressive management.

Metabolic Memory Of ß-cells Controls Insulin Secretion And Is Mediated By Camkii.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) functions both in regulation of insulin secretion and neurotransmitter release through common downstream mediators. Therefore, we hypothesized that pancreatic ß-cells acquire and store the information contained in calcium pulses as a form of metabolic memory, just as neurons store cognitive information. To test this hypothesis, we developed a novel paradigm of pulsed exposure of ß-cells to intervals of high glucose, followed by a 24-h consolidation period to eliminate any acute metabolic effects. Strikingly, ß-cells exposed to this high-glucose pulse paradigm exhibited significantly stronger insulin secretion. This metabolic memory was entirely dependent on CaMKII. Metabolic memory was reflected on the protein level by increased expression of proteins involved in glucose sensing and Ca(2+)-dependent vesicle secretion, and by elevated levels of the key ß-cell transcription factor MAFA. In summary, like neurons, human and mouse ß-cells are able to acquire and retrieve information.