Treatment of inflammatory arthritis via targeting of tristetraprolin, a master regulator of pro-inflammatory gene expression.

OBJECTIVES: Tristetraprolin (TTP), a negative regulator of many pro-inflammatory genes, is strongly expressed in rheumatoid synovial cells. The mitogen-activated protein kinase (MAPK) p38 pathway mediates the inactivation of TTP via phosphorylation of two serine residues. We wished to test the hypothesis that these phosphorylations contribute to the development of inflammatory arthritis, and that, conversely, joint inflammation may be inhibited by promoting the dephosphorylation and activation of TTP. METHODS: The expression of TTP and its relationship with MAPK p38 activity were examined in non-inflamed and rheumatoid arthritis (RA) synovial tissue. Experimental arthritis was induced in a genetically modified mouse strain, in which endogenous TTP cannot be phosphorylated and inactivated. In vitro and in vivo experiments were performed to test anti-inflammatory effects of compounds that activate the protein phosphatase 2A (PP2A) and promote dephosphorylation of TTP. RESULTS: TTP expression was significantly higher in RA than non-inflamed synovium, detected in macrophages, vascular endothelial cells and some fibroblasts and co-localised with MAPK p38 activation. Substitution of TTP phosphorylation sites conferred dramatic protection against inflammatory arthritis in mice. Two distinct PP2A agonists also reduced inflammation and prevented bone erosion. In vitro anti-inflammatory effects of PP2A agonism were mediated by TTP activation. CONCLUSIONS: The phosphorylation state of TTP is a critical determinant of inflammatory responses, and a tractable target for novel anti-inflammatory treatments.

APOE-mimetic peptides reduce behavioral deficits, plaques and tangles in Alzheimer's disease transgenics.

BACKGROUND: After age, the second largest risk factor for Alzheimer's disease (AD) is apolipoprotein E (APOE) genotype, where APOE4 is associated with lower apoE protein levels, more severer brain pathology, enhanced inflammation and disease. Small peptides corresponding to the receptor-binding region of apoE mimic the anti-inflammatory activity of the apoE holoprotein. These apoE mimetics greatly improve behavioral outcomes and neuronal survival in head trauma models that display AD pathology and neuronal loss. OBJECTIVE: To determine whether apoE mimetics change behavior, inflammation and pathology in CVND-AD (SwDI-APP/NOS2(-/-)) transgenic mice. METHODS: Starting at 9 months, apoE peptides were subcutaneously administered 3 times per week for 3 months followed by behavioral, histochemical and biochemical testing. RESULTS: Treatment with apoE mimetics significantly improved behavior while decreasing the inflammatory cytokine IL-6, neurofibrillary tangle-like and amyloid plaque-like structures. Biochemical measures matched the visible pathological results. CONCLUSIONS: Treatment with apoE mimetics significantly improved behavior, reduced inflammation and reduced pathology in CVND-AD mice. These improvements are associated with apoE-mimetic-mediated increases in protein phosphatase 2A activity. Testing in additional AD models showed similar benefits, reinforcing this novel mechanism of action of apoE mimetics. These data suggest that the combination of anti-inflammatory and neuroprotective activities of apoE mimetics represents a new generation of potential therapeutics for AD.

Targeting SET/I(2)PP2A oncoprotein functions as a multi-pathway strategy for cancer therapy.

The SET oncoprotein participates in cancer progression by affecting multiple cellular processes, inhibiting the tumor suppressor protein phosphatase 2A (PP2A), and inhibiting the metastasis suppressor nm23-H1. On the basis of these multiple activities, we hypothesized that targeted inhibition of SET would have multiple discrete and measurable effects on cancer cells. Here, the effects of inhibiting SET oncoprotein function on intracellular signaling and proliferation of human cancer cell lines was investigated. We observed the effects of COG112, a novel SET interacting peptide, on PP2A activity, Akt signaling, nm23-H1 activity and cellular migration/invasion in human U87 glioblastoma and MDA-MB-231 breast adenocarcinoma cancer cell lines. We found that COG112 interacted with SET protein and inhibited the association between SET and PP2A catalytic subunit (PP2A-c) and nm23-H1. The interaction between COG112 and SET caused PP2A phosphatase and nm23-H1 exonuclease activities to increase. COG112-mediated increases in PP2A activity resulted in the inhibition of Akt signaling and cellular proliferation. Additionally, COG112 inhibited SET association with Ras-related C(3) botulinum toxin substrate 1 (Rac1), leading to decreased cellular migration and invasion. COG112 treatment releases the SET-mediated inhibition of the tumor suppressor PP2A, as well as the metastasis suppressor nm23-H1. These results establish SET as a novel molecular target and that the inhibition of SET may have beneficial effects in cancer chemotherapy.

[Potential role of presenilin 1 in regulation of synaptic function].

One of the earliest neuropathological symptoms of Alzheimer's disease is the loss of synapses, which preceed the formation of amyloidosis and neurodegeneration. Although most cases of early-onset familial Alzheimer's disease are caused by mutations in the presenilin 1 (PS1) gene, the functions of PS1 and its role in synaptic disfunction are not yet completely understood. In this paper we analysed of the intracellular and extracellular distribution of PS1 in the cultures of mouse cortical embryonic neurons. We found that PS1 is concentrated on the surface of the growth cone and at neurite contact sites. PS1 was also found in synapses where it is co-localized with synaptophysin. Independent evidense of involvement of PS1 in synaptic function we obtained by transfection of neurons with GFP-PS1 cDNA. GFP was colocalized with synaptophysin in transfected cultures. GFP-immunoprecepitates from transfected neurons contained processed N-cadherin. This result presents an additional proof of involvment PS1 in synapse formation. To evaluate the role of PS1 inactivation in the synaptic functions, we compare synaptic density in neuronal cell cultures from PS1 knockout mice PS1 (-/-) and wild type mice PS1 (+/+). Our results clearly show that PS1 (-/-) displayed a low number of morphological synapses in comparing with wild type culture PS1 (+/+). In summary, our results indicate a role of PS1 in synaptic function.

[Familial Alzheimer's disease mutations in the presenilin 1 gene reduce cell-cell adhesion in transfected fibroblasts].

Experimental evidence has been obtained that mutations in the presenilin 1 (PS1) gene in familial Alzheimer's disease can lead to the disturbance of cell adhesion in model cell cultures. It was shown that, in L fibroblasts of mice with stable expression of GFP-PS1 cDNA containing G209V or E319G mutations, cell-cell interactions and the accumulation of GFP-PS1 cDNA in intercellular contacts are disturbed. Similar results were obtained in transfected human epithelial Hep2 cells. It is assumed that mutations in familial Alzheimer's disease lead to the disturbance of the functions of presenelin 1 in cell adhesion.

Loss of tau elicits axonal degeneration in a mouse model of Alzheimer's disease.

A central issue in the pathogenesis of tauopathy is the question of how tau protein dysfunction leads to neurodegeneration. We have previously demonstrated that the absence of tau protein is associated with destabilization of microtubules and impaired neurite outgrowth (Dawson et al., 2001; Rapoport et al., 2002). We now hypothesize that the absence of functional tau protein may render the central nervous system more vulnerable to secondary insults such as the overexpression of mutated beta amyloid precursor protein (APP) and traumatic brain injury. We therefore crossed tau knockout mice (Dawson et al., 2001) to mice overexpressing a mutated human APP (APP(670,671), A(sw)) (Hsiao et al., 1996) and created a mouse model (A(sw)/mTau(-/-)) that provides evidence that the loss of tau function causes degeneration of neuronal processes. The overexpression of APP(670,671) in tau knockout mice, elicits the extensive formation of axonal spheroids. While spheroids are only found associated with Abeta plaques in mice expressing APP(670,671) on an endogenous mouse tau background (Irizarry et al., 1997), A(sw)/mTau(-/-) mice have spheroids not only surrounding Abeta plaques but also in white matter tracks and in the neuropil. Plaque associated and neuropil dystrophic neurites and spheroids are prominent features of Alzheimer's disease (Masliah et al., 1993; Terry, 1996; Stokin et al., 2005), and our current data suggests that loss of tau function may lead to neurodegeneration.

Dithiolethione compounds inhibit Akt signaling in human breast and lung cancer cells by increasing PP2A activity.

The chemopreventative effects of dithiolethione compounds are attributed to their activation of antioxidant response elements (AREs) by reacting with the Nrf2/Keap1 protein complex. In this study, we show antiproliferative effects of the dithiolethione compound ACS-1 in human cancer cell lines (A549 and MDA-MB-231) by increasing the activity of the tumor suppressor protein phoshatase 2A (PP2A). ACS-1 inhibited epidermal growth factor (EGF)-induced cellular proliferation in a concentration- and time-dependent manner. Akt activation, as determined by serine-473 phosphorylation, was inhibited by ACS-1 in cells stimulated with either EGF or fibronectin. Furthermore, ACS-1 inhibited mammalian target of rapamycin signaling and decreased c-myc protein levels. ACS-1 did not proximally alter EGF receptor or integrin signaling, but caused a concentration-dependent increase in PP2A activity. The effect of ACS-1 on Akt activation was not observed in the presence of the PP2A inhibitor okadaic acid. ACS-1 effects on PP2A activity were independent of ARE activation and cAMP formation. In addition to ACS-1, other dithiolethione compounds showed similar effects in reducing Akt activation, suggesting that this class of compounds may have other effects beyond chemoprevention.

[Protein transduction domain peptide mediates delivery to the brain via the blood-brain barrier in Drosophila].

Protein transduction domain (PTD)-peptides greatly facilitate the delivery of high molecular weight macromolecules across the blood-brain barrier (BBB). This BBB-transport function is highly desirable and helps to enable the development of new therapeutics for treatment of brain disorders. However, the drug discovery process is limited by the generation of a simple and reliable BBB model that is amenable to testing of large number of samples and simultaneously, reproduces the physiological and functional characteristics of the human BBB. To address these challenges, we have studied whether the PTD-peptide penetratin, derived from a Drosophila Antennapedia homeodomain protein, is capable of crossing the BBB in Drosophila while carrying a cargo into the fly brain. An initial in vivo experiment in Drosophila showed that abdominal injection of biotin-tagged penetratin permeated the BBB. The same effect was observed for biotin-tagged penetratin fused with apoE mimetic peptide with demonstrated anti-inflammatory and neuroprotective activities.

Vascular amyloid alters astrocytic water and potassium channels in mouse models and humans with Alzheimer's disease.

The neurovascular unit (NVU) comprises cerebral blood vessels and surrounding astrocytes, neurons, perivascular microglia and pericytes. Astrocytes associated with the NVU are responsible for maintaining cerebral blood flow and ionic and osmotic balances in the brain. A significant proportion of individuals with Alzheimer's disease (AD) have vascular amyloid deposits (cerebral amyloid angiopathy, CAA) that contribute to the heterogeneous nature of the disease. To determine whether NVU astrocytes are affected by the accumulation of amyloid at cerebral blood vessels we examined astrocytic markers in four transgenic mouse models of amyloid deposition. These mouse models represent mild CAA, moderate CAA with disease progression to tau pathology and neuron loss, severe CAA and severe CAA with disease progression to tau pathology and neuron loss. We found that CAA and disease progression both resulted in distinct NVU astrocytic changes. CAA causes a loss of apparent glial fibrillary acidic protein (GFAP)-positive astrocytic end-feet and loss of water channels (aquaporin 4) localized to astrocytic end feet. The potassium channels Kir4.1, an inward rectifying potassium channel, and BK, a calcium-sensitive large-conductance potassium channel, were also lost. The anchoring protein, dystrophin 1, is common to these channels and was reduced in association with CAA. Disease progression was associated with a phenotypic switch in astrocytes indicated by a loss of GFAP-positive cells and a gain of S100 beta-positive cells. Aquaporin 4, Kir4.1 and dystrophin 1 were also reduced in autopsied brain tissue from individuals with AD that also display moderate and severe CAA. Together, these data suggest that damage to the neurovascular unit may be a factor in the pathogenesis of Alzheimer's disease.

[Studying pathogenesis of Alzheimer's disease in a Drosophila melanogaster model: human APP overexpression in the brain of transgenic flies leads to deficit of the synaptic protein synaptotagmin].

Alzheimer's disease (AD) is a progressive neurodegenerative disease whose main pathomorphological sign is synapse degeneration in the cortex and hippocampus. Abnormal synaptogenesis precedes amyloidosis and neurodegeneration and correlates with memory impairment during the early clinical phase. Mutations in the amyloid precursor protein (APP) gene cause familial AD and enhance the secretion of amyloid-beta-protein (Abeta). However, it remains unclear in what way APP and Abeta are involved in synaptic disorder in the absence of visible amyloid structures. In this study, the role of the human APP gene in synaptogenesis in transgenic lines of Drosophila melanogaster whose nerve cells express the human APP695 isoform, truncated APPs, and the presynaptic marker synaptotagmin driving the sequence of the green fluorescent protein. The expression of APP and its truncated forms caused a decrease in the synaptotagmin content of antennal lobes and mushroom lobes of the D. melanogaster brain, as well as neurodegeneration that progressed with age. The results suggest that that abnormal synaptogenesis and neurodegeneration occur in the Drosophila brain in the absence of Abeta. It is assumed that impaired cellular functions of APP and secretion of Abeta independently contribute to the pathogenesis of AD.

APOE genotype affects outcome in a murine model of sepsis: implications for a new treatment strategy.

In this study, we assessed whether apolipoprotein E (APOE) polymorphism affects inflammatory responses and mortality in the caecal ligation and puncture model of peritonitis. In addition, we determined the effects of APOE mimetic peptide administration in this sepsis model. Differences in survival between targeted replacement mice expressing the human APOE3 allele (APOE3TR) and the APOE4 allele (APOE4TR) mice were assessed. In a separate series of experiments, COG1410, an apoE-mimetic peptide, was administered intravenously at 12-hour intervals for 72 hours and compared to vehicle-treated control animals. End-points included mortality and serum levels of interleukin-1beta, interleukin-6, interleukin-12 and tumour necrosis factor-alpha. Mice expressing the human APOE4 allele (n = 16) demonstrated an increase in mortality following caecal ligation and puncture compared with APOE3TR mice (n = 22; P = 0.039). Administration of the apolipoprotein E mimetic COG1410 was well tolerated and APOE3TR mice treated with peptide (n = 20) demonstrated a significant reduction in mortality compared with vehicle treated animals (n = 20; P = 0.007). A similar effect was also observed in APOE4TR animals, in which treatment with COG1410 was associated with reduced mortality compared with vehicle treatment (n =16 animals/group; P = 0.027). COG1410 was also associated with a reduction in TNFalpha, interleukin-1beta, interleukin-6 and interleukin-12 levels in both APOE3TR and APOE4TR (n = 5 animals/group) assessed at 24 hours. Thus, administration of an apolipoprotein E-mimetic peptide is well tolerated, suppresses inflammatory responses, and improves mortality in a caecal ligation and puncture model of sepsis.

[Degeneration of growth cones in a culture of embryonic neurons of mouse with presenilin 1 gene knockout].

A comparative study of growth cone morphology in cultured embryonic neurons derived from wild type PS 1(+/+) and knockout PS 1(-/-) mice has been performed. Growth cones from wild type PS 1(+/+) mice were well spread and usually formed radially continuous and regular lamellar extensions with numerous filopodia. In contrast, most growth cones from knockout PS 1(-/-) mice exhibited small lamellar extensions, short filopodia, and pure adhesion. A significant amount of growth cones from knockout PS 1(-/-) mice collapsed after 3-4 days in culture. It was suggested that PS 1 plays an important role in growth cone structure by stabilizing the integrity of the cytoskeleton. The growth cone collapse may be the main reason for abnormal neuronal migration and impaired synaptic function in PS 1(-/-) mice.

Microvascular injury and blood-brain barrier leakage in Alzheimer's disease.

Thinning and discontinuities within the vascular basement membrane (VBM) are associated with leakage of the plasma protein prothrombin across the blood-brain barrier (BBB) in Alzheimer's disease (AD). Prothrombin immunohistochemistry and ELISA assays were performed on prefrontal cortex. In severe AD, prothrombin was localized within the wall and neuropil surrounding microvessels. Factor VIII staining in severe AD patients indicated that prothrombin leakage was associated with shrinkage of endothelial cells. ELISA revealed elevated prothrombin levels in prefrontal cortex AD cases that increased with the Braak stage (Control=1.39, I-II=1.76, III-IV=2.28, and V-VI=3.11 ng prothrombin/mg total protein). Comparing these four groups, there was a significant difference between control and Braak V-VI (p=0.0095) and also between Braak stages I-II and V-VI (p=0.0048). There was no significant difference in mean prothrombin levels when cases with versus without cerebral amyloid angiopathy (CAA) were compared (p-value=0.3627). When comparing AD patients by APOE genotype (ApoE3,3=2.00, ApoE3,4=2.49, and ApoE4,4=2.96 ng prothrombin/mg total protein) an analysis of variance indicated a difference between genotypes at the 10% significance level (p=0.0705). Tukey's test indicated a difference between the 3,3 and 4,4 groups (p=0.0607). These studies provide evidence that in advanced AD (Braak stage V-VI), plasma proteins like prothrombin can be found within the microvessel wall and surrounding neuropil, and that leakage of the blood-brain barrier may be more common in patients with at least one APOE4 allele.

An apolipoprotein E-based therapeutic improves outcome and reduces Alzheimer's disease pathology following closed head injury: evidence of pharmacogenomic interaction.

Apolipoprotein E (apoE) modifies glial activation and the CNS inflammatory response in an isoform-specific manner. Peptides derived from the receptor-binding region of apoE have been demonstrated to maintain the functional activity of the intact protein, and to improve histological and functional deficits after closed head injury. In the current study, APOE2, APOE3, and APOE4 targeted replacement (TR) mice expressing the human apoE protein isoforms (apoE2, apoE3 and apoE4) were used in a clinically relevant model of closed head injury to assess the interaction between the humanized apoE background and the therapeutic apoE mimetic peptide, apoE(133-149). Treatment with the apoE-mimetic peptide reduced microglial activation and early inflammatory events in all of the targeted replacement animals and was associated with histological and functional improvement in the APOE2TR and APOE3TR animals. Similarly, brain beta amyloid protein (Abeta)(1-42) levels were increased as a function of head injury in all of the targeted replacement mice, while treatment with apoE peptide suppressed Abeta(1-42) levels in the APOE2TR and APOE3TR animals. These results suggest a pharmacogenomic interaction between the therapeutic effects of the apoE mimetic peptide and the human apoE protein isoforms. Furthermore, they suggest that administration of apoE-mimetic peptides may serve as a novel therapeutic strategy for the treatment of acute and chronic neurological disease.

[Expression of presenilin 1 on the cell surface in motile polarized cells].

Most cases of familial early-onset Alzheimer's disease are caused by mutations in the presenilin 1 (PS1) gene. However, the cellular functions of PS1 are not yet completely understood. We showed that endogenous PS1 and the adhesion protein CD44 are redistributed on the surface of cell projections (lamellipodia) in polarized T- lymphocytes (Jurkat cells) after the adhesion to a collagen matrix. This effect was not observed for another surface protein of T lymphocytes, which is not involved in cell adhesion processes, the T cell receptor. In primary cultures of mouse cortical neurons, PS1 was concentrated at the surface of extended growth cones and at the sites of neurite contacts. The concentration of PS1 at the surface of cellular structures that promote cell motility and cell contacts suggests an important role of PSI in cell adhesion in motile polarized cells.

NO synthase 2 (NOS2) deletion promotes multiple pathologies in a mouse model of Alzheimer's disease.

Alzheimer's disease is characterized by two primary pathological features: amyloid plaques and neurofibrillary tangles. The interconnection between amyloid and tau aggregates is of intense interest, but mouse models have yet to reveal a direct interrelationship. We now show that NO may be a key factor that connects amyloid and tau pathologies. Genetic removal of NO synthase 2 in mice expressing mutated amyloid precursor protein results in pathological hyperphosphorylation of mouse tau, its redistribution to the somatodendritic compartment in cortical and hippocampal neurons, and aggregate formation. Lack of NO synthase 2 in the amyloid precursor protein Swedish mutant mouse increased insoluble beta-amyloid peptide levels, neuronal degeneration, caspase-3 activation, and tau cleavage, suggesting that NO acts at a junction point between beta-amyloid peptides, caspase activation, and tau aggregation.

Apolipoprotein E-derived peptides reduce CNS inflammation: implications for therapy of neurological disease.

The apolipoprotein E4 isoform (apoE4) was initially identified as a susceptibility gene for the development of Alzheimer's disease, and has also recently been associated with poor outcome after acute traumatic and ischemic brain injury. One mechanism by which apoE may influence outcome in acute and chronic neurological disease is by downregulating glial activation and the neuroinflammatory response. Because it does not readily cross the blood-brain barrier (BBB), the apoE holoprotein has limited therapeutic potential. However, smaller peptides derived from the receptor binding region of apoE have been developed that mimic the functional anti-inflammatory and neuroprotective effects of the intact apoE protein. These apoE-derived therapeutic peptides cross the BBB and have been demonstrated to improve functional and histological outcomes in murine models of brain injury. Thus, the development of apoE-derived peptides represent a novel therapeutic strategy for the treatment of acute and chronic neurological disease.

Disrupted spermine homeostasis: a novel mechanism in polyglutamine-mediated aggregation and cell death.

Our data suggest a novel mechanism whereby pathological-length polyglutamine (polyQ) proteins promote the spermine synthetic pathway, increasing polyQ-aggregation and cell death. As detected in a cell-free turbidity assay, spermine promotes aggregation of thio-polyQ62 in a dose-dependent manner. Using a stable neuronal cell line expressing pathological-length [polyQ57-yellow fluorescent protein (YFP) (Q57)] or non-pathological-length [polyQ19-YFP (Q19)] polyglutamine protein, we show that multiple steps in the production of polyamines are affected in Q57 cells, suggesting dysfunctional spermine homeostasis. As the building block for spermine synthesis, arginine transport is significantly increased in neuronal cell lines stably expressing Q57. Q57 lines displayed upregulated basal and inducible arginase I activities that were not seen in polyQ19-YFP lines. Normal induction of spermidine/spermine N-acetyltransferase in Q19 lines regulating back-conversion of spermine, thereby reducing spermine levels, however, was not observed in Q57 lines. Pharmacological activation of ornithine decarboxylase (ODC), a key enzyme of the polyamine synthetic pathway, increased cellular aggregates and increased cell death in Q57 cells not observed in Q19 cells. Inhibition of ODC by difluoromethylornithine prevented basal and induced cell death in Q57 cells, demonstrating a central role for polyamines in this process.

Hypothermic treatment restores glucose regulated protein 78 (GRP78) expression in ischemic brain.

Mild hypothermia is a well-known method of reducing brain damage caused by traumatic, hypoxic, and ischemic injury. To elucidate the neuroprotective mechanism induced by hypothermic treatment, we compared gene expression profiles in the hippocampus of gerbils rendered ischemic for 15 min and then reperfused for 3 h under conditions of normothermia (37+/-0.5 degrees C) or hypothermic treatment (34+/-0.5 degrees C). Using the differential display method, we observed significantly reduced expression of the 78 kDa glucose regulated protein (GRP78), in ischemic gerbil hippocampus that underwent normothermic reperfusion, but normal GRP78 expression in animals that underwent hypothermic reperfusion. In situ hybridization and Northern blot analysis showed GRP78 mRNA expression was reduced in the CA1 region of the hippocampus under normothermic conditions, but was not reduced under hypothermic conditions. Western blot analysis also showed the levels of immunoreactive GRP78 protein decreased in neurons of the hippocampal CA-1 region under normothermia, but not under hypothermic treatments. Furthermore, adenovirus-mediated overexpression of GRP78 protects rat hippocampal neurons from cell death and inhibits the rise in intracellular calcium concentration normally induced by hydrogen peroxide. These results suggest that reduction in GRP78 expression contributes to cell damage in the ischemic brain and that hypothermia-mediated restoration of GRP78 expression is one mechanism that enhances neuronal survival.