99mTc- and Re-labeled 6-dialkylamino-2-naphthylethylidene derivatives as imaging probes for β-amyloid plaques

Based on the conjugate strategy, two neutral ^99mTc labeled 2-(1-(6-(dialkylamino)naphthalen-2-yl)ethylidene)malononitrile (DDNP) and 1-(6-(dialkylamino)naphthalen-2-yl)ethanone (ENE) derivatives, and their corresponding rhenium complexes were synthesized. In vitro fluorescent staining indicated that the corresponding rhenium derivatives selectively stained the β-amyloid (Aβ) plaques in the brain sections of AD model mice with low background. Compared with FDDNP and FENE, the affinities of the corresponding rhenium derivatives to Aβ aggregates decreased about 10-14-fold. In vivo biodistribution experiments in normal mice showed that ^99mTc-MAMA-ENE displayed medium initial brain uptake (0.65 %ID/g at 2 min) with a reasonable washout from the brain (0.19 %ID/g at 2 h) while ^99mTc-MAMA-DDNP showed a low brain uptake (0.28 %ID/g at 2 min). Further optimize these ^99mTc-labeled tracers in order to improve their binding affinities to Aβ plaques and diffusion through the blood brain barrier may generate useful imaging agents for SPECT.     

IGF-1 promotes β-amyloid production by a secretase-independent mechanism

β-Amyloid peptide (Aβ) is generated via the sequential proteolysis of β-amyloid precursor protein (APP) by β- and γ-secretases, and plays a crucial role in the pathogenesis of Alzheimer’s disease (AD). Here, we sought to clarify the role of insulin-like growth factor-1 (IGF-1), implicated in the AD pathomechanism, in the generation of Aβ. Treatment of neuroblastoma SH-SY5Y cells expressing AD-associated Swedish mutant APP with IGF-1 did not alter cellular levels of APP, but significantly increased those of β-C-terminal fragment (β-CTF) and secreted Aβ. IGF-1 also enhanced APP phosphorylation at Thr668. Treatment of β-CTF-expressing cells with IGF-1 increased the levels of β-CTF and secreted Aβ. The IGF-1-induced augmentation of β-CTF was observed in the presence of γ-secretase inhibitors, but not in cells expressing β-CTF with a Thr668 to alanine substitution. These results suggest that IGF-1 promotes Aβ production through a secretase-independent mechanism involving APP phosphorylation.     

Amino-caprolactam γ-secretase inhibitors showing potential for the treatment of Alzheimer's disease

Herein we describe the structure-activity relationship (SAR) of amino-caprolactam analogs derived from amino-caprolactam benzene sulfonamide 1, highlighting affects on the potency of γ-secretase inhibition, selectivity for the inhibition of APP versus Notch processing by γ-secretase and selected pharmakokinetic properties. Amino-caprolactams that are efficacious in reducing the cortical Aβ_x-40 levels in FVB mice via a single 100 mpk IP dose are highlighted.     

Novel quinoxaline derivatives for in vivo imaging of β-amyloid plaques in the brain

In a search for new probes to detect β-amyloid plaques in the brain of patients with Alzheimer’s disease (AD), we have synthesized and evaluated a series of quinoxaline derivatives containing a ‘6+6−6’ ring system. These quinoxaline derivatives showed excellent affinity for Aβ_1-42 aggregates with K_i values ranging from 2.6 to 10.7 nM. Autoradiography with sections of brain tissue from an animal model of AD mice (APP/PS1) and AD patients revealed that [¹²⁵I]5 labeled β-amyloid plaques specifically. In biodistribution experiments using normal mice, [¹²⁵I]5 displayed high uptake (6.03% ID/g at 2 min) into and a moderately fast washout from the brain. Although additional refinements are needed to decrease the lipophilicity and improve the washout rate, the quinoxaline scaffold may be useful as a backbone structure to develop novel β-amyloid imaging agents.     

Synthesis and biological evaluation of indole-chalcone derivatives as β-amyloid imaging probe

A series of chaclone derivatives containing an indole moiety were evaluated in competitive binding assays with Aβ_1-42 aggregates versus [¹²⁵I]IMPY. The affinity of these compounds ranged from 4.46 to >1008 nM, depending on the substitution on the phenyl ring. Fluorescent staining in vitro showed that one compound with a N,N-dimethylamino group intensely stained Aβ plaques within brain sections of AD transgenic mice. The radioiodinated probe [¹²⁵I]-(E)-3-(1H-indol-5-yl)-1-(4-iodophenyl)prop-2-en-1-one, [¹²⁵I]4, was prepared and autoradiography in sections of brain tissue from an animal model of AD showed that it labeled Aβ plaques specifically. However, experiments with normal mice indicated that [¹²⁵I]4 exhibited a low uptake into the brain in vivo (0.41% ID/g at 2 min). Additional chemical modifications of this indole-chalcone structure may lead to more useful imaging agents for detecting β-amyloid plaques in the brains of AD patients.     

Proteolytic processing of the ovine prion protein in cell cultures

The cellular compartment and purpose of the proteolytic processing of the prion protein (PrP) are still under debate. We have studied ovine PrP constructs expressed in four cell lines; murine neuroblastoma cells (N2a), human neuroblastoma cells (SH-SY5Y), dog kidney epithelial cells (MDCK), and human furin-deficient colon cancer cells (LoVo). Cleavage of PrP in LoVo cells indicates that the processing is furin independent. Neither is it reduced by some inhibitors of lysosomal proteinases, proteasomes or zinc-metalloproteinases, but incubation with bafilomycin A1, an inhibitor of vacuolar H+/ATPases, increases the amount of uncleaved PrP in the apical medium of MDCK cells. Mutations affecting the putative cleavage site near amino acid 113 reveal that the cleavage is independent of primary structure at this site. Absence of glycosylphosphatidylinositol anchor and glycan modifications does not influence the proteolytic processing of PrP. Our data indicate that PrP is cleaved during transit to the cell membrane.     

Protease digestion indicates that endogenous presenilin 1 is present in at least two physical forms

The membrane-bound protein complex γ-secretase is an intramembranous protease whose substrates are a number of type I transmembrane proteins including the β-amyloid precursor protein (APP). A presenilin molecule is thought to be the catalytic unit of γ-secretase and either of two presenilin homologues, PS1 or PS2, can play this role. Mutations in the presenilins, apparently leading to aberrant processing of APP, have been genetically linked to early-onset familial Alzheimer’s disease. To look for possible molecular heterogeneity in presenilin/γ-secretase we examined the ability of proteinase K (PK) to digest endogenously expressed presenilins in intact endoplasmic reticulum vesicles. We demonstrate the existence of two physically different forms of γ-secretase-associated PS1, one that is relatively PK-sensitive and one that is significantly more PK-resistant. A similarly PK-resistant form of PS2 was not observed. We speculate that the structural heterogeneity we observe may underlie, at least in part, previous observations indicating the physical and functional heterogeneity of γ-secretase. In particular, our results suggest that there are significant differences between γ-secretase complexes incorporating PS1 and PS2. This difference may underlie the more dominant role of PS1 in the generation of β-amyloid peptides and in familial Alzheimer’s disease.     

Comparison of two cellular harvesting methods for primary human oral culture of keratinocytes

The possibility of obtaining transplantable oral epithelia opens new perspectives for oral treatments. Most of them are surgical, resulting in mucosal failures. As reconstructive material this in vitro epithelia would be also useful for other parts of the human body. Many researchers still use controversial methods; therefore it was evaluated and compared the efficiency of the enzymatic and direct explant methods to obtain oral keratinocytes. To this project oral epithelia fragments were used. This work compared: time needed for cell obtainment, best cell amount, life-span and epithelia forming cell capacity. The results showed the possibility to obtain keratinocytes from a small oral fragment and we could verify the advantages and peculiar restrictions. We concluded that under our conditions the enzymatic method showed the best results: in the cells obtaining time needed, cell amount and life-span. Both methods showed the same capacity to form in vitro epithelia.     

Artefacts in cell culture: Pyruvate as a scavenger of hydrogen peroxide generated by ascorbate or epigallocatechin gallate in cell culture media

Ascorbate and several phenolic compounds readily oxidise in cell culture media to generate hydrogen peroxide. However, media containing pyruvate showed much less H₂O₂ production, apparently because pyruvate can scavenge H₂O₂ in the medium. Researchers must be aware that compounds under test can sometimes readily oxidise in cell culture media, that this might not be detected by measurement of H₂O₂ if the media contain pyruvate, and that pyruvate can be substantially depleted in the media as a result.     

ROCK, PAK, and Toll of synapses in Alzheimer's disease

Alzheimer’s disease is a neurodegenerative disorder where the cognitive deficit is the hallmark symptom reflecting the progression of the disease. Synaptic dysfunction is a sensitive parameter of the AD pathology. Rho GTPases and the Rho kinases, ROCK1/2, and PAK1-3, are important regulators of synaptic plasticity, especially in maintaining the actin cytoskeleton of dendritic spines. Recent studies have revealed that β-amyloid oligomers can inhibit PAK and stimulate ROCK-mediated signaling. Both of these effects enhance the disassembly of synaptic actin filaments and ultimately evoke synaptic loss. Brain tissue in AD recognizes the β-amyloid peptide oligomers as foreign protein particles and mounts an inflammatory defense via Toll-like receptor (TLR) signaling which causes synaptic impairment. We will review here the dysfunction of ROCK, PAK, and Toll signaling associated with AD pathology. The protection of synapses in AD may provide new therapeutic approaches to combatting the cognitive impairment in AD.     

Establishment of primary cell culture from the temperate symbiotic cnidarian, Anemonia viridis

The temperate symbiotic sea anemone Anemonia viridis, a member of the Cnidaria phylum, is a relevant experimental model to investigate the molecular and cellular events involved in the preservation or in the rupture of the symbiosis between the animal cells and their symbiotic microalgae, commonly named zooxanthellae. In order to increase research tools for this model, we developed a primary culture from A. viridis animal cells. By adapting enzymatic dissociation protocols, we isolated animal host cells from a whole tentacle in regeneration state. Each plating resulted in a heterogeneous primary culture consisted of free zooxanthellae and many regular, small rounded and adherent cells (of 3–5 μm diameter). Molecular analyses conducted on primary cultures, maintained for 2 weeks, confirmed a specific signature of A. viridis cells. Further serial dilutions and micromanipulation allowed us to obtain homogenous primary cultures of the small rounded cells, corresponding to A. viridis “epithelial-like cells”. The maintenance and the propagation over a 4 weeks period of primary cells provide, for in vitro cnidarian studies, a preliminary step for further investigations on cnidarian cellular pathways notably in regard to symbiosis interactions.     

2-Arylimidazo[2,1-b]benzothiazoles: a new family of amyloid binding agents with potential for PET and SPECT imaging of Alzheimer's brain

We designed and synthesized a small series of 2-aryl-imidazo[2,1-b]benzothiazole, representing a combination of motifs from the two most potent amyloid imaging agents, PIB and IMPY. The binding affinity of the new compounds ranged from 6 to 133 nM. Among the best compounds, 3b (K_i = 6 nM) can be labeled with ¹¹CH₃ for PET imaging whereas 3j (K_i = 10.9 nM) can be labeled with ¹²³I for SPECT imaging.     

A free radical-generating system regulates APP metabolism/processing

Oxidative stress, a risk factor in the pathophysiology of Alzheimer’s disease, is intimately associated with aging. We previously reported that the X-XOD free radical generating system acts as a modulator of lipid metabolism and a mild inducer of apoptotic death. Using the same cell model, the present study examines the metabolism/processing of the amyloid precursor protein (APP). Prior to inducing cell death, X-XOD promoted the secretion of α-secretase-cleaved soluble APP (sAPPα) and increased the level of APP carboxy-terminal fragments produced by α and γ secretase (αCTF and γCTF/AICD). In contrast, it reduced the activity of β-secretase and the level of secreted Aβ. The present results indicate that mild oxidative stress maintained throughout culturing regulates APP metabolism/processing in SK-N-MC human neuroblastoma cells.     

Isolation and primary culture of adult human hepatocytes

Summary Biopsy tissue of adult human liver was gently dissociated with collagenase followed by Dispase. By repeated low g centrifugation, a large number of almost pure, viable hepatocytes was obtained. This is the first report of a successful procedure for obtaining adult human hepatocytes for study in tissue culture. The isolated cells have the typical morphology of liver parenchyma, and these characteristics persist throughout the period of culturing. Evidence of their function is indicated by albumin synthesis. This procedure is now being used to study human hepatocyte functions in vitro and the effects of a variety of agents including carcinogens and viruses.     

Diphenylpropynone derivatives as probes for imaging β-amyloid plaques in Alzheimer's brains

A new series of diphenylpropynone (DPP) derivatives for use in vivo to image β-amyloid (Aβ) plaques in the brain of patients with Alzheimer’s disease (AD) were synthesized and characterized. Binding experiments in vitro revealed high affinity for Aβ (1-42) aggregates at a K_i value ranging from 6 to 326 nM. Furthermore, specific labeling of plaques was observed in sections of brain tissue from Tg2576 transgenic mice stained using one of the compounds, 1. In biodistribution experiments with normal mice, [¹²⁵I]1 displayed moderate uptake (1.55% ID/g at 2 min) and clearance from the brain with time (0.76 ID/g at 60 min). Taken together, DPP can serve as a new molecular scaffold for developing novel Aβ imaging agents by introducing appropriate substituted groups.     

Amino acid sequence determinants and molecular chaperones in amyloid fibril formation

Amyloid consists of cross-β-sheet fibrils and is associated with about 25 human diseases, including several neurodegenerative diseases, systemic and localized amyloidoses and type II diabetes mellitus. Amyloid-forming proteins differ in structures and sequences, and it is to a large extent unknown what makes them convert from their native conformations into amyloid. In this review, current understanding of amino acid sequence determinants and the effects of molecular chaperones on amyloid formation are discussed. Studies of the nonpolar, transmembrane surfactant protein C (SP-C) have revealed amino acid sequence features that determine its amyloid fibril formation, features that are also found in the amyloid β-peptide in Alzheimer’s disease and the prion protein. Moreover, a proprotein chaperone domain (CTC_Brichos) that prevents amyloid-like aggregation during proSP-C biosynthesis can prevent fibril formation also of other amyloidogenic proteins.     

Scleractinian coral cell proliferation is reduced in primary culture of suspended multicellular aggregates compared to polyps

Cell cultures from reef-building scleractinian corals are being developed to study the response of these ecologically important organisms to environmental stress and diseases. Despite the importance of cell division to support propagation, cell proliferation in polyps and in vitro is under-investigated. In this study, suspended multicellular aggregates (tissue balls) were obtained after collagenase dissociation of Pocillopora damicornis coral, with varying yields between enzyme types and brands. Ultrastructure and cell type distribution were characterized in the tissue balls (TBs) compared to the polyp. Morphological evidence of cellular metabolic activity in their ciliated cortex and autophagy in their central mass suggests involvement of active tissue reorganization processes. DNA synthesis was evaluated in the forming multicellular aggregates and in the four cell layers of the polyp, using BrdU labeling of nuclei over a 24 h period. The distribution of BrdU-labeled coral cells was spatially heterogeneous and their proportion was very low in tissue balls (0.2 ± 0.1 %), indicating that suspended multicellular aggregate formation does not involve significant cell division. In polyps, DNA synthesis was significantly lower in the calicoderm (<1 %) compared to both oral and aboral gastroderm (about 10 %) and to the pseudostratified oral epithelium (15–25 % at tip of tentacle). DNA synthesis in the endosymbiotic dinoflagellates dropped in the forming tissue balls (2.7 ± 1.2 %) compared to the polyp (14 ± 3.4 %) where it was not different from the host gastroderm (10.3 ± 1.2 %). A transient (24 h) increase was observed in the cell-specific density of dinoflagellates in individually dissociated coral cell cultures. These results suggest disruption of coral cell proliferation processes upon establishment in primary culture.     

Scavenger receptor control of chromogranin A-induced microglial stress and neurotoxic cascades

Chromogranin A (CgA), a neuroactive glycoprotein, is associated with microglial activation cascades implicated in neurodegeneration. Here we show that CgA-dependent inducible nitric oxide synthase (iNOS) expression and stress responses in microglia involved signalling via scavenger receptors (SR), since SR class-A (SR-A) ligands blocked iNOS expression, mitochondrial depolarisation, apoptosis and glutamate release. Furthermore, block of SR-A ameliorated CgA-induced microglial neurotoxicity. In contrast, block of CD36, or the receptor for advanced glycation end products (RAGE) did not prevent CgA-induced microglial activation and neurotoxicity. Thus, manipulation of specific scavenger receptor-coupled signalling pathways may provide avenues for therapeutic intervention in neurodegenerative diseases implicating microglial activation with chromogranin peptides.     

Impairment of superoxide dismutase activation by N-terminally truncated prion protein (PrP) in PrP-deficient neuronal cell line

Previous studies have reported a neuroprotective role for cellular prion protein (PrP(C)) against apoptosis induced by serum deprivation in an immortalized prion protein gene (Prnp)-deficient neuronal cell line, but the mechanisms remain unclear. In this study, to investigate the mechanisms by which PrP(C) prevents apoptosis, the authors compared apoptosis of Prnp(-/-) cells with that of Prnp(-/-) cells expressing the wild-type PrP(C) or PrP(C) lacking N-terminal octapeptide repeat region under serum-free conditions. Re-introduction of Prnp rescued cells from apoptosis, upregulated superoxide dismutase (SOD) activity, enhanced superoxide anion elimination, and inhibited caspase-3/9 activation. On the other hand, N-terminally truncated PrP(C) enhanced apoptosis accompanied by potentiation of superoxide production and caspase-3/9 activation due to inhibition of SOD. These results suggest that PrP(C) protects Prnp(-/-) cells from apoptosis via superoxide- and caspase-3/9-dependent pathways by upregulating SOD activity. Furthermore, the octapeptide repeat region of PrP(C) plays an essential role in regulating apoptosis and SOD activity.     

beta-Amyloid increases dendritic Ca2+ influx by inhibiting the A-type K+ current in hippocampal CA1 pyramidal neurons

Accumulation of the beta-amyloid peptide (Abeta) is a primary event in the pathogenesis of Alzheimer's disease (AD). However, the mechanisms by which Abeta mediates neurotoxicity and initiates the degenerative processes of AD are still not clear. Recent evidence shows that voltage-gated K+ channels may be involved in Abeta-induced neurodegenerative processes. In particular, a transient A-type K+ current, with a linear increase in its density with distance from soma to distal dendrites in hippocampal CA1 pyramidal neurons, has been shown to contribute to dendritic membrane excitability. Here, I report that Abeta (1-42) inhibits the dendritic A-type K+ current in hippocampal CA1 pyramidal neurons, and this inhibition causes increases in back-propagating dendritic action potential amplitude and associated Ca2+ influx. These results suggest that the persistent inhibition of the A-type K+ current resulting from deposition of Abeta in dendritic arborization will induce a sustained increase in dendritic Ca2+ influx and lead to loss of Ca2+ homeostasis. This may be a component of the events that cause synaptic failure and initiate neuronal degenerative processes in the hippocampus.