Intracellular A-beta amyloid,a sign for worse things to come?

In this review the authors discuss the possible neuropathological role of intracellular amyloid-beta accumulation in Alzheimer's disease (AD) pathology. There is abundant evidence that at early stages of the disease, prior to A-beta amyloid plaque formation, A-beta peptides accumulate intraneuronally in the cerebral cortex and the hippocampus. The experimental evidence would indicate that intracellular amyloid-beta could originate both by intracellular biosynthesis and also from the uptake of amyloidogenic peptides from the extracellular milieu. Herein the aspects of the possible impact of intracellular amyloid-beta in human AD pathology are discussed, as well as recent observations from a rat transgenic model with a phenotype of intracellular accumulation of A-beta fragments in neurons of the hippocampus and cortex, without plaque formation. In this model, the intracellular amyloid-beta phenotype is accompanied by increased MAPK/ERK activity and tau hyperphosphorylation. Finally, the authors discuss the hypothesis that, prior to plaque formation, intracellular A-beta accumulation induces biochemical and pathological changes in the brain at the cellular level priming neurons to further cytotoxic attack of extracellular amyloidogenic peptides.     

Pyroglutamate-modified amyloid beta-peptides--AbetaN3(pE)--strongly affect cultured neuron and astrocyte survival

N-terminally truncated amyloid-beta (Abeta) peptides are present in early and diffuse plaques of individuals with Alzheimer's disease (AD), are overproduced in early onset familial AD and their amount seems to be directly correlated to the severity and the progression of the disease in AD and Down's syndrome (DS). The pyroglutamate-containing isoforms at position 3 [AbetaN3(pE)-40/42] represent the prominent form among the N-truncated species, and may account for more than 50% of Abeta accumulated in plaques. In this study, we compared the toxic properties, fibrillogenic capabilities, and in vitro degradation profile of Abeta1-40, Abeta1-42, AbetaN3(pE)-40 and AbetaN3(pE)-42. Our data show that fibre morphology of Abeta peptides is greatly influenced by the C-terminus while toxicity, interaction with cell membranes and degradation are influenced by the N-terminus. AbetaN3(pE)-40 induced significantly more cell loss than the other species both in neuronal and glial cell cultures. Aggregated AbetaN3(pE) peptides were heavily distributed on plasma membrane and within the cytoplasm of treated cells. AbetaN3(pE)-40/42 peptides showed a significant resistance to degradation by cultured astrocytes, while full-length peptides resulted partially degraded. These findings suggest that formation of N-terminally modified peptides may enhance beta-amyloid aggregation and toxicity, likely worsening the onset and progression of the disease.     

The translocation of focal adhesion kinase in brain synaptosomes is regulated by phosphorylation and actin assembly

Focal adhesion kinase (FAK) and the related proline-rich tyrosine kinase 2 (PYK2) are non-receptor protein tyrosine kinases that transduce extracellular signals through the activation of Src family kinases and are highly enriched in neurones. To further elucidate the regulation of FAK and PYK2 in nervous tissue, we investigated their distribution in brain subcellular fractions and analysed their translocation between membrane and cytosolic compartments. We have found that FAK and PYK2 are present in a small membrane-associated pool and a larger cytosolic pool in various neuronal compartments including nerve terminals. In intact nerve terminals, inhibition of Src kinases inhibited the membrane association of FAK, but not of PYK2, whereas tyrosine phosphatase inhibition sharply increased the membrane association of both FAK and PYK2. Disruption of the actin cytoskeleton was followed by a decrease in the membrane-associated pool of FAK, but not of PYK2. For both kinases, a significant correlation was found between autophosphorylation and membrane association. The data indicate that FAK and PYK2 are present in nerve terminals and that the membrane association of FAK is regulated by both phosphorylation and actin assembly, whereas that of PKY2 is primarily dependent on its phosphorylation state.     

Triacylglycerol-rich lipoproteins trigger the phosphorylation of extracellular-signal regulated kinases in vascular cells

Postprandial triacylglycerol-rich lipoproteins (TRL) have been implicated in the pathophysiology of atherosclerosis, but the intracellular processes by which TRL could affect vascular function are still unknown. Incubation of TRL obtained at 2 h postprandial period with vascular smooth muscle cells (VSMC) produced a tyrosine phosphorylation of the extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) that belong to the mitogen-activated protein kinase (MAPK) family. The activation of ERK1 and ERK2 had a maximum at 15 min, returned to baseline by 60 min, and was partially depleted after incubation of cells with a MAPKK inhibitor (PD 098059). In addition, postprandial TRL did competent VSMC for DNA replication through a MAPK pathway. These effects were dependent of the lipid composition of TRL. Our observations suggest that postprandial TRL can trigger activation of the MAPK pathway and induce a mitogenic response in VSMC in a lipid-dependent fashion.     

FABP4 and omentin-1 gene expression in epicardial adipose tissue from coronary artery disease patients

Omentin-1 and fatty acid-binding protein 4 (FABP4) are adipose tissue adipokines linked to obesity-associated cardiovascular complications. The aim of this study was to investigate epicardial adipose tissue (EAT) omentin-1 and FABP4 gene expression in obese and non-obese patients with coronary artery disease (CAD). Omentin-1 and FABP4 mRNA levels in EAT and paired subcutaneous adipose tissue (SAT) as well as adipokine serum concentrations were assessed in 77 individuals (61 with CAD; 16 without CAD (NCAD)). EAT FABP4 mRNA level was decreased in obese CAD patients when compared to obese NCAD individuals (p=0.001). SAT FABP4 mRNA level was decreased in CAD patients compared to NCAD individuals without respect to their obesity status (p=0.001). Omentin-1 mRNA level in EAT and SAT did not differ between the CAD and NCAD groups. These findings suggest that omentin-1 gene expression in adipose tissue is not changed during CAD; downregulated FABP4 gene expression in SAT is associated with CAD while EAT FABP4 gene expression is decreased only in obesity-related CAD.     

Detection of DNA in Ancient Bones Using Histochemical Methods

We describe histochemical techniques for detecting DNA within the osteocytic lacunae of ancient bones. The bones examined were fragments of femurs from two human individuals found in the Pompeian C. I. Polybius house and fragments of metacarpals from two horses (Equus sp.) found in the Pompeian “Casti Amanti” house. Both buildings were buried by the 79 A. D. Vesuvius eruption. Fragments of femurs from a modern horse, a modern swine and a modern amphibian also were studied as controls. Some bone sections were stained with two different DNA-specific fluorochromes, 4' -' 6-diamidino-2-phenylindole (DAPI) and chromomycin A3 (CMA), while others were stained by the Feulgen reaction. All of the techniques gave a positive reaction within the osteocytic lacunae. Histological analysis of the undecalcified, ground and unstained sections agreed well with results of bone sections stained with either the fluorochromes or the Feulgen reaction. Bones showing good histology also were positive by our DNA-specific stain. Histochemical and histological analyses correlated well with the success of DNA extraction and amplification. Using conventional DNA-specific histochemical techniques in conjunction with histological analysis can be useful in the study of DNA extracted from ancient bone remains while reducing both the amount of time and cost.     

Sex- and tissue-specific expression of aspartic proteinases in Danio rerio (zebrafish)

Full-length zebrafish cDNAs encoding two aspartic proteinases were cloned and sequenced. One of the two cDNAs was a 1708 bp product with an open reading frame of 398 amino acid residues corresponding to a cathepsin D. The other was a 1383 bp product encoding a polypeptide chain of 416 amino acids homologous to nothepsin, an aspartic proteinase first identified by us in the liver of Antarctic Notothenioidei. Gene expression assessed by RT–PCR and northern blot hybridization of RNA from different tissues showed that the expression was tissue- and sex-specific. Whereas the cathepsin D gene was expressed in all the tissues examined independently of the sex, the nothepsin gene was expressed exclusively in female livers.     

Inhibitors of histone deacetylase 6 based on a novel 3-hydroxy-isoxazole zinc binding group

Histone deacetylase 6 (HDAC6) is an established drug target for cancer treatment. Inhibitors of HDAC6 based on a hydroxamic acid zinc binding group (ZBG) are often associated with undesirable side effects. Herein, we describe the identification of HDAC6 inhibitors based on a completely new 3-hydroxy-isoxazole ZBG. A series of derivatives decorated with different aromatic or heteroaromatic linkers, and various cap groups were synthesised and biologically tested. In vitro tests demonstrated that some compounds are able to inhibit HDAC6 with good potency, the best candidate reaching an IC₅₀ of 700 nM. Such good potency obtained with a completely new ZBG make these compounds particularly attractive. The effect of the most active inhibitors on the acetylation levels of histone H3 and α- tubulin and their anti-proliferative activity of DU145 cells were also investigated. Docking studies were performed to evaluate the binding mode of these new derivatives and discuss structure-activity relationships.