Luciano Galeazzi

In vitro peroxidase oxidation induces stable dimers of β-amyloid (1-42) through dityrosine bridge formation

beta-amyloid (A beta) is a normal soluble peptide found in the cerebrospinal fluid (CSF) and other biological fluids. A beta fibrils are associated with Alzheimers disease (AD) senile plaques. We have used purified soluble A beta (1-42) and A beta (12-28) peptides in order to determine the oxidative modification induced in these peptides by exposure to peroxidase and hydrogen peroxide. We have demonstrated that under these in vitro conditions, dimeric forms of A beta (1-42) can be detected by high-resolution polyacrylamide SDS-PAGE electrophoresis. Further experiments performed by reverse-phase high performance liquid chromatography (RP-HPLC), and monitored by fluorescence detection, showed that the dimeric A beta (1-42) forms induced by the peroxidase reaction are the outcomes of dityrosine bridge formation. This cross-link results from the enzyme catalyzed oxidation. During this reaction, phenolic coupling of tyrosine residues of two A beta (1-42) peptides occurs. No detectable peroxidative modifications were observed with the A beta (12-28) peptide which lacks a tyrosine residue. Since oxidative stress is thought to be associated with AD, the experimental model described here can help in understanding the early events leading to chemical, structural and conformational modifications before the conversion of sA beta to amyloid fibrils and eventually the formation of senile plaques in AD.

Albumin protects human red blood cells against Aβ25-35-induced lysis more effectively than ApoE

Inhibition of the lysis of human red blood cells (RBCs) exposed to amyloid peptide A&bgr;25–35 is an in vitro model for screening natural and synthetic substances potentially protective against amyloid damage. In this system, human serum and a component, namely apolipoprotein E (apoE), completely prevent RBC lysis. This report demonstrates that albumin, another serum component, is 8-fold more protective: a concentration of 12.5 μg/ml protects RBCs against 20 μM-A&bgr;25–35, and prevents the formation of fibrillar A&bgr;25–35 aggregates stainable by Congo Red. The biological relevance of these findings is suggested by the following: (1) a large fraction (∼90%) of circulating A&bgr;1–42 is bound to albumin; (2) albumin immunoreactivity is present in brain amyloid plaques; and (3) incubation of A&bgr; with albumin rapidly decreases detectable levels of free A&bgr; suggesting epitope masking. The results add new and important functional consequences to the amyloid-albumin relationship and imply that experimental systems investigating A&bgr; cytotoxicity should consider the protective interaction of albumin.

An in vitro bacterial model of cytotoxicity to living cells caused by dopamine and 6-hydroxydopamine oxidation at physiological pH

The cytotoxicity of dopamine (DA) and 6-hydroxydopamine (6-OHDA) on living cells, in vitro, has been previously deeply investigated in neuroblastoma cells. This study was designed to explore the possibility to use bacteria as targets for studying DA and 6-HODA cytotoxicity. Both DA and 6-HODA oxidize when added to bacteriological media. The rate of autoxidation of 6-HODA was greater than DA within the first hours. The oxidation-dependent cytotoxicity caused bacterial growth-inhibition and killing at concentration of 10(-4)M. All the bacterial strains tested were slightly more susceptible to DA than to 6-HODA. Antioxidants (sodium metabisulfite, cysteine) prevented the oxidation and abolished the growth-inhibitory activity. The addition of exogenous catalase protected the cells against the effect of the oxidation of both the catecholamines up to the concentration of 5 mM, while the addition of exogenous superoxide dismutase protected the cells only at the minimal inhibitory concentrations. Taking into account that some of the results obtained are similar to those previously reported using neuroblastoma cells as targets, the use of bacteria for studying oxygen toxicity from these catecholamines seems to be a potentially useful model system.

Cytochemistry of Intraplatelet Ca++ Spots as a Peripheral Marker of Age‐related Brain Impairment

The etiology and/or the causative events leading to dementing illnesses typical of the third age are still poorly understood. However, an early diagnosis may offer a good opportunity to tackle the problem in due time and, hopefully, to retard the progressive and relentless decline of the senile demented brain. Precocious changes in peripheral cells have been hypothesized to mirror alterations occurring in neurons.1,2 Conceivably these alterations may constitute potential markers of the risk to develop an age-related dementing pathology. In agreement with this rationale, a consistent proliferation of internal membranes has been reported within the platelets of patients affected by Alzheimer’s disease.1,2 These newly synthesized membranes are supposed to be involved in sequestering the free calcium ions escaping impaired homeostatic mechanisms.2,3 With the aim of assessing the calcium content in human platelets, we set up a morphometric procedure to verify whether cytochemically evidenced intraplatelet calcium aggregates can be considered as a predictive risk factor of a dementing pathology of the senile brain. Human platelets were isolated from 10 ml of blood samples obtained from 6 adult healthy volunteers (mean age: 45.3 years). Intraplatelet calcium ions were cytochemically evidenced by the oxalate pyroantimonate (OPA) preferential staining.4,5 Embedding, sectioning, and contrasting were carried out according to conventional electron microscopic procedures. The number and the area of the OPA aggregates/μm2 of the total sampled area, the area of the OPA deposits/μm2 of platelet surface, and the percentage of OPA-positive platelets were measured by random sampling and point counting methods adapted to our image analysis system. Collection of data was terminated when 100 OPA-positive platelets were analyzed. The OPA-Ca++ reaction sites can be easily identified within platelets as discrete dark spots with a sharp membrane (FIG. 1). The histograms reported in FIGURE 2 show

Differential course of HIV-1 infection and apolipoprotein E polymorphism

We studied the course of infection with human immunodeficiency virus type 1 (HIV-1) in relation to apolipoprotein E (APOE) polymorphism found for 209 Italians treated at Infectious Disease Clinics in Rome and Modena. Clinically, patients were classified into four groups according to the yearly rate of decline in CD4+ cell count (LTNP: long-term non-progression; SLOW, ’NORMAL’ or RAPID). Patients at both extremes of the clinical spectrum, i.e. those who rapidly progressed to AIDS and those with stable high CD4 cell counts, had few APOE ɛ4 and ɛ2 alleles (P = 0.04). Detailed clinical information was then used to construct four model-based clinical profiles using grade-of-membership analysis (GoM), predictive of APOE genotypic frequencies: 1. The clinical profile associated with good long-term prognosis lacked ɛ2 (P=0.01); 2. Disease progression to AIDS was associated with ɛ4 and ɛ2, most evident for zidovudine-lamivudine regimens without a protease inhibitor (P = 0.03); and, 3. AIDS patients had low ɛ4 and ɛ2 frequencies, consistent with a high mortality rate among ɛ4+ and ɛ2+ AIDS patients. These findings suggest allele-specific immunomodulatory effects involving inherited APOE isoform important enough to alter the clinical course of HIV infection and, possibly, drug efficacy. They imply a connection between lipid metabolism and immunity potentially relevant to common disorders.

In vitro apolipoprotein E protects human red blood cells against lysis induced by amyloid-beta (Aβ) fragment 25-35

Mattson et al.9 demonstrated lysis of human red blood cells (RBC) exposed to amyloid peptide Aβ25-35′ a new experimental model for amyloid-beta toxicity. Lysis resulted from pore formation in the RBC membranes and was completely prevented by concurrent exposure to Congo red. We demonstrate that human serum, purified ApoE from human plasma, and recombinant isoforms of ApoE neutralize the Aβ25-35 cytotoxicity: the E2 and E4 isoforms were marginally more effective than E3. Second, we demonstrate that Aβ25-35 forms fibrils in the reaction mixtures using electronmicroscopy. Together these results suggest that the RBC model might be useful in preliminary identification of natural and synthetic substances able to protect against amyloid-beta cytotoxic effects due to fibrillar Aβ25-35. Such compounds would be candidate molecules for testing in neuronal systems.

β-amyloid fragment 25-35 induces changes in cytosolic free calcium in human platelets

The beta-amyloid (βA) peptide has a central role in Alzheimer’s disease (AD). Indeed, the major histopathologic hallmarks of AD include βA deposits in brain parenchima (senile plaques) and also around and within the walls of blood vessels (cerebral amyloid angiopathy).1 βA deposits are the final result of an amyloidogenic process triggered by oxidative and conformational modifications and by crosslinking of βA.2–4 βA is a peptide produced by the proteolysis of the amyloid precursor protein (APP). It has been reported that abnormalities of brain APP metabolism may be reflected in platelets, which also possess all the machinery to generate the amyloid β (Aβ)-fragment from APP; moreover, platelets are the primary source of Aβ-peptide in human blood.5–7 In human cortical neurons β-amyloid peptides have been shown to destabilize calcium homeostasis and cause neurodegenerative effects.8,9 Alteration in calcium homeostasis induced by Aβ have been reported also for nonneuronal cells.10,11 Moreover, it has been demonstrated that Aβ25–35 increases cellular APP by inhibiting its secretory processing in human extraneuronal cells.12 The data reported above encourage further exploration about amyloid and platelets as a peripheral laboratory mirroring central amyloid metabolism and activity.1 In the present paper we investigate the effects of neurotoxic Aβ25–35 peptide on platelets and we show that this Aβ-fragment is able to induce dosedependent changes of calcium concentration and degenerative effects in normal human platelets. Blood samples were taken from male healthy donors aged between 31 and 50 years (mean age: 38 ± 7). Citrated blood was centrifuged for 10 min at 200 × g to obtain platelet-rich plasma (PRP). Platelets were separated from PRP by centrifugation at 2000 × g for 20 min and washed twice in phosphate-buffered saline (PBS) 0.1 M, pH 7.4. Platelets were resuspended at a density of 108/ml in Hepes buffer containing 145 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM Hepes, 10 mM glucose, adjusted to pH 7.4. Ten μM prostaglandin E1 (PGE1) was added to prevent aggregation. The platelet suspension was incubated in 1 μM Aβ25–35

Transformation of beta-amyloid (Aβ) (1-42) tyrosine to L-Dopa as the result of in vitro hydroxyl radical attack

A form of beta-amyloid peptide Aβ ending at amino acid 42 (Aβ42) is the major component of senile amyloid plaques in Alzheimers Disease (AD). The Aβ-peptide earliest modifications are extremely important since they constitute the key events in the progression towards further changes finally leading to fibril formation and to Aβ deposits which constitute the core pathological change in AD. Chemical and conformational early modifications of the beta-amyloid peptide are critical steps in AD pathogenesis and have been widely investigated. We now show that a Fenton-type OH-generating system is capable of generating L-Dopa (3, 4-dihydroxyphenylalanine) in the tyrosine residue of Aβ-peptide via aromatic ring hydroxylation, as the result of hydroxyl radical attack on proteins. Since L-Dopa is not a constituent of mammalian proteins and peptides, the formation of L-Dopa in Aβ in vitro constitutes a possible important modification caused by hydroxyl radical attack These results lay the groundwork for further studies on modification and damage associated with the degenerative disorder in AD where oxidative stress and inflammation are known to occur.

β-Amyloid fragment 25-35 selectively damages platelets from patients with Alzheimer's disease

Abstract: In order to analyze the metabolic response of AD patient platelets to β‐amyloid, we have carried out fluorimetric measurements of intracellular calcium and an ultrastructural survey of platelets exposed to the β‐amyloid active fragment 25–35 (βA25‐35). Since it is not possible to analyze directly the damaged neurons in AD, the study of peripheral blood cells, especially platelets, may be of great value for the investigation of the toxic effects of β‐amyloid on AD neuronal cells.

Streptococcus faecalis susceptibility to amiloride depends on medium pH

Amiloride is one of the major molecular probes in basic and applied investigations on the physiology of cation transport in animal cells. In these cells the drug also exerts growth inhibitory activity. Recently, we discovered that amiloride causes growth inhibition also on bacterial cells. In this paper we report that medium pH influences amiloride activity on Streptococcus faecalis. The lowering of external pH causes a drop in the susceptibility of this bacterium to amiloride up to an almost complete resistance. This finding, constitutes a novel aspect of the in vitro experimental pharmacology of this diuretic potentially useful also in clinical pharmacology and in animal cell investigations.