Nadia Angeretti

Apoptosis mediated neurotoxicity induced by chronic application of β amyloid fragment 25-35

To investigate whether and how amyloid-beta protein (A beta) is involved in the neurodegenerative changes characteristic of Alzheimers disease (AD), primary hippocampal neurones from foetal rat brain were exposed acutely and chronically to micromolar concentrations of a synthetic peptide homologous to residues 25-35 of A beta (beta 25-35). A single application of this peptide (25-100 microM) was ineffective but when the neuronal cultures were exposed to beta 25-35 (25-100 microM) repeatedly every two days for ten days, cell survival was dramatically reduced. The structural changes and the DNA fragmentation of cells chronically exposed to the peptide suggested that neuronal death occurred by apoptosis. Furthermore, beta 25-35 showed the intrinsic ability to polymerize into amyloid-like fibrils in vitro. These results confirm the potential pathogenic role of A beta in AD, and indicate that amyloid fibrils may induce neuronal death through a specific programmed process.

Expression of amyloid precursor protein mRNAs in endothelial, neuronal and glial cells: modulation by interleukin-1

The origin of beta-amyloid deposited in senile plaques in Alzheimers disease (AD) is not known. We compared the expression of protein precursor of beta-amyloid (APP) in the cell types involved in plaque formation. The levels of APP mRNA were determined in primary rat neurons and glial cells in culture, human endothelial cells and in a murine brain-derived endothelial cell line. Northern blot analysis was performed using an APP cDNA probe to detect the general APP sequence and an oligonucleotide (40 mer) complementary to the sequence of the Kunitz protease inhibitor (APP-KPI). The APP mRNA transcripts were abundant in all three cell types. The highest level of APP, normalized to beta-actin mRNA content, was expressed in neurons, followed by glial cells, where the APP expression was similar (94%) while in endothelial cells was lower (53%). The proportion between APP-KPI mRNA and total APP mRNA was high in endothelial, intermediate in glial and low in neuronal cells. We compared the effects of exposure to interleukin-1 (IL-1), a cytokine involved in several biological processes and elevated in AD, on APP mRNA expression in neuronal, glial and endothelial cells. In human endothelial and in brain-derived murine endothelial cells we observed a similar increase (50%) of total APP mRNA or APP-KPI mRNA after treatment with human recombinant IL-1 beta. In neuronal cells, IL-1 (200 ng/ml) substantially increased APP mRNA (175%), detected with both probes. In glial cells, the expression of APP mRNA did not appear to be altered by IL-1 (50-400 ng/ml). The results suggest a role of IL-1 in the neuronal mechanisms related to beta-amyloid protein deposition in AD.

Reciprocal control of inflammatory cytokines, IL-1 and IL-6, and β-amyloid production in cultures ☆

To investigate the role of IL-6 in the pathogenesis of Alzheimers disease (AD) its effect on amyloid precursor protein (APP) mRNA expression was evaluated. The levels of APP mRNA were determined by Northern blot analysis in primary cultured rat cortical neurons and glial cells exposed to IL-6 (50-200 ng/ml). The cytokine increased neuronal APP mRNA expression about 100% at the highest dose after 6 h of exposure. APP mRNA expression was unaffected in astroglial cells exposed to IL-6. Since IL-1 beta also increased neuronal APP mRNA, the combination of IL-1 beta and IL-6 was tested. The effects were partially additive. The ability of beta-amyloid fragment 25-35 to induce IL-1 or IL-6 mRNA was also investigated in astroglial cells. IL-1 beta mRNA was strongly induced by beta 25-35 (25-100 microM) while the expression of IL-6 mRNA remaining unchanged. The results suggest roles for both IL-1 and IL-6 in the neuronal mechanisms related to beta-amyloid protein deposition in AD.

Tetracyclines affect prion infectivity

Prion diseases are transmissible neurodegenerative disorders of humans and animals for which no effective treatment is available. Conformationally altered, protease-resistant forms of the prion protein (PrP) termed PrPSc are critical for disease transmissibility and pathogenesis, thus representing a primary target for therapeutic strategies. Based on previous findings that tetracyclines revert abnormal physicochemical properties and abolish neurotoxicity of PrP peptides in vitro, we tested the ability of these compounds to interact with PrPSc from patients with the new variant of Creutzfeldt–Jakob disease (vCJD) and cattle with bovine spongiform encephalopathy (BSE). The incubation with tetracycline hydrochloride or doxycycline hyclate at concentrations ranging from 10 μM to 1 mM resulted in a dose-dependent decrease in protease resistance of PrPSc. This finding prompted us to investigate whether tetracyclines affect prion infectivity by using an animal model of disease. Syrian hamsters were injected intracerebrally with 263K scrapie-infected brain homogenate that was coincubated with 1 mM tetracycline hydrochloride, 1 mM doxycycline hyclate, or vehicle solution before inoculation. Hamsters injected with tetracycline-treated inoculum showed a significant delay in the onset of clinical signs of disease and prolonged survival time. These effects were paralleled by a delay in the appearance of magnetic-resonance abnormalities in the thalamus, neuropathological changes, and PrPSc accumulation. When tetracycline was preincubated with highly diluted scrapie-infected inoculum, one third of hamsters did not develop disease. Our data suggest that these well characterized antibiotics reduce prion infectivity through a direct interaction with PrPSc and are potentially useful for inactivation of BSE- or vCJD-contaminated products and prevention strategies.

Neuroprotective Effect of Somatostatin on Nonapoptotic NMDA-Induced Neuronal Death: Role of Cyclic GMP

Abstract: Somatostatin (SRIF) exerts a modulatory function on neuronal transmission in the CNS. It has been proposed that a reduction of calcium currents is the major determinant of the inhibitory activity of this peptide on synaptic transmission. Because the neurotoxicity induced by activation of the NMDA subtype of glutamate receptor is mediated through excessive Ca2+ influx, we investigated whether SRIF counteracted NMDA‐induced neuronal cell death. Neurons from embryonic rat cerebral cortex were cultured for 7–10 days and then exposed to 0.5 and 1 mM NMDA for 24 h. The neuronal viability, as assessed by the colorimetric method, decreased by 40 and 60%, respectively, compared with the control condition. Morphological and biochemical evidence indicated that cell death occurred by necrosis and not through an apoptotic mechanism. SRIF (0.5–10 µM), simultaneously applied with excitatory amino acid, significantly reduced in a dose‐dependent manner the neurotoxic effect of NMDA but not that of KA (0.25–0.5 mM). GABA (10 µM) partially protected neurons to a similar extent from NMDA‐ or KA‐induced toxicity. SRIF type 2 receptor agonists, octreotide (SMS 201‐995; 10 µM) and vapreotide (RC 160; 10 µM), did not influence the NMDA‐dependent neurotoxicity. The intracellular mechanism involved in SRIF neuroprotection was investigated. Pertussin toxin (300 ng/ml), a G protein blocker, antagonized the protective effect of SRIF on NMDA neurotoxicity. Furthermore, the neuroprotective effect of SRIF was mimicked by dibutyryl‐cyclic GMP (10 µM), a cyclic GMP analogue, whereas 8‐(4‐chlorphenylthio)‐cyclic AMP (10 µM), a cyclic AMP analogue, was ineffective. The cyclic GMP content was increased in a dose‐dependent manner by SRIF (2.5–10 µM). Finally, both specific (Rp‐8‐bromoguanosine 3′,5′‐monophosphate, 10 µM) and nonspecific [1‐(5 isoquinolinylsulfonyl)‐2‐methylpiperazine (H7), 10 µM] cyclic GMP‐dependent protein kinase (cGMP‐PK) inhibitors did not interfere with NMDA toxicity but substantially reduced SRIF neuroprotection. Our data suggest a selective neuroprotective role of SRIF versus NMDA‐induced nonapoptotic neuronal death in cortical cells. This effect is likely mediated by cGMP‐PK presumably by regulation of the intracellular Ca2+ level.

A Neurotoxic and Gliotrophic Fragment of the Prion Protein Increases Plasma Membrane Microviscosity

Prion-related encephalopathies are characterized by astrogliosis and nerve cell degeneration and loss. These lesions might be the consequence of an interaction between the abnormal isoform of the cellular prion protein that accumulates in nervous tissue and the plasma membranes. Previously we found that a synthetic peptide, homologous to residues 106-126 of the human prion protein, is fibrillogenic and toxic to neurons and trophic to astrocytes in vitro. This study dealt with the ability of the peptide to interact with membranes. Accordingly, we compared PrP 106-126 with different synthetic PrP peptides (PrP 89-106, PrP 127-147, a peptide with a scrambled sequences of 106-126, and PrP 106-126 amidated at the C-terminus) as to the ability to increase the microviscosity of artificial and natural membranes. The first three had no effect on nerve and glial cells in vitro, whereas the amidated peptide caused neuronal death. Using a fluorescent probe that becomes incorporated into the hydrocarbon core of the lipid bilayer and records the lipid fluidity, we found PrP 106-126 able to increase significantly the membrane microviscosity of liposomes and of all cell lines investigated. This phenomenon was associated with the distribution of the peptide over the cell surface, but not with changes in the membrane lipid or protein content, or with membrane lipid phase transitions. Accordingly, we deduced that increased membrane microviscosity was unrelated to changes in the membrane native components and was the result of increased lipid density following PrP 106-126 embedding into the lipid bilayer. No control peptides had comparable effects on the membrane microviscosity, except PrP 106-126 amidated at the C-terminus. Since the latter was as neurotoxic, but not as fibrillogenic, as PrP 106-126, we argued that the ability of PrP 106-126 to increase membrane microviscosity was unrelated to the propensity of the peptide to raise fibrils. Rather, it could be connected with the primary structure of PrP 106-126, characterized by two opposing regions, one hydrophilic and the other hydrophobic, that enabled the peptide to interact with the lipid bilayer. Based on these findings, we speculated that the glial and nerve cell involvement occurring in prion-related encephalopathies might be caused by the interaction with the plasma membrane of a PrP 106-126-like fragment or of the sequence spanning residues 106-126 of the abnormal isoform of the prion protein.

Increased tryptophan hydroxylase mRNA in raphe serotonergic neurons spared by 5,7-dihydroxytryptamine

Neurons expressing the tryptophan hydroxylase (TPH) mRNA within the raphe nuclei of control rats showed a distribution similar to that observed using an antibody for TPH. Numerous packed cells expressing the TPH mRNA were observed in the ventral and dorsal zone of the nucleus raphe dorsalis (NDR) and in the pars dorsalis of the nucleus centralis superior (NCS) whereas fewer and more scattered neurons were found in the pars medialis of NCS. Five days after the intracerebroventricular injection of 5,7-dihydroxytryptamine (5,7-DHT), which markedly reduced the serotonin (5-HT) content in the hippocampus, caudate putamen and cortex, the hybridization signal had completely disappeared in the dorsal region of the NDR. In the ventromedial region, above and between the medial longitudinal fasciculus (MLF), which includes the pars dorsalis of NCS, there was a partial decrease of cell number and a marked increase of the grain density over spared neurons. No significant change was noted in the number of TPH-positive cells and hybridization signal in individual neurons of the pars medialis of NCS. Consistent with previous evidence of increased TPH activity in the residual 5-HT terminals, the present study shows that synthesis of the TPH mRNA may be augmented in some neurons surviving the lesion.

Oxidative stress after acute and chronic application of β-amyloid fragment 25-35 in cortical cultures

The aim of this work was to investigate whether free radical reactions play a role in beta-amyloid neurotoxicity. Rat cortical neurons were exposed acutely (24 h) or chronically (3, 7 days) to beta-amyloid biologically active fragment beta 25-35 (50 microM). In these conditions, where only the longest exposure induced neuronal death, superoxide dismutase activity was increased after acute exposure but no change was detected after chronic treatments, whereas a different pattern was observed for glutathione peroxidase. In the basal condition, there was an eight-fold increase in dichlorofluoroscein, used as peroxide production marker, in neuronal cells after 7 days treatment with beta 25-35. Moreover, the intracellular peroxide production induced by Fe2+/ascorbate stimulation was amplified by beta 25-35, increasingly up to 7 days of exposure, by which time the dichlorofluoroscein-stimulated levels were 33 times higher than in controls. In conclusion, our results show that oxidative stress and free radical production are linked to beta 25-35 exposure and may contribute to neurodegenerative events associated with beta-amyloid deposits in Alzheimers disease.

Caspase-3 activation by β-amyloid and prion protein peptides is independent from their neurotoxic effect

Synthetic peptides corresponding to residues 25-35 of beta-amyloid (beta 25-35) and 106-126 of prion protein (PrP 106-126) are amyloidogenic and cause neuronal death by apoptosis in vitro. We evaluated, in rat cortical neurons, the role of caspases activation in the peptides neurotoxicity by measuring of caspase-3 (CPP32) activity and applying a non-selective caspase inhibitor (z-VAD-fmk) or CPP32-specific inhibitor (Asp-Glu-Val-Asp-CHO (DEVD-CHO)). CPP32 was dose-dependently activated by both peptides (2.5-50 microM). The caspase inhibitors completely abolished the CPP32 activation induced by the peptides. However, the neurotoxic effect was partially attenuated with z-VAD-fmk, while no antagonism was found with DEVD-CHO. Thus, although beta 25-35 and PrP 106-126 robustly activated CPP32, their neurotoxic effect was independent of this caspase activation.

Prion Protein Fragment 106–126 Differentially Induces Heme Oxygenase‐1 mRNA in Cultured Neurons and Astroglial Cells

Abstract: Heme oxygenase (HO), which catalyzes the degradation of heme, has two isozymes (HO‐1 and HO‐2). In brain the noninducible HO‐2 isoform is predominant, whereas the inducible HO‐1 is a marker of oxidative stress. Because brain oxidative stress might be present in prion‐related encephalopathies (PREs), as in other neurodegenerative diseases, we investigated whether HO‐1 mRNA was induced in neuronal and astroglial cell cultures by a peptide corresponding to residue 106–126 of human prion protein (PrP). This peptide is amyloidogenic, and when added in vitro to cultured cells it reproduces the neuronal death and astroglial proliferation and hypertrophy occurring in PREs. HO‐1 mRNA did not accumulate in rat cultured neurons from hippocampus or cortex exposed to PrP 106–126 (50 µM for 5 days). PrP 106–126 induced HO‐1 mRNA accumulation in rat astroglial cultures depending on the exposure time and concentration, being maximal (33‐fold) after 7 days of exposure at 50 µM. The nonamyloidogenic amidated or amidated‐acetylated PrP 106–126 was ineffective, as was a scrambled peptide used as control. N‐Acetylcysteine reduced (50%) the accumulation of HO‐1 mRNA in astroglial cells after PrP 106–126 (25 µM) given for 5 days. Thus, oxidative stress is apparently a feature of the toxicity of PrP 106–126, and it might also occur in PREs; induction of HO‐1 could contribute to the greater resistance of astrocytes compared with neurons to PrP 106–126 toxicity.