Native metastable prefibrillar oligomers are the most neurotoxic species among amyloid aggregates.

Many proteins belonging to the amyloid family share the tendency to misfold and aggregate following common steps, and display similar neurotoxicity. In the aggregation pathway different kinds of species are formed, including several types of oligomers and eventually mature fibers. It is now suggested that the pathogenic aggregates are not the mature fibrils, but the intermediate, soluble oligomers. Many kinds of aggregates have been described to exist in a metastable state and in equilibrium with monomers. Up to now it is not clear whether a specific structure is at the basis of the neurotoxicity. Here we characterized, starting from the early aggregation stages, the oligomer populations formed by an amyloid protein, salmon calcitonin (sCT), chosen due to its very slow aggregation rate. To prepare different oligomer populations and characterize them by means of photoinduced cross-linking SDS-PAGE, Energy Filtered-Transmission Electron Microscopy (EF-TEM) and Circular Dichroism (CD) spectroscopy, we used Size Exclusion Chromatography (SEC), a technique that does not influence the aggregation process leaving the protein in the native state. Taking advantage of sCT low aggregation rate, we characterized the neurotoxic potential of the SEC-separated, non-crosslinked fractions in cultured primary hippocampal neurons, analyzing intracellular Ca(2+) influx and apoptotic trend. We provide evidence that native, globular, metastable, prefibrillar oligomers (dimers, trimers and tetramers) were the toxic species and that low concentrations of these aggregates in the population was sufficient to render the sample neurotoxic. Monomers and other kind of aggregates, such as annular or linear protofibers and mature fibers, were totally biologically inactive.

CNF1 improves astrocytic ability to support neuronal growth and differentiation in vitro.

Modulation of cerebral Rho GTPases activity in mice brain by intracerebral administration of Cytotoxic Necrotizing Factor 1 (CNF1) leads to enhanced neurotransmission and synaptic plasticity and improves learning and memory. To gain more insight into the interactions between CNF1 and neuronal cells, we used primary neuronal and astrocytic cultures from rat embryonic brain to study CNF1 effects on neuronal differentiation, focusing on dendritic tree growth and synapse formation, which are strictly modulated by Rho GTPases. CNF1 profoundly remodeled the cytoskeleton of hippocampal and cortical neurons, which showed philopodia-like, actin-positive projections, thickened and poorly branched dendrites, and a decrease in synapse number. CNF1 removal, however, restored dendritic tree development and synapse formation, suggesting that the toxin can reversibly block neuronal differentiation. On differentiated neurons, CNF1 had a similar effacing effect on synapses. Therefore, a direct interaction with CNF1 is apparently deleterious for neurons. Since astrocytes play a pivotal role in neuronal differentiation and synaptic regulation, we wondered if the beneficial in vivo effect could be mediated by astrocytes. Primary astrocytes from embryonic cortex were treated with CNF1 for 48 hours and used as a substrate for growing hippocampal neurons. Such neurons showed an increased development of neurites, in respect to age-matched controls, with a wider dendritic tree and a richer content in synapses. In CNF1-exposed astrocytes, the production of interleukin 1ß, known to reduce dendrite development and complexity in neuronal cultures, was decreased. These results demonstrate that astrocytes, under the influence of CNF1, increase their supporting activity on neuronal growth and differentiation, possibly related to the diminished levels of interleukin 1ß. These observations suggest that the enhanced synaptic plasticity and improved learning and memory described in CNF1-injected mice are probably mediated by astrocytes.

Amyloid oligomer neurotoxicity, calcium dysregulation, and lipid rafts.

Amyloid proteins constitute a chemically heterogeneous group of proteins, which share some biophysical and biological characteristics, the principal of which are the high propensity to acquire an incorrect folding and the tendency to aggregate. A number of diseases are associated with misfolding and aggregation of proteins, although only in some of them-most notably Alzheimer's disease (AD) and transmissible spongiform encephalopathies (TSEs)-a pathogenetic link with misfolded proteins is now widely recognized. Lipid rafts (LRs) have been involved in the pathophysiology of diseases associated with protein misfolding at several levels, including aggregation of misfolded proteins, amyloidogenic processing, and neurotoxicity. Among the pathogenic misfolded proteins, the AD-related protein amyloid ß (Aß) is by far the most studied protein, and a large body of evidence has been gathered on the role played by LRs in Aß pathogenicity. However, significant amount of data has also been collected for several other amyloid proteins, so that their ability to interact with LRs can be considered an additional, shared feature characterizing the amyloid protein family. In this paper, we will review the evidence on the role of LRs in the neurotoxicity of huntingtin, α-synuclein, prion protein, and calcitonin.

Curcumin protects against NMDA-induced toxicity: a possible role for NR2A subunit.

PURPOSE: Curcumin, a phenolic compound extracted from the rhizome of Curcuma longa, was found to attenuate NMDA-induced excitotoxicity in primary retinal cultures. This study was conducted to further characterize curcumin neuroprotective ability and analyze its effects on NMDA receptor (NMDAr). METHODS: NMDAr modifications were analyzed in primary retinal cell cultures using immunocytochemistry, whole-cell patch-clamp recording and western blot analysis. Cell death was evaluated with the TUNEL assay in primary retinal and hippocampal cultures. Optical fluorometric recordings with Fura 2-AM were used to monitor [Ca(2+)](i). RESULTS: Curcumin dose- and time-dependently protected both retinal and hippocampal neurons against NMDA-induced cell death, confirming its anti-excitotoxic property. In primary retinal cultures, in line with the observed reduction of NMDA-induced [Ca(2+)](i) rise, whole-cell patch-clamp experiments showed that a higher percentage of retinal neurons responded to NMDA with low amplitude current after curcumin treatment. In parallel, curcumin induced an increase in NMDAr subunit type 2A (NR2A) level, with kinetics closely correlated to time-course of neuroprotection and decrease in [Ca(2+)](i). The relation between neuroprotection and NR2A level increase was also in line with the observation that curcumin neuroprotection required protein synthesis. Electrophysiology confirmed an increased activity of NR2A-containing NMDAr at the plasma membrane level. CONCLUSIONS: These results confirm the neuroprotective activity of curcumin against NMDA toxicity, possibly related to an increased level of NR2A, and encourage further studies for a possible therapeutic use of curcumin based on neuromodulation of NMDArs.

The reverse transcription inhibitor abacavir shows anticancer activity in prostate cancer cell lines.

BACKGROUND: Transposable Elements (TEs) comprise nearly 45% of the entire genome and are part of sophisticated regulatory network systems that control developmental processes in normal and pathological conditions. The retroviral/retrotransposon gene machinery consists mainly of Long Interspersed Nuclear Elements (LINEs-1) and Human Endogenous Retroviruses (HERVs) that code for their own endogenous reverse transcriptase (RT). Interestingly, RT is typically expressed at high levels in cancer cells. Recent studies report that RT inhibition by non-nucleoside reverse transcriptase inhibitors (NNRTIs) induces growth arrest and cell differentiation in vitro and antagonizes growth of human tumors in animal model. In the present study we analyze the anticancer activity of Abacavir (ABC), a nucleoside reverse transcription inhibitor (NRTI), on PC3 and LNCaP prostate cancer cell lines. PRINCIPAL FINDINGS: ABC significantly reduces cell growth, migration and invasion processes, considerably slows S phase progression, induces senescence and cell death in prostate cancer cells. Consistent with these observations, microarray analysis on PC3 cells shows that ABC induces specific and dose-dependent changes in gene expression, involving multiple cellular pathways. Notably, by quantitative Real-Time PCR we found that LINE-1 ORF1 and ORF2 mRNA levels were significantly up-regulated by ABC treatment. CONCLUSIONS: Our results demonstrate the potential of ABC as anticancer agent able to induce antiproliferative activity and trigger senescence in prostate cancer cells. Noteworthy, we show that ABC elicits up-regulation of LINE-1 expression, suggesting the involvement of these elements in the observed cellular modifications.

Modulation of the bystander effects induced by soluble factors in HaCaT cells by different exposure strategies.

The aim of this investigation was to explore whether the occurrence and the magnitude of radiation-induced, medium-mediated bystander effects could be influenced by the time of transfer of secreted bystander factors. HaCaT cells were exposed to 0.1 and 1.0 Gy of gamma radiation. These doses did not induce a significant reduction in the clonogenic survival of irradiated cells compared to controls. Bystander cells either were co-cultured with irradiated cells or received medium from irradiated cells. The bystander effects analyzed included end points related to survival (clonogenic potential and cell proliferation) and DNA damage (micronucleus induction and gamma-H2AX formation). The bystander effects we investigated either were lacking or varied from potentially protective to detrimental responses in relation to the dose of radiation and the time between irradiation of donor cells and bystander exposure. Our results suggest that the experimental time schedule is important for both the occurrence and the detection of bystander effects in vitro.

Lipid raft disruption protects mature neurons against amyloid oligomer toxicity.

A specific neuronal vulnerability to amyloid protein toxicity may account for brain susceptibility to protein misfolding diseases. To investigate this issue, we compared the effects induced by oligomers from salmon calcitonin (sCTOs), a neurotoxic amyloid protein, on cells of different histogenesis: mature and immature primary hippocampal neurons, primary astrocytes, MG63 osteoblasts and NIH-3T3 fibroblasts. In mature neurons, sCTOs increased apoptosis and induced neuritic and synaptic damages similar to those caused by amyloid beta oligomers. Immature neurons and the other cell types showed no cytotoxicity. sCTOs caused cytosolic Ca(2+) rise in mature, but not in immature neurons and the other cell types. Comparison of plasma membrane lipid composition showed that mature neurons had the highest content in lipid rafts, suggesting a key role for them in neuronal vulnerability to sCTOs. Consistently, depletion in gangliosides protected against sCTO toxicity. We hypothesize that the high content in lipid rafts makes mature neurons especially vulnerable to amyloid proteins, as compared to other cell types; this may help explain why the brain is a target organ for amyloid-related diseases.

Branched-chain amino acids induce neurotoxicity in rat cortical cultures.

The higher risk for amyotrophic lateral sclerosis (ALS) among professional soccer players, recently reported in Italy, has stimulated investigations in the search for environmental factors that may be at the origin of the increased susceptibility to the disease. Here we studied if high concentrations of branched-chain amino acids (BCAAs), widely used among athletes as dietary integrators to improve physical performance, may be related to an excitotoxic neuronal cell damage. Our results show that (i) high concentrations of BCAAs are neurotoxic and increase excitotoxicity in cortical neurons; (ii) neurotoxicity is brain area specific, being detected in cortical, but not in hippocampal neurons; (iii) it is related to NMDA receptor overstimulation, since it is abolished in the presence of MK-801, a specific NMDA channel blocker; (iv) it depends on the presence of astrocytes. We describe here a possible biological link between an environmental factor (high dietary intake of BCAAs) and the increased risk of ALS among soccer players.

Blockade of chloride intracellular ion channel 1 stimulates Abeta phagocytosis.

In amyloid-beta (Abeta)-stimulated microglial cells, blockade of chloride intracellular ion channel 1 (CLIC1) reverts the increase in tumor necrosis factor-alpha and nitric oxide (NO) production and results in neuroprotection of cocultured neurons. This effect could be of therapeutic efficacy in Alzheimer's disease (AD), where microglial activation may contribute to neurodegeneration, but it could reduce Abeta phagocytosis, which could facilitate amyloid plaque removal. Here, we analyzed the CLIC1 blockade effect on Abeta-stimulated mononuclear phagocytosis. In the microglial cell line BV-2, Abeta25-35 treatment enhanced fluorescent bead phagocytosis, which persisted also in the presence of IAA-94, a CLIC1 channel blocker. The same result was obtained in rat primary microglia and in BV2 cells, where CLIC1 expression had been knocked down with a plasmid producing small interfering RNAs. To address specifically the issue of Abeta phagocytosis, we treated BV-2 cells with biotinylated Abeta1-42 and measured intracellular amyloid by morphometric analysis. IAA-94-treated cells showed an increased Abeta phagocytosis after 24 hr and efficient degradation of ingested material after 72 hr. In addition, we tested Abeta1-42 phagocytosis in adult rat peritoneal macrophages. Also, these cells actively phagocytosed Abeta1-42 in the presence of IAA-94. However, the increased expression of inducible NO synthase (iNOS), stimulated by Abeta, was reverted by IAA-94. In parallel, a decrease in NO release was detected. These results suggest that blockade of CLIC1 stimulates Abeta phagocytosis in mononuclear phagocytes while inhibiting the induction of iNOS and further point to CLIC1 as a possible therapeutic target in AD.

Biocompatibility assessment of liquid artificial vitreous replacements: relevance of in vitro studies.

The biocompatibility of liquid artificial vitreous replacements is generally assessed by performing tests in animal models before their clinical use, whereas in vitro experimentation is seldom carried out due to their physico-chemical characteristics. Since their introduction in vitreoretinal surgery, however, the use of some certified vitreous replacements has been discouraged after clinical trials, because of the occurrence of serious side effects. This observation suggests that the tests currently performed for biocompatibility assessment cannot fully guarantee their safety when they are used in humans. Here we review the available literature on in vitro biocompatibility testing of liquid artificial vitreous replacements and survey our own experience on the subject, obtained by using primary retinal cell cultures, seeded on micro-porous inserts. We suggest that in vitro biocompatibility assessment, conducted before experiments in animal models, could improve the required safety evaluation and decrease the risk of undesired side effects, as well as providing a beneficial reduction of animal experimentation.

Induction of apoptosis in rat retinal cell cultures by partially fluorinated alkanes.

PURPOSE: To test whether the partially fluorinated alkanes (PFAs) perfluorobutylbutane (O(44)), perfluorohexylethan (O(62)), and the oligomer OL(62HV), recently proposed as artificial vitreous replacements (AVRs), have pro-apoptotic effect in rat retinal cultures. DESIGN: Laboratory investigation. METHODS: Rat retinal cell cultures were seeded onto microporous inserts to study AVR-cell interaction without impairing cell survival. Cells were treated for 24 hours with O(62), O(44), and OL(62HV). Apoptosis was analyzed by transferase-mediated dUTP-biotin nick end-labeling assay and Hoechst stain. RESULTS: O(44) and O(62) did not affect structural organization and cell survival in retinal cell cultures; however, OL(62HV) induced increased apoptosis compared with control cultures. CONCLUSIONS: OL(62HV), a high-viscosity PFA, induces severe retinal damage in human eyes, although it successfully passed animal experimentation. Our in vitro study showed a remarkable pro-apoptotic effect of OL(62HV) and suggests that in vitro tests can contribute to AVR biocompatibility assessment.

Involvement of the intracellular ion channel CLIC1 in microglia-mediated beta-amyloid-induced neurotoxicity.

It is widely believed that the inflammatory events mediated by microglial activation contribute to several neurodegenerative processes. Alzheimer's disease, for example, is characterized by an accumulation of beta-amyloid protein (Abeta) in neuritic plaques that are infiltrated by reactive microglia and astrocytes. Although Abeta and its fragment 25-35 exert a direct toxic effect on neurons, they also activate microglia. Microglial activation is accompanied by morphological changes, cell proliferation, and release of various cytokines and growth factors. A number of scientific reports suggest that the increased proliferation of microglial cells is dependent on ionic membrane currents and in particular on chloride conductances. An unusual chloride ion channel known to be associated with macrophage activation is the chloride intracellular channel-1 (CLIC1). Here we show that Abeta stimulation of neonatal rat microglia specifically leads to the increase in CLIC1 protein and to the functional expression of CLIC1 chloride conductance, both barely detectable on the plasma membrane of quiescent cells. CLIC1 protein expression in microglia increases after 24 hr of incubation with Abeta, simultaneously with the production of reactive nitrogen intermediates and of tumor necrosis factor-alpha (TNF-alpha). We demonstrate that reducing CLIC1 chloride conductance by a specific blocker [IAA-94 (R(+)-[(6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5yl)-oxy] acetic acid)] prevents neuronal apoptosis in neurons cocultured with Abeta-treated microglia. Furthermore, we show that small interfering RNAs used to knock down CLIC1 expression prevent TNF-alpha release induced by Abeta stimulation. These results provide a direct link between Abeta-induced microglial activation and CLIC1 functional expression.

Astrocyte modulation of in vitro beta-amyloid neurotoxicity.

In Alzheimer's disease brain, beta-amyloid (Abeta) deposition is accompanied by astrocyte activation, whose role in the pathogenesis of the disease is still unclear. To explore the subject, we compared Abeta neurotoxicity in pure hippocampal cultures and neuronal-astrocytic cocultures, where astrocytes conditioned neurons but were not in contact with them or Abeta. In the presence of astrocytes, neurons were protected from Abeta neurotoxicity. Neuritic dystrophy was reduced, synapses were partially preserved, and apoptosis was contrasted. The protection disappeared when astrocytes were also treated with Abeta, suggesting that Abeta-astrocyte interaction is deleterious for neurons. This was supported by comparing Abeta neurotoxicity in pure neurons and neurons grown on astrocytes. In this case, where astrocytes were also in contact with Abeta, neuritic damage was enhanced and expression of synaptic vesicle proteins decreased. Our results suggest that astrocytes can protect neurons from Abeta neurotoxicity, but when they interact with Abeta, the protection is undermined and neurotoxicity enhanced.

Biocompatibility assessment of silicone oil and perfluorocarbon liquids used in retinal reattachment surgery in rat retinal cultures.

The effects of silicone oil and perfluorocarbon liquids used in retinal reattachment surgery were studied in vitro using rat retinal cultures seeded on microporous inserts. These inserts allow the cell layer to be in contact with the material to be tested on the apical side and with the nutrient medium on the basal side. The materials tested were silicone oil, the perfluorocarbons perfluorophenanthrene and perfluoroctane, and hydroxypropylmethylcellulose. Perfluorophenanthrene, the heaviest of the compounds, induced a very precocious detachment of the cell layer. All the other tested biomaterials were compatible with cell survival and did not alter the structural organization of the retinal cultures, as revealed by scanning electron microscopy. By immunocytochemical techniques we evaluated the cell composition and the differentiation state of each of the cultures. In both control and treated samples, neuronal cells were well preserved. The expression of microtubule-associated protein 2, a marker of differentiated neuronal cytoskeleton, was not affected. Amacrine neurons, immunolabeled for gamma-aminobutyric acid, still were detectable after treatment. Synapses, marked by immunoreactivity for synapthophysin, were equally preserved. Vimentin-positive glial cells did not show modifications. The apoptotic rate, as determined by the terminal transferase-mediated dUTP-biotin nick end-labeling assay, was similar in treated and control samples. The results confirm that the use of biomaterials with a specific gravity close to intraocular fluids is compatible with retinal cell survival and differentiation in vitro.