Silvia Paradisi

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. Alzheimers disease, for example, is characterized by an accumulation of β-amyloid protein (Aβ) in neuritic plaques that are infiltrated by reactive microglia and astrocytes. Although Aβ 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 Aβ 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 Aβ, simultaneously with the production of reactive nitrogen intermediates and of tumor necrosis factor-α (TNF-α). 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 Aβ-treated microglia. Furthermore, we show that small interfering RNAs used to knock down CLIC1 expression prevent TNF-α release induced by Aβ stimulation. These results provide a direct link between Aβ-induced microglial activation and CLIC1 functional expression.

Astrocytes contribute to neuronal impairment in βA toxicity increasing apoptosis in rat hippocampal neurons

Astrocytosis is a common feature of amyloid plaques, the hallmark of Alzheimers disease (AD), along with activated microglia, neurofibrillary tangles, and β‐amyloid (βA) deposition. However, the relationship between astrocytosis and neurodegeneration remains unclear. To assess whether βA‐stimulated astrocytes can damage neurons and contribute to βA neurotoxicity, we studied the effects of βA treatment in astrocytic/neuronal co‐cultures, obtained from rat embryonic brain tissue. We found that in neuronal cultures conditioned by βA‐treated astrocytes, but not directly in contact with βA, the number of apoptotic cells increased, doubling the values of controls. In astrocytes, βA did not cause astrocytic cell death, nor did produce changes in nitric oxide or prostaglandin E2 levels. In contrast, S‐100β expression was remarkably increased. Our data show for the first time that βA–astrocytic interaction produces a detrimental effect on neurons, which may contribute to neurodegeneration in AD. GLIA 34:68–72, 2001.

Allelotype of pancreatic acinar cell carcinoma

Pancreatic acinar cell carcinoma (PAC) is a rare pancreatic tumor for which no information about chromosomal and gene anomalies is available. We performed genome‐wide allelotyping of 9 PACs using DNA from 5 frozen and 4 paraffin‐embedded samples and 76 PCR‐amplified, chromosome‐specific microsatellite markers. High degrees of allelic loss were found, with a mean fractional allelic loss of 0.33. Chromosomes 1p, 4q and 17p showed loss of heterozygosity in >70% of cases and chromosomes 11q, 13q, 15q and 16q, in 60% to 70% of cases. Chromosomes 3q, 6q, 8q, 18q and 21q showed loss in 50% to 60% of cases. All of the remaining chromosomes showed no or few allelic losses. The resulting allelotype of PAC is markedly different from that of either ductal or endocrine tumors of the pancreas, and the involvement of chromosomes 4q and 16q appears to be characteristic of this tumor type. High‐resolution mapping of the 12 frequently altered chromosomes in 5 cases with 222 markers permitted subchromosomal localization of regions of consensus loss on 5 chromosomes, including 1p36.31, 3p25.2, 4q26‐31.1, 15q15‐22.1 and 16q21‐q22.1. Our findings suggest that PAC tumorigenesis involves molecular pathways different from those occurring in more common pancreatic tumor types. Int. J. Cancer 88:772–777, 2000.

Nonrandom chromosomal imbalances in primary mediastinal B-cell lymphoma detected by arbitrarily primed PCR fingerprinting.

We used arbitrarily primed polymerase chain reaction (AP‐PCR) fingerprinting to identify chromosomal imbalances in six primary mediastinal B‐cell lymphomas (PMBLs). Seventy‐four chromosomal imbalances were detected, consisting of 49 sequence gains and 25 losses. Amplifications on chromosome X were seen in five cases, four of which involved the same chromosomal locus. Nonrandom gains at the same locus were also identified on chromosomes 2 and 7 in four cases and on chromosomes 5, 9, and 12 in three cases. Five PMBLs were also analyzed by comparative genomic hybridization (CGH), which found chromosome arm 9p amplification as the only nonrandom imbalance. Our data demonstrate that chromosomal amplifications outnumber losses in PMBL. These mainly involve chromosomes 9 and X and may reflect more complex phenomena, such as translocations or other chromosomal rearrangements, as AP‐PCR found coexistent gains and losses on these chromosomes. Comparison between AP‐PCR and CGH suggests that anomalies affecting the same chromosomal regions may occur at much higher frequencies than expected by CGH, suggesting that genomic amplifications are usually confined to DNA segments smaller than the megabase long segments required for detection in CGH. Modest increases in genetic material may be as effective as higher‐level amplifications when affecting sites where a proto‐oncogene resides. Genes Chromosomes Cancer 26:203–209, 1999.

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.

Effects of Clostridium Difficile toxins A and B on cytoskeleton organization in HEp-2 cells: A comparative morphological study

A comparative study on the effects of toxin A and toxin B from Clostridium difficile on HEp-2 cells was carried out. Both toxins caused cell retraction and rounding and seemed to exert their effect on cell morphology via a rearrangement of actin and alpha-actinin microfilaments. Such a rearrangement occurred at an early stage, when no change in microtubular and cytokeratin systems was detectable. Nevertheless, several structural modifications accompanying the cytopathological process induced by toxins A and B appeared to be quite different. In particular, toxin B-treated cells showed an arborized phenotype as a result of cell retraction and rounding, whereas toxin A caused cell rounding without arborization. Moreover, nuclear polarization following disorganization of the microfilament system was only observed in toxin A-treated cells. The structural features distinguishing intoxication processes induced by the two toxins probably reflect a different mechanism of action and suggest the presence of a distinct subcellular component as a primary target for each toxin.

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.

Astrocyte modulation of in vitro β-amyloid neurotoxicity

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

Both UVA and UVB induce cytoskeleton-dependent surface blebbing in epidermoid cells

Data on the morphological changes induced by UVA or UVB irradiation of A431 epidermoid cells in culture are presented. After irradiation with different doses of UVB (120-2400 J m-2) or UVA (10(4)-10(5) J m-2), the membrane and cytoskeleton of these cells were analysed by immunofluorescence and scanning electron microscopy at different times after exposure (0-48 h). Both UVA and UVB alter microtubules and microfilaments and surface blebs are formed after UV irradiation. In particular, UVB induces multiple small blebs on the cells, while UVA induces one single large bleb on each cell. Since cytoskeletal damage and surface blebbing of this type are also induced by oxidative stress, these results add to the body of evidence indicating that UV radiation is capable of pro-oxidant behaviour. Specifically, the morphological changes described in this paper are reminiscent of the modifications which accompany epidermal keratinocytes during their transformation to sunburn cells after UV irradiation. The physiological implications of these findings are discussed.

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 Alzheimers 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 β (Aβ) is by far the most studied protein, and a large body of evidence has been gathered on the role played by LRs in Aβ 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.