Fiorella Malchiodi-Albedi

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.

ALZHEIMER'S DISEASE-ASSOCIATED PRESENILIN 1 IN NEURONAL CELLS : EVIDENCE FOR LOCALIZATION TO THE ENDOPLASMIC RETICULUM-GOLGI INTERMEDIATE COMPARTMENT

The recently identified Alzheimers disease‐associated presenilin 1 and 2 (PS1 and PS2) genes encode two homologous multi membrane‐spanning proteins. Rabbit antibodies to the N‐terminal domain of PS1 detected PS1 in human neuroblastoma SH‐SY5Y wild type and PS1 transfectants (SY5Y‐PS1) as well as in mouse P19, in CHO‐K1 and CHO‐APP770 transfected cells, in rat cerebellar granule and hippocampal neurons, and astrocytes. Immunoblotting detected full‐length protein of 50 kDa, and a major presumptive cleavage product of 30 kDa. The immunofluorescence pattern resembled labeling of the endoplasmic reticulum‐Golgi intermediate compartment (ERGIC) marker protein ERGIC‐53. PS1 distribution showed slight condensation after brefeldin A and more marked condensation after incubation of cells at 16°C, characteristic of the ERGIC compartment. Double labeling showed colocalization of ERGIC‐53 with PS1 in the SY5Y‐PS1 cells. PS1 labeling of SY5Y‐PS1 and P19 cells showed overlap of the cis‐Golgi marker p210 and colocalization with p210 after brefeldin A which causes redistribution of p210 to the ERGIC. Expression of PS1 did not change in level or cellular distribution during development of neurons in culture. Double labeling for the amyloid precursor protein (APP) and PS1 on SY5Y‐PS1 cells and CHO‐APP770 cells showed some overlap under control conditions. These results indicate that PS1 is a resident protein of the ERGIC and could be involved in trafficking of proteins, including APP, between the ER and Golgi compartments. J. Neurosci. Res. 49:719–731, 1997.

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.

Modulation of RhoGTPases Improves the Behavioral Phenotype and Reverses Astrocytic Deficits in a Mouse Model of Rett Syndrome

RhoGTPases are crucial molecules in neuronal plasticity and cognition, as confirmed by their role in non-syndromic mental retardation. Activation of brain RhoGTPases by the bacterial cytotoxic necrotizing factor 1 (CNF1) reshapes the actin cytoskeleton and enhances neurotransmission and synaptic plasticity in mouse brains. We evaluated the effects of a single CNF1 intracerebroventricular inoculation in a mouse model of Rett syndrome (RTT), a rare neurodevelopmental disorder and a genetic cause of mental retardation, for which no effective therapy is available. Fully symptomatic MeCP2-308 male mice were evaluated in a battery of tests specifically tailored to detect RTT-related impairments. At the end of behavioral testing, brain sections were immunohistochemically characterized. Magnetic resonance imaging and spectroscopy (MRS) were also applied to assess morphological and metabolic brain changes. The CNF1 administration markedly improved the behavioral phenotype of MeCP2-308 mice. CNF1 also dramatically reversed the evident signs of atrophy in astrocytes of mutant mice and restored wt-like levels of this cell population. A partial rescue of the overexpression of IL-6 cytokine was also observed in RTT brains. CNF1-induced brain metabolic changes detected by MRS analysis involved markers of glial integrity and bioenergetics, and point to improved mitochondria functionality in CNF1-treated mice. These results clearly indicate that modulation of brain RhoGTPases by CNF1 may constitute a totally innovative therapeutic approach for RTT and, possibly, for other disorders associated with mental retardation.

Protein phosphatase inhibitors induce modification of synapse structure and tau hyperphosphorylation in cultured rat hippocampal neurons

Protein phosphatase inhibitors, okadaic acid and Caliculin A, were used to investigate how perturbation of phosphorylation and dephosphorylation processes might affect neurite and synapse structure in cultures of fetal rat hippocampal neurons. Drug treatments induced neuritic tree modification, with retraction of the processes and the appearance of dilatations along the neurites. The characteristic dotlike pattern of immunoreactivity of synaptic vesicle proteins disappeared. Normal synapses were extremely rare by ultrastructural observation. Vesicles of various diameters accumulated in the dilatations, as did organelles and amorphous material, suggesting impaired axonal transport. Hyperphosphorylation of tau protein was also observed as indicated by the shift in the electrophoretic mobility of a 32P‐labeled 55‐kDa band and by immunoblot with epitope‐specific tau antibody.

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.

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.

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.

Toxic and genotoxic effects of oral administration of furan in mouse liver

In this study, the effects induced in mouse liver by repeated oral exposure to furan were investigated. To this aim, the compound was given for 28 days by daily gavage to male B6C3F1 mice at 2, 4, 8 and 15 mg/kg body weight (b.w.)/day. Twenty-four hours after last administration, animals were sacrificed, liver was excised and the following parameters were evaluated: histological alterations, apoptosis, cell proliferation, polyploidy, overall DNA methylation, gene expression and DNA damage by the immunofluorescence detection of foci of phosphorylated histone H2AX (gamma-H2AX) and by alkaline comet assays, using both standard and modified protocols for the detection of DNA cross links. Liver DNA damage by comet assays was also evaluated in mice receiving furan as a single acute oral dose (15, 100 or 250 mg/kg b.w.). Microscopic analysis of liver sections indicated that repeated oral administration of furan was moderately toxic, producing mild histological alterations with necrotic figures, apoptosis and limited regenerative cell proliferation. The flow cytometric analysis of DNA content in single-cell suspensions of liver cells showed a statistically significant increase in polyploid (8N) cells at the highest dose. No treatment-related changes in overall DNA methylation, gamma-H2AX foci, DNA strand breaks and cross links were observed at the end of the 4-week exposure period. However, several genes involved in DNA damage response, beyond stress and liver toxicity, were over-expressed in mice treated with the highest furan dose (15 mg/kg b.w./day). Acute administration of furan induced evident liver toxicity at the highest dose (250 mg/kg b.w.), which was associated with a significant increase of DNA damage in the alkaline comet assay and with a distinct decrease in gamma-ray-induced DNA migration. Overall, the results obtained suggest that the contribution of genotoxicity to the mechanism of furan carcinogenicity in mouse liver should not be dismissed.