Gianluigi Forloni

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.

Synthetic amyloid-β oligomers impair long-term memory independently of cellular prion protein

Inability to form new memories is an early clinical sign of Alzheimer’s disease (AD). There is ample evidence that the amyloid-β (Aβ) peptide plays a key role in the pathogenesis of this disorder. Soluble, bio-derived oligomers of Aβ are proposed as the key mediators of synaptic and cognitive dysfunction, but more tractable models of Aβ−mediated cognitive impairment are needed. Here we report that, in mice, acute intracerebroventricular injections of synthetic Aβ1–42 oligomers impaired consolidation of the long-term recognition memory, whereas mature Aβ1–42 fibrils and freshly dissolved peptide did not. The deficit induced by oligomers was reversible and was prevented by an anti-Aβ antibody. It has been suggested that the cellular prion protein (PrPC) mediates the impairment of synaptic plasticity induced by Aβ. We confirmed that Aβ1–42 oligomers interact with PrPC, with nanomolar affinity. However, PrP-expressing and PrP knock-out mice were equally susceptible to this impairment. These data suggest that Aβ1–42 oligomers are responsible for cognitive impairment in AD and that PrPC is not required.

Full length α-synuclein is present in cerebrospinal fluid from Parkinson's disease and normal subjects

Several clues suggest that α-synuclein, a presynaptic protein, plays a central role in the pathogenesis of idiopathic Parkinsons disease (PD). To search a peripheral marker of PD, we analyzed presence and amount of α-synuclein in CSF from 12 PD patients and 10 neurologically normal subjects. The protein was extracted from CSF samples through immunoprecipitation and immunoblotting with different specific anti-α-synuclein antibodies. We identified a 19 kDa band that corresponds to monomeric α-synuclein, given its comigration with homologue human recombinant peptide as well as with the protein extracted from cerebral cortex of normal subjects. The amount of CSF 19 kDa α-synuclein did not significantly vary in PD and normal cases. These findings have two implications: (a) full length α-synuclein is released by neurons in the extracellular space; (b) α-synuclein does not appear a peripheral marker of PD pathology.

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.

JNK Signalling: A Possible Target to Prevent Neurodegeneration

The c-Jun N-terminal kinases (JNK) belong to the subfamily of mitogen-activated protein kinase (MAPK). JNK is an important transducing enzyme that is involved in many facets of cellular regulation including gene expression, cell proliferation and programmed cell death. The activation of JNK pathways is critical for naturally occurring cell death during development as well as for pathological death associated with neurodegenerative diseases. Initial research concentrated on defining the components and organization of JNK signalling cascades, but more recent studies see JNK as a target to prevent cell death. Several in vitro and in vivo studies have reported alterations of JNK pathways potentially associated with neuronal death in Parkinsons and Alzheimers disease. So efforts are now aimed at developing chemical inhibitors of this pathway. These have proved effective in vivo, reducing brain damage and some of the symptoms of arthritis in animal models. An alternative cell penetrating peptide approach is now available, with the identification of the JNK permeable peptide inhibitor, which modifies JNK action rather than activation, preventing neuronal death with unprecedented specificity and efficacy in several experimental conditions, including two animal models of ischemia. In this review we examine in detail the role of JNK in neurodegeneration, particularly in Alzheimers and Parkinsons disease. The possibility of intervention on the JNK pathway as a therapeutic approach is also illustrated.

Anti-amyloidogenic activity of tetracyclines: Studies in vitro

Cerebral deposition of β‐amyloid is a major neuropathological feature in Alzheimers disease. Here we show that tetracyclines, tetracycline and doxycycline, classical antibiotics, exhibit anti‐amyloidogenic activity. This capacity was determined by the exposure of β 1‐42 amyloid peptide to the drugs followed by the electron microscopy examination of the amyloid fibrils spontaneously formed and quantified with thioflavine T binding assay. The drugs reduced also the resistance of β 1‐42 amyloid fibrils to trypsin digestion. Tetracyclines not only inhibited the β‐amyloid aggregates formation but also disassembled the pre‐formed fibrils. The results indicate that drugs with a well‐known clinical profile, including activity in the central nervous system, are potentially useful for Alzheimers therapy.

Evaluation of Quinacrine Treatment for Prion Diseases

ABSTRACT Based on in vitro observations in scrapie-infected neuroblastoma cells, quinacrine has recently been proposed as a treatment for Creutzfeldt-Jakob disease (CJD), including a new variant CJD which is linked to contamination of food by the bovine spongiform encephalopathy (BSE) agent. The present study investigated possible mechanisms of action of quinacrine on prions. The ability of quinacrine to interact with and to reduce the protease resistance of PrP peptide aggregates and PrPres of human and animal origin were analyzed, together with its ability to inhibit the in vitro conversion of the normal prion protein (PrPc) to the abnormal form (PrPres). Furthermore, the efficiencies of quinacrine and chlorpromazine, another tricyclic compound, were examined in different in vitro models and in an experimental murine model of BSE. Quinacrine efficiently hampered de novo generation of fibrillogenic prion protein and PrPres accumulation in ScN2a cells. However, it was unable to affect the protease resistance of preexisting PrP fibrils and PrPres from brain homogenates, and a “curing” effect was obtained in ScGT1 cells only after lengthy treatment. In vivo, no detectable effect was observed in the animal model used, consistent with other recent studies and preliminary observations in humans. Despite its ability to cross the blood-brain barrier, the use of quinacrine for the treatment of CJD is questionable, at least as a monotherapy. The multistep experimental approach employed here could be used to test new therapeutic regimes before their use in human trials.

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.

Sporadic human prion diseases: molecular insights and diagnosis

Human prion diseases can be sporadic, inherited, or acquired by infection. Distinct clinical and pathological characteristics separate sporadic diseases into three phenotypes: Creutzfeldt-Jakob disease (CJD), fatal insomnia, and variably protease-sensitive prionopathy. CJD accounts for more than 90% of all cases of sporadic prion disease; it is commonly categorised into five subtypes that can be distinguished according to leading clinical signs, histological lesions, and molecular traits of the pathogenic prion protein. Three subtypes affect prominently cognitive functions whereas the other two impair cerebellar motor activities. An accurate and timely diagnosis depends on careful clinical examination and early performance and interpretation of diagnostic tests, including electroencephalography, quantitative assessment of the surrogate markers 14-3-3, tau, and of the prion protein in the CSF, and neuroimaging. The reliability of CSF tests is improved when these tests are interpreted alongside neuroimaging data.

The SIRT1 activator resveratrol protects SK‐N‐BE cells from oxidative stress and against toxicity caused by α‐synuclein or amyloid‐β (1‐42) peptide

Human sirtuins are a family of seven conserved proteins (SIRT1‐7). The most investigated is the silent mating type information regulation‐2 homolog (SIRT1, NM_012238), which was associated with neuroprotection in models of polyglutamine toxicity or Alzheimer’s disease (AD) and whose activation by the phytocompound resveratrol (RES) has been described. We have examined the neuroprotective role of RES in a cellular model of oxidative stress, a common feature of neurodegeneration. RES prevented toxicity triggered by hydrogen peroxide or 6‐hydroxydopamine (6‐OHDA). This action was likely mediated by SIRT1 activation, as the protection was lost in the presence of the SIRT1 inhibitor sirtinol and when SIRT1 expression was down‐regulated by siRNA approach. RES was also able to protect SK‐N‐BE from the toxicity arising from two aggregation‐prone proteins, the AD‐involved amyloid‐β (1‐42) peptide (Aβ42) and the familiar Parkinson’s disease linked α‐synuclein(A30P) [α‐syn(A30P)]. Alpha‐syn(A30P) toxicity was restored by sirtinol addition, while a partial RES protective effect against Aβ42 was found even in presence of sirtinol, thus suggesting a direct RES effect on Aβ42 fibrils. We conclude that SIRT1 activation by RES can prevent in our neuroblastoma model the deleterious effects triggered by oxidative stress or α‐syn(A30P) aggregation, while RES displayed a SIRT1‐independent protective action against Aβ42.