Can MRI T1 be used to detect early changes in 5xFAD Alzheimer's mouse brain?

OBJECTIVES: In the present study, we have tested whether MRI T1 relaxation time is a sensitive marker to detect early stages of amyloidosis and gliosis in the young 5xFAD transgenic mouse, a well-established animal model for Alzheimer's disease. MATERIALS AND METHODS: 5xFAD and wild-type mice were imaged in a 4.7 T Varian horizontal bore MRI system to generate T1 quantitative maps using the spin-echo multi-slice sequence. Following immunostaining for glial fibrillary acidic protein, Iba-1, and amyloid-ß, T1 and area fraction of staining were quantified in the posterior parietal and primary somatosensory cortex and corpus callosum. RESULTS: In comparison with age-matched wild-type mice, we observed first signs of amyloidosis in 2.5-month-old 5xFAD mice, and development of gliosis in 5-month-old 5xFAD mice. In contrast, MRI T1 relaxation times of young, i.e., 2.5- and 5-month-old, 5xFAD mice were not significantly different to those of age-matched wild-type controls. Furthermore, although disease progression was detectable by increased amyloid-ß load in the brain of 5-month-old 5xFAD mice compared with 2.5-month-old 5xFAD mice, MRI T1 relaxation time did not change. CONCLUSIONS: In summary, our data suggest that MRI T1 relaxation time is neither a sensitive measure of disease onset nor progression at early stages in the 5xFAD mouse transgenic mouse model.

The expression of nicotinamide N-methyltransferase increases ATP synthesis and protects SH-SY5Y neuroblastoma cells against the toxicity of Complex I inhibitors.

NNMT (nicotinamide N-methyltransferase, E.C. 2.1.1.1) catalyses the N-methylation of nicotinamide to 1-methylnicotinamide. NNMT expression is significantly elevated in a number of cancers, and we have previously demonstrated that NNMT expression is significantly increased in the brains of patients who have died of Parkinson's disease. To investigate the cellular effects of NNMT overexpression, we overexpressed NNMT in the SH-SY5Y cell line, a tumour-derived human dopaminergic neuroblastoma cell line with no endogenous expression of NNMT. NNMT expression significantly decreased SH-SY5Y cell death, which correlated with increased intracellular ATP content, ATP/ADP ratio and Complex I activity, and a reduction in the degradation of the NDUFS3 [NADH dehydrogenase (ubiquinone) iron-sulfur protein 3] subunit of Complex I. These effects were replicated by incubation of SH-SY5Y cells with 1-methylnicotinamide, suggesting that 1-methylnicotinamide mediates the cellular effects of NNMT. Both NNMT expression and 1-methylnicotinamide protected SH-SY5Y cells from the toxicity of the Complex I inhibitors MPP+ (1-methyl-4-phenylpyridinium ion) and rotenone by reversing their effects upon ATP synthesis, the ATP/ADP ratio, Complex I activity and the NDUFS3 subunit. The results of the present study raise the possibility that the increase in NNMT expression that we observed in vivo may be a stress response of the cell to the underlying pathogenic process. Furthermore, the results of the present study also raise the possibility of using inhibitors of NNMT for the treatment of cancer.

Statins inhibit the dimerization of beta-secretase via both isoprenoid- and cholesterol-mediated mechanisms.

We have previously reported that protein lipidation in the form of palmitoylation and farnesylation is critical for the production of Abeta (amyloid beta-peptide), the dimerization of beta-secretase and its trafficking into cholesterol-rich microdomains. As statins influence these lipid modifications in addition to their effects on cholesterol biosynthesis, we have investigated the effects of lovastatin and SIMVA (simvastatin) at a range of concentrations chosen to distinguish different cellular effects on Abeta production and beta-secretase structure and its localization in bHEK cells [HEK-293 cells (human embryonic kidney cells) transfected with the Asp-2 gene plus a polyhistidine coding tag] cells. We have compared the changes brought about by statins with those brought about by the palmitoylation inhibitor cerulenin and the farnesyltransferase inhibitor CVFM (Cys-Val-Phe-Met). The statin-mediated reduction in Abeta production correlated with an inhibition of beta-secretase dimerization into its more active form at all concentrations of statin investigated. These effects were reversed by the administration of mevalonate, showing that these effects were mediated via 3-hydroxy-3-methylglutaryl-CoA-dependent pathways. At low (1 microM) statin concentrations, reduction in Abeta production and inhibition of beta-secretase dimerization were mediated by inhibition of isoprenoid synthesis. At high (>10 microM) concentrations of statins, inhibition of beta-secretase palmitoylation occurred, which we demonstrated to be regulated by intracellular cholesterol levels. There was also a concomitant concentration-dependent change in beta-secretase subcellular trafficking. Significantly, Abeta release from cells was markedly higher at 50 microM SIMVA than at 1 microM, whereas these concentrations resulted in similar reductions in total Abeta production, suggesting that low-dose statins may be more beneficial than high doses for the therapeutic treatment of Alzheimer's disease.

The regulation of beta-secretase by cholesterol and statins in Alzheimer's disease.

Epidemiologists have found a decreased risk of developing Alzheimer's disease (AD) in people taking statins (cholesterol biosynthesis inhibitors). We have reported previously that, in cell culture, lovastatin decreases the output of beta-amyloid, a peptide that is toxic to neurones, and is reputably the prime cause of neurodegeneration seen in AD. This report probes the mechanism of statin protection further by finding out how the protease beta-secretase, that releases beta-amyloid from its precursor protein, behaves under changed cholesterol levels induced by statins. We found that, with high cellular cholesterol levels, there is a decrease in glycosylation of mature oligosaccharides in beta-secretase, whereas in the presence of lovastatin, glycosylation progresses further. Moreover, lovastatin does not inhibit beta-secretase in vitro. Thus, the cholesterol and statin effects are due to changes in cellular targeting induced by changed cholesterol gradients. Some of these changes are mimicked by the action of U18666A, a cholesterol-transport inhibitor that produces a defect in cells seen in patients with Neimann Pick's disorder.

The elusive nature and diagnostics of misfolded Aß oligomers.

Amyloid-beta (Aß) peptide oligomers are believed to be the causative agents of Alzheimer's disease (AD). Though post-mortem examination shows that insoluble fibrils are deposited in the brains of AD patients in the form of intracellular (tangles) and extracellular (plaques) deposits, it has been observed that cognitive impairment is linked to synaptic dysfunction in the stages of the illness well before the appearance of these mature deposits. Increasing evidence suggests that the most toxic forms of Aß are soluble low-oligomer ligands whose amounts better correlate with the extent of cognitive loss in patients than the amounts of fibrillar insoluble forms. Therefore, these ligands hold the key to a better understanding of AD prompting the search for clearer correlations between their structure and toxicity. The importance of such correlations and their diagnostic value for the early diagnosis of AD is discussed here with a particular emphasis on the transient nature and structural plasticity of misfolded Aß oligomers.

Gadolinium-complexed Aß-binding contrast agents for MRI diagnosis of Alzheimer's Disease.

MRI contrast agents, containing peptide sequences that bind ß-amyloid and gadolinium ions ligated to DOTA have been synthesized for evaluation in early diagnosis of Alzheimer's Disease in transgenic mice models. A number of brain penetration modifications were incorporated and sufficient amounts of contrast agent in the brain were achieved only by addition of a cationic cell penetration sequence along with the use of microparticle assisted ultrasound activation. In the T1 mode of a MRI scan, the peptide (R2) illuminated areas of brain rich in amyloid plaques.

Pro-domain removal in ASP-2 and the cleavage of the amyloid precursor are influenced by pH.

BACKGROUND: One of the signatures of Alzheimer's disease is the accumulation of aggregated amyloid protein, Abeta, in the brain. Abeta arises from cleavage of the Amyloid Precursor protein by beta and gamma secretases, which present attractive candidates for therapeutic targeting. Two beta-secretase candidates, ASP-1 and ASP-2, were identified as aspartic proteases, both of which cleave the amyloid precursor at the beta-site. These are produced as immature transmembrane proteins containing a pro-segment. RESULTS: ASP-2 expressed in HEK293-cells cleaved the Swedish mutant amyloid precursor at different beta-sites at different pHs in vitro. Recent reports show that furin cleaves the pro-peptide of ASP-2, whereas ASP-1 undergoes auto-catalysis. We show that purified recombinant ASP-2 cleaves its own pro-peptide at ph 5 but not pH 8.5 as seen by mass spectrometry, electrophoresis and N-terminal sequencing. CONCLUSION: We suggest that ASP-2 processing as well as activity are influenced by pH, and hence the cellular localisation of the protein may have profound effects on the production of Abeta. These factors should be taken into consideration in the design of potential inhibitors for these enzymes.

Induction of cellular oxidative stress by the beta-amyloid peptide involved in Alzheimer's disease.

Beta-amyloid, the 39-43 amino acid peptide fragment originating from amyloid precursor protein, is today, generally accepted as the biological entity responsible for causing the debilitating human disorder Alzheimer's disease. Understanding the exact biological effects of beta-amyloid in vitro and in vivo is clearly important to provide therapeutic strategies for the disease. Recent in vitro studies have focused on the production of reactive oxygen species by aggregating beta-amyloid, but the cellular effects of beta-amyloid induced reactive oxygen species production have not been fully elucidated.

Properties of neurotoxic peptides related to the Bri gene.

Familial British dementia, a rare autosomal dominant neurodegenerative disorder, shares features with Alzheimer's disease, including amyloid plaque deposits, neurofibrillary tangles, neuronal loss,progressive dementia, but clinically presents with additional physical defects [1,2]. A mutation in the termination codon of the BRI gene produces a BRI precursor protein 11 amino acids longer than the wild-type protein [3,4]. Mutant and wild-type precursor proteins both may undergo furin cleavage between residues 243 and 244, producing a peptide of 34 amino acids in the case of ABri and 23 amino acids long in the case of the wild type peptide. The ABri 4kDa peptide is the main component of the amyloid deposits found in familial British dementia brains. A decamer duplication in the 3- region of the BRI gene originates the peptide Adan that is associated with dementia in Familial Danish dementia (FDD), similar to BDD clinically, but with additional hearing and eyesight loss [5]. The resulting reading frame is extended to 277 amino acid residues, and cleavage by furin releases a peptide of 34 residues, which is identical to Abri and WT in its N-terminal 22-residues, but contains a distinct C-terminal 10 residues composed of mainly hydrophobic residues. Here we demonstrate that C-terminal extensions of Abri and Adan are required to elongate initially-formed dimers to neurotoxic soluble oligomers and fibrils. In contrast, the shorter wild-type peptide does not aggregate under the same conditions and is not toxic. Conformational analyses indicate triple-beta-sheet structures. Soluble nonfibrillar oligomers of oxidised ABri and reduced Adan were observed in solution (pH7.4) of peptides prior to the appearance of mature fibrils.

Amyloid-ß acts as a regulator of neurotransmitter release disrupting the interaction between synaptophysin and VAMP2.

BACKGROUND: It is becoming increasingly evident that deficits in the cortex and hippocampus at early stages of dementia in Alzheimer's disease (AD) are associated with synaptic damage caused by oligomers of the toxic amyloid-ß peptide (Aß42). However, the underlying molecular and cellular mechanisms behind these deficits are not fully understood. Here we provide evidence of a mechanism by which Aß42 affects synaptic transmission regulating neurotransmitter release. METHODOLOGY/FINDINGS: We first showed that application of 50 nM Aß42 in cultured neurones is followed by its internalisation and translocation to synaptic contacts. Interestingly, our results demonstrate that with time, Aß42 can be detected at the presynaptic terminals where it interacts with Synaptophysin. Furthermore, data from dissociated hippocampal neurons as well as biochemical data provide evidence that Aß42 disrupts the complex formed between Synaptophysin and VAMP2 increasing the amount of primed vesicles and exocytosis. Finally, electrophysiology recordings in brain slices confirmed that Aß42 affects baseline transmission. CONCLUSIONS/SIGNIFICANCE: Our observations provide a necessary and timely insight into cellular mechanisms that underlie the initial pathological events that lead to synaptic dysfunction in Alzheimer's disease. Our results demonstrate a new mechanism by which Aß42 affects synaptic activity.

Development of retro-inverso peptides as anti-aggregation drugs for ß-amyloid in Alzheimer's disease.

Alzheimer's disease (AD) is a devastating degenerative disorder of the brain for which there is no cure or effective treatment. There is much evidence to suggest that ß-amyloid protein (Aß) aggregation in the brain leading to deposits is an important step in the development of AD. Recently, two peptides, RGKLVFFGR (OR1) and RGKLVFFGR-NH(2) (OR2) containing the sequence KLVFF, which is the central region (residues 16-20) of Aß, have been found to be potent inhibitors of Aß aggregate formation. Here we report that retro-inversion of these sequences increases efficacy of the peptides in the inhibition of aggregation and toxicity of ß-amyloid. We describe the synthesis and inhibitory properties of these retro-inverso peptides.

Galantamine inhibits beta-amyloid aggregation and cytotoxicity.

The ability of galantamine (Reminyl) to inhibit the aggregation and toxicity of the beta-amyloid peptide (Abeta) was investigated. Galantamine showed concentration-dependent inhibition of aggregation of both Abeta 1-40 and Abeta 1-42, as determined by an ELISA method. Electron microscope studies of Abeta 1-40 incubated in the presence of galantamine revealed fibrils that were disordered and clumped in appearance. MTT and lactate dehydrogenase assays, employing SH-SY5Y human neuroblastoma cells, showed that galantamine reduced the cytotoxicity induced by Abeta 1-40. Galantamine also dramatically reduced Abeta 1-40-induced cellular apoptosis in these cells. There is some evidence that galantamine may not be acting purely as a symptomatic treatment. Disease-modifying effects of the drug could be due to an additional effect on Abeta aggregation and/or toxicity.

HLA homology within the C5 domain promotes peptide binding by HIV type 1 gp120.

The mechanisms by which HIV-1 induces chronic pathogenic immune activation associated with disease progression remain unclear despite many years of AIDS research. One proposal suggests that sequence and structural mimicry between gp120 and HLA may endow HIV with the capacity to arouse alloreactive and autoimmune responses within the susceptible host, fueling disease progression in a manner similar to graft-versus-host disease (GVHD). Both gp120 and HLA share a common functional interaction with CD4 but also demonstrate peptide binding properties. Here we report the conserved nature of this feature across HIV-1 envelopes, the crucial role of the HLA homologous C5 region for peptide interactions, and the elimination of this property through specific antibody targeting. Given that the C5 domain mimics a HLA activation domain and the reported clinical benefits associated with nonneutralizing antibodies against this region, targeting the C5 domain may have use as a therapeutic vaccine to protect against disease progression.

Designing peptide inhibitors for oligomerization and toxicity of Alzheimer's beta-amyloid peptide.

Convergent biochemical and genetic evidence suggests that the formation of beta-amyloid (Abeta) deposits in the brain is an important and, probably, seminal step in the development of Alzheimer's disease (AD). Recent studies support the hypothesis that Abeta soluble oligomers are the pathogenic species that prompt the disease. Inhibiting Abeta self-oligomerization could, therefore, provide a novel approach to treating the underlying cause of AD. Here, we designed potential peptide-based aggregation inhibitors containing Abeta amino acid sequences (KLVFF) from part of the binding region responsible for Abeta self-association (residues 16-20), with RG-/-GR residues added at their N- and C-terminal ends to aid solubility. Two such peptides (RGKLVFFGR, named OR1, and RGKLVFFGR-NH2, named OR2) were effective inhibitors of Abeta fibril formation, but only one of these peptides (OR2) inhibited Abeta oligomer formation. Interestingly, this same OR2 peptide was the only effective inhibitor of Abeta toxicity toward human neuroblastoma SH-SY5Y cells. Our data support the idea that Abeta oligomers are responsible for the cytotoxic effects of Abeta and identify a potential peptide inhibitor for further development as a novel therapy for AD.

A specific inhibitor of cholesterol biosynthesis, BM15.766, reduces the expression of beta-secretase and the production of amyloid-beta in vitro.

We have previously shown that statins reduce the production of amyloid-beta (Abeta) by both isoprenoid- and cholesterol-dependent mechanisms. These pathways contribute to the regulation of the dimerisation of BACE into its physiologically active form. Statins reduce cellular cholesterol levels by 20-40%; therefore, it is possible that the remaining cholesterol within the cell may play a significant role in the production of Abeta. Incubation of cells with the specific cholesterol biosynthesis inhibitor BM15.766 together with 50 micromol/L simvastatin and 400 micromol/L mevalonate reduced cellular cholesterol levels in a dose-dependent manner with increasing BM15.766 concentration (r = -0.9736, p = 0.0264). Furthermore, decreases in cellular cholesterol levels correlated with reductions in total Abeta production (r = 0.9683, p = 0.0317). A total of 2.5 micromol/L BM15.766 inhibited the dimerisation of BACE, whilst the expression of BACE monomer was reduced by 5 micromol/L BM15.766. BM15.766 treatment localised BACE predominantly within the Golgi, and reduced total BACE expression per cell. Similar changes were observed in the expression of the Golgi marker golgin-97, suggesting that reduced BACE expression may arise from a decrease in protein trafficking and an increase in degradation. By targeting cholesterol synthesis using specific cholesterol biosynthesis inhibitors, it is possible to reduce Abeta production without reducing protein isoprenylation.

Biophysical properties of a synthetic transit peptide from wheat chloroplast ribulose 1,5-bisphosphate carboxylase.

The surface properties of pure RuBisCo transit peptide (RTP) and its interaction with zwitterionic, anionic phospholipids and chloroplast lipids were studied by using the Langmuir monolayer technique. Pure RTP is able to form insoluble films and the observed surface parameters are compatible with an alpha-helix perpendicular to the interface. The alpha-helix structure tendency was also observed by using transmission FT-IR spectroscopy in bulk system of a membrane mimicking environment (SDS). On the other hand, RTP adopts an unordered structure in either aqueous free interface or in the presence of vesicles composed of a zwitterionic phospholipid (POPC). Monolayer studies show that in peptide/lipid mixed monolayers, RTP shows no interaction with zwitterionic phospholipids, regardless of their physical state. Also, with the anionic POPG at high peptide ratios RTP retains its individual surface properties and behaves as an immiscible component of the peptide/lipid mixed interface. This behaviour was also observed when the mixed films were composed by RTP and the typical chloroplast lipids MGDG or DGDG (mono- and di-galactosyldiacylglycerol). Conversely, RTP establishes a particular interaction with phosphatidylglycerol and cardiolipin at low peptide to lipid area covered relation. This interaction takes place with an increase in surface stability and a reduction in peptide molecular area (intermolecular interaction). Data suggest a dynamic membrane modulation by which the peptide fine-tunes its membrane orientation and its lateral stability, depending on the quality (lipid composition) of the interface.

Post-translational processing of beta-secretase in Alzheimer's disease.

Beta-amyloid is released into the brains of Alzheimer's patients, where it aggregates and causes damage to neurons. It is cleaved proteolytically from a large transmembrane glycoprotein amyloid precursor protein by a membrane-bound protease, known as beta-secretase identified previously as the acid protease, Asp-2. We have shown previously that beta-secretase is up-regulated by increased intracellular cholesterol, and down-regulated by cholesterol biosynthesis inhibition. Here we show using mass spectrometry that discrete changes in the glycosylation and palmitoylation of beta-secretase occur when cells expressing it are treated with statins.

Oligomerization and neurotoxicity of the amyloid ADan peptide implicated in familial Danish dementia.

Familial Danish dementia (FDD) is a rare neurodegenerative disorder, which is pathologically characterized by widespread cerebral amyloid angiopathy, parenchymal protein deposits and neurofibrillary degeneration. FDD is associated with mutation in the BRI gene. In FDD a decamer duplication between codons 265 and 266 in the 3' region of the BRI gene originates an amyloid peptide named ADan, 11 residues longer than the wild-type peptide produced from the normal BRI gene. ADan deposits have been found widely distributed in the CNS of FDD cases. The deposits of ADan are predominantly non-fibrillar aggregates. We show here that synthetic ADan forms oligomers in vitro, seen by Tricine-PAGE and gel filtration, and higher aggregates, which are seen by atomic force spectroscopy and electron microscopy as carrot-shaped objects that bunch together. Here we report that oligomeric ADan is toxic to neuronal cell lines. We find that the soluble non-fibrillar oligomeric species of both the reduced and oxidized forms of ADan are toxic. These results support the idea that the non-fibrillar soluble aggregates are the pathogenic species, which may play a central role in the pathogenesis of FDD, and imply that similar mechanism may also be involved in other neurodegenerative diseases associated with amyloid deposits.

Emerging techniques in biomedical research and their application to alcohol toxicity.

This article represents the proceedings of a symposium at the 2002 RSA-ISBRA Meeting in San Francisco. The chairs were Vinood B. Patel and Victor R. Preedy. The presentations were (1) Macromolecular structural analysis, by Vinood B. Patel; (2) Profiling and imaging of proteins in tissue sections using mass spectrometry as a discovery tool in biological research, by Pierre Chaurand and Richard M. Caprioli; (3) The use of SELDI ProteinChip trade mark arrays, by Brian M. Austen, Emma R. Frears, Francesca Manca, and Huw Davies; (4) DNA hybridization array technologies, by Kent E. Vrana; and (5) Adeno- and adeno-associated viral mediated gene transfer approaches for alcoholic liver disease, by Michael Wheeler. Concluding remarks were by Victor R. Preedy.