Characterization of Heparin-induced Glyceraldehyde-3-phosphate Dehydrogenase Early Amyloid-like Oligomers and Their Implication in alpha-Synuclein Aggregation

Lewy bodies and Lewy neurites, neuropathological hallmarks of several neurological diseases, are mainly made of filamentous assemblies of alpha-synuclein. However, other macromolecules including Tau, ubiquitin, glyceraldehyde-3-phosphate dehydrogenase, and glycosaminoglycans are routinely found associated with these amyloid deposits. Glyceraldehyde-3-phosphate dehydrogenase is a glycolytic enzyme that can form fibrillar aggregates in the presence of acidic membranes, but its role in Parkinson disease is still unknown. In this work, the ability of heparin to trigger the amyloid aggregation of this protein at physiological conditions of pH and temperature is demonstrated by infrared and fluorescence spectroscopy, dynamic light scattering, small angle x-ray scattering, circular dichroism, and fluorescence microscopy. Aggregation proceeds through the formation of short rod-like oligomers, which elongates in one dimension. Heparan sulfate was also capable of inducing glyceraldehyde-3-phosphate dehydrogenase aggregation, but chondroitin sulfates A, B, and C together with dextran sulfate had a negligible effect. Aided with molecular docking simulations, a putative binding site on the protein is proposed providing a rational explanation for the structural specificity of heparin and heparan sulfate. Finally, it is demonstrated that in vitro the early oligomers present in the glyceraldehyde-3-phosphate dehydrogenase fibrillation pathway promote alpha-synuclein aggregation. Taking into account the toxicity of alpha-synuclein prefibrillar species, the heparin-induced glyceraldehyde-3-phosphate dehydrogenase early oligomers might come in useful as a novel therapeutic strategy in Parkinson disease and other synucleinopathies.

Impairments of hippocampal synaptic plasticity induced by aggregated beta-amyloid (25-35) are dependent on stimulation-protocol and genetic background

The aggregation of beta-amyloid to plaques in the brain is one of the hallmarks of Alzheimer disease (AD). Numerous studies have tried to elucidate to what degree amyloid peptides play a role in the neurodegenerative developments seen in AD. While most studies report an effect of amyloid on neural activity and cognitive abilities of rodents, there have been many inconsistencies in the results. This study investigated to what degree the different genetic backgrounds affect the outcome of beta-amyloid fragment (25-35) on synaptic plasticity in vivo in the rat hippocampus. Two strains, Wistar and Lister hooded rats, were tested. In addition, the effects of a strong (600 stimuli) and a weak stimulation protocol (100 stimuli) on impairments of LTP were analysed. Furthermore, since the state of amyloid aggregation appears to play a role in the induction of toxic processes, it was tested by dual polarisation interferometry to what degree and at what speed beta-amyloid (25-35) can aggregate in vitro. It was found that 100 nmol beta-amyloid (25-35) injected icv did impair LTP in Wistar rats when using the weak but not the strong stimulation protocol (P <0.001). One-hundred nano mole of the reverse sequence amyloid (35-25) had no effect. LTP in Lister Hooded rats was not impaired by amyloid at any stimulation protocol. The aggregation studies showed that amyloid (25-35) aggregated within hours, while amyloid (35-25) did not. These results show that the genetic background and the stimulation protocol are important variables that greatly influence the experimental outcome. The fact that amyloid (25-35) aggregated quickly and showed neurophysiological effects, while amyloid (35-25) did not aggregate and did not show any effects indicates that the state of aggregation plays an important role in the physiological effects.

In situ atomic force microscopy study of Alzheimer’s β-amyloid peptide on different substrates: New insights into mechanism of β-sheet formation

We have applied in situ atomic force microscopy to directly observe the aggregation of Alzheimer’s β-amyloid peptide (Aβ) in contact with two model solid surfaces: hydrophilic mica and hydrophobic graphite. The time course of aggregation was followed by continuous imaging of surfaces remaining in contact with 10–500 μM solutions of Aβ in PBS (pH 7.4). Visualization of fragile nanoscale aggregates of Aβ was made possible by the application of a tapping mode of imaging, which minimizes the lateral forces between the probe tip and the sample. The size and the shape of Aβ aggregates, as well as the kinetics of their formation, exhibited pronounced dependence on the physicochemical nature of the surface. On hydrophilic mica, Aβ formed particulate, pseudomicellar aggregates, which at higher Aβ concentration had the tendency to form linear assemblies, reminiscent of protofibrillar species described recently in the literature. In contrast, on hydrophobic graphite Aβ formed uniform, elongated sheets. The dimensions of those sheets were consistent with the dimensions of β-sheets with extended peptide chains perpendicular to the long axis of the aggregate. The sheets of Aβ were oriented along three directions at 120° to each other, resembling the crystallographic symmetry of a graphite surface. Such substrate-templated self-assembly may be the distinguishing feature of β-sheets in comparison with α-helices. These studies show that in situ atomic force microscopy enables direct assessment of amyloid aggregation in physiological fluids and suggest that Aβ fibril formation may be driven by interactions at the interface of aqueous solutions and hydrophobic substrates, as occurs in membranes and lipoprotein particles in vivo.

Expression Profile of Rat Hippocampal Neurons Treated with the Neuroprotective Compound 2,4-Dinitrophenol: Up-Regulation of cAMP Signaling Genes

2,4-Dinitrophenol (DNP) is classically known as a mitochondrial uncoupler and, at high concentrations, is toxic to a variety of cells. However, it has recently been shown that, at subtoxic concentrations, DNP protects neurons against a variety of insults and promotes neuronal differentiation and neuritogenesis. The molecular and cellular mechanisms underlying the beneficial neuroactive properties of DNP are still largely unknown. We have now used DNA microarray analysis to investigate changes in gene expression in rat hippocampal neurons in culture treated with low micromolar concentrations of DNP. Under conditions that did not affect neuronal viability, high-energy phosphate levels or mitochondrial oxygen consumption, DNP induced up-regulation of 275 genes and down-regulation of 231 genes. Significantly, several up-regulated genes were linked to intracellular cAMP signaling, known to be involved in neurite outgrowth, synaptic plasticity, and neuronal survival. Differential expression of specific genes was validated by quantitative RT-PCR using independent samples. Results shed light on molecular mechanisms underlying neuroprotection by DNP and point to possible targets for development of novel therapeutics for neurodegenerative disorders.

Each one teaches one: characterizing active forms of proteins by single molecule force spectroscopy

This Ph.D. candidate thesis collects the research work I conducted under the supervision of Prof.Bruno Samor´ı in 2005,2006 and 2007. Some parts of this work included in the Part III have been begun by myself during my undergraduate thesis in the same laboratory and then completed during the initial part of my Ph.D. thesis: the whole results have been included for the sake of understanding and completeness. During my graduate studies I worked on two very different protein systems. The theorical trait d’union between these studies, at the biological level, is the acknowledgement that protein biophysical and structural studies must, in many cases, take into account the dynamical states of protein conformational equilibria and of local physico-chemical conditions where the system studied actually performs its function. This is introducted in the introductory part in Chapter 2. Two different examples of this are presented: the structural significance deriving from the action of mechanical forces in vivo (Chapter 3) and the complexity of conformational equilibria in intrinsically unstructured proteins and amyloid formation (Chapter 4). My experimental work investigated both these examples by using in both cases the single molecule force spectroscopy technique (described in Chapter 5 and Chapter 6). The work conducted on angiostatin focused on the characterization of the relationships between the mechanochemical properties and the mechanism of action of the angiostatin protein, and most importantly their intertwining with the further layer of complexity due to disulfide redox equilibria (Part III). These studies were accompanied concurrently by the elaboration of a theorical model for a novel signalling pathway that may be relevant in the extracellular space, detailed in Chapter 7.2. The work conducted on -synuclein (Part IV) instead brought a whole new twist to the single molecule force spectroscopy methodology, applying it as a structural technique to elucidate the conformational equilibria present in intrinsically unstructured proteins. These equilibria are of utmost interest from a biophysical point of view, but most importantly because of their direct relationship with amyloid aggregation and, consequently, the aetiology of relevant pathologies like Parkinson’s disease. The work characterized, for the first time, conformational equilibria in an intrinsically unstructured protein at the single molecule level and, again for the first time, identified a monomeric folded conformation that is correlated with conditions leading to -synuclein and, ultimately, Parkinson’s disease. Also, during the research work, I found myself in the need of a generalpurpose data analysis application for single molecule force spectroscopy data analysis that could solve some common logistic and data analysis problems that are common in this technique. I developed an application that addresses some of these problems, herein presented (Part V), and that aims to be publicly released soon.

Design and synthesis of novel non peptidomimtic beta-secretase inhibitors in the treatment of Alzheimer's disease

The aspartic protease BACE1 (β-amyloid precursor protein cleaving enzyme, β-secretase) is recognized as one of the most promising targets in the treatment of Alzheimer's disease (AD). The accumulation of β-amyloid peptide (Aβ) in the brain is a major factor in the pathogenesis of AD. Aβ is formed by initial cleavage of β-amyloid precursor protein (APP) by β-secretase, therefore BACE1 inhibition represents one of the therapeutic approaches to control progression of AD, by preventing the abnormal generation of Aβ. For this reason, in the last decade, many research efforts have focused at the identification of new BACE1 inhibitors as drug candidates. Generally, BACE1 inhibitors are grouped into two families: substrate-based inhibitors, designed as peptidomimetic inhibitors, and non-peptidomimetic ones. The research on non-peptidomimetic small molecules BACE1 inhibitors remains the most interesting approach, since these compounds hold an improved bioavailability after systemic administration, due to a good blood-brain barrier permeability in comparison to peptidomimetic inhibitors. Very recently, our research group discovered a new promising lead compound for the treatment of AD, named lipocrine, a hybrid derivative between lipoic acid and the AChE inhibitor (AChEI) tacrine, characterized by a tetrahydroacridinic moiety. Lipocrine is one of the first compounds able to inhibit the catalytic activity of AChE and AChE-induced amyloid-β aggregation and to protect against reactive oxygen species. Due to this interesting profile, lipocrine was also evaluated for BACE1 inhibitory activity, resulting in a potent lead compound for BACE1 inhibition. Starting from this interesting profile, a series of tetrahydroacridine analogues were synthesised varying the chain length between the two fragments. Moreover, following the approach of combining in a single molecule two different pharmacophores, we designed and synthesised different compounds bearing the moieties of known AChEIs (rivastigmine and caproctamine) coupled with lipoic acid, since it was shown that dithiolane group is an important structural feature of lipocrine for the optimal inhibition of BACE1. All the tetrahydroacridines, rivastigmine and caproctamine-based compounds, were evaluated for BACE1 inhibitory activity in a FRET (fluorescence resonance energy transfer) enzymatic assay (test A). With the aim to enhancing the biological activity of the lead compound, we applied the molecular simplification approach to design and synthesize novel heterocyclic compounds related to lipocrine, in which the tetrahydroacridine moiety was replaced by 4-amino-quinoline or 4-amino-quinazoline rings. All the synthesized compounds were also evaluated in a modified FRET enzymatic assay (test B), changing the fluorescent substrate for enzymatic BACE1 cleavage. This test method guided deep structure-activity relationships for BACE1 inhibition on the most promising quinazoline-based derivatives. By varying the substituent on the 2-position of the quinazoline ring and by replacing the lipoic acid residue in lateral chain with different moieties (i.e. trans-ferulic acid, a known antioxidant molecule), a series of quinazoline derivatives were obtained. In order to confirm inhibitory activity of the most active compounds, they were evaluated with a third FRET assay (test C) which, surprisingly, did not confirm the previous good activity profiles. An evaluation study of kinetic parameters of the three assays revealed that method C is endowed with the best specificity and enzymatic efficiency. Biological evaluation of the modified 2,4-diamino-quinazoline derivatives measured through the method C, allow to obtain a new lead compound bearing the trans-ferulic acid residue coupled to 2,4-diamino-quinazoline core endowed with a good BACE1 inhibitory activity (IC50 = 0.8 mM). We reported on the variability of the results in the three different FRET assays that are known to have some disadvantages in term of interference rates that are strongly dependent on compound properties. The observed results variability could be also ascribed to different enzyme origin, varied substrate and different fluorescent groups. The inhibitors should be tested on a parallel screening in order to have a more reliable data prior to be tested into cellular assay. With this aim, preliminary cellular BACE1 inhibition assay carried out on lipocrine confirmed a good cellular activity profile (EC50 = 3.7 mM) strengthening the idea to find a small molecule non-peptidomimetic compound as BACE1 inhibitor. In conclusion, the present study allowed to identify a new lead compound endowed with BACE1 inhibitory activity in submicromolar range. Further lead optimization to the obtained derivative is needed in order to obtain a more potent and a selective BACE1 inhibitor based on 2,4-diamino-quinazoline scaffold. A side project related to the synthesis of novel enzymatic inhibitors of BACE1 in order to explore the pseudopeptidic transition-state isosteres chemistry was carried out during research stage at Università de Montrèal (Canada) in Hanessian's group. The aim of this work has been the synthesis of the δ-aminocyclohexane carboxylic acid motif with stereochemically defined substitution to incorporating such a constrained core in potential BACE1 inhibitors. This fragment, endowed with reduced peptidic character, is not known in the context of peptidomimetic design. In particular, we envisioned an alternative route based on an organocatalytic asymmetric conjugate addition of nitroalkanes to cyclohexenone in presence of D-proline and trans-2,5-dimethylpiperazine. The enantioenriched obtained 3-(α-nitroalkyl)-cyclohexanones were further functionalized to give the corresponding δ-nitroalkyl cyclohexane carboxylic acids. These intermediates were elaborated to the target structures 3-(α-aminoalkyl)-1-cyclohexane carboxylic acids in a new readily accessible way.

New Synthetic Polyamines as Multi-Target-Directed Ligands for the Treatment of Alzheimer's Disease and Cancer: Design, Synthesis and Biological Evaluation

Alzheimer's disease (AD) and cancer represent two of the main causes of death worldwide. They are complex multifactorial diseases and several biochemical targets have been recognized to play a fundamental role in their development. Basing on their complex nature, a promising therapeutical approach could be represented by the so-called "Multi-Target-Directed Ligand" approach. This new strategy is based on the assumption that a single molecule could hit several targets responsible for the onset and/or progression of the pathology. In particular in AD, most currently prescribed drugs aim to increase the level of acetylcholine in the brain by inhibiting the enzyme acetylcholinesterase (AChE). However, clinical experience shows that AChE inhibition is a palliative treatment, and the simple modulation of a single target does not address AD aetiology. Research into newer and more potent anti-AD agents is thus focused on compounds whose properties go beyond AChE inhibition (such as inhibition of the enzyme β-secretase and inhibition of the aggregation of beta-amyloid). Therefore, the MTDL strategy seems a more appropriate approach for addressing the complexity of AD and may provide new drugs for tackling its multifactorial nature. In this thesis, it is described the design of new MTDLs able to tackle the multifactorial nature of AD. Such new MTDLs designed are less flexible analogues of Caproctamine, one of the first MTDL owing biological properties useful for the AD treatment. These new compounds are able to inhibit the enzymes AChE, beta-secretase and to inhibit both AChE-induced and self-induced beta-amyloid aggregation. In particular, the most potent compound of the series is able to inhibit AChE in subnanomolar range, to inhibit β-secretase in micromolar concentration and to inhibit both AChE-induced and self-induced beta-amyloid aggregation in micromolar concentration. Cancer, as AD, is a very complex pathology and many different therapeutical approaches are currently use for the treatment of such pathology. However, due to its multifactorial nature the MTDL approach could be, in principle, apply also to this pathology. Aim of this thesis has been the development of new molecules owing different structural motifs able to simultaneously interact with some of the multitude of targets responsible for the pathology. The designed compounds displayed cytotoxic activity in different cancer cell lines. In particular, the most potent compounds of the series have been further evaluated and they were able to bind DNA resulting 100-fold more potent than the reference compound Mitonafide. Furthermore, these compounds were able to trigger apoptosis through caspases activation and to inhibit PIN1 (preliminary result). This last protein is a very promising target because it is overexpressed in many human cancers, it functions as critical catalyst for multiple oncogenic pathways and in several cancer cell lines depletion of PIN1 determines arrest of mitosis followed by apoptosis induction. In conclusion, this study may represent a promising starting pint for the development of new MTDLs hopefully useful for cancer and AD treatment.

Multi-target-directed ligands: application to the Alzheimer's disease

The MTDL (multi-target-directed ligand) design strategy is used to develop single chemical entities that are able to simultaneously modulate multiple targets. The development of such compounds might disclose new avenues for the treatment of a variety of pathologies (e.g. cancer, AIDS, neurodegenerative diseases), for which an effective cure is urgently needed. This strategy has been successfully applied to Alzheimer’s disease (AD) due to its multifactorial nature, involving cholinergic dysfunction, amyloid aggregation, and oxidative stress. Despite many biological entities have been recognized as possible AD-relevant, only four achetylcholinesterase inhibitors (AChEIs) and one NMDA receptor antagonist are used in therapy. Unfortunately, such compounds are not disease-modifying agents behaving only as cognition enhancers. Therefore, MTDL strategy is emerging as a powerful drug design paradigm: pharmacophores of different drugs are combined in the same structure to afford hybrid molecules. In principle, each pharmacophore of these new drugs should retain the ability to interact with its specific site(s) on the target and, consequently, to produce specific pharmacological responses that, taken together, should slow or block the neurodegenerative process. To this end, the design and synthesis of several examples of MTDLs for combating neurodegenerative diseases have been published. This seems to be the more appropriate approach for addressing the complexity of AD and may provide new drugs for tackling the multifactorial nature of AD, and hopefully stopping its progression. According to this emerging strategy, in this work thesis different classes of new molecular structures, based on the MTDL approach, have been developed. Moreover, curcumin and its constrained analogs have currently received remarkable interest as they have a unique conjugated structure which shows a pleiotropic profile that we considered a suitable framework in developing MTDLs. In fact, beside the well-known direct antioxidant activity, curcumin displays a wide range of biological properties including anti-inflammatory and anti-amyloidogenic activities and an indirect antioxidant action through activation of the cytoprotective enzyme heme oxygenase (HO-1). Thus, since many lines of evidence suggest that oxidative stess and mitochondria impairment have a cental role in age-related neurodegenerative diseases such as AD, we designed mitochondria-targeted antioxidants by connecting curcumin analogs to different polyamine chains that, with the aid of electrostatic force, might drive the selected antioxidant moiety into mitochondria.

Rapid label-free detection of metal-induced Alzheimer's amyloid beta peptide aggregation by electrochemical method

Amyloid beta peptide plays a critical role in the pathogenesis of Alzheimer's disease (AD). Metal ions are highly enriched in cerebral amyloid deposits in AD and are proposed to be able to mediate A beta conformation. Therefore, a rapid, low-cost, and sensitive detection of metal-induced A beta aggregation and their relation to AD is clearly needed for the clinical diagnosis and treatment. In this report, we study metal-induced A beta aggregation by a rapid, label-free electrochemical method and monitor both the aggregation kinetics and the morphology in the absence or presence of Zn (II) and Cu (II).

Identification of the region of non-A beta component (NAC) of alzheimer's disease amyloid responsible for its aggregation and toxicity

The non-beta-amyloid (Aß) component of Alzheimer's disease amyloid (NAC) and its precursor a-synuclein have been linked to amyloidogenesis in several neurodegenerative diseases. NAC and a-synuclein both form ß-sheet structures upon ageing, aggregate to form fibrils, and are neurotoxic. We recently established that a peptide comprising residues 3±18 of NAC retains these properties. To pinpoint the exact region responsible we have carried out assays of toxicity and physicochemical properties on smaller fragments of NAC. Toxicity was measured by the ability of fresh and aged peptides to inhibit the reduction of the redox dye 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) by rat pheochromocytoma PC12 cells and human neuroblastoma SHSY-5Y cells. On immediate dissolution, or after ageing, the fragments NAC(8±18) and NAC(8±16) are toxic, whereas NAC(12±18), NAC(9±16) and NAC(8±15) are not. Circular dichroism indicates that none of the peptides displays ß-sheet structure; rather all remain random coil throughout 24 h. However, in acetonitrile, an organic solvent known to induce ß sheet, fragments NAC(8±18) and NAC(8±16) both form ß-sheet structure. Only NAC(8±18) aggregates, as indicated by concentration of peptide remaining in solution after 3 days, and forms fibrils, as determined by electron microscopy. These findings indicate that residues 8±16 of NAC, equivalent to residues 68±76 in a-synuclein, comprise the region crucial for toxicity.

Monoclonal antibodies inhibit in vitro fibrillar aggregation of the Alzheimer beta-amyloid peptide.

The beta-amyloid peptide, the hallmark of Alzheimer disease, forms fibrillar toxic aggregates in brain tissue that can be dissolved only by strong denaturing agents. To study beta-amyloid formation and its inhibition, we prepared immune complexes with two monoclonal antibodies (mAbs), AMY-33 and 6F/3D, raised against beta-amyloid fragments spanning amino acid residues 1-28 and 8-17 of the beta-amyloid peptide chain, respectively. In vitro aggregation of beta-amyloid peptide was induced by incubation for 3 h at 37 degrees C and monitored by ELISA, negative staining electron microscopy, and fluorimetric studies. We found that the mAs prevent the aggregation of beta-amyloid peptide and that the inhibitory effect appears to be related to the localization of the antibody-binding sites and the nature of the aggregating agents. Preparation of mAbs against "aggregating epitopes," defined as sequences related to the sites where protein aggregation is initiated, may lead to the understanding and prevention of protein aggregation. The results of this study may provide a foundation for using mAbs in vivo to prevent the beta-amyloid peptide aggregation that is associated with Alzheimer disease.

NACP, the precursor protein of the non-amyloid beta/A4 protein (A beta) component of Alzheimer disease amyloid, binds A beta and stimulates A beta aggregation.

NACP, a 140-amino acid presynaptic protein, is the precursor of NAC [the non-amyloid beta/A4 protein (A beta) component of Alzheimer disease (AD) amyloid], a peptide isolated from and immunologically localized to brain amyloid of patients afflicted with AD. NACP produced in Escherichia coli bound to A beta peptides, the major component of AD amyloid. NACP bound to A beta 1-38 and A beta 25-35 immobilized on nitrocellulose but did not bind to A beta 1-28 on the filter under the same conditions. NACP binding to A beta 1-38 was abolished by addition of A beta 25-35 but not by A beta 1-28, suggesting that the hydrophobic region of the A beta peptide is critical to this binding. NACP-112, a shorter splice variant of NACP containing the NAC sequence, bound to A beta, but NACP delta, a deletion mutant of NACP lacking the NAC domain, did not bind A beta 1-38. Furthermore, binding between NACP-112 and A beta 1-38 was decreased by addition of peptide Y, a peptide that covers the last 15 residues of NAC. In an aqueous solution, A beta 1-38 aggregation was observed when NACP was also present in an incubation mixture at a ratio of 1:125 (NACP/A beta), whereas A beta 1-38 alone or NACP alone did not aggregate under the same conditions, suggesting that the formation of a complex between A beta and NACP may promote aggregation of A beta. Thus, NACP can bind A beta peptides through the specific sequence and can promote A beta aggregation, raising the possibility that NACP may play a role in the development of AD amyloid.