Neurodegenerative disorders, including Alzheimer's, Parkinson's, amyotrophic lateral sclerosis and prion diseases are debilitating and so far incurable disorders that demand intensive research. In these diseases, misfolding, aggregation, and precipitation of proteins seem to be directly related to neurotoxicity (Dobson, 2003; Chiti and Dobson, 2006). Specifically, the physiological alterations are associated with the formation of fibrillar aggregates, referred to as amyloid fibrils, that usually accumulate in the extracellular space of tissues or also as intracellular deposits (Stefani and Dobson, 2003; Taylor et al., 2005). Protein molecular assembly is characterized by several events like conformational changes and intermolecular interactions which strongly affect each other. The hierarchy of all these mechanisms and their extent depends on several physical and chemical parameters such as temperature, pH, ionic strength, and addition of denaturants. Until very recently, it was thought that only a small number of polypeptide chains associated with clinical disorders were able to form amyloid fibrils. However, a number of recent studies have shown that proteins unrelated to diseases, under suitable conditions, can form aggregates in vitro with structural and cytotoxic properties that closely resemble those of the amyloid fibrils formed in diseased tissues (Litvinovich et al., 1998; Fandrich et al., 2001; Sirangelo et al., 2004, 2009; Iannuzzi et al., 2013a). These observations have led to the idea that the ability to form amyloid fibrils is a generic property of polypeptide chains irrespective of their amino acid sequence and caused by stable interactions involving primarily the common polypeptide backbone. Despite major differences in the sequences and three-dimensional structures of the peptides and proteins involved, the fibrillar forms of the aggregates share a common ultrastructure (Diaz-Avalos et al., 2003; Nelson et al., 2005; Fitzpatrick et al., 2013). They usually consist of a number (typically 2–6) of protofilaments, each about 2–5 nm in diameter, that are often twisted around each other to form super-coiled ropelike structures typically 7–13 nm in width or that laterally associate to form long ribbons that are 2–5 nm thick and up to 30 nm wide (Serpell et al., 2000). X-ray diffraction analysis has indicated that the characteristic structure, i.e., the β-cross motif, is formed by β-strands oriented perpendicular to the long axis of the fibril, and β-sheets propagating in the fibril direction (Sunde and Blake, 1997; Makin and Serpell, 2002; Maji et al., 2009). These findings suggest that a common molecular mechanism could underlie the aggregation process of the different proteins involved in misfolding diseases (Kopito, 2000; Dobson, 2001).

Three major factors have been identified as important parameters in the conversion of a protein into aggregates; these are high hydrophobicity, high propensity to convert from α-helical to β-sheet structure, and low net charge (Konno, 2001; Ciani et al., 2002; Tjernberg et al., 2002; Chiti et al., 2003; Tartaglia et al., 2008). Protein destabilization favors the formation of partially unfolded conformations that are highly prone to aggregation (Uversky and Fink, 2004). In most cases, protein destabilization is facilitated by amino acid mutations which also increase the structural flexibility of the peptide chain; however, other proteins are amyloidogenic even in the wild type form (Hurle et al., 1994; Goedert et al., 2000; Quintas et al., 2001; Niraula et al., 2002; Iannuzzi et al., 2007; Infusini et al., 2012, 2013). It has been suggested that protein folding and protein aggregation, despite being distinct processes, are in competition each other and the environmental conditions dictate which one is favored for a given polypeptide chain (Tartaglia and Vendruscolo, 2010). On this basis, extensive studies have been carried out in vitro to investigate the nature of the transition between natively folded states and soluble aggregate-precursor states, and between the latter and mature amyloid fibrils and the factors affecting all of these (Wiseman et al., 2005). Recent data indicate that these dangerous aggregation-prone states, although similar to the native conformation, display altered surface charge distribution, alternative β-sheet topologies and increased solvent exposure of hydrophobic surfaces and of aggregation-prone regions of the sequence (De Simone et al., 2011). The propensity of normally folded proteins to form amyloid-like fibrils increases in conditions that allow the protein to break the major unfolding energy barrier, favoring partial unfolding of the native state. These include low pH, high temperature, or the presence of organic solvents (Guijarro et al., 1998; Villegas et al., 2000). However, increasing evidence is now accumulating that folded proteins also retain a significant tendency to aggregate with no need for unfolding as first obligatory step (Plakoutsi et al., 2004; Bemporad and Chiti, 2009).

Protein aggregation begins with the appearance of aggregation nuclei, whose growth is considered the rate-limiting step of the process, which has many characteristics of a nucleation-dependent polymerization mechanism (Kelly, 1998) (Figures 1, 2). These species, generally indicated as protofibrils or soluble oligomeric intermediates, appear as globules of 2.5–5.0 nm in diameter or larger, with an intrinsic tendency to further assemble into pore-like annular and tubular structures (Lashuel et al., 2002; Poirier et al., 2002). Once a nucleus is formed, fibril growth is thought to proceed rapidly by further association of either monomers or oligomers with the nucleus (Cohen et al., 2012).

While insoluble aggregates correlate with disease progression, there are increasing evidences that the initiating and most toxic events are caused by prefibrillar forms rather than mature fibrils. These results have led to the idea that molecular basis of cell and tissue impairment may be related to the transient appearance of prefibrillar assemblies, under conditions where their intracellular levels increase as a consequence of dysfunctions in cellular clearance machineries (Stefani, 2012). The specific mechanism by which these species appear to mediate their toxic effects is not completely understood; probably toxicity is mediated by common structural features shared by prefibrillar precursors (Kayed et al., 2003; Bucciantini et al., 2004; Malmo et al., 2006; Cecchi and Stefani, 2013).

Although the aggregation process of amyloidogenic proteins has been widely studied in vitro and many physiological (environmental and genetic) factors involved have been identified, the molecular mechanisms underlying the formation of aggregates in vivo and in pathological conditions are still poorly understood. The majority of neurodegenerative diseases are sporadic, suggesting that other factors must contribute to the onset and progression of these disorders. Post-translational modifications are known to affect protein structure and function. Some of these modifications might affect proteins in detrimental ways and lead to their misfolding and accumulation. Reducing sugars play an important role in modifying proteins, forming advanced glycation end-products (AGEs) in a non-enzymatic process named glycation. This process is different from glycosylation; indeed these two post-translational modifications affect the structure of the target protein in a different way. Glycosylation is a selective protein modification, driven by specific enzymes, that is generally associated to a gain of function (or stabilization) of the target protein. Non-enzymatic glycation is a non-selective modification and it is generally associated to a loss of function of the target protein due to modifications of its native structure. While glycosylation is a well controlled cellular mechanism, non-enzymatic glycation only depends on the exposure of free amino-groups in the polypeptide chain, concentration of the sugar and oxidative conditions.

Recently, much attention has been devoted to the role played by non-enzymatic glycation of proteins in stimulating amyloid aggregation and toxicity. Proteins in amyloid deposits are found often glycated suggesting a direct correlation between protein glycation and amyloidosis (Miyata et al., 1993; Kikuchi et al., 2000; Munch et al., 2000; Dukic-Stefanovic et al., 2001; Shults, 2006). Glycation reactions are common to all cell types: glycated products slowly accumulate in vivo leading, besides cellular modifications involved in the aging process, to several different protein dysfunctions (Lyons et al., 1991; Miyata et al., 1999; Gul et al., 2009). The process begins with a nucleophilic addition reaction between a free amino group of a protein and a carbonyl group of a reducing sugar, forming a reversible intermediate product (Schiff's base). Side-chains of arginine and lysine residues, the N-terminus amino group of proteins, and thiol groups of cysteine residues, are the main targets of protein glycation. The process depends on several conditions, such as the concentration and reactivity of the glycation agent, the presence of catalytic factors (metals, buffer ions and oxygen), the physiological pH, temperature and the half-life of each protein. All reducing sugars can participate in glycation reactions and, between them, D-ribose is the most active and its intracellular level can be quite high. D-glucose is the least reactive and its intracellular concentration is negligible, while dicarbonyl compounds are far more reactive. The levels of D-ribose in the blood are estimated around 20 mg/L in healthy individuals while D-glucose 6–10 g/L. Once formed, the Schiff's base can turn into a stable ketoamine by Amadori rearrangement (Figure 3). This reaction is reversible depending on the concentration of the reactants. The late-stage of the process is an irreversible cascade of reactions involving dehydration, hydrolysis, and other rearrangements leading to the formation of AGEs. AGEs products are considered to be a marker of several diseases, such as arteriosclerosis, renal failure, Alzheimer disease, or diabetes, although they normally increase in aging (Vlassara, 2005).

Indeed, protein glycation has been considered an age related problem influencing mainly extracellular proteins, such as collagen and elastin, which are located outside the cells and provide strength and flexibility to the tissues. AGEs formation can interfere not only with the regular functioning of the proteins to which they are attached but also induce the formation of covalent cross-links with close proteins. This process is gradual, so that cross-links accumulate over the years on the longest-lived extracellular proteins, which do not get cleared very often; clear evidence of this is found in the extracellular collagen and elastin (Furber, 2010). The observation that proteins in amyloid deposits, such as β-amyloid, tau, prions and transthyretin, are often found glycated in patients suggests a direct correlation between protein glycation and amyloid formation. This is thought to be associated with an increased protein stability through the formation of cross-links that stabilize protein aggregates (Figure 4). Also, glycation affects the structure and the biological activity of proteins as well as their degradation process (Shaklai et al., 1984; Mendez et al., 2005) and, being an abnormal modification, it has been found to induce some proteins to misfold and, thus, promote protein aggregation (Vitek et al., 1994; Chellan and Nagaraj, 1999; Verzijl et al., 2002; Bouma et al., 2003).

Moreover, once proteins become glycated at their exposed lysine residues, clearance by the ubiquitin-proteasome system would be impaired because ubiquitination of lysine residues, a modification that targets proteins to the proteasome for degradation, might be impeded. Thus, accumulation of proteins as aggregates or as depositions or inclusions in tissues might be favored after glycation.

However, in addition to directly affecting protein structure and function, AGEs also exert cellular effects mediated by specific AGEs receptors (RAGE), as well as macrophage scavenger receptors, MSR type II, OST-48, 80K-H, galectin-3, and CD36 (Vlassara et al., 1995; Li et al., 1996; Ohgami et al., 2002; Stern et al., 2002). Indeed, glycation may be responsible, via RAGE, for an increase in oxidative stress and inflammation through the formation of reactive oxygen species and the activation of the nuclear transcription factor-κ B generally associated to amyloid toxicity (Xie et al., 2013).

Several proteins related and not related to misfolding diseases have been so far examined to investigate the effect of glycation on their propensity to aggregate and form amyloid structure.

The above referred considerations make the study of AGEs one of the most important areas of biomedical research today. Several questions remain to be answered: whether glycation of susceptible proteins is a triggering event or just a result of its reactivity toward low-turnover aggregated species, which are highly insoluble and protease-resistant, remains controversial. Several studies suggest that glycation may be an early event promoting or accelerating abnormal protein deposition, followed by increased protease resistance and insolubility. Regardless of the chronology of AGEs formation, it is known that its accumulation is related to sustained inflammatory responses and oxidative stress, which is a common feature in many neurodegenerative disorders. Glycation may then be understood as a dynamic contributor to these multifactorial diseases by promoting, accelerating or stabilizing pathological protein aggregation and inducing responses leading to cell dysfunction, damage and death. Thus, it will be important to further investigate the biochemical effects induced by the interaction of AGEs-modified proteins with cells, such as, the activation of oxidative stress signaling pathway and inflammatory response.