Liquid-liquid phase separation (LLPS) is a physical phenomenon which involves separation of a uniform solution of macromolecules into two distinct liquid phases with different densities of macromolecules, resulting in the formation of coacervate like structures. Phase separation is preceded by accumulation of multiple conformations of α-synuclein monomers some of which are structurally more adapted to develop into protofibrils (Dedmon et al., 2005). Brodie and co-workers used a combination of Molecular Dynamics simulation data substantiated with cross-linking mass spectrometry (MS) and single molecule Förster resonance energy transfer (smFRET) data to generate an ensemble of conformational spaces of such α-synuclein monomers (Brodie et al., 2019). The tendency of α-synuclein to undergo LLPS is facilitated by the presence of low complexity domains (LCDs), one or more sequence stretches with higher abundance of certain amino acids instead of the complete set of 20 amino acids (Mahapatra and Newberry, 2024). These sequences are mostly unstructured representing the IDR regions of proteins and initiate the formation of unique condensates where many molecules of α-synuclein slowly transform into the beta-pleated structure mediated by transient multivalent interactions. This results into a local increase in density of these proteins and marks the initiation of nucleation of the fibres. The process is facilitated by the presence of Polyethylene Glycol, a molecular crowding agent which brings α-synuclein molecules in close proximity by displacing water from the immediate vicinity for enhanced hydrogen bonding, non-covalent interactions and hydrophobic interactions (De Luca et al., 2025). Eventually, further α-synuclein molecules rope in and fibrils are extended leading to the formation of amyloid like hydrogel entities (Mukherjee et al., 2023) (Figure 1). The concentration of α-synuclein in these condensates can reach to as high as 30–40 mM (Dada et al., 2023) which leads to the formation of neurofibrillary tangles or the so-called Lewy bodies. Phase separation of α-synuclein under both cellular and in vitro conditions is guided by the Vesicle Associated Membrane Protein 2 (VAMP2) and its interaction with R-SNARE (Agarwal et al., 2024). The disease pathology is subsequently spread by prion like self-propagation of α-synuclein fibrils (Jan et al., 2021). The indispensable role of the C terminal domain in preventing fibrillation has been demonstrated by an engineered α-synuclein with truncated C terminus which not only displayed accelerated fibrillation kinetics but also promoted the phase separation of WT α-synuclein (Huang et al., 2022). Apart from the intrinsic sequence predisposition and presence of molecular crowding agents, many other extrinsic factors act as mediators of the LLPS; the more significant of these being the pH and the concentration of metal ions in the local microenvironment. At a pH of 7.4, primary nucleation proceeds spontaneously and is the fastest in the absence of other governing factors (Dada et al., 2023) whereas the critical concentration of α-synuclein needed to undergo LLPS is the least at pH 5.5 probably owing to the proximity of the isoelectric point of α-synuclein near that pH assisting separation of proteins from the solution (Hou et al., 2023). A high salt concentration in the medium promotes fibrillation and lowers down the critical concentration of the protein to undergo LLPS by neutralizing the charges at the N and C termini and promoting aggregation of the NAC region (Sawner et al., 2021). In addition to the charge neutralization, it also promotes effective salting out of the protein to induce phase separation.

Under physiological conditions, LLPS of α-synuclein is prevented by β-synuclein, a related protein of the synuclein family which is present at the presynaptic terminals at a similar concentration as that of α-synuclein (around 50 µM). Several independent reports have indicated that β-synuclein exerts its action by associating with α-synuclein and subsequently a) elongating the lag phase of fibrillation (Jain et al., 2018) and b) arresting secondary nucleation (Brown et al., 2016). β-synuclein has a 78% sequence identity with α-synuclein, differs in few critical aspects from the later including a 11 residue shorter NAC region, absence of cysteine and tryptophan residues and an abundance of proline and acidic residues and forms ordered structure more readily than α-synuclein (Hayashi and Carver, 2022). A higher number of proline residues is probably needed by the protein to make up for the folding flexibility curtailed by the shorter NAC region. A particular point mutant of β-synuclein, P123H, results in pathogenic deposits of Lewy Body Dementia (Psol et al., 2021) Li et al. found that β-synuclein associated with α-synuclein employing electrostatic interactions. In an in vitro microenvironment mimicking presynaptic terminal with equimolar amounts of α and β synuclein, the total mean surface area of α/β-Syn condensates was higher than that of α-Syn alone. Although the association of α/β-Syn condensates apparently promoted LLPS of α-synuclein, its corresponding nucleation and fibrillation was effectively prevented (Li et al., 2024). The pivotal role of NAC region in driving the aggregation is also supported by the observation that the second identified pathogenic point mutation V70M (Valine to Methionine) causes predisposition to a sporadic Lewy Body Dementia. Methionine is less hydrophobic than valine and therefore substitution to methionine should have lessened the aggregation propensity; therefore, this observation is probably a testimonial to the indispensability of Valine at the 70th position for NAC mediated aggregation of β-synuclein and its simultaneous association with α-synuclein.

Misfolding of α-synuclein has been linked to a plethora of cognitive ailments arising from the death of dopaminergic neurons and collectively referred to as synucleinopathies. The soluble protofibrils formed from α-synuclein exert their cytotoxicity through disruption of mitochondrial function, promotion of autophagy, lysosomal impairment and loss in regulation of calcium homeostasis (Calabresi et al., 2023). Particularly, the protein has been found to be associated with increased production of Reactive Oxygen Species (ROS) and elicitation of inflammatory response (Van Laar et al., 2020), the two primary reasons accounting for loss in cellular homeostasis.

As stated earlier, α-synuclein fibrillates by addition of monomers after the initial nucleation; during elongation, smaller fragments of oligomers and protofibrils often gets detached from the primary fibril serving as additional points of nucleation thus aggravating the problem. This avalanche of soluble protofibrils is often believed to be more toxic than the stable full length cross-beta fibrils owing to their membrane permeabilization capability and interaction with various cellular organelles (Winner et al., 2011). Mitochondria represent the most vulnerable site of α-synuclein attack primarily owing to the latter’s interaction with cardiolipin, a major mitochondrial phospholipid with simultaneous formation of transient pores in the membrane (Ghio et al., 2019). The first case of α-synuclein independent PD case linked with mitochondria was reported with respect to consumption of banned drugs contaminated with 1-methyl-4-phenyl-1,2,3,6-tetrabydropyridine (MPTP). The toxic bioactive form of this compound 1-methyl-4-phenylpyridinium (MPP+) blocks the functioning of mitochondrial Complex-1 and NADH-ubiquinone oxidoreductase enzyme resulting in release of free electrons with concomitant ROS generation (Javitch et al., 1985). Creation of transient pores by α-synuclein oligomers induce similar effects of uncoupling electron transport from ATP production, resulting in release of free electrons and cytochrome c and concomitant ROS generation. Apart from directly modulating mitochondrial physiology, α-synuclein oligomers also amplify cytosolic peroxide flux resulting in oxidation of glutathiones (Van Laar et al., 2020). A logical explanation behind this observation comes from the fact that elongation of the polymeric fibrillar structure of α-synuclein requires a constant supply of free protons from the solution in order to mask the C terminal negative charges and prevent the interference of the N and C terminal association during fibrillation. In addition, α-synuclein oligomers can also elicit ER stress and consequent induction of Unfolded Protein Response pathway (Puspita et al., 2017). The consequent rise in intracellular calcium levels leads to further augmentation of ROS induced cellular damage. The diverse mechanisms by which misfolded α-synuclein inflict its cellular toxicity has been summarized in Figure 2.

In the reverse perspective, production of cellular ROS is also associated with fibrillation of α-synuclein (Esteves et al., 2008). In a superoxide dismutase 2 (SOD2) transgenic mouse model, development of synucleinopathies was significantly earlier as compared to the control mouse model which proved that a compromise in the capacity to quench free radical led to enhanced fibrillation of α-synuclein (Scudamore and Ciossek, 2018). In related studies, cytochrome c and peroxide radicals also resulted in crosslinking of α-synuclein monomers through the formation 3,3′-dityrosine linkages (Hashimoto et al., 1999; Ruf et al., 2008). Such crosslinking can arrest the formation of conventional cross beta fibrils but at the same time promote off-pathway aggregates as oligomers which are potentially more toxic.

Aggregate formation and fibrillation of α-synuclein is favoured at slightly acidic pH, much like the endosomes and the lysosomes. This is explained by the fact that at neutral pH, the protein bears a net negative charge which is nullified on lowering the pH. The C terminal also gets fully protonated and uncharged at this pH which augments the aggregation process. This facilitates the hydrophobic interactions and subsequent aggregation of the central domain of the protein (Moriarty et al., 2017). These results have also been confirmed by single molecule fluorescence studies which has revealed that a slight excess of protons in the solution is conducive towards aggregation of monomeric α-synuclein (Trexler and Rhoades, 2010). However, at a pH below 6, secondary nucleation is favoured than fibril growth thus shifting the kinetics of fibrillation towards formation of toxic protofibrils (Buell et al., 2014). A more recent dissection of the effect of pH on fibrillation has demonstrated that due to the hyperdynamic nature of α-synuclein surface topology, multiple polymorphs of α-synuclein result, each with a different propensity of fibrillation (Frey et al., 2024). The role of the side chain charges in governing the pH susceptibility has also been demonstrated by β-synuclein which develops fibrils at pH 5.8 but not at pH 7.3. As stated in the earlier section, unlike α-synuclein, β-synuclein has a shorter NAC region to readily undergo fibrillation. It was initially thought that the presence of a particular histidine residue at the 63rd position with a pK_a of around 6.4 probably serves a critical role in mediating this pH dependent switch of the protein. However, substitution of this histidine with an aspartate residue did not alter the mutant β-synuclein’s behaviour as compared to the wild type with respect to this pH dependent conformational change (Moriarty et al., 2017). Therefore, a more plausible explanation is that at pH 5.8, there are probably enough protons in the solution to shield the significant number of negative charges at the C terminus; however, at pH 7.3, the proton concentration in the physicochemical microenvironment probably falls short to negate these charges. In a LLPS experiment carried out at pH 7.4, α-synuclein in presence of equimolar concentrations of β-synuclein showed a significant increase in turbidity as compared to α-synuclein alone; the corresponding sedimentation experiment indicated that the α/β condensate precipitated readily whereas the β -synuclein was left out in the supernatant justifying β-synuclein’s inability to aggregate at neutral pH (Li et al., 2024).

α-synuclein recovered from Lewy bodies of patients affected with sporadic Parkinson’s disease and LBD as well as from glial cytoplasmic inclusion bodies in oligodendrocytes of patients affected with Multiple System Atrophy has been found to be heavily phosphorylated in the 129th serine residue of the C terminal domain (Ghanem et al., 2022). In contrast, in the brains of healthy adults, only 4% of α-synuclein is constitutively phosphorylated (Fujiwara et al., 2002; Hasegawa et al., 2002). Another specific phosphorylation event at S87 found in the brain of patients with synucleinopathies, and the only one at the NAC region has been found to decrease the protein’s membrane binding affinity and also reduces its fibrillation potential (Paleologou et al., 2010). Several important cellular kinases including the G-protein-coupled receptor kinases (GRKs) (Pronin et al., 2000), the casein kinase II (Sano et al., 2021), polo-like kinases (Inglis et al., 2009), and the leucine-rich repeat kinase 2 (LRRK2) (Greggio et al., 2011) mediate phosphorylation of α-synuclein. This suggests a significant role of phosphorylation in regulating fibril formation of α-synuclein. However, contrasting evidences suggest that the timing of phosphorylation is crucial in governing both the initiation nucleation and fibrillation process. Ghanem and coworkers found that efficiency of phosphorylation at S129 increases manifold when protein aggregation has already been initiated (Ghanem et al., 2022). Using state-of-the-art real time quaking induced conversion (Atarashi et al., 2011) of brain homogenates of PD and DLB patients, the authors found that S129 phosphorylation effectively inhibited subsequent fibril formation as well as toxicity associated with α-synuclein. Therefore, it may be safely said that althoughphosphorylation has been demonstrated to augment the fibrillation process; when it occurs at S129 after initiation of the protein fibrillation, both the aggregation propensity as well as resultant cytotoxicity is decreased (Ghanem et al., 2022). A recent MD simulation study has revealed that phosphorylation brings down the average Solvent Accessible Surface Area of the protein thus promoting its hydrophobic core mediated aggregation (de Bruyn et al., 2024).

The process of attachment of sugar molecules on lysine and arginine residues of proteins via formation of Schiff’s base is referred to as glycation. It occurs obligatorily due to the abundance of carbohydrates as metabolic intermediates; the accumulation of sugar molecules in specific physiological milieus such as the serum, observed in case of diabetic and prediabetic individuals leads to the formation of non-native protein-sugar adducts more popularly known as Advanced Glycated End Products (Rungratanawanich et al., 2021). Many of the AGE adducts of proteins which are apparently non-amyloidogenic, such as serum albumins, show amyloid like properties (Das et al., 2020; Prosad Banik, 2023). Glycation of α-synuclein is mostly concentrated in the N terminus due to the abundance of lysine residues and affects its membrane binding, thus leading to accumulation of toxic oligomers (Vicente Miranda et al., 2017). Additionally, glycation also interferes with the growing end of α-synuclein fibrillation but does not affect the nucleation step (Farzadfard et al., 2022b). Therefore, it can have a toxic effect on α-synuclein fibrillation by increasing the number of oligomeric species and protofibrils rather than full length fibrils (Kumari et al., 2022). This unique behaviour of glycated proteins also explains the strong clinical correlation between chronic hyperglycemia and susceptibility to neurodegenerative disorders.

Brain regions affected by neurodegenerative diseases such as PD, Alzheimer’s disease (AD), Amyotrophic Lateral Sclerosis (ALS) and Mass Spectrometry (MS) are found to contain significantly higher concentrations of metal ions such as zinc, iron and copper (Gardner et al., 2017; Miller et al., 2006). The coincidence of cognitive ailments with occupational exposure to these metal ions have been noted on several occasions suggesting a definitive role of the metal ions in disease pathology (Gorell et al., 1997).

α-synuclein fibrillation is strongly affected by metal ions like zinc, copper, iron and calcium (Atarod et al., 2022; Chen et al., 2019). Interestingly, the effect has been significant only in case of divalent/trivalent metal cations (Uversky et al., 2001). Other observations of a high Ferric/ferrous ratio (Zhao et al., 2023) and abundance of oxidised glutathione (Paik et al., 2003) at the SN microenvironment also support the hypothesis that α-synuclein fibrillation is conducive under oxidising conditions. Ferric ions have been shown to bind to a conserved His50 residue in molar equivalents which provides the coordination site for the metal ion. Bound ferric ion decreases lag time and increases rate of fibril formation. However, as reiterated many times in this review, the mechanism of α-synuclein fibrillation is as complex as it can get. Abundance of ferric ions actually halts fibrillation, by inducing formation of spherical oligomers instead of fibrils (Zhao et al., 2023). Probably, the most intriguing effect of metal ions on fibrillation of α-synuclein has been observed with respect to zinc. Unlike the effect of iron, zinc ions are generally known to augment fibrillation. Accumulation of zinc in intracellular milieu has been found to be associated with the death of dopaminergic neurons whereas chelation of zinc reverses the effect (Tamano et al., 2019). However, low concentrations of zinc are actually required by other interacting proteins of α-synuclein to prevent fibrillation (Al-Harthi et al., 2022).

In nutrition, any chemical compound that is required in minute quantity (ranges between 1 and 100 mg/day in adults or constitutes 0%–1% of total body weight) is considered as a “trace element” (Stjernswärd et al., 1996). Zinc is an essential trace element which makes up almost 1.5–2.5 gm of human body weight. Following absorption in small intestine, dietary zinc remains stored in skeletal muscles (60%), bones (30%), skin and liver (5%) and the rest is distributed in different organs like brain, heart, pancreas, and kidney or in mammary glands through blood circulation. In blood, zinc either remains bound with albumins which is exchangeable (75%–85%) or with α-2-macroglobulin which is the non-exchangeable form of serum zinc (15%–20%). Urinary or gastrointestinal excretion of zinc occurs depending on its status in body (Willekens and Runnels, 2022). Zinc, the second most copious trace element after iron in humans has crucial structural, catalytic and regulatory roles as it serves as vital component of more than 2,500 proteins encompassing transcriptions factors and over 100 of enzymes (Kiouri et al., 2023). The spectrum of physiological intervention of zinc is as broad as it gets and includes gene expression and regulation, cell signalling, cell proliferation, apoptosis, DNA metabolism and structural stabilization of chromatin (like sperm chromatin and its decondensation as needed), immune responses, defensive action against oxidants and many other vital physiological processes for sustenance of body homeostasis (Björndahl and Kvist, 2014; Costa et al., 2023) (Table 1).

Zinc serves as co-factor for enzymes involved in defence against oxidative stress. This micro element hinders the activity of pro-oxidant NADPH oxidase, thus maintaining structural stability of the lipid bilayer (Li et al., 2016). Metallothionein (MT) is a cysteine rich protein in cell, which protects from zinc induced toxicity by sequestering the metal. Metallothionine-zinc complex has ROS sequestering activity as it captures and neutralizes the free radicals using cysteine-sulphur ligands; along with its anti-inflammatory and neuroprotective effects (Rodríguez-Menéndez et al., 2018), zinc is also a structural component of the enzyme superoxide dismutase (SOD), found in abundance in the cytoplasm and also a significant enzyme in endogenous antioxidant machineries. SOD contains both copper and zinc and helps to neutralize superoxide radicals thus bringing down the toxicity caused by ROS (Cruz and Soares, 2011). Zinc salts have been also found to be effective in offering protection against lipid peroxidation (Bagchi et al., 1998) by quenching ROS (Bagchi et al., 1997) and also regulates the expression of another cellular antioxidant defence, i.e., glutamate and cysteine ligase, which plays pivotal role in de novo synthesis of glutathione. Therefore, zinc can participate directly in ROS scavenging by glutathione or indirectly by acting as cofactor for the enzyme glutathione peroxidise. Ha et al. has shown that concentration of cellular glutathione can be upregulated by in vitro administration of regulated amount of zinc in epithelial pigment cells of human retina (Tan et al., 2013). Metal Transcription Factor-1 (MTF-1) is dependent on zinc. When the zinc concentration in body rises, it expresses gene for metallothionine and zinc transporter-1 and maintains zinc homeostasis and avert excess metal induced oxidative stress (Günther et al., 2012). MTF-1 also promotes the expression of selenoprotein-1 which binds glutathione and takes part in ROS scavenging. The inertness of zinc towards catalysis of free radical generation reactions additionally boosts its antioxidative efficacy (Oteiza, 2012). Zinc can bind with either thiol or a sulfhydryl group in proteins thus hindering the intra-molecular formation of disulfide (Choi et al., 2018). Zinc alleviates oxidative stress as an anti-inflammatory element by taking part in various molecular signalling pathways which increases the expression of anti-inflammatory protein A20 (otherwise known as TNFAIP3) and downregulates the expression of the pro-inflammatory NF-kB (nuclear factor κB) (Prasad et al., 2010). Zinc has also shown potent effect in attenuation of pro-inflammatory cytokines including Interleukin-6, Tumour Necrosis Factor α (TNF-α), and C-Reactive protein and stimulation of the molecular signalling pathway leading to expression of the A20 protein (Marreiro et al., 2017). Zinc also upregulates the expression of Nrf2, a chief mediator of antioxidant machinery (Olechnowicz et al., 2018).

Zinc has a crucial role in the maintenance of intracellular calcium concentration. Its deficiency causes calcium build-up promoting the release of substance P, a neuropeptide involved in immune response and inflammation resulting in oxidative stress (Weglicki et al., 2011). Deficiency of this micro-element also leads to activation of NADPH oxidase, ROS and reactive nitrogen species (Lee et al., 2021). As zinc is involved in synthesis of the insulin along with its storage and release, it plays a significant role in blood glucose homeostasis with implications in type 2 diabetes, obesity, cardiovascular diseases, altered lipid metabolism and metabolic syndromes (Motamed et al., 2013).

The role of zinc is integral in neuronal health, especially for carrying out routine physiological processes of the Central Nervous System (Li et al., 2023) (Table 2). The hippocampus, amygdala and cortex are the major storage sites of the zinc and a subtly regulated flux of zinc from these regions are vital for carrying out critical functions of the brain including cognition, memory and emotional stability. Scientific evidences have unanimously established the role of zinc ions in modulating glutaminergic synapse transmission (Bertoni-Freddari et al., 2008). The same has been extensively reviewed by Sikora et al. (2020). Physiological concentrations of zinc range between 10 and 50 µM in and around the vicinity of the Substantia nigra (SN) which is typically much higher than that required elsewhere in the body and justifies the diverse integral roles of the metal in the brain. Further rise in concentration of the metal is often met with severe cognitive ailments (Sabouri et al., 2024; Sandstead et al., 2000). Therefore, it is equally essential to control the level of zinc in these cellular micro-niches. ATP13A2 is a P type ATPase generally found in the membranes of acidic vesicles in neurons which, along with the other two more common class of Zinc transporters belonging to the ZnT (SLC30) and ZIP (SLC39) families, is entrusted to serve exactly this role (van Veen et al., 2014). Overexpression of a ATP13A2 orthologue in yeast was shown to impart zinc resistance (Kong et al., 2014); on a different note, an individual with a mutant of ATP13A2 was affected with Kufor-Rakeb syndrome, a disease with shared features of Parkinsonism (van Veen et al., 2014). These observations were subsequently confirmed in a mouse PD model where zinc was shown to increase α-synuclein aggregation; overexpression of ATP13A2 neutralized the effect of zinc thus alleviating α-synuclein pathology whereas its knockout significantly aggravated fibrillation (Gao et al., 2022). It has been confirmed using many independent approaches that the presence of zinc results in increased content of beta structure of α-synuclein.

In contrast to toxic effects of zinc reported by most studies, the metal has been shown to mitigate oxidative stress (Méndez-Álvarez et al., 2002) as well as prevent toxicity of dopaminergic neurons (Ajjimaporn et al., 2008). The physiological role and requirement of zinc is evident from the fact that zinc is stored in within synaptic vesicles at the terminals of glutaminergic neurons especially in the forebrain region. It is released along with glutamate and plays an integral role in mediating transmission of synapse through NMDA and GABA receptors (Anderson et al., 2017; Vergnano et al., 2014). Pathological consequences start occurring when an excess of synaptic zinc leaks in to the post synaptic neurons through the ion channels (Sensi et al., 2011).

As stated previously, many divalent metal cations have the potential to augment the fibrillation of α-synuclein by a common mechanism of shielding the negative charge density on the protein. The effect is more prominent in case of the trivalent lanthanides which can additionally bind to the central NAC region and accelerate fibril formation (Bai et al., 2016). A closer look reveals that different cations vary with regards to their binding affinity to α-synuclein and subsequent effect on the kinetics of nucleation and elongation.

Native nano-electrospray ionisation and ion mobility-mass spectrometry (nESI(-IM)-MS) can differentiate conformations of the protein based on their charged states, relying on the principle that denatured proteins are more extended and therefore are comparatively more protonated than native compact states with a lower Solvent Accessible Surface Area. Therefore, denatured conformations of proteins can readily be differentiated from the native conformers based on their status of protonation. Due to the inherently Intrinsically Disordered nature of α-synuclein, an ensemble of native state monomers of the proteins can be distinguished with a charge ranging between +5 and +18. Using this technique, the binding of zinc to the protein was probed quantitatively and at equimolar ratios of metal ion: protein, zinc was found to exhibit significant binding to α-synuclein along with sodium, copper and lanthanum (Moons et al., 2020). In a subsequent study conducted to understand the effect of zinc on α-synuclein fibrillation, the metal ion was found to decrease the lag time of aggregation (t_lag) with a simultaneous increase in apparent rate constant (k_app). However, the fibrils formed were significantly shorter than those formed in the presence of copper, calcium and magnesium (Atarod et al., 2022). FTIR analysis of amide I vibrational spectrum also confirmed that in presence zinc, additional regions of the protein take up the beta pleated sheet conformation (Li et al., 2022). A combination of single residue resolution level NMR spectroscopy and truncated versions of α-synuclein mutants have revealed the binding sites of zinc. In presence of 100 µM zinc, the most significant binding site has been deciphered to be the Asp121 and Asp135 in the C terminus; however, no additional binding interaction was noted due to the presence of other negatively charged residues in the vicinity including Glu130, Glu131, Glu137 and Glu139. This clearly indicated that the affinity of zinc for the C terminal domain was not solely governed by only electrostatic interactions but was primarily a result of the unique conformation of the protein centred around the Asp121 residue. Binding at a threefold excess molar concentration of zinc revealed the second binding site of the metal at His50 in the N terminus but no additional affinity was noted for the C terminus. The trend of zinc binding to α-synuclein is in unison with the observed effects; at low physiological concentrations, zinc serves a twofold function of both neutralizing the negative charge density at the C terminal of α-synuclein and assists other chaperones which work in a concerted manner to prevent aggregation of α-synuclein, whereas at higher concentrations, zinc promotes fibrillation by binding to His50 at the N terminus and lowering its pK_a, much like that observed in case of iron (Valiente-Gabioud et al., 2012). Given the fact that physiological concentrations of zinc seldom exceed 50 μM at the substantia nigra micromilieu, it is apparent that in vivo, zinc induced α-synuclein toxicity is not an issue of concern. Interestingly enough, the shorter fibrils formed in presence of zinc were not cytotoxic unlike the longer ones formed in presence of the other cations (Atarod et al., 2022). This observation came as a surprise given the fact that oligomers of α-synuclein impart more toxic effects than the fully mature fibrils due to their stronger ability to induce pore formation in the membrane (Zhang et al., 2013). One of the several possible answers to explain this may lie in the fact that zinc ions serve as metal activators of certain chaperonin proteins which bind and neutralize synuclein oligomers (Al-Harthi et al., 2022). Another clue to explain the apparent harmlessness of zinc induced synuclein oligomers comes from a related study in tau proteins implicated in disease pathology of AD. Zinc induced oligomerization of tau occurred in a temperature dependent manner without the necessity of additional cofactors such as heparin. However, these oligomers did not eventually develop into amyloid fibrils and were dissociated on lowering the temperature (Roman et al., 2019) indicating the reversibility in the formation of the oligomers. The results if extrapolatable in case of α-synuclein will imply that oligomers of α-synuclein formed in the presence of zinc are also transient unstable entities not capable of inducing pores in lipid membranes. A similar study conducted by Kim et al. much earlier, had indicated that heat induced aggregation of recombinant α-synuclein was facilitated in presence of zinc (Kim et al., 2000). A possible explanation of these findings is the fact that increasing temperature augments hydrophobic interaction at the NAC region of α-synuclein; however, at the same time temperature lowers the pKa of the acidic side chains in the C terminus thus facilitating the opposite effect of keeping fibrillation at bay. Zinc masks these negative charges even at elevated temperatures to promote fibrillation. As soon as the temperature drops, the hydrophobicity driven factor is neutralized, rendering the proteins into random aggregates which lack a structured fibrillar component and therefore incapable of effecting membrane damage. Zinc is also known to induce liquid-liquid phase separation of proteins implicated in neurodegenerative disorders (Singh et al., 2020; Yatoui et al., 2022). The metal was shown to induce phase separation in tau, the amyloid like protein associated with AD pathology and shift the equilibrium phase boundary of LLPS towards the region of lower protein concentration (Singh et al., 2020). However, there has been little or no study on the effect of zinc in the LLPS of α-synuclein. The dual role of zinc on aggregation and fibrillation of α-synuclein has been summarised in Figure 3.

Apart from zinc, other physiologically significant non-metal micronutrients such as iodine and selenium have also been demonstrated to possess anti-amyloidogenic properties. Iodine is an acclaimed chaotropic agent capable of modulating the conformational stability of a plethora of proteins by reorienting the water structure around the protein’s microenvironment. Iodine has also been shown to interact with amyloid fibrils, amyloid polymorphs and form complex with mature insulin amyloid fibrils (Hiramatsu et al., 2020; Takekiyo et al., 2022). By using a combination of biophysical techniques, Taketiyo et al. demonstrated that ethylammonium iodide (EAI) can inhibit the formation of α-synuclein amyloid aggregate via interaction between I₃ ⁻ ions and Lys residues in the N-terminal domain of the protein (Takekiyo et al., 2022).

Selenium and selenoproteins have also been implicated in modulating α-synuclein fibrillation. Free Selenium can interact with α-Synuclein and induce the formation of protein aggregates (Maass et al., 2020). SelS and SelP are the two major selenoproteins of the body implicated in antioxidant defence and selenium transport respectively (Lu and Holmgren, 2009). Additionally, SelS has been described as the regulator of the protein aggregation; it probably acts by mediating transcriptional downregulation of aggregation prone proteins; however, the exact mechanism is yet to be deciphered. A recent study on PD patients has suggested that serum concentrations of Se and SelP can potentially be used as a biomarker for monitoring α-Synuclein aggregation (Salaramoli et al., 2024).

There are currently very few safe therapeutic strategies to directly degrade and clear amyloid fibrils. One of these is the bacterial enzyme nattokinase which is a serine protease capable of efficiently removing fibrinolytic clots (Ni et al., 2023). A new enzyme from earthworm termed lumbrokinase has been reported by Girigoswami et al. with the potential to degrade fibrin clots (Metkar et al., 2017). The polysaccharide carrageenan isolated from certain species of red algae possess the potential to degrade amyloid fibrils (Makshakova et al., 2023). Girigoswami et al. has recently developed a nanoformulated carrageenan with enhanced degradation potential as compared to the conventional one (Udayakumar et al., 2024).

The ability of zinc to arrest fibrillation by assisting chaperones and to induce formation of non-toxic protofibrils and oligomers have led to the prospects for the development of zinc oxide nanoparticle-based biomarkers. In a study carried out by Asthana et al., zinc oxide nanoparticles showed significant binding affinity with α-synuclein fibrils via a) enthalpy driven interaction with the amphipathic “KA: TKE/QGV” repeats and b) enthalpy-driven interaction with the central NAC region as well as the C terminus; this led to arrest of subsequent fibrillation (Asthana et al., 2020). Based on this observation, a zinc oxide nanoparticle coated aluminium microelectrode has been designed as an efficient tool for early detection of α-synuclein fibrillation (Adam et al., 2021a). A related study based on Surface Enhanced Raman Spectroscopy reported that zinc oxide nanoparticles can effectively bind and sequester α-synuclein oligomers provided they have not been exposed to physiological temperatures (37°C) which would lead to irreversible formation of amyloid fibrils (Slekiene et al., 2022). Further refinement in this regard has been achieved with surface moderated zinc oxide nanoparticle corona which is able to effectively remove flocs or amorphous aggregates of α-synuclein (Jena et al., 2025). In order to maximize the surface area for enhanced sensitivity in detection of amyloids, a zinc nanoflower-based on nano-silver thin film has been developed (Akhtar et al., 2017) and subsequently upgraded to degrade the amyloid fibrils (Girigoswami et al., 2019). As discussed before, Thioflavin-T positive glycated proteins in the bloodstream of diabetic or prediabetic patients can elicit amyloid like complicacies. Similar studies exploring the effect of zinc nanoparticles on glycated proteins need to be conducted to determine whether zinc-oxide nanoparticles can also trap these protein-sugar adducts before they develop into toxic amyloids capable of developing cognitive ailments and other complicacies.