Exploring Protein Misfolding and Aggregate Pathology in Neurodegenerative Diseases: From Molecular Mechanisms to Clinical Interventions

Abstract

Neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease are characterized by progressive neuronal loss, driven mainly by the misfolding, aggregation, and accumulation of each disease’s specific proteins. These pathogenic aggregates, including tau, α-synuclein, TDP-43, and huntingtin, disrupt cellular proteostasis and initiate cascades of neuroinflammation, oxidative stress, mitochondrial dysfunction, and synaptic failure. While protein aggregation has been a long-recognized hallmark of these disorders, growing evidence points towards a more complex interplay of initial molecular pathways with defects in RNA processing, stress granule pathology, and cell-type-specific vulnerability. Notably, such events may manifest differentially with respect to sex and are further modulated by age-related loss of the protein quality control processes like the ubiquitin–proteasome pathway, autophagy–lysosome pathway, and molecular chaperones. This review synthesizes current insights into the structural and functional dynamics of protein aggregation and its significance for neuronal well-being. It highlights the role of post-translational modifications, prion-like transmission, and aggregation kinetics in the regulation of toxicity. The review further discusses promising therapeutic strategies centered on restoring proteostasis, including small molecules that inhibit aggregation, protein clearance pathway enhancers, immunotherapy, antioxidant therapy, and diagnostic prospects such as the identification of reliable molecular signatures in bodily fluids that can reflect pathological changes even before clinical symptoms emerge. Advancements in single-cell transcriptomics and multi-omics platforms, which are changing our understanding of disease onset and progression and opening avenues for precision medicine and personalized treatments, were also discussed. Ultimately, deciphering the molecular logic that distinguishes physiological from pathological protein assemblies and understanding how cellular systems fail to adapt under stress will be key to the development of effective, disease-modifying therapies for these debilitating disorders.

1. Background

Neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Huntington’s disease (HD) are a group of progressive disorders characterized by the progressive loss of structure and function of neurons [1]. While both diseases have distinct clinical presentations, their common pathological characteristic is the misfolding of abnormal proteins in the central nervous system and the aggregation of such misfolded proteins, which disrupts neuronal function and integrity [2].

Misfolding and protein aggregation are hallmark features in a broad range of neurodegenerative diseases. Pathogenic proteins implicated include amyloid-β, tau, α-synuclein, TDP-43, SOD1, mutant huntingtin (polyQ-expanded HTT), and FUS, which form cytotoxic oligomers in various cellular compartments [3]. These misfolded proteins are likely to adopt β-sheet-rich conformations and accumulate into oligomers, protofibrils, and ultimately insoluble fibrils that contribute to the formation of extracellular amyloid plaques, intracellular neurofibrillary tangles, Lewy bodies, and other inclusion bodies [4,5]. These aggregates disrupt cellular homeostasis by disrupting synaptic signaling, intracellular transport, and sequestering vital cellular proteins for transcription, translation, and protein quality control. Their misfolding is induced by conditions including genetic mutation, abnormal post-translational modifications, and persistent cellular stress [6].

Processes like conformational strain diversity, seeding-polymerization, and liquid–liquid phase separation drive aggregation and can facilitate prion-like spreading of pathology between neurons [7]. Cross-seeding between various misfolded proteins also links clinically heterogeneous neurodegenerative diseases. Cellular impacts of aggregation are extended, including from impaired synaptic integrity and mitochondrial dysfunction to oxidative damage, RNA dysregulation (e.g., from TDP-43), and disruption of protein quality control systems such as the ubiquitin–proteasome system and autophagy [7,8]. Proteins such as tau may be recalcitrant to degradation, contributing to their neurotoxicity [9]. In addition, microglial-mediated neuroinflammation and autoantibody responses drive the facilitation of greater neuronal injury and disease progression. Cellular stress pathways, including the unfolded protein response (UPR) induced by endoplasmic reticulum stress, attempt to restore protein folding homeostasis but, in doing so, can drive cell dysfunction if activated chronically [10]. Such molecular mechanisms ultimately drive progressive loss of neurons, brain atrophy, and clinical signs that define neurodegenerative disorders. Consequently, understanding the molecular mechanisms of protein misfolding offers promising avenues for early diagnosis and treatment.

Early and accurate diagnosis of neurodegenerative diseases is crucial for effective intervention and monitoring disease progression. Since misfolded and aggregated proteins are central to the pathology of many neurodegenerative diseases, they represent valuable biomarkers for diagnosis and tracking of disease state [11]. Hence, efforts targeted towards the discovery and development of biomarkers have focused on identifying reliable molecular signatures in bodily fluids and applying advanced imaging technologies that can reflect pathological changes even before clinical symptoms emerge. Furthermore, therapeutic approaches targeting the fundamental molecular basis of protein misfolding and aggregation are being intensively explored. These approaches include enhancing protein clearance pathways, disrupting pathologic protein interactions, and modulating stress response pathways. The use of single-cell transcriptomic techniques to gain insights into cell-type-specific vulnerabilities is also being explored, aimed towards more focused, personalized therapies. Hence, this review provides an overview of the current understanding of protein misfolding in neurodegenerative diseases, including the identification and characterization of key misfolded proteins, their pathogenic mechanisms, the novel therapeutic strategies aimed at restoring protein homeostasis and neuronal function, and future directions for research and clinical applications.

2. Overview of Protein Misfolding

Protein misfolding is central to the molecular pathogenesis of many neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis (ALS) [12]. Proteins are naturally complex macromolecules whose correct three-dimensional structure dictates their biological activity, and folding is fundamentally determined by data from the protein’s primary amino acid sequence [13]. This set of instructions defines the possible conformations of a polypeptide while it folds into its most energy-restricted state. Protein folding, however, is not a linear, error-free process but a dynamic trial-and-error process through which the polypeptide attempts various conformations before settling into its native shape [14]. These thermodynamic forces of hydrophobic interactions and hydrogen bonding drive this process, and molecular chaperones play roles in correct folding and the prevention of inappropriate interactions that result in misfolding, aggregation, and cytotoxicity.

Despite such a coordinated process, errors at the time of folding are relatively common. Under normal physiological conditions, the cell’s quality control mechanisms, such as molecular chaperones, the ubiquitin–proteasome system, and autophagy pathways, all work together to recognize, refold, or degrade any misfolded proteins [15]. This is to prevent compromised or even potentially toxic proteins from accumulating and their constituent amino acids from being recycled to be utilized in new protein synthesis. This balance, however, can be disrupted. Various causes, such as inherited or acquired mutations in the protein coding sequence, contribute to the risk of misfolding by influencing the native folding path or destabilizing the native folding. Equally, extrinsic and intrinsic cellular stressors like oxidative stress, heat shock, and disruption of cellular homeostasis can overwhelm the proteostasis network and folding machinery and lead to an overload of misfolded proteins [16,17]. When the cell’s protective mechanisms are unable to repair these mistakes, misfolded proteins can accumulate and act as pathological seeds. These pathological seeds can seed a nucleation-dependent polymerization event by which small oligomeric assemblies are initially formed. These soluble prefibrillar oligomers (commonly referred to as PAOs, or proto-amyloid oligomers) are believed to be particularly toxic since they are capable of disrupting cellular membranes and interfering with essential cellular functions [14]. These oligomers, with time, may form large, well-ordered fibrillar aggregates with high β-sheet content. Fibrils are formed by growth, typically an autocatalytic process that produces exponential addition and the formation of large proteinaceous deposits that are visible as amyloid plaques or inclusions by the electron microscope. These aggregates are characteristic of most neurodegenerative diseases.

In normal physiological conditions, enzymatic post-translational modifications (PTMs) are highly controlled processes that optimize the function, localization, and interactions of a protein [16]. However, under cellular stress or in disease states, this control is eliminated, often by dysregulation of the upstream signaling pathways that control these modification enzymes. With loss of balance, aberrant or excessive PTMs may ensue, altering the conformation of a protein and increasing its propensity to misfold and aggregate [15]. Other enzymatic PTMs have also been involved in protein misfolding and aggregation, including proteolytic cleavage, glycosylation, sulfation, phosphorylation, and ubiquitination. Among these, proteolytic cleavage is of particular significance to neurodegenerative disease [14]. The production of amyloid-β peptides in Alzheimer’s disease is one such highly characterized instance of how proteolytic processing, when disrupted, results in aggregation. The amyloid precursor protein (APP) is sequentially cleaved by β-secretase and γ-secretase enzymes. The final cleavage by the γ-secretase results in amyloid-β peptides of varying sizes, namely Aβ38, Aβ40, and Aβ42 [18]. Among these, Aβ40 is the most abundant isoform found in the brain; however, abundance alone does not equate to pathogenicity [19]. The Aβ42 isoform, although produced in smaller quantities, is most prone to misfolding and aggregation, resulting in cytotoxic prefibrillar oligomers and fibrils that accumulate in the brain [20,21]. This explains why researchers have primarily focused on Aβ42 in the context of amyloid toxicity and Alzheimer’s disease progression. Although structural characteristics of protein sequences have evolved to reduce the likelihood of misfolding under normal conditions, proteolytic cleavage can remove stabilizing domains or expose hydrophobic surfaces, thereby compromising this evolutionary protection. When such cleavages occur in a deregulated or pathological manner, they can promote the conversion of an otherwise soluble protein to one that seeds disease-causing aggregates [14].

3. Mechanisms of Protein Misfolding in Neurodegenerative Diseases

The formation of protein aggregates typically results from partially unfolded or destabilized conformations of the protein. Partially unfolded states expose the otherwise buried hydrophobic regions to the solvent, enabling favorable intermolecular interactions to facilitate the initial steps of aggregation [22]. However, it is not straightforward to directly access structural information on these intermediate states because they are transient and difficult to isolate experimentally. Transthyretin, a tetrameric protein that must first dissociate into monomers before amyloid fibril elongation can occur, is one such highly documented example [23]. Generally, stabilizers of the native structure of a protein, such as good pH, proper ionic conditions, or molecular chaperones, slow down aggregation, while destabilizers of the native structure promote it. For instance, β-lactoglobulin has shown that its aggregative tendency is at a maximum in the presence of urea concentrations that partially denature the protein, demonstrating how chemical denaturants can induce aggregation by shifting the folding equilibrium [24].

Partial unfolding is induced by various factors, such as pH change, high temperature, chemical modification, genetic mutation, and oxidative stress [13]. Importantly, even intrinsically disordered or natively unfolded proteins like α-synuclein and amyloid-β can readily aggregate under certain conditions like low pH or high temperature [25]. Localized unfolding of specific regions of globular proteins could be enough to trigger aggregation, even if the bulk of the protein is in a near-native conformation. In addition to these factors, metal ions such as iron (Fe), zinc (Zn), and copper (Cu) are also critical modulators of protein folding and misfolding. These transition metals have been found to directly interact with amino acid side chains, especially histidine, cysteine, and aspartate residues, thereby influencing protein conformation and stability [26,27]. Under physiological concentration, these metal ions can stabilize folded states or regulate normal protein function, highlighting their dual role as both essential cofactors and potential aggregation triggers [28]. Iron, for example, is one of the most abundant redox-active metals found in the body that can act as cofactors for multiple proteins and enzymes [29]. However, its connection between α-synuclein was clearly documented in the study by Chen et al. [30]. They stated that iron plays a role in the synthesis of α-synuclein, but iron deposition increases oxidative stress generation, which changes the structural configuration of α-synuclein during synthesis, leading to accumulation and Parkinson’s disease. Also, Zn is a micronutrient for which very little concentration is required to prevent neurotoxic effects. In the case of Alzheimer’s disease, a balanced concentration of Zn and Cu in the brain plays a protective role in dissociating the aggregates. The study by Ryu et al. [31] provided a detailed insight into the defensive function of Zn and Cu ions in Fe-induced aggregates. Results obtained from various photophysical tools in the study imply that the concentration of multiple metal ions, such as Fe, Zn, and Cu, decides the formation or dissociation of ß-amyloid aggregates.

Since amyloid diseases are still prevalent and largely untreatable, understanding how partially unfolded intermediates direct protein self-assembly and amyloid formation is critical. These processes are complex and take many paths but share mechanisms and structural changes that determine the initiation and progression of aggregate formation [13]. Some of these mechanisms are discussed in the following section.

3.1. Self-Assembly of Native Monomeric Protein

Even in their completely folded native form, monomeric proteins can have a native propensity to self-aggregate in some environments [13]. The molecular surface of these monomers often has regions that are inherently self-complementary, and which can thus associate by non-covalent forces and form small, reversible oligomers. With further local concentration of protein, these early oligomers can grow in size by further intermolecular association. Though such oligomerization is generally reversible initially, extended association or alteration of the surrounding environment may favor conversion to more stable, irreversible assemblies. This conversion is commonly enabled by the creation of covalent cross-links, e.g., disulfide bonds, which permanently fix the assemblies into more enduring structures. Insulin, for example, is a therapeutic protein susceptible to reversible oligomer formation under physiological conditions [32]. In neurodegenerative illness, this is especially relevant for proteins like α-synuclein and amyloid-β (Aβ). For instance, native α-synuclein can spontaneously self-assemble into small oligomers under physiological conditions [33]. These initial-stage oligomers are thought to be the seeds from which ultimately grow the Lewy bodies of Parkinson’s disease [34]. Similarly, Aβ peptides released from amyloid precursor protein (APP) are capable of self-aggregating in the monomeric form to create small oligomers that increase in size sequentially to produce the amyloid plaques found in Alzheimer’s disease brains [34,35]. In the very distant past, as early as 1952, it was hypothesized that amyloid aggregate formation is not an absolute requirement for total protein unfolding. Instead, globular proteins aggregate as the monomers make contact in side-by-side or end-to-end conformations, with ordered self-assembly being feasible even if the native fold is largely maintained [36].

3.2. Aggregation of Conformationally Modified Monomeric Proteins

Native monomers can potentially self-associate, but this potential is considerably enhanced if the monomer is caused to adopt a conformational change that positions it in a non-native or partially unfolded state [13]. Such a conformation exposes hydrophobic or aggregation-prone residues that greatly increase the likelihood of stable aggregation. The initial step in this process is therefore a structural departure from the native fold, a variation that distinguishes this mechanism from aggregation in fully native monomers. In neurons, multiple stresses such as heat shock, oxidative stress, or mechanical stress, can trigger destabilization of the native fold to push proteins toward partially unfolded conformations that are prone to aggregation [37]. Under normal circumstances, tau, for example, has a native conformation stabilizing microtubules. But if tau becomes hyperphosphorylated or partially unfolded upon cellular stress or aging, it adopts a form that readily adopts paired helical filaments and neurofibrillary tangles, which are the hallmarks of Alzheimer’s disease and other tauopathies [9]. Likewise, mutant huntingtin proteins with extended polyglutamine tracts have a higher probability of being partially unfolded, with the exposed sticky regions promoting intranuclear inclusion formation in Huntington’s disease [38]. From a therapeutic angle, conditions or additives that can help in stabilizing the native conformation can be useful in minimizing this type of aggregation. For example, chaperone- or small molecule-based therapies are being developed to hold proteins like tau and α-synuclein in their native conformations to avoid the conformational changes that trigger aggregation [39].

3.3. Nucleation and Seeding Process

One of the primary mechanisms of the formation of large, macroscopic protein aggregates in neurodegenerative disorders is the nucleation-dependent pathway, often termed a “nucleation and seeding” process [40]. In this mechanism, native monomers alone generally have no ability to cause fibril formation spontaneously under physiological conditions. Instead, a small aggregate with a critical size (called the “critical nucleus”) must first be created to seed the process [13]. Once this nucleus has been created, it acts as a seed upon which additional monomers are drawn, which adhere to its surface and grow the aggregate into progressively larger fibrillar units [41]. This process typically has a characteristic lag phase, an initial phase during which very little or no aggregated material can be seen, though individual monomers are in a state of supersaturation. After the formation of the nucleus of critical size, the system quickly enters the growth stage, leading to the sudden appearance of big fibrillar or amyloid morphologies. This explains the often unobtrusive beginning of many neurodegenerative disorders, during which toxic oligomers and nuclei are accumulated before wider aggregates become clinically or histologically evident [40].

There are two main types of nucleation: homogeneous nucleation and heterogeneous nucleation. In homogeneous nucleation, the nucleus solely forms from the protein alone in supersaturation conditions. On the other hand, in heterogeneous nucleation, the process of nucleation is activated or accelerated by interaction with foreign particles or surfaces [13]. For instance, impurities such as silica from glass vial leach or metal wear particles from equipment can act as an active nucleation surface [41]. Although more significant in industrial biopharmaceutical manufacturing, the phenomenon is of significance to understanding possible impacts of environmental factors on aggregation in vivo as well. In neurodegenerative illness, nucleation-dependent aggregation is a hallmark of many amyloid-forming proteins. For example, amyloid-β (Aβ) peptides in Alzheimer’s disease, α-synuclein in Parkinson’s disease, islet amyloid polypeptide (IAPP) in type 2 diabetes, and prion proteins in prion diseases all self-assemble by nucleation-dependent mechanisms [13,42]. In each of these conditions, stable nucleus formation by spontaneous self-assembly is rate-limiting, responsible for the lengthy asymptomatic phases of these illnesses.

Furthermore, seeding is a closely related concept of the same importance. If a nucleus or a small fibrillar fragment is introduced into a supersaturated monomer solution, it can bypass the lag phase completely and trigger fibril extension straightaway [13]. These diseases are therefore able to develop so rapidly once aggregation has begun because new aggregates can disassemble, producing multiple seeds that enhance the misfolding process in a self-perpetuating cycle. This is most characteristically described for prion proteins and prion-like disease being seen with tau and α-synuclein, in which seeds of aberrantly folded protein can transmit from cell to cell to induce disease [13,42].

4. The Role of Unfolded Protein Response (UPR) in Protein Misfolding and Neurodegeneration

The endoplasmic reticulum (ER) is a membrane-bound organelle known for its role in secretion and organelle-bound protein production, folding, and modification [10]. Protein synthesis and folding are extremely regulated processes that are vulnerable to disruptions in ER homeostasis, leading to an accumulation of unfolded or misfolded proteins, a condition known as ER stress. Ca²⁺ depletion, hypoxia, altered glycosylation, or viral infection can all contribute to alterations in ER homeostasis [43]. To inhibit the accumulation of unfolded proteins, cells activate a protective signaling network called the unfolded protein response (UPR). Once the UPR detects the accumulation of unfolded proteins, it tries to restore equilibrium by preventing the synthesis of new secretory proteins, encouraging the breakdown of misfolded proteins via mechanisms like ER-associated degradation (ERAD) and lysosomal clearance, and increasing the synthesis of molecular chaperones and foldases that improve the folding ability of the ER [10]. In mammalian cells, the UPR works through three main ER membrane sensors: type I transmembrane protein inositol requiring 1 (IRE1α); eukaryotic initiation factor 2α (eIF2α) kinase (PERK), and activating transcription factor 6 (ATF6) [10,43].

PERK is the first of the three transducers to become activated, followed by IRE1 and ATF6 [44]. Different branches of the transduction pathways for the UPR signal are independently regulated by each transducer. PERK phosphorylates eukaryotic initiation factor 2α (eIF2α) when it is activated, to prevent further increases in the demand for protein folding in the ER and to inhibit cellular protein synthesis by lowering the load of proteins entering the ER. This temporarily inhibits general protein translation within the cell [45,46]. At the same time, specific mRNAs with tiny open reading frames in their 59 untranslated regions, such as activating transcription factor 4 (ATF4), which controls the transcription of UPR target genes, have their translation selectively activated by phosphorylated eIF2α [47]. In ER stress situations, ATF4 and ATF6 work together to restore protein-folding homeostasis. When stress is sensed, ATF6 is directed into transport vesicles and transported out of the ER to the Golgi apparatus, where it is cut by site 1 (S1P) and site 2 (S2P) proteases [46]. This liberates its cytosolic active fragment (ATF6n) that then translocates to the nucleus to enhance the UPR genes that enhance the folding and quality-control capacity of the ER (shown on Figure 1). Conversely, PERK activation leads to phosphorylation of eIF2α that selectively amplifies the translation of ATF4. During mild or transient stress, ATF4 helps cells to adjust by amplifying protective genes. Nevertheless, during severe or sustained ER stress, ATF4 triggers C/EBP homologous protein (CHOP), a crucial pro-apoptotic factor that stimulates death-inducing gene expression [48].

Together, they coordinate transcriptional and translational changes to reduce the burden of misfolded proteins. However, if UPR fails to restore homeostasis due to severe or prolonged stress, the pro-apoptotic signals are triggered, which usually involves kinases like JNK and caspases, ultimately leading to cell death [49,50]. In neurodegenerative diseases, chronic ER stress and persistent UPR activation are common features that contribute to neuronal dysfunction and degeneration, as overwhelmed cells lose their capacity to manage misfolded proteins effectively [10].

4.1. Activation of the UPR via IRE1

There are two predominant models for the IRE1 activation of the UPR as the key ER stress sensor. One hypothesis proposes that unfolded proteins directly bind to the luminal domain of IRE1 [51]. The luminal domain contains structural grooves to selectively bind unfolded protein segments but not correctly folded segments. Binding to misfolded proteins allows for oligomerization of IRE1, which is crucial to activate it [10]. Mutations in these binding grooves or oligomerization interfaces reduce the activity of IRE1 for processing its major RNA substrate, HAC1 mRNA, which is vital for the stimulation of UPR [52]. The second model is the engagement of the ER-resident chaperone BiP (Kar2 in yeast). BiP is bound by IRE1 in its inactive state. Upon accumulation of unfolded proteins, BiP binds selectively to the misfolded proteins and dissociates from IRE1. Released IRE1 molecules form oligomers and activate through trans-autophosphorylation at distinct sites [53,54]. This activation unleashes IRE1’s endoribonuclease function, which specifically splices an intron from HAC1 mRNA (or its mammalian equivalent), producing a mature mRNA that encodes for a transcription factor (Figure 2). This factor, in turn, upregulates chaperones and folding enzymes to help restore ER balance.

Notably, if ER stress is not fixed, active IRE1α also enters pro-apoptotic signaling. Through the activation of proteins like TRAF2 and ASK1, IRE1 signaling cascades into the activation of JNK and p38 kinase, driving the cell toward programmed death [10,50]. In neurons, chronic stress signaling by IRE1 can lead to cell loss in neurodegenerative disease when the protective arm of the UPR does not suppress chronic misfolding.

4.2. PERK Activation and Signaling

Like IRE1, ER stress sensor PERK also shares structural similarities in its luminal domain with a similar mode of activation [10]. In both cases, chaperone BiP is bound to the luminal domain of PERK under normal conditions, deactivating it. With the accumulation of unfolded proteins, BiP dissociates to bind preferentially to the misfolded proteins instead, freeing PERK to oligomerize and undergo trans-autophosphorylation, activating its signaling activity [43]. Although both IRE1 and PERK use this BiP-mediated switch, the domains to which they bind are not the same: in IRE1, the BiP site overlaps directly with the oligomerization domain, but in PERK it does not, which indicates slightly different repression mechanisms [10,55]. Upon activation, PERK phosphorylates eukaryotic initiation factor 2 alpha (eIF2α) (Figure 3). The phosphorylation prevents the normal recycling of eIF2 between its GDP- and GTP-bound forms, which slows general protein translation by disrupting assembly of the ribosomal preinitiation complex. Through the reduction in general protein synthesis, PERK lowers the influx of new proteins into the stressed ER [43,56].

However, not all translations are halted. Phosphorylated eIF2α favors the translation of certain mRNAs, i.e., ATF4 in mammals (GCN4 in yeast) [57]. ATF4 then activates genes for recovery from stress, but also pro-apoptotic pathways during prolonged ER stress. For example, ATF4 activates CHOP, a transcription factor that promotes cell death by inducing pro-apoptotic proteins such as DR5 and TRB3 [58]. ATF3 is induced by ATF4, which can increase GADD34 expression in conjunction with ATF4. GADD34 also forms a complex with protein phosphatase 1 to dephosphorylate eIF2α, providing a feedback response to restore translation when stress resolves [10]. In neurodegeneration, chronic PERK activation and sustained eIF2α phosphorylation can cause reduced protein synthesis required for normal neuronal function, while chronic induction of CHOP increases apoptosis [59]. Both processes result in progressive neuronal loss when the UPR is unresolved.

4.3. ATF6 Signaling

ATF6 is the third route which plays a crucial part in UPR signaling. It is a type II transmembrane protein that is also attached to BiP in its inactive form, just as IRE1 and PERK, and requires BiP dissociation before the activation of the signaling cascade [43]. The transmembrane proteins, like ATF6, are broken down to liberate their cytoplasmic domains, which then enter the nucleus to control gene expression through a process known as regulated intermembrane proteolysis (RIP) [60,61]. The model is the same overall process used by sterol regulatory element-binding proteins (SREBPs) that control cholesterol biosynthesis. Like SREBPs, ATF6 has a cytosol-facing component and a sensor that monitors the ER lumen. Activated ATF6 is transported to the Golgi, where two proteases, site-1 protease (S1P) and site-2 protease (S2P), cleave it sequentially [10]. S1P first cleaves ATF6 at a distinctive recognition sequence to separate its transmembrane domains (Figure 4). S2P then excises the active NH₂-terminal fragment, which translocates to the nucleus to induce genes that enhance the ER’s protein folding capacity (Figure 4). While ATF6 is likely to use the same cleavage pathway as SREBPs, the exact mechanisms of its export to the Golgi (compared to the SREBP escort protein SCAP) are unknown [10].

5. Protein Aggregation: Mechanism and Consequences

Under normal conditions, cells rely on an effective protein quality-control system, which is a combination of molecular chaperones and proteases, to monitor protein folding and repair or degrade misfolded proteins [62]. The system regulates dynamically in response to stress by increasing protective factors that rebuild protein homeostasis. But if more misfolded or degraded proteins are being produced than the cell can refold or destroy, then the balance is lost. This results in the accumulation and formation of aggregates by misfolded proteins. Such failure of quality control can result from a single extreme stress event or from several moderate stress conditions that collectively overwhelm the system in the long term [63,64].

Aggregation is commonly considered a pathological process because it removes functional proteins from circulation and leads to the deposition of misfolded species that can damage cells [62]. One of the most prominent effects is the formation of amyloids, strongly structured protein deposits associated with many devastating diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, systemic amyloidosis, and type 2 diabetes [64]. Notably, not all protein aggregation is harmful. Certain species have evolved functional amyloids, which possess beneficial roles in processes like bacterial biofilm formation or hormone storage in mammals. This suggests that while pathological amyloids are toxic, controlled aggregation could be a normal, targeted cell strategy in certain circumstances [62].

5.1. Definition and Types of Aggregates (Oligomers, Fibrils, Plaques)

Protein aggregates are typically categorized as either soluble aggregates, which are commonly referred to as amyloid oligomers, amorphous aggregates, or amyloid fibrils, which are structures in which the polypeptides are arranged into a cross-β sheet [3]. Fibrils are among the best-characterized aggregates. They are filamentous structures typically 7–13 nm in thickness, consisting of several protofilaments in parallel [5]. The protofilaments, about 2–7 nm in width, share a common structural motif called the cross-β architecture, where the β-strands are perpendicular to the fibril axis and β-sheets stacked parallel to the axis [65]. This highly ordered structure gives amyloid fibrils their remarkable stability. The formation of fibrils follows a nucleation and growth process [66]. Initially, soluble monomers undergo primary nucleation to form small nuclei, which can be single monomers, dimers, or oligomers depending on the protein and conditions [4]. These nuclei then act as seeds that accumulate more monomers to become fully formed fibrils. When a critical quantity of fibrils is reached, secondary nucleation occurs on fibril surfaces, which generates new nuclei and induces fibril growth exponentially until an equilibrium or plateau phase is reached [4,5]. Complete fibril breakage can also provide new fibril ends, further encouraging growth [4].

On the other hand, oligomers are small, water-soluble aggregates of misfolded protein that temporarily occur early in aggregation [3]. These structures have a high content of β-sheet conformation but are less ordered than mature fibrils. Importantly, these oligomers induced by α-synuclein (αS) have been shown to cause neurotoxicity in both species that are released by mature fibrils after they are created and those that originate immediately from the assembly of monomers during the aggregation process (Figure 5) [67]. Specifically, the effects of αS oligomers on neurones have been examined by comparing them to the effects of monomeric proteins and fibrillar species of varying sizes when supplied extracellularly. While unstructured monomers do not cause cellular damage, fibrillar aggregates become toxic over longer exposure times due to the release of structurally similar and β-sheet containing oligomers, even though β-sheet containing oligomers are the most toxic species and cause cellular stress immediately upon addition to the cell culture medium [3]. As shown in Figure 5, it has been found that “forward oligomers” produced directly or “backward oligomers” liberated from fibrils both set off the identical series of biological processes that begin when they meet the cell membrane [3,67].

The terminal composite structure is typically the amyloid plaque, a mass of extracellular aggregate of closely packed fibrils and included oligomers. In Alzheimer’s disease, for example, amyloid plaques consist mainly of β-amyloid fibrils, which over time accumulate in cerebral tissue and result in cellular dysfunction and inflammation initiation [68]. Although plaques and mature fibrils have traditionally been thought of as the culprit of neurotoxicity, current evidence points towards the actual risk being in the dynamic interaction between monomers, oligomers, and fibrils. Studies suggest that the formation of soluble monomers into oligomers and the release of oligomeric fragments from the larger fibrils are initiating events promoting the pathology of disease [67,69]. It is still a major challenge to identify such transient, toxic intermediates, and the assembly of probes, antibodies, and aptamers for selective targeting thereof remains an active area of therapeutic research [5].

5.2. Prion-like Propagation of Misfolded Proteins

Prions are a unique set of infectious misfolded proteins capable of transferring their abnormal conformation to normal counterparts, inducing fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs). These are diseases like Kuru, Creutzfeldt–Jakob disease (CJD), Gerstmann–Sträussler–Scheinker syndrome (GSS), and fatal familial insomnia in humans, bovine spongiform encephalopathy (BSE) in cows, and scrapie in sheep [6]. What makes prions special is their ability to transmit not just within an organism, but also from one individual to another, even across different species, very much like infectious agents, but with no nucleic acids. The key to prion replication is nucleated polymerization, where the healthy prion protein (PrP^C) transforms into the pathogenic form (PrP^Sc) with a dense β-sheet structure [70]. This abnormally folded structure acts as a template and attracts and transforms more normal proteins to the abnormal conformation, replicating itself in a process akin to a chain reaction. Notably, different conformational strains of PrP^Sc can result in different disease presentations, incubation periods, and tissue damage patterns. Inherited forms of prion disease are linked directly with familial mutations within the prion gene, demonstrating how protein misfolding and genetics interact to produce disease [6].

Importantly, the phrase prion-like behavior is not limited to classical prion disease. Even in fungi, it has been identified that non-homologous proteins like Ure2, Sup35, Rnq1, and HET-s also transmit through self-templating conformational changes [71]. They aggregate into infectious filaments that grow by adding the soluble version of the same protein. Deterioration of these filaments, also known as secondary nucleation, creates new “seeds” that accelerate spreading. This closely resembles what happens in mammalian prions, where aggregation stability controls the rate of disease progression [72]. Importantly, many common neurodegenerative disorders, though not technically infectious like prion diseases, in fact display prion-like behavior at the cellular level. Conditions such as Alzheimer’s disease (AD), Parkinson’s disease (PD), frontotemporal dementia (FTD), and Huntington’s disease (HD) are driven by aberrant spreading and aggregation of specific misfolded proteins, including tau, β-amyloid (Aβ), α-synuclein, huntingtin, and others [34]. Even though these illnesses are not passed between people as prions are, their pathology suggests some cell-to-cell transmission within the brain. Tau tangles and α-synuclein inclusions, for example, follow stereotypical patterns through interconnected brain regions as the illness progresses, suggesting intercellular “seeding” and proliferation [6].

Mutations in these proteins’ encoding genes, such as amyloid precursor protein (APP), tau, α-synuclein, superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TDP-43), fused in sarcoma (FUS), and huntingtin, explain familial cases of these conditions [73,74]. Most cases occur sporadically when the quality control within cells fails, and there are misfolded proteins that begin to aggregate and propagate themselves in a prion-like manner. Recent evidence demonstrates that prion diseases could spontaneously develop in the otherwise healthy brains under certain unusual conditions. This has been shown to occur due to exposure to metal surfaces (such as implanted wires or surgical instruments) which has the potential to induce prion formation in two ways: (i) directly converting normal prion proteins (PrP^C) into their infectious state (PrP^Sc) upon interaction with the metal or (ii) adsorbing and concentrating established prion inducers, such as RNA or certain lipids, onto the metal surface, which then promotes misfolding [75,76]. This outcome confirms the hypothesis that prion-like conversion may be seeded by external reagents disrupting protein structure and aggregation kinetics.

5.3. Toxicity of Misfolded Proteins vs. Large Aggregates

Protein aggregation is not static but a dynamic and multi-step process that generates a range of structures, from small transient intermediates to mature amyloid fibrils. This has made it difficult to determine exactly which species are most toxic [77]. It was traditionally thought that large amyloid plaques, such as the amyloid-beta (Aβ) fibrils that are found abundantly in the brains of Alzheimer’s disease (AD) patients, were mostly to blame for the neurodegeneration and cognitive decline [78,79]. These studies reinforced this view, showing that exposure of cultured neurons to Aβ fibrils was enough to trigger AD-like alterations at the cellular level, including disrupted electrical activity, membrane integrity loss, and impaired cell viability [80]. Similar earlier observations in animals showed that intracerebral injection of Aβ fibrils into rats resulted in synaptic transmission impairments and neuronal death [81].

However, the direct correlation of amyloid plaque density with the severity of cognitive symptoms has long been contentious [82]. Evidence is now accumulating to implicate smaller, soluble intermediates, notably oligomers and protofibrils, as more toxic species [83]. Studies have demonstrated that Aβ oligomers cause severe impairment of learning and memory when infused into the brain, whereas mature fibrils are less potent [84,85]. Oligomers are also toxic to synaptic function by blocking critical calcium currents in neurons—a mechanism not observed for monomers or mature fibrils [86]. Some researchers have proposed that the formation of large fibrils serves a protective role by sequestering smaller toxic species in a less reactive state. However, this reservoir may periodically release toxic intermediates back into circulation, further complicating the pathology [87].

The enhanced toxicity of oligomers relative to larger aggregates can be explained by several factors that relate to their different structural arrangement. First, oligomers are likely to have exposed hydrophobic surfaces that promote detrimental interactions with cell membranes, unlike the more stable, tightly packed fibrils [88]. Secondly, because of their compact size, oligomers can diffuse more easily through brain tissue and penetrate cells compared to larger fibrils [89]. Thirdly, oligomers have several exposed active ends that enhance their interactions with cellular targets [90]. Lastly, oligomers are less structured than fibrils and hence more reactive and disruptive [91]. Together, these findings have shifted the traditional amyloid cascade hypothesis to the oligomer hypothesis, which emphasizes that intermediate conformations rather than the visible plaques may be the primary inducers of neurotoxicity in disorders like Alzheimer’s and Parkinson’s [92,93]. However, there is a pressing need to develop in vivo imaging tools to gain a clear and complete understanding of the protein aggregation process and to give a definitive clinical connection of illness stage or severity linked with oligomeric intermediates and mature fibrils.

6. Cellular Consequences of Protein Aggregation

Protein aggregation, a hallmark of numerous neurodegenerative diseases, leads to a cascade of detrimental cellular effects. Misfolded proteins that escape degradation pathways can accumulate into toxic species, disrupting critical cellular functions. The major cellular consequences include loss of proteostasis, dysregulation of key RNA-binding proteins like TDP-43, and synaptic toxicity, each contributing to neuronal dysfunction and cell death [68,94].

6.1. Loss of Proteostasis

Proteostasis (protein homeostasis) involves a tightly regulated network of molecular chaperones, proteasomes, and autophagic systems that ensure proper protein folding, trafficking, and degradation. In neurodegenerative diseases, the accumulation of misfolded proteins overwhelms these systems. It aggregates sequester molecular chaperones and impairs the ubiquitin–proteasome system (UPS) and autophagy–lysosomal pathways. It leads to secondary accumulation of other misfolded or damaged proteins, exacerbating cellular stress. It has been demonstrated that α-synuclein fibrils impair lysosomal function in Parkinson’s disease models by blocking autophagosome–lysosome fusion, causing a backlog of cellular waste and contributing to dopaminergic neuron loss [95]. It restores proteostasis via proteasome activators, chaperone enhancers, or autophagy inducers is a promising therapeutic approach [94].

6.2. TDP-43 Dysfunction

TAR DNA-binding protein 43 (TDP-43) is essential for RNA metabolism, including splicing, transport, and stability. In diseases like ALS and frontotemporal dementia (FTD), TDP-43 mislocalizes from the nucleus to the cytoplasm, forming insoluble aggregates. Loss of nuclear TDP-43 leads to impaired splicing and gene expression regulation. Cytoplasmic aggregates may act as toxic gain-of-function entities, affecting stress granule dynamics and mitochondrial function. Saxton and Kraemer [96] showed that ALS-associated TDP-43 mutations disrupt RNA splicing of critical synaptic and mitochondrial genes, directly contributing to neurodegeneration. Therapies aimed at preventing TDP-43 mislocalization or restoring its nuclear function may protect against RNA dysregulation and neuronal death [97].

6.3. Synaptic Toxicity

Neurons rely heavily on intact synaptic communication for function and survival. Misfolded proteins such as amyloid-β (Aβ), α-synuclein, and tau disrupt synaptic architecture and signaling even before plaque or tangle formation. Oligomeric species interfere with synaptic vesicle release, receptor trafficking, and postsynaptic density integrity. Induce oxidative stress and calcium dysregulation at synapses. Henstridge et al. [98] reported that soluble Aβ oligomers cause synaptic loss in the hippocampus by disrupting NMDA receptor function and reducing dendritic spine density. Synaptic dysfunction is an early marker of neurodegeneration; thus, targeting soluble oligomers could prevent or delay cognitive and motor symptoms [68].

6.4. Escaping Protein Quality Control (PQC)

6.4.1. PQC Failure

Protein quality control (PQC) is a cellular surveillance system that ensures proteins fold correctly and eliminates misfolded or damaged proteins Via molecular chaperones, the ubiquitin–proteasome system (UPS), and autophagy–lysosomal pathways. In neurodegenerative diseases, PQC systems are often overwhelmed or dysfunctional, allowing misfolded proteins to accumulate and aggregate. Mechanisms of failure include: (1) overload, where misfolded proteins accumulate faster than PQC systems can degrade them; (2) impaired proteasome function, where aggregates inhibit proteasome activity, creating a feedback loop of increased misfolding; (3) chaperone sequestration, where molecular chaperones like Hsp70 are trapped by aggregates, impairing their function; (4) autophagy dysfunction where key autophagy proteins (e.g., Beclin-1, LC3) are downregulated or misregulated in disease states [99,100].

Aged neurons have been demonstrated to exhibit a decline in proteasome efficiency and reduced expression of heat shock proteins, leading to accumulation of misfolded α-synuclein and tau in mouse models of neurodegeneration [101]. Targeting proteasome activity pharmacologically helped reduce aggregate burden. Restoring or enhancing PQC pathways through proteasome activators, autophagy inducers, and/or chaperone modulation holds therapeutic promise in early-stage neurodegeneration.

6.4.2. Tau-Specific Mechanisms

Tau, a microtubule-associated protein, becomes pathological when it undergoes hyperphosphorylation, misfolding, and aggregation—central events in Alzheimer’s disease (AD) and other tauopathies. Tau escapes PQC through both generic failure of PQC systems and tau-specific evasion mechanisms. Mechanisms of PQC evasion by tau include: (1) hyperphosphorylation of tau interferes with its recognition by E3 ubiquitin ligases, preventing its tagging for degradation; (2) tau aggregates disrupt autophagosome formation, impeding their own clearance; (3) misfolded tau forms β-sheet-rich structures that are resistant to proteolysis; (4) tau interacts abnormally with stress granules, potentially stabilizing its own aggregation through phase separation [102].

Alquezar et al. [103] found that pathological tau impairs autophagic flux in neurons by interacting with lysosomal membranes, preventing the maturation of autophagosomes into degradative lysosomes. Inhibition of this tau-lysosome interaction restored autophagy and reduced tau pathology in vitro. Kalyaanamoorthy et al. [104] reported that phosphorylation at specific tau residues (Ser202/Thr205) prevents its interaction with Hsp90, a chaperone critical for tau refolding and degradation, thereby stabilizing its toxic conformation. Tau’s ability to evade PQC systems makes it a high-priority therapeutic target. Approaches aiming to enhance tau degradation (e.g., PROTACs, autophagy boosters, dephosphorylation strategies) are actively under investigation.

The failure of protein quality control systems is a key enabler of pathological protein aggregation in neurodegenerative diseases. While general PQC impairment underlies many forms of aggregation, specific proteins like tau possess unique molecular strategies to escape degradation. This dual breakdown of proteostasis—both general and protein-specific—explains the persistence of toxic aggregates and underscores the need for targeted therapies that restore or reinforce PQC function [94]. Neuroinflammation is increasingly recognized as a key contributor to the onset and progression of neurodegenerative diseases. The innate immune system, particularly microglia, the brain’s resident immune cells, plays a dual role: initially protective but later neurotoxic. Additionally, recent studies suggest that autoantibody generation against neuronal proteins may exacerbate pathology. Both processes are intricately linked with protein misfolding and aggregation [105,106].

6.5. Microglial Activation

Microglia serve as the first line of defense in the central nervous system (CNS), constantly surveilling the environment for pathogens, injury, or abnormal proteins. In neurodegenerative diseases, misfolded proteins such as amyloid-β, α-synuclein, and tau act as danger-associated molecular patterns (DAMPs) that chronically activate microglia [107]. Mechanisms of microglial activation include: (1) misfolded proteins bind to pattern recognition receptors (PRRs) such as TLR2, TLR4, and NLRP3 inflammasome; (2) the release of pro-inflammatory cytokines (e.g., IL-1β, TNF-α, IL-6) and reactive oxygen species (ROS) are triggered; (3) prolonged activation leads to a shift from a neuroprotective (M2) to a neurotoxic (M1) phenotype [108,109]. It has been demonstrated that α-synuclein oligomers activate the NLRP3 inflammasome in microglia via TLR2, promoting neuroinflammation in a mouse model of Parkinson’s disease [110]. Additionally, inhibiting NLRP3 reduces dopaminergic neuron loss. Ayyubova [111] reported that phosphorylated tau enhances microglial phagocytic dysfunction, contributing to impaired clearance and sustained inflammation in Alzheimer’s models. Chronic microglial activation creates a self-reinforcing cycle of neurodegeneration and inflammation. Therapies targeting microglial modulators (e.g., NLRP3 inhibitors, TREM2 agonists) are promising for interrupting this feedback loop [107].

6.6. Autoantibody Generation

In addition to cellular innate immunity, humoral responses, especially the generation of autoantibodies against neuronal proteins, are increasingly implicated in neurodegenerative disorders. Although the CNS is considered an immune-privileged site, blood–brain barrier (BBB) breakdown allows peripheral immune components to access the brain. Misfolded proteins such as α-synuclein, tau, or Aβ become exposed to the immune system due to neuronal death or BBB leakage. The adaptive immune system mounts a response, generating IgG or IgM autoantibodies. These antibodies may promote clearance (protective role), trigger complement activation, or Fc receptor-mediated damage (pathogenic role) [112].

Elevated levels of anti-tau and anti-Aβ autoantibodies have been found in cerebrospinal fluid of early-stage Alzheimer’s patients, suggesting an autoimmune component in initial disease stages [113]. Autoantibodies against α-synuclein have also been shown to exacerbate neuroinflammation in PD by promoting complement activation and microglial phagocytosis of healthy neurons [114]. Antibodies represent a double-edged sword in neurodegeneration. While some may help in early clearance of misfolded proteins, others may contribute to neuronal injury and disease progression. These findings open avenues for immunotherapies (e.g., monoclonal antibodies or tolerance induction strategies).

6.7. Oxidative Stress and Mitochondrial Dysfunction

Oxidative stress is a major pathogen of initiation and progression of most chronic disorders, including cancer, cardiovascular diseases, autoimmune diseases, and especially neurodegenerative diseases [1]. Coupled with aging and exposure to environmental or genetic risk factors, the balance between the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and the body’s antioxidant protection is disrupted. This imbalance leads to an augmented accumulation of reactive species, which results in cellular damage [115].

Mitochondria, the cell’s main apparatus for generating energy, are also key sources of ROS via electron leakage from the electron transport chain in the process of oxidative phosphorylation [116]. Excess activation of mitochondrial pathways, particularly under stress, increases ROS production, making neurons highly vulnerable due to their high metabolic activity and limited ability to regenerate [1]. While moderate amounts of ROS/RNS play essential roles in physiological signaling, immunity, and cellular response, their overproduction overpowers antioxidant protection, leading to oxidative damage in proteins, lipids, and nucleic acids, which are all common features of most neurodegenerative diseases [117]. Brain oxidative stress drives such mechanisms as protein misfolding, mitochondrial dysfunction, and synapse loss.

Current evidence indicates that indicators of oxidative damage, such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), are highly elevated in involved brain regions in Alzheimer’s and Parkinson’s disease, i.e., the hippocampus, cortex, and substantia nigra. In addition, protein nitration products (e.g., those found in Lewy bodies) and peroxynitrite-mediated damage are commonly observed in the same regions, suggesting a direct role for oxidative/nitrosative stress in disease pathogenesis [1]. Different reactive species are generated through both metabolic and environmental routes. For example, superoxide anion (O₂⁻) is generated through the one-electron reduction in molecular oxygen and can be reduced further to hydrogen peroxide (H₂O₂) or hydroxyl radicals (OH), which are very reactive [118]. Nitric oxide (NO), while involved in physiological signaling, can interact with superoxide to generate peroxynitrite (ONOO⁻), a cytotoxic molecule with the potential to oxidize lipids, proteins, and DNA [119]. This cascade of radical formation triggers lipid peroxidation, protein malfunction Via nitrosylation, and mutagenesis. These oxidative modifications not only lead to neuronal injury but also impair mitochondrial function, generating a vicious cycle of mounting stress and dysfunction [1].

6.8. Disruption of Synaptic Function

One of the critical mechanisms underlying synaptic dysfunction in neurodegenerative diseases like Alzheimer’s disease is the binding of amyloid-beta (Aβ) oligomers with the cellular prion protein, PrP^c [120]. PrP^c is well defined in prion diseases through its infectious misfolded variant, PrP^sc. Increasing evidence now implicates the same misfolding or signaling processes in Alzheimer’s disease [121]. Studies have shown that soluble Aβ oligomers and not monomers or fibrils selectively bind to a region of PrP^c, namely around amino acid residues 95 to 105 [122,123]. Binding inhibits long-term potentiation (LTP), a synaptic process in learning and memory, particularly in the hippocampus. Experimental findings suggest that antibodies targeted to this 93–109 site on PrP^c can inhibit Aβ oligomer binding and hence prevent Aβ inhibition of LTP both in vitro and in vivo [120].

Specifically, Fab (anti-body fragment) such as D13, which is specific for residues PrP_96–104^c, can prevent Aβ-induced synaptotoxic effects [124]. Notably, antibodies directed to other domains of PrP^c, such as the α-helix domain (not overlapping directly with the Aβ-binding site), also prevent synaptic impairment [123]. This suggests that such antibodies may prevent PrP^c—PrP^c interactions that are necessary for subsequent toxic signaling, perhaps by preventing clustering or dimerization. However, it is important to mention that bivalent antibodies like D13, while protective in some cases, can indeed induce apoptosis in vivo [125]. This action is thought to be due to cross-linking of PrP^c molecules, like what occurs with oligomeric Aβ binding and clustering of PrP^c within the synapse [120]. Such cross-linking may interfere with regular synaptic function, perhaps by interfering with the role of PrP^c in controlling N-methyl-d-aspartate (NMDA) receptor-mediated glutamatergic signaling, to result in eventual excitotoxicity. Additionally, Aβ oligomers may also simultaneously bind other membrane proteins, such as metabotropic glutamate receptor 5 (mGluR5), to form multiprotein complexes that enhance their toxicity [126]. These interactions may enhance disruption of synaptic signaling pathways and underlie cognitive impairment seen in Alzheimer’s disease.

6.9. Impaired Autophagy and Proteasome Activity

Maintaining protein homeostasis (proteostasis) is important for neuronal health. This involves the efficient folding, targeting, and degradation of thousands of proteins in changing cellular conditions [127]. Damaged or misfolded proteins are typically handled by two major degradative pathways: the ubiquitin–proteasome system (UPS) and the autophagy–lysosome pathway [8]. They are also complementary and strictly regulated under coordinated transcriptional programs, which stimulate key enzymes and react to cellular needs. The UPS predominantly breaks down soluble, misfolded proteins by tagging them with ubiquitin and directing them to the proteasome for degradation [127]. Molecular chaperones such as Hsp70 and Hsp90, and co-chaperones such as CHIP, aid in the recognition and targeting of defective proteins [128]. If the proteins are unable to be refolded, they are broken down by proteasomes. Notably, the proteins must be in a soluble state for productive processing by the proteasome, which utilizes ATPase activity to unfold the proteins before their breakdown [127].

However, if the proteins aggregate to a larger, insoluble size, the autophagy–lysosome pathway is required. Cell debris is sequestered into double-membraned structures called autophagosomes, which fuse with lysosomes for degradation during this process [8]. In contrast with bulk autophagy, bound proteins are targeted by selective autophagy, which is mediated through chaperones and adaptor proteins like p62 and Bag-3 [128]. Cells also sequester aggregates into compartments like aggresomes or juxtanuclear quality control centers (JUNQs). These are temporary storage to mitigate the toxicity before the degradation capacity is available. Some misfolded proteins are targeted to lysosomes directly by chaperone-mediated autophagy, which is activated by a KFERQ-like motif within target proteins [127].

In neurodegenerative diseases such as Alzheimer’s, both the proteasome and autophagy systems become compromised. This leads to toxic protein assembly, overwhelming cellular quality control systems, and resulting in neuronal dysfunction and death [8,127].

7. Role of Protein Misfolding and Aggregation in Specific Neurodegenerative Diseases

Protein misfolding, oligomerization, and accumulation in the brain are the primary processes causing pathological abnormalities that lead to neurodegenerative disease, according to strong evidence from genetic, neuropathological, cellular, and biochemical investigations as well as from studies using transgenic mouse models [34,129]. Amyloid-beta (Aβ) in Alzheimer’s disease, tau in Alzheimer’s disease, frontotemporal dementia, corticobasal degeneration, progressive supranuclear palsy, argyrophilic grain disease, and chronic traumatic encephalopathy, alpha-synuclein (α-Syn) in Parkinson’s disease (PD), multiple system atrophy, and dementia with Lewy bodies, TAR DNA-binding protein 43 (TDP-43) in amyotrophic lateral sclerosis and frontotemporal dementia, and prion proteins in prion diseases (i.e., Creutzfeldt–Jakob disease (CJD), bovine spongiform encephalopathy, chronic wasting disease, and scrapie) are the proteins most frequently linked to the buildup of cerebral misfolded aggregates in neurodegenerative diseases [127,129]. These proteins and their mechanisms of toxicity are summarized in

Table 1. Pathological aggregates in neurodegenerative diseases and their mechanisms of toxicity.

Proteins That Aggregate	Main Associated Disease(s)	Mechanism of Toxicity	References
Amyloid-β (Aβ)	Alzheimer’s disease	Synaptic dysfunction, mitochondrial impairment, excitotoxicity, ion channel formation disrupting Ca2+ homeostasis.	[130]
Tau	Alzheimer’s, frontotemporal dementia	Hyperphosphorylation which leads to the formation of neurofibrillary tangles, thereby causing microtubule destabilization, synaptic failure, and trans-neuronal spread.	[131,132]
α-Synuclein	Parkinson’s, Lewy body dementia	Oligomerization into Lewy bodies, mitochondrial and lysosomal dysfunction, calcium dysregulation, neuroinflammation.	[133]
Huntingtin (mHTT)	Huntington’s disease	Expansion of polyglutamine which causes protein misfolding and aggregation, transcriptional dysregulation, impaired axonal transport, excitotoxicity.	[134]
TDP-43	ALS, frontotemporal lobar degeneration (FTLD)	Abnormal aggregation, loss of RNA-binding function, impaired splicing, cytoplasmic inclusions are toxic to neurons.	[135,136]
Prion protein (PrPSc)	Prion diseases (Creutzfeldt–Jakob disease, Kuru, Bovine Spongiform Encephalopathy)	Misfolded prion proteins (PrPSc) act as pathological templates that induce the misfolding of normal cellular prion protein (PrPC), leading to the accumulation of insoluble aggregates in the brain, causing spongiform changes, synaptic loss, and progressive neurodegeneration.	[137,138]

. There are no clear similarities between these disease-associated proteins’ sequence, size, structure, expression level, or function. Nevertheless, in the diseased brain, all of these proteins misfold from their native states to create intermolecular β-sheet-rich structures that range in size from tiny oligomers to massive fibrillar aggregates [129].

7.1. Alzheimer’s Disease

Alzheimer’s disease (AD) is presently one of the most prevalent neurodegenerative disorders that affects almost 10% of the world’s population, particularly among individuals over 60 years of age [139]. It primarily features insidious loss of memory and impairment of cognitive functions, referred to as dementia. The neuropathological characteristics of AD are the formation of neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau protein, extracellular amyloid-beta (Aβ) plaques, and profuse neuronal loss [1].

One of the major causes of neuronal damage in AD is mitochondrial impairment, with resultant excessive generation of reactive oxygen species (ROS). The consequential oxidative stress, together with a disruption of redox homeostasis, sensitizes Aβ and tau-dependent neurotoxicity [1]. Aggregation and deposition of Aβ peptides are believed to initiate a cascade of pathological events. These involve facilitating the abnormal phosphorylation and polymerization of tau protein, hence speeding up the development of NFTs and further promoting neuronal degeneration [140]. Apart from protein aggregation, vascular abnormalities are also important in the progression of AD. These involve decreased cerebral blood flow (CBF), blood–brain barrier (BBB) dysfunction, and cerebral amyloid angiopathy, all of which compromise nutrient and waste exchange in the brain [1]. Among the primary mechanisms causing this dysfunction is the pathological elevation of intracellular calcium (Ca²⁺) concentrations, particularly within mitochondria [141]. This disrupts functions essential to cognition and memory, including proper ion exchanges, such as Na⁺/Ca²⁺ and H⁺/Ca²⁺, thereby disabling synaptic transmission, gene expression, and neural plasticity. Normal neurotransmission, long and short-term plasticity, and gene transcription regulation all depend on Ca²⁺, and any disruption in Ca²⁺ homeostasis exacerbates excitotoxicity (the condition where overstimulation of neurons leads to cell death) [1,141].

Furthermore, ROS levels increase, triggering inflammatory signaling cascades, which boost the production of pro-inflammatory cytokines like interleukin-1 (IL-1), IL-6, tumor necrosis factor-alpha (TNF-α), and other chemokines [142]. This leads to a vicious cycle of chronic neuroinflammation, enhancing stimulation of microglia and astrocytes to produce more ROS, enhancing oxidative stress, and augmenting neuronal damage [1]. The dual effect of chronic inflammation and oxidative stress not only induces the aggregation of Aβ but also forms the basis for the general neurodegenerative process [143]. Some complicating factors include endoplasmic reticulum (ER) stress, disrupted cholinergic neurotransmission, and persistent mitochondrial dysfunction, all of which trigger dementia and apoptosis of neurons [1,143].

7.2. Parkinson’s Disease

Parkinson’s disease (PD), which primarily affects individuals over the age of 60, is currently recognized as the second most prevalent neurodegenerative disorder after Alzheimer’s [144]. PD is characterized by the long-term degeneration of dopaminergic neurons, particularly in the substantia nigra region of the brain. The primary pathological factors involved in PD include oxidative stress, which results from an imbalance in the production of ROS and antioxidant defense mechanisms in the brain [1]. PD involves a tremendous loss of key antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and glutathione peroxidase (GPx) [145]. Downregulation of antioxidants renders the neurons vulnerable to oxidative damage by ROS and other reactive species. Dopamine (DA), the motor control neurotransmitter, is inherently unstable and auto-oxidizes within the nigrostriatal tract to create ROS such as superoxide (O₂⁻), hydrogen peroxide (H₂O₂), and dopamine quinones [1]. Generation of these reactive molecules occurs with age, and thus, individuals of an advanced age are more susceptible.

Dopamine is oxidatively deaminated by monoamine oxidase-B (MAO-B) mainly to major metabolites like 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and minor toxic by-products like ammonia (NH₃), hydrogen peroxide, and quinones formed from dopamine. Such by-products have the potential to cause neurotoxicity, particularly on accumulation in very high amounts. Iron accumulation in dopaminergic neurons may also raise oxidative stress through the Fenton reaction, a process that produces highly reactive hydroxyl radicals. Lipid peroxidation, specifically of cardiolipin, a phospholipid which is found within the mitochondrial membrane, is initiated by these radicals. This damage leads to the release of cytochrome c (Cyt-c) into the cytoplasm, one of the indispensable steps to trigger apoptosis (programmed cell death).

Along with oxidative stress and mitochondrial damage, genetic mutations are also important in PD pathogenesis. Mitochondrial DNA (mtDNA) as well as nuclear gene mutations are implicated in familial and sporadic PD. At least nine nuclear genes are involved in PD, including α-synuclein which is involved in Lewy body formation and can also impair mitochondrial function; parkin which affects mitochondrial quality control; DJ-1 which functions as an antioxidant; PINK1 (PTEN-induced kinase 1) which maintains mitochondria by preventing release of Cyt-c; LRRK2 (Leucine-rich repeat kinase 2) which maintains neuronal function and mitochondrial dynamics; HTRA2 (high-temperature requirement protein A2), a mitochondrial serine protease that is involved in apoptosis; NURR1, tau, and ubiquitin carboxy-terminal hydrolase L1 that are involved in neuronal survival and PD pathology [146,147]. Together, these biochemical disruptions and genetic factors constitute a multi-factorial environment that causes dopaminergic neuronal loss in PD.

7.3. Huntington’s Disease

Huntington’s disease (HD) is a genetic neurodegenerative disease that is defined primarily by progressive motor dysfunction, cognitive disturbance, and psychiatric disturbance [1]. HD typically affects individuals around 30–50 years of age, and is thought to be caused by a CAG trinucleotide repeat expansion in the HTT gene, which results in an abnormally long polyglutamine (polyQ) tract within the huntingtin (Htt) protein [148]. The lengthened polyQ tract induces protein misfolding and synthesis of insoluble aggregates and inclusion bodies in neurons. Htt misfolding interacts with over a hundred cellular proteins, disrupting crucial biological processes such as transcriptional regulation, intracellular trafficking, and proteostasis. The major outcome includes the formation of inclusion bodies (abnormal protein aggregates) that accumulate in axons and dendrites, interfering with synaptic transmission and causing neuronal malfunctioning and death [37,149].

Oxidative stress is a central mechanism of HD pathogenesis. The production of ROS is triggered by misfolded Htt, which results in oxidative damage to cellular components. In HD patients, oxidative stress biomarkers such as lipofuscin (through lipid peroxidation), nitrated proteins, and 8-hydroxy-2′-deoxyguanosine (8-OHdG) are elevated in mitochondrial DNA, which reflects widespread cellular damage [150]. Excitotoxicity, or excess glutamate signaling, contributes to the neurodegeneration of HD as well. The overactivation of the NMDA receptors by glutamine leads to excess calcium buildup and mitochondrial damage, which allows for neuronal injury [151]. Furthermore, the downregulation of the transcriptional coactivator PGC-1α, which regulates genes for mitochondrial biogenesis and oxidative stress resistance, has been seen in HD. This results in decreased expression of components of the respiratory chain, especially complexes II, III, and IV, and lower production of ATP and increased accumulation of lactate [1]. Opening of the outer mitochondrial membrane (OMM) and cytochrome c (Cyt-c) release also contribute to apoptotic cell death. It has also been shown that the expression of the mitochondrial protease HTRA2, which is involved in stress response and apoptosis, is significantly reduced in mutant huntingtin-expressing neurons, particularly in the striatum, which is the most vulnerable brain area in HD [146]. This downregulation further disables subsequent mitochondrial integrity and cellular resilience, leading to the exacerbation of HD.

8. Therapeutic Strategies for the Treatment of Neurodegenerative Diseases

Current evidence demonstrates that intracellular protein oligomer formation (early-stage aggregation intermediates) is the central mechanism underlying cellular toxicity in neurodegenerative disease [152]. These toxic aggregates not only disrupt normal cellular functions within the affected neuron, but they also transmit to neighboring neurons by passing into the extracellular space, seeding further pathological aggregation in neighboring cells. This prion-like activity is the rationale for the progressive nature of neurodegenerative diseases [153,154]. Therefore, therapy has been focused primarily on two fronts: (1) the inhibition or direct reversal of the protein misfolding and aggregation cascade, and (2) the inhibition of external and internal cellular stressors such as oxidative stress, mitochondrial impairment, or inflammation that may induce or aggravate protein aggregation [152]. These approaches are targeted towards preventing disease progression, sustaining neuronal function, and ultimately improving patient outcomes.

8.1. Antibody-Based Immunotherapy: Targeting Extracellular Aggregates

One of the most important therapeutic treatments being studied for neurodegenerative disease is immunotherapy, which is the use of antibodies to clear and eliminate extracellular levels of misfolded protein aggregates. Several clinical trials have aimed at the use of monoclonal antibodies that target aggregating proteins such as amyloid-beta in Alzheimer’s disease and alpha-synuclein in Parkinson’s disease. The core idea is to eliminate these pathological forms from the brain through immune-mediated mechanisms [152]. However, the successful delivery of antibodies to the central nervous system (CNS) is a major problem due to the restrictive nature of the blood–brain barrier (BBB), which limits the diffusion of large molecules. In trying to overcome this, researchers have designed engineered antibodies with the ability to cross the BBB using native transport mechanisms or active transport pathways. Notably, engineered single-chain antibodies such as nanobodies derived from camelids provide encouraging strengths in terms of high solubility, structural integrity, and greater penetration into tissues, making them good candidates for CNS use [155].

Despite these advances, clinical outcomes have been disappointing. Studies such as those by bapineuzumab (directed against the N-terminus of amyloid-beta) and solanezumab (binding to soluble amyloid-beta monomers) were unable to provide evidence of compelling clinical benefits in Alzheimer’s patients [156,157]. These failures suggest that the efficacy of antibody therapy may be extremely sensitive to the form of protein being targeted. Since most of these antibodies attack mature plaques or monomers that are non-toxic rather than the toxic soluble oligomers, they may be missing the key pathogenic species responsible for neuronal damage [152]. Another important consideration is when the intervention should occur. Antibody-based therapies are likely to be more effective earlier, when monomeric proteins initially begin misfolding and forming pathogenic oligomers, rather than later when harmful large fibrils have built up [3,158]. Diagnosing patients in these early phases is still difficult due to the lack of sensitive and specific biomarkers.

Hence, to tackle this, future antibody therapy development must consider several critical issues: enhanced penetration across the BBB, increased specificity towards oligomers, initiating treatment during the early stages of the disease, and developing reliable biomarkers of disease progression and treatment responses, specifically oligomer changes.

8.2. Chaperone Therapy: Optimizing Protein Quality Control Mechanisms

Since misfolding and protein aggregation are the main drivers of toxicity in many neurodegenerative diseases, treatments have moved to adopt the manipulation of cellular machineries responsible for maintaining protein homeostasis. One such approach is chaperone therapy, which involves the enhancement of the activity of molecular chaperones and related degradation machinery such as lysosomal and proteasomal pathways [152]. Molecular chaperones are a class of proteins whose activity is critical to protein quality control. They recognize and bind to the surface-exposed hydrophobic faces of non-native or misfolded proteins, which are regions that are normally hidden within well-folded proteins [159]. This binding either enhances the folding of the protein back into its native, functional conformation or assists in its degradation by pathways such as the ubiquitin–proteasome system (UPS) or autophagy–lysosome pathways [160,161]. These routes are of critical importance in the elimination of misfolded proteins and the prevention of their toxic aggregation.

Studies have demonstrated that certain chaperones, such as Heat Shock Proteins (HSPs), can protect neurons from protein aggregation-induced toxicity in neurodegenerative diseases, by interfering with toxic protein agglomeration. For example, Hsp70 has been shown to suppress the aggregation of toxic oligomers of α-synuclein, which is a primary pathologic protein in Parkinson’s disease [162]. Similarly, Hsp90 has been reported to interact with α-synuclein in an ATP-dependent manner to stabilize and neutralize its aggregates [163]. In addition, chaperones like Hsp27 can convert small toxic amyloid-beta (Aβ) oligomers into large, non-toxic aggregates with reduced surface-to-volume ratios that are more prone to clearance through autophagy [164]. In Alzheimer’s disease models, human prefoldin (hFPD) was observed to stabilize oligomeric Aβ42, with possible reduction in its neurotoxicity, suggesting that chaperones not only repress the initial toxic aggregations but are also capable of influencing the structural content of the aggregates to make them less toxic and easier to clear [165].

Furthermore, extracellular chaperones also support the mitigation of protein toxicity, particularly in AD. Notably, apolipoproteins such as Apolipoprotein E (ApoE) and Apolipoprotein J (ApoJ or clusterin) facilitate the in vivo clearance of extracellular misfolded proteins, such as Aβ, by promoting their uptake via receptor-mediated endocytosis [166]. Clusterin, for instance, sequesters a complex with Aβ and delivers it to be degraded via the megalin receptor, while ApoE binds to low-density lipoprotein receptors to facilitate Aβ uptake and clearance [167]. Similarly, the extracellular chaperone α2-macroglobulin (α2M) interacts with Aβ to create complexes that are internalized and degraded by processes mediated by lipoprotein receptor-related protein (LRP) [168]. These extracellular chaperones, together with endocytosis mechanisms, have been shown repeatedly in vivo to reduce the brain’s toxic protein load and thus are of potential therapeutic interest [152].

Although these mechanisms are promising, the clinical translation of these chaperone-based therapies has faced significant challenges. Hsp90 inhibitors, for example, have been primarily tested in oncology since the late 1990s, but none have gained the approval of the Food and Drug Administration (FDA) due to issues such as dose-limiting toxicity and variable expression of Hsp90 isoforms across tissues, which complicates dosing and safety profiles [169]. Extracellular chaperones such as ApoE4 have been found to be less efficient at forming stable complexes with Aβ compared to ApoE3, and are themselves a major risk factor for Alzheimer’s disease, complicating its potential use as a therapeutic agent [170]. ApoE4 can also disrupt normal endosomal–lysosomal and exosome pathways, thereby impairing neuronal protein clearance and exacerbating disease risk [171]. Lastly, therapies that target autophagy and proteasome activators, though promising in preclinical models, face translational hurdles in human trials due to the complex interplay between autophagy and the UPS and uncertainty about which aggregate forms are accessible to each pathway [172]. Thus, while chaperone therapy is mechanistically sound and biologically validated, its clinical effectiveness is limited, and it will be necessary to overcome delivery, specificity, and safety issues for therapeutic benefit to be realized in the future.

8.3. Antioxidant Therapy

Oxidative stress that involves overproduction of ROS is increasingly recognized as one of the primary drivers of the pathogenesis of neurodegenerative diseases such as AD and PD [1,16]. Numerous studies have shown increased oxidative stress in the brain of patients with Alzheimer’s disease, including increased levels of F2-isoprostane-α in cerebrospinal fluid [173] and frontal and temporal poles [174], and acrolein in amygdala and hippocampus/parahippocampal gyrus [175]. In addition, the frontal, parietal, and temporal lobes of Alzheimer’s disease patients’ brains showed higher levels of nuclear and mitochondrial DNA oxidation as compared to age-matched control subjects [176]. While it remains uncertain whether ROS is a causative primary factor or an epiphenomenon of neurodegeneration, there is substantial evidence to suggest that enhanced ROS plays a major role in mediating neuronal dysfunction and cell death [177]. Antioxidants, either derived from external sources or produced endogenously, play a defensive role by eliminating ROS through multiple mechanisms, including scavenging of free radicals, metal ion reduction, inhibition of lipid peroxidation, and optimization of mitochondrial electron transport efficiency [178,179].

While promising preclinical outcomes, particularly in animal models, have been achieved with antioxidant interventions, clinical translation of these therapies as effective treatments for AD and PD has typically been disappointing [180]. Several reasons may be responsible for this discrepancy. First, oxidative injury can occur early in the course of disease onset, closely linked with protein aggregation processes; hence, interventions delivered at subsequent stages might not exhibit the critical therapeutic effect [152]. Second, many antioxidant molecules suffer from poor bioavailability and cannot reach therapeutic concentrations at the very intracellular sites of ROS production. Lastly, simply scavenging ROS may not be sufficient; instead, strategies that target directly and inhibit the enzymatic origins of ROS production (e.g., mitochondrial complexes, NADPH oxidase) may yield more selective and effective therapeutic outcomes [152,180]. Collectively, these observations suggest that antioxidant therapy is promising, but future treatment will require more selective delivery and earlier intervention to effectively counteract oxidative stress in neurodegenerative disease environments.

9. Diagnostic and Biomarker Potential

Early and accurate diagnosis of neurodegenerative diseases is crucial for effective intervention and monitoring disease progression. Since misfolded and aggregated proteins are central to the pathology of many neurodegenerative diseases, they represent valuable biomarkers for diagnosis and tracking of disease state. Hence, efforts targeted towards the discovery and development of biomarkers have focused on identifying reliable molecular signatures in bodily fluids and applying advanced imaging technologies that can reflect pathological changes even before clinical symptoms emerge.

9.1. Detection of Misfolded Proteins in Cerebrospinal Fluid (CSF) or Blood

The presence and accumulation of misfolded proteins in cerebrospinal fluid (CSF) or peripheral blood have shown significant potential as diagnostic markers of many neurodegenerative diseases. Diagnosis of PD, for instance, remains heavily reliant on clinical evaluation, according to the UK Brain Bank Criteria, along with the patient’s response to dopaminergic medications [181,182]. Although neuroimaging techniques may provide supportive evidence for loss of dopaminergic neurons, they are generally not sufficiently specific to be capable of distinguishing PD from other associated neurodegenerative diseases [11]. Peripheral definitive diagnosis is post-mortem, through histopathological examination of brain tissues for distinctive features such as loss of substantia nigra neurons, Lewy bodies, and neurites [183].

However, the detection of misfolded α-synuclein (αSyn) oligomers (misfolded protein structures implicated in PD pathology) in bodily fluids such as CSF and blood has emerged as a promising new path in biomarker research. This approach has high diagnostic value for several reasons: (1) low-molecular-weight, soluble αSyn oligomers, rather than mature fibrillar complexes, are thought to be the primary toxic species causing neurodegeneration in PD [184,185]; (2) pathological assembly of αSyn is thought to begin years before the onset of clinical symptoms, making it a potentially strong early biomarker [186,187]; (3) these toxic oligomers are suspected to exist in CSF and possibly even blood, providing promising lines of detection [188,189].

Beyond CSF and blood, interstitial fluid (ISF) has also been increasingly recognized as a future diagnostic target. The brain cells are cushioned and supported by two forms of brain-specific fluids, the ISF and the CSF [190]. While the CSF fills the cerebral ventricles and the subarachnoid space, the ISF surrounds neurons and glial cells and therefore provides a more direct reflection of the brain’s extracellular environment [191]. Importantly, misfolded proteins such as amyloid-β and αSyn have been detected in ISF using advanced sampling methods like microdialysis, which allows for the measurement of molecules within the ISF over time, even while the test animals are awake and behaving [192]. This makes it possible to examine dynamic alterations in a molecule under conditions that are near-physiological rather than a single postmortem, static measurement, making ISF a valuable medium for studying protein aggregation dynamics in real time. Since ISF collection is considered less invasive than lumbar puncture for CSF, it holds promise for improving patient comfort and enabling longitudinal monitoring of neurodegenerative disease biomarkers [193].

Recent advancements in biochemical assay engineering have led to highly sensitive platforms for the detection of these elusive protein aggregates such as protein misfolding cyclic amplification (PMCA), where the self-replication characteristic of misfolded αSyn aggregates is used to amplify their presence to a detectable degree [194]. In a proof-of-concept study, Shahnawaz et al. [195] employed the PMCA technology to assess the diagnostic potential of detecting αSyn aggregates in CSF among Parkinson’s disease subjects and related disorders. The inclusion cohorts were 76 PD patients and 97 controls (patients with Alzheimer’s disease and other neurodegenerative illnesses). The PMCA assay demonstrated remarkable diagnostic capabilities with attomole sensitivity for αSyn oligomers at a sensitivity of 88.5% and specificity of 96.9%. Moreover, the PMCA results significantly correlated with disease progression, making it useful not only for diagnosis but also for monitoring. These findings suggest that αSyn-PMCA could be efficiently utilized as a sensitive, non-invasive, and objective biochemical assay for the early diagnosis of neurodegenerative diseases such as Parkinson’s disease.

9.2. Imaging Techniques (e.g., PET Scans for Amyloid)

Neuroimaging plays a key role in early diagnosis and monitoring of neurodegenerative diseases. Unlike traditional imaging, which is structural in nature, molecular neuroimaging techniques allow for the visualization, characterization, and quantification of biological processes at the cellular and molecular levels in humans [196]. This approach can guide disease pathological mechanisms, track disease progression, and evaluate therapeutic intervention effectiveness. In PD, for instance, molecular imaging is aimed at fulfilling a series of relevant goals, including helping clinical diagnosis and trial patient selection by confirming specific pathological alterations; tracking disease progression with time; distinguishing between PD patients and controls in research settings; and establishing the effects of treatment, especially target engagement and biological effect [11,196]. Imaging techniques such as positron emission tomography (PET), magnetic resonance imaging (MRI), and single-photon emission computed tomography (SPECT) have shown potential in this regard [197].

PET is a nuclear imaging technique which relies on the decay characteristics of positron-emitting radionuclides such as fluorine-18 (¹⁸F, t_1/2 = 109 min), carbon-11 (¹¹C, t_1/2 = 20 min), or oxygen-15 (¹⁵O, t_1/2 = 2 min) [196]. PET is a highly sensitive imaging technique that is non-invasive and capable of visualizing and quantifying several physiological and molecular processes within the brain [198]. PET scans detect biologically active compounds, called radiopharmaceuticals, marked with positron-emitting isotopes. For NDDs, PET is applied to assess regional brain function such as metabolism, blood flow, and neurotransmitter activity, and to detect abnormal protein deposits such as amyloid-β plaques [199]. Amongst the most widespread PET tracers is ¹⁸F-FDG PET which measures regional glucose metabolism as a reflection of neuronal activity and function. Distinct patterns of hypometabolism revealed by ¹⁸F-FDG PET are associated with different types of neurodegenerative dementias and potentially useful in their discrimination [200]. In addition, imaging of dopamine transporters (DAT) and vesicular monoamine transporters with the help of dedicated PET tracers aids in the diagnosis and follow-up of Parkinson’s disease by determining the integrity of presynaptic dopaminergic neurons in the striatum [201]. PET radiotracers for amyloid-β (Aβ) plaques have significantly improved the earlier diagnosis and detection of Alzheimer’s disease with the potential for visualizing amyloid pathology in vivo [202]. PET imaging of cholinergic function and microglial activation also provides valuable information regarding the pathophysiology of dementias and other NDDs, allowing for earlier diagnosis and making it easier to develop targeted treatments [203].

In addition to radiopharmaceutical-based imaging, researchers have also developed a wide range of chemical probes specifically designed for the detection of amyloid-β plaques. These include small-molecule fluorescent dyes such as Congo red, thioflavin-T, and newer near-infrared (NIR) probes, which bind selectively to β-sheet-rich amyloid aggregates and allow for sensitive in vitro and in vivo detection [204]. These methods have been increasingly recognized as promising approaches for the early diagnosis of AD due to their real time detection abilities, low cost, lack of radioactive exposure, and high resolution. Congo red, for example, has been found to bind to a variety of Aβ species, from monomers to mature fibrils. Binding to Congo red prevents these Aβ₁ peptides from aggregating normally, which lowers toxicity. For instance, CR has been demonstrated to solubilize toxic Aβ_1–40 species, such as anionic detergents [205], form heterogeneous aggregates with Aβ_1–40 [206], and bind to low-molecular-weight aggregates of Aβ_1–40 to form 1:1 and 1:2 complexes in a nuclear magnetic resonance spectroscopy study [207]. Furthermore, advances in probe chemistry have led to the development of high-affinity, blood-brain-barrier-permeable agents such as Pittsburgh Compound B (PiB, 2-(4-N-[¹¹C]-methylamino phenyl)-6-hydroxybenzothiazole) which has been used as a radiotracer to detect insoluble Aβ aggregates in the brains of live AD patients through PET scans [208]. These probes complement traditional PET and MRI approaches by enhancing signal specificity and providing versatile tools for studying amyloid deposition, tracking disease progression, and evaluating anti-amyloid therapeutic strategies [204].

In addition to its physiological application, the clinical value of PET imaging for amyloid has been extensively validated through numerous large-scale research studies to identify its impact on diagnostic precision and management of patients with neurodegenerative diseases. These trials include the IDEAS (Imaging Dementia–Evidence for Amyloid Scanning) study, the AMYPAD-DPMS (Amyloid Imaging to Prevent Alzheimer’s Disease–Diagnostic and Patient Management Study), the ABIDE (Alzheimer Biomarkers in Daily Practice) study, and a randomized clinical trial [209,210]. In the IDEAS study which was a national US longitudinal study, the impact of amyloid PET imaging on diagnosis and treatment planning was assessed in over 18,000 patients who had been diagnosed with MCI or dementia and met appropriate use criteria at close to 600 specialized clinics [211]. Conducted in partnership with the Centers for Medicare and Medicaid Services (CMS), the study demonstrated that amyloid PET led to a modification of clinical management within 90 days after the scan in over 60% of subjects—i.e., 60.2% in MCI and 63.5% in dementia—well exceeding the hoped-for benchmark of 30%. Common changes included initiation or adjustment of approved Alzheimer’s medications such as cholinesterase inhibitors or memantine. Diagnostic revisions were also significant: approximately 35% of patients were newly diagnosed following PET (25% shifted from Alzheimer’s disease to non-AD, and 10% in the reverse direction). But while diagnostic and treatment decision revisions were substantial, improvements in health outcomes were less spectacular. For instance, the 12-month rate of hospitalization decreased modestly (23.98% among PET patients versus 25.12% among control matches), short of the targeted reduction by 10%. No significant differences were observed regarding the use of emergency departments [210]. These findings demonstrate the potential of imaging techniques to enhance diagnostic accuracy, enable earlier intervention, and support more precise monitoring of disease progression and therapeutic response.

10. Future Directions

Despite major progress in imaging and molecular diagnostics, NDDs remain complex and incurable, highlighting the urgency to pursue novel directions in their etiology and in the development of more effective treatments. Emerging areas of research, particularly those on cellular and molecular dynamics, are about to transform our understanding and management of these disorders.

10.1. Role of Liquid–Liquid Phase Separation (LLPS)

Recent findings in the biophysical behavior of proteins have identified LLPS as a central process contributing to the pathogenesis of different neurodegenerative diseases. LLPS refers to the mechanism by which biomolecules, such as proteins and RNAs, spontaneously self-assembly from the surrounding cellular environment—typically the nucleoplasm or cytoplasm—into dense, membrane-free domains called biomolecular condensates [7]. This dynamic self-assembly creates two coexisting liquid phases: one biomolecule-rich and one relatively dilute. Under physiological conditions, this allows spatial and temporal regulation of cellular processes, including transcription, signaling, and stress response [212]. However, increasing evidence now shows that aberrant phase separation lies at the core of the initiation and propagation of protein misfolding and aggregation, which are features of neurodegenerative disease. These LLPS can serve as a precursor to aggregation, particularly under the circumstances of aging and oxidative stress, in which these environmental or intrinsic stressors destabilize the equilibrium of phase-separated condensates, transforming them from liquid-like, reversible assemblies into solid-like, insoluble aggregates that are difficult to clear. Such protein conformational or stability changes cause the deposition of misfolded protein aggregates, including tau, TDP-43, FUS, α-synuclein, and huntingtin, in certain brain areas and facilitate the progressive damage and dysfunction of neuronal cells as a hallmark of neurodegeneration [7]. Importantly, LLPS offers a unifying mechanistic approach that might account for the selective vulnerability of brain areas in different disorders based on the protein implicated and the cellular environment.

Notably, the phase behavior of LLPS-associated proteins is influenced by genetic mutations and post-translational modifications (PTMs). Such alterations can either enhance or inhibit the ability of proteins to form or maintain condensates. Ding et al. [213] proposed a framework to classify these abnormalities into two classes: loss of phase separation (LoPS) and gain of phase separation (GoPS), recognizing that disease-associated phase behavior is not binary since mutations may both gain and lose function in other cellular contexts. This classification aids in the description of condensates with newly acquired or transitional stages of phase separation, as well as those with limited or even disassembled phase separation capabilities. The richness of pathological protein structures seen in diseases, ranging from amorphous assemblies to fibrillar inclusions, can possibly represent multiple pathways of phase-separated condensates influenced by specific mutations, cellular states, or stage of progression [7]. Elucidating the biophysical principles driving LLPS and its disruption holds much potential for the discovery of early biomarkers and new therapeutic targets. Phase behavior regulated by small molecules or gene manipulation could restore physiological dynamics and prevent pathological aggregation, paving the way to a new therapeutic revolution for neurodegenerative disease prevention and treatment.

10.2. Role of RNA-Binding Proteins and Stress Granules

RNA-binding proteins (RBPs) play crucial roles in RNA metabolism and the dynamic regulation of cellular stress responses. Dysregulation of these proteins in neurodegenerative diseases has progressively been identified as one of the key underlying mechanisms [214]. The majority of RBPs contain low-complexity prion-like domains or poly-glycine repeats, enabling reversible interactions and thus facilitating them to transiently assemble into ribonucleoprotein complexes such as stress granules (SGs) under stress in cells [215]. These granules serve as stress bodies that transiently sequester untranslated mRNAs and bound proteins upon environmental or physiological stress. However, under chronic or pathological conditions such as aging, oxidative stress, or mutation of RBPs, these otherwise reversible complexes transform into stable aggregates. This aberrant phase transition shares numerous characteristics of protein misfolding characteristic of classic neurodegenerative disorders [214]. Both SGs and disease protein aggregates are enriched in ubiquitinated protein, require molecular chaperones to disassemble, and are exacerbated when the protein degradation pathways (e.g., proteasomal and autophagic systems) are compromised [216]. These parallels have led to the hypothesis that stress granules and disease protein aggregates may intersect or even overlap in disease states [214].

Unlike aggresomes, which are big, central inclusions near the microtubule-organizing center, SGs are numerous, small, and distributed throughout the cytoplasm. Despite these architectural differences, SGs have been shown to interact with a variety of disease-causing proteins, including huntingtin (HTT) in Huntington’s disease, the prion protein (PrP) in prion disease, and tau in Alzheimer’s disease and frontotemporal dementia (FTD) [217]. Notably, tau has been discovered to colocalize with SGs, and RNA binding to tau can cause its aggregation, and consequently further engage SGs in tauopathy pathogenesis [218]. Moreover, a strong connection has been established between recurring SGs and pathological inclusions formation in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), particularly with TDP-43 and FUS—two RBPs frequently mutated in familial ALS [219]. Chronic SG formation and dysregulation of neuronal RNA granules may therefore initiate or exacerbate neurodegenerative cascades.

The finding that mutations or malfunctions in some of these proteins can directly cause neurological illnesses is arguably the strongest evidence linking RBPs to the pathogenesis of neurodegeneration [214]. For example, fragile X intellectual disability syndrome (FXS), the most frequent cause of hereditary intellectual disability, is caused by reduced expression of FMRP due to trinucleotide repeat expansions [220]. As people age, FXS also causes a linked neurodegenerative disorder. Spinocerebellar ataxia is caused by expanded trinucleotide repeats in many ataxin genes. Spinal muscular atrophy (SMA) is associated with mutations in survival of motoneuron (SMN-1), while motoneuron illnesses, including ALS, are caused by mutations in TDP-43, FUS, ataxin-2 (ATXN2), optineurin (OPT), and angiogenenin (ANG). These disease processes are now associated with dysregulated or malfunctioning neuronal SGs and RNA granules [221,222]. These findings demonstrate the need for future research to further elucidate the dynamic participation of RBPs and SGs in the pathogenesis of neurodegenerative disease. Since derangements in their normal dynamics, either through genetic mutation, impaired clearance mechanisms, or chronic cellular stress, can initiate the formation of toxic, irreversible aggregates, targeted research must be dedicated to determining their precise molecular mechanisms. This knowledge can uncover new therapeutic strategies to stabilize RBP function, prevent pathological stress granule formation, or enhance aggregate clearance to protect neuronal integrity.

10.3. Advances in Proteomics and Single-Cell Analysis

Neurodegenerative diseases are all characterized by progressive loss of neurons and typically present later in life. Although some have well-recognized genetic causes, the majority are idiopathic or associated with environmental causes [223]. Although traditional molecular and anatomical techniques have been important in identifying the involved brain areas and pathological characteristics, they are not capable of extracting the full cellular and molecular complexities of these diseases. Since NDDs preferentially impact heterogeneous populations of cells in multiple brain areas, emerging technologies such as single-cell analysis and advanced proteomics offer unprecedented resolution to untangle heterogeneity of disease processes [224,225]. Such approaches enable cell-type-specific gene expression profiles and protein signatures to be delineated, which provides new opportunities for mechanistic insight and therapeutic intervention.

Proteomic approaches have become central to neurodegeneration research, as they enable large-scale identification and comparison of proteins, providing detailed insights into distinct cellular states and disease mechanisms [226]. Proteomic techniques such as two-dimensional gel electrophoresis (2-DE) have been recently used to study protease activity by identifying endogenous substrates under physiological and pathological conditions [227]. Since abnormal protease function and dysregulation of their substrates are strongly associated with neurodegenerative diseases, unbiased high-throughput screening of protease targets is essential for understanding how proteolysis drives disease progression [227]. Label-free liquid chromatography with tandem mass spectrometry (LC-MS/MS) has been used to perform comparative proteomic profiling of CSF from sporadic ALS, healthy controls, and other neurological diseases to identify new protein/pathway alterations and candidate biomarkers for ALS [228].

Recently, data-independent acquisition (DIA) mass spectrometry has also emerged as a reliable method for the quantification of the brain proteome, including the analysis of developmental and pathological protein changes [229]. These approaches have enabled the identification of aggregation-prone proteins such as tau, α-synuclein, and amyloid-β and the tracking of their dynamic solubility and conformational changes over the course of the disease. Importantly, mass spectrometry plays a central role in detecting post-translational modifications (PTMs) such as phosphorylation, ubiquitination, acetylation, and glycosylation, which strongly influence protein aggregation propensity and toxicity [230]. More than 95% of current data on PTMs were derived from MS-based proteomic studies, which is likely to increase in the future with the ongoing advances in instrumentation, methods, and bioinformatics [231,232].

Furthermore, single-cell proteomics has increasingly been recognized as a powerful complement to single-cell transcriptomics. While transcriptomics determines gene expression profiles, proteomics captures the functional molecules that bring about pathology. Single-cell proteomics, for example, can determine differential protein abundance, state of aggregation, or PTMs across individual neuronal or glial subtypes and offer a window into cell-type-specific vulnerability that cannot be explained completely by RNA [233,234]. The integration of proteomics with transcriptomics and genomics in multi-omics strategies is being increasingly employed to unravel the complexity of neurodegeneration. Multi-omics pipelines combine genetic risk variants (genomics), transcriptional deregulation (transcriptomics), and proteomic indications of aggregation and PTM states, thereby linking causal variants to functional molecular endpoints [235,236]. Such combined analyses not only improve biomarker discovery but also direct precision therapeutic interventions to the molecular underpinnings of protein misfolding and aggregation.

To characterize the contribution of all cell types of the midbrain to Parkinson’s disease pathology and to assess the cell type-specific risk, Smajić et al. [237] used single-nuclei RNA sequencing to compare over 41,000 transcriptomes from post-mortem midbrain tissue of idiopathic Parkinson’s disease (PD) patients and healthy controls. The findings showed that Parkinson’s pathology is not confined to dopaminergic neurons but also involves glial cells and other regions of the midbrain. Notably, one cluster of dysfunctional dopaminergic neurons was marked by CADPS2 upregulation and TH downregulation. Glial cells also exhibited clear changes: microglia were activated and more numerous, oligodendrocytes were stressed and reduced in number (with upregulated S100B), and astrocytes exhibited reactive features such as CD44 overexpression. Their findings showed that PD genetic risk variants are enriched for cell type-specific expression profiles and are most prominent in glia. Notably, their findings demonstrated the intricacy of PD and the power of single-cell approaches for uncovering discrete cellular contributions to disease pathology, emphasizing the need for glial involvement and cell-type-specific therapeutic targets.

In another study, Mathys et al. [238] performed high-resolution single-nucleus RNA sequencing of 80,660 transcriptomes from 48 human prefrontal cortices with varying amounts of Alzheimer’s pathology that demonstrated pervasive early-stage, cell type-restricted changes in gene expression. The data demonstrated six broad cell types to have transcriptional subpopulations that were distinct and identified prominent regulators of myelination, inflammation, and neuronal survival that play a role in disease-related profiles. Interestingly, global stress response genes were broadly upregulated in late stages, and there was also a sex bias with female cells dominating pathological subtypes. Differential APOE risk gene regulation was also found to be upregulated in microglia but downregulated in astrocytes, which highlighted the limitations of bulk RNA-seq to capture such cell-specific signatures. Importantly, the study demonstrated the power of single-cell transcriptomics in unveiling the intricate molecular and cellular heterogeneity of neurodegenerative diseases. It also shows evidence that the incorporation of single-cell analyses to dissect cell-specific stress responses, RNA metabolism disturbances, and protein aggregation can play a major role in determining disease progression and sex-dependent vulnerability in Alzheimer’s and other associated diseases.

10.4. Personalized Medicine and Gene Editing Approaches

One of the most significant problems in the treatment of NDDs is the time disconnect between the earliest manifestation of genetic mutations and the late onset of clinical symptoms, which often delays correct diagnosis and treatment. This diagnostic delay hinders timely treatment and restricts therapeutic options primarily to management of symptoms, such as behavioral interventions or medication for comorbidities like anxiety and irritability, rather than addressing the pathology of the disorder [239]. Consequently, long-term outcomes for the majority of those impacted by NDDs are suboptimal. Hence, the integration of personalized medicine approaches holds promise in revolutionizing the management and treatment of NDDs. Genetic testing and diagnostic profiling can enable active surveillance and preclinical intervention, even prior to the occurrence of symptoms. Even more importantly, the identification of disease-causing genetic mutations can uncover the specific molecular cascades leading to disease pathogenesis and thereby deliver precision-guided therapeutic interventions using known pharmacological molecules or novel drugs [239].

Genetic insights have already identified most of the dysregulated pathways in NDDs. Dysregulation in protein translation and growth pathways, such as in the mTOR signaling pathway, for instance, has been successfully targeted in preclinical models of diseases such as tuberous sclerosis complex (TSC) [240], PTEN-related macrocephaly [241], and 15q11-13 duplication syndrome [242]. Pharmacologic inhibitors of mTOR, like rapamycin and its derivatives (e.g., sirolimus and everolimus), have proved effective in reversing animal model behavioral and morphological abnormalities and are currently undergoing clinical trials in neurodegenerative and neurodevelopmental disorders [243,244]. Furthermore, modulation of neurotrophic signaling, particularly enhancing insulin-like growth factor 1 (IGF-1) and brain-derived neurotrophic factor (BDNF), has proved to be therapeutically beneficial in Rett syndrome models as well as in early human trials [245,246]. Strategies aimed at rebalancing neuronal excitation-inhibition, such as targeting metabotropic glutamate receptors (mGluRs) or GABAergic receptors, have been in the limelight too, but translation to clinical success has been inconsistent, as evidenced by their limited success in Fragile X syndrome trials [247,248].

New gene-editing and RNA-based technologies are also creating new opportunities for long-term, disease-modifying treatments. Antisense oligonucleotides (ASOs) offer a targeted mechanism for the regulation of gene expression at the level of RNA [239]. In Angelman Syndrome (AS), for example, where loss-of-function of the maternally inherited UBE3A gene causes severe cognitive impairments, ASOs have been used to unsilence the normally silenced paternal allele by blocking the UBE3A antisense transcript and rebalancing functional protein expression [249]. Other approaches are being developed to correct overexpression or underexpression of causative genes in other diseases, such as MeCP2 in Rett syndrome [250]. Hence further research targeted towards the unification of next-generation sequencing, CRISPR-based gene editing, ASOs, and AAV-delivered gene replacement therapies that are tailored to the genetic profiles of patients could drive a shift from reactive symptom management to focused root-cause-directed treatments in the treatment of neurodegenerative disease. Such precision medicine, while still an emerging science, has the capability not just to slow disease progression but to reverse neurological damage, and thus offer new hope for long-term recovery and improved quality of life.

11. Conclusions

Neurodegenerative diseases are increasingly being recognized as disorders rooted in the failure of proteostasis. Gradual accumulation of misfolded proteins such as tau, α-synuclein, TDP-43, and huntingtin identifies a common pathogenic mechanism destabilizing cell homeostasis and triggering neuroinflammation, oxidative damage, and synaptic impairment. Although significant progress has been achieved in the understanding of the structural and biochemical features of amyloidogenic proteins, the complexity and dynamic behavior of protein aggregation continue to present significant challenges to therapeutic intervention. Interestingly, advances over the last few years in single-cell transcriptomics and multi-omics platforms have demonstrated that neurodegenerative pathology inevitably starts with subtle, cell-type-specific molecular changes long before the onset of overt pathology. Interferences with myelination, stress responses, and RNA-binding protein activity add to the complexity of mechanisms at early stages of disease and to the importance of personalized therapeutic intervention.

Protein aggregation is not necessarily disease-inducing; some amyloid-like structures play useful functions in normal physiology. However, the distinction between functional and toxic assemblies in vivo remains difficult since both are based on the same type of intermolecular forces. Hence, understanding of the molecular and evolutionary distinctions between these assemblies, particularly at the aggregation kinetics and pathway regulation levels, will be necessary. Furthermore, unraveling how the proteostasis network becomes overwhelmed—by sequestration of molecular chaperones or loss of degradation capacity due to aging—can explain the self-reinforcing cycle of aggregate formation and proteome imbalance. While several aggregation inhibitors have been discovered in vitro and in vivo, no such therapy is currently available that entirely prevents or reverses disease progression. Therapeutic strategies in the future must not only target the aggregation inhibition but also the restoration of proteostasis by augmenting the vital proteostasis network components, promoting autophagy and proteasome activity, and modulating the stress signal pathways such as the unfolded protein response.