2.1.1 Abnormal SUMOylation of Tau protein in AD

Tau is an essential protein that plays a crucial role in microtubule binding and is highly abundant in neurons, particularly within the axons. The expression patterns of Tau exhibit distinct variations during different stages of development and in specific brain regions. In the adult human brain, six isoforms of Tau are expressed, ranging in length from 352 to 441 amino acids. This diversity in expression arises from the alternative splicing of three exons (2, 3, and 10) within the gene encoding the MAPT. The isoforms of Tau exhibit structural variations in terms of the quantity of N-terminal inserts and C-terminal repeat domains. Consequently, the N-terminal inserts are expressed in different forms at varying levels, designated as 0, 1, and 2 N-terminal inserts (0N, 1N, and 2N, respectively). Furthermore, Tau protein isoforms can be classified based on the number of microtubule-binding repeats (MTBRs) present in their C-terminal domain. Isoforms with four MTBRs are referred to as four-repeat (4R) Tau, while those with three MTBRs are known as three-repeat (3R) Tau. Consequently, any aberrant PTM within Tau domains, especially in the 3R or 4R binding repeats, results in the destabilization of microtubules.^51-53 Tau protein molecular weights also vary, with approximate values ranging from 36.8 to 45.9 kDa.⁵⁴ Therefore, Tau comprises distinct domains, including the N-terminal (also known as the projection domain), the proline-rich, microtubule-binding repeat (MTBR), and the C-terminal. Significantly, the proline-rich domain also assumes a crucial function in mediating the binding of Tau to microtubules.⁵⁵ The different isoforms of Tau play essential physiological roles in regulating microtubules and their functions, such as polymerization and stabilization, which involve several neuronal functions in the brain.^{13, 56} Additionally, Tau protein is known to possess certain characteristics, such as being heat-resistant, highly soluble, and naturally unfolded.²⁸ This abnormal hyperphosphorylation of Tau is associated with various NDDs collectively known as tauopathies.⁵⁷ The Tau protein is susceptible to various PTMs, containing phosphorylation, methylation, acetylation, SUMOylation, and ubiquitination.⁵⁶ While Tau serves various functions in neurons, the abnormal PTMs of Tau, especially hyperphosphorylation, cause its accumulation and subsequent neuronal death.^{53, 58} In AD, the presence of NFts composed of hyperphosphorylated Tau (p-Tau) is accompanied by the presence of ubiquitin. Studies have demonstrated that ubiquitin levels are significantly elevated in the brains of individuals with AD.⁵⁹ Moreover, Tau serves as a substrate for tumor necrosis factor receptor-associated factor 6 (TRAF6), a known ubiquitin E3 ligase. This interaction can lead to the degradation of Tau through ubiquitin-independent proteasomal degradation, highlighting a novel pathway for the regulation of Tau protein levels and potentially offering insights into the development of therapeutic interventions for tauopathies.⁵⁰

Tau can be SUMOylated by SUMO-1, which occurs more frequently than SUMO-2/3 and preferentially attaches at Lys 340. SUMOylation is frequently reported within the fourth microtubule-binding repeat (4R) region, where Tau interacts with microtubules.⁶⁰ Studies suggest that Tau SUMOylation promotes p-Tau at several sites linked to AD. However, this effect can be effectively counteracted by either site-specific mutagenesis of Tau at Lys340Arg (the SUMOylation site) or by concurrently inhibiting SUMO-1 activity.⁶¹ SUMOylation of Lys-340 can lead to an upregulation of Tau phosphorylation, causing an increase in insoluble Tau production and a decrease in Tau ubiquitination and degradation. Consequently, the combined impact of different PTMs may collaborate to regulate the aggregation of Tau through their cooperative interactions. The role of SUMOylation in Tau aggregation remains a topic of ongoing debate, and it may play a part in AD-like Tau accumulation. p-Tau probably promotes its SUMOylation, and, conversely, Tau SUMOylation enhances Tau phosphorylation. This, in turn, can reduce the solubility and ubiquitination of Tau through poly-SUMOylation, ultimately inhibiting the ubiquitination-mediated degradation of Tau.^{29, 61} Hence, when the proteasomal degradation process is inhibited, Tau tends to become more ubiquitinated and less SUMOylated. This suggests that there may be competition between SUMO and ubiquitin for the regulation of Tau stability. As a result, the regulation of Tau modifications may have significant implications for the pathogenic effects of neurodegenerative disorders.³⁸ The C-terminal microtubule domains of Tau protein contain several Lys residues that can be ubiquitinated, which can prevent SUMOylation at that site in AD.⁶² Interestingly, there is evidence to suggest that amyloid pathology may be necessary for Tau SUMOylation. In vivo, research on neuritic plaques in APP-transgenic mice has shown that SUMO-1 and ubiquitin exhibit distinct immunoreactivity in phosphorylated Tau aggregates. Specifically, in APP-transgenic mice, SUMO-1 immunoreactivity colocalized with phosphorylated Tau aggregates in amyloid plaques.⁶² However, in mutant Tau transgenic mice, SUMO-1 immunoreactivity was not detected in Tau aggregates.^{28, 62}

2.1.2 Abnormal SUMOylation of the APP in AD

APP is a type I transmembrane glycoprotein with a molecular weight ranging from 100 to 140 kDa.⁶³ It belongs to a protein family found in mammals, which includes two similar proteins known as APP-like protein 1 (APLP1) and APP-like protein 2 (APLP2). APP695 is predominantly expressed in neurons, while APP751 and APP770 are expressed in most tissues. There are three isoforms of APP, namely APP695, APP751, and APP770, which differ in length and contain 695, 751, and 770 amino acids, respectively.⁶⁴ The APP protein undergoes cleavage by various proteases, resulting in the generation of a wide range of peptides through two distinct and well-defined processing pathways: the amyloidogenic and nonamyloidogenic pathways. One of these peptides, known as the Aβ peptide, is derived from the amyloidogenic pathway and is considered a significant characteristic of AD. In AD, the cleavage of APP and the subsequent production of Aβ aggregates are primarily mediated by two proteases: β-secretase (also known as beta-site APP cleaving enzyme 1 (BACE1)) and γ-secretase.⁶⁵ The amyloidogenic processing of APP involves the cleavage of APP by BACE1, resulting in the production of two fragments: a soluble N-terminal fragment (APPsβ) and a membrane-bound C-terminal fragment (C99). Subsequently, C99 is cleaved by γ-secretase, leading to the generation of Aβ peptides with varying lengths (ranging from 38 to 43 residues) and a cytoplasmic polypeptide called the APP intracellular domain (AICD). AICD functions as a transcriptional factor within the nucleus, while the Aβ peptides are secreted into the extracellular space.^{66, 67} Therefore, AICD plays a crucial role in their functionality. The short cytoplasmic domain of AICD contains multiple functional and binding motifs, which collectively influence the trafficking and metabolism of APP.⁶⁸ The APP protein is proposed to have multifaceted physiological roles, encompassing neural migration, cell adhesion, the facilitation of synaptogenesis,⁶⁹ neurogenesis, neuronal differentiation, neurite outgrowth, axonal outgrowth, and the modulation of synaptic neurotransmission.^{70, 71} Nevertheless, the exact role of APP under normal conditions remains incompletely understood. In humans, APP has been associated with various pathophysiological processes related to neurodegenerative disorders, such as PD and AD.⁷² Likewise, a reduction or elimination of APP has been observed to correlate with neuronal loss and reduced synaptic activity in both in vitro and in vivo settings. Additionally, the reduction of APP in cultured neurons is associated with diminished neurite outgrowth.⁷³ Although APP has been recognized for its potential neuroprotective functions, it has been extensively investigated in the context of AD, primarily because of its role in the generation of Aβ peptides.⁶⁵ The accumulation of Aβ peptides is one of the key features of AD. Specifically, Aβ42 and Aβ43 are toxic due to their strong propensity for aggregation. Moreover, mutations in the APP and presenilin genes often lead to an elevated ratio of Aβ42 to Aβ40, which is a common characteristic observed in early-onset familial AD (FAD) cases. Abnormal processing of APP by β-secretase and γ-secretase can disrupt the equilibrium between the production and clearance of Aβ, leading to the formation of toxic oligomers, fibrils, and SP, especially in the context of AD.⁶⁶ Among the different forms of Aβ, Aβ1–42 has a propensity to rapidly and dynamically oligomerize, eventually forming insoluble fibrils that aggregate into plaques.⁷⁴

APP undergoes various PTMs that can contribute to amyloidogenesis and disease pathogenesis in the context of AD. Some of the known PTMs on APP include palmitoylation, ubiquitination, N-glycosylation, and phosphorylation.⁷⁵ There is some evidence to suggest that other PTMs may exert protective effects against AD, such as O-GlcNAcylation, ubiquitylation, and SUMOylation.^{76, 77} Studies have highlighted the significant involvement of SUMOylation in the regulation of APP. It has been identified as a potential substrate for this PTM, with particular emphasis on two Lys residues located at positions 587 and 595. These Lys residues are considered key targets for SUMOylation, suggesting that this modification may play a crucial role in modulating the functions and processing of APP. Notably, SUMO-1 and SUMO-2 can directly modify these Lys residues, which are located adjacent to the β-secretase cleavage site. Studies have demonstrated that SUMOylation at these sites can attenuate the production of Aβ. Eliminating the two SUMOylations at Lys residues 587 and 595 has been shown to stimulate the high generation of Aβ aggregates, suggesting that both SUMO-1 and SUMO-2 may interfere with the β-secretase cleavage site and inhibit Aβ aggregate formation. This result raises the possibility that the mutation of Lys-595 to asparagine in the Swedish (KM-to-NL) APP mutant could prevent SUMOylation at this residue and contribute to the production of Aβ in the Swedish APP mutant.^{37, 78} The modifications of SUMO-1 and SUMO-2 have demonstrated a significant capability of reducing the levels of Aβ aggregates. However, despite their effectiveness, the exact mechanism of this regulation remains elusive. The attachment of the SUMO protein at Lys 587 and 595 may sterically hinder the binding of β-secretase to APP.⁷⁹ In mouse models of AD, an upregulation of SUMO-1 expression has been observed in association with high levels of BACE1. This increase in BACE1 expression is enhanced in both sporadic and FAD, as well as in ischemic stroke, which is a significant risk factor for AD. This research demonstrates that SUMO-1 interacts with BACE1, leading to BACE1 accumulation and provoking the generation of Aβ peptides. Conversely, the depletion of SUMO-1 in this study resulted in the elimination of BACE1 protein and a reduction in Aβ levels. Furthermore, the available empirical evidence strongly supports the notion that the SUMOylation of BACE1 at the Lys 501 residue is critical for controlling its enzymatic activity and stability, which in turn affects Aβ production and cognitive function in AD mouse models.⁸⁰ These findings highlight the critical role of Lys 501 SUMOylation on BACE1 in regulating its stability and Aβ-generating activity. Notably, overexpression of the non-SUMOylated BACE1 mutant did not affect memory decline in wild-type mice and did not hasten the formation of SP, suggesting that SUMOylation of BACE1 plays a crucial role in AD pathogenesis.^{67, 80} In addition to BACE1’s involvement in the amyloidogenic pathway, it has been implicated in a variety of physiological processes and key pathophysiological mechanisms of AD. BACE1 is widely expressed in the brain, with high levels in specific neuronal cell types, oligodendrocytes, and astrocytes. BACE1 also localizes to various subcellular compartments, such as the plasma membrane and endosomes, indicating its involvement in multiple physiological and pathological processes within the brain.⁶⁷ Moreover, the overexpression of wild-type BACE1 facilitates the formation of SP and aggravates cognitive deficits in AD mouse models.

Interestingly, studies have shown that the non-SUMOylated mutant of BACE1 is susceptible to lysosomal degradation, leading to a decrease in Aβ formation and a reduction in cognitive dysfunction in mouse models of AD.⁸⁰ In contrast to other studies, research on fangchinoline, an alkaloid derived from the traditional Chinese medicine Stephania tetrandra S. Moore, has shown promising therapeutic effects in mouse models of AD. Specifically, fangchinoline has been found to promote the autophagy–lysosomal degradation of BACE1 without affecting its sumoylation. This mechanism contributes to the reduction of amyloidogenic processing of APP and the improvement of cognitive impairment.⁸¹ Moreover, it has been documented that BACE1 SUMOylation promotes the amyloidogenic processing of APP, leading to the production of Aβ aggregates. In the context of AD mouse models, BACE1 SUMOylation has been shown to suppress its phosphorylation at the S498 site. This observation aligns with studies indicating reduced phosphorylation of BACE1 at S498 in AD brain tissues. Furthermore, BACE1 SUMOylation suppresses its ubiquitination in mouse models of AD, similar to the findings regarding Tau SUMOylation and its impact on ubiquitination, which can contribute to Tau aggregation. Additionally, it has been observed that BACE1 phosphorylation inhibits its SUMOylation, promoting BACE1 degradation in vitro. Consequently, BACE1 SUMOylation reciprocally regulates its phosphorylation and ubiquitination processes.⁸²

It should be noted that SUMO-3 overexpression or facilitating SUMOylation can also modulate APP processing by reducing the amyloidogenic pathway and simultaneously enhancing the nonamyloidogenic pathway. SUMO-3 is detected and restricted in neuronal soma in brains from AD or Down's syndrome. The overexpression of SUMO-3 can be associated with the reduction of Aβ levels, although it does not have a direct interaction with APP protein levels.⁸³ In this study, both APP and BACE1 were increased along with the overexpression of SUMO-3. Hence, in contrast to mono-SUMOylation, poly-SUMOylation is responsible for reducing Aβ production.⁸³ Nevertheless, overexpression of SUMO3 leads to increased secretion of both Aβ40 and Aβ42 peptides and the upregulation of BACE1 expression. Conversely, it seems that endogenous sumoylation seems to play only an indirect role in modulating the amyloid processing pathway. Notably, overexpression of monomers of SUMO3 noncovalently leads to Aβ peptide production.⁸⁴ Consequently, the precise action of SUMO-3 in APP processing needs more investigation. Furthermore, SUMOylation of AICD is a protective mechanism in AD by facilitating the degradation of Aβ.⁸⁵ According to all these contradictory data, further investigation is warranted to elucidate the precise mechanisms by which SUMOylation influences APP-related pathways and its implications for various cellular processes and AD progression.

2.2.1 Abnormal SUMOylation of α-syn protein in PD

The α-syn protein is encoded by the α-syn gene, also known as sodium voltage-gated channel alpha subunit 1 (SNCA1). It consists of 140 residues and has a molecular weight of approximately 14 kDa.^{28, 93} α-Syn is part of a protein family called synuclein, which also includes β-syn and γ-syn.⁹³ These three members of the syn family are widely expressed in neurons and are primarily located at presynaptic terminals. Additionally, α-syn constitutes approximately 1% of the total cytosolic protein.^{93, 94} A subgroup of patients with early-onset PD had a mutation in the α-syn gene, SNCA, which led to the association between α-syn and PD. A53T/E, A30P, E46K, H50Q, and G51D are the six SNCA gene variants that have been connected to PD; SNCA duplication and triplication have also been identified to produce early-onset familial PD.⁹⁵ α-Syn is abundantly expressed in various types of neurons in both the peripheral and CNS, including during synapse development. α-Syn plays a multifaceted role in neuronal function. For instance, α-syn is a prominent neuronal protein primarily localized within presynaptic terminals. Its central function lies in the regulation of synaptic vesicle (SV) clustering and trafficking, notably without being found inside the vesicles themselves. Moreover, it can inhibit neurotransmitter release when it is overexpressed and interacts with the integral membrane proteins of SVs. Despite its involvement in various cellular processes, the precise physiological function of α-syn remains unclear.^{96, 97} Moreover, α-syn is typically found in neurons as a membrane-bound α-helical state and as an inherently disordered monomer.⁹⁸ When dysregulated, α-syn may accumulate together to create cross-β amyloid fibrils through less structured oligomeric intermediates, which seem to be especially harmful to neurons.⁹⁹

α-Syn is composed of three main domains, each serving a specific function. The first domain is located in the N-terminal region and is referred to as the amphipathic region. It consists of 60 residues and plays a critical role in membrane binding due to its amphipathic nature. α-Syn is composed of three main domains, each serving a distinct function. The second domain of α-syn is called the nonamyloid component (NAC) region, comprising 34 residues. The NAC region is involved in the aggregation and fibrillation of α-syn, contributing to the formation of pathological aggregates seen in PD. Finally, the C-terminal region of α-syn consists of 44 residues. This region is involved in various interactions with other proteins and cellular components, and it also plays a role in regulating α-syn's physiological function and its aggregation propensity.¹⁰⁰ The α-syn protein is classified as an intrinsically disordered protein due to its heat resistance, high solubility, and natural unfolded state.^{28, 101} However, structural alterations in α-syn have been observed in various studies.^{93, 102} While the molecular pathways underlying the involvement of α-syn in diseases like PD are still not fully understood, it is evident that PD is characterized by the aggregation and accumulation of α-syn.¹⁰³ During this process, α-syn aggregation leads to the formation of off-pathway, nonfibrillar, and soluble oligomers. These transient prefibrillar intermediate species, known as oligomers, eventually transform into insoluble fibrillar aggregates that exhibit a specific cross β-sheet conformation. The abnormal aggregation of α-syn oligomers plays a crucial role in the development of neurodegenerative conditions, collectively referred to as α-synucleinopathies. These conditions encompass PD, dementia with LB, and multiple system atrophy (MSA).¹⁰⁴ In α-synucleinopathies, a prominent pathological feature is the presence of LBs, which are intraneuronal inclusions. These bodies consist of aggregated α-syn that is immunoreactive and include protein inhibitors that spread to neighboring neurons.¹⁰⁵ Furthermore, α-syn aggregates can transmit their pathological conformation to other α-syn molecules in nearby cells, similar to the prion-like spread, resulting in the progression of the disease. In addition to the neurotoxic effects of α-syn oligomers, α-syn fibrils also play a crucial role in the propagation and spread of α-synucleinopathies.^{104, 106} The precise mechanisms underlying the emergence of Lewy pathology in transplanted dopamine neurons are not yet fully understood. However, empirical research suggests that the activation of microglia and the subsequent release of cytokines during immune activation may contribute to the misfolding of α-syn and facilitate its spread.¹⁰⁷ The misfolding and aggregation of α-syn involve a complex, multi-step process that includes various sequential conformational changes. Therefore, to gain a deeper understanding of PD, it is essential to investigate the initial stages of α-syn aggregation, particularly the process of oligomerization.^{93, 104}

Multiple PTMs have been reported for α-syn, including glycation, truncation, acetylation, nitration, phosphorylation, ubiquitination, and SUMOylation.¹⁰³ Abnormal modifications in these PTMs can impact the structure and function of α-syn, leading to protein dysfunction and potentially contributing to the development of PD.^{28, 103} In LBs, various notable modifications have been identified in α-syn. These modifications include phosphorylation at Ser-129, ubiquitination at specific Lys residues (12, 21, and 23), and specific truncations at Asp-115, Asp-119, Asn-122, Tyr-133, and Asp-135. These distinct PTMs contribute to the unique characteristics of the aggregated α-syn protein. Interestingly, this pattern of modifications has been consistently observed not only in sporadic PD but also in familial PD and MSA. This suggests that the preferential accumulation of normally produced Ser-129 phosphorylated α-syn may be a primary factor in the formation of LBs, a pathological hallmark of PD and related disorders.¹⁰⁸ Following the process of inclusion formation, ubiquitination of α-syn may occur subsequently, but it is not a prerequisite for α-syn fibrillization and inclusion formation.¹⁰⁹ A study has shown that the level of ubiquitination in the assembled, filamentous form of α-syn is reduced compared with its soluble form. The major sites of ubiquitination are located at Lys-6, 10, and 12 in the amino-terminal region of the α-syn molecule. These findings suggest that ubiquitination occurs after the formation of α-syn filaments, and the same may hold for α-synucleinopathies observed in the brains of affected individuals.¹¹⁰ The process of α-syn SUMOylation serves as a mechanism to counterbalance the effects of ubiquitination, thereby mitigating its degradation through the proteasome. Several studies have suggested that α-syn SUMOylation is associated with increased steady-state levels and aggregation, as well as potentially enhancing its exosomal release and nuclear translocation.¹⁰³ Consequently, SUMOylation impedes the degradation of α-syn and contributes to its accumulation by inhibiting ubiquitination.¹¹¹ α-Syn possesses two Lys sites, Lys-96 and Lys-102, which are more readily targeted by SUMO-1 than SUMO-2/3. Several E3 ubiquitin ligases, including protein inhibitors of activated STAT 2 (PIAS2), hPc2, and tripartite motif containing 28 (TRIM28), have been identified to SUMOylate α-syn, resulting in either mono-SUMOylation or multi-monoSUMOylation.^{28, 103} Evidence suggests that SUMOylation at the Lys102 residue inhibits the aggregation of α-syn more effectively than at Lys-96, and modification via SUMO-1 is a stronger inhibitor than SUMO-3.¹¹²

The elevated levels of SUMOylated α-syn and SUMOylation machinery components in PD brains indicate a crucial role of SUMOylation in the aggregation of α-syn and formation of LBs. Consequently, the inhibition of SUMOylation could be a promising strategy for preventing the accumulation, aggregation, and propagation of α-syn in PD.¹¹¹ Studies have identified PIAS2 as a SUMO E3 ligase responsible for increasing SUMOylation in the α-syn. Furthermore, ubiquitination of α-syn typically directs it to the proteasome for degradation. This process is reduced by both the E3 ubiquitin-ligase seven in absentia homolog (SIAH) and neural precursor cells that express developmentally downregulated 4 (Nedd4) E3 ubiquitin ligases. However, PIAS2-mediated SUMOylation of α-syn reduces E3 ubiquitin ligases. As a result, α-syn accumulates and aggregates within inclusions. These findings suggest that maintaining a balance between α-syn SUMOylation and ubiquitination is critical for its proper clearance and degradation. Dysregulation of this balance may contribute to the pathogenesis of PD and related disorders.¹¹¹ Additionally, the ubiquitination of α-syn by SIAH-1 does not result in its degradation via the proteasome; rather, it promotes the aggregation of α-syn and enhances its toxicity. This study suggests that SIAH-1-mediated α-syn ubiquitination could significantly influence the development of LBs and the pathogenesis of related conditions.¹¹³ Furthermore, the SUMOylation of α-syn by PIAS2 promotes aggregation both directly and indirectly by impeding degradation and facilitating accumulation through the inhibition of ubiquitin-dependent pathways.¹¹¹ In particular, increased expression of PIAS2 has been detected in the tissues of individuals with PD, providing further evidence of the SUMOylation role in the disease.^{103, 114} Therefore, SUMOylation inhibitors may help reduce α-syn levels and mitigate PD-related aggregation. As a result, there is a decrease in the accumulation, aggregation, and spreading of intracellular α-syn in PD.¹¹¹

2.2.2 Abnormal SUMOylation of Parkin protein in PD

Parkin (also known as PARK2) is a protein of approximately 52 kDa, encoded by the PARK2 gene.^{115, 116} It is primarily expressed in various tissues, with a notable presence in the brain and muscles.¹¹⁷ Parkin serves as a unique multifunctional ubiquitin ligase, functioning as an E3 ubiquitin ligase. Its role involves attaching ubiquitin molecules to specific protein substrates. Thereby, it can play a crucial role in the process of ubiquitination and direct the proteins for degradation through the UPS.^118-120 Remarkably, Parkin has diverse functions, especially within neurons, which are believed to be highly beneficial and serve a protective purpose.¹²⁰ Parkin is known to target a diverse array of over 20 different substrates, some of which play a crucial role in neuronal physiology and pathology. These substrates include O-glycosylated α-syn, synphilin-1, CDCrel-1 (a member of the septin family), Parkin-associated endothelin receptor-like receptor, and dopamine transporter. As an E3 ubiquitin ligase, Parkin functions to prevent protein accumulation.^{119, 121} However, the loss of Parkin's E3 ligase function can lead to substrate accumulation, contributing to neurotoxicity and the development of pathological conditions in NDDs.^{119, 121} Therefore, Parkin plays an essential role in neuronal protection. It acts as a neuroprotective factor by promoting the removal of defective mitochondria, preventing protein accumulation, and shielding cells from excitotoxicity and unfolded protein stress.^{118, 121} Furthermore, Parkin's involvement in various aspects of mitochondrial functioning, including mitochondrial fusion/fission, mitochondrial biogenesis, mitochondrial transport, mitophagy, ER–mitochondrial interactions, cell signaling, and apoptosis, is tightly interconnected. The dysfunction of Parkin has been implicated in neurodegenerative disorders.¹¹⁸ However, recent research has also characterized Parkin as a tumor suppressor.¹²² Mutations in the Parkin gene cause autosomal recessive PD. It may also be involved in sporadic PD. According to a few studies, patients with Parkin mutations indicate dopaminergic neuronal loss in the SN and noradrenergic neuronal loss in the locus coeruleus, with accompanying gliosis.¹¹⁵ Moreover, the pathogenicity of PD appears to be associated with dysfunctional mitochondria, oxidative stress, and protein misfolding and aggregation.^{115, 121} It is noteworthy that activation and/or suppression of Parkin can be regulated through various PTMs, including phosphorylation, ubiquitination, nitrosylation, sulfhydration, sulfonation, SUMOylation, and neddylation.¹²³ Studies suggest that Parkin plays a critical role in PD, as it has been found in LBs, which are the pathological hallmark of the disease. Hence, Parkin dysfunction has been identified as a crucial factor in the pathogenesis of PD.^103,109 Furthermore, reduced proteasomal activity leads to the accumulation of Parkin and a subsequent decrease in its ligase activity. This ultimately results in the formation of noncytotoxic inclusions. These findings provide additional evidence supporting the involvement of Parkin dysfunction in the development of PD.^{124, 125} For instance, nitrosylation can inhibit Parkin's activity as an E3 ubiquitin ligase.¹²³ Additionally, dephosphorylation of Parkin can enhance its activity in cells under ER stress, allowing it to combat unfolded proteins.¹²⁶ Phosphorylation of Parkin has been reported to increase its activity, but the absence of phosphorylation or the presence of an aberrant constitutive form that leads to overactive Parkin can be detrimental.¹²⁷ In addition to these PTMs and their effects, SUMOylation has recently gained attention in drug discovery due to its regulatory role in various processes.^{37, 76} Parkin is a substrate for SUMOylation.¹²⁸ It has been observed that SUMO-1 binds covalently to the Parkin protein in both nonneuronal and neuronal cells.¹²⁸ The SUMOylation of Parkin through SUMO-1 reduces its availability for mitochondrial recruitment by increasing its self-ubiquitination (Ub) and facilitating its translocation from the cytoplasm to the nucleus.¹²⁸ Consequently, the interaction of SUMO-1 with Parkin can alter its intracellular localization and its E3 ubiquitin ligase activity.¹²⁸ Moreover, impaired Parkin function can suppress mitochondrial biogenesis by leading to an accumulation of Parkin interacting substrate (PARIS), subsequently reducing the levels of peroxisomal proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α).^{128, 129} These findings suggest that SUMOylation, specifically through SUMO-1, can influence the activity and localization of Parkin.³⁸ Given Parkin's involvement in various mitochondrial activities and the clearance of substrate accumulation, SUMOylation of Parkin may have implications for PD (Figure 3).¹¹⁸

2.2.3 Abnormal SUMOylation of DJ-1 protein in PD

DJ-1, also known as DJ-1/PfpI, ThiJ/PfpI, or DJ-1/ThiJ/PfpI, is a small protein that belongs to a superfamily. It is expressed in various human tissues, including the brain, skeletal muscle, liver, kidney, and more.^130-132 In the human brain, DJ-1 is predominantly expressed within astrocytes and exhibits sensitivity to oxidative stress situations.¹³³ DJ-1 exists in 10 isoforms in the human brain, consisting of six monomeric forms and four dimeric forms.¹³⁴ DJ-1 is present in both glial and neuronal cells, localized in the cytoplasm and nucleus.¹³⁵ The DJ-1 protein is encoded by the Parkinsonism-associated deglycase gene (PARK7) and consists of 198 residues, weighing approximately 20 kDa.^{130, 136} Mutations in PARK7 have been implicated in familial PD.⁸⁰ Initially identified in 1997, the PARK7 gene was recognized as a mitogen-dependent oncogene associated with a Ras-related signal transduction pathway. DJ-1 is a highly conserved intracellular protein.¹³⁷

The DJ-1 protein serves multiple roles, including functioning as a molecular chaperone, enzyme, and protease. It also regulates mitochondrial homeostasis, transcription pathways, antioxidative stress reactions, and oxidative stress-induced apoptosis. Additionally, DJ-1 acts as a stress sensor and is upregulated in response to various stresses, particularly oxidative stress.¹³⁸ In neurons, DJ-1 not only acts as a sensor and protects neurons against reactive oxygen species (ROS) and oxidative phosphorylation, but it also inhibits the aggregation of α-syn through chaperone-mediated autophagy (CAM). Studies have demonstrated that the suppression of DJ-1 leads to increased levels of aggregated α-syn in experimental models of PD. Conversely, overexpressing DJ-1 reduces the levels of α-syn. These findings highlight the significance of DJ-1 in maintaining the balance of α-syn and suggest that it could be a potential therapeutic target for PD treatment.^{136, 138} There is substantial evidence linking DJ-1 to various types of cancer through the activation of signaling pathways, particularly the Wnt/β-catenin signaling pathway, which promotes proliferation, invasion, and metastasis.^{136, 139} However, the precise contribution of DJ-1 to cell survival in cancer remains unclear.¹³⁹ Notably, research suggests that the DJ-1 protein could serve as a potential biomarker for the diagnosis and prognosis of certain cancers, including acute leukemia. Its overexpression in cancer cells and its presence in blood, secretory fluids, ascites, and pleural effusion make it a promising candidate for diagnostic purposes.^{136, 140} Additionally, DJ-1 may also be an attractive therapeutic target in cancer treatment. Furthermore, DJ-1 has been implicated in the onset and pathogenesis of not only sporadic PD but also familial PD, where it can become inactive due to excessive oxidation.¹³⁸ DJ-1 has been associated with other NDDs, such as HD, amyotrophic lateral sclerosis (ALS), AD, ischemia–reperfusion injury, and autosomal recessive disorders.^{141, 142}

The main cause of PD is the degeneration of dopaminergic neurons in the SN, leading to a reduction in the levels of a neurotransmitter known as dopamine in the striatum.^{143, 144} This deficiency of dopamine is primarily responsible for the manifestation of motor deficits commonly observed in PD. However, it is important to note that dopamine may also play a role in the cognitive impairments observed in some individuals with PD. DJ-1 protein plays a protective role in preventing neurodegeneration in dopaminergic neurons associated with PD, employing various mechanisms.¹⁴³ It regulates adenosine triphosphate (ATP) synthase protein components by interacting with the ATP synthase β subunit, thereby promoting dopaminergic cell metabolism and growth.¹⁴⁵ DJ-1 performs activities like chaperone that can influence α-syn aggregation. Therefore, DJ-1 can suppress α-syn aggregation by taking part in the beginning step of the aggregation process. In vitro experiments suggest that DJ-1 can directly interact with α-syn monomers and oligomers. Overexpression of DJ-1 reduces α-syn dimerization and significantly lowers α-syn levels, resulting in increased aggregation. However, mutations in DJ-1 in familial PD disrupt this process.¹⁴⁶ Loss of DJ-1 expression and transcriptional dysfunction are directly associated with impaired dopamine synthesis, which contributes to the development of PD. Patients with DJ-1 mutations exhibit reduced dopamine uptake, although it remains unclear whether the loss of DJ-1 function affects the survival of dopaminergic neurons. The primary causes of neurodegeneration in PD are believed to be associated with oxidative stress and mitochondrial malfunction.¹³⁸ Reduced or lost DJ-1 function triggers the onset of oxidative stress-related diseases, particularly in PD.¹³⁸

DJ-1 is used as a substrate for SUMOylation, with Lys-130 being the main Lys residue for SUMOylation. This residue becomes conjugated to SUMO-1 through the two E3 SUMO-1 ligases, namely PIASxa or PIASy.¹⁴⁷ Mutations in Lys-130 impair various functions of DJ-1, including its role in anti-UV-induced apoptosis, Ras-dependent transformation, and cell growth promotion. Exposure of DJ-1 to UV radiation leads to an increase in its SUMOylation, causing it to undergo a shift toward a more acidic state.¹⁴⁷ SUMOylation plays a critical role in repressing the transcriptional activity of p53 by DJ-1. In this regard, the non-SUMOylated form of DJ-1, known as DJ-1 (K130R), undergoes translocation from the nucleus to the cytoplasm, resulting in the loss of its protective function against ultraviolet (UV)-induced cell death by failing to repress p53 transcriptional activity.¹⁴⁸ The M26I mutation in DJ-1 disrupts its interaction with SUMO-1, leading to erroneous SUMOylation of DJ-1. As a consequence, it contributes to its insolubility, partial localization in mitochondria, and degradation by the proteasome system.¹⁴⁹ Despite DJ-1's homodimeric nature, a mutation on the Leu-166 residue reduces its stability and causes it to exist as a monomer, thereby interfering with homodimerization.^{150, 151} Consequently, the Lys 166 mutation is considered a fundamental cause of early-onset PD, as it results in the loss of normal DJ-1 activity and promotes its degradation through the UPS.^{150, 151} Improper SUMOylation of the Lys-166 mutant DJ-1 renders it insoluble, which may contribute to the onset of PD.¹⁴⁷

2.2.4 Abnormal SUMOylation of dynamin-related protein 1 in PD

Dynamin-related protein 1 (Drp1) is a multifunctional protein encoded by the DNM1L gene, consisting of 736 amino acids.¹⁵² Drp1 consists of four distinct domains: the middle domain, the variable domain (VR), and the C-terminal GTPase effector domain.¹⁵³ It is primarily located in the cytoplasm, and it has also been observed in various organelles such as mitochondria, Golgi, and peroxisomes.¹⁵⁴ Drp1 plays a crucial role in maintaining mitochondrial dynamics, specifically in processes such as fission, distribution, and potentially peroxisomal fragmentation. Notably, impaired mitochondrial dynamics can occur early in the development of NDDs.¹⁵⁵ This suggests that the proper functioning of Drp1 and mitochondrial dynamics is vital for preserving cellular health and preventing the onset of disease. Generally, a variety of diseases, including neurodegenerative disorders, cardiovascular diseases, and different types of cancer, disrupt mitochondrial dynamics, which play a critical role in sustaining normal cellular function. Therefore, gaining a deeper understanding of the mechanisms governing mitochondrial dynamics and their regulation could pave the way for the development of novel therapies targeting these diseases.^{156, 157}

Neurons have a high demand for energy production, and their proper functioning heavily depends on the activity of mitochondria. As a result, mitochondria are distributed throughout the cytosol to efficiently generate ATP through oxidative metabolism. Any abnormalities in mitochondrial dynamics, particularly impairments in Drp1-mediated mitochondrial fission, can have a profound impact on various neurodegenerative disorders, such as AD, PD, and HD.¹⁵⁸ In mitochondrial dynamics, Drp1 plays a crucial role as a key mediator in the fission processes, and it has a dynamic relationship with mitochondrial fusion.¹⁵⁴ Fission and fusion processes not only regulate the morphology and size of mitochondria but also play important roles in mitophagy, organelle transport, and cell death pathways. These processes are involved in maintaining the overall health and functionality of the organelles as well as controlling the shape and size of mitochondria within the cell.¹⁵⁸ During fission, two mitochondria are generated. Several outer mitochondrial membrane proteins, including fission-1, mitochondrial fission factor, Mid49, and Mid51, act as receptors for Drp1 and recruit it to the site of mitochondrial fission.¹⁵⁴ Therefore, the loss of Drp1 disrupts mitochondrial fission, leading to the formation of mitochondrial aggregates, which can impair neuronal development.¹⁵⁹ Additionally, Drp1 is found within the ER lumen, and ER-associated Drp1 promotes the formation of ER tubules.¹⁵⁹ Given the crucial role of Drp1 in neuronal activities, investigating the PTMs of Drp1 that affect its function and morphology could be a valuable target in both normal conditions and NDDs.^{155, 160} Drp1 activity is tightly regulated through various PTMs, such as phosphorylation, SUMOylation, palmitoylation, ubiquitination, S-nitrosylation, and O-GlcNAcylation, as well as its interaction with other mitochondrial proteins.¹⁶¹ It appears that the Drp1 protein is the only mitochondrial target of SUMOylation.¹⁶² Research indicates that when Drp1 is located at the site of fission, SUMO is found to be localized there. This suggests that SUMOylation may affect the translocation of Drp1 from the cytosol to the mitochondria, which in turn induces mitochondrial fission.³⁷ SUMOylation and deSUMOylation play a pivotal role in regulating mitochondrial dynamics by affecting Drp1 activity.¹⁵⁹ Therefore, SUMOylation of Drp1 occurs through the interaction with the SUMO-conjugating enzyme Ubc9.¹⁵⁵ Drp1 can be modified by all three SUMO isoforms: SUMO-1, SUMO-2, and SUMO-3. SUMOylation happens at two pairs of Lyss or more, particularly Lys532, 535, 558, and 568, which are located on the highly VR domain.^{152, 153} SUMOylation enhances the stability of Drp1, while deSUMOylation reduces its stability.¹⁵⁹ Hence, SUMO-1-mediated SUMOylation of Drp1 promotes the localization of Drp1 on mitochondria and its attachment to mitochondria, the release of cytochrome C, and mitochondrial fragmentation. Conversely, SUMO-2/3-mediated SUMOylation of Drp1 decreases its localization and relocates it to the cytoplasm, leading to a reduction in mitochondrial fission.¹⁵⁹ It has been reported that SUMO-1 promotes the localization of Drp1 on mitochondria, while SUMO-2 and SUMO-3 decrease its localization. Furthermore, the fission process plays a role in apoptosis, and Drp1 is required for this condition. While it is known that Drp1 can undergo SUMOylation at multiple Lys residues on its VR domain, the precise consequences of the modification remain incompletely understood, including its impact on the clearance of damaged mitochondria through mitophagy. Further research is needed to fully elucidate the functional consequences of SUMOylation on Drp1 and its role in mitochondrial biology.^{37, 163}

The inhibition of mitochondrial fission leads to the accumulation of cytochrome C within the mitochondria, thereby hindering the progression of apoptosis.³⁷ Interestingly, research indicates that the upregulation of SUMO-1 is associated with an increase in mitochondrial fission, which could potentially contribute to apoptosis.^{155, 164} It has been suggested that SUMO-1 plays a protective role by preventing Drp1 degradation, stabilizing the Drp1 protein, and promoting its binding to mitochondria. This could explain why the upregulation of SUMO-1 leads to mitochondrial fragmentation and apoptosis.¹⁶⁴ Studies have shown that under oxidative stress conditions like myocardial ischemia/reperfusion (MI/R), Drp1 undergoes SUMOylation, resulting in its relocation from the cytosol to the mitochondria. This, in turn, triggers abnormal mitochondrial fission, leading to impaired morphology and function. Similarly, in the context of MI/R, the upregulation of SENP3 (a SUMO protease) without altering the overall expression of Drp1 is associated with increased mitochondrial Drp1 levels by targeting SUMO-2/3. Consequently, this correlation is linked to infarction.¹⁶⁵ However, other studies demonstrate that oxygen-glucose deprivation degrades SENP3, resulting in the decline of the binding of Drp1 to mitochondria. Following reperfusion, increased SENP3 leads to the deSUMOylation of Drp1, promoting its binding to mitochondria. This, in turn, causes mitochondrial fragmentation and the release of cytochrome C, ultimately leading to neuronal death.¹⁵³ Therefore, Drp1 SUMO modifications play crucial roles in mitochondrial dynamics, and understanding their clear function is essential. In neurodegenerative disorders, extensive mitophagy and dysfunctional mitochondria are consequences of Drp1-mediated mitochondrial fission. In PD, several proteins associated with the disease, such as PINK1, Parkin, and DJ1, have been observed to directly interact with Drp1 and regulate mitochondrial fission and fusion. Specifically, Parkin stimulates the proteasomal degradation of Drp1, leading to a reduction in mitochondrial fission by decreasing the available amount of Drp1.¹⁵² Consequently, the loss of Drp1 function contributes to the death of nigrostriatal dopaminergic neurons, which is a characteristic feature of PD. This is because Drp1 is involved in the survival of dopaminergic terminals in the caudate-putamen region, which is necessary for maintaining their axons.¹²⁹ Furthermore, in patients with AD, there is an increase in Drp1 levels, which interact with Aβ and phosphorylated Tau. This interaction results in the upregulation of mitochondrial fission, leading to neuropathology and cognitive decline.^{152, 166} Considering the role of the fission protein Drp1 in mitochondrial function, it is evident that Drp1 plays a crucial role in NDDs.

2.3.1 Abnormal SUMOylation of Huntington protein in HD

The Huntington protein (Htt protein) in humans is a large protein consisting of 3144 residues (347 kDa) and is involved in many cellular processes.^{170, 173} Htt is abundantly expressed in the nervous system as well as other tissues, including the testes.^{167, 174} A complete understanding of Htt's function in the brain has not yet been achieved. However, it is believed to play a critical role in regulating neuronal and glial function. Among the known functions of Htt are participation in pre- and postsynaptic functions and implications in the regulation of apoptotic signaling, vesicle transport, transcription, and nuclear import.^173-175 Additionally, the Htt protein plays a role in promoting the synthesis and axonal transport of brain-derived neurotrophic factor (BDNF).^{173, 175} BDNF is a critical protein for the growth and survival of neurons, as well as for regulating synaptogenesis and synaptic plasticity, both of which are necessary for learning and memory in the adult CNS.¹⁷⁶ Htt is probably involved in the formation of cilia, which are tiny, hair-like structures that assist in regulating the flow of cerebrospinal fluid (CSF) in the brain and contribute to the movement of this fluid within the ventricular system.^{175, 177} The Htt protein contains several different domains, including a poly-Q tract and a proline-rich region.^{178, 179} Despite some knowledge about the structure of the Htt protein, such as that it is mainly composed of alpha-helices, there is still a lack of understanding of the protein as a whole.^{178, 179} One of the crucial regions of the human Htt protein is located at the N-terminus, which contains a polymorphic glutamine stretch near the proline-rich region and likely affects turnover by preventing protein aggregation through the regulation of protein–protein interactions (PPI).¹⁷⁴ Although the solubility of the Htt protein is not fully understood, studies have detected its soluble and aggregated isoforms, such as soluble mutant Htt (excluding exon 1 Htt) and aggregated Htt. These investigations aim to understand how Htt solubility corresponds to the onset and progression of the disease.^{180, 181} The formation of toxic, soluble oligomers of Htt during the aggregation process is believed to contribute to HD.¹⁸²

Mutations in the Htt gene have been associated with the development of HD.¹⁶⁷ Given that HD is an autosomal dominant disease, the loss of function of the Htt protein has long been recognized as a contributing factor to the complex pathology of the disease.¹⁸³ However, the precise molecular mechanisms underlying the pathology of HD are still largely unknown.¹⁸⁴ The most significant features of HD are the presence of inclusion bodies and the deposition of aggregated protein inside cells. A mutation in the IT15 gene on chromosome 4's short arm causes HD.¹⁸⁵ Therefore, these protein inclusions primarily consist of N-terminal fragments of mHtt. Despite being a characteristic feature of the disease, these inclusions are considered to have a protective role rather than being contributors to neurodegeneration. Soluble oligomeric assemblies of huntingtin formed early in the aggregation process are considered potential toxic agents in HD.¹⁸⁶ The poly-glutamine moiety of the Htt protein is encoded by the cytosine–adenine–guanine (CAG) trinucleotide repeats in exon 1 of the mutated gene. The variety of CAG repeat lengths in normal people is 7–34. With a length inversely correlated with the age at which the disease first manifests, the CAG repeat is both enlarged and unstable in individuals with HD. HD is produced by repeat lengths greater than 40 glutamines, and juvenile-onset is always caused by repeat lengths greater than 70 glutamines.¹⁸⁷ Consequently, as a monogenic disease, one of the key pathogenic causes of HD is associated with the expression of the CAG triplet repeat in the Htt, which leads to the production of mHtt proteins with an expanded poly-Q domain in the N-terminus at exon 1.^{184, 188} Additionally, there is a significant correlation between the length of the polyQ repeat and the aggregation of protein.¹⁸⁶ The fragment of exon 1 within the huntingtin protein (Httex1) has been extensively researched and is identified for its occurrence of improper splicing and proteolytic processing. Notably, the first 17 amino acids (Nt17) of exon 1 undergo PTMs that play roles in regulating the cellular functions of the huntingtin protein in both healthy individuals and those with HD.¹⁸⁹ The length of the poly-Q tract in Htt has an impact on the protein's PTMs, which in turn affects its subcellular distribution, function, cleavage, and stability.^{174, 188} The polyproline (polyP) domain adjacent to the polyQ tract in Htt is responsible for maintaining the protein's solubility, and it is also involved in regulating interactions with vesicle-bound proteins.¹⁶⁷

Therefore, the Htt protein undergoes various PTMs, including SUMOylation, ubiquitination, phosphorylation, acetylation, palmitoylation, and proteolytic cleavage, which are believed to have a significant impact on the protein's PPI flexibility.^{167, 174} Furthermore, PTMs can impact the subcellular localization of Htt.¹⁷⁴ While certain PTMs, such as phosphorylation at S13/16 and S42110, may have a protective effect against the toxic impact of mutant Htt, several PTMs are associated with HD pathogenesis and the increased toxicity of mutant Htt. Additionally, myristoylation is another potential protective PTM in Htt, but its levels decrease in the presence of an expanded polyQ, which may serve as a potential protective PTM.¹⁹⁰ The N-terminal region of Htt contains a site for both SUMOylation and ubiquitination, adjacent to polyQ and polyproline (polyP) tracts.¹⁶⁷ When Htt undergoes ubiquitination, it is degraded by proteasomes, whereas the same Lys residues undergo SUMOylation to protect it from degradation.^{191, 192} The process of ubiquitination on the pathogenic Htt fragment (Httex1p) inhibits neurodegeneration, whereas the SUMOylation of the pathogenic fragment of the protein is believed to contribute to the exacerbation of neurodegeneration.¹⁸² The Htt protein contains three important Lys residues, Lys-6, Lys-9, and Lys-15, which are conjugated to SUMO.^{174, 182} Both SUMO-1 and ubiquitin can modify the same Lys residues on Httex1p. Preventing both modifications through Lys mutations reduces HD pathology, suggesting that the role of SUMOylation in HD pathology is more extensive than simply inhibiting the ubiquitination and degradation of Httex1p.¹⁸² There is evidence suggesting that SUMO-2 modification plays a role in the pathogenic accumulation of mutant Htt and the progression of HD with age. The accumulation of SUMO-2-modified proteins in the insoluble fraction of the postmortem striatum in HD indicates their involvement in the disease. As a result, HD might be influenced in vivo by SUMO-2-modified proteins. The striatum, a region significantly affected by HD, has been observed to exhibit an accumulation of SUMO-2, correlating with the pathological accumulation of Htt.^{170, 191} Moreover, Htt undergoes SUMO-2 modification, and the protein inhibitor of activated STAT 1 (PIAS1) acts as an E3 ligase for Htt SUMOylation. PIAS1, expressed in the brain, specifically enhances SUMO-1 and SUMO-2 modifications of Htt, crucial for the aforementioned protein accumulation.¹⁹¹ Moreover, when Htt is SUMOylated, it can lead to a toxic accumulation of the protein.¹⁹¹ mHtt undergoes SUMOylation via Rhes, resulting in Rhes having a higher efficacy in SUMOylation compared with wild-type, thereby protecting mHtt from degradation. This process elevates the levels of the soluble cytotoxic form of the protein. Rhes, the small guanine nucleotide-binding protein (G-protein), is predominantly found in the striatum.^{174, 193} It plays multiple roles in the SUMOylation process, including acting as a SUMO-E3 ligase, enhancing the efficiency of Ubc9 in SUMOylation mHtt, and promoting cross-SUMOylation between SAE1, SAE2, and Ubc9.¹⁹⁴ As mentioned earlier, the interaction between Rhes and mHtt leads to the SUMOylation of mHtt, which has a cytotoxic nature and ultimately results in cytotoxicity. The interaction between Rhes and mHtt triggers the cytotoxic SUMOylation of mHtt, potentially causing cytotoxicity and contributing to the observed localized neuropathology in HD.¹⁹³ The SUMOylation of Htt may lead to a distinct pathway of aggregation that could potentially impact Htt toxicity.¹⁹⁵ Ubc9, the only identified SUMO-E2 ligase, SUMOylated mHtt at specific Lys sites (6, 9, 15, and 91).¹⁹⁴ The SUMOylation of Htt has been suggested to increase the levels of toxic oligomers, and it is also possible that soluble oligomers may have toxic effects.¹⁸² While most Htt aggregates are found in the cytosol, the accumulation of Htt in the nucleus may enhance its toxic effects. SUMOylation on Htt protein has been found to have paradoxical effects. Subsequently, it reduces the formation of aggregates while its solubility is enhanced.^{191, 192} Therefore, the Htt protein can undergo a pathological fragmentation process, leading to the formation of a fragment known as a pathogenic fragment of Htt (Httex1p), which can also be SUMOylated by SUMO-1. This suggests that SUMOylation might impact the molecular properties and functions of Httex1p. The same Lys residues targeted by SUMOylation might also undergo modification through ubiquitination. The contribution of SUMOylation to HD pathology has been shown to go beyond inhibiting Htt ubiquitination and degradation in a drosophila model of HD. SUMOylation of Httex1p intensifies neurodegeneration, whereas ubiquitination of Httex1p appears to counteract it. Additionally, mutations in Lys (K6,9) and arginine (R15) residues, preventing both SUMOylation and ubiquitination of Httex1p, have been shown to improve HD pathology.¹⁸² Furthermore, research indicates that SUMO-1 exacerbates the solubility and toxicity of Htt. Considering the information, different outcomes are anticipated depending on which SUMO paralog is involved in Htt conjugation.¹⁷⁰ Modifying Htt through either SUMO-1 or SUMO-2/3 has the potential to regulate HD and provide new opportunities for the development of neuroprotective therapies.^{38, 170} Therefore, it is hypothesized that reducing the level of SUMOylated Htt in neurons could be a therapeutic strategy. This reduction could be achieved by lowering the expression of the SUMO-1 precursor, inhibiting SUMO-1 ligases, and enhancing isopeptidase activity to facilitate the removal of SUMO-1.¹⁸²

3.1.1 Abnormal phosphorylation of Tau protein in AD

Among the extensively studied PTMs, Tau phosphorylation is a key focus. It greatly influences Tau's solubility, stability, localization, degradation, aggregation, function, and interactions with microtubules. The phosphorylation of Tau can have a dual effect on its function, leading to both beneficial and detrimental outcomes in physiological and pathological conditions, respectively. Therefore, the specific phosphorylation patterns observed at particular residues of Tau carry substantial importance in the realm of tauopathies, including AD.²⁰⁵ In its normal state, the Tau protein typically carries 2–3 moles of phosphate per mole, which is essential for its function in microtubule assembly, especially within axons.^{206, 207} Consequently, Tau's physiological and pathological roles are intricately linked to the fundamental processes of phosphorylation and dephosphorylation. The complex interplay between Tau's normal and abnormal functions is closely tied to these underlying processes of phosphorylation and dephosphorylation. Preserving the proper functioning of Tau relies on a delicate equilibrium between the enzymes known as kinases and phosphatases, responsible for adding and removing phosphate groups from Tau, respectively. Any disruption in this balance, whether through increased kinase activity or decreased phosphatase activity, culminates in the excessive phosphorylation of Tau. This aberrant phosphorylation subsequently leads to neuropathological consequences in tauopathies.^{58, 208} Tau phosphorylation is regulated by three primary classes of kinases (known as Tau kinases) that target its Ser/Thr and tyrosine residues, including proline-directed kinases (PDPKs), nonproline-directed kinases (non-PDPKs), and tyrosine protein kinases.⁵⁷ Tau undergoes phosphorylation by PDPKs at Ser/Thr-Pro motifs, which refer to Ser/Thr residues positioned immediately before a proline residue. This category encompasses cyclin-dependent kinase 5 (CDK5), glycogen synthase kinase 3 beta (GSK-3β), MAPK, and dual specificity tyrosine-phosphorylation regulated kinase 1A.⁵⁷ Non-PDPKs are responsible for phosphorylating Tau at motifs containing Ser/Thr-X, including MAP/microtubule affinity-regulating kinase 4, casein kinase 1 (CK1), Tau–tubulin kinases, and cyclic AMP-dependent protein kinase A. Tyrosine protein kinases induce Tau phosphorylation through Fyn, spleen tyrosine kinase, Abl kinases, and Src family kinases. In humans, the longest form of the Tau protein consists of around 85 residues that can undergo phosphorylation. This set of residues comprises 45 serine residues and 35 threonine residues, along with five tyrosine residues, all susceptible to phosphorylation. Significantly, the majority of phosphorylation occurrences take place in distinct domains: residues 172–251 within the proline-rich domain, residues 244–369 in the MTBR, and residues 368–441 in the C-terminal tail region.²⁰⁹

As the phosphorylation level of Tau increases, its ability to bind microtubules diminishes. While the Tau protein normally stabilizes microtubule bundles, abnormally phosphorylated Tau at specific residues can interfere with its interaction with tubulin. This disruption ultimately leads to microtubule disassembly, resulting in axonal damage and neuronal apoptosis.²¹⁰ The Tau protein's role in these processes highlights its significance in neuronal injury and dysfunction. In addition to disrupting neuronal transport and causing cytotoxicity, p-Tau can spread to other neurons and different regions of the brain. After becoming hyperphosphorylated, Tau dissociates from microtubules and can be released from their surface. Abnormal phosphorylation of Tau not only disrupts its normal functions but also imparts neurotoxic properties, ultimately leading to neuronal death in AD and other tauopathies. Moreover, abnormally phosphorylated Tau tends to aggregate, potentially forming toxic oligomers and insoluble structures called paired helical filaments (PHFs), eventually contributing to NFts formation.^{211, 212} Specifically, the hyperphosphorylation of Tau at certain residues, including pSer-199, pSer-202, and pThr-205, as well as pThr-212, pThr-231, pSer-262, and pSer-396, has been reported to be implicated in destabilizing microtubules, ultimately leading to neurofibrillary pathology.^{213, 214}

All six isoforms of Tau exhibit hyperphosphorylation, which leads to microtubule destabilization. Subsequently, they are detected within the structures of PHF and NFt.²¹⁵ It is important to note that 3R and 4R Tau isoforms contribute to the composition of PHFs and NFts, further highlighting their significance in pathological Tau aggregation.²¹⁵ These toxic filaments comprise two smaller filaments, each approximately 10 nm in diameter, intertwining to form periodic structures. The crossover distance between the filaments within the PHFs is typically around 65–80 nm. This pattern of intertwining gives rise to the characteristic appearance of PHFs in AD.²¹⁶ Specific phosphorylation residues on Tau proteins have been found to undergo abnormal phosphorylation during the progression of AD and other tauopathies while remaining unphosphorylated in healthy brains. This process indicates a significant increase (at least threefold) in the phosphorylation of Tau compared with its normal state. Around 50 distinct phosphorylation residues involving serine, threonine, and tyrosine have been detected as abnormally phosphorylated in the AD brain, such as Tyr-18, Ser-199, Ser-202, Thr-205, Thr-212, Ser-214, Thr-217, Ser-262, Thr-231, Ser-262, Ser-396 and Ser-422.^{209, 217} Several key residues associated with pathological conditions are prominently located within the MTBR.^{209, 218} These residues are predominantly found within PHFs, NFts, and neuropil threads (NTs).²¹⁶ The phosphorylation of specific residues plays a crucial role in influencing the progression and stages of AD. As a result, various site-specific profiles have been identified in AD, spanning from its early to late stages. Notably, the phosphorylation of Thr-231, Ser-396, and Ser-422 has been recognized as an early event in AD progression. In particular, Ser-422 may act as a significant indicator for the onset of p-Tau-induced pathology and aggregation.^{209, 217} In addition, elevated levels of p-Tau have been observed at key residues, including Ser-199, Ser-202/205, and Ser-422, in the cortex and transentorhinal cortex during advanced Braak stages/VI.²¹⁸ In individuals with AD, the level of phosphorylated Tau at Ser-396 in the frontal cortex is notably elevated. This increased phosphorylation of Tau at the Ser-396 residue is likely associated with Aβ and is considered a crucial factor in the progression of AD. Interestingly, the research revealed that pThr-231 (p-Tau₂₃₁) displayed substantial correlations with AD pathology, demonstrating a notable increase from early stages to advanced AD and a significant connection with Aβ in the disease.²¹⁹ Interestingly, differences in the levels of p-Tau at Tyr-18 or Thr-231 in the transentorhinal region have been observed to vary across different Braak stages of AD. For example, in Braak stages III/IV, phosphorylation at Tyr-18 and Thr-231 is observed in the transentorhinal region, exhibiting a progressive increase. This suggests that Tyr-18 and Thr-231 may play a pivotal role in the early stages of AD onset.²¹⁸

Tau protein naturally maintains a stable conformation with minimal tendencies for misfolding. Its structure is comparable to that of phosphorylation at the Thr-231 paperclip, where both ends of the Tau protein (N- and C-terminal) fold over the MTBR region. This structural configuration acts as a safeguard, helping to inhibit the self-aggregation of the protein.²²⁰ Hyperphosphorylation of the Tau protein brings about a conformational change that facilitates its aggregation. Recent research indicates that phosphorylation at Thr-231 induces the conformational alteration of the Tau protein, leading to a neurotoxic conformation known as cis p-Tau, which is thought to contribute to the early stages of diverse neurodegenerative disorders, including AD. Studies have shown that cis p-Tau, distinct from the normal trans p-Tau conformation, displays exceptional stability and resistance to both microtubule binding and degradation mechanisms.^{208, 221} The misfolded p-Tau, acting as a seed, is associated with specific phosphorylation residues implicated in the pathological seeding of Tau and the progression of AD. Remarkably, Tau seeds exhibit a notable capacity for release from damaged neurons to the extracellular space, utilizing diverse exocytosis mechanisms. This enables them to spread to nearby neurons, displaying prion-like propagation behavior. For instance, neuronal death and/or the secretion of exosomes serve as the means for the release of Tau seeds. Following their release, these Tau seeds are internalized by other neurons or glial cells via different mechanisms, such as membrane fusion of exosomes.²²² One crucial role of the Tau seeds lies in their ability to recruit soluble Tau and contribute to the formation of larger abnormal structures.^{221, 223} Subsequently, the spread of Tau aggregation correlates with cognitive decline, indicating the worsening of the disease state.²²⁴ In AD, Tau aggregates are assumed to initially accumulate in the entorhinal cortex and spread to cortical areas during disease progression.²²⁵ Recent research indicates a substantial accumulation of cis p-Tau, which initiates and propagates p-Tau aggregation, leading to a pathological phenomenon known as cistauosis. This process plays a significant role in neuronal death and the advancement of neurodegeneration, particularly in AD and traumatic brain injury (TBI).^{208, 226}

The abnormal phosphorylation of Tau is closely tied to the progression of insoluble Tau aggregates, which are identified as a hallmark of AD pathology. This interconnection implies that the accumulation rate of insoluble Tau aggregates may indeed play a significant role in the observed cognitive decline in individuals affected by AD.²²⁷ Abnormal phosphorylation of Tau neurotoxicity contributes to cognitive decline through loss of synaptic function and neuronal death.²²⁸ Impaired synaptic function is a crucial factor in the cognitive decline seen in AD and may appear early in the disease process.²²⁹ In healthy human brains, Tau is present in approximately 55 and 70% of presynaptic and postsynaptic compartments, respectively. Its presence actively contributes to regulating the proper functioning of synapses in healthy conditions.²²⁹ Elevated levels of p-Tau have been detected in the postmortem tissues of individuals with AD, particularly in the hippocampus and entorhinal cortex. This accumulation significantly disrupts mitochondrial dynamics, causing a decline in dendritic proteins, loss of dendritic spines, and synaptic function. These effects ultimately contribute to impairments in learning and memory that are associated with the hippocampus. Moreover, p-Tau exhibits a significant propensity to accumulate at synaptic terminals in the hippocampus. This accumulation at synapses can have detrimental effects on synaptic plasticity, eventually resulting in the loss of synapses.^{230, 231} The correlation between the abundance of NFts and the decline of cognitive function, coupled with the loss of synapses, suggests a potential causal role for p-Tau. Additionally, the soluble form of Tau that precedes NFt formation demonstrates a distinct link to synaptic defects, highlighting its ability to interfere with normal synaptic function. Notably, in individuals with AD, elevated pathological Tau could intensify synaptic impairment and accelerate cognitive decline.^{231, 232} On the other hand, phosphorylation of Tau at Ser396/404, referred to as PHF-1, significantly accumulates within mitochondria during the aging process, particularly in synaptic mitochondria in the hippocampal. This accumulation of Tau PHF-1 inside mitochondria leads to extensive damage, implying a potential correlation with synaptic dysfunction and cognitive decline in the elderly individual. Consequently, the occurrence of phosphorylated Tau PHF-1 within mitochondria could potentially serve as an early indicator of neurodegeneration in AD.^{212, 233}

In a physiological state, Tau goes through a continuous cycle of phosphorylation and dephosphorylation to maintain its functionality. However, an imbalance favoring increased phosphorylation diminishes Tau's binding to microtubules, leading to the aggregation of p-Tau.²²¹ Tau undergoes dephosphorylation through the action of several protein phosphatases (PPs). Among these PPs, PP2A is the primary phosphatase responsible for the majority of Tau dephosphorylation. Additionally, other phosphatases such as PP1, PP2B, PP2C, and PP5 also contribute to this process.⁵⁸ In the brains of AD patients, there is a substantial decrease in the activity of PP2A, causing an enhancement of p-Tau levels and NFt in the brain. The reduced activity of PP2A in AD may be attributed to several factors, including changes in the posttranslational state of its catalytic domain and a decrease in PP2A expression.^{234, 235} Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) has been identified as a regulatory factor for PP2A activity, demonstrating its ability to enhance the dephosphorylation of Tau by PP2A. Importantly, Pin1 is actively associated with abnormal phosphorylation patterns observed in AD.^{235, 236} As a result, PP2A and Pin1 are recognized as crucial factors that have the potential to inhibit or reverse the hyperphosphorylation and aggregation of Tau, offering promising insights into the understanding of AD and related disorders.

Pin1 plays a crucial role in modulating the conformation of the Tau protein by isomerizing phosphorylated serine/threonine-proline motifs, particularly Thr-231. Therefore, Pin1 facilitates the dephosphorylation of Tau by PP2A, thereby regulating its phosphorylation state and restoring its functionality. Its catalytic activity enables the isomerization of Tau at pSer/Thr-Pro motifs, restoring Tau's microtubule-binding capability and promoting microtubule assembly. Pin1's catalytic activity facilitates the transition from the conformation of cis p-Tau231 to trans isoform, enabling the restoration of functional Tau that, by restoring its microtubule-binding capability, supports neuronal structure and transport while inhibiting the toxic effects associated with cis p-Tau.^{235, 237} Pin1 has also been reported to exhibit a slightly stronger association with phosphorylation at Ser-202 and Thr-205.²³⁶ Pin1's malfunction in AD is attributed to various factors, including phosphorylation and mutations, resulting in the accumulation of p-Tau231 in a pathogenic cis conformation. Afterwards, this inactivation contributes to the accumulation of p-Tau231 in the neurotoxic cis conformation, initiating cistauosis and neurodegeneration, occurring even before the formation of tangles in AD.^{235, 237} Death-associated protein kinase 1 (DAPK1) exerts a direct impact on Pin1 by phosphorylating Ser-71 within its catalytic active site, leading to the inhibition of its enzymatic function.²³⁸ In addition, DAPK1 also exhibits the capacity to phosphorylate Tau at Thr-231, Ser-262, and Ser-396, which potentially contributes to p-Tau aggregation and neuronal death. DAPK1 is a crucial player in diverse neuronal injury models, and mounting evidence suggests an association between DAPK1 and the risk of AD. Accordingly, the activation of DAPK1 is implicated in the neurodegenerative processes associated with AD in the brain.²³⁹

3.1.2 Abnormal phosphorylation of the APP in AD

Phosphorylation of APP occurs continuously in neurons under normal physiological conditions. APP undergoes phosphorylation at specific residues, involving threonine, serine, and tyrosine, situated across its cytoplasmic and extracellular domains of the protein.²⁴⁰ Phosphorylation plays a regulatory role in modulating the function and localization of APP.²⁴¹ Furthermore, phosphorylation plays a pivotal role in modulating the proteolytic processing of APP and the associated enzymes, which is closely associated with the pathogenesis of AD.^{241, 242} This finding emphasizes the importance of phosphorylation in influencing the regulation of Aβ generation. It not only affects the subcellular distribution of APP but also modulates the enzymatic activities of the relevant secretases involved in its processing.²⁴² Therefore, the modification of APP processing is considered to be a crucial factor in the development of AD pathology.²⁴³ Among the 10 phosphorylated residues recognized in APP695, the cytoplasmic domain exhibits phosphorylation at Tyr-653, Thr-654, Ser-655, Thr-668, Ser-675, Tyr-682, Thr-686, and Tyr-687, while the remaining two phosphorylated residues are located in the ectodomain, including Ser-198 and Ser-206. In its physiological state, Ser-198 stands out as the predominant phosphorylation site in the ectodomain of APP, exhibiting a higher phosphorylation level in comparison with Ser206 site.^{68, 75, 242} The abnormal processing of APP, leading to the accumulation of Aβ peptides, contributes to neurodegeneration and cognitive decline in AD. Numerous phosphorylation sites on APP have been reported to be associated with the development of AD, such as Ser-26, Ser-675, Thr-668, and Tyr-682.^{75, 244, 245} Fyn tyrosine kinase (Fyn TK) has been found to phosphorylate APP at Tyr-682 in AD neurons, indicating its involvement in the phosphorylation process. Following phosphorylation at Tyr-682, APP undergoes relocation into acidic neuronal compartments, leading to the generation of neurotoxic peptides of Aβ. Notably, there is an enhanced interaction between Fyn TK and the Tyr-682 residue of APP. Increased phosphorylation of Tyr-682 has been observed in the neurons of AD patients, and those with elevated phosphorylation levels also exhibit heightened Fyn TK activity.²⁴⁰ The enzymatic noncanonical cleavage of APP through meprin β produces truncated Aβ2–40/42 peptides with a strong tendency to aggregate, suggesting that increased activity of this enzyme might play a role in the Aβ generation in AD. Phosphorylation of APP at Ser675 disrupts APP processing, promoting meprin β and reducing α-secretase activity during APP processing.²⁴³

The significance of phosphorylation at Thr668 becomes particularly noteworthy when observed in models and brain samples of AD. Phosphorylation at Thr668 occurs exclusively in the brain and is regulated by various kinases, including JNK, CDK5, and GSK-3β.^{242, 246} The activation of DAPK1 has also been found to be correlated with the phosphorylation of APP at T668. Heightened levels of DAPK1 exhibited a clear association with the augmentation of APP phosphorylation. Taken collectively, these findings indicate that DAPK1 contributes to the amplification of APP phosphorylation and the amyloidogenic alteration of APP.²⁴⁷ This phosphorylation event is critical for the interaction between AICD and Fe65, suggesting its contribution to neurodegeneration in AD. Fe65 modulates APP processing and contributes to the generation of Aβ peptides. Phosphorylation on residue Thr668 of APP is believed to be crucial for regulating APP's neuronal-related activities, potentially influencing APP metabolism.^{244, 246} The remarkable significance of the phosphorylation of Thr668 residue on APP has garnered significant attention, owing to its prevalent presence in AD and its influential role in modulating the cleavage of APP, highlighting its essential contribution to APP's functionality.^{242, 244} Furthermore, the brains of individuals with AD have been found to have significant upregulation of phosphorylated C-terminal fragments of APP (APP-CTFs) at Thr668, as well as elevated levels of GSK-3β and Tau phosphorylation in the brain.²⁴² Evidence also suggests that Thr-668 phosphorylation plays a regulatory role in APP cleavage by BACE1.²⁴⁴ On the contrary, the phosphorylation of certain residues within some forms of Aβ has been detected in AD. For instance, there is a variant of Aβ that undergoes phosphorylation at the Ser-26 residue. This variant is primarily found within intraneuronal deposits during the initial stages of AD. The phosphorylated Aβ at Ser-26 forms a distinctive oligomeric structure that accumulates within specific intracellular compartments associated with granulovacuolar degeneration. It is worth noting that phosphorylated Ser-26 oligomers display heightened neurotoxicity compared with other forms of Aβ.²⁴⁸

3.2.1 Abnormal phosphorylation of the α-syn protein in PD

The phosphorylation of α-syn stands out as the predominant and pivotal PTM in PD, among the various PTMs identified for the protein. Consequently, it assumes a crucial role in synucleinopathies, specifically PD, influencing the folding of α-syn and leading to aggregation. For instance, around 90% of phosphorylated α-syn was detected within LBs. However, in normal neuronal conditions, only a small portion (under 5%) of soluble α-syn is phosphorylated.²⁵⁶ Recent studies have suggested a strong association between PD and later stages and a notable elevation in the levels of phosphorylated α-syn. These findings highlight the critical significance of phosphorylated α-syn as a potential biomarker for the advanced stages of PD.²⁵⁷ α-Syn is phosphorylated at the C-terminal tail, with specific phosphorylation sites identified at serine residues (Ser-87 and Ser-129) as well as tyrosine residues (Tyr-125, Tyr-133, and Tyr-136). Among these sites, Ser-129 is extensively phosphorylated in synucleinopathies, exhibiting a specific association with PD.^{258, 259} Multiple types of kinases, such as polo-like kinase 2 (PLK2), CK1 and CK2, and the G-protein-coupled receptor kinase 5, are known to phosphorylate α-syn at the Ser129 residue. Among these kinases, PLK2 has been identified as a key kinase in Ser-129 phosphorylation in neurons. Although, the precise involvement of all these kinases in the pathogenesis of α-synucleinopathy and their connection to cellular toxicity resulting from phosphorylation remain incompletely comprehended.^{100, 260} The phosphorylation of Ser129 significantly impacts the conformational structure of α-syn. This modification induces noteworthy changes in the unstructured C-terminus, primarily resulting from the formation of notable salt bridges. These salt bridges connect pS129, which carries a negative charge, with Lys residues carrying a positive charge present in the relevant region.²⁵⁸ Hence, numerous studies have consistently demonstrated a significant correlation between the phosphorylation of α-syn at the serine 129 residue and the formation of LBs. These investigations have unveiled a remarkable increase in phosphorylation levels of Ser-129 (pSer129), rising from approximately 4% under normal physiological conditions to a substantial 90% within the LBs identified in PD. This compelling evidence strongly suggests that pSer129 plays a crucial role in the pathogenesis of the disease and underscores its association with the pathological state.^{258, 261} While the exact connection between Ser-129 phosphorylation and LB formation remains uncertain, various research findings have presented conflicting views on its role in PD. Consequently, studies have shown that phosphorylation at Ser-129 can both impede and promote α-syn aggregation. Phosphorylation at Ser-129 is suggested to possess the capability to enhance the formation of inclusions. Nevertheless, recent research suggests that pSer129 may have the capability to diminish seed aggregation and impede the aggregation process leading to the formation of amyloid structures.^{261, 262} While the majority of phosphorylation sites in α-syn are typically found in the C-terminal region, recent research indicates that a tyrosine residue (Tyr39) located at the N-terminus may also undergo phosphorylation. This phosphorylation event is phosphorylated by the c-Abl protein TK. Phosphorylation of Tyr-39 (pTyr39) has been shown to probably intensify α-syn aggregation, contributing to the pathological process in PD.^{251, 263} Although, the phosphorylation of Tyr-39 residue via c-Abl may have a protective effect on α-syn and potentially protect α-syn from degradation through the autophagy and proteasome pathways within cortical neurons in PD brains.^{251, 264} pTyr39 restructures the fibril core of α-syn by engaging the entire N terminus, leading to pathological fibril formation. Consequently, this alteration modifies the electrostatic characteristics of the N terminus, which in turn stabilizes several Lys residues and creates a hydrophilic channel within the core.²⁵¹

3.2.2 Abnormal phosphorylation of Tau protein in PD

Tau abnormal phosphorylation is indeed a significant contributing factor to neurodegeneration in PD. The neurodegeneration of nigrostriatal dopaminergic cells is a key feature in PD, and the onset of this degeneration may be linked to the aggregation of p-Tau. Therefore, p-Tau could potentially play a crucial role in both the early and late stages of PD development.^{265, 266} As a result, numerous studies have established a correlation between p-Tau and neuronal damage, suggesting its involvement in the mechanisms of degeneration in PD. Accumulating evidence also demonstrates the involvement of p-Tau and the formation of NFts in the postmortem brain samples of individuals with PD. Furthermore, in PD, the extent of Tau pathology has been associated with cognitive decline. NFts have been noted to manifest varying occurrences in postmortem examinations of individuals diagnosed with PD. It is crucial to emphasize that not all examined samples exhibited NFts. Consequently, the presence of NFts has been possibly correlated with the onset of cognitive impairment and dementia in certain cases.²⁶⁵ Extensive research suggests that phosphorylated Tau, characterized by prion-like features, exhibits a broader distribution across multiple regions of the brain compared with α-syn. This observation implies that pathogenic Tau may play a pivotal role in steering the overall neurodegeneration observed in PD.²⁶⁷

Hyperphosphorylated Tau proteins not only contribute to protein aggregation but also exhibit a tendency to interact with α-syn. This interaction leads to mutual fibrillization, eventually resulting in the formation of LBs. Therefore, the presence of hyperphosphorylated Tau is closely associated with the pathological development of LB formation.²⁶⁸ Hence, the accumulation of phosphorylated-Tau has gained recognition as a significant and possibly initial pathogenic determinant in PD, playing an essential role in its early stages and overall development.²⁶⁶ Multiple studies have detected a few specific residues on the Tau protein phosphorylated in postmortem samples from individuals with PD, particularly Ser-202, Ser-262, Ser-396, and Ser-404, as well as Thr-205. Moreover, compelling research indicates a significant enhancement in hyperphosphorylated Tau at Ser-202, Ser-262, and Ser-396/404 levels in the striatum of individuals affected by PD. Furthermore, earlier investigations have established the presence of hyperphosphorylated Tau at Ser-396 in synaptic-enriched fractions in the fractions in the frontal cortex of PD samples.^{91, 269} Recent evidence suggests that there is a significant increase in the concentration of hyperphosphorylated Tau at Thr-181 (p-Tau181) in the plasma of individuals diagnosed with PD. This observation indicates that the elevated levels of p-Tau181 have the potential to serve as a valuable biomarker for cognitive decline in PD. Moreover, it shows promise as a noninvasive and reliable way to track the progression of cognitive impairment in PD patients. Nonetheless, these initial results provide a foundation for future investigations and potential therapeutic interventions.^{270, 271} Although abnormal Tau phosphorylation is commonly recognized as a key phosphorylated protein implicated in the pathology of PD, other essential proteins such as ubiquitin, Rab10, 14-3-3 proteins, and Drp1 have also been identified in diverse research investigations (refer to Table 1 for further details). Therefore, understanding the mechanisms underlying the impact of p-Tau and its interaction with other pathological proteins, as well as the phosphorylation of other proteins associated with PD, could provide valuable insights into PD pathology.

3.3.1 Abnormal phosphorylation of the Huntingtin protein in HD

Multiple phosphorylation sites have been detected within the Htt protein, such as Thr-3, Ser-13, Ser1-6, Ser-116, Ser-421, Ser-536, Ser-434, Ser-1181, and Ser-1201.^{173, 282, 283} Some of the phosphorylation sites have a beneficial role in protecting cells from the harmful effects of expanded polyQ-induced toxicity and modulating neuronal toxicity. However, any disturbance in the phosphorylation pattern of these sites triggers the aggregation of mHtt, thereby inducing alterations in phosphorylation patterns associated with the disease. The phosphorylation of the Httex1 region has been shown to play crucial regulatory roles.²⁸³ Extensive research into the Nt17 domain of Httex1 has yielded compelling evidence highlighting phosphorylation events at specific serine and threonine residues. Particularly noteworthy are Thr-3, Ser-13, and Ser-16, which have been identified as pivotal phosphorylation sites within this domain. Phosphorylation at Ser13 and Ser16 not only affects the structure of the protein but also plays a crucial role in influencing its aggregation propensity and subcellular localization. These phosphorylation events hold considerable importance in the context of the disease.²⁷⁹ Recent studies have focused on investigating the effects of phosphorylation at positions 13 and 16 on the Httex1 region. These investigations have consistently revealed compelling evidence that phosphorylated serine residues at positions 13 and 16 actively mitigate protein aggregation. Moreover, the phosphorylation of these serine residues, either individually or in combination, leads to mutant Httex1 fibrils exhibiting a heightened propensity for localization within the nuclear compartment.^{279, 284} Studies on clinical samples of HD have revealed a significant decrease in the levels of phosphorylation at residue Thr-3 in mHtt. When Thr-3 is phosphorylated, it has a stabilizing effect on the α-helical conformation of Nt17. Research indicates that phosphorylated Thr-3 predominantly occurs on full-length Htt, influencing its aggregation tendencies and pathogenic characteristics. In other words, phosphorylation at Thr-3 actively inhibits the aggregation of mHttex1. Nevertheless, this suggests that phosphorylation at Thr-3 plays a significant role in reducing these pathological protein structure processes in Httex1.^{285, 286}

The IκB complex (IKK) is detected as a crucial kinase in Htt phosphorylation. Specifically, IKK is responsible for directly phosphorylating the Ser-13 residue of Htt, which could potentially lead to the subsequent phosphorylation of the Ser-16 residue via IKK. This suggests a potential cross-talk between these residues’ phosphorylation, indicating their probable interconnected roles in the regulation of Htt pathology. Upon phosphorylation of these residues, a cascade of events is initiated, including acetylation of nearby Lys residues, ultimately facilitating the clearance of Htt via both the proteasome and lysosome.^{282, 286} However, the expansion of the polyQ repeat length in mHtt adversely affects the efficiency of phosphorylation at Ser-13, which is associated with HD. This reduced efficiency could have significant implications for PPI and directly contribute to the accumulation of mHtt.^{285, 286}

3.3.2 Abnormal phosphorylation of Tau protein in HD

Nowadays, HD could potentially be categorized as a secondary tauopathy, as evidenced by the identification of essential tauopathic features in the postmortem brain tissue of HD patients. These features include misfolding, hyperphosphorylation, and the presence of NFts and NTs. Notably, the advanced stages of HD show the additional presence of p-Tau insoluble aggregation.^{280, 287} Specifically, the presence of sarkosyl-insoluble Tau and its abnormal phosphorylation patterns in HD displayed a strikingly different isoform.^{281, 288} Recent studies have revealed notable increases in Tau phosphorylation throughout the progression of HD. This abnormal phosphorylation of Tau has been observed in postmortem samples of individuals with HD, specifically at sites including Ser199, Thr205, and Ser-396/Ser-404. Interestingly, some of these phosphorylation sites are also found to be implicated in AD.²⁸⁹ In recent studies, investigations into HD postmortem samples have revealed a noteworthy observation despite: the presence of p-Tau abnormalities, the level of phosphorylation in HD appears to be relatively weak compared with the extensive phosphorylation typically observed in AD.^{280, 289}. In the context of HD, it has been observed that Tau pathology in transplanted neural tissue exhibits impaired Tau splicing in exons 2, 3, and 10. Specifically, impairment in Tau splicing in exon 2 was detected in the putamen region of HD brain samples. These abnormal splicing events, along with hyperphosphorylation of Tau, occur early in the disease and may influence the aggregation of mHtt.^{281, 288, 290} During a study, it was observed that Tau pathology could appear in previously healthy neural tissue transplanted into the brains of individuals with HD. Specifically, two patients with HD exhibited the presence of hyperphosphorylated Tau in the transplanted tissue, specifically at the phosphorylation sites of Ser-202 and Thr-205 residues. It is interesting to note that hyperphosphorylated Tau was found to spread to healthy neurons.^{289, 291} Hence, Tau may have a high propensity to spread to other regions of the brain. The localization of phosphorylated Tau oligomers displayed distinct patterns in HD brains. Tau hyperphosphorylation was detected in specific brain regions, including the putamen, striatum, cortex, and hippocampus, within HD. Notably, a significant elevation of p-Tau was specifically observed in the putamen.^{287, 289}

4.2.1 Acetylation of histones in the tissues of Parkinson patients

Histone acetylation, alone or in conjunction with other PTMs, plays a crucial role in governing gene expression and regulating other genomic activities by attracting or repelling chromatin regulatory protein complexes. This significantly effects on some critical cellular processes and the progression of illnesses.³¹⁹ KATs normally acetylate histones to decrease their binding to DNA, relax chromatin, and switch on gene transcription; HDACs deacetylate histones to cause chromatin condensation and inhibit gene transcription.³²⁰ Furthermore, histone acetylation permits it to interact with TFs and proteins containing the bromodomain, which increases the number of regulatory factors.³²¹ A new investigation compared two sets of fibroblasts from individuals with hereditary PDLRRK2 G2109S and idiopathic PD.³²² G2109S and R1441G mutations in the LRRK2 gene are responsible for an autosomal dominant form of familial PD.⁸⁹ Histone acetylation is reduced in idiopathic PD fibroblasts as opposed to controls, despite an increase in the acetylation levels of total proteins. Anacardic acid (AA), a HAT inhibitor, is advantageous for idiopathic PD fibroblasts, whereas the nonspecific HDAC inhibitor trichostatin A is detrimental.³²² Idiopathic PD cells may have hyperacetylation of proteins due to impaired mitophagy and defective mitochondrial accumulation, which leads to reduced nicotinamide adenine dinucleotide (NAD⁺) synthesis and subsequently lowers SIRT activity. Upon SIRT inhibition, class I and II KDACs such as HDAC2, HDAC3, and HDAC4 become more active; overall HDAC activity, particularly HDAC6 activity, is reduced. Therefore, to restore the balance of histone acetylation levels and reduce cell damage in PD, HAT inhibitors or selective inhibitors targeting HDAC2, HDAC3, or HDAC4 are preferable to nonspecific HDAC inhibitors.³²³

4.2.2 PD-related neurotoxins and acetylation of histones

When various PD-related neurotoxins such as 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine/1-methyl-4-phenylpyridinium (MPTP/MPP+), dieldrin, rotenone, and paraquat are applied to DA neurons, one of the main epigenetic changes that occur is hyperacetylation of histones H3 or H4. For example, treatment with MPTP in mice or MPP+ in neuronal cells leads to increased Lys acetylation of histones, accompanied by reductions in the levels of HDAC1, HDAC2, HDAC4, HDAC6, and SIRT1.³²⁴ MPP+ treatment reduces SIRT1 expression, increases H3K14 acetylation, and triggers transcription of the hypoxia-inducible factor-1 promoter.³²⁵ Mice treated with MPTP also exhibit increased H3 acetylation in the striatum, consistent with the study's findings. Dieldrin, a toxin linked to the pathophysiology of PD, induces time-dependent hyperacetylation of H3 and H4 via CBP accumulation, with hyperacetylation serving as a precursor to dieldrin-induced neurotoxicity. Additionally, the HAT inhibitor AA significantly reduces dieldrin-mediated DA neuronal death.³²⁶ Paraquat induces histone acetylation by downregulating HDAC4 and HDAC7, leading to increased H3, rather than H4, hyperacetylation, and subsequent death of DA neuronal cells.³²⁷ Rotenone triggers neurodegeneration by boosting p53 expression and elevating H3K9 acetylation through decreased SIRT1 levels. Resveratrol, a SIRT1 activator, mitigates p53 production and cell damage induced by rotenone.³²⁸ HAT inhibitors such as curcumin, AA, and garcinol have been shown to alleviate l-DOPA-induced dyskinesia in 6-hydroxydopamine-induced PD animal models. This suggests that a combination of l-DOPA with a HAT inhibitor may hold therapeutic potential for managing l-DOPA-induced dyskinesia in PD patients.³²⁹

4.3.1 Transcriptional dysregulation in HD

Strong evidence from recent research indicates that transcriptional dysregulation is a key underlying mechanism in the pathophysiology of HD.^{332, 333} Throughout several HD models, transcriptional dysregulation is a key early event in HD pathogenesis.³³⁴ The structure of the Htt protein is comparable to that of known TFs because of the poly-Q repeats in its N-terminal region. The expansion of these repeats causes abnormal cleavage by caspases. The fragments penetrate the nucleus and create nuclear aggregates that could interfere with transcription. Since mutant Htt interacts with many TFs, including p5327, TATA-binding protein (TBP), CBP, and Sp1, Htt nuclear aggregates may sequester TFs and cause transcriptional dysregulation.³³⁵

4.3.2 HDAC inhibitors in HD

Histone-modifying activity is possessed by various Htt-interacting proteins. CBP is a coactivator at different promoters and carries an acetyltransferase domain. It has been demonstrated that CBP and other TFs are confined to Htt aggregates in the brains of HD patients and transgenic mice.³³⁶ Moreover, CBP overexpression reduces cell mortality caused by poly-Q.³³⁷ It was shown by Steffan and colleagues that the acetyltransferase domain of CBP and p300/CBP-associated factor are directly bound by Htt exon 1 with 51 glutamines that contain the poly proline domain (Httex1p 51Q).³³⁷ The acetylation of histone H4 is reduced in the presence of mutant Htt, according to in vitro research, indicating that Htt's direct interaction with the acetyltransferase domains suppresses the acetylation of histone proteins. Gardian et al.³³⁸ treated another transgenic mouse strain, HD-N171-82Q, with phenylbutyrate, an HDAC inhibitor, when symptoms appeared. When given to 75-day-old HD-N171-82Q mice, phenylbutyrate boosted survival and reduced ventricular hypertrophy and striatal atrophy. On motor function, weight loss, or the generation of Htt aggregates, there was no difference. After being treated with phenylbutyrate, the striatum showed an increase in histone H3 and H4 acetylation and a decrease in histone H3 methylation. The HD-N171-82Q striatum treated with phenylbutyrate was subjected to microarray analysis, which revealed that certain genes were elevated and others were downregulated. Given that treatment was started after symptoms appeared, it is encouraging that transgenic mice displayed an overall improvement in their condition, even if phenylbutyrate did not increase the expression of mutant-Htt-downregulated genes. It is unclear how HDAC inhibitors work therapeutically. The most straightforward explanation that can be imagined is one in which the downregulation of particular genes brought on by mHtt is rectified by the injection of HDAC inhibitors. However, limited research using HDAC inhibitors on transgenic mice suggests that this is not the case. Alternatively, the advantages of HDAC inhibitors could come from higher global gene expression. If so, HDAC inhibitors may have advantages not only for HD but also for other NDDs. HDAC inhibitors have also demonstrated promise in AML transgenic animal models, schizophrenia, and ischemia.³³⁹