Phosphorylation-driven aggregative proteins in neurodegenerative diseases: implications and therapeutics

Protein aggregation is related to a large number of neurodegenerative disorders. Particularly in some cases, aggregation is induced by hyperphosphorylation of a given protein. This is the case of tau, TAR DNA-binding protein 43 (TDP-43), amyloid-beta peptides (Aβ) and alpha-synuclein (α-syn), which play a key pathogenic role in Alzheimer’s disease (AD), Parkinson’s disease (PD) and amyotrophic lateral sclerosis, among others (Tenreiro et al., 2014; Chiti and Dobson, 2017). In this perspective, we will discuss both the relationship between phosphorylation and amyloid aggregation as well as current and future therapeutic strategies aimed at inhibiting specific kinases involved in the phosphorylation step or inhibiting subsequent protein aggregation. Moreover, we will address novel approaches that are based on chemical-inducing proximity, to recruit the proteasome, the autophagy-lysosome system or specific phosphatases to degrade or dephosphorylate the target proteins. We want to focus our interest on proteolysis-targeting chimeras (PROTACs) systems, which represent one of the newest and most interesting therapeutic approaches that could be applicable to phosphorylation-driven aggregative proteins in the near future (Ciechanover and Kwon, 2015; Esposto and Martic, 2021; Jangampalli et al., 2021; Jiang et al., 2021).

Conformational diseases are a group of disorders caused by misfolded or abnormally folded proteins. In these diseases, the proteins do not adopt the correct three-dimensional structure required for their normal function. Instead, they can aggregate and form clumps, known as amyloid fibrils, which can accumulate in tissues and organs throughout the body, leading to cell damage and dysfunction. Examples of conformational diseases include AD, PD, diabetes mellitus type II, Huntington’s disease, amyotrophic lateral sclerosis, frontotemporal dementia and prion diseases viz. Creutzfeldt-Jakob disease in humans. These disorders are typically progressive and degenerative and have a significant impact on an individual’s quality of life (Chiti and Dobson, 2017).

Phosphorylation is a post-translational modification that can alter the activity, localization, and stability of proteins. Over the last two decades, it has been revealed that the phosphorylation of certain proteins involved in conformational diseases could be one of the key triggering factors in their onset and progression (Box 1; Tenreiro et al., 2014; Kawahata et al., 2016; Rezaei-Ghaleh et al., 2016). Among these, a subgroup is phosphorylation-driven aggregative proteins that undergo self-aggregation as a result of changes in their phosphorylation state (as indicated in bold in Box 1).

Box 1.

Phosphorylation-driven proteins involved in neurodegenerative diseases

Disease	Protein
Alzheimer’s disease	Adenylate cyclase associated protein 1 (CAP1); AMPA receptor subunit GluR2; AMP activated protein kinase (AMPK); amyloid-β peptides (Aβ); apolipoprotein E (APOE); amyloid precursor protein (APP); Bcl2 associated death promoter (BAD); β-catenin; c-JUN N-terminal kinase (JNK); casein kinase 1 delta (CK1δ); cyclin dependent kinase 2 (CDK2); cyclin dependent kinase 5 (CDK5); dynamin; Fyn; glial fibrillary acidic protein (GFAP); glycogen synthase kinase 3α (GSK3α); glycogen synthase kinase 3β (GSK3β); insulin receptor substrate 1 (IRS1); lysosome associated membrane protein 2A (LAMP2A); MAP kinase phosphatase 1 (MKP1); mTOR; myelin basic protein (MBP); neural cell adhesion molecule (NCAM); neurogranin; nicastrin; nuclear factor kappa B (NF-κB); Parkin; phosphatase and tensin homolog (PTEN); presenilin 1 (PS1); receptor interacting protein 1 (RIP1); prion protein (PrP); receptor for activated C kinase 1 (RACK1); secreted protein acidic and rich in cysteine (SPARC); serine/threonine kinase 11 (LKB1); signal transducer and activator of transcription 3 (STAT3); superoxide dismutase 2 (SOD2); synapsin; Tau (τ); Tau tubulin kinase 1 (TTBK1); TAR DNA-binding protein 43 (TDP-43); E3 ubiquitin-protein ligase TRIM9 (TRIM9); ubiquitin; vimentin
Parkinson’s disease	CAP1; α-synuclein (α-syn); BAD; JNK; CK1δ; CDK5; DJ1; GFAP; GSK3β; heat shock protein 27 (HSP27); LRRK2; mTOR; NFκB; Parkin; PTEN; PTEN-induced kinase 1 (PINK1); postsynaptic density protein 95 (PSD-95); SOD2; tyrosine hydroxylase (TH); ubiquitin; vimentin; vacuolar protein sorting ortholog 35 (VPS35)
Amyotrophic lateral sclerosis	C9orf72; CDK5; DCTN1; fibrillarin; fused in Sarcoma RNA-binding protein (FUS); hnRNPA1; neurofilament light chain (NFL); peripherin; superoxide dismutase 1 (SOD1); TAF15; TDP-43; ubiquitin; vesicle-associated membrane protein-associated protein B/C (VAPB); valosin-containing protein (VCP)
Huntington’s disease	CAP1; CK1δ; CDK5; GSK3β; hnRNPA1; huntingtin (htt); ubiquitin
Frontotemporal dementia	C9orf72; CDK5; FUS; Tau; TDP-43; ubiquitin; VCP
Spinocerebellar ataxia	Ataxin-1 (ATXN1) for ataxin-1 for spinocerebellar ataxia type 1; (ATXN2) for spinocerebellar ataxia type 2; ataxin-3 (ATXN3) for Machado-Joseph disease; ataxin-7 (ATXN7) for spinocerebellar ataxia type 7
Creutzfeldt-Jakob disease	PrP; Tau; α-syn; TDP-43; FUS

Phosphorylation-driven aggregative proteins play crucial roles in the pathogenesis of the main neurodegenerative diseases, leading to the formation of toxic protein aggregates and contributing to neuronal dysfunction and death. However, this is not limited to neurodegenerative diseases, other proteins have been identified that can aggregate due to changes in their phosphorylation state. Understanding the aggregation mechanism of these proteins under phosphorylated conditions may provide insights into the development of new treatments for diseases associated with protein aggregation (Tenreiro et al., 2014).

As shown in Box 1, phosphorylation-driven proteins are prominently present in neurodegenerative illnesses. Additionally, it is of special relevance that all the key amyloid-prone proteins, including Aβ, tau, α-syn, TDP-43, superoxide dismutase 1 (SOD1), huntingtin (htt), and prion protein (PrP) (as indicated in bold in Box 1), which cover the vast majority of dementia cases, are phosphorylation-driven aggregative proteins. This reveals a likely relationship between phosphorylation and amyloid aggregation. Interestingly, AD, which is the cause of 70% of dementia cases worldwide, shows the longest list of phosphorylation-driven aggregative proteins (Martin et al., 2013; Tenreiro et al., 2014). These findings suggest that protein phosphorylation could be a key factor in the amyloid aggregation observed in conformational diseases.

Some of the most relevant phosphorylation-driven aggregative proteins are phosphorylated at various protein residues and sites (Box 2) affecting their localization, stability and/or aggregation propensity in several manners. Phosphorylation has a dual effect on proteins. On the one hand, it can alter their conformation or shape, thereby affecting their propensity to aggregate. This is particularly evident in the case of tau (Tenreiro et al., 2014). On the other hand, phosphorylation can also impact protein-protein interactions, which in turn can influence aggregation. For instance, research has demonstrated that the phosphorylation of htt affects its interaction with other proteins, which can, in turn, affect its likelihood to aggregate (DeGuire et al., 2018). The relationship between phosphorylation and protein aggregation is complex and context-dependent. In some cases, it remains to be determined whether phosphorylation is a cause or a consequence in the aggregation process.

Box 2.

Protein phosphorylation sites in proteins linked to neurodegenerative illnesses

Protein	Protein residue	Kinases involved
α-Synuclein	18 putative sites	Several kinases implicated
	Y39, S87, Y125, S129, Y133, Y136	CK1 and 2, PLK2, c-Abl, GRKs (1, 2, 5 and 6), PLKs (1, 2 and 3), LRRK2
Amyloid precursor protein (APP)	10 potential phosphosites	Several kinases involved
	Y653, T654, S655, T668, S675, Y682, T686, Y687	CDC2, GSK-3β, CDK5, JNK, JIP-3-JNK, DAPK1, LRRK2, PLK2, CK1 and 2
Amyloid-β peptides	S8, Y10, S26	PKA, CDC2
Ataxins
ATXN1	S776, T777	PKA
ATXN3	S12, S256, S347	GSK-3β, CK2
Fused in Sarcoma RNA-binding protein (FUS)	17 putative phosphosites	PIKKs, DNA-PK
	T7, S26, S30, S42, S57, S61, T71, S77, S84, S96, S131
Huntingtin (htt)	Multiple phosphosites	IKK, CK2, SGK, CDK5
	S13, S16, S421, S434, S513, S536
RNA-binding protein (TDP43)	64 potential residues	CK1 and 2, CDC7, TTBK1 and 2
	S369, S379, S403, S404, S409, S410
Prion protein (PrP)	Multiple sites	PKC, CK2, Lyn, c-Fgr
	S43, S154, S231, S237
Superoxide dismutase 1 (SOD1)	12 phosphorylation sites	MAPKs, CK1, Dun1
	T2, T58, S59
Tau protein	80 putative phosphorylation sites	Several kinases implicated
	Hyperphosphorylated	GSK-3β, CDK5, LRRK2, MAPK2 and 4, DAPK1, PKA

c-Abl: Abelson tyrosine-protein kinase; CDC: cell division control protein; CDK: cyclin-dependent kinase; c-Fgr: gardner-Rasheed feline sarcoma viral (v-Fgr) oncogene homolog tyrosine-protein kinase; CK: casein kinase; DAPK: death-associated protein kinase; DNA-PK: DNA-dependent protein kinase; Dun: DNA damage response kinase; GRK: G protein-coupled receptor kinase; GSK: glycogen synthase kinase; IKK: IkappaB kinase; JIP: JNK-interacting protein; JNK: c-Jun N-terminal kinase; LRRK: leucine-rich repeat kinase; Lyn: tyrosine-protein kinase Lyn; MAPK: mitogen-activated protein kinase; PIKK: phosphatidylinositol 3-kinase-related kinase; PKA: protein kinase A; PKC: protein kinase C; PLK: polo-like kinase; SGK: serum/glucocorticoid-regulated kinase; TTBK: tau tubulin kinase.

Several kinases, enzymes that catalyze the transfer of phosphate group onto an amino acid residue (typically serine, threonine or tyrosine), are involved in the phosphorylation of various proteins that are associated with neurodegenerative diseases, becoming potential targets for the treatment of amyloid-related diseases (Martin et al., 2013; Tenreiro et al., 2014). Some of the key enzymes involved in these processes are included in Box 2.

There are currently no approved treatments that specifically target phosphorylation-driven aggregative proteins. However, there are several approaches being studied that aim to prevent or reduce protein aggregation in neurodegenerative diseases such as AD and PD, where phosphorylation-driven aggregation plays a role. Thus, the use of small molecules that target specific protein aggregates (Jangampalli et al., 2021), and antibodies that specifically recognize and bind to protein aggregates allowing for their clearance by the immune system, represent two promising approaches including the phosphorylation pathway (Esposto and Martic, 2021).

Figure 1 illustrates the proposed approaches to combat protein aggregation by targeting phosphorylation as a therapeutic strategy. In certain neurodegenerative disorders, the dysregulation of phosphatase activity is a significant contributing factor. Therefore, inhibiting the activity of specific kinases involved in protein misfolding and aggregation, and/or restoring or enhancing the activity of specific phosphatases through the use of small molecules, are promising therapeutic strategies. However, the effectiveness of kinase inhibitors is not entirely clear, since many of the kinases related to these amyloid proteins have a ubiquitous distribution and the same residues can be phosphorylated by several different kinases. In contrast, although phosphatases are considered less specific than kinases (Tenreiro et al., 2014) having a lower level of redundancy can be seen as an advantage, suggesting that they could be potential therapeutic targets.

Figure 1.

Therapeutic strategies against phosphorylation-driven proteins.

Kinase inhibitors: Small molecules that target the enzymes responsible for the phosphorylation of proteins and can reduce the accumulation of phosphorylated proteins and prevent their aggregation (e.g., nilotinib, a specific inhibitor of c-Abl, that increases α-syn degradation (Mahul-Mellier et al., 2014)). Phosphatase activators: Drugs activate the enzymes that dephosphorylate proteins, which can reduce the levels of phosphorylated proteins and prevent their aggregation (e.g., eicosanoyl-5-hydroxytryptamine, an inducer of PP2A, results in the dephosphorylation of α-syn at S129 (Lee et al., 2011)). Autophagy enhancement: Chaperone-mediated autophagy sequences (e.g., rapamycin, an autophagy’s inducer that promotes the clearance of pathogenic protein aggregates of tau, mhtt and α-syn (revised in Ciechanover and Kwon, 2015 and Zhang et al., 2021)). Spreading modulation: Inhibition of the spread of misfolded proteins in the brain (e.g., anti-tau and anti-Aβ antibodies that may inhibit spreading (Giacobini and Gold, 2013)). RNA interference: Small interfering RNA (siRNA) can be designed to specifically target the expression of proteins involved in phosphorylation and aggregation, reducing their production and accumulation (e.g., the inhibition of GSK-3β activity by anti-sense RNAs decrease the phosphorylation of tau (Tenreiro et al., 2014)). Protein clearance: Enhancing of clearance mechanisms such as ubiquitin-proteasome system (e.g., P-tau PROTAC-based small molecule (Jangampalli et al., 2021)). Phosphorylation-specific antibodies: Antibodies that specifically target phosphorylated proteins can help to clear them from the brain and prevent their aggregation (e.g., the inhibition of phosphorylated TDP-43 aggregation using specific anti-TDP-43 antibodies (Esposto and Martic, 2021)). Created with Microsoft PowerPoint. Peptide inhibitors: Peptides that mimic the phosphorylated protein can bind to the proteins responsible for their aggregation, preventing their interaction and subsequent aggregation.

Other attractive therapeutic strategies are to enhance autophagy (the cellular process by which damaged proteins are degraded and recycled) and modulate protein clearance. Recent studies suggest a relationship between the progression of neurodegenerative diseases, such as AD and PD, and the cell-to-cell transmission of amyloidogenic species. Therefore, the removal of these toxic species may help slow or contain neurodegeneration by reducing the spread of pathology caused by phosphorylation (Tenreiro et al., 2014; Ciechanover and Kwon, 2015).

Proximity-induced pharmacology (PIP) is an emergent strategy that uses several potential approaches to treat neurodegenerative diseases in an innovative and highly interesting manner. PIP is a technique that uses small molecules or antibodies to bring two specific proteins into close proximity, inducing a biological response. The two proteins involved are the protein that is being modulated and the protein that is performing the action. Examples of PIP include PROTACs, phosphorylation-targeting chimeras (PhosTACs), and autophagy-targeting chimera (AUTACs).

PROTAC works by recruiting an E3 ubiquitin ligase to a target protein, resulting in its ubiquitination and subsequent degradation by the proteasome. This allows for the targeted degradation of disease-causing proteins. In neurodegenerative diseases, PROTACs have shown particular success in the treatment of tau. Tau PROTACs are unique in that they can degrade both phosphorylated and non-phosphorylated tau, both of which are implicated in the development of AD. Phosphorylated tau is a hallmark of the disease and is known to form toxic aggregates in the brain. However, non-phosphorylated tau has also been shown to contribute to the disease process, making it an important target for drug development. By degrading both forms of tau, Tau PROTACs have the potential to be more effective than other drugs that only target one form of the protein. This makes them an exciting new avenue for the treatment of neurodegenerative diseases, and researchers are actively exploring their therapeutic potential (Jangampalli et al., 2021; Jiang et al., 2021).

PhosTAC, on the other hand, works by recruiting phosphatases to the target proteins and inducing dephosphorylation. Studies have shown that PhosTAC tau is a promising new approach to treating tauopathies, including AD, to specifically induce tau dephosphorylation (Hu et al., 2023).

AUTAC is a novel therapeutic approach that utilizes the autophagy pathway to selectively degrade disease-causing proteins. It involves using small molecules to induce the formation of autophagosomes around the target protein, which is then degraded by the lysosome. Autophagy plays a critical role in maintaining cellular homeostasis, including clearing damaged or misfolded proteins. Dysfunctional autophagy has been linked to neurodegenerative diseases. Therefore, the use of AUTACs to restore proper autophagy function may provide therapeutic benefits. In neurodegenerative diseases, AUTACs have been designed to selectively target and degrade abnormal proteins, such as tau and α-syn, implicated in the development and progression of these diseases (Zhang et al., 2021).

After demonstrating clinical proof-of-concept in 2020 for PROTAC molecules targeting two established cancer targets, the field is now ready to pursue targets that were previously deemed undruggable. As a result, there are currently 18 protein degraders undergoing phase I or phase I/II clinical trials involving patients with different types of tumors. Additionally, a phase III trial for one of these degraders was initiated in 2022. The encouraging initial outcomes of PROTAC systems in cancer indicate the possibility of extending their application to conformational diseases within the next few years. In the realm of neurodegenerative disease treatment, PROTACs, PhosTACs, and AUTACs have emerged as promising approaches, albeit still in the early stages of development. At present, these and other PIP approaches have primarily been tested in vitro, in cellulo and animal models, with no preclinical or clinical trials conducted thus far. However, the potential of leveraging phosphorylated proteins as central contributors to the pathogenesis of various neurodegenerative disorders has garnered significant attention. Particularly, recent advancements in the development of tau PhosTACs have fostered a strong belief in the prospective therapeutic utility of targeting the phosphorylation pathway in the coming years, specifically for the treatment of neurodegenerative diseases associated with tau phosphorylation.

This work was funded by Ministerio de Ciencia e Innovación, Grant Number PID2021-127863OB-I00 (to AE and RS).

Additional file: Open peer review report 1 ^{(81.2KB, pdf)} .