The Proteostasis Network in Proteinopathies

Abstract

Proteinopathies are neurodegenerative disorders that are characterized by accumulation of misfolded toxic protein aggregates that lead to synaptic and neuronal dysfunction. Although genetically, clinically, and pathologically distinct, a common feature of these diseases is disruption of protein homeostasis (proteostasis), which causes accumulation of misfolded proteins. The machinery mediating proteostasis exquisitely balances and interlaces protein synthesis, protein folding and trafficking, and protein degradation processes within the proteostasis network to maintain homeostasis. The proteostasis network governs a functional and dynamic proteome by modulating the timing, location, and stoichiometry of protein expression, surveillance, and maintenance of protein folding and removal of misfolded or excess proteins. Although a functional proteome is essential for the health of all cell types, this is especially true for neurons, which are prone to enhanced cellular stress. Aging is the most important risk factor for proteostasis decline and the development of proteinopathies. However, germline and somatic mutations can also functionally impair components of the proteostasis network. Post-mitotic cells, particularly neurons, are rendered further susceptible to proteostasis dysfunction because of their extended lifespan. This review discusses the interconnections between the functional components mediating proteostasis in neuronal cells and how aberrations in proteostasis contribute to neuronal dysfunction and disease.

Graphical abstract

Proteostasis orchestrates a network of cellular processes to ensure a balanced functional proteome at the organismal, tissue, cellular and subcellular levels with temporal specificity.1, 2, 3, 4, 5, 6, 7 Compromised proteostasis leads to an accumulation of dysfunctional proteins (aggregates) that slowly disrupt normal cellular function before neuronal loss; these changes are an early hallmark of several neurodegenerative diseases collectively known as proteinopathies.^5,8 Understanding the molecular and functional mechanisms of proteostasis offers critical insights into the pathogenesis of proteinopathies, as well as therapeutic approaches to extending brain health.

The fundamental processes involved in proteostasis include three interconnected arms or domains known as protein synthesis, protein folding and trafficking, and protein degradation. Approximately 3000 genes encode the components of this integrated network and collectively it is called the proteostasis network.9, 10, 11 These components do not function with mutual exclusivity; they function cooperatively across all three processes to provide surveillance of proteome integrity and limit the accumulation of toxic proteins (Figure 1).12, 13, 14 These components act within nine organelle or process-specific branches: proteostasis network regulation and protein translation, nuclear, mitochondrial, endoplasmic reticulum (ER), extracellular and cytonuclear proteostasis, ubiquitin-proteasome system (UPS), and autophagy-lysosome pathway (ALP; for a full review, see the studies by Elsasser et al).^9,10 Proteostasis mediates protein functionality globally, spatially, and temporally by ensuring proper protein folding and post-translational modifications (SUMOylation, glycosylation, phosphorylation, ubiquitination, palmitoylation, and disulfide bond formation), and protein disposal through collaborative organelle (ER, mitochondria, and Golgi apparatus) proteostasis efforts.^4,15, 16, 17, 18, 19 These components mediate adaptive cellular responses to restore balance and limit cellular damage. Adaptive cellular responses, like the unfolded protein response (UPR), are cytoprotective defense mechanisms activated by cells under different types of stress to restore homeostasis.²⁰ Examples of intrinsic cellular stress include increased protein biosynthetic demand, glycosylation errors, abnormal protein folding, and ribosome stalling (causes ER stress), ER stress (causes an increase in misfolded proteins, hypoglycosylation, organelle dysfunction, and calcium imbalance), oxidative stress (causes glycosylation errors, and lipid, protein, and DNA damage), iron deprivation (causes mitochondrial and oxidative stress), or gene mutations that affect a protein's aggregation potential, function, or expression levels.^18,21, 22, 23, 24, 25, 26, 27 Extrinsic or environmental cellular stressors include nutrient/glucose deprivation (causes oxidative and ER stress), viral infections (causes oxidative and inflammatory stress), and hypoxia (causes energy depletion, and oxidative, ER, and inflammatory stress).^18,21,26,27 Neurons are constantly exposed to these stressors, and failure of the proteostasis network to remedy stress is associated with accumulated misfolded proteins and loss of synaptic plasticity, which contributes to the onset and progression of proteinopathies.

Figure 1.

The proteostasis network mechanisms and machinery. Proteostasis is composed of three main modules, protein synthesis/translation, protein folding and trafficking, and protein degradation. The components functioning within these modules are divided into nine branches. Highlighted in the figure are key molecular mechanisms and protein components. There is significant crosstalk and functional redundancy within the network, as the mechanisms within each module are interconnected. AKT1, AKT serine/threonine kinase 1; ALP, autophagy-lysosome pathway; AMFR, autocrine motility factor receptor; AMPK, AMP-activated protein kinase; ATF, activating transcription factor; C/N, cytonuclear; DNAJC2, DnaJ heat shock protein family (Hsp40) member C2; DUB, deubiquitinating enzyme; EC, extracellular; eIF1/2/3/4, eukaryotic translation initiation factor 1, 2, 3, and 4; eIF4E2/5A/3J, eIF4E family member 2, eIF5A eIF3 subunit J; ER, endoplasmic reticulum; ERAD, ER-associated degradation; GCN1, GCN1 activator or EIF2AK4; GSPT1/2, G₁ to S phase transition 1 and 2; HSF, heat shock transcription factor; HSP14, heat shock protein family A (Hsp70) member 14; M, mitochondrial; MitoTAD, mitochondrial translocation-associated degradation; MRPL, mitochondrial ribosomal protein L family member; MRPS, mitochondrial ribosomal protein s family member; MTIF2/3, mitochondrial translational initiation factor 2 or 3); mTORC1, mammalian target of rapamycin complex 1; MTRF1/L1/R, mitochondrial translation release factor F1, like factor F1, L1, or R; N, nuclear; NAAs, N-alpha-acetyltransferases; NR, network regulation; PERK, protein kinase R (PKR)–like ER kinase; PI3K, phosphatidylinositol 3-kinase; RPL, ribosomal protein L family member; RPS, ribosomal protein Ss family member; T, translation regulation; TRAPPC, trafficking protein particle complex 1, 3, 4, 5, 8, 11, 12, and 13; Ub, ubiquitin; UBL, ubiquitin-like protein; ULK1, unc-51–like autophagy-activating kinase 1; UPS, ubiquitin-proteasome system; VCP, valosin-containing protein; XBP1, X-box binding protein 1.

A functional proteostasis network is particularly critical for neurons for several reasons. First, the high metabolic demands of neurons are themselves stressful, meaning any additional stress increases the likelihood of disrupted proteostasis.²⁸ Neurons are highly polarized cells with compartmentalized and spatially specific signaling demands (eg, synapses and axonal growth cones) (Figure 2, A and B).29, 30, 31, 32 Neurons constantly require efficient neuronal and synaptic protein turnover to provide on-demand signaling that is required for long-term memory formation and neuronal plasticity, albeit at a high energy and functional cost.^7,16,19,33, 34, 35, 36, 37 These high-energy demands result in the brain consuming >20% of the body's total oxygen, making neurons highly susceptible to oxidative damage that impacts proteins, DNA, and lipids.^38,39 Furthermore, although neurons tolerate excessive oxidative stress, over time that tolerance wanes, partially because of age-related reductions in stress-induced chaperones (eg, heat shock proteins).⁴⁰ Oxidative stress is thus intimately tied to many proteinopathies. Furthermore, neurons are long-living post-mitotic cells that cannot dilute cellular damage (eg, DNA damage, reactive oxygen species, or misfolded protein accumulation) by cellular division to prevent it from reaching cytotoxic levels.^23,41 Biological failures are inevitable when generating functional proteins, and because neurons are normally in a stressful environment, they depend heavily on systems that surveille protein folding and degradation to avoid cellular damage (Figure 2, C and D).^5,28 However, whether aggregate formation is a cause or consequence of inadequate chaperone or degradation activity remains elusive and likely case dependent.⁴²

Figure 2.

Neuronal proteostasis. A: The complexity of the neuronal proteostasis network is represented, with each arm of proteostasis highlighted in distinct inset boxed areas (B–E). For simplicity, only key components for each arm of the network are highlighted. B: Synthesis occurs in the soma and locally in axons, dendrites, and dendritic spines to generate unique subcellular proteomes. Free ribosomes, ribonucleoprotein (RNP) granules, and mitochondria maintain local compartmental proteomes for growth cones, dendritic growth, and spine formation. Dendritic endoplasmic reticulum (ER) is crucial for fine branching of dendrites (arborization). Golgi assist in sorting and glycosylating lipids and proteins destined for inside or outside of the neuron. C: Trafficking/folding occurs throughout the neuron and dendrites and is mainly maintained by chaperones and cytoskeletal proteins (microtubules, actin, and microfilaments) and small heat shock protein (HSP). The ER and mitochondria also play a role in trafficking of proteins. These proteins not only support the organization and structure of dendrites but also aid in trafficking of small organelles, granules, proteins, and RNA molecules. Chaperones also facilitate degradation by clearing aggregates. D: Degradation is crucial for neuronal health, which depends on proper dendritic spine and synapse pruning and removal of ubiquitinated labeled proteins, toxic aggregates, and faulty organelles. Proteasome, chaperone-mediated autophagy, lysosome autophagy, exosome, and endosome components cooperate with proteases to degrade and/or recycle local proteins or aggregates, an essential process to promote plasticity. Ubiquitinated proteins and synaptic pruning are essential for long-term depression. When these degradative processes become overwhelmed because of failures of the other modules of proteostasis, aging, mutations, or are faulty themselves, aberrant proteostasis results. E: Aberrant proteostasis pathologically is an accumulation of ER stress and toxic misfolded proteins, tau aggregates (neurofibrillary tangles), Aβ plaques, and stress granules. Functional proteins and chaperones get trapped in aggregates. This eventually results in a loss of functional dendritic spines and eventually neuronal degeneration. HSR, heat shock response; ISR, integrated stress response; Ub, ubiquitin; UPR, unfolded protein response.

Neuronal complexity involves intracellular and intercellular signaling under both physiological and stressful conditions.³⁶ This requires neurons to depend heavily on proteostasis to maintain their dynamic nature and plasticity.³⁶ There are approximately 15,000 proteins in the neuronal proteome, and hippocampal CA1 and CA3 neurons have approximately 50,000 synapses per neuron, with hundreds to thousands of copies of different proteins.^23,38,43 Billions of dynamically interacting neuronal and glial cells contribute to this complexity.³⁸ To facilitate and maintain neuronal plasticity, dendrites and synapses require efficient protein trafficking and a high rate of local protein synthesis and degradation (Figure 2, B–D).^19,37 Neurons set up remote organelles to meet synthesis needs and to conserve energy in several ways; satellite ER exists at dendrites and branching points, whereas Golgi apparatuses are concentrated in dendrites to nucleate microtubules for protein trafficking, and at dendritic branching points to provide local post-translational modifications (Figure 2C).^13,33,43 Defects in microtubules or blockages in axonal transport due to aggregates can cripple this entire system and contribute to proteostasis failures.^13,44

Interaction or aggregation with other proteins is an intrinsic property of proteins, and a protein's aggregation propensity, or insolubility, is tied to its amino acid sequence, the presence of cysteine residues (disulfide bond formation), and whether its expression is supersaturated.^5,45, 46, 47 Many neuropathologic proteins have a high intrinsic aggregation propensity, making neurons especially vulnerable to oxidative stress and aggregate formation.⁴⁷ Oxidation of cysteines in aggregate-prone proteins promotes protein unfolding and inappropriate intrachain and interchain disulfide bond formation, resulting in aggregate nucleation, trapped chaperone proteins, and chronic ER stress.^45,46,48, 49, 50, 51 Aggregate propensity is also influenced by a neuron's high protein turnover rate, crowded intracellular environment, protein trafficking distances to dendrites and synapses, and inadequate chaperone activity.^{7,14,31,34,41} Neurons in particular are overreliant on chaperone activity to protect nascent, partially unfolded proteins from inappropriate interactions while they assume their functional conformation or as they travel long distances along microtubules to their destination.⁷ Misfolded, partially folded or oligomeric proteins are toxic because they interact inappropriately with other proteins and organelles (compromising organelle integrity), or form aggregates that sequester essential proteostasis network proteins, such as degradation machinery and molecular chaperones (Figure 2E).^34,47 As with microtubules, chaperone expression and activity are commonly deficient with age and in proteinopathies.^24,44

Neuronal proteostasis must balance a tolerable accumulation of toxic aggregates with preservation of synaptic plasticity. Unfortunately, maintaining this balance is challenging as proteostasis pathways progressively and nonuniformly decline with age for poorly understood reasons.^2,5,18,52, 53, 54, 55 Proteostasis network components can also be genetically disrupted, rendering neurons prone to misfolded or abnormal proteins, which ultimately leads to imbalanced local proteomes and synaptic failure.^18,29,30,56, 57, 58 Proteinopathies are also caused by age-related declines in expression or activity or mutational disruption (sporadic or familial) of proteostasis components. Alzheimer disease (AD), Huntington disease (familial only), prion diseases, amyotrophic lateral sclerosis (ALS), frontotemporal lobar degenerations (FTLDs), polyQ diseases, multisystem proteinopathy, and synucleinopathies [Parkinson disease (PD), dementia with Lewy bodies, and multiple system atrophy] are all proteinopathies that demonstrate pathologic aggregates.^8,59 The toxic aggregates most commonly encountered in proteinopathies have high aggregation propensity and include hyperphosphorylated tau (p-tau; associated with AD and some forms of FTLD); β-amyloid peptide (Aβ; associated with AD); α-synuclein (associated with PD, dementia with Lewy bodies, and multiple system atrophy); transactive response (TAR) DNA-binding protein 43 (TARDBP/TDP-43; associated with AD, FTLD, and ALS); superoxide dismutase 1 (SOD1; associated with ALS); fused in sarcoma (FUS; associated with ALS and FTLD); huntingtin (associated with Huntington disease); and TATA-box binding protein associated factor 15 (associated with FTLD).^8,24,60, 61, 62, 63 Soluble monomers (eg, Aβ and p-tau) and oligomers (eg, α-synuclein) of these partially folded proteins are aggregation prone because they stably bind off-target proteins, with some forming insoluble fibrils.^2,34,64 Genetic variants associated with low baseline proteostasis activity and environmental exposures are other risk factors for aggregate formation and proteinopathy development.^32,65, 66, 67 Loss of synaptic plasticity (synaptic failure) is an early and key contributor to the gradual decline of cognitive function observed as proteinopathies progress.^37,68,69

In summary, proteostasis is a network of cellular and molecular components that work together to safeguard neuronal function by ensuring adequate functional protein pool fluxes, at the right time, in the right place, and in response to cellular and environmental stressors.^16,34,70,71 The brain is unique in that its proteostasis demands involve the coordination of dynamic communication between trillions of synapses within a cellularly heterogeneous and stressful environment. Although the molecular mechanisms driving proteinopathies are still poorly understood, neuronal accumulation of misfolded protein aggregates is definitively a failure of the proteostasis network. This review focuses on the contribution of dysfunctional proteostasis to the development of proteinopathies.

The Components of the Proteostasis Network

Protein Synthesis

Protein synthesis is the biological process by which proteins are made or translated. It is managed by many quality control mechanisms within the Synthesis module that modulate transcriptional and translational control of genes. The molecular components are divided into two functional branches: one branch orchestrates the translational machinery to maintain proteome biosynthesis (translational regulation), and the other branch mediates the transcriptional regulation of genes necessary for cells to respond to cellular stress and restore homeostasis (network regulation) (Figure 1).

The translation regulation branch includes all the basic translational components for global cytosolic and mitochondrial protein synthesis. This includes ribosomal subunits, tRNA synthetases, and other factors mediating translational initiation, elongation, and termination, the biogenesis of ribosomes and mitoribosomes, ATPases, and ribosome quality control (RQC)–associated proteins.^9,10 Because meeting the spatial-specific proteome needs of dendrites and synapses requires immense ATP consumption, neurons establish protein synthesis hubs outside of the soma to conserve energy.^72,73 Local versus dendritic mRNA localization depends on the half-life of the protein or mRNA, the ratio of coding versus noncoding nucleotides in the mRNA, and the protein size.⁷² Ribonucleoprotein granules and ribosome-associated vesicles bind mRNAs in the nucleus and transport them to dendrites and other subcellular compartments for local translation.^73,74 Valosin-containing protein (VCP; alias p97) and mechanistic target of rapamycin complex 1 (mTORC1) are essential mediators in global and spatial proteome generation and quality.^9,10,74 VCP/p97 is a major regulator of RQC and the induction of translation regulatory factors. Ribosome function is error prone, with an error rate of 1 in every 20 amino acids, which can lead to a misfolded protein if RQC is not properly engaged.¹¹ The RQC machinery provides surveillance of ribosome errors, stalled ribosomes, and post-transcriptional splicing errors that can result in defective protein products.¹¹ Neuronal vulnerability increases when the RQC system becomes overwhelmed by cellular stress, aging, mutations in RQC pathway genes (eg, VCP/p95 in inclusion body myopathy associated with Paget disease of bone with or without frontotemporal dementia or nuclear export mediator factor in AD), or a lack of available tRNAs.⁷⁵ Consequently, global protein synthesis rates can become too low and compromise protein quality control (PQC) mechanisms, leading to an increase in the number of faulty proteins.²⁵

The network regulation branch includes the components directly responsible for orchestrating proteome changes in response to specific cellular stressors. These components function at transcriptional and translational levels to provide on-demand gene expression and restore proteostasis.^{9,10,18,20,26} They include transcription factor regulators (ie, heat shock transcription factors) and translational regulators [ie, eukaryotic translation initiation factors (EIF2AK)].^9,10 Cellular stressors include hypoxic stress response, oxidative stress response, heat shock response, inflammatory response, integrated stress response (ISR), and UPR.^18,20 Activation of heat shock response, oxidative stress response, or inflammatory response transcriptionally reprograms target genes in response to compartment-specific stress. In contrast, ISR or UPR activation translationally (ie, ISR and UPR) or transcriptionally (ie, UPR) reprograms the proteome in response to simultaneous diverse cellular stresses.^1,21,76 In other words, if individual adaptive cellular responses (heat shock response, oxidative stress response, or inflammatory response) fail, they activate integrated adaptive responses (ISR or UPR) and when those fail, apoptosis (maladaptive response) is activated.⁶⁵ ISR senses stress in the cytosol (viral infection, amino acid imbalance, uncharged tRNAs, and heme depletion), ER (protein load), or mitochondria (protein load and heme deficiency), whereas the heat shock response senses and responds to misfolded proteins in the cytosol, the oxidative stress response senses stress in the mitochondria, and the UPR senses stress in the lumen of the ER or mitochondria (not discussed here) via transmembrane sensors that detect misfolded proteins (Figure 2A).^21,26,77 UPR activation specifically leads to translation and ER translocation attenuation, transcriptional up-regulation of specific ER-associated degradation (ERAD) factors, and up-regulation of lipid synthesis, molecular chaperones, and post-translational modifying enzymes to increase ER volume and folding capacity ER^1,26,78, 79, 80 (Figure 3).

Figure 3.

Activation of the unfolded protein response (UPR) and its involvement in proteinopathies. The UPR pathways, including three key transmembrane proteins [protein kinase R (PKR)–like endoplasmic reticulum (ER) kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1)], can be activated in response to ER stress-induced UPR activation, which can restore homeostasis. The figure represents UPR activation as an adaptive cellular response. Dysregulation of these pathways has been implicated in various neurodegenerative diseases, such as Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, frontotemporal lobal degeneration, and Huntington disease. EIF2AK3, eukaryotic translation initiation factor 2 alpha (alias PERK); ERAD, ER-associated degradation; ISR, integrated stress response; XBP1, X-box binding protein 1.

Activation of ISR and the UPR attempts to prevent cytotoxicity by halting global protein synthesis and prioritizing the production of stress response genes (ie, chaperones and amino acid biosynthesis) that are needed in the affected organelle(s).^{7,11,21,25,64,77,81} Crosstalk between the ISR and proteotoxicity adaptive pathways (ie, UPR, UPS, and autophagy) occurs through activation of the protein kinase R (PKR)–like ER kinase (PERK/EIF2AK3)/eIF2α/activating transcription factor 4 (ATF4) signaling axis (Figure 3).^{1,26,80,82,83} In the case with ISR and UPR, PERK activation and subsequent mTORC1 inhibition are shared signaling events intended for temporary inhibition of global protein synthesis and increases in autophagy.^{1,25,26,41,64} Persistent activation of ISR and UPR, as is seen in proteinopathies, becomes maladaptive because of these effects. ISR and UPR mechanisms are still ill defined but are being explored as therapeutic targets using new methods like HOTag, a fluorescent labeling technology that marks unfolded proteins for tracking.⁸⁴

Protein Folding and Trafficking

Protein folding and trafficking are biological processes by which proteins are assembled into their functional shape and transported to their destination. Folding and trafficking components work collaboratively and include chaperones, co-chaperones, ubiquitin-related proteins and isomerases that assist the folding process.¹⁰ The folding and trafficking module also includes transport proteins associated with organelle-specific degradation, glycoproteostasis, and protein maturation. Protein folding is highly prone to error, with up to 30% of newly synthesized proteins prone to misfolding, unfolding, or aggregating.^41,81,85 Molecular chaperones are indispensable for the folding and refolding of nascent polypeptides, general protein transport, or transport to the proteasome for degradation or recycling.^41,81,85 Chaperones, including stress-induced heat shock proteins (HSPs), are a large family of structurally and functionally diverse proteins that reside throughout the cell and function under normal and/or stress conditions.⁸⁶ For example, small HSPs function in the cytoplasm, mitochondria, nucleus, and extracellular space to assist in folding to initiate stress response cascades that prevent protein aggregation.^86,87 HSPs bind vulnerable exposed hydrophobic regions of partially or misfolded proteins to prevent their aggregation.^34,41 Collectively the components in this network can be categorized into five functional branches: nuclear, mitochondria, cytonuclear, extracellular, and ER¹⁰ (Figure 1) [for an updated and exhaustive list of components, please see the Proteostasis Consortium website (https://www.proteostasisconsortium.com/pn-annotation, last accessed June 2025)]. Briefly, components within the nuclear proteostasis branch mediate chaperone-mediated histone folding, nuclear pore complex formation, and nuclear protein transport. Components within the mitochondria proteostasis branch mediate mitochondrial-associated physiological and stress response protein folding and refolding, including mitochondrial-specific protein maturation, protein import, and degradation. Components within the cytonuclear proteostasis branch function in both the cytoplasm and nucleus to mediate folding processes. Components within the extracellular proteostasis branch include proteases and chaperones that mediate cell adhesion, transsynaptic communication, and inhibition of aggregates of extracellular misfolded proteins, like Aβ.88, 89, 90, 91 Components within the ER proteostasis branch mediate ER-associated physiological and stress response protein folding and refolding processes, including glycoproteostasis, ER-specific protein maturation, protein transport, and degradation (ie, ERAD). VCP, ubiquitin-related proteins, and HSPs are key players in ER proteostasis, and they also function across all three arms of the proteostasis network.¹⁰

The ER deserves special attention because it not only generates 30% to 40% of the total proteome, it is central to connecting all three modules of proteostasis by sensing protein synthesis levels and protein integrity, and activating degradation mechanisms.^{34,58,81,91,92} Like neurons, the ER is normally in a stressful environment. The ER and mitochondria are the only organelles that can perform the most challenging type of folding (oxidative folding), and approximately 80% of secreted proteins and approximately 41% of membrane proteins are cysteine rich with disulfide bonds.^93,94 Cysteine-containing proteins are the most difficult to fold because of the number of chaperones required for proper folding, and the large number of disulfide-bonded (oxidative folding) isomer iterations that occur.¹⁸ ER stress occurs when chaperone proteins can no longer manage the protein folding load and misfolded proteins pileup in the ER. ER or mitochondria-containing clogged translocation-arrested precursor proteins will activate ERAD or mitochondrial translocation-associated degradation, respectively. ERAD and mitochondrial translocation-associated degradation contribute to organelle integrity by promoting the removal of mislocalized membrane proteins and distinguishing terminally misfolded proteins that should be degraded from nascent proteins that should be refolded.⁹⁵ ERAD is a well-studied but still poorly understood molecular process that weaves through both the folding and trafficking and degradation modules of the proteostasis network, albeit with distinct functional roles. ERAD's functional role in the folding and trafficking module involves VCP/p97-mediated ER retrotranslocation, unfolding, and cytosolic handling of substrates (deglycosylation and substrate association with proteasome components).^9,10 However, in the degradation branch, ERAD functions within the UPS system to mediate PQC through association with ERAD cofactors (VCP/p97) and ubiquitinated proteins for their delivery to the proteasome, and in persistent cases to the autophagy machinery.^80,96,97 Secreted and transmembrane proteins, including most synaptic proteins, are synthesized, folded, and post-translationally modified inside the ER.^18,58 The ER is the predominant trigger of cellular degradation pathways.⁵⁸

Protein Degradation

Protein degradation is a core process for protein homeostasis in which superfluous proteins are broken down to recycle their amino acids or to remove aberrant proteins or organelles. The degradation module of the proteostasis network is made up of two functional branches, UPS and ALP (Figure 1). It contains the largest number of protein components and is a crucial modulator of synaptic plasticity.⁸ The UPS and ALP degradation branches have unique and overlapping functional components, but their activation is triggered by different events and by different types of aggregates.^64,98 For example, the proteasome regulates synaptic plasticity by controlling the degradation of presynaptic and post-synaptic proteins, whereas autophagy modulates hyperexcitability by degrading postsynaptic receptors and disrupting the excitatory-inhibitory balance in neuronal circuits.⁸

The UPS branch of the degradation module is a fundamental PQC system that orchestrates protein disaggregation via the 26S proteasome.^11,99 The UPS branch of the degradation module is made up primarily of proteasomes and their associated proteins, VCP/p97-containing complexes, deubiquitinating enzymes (DUBs), ubiquitin and ubiquitin-like protein modifiers, ligases, and binding proteins that target excess soluble proteins, or nascent and mature misfolded proteins (Figure 1).^9,10,100 There are both nuclear and cytoplasmic proteasome pathways, but here we focus on the cytoplasmic UPS. The UPS system is the first responder to misfolded ubiquitin-labeled protein accumulation mediated by ERAD components and accounts for approximately 80% of protein turnover.¹¹ To alleviate stress, ERAD is first activated to remove misfolded or unfolded nascent proteins. Misfolded proteins in the ER are retrotranslocated out of the ER by VCP/p97; they then are ubiquitinated and targeted for proteasomal degradation.⁹⁶ Misfolded cytosolic proteins or the cytosolic faces of misfolded proteins in the ER are also ubiquitinated; when four or more ubiquitin substrates are added, the protein is targeted for degradation by the 26S proteasome.³⁴

The ALP branch of the degradation module functions as an adaptive cellular process to complement the UPS or when dysfunctional organelles require disposal.⁹⁹ Although most soluble and misfolded cytosolic proteins are degraded by the proteasome, most ER-resident protein aggregates are cleared by autophagy.⁹⁹ The autophagy-lysosomal system has three distinct canonical processes: microautophagy, chaperone-mediated autophagy, and macroautophagy.101, 102, 103 More recently, the noncanonical processes called conjugation of autophagy-related-gene (ATG) 8 to single membranes or autophagy-related extracellular secretion have been identified.101, 102, 103

Microautophagy occurs when late endosomes and lysosomes engulf misfolded proteins. Chaperone-mediated autophagy is an HSP assisted nonvesicular, nonubiquitin, non–ATG-dependent cytoplasmic lysosomal process for degrading specific soluble proteins.^64,104 Macroautophagy, or autophagy, is a multistage lysosomal-based degradation process involving autophagosomes, in which proteins encoded by ATGs engulf large organelles under normal conditions [eg, redundant peroxisomes (perophagy)], pathogenic organisms (xenophagy), dysfunctional mitochondria (mitophagy), dysfunctional lysosomes (lysophagy), aberrant or excess ER components (ER-phagy/reticulophagy), insoluble misfolded protein aggregates, and larger inclusions (aggrephagy).¹⁰⁵ Autophagy involves the conjugation of ubiquitin-like ATG8 proteins (ATG8ylation) on double-membrane autophagosomes (immature) and their fusion to lysosomes (mature), a process that is dependent on VCP/p97.^{57,97,106,107} In noncanonical autophagy (eg, conjugation of ATG8 to single membranes), ATG8ylation occurs on nonautophagic single membranes, such as lysosomes, Golgi, ER, endosomes, phagosomes, and lipid droplets, via at least two distinct mechanisms: V-ATPase-ATG16L1–induced LC3 lipidation or sphingomyelin-TECPR1–induced LC3 lipidation.¹⁰⁸ Although different types of noncanonical autophagy are still being identified, several forms of this have been described, including LC3-associated phagocytosis, LC3-associated endocytosis, LC3-dependent extracellular vesicle loading and secretion, LC3-associated micropinocytosis, and retromer-dependent trafficking, RAS-induced noncanonical autophagy via ATG8ylation.101, 102, 103^,109,110 This is an emerging area of research, but Parkinson and Alzheimer disease have already been found to have failures in at least LC3-associated endocytosis.^101,102

Autophagy is a core function of the ALP and is usually activated by starvation or cellular stress (eg, ER stress, hypoxia, toxic protein aggregates, oxidative stress, and damaged organelles).^58,104 Autophagy and the ER stress response are both protective mechanisms that can function independently or via interconnections that are still poorly understood.^80,111 Prolonged ER stress activates UPR-triggered autophagy via PERK/ATF4-mediated inhibition of mTORC1 and activation of AMP-activated protein kinase (AMPK)⁸⁰ (Figure 3). mTORC1 negatively regulates autophagy by inhibiting the key initiation kinase, unc-51–like autophagy-activating kinase 1 (ULK1), and AMPK promotes autophagy by activating ULK1, making ULK1 a key signaling axis between mTORC1 and AMPK.⁶⁴ It has been difficult to parse out the specific molecules responding to each of these cellular stressors because of their interconnected nature, as failure in one proteostasis module can cause failure in another module, expediting proteinopathy progression. This is especially true in the context of familial or sporadic mutations associated with proteinopathies.

Disrupted Proteostasis in Proteinopathies

Disrupted proteostasis can result from a plethora of aging-related declines in network component expression or activity (eg, chaperones) or by familial or somatic mutations that compromise the function of a network component (eg, VCP/p95), or by familial or somatic mutations that chronically stress proteostasis (eg, SOD1). Mutational effects are further compounded by aging-related declines in neuronal proteostasis components.

Although familial gene mutations can contribute to the development of proteinopathies, age is the major risk factor for cognitive decline, and there are still no cures for these progressive diseases.¹¹² Why intrinsic proteostasis network mechanisms fail with aging is up for debate, but several lines of evidence suggest that a lack of genetic programming designed to keep protein homeostasis intact through post-reproductive years is a key contributor.¹⁴ Cumulative intrinsic events that promote proteostasis failure include DNA damage that accumulates over time (somatic mutations), progressive shortening of telomeres, epigenetic changes, global reductions in protein synthesis (especially those involved with heat shock proteins/chaperones), increased iron deposition in the brain (inhibits autophagy clearance), reduction in degradation efficiency (UPS and ALP), decreased solubility of proteins (thereby trapping chaperones), and increases in oxidative stress.^{5,8,14,22,53,113}

In addition, some gene variants negatively impact the proteostasis network. For example, the most significant germline risk factor for late-onset AD is apolipoprotein E ε4 haplotype, which disrupts autophagy and Aβ clearance.¹¹⁴ In familial forms of proteinopathies, disease-causing gene mutations indirectly or directly affect proteostasis network components; mutations in C9orf72 and FUS negatively impact vesicular transport and autophagy.⁹⁷ Additional genetic risk factors are still being identified.

Synthesis Failures in Proteinopathies

Translational, ribosomal, and PQC capacity decreases as we age, but along with age-related drivers of proteinopathies, sporadic mutations in quality control–associated genes also contribute to disease. For example, mutations in tRNA synthetases affect tRNA charging, whereas mutations affecting RNA-binding proteins [eg, TARDBP/TDP-43, FUS, T-cell-restricted intracellular antigen-1 (TIA1), and heterogeneous nuclear ribonucleoprotein A (hnRNPAs)] impair RNA splicing and stress granule formation, which promotes the accumulation of misfolded proteins and proteinopathies.²⁴ Increased mTORC1 activity is associated with a pathogenic form of α-synuclein in PD, leading to increased ribosomal elongation and neuronal toxicity through a mechanism that overloads the protein folding machinery and inhibits autophagy clearance.¹¹⁵ Aberrant increases in mTORC1 activity can promote an increase in translation and translational errors, which, in turn, promotes ER stress and misfolded protein accumulation.¹¹⁶ Reductions in mTORC1 activity, however, are frequently observed in proteinopathies that display chronic UPR activation. When misfolded proteins accumulate in the ER because of age-related declines or germline or somatic mutations in quality control genes, like VCP in FTLD and ALS, the UPR or ISR stress response can become chronically activated. Prolonged UPR or ISR activation of PERK/eIF2α is unsustainable for neurons because chronically low protein synthesis rates become maladaptive; in animal models of proteinopathies, this ultimately leads to a loss of synaptic plasticity and activation of cell death pathways.^3,83,117, 118, 119, 120 In AD, for example, Aβ or tau deposition activates ER stress. As the disease progresses, irreversible ER stress and excessive UPR activation switches cellular responses from adaptive to maladaptive. What determines this switch is ill defined but is dependent on the length and intensity of PERK activation.²⁸ Alleviating sustained repression of protein synthesis by modulating mTORC1 activity has been explored therapeutically.^{8,34,41,56,115,121}

Folding and Trafficking Failures in Proteinopathies

When folding and trafficking components fail, misfolded or improperly post-translationally modified proteins can accumulate and progressively lead to chronic UPR and ALP activation, organelle dysfunction, proteotoxicity, synaptic failure, and eventually neuronal death.¹²² The ER depends heavily on chaperones to avoid toxic accumulations, and consequently chaperone dysfunction resulting from aging or mutations has a major impact on neurons.¹²³ Aging-impaired induction of chaperones leads to improper folding, interactions, and aberrant complex formation, ultimately resulting in the formation of protein aggregates.¹⁴ Somatic mutations can also impair folding and trafficking. For instance, somatic loss-of-function mutations in PIN1, which encodes the folding-related protein peptidylprolyl cis/trans isomerase, never in mitotis A1 (NIMA)–interacting 1 leads to increased p-tau aggregation AD.¹²⁴ In proteinopathies, oxidative damage is a major age-related driver of somatic mutations.¹²⁴

Disease-causing mutations can also indirectly impair chaperone function by sequestering chaperones into dysfunctional aggregates. In ALS, familial mutations in SOD1 affect its solubility, causing it to aggregate with other proteins, including chaperones.^12,51 In ALS and FTLD, sporadic and familial C9ORF72 (GGGGCC repeat) mutations also encode insoluble peptides that sequester chaperones, which disrupts vesicular trafficking, mitochondrial function, and autophagy.^24,97 In early-onset AD, familial presenilin 1 or presenilin 2 mutations promote folding and trafficking dysfunction by generating aggregates of misfolded amyloid peptides, which triggers prolonged ER stress and maladaptive UPR activation.²⁸ In PD, familial mutations in chaperones like DnaJ HSP family (Hsp40) member C6 (DNAJC6) promote proteinopathy by disrupting synaptic vesicle recycling.⁴² Somatic mutations in cellular trafficking genes [eg, phosphatidylinositol-binding clathrin assembly protein (PICALM) and sortilin-related receptor 1 (SORL1)] impair the trafficking of aggregates in late-onset AD.¹¹⁴

ER proteostasis failures also indirectly cause other organellar (eg, mitochondria, Golgi apparatus, lysosomes, and peroxisomes) and synaptic failures when the ER can no longer provide essential components, such as lipids, leading to compromised organelle integrity.

Degradation Failures in Proteinopathies

The UPS (proteasome) and ALP (autophagy) are the fundamental processes that neurons use to maintain proteostasis and prevent proteinopathies. When intrinsic or extrinsic cell stresses come into play, aggregation-prone proteins must be degraded to prevent proteotoxicity.^{32,54,104,124} Over time, the accumulated effects of oxidative injury result in protein aggregation, which increases the demand for UPS and ALP function. Unfortunately, however, these mechanisms are impaired during aging, which leads to maladaptive processes and increased risk for the development of a proteinopathy.³² Furthermore, many acquired or inherited proteinopathy-associated mutations impair the degradation machinery. When the protein degradation machinery is rendered dysfunctional by mutations and/or aging, its ability to combat proteotoxicity is impaired.

The proteasome degrades proteinopathy-associated proteins, like Aβ, tau, SOD1, and α-synuclein. Although the process is poorly understood, Aβ and p-tau accumulation inversely correlates with proteasomal activity, and decreased UPS activity inversely correlates with plaque and tangle formation and neuronal death.¹²⁵ Genetic variants associated with low baseline proteasome activity have been identified as risk factors for AD and PD, including variants affecting proteasome subunits, DUBs, ubiquitin ligases [autocrine motility factor receptor (AMFR)], HSP (DNAJB14), and vesicle trafficking [SEC24 homolog D, COP11 coat complex component (SEC24D)].³²

Accumulation of ubiquitin-marked proteins and certain mutant proteins can cause UPS overload, leading to activation of the UPR and ALP in an mTORC1- and AMPK-dependent manner as the neuron desperately attempts to restore protein homeostasis via the PQC system.^27,29,64,126 Notably, mTORC1, eIF2α phosphorylation, and VCP/p97 play another role in PQC through membrane-less structures called stress granules. Stress granules form in the soma when the UPS is shut down or during translation and cellular stress, and when persistent, play a progressive role in AD, FTLD, and ALS.¹²⁷ Proteasome and autophagy failure (eg, in a VCP/p97 mutant background) leads to impaired removal of stress granules, which directly contributes to persistent neuronal stress and disease severity.¹²⁷ TARDBP/TDP-43 and FUS mutations promote excessive stress granule formation, which contributes to the progression of ALS and FTLD-TDP type D.¹²⁷ Autophagy and UPS can compensate and cooperate with each other for degradation of unwanted or toxic molecules, like p-tau.¹²⁸ P-tau blocks the autophagy retrograde trafficking system by destabilizing microtubules, leading to an accumulation of immature autophagosomes in axons.¹²⁸ P-tau can accumulate when the UPS becomes overwhelmed, resulting in the development of pretangles and neurofibrillary tangles in diseases such as AD.

Although healthy neurons have low levels of immature autophagosomes, excessive autophagosomes are common in dystrophic neurites and neuronal cell bodies in patients with AD.⁵⁶ Aggrephagy is a substrate-specific autophagy pathway that occurs only in response to protein aggregates or large inclusions (themselves often caused by impaired autophagy). Like the UPS, aggrephagy functions as an Aβ scavenger by both lowering amyloid precursor protein (APP) levels and mediating extracellular disposal of Aβ. Aggrephagy also eliminates toxic intracellular aggregates through lysosomal degradation or extracellular release. Proteinopathies, such as AD and inclusion body myopathy associated with Paget disease of bone with or without frontotemporal dementia, have immature autophagic vacuoles in neuronal dendrites and defective autophagy.^56,129 Age-related loss of ATG expression can also be responsible for dysfunctional autophagy in proteinopathies. ApoE4, a major risk factor for late-onset AD, promotes membrane disruption of lysosomes, thereby destabilizing autophagy.¹²⁵ Other AD- and ALS/FTLD-associated gene variants that affect the ALP network include PICALM, CLU, GRN, TMEM106B (AD), and TARDBP/TBP-43 (ALS/FTLD).^24,85,114 Approximately 10% to 15% of PD cases have familial or sporadic mutations that disrupt autophagy; affected genes include LRRK2, VPS35, and RIT2 (autophagy); Parkin/PINK1, KAT8/KANSL1, SNCA, PRKN/PARK2, and LRRK2 (mitophagy); and SNCA (PARK1/4), LRRK2, UCHL1, and VPS35 (chaperone-mediated autophagy).101, 102, 103 Age-related declines and LRRK2 mutations can also impact noncanonical autophagy.¹⁰² Furthermore, in ALS, mutant SOD1 impairs autophagy by sequestering Hsp70/Hsp40 chaperones into nonfunctional aggregates, and in ALS/FTLD, mutant VCP disrupts autophagy by impairing autophagosome formation and trafficking.⁸⁵

Extracellular disposal of misfolded proteins or partially degraded or undegraded protein aggregates (ie, α-synuclein, Aβ, and p-tau) is used to maintain cellular homeostasis via unconventional UPS and autophagy-related pathways.^130,131 Extracellular cargo release can involve many different methods, including exosomes, the misfolding-associated protein secretion pathway, nanotubes, exophagy, or release as free molecules.^{105,106,130,132} Ironically, relieving intracellular accumulation via extracellular Aβ deposition can exacerbate proteinopathies when autophagy is impaired; this is because exosomal release of aggregates inhibits autophagy further by maintaining their seeding potential through undefined mechanisms.¹²⁵ Meanwhile, while microglia and astrocytes initially facilitate extracellular clearance of aggregated proteins, excessive uptake of these toxic aggregates ultimately impairs microglial and astrocytic function and promotes proteinopathies.^106,133 Despite their ability to internalize aggregates, astrocytes have limited degradation capabilities, and excessive internalization impairs astrocytic synaptic pruning and mitochondrial function, which promotes an inflammatory response.¹⁰⁶ Microglia, on the other hand, internalize and degrade extracellular Aβ, α-synuclein, and p-tau via digestive exophagy, which facilitates seed growth and spreading. Microglial cells also use LC3-associated endocytosis to internalize tau aggregates and limit its spreading in AD, which is beneficial mechanism in the early stages of the disease.¹⁰¹ Enhancing degradation pathways has been explored as a therapeutic approach in proteinopathies, but a better mechanistic understanding is necessary for this to be clinically useful.¹²

Lifestyle and Therapeutic Approaches

Although protein homeostasis declines during aging, there are strategies that can be used to reset proteostasis, like lifestyle changes, avoidance of environmental risk factors, and therapeutic approaches to slow down proteostasis dysfunction.² Prevention and early detection are key to all neurodegenerative diseases. Although average lifespans have doubled in the past century, these extended lifespans are associated with an increase in chronic diseases, such as proteinopathies.¹³⁴ This is due, in part, to poor diet and exercise, environmental toxin exposures, and genetics.¹³⁴ Alleviating cellular and environmental stressors is a reasonable first line of defense to prevent and reduce proteinopathies.

Lifestyle: Diet and Exercise

A lifelong proper diet and exercise are crucial for health and longevity.¹³⁵ Fortunately, the science behind the beneficial effects of a proper diet and exercise and how they affect proteostasis is becoming better understood. mTORC1, which plays multiple roles in regulating proteostasis, is activated by growth factors, neurotransmitters, and dietary nutrients (glucose and amino acids).⁵⁵ However, mTORC1 is hyperactivated in the hippocampus of some post-mortem AD brains.⁵⁵ This suggests that lowering mTORC1 activation may be neuroprotective in some types of AD by restoring autophagy, because prolonged inhibition of autophagy drives aggregate accumulation.^136,137 In animal models, mTORC1 inactivation improves long-term potentiation, and synaptic plasticity by prioritizing the synthesis of key synaptic and structural proteins.^34,138 Inactivation of mTORC1, activation of AMPK, and moderate ketone body production occur when glucose pools are extremely low. Low glucose pools and mTORC1 inactivation can be achieved by nutrient restriction or intermittent fasting, which increases the production of ketone bodies, lowers the risk of proteinopathies, and improves longevity in many species.^138,139 This type of ketone production is a normal metabolic process and is different from diabetic ketoacidosis. β-Hydroxybutyrate and acetoacetate are two neuroprotective ketone bodies. When produced endogenously by nutrient restriction or taken as supplements, β-hydroxybutyrate and acetoacetate improve memory and learning in AD and PD animal models (Figure 4).138, 139, 140, 141 Although not all risk factors can be avoided, a lifelong healthy lifestyle is crucial for prevention and protection against proteinopathy pathogenesis.

Figure 4.

Therapeutic targeting of proteostasis in proteinopathies. The figure represents promising approaches for targeting chronic activation of endoplasmic reticulum (ER) stress, unfolded protein response (UPR), and integrated stress response (ISR) in proteinopathies. These potential neuroprotective therapies target key and overlapping signaling pathways in proteostasis, including UPR, protein kinase R (PKR)–like ER kinase (PERK), mammalian target of rapamycin complex 1 (mTORC1), and AMP-activated protein kinase (AMPK). AcAc, acetoacetate; ALP, autophagy-lysosome pathway; BHB, β-hydroxybutyrate; DBM, dibenzoylmethane; DUB, deubiquitinating enzyme; HDAC, histone deacetylase; HSF, heat shock transcription factor; HSP, heat shock protein; p-eIF2, phosphorylated eukaryotic initiation factor 2; pPERK, phosphorylated PERK; SOD1, superoxide dismutase 1; TARDBP, transactive response (TAR) DNA-binding protein 43; UPS, ubiquitin-proteasome system.

Along with diet, multiple studies have shown that exercise has significant beneficial effects on the aging brain partly through its inhibitory effect on the mTOR pathway and activation of autophagy (Figure 4).^112,135,141, 142, 143, 144 Furthermore, exercise alone reduces neuroinflammation, alters mitochondrial function, and promotes neurogenesis via release of growth factors, anti-inflammatory cytokines, and neurotransmitters.^8,112 There is strong evidence that exercise improves memory and cognition and delays the progression of diseases, like ALS, AD, PD, and Huntington disease.^140,144,145 Although the lifestyle approaches discussed here are widely accessible, they are often not followed, which has led to searches for a pharmaceutical solution to proteinopathies. Unfortunately, bench-to-bedside pharmaceutical approaches have not yet produced safe and effective therapies.

Environmental Risk Factors

Nonbiodegradable microplastics and nanoplastics (MNPs) are a recently identified contributor to proteinopathies as they can cross the blood-brain and placental barriers.¹⁴⁶ Over time, plastic breaks down to form MNPs (10 nm to 5 mm in size), which have ubiquitously contaminated our environment (air, water, soil, and food), where they produce detrimental effects on many aspects of our health.146, 147, 148, 149, 150 The two major chemical culprits are polypropylene (38%) and polytheylene (56%). The major contributing sources of MNPs are synthetic fiber production (35%), car tire production (29%), and city dust (24%).^146,151 At least 8 million tons of plastics contaminate our oceans annually, and it is projected that by 2050, plastic accumulation in our environment will reach approximately 13 billion tons.^76,79 At autopsy, high concentrations of MNPs are found in multiple organs, with the brain having the highest concentration of polyethylene MNPs. Brains of patients with dementia (AD, vascular dementia, and other) had even higher polyethylene MNP levels than patients without dementia.¹⁴⁹ MNPs induce blood-brain barrier disruption and deposit in the brain, as well as disrupting the gut-brain axis.^146,147,150 In a mouse model, MNPs were shown to enter the blood stream and block cerebral flow, causing neurotoxic effects.¹⁵² Accumulation of MNPs in tissues leads to oxidative stress, immune inflammation, cellular damage, increased calcium levels, mitochondrial damage, autophagy impairment, improper protein folding, neuron loss, DNA damage, neurotoxicity, and metabolic disorders; these MNP-induced changes are thought to be a contributing factor to AD, PD, and other dementias.^146,147 More research is needed in this area to understand MNP deposition, how to recycle plastics safely and properly, and to understand the contribution that MNPs make to proteinopathies.

Pharmaceutical Approaches

Restoring protein homeostasis has been a key therapeutic strategy for proteinopathies. Different model systems and experimental approaches have been used over the decades to study and develop therapies for complex multifactorial diseases, like proteinopathies. Thus far, bench-to-bedside success has been lacking, in part because no single disease model or experimental approach recapitulates the human disease phenotype, pathologic features, symptoms, spatiotemporal gene effects, cell autonomous and nonautonomous mechanisms, environmental exposures, and molecular mechanisms. Historically, mouse models and targeted experimental approaches have been the norm, but increasingly investigators are using advanced in vitro model systems and multitargeted network-based (systems biology) experimental approaches to develop therapies targeting aberrant proteostasis. Network-based approaches combine data from different functional gene networks and/or research model systems to determine a system-wide consequence of potential drug targets, and have been more successful than classic monotherapy approaches.^153,154 The most common models for therapeutic development include in vitro two-dimensional cultures, ex vivo cultured tissue or three-dimensional induced pluripotent stem cell organoids, in vivo vertebrates, in vivo invertebrates, and yeast models, each with their pros and cons. Many recent studies suggest that in vitro human induced pluripotent stem cell models and network-based approaches are key to the future of precision medicine in proteinopathies.^14,153, 154, 155, 156, 157

The experimental model noted above has been used to identify therapeutically targetable molecules involved in key pathways driving proteinopathies (protein folding and degradation). Enhanced protein folding was investigated therapeutically with small molecules (eg, geldanamycin) that either inhibit HSP90 or activate heat shock transcription factor 1 to increase the overall functional output of the molecular chaperones HSP40 and HSP70 (Figure 4).¹⁴ This approach was shown to prevent TARDBP/TDP-43 aggregation and alleviate activation of the UPR by prolonged ER stress in cellular and animal models.^11,29 Chemical chaperones, like sodium phenylbutyrate or 4-phenylbutyrate, enhance protein folding and clearance of SOD1, α-synuclein, and TARDBP/TDP-43 aggregates, alleviating ER stress-induced prolonged UPR activation in in vitro and in vivo models^{12,122,158,159} (Figure 4). These approaches have failed in clinical trials for ALS because of adverse effects or poor effectiveness.

Enhancing the UPS degradation branch by modulating DUBs has been explored as an approach to reduce the load of aggregates in AD, PD, ALS, Huntington disease, and FTLD because the UPS targets and degrades 80% to 90% of all cellular proteins.^160,161 In AD, the expression of many DUBs is dysregulated, thereby preventing the degradation of toxic proteins like Aβ, p-tau, and TDP-43.¹⁶¹ Several DUB inhibitors have been developed and shown to be successful in vitro. However, only a few have made it to preclinical trials because of cytotoxicity or failure to cross the blood-brain barrier (AZ1, IU1, LDN-57444, and PR-619) in patients with AD (Figure 4).¹⁶¹

There has been recent interest in using repurposed drugs or natural molecules to treat proteinopathies. These include US Food and Drug Administration–approved drugs, such as rapamycin, metformin, trazodone, diazoxide, and the β-hydroxybutyrate agonist dibenzoylmethane (DBM) (Figure 4). Rapamycin was investigated for its ability to activate autophagy via inhibition of mTORC1. Preclinical studies indicate that rapamycin maybe be beneficial as a preventative treatment for AD, and this is currently being tested clinically.^162,163 Like rapamycin, nutrient deprivation and regular physical exercise can also activate autophagy by modulating mTOR to reverse proteostasis dysfunction linked with aging and proteinopathies.⁸ Another repurposed therapeutic approach for AD involved metformin, a widely prescribed drug for the treatment of type 2 diabetes. Metformin was studied as an AD treatment because of its ability to promote autophagy via activation of AMPK and inhibition of mTORC1 (Figure 4). There are conflicting reports of metformin's effectiveness, with some investigators reporting protective effects and others reporting detrimental effects in human studies and mouse AD models.164, 165, 166, 167 Trazodone and a nontoxic molecule derived from licorice root called DBM were identified in unbiased drug screens for compounds that restore protein synthesis by reversing the effects of overactive UPR and ISR stress mechanisms (Figure 4).^12,119 In animal models, trazodone and DBM reduced p-tau burden, restored synaptic and mitochondrial protein synthesis, improved synaptic plasticity resilience, memory and learning-associated behaviors, and increased global protein synthesis within the hippocampus of prion, FTLD-tau, and AD animal models (Figure 4).^118,119 Trazodone's efficacy is currently being tested in human clinical trials. Testing DBM in combination with other molecules, like diazoxide, has been proposed as a novel approach awaiting clinical trial investigation.¹⁶⁸ In preclinical rat models, combinatorial therapy with DBM and diazoxide restored protein synthesis, resulting in improved spatial working deficits, and reduced hippocampal Aβ plaque and neurofibrillary tau-tangle burden.¹⁶⁸ Salubrinal is an α-synuclein therapy that, like trazodone and DBM, functions downstream of pEIF2α.¹²

Summary

Protein accumulation pathology, which is the hallmark of proteinopathies like AD, ALS, PD, and FTLD, results from age, mutation-related protein folding and clearance deficiencies, and multifactorial risk factors.⁸ Over time, impaired proteostasis leads to cytotoxic levels of protein aggregation, a decline in synaptic plasticity and density, and ultimately cell death. Using approaches, such as systems biology, to identify novel drug targets and pathway interactions that function across multiple proteostasis branches will be crucial for translational success in complex diseases, like proteinopathies. Understanding the mechanisms behind the initiation and progression of these diseases and determining the critical time point during disease progression where a disease causing gene exerts its effects are currently lacking, which is impeding therapeutic advancement.¹⁵⁷ Furthermore, understanding the molecular and functional mechanisms of proteostasis failure is key for improving the longevity of healthy neurons.

Disclosure Statement

None declared.