Cells have a protein quality control (PQC) system to maintain protein homeostasis. Preserving protein homeostasis involves the coordinated action of several pathways that regulate biogenesis, stabilization, correct folding, trafficking, and degradation of proteins, with the overall goal to prevent the accumulation of misfolded proteins and to maintain the integrity of the proteome.

One of the cellular mechanisms that copes with misfolded proteins is the chaperone machinery. A molecular chaperone is defined as a protein that interacts with, stabilizes or assists another protein to gain its native and functionally active conformation without being present in the final structure (Ellis, 1987). Many members of the chaperone protein family are referred to as heat shock proteins (HSP), as they are upregulated during stress conditions such as heat shock (Ellis and Hartl, 1999; Kim et al., 2013). In addition to folding of misfolded proteins, molecular chaperones are also involved in a wide range of biological processes such as the folding of newly synthesized proteins, transport of proteins across membranes, macromolecular-complex assembly or protein degradation and activation of signal transduction routes (Kim et al., 2013; Kakkar et al., 2014). Under the denomination of “molecular chaperones” there are a variability of proteins that have been classified into five different families according to sequence homology, common functional domains or subcellular localization: the HSP100s, the HSP90s, the HSP70/HSP110, HSP60/CCTs, and the a-crystallin-containing domain generally called the “small HSPs” (Lindquist and Craig, 1988; Sharma and Priya, 2016). Typically, molecular chaperones recognize exposed hydrophobic domains in unfolded or misfolded proteins, preventing their self-association and aggregation (Hartl et al., 2011; Kim et al., 2013). The regulation of chaperones can be divided into three categories, (1) constitutively expressed, (2) induced upon stress, and (3) constitutively expressed and induced upon stress (Morimoto, 2008). Under normal conditions the HSP levels match the overall level of protein synthesis, but during stress when mature proteins are unfolded the chaperone machinery is challenged and the expression of specific HSPs increases (Kakkar et al., 2014).

Next to their function under normal cellular conditions, chaperones play an important part during neurodegeneration when there is an overload of the PQC system by unfolded proteins (Kim et al., 2013; Kakkar et al., 2014; Lindberg et al., 2015). Each neurodegenerative disease is associated with a different subset of HSPs that can positively influence the overload of unfolded proteins (Kakkar et al., 2014). One example is the molecular chaperone DNAJB6b that can suppress polyglutamine (polyQ) aggregation and toxicity in a cell model for polyQ diseases (Hageman et al., 2010; Gillis et al., 2013), and suppress the primary nucleation step by a direct protein-protein interaction with polyQ proteins (Månsson et al., 2014b) and Aβ42 (Månsson et al., 2014a). Overexpression of DNAJB6 in a mouse model for HD results in reduction of the disease symptoms and increase life span (Kakkar et al., 2016). In PD, the overexpression of HSP70 can prevent α-synclein-induced cell death in yeast, Drosophila and mouse models of this disease (Auluck and Bonini, 2002; Klucken et al., 2004; Flower et al., 2005; Sharma and Priya, 2016). HSP70 has been shown to bind prefibrillar species of α-synclein and to inhibit the fibril formation (Dedmon et al., 2005). There is also a role for molecular chaperones in AD, where the overexpression of heat shock factor 1 (HSF-1), main regulator of HSPs expression, in an AD mouse model diminished soluble Aβ levels (Pierce et al., 2013), and multiple HSPs alleviated Tau toxicity in cells (Kakkar et al., 2014).

Additionally to the inhibition of protein aggregation of misfolded proteins, a disaggregase activity has been described for some molecular chaperones that can solubilize aggregated proteins (Glover and Lindquist, 1998; Tyedmers et al., 2010; Winkler et al., 2012). In bacteria, yeast, fungi and plants the HSP100 disaggregases are highly conserved (Tyedmers et al., 2010; Torrente and Shorter, 2013). In yeast, HSP104 collaborates with the other HSPs, to effectively disaggregate and reactivate proteins trapped in disordered aggregates (Glover and Lindquist, 1998; Shorter, 2011; Torrente and Shorter, 2013; Lindberg et al., 2015). Metazoans entirely lack HSP100 disaggregases in the cell, however, it has recently shown that in mammalians the disaggregase function is performed by the HSPH (Hsp110) family in cooperation with the HSP70-40 machine (Rampelt et al., 2012; Gao et al., 2015; Nillegoda and Bukau, 2015). This machinery has been shown to fragmentize and depolarize large α-synclein fibrils within minutes into smaller fibrils, oligomers and monomeric α-synclein in an ATP-dependent fashion (Gao et al., 2015).

Chaperones are also involved in other pathways of PQC. As discussed below they can mediate the degradation of misfolded proteins or their sequestration in cellular compartments.

Together, this shows the important direct role chaperones play in the formation of amyloids and thereby making chaperones an interesting therapeutic target for neurodegenerative diseases.

Protein degradation is another key mechanism to deal with misfolded proteins. Three pathways have been described, i.e., the ubiquitin (Ub)-proteasome system (UPS), chaperone mediated autophagy (CMA), and macroautophagy (Ciechanover, 2006; Ciechanover and Kwon, 2015). Soluble misfolded proteins are degraded by the UPS, a system that is dependent on a cascade of three enzymes E1, E2, and E3 ligase that conjugate ubiquitin to the misfolded proteins. The ubiquitinated protein is transported by molecular chaperones to the proteolytic system, where the protein is unfolded and passed through the narrow chamber of the proteasome that cleaves it into short peptides (Ciechanover et al., 2000). The CMA degrades proteins that expose KFERQ-like regions, these regions are recognized by the chaperone heat-shock cognate 70 (Hsc70) and delivered to the lysosomes and degraded by lysosomal hydrolases into amino acids (Kiffin et al., 2004; Rothenberg et al., 2010). Protein aggregates or proteins that escape the first two degradation pathways are directed to macroautophagy, a degradation system where substrates are segregated into autophagosomes which in turn are fused with lysosomes for degradation into amino acids (Koga and Cuervo, 2011). The proteins involved in neurodegenerative disease can rapidly aggregate and can thereby escape degradation when they are still soluble, the aggregates, and intermediate forms are partly resistant to the known degradation pathways (reviewed in Ciechanover and Kwon, 2015).

In the endoplasmic reticulum (ER), the unfolded protein response (UPR), induced during periods of cellular and ER stress, aims to reduce unfolded protein load, and restore protein homeostasis by translational repression. ER stress can be the result of numerous conditions, including amino acid deprivation, viral replication and the presence of unfolded proteins, resulting in activation of the UPR. The UPR has three pathways activated through kinases, (1) protein kinase RNA (PKR)-like ER kinase (PERK), (2) inositol-requiring enzyme 1 (IRE1), and (3) activating transcription factor 6 (ATF6; Halliday and Mallucci, 2015). These kinases are kept in their inactive state by the binding immunoglobulin protein (BiP), during ER stress this protein binds to exposed hydrophobic domains of unfolded proteins and thereby allowing activation of these factors (Gething, 1999). In neurodegenerative diseases markers of the UPR, like PERK-P and eIF2α-P, have been reported in the brain of patients with neurodegenerative disease and in mouse models of neurodegeneration (Hetz and Mollereau, 2014; Scheper and Hoozemans, 2015).

In the cell, misfolded proteins can be sequestered in distinct protein quality control compartments by chaperones and sorting factors. These compartments function as temporary storage until the protein can be refolded or degraded by the proteasome. Different compartments have been described in the literature that sequester different kind of misfolded proteins at various conditions, these include JUNQ, IPOD, Q-body, and aggresome (Sontag et al., 2014). Insoluble proteins are sequestered into insoluble protein deposit (IPOD) compartments that are located near the periphery of the cell (Kaganovich et al., 2008; Specht et al., 2011). If the proteasome is impaired these insoluble proteins can also be sequestered in aggresomes (Johnston et al., 1998), whereas, soluble misfolded proteins can be sequestered into ER-anchored structures named Q-bodies (Escusa-Toret et al., 2013). However, when the proteasome is impaired soluble ubiquitinated misfolded proteins are sequestered into ER-associated juxtanuclear quality control compartments (JUNQ) compartments (Kaganovich et al., 2008; Specht et al., 2011).

The JUNQ and Q-bodies concentrate misfolded proteins in distinct compartments together with chaperones and clearance factors, which makes processing them easier and more efficient. The IPOD and aggresomes are thought to protect the cell from toxic misfolded species, they do however also contain some disaggregases and autophagy related proteins and might therefore be recovered from these compartments (Kaganovich et al., 2008; Specht et al., 2011).

Most studies on neurodegenerative diseases focus on either the toxic mechanisms or on the PQC system as possible targets for treatment. Only a few studies so far have focused directly on modifiers of the protein aggregation pathway. One example is the study that focused on a reduced insulin/insulin-like growth factor 1 signaling (IIS), which induces the assembly of Aβ into densely packed and larger fibrillar structures (Cohen et al., 2009). The exact mechanisms behind the formation of these tightly packed amyloid structures by IIS signaling remains to be unraveled.

MOAG-4 (modifier of aggregation 4) was found in a forward genetic screen using C. elegans models for neurodegenerative diseases, as an enhancer of aggregation and toxicity of several aggregation-prone disease proteins, including polyQ, α-synuclein, and Aβ (van Ham et al., 2010). MOAG-4 is a small protein of unknown function that is evolutionarily highly conserved. It contains a 4F5 domain of unknown function and is predicted to have a helix-loop-helix secondary structure. MOAG-4 itself was excluded from the polyQ aggregates in the C. elegans model. Based on biochemical experiments with worm extracts, MOAG-4 has been suggested to act on the formation of a compact aggregation intermediate. Furthermore, in vitro studies with mutant HTT exon 1 and MOAG-4 show a direct increase in aggregation (Unpublished data). Moreover, it was shown that the effect on aggregation works independent from DAF-16, HSF-1, and chaperones.

The human orthologs of MOAG-4 were found to be a two small proteins with unknown function, i.e., Small EDKR Rich Factor (SERF) 1A and 2. These two orhologs are 40% identical and 54% similar to MOAG-4 (van Ham et al., 2010). It was found that SERF1a (Falsone et al., 2012) is able to directly drive the amyloid formation of mutant HTT exon 1 and alpha-synuclein in an in vitro assay. It has been suggested that SERF1a directly affects the amyloidogenesis of alpha-synuclein by catalyzing the transition of an alpha-synuclein monomer into a amyloid-nucleating species (Falsone et al., 2012). From cell culture experiments we know that overexpression of SERF1a or SERF2, together with mutant HTT exon 1 results in an increase in toxicity and aggregation of the polyQ protein. Whereas, knock down of SERF using siRNA results in reduced toxicity and aggregation (van Ham et al., 2010).

Neuroinflammation or gliosis, a reactive change of the glial cells in response to damage, is a common pathological feature in neurodegenerative diseases like AD and HD (Perry et al., 2010). However, whether inflammation plays an active or consequential role in disease is still a topic for debate. Glial cells are divided into two major classes: microglia and macroglia, where microglia are the phagocytes that are ubiquitously distributed in the brain and are mobilized after injury, disease, or infection. Pathological triggers, such as neuronal death or protein aggregates, activate the migration of microglia, which accumulate at the site of injury. This migration and recruitment is followed by the initiation of an innate immune response, which is a non-specific reaction resulting in the release of pro-inflammatory chemo- and cytokines (Gordon and Taylor, 2005; Hanisch and Kettenmann, 2007; Perry et al., 2010). The importance of glial cells in neurodegeneration is supported by the association found in genome wide association studies of immune receptors like TREM2 (Guerreiro et al., 2013; Jonsson et al., 2013) and CD33 (Griciuc et al., 2013) in AD. Gliosis has also been described for other neurodegenerative diseases as PD (Gerhard et al., 2006) and HD (Shin et al., 2005), but as the main aggregates are intracellular the response from microglia is not as strong as in AD.

Prion diseases (PrD) are a group of fatal neurodegenerative disorders caused by infectious proteins called prions. In humans most PrD can be identified under the name Creutzfeldt-Jakob disease (CJD), and in animals under the name bovine spongiform encephalopathy (BSE; Collinge, 2001). In PrD, the cellular form of the prion protein (PrP^C) undergoes a conformational conversion into a β-sheet enriched isoform denoted as PrP^Sc. This occurs when the PrP^Sc comes in contact with the mostly α-helical PrP^C, as a result the PrP^C is misfolded into pathogenic PrP^Sc, which in turn can become a template for conversion of other PrP^C. The PrP^Sc form can form protein aggregates, prion deposits, often present as amyloid structures, which can propagate and possibly cause cell death (Collinge and Clarke, 2007; Collinge, 2016). PrDs are well-known to be able to spread throughout the brain via infectious prions. By the conversion of the protein into “seeds” due to stress, mutations or when PrP^C comes in contact with PrP^Sc, it incites a chain reaction of PrP misfolding (Halliday et al., 2014). Prions are out of scope for this review, although they are one of the most relevant topics in neurodegenerative diseases especially due to their infectivity. This “prion-like” character of other neurodegenerative disease proteins has been proposed.

Spreading of Aβ in AD was first observed in a marmoset injected with brain extract from AD patients or AD affected marmosets, leading to AD pathology 6–10 years after injection (Baker et al., 1993; Ridley et al., 2006). Injection with only cerebrospinal fluid of AD patients or synthetic Aβ did not result in AD pathology in the marmoset (Ridley et al., 2006). As studies with marmosets are limited, these studies were replicated in mice to further investigate the spreading of Aβ. Brain extracts from AD patients or transgenic mouse models can initiate AD pathology in the brains of transgenic mice overexpressing the Swedish-mutated human APP (Meyer-Luehmann et al., 2006). Injection of synthetic human Aβ fibrils can induce AD pathology in mice, however the potency is lower than with AD brain extract (Stöhr et al., 2012). In mice depleted of amyloid-beta precursor protein (APP) there is no spreading of the disease, however if you take brain extracts of APP depleted mice inoculated with Aβ seeds, this can lead to propagation after second transmission for up to 180 days, suggesting extreme longevity of the Aβ “seeds” (Ye et al., 2015). Infectiousness of AD in humans has not yet been proven, though possible spreading of Aβ in humans was observed in two individual studies. The first study described four individuals with infectious Creutzfeldt-Jakob disease (CJD) who also showed moderate to severe AD pathology, they were injected as children with human growth hormone from cadaveric pituitary glands that contained PrP (Jaunmuktane et al., 2015). Another study observed infectious CJD in patients who received a dura mater transplant as a result of brain trauma or tumor, in five patients AD pathology was observed (Frontzek et al., 2016). As the patients in both studies did not carry pathogenic AD mutations or risk alleles and were too young to develop sporadic AD, the studies suggested that the treatment samples contained Aβ peptides.

Spreading of the PD pathology was first suggested when healthy dopaminergic neurons injected into the brain of PD patients showed Lewy body formation 11–16 years after transplantation (Kordower et al., 2008; Li et al., 2008). Follow-up studies in PD mouse models show that injection of brain extracts of PD transgenic mice results in the formation of PD pathology and increased mortality (Luk et al., 2012b; Mougenot et al., 2012). Furthermore, injection of synthetic α-synuclein (Luk et al., 2012a) or dementia with Lewy bodies (DLB) patient brain extract (Masuda-Suzukake et al., 2013) also results in PD pathology and neuronal death in healthy mice.

Proteins or peptides of most neurodegenerative diseases are intrinsically disordered in their free soluble form, like the Aβ peptide in AD and α-synclein in PD (Chiti and Dobson, 2006, 2009). Mutations in these disease proteins can make the protein even more prone to aggregate. For example, the A53T and A30P mutation of α-synclein found in early onset PD, promotes the acceleration of amyloid fibrils in vitro (Conway et al., 1998, 2000).

Furthermore, having too many copies of an aggregation-prone protein itself can lead to disease by increasing protein concentrations in the cell (Chiti and Dobson, 2006, 2009). This increase in protein concentration can switch the stability of the soluble state toward the amyloid state. For examples trisomy 21 patients (Down's syndrome) who have an extra copy of the APP protein and a highly increased risk of developing early onset AD (Wiseman et al., 2015). In addition, duplication or triplication of the α-synuclein gene (SNCA) results in early onset PD (Singleton et al., 2003), besides, the onset, progression, and severity of the disease phenotype increases with the number of copies of the SNCA gene (Chartier-Harlin et al., 2004). To this end, also proteins that regulate expression levels of disease proteins can cause or influence diseases, an example is the RNA binding protein Pumilio1 that regulates the mRNA levels of Ataxin1 RNA. Pumilio1 haploinsufficiency accelerates the SCA1 disease progression in a mouse model for disease due to increase of the Atxn1 mRNA and protein levels (Gennarino et al., 2015). If protein levels strongly influence the toxicity and disease phenotype this would suggests that lowering the protein load could be a therapeutic strategy. This was shown in an AD mouse model where the APP transgenes could be turned off with a tet-off system, when the APP levels were halted there was an arrest of the AD pathology without clearance of the excising plaques (Jankowsky et al., 2005), resulting in a significant effect on cognitive function (Fowler et al., 2014). Indicating that the concentration of disease proteins influences the disease progression, thereby affecting the development of disease.

That structural differences between amyloid “strains” can influence disease phenotype was first described for PrD, where isolated strains of PrP aggregates from different sources propagated different in mice showing distinct incubation times and patterns of neuropathology (Fraser and Dickinson, 1973). Furthermore, different human PrP strains have been associated with differences in proteinase K digestion and distinct phenotypes of neuropathology (reviewed in Collinge and Clarke, 2007). More recently, investigation of two familial human AD patients with different disease symptoms, showed a structural difference in amyloid fibril structure (Lu et al., 2013). Furthermore, Arctic and Swedisch familial AD patients brain homogenate results in distinct disease phenotypes in transgenic mice even after serial passage (Watts et al., 2014). Comparable results were found for Tau, another aggregation-prone protein involved in AD. Injection of two distinct in vitro generated Tau strains into transgenic mice resulted in distinct pathologies up to three generations (Sanders et al., 2014). These studies suggest that variations in the properties of amyloid fibrils could affect disease pathology and symptoms. How these different strains are formed and how they contribute to the disease pathology is still unknown. It was however found that reduced IIS signaling in the APP/PS1 AD mouse model induces the assembly of Aβ into densely packed and larger fibrillar structures later in life, resulting in reduced AD symptoms (Cohen et al., 2009). Suggesting that altering the structure of the amyloid fibrils could be beneficial for patients, as certain structures appear to be more toxic than others.

Amyloids structures are known to have biological functions in Escherichia coli, silkworms, fungi, and mammals (Fowler et al., 2007). One example in mammals is Pmel17 (Figure 3), a highly aggregation-prone protein that forms functional amyloid structures that are the main component of melanosome fibrils, membrane-bound organelles in pigment cells that store and synthesize melanin. Plem17 contains a partial repeat sequence that is essential for amyloid formation that can only be formed in the mildly acid pH of melanosomes (McGlinchey et al., 2009). The exact function of Pmel17 in melanosomes is unknown, although a role in protection against oxidative damage has been suggested, as well as a role in concentrating melanins to facilitate intra- and extracellular transport (Watt et al., 2013).

More functional amyloids in mammals can be found in hormone release, it was shown that certain hormones can be stored in amyloid-like aggregates in the secretory granules of the cell. These secretory granules have a β-sheet rich structure that is Thioflavin S and Congo Red positive and are able to release functional monomeric hormone structures upon dilution, and show only moderately toxicity on cell cultures, possibly due to their membrane-encapsulated state in the granules (Maji et al., 2009).

Interestingly, the formation of amyloids has recently been associated with long-term memory. The cytoplasmic polyadenylation element-binding protein (CPEBs) is a regulator of activity dependent synthesis in the synapse. The fly homolog Orb2 (Majumdar et al., 2012) and mouse homolog CPEB3 (Fioriti et al., 2015) are present in the brain as a monomer and SDS-resistant oligomer. Activation of the fly or mouse brain results in increase of the oligomeric Orb2/CPEB3 species. Selectively disrupting the oligomerization capacity of Orb2 by a genetic mutation resulted in long-term memory loss in flies (Majumdar et al., 2012) and loss of CPEB3 in the mouse brain resulted in impaired long term memory (Fioriti et al., 2015). Orb2 alters protein composition of the synapse by a mechanism in which the oligomeric Orb2 stimulates translation by elongation and protection of poly(A) tail, whereas the monomeric Orb2 does the contrary (Khan et al., 2015).

These functional amyloids point toward the origin of amyloid-prone sequences and their suppressors and enhancers. Even though, these functional amyloids have not been linked to human diseases, a functional role might be the case for the amyloid domains of disease proteins with unknown functions. More studies toward understanding the functionality of these amyloids and the difference with the disease amyloids are required to have a better understanding of why certain amyloids are toxic while others are not.

It was recently found that proteins with prion-like domains can form functional non-membrane-bound organelles like ribonucleoprotein (RNP) bodies, that behave like liquid droplets which can rapidly assemble and disassemble in a response to changes in the cellular environment (Han et al., 2012; Kato et al., 2012). The RNP bodies include processing bodies and stress granules in the cytoplasm, and nucleoli, Cajal bodies and PML bodies in the nucleus. Due to the dynamic structures of RNPs there is free diffusion within the bodies and rapid exchange with the external environment. Like in liquid-liquid phase separation (LLPS) the RNP bodies exhibit liquid-like behaviors such as wetting, dripping, and relaxation to spherical structures upon fusion (Chong and Forman-Kay, 2016; Uversky, 2017). These properties can facilitate their function, by allowing for high concentration of molecular components that nonetheless remain dynamic within the droplet. Interestingly many of the proteins known to segregate into RNP bodies contain repetitive putatively prion-like domains, that can reversibly transform from soluble to a dynamic amyloid-like state (Kato et al., 2012). Furthermore, dysregulation of these RNP bodies by RNA-binding proteins have been associated with neurodegenerative diseases as ALS (Ramaswami et al., 2013).

The link for these RNP bodies in disease was first found for the FUS protein, mutations in the N-terminal prion-like domain have been associated with ALS, and FTD. This protein plays an important role in RNA processing and localizes to both cytoplasmic RNP bodies and transcriptionally active nuclear puncta, the prion-like domain is essential for forming these liquid-like compartments (Shelkovnikova et al., 2014). The N-terminus of FUS is structurally disordered both as a monomer and in its liquid state (Burke et al., 2015). In vitro, these droplets convert with time from a liquid to an aggregated state (Figure 4C), and this conversion is accelerated by patient-derived mutations (Patel et al., 2015). Furthermore, concentrated liquid droplets increase the probability of aggregation events of RNA-binding proteins in the RNP bodies in a concentration dependent manner (Molliex et al., 2015). mRNA itself can drive its phase transition of the disordered RNA binding-protein Whi3, and thereby altering the droplet viscosity, dynamics, and propensity to fuse. Suggesting that, mRNA contains biophysical properties of phase-separated compartments. Like FUS droplets the Whi3 droplets mature over time and appear to be fibrillar (Zhang et al., 2015).

This new line of research indicates another possible function for prion-like domains of various proteins and the proteins it interacts with. Furthermore, research to these RNP bodies shows possible reasons why these proteins form amyloids. However, much is still unknown about the exact mechanisms of the amyloid like domains and the RNP bodies that have to be investigated.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.