Thinking laterally about neurodegenerative proteinopathies (original) (raw)
Prionoid mechanisms? Amyloid formation is a concentration-dependent, seeded, polymerization reaction (15, 16). In this reaction, formation of an initial assembly, referred to as a nucleation event or seed, catalyzes the conversion of the normal protein into the pathological amyloid state. Once seeded, growth of amyloid and amyloid-like structures is typically exponential, resulting in rapid formation of macromolecular structures that appear as intracellular inclusions or extracellular deposits used to pathologically define the disease. Building on seminal studies in prion diseases, in which the data unequivocally demonstrate that conversion of the normal cellular prion protein (PrPc) into the abnormal disease-causing prion (PrPsc) results in pathology spread within the infected CNS and enables disease transmissions (17, 18), there is now abundant evidence that many CNS proteinopathies can spread via a prion-like mechanism (reviewed in refs. 19–21). Indeed, this evidence has been used to support human pathological studies that suggest that neurofibrillary tau pathology spreads into the neocortex in AD (22) and α-synuclein pathology spreads into the brain from enteric nerves in PD (23, 24). However, unlike prion disease, there is no evidence that these and other human CNS proteinopathies are transmissible, and, thus, they have been termed by some as prionoids (19).
Despite the emergence of the prionoid hypothesis to explain proteinopathy spread within the brain, at present, there are many unanswered questions regarding the importance and relevance of prionoid mechanism in most human CNS proteinopathies. Answering these questions will enhance our understanding of disease progression and provide insight into the therapeutic tractability of targeting this pathway. One of the more elusive challenges for the field has been to define a seed. Though it is hypothesized that seeds contain structurally altered forms of the normal proteins, a precise structural understanding is lacking (25). Even for prions and Aβ, the presence of seeds is empirically defined by showing that some extract or preaggregated solution of the purified protein can catalyze aggregate formation, without a nucleation or lag phase. Given the likelihood that seeds are present in trace quantities in initial disease states, the seeds theoretically represent attractive, albeit elusive, targets.
A second perplexing question for intracellular proteinopathies relates to how cell-to-cell transmission occurs. Proteins implicated in intracellular CNS proteinopathies are largely localized to the cytoplasm, nucleus, or part of the scaffolding network of the cell, but there is growing evidence that these proteins can be secreted and are also present at low levels in interstitial and cerebrospinal fluid (19, 20, 26, 27). Proteins such as tau and α-synuclein may be secreted in exosomes and containment within exosomes might enhance cellular uptake, but the contribution of this pathway in vivo remains unclear (28–30). Furthermore, there are little data addressing (a) whether aggregated proteins are preferentially released, (b) whether secretion of these proteins has a normal physiologic role or is a disposal mechanism, or (c) whether the secreted proteins present in interstitial fluid or cerebrospinal fluid contain seeds capable of inducing pathology. Notably, the recognition that tau and α-synuclein aggregates may be secreted already provides one explanation for the apparent preclinical efficacy of immunotherapy targeting these proteins, and further elucidation of the mechanism of cell-to-cell transmission may provide a basis for novel therapies (31, 32).
Another unknown relates to the difference between prionoid and prion mechanisms. By definition, prions are transmissible; prionoids are not (33). Though some experiments raise the specter that certain proteinopathies could be transmitted from one genetically manipulated mouse to another, there is no human data to support human-to-human transmission of any CNS proteinopathy other than prions. So why are PrPsc prions uniquely transmissible? Indeed, many of the normal proteins involved in CNS proteinopathies are quite easily converted into aggregates that can behave as seeds, in contrast to PrPc (17). Speculatively, this difference could be explained by the kinetics of the templating reaction that follows seeding rather than the initial kinetic barriers to conversion. De novo conversion of PrPc into PrPsc is extremely challenging, but there is evidence that PrPsc may rapidly convert PrPc — in the order of minutes or seconds (34). In contrast, it is quite easy to nucleate Aβ, but the subsequent templating still takes hours to days to reach completion (15). Differences in peripheral expression levels might also play a role; if the normal protein is not present at sufficient levels to support templating, then the disease will not be transmitted peripherally. A final issue may relate to immunogenicity. Perhaps, in the periphery, PrPsc evades the immune system, whereas prionoids do not. If a seed or aggregate is easily recognized by the immune systems, a formidable barrier to transmission would exist.
A final question that serves as an excellent reminder that other mechanisms may account for the apparent spreading of pathology is whether spreading and cell-to-cell transmission of pathology can occur under physiologic levels of protein expression. The vast majority of data generated to date supporting prionoid mechanisms for tau, Aβ, superoxide dismutase-1 (SOD1), TDP-43, and α-synuclein use cell or animal models that have substantial overexpression of the protein and exogenous administration of the seed (21, 35–43). Whether pathology spread can occur under physiological expression levels is not as clear. Except for a recent study of α-synuclein seeding in nontransgenic mice (44), in most other instances induction of pathology is quite limited in the absence of overexpression (45–47). Furthermore, human studies, such as those regarding pathology in transplanted neurons in human patients with PD, cannot exclude other mechanisms such as those discussed below (48, 49). Though the physiologic relevance remains to be definitively established, the ability to rapidly induce seeding of pathological inclusions that phenocopy human proteinopathies is a major advance for the field. Indeed, armed with such models, it should be feasible to not only dissect out the consequences of inclusion formation in various cell types, but also to screen for both genetic and pharmacologic modifiers of pathology. Collectively, such studies may reveal novel strategies for therapy.
Prionoids as danger-associated molecular patterns that propagate via a toxic environment. One of the hallmarks of a proteinopathy is that the proteins that accumulate are either alternatively folded or misfolded and are found in a substantial, ordered assembly. Thus, there is significant potential for recognition of repetitive, pathological, conformational epitopes in the aggregate as non-self antigens (50, 51). Prionoid self-protein aggregates represent what are referred to immunologically as danger-associated molecular patterns (DAMPs) that are capable of inducing a robust immune response (52). Notably, a large number of studies show that prionoids associated with CNS proteinopathies, when applied exogenously to glial cells, activate innate immunity through pattern recognition receptors and induce a proinflammatory response (50, 53). Furthermore, there is some evidence that the resulting response to the DAMPs could modify the protein aggregate via various posttranslational modifications, such nitration, oxidation, or proteolysis, enhancing toxicity or promoting additional aggregation (54–56). Though studies of Aβ aggregates acting as DAMPs have until recently dominated this area of investigation, the finding that intracellular protein aggregates can be secreted provides a mechanism whereby even tau, α-synuclein, and other intracellular aggregates could activate the innate immune system upon secretion (57–59). Indeed, recent identification of AD risk alleles within the triggering receptor expressed on monocytes 2 (TREM2) gene highlight the potential relevance of this pathway to neurodegenerative disease (60, 61).
It is likely that innate immune activation can have positive or negative effects on proteostasis, behavior, and neurodegeneration, with the balance between positive and negative effects dependent on the nature, timing, duration, and strength of the specific signals. For example, there are conflicting data regarding the relationship between alterations in innate immune activation states and effects on extracellular Aβ accumulation and behavior (53, 62–64). There are more consistent data that indicate that proinflammatory stimuli may promote tau and α-synuclein pathology but also many fewer studies in this area (53). For example, LPS and various other proinflammatory stimuli have been shown to induce tau and α-synuclein pathology (65–67). Thus, at least in tau- and α-synuclein-opathies, there is evidence that a proinflammatory neurotoxic environment could induce or promote spread of pathology. Extracellular aggregates acting as both DAMPs and “seeds” may set off a vicious cycle of aggregate secretion, inflammation, and reseeding that propagates pathology. Inflammatory factors may enhance cell permeability, resulting in enhanced remodeling or destruction of synapses and other cellular processes. Further, such factors may lead to increased levels of extracellular aggregates, seeds, and other types of DAMPs (cell debris, nucleic acids, etc.) that in turn trigger more inflammation and reinforce this positive feedback loop (Figure 2).
Schematic of mechanism of possible spread of neurodegenerative proteinopathies and contribution to cellular demise. In this scheme, initiation of a proteinopathy can trigger a series of events that illicit feedback that contributes to spread of pathology and cellular demise. Danger signals might include DAMPs but also other signals indicative of cellular stress (ATP release, expression of MHC, etc.). Although there is strong evidence that microglia (and possibly astrocytes) may secrete neurotoxic factors, these factors have not been definitively identified in human neurodegenerative proteinopathies. Although most in the field have focused on mechanisms of neuronal decline, it is also important to consider the possibility that proteinopathies might result in functional decline and death of other CNS cells, including microglia, astrocytes, and oligodendrocytes (not shown). Indeed, in multiple system atrophy, oligodendrocytes are the primary cell affected by α-synuclein inclusions (97), and there is evidence for microglial dystrophy in AD (98).
An area that has received much less attention is whether cytoplasmic or organelle-bound intracellular aggregates can also act as DAMPs and activate intracellular immune pathways. Both fibrillar and oligomeric assemblies of proteins resemble intracellular pathogens. As there are established links among intracellular innate immunity, autophagic pathways, and the induction of other stress responses, further investigation of the action of intracellular protein aggregates as DAMPs might provide novel insights into how these protein aggregates alter cellular function (68–70). Extending this concept further, many of the responses of neurons to intracellular proteinopathies are highly similar to responses observed following sublytic viral infection of a neuron — including both induction of various elimination signals and general suppression of protein synthesis pathways (71). Furthermore, evidence from ALS models suggests that expression of mutant SOD1 in astrocytes and microglia plays a key role in mediating disease progression, implicating some role for these aggregates in activating intrinsic pathways that may contribute to neurotoxicity (72).
Gliosis and altered CNS immune activation states have long been recognized as an invariant accompanying feature of CNS proteinopathies, but the role of altered innate immune activation remains poorly understood (53). Though epidemiological studies have suggested that antiinflammatory strategies might be useful in AD, PD, and ALS, testing of antiinflammatory therapies in various disease states has not shown much promise to date (73, 74). However, as with therapies targeting the triggering proteinopathy, there is some concern that these therapies may have been tested in disease states that are too far advanced (13). Despite this lack of success to date, the notion that the immune system could be therapeutically harnessed to clear the underlying proteinopathy is attractive. In this light, we would simply point out that coordinated innate immune activation can clear a virus from the brain (71, 75). Given the evidence for prionoid- and DAMP-like properties of these protein aggregates, one might hypothesize that appropriate activation of the innate immune system could be used to restore normal proteostasis.
As with prionoid-like mechanisms, more insight is needed in a number of areas to fully comprehend the role of innate immunity in CNS proteinopathies. First, we need a better understanding of the temporal sequence of innate immune activation in the brain, and even the periphery, during disease progression. Such immunophenotyping might not only identify novel therapeutic targets but also novel biomarkers for disease. Indeed, a recent study in SOD1 mutant mice and human patients with ALS illustrates how detailed temporal immunophenotyping can inform both biomarker and therapeutic development (76). Second, there needs to be a more thorough understanding of the pattern recognition receptors activated by prionoids as well as the signaling cascades induced (77). Such studies should not only focus on glia as the resident immune cells of the brain, but also explore whether neurons activate innate immune pathways in response to prionoid aggregates. Third, there needs to be a much more intensive study of various protein aggregates acting as DAMPs. At this time, it is not clear whether all protein aggregates as well as the various types of assemblies formed act as DAMPs and, if they do, whether they behave in similar or distinct fashions. Finally, it will be important, both conceptually and from a therapeutic development perspective, to establish the relative contribution of prionoid spread compared with that of induction of the toxic environment for all of the CNS proteinopathies.
Spread via intrinsic disruptions of proteostatic mechanisms? The proteostasis network is a concept used to define the myriad of activities and functions that work in concert to maintain the proteome (78). A major conceptual premise of the proteostasis network is that the system is tightly balanced and regulated to optimize efficient use of cellular resources (79). Components of the proteostasis network (e.g., protein synthesis, protein chaperones, and protein degradation activities) are proposed to be present in sufficient, but not excess, levels; insults that diminish or burden the function of one or more elements of the network could create a condition of insufficiency and an environment that is unable to prevent the accumulation of misfolded proteins. Nonneuronal cells may counteract this insult by an upregulation of chaperones (for example, heat-shock response). However, aspects of this response are largely absent in the CNS of neurodegenerative disease models (80, 81). The proteostasis network is often conceptualized only as the protein machinery and intracellular compartments that assist in the folding of intracellular proteins, recognize inappropriately folded or modified proteins, and target them for degradation via the proteosome or autophagic pathways (82, 83). Given the role of extracellular clearance pathways and innate immunity in mediating detection and removal of protein DAMPs, we believe that it may be appropriate to more critically consider the role of the immune system in proteostasis. Nevertheless, proteostasis provides a conceptual framework to explain not only the origin of some of the pathologic features in neurodegenerative disease, but possibly both spread of pathology and cellular demise.
Age-related disruptions of the proteostasis network have often been invoked to explain why many CNS proteinopathies are late-onset diseases and some show dramatic, continuous increases in prevalence with age (80, 83). A number of elegant studies in model organisms link various pathways that perturb the proteostasis network to both life span and predisposition to develop a proteinopathy (84). But, it has been challenging to definitively identify any aging-related changes within the proteostasis networks that clearly trigger a CNS proteinopathy in mammalian model, let alone in humans (84). Furthermore, genetic alterations, such as triplet expansion in Huntington’s disease and various spinocerebellar ataxias, more aggressive presenilin mutations associated with high levels of Aβ42 production, and increased copy number of normal α-synuclein, all demonstrate that intrinsic factors that promote self-aggregation of proteins can trump any aging effects, causing disease in early life. Thus, triggering events in most proteinopathies may reflect aging-related disturbances in proteostasis but may simply be stochastic in nature.
Ultimately, the presence of the proteinopathy demonstrates a failure of the proteostasis network to handle the altered protein. This intrinsic failure can the set off the cycle illustrated in Figure 2 that promotes spread and induces a toxic environment. Indeed, even for prion disease, the vast majority of the cases are not caused by exogenously transmitted prions but are sporadic cases or arise from genetic mutations that predispose PrPc to intrinsically convert to PrpPsc. In either case, the accumulation of one protein may then enhance the propensity of other aggregation-prone, metastable proteins to aggregate by depleting chaperones required for proteostasis. If such disruptions occur, then one might predict the consequence would be induction of multiple proteinopathies. Indeed, there is a great deal of pathological evidence to suggest that many CNS proteinopathies are, at least at end stage, mixed. AD, for example, is characterized not only by accumulation of Aβ and tau pathology but, in a subset of cases, also α-synuclein and TDP-43 pathology (see Table 1 and Figure 3). In addition, studies in model organisms support the idea that an initiating proteinopathy can trigger induction of other proteinopathies (80, 85). However, it remains an open question as to why various secondary proteinopathies are induced to varying extents in AD and other neurodegenerative diseases? An alternative explanation for these observations is that cross-seeding via a prionoid-like mechanisms could result in one proteinopathy triggering another. For example, “amyloidogenic” intermediates or oligomers formed from different proteins may share sufficient structural homology to cross-seed each other into the amyloid pathway (39, 86, 87). Curiously, the intracellular proteins predominantly do not make heterogeneous inclusions; individual proteins accumulate in distinct inclusions (88). Further, some in vitro studies show that many amyloidogenic proteins prefer to form homopolymers (39, 88). On the surface, such an observation argues against a cross-seeding mechanism. However, there is ample evidence for cross-seeding in experimental systems, and it appears that once cross-seeding occurs, subsequent templating preferentially results in aggregation of each distinct protein. Therefore, structurally homologous intermediates may be involved in cross-seeding, but most proteins are kinetically inclined to assemble as homopolymers, which reflects the unique primary sequence characteristic of each protein.
Schematic of the interrelated neurodegenerative proteinopathies. Diseases are organized in color blocks that indicate their primary proteinaceous aggregate. AD has primary proteinaceous aggregates of both Aβ (yellow) and tau (red) and is therefore designated orange. Diseases are connected to proteinaceous aggregates that can be observed in at least some cases of the disease with lines. AGD, argyrophilic grain disease; CBD, corticobasal degeneration; DLB, dementia with Lewy bodies; FTD, frontotemporal dementia; HD, Huntington’s disease; MSA, multiple system atrophy; Perry synd., Perry syndrome; PDC, parkinsonism-dementia complex; PiD, Pick’s disease; PSP, progressive supranuclear palsy; αSyn, α-synuclein.
Irrespective of the role of disruptions in the proteostasis networks in disease induction and spread, the notion that one can treat CNS proteinopathies by augmenting the proteostasis network is highly intriguing. One might envision that augmenting chaperone systems or clearance pathways might be applicable to multiple CNS proteinopathies, especially those involving intracellular proteins. Again, several seminal studies suggest the feasibility of such an approach, but the long-term consequences of perturbing such networks in humans are largely unknown (89–93).