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The misfolding and aggregation of specific proteins is a seminal occurrence in a remarkable variety of neurodegenerative disorders. In Alzheimer’s disease (the most prevalent cerebral proteopathy), the two principal aggregating proteins are β-amyloid (Aβ) and tau. The abnormal assemblies formed by conformational variants of these proteins range in size from small oligomers to the characteristic lesions that are visible by optical microscopy, such as senile plaques and neurofibrillary tangles. Pathologic similarities with prion disease suggest that the formation and spread of these proteinaceous lesions might involve a common molecular mechanism – corruptive protein templating. Experimentally, cerebral β-amyloidosis can be exogenously induced by exposure to dilute brain extracts containing aggregated Aβ seeds. The amyloid-inducing agent probably is Aβ itself, in a conformation generated most effectively in the living brain. Once initiated, Aβ lesions proliferate within and among brain regions. The induction process is governed by the structural and biochemical nature of the Aβ seed, as well as the attributes of the host, reminiscent of pathogenically variant prion strains. The concept of prion-like induction and spreading of pathogenic proteins recently has been expanded to include aggregates of tau, α-synuclein, huntingtin, superoxide dismutase-1, and TDP-43, which characterize such human neurodegenerative disorders as frontotemporal lobar degeneration, Parkinson’s/Lewy body disease, Huntington’s disease, and amyotrophic lateral sclerosis. Our recent finding that the most effective Aβ seeds are small and soluble intensifies the search in bodily fluids for misfolded protein seeds that are upstream in the proteopathic cascade, and thus could serve as predictive diagnostics and the targets of early, mechanism-based interventions. Establishing the clinical implications of corruptive protein templating will require further mechanistic and epidemiologic investigations. However, the theory that many chronic neurodegenerative diseases can originate and progress via the seeded corruption of misfolded proteins has the potential to unify experimental and translational approaches to these increasingly prevalent disorders.
It is a concept that is compelling in its power and simplicity: the misfolding and aggregation of specific proteins underlies many of the chronic neurodegenerative diseases that afflict aging humans. But what initiates this injurious cascade, and how does the pathology ramify throughout the nervous system? A wave of recent research, initially driven by tantalizing pathologic similarities between prion diseases and Alzheimer’s disease (AD)1-3, has begun to address these questions, and the findings increasingly implicate corruptive protein templating, or seeding, as a prime mover of the neurodegenerative process. The prion-like corruption of proteins may also be involved in the pathogenesis of such clinically and etiologically diverse neurological disorders as Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and chronic traumatic encephalopathy. Currently there is no evidence that these maladies are infectious in the same sense as are prion diseases. However, understanding protein misfolding and aggregation could reveal general principles, and thus similar therapeutic targets, of a pathogenic mechanism that impels some of the most devastating diseases of the elderly, AD foremost among them.
Alois Alzheimer linked senile plaques and neurofibrillary tangles to the dementia of AD over a century ago, but fundamental insights into the pathogenesis of the disorder emerged only with the identification of the principal proteins that comprise these lesions: Aβ in senile plaques and cerebral amyloid angiopathy (CAA), and tau in neurofibrillary tangles4 (for historical references see5). Whereas the degree of tauopathy correlates strongly with cognitive decline in AD6-8, genetic, pathologic and biochemical evidence implicates the aggregation of Aβ as a critical, early trigger in the chain of events that leads to tauopathy, neuronal dysfunction and dementia9.
Advancing age is the most prevalent risk factor for the accumulation of Aβ in the brain10, 11, possibly because of the decline of cellular protein quality control processes12. All firmly established genetic risk factors for AD promote the buildup of Aβ, either by increasing its production, promoting its aggregation, or impeding its elimination9, 13. Recent longitudinal imaging studies indicate that cerebral Aβ deposition precedes the clinical symptoms of AD by a decade or more4. How Aβ aggregates impair neuronal function remains uncertain, but evidence is growing that oligomeric forms of the protein, which can range in size from dimers to dodecamers or larger14-18, are more deleterious to brain function than are histologically obvious Aβ lesions such as senile plaques and CAA. Moreover, at least some of the Aβ toxicity appears to be tau-dependent19, 20.
Cross-sectional analyses of postmortem human brains reveal a characteristic progression of β-amyloid plaques and a highly stereotypical appearance of neurofibrillary tangles (Fig 1). β-amyloid plaques develop first in the neocortex, followed by the allocortex and then the subcortex, and the progression of their appearance often corresponds to functionally and anatomically coupled brain regions21-23. Neurofibrillary tangles first arise in the locus coeruleus and entorhinal/limbic brain areas, and then spread to interconnected neocortical regions8, 24. The pattern that emerges from these studies implicates neuronal transport and synaptic exchange mechanisms in the spread of AD lesions within the brain24-26. Overall, the incidence of plaques and tangles correlates positively in AD, but a consistent anatomical relationship between the lesions is not apparent. Imaging ligands that bind selectively to pathogenic protein deposits in vivo, such as the β-amyloid binding agent Pittsburgh Compound B27, will increasingly enable the longitudinal analysis of lesion spread in AD patients28.
In humans, the prion diseases (spongiform encephalopathies) are relatively uncommon but uniformly fatal neurodegenerative disorders that include Creutzfeldt-Jakob disease (CJD), variant CJD, fatal familial insomnia, Gerstmann-Sträussler-Scheinker Syndrome and Kuru. In other mammals, spongiform encephalopathies can be more prevalent; the nonhuman prionoses include scrapie, chronic wasting disease, bovine spongiform encephalopathy (BSE), transmissible mink encephalopathy, and others29-31. Prion diseases are unusual in that they can be genetic, idiopathic, or infectious (transmissible) in origin29. Experimentally, prion disease has been transmitted to a broad range of species and models29, 32, 33. Fortunately, the transmission of prion disease to humans is unusual, having occurred in fewer than 700 known cases, most under extraordinary circumstances such as treatment with human growth hormone derived from cadaveric pituitary glands, or in conjunction with the BSE outbreak that peaked in the late 20th century34.
Prions ‘infect’ via an unconventional mechanism whereby misconformed, β-sheet-rich prion protein induces the templated misfolding of other prion protein molecules29, 30, 35-37. The biological functions of the normal (‘cellular’) prion protein (PrPC) remain indeterminate, but it is not essential for survival38. In the disease state, the replication of infectious particles is sustained because cells continually produce PrPC 29, 30, which serves as the raw material for templated conversion to the pathogenic form (‘PrP Scrapie’, or PrPSc). The conformationally corrupted molecules are predisposed to self-aggregation, in which form they can become injurious to neurons29, 30. Though the mechanism of spread remains uncertain, there is evidence that prions can be conveyed between neurons by trans-synaptic transport39. Clinically, pathologically, and molecularly, the prion diseases exhibit variability suggestive of polymorphic and polyfunctional strains of the agent35-37, 40. While prion disease is the only demonstrably infectious cerebral proteopathy, mounting evidence implicates prion-like molecular mechanisms in the initiation and spread of a variety of neurological and systemic diseases39, 41-45. This emerging principle of pathogenesis has the potential to unify experimental and therapeutic approaches to these seemingly disparate disorders.
The pathologic similarities between prion disease and AD have long engendered speculation that AD might be inducible in a prion-like manner1-3. Early, long-term studies in nonhuman primates reported evidence both against46 and for47, 48 the exogenous inducibility of senile plaques. The introduction of transgenic mouse models of β-amyloidosis has provided a more efficient and definitive means of testing the hypothesis that AD-like lesions can be seeded in vivo. Studies in our laboratories and others have shown that the deposition of Aβ can be instigated in the brains of Aβ precursor protein (APP)-transgenic mice by the intracerebral infusion of brain extracts containing minute amounts of aggregated (multimeric) Aβ49-53 (Fig 2).
Several lines of evidence49-52 collectively argue that aggregated Aβ in the brain extract is critical for in vivo seeding: (1) The extract is able to seed only if it contains aggregated, human-sequence Aβ; (2) Immunoneutralization/depletion of Aβ in the extract by anti-Aβ antibodies impedes seeding; (3) Seeding of Aβ deposition is ineffective in non-transgenic mice, which (because of three amino acid differences from human-sequence Aβ) do not develop cerebral β-amyloidosis; (4) The phenotype of the induced Aβ deposits mirrors that of the deposits in the extract, suggesting an Aβ-templating mechanism; (5) Aβ-rich brain extracts from transgenic mice seed as effectively as do AD brain extracts, ruling out such factors as a cross-species immune reaction or human-specific microbes; and (6) Seeding can be abrogated by denaturation of the extract with formic acid. Recent findings reveal that the β-amyloid-inducing seeds do not consist of a single type of Aβ aggregate, but rather can occur as proteinase K (PK)-resistant species in the pellet fraction and as a soluble, PK-sensitive species in the 100,000×g soluble fraction54. Sonication of the extract, and thus presumed fragmentation of the “insoluble” Aβ seeds into smaller “soluble” Aβ multimers, enhances seeding, and thus hints at a continuum of Aβ aggregates of various sizes that can act as amyloid-inducing agents54.
The induction of Aβ deposits following injection of Aβ-rich brain extracts initially is most evident within the injected brain area. However there is also spreading between non-contiguous but axonally interconnected regions50, 52 (Fig 2), suggesting that seeds can migrate along defined neuronal pathways. Interestingly, seed placement in one region also can foster CAA in separate locations51, implicating perivascular fluid drainage channels55 and/or vascular transport mechanisms in the dissemination of the seeds51. Moreover, the intraperitoneal injection of Aβ-rich brain extracts into APP-transgenic mice induces β-amyloidosis in the brain after prolonged incubation56. How the seeds travel from the periphery to the brain in this model is an important open question.
Aggregated synthetic Aβ thus far has not induced significant cerebral Aβ-deposition in APP-transgenic mice51. The failure of seeding by synthetic Aβ was not unanticipated, in that the induction of prion disease also has been difficult to achieve with PrP generated in vitro57. It is possible that the aggregation of synthetic Aβ under suitable (but as yet undefined) conditions will yield a more effective in vivo seed, as was recently demonstrated for PrP58. The differential seeding ability of synthetic vs. natural Aβ aggregates (which may contain additional factors) suggests the possibility that Aβ, like prions, can misfold into polymorphic and polyfunctional strains51, 59-62.
Using a paradigm similar to in vivo Aβ-seeding, neurofibrillary (tau) tangles can be exogenously induced by the intracerebral infusion of brain extract containing abnormal tau filaments into mice bearing a human tau transgene63. This finding is remarkable in that the host (Alz17 mouse) expresses non-mutant human tau and does not normally develop tau filaments; moreover, the seeded tangles, unlike Aβ plaques and CAA, are intracellular. The induction of tauopathy is time- and brain region-dependent, and immunodepletion of tau from the donor brain extract prevents seeding63. Initially, the induced tau filaments are confined to the injected brain region, but over time, they extend to neighboring and/or axonally connected areas, suggesting directed spreading of seeds by neuronal transport processes. Although final evidence is still lacking that the in vivo induction of tau occurs via prion-like corruptive protein templating (versus, for example, activation of a signaling cascade that promotes tau aggregation), in vitro studies favor a prion-like mechanism64-66. Specifically, (1) aggregated tau is taken up via endocytosis from the cell medium and can induce the aggregation of soluble, endogenous tau in cells; (2) tau aggregates can transfer among co-cultured cells; and (3) distinct conformational properties of recombinant tau fibrils can be propagated. The latter finding, albeit achieved in a cell-free system65, is intriguing because tau inclusions characterize a variety of sporadic and genetic neurodegenerative diseases in which the aggregates display polymorphic conformations67, 68. Thus, as with other proteopathies, distinct tau strains may explain the pathogenic and phenotypic variations among the tauopathies45.
The coexistence of Aβ- and tau-lesions in AD still lacks a mechanistic explanation. Experiments in transgenic mice have shown that tauopathy can be augmented by aggregated, synthetic Aβ69 or by Aβ-rich brain extracts70. While this phenomenon may result from the direct cross-seeding of tau by aggregated Aβ71, 72, indirect pathways such as Aβ-induced tau phosphorylation, inflammation, and/or disruption of proteostasis20, 73-75 have not been ruled out.
Increasing evidence implicates the templated corruption of disease-specific proteins in other neurodegenerative diseases. In the brain, the α-synuclein-rich lesions that typify Lewy body disease/Parkinson’s disease first arise in the lower brainstem (notably the dorsal motor nucleus of the vagus nerve), and in the anterior olfactory nucleus and the olfactory bulb; they subsequently appear in a predictable sequence in mesencephalic and neocortical regions76, 77. The concept that α-synuclein lesions ramify within the CNS by a seeding-like process is bolstered by the observation that fetal dopaminergic neural transplants in the striatum of Parkinsonian patients can eventually exhibit α-synuclein-positive Lewy bodies in some cells, implying that synuclein seeds propagate from the host to the graft78, 79. In support of this observation, neural grafts placed into transgenic mice expressing human α-synuclein take up the human protein and form synuclein-positive aggregates80-82. The in vivo approaches in these studies could not discriminate between a prion-like corruptive templating mechanism, i.e. host-derived, misfolded α-synuclein inducing the misfolding of α-synuclein generated in the graft, versus the simple translocation of aggregated synuclein from the host to the graft. In cell culture, however, the prion-like propagation of α-synuclein lesions has been demonstrated80, 81, as has the induction of proteinaceous lesions associated with other neurodegenerative diseases, such as aggregates of superoxide dismutase 1 (SOD1)83, 84, which are characteristic of SOD1-mutant and some idiopathic cases of amyotrophic lateral sclerosis (ALS), cytosolic aggregates of TDP-4385, which are present in ALS and frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP), and aggregates of polyglutamine86, which typify Huntington’s disease and spinocerebellar ataxias.
These studies, along with those of Aβ and tau (above), imply that disease agents can be disseminated by cells, but the means whereby protein aggregates travel between cells, and the cellular domain(s) in which the templated conversion occurs, remain poorly understood39, 43, 44, 87. In vitro, aggregates of tau, α-synuclein, polyglutamine, and SOD1 all can be taken up by endocytosis and induce the misfolding of the corresponding intracellular proteins; moreover, cytoplasmic protein aggregates can translocate from one cultured cell to another64, 66, 80, 81, 83, 84, 86, 88. The mode of cell-to-cell transfer is unclear39, but at least α-synuclein and SOD1 are secreted into the cell medium89, 90. Furthermore, tau and α-synuclein have been measured at robust levels in the cerebrospinal fluid, suggesting secretion of these proteins in vivo91. The intercellular transfer of cytosolic protein aggregates may also occur through nanotubes, exosomes or microvesicles39. Like other pathogenic proteins, Aβ can be taken up, modified and secreted by cells in vitro92, 93, and it also is present in the CSF94.
The induction and proliferation of proteinaceous aggregates by corruptive protein templating appears to be a common feature of multiple, clinically diverse disorders, although many questions remain to be addressed (Table 1). The unexpected prevalence of this pathogenic mechanism raises a number of clinical and practical issues, and highlights the corruptive seeds as both potential biomarkers and therapeutic targets44 (Table 1).
In AD, cerebral β-amyloid deposition begins in humans at least a decade prior to the onset of cognitive decline, and hence is an early and predictive indicator of the disease4. It is likely, therefore, that an effective disease-modifying therapy must be initiated prophylactically, before the disease has inflicted irreversible damage on the brain. Early intervention will require an early and informative biomarker4, 95. In vitro, the formation of corruptive seeds is a relatively slow, stochastic process96, though once a seed is present, aggregation proceeds quite rapidly. In vivo, then, the appearance of soluble Aβ seeds may precede appreciable Aβ-deposition in plaques and blood vessels. Because the most potent Aβ seeds, like prions97, appear to be relatively small54, soluble Aβ seeds could serve as informative biomarkers in bodily fluids.
The ability of Aβ-rich brain extracts injected into the peritoneal cavity to induce Aβ deposition in the brain56 indicates that Aβ seeds resemble prions in their ability to reach the central nervous system from the periphery. In the absence of direct evidence linking non-prion neurodegenerative diseases to seeds arising outside the central nervous system or taken up from the environment (e.g. in food or air), the practical implications of this finding are uncertain. It is probable, however, that a fuller understanding of the trafficking of pathogenic seeds will yield insights into the endogenous progression of disease, and hence denote novel points of intervention. For example, the early appearance of α-synuclein-containing Lewy bodies in the peripheral nervous system, and their relatively systematic spread within the brain98, 99, suggest that seeds-in-transit (i.e., those traveling between cells or from one region to another) might be profitable objectives for therapeutic interference44.
In some cases, protein misfolding and aggregation can be initiated by heterologous, β-sheet-rich proteins41, 100-102. This ‘cross-seeding’ is generally less potent than is homologous seeding, but the potential corruption of proteins by exogenous nanoscale materials, some of which may feature amyloid-like structural properties103, 104, should be factored into safety evaluations of such materials41. Furthermore, the cell-to-cell transfer of seeds and the induction of pathogenic protein aggregates in cellular grafts44, 79, 105 underscores the need for measures to protect grafted cells from host-induced corruptive protein templating, e.g., by selectively engineering the cells so that vulnerable proteins are either absent or resistant to seeding.
Beyond its importance as a therapeutic objective, there is a clear need for studies on the clinical and epidemiological implications of corruptive protein templating as a disease mechanism (Table 1). Given our current state of knowledge, we feel that it is unlikely that non-prion proteopathies are communicable under everyday circumstances. However, it is worth considering the possibility that non-prion proteopathies can be promoted under certain extraordinary circumstances. The most efficient induction of prion disease and Aβ deposition is achieved by direct introduction of the seeding agent into the brain30, 56. In rare instances, prion disease has been transmitted to humans by contaminated neurosurgical instruments34. Experimentally, β-amyloid induction can be triggered in transgenic mice by intracerebrally implanted stainless steel wires coated with minute amounts of brain extract rich in aggregated Aβ52 (Fig 2). While the transmission of non-prion proteopathies by tainted instruments thus is a theoretical possibility, proof of such a phenomenon (which could be obscured by a protracted incubation period) has not been demonstrated in humans. Nevertheless, when considered alongside the (slight) risk of prion transmission by instruments used on patients with undiagnosed prion disease106, the hypothetical risk that non-prion proteopathies might be similarly induced suggests a need for more research into the epidemiology of such disorders in long-term, post-neurosurgical patients. In addition, risk analysis of the incidence of proteopathic diseases in the recipients of donated organs, tissues, extracts or fluids is needed to establish with confidence the inclusion criteria for donors. For example, inasmuch as advancing age is a salient risk factor for most human neurodegenerative diseases, should there be an age-limit for organ and tissue donation? It is still premature to provide answers to these questions, but the growing prevalence of cerebral proteopathies necessitates a comprehensive quest to illuminate the causes and consequences of corruptive protein templating in human disease.
The seeded proliferation of misfolded proteins, a concept that arose and evolved in the prion field, holds considerable explanatory power for the pathogenesis of many of the neurodegenerative diseases that afflict our burgeoning elderly population. The theoretical framework underlying this paradigm recently has expanded to include an extraordinary array of neurological and systemic disorders. Moreover, the templated modification of protein structure also subserves the transfer and storage of useful biological information in systems ranging from microbes to mammals107-110. Unfortunately, much of the research on this far-reaching phenomenon is fragmentary, and the clinical implications remain uncertain. A concerted inquiry into the biophysics, biochemistry, and cell biology of protein aggregation is needed to decipher the molecular underpinnings of a large and growing constellation of age-related disorders of the nervous system, and thereby accelerate the discovery of efficacious therapies.
We thank Yvonne Eisele (Tübingen, Germany), Rebecca Rosen (Washington, D.C.), Harry LeVine III (Lexington, Kentucky) and other members of our laboratories for experimental support and comments on this manuscript. The contributions of H. Braak and D. Thal (Ulm, Germany), M. Tolnay (Basel, Switzerland), and S. Eberle (Tübingen) to figures and text are greatly appreciated. Supported by the BMBF in the framework of ERA-Net NEURON (MIPROTRAN), Competence Network on Degenerative Dementias (BMBF-01GI0705), NIH RR-00165, PO1AG026423, P50AG025688, and the CART Foundation.