# data[].attributes.body
Antagonistic Pleiotropy - a gene that provides a fitness benefit early in life (good function of the protein) is selected for despite its fitness cost later in life (misfolding and aggregation of the protein)

# data[].attributes.body
Protein is the infectious agent - no genetic material!

# data[].attributes.body
Protein degradation and chaperone activity become less-effective with time --> increased risk of aggregation

# data[].attributes.headline
Despite differences in sequence and native structure, all amyloid fibres are overall very similar 

# data[].attributes.subject
Despite differences in sequence and native structure, all amyloid fibres are overall very similar 

# data[].attributes.body
Virtually any protein can form amyloid fibres at sufficient concentration and under denaturing conditions

# data[].attributes.body
Time is also required for the protein to accumulate to high enough conc. to form toxic aggregates

# data[].attributes.body
Bacteria and fungi use the amyloid pathway for biofilm formation, protection and interaction

Amyloid pathway in the human brain may have a role in the inflammatory response to infection, trapping pathogens and disrupting their membranes

# data[].attributes.body
Age-related neurodegenerative disease

# data[].attributes.body
Three alleles due to two non-coding SNPs: ε2, ε3, and ε4

ε4 --> increased risk of developing sporadic AD (especially if homozygous)

BUT not determinative (other factors involved)

Could affect oligomerization or clearance of Aβ and facilitates entry of some pathogens (e.g. HSV1) into the cell

# data[].attributes.body
Different prion strains with distinct structures are associated with different clinical and pathological phenotypes

--> strain specificity e.g. sheep Scrapie has never infected humans

# data[].attributes.headline
Diseases where tau protein misfolds and aggregates into cytotoxic β-rich filaments --> neural death, loss of synapses --> neurodegeneration

# data[].attributes.subject
Diseases where tau protein misfolds and aggregates into cytotoxic β-rich filaments --> neural death, loss of synapses --> neurodegeneration

# data[].attributes.body
β-sheets contribute to the aggregation and stability of the filaments

Native tau contains some β-sheets, but it is not β-rich

# data[].attributes.body
Accumulation of unfolded proteins in the ER --> ERstress --> UPR

Up-regulation of chaperones --> assist folding

Translational attenuation --> reduced protein load

Up-regulation of ERAD and autophagy --> removal of dnagerous unfolded proteins

# data[].attributes.headline
Off-pathway aggregates of unfolded proteins are highly cytotoxic

# data[].attributes.subject
Off-pathway aggregates of unfolded proteins are highly cytotoxic

# data[].attributes.body
Misfolded proteins trapped at local free energy minima posses exposed hydrophobic surfaces --> prone to aggregation in amyloid fibrils --> toxic to the cell

# data[].attributes.body
APOE ε4 allele antagonistic pleiotropy

ε4 allele increases risk of developing Alzheimer's later in life BUT correlates with cognitive benefits early in life --> selected for!

Young ε4 carriers have higher IQs than noncarriers

ε4 carriers may recruit additional right hemisphere frontal regions in order to achieve cognitive advantage

25% of people carry the ε4 allele

# data[].attributes.headline
Aβ acts as an antimicrobial peptide that protects the brain against pathogens

# data[].attributes.subject
Aβ acts as an antimicrobial peptide that protects the brain against pathogens

# data[].attributes.body
AD is a good example how an immune response initially aiming at maintaining the integrity of the body may fail and consequently lead to tissue destruction and neuronal loss

It is likely that AD prevention and treatment must also address the infection and inflammatory response that triggers Aβ production

# data[].attributes.body
Amyloid-β precursor protein (AβPP) cleaved by β- and γ-secretases --> amyloid-β peptide (Aβ)--> oligomerisation into fibrils --> aggregation into neurotoxic amyloid plaques --> neural death --> mental deterioration, death

# data[].attributes.body
De-methylation of AβPP --> increased AβPP expression --> increased amyloid-β --> increased aggregation into neurotoxic fibrils

# data[].attributes.body
Tau - tubulin associated subunit

Normally found in CNS neurons where it plays a crucial role in stabilizing microtubules in axons

Mutations in MAPT on chromosome 17 (encodes tau) --> Alzheimer's (dysfunction in tau is sufficient to cause Alzheimers)

Six tau isoforms of varying length (produced by alternate splicing) are expressed in the human brain

# data[].attributes.body
Prolonged ER stress --> pro-apoptotic UPR --> neuronal loss --> implicated in neurogenerative diseases e.g. Alzheimer's

# data[].attributes.body
Individuals with age-related neurodegenerative diseases such as Alzheimer's or Parkinson's show lower rates of cancer than the general population - impaired chaperone activity likely limits cancer progression

# data[].attributes.body
α-Synuclein (αS) regulates the trafficking of synaptic vesicles for neurotransmitter release

Associates with vesicle membranes through protein-membrane interactions and acts as a chaperone of SNARE complex assembly through protein-protein interactions

# data[].attributes.body
Tau protein must stay for years --> good substrate for protein aggregation (time required to aggregate)

# data[].attributes.body
Inflammatory proteins increase AβPP expression and stimulate β-secretase --> stimulate Aβ production and fibril formation

Likely mediated by pro-inflammatory cytokines TNF and IF-γ

Aβ may also stimulate inflammatory protein production by microglial cells and astrocytes --> positive feedback likely involved in mediating an inflammatory response

# data[].attributes.body
α-secretase cleavage --> non-amyloidgenic Aβ

β- and γ-secretase cleavage --> amyloidgenic pathway

# data[].attributes.body
AβPP A2T (Icelandic) mutation

Thr more polar than Ala --> disrupts hydrophobic cluster stabilising fibril --> may protect against AD

# data[].attributes.body
280 aa, soluble, α-rich protein (40% α-helix)

Flexible, disordered, N-terminus

N-glycosylation

GPI-anchored to the cell membrane at its C-terminus

Widespread in mammals (also in Drosophila)

Predominantly found in the brain (but also expressed in other tissues)

Protease-sensitive

Function unknown but likely plays roles in cell signalling and neural health

Mouse knockouts are normal until old age (display age-related chronic demyelinating)

# data[].attributes.headline
Abnormal tau hyperphosphorylation reduces its microtubule binding and leads to toxic neurofibrillary tangle formation 

# data[].attributes.subject
Abnormal tau hyperphosphorylation reduces its microtubule binding and leads to toxic neurofibrillary tangle formation 

# data[].attributes.body
Tau aggregation --> neurofibrillary tangles --> neural death

Loss of tau from MTs --> MT destruction --> synaptic/axon destruction --> neural death

# data[].attributes.body
140 aa protein enriched at pre-synaptic butons at the ends of neuronal axons in vertebrates

BUT not present at all synaptic terminals (--> selective expression, targeting, and pathogenic vulnerability in certain neuronal populations) and expression also not confined to neurons (e.g. present in RBCs)

# data[].attributes.attachment_link.title
The Unfolded Protein Response and the Role of Protein Disulfide Isomerase in Neurodegeneration

# data[].attributes.attachment_link.description
The maintenance and regulation of proteostasis is a critical function for post-mitotic neurons and its dysregulation is increasingly implicated in neurodegenerative diseases. Despite having different clinical manifestations, these disorders share similar…

# data[].attributes.body
Aβ oligomerisation may trap pathogens (agglutination) and prevent them from entering neurons, spreading or replicating

Aβ fibrils may disrupt pathogen membranes (but may also disrupt neuronal membranes)

# data[].attributes.body
AβPP A2V mutation

Val more hydrophobic than Ala --> strengthens hydrophobic interaction leading to fibril stability --> pathogenic

# data[].attributes.body
Chemically identical

Insoluble β-rich protein (45% β-sheet), structurally distinct from PrpC

Protease-resistant after proteolysis of 67 N-terminal residues

# data[].attributes.headline
Tau fibril formation may be stimulated by Aβ and neuroinflammation

# data[].attributes.subject
Tau fibril formation may be stimulated by Aβ and neuroinflammation

# data[].attributes.body
Hyperphosphorylated tau may have intracellular antimicrobial activity?

# data[].attributes.headline
Upon binding to membranes of synaptic vesicles, the N-terminal region of αS adopts a helical structure

# data[].attributes.subject
Upon binding to membranes of synaptic vesicles, the N-terminal region of αS adopts a helical structure

# data[].attributes.body
N-terminal repeats of KTKGEV consensus sequence (residues 1–95)--> form a three-turn amphipathic α-helix --> mediates association of α-synuclein with lipid membranes believed to be key for α-synuclein function

This region also contains a NAC domain (residues 60–95) implicated in aggregation

Most identified mutations associated with synucleinopathies are located in this region --> lipid binding or lack of may be associated with αS pathology

# data[].attributes.body
Aβ normally degraded by microglial cells and T-cells --> natural defence against Aβ accumulation

Aβ degradation mechanisms are often impaired in patients with AD

# data[].attributes.body
Aβ fibril consists of two β-rich intertwined protofilaments of regular helical geometry

'LS' topology of individual subunits

N-terminus L-shaped

C-terminus S-shaped

Staggered arrangement of non-planar subunits in a zipper-like manner

Dimer interface shields hydrophobic C-termini from the solvent

Different fibril ends ('groove' and 'ridge') with different binding pathways (and possibly energy barriers)

# data[].attributes.body
PrPSc can induce the misfolding of PrPC to PrPSc --> aggregate into fibrils and plaques --> neural death --> spongy degeneration of brain and nervous tissue

No nucleic acid involved in 'infection' - all disease information comes from the misfolded protein's ability to convert the normal protein into the pathogenic form

# data[].attributes.headline
Tau fibrils are primarily intraneuronal 

# data[].attributes.subject
Tau fibrils are primarily intraneuronal 

# data[].attributes.headline
Membrane-binding of αS is associated with multimerization, which is essential for its physiological function at the synapse

# data[].attributes.subject
Membrane-binding of αS is associated with multimerization, which is essential for its physiological function at the synapse

# data[].attributes.body
May act as a non-classical chaperone to facilitate SNARE complex assembly through interaction with synaptobrevin

May stabilise synaptic vesicle docking and fusion

# data[].attributes.attachment_link.title
Unfolded protein response (UPR) mediated under heat stress in plants - Plant Physiology Reports

# data[].attributes.attachment_link.description
Drastic fluctuation in temperature during crop growth challenges its yield to a greater extent, thus diminishing the agricultural productivity globally. At the cellular level, protein homeostasis is altered or disturbed directly or indirectly by the high temperature. Improper protein folding due to heat stress creates ER stress in the endoplasmic reticulum (ER). In order to balance the protein folding demand and its capacity and to maintain ER homeostasis, cells activate unfolded protein response (UPR), as a homeostatic mechanism. The transcription factors, bZip60 and bZip28/17 have been reported as the key modules of UPR for alleviating ER stress. Despite UPR mechanisms has been extensively investigated, the intervention of heat stress with ER homeostasis remains unclear. This review summarizes the key findings on this potential nexus. Additionally, the link between ER associated degradation and plant growth regulators during heat stress were also addressed.

# data[].attributes.body
Prolonged or excessive immune response and ineffective Aβ degradation --> AD pathogenesis

# data[].attributes.headline
Misfolded Aβ fibrils exhibit 'infectious' piron-like properties

# data[].attributes.subject
Misfolded Aβ fibrils exhibit 'infectious' piron-like properties

# data[].attributes.body
Misfolded conformation can propagate

# data[].attributes.attachment_link.readable_text
Joel C. Watts, a, b, ¹ Carlo Condello, a Jan Stöhr, a, b Abby Oehler, c Joanne Lee, a Stephen J. DeArmond, a, c Lars Lannfelt, d Martin Ingelsson, d Kurt Giles, a, b and Stanley B. Prusiner a, b, ²

Significance

The amyloid-β (Aβ) peptide, which plays a central role in Alzheimer's disease (AD) pathogenesis, exhibits many properties that are reminiscent of prions (self-propagating proteins that cause neurodegenerative disorders, such as mad cow disease). In the human prion diseases, distinct strains of prions can be distinguished, and therefore, we asked whether different strains of Aβ aggregates might exist in the brains of AD patients. Inoculation of transgenic mice with brain samples from patients with two different heritable forms of AD produced two distinct patterns of cerebral Aβ deposition, and these differences were maintained on serial passage. We conclude that distinct strains of Aβ can be discerned in AD patients, which may help to explain the clinical heterogeneity observed in the disease.

Keywords: neurodegeneration, bioluminescence imaging, seeding, proteinopathies

Abstract

An increasing number of studies argues that self-propagating protein conformations (i.e., prions) feature in the pathogenesis of several common neurodegenerative diseases. Mounting evidence contends that aggregates of the amyloid-β (Aβ) peptide become self-propagating in Alzheimer's disease (AD) patients. An important characteristic of prions is their ability to replicate distinct strains, the biological information for which is enciphered within different conformations of protein aggregates. To investigate whether distinct strains of Aβ prions can be discerned in AD patients, we performed transmission studies in susceptible transgenic mice using brain homogenates from sporadic or heritable (Arctic and Swedish) AD cases. Mice inoculated with the Arctic AD sample exhibited a pathology that could be distinguished from mice inoculated with the Swedish or sporadic AD samples, which was judged by differential accumulation of Aβ isoforms and the morphology of cerebrovascular Aβ deposition. Unlike Swedish AD- or sporadic AD-inoculated animals, Arctic AD-inoculated mice, like Arctic AD patients, displayed a prominent Aβ38-containing cerebral amyloid angiopathy. The divergent transmission behavior of the Arctic AD sample compared with the Swedish and sporadic AD samples was maintained during second passage in mice, showing that Aβ strains are serially transmissible. We conclude that at least two distinct strains of Aβ prions can be discerned in the brains of AD patients and that strain fidelity was preserved on serial passage in mice. Our results provide a potential explanation for the clinical and pathological heterogeneity observed in AD patients.

Alzheimer's disease (AD) is the most common human neurodegenerative disease, and it is characterized by the accumulation of extracellular amyloid plaques composed of aggregated amyloid-β (Aβ) peptide as well as intracellular neurofibrillary tangles composed of aggregated and hyperphosphorylated tau protein in the brain. The Aβ peptide is generated by the sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretase enzymes. The amyloid cascade hypothesis posits that the accumulation and subsequent deposition of Aβ in the brain are the initiating pathological events in AD that lead to the downstream aggregation of tau ( 1). Although most AD cases are sporadic, a minority results from mutations in the genes encoding APP or γ-secretase components.

Mounting evidence argues that the progressive nature of AD as well as many other neurodegenerative illnesses may stem from the formation and subsequent spread of self-propagating, β-sheet-rich protein conformations (i.e., prions) in the brain ( 2- 4). The term prion, derived from the words "protein" and "infectious," was introduced to define a novel pathogen lacking nucleic acids ( 5). Because the mechanism of prion propagation was shown to involve template-directed conformational change, the prion paradigm was recognized to apply more broadly in biology, including non-Mendelian phenotypic inheritance in yeast ( 6) and maintenance of synapse-specific changes in neurons ( 7). Diverse studies have converged to argue that prions, formed from normal proteins, cause many, if not most, neurodegenerative diseases ( 8). Some investigators prefer to use other terms for these self-propagating protein aggregates, including prion-like protein aggregates, prionoids, and proteopathic seeds ( 9, 10).

A wealth of studies argues that the formation of Aβ prions is involved in the pathogenesis of AD. In AD, cerebral Aβ deposition follows a stereotypical progression, in which the neocortex is targeted first, followed by spreading to subcortical regions of the brain ( 11). Moreover, inoculation of susceptible transgenic (Tg) mice or rats expressing mutant or WT human APP with brain homogenate containing Aβ aggregates, purified Aβ amyloid fibrils, or synthetic Aβ aggregates induced widespread cerebral Aβ deposition, revealing that Aβ aggregates are self-propagating and hence, prions ( 12- 17).

An important characteristic of prions is their ability to replicate distinct strains, which can be distinguished by their transmission behavior as well as their biochemical and pathogenic properties. Prion strain-specific biological information is enciphered within different conformations of protein aggregates ( 18- 20). Distinct conformations of Aβ aggregates have been described as formed either spontaneously from synthetic Aβ ( 21- 23) or after seeding of synthetic Aβ by Aβ aggregates from AD brains ( 24, 25). Furthermore, AD is a clinically heterogeneous disease ( 26), which could potentially be explained by the existence of multiple Aβ strains.

Based on the observation that different mutations in the human prion protein (PrP) encipher distinct strains of PrP prions ( 19), we hypothesized that heritable AD caused by different APP mutations (particularly mutations that result in the production of mutant Aβ) might result in the formation of distinct Aβ prion strains. The Arctic mutation in APP (E693G) occurs within the sequence of Aβ (E22G), causes enhanced Aβ protofibril formation, produces distinct fibril morphology, and results in a distinct AD pathology ( 27- 30). In contrast, the Swedish mutation (K670M/N671L) occurs outside the Aβ sequence but results in the overproduction of WT Aβ and typical AD pathology ( 31- 33). Here, we report that brain homogenates from Arctic and Swedish AD patients induced distinct disease phenotypes after multiple passages in susceptible Tg mice, suggesting that distinct Aβ strains form in the brains of AD patients.

Results

To determine whether the brains from an Arctic AD patient and a Swedish AD patient contain distinct conformations of Aβ aggregates, we performed conformational stability assays, which have been used to distinguish different strains of PrP prions ( 34). Brain homogenates prepared from AD patients were subjected to increasing concentrations of guanidine hydrochloride (GdnHCl) to determine the resistance of aggregates to denaturation. Samples were then digested with proteinase K (PK), and the residual fraction of PK-resistant Aβ was analyzed by Western blotting ( Fig. 1A ). We then calculated [GdnHCl] 1/2 values (the GdnHCl concentration at which 50% of Aβ is denatured) and found that the Aβ aggregates in the Arctic AD sample were significantly less resistant to GdnHCl denaturation than the Aβ aggregates in the Swedish AD sample ( Fig. 1B ), arguing that they are conformationally distinct. We also analyzed brain homogenates from two sporadic cases of AD, which yielded intermediate conformational stabilities.

To test if the distinct Aβ conformations in the brains of Arctic and Swedish AD patients are self-propagating, we performed transmission studies in bigenic Tg(APP23: Gfap-luc) mice ( Fig. 2A ) that express a Swedish mutant human APP transgene that produces WT Aβ peptide as well as a luciferase reporter under the control of the GFAP promoter ( 35, 36). This system permits the kinetics of induced cerebral Aβ accumulation to be monitored in living animals using bioluminescence imaging (BLI) of astrocytosis stimulated by the deposition of Aβ in amyloid plaques ( 14). Tg(APP23: Gfap-luc) mice were intracerebrally inoculated with brain homogenates from the four AD cases as well as a control homogenate from an age-matched individual free of any neurodegenerative illnesses. Levels of both formic acid-extractable and PK-resistant Aβ were much higher in the AD homogenates compared with the control homogenate ( Table S1 and Fig. S1A). Inoculation with each of the AD homogenates accelerated the onset of the brain BLI signal increase compared with inoculation with the control homogenate ( Fig. S1A). Moreover, Aβ levels, cerebral Aβ deposition, and Aβ plaque-associated astrocytic gliosis were significantly greater in the mice inoculated with AD brain homogenate compared with control-inoculated and uninoculated animals ( Fig. S1 B-R), indicating that the four AD homogenates contained Aβ prions.

Remarkably, the Arctic AD sample induced a distinct disease phenotype in the bigenic mice that could be distinguished from both the Swedish and sporadic AD inocula. Incubation periods for Aβ aggregate-containing samples on inoculation into Tg(APP23: Gfap-luc) mice can be assigned using BLI by measuring the time interval from inoculation to when a sustained increase in brain bioluminescence is observed ( 16). For these calculations, male and female mice were considered separately, because the kinetics of Aβ accumulation in Tg(APP23) mice are sex-dependent ( 37). The incubation period for the Arctic AD sample in both male and female mice was significantly shorter than for the other AD samples ( Fig. 2B ), despite lower levels of formic acid-extractable Aβ in the inoculum ( Table S1). Furthermore, the ratio of Aβ40 to Aβ42 in the Arctic AD-inoculated mice was significantly lower than in the mice inoculated with the other three samples ( Fig. 2C ). Different Aβ peptide ratios were also observed after inoculation of Tg(APP23) mice with brain extracts from two different Tg mouse models of AD ( 38).

Despite harboring parenchymal Aβ deposits of similar morphology to other AD-inoculated mice ( Fig. S1), the Arctic AD-inoculated animals showed a striking difference in the morphology of Aβ cerebral amyloid angiopathy (CAA) in the thalamus. Much of the thalamic CAA in Arctic AD-inoculated mice at 330 d postinoculation appeared furry in nature, with Aβ deposits radiating outward from the blood vessels ( Fig. 2D and Fig. S2A). A similar CAA morphology was observed in the thalamus of Tg mice expressing Arctic mutant APP ( 39). In contrast, mice inoculated with sporadic AD case i, Swedish AD, brain homogenate from an aged Tg(APP23) mouse, Aβ fibrils purified from Tg(APP23) mice ( 16), or synthetic Aβ(1-40) peptide as well as 635-d-old uninoculated mice all exhibited a similar pattern of CAA: a thin, compact layer of Aβ deposition surrounding blood vessels ( and Fig. S2 B-E). Interestingly, mice inoculated with sporadic AD case ii exhibited both CAA morphologies ( Fig. S2 F and G). Uninoculated Tg(APP23: Gfap-luc) mice at 390 d of age did not exhibit any spontaneous CAA in the thalamus ( Fig. 2G ). Significantly greater numbers of furry Aβ CAA deposits were observed in the Arctic AD-inoculated animals ( Fig. 2H ). Because the incubation periods for female mice inoculated with Arctic AD and purified Aβ fibrils were similar (∼170 d) ( 16), the unique furry vessel phenotype in Arctic AD-inoculated animals cannot be attributed to faster Aβ replication kinetics. No CAA was found in any of the inoculated mice at 33 d postinoculation ( Fig. S3), indicating that the induced CAA was not composed of residual inoculated material. Staining for collagen IV, which labels the basement membrane of blood vessels, revealed that many of the thalamic blood vessels surrounded by the furry Aβ deposits in Arctic AD-inoculated mice, but not Swedish AD-inoculated mice, were undergoing atrophy ().

A key feature that distinguishes Arctic AD cases from most sporadic AD cases and cases of heritable AD in which the mutation does not occur within the sequence of Aβ is the preferential deposition of Aβ38 peptide, particularly surrounding blood vessels ( 40). Indeed, PK-resistant Aβ38 was abundant in the Arctic AD sample and present in sporadic AD case ii but absent in the Swedish AD and sporadic AD case i samples ( Fig. 3A ). We used mAb BA1-13, which is specific for the Aβ peptide ending at residue 38 ( Fig. S4A), to assess Aβ38 deposition in AD-inoculated mice. Mice inoculated with either Arctic or Swedish AD samples showed similar levels of formic acid-extractable Aβ38 in their brains ( Fig. S4B) and amyloid plaques ( Fig. S4 C and D). However, abundant Aβ38-positive CAA within the thalamus was observed in mice inoculated with Arctic Aβ ( Fig. 3B ) but not in mice inoculated with Swedish AD or sporadic AD case i. Mice inoculated with sporadic AD case ii also exhibited some thalamic Aβ38-positive CAA ( Fig. 3C ). The quantity of Aβ38-positive blood vessels in the thalamus, leptomeninges, and the frontal cortex was significantly greater in Arctic AD-inoculated mice compared with sporadic AD- and Swedish AD-injected mice ( Fig. 3D and Fig. S4 E and F).

To confirm these findings, we analyzed a second cohort of Arctic AD- and Swedish AD-inoculated mice by quantitative confocal fluorescence microscopy using a different Aβ38-specific antibody (7-14-4) ( Fig. S4A). Significantly higher levels of Aβ38 staining were observed in thalamic blood vessels in Arctic AD-inoculated mice compared with Swedish AD-inoculated animals (). Moreover, codeposition of Aβ38 and Aβ40 peptides in thalamic blood vessels was observed in mice inoculated with Arctic AD or sporadic AD case ii but not in mice inoculated with Swedish AD, sporadic AD case i, or other Aβ aggregate-containing preparations ( and Fig. S5 A-G). Inoculation of mice with brain homogenate from aged Tg(APP23) mice, which also contained elevated levels of PK-resistant Aβ38 peptide ( Fig. S5 H-J), did not result in Aβ38-positive CAA within the thalamus ( Fig. S5E), suggesting that the conformation of Aβ in the inoculum, not the absolute levels of Aβ38, determines the phenotype in inoculated mice. These observations argue that the Arctic AD sample induced the preferential deposition of Aβ38 in blood vessels in mice that do not express the Arctic mutation. That induced Aβ38 deposition within plaques was observed in mice injected with Swedish or Arctic AD ( Fig. S4 C and D), despite the absence of PK-resistant Aβ38 in the Swedish AD inoculum ( Fig. 3A ), may reflect a predisposition of Tg(APP23) mice to depositing Aβ38 in amyloid plaques ( Fig. S5I).

An important characteristic of prion strains composed of PrP is that their properties are generally maintained after repeated passages in mice. To test if Aβ strains can also be serially propagated in mice, we performed a second passage experiment. In these experiments, both the passaged Swedish and Arctic inocula contained identical Aβ sequences, because they both underwent initial passage in Tg(APP23: Gfap-luc) mice producing WT Aβ peptide ( Fig. 4A ). On second passage, the Swedish and Arctic AD samples as well as sporadic AD case i accelerated the onset of the brain BLI signal increase and the elevation of cerebral Aβ deposition compared with passaged control brain homogenate ( Fig. S6). Second passage of all AD samples resulted in incubation periods similar to their respective first passages, with the Arctic AD inoculum showing the shortest incubation periods ( Fig. 4B ). Similar to first passage, furry Aβ CAA deposits were observed in the thalamus of mice inoculated with the passaged Arctic AD sample but not in mice inoculated with the passaged Swedish AD sample (). Likewise, mice inoculated with the passaged Arctic AD sample, but not mice inoculated with the passaged Swedish or sporadic AD samples, exhibited prominent CAA deposition of Aβ38 in the thalamus () and other areas of the brain ( Fig. S7 A and B) as well as codeposition of Aβ38 and Aβ40 in thalamic blood vessels (). Levels of formic acid-extractable Aβ38 were not significantly different in mice inoculated with the passaged Arctic or Swedish AD samples ( Fig. S7C).

Discussion

In the studies reported here, we found evidence for distinct strains of Aβ prions in brain homogenates prepared from patients with heritable AD. At least certain properties of the Aβ prion strains, such as the relative amounts of cerebrovascular Aβ38 deposition, were maintained after multiple passages in Tg mice. The existence of distinct Aβ strains and their ability to be serially propagated in mice contend that the Aβ aggregates in the brains of AD patients show all of the attributes of prions.

By melting the conformation of Aβ, we found that the Aβ prions in the brain of a patient carrying the Arctic mutation and producing mutant Aβ (E22G) were substantially less resistant to GdnHCl denaturation than those in the brain of a patient with the Swedish mutation, which does not alter the sequence of WT Aβ. We note that Arctic AD patients are heterozygous for the mutation, resulting in a mixture of WT and mutant Aβ peptides in the brain. Although we did not determine the allelic origin of the protease-resistant Aβ isoforms, studies of mutant PrPs may be informative: mutant PrPs in patients harboring the P102L, D178N, or E200K mutations were preferentially converted into the PrP Sc isoform ( 41- 43). Assuming that Arctic Aβ was preferentially converted into prions as manifest by protease resistance, it seems likely that WT Aβ did not form prions, because a monophasic melting curve was observed ( Fig. 1B ). If WT Aβ in Arctic AD patients had become a prion and adopted a conformation similar to the WT Aβ prions in the brain of the Swedish AD patient, then the melting curve should have been biphasic. Alternatively, WT Aβ may have adopted the same conformation as Arctic mutant Aβ.

The analysis of mutant and WT Aβ prions is further complicated by heterogeneous γ-secretase cleavage that generates Aβ peptides varying in length from 37 to 43 aa ( 44). Each of these peptides is likely to possess a different thermodynamic landscape with respect to conversion into a prion. The difficulties surrounding studies of Aβ prion strains are highlighted by a study using Tg(APP23) and Tg(APPPS1) mice; the former produces much more Aβ40 than Aβ42, whereas the latter produces the opposite ( 38). In our AD-inoculated mice, the most prominent strain-specific differences, including the relative amount of Aβ38 deposition, were observed in cerebral blood vessels but not parenchymal amyloid plaques. However, this difference was not inherent to the Tg(APP23) line, because mice inoculated with distinct strains of synthetic Aβ prions exhibited strain-specific differences in the ratios of Aβ40 and Aβ42 within amyloid plaques ( 45).

Our studies suggest that the Arctic AD mutation may encode a strain of Aβ prions that is distinct from that enciphered by the Swedish mutation, although this result will need to be confirmed using additional cases. Based on our transmission studies, it seems likely that the Aβ strains present in the Swedish AD and sporadic AD case i samples are similar and that the sporadic AD case ii sample contains a mixture of the Swedish-like and Arctic-like strains. Interestingly, the Dutch mutation in Aβ (E22Q) ( 46), which occurs at the same residue as the Arctic mutation, leads almost exclusively to cerebrovascular Aβ deposition and cerebral hemorrhages, implying the existence of additional Aβ strains and suggesting that residue 22 in Aβ may be critical for determining the conformation of Aβ prions. However, mutations within the Aβ peptide sequence may not be necessary for the genesis of distinct strains, because different conformations of Aβ aggregates have been observed in two different cases of sporadic AD ( 25). Moreover, different subtypes of sporadic AD, such as rapidly progressive AD ( 47), may stem from the propagation of unique strains of WT Aβ prions in the brain.

Because the Tg(APP23: Gfap-luc) mice used herein produce WT Aβ peptide, we conclude that the conformation of Aβ aggregates (not the presence of a mutation per se) enciphers the properties of Aβ strains. These findings parallel the maintenance of strain-specified properties observed on transmission of different inherited forms of human PrP prion disease, each expressing a different mutant PrP molecule, to Tg mice expressing chimeric mouse/human PrP ( 19). The phenotypic differences observed from the Arctic and Swedish AD transmissions may be caused by the Arctic mutation favoring the formation of a self-propagating Aβ conformation in which Aβ38 is stabilized, leading to its accumulation and deposition in the brain. However, a similar conformation may infrequently arise spontaneously in the absence of a mutation in Aβ, which was observed with sporadic AD case ii.

Distinct self-propagating strains of protein aggregates have also recently been described for both the human synucleinopathies and tauopathies ( 48- 50). Experimental transmission of multiple system atrophy to Tg mice revealed a particularly aggressive strain of WT α-synuclein prions ( 49). The existence of multiple prion strains causing neurodegenerative diseases poses a challenge for the development of therapeutics. Studies with PrP prions have revealed that therapeutic compounds identified using mouse-passaged strains were efficacious against mouse and chronic wasting disease prions but not against human strains ( 51). Because the conformation of Aβ aggregates in Tg mice seems distinct from that in AD patients ( 52), the translational use of such mouse models for anti-Aβ therapeutics seems questionable. Our ability to propagate distinct AD-associated Aβ strains in the brains of Tg(APP23: Gfap-luc) mice may, therefore, provide a superior tool for assessing drug efficacy. More importantly, heterogeneity in Aβ aggregate conformations suggests that passive Aβ immunotherapy approaches for treating AD may only work for certain strains of Aβ that exhibit conformations similar to those against which the mAb was raised. Several clinical trials involving Aβ mAbs have produced disappointing results ( 53). Although these antibodies may have been administered too late in the disease course to produce beneficial effects, Aβ strain heterogeneity in AD patients may have also contributed to their failure.

Materials and Methods

Additional methods are provided in SI Materials and Methods.

Human Tissue Samples.

Frozen brain tissue samples (temporal cortex) were obtained from neuropathologically confirmed cases of AD. The Swedish mutant sample (Braak stage V-VI with CAA and a minor cerebellar hemorrhage) was obtained from a 61-y-old female, and the Arctic mutant sample (Braak stage V-VI with CAA and no other copathology) was obtained from a 64-y-old male. Two sporadic AD samples were used: case i (Braak stage V-VI with Lewy body copathology) was obtained from a 62-y-old male, and case ii (Braak stage V-VI with no other copathology) was obtained from an 85-y-old female. Control brain tissue was obtained from the frontal cortex of a 79-y-old male who did not exhibit any clinical or pathological signs of a neurodegenerative disease. Levels of various Aβ peptides in the individual brain samples are shown in Table S1.

Mice.

Tg(APP23) mice, which express human APP (751-aa isoform) containing the Swedish mutation under the control of the Thy-1.2 promoter ( 35), were maintained on a C57BL/6 background. Tg( Gfap-luc) mice, which express firefly luciferase under the control of the murine Gfap promoter ( 36), were a gift from Caliper Life Sciences (Alameda, CA) and maintained on an FVB/N background. To create bigenic Tg(APP23: Gfap-luc) mice, hemizygous Tg(APP23) mice were crossed with homozygous Tg( Gfap-luc) animals. All animal experiments were performed under protocols approved by the Institutional Animal Care and Use Committee at the University of California.

Neuropathology.

Female mice were euthanized but not perfused before brain removal. Brains were then immersion-fixed in 10% (vol/vol) buffered formalin followed by embedding in paraffin using standard procedures. Sections were cut at 8 μm, mounted on glass slides, deparaffinized, and then processed for immunohistochemistry. Endogenous tissue peroxidases were inhibited by incubating the slides in 3% hydrogen peroxide solution for 30 min, and sections to be stained with anti-Aβ antibodies were pretreated with formic acid for 5 min. Slides were blocked with 10% (vol/vol) normal goat serum for 1 h and then incubated with primary antibody overnight at 4 °C. The following primary antibodies were used: anti-Aβ mouse mAb 4G8 (1:250 dilution; Covance), anti-Aβ40 mouse mAb 11A50-B10 (1:200 dilution; Covance), anti-Aβ38 rabbit mAbs BA1-13 (1:200 dilution; Covance) and 7-14-4 (1:250 dilution; Covance), anti-collagen IV rabbit polyclonal antibody ab6586 (1:500 dilution; Abcam), and anti-GFAP rabbit polyclonal antibody Z0334 (1:500 dilution; Dako). Bound antibody was detected using a Vectastain ABC Peroxidase Kit (Vector Laboratories) and visualized using 3-3′-diaminobenzidine. Slides were counterstained with hematoxylin and then photographed using an AxioImager.A1 microscope (Carl Zeiss). For fluorescent double-labeling experiments, secondary antibodies conjugated to AlexaFluor 488, 568, or 647 (Life Technologies) were used. For Aβ/collagen IV double stains, sections were first pretreated with formic acid and then autoclaved in citrate buffer for epitope retrieval. All fluorescently labeled samples were visualized using a Leica SP8 confocal microscope.

Acknowledgments

We thank the staff at the Hunter's Point animal facility for assistance with the animal experiments, Marta Gavidia for mouse genotyping, and Smita Patel for brain homogenization. The Tg( Gfap-luc) mouse line was a gift from Caliper Life Sciences. This work was supported by National Institutes of Health Grants AG002132, AG010770, AG021601, AG031220, and AG042453 and gifts from the Sherman Fairchild Foundation; the Rainwater Charitable Foundation; and the Glenn Foundation for Medical Research. J.C.W. was supported by National Institute on Aging K99 Grant AG042453.

References

6. Wickner RB. [URE3] as an altered URE2 protein: Evidence for a prion analog in [ Saccharomyces cerevisiae. Science. 1994;264(5158):566-569. [ PubMed] Google Scholar]

15. Morales R, Duran-Aniotz C, Castilla J, Estrada LD, Soto C. [ De novo induction of amyloid-β deposition in vivo. Mol Psychiatry. 2012;17(12):1347-1353. [ PubMed] Google Scholar]

40. Moro ML, et al. [ APP mutations in the Aβ coding region are associated with abundant cerebral deposition of Aβ38. Acta Neuropathol. 2012;124(6):809-821. [ PubMed] Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

# data[].attributes.attachment_link.title
Serial propagation of distinct strains of Aβ prions from Alzheimer's disease patients

# data[].attributes.attachment_link.description
The amyloid-β (Aβ) peptide, which plays a central role in Alzheimer's disease (AD) pathogenesis, exhibits many properties that are reminiscent of prions (self-propagating proteins that cause neurodegenerative disorders, such as ...

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The function of the cellular prion protein in health and disease

# data[].attributes.subject
The function of the cellular prion protein in health and disease

# data[].attributes.body
Tau neurofibrillary fibres have two ultrastructural polymorphs

Paired helical fibre (PHF) - filaments twist around each other like a double helix

Straight fibre (SF) - straighter and slightly thicker

# data[].attributes.attachment_link.title
Figure 2: Cross-sections of the PHF and SF cryo-EM structures. | Nature

# data[].attributes.attachment_link.description
Cryo-EM density and atomic models of PHFs (a) and SFs (b). Overviews of the helical reconstructions (left) show the orientation of the cross-sectional densities (right). Sharpened, high-resolution maps are shown in blue (PHFs) and green (SFs). Red arrows indicate additional densities in contact with K317 and K321. Unsharpened, 4.5 Å low-pass filtered density is shown in grey. Unsharpened density highlighted with an orange background is reminiscent of a less-ordered β-sheet and could accommodate an additional 16 amino acids, which would correspond to a mixture of residues 259–274 (R1) from 3R tau and residues 290–305 (R2) from 4R tau.

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Highly acidic, largely unstructured C-terminus

Target of PTMs

Interacts with proteins

Modulates membrane-binding

Protects against aggregation

# data[].attributes.attachment_link.title
Infectious origin of Alzheimer’s disease: Amyloid beta as a component of brain antimicrobial immunity

# data[].attributes.attachment_link.description
Author summary More than a century after its discovery, Alzheimer’s disease (AD) remains incurable and mysterious. The dominant hypothesis of amyloid cascade has succeeded in explaining the key pathological mechanism, but not its trigger. Amyloid beta has been traditionally considered a pathological peptide, and its physiological functions remain poorly known. These knowledge gaps have contributed to repeated failures of clinical studies. The emerging infectious hypothesis of AD considers central nervous system (CNS) infection the primary trigger of sporadic AD. A closely connected hypothesis claims that amyloid beta is an antimicrobial peptide. In this review, we discuss the available evidence for the involvement of infections in AD, coming from epidemiological studies, post mortem analyses of brain tissue, and experiments in vitro and in vivo. We argue there is no unique “Alzheimer’s germ,” instead, AD is a general reaction of the CNS to chronic infections, in the milieu of an aged immune system. The pathology may become self-sustained even without continuous presence of microbes in the brain. Importantly, the infectious hypothesis leads to testable predictions. Targeting amyloid beta should be ineffective, unless the triggering pathogen and inflammatory response are addressed as well. Meticulous control of selected infections might be the best near-term strategy for AD prevention.

# data[].attributes.body
Aβ fibrils are highly heterogeneous (varying width and helical pitch, differences in interactions, different cross section profiles etc.)

Strain-like properties: Aβ fibril polymorph may correlate with AD phenotype (e.g. rapidly progressive vs. prolonged-duration)

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Variant CJD - associated with transmission from cows (linked to BSE)

Familial CJD - genetic predisposition

Sporadic CJD

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Nature 547, 185-190 (2017) Cite this article

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Abstract

Alzheimer's disease is the most common neurodegenerative disease, and there are no mechanism-based therapies. The disease is defined by the presence of abundant neurofibrillary lesions and neuritic plaques in the cerebral cortex. Neurofibrillary lesions comprise paired helical and straight tau filaments, whereas tau filaments with different morphologies characterize other neurodegenerative diseases. No high-resolution structures of tau filaments are available. Here we present cryo-electron microscopy (cryo-EM) maps at 3.4-3.5 Å resolution and corresponding atomic models of paired helical and straight filaments from the brain of an individual with Alzheimer's disease. Filament cores are made of two identical protofilaments comprising residues 306-378 of tau protein, which adopt a combined cross-β/β-helix structure and define the seed for tau aggregation. Paired helical and straight filaments differ in their inter-protofilament packing, showing that they are ultrastructural polymorphs. These findings demonstrate that cryo-EM allows atomic characterization of amyloid filaments from patient-derived material, and pave the way for investigation of a range of neurodegenerative diseases.

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References

1. Ghetti, B. et al. Frontotemporal dementia caused by microtubule-associated protein tau gene ( MAPT) mutations: a chameleon for neuropathology and neuroimaging. Neuropathol. Appl. Neurobiol. 41, 24-46 (2015)

2. Berriman, J. et al. Tau filaments from human brain and from in vitro assembly of recombinant protein show cross-β structure. Proc. Natl Acad. Sci. USA 100, 9034-9038 (2003)

3. Kaufman, S. K. et al. Tau prion strains dictate patterns of cell pathology, progression rate, and regional vulnerability in vivo. Neuron 92, 796-812 (2016)

4. Åslund, A. et al. Novel pentameric thiophene derivatives for in vitro and in vivo optical imaging of a plethora of protein aggregates in cerebral amyloidoses. ACS Chem. Biol. 4, 673-684 (2009)

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Acknowledgements

These findings mark the culmination of a conversation at the MRC Laboratory of Molecular Biology 34 years ago between A. Klug, the late M. Roth and R.A.C. about the structural analysis of Alzheimer filaments. We thank the patient's family for donating brain tissue; M. R. Farlow for clinical evaluation; F. Epperson, R. M. Richardson and U. Kuederli for human brain collection and analysis; P. Davies, M. Hasegawa and M. Novak for antibodies MC-1, TauC4 and MN423, respectively; H. Zhou for use of the Titan Krios at UCLA EICN; S. Chen, C. Savva and G. Cannone for support with electron microscopy at the MRC Laboratory of Molecular Biology; T. Darling and J. Grimmett for help with computing; and M. Skehel for support with mass spectrometry. M.G. is an Honorary Professor in the Department of Clinical Neurosciences of the University of Cambridge. This work was supported by the UK Medical Research Council (MC_UP_A025_1012 to G.M., MC_U105184291 to M.G. and MC_UP_A025_1013 to S.H.W.S.), the European Union (Marie Curie International Outgoing Fellowship to A.W.P.F., Joint Programme-Neurodegeneration Research to M.G. and B.F., and Horizon 2020 IMPRiND to M.G. and A.W.P.F.), the US National Institutes of Health (grant P30-AG010133 to B.G.) and the Department of Pathology and Laboratory Medicine, Indiana University School of Medicine (to B.G.).

Author information

Author notes

Michel Goedert and Sjors H. W. Scheres: These authors jointly supervised this work.

Authors and Affiliations

1. MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK

Anthony W. P. Fitzpatrick, Benjamin Falcon, Shaoda He, Alexey G. Murzin, Garib Murshudov, R. Anthony Crowther, Michel Goedert & Sjors H. W. Scheres

2. Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, 46202, Indiana, USA

Holly J. Garringer & Bernardino Ghetti

Contributions

B.G. performed neuropathology; H.J.G. performed genetic analysis; B.F. conducted filament extraction and immunolabelling; A.W.P.F. performed cryo-EM; S.H. and S.H.W.S. provided cryo-EM software; A.W.P.F. and S.H.W.S. analysed cryo-EM data; A.W.P.F., A.G.M. and G.M. built the atomic model; R.A.C. contributed to the inception of the study; M.G. and S.H.W.S. supervised the project; all authors contributed to writing the manuscript.

Corresponding authors

Correspondence to Michel Goedert or Sjors H. W. Scheres.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks E. Egelman, D. Eisenberg and B. Meier for their contribution to the peer review of this work.

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Extended data figures and tables

a, b, Immunolabelling of the sarkosyl-insoluble fraction from the patient's temporal cortex. a, Immunoblots using anti-tau antibodies BR133 (N terminus), BR135 (R3), TauC4 (R4), BR134 (C terminus), AT8 (pS202/pT205) and MC-1. b, Immunogold negative-stain electron microscopy of PHFs and SFs with BR133, BR135, TauC4, BR134, AT8 and MC-1. Scale bar, 500 Å. c, Light microscopy of sections from the temporal cortex showing staining of neurofibrillary tangles, neuropil threads and plaque neurites using RD3 (3R), anti-4R (4R), AT8 and AT100 (pT212/pS214/pT217). Nuclei are counterstained blue. Scale bar, 50 μm.

a- c, Cryo-EM micrographs and reference-free 2D class averages for PHFs (blue insets) and SFs (green insets) for the tau sample after the sucrose step ( a), gel filtration ( b), and pronase treatment ( c). Examples of PHFs and SFs in the micrographs are indicated with blue and green arrowheads, respectively. Scale bars, 500 Å. d, Western blots with antibody HT7 (Thermo; catalogue nr. MN1000) of the total lysate, sarkosyl-soluble and sarkosyl-insoluble fractions of HEK 293T cells expressing wild-type 0N4R human tau and treated with (+) or without (−) the sarkosyl-insoluble fraction from the patient's temporal cortex following gel filtration show that the cryo-EM sample is capable of seeding aggregation of human tau. e, Densitometric analysis (mean ± s.e.m., n  = 3) of HT7 blots of sarkosyl-insoluble fractions from cells.

a, Fourier shell correlation (FSC) curves between two independently refined half-maps for the full-length (FL) PHFs (blue, solid); FL SFs (green, solid); pronase-treated (PT) PHFs (blue, dashed) and PT SFs (green, dashed). b, FSC curves between the cryo-EM reconstructions and the refined atomic models, using the same colour coding as in a. c, Local resolution estimates for the four cryo-EM reconstructions. d, Comparison of power spectra (the squared amplitudes of the Fourier transform, FT) of reference-free 2D class averages with those of corresponding projections of the atomic models. In PHFs the approximate 2 ₁ screw symmetry between subunits on the two protofilaments leads to off-meridional n = 1 Bessel function peaks on the 1/(4.7 Å) layer line (blue arrows). For SFs, in which the asymmetric unit consists of two subunits at the same level, one from each protofilament, there is a meridional n  = 0 Bessel function peak on the 1/(4.7 Å) layer line (green arrows).

The R3-R4 model provides the best fit to cryo-EM densities in the β-helix region (top row) and the cross-β region near the termini (second row) of the PHF protofilaments. The same model also provides the best fit to densities at the cross-β regions near the termini of the two protofilaments (third and fourth row) in SFs. Major discrepancies between the cryo-EM density and alternative atomic models (consisting of R2-R3, R1-R3 and R1-R2 cores) are highlighted with red dashed outlines.

a, The PHF backbone atoms are shown in blue and the SF backbone atoms in green. The r.m.s.d. between the backbone atoms is 1.2 Å. b, As in a, but showing all atoms.

Sharpened maps are shown in blue for PHFs ( a) and green for SFs ( b). Additional densities in contact with lysines 317 and 321 are indicated with red arrows. Unsharpened density, lowpass filtered to 4.5 Å resolution for the PHFs, is shown in grey. The unsharpened density that is highlighted with an orange background is reminiscent of a less ordered β-sheet and could accommodate an additional 16 amino acids, which would correspond to a mixture of residues 259-274 (R1) from 3R tau and residues 290-305 (R2) from 4R tau.

a, Relative abundance of identified peptides from the pronase-treated filaments by mass spectrometry (shown in green). The peptide used to produce anti-4R tau is shown in blue (with a D instead of an N at position 279). The epitope of MN423 is shown in orange. b, Immunogold negative-stain electron microscopy of PHFs and SFs with anti-4R tau (top) and MN423 (bottom) with (+) and without (−) pronase treatment. Scale bar, 100 nm.

a, Overlay of the backbones of the β-helix in tau (cyan) and the HET-s β-helices (orange and purple). The r.m.s.d. between the backbone atoms is 1.3 Å. b, c, All-atom overlays of the tau and HET-s β-helices.

Schematic views of hypothetical core structures of R1-R3 from 3R tau ( a), R2-R3 from 4R tau ( b) and R1-R2 from 4R tau ( c). Note that in these conformations, the β-helices would only be 18 instead of 19 residues long, because of an additional residue (D ₃₄₈) in R4. Still, the β-helices would have mainly hydrophobic residues in the core, polar residues on the outside, and the pivotal glycine closing the motif. Each hypothetical core structure also contains the first 10 amino acids of the next repeat (R4 for R1-3 and R2-R3; R3 for R1-R2). It could also be that there are cores made of three or more repeats.

Extended Data Table 1 Cryo-EM structure determination and model statistics for full-length (FL) and pronase-treated (PT) filaments

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Fitzpatrick, A., Falcon, B., He, S. et al. Cryo-EM structures of tau filaments from Alzheimer's disease. Nature 547, 185-190 (2017). https://doi.org/10.1038/nature23002
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# data[].attributes.attachment_link.title
Cryo-EM structures of tau filaments from Alzheimer's disease - Nature

# data[].attributes.attachment_link.description
Alzheimer's disease is the most common neurodegenerative disease, and there are no mechanism-based therapies. The disease is defined by the presence of abundant neurofibrillary lesions and neuritic plaques in the cerebral cortex. Neurofibrillary lesions comprise paired helical and straight tau filaments, whereas tau filaments with different morphologies characterize other neurodegenerative diseases.

# data[].attributes.headline
Cytosolic αS is monomeric and natively unfolded/disordered

# data[].attributes.subject
Cytosolic αS is monomeric and natively unfolded/disordered

# data[].attributes.body
Pathogen or inflammatory molecule invasion of brain via disrupted blood-brain barrier, disrupted oral or olfactory mucosa, trigeminal nerve --> triggers CNS immune response

Herpes viruses HSV1, HHV6 and HHV7 implicated in AD pathogenesis

Opportunistic infection of commensal microbes (e.g. candida, chlamydia) implicated in AD pathogenesis

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Fibril structure of amyloid-β(1–42) by cryo–electron microscopy

# data[].attributes.subject
Fibril structure of amyloid-β(1–42) by cryo–electron microscopy

# data[].attributes.body
Solution conditions for cryo-EM had to be adjusted to achieve a homogenous sample (low pH, organic solvent)

EM data augmented by solid-state NMR and X-ray diffraction

# data[].attributes.headline
Mutations in PrP are also linked to disease

# data[].attributes.subject
Mutations in PrP are also linked to disease

# data[].attributes.body
May increase the likelihood of spontaneous conversion --> propagated to other proteins

e.g. Familial CJD, fatal familial insomnia

# data[].attributes.body
Taupathies include Alzheimer's, angle-only dementia, chronic traumatic encephalopathy, argyrophilic grain disease, progressive supranuclear palsy, corticobasal degeneration, globular glial tauopathy and Pick’s disease/frontotemporal dementia

Diseases other than Alzheimer's lack Aβ plaques

'Strain-like' properties: different diseases display different compositions of tau isoforms and have distinct filament morphologies

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Pathogenesis: unfolded αS in the cytosol --> converts to β-rich greek-key oligomers (protofibrils) --> amyloid fibrils --> cytotoxic inclusions

Oligomeric intermediates are likely the most cytotoxic species and may permeabilise membranes (including mitochondrial membranes --> release of cytochrome C --> caspase activation --> apoptosis

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Nature 541, 217-221 (2017) Cite this article

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Abstract

Aggregation of amyloid-β peptides into fibrils or other self-assembled states is central to the pathogenesis of Alzheimer's disease. Fibrils formed in vitro by 40- and 42-residue amyloid-β peptides (Aβ40 and Aβ42) are polymorphic, with variations in molecular structure that depend on fibril growth conditions1,2,3,4,5,6,7,8,9,10,11,12. Recent experiments1,13,14,15,16 suggest that variations in amyloid-β fibril structure in vivo may correlate with variations in Alzheimer's disease phenotype, in analogy to distinct prion strains that are associated with different clinical and pathological phenotypes17,18,19. Here we investigate correlations between structural variation and Alzheimer's disease phenotype using solid-state nuclear magnetic resonance (ssNMR) measurements on Aβ40 and Aβ42 fibrils prepared by seeded growth from extracts of Alzheimer's disease brain cortex. We compared two atypical Alzheimer's disease clinical subtypes-the rapidly progressive form (r-AD) and the posterior cortical atrophy variant (PCA-AD)-with a typical prolonged-duration form (t-AD). On the basis of ssNMR data from 37 cortical tissue samples from 18 individuals, we find that a single Aβ40 fibril structure is most abundant in samples from patients with t-AD and PCA-AD, whereas Aβ40 fibrils from r-AD samples exhibit a significantly greater proportion of additional structures. Data for Aβ42 fibrils indicate structural heterogeneity in most samples from all patient categories, with at least two prevalent structures. These results demonstrate the existence of a specific predominant Aβ40 fibril structure in t-AD and PCA-AD, suggest that r-AD may relate to additional fibril structures and indicate that there is a qualitative difference between Aβ40 and Aβ42 aggregates in the brain tissue of patients with Alzheimer's disease.

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References

Safar, J. et al. Eight prion strains have PrP Sc molecules with different conformations. Nat. Med. 4, 1157-1165 (1998)

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Acknowledgements

This work was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases of the US National Institutes of Health, by the UK Medical Research Council and by the National Institute of Health Research (NIHR) UCLH/UCL Biomedical Research Centre. We are grateful for the assistance of S. Mead, O. Avwenagha, and J. Wadsworth at the MRC Prion Unit in selection and processing of tissue samples. We thank UK neurologists for referral of rapidly progressive dementias to the NHS National Prion Clinic, National Hospital for Neurology and Neurosurgery (NHNN), University College London Hospitals NHS Foundation Trust (UCLH). We thank the Queen Square Brain Bank for Neurological Disorders (supported by the Reta Lila Weston Trust for Medical Research, the Progressive Supranuclear Palsy [Europe] Association and the Medical Research Council) at the UCL Institute of Neurology, for provision of the human brain tissue samples. We thank all patients and their families for consent to use tissues in research.

Author information

Author notes

Wei Qiang & Jun-Xia Lu

Present address: †Present addresses: Department of Chemistry, Binghamton University, State University of New York, Binghamton, New York 13902, USA (W.Q.); School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China (J.-X.L.).,

Authors and Affiliations

1. Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, 20892, Maryland, USA

Wei Qiang, Wai-Ming Yau, Jun-Xia Lu & Robert Tycko

2. MRC Prion Unit and Department of Neurodegenerative Disease, UCL Institute of Neurology, London, 3BG, WC1N, UK

John Collinge

Contributions

W.Q., J.-X.L., J.C., and R.T. designed experiments, including selection of tissue samples, development of protocols for preparation of brain-seeded fibrils, and selection of ssNMR measurements. W.Q., J.-X.L., and R.T. prepared fibril samples and acquired TEM images and ssNMR data. W.-M.Y. synthesized isotopically labelled peptides and performed ELISA measurements. W.Q. and R.T. analysed ssNMR data. J.C. and R.T. wrote the manuscript, with contributions from all other authors.

Corresponding authors

Correspondence to John Collinge or Robert Tycko.

Extended data figures and tables

a, TEM grids were prepared 4 h after the addition of solubilized Aβ40 or Aβ42 to sonicated brain extract and were negatively stained with uranyl acetate. Collagen fibrils in the extract (40-100 nm width, with characteristic transverse bands) appear in some images. Material with an amorphous appearance are non-fibrillar, non-Aβ components of the brain extract. Yellow arrows indicate Aβ40 fibrils with an apparent width modulation, attributable to an approximately periodic twisting of the fibril structure about the fibril growth direction. TEM images of all 37 brain-seeded Aβ40 and all 33 Aβ42 fibril samples are available at http://dx.doi.org/10.17632/whgp9r7tkd.1. b, Histogram of distances between width minima for Aβ40 fibrils with apparent width modulation. The Gaussian fit to this histogram (red curve) has a mean value of 107.2 nm ( n  = 65) and a full-width-at-half-maximum of 46.1 nm.

a, 2D ¹³C- ¹³C spectra of fibrils seeded with extract from t-AD, PCA-AD, r-AD, or non-dementia (ND) tissue. Aliphatic regions are shown, with 15 contour levels (increasing by successive factors of 1.3, and with the highest contour at the maximum signal in each 2D spectrum). b, 2D ¹⁵N- ¹³C spectra of fibrils seeded with extract from t-AD, PCA-AD, r-AD, or non-dementia tissue. Regions containing intra-residue ¹⁵N- ¹³C α cross-peaks are shown, with 11 contour levels (increasing by successive factors of 1.3, with the highest contour at the maximum signal in each spectrum). ¹⁵N- ¹³Cβ cross-peaks from L34 appear in some spectra. Positions of cross-peaks from the predominant Aβ40 fibril structure are indicated by colour-coded circles (F19, blue; V24, cyan; G25, pink; S26, orange; A30, purple; I31, red; L34, green; M35, magenta). Only 2D spectra that were included in the analyses in Figs. 3 and 4 are shown. The full set of 42 2D ¹³C- ¹³C spectra and 40 2D ¹⁵N- ¹³C spectra, including those with lower signal-to-noise ratios, controls, and technical replicates, is available on-line at http://dx.doi.org/10.17632/tbp45pm92x.1.

a, 2D ¹³C- ¹³C spectra of fibrils seeded with extract from t-AD, PCA-AD, r-AD, or non-dementia (ND) tissue. Aliphatic regions are shown, with 15 contour levels (increasing by successive factors of 1.3, and with the highest contour at the maximum signal in each spectrum). b, 2D ¹⁵N- ¹³C spectra of fibrils seeded with extract from t-AD, PCA-AD, r-AD, or non-dementia tissue. Regions containing intra-residue ¹⁵N- ¹³C α cross-peaks are shown, with 11 contour levels (increasing by successive factors of 1.3, with the highest contour at the maximum signal in each spectrum). Only 2D spectra that were included in the analyses in Figs. 3 and 4 are shown. The full set of 33 2D ¹³C- ¹³C spectra and 23 2D ¹⁵N- ¹³C spectra, including those with lower signal-to-noise, controls, and technical replicates, is available at http://dx.doi.org/10.17632/tbp45pm92x.1.

a, Comparison of TEM images of control tissue extract and r-AD2p′ extract after incubation for 4 h with solubilized Aβ40, under conditions identical to those that led to fibrils shown in Extended Data Fig. 1a. Fibrils associated with brain material were abundant on the TEM grid of the r-AD2p′-seeded sample, but were not observed in an extensive search over the TEM grid of the control sample. b, TEM images of control tissue extract and r-AD2p′ extract after incubation for 4 h with solubilized Aβ42, under conditions identical to those that led to fibrils shown in Extended Data Fig. 1b. Fibrils associated with brain material were abundant on the TEM grid of the r-AD2p′-seeded sample, but were not observed in an extensive search over the TEM grid of the control sample. c, 2D ¹³C- ¹³C and ¹⁵N- ¹³C spectra of Aβ40 fibrils (blue) and Aβ42 fibrils (red) that developed in control samples after 168 h or 48 h incubation, respectively, followed by 24 h intermittent sonication (see Supplementary Methods) and a further 72 h of additional incubation. Contour levels increase by successive factors of 1.4. d, r.m.s.d. values between 2D spectra of control fibrils and 2D spectra of AD brain-seeded fibrils, with dashed lines at values corresponding to white shades in Fig. 3. Occipital tissue of a female who died from cardiac arrest at age 86 was used as a control.

a, The first five principal components (PC1-PC5) of the 32 experimental 2D ¹³C- ¹³C spectra shown as contour plots, with positive contours in blue and negative contours in red. Principal component spectra were obtained by singular-value decomposition of the experimental spectra, considering only the aliphatic region and excluding points within 5 p.p.m. of the diagonal. Contour levels increase (or decrease, in the case of negative contours) by successive factors of 1.5. b, Experimental 2D ¹³C- ¹³C spectrum of t-AD4f Aβ40 fibrils (left) and 2D spectrum constructed as a linear combination of PC1-PC5 (right, with coefficients of PC1-PC5 shown in parentheses). c, Experimental 2D ¹³C- ¹³C spectrum of r-AD1f Aβ40 fibrils (left) and 2D spectrum constructed as a linear combination of PC1-PC5 (right). d, The first five principal components of the 29 experimental 2D ¹⁵N- ¹³C spectra. e, Experimental 2D ¹⁵N- ¹³C spectrum of PCA2p Aβ40 fibrils (left) and 2D spectrum constructed as a linear combination of PC1-PC5 (right). f, Experimental 2D ¹⁵N- ¹³ C spectrum of r-AD2o Aβ40 fibrils (left) and 2D spectrum constructed as a linear combination of PC1-PC5 (right).

a, The first five principal components (PC1-PC5) of the 17 experimental 2D ¹³C- ¹³C spectra, plotted as in Extended Data Fig. 5. b, Experimental 2D ¹³C- ¹³C spectrum of t-AD1p Aβ42 fibrils (left) and 2D spectrum constructed as a linear combination of PC1-PC5 (right, with coefficients of PC1-PC5 shown in parentheses). c, Experimental 2D ¹³C- ¹³C spectrum of r-AD2f Aβ42 fibrils (left) and 2D spectrum constructed as a linear combination of PC1-PC5 (right). d, The first five principal components of the 15 experimental 2D ¹⁵N- ¹³C spectra. e, Experimental 2D ¹⁵N- ¹³C spectrum of t-AD3f Aβ42 fibrils (left) and 2D spectrum constructed as a linear combination of PC1-PC5 (right). f, Experimental 2D ¹⁵N- ¹³ C spectrum of r-AD2p Aβ42 fibrils (left) and 2D spectrum constructed as a linear combination of PC1-PC5 (right).

a, Examples of 2D spectra (of the 29 Aβ40 and 15 Aβ42 spectra with adequate signal-to-noise ratios presented in Table 1), with fitted cross-peak positions indicated by crosses. Red and blue crosses indicate cross-peaks for chemical shift sets 'a' and 'b', respectively (see Supplementary Methods, Supplementary Discussion and Extended Data Fig. 8). Cyan crosses indicate additional cross-peaks. Contour levels increase by successive factors of 1.4. b, Pairwise differences among fitted cross-peak volumes for spectra of Aβ40 fibrils (left) and Aβ42 fibrils (right), with colour scales representing r.m.s.d. values. Total cross-peak volumes in each spectrum were normalized before calculation of r.m.s.d. values. Results from this cross-peak-fitting analysis are similar to those in Fig. 3, in which the same experimental data were analysed by direct comparisons of signal amplitudes in 2D spectra without fitting the signals with cross-peaks at specific positions. c, Fractions of the total fitted cross-peak volumes at 'a' and 'b' chemical shifts, with mean values indicated by horizontal bars. For Aβ40, mean values of 'a' volumes in spectra of t-AD ( n = 12) or PCA-AD ( n = 6) samples are significantly greater than the mean value ( n = 10) in spectra of r-AD samples ( P < 0.02, Welch's t-test; P < 0.02, Mann-Whitney-Wilcoxon test).

a, ¹⁵N and ¹³C chemical shifts (p.p.m.) from spectra of brain-seeded samples in Table 1 (grouped into sets 'a', 'b', and so on, based on correlations of the corresponding signal amplitudes over multiple 2D spectra) are compared with chemical shifts from previous ssNMR studies of Aβ40 and Aβ42 fibrils, as deposited in the Biological Magnetic Resonance Bank ( http://www.bmrb.wisc.edu/) with the indicated BMRB accession numbers. b, Differences in chemical shift after adjustments of chemical shift referencing in each set to make the average ¹³C α shifts and the average ¹⁵ N shifts equal in all sets.

Extended Data Table 1 Statistical significance of analyses in Figs. 3 and 4

Extended Data Table 2 Quantification by ELISA of Aβ40/Aβ40 molar ratios in amyloid-enriched brain extracts

Supplementary information

This file contains Supplementary Methods, a Supplementary Discussion and Supplementary References. The Supplementary Methods describe the selection of brain tissue samples, preparation of brain-seeded fibrils, conditions for ssNMR and TEM measurements, determination of Aβ40/Aβ42 ratios, RMSD, principal component, and crosspeak fitting calculations, statistical tests, and code availability and the Supplementary Discussion describes control experiments, chemical shift comparisons, ssNMR linewidths, and consistency among samples. (PDF 361 kb)

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Qiang, W., Yau, WM., Lu, JX. et al. Structural variation in amyloid-β fibrils from Alzheimer's disease clinical subtypes. Nature 541, 217-221 (2017). https://doi.org/10.1038/nature20814
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# data[].attributes.attachment_link.title
Structural variation in amyloid-β fibrils from Alzheimer's disease clinical subtypes - Nature

# data[].attributes.attachment_link.description
Structural differences in 40- and 42-residue-long amyloid-β fibrils seeded in vitro from the cortical tissue of patients with different clinical subtypes of Alzheimer's disease suggest that different fibril structures form in different disease variants and with different peptide lengths.

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Misfolded tau fibrils exhibit 'infectious' piron-like properties

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Misfolded tau fibrils exhibit 'infectious' piron-like properties

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Misfolded conformation can propagate

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Also Lewy neurites, glial cytoplasmic inclusions

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Two other synuclein family members (β and γ) but not present in Lewy bodies

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Impaired autophagy has been implicated in PD pathogenesis and could allow αS accumulation

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Parkin is an E3 ubiquitin ligase involved in targeting aggregated proteins and damaged mitochondria for degradation

Mutations in PARK2, which encodes parkin --> accumulation of protein aggregates (including αS fibrils) and damaged mitochondria --> cellular damage --> early onset PD

Parkin dysfunction leading to impaired mitophagy is especially detrimental in neurons due to their high energy needs

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αS mutants may be less prone to degradation (inhibit targeting by Parkin E3 ubiquitin ligase)

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αS is a critical mediator of inflammatory and immune responses required for normal immune function

May accumulate within the nervous system of PD individuals bc. of an inflammatory/immune response

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Affiliations

¹ Cellular Immunology Section, Laboratory of Cancer ImmunoMetabolism, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702-1201, USA.

² Cellular Immunology Section, Laboratory of Cancer ImmunoMetabolism, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702-1201, USA. Electronic address: yangd@mail.nih.gov.

³ Molecular Histopathology Laboratory, Leidos Biomedical Research, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702-1201, USA.

⁴ Enterin Research Institute, Philadelphia, PA 19103, USA.

⁵ Enterin Research Institute, Philadelphia, PA 19103, USA; MedStar Georgetown Transplant Institute, Georgetown University Hospital, Washington, DC 20007, USA.

⁶ Cellular Immunology Section, Laboratory of Cancer ImmunoMetabolism, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702-1201, USA. Electronic address: oppenhej@mail.nih.gov.

Free PMC article

Free PMC article

Affiliations

¹ Cellular Immunology Section, Laboratory of Cancer ImmunoMetabolism, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702-1201, USA.

² Cellular Immunology Section, Laboratory of Cancer ImmunoMetabolism, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702-1201, USA. Electronic address: yangd@mail.nih.gov.

³ Molecular Histopathology Laboratory, Leidos Biomedical Research, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702-1201, USA.

⁴ Enterin Research Institute, Philadelphia, PA 19103, USA.

⁵ Enterin Research Institute, Philadelphia, PA 19103, USA; MedStar Georgetown Transplant Institute, Georgetown University Hospital, Washington, DC 20007, USA.

⁶ Cellular Immunology Section, Laboratory of Cancer ImmunoMetabolism, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702-1201, USA. Electronic address: oppenhej@mail.nih.gov.

Abstract

Alpha-synuclein (αS) is causally involved in the development of Parkinson disease (PD); however, its role in normal vertebrate physiology has remained unknown. Recent studies demonstrate that αS is induced by noroviral infection in the enteric nervous system of children and protects mice against lethal neurotropic viral infection. Additionally, αS is a potent chemotactic activator of phagocytes. In this report, using both wild-type and αS knockout mice, we show that αS is a critical mediator of inflammatory and immune responses. αS is required for the development of a normal inflammatory response to bacterial peptidoglycan introduced into the peritoneal cavity as well as antigen-specific and T cell responses following intraperitoneal immunization. Furthermore, we show that neural cells are the sources of αS required for immune competence. Our report supports the hypothesis that αS accumulates within the nervous system of PD individuals because of an inflammatory/immune response.

Keywords: Parkinson disease; alarmins; alpha-synuclein; dendritic cells; immune response; immunization; inflammation; macrophage; peritoneal cavity; αS.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

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national center for biotechnology information

# data[].attributes.attachment_link.title
Alpha synuclein, the culprit in Parkinson disease, is required for normal immune function - PubMed

# data[].attributes.attachment_link.description
Alpha-synuclein (αS) is causally involved in the development of Parkinson disease (PD); however, its role in normal vertebrate physiology has remained unknown. Recent studies demonstrate that αS is induced by noroviral infection in the enteric nervous system of children and protects mice against let ...

# data[].attributes.attachment_link.readable_text
Despite their potential importance as therapeutic targets, the initial events in neurodegenerative diseases are poorly understood. Emerging evidence suggests that presynaptic dysfunction might be an early event in these pathologies, and three papers now link dysregulation of SNARE protein levels and function caused by the absence of synuclein or cysteine string protein (CSP) to activity-dependent neurodegeneration.

Neurodegenerative diseases are a major health issue, and the urgency of understanding their cause and developing potential treatments is increasing as life expectancy is extending. A common pathological feature of many of these diseases is the progressive appearance of insoluble protein aggregates, or inclusions, followed by neuronal death. Much work has concentrated on potential mechanisms for the prevention of protein aggregation, although it has long been questioned whether protein aggregates are themselves causative of neuronal death or are instead even neuroprotective. The development of effective therapeutic interventions to prevent neurodegeneration therefore requires a better understanding of the early events that precede neuronal loss. Recent work in diverse disease models has begun to highlight the importance of presynaptic dysfunction as an early event that occurs before neurodegeneration. Three recent papers suggest that maintaining SNARE protein function in neurotransmitter release is important for the prevention of neurodegeneration.

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Authors and Affiliations

Robert D. Burgoyne and Alan Morgan are in the Department of Cellular and Molecular Physiology, The Physiological Laboratory, Institute of Translational Medicine, Crown Street, University of Liverpool, Liverpool, L69 3BX, UK. burgoyne@liv.ac.uk,

Robert D. Burgoyne & Alan Morgan

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The authors declare no competing financial interests.

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Burgoyne, R., Morgan, A. Chaperoning the SNAREs: a role in preventing neurodegeneration?. Nat Cell Biol 13, 8-9 (2011). https://doi.org/10.1038/ncb0111-8
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Chaperoning the SNAREs: a role in preventing neurodegeneration? - Nature Cell Biology

# data[].attributes.attachment_link.description
Despite their potential importance as therapeutic targets, the initial events in neurodegenerative diseases are poorly understood. Emerging evidence suggests that presynaptic dysfunction might be an early event in these pathologies, and three papers now link dysregulation of SNARE protein levels and function caused by the absence of synuclein or cysteine string protein (CSP) to activity-dependent neurodegeneration.

# data[].attributes.body
Amyloid fibrils are sequence-specific --> requires a lot of the same peptide

# data[].attributes.body
Transient, unstructured --> difficult to study, but can use NMR

Normally non-toxic

Potential therapeutic targets to prevent oligomerisation

# data[].attributes.body
Misfolded intermediates can often be quite compact, rather than substantially unstructured and highly dynamic, and may contain misassembled structural elements along with more extended correctly folded regions

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Difficult to isolate and structurally characterise

Most cytotoxic because detergent-like properties cause membrane lysis

Lyse mitochondria --> release cytochrome C --> trigger apoptosis

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Double β-sheet in which each sheet is formed from parallel segments stacked in register

Backbone-backbone hydrogen bonding interactions in the cross β-spine are important for fibril growth

Steric zipper model: side chains of monomers intercalate to form a dry interface that excludes water (hydrophobic core) --> entropically favourable fibrillation, strength and stability

Highly resistant to proteases, detergents and denaturants

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Expansion of CAG repeats in HTT gene (>35) --> abnormally long HTT protein with 'sticky' poly-Gln tract --> aggregation --> neural dysfunction and death

'Anticipation' - number of CAG repeats correlates with earlier disease onset and increased severity

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TRiC chaperonin binds the poly-Gln tract and isolating the proteins in its internal chamber --> prevents toxic aggregation

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In Huntington's disease (HD), an abnormal increase in the number of CAG repeats in the mutant Huntington gene corresponds to a long tract of glutamine amino acids in the huntingtin protein ( Htt). This excessively long glutamine tract is sticky and leads to the formation of protein aggregates in brain cells. Whether these aggregates are toxic only upon formation or are formed as a cellular defense mechanism against free toxic mutant huntingtin protein (mHtt) is still hotly debated (more information about huntingtin protein aggregation can be found here); however, what is certain is that these protein aggregates are directly linked to neuron dysfunction and death. Moreover, a larger number of CAG repeats is clearly correlated with earlier disease onset and increased severity.

Although these findings suggest that HD is solely a result of the CAG expansion and subsequent protein aggregation, several observations indicate that other factors are also at play. For example, patients with the same number of CAG repeats may exhibit different disease patterns. In addition, there are genes other than the Huntington gene that also contain a CAG repeat region. Some of these genes may have a long region of upwards of 50 CAG repeats, but still produce functional non-harmful proteins (recall that HD can occur in patients with as few as 36 CAG repeats in the Huntington gene). Evidently, there are other cellular mechanisms, though not well understood, that govern how the expanded number of CAG repeats translates into the toxic behavior of mHtt and aggregates seen in the specific case of HD.

Recently, researchers have found increasing evidence that the cellular mechanisms contributing to neurodegenerative diseases resulting from protein aggregates involve disruptions to the quality control machinery in the cell responsible for identifying and eliminating these toxic proteins. In particular, a protein known as TRiC (TCP-1 ring complex) has been found to be involved in controlling whether proteins with a long glutamine tract like Htt will fold and aggregate. This article will first review how certain classes of proteins can control protein folding, then go over the case of TRiC and its role in Htt folding, and finally discuss how this knowledge might be helpful for researching HD therapeutics.

Protein Folding and Molecular Chaperones^

However, proteins are not functional as a linear chain; they need to take on a certain shape or conformation in order to exert their proper functions. To assist in this process known as protein folding, proteins known as molecular chaperones bind to chains of amino acids that are newly made by ribosomes. Molecular chaperones, as their name suggests, are present during the protein-making process to ensure that newly made proteins behave normally and appropriately for their intended function. They do this by binding to the amino acid chain, thereby stabilizing it and facilitating proper folding. Another function that chaperones perform is to prevent folding errors and inappropriate aggregation, by recognizing incorrectly folded proteins and targeting them for repair or elimination. More information about molecular chaperones and proper protein folding can be found here.

Proteins folding incorrectly can lead to protein aggregation and disease. Moreover, there is a natural decline in a cell's ability to continually make and fold protein correctly as one ages. As a result, in the study of HD, many scientists have focused their attention to the study of molecular chaperones and their role in responding to protein aggregation. TRiC is a molecular chaperone that has recently been found to be directly involved in regulating aggregation seen in proteins containing a long tract of glutamine amino acids, such as Htt.

What is TRiC and how is it involved in protein folding?^

Although roundworm models are easy to manipulate in the lab, it is much more relevant to study Htt aggregation in human cells. Consistent with the previous result, it has also been found that blocking TRiC in human cells will result in increased aggregation of proteins with an expanded glutamine repeat region.

How does TRiC actually suppress huntingtin aggregation?^

So far, we've discussed how TRiC is involved in folding newly formed proteins, but how does TRiC prevent already formed mutant huntingtin (mHtt) from aggregating? Studies in this area of research first found that TRiC is able to directly bind to the Htt protein. This result aligns with what we previously knew about TRiC - that it acts as a molecular chaperone by assisting in protein folding. In particular, scientists have found, through several different experiments, that CCT1, one of the eight subunits forming TRiC, is especially important for binding to Htt. First, an study of the brain tissue of a transgenic mouse expressing mHtt revealed that TRiC and, specifically, CCT1 directly interacts with the Htt protein. This is very strong evidence indicating that TRiC regulates Htt activity in the brain in some way. Furthermore, by suppressing the CCT1 subunit alone, researchers still observe the same increase in Htt aggregation as when TRiC is suppressed. Even more significantly, expressing more CCT1 in cells can actually decrease the amount of Htt aggregation and toxicity in human cell culture models. All of these experiments suggest that TRiC can somehow control Htt aggregation through the CCT1 subunit.

How can these findings about TRiC translate to an HD therapeutic?^

Research about TRiC is still in its preliminary stages, since much of the mechanisms behind TRiC's ability to suppress huntingtin ( Htt) aggregation remains to be understood. However, the fact that we do know TRiC and specifically its CCT1 subunit are potent inhibitors of aggregation is useful for researchers who are thinking of ways to stop the formation of dangerous aggregates that are toxic to brain cells.

A study published in 2012 by Sontag et al. took this knowledge and asked the following question: because cells that express more of the CCT1 subunit had less Htt protein aggregation, can this CCT1 subunit be applied as a drug and be directly delivered to brain cells? In order to answer this question, the researchers used cell models of HD and first showed that a modified fragment of CCT1 can penetrate the cell membrane and enter the cell to exert its effects. With regards to therapeutic benefits, the researchers observed that when higher doses of CCT1 were applied to rat cells engineered to express mHtt, there was a corresponding decrease in the amount of visible Htt aggregates formed. The scientists followed up with another experiment where they applied CCT1 to cells of HD mice that were removed from the striatum, the brain area most affected in HD. They obtained perhaps an even more encouraging result: the cells receiving CCT1 treatment exhibited a higher survival rate compared to cells without treatment. This suggests that CCT1 may exert therapeutic benefits for cells expressing mHtt beyond just suppressing protein aggregation.

Conclusion^

The above research is simply a small-scale experiment that demonstrates the potential that this line of research can have for HD therapeutics. Evidently, there is a still a lot that we don't know about how molecular chaperones like TRiC can suppress huntingtin ( Htt) aggregation. The experiments up to this point have all been performed with cells in laboratory settings, and these results will have to be verified in subsequent animal studies before any conclusions can be made about their actual therapeutic benefit. However, studying TRiC has emphasized the importance of looking beyond the infamous glutamine repeats in Htt and looking at regions such as N17 to understand the mechanism of Htt aggregation and toxicity. Furthermore, the fact that direct protein delivery to the brain is already being done in mice for Parkinson's disease research could lead the way for testing CCT1 in HD mouse models as well.

Further Reading^

Meyer AS, Gillespie JR, Walther D, Millet IS, Doniach S, Frydman J. (2003) Closing the Folding Chamber of the Eukaryotic Chaperonin Requires the Transition State of ATP Hydrolysis. 113(3): 369-381.

Behrands C, Langer CA, Boteva R, Böttcher UM, Stemp MJ, Schaffer G, Rao BR, Giese A, Kretzschmar H, Siegers K, Hartl FU. (2006). Chaperonin TRiC Promotes the Assembly of polyQ Expansion Proteins into Nontoxic Oligomers. . 23(6): 887-897.

Tam S, Geller R, Spiess C, Frydman J. (2006). The chaperonin TRiC controls polyglutamine aggregation and toxicity through subunit-specific interactions. Nat. Cell Biol. 8(10): 1155-1162.

Tam S, Spiess C, Auyeung W, Joachimiak L, Chen B, Poirier MA, Frydman J. (2009) The Chaperonin TRiC Blocks a Huntingtin Sequence Element that promotes the Conformational Switch to Aggregation. Nat. Struct. Mol. Biol. 16(12): 1279-1285.

Shen K, Frydman J. (2013). The interplay between the chaperonin TRiC and N-terminal region of Huntingtin mediates Huntington's Disease aggregation and pathogenesis. Protein Quality Control in Neurodegenerative Diseases. Eds. Morimoto RI, Christen Y. 121-132.

Shahmoradian SH, Galaz-Montoya JG, Schmid MF, Cong Y, Ma B, Spiess C, Frydman J, Ludtke SJ, Chiu W. (2013). TRiC's tricks inhibit huntingtin aggregation. eLife. 2:e00710.

Sontag EM, Joachimiak LA, Tan Z, Tomlinson A, Housman DE, Glabes CG, Potkin SG, Frydman J, Thompson LM. (2012). Exogenous delivery of chaperonin subunit fragment ApiCCT1 modulates mutant Huntingtin cellular phenotypes. Proc. Natl. Acad. Sci. USA. 110(8): 3077-3082.

- J. Choi, 02-03-14

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TRiC and Huntingtin Protein Aggregation - HOPES Huntington's Disease

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In Huntington's disease (HD), an abnormal increase in the number of CAG repeats in the mutant Huntington gene corresponds to a long tract of glutamine amino acids in the huntingtin protein (Htt). This excessively long glutamine tract is sticky and leads to the formation of protein aggregates in brain cells.

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Incorrectly folded states transiently populated during the folding process are potentially prone to aggregation and have been implicated in a range of misfolding disorders including Alzheimer's, Parkinson's and Type II diabetes

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Sometimes non-productive pathways are taken rather than the correct folding pathway

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D: denatured, N: native, I: off-pathway intermediate, TSI: transition state D -> I, TSN: transition state D -> N

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Biphasic folding is caused by kinetic partitioning between the two pathways, one leading to a more stable state and the other to a less-stable off-pathway intermediate

Kinetic competition between the native state and a misfolded off-pathway intermediate may be common for large proteins with complex topologies

Mutation destabilises N more than I --> increased stability of I relative to N --> I more likely/longer lifetime (shifts equilibrium towards I)--> high risk of misfolding and aggregation

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Unusual φ values (φ>1 or φ<0) are indicative of non-native contact formation in I or TS

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Unusual φ values (φ>1 or φ<0) are indicative of non-native contact formation in I or TS

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φ value analysis allows one not just to define the structure of the misfolded intermediate but also to understand the molecular events that lead to its formation and are distinct from those leading to correct folding into the native state

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The off-pathway intermediate represents a 'kinetic trap' that needs to unfold before the protein process can proceed to the native state

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Alzheimer's/Parkinson's - kinetic competition between correctly-folded amyloid-β or α-synuclein proteins and misfolded oligomer/fibrillar aggregates

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Author notes

1. Ylva Ivarsson

Present address: Present address: Department of Human Genetics, Katholieke Universiteit Leuven, Herestraat, Leuven, Belgium.,

2. Stefano Gianni and Ylva Ivarsson: These authors contributed equally to this work.

Authors and Affiliations

1. Dipartimento di Scienze Biochimiche 'A. Rossi Fanelli', Istituto Pasteur-Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia Molecolari del CNR, Università di Roma 'La Sapienza', Rome, Italy

Stefano Gianni, Ylva Ivarsson, Carlo Travaglini-Allocatelli & Maurizio Brunori

2. Department of Chemistry, University of Cambridge, Cambridge, UK

Alfonso De Simone & Michele Vendruscolo

Contributions

S.G., Y.I., C.T.-A. and M.B. conceived and designed the experimental work; A.D.S. and M.V. conceived and designed the computational work; Y.I. expressed and purified the protein samples; S.G. and Y.I. conducted the experiments; A.D.S. did the simulations; S.G., Y.I., A.D.S., C.T.-A., M.B. and M.V. analyzed the data and wrote the paper.

Corresponding author

Correspondence to Michele Vendruscolo.

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Structural characterization of a misfolded intermediate populated during the folding process of a PDZ domain - Nature Structural & Molecular Biology

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A high-resolution structure of an off-pathway misfolded intermediate state of a PDZ domain is now obtained, through a combination of phi-value analysis and computational modeling. The structure reveals that a misfolded intermediate can look quite similar to the native state.

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Distinct demands of folding and function can shape a free energy landscape with local minima 'kinetic traps' of sufficient depth that the protein can get stuck as a misfolded intermediate and cannot escape by thermal motion alone

These kinetic traps are not 'smoothed' by evolution bc. doing so would disrupt protein function

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Functional demands in shaping the ligand-binding pocket may compromise an otherwise robust folding process

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Highly frustrated regions often correspond to binding sites

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e.g. APOE ε4 allele and Alzheimer's

ε4 allele increases risk of developing Alzheimer's later in life BUT correlates with cognitive benefits early in life --> selected for!

Young ε4 carriers have higher IQs than noncarriers

ε4 carriers may recruit additional right hemisphere frontal regions in order to achieve cognitive advantage

25% of people carry the ε4 allele