Protein Aggregation

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cls May 22, 2019 14:27
Annual Review of Biochemistry

Propagation of Protein
Aggregation in
Neurodegenerative Diseases
Annu. Rev. Biochem. 2019.88:785-810. Downloaded from www.annualreviews.org
Jaime Vaquer-Alicea and Marc I. Diamond

Access provided by 187.190.155.179 on 07/16/19. For personal use only.
Center for Alzheimer’s and Neurodegenerative Diseases, University of Texas Southwestern

Medical Center, Dallas, Texas 75390, USA; email: [email protected]
Annu. Rev. Biochem. 2019. 88:785–810 Keywords

First published as a Review in Advance on
amyloid, tau, prion, propagation, strain, aggregation
March 27, 2019
The Annual Review of Biochemistry is online at Abstract

biochem.annualreviews.org
Most common neurodegenerative diseases feature deposition of protein
https://doi.org/10.1146/annurev-biochem-061516-
amyloids and degeneration of brain networks. Amyloids are ordered pro-
045049
tein assemblies that can act as templates for their own replication through
Copyright © 2019 by Annual Reviews.
monomer addition. Evidence suggests that this characteristic may underlie
All rights reserved
the progression of pathology in neurodegenerative diseases. Many differ-
ent amyloid proteins, including Aβ, tau, and α-synuclein, exhibit properties
similar to those of infectious prion protein in experimental systems: discrete
and self-replicating amyloid structures, transcellular propagation of aggre-
gation, and transmissible neuropathology. This review discusses the contri-
bution of prion phenomena and transcellular propagation to the progression
of pathology in common neurodegenerative diseases such as Alzheimer’s and
Parkinson’s. It reviews fundamental events such as cell entry, amplification,
and transcellular movement. It also discusses amyloid strains, which produce
distinct patterns of neuropathology and spread through the nervous system.
These concepts may impact the development of new diagnostic and thera-
peutic strategies.
785
BI88CH29_Diamond ARjats.cls May 22, 2019 14:27
Contents
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786
CELL BIOLOGY OF AGGREGATE PROPAGATION . . . . . . . . . . . . . . . . . . . . . . . . . . . 788
AGGREGATE PROPAGATION INTO CELLS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 788
UPTAKE AND RELEASE MECHANISMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 789
SEEDING ASSAYS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 792
MOUSE MODELS OF SPREADING PATHOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 792
THERAPEUTIC MECHANISMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794
Blocking Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794
Immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794
Cell Uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796
Intracellular Clearance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796
INHIBITION OF INTRACELLULAR SEEDING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796

THE ROLE OF PRION STRAINS AND NETWORKS

IN NEURODEGENERATIVE DISEASES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797
YEAST PRION STRAINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797
Aβ, TAU, AND α-SYNUCLEIN STRAINS AND STRAIN-LIKE BEHAVIOR . . . . 798
NETWORKS IN PROPAGATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800
THE PRION MODEL ADVANCES THERAPEUTIC
AND DIAGNOSTIC OPPORTUNITIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 801
INTRODUCTION
Neurodegenerative diseases are a major health problem for the world’s aging population. The
most common are linked to the accumulation of protein amyloids inside or outside cells. We use
the term amyloid to refer to long, unbranched protein fibrils that display cross-β fiber diffrac-
tion when examined with X-rays (1, 2). Much like crystallization of small molecules or proteins,
amyloids serve as templates for their own replication in vitro. Several models have been proposed
to explain the mechanisms of amyloid growth. The first model proposes a nucleated polymer-
ization event, in which a monomer is converted to a seed from which fibrils grow via monomer
addition (2–8). Recent work indicates that for tau the pathogenic seed can be a single molecule
(9). The second model proposes an induced fit, whereby monomers convert into aggregates that
are unable to grow into ordered amyloid fibrils. Instead, these aggregates can serve as substrates
for other proteins to bind and undergo a conformational change that enables subsequent amy-
loid growth (2, 4). In the case of both models, after fibrils begin to grow, they are subject to
fragmentation and secondary nucleation events that rapidly amplify protein amyloids. This self-
replication mechanism based on template formation has provided a conceptual framework for
understanding the origin and progression of multiple neurodegenerative diseases. Importantly, in
most cases, mutations that cause dominantly inherited neurodegenerative diseases typically alter
the very same proteins that accumulate in sporadic cases, usually via structural destabilization that
promotes amyloid formation, or via overproduction. Although the triggers of protein aggrega-
tion in sporadic diseases remain unknown, many groups have reported diminished protein quality
control in aging organisms (10), which could conceivably play a role. In the case of the most com-
mon age-related neurodegenerative disease, Alzheimer’s disease (AD), branches of the ubiquitin–
proteasome system and the endosomal/lysosomal pathways are particularly important for keeping
786 Vaquer-Alicea • Diamond

aggregation-prone proteins under control (11). Perhaps unsurprisingly, genome-wide association

studies have reported that many proteins involved in these pathways are linked to AD. Both spo-
radic and dominantly inherited AD show a primary pathogenic role for protein aggregation and
Prion: a structured
accumulation. protein assembly that
Prions are infectious protein assemblies that can transmit disease between individuals by serv- self-replicates in living
ing as templates that convert normal protein to a pathogenic conformation (12–14). The eluci- systems and whose
dation of this novel and frightening basis of neurodegenerative disease led to the Nobel Prize in conformation controls
its biological activity
Physiology or Medicine in 1997 for Stanley Prusiner, who identified the causative agent, the prion
and potential for
protein (PrP). However, with the predominant effort to confirm protein-based infectious propa- transmission between
gation of pathology, in some respects the field was distracted from investigation into the molec- individuals
ular and cellular mechanisms of pathogenesis, in which a tiny inoculum progressively amplifies
Strain: a unique prion
its structure within individual cells and then spreads throughout the nervous system. Importantly, conformation that
most prion disease cases are sporadic, not infectious, indicating that this process can begin and replicates faithfully in
progress on the basis of endogenous mechanisms. living systems and

In most respects, prion disease resembles noninfectious diseases such as AD, Parkinson’s dis- confers specific
biological effects
ease (PD), and amyotrophic lateral sclerosis (ALS), as virtually all cases include progressive neu-
rodegeneration, deposition of protein amyloids, and genetic as well as sporadic causes. In the Transcellular
last decade compelling basic research has linked prion mechanisms to common amyloid diseases propagation: growth
of a specific amyloid
(15–21). New studies make clear that parallels extend to fundamental aspects of propagation and
structure within a cell,
the role of strains, which are faithfully self-replicating amyloid assemblies that produce unique followed by its
patterns of disease (Figure 1). Elucidation of basic mechanisms involved in the cell–cell propaga- movement to another
tion of pathology promises to introduce new therapeutic and diagnostic strategies. Additionally, cell and subsequent
by learning from prion biology about the role of discrete amyloid structures in driving certain amplification based on
interaction with native
patterns of pathology, we may gain additional insights into pathogenic mechanisms. This review
protein
focuses on two aspects of prion mechanisms in the spreading pathology of disease: (a) transcellular
propagation and disease progression and (b) the role of strains in dictating neuronal vulnerability
and patterns of spread through the nervous system.
Strains
Alternative
conformations
Native
monomer
Unique Biological
interactions effects
Figure 1
Prions and strains. Prion formation begins with conversion of native monomer to a form that can
self-associate into ordered assemblies. These amplify through contact with free monomer, preserving their
original structure by acting as templates. Strains are distinctly defined structures that lead to unique
biological consequences, such as neurotoxicity, variation in the rate of spread through the nervous system,
and specific patterns of cell vulnerability within brain networks. This presumably occurs because of
strain-dependent variation in uptake, intracellular seeding, and contact with other cellular components.
www.annualreviews.org • Protein Aggregation in Neurodegeneration 787

CELL BIOLOGY OF AGGREGATE PROPAGATION

Studies of PrP have long implicated propagation of pathological protein aggregation by way of
Seeding: the ability of neural connections, as inoculation of the eye leads to spread of pathology along the visual network
an ordered protein or of the central nervous system (22–24). In 2000, Walker and colleagues (25) described the induc-
protein assembly to act tion of plaque pathology in transgenic mice overexpressing amyloid precursor protein (APP) five
as a template to trigger
months after infusion of brain homogenates containing amyloid-β (Aβ), but not homogenates of
subsequent growth of
a homotypic amyloid young, nondiseased brain. A follow-up study (26) described the appearance of histopathological
fibril markers in the contralateral hemisphere, suggesting spread of pathology. Subsequent work indi-
cated that Aβ can propagate a distinct structure in vitro (27). Jucker and colleagues (18) found
that inoculation of transgenic mice that overexpress APP with various preparations of Aβ fibrils
can induce plaque pathology, and they observed that Aβ immunodepletion, protein denaturation,
or immunization effectively abolished the seeding ability of the extracts. These pioneering studies
clearly indicated that Aβ fibrils could be infectious in animal models (i.e., they could be propa-
gated on the basis of inoculation) and suggested that template-induced aggregation might play a
role outside of classical prion diseases (18).

Induction of Aβ pathology occurs via multiple routes, including direct inoculation (18), inser-
tion into the brain of metal wires coated with Aβ fibrils (28), and even peripheral administration
(29). These and other studies also highlighted the presence of distinct Aβ conformations that
could be detected in human tissue (30) or could produce distinct pathological patterns in animals
(31). This work followed up predictions made by Prusiner (32) in 1984 linking AD and prion dis-
ease, which were based on his observation of PrP fibril rods that were similar to Aβ fibrils seen
in AD. Unlike most amyloids associated with neurodegeneration, Aβ accumulates outside cells,
which is presumably where growth of fibrils occurs, and thus progressive deposition need not
involve transcellular propagation.
AGGREGATE PROPAGATION INTO CELLS

In 2008, two landmark studies documented the occurrence of α-synuclein inclusions, or Lewy
bodies, in cells transplanted into the striatum of patients with PD (20, 33). In an effort to ameliorate
their PD symptoms, these patients had received fetal dopaminergic transplants up to 14 years be-
fore autopsy. The investigators observed α-synuclein protein inclusions in the transplanted cells,
which expressed tyrosine hydroxylase, and could thus be discriminated from surrounding neurons.
These observations suggested either that pathological synuclein had transferred from the host to
the grafted cells, or that something toxic in the brains of patients with PD led to aggregation
of synuclein in the relatively young transplanted cells. Follow-up studies by S.-J. Lee and col-
leagues (19) indicated that aggregated α-synuclein transfers between cultured cells, and from host
to engrafted cells, and work from V. Lee and colleagues (34) indicated that α-synuclein directly
transduced into cultured cells triggers accumulation of phosphorylated α-synuclein aggregates.
Investigations of tau protein provided clear evidence of induced misfolding that could trans-
mit across membranes. Tolnay and colleagues (17) injected brain lysates containing tau aggregates
into the brains of tau transgenic mice. They observed induction of local tau pathology, and appar-
ent spread to connected cells, albeit across relatively short distances (17). Concurrently, our group
(21) determined that exposure of cultured cells expressing tau protein to extracellular fibrils would
directly induce intracellular amyloid formation and that induced intracellular aggregates would
transfer to cocultured cells. Additionally, the Kopito laboratory (35) determined that polygluta-
mine aggregates driven into cultured cells expressing a wild-type form of the protein huntingtin
can trigger a persistent state of aggregation that is maintained in a few cells over many cell di-
visions. Other groups have made similar observations with tau (36), α-synuclein (34), superoxide

Table 1 Evidence for prion mechanisms in neurodegenerative diseases

Evidence for prion-based
Disease Protein mechanism of spread Reference(s)
Alzheimer’s disease Microtubule-associated protein Cells 21, 36, 41, 42
tau (tau) Rodent 17, 43–45
Amyloid-β (Aβ) Cells 46, 47
Rodent 18, 25, 29
Human 48
Parkinson’s disease α-Synuclein Cells 34, 36, 49, 50
Rodent 49, 51, 52
Human 20, 33
Amyotrophic lateral sclerosis Superoxide dismutase 1 (SOD1) Cells 37, 38, 53
Rodent 54, 55
Transmissible spongiform Prion protein (PrP) Cells 56, 57
encephalopathy Rodent 58, 59

Human 60, 61
Huntington’s disease Huntingtin (Htt) Cells 35, 62
Rodent 63
Human 64
dismutase 1 (SOD1) (37, 38), and other proteins associated with neurodegeneration (39) (Table 1).
Taken together, a broad consensus has rapidly emerged regarding fundamental cellular events that
could underlie transcellular propagation: Protein aggregates move between cells in culture and
in vivo, and aggregates taken into cells can trigger intracellular misfolding. It remains unknown
whether these mechanisms truly underlie progressive propagation of pathology in humans, and
other models are still proposed (40).
UPTAKE AND RELEASE MECHANISMS

Several aggregate internalization mechanisms for Aβ, α-synuclein, and tau have been proposed
(Figure 2). Evidence for endocytosis of α-synuclein stems from original observations of a reduc-
tion in uptake and toxicity of fibrils exogenously added to neurons expressing GTPase-deficient
Rab5A, which mediates both clathrin-dependent and clathrin-independent endocytosis (65). In-
ternalization of α-synuclein via clathrin-mediated endocytosis is supported by proteomic evidence
that identifies clathrin as a necessary component for microglial activation following uptake of
α-synuclein fibrils (66). A recent report proposed lymphocyte-activation gene 3 as the receptor
that mediates internalization of α-synuclein (67). Exogenous Aβ has been proposed to be inter-
nalized via heparan sulfate proteoglycans (HSPGs) (68); via endocytosis mediated by multiple
factors, including dynamin, caveolin-1, and GM1 (69); and via receptor-mediated endocytosis in-
volving lipoprotein receptor-related protein 1/apolipoprotein E, sortilin, and alpha-7-nicotinic
acetylcholine receptors (70, 71), NMDA receptors (72–75), and the cellular form of PrP (76). Fi-
nally, a mechanism involving tunneling nanotubes has been suggested for internalization of Aβ
(77). Given the uncertainty about where Aβ seeding might occur, the relevance of its uptake to
aggregate growth must be studied further.
Macropinocytosis is the best-characterized uptake mechanism for tau fibrils in cells and ani-
mals (78). This involves binding and uptake of free protein aggregates. Our laboratory initially
observed that tau aggregates are taken up via fluid-phase endocytosis (21). Subsequent work de-
termined that tau monomer and aggregates bind directly to HSPGs on the cell surface (78) on

HSPG-mediated Exosome Receptor-mediated

Phagocytosis
endocytosis fusion endocytosis
Figure 2
Mechanisms of uptake. Multiple mechanisms of uptake for pathogenic amyloid seeds have been proposed. Macropinocytosis has been
clearly linked to tau and α-synuclein aggregate uptake and seeding in diverse cells, including neurons. This is based on binding to
heparan sulfate proteoglycans (HSPGs) on the cell surface, which triggers the formation of large endocytic vesicles (macropinosomes)
that bring aggregates into the cell. Although not clearly defined, receptor-mediated endocytosis, based on binding of aggregates to
specific proteins at the cell surface, could occur. Exosome fusion and phagocytosis might also play a role.
the basis of specific sulfation patterns (79). HSPGs are transmembrane and glycolipid-anchored
proteins that are heavily glycosylated and sulfated during their maturation. They coat all cells
and mediate interactions with other cells, signaling molecules, and the extracellular matrix (80).
Tau uptake into cells and neurons is blocked by genetic disruption of HSPG synthesis, enzymatic
cleavage of heparan moieties, or interference with proper HSPG sulfation (78, 79). Finally, gly-
cosaminoglycan mimetics such as heparin mask the HSPG binding site on tau, which blocks its
binding to the cell surface, uptake, and seeding. We found that a synthetic heparin-like compound
termed F6 binds tau and prevents its uptake into cells and neurons in mouse brain (78). Elucidation
of this molecular mechanism of cell uptake requires further study, including development of more
effective compounds that directly bind tau, and potentially the identification of HSPG-related
synthetic genes that might be targeted to block uptake in neurons. Interestingly, we observed that
α-synuclein aggregates utilize HSPG-mediated uptake in a manner similar (78), but not identi-
cal (79), to that of tau. Other studies have implicated release of aggregates in ectosomes (81) and
exosomes (82), which could mediate uptake upon fusion to secondary cell membranes.
The precise size of aggregates that mediates transcellular propagation of neuronal pathology
in vivo is unknown. One study reported that low-molecular-weight aggregates and short fibrils,
but not monomers or long fibrils, are competent to seed into cells, although precise sizes of the
species were not defined (83). We have detected several soluble tau aggregates in the brains of
patients with AD (84). Fractionation of recombinant protein indicated that three units of tau are
the minimal size of an aggregate that is sufficient to trigger uptake and seeding into HEK293 cell
and primary neurons (84). Remarkably, this is the same size of PrP assembly determined almost

Membrane Tunneling
Exocytosis Exosome release
breakdown nanotubes
Figure 3
Mechanisms of release. Multiple mechanisms of aggregate release have been proposed. Membrane breakdown could allow passive,
transient efflux of aggregates from the cell. Alternatively, aggregates could be packaged into vesicles and released via exocytosis.
Ectosome formation also could occur, in which aggregates are packaged in smaller vesicles and subsequently released by fusion of
a larger multivesicular body. Finally, aggregates might transfer via tunneling nanotubes that physically link nearby cells.
30 years ago to be the minimal unit of infectivity (85). Others have also implicated tau trimers as
toxic agents (86). We observed no clear upper limit of assembly size for seeding into cells (84).
These observations directly bear on other cellular studies of tau secretion, which have quanti-
fied the release of tau monomer from cultured cells into the medium or into interstitial fluid or
cerebrospinal fluid (CSF) (87–93). The secretion of tau monomer may have a physiologic role.
However, our studies of aggregate uptake and seeding indicate that tau monomer is unlikely to
mediate transcellular propagation of aggregation.
Multiple studies have now also studied aggregate uptake and movement in primary neurons
(83). Microfluidic systems have enabled the study of axonal trafficking and have documented both
anterograde and retrograde transport of aggregates, with uptake by secondary cells (94). In mouse
models there is now clear evidence for transneuronal movement of tau protein (43, 45, 94), al-
though it is unclear whether this is mediated by aggregates. Many studies have evaluated release
of α-synuclein from cells and have implicated a variety of release mechanisms that include direct
membrane penetration, exosomes, and exocytosis (95) (Figure 3). Recent work also suggests that
synaptic activity might play a role in stimulating tau release (91, 96). It is unknown whether patho-
logical aggregates can be released in vesicles or whether aggregates enter the extracellular space
without an enclosing membrane. Studies of cell–cell propagation must also take into account the
fact that aggregates will move even between nonneural cells (21). It may be that a synapse, from
the point of view of an aggregate, is merely a place where two plasma membranes come into
close approximation. This idea is supported by the findings of Moechars and colleagues (97), who
observed that creation of an artificial synapse through expression of neuroligin 1 and leucine-
rich repeat transmembrane protein 2 on aggregate donor cells, which facilitated a connection to

primary neurons, increased induction of tau aggregation in the neurons. Negative control expres-
sion of N-cadherin, which also increased cell contact without creating a synapse-like structure,
did not facilitate propagation (97). Direct aggregate release, or loss of integrity of even a small
component of the cell membrane, could lead to extracellular aggregates that subsequently bind
the surface of a connected or neighboring cell and trigger their own uptake. Although neuronal
signaling may enhance tau aggregate propagation (97) or release in vitro (98), the observation of
transcellular movement in the absence of synapses implies a mechanism distinct from a classical
transmission process linked to synaptic vesicle release. With this in mind, atypical release via ecto-
somes could play a role (81). While it is unknown whether distinct release mechanisms will apply
to each amyloid protein, we favor a unifying model involving release of free aggregates into the
extracellular space, especially given the effectiveness of antitau antibodies to modulate cell uptake
in vitro (99) and toxicity in vivo (100, 101) and the conspicuous absence of tau from the proteome
of synaptic vesicles (102).
SEEDING ASSAYS
The detection of PrP prions based on in vitro seeding assays has advanced to extremely high sen-
sitivity (103, 104). In 2017, researchers described an in vitro detection system (real-time quaking-
induced conversion, or RT-QuIC) for biological fluids that detects three-repeat tau seeding
activity in patient-derived CSF using minute amounts of material (105). RT-QuIC is similar to
previously described methods (106, 107), but the sensitivity appears higher. Other researchers have
exploited similar techniques with high sensitivity and specificity to detect Aβ oligomers in CSF
from patients with AD (108) and α-synuclein (109) in patients with PD and dementia with Lewy
bodies (Figure 4).
To study the biology of transcellular propagation of tau and α-synuclein pathology, and that
of other propagating amyloid disorders, researchers required new tools to identify and quantify
seeding activity in biological samples. Consequently, we developed a biosensor cell line based
on stable expression of tau repeat domain containing a single disease-associated mutation (P301S)
fused to cyan and yellow fluorescent proteins (RD-CFPs/YFPs) (110). Upon aggregation, quench-
ing of CFP by YFP leads to fluorescence resonance energy transfer (FRET). This can be quantified
most accurately by flow cytometry (110, 111) (Figure 4). This assay is highly sensitive and specific,
detects tau seeds to the level of ∼300 femtomolar (monomer equivalent), and can be applied to
fresh frozen or fixed brain material (112). When used to study brain tissue from a transgenic mouse
model (PS19) that expresses full-length human tau containing the P301S mutation, this assay de-
tected tau seeding activity at 6 weeks of age, several months before the earliest neuropathology
could be detected at approximately 16 weeks (110). The biosensor assay has proved useful for
monitoring tau seeding activity in localized brain tissue in patients and in transgenic mouse mod-
els of propagating pathology (113); it also works with formalin-fixed tissue (112). Recent work
based on this assay indicates that seeding activity in human AD samples anticipates the develop-
ment of classical neurofibrillary pathology, as it occurs widely throughout the brain at relatively
early Braak stages, whereas AT8-positive (phospho-tau) inclusions become apparent only at later
stages (114). The introduction of a rapid, quantitative metric to monitor pathology could have
important implications for tracking disease progression in mouse models and patients.
MOUSE MODELS OF SPREADING PATHOLOGY

Many mouse models exist for the major neurodegenerative diseases (Table 1). Transgenic mice
typically feature abnormal protein expression throughout the brain, making it difficult to monitor

a
Sonication
or quaking
+
Amyloid fibrils Disrupted fibrils Native monomer Amplified fibrils
b Seeding assay Detection via FRET

Figure 4
Seeding assays. Two distinct approaches quantify seeding activity. (a) In the first approach, a sample
containing seeds is exposed to recombinant cognate monomer. A cycle of incubation, quaking (agitation), or
sonication is repeated to encourage fibril breakage and exponential growth. Large assemblies can be detected
by a variety of means, typically through incorporation of a fluorescent dye (e.g., thioflavin). (b) In the second,
cell-based approach, a biosensor line is created with stable expression of cognate protein fused to cyan and
yellow fluorescent proteins, or a similarly complementary fluorescent pair. Upon exposure to a sample
containing seeds, often with a reagent such as a cationic liposome to increase cell uptake, seeds are brought
into the cell, where they interact with the labeled protein and trigger intracellular aggregation. This is
quantified in a defined population of cells either by fluorescence microscopy or FRET flow cytometry.
Abbreviation: FRET, fluorescence resonance energy transfer.
spreading pathology without specific control of gene expression. One conditional model uses the
Tet-Off factor driven by the neuropsin promoter to activate mutant tau expression in one brain
region. At early ages, this activates gene expression predominantly in layer II of the entorhinal
cortex (43, 45). Tau pathology subsequently becomes apparent in hippocampal cells that receive
input from the entorhinal cortex. Results from two independent laboratories were consistent with
the spreading model of neurodegeneration, as they described the movement of pathological tau
protein from one region to another. It is less clear that this movement actually represents true
transcellular propagation of pathology, in which a seed moves between cells and then corrupts na-
tive protein to amplify an aggregated state. Two reports cast doubt on propagation in this mouse
model. The first study found that activity of the neuropsin promoter, which was used to drive the
Tet-Off factor, may not be anatomically restricted to the entorhinal cortex and that it expresses tau
more widely over time, especially within the hippocampus (115). Thus, during the long incubation
periods used for the studies, mutant tau may have been expressed at low levels in secondary cells
and may not have moved there. In a second study, propagation of entorhinal tau was observed in a
tau knockout background (116). In the absence of endogenous tau, it is technically impossible to
propagate pathology, but nonetheless progressive tau pathology was observed. These results sug-
gest that earlier propagation may simply have represented transcellular movement of pathological

proteins. Similar results have been obtained with virus-mediated expression of mutant tau, which
also transfers between neurons in vivo (94).
Other mouse models have used inoculation of virus or proteopathic seeds directly into the
brain. Inoculation of synuclein fibrils derived from either patients or recombinant sources leads
to progressive pathology (51). In fact, even wild-type mice inoculated with α-synuclein fibrils de-
velop progressive disease (52). Many studies of tau indicate that progressive pathology develops
from local inoculation of seeds (15, 17). In all such studies it is important to rule out simple move-
ment of inoculum from one cell to another, as this does not represent propagation. Our laboratory
has observed that distinct tau aggregate structures, or strains, replicate and propagate through-
out the brain at different rates. This is independent of the seeding activity of each strain and
thus appears to depend on tau prion structure (113). Other laboratories have used virus-mediated
expression of full-length, mutant tau to drive tauopathy in different brain regions (94). In most
cases, documentation of the spreading pathology, but not extraction and documentation of fib-
rillar material or tau seeding activity, has been based on immunostains for hyperphosphorylated
tau (17, 43, 94). Consequently, it is difficult to know at this time whether the presence of tau or
α-synuclein seeding activity directly correlates with standard histopathological markers. Taken
together, multiple useful mouse models exist to study propagation of pathology. We do not yet
know their validity for studying basic cellular mechanisms of propagation. But it is clear that these
models can be used to study transport of protein monomer and aggregates and seeded induction
of protein aggregation.
THERAPEUTIC MECHANISMS
As we learn more about the cellular mechanisms by which aggregates bind the cell surface, trigger
uptake, seed intracellular aggregation, and move between cells, we anticipate this will facilitate the
development of mechanism-based therapies to slow down or stop disease progression (Figure 5).
Blocking Release
Blocking the release of pathological aggregates from the cell could in theory prevent the transcel-
lular propagation of pathology. It is unknown how release of pathological seeds occurs in patients.
On the one hand, this process could be initiated upon cell death or local membrane breakdown, in
which case it seems unlikely that any biological target will emerge. On the other hand, if uncon-
ventional secretion of protein aggregates forms the basis of cell release, this could theoretically
be a viable therapeutic mechanism. Katsinelos et al. (117) reported that tau is released via an un-
conventional pathway that involves binding to the inner leaflet plasma membrane followed by
direct translocation mediated by interaction with sulfated proteoglycans. Although intriguing, it
remains to be understood how this mode of secretion may enable neuronal activity–dependent
release of tau as reported previously (91). Currently, we do not know enough about fundamental
release mechanisms to design specific therapeutic inhibitors.
Immunotherapy
Immunotherapy is a very near-term therapeutic option, as clinical trials are under way to evaluate
antibodies against tau and Aβ (NCT02494024, NCT02760602, NCT02353598, NCT02051608,
NCT01998841). Since the initial reports of the effectiveness of immunotherapy against the Aβ
protein (118), this approach has been explored to treat myriad neurodegenerative diseases. Initial
attempts to vaccinate mouse models against α-synuclein were successful (119). At that time the

tau
7
2
4
8
6
Figure 5
Therapeutic mechanisms. Given the knowledge about propagation of amyloid pathology, several
mechanisms could be exploited to block this process. (● 1 ) Reduction in expression of tau could be mediated
by antisense oligonucleotides or shRNAs. (● 2 ) Prevention of monomer conversion could be effected by
compounds that bind and stabilize native tau or by prevention of events that lead to its conformational
change. (● 3 ) Blocking aggregate formation could be achieved by small molecules to prevent self-assembly.
(●4 ) Blocking aggregate release might prevent transcellular propagation. (● 5 ) Small-molecule inhibitors of tau
binding and uptake could be used to prevent transcellular propagation. (● 6 ) Heparin mimetics to block
HSPG binding could prevent transcellular propagation. (● 7 ) Antibodies could be used to target extracellular
tau, promoting clearance or preventing uptake. (● 8 ) Blocking uptake mechanisms could prevent subsequent
intracellular seeding. (●9 ) Blocking aggregate amplification could block propagation. Abbreviations: HSPG,
heparan sulfate proteoglycan; shRNA, small hairpin RNA.
effectiveness of the vaccine was not understood in the context of spreading protein pathology.
Since then, multiple studies have reported vaccine therapies against α-synuclein and tau (100,
120–122). These have included active (121) and passive (100, 120) vaccination strategies. In our ex-
perience, peripheral administration typically has more modest effects on transgenic mouse models
(122), whereas central administration of antibodies has been more effective (100). The mechanisms
of immunotherapy are not well defined. Antibodies could promote the clearance of pathogenic
proteins from the interstitial space to the periphery (122). Alternatively, antibodies may directly
affect brain clearance. Intraneuronal mechanisms of action for therapeutic antibodies have even
been proposed (123, 124). We found that two antibodies directed against the amino terminus of
the tau protein promoted uptake into microglia-like cells in vitro, while another antibody that

targeted the repeat domain of tau directly inhibited uptake of protein aggregates into neurons but
did not affect their uptake into microglia-like cells (99). Overall, we observed multiple mecha-
nisms for different antitau antibodies, including differential effects based on aggregate size (99).
Immunotherapy could thus have multiple mechanisms of action that depend on particular epitopes
as well as the size of the targeted aggregate.
Cell Uptake
Several modes of tau uptake into neurons have been proposed, but data are limited for most mech-
anisms with the exception of macropinocytosis, also termed bulk or fluid phase endocytosis. Cell
uptake mediated by HSPGs has been clearly defined in vitro and is required for efficient seed-
ing. We have previously observed that HSPGs mediate the binding and uptake of both tau and
α-synuclein seeds (78). Targeting the HSPG pathway could involve small-molecule inhibitors of
HSPG synthesis or molecules such as F6 that bind tau and inhibit binding to the cell surface.
Functional HSPGs require enzyme-mediated maturation within the secretory pathway. This in-
cludes sulfation and glycosylation patterns that lend specificity to ligand binding. Recently, we
and others have reported that tau and α-synuclein require specific but distinct sulfation patterns
of heparan sulfate chains to bind glycans and enter cells (79, 125). However, this pathway has not
been studied extensively enough for tau or other propagating amyloids to know whether it holds
viable therapeutic enzyme candidates. Clathrin-mediated endocytosis has also been implicated for
α-synuclein (126), possibly suggesting an alternative therapeutic approach.
Intracellular Clearance
Increased clearance of protein aggregates has long been a therapeutic goal (127, 128). The knowl-
edge that small molecules can upregulate protein clearance has raised the hope of shifting the
cellular balance from aggregate accumulation to degradation (129–133). We do not yet know the
effects of chronic administration of compounds such as rapamycin, which upregulate autophagy,
to patients with neurodegenerative diseases. However, it may be possible to avoid chronic ad-
ministration through drug holidays, whereby patients receive brief courses of therapy designed
to promote clearance of protein aggregates, followed by gaps in treatment. In theory, such an ap-
proach could allow recovery of injured neurons and reset their protein aggregate load. We are
still learning about distinct pathways of protein aggregate degradation, and as these data emerge,
along with the development of compounds that activate them specifically, it may be possible to
better direct aggregate clearance.
Antisense oligonucleotides (ASOs) and RNA interference also represent important approaches
to reduce intracellular toxic protein expression. Clinical trials of intrathecally administered ASOs
are now under way to evaluate the efficacy of SOD1 knockdown in familial forms of ALS
(134) (NCT01041222, NCT02623699). An ASO targeting tau in mouse models of tauopathy
has also reduced pathology and seeding activity (135), and another is now in early clinical trials
(NCT03186989). The use of ASOs could revolutionize therapy of myriad neurodegenerative dis-
eases linked to intracellular amyloid accumulation, as a common mode of therapy (ASO) could be
used against many genetic targets.
INHIBITION OF INTRACELLULAR SEEDING

Upon cell entry, pathological aggregates act as templates for conversion of native protein. This
has sparked many attempts to develop therapies based on preventing fibril growth (101). So far

none have been approved for clinical use. With more potent compounds it may be possible to
achieve greater efficacy, although most still must function stoichiometrically. In theory, approaches
to modulate aggregation directly could employ multiple mechanisms, including stabilization of
monomer; inhibition of fibril growth; acceleration of aggregation to form larger, less toxic assem-
blies; or reduction of fibril fragmentation (136). Indeed, some groups are exploring small-molecule
and antibody approaches to inhibit primary and secondary nucleation steps of amyloid aggregation
(3, 137, 138).
New approaches may also come from better elucidation of the mechanisms of intracellular ag-
gregation. In contrast to seeded polymerization of amyloids in vitro, within a cell the complexity of
the environment and dedicated degradation systems would appear to mitigate against spontaneous
fibril assembly following uptake of a limited number of seeds, suggesting a role for a replication
machinery. For example, the heat shock protein 70/40 (Hsp70/40) chaperone system has been
proposed to fragment fibrils and thus enable subsequent growth (139). Other cellular chaperones,
however, inhibit microscopic steps in the process of Aβ aggregation, such as secondary nucleation,
and could thus be important therapeutic targets (140). We do not yet know which of the many
subtypes of chaperones could be therapeutic targets for preventing amyloid formation of other
proteins. Functional genomics will play a key role in identifying these and other regulators of
aggregation.
THE ROLE OF PRION STRAINS AND NETWORKS

IN NEURODEGENERATIVE DISEASES
Prion strains are pathological assemblies of unique conformation that faithfully replicate in living
systems and produce predictable patterns of neuropathology. We discriminate true strains from
amyloid conformers that can be produced in vitro because only certain conformations replicate
stably in living systems, account for specific biological activity, and thus have clinical significance.
Tau strains clearly have unique effects on cells and are linked to specific neuropathological syn-
dromes (15). Work from our laboratory also indicates that strain conformation alone is sufficient
to account for enormous variation in neuropathology (113). Understanding strains in neurode-
generative diseases will help us account for neuronal vulnerability, rates of progression, responses
to future therapies, and more accurate diagnoses.
YEAST PRION STRAINS

Yeast prions were identified in the 1960s as mediators of epigenetic inheritance (141). Their
study has greatly informed our knowledge of mammalian PrP prions. It was not clear initially
how yeast prions served the organism, but they now appear to play a fundamental role in
regulating metabolism (142–145). Using computational and functional approaches, investigators
have now identified multiple proteins within the yeast proteome that are putative prions (142).
The best-studied yeast prion, Sup35, normally functions as a translation termination factor
in the setting of nonsense mutations (146). Under certain circumstances, Sup35 transitions
to an amyloid structure that recruits and depletes free monomer from the cytosol, permitting
read-through of genetic errors. This is presumably beneficial during organismal stress. In this
sense Sup35 functions as a binary switch. However, multiple strains of Sup35 have been identified
that aggregate to variable degrees. How much they leave free monomer to function in the cell
is inversely proportional to their strength as a prion. Sup35 and fusion proteins created from
its prion domain have enabled the development of synthetic phenotypes in yeast cells to dissect
regulatory factors. Principal among these is the chaperone Hsp104, which plays an essential

role in prion replication (147) and helps break amyloid fibrils (148). At levels optimal for prion
replication, Hsp104 enables efficient fibril severing to create new amyloid seeds. These seeds
are efficiently inherited by dividing yeast cells, maintaining mother-to-daughter transmission of
the prion state. Somewhat paradoxically, either under- or overexpression of Hsp104 blocks prion
maintenance. Low levels lead to diminished fibril severing with inefficient inheritance, whereas
high levels lead to fibril degradation that exceeds growth. This has led to a model of yeast prion
strain maintenance that takes into account fibril growth rate versus stability (149) and may be
applicable to mammalian prions (150). There is no known mammalian homolog of Hsp104, but
other factors, such as Hsp70 and Hsp40, may play similar roles (139). Given the evolutionary
conservation of yeast signaling mechanisms in metazoans, it seems likely that functional prion
strains will have widespread importance in cell and organismal biology (16).
Aβ, TAU, AND α-SYNUCLEIN STRAINS AND STRAIN-LIKE BEHAVIOR

Walker and colleagues (18, 31) first described the effects of distinct amyloid conformations on
neuropathology following inoculation in mice. These studies were highly reminiscent of prior
studies of PrP strains that had spanned decades (151, 152). The investigators found that both the
source of Aβ fibrils and the strains of mice into which they were injected dictated unique patterns
of neuropathology (18). Because Aβ accumulates predominantly in the extracellular space, there
was no study of transcellular propagation. However, these investigators were the first to observe
unique pathologies arising from amyloid proteins other than PrP prion strains. Subsequent studies
of Aβ propagation from animal to animal have reported consistent strain-like protein behavior
(153, 154). Although Aβ conformers have not been demonstrated to propagate indefinitely in
vivo, it is clear that distinct amyloid structures produce unique patterns of neuropathology upon
inoculation, which is consistent with Aβ strains. This idea is supported by recent reports that
patients infected with pathogenic prions also developed unique Aβ deposition patterns, consistent
with the idea that Aβ transmits pathology in humans under the right circumstances (48) and that
unique Aβ conformers are associated with AD subtypes (155).
Multiple reports now additionally describe distinct tau and α-synuclein aggregate conform-
ers in experimental systems (156–158), and reports indicate that different strains may exist in
patients (15, 30, 159, 160). The clearest evidence to date is the elucidation of the cryo–electron
microscopy structure of ultrastructurally distinct tau fibrils from a tauopathy patient (160). In
2013, Lee and colleagues (157) created uniquely structured synuclein fibrils in vitro that trig-
gered distinct patterns of tau pathology in cultured neurons and upon inoculation into mice. In
this study, α-synuclein was iteratively fibrillized with successive seeding reactions. The investi-
gators noticed that the conformation of the α-synuclein fibril preparations changed over succes-
sive reactions, and fibrils created after 10 generations of seeding had biological effects different
from those of the initial preparation (157). Although not meeting the criteria of strains as defined
above, this investigation clearly showed variance in the biological activities of amyloids of distinct
structure.
In other studies of α-synuclein, Melki and colleagues (156) created two distinct types of fibril
conformations based on different fibrillization conditions. When inoculated into cells, the two
types replicated the biochemical characteristics of the original fibrils (156). A subsequent publi-
cation described inoculation of distinct synuclein fibril conformers into animals, which produced
unique patterns of neural pathology (161). Prusiner and colleagues (159, 162) have also studied α-
synuclein and have observed differential seeding from human synucleinopathy brains into cultured
cell and animal models. In both cases, they found that PD brain had no detectable seeding activ-
ity, whereas multiple system atrophy (MSA) brain exhibited seeding activity in cells and animals.

Moreover, they recently reported that familial PD mutations abolish the ability of MSA prions
to replicate (163). They interpreted these data to mean that distinct α-synuclein prion strains ex-
ist in the two diseases. Taken together, these studies are consistent with the idea of α-synuclein
prion strains, but they have not conclusively demonstrated their existence through isolation and
characterization of synuclein prion assemblies of distinct structure to fulfill Koch’s postulates for
transmission.
Our laboratory (15) studied tau prion strains using a reductionist system based on expression of
the tau repeat domain containing two disease-associated mutations (P301L, V337M). This protein
was fused to YFP to enable visualization of intracellular inclusions. We exposed cells expressing
this protein to recombinant fibrils and measured the retention of inclusions over time by visual
inspection. We observed a rapid reduction of cells with inclusions, but even after 50 days in culture
the cells that had been exposed to an inoculum maintained approximately 1–2% of the population
with inclusions (15). This finding was similar to a prior report of maintenance of polyglutamine in-
clusions (35). We hypothesized that the maintenance of inclusions came from mother-to-daughter
aggregate transmission during mitosis. We tested this hypothesis by deriving multiple monoclonal
cell lines that stably propagated aggregates, and observed two dominant intracellular inclusion
morphologies associated with distinct patterns of seeding activity, detergent solubility, and pro-
tease sensitivity. Remarkably, aggregation patterns were transferable by extraction of tau from one
cell line and seeding into a naïve line. Thus, in a cell culture system, tau met the criteria of a prion:
It produced aggregates of distinct morphology and biological effects and had a structure that was
maintained indefinitely through mother-to-daughter transmission or through successive cellular
seeding reactions. We extended these studies to mice and observed that lysates from these distinct
cell lines produced unique patterns of neuropathology. This work involved creation of tau strains
from recombinant protein and faithful propagation through living systems, with the induction of
conformation-dependent pathology. Thus, tau satisfies the criteria of a bona fide prion in most
respects, save for spontaneous transmission between individuals.
Finally, we evaluated human tauopathies to test for distinct strain composition. We isolated
several hundred monoclonal lines that stably propagated human tau prions. A blind analysis of
these lines indicated that different syndromes had clearly distinct strain compositions, often with
multiple strains in a single individual (15). Altogether, we observed a surprising fidelity of tau strain
propagation in cells and animals and, further, that human tauopathies appear to be comprised of
clouds of prion strains, as has been previously described for PrP prions (151).
Although individuals with tauopathy can manifest multiple strains, the basis of variation in
clinical and neuropathological findings observed in tauopathy patients has remained unclear. We
addressed this problem by isolating and characterizing 18 individual tau strains passaged in cul-
tured cell lines, each with a unique seeding, toxicity, and proteolytic digestion pattern (113). We
inoculated extracts from each line individually into a single mouse model that expresses full-length
tau containing a disease-associated mutation (P301S). Each strain produced a completely distinct
neuropathological syndrome. This included unique patterns of intracellular pathology, differential
rates of propagation of pathology throughout the brain, and distinct patterns of regional pathol-
ogy. We directly tested for regional vulnerability by inoculating a subset of strains into multiple
brain regions and observed striking differences. Some strains were promiscuous and would in-
duce pathology in any region to which they were introduced. Other strains were highly restricted
in their tropism to specific brain regions. Taken together with the preceding studies, this work
indicates that strain identity alone, independent of genetic background, is sufficient to account
for tremendous phenotypic diversity (113) (Figure 6). Clearly, genetic and environmental fac-
tors could also influence presentation, but these data indicate that study of isolated strains might

Native protein
conformation
Alternative seed
formation
Monomer addition
and amplification
Distinct cell
aggregate patterns
Strain-dependent
neuronal vulnerability
Extract seeds
Reintroduce
to cells
Strain-dependent
Recreate same regional atrophy
cell lines
Indefinite mother-to-daughter
propagation of aggregation
Figure 6
Strains and patterns of neuronal degeneration. Strains for amyloid proteins such as tau have been studied in
depth. Distinct strains of tau will replicate a defined structure when introduced into a previously unexposed
cell line. In many cases, this structure is maintained indefinitely in cultured cells by mother-to-daughter
transmission. If tau aggregates are extracted from a cell line and reintroduced to a naïve cell line, they will
recreate the same structure. When inoculated into animals, cell lysates containing the tau strains produce
defined pathology that can be transmitted from animal to animal, and even back into cultured cells. Each
distinct strain produces a unique pattern of cellular pathology, rate of progression, and involvement of brain
networks. Strains thus can account for enormous variation in the presentation of neurodegenerative diseases
due to amyloid accumulation.
provide molecular tools to understand the principles of specific neuronal vulnerability and pro-
gression rates in neurodegenerative diseases.
NETWORKS IN PROPAGATION
The association of neural networks with neurodegeneration syndromes has been recognized for
some time. In experimental models of prion disease, inoculation into the eye leads to progressive
degeneration that follows the optic tracts, which is consistent with a transneuronal propagation
mechanism (86). In noninfectious human disorders such as ALS, there is combined degeneration
of the upper and lower motor neurons that comprise a network (164). ALS progression can involve
local spread within either the cortex or the spinal cord (165). Braak & Braak (166) carried out
meticulous studies of hundreds of patients with AD to create a staging system to characterize
AD progression. Their system groups patients into six distinct stages that are consistent with a
progressive pattern of tau deposition in regions known to share synaptic connectivity, such as the
entorhinal cortex, transentorhinal cortex, hippocampus, and neocortex (166–168).
Human pathological studies are by necessity cross-sectional, but imaging of living patients
offers new insights into progression within individuals (169). Human brain imaging has advanced
considerably in the last decade, especially in the realm of positron emission tomography (PET)

and functional connectivity magnetic resonance imaging (fcMRI). fcMRI uses activity-dependent
variations in blood flow patterns to infer connectivity among groups of neurons, although the
timescale in which blood flow varies (seconds) is much slower than neuronal signaling (millisec-
onds). Nonetheless, fcMRI has largely recapitulated known local networks and has suggested
the existence of many distributed networks in the brain (170–172). A series of important studies
have compared networks defined by fcMRI to patterns of progressive atrophy in patients with
neurodegenerative diseases. Networks were first defined in normal patients. Progressive atrophy
was then determined on the basis of sequential structural MRI scans that analyzed brain volume.
When the atrophy patterns were superimposed on known networks, the investigators found a
significant correlation, with distinct syndromes tracking to particular networks (173). Subsequent
work tested the likelihood of neurodegeneration that follows atrophy in one region of brain
against network predictions (174, 175). Putatively connected regions tended to degenerate
together, independent of geographical distance between them. Coupled with the original patho-
logical studies of Braak & Braak, these imaging studies have strongly supported the idea that
neuronal connections underlie patterns of neurodegeneration.
THE PRION MODEL ADVANCES THERAPEUTIC

AND DIAGNOSTIC OPPORTUNITIES
Fortunately, we have not yet encountered cases in which direct exposure of tau, α-synuclein, or
other proteins that account for common, sporadic neurodegenerative diseases clearly causes a neu-
rodegeneration syndrome in the manner of infectious prions. Nonetheless, a recent study docu-
mented highly unusual patterns of Aβ pathology in cases of transmitted Creutzfeldt-Jakob disease
(CJD) that could indicate infection by this protein (because patients died of CJD, it is unknown
whether they would ever have become symptomatic from the putative Aβ exposure) (48, 176).
Newer reports independently confirmed an increase in coincident Aβ pathology with iatrogenic
CJD (177) and implicated transplanted dura mater as a source of infectious Aβ (178). We still
do not know whether transcellular propagation underlies the progression of common neurode-
generative diseases. Yet the transcellular propagation model has many important implications. As
cellular mechanisms of propagation are defined, specific interventions designed to interrupt key
steps such as cell entry or intracellular seeding will provide critical tests of this hypothesis in ani-
mal models and, if successful, in patients. The use of therapeutic antibodies against tau, which are
currently in early clinical trials, provides an excellent example.
This concept of prion strains has important implications for categorization of amyloid dis-
eases. Strains can explain many aspects of phenotypic diversity and network involvement. Small
molecules or antibodies designed to bind tau or inhibit specific aspects of replication of tau or
α-synuclein may have strain specificity. Thus, it may be crucial to define the composition of strains
within a given patient to predict efficacy of a certain drug. At a minimum, an ability to classify
strain composition in patients may provide more accurate correlation of clinical and neuropatho-
logical phenotypes, which can be divergent. The existence of strains makes it risky to treat all
proteinopathies equivalently, and we must recognize the potential diversity of pathogenic targets.
From the standpoint of diagnosis, it is then critical to discriminate distinct amyloid structures in
patients. This could be done through biochemical approaches to define seed structure or through
novel PET imaging that exploits conformational differences in protein amyloids. In this light,
it is remarkable that multiple tau imaging agents have been developed and used in patients, but
there is little uniformity across syndromes with regard to PET ligand binding (179). Clearly, more
work is required to understand why one agent binds tau in some syndromes but not in others. We
anticipate that the prion model will ultimately help lead the field to more effective therapy and
diagnosis for neurodegenerative diseases.
SUMMARY POINTS
1. Multiple proteins now exhibit many characteristics of prions, except for spontaneous
transmission of pathology between individuals.
2. Multiple mechanisms have been proposed for cell uptake and cell release.
3. Although the best evidence suggests macropinocytosis as a primary mechanism of up-
take, no studies in vivo have directly tested this or any uptake or release mechanism.
4. The ability of antibody therapies to reduce pathology in animal models strongly suggests
that free extracellular aggregates play some role in pathogenesis.
FUTURE ISSUES
1. Clinical trials are planned or underway for multiple vaccine and genetic therapies.
2. Direct targeting of aggregate formation or growth is a promising therapeutic strategy.
3. Better knowledge of cell biology and biochemistry of aggregate formation and growth
may yield new enzymatic targets.
4. Experimental data suggest that distinct amyloid structures target specific cells and
networks, providing a framework for understanding diversity of neurodegeneration
syndromes.
5. Identification of strains in living patients may improve diagnostic accuracy within syn-
dromes. This might bridge the gaps between clinical and neuropathological diagnoses.
6. Better classification of protein amyloid diseases according to strain composition may
lead to improved decision-making for therapies.
DISCLOSURE STATEMENT
M.I.D. is the coinventor of a therapeutic antibody that was licensed from Washington University
in St. Louis by C2N Diagnostics and is in clinical trials.
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BI88_FrontMatter ARI 22 May 2019 14:41
Annual Review of
Biochemistry
Contents Volume 88, 2019
Moving Through Barriers in Science and Life

Judith P. Klinman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
Biophysical Techniques in Structural Biology
Christopher M. Dobson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p25
X-Ray Free-Electron Lasers for the Structure and Dynamics of

Macromolecules
Henry N. Chapman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p35
Bacteriorhodopsin: Structural Insights Revealed Using X-Ray Lasers
and Synchrotron Radiation
Cecelia Wickstrand, Przemyslaw Nogly, Eriko Nango, So Iwata, Jörg Standfuss,
and Richard Neutze p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p59
Membrane Protein–Lipid Interactions Probed Using Mass
Spectrometry
Jani Reddy Bolla, Mark T. Agasid, Shahid Mehmood, and Carol V. Robinson p p p p p p p p p p p85
Integrative Structure Modeling: Overview and Assessment
Merav Braitbard, Dina Schneidman-Duhovny, and Nir Kalisman p p p p p p p p p p p p p p p p p p p p p 113
Eukaryotic Base Excision Repair: New Approaches Shine Light on
Mechanism
William A. Beard, Julie K. Horton, Rajendra Prasad, and Samuel H. Wilson p p p p p p p p p 137
Redox Chemistry in the Genome: Emergence of the [4Fe4S] Cofactor
in Repair and Replication
Jacqueline K. Barton, Rebekah M.B. Silva, and Elizabeth O’Brien p p p p p p p p p p p p p p p p p p p p p p 163
Evaluating and Enhancing Target Specificity of Gene-Editing
Nucleases and Deaminases
Daesik Kim, Kevin Luk, Scot A. Wolfe, and Jin-Soo Kim p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 191
The BRCA Tumor Suppressor Network in Chromosome Damage
Repair by Homologous Recombination
Weixing Zhao, Claudia Wiese, Youngho Kwon, Robert Hromas,
and Patrick Sung p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 221
Cancer Treatment in the Genomic Era
Gary J. Doherty, Michele Petruzzelli, Emma Beddowes, Saif S. Ahmad,
Carlos Caldas, and Richard J. Gilbertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 247
v
Eukaryotic Ribosome Assembly

Jochen Baßler and Ed Hurt p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 281
The Organizing Principles of Eukaryotic Ribosome Recruitment
Jerry Pelletier and Nahum Sonenberg p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 307
Mechanisms of Cotranslational Maturation of Newly Synthesized
Proteins
Günter Kramer, Ayala Shiber, and Bernd Bukau p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 337
Lysine-Targeted Inhibitors and Chemoproteomic Probes
Adolfo Cuesta and Jack Taunton p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 365
Horizontal Cell Biology: Monitoring Global Changes of Protein
Interaction States with the Proteome-Wide Cellular Thermal Shift

Assay (CETSA)
Lingyun Dai, Nayana Prabhu, Liang Ying Yu, Smaranda Bacanu,

Anderson Daniel Ramos, and Pär Nordlund p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 383
Soluble Methane Monooxygenase
Rahul Banerjee, Jason C. Jones, and John D. Lipscomb p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 409
Glycoengineering of Antibodies for Modulating Functions
Lai-Xi Wang, Xin Tong, Chao Li, John P. Giddens, and Tiezheng Li p p p p p p p p p p p p p p p p p 433
Lysosomal Glycosphingolipid Storage Diseases
Bernadette Breiden and Konrad Sandhoff p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 461
Exosomes
D. Michiel Pegtel and Stephen J. Gould p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 487
Structure and Mechanisms of F-Type ATP Synthases
Werner Kühlbrandt p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 515
ECF-Type ATP-Binding Cassette Transporters
S. Rempel, W.K. Stanek, and D.J. Slotboom p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 551
The Hippo Pathway: Biology and Pathophysiology
Shenghong Ma, Zhipeng Meng, Rui Chen, and Kun-Liang Guan p p p p p p p p p p p p p p p p p p p p p p 577
Small-Molecule-Based Fluorescent Sensors for Selective Detection of
Reactive Oxygen Species in Biological Systems
Xiaoyu Bai, Kenneth King-Hei Ng, Jun Jacob Hu, Sen Ye, and Dan Yang p p p p p p p p p p p p 605
Single-Molecule Kinetics in Living Cells
Johan Elf and Irmeli Barkefors p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 635
Molecular Mechanism of Cytokinesis
Thomas D. Pollard and Ben O’Shaughnessy p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 661
Mechanism and Regulation of Centriole and Cilium Biogenesis
David K. Breslow and Andrew J. Holland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 691
vi Contents
The Structure of the Nuclear Pore Complex (An Update)

Daniel H. Lin and André Hoelz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 725
Propagation of Protein Aggregation in Neurodegenerative Diseases
Jaime Vaquer-Alicea and Marc I. Diamond p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 785
Botulinum and Tetanus Neurotoxins
Min Dong, Geoffrey Masuyer, and Pål Stenmark p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 811
Errata
An online log of corrections to Annual Review of Biochemistry articles may be found at

http://www.annualreviews.org/errata/biochem
Contents vii

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BI88CH29_Diamond ARjats.

cls May 22, 2019 14:27

Annual Review of Biochemistry

Jaime Vaquer-Alicea and Marc I. Diamond

Center for Alzheimer’s and Neurodegenerative Diseases, University of Texas Southwestern

Annu. Rev. Biochem. 2019. 88:785–810 Keywords

The Annual Review of Biochemistry is online at Abstract

INHIBITION OF INTRACELLULAR SEEDING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 796

THE ROLE OF PRION STRAINS AND NETWORKS

786 Vaquer-Alicea • Diamond

aggregation-prone proteins under control (11). Perhaps unsurprisingly, genome-wide association

progress on the basis of endogenous mechanisms. living systems and

www.annualreviews.org • Protein Aggregation in Neurodegeneration 787

CELL BIOLOGY OF AGGREGATE PROPAGATION

role outside of classical prion diseases (18).

AGGREGATE PROPAGATION INTO CELLS

788 Vaquer-Alicea • Diamond

Table 1 Evidence for prion mechanisms in neurodegenerative diseases

encephalopathy Rodent 58, 59

UPTAKE AND RELEASE MECHANISMS

www.annualreviews.org • Protein Aggregation in Neurodegeneration 789

HSPG-mediated Exosome Receptor-mediated

790 Vaquer-Alicea • Diamond

www.annualreviews.org • Protein Aggregation in Neurodegeneration 791

MOUSE MODELS OF SPREADING PATHOLOGY

792 Vaquer-Alicea • Diamond

Amyloid fibrils Disrupted fibrils Native monomer Amplified fibrils

b Seeding assay Detection via FRET

www.annualreviews.org • Protein Aggregation in Neurodegeneration 793

794 Vaquer-Alicea • Diamond

by antisense oligonucleotides or shRNAs. (● 2 ) Prevention of monomer conversion could be effected by

heparan sulfate proteoglycan; shRNA, small hairpin RNA.

www.annualreviews.org • Protein Aggregation in Neurodegeneration 795

INHIBITION OF INTRACELLULAR SEEDING

796 Vaquer-Alicea • Diamond

THE ROLE OF PRION STRAINS AND NETWORKS

YEAST PRION STRAINS

www.annualreviews.org • Protein Aggregation in Neurodegeneration 797

Aβ, TAU, AND α-SYNUCLEIN STRAINS AND STRAIN-LIKE BEHAVIOR

798 Vaquer-Alicea • Diamond

www.annualreviews.org • Protein Aggregation in Neurodegeneration 799

800 Vaquer-Alicea • Diamond

THE PRION MODEL ADVANCES THERAPEUTIC

802 Vaquer-Alicea • Diamond

www.annualreviews.org • Protein Aggregation in Neurodegeneration 803

rodegeneration. Nat. Rev. Neurosci. 17(4):251–60

804 Vaquer-Alicea • Diamond

mutant huntingtin protein. Acta Neuropathol. 132(4):577–92

www.annualreviews.org • Protein Aggregation in Neurodegeneration 805

806 Vaquer-Alicea • Diamond

www.annualreviews.org • Protein Aggregation in Neurodegeneration 807

808 Vaquer-Alicea • Diamond

passaged scrapie agents, 22A and ME7. Genet. Res. 13(2):213–25

www.annualreviews.org • Protein Aggregation in Neurodegeneration 809

810 Vaquer-Alicea • Diamond

Moving Through Barriers in Science and Life

X-Ray Free-Electron Lasers for the Structure and Dynamics of

Eukaryotic Ribosome Assembly

Interaction States with the Proteome-Wide Cellular Thermal Shift