Christopher A Ross & Michelle A Poirier

Protein aggregation and neurodegenerative

disease
Nature Medicine 10, S10–S17 (2004)

The Division of Neurobiology, Department of Psychiatry, and Departments of Neurology and

Neuroscience, Johns Hopkins University School of Medicine, Ross Research Building, Room 618, 720
Rutland Avenue, Baltimore, Maryland 21205, USA.
Why?
• Neurodegenerative diseases

How?
• Protein aggregation
Neurodegenerative diseases
• Alzheimer's disease (AD),
• Parkinson's disease (PD),
• Huntington's disease (HD),
• amyotrophic lateral sclerosis (ALS),
• prion diseases

Neurodegenerative diseases are increasingly being realized to have common

cellular and molecular mechanisms including protein aggregation and
inclusion body formation.

All of the diseases discussed here involve selective neuronal vulnerability

with degeneration in specific brain regions, and deposits of abnormal
proteins in neurons and other cells or extracellularly.
Alzheimer's disease. AD is a late-onset dementing illness, with
progressive loss of memory, task performance, speech, and
recognition of people and objects. There is degeneration of neurons
(particularly in the basal forebrain and hippocampus), but at least as
important for pathogenesis may be synaptic pathology and altered
neuronal connections.
The disease is age-sensitive, afflicting about 3% of people between
65 to 74 years of age, and rising to an incidence of nearly 50% in
those over age 85.
Alzheimer’s disease involves a defect in a protein normally found in
the membrane of neurons. The protein, called beta-amyloid precursor
protein (BAPP), is a transmembrane protein.
A slightly truncated, soluble form is also found secreted from cells and is
found in extracellular fluid (such as cerebrospinal fluid and blood).
The normal function of these BAPP proteins is not yet clear.

An endoprotease cleaves a small 40-42 amino acid fragment from this

protein, forming the amyloid beta (Ab) protein. It is this protein or a
mutant form of it which aggregates to form beta-sheet containing fibrils in
Alzheimer’s disease.

In a healthy brain, these protein fragments would be broken down and

eliminated. In Alzheimer's disease, the fragments accumulate to form
hard, insoluble plaques.
AMYLOID PLAQUES
One of the hallmarks of Alzheimer's disease is the accumulation of
amyloid plaques between nerve cells (neurons) in the brain. These
deposited plaques are extracellular, and have been shown to cause
neuronal damage. The are found in areas of the brain required for memory
and cognition.
AD involves two major kinds of protein aggregates. Extracellular
aggregates known as neuritic plaques have as their major constituent the
A-peptide.
There are also intracellular aggregates of the microtubule-associated
protein tau, called neurofibrillary tangles.
Tau is an axonal protein involved in microtubule assembly and stabilisation
of the microtubules. When tau detaches from the microtubules, they can
clump together in neurofibrillary tangles.
The mechanism by which tau is
damaged seems to involve the addition
of excess phosphate groups to the
protein, technically called
hyperphosphorylation. Researchers
have shown that this
hyperphosphorylated tau with its excess
number of attached phosphates detaches
from microtubules.
A more recent theory for the pathogenesis of Alzheimer’s disease is its
possible cause by inflammatory processes associated with ageing, and not by
amyloid plaque deposits as is most widely believed. Although ß amyloid is still
very much involved, the cause of the disease is thought to be the formation of
toxic proteins, as opposed to a build up of amyloid plaques and neurofibrillary
tangles within brain nerve cells.

One suggestion was the possibility of organophosphorous (OP) molecules being

the fundamental cause as there is evidence that OP molecules attach to Tau
proteins and cause them to tangle.

Researchers have found oxidative damage to brain cells to be a potential

indicator of Alzheimer disease activity. Oxidative damage to cells is a result of
free radical activity, free radicals being released during normal cell processes. It is
thought that this leads to oxidative stress, which causes tissue inflammation - a
suspected cause of Alzheimer’s disease.
Parkinson’s disease
Parkinson’s disease is a brain disorder. It occurs
when certain nerve cells (neurons) in a part of the brain
called the substantia nigra die or become impaired.
Normally, these cells produce a vital chemical known
as dopamine. Dopamine allows smooth, coordinated
function of the body's muscles and movement. When
approximately 80% of the dopamine-producing cells
are damaged, the symptoms of Parkinson disease
appear.
The key signs of Parkinson’s disease are:
•Tremor (shaking)
•Slowness of movement
•Rigidity (stiffness)
•Difficulty with balance
The pathological hallmark of adult-onset PD is the Lewy body, an
inclusion body found in the cytoplasm of neurons, often near the nucleus.
Lewy bodies are circular and have a dense protein core surrounded by a
peripheral halo.

Their presence in the brain disrupts the brain's normal functioning,

interrupting the action of the important chemical messenger's, including
acetylcholine and dopamine.

There are also aggregates in neurites,

which are referred to as Lewy neurites.

A major constituent of Lewy bodies

is aggregated -synuclein protein.
 Lewy body - synuclein immunostain Lewy body substantia nigra

Lewy neurites -ubiquitin immunostain amyloid plaque

Figure 1. Characteristic
neurodegenerative disease
neuropathological lesions involve
deposition of abnormal proteins, which
can be intranuclear, cytoplasmic or
extracellular.
All are labeled with antibodies (except d).
(a) HD, intranuclear inclusion labeled for ubiquitin
(cerebral cortex)
(b) HD, intranuclear inclusion labeled for
huntingtin (cerebral cortex).
(c) AD, neuritic plaque.
(d) AD, neuritic plaque, silver stained.
(e) PD, Lewy bodies labeled for alpha-synuclein
(fine granular brown label in this and the next panel
represent neuromelanin) (substantial nigra).
(f) PD, Lewy body labeled for phosphorylated
alpha-synuclein (substantia nigra).
(g) ALS, cytoplasmic skein of neurofilaments
labeled with ubiquitin (medulla oblongata). (h)
ALS, cytoplasmic skein of neurofilaments labeled
with neurofilament (medulla oblongata).
Prion Diseases
Neurodegenerative diseases caused by prions can be sporadic or can be
acquired either by environmental transmission or via genetic mutations.
The diseases are characterised by loss of motor control, dementia, paralysis
wasting and eventually death, typically following pneumonia.

Environmental pathways include eating prion particles derived from infected

brain tissue or surgical implantation via infected instruments.
Prion disease can also be caused by point mutations in the prion gene, leading
to alterations of the prion protein.

Pathology can include amyloid plaques that

appear similar to those of AD.
In all cases, disease is caused by abnormally
folded prion proteins. Prion aggregation can
take place both extracellularly and intracellularly.

Structure of a portion of the bovine prion protein,

a molecule associated with mad cow disease.
PrPC
The normal protein
•is called PrPC (for cellular)
•is a glycoprotein normally found at the cell surface inserted in the plasma membrane
•has its secondary structure dominated by alpha helices (probably 3 of them)
•is easily soluble
•is easily digested by proteases
•is encoded by a gene designated (in humans) PRNP located on our chromosome 20.
PrPSc
The abnormal, disease-producing protein
•is called PrPSc (for scrapie)
•has the same amino acid sequence as the normal protein;
•its secondary structure is dominated by beta-conformation
•is insoluble in all but the strongest solvents
•is highly resistant to digestion by proteases
•When PrPSc comes in contact with PrPC, it converts the PrPC into more of itself (even in the
test tube).
•These molecules bind to each other forming aggregates.
•It is not yet clear if these aggregates are themselves the cause of the cell damage or are
simply a side effect of the underlying disease process.
 Prions are a "protein-only" phenomenon
A few molecules of a PrPSc form of the Sup35 protein, when introduced into
yeast cells, convert the yeast cell's own Sup35 protein into prion aggregates. The
resulting "disease" phenotype is then passed on to the cell's daughters.
The introduced protein was synthesized in bacteria making it unlikely that it
could be contaminated by any gene-containing infectious agent of yeast.

PrPsc acts as an anti-chaperone, catalyzing the transformation

of PrPc into PrPsc.
 Mad Cow Disease
KURU - cannibalism
Rudolph Virchow, in 1854, introduced and popularized the term amyloid
to denote a macroscopic tissue abnormality that exhibited a positive iodine
staining reaction.

Amyloid deposits at low magnification;

(a) attributed to Frerichs, 1862
(a) Amyloid fibrils from human amyloidotic spleen isolated by homogenization
in physiological saline followed by sucrose gradient centrifugation. Shadowed
with platinum–palladium, original magnification 370 000 (after Shirahama and
Cohen, 1967).
 Commonalities of amyloid structure
Amyloid fibrils are filamentous structures with a width of 10 nm and a
length of 0.1–10 m.
On the molecular level a conformational
change is induced from primarily -helix and
random coil (in the ‘healthy’ protein) into ß-
strand structure (in the wrongly folded
protein). These β-strands can interact with each
other, forming a ß-sheet structure.
In this structural motif, ribbonlike b-sheets are
formed by b-strands running nearly
perpendicular to the long axis of the fibril and
hydrogen bonds that run nearly parallel to the
long axis.
In this fashion the proteins stick together to form
a dimer, trimer, tetramer, and so on, and
eventually form large aggregates, named A representation of a fibril.
fibrils. The colored arrows indicate
the ß-strands.
 Figure 2. b-sheet, b -turn models for
expanded polyglutamine and Ab
amyloid suggest commonalities in
amyloid structure in different
neurodegenerative diseases.

(a) Sketch of expanded polyglutamine, with -

turns constrained by proline-glycine insertions
every nine glutamines. This is proposed to be
similar to the structure of expanded pure
polyglutamine, in which side chains may
participate in the hydrogen bonding. Light blue,
carbon; dark blue, nitrogen; red, oxygen.
(b) Model for an Ab (1–40) fibril with -sheets
formed by residues 12–24 and 30–40. Residue side
chains: green, hydrophobic; magenta, polar; blue,
positive; red, negative.

-sheet plus -turn structure may be a common

form of neurodegenerative disase-related amyloid
 Three-dimensional reconstructions and
contoured density sections of the 610 Å (a
and c) and the 580 Å form (b and d).

The fibrils are shown as rendered surfaces in (a)

and (b) (surface is 3 above the mean density),
and as contoured density cross-sections in (c) and
(d). The two independent reconstructions are very
similar and both show four protofilaments
winding around a hollow core, with protruding
edge regions. The 27-Å subunit repeat is most
pronounced on the edge structures. The subunit
repeat was clearly observable in axial 1D
projections of the class averages after square root
amplitude filtering (Figure 2c and g). The repeat
was determined as 27 Å in both cases, and the
exact value used was chosen to give an integral
number of subunits in the 580- and 610-Å repeats
(21 and 22 subunits respectively). (a) and (b) were
produced using AVS (Advanced Visualization
System).

José L. Jiménez, J.Iñaki Guijarro, Elena Orlova, Jesús Zurdo, Christopher M. Dobson, Margaret Sunde and Helen R. Saibil
Cryo-electron microscopy structure of an SH3 amyloid fibril and model of the molecular packing. The EMBO Journal (1999) 18,
815–821, doi: 10.1093/emboj/18.4.815
 Modelling the polypeptide
fold in the fibril density.
(a) Overview of the fibril structure,
showing the outer surface as green wire
mesh and the protofilaments as solid
blue surfaces, contoured at a higher
density level (4 above the mean
density). The ribbon-like protofilaments
form the skeleton of the fibril structure.
A model for the molecular packing is
shown in (b–d), with the EM map as a
transparent rendered surface.
(b) Side view of a single protofilament,
(c) cross-section of the fibril and
(d) slightly tilted side view of the
fibril.

José L. Jiménez, J.Iñaki Guijarro, Elena Orlova, Jesús Zurdo, Christopher M. Dobson, Margaret Sunde and Helen R. Saibil
Cryo-electron microscopy structure of an SH3 amyloid fibril and model of the molecular packing. The EMBO Journal (1999) 18,
815–821, doi: 10.1093/emboj/18.4.815
Typical morphology of amyloid fibrils.

The twisted structure is commonly observed in amyloid fibrils, and represents

several (usually two to five) protofilaments interacting to form a rope-like
structure. All fibrils have similar morphologies, and mature fibrils usually have a
twisted, rope-like structure, reflecting a filamentous substructure.
General mechanism of aggregation of disease-associated proteins.
Misfolded proteins form intermediates, which then go on to form clumps
(sometimes called protofibrils), and then strand-like fibrils.
Recent reports give strong support to the idea that amyloid fibril formation and the
subsequent development of protein deposition diseases originate from conformational
changes in corresponding amyloidogenic proteins. Recent findings are surveyed to
illustrate that protein fibrillogenesis requires a partially folded conformation. This
amyloidogenic conformation is relatively unfolded, and shares many structural
properties with the pre-molten globule state, a partially folded intermediate frequently
observed in the early stages of protein folding and under some equilibrium conditions.
The inherent flexibility of such an intermediate is essential in allowing the
conformational rearrangements necessary to form the core cross-beta structure of the
amyloid fibril.
 A.Fink: General model of protein aggregation
1: Aggregation is initiated by a structural transformation in the protein
into a partially folded conformation. Proteins with different types of
structure (a-helical, b-structural, natively unfolded, a + b or a/b; single-
domain or multi-domain; etc.,) transform into the partially folded
conformation;
2: partially folded molecules can assemble into specific oligomers
(nucleus, or protofibrils).
3: Depending on the peculiarities of
the amino acid sequence and the
environmental conditions, the
protein may end up as amyloid
fibril, soluble oligomer or
amorphous aggregate.
Serrano et. al. /Nature 2004/:

Intermolecular beta-sheet formation as a widespread

underlying mechanism of protein aggregation.
 Figure 3. Flowchart for therapeutic
intervention in a hypothetical several-
step pathway of protein aggregation.
An initiating event in aggregation may be covalent
modification of the disease protein, for example by
cleavage or phosphorylation, facilitating conversion
of the protein to an abnormal conformation.
Oligomeric (globular) intermediates may form, and
then protofibrillar structures are assembled.
Amyloid fibers can then form, possibly through
association of protofibrillar intermediates, resulting in
aggregates or inclusions visible in the light
microscope.
The intermediate species are hypothesized to be more
toxic than either the precursor protein or the
aggresomes and inclusions.

Inhibition early in the pathway would be beneficial to the cell, because it may prevent the
formation of potentially toxic oligomeric or other intermediates. By contrast, inhibition at later
stages could be detrimental, because it may result in accumulation of toxic intermediates.
Initiation of aggregation
Neurodegenerative disease proteins often appear to be natively unfolded.
There may be several kinds of aggregates, including disordered or
'amorphous' aggregates, but amyloid fibrils are most characteristic.

What might initiate the aggregation process?

The initiation of misfolding in a particular cell may be a stochastic event,
with a constant risk over the life of the individual.
Amyloid formation may proceed via a process of ‘seeded polymerization’.

The likelihood of aggregation could be increased by increasing protein

concentration
• genetic dosage alterations;
• an extra copy of the APP locus on chromosome 21;
• polymorphisms in promoter sites of disease-associated genes.
Aging
• oxidative modifications of proteins;
• decrease the ability of the cell to clear misfolded proteins.
Protein phosphorylation
Intermediates in the aggregation process
It is becoming increasingly clear that protein aggregation is a complex process,
involving several kinds of intermediates and resulting in different kinds of fibers
or amorphous aggregates.
Many of the studies to date have been done in vitro and may not mimic the
situation in human diseases, so there is much to be learned.
Therapeutic strategies
• to enhance cellular defence mechanisms;

The cell has developed mechanisms to defend against misfolded and aggregated
proteins. The first line of defense involves the many molecular chaperones that
aid in normal folding and also in refolding of abnormal conformations back to
the native state.
If this fails, abnormal proteins can be targeted for degradation by covalent
attachment of polyubiquitin followed by targeting to the proteasome and
degradation.

• to modulate and enhance chaperone levels;

• to stimulate proteasome activity,
• to target the protein misfolding pathway
Congo red binds to proteins with  -sheet structure and may alter the protein
misfolding pathway and reduce toxicity in vivo.