Download Pathogenicity and Effects of Prions Misfolding

Pathogenicity and Effects of Prions Misfolding
Patrick Stevens
Abstract
Prions are proteins found naturally in the human body and also in other species.
Prions are ubiquitous and have been found in everything from plant and mammal
cells to single celled organisms such as the bacterium, Escerichia coli. The natural
function of prions is largely unknown, however it is thought that they are involved
in several functions such as cellular adhesion, protection, and cell signaling. The
pathogenicity of prions stems from an altered pattern of folding of the protein.
The altering event that triggers change is a single amino acid replacement which
converts one of the alpha helices in the cellular prion protein into a beta sheet
confirmation. This specific mutation induces the pathogenic isoform This isoform
can interact with the normal celular versions of prion to convert them into the
pathenogenic forms. This paper will look at prion diseases and how they affect the
brain as well as what contributes to their pathogenicity and transmission.
Introduction
Prions are responsible for a number of degenerative neurological diseases called
transmissable spongiform encephalopathies or TSEs. Examples of TSEs include
Scrapie, fatal familial insomnia, sporadic familial insomnia (sFI), CrutzfeldtJakob disease, bovine spongiform encephalopathy and kuru to name a few. These
diseases are severely dehabilitating and eventually fatal for those affected. TSEs
are characterized by brain shrinkage caused by the deterioration of neurons. The
deterioration of neurons gives a cross section of the brain a "spongifom" appearance.
TSEs can be diagnosed by looking at a stained cross section of the thalumus, part
of the brain that normally accounts for sleep patterns. As can be imagined this
presents some difficulty when trying to diagnose these diseases in vivo. Another
more practical way to diagnose TSEs, the electroencephalogram (EEG), has
emerged within the past few years. EEGs can potentially be an instrument used
to diagnose TSEs early and prepare a more targeted care plan and treat affected
patients more effectively.
In particular this is the process of how prion misfolding leads to neuronal
death which subsequently leads to a characteristic EEG pattern.
Some TSEs are fairly common while others are more rare. One TSE that is
commonly known is Creutzfeldt-Jakob disease (CJD), the human variant of bovine
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spongiform encephalopathy (BSE), more commonly known as mad cow disease.
According to the Center for Disease Control, CJD has increased with population
over the past 30 years as expected. While contrastingly, the rates of fatal familial
insomnia, another TSE, have remained low. The low rates are due to the genetic
transfer of the fatal familial insomnia genes and the fact that fatal familial insomnia
is not transmitted through animal vectors. Fatal familial insomnia is illustrated
and explained best with a case study which shows the severity of the symptoms.
The case study described by Moody et al (2011) entitled, "Sporadic Fatal
Insomnia in a Young Woman: A Diagnostic Challenge: Case Report;' will be used
to understand the progression of symptoms in a person afflicted with a TSE. In this
case study, a 31-year-old female initially presents with decreased ability to maintain
attention, progressive memory loss, and difficulty sleeping. The progression of her
symptoms correlates closely to the four established phases of sporadic fatal insomnia
(sFI) (Moody et al 2011). The first phase of sFI presents with increasing insomnia,
panic attacks, paranoia and phobias. Although this is characteristic for the onset
of this disease, it is also characteristic for a number of other neurodegenerative
diseases. However, with sFI the patients condition rapidly deteriorates. The second
phase shows progression of the disease in hallucinations and apparent panic attacks
which are described as "acting out of the patient's dreams while awake". The third
phase includes substantial weight loss and increased or a complete inability to sleep.
In the fourth phase, the disease progresses to dementia and eventual death. After
the initial onset of symptoms in Moody's (2011) case study, the patient progressed
to the second phase within six months by displaying bizarre behavior. Another
notable observation that occurred during the progression of the woman's disease
is seen eleven months after the onset of her symptoms in the EEG ordered by her
physician. The EEG showed bilateral periodic eliptiform discharges which was
deemed an abnormal EEG pattern. The disease later progressed approximately one
year and seven months after onset of her symptoms to the third phase where she
experienced near complete inability to sleep and was awake most of the time. One
year and ten months after the onset of her symptoms, the woman in the case study
died at the age of 33. By the time of her death, she had not been able to sleep for a
total of five months.
Although the patient's disease was not diagnosed as fatal familial
insomnia until after her death, it has come to light that excitatory EEG patterns,
called periodic sharp wave complexes, or PSWCs, can be useful in diagnosing
transmissible spongiform encephalopathy. These wave complexes, seen on the
patient's EEG, can now be used to more quickly diagnose TSE, allowing for an
earlier and more targeted care plan for the patient. A contrast between a normal
EEG and the patients EEG, respectively, can be seen in Figure 1.
10
Indiana University South Bend
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Figure 1. Comparision between normal and abnormal EEG. Abnormal EEG shows
PSWCs.(Moody et al 2011).
Although prions are generally given a bad reputation in popular news outlets
due to the debilitating diseases they have been preported to have caused, prions are
naturally found throughout the body. Although much about the specific functions
prions is unknown, what is known is that they serve a functional role in several
natural cellular processes, including adhesion, protection, and cell signaling
(Martins and Brentani, 2002). Prions are made pathogenic by genetic mutation
causing placement of abnormal amino acids certainn places in the peptide
chain. Another way for prions to become pathogenic is through acquisition of
pathogenic prion via animal vectors. Normal prion is noted in literature as PrP for
Proteinacious Particle and pathogenic prions are noted as either PrPres or PrPsc
(Ma and Lindquist 2002). The denotation of sc is an abbreviated form of the word
scrapie which is the namesake disease in which prions were discovered to be the
primary pathogenic particles involved. Scrapie gets its name from affected sheep
which would rub up upon trees or fences, effectively "scraping" off their wool
(Lacroux et al 2008). The replacement of several amino acids in the sequence of
the prion protein induces a confirmational change in which a beta sheet is induced
in the pathogenic prion where an alpha helix would have been expressed in the
cellular prion (Pan et al 1993). The pathogenic prion then has the ability to induce
confirmational change in other normal cellular prion proteins. The exposure to
one pathogenic prion can induce the disease symptoms simply from converting
the normal host prion. Pathenogenic prions are able to cause aggregates of many
prions through conversion and attachment. These large prion aggregates effectively
clog neurons and cause inhibition of neuronal firing. This is similar to these sorts
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of protein aggregates found in diseases such as Alzheimer's and other forms of
dementia.
Review and Synthesis
What behavioral abnormalities are consistently attributed with FFI?
In a study by Jackson et al (2009), entitled "Spontaneous Generation of
Prion Infectivity in Fatal Familial Insomnia Knock-in Mice;' knock-in mice were
genetically engineered to display human genes for fatal familial insomnia (FFI).
Knock-in mice are mice used by geneticists to express specific genes, which they
"knock-in" to the mouse genome. Also added to the genome of the mice was an
antibody binding site 3F4, which is a common and established antibody binding
site on the FFI gene. The gene for the human version of FFI can be found on the P
arm of the 20th chromosome in position 20Pl3.
A number of "normal" and "erratic" behaviors were tested to determine if the
genetic mutation correlated with a change in the mouse behavior. Figure 2 shows
a plot with several different common mouse behaviors as well as abnormal ones
and which mice demonstrated them. It may be seen in Figure 2 that the behaviors
of twitching during rest and awakening show increases with age progression. This
increase can be attributed to the progression of the stages of the disease. Other
information which may be obtained from this figure is that hanging, jumping,
stretching and sniffing behaviors have all decreased overtime. This decrease in
common mouse behavior correlates to a progression in the course of the disease.
The conclusion drawn from Figure 2 is that the introduction of the point mutations
on the prion protein (PRNP) gene introduces behaviors that are consistent with FFI
in mice (Jackson et al 2009).
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Indiana University South Bend
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Figure 2. Common and uncommon mouse behaviors and frequency at which they
are expressed in relation to mice genetically engineered to express with the human
FFI genes (Jackson et al 2009).
Also tested by Jackson et al (2009) was the survivorship of mice that are
injected with pathogenic prion protein. As may be seen in Figure 3A, wild type
mice were injected with wild type brain homogenate which caused no change in
the survivorship of the mice, resulting in the survival of all mice. In Figure 3B,
TGA20 mice were used. TGA20 mice are a special strain of mice that are often
used to demonstrate neurodegenerative diseases (Mehta et al 2008). TGA20 mice
have large amounts of prion present in the brain. Figure 3B shows that TGA20
mice were injected with brain. homogenates from the mice affected with FFI. It
may be gathered from Figure 3B that the TGA20 mice showed a drastic decrease
in survivorship after approximately 300 days. All mice injected with the FFI brain
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homogenate experienced early mortality. In Figure 3C wild type mice are injected
with FFI brain homogenate. Similarly to the Figure 3B, each mouse was infected
and experienced early mortality, however the onset of the infection was earlier and
the last affected mouse died later. The sharp contrast between Figures 3B and 3C
can largly be attributed to the increased amount of prion present in the brains of
TGA20 mice, which correlates to a faster onset of the disease. In Figure 30, mice
were engineered which knocked out the PRNP gene from the mouse genome. Each
of these were then infected with FFI brain homogenate and as may be seen in Figure
30 only the mice that had the PRNP gene died. Contrastingly, the knock-out mice
which did not display the PRNP gene survived.
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Figure 3. Survivorship plots of mice infected with wild type and FFI brain
homogenates (Jackson et al 2009).
What organisms can TSEs be transmitted into?
Another study, done by Caughey (2000), entitled "Formation of Protease
Resistant Prion Protein in Cell Free Systems;' looks in particular at what organisms
can be infected by TS Es. At the time of this study, bovine spongiform encephalopathy
was a hot topic due to recent outbreaks. Caughey specifically looked at the prions
which caused BSE which he termed PrPbse. Prior knowledge is taken into account
with this study that certain strains of sheep and hamsters are not able to be affected
by BSE. As can be seen in figure 4, cattle, sheep, mice and hamsters were all subjects
that have been tested before to see whether Pr Pbse was transmissable. At the time
of the study it was questionable whether humans could be affected by BSE, because
of exposure to PrPbse or not.
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Figure 4. Graphical representation of prior knowledge to display the organisms
tested in the western blot and expected results (Caughey 2000)
In Caughey's study, a western blot was done using radiolabeled antibodies that
attached to the 3F4 site and provide visualization of natural PrP. A western blot
is a anaylitical technique used to detect specific protiens in a tissue. As may be
seen in Figure 5, the homogenates of five different organisms in several different
genetic variations are incubated with a minute amount of PrPbse and subsequently
treated with a generic protease. This study takes advantage of the fact that one of
the pathogenic qualities of PrPsc is its resistance to proteases (Caughey 2000). All
undigested PrP is assumed to be natural cellular PrP converted to PrPbse. As was
expected, the results confirm that PrPbse cannot be transmitted into one strain
of sheep cells with the amino acid makeup of Al36, Rl 71 and also cannot be
transmitted to the two strains of hamster that
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were tested. An interesting fact to note is that
the converted PrPbse has Lhe molecular weight
of approximately 19 kDa which seems to be
characteristic of pathenogenic prion protiens
(Caughey 2000).
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Figure 5. Western blot of brain homogenates
from different organisms with radiolabeled
antibodies before and after incubation with
PrPbse and addition of a generic protease
(Caughey 2000).
For comparison, we return to .Moody's (2011) case study in which a Western
blot was done to test for presence of pathogenic prion in different sections of the
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brain. As can be seen from Figure 6, a radiolabele.d antibody was added to the brain
homogenate of Moody's patient from different regions of the brain. Both normal
cellular prion and pathogenic prion were found in different regions of the brain
as expected. However, initially no prion was recovered from the expected region
where the women's disease symptoms would have stemmed from, the thalamus.
Only upon further concentration of the homogenate was both normal cellular prion
and pathogenic prion resolved from the thalamus. Perhaps, the most interesting
quality found by this study is that pathogenic prion was found at approximately 19
kDa which is exactly the same size pathogenic prion in each of the organisms in
Figure 5.
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Figure 6. Radiolabed PrP from differing sections of the brain homogenate of the
case study (Moody et al 2011).
Further examination of the patient's brain was done by Moody et al (2011)
by taking cross-sections of the brain to study neuronal death. Figures 7A and 7C
represent cross-sections of the patient's brain while Figures 7B and 7D represent
cross-sections of a normal brain. The circles represent areas of neuronal death.
The circles are largely absent from Figure 7B. The arrows in Figure 7A represent
areas of gliosis. Gliosis is a nonspecific reactive change in glial cells as a result of
damage of the central nervous system. This is usually presented in hypertrophy or
proliferation of glial cells. In Figure 7A these reactive changes in glial cells can be
clearly visualized, while in Figure 7B the arrows point to normal glial cells. Figure
7C shows a closer look at a proliferated glial cell. The box highlights an astrocyte
of erratic form which is been labeled by CNPase, a protein that is naturally found
in astrocytes and no other glial cell. Figure 7D shows a normal brain without the
presence of proliferated glial cells.
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Figure 7.
Immunohistological
study of the case
studies brain contrast
with a normal brain to
highlight neuronal death
and reactive gliosis
(Caughey 2000).
100µm
What specific regions in the brain and cell types are affected by pathogenic PrP?
In a study done by Moleres and Valleyos (2005) entitled, "Expression of Pr PC
in the Rat Brain and Characterization a Subset of Cortical Neurons;' fifteen age
and sex matched Wister rats were used for immunohistological studies to identify
specific regions of the brain and cell types that are affected by TSEs. In Figure 8,
a number of regions in the brain are shown through use of histological slides and
immunofluorescent antibody labeling which targets PrP. The rationale for the study
done by Moleres and Valleyos is that the place where the most cellular prion protein
is found will be the place where the pathogenic prion will have the most effect.
In Figures 8A-8H, histological slides are made from eight sections of the brain,
the olfactory bulb, cerebral cortex, lateral globulus pallidus, mediodorsal thalmic
nucleus, reticular thalmic nucleus, hippocampus, cerebellum, and corpus callosum.
The top six slides represent gray matter
which are the areas in which axonal
projections are not normally located,
while the bottom two slides represent
white matter where axonal projections
are normally found. The conclusion
drawn from these immunohistological
studies is that prions are ubiquitous,
but present in differing concentrations
throughout the brain.
Figure 8. Immunohistological studies
of cross sections of several different
sections of whiteand grey matter of the
rat brain (Moleres & Velayos 2005).
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Figure 9 shows a closer look at glial cells, specifically astrocytes and
oligodendrocytes, in relation to prion protein .. It is believed that prion may be
initially produced by glial cells and subsequently transported to neurons. Figure 9
shows an immunofluorescent co-location study of astrocytes and oligodendrocytes
with immunofluorecent PrP. PrP is represented by green while astrocytes and
oligodedrocytes fluoresce in red (Moleres &Valeyos 2005). It is found that prion
co-locate with both astrocytes and oligodendrocytes. The conclusion drawn
from Figure 9 is that it is possible that prions are produced in the glial cells and
transported to other neurons via axonal projections (Taraboulos 1992).
Figure 9. Immunofluorescent
co-location study of astrocytes
and oligodendrocytes with
PrP (Moleres & Velayos 2005).
How are pathogenic prions transmitted and spread post production?
In the next set of histological slides Moleres and Valleyos (2005) took a closer
look at several calcium binding proteins present in a subset of neurons. Calcium
binding proteins are used because they are consistently expressed in nonoverlapping
populations of GABAergic neurons (Bouzamondo-Bernstein 2004). GABAergic
neurons are focused upon because they are a specific type of inhibitory neuron
which is known to be affected in CJD. The calcium binding proteins that are looked
at in depth are parvalbumin (PV), calbindin (CB), and calrectin (CR). The first
column of Figure 10 represents the marker protein or calcium binding protein.
The second column of Figure 10 represents immunofluorescent PrP. The third
column of Figure 10 represents a merged study of the marker in PrP. As seen in
Figure 10, the GABAergic cells marked with parvalbumin are scattered throughout
the cerebral cortex and show a significant amount of co-location with PrP. The
GABAergic cells marked with calrectin and calbindin are located more towards the
top few layers of the cerebral cortex and show very little co-location with PrP. This
is significant because it shows a possible specific cell type where prion is most likely
to be produced.
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markers
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PrP (Moleres & Velayos 2005).
How does cell death translate into symptoms and findings characteristic of TSEs?
The last of the immunohistological studies done by Moleres and Velayos
(2005) show perineuronal nets (PNN) in a co-location study with PrP. PNNs are
a protective protinacious structure found to surround GABAergic cells. Again,
the marker protein PNNs are shown in red while the PrP is shown by green by
immunofluorescence. The first row represents an overall look at the cerebral cortex
while the second row shows a close up look at the deeper layers of the cerebral
cortex. As may be seen in Figure 11, there is very little co-location throughout the
cerebral cortex, but there is some co-location on the outside of the PNN. This is
visulized easier by the second, close-up row. These data were subsequently used by
Moleres and Velayos to form a hypothesis as to what mechanism drives cell death
and subsequently causes symptoms which are characteristic of the abnormal EEG
found in patients affected by TSEs.
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Undergraduate Research journal #14
19
Conclusion
Moleres and Valleyos (2005) propose a mechanism as to how cell death
occurs in GABAergic neurons as well as how this translates into characteristic
EEG findings. It is proposed that pathogenic prions do not directly interact with
PNNs or GABAergic cells. It is instead proposed that pathogenic prions interact
with a factor cited as factor X, which is necessary to maintain the PNNs (Telling
et al 1995). Nonpathogenic cellular prions do not have a binding site for factor
X. Pathogenic prions have a higher affinity for factor X then do PNNs. Hence it
is proposed that the pathogenic prions interact with this factor which leaves no
ability for maintenance of the PNNs. Subsequently, the PNN breaks down leaving
the neuron exposed to the extracellular matrix. This causes the degradation of the
neuron and eventual cell death illustrated in Figure 12. Death of an inhibitory
neuron causes PSWCs which are the findings characteristic on an EEG of a patient
affected by TSEs. It is not currently known what factor X is used for by pathogenic
prion, but it is thought it may play a role in replication. It is concluded by Moleres
and Valleyos (2005) that without the aid of factor X to maintain the PNNs, the PNN
deteriorates which causes neuronal death and excitatory EEG findings (Moleres
&Valeyos 2005).
Figure 12. Proposed pathenogenic mechanism for misfolded prions to cause cell
death and excitatory EEG symptoms (Moleres & Velayos 2005).
Hopefully, with this new knowledge that has come to light, physicians will
more effectively and effeciently be able to diagnose and subsequently treat TSEs.
Although the prognosis is negative for those whom are infected by prion diseases,
this is a step towards being able to treat the disease in vivo.
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