An Internship project review on the topic "Immunohistochemistry based approach to detect synucleinopathy conformations in parkinson"

1. Immunohistochemistry based approach to detect
Synucleinopathy conformations in Parkinson’s diseases: A
Review
Joel John a*
a
B.Tech Biotechnology, Department of Biotechnology, Manipal Institute of Technology, MAHE, Manipal.
A B S T R A C T
Parkinson's diseases (PD) is a standout amongst the most widely recognized neurodegenerative
issue. The condition causes an overwhelming burden both on those influenced, and additionally to
their families. The oligomerizaton of α-synuclein marks the onset of Parkinson’s diseases. α-
synuclein is the potential biomarker that facilitates the prognosis towards Parkinsonism. The
diagnostic approach needs to be accurate, and is critical as it remains established on clinical
grounds as no particular symptomatic test is accessible up until now. Various clinical trials have
given proof that wellbeing of life can be considerably enhanced with early diagnosis. This review
discusses how invitro immunohistochemical approach utilizing monoclonal and polyclonal
antibodies aids in the detection of proteopathological conformations showcased by α-synuclein
and thereby facilitates in the diagnosis of Parkinson’s.
K E Y W O R D S
Parkinson’s diseases (PD), synucleinopathy, α-synuclein, synuclein aggregation, Lewy body (LB),
Immunohistochemistry, monoclonal antibody, polyclonal antibody, and oligomerization.
1. INTRODUCTION
Neurological disorders that affects the Central Nervous system (CNS), includes Alzheimer's,
Parkinson's, and amyotrophic lateral sclerosis (ALS), Dementia with lewy body (DLB) and stroke
are issues that both the patient and clinician has to face since these diseases are literally hard to
treat utilizing the conventional pharmacological methodologies. Patients with these diseases
endures effects from the two sides, the symptoms and sequelae in their remaining lifetime [1]
. New
procedures are dynamically created to defeat the neglected therapeutic needs in regular treatment.
Now focusing on Parkinson’s disease, it’s the standout amongst the neurological disorders that
affects the CNS, and also is considered as a long term neurological disease. In all the
proteopathological disorders, PD is well known for its most potent biomarker, α-synuclein. The
initiation of PD and its progression could be backed by genetic and environmental factors [2]
.
Being 17kDa in molecular weight, and consisting of 140 amino acids, this protein the product of
the SNCA gene acts as the ultimate potential biomarker for PD. Throughout the progression of the
disease, the aggregation of the monomers of α-synuclein within the neural cytosol to leads to the
formation of lewy bodies, that distorts the ion channel. Even though the exact function of the
protein is unclear it’s proposed to function as a transmembrane protein aiding in axonal transport

2. and in dopamine homeostasis. The α-synuclein dimers forms a transmembrane ion channel that
increases the uptake of Ca2+
ions into the neural networks thereby disrupting the electron transport
chain, leading to the loss of motor control within the patients. The accumulation of α-synuclein is
predominantly observed at the Substantia nigra, a region within the mid-brain. The
oligomerization of α-synuclein monomers to dimers and fibrils by undergoing conformational
changes once in contact with α-synuclein seeds leads to the formation of lewy bodies [3]
. At the
final stages, a greater distribution of LBs are observed within the central nervous system and the
peripheral nervous system that has staged Parkinsonism [4][5]
.
The immunohistochemistry based diagnostics is determined to help in analyzing the stage of
progression of the diseases. This diagnostic methods utilizes engineered primary and secondary
antibodies that are specific to this oligomerized antigenic, proteopathic α-synuclein conformations.
The immunohistochemistry based diagnostic techniques is still in the research phase as an in-vivo
clinical trial faces issues regarding the inability of the antibody to cross the blood brain barrier.
Therefore we would be discussing on the aspect of detecting α-synuclein conformations using
antigen-epitope specific antibody based detection through an in-vitro approach. The binding could
be visualized by utilizing either fluorescence or through chemiluminescence imaging (at
pico/femto concentrations of chemiluminol). For this structure specific targeting technique,
epitope specific monoclonal and polyclonal antibodies are being engineered such that its capable
of crossing the blood brain barrier and then binding towards the structure specific antigens. This
review would discuss upon the oligomerization of α-synuclein monomers to take various structural
conformations like dimers, aggregates, seeds and LBs, in addition this paper showcases the
different types of engineered antibodies that are constructed to aid in the efficient detection of the
various protein conformations.
2. THE TRANSITION THROUGH OLIGOMERIZATION
In most of the proteopathological disorders which could be visualised as a protein misfolding, error
in folding occurs because of either an undesirable mutation in the polypeptide or, due to some
unknown reasons. In the case of PD As
discussed above the neuro-degenerative
disease of the CNS, Parkinson’s disease, is a
disorder in which there is an excessive
accumulation of α-synuclein seeds within the
neurons leading to the formation of lewy
bodies. α-synuclein is a natively unfolded α-
helical structure, on contact with seeds it
undergoes conformational modification and
folds down to take up a β sheet configuration
forming a highly unstable dimer.
Fig 1. Natively unfolded, α helical structure of α-
synuclein monomers [3]
.

3. The change in configuration of this proteins shifts it from the cytosol towards the transmembrane
facilitating axonal transport, leading to increase in the intake of ions into the neurons causing
electron imbalance within the neurons. The harmful effects of this protein misfolding could be due
to the deleterious gain of function concept which is obserered in various other proteopathological
neurodegenerative disorders [3][5]
. As stated above the initiation of the protein misfolding and the
progression of this disease could be due to genetic as well environmental factors.
Fig 2. The oligomerization process is depicted above. The α-helical structure in the polypeptide chain of a native
protein are converted into β-pleated sheets. (A) Native polypeptide chain composed of α-helical structure. (B)
Misfolding causes the conversion of α-helical structure to β-pleated sheets and (C) final misfolded structure of
polypeptide chain contains β-pleated sheets [3]
.
The formation of this β sheet promotes the gain of function for the dimeric and oligomeric proteins
and displaces them towards the transmembrane, initiating neuronal degradation by creating an ion
imbalance within the system.
Fig 3. Protein oligomerization. Protein misfolding occurring to new monomeric α-synuclein proteins leading to
formation of β pleated sheets, acting as a transmembrane ion channel [3]
.

4. Progression of the synucleiopathy is determined
by the aggregation induced onto the monomeric
α-synuclein by these oligomers to form
intermediate (called aggregates), which further
leads to the formation of fibrils (seeds). The
aggregation of these fibrils leads to the
formation of a dense, high molecular weight,
protein deposits within the neurons called as
Lewy bodies (LB). The development and
increase in the number of these deposits leads to
the initiation of symptoms displayed by
Parkinson’s diseases i.e. the loss of motor
control.
Fig 4: α-synuclein seeds (α-synuclein fibrils)
Fig 5. Depicts the “Gain of Function” concept of the oligomeric protein complex [6]
.
3. IN-VITRO IMMUNOHISTOCHEMISTRY BASED DETECTION OF SYNUCLEIOPATHY
PROTIEN CONFORMATIONS
Immunohistochemistry based detection utilizes both monoclonal and polyclonal antibodies. These
engineered antibodies are highly specific towards different conformations that α-synuclein exists
in. The collected cell lysates for in-vitro based detection may contain all the different types of
conformations as discussed and the antibody indeed need to be highly specific to each of these
conformation to deliver accurate and high sensitive results. There have been recorded data where
monoclonal antibodies (mAbs) being less specific in identifying all the synucleiopathy
conformations present within the sample, as mAbs is highly specific to only one type of epitope.
Whereby utilizing multiple epitope specific polyclonal antibodies (pAbs) has displayed a more
appropriate, and resolute detection of the various conformations [7][10]
. Each epitope specific

5. polyclonal antibody acts as monoclonal antibodies, The sensitivity of utilizing antibodies to detect
biomarkers within the cells have been utilized to the fullest over the past few decades in the field
of medicine. There are monoclonal antibodies that are highly specific to α-synuclein monomers
and aggregates, but are unable to detect the both simultaneously, since each conformations would
be at a different state of progression, therefore pAbs are more reliable in terms of detecting
complex conformations, of different epitopes, simultaneously without the loss of accuracy and
sensitivity.
Majorly engineered monoclonal
antibodies (mAbs) being used are
SnO1, SnO2, Sn03, and SnF1[7]
series of mAbs that are highly
sensitive towards the detection of
α-synuclein aggregates. Each of
them are engineered to cross the
blood brain barrier and accurately
attach to its specific epitopes.
Fig 6. In-vitro analysis of α-synuclein samples at NDRC-QBRI
©
.
Various polyclonal
antibodies (pAbs) are
engineered to
overcome the less
sensitive approach
given by mAbs.
Engineered pAbs like
pAb13, pAb17 are
studied in order to
detect all the possible
conformation of α-
synuclein within the
given cell lysates.
Fig 7. In-vitro analysis of α-synuclein samples detected using pAbs at NDRC-QBRI
©
.
4. DISCUSSIONS
Antibody based detection techniques have shown positive results towards gathering data regarding
various synucleiopathy conformations expressed within Parkinson’s disease. Antibody based tags
could be engineered and developed in the future to prevent the aggregation of these protein
complexes thereby reducing the rate of progression of neuro-degenerative disorders. Future
researches could be conducted on studying the functionality of the native α-synuclein, the post
translational modifications that induces oligomerization, and other therapeutic techniques to
Recombinant Monomers
Detected using mAbs
Α synuclein conformations detected using engineered pAbs

6. regulate the expression of SNCA gene through the most advanced form of gene therapy –
peptide/lipid mediated gene therapy.
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