PD neurological signs (motor + non-motor), Animal model / CROs / Database, Antibody monoclonal vs polyclonal, Epitope mapping, Affinity Maturation, APOE start

PD — Neurological signs

		sign	description	note
Motor	DA	bradykinesia,	slowness of movement that is ongoing
		Rest tremor	Asymmetric 4-6 Hz moderate amplitude tremor which usually involves the thumb (pill-rolling tremor)	the classical model of the prevalence of the indirect pathway over the direct one is challenged. dopaminergic loss in the midbrain retrorubral A8 field, which projects to the pallidum and is separate from the nigrostriatal pathways Thalamocortical relay neurons (ie in thalamus) have ion channel properties that support pacemaking at approximately rest tremor frequency and may be modulated through hyperpolarization by reducing excitatory drive or excitatory input from the cerebellum [79]. Other models suggest a role of the recurrent loop between GPe and the STN as primary oscillator [80] and the STN-cortical oscillatory coupling [81]. The cerebellum seems to have a central role in PD tremor pathogenesis, because rest tremor disappears following lesions of the ventralis intermedius (VIM) thalamic nucleus, which receives cerebellar input, and cerebellar stimulation alter the timing of peripheral tremor. An emergent model indicates abnormally synchronized BG-thalamocortical (BGTC) loop, a GPe-STN pacemaker, and the cerebellar dentate thalamocortical (CTC) circuit as the main actors producing rest tremor [32, 77]. According to this hypothesis, the GPe-STN pacemaker and the BGTC loop trigger tremor episodes, and the CTC circuit maintains and modulates their amplitude [77].
	rigidity	Increased muscle tone felt during examination by passive movement of the affected segment, involving both flexor and extensor muscles and not increased with higher mobilization speed (in contrast with spasticity)	대체로, 적어도 BG vs CbII 은 구분 안 되나? • Unlike rigidity & bradykinesia, tremor is only weakly correlated with DATSCAN (Birman)
Non-DA		Postural instability	Impaired postural adjustment due to decrease or loss of postural reflexes	- early stage: manifestation of bradykinesia - late stage: NE, HT, Pedunclopontine (Ach)
		Postural sway	decreased PPN thalamic innervation (but not NBM cortical cholinergic terminal or striatal DA terminal deficits).
Non-motor		Cognitive control of gait	NBM cortical terminals, (which also control cognitive functioning)
		cognition	Cortical cholinergic neuron loss

Animal model

CROs

CRO	note
Inotiv	Sage → Horizon → Envigo → Inotiv. Serious issues → terminated by MJF
Taconic	MJF associated
RRRC (Rat Resource and Research Center)	, University of Missouri. Mjf associated
Mutant Mouse Regional Resource Center (MMRRC)	University of Missouri, Mjf associated

Database

	content
MGI	http://www.informatics.jax.org/mgihome/projects/aboutmgi.shtml

Antibody

	monoclonal	polyclonal
generation	generated by identical immune cells which are clones of a single parent cell.	produced by multiple immune cells (plasma B cells)
system	hybridoma	antisera
specificity	the antibody recognizes only a single epitope of an antigen and is extremely specific. Note: each clone reacts to different epitopes on the same antigen	Polyclonal antibodies bind to the same antigen, but different epitope
composition	composed of a single IgG against one epitope	contain a heterologous mixture of IgGs against the whole antigen,
affinity	Monoclonal antibody reacts to a singleepitope. Therefore, only one antibody molecule can bind toan antigen molecule. In contrast, polyclonal	Higher overall antibody affinity against the antigen due to the recognition of multiple epitopes. Have a high sensitivity for detecting low-quantity proteins.
multimeric form	must be detecting the multimeric form (monomer 도 당연히 detect 할 거지), as an oligomer would have only one binding epitope	이것도 당연히 monomer & oligomer 다 detect 하지 않나?
binding		(한번에) multiple antibody molecules bind to an antigen molecule.
secondary use	Similarly, only one antibody molecule can bind to a primary antibody molecule if the secondary antibody is a labeled monoclonal antibody. Whereas multiple antibodies can bind to a primary antibody molecule if the secondary antibody isa polyclonal antibody. Consequently, polyclonal antibodies provide a higher sensitivity of detection (amplification of the signal) and, therefore, are commonly used as a secondary antibody.
filament, fibril?		Possible: as antibodies are usually able to bind proteins in different forms/complexes, as long as the epitope is accessible
variation		commercial polyclonal antibodies have high batch-to-batch variation

EPITOPES

The small site on an antigen to which a complementary antibody may specifically bind is called an epitope or antigenic determinant. This is usually one to six monosaccharides or five to eight amino acid residues on the surface of the antigen

If the target molecule is denatured, e.g., through fixation, reduction, pH changes, or during preparation for gel electrophoresis, the epitope may be altered and this may affect its ability to interact with an antibody. For example, some antibodies are ineffective in Western blotting (WB) but are suitable for immunohistochemistry (IHC) applications, because, in the IHC procedure, a complex antigenic site might be maintained in the tissue, whereas in the WB procedure, the procedure of sample preparation alters the protein conformation sufficiently to destroy the antigenic site, and hence eliminates antibody binding

In a denatured protein, only the linear epitope may be recognized. Hence, in protocols where a denatured protein is used, such as in Western blotting, an antibody that recognizes a linear epitope is preferred. Sometimes an epitope is on the interior of a folded protein. The epitope is then inaccessible to the antibody in a nondenaturing protocol, such as immunoprecipitation. A conformational epitope, by definition, is on the outside of the folded protein. An antibody that recognizes the conformational epitope is suitable for mild, nondenaturing procedures such as immunoprecipitation or flow cytometry.

Optimally, an antibody that recognizes a linear epitope on the surface of a normally folded protein will work well in both nondenaturing and denaturing protocols. Thus, the epitope may be present in the antigen’s native, cellular environment, or it may be exposed only when denatured. In their natural form, antigens may be cytoplasmic (soluble), membrane-associated, or secreted. The number, location and size of the epitopes depend on how much of the antigen is presented during the antibody-making process.

Epitope mapping

method	note	disadvantage
X-ray co-crystallography
Cryogenic electron microscopy (Cryo-EM)
Array-based oligo-peptide scanning	library of oligo-peptide sequences from overlapping and non-overlapping segments Importance for intellectual property (IP) protection Methods Provided by	The main disadvantage of this approach is that discontinuous epitopes are deconstructed into smaller peptides, which can cause lower binding affinities.
	Takeda PharmaceuticalCompany Limited overlapping segments of a target protein, and tests for their ability to bind the antibody of interest.

Affinity Maturation

methods

• untargeted mutagenesis
• oligonucleotide-directed mutagenesis

APOE

• GWAS
  • See GWAS section
• ANIMal model
  • ((Davis, 2020 #1522)
• Patient cohort data (longitudinal)
  • ((Davis, 2020 #1522): APOE e4 was associated with a faster rate of decline in MoCA score over time (Table 1), MMSE
• Patient cohort data (cross-sectional)