This document describes an experiment examining the effects of three antibodies (A10, 181, 396) on the aggregation kinetics of the Tau protein. It found that only antibody 396 significantly increased both the nucleation and elongation rate constants of Tau aggregation. While aggregation rates increased, electron microscopy revealed that aggregates retained more oligomeric character and fibrils were less dense compared to the untreated control. The addition of antibody 396 therefore altered Tau aggregation kinetics in a way that may represent a less toxic form of Tau inclusions.

1. Component Amount Function
Tau (383) 16 µM Protein
Antibody 5-150 nM Experimental Variable
Thioflavin T 5 µM Fluorescence
EDTA 10 mM Chelation of trace metals
NaCl 100 mM Maintenance of ionic strength
Hepes, pH 7.5 10 mM pH Buffer
Heparin Sulfate 5 µM Facilitate Aggregation
Human microtubule associated protein Tau (MAPT) is an
important factor in the pathophysiology of Alzheimer’s Disease.
Dissociation of MAPT from neuronal microtubules culminates in
the formation of intracellular filamentous inclusions known as
neurofibrillary tangles, a common feature in a class of
neurodegenerative diseases collectively known as Tauopathies.
The fluorescence of the fibril-binding benziothiazole dye
thioflavin T was used to measure Tau aggregation in a 96-well
plate in an Infinite M200 Pro microplate reader. Varying
nanomolar concentrations of antibody were mixed with an
aggregation master mix . The samples were loaded in triplicates
of 100 µl. Fluorescence intensity was measured every 4
minutes with continuous shaking at 280 rpm using 444 nm
excitation and 485 nm emission filters.
MethodsBackground
Data was fitted using Sigma Plot statistical software. Sigmoidal
curves were estimated with four parameters obtained from raw,
in-vitro Tau aggregation data as variables in the 4 parameter
sigmoid curve formula:
Aggregation Master Mix
Data Fitting
Results
Elongation Rate Constant
Time (Hours)
Nucleation Rate Constant
Concentration of Antibody (nM)
Concentration of Antibody (nM)
Fluorescence
Kinetics of Tau Aggregation in the Presence of Antibody 396
Among three experimental antibodies A10, 181 and 396, only antibody
396 produced a dramatic increase in the nucleation rate constant,
corresponding to a shortened lag phase across all concentrations of
antibody. While addition of 396 significantly altered the nucleation rate
constant, increases in concentration of the antibody showed no
discernible influence.
Among three experimental antibodies A10, 181 and 396, only
antibody 396 produced a dramatic increase in the elongation
rate constant, corresponding to an increase in the rate of fibril
formation across all concentrations of antibody. While addition
of the 396 antibody significantly altered the elongation rate
constant, increases in concentration of the antibody showed no
discernible influence.
Antibody MediatedTherapy
Elements of active and passive immunization have been the
subject of experimentation against Tau aggregation, with varying
degrees of success . Passive immunization with engineered
antibodies directed at peptide sequences on dissociated Tau has
been shown to be efficacious in mitigating the onset of
neurocognitive deficits associated with tauopathies and
mitigating the cell to cell transmission of protein aggregates, also
known as interneuronal seeding.
The most eminent challenge to antibody therapy is the existence
of a physiological blood-brain barrier which safeguards against
the passage of macromolecules from the bloodstream into the
extracellular fluid of the brain by tightly regulating vascular
permeability. Engineering blood-brain barrier permeable
antibodies and direct intrathecal injectons have enhance the
likelihood of antibody penetration, nevertheless, cerebrospinal
fluid concentration.
Objectives
The mechanism of antibody-mediated Tau therapy remains
elusive. In-vitro kinetic aggregation assays containing very low
concentrations of antibodies may help facilitate the
understanding of antibody-Tau interactions at physiological
levels. We aim to examine the kinetic effect of three antibodies
(A10, 181, and 396) on the formation of Tau fibrils.
Antibody Binding Sites onTau
Kinetic curves exhibiting antibody 396-mediated Tau aggregation
reflect decrease in the lag phase, corresponding to an increase
in the nucleation rate constant.
The addition of antibody also produced an increase in the
elongation rate constant, resulting in a hastened rate of fibril
formation per unit of time.
Dissociated Tau monomers are thermodynamically unstable and
form oligomeric complexes with other Tau monomers to lower
their free energy. These complexes recruit nearby oligomers in a
process known as nucleation.
When a critical threshold of nucleation is reached, the oligomers
begin to actively form fibrils in a process known as elongation.
The final outcome of this process is the formation of
neurofibrillary tangles.
Nucleation
Dimer
Protofibrils
Mature Fibrils
Monomer
Oligomer
Four Parameter Sigmoid Curve
Time (Hours)
Fluorescence
Xo
Yo
a
The kinetics of tau fibril formation can be illustrated through a
four parameter sigmoid curve, whereby four variables contribute
to the understanding of common attributes found in tau
aggregation. In this case Yo represents the thioflavin T
fluorescence (as a measure of aggregation) values at time = 0,
Xo reflects the time at which fluorescence (as a measure of
aggregation) is at half of its peak value a.
The lag phase (Xo-2b) describes the period of time prior to the
onset of fibrillation when nucleation occurs. The reciprocal of the
lag phase (1/[Xo-2b]) denotes the nucleation rate constant, or
the rate at which nucleation occurs per unit of time.
The elongation constant (1/b) describes the rate at which fibril
formation occurs during active aggregation.
Electron Microscopy
Samples treated with antibody 396 retained some oligomeric
character compared to their untreated counterparts. Fibril
formations in treated groups were visibly less dense and more
diffuse compared to control groups.
Conclusions
We observed that addition of the antibody 396 to the microtubule
associated protein Tau in the process of its aggregation resulted
in an increase in the rate of lag phase nucleation paralleled by
an increase in the rate of active elongation/fibrillation.
Although aggregation rates had undergone a sizable increase,
electron microscopy revealed retention of oligomers and a
reduction in the density of end product fibrils. Perhaps these
states represent a less toxic form of Tau inclusions that form in
the presence of the antibody.
Control Control Control
35 nM,
Ab 396
65 nM,
Ab 396
110 nM,
Ab 396
Targeting Alzheimer’s Disease: Insights on Immunotherapy
Against Tauopathies in Neurodegenerative Disorders
Konstantin M. Ravvina*, Leonid Breydoa, b, Vladimir N. Uverskya, b
a Department of Molecular Medicine and bByrd Alzheimer's Research Institute, Morsani College of Medicine, University of South Florida, Tampa, FL
Elongation/Fibrillation