Targeting Alzheimers 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

Background

Methods

Results

Human microtubule associated protein Tau (MAPT) is an

important factor in the pathophysiology of Alzheimers 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 l. Fluorescence intensity was measured every 4
minutes with continuous shaking at 280 rpm using 444 nm
excitation and 485 nm emission filters.

Nucleation Rate Constant

Aggregation Master Mix

Component

Amount

Function

Tau (383)

16 M

Protein

Concentration of Antibody (nM)

Antibody

5-150 nM

Experimental Variable

Thioflavin T

5 M

Fluorescence

EDTA

10 mM

Chelation of trace metals

Monomer
Dimer

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.

Elongation Rate Constant

Oligomer

NaCl

100 mM

Maintenance of ionic strength

Hepes, pH 7.5

10 mM

pH Buffer

Heparin Sulfate

5 M

Facilitate Aggregation

Protofibrils
Mature Fibrils

Nucleation
Antibody Binding Sites on Tau

Concentration of Antibody (nM)

Elongation/Fibrillation

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.

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.

Kinetics of Tau Aggregation in the Presence of Antibody 396

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:

Antibody Mediated Therapy

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.

Time (Hours)

Kinetic curves exhibiting antibody 396-mediated Tau aggregation

reflect decrease in the lag phase, corresponding to an increase
in the nucleation rate constant.

Fluorescence

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.

Fluorescence

Data Fitting

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.

Electron Microscopy

Yo
Xo
Four Parameter Sigmoid
Curve
Time (Hours)

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.

Control

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.

35 nM,
Ab 396

Control

65 nM,
Ab 396

Control

110 nM,
Ab 396

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.