Component Amount Function

Tau (383) 16 M Protein

Antibody 5-150 nM Experimental Variable

Thioflavin T 5 M 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 M Facilitate Aggregation

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 dyethioflavin T was used to measure Tau aggregation in a 96-wellplate in an Infinite M200 Pro microplate reader. Varyingnanomolar concentrations of antibody were mixed with anaggregation master mix . The samples were loaded in triplicatesof 100 l. Fluorescence intensity was measured every 4minutes with continuous shaking at 280 rpm using 444 nmexcitation 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)

Flu

ore

sce

nc

e

Kinetics of Tau Aggregation in the Presence of Antibody 396

Among three experimental antibodies A10, 181 and 396, only antibody396 produced a dramatic increase in the nucleation rate constant,corresponding to a shortened lag phase across all concentrations ofantibody. While addition of 396 significantly altered the nucleation rateconstant, increases in concentration of the antibody showed nodiscernible influence.

Among three experimental antibodies A10, 181 and 396, onlyantibody 396 produced a dramatic increase in the elongationrate constant, corresponding to an increase in the rate of fibrilformation across all concentrations of antibody. While additionof the 396 antibody significantly altered the elongation rateconstant, increases in concentration of the antibody showed nodiscernible influence.

Antibody Mediated Therapy

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 on Tau

Kinetic curves exhibiting antibody 396-mediated Tau aggregationreflect decrease in the lag phase, corresponding to an increasein the nucleation rate constant.

The addition of antibody also produced an increase in theelongation rate constant, resulting in a hastened rate of fibrilformation 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)

Flu

ore

sce

nc

e

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 39665 nM,

Ab 396

110 nM,

Ab 396

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

Elongation/Fibrillation