AD CSF HC CSF

0

20

40

60

80

100

Ca

lciu

m in

flu

x (

%)

n.s.

Suman De1, Patrick Flagmeier1, D. C. Wirthensohn1, Daniel Whiten1, Cecile Vincke2, Serge Muyldermans2, Steven F Lee1, Tuomas

P.J. Knowles1, Sonia Gandhi3, Christopher M. Dobson1, David Klenerman1

1

2

3

45

13

12

11

10

6

78

9

1

2

3

45

13

12

11

10

6

78

9

1

2

3

45

13

12

11

10

6

78

9

Protein aggregate

Fie

ld o

f vie

w

(exp

erim

en

tal)

Blank Ionomycin

1 3 5 7 9 11 13

0

10

20

30

40

Spot number

(corresponding to field of view)

Ca

2+

influx into

indiv

idual v

esic

les (

%)

0 20 40 60 80 100 1200

50

100

150

200

250

Nu

mb

er

of

ind

ivid

ua

l

flu

ore

sce

nt

sp

ots

Ca2+ influx (%)

Particles Analysed = 744

Mean Ca2+ influx = 19.4 %

Acknowledgements

1 Department of Chemistry, University of Cambridge, Cambridge, UK,. 2 Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Belgium, 3 Sobell Department of Motor Neuroscience and Movement Disorders, University College London, London, UK

Measuring the membrane permeability induced by human cerebrospinal fluid

In order to quantify and characterize the potentially toxic aggregates associated with

neurodegenerative disease, a high throughput assay based on measuring the extent of

aggregate induced Ca2+ entry into individual vesicles has been developed.

(1) This approach was implemented by tethering vesicles containing a Ca2+ sensitive

fluorescent dye to a passivated surface and measuring changes in the fluorescence as

a result of membrane disruption using total internal reflection microscopy.

(2) Pico-molar concentrations of Aβ42 oligomers could be observed to induce Ca2+ influx,

(3) which could be inhibited by the addition of a naturally occurring chaperone and a

nanobody designed to bind to the Aβ peptide. The assay can be used to study aggregates

from other proteins and to probe the effects of complex biofluids, such as cerebrospinal fluid,

and thus has wide applicability. Potentially, our method enables the quantitative measurement

of any biochemical process which involves membrane permeabilization and subsequent

Ca2+ influx.

Abstract

1. Methodology

Ca2+ influx into individual vesicles can be measured based on the increased fluorescence

intensity and the Ca2+ influx caused by a solution containing protein aggregate can be

quantified relative to the Ca2+ influx caused by ionomycin.

0

0.5

1

1.5

2

[Fib

ril] (

μM)

Time (h)

0 1 2 43 5

t1 t2 t3

0

20

40

60

80

100

t1 t2 t3

Ave

rag

e C

a2

+ in

flu

x (

%)

0 10 20 30 40 50

0

20

40

60

80

100

0 0.1 0.2 0.3 0.4

10

20

30

40

50

[Aβ42] (nM) at t2

0

0.5

Ca2+

influx (

%)

[Aβ42] (nM) at t2

Ave

rag

e C

a2

+ in

flu

x (

%)

2. Measurement of Ca2+ influx caused by solutions of Aβ42

2.1. Characteristic time-points of an Aβ42 aggregation reaction, such as the

monomeric protein, the end of the lag-phase and the plateau phase differ in

their ability to permeate the membrane.

2.2. Ca2+ influx caused by solutions of Aβ42 can be detected in the range of

pico-molar concentrations and in a highly reproducible manner.

Replicate 1

Replicate 2

Replicate 3

Replicate 4

0.1 1 10

0

20

40

60

80

100

[Aβ42] (nM)

Ave

rag

e C

a2

+ I

nflu

x (

%)

3. Human cerebrospinal fluid (CSF) of individual with AD and

healthy individual, both can induce Ca2+ influx

0

40

20

60

80

100

Aβ4

2

+ C

lusterin

+ N

b3

+ a

nti-

GFP

Ave

rag

e C

a2

+ in

flu

x (

%)

ns

*** ***

2.3. Chaperones and nanobodies can be tested to whether they are able to counteract

the Ca2+ influx caused by Aβ42 oligomers.

3.1. We have found that CSF of individuals with AD and healthy controls can induce Ca2+ influx

and Nb3 nanobody designed to bind to the Aβ peptide can partially inhibit this influx.

Cal-520 dye

filled vesiclesBiotin Neutravidin Ca2+ ion Ca2+ bound fluorescent dye

Protein

solution Ionomycin

1. Ultrasensitive Measurement of Ca2+ Influx into Lipid Vesicles Induced by

Protein Aggregates. Angew Chem Int Ed Engl. 2017, 56, 7750

2. Inhibiting the Ca2+ influx induced by human CSF; (under review)

4. Conclusions

Reference

4.2. We show that Aβ42 oligomers are responsible for the lipid bilayer permeabilisation.

The developed method is quantitative and reproducible.

Antibodies and chaperones can be evaluated according to their ability to bind to and

prevent the oligomer induced membrane permeation.

We have found that CSF of individuals with AD and healthy controls can induce

Ca2+ influx and the Nb3 and Bapineuzumab nanobody designed to bind to the Aβ peptide can partially inhibit this influx.

4.1. We have developed a method that enables the quantification of the ability of

protein aggregation reactions to permeabilise lipid bilayers.

4.3. More than fifty protein misfolding diseases were identified to which our method may be

applied. In addition the method may assist in the development of therapeutic agents.

0

40

20

60

80

100

Ave

rag

e C

a2

+ in

flu

x (

%)

0 4020 60 80 100

[Nb3] (nM)

0 20 40 60 80 100

0

0.2

0.4

0.6

0.8

1

[Nb3] / (nM)

Norm

alised C

a2+

influx

AD CSF HC CSF

0

20

40

60

80

100

n.s.

Ave

rag

e C

a2

+ in

flu

x (

%)

0

20

40

60

80

100

****

+ 150 nM

Nb3

CSF

Ave

rag

e C

a2

+ in

flu

x (

%)

Marie Skłodowska-Curie Individual Fellowships

0

20

40

60

80

100

+ 150 nM

Bapineu

-zumab

CSF

*

+ 1 mM

Bapineu

-zumab

***

Ave

rag

e C

a2

+ in

flu

x (

%)

0 200 400 600 800 1000

0

20

40

60

80

100

0 200 400 600 8001000

0.0

0.2

0.4

0.6

0.8

1.0

Conc of Bapineuzumab (nM)

Ave

rag

e C

a2

+ in

flu

x (

%)

Norm

alised C

a2+

influx

3.2. We probed Bapineuzumab, which is bivalent and binds soluble Aβ monomer and synthetic oligomers at the N terminus. We first measured that ~90 nM Bapineuzumab was required at

half the Ca2+ influx caused by Aβ42 oligomers.