Universitas  21  Undergraduate  Research  Conference  The  University  of  Amsterdam  8-­‐13th  July  2013  

By  Charlotte  Dewdney    I   was   fortunate   enough   to   have   been   selected   to   attend   the   9th   Universitas   21  Undergraduate   Research   Conference,   held   at   the   University   of   Amsterdam   in   July  2013.  Having  just  completed  my  Neuroscience  BMedSci  (Hons),  this  was  a  fantastic  opportunity   for   me   to   be   able   to   present   research   arising   from   my   time   spent  working   in   the   Horsburgh   lab   at   the   centre   for   Neuroregeneration   as   part   of   my  Honours  dissertation.      Before   leaving   for   Amsterdam   I   was   a   little   nervous   and   not   quite   sure   what   to  expect,  but  all  of  my  fears  were  quickly  allayed.  As  soon  as   I  arrived   I  was  greeted  with  a  warm  smile  and  told  to  go  and  choose  my  bike!  It  transpired  that  we  would  each   have   our   own   bicycle   for   the   duration   of   the   conference,   which   provided   a  brilliant  way  to  get  around  Amsterdam.    The  first  day  was  set  aside  as  an  ice-­‐breaker  and  consisted  of  various  networking  events  including  a  “speed  dating”  session,  aimed  at  finding  out  about  one  another’s  research,  and  the  chance  to  explore  Amsterdam.  The  weather  was  in  our  favour  and  we  were  able  to  enjoy  the  glorious  sunshine  whilst  taking  in  this  beautiful  city  by  bicycle,  boat  and  foot.  We  visited  the  new  Rijksmuseum  and  relaxed  by  the  “I  Amsterdam”  sign.  By  the  end  of  the  day  I  had  talked  to  delegates  from  21  different  universities  from  13  different  countries,  each  with  a  different  story  to  tell.  It  was  a  hugely  beneficial  day  both  in  making  everyone  more  at  ease  with  their  upcoming  oral  and  poster  presentations  and  in  helping  everyone  to  settle  in.      

   The  theme  of  the  conference  was  “urban  challenges”  and  we  were  each  asked  what  we  hoped   to   learn  by   the  end  of   the  week.  After   reading  all   of   the  project   titles   I  found  that  there  was  an  immense  range  of  research  to  be  presented.  Initially  I  was  sceptical   that   everyone   would   take   an   interest   in   my   research:   what   use   would  research   in   the   Alzheimer’s   field   be   to   a   delegate   speaking   about   recycling   in  Singapore?   As   soon   as   the   presentations  were   underway   however,   I   realised   how  wrong  I  had  been.      

The  conference  delegates  outside  the  “I  amsterdam”  sign

 

Charlotte  Dewdney  

We   had   three   days   of   oral   and   poster   presentations   on   a   vast   variety   of   topics  including   geography,   economics,   music,   languages   and   health.   Being   a   medical  student,   I   have   found   that   one   can   become   slightly   narrow-­‐minded   whilst  completing  a  medical  degree  and   for  me   this   conference   really   reinforced   the   fact  that   research   exists   outside   that   focussed   on   the   treatment   of   diseases   or   indeed  outside   the   domain   of   science   altogether,   truly   enabling   me   to   appreciate   the  “bigger  picture”.  In  fact,  I  think  that  it  is  important  that  medical  students  are  able  to  experience  conferences  such  as  this  one  to  be  able   to   appreciate   the   research   that   is  occurring  across  multiple  different  domains.      On   presenting   my   poster,   the   greatest  challenge  was  to  ensure  that  my  research  was  accessible   for   both   the   layperson   and   the  expert   alike.   The   title   of   my   project  “Cerebrovascular   Amyloid   β   Deposition   is  Associated  with  Astrocytic   Changes   in   the   Tg-­‐SwDI   Mouse   Model   of   Alzheimer's   Disease”  was  a  bit  of  a  mouthful,   so   I   started  by  giving  some   background   to   my   research   and  explaining  how  Alzheimer’s  disease  is  an  urban  challenge.  Once   I   had   stated   that   Alzheimer’s  disease   now   affects   35   million   people  worldwide   and   that   it   has   been   deemed   a  public   health   priority   by   the   World   Health  Organisation,   people   quickly   came   to   appreciate   that   research   in   this   field   is   as  important   as   that   into   sustainable   energies  when   considering   the   theme   of   urban  challenges.   The   other   delegates   and   staff   asked   me   some   testing   but   extremely  interesting  questions,  some  of  which  really  made  me  think  about  my  research   in  a  different  light.  Some  of  the  conversation  was  very  specific  to  my  research  in  terms  of  the  link  between  astrocytes  and  the  aetiology  of  Alzheimer’s  disease,  and  the  exact  methods   that   I   had   used   throughout   my   study.   However,   the   conversation   that   I  most   enjoyed   and   feel   that   I   learnt   the  most   from  was   that   from   a   social   science  aspect,   in   terms   of   what   Alzheimer’s   disease  will   mean   for   the   economy   and   our  future  generations.      Throughout  the  week  we  received  daily  keynote  speeches  from  respected  academics  at  the  University  of  Amsterdam.  I  found  these  to  be  truly  inspiring  and  they  provided  great  insight  into  the  history  of  Amsterdam,  future  challenges  that  both  Amsterdam  and   cities   around   the   world   are   going   to   face,   and   how   research   such   as   ours   is  indispensible   for  “building  healthy,   smart  and  &  creative  cities   for   the   future”   (the  slogan  of  the  conference).      For  me,  the  conference  was  immensely  beneficial  on  a  number  of  levels.  It  enabled  me   to   present   my   research   on   an   international   level   whilst   communicating   with  people  who  took  a  real  interest  in  my  work.  This  opportunity  will  stand  me  in  great  stead  for  future  presentations  I  will  have  to  give  and  will  be  of  considerable  benefit  

Presenting  my  poster  

 

Charlotte  Dewdney  

to  my  future  medical  career.  The  conference  was  a  fantastic  learning  experience;  it  gave  me  the  opportunity  to  learn  about  indigenous  migration  in  Bolivia  and  Peru;  the  healthcare   system   in   China;   and   the   life   of   slum   dwellers   in   India,   Tanzania   and  Kenya   (to   name   only   a   select   few   topics).   Having   only   ever   attended   conferences  within   the   scope   of   Edinburgh   Neuroscience   this   was   a   real   eye-­‐opener   and   has  helped  to  broaden  my  appreciation  of  the  research  that  is  going  on  around  the  world  across   multiple   disciplines.   This   may   sound   like   a   cliché,   but   I   met   some   truly  inspirational  people  and  I  hope  to  keep  in  contact  with  a  number  of  them  via  email  and  social  networking  sites.  Finally,  and  perhaps  most  importantly,  it  has  inspired  me  to  pursue  some  form  of  academic  research  in  my  future  career.      

   I   would   like   to   finish   by   thanking   the   University   of   Edinburgh   for   giving   me   this  wonderful   opportunity   and   providing   generous   funding.   I  would   also   like   to   thank  everyone   in   the   Horsburgh   lab   for   their   patience   and   assistance   throughout   my  project,  in  particular  my  supervisor  Professor  Karen  Horsburgh  for  her  guidance  and  encouragement.      Finally,   I   would   urge   all   undergraduates   to   take   advantage   of   any   opportunity  presented  to  them  to  share  their  research  with  others  on  whatever  level.  Equally,  if  you  ever  visit  Amsterdam  (which  you  most  definitely   should),   you  should  see   it  by  bicycle!    Research  acknowledgements: Supervisor:  Prof.  Karen  Horsburgh Contributors:  Natalia  Salvadores,  Luke  Searcy  and  Fiona  Scott    

         

Edinburgh  students.  A:  Receiving  our  certificates.  B:  At  the  gala  dinner  

B A

 

•  There have been over 90,000 publications within the Alzheimer’s field yet, in the large majority of sporadic cases, the cause remains unknown •  The present findings indicate that there is a temporal increase in Aβ deposition and astrocyte reactivity within the thalamus. Although Western blot analysis did not demonstrate changes in astrocytic protein expression, crucially confocal analysis revealed that cerebrovascular Aβ load increased between 5 and 7 months of age whereas astrocyte colocalisation with blood vessels decreased •  These results suggest that astrocytic endfeet were retracting from the blood vessels. Future work is needed to ascertain how Aβ deposition, astrocyte activation and endfeet retraction integrate, focusing on the implications for CBF and the BBB •  It will be important to question the role of predisposing vascular risk factors in accelerating cerebrovascular AD pathology, with the ultimate goal of opening up new avenues for the treatment of Alzheimer’s disease

Cerebrovascular Amyloid β Deposition is Associated with Astrocytic Changes in the

Tg-SwDI Mouse Model of Alzheimer's Disease

Authors: Charlotte Dewdney, MBChB with BMedSci (Neuroscience), Salvadores N, Searcy L, and Horsburgh K

College of Medicine and Veterinary Medicine, University of Edinburgh, UK. Email: C.Dewdney@sms.ed.ac.uk

•  Alzheimer’s Disease (AD) affects more than 35 million people worldwide and, according to the World Health Organisation, it is a public health priority

•  The cause of AD is still largely unknown but mounting evidence suggests that cerebrovascular amyloid-β (Aβ) deposition is a critical part of the disease

•  Astrocytes, whose endfeet form the blood-brain barrier (BBB), are crucial in maintaining cerebral blood flow (CBF)

•  While the role of astrocytes under normal physiological conditions is relatively well characterised, questions regarding their function and relationship with the cerebrovasculature during AD remain virtually unexplored •  Transgenic mouse models of AD are critical to understanding the pathophysiology of AD. One such model is the Tg-SwDI mouse, which expresses mutant human Aβ precursor protein

Aims 1.  Characterise the temporal accumulation of Aβ in Tg-

SwDI mice 2.  Determine whether the accumulation of

cerebrovascular Aβ in Tg-SwDI mice is associated with pathological changes of astrocytes

3.  Relate changes in astrocytes to alterations in the neurovascular unit

Hypothesis Pathological astrocytic changes are caused by cerebrovascular Aβ deposition in the Tg-SwDI mouse model of AD Methods Using Tg-SwDI mice. Immunohistochemistry: light & confocal microscopy; Western blotting: vessel-enriched fractions; ELISA: insoluble Aβ-40 [C(pg/mg)]. Data analysed using GraphPad Prism, significance: p<0.05

AIMS, HYPOTHESIS & METHODS

CONCLUSIONS & FUTURE WORK

Key reference (Tg-SwDI model): Davis J et al. (2004). The Journal of biological chemistry vol. 279 (19) p. 20296-306. Acknowledgments: I would express my gratitude to all at the Horsburgh lab, CNR, UoE

1.  Aβ deposition increases with age in the thalamus of Tg-SwDI mouse brain

2. Astrocyte reactivity is increased in the thalamus of 9 month old Tg-SwDI mice

RESULTS

Figure 1. An astrocyte at the blood-brain barrier

3"Months"Old""

9"Months"Old""

Aβ

Dep

ositi

on (%

) per

12

6 x

104 µ

m2

3 Month

s

6 Month

s

9 Month

s0

1

2

3

4C57BL/6JTg-SwDI

Age

Aβ Deposition

"! ****!

Figure 2. Representative Tg-SwDI thalamic sections immunostained for Aβ with 6E10 and visualised at 10x magnification. Scale bars = 200µm. The graph displays quantification of Aβ deposition. ****, p < 0.0001 by two-way ANOVA, Bonferroni’s post-hoc test. C57BL/6J: 3M n=8; 6M, n=9; 9M; n=14. TgSwDI: 3M, n=8; 6M, n=10; 9M; n=13

INTRODUCTION

3"Months"Old""

9"Months"Old""

GFAP staining

Age

GFA

P st

aini

ng (%

) per

27

4 x

104 µ

m2

3 Month

s

6 Month

s

9 Month

s0

5

10

15

20

25 "!

****!

"! ****!

Figure 3. Representative Tg-SwDI thalamic sections immunostained for astrocytes with anti-GFAP, visualised at 10x magnification. Scale bars = 200µm. The graph displays quantification of Aβ deposition. ****, p < 0.0001 by one-way ANOVA, Tukey’s post-hoc test. 3M, n=8; 6M, n=10; 9M; n = 13.

3.  No significant correlation between astrocytic protein expression and insoluble Aβ-40 load in 5 and 7 month old Tg-SwDI mice

4.  Cerebrovascular Aβ deposition is increased in 7 month old Tg-SwDI mice

5.  Colocalisation between astrocytes and blood vessels is decreased in the thalamus of 7 month old Tg-SwDI mice

6E10! Anti-collagen IV! 6E10/Anti-collagen IV!

5-m

onth

s-ol

d!7-

mon

ths-

old!

Aβ and blood vessel colocalisation

Thre

shol

ded

Man

der'

s co

loca

lisat

ion

(%)

5 Month

s

7 Month

s0

2

4

6

Age

"!

****!

! Figure 5. Representative confocal photomicrographs showing double labelling with anti-collagen IV antibody (red) and 6E10, anti-Aβ antibody, (green) in the thalamus of Tg-SwDI mice sacrificed at 5 and 7 months of age. The data presented are the mean ± S.E.M (T-bars) of the colocalisation between 6E10-posi t ive Aβ and collagen IV-positive blood vessel. ****, p<0.0001 by an unpaired Student’s t-test. 5M, n=8; 7M, n=8

Astrocytes and blood vessel colocalisation

Thre

shol

ded

Man

der's

co

loca

lisat

ion

(%)

5 Month

s

7 Month

s0

1

2

3

4

5

Age

"!

**!

!Figure 6. Representative confocal photomicrographs showing double labelling with anti-collagen IV antibody (red, visualises blood vessels) and anti-GFAP (green, visualises astrocytes) in the thalamus of Tg-SwDI mice sacrificed at 5 and 7 months of age. T h e d a t a p r e s e n t e d a r e t h e colocalisation between 6E10-positive Aβ and collagen IV-positive blood vessel. **, p<0.0001 by an unpaired Student’s t-test. 5M, n=8; 7M, n=8

β-dystroglycan

Age

Rel

ativ

e ba

nd in

tens

ity o

f β-d

ystr

ogly

can

(nor

mal

ised

by

tubu

lin)

5 month

s

7 month

s0.0

0.5

1.0

1.5

2.0

AQP4

Age

Rel

ativ

e ba

nd in

tens

ity o

f AQ

P4

(nor

mal

ised

by

tubu

lin)

5 month

s

7 month

s0.0

0.5

1.0

1.5

2.0

GFAP

Age

Rel

ativ

e ba

nd in

tens

ity o

f GFA

P (n

orm

alis

ed b

y tu

bulin

)

5 Month

s

7 Month

s0.0

0.2

0.4

0.6

0.8

1.0

!""

Anti – GFAP!

50kDa!

44kDa!

"!Anti – tubulin!

"!

43kDa! Anti - β dystroglycan!

37kDa! Anti - AQP4!

"!"!

"!

5M 7M!!

Figure 4. Astrocytic protein expression: β–dystroglycan, AQP4 and GFAP; within vessel-enriched fractions from Tg-SwDI mice aged 5 and 7 months old was determined using Western blotting. Representative blots from 5-month-old and 7 month old Tg-SwDI mice are demonstrated. Protein expression was quantified as the ratio of the intensity of its band to the intensity of the anti-tubulin signal. No significant differences between ages (p>0.05; Mann-Whitney test). These values were correlated with insoluble Aβ-40 load. There was no significant correlation between any of the markers and Aβ-40. p>0.05; r = Pearson’s correlation coefficient. 5M, n=10; 7M, n=6

Anti-GFAP! Anti-collagen IV! Anti-GFAP/Anti-collagen IV!

5-m

onth

s-ol

d!7-

mon

ths-

old!

Figure 7. Working model illustrating a putative link between vascular Aβ, astrocyte reactivity and AD. Future studies should determine whether astrocytic endfoot retraction leads to dysregulation of CBF and/or BBB dysfunction. Based on Zlokovic 2011.!

ns ns

ns

β-dystroglycan

Aβ40 load C (pg/mg)Rel

ativ

e ba

nd in

tens

ity o

f β-d

ystr

ogly

can

(nor

mal

ised

by

tubu

lin)

0 10000 20000 30000 400000.8

1.0

1.2

1.4

1.6

1.8

2.0 Black: 5-months-old!Red: 7-months-old!r=0.01, !p=0.98!

AQP4

Aβ40 load C (pg/mg)

Re

lativ

e b

an

d in

ten

sity

of A

QP

4

(no

rma

lise

d b

y tu

bu

lin)

0 10000 20000 30000 400001.0

1.2

1.4

1.6

1.8

2.0 r=0.10, p=0.70!

BBB dysfunction ↑Aβ

Neuronal dysfunction

AD

p-tau

Cerebrovascular Aβ Deposition

Reduced CBF

Microglial activation

Classical complement

pathway e.g. IL-1

Astrocyte activation

Endfeet retraction

? ?

GFAP

Aβ40 load C (pg/mg)

Re

lativ

e b

an

d in

ten

sity

of G

FA

P (n

orm

alis

ed

by

tu

bu

lin)

0 10000 20000 30000 400000.0

0.5

1.0

1.5r=-0.23, p=0.40!

Synapse

Astrocyte cell body

Astrocytic endfoot

Capillary lumen

Endothelium

Jjj##

AQP4

Kir4.1

•  There have been over 90,000 publications within the Alzheimer’s field yet, in the large majority of sporadic cases, the cause remains unknown •  The present findings indicate that there is a temporal increase in Aβ deposition and astrocyte reactivity within the thalamus. Although Western blot analysis did not demonstrate changes in astrocytic protein expression, crucially confocal analysis revealed that cerebrovascular Aβ load increased between 5 and 7 months of age whereas astrocyte colocalisation with blood vessels decreased •  These results suggest that astrocytic endfeet were retracting from the blood vessels. Future work is needed to ascertain how Aβ deposition, astrocyte activation and endfeet retraction integrate, focusing on the implications for CBF and the BBB •  It will be important to question the role of predisposing vascular risk factors in accelerating cerebrovascular AD pathology, with the ultimate goal of opening up new avenues for the treatment of Alzheimer’s disease

Cerebrovascular Amyloid β Deposition is Associated with Astrocytic Changes in the

Tg-SwDI Mouse Model of Alzheimer's Disease

Authors: Charlotte Dewdney, MBChB with BMedSci (Neuroscience), Salvadores N, Searcy L, and Horsburgh K

College of Medicine and Veterinary Medicine, University of Edinburgh, UK. Email: C.Dewdney@sms.ed.ac.uk

•  Alzheimer’s Disease (AD) affects more than 35 million people worldwide and, according to the World Health Organisation, it is a public health priority

•  The cause of AD is still largely unknown but mounting evidence suggests that cerebrovascular amyloid-β (Aβ) deposition is a critical part of the disease

•  Astrocytes, whose endfeet form the blood-brain barrier (BBB), are crucial in maintaining cerebral blood flow (CBF)

•  While the role of astrocytes under normal physiological conditions is relatively well characterised, questions regarding their function and relationship with the cerebrovasculature during AD remain virtually unexplored •  Transgenic mouse models of AD are critical to understanding the pathophysiology of AD. One such model is the Tg-SwDI mouse, which expresses mutant human Aβ precursor protein

Aims 1.  Characterise the temporal accumulation of Aβ in Tg-

SwDI mice 2.  Determine whether the accumulation of

cerebrovascular Aβ in Tg-SwDI mice is associated with pathological changes of astrocytes

3.  Relate changes in astrocytes to alterations in the neurovascular unit

Hypothesis Pathological astrocytic changes are caused by cerebrovascular Aβ deposition in the Tg-SwDI mouse model of AD Methods Using Tg-SwDI mice. Immunohistochemistry: light & confocal microscopy; Western blotting: vessel-enriched fractions; ELISA: insoluble Aβ-40 [C(pg/mg)]. Data analysed using GraphPad Prism, significance: p<0.05

AIMS, HYPOTHESIS & METHODS

CONCLUSIONS & FUTURE WORK

Key reference (Tg-SwDI model): Davis J et al. (2004). The Journal of biological chemistry vol. 279 (19) p. 20296-306. Acknowledgments: I would express my gratitude to all at the Horsburgh lab, CNR, UoE

1.  Aβ deposition increases with age in the thalamus of Tg-SwDI mouse brain

2. Astrocyte reactivity is increased in the thalamus of 9 month old Tg-SwDI mice

RESULTS

Figure 1. An astrocyte at the blood-brain barrier

3"Months"Old""

9"Months"Old""

Aβ

Dep

ositi

on (%

) per

12

6 x

104 µ

m2

3 Month

s

6 Month

s

9 Month

s0

1

2

3

4C57BL/6JTg-SwDI

Age

Aβ Deposition

"! ****!

Figure 2. Representative Tg-SwDI thalamic sections immunostained for Aβ with 6E10 and visualised at 10x magnification. Scale bars = 200µm. The graph displays quantification of Aβ deposition. ****, p < 0.0001 by two-way ANOVA, Bonferroni’s post-hoc test. C57BL/6J: 3M n=8; 6M, n=9; 9M; n=14. TgSwDI: 3M, n=8; 6M, n=10; 9M; n=13

INTRODUCTION

3"Months"Old""

9"Months"Old""

GFAP staining

Age

GFA

P st

aini

ng (%

) per

27

4 x

104 µ

m2

3 Month

s

6 Month

s

9 Month

s0

5

10

15

20

25 "!

****!

"! ****!

Figure 3. Representative Tg-SwDI thalamic sections immunostained for astrocytes with anti-GFAP, visualised at 10x magnification. Scale bars = 200µm. The graph displays quantification of Aβ deposition. ****, p < 0.0001 by one-way ANOVA, Tukey’s post-hoc test. 3M, n=8; 6M, n=10; 9M; n = 13.

3.  No significant correlation between astrocytic protein expression and insoluble Aβ-40 load in 5 and 7 month old Tg-SwDI mice

4.  Cerebrovascular Aβ deposition is increased in 7 month old Tg-SwDI mice

5.  Colocalisation between astrocytes and blood vessels is decreased in the thalamus of 7 month old Tg-SwDI mice

6E10! Anti-collagen IV! 6E10/Anti-collagen IV!

5-m

onth

s-ol

d!7-

mon

ths-

old!

Aβ and blood vessel colocalisation

Thre

shol

ded

Man

der'

s co

loca

lisat

ion

(%)

5 Month

s

7 Month

s0

2

4

6

Age

"!

****!

! Figure 5. Representative confocal photomicrographs showing double labelling with anti-collagen IV antibody (red) and 6E10, anti-Aβ antibody, (green) in the thalamus of Tg-SwDI mice sacrificed at 5 and 7 months of age. The data presented are the mean ± S.E.M (T-bars) of the colocalisation between 6E10-posi t ive Aβ and collagen IV-positive blood vessel. ****, p<0.0001 by an unpaired Student’s t-test. 5M, n=8; 7M, n=8

Astrocytes and blood vessel colocalisation

Thre

shol

ded

Man

der's

co

loca

lisat

ion

(%)

5 Month

s

7 Month

s0

1

2

3

4

5

Age

"!

**!

!Figure 6. Representative confocal photomicrographs showing double labelling with anti-collagen IV antibody (red, visualises blood vessels) and anti-GFAP (green, visualises astrocytes) in the thalamus of Tg-SwDI mice sacrificed at 5 and 7 months of age. T h e d a t a p r e s e n t e d a r e t h e colocalisation between 6E10-positive Aβ and collagen IV-positive blood vessel. **, p<0.0001 by an unpaired Student’s t-test. 5M, n=8; 7M, n=8

β-dystroglycan

Age

Rel

ativ

e ba

nd in

tens

ity o

f β-d

ystr

ogly

can

(nor

mal

ised

by

tubu

lin)

5 month

s

7 month

s0.0

0.5

1.0

1.5

2.0

AQP4

Age

Rel

ativ

e ba

nd in

tens

ity o

f AQ

P4

(nor

mal

ised

by

tubu

lin)

5 month

s

7 month

s0.0

0.5

1.0

1.5

2.0

GFAP

Age

Rel

ativ

e ba

nd in

tens

ity o

f GFA

P (n

orm

alis

ed b

y tu

bulin

)

5 Month

s

7 Month

s0.0

0.2

0.4

0.6

0.8

1.0

!""

Anti – GFAP!

50kDa!

44kDa!

"!Anti – tubulin!

"!

43kDa! Anti - β dystroglycan!

37kDa! Anti - AQP4!

"!"!

"!

5M 7M!!

Figure 4. Astrocytic protein expression: β–dystroglycan, AQP4 and GFAP; within vessel-enriched fractions from Tg-SwDI mice aged 5 and 7 months old was determined using Western blotting. Representative blots from 5-month-old and 7 month old Tg-SwDI mice are demonstrated. Protein expression was quantified as the ratio of the intensity of its band to the intensity of the anti-tubulin signal. No significant differences between ages (p>0.05; Mann-Whitney test). These values were correlated with insoluble Aβ-40 load. There was no significant correlation between any of the markers and Aβ-40. p>0.05; r = Pearson’s correlation coefficient. 5M, n=10; 7M, n=6

Anti-GFAP! Anti-collagen IV! Anti-GFAP/Anti-collagen IV!

5-m

onth

s-ol

d!7-

mon

ths-

old!

Figure 7. Working model illustrating a putative link between vascular Aβ, astrocyte reactivity and AD. Future studies should determine whether astrocytic endfoot retraction leads to dysregulation of CBF and/or BBB dysfunction. Based on Zlokovic 2011.!

ns ns

ns

β-dystroglycan

Aβ40 load C (pg/mg)Rel

ativ

e ba

nd in

tens

ity o

f β-d

ystr

ogly

can

(nor

mal

ised

by

tubu

lin)

0 10000 20000 30000 400000.8

1.0

1.2

1.4

1.6

1.8

2.0 Black: 5-months-old!Red: 7-months-old!r=0.01, !p=0.98!

AQP4

Aβ40 load C (pg/mg)

Re

lativ

e b

an

d in

ten

sity

of A

QP

4

(no

rma

lise

d b

y tu

bu

lin)

0 10000 20000 30000 400001.0

1.2

1.4

1.6

1.8

2.0 r=0.10, p=0.70!

BBB dysfunction ↑Aβ

Neuronal dysfunction

AD

p-tau

Cerebrovascular Aβ Deposition

Reduced CBF

Microglial activation

Classical complement

pathway e.g. IL-1

Astrocyte activation

Endfeet retraction

? ?

GFAP

Aβ40 load C (pg/mg)

Re

lativ

e b

an

d in

ten

sity

of G

FA

P (n

orm

alis

ed

by

tub

ulin

)

0 10000 20000 30000 400000.0

0.5

1.0

1.5r=-0.23, p=0.40!

Synapse

Astrocyte cell body

Astrocytic endfoot

Capillary lumen

Endothelium

Jjj##

AQP4

Kir4.1