Science - USA (2022-02-18)

of interaction with the widely expressed Fz
receptors.
In contrast to Wnt7a, the uncovered Gpr124/
Reck agonists were well tolerated in vivo de-
spite the broad expression strategies adopted
in this study. InXenopusand zebrafish, their
ubiquitous expression during early develop-
ment did not cause morphological alterations.

In mice, CNS-wide expression of Gpr124/Reck

agonists did not trigger ectopic Wnt activation

or detectable adverse phenotypes. In addition

to their strict signaling specificity, we suspect

that homeostatic feedback loops maintain Wnt

signaling within carefully controlled physiolog-

ical activity windows. Accordingly, we detected

increased endothelial Wnt signaling only in

dysfunctional BBB vessels, leaving healthy pa-

renchymal vessels unaffected.

Alternative strategies are being pursued to

restore BBB function in disease, including

those relying on protein CÐmediated activa-

tion of endothelial PAR-1 signaling ( 36 ) or

PDGF-C inhibition within the neurovascular

unit ( 37 ). As a contrasting strategy to these

Martinet al.,Science 375 , eabm4459 (2022) 18 February 2022 9 of 11

Fig. 6. Gpr124/Reck agonists
as BBB repair agents in
glioblastoma and stroke
models.(A) MRI monitoring
of tumor volumes after
implantation of 1 × 10^5 Tet-Off
Dkk1 GL261 cells, in the
absence of doxycycline
(Dkk1+,–dox) or the presence
of doxycycline (Dkk1–, +dox).
Doxycycline-exposed mice
were injected intravenously
with AAV-EGFP or AAV-K190A
as indicated. (B) Quantifica-
tion of fibrinogen leakage
into the tumor. Data for WT
GL261 are the same as in
Fig. 5G. (C) Leakage of
transcardially perfused sulfo-
NHS-biotin within the
tumor (Tum.) and the
healthy parenchyma (Par.).
The dashed lines highlight the
tumor margin. (D) Electron
micrographs of tumor and
cortical sections of HRP-
injected mice. White
and black asterisks indicate
tracer within the vessel
lumen and vascular basement
membrane, respectively.
Proportions of vessels with high
lumen–basement membrane
HRP ratios (L/BM) and func-
tional tight junctions (TJ) are
indicated. Arrowheads label the
tight junctions kissing points.
(EtoG) Coimmunostaining of
CD31 together with claudin-5
(E), Mfsd2a (F), or desmin (G).
(HandI) Infarct volumes of
AAV-injected mice subjected to
tMCAO [(H), TTC-stained
sections, 24 hours after stroke],
transient focal endothelin-
1stroke[(I),ET-1,MRIscans,
48 hours after stroke] or per-
manent bengal rose photo-
thrombotic stroke [(I), PT, MRI
scans, 48 hours after stroke].
Data are means ± SD. P<
0.05, P< 0.01, P< 0.001.

CD31

Streptavidin

50 μm 100 μm

**

**

**

0

20

60

40

+ --

Dkk1

+ --

Dkk1

Striatal parenchTumor

Mfsd2a intensity (10^3 a.u)

AAV-EGFP AAV-K190A

Tet-Off GL261

CD31

Claudin-5

Claudin-5

Mfsd2a

Mfsd2a

100 μm

*

**

**

AAV-

EGFP

AAV-

K190A

FHI

*

*

0

4

2

6

8 ET-1

0

5

10

(^15) ns
PT
ET-1 PT
0
0.1
0.2
0.3
Stroke volume
(cm³)
Stroke volume
(mm³)
tMCAO
AAV-EGFP
AAV-K190A 5 mm
E Claudin-5 intensityG
(10^6 a.u)
500 nm 500 nm
500 nm 500 nm
Desmin coverage
(%)

• 0
  5
  10
  15
  20

• --
  Dkk1


• --
  Dkk1
  0
  20
  40
  60


• --
  Dkk1


• --
  Dkk1
  AAV-EGFP AAV-K190A
  Tet-Off GL261
  CD31
  100 μm
  AAV-EGFP AAV-K190A
  Tet-Off GL261
  CD31
  Desmin
  Desmin
  100 μm
  Striatal
  parench Tumor
  Striatal
  parench Tumor


• 
  

  Biotin leakage intensity (10 a.u) 0
  1
  2
  3
  4
  5
  6

  
  

  ---

  
  

  ParTum.
  .
  ParTum.
  .
  PaTum.
  r.
  PaTum.
  r.
  PaTum.
  r.
  --Dkk1
  Parenchyme
  WT GL261 Tet-Off GL261
  AAV-EGFP AAV-K190A AAV-EGFP AAV-K190A
  WT GL261 Tet-Off GL261
  Streptavidin
  Sulfo-NHS-
  Biotin

  
  

• 
  
  Dkk1

• Dkk1

• Dkk1


• 
  

D

Days post implantation (dpi)

Tumor volume (mm³)

0

40

20

60

80

100

120

140

160

0

40

20

60

80

100

120

140

160

0

40

20

60

80

100

120

140

160

14 16 18 20 2224

24%

38%

38%

Days post implantation (dpi)

Tumor volume (mm³)

14 16 1820 22 24

0%

36%

64%

Days post implantation (dpi)

Tumor volume (mm³)

14 16 18 2022 24

4%

96%

0%

A Dkk1+ Dkk1-+ AAV-EGFP Dkk1-+ AAV-K190A

C

B Fibrinogen intensity

(10^3 a.u)

0

5

10

15

20

25

+ -- WT

GL261

AAV-EGFP

AAV-K190A

Dkk1

Dkk1+ Dkk1-

Dkk1+ Dkk1+

Dkk1-

AAV-EGFP

500 nm

Tumor

Cortex

Tumor

Cortex

AAV-K190A

Tumor

500 nm Cortex

500 nm 500 nm

500 nm 500 nm

500 nm 500 nm

Dkk1- Dkk1- Dkk1- Dkk1-

Dkk1-

Dkk1+

Dkk1-

L/BM: 52/52

TJ: 17/17

L/BM: 0/7

TJ: 1/8

L/BM: 71/71

TJ: 29/29

L/BM: 8/18

TJ: 3/10

L/BM: 66/66

TJ: 37/37

L/BM: 27/29

TJ: 20/23

***

***

***

***

RESEARCH | RESEARCH ARTICLE