Brainwide delivery of Gpr124/Reck agonists
in mice
We next evaluated Gpr124/Reck agonists as
BBB-protective agents in mammals. To deliver
the agonists widely throughout the mouse
CNS, we generated PHP.eB adeno-associated
viruses expressing enhanced green fluores-
cent protein (AAV-EGFP), Wnt7a-P2A-EGFP
(AAV-Wnt7a), or Wnt7aK190A-P2A-EGFP (AAV-
K190A) under the control of a constitutive
CAG promoter. Upon intravenous injection of
4×10^11 vg (viral genomes) in 8-week-old mice,
CD31-positive brain vessels were surrounded
by EGFP+cells (Fig. 4A), exposing ECs to local
sources of Wnt7a or Wnt7aK190A. The AAV-
PHP.eB capsid drives expression of the trans-
genes in ~25% of ECs, ~30% of astrocytes, and
~45% of NeuN+neurons (fig. S12A), as reportedpreviously ( 26 ). More than 95% of the desmin+
pericytes were negative for EGFP. Pericytes are
thus, at best, marginal sources of Wnt7a in this
approach (fig. S12B).
Although expressed from widely distributed
cells, Wnt7a and Wnt7aK190Ashowed a more
discrete distribution, with preferential accu-
mulation at the level of CD31-positive vessels,
as well as some scattered parenchymal cellsMartinet al.,Science 375 , eabm4459 (2022) 18 February 2022 4 of 11
Fig. 2. Gpr124/Reck
agonists are unable
to bind Fz autonomously.
(A)GPR124−/−;RECK−/−
HEK293 cells transiently
expressing Wnt7a,
Wnt7aNTD, or Wnt7aK190A
together with Fz5, Reck, or
the empty pCS2 vector
were submitted to Wnt7a
double immunostaining,
before (Wnt7a extracellular,
green/black) and after
(Wnt7a intracellular, pink)
cell permeabilization.
(B) Quantification of (A), as
described in the supple-
mentary materials. (Cand
D) Anti-HA coimmunopreci-
pitation assays after
membrane-impermeable
cross-linking in total lysates
ofRECK−/−HEK293T
cells expressing HA-Reck
(C) or HA-Fz5 (D) together
with Wnt7a, Wnt7aNTD, or
Wnt7aK190A, with or without
expression of untagged
Reck (D). (E) Gpr124/Reck/
Fz1 and Fz5 activity
correlations across the
Wnt7a single-residue variants
collection. (F) Relative STF
luciferase activity in the
presence of Gpr124/Reck/
Fz1 or Fz5 in response to
Wnt7a or Wnt7aK190Ain
autocrine monoculture or
paracrine coculture variants
settings. (GtoI) Gpr124/
Reck/Fz1 or Fz5 STF
activities of Wnt7aK190Awith
combined agonistic muta-
tions (G), activity-increasing
mutations (H), or
alternative substitutions
of Lys^190 (I). Wnt7a and
variants are V5-tagged.
Data are means ± SD.
***P< 0.001; ns,
nonsignificant.
WT
K190AK190GK190SK190LK190PK190DK190EK190R
01.00.5Gpr124/Reck/Fz1 Fz5H Relative luciferase activity+K95A,K255A,K273A,K296A
Gpr124/Reck/Fz1 Fz5(^0) - ++-
4
1
3
5
Relative luciferase activity
2
Wnt7a Wnt7aNTD Wnt7aK190A
Fz5
- ++
-- +- ++
-- +- ++
Reck --
Fz5
(HA)
- ++
- ++
IP HA Wnt7InputsFz5
(HA)Wnt770kDa
55kDa70kDa
55kDa40kDa
35kDa40kDa
35kDan=19 n=3 n=13 n=112Gpr124/Reck/Fz1 activity classesFz5 activitydistributionvery low
<10%low
10<35%moderate
35<70%high
>70%Fz5 activity classesGpr124/Reck/Fz1activity distributionvery low
<10%low
10<35%moderate
35<70%high
>70%n=54 n=23 n=24 n=46FWTK190A +K190AK212AR222AP263AT266AW322ARelative luciferase activityGpr124/Reck/Fz1 Fz501.00.5GAB CDWnt7aWnt7aNTDWnt7aK190ApCS2 Fz5 Reck
Wnt7 ext
Wnt7 intWnt7a Wnt7aNTD Wnt7aK190AExtracellular vs Intracellular signal
(normalized ratio)Fz5
pCS2 Reck
Fz5
pCS2 Reck
Fz5
pCS2 Reck0268104Wnt7aWT WTReck - ++- ++IP HAInputsReck
(HA)Wnt7Reck
(HA)Wnt7130kDa
100kDa130kDa
100kDa40kDa
35kDa40kDa
35kDaNTD WT WTK190AWnt7 extDAPI
Wnt7 int50 μmRelative luciferase activitymono-culture settingWnt secreting cell
=
Wnt reporter cellWnt secreting cell
=
Wnt reporter cellco-culture settingWnt7a or Wnt7aK190A Wnt7a or Wnt7aK190AWnt7a Wnt7aK190AWnt7a Wnt7aK190AWnt7aWnt7aK190A01.00.501.00.5Gpr124/Reck/Fz1 Fz5mono-cultureco-culture
mono-cultureco-culture
mono-cultureco-culture
mono-cultureco-cultureEI****** ns*** ns***RESEARCH | RESEARCH ARTICLE