Cell Stem Cell

ArticleMetformin Activates an Atypical PKC-CBP Pathway
to Promote Neurogenesis
and Enhance Spatial Memory Formation
Jing Wang,1,2 Denis Gallagher,1,2,4,9 Loren M. DeVito,3,9 Gonzalo I. Cancino,1,2 David Tsui,1,5 Ling He,8 Gordon M. Keller,4Paul W. Frankland,3,5,6 David R. Kaplan,2,5,7 and Freda D. Miller1,4,5,6,7,*
1Programs in Developmental and Stem Cell Biology
2Cell Biology
3Neurosciences and Mental HealthHospital for Sick Children, Toronto, ON M5G 1X8, Canada
4McEwen Center for Regenerative Medicine, University Health Network, Toronto, ON M5G 1L7, Canada
5Institute of Medical Science
6Department of Physiology
7Department of Molecular Genetics
University of Toronto, Toronto, ON M5SG 1A8, Canada
8Department of Pediatrics and Medicine, Johns Hopkins Medical School, Baltimore, MD 21287, USA
9These authors contributed equally to this work
*Correspondence: fredam@sickkids.ca
DOI 10.1016/j.stem.2012.03.016

SUMMARY

Although endogenous recruitment of adult neuralstem cells has been proposed as a therapeuticstrategy, clinical approaches for achieving this arelacking. Here, we show that metformin, a widelyused drug, promotes neurogenesis and enhances
spatial memory formation. Specifically, we showthat an atypical PKC-CBP pathway is essential forthe normal genesis of neurons from neural precursors and that metformin activates this pathway topromote rodent and human neurogenesis in culture.
Metformin also enhances neurogenesis in the adultmouse brain in a CBP-dependent fashion, and in sodoing enhances spatial reversal learning in the water
maze. Thus, metformin, by activating an aPKC-CBP
pathway, recruits neural stem cells and enhances
neural function, thereby providing a candidate pharmacological approach for nervous system therapy.
INTRODUCTION
Adult neural stem cells and the new neurons that they generate
play key functional roles in the mammalian brain (Zhao et al.,2008). Intriguingly, growing evidence indicates that neural stem
cells are also recruited in an attempt at endogenous repair in
the injured or degenerating brain (Kernie and Parent, 2010), findings that suggest that if we could exogenously recruit adultneural stem cells, this might provide a novel therapeutic strategy.
Some studies have attempted to do this with growth factors, but
this approach has not been successful in part because of difficulties in delivering growth factors to the nervous system and in part
because these are broadly active and highly potent biologicalentities (Miller and Kaplan, 2012).

One alternative to growth factors are small molecules that
promote stem cell self-renewal and/or differentiation either as
identified by high throughput screens (Li et al., 2012) or bydefining relevant signaling pathways. With regard to the latter,
we recently showed that the CBP transcriptional coactivator
was necessary for optimal differentiation of embryonic neuralprecursors and that CBP ability to promote differentiation
required phosphorylation by atypical protein kinase C (aPKC)(Wang et al., 2010). Intriguingly, in liver cells, the aPKC-CBP
pathway is downstream of AMP kinase (AMPK) and is activatedby the AMPK-activating drug metformin (He et al., 2009), which is
widely used to treat type II diabetes and other metabolic
syndromes. These findings therefore suggest that metformin
might activate aPKCs in neural stem cells and, in so doing, allow
their recruitment in the adult brain.
Here, we have tested this idea. We define distinct roles for
aPKCs i and z in regulating the neural precursor to neuron transition and show that aPKCz regulates the genesis of neurons in
a CBP-dependent fashion. Moreover, we show that metformin
can activate this aPKC-CBP pathway to promote rodent andhuman neurogenesis in culture. Finally, we show that metforminhas similar actions in the adult mouse CNS in vivo, acting toincrease the genesis of neurons in the hippocampus in a CBPdependent fashion and, in so doing, to enhance spatial reversal
learning in a water maze task. Thus, metformin represents a
candidate pharmacological approach for recruitment of neuralstem cells in the adult human brain, a strategy that might be oftherapeutic value for the injured or degenerating nervous system.
RESULTS
Atypical Protein Kinase C Isoforms Differentially
Regulate the Radial Precursor to Neuron Transition
during Embryonic Cortical Neurogenesis
We previously showed that two aPKC isoforms, i and z, areexpressed in the developing murine cortex and that aPKCz isCell Stem Cell 11, 23–35, July 6, 2012 ª2012 Elsevier Inc. 23

Cell Stem CellCD50
% CC3+ve cells

50

scr shaPKCζ shaPKCι sh

EGFP/Hoechst- + + +

B
% condensed nuclei

(-)
(-)

A

Scr sh
aPKCζ
sh #1
aPKCζ
sh #2

Metformin Enhances Neurogenesis via aPKC-CBP

scr shaPKCζ shaPKCι shFigure 1. aPKCs z and i Regulate Cortical
Precursor Differentiation into Neurons in
Culture40

40

βIII tubulin+ve cells(% of control)βIII tubulin+ve cells(% of control)% βIII tubulin+ve cells% βIII tubulin+ve cells% βIII tubulin+ve cells% p-H3+ve cells

Scr sh
aPKCι
sh #1
aPKCι
sh #2

(-)
(-)

+

CC3/EGFP(A) Western blot analysis of HEK293 cells cotransfected with a flag-tagged expression con30
30
flag
struct for murine aPKCz (top) or aPKCi (bottom)
and either a scrambled shRNA (Scr) or one of two
20
20
ERK
shRNAs specific for aPKCz (top) or aPKCi
10
10(bottom). Blots were probed for the flag tag (top) or0
aPKCi (bottom) and reprobed for total ERK as00
a loading control.
(B–D) Precursors were cotransfected with plasaPKCι - + + + +EFmids encoding EGFP and aPKCz, aPKCi, or
scr shaPKCι
scrambled shRNAs for 2 days, immunostained for50
EGFP/Hoechst βIII tubulin/EGFP
aPKCζ sh
EGFP (C, green) and cleaved caspase-3 (C, red),aPKCι sh40
ERK
and counterstained with Hoechst 33258 (C, blue)30
and the percentage of transfected cells with condensed, apoptotic morphology (B) or expressing
20
cleaved caspase-3 (CC3) (D) was determined. In
10(C) the same field is shown in both panels, and0arrows denote a double-labeled cell with a
scr shscr shG
H
I
condensed nucleus. p > 0.05; n = 3 independent
aPKCι
sh
aPKCζ
sh
scr shexperiments, pooled. Scale bars represent 25 mm.
*
120
*
80
120
aPKCζ sh(E) Percentage of transfected, phospho-histone**aPKCι sh**H3-positive cells in cultures cotransfected with
90
90
60
EGFP and aPKCz, aPKCi, or scrambled shRNAs
for 2 days. p > 0.05; n = 3 independent experi60
60
40
ments, pooled.
(F) Precursors cotransfected with plasmids en30
30
20
coding EGFP and scrambled shRNA for 3 days,
immunostained for EGFP (green) and bIII-tubulin000(red), and counterstained with Hoechst 33258
EV
haPKCζEV
haPKCι
(blue). The same field is shown in both panels, andarrows denote double-labeled cells. Scale bars
JK
scr shControl
represent 50 mm.
8080aPKC ζ/ι shaPKCi(G) Percentage of transfected, bIII-tubulin-positive
cells in precursor cultures cotransfected with
60
60*
EGFP and aPKCz, aPKCi or scrambled shRNAs
for 3 days, as shown in (F). **p < 0.01; n = 4–5
40
40*
independent experiments, pooled.
20
20(H and I) Percentage of bIII-tubulin-positive transfected neurons in precursor cultures 3 days after
00cotransfection with aPKCi (H) or aPKCz (I) shRNAand expression plasmids for EGFP and humanaPKCi (H) or aPKCz (I). The number of transfected neurons under control conditions was normalized to 100%. *p < 0.05; n = 3 independent experiments
each, pooled.
(J) Percentage of bIII-tubulin-positive cells in precursor cultures treated with myristoylated aPKC pseudosubstrate peptide inhibitor (2 mM) or water, as a control,for 3 days. *p < 0.05; n = 4 independent experiments, pooled.
(K) Percentage of transfected, bIII-tubulin-positive cells in precursors cotransfected with EGFP and aPKCz plus aPKCi shRNAs, or with scrambled shRNA for
3 days, and analyzed as shown in (F). *p < 0.05; n = 3 independent experiments, pooled.
Error bars represent SEM. See also Figure S1.

aPKCζ
flag

essential for the genesis of glial cells from cultured neural precursors (Wang et al., 2010). To ask whether aPKCs were also essential for neurogenesis, we studied cultured radial precursors fromthe E12.5 murine cortex that generate neurons first and glia
second. We knocked down the aPKC isoforms in these precursors with previously characterized shRNAs (He et al., 2009;
Wang et al., 2010), confirming their efficacy with two experiments. First, we cotransfected them individually into HEK293
cells along with aPKCi or z expression plasmids, as relevant;western blots confirmed that the shRNAs effectively knocked
down aPKC isoform expression relative to either a scrambled
shRNA or no shRNA (Figure 1A). Second, we cotransfectedeach shRNA with an EGFP expression plasmid into cultured
cortical precursors and immunostained them 2 days later for
24 Cell Stem Cell 11, 23–35, July 6, 2012 ª2012 Elsevier Inc.

aPKCi or z; the proportion of EGFP-positive cells expressing
no or low aPKCi or z was increased by approximately 2-foldand 1.7-fold, respectively (Figures S1A and S1B available
online).
We then used these shRNAs to ask what role aPKCs played inneural precursors. Cortical precursors were cotransfected with
an EGFP plasmid and either aPKCz shRNA#1 or aPKCishRNA#1 and analyzed 3 days later when many precursors
had differentiated into neurons. As a control, we used a scrambled shRNA that had no effect on cortical precursor biology(Figure S1C). Quantification of condensed, apoptotic nuclei(Figures 1B and 1C) and immunostaining for the apoptotic
marker cleaved caspase-3 (Figures 1C and 1D) indicated thatneither aPKC isoform was essential for cell survival. Similarly,

Cell Stem Cell
Metformin Enhances Neurogenesis via aPKC-CBP

Figure 2. Genetic Knockdown of aPKCs
Differentially Regulates Neural Precursor
Differentiation In Vivo

B

A

C
D

E

F

G

H

I

J

K

L

M

N

immunostaining of transfected cultures for two markers of proliferating cells, phosphohistone H3 (Figure 1E) and Ki67 (Figure S1D), demonstrated that cell division was not altered byeither shRNA. In contrast, both aPKC isoforms were necessary
for neurogenesis; knockdown of either aPKCz or aPKCi significantly decreased the proportion of bIII-tubulin-positive newlyborn neurons (Figures 1F and 1G).
We confirmed the specificity of this neurogenesis phenotype inthree ways. First, we showed that aPKCi shRNA#2 and aPKCzshRNA#2 also decreased neurogenesis without affecting proliferation (Figures S1E and S1F). Second, we rescued thedecreased neurogenesis by coincidently expressing humanaPKCi or z, which are not targeted by the murine shRNAs (Figures
1H and 1I). Third, we inhibited total aPKC activity by using a(A) Immunostaining with an antibody for total aPKC
(red; Hoechst 33258 counterstain is blue) on
coronal E12 cortical sections. The boxed area isenlarged at right to show aPKC immunoreactivity
adjacent to the ventricle (V). Control is immunostained with secondary antibody alone. Scale bars
represent 50 mm (left) and 25 mm (right).
(B–N) E13/14 cortices were electroporated withplasmids encoding EGFP and aPKCz (E–J), aPKCi(K–N), or scrambled shRNAs and sectioned coronally for analysis 3 days later.
(B) Immunostaining for EGFP (white) in sections
electroporated with scrambled (left) or aPKCz(right) shRNAs. White lines denote region boundaries. V denotes the ventricle. Scale bars represent
250 mm.
(C) Percentage of EGFP-positive cells within
VZ/SVZ, IZ, and CP as shown in (B). *p < 0.05;***p < 0.001; n = 4–6 brains each.
(D) Immunostaining for EGFP (green) and CC3(red; blue is Hoechst 33258) in the section electroporated with scrambled shRNA. The boxed area
at left is shown at higher magnification at right.
Arrows denote a CC3-positive cell. V denotes theventricle. Scale bars represent 100 mm (left) and25 mm (right).
(E) Confocal images of cortical sections electroporated with aPKCz shRNA (top) or scrambled
shRNA (all other panels) and double-labeled for
EGFP (green) and Ki67, HuD, Pax6, Sox2, or Tbr2,
as indicated (all red). In all cases, the same field isshown in left and right panels, and the arrows
denote double-labeled cells. All panels show the
VZ/SVZ except for the HuD panel, which shows the
CP. Scale bars represent 20 mm.
(F–J) Percentage of transfected Ki67-positive (F),
HuD-positive (G), Pax6-positive (H), Sox2-positive(I), or Tbr2-positive (J) cells in cortices electroporated with aPKCz or scrambled shRNAs andimmunostained as in (E). **p < 0.01; ***p < 0.001;
n = 4–6 brains each.
(K–N) Percentage of transfected, HuD-positive(K), Pax6-positive (L), Sox2-positive (M), or Tbr2positive (N) cells in cortices electroporated with
aPKCi or scrambled shRNAs and immunostained
as in (E). *p < 0.05; **p < 0.01; n = 4–6 brains each.
Error bars represent SEM. See also Figure S2.

myristoylated aPKC pseudosubstrate peptide inhibitor that is
efficacious in cortical precursors (Wang et al., 2010) and thatblocks the activity of both isoforms. This inhibitor significantly
reduced neurogenesis at 3 days (Figure 1J), and the magnitudeof this decrease was similar to that seen when precursors were
cotransfected with aPKCi and z shRNAs together (Figure 1K).
Thus, aPKCs normally regulate cortical neurogenesis in culture.
We therefore asked whether aPKCs also regulate cortical neurogenesis in vivo; our previous biochemical analysis showedcortical expression of both aPKC isoforms from E12, the earliest
time point examined, until birth (Wang et al., 2010). Immunostaining with a pan-aPKC antibody demonstrated that aPKCs
were apically localized in precursors that line the ventricles inthe E12 cortex (Figure 2A), as previously reported (Imai et al.,Cell Stem Cell 11, 23–35, July 6, 2012 ª2012 Elsevier Inc. 25

Cell Stem Cell
Metformin Enhances Neurogenesis via aPKC-CBP

2006; Costa et al., 2008), and consistent with an association
between aPKCi and the apical polarity complex in radial precursors (Costa et al., 2008; Bultje et al., 2009). We therefore
knocked down each aPKC isoform individually in radial precursors by in utero electroporation of the shRNAs together witha cytoplasmic EGFP expression plasmid into E13/14 cortices(Wang et al., 2010). This approach electroporates radial precursors that line the ventricle, many of which will then differentiate
into neurons in the ventricular zone/subventricular zone (VZ/
SVZ), prior to their migration through the intermediate zone (IZ)
to the cortical plate (CP). At later time points the same precursor
pool generates glial cells and adult neural stem cells.
Analysis of cortices 3 days postelectroporation demonstrated
that both aPKC isoforms regulated the radial precursor to neurontransition but that they did so in different ways. For aPKCz, analysis of the location of electroporated cells showed a decrease in
EGFP-positive cells within the CP and a small increase in the IZ(Figures 2B and 2C). To ask about the cellular basis of thisphenotype, we characterized the survival, proliferation andidentity of the electroporated cells. Immunostaining for cleaved
caspase-3 (Figure 2D) showed that survival was unaffected,
with only five to ten positive cells per cortical section with orwithout aPKCz knockdown. Immunostaining for Ki67 (Figure 2E)
showed that the percentage of proliferating cells was alsounchanged (Figure 2F). In contrast, immunostaining for theneuron-specific protein HuD demonstrated that aPKCz knockdown reduced the number of EGFP-positive neurons (Figures
2E and 2G), thereby explaining the decrease in EGFP-positive
cells in the CP. To ask whether undifferentiated precursors
were coincidentally increased, we immunostained sections forthe radial precursor markers Pax6 and Sox2 (Figure 2E); aPKCz
knockdown increased both populations (Figures 2H and 2I). In
contrast, immunostaining for Tbr2, which marks the basal
progenitor transit-amplifying cells in this system, showed thatthey were unaffected (Figures 2E and 2J). Thus, aPKCz promotes differentiation of radial precursors into neurons in vivo.
A similar analysis for the aPKCi knockdown showed that it did
not affect the distribution of EGFP-positive transfected cells(Figures S2A and S2B), nor did it affect cleaved caspase-3positive cells. There was also no significant change in HuD-positive electroporated neurons, although there was a trend toward
a decrease (Figure 2K). In contrast, aPKCi knockdown significantly altered precursor populations. Pax6-positive and Sox2positive radial precursors were decreased (Figures 2L and 2M),
and Tbr2-positive basal progenitors were correspondingly
increased (Figure 2N). Thus, aPKCi functions to maintain radial
precursors and to inhibit their transition to basal progenitors,
consistent with its role in the polarity complex.
These data argue that aPKCs z and i control cortical neurogenesis via distinct mechanisms. In this regard, we previously
showed that aPKCz but not i regulated cortical gliogenesis byphosphorylating and activating CBP (Wang et al., 2010). We
confirmed this previous result (Figures S1G and S1H) and thendetermined whether CBP was also downstream of aPKCz withregard to neurogenesis. We did this genetically, asking whether
a CBP phosphomimic that was mutated at the aPKC phosphorylation site could rescue the decrease in neurogenesis caused
by aPKCz knockdown. For comparison, we examined aPKCiknockdown. Quantification of cotransfected precursor cultures
26 Cell Stem Cell 11, 23–35, July 6, 2012 ª2012 Elsevier Inc.

showed that the CBP phosphomimic rescued the decreased
neurogenesis seen with aPKCz but not aPKCi knockdown (Figure 3A). Thus, aPKCz enhances neurogenesis by activating CBP.
Metformin Activates Atypical PKC and Promotes
Neurogenesis in Rodent Cortical Precursors
Our data indicate that the aPKC-CBP pathway promotes neurogenesis. Because metformin activates this pathway in liver cells(He et al., 2009), we asked whether it would also do so in corticalprecursors. Precursors were cultured for 1 day and exposed to
15 mM metformin for 15 min, and lysates were probed with antibodies for total aPKC and for aPKC phosphorylated at threonine
403, which is critical for kinase activity (Messerschmidt et al.,2005). Metformin caused a modest but reproducible increase
in aPKC threonine 403/410 phosphorylation (Figure 3B). We
then cultured precursors in 500 mM metformin and asked how
this affected their biology. Analysis of cleaved caspase-3 andcells with condensed nuclei at 2 days showed that cell survival
was unaffected by metformin (Figures 3C and 3D). Similarly,
cell division, as monitored by phosphohistone H3-positive cells
at 2 days, was unchanged (Figure 3E). In contrast, metformin
almost doubled the percentage of bIII-tubulin-positive neurons
present at 3 days (Figures 3F and 3G), an increase that was
accompanied by a corresponding decrease in the proportionof Pax6-positive (Figure 3H) and Sox2-positive (Figure 3I)
precursors.
We next asked whether the metformin-induced increase inneurogenesis required the aPKC-CBP pathway. Initially, we
asked about the aPKCs, transfecting precursors with shRNAs
for both isoforms at levels that did not significantly decrease
basal neurogenesis (Figure 3J), presumably because the remaining aPKC levels were sufficient for maintenance. We thencultured these transfected precursors with or without metformin.
Analysis of transfected bIII-tubulin-positive neurons after 3 days
(Figure 3J) showed that metformin increased neurogenesis andthat the aPKC shRNAs inhibited metformin-induced neurogenesis. We then asked about CBP, by using previously characterized siRNAs (Wang et al., 2010). Precursors were transfectedwith CBP or scrambled siRNA and were cultured in metforminfor 3 days. CBP knockdown completely inhibited the metformin-induced increase in neurogenesis (Figure 3K).
These results indicate that metformin activates the aPKC-CBP
pathway in cortical precursors, thereby enhancing neurogenesis. We therefore asked whether it also promotes gliogenesis,
as would be predicted by our previous results (Wang et al.,2010). To do this, we added metformin to precursor cultures
on day 2 and analyzed them on day 7, when endogenous gliogenesis has commenced. Metformin increased genesis of both
GFAP-positive astrocytes and A2B5-positive oligodendrocyte
precursors (Figure 3L), suggesting that metformin activates an
aPKC-CBP pathway to generally promote cortical precursor
differentiation.
Finally, we asked whether metformin affected cortical precursor differentiation in vivo by electroporating E13/14 embryoswith an EGFP expression construct and by treating their motherswith metformin daily for 4 days, starting 1 day prior to electroporation. Analysis 3 days postelectroporation showed that thelocation of EGFP-positive cells was similar in progeny of control
versus metformin-treated mothers (Figures S3A and S3B).

Cell Stem Cell
Metformin Enhances Neurogenesis via aPKC-CBP

scr shaPKCζ shaPKCι shMet

B
Control

A

Met
Control

Figure 3. Metformin Activates an aPKC-CBP
Pathway to Promote Genesis of Neurons
from Murine Cortical Precursors**(T410/403)
p-aPKCζ/ι

160*% p-H3+ve cells% condensed nuclei

βIII tubulin+ve cells(% of control)% CC3+ve cells**% Sox2+ve cells+ve cells(% of control)βIII tubulin+ve cells(% of control)% βIII tubulin+ve cells% Pax6+ve cells

Metformin% βIII tubulin+ve cells

Control*relative densitometry(% of control)(A) Percentage of transfected, bIII-tubulin-positive
cells in precursor cultures 3 days after cotransfection with EGFP and aPKCz, aPKCi, or scrambled
120
90
aPKC
shRNAs plus or minus a CBP phosphomimic. The
number of neurons generated with the scrambled
80
60
ERK
shRNA was normalized to 100%. *p < 0.05;
40**p < 0.01; n = 3 independent experiments.
30(B) Western blot for aPKC z/i phosphorylation at
00T410/403 in cortical precursors treated with waterEV
CBPS436D(as a control) or metformin (15 mM) for 15 min. Blot
was reprobed for total aPKC and for ERKs as aCD
E 50loading control. The graph shows the relative level
50
Met
Met
Met
50
Control
Control
Controlof phosphorylation of aPKC normalized to total
40
40
40
aPKC, as determined by densitometry. *p < 0.05;
n = 3 independent experiments.
3030
30(C–I) Cultured cortical precursors were treated with
20
20
20
500 mM metformin (Met) or water (Control) for 2
10(C–E) or 3 (F–I) days.
10
10(C and D) Percentage of transfected cells that were000positive for cleaved caspase-3 (CC3) (C) or that
F βIII tubulin/Hoechst
displayed condensed, apoptotic nuclei (D). p > 0.05;
n = 3 independent experiments, pooled.
HI
G(E) Percentage of cells positive for the proliferation
Met
Met
Met
Control
Control
Controlmarker phospho-histone H3. p > 0.05; n = 3 independent experiments, pooled.
200
50
50
*
(F) Immunostaining (red; blue is Hoechst 33258 to40
40
show nuclei) for bIII-tubulin in precursor cultures150*
treated with water or metformin for 3 days. Scale
30
30
100
bars represent 25 mm.
**
2020(G) Percentage of bIII-tubulin-positive neurons in
50
1010
precursor cultures as shown in (F), where thenumber of neurons under control conditions was000
normalized to 100%. *p < 0.05; n = 5 independent
experiments, pooled.
(H and I) Percentage of Pax6-positive precursors
K
L
J
Met
Met
Met
(H) or Sox2-positive precursors (I) in cultures
Control
Control
Control
treated as in (F). *p < 0.05; **p < 0.01; n = 4 inde80
200
80
**
*
pendent experiments, pooled.
*(J and K) Percentage of bIII-tubulin-positive
60
150
60
*
neurons in cultures treated for 2 days with 500 mM
metformin or water (Control) 18 hr after co40100
40transfection with EGFP and either (J) aPKCz plus
20
50
20
aPKCi shRNAs or (K) CBP siRNAs. As controls,
cells were cotransfected with scrambled shRNA or000siRNA. In (J), only half as much aPKC shRNA was
GFAP
A2B5
Scr-siRNA CBP-siRNA
Scr sh aPKC ζ/ ι shused as in Figure 1K. *p < 0.05; **p < 0.01; n = 3 (J)or 4 (K) independent experiments, pooled.
(L) Percentage of GFAP-positive and A2B5-positive cells in precursor cultures treated with water or 500 mM metformin from day 2 to day 7, where the numbers ofastrocytes or oligodendrocyte precursors under control conditions were normalized to 100%. *p < 0.05; n = 3 independent experiments, pooled.
Error bars represent SEM. See also Figure S3.

120

Although the proportion of Pax6-positive precursors was
unchanged (Figure S3D), there was a small but significant
increase in HuD-positive neurons (Figure S3C), consistent withthe culture data.
Metformin Enhances Neurogenesis from Human
ESC-Derived Neural Precursors
If metformin could promote neurogenesis from human neuralstem cells as it does from murine, then this might serve as thebasis for a potential therapeutic strategy. To address this possibility, we developed a method for generating forebrain neuralprecursors from human embryonic stem cells (hESCs). Embryoid
bodies (EBs) were generated from hESCs and were treated for
12 days with Dorsomorphin, an inhibitor of BMP signaling, andSB43152, an inhibitor of Lefty/Activin/TGF-b signaling (Chambers et al., 2009; Zhou et al., 2010). These differentiated EBs
were dissociated and adhered to a laminin-coated surface.
Immunostaining showed that approximately 60%–70% of the
freshly plated cells expressed Pax6 (Figure 4A). In addition,
they expressed other markers consistent with a forebrain precursor identity including nestin (Figure 4B), the radial precursor
marker BLBP (data not shown), and the cortical marker Emx1
Cell Stem Cell 11, 23–35, July 6, 2012 ª2012 Elsevier Inc. 27

Cell Stem Cell
Metformin Enhances Neurogenesis via aPKC-CBP

A

Hoechst

Pax6

Merge

aPKCi
ControlG

B

Hoechst

NestinβIII tubulin+ve cells(% of control)

100

Merge

80
60

*

40
20
0

C

Hoechst

Emx1

Merge

Met
ControlH% βIII tubulin+ve cells

30

*

24

D

HoechstβIII tubulin

18

Merge

12
6

Figure 4. Metformin Activates aPKCs to
Enhance the Genesis of Neurons fromhESC-Derived Forebrain Neural Precursors(A–C) hESCs were cultured as embryoid bodies in
the presence of 2 mM Dorsomorphin and 10 mM
SB43152 for 12 days, plated, and immunostainedfor Pax6 (A), nestin (B), and Emx1 (C) (all red; blue
is Hoechst 33258). Scale bars represent 50 mm.
(D–F) Cells were cultured adherently for a further
10 days and immunostained for bIII-tubulin (D) andTbr1 (E) or V-GLUT1 (F). Scale bars represent
50 mm.
(G–I) Neuralized embryoid bodies were plated withand without 2 mM aPKC pseudosubstrate inhibitor
peptide (G), with and without 500 mM metformin(H), or with 500 mM metformin plus or minus theaPKC inhibitor (I) for 4 days, immunostained forbIII-tubulin, counterstained for Hoechst 33258,
and the relative proportion of bIII-tubulin-positive
neurons was determined. In (G), numbers ofcontrol neurons in each experiment were normalized to 100%. *p < 0.05, ***p < 0.001; n = 3 independent experiments.
Error bars represent SEM.

0E

βIII tubulin

Tbr1

Merge

Met+aPKCi
Met
ControlI% βIII tubulin+ve cells

40
30

* ***

(Figure 4I). Thus, as seen with murine
precursors, metformin acts via aPKCs topromote human neurogenesis in culture.

Metformin Promotes Neurogenesis
from Adult Forebrain Neural
20
Precursors
To determine whether metformin also
10
regulates adult neurogenesis, we studied
neural precursor cells (NPCs) in the lateral
0
ventricle SVZ, because some of thesederive from embryonic cortical precur(Figure 4C). To ask whether these precursors would generate sors (Merkle et al., 2004). Initially, we confirmed that forebrainneurons, we differentiated them for 10 days in neural induc- NPCs express aPKCs, taking advantage of the fact that theytion medium (see Experimental Procedures). Immunostaining will expand as neurospheres in culture. Immunostaining of
showed that the Pax6-positive cells were replaced by morpho- primary SVZ neurospheres showed that most cells displayed
logically complex glutamatergic neurons that expressed bIII- aPKC immunoreactivity in both the cytoplasm and nucleus
tubulin (Figure 4D), the glutamatergic neuron marker VGLUT1 (Figure 5A). We confirmed that this was also true in vivo by(Figure 4F), and the cortical neuron marker Tbr1 (Fig- immunostaining coronal sections through the adult cortex.
ure 4E). Thus, as reported for murine ESCs (Bibel et al., 2007), Detectable aPKC immunoreactivity was seen in many neurons
in the absence of specific inductive signals, neuralized hESCs and white matter tracts, as previously reported (Naik et al.,make forebrain radial precursors that generate glutamatergic 2000; Oster et al., 2004) and was also observed in GFAPneurons.
positive cells within the SVZ (Figures S4A and S4B), probably
To ask whether aPKCs were important for human neurogene- type B NPCs.
sis, we cultured dissociated hESC-derived neural precursors
Forebrain NPCs normally generate interneurons for the olfacwith or without the myristoylated aPKC peptide inhibitor for tory bulb. We therefore asked whether metformin increased
4 days. This inhibitor had no effect on total cell number (quanti- olfactory neurogenesis; adult mice were injected with BrdU five
fied as cells/field; p > 0.05), but it decreased bIII-tubulin-positive times at 3 hr intervals to label dividing cells and were then treatedneurons by almost 2-fold (Figure 4G). We then asked whether with 200 mg/kg metformin daily for 21 days. Immunostaining of
metformin enhanced neurogenesis by activating aPKC in human serial sections through the main olfactory bulbs of these mice
forebrain precursors. Cells were grown for 4 days with or without (Figure 5B) showed that metformin significantly increased themetformin plus or minus the myristoylated aPKC inhibitory number of BrdU-positive, NeuN-positive olfactory neurons in
peptide. This analysis showed that 500 mM metformin signifi- the granule cell layer (Figure 5C). We then asked whether metforcantly increased bIII-tubulin-positive newly born neurons (Fig- min had any impact on the number of SVZ NPCs. Mice were inure 4H) and that inhibition of the aPKCs abolished this increase jected with metformin daily for 7 days and coinjected withF

βIII tubulin

V-GLUT1

Merge

28 Cell Stem Cell 11, 23–35, July 6, 2012 ª2012 Elsevier Inc.

Cell Stem Cell
Metformin Enhances Neurogenesis via aPKC-CBP

100 mg/kg BrdU on the final day. Analysis of coronal forebrain
sections 24 hr later demonstrated that metformin significantly
enhanced the number of BrdU-labeled cells (Figures 5D and
5E) and the number of cells double-labeled for BrdU and Ki67
(Figures 5D and 5F) over this time frame. Thus, metforminenhanced adult forebrain neurogenesis.

A

B
C

E
D

F

Figure 5. Metformin Promotes Neurogenesis in the Adult Murine
Olfactory Bulb
(A) Primary passage adult SVZ neurospheres immunostained for total aPKCs(red; blue is Hoechst 33258). Control was immunostained with normal rabbit
IgG and the same secondary antibody. Arrows denote aPKC-positive cells.
Scale bar represents 20 mm.
(B) Immunostaining for BrdU (green) and NeuN (red) in coronal sections
through the main olfactory bulb of mice injected with BrdU five times at 3 hr
intervals and treated with 200 mg/kg metformin or PBS (Control) daily for
21 days. Right panels are higher magnifications of the boxed areas. Arrows
denote double-labeled cells. Scale bars represent 200 mm (left) and 50 mm(right).
(C) Total number of BrdU-positive, NeuN-positive newborn neurons in control
versus metformin-treated olfactory bulbs as determined from sections similar
to those in (B). **p < 0.01; n = 5 animals each.
(D) Immunostaining for BrdU (green) and Ki67(red) in coronal SVZ sections ofmice injected with 200 mg/kg metformin or PBS (Control) daily for 7 days, andcoinjected with BrdU on the seventh day, 24 hr prior to analysis. Right panels
show higher magnifications of the boxed areas. Arrows denote double-labeledcells. Scale bars represent 200 mm (left) and 50 mm (right).
(E and F) Quantification of BrdU-positive (E) or BrdU and Ki67 double-positive(F) cells in the SVZ of control versus metformin-treated animals, determinedfrom four anatomically matched coronal sections similar to those shown in (D).
**p < 0.01; n = 5 animals each.
Error bars represent SEM. See also Figure S4.

Metformin Enhances Hippocampal Neurogenesis
in a CBP-Dependent Fashion
Because metformin enhanced the genesis of olfactory neurons,
we asked whether it also increased adult hippocampal dentate
gyrus neurogenesis. Immunostaining of hippocampal sections
showed that aPKCs were detectably expressed in most dentate
gyrus cells (Figure S4C) including some GFAP-positive cells
within the subgranular zone (SGZ; Figure 6A), probably adult
NPCs. We therefore asked whether metformin enhanced
hippocampal neurogenesis; adult mice were injected daily with
metformin and BrdU for 3 days, and then were injected with metformin alone for 9 additional days. Immunostaining of hippocampal sections (Figure 6B) demonstrated that metforminsignificantly increased the number of BrdU-positive, NeuNpositive newly born granule neurons by approximately 30% (Figure 6C). Consistent with this, we observed an increase in cells
expressing doublecortin, a marker of newborn neurons (Figures
6D and 6E). Thus, metformin enhanced adult hippocampal
neurogenesis.
We also asked whether metformin had an impact on the hippocampal NPC pool, immunostaining sections from the same
experiments for BrdU and for GFAP, which is expressed in
SGZ NPCs and in astrocytes in the hilus. This analysis showedthat metformin did not significantly change GFAP-positive,
BrdU-positive NPCs in the SGZ (Figure 6F). It also showed that
virtually all of the GFAP-positive, BrdU-positive cells werelocated in the SGZ, with very few double-labeled hilar astrocytes
in either controls or metformin-treated animals (an average oftwo versus seven cells/dentate gyrus in control versus metformin-treated mice). We did not detect BrdU-positive cells coexpressing the oligodendrocyte marker GalC in either control or
metformin-treated hippocampi. To confirm that metformin did
not alter NPC pools, we injected mice with metformin daily for
7 days and coinjected them with BrdU on the final day (Figure 6G). Immunostaining of serial sections showed that thistreatment did not alter the number of proliferating BrdU- andKi67-positive cells (Figure 6H).
Our culture data indicate that metformin promotes neurogenesis via an aPKC-CBP pathway. To ask whether the same
pathway is important in vivo, we used mice that are heterozygous for CBP (Kung et al., 2000). Previous work indicates thatthe adult brain in cbp+/ mice is morphologically normal (Alarcón
et al., 2004; Wang et al., 2010), as is basal hippocampal neurogenesis (Lopez-Atalaya et al., 2011). We first confirmed this latter
finding by administering BrdU for 3 days to 2-month-old cbp+/+
and cbp+/ littermates. Quantification of BrdU/NeuN-positive
newborn neurons 9 days later showed that basal hippocampal
neurogenesis was similar in mice of both genotypes (Figure
6J), as reported (Lopez-Atalaya et al., 2011). We then asked
whether metformin required normal levels of CBP to enhancehippocampal neurogenesis. Seven-month-old cbp+/+ andcbp+/ littermates were treated with metformin and BrdU for
Cell Stem Cell 11, 23–35, July 6, 2012 ª2012 Elsevier Inc. 29

Cell Stem Cell
Metformin Enhances Neurogenesis via aPKC-CBP

A aPKCζ/ι/
HoechstSGZ

aPKCζ/ι/GFAP/
Control
HoechstFigure 6. Metformin Promotes Neurogenesis in the Adult Murine Hippocampus ina CBP-Dependent Fashion

B NeuN/BrdU

Control
Metformin

total BrdU/GFAP+ve
cells within SGZ
Control

total DCX+cells/hippocampus

Metformin

total Brdu/NeuN+cells/hippocampus

total Brdu/NeuN+
cells/hippocampus

total BrdU/Ki67+ve
cells/hippocampus

total DCX+ve cells/hippocampus

total BrdU/NeuN+ve
cells/hippocampus(A) Confocal image of a coronal adult dentate
gyrus section immunostained for total aPKC (red)and GFAP (green). Arrows denote a doublelabeled cell in the SGZ. The control was immunostained with normal rabbit IgG and the same
Met
Control
Metformin
C
D
Controlsecondary antibody. Scale bar represents 20 mm.
2500***(B) Coronal hippocampal dentate gyrus sections
2000
from mice injected with metformin or PBS (control)
1500
daily for 12 days, with BrdU coinjection for the first1000
3 days. Sections were immunostained for BrdU
(green) and NeuN (red), and insets show higher
500
magnifications of the boxed areas. Scale bar
Control
Metformin0
represents 200 mm.
Ki67/BrdU
F
G
E
Met
Met
(C) Total number of BrdU-positive, NeuN-positive
Control
Control
newborn neurons in PBS (Control)- versus met120
5000*
formin (Met)-treated hippocampi determined from4000
sections as in (B). ***p < 0.001; n = 9 animals each.
90(D) Hippocampal dentate gyrus coronal sections
3000
60
from adult mice treated as in (B) and immuno2000
stained for doublecortin (DCX). Arrows denote
30
1000
positive cells. Scale bar represents 50 mm.
(E) Total number of doublecortin-positive neurons
00
in PBS (Control)- versus metformin (Met)-treated
NeuN/BrdU
I
hippocampi determined from sections as in (D).
H
Met
*p < 0.05; n = 9 animals each.
Control(F) Total number of BrdU-positive, GFAP-positive1000
cells within the SGZ of PBS (Control)- versus
800
metformin (Met)-treated hippocampi determined
600
from sections similar to those in (B) that were
immunostained for GFAP rather than NeuN. p >400
0.05; n = 5 animals each.
200(G) Immunostaining for BrdU (green) and Ki67 (red)0in coronal dentate gyrus sections of mice injected+/++/with PBS (Control) or metformin (Met) daily for
Met
Met
J
K
L
Control
Control
7 days and coinjected with BrdU on the last day.
+/+/+
The insets show the boxed areas at higher
1600
4500
*
*
2500
magnification. Scale bar represents 100 mm.
3600
1200
n.s.
2000
(H) Total number of BrdU and Ki67 double-positive
n.s.
2700
cells in the SGZ of PBS (Control) and metformin
1500
800(Met)-treated mice, determined from sections as in
1800
1000
(G). p > 0.05; n = 5 animals each.
400
900
500
(I) Coronal dentate gyrus sections of 7-month-old000
cbp+/+ and cbp+/ mice injected with metformin or
+/+/+
+/+
+/PBS (control) daily for 12 days, with BrdU coinjection for the first 3 days. Sections were immunostained for BrdU (green) and NeuN (red). The insets show the boxed areas at higher magnification. Scale bar
represents 200 mm.
(J) Total number of BrdU-positive, NeuN-positive newborn neurons in hippocampi of 2-month-old cbp+/+ versus cbp+/ littermates. Mice were injected with BrdU
for 3 consecutive days and analyzed as in (I) 9 days later (n = 4 animals each, p > 0.05).
(K and L) Total number of BrdU-positive, NeuN-positive (K), and doublecortin-positive (L) newborn neurons in PBS (Control)- versus metformin (Met)-treatedcbp+/+ versus cbp+/ hippocampi from 7-month-old mice, determined from sections as in (I) and (D). *p < 0.05; n = 4–5 animals each.
Error bars represent SEM.

3 days and then metformin alone every day for 9 additional days.
Immunostaining (Figure 6I) showed that although basal neurogenesis was decreased at this age, metformin still enhanced
hippocampal neurogenesis in the wild-type cbp+/+ mice (Figure 6K). In contrast, metformin did not enhance neurogenesis
in mice that were haploinsufficient for CBP (Figure 6K). Thisconclusion was confirmed by quantifying doublecortin-positive
newly born neurons in these hippocampi (Figure 6L). Thus, metformin promotes neurogenesis by activating an aPKC-CBP
pathway both in culture and in vivo.
30 Cell Stem Cell 11, 23–35, July 6, 2012 ª2012 Elsevier Inc.

Metformin Enhances Spatial Memory Formation
Adult neurogenesis in the hippocampus plays a key role in spatial
memory function, regulating acquisition of a spatial memory andsubsequent flexible use of that spatial memory (Dupret et al.,2007; Garthe et al., 2009; Stone et al., 2011). We therefore developed a hidden platform version of the water maze task where
mice were asked to acquire an initial platform location andthen to rapidly learn a second platform location (Figure 7A). To
determine whether metformin had any effect on learning in thisparadigm, we injected adult mice with metformin (200 mg/kg)

Cell Stem Cell
Metformin Enhances Neurogenesis via aPKC-CBP

Figure 7. Metformin Treatment Enhances
Spatial Memory Formation in Adult MiceA(A) Schematic of the BrdU, metformin, and TMZ
injections relative to the time course of training
BrdU injection
and testing protocols for the Morris water mazeexperiments. Metformin or sterile saline (as a
Met injection
control) were injected daily for the 38 day
Day 1
5 7
22
38 Sacrificeperiod and a subset of mice were coinjected with
TMZ injection
BrdU as indicated. Some mice also received
BC
injections of TMZ or vehicle at the indicated time
ReversalAcquisition
points.
Met
Met(B) Acquisition of the initial platform location
60
60
Control
Controlacross 5 days of training with latency to reach theplatform as a measurement of learning. Metformin
40
40
and control groups were similar in the initialacquisition of the task (2-way ANOVA: group20
20
F(1,279) = 0.662, p = 0.42; training day F(1,279) =34.311, p < 0.001; group X training day interaction00
F(4,279) = 0.734, p = 0.57; n = 56).
(Day)
(Day)
(C) Acquisition of the platform location after it was
moved to the opposite quadrant of the pool. Both
Reversal Probe
metformin and control groups acquired the new
D
F
Adj L
E
location at equivalent rates (2-way ANOVA: group
Training
Met
Met
F(1,111) = 1.752, p = 0.19; training day F(1,111) =Adj R
Control
Control
Opposite
17.727, p < 0.001; group X training day interaction
50
1000
50
F(1,111) = 0.325, p = 0.57; n = 56).
*
**
***
n.s.
(D) After acquisition of the new location, the
40
800
40
#
platform was removed and mice were given
*
30
600
30
a 60 s probe trial. Both the control and the-------------------------------------------------------------20
20
400
combined metformin- and metformin/vehicletreated groups showed significant differences
10
200
10
between the time spent in the different quadrants000
by one-way ANOVA. Post-hoc analysis showed
Training Opposite
Met/TMZ
ControlMet &that the metformin/metformin plus vehicleMet/Veh
treated mice searched selectively in the target
GJ
H
quadrant relative to all the other quadrants (oneIMet
Met/TMZMet
Met
way ANOVA, F(3,111) = 11.6, ***p < 0.0001;
Control
Met/VehControl
Control
2000
training quadrant > all other quadrants, p < 0.05;
8000
400
1000
*
n = 14 each of metformin and metformin plus
***
1500
6000
800
300
vehicle, combined because they were not sig**
nificantly different) while the control mice only
600
1000
4000
200
searched significantly more in the target quad400
rant relative to the adjacent left quadrant (one500
100
2000
200
way ANOVA, F(3,111) = 3.202, *p = 0.02; training
quadrant > adj l quadrant, p < 0.05; n = 28). In0000contrast to these two groups, the Met/TMZ group
showed no significant preference for the training
quadrant relative to the other three quadrants (one-way ANOVA, F(3,51) = 2.4, p = 0.07; for the training quadrant relative to all other quadrants, n.s. p > 0.05;
n = 13).
(E) Relative to control mice, the metformin-treated mice also spent a significantly greater amount of time searching in the quadrant containing the new platform
location (t(55) = 4.277, p = .04; n = 28 in each group) and significantly less time in the opposite quadrant where the target had been previously located (t(55) = 4.036,
p = 0.05; n = 28 in each group).
(F–H) Coronal dentate gyrus sections of mice treated with metformin or sterile saline (Control) as in (A) and injected with BrdU at the start of the experiment wereanalyzed anatomically by immunostaining for BrdU and NeuN (as in Figure 6B), and these sections were quantified for the total number of BrdU-positive cells (F),newly born BrdU-positive, NeuN-positive neurons (G), and BrdU-positive, NeuN-negative cells (H). **p < 0.01, ***p < 0.001; n = 5 animals each.
(I) Coronal dentate gyrus sections similar to those in (F)–(H) were analyzed by immunostaining for doublecortin (as in Figure 6D), and these sections were
quantified for the total number of doublecortin-positive neurons. *p < 0.05; n = 5 animals each.
(J) Mice treated with metformin plus vehicle or metformin plus TMZ as in (A) were injected with BrdU at day 38 and coronal dentate gyrus sections were analyzed
1 day later by immunostaining for BrdU. The graph shows quantification of the number of BrdU-positive proliferating cells in these sections. **p < 0.01; n = 5
animals each.
Error bars represent SEM. See also Figure S5.
Reversal
Probe Test
3 trials /day for 2 days

or control (sterile saline) daily for 38 days, coinjecting someanimals with BrdU on days 5, 6, and 7 to assess neurogenesis.
From 22 to 38 days, mice were trained and assessed in this water
maze task (Figure 7A). Metformin did not affect body weight overthis time course (Figure S5A). Behavioral analysis showed that

total Brdu+cells/hippocampus

total BrdU+ve cells/
hippocampus

2

1

total DCX+ve cells/hippocampus% time

0

3

4

2

1

5

BrdU+/NeuN-ve cells/
hippocampus

BrdU/NeuN+ve cells/
hippocampus% time0latency to platform (s)latency to platform (s)Acquisition
3 trials /day for 5 days

the metformin and control groups were similar in their acquisitionof the initial platform location (Figure 7B) and that this initial
spatial memory was stable in both groups, as assessed by probe
tests 1 day and 1 week after completion of training (data notshown). Mice were then assessed on their ability to learn the
Cell Stem Cell 11, 23–35, July 6, 2012 ª2012 Elsevier Inc. 31

Cell Stem Cell
Metformin Enhances Neurogenesis via aPKC-CBP

new platform location in the reversal phase of this task. Bothgroups were similar with regard to their rate of learning (Figure 7C), and both spent significantly longer in the target zone
than in the other zones (Figure 7D). However, during the probe
test, the metformin-treated mice spent significantly more timein the quadrant containing the new target (Figure 7E) while thecontrol mice spent more time in the quadrant that had contained
the platform in the initial training phase (the opposite quadrant)(Figure 7E). Both groups spent a similar amount of time in quadrants that had never contained either target (Figure 7D). Importantly, there was no difference between the two groups in the
distance traveled (Figure S5B) or swimming speed (Figure S5C).
Thus, metformin treatment selectively enhanced the ability to
update spatial memory, consistent with previous studies demonstrating that neurogenesis is necessary for flexible memory
processes (Dupret et al., 2007; Garthe et al., 2009).
We then analyzed a subset of these animals anatomically to
ask whether the enhanced spatial memory formation was coincident with a long-term increase in dentate gyrus neurons. Immunostaining demonstrated that 1 month after BrdU injection, at thecompletion of behavioral training, the large majority of the BrdUpositive cells in the dentate gyrus were NeuN-positive neurons(compare Figures 7F and 7G). Moreover, the total number of
BrdU-positive cells was increased by the metformin treatment(Figure 7F) as was the total number of BrdU-positive, NeuNpositive neurons (Figure 7G). In contrast, the number of BrdUpositive, NeuN-negative cells (presumably primarily NPCs) was
unaltered (Figure 7H). Consistent with these findings, the numberof doublecortin-positive cells was also increased (Figure 7I).
Thus, metformin promotes the genesis of new hippocampal
neurons and these neurons survive in the long term.
These results demonstrate a correlation between a metformininduced increase in hippocampal neurogenesis and enhanced
spatial learning. To ask whether this was a causal relationship,
we took advantage of the fact that the only rapidly proliferating
cells in the brain are NPCs. We therefore used the drug temozolomide (TMZ), a DNA alkylating agent that selectively induces thedeath of replicating cells, to decrease NPC numbers and ask
whether this inhibited the metformin-induced increase in spatial
memory. We and others have previously used this approach inadult neurogenesis studies (Garthe et al., 2009; Stone et al.,2011). To perform these experiments, we used the same metformin administration and training regimen but injected mice with
25 mg/kg TMZ for 6 cycles of 3 days on and 4 days off, startingfrom the first day of metformin injection throughout the training
regimen (Figure 7A). As controls, mice were treated in the same
way but were injected with vehicle rather than TMZ. Both of thesegroups were able to acquire the initial platform location in thewater maze, and this initial memory was stable, as indicated byprobe tests 1 day and 1 week later (Figures S5D and S5E). Analysis of the reversal phase of the water maze task demonstrated
that the performance of metformin/vehicle-treated mice wassimilar to mice treated with metformin alone (p > 0.05). In
contrast, mice treated with metformin and TMZ were impairedwith regard to learning the new platform location, showing nopreference for the training quadrant over the other quadrants(Figure 7D). Thus, although TMZ did not affect the ability ofmice to learn the initial platform location, it blocked metformin’s
ability to promote enhanced learning on the reversal task.
32 Cell Stem Cell 11, 23–35, July 6, 2012 ª2012 Elsevier Inc.

To ensure that the TMZ regimen decreased the proportionof proliferating precursors in the hippocampus, we injecteda subset of mice with BrdU immediately after the reversal taskprobe test. Analysis of hippocampi 24 hr later demonstrated
that metformin/TMZ-treated mice had only approximately half
as many BrdU-positive cells as did metformin/vehicle-treatedmice (Figure 7J), confirming the efficacy of this treatment regime.
Thus, inhibition of adult neurogenesis impairs metformin’s ability
to enhance spatial learning.
DISCUSSION
The findings presented here demonstrate that the widely useddiabetes drug metformin is sufficient to activate the aPKC-CBP
pathway in neural precursors and to thereby enhance neurogenesis and raise the possibility that metformin could provide thebasis for a therapeutic strategy for the human nervous system.
Specifically, these findings support five major conclusions. First,
this work demonstrates that aPKCs z and i play essential but
distinct roles in regulating the genesis of neurons from radial
precursors during embryonic development. aPKCz regulates
the differentiation of radial precursors into neurons by activating
CBP, whereas aPKCi regulates the transition from radial precursors to basal progenitors, presumably by acting as part of theapical polarity complex (Costa et al., 2008). Second, by using
a hESC-based approach for generating developing neuralprecursors, we show that aPKCs are also essential for humanforebrain neurogenesis, at least in culture. Third, we show that
metformin activates the aPKC-CBP pathway in neural precursors and that this activation promotes genesis of both humanand rodent neurons in culture. Fourth, we show that metformin
also promotes neurogenesis in the adult brain, enhancing thenumber of newly born neurons in both the olfactory bulb andthe hippocampus. This adult neurogenesis requires CBP; when
CBP is haploinsufficient, basal neurogenesis is unaffected, but
metformin-induced neurogenesis is completely inhibited. Fifth,
our behavioral studies demonstrate that metformin enhances
spatial memory function coincident with a long-term increasein the number of newly born adult dentate gyrus neurons.
When this metformin-induced neurogenesis is inhibited withthe pharmacological agent TMZ, this also prevents the metformin-associated facilitation of spatial memory. Thus, metformin,by activating an aPKC-CBP pathway, recruits adult neuralprecursors and enhances neural function, thereby providing
a candidate pharmacological approach for nervous systemtherapy.
There is considerable interest in the idea that recruitment ofendogenous neural stem cells might provide a therapeuticstrategy, based upon two lines of investigation. First, it is now
clear that newly born adult neurons play a key role in the functionality of both the olfactory system and the hippocampus(Zhao et al., 2008). These findings are particularly relevant withregard to the hippocampus, because this structure plays a keyrole in normal cognition and is preferentially vulnerable in disorders such as ischemic stroke and Alzheimer’s disease. Second,growing evidence indicates that adult neural stem cells are recruited in the injured and degenerating brain and that this represents an attempt at endogenous repair. Although the functional
importance of this recruitment is still an open question, it is clear

Cell Stem Cell
Metformin Enhances Neurogenesis via aPKC-CBP

that adult neural stem cells generate new neurons and oligodendrocytes in a number of neural injury paradigms (reviewed in
Kernie and Parent, 2010), raising the hope that if this response
could be enhanced, then perhaps it would promote repair.
However, clinical approaches for recruiting adult neural stem
cells in humans are currently lacking.
The search for pharmacological agents that can activate
endogenous adult stem cells has led to a number of recently
published chemical screens (reviewed in Li et al., 2012). Here,
we have taken a different approach; we have defined an
aPKC-CBP signaling pathway that is important for neuralprecursor differentiation (Wang et al., 2010; data presentedhere) and then focused upon a widely used drug, metformin,that activates this pathway in liver cells (He et al., 2009). In thisregard, our data indicate that metformin does indeed activate
the aPKC-CBP pathway to promote neurogenesis and thatit coincidentally enhances hippocampus-dependent spatial
memory formation, something that is thought to depend upon
adult neurogenesis (Garthe et al., 2009). Equally importantly,
we have shown that metformin has a similar activity on humanneural precursors, increasing the likelihood that it might enhanceneurogenesis in the human brain, as it does in the rodent.
Our findings also provide insights into the molecular mechanisms regulating neurogenesis. Our previous work demonstrated that CBP, a transcriptional coactivator and histone acetyltransferase, enhanced differentiation of developing radial
precursors by binding to and acetylating genes that were important for neuronal and glial differentiation. This required CBPphosphorylation by aPKCz, presumably when this kinase wasitself activated in response to extrinsic stimuli. Here, we showthat aPKCz knockdown decreases neurogenesis and increases
the maintenance of radial precursors. Moreover, we show that
this phenotype can be rescued by expression of a CBP phosphomimic. Thus, activation of an aPKCz-CBP pathway is essential for radial precursor differentiation, and our inhibition andknockdown studies indicate that metformin mediates its proneurogenic effects by activating this pathway. In contrast, foraPKCi, we show that it regulates neurogenesis but not gliogenesis in a CBP-independent fashion and that it enhances the transition from radial precursors to basal progenitors in vivo, thereby
indicating a different role for this isoform. These findings are
consistent with previous studies (Imai et al., 2006; Costa et al.,2008; Bultje et al., 2009) indicating that aPKCi is an essential
component of an apical adherens/polarity complex that serves,in part, to maintain radial precursors. Thus, aPKCs z and i play
distinct and important roles in neural precursors.
Our in vivo studies demonstrate that metformin enhances neurogenesis in a CBP-dependent fashion in adult mice and our
behavioral work suggests that the increase in hippocampal
neurons is functionally relevant. In particular, we show that
metformin preferentially enhances the reversal phase of thewater maze task, and our data using TMZ to pharmacologicallydeplete adult precursors suggests that it does so by promoting
genesis of new hippocampal neurons. In this regard, the specificity of the behavioral response is intriguing, because it raises
the possibility that the initial acquisition of a spatial memory
might have a somewhat different underlying neural substrate
than the acquisition of a second, related spatial memory, withthe latter involving newborn neurons.

Metformin is widely used in humans as a treatment for type II
diabetes and other metabolic disorders. Intriguingly, there is
widespread interest in using metformin in individuals withearly-stage Alzheimer’s disease because an increasing proportion of these individuals are also diabetic, and hyperinsulinemia
may enhance the onset and progression of neurodegeneration.
In this regard, a recent retrospective study indirectly suggested
that metformin might improve some of the adverse neuroanatomical outcomes associated with AD (Beeri et al.,2008). Moreover, metformin has been shown to increase lifespan and delay the onset of cognitive impairment in a mousemodel of Huntington’s disease (Ma et al., 2007). Thus, while
metformin may have other actions in the nervous system, ourfindings raise the possibility that its ability to enhance neurogenesis might have a positive impact in at least some nervous
system disorders.
EXPERIMENTAL PROCEDURES
Animals
All animal use was approved by the Animal Care Committee of the Hospital for
Sick Children in accordance with the Canadian Council of Animal Care policies. CD1 and C57BL/6 mice were obtained from Charles River Laboratory.
C57/129J mice, which were used for the behavioral tests, were bred at the
Hospital for Sick Children. All mice were maintained on a 12 hr light/12 hr
dark cycle with ad libitum access to food and water. Mice were used at
2 months of age unless indicated otherwise, and behavioral procedures
were conducted during the light phase of the cycle. B6.126S6-Crebbptm1Dlimice (cbp+/ mice) were obtained from The Jackson Laboratory and maintained as described (Wang et al., 2010).
Cell Culture Analysis
E11.5–E12.5 cortical precursors were isolated from CD1 embryos andcultured, transfected, and immunostained as described (Wang et al., 2010;
Supplemental Information) at a plating density of 150,000–180,000 cells/ml.
CNTF (50 ng/ml; Cedarlane Laboratories) and neuregulin-b (100 ng/ml;
R&D Systems) were added 1 day after plating. Myristoylated aPKC peptide(2 mM; Calbiochem) and metformin (Sigma-Aldrich) were used as indicated.
For quantification, more than 400 cells per condition in at least 8 randomly
chosen fields per experiment were counted. Primary adult SVZ neurospheres were cultured as described (Fujitani et al., 2010). HEK293 cells
were grown and transfected as described (Fujitani et al., 2010; Supplemental
Information).
In Utero Electroporation
In utero electroporations and immunostaining were performed as described(Wang et al., 2010) with E13/E14 CD1 mice. For quantification, three to four
fields per coronal section from each of four anatomically matched sections
per brain were analyzed on a Zeiss Pascal confocal microscope, as previously
described (Wang et al., 2010). A mean of four scans taken with a 403 objective
were computed for each image.
Maintenance and Differentiation of hESCs
Undifferentiated HES2 cells were maintained in hESC medium as described(Kennedy et al., 2007) and prior to neural differentiation were depleted offeeder cells by culturing on Matrigel (BD Biosciences)-coated plates for 24
to 48 hr. Neuralized embryoid bodies (EBs) were generated by culturing small
aggregates of feeder-depleted hESCs in low cluster plates (Corning Inc.) inknockout DMEM, supplemented with penicillin/streptomycin, 2 mM glutamine, 4 mM monothioglycerol, 50 mg/ml ascorbic acid (Sigma Aldrich), 5%
N2 supplement (GIBCO), 5% B27 (GIBCO), and 20 ng/ml hbFGF at 37 C inan environment of 5% CO2, 5% O2, and 90% N2 for 24 hr. EBs were harvested,washed, and cultured in the same medium with 2 mM Dorsomorphin and 10 mM
SB43152 for a further 12 days and then gently dissociated and plated on polyd-lysine/laminin-coated plates in the same medium plus 20 ng/ml bhFGF,

Cell Stem Cell 11, 23–35, July 6, 2012 ª2012 Elsevier Inc. 33

Cell Stem Cell
Metformin Enhances Neurogenesis via aPKC-CBP

10% N2 supplement, and 5% B27 supplement for 10 more days. For quantification, an average of 700 cells was counted per condition in 5–6 random
fields per independent experiment.
BrdU LabelingMice were treated with BrdU (Sigma-Aldrich) as described for the SVZ(Morshead et al., 1998) and the hippocampus (Fujitani et al., 2010; Supplemental Information). For the hippocampus and main olfactory bulb, every tenthsection was immunostained and quantitatively analyzed as described (Wojtowicz and Kee, 2006; Fujitani et al., 2010) and counts were normalized by multiplying for the total number of sections. For the SVZ, we quantified all cells
within two to three cell diameters of the ventricular surface from four anatomically matched coronal sections from each animal.
Statistics
Statistical analyses were performed with a two-tailed Student’s t test or
ANOVA with Tukey’s multiple comparison post hoc analysis, unless otherwise
indicated. Error bars indicate the standard error of the mean (SEM).
Western Blot Analysis and Densitometry
Western blots were performed as previously described (Wang et al., 2010).
Antibodies are in Supplemental Information. Densitometry was performed
with Adobe (San Jose, CA) Photoshop.
Morris Water Maze
Two-month-old female C57/129J mice were used for all analyses and were
injected daily with metformin (200 mg/kg) or sterile saline for the entirety ofthe experiment (Figure 7A). Some mice were also injected with 25 mg/kg
TMZ or 10% DMSO as indicated (n = 28 sterile saline alone, n = 14 metformin
alone, n = 14 metformin plus DMSO, and n = 13 metformin plus TMZ). Somewere also injected with BrdU as indicated. Mice were trained on the hidden
platform version of the water maze using a circular tank (120 cm diameter,
50 cm depth, 28 C) filled with 40 cm of water made opaque with nontoxic
white paint. All testing was conducted under dim light. Extramaze visualcues were located on curtains surrounding the maze. The escape platform(10 cm diameter) was submerged 0.5 cm below the water surface. Acquisition was measured as latency to reach the platform and four possible startlocations were pseudorandomly assigned to each trial. Actimetrics tracking
software was used to record escape latency, swim speed, and distancetraveled. Each animal was given three 60 s trials to find the platform witha 15 s intertrial interval across 5 days. After 5 days of training, mice wereadministered immediate (1 day) and remote (1 week) memory probe tests
where the platform was removed and they searched for 60 s. Immediately
after these tests, the platform was moved to the opposite location andanimals were trained in the same manner for 2 additional days. The day after
this reversal training, mice were given a probe test where the platform wasremoved and the amount of time spent searching in each quadrant wasmeasured.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Proceduresand five figures and can be found with this article online at doi:10.1016/
j.stem.2012.03.016.
ACKNOWLEDGMENTS
The authors would like to thank Dennis Aquino, Sibel Naska, and Mark
Gagliardi for technical support, advice, and discussions and Fred Wondisford
for valuable insights and support. J.W. was supported by fellowships from theMultiple Sclerosis Society of Canada and the Three to Be Foundation, D.G. by
a McEwen Center McMurrich fellowship, L.M.D. by an Alzheimer Society ofCanada fellowship, G.I.C. by a Heart and Stroke Foundation of Canada
fellowship, and D.T. by a CIHR Michael Smith studentship. P.W.F., D.R.K.,
and F.D.M. are Canada Research Chairs. This work was supported by funding
from the CIHR (MOP86762 and MOP13958), the McEwen Center for Regenerative Medicine, the Canadian Stem Cell Network, and the Three to Be
Foundation.

34 Cell Stem Cell 11, 23–35, July 6, 2012 ª2012 Elsevier Inc.

Received: February 8, 2011
Revised: March 9, 2012
Accepted: March 29, 2012
Published: July 5, 2012
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