Enzimas Alostericas

Instituto Tecnolgico
de Veracruz
ING. BIOQUMICA
PROFESORA: ZAIDA ORTA FLORES
BIOQUMICA 1
UNIDAD 2
TRADUCCIN DE ARTCULO DE INVESTIGACIN:

El estado bionergtico y antioxidante de las neuronas es
controlado por la continua degradacin de una llave enzima
glicoltica por APC/C-Cdh1
ESTUDIANTE: LUZ MARA VIAS CERN
LETTERS
m in GFP+ neurons (percentage)
El estado bionergtico y antioxidante de las neuronas

LETTERS
es controlado por la continua degradacin de una
llave
enzima glicoltica
por APC/C-Cdh1
The bioenergetic
and antioxidant
status of neurons is
120
120
controlled by continuous degradation of a key glycolytic

enzyme by APC/CCdh1
100
Angel Herrero Mendez, 100

Angeles Almeida, Emilio Fernndez, Carolina Maestre, Salvador Moncada
y Juan P. Bolaos.
80
80
GI Pfkfb
siR 3
NA
+P
Pfk
fb3
ntr
ol
Co
+P
GI Pfkfb
siR 3
NA
Pfk
fb3
Co
ntr
ol
Apoptotic GFP+ neurons (percentage)
*
Las
neuronas
son conocidas
por tener una
que K6 fue el ms abundante variante de
*
60
60
c
(2 h) neurons
1
1,2
1
1,2
tasa
menor
que los
astrocitos,
y Fernndez
empalme
de NO-treated
pfkfb3
mRNA,
alcanzamos
Angelglicoltica
Herrero-Mendez
, Angeles
Almeida
, Emilio
, Carolina
Maestre
, Salvador
Moncada3,4un
24
120
*
1,4
cuando
estresados
son incapaces de
anticuerpo *en contra de su dominio carboxi
and
Juan P.son
Bolaos
40
40
*
sobreregular la glicolisis
por una baja
terminal (Informacion adicional en fig s1b,c) y
and astrocytes.
Pfkfb3 was la
undetectable
by western
Neurons
are
known
to
have
a
lower
glycolytic
rate
than
actividad
de
Pfkfb3
(6fosfofructo2
loneurons
utilizaron
para evaluar
expresin
de lablotting
20
20
18
90
Pfkfb3
Control
in
neurons,
whereas
it
was
present
in
astrocytes
(Fig.
1a,
lower panels;
astrocytes
and
when
stressed
they
are
unable
to
upregulate
cinasa/fructuosa-2,
6 bifosfatasa-3).
Esta
protena Pfkfb3 en neuronas* y atrocitos.
Pfkfb3 + NO
Control + NO
1
Supplementary
Information,
Fig.
S1d,
e).
Immunohistochemistry
in
glycolysis
because
of
low
Pfkfb3
(6-phosphofructo-2-kinase/
enzima
genera fructuosa-2,
6-bifosfatasa
Pfkfb3 fue indetectable para el secante oeste
0
0
22
0
4enzyme
6
8
0
2 6-bisphosphatase-3)
4
6
8
coronal
sections
of
rat
brain
cortex
showed
that
Pfkfb3
did
not
colocalfructose-2,
activity
.
This
(F2,6P2), Time
el ms
potente activador
de 6en las
neuronas, mientras
estuvo presente en
12
60
(h)
ize with the neuronal
nuclear
marker NeuN
it did colocalize
generates
fructose-2,6-bisphosphate
(F2,6PTime
)3, (h)
the most
2
fosfofructo-1cinasa
(Pfk1;
ref.4),
un
astrocitos
(fig
1,
paneles
msbutbajos;
info with
b 60
60
the astrocyte marker GFAP (Fig. 1b). Thus, although Pfkfb3 mRNA is
potent activator of 6-phosphofructo-1-kinase
(Pfk1; ref. 4),
regulador maestro
de glicosis. Aqu
adicional fig 1Sd y e). La hinmunohistoquimica
* 5. Here, we show that Pfkfb3
present in neurons, Pfkfb3 protein is absent, suggesting that the enzyme
a master regulator of glycolysis
mostramos
que
el
Pfkfb3
est
ausente
en
en secciones
coronales 6de la corteza cerebral
20
50absent from neurons in the brain
50 cortex and that Pfkfb3 in
is downregulated
post-transcriptionally in these cells.
is
las neuronas de la corteza cerebral y ese*
de la rata mostr que el Pfkfb3 no colocaliz
Incubation of neurons with the proteosome inhibitors MG132 or
neurons is constantly subject to proteasomal degradation by
Pfkfb3
es
constantemente
con
el marcador neuronal nuclear NeuN pero
6 objeto de una
40
40
lactacystine for 1 h resulted in accumulation of Pfkfb3 protein (Fig. 1c,
the action of the E3 ubiquitin ligase , anaphase-promoting
0
degradacin* proteasomal
por
la accin
de
siupper
colocaliz
con el marcador
astrocito
GFAP
0 panel). Furthermore,
immunoprecipitation
of Pfkfb3
in either
complex/cyclosome
(APC/C)Cdh1. By
contrast,
astrocytes
la
E3
ubiquitina
ligasa,
promovedor
de
la
(Fig
1b).
As,
aunque
Pfkfb3
mRNA
estblotting
30
30
MG132- or lactacystine-treated neurons, followed by western
have low APC/CCdh1 activity and therefore Pfkfb3 is present
anafase
ciclosoma
presente
en las neuronas,
la protena
* compleja
using an anti-ubiquitin
antibody, revealed
an increase Pfkfb3
in Pfkfb3 ubiqin
these cells.
Upregulation
of Pfkfb3 by(APC/C)-Cdh1.
either inhibition of
20
En
contraste,
los
astrocitos
tienen
baja
est
ausente,
sugiriendo
que
la indicate
enzima
esneurons
20in neurons resulted in the
uitylation (Fig. 1c, lower panel). These results
that in
Cdh1 or* overexpression of Pfkfb3
actividad
APC/C-Cdh1
y por
Pfkfb3
regulada
a la through
baja post
transcripcionalmente
Pfkfb3 is degraded
the ubiquitinproteosome
pathway, which
activation
of glycolysis.
This, however,
wastanto
accompanied
by
10marked
hasestas
been described
in myogenic cells during differentiation8. We then
ae
s t pdecrease
r eControl
s e nint ethe eoxidation
n e10s aofsglucose
c lPfkfb3
uthrough
l a s . the
La
en
clulas.
pentose
phosphate
(a metabolic
involved
investigated
possible
targetingcon
Pfkfb3
for ubiquitylation
regulacin
a la+pathway
alza de
Pfkfb3 route
ya Pfkfb3
sea
lathe La
incubacin
demotifs
neuronas
inhibidores
de and
Control
NO
+por
NO in
7
0
regeneration
ofde
reduced
glutathione
found that Pfkfb3,MG132
but not theo1, 2lactasistina
or 4 isomers, contains
a una
KEN box at
) resulting
in oxidative
0 la
inhibicin
Cdh1
o
por
sobreexpresin
proteosoma
por
0
2
4
6
8
0
2
4
6
8
position
142 (Supplementary
Information, Fig. S1f).
KEN box targets
stress
and apoptotic
death.neuronas
Thus, by actively
downregulating
de Pfkfb3
en
result
hora
result
en la acumulacin
de Aprotena
Time
(h) las
Time
(h) en la
9
proteins
for
ubiquitylation
by
the
APC/C
glycolysis
by
APC/CCdh1,
neurons
use
glucose
to
maintain
.
Activation
of
APC/Clarequires
activacin de glicolisis. Esto, sin embargo,
Pfkfb3 (Fig 1c. panel superior). Adems,
Figure 4 Expression
of Pfkfb3 transiently
protects
neurons
from
loss
of
death was assessed
in neurons
treated
as inwith
a. NO
triggered
apoptotic
death
their
antioxidant
status
at
the
expense
of
its
utilization
for
the
formation
of
a
complex
Cdc20
or
Cdh1
(ref.
10);
however, Cdh1
acompaado
por un marcado descenso
inmunoprecipitacin
de Pfkfb3
cualquiera
mitochondrialfue
membrane
potential ( ) and
of neurons transfected
with control plasmid vector
(left panel).en
in
bioenergetic
purposes. apoptotic death triggered by
is the only
possible activator of APC/C
in theHowever,
terminally
differentiated
nitric oxide (NO).
neurons were
with control
plasmid de neurons
de(a)laPrimary
oxidacin
detransfected
la glucosa
a travs
la transfected
de ambas
MG132
por
with Pfkfb3,
the effectooflactasistina,
NO was delayed forseguido
4 h (right
11
neurons
used
in
this
study
.
Cdh1
was
knocked
down
in
neurons
using
(left
vector (left panel)
or
Pfkfb3
(right
panel)
and
then
exposed
to
NO
(released
panel).
(c)
The
protective
effect
of
Pfkfb3
against
NO-mediated
loss
of
va pentasa fosfato (una ruta metablica

la secante oeste usando un anticuerpo anti
,
panel) in
and apoptotic
death (rightRNA
panel)
was prevented
by PGIprotein
siRNA. Results
from the NO donor
0.5 mM).polymerase-chain
NO triggered a rapidreaction
loss of (RTPCR)
UsingDETANONOate,
reverse-transcriptase
short interfering
(siRNA)
and Pfkfb3
was found to accuinvolucrada
en
la
regeneracin
de
glutatin
ubiquitina,
revel
incremento
en la
which was initially
neurons
transfected
with Pfkfb3. rat
(b)
Apoptotic
are means
s.e.m. (n(Fig.
= 3).1d).
*P <Cdh1
0.05 protein
versus un
the
corresponding
RNAprevented
extracts in
from
terminally
differentiated
cortical neurons
and mulate
abundance
(Fig.control.
1d) and APC/C activity
reducido)
resultando
en
un
estrs
oxidativo
ubiquitylation
de
Pfkfb3
(fig
1c
panel
ms
astrocytes, we established that Pfkfb mRNA is expressed in neurons and (Fig. 1e) were lower in astrocytes than in neurons, and overexpression
only limited
protection,
as glycolysis
diverts
glucose
away
transfected y
withmuerte
Pfkfb3 is consequent
to the diversion
of G6P
from this produces
apopttica.
As,
regulando
bajo).
Este
resultado
indica
que
en
neuronas
that isoform 3 (Pfkfb3) mRNA is the most abundant in both cell types of Cdh1 in astrocytes decreased Pfkfb3 protein (Fig. 1f). Transfection
from the PPP, resulting
in oxidative stress andadeath.
the PPP to glycolysis.
activamente
a la baja
por APC/
dein la
via
(Supplementary
Information,
Fig. la
S1a).gliclisis
This was confirmed
by northern Pfkfb3
of neuronses
withdegradada
wild-type Pfkfb3 or atravs
mutant form
which
the KEN
Thus,
Pfkfb3
is
degraded
by an active mechanism
thatsido
seemsdescrita
to be
TreatmentC-Cdh1,
of neurons with
the
cytochrome
c
oxidase
inhibitor
nitric
las
neuronas
glucosa
para
la cualmutagenesis,
ha
blotting (Fig. 1a,
upper
panel). Havingusan
demonstrated
by RTPCR
that K6 ubiquitina-proteosoma,
box was altered to AAA by site-directed
followed by flow
oxide (NO) is known to cause a marked drop in mitochondrial mem- physiological as the enzyme is absent in neurons in the normal rat+
mantener
susplice
estado
antioxidante
a anti- en
las sorting
clulas
durante
la
was
the most abundant
variant of Pfkfb3
mRNA, we raised an
cytometry
(using miogenicas
green fluorescent protein
(GFP) labelling)
brane potential (m), which is associated with an increase in apoptosis1. brain. This explains the lower rate of glycolytic metabolism in neurons
body
against its carboxy-terminal
domain (Supplementary
Information, diferenciacin.
expensas
de
su
utilizacin
para
propsitos
Investigamos
entonces
revealed
greater
accumulation
of
the
mutant
Pfkfb3
form
than
of the
This does not occur in astrocytes because they are able to upregulate than in astrocytes1. Furthermore, under stress conditions, this mechaFig.
S1b,
c)
and
used
it
to
assess
the
expression
of
Pfkfb3
protein
in
wild-type
(Fig.
1g).
These
results
demonstrate
that
although
Pfkfb3
bioenergticos.
posibles
motivos
orientando
Pfkfb3
para
glycolysis and
can use glycolytically generated ATP to maintain their nism prevents
upregulation
of glycolysis,
which is normally
observed
ubiquitylation
y
hallamos
ese
Pfkfb3,
pero
2
m. We therefore investigated the effect of Pfkfb3 expression (at a con- in astrocytes as part of their defence response . Indeed, we show that no
Usando
una
reaccin
cadena
de
los de
2 yleads
4,
contiene
una Spain.
caja
de de
centration that
did not itself
cause
apoptosis)
on Molecular,
thede
response
of neurons
enhancement
ofismeros
glycolysis in1,
neurons
to
theirSalamanca,
apoptotic
deathUnidad
Departamento
de
Bioquimica
y Biologia
Universidad
de Salamanca,
Instituto
Neurociencias
de
Castilla
Leon,
37007
Hospital
UniversitarioTreatment
de Salamanca,
de Estudios
de Ciencias
de lastress,
Saluden
de
Castilla
y Leon,
Salamanca,
Spain.
WolfsonofInstitute
polimerasa
transcriptasa
inversa
(RT-PCR)
en
KEN
la posicin
142
adicional
fig for
to inhibitionInvestigacion,
of mitochondrial
respiration.
withInstituto
NO,
adminfrom
oxidative
consequent
to a37007
decrease
in(Info
the regeneration
Biomedical Research, University College London, Gower Street, London WC1E 6BT, UK.
istered as DETANONOate,
resulted
in
a
marked
drop
in
reduced
glutathione.
(Fig.
4a,
RNA
extrado
neuronas
corticales
de ratas
S1f).
Correspondence
shouldde
be addressed
either to
J.P.B. or S.M.
(e-mail:
jbolanos@usal.es;
s.moncada@ucl.ac.uk)
m
also suggest
neuronal
consumption
of glucose by the
left panel) and
an increase in apoptosis
(Fig. 4b, left establecimos
panel). Transfection queOur results
terminales
y astrocitos,
Una
caja that
KEN
escoge
protenas
para
12 December 2008; accepted 17 February 2009; published online 17 May 2009; DOI: 10.1038/ncb1881
PPP
to
maintain
their
antioxidant
status
may
take
priority
over the use
with Pfkfb3 Received
transiently
delayed,
but
did
not
prevent
the
onset
of
the
Pfkfb m RNA es expresado en neuronas y ese
ubiquitylation por la APC/C. La activacin
de la
of glucoseAPC/C
to fulfill their
bioenergetic
requirements, de
which
be met
NO-inducedisoforma
fall in m (Fig.
right panel) and
apoptosis
3 4a,(Pfkfb3)
mRNA
es(Fig.el4b, ms
requiere
la formacin
uncancomplejo
right panel),NATURE
an effectCELL
that could
be abolished
by ONLINE
silencingPUBLICATION
4c). by other sources. Increasing evidence indicates that neurons can use
BIOLOGY
ADVANCE
1
abundante
en
ambos
tipos PGI
de(Fig.clulas.
de Cdc20 o Cdh1 (ref 10); sin embargo, Cdh1
Similar results were obtained when antimycin A was used to inhibit the lactate generated by astrocytes to produce energy 17 and that this is not
(Informacin adicional en figura S1a). Esto fue
es el nico activador posible de APC/C en las
electron transport chain (Supplementary Information, Fig. S3fh). Thus, a uniform process but varies as a result of glutamatergic activation18.
confirmado
por
el
secante
norte
(Fig
1
panel
neuronas
terminalmente
usadas
following cellular stress induced by inhibition of mitochondrial respira- The fact that
Pfkfb3 is subject
to proteasomal diferenciadas
degradation suggests
that
superior).
Teniendo
demostrado
por
RT-PCR
en
este
estudio.
Cdh1
fue
derribadaen
tion, transfection of neurons with Pfkfb3 activates glycolysis; however this mechanism is amenable to modulation, under conditions that now
m
NATURE CELL BIOLOGY
NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION
ADVANCE ONLINE PUBLICATION
2009 Macmillan Publishers Limited. All rights reserved.
4
Time (h)
60
*
Pfkfb3
280
50
GFAP
Map2
60
20
Pfkfb3
12
6
Merge
0
Pfkfb3
4
6
8 Cdh1 siRNA
0
Time (h) GAPDH
Mr(K)
Pfkfb3
Pfkfb3 + NO
63
4
+
Time (h)
6
Pfkfb3
Mr(K)
Neurons
Long
+P
g
Neurons
Astrocytes
0 20 60 0 20 60 min
GI Pfkfb
siR 3
NA
ntr
ol
Co
f
Astrocytes
Neurons
+ Cdh1
Pfkfb3
Pfkfb3mut
10
Pfkfb3
ct
No
ne
37
Control
Control + NO
La
10
Astr
20
+P
GI Pfkfb
siR 3
NA
30
S-Cyclin B1
GAPDH
Mr(K)
0
0 63
DAPI
Merge
18
Pfk
fb3
GFAP
37
20
DAPI
90
40
50
30
Pfkfb3
Pfkfb3
Pfkfb3 + NO
Pfk
fb3
Protein
Cyclophilin
Mr(K)
63
50
40
4
6
Time (h)Pfkfb3
NeuN
24
Co
ntr
ol
60
mRNA
20 b
Control
Control + NO
120
40
Ne
uro
ns
As
tro
cy
tes
20
protena verde fluorescente etiquetada como

GFP) revel mayores acumulaciones de forma
Pfkfb3 mutante que el tipo salvaje (fig 1g).
LETTERS
Estos resultados demuestran que aunque
Pfkfb3 esta sujeta a degradacin por la accin
de APC/C-Cdh1 en neuronas, Pfkfb3 protena
es estable en astrocitos como un resultado de
su baja actividad de
APC/C-Cdh1. Los
resultados tambin indican que represin posttranscripcional de Pfkfb1,2 y 4 en neuronas, si
esto ocurre, es a travss de un mecanismo
cque es independiente
NO-treated (2 de
h) neurons
APC/C-Cdh1.
LETTERS
40
MG
neuronas usando interferencias cortas RNA

(siRNA) y la protena Pfkfb3 fue encontrada
para acumular. La abundancia de protena
Cdh1 (fig 1d) y la actividad de APC/C (Fig 1e)
fue mas baja en astrocitos que en las
120
neuronas, y una sobre 120
expresin de Cdh1 en
astrocitos decrementa la protena Pfkfb3 (Fig
100
1f). La transfeccin de100neuronas con Pfkfb3
tipo salvaje o una forma mutante en la que la
*
80
80
caja
de KEN fue alterada
a AAA por
*
*
mutagenesis* directa en sitio seguida
de
60
60
flujocitometra
de clasificacin
(usando
35
Figure 4 Expression180
of Pfkfb3 transientlyIP:protects
neurons from
loss of
death
anti-Pfkfb3
55 was assessed in neurons treated as in a. NO triggered
Cdh1 apoptotic death
Pfkfb3
54
Cdh1
mitochondrial membrane
of neurons transfected with control plasmid vector (left panel). However, in
115 potential (m) and apoptotic death triggered by
37
GAPDH
nitric oxide (NO). (a) Primary neurons were
with control plasmid GAPDH
neurons transfected with Pfkfb3, the effect of NO was
delayed for 4 h (right
GAPDH
WB:transfected
anti-Ub
Short
82
vector (left panel) or Pfkfb3 (right panel) and then exposed to NO (released
panel). (c) The protective effect of Pfkfb3 against NO-mediated loss of m (left
63
panel) and apoptotic death (right panel) was prevented by PGI siRNA. Results
from the NO donor DETANONOate,
0.5 mM). NO triggered a rapid loss of m,
which was initially prevented in neurons transfected with Pfkfb3. (b) Apoptotic
are means s.e.m. (n = 3). *P < 0.05 versus the corresponding control.
Figure 1 Pfkfb3 protein is degraded through the ubiquitinproteasome
Pfkfb3 accumulation, shown by western blotting. Immunoprecipitation of
proteinonly
extracts
with an
anti-Pfkfb3as
antibody,
followed
by western
pathway
mediated
by APC/CCdh1
in ratdiversion
cortical neurons
but not
in
this produces
limited
protection,
glycolysis
diverts
glucoseblotting
away
transfected with
Pfkfb3
is consequent
to the
of G6P
from
ubiquitin (Ub)
showed increased
Pfkfb3
protein ubiquitylation in the
astrocytes. (a) Upper panel: northern blotting of total RNA extracts fromfrom theagainst
PPP, resulting
in oxidative
stress and
death.
lactacystine- and MG132-treated neurons. (d) Silencing Cdh1 in neurons
terminally differentiated rat cortical neurons and astrocytes showed
Pfkfb3
byinan
active mechanism
Treatment that
of neurons
with the
cytochrome
c oxidase
inhibitor
3 days is
in degraded
vitro resulted
significant
knockdown of that
Cdh1seems
protein to
andbe
similar amounts
of Pfkfb3
mRNA were
expressed
in eachnitric
cell type. Thus, at
accumulation
Pfkfb3 (after
3 days)in
to approximately
of the level
as theofenzyme
is absent
neurons in 50%
the normal
rat
oxide (NO) isCyclophilin
known towas
cause
in marker.
mitochondrial
memusedaasmarked
an mRNAdrop
loading
Lower panel:
westernphysiological
found
in astrocytes,
as assessed
densitometry;
astrocytes express
very
1
blotting
an anti-Pfkfb3
antibody
the absence
of Pfkfb3
explains
the lower
rate ofbyglycolytic
metabolism
in neurons
brane potential
(musing
), which
is associated
with revealed
an increase
in apoptosis
. brain. This
little Cdh1 1protein. (e) APC/C activity, measured as the ability to ubiquitylate
protein expression in neurons, whereas Pfkfb3 was expressed abundantly in
This does notastrocytes.
occur inMap2
astrocytes
because
they marker,
are ableGFAP
to upregulate
. Furthermore,
under
stress
conditions,
thisindicates
mecha-the
35
S-cyclin B1,
is lower in astrocytes
than
in neurons
(the arrow
was used
as a neuronal
as a glial markerthan
and in astrocytes
most abundant
ubiquitylated
form of cyclin
B1; long
and short
indicate
glycolysis andGADPH
can use
ATPmicrophotographs
to maintain their
nism prevents
upregulation
of glycolysis,
which
is normally
observed
as aglycolytically
loading control.generated
(b) Fluorescence
of coronal
the exposure
of the defence
film: shortresponse
exposure allows
visualization
of the
2
sections
of rat brain cortex
show of
immunohistochemically
the aabsence
m. We therefore
as parttime
of their
investigated
the effect
Pfkfb3 expression (at
con- ofin astrocytes
.
Indeed,
we
show
that
corresponding decrease in 35S-cyclin B1). (f) Overexpression of Cdh1 in
Pfkfb3 in neurons and its presence in astrocytes. Neurons and glial cells were
centration thatidentified
did notusing
itselfthe
cause
apoptosis)
on
the
response
of
neurons
enhancement
of
glycolysis
in
neurons
leads
to
their
apoptotic
death
astrocytes
decreases
Pfkfb3
protein.
(g)
Wild-type
Pfkfb3,
but
not
a
sitespecific markers NeuN and GFAP, respectively. Nuclei
Pfkfb3in
),the
is degraded
when of
directed stress,
mutant consequent
form (142KEN to
to inhibition of
mitochondrial
Treatment
NO, adminfrom oxidative
to142aAAA,
decrease
regeneration
were
identified usingrespiration.
DAPI. Scale bars,
20 m. with
(c) Incubation
of neurons
mut
expressed
in
neurons
using
low
amounts of cDNA.
with
the
proteasome
inhibitors
lactacystine
or
MG132
for
1
h
resulted
in
istered as DETANONOate, resulted in a marked drop in m (Fig. 4a, reduced glutathione.
Our results also suggest that neuronal consumption of glucose by the
left panel) and an increase in apoptosis (Fig. 4b, left panel). Transfection
Next we
investigated
whether
anmay
increase
the basal
glycolytic
is subject todelayed,
degradation
by the
of APC/CCdh1
in neurons,
PPP to maintain
their
antioxidant
status
takein
priority
over
the userate
with Pfkfb3 transiently
but did
notaction
prevent
the onset of the
Pfkfb3 protein is stable in astrocytes as a result of their low APC/CCdh1 would render neurons more resistant to stress. Glycolysis was increased
NO-induced fall in m (Fig. 4a, right panel) and apoptosis (Fig. 4b, of glucose to fulfill their bioenergetic requirements, which can be met
activity. The results also indicate that post-transcriptional repression of initially by silencing Cdh1; however, this treatment enhanced neuronal
use
right panel), an effect that could be abolished by silencing PGI (Fig. 4c). by other sources. Increasing evidence indicates that neurons can
Pfkfb1, 2 and 4 in neurons, if it occurs, is through a mechanism that is apoptotic death, as assessed by flow cytometry of annexin V+/7-AAD
Similar results were obtained when antimycin A was used to inhibit the lactate generated by astrocytes to produce energy 17 and that this is not
independent of APC/CCdh1.
cells (Fig. 2c; Supplementary Information, Fig. S2e). The apoptosis
electron transport chain (Supplementary Information, Fig. S3fh). Thus, a uniform process but varies as a result of glutamatergic activation18.
We then investigated whether Cdh1 regulates glycolysis in neurons. caused by silencing Cdh1 was partially prevented by silencing PGI
following cellular
stress induced by inhibition of mitochondrial respira- The fact that Pfkfb3 is subject to proteasomal degradation suggests that
Silencing Cdh1 increased both the glycolytic flux (Fig. 2a; Supplementary (Fig. 2c) or Pfkfb3 (Fig. 2d). Cyclin B1 also mediates, in part, apoptotion, transfection
of neurons
glycolysis;
however lactate
this mechanism
is amenable
to modulation,
under
11 conditions that now
Information,
Fig.with
S2a)Pfkfb3
and theactivates
concentration
of intracellular
sis induced
by silencing
Cdh1 in neurons
(Fig. 2d), but co-silencing
(Fig. 2b; Supplementary Information, Fig. S2b). These effects were abol- cyclin B1 and Pfkfb3 fully abolished apoptosis in Cdh1 siRNA neurons
8
ADVANCE
ONLINE(Fig.
PUBLICATION
(PGI),
the glycolytic
2d). Thus, silencing Cdh1 in these cells triggers apoptosis through
5
enzyme responsible for the formation of fructose-6-phosphate
(F6P), Limited.
both cyclin
B1 (ref.
11) and Pfkfb3. Neurons were then transfected with
2009 Macmillan Publishers
All rights
reserved.
which is the substrate of Pfk1 (Fig. 2a; Supplementary Information, the full-length Pfkfb3 cDNA and sorted by flow cytometry using GFP
Fig. S2c,d). The increase in the glycolytic flux induced by silencing Cdh1 (Supplementary Information, Fig. S2f, g). This resulted in an increase in
NATURE CELL BIOLOGY
by co-silencing
phosphoglucose
isomerase
NATURE CELLished
BIOLOGY
ADVANCE
ONLINE PUBLICATION
10
0
10
Control
Control + NO
0
4
Time (h)
GI Pfkfb
siR 3
NA
Pfk
fb3
ntr
ol
20
+P
Co
*
20
+P
GI Pfkfb
siR 3
NA
60
Pfk
fb3
60
*
Despus,
nosotros
investigamos si un
50
incremento en la proporcin
basal glucoltca
50
*
hara a las neuronas ms resistentes al estrs.
40 gluclisis fue incrementada
40
La
inicialmente por
*
Cdha1 disminuido
; sin embargo, este
30
tratamiento
mejor la 30muerte apopttica
Co
ntr
ol
neuronal, segn lo evaluado por la cotometra

de flujo de la anexina V+/7-AAD- celular (Fig.
2c; Informacin Suplementaria, Fig. S2e). La
L E T T E R Sfue
apoptosis causada por Cdh1 disminuido
parcialmente impedida por PGI disminuido
(Fig. 2c) o Pfkfb3 (Fig. 2d). Ciclina B1 tambin
mediada, en parte , indujo apoptosis por Cdh1
disminuido en neuronas11 (Fig. 2d), pero codisminuyendo ciclina B1 y Pfkfb3 fue
completamente abolida la apoptosis en
neuronas Cdh1 siRNA (Fig. 2d). As, el Cdh1
estas
clulas desencadena
cdisminuido en
NO-treated
(2 h) neurons
apoptosis
a
travs
de
24 ambos, ciclina B1y
120
*
Pfkfb3. Las neuronas fueron transfectadas con
Pfkfb3 cDNA completa y ordenadas por
citometra
de flujo usando
GFP (Informacin
18
90
* resulto en un
Suplementaria, Fig. S2f, g). Esto
incremento en el flujo glucoltico (Fig. 2a) y
12
una60acumulacin de lactato,
que fue impedida
por el PGI disminuido (Fig. 2e). Expresiones
de Pfkfb3 aument la apoptosis en neuronas
6
20
en una
concentracin -(Fig.
2f) y tiempo- (Fig.
2g) de manera dependiente, pero tambin
tuvo efecto sobre apoptosis en astrocitos
0
0
(Informacin
Suplementaria,
Fig. S3a).
Entonces nosotros investigamos si Cdh1

regulada la gluclisis en neuronas.
Silenciando Cdh1 incremento tanto el flujo
glucoltico (Fig. 2a; Informacin
Suplementaria, Fig. S2a) como la
concentracin del lacato
intracelular (Fig. 2b;
120
120
Informacin Suplementaria, Fig. S2b). Estos
efectos son abolidos
100
100 por co-reducida
fosfoglucosa isomerasa (PGI), la enzima
glucoltica
responsable 80
para la formacin de
*
80
*
fructosa-6-fosfato
(F6P),
que es el sustrato
de
*
* Informacin
60
Pfk1
(Fig. 2a;
60 Suplementaria, Fig.
S2c,d). El incremento en el flujo glucoltico
*
40
40 (o reducido) Cdh1
inducido
por el disminuido
*
fue tambin abolido por la co-reducida Pfkfb3
20
(Fig.
2a). Control
As el bajo20 valor glucolitico
en
Pfkfb3
NOr l a
n e u r o n aControl
s p +uNO
e d e e x p l i c a r sPfkfb3
e p+ o
0
0
degradacin
de6 APC/C-Cdh1-mediada
a
0
2
4
6
8
0
2
4
8
Pfkfb3. Time (h)
Time (h)
Pfkfb3
Pfkfb3 + NO
0
4
Time (h)
Figure 4 Expression of Pfkfb3 transiently protects neurons from loss of

mitochondrial membrane potential (m) and apoptotic death triggered by
nitric oxide (NO). (a) Primary neurons were transfected with control plasmid
from the NO donor DETANONOate, 0.5 mM). NO triggered a rapid loss of m,
death was assessed in neurons treated as in a. NO triggered apoptotic death

neurons transfected with Pfkfb3, the effect of NO was delayed for 4 h (right
transfected with Pfkfb3 is consequent to the diversion of G6P from

Treatment of neurons with the cytochrome c oxidase inhibitor nitric
oxide (NO) is known to cause a marked drop in mitochondrial membrane potential (m), which is associated with an increase in apoptosis1.
This does not occur in astrocytes because they are able to upregulate
glycolysis and can use glycolytically generated ATP to maintain their
m. We therefore investigated the effect of Pfkfb3 expression (at a concentration that did not itself cause apoptosis) on the response of neurons
to inhibition of mitochondrial respiration. Treatment with NO, administered as DETANONOate, resulted in a marked drop in m (Fig. 4a,
with Pfkfb3 transiently delayed, but did not prevent the onset of the
NO-induced fall in m (Fig. 4a, right panel) and apoptosis (Fig. 4b,
right panel), an effect that could be abolished by silencing PGI (Fig. 4c).
Similar results were obtained when antimycin A was used to inhibit the
electron transport chain (Supplementary Information, Fig. S3fh). Thus,
following cellular stress induced by inhibition of mitochondrial respiration, transfection
of neurons with Pfkfb3 activates glycolysis; however
this produces only limited protection, as glycolysis diverts glucose away

from the PPP, resulting in oxidative stress and death.
Thus, Pfkfb3 is degraded by an active mechanism that seems to be
physiological as the enzyme is absent in neurons in the normal rat
brain. This explains the lower rate of glycolytic metabolism in neurons
than in astrocytes1. Furthermore, under stress conditions, this mechanism prevents upregulation of glycolysis, which is normally observed
in astrocytes as part of their defence response2. Indeed, we show that
enhancement of glycolysis in neurons leads to their apoptotic death
from oxidative stress, consequent to a decrease in the regeneration of
reduced glutathione.
PPP to maintain their antioxidant status may take priority over the use
of glucose to fulfill their bioenergetic requirements, which can be met
by other sources. Increasing evidence indicates that neurons can use
lactate generated by astrocytes to produce energy 17 and that this is not
a uniform process but varies as a result of glutamatergic activation18.
The fact that Pfkfb3 is subject to proteasomal degradation suggests that
this mechanism is amenable to modulation, under conditions that now
NATURE CELL BIOLOGY

LETTERS
4
Time (h)
60
20
50
0.5
*
10
40
30
0.8
1.6
Cdh1 siRNA
NA
l
ntr
o
30
siR
24
h1
90
Control
Pfkfb3
*
60
20
20
10
0
0
0
Pfkfb3 (g mL1)
10
1
2
3
Days post-transfection
20
40
18
+ +
+
+
+
+
+
Control siRNA
PGI siRNA*
30 12
20
10
0
0
+P BPPf
GI askfb
siR e 3
NA
30
20
o
Pfk ntro
fb3 l
KinPf
askfb
e 3
GFP
40
#*
#*
Cyclin B1 shRNA
Pfkfb3 shRNA
Pfkfb3
GFP
+ NO
Pfkfb3
+
40
30
Co
ntr
ol
20
f
120
LETTERS
*
0
NO-treated (2 h) neurons
0
Co
10
1.0
Co
ntr
o
40
#*
20
0
l
h1
siR
NA
Cd
+ P h1 s
GI iRN
siR A
NA
Cd
4
Time (h)
* +
+P
GI Pfkfb
siR 3
NA
30
20
0.5
30
Pfk
fb3
40
*
1.0
50
+
+
ntr
ol
60
60
Control *
Control + NO
Co
1.5
Pfk
40
PGI siRNA
Cd
80
Control siRNA
Co
ntr
ol
0.3
40
Apoptotic neurons (percentage)
100
0
Cdh1 siRNA
PGI siRNA
Pfkfb3 shRNA
Pfkfb3
40
20
120
0.6
1.5
60
Intracellular lactate (mol/106 neurons)
80
100
0.9
fb3
P
PG fkfb
Is 3+
iRN
A
120
Intracellular lactate (mol / 106 neurons)
6-14C-Glucose conversion to 14C-lactate

(nmol / h x 106 neurons)
Figure 2 Cdh1 downregulates glycolysis and protects against apoptotic death
(e) Transfection of full-length Pfkfb3 cDNA in neurons significantly increased
co-silencing PGI. (c) Cdh1 siRNA increased the proportion of apoptotic (annexin
a truncated form of Pfkfb3 containing the kinase domain. Transfection of
10through Pfkfb3 degradation in neurons. (a) Transfection

10
with Cdh1 siRNA in
intracellular lactate concentrations, as assessed in GFP+ neurons after sorting
Control
Pfkfb3
by flow cytometry; this effect was prevented by PGI siRNA. (f, g) Transfection
neurons significantly increased the rate of glycolysis, measured as the rate of
Control + NO
Pfkfb3 + NO
of neurons with Pfkfb3 induced apoptotic death in a concentration- (f) and
6-14C-glucose conversion to 14C-lactate; this effect was prevented by co-silencing
0PGI or Pfkfb3, and was mimicked by overexpression
0
of Pfkfb3. Left histogram
time- (g) dependent manner. Apoptosis was assessed by the presence of annexin
0
2
4
6
8
0
2
4
6
8 +
bar corresponds to control (luciferase) siRNA/shRNA. (b) Silencing Cdh1
V /7-AAD cells measured by flow cytometry within the GFP+ population. (h)
Time
(h)
Time
(h)
Apoptotic death was also observed following transfection of Pfkfb3 and of
increased intracellular lactate concentrations, an effect that was prevented by
) neurons,
an effectprotects
that was partially
prevented
by co-silencing
bisphosphatase
domain
did not
affect
apoptosis.
The apoptotic
increase death
V+/7-AAD
Figure 4 Expression
of Pfkfb3
transiently
neurons
from loss
of
deaththe
was
assessed inPfkfb3
neurons
treated
as in
a. NO
triggered
PGI. (d) Apoptotic
death
in Cdh1-silenced
neurons
was
partially prevented
in apoptotic
death in with
neurons
caused
by Pfkfb3
or the(left
kinase
domainHowever,
was
) and apoptotic
death
triggered
by
mitochondrial membrane
potential
(
of neurons
transfected
control
plasmid
vector
panel).
in
m
co-silencing
cyclin
B1 transfected
or Pfkfb3 andwith
was abolished
by silencing
prevented
by silencing
Results
are
meanof
s.e.m.
(n =delayed
3). *P <for
0.05
nitric oxide (NO).by(a)
Primary either
neurons
were
control plasmid
neurons
transfected
withPGI.
Pfkfb3,
the
effect
NO
was
4 versus
h (right
both. Left histogram bar corresponds to control (luciferase) siRNA/shRNA.
the corresponding control; #P < 0.05 versus Cdh1 siRNA (d).
pentose-phosphate
pathway
(PPP),
which
metabolically control.
linked to
manner,
but had
no effect
on apoptosis
astrocytes
(Supplementary
which was initially
prevented
in neurons
transfected
withinPfkfb3.
(b) Apoptotic
are means
s.e.m. (n = 3).
*P < 0.05
versus
theiscorresponding
Expresin
deS3a).
la cinasa,
pero
no la
Information, Fig.
Expression of
the kinase,
butbisfosfatasa
not the bisphos-
gluclisis,
decidimos
investigar
glycolysis
through the nosotros
common intermediate
glucose-6-phosphate
si tal
(G6P) activacin
phatase
domain
of Pfkfb3 mimicked
effect
apoptosis
in neurons
. Toonly
establish
whether
modulation
of glycolysis
in glucose
neurons
de
Pfkfb3
imitado
esteonefecto
sobre
la produces
afecta
el asvalor
de diverts
glucosa
utilizado
this
limited
protection,
glycolysis
away
transfected domino
with
Pfkfb3
is consequent
to the its
diversion
of G6P from
(Fig. 2h). Silencing
abolished the apoptosis
triggered
by expression affectspor
the rate
oxidation
through the PPP, (PPP),
neurons were
apoptosis
en PGI
neuronas.
(Fig. 2h).
Silenciando
laof G6P
va
pentosa-fosfato
que
es
from the PPP, resulting
in oxidative
stress
and
death.
of either the full-length or the kinase domain of Pfkfb3 (Fig. 2h). The transfected with Pfkfb3 and GFP+ cells were sorted by flow cytomabolimos
la
apoptosis
disparada
por
la
metablicamente
ligada
a
la
gluclisis
a
travs
Thus,
Pfkfb3
is
degraded
by
an
active
mechanism
that
seems
to
be
TreatmentPGI
ofapoptosis
neurons
with
the
cytochrome
c
oxidase
inhibitor
nitric
in neurons resulting from expression of Pfkfb3 was caused by etry to assess PPP activity 13,14. In control neurons the rate of glucose
physiological
the enzyme
is (Fig.
absent
in neurons
inthrough
the normal
oxide (NO) expresin
is activation
known toofcause
acualquiera
marked
drop
in mitochondrial
memde
de
las
completas
ouse
de
del as through
intermediario
comn
the
intrinsic
apoptotic
pathway,
as demonstrated
by the
consumption
the PPP
3a)
was
double glucosa-6-fosfato
that
gly- rat
1
12
brain.
This
explains
the
lower
rate
of
glycolytic
metabolism
in
neurons de
brane potential
(
),
which
is
associated
with
an
increase
in
apoptosis
.
selective
inhibitors
of caspases (Supplementary
Fig. S3b).
(Supplementary
Information,
Fig. S2a).si
Pfkfb3
transfection
laof cinasa
dominante
de Pfkfb3Information,
(Fig. 2h)
La colysis(G6P)
. Para
establecer
la modulacin
m
1
Thus,
although
overexpression
of
Pfkfb3
in
neurons
activates
glycolysis,
inhibited
glucose
consumption
through
the
PPP
in
neurons
by
about
than
in
astrocytes
This does not
occur
in
astrocytes
because
they
are
able
to
upregulate
.
Furthermore,
under
stress
conditions,
this
mechaapoptosis en neuronas resultando desde la
la gluclisis en neuronas affecta el valor
de la
concomitantly
triggers apoptotic
death.
(Fig. 3a);
this effect was
bywhich
silencing
PGI, indicating
glycolysis anditcan
use glycolytically
generated
ATP to maintain their nism50%
prevents
upregulation
of abolished
glycolysis,
is normally
observed
expresin
de
Pfkfb3
es
causada
por
la
oxidacion
de
G6P
a
travs
del
PPP,
las
To investigated
understand the
reason(s)
apoptotic
death of neurons
followthat such inhibition
a consequence
of the activation
glycoly2
m. We therefore
in astrocytes
as part ofwas
their
defence response
the
effect offorPfkfb3
expression
(at a con. Indeed,ofwe
show that
activacin
de activation
la vaof glycolysis,
intrnseca
apopttica,
neuronas
fueron
conforPkfkfb3
y
ing Pfkfb3-mediated
we decided
to investigate sis (Fig.
3a). Overexpression
of the transferidas
full-length cDNA coding
glucentration that did not itself cause apoptosis) on the response of neurons enhancement of glycolysis +in neurons leads to their apoptotic death
whether such
activation affected
the
rate
of glucose
utilization
by the cose-6-phosphate
dehydrogenase
(G6PD;
the rate-limitingpor
step ofcitometra
the
como
demostr
por
el
uso
de
inhibidores
clulas
GFP
son
ordenadas
to inhibition of mitochondrial respiration. Treatment with NO, adminfrom oxidative stress, consequent to a decrease in the regeneration of
s e l e c t i v o sresulted
d e inc a
sposa
s in( I
n f o(Fig.
r m4a,
a c i reduced
n
de flujo para accesar la actividad PPP13,14.
a marked
drop
glutathione.
m
NATURE CELL BIOLOGY
ADVANCE
ONLINE
PUBLICATION
3
Suplementaria,
Fig.
S3b).
panel).
Dentro
del that
control
deconsumption
neuronas
el valor
Our results
also suggest
neuronal
of glucose
by thedel
left panel) and
an increase in apoptosis
(Fig.
4b,
left
Transfection
aunque
la but
sobreexpresin
de Pfkfb3
consumo
de glucosa
a travs
deover
PPP
(Fig.
PPP to maintain
their antioxidant
status may
take priority
the use
with Pfkfb3 As,
transiently
delayed,
did not prevent the onset
of the en
activa
gluclisis,
fuebioenergetic
dobladarequirements,
a travswhich
decangluclisis
to fulfill their
be met
NO-inducedneuronas
fall in m (Fig.
4a, rightla
panel)
and apoptosisdisparando
(Fig. 4b, of glucose3a)
Increasing evidence
indicates that Fig.
neuronsS2a).
can use La
right panel),concomitantemente
an effect that could be abolished
by silencing
PGI (Fig. 4c). by other sources.
la muerte
apopttica.
(Informacin
Suplementaria,
17
lactate generated
by astrocytes
produce energy
Similar results
were obtained
whenla
antimycin
was used
inhibit the por
and
this is not de
Para
entender
raznA o
lastorazones
transfeccin
detoPfkfb3
inhibi
el that
consumo
electron transport
chainapopttica
(Supplementary
Information,
Fig. S3fh).siguiendo
Thus, a uniformglucosa
process but travs
varies as a de
resultPPP
of glutamatergic
activation18.por
muerte
de
las neuronas
en neuronas
following cellular stress induced by inhibition of mitochondrial respira- The fact that Pfkfb3 is subject to proteasomal degradation suggests that
la Pfkfb3- mediada en la activacin de la
aproximadamente el 50% (Fig. 3a); este efecto
tion, transfection of neurons with Pfkfb3 activates glycolysis; however this mechanism is amenable to modulation, under conditions that now
12
NATURE CELL BIOLOGY
1.2
1.6
G6PD (g mL )
Merge
30
4
Pfkfb3
Time (h)
+ PGI siRNA
4
Time (h)
20
nmol mg1 protein
None
+GSH-EE
30
*
20
10
10
death was assessed
in neurons treated as in a. NO triggered
apoptotic death
panel) and apoptotic
death (right panel) was prevented 0by PGI siRNA. Results
0
Co

nitric oxide (NO). (a) Primary Pfkfb3
neurons were transfected with control plasmid
G6PDpanel) and then exposed to NO (released
vector (left panel) or Pfkfb3 +(right
b3
b3
+P
P
f
GI
k
siR fb3
NA
Pfk
fb3
+G
6P
D
Pfkfb3
Pfkfb3 + NO
Pfk
f
Control
Control + NO
ntr
ol
10
Pfkfb3
MitoSox Red fluorescence in

GFP+ neurons (arbitrary units)
*
10
0 0
on
t
0.8
rol
GI PfPkfb
siR fkf3b
NA 3
0.4
+P
0.5
l3
20
10
10
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Pfk
C fb3
12
Pnftkr
fob
nmol mg1 protein
60 *
20
15
Pfk
f
HEt
ntr
ol
20
GFP
Co
ntr
o
30
20
18
1.5
+P
GSH
Co
90
GSSG
2.0
Co
GFPGFP+
l
ntr
o
0.6
50
40
Co
30
+P
GI Pfkfb
siR 3
NA
0.4
Time (h)
60
40
20
1.24
ntr
ol
Pfk
fb3
Pfk
fb
G6 3 +
PD
50
40
30
24
Co
60
4
Time (h)
GI Pfkfb
siR 3
NA
G6
PD
20.8
*
Pfkfb3
Pfkfb3 + NO
Pfk
fb3
20
*
Control
Control + NO
nmol / 106 neurons
20
1.8
PPP activity
1.2
Intracellular
lactate
Pfk
fb3
40
120
Co
ntr
ol
GL
UT
40
60
LETTERS
3b, c). Estos resultados sugieren que dentro

de las neuronas una porcin significativa de
G6P es dirigida hacia el PPP LyEentonces
T T E R S la
regulacin por incremento de Pfkfb3 es
desviada hacia la gluclisis. En astrocitos, sin
embargo, a pesar de la considerable actividad
del PPP, hay un sustancial metabolismo de
glucosa por gluclisis (Informacin
Suplementaria Fig. S3c), consistente con la
mayor expresin observada de ambos G9PD
(Informacin Suplementaria, Fig. S3d) y Pfkfb3
60
nmol / h x 106 neurons
fue abolido por el PGI silenciado, indicando

que tal inhibicin fue una consecuencia de la
activacin de la gluclisis (Fig. 3a). La
sobreexpresin completa del cDNA
c o d i fi c a n d o p o r g l u c o s a - 6 - f o s f a t o
120
120 el valor-limitante al
deshidrogenasa (G6PD;
paso de PPP) incremento significativamente la
100
100
actividad PPP, como
como lo hizo la
sobreexpresin de la glucosa transportadora
*
80
80
GLUT1 (Fig. 3a.).
*
transfected with Pfkfb3 is consequent to the diversion of G6P from this produces only limited protection, as glycolysis diverts glucose away
Thus, Pfkfb3
degraded
by an(d)
active
that seems
Treatment of neurons
with
the
cytochrome
c
oxidase
inhibitor
nitric
Figure 3 Pfkfb3 expression in neurons triggers a decrease in glucose oxidation
those thatiswere
not transfected.
Pfkfb3mechanism
expression in neurons
followedto be
revealed an
increase
of aboutrat
through
PPP, acausing
oxidative
(a) The rate of PPPmemactivity, measured
by flowas
cytometric
sorting ofisthe
GFP+ neurons
physiological
the
enzyme
absent
in
neurons
in
the
normal
oxide (NO) is known
tothe
cause
marked
dropstress.
in mitochondrial
was
4-fold in the intracellular concentration of GSSG and a corresponding decrease
as the difference in the rates of oxidation of 1-14C- and 6-14C-glucose,
1
brain.
This
explains
the
lower
rate
of
glycolytic
metabolism
in
neurons
brane potential (
),
which
is
associated
with
an
increase
in
apoptosis
.
reduced
in neurons transfected with Pfkfb3; this effect was prevented by PGI
in reduced glutathione (GSH). (e) Pfkfb3 expression in neurons increased
m
siRNA.
of abecause
plasmid vector
theto
full-length
cDNA of G6PD,
the intensity1.of
hydroethidine (HEt)
fluorescence
in the GFP+ population,
than in astrocytes
This does not occur
in Expression
astrocytes
theyencoding
are able
upregulate
Furthermore,
under
stress conditions,
this mechabut not in the GFP cells. Increased HEt fluorescence was abolished either
the glucose transporter GLUT1 increased the rate of 1-14C-glucose oxidation.
glycolysis and canor
use
glycolytically generated ATP to maintain their nism prevents
upregulation of glycolysis, which is normally observed
These measurements were performed in GFP+ cells sorted by flow cytometry.
by silencing PGI (PGI siRNA) or by G6PD expression. Scale bars, 20 m. (f)
2
m. We therefore(b)
in astrocytes
asexpression
part of in
their
defence
response
investigated
effect
of Pfkfb3
expression
(atintracellular
a con- lactate
. Indeed,
wefluorescence
show that
Transfection ofthe
Pfkfb3
in neurons
resulted
in increased
Pfkfb3
neurons
increased
the intensity
of MitoSox
+
population,
but
not
in
the
GFP
cells;
this
effectapoptotic
was abolished
concentrations,
an
effect
that
was
prevented
by
co-expressing
G6PD.
These
in
the
GFP
centration that did not itself cause apoptosis) on the
response
of
neurons
enhancement
of
glycolysis
in
neurons
leads
to
their
death
either by silencing PGI (PGI siRNA) or by G6PD expression. (g) Incubation of
measurements were performed in GFP+ cells sorted by flow cytometry. (c)
to inhibition of mitochondrial
respiration.
Treatment
from
oxidative
stress,
consequent
to
a
decrease
in
the
regeneration
+
with NO, adminNeuronal apoptotic death (annexin V /7-AAD ) caused by Pfkfb3 expression was
neurons with a plasma-permeable form of glutathione (glutathione ethyl ester,of
+
neurons
indicate
those
efficiently
prevented by co-expression
GFPdrop
GSH-EE) prevented apoptotic death caused by Pfkfb3 expression. Results are
resulted inofaG6PD.
marked
in
reduced
glutathione.
(Fig.
4a,
m
transfected with the Pfkfb3 cDNA construct, whereas GFP neurons represent
means s.e.m. (n = 3). *P < 0.05 versus the corresponding control.
Our results
also suggest that neuronal consumption of glucose by the
left panel) and an increase
in apoptosis (Fig. 4b, left panel). Transfection
with Pfkfb3 transiently delayed, but did not prevent the onset of the PPP to maintain their antioxidant status may take priority over the use
A major function of the PPP is the regeneration of reduced gluPPP) significantly increased PPP activity, as did overexpression of the
of
to fulfill
their
bioenergetic
requirements,
which neuroproteccan con
be met las
NO-inducedMfall
(Fig.
4b,
+ 7,13
at1a),
the expense
of NADPH(H
sin
a mdtransporter
e l a4a,
n t right
e GLUT1
, panel)
e l (Fig.
i nand
c3a).rapoptosis
eFurthermore,
m e n t(Fig.
o the
e nincrease
l aglucose
(Fig.
cuando
se
glucose
in tathione
) compar
to provide
1416
by other tion
sources.
Increasing
evidence
indicatesincreased
that neurons
can useof
right panel), an effect
that
could be abolished
by silencing
PGIby(Fig.
4c).
lactate
accumulation
and
the
apoptosis
induced
Pfkfb3
transfection
.
We
found
that
Pfkfb3
transfection
the
oxidation
acumulacin de lactato y la apoptosis inducida
neuronas.
17
by (Fig.
astrocytes
to produce
energy
Similar results were
obtained
when by
antimycin
A was
inhibit
and
this ismeasnot
prevented
co-expression
of used
G6PDto
(Fig.
3b, c).the
Theselactate
results generated
glutathione
3d), indicating
oxidative
stress.
Wethat
therefore
por could
la betransfeccin
de Pfkfb3
puede
ser
18
electron transport suggest
chain (Supplementary
Fig. S3fh).
a uniformured
process
but varies
as a result
of glutamatergic
activation
.
that in neurons Information,
a significant proportion
ofThus,
G6P is directed
the formation
of reactive
oxygen
species (ROS). Pfkfb3
expresprevenida
por
la
co-expresin
de
G6PD
(Fig.
towards
the
PPP
and
that
Pfkfb3
upregulation
diverts
it
towards
glycosion
enhanced
the
formation
of
ROS
in
neurons,
an
effect
that
was
following cellular stress induced by inhibition of mitochondrial respira- The fact that Pfkfb3 is subject to proteasomal degradation suggests that
In astrocytes,
however,
despite considerable
PPP activity 14this
prevented
either by silencing
PGI or by co-expressing
G6PDthat
(Figsnow
3e, f;
, there
tion, transfection lysis.
of neurons
with Pfkfb3
activates
glycolysis; however
mechanism
is amenable
to modulation,
under conditions
is substantial metabolism of glucose by glycolysis (Supplementary Supplementary Information, Fig. S3e). Moreover, incubation of neuInformation, Fig. S3c), consistent with the observed higher expres- rons with a plasma-membrane-permeable form of glutathione (gluNATURE
CELL
BIOLOGYInformation,ADVANCE
ONLINE PUBLICATION
NATURE CELL BIOLOGY
ADVANCE
ONLINE PUBLICATION
5
sion of both
G6PD (Supplementary
Fig. S3d) and Pfkfb3 tathione ethyl ester) prevented apoptosis caused by Pfkfb3 expression
2009
Macmillan
Publishers
Limited.
All These
rights results
reserved.
(Fig. 3g).
indicate that production of ROS in neurons
(Fig. 1a) when compared with neurons.
4
40
40
30
20
20
30
0
2
4
Time (h)
8
20
60
4
Time (h)
*10
Control
Control + NO
50
4
Time (h)
40
4
Time (h)
Control
10
90
60
Pfkfb3
Pfkfb3 + NO
50

30
30 triggered by
mitochondrial membrane potential (m) and apoptotic death
nitric oxide (NO). (a) Primary neurons
were transfected with control plasmid
*
vector (left panel) or20
Pfkfb3 (right panel) and then exposed20
to NO (released
*
10
60
10
0
40
Pfkfb3
Pfkfb3 + NO
6
24
Control
Control + NO
120
Pfkfb3
12
20
18

fb3
P
GI fkfb
siR 3
NA
20
20
+P
12
Pfk
40
60
GI Pfkfb
siR 3
NA
+P
Pfk
fb3
50
40
4
Time (h)
60
50
60
60
0
80
ntr
ol
Co
18

Co
ntr
ol
* Time (h)
90
fb3
+P
GI Pfkfb
siR 3
NA
60
0
0
80
Pfkfb3
Pfkfb3 + NO
100
Pfk
Control
Control + NO
100
+P
GI Pfkfb
siR 3
NA
120
20
ntr
ol
120
20
LETTERS
24
Pfk
fb3
40
120
Co
m in GFP neurons (percentage)

Co
ntr
ol
40
60
60
fue impedido por el silenciado de PGI o por

co-expresado G6PD (Figs 3e,f; Informacin
Suplementaria, Fig. S3e). Mas all, la
LETTERS
incubacin de neuronas con una membrana
de plasma permeable a partir de Glutatin
(glutaninetilester) impidi la apoptosis
causada por la expresin de Pfkfb3 (Fig. 3g).
Estos resultados indican que la produccin de
ROS en neuronas transfectadas con Pfkfb3
son consecuencia de la desviacin de G6P
desde el PPP hasta la gluclisis.
Una mayor funcin de el PPP es la

regeneracin reducida de glutatin a expensas
d e N A D P H ( H+)7,13 p a r a p r o v e e r
neuroproteccin14-16. Encontramos que la
transfeccin de Pfkfb3 increment la oxidacin
a
120 glutatin (Fig. 3D),
120 indicando estrs
de
oxidativo. Por lo tanto, medimos la formacin
100de especies reactivas 100
de
al oxigeno (ROS). La
expresin de Pfkfb3 mejor la formacin de
80
ROS
en* las neuronas, un80efecto que tambin

transfected with Pfkfb3 is consequent
to the diversion of G6P from
Control + NO
Pfkfb3 + NO
0
0
0
2
4
6
8
0
2
4
6
8
Treatment of neurons with the
cytochrome
c
oxidase
inhibitor
nitric
Time (h)
Time (h)
oxide (NO) is known to cause a marked drop in mitochondrial mem- physiological as the enzyme is absent in neurons in the normal rat
4 Expression
of Pfkfb3
transiently with
protects
loss of 1.
death
assessed
in lower
neuronsrate
treated
as in a. NOmetabolism
triggered apoptotic
death
brain.
Thiswas
explains
the
of glycolytic
in neurons
braneFigure
potential
(m), which
is associated
an neurons
increasefrom
in apoptosis
)
and
apoptotic
death
triggered
by
mitochondrial
membrane
potential
(
transfected
with controlunder
plasmid
vector
(left panel).this
However,
in
1
m
thanofinneurons
astrocytes
This does not occur in astrocytes because
they are able to upregulate
. Furthermore,
stress
conditions,
mechanitricEl
oxide
(NO). (a) Primaryde
neurons
were transfected
with
control
plasmid c
neurons
transfected
withen
Pfkfb3,
the
effect of NO
was
delayed for 4 hen
(rightla
tratamiento
neuronas
con
el
citocromo
no
causara
si
mismo
la
apoptosis)
glycolysis
and can use glycolytically generated ATP to maintain their nismpanel).
prevents
upregulation of glycolysis, which is normally observed
(c) The protective effect of Pfkfb3 against NO-mediated loss of m (left
2la inhibicin de la
oxidasa
inhibidor
oxido
nitrico
(NO)
esmin
repuesta
neuronas
para
m. from
part de
ofdeath
their
defence
response
We therefore
investigated
the effect
Pfkfb3
expression
(at
aloss
con. Indeed,
wesiRNA.
showResults
that
, astrocytes
panel)
andas
apoptotic
(right
panel)
was
prevented
by PGI
the NO donor
DETANONOate,
0.5of
mM).
NO
triggered
a rapid
of
which
was
initially
prevented
in
neurons
transfected
with
Pfkfb3.
(b)
Apoptotic
are
means
s.e.m.
(n
=
3).
*P
<
0.05
versus
the
control.
porcause
causar
una
marcado
respiracin
mitocondrial.
tratamiento
con
centrationconocido
that did not itself
apoptosis)
on the
response ofdescenso
neurons enhancement
of glycolysis
in neurons leads El
tocorresponding
their
apoptotic
death
to inhibition
Treatment withmitocondrial
NO, adminfrom oxidative
consequent to como
a decreaseDETA-NONOate,
in the regeneration of
enoflamitochondrial
potencialrespiration.
de la membrana
NO, stress,
administrado
thisresult
produces
only
limited
protection,
as glycolysis
diverts glucose
transfected
with Pfkfb3 is
consequent
to thedrop
diversion
G6P
istered
as (
DETANONOate,
in a marked
in mof
glutathione.
(Fig.
4a,from
asociado
con
un
incremente
enreduced
dentro
del
descenso
marcado
en away
m), que esresulted
m
from
the PPP,
resulting
in oxidative
stress
and death.
the PPP
to
glycolysis.
Our
results
also
suggest
that
neuronal
consumption
of
glucose
by the
left panel)
and
an
increase
in
apoptosis
(Fig.
4b,
left
panel).
Transfection
apoptosis. Esto
no ocurre en astrocitos
(Fig. 4a, panel izquierdo), un efecto que podra
Pfkfb3
is antioxidant
degraded bystatus
an active
to be
Treatment
of neurons
withbut
the cytochrome
c oxidase
inhibitor nitricPPP toThus,
maintain
their
may mechanism
take prioritythat
overseems
the use
with Pfkfb3
the onset
p otransiently
r q u eknown
edelayed,
l lto
o cause
s o andid
cnot
a pprevent
a c einsmitochondrial
d e r eofgthe
umeml a r physiological
ser abolido
por
el PGI silenciado
(Fig.
4c).
the bioenergetic
enzyme is absent
in neurons
in the
oxide (NO)
marked
of glucose to fulfillastheir
requirements,
which
cannormal
be met rat
NO-induced
fall is
in m (Fig.
4a, right
panel)drop
and apoptosis (Fig. 4b,
la gluclisis
y pueden
usar
ATP1. brain.
Resultados
similares
obtenidos
cuando
This explains
the lower
ratefueron
ofindicates
glycolytic
metabolism
in neurons
branepositivamente
potential
which
associatedby
with
an increase
in apoptosis
other sources.
Increasing
evidence
that
neurons can
use
right panel),
an effect(
thatm),could
beisabolished
silencing
PGI (Fig.
4c). by
1 A (antimycin A) fue
glucolticamente
generado
para
mantener
su
antimicina
usado
than
in astrocytes
This
doeswere
not occur
in astrocytes
because
are able
to upregulate
. Furthermore,
underenergy
stress17conditions,
thisispara
mechagenerated
by astrocytes
to produce
Similar
results
obtained
when antimycin
A they
was used
to inhibit
the lactate
and that this
not
m.and
Por
investigamos
el
detheir
laa uniform
inhibir
laupregulation
cadena
electrones
18
glycolysis
can tanto,
use glycolytically
generated ATP
to maintain
nism
prevents
of
glycolysis,
which isde
normally
observed
electron
transport
chain
(Supplementary
Information,
Fig.efecto
S3fh).
Thus,
process
but
varies astransportadora
a result
of glutamatergic
activation
.
2
de Pfkfb3
una
concentracin
(Informacin
Suplementaria,
Fig.. Indeed,
S3f-h).
m.expresin
as is
part
of
their
defence response
We therefore
investigated
the(a
effect
of
expression
(at aque
con-The in
we show
following
cellular
stress induced
by inhibition
of Pfkfb3
mitochondrial
respirafactastrocytes
that Pfkfb3
subject
to proteasomal
degradation
suggests
thatthat
centration that
not itself
cause
apoptosis)
onglycolysis;
the response
of neuronsthis mechanism
enhancement
of glycolysis
in neurons leads
their apoptotic
death
tion, transfection
ofdid
neurons
with
Pfkfb3
activates
however
is amenable
to modulation,
undertoconditions
that now
to inhibition
of mitochondrial respiration. Treatment with NO, adminfrom oxidative stress, consequent to a decrease in the regeneration of
istered
as DETANONOate,
resulted in a marked drop
in m (Fig.ONLINE
glutathione.
4a, reduced
NATURE
CELL BIOLOGY
8
ADVANCE
PUBLICATION
5
with Pfkfb3 transiently delayed, but did not prevent the onset of the PPP to maintain their antioxidant status may take priority over the use
NO-induced fall in m (Fig. 4a, right panel) and apoptosis (Fig. 4b, of glucose to fulfill their bioenergetic requirements, which can be met
10
0
10
Control
Control + NO
0
20
4
Time (h)
GI Pfkfb
siR 3
NA
Pfk
fb3
+P
20
ntr
ol
30
Co
30
+P
GI Pfkfb
siR 3
NA
*
80 consecuencia,
En
Pfkfb380es degradada por un
*
mecanismo
activo que parece ser *fisiolgico
*
60
60
donde la enzima esta ausente
en neuronas
dentro de los valores normales cerebrales.
*
40
40
Esto
explica el bajo* valor
del metabolismo
glucolitico en neuronas que estn en
20
20
astrocitos1Control
. Adems, bajo condiciones
de
Pfkfb3
Pfkfb3 + NO
Control + NO
estrs,
este
mecanismo
impide
la
regulacin
0
0
por 0 incremento
de
0
2 positiva)
4
6
8
2
4
6( o 8 regulacin
Time (h)
gluclisis, Time
que(h) es normalmente observada
en
60
60 su respuesta de
astrocitos
como parte de
defensa2. En efecto, *mostramos que el realce
50
50 lleva a su muerte
de
gluclisis en neuronas
*
apopttica a partir de estrs oxidativo,
40
c o n s e c u e n t e a u n 40d e s c e n s o e n l a
regeneracin* del glutanin reducido.
Pfk
fb3
100
Co
ntr
ol
100
Nuestros resultados tambin sugieren que el

consumo neuronal de glucosa por PPP para
L E T T E puede
RS
mantener sus estado antioxidante
adoptar prioridad sobre el uso de glucosa para
cumplir sus requerimientos de bioenergticos,
que pueden ser tomados por otras fuentes.
Creciente evidencia indica que las neuronas
pueden usar lactato generado por astrocitos
para producir energa17 y que esto no es un
proceso uniforme pero varia como resultado
cde la activacin
NO-treated
(2 h) neurons 18.
glutamatrgica
24
120
*
El hecho que Pfkfb3 este sujeto a degradacin
proteasomal sugiere que el mecanismo es
18
90
susceptible
a modulacin,
bajo condiciones
*
que por ahora requieren investigacin.
Ciertamente, es probable que el aumento del
12
uso60de lactato por neuronas es acoplado a un
incremento en su regeneracin para reducir
glutatin a partir de glucosa. La regulacin de
6
20
la estabilidad de Pfkfb3 por la APC/C-Cdh1,
va que nosotros ahora describimos, podra
subrayar este proceso. 0
As, siguiendo el estrs celular inducido por la

inhibicin de la respiracin mitocondrial, la
transfeccin de neuronas con Pfkfb3 activa la
gluclisis; sin embargo esto produce
solamente una proteccin limitada, como la
gluclisis desva glucosa
PPP,
120
120 lejos de la
resultando en estrs oxidativo y muerte.
Pfkfb3
Pfkfb3 + NO
0
4
Time (h)


transfected with Pfkfb3 is consequent to the diversion of G6P from

following cellular stress induced by inhibition of mitochondrial respiration, transfection of neurons with Pfkfb3 activates glycolysis; however

in astrocytes as part of their defence response2. Indeed, we show that
NATURE CELL BIOLOGY
Leica, Mannheim, Germany).

Accession numbers. Pfkfb1 (NM_012621), Pfkfb2 (NM_080477), Pfkfb3
(NM_057135), Pfkfb4 (NM_019333), PGI (NM_207192), Cdh1 (NM_016263), and
cyclin B1 (AY338491).
REFERENCIAS
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& Kemp, R. G. Isotope
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90
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of Tritiated
J Biol Chem 240, PC1484-6 (1965). *
+ NO
Pfkfb3 Glucose.
Control +Metabolism
NO
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0 D., Quaade, C., Johnson, J. H., Ferber, S. & Newgard, C. B.
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Gutmann,
I.
&
Wahlefeld,
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Control Bergmeyer, H. U.) 1464-1468
Pfkfb3
(Verlag Chemie GmbH, Weinheim, 1974).
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Pfkfb3 + NO
11.
Van
Schaftingen,
E.,
Lederer,
B.,
Bartrons, R. & Hers, H. G. A kinetic study of
0
0
0
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8
pyrophosphate:
fructose-6-phosphate
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Time (h)
Time (h)
Application to a microassay of fructose 2,6-bisphosphate. Eur J Biochem 129,
Figure 4 Expression of Pfkfb3 transiently
protects neurons
from loss of
191-195
(1982).
mitochondrial membrane potential ( ) and apoptotic death triggered by
12.
Tietze,
F.
Enzyme
method
for
quantitative determination of nanogram amounts
ofand
total
oxidized
glutathione:
application
to effect
mammalian
blood
and other
vector (left panel) or Pfkfb3 (right panel)
thenand
exposed
to NO (released
panel).
(c) The protective
of Pfkfb3 against
NO-mediated
loss of (left
panel) and
apoptotic death (right panel) was prevented by PGI siRNA. Results
from the NO donor DETANONOate, 0.5
mM). NOAnal.
triggeredBiochem.
a rapid loss of27,
,502-522
tissues.
(1969).
13.
Kimata, Y. et al. A mutual inhibition between APC/C and its substrate Mes1
required
meiotic
progression
fissiononly
yeast.
Dev
Cell 14,
446-54diverts
(2008).
produces
limited
protection,
as glycolysis
glucose away
transfected with Pfkfb3 is consequent
to thefor
diversion
of G6P
from thisin
GI Pfkfb
siR 3
NA
+P
Pfk
fb3
Co
ntr
ol
+P
GI Pfkfb
siR 3
NA
Pfk
fb3
Co
ntr
ol
1.

following cellular stress induced by inhibition of mitochondrial respiration, transfection of neurons with Pfkfb3 activates glycolysis; however
NATURE CELL BIOLOGY

5 that
in astrocytes as part of their defence response2. Indeed, we show

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Instituto Tecnolgico

TRADUCCIN DE ARTCULO DE INVESTIGACIN:

ESTUDIANTE: LUZ MARA VIAS CERN

m in GFP+ neurons (percentage)

El estado bionergtico y antioxidante de las neuronas

controlled by continuous degradation of a key glycolytic

Angel Herrero Mendez, 100

Apoptotic GFP+ neurons (percentage)

m in GFP+ neurons (percentage)

Apoptotic GFP+ neurons (percentage)

va pentasa fosfato (una ruta metablica

NATURE CELL BIOLOGY

NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION

ADVANCE ONLINE PUBLICATION

2009 Macmillan Publishers Limited. All rights reserved.

Apoptotic GFP+ neurons (percentage)

Apoptotic GFP+ neurons (percentage)

protena verde fluorescente etiquetada como

m in GFP+ neurons (percentage)

neuronas usando interferencias cortas RNA

Pfkfb3 accumulation, shown by western blotting. Immunoprecipitation of

NATURE CELL BIOLOGY

Apoptotic GFP+ neurons (percentage)

Apoptotic GFP+ neurons (percentage)

neuronal, segn lo evaluado por la cotometra

m in GFP+ neurons (percentage)

m in GFP+ neurons (percentage)

Entonces nosotros investigamos si Cdh1

Figure 4 Expression of Pfkfb3 transiently protects neurons from loss of

death was assessed in neurons treated as in a. NO triggered apoptotic death

transfected with Pfkfb3 is consequent to the diversion of G6P from

this produces only limited protection, as glycolysis diverts glucose away

NATURE CELL BIOLOGY

ADVANCE ONLINE PUBLICATION

NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION

Apoptotic GFP+ neurons (percentage)

Apoptotic GFP+ neurons (percentage)

Apoptotic GFP+ neurons (percentage)

Apoptotic GFP+ neurons (percentage)

Apoptotic GFP+ neurons (percentage)

Apoptotic neurons (percentage)

m in GFP+ neurons (percentage)

Intracellular lactate (mol/106 neurons)

Apoptotic neurons (percentage)

Intracellular lactate (mol / 106 neurons)

m in GFP+ neurons (percentage)

6-14C-Glucose conversion to 14C-lactate

Figure 2 Cdh1 downregulates glycolysis and protects against apoptotic death

(e) Transfection of full-length Pfkfb3 cDNA in neurons significantly increased

a truncated form of Pfkfb3 containing the kinase domain. Transfection of

10through Pfkfb3 degradation in neurons. (a) Transfection

NATURE CELL BIOLOGY

NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION

ADVANCE ONLINE PUBLICATION

2009 Macmillan Publishers Limited. All rights reserved.

Apoptotic GFP+ neurons (percentage)

nmol mg1 protein

Figure 4 Expression of Pfkfb3 transiently protects neurons from loss of

MitoSox Red fluorescence in

nmol mg1 protein

Apoptotic GFP+ neurons (percentage)

Apoptotic GFP+ neurons (percentage)

nmol / 106 neurons

m in GFP+ neurons (percentage)