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CONTENTS
Introductions
2 An Age-Old Problem
3 Brain Science Today: A “Synapse-is” of Old and New Technologies
Sean Sanders
Kevin D. Long
Articles
4 Parkinson’s—Divergent Causes, Convergent Mechanisms
J. Timothy Greenamyre and Teresa G. Hastings
21 May 2004, p. 1120
7 A Century of Alzheimer’s Disease
15 Hereditary Early-Onset Parkinson’s Disease Caused by Mutations in PINK1
19 Endothelial Cells Stimulate Self-Renewal and Expand Neurogenesis of Neural Stem Cells
24 Mosaic Organization of Neural Stem Cells in the Adult Brain
Michel Goedert and Maria Grazia Spillantini
3 November 2006, p. 777
Enza Maria Valente, Patrick M. Abou-Sleiman, Viviana Caputo, et al.
21 May 2004, p. 1158
Qin Shen, Susan K. Goderie, Li Jin, et al.
28 May 2004, p. 1338
Florian T. Merkle, Zaman Mirzadeh, Arturo Alvarez-Buylla
20 July 2007, p. 381
Technical Note
30 Detecting the Future of Neuroscience
Editor: Sean Sanders, Ph.D.; Copy Editor: Robert Buck; Designer: Amy Hardcastle
Cover Image: IStockphoto.com/Eraxion
© 2009 by The American Association for the Advancement of Science. All rights reserved.
27 November 2009
AN AGE-OLD PROBLEM
Advances in medical science have allowed us to treat and often cure previously fatal
diseases—think childhood leukemia and HIV/AIDS—as well as prevent others
through diet, surgery, or prophylactic medication, such as heart disease, and prostate
and breast cancer.
Since many are now surviving previously fatal conditions through these advances,
the result is an extension of the average life-span such that, according to the US
Census, those aged 90 and over now comprise the most rapidly growing demographic
group. It is therefore not unexpected that a concomitant increase would be seen
in diseases associated with old age: neurodegenerative diseases like Alzheimer’s,
Parkinson’s, and amyotrophic lateral sclerosis (ALS, or Lou Gehrig’s disease).
The numbers support this expectation: an estimated 5.3 million Americans suffer
the effects of Alzheimer’s, the most common neurodegenerative disease and the sixth
leading cause of death in the United States.1 Worldwide, the incidence of Parkinson’s
disease is expected to double over the next 20 years according to a 2007 report,2 while
the number of cases of Alzheimer’s is predicted to quadruple by 2050.3
In order to treat these devastating diseases, we need to better understand the molecular mechanisms underlying the disease pathogenesis. This will enable the identification
of possible pathways for clinical intervention while pointing to putative biomarkers for
early diagnosis of these disorders.
One exciting and potentially groundbreaking avenue for treating neurodegeneration
is the use of stem cells. Although stem cell implantation is still in its early days, there is
great promise in using multipotent or pluripotent stem cells to replace diseased cells in
the brain.
This booklet brings together both of these topics: elucidating neurodegenerative
pathways and developing the cures. We start with a perspective from Greenamyre
and Hastings and a review article from Goedert and Spillantini. The latter looks back
at the previous 100 years of Alzheimer’s research, and reviews the roles of both tau
and β-amyloid protein in Alzheimer’s progression; the former provides a similar
retrospective of Parkinson’s disease, discussing the manifold pathways to causation. It
further emphasizes the importance of a recent research paper, also reprinted here, from
Valente and colleagues, which demonstrates the involvement of a mitochondrial protein
kinase (PINK1) in a form of Parkinson’s. The final two papers from Shen and Merkle
highlight recent advances in neural stem cell research that underline the potential for
these cells to treat and even cure neurological disorders.
The use of stem cells is but one weapon in an arsenal that researchers are developing
to address the growing need for treatments to combat neurodegeneration. Numerous
drug-based therapies are in development, while changes in life-style and the role of
environmental factors are also being examined. Through these multiple avenues, steady
progress is being made toward solving this age-old problem.
Sean Sanders, Ph.D.
Commercial Editor, Science
Alzheimer’s Association. 2009 Alzheimer’s Disease Facts and Figures (Washington, D.C.:
Alzheimer’s Association, 2009). Accessible at www.alz.org.
2
Dorsey, E.R. et al. Projected number of people with Parkinson disease in the most populous
nations, 2005 through 2030. Neurology 68:384-386, 2007.
3
Brookmeyer, R. et al., Forecasting the global burden of Alzheimer’s disease. Alzheimer’s &
Dementia 3:186-191, July 2007.
1
BRAIN SCIENCE TODAY:
A “Synapse-is” of Old and New Technologies
Understanding our complex brain requires dedicated research from overlapping
but diverse fields of science. From biophysics to molecular biology, physiology
to cognitive neuroscience, researchers are using a broad range of tools to advance
brain science. Though the difficulties in studying neural systems are rooted in their
complex and delicate configurations, many experimental challenges have been
surmounted by research tools that are not quantum-leap advancements but are rather
amalgams of old and new technologies.
This synergy of old and new is epitomized by modern applications of the decadesold chemistry underlying immunodetection. Immunochemistry depends on an
antibody combining with its specific antigen to form a unique antibody-antigen
complex. Developing antibodies to recognize macromolecular epitopes of interest
is particularly tractable because antibodies can be generated, sans molecular
engineering, by a host immunological response. The immutable tenet of antibodyantigen specificity has laid the foundation for both simple immunodetection assays
as well as more complex multiparametric biomarker detection and whole
cell analysis.
Antibody-based biomarker detection reagents are now available for numerous
cell and tissue types and disease states, and for measuring responses to targeted
therapies. These extremely specific, robust immunoprobes are now used with
high-tech fluorescent secondary antibodies and powerful microscopes, keeping the
classic technique of immunocytochemistry a workhorse in neuroscience research.
Western blotting, another established immunodetection technique for cell and tissue
lysates, has been vastly improved by membrane (and probe) technology. From the
old technique of immunoprecipitation has evolved chromatin immunoprecipitation
(ChIP), in which antibodies recognize specific proteins bound to DNA. The field of
epigenetics owes most of its growth to improvements in ChIP. The nascent method
RIP-chip (RNA-binding protein immunoprecipitation coupled with microarray
analysis) promises to be equally valuable.
Multiparametric biomarker detection also combines old and new technologies.
Enzyme-linked immunosorbent assays (ELISA), long favored for biomarker
quantification, have also experienced technological advances, as evidenced by
the proliferation of multiplex assays that exploit antibody-fluorophore conjugates
for efficient, precise measurement of nearly 100 analytes per sample. Even the
technique of flow cytometry, historically limited to users with specialized expertise,
has vastly broadened with the availability of precision antibody-based reagents for
biomarker detection. New, inexpensive systems and good neurospecific biomarkers
have granted flow cytometry access to neuroscientists in cancer research and
regenerative medicine—no longer is a comprehensive picture of a patient’s status
limited to the futuristic realm of Star Trek.
Neuroscience is a heterogeneous field, requiring integrated analysis of
multiple cell types, tissues, and organs using diverse techniques. With which of
these techniques will the next breakthrough be made? Given that researchers
tend to build upon traditional technologies rather than abandon them, the next
advance in neuroscience will likely rely on antibodies and immunodetection.
Kevin D. Long, M.S., Ph.D.
Scientist, Technical Information
Bioscience Division, Millipore Corporation
their work are in excellent agreement. The distinctive signatures regarding the strucvibrational transitions probed by both studies ture of the clusters. The interpretation of
are the OH stretch fundamentals of the clus- these signatures is aided greatly by theoretter, which show exquisite sensitivity to the ical calculations of co-authors Christie and
hydrogen-bonding environment of water’s Jordan (1). In particular, the frequency of
OH groups and the chemical structure of the the free OH stretch fundamental shifts charcore ion. The two OH bonds in an isolated acteristically, depending on whether the wawater molecule produce two OH stretch fun- ter molecule is two- or three-coordinated.
damentals that appear at 3657 and 3756 cm 1, Over much of the cluster size range studied
J.
Timothy
Greenamyre
Teresa G. Hastings
which
are in-phase
andand
out-of-phase
vibra- (n = 10 to 20 and 23 to 27), there are welltions, respectively, of the two OH bonds. resolved transitions due to both types, indiWhenarkinson’s
an OH group
is placed
that thesechemicals,
clusters possess
complidisease
(PD)inisaahydrogen
progres- cating
environmental
such as apesticides,
… O),
bond sive
to another
water molecule
(OH
containing
neurodegenerative
illness
that
af- cated
mighthydrogen-bonded
be crucial factors network
in PD pathogenesis.
the frequency
of 1themillion
hydrogen-bonded
OH both
fects about
people in North
The two-coordinate
discovery that (single
MPTP acceptor–single
inhibits the first
stretch vibration
is lowered and
intensity donor,
AD)
and three-coordinate
(AAD elecand
America.
PD is associated
withthe
a profound
enzyme
complex
of the mitochondrial
of itsselective
IR fundamental
is increased. In
bulk ADD)
water molecules.
However,
n = 21,
and
loss of dopaminergic
neurons
tron-transfer
chain (complex
I) atprompted
liquid
water or ice,pathway
almost all
moleabsorption
to two-coordinated
wain
the nigrostriatal
of water
the brain,
as the
several
groups due
to uncover
complex I mitocules are involved in four hydrogen bonds ter molecules disappears, pointing to a
well as a more widespread (but inadequately chondrial defects in the brains and platelets
with neighbors: Each molecule donates its highly symmetric structure for the n = 21
characterized) neuronal loss in other brain of people with PD (4). But could an apparent
OH group to two hydrogen bonds and uses cluster. Other features of the spectrum, supregions.
Clinicalpairs
manifestations
include
systemicbydefect
in a mitochondrial
enzyme
its lone electron
on the oxygen
atommoto ported
calculation,
suggest that
this
tor
abnormalities
(tremor,
rigidity,
slowness,
cause
the
selective
neurodegeneration
that
accept two hydrogen bonds from its neigh- structure consists of a dodecahedral cage
balance
problems),
autonomic
characterizes
PD? The
puzzle was resolved
bors. However,
in clusters
of thedisturbances,
size report- with
a single interior
water.
psychiatric
sequelae
(usually
depression),
with
the so
discovery
chronicproblems
systemic aded here, many
of the water
molecules
in the
Like
many that
important
in
and
cognitive
impairment.
ministration
of the lipophilic
I incluster
are on the
surface of Although
the cluster,enviand science,
the present
chapter oncomplex
protonated
ronmental
risk
for or
PDthree
havehydrogen
received water
hibitor,clusters
rotenone,
reproduce
manyonof
are involved
in factors
either two
doescould
not close
the book
bonds. When attention,
only a single
OH group is
fact,including
these firstthe
spectral
data
considerable
the importance
of the
the subject.
features In
of PD,
cytoplasmic
hydrogen
bonded,
the two susceptibility
OH bonds on to
a on
large protonated
water
clusters
the
genetics
underlying
proteinaceous
inclusions
called
Lewyraise
bodgiven
become
decoupled,
forming
questionsofasthe
they
answer.
PD
is molecule
increasingly
recognized.
Despite
the nearly
ies thatasaremany
characteristic
disease
(5).
overall rarity of the familial forms of PD Together, these studies suggest that environB I O M of
E Dcases),
ICINE
(<10%
the identification of single mental chemicals, disrupted mitochondrial
genes linked to the disease has yielded cru- complex I activity, and oxidative stress may
cial insights into possible mechanisms of all participate in the killing of dopaminergic
PD pathogenesis. Now, on page 15 of this neurons in PD.
booklet, Valente et al. (1) report that a newly
Genetic studies of PD have led in other
identified familial form of PD is caused by directions. The first causative but rare mutaa mutation in a putative mitochondrial pro- tion was found in the α-synuclein gene (6).
tein kinase called PINK1
(PTEN-induced
Subsequently,
α-synuclein, a phosphoprotein
J. Timothy
Greenamyre and
Teresa G. Hastings
kinase 1).
of uncertain function, was found to be a maPostmortem
have
consistently
im- for
jor component
of Lewyconsiderable
bodies, evenattenin the
arkinson’sstudies
disease
(PD)
is a progresPD have received
plicated
damage (2)illness
in PDthat
pathomorethe
common
sporadic
cases
of PDunderwhere
siveoxidative
neurodegenerative
af- tion,
importance
of the
genetics
fects
1 million
in North
susceptibility
PD isMoreover,
increasingly
genesis,
butabout
the source
of people
this damage
has lying
no mutation
has beentofound.
overAmerica.
PD is Leading
associated
with a profound
Despite
the overall
raritycan
of the
not
been clear.
candidates
for pro- recognized.
expression of
wild-type
α-synuclein
also
and selective
loss ofoxygen
dopaminergic
ofPD
PDpatients,
(<10% of
duction
of reactive
speciesneurons
include familial
cause PDforms
(7). In
thecases),
proteinthe
apin the nigrostriatal
pathway
the brain, as
of single and
genes
linked tomodithe
dopamine
metabolism
and of
dysfunction
of identification
pears to be oxidatively
nitratively
well as a more
widespread
yielded in
crucial
insights
into
mitochondria.
In 1982,
after a (but
groupinadeof in- disease
fied and has
cross-linked
insoluble
aggregates.
quately
characterized)
neuronal
loss
in
othpossible
mechanisms
of
PD
pathogenesis.
travenous drug users developed acute, per- The formation of dopamine-quinone adducts
er brainparkinsonism
regions. Clinical
manifestations
on page
1158 in
of this
this issue,
Valente
et
manent
from injecting
a con- Now,
may be
important
process.
Another
include motor abnormalities (tremor, rigid- al. (1) report that a newly identified familtaminant (MPTP) of a synthetic opiate (3), familial PD mutation affects ubiquitin carity, slowness, balance problems), autonom- ial form of PD is caused by a mutation in a
iticbecame
clear that
“environmental”
boxyl-terminal
hydrolase-1protein
(UCHL1)
(8)—
disturbances,
psychiatric
sequelaechemi(usu- putative
mitochondrial
kinase
cals
might
be
the
culprits
in
some
cases.
a
component
of
the
cell’s
ubiquitin-proteaally depression), and cognitive impair- called PINK1 (PTEN-induced kinase 1).
Epidemiological
studies also suggested
that some
system (UPS)
that have
degrades
damaged
ment. Although environmental
risk factors
Postmortem
studies
consistently
proteins; UCHL1
hasdamage
both hydrolase
and
implicated
oxidative
(2) in PD
ubiquitin ligase
A third,
anddammuch
J. T. Greenamyre is in the Department of Neurology,
pathogenesis,
butactivities.
the source
of this
Emory University School of Medicine, Atlanta, GA
more
mutation,
affects
age
hascommon
not beencausative
clear. Leading
candidates
30322, USA. T. G. Hastings is in the Department of
another
component
of theoxygen
UPS, species
a ubiqfor
production
of reactive
Neurology, University of Pittsburgh School of
include
metabolism
andFinally,
dysuitin E3dopamine
ligase called
parkin (9).
Medicine, Pittsburgh, PA 15213, USA. E-mail:
function
of mutations
mitochondria.
1982,
after ain
[email protected]
pathogenic
haveInbeen
reported
Parkinson’s—Divergent Causes,
Convergent Mechanisms
−
P
Parkinson’s—Divergent Causes,
Convergent Mechanisms
P
1120
21 MAY 2004
VOL 304
SCIENCE
stretch t
not obse
of the H
ing up a
Furtherm
no meas
despite t
ecule th
hedral c
spectra r
from mo
given cl
these gr
ingly so
tions, w
importa
the futur
Refer
1. J.-W. S
online
2. M. Miy
1134
(10.11
3. M. Eige
4. G. Zun
5. J. Q. Se
6. S. Wei,
3268 (
Published o
10.1126/sc
Include thi
group of
acute, p
jecting a
thetic op
vironme
prits in s
ies also
chemica
crucial
discover
zyme co
tron-tran
several g
chondria
of peopl
ent syste
zyme ca
tion that
resolved
systemic
complex
duce ma
the cyto
called L
of the d
suggest
rupted m
and oxid
the killin
www.sciencemag.org
(16) and increase
oxidative
stress.oxygen
Inhibi- mutations
can bring (see
into the
playfigure).
the gene
products PD
and
n of
hasLewy
been bodies,
found. apparently
by yielding
reactive
Conversely,
mon
cases
tion of that
complex
can also impair
pathways may
implicated
disease-causing
n ofsporadic
wild-type
- species
causeI oxidative
damagethe
to UPS,
pro- mutations
lead to by
mitochondrial
imon
(see the and
figure).
Conversely,
PDhas
(7).been found. apparently by yielding reactive oxygen mutationspairment
oxidative
stress. PD
on
of
wild-type
species
that
cause
oxidative
damage
to
promutations
may
lead
to
mitochondrial
imein apSo,a where
DJ-1,
proteindoes
that PINK1
par- fit in?
(7).
pairment
andnuclear-encoded
oxidative stress.miNuclear mutations
ndPD
nitraIt
is
the
first
ticipates
in the does
oxidative
tein apSo, where
PINK1
fit in?
s-linked
tochondrial
protein(10).
to be unamstress
response
Nuclear mutations
nd
nitraIt
is
the
first
nuclear-encoded
miThe forbiguously
implicated
in
PD
Thus,
disease-causing
s-linked
tochondrial
protein
to discovery
be unamquinone
pathogenesis.
But
this
mutations
implicate
ab- in PD
The
biguously
implicated
tant forin
prompts
many
new questions.
Is
errant
protein
handling
quinone
pathogenesis.
But
this
discovery
familial
the localization of PINK1
excluand
oxidative
stress
as
rtant in
prompts
many new questions.
biquitin
sively
mitochondrial?
And are theIs
DJ-1
-synuclein
UCHL1
Parkin
PINK1
key
in PD of
pathofamilial
the events
localization
PINK1mutant
excluolase–1
mitochondria
where
biquitin
sively mitochondrial?
And are efthe
genesis.
nent
of
PINK1
exerts its pathogenic
DJ-1
-synuclein
UCHL1
Parkin
PINK1
olase–1
mitochondria
where
mutant
The
diverse
causes
easome
fects? Is PINK1 really a protein
onent
of
PINK1
exerts
itsrelated
pathogenic
efof
PD may
beare
egrades
kinase?
What
its
substrates?
teasome
fects?does
Is PINK1
really
aputative
protein
Oxidative
Protein
mechanistically
(see
the
HL1 has
How
the
loss
of
its
stress
cross-linking
egrades
kinase? What
arekinase
its substrates?
figure).
As expected,
itin
ligserine/threonine
activity
Oxidative
aggregation
Protein
HL1
has
How does
the loss of its
putative
stress
normal
mitochondrial
d much
affect
mitochondrial
function?
cross-linking
uitin
ligserine/threonine
kinase
activity
aggregation
Oxidative
e mutaValente may
and be
colleagues
activity
affected hypothend much
affect
function?
stress
mponent
size
thatmitochondrial
“PINK1chemmay phosphoby
environmental
Oxidative
mutaValentemitochondrial
and colleagues
hypothe3e ligase
rylate
proteins
in
icals
(both
natural may
and
stress
mponent
size
that
“PINK1
phospho, pathoresponse toand
cellular
stress, prosynthetic),
by
miUPS
E3
rylate mitochondrial
proteins in
eenligase
retecting
against
mitochondrial
tochondrial
DNA
polyy,
pathoresponse
to
cellular
stress,
proein that
dysfunction.” (11)
However,
UPS
morphisms
and current
been retecting
against
mitochondrial
xidative
data
seem
just
as
compatible
with
nuclear
genes. Less
ex- current
ein disthat
dysfunction.”
However,
us,
the
idea that loss
of the phosphoDopamine
pected
are just
the as
findings
mtDNA
xidative
data
seem
compatible
with
oxidation
implirylation of normal mitochondripolymorphisms
that
α-synuclein
overhus,
distheproteins
idea
that might
loss of
the phosphoDopamine
andling
al
cause
mitomtDNA
oxidation
rylation ofdysfunction,
normal
mitochondriexpression
and inacpolymorphisms
asimplikey
chondrial
perhaps
DA
handling
al
proteins
might
cause mitotivation
of
parkin
can
DA
DA
is.
through increased production
of
as
key
chondrial
dysfunction,
also
causeoxygen
mitochondriDA
of PD
reactive
species. perhaps
In this
-synuclein
DA
DA
is.
through
production
of
al
dysfunction
(12, 13).
istically
regard,
it increased
is noteworthy
that sevof PD
reactive
oxygen
species. In
-synuclein
A
common
by-product
xpected,
eral
subunits
of complex
I, this
inEnvironmental toxins
?
istically
regard,
is noteworthy
that sevof
many itone
types
of is
mitoactivity
cluding
that
essential
for
xpected,
eral
subunits
of
complex
I, inEnvironmental toxins
?
onmen- Common themes in PD. Five nuclear genes are known to carry muta- chondrial
proper assembly
of the complex,
impairment
activity
cluding
one
that
is essential
for
ral
and tions that cause PD. These genes encode -synuclein, UCHL1, parkin, isare
known
toproduction
be reversibly
phosincreased
ronmenproper
assembly
of
the
complex,
Common
themes
in
PD.
Five
nuclear
genes
are
known
to
carry
mutahondrial DJ-1, and PINK1. Mutations or altered expression of these proteins con- of
phorylated
(18, 19).speThere is also
reactive
ural and
and tributes
known oxygen
to
bephosphorylationreversibly phostions that
cause
PD. Thesethrough
genes encode
UCHL1,
parkin,
11)
aare
precedent
for
to PD
pathogenesis
common -synuclein,
mechanisms
that result
in cies
(“free
radicals”),
hondrial
phorylated
(18,
19).
is also
DJ-1, and PINK1.
Mutations oxidative
or altered stress,
expression
these mishandling.
proteins con- dependent control ofThere
impairment,
and of
protein
cted are mitochondrial
and
this may
be
themitochon11)
and
a
precedent
for
phosphorylationtributes
to
PD
pathogenesis
through
common
mechanisms
that
result
in
nuclein Similar mechanisms also may be at work in toxin or pesticide-induced source
drial respiration
(20) and of reacof thecontrol
oxidative
mitochondrial
oxidative
stress, and protein,
protein is
mishandling.
ected
are PD.
dependent
of production
mitochon-Synuclein,impairment,
a cytosolic and
vesicle-associated
oxidative- tive
tivation
oxygen
species
damage
found
in
PD
Similar
mechanisms
also
may
be
at
work
in
toxin
or
pesticide-induced
ynuclein
drial
respiration
(20)
and
reacly
and
nitratively
modified
and
may
form
adducts
with
dopamine
e mito- PD. -Synuclein, a cytosolic and vesicle-associated protein, is oxidative- by the electron-transferofchain
Inhibition
ctivation
tive oxygen
species ofproduction
all of which accelerate its aggregation. UCHL1 and parkin are brains.
12,
13). quinones,
(21).
ly and nitratively
and may form
adducts
se many
mito- components
by There
the electron-transfer
chain
of the modified
ubiquitin-proteasome
system
(UPS)with
that dopamine
degrades mitochondrial
of
seem complex
to be multiple,
diquinones, or
all damaged
of which accelerate
its aggregation.
UCHL1
and parkin
are I (21).
12,
13).
leads
to
increased
misfolded
proteins.
Inactivation
of
parkin
results
in
mitoimpair- components of the ubiquitin-proteasome system (UPS) that degrades vergent causes of PD, yet the
of many
There seem
be multiple,
diimpairment, and parkin may be S-nitrosylated and inactivated production
and
aggrection
of chondrial
oftothis
disease apdamaged
proteins.
Inactivation
of may
parkinbecome
resultslocalized
in mito- pathogenesis
impairvergent
causes
of
PD,
yet
the
inmisfolded
PD. DJ-1 or
protects
against
oxidative
stress and
gation
of
α-synuclein
(“free chondrial impairment, and parkin may be S-nitrosylated and inactivated pears to be converging on comction
of in mitochondria during times of oxidative stress. PINK1 is a nuclear- (14).
pathogenesis
of this disease apIn dopaminergic
be the
mon
mechanisms—mitochondriin PD. DJ-1
protects against
oxidative
and may become
mitochondrial
protein
kinase,stress
the substrates
of whichlocalized
remain pears to be converging on comsdamage
(“free encoded,
neurons,
aggregation
al
impairment,
oxidative stress,
in be
mitochondria
during timesinofmitochondrial
oxidative stress.
is a nuclearto
defined. Polymorphisms
genesPINK1
dramatically
alter mon mechanisms—mitochondriy be the
may
be
promoted
by dobition
of the
and
protein
mishandling,
of
encoded,
mitochondrial
protein
kinase,
the
substrates
of
which
remain
risk of developing PD. Environmental toxins, such as rotenone, impair al impairment, oxidative all
stress,
Idamage
leads mitochondrial
which are
tightly linked.
to be defined. function,
Polymorphisms
in mitochondrial
genes
dramatically
alter pamine
metabolites
and, At this
cause oxidative
stress, and
lead
to aggregation
bition
of
and
protein
mishandling,
all
of
theproteins,
risk of developing
Environmental
toxins,
such as(DA)
rotenone,
impair
and ag- of
point, exactly
howformaPINK1 fits inby the
including PD.
-synuclein.
Cytosolic
dopamine
may be
ox- perhaps,
I
leads
which
are
tightly
linked.
At
this
mitochondrial
oxidative
stress,
andthis
lead
to help
aggregation
(14). In idized
to theof pathogenic
cascade is unhighlyhow
reactive
to highlyfunction,
reactive cause
dopamine
quinones,
and
may
to de- tion
and ag- of proteins,
point,Nevertheless,
exactly
PINK1
fits inincluding
-synuclein.
Cytosolic
dopamine (DA)
may in
be PD.
ox- dopamine-quinones.
ggregaclear.
the discovery
termine
the
selective
loss
of
nigrostriatal
dopaminergic
neurons
The
(14).
In
to
the
pathogenic
cascade
is unidized
to
highly
reactive
dopamine
quinones,
and
this
may
help
to
deed by Red arrows indicate putative primary causes of PD; dashed arrows indi- latter
of PINK1
as
a putative
mitochoncan
form
adducts
aggregaclear.protein
Nevertheless,
the is
discovery
termine
thethat
selective
loss of nigrostriatal
dopaminergic
neurons
in PD. drial
nd, per- cate
kinase
that
imporeffects
are probably
secondary. Blue
arrows indicate
mechaproteins,a putative
such asmitochonby Red arrows indicate putative primary causes of PD; dashed arrows indi- with
of PINK1
fted
highly
tant
in PDas(15),
pathogenesis
opens
nisms of PD that are probably secondary to primary genetic or environ- α-synuclein
crossnd,
perdrial
protein
kinase
that is imporcate
effects
that
are
probably
secondary.
Blue
arrows
indicate
mechainones. mental causes. mtDNA, mitochondrial DNA.
new avenues of investigation
into
andpathogenesis
facilitate
f highly nisms of PD that are probably secondary to primary genetic or environ- link
tantthem,
in PD
opens
uinones. mental causes. mtDNA, mitochondrial DNA.
new aggregation.
avenues of investigation
into
their
In adα
α
α
α
α
SOURCE: KATHARINE SUTLIFF/SCIENCE
α
α
α
www.sciencemag.org
www.sciencemag.org
SCIENCE
SCIENCE
VOL 304
VOL 304
1
21 MAY 2004
21 MAY 2004
1
dition to aberrant protein modification, dopamine oxidation can affect mitochondrial
function (16) and increase oxidative stress.
Inhibition of complex I can also impair the
UPS, apparently by yielding reactive oxygen
species that cause oxidative damage to proteins, perhaps including the components of
proteasomes (17). The normal version of DJ1 protects against oxidative stress, but the
mutant protein does not. Thus, mitochondrial
dysfunction and oxidative stress can bring
into play the gene products and pathways
implicated by disease-causing mutations
(see the figure). Conversely, PD mutations
may lead to mitochondrial impairment and
oxidative stress.
So, where does PINK1 fit in? It is the first
nuclear-encoded mitochondrial protein to
be unambiguously implicated in PD pathogenesis. But this discovery prompts many
new questions. Is the localization of PINK1
exclusively mitochondrial? And are the mitochondria where mutant PINK1 exerts its
pathogenic effects? Is PINK1 really a protein kinase? What are its substrates? How
does the loss of its putative serine/threonine
kinase activity affect mitochondrial function? Valente and colleagues hypothesize
that “PINK1 may phosphorylate mitochondrial proteins in response to cellular stress,
References
1. E. M. Valente et al., Science 304, 1158 (2004);
published online 15 April 2004 (10.1126/
science.1096284).
2. P. Jenner, Ann. Neurol. 53 (suppl. 3), S26 (2003).
3. J. W. Langston, P. Ballard, J. W. Tetrud, I. Irwin,
Science 219, 979 (1983).
4. T. M. Dawson, V. L. Dawson, Science 302, 819
(2003).
5. R. Betarbet et al., Nature Neurosci. 3, 1301
(2000).
6. M. H. Polymeropoulos et al., Science 276, 2045
(1997).
7. A. B. Singleton et al., Science 302, 841 (2003).
8. E. Leroy et al., Nature 395, 451 (1998).
9. T. Kitada et al., Nature 392, 605 (1998).
10. V. Bonifati et al., Science 299, 256 (2003).
11. J. M. van der Walt et al., Am. J. Hum. Genet. 72,
804 (2003).
protecting against mitochondrial dysfunction.” However, current data seem just as
compatible with the idea that loss of the
phosphorylation of normal mitochondrial
proteins might cause mitochondrial dysfunction, perhaps through increased production of reactive oxygen species. In this
regard, it is noteworthy that several subunits
of complex I, including one that is essential for proper assembly of the complex,
are known to be reversibly phosphorylated
(18, 19). There is also a precedent for phosphorylation-dependent control of mitochondrial respiration (20) and of reactive oxygen
species production by the electron-transfer
chain (21).
There seem to be multiple, divergent
causes of PD, yet the pathogenesis of this
disease appears to be converging on common mechanisms—mitochondrial impairment, oxidative stress, and protein mishandling, all of which are tightly linked.
At this point, exactly how PINK1 fits into
the pathogenic cascade is unclear. Nevertheless, the discovery of PINK1 as a putative mitochondrial protein kinase that is
important in PD pathogenesis opens new
avenues of investigation into both basic
mitochondrial biology and neurodegenerative disease.
12. J. J. Palacino et al., J. Biol. Chem. 279, 18614
(2004).
13. M. F. Beal, Exp. Neurol. 186, 109 (2004).
14. T. B. Sherer et al., J. Neurosci. 22, 7006 (2002).
15. K. A. Conway et al., Science 294, 1346 (2001).
16. S. B. Berman, T. G. Hastings, J. Neurochem. 73,
1127 (1999).
17. M. Shamoto-Nagai et al., J. Neurosci. Res. 74,
589 (2003).
18. R. Chen, I. M. Fearnley, S. Y. Peak-Chew, J. E.
Walker, J. Biol. Chem., 10.1074/jbc.M402710200
(2004).
19. B. Schulenberg et al., J. Biol. Chem. 278, 27251
(2003).
20. P. Pasdois et al., J. Bioenerg. Biomembr. 35, 439
(2003).
21. I. Lee, E. Bender, B. Kadenbach, Mol. Cell.
Biochem. 234, 63 (2002).
A Century of Alzheimer’s Disease
Michel Goedert1* and Maria Grazia Spillantini2
A Century of Alzheimer’s Disease
One hundred years ago a small group of psychiatrists described the abnormal protein deposits in
the brain that define the most common neurodegenerative diseases. Over the past 25 years, it has
become clear that the proteins forming the deposits are central to the disease process. Amyloid-b
1* the plaques and tangles of Alzheimer’s
and tauGoedert
make up
disease, where these normally soluble
Michel
and Maria Grazia Spillantini 2
proteins assemble into amyloid-like filaments. Tau inclusions are also found in a number of related
One
hundred
yearsstudies
ago a small
psychiatrists
the abnormal
protein deposits
disorders.
Genetic
have group
shown of
that
dysfunctiondescribed
of amyloid-b
or tau is sufficient
to cause
in
the brainThe
that
define molecular
the most common
diseases.
Over the
past 25 years,
dementia.
ongoing
dissectionneurodegenerative
of the neurodegenerative
pathways
is expected
to lead
ittohas
become
clear that the
proteins
forming the deposits are central to the disease process.
a true
understanding
of disease
pathogenesis.
Amyloid-ß and tau make up the plaques and tangles of Alzheimer’s disease, where these
normally soluble proteins assemble into amyloid-like filaments. Tau inclusions are also found
pathological
process
remainedof unknown.
n 3 November
at theGenetic
37th meeting
in a number
of related 1906,
disorders.
studies have
shown that
dysfunction
amyloid-ßOver
or
the past dissection
25 years, of
a the
basicneurodegenerative
understanding has
of the to
Society
Southwest
tau is sufficient
causeof
dementia.
TheGerman
ongoing molecular
emerged
from pathogenesis.
the coming together of two inin lead
Tübingen,
pathwaysPsychiatrists
is expected to
to a trueGermany,
understanding
of disease
O
O
Alois Alzheimer presented the clinical and
neuropathological characteristics of the disease
3 November
1906,subsequently
at the 37th meet(1, 2) nthat
Emil Kraepelin
named
ing (3).
of the
Society disease
of Southwest
after him
Alzheimer’s
(AD) isGernow
the most
neurodegenerative
mancommon
Psychiatrists
in Tübingen,disease,
Gerwith more
20 million
cases worldwide.
At
many,
Aloisthan
Alzheimer
presented
the clinical
the time
of his lecture, Alzheimer
was headofofthe
the
and
neuropathological
characteristics
Anatomical
at theKraepelin
Royal Psychiatric
disease
(1, Laboratory
2) that Emil
subseClinic ofnamed
the University
Munich.
He had
quently
after himof(3).
Alzheimer’s
moved there
spent 14 neuyears
disease
(AD)inis1903
nowafter
the having
most common
at the Municipal Institution for the Mentally Ill
rodegenerative disease, with more than 20
and Epileptics in Frankfurt, where Franz Nissl
million
cases worldwide.
Athistopathology.
the time of his
had introduced
him to brain
In
lecture,
Alzheimer
was head admitted
of the AnatomiNovember
1901, Alzheimer
Auguste
cal
atpatient,
the Royal
Psychiatric
Clinic
D.,Laboratory
a 51-year-old
to the
Frankfurt hospital
of
the University
of Munich.
He had
moved
because
of progressive
memory
loss,
focal
there
in 1903
after having
spent 14 years
at
symptoms,
delusions,
and hallucinations.
After
the
Municipal
Institution
for 1906,
the Mentally
the death
of Auguste
D. in April
her brain
was
sent
to MunichinforFrankfurt,
analysis. Alzheimer’s
use
Ill
and
Epileptics
where Franz
of the had
silverintroduced
staining method
developed
by Max
Nissl
him to
brain histopaBielschowsky
4 years earlier
was crucialadfor
thology.
In November
1901,(4)Alzheimer
the identification
of neuritic
plaquespatient,
and neuromitted
Auguste D.,
a 51-year-old
to
fibrillary
tangles,
the defining
the
Frankfurt
hospital
becauseneuropathological
of progressive
characteristics
the disease.
Whereas
plaques
memory
loss, offocal
symptoms,
delusions,
had been reported before, first by Blocq and
and hallucinations. After the death of AuMarinesco in an elderly patient with epilepsy (5),
guste D. in April 1906, her brain was sent to
Alzheimer was the first to describe the tangle
Munich
forInanalysis.
use type
of the
pathology.
1911, heAlzheimer’s
found a different
of
silver
method
developed
by Max
nerve staining
cell inclusion
in two
cases with
focal
Bielschowsky
years
earliercortex
(4) was
degeneration of4 the
cerebral
(2).crucial
This is
for
identification
neuritic
nowthe
known
as the Pickof
body
(even plaques
though itand
was
neurofibrillary
theand
defining
neurofirst described bytangles,
Alzheimer)
the clinicopathpathological
of the
disease.
ological entitycharacteristics
is known as Pick’s
disease,
after
Arnold Pick,
who first
it in 1892
(6).
Whereas
plaques
had described
been reported
before,
Pick’s
belongs
to theinspectrum
of
first
by disease
Blocq and
Marinesco
an elderly
frontotemporal
lobar degeneration
(FTLD). was
patient
with epilepsy
(5), Alzheimer
The presence
of the
abnormal
helped
the first
to describe
tangledeposits
pathology.
In
greatlyhewith
disease
classification
(7).nerve
However,
1911,
found
a different
type of
cell
their molecular composition and role in the
1
Laboratory of Molecular Biology, Medical Research
Council, Cambridge CB2 2QH, UK. 2Cambridge Centre
for Brain Repair, Department of Clinical Neurosciences,
University of Cambridge, Cambridge CB2 2PY, UK.
*To whom correspondence should be addressed. E-mail:
[email protected]
dependent lines of research. First, the molecular
study of the deposits led to the identification of
inclusion
two cases with
focalthedegeneratheir majorincomponents.
Second,
study of
tion
the cerebral
cortex
(2).resulted
This isinnow
rare, of
inherited
forms of
disease
the
discovery
causative
known
as of
thethePick
body gene
(evendefects.
thoughInit most
was
cases,described
the defective
genes encode
first
by Alzheimer)
and the
the major
clinicomponents of the
pathological
lesions
or factors
copathological
entity
is known
as Pick’s
disthat change
their levels.
follows
a toxic
ease,
after Arnold
Pick,Itwho
firstthat
described
thePick’s
proteins
that belongs
make up
the
itproperty
in 1892of(6).
disease
to the
filamentous
underlies the
inherited
disspectrum
of lesions
frontotemporal
lobar
degeneraease cases. A similar toxic property may also
tion (FTLD).
cause the much more common sporadic forms of
The presence
of abnormal
deposits
helped
disease.
Here we review
the evidence
implicating
greatly
with
classification (7). Howamyloid-b
anddisease
tau in neurodegeneration.
ever, their molecular composition and role in
the
pathological
process remained unknown.
Abnormal
Filaments
Over
past 25microscope,
years, a basic
understanding
In thethe
electron
plaques
and tangles
has
emerged
from
the coming
of two
contain
abnormal
filaments
(8, 9).together
Plaque filaments
are extracellular
and of
have
the molecular
independent
lines
research.
First,fine
thestrucmoture of amyloid.
refersled
to filaments
with a
lecular
study ofThis
theterm
deposits
to the identidiameter of
of their
around
10 nm
that have aSecond,
cross-b
fication
major
components.
structure
dye-binding
the
studyand
ofcharacteristic
rare, inherited
forms ofproperties.
disease
Most tangle
filaments
haveofa the
paired
helical
resulted
in the
discovery
causative
morphology
alsocases,
amyloid-like.
Paired
gene
defects.andInare
most
the defective
helical filaments are present in nerve cell bodies,
genes encode the major components of the
as well as in neurites in the neuropil and at the
pathological lesions or factors that change
periphery of neuritic plaques. After the identificatheir
It follows
toxic property
tion oflevels.
filaments
(8, 9), itthat
tooka another
20 years
of
the their
proteins
make up were
the filamentous
before
majorthat
components
known. The
lesions
underlies
the inherited
identification
of amyloid-b
as thedisease
major cases.
plaque
A
similar toxic
maytangle
also component
cause the
component
and tauproperty
as the major
much
sporadic
forms
of (Fig.
disusheredmore
in thecommon
modern era
of research
on AD
ease.
Here we review
the evidence
implicat1A). Filamentous
tau deposits
are also present
in a
number
of other
disorders,
ing
amyloid-ß
andneurodegenerative
tau in neurodegeneration.
including Pick’s disease (Fig. 1B).
Abnormal Filaments
Amyloid-b
In
the electron microscope, plaques and
Amyloid-b
is 40 toabnormal
42 amino filaments
acids in length
tangles
contain
(8, and
9).
is
generated
by
proteolytic
cleavage
of
the
Plaque filaments are extracellular and much
have
larger amyloid precursor protein (APP), a transthe molecular fine structure of amyloid. This
membrane protein of unknown function with a
term refers to filaments with a diameter of
single membrane-spanning domain (10–13)
around
10 nm
have a cross-ß
structure
(Fig. 2A).
The that
N terminus
of amyloid-b
is
and
characteristic
dye-binding
properties.
located in the extracellular domain of APP, 28
Most
filaments
have a pairedregion,
heliamino tangle
acids from
the transmembrane
www.sciencemag.org
SCIENCE
VOL 314
a-secreta
precludi
species
long, an
acid form
amino a
three-dim
fibril, re
residues
strand m
formed b
Map
21, toge
tangles i
syndrom
gested a
ever, di
came fro
rhage wi
a rare co
rhages re
in cereb
caused b
portion o
of amylo
brains (1
blood ve
advancin
HCHWA
posits in
differs fr
present,
more, pl
In th
tations i
familial
linked t
such mu
(Fig. 2,
creted p
has a (
APP mu
or lead t
ending a
flank th
peptide
mutation
HCHWA
have litt
the prop
Missens
vascular
Twenty
have be
creased
cause of
gene giv
neuropil
with clin
hemorrh
3 NOVEMBER 20
cal morphology and are also amyloid-like.
Paired helical filaments are present in nerve
cell bodies, as well as in neurites in the neuropil and at the periphery of neuritic plaques.
After the identification of filaments (8, 9),
it took another 20 years before their major
components were known. The identification
of amyloid-ß as the major plaque component and tau as the major tangle component
ushered in the modern era of research on
AD (Fig. 1A). Filamentous tau deposits are
also present in a number of other neurodegenerative disorders, including Pick’s
disease (Fig. 1B).
Amyloid-ß
Amyloid-ß is 40 to 42 amino acids in length
and is generated by proteolytic cleavage of
the much larger amyloid precursor protein
(APP), a transmembrane protein of unknown
function with a single membrane-spanning
domain (10–13) (Fig. 2A). The N terminus
of amyloid-ß is located in the extracellular
domain of APP, 28 amino acids from the
transmembrane region, and its C terminus is
in the transmembrane region. The enzymes
whose activity gives rise to the N and C termini are called ß-secretase and γ-secretase,
respectively. A third enzyme, α-secretase,
cleaves between residues 16 and 17, precluding amyloid-ß formation. The major species
of amyloid-ß are 40 or 42 amino acids long,
and it is the more amyloidogenic 42–amino
acid form (with its two additional hydrophobic amino acids) that is deposited first (14).
In the three-dimensional structure of the
amyloid-ß fibril, residues 1 to 17 are disordered, with residues 18 to 42 forming a ßstrand–turn–ß-strand motif that contains two
parallel ß sheets formed by residues 18 to 26
and 31 to 42 (15).
Mapping of the APP gene to chromosome
21, together with observation of plaques
and tangles in most elderly individuals with
Down’s syndrome (trisomy of chromosome
21), suggested an important role for amyloid-ß. However, direct genetic evidence was
lacking. It came from work on hereditary cerebral hemorrhage with amyloidosis–Dutch
type (HCHWA-D), a rare condition characterized by recurrent hemorrhages resulting
from the deposition of amyloid-ß in cerebral
blood vessel walls. HCHWA-D is caused by
a missense mutation in the amyloid-ß portion
of APP (16). Six years after the purification
of amyloid-ß from meningeal blood vessels of AD brains (10), this was the second
time that cerebral blood vessels were found
to play a crucial role in advancing the understanding of AD. Although HCHWA-D is
characterized by amyloid-ß deposits in the
walls of cerebral microvessels, it differs from
AD in several respects. Thus, when present,
dementia is vascular in origin. Furthermore,
plaques are sparse and tangles absent.
In the late 1980s, it was speculated that
mutations in the APP gene would also be
found in familial AD, some cases of which
had been linked to chromosome 21 (17, 18).
The first such mutations were soon identified (19–21) (Fig. 2, B and C). Amyloid-ß
is a normal, secreted product (22–24), which
suggests that it has a (still unknown) physiological function. APP mutations increase
amyloid-ß production or lead to an increased
proportion of amyloid-ß ending at residue 42
(25, 26). Most mutations flank the amyloidß region, with the secreted peptide being the
wild type. However, several mutations are
within amyloid-ß itself. Like the HCHWA-D
mutation, some of these mutations have little
effect on APP processing but increase the
propensity of amyloid-ß to form fibrils (27).
Missense mutations in amyloid-ß lead to
vascular deposits, parenchymal plaques, or
both. Twenty missense mutations in the APP
gene have been described (Fig. 2C). Recently, increased gene dosage was identified as
another cause of disease (Fig. 2B). Duplication of the APP gene gives rise to amyloid-ß
deposition in brain neuropil, cerebral blood
vessels, or both locations, with clinical pictures of AD or recurrent brain hemorrhages
(28, 29). These findings are reminiscent of
Down’s syndrome, although brain hemorrhages are only rarely observed. They underscore the need to understand more about the
factors that determine whether amyloid-ß is
deposited in brain or vasculature. Neuronally
derived amyloid-ß is transported to the vasculature, where it is cleared via transport into
the blood or via the perivascular fluid drainage pathway (30). These findings have been
replicated to some extent in transgenic mice.
Expression of mutant human APP in nerve
cells leads to amyloid plaque and blood ves-
and blood vessel wall deposits (31, 32). However, human brain, similar levels of three- and four- the developme
tangles and extensive nerve cell loss have not repeat isoforms are expressed. In tau filaments at reducing tau
the phos
been observed in these mouse lines.
in the di
Mutations in the APP gene account for
sel wall deposits (31, 32). Howposition
only a minority of familial AD cases. Linkever, tangles and extensive nerve
CBD, an
age studies established the presence of a
loss locus
have on
notchromosome
been observed
are prese
major cell
disease
14
in these
mouse
lines.
repeats a
(33), and
positional
cloning
led to the identhepresenilin-1
APP gene
acisoforms
tificationMutations
of mutations in
in the
gene,
fora polytopic
only a minority
fadementia
whichcount
encodes
membraneofpromilial
AD cases.
Linkage are
studies
dementia
tein (34).
Mutations
in presenilin-1
the
the presence
of aAD.
matau depo
most established
common cause
of familial
tau filam
Mutations
in the related
gene
jor disease
locus presenilin-2
on chromosome
their com
also give
rise toand
ADpositional
(35, 36). cloning
More than
14 (33),
led
the mech
160 mutations
in the presenilin
genes
to the identification
of mutations
However
have in
been
Presenilins
are
the identified.
presenilin-1
gene, which
for the e
centralencodes
components
of
the
atypical
asa polytopic membrane
and relat
partylprotein
protease
complexes
(34).
Mutationsresponsible
in preseniSuch a
for the g-secretase cleavage of APP
lin-1 are the most common cause
because
(37, 38), but other transmembrane proof familial AD. Mutations in the
tau patho
teins are also g-secretase substrates.
related
presenilin-2
also give
degenera
Presenilin
gene
mutationsgene
increase
the
riseamyloid-b
to AD (35,
More than
However
ratio of
42 to36).
amyloid-b
40,
160appears
mutations
in from
the presenilin
the genes
and this
to result
a change
genes (39)
havethat
been
identified.
the prese
in function
manifests
itselfPrein
senilins
are central
components
apparentl
reduced
g-secretase
activity.
In preof cases
the atypical
aspartyl protease
on the im
clinical
with presenilin-1
mutaThe
tions,complexes
deposition responsible
of amyloid-bfor
42 the
is anγgene cau
early secretase
event (40,cleavage
41). The ofphenotypic
APP (37,
dementi
spectrum
with transmembrane
presenilin gene
38),associated
but other
chromos
mutations
mayare
extend
beyond ADsubto
proteins
also γ-secretase
encompass
of FTLD
with tau Fig. 1. The abnormal deposits that Alzheimer described. (A) doubt (6
strates.cases
Presenilin
gene mutations
Neuritic plaques made of amyloid-b (blue) and neuro- have bee
deposits
(42). If
this would
increase
theconfirmed,
ratio of amyloid-ß
42
indicate that these mutations can cause fibrillary tangles made of tau (brown) in Alzheimer’s disease. which b
to amyloid-ß 40, and this appears (B) Pick bodies and neurites made of tau (brown) in Pick’s
diseases,
disease through amyloid-b–independent
to Support
result from
a change
func- disease.
predomin
effects.
for this
notionincomes
tion
(39)
that
manifests
itself
in
parkinso
from transgenic animal models, which
reduced γ-secretase
activity.
In preclinical
tangle
formation,
nerve are
cellpresent
degeneration,
from AD
brains,
all six isoforms
in motor neuron d
have suggested
that a reduction
in g-secretase
cases
presenilin-1
mutations, deposition
and dementia
being
events. ical syndromes
similar to
thosedownstream
in normal brains.
activity
can with
lead to
the hyperphosphorylation
of proportions
is an early
event(43).
(40, 41).
Filamentous tau deposits are also found in a disease have a
tau inoftheamyloid-ß
absence of42amyloid-b
deposits
Thethephenotypic
associated with
Tau
number
of other neurodegenerative diseases, tau inclusions
Unlike
presenilins,spectrum
no disease-causing
presenilin
gene identified
mutationsin may
extend beIt took
severalsupranuclear
years of work
absence of am
including
progressive
palsybefore
(PSP),it was
mutations
have been
the aspartyl
the mutations,
corticobasal
(CBD),
Pick’sfilaments
disease, are
protease
which is cases
identical
with with
yondBACE1,
AD to encompass
of FTLD
clear degeneration
that the paired
helical
argyrophilic
grain
disease (AGD),
and the cells or in ner
b-secretase
(44). (42). If confirmed, this would
tau deposits
made of
full-length,
hyperphosphorylated
three-repeat t
Parkinson-dementia
(55). InassemTaken
as a whole,
the mutations
work on familial
AD disindicate
that these
can cause
tau, a protein complex
involvedof
in Guam
microtubule
isoforms. The
PSP, CBD,
and stabilization
AGD, the deposits
are present
formsease
the through
bedrockamyloid-ß–independent
of the amyloid cascade
effects.
bly and
(46–53).
In theinhuman
nerve brain,
cells and
glialisoforms
cells, whereas
in AD,fromarea reflected i
hypothesis
(45),
an increase
Support
forwhich
this holds
notionthat
comes
from transsix tau
are produced
Tau mutations
disease,
andthrough
the Parkinsonism-dementia
in amyloid-b
42 triggers
all cases
AD, suggested
with Pick’s single
genic animal
models,
whichofhave
gene
alternative mRNA splictangle formation, nerve cell degeneration, and complex of Guam they are largely confined to the intron flank
that a reduction in γ-secretase activity can ing (54) (Fig. 3A). They fall into two groups
nerve cells. Unlike AD, these diseases all lack The latter en
dementia being downstream events.
lead to the hyperphosphorylation of tau amyloid-b
in on the
basis Besides
of numbers
of microtubulepathology.
AD, several
other repeat included
binding repeats,
witharethree
isoforms
Tau the absence of amyloid-ß deposits (43). Unmutations fall
neurodegenerative
diseases
associated
withhaving
the presenilins,
disease-causing
three repeats
and such
threeas
isoforms
categories: tho
extracellular
protein each
deposits,
the Abrihaving
It tooklike
several
years of worknobefore
it was clear mutations
identified
in the
aspartyl
presence
absence
splicing of tau
peptidefour
in repeats
familial each.
BritishThe
dementia
andorthe
that the
pairedhave
helicalbeen
filaments
are made
of fullprotease BACE1, which is identical with of N-terminal inserts distinguishes the three
ß-secretase (44).
isoforms in each group. In the normal human
778
314 levels
SCIENCE
www.sciencemag.org
Taken as a whole, the work3onNOVEMBER
familial AD 2006
brain,VOL
similar
of threeand four-reforms the bedrock of the amyloid cascade peat isoforms are expressed. In tau filaments
hypothesis (45), which holds that an increase from AD brains, all six isoforms are presin amyloid-ß 42 triggers all cases of AD, with ent in proportions similar to those in normal
KPI ax2
A
1
disease
shown t
the exp
suggests
from H1
this coul
four-repe
The
velopme
that repl
features
hyperph
extensiv
of lines
pressing
patholog
βα γ
770
APP
α-secretase
APPsα
β-secretase
α-stub
APPsβ
β-stub
γ-secretase
Aβ
B
Allele 1
Allele 2
Gene
dosage
Missense
mutation
C
β-secretase
α-secretase
668
γ-secretase
726
EVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKK
KM670/671NL
D678N
A692G E693Q D694N L705V A713T T714I V715M 1716V V717I L723P
E693K
T714A V715A 1716T V717F
E693G
V717G
V717L
Fig. 2. Amyloid-b. (A) Generation of amyloid-b (Ab) from the amyloid precursor protein (APP).
Cleavage by b-secretase generates the N terminus and intramembranous cleavage by g-secretase
gives rise to the C terminus of amyloid-b. Cleavage by a-secretase precludes Ab formation. (B)
Duplication of the APP gene and missense mutations (black box) in the APP gene cause inherited
forms of Alzheimer’s disease and cerebral amyloid angiopathy. (C) Twenty missense mutations in
APP are shown. Single-letter abbreviations for amino acid residues: A, Ala; C, Cys; D, Asp; E, Glu; F,
Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val;
W, Trp; Y, Tyr.
brains. Filamentous tau deposits are also
primaryin effect
is at oftheother
protein
level. In acfound
a number
neurodegeneracordance
with including
their location
in the microtubuletive
diseases,
progressive
supranubinding
region,
mostcorticobasal
missense mutations
reduce
clear
palsy
(PSP),
degeneration
the ability
of taudisease,
to interact
with microtubules
(CBD),
Pick’s
argyrophilic
grain
(63). Some
mutations
aggregation
disease
(AGD),
and also
the promote
Parkinson-demenintocomplex
filaments.
mutations
andCBD,
most
tia
of Intronic
Guam (55).
In PSP,
coding region mutations in exon 10 increase the
and AGD, the deposits are present in nerve
splicing of exon 10, leading to the relative
cells and glial cells, whereas in AD, Pick’s
overproduction of four-repeat tau (61, 62, 64).
disease,
andbrain,
the a correct
Parkinsonism-dementia
In the normal
ratio of three-repeat
complex
of Guam
they areislargely
to four-repeat
tau isoforms
essentialconfined
for preto
nerve
cells.
Unlike
AD,
these diseases
venting neurodegeneration and dementia.
Multiall
lack amyloid-ß
Besides
AD,
plications
of Tau havepathology.
so far not been
reported.
several
neurodegenerative
diseases
Althoughother
the pathway
leading from a mutation
in
are
associated
with extracellular
protein
Tau to
neurodegeneration
is only incompletely
known, it appears likely that a reduced ability to
10
interact with microtubules is necessary for setting
in motion the gain of toxic function that will
deposits, such as the Abri peptide in familis genetically
ial FTLD
Britishitself
dementia
and the heterogeneous,
prion protein
with a substantial
number of cases exhibiting
(PrP)
in Gerstmann-Sträussler-Scheinker
tau-negative,
ubiquitin-positive
nervedeposits
cell in(GSS)
disease.
As in AD, abundant
clusions.
Mutations
the bodies
genes encoding
the
of
tau form
in nerveincell
and around
apparently
unconnected
p97
(65),
CHMP2B
plaques in familial British dementia and in
(charged
multivesicular
body
proteinPrP
2B) gene
(66),
GSS
disease
caused by
certain
and, in particular, progranulin (67, 68) cause
mutations (56, 57).
these forms of FTLD. In contrast to Tau mutaHyperphosphorylation of tau is common
tions, they all appear to lead to disease through
to
filaments
lossallofdiseases
function with
of thetau
mutant
allele.and may be
required
for toxicity.
is knownthe
about
Haplotypes
H1 and Much
H2 characterize
Tau
phosphorylation
sites
and
candidate
protein
gene in populations of European descent (69).
kinases
auguring
well
for
They areand
the phosphatases,
result of a 900-kb
genomic
inverthe
of preventive
sion development
polymorphism that
encompassesstrategies
Tau (70).
aimed
at reducing
tau phosphorylation
Heterozygous
microdeletions
in this region(58).
give
rise to a form of mental retardation (71–73).
These findings point to a possible role for tau in
brain development and are consistent with the
Sporadi
Most ca
nantly in
1% of th
apolipop
establish
(81), bu
Amyloi
e4-posit
addition
of other
patholog
chondria
function
Age
numbers
individu
ogy app
from wh
amygda
Amyloid
neocorte
independ
later sta
the neo
severe t
suggestio
erbate ag
consisten
APP ge
overprod
tau dys
other ha
degener
rise to
question
neurotox
has accu
species
culprits
mere pr
cell proc
cell, if o
Whereas the phosphorylated sites in tau are
similar in the different diseases, the isoform
composition of tau filaments differs. In PSP,
CBD, and AGD, four-repeat tau isoforms
are present, whereas tau isoforms with three
repeats are found in Pick’s disease. All six
isoforms are present in Parkinsonism-dementia complex of Guam, familial British
dementia, and cases of GSS disease with
tau deposits. The molecular dissection of
tau filaments gave a complete description of
their composition and provided clues about
the mechanisms underlying their formation.
However, the relevance of tau dysfunction
for the etiology and pathogenesis of AD and
related disorders had remained unclear. Such
a connection had been suspected because the
distribution and abundance of tau pathology
correlated well with nerve cell degeneration and clinical symptoms (59). However,
the identification of mutations in the genes
encoding APP and presenilin, and the presence of tau deposits in a number of apparently unrelated disorders, cast doubt on the
importance of tau.
The finding that mutations in the Tau gene
cause the inherited frontotemporal dementia
and parkinsonism linked to chromosome 17
(FTDP-17) removed this doubt (60–62). To
date, 39 such mutations have been described
(Fig. 3B). FTDP-17, which belongs to the
FTLD spectrum of diseases, is quite varied.
It can present predominantly as a dementing
disorder, a parkinsonian disease, or a condition with motor neuron disease–like symptoms. Neurological syndromes similar to
PSP, CBD, and Pick’s disease have also been
described. Filamentous tau inclusions are invariably present in the absence of amyloid-ß
deposits. Depending on the mutations, the inclusions are present in nerve cells or in nerve
cells and glia, and consist of three-repeat
tau, four-repeat tau, or all six isoforms. The
different isoform compositions are reflected
in varied filament morphologies. Tau mutations are located in the coding region or the
intron flanking alternatively spliced exon 10.
The latter encodes the microtubule-binding
repeat included in four-repeat tau. Functionally, mutations fall into two largely nonoverlapping categories: those that influence the
alternative splicing of tau pre-mRNA, and
those whose primary effect is at the protein
level. In accordance with their location in the
microtubule-binding region, most missense
mutations reduce the ability of tau to interact
with microtubules (63). Some mutations also
promote aggregation into filaments. Intronic
mutations and most coding region mutations
in exon 10 increase the splicing of exon 10,
leading to the relative overproduction of fourrepeat tau (61, 62, 64). In the normal brain, a
correct ratio of three-repeat to four-repeat tau
isoforms is essential for preventing neurodegeneration and dementia. Multiplications of
Tau have so far not been reported. Although
the pathway leading from a mutation in Tau
to neurodegeneration is only incompletely
known, it appears likely that a reduced ability to interact with microtubules is necessary
for setting in motion the gain of toxic function that will cause neurodegeneration. This
work is relevant beyond FTDP-17, because
it shows that whenever filamentous tau inclusions form in the brain, abnormalities in
tau are directly involved in the ensuing neurodegeneration.
FTLD itself is genetically heterogeneous,
with a substantial number of cases exhibiting tau-negative, ubiquitin-positive nerve
cell inclusions. Mutations in the genes encoding the apparently unconnected p97 (65),
CHMP2B (charged multivesicular body protein 2B) (66), and, in particular, progranulin (67, 68) cause these forms of FTLD. In
contrast to Tau mutations, they all appear to
lead to disease through loss of function of the
mutant allele.
Haplotypes H1 and H2 characterize the
Tau gene in populations of European descent
(69). They are the result of a 900-kb genomic
inversion polymorphism that encompasses
Tau (70). Heterozygous microdeletions in
this region give rise to a form of mental
retardation (71–73). These findings point
to a possible role for tau in brain development and are consistent with the notion that
FTDP-17 is caused by a gain of toxic function of tau. Inheritance of H1 is a risk factor
for PSP and CBD (69, 74, 75). An association has also been described between H1 and
idiopathic Parkinson’s disease (76), a disease
without tau pathology. H1 has been shown to
be more effective than H2 at driving the expression of a reporter gene, which suggests
that higher levels of tau are expressed from
11
H1 (77). However, it remains unclear how
this could explain the preferential deposition
of four-repeat tau in PSP and CBD.
The work on FTDP-17 has led to the development of robust transgenic mouse models that replicate the essential molecular and
cellular features of the human tauopathies,
including tau hyperphosphorylation, filament
formation, and extensive nerve cell loss (78,
79). The crossing of lines expressing mutant
tau with lines expressing mutant APP results
in enhanced tau pathology (80).
Sporadic Alzheimer’s Disease
Most cases of AD are sporadic, with
dominantly inherited forms accounting for
less than 1% of the total. Inheritance of the
ε4 allele of apolipoprotein E (APOE) is the
only well-established genetic risk factor for
sporadic AD (81), but its mode of action
is unknown. Amyloid-ß deposits are more
abundant in ε4-positive than in ε4-negative
cases (82). In addition, apoE4 is associated
with a number of other factors that may
contribute to AD pathology, including low
glucose usage, mitochondrial abnormalities,
and cytoskeletal dysfunction (83).
Age is a major risk factor for AD, and
small numbers of plaques and tangles form
in most individuals as they grow older (59).
Tau pathology appears first in the transentorhinal region, from where it spreads to the
hippocampus and amygdala, followed by
neocortical areas. Amyloid-ß deposits tend
to appear first in the neocortex. Both types
of inclusion seem to form independently,
with tangles appearing first. At later stages,
extensive amyloid-ß deposition in the neocortex has been reported to precede severe
tangle pathology (84), leading to the suggestion that amyloid-ß deposition may exacerbate age-related tau pathology. This would
be consistent with what is known from cases
with APP gene mutations and duplications,
where overproduction of amyloid-ß 42 is
upstream of tau dysfunction. Mutations in
Tau, on the other hand, lead to filament formation, neurodegeneration, and dementia
but do not give rise to amyloid-ß deposits.
An outstanding question relates to the molecular nature of the neurotoxic species. In
recent years, evidence has accumulated that
suggests that oligomeric species of amyloid12
ß and tau may be major culprits (85). For
tau, it appears likely that the mere presence
of abnormal filaments in nerve cell processes is also detrimental to the parent cell,
if only because they are space-occupying
lesions that are bound to interfere with axonal transport.
In AD, neurodegeneration is estimated to
start 20 to 30 years before the appearance of
the first clinical symptoms. The early clinical
phase is often called amnestic mild cognitive
impairment (aMCI) (86). The neuropathological features of aMCI are intermediate
between those of normal aging and AD, in
that tau deposits are abundant in the entorhinal cortex and hippocampus and some amyloid-ß deposits are present in the neocortex
(87). For aMCI, the regional distribution of
tau deposits correlates better with the degree
of cognitive impairment than does the amyloid-ß load. It has been suggested that the
transition from aMCI to AD occurs when tau
pathology spreads beyond the medial temporal lobe. Work has so far concentrated on
the presence of deposits. In the future, it will
be important to measure levels of amyloid-ß
and tau oligomers in aMCI.
The long presymptomatic phase of AD
augurs well for the development of preventive strategies. To test their effectiveness, it
will be necessary to identify neuropathological abnormalities before the development of
cognitive changes. Use of Pittsburgh compound B (PIB), a thioflavin T derivative, has
already resulted in the visualization of amyloid-ß deposits in patients with AD and in
some nondemented elderly individuals (88),
which suggests that imaging with PIB can
detect clinical and preclinical disease. In the
future, it may also become possible to image
tau deposits in the living human brain.
Closing Remarks
The protein deposits described by Alzheimer
are at the center of current work. Although
much has been learned, major questions
remain. Perhaps the greatest unknown relates to the links between amyloid-ß and
tau. Another important question concerns
the mechanisms that determine the selective vulnerability of defined neuronal and
glial populations. A related issue has to do
with the molecular species that cause nerve
REVIEWS
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
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20.
21.
22.
23.
24.
25.
26.
27.
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Fig. 3. Tau. (A) The six tau isoforms expressed in adult human brain. Alternatively spliced exons
are shown in red, green, and yellow, respectively, and the microtubule-binding repeats are
indicated by black bars. (B) Mutations in the Tau gene in frontotemporal dementia and
parkinsonism linked to chromosome 17 (FTDP-17). Thirty-one coding region mutations in exons (E)
1, 9, 10, 11, 12, and 13 and eight intronic mutations flanking E10 are shown.
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10.1126/science.1132814
Hereditary Early-Onset
Parkinson’s Disease
Caused by Mutations in PINK1
Enza Maria Valente,1*‡ Patrick M. Abou-Sleiman,2* Viviana Caputo,1,3† Miratul M. K. Muqit,2,4†
Kirsten Harvey,5 Suzana Gispert,6 Zeeshan Ali,6 Domenico Del Turco,7 Anna Rita Bentivoglio,9
Daniel G Healy,2 Alberto Albanese,10 Robert Nussbaum,11 Rafael González-Maldonado,12 Thomas
Deller,7 Sergio Salvi,1 Pietro Cortelli,13 William P. Gilks,2 David S. Latchman,4,14 Robert J. Harvey,5
Bruno Dallapiccola,1,3 Georg Auburger,8 ‡ Nicholas W. Wood2 ‡
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by degeneration of dopaminergic
neurons in the substantia nigra. We previously mapped a locus for a rare familial form of PD to chromosome 1p36 (PARK6). Here we show that mutations in PINK1 (PTEN-induced kinase 1) are associated
with PARK6. We have identified two homozygous mutations affecting the PINK1 kinase domain in three
consanguineous PARK6 families: a truncating nonsense mutation and a missense mutation at a highly
conserved amino acid. Cell culture studies suggest that PINK1 is mitochondrially located and may exert a
protective effect on the cell that is abrogated by the mutations, resulting in increased susceptibility to cellular stress. These data provide a direct molecular link between mitochondria and the pathogenesis of PD.
P
arkinson’s disease (PD) is a common
neurodegenerative disorder that is
characterized by the loss of dopaminergic neurons in the substantia nigra and the
presence of cytoplasmic protein inclusions
transition
exonbodies.
4 [nucleotide
(nt) 11185
in
known
as in
Lewy
The majority
of PD
NT_004610],
resulting
in an amino
substicases
are sporadic;
however,
the acid
identifica1
CSS IRCCS, Mendel Institute, viale Regina Margherita
261, 00198 Rome, Italy. 2Department of Molecular Neuroscience, Institute of Neurology, Queen Square, London, WC1N 3BG, UK. 3Department of Experimental
Medicine and Pathology, University La Sapienza, Viale
Regina Elena 324, 00187 Rome, Italy. 4Medical Molecular Biology Unit, Institute of Child Health, 30 Guilford
Street, London WC1N 1EH, UK. 5Department of Pharmacology, The School of Pharmacy, 29/39 Brunswick
Square, London WC1N 1AX, UK. 6Institute for Experimental Neurobiology; 7Institute of Clinical Neuroanatomy; 8Section of Molecular Neurogenetics, Clinic for
Neurology; J.W. Goethe University, Theodor Stern Kai 7,
60590 Frankfurt/M, Germany. 9Institute of Neurology,
Catholic University, largo A. Gemelli 8, I-00168 Rome,
Italy. 10National Neurologic Institute Carlo Besta, via
Celoria 11, 20133 Milan, Italy. 11National Human Genetics Research Institute, National Institutes of Health,
49 Convent Drive, Bethesda, MD 20892, USA. 12Department of Neurology, Hospital Universitatio San Cecilio,
Avenida Dr. Olóriz s/n, 18012 Granada, Spain. 13Department of Neurosciences, University of Modena and
Reggio Emilia, via del Pozzo 71, 41100 Modena, Italy.
14
Birkbeck, University of London, Malet Street, London WC1E 7HX, UK.
*These authors contributed equally to this work.
†These authors share joint second authorship.
‡To whom correspondence should be addressed.
E-mail: [email protected] (N.W.W.); auburger@em.
uni-frankfurt.de (G.A.); [email protected] (E.M.V.)
Fig. 1. PINK1 is localized to the mitochon-
tion of a number of genes responsible for
rare familial forms of PD has provided important insights into the underlying mechanisms of the disease. These genes, encoding
α-synuclein, parkin, UCH-L1, and DJ-1,
tution
(G309D) (5)protein
at a highly
conserved
posihave implicated
misfolding,
impairtion
putative
kinase domain (fig.
S1).
mentinofthethe
ubiquitin-proteasome
system,
Both Italian families carried the same G3 A
and oxidative stress in the pathogenesis of
transitions in exon 7 (nt 15600 in NT_004610),
the disease (1, 2).
which results in a W437OPA substitution, trunWe the
previously
mapped
PARK6,
locus
cating
last 145 amino
acids
encodinga the
C
linked to
autosomal
recessive,
terminus
of the
kinase domain.
Theseearly-onfamilies
set PD,a common
to a 12.5-centimorgan
(cM)
region
shared
haplotype, implying
common
on chromosome
ancestry
(table S1).1p35-p36 by autozygosity
mapping
in a gene
largecontains
consanguineous
The PINK1
eight exonsfamily
spanfrom1.8
Sicilykilobases
(3). Subsequent
identification
of
ning
and encodes
a 581–amino
two additional
consanguineous
families [one
acid
protein. The
transcript is ubiquitously
expressed
(6) and
is (family
predictedIT-GR)
to encode
a 34
–
from central
Italy
(4) and
one
amino
acid mitochondrial
targetingevidence
motif (the
from Spain]
provided additional
of
cleavage
for the A
mitochondrial
processing
linkage tosite
PARK6.
critical recombination
peptidase
between
residues
34refined
and 35)theand
a
event in the
Spanish
family
canhighly
kinase
domain
(resididate conserved
region to protein
a 3.7-cM
interval
between
dues
156 markers
to 509) that
shows high
degree of
flanking
D1S2647
and D1S1539.
homology to the serine/threonine kinases of the
Fine
mapping
of
single-nucleotide
polymorCa2/calmodulin family.
phisms and newly generated short tandem
To investigate the consequences of the
repeat markers
in the
families
defined
missense
mutation
at three
the cellular
level,
we
atransiently
2.8-megabase
region
of
homozygosity
transfected wild-type or G309D
within contig NT_004610,
containing
apc-Myc–tagged
PINK1 cDNA
constructs
proximately
genes.COS-7 cells. The muinto
monkey40
kidney
Candidate
genes
were
prioritizedofonmature
the batation
did not
alter
production
sis of their putative
and expression
full-length
protein, function
which suggests
that it
in thenot
central
nervous system,
as assessed
by
does
significantly
affect protein
stabil15
ity (fig
PINK1
sessed
of trans
human
S3). Fu
ization
ern blot
tions ob
transfec
PINK1
We n
mutation
fluoresc
based as
tential
tion of
(TMRM
tochond
mitocho
port of i
vides the
rylation
G3 A (N.W.W.);
sessed by
immunofluoresence
microscopy
full-length
protein, which suggests
@ion.ucl.ac.uk
auburger@em.
04610),
of transfected(E.M.V.)
COS-7 does
cellsnot
(Fig.
1) and affect protein
(G.A.);
[email protected]
significantly
on, trunng
C
is the
localfamilies
tochonommon
mmalian
ocaliza-
affected
ns
span9D mu1–amino
7 cells
uslywith
exetoa C)
34–
or
otif
to (the
F)
d PINK1
ocessing
)shown.
and a
escence
in (resiout with
greeand
of
ody
esasof folthe
human neuroblastoma SH-SY5Y cells (fig.
S3). Furthermore, the mitochondrial localization of PINK1 was confirmed by Western blotting of mitochondrial enriched fractions obtained from COS-7 cells transiently
transfected with c-Myc–tagged wild-type
PINK1 cDNA (Fig. 2).
We next investigated the effect of PINK1
mutations on mitochondrial function using a
fluorescence-activated cell sorting (FACS)–
based assay of mitochondrial membrane potential (m) by examining the distribution of tetramethylrhodamine methyl ester
(TMRM), a fluorescent lipophilic cation. Mitochondrial membrane potential is central to
mitochondrial biology: It defines the transport of ions, including Ca2 uptake, and provides the driving force for oxidative phosphorylation (7). SH-SY5Y cells were transiently
c–PINK1
ndof (D)];
the
red, we
(B)
vel,
c-Myc–
G309D
mitonstructs
ed [(C)
he
mue bar,
8.0 m.
mature
that it
stabil-
Fig. 1. PINK1 is localized
to the mitochondria in
mammalian cells, and its
localization is not affected
by the G309D mutation.
COS-7 cells transfected
with wild-type (A to C)
or G309D (D to F) c-Myc–
tagged PINK1 protein are
Fig. 2. PINK1
is localized to the mi
shown.
Immunofluoresenriched
fraction
mammalian
cence
was carried
out of
with
cells were transiently transfe
c-Myc
antibody
and
mitoc-Myc–tagged wild-type PINK1. C
tracker
c-Myc–
(lane as
1)follows:
and mitochondria-enrich
PINK1
[green,
(A) and
(D)]; and probe
fractions
were
obtained
PINK1 expression
by Western b
mitotracker
[red, (B) and
using
antibody to
(E)];
and an
c-Myc–PINK1
andc-Myc. The
brane wasmerged
stripped[(C)
and reprobed
mitotracker
bodies to heat shock protein 6
and (F)]. Scale bar, 8.0 µm.
complex I, and glyceraldehyde-3
dehydrogenase (GAPDH) to det
relative purity of fractions analy
Fig. 2. PINK1 is localized to the mitochondria-enriched fraction
of mammalian cells. COS-7 cells were transiently transfected
with c-Myc–tagged wild-type PINK1. Cytoplasmic (lane 1) and
mitochondria-enriched (lane 2) fractions were obtained and
probed for c-Myc PINK1 expression by Western blot analysis
using an antibody to c-Myc. The same membrane was stripped
and reprobed with antibodies to heat shock protein 60 (HSP60),
complex I, and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) to determine the relative purity of fractions analyzed.
thatPINK1
it
ype
but not mutant
stabilprotects against stress-inondrial dysfunction and apoB) Effect of wild-type and
1 on mitochondrial memial (m) determined by
-gated events of SH-SY5Y
with vehicle (basal value) or
Basal TMRM values normalmean  SEM percent TMRM
of vector. (B) Mean  SEM
e in median TMRM fluoresV-PE–positive GFP-gated events of SH-SY5Y cells treated w
e basal value for each construct after treatment with 15
sulfoxide vehicle (open bars) or MG-132 (solid bars). The m
*P 0.01 for G309D versus wild-type PINK1; ANOVA with
Fig.
2.
PINK1
is
localized
to
the
mitochondriapercent apoptotic cell death for each construct after trea
erroni correction; n  8 data sets from three independent
enriched
fraction
of
mammalian
cells.
COS-7
vehicle or 15 M MG-132 is shown. *P  0.05; ANOVA, n 
erformed in duplicate or triplicate. (C) Effect of wild-type
cells
were
transiently
transfected
with
bioinformatic
analysis and
by exon
amplifi- from
the kinase
domain. These
familiesperformed
shared ain triplicate
four independent
experiments
PINK1 on apoptosis,
as determined
by FACS
of annexin
c-Myc–tagged
wild-type
PINK1.
Cytoplasmic
cation from a human substantia nigra cDNA common haplotype, implying common an(lane 1) and mitochondria-enriched (lane 2)
library.
analysis
candidate
genes
www.sciencemag.org
SCIENCE cestry
VOL (table
304 S1).
21 MAY 2004
fractionsSequence
were obtained
andofprobed
for c-Myc
in
affected
members
each blot
family
led to
The PINK1 gene contains eight exons
PINK1
expression
by from
Western
analysis
using
an antibody of
to two
c-Myc.
The same memthe
identification
homozygous
muta- spanning ~1.8 kilobases and encodes a
brane in
was
and reprobed
withkinase
antitions
thestripped
PTEN-induced
putative
581–amino acid protein. The transcript is
bodies to heat shock protein 60 (HSP60),
1complex
(PINK1)
gene.
The
mutations
segregated
ubiquitously expressed (6) and is predicted
I, and glyceraldehyde-3-phosphate
with
the disease(GAPDH)
phenotypetoindetermine
the three conto encode a 34–amino acid mitochondrial
dehydrogenase
the
relative purityfamilies,
of fractions
sanguineous
wereanalyzed.
confirmed in the targeting motif (the cleavage site for the
cDNA, and were absent from 400 control
chromosomes, including 200 chromosomes
from Sicilian individuals. The Spanish family carried a G→A transition in exon 4 [nucleotide (nt) 11185 in NT_004610], resulting
in an amino acid substitution (G309D) (5) at
a highly conserved position in the putative
kinase domain (fig. S1). Both Italian families
carried the same G→A transitions in exon 7
(nt 15600 in NT_004610), which results in
W437OPA
substitution,
truncating
the last
GFP-gated aevents
of SH-SY5Y
cells treated
with dimethyl
145 or
amino
acids(solid
encoding
terminus
of
icle (open bars)
MG-132
bars). the
TheCmean
 SEM
totic cell death for each construct after treatment with
M MG-13216is shown. *P  0.05; ANOVA, n  12 data sets
ependent experiments performed in triplicate.
mitochondrial processing peptidase between
residues 34 and 35) and a highly conserved
protein kinase domain (residues 156 to 509)
that shows high degree of homology to the
serine/threonine kinases of the Ca2+/calmodulin family.
To investigate the consequences of the
missense mutation at the cellular level, we
transiently transfected wild-type or G309D
c-Myc–tagged PINK1 cDNA constructs into
monkey kidney COS-7 cells. The mutation
did not alter production of mature full-length
dehydrogenase (GAPDH) to determine the
relative purity of fractions analyzed.
mutant
tress-inand apoype and
l memined by
SH-SY5Y
value) or
normalnt TMRM
 SEM
fluores3. Wild-type
but not
mutant G309D
PINK1 protects
stress-induced
V-PE–positive
GFP-gated
events ofagainst
SH-SY5Y
cells treatedmitochondrial
with dimethyl
ach constructFig.
after
treatment PINK1
with 15
dysfunction
apoptosis.
B) Effect
of wild-type
and mutant
PINK1(solid
on mitochondrial
membrane
sulfoxide
vehicle
(open bars)
or MG-132
bars). The mean
 SEM
versus wild-type
PINK1;and
ANOVA
with (A and
death
for each cells
construct
with
 8 data sets
from three
potential
(ΔΨindependent
m) determined bypercent
FACS ofapoptotic
GFP-gatedcell
events
of SH-SY5Y
treatedafter
withtreatment
vehicle (basal
vehicle
or normalized
15 M MG-132
is shown.
0.05;
ANOVA,
n
12 data sets
te or triplicate.
(C)orEffect
of wild-type
value)
MG-132.
(A) Basal TMRM
values
to vector,
mean*P± 
SEM
percent
TMRM
fluorescence
from four independent experiments performed in triplicate.
as determined by FACS of annexin
of vector. (B) Mean ± SEM percent change in median TMRM fluorescence from the basal value for each
construct after treatment with 15 µM MG-132. *P >0.01 for G309D versus wild-type PINK1; ANOVA with
www.sciencemag.org
SCIENCEn = VOL
21 MAY
post-hoc Bonferroni correction;
8 data304
sets from
three 2004
independent experiments performed in duplicate 1159
or triplicate. (C) Effect of wild-type and mutant PINK1 on apoptosis, as determined by FACS of annexin
V-PE–positive GFP-gated events of SH-SY5Y cells treated with dimethyl sulfoxide vehicle (open bars) or
MG-132 (solid bars). The mean ± SEM percent apoptotic cell death for each construct after treatment with
vehicle or 15 µM MG-132 is shown. *P < 0.05; ANOVA, n = 12 data sets from four independent experiments
performed in triplicate.
protein, which suggests that it does not significantly affect protein stability (fig. S2).
Both wild-type and mutant PINK1 localized
to mitochondria, as assessed by immunofluoresence microscopy of transfected COS-7
cells (Fig. 1) and human neuroblastoma SHSY5Y cells (fig. S3). Furthermore, the mitochondrial localization of PINK1 was confirmed by Western blotting of mitochondrial
enriched fractions obtained from COS-7 cells
transiently transfected with c-Myc–tagged
wild-type PINK1 cDNA (Fig. 2).
We next investigated the effect of PINK1
mutations on mitochondrial function using a fluorescence-activated cell sorting
(FACS)–based assay of mitochondrial membrane potential (ΔΨm) by examining the
distribution of tetramethylrhodamine methyl
ester (TMRM), a fluorescent lipophilic cation. Mitochondrial membrane potential is
central to mitochondrial biology: It defines
the transport of ions, including Ca2+ uptake,
and provides the driving force for oxidative
phosphorylation (7). SH-SY5Y cells were
transiently cotransfected with wild-type
and mutant PINK1 cDNA and a green fluorescent protein (GFP) reporter plasmid and
then stressed with the peptide aldehyde Cbzleu-leu-leucinal (MG-132), which inhibits
the proteasome and induces apoptosis via
distinct mechanisms, including mitochon-
drial injury (8). Analysis of TMRM fluorescence in GFP-positive cells revealed that the
PINK1 mutation had no significant effect
on ΔΨm under basal conditions (Fig. 3A).
However, after stress with MG-132, there
was a significant decrease in ΔΨm from
basal levels in cells transfected with G309D
PINK1 as compared with wild-type PINK1
(G309D, –44.1% ± 8.1; versus wild-type
PINK1, 13.0% ± 13.7; P < 0.01; n = 8 data
sets) (Fig. 3B and fig. S4).
We next studied apoptosis of MG-132–
stressed SH-SY5Y cells transfected with
either wild-type or G309D PINK1 by FACS
using annexin V conjugated to the fluorochrome phycoerythrin (annexin V-PE). Annexin V has a high binding affinity for the
membrane phospholipid, phosphatidylserine,
that is exposed on the surface of apoptotic
cells. Consistent with the changes in ΔΨm
after stress, overexpression of wild-type
PINK1 but not mutant PINK1 significantly
reduced the level of apoptotic cell death induced by MG-132 in GFP-positive cells [vector, 45.4% ± 5.0; wild-type PINK1, 32.7% ±
4.0; G309D, 45.8% ± 5.0; P > 0.05; analysis
of variance (ANOVA), n = 12 data sets] (Fig.
3C and fig. S5). These preliminary findings
suggest that wild type PINK1 may protect
neurons from stress-induced mitochondrial
dysfunction and stress-induced apoptosis
17
and that this effect is abrogated by the
G309D mutation.
Several lines of evidence suggest that
impairment of mitochondrial activity could
represent an early critical event in the pathogenesis of sporadic PD (2). Environmental
toxins such as 1-methyl-4-phenyl-1,2,3,6tetrahydro-pyridin and the pesticide rotenone induce selective death of dopaminergic
neurons through inhibition of complex I
activity (9, 10). Complex I deficiency and a
variety of markers of oxidative stress have
been demonstrated in postmortem brains
of PD patients (2, 11, 12). In addition, several reports have shown that mitochondrial
dysfunction associated with oxidative stress
can trigger α-synuclein aggregation and accumulation, although the exact mechanisms
remain unclear (13).
The PINK1 mutations described here occur in the putative serine/threonine kinase
domain and thus conceivably could affect
kinase activity or substrate recognition. Al
References and Notes
1. W. Dauer, S. Przedborski, Neuron 39, 889 (2003).
2. T. M. Dawson, V. L. Dawson, Science 302, 819
(2003).
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(2001).
4. E. M. Valente et al., Ann. Neurol. 51, 14 (2002).
5. Single-letter abbreviations for the amino acid
residues are as follows: A, Ala; D, Asp; G, Gly;
W, Trp.
6. M. Unoki, Y. Nakamura, Oncogene 20, 4457
(2001).
7. M. R. Duchen, A. Surin, J. Jacobson, Methods
Enzymol. 361, 353 (2003).
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36, 2503 (1985).
10. R. Betarbet et al., Nature Neurosci. 3, 1301
(2000).
11. A. H. Schapira et al., Lancet 2, 1269 (1989).
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(1998).
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(2002).
17. D. S. Gary, M. P. Mattson, Neuromol. Med. 2,
261 (2002).
18. We thank the patients and families who participated in this study, J. Sinclair for technical
assistance with FACS experiments, G. Howell
for bioinformatic support, M. Duchen for useful
18
tered phosphorylation has been reported as a
pathogenetic mechanism in other neurodegenerative diseases, including Alzheimer’s disease, tauopathy, and spinocerebellar
ataxia (14, 15). The recent demonstration that
phosphorylation of α-synuclein at serine 129
occurs in Lewy bodies in a variety of brains
from humans with synucleinopathy (16) suggests that altered phosphorylation may also
play a role in PD. We hypothesize that PINK1
may phosphorylate mitochondrial proteins in
response to cellular stress, protecting against
mitochondrial dysfunction. PINK1 was
originally shown to be upregulated by the
tumor suppressor gene PTEN in cancer cells
(6). In neurons, the PTEN signaling pathway
is involved in cell cycle regulation and cell
migration and promotes excitotoxin-induced
apoptosis in the hippocampus (17). However,
PINK1 has not been shown to have any effects on PTEN-dependent cell phenotypes
(6), and its role in the PTEN pathway therefore requires further investigation.
discussion, S. Eaton for assistance with mitochondrial fractionation, and Y. Nakamura and
M. Unoki for the PINK1 plasmid. Supported by
grants from Telethon, Italy (E.M.V.); the Italian
Ministry of Health (E.M.V. and B.D.); MURST
(B.D.); the Parkinson’s Disease Society, UK
(N.W.W., D.S.L., R.J.H., and D.G.H.); the Brain
Research Trust (P.M.A.S. and N.W.W.); and
the Deutsche Forschungsgemeinschaft (G.A.
and S.G.). M.M.K.M. is a Medical Research
Council Clinical Research Training Fellow.
GenBank accession numbers are as follows:
PINK1 genomic sequence, AL391357; PINK1
mRNAs, AB053323, AF316873, AK075225,
BC009534, and BC028215; and PINK1 protein,
BAB55647 AAK28062 BAC11484 AAH09534
and AAH28215.
Supporting Online Material
www.sciencemag.org/cgi/content/full/1096284/DC1
Materials and Methods
SOM Text
Figs. S1 to S5
Tables S1 and S2
References
2 February 2004; accepted 1 April 2004
Published online 15 April 2004;
10.1126/science.1096284
Include this information when citing this paper.
Endothelial Cells Stimulate
Self-Renewal and Expand
Neurogenesis of Neural Stem Cells
Qin Shen,1 Susan K. Goderie,1 Li Jin,1 Nithin Karanth,1 Yu Sun,1 Natalia Abramova,1
Peter Vincent,2 Kevin Pumiglia,3 Sally Temple1*
Neural stem cells are reported to lie in a vascular niche, but there is no direct evidence for a functional
relationship between the stem cells and blood vessel component cells. We show that endothelial cells but
not vascular smooth muscle cells release soluble factors that stimulate the self-renewal of neural stem
cells, inhibit their differentiation, and enhance their neuron production. Both embryonic and adult neural
stem cells respond, allowing extensive production of both projection neuron and interneuron types in
vitro. Endothelial coculture stimulates neuroepithelial cell contact, activating Notch and Hes 1 to promote
self-renewal. These findings identify endothelial cells as a critical component of the neural stem cell niche.
S
tem cell expansion and differentiation lial cells were never found in the lower comare regulated in vivo by environmental partment when BPAE or MbEND cells were
factors encountered in the stem cell plated in the transwell upper compartment
niche (1). In the adult, neural stem cells lie (Fig. 1B), confirming that the feeder cells
REPORTS
close to blood vessels: in the hippocampus could not migrate through the 0.4-µm-diam(2), the subventricular zone (SVZ) (3), and eter membrane pores.
the songbird higher vocal center (4). In the
As expected (6), embryonic stem cell
developing central nervous system (CNS), clones cocultured with CTX began proventricular zone cells produce vascular en- ducing neurons within a day. Most neuron
dothelial growth factor, which attracts vessel production was over by 7 days, and growth
growth toward them (5). Thus, vascular cells after this time was largely in glial lineages.
are close to CNS germinal zones throughout Clones cocultured with BPAE or MbEND
life (fig. S1), and it1has been suggested that
cells behaved differently (Fig. 1D and fig.
Qin Shen, Susan K. Goderie,1 Li Jin,1 Nithin Karanth,1 Yu Sun,1
they form a niche for neural 1stem cells (2).
S2), growing into sheets
of largely flattened
3
1
Peter interacVincent,2progeny
Kevin Pumiglia,
Sally
Temple
To Natalia
examine Abramova,
a possible functional
that maintained
tight
cell-cell* contion, weNeural
cocultured
andreported
vasculartocells
(illustrated
1Cis by
strong juncstem neural
cells are
lie in atact
vascular
niche, in
butFig.
there
no direct
(Fig. 1A).
Neuralforstem
cells fromrelationship
mouse ce- between
tional the
ß-catenin
staining),
withvessel
only a few
evidence
a functional
stem cells
and blood
rebral cortex
from cells.
embryonic
day 10
to endothelial
11 immature,
cells appearing
component
We show
that
cells neuron-like
but not vascular
smooth on top
(E10-11)
werecells
plated
at clonal
density
the sheets.
stem cell
muscle
release
soluble
factorsonthat of
stimulate
theNeural
self-renewal
of clones
neural grown
the base
of cells,
culture
wells.
Thedifferentiation,
upper tran- and
withenhance
endothelial
wereproduction.
larger, with more
stem
inhibit
their
theircells
neuron
swell compartment
wasand
seeded
primitive
progeny
[expressing
progeniBoth embryonic
adult with
neuralpuristem cells
respond,
allowing
extensivethe
production of both projection
neuron
and interneuron
in and
vitro.LeX
Endothelial
fied vascular-associated
or other feeder
cells:
tor markerstypes
Nestin
(3)] and fewer
stimulates neuroepithelial
and Hes1than
to were
primarycoculture
bovine pulmonary
artery endothe-cell contact,
neurons activating
(expressingNotch
ß-tubulin-III),
promote
These
findings identify
endothelial
a critical
lial (BPAE)
cells,self-renewal.
a mouse brain
endothelial
clones grown
with cells
CTXas(Fig.
1, D to G).
component
the neural
stemmuscle
cell niche.
(MbEND)
cell line,ofvascular
smooth
Hence, endothelial factors facilitate expan(VSM) cells, NIH3T3 fibroblasts, or as a sion of cortical stem cell clones and inhibit
Stem cell
expansionage-matched
and differentiation
In thedifferentiation.
adult, neural VSM
stem and
cellsNIH3T3
lie closecells
to
control,
high-density
cortical their
are
regulated
in
vivo
by
environmental
facblood
vessels:
in
the
hippocampus
(2),
the
+
cells (CTX). CD31 (platelet endothelial cell also promoted neural stem cell proliferation
tors encountered in the stem cell niche (1).
subventricular zone (SVZ) (3), and the
adhesion molecule c-1, PECAM-1) endothe- (Fig. 1F), but clones were less cohesive and
songbird higher vocal center (4 ). In the
included
more
glial-like progeny than those
developing
central nervous system (CNS),
1
Center for Neuropharmacology and Neuroscience,
in
endothelial
coculture.
ventricular
zone
cells produce vascular en2
Center for Cardiovascular Sciences, 3Center for Cell
When thegrowth
transwells
were which
removed,
endodothelial
factor,
attracts
Biology and Cancer Research, Albany Medical College,
thelial-expanded
stem cell
continued
Albany, NY 12208, USA.
vessel growth toward
themclones
(5). Thus,
vasto
proliferate
also
to differentiate
cular
cells arebut
close
to began
CNS germinal
zones
*To whom correspondence should be addressed. E(Fig.
2A), and
4 days
produced
mail: [email protected]
throughout
lifewithin
(fig. S1),
andthey
it has
been
Endothelial Cells Stimulate
Self-Renewal and Expand
Neurogenesis of Neural Stem Cells
Fig. 1. Endothelial cell–derived soluble factors
stimulate cortical stem cell expansion and delay
19
suggest
stem ce
To e
action,
cells (F
mouse c
10 to 1
density
upper t
with pu
feeder c
tery en
brain en
cular sm
fibrobla
age-ma
(platele
c-1, PE
er foun
BPAE
the tran
confirm
migrate
membra
As e
clones c
ing neu
product
after thi
Clones
cells be
S2), gr
cular cells are close to CNS germinal zones
throughout life (fig. S1), and it has been
ddressed. E-
cells behaved differently (Fig. 1D and fig.
S2), growing into sheets of largely flat-
ble factors
and delay
m. (B to G)
s. (B) CD31
s (top), but
the cortical
25 m. (C)
, cohesive
onger juncal (bottom)
cale bar, 50
for 7 days
et-like and
fewer ones. Scale
ze (defined
t day 7 in
rent feeder
of variance
post-hoc
progenitor
cell clone
Fig. 1. Endothelial cell–derived soluble factors stimulate cortical stem cell expansion and delay differentiation.
(A) The
coculture
(B to G) Results
from E10-11 cortical stem cells. (B) CD31 stains endothelial cells
28 MAY
2004
VOL system.
304 SCIENCE
www.sciencemag.org
in the transwells (top), but no CD31+ cells are detected below in the cortical cell compartment (bottom).
Scale bar, 25 µm. (C) Cortical stem cells generate larger, cohesive clones of flattened progeny with stronger
junctional ß-catenin staining in endothelial (bottom) compared to cortical (top) coculture. Scale bar, 50 µm.
(D) Neural stem cell clones grown for 7 days with endothelial (Endo) cells were sheet-like and had more
Nestin+ and LeX+ and fewer ß-tubulin-III+ progeny than control clones. Scale bar, 100 µm. (E) Histogram of
clone size (defined by number of progeny) frequency at day 7 in culture. (F) Mean clone size with different
feeder layers at 7 days in culture [analysis of variance (ANOVA), * indicates P < 0.01 by post-hoc tests]. (G)
Percentages of cells with progenitor and neuronal markers per stem cell clone (*, P < 0.05, t test).
ß-tubulin-III+ neurons (Fig. 2B), which were
almost all microtubule-associated protein 2
(MAP-2+). About 30% of the neurons had
acquired the later neuronal marker NeuN (7).
The clones contained up to ~10,000 progeny,
and on average 31% were neurons. In contrast, in control CTX cocultures 4 days after
transwell removal, stem cell clones ranged
up to 4350 cells, and on average only 9%
were neurons. Similarly, E11 cortical cells
cultured as neurospheres for 7 days then
differentiated in adherent culture for 4 days
produced only 7% neurons. Many more stem
cell clones growing in BPAE cocultures con20
tained a high percentage of neurons, up to
64%, compared to clones grown in CTX coculture (Fig. 2, D and E), and neuron production was prolonged (supporting online text
and fig. S3). Increased neurogenesis from
endothelial cocultured neural stem cells did
not occur at the expense of gliogenesis: The
percentage of glial fibrillary acidic protein
(GFAP+) astrocytes generated was similar,
and although oligodendrocyte differentiation
(indicated by staining with the early oligodendrocyte marker O4) was reduced in BPAE
cocultures compared to CTX cocultures, the
difference could not account for the enhance-
less cohesive and included
ke progeny than those in endoure.
tured as neurospheres for 7 days then differentiated in adherent culture for 4 days
produced only 7% neurons. Many more
ial-expanded
w enhanced
uction. (A)
the experiResults from
stem cells.
al-cocultured
(Endo) had
n production
o controlells (CTX),
ubulin-III exy 11 (D11).
m. (C) Oliand astroby O4 (red)
en) labeling,
e present in
al and CTX
nes at D14.
m. (D) Hisuron generacell clone. (E)
of neuron
m stem cells
r at the excell producproduction is
anced by encoculture,
oculture with
(*, P  0.05;
01; ANOVA,
test). (F to
em cells from
ic and adult
hanced neuendothelial
(F) Neurons
ical cell adCTX) (*, P 
NewmanNeurons per
neurosphere
001, ANOVA,
test). (H)
adult SVZ
(*, P  0.01,
ference could not account for the enhancement of neuron generation (Fig. 2, C and
E). NIH3T3 cells enhanced oligodendrocyte generation. Coculture with VSM or
NIH3T3
reduced neurogenesis comFig.
2. cells
Endothelial-expanded
pared cells
to CTX
(Fig.
2E), showing
stem
show
enhanced
neu- that the
endothelial effect is cell-type specific.
ron
production. (A) Schematic of
Endothelial cells stimulate proliferation
the
experiment. (B to E) Results
and neurogenesis of neural stem cells from a
from
cortical CNS
stem regions
cells. (7) and
varietyE10-11
of embryonic
(B)
stem and adult
fromEndothelial-cocultured
different stages. E15.5 cortical
cells
hadgrown
massive
neuron
SVZ (Endo)
stem cells
in endothelial
cocul
, Nestin cells.
ture generated
sheets ofto
LeX
production
compared
controlAfter differentiation,
E15.5 by
endothelialcocultured
cells (CTX), shown
expanded cortical
cells and
adult
ß-tubulin-III
expression
at day
11 SVZ cells
produced
more
(D11).
Scale
bar, neurons
200 µm. compared
(C) Oligo- to control
cells (Fig. 2, F and H).
dendrocytes
and astrocytes, shown
Neurosphere-expanded stem cells reby
O4 (red) and GFAP (green) lasponded to endothelial factors. E15.5 cortical
beling,
respectively,
are present inin fibroblast
cells grown
as neurospheres
both
endothelial
CTXfor
coculgrowth
factor 2 and
(FGF2)
7 days were
tured
at D14.conditions
Scale bar,and
100cocultured
platedclones
in adherent
for 3(D)days
with endothelial
cells or with
µm.
Histogram
of neuron genage-matched
cortical
differentiated
eration
per stem
cell cells,
clone.then
(E) Enby withdrawal
feeder production
cells for 4 days. Stem
hancement
of ofneuron
cells exposed
to does
endothelial
factors
from
stem cells
not occur
at produced
22% neurons, compared to 2% neurons in
the expense of glial cell production.
control CTX cocultures (Fig. 2G).
Neuron
production
is specifically
In vivo,
most projection
neurons are
enhanced
byearly
endothelial
cell coculborn in the
embryonic
period, whereture,
compared
to
coculture
with
as glia and interneurons arise later; adult
other
cell types
(*, P <to0.05;
**, P interneustem cells
are primed
generate
(8, 9).
To examine
the neuron subtypes
<rons
0.001;
ANOVA,
Newman-Keuls
generated
E10-11stem
cortical
test).
(F to from
H) Forebrain
cellsstem cells
expanded
endothelial
from
olderinembryonic
andcoculture,
adult differentiatedshow
clones
were stained
for glutamic
stages
enhanced
neurogenacid decarboxylase (GAD67), a GABAeresis with endothelial cell coculture.
gic marker typically expressed in interneu(F)
Neurons
per an
E15.5
cortical
cell neuron
rons,
or Tbr1,
early
pyramidal
adherent
clone
(CTX) (*, P <
0.05,projection
marker that
preferentially
labels
ANOVA,
(G) stem cell
neurons Newman-Keuls
(10) (Fig. 3A).test).
More
Neurons
per E15.5incortical
clones growing
BPAE neurococulture made
Tbr1 projection
sphere
(NS) (*, P neurons,
< 0.001, compared
ANOVA, to CTXcocultured clonestest).
(Fig.(H)
3B).Neurons
BPAE-cocultured
Newman-Keuls

neurons than
stem adult
cells generated
more Tbrclone
per
SVZ adherent
neurosphere-expanded
E10
cells
that were sub(*, P < 0.01, t test).
sequently differentiated in adherent culture
(9.95% versus 2.41%). Thus, endothelial cell
coculture supports development of both projection neurons and interneurons.
www.sciencemag.org SCIENCE VOL 304 28 MAY 2004
ment of neuron generation (Fig. 2, C and E).
NIH3T3 cells enhanced oligodendrocyte
generation. Coculture with VSM or NIH3T3
cells reduced neurogenesis compared to CTX
(Fig. 2E), showing that the endothelial effect
is cell-type specific.
Endothelial cells stimulate proliferation
and neurogenesis of neural stem cells from
a variety of embryonic CNS regions (7) and
from different stages. E15.5 cortical and
adult SVZ stem cells grown in endothelial
coculture generated sheets of LeX+, Nestin+
cells. After differentiation, E15.5 endothe-
lial-expanded cortical cells and adult SVZ
cells produced more neurons compared to
control cells (Fig. 2, F and H).
Neurosphere-expanded stem cells
responded to endothelial factors. E15.5
cortical cells grown as neurospheres in
fibroblast growth factor 2 (FGF2) for 7
days were plated in adherent conditions
and cocultured for 3 days with endothelial
cells or with age-matched cortical cells,
then differentiated by withdrawal of feeder
cells for 4 days. Stem cells exposed to
endothelial factors produced 22% neurons,
21
13
R RE EP POOR RT TS S
Fig.
3.
Endothelial-
3. E10
EndothelialFig. 3.Fig.
Endothelial-expanded
expanded
stem cell
expanded
E10
stem
cell
clones
theretain
ability
E10 stem
cellretain
clones

clones
retain
the
ability
+
to generate
Tbr1
prothe ability
to generate
Tbr1
tojection
generate
Tbr1
proneurons as well
projection
neurons
as
well

jection
neurons
as
well
as
GAD interneurons.
+

as GAD
interneurons.
(A)
as(A)
GAD
GADinterneurons.
(cytoplasmic,
(A)
GAD
red), Tbr1(cytoplasmic,
(nuclear
GAD (cytoplasmic,
red), markTbr1
red),
Tbr1
(nuclear
marker,marker,
red) and
-tubulin-III
(nuclear
red)
and
ßer,(green)
red) and
-tubulin-III
staining.
(B) Histubulin-III
(green)
staining.
tograms
showing
the
(green)
staining.
(B) His(B) Histograms
showing
the
frequencyshowing
of GADthe
and
tograms
+
Tbr1
Tbr1
neurons
stem
frequency
ofGAD
andin
frequency
of +GAD
and
cell
clones.
Tbr1
neurons
in stem
neurons
instem
cell clones.
cell clones.
Fig. 4. Endothelial factors stimu-
late
of
neural
stem
Fig. 4.Fig.
Endothelial
factorsfactors
stimulate
self4. self-renewal
Endothelial
stimucells.
(A) Comparison
between
late
ofcells.
neural
stem
renewal
ofself-renewal
neural
stem
(A)
Comtypical
lineage
trees
reconstructcells.
(A) Comparison
between
parison
between
typical lineage
trees
ed
from
time-lapse
video recordtypical
lineage
trees reconstructings
of
single
E10
cortical
stem
reconstructed
from
time-lapse
video
ed from time-lapse video recordcellsofgrown
with
cells
recordings
single
E10endothelial
corticalstem
stem
ings
of single
E10
cortical
and those grown under control
cells cells
grown
with
endothelial
cells
and
grown
with
endothelial
cells
conditions. In endothelial coculthose
grown
under
control
thoseand
grown
under
control
conditions.
In
ture,
the
cortical
stem
cell
dividconditions.
In endothelial
coculendothelial
coculture,
the and
cortical
stem
ed symmetrically
did
not
ture,
theneurons
corticalduring
stem the
cell recorddividmake
cell divided
symmetrically
anddid
didnot
not
edingsymmetrically
and
period (all progeny
were
make make
neurons
during
the
recording
pe
neurons
duringinthe
recordNestin
as shown
the
fluores+
riod (all
progeny
were
Nestin
as of
shown
ingcence
period
(all
progeny
were
and
phase
images
the

as shown
in theimages
fluoresin theNestin
fluorescence
and
phase
of
final clone).
In contrast,
a corticence
and cell
phase
images
the
cal
stem
grown
under
conthe final clone).
In contrast,
a of
cortical
final
clone).
In contrast,
a cortitrol
conditions
generated
an
stem cell
grown
under
control
conditions
calasymmetric
stem cell
grown
conlineage under
tree, genergenerated
an
asymmetric
lineage
tree,
trol
an
atingconditions
neurons generated
[-tubulin-III
+
generating
neurons
[ß-tubulin-III
(red),
(red),
designated
astree,
N in generthe
linasymmetric
lineage

eageasneurons
tree]
as Nestin
ating
[-tubulin-III
designated
N in as
thewell
lineage
tree] as
progenitor
cells as
(green).
Neuro+
(red),
designated
Ncells
in the
linwell as
Nestin
progenitor
(green).
nal tree]
progeny
are numbered
to
eage
as well
as Nestin
Neuronal
progeny
are numbered
toinshow
show
the cells
match
of cellsNeurothe
progenitor
(green).
the match
of
cells
in
the
final
clone
final
clone
to
the
lineage
tree.
nal progeny are numbered toto
Arrows
indicate
neurons
the
the lineage
tree.
Arrowsof
indicate
show
the
match
cells inin neuthe
field
that that
did
not
fromfrom this clone. (B) After treatment of 4-day-old cocultures with
final
to the
lineage
tree.
rons in
the clone
field
did originate
not
originate
this clone.
(B) After
treatment
cocultures with -secretase inhibitor II for 6 hours,
Arrows
indicate
neurons
in ß-catenin
the of 4-day-old
γ-secretase
inhibitor
II for
6is hours,
staining is significantly decreased and ß-tubulin-III staining
-catenin
significantly
field
that didstaining
not originate
from decreased and -tubulin-III staining significantly increased in
significantly
increased
inclones,
BPAE cocultured
clones,
there
was no
effect onclones
CTX cocultured
clones
BPAE
cocultured
whereas
waswhereas
no
effect
onwith
CTX
cocultured
(ANOVA;
P
this
clone.
(B) After
treatment
of there
4-day-old
cocultures
-secretase
inhibitor
II for *,
6 hours,
0.01
post-hoc
DMSO,
dimethyl
sulfoxide.
(C) (C)
Hes1
is isup-regulated
after
(ANOVA;
*, P by
< 0.01
by post-hoc
tests).
DMSO,
dimethyl
sulfoxide.
Hes1
up-regulated
afterendothelial
endothelialin
-catenin
staining
istests).
significantly
decreased
and
-tubulin-III
staining
significantly
increased
coculture,
Hes5 expression
wastosimilar
to that incoculture.
control coculture.
Reverse transcription–
coculture,
but
Hes5but
expression
was similar
Reverse transcription–polymerase
BPAE
cocultured
clones, whereas
there that
was in
nocontrol
effect on CTX cocultured
clones (ANOVA; *, P 
polymerase chain reaction gel band densities were normalized to expression levels of glyceraldebyphosphate
post-hoc
tests). DMSO,
dimethyl to
sulfoxide.
(C)levels
Hes1ofisglyceraldehyde
up-regulated phosphate
after endothelial
chain 0.01
reaction
gel band densities
were normalized
expression
dehyhyde
dehydrogenase
(GAPDH).
coculture,
but Hes5 expression was similar to that in control coculture. Reverse transcription–
drogenase
(GAPDH).
polymerase chain reaction gel band densities were normalized to expression levels of glyceraldehyde phosphate
dehydrogenase typical
(GAPDH).
projection
of the
tical stem cells cocultured with endothelial
comparedThat
to 2%
neuronsneurons
in control CTX
stem
cell clones growing in BPAE coculture
early embryo arise in E10-11 cocultures
cells for +4 days generated more secondary
cocultures
(Fig.
2G).
made
Tbr1 clones,
projection
neurons,and
compared
after
many
cell divisions
suggests
that
enstem
neurospheres,
neuronThat
projection
neurons
typical
of the
tical cell
stem cells cocultured
with
endothelial
In vivo,
most
projection
neurons
arecell
born
clones
(Fig.
3B).
dothelial
factors
promote
stem
self- to CTX-cocultured
generating progenitor
cells
than
didBPAEthose
early
embryo
arise
in
E10-11
cocultures
cells
for
4
days
generated
more
secondary
in the early
embryonic
whereas
glia cocultured
generated
more Tbr+
renewal
and inhibitperiod,
the normal
progression
coculturedstem
with cells
CTX cells
(fig. S4).
after many cell divisions suggests that enstem cell clones, neurospheres, and neuronin which older
stem
cellsadult
preferentially
The than
most obvious
effect of endothelial
and interneurons
arise
later;
stem cellspro- neurons
neurosphere-expanded
E10
dothelial factors promote stem cell selfgenerating progenitor cells than did those
duce to
gliagenerate
or interneurons.
We found
factors
that they
promote neural
stem cell
are primed
interneurons
(8, 9).few cells
that iswere
subsequently
differentiated
renewal
cocultured
with
CTX
cells
(fig.
S4).
 and inhibit the normal progression
neurons
produced
fromgenerated
E15.5 stem in growth
Tbr1 the
as culture
epithelial
sheets versus
with extensive
To examine
neuron
subtypes
adherent
(9.95%
2.41%).
incells
which
older
cells
preferentially
proThe most
obvious
endothelial
and
nonestem
from
adult
SVZ cells, indijunctional
contacts
(Fig.effect
1C), of
which
could
fromduce
E10-11
cortical
stem
cells
expanded
in
Thus,
endothelial
cell
coculture
gliathat
or endothelial
interneurons.
We are
found
few
factors is self-renewal
that they promote
neuralsupports
stem cell
cating
factors
permispromote
by
influencing

endothelial
coculture,
differentiated
clones
bothpathways
projection
neurons
and
produced
stem development
Tbr1
growth signaling
as of
epithelial
sheets(12,
with
extensive
sive, neurons
not instructive,
forfrom
this E15.5
fate:
They
catenin
13),
mode
andfor
none
from
adultdecarboxylase
SVZ cells, indi- interneurons.
junctional
contacts
1C), which
could
werecells
stained
glutamic
acid
cannot
reverse
the restriction.
of
cell division
(14 ),(Fig.
and Notch
activation
catingSupporting
endothelial
factors
permispromote
self-renewal
bycocultured
influencing
(GAD67),
athat
GABAergic
marker
typically
exThat
neurons
of the early
the hypothesis
thatare
endothelial
(15
).projection
Indeed,
stem
cellstypical
withsive,
notpromote
instructive,
forself-renewal,
this
fate: pyThey
catenin
signaling
pathways
(12,after
13),to
mode
factors
stem
time- embryo
endothelial
and
then exposed
pressed
in interneurons,
or cell
Tbr1,
an early
arise
incells
E10-11
cocultures
many
cannot
reverse
the that
restriction.
ofdivisions
cell division
(14 that
), II,
and
Notch activation
lapse
video
recording
of
dividing clones
inhibitor
which
inhibits
ramidal
neuron
marker
preferentially
la- re- cellsecretase
suggests
endothelial
factors
Supporting
thecells
hypothesis
that
endothelial
(15 ). Indeed,
cocultured
with
veals
thatneurons
stem
grown
endothelial
Notch1
activation
(16cells
), showed
a similar
bels projection
(10)
(Fig.with
3A).
More promote
stem
cellstem
self-renewal
and
inhibit
factors
promote stem
cell self-renewal,
timeendothelial
cells and
then division,
exposed to
cells underwent
symmetric,
proliferative
diextent
of cell-cell
contact,
and
progeny,
in convisions
generating
Nestin
differentiation
to those II,
in CTX
cocultures
lapse
video
recording
of dividing
clones
resecretase inhibitor
which
inhibits
22
trastthat
to the
asymmetric
division
seen
(Fig.
4B activation
and fig. S5).
cells
veals
stem
cells grown
withpatterns
endothelial
Notch1
(16In
), neural
showedstem
a similar
in control
conditions
(6, 11)proliferative
(Fig. 4A). Corcultured
endothelial
factors,
the Notch
cells
underwent
symmetric,
diextent ofwith
cell-cell
contact,
division,
and
effector
effecto
was not
was of
no
ment
ment
renewal
renew
Our r
Ouc
critical
critica
niche,
as
niche,
maintain
mainta
neurogen
neurog
promotes
promoa
cannot
cannot
endothel
accompl
endoth
In the
accom
ral stem
In t
tive
ral divi
stem
stem
tivecell
d
tency
stemanc
generate
tency
oligoden
genera
tected
in
oligod
these
circ
tected
generate
these c
Grow
genera
tors may
Gro
neural st
tors m
esis, allo
neural
stem
cell
useesis,
in rea
stem c
use
in
Refere
1. F. Doet
2. T. Ref
D. P
Neurol
1.
F. D
3. 2.
A. Cape
T. D
4. A. Loui
Neu
Neuron
3. A. C
5. G. Brei
4.
A. L
ment 1
Neu
6. X. Qian
7. 5.
Q. G.
SheB
men
data.
6.
X.
8. J. O. Q
S
7.
Q. S
Nature
data
9. D. G.
H
8.
J. N
O
Ann.
Natu
10. R. F.
H
D. G
11. 9.
X. Qian
Ann
Develo
12.10.
A. Chen
R. F
13.11.
A. Chen
X. Q
14. B. Lu,
DevF
(2001)
12.
A. C
15.13.
S. Hito
A. C
16.14.
A. Cho
B. Lu
S. Weis
(200
17.15.
Y. Nak
S. H
18.16.
T. Ohts
A. C
J. Biol.
S. W
19.17.
WeY.tha
N
Tbr1 an
18. T. O
K. Kirch
J. Bi
ported
19.
We
orders
Tbr1
researc
K. K
Supporting
port
www.scienc
orde
Materials
a
rese
SOM Text
Support
Figs.
S1 to
www.sci
References
Material
9 January
SOM Te2
Published
Figs. S1o
10.1126/sc
Referenc
Include this
the normal progression in which older stem
cells preferentially produce glia or interneurons. We found few Tbr1+ neurons produced
from E15.5 stem cells and none from adult
SVZ cells, indicating that endothelial factors
are permissive, not instructive, for this fate:
They cannot reverse the restriction.
Supporting the hypothesis that endothelial
factors promote stem cell self-renewal, timelapse video recording of dividing clones reveals that stem cells grown with endothelial
cells underwent symmetric, proliferative divisions generating Nestin+ progeny, in contrast to the asymmetric division patterns seen
in control conditions (6, 11) (Fig. 4A). Cortical stem cells cocultured with endothelial
cells for 4 days generated more secondary
stem cell clones, neurospheres, and neurongenerating progenitor cells than did those cocultured with CTX cells (fig. S4).
The most obvious effect of endothelial
factors is that they promote neural stem cell
growth as epithelial sheets with extensive
junctional contacts (Fig. 1C), which could
promote self-renewal by influencing ßcatenin signaling pathways (12, 13), mode of
cell division (14), and Notch activation (15).
Indeed, stem cells cocultured with endothelial
cells and then exposed to γ-secretase inhibitor
II, which inhibits Notch1 activation (16),
showed a similar extent of cell-cell contact,
division, and differentiation to those in CTX
cocultures (Fig. 4B and fig. S5). In neural
stem cells cultured with endothelial factors,
the Notch effector Hes1 was up-regulated,
but Hes5 was not (Fig. 4C), consistent with
involvement of Hes1 in neural stem cell selfrenewal (17, 18).
Our results identify endothelial cells as critical components of the neural stem cell niche,
as they secrete soluble factors that maintain
CNS stem cell self-renewal and neurogenic
potential. Thus, although FGF2 promotes
neural stem cell proliferation, it cannot alone
maintain their self-renewal; endothelial factors acting with FGF2 accomplish this.
In the presence of endothelial cells, a neural
stem cell undergoes symmetric, proliferative
divisions to produce undifferentiated stem
cell sheets that maintain their multipotency
and, upon endothelial cell removal, generate
neurons as well as astrocytes and oligodendrocytes. No CD31+ cells were detected in
clones, showing that, at least under these circumstances, neural stem cells do not generate
endothelial progeny.
Growth with endothelial cell–derived factors may be an important tool for promoting
neural stem cell self-renewal and neurogenesis, allowing efficient production of neural
stem cells and a variety of CNS neurons for
use in replacement therapies.
References and Notes
1. F. Doetsch, Curr. Opin. Genet. Dev. 13, 543
(2003).
2. T. D. Palmer, A. R. Willhoite, F. H. Gage, J.
Comp. Neurol. 425, 479 (2000).
3. A. Capela, S. Temple, Neuron 35, 865 (2002).
4. A. Louissaint Jr., S. Rao, C. Leventhal, S. A.
Goldman, Neuron 34, 945 (2002).
5. G. Breier, U. Albrecht, S. Sterrer, W. Risau,
Development 114, 521 (1992).
6. X. Qian et al., Neuron 28, 69 (2000).
7. Q. Shen, L. Jin, S. K. Goderie, S. Temple, unpublished data.
8. J. O. Suhonen, D. A. Peterson, J. Ray, F. H.
Gage, Nature 383, 624 (1996).
9. D. G. Herrera, J. M. Garcia-Verdugo, A. Alvarez-Buylla, Ann. Neurol. 46, 867 (1999).
10. R. F. Hevner et al., Neuron 29, 353 (2001).
11. X. Qian, S. K. Goderie, Q. Shen, J. H. Stern, S.
Temple, Development 125, 3143 (1998).
12. A. Chenn, C. A. Walsh, Science 297, 365 (2002).
13. A. Chenn, C. A. Walsh, Cereb. Cortex 13, 599
(2003).
14. B. Lu, F. Roegiers, L. Y. Jan, Y. N. Jan, Nature
409, 522 (2001).
15. S. Hitoshi et al., Genes Dev. 16, 846 (2002).
16. A. Chojnacki, T. Shimazaki, C. Gregg, G. Weinmaster, S. Weiss, J. Neurosci. 23, 1730 (2003).
17. Y. Nakamura et al., J. Neurosci. 20, 283 (2000).
18. T. Ohtsuka, M. Sakamoto, F. Guillemot, R.
Kageyama, J. Biol. Chem. 276, 30467 (2001).
19. We thank H. Singer for VSM cells, Y.-P. Hseuh
for Tbr1 antibody, and C. Fasano, Y. Wang, C.
Butler, and K. Kirchofer for help in manuscript
preparation. Supported by the National Institute
of Neurological Disorders and Stroke and the
New York State spinal cord research program.
Supporting Online Material
www.sciencemag.org/cgi/content/full/1095505/DC1
Materials and Methods
SOM Text
Figs. S1 to S7
References and Notes
9 January 2004; accepted 19 March 2004
Published online 1 April 2004;
10.1126/science.1095505
Include this information when citing this paper.
23
Mosaic Organization
Organization of
of Neural
Neural Stem
Mosaic
Cells Cells
in theinAdult
BrainBrain
Stem
the Adult
Florian T. Merkle, Zaman Mirzadeh, Arturo Alvarez-Buylla*
Florian T. Merkle, Zaman Mirzadeh, Arturo Alvarez-Buylla*
The in vivo potential of neural stem cells in the postnatal mouse brain is not known, but
The
in vivo
potential
neural
stem cells
theneurons,
postnatal
mouse
is notvery
known,
but because
because
they
produceofmany
different
typesin of
they
mustbrain
be either
versatile
or very
they produce many different types of neurons, they must be either very versatile or very diverse. By
diverse. By specifically targeting stem cells and following their progeny in vivo, we showed that
specifically targeting stem cells and following their progeny in vivo, we showed that postnatal stem
postnatal stem cells in different regions produce different types of neurons, even when
cells in different regions produce different types of neurons, even when heterotopically grafted or
heterotopically
or grown
culture.
suggests
than being
plastic
grown
in culture.grafted
This suggests
thatinrather
thanThis
being
plasticthat
and rather
homogeneous,
neural
stemand
cells are
cells are
a restricted and diverse population of progenitors.
ahomogeneous,
restricted and neural
diverse stem
population
of progenitors.
EE
very day,
day, thousands
neurons
are
very
thousandsofofnew
new
neurons
generated by astrocyte-like stem cells reare
generated by astrocyte-like stem
siding in the subventricular zone (SVZ), a
cells residing in the subventricular zone
thin but extensive layer of cells lining the lateral
(SVZ),
thin
but extensive
layer
cells
linwall of athe
lateral
ventricle in
the of
adult
mouse
ing
the
lateral
wall
of
the
lateral
ventricle
in
brain (1). These newly born neurons (neurothe
adult
mouse
brain
(1). These
newly
born
blasts)
migrate
to the
olfactory
bulb in
a complex
neurons
(neuroblasts)
migrate to
the toolfacnetwork of
chains that eventually
merge
form
tory
bulb in
a complex
network
of On
chains
that
the rostral
migratory
stream
(RMS).
reaching
eventually
form the integrate
rostral migratothe olfactorymerge
bulb, to
neuroblasts
and maturestream
into several
distinct
cell typesthe
(2).olfactory
It is not
ry
(RMS).
On reaching
knownneuroblasts
how this diversity
neurons
is into
genbulb,
integrateofand
mature
erated, largely
it (2).
is difficult
study
several
distinctbecause
cell types
It is nottoknown
individual
stem cellsofinneurons
vivo. Indeed,
our unhow
this diversity
is generated,
derstanding
of stem
is strongly
influenced
largely
because
it iscells
difficult
to study
indiby the stem
in vitro
techniques
whichour
they
were
vidual
cells
in vivo.with
Indeed,
underfirst isolated and later defined by their ability to
standing of stem cells is strongly influenced
be passaged (demonstrating self-renewal) and
by the in vitro techniques with which they
differentiated into astrocytes, neurons, and oligowere
first isolated
and later
defined by (3,
their
dendrocytes
(demonstrating
multipotency)
4).
ability
to be led
passaged
(demonstrating
selfThese results
to the widely
held assumption
renewal)
andstem
differentiated
astrocytes,
that neural
cells are into
a homogeneous
neurons,
(demonstratpopulationand
of oligodendrocytes
multipotent, plastic
progenitors
ing
results
led to
(Fig.multipotency)
1A). Similarly,(3,it 4).
wasThese
thought
that neurothe
widely
held
that
neural stem
blasts
born in
the assumption
SVZ might be
equivalent
until
they reach
the olfactory bulb
and beginoftomuldifcells
are a homogeneous
population
ferentiate.plastic
However,
recent evidence
tipotent,
progenitors
(Fig. 1A).suggests
Simithat neuroblasts
are heterogeneous
before
reachlarly,
it was thought
that neuroblasts
born
in
ing SVZ
the olfactory
(5, 6). We
hythe
might bebulb
equivalent
untiltherefore
they reach
pothesized
thatbulb
stemand
cellsbegin
are not
and
the
olfactory
to equivalent
differentiate.
that they specify
the fate ofsuggests
the neurons
they
However,
recent evidence
that neuproduce (Fig. 1B). To test this hypothesis, we
roblasts
are heterogeneous before reaching
labeled stem cells in different regions and folthe olfactory bulb (5, 6). We therefore hylowed their progeny in vivo.
pothesized
stem cells
are that
not adult
equivalent
We havethat
previously
shown
neural
and
specifyfrom
the fate
thepresent
neurons
stemthat
cellsthey
are derived
radialofglia
in
they produce (Fig. 1B). To test this hypotheDepartment of Neurosurgery and Developmental and Stem
Cell Biology Program, University of California, San Francisco,
San Francisco, CA 94143–0525, USA.
*To whom correspondence should be addressed. E-mail:
[email protected]
24
the
neonatal
(P0)stem
mouse
(7). Radial
glia,
sis, we
labeled
cellsbrain
in different
regions
which are now recognized as the principal stem
and followed their progeny in vivo.
cell of the embryonic and early postnatal mouse
We have previously shown that adult neubrain (8, 9), have a unique morphology that
ral stem
cells
derived
from radial
allows
them
to beare
targeted
specifically.
Their glia
cell
present
in
the
neonatal
(P0)
mouse
bodies line the ventricles and they send a brain
long,
(7). Radial
which
are nowAdenoviruses
recognized
radial
processglia,
to the
brain surface.
as the infect
principal
cell of
readily
thesestem
processes
andthe
are embryonic
transported
and
postnatal
brain (8,expressing
9), have
to
theearly
cell body.
Whenmouse
an adenovirus
a unique
morphology
thatis allows
to be
Cre
recombinase
(Ad:Cre)
injectedthem
into green
fluorescent
protein (GFP)
reporter
targeted specifically.
Their
cell (Z/EG)
bodies mice
line
(10),
infected radial
glia send
and atheir
progeny
the ventricles
and they
long,
radial
become
permanently
GFP (7).
process to
the brain labeled
surface.with
Adenoviruses
Because
adenoviruses
do not diffuse
readily
in
readily infect
these processes
and are
transthe
brain,
injection
small volported
to the
the localized
cell body.
When of
anaadenovirus
ume
(20 nl) of
Ad:Cre
labels a spatially
restricted
expressing
Cre
recombinase
(Ad:Cre)
is inpatch of neural stem cells.
jected into green fluorescent protein (GFP)
To label radial glia in a regionally specific
reporter (Z/EG) mice (10), infected radial
manner, we developed a method to stereotaxiglia and
their
progeny
permanently
cally
inject
Ad:Cre
in P0become
mice (Fig.
1E) (11).
labeled with
GFPin(7).
adenoviruses
Injections
resulted
the Because
reproducible
labeling of
do not diffuse
readily
in the
brain,glia
the(Fig.
localspatially
segregated
patches
of radial
1,
ized
smallSVZ
volume
of
C
andinjection
D) and of
theaadult
stem (20
cellsnl)they
Ad:Cre labels
patch
of
generate
(Fig. 1,a spatially
F and G).restricted
In contrast,
cells
neural stem
labeled
locallycells.
at the injection site do not give rise
to To
olfactory
stem cells
(7).
label bulb
radialneurons
glia inora neural
regionally
specific
By
systematically
varyingathe
injection
manner,
we developed
method
to location
stereo(Fig.
1H), inject
we targeted
15 different
populations
of
taxically
Ad:Cre
in P0 mice
(Fig. 1E)
radial
glial cells resulted
at six different
rostrocaudal
(11). Injections
in the reproducible
levels
(i to
(11). We
targeted patches
the dorsal
labeling
of vi)
spatially
segregated
of(D)
raor
ventral
(V) lateral
lateral
ventricle
at
dial
glia (Fig.
1, C wall
and of
D)the
and
the adult
SVZ
four different rostrocaudal levels (ii to v) and the
stem cells they generate (Fig. 1, F and G). In
entire dorsoventral extent of the lateral wall at
contrast, cells labeled locally at the injection
level vi. We also targeted the RMS (i), the medial
site do not
riseand
to olfactory
bulb
neurons
(septal)
wallgive
(iiM),
the cortical
wall
of the
or neural
stem(iiC
cells
(7).because
By systematically
lateral
ventricle
to vC)
these regions
varying
injectiontolocation
(Fig. 1H),
we
have
beenthe
suggested
contain neural
progenitargeted
different
of brains
radial
tors
(1, 4, 15
12, 13).
Whenpopulations
we analyzed the
glial
cells
at six after
different
rostrocaudal
levels
of
mice
4 weeks
Ad:Cre
injection, we
ob(i to vi)a (11).
the dorsal
or
served
patchWe
of targeted
labeled cells
in the(D)
same
anatomical
the of
targeted
radial venglial
ventral (V)location
lateral as
wall
the lateral
cell
indicating
neural stemlevels
cells do
triclebodies,
at four
differentthat
rostrocaudal
(ii
www.sciencemag.org
SCIENCE
VOL 317
not dispe
G, show
targeting
respectiv
We t
neurons
types of
ular cells
modulate
olfactory
divided i
cells: cal
(CalB+)
(TH+) d
include d
2A) (15,
distinct f
circuitry
Olfac
from all
and iiC
wall of t
ary of th
region g
interneur
produced
S2, A to
was prod
A and B
produced
regions
percenta
to C), wh
ly ventra
fig. S3, D
less frequ
targeted
targeting
S3, G to
ule cells
relatively
Dorsal r
produce
regions (
cells (Fi
granule
anterior
many C
fig. S3,
study sho
are deriv
embryon
Each
produce
the conti
S2, B, D
into SVZ
20 JULY 2007
t they
ogeniwork
ogenallosal
more,
d deep
TH+,
ly the
from
s that
ferent
over
inadntially
ons at
s only
nance
pment
neural
oughanner.
mental
tween
onatal
11). If
wborn
their
s and
on. To
radial
cribed
ciated
ally or
es of
ed the
s after
glia
priate
C). To
their
f any
dorsal,
itions
enesis
tiated
type–
moved
sed to
cells
(fig.
we microdissected progenitors from different
regions of neonatal wild-type or ActB-GFP mice
intermediate progenitors. Again, heterotopically
grafted stem cells produced neuronal subtypes
Fig.1.1.Specific
Specific regional
regional targeting
Fig.
targetingof
ofneural
neuralstem
stemcells.
cells.(A
(Aand
andB)B)InInthe
thetraditional
traditionalmodel
modelofofSVZ
SVZstem
stemcell
cell
potential
(A),
equivalentstem
stemcells
cells(black
(blackdots)
dots)
generatemultiple
multipleneuron
neurontypes,
types,which
whichare
areproduced
producedby
bya
potential
(A),
equivalent
generate
a diverse
population
in the
proposed
model(B).
(B).(C(Cand
andD)
D)Diagram
Diagram of
of aa neonatal
neonatal mouse
diverse
stemstem
cell cell
population
in the
proposed
model
mouse brain
brain
showing
targetingofofdorsal
dorsal(C)
(C)ororventral
ventral(D)
(D)radial
radial glia
glia (green)
(green) with
showing
targeting
with virus
virus deposited
depositedatatthe
theinjection
injectionsite
site
andalong
alongthe
the needle
needle tract
andand
bar).bar).
(E) Stereotaxic
setup showing
an acrylic
a neonatal
and
tract(gray
(graycircle
circle
(E) Stereotaxic
setup showing
anmodel
acrylicofmodel
of a
pup positioned
in a customized
head mold
viralfor
injection
parallel parallel
to radialtoglial
processes.
(F and G)(F
neonatal
pup positioned
in a customized
headformold
viral injection
radial
glial processes.
Photomicrograph
of a P28
at P0 to at
target
dorsal
(F)dorsal
or ventral
(G)ventral
radial glia,
visualized
and
G) Photomicrograph
ofZ/EG
a P28brain
Z/EGinjected
brain injected
P0 to
target
(F) or
(G) radial
glia,
with immunoperoxidase
staining for
GFP. (H)
brain regions
targeted.
frontal
visualized
with immunoperoxidase
staining
for Diagram
GFP. (H)ofDiagram
of brain
regionsRepresentative
targeted. Representsections
of the
right hemisphere
traced
from the adult
arethe
shown
relative
a photomicrograph
ative
frontal
sections
of the right
hemisphere
tracedbrain
from
adult
brainto are
shown relativeoftoana
adult lateral ventricular
walllateral
whole ventricular
mount outlined
bluemount
(30). Targeted
by green
dots,
photomicrograph
of an adult
wall in
whole
outlinedregions,
in blueindicated
(30). Targeted
regions,
are named
theirdots,
anterior-posterior
(i to
vi) followed by the
location
within
that level,
where
C is
indicated
by for
green
are named forlevel
their
anterior-posterior
level
(i to vi)
followed
by the
location
cortical,
is medial,
D is
and VMisisventral.
within
thatMlevel,
where
C dorsal,
is cortical,
medial, D is dorsal, and V is ventral.
0 JULY 2007
VOLthe
317
to v) and
entireSCIENCE
dorsoventralwww.sciencemag.org
extent of the gested to contain neural progenitors (1, 4, 12,
lateral wall at level vi. We also targeted the 13). When we analyzed the brains of mice 4
RMS (i), the medial (septal) wall (iiM), and weeks after Ad:Cre injection, we observed a
the cortical wall of the lateral ventricle (iiC patch of labeled cells in the same anatomical
to vC) because these regions have been sug- location as the targeted radial glial cell bod25
REPORTS
production
born inter(A) Colorucida traces
neuron subsed over a
of the olL, external
PL, internal
GC, granule
rular layer;
cell layer;
rular cell.
of labeled
bulb neuiglomerular
s was perafter stem
d targeted
ed as in Fig.
entage of labeled periglomerular cells immunopositive for TH (C), CalB
Fig. 2. Regional production of postnatally
F) Position of labeled granule cells within the GRL. Cell distributions that
interneuron
subtypes.
Color- in
nitively classifiedborn
as superficial
(green) or
deep (blue)(A)
are indicated
coded
lucida for
traces
of analyzed
ge of labeled granule
cellscamera
immunopositive
CalR. Error
bars represent
an, except in (F),interneuron
where they represent
the SEM.superimposed
subtypes
over a photomicrograph of the olfactory
bulb. EPL, external plexiform layer; IPL, internal plexiform layer; GC, granule cell; GL, glomerular layer; GRL,
otential of neural stem cells is
granule cell layer; PGC, periglomerular cell. (B) Percentage of labeled (GFP+) olfactory bulb neurons that
the adult. (A to D) Photomicroare periglomerular
tory bulbs of Z/EG
mice injected cells. All analysis was performed 28 days after stem cell labeling, and targeted regions
e 28 days earlier
at P60
in region
are
named
as ini Fig. 1H. (C to E) Percentage of labeled periglomerular cells immunopositive for TH (C),
(C), or vC (D), with
CalBcells
(D),visualized
or CalR (E). (F) Position of labeled granule cells within the GRL. Cell distributions that could not
oxidase staining for GFP. The
be definitively classified as superficial (green) or deep (blue) are indicated in teal. (G) Percentage of labeled
ranule cells within the granule cell
immunopositive for CalR. Error bars represent the SD of the mean, except in (F), where they
the continued granule
presence cells
of neuroactory bulb corerepresent
(arrowheads)
theare
SEM.
nd F) Quantification of labeled
ition in the GRL (E) and TH+ (red),
and CalR+ (gold) cell production
ies,orindicating
r radial glial (P0)
adult (P60) that neural stem
ng.
disperse tangentially (fig. S1).
cells do not the lateral wall of the lateral ventricle, the
Figure 1, F accepted boundary of the neurogenic adult
and G, show examples of SVZ labeling after SVZ. Notably, each region gave rise to only
neonatal targeting of dorsal and ventral SVZ a very specific subset of interneuron subradial glia, respectively.
types. Anterior and dorsal regions produced
We then examined the mature GFP-la- periglomerular cells (Fig. 2B and fig. S2, A
beled neurons in the olfactory bulb. The two to D). Interestingly, the highest percentage
principal
adult-born
olfactory
neuwas produced
in region
iiMthe
(Fig.
2B and
progenitors
also raises
possibility
thatfig.
the
cells
are diverse and
are organized
in an intricate
egional specificity
observedtypes
by in of
activity
stem cells is regionally
in theand
postnatal
germinal
zone. S2, A and
cing (Fig. 4, Brons,
to E). The
lack of mosaic
periglomerular
cells
granule
cells,
B).ofPeriglomerular
cell modulated
subtypesin
order
to regulate
production of different
These
findings suggest
that, as during
em-also
en partial respecification
indicates
are interneurons
that
modulate
the activity
were
produced
in athe
region-specific
mans behaved like a single population bryonic brain development (27–29), the poten- types of neurons. This may provide a mechaof
neurons
that
project
to
olfactory
cortex.
ner.
Dorsal
regions
(iiC
to
vC) produced
fine tunethe
the
nt to respecification. However, we tial of postnatal neural stem cells is determined nism for the brain to dynamically
can be
subdivided
into
TH+ cells (Fig. 2C and
olfactory bulbof
circuitry.
by a spatial
code,
supporting the
model highest
shown percentage
the possibilityPeriglomerular
that some envi- cells
Stem cells doofnotcells:
appear to migrate
rs were not totally
in in Fig. 1B.
threeeliminated
nonoverlapping
populations
fig. S3, A to C), whereas CalB+ cells were
s. Neural stem cell potential could tangentially as they mature from radial glia into
Referencesventrally,
and Notes in regions iiV
calretinin-(CalR+)
and
calbindin-expressing
produced mainly
by factors not included in our astrocyte-like adult cells and must have
1. F. Doetsch, I. Caille, D. A. Lim, J. M. García-Verdugo,
(CalB+)
cells,
tyrosine
hydroxylaseand
iiiV (Fig.
2D and fig.
S3,
to F). CalR+
A. Alvarez-Buylla,
Cell 97,
703D
(1999).
integrated
positional
information at some
point
ons or after extended
periods
of and
2. F. H. Gage, Science 287, 1433 (2000).
in development.
We suggest
this positional
Previous studies
have suggested
expressing
(TH+)
dopaminergic
cells that
(14).
periglomerular
cells were less frequently
3. B. A. Reynolds, S. Weiss, Science 255, 1707 (1992).
campal or spinal cord progenitors information becomes encoded in the progeniC. M. Morshead
al., Neuron 13,
1071 targeted,
(1994).
Granule cells include deep, superficial, and observed 4.when
theseetregions
were
ified when heterotopically grafted tors, perhaps by expression of a transcription
5. M. A. Hack et al., Nat. Neurosci. (2005).
cells
(Fig.factor
2A)code,
(15,and16).
TheseintosubbutThiswere 6.frequently
labeled
withA. targeting
M. Kohwi, N. Osumi,
J. L. Rubenstein,
Alvarez-Buylla,
maintained
adulthood.
ndings suggest CalR+
that although
SVZ
Neurosci.
25, 6997
insight
step toward
understanding
the
ls retain the potential
produce
types toare
thought
to isbea key
distinct
functional
of regions
iJ. and
iiM
(Fig.(2005).
2E and fig. S3, G
7. F. T. Merkle, A. D. Tramontin, J. M. Garcia-Verdugo,
molecular mechanisms
of neural
cell Each
odendrocytes, elements
and neurons,ofthey
the olfactory
bulb circuitry
(14, stem
to I).
targeted
region
produced
granule
A. Alvarez-Buylla, Proc. Natl.
Acad. Sci. U.S.A.
101,
n the types of neurons they can potential and for future efforts to use these cells
17528 (2004).
15,
17).
cells
(Fig.
2F),
though
region
iiM
produced
nclude that postnatal neural stem for brain repair. The mosaic distribution of
8. S. C. Noctor et al., J. Neurosci. 22, 3161 (2002).
Olfactory bulb interneurons were produced relatively few (Fig. 2B and fig. S2, A and B).
from all labeled regions including regions Dorsal regions (iiC to vC, iiD, iiiD) tended
383
www.sciencemag.org SCIENCE VOL 317 20 JULY 2007
i, iiM, and iiC to vC, which extend beyond to produce superficial granule cells, whereas
26
entage of labeled granule cells immunopositive for CalR. Error bars represent
e mean, except in (F), where they represent the SEM.
3. The
e potential ofFig.
neural
stempotencells is
in the adult. (A
PhotomicrotialtoofD)
neural
stem
lfactory bulbs of Z/EG mice injected
cells at
is maintained
P-Cre 28 days earlier
P60 in region i
in the
(A to
iiiV (C), or vC (D),
with adult.
cells visualized
operoxidase staining
for GFP. The
D) Photomicroof granule cells within the granule cell
graphs of olfacand the continued presence of neurobulbs of Z/
olfactory bulb tory
core (arrowheads)
are
E and F) Quantification
labeled
EG mice of
injected
position in the with
GRL (E)Ad:GFAP-Cre
and TH+ (red),
le), and CalR+ (gold) cell production
28(P0)
daysor earlier
at
after radial glial
adult (P60)
P60 in region i (A),
geting.
iiiD (B), iiiV (C), or
vC (D), with cells
visualized by immunoperoxidase
staining for GFP.
The distribution of granule cells within the granule cell layer (GRL) and the continued presence of neurome regional specificity observed by in cells are diverse and are organized in an intricate progenitors also raises the possibility that the
blasts in the olfactory bulb core (arrowheads) are indicated. (E and F) Quantification of labeled granule cell
activity of stem cells is regionally modulated in
e tracing (Fig. 4, B to E). The lack of mosaic in the postnatal germinal zone.
position in indicates
the GRL (E) and
TH+
(red),suggest
CalB+ that,
(purple),
and emCalR+order
(gold)to cell
production
(F) 28 days
after
regulate
the production
of different
These
findings
as during
r even partial respecification
radial
glialpopulation
(P0) or adult
(P60)brain
stemdevelopment
cell targeting.
bryonic
(27–29), the poten- types of neurons. This may provide a mechacells behaved like
a single
stant to respecification. However, we tial of postnatal neural stem cells is determined nism for the brain to dynamically fine tune the
ard the possibility that some envi- by a spatial code, supporting the model shown olfactory bulb circuitry.
Fig.vV)
1B. Stem
cells domostly
not appear tofinding
migrate agrees with previous work suggestactors were notventral
totally eliminated
regionsin(iiVinto
produced
they mature
fromEradial ing
glia into
ents. Neural stem
cell potential
References
Notes
deep
granulecould
cellstangentially
(Fig. 2Fas and
fig. S2,
the presence
of aandneurogenic
progenitor
ered by factors not included in our astrocyte-like adult cells and must have
1. F. Doetsch, I. Caille, D. A. Lim, J. M. García-Verdugo,
and
H).
CalR+
granule
cells
were
produced
in
the
adult
RMS
(5,
20,
21)703and
subcallosal
A.
Alvarez-Buylla,
Cell
97,
(1999).
ditions or after extended periods of integrated positional information at some point
2. F. H.
Gage,
Science
287,(12).
1433 (2000).
in development.
that this positional
o. Previous studies
havefrom
suggested
mostly
the anterior
regionsWe(isuggest
and iiM)
zone (regions
ivC
and
vC)
Furthermore,
3. B. A. Reynolds, S. Weiss, Science 255, 1707 (1992).
information
becomes
encoded in the these
progeni-labeled
ppocampal or spinal
4. C. M.
Morshead
et al., Neuronsuperficial
13, 1071 (1994). and
that cord
alsoprogenitors
produced many
CalR+
periglomerucells
produced
pecified when heterotopically grafted tors, perhaps by expression of a transcription
5. M. A. Hack et al., Nat. Neurosci. (2005).
larthat
cells
(Fig.SVZ
2G and
fig.
S3,
to L). This
is deep
cellsN.(Fig.
A to E),
as well
6. M. Kohwi,
Osumi, J.3,
L. Rubenstein,
A. Alvarez-Buylla,
factor
code,
andJmaintained
into adulthood.
Thisgranule
r findings suggest
although
J. Neurosci.
6997 (2005).
insight isstudy
a key step
toward that
understanding
the CalB+,
cells retain theconsistent
potential to produce
with a recent
showing
as TH+,
and25,CalR+
cells (Fig. 3F) in
7. F. T. Merkle, A. D. Tramontin, J. M. Garcia-Verdugo,
molecular
mechanisms
of neural stem
cell the A.same
oligodendrocytes,
and neurons,
they bulb
CalR+
olfactory
neurons
are derived
virtually
region-specific
pattern
obAlvarez-Buylla,
Proc. Natl. Acad. Sci.
U.S.A. 101,
d in the types of neurons they can potential and for future efforts to use these cells
17528 (2004).
from
SP8+
cells,
which
are
found
embryonitained
neonatal
labeling.
study
e conclude that postnatal neural stem for brain repair. The mosaic distribution of from
8. S.
C. Noctor et al.,
J. Neurosci. A
22,recent
3161 (2002).
cally in regions i and iiM (18).
suggests that the potential of progenitors to
Each neonatally
targeted region
continued
types of periglomerular 383
www.sciencemag.org
SCIENCE
VOL 317produce
20 JULYdifferent
2007
to produce neuroblasts in the adult brain, sug- cells changes over development (22). This
gesting the continued presence of a neural study might have inadvertently examined
stem cell (fig. S2, B, D, F, and H). Neonatal progenitors and tangentially migrating neuradial glia convert into SVZ astrocytes that roblasts from different regions at different
express glial fibrillary acidic protein (GFAP) ages, whereas our technique targets only priand function as the adult neural stem cells mary progenitors. The apparent maintenance
(1, 7, 19). To test whether adult neural stem of stem cell potential over postnatal develcells were also regionally specified, we con- opment suggests that the factors specifying
structed an adenovirus that expresses Cre un- neural progenitors in the SVZ are maintained
der the control of the murine GFAP promoter through-out development in a regionally spe(Ad:GFAP-Cre). This virus induces recom- cific manner.
bination in GFAP-expressing cells including
These factors could be either environmenadult neural stem cells, but not the more dif- tal or intrinsic to stem cells. To distinguish
ferentiated cells they give rise to (fig. S4, A to between these two possibilities, we chalC) (11). We injected P60 Z/EG mice with Ad: lenged neonatal stem cells by heterotopic
GFAP-Cre in regions i, iiiD, iiiV, and vC (fig. transplantation (11). If environmental factors
S4, D to G) and killed the animals 28 days specify the fate of newborn neurons, grafted
later to examine the olfactory bulb cell types (donor) stem cells and their progeny should
produced. Each targeted region produced ol- respond to these factors and make cell types
factory bulb neurons and neuroblasts (Fig. 3, produced in the host region. To obtain labeled
A to D), suggesting that they contain long- neural stem cells, neonatal radial glia were
lived GFAP+ neurogenic progenitors. This infected with Ad:Cre as described above,
27
REPORTS
Fig. 4. Neural stem cell potential is cell-intrinsic. (A) Neonatal SVZ progenitors from
GFP+ and wild-type mice are
cultured for multiple passages, mixed 1:10, and grafted
into the SVZ of a wild-type
P0 host. (B and C) Quantification of granule cell distribution in the granule cell layer
(B) and of TH+ (red) or CalB+
(purple) periglomerular cell
production (C) by GFP+ cells
from dorsal or ventral regions
grafted hetero- or homotopically. (D and E) Quantification of periglomerular cell
production (D) or CalR+ periglomerular (orange) and granule cell (gold) production (E)
by GFP+ cells from anterior or
posterior regions grafted hetero- or homotopically.
Fig. 4. Neural stem cell potential is cell-intrinsic. (A) Neonatal SVZ progenitors from GFP+ and wild-type
mice are cultured for multiple passages, mixed 1:10, and grafted into the SVZ of a wild-type P0 host. (B
C) Quantification
of granule
distribution
in the
granuleeliminate
cell layer (B)the
and possibility
of TH+ (red) or CalB+
microdissected after 2and
hours,
dissociated
to cellbrain,
but we
cannot
(purple)
cell production
by GFP+ cellscells
from grafted
dorsal or ventral
regions labeled
grafted hetero- or
a single-cell suspension,
andperiglomerular
homotopically
that(C)unlabeled
alongside
homotopically. (D and E) Quantification of periglomerular cell production (D) or CalR+ periglomerular
or heterotopically grafted
intoand
thegranule
littermates
stem cells
be carrying
factors
from
the grafted
(orange)
cell (gold) production
(E) bycould
GFP+ cells
from anterior
or posterior
regions
or homotopically.
of donor animals (fig.heteroS5A).
We then ana- donor environment into the host graft site.
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Sup
www
Mat
Figs
Refe
lyzed the olfactory bulb9. cell
typesC.produced
Therefore,
T. E. Anthony,
Klein, G. Fishell, N. Heintz,
Neuron 41, we11. microdissected
Materials and methods are progenitors
available as supporting
10
(2004). that labeled
material on
Online. wild-type
Pub
40 days after grafting and881
found
from different regions
ofScience
neonatal
10. A. Novak, C. Guo, W. Yang, A. Nagy, C. G. Lobe, Genesis
12. B. Seri et al., Cereb. Cortex 16 (suppl. 1), i103
10.1
radial glia continued to produce
the cell types or ActB-GFP mice
28, 147 (2000).
(2006).(24) and cultured them
Incl
appropriate to their region of origin (fig. S5, for three passages over 2 weeks. To ensure
B and C). To determine if progenitors would that grafted cells were surrounded by foreign
bui
maintain their region-specific potential in the environmental cues, we mixed GFP+ cells
opm
absence of any environmental cues, we cul- from the donor region with a 10:1 excess
beh
tured anterior, dorsal, or ventral progenitors of unlabeled wild-type cells from the host
lev
under adherent conditions that recapitulate region before grafting them into wild-type
sio
Vanina Vergoz,
A. Schreurs,
Alison R.mice
Mercer*
postnatal SVZ neurogenesis
(23). Haley
Cultures
neonatal
(Fig. 4A). Four weeks later,
alte
were expanded, differentiated (fig. S5D), and grafted brains contained labeled astrocyteQueen mandibular pheromone (QMP) has profound effects on dopamine signaling in the brain of QM
con
immunostained for cellyoung
type–specific
like cells
at the
site (fig.
S7, A,in C,
E,
worker honey markbees. As dopamine
in insects
has graft
been strongly
implicated
aversive
we examinedfrom
QMP’s impact
associative
olfactory learning
We found
ers (fig. S6). Despite learning,
being removed
andonG)
and mature
neuronsin bees.
in the
olfac-that QMP and
blocks aversive
learningto
in ayoungtory
workers,
but (fig.
leavesS7,
appetitive
We postulate that ess
their complex environment
and exposed
bulbs
B, D,learning
F, andintact.
H). Because
rais
QMP’s effects on aversive learning enhance the likelihood that young workers remain in close
cocktail of growth factors,
stem
cells
grafted
continued
to produce
neuroits
contactneural
with their
queen
by preventing
themcells
from forming
an aversion
to their mother’s
wo
maintained their region-specific
potential
as interesting
efficiently
pheromone bouquet.
The results blasts
provide an
twistastodirectly
a story of targeted
success andprisurvival.
olf
(fig. S5E). We confirmed this result by tar- mary progenitors (fig. S8), grafted cells were
geting dorsal or ventral radial
glia, culturing
likely stem
and not
intermediate
blend cells
of substances
known
as queen mandibular olf
o advertise
her presence most
in the colony
pheromone
(QMP) (1). Young workers,
and tothem
exert influence
its members, Again,
them for 2 weeks, and grafting
hetero- overprogenitors.
heterotopically
graftedattracted the
a honey bee queen produces a complex to the queen by QMP are enticed not only to feed age
topically into wild-type neonatal
mice (fig. stem cells produced neuronal subtypes with
her, but also to lick and to antennate her body. As wit
S5F). Again, grafted cells produced cell types the same regional
specificity
observed
in which To
they do
so, they gather
samples ofby
QMP,
Department of Zoology, University of Otago, Dunedin, New
they distribute
the colony siv
appropriate to their region
of origin, not their vivo lineage tracing
(Fig. to
4, other
B to members
E). The oflack
Zealand.
(2, 3).
At the
colonyrespecification
level, QMP inhibits the fer
*To whom correspondence should be addressed.
E-mail: for
grafted location (fig. S5G).
of evidence
even
partial
rearing of new queens (4), influences comb- bee
[email protected]
The above experiments suggest that neural indicates that grafted cells behaved like a sinstem cells are not readily respecified by en- gle population that was resistant to respecifi384
20 JULY 2007 VOL 317 SCIENCE www.sciencemag
vironmental factors
present in the postnatal cation.
However, we cannot discard the pos-
Queen Pheromone Blocks Aversive
Learning in Young Worker Bees
T
28
sibility that some environmental factors were
not totally eliminated in our experiments.
Neural stem cell potential could also be altered by factors not included in our culture
conditions or after extended periods of time
ex vivo. Previous studies have suggested that
adult hippocampal or spinal cord progenitors
might be respecified when heterotopically
grafted (25, 26). Our findings suggest that
although SVZ neural stem cells retain the
potential to produce astrocytes, oligodendrocytes, and neurons, they are restricted in
the types of neurons they can generate. We
conclude that postnatal neural stem cells are
diverse and are organized in an intricate mosaic in the postnatal germinal zone.
These findings suggest that, as during
embryonic brain development (27–29), the
potential of postnatal neural stem cells is
determined by a spatial code, supporting the
model shown in Fig. 1B. Stem cells do not
appear to migrate tangentially as they mature
from radial glia into astrocyte-like adult cells
and must have integrated positional information at some point in development. We suggest that this positional information becomes
encoded in the progenitors, perhaps by expression of a transcription factor code, and
maintained into adulthood. This insight is a
key step toward understanding the molecular
mechanisms of neural stem cell potential and
for future efforts to use these cells for brain
repair. The mosaic distribution of progenitors
also raises the possibility that the activity of
stem cells is regionally modulated in order to
regulate the production of different types of
neurons. This may provide a mechanism for
the brain to dynamically fine tune the olfactory bulb circuitry.
References and Notes
1.F. Doetsch, I. Caille, D. A. Lim, J. M. García-Verdugo, A. Alvarez-Buylla, Cell 97, 703
(1999).
2.F. H. Gage, Science 287, 1433 (2000).
3 B. A. Reynolds, S. Weiss, Science 255, 1707
(1992).
4.C. M. Morshead et al., Neuron 13, 1071 (1994).
5.M. A. Hack et al., Nat. Neurosci. (2005).
6.M. Kohwi, N. Osumi, J. L. Rubenstein, A.
Alvarez-Buylla, J. Neurosci. 25, 6997 (2005).
7.F. T. Merkle, A. D. Tramontin, J. M. GarciaVerdugo, A. Alvarez-Buylla, Proc. Natl. Acad.
Sci. U.S.A. 101, 17528 (2004).
8.S. C. Noctor et al., J. Neurosci. 22, 3161
(2002).
9.T. E. Anthony, C. Klein, G. Fishell, N. Heintz,
Neuron 41, 881 (2004).
10.A. Novak, C. Guo, W. Yang, A. Nagy, C. G.
Lobe, Genesis 28, 147 (2000).
11. Materials and methods are available as supporting material on Science Online.
12.B. Seri et al., Cereb. Cortex 16 (suppl. 1), i103
(2006).
13.R. E. Ventura, J. E. Goldman, J. Neurosci. 27,
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28.K. Campbell, Curr. Opin. Neurobiol. 13, 50
(2003).
29.F. Guillemot, Curr. Opin. Cell Biol. 17, 639
(2005).
30.F. Doetsch, A. Alvarez-Buylla, Proc. Natl.
Acad. Sci. U.S.A. 93, 14895 (1996).
31.We thank C. Lois for supplying Ad:Cre virus
and C. Yaschine and R. Romero for assistance
with cell culture and tissue processing. This
work was supported by grants from the NIH
and by a fellowship from the NSF for F.T.M.
Supporting Online Material
www.sciencemag.org/cgi/content/full/1144914/DC1
Materials and Methods
Figs. S1 to S8
References
10 May 2007; accepted 19 June 2007
Published online 6 July 2007;
10.1126/science.1144914
Include this information when citing this paper.
29
Detecting the Future of Neuroscience
M
odern neuroscience
relies on cutting edge
techniques, often
confounded by complex
cell culture and intractable
membrane-associated
proteins. To meet these
challenges, Millipore offers
a single source for validated
neuroscience antibodies,
specialized kits and assays,
and robust platforms
for quantitative protein
expression analysis. As a
result, Millipore has emerged
as the leading research
partner for measuring
protein biomarkers of the
central, peripheral, and
developing nervous systems.
The importance of
developing and using
biomarkers in neuroscience
research is twofold. First,
biomarkers that differentiate
between healthy nervous
systems and those afflicted
by neurodegenerative
diseases can accelerate
target validation, drug
development, and clinical
diagnostics. Second, protein
biomarkers that provide
structural and functional
information about specific
nervous system cell types
can elucidate mechanisms of
development, differentiation,
therapeutic activity, and
disease progression.
Neuroscience researchers
at Millipore have developed
multiple biomarker detection
assays of varying resolution,
spatiotemporal information, and quantitativeness,
in order to fit the specific
requirements for customers at every part of their
experimental workflow.
Three of these assay
platforms are the MilliMark™ pan-neuronal marker, the MILLIPLEX® MAP
neurodegenerative disease
panel, and the FlowCellect™
stem cell assay kit
for studying lineagespecific differentiation
of neural progenitors.
30
Figure 1: Neuron type-specific antibodies reveal
localization of individual proteins (a-d). Milli-Mark
pan-neuronal marker brightly illuminates all parts
of all neurons (e). For immunofluorescence (a, d,
e), cultured neurons were fixed and incubated
with primary antibodies and fluorescently labeled
secondary antibody. For immunohistochemistry (b,c),
neurons were embedded, sectioned, deparaffinized
and incubated with primary antibodies, biotinylated
secondary antibody and DAB/nickel stain.
Glutamatergic neurons
Anti-VGLUT2
(Millipore #MAB5504)
GABAergic neuron
Anti-GAD67
(Millipore #MAB5406)
Dopaminergic neurons
Anti-TH (tyrosine hydroxylase)
(Millipore #MAB5280)
Serotonergic neurons
Anti-SERT (serotonin transporter)
(Millipore #MAB1772)
All neurons
Milli-Mark pan-neuronal marker
(Millipore #MAB2300)
The Milli-Mark antibody cocktail
illuminates neuron cytoarchitecture
Multiparametric, singlecell tracking of lineagespecific differentiation
Antibodies to neuronal proteins have become critical tools
for identifying neurons and discerning morphological
characteristics in culture and complex tissue. While Golgi
staining and fluorescent protein-fused constructs yield
excellent cytoarchitectural detail, these approaches are
technically challenging and often impractical. Neuron-specific
antibodies can reveal cytoarchitecture, but are limited to the
target protein’s distribution within the neuron, which may
differ greatly from nucleus to soma to dendrite and axon.
Millipore’s FlowCellect neural
stem cell characterization
kit, used with the guava®
benchtop flow cytometry
systems, has proved to be
a powerful tool for neural
stem cell (NSC) research.
By measuring multiple
parameters on hundreds
of cells per second, flow
cytometry delivers robust,
high content information
in far less time than
other methods. Guava
flow cytometry systems
are designed for low
cell numbers and small
volume cell populations,
helping to preserve
precious NSC samples.
The Milli-Mark pan-neuronal marker achieves as complete
a morphological staining as possible across all parts
of neurons. This monoclonal antibody blend can then
be detected by a single secondary antibody. The MilliMark reagent has been validated in diverse fixations, cell
culture and immunohistochemistry protocols, including
archival brain tissue, and thus yields specific, highresolution information about neuronal morphology.
MILLIPLEX MAP multiplex analyte panels
quantify disease biomarkers
Millipore is the first to develop multiplex analyte panels
for neurodegenerative disease; the Luminex®-based
MILLIPLEX MAP human neurodegenerative panels help
unravel the complexities of the nervous system and the
pathobiology of disease. Accurate measurement of key
biomarkers is fundamental to determining the pathogenesis
of devastating neurodegenerative diseases, such as
Alzheimer’s disease, Parkinson’s disease, Lewy body
disease, amyotrophic lateral sclerosis, and neurological
disorders. However, conventional methods, including
RIAs and ELISAs, are not able to simultaneously measure
multiple biomarkers with small sample volume as is
required when analyzing valuable patient samples.
The FlowCellect kit provides
rapid, sensitive assessments
of NSC phenotypes at
various stages of lineagespecific differentiation.
Elucidating the pathways by
which neurons, astrocytes,
and oligodendrocytes arise
from NSCs has important
clinical applications for
central nervous system
diseases like Parkinson’s
disease. Successful
engraftment of NSCs into the
brain of rodent models has
demonstrated the therapeutic
potential of this cell type.
Ultimately, the MILLIPLEX MAP disease biomarker panels will
help identify people with these disorders before the onset of
symptoms and potentially provide new therapeutic tools.
MILLIPLEX¨ MAP
Human Neurodegenerative Disease Panel 3
LmZg]Zk]<nko^l
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*%)))
Concentration (ng/mL)
Figure 2: As shown, MILLIPLEX MAP 10-plex Neurodegenerative Disease Panel
3 can accurately measure analyte levels over a wide range of concentrations. The
overnight assay requires only 25 μL of sample, and is compatible with serum,
plasma, CSF (cerebrospinal fluid), and cell/tissue extract or culture samples.
31
Millipore: Detecting the whole
spectrum of neuroscience
The future of neuroscience depends on detecting biomarkers
at multiple points on the spectrum of resolution:
•Qualitative, subcellular localization in
individual cells (neuron-specific and panneuronal markers – Milli-Mark reagent)
•Quantitative detection of mean biomarker
levels in cell populations (multiplex beadbased detection—MILLIPLEX MAP panels)
•Multiparametric biomarker detection in single cells
(flow cytometry—guava FlowCellect kits)
As a single source providing solutions to each and
all of these needs, Millipore is uniquely positioned
to advance neuroscience research on all fronts.
<'
A
Count
2)
/)
54%
,)
*)^*
*)^+
*)^,
*)^-
½β-Tubulin III-PE/Cy5
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)
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Ng]b__^k^gmbZm^]G^nkZeLm^f\^eel
Figure 3: The FlowCellect neural stem cell kit and
the guava flow cytometry system were used to track
differentiation of rat neural stem cells into either neurons
or astrocytes. Beta tubulin III, a neuronal marker, was
upregulated in the neuronal-differentiated cells (a). GFAP,
an astrocyte-specific marker, was upregulated in the
astrocyte-differentiated cells (b).
Learn more at www.millipore.com/neuromarkers
32
Millipore’s biomarker
expertise goes
beyond the nervous
system, offering
validated antibodies
for Epigenetics,
Immunology,
Inflammation, Cell
Structure, Protein
Trafficking and more.
Other MILLIPLEX MAP
analyte panels cover:
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)
*)^)
BEYOND
NEUROSCIENCE
•Toxicity
•Cancer
•Metabolic Syndrome
•Cardiovascular Disease
•Bone Metabolism
•Cell Signaling
•Chemokine Analysis
Some members of the
growing portolio of
FlowCellect assay kits:
•Apoptosis & Cancer
•Chemokine Analysis
•Cell Counting & Viability
•Cell Cycle
•HIV Monitoring
•Signaling
Neuroscience
MILLIPLEX® MAP.
Hot new targets
for a growing
body of work.
Inflammation
Allergies, Asthma,
Autoimmune
Endocrine
Isotyping
Toxicity
Metabolism
Skin
Reproduction
Cancer
Cell Signaling
Bone Metabolism
Immunology
The multiplex resource as dynamic as your research.
MILLIPLEX map offers the broadest selection of biomarker immunoassay kits and reagents in a wide range of
therapeutic areas, providing over 250 analytes to run on the Luminex® platform—plus the instruments, software
and services you need, all in one place. And as your research grows, so will we.
Featured Products
• Toxicity: Kidney Toxicity Panels—based on FDA/EMEA guidelines for identifying drug-induced renal damage
• Metabolic: Metabolic Multiplex Panels—the right analytes for studying metabolic pathways across multiple
organ systems
• Neuroscience: Human Neuropeptide Panel—the premier panel in a unique neuroscience multiplex offering
ADVANCING LIFE SCIENCE TOGETHER™
Research. Development. Production.
Millipore and MILLIPLEX are registered trademarks of Millipore Corporation.
Luminex is a registered trademark of Luminex Corporation.
Advancing Life Science Together and the Millipore logo are trademarks of Millipore Corporation.
©2009 Millipore Corporation. All rights reserved.
STEM
CELL
RESEARCH:
Discover
one-stop
multiplexing
STEM
CELL
RESEARCH: at
www.millipore.com/stemcell
www.millipore.com/milliplexC
www.millipore.com/stemcell
FROM CORE LAB
TO YOUR LAB.
Introducing the NEW easyCyte™ 8HT system
COMPLETE - the world's first end-to-end
bench top flow cytometry solution.
OPTIMIZED - multiparametric kits and assays.
INTUITIVE - software and analysis packages.
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Millipore brings the power of flow cytometry to your bench top.
Imagine running your most complex assays right on your bench top. No core labs. No outside expertise necessary. With
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ADVANCING LIFE SCIENCE TOGETHER™
Research. Development. Production.
Millipore is a registered trademark of Millipore Corporation.
Guava is a registered trademark of Guava Technologies, Inc.
Advancing Life Science Together and the Millipore logo are
trademarks of Millipore Corporation.
easyCyte is a trademark of Guava Technologies, Inc.
©2009 Millipore Corporation. All rights reserved.
Make the move to Guava at
www.millipore.com/flowcytometryE