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Cerebral neurogenesis is induced by intranasal administration of growth factors.

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Cerebral Neurogenesis Is
Induced by Intranasal
Administration of
Growth Factors
Kunlin Jin, MD, PhD, Lin Xie, BS, Jocelyn Childs, BA,
Yunjuan Sun, MD, Xiao Ou Mao, MD,
Anna Logvinova, MD,
and David A. Greenberg, MD, PhD
Neurogenesis persists in the adult brain, where it may contribute to repair and recovery after injury, but the lack of
methods for noninvasive stimulation of cerebral neurogenesis limits its potential for clinical application. We report
that intranasal administration of either fibroblast growth
factor–2 or heparin-binding epidermal growth factor–like
growth factor increases neurogenesis, measured by the incorporation of bromodeoxyuridine into cells that express
the early neuronal marker protein doublecortin in the subventricular zone of mouse brain. These findings indicate
that intranasal growth factors may have potential as
neurogenesis-promoting therapeutic agents.
Ann Neurol 2003;53:405– 409
Neurogenesis, which is critical in brain development
and continues into adulthood, can be stimulated by injury and may have a role in brain repair and associated
From the Buck Institute for Age Research, Novato, CA.
Received Oct 16, 2002, and in revised form Nov 21. Accepted for
publication Nov 21, 2002.
Address correspondence to Dr Greenberg, Special Research Programs, Buck Institute for Age Research, 8001 Redwood Boulevard,
Novato, CA 94945. E-mail:
© 2003 Wiley-Liss, Inc.
functional recovery.1 Therefore, the ability to augment
injury-induced neurogenesis could have therapeutic
consequences for acute and chronic neurodegenerative
disease. Growth factors, usually given by the intracerebroventricular route, are among the best characterized
stimuli to neurogenesis in rodents,2 but their clinical
usefulness is restricted by their limited access to the
brain after systemic administration. On the basis of reports that growth factors can enter the brain after intranasal delivery,3,4 we investigated the effects of intranasal administration of two growth factors, fibroblast
growth factor–2 (FGF-2) and heparin-binding epidermal growth factor–like growth factor (HB-EGF), on
neurogenesis in the principal neuroproliferative regions
of the adult mouse brain, the rostral subventricular
zone (SVZ), and the subgranular zone (SGZ) of the
hippocampal dentate gyrus.
Materials and Methods
Male CD1 mice weighing 30 to 40gm were given 20␮l of a
10␮g/ml solution of FGF-2 or HB-EGF into the right naris,
five times at 5-minute intervals, and this was repeated twice
daily for 1 week. Bromodeoxyuridine (BrdU; 50mg/kg;
Sigma, St. Louis, MO) was dissolved in saline and given by
the intraperitoneal route, twice daily with doses spaced 8
hours apart, for the same week, and animals were killed 24
hours after the final BrdU dose.
For BrdU immunohistochemistry, brains (five per condition) were removed after perfusion with saline and 4% paraformaldehyde in phosphate-buffered saline (PBS). Adjacent
50␮m sections, corresponding to coronal coordinates interaural 3.94 to 4.9mm, bregma ⫺0.14 to bregma ⫺1.10mm
(SVZ) and interaural 1.26 to 2.46mm, bregma ⫺1.34 to
bregma ⫺2.54 (dentate gyrus), were cut with a cryostat and
stored at ⫺80°C. Sections were pretreated with 50% formamide, 280mM NaCl and 30mM sodium citrate at 65°C for
2 hours, incubated in 2M HCl at 37°C for 30 minutes, and
rinsed in 0.1M boric acid (pH 8.5) at room temperature for
10 minutes. Sections were incubated in 1% H2O2 in PBS
for 15 minutes, in blocking solution (2% goat serum, 0.3%
Triton X-100, and 0.1% bovine serum albumin in PBS) for
2 hours at room temperature, and with 2␮g/ml of mouse
monoclonal anti–BrdU antibody (Roche, Indianapolis, IN)
at 4°C overnight. Sections were washed with PBS, incubated
with biotinylated goat anti–mouse secondary antibody
(1:200; Vector Laboratories, Burlingame, CA) for 2 hours at
25°C, washed, and placed in avidin-peroxidase conjugate
(Vector) solution for 1 hour. The horseradish peroxidase reaction was detected with 0.05% diaminobenzidine and
0.03% H2O2. Processing was stopped with H2O and sections were dehydrated through graded alcohols, cleared in
xylene, and coverslipped in permanent mounting medium
(Vector). Sections were examined with a Nikon E300 epifluorescence microscope.
BrdU-positive cells in SGZ and SVZ were counted blindly
in five to seven diaminobenzidine-stained, 50␮m coronal
sections per animal, spaced 200␮m apart. Cells were counted
under high power (⫻200) on a Nikon E300 microscope
with Magnifire digital camera (ChipCoolers, Warwick, RI),
© 2003 Wiley-Liss, Inc.
and the image was displayed on a computer monitor. Results
were expressed as the average number of BrdU-positive cells
per section (mean ⫾ standard error of the mean) from at
least three experiments. Student’s t test was used for statistical analysis, with p value less than 0.05 considered
For double-immunolabeling studies, sections were fixed
with 4% paraformaldehyde in PBS for 1 hour at room temperature, washed twice with PBS, and incubated in 2M HCl
at 37°C for 1 hour. After washing again, sections were incubated with blocking solution, then with primary antibodies
at 4°C overnight, and with secondary antibodies in blocking
solution at room temperature for 2 hours. The primary antibodies used were mouse monoclonal anti-BrdU (Roche;
2␮g/ml) and affinity-purified goat polyclonal anti-Dcx (1:
100; Santa Cruz Biotechnology, Santa Cruz, CA); the secondary antibodies were rhodamine-conjugated rat-absorbed
donkey anti–mouse IgG (1:200; Jackson ImmunoResearch,
West Grove, PA) and fluorescein isothiocyanate–conjugated
pig anti–goat IgG (1:200; Jackson ImmunoResearch). Sections were mounted with Vectashield (Vector) and fluorescence signals were detected with a Nikon E800 microscope
at excitation/emission wavelengths of 535/565 nm (rhodamine, red) and 470/505 nm (fluorescein isothiocyanate,
green). Results were recorded with a Magnifire digital camera
(ChipCoolers). For confocal microscopy, a Nikon PCM2000 laser-scanning confocal microscope and Simple PCI
imaging software (Compix, Cranberry Township, PA) were
used. Three-dimensional reconstructions were produced from
16nm slices using Imaris software (Bitplane AG, Zurich,
Results and Discussion
Control mice showed incorporation of BrdU into a
modest number of cells in the SVZ and SGZ (Fig 1),
but after intranasal administration of FGF-2 or HBEGF, BrdU labeling in the SVZ increased by approximately 70% and approximately 40%, respectively,
whereas labeling in the SGZ was unchanged. We also
have observed more pronounced stimulation of neurogenesis in SVZ than in SGZ after intracerebroventricular administration of HB-EGF, which may be related
to the greater concentration of EGF receptors in SVZ,5
although differences in the accessibility of intranasally
administered growth factors to SVZ and SGZ could
contribute to this disparity as well.
To determine the phenotype of cells that incorporated BrdU in response to FGF-2 or HB-EGF, we
stained sections through the SVZ with antibodies
against BrdU and against the immature neuronal
marker, Dcx.6,7 Dcx colocalized with BrdU in this region to a marked extent (Fig 2), indicating that many
recently divided cells labeled with BrdU were of neuronal lineage. To confirm that growth factor treatment
increased the absolute number of BrdU- and Dcximmunopositive cells, we counted cells and calculated
the percentage of BrdU-labeled cells that also expressed
Dcx in SVZ from control and treated mice. Dcx-
immunopositive cells accounted for 45 ⫾ 5% (n ⫽ 3)
of BrdU-labeled cells in control and 40 ⫾ 3% (n ⫽ 5)
of BrdU-labeled cells in treated mice (P ⫽ 0.39). Taking into account the approximately 70% increase in
BrdU labeling induced by FGF-2 and the approximately 40% increase in labeling induced by HB-EGF
(see above), this translates to increases of approximately
50% (for FGF-2) and approximately 25% (for HBEGF) in the absolute number of BrdU- and Dcximmunopositive cells in SVZ of growth factor–treated
Neurons that arise in the SVZ of adult rodent brain
Fig 1. Effect of intranasal growth factors on bromodeoxyuridine (BrdU) labeling in neuroproliferative zones of adult
mouse brain. Intranasal saline (Control), fibroblast growth
factor–2 (FGF-2), or heparin-binding epidermal growth
factor–like growth factor (HB-EGF) and intraperitoneal BrdU
were given, and BrdU labeling was detected with mouse
monoclonal anti-BrdU in (a) subventricular zone (SVZ) and
(b) subgranular zone (SGZ). The number of BrdUimmunopositive cells was increased by both FGF-2 or HBEGF in SVZ but not SGZ (c). Data shown are representative
fields (a, b) or mean values ⫾ SEM, n ⫽ 3 (c). (asterisks)
p ⬍ 0.05 compared with control (ANOVA and post hoc
Student-Newman-Keuls test). Bars ⫽ 100␮m (a) and
200␮m (b).
Fig 2. Colocalization of bromodeoxyuridine (BrdU) and Dcx
in subventricular zone (SVZ) of adult mouse brain after intranasal administration of fibroblast growth factor–2 (FGF-2)
(a) or heparin-binding epidermal growth factor–like growth
factor (HB-EGF) (b). Brain sections from mice treated with
growth factors and BrdU were stained with antibodies against
BrdU (red nuclei) and Dcx (green cytoplasm) and examined
by double-label fluorescence immunohistochemistry with confocal imaging and three-dimensional reconstruction, which
showed colocalization of BrdU and Dcx to the same cells.
Data shown are representative fields (n ⫽ 3). Bars ⫽ 10␮m.
normally migrate via the rostral migratory stream to
the olfactory bulb (OB), where they replace interneurons undergoing rapid turnover.8,9 To determine
whether the increased neurogenesis observed in the
SVZ after growth factor administration was effectual,
we counted BrdU-immunopositive neurons in the OB
after treatment with intranasal FGF-2 or HB-EGF.
BrdU-immunopositive cell counts in the OB increased
by approximately 40% after FGF-2 and by approximately 30% after HB-EGF administration (Fig 3),
consistent with functional neurogenesis.
Jin et al: Neurogenesis and Intranasal Growth Factors
Fig 3. Effect of intranasal growth factors on bromodeoxyuridine (BrdU) labeling in olfactory bulb (OB) of adult mouse brain. Intranasal saline (Control), fibroblast growth factor–2 (FGF-2), or heparin-binding epidermal growth factor–like growth factor
(HB-EGF) and intraperitoneal BrdU were given, and BrdU labeling in OB was detected by immunohistochemistry (a). Cell counts
showed an increase in BrdU-immunopositive cells in OB induced by either FGF-2 or HB-EGF (b). Data shown are representative
fields (a) or mean values ⫾ SEM, n ⫽ 6 –12 (b). (asterisks) p ⬍ 0.05 compared with control (ANOVA and post hoc StudentNewman-Keuls test). Bar ⫽ 100␮m.
These findings demonstrate that growth factors administered by the intranasal route can stimulate neurogenesis in the adult mammalian brain. The persistence
of neurogenesis in the adult brain suggests that newborn neurons might provide a source for the replacement of neurons destroyed by acute neurological
catastrophes, such as stroke, or more insidious neurodegenerative processes, including Alzheimer’s disease or
Parkinson’s disease.1 Several forms of experimental cerebral injury, including trauma,10 seizures,11 and ischemia,12,13 stimulate neurogenesis, which could constitute an endogenous mechanism that promotes brain
repair. Adult neurogenesis is responsive to a variety of
growth factors, but many of these large (approximately
5–30kDa) molecules penetrate only poorly from the
systemic circulation into the brain, and their limited
bioavailability is a major impediment to therapeutic
application.14 For some growth factors, systemic administration is possible15,16 but could present a risk of
adverse systemic effects such as tumor angiogenesis or
Growth factors can pass from the nasal cavity directly into the brain or cerebrospinal fluid by slow
transneuronal or more rapid perineuronal pathways
created by bipolar olfactory sensory neurons, axons of
which penetrate the cribriform plate of the ethmoid
bone to enter the OB.18 For example, intranasal IGF-I
enters the brain and improves outcome after focal cerebral ischemia in rats,3 and melanocortin, vasopressin,
and insulin enter the cerebrospinal fluid, without first
entering the bloodstream, in humans.4 Which of these
Annals of Neurology
Vol 53
No 3
March 2003
routes is used by FGF-2 or HB-EGF to stimulate neurogenesis remains to be determined. However, the ability to enhance neurogenesis in the adult brain by noninvasive means may have therapeutic implications for
neurological disease.
This work was supported by the National Institutes of Health
(NS44921, D.G.) and the Buck Institute for Age Research (D.G.).
1. Horner PJ, Gage FH. Regenerating the damaged central nervous system. Nature 2000;407:963–970.
2. Cameron HA, Hazel TG, McKay RD. Regulation of neurogenesis by growth factors and neurotransmitters. J Neurobiol 1998;
3. Liu X-F, Fawcett JR, Thorne RG, et al. Intranasal administration of insulin-like growth factor-I bypasses the blood-brain
barrier and protects against focal cerebral ischemic damage.
J Neurol Sci 2001;187:91–97.
4. Born J, Lange T, Kern W, et al. Sniffing neuropeptides: a
transnasal approach to the human brain. Nat Neurosci 2002;5:
514 –516.
5. Jin K, Mao XO, Sun Y, et al. Heparin-binding epidermal
growth factor-like growth factor (HB-EGF): hypoxia-inducible
expression and stimulation of neurogenesis. J Neurosci 2002;
6. Francis F, Koulakoff A, Boucher D, et al. Doublecortin is a
developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons. Neuron 1999;
7. Gleeson JG, Lin PT, Flanagan LA, Walsh CA. Doublecortin is
a microtubule-associated protein and is expressed widely by migrating neurons. Neuron 1999;23:257–271.
8. Luskin MB. Restricted proliferation and migration of postnatally generated neurons derived from the forebrain subventricular zone. Neuron 1993;11:173–189.
9. Lois C, Garcia-Verdugo JM, Alvarez-Buylla A. Chain migration
of neuronal precursors. Science 1996;271:978 –981.
10. Gould E, Tanapat P. Lesion-induced proliferation of neuronal
progenitors in the dentate gyrus of the adult rat. Neuroscience
1997;80:427– 436.
11. Parent JM, Yu TW, Leibowitz RT, et al. Dentate granule cell
neurogenesis is increased by seizures and contributes to aberrant
network reorganization in the adult rat hippocampus. J Neurosci 1997;17:3727–3738.
12. Liu J, Solway K, Messing RO, Sharp FR. Increased neurogenesis in the dentate gyrus after transient global ischemia in gerbils. J Neurosci 1998;18:7768 –7778.
13. Jin K, Minami M, Lan JQ, et al. Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral
ischemia in the rat. Proc Natl Acad Sci USA 2001;98:
4710 – 4715.
14. Thorne RG, Frey WH II. Delivery of neurotrophic factors to
the central nervous system: pharmacokinetic considerations.
Clin Pharmacokinet 2001;40:907–946.
15. Wagner JP, Black IB, DiCicco-Bloom E. Stimulation of neonatal and adult brain neurogenesis by subcutaneous injection of
basic fibroblast growth factor. J Neurosci 1999;19:6006 – 6016.
16. Aberg MA, Aberg ND, Hedbacker H, et al. Peripheral infusion
of IGF-I selectively induces neurogenesis in the adult rat hippocampus. J Neurosci 2000;20:2896 –2903.
17. Talapatra S, Thompson CB. Growth factor signaling in cell
survival: implications for cancer treatment. J Pharmacol Exp
Ther 2001;298:873– 878.
18. Illum L. Transport of drugs from the nasal cavity to the central
nervous system. Eur J Pharm Sci 2000;11:1–18.
PRNP Val129 Homozygosity
Increases Risk for EarlyOnset Alzheimer’s Disease
Bart Dermaut, MD,1 Esther A. Croes, MD, PhD,2
Rosa Rademakers, MSc,1 Marleen Van den Broeck,1
Marc Cruts, PhD,1 Albert Hofman, MD, PhD,2
Cornelia M. van Duijn, PhD,1,2
and Christine Van Broeckhoven, PhD, DSc1
We analyzed the PRNP M129V polymorphism in a
Dutch population-based early-onset Alzheimer’s disease
sample. We observed a significant association between
early-onset Alzheimer’s disease and homozygosity of
M129V (odds ratio [OR], 1.9; 95% confidence interval
[CI], 1.1–3.3; p ⴝ 0.02) with the highest risk for V homozygotes (OR, 3.2; 95% CI, 1.4 –7.1; p < 0.01). In
patients with a positive family history, these risks increased to 2.6 (95% CI, 1.3–5.3; p < 0.01) and 3.5 (95%
CI, 1.3–9.3; p ⴝ 0.01), respectively.
Ann Neurol 2003;53:409 – 412
Alzheimer’s disease (AD) is the most common form of
dementia. Genetic factors are important in both earlyonset AD (EOAD; onset ⬍65 years) and late-onset
AD (LOAD; onset ⬎65 years).1 We have estimated
that mutations in the amyloid precursor protein (APP)
and the presenilins (PSEN1, PSEN2) account for 20%
of autosomal dominant EOAD.2 On the population
level, the ε4 allele of apolipoprotein E (APOE) is an
important risk factor for AD.3
The prion protein gene (PRNP) is an established risk
gene for Creutzfeldt–Jakob disease (CJD), a rapidly
progressive dementia with deposition of proteinase resistant prion proteins in brain. Individuals homozygous
for the PRNP M129V polymorphism are at increased
risk of developing sporadic, iatrogenic, and variant
Several lines of evidence suggest that a molecular genetic study of PRNP in AD is relevant. First, epidemi-
From the 1Department of Molecular Genetics, Flanders Interuniversity Institute of Biotechnology (VIB8), University of Antwerp
(UIA), Antwerpen, Belgium; and 2Department of Epidemiology and
Biostatistics, Erasmus Medical Center, Rotterdam, The Netherlands
Received Aug 5, 2002, and in revised form Nov 30. Accepted for
publication Dec 1, 2002.
Address correspondence to Dr Van Broeckhoven, Department of
Molecular Genetics (VIB8), University of Antwerp (UIA), Universiteitsplein 1, B-2610, Antwerpen, Belgium.
© 2003 Wiley-Liss, Inc.
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factors, neurogenesis, induced, growth, administration, intranasal, cerebral
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