Full article of Madelyn's former abstract.

Experimental Neurology 202 (2006) 336 –347
www.elsevier.com/locate/yexnr

A partial GDNF depletion leads to earlier age-related deterioration of motor
function and tyrosine hydroxylase expression in the substantia nigra
H.A. Boger a, L.D. Middaugh a,c, P. Huang b, V. Zaman a, A.C. Smith d, B.J. Hoffer e,
A.C. Tomac e, A.-Ch. Granholm a,.
a

Department of Neurosciences, Center on Aging, MUSC, 26 Bee Street, Charleston, SC 29425, USA
b Department of Biometry and Epidemiology, Medical University of South Carolina, SC, USA

c

Department of Psychiatry, Medical University of South Carolina, SC, USA
d Department of Comparative Medicine, Medical University of South Carolina, SC, USA


e

Intramural Research Program, NIDA, Baltimore, MD, USA

Received 15 February 2006; revised 1 June 2006; accepted 19 June 2006
Available online 2 August 2006


Abstract

Glial cell line-derived neurotrophic factor (GDNF) is a trophic factor for peripheral organs, spinal cord, and midbrain dopamine (DA)
neurons. Levels of GDNF deteriorate in the substantia nigra in Parkinson's disease (PD). A heterozygous mouse model was created to assess
whether chronic reductions in this neurotrophic factor impact motor function and the nigrostriatal dopamine system during the aging process.
Due to the important role GDNF plays in kidney development, kidney function and histology were assessed and were found to be normal in
both wild-type (WT) and GDNF+/- mice up to 22 months of age. Further, the animals of both genotypes had similar weights throughout the
experiment. Locomotor activity was assessed for male WT and GDNF+/- mice at 4-month intervals from 4 to 20 months of age. Both GDNF+/-
and WT mice exhibited an age-related decline in horizontal activity, although this was found 4 months earlier in GDNF+/- mice, at 12 months of
age. Comparison of young (8 month old) and aged (20 month old) GDNF+/- and WT mice on an accelerating rotarod apparatus established a
deficiency for aged but not young GDNF+/- mice, while aged WT mice performed as well as young WT mice on this task. Finally, both WT and
GDNF+/- mice exhibited an age-related decrease in substantia nigra TH immunostaining, which was accelerated in the GDNF+/- mice. These
behavioral and histological alterations suggest that GDNF may be an important factor for maintenance of motor coordination and spontaneous
activity as well as DA neuronal function during aging, and further suggest that GDNF+/- mice may serve as a model for neuroprotective or
rescue studies.
© 2006 Elsevier Inc. All rights reserved.

Keywords: Dopamine; Substantia nigra; Parkinson's disease; Animal models; Movement disorders; Neurodegeneration

Introduction

Recent information indicates that the incidence of extrapyramidal
symptoms increases significantly with age, to
involve up to 50% of the population over 85 years of age
(Bennett et al., 1996; Kluger et al., 1997). Epidemiological data
suggest that this age-related motor impairment results from a
multifactorial process. Many studies suggest that the dopaminergic
(DA) nigrostriatal system, although not the only system,

. Corresponding author. Fax: +1 843 792 0679.
E-mail address: granholm@musc.edu (A.-Ch. Granholm).


0014-4886/$ -see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.expneurol.2006.06.006

is directly involved in age-related motor dysfunction (see e.g.

Volkow et al., 1998; Naoi and Maruyama, 1999; Palmer and
DeKosky, 1993; see also Ingram, 2000). In human brain, the
number of midbrain DAergic neurons is reduced 4.7–6.0% per
decade after the age of 50 (Fearnley and Lees, 1991). In
addition, imaging studies have shown a substantial age-related
decline in brain DA D2 receptors (Volkow et al., 1996), DA D1
receptors (Suhara et al., 1991), and DA transporters (Volkow et
al., 1996; see also Kaasinen and Rinne, 2002, for review).
Specific age-related deterioration in motor function has also
been related to the DA transmitter system in animal models (see

e.g. Hebert and Gerhardt, 1998; Cass et al., 2005; Yurek and

H.A. Boger et al. / Experimental Neurology 202 (2006) 336–347
Fletcher-Turner, 2000). Studies have shown alterations in DA
uptake in the aged Rhesus monkey (Dejesus et al., 2001),
reduced DA release in aged F344 rats (Hebert and Gerhardt,
1998), as well as a reduction in DA receptor binding in aged
mice (see e.g. Ingram, 2000 for review). Additional studies
indicate that aged rodents have an increased vulnerability to
DA-specific neurotoxins such as 6-hydroxydopamine in rats (6OHDA;
see Cass et al., 2002) and 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine
(MPTP) in mice (Mandavilli et al., 2000).
Even though the mechanisms for age-related nigral neuron
vulnerability are unknown, it has been suggested that a
reduction in support systems such as growth factors increases
the susceptibility of the substantia nigra (SN) DA neurons to
external stressors and toxins (see e.g. Yurek and Fletcher-
Turner, 2000, 2001).

GDNF is a member of the transforming growth factor-ß
superfamily of neurotrophic factors (Krieglstein et al., 1995;
Saarma, 2000). Organs and cells dependent on GDNF for
their survival include spermatogenesis (Naughton et al.,
2006), kidneys, spinal cord motor neurons, sensory neurons,
and cranial nerve motor neurons (for review, see Bates, 2000;
Saarma, 2000; Malcangio, 2003; Sah et al., 2005). GDNF is
also required for the survival, high-affinity DA uptake, and
neurite outgrowth of cultured midbrain DAergic neurons (Lin
et al., 1993) and promotes recovery of behavior and morphology
in rodent and nonhuman primate models of
Parkinson's disease (PD; Bowenkamp et al., 1995; Gash et al.,
1996; Lindner et al., 1995; Björklund et al., 1997; Mandel et al.,
1997; Kordower et al., 2000; Cass and Manning, 1999;
Granholm et al., 1997a,b; Dowd et al., 2005). Importantly,
GDNF levels are decreased in the SN of PD patients (Jenner and
Olanow, 1998) and also in normal aged rodents (Yurek et al.,
2000) suggesting its involvement in motor dysfunction and
DA neuron degeneration. GDNF has therefore recently been
used as an experimental therapeutic agent for patients with PD
(Gill et al., 2003). The cellular responses to GDNF family
ligands are mediated via a multicomponent receptor consisting
of the RET receptor tyrosine kinase and a GPI-linked ligand-
binding subunit known as GFR alpha (see e.g. Harvey et al.,
2005; Airaksinen and Saarma, 2002). Recent studies have
demonstrated that GDNF-mediated effects require Src kinase
and phosphatidylinositol 3-kinase (PI3K)/Akt activation to
increase intracellular glutathione and enhance survival-promoting
cell signaling (Onyango et al., 2005). In PC12-GFRalpha1
cell, the phosphatidylinositol 3-kinase pathway seems to
mediate the survival-promoting effect of GDNF, while the
activation of the mitogen-activated protein kinase pathway
could be an important step in mediating PC12-GFRalpha1 cell
differentiation induced by this growth factor (Chen et al., 2001).
It has not been fully determined, to our knowledge, which
intracellular signaling pathways are involved in the protective
effects of GDNF on SN DA neurons during aging and
neurodegeneration.

A GDNF knockout model was developed to explore the
role of GDNF in fetal and postnatal development (see Pichel
et al., 1996; Sanchez et al., 1996; Moore et al., 1996). Early
development of mesencephalic DA neurons was unaffected in

GDNF-/- mice possibly because the major apoptotic waves for
midbrain DA neurons occur on postnatal days 2 and 14 in the
mouse (Oo and Burke, 1997; Mahalik et al., 1994). Since
GDNF-/- mice die at birth due to renal agenesis, studies from
our laboratory have used fetal grafts from GDNF-/- mice to
demonstrate that postnatal survival of mesencephalic DA
neurons is dependent on GDNF (Granholm et al., 2000). In
order to examine the role of endogenous GDNF in a mouse
model, we have examined long-term effects of a partial GDNF
depletion (GDNF+/-) on the nigrostriatal DA system throughout
the life-span. Due to the fact that GDNF is also involved in renal
development, we performed routine assessments of kidney size,
morphology, and function throughout this longitudinal study of
male GDNF+/- mice.

Materials and methods

Animals

Male GDNF heterozygous (GDNF+/-) mice were compared
to their wild-type (WT) littermates in all experiments. The
nonfunctional allele for the GDNF gene was generated by
replacing part of the third exon that encodes GDNF protein with
a cassette expressing the selectable marker neomycin phophotransferase,
as described previously in detail (Pichel et al., 1996;
Granholm et al., 1997a,b, 2000). Heterozygous offspring are
viable and fertile, whereas mice homozygous to the mutant
GDNF allele (GDNF-/-) die within 24 h of birth. The mice for
this study were bred locally at the Medical University of South
Carolina, on a C57BL/6J background according to NIH
approved protocols. The mice were housed in groups of 3–4
to a cage and had free access to food and water. Food intake and
body weight were monitored throughout the study. They were
maintained under 12-h light: 12-h dark cycle and at an ambient
temperature of 20–22°C and were given water and mouse chow
ad libitum.

PCR genotyping

Genomic DNA was prepared from a 1 cm sample of tissue
from the tail. The tissue was homogenized in 0.2 M NaCl,
5 mM EDTA, 100 mM Tris, pH 8.5, 0.2% SDS and 400 µg/
ml proteinase K overnight at 55°C with agitation. After
centrifugation (12,000 rpm), DNA was precipitated from the
supernatant with one volume isopropanol, pelleted, washed
twice with 70% ethanol, and re-suspended in 500 µl sterile
water. The DNA was assayed for the presence of the WT or
knockout allele in a single PCR reaction using WT primer
mix and knockout primer mix. PCR was performed in a 50 µl
reaction volume containing 2 µl of genomic DNA, 2 µMof
each primer, 5 mM MgCl2, 200 µM each of dATP, dGTP,
dCTP, and dTTP, and one unit Taq polymerase. The genomic
DNA was amplified for a total of 35 cycles and the products
analyzed for the presence of the WT or -/- allele on a 1.3%
agarose gel. Amplification of the WT allele gave a band of
344 base pairs, while the mutant allele gave a band of 255
base pairs (Pichel et al., 1996).


H.A. Boger et al. / Experimental Neurology 202 (2006) 336–347
Urea and creatinine assays

Since GDNF-/- die at birth due to kidney agenesis, we
wanted to examine if kidney dysfunction would also occur in
GDNF+/- mice with age, and if such alterations may interfere
with alterations in brain parameters or behavior. Therefore,
Quantichrome Urea Assay (DIUR-01K) and Quantichrome
Creatinine Assay (DICT-500) were utilized to determine the
levels of urea and creatinine, respectively, in serum of all groups
at 8, 12, and 22 months of age. Urea measurements reflect the
glomerular filtration rate, which is often used as a criterium for
kidney function. The urea assay measures urea in blood without
any pretreatment in 5 min. This assay uses a chromogenic
reagent that forms a colored complex specifically with urea and
the intensity of the color (measured at 520 nm) is directly
proportional to the concentration of urea in the sample. As little
as 5 µl of sample is needed to perform the assay and the linear
detection range is 0.006 mg/dl to 100 mg/dl (17 mM). The
creatinine assay is also a direct and simple procedure that
assesses kidney function by measuring creatinine directly in
blood. The procedure takes 20 min. This procedure also involves
a chromagenic reagent that forms a colored complex specifically
with creatinine and the intensity of the color (measured at
510 nm) is directly proportional to the concentration of
creatinine in the sample. The same amount (5 µl) is needed for
this assay and the detection range is 0.10 mg/dl to 100 mg/dl (8–

8.8 mM). Trunk blood was obtained from both genotypes at the
time of sacrifice at 8, 12, and 22 months of age (n=10 per group).
In addition, kidneys were dissected and measured along the
longitudinal axis (Fig. 1A), and were fixed with 10%
formaldehyde for sectioning and pathological evaluation.
Enzyme linked immunosorbent assay (ELISA)

GDNF levels were assessed using a commercially available
assay kit from Promega according to our standard protocol
(Albeck et al., 2003). Animals were sacrificed using a halothane
overdose, and the entire striatum dissected out (including medial
and lateral striatum, but excluding the nucleus accumbens) by
gently peeling back the overlying cortex and accessing the tissue
via coronal sections. In brief, flat-bottom plates were coated
with the corresponding capture antibody, which binds the
soluble captured neurotrophin. The captured neurotrophin was
bound by a second specific antibody, which was detected using a
species-specific antibody conjugated to horseradish peroxidase
as a tertiary reactant. All unbound conjugates were removed by
subsequent wash steps according to the Promega protocol. After
an incubation period with chromagenic substrate, color change
was measured in an ELISA plate reader at 450 nm. Using these
kits, GDNF can be quantified in the range of 7.8–500 pg/ml. For
each assay kit, cross-reactivity with other trophic proteins is <2–
3%. GDNF was expressed as pg/mg tissue.

Locomotor testing

GDNF+/- and WT mice were tested at various ages (4, 8, 12,
16, and 20 months of age) to evaluate the effects of a partial

GDNF deletion on motor activity throughout the animal's life
span. Locomotor activity (total distance traveled) was assessed
in a Digiscan Animal Activity Monitor system for 1 h
(Omnitech Electronics Model RXYZCM(8) TAO, Columbus,
OH). The details of the apparatus have been described previously
(Halberda et al., 1997). On the day of testing, the mice
were transferred from the animal colony into the laboratory in
groups of six and tested in a darkened environment. Data were
collected in 5-min intervals for 1 h at the same time of day (8 am
to 12 am) for each test period.

Accelerated rotarod

Motor coordination was evaluated using an accelerating
rotarod treadmill (Ugo Basile, Verese, Italy). The mice were
trained to remain on the rotarod for 10 min at a set speed of
4 rpm over a period of 2 days prior to actual testing. Mice
were then tested for their ability to remain on the rotarod at
increasing rotation speeds of 4, 8, 16, 24, 32, and 40 rpm.
During these tests, the animal was allowed a maximum of
5 min at each rotation speed and 5 min rest between each test.
Mice were tested for 3 consecutive days, and the results
averaged to obtain a single value for each animal at a given
rotational speed on a given day (as adapted from Rozas et al.,
1997).

Immunohistochemistry

For histochemical analysis, mice were anesthetized with
halothane and perfused transcardially with saline followed by
4% paraformaldehyde in phosphate buffer (0.1 M, pH 7.4). The
brains were removed, postfixed in 4% paraformaldehyde for 24
h, and then transferred to 30% sucrose in 0.1 M phosphate
buffered saline (PBS) for at least 24 h before sectioning. The
midbrain was sectioned on a cryostat (Microm, Zeiss, Thorn-
wood, NY, USA) at 45 µm. Every 3rd section throughout the
nigra region was processed for free-floating immunohistochemistry
using a polyclonal antibody against tyrosine hydroxylase
(TH, Pel-Freeze Inc., Roger, AZ, USA). Immunohistochemistry
was performed according to our standard protocol (Granholm
et al., 1997a,b; Zaman et al., 2003). Briefly, free-floating serial
sections were treated with H2O2, methanol, and 0.01 M Tris
buffer saline (TBS, pH 7.6; 1:2:7 respectively) for 15 min to
quench endogenous peroxidase activity. Sections were permeabilized
in TBST (tris buffer with 0.9% sodium chloride
and 0.25% TritonX-100) and treated for 20 min with sodium
m-peroxidate (0.1 M) in TBS. To block nonspecific binding
sites, sections were incubated in 10% normal goat serum
(NGS, Sigma) in TBST for 30 min at room temperature.
Sections were incubated with the primary antibody (1:1000) in
TBST with 3% NGS at room temperature for 24 h. Following
washing in TBST, sections were incubated with an appropriate
secondary antibody (1:200, Vector Labs, Burlingame, CA) and
incubated subsequently in the avidin–biotin complex (ABC
kit, Vector Labs). 3'3'-Diaminobenzidine (DAB, Sigma) was
used as a chromagen to develop the reaction using 0.05% of
3% H202. Nickel ammonium sulfate (2.5%, Sigma) was used


H.A. Boger et al. / Experimental Neurology 202 (2006) 336–347
Fig. 1. Kidney function and striatal GDNF levels with aging in GDNF+/- vs. WT mice. (A) Measurement of kidney sizes in 16 GDNF+/- and 13 wild-type control mice
at 12 months of age. There was no genotype difference. This included both left and right kidney. None of the animals lacked one kidney, suggesting that heterozygosity
for the GDNF gene is not detrimental to kidney function (homozygous mice lack both kidneys). (B) Creatinine levels in 8, 12, and 22 month old GDNF+/- and WT
mice (total n =58). There were no genotype differences at any age. (C) BUN levels in the same mice, showing that there was no alteration in BUN levels at any age
tested. (D) GDNF protein levels were decreased in GDNF+/- mice at all ages tested. GDNF+/- mice had a significant reduction in striatal GDNF protein levels
compared to age-matched WT mice (p <0.05). (E) Body weights (gram) for both genotypes at 4–21 months of age. As can be seen in this graph, there was no difference
between the groups in terms of initial growth or maintenance of body weight with age (*p < 0.05).

to enhance the reaction. Sections were mounted on glass slides
and coverslipped with DPX.

Stereological cell counts

Quantitative estimates of the total number of TH-ir neurons
in the substantia nigra (SN) were achieved using an unbiased,
stereological cell counting method (see Gundersen and Jensen,
1987; Granholm et al., 2002; Hunter et al., 2004a,b,c). It is
important to note that the cell counting included the entire A9
nucleus, but did not include A10 (ventral tegmental area,
VTA). Briefly, the optical fractionator system consists of a
computer-assisted image analysis system including a Nikon
Eclipse E-600 microscope hard-coupled to a Prior H128
computer controlled x–y–z motorized stage, an Olympus-750
video camera system, a Micron Pentium III 450 computer, and

stereological software (Stereoinvestigator®, MicroBrightField
Inc.; Colchester, VT). The SN was outlined under low
magnification (10×) on every 3rd section through the rostrocaudal
extent of the midbrain, and the outlined region was
measured with a systematic random design of disector counting
frames (100×100 µm). Actual mounted section thickness was
found to be 35–37 µm, and a 2 µm guard zone was set for the
top and bottom of each section. A 40× objective lens with a 1.4
numerical aperture was used to count cells within the counting
frames. Data were analyzed using 4×2 ANOVA with Fisher's
post hoc analysis.

Densitometry of the fiber density in pars reticulata

Since qualitative assessment of the SN region suggested
that there was a reduction in TH-immunoreactive neurites in


H.A. Boger et al. / Experimental Neurology 202 (2006) 336–347
the pars reticulata region in the older GDNF+/- and WT
groups, we performed densitometry measurements on subjects
from the 12 and 20 month old groups. Briefly, staining intensity
of TH immunostained neurites in the SN pars reticulata was
determined using NIH Image Software to measure a gray scale
value within the range of 0–256, where 0 represents white and
256 black. A template was created and used on all brains
similarly, and images were captured with a Nikon Eclipse E-600
microscope, an Olympus-750 video camera system, and a Dell
Pentium III computer. Measurements were performed blinded
and measurements from 4–6 sections per brain were averaged to
obtain one value per subject. The template used is outlined in
Fig. 3A. Staining density was obtained when background
staining was subtracted from mean staining intensities on every
6th section through the SN. Data were analyzed using a two-
way ANOVA with Fisher's post hoc analysis.

Statistical methods

The effects of age and genotype on motor activity were
assessed with linear regression models that included age,
genotype, and their interaction as covariates, and horizontal
activity as the dependent variable. One regression model treated
activity scores at ages 4, 8, 12, 16, and 20 months as repeated
measures. Since a primary interest was the impact of GDNF
reduction on changes in motor activity across age, a second
regression model was completed adjusting for genotype-related
differences at 4 months as a confounding covariate. Rotarod
data were analyzed using a random-effects regression model
that included age, genotype, rotarod speed, test day, and all
possible two-way and three-way interactions as fixed covariates,
and intercept as random effect in order to account for
correlation among repeated measures. A step-wise regression
procedure was used in the random-effects model fitting to
identify most influential covariates. Significant 3-way interactions
were resolved by first fixing one covariate involved in the
3-way interaction at any one of its levels, and re-fitting the
model that included only the 2-way interaction of the other two
covariates. This model fitting procedure was repeated for all
different levels of that fixed covariate to resolve the 3-way
interaction.

Results

Kidney function, weight gain, and survival rates of GDNF+/-
mice

Since GDNF is known to affect kidney function and
GDNF-/- mice suffer from kidney agenesis (see e.g. Pichel et
al., 1996), kidney morphology and function were examined at
different ages in GDNF+/- mice and controls. Kidney size
(Fig. 1A) and weight (Fig. 1E) were not found to be
significantly different between the groups. Parameters indicative
of kidney function (creatinine (CRT) and blood urea
nitrogen (BUN)) were compared between the two genotypes
at different ages (Figs. 1B and C), and were also found to be
normal, and within the normal range of C57BL/6J mice. In

terms of general weight gain and survival rates of GDNF+/-
compared to WT mice, we found that the two genotypes did
not differ from each other in body weight throughout the
study (see Fig. 1E). Both WT and GDNF+/- mice increased in
weight over the first 12 months and then leveled off to
maintain their weight for the remainder of the experiment (21
months of age, see Fig. 1E). Further, the mortality rate was
similar between the two groups and attrition reached a mean
of 16% in both groups by the age of 24 months. Thus, there
was no increased attrition rate or altered body weight with the
partial deletion of the GDNF gene.

GDNF protein levels

In order to determine whether GDNF+/- mice did indeed
have lower GDNF levels. We examined GDNF protein levels
using ELISA in the striatum of 4, 12, and 18–21 month old male
mice of both genotypes (Fig. 1D). At 4 and 12 months of age,
GDNF+/- mice exhibited a significant reduction (34–35%) in
striatal GDNF protein levels compared to age-matched WT
controls. In addition, striatal GDNF levels in mice ranging in
age from 18 to 21 months of age were significantly reduced in
GDNF+/- mice by displaying a 42% reduction compared to age-
matched WT mice. No age-related decreases in GDNF levels
were seen in either genotype.

Effects of a partial GDNF deletion on spontaneous motor
activity

Spontaneous motor activity for male GDNF+/- and WT
mice at four-month intervals from 4 to 20 months of age is
summarized in Fig. 2A, showing longitudinal measures of 10
GDNF+/- and 8 WT mice. At 4 and 8 months of age,
spontaneous locomotion of GDNF+/- mice was comparable to
that of age-matched WT mice (Fig. 2A). Between 8 and 12
months of age, GDNF+/- mice exhibited a 50% decline in
activity and were 54% below activity of age-matched WT
mice. WT mice did not exhibit a significant decline in motor
activity until 16 months of age, where they displayed a 36%
decline in activity. Two linear regression models were fitted
that included age, genotype, and age by genotype interaction
as covariates and motor activity as dependent outcome. The
first model treated motor activity measures generated by each
animal across time as a repeated measure. The second model
treated total motor activity measures at age 4 months as a
covariate and was adjusted in the model to explore the rate of
decline as a factor of aging and genotype. Neither model
yielded a significant age by genotype interaction (p >0.65 in
both models); however, motor activity was significantly lower
for GDNF+/- than for WT mice for each model (p < 0.017 in
both models). The absence of a significant interaction was
due to the large variation among different subjects within
groups. Regardless of age, WT mice had significantly higher
mean activity (393.0 ± 10.6 cm traveled over a 15-min
interval) compared to GDNF+/- mice (276.7±9.2 cm traveled
over a 15-min interval). A two-sided t test to compare these
two means yielded a p value <0.001.


H.A. Boger et al. / Experimental Neurology 202 (2006) 336–347
Fig. 2. Horizontal activity and accelerating rotarod performance was reduced
with aging in GDNF+/- vs. WT mice. (A) No difference existed in spontaneous
locomotion at 4 and 8 months of age between the genotypes. However, at 12
months of age, GDNF+/- mice displayed a reduction in locomotion compared to
the WT mice (p <0.01). Also, GDNF+/- mice exhibited a decrease in
spontaneous locomotion between ages 8 and 12 months (p <0.01). This
decrease in performance for GDNF+/- mice continued through the 20 month
time-point, suggesting an age-related deficit (p <0.01). (B) A three-way
interaction existed among age, genotype, and rotarod speed (p <0.0001). No
genotypic effect existed in the young animal group but a significant interaction
of genotype and rotarod speed did exist in the aged animal group (p =0.004).
After daily testing, the decline of time spent on the rotarod at the various speeds
became significantly less than earlier test days (p <0.001 for both age groups).
Both GDNF+/- and WT mice increased their performance at a similar rate
(p =0.2180), suggesting no effect of genotype on learning. However, a
significant speed by genotype interaction was still present on the last day of
testing in the aged mice (p =0.043) (*p <0.05, **p <0.01).

Accelerating rotarod performance in GDNF+/- mice

To assess the impact of a partial GDNF deletion on motor
coordination and learning, young (8 month old) and aged (20
month old) male GDNF+/- and WT mice were compared on
their ability to remain on the accelerating rotarod device (see
Materials and methods, Fig. 2B). These data were fitted with a
random effects model that included age, genotype, rotarod
speed, test day, and all their two-way and three way interactions
as fixed covariates and intercept as random effect. The three-
way interaction among age, genotype, and rotarod speed was
highly significant (p <0.0001). To further explore the impact of
age, speed, and genotype on the rotarod time, we fitted
separated random-effects models to different subsets of animals

(i.e. to fix the level of age). The subsets were the young (8
months) and aged (20 months) mice. A Step-wise procedure
was used in model selection. In the final models, the genotype
effect was not significant (p =0.60) for the young group. In
contrast, for the aged animal group, genotype was significant
through its interaction with the rotarod speed (p =0.004). The
time mice remained on the rotarod as the speed increased
declined more rapidly for GDNF+/- compared to WT mice.
Additional analysis indicated that with continued practice (Day
3, Fig. 2B), the GDNF+/- mice overcame this deficiency to
some extent (i.e. on the third test day GDNF+/- mice remained
on the rotarod for a longer period of time as speed increased
than was observed on earlier test days (p <0.001 for both age
groups). Both GDNF+/- mice and WT mice increased their
performance at a similar rate since the genotype did not interact
with the test days (p =0.2180). However, when we examined
rotarod time at the last test day, significant speed by genotype
interaction was still present in aged animals (p =0.043) but not
in young animals. Again, no genotype effect was found in
young animals (p =0.37).

Effects of GDNF loss on TH immunohistochemistry

The SN from male GDNF+/- and WT mice was processed
for TH immunohistochemistry at ages 4, 8, 12, and 20 months
in order to quantify the morphological effects of a partial GDNF
loss. Since the motor activity study in Fig. 2A was longitudinal,
morphological studies included different sets of mice for each
age (10 WT, 10 GDNF+/-) except for the oldest group. Sample
morphology from each group is shown in low magnification
and in high magnification (Fig. 3), and cell counts of total
number of TH-ir neurons per animal and density of TH-ir
neurites in the SN pars reticulata are summarized in Fig. 4.

Morphological alterations in SN

Inspection of Fig. 3 suggests that there was no loss of TH-ir
in the nigra region in young GDNF+/- mice compared to WT
mice at the two earliest ages studied (4 and 8 months of age;
Figs. 3A–H). However, beginning at 12 months of age and
continuing in the oldest group, there was a more pronounced
TH cell loss with aging in GDNF+/- than in WT mice, even at
the oldest age (20 months) (Figs. 3I–P). At higher magnification
(Fig. 3), signs of degeneration can be observed in the nigra of
GDNF+/- mice at the older ages, such as cytoplasmic
displacement, dystrophic neurites, and axonal swellings in
some cases (see especially Figs. 3O, P). The total number of
TH-positive neurons decreased between 8 and 12 months of
age, in the male GDNF+/- mice, a change not observed to the
same extent in male WT mice. This reduction in TH positive
neurons was more apparent in the medial than the lateral SN,
even though the low magnification micrographs indicate an
overall loss of fiber density and cell bodies in both medial and
lateral aspects of SN (Fig. 3). WT mice also exhibited an age-
related loss of TH-positive neurons in the SN, but to a lesser
extent than the heterozygous mice (Fig. 4A).

Stereological cell counts of TH-ir neurons

There was no significant difference in the total number of TH
positive neurons in the SN between genotypes in the 4 month


H.A. Boger et al. / Experimental Neurology 202 (2006) 336–347
Fig. 3. Dopamine neuron degeneration in 12–20 month old GDNF+/- mice. Tyrosine hydroxylase (TH) immunohistochemistry of midbrain sections from WT (A, E, I,
M) and GDNF+/- (C, G, K, O) mice at low magnification. Note that at 4 months (A, B) and 8 months (C, D) of age, no morphological differences exist between the two
genotypes. However, at 12 months of age (I–L), GDNF+/- mice exhibit a decrease in TH-ir neurons in the SNpc as well as a decrease in fiber density of the SNpr
compared to WT mice. The effects of a partial depletion of GDNF are further evident at 20 months of age (K, L, O, P). The boxes indicate the high magnification
images shown in panels B, D, F, H, J, L, N, and P and the tracing in panel A represents the template used to measure density of TH staining in the SNpr (see Fig. 4B).
From the images in this figure of higher magnification, degenerative alterations both in fiber morphology and cell body morphology are evident, particularly in panels
L and P (12 and 20 month old GDNF+/- mouse) (scale bar in panel O: 200 µm; scale bar in panel P: 50 µm).

old mice and 8 month old mice (Fig. 4A). However, the 12
month old GDNF+/- mice had a significant decrease in SN TH
positive neurons compared to the WT controls, resulting in a
mean decline of 19% (Fig. 4A, n =10, p <0.01). The decrease in
the number of TH positive neurons between genotypes
continued at the 20 month old time point with a 15% reduction
(n =10, p <0.05). In addition, the 12 month old GDNF+/- mice
had a 19.7% reduction of total TH positive neurons compared to
8 month old GDNF+/- mice (n =10, p <0.05), unlike the WT
mice where there was no reduction in total TH cell counts
between these two ages. The number of TH positive neurons
was further decreased 14.3% in 20 month old GDNF+/-
compared to the 12 month old GDNF+/- mice (p <0.05), thus
suggesting a progressive aging effect (Fig. 4A). However, the
WT mice did not display a decrease in total TH cell counts until
20 months of age with a 20% reduction (p <0.01). Overall, with
aging, the GDNF+/- mice exhibited a 35.71% decrease in total

SN TH positive neurons from 4 to 20 months of age, whereas
the WT mice demonstrated a 28.6% decrease in total cell
counts. Data fitted according to linear regression using age and
genotype as covariates revealed no age by genotype interaction
(p =0.956); however, TH positive neuron cell count decreased
across age (p < 0.01), and was lower for GDNF+/- mice than for
WT mice (p <0.01).

Densitometry of TH-ir fibers in the substantia nigra pars
reticulata

The staining density for TH immunoreactivity was measured
in the pars reticulata of the SN in 12 and 20 month old WT and
GDNF+/- mice. These age groups were chosen since there was a
visible difference in fiber density in this brain region compared
to the younger animals (see Fig. 3). We found that there was a
significant decrease in staining density in the GDNF+/- mice


H.A. Boger et al. / Experimental Neurology 202 (2006) 336–347
Fig. 4. Reduction in total number of TH immunoreactive neurons and nerve fiber
densities in SN of GDNF+/- mice. (A) Unbiased stereological cell counts of
midbrain TH immunoreactive neurons. No difference existed in the number of
TH-ir neurons between GDNF+/- and WT mice at 4 and 8 months of age.
Between 8 and 12 months, the GDNF+/- mice displayed a 19.7% decline
(p <0.05) in SN TH-ir neurons. The number of TH-ir neurons was further
decreased 14.3% in 20 month old GDNF+/- mice (p <0.05), whereas, the WT
mice displayed an age-related loss between 12 and 20 months of age with a 20%
reduction (p <0.01). In addition, GDNF+/- mice at 12 months had a 19% decline
of TH-ir neurons when compared to age-matched WT mice (p <0.01). The
decreased number of TH-ir cells between genotypes continued through the 20
month time-point with a 15% decline (p <0.05). (B) TH densitometry in the
SNpr in 12 and 20 month old mice revealed a significant decrease in relative TH
density in GDNF+/- mice compared to WT mice at 12 months of age (p<0.01).
Both groups were significantly reduced compared to 12 month WT mice at the
20 month time point (*p <0.05, **p <0.01).

compared to age-matched WT mice (p <0.01; see Fig. 4B).
However, both aged groups (20 months) were reduced in
staining density compared to the WT 12 month old group
(p <0.05). Mean relative TH staining density values for the 12
month old WT and GDNF+/- mice were 55±2 and 41±4,
respectively and for the 20 month old animals they were 33±4
and 36±6, respectively.

Discussion

In the present study, we found that male GDNF+/- mice
exhibited a progressive decline in spontaneous motor activity,
motor coordination, and SN TH-immunoreactive neurons, over
the course of a year that differed from WT mice. The
deterioration in these two measures was beyond that observed
with normal aging in WT mice. In addition to the abnormalities
noted in the present experiment, GDNF+/- mice have a loss of
locus coeruleus noradrenergic (LC-NE) neurons (Zaman et al.,
2003), as well as a cognitive decline with age reported by others
(Gerlai et al., 2001). Other investigators (Airavaara et al., 2004)

demonstrated increased dopaminergic postsynaptic activity in
GDNF+/- mice at an early age, suggesting compensatory
alterations in dopaminergic pathways. In contrast to these deficiencies,
we did not find altered kidney function in GDNF+/-
at any age, and weight gain and survival rate were similar
between the two genotypes as well.

GDNF is required for ureter bud formation and branching
during metanephros development of the kidney, and is essential
for proper innervation of the gastrointestinal tract. Thus, mice
lacking both copies of GDNF lack kidneys and die at birth
(Pichel et al., 1996), whereas the loss of one allele for GDNF
results in approximately 30% fewer but normal sized glomeruli
in young mice at postnatal day 30 (Cullen-McEwan et al.,
2001). In 14 month old GDNF+/- mice, the same authors found
that the animals continued to have 30% fewer glomeruli but that
the remaining glomeruli were larger. This increase in glomeruli
size provided an adaptation such that kidney filtration rate was
normal in these animals (Cullen-McEwan et al., 2003). Data
from the current experiment demonstrate that both kidney size
and BUN/creatinine levels in serum from mice of all ages
confirmed these findings by Cullen-MCEwan et al. (2003), and
further demonstrated that normal kidney function was sustained
throughout this experiment (up to 22 months of age), possibly
by adapting to the decreased glomerular size reported in fetal
heterozygous mice (Cullen-McEwan et al., 2001). We previously
reported that one kidney was often hypotrophic in fetal
GDNF heterozygous mice (Pichel et al., 1996), but our data and
those from Cullen-McEwan (2001, 2003) suggest that this
atrophy is compensated for in continued development, to lead to
two full-sized kidneys in adult life. In addition to the kidney
abnormalities observed in fetal GDNF knockout mice (Pichel et
al., 1996), the absence of GDNF prevents differentiation of
ameloblasts and odontoblasts, and the enamel matrix and
predentin layers are absent (de Vicente et al., 2002). A recent
study by Shen et al. (2002) has also shown that some GDNF+/-
mice may suffer from hypoganglionosis in the enteric ganglia
and intestinal obstruction. Curiously, our mouse strain does not
appear to have long-lasting detrimental effects of this defect
since the weight gains the same between both groups through 22
months of age, when the last animals were sacrificed. We have
also studied food intake in our mice and not seen any reduction
in food consumption between WT and GDNF+/- mice, thus
suggesting that they may overcome these difficulties later in
life. In addition, GDNF+/- mice have no increased mortality at
least up to 24 months of age, compared to WT littermates
(unpublished observations). Thus, it is unlikely that abnormal
kidney function or other peripheral dysfunctions can account for
the alterations in TH expression and motor behavior observed in
the present experiment, even though these peripheral neuropathies
are interesting and deserve attention in future studies.
Another possible mechanism for the motor dysfunction
observed here is altered function of spinal cord and brainstem
motor neurons, since these have GDNF receptors and are
susceptible to GDNF treatment after various injuries (see e.g.
Klein et al., 2005). Although this possibility is interesting and
cannot be excluded, it will have to be explored in future studies.
One would assume that if deficient motor neurons gave rise to


H.A. Boger et al. / Experimental Neurology 202 (2006) 336–347
the altered motor behavior reported here, this difference
between GDNF+/- mice and controls would remain throughout
life, and this is not the case in our studies. In addition, our
findings regarding spontaneous locomotion and rotarod performance
are quite comparable to the deficits in these behavioral
measures observed after a targeted MPTP lesion in mice (see

e.g. Rozas et al., 1997, 1998), suggesting at the very least that
the decrease in TH expression observed here and the reduction
of DA levels (Boger et al., submitted for publication) may play a
role in these behavioral deficits which occur in the GDNF+/-
mouse.
Earlier reports indicate that locomotor activity declines in
both rodents and primates during normal aging (see e.g.
Emborg et al., 1998; Emerich et al., 1993; Hebert and
Gerhardt, 1998; Schulz and Huston, 2002). The age-related
reduction of spontaneous locomotion observed in the WT mice
in the present study is similar to what has been reported for this
mouse strain (C57Bl/6J) in previous publications (Dean et al.,
1981). GDNF+/- mice exhibited a greater rate of motor decline
than WT mice in the present study. Their activity declined
several months earlier than that of WT mice. The data suggest
that GDNF is involved in the regulation of spontaneous motor
function at least in the GDNF+/- mice, either via one or several
transmitter systems, and that this influence becomes more
pronounced with age. It is not known yet why the WT mice
also declined in terms of their motor function at the later age
(20 months) to reach the activity levels seen in GDNF+/- mice.
It is plausible that this decline in motor activity may relate to
the loss of TH immunoreactivity, and hence also DA neuron
dysfunction, which occurs also in WT mice in the oldest group,
or a loss of other factors which naturally decline with aging.
Our data do not suggest that this age-associated decline in
motor activity and TH immunoreactivity in WT mice is
directly associated with a loss of striatal GDNF in the WT
mice, since GDNF levels were similar to those seen in the
young WT mice throughout the ages studied. It is of course
possible that with continued aging (>22 months of age), wild-
type mice will also undergo a significant decrease in GDNF
levels, similar to what has been seen in aged rats previously
(Yurek et al., 2001) and in other mouse models, such as the
senescence accelerated mouse (Miyazaki et al., 2003). However,
other investigators have not seen altered striatal GDNF
mRNA levels in 24 month old mice as part of normal aging,
supporting the findings in the present study (Blum and
Weickert, 1995). Further studies should include the relationship
between age-associated decrease in TH immunoreactivity,
motor dysfunction, and other factors which may be altered in
the aged WT mouse.

The rotarod is widely used to assess motor coordination in
experiments to evaluate motor injury, pharmacological treatments,
or genetic manipulations (see e.g. Fenton et al., 1994;
Wagner and Walsh, 1987; Hamm, 2001; Smith and Stoops,
2001). In the present study, rotarod performance was influenced
by a three-way interaction of age, genotype, and rotarod speed.
Motor coordination deteriorated with age and speed of rotation
for both genotypes, although significantly more so in the
GDNF+/- than in the WT mice. Performance improved across

days of testing suggesting that motor learning could occur in all
groups, albeit to a lesser degree in 20 month old GDNF+/- mice.
The highly significant reduction in performance is not
surprising, since motor coordination is one of the first
behavioral components known to deteriorate with normal
aging in rodents (see e.g. Bickford et al., 1992). However, it
is important to note that aged WT mice were able to learn the
task by day 3 to the same extent as the young WT mice, leaving
a defect in the highest speed only in aged GDNF+/- mice. It is
possible that impairment in this particular behavioral task
occurs at a later age in normal WT mice than that studied in the
present experiments (20 months of age). The fact that GDNF+/-
mice were able to learn at least to some extent suggests that they
were motivated enough to perform the task, and had the basic
abilities related to the task. Thus, if the GDNF+/- mice had
major peripheral disabilities that prevented them from performing
this task, they would not have improved on days 2 and 3.
The neuronal substrate for this deficit could be related to
noradrenergic as well as DAergic neurotransmission, since both
of these systems have been implicated in this particular
behavioral task (Bickford, 1995; McEntee et al., 1987; Mason
and Iversen, 1977). Since this mouse model exhibits loss of both
LC-NE neurons (Zaman et al., 2003) and TH immunoreactivity
in the SN (present study), either, or both of these transmitter
systems could mediate the motor deficit. Targeted delivery of
GDNF to either LC or SN neurons in GDNF+/- mice would
reveal if one or both of these systems are involved, and this will
be the focus for future studies.

DAergic pathways are especially susceptible to the effects of
normal aging (McGeer et al., 1977; Carlsson, 1987; Seeman
et al., 1987; Kish et al., 1992). In the present study, WT mice
exhibited a small but significant decline (20%) of TH-ir neurons
with age, as previously reported for this mouse strain (McNeill
and Koek, 1990; Tatton et al., 1991). GDNF+/- mice were
distinguishable from WT mice with a greater overall decline
(35.7%) of TH-ir neurons which occurred earlier in the life
span, between 8 and 12 months of age rather than 12–20
months as seen in WT mice. In further studies, we have now
also demonstrated that GDNF+/- mice have a significant
reduction in striatal DA levels at 12 months of age as evidenced
by HPLC, further supporting the findings reported here for TH
immunoreactivity (see Boger et al., submitted for publication),
indeed suggesting that SN DA neurons are severely dysfunctional
in mice with a partial GDNF deletion. Thus, TH-
immunoreactive cell bodies decline; TH-immunoreactive neurites
become more sparse in the pars reticulata of the SN and DA
levels are reduced in the striatum. All of these are hallmarks for
DA neurodegeneration. Together, these results suggest that
GDNF may be involved in maintenance of this neuronal
population, especially during aging. Future studies will
determine if neurons eventually die or actually just loose their
phenotype.

Collectively, the GDNF heterozygous mouse has alterations
in motor function and DA morphology as aging progresses, and
eventually these alterations are also evidenced with normal
aging in the WT mice. We propose that this genetic predisposition
may set individuals up for increased vulnerability when


H.A. Boger et al. / Experimental Neurology 202 (2006) 336–347
presented with an environmental toxin and that a combination of
a genetic predisposition and extrinsic factors when an individual
is young can increase aging-related vulnerability of midbrain
dopamine neurons. Other research groups have also proposed a
multifactorial ethiology of PD (see e.g. Steece-Collier et al.,
2002). Thus, we hypothesize a “dual-hit” hypothesis of
neurodegeneration in which a combination of factors during
early pre-or postnatal development enhances the vulnerability
of DA neurons leading to altered neuronal functioning during
aging. In line with this conjecture, we recently obtained
evidence that a toxic methamphetamine binge (10 mg/kg IP X
4, every 2 h) in 2.5 month old animals leads to more detrimental
long-term behavioral and morphological problems in GDNF+/-
mice compared to WT controls (Boger et al., submitted for
publication). This animal model will thus provide an opportunity
to determine if a genetic predisposition coupled with a
neurotoxin may further increase the vulnerability of this
neuronal population, resulting in motor disorders during
middle-age or aging. This becomes important as our population
ages, and it has been shown that GDNF levels are decreased in
SN neurons of PD patients (Jenner and Olanow, 1998).
Perhaps decreased efficiency of the GDNF system represents a
genetic predisposition for some individuals for development of
clinical PD. Further studies in humans will be needed to
explore this possibility.

In conclusion, the present study demonstrated that a life-long
reduction of GDNF leads to progressive neurodegenerative
alterations in the nigrostriatal system and accelerated age-
associated deficits. Evaluation of open-field locomotor testing
and performance on the accelerating rotarod revealed that the
GDNF+/- mice had deficient motor function and coordination
with age compared to WT mice. Animal models of PD with
acute DA lesion paradigms, such as 6-OHDA and MPTPlesioned
animals, also exhibit lower spontaneous locomotion,
even though this deficit develops rapidly after the lesion
(Przedborski et al., 2004; Cass et al., 2005; Tamas et al., 2005;
Deumens et al., 2002). Thus, the present transgene may
represent an interesting model for motor dysfunction in animals,
as the deficit developed much slower than that seen in the lesion
models, and can thus be used for prevention or rescue studies
that extend over several months, mimicking the situation in
humans more closely. Stereological cell counts and measurements
of TH-ir fiber densities and DA levels indicated that with
aging, the GDNF+/- mice had an accelerated decrease in nigral
TH expression and reduced levels of DA in the striatum (Boger
et al., submitted for publication). These data provide evidence
for a role for GDNF in the maintenance of the nigrostriatal
DAergic system, and also provide a novel animal model for
therapeutic intervention, both in terms of prevention and rescue,
of aging DA neurons.

Acknowledgments

This work was made possible by USPHS grant AG023630
and a grant from the US Army (DAMD#17-99-1-9480). The
authors would like to thank Mr. Alfred Moore and Ms. Jeanetta
Smith for expert technical assistance.

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here's another about protein -

Protecting Neurons from Parkinson’s
New insights into the disease’s protein culprit
Katherine Bourzac, SM
November 14, 2006(Technology Review) - MIT researchers led by Susan Lindquist, a biology professor and member of the Whitehead Institute for Biomedical Research, have developed a way to protect neurons from degeneration and death in animal studies of Parkinson’s disease. The research, which focused on a protein called alpha-synuclein, could lead to therapies for human Parkinson’s.
The disease’s characteristic tremors and muscle rigidity are caused by damage to and the death of neurons that use the neurotransmitter dopamine to communicate with neighboring neurons. Alpha-synuclein was known to be one of the main causes of that damage; large clumps of it, in a misfolded form, are found in the brains of Parkinson’s patients. But researchers did not know what alpha-synuclein’s normal role is, why Parkinson’s neurons accumulate too much of it, or how it causes disease. Lindquist’s team used a yeast model of Parkinson’s to study these questions.

Their research suggests that alpha-synuclein plays a role in the process cells use to shuttle proteins between two internal compartments in which critical refinements to proteins are made. Before being shipped off to different parts of the cell, protein strings often need to be cut or folded into three-dimensional shapes, and sometimes groups such as carbohydrates must be added to them. During these processes, the young proteins are sheltered within protective lipid bubbles. The bubbles also protect the neurons that produce dopamine from damage that can occur if too much dopamine leaks out.

"Dopamine must be packaged in these membranes and sequestered from [the insides of the cell], where it can cause oxidative damage," says Aaron Gitler, a postdoc in Lindquist’s lab.

The researchers aren’t sure exactly how buildup of misfolded alpha-*synuclein disrupts protein trafficking but suspect it disturbs these lipid *bubbles. Gitler and Lindquist suggest that as a result, neurons in Parkinson’s patients are unprotected from their own dopamine, which thus becomes toxic.

The scientists searched for a way to interfere with this effect. Gene screening showed that activating the gene ypt1, which makes a protein that helps shepherd other, freshly made proteins from one part of the cell to another, did the job: the Parkinson’s yeast lived. Rab1, the equivalent shepherding protein in nematode, fly, and rat neurons, also countered alpha-synuclein’s toxicity. Rab1 did not completely eliminate neuron death in some of these higher organisms, but it was protective.

Much remains to be done, validation in tests on mice being the most important step. But the Whitehead results have left researchers optimistic about getting at the molecular details of Parkinson’s. A complex disease with few treatment options, Parkinson’s affects about a million people in the United States. This research represents an important step toward understanding and curing it.