1 Unité de recherche en génétique moléculaire,
Institut de recherches cliniques de Montréal (IRCM), Montréal,
QC H2W 1R7, Canada
2 Cone Laboratory, Department of Neurology and Neurosurgery, Montréal
Neurological Institute, McGill University, Montréal, QC H3A 2B4,
Canada
3 Laboratoire de neurobiologie, Centre de recherche, CHUM Hôpital
Notre-Dame, Université de Montréal, Montréal QC H2L 4M1,
Canada
4 Centre de recherche, CHUM Hôpital Saint-Luc, Université de
Montréal, Montréal QC H2X 3J4, Canada
Authors for correspondence (e-mail:
drouinj{at}ircm.qc.ca
and
sadikot{at}bic.mni.mcgill.ca)
Accepted 4 March 2003
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SUMMARY |
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Key words: Homeobox, Transcription factor, Pitx3 (Ptx3), Midbrain, Dopamine
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INTRODUCTION |
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MesDA neurons are located in the ventral midbrain and form the substantia
nigra (SN) and ventral tegmental area (VTA). Differentiation and anatomical
localization of MesDA neurons are dependent on the action of various
diffusible factors and transcription factors. MesDA neurons develop at sites
where the signals of sonic hedgehog (Shh) and Fgf8 intersect, both being
necessary and sufficient for induction of DA neurons
(Ye et al., 1998). Before
expression of DA-specific markers, early ventral midbrain markers like
En1/2, Lmx1b, Pax2/5 and Wnt1 are expressed in these cells
(Hynes and Rosenthal, 1999
;
Smidt et al., 2000
). The
appearance of the key enzyme in DA synthesis, tyrosine hydroxylase (TH) at
embryonic day 11.5 (E11.5) of mouse development shortly follows expression of
the orphan nuclear receptor Nurr1 (Nr4a2 Mouse Genome Informatics)
(E10.5) and of the homeobox gene Pitx3 (E11). The expression of
Nurr1 is not restricted to MesDA neurons and extends in a large field
in the mesencephalon and diencephalon
(Zetterstrom et al., 1996
).
Nurr1-null mice fail to induce TH in MesDA progenitor neurons and die
soon after birth (Zetterstrom et al.,
1997
). Whether these progenitors are lost during late fetal
development or maintained postnatally is not entirely clear yet
(Saucedo-Cardenas et al.,
1998
; Witta et al.,
2000
).
Pitx3 expression is, at the brain level, confined to MesDA neurons
and is maintained throughout adult life in both rodents and humans
(Smidt et al., 1997).
Extraneural Pitx3 expression was shown in the eye, where it is
present in the developing lens (Semina et
al., 1997
). In humans, mutations of the PITX3 gene have
been found in two families with inherited forms of cataracts and anterior
segment mesenchymal dysgenesis (Semina et
al., 1998
). Similarly, abnormal eye lens development was observed
in a naturally occurring mouse mutant, the aphakia (ak)
mouse, which has two 5' deletions in the Pitx3 gene
(Rieger et al., 2001
),
including one that deletes exon 1.
We show that Pitx3 is only expressed in the ventral tier of the SN pars compacta (vSNc) and in about half of the VTA DA neurons. In ak mice, we show undetectable midbrain Pitx3 expression, selective degeneration of vSNc DA neurons, as well as of roughly half VTA neurons and greater than 90% decrease in dorsal striatal DA levels in association with marked reduction in spontaneous locomotor activity. The strong correlation between Pitx3-expressing TH neurons and neuronal losses in ak mice or in individuals with PD suggests that Pitx3 defines the neuronal population that is more susceptible to degeneration in PD. ak mice thus represent a highly specific mouse model of neuronal loss in human PD.
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MATERIALS AND METHODS |
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Brain preparation and immunohistochemistry
Male P1, P21, P50 and P100 wild-type and ak mice were
transcardially perfused with buffered 4% paraformaldehyde. Brains were
collected, postfixed for 24 hours and embedded in paraffin (P50) or
cryoprotected in 30% sucrose for an additional 48 hours (P1, P21 and P100).
P50 midbrain-containing sections (5 µm) were mounted and immunostained for
TH and Pitx3. P1, P21 and P100 brains were cut into 50 µm coronal sections
encompassing the entire striatum and midbrain using a freezing microtome.
Free-floating sections were collected for immunohistochemistry as separate
sets so that each set contained every third serial section. One set of
sections was immunostained for TH, another set was processed using 0.1% Cresyl
Violet as a Nissl stain. Rostrocaudal position of sections was assessed with
the aid of the mouse brain atlas of Franklin and Paxinos
(Franklin and Paxinos, 1997).
For embryos, pregnant mothers were perfused transcardially with 4%
paraformaldehyde. Embryos were dissected and their heads were postfixed for 24
hours and embedded in paraffin wax. Midbrain-containing sections (5 µm)
were mounted and immunostained for TH.
Immunostaining was performed using an avidin-biotin-peroxidase complex
(ABC) method and a fluorescein/rhodamine-fluorochrome labeling method.
Antibodies and dilutions used: anti-Pitx3
(Lebel et al., 2001), 1:10;
anti-TH (Chemicon polyclonal 1:100); anti-TH (Immunostar monoclonal 1:1000).
Confocal microscopy was performed using a Zeiss LSM510 instrument. Apoptotic
cells were identified using the Apoptag kit from Intergen according to the
manufacturer's recommendations. Percentage apoptotic cell was calculated
relative to nuclei counted on Nissl-stained sections.
Stereology and quantitative morphology
Unbiased estimates of midbrain DA neurons were obtained using the optical
dissector method of West and Gundersen
(West and Gundersen, 1990;
West, 1993
). The entire
rostrocaudal extent of the midbrain was examined in a 1:3 series of TH-stained
coronal sections using an Olympus BX-40 microscope equipped with a motorized
XYZ stage and StereoInvestigator software (Microbrightfiel). The SN and VTA
were traced at low power (10x). TH cell counts were performed at
100x magnification (oil, NA 1.3) using a 60x60 µm counting
frame. A 10 µm dissector was placed 2 µm below the surface of the
section at counting sites located at 150 µm intervals after a random
start.
Cell densities within SNc and VTA were determined in Cresyl Violet stained sections delineated according to adjacent TH-stained sections. Nissl-stained profiles greater than 7 µm in diameter were counted. Total profile counts were then divided by SNc or VTA surface area estimated with the StereoInvestigator software.
Locomotor activity measurements
Male wild-type and ak mice of 115 days of age were maintained
in standard animal housing conditions with a 12 hour light-dark cycle and
lights on at 6 am. Tests were carried out between 4 pm and 3 pm the next day.
At 3.30 pm, mice were placed in the 43x43 cm Plexiglas arena of the
Opto-Varimex-3 photocell-base monitor (Columbus Instruments) with water and
food freely available, and recordings started 30 minutes later. The
Opto-Varimex-3 animal activity monitor employs a 15x15 photocell beam
grid to measure spontaneous ambulatory and stereotypic activities like
grooming, scratching and other non-ambulatory activities (as well as the
amount of time spent on these activities) by separating beam interruptions
associated with ambulatory activity from total activity.
Dopamine quantitation
Male wild-type and ak mice of 130 days of age were analyzed
for postmortem tissue content of DA. After cervical dislocation, brains were
cut into 1 mm sections on an ice-cold dissection plate; dorsal and ventral
striatum were collected from two sections per brain with a biopsy punch (0.5
mm diameter). Homogenization of brain samples and DA quantitation by
reverse-phase HPLC with electrochemical detection were done as described
previously (Ste-Marie et al.,
1999
). Protein content was determined using the BCA assay in order
to normalize dopamine context.
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RESULTS AND DISCUSSION |
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These data indicate that early differentiation of MesDA neurons is not highly dependent on the Pitx3 gene, as shown in ak mice that carry a strongly hypomorphic (and possibly null) allele of this gene. However, survival of Pitx3-expressing MesDA neurons requires significant Pitx3 expression. Most sensitive are the vSNc neurons that are severely depleted by birth in ak mice, in contrast to those of the VTA that are lost later.
Striatal dopamine deficiency
SN dopaminergic neurons project primarily to the dorsal striatum to
regulate motor control, whereas VTA dopaminergic neurons project to the
ventral striatum and modulate emotional behavior
(Björklund and Lindvall,
1984). The impact of MesDA neuronal depletion in ak mice
was assessed by immunohistochemical staining of striatal TH fibers
(Fig. 3A,B) and high pressure
liquid chromatography (HPLC) measurement of striatal DA levels
(Fig. 3C,D). A dramatic
reduction of dopamine-mediated innervation was observed in the dorsolateral
striatum of ak mice, with relative sparing in the ventral striatum
(compare Fig. 3B with 3A).
Corresponding striatal DA levels were reduced by 93% in the dorsal striatum
and by 69% in the ventral striatum (Fig.
3C,D).
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Reduced spontaneous locomotor activity
We then determined whether ak mice display altered locomotor
behavior by measuring spontaneous ambulatory and stereotypic activities over
23 hour periods using a photocell grid counter. During the day when mice are
normally less active, no differences were observed between groups
(Fig. 4A-E). However,
ak mice showed a marked reduction in ambulatory
(Fig. 4A) and stereotypic
(Fig. 4D) activities during the
night, as they walked 71% less than wild type
(Fig. 4B), spent 69% less time
walking (Fig. 4C), made 53%
less stereotypic movements (Fig.
4E) and spent 44% less time making stereotypic movements
(Fig. 4F). Conversely, they
spent 38% more time resting (Fig.
4G,H). In view of the ak mice eye defect, it is
interesting to contrast the reduction of spontaneous movement in ak
mice with the effects of gene mutations that eliminate circadian rhythms, such
as mutations of the Clock or Per1 and Per2 genes
(King et al., 1997;
Zheng et al., 2001
). The
latter result in loss of diurnal rythmicity, but not in reduction of total
movement per 24 hour period as observed in ak mice. Moreover, the
speed of spontaneous ambulatory movements was not different in ak
compared with wild type (Fig.
4I), suggesting that the ak mutation and the associated
blindness do not impair peripheral motor function. These results indicate that
ak mice display marked akinesia.
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Finally, the dependence on Pitx3 for survival of Pitx3-positive TH neurons
and the sensitivity of Pitx3-positive MesDA cells to degenerate in PD
(Smidt et al., 1997) suggest
that Pitx3-dependent function(s) may relate to the pathogenesis of human PD.
Such function or downstream target gene(s) may contribute to control cell
survival/death in development and/or in pathogenesis of the MesDA system.
Further investigation of developmental defects resulting from Pitx3 deficiency
may provide novel insight into disease pathways involved in PD.
Pitx3 gene mutations may be involved in the etiology of diseases
that affect the MesDA system. So far, two PITX3 mutations have been identified
in families with autosomal-dominant cataracts and autosomal-dominant anterior
segment mesenchymal dysgenesis (Semina et
al., 1998). These individuals are not known to have parkinsonian
symptoms. It is noteworthy, however, that both mutations have dominant effects
in individuals that still have an intact PITX3 allele. Because a midbrain
phenotype may not be expected in hemizygous carriers, as heterozygous
ak mice do not exhibit any phenotype
(Varnum and Stevens, 1968
)
(data not shown), it is likely that these human mutations cause a dominant
effect that may for example, impair eye-specific protein:protein interactions
(Semina et al., 1998
). This
would be consistent with their position in the N or C termini of PITX3 rather
than in the homeodomain that has been implicated in many loss-of-function
mutations in the related PITX2 gene
(Amendt et al., 1998
). Thus, it
would be worthwhile to investigate whether PITX3 allelic polymorphism can be
detected in families with Parkinson's disease.
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ACKNOWLEDGMENTS |
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Footnotes |
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