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Developmental Brain Research 149 (2004) 131–142

Research report

Parkin expression in the developing mouse

Kati Kuhna, Xin-Ran Zhua, Hermann Lubberta,b, Christine C. Stichelb,*

aDepartment of Animal Physiology, ND5/132, Ruhr-University of Bochum, D-44780 Bochum, GermanybBiofrontera Pharmaceuticals GmbH, D-51377 Leverkusen, Germany

Accepted 18 February 2004

Abstract

Parkin is an E3 ubiquitin ligase causally involved in the pathogenesis of autosomal recessive juvenile parkinsonism. In this paper,

we analysed the formation of alternative splice products and the spatio-temporal expression pattern of parkin during pre- and postnatal

mouse development. Using RT-PCR, Northern blot, in situ hybridization, Western blot analysis, and immunohistochemistry we found (i)

alternative splice forms of parkin; (ii) an early and widespread expression of parkin mRNA and protein in the CNS and several organs,

already at E10/12; (iii) a marked increase in expression level during midgestational development (E15–18) in the CNS, followed by a

steady increase until adulthood; (iv) an ubiquitous distribution throughout CNS ontogeny. Our results show that parkin expression is

correlated with cell maturation and suggests an important physiological role of parkin in neurons that is at no time limited to the

dopaminergic system.

D 2004 Elsevier B.V. All rights reserved.

Theme: Cellular and molecular biology

Topic: Gene structure and function: general

Keywords: Parkinson’s disease; Ontogeny; Gene expression

1. Introduction type ubiquitin (Ub) protein ligase (E3) collaborating with a

The most common forms of familial Parkinson’s disease

are autosomal recessive (AR-JP) and characterized by selec-

tive and massive loss of dopaminergic neurons in the sub-

stantia nigra of the midbrain, and absence of Lewy bodies

[1,2,21,26].

Mutations in the parkin gene are responsible for many

cases of AR-JP. The gene consists of 12 exons encoding a

protein of 465 amino acids. The 52 kDa protein is a RING-

0165-3806/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.devbrainres.2004.02.001

Abbreviations: Ad, adult; AFG, Aldehydfuchsin–Goldner procedure;

AR-JP, autosomal recessive form of Parkinson’s disease; BSA, bovine

serum albumine; CASK, calcium/calmodulin-dependent serine protein

kinase; Cb, cerebellum; Cx, cerebral cortex; DG, dentate gyrus; E,

embryonic; Hi, hippocampus; IHC, immunohistochemistry; ISH, in situ

hybridization; PBS, phosphate-buffered saline; PD, Parkinson’s disease;

PFA, paraformaldehyde; Pi, piriform cortex; Pn, pons; SDS, sodium dodecyl

sulfate; SSC, saline sodium citrate; ST, striatum; TBS, Tris-buffered saline;

TDL, triple-detergent-lysis buffer; Te, tectum; Th, thalamus

* Corresponding author. Tel.: +49-234-32-25829; fax: +49-234-32-

14189.

E-mail address: stichel@biofrontera.de (C.C. Stichel).

Ub-conjugating enzyme (E2) belonging to a cognate class of

UbcH7 or UbcH8 [28,34]. To date, various deletions, point

and intronic splice site mutations along with a promoter

variation have been described in AR-JP patients [16,20,

33,35]. Analysis of these parkin mutations in AP-JP patients

reveals that the functional loss of parkin as an E3 enzyme is

the molecular basis of AR-JP [15].

Recent studies suggest that parkin is expressed ubiqui-

tously in the adult brain of both vertebrates and inverte-

brates [6,7,12,27,29,31,32,38]. Immunohistochemistry and

in situ hybridization analysis revealed that parkin is pre-

dominantly found in neurons and only single glial and

endothelial cells express the protein within the central

nervous system. Interestingly, parkin expression is not

confined to the nervous system. Northern blot analysis

identified parkin transcript in systemic organs like heart,

testis, liver and kidney [18]; the cellular distribution patterns

in these organs remains unknown.

Parkinson’s disease (PD) is a slowly progressing disease

and neurodegeneration starts much earlier than the onset of

symptoms in adulthood. Since the neurodegenerative pro-

cess might start in development, it is crucial to study the

K. Kuhn et al. / Developmental Brain Research 149 (2004) 131–142132

expression of genes that are already causally related to the

disease during ontogeny. But detailed knowledge of parkin’s

expression and localization during brain and organ devel-

opment is not yet available. To date there is only one report

about the regional and cellular distribution of parkin mRNA

in the developing rat brain [36]. In developing mice, one

study has analysed the distribution pattern of parkin protein

[14] and a short paper has described the transcription in total

brain using Northern blot analysis [18].

In order to further analyse the kinetics and distribution of

parkin gene expression from early developmental stages to

adulthood, we studied the expression patterns of parkin

during mouse ontogeny. This knowledge may contribute

to clarify potential physiological functions and roles in the

pathogenesis of PD.

2. Materials and methods

2.1. Animals

Experiments were conducted on C57BL/6 mice (Janvier,

France) and all animal experiments were conducted and

approved using the German guidelines of the animal care

and use committee. Developmental ages stated in the paper

follow the convention that refers to the day after insemina-

tion as embryonic day 1 (E1) and the day of birth as

postnatal day 1 (P1).

Mice were analysed at embryonic (E) days E10, E12,

E14, E15, E18 and postnatal (P) days P1, P7, P14, P28 and

8 weeks (Ad). Male mice were preferred for analysis.

The animals were housed in a 12:12 h light/dark cycle

with free access to food and water. Ambient temperature

was maintained at 24 jC.

2.2. Tissue preparation

For in situ hybridization (ISH) either brains (P1, P7, P14,

P28, Ad) or whole embryos/fetuses (E10, E12, E14, E15,

E18) were quickly removed and frozen in methylbutane at

� 50 to � 60 jC. Native tissue was cryocut and 30 Amsections were mounted on Superfrost slides (Menzel-

Glaeser, FRG). After postfixation for 1 h in 4% parafor-

maldehyde, sections were washed twice in phosphate-buff-

ered saline (PBS), dehydrated, air dried and stored until

usage at � 80 jC.For histological stainings whole embryos/fetuses were

postfixed overnight (ov) in 4% paraformaldehyde (PFA) in

PBS and embedded in paraffin. Sagittal paraffin sections (30

Am) were mounted on slides and stored at room temperature

(RT).

2.3. Histology

To identify organs and define brain areas, some sec-

tions of each developmental stage were stained by an

Aldehydfuchsin–Goldner (AFG) procedure according to

Blum et al. [3].

2.4. Total RNA isolation

Brains and bodies of prenatal stages E10, E12, E14 and

E15 as well as brains of developmental stages E18, P1, P7,

P14, P18 and Ad were used for total RNA preparation.

Tissues were homogenized with 1 ml TRIzol (Invitrogen,

FRG) per 100 mg tissue and incubated for 5 min at RT.

After adding 0.2 ml chloroform per ml TRIzol, the organic

TRIzol-containing phase and the phase containing total

RNA were separated by centrifugation in a precold centri-

fuge for 10 min at 13.000 rpm. The colourless supernatant

was removed and total RNAwas precipitated by adding iso-

propanol 1:1 followed by a second centrifugation step. The

RNA pellet was washed in 70% ethanol, air-dried and

dissolved in DEPC-water for subsequent reaction.

To remove DNA contamination from the total RNA

preparation, DNase digestion was done for 15 min at 37

jC in a 500 Al reaction containing 17.5 Al 1 M Tris–HCl pH

8.0; 7.5 Al 1 M Tris–HCl pH 7.0; 200 units RNase inhibitor

(Promega, FRG), 50 units RNase-free DNase (Roche,

FRG), and 5 Al 1 M MgCl2.

2.5. Northern blot

Total RNA, isolated from the head of E10, E12, E14,

E15 or the brain of E18, P1, P7, P14, P28 and Ad mice as

well as from the body of E10, E12, E14 and E15 mice, was

electrophoretically separated (25 Ag per lane) on a 1.5%

agarose gel containing 2.2% formaldehyde. After transfer to

a nitrocellulose membrane (Macherey and Nagel, FRG), the

RNAwas covalently linked to the dried membrane at 80 jCfor 2 h. The membrane was prehybridized for at least 3

h with hybridization buffer (50% formamide, 5� saline

sodium citrate (SSC), 5� Denhardt’s, 0,2% sodium

dodecyl sulfate (SDS)) containing 200 Ag/ml denaturated

salmon sperm DNA at 42 jC. Approximately 50 ng parkin

PCR product (corresponding to nucleotides 55–1478, acc.

No.AF250293) was labeled with 50 ACi [a32P]dCTP

(Amersham, USA) for 15–30 min at 37 jC following the

manual instructions of Amersham Megaprime DNA Label-

ing Kit (Amersham). Free nucleotides were removed by

Sephadex G-50 columns (Amersham). Hybridization was

carried out at 42 jC overnight. After hybridization, mem-

brane was washed several times in washing buffers 2�SSC, 0.1% SDS and 0.2� SSC, 0.1% SDS. The nitrocel-

lulose membrane was exposed to Kodak MS film.

2.6. RT-PCR analysis

For cDNA synthesis 3 Ag of total brain RNAwas reverse

transcribed using oligo-dT primers following the manual

instructions of the ‘‘First strand cDNA synthesis kit’’ (MBI

Fermentas, FRG).

Fig. 1. Schematic presentation of parkin splice variants detected by RT-

PCR. (A) Parkin gene. Exons are indicated as boxes and are numbered 1–

12. Numbers above exonic structure indicate nucleotide position. (B) Parkin

splice variant (Stichel et al. [31]) with exon 1–6 and PA2 cassette (striped

box). Lines below gene structures represent the positions and expected

fragment lengths of PCR products PP1, PP2, PP3 (A) and PP4 (B).

K. Kuhn et al. / Developmental Brain Research 149 (2004) 131–142 133

The composition of PCR reactions was identical in

probes of all developmental stages independent of primer

pairs (PP1–4; Fig. 1) and annealing temperature. A typical

20 Al PCR reaction contained 2 Al cDNA, 2 Al 10� PCR

buffer with ammonium sulfate (MBI Fermentas), 1.5 mM

magnesium chloride, 2 AM of each primer, 0.4 AM dNTP

mix and 0.0625 U Taq-DNA polymerase (MBI Fermentas).

We performed the PCR with 35 cycles.

To avoid generation of unspecific PCR products that

often appear during long extension steps, we divided parkin

cDNA in three parts. Four primer pairs were used at four

different annealing temperatures to amplify short fragments

of parkin cDNA (see Table 1). These fragments overlap and

therefore allow us to detect all potential deletions between

exons 1 and 12, and the generation of a transcript with the

mPA2 cassette.

Electrophoretical separation of the RT-PCR fragments

was performed on 1.5% agarose gels running at 60–100 mA

for 1–1.5 h.

2.7. Generation of riboprobes

Riboprobes for ISH were directly generated from RT-PCR

products. The amplified cDNA sequence (corresponding to

nucleotides 391–839 acc. No.AF250293) was cloned and

checked by sequence analysis. Primers mPA-ISH-7 and

mPA-ISH-5 contained the T7 promoter sequence (Table 2).

Table 1

Primers used for RT-PCR analysis

Fragment Accession

numbers

Sense primer

PP1 AF250293 exon 1 for 5V-cgcgtaggtccttctcgacc-3VPP2 AF250293 mPA-ISH-6 5V-gagtccaggagcttgacacgagt-3VPP3 AF250293 exon6 for 5V-gcatagcgtgcacagatgtc-3VPP4 AF250295 mPA-ISH-6 5V-gagtccaggagcttgacacgagt-3V

Labeled riboprobes were generated with T7 polymerase

(Roche) in the presence of digoxygenin (DIG)-labeled

nucleotides.

The quality of the DIG-labeled riboprobes, their approx-

imate size and the transcription efficiency was checked by

agarose gel electrophoresis followed by Northern blotting

and subsequent immunodetection using the anti-DIG anti-

body tagged with alkaline phosphatase (anti-DIG-AP;

1:5000; Roche, FRG).

2.8. In situ hybridization (ISH)

To localize parkin transcripts, in situ hybridization (ISH)

was performed on native cryostate sections as described by

Wisden and Morris [37].

Native cryosections (30 Am) were quickly unthawed and

rehydrated followed by proteinase K digestion (10 Ag/ml

proteinase K in 20 mM Tris–HCl pH 7.0, 5 mM EDTA pH

8.0) for 10 min at 37 jC and fixation in 4% PFA for 10 min

on ice. After several washing steps in DEPC-water, sections

were acetylated in 0.1 M triethanolamine hydrochloride,

0.25% acetic anhydrate and 25 mM NaOH for 10 min at RT.

Hybridization was carried out on dehydrated air dried

sections overnight at 45 jC with parkin riboprobes diluted

1:200 in hybridization buffer containing 50% formamide,

4� SSC (1� SSC = 0.15 M NaCl and 0.015 M sodium

citrate, pH 7.0), 50 mM NaH2PO4 pH 6.5, 250 Ag/ml

denaturated salmon sperm DNA, 100 Ag/ml tRNA, 5%

dextransulfate and 1� Denhardt’s solution. After several

washing steps (2� SSC two times at RT, 2� SSC con-

taining 50% formamide for 15 min at 50 jC, 0.1� SSC

containing 50% formamide for 15 min at 50 jC, two times

0.1� SSC for 15 min and a blocking step in 1% blocking

reagent (Roche) for 1 h at RT, slides were incubated with

sheep anti-DIG-AP antibody (1:2000) overnight at 4 jC.Digoxygenin detection was performed by incubation with

nitroblue tetrazolium (NBT 0.24 mg/ml, Roche) and 5-

bromo-4-chloro-3-indolyl phosphate (BCIP 0.125 mg/ml,

Roche) in 0.1 M Tris, 0.1 M NaCl, 0.05 M MgCl2 at pH 9.5.

The reaction was stopped in TE buffer (0.01 M Tris, 0.01 M

EDTA pH 8.0). Antisense and sense probes were used under

identical conditions. Significant differences in hybridization

signals were observed between the signal detected with

antisense probes and the signal with sense probes in all

developmental stages and all regions examined. The signal

of the sense probes almost matched that of the background.

Antisense primer Annealing

temperature (jC)

exon6 rev 5V-aagggatgctgcgcctgttgc-3V 65

exon7 rev 5V-catcgtggagaaactgccgatc-3V 64

exon12 rev 5V-tgatctcccatgcaggctcg-3V 62

mPA2 5V-tcttggagaagctaagcggt-3V 58

Table 2

Primers used for the generation of riboprobes

Probe Sense primer Antisense primer

Parkin as mPA-ISH-6 5V-gagtccaggagcttgacacgagt-3V mPA-ISH-7 5V-ttgtaatacgactcactatagggtacagggctcctgacatctgtg-3VParkin s mPA-ISH-5 5V-ttgtaatacgactcactatagggtgagtccaggagcttgacacgagt-3 mPA-ISH-8 5V-acagggctcctgacatctgtg-3V

K. Kuhn et al. / Developmental Brain Research 149 (2004) 131–142134

2.9. Protein extraction and Western blots

Mouse tissues were homogenized at 4 jC either in lysis

buffer 1 (0.32 sucrose, complete TM Protease inhibitor,

Roche) for h-actin, parkin and calcium/calmodulin-depen-

dent serine protein kinase (CASK) or in Triple-Detergent-

Lysis (TDL) buffer (50 mM HEPES, 150 mM NaCl, 25 mM

EDTA, 1% Nonidet P40, 0.5% Na-deoxycholate, 0.1% SDS

and protease inhibitor cocktail tablets (Roche) for h-actinand synphilin-1 Western blot. Samples were chilled on ice

for 30 min and centrifuged at 14.000 rpm in a precooled

centrifuge. Supernatant was diluted 1:1 in modified 2�Laemmli sample buffer (125 mM Tris–HCl pH 6.8, 6%

SDS, 10% h-mercaptoethanol, 30% glycerin) and boiled for

5 min at 100 jC.For electrophoretical separation, 50 Ag protein for h-

actin, parkin, CASK or 5 Ag protein for h-actin and

synphilin-1 were loaded per lane on 8% or 10% polyacryl-

amide gels and electrophoresed at 20 mA for 1–2 h.

Proteins were transferred to polyvinylidene difluoride mem-

branes (PVDF; Millipore, UK) and unspecific binding was

blocked with 3% milk powder containing 2% BSA in TBS-

Tween buffer (0.1% Tween, 20 mM TBS, 137 mM NaCl,

pH 7.6) for 1 h.

Incubation with the first antibody (rb anti-parkin 1:2000;

ms anti-h-actin 1:100, (Sigma); rb anti-synphilin-1 1:1000

(Abcam, UK), or ms anti-CASK 1:1000 (BD Transduction,

UK)) diluted in blocking buffer took place overnight at 4 jCand was followed by several washing steps in TBS-Tween

buffer. The rb anti-parkin antibody was raised against a

synthetic peptide corresponding to residues 71–91 (exon 3)

of predicted murine parkin [31]. Incubation with the sec-

ondary HRP-coupled antibody (Amersham; 1:25.000 in 1%

milk powder, 0.5% BSA in TBS-Tween buffer) took place

for 1.5 h at RT. Finally the membrane was washed and

developed by using the ECL + plus detection system (Amer-

sham). In some cases, the same membrane was used for

incubation with different primary antibodies. For stripping,

membranes were incubated in Restore Western Blot Strip-

ping buffer (Pierce, USA) prior to incubation with the next

primary antibody.

2.10. Immunohistochemistry (IHC)

For single labeling, native 30 Am cryostat sections were

fixed in 4% PFA. After washing in 0.01 M PBS buffer, tissue

sections were incubated with 3% normal goat serum (NGS)

at RT for 1 h, followed by an incubation with the first

antibody rb anti-parkin (1:2000, overnight; [31]). Primary

antibody was detected using biotinylated secondary antise-

rum (1:300 in 0.01 M PBS; 45 min/RT; Vector Laboratories,

Burlingame, CA) and the avidin-biotinylated peroxidase

complex (1:200 in 0.01 M PBS; 45 min/RT, Vector Labora-

tories) and visualized with 7% 3,3Vdiaminobenzidine tetra-

hydrochloride (DAB, Sigma). For enhancement of the

staining, a silver intensification method was performed as

described [30]. Primary antibody specificity was confirmed

by antigen absorption tests and negative controls, as

published [31].

2.11. Data analysis

Brain sections were visualized at the microscopic level

(Axioskop 2; Zeiss, FRG) under brightfield illumination.

Images were captured with an imaging system (Sony

MC3255 camera) connected to a computer equipped with

an image program (KS100 Rel.3.0). For final output, images

were processed using the Photoimpact 4 software.

Structures were described according to the main subdi-

visions of the brain and were identified with the aid of the

Paxinos and Franklin [22] and Kaufman [17] atlases for

adult and developmental neuroanatomy, respectively.

3. Results

3.1. RT-PCR analysis of parkin splice variants

Ten different developmental stages were analysed for the

onset of parkin expression and the formation of alternative

splice variants using RT-PCR analysis. With all primer pairs

we were able to amplify parkin transcript from E10 onwards

until adulthood (Fig. 2). DNA sequence analysis of the PCR

products confirmed the expected nucleotide sequence de-

fined by the primer pairs.

Independent of the primer pair used, parkin expression

levels increased from E10/E12 (head preparations) to much

higher levels in the brain preparations of the older devel-

opmental stages (Fig. 2).

Besides a large parkin transcript, smaller fragments

showed up in some developmental stages (Fig. 2). Primer

pair 1 (PP1, Fig. 2) amplified several smaller fragments of

513–700 bp (a, h, g) at the postnatal stages. DNA se-

quencing determined that two of these fragments (h, g)

carry deletions of exon 2 or exon 3, respectively (Table 3).

Reamplification of the a-fragment resulted in two PCR

products fragments (h, g), indicating that the a-fragment

is a complex between the h and g fragments. Primer pairs 2

Fig. 2. RT-PCR analysis of alternative splicing during pre- and postnatal brain development. Upper bands represent the full-length PCR products, while splice

variants below are faint and marked by Greek symbols with fragment sizes in brackets.

Table 3

Parkin mRNA isoforms detected by RT-PCR

Primer pair Fragment

(size, bp)

cDNA

size (bp)

Alternative

spliced form

PP1 h (589) 3135 without exon 2

PP1 g (513) 3060 without exon 3

PP2 y (433) 3183 without exon 6

PP3 q (522) 3146 without exon 9

PP4 A (664) 1420 full-length mPA2,

[31]

PP4 k (548) 1303 without exon 6

K. Kuhn et al. / Developmental Brain Research 149 (2004) 131–142 135

and 3 (PP2, PP3; Fig. 2) amplified an additional very weak

fragment of 433 (y) and 522 bp (q), respectively, in P1

preparations. The 433 bp fragment lacked exon 6, while the

522 bp fragment represented the parkin sequence without

exon 9 (Table 3). Primer pair 4 was used to analyse the

expression of a previously described alternative, very short

transcript (mPA2, AF250294; [31]) during ontogeny. Using

this primer pair, we detected two fragments (A, k), whichconsist of exons 1–5 and 1–6, respectively, followed by a

putative new exon. The intensity of the lower band was

weak in prenatal stages but increased around birth and

remained high during postnatal development.

3.2. Northern blot analysis of parkin mRNA expression

To quantify changes in expression of parkin mRNA and

its potential splice variants, we performed Northern blot

analysis.

Labeled parkin DNA probe revealed a band of approx-

imately 3.3 kb from E12 onwards (Fig. 3). This band

corresponds to the native full-length transcript of mouse

parkin mRNA. Although the expression level was low until

E15, we noted two periods of increase in the parkin mRNA

signal. First, the parkin mRNA level increased abruptly

between E15 and E18, followed by a smaller gradual

increase postnatally. Thereafter, the parkin mRNA signal

intensity remained high until adulthood. No smaller bands

were detected at either developmental stage.

3.3. Western blot analysis of parkin protein and its binding

partners

To complement the mRNA expression pattern with

protein data, we performed Western blots of protein

extracts from head or brain tissues with a polyclonal

antibody against parkin [31]. Blots were stripped and

incubated with h-actin antibody to control protein loading

and with anti-synphillin1 or anti-CASK to follow the

developmental expression of a substrate and a binding

partner of parkin.

Fig. 3. Northern blot analysis of parkin gene expression during pre- and postnatal development. Total RNAwas prepared from brain (br), head (h) or body (b) at

different ontogenetic stages. Equal amounts of RNA (25 Ag) were loaded per lane. Parkin full-length transcript has a size of approximately 3.3 kb. Parkin gene

expression appeared as early as E12 in head as well as in body preparations with a strong increase between E15 and E18 in head, followed by a slight increase

during postnatal stages in brain. Northern blots showed no splice variant.

K. Kuhn et al. / Developmental Brain Research 149 (2004) 131–142136

The parkin antibody detected a major band of about 50

kDa and some fainter bands of higher and lower molecular

weights (Fig. 4A). The strong band corresponds to the

predicted molecular weight of mouse parkin, while the faint

bands may represent either post-translational modifications

of the protein [11] or unspecific binding of the antiserum.

Western analysis revealed that parkin protein appears at

embryonic day E14. The protein level increased significant-

ly between E15 and E18 and remained high until adulthood

(Fig. 4A). The h-actin Western blots confirmed that constant

amounts of protein were loaded per lane (Fig. 4B). The

slight decrease in h-actin during early development is a

well-described phenomenon [19] and does not indicate

variations in protein loading.

Fig. 4. Western blot analysis of parkin (A), h-actin (B), synphilin (C) and CASK (D

with the primary antibodies, the blot was stripped and incubated in antibody to h

Synphilin1, an a-synuclein interacting protein [9], which

is ubiquinated by parkin [5], appeared much later during

development. We detected a strong band at the expected size

of 90 kDa from postnatal day 7 onwards (Fig. 4C). The

expression level at earlier stages was very low.

CASK is a PDZ-protein that may function in targeting or

scaffolding parkin [10]. This protein is constantly expressed

from E14 onwards (Fig. 4D).

3.4. Developmental expression patterns of parkin mRNA

and protein

To examine the regional distribution of parkin mRNA

and protein during ontogeny we used in situ hybridization

) expression during pre- and postnatal brain development. After incubation

-actin to check for equal loading.

Fig. 5. Expression profiles of parkin mRNA (ISH) and protein (IHC) in different organs. Lung and liver displayed strong hybridization signal already at E12.

While in these two organs, the signal disappeared before birth, in kidney and testis, parkin mRNA was expressed until adulthood. Hybridization with sense

probe resulted only in low background staining. While mRNA expression data coincided with those of protein expression in testis and kidney, lung failed to

display parkin immunostaining, and the liver showed protein expression at time points when mRNA expression had disappeared. Scale bars, 200 Am.

K. Kuhn et al. / Developmental Brain Research 149 (2004) 131–142 137

K. Kuhn et al. / Developmental Brain Research 149 (2004) 131–142138

and immunohistochemistry. We stained whole mice of

embryonic stages (E12, E14, E15 and E18), some organs

of adult mice, and brains at all postnatal stages (P1–Ad).

Every tenth section was processed with an AFG-staining for

the identification of brain regions and organs.

Already at embryonic day 12 (E12), we noted parkin

mRNA expression in all organs analysed (Figs. 5 and 6).

In the periphery, liver and lung displayed a strong but

transient hybridization signal between E12 and E15 (Fig. 5),

while the signal in kidney and testis persisted until adult-

hood (Fig. 5). The latter also displayed a strong parkin

protein staining in adulthood (Fig. 5). For liver and lung, a

discrepancy between mRNA and protein expression was

Fig. 6. In situ hybridization (ISH) and immunohistochemistry (IHC) show the dis

postnatal development. Native cryosections were processed for non-radioactive

respectively, or reacted with a rb anti-parkin antibody. Note that mRNA and protein

the white matter regions. In the adult brain, both mRNA and protein became promi

under identical conditions. Scale bars, 1000 Am. Cb, cerebellum; Cx, cerebral cort

striatum; Te, tectum; Th, thalamus.

observed. Although in early prenatal development, parkin

mRNA was prominent in both organs, no immunostaining

of parkin protein was detected at these developmental stages

(Fig. 5). Parkin protein was undetectable in lung cells even

at later developmental stages, while the liver displayed

distinct parkin immunoreactivity from E15 onwards when

mRNA expression declines.

In the brain, parkin mRNA expression was faint at E12,

increased in intensity at E14 and was prominent until

adulthood (Figs. 6 and 7). The mRNA expression was

accompanied by a strong parkin immunoreactivity from

E12 onwards (Figs. 6 and 7). Parkin mRNA and protein

were widely distributed at all stages examined. However,

tribution of parkin mRNA and protein in the mouse brain during pre- and

in situ hybridization with parkin-specific antisense and sense riboprobes,

were distributed widespread in grey matter regions of the brains, but spared

nent in certain brain nuclei. The sections were photographed and reproduced

ex; DG, dentate gyrus; Hi, hippocampus; Pi, piriform cortex; Pn, Pons; ST,

Fig. 7. Expression patterns of parkin mRNA (ISH) and protein (IHC) in four brain regions at different postnatal ages. In the substantia nigra, the striatum, the

cerebral cortex and the hippocampus, many cells displayed strong hybridization signal and immunostaining. At birth, the signals appeared diffuse but became

more cellular in later postnatal stages. Scale bars, 100 Am.

K. Kuhn et al. / Developmental Brain Research 149 (2004) 131–142 139

the density of mRNA signal varied among the brain regions.

At E12, high levels of signals appeared in germinative

ventricular zones of the basal forebrain and in the thalamus

(Fig. 6). From E14 onwards, discrete nuclei appeared in the

fore- and midbrain (Fig. 6). At the same time points, parkin

protein was detected. Similar to mRNA distribution parkin

protein was prominent in grey matter regions (Figs. 6 and

7). However, in contrast to mRNA the protein level was

K. Kuhn et al. / Developmental Brain Research 149 (2004) 131–142140

homogeneous in all brain sub-regions during prenatal de-

velopmental. At postnatal stages, the patterns of mRNA and

protein expression were almost identical to those in adult

brain (Figs. 6 and 7). During this time period they did not

show conspicuous variations in regional distribution or

expression level. Stainings were prominent in grey matter

compartments and absent in fiber tracts. In general, parkin

transcript and protein localization coincided. Both mRNA

and protein were particularly prominent in the hippocampus,

in the cerebellar nuclei, in several brain stem nuclei, the red

nucleus and in the piriform cortex (Figs. 6 and 7); regions

with either high neuronal density and/or large neurons.

4. Discussion

Information about the expression pattern of the parkin

gene is a requisite for studying the role of this gene in the

pathogenesis of PD. Therefore, we analysed the expression

of parkin during pre- and postnatal development of the CNS

and of some organs in the mouse. The main findings of this

study are: (i) the formation of small amounts of alternative

parkin splice variants; (ii) an early and widespread expres-

sion of parkin mRNA and protein in the CNS and several

organs already at E10/12; (iii) a marked increase in expres-

sion level during midgestational development (E15–18) in

the CNS followed by a steady increase until adulthood; (iv)

an ubiquitous distribution throughout CNS ontogeny.

4.1. Splice variants

By use of RT-PCR with various different primer pairs, we

recovered seven potential parkin mRNA splice variants at

very low abundances. Four of them (h, g, y, q) are the

results of exons missing, while the a PCR product could not

be cloned. Since the reamplification of the a-band revealed

PCR products of previously reported fragment sizes (h, g),we assume the formation of strong secondary structures

between the smaller fragments h and g. Another possibility

might be that the alpha PCR product is contaminated by

trace amounts of beta and gamma products. PCR products Aand k represent short truncated mRNAs consisting of exons

1–5 and 1–6, respectively, followed by a new exon. In the

Northern blot, we nevertheless detected only one band of

3.3 kb. Since all isoforms except two (A and k) give rise tomRNAs that are similar in size to the full-length parkin

mRNA (Table 3) and hence could be hidden by the major

band in the Northern blot, and all are present at very low

abundances which may be undetectable by Northern blot-

ting, they may be difficult to visualize by this technique.

Similar to our results, a recent publication [4] described a

large number of alternative splice forms in rat brain, but

neglected to perform Northern blot analysis.

Similarly, we could not unequivocally demonstrate the

existence of splice variants on the protein level. The parkin

band was clearly visible, and upon overexposure of the blot,

additional bands appeared as well, but most of them were

larger than the parkin band which rendered it difficult to

assign these bands to the differentially spliced mRNAs. On

the other hand, it is known that large heteronuclear (hn)

RNA is especially susceptible to splicing errors. The parkin

gene spans a region of approximately 1 mbp on chromo-

some 6 and includes small exonic and very large intronic

sequences. It must therefore be taken into consideration that

the multitude of differentially spliced transcripts described

by different authors may simply be due to splicing errors

[4,18,31,32].

Taken together, our results implicate the formation of

various alternative splice variants of parkin at very low

levels. It is not clear to which extent the differentially

spliced transcripts give rise to proteins and whether this

has a functional significance.

4.2. Spatio-temporal expression pattern

Both RT-PCR and in situ hybridization (ISH) revealed

that parkin mRNA is expressed in early gestational brain

development. RT-PCR showed a strong band already at E10,

while Northern blotting exhibited a distinct band from E12

onwards. This discrepancy might result from differences in

method sensitivity. In accordance with the mRNA data, we

detected parkin protein on the Western blot already at E10.

These results are in agreement with the Northern data

published by Kitada et al. [18] and the Western/IHC analysis

by Huynh et al. [14] in the mouse brain. The early onset of

expression of both mRNA and protein may indicate that

parkin is relevant for neuronal development.

Northern and Western blotting exhibited two phases of

expression changes. Between E15 and E18, parkin mRNA

levels undergo a steep increase, followed by a gradual,

slight upregulation postnatally. The phase of steep mRNA

increase appears to coincide with the most expansive phase

of proliferation in the brain [17,25]. It is paralleled by a

significant increase of parkin protein, as shown in the

Western blot. These findings support previous observations

in the mouse brain that showed a significant increase in

mRNA [18] and protein [14] at the same developmental

period.

The period of mRNA upregulation was not accompanied

by a change in distribution pattern. In every developmental

stage, parkin transcript was widely distributed in all brain

regions and almost exclusively expressed in neurons. More-

over, the intense staining of the neuroepithelial cells sur-

rounding the ventricles provides evidence that parkin

already appears in proliferating neurons. The latter obser-

vation, however, does not correspond to our IHC data and

those described by Huynh et al. [14]. This discrepancy

between transcript and protein levels might be explained

by the initially low level of protein that impedes detection

via immunohistochemistry.

Neurons continue to express parkin mRNA and protein

throughout postnatal development up to adulthood. More-

K. Kuhn et al. / Developmental Brain Research 149 (2004) 131–142 141

over, the parkin level increased slightly in postnatal ontog-

eny, a period when neuronal proliferation is already com-

pleted [25]. During the same period, our Western analysis,

in agreement with previous immunohistochemical studies in

rats [13,24], indicate a strong upregulation of synphilin1, a

parkin substrate, and a slight increase of CASK protein, an

interacting partner of parkin. These data underline the

functional interaction of these proteins.

It is interesting to note that parkin expression levels vary

among different organs. In kidney, testis and brain, parkin

mRNA and protein expression persisted up to adulthood,

while parkin mRNA expression in lung and liver ceased

before birth. Surprisingly, we failed to detect any parkin

immunoreactivity in the lung at any developmental stage.

Reasons for this lack of protein staining might be the

masking of the epitope or expression of the g splicing

variant, which lacks exon 3, the binding site of our poly-

clonal antibody. In the liver, parkin protein was detectable

by immunohistochemistry at a time when in situ hybridiza-

tion staining had already disappeared. This phenomenon

was also described for a-synuclein and synaptic vesicle

proteins in the developing rat brain [8,23] and indicates a

long half-life of the protein, at least at this time point.

Taken together, the spatio-temporal expression patterns

during ontogeny suggest a role for parkin in neuronal and

also in peripheral organ maturation. The strong but ubiqui-

tous distribution in the brain at all developmental stages

implicates an important physiological role in neurons that is

at no time point limited to the dopaminergic system.

Acknowledgements

The authors thank Petra Jergolla, Holger Schlierenkamp

and Katrin Schuster for their expert technical assistance.

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