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Human Molecular Genetics Advance Access originally published online on November 20, 2006
Human Molecular Genetics 2007 16(1):50-60; doi:10.1093/hmg/ddl439
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© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Pael receptor induces death of dopaminergic neurons in the substantia nigra via endoplasmic reticulum stress and dopamine toxicity, which is enhanced under condition of parkin inactivation

Yasuko Kitao1,*,{dagger}, Yuzuru Imai2,{dagger}, Kentaro Ozawa1, Ayane Kataoka2, Toshio Ikeda3, Mariko Soda2, Kazuhiko Nakimawa4, Hiroshi Kiyama4, David M. Stern7, Osamu Hori1, Kazumasa Wakamatsu6, Shosuke Ito6, Shigeyoshi Itohara3, Ryosuke Takahashi2,5,{dagger} and Satoshi Ogawa1,{dagger}

1 Department of Neuroanatomy, Kanazawa University Medical School, 13-1, Takara-machi, Kanazawa City, 920-8640 Ishikawa, Japan, 2 Laboratory for Motor System Neurodegeneration and 3 Laboratory for Behavioral Genetics and RIKEN Brain Science Institute (BSI), Saitama 351-0198, Japan, 4 Department of Anatomy and Neurobiology, Osaka City University, Graduate School of Medicine, Osaka, Japan, 5 Department of Neurology, Kyoto University Medical School, Kyoto, Japan, 6 Department of Chemistry, Fujita Health University School of Health Sciences, Aichi 470-1192, Japan and 7 Dean's Office, College of Medicine, University of Cincinnati, Cincinnati, OH 45267, USA

* To whom correspondence should be addressed. Tel: +81 762652162; Fax: +81 762344222; Email: kitao{at}nanat.m.kanazawa-u.ac.jp

Received August 2, 2006; Revised October 19, 2006; Accepted November 13, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Selective loss of dopaminergic neurons is the final common pathway in Parkinson's disease. Expression of Parkin associated endothelin-receptor like receptor (Pael-R) in mouse brain was achieved by injecting adenoviral vectors carrying a modified neuron-specific promoter and Cre recombinase into the striatum. Upregulation of Pael-R in the substantia nigra pars compacta of mice by retrograde infection induced endoplasmic reticulum (ER) stress leads to death of dopaminergic neurons. The role of ER stress in dopaminergic neuronal vulnerability was highlighted by their decreased survival in mice deficient in the ubiquitin-protein ligase Parkin and the ER chaperone ORP150 (150 kDa oxygen-regulated protein). Dopamine-related toxicity was also a key factor, as a dopamine synthesis inhibitor blocked neuronal death in parkin null mice. These data suggest a model in which ER- and dopamine-related stress are major contributors to decreased viability of dopaminergic neurons in a setting relevant to Parkinson's disease.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Though Parkinson's disease (PD) is a major contributor to disability and death in the aging population, the molecular basis of selective dopaminergic neuronal toxicity is still under investigation. Progression of the clinical syndrome associated with PD, which includes a well-characterized movement disorder, correlates closely with inexorable loss of neurons in the substantia nigra pars compacta (SNpc) (1).

Mutations in the Parkin gene (2) have been identified in the autosomal recessive form of PD (AR-JP), a major cause of juvenile PD. Parkin is a 465 amino acid polypeptide with properties of an ubiquitin-protein ligase (E3) whose N-terminus displays homology to ubiquitin and C-terminus is comprised of two RING fingers flanking a cysteine-rich domain, termed in between RING fingers (IBR) (3–5). Consistent with this view, AR-JP-linked parkin mutants are defective in E3 activity (6–8).

A putative G protein-coupled transmembrane polypeptide, Pael Receptor [Parkin-associated endothelin-receptor like receptor (Pael-R)], has been identified as a Parkin substrate (9). Expression of Pael-R in cultured cells results in accumulation of unfolded, insoluble and ubiquinated Pael-R in the ER, eventuating in ER stress, as indicated by upregulation of chaperones, such as GRP78/BiP, and subsequent neuronal death (10). Overexpression of Parkin in this in vitro model resulted in removal/degradation of accumulated Pael-R and increased cell viability. The relevance of ER stress in the central nervous system to pathologic situations and normal neuronal development is suggested by induction of the unfolded protein response (UPR) in cell stress associated with cerebral ischemia (11,12) and exposure to excitatory amino acids (13), as well as during rapid neuronal growth in the neonatal period (14). Salient features of the UPR include upregulation of ER chaperones, suppression of general translation, and activation of the upiquitin-proteasome pathway.

A key facet of the pathology of PD is limitation of cell loss to dopaminergic neurons, especially in the SNpc. In this context, studies of Pael-R might be especially relevant. Pan-neuronal expression of Pael-R in Drosophila brain caused selective, age-dependent degeneration of dopaminergic neurons (10). Thus, increased levels of Pael-R render dopaminergic neurons vulnerable to cell death. In the current study, we have found that increased expression of Pael-R in mice in the SNpc results in neuronal death which is accentuated by suppression of Parkin (in Parkin–/– mice) or an ER chaperone (in Orp150+/– mice). In contrast, a dopamine (DA) synthesis inhibitor had neuroprotective properties in this system. Thus, we propose a unified model for cytotoxicity in PD through which a combination of ER stress and dopamine toxicity, potentially acting in concert with mitochondrial dysfunction, result in an ascending cycle of cellular perturbation and, ultimately, death of dopaminergic neurons.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Retrograde and neuron-specific gene expression by adenoviral vectors in the SNpc
It was essential to develop a system with cell-specific protein expression which could be targeted to the SNpc. For this purpose, we established two adenoviral vectors in which nuclear Cre-recombinase was driven by cell-specific promoters; for neuronal expression, we employed AxS2NPNCre (abbreviated as S2NPNCre) with a modified SCG10 promoter (superior cervical ganglia neural-specific 10 protein), and for glial-specific expression, we utilized AxGFAPNCre (abbreviated as GFAPNCre) with the glial fibrillary acidic protein promoter (Fig. 1A). When either of these adenoviral vectors was co-infected with AxCALNLEGFP (abbreviated as LoxEGFP), specific expression of EGFP occurred in neurons (with S2NPNCre) or astrocytes (with GFAPNCre) in a cell culture system (not shown). In vivoexpression studies were performed in mice by co-infecting either S2NPNCre with LoxEGFP, or GFAPNCre with LoxEGFP. Spatial limitation of gene expression was achieved by injecting viral vectors into the striatum (Fig. 1B), resulting in expression of EGFP protein in the ipsilateral SNpc (Fig. 1C, top panel). When S2NPNCre was injected with LoxEGFP, expression of EGFP (marking neurons) and tyrosine hydroxylase [TH; marking dopaminergic neurons in the SNpc (15)] overlapped (Fig. 1C; middle set of panels); quantitation of this overlap by image analysis, based on studying multiple fields, confirmed extensive coexpression of EGFP with TH (Fig. 1D). In contrast, there was no overlap of EGFP, following injection of S2NPNCre with LoxEGFP, when immunostaining was performed to visualize GFAP (Fig. 1C, lowest set of panels and Fig. 1D). These data indicate that co-infection of the striatum with recombinant adenoviral vectors encoding cell-specific Cre recombinase and EGFP (or other transgenes) flanked by lox P sites, leads to retrograde transport of adenovirus in the nigrostriatal system resulting in gene expression in the SNpc.


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  		Figure 1. Neuron-specific expression of a transgene in the SNpc after adenoviral infection of the striatum. Two adenoviral vectors were constructed to drive cell type-specific expression of Cre recombinase in neurons (AxS2NPNCre also termed S2NPNCre) and astrocytes (AxGFAPNCre also termed GFAPNCre) (A). S2NPNCre was injected unilaterally along with LoxEGFP into the striatum (109 p.f.u., in each case) as shown (B, upper panel). 7 days after injection, brain slices corresponding to the striatum (B, upper panel) or SNpc (B, lower panel) were analyzed by Nissl staining. On the contralateral side, GFAPNCre was injected along with LoxEGFP (109 p.f.u., in each case). SNpc sections were analyzed by fluorescence microscopy using antibodies to TH or GFAP (C). Merged images are shown on the top and the far right (marker bar corresponds to 200 µm; note that magnification is greater in the lower panels compared with the top panel), and are representative of six repeat experiments, obtained at –3.1 mM from the Bregma. (D) Combinations of adenoviral vectors at the indicated concentrations were injected into the striatum of wild-type mice, and, 7 days later, animals were sacrificed and sections of SNpc were analyzed at five different levels (–3.16, –3.28, –3.40, –3.52 and –3.64 mM from the Bregma). Sections were subjected to image analysis in order to determine the area of TH-positivity [TH(+)] as a percentage of the area of EGFP-positivity [%TH(+)/EGFP(+)]. The mean±SD is shown (n = 6), and ** denotes P < 0.01, compared to S2NPNCre infection alone.

 
Expression of Pael-R in the SNpc activates the unfolded protein response
Using this adenoviral system, we sought to express Pael-R in the SNpc. For this purpose, an adenoviral vector was made with expression of Pael-R under control of the SCG10 promoter regulated by the LoxP system (16), AxCALNLPael-R (abbreviated as LoxPael-R; Fig. 2A). Co-injection of the two adenoviral vectors, S2NPNCre and LoxPael-R, into the striatum increased expression of Pael-R in the SNpc (Fig. 2B, referred to as ipsilateral side). Enhanced expression of Pael-R required both vectors to be co-injected, and was specific for neurons.


Figure 4392
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  		Figure 2. Transfection of Pael-R induces the ER stress in the SNpc. (A) Schematic depiction of the construct pAxCALNLPael-R to yield the adenoviral vector AxCALNLPael-R (LoxPael-R). A stuffer sequence (gray box) flanked by two lox-P sequences (arrowhead) was inserted between the promoter and Pael-R gene. (B) Adenoviral vectors carrying either S2NPNCre, GFAPCre or LoxPael-R were injected into the striatum as described in Figure 1, and, 7 days later, expression of Pael-R was assessed by Western blotting (upper panel), together with the levels of ß-actin as an internal control. Intensity of the corresponding bands was semi-quantitatively assessed by densitometric analysis and expressed in terms of fold-increase versus control samples where no adenovirus was injected (lower panel; n = 6, mean±SD, and ** denotes P < 0.05 compared to LoxPael-R infection alone). (C) A mixture (total 2 µl) of adenoviral vectors including S2NPNCre (109 p.f.u.), LoxEGFP (5 x 108 p.f.u.), and AxCALNLNZ (LoxLacZ; 109 p.f.u.) was injected unilaterally in the striatum of C57Black/6J mice (upper panels). The same mixture was injected on the contralateral side, except that LoxLacZ was replaced by LoxPael-R (109 p.f.u.; lower panels). Seven days later, animals were perfusion-fixed and brainstem sections were immunostained using anti-ORP150 antibody (red). Merged images with EGFP signals (green) are shown in yellow. Open boxes indicated in the far left panels are magnified in the three panels on the right. Images are representative of six repeat experiments and the marker bar indicates 200 µm. (D) S2NPNCre+LoxLacZ (109 p.f.u., each) were injected unilaterally (termed ipsilateral) into the striatum, and S2NPNCre+LoxPael-R (109 p.f.u., each) were injected on the contralateral side. At the indicated time points, the brainstem was removed. The SNpc was separated and Western blotting was performed using antibodies to ORP150, GRP78, HSP70 and ß-actin (images are representative of six repeat experiments). (E) Expression of Pael-R (left panel), ORP150 (middle panel), and GRP78 (right panel) on the ipsilateral (open bars) or contralateral side (closed bars) was assessed by densitometric analysis and is expressed as fold-increase versus control (day 0 indicates the day of the operation without injection of adenoviral vectors (n = 6, mean±S.D is shown). ** denotes P < 0.05 by multiple comparison analysis compared to day 0 on the ipsilateral side.

 
Based on previous in vitro findings, increased expression of Pael-R in dopaminergic neurons might result in ER stress and diminished cell viability (9). Control experiments were performed by co-injecting three adenoviral vectors, S2NPNCre, LoxEGFP and LoxLacZ, into the striatum on one side of the brain, referred to as contralateral (Fig. 2C, upper panels). Expression of an ER chaperone, ORP150 (oxygen-regulated protein 150), known to increase with ER stress (11), was assessed in neurons expressing EGFP (Fig. 2C, upper panels). ORP150 levels, assessed by Western blotting, were unchanged in the SNpc following injection of this combination of adenoviral vectors (Fig. 2D, left portion and Fig. 2E, middle panel, open bars). Faint ORP150 staining was demonstrated in multiple cells and a much stronger signal for EGFP was observed (Fig. 2C, upper panels). ORP150 has a putative ATPase domain and protein binding site, suggesting that it may have a chaperone-like role in maintaining ER function under stress [(17), and see below]. In this context, we have demonstrated that overexpression of ORP150 rescues neurons from cell death mediated by ischemia (11,12) and excitatory amino acids (13). When S2NPNCre, LoxEGFP and LoxPael-R were injected into the striatum on the ipsilateral side of the brain (the vectors with LoxLacZ in place of LoxPael-R were injected on the contralateral side), a prominent increase in Pael-R in the SNpc (Fig, 2D, right portion) was accompanied by increased expression of ORP150 (Fig. 2C, lower panels) in Pael-R expressing neuron (labeled with EGFP). Western blotting indicated that increased expression of ER chaperones, GRP78 and ORP150, reached a maximum by 7–12 days after infection (Fig, 2D, right portion). In contrast, there was no increase in these chaperones on the contralateral side (i.e. the side where the LoxPael-R adenoviral vector was replace by a LoxLacZ vector; Fig. 2D and E). Levels of a cytoplasmic chaperone, the 70 kDa heat shock protein, remained unchanged on both sides of the brain (Fig. 2D). These data indicate that co-infection of LoxPael-R and S2NPNCre in the striatum caused expression of Pael-R in SNpc neurons, and triggered the ER stress.

Expression of Pael-R in SNpc of parkin–/– mice resulted in neuronal death
Parkin null mice (Parkin–/–) were generated by replacing the proximal exon 3 with a neo cassette (PGK-neo) and two lox P sites (Supplementary Material, Fig. S1A). Southern blotting revealed homologous recombination of the mutant allele (Supplementary Material, Fig. S1B). Reverse transcriptase–polymerase chain reaction (RT–PCR) to detect Parkin transcripts confirmed the absence of normal transcripts in homozygous mutant mice (Supplementary Material, Fig. S1C). Sequencing of RT–PCR products confirmed complete deletion of exon 3 and a frame-shift downstream of exon 2 in mutant mice (Supplementary Material, Fig. S1D). Consistent with these data, western blotting with anti-Parkin antibodies showed the absence of Parkin antigen in brain samples (Supplementary Material, Fig. S1E). Parkin homozygous mutant mice were born at the expected mendelian ratio, showed no overt abnormalities (except a slight decrease in body weight) and had normal lifespans (18,19). Moreover, Parkin–/– mice were comparable, in terms of the number of TH-positive neurons in the SNpc, compared to wild-type littermates. However, Parkin–/– animals displayed a slight increase in striatal dopamine content, compared with wild-type littermates (not shown).

Parkin has been shown to ubiquinate Pael-R, thereby promoting degradation of insoluble and toxic Pael-R overexpressed in cell culture systems (6). Thus, we hypothesized that similar overexpression of Pael-R in Parkin–/– mice might result in severe ER stress and cell death in the SNpc. Using Parkin+/+ and Parkin–/– mice, LoxPael-R was co-injected unilaterally into the striatum with LoxEGFP (as a neuronal marker) and S2NPNCre. Whereas up-regulation of Pael-R was confirmed by immunohistochemical analysis in the ipsilateral SNpc of both types of mice (white arrowheads in the lower panels of Fig. 3A indicate neurons positive with both TH and Pael-R), a marked decrease in the number of Nissl-positive neurons was demonstrated in Parkin–/– mice, (Fig. 3A and B). Neurodegeneration in the SNpc was accompanied by loss of TH immunointensity and decreased dopamine content in the striatum (Fig. 3C and D). No significant neuronal damage was observed in Parkin+/+ mice after the overexpression of Pael-R using these methods.


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  		Figure 3. Enhanced neuronal death in the SNpc of parkin–/– mice by the up-regulation of Pael-R. (A) Adenoviral vectors (2 µl), including LoxEGFP (5 x 108 p.f.u.), S2NPNCre (109 p.f.u.) and LoxPael-R (109 p.f.u.) were injected unilaterally in the striatum of either parkin+/+ or parkin–/– mice, as described in Figure 1. As a control, LoxPael-R was replaced by LoxLacZ (109 p.f.u.), and S2NPNCre+LoxLacZ was injected on the contralateral side. Brains of animals were then perfusion-fixed 7 days later, and midbrain sections were stained using the Nissl method (upper two panels). One of consecutive sections was also immunostained with anti-Pael-R antibody (lower panels). Images at –3.52 mM from the Bregma are shown. The open boxes in the upper panel are magnified in lower two panels. Typical examples of neurons positive with both TH and Pael-R are indicated by white arrowheads in the lower panels. Typical examples of degenerating neurons are identified by the open arrowheads in the middle panels. (B) Nissl positive neurons were counted on the ipsilateral (closed bars) and contralateral sides (open bars) as described in the text, 7 and 14 days after the injection. In each case, n = 6, and the mean±SD is shown. ** denotes P < 0.01 compared to parkin+/+ mice injected with LoxLacZ. (C) 10 days after the injection, midbrain sections were stained with anti-TH antibody as described in text. Images at –3.28 mM (upper panels) and –3.52 mM from the Bregma (lower panels) are shown. (D) TH positive neurons were counted 7 days after the injection (left panel). DA content was also measured by the HPLC-EC method 7 days after the injection (right panel). In each case, closed and open bars correspond to the ipsilateral (closed bars) or contralateral striatum (open bars) of parkin–/– or parkin+/+ mice, respectively. In each case, n = 6, and the mean±SD is shown. ** denotes P < 0.01 compared to parkin+/+ mice injected with LoxLacZ.

 
To determine whether loss of TH staining in the SNpc, associated with increased expression of Pael-R, was due to neuronal death, we performed TUNEL analysis and monitored expression of a neo-epitope for activated caspase-3 (20). Increased expression of Pael-R in the SNpc, achieved as above (by unilateral injection of S2NPNCre, LoxPael-R and LoxEGFP; the contralateral side was injected with S2NPNCre, LoxLacZ and LoxEGFP as a control), demonstrated a small increase in DNA fragmentation even in the ipsilateral SNpc of wild-type mice (Fig. 4A and C). Increased DNA fragmentation was more striking in the ipsilateral SNpc of Parkin–/– mice, with maximal intensity 7 days after infection (Fig. 4B and C). The number of EGFP-and TUNEL-positive cells is significantly increased in the SNpc of Parkin–/– mice (Fig. 4B and C). It should be noted that there was a small increase in TUNEL staining on the contralateral side (Fig. 4C) observed most consistently in caudal sections (–3.4 and –3.6 from the Bregma). This might be due, at least in part, to crossed projections from the striatum to the contralateral SNpc. Pilot studies showed EGFP expression in the contralateral SNpc after unilateral injection of S2NPNCre and LoxEGFP, though this was <5% of the EGFP expression observed in the ipsilateral SNpc (not shown). Increased expression of Pael-R in the SNpc, achieved as above, demonstrated a small increase in the intensity of activated caspase-3 staining in the ipsilateral SNpc of wild-type mice (Fig. 4A and D). Expression of activated caspase-3 antigen was more striking in the ipsilateral SNpc of Parkin–/– mice (Fig. 4B and D), with maximal intensity 5 days after infection (not shown). The number of EGFP-positive cells costaining with activated caspase-3 is significantly increased in the SNpc of Parkin–/– mice (Fig. 4B and D).


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  		Figure 4. Assessment of neuronal cell death in parkin–/– mice (A,B) After infection of adenoviral vectors by the same protocol as used in Figure 3, brains of animals were perfusion-fixed 7 (upper panels for TUNEL) and 5 (lower panels for activated caspase-3 staining) days later, and midbrain sections were stained using either the TUNEL method or anti-activated caspase-3 antibody ({alpha}Casp3). Images at –3.52 mM from the Bregma were obtained to visualize the indicated antibody and the EGFP signal (green). In each panel, areas indicated by arrowheads are magnified in the insets at the lower corner of the panel. Note that TUNEL-positive and activated caspase-3-positive cells are increased in the EGFP positive area. Scale bar: 200 µm. Images shown in this figure (panels A,B) are representative of six repeated experiments. (C,D) Quantitation of TUNEL-positive signals (number of positive signals) 7 days after injection (panel C) or the percentage of cells positive for activated caspase-3 ({alpha}Casp3) in the population of EGFP-positive neurons 5 days after injection (panel D) on the ipsilateral (closed bars) or contralateral SNpc (open bars) of either parkin–/– or parkin+/+ mice. In each case, n = 6, and the mean±SD is shown. ** denotes P < 0.01 compared to parkin+/+ mice injected with LoxLacZ.

 
Since there was no apparent damage, due to experimental manipulation or other factors, in the ipsilateral striatum (this side received injection of S2NPNCre, LoxPael-R and LoxEGFP) based on EGFP fluorescence or Nissl staining (Supplementary Material, Fig. S2B), diminished dopamine content most likely reflects loss of functional dopaminergic neurons in the ipsilateral SNpc. Moreover, injection of S2NPNCre and LoxPael-R resulted in the expression of Pael-R (marked by expression of EGFP) also in the motor cortex by retrograde infection (Supplementary Material, Fig. S2A). In contrast to the SNpc, no neuronal death was observed in either motor cortex or striatum (Supplementary Material, Fig. S2C). In other brain sublesions which have the connection to the striatum (i.e. the thalamus, the interpeduncular nucleus, the locus coeruleus and the raphe nucleus), no EGFP signals were detected, which may be due to the fewer communication to the striatum (not shown). The loss of TH immunointensity in the SNpc mainly occurred in neurons expressing Pael-R (Supplementary Material, Fig. S3A–C). These data suggest that Pael-R overexpression caused neuronal cell death, rather selectively in dopaminergic neurons in the SNpc.

ORP150 suppresses Pael-R-mediated neuronal cell death
These observations led us to hypothesize that ER stress might trigger loss of dopaminergic neurons due to accumulation of toxic Pael-R. To gain further insight into mechanisms underlying this observation, we focused on the function of an ER chaperone, ORP150, which promotes protein folding/degradation (17). We reasoned that if the ER stress is the cause of Pael-R-mediated dopaminergic neuron death, even in the presence of wild-type Parkin, decreased levels of ORP150 should accentuate Pael-R-induced neuronal death in the SNpc, whereas overexpression of ORP150 might be protective. Using targeted injection of adenoviral vectors (as above), Pael-R was overexpressed in the SNpc of either heterozygous Orp150 truncation mutants [Orp150+/– mice; note, homozygous Orp150–/– mice have a developmental lethal phenotype 13)], strain-matched controls (Orp150+/+ mice) or Orp150 overexpressing wild-type transgenics [Orp150 TG mice, driven by pCAGGS promoter (21)]. Elevated levels of ORP150 were confirmed in SNpc neurons of Orp150 TG mice by immunohistochemical analysis (Supplementary Material, Fig. S4 upper panels; levels of ORP150 in strain-matched normal animals are also shown in lower panels). Degeneration of dopaminergic neurons was assessed by TUNEL staining and expression of activated caspase-3 (Fig. 5A and B), since these methods appeared to have adequate sensitivity, as shown in Figure 4. A gene dosage effect was observed; increased levels of ORP150 afforded protection to dopaminergic neurons. Orp150+/– mice, with the lowest levels of functional ORP150, displayed exaggerated damage to dopaminergic neurons; TUNEL staining and activated caspase-3 were enhanced on the ipsilateral SNpc (the side in which injection of adenoviral vectors resulted in over-expression of Pael-R; Fig. 5A and B), whereas no significant cell death was observed on the contralateral side where LoxLacZ was expressed (data not shown). Each of these indices of neuronal stress/toxicity in this setting was decreased, in a manner dependent on the ‘dose’ of Orp150, when the same experiment was performed in wild-type (Orp150+/+) or mice overexpressing ORP150 (Orp150 TG mice) (Fig. 5A and B).


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  		Figure 5. Effect of ORP150, Parkin, and GRP78 on Pael-R-mediated cell death in the SNpc. (A) Adenoviral vectors (2 µl), including LoxEGFP (5 x 108 p.f.u.), S2NPNCre (109 p.f.u.), and LoxPael-R (109 p.f.u.), were injected unilaterally into the striatum of either ORP150+/– (left panels), ORP150+/+ (middle panels) or ORP150 transgenic mice (ORP150 TG; right panels) mice. Where indicated, LoxPael-R was replaced with LoxLacZ (109 p.f.u.), and the latter mixture was injected on the contralateral side. Brains were then perfusion fixed at 7 (for TUNEL analysis; upper panels) and 5 (for activated caspase-3 staining; {alpha}Casp3, lower panels) days after the injection. Images were overlapped with the EGFP signal (green). In each panel, the area indicated by the arrowhead is magnified in a small inset in the lower corner. Note the increase in cells staining positively by TUNEL analysis and for activated caspase-3 antigen in the EGFP positive area. This was most apparent in ORP150+/– mice. Scale bar: 200 µm. All images shown in this figure are representative of six repeated experiments. (B) Total number of TUNEL-positive signals (left panel) and the percentage of activated caspase-3 ({alpha}Casp3) positive cells in the population of EGFP-positive neurons (right panels) determined in the ipsilateral SNpc. In each case, n = 6, and the mean±SD is shown. ** denotes P < 0.01 compared to ORP150+/+ (wild type) mice. (C) A mixture (total 2 µl) of LoxEGFP (5 x 108 p.f.u.), S2NPNCre (109 p.f.u.), LoxPael-R (109 p.f.u.), and LoxLacZ (1.5 x 109 p.f.u.) was injected unilaterally into the striatum of parkin–/– mice. On the contralateral side, LoxLacZ was replaced with either AxCMParkin (Parkin; 1.5 x 109 p.f.u.) or AxCAGRP78 (GRP78; 1.5 x 109 p.f.u.). Midbrain sections were stained with anti-activated caspase-3 antibody (5 days later). The percentage of activated caspase-3-positive neurons was determined on the ipsilateral (closed bars) and contralateral sides. In the latter case, data is shown following injection of the vector expressing parkin (gray bars) and GRP78 (open bars) are shown. In each case, n = 6, and the mean±S.D is shown. Midbrain samples collected from parkin–/– mice as described in text were also subjected to Western blot analysis using either anti-ß-galactosidase, anti-GRP78, anti-Parkin or anti-ß-actin antibody (for an internal control). A typical example of five repeated experiments is shown. * denotes P < 0.01 compared to the group injected with the vector expressing LacZ.

 
These data suggest an essential contribution of ER function in protecting neurons from lethal toxicity when Pael-R is overexpressed. According to this concept, we further reasoned that such neurons in Parkin–/– mice might be rescued by either expression of Parkin or ER chaperones capable of promoting protein folding/renaturation, such as GRP78. Though adenoviral expression of LacZ in neurons failed to rescue SNpc neurons from Pael-R-mediated cell death, overexpression of Parkin minimized neuronal damage (Fig. 5C). Similarly, overexpression of GRP78 could substitute for Parkin in preventing Pael-R-mediated neuronal death in Parkin–/– mice. Western blot analysis of brain stem samples confirmed the expression of transfected gene products (Fig. 5C, right panel). These data indicate that the ER chaperones, such as GRP78 and ORP150, have the capacity to relieve ER stress due to increased expression of Pael-R, thereby exerting a protective effect on dopaminergic neurons in the SNpc.

Suppression of Pael-R-mediated cell death by inhibition of dopamine synthesis
Increased dopamine content in the striatum of Parkin–/– mice has been noted by some investigators, though the increase is small (18,19). Furthermore, Pael-R has been implicated in the regulation of dopamine levels; increased Pael-R is associated with increased dopamine content in the striatum (Imai, Y. et al., manuscript in preparation). Based on these observations, we reasoned that Pael-R-mediated cell death in the SNpc of Parkin–/– mice might be associated with elevated levels of dopamine. Since dopamine has considerable potential toxicity (8,22), we further hypothesized that dopamine-derived metabolites/catabolites might contribute to loss of TH-positive (+) neurons. To address this issue directly, we determined whether suppression of DA synthesis by AMPT, a specific inhibitor of TH (15), would have a neuroprotective effect on TH(+) neurons overexpressing Pael-R in the SNpc of Parkin–/– mice. Accidental death of mice occurred in {approx}4% of animals within 36 h after the first administration of AMPT. This might be due to the shifts of circadian temperature rhythms (23). The systemic toxicity of AMPT was not observed at later phase. Repeated administration of AMPT over a 7 day period lowered dopamine content of the striatum to {approx}30% of that observed in untreated controls (Supplementary Material, Fig. S5). Using this protocol of AMPT treatment, adenoviral vectors were injected into the striatum to increase neuronal Pael-R levels in Parkin–/– mice. Compared with Parkin–/– mice treated with phosphate-buffered saline (PBS), animals receiving AMPT displayed striking neuroprotection. Inhibition of TH in Parkin–/– mice overexpressing Pael-R in the SNpc suppressed the number of TUNEL-positive nuclei and generation of activated caspase-3 epitopes, compared with animals treated with PBS alone (Fig. 6A–C). AMPT treatment was also effective in preventing Pael-R-mediated cell death in the SNpc of Orp150+/– mice, compared with animals treated with saline (Fig. 6C), suggesting a toxic effect of DA in ER dysfunction. These data suggest that dopamine enhances neurotoxicity associated with overexpression of Pael-R, especially in the absence of Parkin.


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  		Figure 6. Suppression of neuronal death in the SNpc of mice overexpressing Pael-R by inhibition of dopamine synthetase using AMPT. (A) Parkin–/– mice were treated with either PBS or AMPT up to 7 days after unilateral injection of adenoviral vectors, including LoxEGFP (5 x 108 p.f.u.), S2NPNCre (109 p.f.u.) and LoxPael-R (109 p.f.u.). As a control, the same vectors, with LoxPael-R replaced by LoxLacZ (109 p.f.u.), were injected on the contralateral side. Brains were perfusion-fixed at 7 (upper panels; for TUNEL analysis) and 5 (lower panels; staining for activated caspase-3, {alpha}Casp3) days after injection of the vectors, and were then subjected to histochemical analysis as described above. The above images were overlapped with EGFP (green). Scale bar: 200 µm. All images shown in this figure are representative of six repetitions of the experimental protocol. In each panel, the area indicated by the arrowhead is magnified in a small inset in the lower corner. Note that TUNEL-positive and activated caspase-3-positive cells were diminished by AMPT treatment (i.e. in the presence of the latter, the area/level of apoptotic cells approximated that observed in controls overexpressing LacZ). (B,C) Statistical analysis was performed as described above, in either Parkin–/– mice (B) or Orp150+/– mice (C). The number of TUNEL-positive signals (left panels), and % of activated caspase-3-positive cells in the population of EGFP-positive neurons (right panels) in the ipsilateral SNpc are shown (n = 6; the mean±SD). P-values,obtained by Student's t-analysis, are shown in each panel.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Our data indicate that in vivo overexpression of Pael-R in neurons of the SNpc results in enhanced ER stress, which, especially in a setting with decreased functional Parkin, targets TH-positive neurons for accentuated cytotoxicity. Whereas expression of a protein as difficult to properly fold as Pael-R has been shown to cause ER stress in a range of cell types in vitro (9), we believe that cellular vulnerability in this situation is critically exaggerated in dopaminergic neurons in vivo due to the superimposed toxicity of dopamine (DA) itself. These data also emphasize the potential relevance of parkin targets, such as Pael-R, in addition to the aminoacyl-tRNA synthetase cofactor p38, to neurotoxicity in dopaminergic neurons (24).

The technical approach employed in our experiments, injection of adenovirus under stereotactic guidance into the striatum with selective neuronal expression of gene products, is quite unique. First, such results have been difficult to achieve in the mouse because of anatomic limitations. Though adenovirus vectors are more immunogenic than adenovirus associated virus, they still have greater merits in retrograde transfection (25). We have taken this advantage into our experimental system, to achieve an efficient gene transfer to SNpc, where direct injection of viral vectors will be inapplicable because of anatomical limitations. Second, adenovirus infection predominately affects glia, rather than neurons. Modification of the SCG10 (superior cervical ganglion) promoter by tandem insertion of two neuron-restrictive silencers produced almost 100% expression of transgenes in neuronal cultures, and considerably lower expression in astrocytes (not shown). Coinjection of S2NPNCre with LoxEGFP into the striatum resulted in expression of transgenes in neurons of the mouse SNpc. This approach allowed us to obtain neuron-specific expression of Pael-R enabling study of its effect(s) on neuronal physiology in vivo. The proximal result of such Pael-R expression included upregulation of ER chaperones, such as GRP78 and ORP150, whereas the distal result was loss of TH-positive neurons, especially in Parkin–/– mice. In contrast, expression of Pael-R had no effect on the constitutively expressed form of HSP70, important for housekeeping functions in the cytoplasm (Fig. 2D).

Several observations are consistent with the importance of ER stress as a mechanism underlying Pael-R-induced cellular toxicity. In previous studies using cultured neuroblastoma cells, Pael-R has been identified as a substrate of Parkin, an E3 ubiquitin ligase (6). Thus, in the absence of Parkin, Pael-R may accumulate since it is not subject to efficient removal. In contrast, no detectable change of Pael-R levels has been reported in SNpc of Parkin null mice (24). Since there is no evidence of progressive neuronal cell death in Parkin null mice, there would appear to be redundant mechanisms able to compensate for loss of Parkin under physiologic conditions. The ability of neurons to withstand ER stress imposed by expression of Pael-R is likely to be dependent on the effectiveness of the ER-stress response; higher levels of Pael-R would require a facile ER-stress response (i.e. adequate or increased levels of parkin, ORP150, GRP78 etc), whereas diminished levels of ORP150 or GRP78 would render neurons vulnerable to toxicity because of a compromised stress response. These predictions have been borne out by our experimental results. Therefore, the possible toxicity of Pael-R cannot be completely ignored, especially in pathological conditions. Increasing expression of GRP78 had a protective effect in Parkin–/– mice overexpressing Pael-R. Furthermore, expression of ORP150, an important factor modulating ER stress even in the presence of Parkin, modulated the toxicity of Pael-R for dopaminergic neurons; increased levels of ORP150 in transgenic mice were neuroprotective, whereas diminished levels in ORP150 in Orp150+/– mice resulted in enhanced cell death (i.e. increased TUNEL staining and immunoreactivity for activated caspase-3 in the SNpc).

Our experimental system produced Pael-R overexpression and subsequent cell death in the SNpc of mouse, whereas no apparent cell death occurred either in the striatum or in the motor cortex (Supplementary Material, Fig. S2C). Furthermore, we observed a neuroprotective effect of a TH inhibitor in the SNpc of mouse, suggesting that DA also contributes to Pael-R-induced cell death of dopaminergic neurons. Consistent with these results in our mouse models, studies in transgenic flies overexpressing Pael-R in most of neuronal populations also showed selective loss of dopaminergic neurons, though the mechanism of cellular degeneration is not fully understood (10). One plausible explanation for focusing toxicity on dopaminergic neurons is the oxidative stress caused by the presence of DA and its derivatives (1). Another possibility that DA might compromise the survival of dopaminergic neurons is a chemical inactivation of cellular proteins by addition of DA to sulfhydryl group of proteins. Recently, it has been reported that dopamine covalently modifies Parkin, a protein rich in sulfhydryl group, in dopaminergic cells, leading to increased Parkin insolubility and inactivation of its E3 function (22). Vulnerability of Parkin to modification by DA further impairs degradation of Pael-R. Thus, even in sporadic PD, DA might interfere in the degradation of certain proteins, such as Pael-R, by the inactivation of Parkin. Collectively, these data propose a model in which a combination of ER stress and DA-related stress plays an important role in degeneration of dopaminergic neurons in sporadic PD as well as PD caused by parkin mutations.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Targeted disruption of the mouse parkin gene
Parkin–/– mice were generated using standard gene targeting techniques (26). A targeting vector was constructed with a 15.7 kb genomic DNA fragment containing exon 3 of the parkin gene (Supplementary Material, Fig. S1). The region containing exon 3 was replaced with a Floxed pgk-neo cassette. A DT-ApA cassette was flanked at the 5'-end of the homologous arm for negative selection (27). The linearized targeting vector was transfected into E14 (129sv) ES cells. Positive clones were selected by Southern blotting, and then injected into C57Bl/6J (B6) blastocysts. Offspring harboring the targeted allele were generated by crossing chimeric mice with B6 mice. Results of such crosses were confirmed by Southern analysis.

Reverse transcription–polymerase chain reaction analysis of parkin null mice
Total RNA was extracted from whole brain tissue using Isogen (Nippon gene). RT reactions containing 1 µg of total RNA were performed using the SuperScript II First-Strand Synthesis System for RT–PCR (Invitrogen). The resulting cDNA was added to PCR reactions containing 1 unit of Ex Taq DNA Polymerase (Takara) for 35 PCR cycles. PCR products were separated on a 1% agarose gel. Primers were as follows: RT primer, 5'-agt ttc cct tga ggt tgt gc; Primer A, 5'-cgt agg tcc ttc tcg acc; Primer B, 5'-ttg agg ttg tgc gtc cag g; Primer C, 5'-acc tca gag ggc tcc ata tg; and, Primer D, 5'-ctc tct cta cac gtc aaa cca gtg.

Construction of adenoviral vectors
Modified SCG10 (S2NP10) promoter [2 kb (28)] and mouse GFAP promoter region (2.5 kb; kindly provided by Dr Ikenaka, National Institute for Physiological Science) were cloned into pAxAwNCre (kindly provided by Dr Saito, Tokyo University), a promoter-less cosmid vector for preparing cell type-specific Cre-recombinase expressing adenovirus (Fig. 2A; AxS2NPNCre and AxGFAPNCre). Human Pael-R (9) was cloned into pAxCALNLw (Takara Bio Inc., Shiga, Japan) in order to control Pael-R expression using Cre reconbinase (AxLNLPael-R). The Pael-R gene is silenced because of the presence of the staffer of the neo-resistant gene, and is activated by Cre-mediated excisional deletion of the stuffer when a sufficient amount of Cre recombinase is expressed (Fig. 3). To prepare an adenoviral vector of parkin (AxCMParkin), the human parkin gene was cloned into pAxCMwt. Recombinant adenovirus was generating using the COS-terminal protein complex (TPC) method and the Takara adenovirus expression kit (Takara Bio Inc.). AxLNLNZ (Takara Bio Inc.), which overexpresses LacZ with a nuclear localization signal mediated by Cre recombinase, was used for control experiments. Cre-mediated EGFP-expressing adenovirus, AxLNLEGFP, was kindly provided by Dr Okado (Tokyo Metropolitan Institute of Neuroscience). An adenoviral vector for overexpression of GRP78/Bip (AxCAGRP78) was generously provided by Drs S. Tanaka and T. Koike [(29) Graduate School of Science, Hokkaido University]. Each adenovirus was amplified in HEK293 cells and purified using VIRAPREP Adenovirus Purification Kit (Virapur LLC., San Diego, CA, USA). Viral titers were determined by a plaque-forming assay in HEK293 cells.

Western blotting
Levels of Pael-R in tissue extracts were determined by Western blotting as described (9). Mouse brain was quickly removed and placed on a cold plate. Brain slices (200 µm) were obtained at –3.5 mM from the Bregma on a vibratome. Substantia nigra was then carefully removed under guidance of a stereoscopic microscope according to the mouse brain atlas (Paxinos and Franklin, Academic Press, 1997, San Diego) using the cerebral peduncle and medial lemniscus as landmarks. Tissue extracts were prepared from SNpc in PBS containing NP-40 (1%). Proteins were separated by SDS-PAGE, and transferred to PVDF. PVDF was then incubated with antibody to either human Pael-R or ß-actin, the latter as an internal control (1000xdilution, Sigma, St Louis, MO, USA). Levels of chaperones in tissue extracts were determined by Western blotting, as described (30). PVDF was incubated with either anti-human ORP150 antibody (1 µg/ml), anti-GRP78 monoclonal antibody (Stressgen, Canada; 0.2 µg/ml), or anti-HSP73 antibody (0.1 µg/ml; Stressgen). Where indicated, either anti-ß-galactosideas antibody (1000xdilution, Sigma) or anti-Parkin antibody (1000xdilution, Cell Signaling Technologies Inc.) was used to access the level of these proteins. Images were further subjected to densitometric analysis using NIH image software.

Injection of adenoviral vectors into the striatum and treatment of animals
Animals were housed and treated according to institutional and national guidelines. Mice were stereotactically positioned under deep anesthesia, and the indicated combination of adenovirus vectors was injected unilaterally (a different combination, including a LacZ control, was injected on the contralateral side) into the striatum at six points (AP/ML/DV/ = 0.8/1.2/–2.5, 0.8/1.2/–3.2, 0.8/1.7/–2.5, 0.8/1.7/–3.2, 0.8/2.2/–2.5 and 0.8/2.2/–3.2; units are mm), followed by free access to food and water. Retrograde passage and infection of adenoviruses was confirmed 5–12 days after the injection by detection of a green fluorescent signal in the SNpc by fluorescence microscopy. Where indicated, animals were pretreated with {alpha}-methyl-DL-Tyrosine (AMPT) to suppress DA synthesis. AMPT-HCl (150 mg/kg, Sigma) was intra-peritoneally administrated twice per day for 5 days before injection of adenoviral vectors, as well as after the latter and until the day of sacrifice.

Assessment of neuronal death in vivo
At the indicated time points, animals were perfused with 4% paraformaldehyde under deep anesthesia, the brain was excised and coronal brain sections (14 µm) were cut on a cryostat. Sections were processed for cresyl violet staining or immunohistochemistry using either mouse anti-tyrosine hydroxylase antibody (TH; 1 µg/ml, Sigma), rabbit anti-human ORP 150 antibody [5 µg/ml (30)], rabbit anti-activated casepase-3 antibody(0.1 µg/ml, Genzyme/Teche), rabbit anti-Pael-R antibody [10 µg/ml, (9)] or goat anti-GFAP antibody (4 µg/ml. Santa Cruz Biotechnology, Santa Cruz, CA). Sections were also subjected to TUNEL staining using a commercially available kit (ApopTag, Intergen, Purchase, NY). To evaluate neuronal death in the SNpc, either Nissl or EGFP positive cells were counted in each coronal slice obtained at five different levels (–3.16, –3.28, –3.40, –3.52 and –3.64 mM from the Bregma), as described (31) by acquiring digital images using a CCD camera (Nikon, Coolscope). Cell death was semi-quantitatively assessed by counting TUNEL-positive cells/nuclei in the EGFP-positive area, and evaluating the percentage of activated caspase-3-positive cells in the population of EGFP-positive cells. Sections were analyzed using a laser scanning confocal microscope system (Leica, TCS SP2). In each case, two observers without knowledge of the experimental protocol evaluated sections and experiments were repeated at least three times.

DA content in the striatum was measured in the SNpc as described (32). In brief, striatal tissue was homogenized in solution H (0.4 M HClO4 containing 4 mM Na2S2O5, 4 mM diethylenetriaminepentaacetic acid, and 5 mM 1,4-dithiothreitol). Crude tissue lysate was separated on a C-18 reversed-phase column (MCM HPLC, 4.6 mMx15 cm, 5 ml, ESA, Chelmsford, MA, USA), followed by electrochemical detection (Coulochem III, ESA).

Statistical analysis
Data shown represent the mean ±SD Multiple group comparisons were performed by one-way ANOVA, followed by Newman-Kuels test as a post hoc analysis. Comparison between two groups was analyzed by two-tailed Student's t-test.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We thank Dr Martin Hooper (Western General Hospital, Edinburgh) for generous contribution of E14 cell line, and Drs Yumi Onodera, Yoshikazu Saito, Hitomi Suzuki (RIKEN, Wako, Japan) for technical assistance for establishing chimera mice. The authors also appreciate the generous gifts of AxCALNLEGFP, GAG promoter and Ax1CAGRP78 from Drs Haruo Okado (Tokyo Metropolitan Institute for Neuroscience), Jun-Ichi Miyazaki (Osaka University Medical School), and Shuuitsu Tanaka and Tetsuro Koike (Hokkaido University), respectively. This work was partially supported by the Ministry of Education, Science, Sports and Culture, a Grant-in-Aid for Scientific Research on Priority Areas—Advanced Brain Science Project—#15016120 to R.T., for Scientific Research (A) #14207032 to R.T., and for Young Scientists (A) #15680011 to Y.I. and a grant from the Special Postdoctoral Researcher Program of RIKEN to Y.I.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} These authors equally contributed to this work. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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