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Cell Reports, Volume 3, Issue 1, 186-199, 17 January 2013
SorLA Controls Neurotrophic Activity by Sorting of GDNF and Its Receptors GFRα1 and RET
Simon Glerup1, 6, , , Maria Lume4, 6, Ditte Olsen1, Jens R. Nyengaard2, Christian B. Vaegter1, Camilla Gustafsen1, Erik I. Christensen1, Mads Kjolby1, Anders Hay-Schmidt5, Dirk Bender3, Peder Madsen1, Mart Saarma4, Anders Nykjaer1 and Claus M. Petersen1
1 MIND Centre, Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark
2 MIND Centre, Stereology and Electron Microscopy Laboratory, Aarhus University, DK-8000 Aarhus C, Denmark
3 MIND Centre, PET Centre, Aarhus University, DK-8000 Aarhus C, Denmark
4 Institute of Biotechnology, University of Helsinki, Viikki Biocenter, FIN-00014, Helsinki, Finland
5 Department of Neuroscience and Pharmacology, University of Copenhagen, DK-2200, Copenhagen N, Denmark
6 These authors contributed equally to this work
Graphical Abstract
► SorLA is a sorting receptor for GDNF and its signaling receptors GFRa1 and RET ► The SorLA/GFRa1 complex targets GDNF for lysosomal degradation, while GFRa1 is recycled ► SorLA/GFRa1 targets RET for endocytosis and influences GDNF-induced neurotrophic effects ► SorLA knockout mice display altered dopaminergic function and an ADHD-like phenotype
- Glial cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor that has reached clinical trials for Parkinson's disease. GDNF binds to its coreceptor GFRα1 and signals through the transmembrane receptor tyrosine kinase RET, or RET independently through NCAM or syndecan-3. Whereas the GDNF signaling cascades are well described, cellular turnover and trafficking of GDNF and its receptors remain poorly characterized. Here, we find that SorLA acts as sorting receptor for the GDNF/GFRα1 complex, directing it from the cell surface to endosomes. Through this mechanism, GDNF is targeted to lysosomes and degraded while GFRα1 recycles, creating an efficient GDNF clearance pathway. The SorLA/GFRα1 complex further targets RET for endocytosis but not for degradation, affecting GDNF-induced neurotrophic activities. SorLA-deficient mice display elevated GDNF levels, altered dopaminergic function, marked hyperactivity, and reduced anxiety, all of which are phenotypes related to abnormal GDNF activity. Taken together, these findings establish SorLA as a critical regulator of GDNF activity in the CNS.
Neurotrophic factors are key molecules in sculpting the developing nervous system as well as regulating adult neuronal maintenance and plasticity. Glial cell line-derived neurotrophic factor (GDNF) is essential for the development of several neuronal populations (Airaksinen and Saarma, 2002), and a critical survival factor for a number of neuronal subtypes, most notably midbrain dopaminergic (DA) neurons (Lin et al., 1993). More specifically, GDNF protects and repairs nigral DA neurons in neurotoxin-lesioned animal models of Parkinson's disease (Kirik et al., 2004), and phase I clinical trials with parkinsonian patients, GDNF gave highly promising results that were unfortunately hampered by conflicting outcome from phase II studies (Gill et al., 2003; Lang et al., 2006).
GDNF is a homodimeric neurotrophic factor belonging to the GDNF family ligands (GFLs) together with neurturin, persephin, and artemin (Airaksinen and Saarma, 2002). GDNF signaling is conventionally mediated via two receptors. First, the GDNF dimer binds GDNF family receptor α1 (GFRα1), which is linked to the plasma membrane through a glycosylphosphatidylinositol anchor. The resulting tetrameric (2:2) complex interacts with Rearranged During Transfection (RET) receptor tyrosine kinase and activates Erk, Akt, Src, and PLCγ pathways (Airaksinen and Saarma, 2002). Although RET is the established GDNF signaling receptor, many cells responding to GDNF and expressing GFRα1 do not express RET (Trupp et al., 1997; Yu et al., 1998). To date, two alternative receptors have been discovered: neural cell adhesion molecule N-CAM and syndecan-3 (Bespalov et al., 2011; Paratcha et al., 2003). In both cases, the ligand-receptor interaction leads to the activation of Src family kinases, modulating cell migration, neurite outgrowth, and synapse formation (Bespalov et al., 2011; Ibáñez, 2010).
Despite the progress in characterizing GDNF receptors and their signaling pathways, still very little is known about their trafficking and how GDNF activity is regulated. Due to alternative splicing, GDNF is synthesized in two precursor forms (α)- and (β)proGDNF that are sorted differentially through the secretory pathway prior to propeptide cleavage by a proprotein convertase (Lonka-Nevalaita et al., 2010), a process proposed to involve the sorting receptor SorLA (Geng et al., 2011; Westergaard et al., 2004). SorLA is one of five members of the sortilin-related receptor family (sortilins), unified by the vacuolar protein sorting protein 10p (Vps10p) domain (Jacobsen et al., 1996; Willnow et al., 2008). The cytoplasmic tail of SorLA contains several consensus binding sites for adaptor proteins that mediate internalization from the cell surface, Golgi-endosome transport, and retrograde sorting to the TGN (Nielsen et al., 2007). In the present study, we find that SorLA conveys internalization of GFRα1 alone and in complex with GDNF and mediates their sorting to endosomal compartments. GFRα1 bypasses lysosomal degradation, whereas GDNF does not. The SorLA/GFRα1 complex further mediates subcellular sorting of RET, influencing GDNF-induced neurotrophic activity including the survival of primary DA neurons. SorLA-deficient mice have increased GDNF levels in the striatum and midbrain and exhibit a behavioral phenotype characterized by reduced anxiety, hyperactivity, and insensitivity to amphetamine, symptoms reminiscent of attention deficit hyperactivity disorder (ADHD) in humans.
SorLA Selectively Binds GDNF
GDNF bound to the extracellular domain of SorLA in a concentration-dependent manner as assessed using Biacore (Figure 1A). The interaction was of high affinity with a Kd of 3 nM, and selective as no other GFL showed binding to SorLA (Figure 1B). GDNF is unique among the GFLs by containing an extended N terminus of 38 amino acids (Baloh et al., 2000). As this region might contribute to SorLA binding, we generated a glutathione S-transferase (GST) fusion peptide encompassing the GDNF propeptide followed by the N-terminal extension (GDNF aa 20–115). This peptide bound SorLA with a Kd of 76 nM, whereas neither GST-GDNF propeptide (GDNF aa 20–77) nor GST-neurturin propeptide displayed any binding, clearly indicating that the GDNF N-terminal region is involved in binding of SorLA (Figure 1C and data not shown). The ∼25-fold difference in the affinity of GDNF to SorLA compared to the N-terminal peptide could be due to additional degrees of freedom of the peptide fragment when not covalently associated with the folded structure of GDNF. However, at present, the involvement of other GDNF regions in SorLA binding cannot be excluded either. SorLA is a large mosaic receptor containing several potential ligand binding domains, one of which is the Vps10p domain (Figure 1D). Prior to its removal in late Golgi compartments, the SorLA propeptide inhibits access to this domain (Jacobsen et al., 2001). We found that excess SorLA propeptide prevented the binding of GDNF to SorLA (Figure S1A). In analogy, 200-fold molar excess of the small neuropeptide neurotensin, predicted to bind inside the tunnel-like cavity of the SorLA Vps10p domain (Quistgaard et al., 2009), also abolished GDNF binding to SorLA in Biacore experiments (Figure S1B). The SorLA-GDNF interaction was further confirmed in HEK293 cells transfected with an endocytosis-deficient SorLA mutant where the binding of 125I-GDNF (100 pM) was displaced by increasing concentrations of unlabelled GDNF (Figure S1C). The combined data demonstrate that the SorLA Vps10p domain specifically and selectively binds to the N-terminal region of GDNF.
SorLA and GFRα1 Direct GDNF to Lysosomes
SorLA rapidly traffics from the cell surface to endosomes (Nielsen et al., 2007). Thus, to study SorLA-mediated GDNF endocytosis and how this was affected by the presence of the GDNF signaling receptors, we used HEK293 cells transfected with combinations of SorLA, GFRα1, and RET. Cells were incubated with GDNF (3 nM) for 2 hr on ice to allow cell surface binding before changing to 37°C medium without GDNF to commence internalization for 30 min. Analysis using confocal microscopy revealed GDNF in vesicular structures of SorLA cells, whereas faint spots of GDNF lined the surface of cells transfected with RET or empty vector (Figure 1E). In contrast, GFRα1 or GFRα1/RET-expressing cells showed intense GDNF staining lining the plasma membrane, but no endocytosis was apparent (Figure 1E). GDNF endocytosis by RET/SorLA cells was comparable to SorLA alone (Figure 1E). Strikingly, coexpression of SorLA and GFRα1 resulted in a dramatic increase in GDNF endocytosis independent of RET (Figures 1E and S1D). In fact, SorLA and GFRα1 together resulted in detectable internalization at a 100-fold lower GDNF concentration compared to SorLA alone (Figures S1E and S1F), demonstrating a cooperative effect. Furthermore, GDNF surface staining was reduced by 75% during 45 min in SorLA/GFRα1 cells compared to only ∼30% in GFRα1 cells (Figures S2A and S2B).
Interestingly, SorLA cells metabolized GDNF from the culture medium to a much greater extent than mock-transfected cells (Figure S2C), and 125I-GDNF bound to the surface of cells expressing SorLA, decreased from 3,214 ± 450 to 1,046 ± 72 cpm during 60 min, and was accompanied by an equivalent accumulation of nonprecipitable radioactivity released into the culture medium, possibly derived from degraded GDNF (Figure S2D). Importantly, GDNF did not remain associated with SorLA and GFRα1 but colocalized with the endosomal marker EEA1 (Figure 1F), and the intensity of the GDNF staining decreased over time (from 30 to 60 min) (Figures 1Fand 1G). Lysosome inhibition by leupeptin and pepstatin (leu/pep) clearly augmented GDNF immunoreactivity in structures of SorLA/GFRα1 cells also positive for the lysosomal marker Lamp-1 (Figure 1G). In contrast, no change in GDNF staining upon leu/pep treatment was evident in GFRα1 cells, where abundant GDNF was associated with the plasma membrane (Figure 1H). Taken together, the data show that SorLA alone and in cooperation with GFRα1 of the GFRα1-RET complex binds extracellular GDNF and targets it for lysosomal degradation.
GFRα1 and SorLA Forms GDNF Sorting Complex
To study how GFRα1 delivers GDNF to SorLA, we probed for a receptor-receptor complex by coimmunoprecipitation (co-IP). Indeed, SorLA and GFRα1 co-IPed both in the presence and absence of GDNF (Figure 2A), whereas we were unable to co-IP SorLA and RET (data not shown). The SorLA/GFRα1 interaction was direct as assessed using Biacore (Kd = 6 nM) (Figures 2B and S3A), whereas we observed no binding between the SorLA and RET (Figure 2B). GFRα1 binding was inhibited by excess SorLA propeptide (Figure S3B), but, unlike for GDNF, neurotensin did not affect the SorLA-GFRα1 interaction (Figure S3C). We speculated whether SorLA-GFRα1-GDNF might interact simultaneously and tested this in a Biacore experiment where immobilized SorLA was first saturated with GDNF and subsequently tested for its ability to bind GFRα1. No reduction in the affinity for GFRα1 was observed for the SorLA-GDNF complex compared to SorLA alone (Figure S3D). In fact, picomolar GDNF concentrations increased SorLA/GFRα1 co-IP, indicating the potential formation of a ternary complex (Figure S3E). Finally, by crosslinking followed by sequential co-IP of GDNF and GFRα1 as described in Figure S3F, SorLA was pulled down specifically as part of a ternary SorLA/GFRα1/GDNF complex, confirming its existence.
SorLA Sorts GFRα1 from the Cell Surface to the TGN
To monitor SorLA-mediated sorting of GFRα1, we labeled surface receptor with antibodies. In the presence of SorLA, the majority of GFRα1 accumulated in paranuclear compartments within 45 min, whereas no endocytosis was observed for GFRα1 alone (Figures 2C and 2D). To determine if SorLA destines GFRα1 for lysosomal degradation, we studied its turnover by metabolic labeling of cells expressing GFRα1 alone or coexpressing SorLA. Surprisingly, SorLA appeared to prolong GFRα1 half-life, ruling out lysosomal sorting (Figures 2E, S3G, and S3H). GFRα1 alone localized mainly to the plasma membrane, but coexpression with SorLA shifted a substantial fraction of GFRα1 to vesicular structures (Figure S3I). SorLA and GFRα1 partially colocalized with the TGN marker TGN46 (Figure 2F) and were also found together in endosomes and multivesicular bodies as shown by immune electron microscopy (Figure 2G). Although SorLA does not bind the additional GFLs, it is tempting to speculate that it may sort neurturin coreceptor (GFRα2), artemin coreceptor (GFRα3) and persephin coreceptor (GFRα4) in a similar manner to GFRα1. Indeed, SorLA was specifically pulled down by co-IP with the individual GFRαs (Figure S4A). Furthermore, SorLA directed GFRα2 and -4 from the cell surface into vesicles (Figures S4B–S4D), suggesting that it may function as a general GFRα sorting receptor.
SorLA/GFRα1 Mediates RET Endocytosis
We next tested if SorLA affects RET/GFRα1 complex formation. Intriguingly, SorLA did not inhibit pulldown of RET and GFRα1; rather, it appeared to increase their interaction slightly (Figure 3A). Of note, the presence of SorLA mainly induced co-IP of GFRα1 with the lower molecular weight form of RET, likely representing immature intracellular receptor. Similarly, co-IP of SorLA by GFRα1 was also somewhat increased by RET despite the fact that we were unable to demonstrate an interaction between SorLA and RET (Figure 3A and data not shown). This was not altered by the presence of GDNF (3 nM) (data not shown). To study if RET trafficking is affected by SorLA, we labeled surface RET with antibodies and studied its internalization. At 30 min the vast majority of labeled RET remained at the surface of cells expressing RET alone, or RET together with GFRα1 or SorLA (Figures 3B and 3C). However, in SorLA/GFRα1/RET cells, RET was directed to EEA1-positive endosomes (Figures 3C and 3D), an observation that was independent of GDNF (Figure 3D, left panel). We speculated if the SorLA/GFRα1 complex sorts endogenous RET and tested this in SY5Y cells, which express all three receptors endogenously, and in SY5Y cells overexpressing SorLA (SY5Y/SorLA) (Figure S5A). To separate intracellular structures, we employed a two step gradient centrifugation protocol (Nielsen et al., 2007) and analyzed fractions by western blotting (Figures 3E and S5B). This experiment revealed a marked shift in RET localization in SY5Y/SorLA cells to fractions of higher density also enriched in SorLA (Figure 3E), strongly indicating that RET undergoes intracellular trafficking together with SorLA/GFRα1. Yet, such sorting does not affect RET degradation as newly synthesized receptor rapidly disappeared independent of SorLA overexpression (Figure 3F).
SorLA Inhibits GDNF-Induced Neurotrophic Activity
As SorLA targets all three components of the extracellular GDNF signaling machinery, we tested its impact on the activation of intracellular cascades. We treated SY5Y cells for 2 hr with SorLA antibodies that block GDNF/GFRα1 endocytosis (Figure S5C) and observed a marked increase in GDNF-induced Erk and Akt phosphorylation (3 nM, 15 min), suggesting an inhibitory role of endogenous SorLA (Figure 4A). Serum-depleted SY5Y cells depend on autocrine GDNF stimulation as anti-GDNF reduced survival by ∼35% (Figure 4B). In contrast, survival was potentiated by the addition of exogenous GDNF (Figure 4C). The SorLA propeptide (SorLApro) can be used as a SorLA antagonist as illustrated in Figures S5D and S5E. Accordingly, SorLApro induced survival as efficiently as exogenously added GDNF, and their combination was even more effective (Figure 4C). In contrast, SorLA overexpression (SY5Y/SorLA) reduced survival to a similar extent as anti-GDNF (Figure 4D). Notably, SY5Y/SorLA cells also displayed markedly increased clearance of surface bound GDNF (3 nM) during 30 min compared to mock-transfected SY5Y cells (Figure 4E). GDNF-GFRα1-RET signaling is further known to promote proliferation of SY5Y cells (Hirata and Kiuchi, 2003), and in analogy the addition of SorLApro markedly increased the number of cells/well after 24 hr (Figure 4F). Proliferation could not be potentiated further by addition of both GDNF and SorLA propeptide, suggesting that it was already proceeding at its highest rate (data not shown). Cell differentiation, e.g., neurite outgrowth is a third downstream effect of GDNF. Addition of GDNF to the culture medium resulted in approximately 30% process-bearing SY5Y cells (Figure 4G). However, SorLA overexpression blocked GDNF-induced process outgrowth but did not significantly affect outgrowth induced by retinoic acid (RA) that initiates a different signaling cascade (Figure 4G). In summary, SorLA negatively modulates downstream effects of GDNF signaling likely by controlling both GDNF levels and the subcellular distribution of GFRα1-RET.
SorLA Controls GFRα1 Localization and GDNF Uptake in Neurons
Using western blotting, we found SorLA expressed in all CNS tissues analyzed (Figure 5A), and immunohistochemistry of mouse brain sections revealed the presence of SorLA in distinct neuronal vesicular structures as exemplified in the cortex (Figure 5B) but also in GFAP-positive glia (Figures S6A and S6B). GFRα1 is highly expressed in the developing cortex and hippocampus where it acts independent of RET (Bespalov et al., 2011; Ledda et al., 2007; Pozas and Ibáñez, 2005). In primary hippocampal neurons, we detected high levels of endogenous GFRα1 but not RET, and all neurons also expressed variable levels of SorLA (Figure 5C and data not shown). Interestingly, GFRα1 was markedly enriched in the initial segment of filaments (Figure 5C). As SorLA-positive vesicles are abundant in the soma and protrude gradually through the initial segment of filaments (Figure 5C), we speculated if it might function here as sorting hub for GFRα1 targeted for axons and dendrites. Indeed, such GFRα1 enriched initial segments displayed much stronger surface immunoreaction in neurons derived from SorLA knockout (KO) mice (Figure 5D) albeit total GFRα1 levels were unaltered in KO neurons compared to wild-type (WT) (Figure 5F). We therefore performed GFRα1 immunostainings of nonpermeabilized neurons from both genotypes and quantified the relative intensity of surface staining in the initial segments as illustrated in Figure 5D. Notably, lack of SorLA resulted in a marked increase in surface GFRα1 in the soma and at the initial segment of filaments (Figure 5E), showing that SorLA regulates GFRα1 subcellular localization in neurons. We subsequently tested the ability of hippocampal neurons to bind and metabolize GDNF. Exogenous GDNF (3 nM) avidly bound to the surface of neurons derived from WT and KO mice upon incubation for 2 hr at 0°C (Figures 5G and 5H). Following 30 min incubation in 37°C culture medium, the GDNF signal in the soma markedly decreased and was now found in a few faint vesicles of WT neurons (Figure 5G). In contrast, GDNF remained associated with the surface of SorLA KO neurons, suggesting that endogenous SorLA/GFRα1 complex mediates its endocytosis (Figure 5H).
SorLA Regulates In Vivo GDNF Levels and GDNF-Induced Survival of DA Neurons
The survival of midbrain dopaminergic (DA) neurons in culture depends on exogenous GDNF in a RET-dependent manner, as well as on a supporting layer of glial cells (Burke et al., 1998; Lin et al., 1993). Interestingly, both glia and DA neurons were found to express high levels of SorLA (Figures 6A–6C, S6A, and S6B). Hence, SorLA might regulate DA survival by sequestering extracellular GDNF and altering the subcellular distribution of its receptors. Accordingly, we found that when GFAP-positive cortical glia were incubated with GDNF (3 nM), it accumulated in vesicular structures within 15 min (Figures 6D and 6E). In contrast, no GDNF internalization was observed in cells derived from SorLA KO mice (Figure 6F). In addition, GFRα1 appeared to be increased at the surface of DA neurons from SorLA KO compared to WT neurons where it was more pronounced in vesicular structures (Figure 6G). We therefore cultured rat DA neurons with anti-SorLA or unspecific immunoglobulin G (IgG) in the culture medium. In the absence of GDNF, anti-SorLA did not affect survival compared to the control (data not shown). However, in the presence of GDNF (0.3 nM), SorLA inhibition potentiated survival by nearly 50% (Figure 6H). Similarly, the GDNF-induced survival of SorLA knockout DA neurons was markedly increased compared to that of WT neurons (Figure 6I). To determine if SorLA regulates the amount of GDNF available to DA neurons in vivo, we first examined SorLA expression in the adult DA system by immunohistochemistry. A clear SorLA staining was observed in vesicle-like structures in the soma of tyrosine hydroxylase (TH)-positive midbrain neurons, and also in the surrounding cells (Figure 6J). In striatum, we observed abundance of SorLA-expressing cells (Figure S6E), some of which were also GFAP positive (Figure S6F). We next analyzed adult striatal and midbrain homogenates for their content of GDNF by ELISA and found it be highly elevated in KO mice compared to WT (Figure 6K).
SorLA Deficiency Affects the DA System and Anxiety-Related Behavior
We speculated that the absence of SorLA, and thus potentially altered GDNF activity, might lead to abnormal functionality of the DA system. The overall appearance of the midbrain DA system of SorLA knockouts was normal compared to WT mice as shown by TH staining (Figure S7A). Stereological counting showed that the number of midbrain TH+ neurons in the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) was unchanged in both young (5 weeks, n = 6 in each group) and older (45 weeks, n = 3 in each group, data not shown) Sorl1−/− mice compared to WT mice (Figures 7A and 7B). Also the length density of TH+ nerve fiber protruding from the SNpc and VTA from sections of 10-week-old mice was similar between genotypes (Figures 7C and 7D). To assess DA connectivity in the nigrostriatal system, we performed unilateral intrastriatal injections of the retrograde tracer cholera toxin β subunit (CTb). CTb-labeled TH+ neurons in the SNpc were then identified by double immunofluorescence (IF) and counted. Remarkably, the number of CTb-labeled DA neurons in knockouts was less than one-fourth of that of WT mice (Figures 7E, S7B, and S7C), showing that nigrostriatal connectivity was severely perturbed. The reduction in nigrostriatal connectivity was accompanied by approximately 50% reduction in TH protein levels in striatum of SorLA knockouts as assessed by western blotting (Figures 7F and 7G), and a slight reduction in dopamine levels as measured by high-performance liquid chromatography (HPLC) (Figures 7H and S7D–S7K).
The activity of midbrain DA neurons is instrumental in the behavioral response to phychostimulants. We therefore tested the role of SorLA in amphetamine-induced hyperlocomotion by injecting WT and SorLA knockouts with either saline or amphetamine and monitoring their behavior in an open field. The locomotor activity of WT mice was increased approximately three times upon amphetamine (10 mg/kg) administration and the average distance traveled during 40 min augmented from 58.28 ± 4.19 m to 172.51 ± 19.88 m (p = 2E-4). In contrast, saline-injected KO mice were already hyperactive compared to WT controls (p = 0.006) and traveled a distance of approximately 108.62 ± 14.52 m, but no increase was observed following injection of amphetamine (104.33 ± 6.76 m) (Figures 7I–7J), strongly suggesting impaired DA functionality in SorLA KO mice. The pronounced hyperactivity of saline-treated knockouts led us to study their anxiety-related behavior. In the elevated plus maze, knockouts displayed markedly reduced anxiety levels evidenced by more entries and increased time spent in the open arms of the maze compared to controls (Figures 7K–7M). In striking contrast, Gdnf+/− mice displayed markedly increased anxiety characterized by reduced entries and time in the open arms (Figures 7N and 7O). The combined data show that SorLA is critical for DA function and anxiety-related behavior. Previous studies have shown that SorLA affects processing of amyloid precursor protein (APP) (Andersen et al., 2005) and sorting of lipoprotein lipase (LpL) to lysosomes (Klinger et al., 2011). However, neither APP nor LpL has been implicated in DA function so far. It is therefore tempting to speculate that the impaired DA function in SorLA KO is mainly caused by altered activity of GDNF and potentially of other GFLs.
We here identify SorLA as a sorting receptor for GDNF and GFRα1 that traffics a ternary SorLA/GFRα1/GDNF protein complex from the cell surface to endosomal compartments, thereby mediating uptake and clearance of extracellular GDNF as well as redistribution of the GFRα1 receptor pool. The SorLA/GFRα1 complex further targets RET for endocytosis independent of GDNF, thereby altering its subcellular distribution and signaling. Thus, SorLA represents an example of a transmembrane protein that actively mediates trafficking of complexes of a neurotrophic factor and its receptors. The SorLA/GFRα1 complex is most likely stable once formed, as the affinity is unusually high for a receptor-receptor interaction (Kd = 6 nM) considering the restricted diffusion in the cell membrane. For comparison, this affinity is approximately 5- to 30-fold higher than what has been reported for Eph-ephrin interactions (Himanen et al., 2004) and at least 70-fold higher than for neurexin-neuroligin binding in Biacore experiments (Koehnke et al., 2010). As GFRα1 itself has no cytosolic link to the intracellular sorting machinery, the docking of the SorLA tail to the adaptors AP-1 or the retromer complex (Nielsen et al., 2007) also results in retraction of GFRα1 from late endosomes, subsequent return to the trans-Golgi and then re-exit to the cell surface for another round of GDNF capture. The physiological role of SorLA/GFRα1-mediated trafficking of RET is unclear, but, in exerting its neurotrophic function, the RET/GFRα1/GDNF complex is well known to undergo internalization and retrograde transport from axon terminals to the soma. This is unlikely to involve SorLA, which is largely absent from nerve endings (Klinger et al., 2011), and because the SorLA/GFRα1-mediated RET endocytosis is independent of GDNF. However, it is evident, at least in overexpressing cells, that the SorLA/GFRα1-mediated endocytosis of GDNF and RET is quantitatively more important than internalization of the RET/GFRα1/GDNF complex.
Through this mechanism, SorLA inhibits downstream effects of GDNF-induced signaling such as cell survival, proliferation, and differentiation of neuronal cells, notably the survival of DA neurons, by lysosomal targeting of GDNF, and by altering GFRα1/RET subcellular distribution in both neurons and glia. Future studies will be carried out to dissect how SorLA/GFRα complexes affect sorting of neurturin, artemin, and persephin, and what role SorLA might play in other neuronal systems that depend on GFL activity. Finally, it would be important to address whether SorLA differentially affects GDNF-induced RET signaling versus RET-independent signaling.
GDNF has received massive attention as a potential treatment for Parkinson's disease due to its ability to protect and repair lesioned nigrostriatal pathway in vivo, but only very little is known about its role in the intact DA system. In both animal models of neurotoxicity and human patients, GDNF has been found to initially potentiate the DA system through increasing TH mRNA and protein levels, dopamine synthesis, and uptake, and by enhancing DA target innervation. However, long-lasting overexpression of GDNF in rats with lesioned nigrostriatal system led to downregulation of TH. Similarly, long-term GDNF overexpression in the striatum of rats with intact nigrostriatal pathway led to compensatory downregulation of TH with no effect on dopamine levels (Georgievska et al., 2002, Georgievska et al., 2004; Rosenblad et al., 2003) while another study reported reductions in both striatal TH and dopamine (Sajadi et al., 2005). These observations have been proposed to reflect compensatory effects of increased GDNF actions on the DA synapse and at the level of TH expression (Georgievska et al., 2004; Rosenblad et al., 2003). Contrary, dopamine levels are normal in the striatum and even increased in the nucleus accumbens of Gdnf+/− mice compared to their control littermates (Gerlai et al., 2001).
In the present study, we find that SorLA deficiency results in increased in vivo levels of GDNF that, as may be expected from the above, translates into reduced striatal TH albeit unaltered number of TH+ neurons in the midbrain and normal nigral fiber length in SorLA KO mice. Increased GDNF activity in the VTA or nucleus accumbens has been reported to diminish the behavioral response to drugs of abuse (Carnicella and Ron, 2009). Conversely, infusion of GDNF inhibitory antibodies into the VTA or lack of one GDNF allele in Gdnf+/− mice increases the drug response (Messer et al., 2000). Intriguingly, SorLA knockouts display completely blunted response to amphetamine, which is most likely explained by a dramatic reduction in nigrostriatal connectivity as determined by retrograde tracing. Such reduced sensitivity to amphetamine is a characteristic of the dopamine transporter (DAT) knockout (Giros et al., 1996) and mice lacking α-synuclein (Abeliovich et al., 2000), both molecules involved in DA synaptic function. Blunted response to amphetamine has also been reported in mice transgenic for molecules involved in shaping DA connectivity such as the netrin-1-receptor-deficient (Grant et al., 2007) or EphA5-overexpressing mice (Sieber et al., 2004). The insensitivity toward amphetamine of SorLA knockouts stands somewhat in contrast to the previously reported increased response to amphetamine 4 days after a single dose of GDNF or neurturin into the striatum of mice (Horger et al., 1998). However, the SorLA KO phenotype may instead reflect an adaptive response to a long-term increase in GFL activity. SorLA KO mice were further characterized by marked hyperactivity and reduced anxiety levels, in striking contrast to mice lacking one GDNF allele, suggesting that the two could operate together to regulate anxiety-related behavior. This is interesting, as epigenetic regulation of GDNF expression was recently proposed to affect susceptibility to stressful events with its repression correlating with increased anxiety (Uchida et al., 2011). Thus, SorLA KO animals exhibit behavioral traits that may model symptoms of psychiatric disorders, in particular, ADHD, and further studies should validate the potential of SorLA-deficient mice as an animal model of ADHD. Interestingly, components of the GDNF system show no genetic association with the development of neurodegenerative diseases, but linkage has been suggested to ADHD, schizophrenia, and other neuropsychiatric disorders related to dysfunction of the DA system (Michelato et al., 2004; Souza et al., 2010; Syed et al., 2007).
We conclude that the interaction among SorLA, GFRα1, and GDNF is a key regulatory element in the GDNF signaling through GFRα1-RET. Our results also suggest that SorLA is a potential target for the treatment of pathological conditions related to the DA system such as drug abuse and Parkinson's disease.
Experimental Procedures
Biacore was performed as described (Jacobsen et al., 2001). Binding was expressed in relative response units (RUs), the difference in response between the immobilized protein flow cell and the corresponding control flow cell. Kinetic parameters were determined using BIAevaluation 4.1.
Internalization Assay
Cells were cultured on poly-L-lysine coated coverslips and incubated with cold medium containing GDNF (3 nM), anti-GFRα1 (0.1 μg/ml), or anti-RET (0.1 μg/ml) for 2 hr on ice. The culture medium was then changed to 37°C normal medium and at specific time points, and the cells were fixed in 4% paraformaldehyde (PFA) (pH 7.4) and permeabilized with 0.1% Triton X-100 unless indicated otherwise. Internalized ligand and subcellular markers were subsequently visualized using IF on a LSM 710 (Carl Zeiss). In different experiments, cells were incubated with GDNF added to 37°C medium for defined time periods. Internalization was quantified from nonsaturated immunofluorescent images using ImageJ. Regions of interest were randomly selected along the plasma membrane of at least 120 cells for each time point. Inhibition of lysosomal proteinases was achieved by adding fresh medium containing leupeptin and pepstatin (50 μg/ml, Sigma-Aldrich) every 6 hr, starting 24 hr before the experiments.
Metabolic Labeling
Transfected HEK293 or SY5Y cells were labeled using ∼200 μCi/ml L-[35S]-cysteine and L-[35S]-methionine (Pro-mix; GE Healthcare) in medium without methionine and cysteine in the presence of Brefeldin A (Pierce). After 4 hr, the cells were washed and changed to normal culture medium, and newly synthesized GFRα1 or RET were chased for specific time periods. GFRα1 or RET was subsequently immunoprecipitated from cell lysates, separated by SDS-PAGE, and visualized using phosphoimaging.
DA Cultures
Cultures of DA neurons were prepared from the midbrain of P0 rats or mice and grown on a layer of cortical glial cells as described (Burke et al., 1998). Culture medium was supplemented with 0.3 nM GDNF and changed every 3 days. The effect of endogenous SorLA in rat DA neurons was assessed by adding 10 μg/ml SorLA antibodies (rabbit anti-human SorLA) or control antibodies to the medium (rabbit IgG, R&D Systems). The number of surviving DA neurons was counted at 7 DIV (for rats) or at 4 DIV (for mice) after TH staining.
Behavioral tests were performed during the light phase (9 a.m. to 4 p.m.) Mice were tested for anxiety levels in an elevated plus maze essentially as described (Chen et al., 2006). In the open-field test, wild-type and Sorl1−/− mice were administered amphetamine (10 mg/kg) or saline by intraperitoneal (i.p.) injection, and placed in the corner of a (40 × 40 × 35 cm) clear Plexiglas arena and their activity was recorded over a 40 min session and analyzed using the Any-maze tracking software.
The SorLA KO mouse was first described in Andersen et al., 2005 and has been backcrossed for ten generations into C57/BL6J. Behavioral studies were done with the backcrossed homozygous mice compared to the same C57/BL6J substrain that was used for backcrossing. The behavioral studies have subsequently been recapitulated using KO and WT offspring from heterozygous breedings. Gdnf+/+ and Gdnf+/− littermates (Pichel et al., 1996) were obtained by heterozygous breeding using a GDNF KO line that has been backcrossed for five generations into C57/BL6J. Male mice were used for all experiments shown, but similar results were obtained using both genders (data not shown). Animal experiments were performed according to institutional and national regulations.
For further details, please see the Extended Experimental Procedures.
DNA Constructs, Proteins, and Antibodies
Human full length WT SorLA and endocytosis-deficient SorLA mutant inserted into pcDNA3.1(-)/zeo have previously been described in Jacobsen et al., 2001; and Nielsen et al., 2007. Rat WT GFRα1 cDNA was inserted in pcDNA3 and human wild-type RET cDNA in pCR3.1 as described (Virtanen et al., 2005). The prodomain of human GDNF (F20-R77) and the prodomain including the N-terminal 38 amino acids of mature GDNF (F20-N115) were amplified from cDNA generated from fetal brain by RT-PCR and ligated into pGEX4T-1 (GE Healthcare). The prodomain of human neurturin (I20-R97) was generated as described (Westergaard et al., 2005). The peptides were expressed in E. coli BL21 as GST-fusion proteins and purified using glutathione-Sepharose beads (GE Healthcare). A truncated soluble variant of GFRα1 (isoform 2; amino acids M1-L420) was cloned into site A of pIRES (Clonetech) with zeocine resistance gene in site B and expressed in CHO-K1. Purification was performed by IMAC on Ni-NTA Superflow Cartrigde (5mL, QIAGEN). Purified recombinant human GDNF was from ProSpec. Neurturin, artemin, and persephin were from R&D systems. Anti-SorLA monoclonal and rabbit polyclonal antibodies were generated by immunization with purified SorLA extracellular domain as described in Klinger et al., 2011. Mouse anti-pErk, anti-pAkt, and Akt and Erk antibodies were from Cell Signaling. Goat anti-GDNF, goat anti-GFRα1-4, and goat anti-RET were all from R&D systems. Mouse anti-TH for Western blotting was from BD laboratories. Rabbit anti-TH for immunostainings was from Pel-Freez. Dopamine transporter (DAT) mAb was from Santa Cruz. Sheep anti-TGN46 was from AbDSerotec. Rabbit anti-GFAP was from Dako. EEA1 and Lamp1 mAb were from BD laboratories. Rabbit anti-iba1 (019-19741) was from Wako. Further details on our use of these antibodies can be found at http://www.pabmabs.com.
Cell Cultures
All cell lines were cultured at 37°C in a humidified atmosphere containing 5% CO2. Human embryonic kidney HEK293 cells and SY5Y neuroblastoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM) or DMEM-F12, respectively, supplemented with 10% fetal calf serum (FCS). All culture media were supplemented with penicillin (50 U/ml) and streptomycin (50 μg/ml). Transfection of the cells was carried out using FUGENE 6 (Roche) or Turbofect (Fermentas) as transfection agent. 48 hr post transfection, stable transfectants were selected at 300 μg/ml zeocin and/or 400 μg/ml G418 (GIBCO-BRL) and identified by Western blotting and immunocytochemistry.
Binding Studies in Cells
For the cell-based homologous binding assay, GDNF was iodinated with [125I]Na (PerkinElmer Life Sciences) by lactoperoxidase method (Laurikainen et al., 2000). HEK293 cells were plated on poly-lysine coated 35mm culture dishes and transfected with endocytosis-deficient SorLA mutant or GFRα1 plasmid. At 48 hr, cells were blocked using binding buffer (Dulbecco's modified Eagle's medium (DMEM), 0.5% skimmed milk, 0.2% BSA, 15 mM HEPES, pH 7.2) for 30 min on ice. Next, cells were incubated on ice for 2 hr with 100pM [125I]-GDNF in binding buffer in the presence of increasing concentrations of unlabelled WT GDNF (0 to 50nM). Thereafter, cells were washed four times with DMEM/15 mM HEPES, pH7.2 and lysed with 1M NaOH. Samples were counted on a 1214 RackBeta (Wallac/LKB) scintillation counter. The background level was assessed using GFP-transfected cells.
Western Blotting
Protein samples were separated to SDS-PAGE and electro-blotted for 2 hr onto polyvinylidenedifluoride (PVDF) filters (GE Healthcare) in 192 mM glycine, 25 mM Tris-HCl, pH 8.0. Membranes were subsequently blocked in 0.05 M Tris-base, 0.5 M NaCl supplemented with 2% skimmed milk powder and 2% Tween-20. After blocking, membranes were washed in 10 mM HEPES pH 7.8, 140 mM NaCl, 2 mM CaCl2, 1 mM mgCl2, 0.2% skimmed milk powder, 0.05% Tween-20, and incubated with primary antibodies diluted in washing buffer as follows. Rabbit anti-SorLA serum 1:1000, rabbit anti-pErk, anti-pAkt, anti-Akt and anti-Erk (Cell Signaling) 1:1000, mouse anti-TH (BD laboratories) 1:2000, goat anti-RET and goat anti-GFRα1 (R&D systems) 0.1 μg/ml. Anti-DAT (Santa Cruz) 1:1000. Following another washing step, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Following a final washing step, blots were visualized with the ECL plus Western Blotting detection system (GE Healthcare) and Fuji film LAS1000.
Cells were treated with 2 nM reducible protein cross-linker dithiobis-(succinimidylpropionate) (DSP) (Pierce). After washing, cells were lysed on ice for 10 min in TNE buffer supplemented with protease inhibitors. Samples were immunoprecipitated overnight at 4°C by use of Gammabind G-Sepharose beads (GE Healthcare) coupled with antibodies. Unspecific binding was removed by washing 5 times in Tris-buffered saline containing 0.05% Tween-20, and proteins eluted by boiling samples in reducing sample buffer (20 mM dithioerythreitol (DTE), 2.5% SDS).
Coronal sections of the brain were obtained from Sorl1−/− and wild-type mice perfusion fixated in 4% PFA. The brains were harvested and prepared for paraffin embedding or cryoembedding in O.C.T. (Sakura Finetek, Denmark). The paraffin embedded mouse brain sections were deparaffinated, and then demasked using pronase treatment. The cryo- or deparaffinated sections were then incubated with primary antibodies for 24 hr at 4°C. The deparaffinated sections were developed with FastRed. The cryo embedded sections were incubated for 2 hr at RT with fluorescently labeled secondary antibodies. Samples were analyzed on a Zeiss confocal LSM 710 microscope.
Cellular Assays
SY5Y cells were seeded into poly-lysine coated black 96-well tissue culture plates (PerkinElmer Life Sciences) at a density of 10.000 cells/well. After 24 hr, cells were washed three times in DMEM without phenol red, supplemented with antibiotics, 1% glutamax (GIBCO), and incubated with or without 3 nM GDNF. At 72 hr, the number of live cells was scored with fluorescent-based assay (Promega) on a Victor3 1420 multilabel counter. The signal obtained using this assay depends linearly on the number of cells per well. Cell proliferation was assessed similarly but in normal culture medium. Cell differentiation was studied by scoring the number of process-bearing cells in a culture treated with GDNF (3 nM) for 72 hr. A process was defined as an extension when at least twice the length of the cell body diameter.
Hippocampal Cultures
Cultures of hippocampal neurons were derived from P0 neonate hippocampus that were dissected in cold PBS and placed in ice-cold L-15 medium (Invitrogen). Papain (Worthington) was diluted in L-15 (20 units/ml, EDTA 2 mM) and activated at 37°C for 20 min. Tissues were digested in the papain solution for 30 min at 37°C and the enzyme reaction quenched with DMEM + 10% FCS. After a brief spin down, the tissue pellet was resuspended by DMEM (10% FCS and DNaseI 10 units/ml) and triturated with fire-polished plastic pipettes. Dissociated cells were spun down again and resuspended in Neurobasal (Invitrogen) supplemented with B27 (Invitrogen), Primocin (Invivogen) and flouro-uridin (Sigma) for plating. Hippocampal cells were cultured in 12-well dishes (approx. 1.5 mio cells per well) or 12mm coverslips (approx. 0.1 mio cells per coverslip) coated with poly-L-lysine (Sigma) and laminin (Invitrogen). Half of the medium was replaced twice a week and the neurons were fixed or used for experiments at 11DIV.
Immuno Electron Microscopy
For electron microscopy, cells were fixed with 4% formaldehyde for 30 min in 0.01 M phosphate-buffered saline (PBS), pH 7.4, at room temperature. The cells were washed x3 and removed from the support with a rubber policeman in the same buffer containing 1% gelatine (Merck), pelleted and embedded in 12% gelatine, infiltrated with 2.3 M sucrose in PBS for 30 min, and frozen in liquid nitrogen. Ultrathin (70 ± 90 nm) cryosections were sectioned at −100°C on a FCS Reichert Ultracut S cryoultramicrotome and collected on 200 mesh Ni grids. The sections were preincubated with 0.01 M PBS, 1% BSA, 0.05 M glycine, pH 7.4, followed by incubation over night with primary antibodies (mouse anti-SorLA (mAb 20C11),1:50 and goat anti-GFRα1 (R&D Systems), 1:100) prior to 2 hr incubation with donkey anti-mouse gold (10nm) and donkey anti-goat gold (6 nm) (BioCell, Cardiff, UK). All incubations were performed at 4°C. The sections were finally contrasted with methyl cellulose containing 0.3% uranylacetate and studied in a FEI CM100 electron microscope.
Subcellular Fractionation
Post nuclear supernatants (PNS) were prepared from SorLA transfected or mock transfected SY5Y neuroblastoma cells and submitted to a two-step subcellular fractionation protocol. In brief, three confluent T-160 tissue culture flasks with SY5Y and SY5Y/SorLA cells were washed, harvested, and pelleted in Tris-buffered saline (TBS) supplemented with 0.4 mM phenylmethylsulfonyl fluoride (PMSF) at 4°C. Cells were resuspended in homogenization buffer (0.25 M sucrose, 10 mM HEPES-KOH pH 7.2, 1 mM EDTA pH 7.5, 1 mM magnesium acetate (MgOAc), and 0.4 mM PMSF) and centrifuged at 1800 rpm for 7 min at 4°C. Following resuspension in 1 ml homogenization buffer, cells were disrupted by passage through a 21-gauge needle followed by five times passage through a metal cell cracker with a 9-μm gap at 4°C. In order to remove nuclei and unbroken cells, the homogenate was centrifuged at 3200 rpm for 7 min at 4°C. For velocity gradient centrifugation, the supernatant was overlaid a continuous 0.3 – 1.2 M sucrose gradient (in 10 mM HEPES-KOH pH 7.2) prepared by mixing at 45° for 10 min followed by 80° for 1 min with a Biocomp gradient master. Samples were centrifuged at 25.000 rpm for 18 min using a SW40 rotor. Collection of fractions was done using a Biocomp piston gradient fractionator. For equilibrium gradient centrifugations, selected fractions were pooled and centrifuged (overnight at 25.000 rpm using a SW40 rotor) over a 0.8 – 1.6 M sucrose step gradient. The resulting fractions were collected as above and analyzed by Western blotting using anti-SorLA and anti-RET.
Tissue Preparation
Mice were killed by cervical dislocation. Tissue was lysed in 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.8, 140 mM NaCl, and 1% Triton X-100 containing protease inhibitors (Complete, Roche) using a pellet pestle and centrifuged at 10.000 x g for 10 min at 4°C. Samples were kept on ice at all times and protein concentration was determined using the Bicinchoninic Acid kit from Sigma-Aldrich.
Tissue GDNF protein content was measured in samples obtained from wild-type and SorLA knockout mice using a commercial ELISA kit (GDNF Emax Immunoassay System; Promega). All of the tissues were removed and immediately frozen on dry ice. The tissues were afterward homogenized using a motor driven pellet pestle. We pooled the tissue from three mice into one sample. Protein concentration was determined using a BCA protein assay (Sigma-Aldrich). The protein extraction and the ELISA were carried out following the instructions of the manufacturer. Absorbance at 450 nm was measured in a plate reader (Wallac VICTOR3 1420 Multilabel Counter, PerkinElmer Life sciences).
Estimation of Total Number of TH+ Neurons in SNpc and VTA
Mice brains were perfusion fixed with phosphate-buffered 4% formaldehyde through the left ventricle of the heart for 5 min and post-fixed overnight in the same fixative. Following sucrose-treatment, the brains were frozen in isopentane and cut exhaustively in 65 μm thick sections on a cryostat. Every third section was sampled systematically and stained with rabbit anti-TH antibody (Pel-freez Biologicals). The total number of TH+ neurons were estimated by the optical fractionator with varying height sampling fraction (Dorph-Petersen and Lewis, 2011) using an Olympus BX 50 light microscope equipped with a 60x oil lens (NA = 1.40), a Prior motorized stage, a Heidenhain microcator, and an Olympus DP70 digital camera controlled by newCAST (Visiopharm, Hørsholm, Denmark) software. The step lengths in x- and y-direction were 60 μm, the area of the 2D unbiased counting frame was 1615 μm and the disector height was 10 μm. The TH+ neurons were counted in substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) in one side of the brain chosen at random. The coefficient of errors of the two number estimates was both approximately 9% (Gundersen et al., 1999).
Estimation of Total Length of TH+ Nerve Fibers in SNpc and VTA
Six WT and six SorLA KO mice aged 10 weeks were perfusion fixed through the left ventricle with 4% phosphate-buffered formaldehyde. Brains were cut in 60 μm thick sections on a vibratome and every third section was sampled systematically and TH+ nerve fibers were stained with primary rabbit-anti-TH (Millipore, AB 152) 1:2000, secondary biotinylated goat-anti-rabbit (Vector, BA1000) 1:200 and visualized by 3,3-diaminobenzidine (DAB). As length estimation using thin sections requires isotropic sections, we instead used thick sections and isotropic virtual planes (Larsen et al., 1998) together with the microscope described above and newCAST (Visiopharm, Hørsholm, Denmark) software. The distance between virtual planes was 8 μm, there were five planes per box, which had an area of 500 μm2, and step lengths were 600 μm. The length density of TH+ nerve fibers was then estimated in SNpc and VTA. The Cavalieri estimator was used to estimate the volume of the two regions using the 2D nucleator. The product of the length density and volume of the reference regions was then used to estimate the total length of TH+ nerve fibers in substantia nigra and ventral tegmental area.
Estimation of Retrogradely Labeled TH+ Neurons in Substantia Nigra
SorLA KO mice and WT mice were anesthetized with Hypnorm and midazolam, placed in a stereotaxic frame (Kopf Instruments, Model 1404, Turjunga, CA, USA). Mice were iontophoretic injected with a 4% solution of the retrograde tracer Cholera Toxin β-subunit (CTb) (Cat No. 104, LIST Biological Laboratories, Campbell, CA) in 0.9% saline, pulse 10mV 7 s on; 7 s off for 10minutes as described in (Hale et al., 2008). As coordinates were used Bregma level 0.38mm, lateral 2.00mm; and 3.5mm below dura adopted from Paxinos and Watson mouse brain atlas (The mouse brain in stereotaxic coordinates; 2nd edition, 2001 AP, George Paxinos and Keith J. Franklin). The procedure was followed by postoperative antibiotic and pain treatment (BaytrilH vet, 5 mg/kg and Rimadyl 5 mg/kg). By using double immunostaining with antibodies against TH and CTb, a portion of the retrogradely labeled neurons could be observed and quantified to co-store TH. One week after the injection of CTb in striatum, mice were anesthetized with Hypnorm and midazolam and perfused transcardially with PBS for 30 s followed by a solution of 4% phosphate-buffered formaldehyde for 6 min and post-fixed for 4 hr in same fixative. Brains were cryoprotected for 3 days in 30% sucrose in PBS, frozen in isopentane and 60 μm-thick sections were cut on a cryostat. Sections were divided into three series by systematic sampling, and one set of slices was used for double immunostaining using primary antibodies directed against CTb (goat anti-CTb, Cat No. 703, 1:1000, List Biological Laboratories) and primary rabbit-anti-TH (Millipore, AB 152) 1:2000 overnight. Cryo-sections containing SNpc was observed in the above-mentioned light microscope system with a fluorescent attachment and an Olympus U-M51006 Dual band filterset for FITC/Texas Red. The optical fractionator with varying height sampling fraction (see above) was used to count the double-stained neurons.
Dopamine Levels
To determine the concentrations of 3,4-dihydroxyphenylalanine (DOPA), dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 3-methoxythyramine (3-MT), the striatal samples were homogenized with a teflon pestle adding 0.2 ml ice-cold 0.5 M/l perchloric acid containing 0.05% w/v sodium thiosulfate (Pharmacy, Aarhus University Hospital). The homogenate was centrifuged for 5 min at 13.200 rpm. The supernatant was analyzed by HPLC using Perkin-Elmer Series 200 HPLC pump (Titan) connected in series with a Rheodyne 7025 injector (20 μl loop), a Phenomenex Sphereclone 5μ ODS (2) (250 × 4.6 mm, Phenomenex), a Coulochem III (ESA) electrochemical detector in amperometry mode (400mV). For data acquisition Dionex Chromeleon Software 6.8 was used. The mobile phase was a mixture of 90% buffer containing 37 mM NaH2PO4 x H2O (4.55 g/l), 1 mM octanesulphonic acid sodium salt (195 mg/l), 0.04 mM EDTA (14 mg/l), pH 3.3 adjusted with acetic acid and 10% ethanol. At a flow rate of 1 ml/min DOPA eluted around 4 min, DOPAC around 8 min, dopamine around 10 min, HVA around 15 min and 3-MT around 20 min. Metabolite concentrations were determined by comparing the signals with those of the respective compound, concentration 1 μg/ml each. For details, see (Gamache et al., 1993; Mikkelsen et al., 1999).
Error bars are standard error of mean. Significance was evaluated using two-tailed t test.
This study was funded by the Lundbeck Foundation (C.M.P., A.N., J.R.N., M.S.), Danish Medical Research Council (S.G., E.I.C.), EU/Marie Curie fellowship (MEST-CT-2005-019729) (M.L.), and Academy of Finland (grant no 11411226) (M.S.). Jan Jacobsen is greatly acknowledged for dopamine and metabolite measurements. Anja Aagaard Pedersen, Benedicte Vestergaard, Helene Andersen, Hanne Sidelmann, and Hang Pham Jensen are thanked for excellent technical assistance.
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Publication Information
Received: January 22, 2012
Revised: October 23, 2012
Accepted: December 14, 2012
Published: January 17, 2013


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Martin Lawrence Interview BIG MOMMA'S: LIKE FATHER, LIKE SON; Updates on BAD BOYS 3 and THE SKANK ROBBERS With Jamie Foxx
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In the comedy film Big Momma's: Like Father, Like Son, FBI Agent Malcolm Turner (Martin Lawrence) is back undercover as alter-ego Big Momma, but this time he is joined by his teenage stepson Trent (Brandon T. Jackson). After Trent witnesses a murder, he does a little undercover work of his own, as the hefty Charmaine, as the two go to an all-girls performing arts school to find the killer before he finds them.
During the film's recent press day, actor/comedian Martin Lawrence talked about returning to the character of Big Momma for the third time, dreading the make-up process to become the larger-than-life matriarch, the possibility of Big Momma teaming up with Tyler Perry's Madea, and how he'd definitely put on the fat suit again, if the audience demanded it. He also talked about the status of Bad Boys 3 and his desire to re-team with co-star Will Smith, along with returning to the character of Sheneneh, opposite Jamie Foxx as Wanda, for The Skank Robbers. Check out what he had to say after the jump:
Before getting to the full interview, for all the fans of Bad Boys that are waiting for a third film, Lawrence says:
"they're writing the script, as we speak. We're just waiting for it to come together. I think Jerry Bruckheimer is meeting with some of my people. Will came down to see me, when I was filming Death at a Funeral, and I was like, "What do you think? Do you think we can do ...
<< Big Mommas: Like Father, Like Son: Generic Interview – Martin Lawrence

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NO, I am not yet 21,
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HMRC advertises for new CIO
The new chief information officer of HM Revenue and Customs will initially earn up to £180k, according to the advertisement for the role. The job, initially a three year fixed term appointment, is in a salary band paying £100,000 to £205,000 annually. It is open to UK nationals with "extensive information technology leadership …
I'll do it
Just place all tenders of interest in a plain, brown (preferably bulging) envelope and I am sure I will have no trouble in selecting the "best" partner within which to co-align or core synergies and set the standard of homologous revenue empowerment.
No joke icon because, frankly, it's not funny; it's reality. :o(
This should be an easy job to get
"I will ensure only encrypted media will be lost in car parks, on trains and in airports"
"I will ensure secure network transfers of sensitive data are made. The Royal Mail can kiss goodbye to transporting our CDs"
"I will ensure that all coats are patted-down when people are leaving the office....."
HMRC certainly needs new leadership
Two thirds of the hassle of filling in the Self Assessment pages could be avoided if the web site made proper use of information ALREADY known to HMRC instead of asking the taxpayer to fill it in over and over again.
As an absolute minimum, that means:
a) tax returns for previous years remaining viewable for the statutory SIX years
b) ability to automatically COPY last year's answer as default in all questions where the answer is still likely to be correct (eg employer's name, bank account number, etc)
c) Not asking for information already known to HMRC. Use the information submitted by the third party (UK bank interest, share dividents, wages, pensions etc) and ask for confirmation that the values are correct instead of entering them from scratch).
d) Already know about, and *USE*, details of my overpayments and overpayments. You should be TELLING me about them, not asking me to tell you what you already should know.
180k for ...
.. just sprinkling random DvD / CD/ Flash disk copies/backups on various forms of public transport?! Where do I apply?
@AC HMRC certainly needs new leadership
...But then it would be much harder to fill it in 2 years late. If they used all the info they already have they'd be chasing you for an amount when you go over the deadline as opposed to just fining you 100 quid and asking you nicely to remember it and get it in soon. Even though they charge interest and fine you, sometimes you can be much better off leaving it until you actually have the 20K you owe them before telling them that's how much you owe!
I'll bet my left ball
That the only qualifications needed are the ability to use the words ''process'', ''synergy'' and ''cost rationalism'' as if you really knew what they meant. Hell painting your nose brown would be a sure shot way of breezing through the personal round.


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Due to its contents, this product cannot be shipped via our Priority Service or sent to Alaska, Hawaii, and/or APO/FPO military addresses.
Promotional offers available online at Kohls.com may vary from those offered in Kohl's stores.


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Some information is missing or invalid below.
Catch all your cooking spills with this Range Kleen drip pan set.
- Style A design fits most electric ranges with one large and three small plug-in burners.
- Original equipment manufacturing (OEM) guarantees quality, fit and performance.
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- Details:
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- Set includes: 1 large & 3 small drip pans
- Fits most Amana, Crosley, Frigidaire, Maytag and Whirlpool stoves
- Hand wash
- Manufacturer's 1-year limited warranty
- Model no. P10124XZ
- Promotional offers available online at Kohls.com may vary from those offered in Kohl's stores.


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This foot of poetry is made up of three syllables: two unstressed syllables followed by a stressed syllable: dadaDUM.
Want a famous example? Clement C. Moore's poem "Twas The Night Before Christmas" is made up almost entirely of anapests. When you read it aloud, it sounds like a bunch of dadaDUMs strung together.
For anapestic poems on Shmoop, check out our analysis of William Blake's "The Sick Rose" and W.H. Auden's "The Unknown Citizen".


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The Beachfront house at Casuarina bay in Bahamas
- bedrooms 4
- baths 2
- sleeps 8
- pool no
- hot tub no
- pets allowed yes
- type House
- community
rates (USD)
/ night
$ 2,950 - $ 3,950 / week
vacation rental description
The Beachfront House at Casuarina bay was designed and built to savor the incredible natural
unspoiled beauty of Eleuthera.
It is only 50 feet from its own beautiful sand beach, so wherever you are in the house you have
breathtaking views in all directions.
To the east you see the turquoise ocean and glistening beach. Looking to the west your eyes rest on
untouched green hills and colorful Caribbean sunsets. At night the brilliance of the stars and moon
are unforgettable.
This hand crafted cedar house sleeps 6-8, with 3 main bedrooms (1 king, 1 queen, 2 twins), and one garden bedroom that is accessable from the outside of the house only. The garden bedroom and twin bedroom have A/C.
There are 2 full bathrooms, living and dining room, kitchen and decks on 3 sides plus a private balcony on the second floor
overlooking the ocean.
The house is built on a 14 acre estate stretching across to the ocean on other side of the island.
The property is filled with lush plants and fragrant tropical flowers. You can relax on our many
decks, private beach or in the thatched cabana, where there is a freshwater shower.
This beautiful house is perfect for a family vacation or group of friends looking to relax and enjoy
the spectacular beauty of this is magical island.
property availability
Last modified on 02-4-2014 at 12:14 PM.
email owner view full calendar
Taja McKinney 310 999 1677
house rental amenities
The house has free wireless Internet. There is a BBQ area over looking the water and outdoor dinning areas on the deck, in the garden and
on the beach.
The house has a fully equipped modern kitchen including a dishwasher, stove, large fridge, microwave,
coffee maker, blender etc. There is satellite TV plus a DVD player and selection of videos and DVDs. We also have free wireless Internet.
There is a phone, CD player and tape deck.
We supply all towels and linens for your stay and there are laundry facilities you are welcome to
use. We provide ocean kayaks, boogie boards and other fun beach toys.
activities and attractions in Bahamas Caribbean
Ocean-lovers will find some of the best snorkeling, scuba diving and swimming in the world.
Underwater visibility is up to 100 feet, with water temperatures ranging between 70 and 80 degrees
Fahrenheit. One can snorkel from right in front of the house or explore the miles of exceptional
beaches with our ocean kayaks.
The adventurous traveler can enjoy nearby islands and virgin coral reefs teeming with exotic tropical
fish, or charter boats for world-class bone or deep-sea fishing. Other sports available on the island
include golf, tennis and surfing.
The stars at night and the moonrise out of the ocean are strikingly beautiful on this remote island
Easily accessible, Casuarina Bay is just 1 hour by air from Fort Lauderdale or Miami and 15 minutes
from Nassau. Casuarina bay is only seven miles from Governors Harbour airport. Our 14 acre sea to sea
estate is located 1.5 miles northeast of the charming village of Governors Harbour, one of the oldest
settlements in the Bahamas. There you will find food and gift stores, restaurants, bars, bank,
clinic, dive shop, bike rentals, library with internet etc.
Eleuthera, a 110 mile long island, offers many fascinating places and deserted beaches to explore and we have a guide in the house for when you arrival with our suggestions on beaches to visit, day trips, restaurants, maps etc.
vacation rental rates
Rates are for 6 guests and include use of towels, linens, ocean kayaks, wireless Internet etc.
Rate includes all cleaning fees, taxes etc.
Additional guests are $200 per week. No charge for children under 6.
5 night minimum stay.
$700 refundable damage deposit required.
$100 surcharge for Sunday check-in
Pet fee $75.
SPECIALS- Please inquire for last minute specials.
We accept personal checks, credit cards and paypal payments.
Low Season rate:
July 1st -Oct 31st : $2,950/Week or $460 per night
High Season rate:
Nov 1st- Dec 20th and Jan 5th 2011 to June 30th: $3250/week or $495 per day
Christmas/New Years rate:
Dec 21st- Jan 4th: $3,950/Week or $600 per night
Taja McKinney 310 999 1677
accepted payment methods
Prior to paying a reservation deposit, always verify payment instructions by calling the phone number displayed on this listing page. By contacting the owner directly and paying by an approved method - credit card, paypal, check or bank transfer - your reservation payment will be protected up to $1,000.
Learn more in our Security CenterTop things to do in Bahamas
- Snorkel around beautiful coral reefs
- Sample excellent island cuisine and tropical drinks
- Relax!
- go fishing
Top things to do on a budget
- Kayak and explore the island coastline
- Enjoy exquisite sunsets and watch the moon rise from the ocean
- Star gaze under the spectacular night sky
- Pic Nic on stunning untouched beaches
- Swim and snorkel
Top things to do with a family
- Explore beaches and tidepools
- Build sandcastles
- Snorkel and kayak
Taja McKinney , owner


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View trusted insights from KidsHealth.org, the No. 1 most viewed health site for children, created by the experts at Nemours. We've also provided information from the most-respected non-profit organizations.
From Nemours' KidsHealth
- Growth and Your Newborn
- Medical Care and Your Newborn
- Medical Care and Your 8- to 12-Month-Old
- How to Talk to Your Child's Doctor
- Preparing Your Child for Visits to the Doctor
- Finding a Doctor for Your New Baby
- Medical Care and Your 1- to 2-Year-Old
- Medical Care and Your 4- to 5-Year-Old
- Medical Care and Your 6- to 12-Year-Old
- Medical Care and Your 13- to 18-Year-Old
- Medical Care and Your 2- to 3-Year-Old
- Growth Charts
- Looking at Your Newborn: What's Normal
- Your Child's Immunizations
- Newborn Screening Tests
- Common Cold
- What's a Nurse Practitioner?
- Frequently Asked Questions About Immunizations
- Immunization Schedule
- Medical Care and Your 1- to 3-Month-Old
- Failure to Thrive
- Medical Care and Your 4- to 7-Month-Old
- Influenza (Flu)
- What Can I Do to Ease My Child's Fear of Shots?
- Your Child's Checkups
- Sports Physicals
- The Risks of Postponing or Avoiding Vaccinations
Trusted External Resources
Trusted Resources
- Delaware's Department of Services for Children, Youth, and Their Families (DSCYF)
- 2012 Child & Adolescent Immunization Schedules (from the Centers for Disease Control & Prevention; to help foster parents know which vaccines are recommended and when)
- Child Welfare League of America (CWLA)
- Healthy Foster Care America (from the American Academy of Pediatrics)
- Delaware's Department of Services for Children, Youth, and Their Families (DSCYF)
Medical Care and Your 8- to 12-Month-Old
As your baby becomes more independent and mobile, your questions for your child's doctor may have more to do with bumps, bruises, and behavior than with anything else.
You can't protect your baby from every knee-bump suffered while learning to walk. But you can make sure poisons and medicines are kept where kids can't possibly get to them and provide a safe environment for exploration.
Your baby is probably hearing "no" a lot these days while exploring boundaries; soon, you'll hear that word back from your little one! Be consistent but loving while teaching the difference between acceptable and unacceptable behavior.
When Will We See the Doctor?
Doctors often have their own schedules for well-baby visits, but most will generally see a baby twice during this stage, once at 9 months and again at 12 months.
If you have missed any immunizations, or if a problem has been detected that needs special attention, additional visits may be scheduled.
What to Expect During the Office Visit
The well-baby visits at 9 and 12 months are pretty similar to the exams that have taken place so far, although your discussions with your doctor about behavior and habits may become more frequent.
Expect these common procedures and questions:
- Measurement of your baby's length, weight, and head circumference. Growth will be plotted on the growth chart and you will be advised of your little one's progress.
- A physical examination checking for normal function of the eyes, ears, heart, abdomen, hands and feet, reflexes, etc. The doctor may check your baby's soft spot (the fontanel), which may be closed or much smaller (the size of a fingertip). The doctor will check your baby's mouth for new teeth and signs of others to come.
- A review of your baby's physical and emotional development through both observation and your report: Can your baby sit up alone? Pull up on things to stand? Recognize his or her own name and the names of family members? Enjoy games like "peekaboo"? Your doctor may ask you these questions and others.
- You may be asked how you're doing with your baby and how the rest of the family is functioning. Your doctor may review safety with you: Have you babyproofed your home? Is your baby in an appropriate car seat while riding in the car?
- A discussion of eating habits: Is your baby eating more table foods? Interested in finger foods on the tray of the highchair? Able to use a cup? Being weaned from the breast or bottle? Most doctors advise a switch from bottle to cup by the first birthday to be sure the bottle doesn't interfere with normal tooth development and to avoid struggles with a determined toddler later.
By age 1 year, most babies can be given foods that were off-limits before, such as cow's milk, citrus fruits, and eggs. Your doctor can discuss these dietary additions with you. - Advice on what to expect in the coming months.
- Your baby will receive immunizations during some visits (see below).
Depending on where they live and the potential risk of tuberculosis, sometimes babies at about 1 year of age undergo a tuberculin skin test. You'll be given instructions on how to monitor the test and asked to return to the office for the nurse or doctor to check the results of the test. Discuss possible vaccine reactions with your doctor and when to call with unusual problems.
During appointments, raise any questions or concerns you have and jot down any instructions the doctor gives you about special baby care. Keep updating your child's permanent medical record, listing information on growth and any problems or illnesses.
Immunizations Your Baby Will Receive
If your baby missed immunizations at previous visits because of illness or scheduling problems, he or she will probably be brought up-to-date now.
Because your baby is becoming more and more mobile and is in contact with other kids more often, you'll want to make sure immunizations are given as close to the recommended times as possible. This is especially true if your baby goes out of your home for childcare.
Because more immunizations than ever are given to kids by 2 years of age, doctors are spacing vaccinations so that they won't need more than three to four shots per well-baby visit.
From the Recommended Childhood Immunization Schedule of the American Academy of Pediatrics (AAP):
- At the 12-month visit, your baby may receive the first measles, mumps, and rubella (MMR) vaccine (it's given between 12-15 months of age).
- The first dose of chickenpox (varicella) vaccine is given as a single injection between 12-15 months of age.
- The fourth pneumococcal conjugate vaccine (PCV) is given between 12-15 months of age.
- The fourth Haemophilus influenzae type B (Hib) vaccine is given between 12-15 months of age.
- The first dose of hepatitis A vaccine may be given at 12 months of age or older.
Your baby also may receive:
- the third hepatitis B vaccine (Hep B), which can be given at any time during 6-18 months of age
- the third polio vaccine (IPV), which can be given at any time during 6-18 months of age
- a flu shot
- the meningococcal vaccine (MCV4), which can be given from 9 months. This is given to children at higher risk of developing meningococcal disease, which can lead to bacterial meningitis.
This immunization schedule can vary depending on what combined vaccines your doctor uses.
When to Call the Doctor
You should feel comfortable enough with your doctor to call with questions and concerns that can't wait until the next scheduled visit. If your questions can wait, write them down so you don't forget. Of course, call the doctor immediately if your child has an injury or illness that needs attention.
Call the doctor right away if your baby seems especially sluggish, is refusing food or drink, is vomiting or has diarrhea, or has a temperature of 102.2ºF (39ºC) or higher.
At this age, developmental delays may cause concern, so contact your doctor if you suspect your child is not developing within the range of normal. Kids have their own timetable for crawling, talking, and walking, so keep that in mind when checking for these signs of developmental progress by the first birthday.
Make sure your child:
- has said a first single word (mama, dada)
- uses gestures (waves bye-bye, shakes head no)
- responds to familiar pictures or toys
- stands when supported and pulls up on things to stand
Again, the absence of any of these signs may or may not be cause for concern. Share them with your doctor, though, because problems caught early can be treated more successfully.
Reviewed by: Steven Dowshen, MD
Date reviewed: September 2011
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SRAM XG-1195 XO1 X-Glide Cassettes
Customer Reviews
Average Customer Rating:
(from total reviews)
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Gear Range: 10X42
Weight: 275 g.
Ranging from 10- to 42-teeth, the 11-speed SRAM XG-1195 XO1 X-Glide Cassette delivers an incredibly wide gear range while maintaining even, optimized steps. The single-unit cassette combines with the XD driver body for a superior connection to the wheel. (Wheels equipped for XD driver body available from SRAM, Mavic and DT Swiss.)
Cog Sizes:
10-42: 10-12-14-16-18-21-24-28-32-36-42
Item Code: SRAEEM65


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Summary (Masterplots II: Short Story Series, Revised Edition)
Even as a child, Owen Warland enjoyed carving intricate figures of birds and flowers and showed mechanical ability. Hence, he is apprenticed to Peter Hovenden, a master watchmaker, with whom, his relatives hope, he will be able to make practical use of his delicate talents.
Peter, however, is not impressed with Owen's character. He recognizes his apprentice's considerable talents but senses that Owen does not care to apply them in a conventional way. When failing eyesight forces Peter to surrender his shop to Owen, the young man confirms his master's fears. Owen's business declines because his customers do not appreciate the way he trifles with their beloved timepieces, which he tends to embellish fancifully.
Far from regretting this lack of customers, Owen rejoices in the free time he now has to pursue his goal of creating an object so like its natural original that it will be indistinguishable from it. The first attempt fails after Robert Danforth comes to deliver a small forge ordered from the blacksmith. Danforth's brute strength so disturbs Owen that he carelessly demolishes the artifact.
For some months Owen returns to watchmaking, abandoning any artistic pretense. Slowly, however, he recovers his interest in his project and is about to begin again when Peter visits him. His former master's skepticism toward anything lacking utilitarian value so upsets Owen that he relinquishes his dream.
In the summer...
(The entire section is 464 words.)
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"Think about death make us live better.
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i have been looking for this video for ages and i finally found it
here it is enjoy :)
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For once, I'ma agree with Hugh Lovric.
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Statement from Minister
Thank you for visiting the online consultation website for Canada's first-ever digital economy strategy.
The consultation period has now ended, and I would like to thank everyone who took the time to consider this important subject—to share your thoughts and opinions on digital technology and on how Canada's digital future should take shape.
For more than 60 days we've seen a constant stream of visitors to our consultation site. I thank you for all your many formal submissions and for your contributions to the ideas forum. It's clear that a lot of thought has gone into this input.
Now we begin reviewing your submissions, ideas, and tweets. Once we have reviewed all of the public input, as well as the results of meetings with industry as well as federal, provincial, and territorial representatives, we will develop an action plan for regaining Canada's leadership as a digital economy.
The work is underway, but I ask your patience.
We want to get this right. It's important to Canada's economic and competitive future. We want to make sure that the resulting strategy for Canada's digital economy reflects what we've heard from you over the past 60-plus days.
Other countries have typically taken between 6 and 18 months to develop their strategies, so it's reasonable to expect that Canada's strategy will be developed in a similar time frame.
This website is now closed to any further submissions, but I invite you to check back here for news and further developments as we put together our strategy for Canada's digital economy.
Of course, we didn't put everything else on hold while the consultation was taking place.
In recent weeks the Government of Canada announced investments under the Broadband Canada program, introduced new copyright and anti-spam legislation, and updated our privacy laws for the digital age. All of these measures are critical cornerstones of the digital economy.
The Government of Canada is looking at realigning resources and continuing to invest in initiatives that yield the greatest return, that are vital to Canada's long-term competitiveness.
Let me thank you again for taking part in this consultation on Canada's digital future.
Thank you.
- Date modified:


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Call For More Information
For questions regarding Undergraduate Programs call 215-437-2219.
For questions regarding Graduate Programs call 267-341-3327.
NUR 452 Leadership in Nursing Practice
3 credits
Four classroom hours per week.
This course provides students with the opportunity to apply and evaluate the leadership and management roles of a professional nurse to improve safety and quality of patient care. Students critically appraise current issues and trends in nursing and health care and their effects on the health care delivery system.


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Originally posted by: want2fishmore on 10/29/2006 8:36:36 PM
Just got two new d-riggers at a great buy this week. We plan to go stiper fishing alot at lake Texoma. The quick release clip has a short cable with no swivel on it. Question one, Do you clip your bait line close to the release or do you cast out so many feet out then clip on to the quick release? Question two, Do you need a swivel on the quick release cable to prevent line twist? We can use any pointers on trolling speeds, or hook-up, crank baits with or without lips,ball weight sizes (4,6,8,10 lbs.) just about any thing usefull. Thanks Bill


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A state law barring registered sex offenders from loitering near schools, public parks and public playgrounds is constitutional because it provides sufficient warning of what conduct is prohibited, the South Dakota Supreme Court ruled Thursday.
The high court's unanimous ruling upheld the conviction of a Sioux Falls man who argued the law is unconstitutional because it is too vague.
Kenneth Dale Stark, 61, was convicted of two counts of loitering in a community safety zone after law officers observed him near two public parks in Sioux Falls. A circuit judge then sentenced him to six years in prison.
Stark had registered as a sex offender after a prior conviction.
After a woman reported that someone in a vehicle registered to Stark was watching or following children going to swimming lessons in April 2009, law officers followed Stark after he left work the following day. They testified in his trial that they saw Stark circle one park for about 20 minutes and later saw him drive away after being parked near another park.
Stark's lawyer declined to comment on the case Thursday. South Dakota Attorney General Marty Jackley was not immediately available to comment because he was attending a funeral.
'Vague' argument
During his trial and in the appeal, Stark argued the law was too vague and violated his constitutional freedom to loiter for innocent purposes. He contended he just drove past the first park and pulled over near the second park to yield to an oncoming car and allow some children to cross the street.
The Supreme Court said criminal laws must provide sufficient notice of what conduct is prohibited and must give law officers sufficient guidelines for enforcement.
South Dakota law specifically prohibits registered sex offenders from loitering within 500 feet of any school, public park or public playground for the purpose of observing or contacting minors, the Supreme Court ruled. That language limits application of the law to loitering that has an apparently harmful purpose or effect, the justices said.
''The statutes at issue were sufficient to provide Stark with notice that his conduct was prohibited, and they did not authorize the law enforcement officers in this case to act in an arbitrary or discriminatory manner,'' Justice Glen Severson wrote for the court.
Prosecutors provided sufficient evidence for the jury to find Stark's primary purpose for remaining near the parks was to observe or contact minors, the Supreme Court said.