Abstract
Delivery of exogenous glial cell line-derived neurotrophic factor (GDNF) increases locomotor activity in rodent models of aging and Parkinson’s disease in conjunction with increased dopamine (DA) tissue content in substantia nigra (SN). Striatal GDNF infusion also increases expression of GDNF’s cognate receptor, GFRα1, and tyrosine hydroxylase (TH) ser31 phosphorylation in the SN of aged rats long after elevated GDNF is no longer detectable. In aging, expression of soluble GFRα1 in the SN decreases in association with decreased TH expression, TH ser31 phosphorylation, DA tissue content, and locomotor activity. Thus, we hypothesized that, in aged rats, replenishing soluble GFRα1 in SN could reverse these deficits and increase locomotor activity. We determined that the quantity of soluble GFRα1 in young adult rat SN is ~3.6 ng. To replenish age-related loss, which is ~30 %, we infused 1 ng soluble GFRα1 bilaterally into SN of aged male rats and observed increased locomotor activity compared to vehicle-infused rats up to 4 days following infusion, with maximal effects on day 3. Five days after infusion, however, neither locomotor activity nor nigrostriatal neurochemical measures were significantly different between groups. In a separate cohort of male rats, nigral, but not striatal, DA, TH, and TH ser31 phosphorylation were increased 3 days following unilateral infusion of 1 ng soluble GFRα1into SN. Therefore, in aged male rats, the transient increase in locomotor activity induced by replenishing age-related loss of soluble GFRα1is temporally matched with increased nigral dopaminergic function. Thus, expression of soluble GFRα1 in SN may be a key component in locomotor activity regulation through its influence over TH regulation and DA biosynthesis.
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Introduction
Glial cell line-derived neurotrophic factor (GDNF) signaling is critical for dopamine (DA) neuron survival and function [1–3] and is involved in the regulation of locomotor activity. A decrease in GDNF signaling precipitates locomotor impairment while increasing GDNF improves movement. For instance, age-related locomotor impairment is accelerated in mice with heterozygous knockout of either the ligand, GDNF, or its cognate receptor, GDNF family receptor α1 (GFRα1) [4–6]. Conversely, direct delivery of exogenous GDNF improves locomotor functions in animal models of aging [7–11], models of Parkinson’s disease (PD) [12–16], and, in some studies, the PD patient [17, 18].
The locomotor benefits afforded by GDNF therapy are thought to be mediated primarily by effects on DA and DA-regulating proteins, such as tyrosine hydroxylase (TH), in the nigrostriatal pathway. In PD models, GDNF increases expression of striatal DA tissue content and TH [16]. However, in aging models or the otherwise “intact” nigrostriatal pathway, GDNF actually decreases striatal TH expression [19–21]. Interestingly, despite these alternate effects in the striatum, GDNF similarly increases DA tissue content in the dopaminergic cell body region of the substantia nigra (SN) in rodent models of aging and PD [7, 9, 11–13, 22]. In addition, GDNF increases TH phosphorylation, a major means of TH activation, in the SN [20, 21]. These effects of GDNF in the SN may be directly linked to the locomotor benefit of this neurotrophic factor since modulation of nigral DA alone has been shown to influence locomotor activity generation [23–26]. Furthermore, GDNF can induce locomotor improvement even when there is no change in striatal DA tissue content [12, 13]. Thus, the effects of GDNF on DA regulation in the SN could play some role in locomotor improvement.
Unfortunately, despite the demonstrated benefits of GDNF, controversy remains as to whether GDNF should continue to be pursued as a therapeutic option due, in part, to the outcomes of studies challenging GDNF’s efficacy in the clinic [27] or in particular PD models [28]. Given the current roadblocks to GDNF therapy, targeting other molecular entities involved in GDNF signaling may prove beneficial in the treatment of locomotor impairment. One such molecular entity is GDNF’s own receptor, GFRα1. After binding GDNF, GFRα1 mediates downstream signal transduction through interactions with signaling partners such as Ret [29, 30], which is known to affect TH and DA function [31]. GDNF signaling through GFR α1 may also be RET-independent in some cases, but does require the presence of heparin sulphate glycosaminoglycans, even in RET-dependent signaling [32]. GFRα1 expression may even be the limiting factor in GDNF signaling [33, 34] and may mediate the long-lasting dopaminergic and locomotor improvements induced by GDNF administration. For instance, GDNF delivery induces a sustained increase in nigral GFRα1 expression that matches long-term increases in nigral DA, TH, and TH phosphorylation [20, 21]. Notably, increased nigral GFRα1 expression and TH phosphorylation remain elevated long after GDNF levels in the SN are no longer elevated [21].
GFRα1 is widely expressed in the brain, with notably intense immunohistochemical staining in the SN [35]. It exists primarily in two forms: a glycosyl-phosphatidylinositol (GPI)-linked receptor attached to the outer membrane of cell surfaces with a molecular weight of ~52 kDa [29, 30, 35], and a cleaved form of ~48 kDa [35, 36] that is a fully functional and soluble receptor [33, 36]. While both forms of GFRα1 share similar downstream signaling mechanisms, the primary difference between the two forms is that signaling via soluble GFRα1 is slower in onset but much more sustained compared to signaling via the GPI-linked receptor [36]. The cleaved form of GFRα1 in the SN declines with aging in association with reduced nigral TH, ser31 TH phosphorylation, DA tissue content, and locomotor activity [26, 37]. Here, we determined whether replacing the quantity of soluble GFRα1 in the SN normally lost in aging could, at least, transiently improve locomotor activity and, in a temporally matched fashion, increase nigral TH protein and phosphorylation as well as DA tissue content.
Methods
Test Subjects
Male Brown–Norway/Fischer 344 F1 hybrid (BNF) rats were utilized for all three of the major experiments. Four 11-month-old BNF rats were used to characterize levels of GFRα1 in the nigrostriatal pathway against a soluble GFRα1 standard curve. Ten male BNF rats were used to determine the locomotor effects of bilateral infusion of soluble GFRα1. The locomotor activity of these rats was first characterized at 25 months of age. Bilateral infusions and locomotor testing were conducted when the rats were 29–31 months old; neurochemistry was determined 5 days following infusion. Five male BNF rats ranging in age from 18 to 25 months old were used to characterize the neurochemical effects 3 days after unilateral soluble GFRα1 infusion to the SN. For the purpose of increasing n for determination of GFRα1 differences 3 days following infusion, tissue from five female BNF rats was utilized.
HPLC Analysis
DA and DOPAC tissue content were measured by HPLC analysis as previously described [26, 38, 39]. Our technique allows for the processing of the protein pellet precipitated during the HPLC preparation for Western blot analysis of proteins and protein phosphorylation.
Western Blot Analysis
Tissue protein precipitates were prepared as previously described [38]. TH protein content and TH phosphorylation stoichiometry (p.s.) at ser19 and ser31 were determined by quantitative western blot against standard curves of known TH and phosphor-TH quantities from in house standards (calibrated PC12 cell extract) as previously described [26, 39]. The sources for primary antibodies were as follows: TH protein, Millipore (Cat. # AB152), ser19 phosphoTH, Phosphosolutions (Cat. # p1580-19), ser31 phosphoTH, 21st Century Biochemicals and validated previously [26, 39]. Table S1 contains information on all antibodies used. Ser40 TH phosphorylation was not assessed as the unilateral tissue dissection used for the 3-day neurochemical profile determination yielded low sample availability. We prioritized analysis first to ser31, because this site is phosphorylated by ERK1 and ERK2 [40], and these kinases are downstream of Ret [41, 42], which is a major signaling partner of GFRα1 [29, 30]. We also prioritized TH phosphorylation analysis to ser19, because this site is linked to Ca2+-dependent depolarizing stimuli [43, 44], and GDNF itself can increase membrane depolarization [45] and ser19 phosphorylation in the SN [21].
GFRα1 levels were also determined comparing sample aliquots of SN and striatum tissue (40 μg nominal protein load) collected from 11-month-old BNF rats (n = 4) against a soluble GFRα1 standard curve. Soluble GFRα1 was acquired as a custom order from R&D Systems (Recombinant Rat GFRα1). The resulting soluble GFRα1 has a MW of ~47 kDa and the amino acid sequence of rat GFRα1 from Asp25–Leu445 with the addition of the following sequence of amino acids at the C terminus: CSIEGR. The soluble GFRα1 was provided in PBS (pH 7.4) at a concentration of 0.106 mg/ml. Dilutions of soluble GFRα1 were made in Krebs bicarbonate buffer. The soluble GFRα1 standard curve ranged from 25 to 1,600 pg. For the standard curve, 10 μg of protein from a VTA sample from an 11-month-old BNF rat was loaded with standards to serve as a protein carrier and background GFRα1 immunoreactivity from this VTA sample was subtracted from the standard curve.
Soluble GFRα1 Infusion Procedure
Soluble GFRα1 was diluted in Krebs buffer to 0.5 ng/μl and infused at 1 μl/min directly into the SN (relative to bregma: −5.7 AP, ±2.5 ML, −8.5 DV) of anesthetized (pentobarbital sodium, 40 mg/kg, i.p.) BNF rats. For bilateral infusions, either 2 μl of the 0.5 ng/μl soluble GFRα1 solution or vehicle (Kreb’s buffer) were infused into each SN. For unilateral infusions, 2 μl of the 0.5 ng/μl soluble GFRα1 solution was infused into one SN, while an equal volume of vehicle (Krebs buffer) was infused into the contralateral SN. The infusion of an equal volume of vehicle into the contralateral hemisphere or bilaterally in rats designated as controls was intended to represent inherent DA regulation in the tissue and control for any inflammatory responses arising from the penetration of the needle and the 2 μl infusion volume.
Locomotor Testing
Open-field locomotor activity was assessed using automated activity chambers as previously described [26]. Locomotor activity was recorded for 1 h, once per day. Prior to assigning rats to experimental groups, baseline locomotor activity was measured in ten BNF rats. Five consecutive daily trials were performed when the rats were 25 months of age and another four daily trials were conducted at 28–29 months of age. These two periods of locomotor assessment were used to determine baseline locomotor activity for each animal. The rats were then placed into two groups (GFR or vehicle) which had no baseline difference in horizontal activity, movement number, total distance, and time spent moving, as these parameters correlate to nigral DA levels [26]. Rats then underwent surgery to deliver either soluble GFRα1 or vehicle (Krebs buffer) bilaterally to the SN after one final baseline locomotor assessment and had daily locomotor assessments for 5 consecutive days.
Statistics
To analyze differences in total recovery of GFRα1 in each of the three immunoreactive bands between the SN and the striatum, two-way ANOVA followed by Bonferroni posttests were utilized. Two-way ANOVA followed by Bonferroni posttests were also used to analyze both time- and group-dependent differences in locomotor activity measures. To determine the effect of bilateral infusion of soluble GFRα1 into the SN on neurochemical parameters 5 days following infusion, an unpaired two-tailed t-test was used comparing the values of each neurochemical parameter within each brain region between the group receiving bilateral soluble GFRα1 infusions to the SN and the group receiving bilateral vehicle infusions to the SN. To determine the effect of unilateral infusion of soluble GFRα1 into the SN on neurochemical parameters 3 days following infusion, a paired two-tailed t-test was used comparing the values of each neurochemical parameter within each brain region between the soluble GFRα1-infused hemisphere and the vehicle-infused hemisphere for each rat.
Results
Quantification of GFRα1 Levels In Vivo
To determine the quantity of cleaved GFRα1 (~48 kDa) receptor lost in the SN during aging [37], we measured nigrostriatal GFRα1 immunoreactivity in 11-month-old BNF rats against a soluble GFRα1 standard curve ranging from 25 to 1,600 pg soluble GFRα1 (Fig. 1a, b). Three immunoreactive bands were detected at ~48, 52, and 60 kDa (Fig. 1a). Immunoreactivity of each band was compared to the soluble GFRα1 standard curve (Fig. 1b) to determine the amount of GFRα1 (Fig. 1c), which was similar between the striatum and the SN (Fig. 1c). The total recovery of the GFRα1-immunoreactive band at ~48 kDA, representing the cleaved and soluble form of GFRα1 [37], in the SN was 0.074 ± 0.0120 pmol or ~3.6 ng. Therefore, because expression of this form of GFRα1 is reduced in the SN by ~30 % between 12 and 30 months of age, a period in the lifespan wherein bradykinesia becomes manifest in this rat strain [26, 37], we deduced that the amount of soluble GFRα1in the SN lost with aging was likely ~1 ng.
Locomotor Effects of Bilateral Infusion of Soluble GFRα1 into the SN of Aged Rats
Decreased nigral GFRα1 is linked to reduced TH and DA in the SN [37] and reduced nigral DA or DA signaling is associated with decreased locomotor activity [23–26]. More specifically, nigral DA levels correlate to horizontal activity, movement number, total distance, and time spent moving, but not locomotor speed [26]. Therefore, we hypothesized that replenishing the age-related loss of soluble GFRα1 would increase these locomotor measures in aged rats compared to age-matched controls of equal overall locomotor capability (Table 1). Thus, 1 ng of soluble GFRα1, the approximate amount of soluble GFRα1 predicted to be lost in aging (Fig. 1), was infused bilaterally to each SN, while control rats received bilateral vehicle infusions into each SN. In the first 4 days after surgery, the group receiving soluble GFRα1 had significantly greater horizontal activity (p < 0.0001), movement number (p = 0.033), total distance (p = 0.0096), time spent moving (p = 0.018), and vertical activity (p = 0.0074) than the vehicle-treated group (Table 1, Fig. 2). In particular, there was a significant separation between the two groups’ horizontal activity on day 3 (Fig. 2a) and movement number on day 4 following infusion.
Locomotor increases induced by soluble GFRα1 infusion into the SN were transient and were no longer uniformly present on the fifth day following the infusion surgery (Fig. 2b, Table 1). In agreement with our previous finding that movement speed does not correlate with nigral DA levels [26], no significant difference in movement speed was observed after GFRα1 infusion (Table 1, Fig. 2c).
Neurochemical Effects 5 Days After Bilateral Nigral Infusion of Soluble GFRα1
Immediately after the locomotor trial performed on the fifth day following infusion surgery, dopaminergic tissue was collected. DA levels, TH levels, DA per TH, and DA turnover were not significantly different between the two treatment groups for any of the dopaminergic brain regions collected (Table 2). This lack of a difference in any DA parameter 5 days following infusion of 1 ng soluble GFRα1 to the SN temporally matches the lack of a difference in locomotor activity between the solubleGFRα1- and vehicle-infused rats at day 5 (Fig. 2).
Neurochemical Effects 3 Days After Unilateral Nigral Infusion of Soluble GFRα1
In a different cohort of 18- to 25-month-old BNF rats, we infused 1 ng soluble GFRα1 unilaterally into the SN (an equal volume of vehicle was infused into the contralateral SN) to determine if the maximal differences in horizontal activity seen 3 days after bilateral infusion of soluble GFRα1 into the SN (Fig. 2a) could be temporally matched to increased nigral DA and TH expression. Unilateral nigral infusion of 1 ng soluble GFRα1 increased DA levels by 39 % (Fig. 3a) and DOPAC levels (Fig. 3b) in the SN compared to the contralateral vehicle-infused SN, but was without effect in the striatum, NAc, or adjoining VTA. Increased nigral DA was matched by a ~47 % increase in nigral TH protein (Fig. 4b, d), with no effect in striatum (Fig. 4a, c).
GFRα1 Quantification After Nigral Infusion
Because of the impact of nigral GFR α-1 infusion on locomotor activity and DA modulation in the SN, we sought to determine if the time course of increased locomotor activity and nigral DA matched increased recovery of the receptor following its infusion. A trend toward an increase in the ~48 kDa band of GFRα1 was observed in the GFRα1-infused SN 3 days following infusion, and with the addition of nigral samples from a separate cohort of rats, there was significantly greater (~27 %) GFRα1 recovery in the GFRα1-infused SN compared to endogenous levels in the vehicle-infused SN 3 days following infusion (Fig. 5a). However, GFRα1 levels were not significantly different between the groups 5 days following infusion (Fig. 5b), indicating that soluble GFRα1 was either washed out or degraded by this time point.
TH Phosphorylation
TH phosphorylation at ser19 is a Ca2+-dependent process tied to neuronal excitability [43, 44], and GDNF can increase DA neuron excitability and Ca2+ influx [45]. Infusion of 1 ng soluble GFRα1 into the SN was without effect on p.s. at ser19 in either the striatum or SN (Fig. 6a). Thus, the infusion of soluble GFRα1 likely did not cause any changes in Ca2+ influx or excitability of DA neurons.
Soluble GFRα1 infusion did significantly increase TH p.s. at ser31 in SN ~23 % (Fig. 6b). TH is phosphorylated at ser31 by ERK1 and ERK2 [40], which is downstream of Ret activation [41, 42]. Thus, the increase in nigral ser31 p.s. is likely to be indicative of increased activation of Ret due to interactions with the infused soluble GFRα1. Lack of sample availability prevented the assessment of TH phosphorylation at ser40.
Discussion
Locomotor and Neurochemical Effects of Replenishing Soluble GFRα1 in the Aged SN
Our previous work indicates that age-related reductions in initiated locomotor activity are associated with decreased nigral DA tissue content, TH protein, and ser31 TH phosphorylation [26]. These decreases, in turn, are linked to an age-related reduction in the cleaved form of GFRα1 in the SN [37]. We, therefore, hypothesized that decreased expression of the cleaved and soluble form of GFRα1 in the SN is a deficient molecular link upstream of age-related reductions in nigral DA and, consequently, locomotor activity. We now demonstrate that locomotor activity in aged rats can be transiently increased following nigral infusion of soluble GFRα1 in the quantity normally lost with aging. This locomotor effect was temporally matched with increased nigral DA along with increased TH protein expression and ser31 phosphorylation (Figs. 3, 4b, d and 6b). Furthermore, not only did infusion of soluble GFRα1 into the SN increase nigral DA, TH, and ser31 phosphorylation of TH, measures which all normally decline with aging [26, 39], it increased these measures to nearly the same magnitude that aging has been shown to reduce them (Table 3). These observations suggest GFRα1 may normally maintain DA biosynthesis capacity in the SN, and adequate expression may preserve normal locomotor activity. It also raises questions as to why expression of the soluble form of GFRα1 declines with aging.
Bilateral infusion of 1 ng soluble GFRα1 to the SN maximally increased horizontal activity 3 days following infusion (Fig. 2a). Out to 4 days past infusion, movement number, total distance, and time spent moving, measures previously shown to correlate with nigral DA levels [26], as well as vertical activity were also significantly increased compared to vehicle-infused controls (Table 1). On the other hand, movement speed, a movement parameter shown not to correlate with nigral DA levels [26], was not significantly different at any time point after soluble GFRα1 infusion (Table 1, Fig. 2c). The transient locomotor effects induced by soluble GFRα1 infusion were temporally correlated with increased nigral DA levels (Figs. 2 and 3), both with regard to the increases seen in each at 3 days and eventual lack of effects 5 days following infusion (Fig. 2; Tables 1 and 2). Furthermore, these effects coincided with the time course during which nigral GFRα1 levels were themselves elevated after infusion (Fig. 5). The transient nature of the effects induced by nigral GFRα1 infusion suggests that sufficient and sustained expression of GFRα1 in the SN may be required for continued locomotor activity improvement or maintenance during aging.
Comparison of Soluble GFRα1 Effects Versus GDNF Administration
Both GDNF and soluble GFRα1, as seen in our work, similarly increase nigral DA and locomotor activity. In aged rats and non-human primates, GDNF administration increases locomotor activity [7–11]. In fact, 10 μg GDNF to the SN can increase total distance out to one [7] or 3 weeks [9]. Furthermore, GDNF-mediated increases in locomotor activity are associated with elevated DA tissue content in the SN but not the striatum [7, 9, 11–13]. The increase in nigral DA induced by either GDNF or soluble GFRα1 likely arises, at least in part, through increased ser31 TH phosphorylation, which is seen following nigral infusion of soluble GFRα1 (Fig. 6b) or striatal delivery of GDNF [20, 21].
There is, however, a notable contrast between the effects of GDNF compared to GFRα1 on TH expression in the “intact” nigrostriatal pathway. While nigral TH levels were increased following unilateral infusion of 1 ng soluble GFRα1 (Fig. 4d), GDNF infusion to intact nigrostriatal pathways (such as in aged rats) decreases striatal TH expression [19–21, 46]. In models of Parkinson’s disease, though, wherein there is substantial loss of TH, GDNF does increase striatal TH expression [13–16], suggesting that basal TH expression levels may predict responsiveness to GDNF such that TH is only increased by GDNF when basal TH levels are below normal. Since little if any loss of TH protein has been reported in the striatum of aged rats [26, 39] or humans [47], this may explain why delivery of GDNF to intact systems reduces striatal TH expression. However, the BNF rats utilized in this study were at ages for which nigral TH is reduced [26, 39]. Thus the increase in nigral TH seen following infusion of GFRα1 in this study may be more analogous to the effect GDNF has of increasing TH expression in Parkinson’s disease models.
Another major difference between GDNF and soluble GFRα1 administration is the longevity of their effects. Namely, the neurochemical and locomotor effects following infusion of 1 ng soluble GFRα1 to the SN occur for a much shorter period of time than those following GDNF administration. However, this temporal difference may be accounted for by the long-lasting impact GDNF has upon GFRα1 expression and the difference in the quantities of GDNF typically infused into brain compared to the amount of soluble GFRα1 infused in this study. Normal levels of GDNF in the SN are 0.003 ng per mg protein in the rhesus monkey [48] and in the range of 0.01–0.06 ng per mg protein in the rat [19, 49], while levels of cleaved GFRα1 in the rat SN are ~100-fold greater, being 5.7 ± 0.50 ng per mg protein by our calculation (Fig. 1). Given that the average total protein recovery from the SN samples used to quantify GFRα1 levels was 0.63 ± 0.098 mg, this suggests that the quantity of GDNF in the rat is ~ 0.01–0.04 ng per SN, compared to the total amount of cleaved GFRα1, which is ~3.6 ng. As a point of reference to this in vivo data on GDNF expression, GDNF studies often infuse 10–100 μg GDNF [7–9, 20, 21], which is 250,000–10,000,000 times greater than endogenous levels. Conversely, in this study we infused 1 ng soluble GFRα, which is only a third of the total amount of cleaved GFRα1 recovered from the rostral–caudal extent of the SN from 11-month-old BNF rats. Thus, our infusion of soluble GFRα1 is within physiological range and was intended to transiently increase nigral GFRα1 levels to levels seen in younger rats.
A potentially unique feature of GDNF is that it may induce a long-term increase in GFRα1-mediated signaling. Notably, GDNF increases the expression of GFRα1 [20, 49] specifically within the SN up to 30 days following a 100-μg infusion of GDNF into the ipsilateral striatum [20]. This sustained elevation of nigral GFRα1 levels is seen despite the fact that nigral GDNF levels themselves are elevated at 1 day, but not 7 days, following striatal GDNF infusion [21]. Thus, the prolonged increases in DA function and locomotor activity induced by supra-physiological levels of GDNF may result primarily from a sustained increase in GFRα1 expression.
Implications
Age-related loss of soluble GFRα1 in the SN could be a critical molecular event precipitating bradykinesia in aging, particularly since levels of striatal GDNF [50] and nigral RET [51] are maintained with aging. Our findings show that a rather narrow window of receptor quantity (~1 ng) is associated with TH protein expression, TH phosphorylation at ser31, and DA tissue content in the SN. Given that nigral DA may affect locomotor capabilities [23–26], it stands to reason that sustaining GFRα1 expression in the SN could represent a new strategy for treating locomotor impairment. However, we point out that differentiating between the expression of soluble and GPI-linked forms of GFRα1 may be critical in this regard, particularly since a recent study suggests that there is no loss of nigral GFRα1 in a population of aged Asian Indians [52]. While this may seem at odds with our finding of reduced soluble GFRα1 in the SN with aging [37], one reason for this apparent dissimilarity may be that in this study GFRα1 expression was determined by immunohistochemistry, which may have only measured the GPI-linked form of GFRα1 as soluble GFRα1 could potentially be washed out during tissue preparation. We also have reported no age-related loss of the full-length (~52 kDa), presumably GPI-linked, form of GFRα1 in the SN [37]. Also, this particular population of Asian Indians is less prone to Parkinson’s disease [52]. As such, preservation of nigrostriatal function with aging may be unique to this population. Indeed, while bradykinesia becomes increasingly prevalent with aging, half of the population over age 80 years does not exhibit bradykinesia [53, 54]. Thus, loss of soluble GFRα1 may represent a molecular deficit, which predisposes the aging population to developing bradykinesia.
Recent work by Bartus et al. [55] has prompted consideration of targeting growth factors to the SN in order to overcome the deficits in the retrograde transport of proteins that occur in neurodegenerative disorders. Here, we show that targeted infusion of the soluble GDNF receptor, GFRα1, to the SN can transiently increase locomotor activity in a manner that is temporally matched with increased DA tissue content, TH expression, and TH ser31 phosphorylation in the SN and, notably, independent of any such increases in the striatum. However, there are questions that remain unanswered going forward. First and foremost, we stress that our study took place in the temporal window immediately following infusion surgery wherein locomotor activity was decreased below the presurgery baseline level (Fig. 2), and during this time frame, deficits in locomotion and nigral DA function were only transiently increased following a one-time infusion of soluble GFRα1. Therefore, our future work will examine the impact of GFRα1 infusion on a more protracted basis, wherein post-surgical effects on locomotor function are diminished in both control and treatment groups. Second, the cellular source of soluble GFRα1 in the SN is unknown as is the mechanism of how expression of this receptor decreases with aging. Both dopaminergic and GABAergic neurons [56] as well as astrocytes [57, 58] and microglia [59] can express this receptor, so determining if any of these cell types are the primary source of soluble GFRα1 in the SN will be a necessary next step. Third, we need to determine if the transient increase in nigral TH expression induced by soluble GFRα1 infusion is mediated by an increase in the number of TH-positive neurons or simply an upregulation of TH in those neurons already expressing TH. Indeed, increasing TH expression or function in the SN could be a means to increase locomotor activity, possibly through its impact upon nigral TH expression or activity and resulting increased DA [60]. Our findings have revealed soluble GFRα1 as a molecular substrate involved in the maintenance of nigral DA levels, demonstrated that increasing its expression in the SN can increase locomotor activity, and identified this soluble receptor as a possible target to treat impaired locomotor activity.
References
Granholm AC, Reyland M, Albeck D (2000) Glial cell line-derived neurotrophic factor is essential for postnatal survival of midbrain dopamine neurons. J Neurosci 20:3182–3190
Pascual A, Hidalgo-Figueroa M, Piruat JI et al (2008) Absolute requirement of GDNF for adult catecholaminergic neuron survival. Nat Neurosci 11:755–761
Nevalainen N, Chermenina M, Rehnmark A et al (2010) Glial cell line-derived neurotrophic factor is crucial for long-term maintenance of the nigrostriatal system. Neuroscience 171:1357–1366
Boger HA, Middaugh LD, Huang P et al (2006) A partial GDNF depletion leads to earlier age-related deterioration of motor function and tyrosine hydroxylase expression in the substantia nigra. Exp Neurol 202:336–347
Boger HA, Middaugh LD, Zaman V et al (2008) Differential effects of the dopamine neurotoxin MPTP in animals with a partial deletion of the GDNF receptor, GFR alpha1, gene. Brain Res 1241:18–28
Zaman V, Boger HA, Granholm AC et al (2008) The nigrostriatal dopamine system of aging GFRalpha-1 heterozygous mice: neurochemistry, morphology and behavior. Eur J Neurosci 28:1557–1568
Hudson J, Granholm AC, Gerhardt GA et al (1995) Glial cell line-derived neurotrophic factor augments midbrain dopaminergic circuits in vivo. Brain Res Bull 36:425–432
Bowenkamp KE, Lapchak PA, Hoffer BJ, Bickford PC (1996) Glial cell line-derived neurotrophic factor reverses motor impairment in 16–17 month old rats. Neurosci Lett 211:81–84
Hebert MA, Gerhardt GA (1997) Behavioral and neurochemical effects of intranigral administration of glial cell line-derived neurotrophic factor on aged Fischer 344 rats. J Pharmacol Exp Ther 282:760–768
Maswood N, Grondin R, Zhang Z et al (2002) Effects of chronic intraputamenal infusion of glial cell line-derived neurotrophic factor (GDNF) in aged Rhesus monkeys. Neurobiol Aging 23:881–889
Grondin R, Cass WA, Zhang Z et al (2003) Glial cell line-derived neurotrophic factor increases stimulus-evoked dopamine release and motor speed in aged rhesus monkeys. J Neurosci 23:1974–1980
Hoffer BJ, Hoffman AF, Bowencamp KE et al (1994) Glial cell line-derived neurotrophic factor reverses toxin-induced injury to midbrain dopaminergic neurons in vivo. Neurosci Lett 182:107–111
Gash DM, Zhang Z, Ovadia A et al (1996) Functional recovery in parkinsonian monkeys treated with GDNF. Nature 380:252–255
Aoi M, Date I, Tomita S, Ohmoto T (2000) The effect of intrastriatal single injection of GDNF on the nigrostriatal dopaminergic system in hemiparkinsonian rats: behavioral and histological studies using two different dosages. Neurosci Res 36:319–325
Connor B, Kozlowski DA, Unnerstall JR et al (2001) Glial cell line-derived neurotrophic factor (GDNF) gene delivery protects dopaminergic terminals from degeneration. Exp Neurol 169:83–95
Grondin R, Zhang Z, Ai Y et al (2002) Chronic, controlled GDNF infusion promotes structural and functional recovery in advanced parkinsonian monkeys. Brain 125:2191–2201
Gill SS, Patel NK, Hotton GR et al (2003) Direct brain infusion of glial cell line-derived neurotrophic factor in Parkinson disease. Nat Med 9:589–595
Slevin JT, Gerhardt GA, Smith CD et al (2005) Improvement of bilateral motor functions in patients with Parkinson disease through the unilateral intraputaminal infusion of glial cell line-derived neurotrophic factor. J Neurosurg 102:216–222
Georgievska B, Kirik D, Bjorklund A (2004) Overexpression of glial cell line-derived neurotrophic factor using a lentiviral vector induces time- and dose-dependent downregulation of tyrosine hydroxylase in the intact nigrostriatal dopamine system. J Neurosci 24:6437–6445
Salvatore MF, Zhang JL, Large DM et al (2004) Striatal GDNF administration increases tyrosine hydroxylase phosphorylation in the rat striatum and substantia nigra. J Neurochem 90:245–254
Salvatore MF, Gerhardt GA, Dayton RD, Klein RL, Stanford JA (2009) Bilateral effects of unilateral GDNF administration on dopamine- and GABA-regulating proteins in the rat nigrostriatal system. Exp Neurol 219:197–207
Gerhardt GA, Cass WA, Huettl P et al (1999) GDNF improves dopamine function in the substantia nigra but not the putamen of unilateral MPTP-lesioned rhesus monkeys. Brain Res 817:163–171
Trevitt JT, Carlson BB, Nowend K, Salamone JD (2004) Substantia nigra pars reticulata is a highly potent site of action for the behavioral effects of the D1 antagonist SCH23390 in the rat. Psychopharmacology 156:32–41
Bergquist F, Shahabi HN, Nissbrandt H (2003) Somatodendritic dopamine release in rat substantia nigra influences motor performance on the accelerating rod. Brain Res 973:81–91
Andersson DR, Nissbrandt H, Bergquist F (2006) Partial depletion of dopamine in substantia nigra impairs motor performance without altering striatal dopamine neurotransmission. Eur J Neurosci 24:617–624
Salvatore MF, Pruett BS, Spann SL, Dempsey C (2009) Aging reveals a role for nigral tyrosine hydroxylase ser31 phosphorylation in locomotor activity generation. PLoS One 4:8466
Lang AE, Gill SS, Patel NK et al (2006) Randomized controlled trial of intraputamenal glial cell line-derived neurotrophic factor infusion in Parkinson's disease. Ann Neurol 59:459–466
Decressac M, Ulusoy A, Mattson B et al (2011) GDNF fails to exert neuroprotection in a rat a-synuclein model of Parkinson's disease. Brain 134:2302–2311
Jing S, Wen D, Yu Y et al (1996) GDNF-induced activation of the ret protein tyrosine kinase is mediated by GDNFR-alpha, a novel receptor for GDNF. Cell 85:1113–1124
Treanor JJ, Goodman L, de Sauvage F et al (1996) Characterization of a multicomponent receptor for GDNF. Nature 382:80–83
Mijatovic J, Airavaara M, Planken A et al (2007) Constitutive Ret activity in knock-in multiple endocrine neoplasia type B mice induces profound elevation of brain dopamine concentration via enhanced synthesis and increases the number of TH-positive cells in the substantia nigra. J Neurosci 18:4799–4809
Sariola H, Saarma M (2003) Novel functions and signaling pathways for GDNF. J Cell Sci 116:3855–3862
Tomac AC, Widenfalk J, Lin LFH et al (1995) Retrograde axonal transport of glial cell line-derived neurotrophic factor in the adult nigrostriatal system suggests a trophic role in the adult. Proc Natl Acad Sci A 92:8274–8278
Tomac AC, Grinberg A, Huang SP et al (2000) Gial cell line-derived neurotrophic factor receptor α1 availability regulates glial cell line-derived neurotrophic factor signaling: evidence from mice carrying one or two mutated alles. Neuroscience 95:1011–1023
Matsuo A, Nakamura S, Akiguchi I (2000) Immunohistochemical localization of glial cell line-derived neurotrophic factor family receptor α-1 in the rat brain: confirmation of expression in various neuronal systems. Brain Res 859:57–71
Paratcha G, Ledda F, Baars L et al (2001) Released GFRalpha1 potentiates downstream signaling, neuronal survival, and differentiation via a novel mechanism of recruitment of c-Ret to lipid rafts. Neuron 29:171–184
Pruett BS, Salvatore MF (2010) GFR alpha-1 receptor expression in the aging nigrostriatal and mesoaccumbens pathways. J Neurochem 115:707–715
Salvatore MF, Pruett BS, Dempsey C, Fields V (2012) Comprehensive profiling of dopamine regulation in substantia nigra and ventral tegmental area. JOVE (66), e4171, DOI: 10.3791/4171
Salvatore MF, Pruett BS (2012) Dichotomy of tyrosine hydroxylase and dopamine regulation between somatodendritic and terminal field areas of nigrostriatal and mesoaccumbens pathways. PLoS One 7(1):29867
Haycock JW, Ahn NG, Cobb MH, Krebs EG (1992) ERK1 and ERK2, two microtubule-associated protein 2 kinases, mediate the phosphorylation of tyrosine hydroxylase at serine-31 in situ. Proc Natl Acad Sci A 89:2365–2369
Trupp M, Scott R, Whittemore SR, Ibanez CF (1999) Ret-dependent and -independent mechanisms of glial cell line-derived neurotrophic factor signaling in neuronal cells. J Biol Chem 274:20885–20894
Besset V, Scott RP, Ibanez CF (2000) Signaling complexes and protein-protein interactions involved in the activation of the Ras and phosphatidylinositol 3-kinase pathways by the c-Ret receptor tyrosine kinase. J Biol Chem 275:39159–39166
Salvatore MF, Waymire JC, Haycock JW (2001) Depolarization-stimulated catecholamine biosynthesis: involvement of protein kinases and tyrosine hydroxylase phosphorylation sites in situ. J Neurochem 79:349–360
Salvatore MF, Davis RW, Arnold JC, Chotibut T (2012) Transient striatal GLT-1 blockade increases EAAC1 expression, glutamate reuptake, and decreases tyrosine hydroxylase phosphorylation at ser(19). Exp Neurol 234:428–436
Wang C-Y, Yang F, He X et al (2001) Ca2+ binding protein frequenin mediates GDNF-induced potentiation of Ca2+ channels and transmitter release. Neuron 32:99–112
Rosenblad C, Georgievska B, Kirik D (2003) Long-term striatal overexpression of GNDF selectively downregulates tyrosine hydroxylase in the intact nigrostriatal dopamine system. Eur J Neurosci 17:260–270
Haycock JW, Becker L, Ang L et al (2003) Marked disparity between age-related changes in dopamine and other presynaptic dopaminergic markers in human striatum. J Neurochem 87:574–585
Salvatore MF, Ai Y, Fischer B et al (2006) Point source concentration of GDNF may explain failure of phase II clinical trial. Exp Neurol 202:497–505
Lei Z, Jiang Y, Li T et al (2011) Signaling of glial cell line-derived neurotrophic factor and its receptor GFRalpha1 induce Nurr1 and Pitx3 to promote survival of grafted midbrain-derived neural stem cells in a rat model of Parkinson disease. J Neuropathol Exp Neurol 70:736–747
Collier TJ, Ling ZD, Carvey PM et al (2005) Striatal trophic factor activity in aging monkeys with unilateral MPTP-induced parkinsonism. Exp Neurol 191(Suppl 1):S60–S67
Dass B, Kladis T, Chu Y, Kordower JH (2006) RET expression does not change with age in the substantia nigra pars compacta of rhesus monkeys. Neurobiol Aging 27:857–861
Alladi PA, Mahadevan A, Shankar SK et al (2010) Expression of GDNF receptors GFRalpha1 and RET is preserved in substantia nigra pars compacta of aging Asian Indians. J Chem Neuroanat 40:43–52
Bennett DA, Beckett LA, Murray AM et al (1996) Prevalence of parkinsonian signs and associated mortality in a community population of older people. N Engl J Med 334:71–76
Prettyman R (1998) Extrapyramidal signs in cognitively intact elderly people. Age Ageing 27:557–560
Bartus RT, Brown L, Wilson A et al (2011) Properly scaled and targeted AAV2-NRTN (neurturin) to the substantia nigra is safe, effective and causes no weight loss: support for nigral targeting in Parkinson's disease. Neurobiol Dis 44:38–52
Sarabi A, Hoffer BJ, Olson L, Morales M (2001) GFRalpha-1 mRNA in dopaminergic and nondopaminergic neurons in the substantia nigra and ventral tegmental area. J Comp Neurol 441:106–117
Franke B, Figiel M, Engele J (1998) CNS glia are targets for GDNF and neurturin. Histochem Cell Biol 110:595–601
Remy S, Naveilhan P, Brachet P, Neveu I (2001) Differential regulation of GDNF, neurturin, and their receptors in primary cultures of rat glial cells. J Neurosci Res 64:242–251
Honda S, Nakajima K, Nakamura Y et al (1999) Rat primary cultured microglia express glial cell line-derived neurotrophic factor receptors. Neurosci Lett 275:203–206
Salvatore MF (2012) Targeting tyrosine hydroxylase to improve bradykinesia. In: Dushanova J (ed) Mechanisms in Parkinson’s disease—models and treatments. InTech http://www.intechopen.com/books/mechanisms-in-parkinson-s-disease-models-and-treatments/targeting-tyrosine-hydroxylase-to-improve-bradykinesia
Acknowledgments
This work was funded in part by an NIH grant award to MFS, 1R01AG040261-01A1, and The Ike Muslow Predoctoral Fellowship to BSP. The authors also wish to thank Victoria Fields and Charles Dempsey for outstanding technical support.
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Pruett, B.S., Salvatore, M.F. Nigral GFRα1 Infusion in Aged Rats Increases Locomotor Activity, Nigral Tyrosine Hydroxylase, and Dopamine Content in Synchronicity. Mol Neurobiol 47, 988–999 (2013). https://doi.org/10.1007/s12035-013-8397-7
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DOI: https://doi.org/10.1007/s12035-013-8397-7