Role of heparin binding growth factors in nigrostriatal dopamine system development and Parkinson's disease

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Role of heparin binding growth factors in nigrostriatal dopamine system development and Parkinson's disease Deanna M. Marchionini a,⁎, Elin Lehrmann b , Yaping Chu a , Bin He a , Caryl E. Sortwell a , Kevin G. Becker c , William J. Freed b , Jeffrey H. Kordower a , Timothy J. Collier a a

Rush University Medical Center, Dept. Neurological Sciences, Chicago, IL 60612, USA Cellular Neurobiology Research Branch, National Institute on Drug Abuse, National Institutes of Health, DHHS, Baltimore, MD 21224, USA c Gene Expression and Genomics Unit, Research Resources Branch, National Institute on Aging, National Institutes of Health, DHHS, Baltimore, MD 21224, USA b

A R T I C LE I N FO

AB S T R A C T

Article history:

The developmental biology of the dopamine (DA) system may hold important clues to its

Accepted 6 February 2007

reconstruction. We hypothesized that factors highly expressed during nigrostriatal

Available online 22 February 2007

development and re-expressed after injury and disease may play a role in protection and reconstruction of the nigrostriatal system. Examination of gene expression in the

Keywords:

developing striatum suggested an important role for the heparin binding growth factor

Parkinson's disease

family at time points relevant to establishment of dopaminergic innervation. Midkine,

Striatum

pleiotrophin (PTN), and their receptors syndecan-3 and receptor protein tyrosine

Human

phosphatase β/ζ, were highly expressed in the striatum during development.

HB-GAM

Furthermore, PTN was up-regulated in the degenerating substantia nigra of Parkinson's

Microarray

patients. The addition of PTN to ventral mesencephalic cultures augmented DA neuron

Development

survival and neurite outgrowth. Thus, PTN was identified as a factor that plays a role in the nigrostriatal system during development and in response to disease, and may therefore be useful for neuroprotection or reconstruction of the DA system. © 2007 Elsevier B.V. All rights reserved.

1.

Introduction

Parkinson's disease (PD) is characterized by the progressive loss of dopamine (DA) in the nigrostriatal system. Infusion of trophic factors, such as glial cell line-derived neurotrophic factor (GDNF), offer some neuroprotection from nigrostriatal

degeneration induced by neurotoxins in animal models (Sauer et al., 1995; Gash et al., 1998; Kordower et al., 2000). Whether GDNF provides clinical benefits in PD patients, however, remains controversial (Gill et al., 2003; Nutt et al., 2003; Love et al., 2005; Patel et al., 2005; Slevin et al., 2005). Therapeutic effects of GDNF were minimal in a recent double-blind clinical

⁎ Corresponding author. Department of Neurological Sciences, Rush University Medical Center, 1735 West Harrison Street, Suite 300, Chicago, IL 60612, USA. Fax +1 312 563 3571. E-mail address: [email protected] (D.M. Marchionini). Abbreviations: DA, dopamine; PTN, pleiotrophin; RPTP β/ζ, receptor protein tyrosine phosphatase β/ζ; PD, Parkinson's disease; GDNF, glial cell line-derived neurotrophic factor; E, embryonic day; VM, ventral mesencephalon; P, postnatal day; SN, substantia nigra; LGE, lateral ganglionic eminence; CMF, calcium–magnesium-free buffer; AD, Alzheimer's disease; PSP, progressive supranigral palsy; PTNir, pleiotrophin immunoreactive; PFA, paraformaldehyde; BSA, bovine albumin serum; DAB, 3,3-diaminobenzidine; TH, tyrosine hydroxylase; HSSF, hormone-supplemented serum-free; PBS, phosphate-buffered saline; CASK, calmodulin-associated serine/threonine kinase; ALK, anaplastic lymphoma kinase; 6-OHDA, 6-hydroxydopamine 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.02.028

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trial in PD (Lang et al., 2006). Nevertheless, the general concept of using neuroprotective factors to treat PD may still be viable. Examination of the developing nigrostriatal system may provide clues to mechanisms that can be exploited to promote repair or recovery from disease. Molecules that are highly expressed during development are often re-expressed in response to injury and disease (Zhou et al., 2000; Ikeda et al., 2001; Marco et al., 2002). We hypothesized that exploitation of normal developmental and repair mechanisms might be used to promote restoration of the nigrostriatal DA system. Development of the nigrostriatal system occurs during the mid-embryonic period in rodents. The majority of nigral DA neurons have migrated to the ventral mesencephalon (VM) by embryonic day (E) 14 (Voorn et al., 1988). Striatal DA is first detected at E16, indicating that the first nigral neurons have extended processes to their target at this time (PerroneCapano and Di Porzio, 2000). Therefore, striatal cues that prompt establishment of nigrostriatal circuitry are likely highly expressed between E15 and E17. In addition, there are peaks of developmental nigral cell death at postnatal day (P) 2 and P14 (Janec and Burke, 1993). For other neural systems, this programmed cell death accompanies competition for stable synaptic connections (Hamburger and Oppenheim, 1982; Oppenheim, 1991). Increasing or decreasing the amount of striatal tissue available for innervation alters the magnitude of developmental nigral cell death (Burke et al., 1992). We examined the striatum at E15, a time point immediately prior to first detectable DA, and P1, just prior to nigral programmed cell death, based on the hypothesis that these time points are associated with expression of cues that attract nigral neurites to the striatum and stabilize dopaminergic innervation. Here we report that midkine and pleiotrophin (PTN), their receptors syndecan-3 and receptor protein tyrosine phosphatase β/ζ (RPTP β/ζ), and associated intracellular signaling molecules are highly expressed in the striatum during nigrostriatal development. PTN has previously been shown to be up-regulated in the injured brain, including nigrostriatal degeneration, in experimental animals (Takeda et al., 1995; Yeh et al., 1998; Poulsen et al., 2000; Hida et al., 2003). We also found that PTN is elevated in the substantia nigra (SN) of patients with PD. Previous studies have demonstrated a trophic effect of PTN on DA neurons in serum-containing conditions (Hida et al., 2003). In the present study, we found that PTN enhanced survival and neurite extension from DA neurons in serum-free conditions, suggesting a direct effect of PTN on DA neurons. These data suggest that PTN and midkine are involved in development of the nigrostriatal dopaminergic pathway as well as in PD, a degenerative disorder involving the nigrostriatal dopamine system.

2.

Results

2.1.

Microarray analysis

Transcripts that showed a greater than 1.5 z-ratio increase in the E15 and P1 striatum, as compared to the adult striatum were selected. A total of 139 of the 2700 transcripts (5.1%) were significantly increased at E15 and P1. We subsequently

classified these genes into functional groups (Table S1). The most highly up-regulated genes on our array were related to cell signaling, gene transcription, cell adhesion or extracellular matrix molecules in the developing striatum, compared to adult. Midkine gene expression showed a marked increase, with a 4.03 z-ratio increase in expression at E15 as compared to adulthood. Several members of the syndecan family of receptors were increased by z-ratio greater than 2.0 at E15 as compared to adult. Several isoforms of the receptor protein tyrosine phosphatases (RPTP), as well as the associated intracellular signaling molecules cadherin, actin, tubulin, cortactin and calmodulin-associated serine/threonine kinase (CASK) had at least 1.5 z-ratio increases in E15 and P1 striatum, compared to adult (Table S1). Fig. 1 outlines proposed signaling mechanisms of the heparin binding growth factor family.

2.2.

Verification of the microarray results

Our analysis of gene expression provided a potential link between the heparin binding growth factor family, their receptors and intracellular signaling pathways in the development of dopaminergic innervation of the striatum. However, not all of the relevant genes encoding molecules of this group were represented on the microarray. Thus, we expanded our analysis of this system using quantitative real-time reverse transcriptase–polymerase chain reaction (qPCR) and Western blot analyses. Consistent with microarray results, qPCR revealed a significant increase in midkine mRNA in the striatum at E15 as compared to the adult (3.29 ± 0.46 and 0.72 ± 0.14, respectively; [F(2,6) = 24.14, p = 0.001] Fisher's PLSD, p < 0.001; Fig. 2). Western blotting showed that there were significant changes in protein expression of midkine in the striatum during time points relevant to nigrostriatal development [F(2,6) = 21.58, p = 0.002]. Midkine was highly expressed in the striatum at E15, while at P1 and young adulthood it was down-regulated to an undetectable level (p = 0.001; Fig. 3). While a cDNA for PTN was not on the array, it shares a 50% homology with midkine and signals through similar receptors and intracellular cascades (see Muramatsu, 1993). Western blots revealed that PTN was highly expressed in the striatum at selected time points during development of DA innervation

Fig. 1 – Proposed mechanisms of heparin binding growth factor family signaling.

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Fig. 2 – To verify microarray data, midkine mRNA was measured in the striatum during development using qPCR. Midkine was most highly expressed at E15, and significantly down-regulated in the P1 and adult striatum (*p < 0.05). [F(2,6) = 15.97, p = 0.004], with significant down-regulation in adulthood (p = 0.001; Fig. 3). The PTN receptor RPTPβ/ζ followed a similar protein expression pattern in the striatum ([F(2,6) = 159.34, p < 0.0001]; Fig. 3). Syndecan-3 appeared to be expressed at similar levels in the striatum at all time points examined (p > 0.05; Fig. 3).

2.3.

PTN in the human SN

We tested the hypothesis that PTN would be up-regulated in the parkinsonian SN. Three distinct populations of cells in the

Fig. 3 – Western blotting of proteins encoded by genes-of-interest from the microarray analysis. Western blotting in the E15, P1 and adult striatum (n = 3) demonstrated that midkine (18 kDa), was highly expressed at E15, and down-regulated to an undetectable level by P1. PTN (18 kDa) was most highly expressed at P1, compared to E15 and adult (p < 0.05). Syndecan-3 (43 kDa) was expressed at statistically similar levels in the striatum throughout development and young adulthood. RPTP β (250 kDa) was most highly expressed in the E15 and P1 striatum, compared to adult (p < 0.05). β-Actin (42 kDa) was used as a control to verify that equal amounts of protein were added to each lane. # Significant difference from E15, ♦significant difference from P1, *significant difference from adult.

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SN were quantified by stereology: neuromelanin only, pleiotrophin immunoreactive (PTNir) only, and neuromelanin + PTNir. PTN immunolabeling was evenly distributed throughout subregions of the SN and its expression appeared to localize with specific cell morphology. Cells that stained positive for PTN had small soma and few neuritic processes, relative to PTN-negative cells. Stereological counts showed that there were significant differences in the number of neuromelanin cells between groups [F(3,17) = 11.73, p < 0.001; Fig. 4. As expected, there were significantly fewer neuromelanin only containing cells in PD and progressive supranuclear palsy (PSP) cases, as compared to normal aged and Alzheimer's disease (AD) cases, since the former diseases are characterized by nigral degeneration (p < 0.01). Similar numbers of neuromelanin + PTNir cells were detected in all brains examined [F(3,17) = 2.21, p > 0.05]. Significant differences in the number of PTNir only cells were observed between groups [F(3,17) = 3.70, p < 0.05]. There was a significant increase in the number of PTNir only cells in the SN of PD cases, as compared to all other groups (p < 0.03). There were no significant differences in staining intensity in any of the cell populations examined or between any of the diseases [F(7,28) = 1.49, p > 0.05; Fig. 5. Previous studies suggest that the majority of neuromelanin containing cells are dopaminergic (Bogerts et al., 1983; Hirsch et al., 1988). Since there was a significant population of cells that were neuromelanin + PTNir, we examined those cells for the DA phenotype. Several sections of normal aged and PD cases were double labeled with antibodies against PTN and tyrosine hydroxylase (TH) in order to identify the expression of PTN in DA neurons. Very few tyrosine hydroxylase immunoreactive (THir) + PTNir cells were identified in the SN of aged and PD cases. However, there was an abundance of PTN + neuromelanin (TH-negative) cells consistent with the presence of DA neurons that potentially have lost THir phenotype and have upregulated PTN (Fig. 6). We further evaluated

Fig. 4 – Stereological analysis of PTNir immunolabeled cells and naturally pigmented neuromelanin (NM) cells. Stereological analysis of NM only, PTNir + NM and PTNir only cells in the post-mortem SN from normal aged control, AD, PD and PSP cases. There was a significant decrease in the number of NM only containing cells in PD (*p < 0.001) and PSP (♦p < 0.01) cases, compared to normal aged control with no cognitive impairment (NCI) and AD cases. PD cases had a significant up-regulation in the number of PTNir only cells than all other cases (#p < 0.03).

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10, 50, 100 or 250 ng/ml PTN significantly increased THir neurite length by 155 ± 15.5%, 156 ± 21.5%, 163 ± 14.7% and 124 ± 17.8% of control (p < 0.03), while heat-inactivated PTN failed to promote THir neurite outgrowth (88 ± 17.2% of control, p > 0.05). Although the addition of 100 ng/ml GDNF resulted in a significant increase in THir neurite length (173 ± 34% of control, p < 0.02), the addition of 10, 50 or 150 ng/ml GDNF did not result in significant effects on the length of THir neurites (p > 0.05). Therefore, our evidence suggests that PTN was more potent than GDNF in stimulating neurite outgrowth. Fig. 5 – The SN from normal aged control, PD, AD and PSP were immunolabeled with an antibody against PTN. The optical density of PTN immunoreactivity in PTN only ( ) and PTN + neuromelanin-containing cells ( ) was measured. There were no significant differences in staining intensity in any of the groups examined.

▪

sections double labeled with antibodies against PTN and GFAP. PTN did not co-localize to astrocytes (Fig. 7). We therefore concluded that in the human SN, PTN is expressed in neurons, as evidenced by PTNir cell morphology, colabeling with TH and neuromelanin, and the absence of GFAP immunoreactivity. No PTNir was found in the striatum of normal control or PD cases (data not shown).

2.4.

Trophic effects in vitro

In order to evaluate the biological effects of midkine and PTN on DA neurons, these proteins were added to E14 VM cultures immediately after plating. Cultures were examined on day in vitro (DIV) 5 and THir neuron counts were made. Trophic effects of midkine and PTN were compared to the known DA trophic factor GDNF. Midkine (0, 10, 50, 100 or 250 ng/ml) had no significant effects on the number of THir neurons in VM cultures (data not shown). The addition of PTN or GDNF to E14 VM cultures significantly increased the number of THir neurons [F(9,90) = 2.568, p = 0.01; Fig. 8. The addition of 10, 50, 100 or 250 ng/ml PTN or GDNF significantly increased the number of THir neurons to 128 ± 5.3%, 125 ± 6.5%, 123 ± 5.5% and 120 ± 6.7% of control value, respectively, for PTN (p < 0.01). For GDNF, increases to 130 ± 6.6%, 118 ± 6.1%, 125 ± 8.9% and 121 ± 7.1% of control, respectively, were observed (p < 0.05). The percent increases for GDNF are consistent with trophic effects previously documented in microisland cultures (Collier et al., 2003). Based on previous reports in the literature (see Collier and Sortwell, 1999 for review), a wide range of doses of GDNF and PTN was tested. However, the lack of dose-related effect on survival in the range of 10–250 ng/ml suggests that all of these doses may saturate receptors for these molecules and that exploration of lower doses is warranted in future studies. Heat-inactivated PTN did not influence the number of THir neurons (105 ± 6.2% of control, p > 0.05). VM cultures grown in serum-free conditions do not support astrocyte growth. Our conditions yielded 16.1 ± 6.1 GFAP-positive cells per well, and the addition of PTN to cultures did not increase glial cell proliferation (16.6 ± 5.8 GFAP-positive cells). PTN and GDNF also significantly increased THir neurite length in VM cultures [F(9,95) = 2.062, p = 0.03]. The addition of

3.

Discussion

In response to damage the brain often re-expresses developmental cues as part of the recovery and repair process. Thus, we hypothesized that examination of molecules involved in differentiation and growth of the dopaminergic system might provide clues relevant to its repair following injury or disease. The present study addresses this possibility. We found that: (1) PTN and midkine, members of the heparin binding growth factor family, and their receptors syndecan-3 and RPTPβ/ζ were highly expressed in the striatum at selected time points during initiation and maturation of dopaminergic innervation; (2) The number of PTNir cells was increased in the degenerating SN in PD, apparently in DA neurons which had downregulated expression of TH; and (3) Addition of PTN to VM cultures significantly augmented the survival of THir neurons and promoted neurite outgrowth in serum-free medium microisland conditions. These data suggest that PTN is a natural participant in the development and response to damage in the DA system, and may be a promising candidate for use in therapy to protect or reconstruct the nigrostriatal system in PD. Midkine and PTN constitute the family of heparin binding growth and differentiation factors (Li et al., 1990; Nakamoto et al., 1992). PTN is expressed on axons, radial glial fibers, reactive astrocytes and axonal tracts during development (Takeda et al., 1995; McKeon et al., 1999; Rauvala and Peng, 1997; Rauvala et al., 1994). There are at least 3 receptors for PTN, including RPTPβ/ζ (Maeda and Noda, 1998; Maeda et al., 2003), syndecan-3 (Kinnunen et al., 1996; Rauvala et al., 2000) and anaplastic lymphoma kinase (ALK) (Powers et al., 2002; Stoica et al., 2002; Bowden et al., 2002). On the microarray, a number of transcripts related to the heparin binding growth factor family were highly up-regulated in the E15 and P1 striatum, as compared to the adult. The related downstream signaling transcripts cadherin, actin, tubulin, cortactin and CASK have been linked to the processes of cell division, migration, neurite outgrowth and synaptogenesis (for reviews see Rauvala et al., 2000; Kiryushko et al., 2004). Specifically, there is evidence that syndecan-3 promotes neurite outgrowth through the Src kinase–cortactin pathway (Kinnunen et al., 1998). PTN appears to be an important ligand for initiating the signaling cascade that promotes neurite outgrowth, which is critical during periods of plasticity, such as the remodeling that occurs during development and repair. In this context, it may be counterintuitive that PTN expression is high at P1 immediately prior to a wave of DA neuron developmental cell death. Yet there is no evidence to suggest that this level of

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protein does not represent a limiting supply of growth factor that may stabilize innervation of some DA neurons while participating in developmental death of other DA neurons. In vitro studies reveal that PTN promotes neurite outgrowth, cell migration, differentiation, mitogenicity, cell survival and angiogenesis in several cell populations (Raulo et al., 1992; Fang et al., 1992; Chauhan et al., 1993; Laaroubi et al., 1994; Nolo et al., 1996; Souttou et al., 1997, 1998, 2001; Stoica et al., 2001; Wang et al., 2002; Hida et al., 2003). The present results confirm previous findings of Hida et al. (2003) showing that PTN enhanced survival of cultured DA neurons. Confounding in vitro results regarding the same molecules can be found due to variations in the cell culture experimental paradigm. Therefore, verification of PTN's trophic effects on the DA system, utilizing varying cell culture methods, is important. Our experiments utilized microisland cultures,

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Fig. 7 – PTN expression was not found on glial cells. Doubled immunolabeling with antibodies against PTN (blue) and GFAP (red) demonstrated that PTN did not co-localize to GFAP immunoreactive cells. Scale bar = 25 μM.

which produce high-density, uniform cell cultures. Cell survival is directly influenced by cell density in culture (Barbin et al., 1984; Dal Toso et al., 1988; O'Malley et al., 1991; Sasaki et al., 1998; Marchionini et al., 2004). This density-related survival phenomenon is attributable to cell–cell contacts and release of autocrine and paracrine soluble factors in highdensity cultures (Sasaki et al., 1998; Marchionini et al., 2003). Low-density cultures, as used by Hida et al. (2003), experience greater baseline cellular stress and therefore are more susceptible to insult and potential rescue than high-density cultures. In fact, we have found that the magnitude of trophic effects provided by many known DA neuron growth factors is significantly diminished when tested in the microisland culture paradigm (Collier et al., 2003). Thus, the capacity of PTN to maintain trophic effects for DA neurons in microisland cultures argues that this molecule has potent effects on these neurons. In addition, our culture paradigm provides information suggesting that PTN has direct trophic effects on cultured DA neurons. It is difficult to ascertain the direct effects of a molecule on neuron survival in the presence of glia (serumcontaining conditions), since the molecule may act indirectly Fig. 6 – Localization of PTNir cells in postmortem SN. Neuromelanin has a natural brown pigment (black arrowhead). Neuromelanin appeared as blocks of brown granules localized to discrete portions of the cytoplasm. Double immunolabeling with antibodies against PTN (blue) and TH (red) demonstrated neuromelanin + PTNir cells (3 and 4), THir cells (1) and PTNir only cells (5). THir presented as diffuse pale red staining that homogeneously filled the neuronal soma and processes (A). Double-labeled PTNir and THir cells (2) appeared to have a purplish appearance of the cell body, suggesting co-localization of PTN and TH, while more distinct red (THir) and blue (PTNir) staining is present in the processes (B). Preadsorption of the PTN antibody solution with PTN protein abolished PTNir (C), demonstrating the specificity of this antibody for human PTN. The numbered arrows detail our hypothesized progression of the neurodegenerative process accompanied by the expression of PTNir, as detailed in the discussion. Scale bar = 50 μM.

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Fig. 8 – Photomicrographs and graphs illustrating the response of microisland VM cultures to treatment with PTN or GDNF. Microisland VM cultures were flooded with HSSF containing 0 (□), 10, 50, 100 or 250 ng/ml PTN ( ) or GDNF ( ), or 100 ng/ml heat-inactivated PTN. At DIV 5, cells were fixed and immunolabeled with TH. Photomicrograph with cells treated with 0 (A), 100 ng/ml PTN (B) or 100 ng/ml GDNF (C). PTN and GDNF similarly augmented the number of surviving THir neurons (D) (p < 0.05). PTN significantly promoted THir neurite outgrowth (E), and was more potent than GDNF at lower doses. *Significant from 0 (p < 0.05).

▪

on neurons by stimulating glial cells to produce undefined supportive factors. Since the present data employed cultures grown in serum-free medium, which does not support glial cell survival, PTN is likely to have a direct effect on DA neurons. Indeed, a recent study demonstrates that the PTN receptors RPTPβ/ζ and syndecan-3 are expressed by DA neurons in vitro (Mourlevat et al., 2005). Also, Jung et al. (2004) have demonstrated that PTN is highly expressed in VM progenitor cells, and the addition of PTN to progenitor cells promoted DA neuron differentiation. Furthermore, PTN regulates catecholaminergic biosynthetic enzymes in the aorta (Ezquerra et al., 2004). Collectively, these studies show that there is a relationship between PTN and the DA phenotype, which may be important in the development of the nigrostriatal system. By their nature, our culture experiments and in vivo development study demonstrate a relationship between PTN and the developing DA system. However, further studies should evaluate PTN's effects on cultured postnatal DA neurons and on the adult DA system to assess the generalization of trophic effects to these more mature neurons. PTN, syndecan-3 and RPTPβ/ζ are up-regulated following brain injury (Takeda et al., 1995; Yeh et al., 1998; Poulsen et al., 2000). Moreover, Hida et al. (2003) showed that there is an upregulation of PTN, syndecan-3 and RPTPβ/ζ mRNA in the striatum following 6-hydroxydopamine (6-OHDA)-mediated dopaminergic denervation in rats. This suggests that PTN is activated in response to injury, and that the degenerating DA system may be responsive to exogenous administration of the growth factor PTN. Our study detected an increase in PTNir

only cells in the SN of PD cases. While we hypothesize that this population of cells represents defunct DA neurons, it is possible that PTN co-localized to non-dopaminergic neurons, which make up approximately 5% of the population of the human SN. It is well documented that the normal SN contains abundant DA neurons, with aggregated neuromelanin that also are THir (Bogerts et al., 1983; Hirsch et al., 1988). We observed co-expression of TH and PTN in neurons with large soma and long neuritic processes. It is tempting to speculate that these are mildly stressed cells which have begun to upregulate PTN expression. Similarly, the population of cells significantly increased in the PD SN were intensely PTNir with diffuse cytoplasmic neuromelanin and small soma. These may be dysfunctional DA neurons, which have up-regulated PTN in response to severe stress or injury. Up-regulation of PTN expression appears to be specific to PD-induced nigral degeneration, since up-regulation of PTN was not found in the degenerating SN of PSP cases. It should, however, be noted that all PD patients included in this study were treated with levodopa, which has been shown to promote plasticity in the DA system (Murer et al., 1998). Moreover, up-regulation of PTN has been reported in the denervated striatum of levodopatreated rats (Ferrario et al., 2004). Manipulation of the host environment may prove a valuable tool in protection or restoration of the nigrostriatal system. This is particularly critical for cell replacement therapies. Transplantation of embryonic VM tissue is plagued by a meager 5–15% survival rate and a limited capacity of transplanted cells to reinnervate the striatum (Freed et al.,

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1987; Brundin and Bjorklund, 1987; Kordower et al., 1996). Stem cells face similar challenges, in addition to difficulties maintaining the dopaminergic phenotype (Zeng et al., 2004). The aged striatum, like that found in PD patients, is a particularly poor environment for support of grafted DA neurons (Collier et al., 1999; Sortwell et al., 2001). In fact, one clinical trial of cell replacement suggested that transplants of DA neurons provided no benefit to PD patients over 60 years of age (Freed et al., 2001). Recapitulation of the developmental environment may promote graft-derived functional recovery in aged PD patients. Administration of PTN into the denervated striatum may promote neurite outgrowth from VM grafts, thereby promoting a more homogenous and complete reinnervation of the striatum. This is especially important considering that novel dyskinesias in transplanted PD patients have been attributed to incomplete striatal reinnervation (Obeso et al., 2000; Ma et al., 2002; Olanow et al., 2003). PTN has properties consistent with its use as a therapeutic growth factor. GDNF is the current model for trophic factor therapy for PD, but it has to date failed to provide significant benefits in clinical trials (Nutt et al., 2003). GDNF is highly expressed in the developing nigrostriatal system (Schaar et al., 1993), supports the survival of DA neurons in culture (Lin et al., 1993), and protects the nigrostriatal system from DA-specific neurotoxins (Björklund et al., 1997; Kordower et al., 2000). We found that PTN and GDNF had similar effects on cultured DA neurons; however, PTN was substantially more potent in stimulating neurite extension. Since GDNF and PTN act through different mechanisms, they may have synergistic effects on the nigrostriatal system in vivo, as demonstrated in culture by Hida et al. (2003). To date, PTN has not been evaluated for its effects on the nigrostriatal DA system in vivo. Ongoing studies are evaluating the effects of PTN in neuroprotection and repair of the injured adult DA system, as well as its capacity to promote VM graft-derived reinnervation of the striatum, in animal models of PD.

4.

Experimental procedures

4.1.

Animals

Timed pregnant female and three month old male Fischer 344 rats were purchased from Harlan (Indianapolis, IN) and housed in the vivarium at Rush University Medical Center, which is fully AAALAC approved. All procedures were approved by the Rush University Medical Center Institutional Animal Care and Use Committee.

4.2.

Tissue dissection

Animals were anesthetized by CO2 inhalation, followed by rapid decapitation. Fischer 344 rat P1 and adult striatum were dissected on ice and pooled. Timed pregnant Fischer 344 rats provided E14 or E15 pups, from which the VM or lateral ganglionic eminence (LGE), respectively, were dissected (Nakao et al., 1994; Dunnett and Bjorklund, 1997). All tissue was collected in RNAlater (Ambion, Austin, TX) or calcium– magnesium-free buffer (CMF), for RNA and protein or culture studies, respectively.

4.3.

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Microarray experiments and analysis

The procedures for RNA extraction, sample labeling and microarray hybridization and analysis were described previously (Lehrmann et al., 2003). The human focused array contained 2700 cDNA clones in duplicate. In brief, total RNA was extracted per the manufacturer's protocol using TRIzol (Invitrogen, Carlsbad, CA). Two replicates were performed using the same starting RNA. For each sample, 5 (g total RNA was reverse transcribed in the presence of 33P-dCTP. The radiolabeled cDNA sample was purified through spin columns, denatured and allowed to hybridize to the array membrane at 55 °C for 16–18 h with rotation. The arrays were washed once with 2× SSC buffer at room temperature, followed by two rinses in 2× SSC with 0.1% SDS at 60 °C. The arrays were exposed to a low-energy phosphor screen (Molecular Dynamics, Sunnyvale, CA) for 7 days, then scanned in a Phosphorimager 860 (Molecular Dynamics) at 50 μm resolution, and quantified using ImageQuant software (Molecular Dynamics). Data were analyzed using background subtracted hybridization intensities, which were log-transformed and normalized to yield z-scores. These z-scores were used to calculate z-ratio values by taking the difference between the observed gene z-scores for the experimental and control comparisons and dividing by the standard deviation of these differences (Cheadle et al., 2003). Two replicates were performed for each sample using the same starting RNA, and since each membrane contained duplicate copies of each cDNA clone, the array data reflects the average intensities from four data points for each cDNA clone.

4.4. Quantitative real-time reverse transcriptase–polymerase chain reaction (qPCR) Total RNA was treated with 10 U DNase I (Ambion) to remove DNA contamination. Total RNA was reverse transcribed to cDNA using SuperScript II, oligo(dT)20 primers (Invitrogen, Carlsbad, CA). Primers were designed using MacVector 4.1 software (Accelrys, San Diego, CA) and specificity was ensured using BLAST: midkine sense (5′-GTTTGGAGCCGACTGCAAATAC-3′), antisense (5′-CATTGTACCGAGCCTTCTTCAGG-3′), while primers for 18S rRNA (Eurogentec, San Diego, CA) were used as an endogenous control. As described previously (Dickey et al., 2003), each sample was mixed with 50 μl QuantiTect SYBR Green (Qiagen, Valencia, CA). PCR reactions were run in triplicate at 1 cycle of 95 °C for 15 min, and 50 cycles of 95 °C for 15 s, 55.4 °C for 30 s, followed by 72 °C for 30 s. Primer pairs amplified a single peak of fluorescence by melt curve analysis. The standard curve was calculated by plotting the threshold cycle against the log nanogram quantity of RNA added to the real-time reactions. A linear regression was performed and the slope was calculated to determine the mass quantity of RNA. Quantities of RNA were divided by 18S mass values of the same real-time reaction to determine fold change in expression relative to the standard pool of RNA. Real-time PCR product was run on a 1% agarose gel by electrophoresis.

4.5.

Western blots

Protein expression for selected genes of interest was verified using Western blotting. Tissue was homogenized in ice-cold

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lysis buffer (0.1 M Tris, 0.9% NaCl, 0.5% NP-40, 1 mM EDTA, 0.01 M aprotinin, 0.5 mM leupeptin, 0.7 mM pepstatin and 2 mM PMSF). Protein concentration was determined using the Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA). For each sample, 20 μg total protein was denatured in Laemmli buffer with reducing agent (2% 2-mercaptoethanol; Bio-Rad) then separated on a 4–20% gradient sodium dodecyl sulphate– polyacrylamide gel and electrophoretically transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA). Nonspecific binding was blocked using 5% non-fat dried milk in Tris-buffered saline (TBS)–Tween (50 mM Tris–HCl, 200 mM NaCl, 0.05% Tween 20). Blotted membranes were incubated for 12 h at 4 °C with antibodies against midkine (0.1 μg/ml; R&D Systems, Minneapolis, MN), pleiotrophin (0.1 μg/ml; R&D Systems), RPTPβ (1:250; BD Biosciences, San Diego, CA), syndecan-3 (1:150; Zymed, South San Francisco, CA) or βactin as a loading control (1:5000; Abcam, Cambridge, MA). After washing, membranes were incubated for 1 h in horseradish peroxidase-conjugated donkey anti-goat (1:5000; Santa Cruz), goat anti-mouse or goat anti-rabbit (1:5000; Chemicon, Temecula, CA) secondary antibodies, respectively, followed by visualization with an ECL chemiluminescent substrate kit (Amersham Biosciences, Buckinghamshire, England) using BioMax XAR film (Eastman Kodak, Rochester, NY). Film was developed in a Kodak processor. All blots were repeated 3 times with independent samples. Band intensity was quantified using SigmaScan Pro (San Rafael, CA); values were normalized to β-actin signals for quantitative analysis.

4.6.

Human tissue

Subjects included in the study had a clinical and neuropathological diagnosis of idiopathic PD (n = 6), AD (n = 5), PSP (n = 4) or age-matched control (n = 6). Diagnoses were made as described previously (Litvan et al., 1996; Chu et al., 2001; Cubo et al., 2002). The average age was 79± 1.7 years old. The postmortem interval was 6.5 ± 1.2 h (Table 1). There were no statistical differences in the age at time of death or postmortem interval between the groups examined (p > 0.05). Each brain was cut into 1-cm-thick coronal slabs, then hemisected. The right side brain slabs were fixed in 4% PFA. Slabs containing the SN were sectioned on a freezing microtome into 40 μM transverse sections.

4.7.

Immunocytochemistry

PTN expression was quantified immunohistochemically in the SN of normal aged, PD, AD and PSP brains. AD cases served as a control for the presence of neurodegenerative disease. PSP cases served as a control for SN degeneration with pathology distinct from that seen in PD. The polyclonal PTN antibody (R&D Systems) was produced by immunizing goats with purified, insect cell line Sf21-derived, recombinant human pleiotrophin. PTN-specific IgG was purified by human PTN affinity chromatography. The antibody was selected for its ability to recognize PTN in direct ELISA and Western blot assays, and has less than 1% cross-reactivity with midkine, according to the manufacturer. Every 18th section was immunolabeled using the free-floating method for PTN. First, antigens were unmasked by heating sections to 95 °C in citric acid buffer (10 mM, pH = 6.0) for 20 min. After rinsing the

Table 1 – Case demographics Case and diagnosis PD-1 PD-2 PD-3 PD-4 PD-5 PD-6 NCI-1 NCI-2 NCI-3 NCI-4 NCI-5 NCI-6 AD-1 AD-2 AD-3 AD-4 AD-5 PSP-1 PSP-2 PSP-3 PSP-4 PSP-5

Gender

Age (years)

PMI (h)

M M M F M M M M M F M F M F F F M F M M M M

76 77 87 84 63 81 71 84 83 91 61 87 80 84 83 83 97 66 83 79 77 80

4 5.8 29 3.3 4.5 3.1 4 4 6 11 6 4 3 4.1 7.5 5.5 4 13 4 6 7 6

PMI – post-mortem interval in hours, PD – Parkinson's disease, NCI – normal aged control with no cognitive impairment, AD – Alzheimer's disease, PSP – progressive supranuclear palsy.

sections in TBS, endogenous peroxidases were quenched with 0.3% hydrogen peroxide for 20 min. Sections were rinsed in TBS, followed by blocking for 1 h in 10% horse serum, 2% bovine albumin serum (BSA) and 0.5% Triton-X in TBS. Sections were incubated in primary antibody against PTN (0.5 μg/ml; R&D Systems) for 18 h at room temperature. Additional control sections were tested, omitting the primary antibody or using PTN antibody preadsorbed with PTN protein (1:10). After TBS rinses, cells were incubated for 2 h in biotinylated goat antigoat IgG antibody (1:400; Chemicon), rinsed again, followed by 2 h in avidin–biotin–peroxidase complex reagent (Vector, Burlingame, CA). PTN immunoreactivity was visualized using the chromagen 3,3-diaminobenzidine (DAB), enhanced with 0.04% nickel ammonium sulphate and 0.03% hydrogen peroxide. Sections were mounted on subbed slides, dehydrated through graded alcohol, cleared in xylene and coverslipped with DPX (Electron Microscopy Sciences, Hatfield, PA).

4.7.1.

Double immunolabeling

The method described above was followed with the following exceptions. PTNir was visualized using a Vector SG substrate kit according to manufacturer's instructions (Vector). Afterwards, sections were incubated with antibody against TH (1:20,000; IncStar, Stillwater, MN) or glial fibrillary acidic protein (GFAP; 1:1000, Chemicon) as described above and visualized with Vector Red according to manufacturer's instructions (Vector).

4.8.

Stereology

Numbers of neuromelanin-containing, neuromelanin + PTNir and PTNir (neuromelanin-negative) cells within the SN were estimated using the optical fractionator method by an investigator blind to the experimental design (West, 1993). Cells

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were counted using StereoInvestigator software (MicroBrightField, Colchester, VT), as described in detail previously (Chu et al., 2002). The center of the SN, at the level of the ocular motor tract was sampled. Approximately 8 equi-spaced sections through this area were sampled for each case. The average area sampled for each case was 39.98 ± 1.69 mm3. The section sampling fraction (ssf) was 1/0.055. The area sampling fraction (asf) was 1/0.03. Section thickness was determined empirically and averaged 14.5 ± 2.0 μm in the midbrain and the thickness sampling fraction (tsf) was 1/0.57. The total number of neuromelanin, neuromelanin + PTNir and PTNir (neuromelanin-negative) cells within the SN was calculated using the following formula: N = Q − (1 / ssf)(1 / asf)(1 / tsf). Q was the total number of raw counts. The coefficients of error (CE) were calculated according to the procedure of Gundersen (Gundersen, 1977; Gundersen and Jensen, 1987). The values of CE ranged from 0.06 to 0.10.

4.9.

Quantification of PTN immunoreactivity

For quantification of PTNir, cells were randomly sampled throughout the extent of the SN that was sampled for stereological counts. PTNir was measured in both PTN only and neuromelanin + PTN cells using 60× magnification. The optical density of PTN immunoreactivity was measured using SigmaScan Pro, and values represent the total number of pixels/in.2. A small square was used to sample the PTN immunoreactivity in the cytoplasm of the soma, and caution was used to avoid sampling neuromelanin. Samples were also taken in the white matter of the ocular motor tract, and this value was subtracted from cell optical density to give the final reported value, used to compare cases.

4.10.

85

peroxide. THir neurons were counted in each of nine 20× fields at the center of each well, summed and used for statistical analysis. The center of each well was also digitally captured and analyzed using Image Analysis Software (SigmaScan Pro, Aspire Software International, Leesburg, VA). Every THir neurite in the field of view was measured, and the average neurite length for each well was used for statistical analysis. Each experiment consisted of at least 4 wells per treatment, and each experiment was repeated at least twice.

4.11.

Statistics

All comparisons between groups were done using a two-way ANOVA, and if significant, followed by a Fisher's PLSD posthoc analysis. Comparisons of cultures treated with 0 or 100 ng/ ml PTN were analyzed with a Student's t-test.

Acknowledgments We would like to thank Diane Teichberg and William H. Wood III for help with the microarray experiment and analysis. Research was supported by NIH grant NS42125 (TJC).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.brainres.2007.02.028.

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VM tissue was rinsed several times in CMF, then incubated in hormone-supplemented serum-free medium (HSSF; equal volumes of DMEM and Ham's F-12 supplemented with 1.0 mM glutamine, 1.0 mg/ml bovine albumin fraction V, 0.1 mg/ml apo-transferrin, 5 μg/ml insulin, 10 nM L-thyroxin, 20 nM progesterone, 30 nM sodium selenite, 10 U/ml penicillin, and 2.5 μg/ml fungizone) with 0.125% trypsin for 10 min at 37 °C. After several rinses with CMF, followed by HSSF/0.004% DNase, cells were dissociated into a single-cell suspension by trituration through a fire-polished Pasteur pipette. Cells were counted with a hemocytometer and verified to be greater than 95% viable using the trypan blue exclusion test. VM cells were dry-plated into 30,000 cell microislands on poly-D-lysinecoated plates (Takeshima et al., 1996). After 1 h at 37 °C, wells were flooded with HSSF containing 0, 10, 50, 100 or 250 ng/ml PTN, midkine or GDNF, or 100 ng/ml heatinactivated PTN. Cells were fixed on DIV 5 with 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PBS) for 30 min, then rinsed in TBS. Cells were incubated in primary antibody against TH (1:4000; Chemicon) for 12 h at 4 °C. After TBS rinses, cells were incubated for 2 h in biotinylated goat anti-mouse IgG antibody (1:400, Chemicon), rinsed again, and followed by 2 h in avidin–biotin–peroxidase complex reagent (Vector, Burlingame, CA). TH immunoreactivity (THir) was visualized using the chromagen DAB with 0.03% hydrogen

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