Neuroprotective Utility and Neurotrophic Action of Neurturin in Postnatal Motor Neurons: Comparison with GDNF and Persephin

MCN

Molecular and Cellular Neuroscience 13, 326–336 (1999) Article ID mcne.1999.0756, available online at http://www.idealibrary.com on

Neuroprotective Utility and Neurotrophic Action of Neurturin in Postnatal Motor Neurons: Comparison with GDNF and Persephin Masako M. Bilak, David A. Shifrin, Andrea M. Corse, Stephan R. Bilak, and Ralph W. Kuncl1 Department of Neurology, Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Baltimore, Maryland 21287-7519

Neurturin and persephin are recently discovered homologs of glial cell line-derived neurotrophic factor (GDNF). Here, we report that neurturin, like GDNF, increases the choline acetyltransferase activity of normal postnatal motor neurons, induces neurite outgrowth in spinal cord, and potently protects motor neurons from chronic glutamatemediated degeneration. Persephin, in contrast, does not appear to have neurotrophic or neurite-promoting effects on mature motor neurons and may instead worsen the glutamate injury of motor neurons. This pattern in the TGF-␤ family suggests certain receptor specificities, requiring at least the Ret/GFR␣-1 receptor complex. The results predict potential benefit of neurturin, but not persephin, in the treatment of motor neuron disorders and spinal cord diseases.

INTRODUCTION Amyotrophic lateral sclerosis (ALS) is a progressive neurological disorder characterized by selective degeneration of motor neurons. Slow, or ‘‘weak,’’ glutamate toxicity, resulting from abnormal synaptic clearance of glutamate, is one of several processes implicated in the pathogenesis of ALS (Rothstein et al., 1992). In this regard, neurotrophic factors could be beneficial in ALS by exerting trophic actions on surviving neurons or by decreasing neuronal sensitivity to glutamate neurotoxicity (Mattson et al., 1989; Schubert et al., 1992). Many neurotrophic factors exert specific trophic actions on embryonic spinal motor neurons. For example, glial cell 1 To whom correspondence should be addressed at the Department of Neurology, Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Baltimore, MD 21287-7519. Fax: (410) 614-9003. E-mail: [email protected].

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line-derived neurotrophic factor (GDNF), a member of the transforming growth factor-␤-related neurotrophic factor (TRN) family (Creedon et al., 1997), increases choline acetyltransferase (ChAT) activity of embryonic motor neurons (Zurn et al., 1994) and is perhaps the most potent factor in rescuing developing motor neurons from natural and axotomy-induced cell death (Henderson et al., 1994; Zurn et al., 1994; Oppenheim et al., 1995; Yan et al., 1995). GDNF has also been shown to protect adult motor neurons from avulsion-induced cell death (Li et al., 1995) and to enhance survival of postnatal motor neurons from chronic glutamatemediated degeneration (Corse et al., 1995, 1999). Neurturin and persephin are recently identified homologs of GDNF (Kotzbauer et al., 1996; Milbrandt et al., 1998). Both neurturin and persephin enhance the survival of developing motor neurons from natural and axotomyinduced cell death (Kotzbauer et al., 1996; Milbrandt et al., 1998). It is not known whether neurturin and persephin are neurotrophic for mature motor neurons or able to protect against chronic motor neuron degeneration. In the present study, we tested first whether neurturin and persephin can increase ChAT activity in postnatal motor neurons. Second, we examined whether neurturin and persephin can induce neurite outgrowth in the spinal cord. Finally, we investigated the possibility that neurturin or persephin may protect motor neurons from cell death in a culture model of ALS. The unique test system is the organotypic culture of postnatal rat spinal cord (Corse and Rothstein, 1995). This system offers several combined advantages: long-term survival (⬎60 days) of stable motor neurons, the maturity of postnatal rather than embryonic modeling, partially preserved synaptic connections among local neurons, and a glial 1044-7431/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.

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environment as well. Furthermore, long-term incubation of spinal cord cultures in the presence of threohydroxyaspartate (THA), a potent glutamate transport inhibitor, reproduces relatively selective glutamatemediated motor neuron degeneration, thus yielding a robust model of chronic motor neuron degeneration which has relevance for ALS (Rothstein et al., 1993). We have used this model already to successfully predict efficacy of motor neuron protection by riluzole and neurontin (Rothstein and Kuncl, 1995) and insulin-like growth factor-I (IGF-I) (Corse et al., 1993), all of which are being used for human motor neuron disease (Lai et al., 1997); similarly, the model (Corse et al., 1995) correlates with the failure of ciliary neurotrophic factor and brain-derived neurotrophic factor in clinical trials (ALS CNTF Treatment Study Group, 1996; Cedarbaum et al., 1998).

an increase in the median numbers of surviving motor neurons in neurturin (200 ng/ml)- or GDNF (100 ng/ml)treated cultures, which were not statistically different from age-matched untreated controls by Mann–Whitney U test (PB ⬎ 0.5). In contrast, addition of persephin at any dose (0.1–500 ng/ml) had no significant trophic or toxic effect on ChAT activity (Fig. 1B). Quantitation of total proteins showed no significant change in neurturin-, persephin-, or GDNF-treated cultures compared to untreated cultures, implying that the neurotrophic effects of neurturin and GDNF may be specific for motor neurons, as ChAT is a marker for motor neurons. By phase-microscopic examination, cultures treated with neurturin, persephin, or GDNF appeared morphologically identical to untreated controls (Figs. 2A and 2B).

Neurturin Induces Prolific Neurite Outgrowth in the Spinal Cord

RESULTS Neurturin Increases ChAT Activity of Motor Neurons We tested whether neurturin or persephin can upregulate the activity of ChAT in normal postnatal motor neurons, as one of their possible neurotrophic actions. Incubation of cultures with neurturin for 2 weeks caused a significant, dose-dependent increase in ChAT activity compared to age-matched untreated cultures (Fig. 1A). GDNF similarly increased ChAT activity at comparable doses (Fig. 1C). This effect did not represent

Neurotrophic factors could be beneficial for spinal cord diseases by inducing neurite outgrowth from normal surviving neurons. Previous studies have shown that GDNF induces robust neurite outgrowth in dopaminergic neurons. We examined the possibility that neurturin and persephin may induce neurite outgrowth in the spinal cord in our explant cultures. Immunostaining for neurofilament-H revealed abundant neurite outgrowth in both GDNF-treated cultures (1 to 100 ng/ml) (Fig. 2F) and neurturin-treated cultures (100 and 200 ng/ml) (Fig. 2D), and this was reflected in the

FIG. 1. Neurotrophic effects of neurturin on motor neuron ChAT. ChAT activity is expressed as a percentage of untreated control. (A) Neurturin treatment of cultures significantly increases ChAT activity of normal motor neurons after 21–23 days in culture (F ⫽ 51.1 by ANOVA; PB ⬍ 1 ⫻ 10⫺23, where PB equals calculated P value ⫻ 3). (B) Persephin, in contrast, does not affect ChAT activity between 0.1 and 500 ng/ml doses. (C) Like neurturin, GDNF increases motor neuron ChAT activity at similar doses. In each case, the diluents HCl and urea had no effect of their own. For significance level of individual comparisons with t tests, PB equals calculated P value ⫻ 15, the number of comparisons made in this graph. *Statistically significant increase from untreated control, PB ⫽ 0.026; **PB ⬍ 1 ⫻ 10⫺19.

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FIG. 2. Neurturin induces neurite outgrowth in the normal spinal cord. (A and B) Phase-contrast photographs illustrate preserved organotypic structures of untreated control (A) and neurturin (200 ng/ml)-treated cultures (B) (identically appearing persephin and GDNF-treated cultures are not illustrated). Neither neurturin or persephin appears toxic to the spinal cord. (C–F) Immunostaining of cultures for NF-H reveals neurons and their neurites (C, untreated control). Cultures treated with 200 ng/ml neurturin (D) or 10 ng/ml GDNF (F) contain somewhat larger motor neurons and abundant neurites which cross the midline or extend from the gray matter to the edge of the glial scar in ventral (arrowheads) and dorsal horns. Cultures treated with 200 ng/ml persephin (E), in contrast, do not appear distinguishable from untreated cultures (C). VH, ventral horn; DH, dorsal horn. Scale bars, 500 µm.

overall ratings and quantitative assessment of neurite outgrowth, masked to treatment assignment (Table 1). Analysis of variance indicated that neurite outgrowth across the glial scar was attributable to drug treatment and was highly significant statistically (Table 1). Neurturin evoked neurites to grow across the glial scar from both ventral and dorsal horn neurons, comparable to GDNF dorsally, but less often than GDNF ventrally (P ⫽ 0.036). Persephin at the same doses (100 or 200 ng/ml) did not appear to cause significant neurite outgrowth (Fig. 2E) compared to untreated controls (Fig. 2C), and this was reflected quantitatively on masked slides. Neurites crossing the midline were more variable and less frequent, visible mainly after GDNF treatment (data not shown). Independent counts of neurites on 57 masked photographs (covering the entire experimental data range) by two investigators were highly intercorrelated, indicating the significant reliability of the technique: correlation coefficients by Spearman’s ␳ test ⫽ 0.96 for dorsal midline crossing, 0.92 for ventral midline crossing, 0.93 across dorsal glial scar, 0.95 across ventral glial scar, and 0.721 for the more subjective overall rating of neurite outgrowth.

TABLE 1 Quantitation of Neurite Outgrowth

Factor (dose) Control Persephin (100 ng/ml) (200 ng/ml) Neurturin (100 ng/ml) (200 ng/ml) GDNF (1 ng/ml) (100 ng/ml) P B by ANOVA c

Neurites extending across glial scar a Dorsal

Ventral

Overall rating b

4.0 (0–14)

0.0 (0–5)

0.0 (0–1)

5.0 (0–16) 3.5 (0–8)

1.0 (0–8) 0.0 (0–3)

1.0 (0–1) 0.0 (0–1)

18.5 (9–39)** 11.5 (0–21)

3.0 (1–18)* 6.0 (2–13)**

1.0 (0–2)** 2.0 (1–2)**

25.5 (8–39)*** 25.5 (5–36)**

12.5 (2–45)*** 9.5 (4–22)***

2.0 (1–2)*** 2.0 (1–2)***

⬍1 ⫻ 10⫺10

⬍1 ⫻ 10⫺12

⬍1 ⫻ 10⫺12

Note. For significance level of individual comparisons with control by Mann–Whitney U tests, P B equals calculated P value ⫻ 18, the number of comparisons made: *⬍0.05, **⬍5 ⫻ 10⫺3, ***⬍5 ⫻ 10⫺6. aMedian number of neurites, per spinal cord slice (range in parentheses). bScoring: 0, none or very rare; 1, mild to moderate, but definite; 2, abundant. cKruskall–Wallis analysis of variance, where P B is the calculated P value ⫻ 3.

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Neurturin Protects Motor Neurons from Chronic Glutamate-Mediated Degeneration, whereas Persephin Potentiates the Glutamate Toxicity We tested the neuroprotective efficacy of neurturin and persephin in a culture model of chronic motor neuron degeneration mediated by glutamate (Rothstein et al., 1993). Long-term exposure of cultures to 100 µM THA significantly decreased ChAT activity by 40 ⫾ 5.8% (PB ⬍ 5 ⫻ 10⫺23 compared to untreated control). Neurturin consistently and potently overcame THA toxicity and increased ChAT activity compared to the THAintoxicated controls (Fig. 3A). The neuroprotective effect of neurturin was potent and dose-dependent over the tested range of 10 to 200 ng/ml, maximal at 200 ng/ml. The protective efficacy of 100 ng/ml neurturin on motor neurons (257 ⫾ 24% of THA control) was comparable to that of 1 ng/ml GDNF (249 ⫾ 40% of THA control) (Fig. 3C). Although both drugs have powerful neuroprotective effects, GDNF is roughly 100-fold more potent than neurturin on a dose–response basis in this model of motor neuron degeneration. Persephin did not repair THA toxicity (Fig. 3B); quite the contrary, 100 ng/ml persephin significantly and substantially added to toxicity (44 ⫾ 6% of THA control). Persephin at very low dose (0.1 ng/ml) also appeared to decrease ChAT activity. The significance of this apparent biphasic effect is unknown. Phase-contrast microscopy also revealed the neuroprotective effect of neurturin. Cultures treated with 100 µM

THA became thin and granular in appearance after approximately 2–3 weeks of treatment (Fig. 4A). However, cotreatment with neurturin (100–200 ng/ml) reproducibly and completely preserved the gross organotypic features (Fig. 4B). In contrast, cultures cotreated with persephin at a high dose (100 ng/ml) appeared even thinner and more granular than their THA-treated controls (Fig. 4C). To analyze the neuroprotective effects of neurturin and persephin on motor neurons in particular, we counted neurofilament-H (NF-H)-immunostained motor neurons in cultures that had been treated with either control medium or medium containing THA alone (Fig. 4D), neurturin plus THA (Fig. 4E), or persephin plus THA (Fig. 4F). The variance in the number of surviving motor neurons among groups attributable to neurturin or persephin treatment was highly significant by the Kruskal–Wallis ANOVA (PB ⬍ 1.0 ⫻ 10⫺4) in four experiments in which THA killed a median of 41% of motor neurons (Fig. 5). Neurturin treatment of THAintoxicated cultures completely spared motor neurons. Persephin was nonprotective. In fact, 100 ng/ml persephin treatment killed 71% of motor neurons, significantly worse than parallel THA-controls.

DISCUSSION This study extends to postnatal motor neurons the finding that neurturin has both neurotrophic actions (increased neurite outgrowth and ChAT activity) and

FIG. 3. Motor neuron protection with neurturin by ChAT analysis. ChAT activity is expressed as a percentage of THA-treated control. (A) Compared to THA-intoxicated cultures, cultures cotreated with neurturin have significantly higher ChAT activity after 40–47 days in culture (F ⫽ 38.96, PB ⬍ 1 ⫻ 10⫺23, by ANOVA, where PB equals calculated P value ⫻ 2). The response is dose dependent. For significance level of individual comparisons against THA control using t tests, PB equals calculated P value ⫻ 8, the number of comparisons made. *Statistically significant increase (PB ⬍ 4.5 ⫻ 10⫺13). (B) Persephin treatment of cultures does not increase motor neuron ChAT activity, but instead, significantly potentiates THA toxicity (†PB ⫽ 0.015, ‡PB ⫽ 3.2 ⫻ 10⫺7). (C) GDNF cotreatment of THA-intoxicated cultures, shown for comparison, has been previously reported to be significantly neuroprotective in this model (data adapted from Corse et al., 1995).

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FIG. 4. Morphologic evidence of neuroprotective and neurite-promoting effects of neurturin. (A–C) Phase-contrast micrographs of 40- to 47-day-old spinal cord cultures. Cultures exposed to 100 µM THA demonstrate marked thinning and granularity (compare Fig. 2A). The addition of neurturin (200 ng/ml) to THA dramatically and reproducibly preserves gross organotypic features (B). In sharp contrast, the addition of persephin (100 ng/ml) severely worsens the disruption of gross organotypic features, especially in the ventral horn (C). (D–F) Immunocytochemistry of 40- to 47-day-old organotypic cultures stained for nonphosphorylated NF-H in neuronal cell bodies and their processes. In cultures exposed to 100 µM THA, NF-H immunostaining reveals severe loss of motor neurons and a paucity of neurite outgrowth throughout the cord (D). Concurrent treatment of THA-intoxicated cultures with neurturin (200 ng/ml) dramatically and reproducibly preserves motor neurons and also induces prolific neurite outgrowth (E). Compare with THA-treated spinal cord in D and with untreated controls (Fig. 2D). Persephin treatment, in contrast, worsens the THA-mediated loss of motor neurons and fails to induce neurite outgrowth (F). Persephin particularly appears to damage the ventral horn. VH, ventral horn; DH, dorsal horn. Scale bars, 500 µm.

neuroprotective effects resembling the actions of GDNF. Persephin has opposite effects. Members of the TRN family are thought to signal via a two-component receptor complex consisting of a glycosylphosphatidylinositol-linked ␣ subunit (GDNF family receptor ␣ or GFR␣) and a transmembrane ␤ subunit (the receptor tyrosine kinase, Ret) (Durbec et al., 1996; Jing et al., 1996; Treanor et al., 1996; Trupp et al., 1996). GDNF and neurturin share GFR␣-1 and GFR␣-2 in complex with Ret, implying that GDNF and neurturin may have similar biological actions, although some preference, i.e., GDNF for GFR␣-1 and neurturin for GFR␣-2, has been demonstrated (Baloh et al., 1997; Jing et al., 1997; Klein et al., 1997; Trupp et al., 1998). In motor neurons, Ret and GFR␣-1 mRNAs are highly expressed during developing and adult stages, whereas GFR␣-2 mRNA is present only in developmental stages (Widenfolk et al., 1997; Yu et al., 1998). Thus, neurotrophic and neuroprotective effects of neurturin and

GDNF in mature motor neurons in our system may be mediated via Ret/GFR␣-1. A recent report suggested that Ret/GFR␣-1 or -2 receptor complexes are inadequate for persephin signaling (Milbrandt et al., 1998). Instead, persephin signals through Ret in combination with another new member of the GFR␣ receptor family, GFR␣-4, at least in the chicken (Enokido et al., 1998; Thompson et al., 1998). The presence of GFR␣-4 in mammalian cells is still unknown. In postnatal spinal cord motor neurons, neurturin but not persephin increased the activity of ChAT. GDNF, another member of the TRN family, also increases the activity of ChAT at similar doses. These effects seem to represent an upregulation of ChAT activity per motor neuron, because neither neurturin nor GDNF increases the median number of normal surviving motor neurons (in the absence of THA). The ability of neurturin to increase motor neuron ChAT has not been previously reported. GDNF has been previously reported to in-

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FIG. 5. Motor neuron survival after neurturin treatment. We identified and counted large (⬎25 µm) ventral horn neurons in cultures immunostained for NF-H (see Figs. 4D–4F for examples). This ‘‘box-and-whisker’’ plot summarizes the number of surviving motor neurons per culture slice. Bar indicates the median, ‘‘box’’ represents 25th and 75th percentiles, and ‘‘whiskers’’ represent 5th and 95th percentiles. (A) Untreated control cultures. (B) Chronic glutamate toxicity model, using 100 µM THA. Neurturin cotreatment, examined at two doses of 10 and 100 ng/ml, was highly significantly neuroprotective: 185% of the median THA-treated controls. In contrast, treatment with equal doses of persephin significantly reduced the number of motor neurons, apparently potentiating the toxicity of THA. *Significantly increased (PB ⬍ 0.006) compared to THA controls; †Significantly decreased compared to THA controls (PB ⫽ 0.00024); N indicates number of culture slices used for the analysis. For significance level of individual comparisons with Mann–Whitney U tests, PB equals calculated P value ⫻ 4, the number of comparisons made.

crease ChAT activity of embryonic motor neurons (Zurn et al., 1994). A structurally unrelated neurotrophic factor, IGF-I, has also been shown to upregulate ChAT activity of postnatal motor neurons (Corse et al., 1993). These observed neurotrophic effects of neurturin, GDNF, and IGF-I might be mediated through a common intracellular signaling pathway in motor neurons. To look at another trophic action, we used semiquantitative assays to demonstrate the relative neuritogenic properties of the TRN family members. Neurturin effectively induced neurite outgrowth from spinal cord neurons to approximately the same extent as did GDNF. However, comparing threshold doses for neurite outgrowth, GDNF appeared 100-fold more potent than neurturin on a per-milligram basis. Sprouting occurred approximately equally from dorsal and ventral horn neurons. Persephin did not significantly affect neurite outgrowth in this culture model. Our finding is consistent with reports showing that transgenic overexpression of GDNF causes hyperinnervation of neuromuscular junctions farther downstream at nerve terminals

(Nguyen et al., 1998) and that GDNF promotes neurite outgrowth from dopaminergic neurons (Hou et al., 1996; Krieglstein et al., 1995). GDNF and neurturin could be beneficial for spinal cord diseases by inducing prolific neurite outgrowth from normal surviving neurons. One caveat is that dose–response curves do not translate directly to in vivo experiments or single-cell systems probably because of the thickness of organotypic spinal cord slices (Klein et al., 1997). The pattern of potency, GDNF ⬎ neurturin ⫽ persephin, suggests that the neuritogenic effect of GDNF and neurturin may be mediated by a common cell surface receptor complex, Ret/GFR␣-1 (Jing et al., 1997; Klein et al., 1997; Widenfolk et al., 1997; Yu et al., 1998), as discussed above. Intracellularly, the neuritogenic action of GDNF and neurturin may be signaled via mitogen-activated protein kinase, as previously observed in superior cervical ganglion neurons (Creedon et al., 1997). We have shown that GDNF rescues postnatal rat motor neurons from chronic glutamate-mediated degeneration (Corse et al., 1995, 1999). Potent neuroprotective

332 actions of GDNF on motor neurons have been well documented previously. GDNF rescues motor neurons from programmed and axotomy-induced cell death in neonatal rats (Henderson et al., 1994; Milbrandt et al., 1998; Yan et al., 1995; Zurn et al., 1994) and neonatal mice (Oppenheim et al., 1995). In adult animals, GDNF can markedly reduce the axotomy-induced decrease of ChAT immunoreactivity in the rat facial nucleus (Yan et al., 1995) and avulsion-induced motor neuron death in the mouse (Li et al., 1995). In the present study, we show that neurturin is as neuroprotective as GDNF. We used two independent methods to show this. First, we used a radiochemical assay to measure ChAT enzymatic activity and showed that neurturin prevents glutamateinduced loss of ChAT activity. ChAT is a reliable marker largely restricted to ␣-motor neurons in the lower lumbar cord (Armstrong et al., 1991; Burnett et al., 1995; Fonnum, 1975; Phelps et al., 1984) and in organotypic spinal cord slices the level of enzyme activity is comparable to that in age-matched uncultured lumbar spinal cord tissue (Rothstein et al., 1993). But the neurturininduced sparing of motor neuron ChAT is not merely a neurotrophic upregulation of ChAT but a separate neuroprotective ability, as shown by the following. The second method, counting the number of motor neurons in organotypic cultures fixed and immunostained for NF-H, showed that neurturin increases the number of postnatal motor neurons surviving chronic glutamate excitotoxicity. This raises a methodologic issue: it is important to consider the incomparability of different methods for motor neuron counting. Other studies have rarely counted motor neurons in animals older than the first postnatal week, i.e., after the process of massive natural cell death is complete. We have previously reported motor neuron counts in normal postnatal rat organotypic cultures immunostained for ChAT to be ⬃20 per 350-µm length of cord (Rothstein et al., 1993; Corse and Rothstein,1995). Another group (Mitsumoto et al., 1994) used ChAT immunostaining of the adult mouse cervical spinal cord (which should be higher than lumbar spinal cord and may include autonomic neurons); one can extrapolate from their data an estimate of at least ⬃24–72 motor neuron counts per 360-µm length of cord. Thus, the counts of motor neurons we report in this study by NF-H immunostaining (7–44 per slice in normals) are at least roughly comparable to those reported previously by ChAT immunostaining. Two other reports provide normative data obtained by routine histology. In less mature postnatal day (PND)-5 rat spinal cord (Oppenheim, 1986), one can estimate ⬃170 ␣-motor neurons per culture slice. Or, using normative data from serial

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sections of PND-12 mouse lumbar spinal cord, one would predict ⬃90 motor neurons per 350-µm slice (Li et al., 1994). Our counts probably represent a partial underestimate of motor neurons actually present in these cultures. We used narrow operational definitions to identify and count motor neurons apart from other neurons after NF-H-immunostaining; neurons that did not meet our conservative criteria, i.e., large neurons with no visible thick processes or those located in the dorsal half of the ventral horn, were excluded. In addition, our organotypic slices are thick (350 µm), and the penetration of antibody is not likely to be nearly complete, which may also lead to underestimation. Also, cells that were split by the knife or physically damaged during the culture preparation would be lost. Nevertheless, we handled controls and experimental groups identically, and we removed bias by using masked slides. The counts are therefore probably representative of the majority of motor neurons. Most importantly, the two independent methods we used to assess neuroprotection (ChAT activity and motor neuron counts) are in agreement that neurturin provides virtually complete protection for postnatal motor neurons from chronic motor neuron degeneration. Neurturin’s protective effect is biologically important and extends beyond motor neurons, as it significantly spared gross organotypic structure and preserved both dorsal and ventral horn. The magnitude of the effect is comparable to that of the most potent neuroprotectants reported previously in this model, i.e., IGF-I (Corse et al., 1993) and GDNF (Corse et al., 1995, 1999). In contrast, persephin had no such effect. Even though it shares ⬃40% sequence identity with GDNF and neurturin, and reportedly supports the survival of embryonic and axotomized PND-2 neonatal motor neurons (Milbrandt et al., 1998), it seems to lack protective action on motor neurons in our postnatal organotypic cultures. Instead, in this model it actually causes injurious potentiation of glutamate toxicity at high doses. These opposite neuroprotective effects of neurturin and persephin may not be surprising. Persephin has previously been reported to lack survival-promoting effects on peripheral neurons such as sympathetic, parasympathetic, sensory, and enteric neurons in contrast to potent effects of neurturin and GDNF on these neurons (Buj-Bello et al., 1995; Ebendal et al., 1995; Trupp et al., 1995; Kotzbauer et al., 1996; Heuckeroth et al., 1998; Milbrandt et al., 1998). While the observed worsening of glutamate toxicity by persephin could have been due to contaminants in the persephin preparation, this seems highly unlikely, as persephin treatment by itself did not have any toxicity on motor neurons (see Fig. 1), and furthermore, each

Protective Effects of Neurturin on Motor Neurons

batch of persephin was purified to obtain a single band in Coomassie blue-stained SDS–polyacrylamide gels. Since the worsening of survival was seen only at a high dose, far above doses proven to be bioactive for cholinergic and dopaminergic neuron survival in other systems, the effect is probably indirect, not receptor-mediated. From the discussion above, individual members of the TRN family of neurotrophic factors may have unique actions on postnatal motor neurons depending on coreceptor profiles (Milbrandt et al., 1998). On the other hand, intracellular signaling may also be explanatory. The ability of neurturin, GDNF, and IGF-I to protect mature motor neurons from glutamate injury may correspond with their ability to activate the phosphatidylinositol 3-kinase (PI-3-K) pathway in other types of neurons, as PI-3-K has been implicated in neuronal survival (Creedon et al., 1996, 1997; Miller et al., 1997). Whether persephin stimulates PI-3-K activity is not known. The mechanism of action of these factors must await a better characterization of the receptor complexes and signaling pathways they utilize.

CONCLUSION Preclinical tests of the potential neurotrophic and neuroprotective effects of neurturin and persephin on lower motor neurons show that: (1) neurturin, but not persephin, trophically increases ChAT activity of postnatal motor neurons and promotes neurite outgrowth in the spinal cord, very much like GDNF although less potently; and (2) neurturin protects motor neurons virtually completely from slow glutamate toxicity caused by THA, whereas persephin actually potentiates the toxicity. Neither neurturin nor persephin alone has any inherent toxic effect on motor neurons. The neuroprotective effect of neurturin is concentration-dependent. Taken together with recent reports that Ret and at least GFR␣-1 mRNAs are highly expressed in motor neurons during developmental and adult stages, neurturin may be beneficial in the treatment of motor neuron diseases including ALS and spinal muscular atrophy. The results may extend to spinal cord trauma and other myelopathies.

EXPERIMENTAL METHODS Experimental paradigms. Organotypic spinal cord cultures were prepared from lumbar spinal cord of PND-8 rat pups as described previously (Corse and Rothstein, 1995). Neurturin and persephin were pre-

333 pared by Eugene Johnson and Jeffrey Milbrandt (Washington University, St. Louis, MO) as described (Kotzbauer et al., 1996; Milbrandt et al., 1998). In brief, a synthetic gene for the murine neurturin coding sequence and a fragment encoding the mature murine persephin protein were each expressed in Escherichia coli (Creedon et al., 1997; Milbrandt et al., 1998). Each batch of neurturin or persephin was purified by urea column chromatography, then renatured and tested for bioactivity in superior cervical ganglion cells and/or dopaminergic and motor neurons (Creedon et al., 1997; Milbrandt et al., 1998). Purity of neurturin and persephin was routinely confirmed by the presence of a single band in Coomassie blue-stained SDS–polyacrylamide gels. GDNF was commercially obtained (Pepro Tech, Rocky Hill, NJ). Cultures were allowed an 8-day recovery period after initial preparation, prior to the addition of drugs. We have previously shown that the initial drop in ChAT enzyme activity is a typical transient response of motor neurons to partial axotomy, not a loss of cells (Rothstein et al., 1993). During the postrecovery test period ChAT activity returns to a baseline value which is comparable to age-matched, uncultured lumbar spinal cord. Thus, this culture system does not closely resemble motor neuron axotomy models. Two paradigms were used: (1) Neurotrophic. To examine constitutive effects of neurturin and persephin on motor neurons, we treated cultures (starting on culture day 8) with either neurturin (1,10, 100, 200, or 500 ng/ml) or persephin (0.1, 1, 10, 100, 200, or 500 ng/ml) for 2 weeks. The lowest doses were initially chosen on the basis of effects in previous reports (Kotzbauer et al., 1996; Milbrandt et al., 1998). Each data point represented 1 culture well (five spinal cord slices per well). Experimental groups included 6–22 culture wells and controls included 60 wells. Neurturin and persephin were compared with GDNF in parallel experiments. We used assays of ChAT activity as a reliable marker for large motor neurons (Fonnum, 1975; Phelps et al., 1984; Armstrong et al., 1991), as ChAT is largely restricted to motor neurons in the rat lumbar spinal cord at L2–L5 (Burnett et al., 1995). (2) Neuroprotective. We have previously shown in this system that inhibition of glutamate/aspartate transport with 100 µM THA elevates extracellular glutamate and injures large motor neurons in the ventral horn with a morphology typical of excitotoxic degeneration (Rothstein et al., 1993). The time course is slow, and the process is mediated by non-NMDA receptors (Rothstein et al., 1993). To determine the neuroprotective potentiality of neurturin and persephin against chronic glutamate neurotoxicity, THA-intoxicated culture slices were treated (starting on culture day 8) with either neurturin

334 (1, 10, 100, or 200 ng/ml) or persephin (0.1, 1, 10, or 100 ng/ml) for 31–39 days. The number of culture wells used for ChAT assays in neuroprotection experiments was approximately 50 for both untreated control and THA-treated controls and ranged from 12 to 21 for experimental groups. At each medium change, we added doses of either THA alone or THA plus neurturin or persephin to the medium. At the end of the incubation, we compared these treatment groups with the THA control by ChAT radioassay, by phase microscopy, and by directly counting motor neurons in immunostained cultures. Cultures treated with GDNF (1 ng/ml) served as the comparison control. Choline acetyltransferase assay and total protein content of cultures. ChAT activity was measured radiometrically using [3H]acetyl coenzyme A (Dupont NEN, Boston, MA) as described (Corse and Rothstein, 1995). For statistical analyses of ChAT data from cultures, identical treatment groups from multiple experiments were combined after normalization to the mean of untreated controls within an individual experiment. Total protein content of culture homogenates was determined using the Coomassie Plus Protein Assay Kit (Pierce, Rockford, IL). Morphological analysis and cell counts. The gross morphology of cultures was monitored daily by inverted phase microscopy. Immunocytochemistry was used to visualize neurons with their processes in order to determine the number of motor neurons in spinal cord cultures. After neuroprotective treatments, cultures were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 20 min at room temperature, permeabilized with methanol for 20 min at 4°C, rinsed with Tris-buffered saline, and blocked with 10% normal goat serum for 1 h at room temperature. Cultures were then incubated with a well-characterized monoclonal antibody (SMI-32; Sternberger Monoclonals, Inc., Baltimore, MD) against nonphosphorylated NF-H (Sternberger and Sternberger, 1983) at 1:8000 dilution overnight at 4°C. Cultures were processed with the VectaStain ABC Kit (Vector Laboratories, Burlingame, CA) and diaminobenzidine (Polyscience, Warrington, PA). Previous experience has shown that ChAT activity correlates strongly with the number of motor neurons in the ventral horn (Corse et al., 1993; Rothstein et al., 1993). To determine the number of motor neurons in spinal cord cultures, we counted neurons in the NF-H-immunostained cultures meeting the following criteria: (1) size (⬎25 µm), (2) possession of at least one thick process, and (3) location in the ventral half of the ventral gray matter. The size of neurons was estimated by comparison with a 50-µm crosshair in the eyepiece, using a 10⫻

Bilak et al.

bright-field objective lens. In the neuroprotection paradigm, cultures were 40–47 days old; at least 18 cultures from each experimental group were used for counting motor neurons on masked slides by a single investigator. Quantitation of neurites in spinal cord cultures. The amount of neurite outgrowth in organotypic spinal cord cultures was analyzed using immunostaining for NF-H in the growing neurites (see above), as the staining is robust and provides extraordinarily high resolution. Because of obvious difficulty in tracking individual neurites throughout a thick, complex section, the following operational definitions were used. In untreated control cultures, neurites rarely grow beyond the margin of the surviving gray matter of culture slices to cross the surrounding ‘‘glial scar’’ which results from degeneration of axotomized tracts beginning at the time of culture preparation. Similarly, neurites rarely grow across the midline into the other hemicord. To quantitate neurites in cultures, we counted neurites that extended beyond the gray matter through the entire glial scar or those that crossed the midline. In each case, we considered separately whether neurites came from the ventral or the dorsal cord, defined by the lateral sulcus. Neurites observed contacting the outer edge of the glial scar at multiple sites or crossing the midline multiple times were given only one count. Cultures were treated with either neurturin (100 or 200 ng/ml) or persephin (100 or 200 ng/ml) for 23 days and compared to (1) an untreated control and (2) a positive control, i.e., GDNF-treated cultures (1 or 100 ng/ml), for which more is known about sprouting, at least in dopaminergic neurons (Krieglstein et al., 1995; Hou et al., 1996). Neurites were counted directly using a loupe and 5 ⫻ 8-in. digital photomicrographs taken from neurotrophic factor-treated and untreated control cultures. We also estimated qualitatively the neurite growth in each culture by grading on a 3-point scale (0, none or rare; 1⫹, mild to moderate but definite; 2⫹, abundant). For all these measures of neurite growth, at least 10 cultures from each experimental group were analyzed by a single investigator who was masked to treatment. Statistical analyses. Statistical analysis of continuous data (ChAT activity) was performed by one-way analysis of variance for each drug at its various dosages, followed by two-tailed Student’s t tests to compare each drug condition with either the appropriate untreated control (constitutive effects of trophic factors) or THAtreated control (neuroprotective paradigms). We considered the quantitative analyses of both motor neurons and neurites to be near-ordinal data, which required the nonparametric Kruskal–Wallis one-way analysis of vari-

Protective Effects of Neurturin on Motor Neurons

ance to analyze multiple tested drugs at multiple doses. This was followed by the Mann–Whitney U test to compare each drug dose with its appropriate control. Two-tailed analyses were always performed. In order to justify the use of a 0.05 significance level and because of the large number of ANOVAs and the large number of possible comparisons by t and Mann–Whitney U tests, we applied the most conservative correction for multiple comparisons, the Bonferroni inequality correction (Fisher et al., 1993). This has the effect of multiplying the calculated P values by the number of comparisons performed in each type of experiment, yielding PB (see figure legends). Data are presented as means ⫾ standard errors (ChAT) or medians (counts of neurons or neurites).

ACKNOWLEDGMENTS We especially thank E. Johnson, J. Milbrandt, and P. Osborne (Washington University, St. Louis, MO) for providing neurturin and persephin and P. Kotzbauer for arranging the collaboration. M. Lehar provided technical help. This work was supported by research grants from the NINDS and the Muscular Dystrophy Association, an NINDS clinician investigator development award (to A.M.C.), the Cal Ripken/ Lou Gehrig Fund for Neuromuscular Research, and the Jay Slotkin Fund for Neuromuscular Research.

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