JOURNAL OF THE

NEUROLOGICAL SCIENCES

ELSEVIER Journal of the Neurological Sciences 140 (I 996) 1 I7- I22

Expression of ciliary neurotrophic factor is maintained in spinal motor neurons of amyotrophic lateral sclerosis

Michael Schorr, Lepu Zhou, Karl Schwechheimer * Institutfir Neuropatholoyie, Univzrsitiit Essen. Hufelandstr. 55. 45122 Essen, Germany

Received 30 October 1995; revised 27 February 1996; accepted 19 March 1996

Abstract

Ciliary neurotrophic factor (CNTF) was originally identified as a potent survival factor for a variety of neuronal cell types in vitro and in vivo and in particular in spinal motor neurons of embryonic chick and rat. Using a monoclonal antibody against CNTF (clone 4-68) we analysed the expression of CNTF in paraffin sections of seven human brains and spinal cords immunocytochemically using the ABC method and compared these results with sections of the spinal cords of patients suffering from amyotrophic lateral sclerosis (ALS). In normal human tissue of the central nervous system CNTF immunoreactivity was found in most of the motor neurons of the motor cortex and ventral horn, neurons of the nucleus oculomotorius, intermediolateralis, thoracicus, ependymal cells as well as in smooth muscle cells and endothelial cells of small arteries. A reduced number of astrocytes showed a positive immunocytochemical reaction. In peripheral nerves and nerve roots of the spinal cord we also found a positive staining of Schwann cells and some axons. These immunoreactions could be confirmed by Western blot analyses. Next we analysed postmortem paraffin sections of the spinal cord of seven patients suffering from ALS (age range 30-76 years, median age 46 years, female/male = 4:3). We found CNTF immunoreactivity in most of the motor neurons of the ventral horn in 5 cases. In two cases the number of positively stained motor neurons was less. From these results we conclude that CNTF is expressed in a high number of upper and lower motor neurons in the human CNS and that its expression is maintained in ALS patients.

Keywords: CNTF: ALS; Neuron; Immunoreaction; Western blot

1. Introduction

Ciliary neurotrophic factor (CNTF), a cytoplasmatic 200 amino acid protein with a molecule weight of 23 kDa (Stiickli et al., 1989; Masiakowski et al., 19911, is ex- pressed in neurons and astrocytes of the adult rat periph- eral and central nervous system (Stiickli et al., 1991; Henderson et al., 1994). CNTF has been described as a neurotrophic factor that supports the survival of parasym- pathetic neurons from the ciliary ganglion of chick em- bryos in vitro (Adler et al., 1979; Barbin et al., 1984). It is involved in the proliferation and differentiation of embry- onic chick sympathetic neurons in vitro (Emsberger et al., 1989). In vitro, CNTF promotes the differentiation of rat 02A progenitor cells to type-2 astrocytes (Hughes et al., 1988). In vivo, CNTF prevents spinal motoneurons from

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entering programmed cell death (Oppenheim et al., 19911, degeneration of motoneurons in a mutant mouse model of motoneuron desease (Sendtner et al., 19921, lesion induced degeneration of rat facial motoneurons (Sendtner et al., 19901, as well as lesion induced degeneration of adult rat substantia nigra dopaminergic neurons (Hagg and Varon, 1993). After peripheral nerve injury the retrograde axonal transport of CNTF is increased (Curtis et al., 1993). It has also been described that the disruption of the CNTF gene results in a loss of motoneurons and progressive muscular atrophy in adult mice (Masu et al., 1993).

The CNTF receptor (CNTFR) is a tripartite receptor which includes CNTFR alpha, LIFR beta and gp 130 (Davis et al., 1991; Davis et al., 1993). It shares its system with the cytokines leukemia inhibitory factor (LIF) and interleukine 6 (IL6), which mediates its signals via the gp 130 receptor (Ip and Yancopoulos, 1992). CNTFR is widely distributed in the CNS of the adult rat (Ip et al., 1993).

Here we describe the regional distribution of CNTF in normal human brain and spinal cord. The focus of our

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118 M. Schorr et al. /Journal of the Neurological Sciences 140 C1996) 117-122

study is the description of CNTF immunoreactvity in spinal cord sections of patients suffering from ALS.

2. Material and methods

2.1. Human brain tissue

Formaldehyde-fixed and paraffin-embedded autopsy material (4- 16 h post mortem) from seven brains and spinal cords without morphological alterations were used as a control group to study the distribution of CNTF immunomorphologically. Areas of the motor cortex, the midbrain and the cervical and lumbar spinal cord were selected (age range 47-80 years, median age 69 years, male/female = 4:3). 4-6 pm paraffin slides were stained with hematoxylin eosin and according to Khiver-Barrera and histologically examined.

2.2. Spinal cord of ALS patients

Seven spinal cord sections of post mortem tissue from patients suffering from ALS were analysed (age range 30-70 years; median age 46 years; male: female = 3:4). Age and postmortem delay of cases were closely matched within the normal human brain and spinal cord tissues of ALS patients.

2.3. Immunomorphology

4-6 pm paraffin sections were fixed on poly(L-lysine)- coated slides, dewaxed and rehydrated. For antigen re- trieval the sections were wet autoclaved in 0.01 M citrate buffer (pH 6.0) (BankfIavi et al., 1994). Endogenous per- ox&se activity was blocked with 3% hydrogen peroxide in aqua bidest. After blocking with rabbit serum (1:20; 30 minutes at room temperature; Dako, Hamburg, Germany) the CNTF monoclonal mouse antibody (1:500; IgGl, clone 4-68; hybridoma supematant diluted 1:2 in PBS, a kind gift from Dr. Sendtner, Wiirzburg, Germany) was applied for 12 h at 4°C. After rinsing in Tris-HCl buffer (pH 7,6) a rabbit anti-mouse antiserum (1:200; 1 h at room tempera- ture; Dako, Hamburg, Germany) was applied and after incubation with streptavidin (1:300; Dako, Hamburg, Ger- many) for 30 min at room temperature the peroxidase activity was visualised with 3-amino-9-ethylcarbazole (Hsu et al., 1981).

2.4. Protein preparation and Western blotting

Prepared human autopsy tissue from the motor cortex, ventral horn from the spinal cord and from sciatic nerves was homogenized in hypotonic lysis buffer (pH 7.0) and ultracentrifuged at 100000 X g for 30 min in an Optima L 70 ultra-centrifuge (Beckman Instruments, Palo Alto, CA,

Table 1 Distribution of CNTF immunoreaction in normal brain

Localisation Normal brain

Motor cortex Motor neurons (Stratum ganglionaris) +++/++++ Stratum pyramidalis +++ Stratum granularis intema +++ Striatum Large neurons -

Small neurons -

Ependymal cells +++ Midbrain Neurons of substantia nigra -

Neurons of nucleus ruber ++/+++ Neurons of nucleus oculomotorius +++ Ependymal cells +++ Neuropil/axons (+I Astrocytes + Oligodendrocytes -

Aterial vessels Endothelial cells +++ Smooth muscle cells ++++

+ = A few, + + = about 50%, + + + = most of, + + + + = all cells stained.

USA) and the supematant was removed. Protein concentra- tions were determined using the folin phenol reagent as described by Lowry et al. (195 1).

For Western blot analyses 50 pg protein per lane were run on a lo-20% polyacrylamide gel for 45 min. Follow- ing blotting onto a nitrocellulose membrane (Schleicher and Schull Inc., Dassel, Germany) for 12 hours at 150 mA (Wet Blot, Mini, Sigma, St. Louis, MO, USA) the mem- brane was blocked with 0,1% dry milk powder, 1 kg NaN,, 20 ~1 Tween 20 (Sigma) and 1 ~1 Antifoam A (Sigma) in aqua bidest for 12 h at room temperature. Then, the membrane was incubated with the monoclonal CNTF antibody (1:500) overnight at 4°C. After rinsing in 0.05 M Tris-HCl buffer (pH 7.6) the following steps were carried out as described above.

Table 2 Distribution of CNTF immunoreaction in normal spinal cord and spinal cord of ALS

Localisation Normal ALS

Motor neurons ++++ + + + + (5/7)+ + (2/7) Neurons of nucleus +++/++++ +++ intermediolateralis Neurons of nucleus + + + / + + + + +++/++++ thoracicus Neuropil/axons + + Astrocytes + +/+ + Oligodendrocytes - -

Ependymal cells +++ ++ Schwann cells ++/+++ +++

+ = A few, + + = about 50%, + + + = most of, + + + + = all cells stained.

M. Schorr et al. /Journal of the Neurological Sciences 140 (19961 117-122 119

As a negative control a nonspecific mouse IgGl was used (1: 100; Camon, Wiesbaden, Germany) and consis- tently negative results were shown.

3. Results

Using the immunocytochemical method with a mono- clonal mouse CNTF-antibody we first investigated the distribution of CNTF in paraffin sections from different parts of the normal human brain and spinal cord (Table 1) and compared the results with spinal cord sections of patients suffering from ALS (Table 2). We found positive

cytoplasmic immunoreactivity in most motor neurons (Fig. la), neurons of the stratum ganglionaris, the stratum pyra- midalis cell layers and in a lower part of astrocytes of the motor cortex.

The granular neuron layers, the neuropil and oligoden- drocytes were negative. In the corpus striatum we could only find a positive staining in a part of the astrocytes and in ependymal cells. In midbrain sections at the altitude around the colliculus superior neurons of the nucleus ruber (pars parvicellularis and magnacellularis), nucleus occulo- motorius (Fig. lb), ependymal cells, a lower part of the astrocytes and few axons showed a positive immuno- reactivity.

Fig. I Positive CNTF immunoreaction of (a) neurons of the human motor cortex, (b) neurons of the Nucleus oculomotorius and (d) Schwann cells of the nervus suralis. (c) Negative CNTF immunoreaction in neurons of the human motor CortexParaffin sections; monoclonal CNTF antibody, clone 4-68; ABC method, original magnification X 25.

120 M. Schorr et al. /Journal of‘thr Neurological Sciences 140 (1996/ I I7- 122

Fig, 2. Positive CNTF immunoreaction in the ventral horn motoneurons of (a) the human spinal cord in comparison to (b) the motonettrons of the same region in patients suffering from ALS. (a) insert: Negative CNTF immunoreaction in motor neurons of the spinal cord.Paraffin sections; monoclonal CNTP antibody, clone 4-68; ABC method; original magnification X 25.

In the spinal cord most motoneurons (Fig. 2a), neurons of the nucleus intermediolateralis, the nucleus thoracicus and ependymal cells were CNTF-positive. A small number of the astrocytes and a few axons were also positive. CNTF positivity could also be found in Schwann cells of the radices (Table 2).

In Schwann cells of the nervi suralis and ischiadicus positive immunoreactivity could be found too (Fig. Id).

In all sections of the CNS and peripheral nerves smooth muscle and endothelial cells of small arterial vessels a positive immunoreaction was observed.

Fig. 3. Western blot analysis demonstrating a 23 kDa immunoreactive band correlating to CNTF in human ischiadic nerve (a), motor cortex (h) and spinal cord (c). Dots at the right site denote molecular mass standards lfrom the top to the bottom: 39.2 kDa, aldolase; 26.6 kDa, triose phosphate isomerase; 21.5 kDa. trypsin inhibitor; 14.4 kDa, lysozyme. Triangle at the left site shows a 23.kDa band which corresponds to the molecular mass of CNTF.

In paraffin sections of the spinal cords of seven patients suffering from ALS a strong positive immunoreaction in motor neurons as well as some astrocytes was observed (Fig. 2b; Table 2). Th e positive CNTF-immunoreaction has been seen in both microscopically healthy large motor neurons and degenerated neuons of smaller size.

We found CNTF immunoreactivity in most of the motor neurons of the ventral horn in five cases. In two cases, the number of positively stained motor neurons was less.

A non-specific monoclonal IgGl antibody and omission of the first specific antibody, as controls, gave consistently negative results (Fig. lc, Fig. 2a, insert).

To support the reliability of the immunomorphological results we used Western blot analysis. For this we prepared the protein fractions from the motor cortex, ventral horns of the spinal cord and sciatic nerves. In all preparations we found a 23 kDa reactive band (Fig. 3).

4. Discussion

This is the first report demonstrating CNTF immuno- reactivity in the human central nervous system. CNTF immunoreaction in the human brain could be found in various cell types. Reaction was strongly and consistently positive in neurons such as motoneurons of the central nervous system. Positive reactions were also observed in a subpopulation of astrocytes and in ependymal cells. In radices and peripheral nerves, Schwann cells and some axons a positive immunoreaction was exhibited.

In a recent immunomorphological study of adult rat and mouse brain, spinal cord, and sciatic nerve CNTF im-

M. Schorr et ~~I./Journal of the Neurologiccrl Sciences 140 (19961 I1 7-122 I21

munoreactions were reported to be found in neurons, glia, and Schwann cells (Henderson et al., 1994). Using an affinity purified anti-CNTF antibody, the authors found cytoplasmic and - in contrast to our data - nuclear immunostaining within neurons.

Our immunomorphological results have been confirmed by Western blot analyses. The 23 kDa immunoreactive band is obviously due to the presence of CNTF.

CNTF has a potent survival effect in tissue culture of various neuron populations, in particular, neurons isolated from chick ciliary, dorsal root sensory and sympathetic neurons, as well as chick embryonic motoneurons (Barbin et al., 1984; Arkawara et al., 1990). The neurotrophic effects of CNTF have not only been observed in cultured motoneurons but also during the embryogenesis of verte- brate motoneurons (Oppenheim et al., 1991). In addition, CNTF has been shown to slow the progression of neuro- muscular dysfunction in several mutant mice strains in- cluding the progressive motor neuron desease mouse (Friedmann et al.. 1992; Sendtner et al., 1992) and the wobbler mouse (Ikeda and Mitsumoto, 1993; Mitsumoto et al., 1994). Recently it has been reported that the disruption of the CNTF gene expression by homologous recombina- tion results in progressive atrophy and loss of motor neurons in adult mice which is functionally reflected by a small but significant reduction in muscle strength (Masu et al., 1993). On the basis of these experimental results, it was interesting to study the expression of CNTF in a human neurodegenerative disease affecting the motor ner- vous system such as amyotrophic lateral sclerosis (ALS).

ALS is an age-dependent neurodegenerative disorder. It typically afflicts humans in middle adult life leading to paralysis and death within 3 to 5 years (Williams and Windebank, 199 1). Pathologically, ALS is distinguished by atrophy and death of the affected neurons. with dissolu- tion of both cytoplasm and nuclei. The motor neuron death process appears to be cell autonomous. According to our immuncytochemical results CNTF expression, which has been found in most of the motoneurons of the motor cortex and the ventral horn of the spinal cord, is maintained in patients suffering from ALS. In the ventral horns of these patients many motoneurons were degenerated. In the re- maining motoneurons, however, CNTF immunoreaction in neuronal cell bodies and in astrocytes was as strong as in normal cells. Duberley et al. (1995) recently demonstrated a marked increase in the hybridisation signal for the CNTF receptor mRNA in the spinal cord of ALS patients in contrast to healthy control patients. In combination with our results these data indicate, that the CNTF receptor, but not CNTF may be associated with the degenerative effect of ALS.

In ELISA studies, however, Anand et al. (1995) re- ported a marked decrease of CNTF in the ventral horn of post mortem spinal cords in ALS, with no change in the cerebral motor cortex. ELISA data cannot be directly compared to our immunocytochemical results. Using im-

munocytochemistry, CNTF expression is observed in sin- gle cells independent of the fact, that as the result of the ALS disease process many neurons have degenerated and died. In addition, these authors observed much lower CNTF levels in adult human motor and occipital cortex with no significant change to ALS and conclude that this finding argues against a primary and secondary role for endogenous CNTF in upper motor neuron degeneration in ALS.

In conclusion CNTF seems not to be involved in a human neurodegenerative process affecting the motoneu- ron system such as ALS. However, we cannot exclude the possibility that the function of CNTF is lost due to an insufficient or reduced CNTF receptor complex which could be the result of a defective or lost receptor.

References

Adler. R., Landa, K.B., Manthrope. M. and Varon, S. (1979) Cholinergic neuronotrophic factors: II. Selective intraocular distribution of soluble trophic activity for ciliary ganglionic neurons. Science, 204: 1434- 1436.

Anand, P.. Parrett, A., Martin, J., Zeman. S., Foley. P.. Swash, M., Leigh, P.N.. Cedarbaum. J.M.. Lindsay. R.M., Williams-Chestnut. R.E. and Sinicropi, D.V. (1995) Regina1 changes of ciliary neurotrophic factor and nerve growth factor levels in post mortem spinal cord and cerebral cortex from patients with motor disease. Nature Med.. 2: 168-173.

Arkawara. Y.. Sendtner, M. and Thoenen, H. (1990) Survival effect of ciliary neurotrophic factor (CNTF) on chick embryonic motoneurons in culture: Comparison with other neurotrophic factors and cytokines. J. Neuroxi.. 10: 3507-3515.

Bankflavi. A., Riehemann. K.. Gfner, D.. Checci. R., Morgan, J.M.. Piftko, J.. Biicker, W.. Jasani. B. and Schmid, K.W. (1994) Der einfachere Weg zur Antigendemaskierung. Pathologe. 15: 345-349.

Barbin. G.. Manthrope, M. and Varon, S. (1984) Purification of the chick eye ciliary neuronotrophic factor, J. Neurochem.. 43: 1468-1478.

Curtis. K.. Adryan, K.M.. Zhu. Y.. Harknesa, P.J., Lindsay. R.M. and DiStefano. P.S. (1993) Retrograde axonal transport of ciliary neu- rotrophic factor is increased by peripheral nerve injury. Nature. 365: 253-255.

Davis. S., Aldrich, T.H., Valenzuela. D.M., Won&. V.. Furth. M.E.. Squint,. S.P. and Yancopoulos, G.D. (1991) The receptor for ciliary neurotrophic factor. Science, 253: 59-63.

Davis, S., Aldrich, T.H., Stahl. N., Pan. L.. Taga. T.. Kishimoto, T.. Ip. N.Y. and Yancopoulos. G.D. (1993) LIFRP and gpl30 as het- erodimerizing signal transducers of the tripartite CNTF receptor. Science, 260: 1805- 1808.

Duberley. R.M., Johnson. I.P., Anand. P.. Swash. M., Martin. J.. Leigh, P.N. and Zeman. S. (1995) Ciliary Neurotrophic factor receptor expression in spinal cord an motor cortex in amyotrophic lateral sclerosis. J. Neural. Sci.. 129 (SuppI.): 109-l 13.

Emsberger. U.. Sendtner. M. and Rohrer, H. (1989) Proliferation and differentiation of embryonic chick sympathetic neurons: effects of ciliary neurotrophic factor. Neuron, 2: l275- 1284.

Friedmann. B.. Scherer. S.S., Rudge, J.S., Helgren. M., Morrisey, D., McClain. J.. Wang. D., Wiegand, S.J.. Fiirth. M.E.. Lindsay. R.M. and Ip. N.Y. (1992) Regulation of ciliary neurotrophic factor expres- sion in myelin-related Schwann cells in viva. Neuron, 9: 295-305.

Hagg. T. and Varon, S. (1993) Ciliary neurotrophic factor prevents degeneration of adult rat substantia nigra dopaminergic neurons in viva. Proc. Nat]. Acad. Sci. USA, 90: 6315-6319.

122 M. Schorr et al. /Journal of the Neurological Sciences 140 (19961 I 17-122

Henderson, J.T., Seniuk, N.A. and Roder, J.C. (1994) Localization of Masu, Y., Wolf, E., Holtmann, B., Sendtner, M., Brem, G. and Thoenen, CNTF immunoreactivity to neurons and astroglia in the CNS. Mol. H. (1993) Distribution of the CNTF gene results in motor neuron Brain Res., 22: 151-165. degeneration. Nature, 365: 27-32.

Hsu, SM., Raine, L. and Fanger, H. (1981) Use of a avidin-biotin complex (ABC) in immunoperoxidase techniques. J. Histochem. Cy- tochem., 29: 577-580.

Hughes, S.M., Lillien, L.E., Raff, M.C., Rohrer, H. and Sendtner, M. (19881 Ciliary neurotrophic factor induces type -2 astrocytes differen- tiation in culture. Nature, 335: 70-73.

Ikeda, K. and Mitsumoto, H. (19931 Morphological changes correlate with functional improvement following ciliary neurotrophic factor (CNTF) treatment of motor neuron disease in the wobbler mouse. Neurology, 43 (Suppl.1: 32 I.

Mitsumoto, H., Ikeda, K., Klinkosz, B., Cedarbaum, J.M., Wong, V. and Lindsay, R.M. (19941 Arrest of motor neuron disease in wobbler mice cotreated with CNTF and BDNF. Science, 265: 1107-l 110.

Oppenheim, R.W., Prevette, D., Qin-Wei, Y., Collins, F. and MacDonald, J. (I 99 1) Control of embryonic motoneuron survival in viva by ciliary neurotrophic factor. Science, 251: 1616-1618.

Sendtner, M., Kreutzberg, G.W. and Thoenen, H. (1990) Ciliary neu- rotrophic factor prevents the degeneration of motor neurons after axotomy. Nature, 345: 440-441,

Ip, N.Y. and Yancopoulos, G.D. (1992) Ciliary neurotrophic factor and its receptor complex. Prog. Growth Factor Res., 4: 139- 154.

Ip, N.Y., McClain, J., Barrezueta, N.X., Aldrich, T.H., Pan, L., Li, Y., Wiegand, S.J., Friedman, B., Davis, S. and Yancopoulos, G.D. (1993) The component of the CNTF receptor is required for signaling and defines potential CNTF targets in the adult and during development. Neuron, 10: 89-102.

Sendtner, M., Schmalbruch, H., Stockli, K.A., Carroll, P., Kreutzberg, G.W. and Thoenen, H. (1992) Ciliary neurotrophic factor prevents degeneration of motor neurons in mouse mutant progressive motor neuronopathy. Nature, 358: 502-504.

Stockli, K.A., Lottspeich, F., Sendtner, M., Masiakowski, P., Carroll, P., G&z, R., Lindholm, D. and Thoenen, H. (1989) Molecular cloning. expression and regional distribution of rat ciliary neurotrophic factor. Nature, 342: 920-923.

Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein mesurement with the folin phenol reagent. J. Biol. Chem.. 193: 265-275.

Masiakowski, P., Haoxing, L., Radziejewski, C., Lottspeich, F., Oberthuer, W., Wong, V., Lindsay, R.M., Furth, M.E. and Panay- otatos. N. (1991) Recombinant human and rat ciliary neurotrophic factors. J. Neurochem., 57: 1003-1012.

Stdckli, K.A., Lillien, L.E., Naher-Not?, M., Breitfeld. G., Hughes, R.A., Raff, M.C., Thoenen, H. and Sendtner, M. (1991) Regional distribu- tion. developmental changes, and cellular localization of CNTF- mRNA and protein in the rat brain. J. Cell Biol., 115: 447-459.

Williams, D.B. and Windebank, A.J. (1991) Motor neuron disease (amyotrophic lateral sclerosis). Mayo Clin. Proc., 66: 54-82.