Neurite promoting activity of insulin, insulin-like growth factor I and nerve growth factor on spinal motoneurons is astrocyte dependent

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Del,elopmental Brain Research, 74 (1993) 83-88 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-3806/93/$06.00

B R E S D 51652

Neurite promoting activity of insulin, insulin-like growth factor I and nerve growth factor on spinal motoneurons is astrocyte dependent L e e C. A n g a, B a n a n i B h a u m i c k b a n d B e r n h a r d H.J. J u u r l i n k c Departments of " Pathology (Neuropathology),

t, Medicine and ' Anatomy, Unirersity of Saskatchewan and Royal Unit'ersity Hospital, Saskatoon, Sask. (Canada) (Accepted 26 January 1993)

Key words: Spinal motoneuron; N e u r o n - g l i a interaction; Insulin; Insulin-like growth factor I; Nerve growth factor: Ncurite outgrowth

Mouse motoneurons were isolated from dissociated E l 5 mouse spinal cord and grown on polyornithine-coated round coverslips in a growth medium ( D M E M / F I 2 ) supplemented with progesterone, trans-ferrin, selenium, horse serum and muscle extract. Astrocytes from newborn mouse neopallium were grown on rectangular coverslips. T h e motoneuron neurite growth was determined at day 8 of culture by counting, using the light microscope, the intersections produced by neurites radiating from the perikaryon placed centrally in a graticule eyepiece of concentric circles. The mean intersections for cultures without addition of astrocytes, insulin, insulin-like growth factor I (IGF-I) or nerve growth factor (NGF) was 12.6±0.8. W h e n astrocytes on a separate coverslip were introduced from day 1, there was a small increase in neurite growth (16.3 _+0.9). The neurite growth was further increased significantly with the addition of insulin (27.3 ± 1.4), 1GF-I (31.5 + 1.4) or N G F (21.8 + 1.1) to cultures with astrocytes. Insulin, IGF-I or N G F in the absence of astrocytes did not greatly increase the neuritc growth. We conclude that insulin, IGF-I and N G F promote neurite growth through some interactions with astrocytes.

INTRODUCTION

Previous studies have shown that growth factors such as insulin, insulin-like growth factor I (IGF-I) and nerve growth factor (NGF) promote neurite develo p m e n t in various populations of central neurons2'3'8"15'~8'29; however, since most of the culture systems used in these studies contained neurons as well as astrocytes, it was not possible to determine whether these factors acted directly on neurons or whether the neurite promoting effects were mediated by astrocytes. Our culture system 4, consisting of neurons and astrocytes grown on separate coverslips enables one to determine whether the neurite promoting effects of growth factors are mediated by astrocytes. In the present study we demonstrate that insulin, IGF-I as well as N G F promote neurite development in mouse spinal motoneurons but only when astrocytes are introduced into the same culture dish, demonstrating that in some manner astrocytes mediate the neurite-promoting ac-

tivity of these growth factors. Some of this work has previously been presented in an abstract form 5. MATERIALS

AND METHODS

Astroeyte culture preparation The neopallia of newborn mice was aseptically isolated, minced and forced gently through a 75 p~m2 nylon mesh and planted onto sterile 11 × 22 m m glass coverslips contained in a 100-ram Petri dish. The growth medium used was a modified Eagle's m i n i m u m essential medium containing 5% horse serum ~'~'2t. The medium was changed every 3 days. Such cultures have been shown to consist of more than 95% astrocytes as determined by glial fibril[ary acidic protein immunocytochemistry 20.

Motoneuron isolation Postmitotic moloneurons were isolated from K15 mouse embryos essentially as outlined previously ~2. Spinal cords were isolated, freed of meninges and dorsal root ganglia and then incubated at 37°C fl~r 30 rain in 0.025% trypsin in a CaX~-Mg2+-free balanced salt solution. The solution was then replaced with D M E M / F I 2 ( 1 / I ) containing 0.02% deoxyribonuclease (DNase) and spinal cords were mechanically dissociated using a Stomacher Lab Blender 8(l TM. The remaining steps were done at 4°C. Larger fragments were allowed to settle and the supernatant consisting of dissociated spinal cells was cen-

Correspondence." L.C. Ang, D e p a r t m e n t of Pathology, Sunnybrook Health Science Centre, 2075 Bayview Avenue, North York, Ont., Canada M4N 3M5. Fax: (1)(416)480-4271.

84 Culture Paradigm

Group I

Concentric Circles

Groupll

(MN= motoneuron) Fig. 1. Group 1 cultures consisted of motoneurons grown on 2 separate circular coverstips placed in a Petri dish containing growth medium. Group II cultures consisted of Petri dishes containing 2 circular coverslips of motoneurons and 1 rectangular coverslip of astrocytes. trifuged at 180 g for 10 min. The cell pellet was resuspended in 5 ml D M E M / F I 2 / 0 . 0 2 % DNase and centrifuged through 3.5%, bovine serum albumin (15 min at 100 g) to remove debris. This cell pellet was resuspended in 5 ml D M E M / F 1 2 / 0 . 0 2 % DNase, placed upon a 6.2% metrizamide solution and centrifuged at 500 g for 30 min. The motoneuron fraction situated at the medium-metrizamide interface was isolated, diluted in D M E M / F 1 2 / 0 . 0 2 % DNase, centrifuged at 180 g for 10 min and resuspended in chemically defined medium (CDM) of D M E M / F 1 2 containing 3 . 1 0 - S M sodium selenite, 2. 10-SM progesterone and 5 / z g / m l transferrin.

Motoneuron culture The neurons were planted onto polyornithine (PORN) coated round 12 mm coverslips at a low density of 40 cells/mm 2. Two coverslips of motoneurons were placed into each 35-ram Petri dish (Falcon). The growth medium consisted of CDM containing 10% (v/v) heat-inactivated horse serum plus 50 /zg muscle extract prot e i n / m l prepared as outlined by Dohrmann et al) 2. The cultures were fed after 1 day (24 h) and maintained for another 7 days.

Experimental design Motoneurons were divided into two basic groups and grown for 8 days in growth medium outlined above. Group I cultures consisted of two motoneuron coverslips per Petri dish. Group II cultures contained in addition to the motoneuron coverslips, one coverslip of 2-week-old astrocytes (Fig. 1). Both group I and group II cultures were given one of the following supplementations: (i) no additional supplementation; (ii) 5 / z g / m l insulin; (iii) 10 n g / m l IGF-I; (iv) 100 n g / m l IGF-I; (v) 10 n g / m l NGF; (vi) 100 n g / m l NGF. The cultures were given the above supplements at the beginning of the culture period as well as after 24 h when the cultures were fed. Bovine insulin was purchased from Sigma Chemical (St. Louis, MO). The recombinant tGF-I was obtained from Amgen Biologicals (Thousand Oaks, CA) and 7S NGF from Collaborative Research (Bedford, MA).

Fig. 2. View of a graticule eyepiece of concentric circles with perikaryon of motoneuron placed in the centre. The intersections of neurites radiating from the perikaryon with lines of the circles were counted with light microscope at 250 times magnification. centrally in a graticule eyepiece of concentric circles 4l (Fig. 2). Counts were performed on viable motoneurons on one horizontal strip extending from one edge through the center to the opposite edge of a coverslip and the mean intersections per neuron was worked out. The mean number of neurons counted per reading was 54.0+6.2. A total of 48 coverslips were counted. The data were analyzed using a one way A N O V A with a post hoc Scheff~ analysis with a = 0.05 considered to be significant.

RESULTS The results of the quantification of neurite growth with the various supplementations are summarized in Fig. 3. [-]a~roc~es

40

~es

== O O

30

0

Z

Quantification of neuritic outgrowth Cultures were fixed in 4% paraformaldehyde containing 0.2% picric acid in phosphate-buffered saline 46 and stained with iron hematoxylin. These cultures consisted of 95% neurons and 5% astro-cytes. Approximately 90% of the neurons in these cultures are motoneurons as determined by calcitonin gene-related peptide. Our previous work has demonstrated that CGRP can be used as a marker of motoneurons in culture 4'22. In avian and mammalian spinal cord, CGRP is localized to the axons and somata of preganglionic autonomic and somatic motoneurons 6'7J6'24. The neuritic growth was determined at day 8 of culture by counting, using the light microscope (magnification, × 250), the intersections produced by neurites radiating from the perikaryon placed

0 CONTROL

INSULIN

IGF-1 10 ng/ml

IGF-1 100 r~/ml

NGF 10 ng/ml

NGF 100 r~/ml

Fig. 3. Quantification of neurite outgrowth at 8 days with~various supplementations. The neurite outgrowth was represented by the mean intersections per motoneuron for the different supplementations. Each bar represents the mean intersections (_+SEM) of an average of 54 neurons. Data were analyzed with a one way A N O V A with a post hoc Scheff6 analysis. Single asterisks indicate that these values are significantly greater than the value of the control motoneurons grown in the presence of astrocytes alone. The double asterisk indicates that this value is significantly greater than the value of the control motoneurons grown in the absence of astrocytes.

85 achieved by adding either insulin, IGF-I or NGF to motoneurons co-cultured with astrocytes. The greatest increase in neurite outgrowth compared to cultures with astrocytes alone was observed with addition of either 5 / x g / m l of insulin or 100 ng/ml of IGF-I, both increases are significant according to the post hoc Scheff6 analysis. Morphologically these spinal motoneurons not only showed an increased number of neurites but also these neurites were elongated and more complex with secondary branching (Fig. 4C,D). The addition of a lower dose of IGF-I at 10 ng/ml had no significant effect on the neurite growth (18.7 + 0.8) of motoneurons grown with astrocytes. The neurite

After 8 days of culture, the mean intersections per viable neuron for control cultures without the addition of either astrocytes, insulin, NGF or IGF-I was 12.6 + 0.8. There were many spinal motoneurons showing degenerative changes with retraction of neurites (Fig. 4A). When astrocytes on separate coverslips were introduced from day 1 into the Petri dish containing coverslips of motoneurons, there was a small increase in neurite branching as indicated by the intersections per neuron (16.3 _+0.9) in the 8 day culture. Most of the neurites radiating from the perikaryon appeared healthy and straight with little secondary branching (Fig. 4B). Further increase of neuritic outgrowth was

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Fig. 4. Motoneurons grown (A) without astrocytes showing degenerative changes in somata and neurites; (B) with astrocytes alone showing neurons having healthy and straight neurites with few secondary branches; (C) with astrocytes in growth medium containing 5/xg/ml of insulin showing a great increase number of neurites and secondary branching and D)with astrocytes in growth medium containing 100 ng/ml of IGF-1. There is an increase in the number of neurites that are more complex with secondary branching. Iron-hematoxylin stain.

86 ou'tgrowth obtained using NGF at either 10 n g / m l (21.8_+ 1.1)or 100 n g / m l (21.7_+ 1.2) in motoneurons co-cultured with astrocytes was also significantly greater than neuronal cultures with astrocytes alone (Fig. 3). However, addition of insulin, N G F or lower dose of IGF-I (10 n g / m l ) to cultures without astrocytes had minimal effect on neuritic branching. In fact without astrocytes, significant degenerative changes were observed in the neurites even in the presence of insulin, IGF or NGF. Interestingly, a small but apparently significant increase in neuritic outgrowth of motoneurons was achieved in cultures with IGF-I at 100 n g / m l without astrocytes (17.9 _+ 1.3) when compared to control with growth medium alone. DISCUSSION Various experiments have demonstrated that astrocytes exert a trophic effect on the survival of cultured motoneurons isolated from chick, rat and mouse spinal cords 13'4°. Astrocytes have been known to be the likely source of a number of neurotrophic and neurite elongation factors T M . Our previous experiment suggests that the significant effect of astrocyte on motoneuron survival in vitro is most likely mediated by diffusible factors and the neurotrophic effects of insulin and IGF-I on motoneuron survival were dependent on astrocytes 4. Using the same experimental design as previously described 4, we have found that astrocytes alone have a small effect in promoting neurite outgrowth and branching. However, astrocytes are essential for maintaining the survival of neuronal perikarya with fewer but healthy neurites which would otherwise have undergone degeneration. Insulin and IGF-I have been shown to enhance neuronal survival and neurite outgrowth in a variety of neuronal population in vitro including that of spinal motoneurons 2'3"s. In most neuronal cell lines and primary neuronal cultures, IGF-I could produce a similar degree of trophic response as insulin though in much smaller dose closer to physiological range, suggesting that it is a more potent neurotrophic factor than insulin 3'36. Insulin is also known to cross react with IGF-I receptors and it is possible that some of the neurotrophic effects of insulin could be mediated by IGF-I receptors ~. IGF-I administered locally around crushed lesion of rat sciatic nerve stimulates regeneration and this effect could be blocked by anti-lGF-I antibodies ~. Moreover exposure of adult rat or mouse gluteus muscle to low quantities of IGF-I in vivo has induced a tenfold increase in nerve sprouting compared to untreated controls 23. It is postulated that insulin and IGF-I stimulate the phosphorylation of the beta sub-

units of their respective receptors in neuronal cells of primary cultures triggering off molecular events essential to neurite growth and regeneration :'~6"32'~s. It is known that IGF-I and IGF-II could affect the astrocytes either through their mitogenic effect or actions on specific receptors identified in these cells 9"4725. Perhaps, the astrocytes, in turn, could then provide trophic support for various neuronal populations m,3°'~5. Our data indicate that the presence of astrocytes is vital for both insulin and IGF-I to exert a significant effect on neurite extension in purified moto-neuron cultures. Furthermore, the effect of IGF-I is dose-dependent as the neurite extension is more marked with 100 n g / m l than with 10 n g / m l of IGF-I. It should be noted that all experiments were carried out in the presence of 10% serum and 50 ~zg/ml muscle extract protein and this is likely why such a high concentration of IGF-1 is required to demonstrate a significant effect. While the trophic effect of N G F on cholinergic neurons of the basal forebrain has been quite well documented it is generally considered that the response of cholinergic motoneurons from chick and rat spinal cord to NGF is transient 42"43. McMamanam et al. :~ have also shown that NGF failed to rescue spinal motoneurons of chick embryos from death in vivo. However, the expression of /3-NGF receptor and its m R N A have been documented in chick and rat embryos implicating some functional role of NGF during development ~4'37. It has also been found that although low-affinity N G F receptors appear to be relatively evenly distributed in embryonic rat spinal cord cell, high-affinity NGF receptors are found primarily on motoneurons 2s. We have not been able to demonstrate the trophic effect of NGF on survival of spinal motoneuron even in the presence of astrocytes (unpublished data). But definitely N G F is shown here to have a significant effect on neurite extension and branching when introduced into cultures containing both motoneurons and astrocytes. Although astrocytes by themselves appear to have only a small effect on the neurite outgrowth and branching of motoneurons in vitro their presence is necessary for growth factors such as insulin, IGF-I and N G F to produce such an effect. Our study has highlighted two important aspects involving in vitro experiments used for studying growth factors in neuronal cells. First, the important influences of astrocytes in promoting responses from neurons to some trophic factors, without such influences there may be no responses from these cells. Second the complex interactions between astrocytes, neurons and growth factors should always be taken into consideration when interpreting the results of experiments in which explants,

87

mixed or non-purified primary neuronal cultures are used 23'45. Often it is assumed that the observed responses of certain neuronal populations to growth factors are the results of direct actions of such factors on the neurons themselves. Our data indicate that the effect of insulin, IGF-I and NGF on neurite outgrowth could be either mediated by astrocytes or alternately, insulin, IGF-I and N G F could interact synergistically with certain factors secreted by astrocytes. As there was no physical contact between astrocytes and spinal motoneurons in our experiment it is most certainly that diffusible factors are involved. Perhaps astrocytes could secrete some factors that induce IGF-I and NGF receptor expression in motoneurons, thus enabling these neurons to be responsive to IGF-I and NGF. Even more important may be that astrocytes are necessary for survival of motoneurons and without them neurons will degenerate and growth factors would not be able to produce any effect on neurite growth. Our findings suggest that astrocytes are a major participant in the interaction between growth factors and neuronal cells. Recognition of its role in such interaction could provide further insight into the pathobiology of neuronal degeneration and regeneration in the CNS. Acknowledgements'.

We would like to acknowledge the Saskatchewan Health Research Board for supporting this research. We also thank Connie Wong and Wendy Kao for the excellent technical assistance and Robert van den Beuken, Todd Reichert and Deanna Turetski for preparation of photographs and manuscript.

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