Original article
Survival motor neuron – motor neuron insurance for a whole lifespan?

Introduction

Among many neurological diseases spinal muscular atrophy (SMA) is one of the most dramatic illnesses. It is characterized by immaturity of spinal motor neurons and muscle cells. The immature cells do not attain maturity and die, which is manifested clinically as weak­ness and atrophy of skeletal muscles. There are several forms of SMA. The most se­vere form is called Werdnig-Hoffman disease (group 1) and its symptoms are observed directly after delive­ry (floppy children). It is characterized by disturban­ces in milk sucking and weak motor activity of newborns. In group 2, the course of the disease is more benign. In group 3 with clinical onset in young adults, SMA course is the least devastating.

At the end of the twentieth century it was disco­vered that forms 1, 2, and 3 of SMA are caused by mutations in exon 7 of the Survival Motor Neuron (SMN) gene mapped on chromosome 5q11.2-q13.3 [9]. In hu­mans, the SMN gene possesses two copies – telomeric (SMN1) and centromeric (SMN2). SMA de­velops as a result of mutations only in the SMN1 gene. Lack of SMN-delta7 mRNA in SMN2 causes that mutations in exon 2 are not pathogenic. In other mammals only one copy of the SMN gene exists. An elegant study performed by Rochette et al. [13] did not reveal presence of SMN2 on sub-human mon­keys. The authors cited above suggested that SMN duplication appeared over 3 million years ago, before division into anthropoids and Homo sapiens. The SMN- gemin complex is present not only in mammals [3]. Presence of SMN protein and gemins 2, 3 and 5 has been found in Drosophila [4].

Physiologically, SMN binds to 7 multifunctional proteins called gemins (gemins 2-8), forming an oligomeric complex [12,15]. Functions of gemins are different. Gemin 2 stabilizes the SMN-gemin complex [12] while gemins 4 and 5 transmit the oligomeric complex from and to the cell nucleus [1,6]. Gemins 2 and 3 are colocalized with SMN protein and are pre­sent in all cell compartments (cell body, dendrites, axons and growth cones [18]), while gemins 6 and 7 are localized only in the cytoplasm.

The SMN-gemin complex together with small nu­clear ribonucleoproteins takes part in RNA splicing. It is known that mutations in the SMN gene lead to alterations in SMN oligomerization and formation of SMN-gemin complex [7], resulting in disturbances in proliferation, migration and development of nerve cells [17]. In SMN-depleted mice, increased proliferation and morphological changes in nerve cells and developmental disturbances in stem cells were ob­served [14]. The transgenic animals also revealed regionally selected abnormalities in CNS morphology manifested as decreased proliferation and cell density in the hippocampus [17]. But the SMN gene acts not only in early ontogenesis. In experimental SMA it was shown that postnatal activity of SMN protein was neuroprotective [8,11,18,20]. These data raise some new questions:

1. How long during a lifespan is the SMN gene active?

2. Are only anterior horn motor neurons protected by SMN?

3. Does the SMN gene protect motor neurons only from spinal muscular atrophy or also from other disorders proceeding with degeneration of spinal motor neurons such as ALS?

Material and methods

The material consisted of spinal cords from 27 Wi­star rats at the age of 1-350 days (9 groups compos­ed of 3 rats at the age of 1, 10, 20, 30, 60, 150, 200, 250 and 350 days).

In rat spinal cords fixed in formalin and embedded in paraffin, expression of SMN and gemin 2, 3 and 4 was assessed in light and immunofluorescence (only SMN and gemin 3) microscopy. Immunohistochemistry for light microscopy was performed ac­cording to the avidin-biotin-peroxidase method. Tissue slides were dehydrated in alcohol, pre-treated with heat retrieval using a microwave for 3 × 10 min in 10 mM citrate buffer (pH 6.0), and immunostained with primary antibodies against SMN (Santa Cruz Biotechnology, 1 : 250), gemin 2 (Santa Cruz Biotechnology, 1 : 500), gemin 3 (Santa Cruz Biotechnology, 1 : 1000), and gemin 4 (Santa Cruz Biotechnology, 1 : 1000) using Goat F(ab)2 Fragment anti-mouse IgG-biotin (Beckman Counter, 1 : 1500) and Goat F(ab)2 Fragment anti-rabbit IgG-biotin (Beckman Coun­ter, 1 : 1500) respectively and diaminobenzidine as the chromogen.

For fluorescent microscopy double immunolabell­ing of SMN and gemin 3 was performed. The first part of the procedure was the same as the procedure of single immunohistochemistry but then simultaneous incubation with two primary antibodies (Abs), monoclonal mouse anti-gemin 3 Ab and polyclonal rabbit anti-SMN Ab, was performed. Next, the sections were washed and incubated for 1 h at 37°C with secondary antibodies: goat anti-mouse Alexa Fluor 594 (Invitrogen – Molecular Probes, 1 : 100) and goat anti-rabbit Alexa Fluor 488 (Invitrogen – Molecular Probes, 1 : 100). Then, they were washed in PBS, dried and mounted with Vectashield Mounting Medium (Vector Laboratories Inc) for fluorescen­ce microscopy. Sections were analysed and captured with an Optiphot-2 Nikon microscope (Japan) equipp­ed with the appropriate filters and a DS-L1 Nikon camera (Japan).

Specificity of the immune reactions was verified by performing a “negative control” staining procedu­re with primary antibodies omitted in the incubation mixture.

Apart from immunohistochemistry, computer-based image processing methods were applied for graphical visualization of the SMN staining intensity within ventral horn areas of the investigated sections. The image processing software Scion Image (Scion Image for Windows Scion Corporation) was used for the analysis. Sections were converted to greyscale, calibrated on an 8-bit scale and surface plot profiles were performed. The height of plots pointed to the level of intensity of the immune reaction.

Results

Expression of SMN in rats at the age of 10 days was absent or very weak and only a part of cells revealed the positive immune reaction within neuronal cytoplasm (Fig. 1A). In 20-day old rats SMN-immunopositive neurons were more numerous and the immune reaction was more pronounced than in the younger animals (Fig. 1B). Nuclear localization of SMN was observed in rats starting from the 30th day of life (Fig. 1C). In rats at the age of 30-350 days pronounced SMN immunoreactivity was present in all the examined animals (Figs. 1D-F, Figs. 2A-C).

Graphic visualization of the SMN immunolabel within rat ventral horns (shown in Figs. 1D-F and Figs. 2A-C) is demonstrated in Figs. 5A-C, in which the height of plots indicates the level of intensity of the immune reaction. Data obtained in the graphic method confirmed our results from the immunohistochemical studies and showed increase of intensity of the SMN-immunoreactive signal in sections de­rived from older rats at the age of 30-350 days in comparison to younger rats at the age of 1-20 days.

Assessment of gemins 2, 3 and 4 revealed immu­noreactivity pattern of their expression similar to SMN. Analogous to SMN immune reaction, nuclear localization of gemin 2 was also observed in rats starting from the 30th day of life (Fig. 1C).

In rats at the age of 10 days we noted a very inte­resting phenomenon. Contrary to very weak immu­ne reactions to SMN and gemin 2 in that period, expression of gemins 3 and 4 was very prominent and found both in neuron cytoplasm and nucleus (Fig. 1A).

The immune label for SMN and gemins was observed in neurons both in anterior and posterior spinal cord horns (Figs. 3A-C) as well as in vegetative neurons within lateral horns in the thoracic spinal cord (Fig. 3D) and in neurons in sensory spinal ganglia (Fig. 3E).

In the immunofluorescent method, colocalization of gemin 3 with SMN protein in the same neurons was demonstrated (Figs. 4A-B). The cell membrane was especially very intensively decorated.

Discussion

In the literature there are scarce data concerning activity and expression of the SMN gene and they refer mainly to experimental animals. In animals SMN was found in the CNS, liver, kidneys, lungs and muscles [7]. The protein expression was maximal in the embryonic and early postnatal period and de­creas­ed after ontogenesis [2,7]. In humans, expression of SMN in fetuses and adults was also investigated [5,16]. In spite of these studies, the dynamics of SMN expression during the lifespan remain un­known.

In our experimental rat material involving a long period of the animal’s life (from birth to old age), very weak expression of SMN protein was already visible in a part of neurons in rats at the age of 20 days and the expression increased with the animal’s age. It was confirmed both in light and immu­nofluorescent studies. Surface plot profiles of the SMN immunolabel in rat ventral horn areas also showed enhancement of the immunostaining intensity in the postnatal period. Contrary to humans, in neonatal rats and mice both anterior and posterior horn cells are immature. Our results suggest that the immune expression of SMN is parallel with cell maturity. The clinical improvement after postnatal induction of SMN expression observed in the mice model of SMA [8] might be connected with this phenomenon and confirms our supposition. Also nuclear localization of SMN found in our material in rats at the age of 30 days and older may indicate cell “maturity”.

Our observations of SMN expression in rats aged 30-350 days revealed its various immunoreactivity in spinal cord neurons and glial cells. This suggests that SMN expression may be dependent on the level of the protein synthesis and associated with the fun­ctional stage of cells. Various SMN expression may also be dependent on neuron size and individual differences among the cells, although it is not clear whether such relationships really exist. Graphical visualization of the SMN immunostaining data in cells in rat ventral horns showed the intensification of individual pixel labelling. These results demonstrate that during maturation not only the number of cells immunoreactive to SMN (motoneurons, inter­neurons) increases but also intensity of the immune signal. The data point to the presence of SMN in many cells during the lifespan and indicate that in rats the SMN gene is probably active during the whole lifespan of the animals.

Estimation of gemin expression in rats at the age of 10 and 20 days revealed pronounced immunoreactivity of gemin 3 and 4 while expression of gemin 2 was poor. Since SMN expression at that period was also very weak or absent, it may suggest that in the SMN complex gemins 3 and 4 appear earlier than SMN protein and gemin 2. Moreover, since gemin 2 stabilizes the SMN complex, perhaps full activity of the complex occurs later, after complex stabilization. Poor reactivity to gemin 2 observed in 350-day old rats may also suggest that its expression decreases with aging.

Nuclear localization of SMN and the investigated gemins was observed in rats starting from the 30th day of life. This finding may indicate that transport of the SMN complex to the nucleus by gemin 4 [10] and from the nucleus by gemin 5 [16] (not investigated in our material) probably is already present.

The previously observed colocalization of gemin 3 with SMN protein in neurons [18] was confirmed in our immunofluorescent study. It was interesting that in our investigation the cell membrane was especially very intensively decorated. It is not clear what was responsible for it – maybe numerous immunoreactive receptors?

The name Survival Motor Neuron suggests connection of the gene/protein only with motor neurons. But in our material apart from motoneurons alpha and gamma, also interneurons in spinal posterior horns expressed SMN and gemins. In addition, neurons in Clarke’s columns and even sympathetic nerve cells in thoracic lateral horns and intervertebral ganglia were evidently immunopositive to SMN and the investigated gemins. This finding implies that the SMN gene may influence not only moto­neurons but also sensory and vegetative neurons. In other words, all nerve cells in rat spinal cord may be under the influence of the SMN gene.

Since expression of the SMN gene essential for neuron survival is preserved after ontogenesis and its mutations result in SMA development, there is a hypothesis that the gene may play a protective role in other neurodegenerative disorders involving spinal cord neurons such as amyotrophic lateral sclerosis (ALS). The first reports which seem to confirm that supposition have been published. It was demonstrated that in a rat ALS model with SOD-1 mutation, SMN protein acts neuroprotectively [19]. Its neuroprotective influence was also observed in vitro in neuroblastoma cells with SOD-1 mutation [20]. But this interesting hypothesis requires further investigations.

Conclusions

1. In rat spinal cord the immune expression of SMN and gemins 2, 3, and 4 was present from the early postnatal period to old age.

2. The expression of the investigated proteins was observed both in motoneurons alpha and gamma, as well as in interneurons and glia.

3. In rat spinal cord the immune expression of SMN and gemins 2, 3, 4 was also present in sensory and autonomic neurons.

4. Since the SMN and gemin expression was found in rat spinal cord neurons it means that not only mice but also the rat experimental model may be useful in SMA studies.

Acknowledgments

This study was supported by the Ministry of Science and Higher Education grant NN 401 014640.

References

 1. Baccon J, Pellizzoni L, Rappsilber J, Mann M, Dreyfuss G. Identification and characterization of Gemin7, a novel component of the survival of motor neuron complex. J Biol Chem 2002; 277: 31957-31962.  

2. Battaglia G, Princivalle A, Fortli F, Lizier C, Zeviani M. Expression of the SMN gene, the spinal muscular atrophy determining gene, in the mammalian central nervous system. Hum Mol Genet 1997; 6: 1961-1971.  

3. Cauchi RJ. SMN and Gemins: “we are family”... or are we?: insights into the partnership between Gemins and the spinal muscular atrophy disease protein SMN. Bioessays 2010; 32: 1077-1089.  

4. Cauchi RJ, Sanchez-Pulido L, Liu JL. Drosophila SMN complex proteins Gemin2, Gemin3, and Gemin5 are components of U bodies. Exp Cell Res 2010; 316: 2354-2364.  

5. Giavazzi A, Setola V, Simonati A, Battaglia G. Neuronal-specific roles of the survival motor neuron protein: evidence from survival motor neuron expression patterns in the developing hu­man central nervous system. J Neuropathol Exp Neurol 2006; 65: 267-377.  

6. Hao le T, Fuller HR, Lam le T, Le TT, Burghes AH, Morris GE. Absence of gemin5 from SMN complexes in nuclear Cajal bodies. BMC Cell Biol 2007; 18: 8-28.  

7. La Bella V, Cisterni C, Salaün D, Pettman B. Survival motor neuron (SMN) protein in rat is expressed as different molecular forms and is developmentally regulated. Eur J Neurosci 1998; 10: 2913-2923.  

8. Le TT, McGovern VL, Alwine IE, Wang X, Massoni-Laporte A, Rich MM, Burghes AH. Temporal requirement for high SMN expression in SMA mice. Hum Mol Genet 2011; 20: 3578-3591.  

9. Lefebvre S, Bürglen L, Reboullet S, Clermont O, Burlet P, Viollet L, Benichou B, Cruaud C, Millasseau P, Zeviani M, et al. Identification and characterisation of a spinal muscular atrophy-determining gene. Cell 1995; 80: 155-165.

10. Lorson MA, Dickson AM, Shaw DJ, Todd AG, Young EC, Morse R, Wolstencroft C, Lorson CL, Young PJ. Identification and characterisation of a nuclear localisation signal in the SMN associated protein, Gemin4. Biochem Biophys Res Commun 2008; 375: 33-37.

11. Lutz CM, Kariya S, Patruni S, Osborne MA, Liu D, Henderson CE, Li DK, Pellizzoni L, Rojas J, Valenzuela DM, Murphy AJ, Winberg ML, Monani UR. Postsymptomatic restoration of SMN rescues the disease phenotype in a mouse model of severe spinal muscular atrophy. J Clin Invest 2011; 121: 3029-3041.

12. Ogawa C, Usui K, Aoki M, Ito F, Itoh M, Kai C, Kanamori-Katayama M, Hayashizaki Y, Suzuki H. Gemin 2 plays an important role in stabilizing the survival of motor neuron complex. J Biol Chem 2007; 282: 11122-11134.

13. Rochette CF, Gilbert N, Simard LR. SMN gene duplication and the emergence of the SMN2 gene occurred in distinct homi­nids: SMN2 is unique to Homo sapiens. Hum Genet 2001; 108: 255-266.

14. Shafey D, MacKenzie AE, Kothary R. Neurodevelopmental ab­nor­malities in neurosphere-derived neural stem cells from SMN-depleted mice. J Neurosci Res 2008; 86: 2839-2847.

15. Shpargel KB, Matera AG. Gemin proteins are required for efficient assembley of Sm-class ribonucleoproteins. Proc Natl Acad Sci USA 2005; 102: 17372-17377.

16. Soler-Botija C, Cuscó I, Caselles L, López E, Baiget M, Tizzano EF. Implication of fetal SMN2 expression in type I SMA pathogenesis: protection or pathological gain of function? J Neuropathol Exp Neurol 2005; 64: 215-223.

17. Wishart TM, Huang JP, Murray LM, Lamont DJ, Mutsaers CA, Ross J, Geldsetzer P, Ansorge O, Talbot K, Parson SH, Gillingwa­ter TH. SMN deficiency disrupts brain development in a mouse model of severe spinal muscular atrophy. Hum Mol Genet 2010; 19: 4216-4228.

18. Zhang H, Xing L, Rossoll W, Wichterle H, Singer RH, Bassell GJ. Multiprotein complexes of the survival of motor neuron protein SMN with Gemins traffic to neuronal processes and growth cones of motor neurons. J Neurosci 2006; 26: 8622-8632.

19. Zhang Z, Lotti F, Dittmar K, Younis I, Wan L, Kasim M, Dreyfuss G. SMN deficiency causes tissue-specific perturbations in the repertoire of snRNAs and widespread defects in splicing. Cell 2008; 133: 585-600.

20. Zou T, Ilangovan R, Yu F, Xu Z, Zhou J. SMN protects cells against mutant SOD1 toxicity by increasing chaperone activity. Biochem Biophys Res Commun 2007; 364: 850-855.