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Analysis of developing chick Gallus domesticus spinal cord proteins using two dimensional gel electrophoresis Ethell, Douglas Wayne 1990

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ANALYSIS OF DEVELOPING CHICK Gallus domesticus SPINAL CORD PROTEINS USING TWO DIMENSIONAL GEL ELECTROPHORESIS by DOUGLAS WAYNE ETHELL B.Sc, The University of British Columbia, 1987. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA JANUARY, 1991 ©Douglas Wayne Ethell, 1990. In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Colurnl Vancouver, Canada Department Date DE-6 (2788) ABSTRACT Several recent experiments on developing chick spinal cord have established a time window when the developing spinal cord changes from a permissive to a restrictive environment for regeneration. This time window occurs during embryonic days 13-14 (E13-E14) of chick development. Recent experiments in adult rat, have found two proteins that actively inhibit axonal regeneration. This study has sought possible inhibitory proteins, in chicks, correlating to this temporal change. Proteins continuously present after this change (E14-E20) but not before (Ell) were identified. Two-dimensional gel electrophoresis was used for separatation of the proteins. Seven protein spots of interest demonstrated this correlative late-expressing neural protein (LNP) profile. Although the functions of these proteins could not be ascertained in this study, further investigation is warranted. i i TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iii LIST OF TABLES iv LIST OF FIGURES iv ACKNOWLEDGEMENTS v INTRODUCTION 1 METHODS 8 RESULTS 10 DISCUSSION 19 CONCLUSIONS 25 REFERENCES 26 i i i LIST OF TABLES I Approximate molecular weights and isoelectric points for protein spots of interest identified in this study 18 LIST OF FIGURES 1. Photograph of an E14 two-dimensional protein gel 12 2. Photograph of an E l 1 two-dimensional protein gel 13 3. Photograph of an E16 two-dimensional protein gel 14 4. Photograph of an El8 two-dimensional protein gel 15 5. Photograph of an E20 two-dimensional protein gel 16 6. Photograph of an E14 two-dimensional protein gel with all spots of interest labelled 17 i v ACKNOWLEDGEMENTS I owe a huge debt of gratitude to my parents of their never ending moral and financial support. As well, I would like to thank Dr. John Steeves for allowing me the freedom to pursue my own ideas, even if it meant going down a few blind alleys. I am also grateful to Drs. Deirdre Webster and John Brown who let me pick their brains. Special appreciation goes out to all of the people in Zoology who have stimulated me to continue over the years. v I N T R O D U C T I O N Spinal cord injuries account for an increasingly large proportion of extended medical care in Canada each year (Statistics Canada, 1990). Currently, victims of spinal cord injuries have little hope for complete functional recovery, due to the innate lack of regenerative ability within the central nervous system (CNS). Treatment is conservative and symptomatic, being primarily dependent upon minimizing trauma to the spinal column (Sabra & Adams, 1977). The ultimate goal of research in this area is to be able to bring about functional recovery of spinal cord injured patients. A task not without complexity; essentially we want to cause something to happen in the spinal cord that does not normally occur. To this end, it is of the utmost importance that we completely understand the fundamental processes of uninjured spinal cord development and function. Focused investigations into the rudimentary aspects of the spinal cord, must lay the foundations for a comprehensive and methodical approach to this problem. This thesis has taken such an approach, in a specific search for proteins which may be involved in the restriction of regenerative ability within the spinal cord. * * * Vertebrate locomotion is initiated and controlled by complex neural networks within the brainstem and spinal cord (Sherrington, 1910; ten Cate, 1960; Gallistel, 1980; Steeves & Jordan, 1980; Shefchyk et al., 1984;Sholomenko & Steeves, 1987). Spinal cord contains the neural circuitry responsible for basic locomotor patterns (Sherrington, 1910; ten Cate, 1960; Sholomenko & Steeves, 1987). These intrinsic "pattern generators", of the spinal cord are controlled via descending pathways from the brainstem (Steeves & Jordan, 1980; Shefchyk et al., 1984; Steeves et al., 1987). Bipedal vertebrates, such as humans and birds, are 1 primarily dependant on pattern generation in the caudal spinal cord for walking (Sholomenko & Steeves, 1987). Evolutionary conservation of descending spinal pathways among "phylogenetically higher" vertebrates (eg. birds and mammals) has been very strong (Steeves & Jordan, 1980; Shefchyk et a l . , 1984; Steeves et al., 1987; Webster & Steeves, 1988; Ethell et a l . , 1988; Hasan et al.,1990). Anatomically, brainstem structure and descending brainstem-spinal pathways are virtually identical, with the exception of the pyramidal system (Webster & Steeves, 1988); As well, brainstem centers from which locomotion can be evoked, are very similar (Steeves & Jordan, 1980; Shefchyk et a l . , 1984; Steeves et a l . , 1987; Ethell et a l . , 1987; Hasan et al., 1990). Injuries to the rostral spinal cord (thoracic or cervical) often disrupt descending control, resulting in partial or complete paraplegia (Steeves & Jordan, 1980; Shefchyk et al, 1984; Steeves et a l . , 1987). Peripheral nervous system (PNS) has long been recognized as having a remarkable capacity for regeneration (Waller, 1852; Ranvier, 1871; Ranvier, 1873; Cajal, 1928). Subsequent to axotomy both the proximal (still attached to the soma) and distal (unattached) stumps undergo immediate traumatic degeneration. Distal axon stumps continue with Wallerian (or secondary) degeneration (Waller, 1952; Ranvier, 1871; Ranvier, 1873), due to their lack of "trophic" support (Cajal, 1928). However, proximal axon stumps develop new growth cones, or "clubs", as referred to by Cajal (1928), within one day of injury. Regeneration proceeds at a rate of approximately 1mm per day (see: Cajal, 1928). Although regeneration is greatly facilitated by proximity to remnants of the distal nerve portion (ie. Schwann cell tube), modest regeneration can occur in its absence (Cajal, 1928). Near normal functional recovery can occur in a relatively short time of weeks or months following injury to the PNS (Cajal, 1928). Paradoxically, repair of CNS injuries is poor in "phylogenetically higher" vertebrates (Cajal, 1928; Barnard & Carpenter, 1950; Bjorklund et al., 1971). Again, both proximal and distal stumps undergo immediate traumatic degeneration, and distal axon stumps undergo 2 Wallerian degeneration (Cajal, 1928; Bjorklund et al., 1971). Although proximal axon stumps develop new growth cones, they will only elongate l-2mm before degenerating (Cajal, 1928; Bjorklund et al., 1971). Thus, functional recovery of CNS injury is rare and usually accomplished via compensatory mechanisms, such as collateral sprouting of any surviving intact fibers (Cajal, 1928). Generally, axotomized fibers in adult vertebrate CNS fail to regenerate (or even substantially elongate), with few exceptions (Cajal, 1928; Barnard & Carpenter, 1950; Bjorklund, et al., 1971). In 1981, David and Aguayo reported that cut CNS axons will regenerate if their proximal stumps were permitted access to a PNS environment; thereby dispelling previous suggestions that the lack of adult CNS regeneration was due to irreversible suppression of intrinsic neuronal properties. Rats were subjected to mid-thoracic spinal cord transections, and a "PNS bridge", of sciatic nerve was grafted to both sides of the injury. Fiber growth through the PNS bridge was assessed by focal injection of the retrograde tracer, Horseraddish Peroxidase (HRP), into the caudal end of the bridge. Histologically processed sections of brainstem and spinal cord, rostral to the transection, were examined for labelled neuronal cell bodies. Cells in these areas could only be labelled if they had grown axonal processes through the PNS bridge (David & Aguayo, 1981). Primarily because of this eloquent experiment, it is now generally accepted that, providing environmental conditions are "favorable", CNS axons are capable of significant regeneration. Environmental considerations for axonal regeneration are factors, present in the adult CNS, which may have detrimental effects for neurite1 outgrowth (Schwab & Thoenen, 1985; Schwab & Caroni, 1988; Caroni & Schwab, 1988; Schnell & Schwab, 1990). The inhibitory effects of myelination are of particular interest with the recent discovery of two proteins expressed by oligodendrocytes (Schwab & Caroni, 1988; Caroni & Schwab, 1988). These two proteins, one of molecular weight -35,000 Daltons (35kDa) and the other 1 Neurite i s a term used t o describe both, growing axons and d e n d r i t e s . 3 ~250kDa, have both been found to inhibit neurite outgrowth in vitro (Schwab & Caroni, 1988; Caroni & Schwab, 1988). As well, in vivo studies have successfully blocked the neurite inhibiting properties of these two proteins and enhanced axonal regeneration after CNS injury (Schnell & Schwab, 1990). Using ventricular injections of hybridoma cells, producing antibodies to these proteins, Schnell and Schwab (1990) demonstrated extensive regeneration of corticospinal neurons in spinally injured, adult rats. A recently discovered exception to ineffective CNS regeneration is the embryonic spinal cord (Nelson & Steeves, 1987; Ethell et al., 1988; Shimizu et al., 1990; Hasan et al, 1990). Since regeneration is, in part, a recapitulation of development (Holder & Clark, 1988), these findings are not surprising. An environment capable of supporting the extensive growth cone migrations, during spinal cord development, should also be supportive of regenerating axons. Embryonic spinal cord, permissive for regeneration, develops into late embryonic and then adult spinal cord, both restrictive of regeneration (Nelson & Steeves, 1987; Ethell et al., 1988; Shimizu et al., 1990; Hasan et al., 1990). As such, there must be a time during development when the transition from a permissive to a restrictive environment occurs. When this change occurs, and what determines permissive versus restrictive environments has been of particular interest in recent years. Chicken (Gallus domesticus) may be one of the better higher vertebrate systems for use in the study of spinal cord development, injury, and repair (Hamburger & Hamilton, 1951; Nelson & Steeves, 1987; Ethell et al., 1988; Webster & Steeves, 1988; Shimizu et al., 1990; Hasan et al., 1990). First, developing chick is a classic model of vertebrate embryology; as such its stages of development are thoroughly described and well known. Second, all birds are bipedal walkers, resulting in the neural circuitry between brainstem and caudal spinal cord to be nearly identical to that of humans (Nelson & Steeves, 1987; Webster & Steeves, 1988). Third, embryonic chick development is easily timed and embryonic manipulations do not require consideration of maternal effects, such as spontaneous abortion in mammals (Hamburger & Hamilton, 1951). 4 The development of descending brainstem-spinal pathways in chick has been investigated by Okado and Oppenheim (1985). Using the retrograde tract-tracers wheat germ agglutinated-horseraddish peroxidase (WGA-HRP), they injected lumbar spinal cords of chicks from all 21 embryonic days of age. Subsequent sectioning and analysis of brainstem nuclei, for labelled neuronal cell bodies, demonstrated when the developmental onset and completion of various pathways occurred. Two of the most critical pathways for evoked locomotion, via brainstem stimulation, are the medullary reticulospinal, and the rubrospinal tracts (Sholomenko & Steeves, 1987). The rubrospinal, originating from the red nucleus, begins descending axons on E5 and is completed by E12. The nucleus reticularis medialis, giving rise to part of the reticulospinal tract, begins descending axons on E4 and is complete by E10. By E14 all brinstem-spinal pathways have completely descended their axons to the lumbar cord (Okado & Oppenheim, 1985). Several recent experiments have focused on determining the duration of development when the embryonic chick is supportive of CNS axonal repair (Nelson & Steeves,1987; Ethell et al., 1988; Shimizu et al., 1990; Hasan et al., 1990). Anatomical and functional recuperative capacities were assessed following complete transection of the mid-thoracic spinal cord. Anatomical recovery was detected using the neuroanatomical tracers, WGA-HRP or fluorescent dextran amines (Nelson & Steeves, 1987; Shimizu et al., 1990; Hasan et al, 1990). One of these tracers was focally injected caudal to the transection site 5-8 days post-transection, followed by histological examination for labelled descending brainstem-spinal neuronal cell bodies. Functional spinal recovery was confirmed by in ovo brainstem stimulation of previously transected embryos to evoke locomotor patterns by the legs (Ethell et al., 1988; Hasan et al., 1989). A capacity for anatomical and physiological repair of descending pathways was maintained at least as late as E12 (Nelson & Steeves, 1987; Ethell et al., 1988; Shimizu et al., 1990; Hasan et al., 1990). Anatomical recovery was robust in embryos spinally transected on, or before, E12 (Nelson & Steeves, 1987; Hasan et al., 1990; Shimizu et al., 1990). 5 Brainstem nuclei projecting to the spinal cord showed statistically similar numbers of labelled cells in E12 transects as untransected control embryos. There was little evidence for anatomical recovery in El3 transects, with markedly reduced numbers of labelled cells. Virtually no labelled cells were detected in the brainstem nuclei of embryos undergoing spinal transection after E13 (Nelson & Steeves, 1987; Hasan et al., 1990; Shimizu et al., 1990). In agreement with anatomical findings, there was evidence physiological recovery in embryos transected on or prior to E12 (Ethell et al., 1988; Hasan et al., 1990). Brainstem stimulation of E12 transects evoked both bipedal kicking (hatching response) and alternating stepping activities in the legs. E13 transects showed poor physiological recovery; although bipedal kicking was evoked, only very poor alternating stepping activities could be evoked. Embryos transected on E13 or after showed no evoked locomotor responses to brainstem stimulation (Ethell et al., 1988; Hasan et al., 1990). Finally, there is a system in which to study permissive and restrictive environments for axonal regeneration in the same tissue (Nelson & Steeves, 1987; Ethell et al., 1988; Shimizu et al., 1990; Hasan et al., 1990). Using the temporal discrepancy in embryonic chick spinal cord it is possible to examine developmental changes leading to the loss of regenerative ability. These findings (Nelson & Steeves, 1987; Ethell et al., 1988; Shimizu et al., 1990; Hasan et al., 1990) suggest that developmental events occur in the spinal cord around E13 leading to a loss of axonal regenerative capacity. What changes in E13 spinal cord can account for the loss of regenerative ability? Although morphological changes in the spinal cord at E13-E14 are quite dramatic, it is proteins which govern neurite activity, as previously discussed. As such changes in protein expression corollary to this developmental time (E13-E14) frame are the focus of this thesis. Of particular interest were proteins first expressed during or after this transition period. It is possible that some of these proteins may have inhibitory effects on elongating neurites attempting to regenerate. 6 For this dissertation, I have identified proteins correlative to the restrictive period for spinal cord repair in embryonic chick. In brief, proteins commencing expression immediately at or after the loss of the embryonic spinal repair capacity (at or after El3), and consistently expressed after that time. I examined the developmental expression of soluble proteins from the upper thoracic spinal cord (segments Tl-5) of embryonic chicks. Soluble protein fractions were purified from embryonic chicks of various developmental ages and then two-dimensional gel (2D gel) electrophoresis was employed to separate the proteins. Gels from different developmental ages were compared to establish the expression timing of each spot throughout later embryonic development. Protein spots of primary interest were identified as appearing first around E14 and constantly present after that time. Seven protein spots of interest were identified. Isoelectric points and molecular weights were estimated for each spot. Identification and characterization of these proteins may provide insight into some known and/or unknown molecular events occurring at the time embryonic spinal cord loses its ability to regenerate axotomized fibers. 7 METHODS Fertilized White Leghorn (DeKalb strain) chicken eggs were obtained from B & J Farm (Surrey, B.C.) and placed in a humid incubator set at 39°C. Staging was checked as described by Hamburger and Hamilton (1950). Upper thoracic spinal cord segments (Tl-5) were micro-dissected from embryonic chicks. Samples were taken from chicks of ages embryonic day 11 (El 1), E14, ,E16, E18, & E20. At least two spinal cords, of each age, were pooled for consistency. Younger embryos provided so little tissue that up to eight spinal cords were pooled for E l 1. Isolated spinal cords were immediately placed in ice-cold homogenization buffer (300mM sucrose; 50mM Tris-HCl, pH=7.4; 3mM DTT; lOmM DNase; 5mM EDTA; 5mM MgCl2; modified from Benowitz & Lewis, 1983). Tissue was homogenized with a micro-glass mortar and pestle, then centrifuged for ten minutes at 1000 X g. The supernatant was centrifuged for 90 minutes at 100,000 X g, and then dialyzed against 50mM Ammonium acetate three times for 12 hours each, max. pore size of ~3,500 Daltons (Da). Purified protein fractions were precipitated using five times volumes of ice-cold acetone, and vacuum dried. Protein fractions were run on two dimensional electrophoresis according to O'Farrell (1975), with modifications. Isoelectric focusing (IEF) was run with ampholines pH=2.5-5: pH=3-10; pH=5-8 mixed in concentrations of 2%:2%:2%. IEF runs were 16 hours at 400 Volts, followed by 2 hours at 800 Volts. Tube gels were frozen for easy extrusion from glass tubes, and equilibrated for ten minutes in 5ml of equilibration buffer (60mM Tris-HCl, pH=6.8; 5% SDS; 4% 2-Mercaptoethanol; 10% glycerol; 0.01% bromophenol blue) immediately prior to application to the second dimension. The second dimension was run using Laemmli's (1970) system, with Ornstein (1964)/Davis (1964) discontinuous buffers. SDS-PAGE gels were 7-12% linear acrylamide (29.2:0.8 % w/v of acrylamide:bis-acrylamide stock) gradients (pH=8.8), with a 4% 8 acrylamide stacking gel (pH=6.8). Tube gels were held in position with 1% molten agarose made from upper gel buffer. SDS-PAGE gels were run at 13mA/gel until the bromophenol dye reached the resolving gel, when the current was changed to 18mA/gel until completion. Completed gels were fixed and silver stained using a high resolution method described by Merril et al. (1981). All gels were photographed and large prints made. Two dimensional gels were prepared such that a minimum of 240 spots were counted between molecular weights of 14 and lOOkDa for the gel to be acceptable. Protein spots consistently appearing on 2D gels from even numbered days E14 through E20 were identified. Those spots were taken as representative of proteins present during the restrictive period. Consistent spots were then compared with several high resolution gels of tissue from embryos immediately prior to the cutoff period at E l 1. Thus spots not appearing in E l 1, but appearing on E14 and later, were identified as the spots of interest. Approximate molecular weights of proteins were determined by running known molecular weight marker proteins in the second dimension along side tube gels. Marker proteins (MW-SDS-70L kit from Sigma) consisted of 14.4,20,24,29, 36,45, & 66 kDa marker proteins. Isoelectric points were extrapolated from pH measures of blank IEF tube gels run with experimental gels. Blank tube gels were immediately frozen after the run and cut into one cm lengths. Pieces were placed into separate scintillation vials for equilibration with two mLs of distilled, deionized, and freshly degassed water containing 9.5 M urea. After two hours of equilibration, pH was measured with a cathode & pH meter. 9 RESULTS An attempt was made to limit this investigation as much as possible. A relatively small portion of spinal cord was used (Tl-5) in an effort to limit possible regional variations in the tissue. Due to the small size of the early embryonic spinal cords, 5 segments were taken. Purification of the proteins limited the investigation to soluble proteins as detergent was not used in homogenization. Protein spots of interest were selected from a stringent protocol. Their appearance had to be consistently shown in later embryonic development (E14-E20), and absent on E l 1. Thus, appearance of these protein spots correlate with the loss of regenerative ability. Seven protein spots were identified as conforming to the described criteria. Figures 1-5 show representative 2D gels of mid-thoracic spinal cord tissue from each developmental day studied. The E l 1 gels contained at least 280 distinguishable spots. In E l 1A 281 spots (fig. 1) and in E l IB 285 spots were counted. Over 421 spots were counted on the highest resolution gel, E16A (fig. 3). The gel with the least number of spots (244) was E20. Figure 1 shows an E14 gel. Sixteen spots corresponding to proteins present in E14 but not E l 1 gels have been marked with arrows and labelled "a" through "p". Although spot "a" is present in E l l , its dramatic increase in later days made it worth noting. Figure 2 shows an E l l gel with arrows indicating where the appropriate spots, "a" through "p", would be located, if present. Figure 3 shows an E16 gel with the same proteins labelled as in figures 1 and 2. Note that spot "n" is absent, and was eliminated as a possible spot of interest. An E18 gel is shown in figure 4, again possible spots of interest are labelled. Spots "b, c, h", and "k" are absent. The final screen for the spots of interest was E20 (fig 5). Spots "a, d, e, f, g, j", and "1" are indicated. These spots are all defined, in this study, as spots of interest. 10 Spots of interest were named as, "Late-expressing Neural Proteins-" (LNP-) 1 through 7. Figure 6 shows an E14 gel with all spots of interest labelled accordingly. See table I for a summary of estimated molecular weights and isoelectric points for each spot. 11 Figure 1. Photograph of an E14 two-dimensional protein gel; Arrows indicate protein spots shown on E14 gels but absent on two-dimensional protein gels of E l 1 tissue. 12 Figure 2. Photograph of an E l l two-dimensional protein gel; Arrows indicate the locations of protein spots present on E14 gels, but absent on E l 1 gels. 13 Figure 3. Photograph of an E16 two-dimensional protein gel; Arrows indicate protein spots present at both E14 and E16 but absent on E l l . 14 Figure 4. Photograph of an E18 two-dimensional protein gel; Arrows indicate protein spots present on E14, E16, & E18 gels, but absent on E l 1 gels. 15 Figure 5. Photograph of an E20 two-dimensional protein gel; Arrows indicate protein spots present on E14, E16, E18, & E20 gels, but absent on E l 1 gels. 16 Figure 6. Photograph of an E14 twc-dimensional protein gel with all spots of interest labelled. 17 TABLE I Approximate molecular weights and isoelectric points for protein spots of interest identified in this study. Alphabetical letter Molecular weight (kiloDaltons) Isoelectric point (pH) LNP-1* a 43 6.8 LNP-2 d 60.5 8.2 LNP-3 e 60 8.3 LNP-4 f 39 8.2 LNP-5 g 34 8.1 LNP-6 j 27 7.5 LNP-7 1 20.5 7.9 LNP=Late-expressing Neural Protein 18 DISCUSSION Seven protein spots of interest were identified in this study. Their appearance correlates with a loss of regenerative ability in the embryonic spinal cord. LNP-2 through -7 were present only after the loss of regenerative ability and not before. LNP-1 demonstrated a similar developmental profile, except that there was appearance on E l 1; however, LNP-1 showed such a dramatic increase in E14-E20 that an exception to the criteria was made and this was included as an LNP of interest. Although this expressional association is close, correlation does not imply causality. The function of these proteins cannot be deciphered from analysis on 2D gels alone. In fact, it is entirely possible that none of the seven protein spots identified have any direct consequence on the loss of regenerative ability. However, these proteins spots may be involved in the inhibition of spinal cord regeneration, and are worthy of further investigation. Spinal cord development and plasticity are very precise and complex processes. Attempts to mix up a "witches brew", to repair spinal cord using a mixture of things, not knowing what they do or how, has been tried for hundreds, possibly thousands, of years. Recent attempts are much more complex and involve direct manipulation of the spinal cord, but the reasoning is the same with a primary emphasis on luck. To properly manipulate such a complex entity as the CNS it is critical to thoroughly understand the subject. Thus, the results of this study, and any further studies arising from it, must be considered in the context of many other factors involved in spinal cord development. As such, I will describe some of the critical considerations for spinal cord development and plasticity. Three possible explanations may account for the lack of regenerative ability in adult CNS. First, there may be insufficient amounts of factors necessary for sustained elongation. These factors, that promote growth, may be produced by cells accessible to the PNS, such as Schwann cells, but not accessible to the CNS due to the blood-brain barrier. Cajal's (1928) 19 "trophic" theory fits within this explanation. Second, there may be factors in adult CNS that inhibit growth cone elongation. Schwab and his colleagues have demonstrated two such proteins in the rat (Caroni & Schwab, 1988; Schwab & Caroni, 1988; Schnell & Schwab, 1990). The purpose of these proteins is not entirely clear, but their presence has been well established. Third, a combination of the first two possibilities. This is the most attractive explanation because of the strong evidence supporting both of the first two explanations. In vitro studies of neurite growth, motility, and inhibition are laying the foundations for a comprehensive understanding of molecular interactions between neuronal growth cones and their environments. Using in vitro systems, it is possible to isolate factors and investigate them in a controlled environment, separate from the complexities of the spinal cord. These techniques have been used most extensively to examine factors that promote neurite outgrowth. Growth Promoting Factors Three major environmental factors are required by regenerating growth cones. First, adhesion to other cells, neural, glial, and otherwise. Second, trophic factors; the existence of trophic factors was initially hypothesized by Cajal (reviewed in 1928). Third, growth cones, like most cellular processes, in the CNS, may use the extracellular matrix (ECM) for anchorage. Adhesion to other cells is mediated by cell adhesion molecules (CAMs) (Rutishauser et al., 1988;Kemler et al., 1989). CAMs are membrane glycoproteins with homotopic and/or heterotopic binding properties. Briefly, there are two major classes of CAMs, depending upon their calcium binding requirements. Highly calcium dependent proteins include Cadherin and NgCAM (see: Kemler et al., 1989), whereas NCAM is an example of a calcium independent adhesion protein (see: Rutishauser et al., 1988). Trophic factors, such as Nerve Growth Factor (NGF) and Brain Derived Neurotrophic Factor (BDNF), are responsible for altering gene expression and sustaining the 20 metabolism of neurons such that they can support the cellular machinery required to build axons (Levi-Montalcini & Angeletti, 1968;Greene & Tischler, 1976;Thoenen & Barde, 1980). For a review of NGF receptors in the CNS see Springer (1988). A large number of trophic factors have been identified, they are commonly, but not necessarily, protein in nature. Interestingly, development of the blood-brain barrier (Wakai & Hirokawa, 1978;Risau & Wolburg, 1990) both begin around E13 of chick development. This raises the question of whether systemic trophic factors are facilitating early embryonic spinal cord repair. Extracellular matrix (ECM) components such as laminin, collagen, and fibronectin provide a scaffolding that is commonly used for cell migration and anchorage (see: Bernfield, 1989;Carbonetto, 1984). Cellular interaction with the ECM is accomplished using specific membrane protein receptors, such as integrins (Bernfield, 1989). Membrane-bound receptors, such as integrins, specific for these ECM structures have been found in abundance on growth cones (Carbonetto, 1984). All CAMs, trophic factors, and ECM components all play active ontogenic roles in the CNS. Many are down-regulated upon completion of spinal cord development; whether or not they can be up-regulated following CNS injury is unknown. Permanent down-regulation of these environmental factors may be required for efficacy of the spinal cord. The presence of these molecules at embryonic levels, in the adult, may be chaotic for the spinal cord. As such, the possiblity that they may be up-regulated may be actively discouraged by the spinal cord itself. Growth Inhibiting Factors Factors inhibiting neurite sprouting may do so by either physically blocking growth cones, as with a glial scar, or physiologically altering growth cone motility and elongation via biochemically transduced signals. Glial scarring is common following CNS injury (Reier & Houle, 1988). Cells dying as a result of the injury are endocytosed by mobile microglia, 21 and digested. Cavities resulting from necrosis are filled by reactive astrocytes which then form glial scars. It has been suggested that this physical barrier is a major impediment to successful axon regeneration within CNS (for an excellent review see Reier & Houle, 1988). Minimizing or eliminating glial scar formation has been found to gready improve axonal outgrowth, but successful, functional regeneration will still not occur in adult CNS by the elimination of glial scarring alone (Reier & Houle, 1988). Endogenous CNS proteins inhibitory to neurite sprouting are a relatively recent discovery (Caroni & Schwab, 1989; Schwab & Caroni, 1989; Schnell & Schwab, 1990). One 35kDa and one 250kDa protein have been isolated from myelin fractions; both proteins demonstrate active inhibition of neurite outgrowth in vitro (Schwab & Caroni, 1989). Even very small amounts of the 35kDa protein cause growth cone collapse and complete neurite inhibition (Schwab, personal communication) Two specific monoclonal antibodies (IN-1 & IN-2) have been produced that functionally block the inhibitory properties of both proteins (Schnell & Schwab, 1990). Schnell and Schwab (1990) have reported that blockage of these inhibitory proteins, in vivo, results in extensive regeneration of injured adult rat spinal cord. Hybridoma cells producing the IN-1 and IN-2 antibodies were implanted into the lateral ventricles of adult rats. Following axotomy in the spinal cord, hybridoma implanted rats showed extensive anatomical regeneration of axons whereas controls showed none. The 35kDa inhibitory protein has been intensively studied. In vitro experiments using Fura-2 Calcium imaging with confocal microscopy have investigated the physiological effects of the 35 kDa protein on growth cones. Filopodial contact with membranes containing the inhibitory proteins causes a large Calcium influx into the growth cone (Schwab, personal communication; Kater, personal communication). Resultant high Calcium concentrations are thought to disrupt the stability of actin and tubulin polymers causing retraction of the growth cone (Kater et al., 1988). 22 Goldfish optic nerve is a classical model of successful CNS regeneration (Sperry, 1944; Attard & Sperry, 1963; Gaze, 1970; Steurmer, 1988). Interestingly, neither inhibitory protein has an apparent homologue in goldfish optic nerve (Schwab, personal communication). In fact, myelin fractions from goldfish optic nerve are excellent substrata for neurite outgrowth (Steurmer, personal communication). Such findings, in goldfish, support the view that these two inhibitory proteins are made specifically to inhibit neurite outgrowth in the adult spinal cord of higher vertebrates. The reasons why adult spinal cord may actively discourage axon outgrowth and regeneration have been of recent controversy. A developing consensus on the function of these proteins suggests that they prevent short-circuiting of CNS fibers (Schwab, personal communication). Spontaneous sprouting of axon collaterals may be virtually eliminated by inhibiting all neurite outgrowth. Once the CNS has completed most tract formation, myelination with inhibitory proteins may prevent superfluous axo-axonic and axo-dendritic connections within long ascending and descending pathways of the spinal cord. Several recent experiments have examined the repair capacity of spinal cord throughout embryonic development (Nelson & Steeves, 1987; Ethell et al, 1987; Shimizu et al, 1990; Hasan et al, 1990). Complete mid-thoracic spinal cord transections showed differing anatomical and physiological repair capacities. Anatomical recovery was detected, using HRP and fluorescent retrograde tracers, in embryos transected up to and including E13. Whereas restored functional connectivity, assessed using in ovo brainstem stimulation, was detected only in embryos transected before El3. This time discrepancy may be due to the spinal cord changing into a restrictive environment; axons detected anatomically may have been enroute, but failed to reach their targets before the change occurred. Using this axiom, the time period when spinal cord changes from a permissive to a restrictive environment for axonal regeneration was limited; change was estimated to occur around E13. Thus proteins from embryonic spinal cord just before that time, that is E l l , were compared with those from embryos immediately after that time period, that is E14 to E20. 23 Directional guidance of regenerating CNS growth cones is a future consideration. If we can eventually manipulate adult spinal cord to support the extensive neurite elongations required for regeneration, will the growth cones know where they are supposed to go? With the exception of pioneering growth cones, spinal pathways are fasciculated by neurites growing alongside previously established axons. Fasciculation may be sufficient in the adult; however, essential directional cues may no longer present. Myelination may block fasciculation, except there are probably just as many unmyelinated as myelinated fibers in tracts of the spinal cord (R. Bunge, personal communication). Further, growth cones regenerating in the adult must migrate distances, orders of magnitude, longer than in developing spinal cord. Regardless of these considerations, the ability of regenerating CNS axons to find their appropriate targets is only speculative until the first obstacle of axon elongation is overcome. Proteins identified in this thesis provide an avenue of research from a slightly different perspective on spinal cord development and plasticity. Close examination of biochemical and histological events at this critical time in spinal cord development, may ellucidate much about the loss of regenerative ability. Even if none of the proteins, identified in this study, play either direct or indirect roles in the inhibition of axonal regeneration, they undoubtedly serve some function in the upper thoracic spinal cord, sometime after E l l . Since a more thorough assessment of spinal cord development can only help our understanding of the environments axons encounter, in vivo, it is my opinion that protein spots LNP-1 through -7, warrant further investigation. One possible avenue of investigation is to isolate protein spots directly from 2D gels and attempt to obtain partial amino acid sequences using an automated amino acid sequencer. This sequence information could be run on databases to look for homology with known proteins, or used to synthesize specific oligonucleotide probes for the cloning of cDNAs, and in situ hybridization. 24 CONCLUSIONS Seven proteins (LNP-1 through -7) have been identified, any of which may play an inhibitory role in spinal cord regeneration. The appearance of these proteins has been correlated with a loss of regenerative ability. However, correlation does not indicate causality. Only further studies can determine if these proteins are involved in neurite inhibition. Even if none of LNP-1 through -7 are directly involved in regenerative suppression, their expressions still change at a critical time in spinal cord development. 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