Tubulin expression and axonal transport in injured ...

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Tubulin expression and axonal transport in injured and regenerating neurons in the adult mammalian central nervous system Alyson E. Fournier and Lisa McKerracher

Abstract:Microtubules are essential components of the cytoskeleton required for axonal growth. To investigate how changes in tubulin transport and expression may affect axon regeneration, injury in the adult mammalian central nervous system was studied. Axotomized retinal ganglion cells (RGCs) that do not regenerate were compared with RGCs that regenerate their axons when the optic nerve is replaced with a peripheral nerve graft. When RGC axons regenerated through peripheral nerve grafts, the rate of slow transport increased but decreased when no regrowth occurred. To investigate the molecular mechanisms that mediate these responses, alterations in tubulin rnRNA levels after injury were examined. Total tubulin mRNA levels fell after injury in the optic nerve but increased in those RGCs that regenerated their axons into a peripheral nerve graft. Further, the expression of four separate P-tubulin isotypes in injured rat RGCs was characterized. mRNA levels for all four isotypes decreased in RGCs after injury in the optic nerve. How the autoregulation of tubulin expression may contribute to the changes in P-tubulin isotype expression after injury is discussed. Key words: tubulin, retinal ganglion cell, axotomy, axonal transport, in situ hybridization.

Resume : Les microtubules sont des constituants essentiels du cytosquelette requis pour la croissance de l'axone. Afin de vkrifier comment les changements de l'expression et du transport de la tubuline affectent la rCgCnCration de l'axone, nous avons CtudiC les effets d'une lCsion du systkme nerveux central chez un mammifere adulte. Nous avons compark les cellules ganglionnaires de la rCtine dont l'axone ne rCgCnkre pas aprks axotomie aux cellules ganglionnaires de la rktine dont l'axone rkg6nkre lorsque le nerf optique est remplacC par une greffe de nerf pCriph6rique. Lorsque les axones des cellules ganglionnaires de la rCtine rtgCnkrent la suite d'une greffe d'un nerfpCriphCrique, le taux du transport axonal lent augmente, alors que ce taux diminue si aucune rCgCnkration ne se produit. Afin d'Ctudier les mtcanismes molCculaires intervenant dans ces rkponses, nous avons dCtermin6 les modifications du taux de 1'ARNm de la tubuline i la suite d'une 16sion. Le taux de 1'ARNm total de la tubuline diminue aprks la lCsion du nerf optique, mais il augmente dans les cellules ganglionnaires de la rktine dont l'axone rCgCn8re lors d'une greffe de nerf pCriphCrique. De plus, nous avons CtudiC l'expression de quatre isotypes distincts de la P-tubuline dans les cellules ganglionnaires de la rCtine aprks la 1Csion. Nous avons not6 que les taux des ARNm des quatre isotypes diminuent dans ces cellules aprks la lesion du nerf optique. Nous discutons du mCcanisme par lequel I'autorCgulation de l'expression de la tubuline contribuerait aux changements de l'expression des isotypes de la P-tubuline aprks la Itsion. Mots clis : tubuline, cellule ganglionnaire de la rktine, axotomie, transport axonal, hybridation in situ. [Traduit par la rkdaction]

Received April 28, 1995. Accepted June 12, 1995.

Abbreviations:RGC, retinal ganglion cell; CNS, central nervous system; BDNF, brain-derived neurotrophic factor; SCa, slow component a; SCb, slow component b; PNS, peripheral nervous system; SDS-PAGE, sodium dodecyl sulfate - polyacrylarnide gel electrophoresis. A.E. ~ournier'and L. McKe~acher.Centre for Research in Neuroscience, MontrCal General Hospital Research Institute, 1650 Cedar Avenue, Montreal, QC H3G 1A4, Canada.

Biochem. Cell Biol. 73: 659464 (1995). Printed in Canada 1 Imprime au Canada

Biochem. Cell Biol. Vol. 73, 1995 Fig. 1. (A) Schematic representation of an injury to an RGC axon. After axon transection, there is no substantial regrowth from the remaining proximal segment of the axon. (B) A periph-


eral nerve grafted near the injured axon provides a permissive growth substrate into which some RGCs regenerate an axon.

A

Axotomy

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Axotomy 1 PN graft

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7 growth factors substrate molecules

Introduction The ability of mammalian CNS neurons to survive and regrow after axonal injury is dependent on both the site of the injury, and the availability of appropriate growth factors and guidance molecules (Bray et al. 1991). The visual system has been particularly well studied as a model of injury and regeneration in the CNS because the projection of RGC axons in the optic nerve does not branch and because RGCs represent a fairly homogeneous population of cells. Using this model it is possible to specifically axotomize RGC fibers by cutting the optic nerve, without directly damaging other cells in the retina. While RGCs do not spontaneously regenerate after such an injury (Fig. 1A), replacement of the degenerating optic nerve with a living peripheral nerve graft enhances the survival of injured RGCs (Villegas-Perez et al. 1988), and provides a permissive substrate for regrowth of injured axons (Vidal-Sanz et al. 1987). RGCs extend processes into the graft (Fig. 1B), and demonstrate dramatic changes in cytoskeletal transport and gene expression, while neighboring cells that do not regrow, do not show these changes (McKerracher et al. 1993b). These studies indicate that neurons in the CNS are capable of growth, given the appropriate environment. Many studies have focussed on the identification of factors within the peripheral nerve that support the in vivo regeneration of RGC axons. Living components of peripheral nerve grafts, such as Schwann cells, secrete multiple soluble factors and make several substrate molecules that support the survival and growth of RGCs (Hopkins and Bunge 1991). Soluble factors present in Schwann cell conditioned media allow adult rat RGCs placed in culture to grow neurites when placed on certain substrata (Thanos et al. 1989). Some of the molecules present in such conditioned media are likely to be known neurotrophic factors. One such trophic factor, BDNF, supports the survival of RGCs in culture (Thanos et al. 1989). Furthermore, in vivo injections of BDNF foster the survival of injured RGCs and may elicit some intraretinal axonal sprouting (Mansour-

Robaey et al. 1994). However, it is not clear if BDNF directly stimulates axonal regrowth or if surviving cells are better able to respond to other secreted factors or extracellular substrate molecules. Schwann cells also express cell surface molecules that directly support the growth of RGCs (Hopkins and Bunge 1991). The Schwann cell basal lamina provides a supportive surface for axon growth and is enriched in laminin, an extracellular matrix molecule that supports profuse neurite growth from RGCs placed in tissue culture (Cohen and Johnson 1991). A larninin receptor, the a6p1 integrin heterodimer, is expressed by RGCs and mediates the interaction between RGC growth cones and laminin (Cohen and Johnson 1991; de Curtis and Reichardt 1993). Furthermore, while adult RGCs do not show robust expression of this laminin receptor, injured RGCs that regenerate their axons into a peripheral nerve graft reexpress high levels of a6Bl integrins (Knoops et al. 1993). This in vivo observation suggests that the interaction of RGC axons with larninin present in the graft is an important determinant of RGC regeneration. Although a peripheral nerve graft provides many factors that enhance RGC regeneration, only a small number of RGCs extend processes into the graft (Vidal-Sanz et al. 1987). To elucidate why some axons regrow and others do not, a better understanding of the neural cell response to injury and of the cellular mechanisms responsible for axonal growth is needed. A critical aspect of successful axonal growth is appropriate cytoskeletal rearrangement and extension into the newly forming axon. To determine how the neuronal cytoskeleton is affected by injury and regeneration, we have compared the neuronal response of injured RGCs with RGCs that regenerate their axons into a peripheral nerve graft (Fig. 1). Through these studies, it is becoming increasingly clear that some of the growth-promoting factors in a peripheral nerve graft act to modify the transport and expression of a number of cytoskeleta1 components. In particular, tubulin, the principle component of microtubules, which is essential for axonal elongation and axon stabilization, must be appropriately regulated for successful axon regrowth. Some of our studies that demonstrate the importance of the changes in tubulin transport and tubulin expression for the successful regeneration of adult rat RGC axons are described below. Moreover, some new data on how axon injury affects the expression of the individual ptubulin isotype genes is provided and the possible changes that may be essential for successful axonal elongation in the CNS are discussed.

Changes in slow axonal transport in injured and regenerating RGCs Protein synthesis occurs exclusively in the somatodendritic compartment of mature neurons; therefore, proteins that travel into the axon must rely exclusively on an axonal transport system. Two major rates of axonal transport have been identified, fast and slow. The fast axonal transport rate corresponds to the transport of vesicular organelles, while the slow axonal transport rate corresponds to the translocation of the cytoplasmic matrix (Lorenz and Willard 1978). By radioactively labelling proteins within the cell body, cytoskeletal proteins are observed to move together at approximately 1 mmlday and they appear at increasing distances along the axon over time

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Fig. 2. Slow transport in normal and axotomized RGC axons. Autoradiographs of axonally transported proteins in normal (left) and crushed (right) optic nerves. The proteins were prelabelled by injection of [35~]methionine1 week before optic nerve injury. Eight days after optic nerve crush, the optic nerves were divided into 2-mm pieces and separated by SDS-PAGE, and fluorographs were prepared. The tubulin and neurofilament subunits extend further distally in the control optic nerve compared with the axotomized nerve. NF-H, NF-M, NF-L: neurofilament heavy, medium, and light, respectively.

SPECTRIN NF-H NF-M

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9 rr *r--

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rr

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I

.

DISTANCE FROM EYE (mm) (Lasek et al. 1984; Oblinger and Lasek 1988). Two major rates of slow axonal transport have been defined in adult neurons. SCa is composed of the major cytoskeletal proteins, including tubulin, neurofilament, and actin. SCb represents a more heterogeneous mixture of proteins, and the attributes of SCb vary in different neuronal populations. Perhaps significantly, a substantial amount of tubulin is carried in SCb of peripheral nerves, while in neurons of the CNS, tubulin is not transported in SCb. Changes in the rate of tubulin transport seem to be important for axonal growth in developing and regenerating neurons because altered dynamics of tubulin transport are consistently observed when injured neurons regenerate their axons (McKerracher and Hirscheimer 1992). The differences in slow axonal transport profiles in the CNS and PNS correlate with their disparate regenerative responses to injury. When injured peripheral nerves regenerate, the SCb tubulin transport rate increases to the same rate as axonal extension (Hoffman and Lasek 1980; McKerracher and Hirscheimer 1992). This increase in the slow transport rate of tubulin is consistently observed in all spontaneously regenerating nerves, including those of various fish species (Grafstein 1986). In contrast with these observed changes, after optic nerve crush of adult rats, the rate of slow axonal transport decreases from its normal rate of 0.5 m d d a y to 0.06 mmlday . change in rate is observed as (McKerracher et al. 1 9 9 0 ~ )This

a failure of the tubulin and neurofilament proteins to move into distal segments of crushed optic nerves (Fig. 2). Importantly, the axonal projection in the optic nerve remains intact early after axotomy when the changes in transport rates are first observed (McKerracher et al. 1990b; Minzenberg et al. 1995). Therefore, a dramatic decrease in the rate of axonal transport follows injury of these CNS neurons. However, when these same RGCs regenerate their axons in a peripheral nerve graft, the slow transport rate increases to 1 mmlday (McKerracher et al. 1990b). Also, in regenerating rat RGCs there is only one slow transport rate and this is consistent with other regenerating neurons in which it has been shown that SCa and SCb components may overlap in regions of new growth but remain distinct in the proximal segment of the axotomized nerves. Therefore, an accelerated slow axonal transport rate always accompanies successful axonal regrowth, even when CNS neurons regenerate their axons. Changes in the rate of cytoskeletal protein transport suggest that either the composition or dynamics of the neuronal cytoskeleton are a critical component for axonal regrowth. Such a contention is supported by recent observations that a specific isotype of tubulin, PI11 tubulin, is enriched in SCb of axonal transport in response to crush injury of the distal sciatic nerve (Moskowitz and Oblinger 1995). The increased rate of slow transport during regeneration may be, therefore, a reflec-

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tion of the changing composition of proteins carried by slow axonal transport. To understand if changes in tubulin expression are truly important determinants of axonal growth, we sought to determine at the molecular level how injury affected tubulin expression in RGCs and how a peripheral nerve graft might modify these changes.

tubulin mediates axonal elongation, growth cone turning, and axon stabilization (Bentley and O'Connor 1994; Tanaka et al. 1995), and these are all processes that would be required for the regenerative process.

Changes in tubulin expression after injury

Different p=tubulinisotypes expressed in RGCs

Several studies have examined the molecular correlates of changes in the slow axonal transport of tubulin. The levels of mRNA for several cytoskeletal proteins, including tubulin, actin, and peripherin, increase when injured peripheral nerves regenerate their axons (Miller et al. 1989; Mizobuchi et al. 1990; Tetzlaff et al. 1991; Chadan et al. 1994). When examining such changes in gene expression in the visual system, the analysis is often complicated by the cell death that follows injury. To circumvent this problem and analyze how injury affects RGCs at the cellular level, we have used in situ hybridization to analyze tubulin mRNA levels. With this procedure, we compare mRNA levels in axotomized RGCs with the levels in contralateral intact uninjured RGCs. To allow a quantitative comparison, the injured and control retinas are processed together to control for section thickness, hybridization conditions, and emulsion thickness. Total tubulin mRNA levels were examined after both intraorbital and intracranial axotomy of RGCs. In both cases, the hybridization signal was reduced in the axotomized RGCs by 2 weeks after axotomy, and the level remained low for at least 6 months. An image-analysis system (Universal Imaging, West Chester, Pa.) was used to count the number of autoradiographic silver grains over RGCs of treated and control retinas. The average number of grains per RGC were counted and expressed as a percentage of those in the intact contralateral retina. Following intracranial RGC axotomy, total tubulin mRNA levels increase initially and then decrease to 70% of control levels by 1 week (McKerracher et al. 1993a). The transient increase may be related to the short period of abortive sprouting that is seen after RGC axotomy (Richardson et al. 1982); however, this response is not maintained and tubulin mRNA levels soon drop. This decrease in tubulin mRNA levels is distinctly different from injured peripheral nerves, which show a robust increase in tubulin mRNA expression after injury. To determine how a peripheral nerve graft might modify the injury response of CNS neurons, we examined RGCs 2-3 weeks after injury and grafting, a time when the RGCs are actively elongating their axons into the graft. Surprisingly, when the optic nerve was replaced with a peripheral nerve graft, a similar decrease in tubulin mRNA levels was observed over most RGCs. However, a small number of RGCs showed a dramatic increase in the number of autoradiographic grains that appeared as hotspots of hybridization. Retrograde labelling studies demonstrated that the increased tubulin mRNA signal was restricted to RGCs that regenerated their axons (McKerracher et al. 1993b), while most RGCs that did not regrow did not exhibit this increase. Rather, their tubulin mRNAs decreased as they would after axotomy and in the absence of a peripheral nerve graft (McKerracher et al. 1 9 9 3 ~ )The . striking correlation between total tubulin mRNA levels and RGC regeneration suggests that appropriate changes to tubulin expression may make a significant contribution to axonal regrowth. Many studies have indicated that

Tubulin is a heterodimeric protein composed of an a-and Bprotein subunit, and different subunits are members of closely related but separate gene families. Proteins in the p-tubulin family are structurally distinct, distinguished mainly by a highly variable region in the extreme carboxyl-terminal region of the sequence (Sullivan and Cleveland 1986). For our studies, we have focussed on the p-tubulin isotypes because they are well characterized in rodents (Lewis et al. 1985) and because P-tubulin expression is known to change when peripheral nerves regenerate their axons (Hoffman and Cleveland 1988; Moskowitz et al. 1993; Jiang et al. 1994) in a manner that resembles the expression patterns in brain development (Lewis et al. 1985; Hoffman and Cleveland 1988). To understand how injury in the optic nerve affects tubulin isotype expression and to determine if these changes are important for regrowth of injured neurons in the CNS, we have begun to analyze p-tubulin isotype expression in injured and regenerating RGCs. There are five p-tubulin isotypes expressed in mammalian brain. The class I isotype is a major constitutively expressed isotype, expressed at greater levels in the immature nervous system than in the adult. The class LT isotype is the major neuronal isotype, expressed predominantly in the lung and in the brain where levels are enhanced during development (Sullivan and Cleveland 1986). Class I11 P-tubulin is the minor neuronal isotype found specifically in neurons in the CNS (Burgoyne et al. 1988; Joshi and Cleveland 1989), with limited expression in the testis (Jiang and Oblinger 1992). Class IV p-tubulin is controlled by two genes that encode almost identical proteins (Luduena 1993). Class IV, is neural specific and its levels are highest in the adult brain, while class IV, is constitutively and ubiquitously expressed (Lewis et al. 1985; Sullivan and Cleveland 1986). We have begun to characterize the expression of the ptubulin isotypes in normal and injured rat retina. The cDNA probes to the unique 3' regions were obtained from Dr. Nicholas Cowan (New York University Medical Center, New York). Northern blots of total retinal RNA demonstrated that the mouse cDNA probes recognized specific bands of rat mRNA that corresponded in size to the predicted isotype mRNAs (Lewis et al. 1985). The four isotypes that we examined, classes I, 11, 111, and IV, were all present in rat retina. In situ hybridization revealed that all isotypes were expressed by RGCs. Therefore, we have examined how optic nerve transection affects the expression in injured R&S compared with the intact contralateral control RGCs. The retinas were examined 2 weeks after intraorbital axotomy and hybridization with 35~-labelledcDNA probes recognizing each of four p-tubulin isotypes: classes I, 11, 111, and IV. Preliminary observations indicate that by 2 weeks after intraorbital axotomy, all tubulin isotypes showed a similar decrease in the hybridization signal in injured RGCs. An example of such a change is shown in Fig. 3. The decreased

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Fig. 3. Reduced tubulin mRNA levels in axotomized RGCs. Samples were processed together as previously described (McKerracher et al. 1 9 9 3 ~and ) hybridized in situ with a probe specific for the type-I tubulin isotype. Bright field (A, B) and corresponding dark field (C, D) micrographs of matched axotomized (A, C) and control (B, D) retinas are shown. Two weeks after axotomy, the hybridization signal decreases in the RGC layer of the retina relative to the signal in the RGC layer of the control retina. White arrows indicate the retinal ganglion cell layer. Bar, 100 pm.


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signal was restricted to the RGC layer, indicating that only the RGCs, not the other neurons in the retina, showed a change in tubulin expression after axotomy. These results suggest that the four p-tubulin isotypes are regulated in a similar fashion following RGC injury. The overall decrease in all isotype mRNA levels could reflect an increase in the unassembled p-tubulin subunit concentration. The mRNA levels for p-tubulin are autoregulated by the tubulin monomer pool (Cleveland et al. 1981) by a sequence present in all neuronal O-tubulin isotypes (Lewis et al. 1985; Bachurski et al. 1994). When the tubulin monomer levels rise in the cell body, tubulin mRNA is degraded and the mRNA levels drop. Therefore, we speculate that the general decrease in all tubulin isotype mRNA levels observed after RGC injury may reflect an increase in free tubulin monomers in the RGC bodies. This possibility is in keeping with the observed decrease in slow axonal transport, although it is not clear why microtubules may not be assembled and exported into RGC axons at the normal rate after axotomy in the optic nerve. Posttranscriptional events responsible for the assembly or transport of the axonal cytoskeleton may be altered by axonal transection. Our preliminary results of regenerating RGCs suggest

that not all isotypes are equally upregulated when RGC axons regrow into a peripheral nerve graft. Therefore, it is likely that a peripheral nerve graft modifies the microtubule dynamics in injured RGCs, at least indirectly. Changes in isotype expression that accompany axonal regeneration could have dramatic consequences at the microtubule structural level. Our characterization of the changes that occur in vivo after injury and during regeneration in the CNS will suggest the changes critical for successful axonal regrowth. The enhanced axonal transport and increased total tubulin expression observed in regenerating RGCs underlies the importance of the microtubule cytoskeleton for successful axonal regrowth. Future studies on tubulin isotype expression when RGC axons regenerate should provide a good framework to help understand the environmental factors affecting the regulation of the neuronal microtubule cytoskeleton and its role as a determinant for axonal regeneration.

Acknowledgment This work was supported by the Natural Sciences and Engineering Research Council.

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