Clusterin Upregulation Following Rubrospinal Tract Lesion in the Adult Rat

Experimental Neurology 157, 69–76 (1999) Article ID exnr.1999.7046, available online at http://www.idealibrary.com on

Clusterin Upregulation Following Rubrospinal Tract Lesion in the Adult Rat Li Liu,* Mikael Svensson,† and Håkan Aldskogius* *Department of Neuroscience, Uppsala University, P.O. Box 571, SE-751 23 Uppsala, Sweden; †Department of Clinical Neuroscience, Section for Neurosurgery, Karolinska Institutet, Karolinska Hospital, SE-17176, Stockholm, Sweden Received May 21, 1998; accepted January 19, 1999

cytes following motor axon injury (28), in spinal cord dorsal horn astrocytes following sensory axon injury (21), as well as in glial cells along Wallerian degeneration of central pathways (20). Despite a substantial body of information on the conditions regulating clusterin expression, its functional role is still obscure. The response to axotomy is markedly different between neurons projecting to a peripheral nerve and those with axons entirely within the central nervous system (CNS). While the former undergo a robust growth response to axotomy and are able to regenerate their axons and at least partially restore function, injury to axons in the CNS results only in a transient growth response and regrowth of the injured axons does not occur (3). Furthermore, there is evidence indicating that the glial cell responses in the vicinity of these two kinds of neurons is also markedly different, the former inducing a prompt and intense reaction by microglia and astrocytes (2), while injury to intrinsic CNS neurons is associated with minimal reactions by these cells (3, 19, 30, 31). The aim of the present study was to examine clusterin expression in an intrinsic CNS system at all levels where significant biological responses to axotomy occurs. We have selected the rubrospinal tract as experimental model for our experiments. Lesioning this pathway, which is located in the lateral funiculus of the cervical cord, induces a retrograde response in contralateral red nucleus neurons. This lesion also allows us to explore the changes at the site of the injury as well as in the white matter caudal to the lesion, where Wallerian degeneration takes place. The rubrospinal system has been explored in previous studies concerning neuronal responses to axon injury, making it possible to integrate our findings into the existing context of axotomy-induced reactions in CNS neurons.

We have examined the expression of the multifunctional protein clusterin in the axotomized red nucleus, at the lesion site in the lateral funiculus of C3, as well as along the Wallerian degeneration in the lateral funiculus of T1. There was a marked increase in clusterin-immunoreactivity (IR) and clusterin mRNA in red nucleus nerve cell bodies. An early, transient occurrence of large, heavily clusterin-IR globules were found in axons in the spinal cord at the lesion site in C3 as well as a marked upregulation of mRNA for clusterin, presumably associated with reactive astrocytes and oligodendrocytes from 1 to 4 weeks postoperatively. Clusterin-IR and its mRNA were markedly increased in the zone of Wallerian degeneration at T1, where some strongly expressing cells were identified as oligodendrocytes. Taken together with previous changes in clusterin expression following peripheral nerve and dorsal root injury, we suggest that this protein is involved in regenerative as well as degenerative neural responses. r 1999 Academic Press Key Words: nerve degeneration; nerve regeneration; neuroglia; spinal cord; Apo-J.

INTRODUCTION

Clusterin (apolipoprotein J) is a glycoprotein that has been implicated in a number of cellular processes, such as lipid transfer, cell–cell interactions, programmed cell death, and inhibition of the terminal complement complex formation (24). It is expressed in low amounts in astrocytes and many neuronal populations in the intact CNS (8, 23). Its functions can be exerted via binding to the gp330/megalin receptor (4) or it may bind directly to other proteins or protein complexes, thereby regulating their specific activities. Interest in the functional role of this protein in the nervous system stems largely from its association with neuritic plaques in the brains of Alzheimer patients (22). Upregulation of clusterin and its mRNA has also been demonstrated in areas of cerebral ischemia (32) and cortical contusions (5), in axotomized motoneurons and astro-

MATERIAL AND METHODS

Altogether 24 adult female Sprague–Dawley rats (200–250 g) were used for the study. The experimental procedures on animals followed the guidelines of the 69

0014-4886/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.

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National Institute of Health, and were approved by the local animal ethics committee. All operative procedures were done under deep anesthesia following intraperitoneal injection of chloral hydrate (35 mg/100 g body weight). Surgery The C3 vertebra was removed on the left side to expose the spinal cord, and the left lateral funiculus of the spinal cord was transected at this level after 1, 2, 4, 7, 14, 28 days, and 3 months (two animals at each postoperative survival time). At the appropriate postoperative survival time, the rats were reanesthetized and the animals were perfused with saline (37°C) followed by 4% (w/v) paraformaldehyde in 0.15 M phosphate buffer (pH 7.3–7.4; 4°C) containing 14% (v/v) of a saturated picric acid solution. One intact rat was used as control. Immunocytochemistry The midbrain, C3, and T1 segments of the spinal cord were removed from each animal and postfixed in the same fixative for 1 to 2 h and 14-µm serial sections were cut on a cryostat. Sections were incubated in 1% Bovine Serum Albumin (BSA, Amersham, UK) and 0.3% Triton-X 100 (Sigma) for 1 h at room temperature, followed by one of the primary antisera (cf. Table 1) overnight (4°C). For the ABC method, the sections were incubated in biotinylated secondary antisera (horse anti-mouse or goat anti-rabbit) for 1 h (1:200, Vector), washed, and incubated with avidin–biotin complex for 1 h at room temperature (1:50, ABC, Eukitt, Vector). The immunoreactivity (IR) was visualized by incubating the sections in diaminobenzidine (Sigma, 50 mg/100 ml, 0.1 M Tris–HCl buffer, pH 7.4). The sections were dehydrated and mounted in a nonaqueous medium (Eukitt, D. Kinder GmbH, Germany). Indirect immunofluorescence was used for double labeling to define the cellular

identity of clusterin expressing profiles. Anti-clusterin was incubated with either antibodies to glial fibrillary acidic protein (GFAP; astrocytes), ED1 (microglia/ macrophages), anti-transferrin (oligodendrocytes), or anti-PGP9.5 (axons). Two kinds of control incubations were made (1) omission of primary antibodies, and (2) preabsorption with cow GFAP (0.01 mg/ml). In Situ Hybridization In nine rats, the left rubrospinal tract was transected at the level of the C3 segment of the spinal cord as described above. These rats were sacrificed at 7, 14, and 28 days (three in each group) after injury, respectively. The midbrain, C3, and T1 segments of the spinal cord were removed and frozen on dry ice. Fourteen-micrometer sections were cut on a cryostat and mounted on sterilized slides coated with poly-l-lysine (50 µl/ml; Sigma). All sections were fixed in sterile 4% (w/v) paraformaldehyde in 0.1 M phosphate buffer saline (PBS, pH 7.4, 4°C) for 30 min followed by a wash in PBS. Antisense oligonucleotide probes (48 mer) complementary to nucleotides 40–87 or 323–370 of rat clusterin (6) were synthesized (Scandinavian Gene Synthesis AB, Sweden). A 48-mer sense probe described previously was used as control (27). Generation of the probe and hybridization of tissue sections were done as described previously (27). Briefly, sections were incubated with oligonucleotides labeled at the 38 end with (alpha-35S)dATP overnight at 42°C in a sealed chamber. Following rinsing, the sections were dehydrated and air-dried prior to mounting in an X-ray cassette together with Beta-max Hyperfilm (Amersham) for 1 to 2 weeks, whereafter the sections were dipped in Kodak NTB-2 photoemulsion diluted 1:1 in water. The sections were exposed for 2 weeks at 4°C, developed in Kodak D19, and counterstained with cresyl violet.

TABLE 1 Antibodies and Probes Used in the Present Study Antigen/mRNA Rat CR3 Rat phagosome Membrane protein Cow GFAPa Bovine GFAPa Rat transferrin Human PGP 9.5a Rat clusterin Rat clusterin Clusterin mRNA a

Antibody/probe

Species

Source

Titer

OX42 Monoclonal ED1 Monoclonal

Mouse Mouse

Sera-lab, UK Serotec, UK

1:800 1:500

Polyclonal Monoclonal Polyclonal Polyclonal Polyclonal Monoclonal 48-mer oligonucleotide

Rabbit Mouse Rabbit Rabbit Rabbit Mouse

Dako, Sweden Boehringer-Mannheim, Germany Sigma, USA UltraClone Ltd, UK Gift from Prof. Griswold KeLab, Sweden Scand. Gene Synthesis AB

1:1000 1:200 1:2000 1:1000 1:200 1:100 5 ⫻ 10 cpm/ml

Known to cross-react with rat equivalent.

CLUSTERIN IN RUBROSPINAL SYSTEM

RESULTS

Red Nucleus Low levels of clusterin-IR were observed in rubral motor neurons as well as in surrounding nonneuronal cells, presumably astrocytes, in nonoperated animals, and on the uninjured side of experimental animals (Fig. 1A). No obvious differences were found between the operated and nonoperated sides at 1 and 2 days after the lesion. However, clusterin-IR increased markedly in injured rubral motor neurons at 4 (Fig. 1B), 7 (Fig.

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1C), 14, and 28 (Fig. 1D) days postlesion. The operated/ nonoperated difference was less prominent 3 months following injury. Labeling with the two antisense clusterin mRNA probes showed a marked increase in the injured red nucleus at 7 (Fig. 2), 14, and 28 days. The Lesion Area in the C3 Spinal Cord Segment Small clusterin-IR profiles colocalized with GFAP-IR in the intact C3 segment of the spinal cord. After injury, clusterin-IR appeared in two distinctly different forms in the injured lateral funiculus. Large, globular clus-

FIG. 1. Clusterin immunoreactivity (IR; A–D) in the red nucleus after unilateral lateral funiculus lesion at C3. (A) Low levels of clusterin-IR are observed in rubral neurons (arrows) as well as in surrounding nonneuronal cells (arrowheads) in intact animals as well as on the nonoperated side (A) of operated animals. Clusterin-IR is increased in neurons and nonneuronal cells on the operated side 4 days (B), 7 days (C), and 28 days (D) postlesion. Bar, 50 µm.

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expression appeared as smaller, granular profiles (cf. Figs. 4B–4D). These first appeared 14 days after injury and continuously increased in number throughout the entire postoperative survival period, 3 months. Some of this labeling colocalized with an antibody to transferrin, a marker for oligodendrocytes (Fig. 4D). Labeling with the antisense clusterin mRNA probe showed a marked increase from 7 to 28 days postlesion (Figs. 5A and 5B). Wallerian Degeneration in the Lateral Funiculus of the T1 Segment of the Spinal Cord

FIG. 2. Clusterin mRNA in the red nucleus is increased on the operated side (left) compared to the nonoperated side. Seven days postoperative survival. *Cerebral aqueduct. Bar, 1 mm.

terin-IR profiles were observed at 1 day postlesion. Similar profiles were heavily labeled at 2 days postoperatively (Figs. 3, 4A, and 4B), to a lesser extent at 4 days and almost absent at 7 days. This labeling coexisted with an antibody to the axon-specific protein PGP9.5 (Fig. 4A). The other major type of clusterin

Clusterin expression appeared as strongly labeled granular profiles from 14 days to 3 months (Figs. 4C and 4D). These profiles could in many cases also be labeled with anti-transferrin (Fig. 4D). There was a marked increase in labeling with the antisense clusterin mRNA probe 7 to 28 days after injury (Figs. 5C–5E). Controls The control experiments involving omission of primary antibody in the immunohistochemistry protocol resulted in absence of labeling. Hybridization with the sense probe produced only background level of radioactivity (Fig. 5F).

FIG. 3. Clusterin-IR at the lesion site in C3 2 days postlesion. The IR appears to be strictly confined to the actual lesion site in the lateral funiculus. Bar, 0.66 µm.

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FIG. 4. Clusterin-IR at the lesion site in C3 2 days (A, B) and in T1 1 month (C, D) after injury. (A) Numerous clusterin-IR globules (green), which also stain with anti-PGP9.5 (red). (B) Red clusterin-IR globules, which are not double labeled with anti-GFAP (green). However, there is some colocalization of clusterin- and GFAP-IR in smaller profiles (arrows). (C) Clusterin (red)- and ED1 (green)-IR with no overlap between these two markers. (D) Shows that there is some coexistence (arrows) between clusterin (green) and the oligodendrocyte marker transferrin (red). Bar, 100 µm.

DISCUSSION

Clusterin (apolipoprotein J) immunoreactivity and labeling for clusterin mRNA were increased in axotomized red nucleus neurons in a temporal pattern with marked similarities to the response in peripherally injured motoneurons (20, 28). In contrast to the situa-

tion after peripheral nerve injury, however, the upregulation in red nucleus neurons took place without activation of complement (Liu, Svensson, and Aldskogius, unpublished observations). This finding gives additional support to the previous suggestion that clusterin upregulation in the CNS is independent of complement activation (20). Clusterin may rather be an inherent

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FIG. 5. Labeling with an antisense clusterin mRNA probe in the lateral funiculus at the lesion level C3 (A, B) and thoracic level T1 (C–E) of the spinal cord. Clusterin mRNA labeling is increased in the lesion area (A, seven days; B, 28 days) as well as at the T1 level (C, seven days; D, 14 days; E, 28 days) after injury of the ipsilateral lateral funiculus at C3. There is no labeling with a sense probe (F). Operated side to the right. Bar, 0.8 mm.

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part of the nerve cell body response to axotomy, subserving, e.g., lipid transport or mediating cell–cell interactions, which occur in all neuronal systems following axon injury (16). At the lesion site in C3 there was an increased expression of clusterin accompanying the gradual upregulation of GFAP, typical for the ‘‘scar’’ formed after a direct CNS lesion. In addition, an intense clusterin expression occurred transiently also in the expanded injured axons, as shown by colocalization of clusterinand PGP 9.5-IR, an ubiquitin carboxyl-terminal hydrolase (33), selectively located in axons (29). There was also a markedly increased labeling of clusterin mRNA in this area, but it was not possible to determine whether specific labeling was present over these expanded axons. Presumably, the axonal clusterin was either taken up from the environment, where it might have been released by astrocytes, or transported anterogradely from supraspinal neuronal perikarya (9, 32). The stumps of injured central axons rapidly undergo expansion and accumulate a variety of subcellular components, such as vesicles, filaments, mitochondria, and lysosome-associated structures (18). However, the elucidation of the precise relationship between clusterin expression and the subcellular adaptations in injured CNS axons requires ultrastructural analysis of immunostained material. In the zone of Wallerian degeneration in the lateral funiculus, clusterin displays an intriguing pattern of expression with the appearance of intensely clusterinpositive profiles, which largely colocalized with the oligodendrocytic marker, transferrin (18). We have previously suggested that this clusterin expression is associated with degeneration of oligodendrocytes as a result of loss of all internodal segments associated with these particular oligodendrocytes (20). Evidence for oligodendrocytic degeneration has been demonstrated previously during whiter matter Wallerian degeneration (1, 11, 12). However, recent studies indicate that clusterin may be expressed in surviving rather than dying cells (13, 17), suggesting that the clusterinpositive oligodendrocytes are actually recovering rather than degenerating from the partial loss of their cytoplasm. Taken together, the results of the present study demonstrate a prominent upregulation of clusterin expression at virtually all levels of the injured rubrospinal system. Red nucleus neurons showed a marked increase from their low baseline levels, replicating what we previously found in axotomized motoneurons (28). An intriguing finding, however, was the very strong and transient induction of clusterin in the proximal stump of the expanded axons at the lesion site. This expression may reflect a significant role of clusterin in the injury-induced reorganization of the intracellular structure and molecular pathways of axons

undergoing abortive or effective sprouting. Further caudally, nonneuronal cells, presumably oligodendrocytes in the degenerating white matter, showed a strong and persistent upregulation of clusterin (see also Ref. 20), which may also be associated with the adaptation of these cells to the loss of all or most of their internodes. Clusterin upregulation has been demonstrated in a variety of pathological tissues, such as Alzheimer (14), myocardial infarction (26), and cellular oxidative stress (25). Circumstantial evidence suggest that clusterin promotes repair processes in these conditions. Clusterin also appears to provide increased resistance to tumor necrosis alpha mediated cell death (15). In the light of these data, it is possible that clusterin upregulation is one of the components in the axon reaction which protects axotomized neurons from dying, i.e., an intrinsic neuroprotective compound. ACKNOWLEDGMENTS The technical assistance by Mss. Ingmarie Olsson, Marianne Ljungkvist, and Britt Meijer are gratefully acknowledged. We are grateful to Dr. M. Griswold for the generous gift of antibodies to clusterin. Supported by grants from the Swedish Medical Research Council project 5420, Swedish Medical Association, the Medical Faculty Uppsala University, Magn. Bergvalls Foundation, and the Alzheimer Society.

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