J. Anat.

(2004) 204, pp311

Blackwell Publishing, Ltd.

REVIEW

Strategies for identifying genes that play a role in spinal cord regeneration
M. Wintzer,* M. Mladinic, D. Lazarevic, C. Casseler, A. Cattaneo and J. Nicholls
Sissa, Trieste, Italy

Abstract
A search for genes that promote or block CNS regeneration requires numerous approaches; for example, tests can be made on individual candidate molecules. Here, however, we describe methods for comprehensive identification of genes up- and down-regulated in neurons that can and cannot regenerate after injury. One problem concerns identification of low-abundance genes out of the 30 000 or so genes expressed by neurons. Another difficulty is knowing whether a single gene or multiple genes are necessary. When microchips and subtractive differential display are used to identify genes turned on or off, the numbers are still too great to test which molecules are actually important for regeneration. Candidates are genes coding for trophic, inhibitory, receptor and extracellular matrix molecules, as well as unknown genes. A preparation useful for narrowing the search is the neonatal opossum. The spinal cord and optic nerve can regenerate after injury at 9 days but cannot at 12 days after birth. This narrow window allows genes responsible for the turning off of regeneration to be identified. As a next step, sites at which they are expressed (forebrain, midbrain, spinal cord, neurons or glia, intracellular or extracellular) must be determined. An essential step is to characterize proteins, their levels of expression, and their importance for regeneration. Comprehensive searches for molecular mechanisms represent a lengthy series of experiments that could help in devising strategies for repairing injured spinal cord. Key words CNS lesions; CNS repair; molecular analysis.

Introduction
The adult CNS of birds and mammals has little or no capacity for functionally useful regeneration. Although it has been known since the time of Ramon y Cajal that some damaged CNS neurons can send out sprouts (Ramon y Cajal, 1928), they do not grow through lesions or make connections. By contrast, peripheral nerves regenerate successfully after injury, as do connections in the CNS of fish, amphibians, reptiles and invertebrates. To try to repair the injured spinal cord has become a challenging problem in neuroscience. Strategies employed to enhance regeneration in the mammalian

CNS include the neutralization of potential growth inhibitory molecules (Caroni & Schwab, 1988; Sims & Gilmore, 1994; Dyer et al. 1998), the transplantation of cells or tissue that support axonal elongation (BernsteinGoral & Bregman, 1997; McDonald et al. 1999; RamonCueto et al. 2000; Ito et al. 2001), and the delivery of factors that are known to promote axonal growth, such as neurotrophic factors (Xu et al. 1995; Nakahara et al. 1996; Zhang et al. 1998). Those approaches are showing promising results, although the growth and functional recovery that can be observed are often limited. Knowledge of the molecules that are involved and the way they interact to promote or prevent regeneration is far from complete.

Correspondence Dr J. Nicholls, Sissa, Via Beirut 2, Trieste 34014, Italy. E: nicholls@sissa.it *Present address: Department of Neuroscience, Karolinska Institute, Stockholm 171 77, Sweden. Accepted for publication 28 October 2003 Anatomical Society of Great Britain and Ireland 2004

Two different strategies

Sustained growth of axons involves participation by the neuronal cell body. Axon regeneration might require that injured neurons up-regulate a specific set of

4 Genes for CNS regeneration, M. Wintzer et al.

growth-associated genes. Some of the genes that are up-regulated or constitutively expressed in association with axonal growth have been identified. Their products include: (1) transcription factors, such as c-jun, which mediates subsequent gene expression (Jenkins et al. 1993; Herdegen et al. 1997); (2) cytoskeletal proteins involved in axonal extension, such as T1-tubulin (Miller et al. 1989; Fernandes et al. 1999); (3) cell adhesion molecules, such as N-CAM involved in growth cone guidance (Jung et al. 1997); and (4) cytoplasmic growth cone proteins involved in mediating signal transduction, such as GAP-43 (Frey et al. 2000). One widely used strategy consists of testing individual candidates for their potential to enhance fibre outgrowth. Such experiments aim to find the gene that will promote or prevent spinal cord regeneration as a first step toward a cure for injury in patients. Studying genes one by one can provide interesting cues as to their role in the process of regeneration. Because regenerating growth cones make complex interactions with their local environment, it seems unlikely that a single gene is responsible for promoting or preventing growth. For example, regeneration of spinal-cord axons of dorsal root ganglia occurs after transgenic expression of GAP-43 and CAP-23 in combination, but not when either one of these genes was expressed on its own (Bomze et al. 2001). Nevertheless, the existence of a Master gene switching on a specific genetic program cannot be excluded. A more ambitious approach is to make a survey of all the genes that are expressed in regenerating and nonregenerating tissue. Differentially expressed genes can then be fished out by comparing various samples, such as adult and immature spinal cord, PNS and CNS, injured and uninjured tissue. Such comparisons provide a global view of the genes that might enhance or inhibit neurite outgrowth.

expressed by a single cell is made up of 20 00030 000 distinct mRNAs, with approximately 99% being rare (Hahn & Laird, 1971; Grouse et al. 1972; Hahn et al. 1978; Croizat et al. 1979). These 99% represent less than 30% of the total mRNA mass. Most of the mRNA species that are isolated from a brain or a spinal-cord sample are therefore medium- to high-abundance transcripts. Their corresponding genes are likely to encode proteins necessary for basic biochemical pathways, housekeeping proteins that are common to every cell in the body. Thus, it is reasonable to suppose that genes involved in the process of regeneration are of low abundance. A further dilution of the genes of interest is likely to occur when experiments are performed in immature preparations (see below). Tremendous changes in gene expression occur in the developing CNS. They are responsible for the myriad processes that may again be different from regeneration.

Methods for studying differential gene expression

A number of techniques have been developed in recent years for the identification of differentially expressed genes that could be involved in CNS regeneration. In the following we describe briefly the underlying principles for techniques such as: differential message display, serial analysis of gene expression (SAGE), large-scale generation of expressed sequence tags (ESTs) from cDNA libraries, cDNA microarray screening, and various subtractive hybridization strategies. Differential display (DD) has been widely used to detect and isolate genes that respond to growth factors, developmentally regulated genes and genes whose expression correlates with certain diseases (Pazman et al. 2000; Fu, 2002). The general strategy is to amplify partial cDNA sequences from subsets of mRNA by reverse transcription and PCR, and then to display these short cDNA fragments on a sequencing gel to obtain an mRNA fingerprint. Differential display is one of the most suitable methods for tracking novel genes (Gratsch, 2002), although the generation of false positives remains one of its major drawbacks (Broude, 2002). Su et al. (1997) used this method to demonstrate the up-regulation of axonal transport molecules during motor nerve regeneration in the mouse. Large-scale sequencing of expressed sequence tags (ESTs) is another approach for studying mRNA
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Rarity of genes involved in regeneration

Although it may seem to be a rather simple procedure, in practice tracking those genes that play a part in CNS regeneration is like looking for a needle in a haystack; indeed it is worse because neither the shape of the needles or their numbers are known. The human brain and spinal cord have the greatest complexity of gene expression of any region of the body, reflecting the diverse functions of neurons and glia. Saturation and kinetic studies indicate that the mRNA population

Genes for CNS regeneration, M. Wintzer et al. 5

expression (Okubo et al. 1992). ESTs are randomly selected clones sequenced from cDNA libraries. They are short, partial DNA sequences, as opposed to full-length clones. The first collection of ESTs was initiated in 1991 as part of the human genome project (Adams et al. 1991). Each cDNA library is constructed from total RNA or poly(A)+ RNA derived from a specific tissue or cell, and thus the library represents genes expressed in the original cellular population. Overlapping EST sequences can be assembled, allowing the complete mRNA sequence to be determined. The analysis of EST data revealed answers to fundamental questions such as which genes are most abundantly expressed (e.g. -actin, - and -tubulin) and the amount of regional variation of expression within brain regions. There are currently over 1.5 million human EST sequences deposited in the publicly available database of ESTs provided by the National Center for Biotechnology Information (NCBI). Serial analysis of gene expression (SAGE) is a sequencebased strategy that allows the simultaneous analysis of a large number of transcripts (Velculescu et al. 1995; Scott & Chrast, 2001; Yamamoto et al. 2001). Typically, cDNA fragments are generated with short tags (usually from 8 to 11 base pairs) at the 3-end of each transcript, concatenated and sequenced. Analysis of tags allows the cataloging of thousands of genes expressed from a tissue source, including a quantitative estimate of gene expression. However, this straightforward technique requires high-throughput sequencing facilities. SAGE has been successfully applied to analyse changes in gene expression in hippocampus during the early epileptic phase in the rat (Hendriksen et al. 2001). Reports of its use to isolate genes differentially expressed after spinal cord injury are few to date. DNA microarrays represent a highly effective tool for studying gene expression profiles and genome composition. The principle is to immobilize hundreds or thousands of cDNAs or nucleotides corresponding to ESTs or known genes on a solid support, such as nylon membrane or glass microscope slide (Lockhart et al. 1996; Freed & Vawter, 2001; Lobenhofer et al. 2001). To determine the difference in gene expression, labelled cDNAs or oligonucleotides are hybridized to the array. After hybridization and imaging, the pattern of gene expression is quantified. The principal advantages of this approach are the immediate interpretation of the results and the possibility of clustering genes into temporal and spatial expression patterns. One drawback is
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the difficulty in calibrating and interpreting hybridization signals of weakly expressed genes. Microarray analysis after spinal cord injury (Carmel et al. 2001; Fan et al. 2001) or brain injury (Matzilevich et al. 2002) has proven useful to describe global patterns of gene expression and to cluster genes into temporal and spatial expression groups. Subtractive hybridization (SH) is a valuable technique for the isolation of differentially expressed genes. Although there are numerous protocols for SH, the principles remain the same (Diatchenko et al. 1996). Two cDNA populations are generated from two different RNA sources. For example, in suppressive PCRbased subtractive hybridization, adaptors are ligated to a tester pool of cDNA. A second pool (driver cDNA) is added in excess to hybridize, and only genes that are up-regulated in the tester pool can then be selectively amplified by PCR. This technique requires only small amounts of RNA, and is well suited for identifying rare transcripts and novel genes. Subtractive hybridization is one of the most commonly used methods for identifying differentially expressed mRNAs and has been successfully applied in studies of development, cancer, growth-factor-stimulated cells (Feng & Liau, 1993; Cho et al. 1998; Davidson & Swalla, 2001; Shridhar et al. 2002). Few laboratories have used it to analyse genes expressed after injury to the CNS. An exception is the series of subtractions made from regenerating identified nerve cells in the leech. Blackshaw and colleagues (Korneev et al. 1997) have made a subtractive library from regenerating Retzius cells of the leech, which revealed novel sequences as well as genes such as alpha-tubulin, synapsin and calmodulin, up-regulated in other species, during nerve regeneration. A remarkable feature of this study was that the differences in expression were analysed in individual identified neurons from lesioned and unlesioned nerve cords. A common feature of all methods for analysis of differences in gene expression is the generation of large amounts of data (often hundreds or thousands of genes). The over- or under-expressed sequences are then compared with sequences from public databases. Genome sequencing programmes carried out for several organisms including human and mouse have provided an immense amount of sequence information in databanks. Techniques are now available for studying the expression profiles of previously insufficiently characterized genes.

6 Genes for CNS regeneration, M. Wintzer et al.

Methods for selecting genes that are relevant for regeneration

A major difficulty arises once the genes that are differentially expressed have been identified. Which of the genes that stand out are actually important and how can one distinguish genes that play a part in regeneration from unrelated genes that have slipped through the subtraction procedure? With several hundred candidates, the number to be tested experimentally must be narrowed down. Procedures for focusing on relevant genes include multiple screenings and hybridizations, or repeated experiments under various conditions and with different tissue samples. For example, a specific sequence may become significant if it appears in screens made of an invertebrate and a vertebrate, or in regenerating spinal cord and optic nerve. An interesting approach for focusing on putative candidates is to combine some of the techniques described above. For example, the hundreds of clones obtained after subtractive hybridization can be spotted onto microchips and can then be analysed using microarray procedures. This strategy has proved to be useful in identifying genes in the nervous system (Colantuoni et al. 2001; Kornblum & Geschwind, 2001; Dougherty & Geschwind, 2002). We describe in greater detail methods for narrowing the search in a later section devoted to work on the neonatal opossum.

comparing patterns obtained from two-dimensional (2D) protein gels. One strength of 2D gel electrophoresis is its ability to measure quantitatively several thousand proteins in a single sample, with subsequent identification by mass spectrometry (reviewed by Fey & Larsen, 2001; Lilley et al. 2002). In this way one can compare quantities of proteins in related samples, such as injured and uninjured tissue. A major difficulty that remains is the detection of low-abundance proteins by 2D gels, until the resolution and sensitivity of the method are improved. In the near future one can expect to have available protein microarrays to measure the properties of thousands of proteins simultaneously. Proteinprotein interactions analysed with fluorescent probes will be analogous to DNA microarrays. An important application of this powerful technique will be to profile the proteins in cells under different conditions, as for example in regenerating and non-regenerating neurons.

Localization in CNS of candidate genes relevant for regeneration

Analysis of differential gene (or protein) expression is a first step in the search for genes that play a part in promoting or blocking regeneration. Identification of putative candidates does not, however, indicate at what sites they are expressed in the original tissue. In situ hybridization is important for answering fundamental questions of distribution: Does the gene show a widespread expression? If not, in which areas of the CNS can the product be found? Is it expressed by neurons or by glial cells and in which structures (membranes, extracellular space, internal organelles) does the pattern of expression change after an injury? The procedure for in situ hybridization involves screening sections or the entire CNS by labelled probes complementary to the mRNA of interest. A risk is that one might discard genes that at first seem uninteresting. This is exemplified by Nogo, a member of the reticular family of transmembrane proteins. Nogo mRNA is widely expressed in fetal, developing and adult nervous system of rat and human (Josephson et al. 2001), and its levels of expression are not significantly changed after lesions to the cortex or spinal cord (Huber et al. 2002). From those characteristics one might exclude Nogo from a list of interesting candidates, and yet it is known to be an inhibitor of regeneration in the CNS (Huber & Schwab, 2000; Woolf, 2003).
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Possible discrepancies between RNA and protein levels

Differences in abundance of a specific mRNA between two samples do not necessarily reflect equivalent quantitative differences in protein levels. Although it is generally true that the level of a protein follows a change of its encoding mRNA, the extent and kinetics are often unpredictable. The activity of the proteins encoded by mRNAs is regulated at several levels beyond their mRNA or protein expression by their subcellular localization and by the extent to which they are post-translationally modified. Neither of these parameters is revealed by measurements of mRNA abundance. Twiss et al. (2000) showed that under some circumstances neurons can be primed to rapidly regenerate injured processes independently of new gene expression, translating already existing messengers. Such regulation at the level of translation would not be detected in assays of differential gene expression. Large-scale analysis similar to that made at the mRNA level can be undertaken directly with proteins, by

Genes for CNS regeneration, M. Wintzer et al. 7

Assessment of the functional role of unknown genes and proteins

Many genes that are detected by screening of regenerating and non-regenerating preparations have no known function. The analysis of the full-length sequence of such candidates with bioinformatic techniques is a first step towards understanding what role they might play in regeneration. Valuable information can be obtained from the gene sequence that is responsible for directing the encoded protein to its cellular or extracellular location (e.g. nucleus, organelles, plasma membrane). Translation of the DNA sequence of a gene into the amino acid sequence of the encoded protein can provide clues to its structure and function. To gain further insight into the functional role of a candidate, numerous tests can be performed on cells in culture. One approach is to measure effects of a candidate gene on growth, either by over-expressing genes that promote regeneration (gain of function) or by blocking expression of genes that inhibit regeneration (loss of function). For example, immortalized cell lines such as PC12 cells can be engineered to produce a protein by introducing its coding gene into cells in culture. One method for transfecting cells is to use a gene gun, which bombards them with small gold particles coated with the DNA of interest (McAllister, 2000; Sato et al. 2000). Alternatively, genes can be delivered by trapping the DNA into liposomes, which are taken up by the cell (Kofler et al. 1998), or by precipitating DNA with calcium phosphate. Proteins can be studied in greater detail by introducing the recombinant gene into cells together with green fluorescent protein (GFP) (reviewed by Van Roessel & Brand, 2002). The label makes it possible to follow the gene fusion product in the living cell. Specific localization and co-localization with other known proteins can provide information about the role of the protein in specific pathways of neurons and glial cells. For example, a recombinant protein expressed in a defined cell line may promote cell cycle arrest or loss of motility. Methods by which to block the expression of a gene include the introduction of antisense oligonucleotides that hybridize with the RNA in the cell and hinder their translation into proteins (reviewed by Lebedeva et al. 2000; Sazani et al. 2002). Whenever they are available, antibodies that specifically block the action of an identified protein can provide clear evidence for function. A recent effective method for silencing the expression
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of a target gene is to introduce vectors that express small interfering RNAs (known as siRNAs). siRNAs are short (about 20 nucleotides), double-stranded molecules. Once inside the cell the siRNA strands unwind and become associated with complementary RNA molecules. This provokes the cleavage and destruction of target mRNA and inhibition of protein synthesis by the cell (Scherr et al. 2003; Sorensen et al. 2003). A further step is modification of specific sequences to identify functional domains of the protein. For example, deletions or mutations can define regions of genes important for nuclear importexport or degradation. A two-hybrid system in yeast can also be used to provide information about function. By this method it is possible to investigate proteinprotein interactions and to identify molecular partners (reviewed by Causier & Davies, 2002). The two-hybrid system exploits properties of transcription factors that have two separate functional domains. One is a DNA binding domain that binds to the DNA of the promoter and the other an activation domain that binds to the transcription apparatus. Each separate domain can be fused as a hybrid to a second protein without changing the basic properties of the transcription factor. When an interaction occurs, the DNA binding domain and the activation domain of the transcription factor come close together and can activate transcription of a reporter gene encoding for a selection marker. In this way the protein of interest can be tested for interactions with several possible partners. Experiments performed on cells in vitro are indispensable to assay the role of candidate molecules and their possible involvement in regeneration. Observation of fibre outgrowth in vitro does not, however, imply that similar events will occur in the animal, let alone that growth will lead to functional recovery. Although most tests for regeneration are made in vivo in rats and mice, experiments performed in Monodelphis have demonstrated the usefulness of this mammal for studying regeneration.

The opossum as a preparation for molecular studies of regeneration

Properties of neonatal opossum CNS In the following sections we show how the approaches described above have been applied to the CNS of the newborn opossum, Monodelphis domestica. The emphasis

8 Genes for CNS regeneration, M. Wintzer et al.

is on work in progress with certain clear advances but no defined end-point in sight. In newborn opossum pups the immature CNS is so small that it can be removed in its entirety and maintained in tissue culture for up to 2 weeks (Stewart et al. 1991; Treherne et al. 1992; Mllgard et al. 1994). During that time in culture, cell division goes on, the isolated CNS maintains reflex responses to electrical stimulation, and the fine structure of the nervous system is preserved (Nicholls et al. 1990; Eugenin & Nicholls, 1997). Axons regenerate rapidly and reliably through spinal cord lesions (Nicholls et al. 1994; Saunders et al. 1995; reviewed by Nicholls & Saunders, 1996). Amino acids, transmitters and larger molecules such as antibodies rapidly penetrate the isolated tissue from the bathing fluid (Zou et al. 1991; Varga et al. 1995). These properties facilitate detailed analysis of mechanisms that promote or block regeneration. After a lesion to the spinal cord in the immature animal, regeneration is so complete that functions such as coordinated walking and swimming are virtually normal (Saunders et al. 1998). In adult opossums, as in other mammals, the CNS does not regenerate. The transition point has been shown to occur in the early days of postnatal life. At 9 days of age regeneration occurs reliably. However, it is no longer possible in cervical spinal cord segments of animals aged 12 days or more. Owing to a rostro-caudal gradient in development, the less mature lumbar segments still show regeneration in animals up to 17 days. The narrow window of time during which regeneration stops being possible is particularly useful for studies of differences in gene expression. The space of time between 9 and 12 days after birth is long enough for expression of differences in genes of interest, yet short enough to avoid the over-representation of genes related to development. The presence of both mature (cervical) and immature (lumbar) parts in the spinal cord at 12 days enables one to perform additional comparisons between regenerating and nonregenerating tissue of a single animal. In this way differences in gene expression related to regeneration can be analysed at different times (9 days vs. 12 days) and in different places (cervical vs. lumbar at 12 days).

Table 1. Subtractions to reveal genes that are differentially expressed in opossum spinal cord regions that do and do not regenerate

opossum at different ages and in different parts of the spinal cord. Both forward and backward subtractions were done between cervical spinal cord at 9 days (can regenerate) and at 12 days (cannot regenerate). Other subtractions were done between 12-day lumbar (can regenerate) and cervical parts at the same age (cannot regenerate). Table 1 provides a summary of the various subtractions. cDNAs from the two first subtractions (C9C12 and C12C9) were cloned in E. coli, resulting in about 3000 recombinant clones. A few clones were chosen at random from those libraries and sequenced. About half of these represented novel genes whose function is as yet unknown. Sequencing also revealed numerous interesting genes that could be involved in regeneration. They coded for transcription factors, receptors, signal transducing molecules, structural proteins and extracellular matrix proteins, among others. Other genes, as expected, were responsible for housekeeping functions (for example, enzymes and structural proteins).

Narrowing down the search To separate non-relevant genes from those involved in regeneration we designed experiments based on crosshybridizations between different subtractions. The aim of those experiments was to find common clones in the various subtractions. All clones from a pair of subtractive libraries were arrayed onto nylon membranes and hybridized with radioactively labelled probes derived
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Analysis of genes expressed in regenerating and nonregenerating spinal cords We conducted subtractive hybridization experiments to detect genes that are up- or down-regulated in the

Genes for CNS regeneration, M. Wintzer et al. 9

from the other subtractions. This process reduced the number of potentially involved genes by a factor of about 10. Those remaining included (among others): reelin, laminin receptor, TATA-box binding protein, MAP1b, and these were all more abundant at 9 days. At 12 days, genes selectively expressed included PAX-6, semaphorin receptor and GABA(A) receptor-associated protein. Novel genes were represented in both categories. In contrast with other studies focusing on one gene or protein, this represents an approach based on broad screenings that can lead to unbiased identification of potentially interesting genes in the spinal cord at different ages. Comparison with other databases can help in the functional identification of relevant molecules. A striking feature is that opossum gene sequences show high similarities to those of mouse, rat or human deposited in databanks. EST and microarrays are now available for another marsupial, the Tammar wallaby. What remains is to determine for each gene: the location of expression, its abundance, the level of protein and its effect on the regeneration of intact spinal cord axons after injury.

Acknowledgements
We thank Dr Marco Stebel and Mr Salvatore Guarino for their diligent and skilled care of the animal colony.

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Concluding remarks
Studies of differential gene expression in injured spinal cord provide a picture of molecular events associated with processes of regeneration. Many steps, however, are necessary before it can be known whether this approach will lead to cures for patients with spinal cord lesions. Overoptimistic claims of cures that are just around the corner can have devastating consequences for patients with spinal cord injuries (Pearson, 2003). Animal tests, and extensive clinical trials for efficacy and side-effects involve expenditure of immense sums of money and large numbers of skilled manpower hours. It seems possible that treatments might arise sooner from the approach not discussed in detail here, namely the testing of likely molecules without any comprehensive screen. Although the large-scale screening described here may well produce no miracle gene, what it will do is increase basic knowledge. Our underlying assumption is that, just as it is easier to repair a watch once one knows the way it works, analysis of mechanisms responsible for failure of regeneration in adult mammalian spinal cord will be facilitated by an understanding of molecular mechanisms that promote and inhibit neuron outgrowth.
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