Role of L1 in Neural Development: What the Knockouts Tell Us

MCN

Molecular and Cellular Neuroscience 12, 48–55 (1998) Article No. CN980702

REVIEW Role of L1 in Neural Development: What the Knockouts Tell Us Hiroyuki Kamiguchi,* Mary Louise Hlavin,† and Vance Lemmon*,1 *Department of Neurosciences and †Department of Neurological Surgery, Case Western Reserve University, Cleveland, Ohio 44106

Mutations in the cell adhesion molecule L1 cause severe developmental anomalies in the human nervous system. Recent descriptions of L1 gene knock-out mice from three research groups demonstrate that these mice are strikingly similar to humans with mutations in the L1 gene. In both humans and mice there are defects in the development of the corticospinal tract and cerebellar vermis, hydrocephalus, and impaired learning. The production of a viable animal model for X-linked hydrocephalus suggests that unanswerable questions posed by the human disease will finally be approachable using modern experimental methods.

INTRODUCTION It is not often that the interests of pediatric neurologists, clinical geneticists, and developmental neurobiologists converge. Nonetheless it does happen occasionally, to the mutual benefit of all. The recent description of L1 knock-out mice (Cohen et al., 1997; Dahme et al., 1997; Fransen et al., 1998a) is an exciting focal point where these diverse groups are converging to try to understand how mutations in a cell adhesion molecule can lead to drastic disruptions in normal brain development. During nervous system development, migrating neurons and growing axons search for their specific destinations by interacting with their microenvironment. Proper pathways are selected through a variety of guidance cues present in the extracellular matrix (ECM) or on glial cells or preexisting axons. Cell–cell and cell–ECM interactions are mediated by a variety of factors which 1 To whom correspondence should be addressed. Fax: (216) 368– 4650. E-mail: [email protected].

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include cell adhesion molecules (CAMs), ECM molecules, and diffusible factors. CAMs can act both as ligands and as cell surface receptors, thus regulating intracellular events. Neural CAMs are divided into three major classes: cadherins, integrins, and members of the immunoglobulin (Ig) superfamily. A subfamily of the third category is the L1 subfamily whose members have six Ig-like domains, at least four fibronectin type III repeats, and highly conserved cytoplasmic domains (Bru¨mmendorf and Rathjen, 1994). L1 is predominantly expressed in the developing central and peripheral nervous systems (CNS and PNS). Two alternatively spliced forms of L1 are known: a neuronal form and a nonneuronal form expressed by Schwann cells (Miura et al., 1991; Takeda et al., 1996). L1 has been implicated in a variety of important processes including neuronal migration (Lindner et al., 1983), neurite growth and fasciculation (Stallcup and Beasley, 1985; Lagenaur and Lemmon, 1987), myelination (Wood et al., 1990), and synaptic plasticity (Lu¨thl et al., 1994). Homophilic L1–L1 binding between adjacent membranes of neurons is probably the most common mode of action, and the second Ig-like domain in the L1 extracellular domain (L1ED) is sufficient to mediate homophilic adhesion (Zhao and Siu, 1995). However, a variety of heterophilic binding partners for L1 have also been identified (Hortsch, 1996). For example, the RGD (Arg-Gly-Asp) motif in the sixth Ig-like domain binds b1 and b3 integrins (Ruppert et al., 1995; Montgomery et al., 1996; Felding-Habermann et al., 1997) and can promote neurite growth from embryonic chick dorsal root ganglion neurons via an interaction with aVb3 integrin (Yip et al., 1998). It is also clear that L1 functions not only as an adhesive molecule but as a signal-transducing receptor (Schuch et al., 1989; von Bohlen und Halbach et al., 1044-7431/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.

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L1 Mutations in Mice and Humans

1992). In addition to trans-binding, the L1ED interacts in the same membrane (cis interaction) with axonin-1 and probably with the fibroblast growth factor receptor (FGFr), both of which are involved in L1-dependent neurite growth (Buchstaller et al., 1996; Doherty and Walsh, 1996; Saffell et al., 1997). Although deletion of the cytoplasmic domain from L1 does not alter its homophilic adhesion (Wong et al., 1995), it is possible that L1 adhesivity is altered by phosphorylation or interactions with intracellular binding partners (Hortsch et al., 1998). In any case, the L1 cytoplasmic domain (L1CD) plays a significant role in signal transduction and interactions with the cytoskeleton (Davis and Bennett, 1994; Wong et al., 1996; Dahlin-Huppe et al., 1997). Thus, L1 can influence growth cone and cell migration behavior in response to ligand binding. While it has been known that mutations in the L1 gene are associated with neural abnormalities in humans, it has only recently been shown that similar defects occur in L1 knock-out mice. The loss of L1 function alters both axon guidance and neural morphogenesis but probably by different mechanisms.

HUMAN DISEASES CAUSED BY L1 MUTATIONS In humans, the gene encoding L1 is located near the telomere of the long arm of the X chromosome (Xq28) (Djabali et al., 1990). Mutations in this gene cause a wide spectrum of nervous system malformations. Since first associated with X-linked hydrocephalus (XLH) (Rosenthal et al., 1992; Van Camp et al., 1993), 85 different L1

mutations have been identified (L1CAM Mutation Web Page maintained by Willems and associates (http:// dnalab-www.uia.ac.be/dnalab/l1/)). Due to the interand intrafamilial variability of clinical features, patients with L1 mutations have been diagnosed with XLH, MASA (mental retardation, aphasia, shuffling gait, and adducted thumbs) syndrome, X-linked spastic paraplegia, or X-linked corpus callosum agenesis (Boyd et al., 1993; Jouet et al., 1994). It is now recognized that they represent a single clinical entity (Fransen et al., 1995). Although the linkage between L1 mutations and XLH was well established, it was unclear until recently whether different mutations altered the phenotypic severity. To study genotypic–phenotypic correlation, Yamasaki et al. (1997) categorized L1 mutations into three classes based on how the mutations affected the expression or structure of L1. Class 1 included any mutation that affected only the L1CD. Class 2 consisted of missense point mutations in the L1ED. Class 3 included nonsense or frameshift mutations that produced a premature stop codon in the L1ED. Mutant L1 molecules in this class would not remain integrated in the plasma membrane due to loss of their transmembrane domain. Table 1 summarizes the correlation between the different L1 mutations and the severity of XLH (Yamasaki et al., 1997). These observations were confirmed recently by Fransen et al. (1998b). Patients with the Class 3 mutation almost always have a massive and progressive ventricular enlargement that often requires shunting or drainage of the cerebrospinal fluid (CSF). Most patients with the Class 1 mutation have no hydrocephalus, but about 20% in this class have slight ventricular dilatation that does not

TABLE 1 Nervous System Anomalies Associated with L1 Mutations in Humans and Mice Human L1 mutations Class 1

Class 2

Class 3

Ventricular dilatation

Usually absent

Moderate or severe

Severe

Survival Learning and memory

All live beyond 1 year 25% have IQ ,50

30% die before 1 year 25% have IQ ,50

50% die before 1 year All have IQ ,50

Spastic paraparesis (corticospinal tract hypoplasia) Cerebellar vermis Corpus callosum Adducted thumbs

Almost always present

Almost always present

Almost always present

Hypoplastica Hypoplastic or aplastica Almost always present

Hypoplastica Hypoplastic or aplastica Almost always present

Hypoplastica Hypoplastic or aplastica Almost always present

aIncidence

in each class of mutations has not been determined.

L1 knock-out mice null mutations Degree of ventriculomegaly depends on strain background Significantly worse than wild type Decreased performance on Morris water maze Weakness of hind limbs; impaired decussation and hypoplasia of corticospinal tract Hypoplastic No anomaly detected ?

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FIG. 1. Representative brain CT scans of XLH and MASA syndrome patients showing no (A), moderate (B), or severe hydrocephalus (C). (B) Reprinted, with permission of the publisher, from Wong et al. (1995b) Trends Neurosci. 18:168–172.

require CSF diversion. Examples of severe, moderate, or no hydrocephalus are shown (Fig. 1). Survivorship and mental retardation show similar patterns, where the Class 3 mutations have the worst outcome while the Class 2 mutations cause poorer survival than the Class 1. The Class 2 mutations can be divided into two subclasses based on molecular modeling studies in which amino acid residues in the L1ED were differentiated between key residues responsible for maintaining the conformation of the domains and residues on the surface of the domains (Batemann et al., 1996). As one might expect, mutations in key residues are more likely (78%) to cause severe hydrocephalus than mutations in surface residues (28%), suggesting that the overall conformation of the L1ED is critical for L1 interactions with its binding partners (Kamiguchi et al., 1998a). The high incidence of severe hydrocephalus, severe mental retardation, and mortality with the Class 3 mutations suggests that severe hydrocephalus causes mental retardation and early mortality to some extent. However, additional factors, for example, failure in the establishment of proper neural circuits, must also lead to mental retardation, because some patients without hydrocephalus have severe mental retardation and cognition does not improve after CSF shunting in many patients. A detailed listing of the phenotype–genotype correlation is available (http://neurowww.neur.cwru.edu/faculty/ vpl/ytl.html). In contrast to the strong correlation between the type of L1 mutations and the severity of ventricular enlargement, abnormal development of major axonal tracts, such as the corticospinal tract and corpus callosum, is commonly found in XLH regardless of the type of mutations (Yamasaki et al., 1997). In addition, almost all

patients in each class of mutations show adducted thumbs (Yamasaki et al., 1997), which is caused by loss of innervation of the extensor pollicis muscle (Holtzman et al., 1976). Other neuropathologic findings associated with XLH include fused thalami, flattening of the quadrigeminal plate, and atrophy of the anterior cerebellar vermis (Yamasaki et al., 1995). These defects are associated with midline development of the CNS, perhaps involving abnormal neuronal migration. In some cases, the region around the ventricular proliferative zone of developing brain contains a huge number of cells that have failed to migrate properly (Lemmon et al., unpublished observations). This correlates well with the in vitro findings that neuronal migration from the subependymal zone in songbird brain depends on the heterophilic interaction between neuronal L1 and a radial glial cell receptor (Barami et al., 1994). Interestingly, periventricular heterotopia that consists of well-differentiated cortical neurons filling the adult subependymal zone is linked to markers in distal Xq28 (Eksioglu et al., 1996; Fink et al., 1997), the region where the L1 gene is located. It will be important to determine whether L1 mutations underlie periventricular heterotopia by disrupting neuronal migration. Hirshsprung’s disease, which involves failure of colonic neural migration, is also found in some cases of XLH (Kaplan, 1983).

L1 KNOCK-OUT MICE Currently, three research groups have analyzed the phenotype of two independent L1 knock-out mouse lines (Cohen et al., 1997; Dahme et al., 1997; Fransen et al.,

L1 Mutations in Mice and Humans

1998a). In both lines, the L1 gene was disrupted in the Ig domain coding regions. Therefore, L1 mutations in both mouse lines correspond to the Class 3 mutations in humans. As summarized in Table 1, there are a number of similarities between human mutants and L1 knockout mice. L1 knock-out mice have enlarged lateral ventricles as revealed by high-resolution magnetic resonance imaging (Fransen et al., 1998a). The lateral ventricles are much more enlarged with a strong dependence on strain background (Dahme et al., 1997), suggesting that genetic background modifies the progression of ventricular dilatation. This probably explains the intrafamilial variability and interfamilial variability of the severity of hydrocephalus in humans. L1 knock-out offspring are born at a frequency that is approximately 50% of what would be expected by Mendelian genetics, suggesting intrauterine demise (Cohen et al., 1997; Dahme et al., 1997; Fransen et al., 1998a), and they have a shorter life span than wild-type mice (Dahme, 1997). L1 knock-out mice exhibit decreased performance on Morris water maze but not on Morris water maze with visible platform, indicating deficits in spatial memory (Fransen et al., 1998a). They also have delayed motor response and weakness of the hind-limbs (Dahme et al., 1997), which is reminiscent of the spastic paraplegia in XLH. Histologically, dramatic malformations in the corticospinal tract were found in L1 knock-out mice (Figs. 2A and 2B). In normal mice, the corticospinal tract is formed by descending axons which, in the hindbrain, pass through the ventral pyramids. At the level near the hindbrain–spinal cord boundary, the axons turn dorsally and cross the midline, forming the pyramidal decussation. Then, they extend caudally in the contralateral dorsal column of the spinal cord. However, in L1 knock-out mice, a significant number of axons fail to cross the midline and instead pass ipsilaterally into the dorsal column (Cohen et al., 1997). The mutant mice have reduced size of the ventral pyramid in the hindbrain (Dahme et al., 1997) and no corticospinal axons detected in the spinal cord caudal to the cervical level (Cohen et al., 1997). These observations suggest that some populations of corticospinal neurons that would normally reach the distal spinal cord cannot survive due to impaired connections with their targets. The failure of axons lacking L1 to follow the proper pathway in the pyramidal decussation indicates that L1 plays a critical role in axon guidance rather than axonogenesis in the corticospinal system. Interestingly, no abnormalities of decussation were found in the corpus callosum, optic chiasm, or spinal commissural projection of L1 knockout mice (Cohen et al., 1997). This suggests that the

51 ventral midline at the point of pyramidal decussation expresses a specific guidance cue which functions as a ligand for L1, regulating crossing of axons over the midline. Another important feature in L1 knock-out mice is that of a hypoplastic cerebellar vermis leading to an enlarged fourth ventricle (Figs. 2C and 2D) (Fransen et al., 1998a). This is similar to anomalies found in humans with L1 mutations. Although the molecular cause of this malformation remains unclear, these observations indicate that L1 plays a significant role in vermian development, perhaps by directing neuronal migration. Interestingly, L1 knock-out mice showed abnormal exploratory behavior as characterized by stereotypic peripheral circling reminiscent of that of rats with induced cerebellar lesions (Fransen et al., 1998a). While L1 was implicated in neuronal migration in the cerebellum in vitro (Lindner et al., 1983), there was conflicting evidence as to its importance in cerebellar development (Fishell and Hatten, 1991). Indeed, no abnormal cytoarchitecture of the cerebellar cortex was observed in L1 knock-out mice (Dahme et al., 1997; Fransen et al., 1998a). In the PNS of L1 knock-out mice, nonmyelinating Schwann cells show impaired interactions with axons, forming processes not associated with axons (Dahme et al., 1997). It remains unknown whether this is due to a defect in the Schwann cells or axons.

MOLECULAR BASIS FOR NERVOUS SYSTEM MALFORMATIONS ASSOCIATED WITH L1 MUTATIONS In contrast to the significant advances in understanding the phenotype of L1 mutations, it remains unclear what molecular mechanisms underlie the genesis of nervous system anomalies associated with L1 mutations. However, the genotype–phenotype correlation in XLH has demonstrated that disruption of different functional aspects of L1 leads to different phenotypes. Since the mutations of the L1CD result in axonal tract defects, protein interactions or intracellular signaling events involving the L1CD are likely to be critical for normal development of the axonal tracts. Because deletion of the L1CD does not alter L1-mediated homophilic adhesion (Wong et al., 1995), the L1CD clearly must serve other important roles which are critical in the formation of axonal pathways. A number of in vitro studies have shown that L1-dependent axon growth involves second-messenger systems (Williams et al., 1992), L1-associated kinases (Wong et al., 1996), the nonreceptor tyrosine kinase pp60c2src (Ignelzi et al.,

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FIG. 2. (A and B) Abnormal development of the corticospinal tract in L1 knock-out mice. Coronal sections through the pyramidal decussation of adult wild-type (A) and mutant (B) mice after unilateral injection of horseradish peroxidase (HRP) into the cerebral cortex are shown. In the wild-type mouse, the HRP-labeled corticospinal axons project normally from the ventral pyramid (P), across the midline (arrow) to the contralateral dorsal column (DC). In the mutant mouse, there is disruption of the normal pattern of decussation with many axons failing to cross the midline and instead following a mirror symmetrical course to the ipsilateral dorsal column (iDC). Reprinted, with permission of the publisher, from Cohen et al. (1997) Curr. Biol. 8: 26–33. (C and D) Hypoplastic cerebellar vermis and enlarged fourth ventricle (arrow) in L1 knock-out mice. Cresyl violet staining of midsagittal sections through the vermis of wild-type (C) and mutant (D) mice is shown. Reprinted, with permission of the publisher, from Fransen et al. (1998a) Hum. Mol. Genet. 7: 999–1009.

1994), the FGFr (Saffell et al., 1997), and probably interactions with the cytoskeleton (Davis and Bennett, 1994; Dahlin-Huppe et al., 1997). In addition, the L1CD plays a crucial role in regulating intracellular L1 trafficking that is required for L1-dependent axon growth

(Kamiguchi and Lemmon, 1998; Kamiguchi et al., 1998b). Mutations of the L1CD must impair one or more of these L1 functions depending on the site of mutation, leading to abnormal development of major axonal tracts such as the corticospinal tract and corpus callosum.

L1 Mutations in Mice and Humans

The fact that the mutations of the L1ED more frequently cause severe ventriculomegaly than those of the L1CD argues that loss of L1-mediated adhesion is responsible for the genesis of ventricular enlargement (Yamasaki et al., 1997; Fransen et al., 1998b). There are several ways the dilation might be produced. A rather simple explanation is that the increase in ventricular size is a direct result of loss of brain parenchyma (hydrocephalus ex vacuo), which might result from the failure of establishment of proper synaptic connections and subsequent neuronal death. For example, the enlarged fourth ventricle in L1 knock-out mice is probably due to the decreased size of the cerebellar vermis. An additional explanation comes from the theory that mechanical tension generated by axons, dendrites, and glial processes plays a significant role in the morphogenesis of the CNS (Van Essen, 1997). Since ventricular size is determined by the balance between hydrostatic pressure of the CSF and brain compliance, loss of cell–cell adhesion that increases the compliance of developing brain would result in ventricular dilatation. Figure 3 illustrates a possible role for L1-mediated adhesion in generating and maintaining brain compliance. In the normal developing brain, axon–axon adhesion generates tension in the white matter that is sufficient to balance the hydrostatic pressure of the CSF (Fig. 3A). However, with loss of adhesion, axons could slide along each other, resulting in increased brain compliance (Fig. 3B). Furthermore, loss of axons in the corpus callosum and corticospinal tract would contribute to the increase

53 in compliance. Both hydrocephalus ex vacuo and increased brain compliance might be initial mechanisms for the genesis of ventricular dilatation in mild XLH. To produce severe ventriculomegaly or true hydrocephalus with elevated pressure, the CSF outflow or resorption has to be impaired. In some cases, it is thought that secondary obstruction, such as at the aqueduct of Sylvius, can be produced by the compression from the enlarged lateral ventricles (Fig. 3C) (Landrieu et al., 1979; Renier et al., 1982). However, since total loss of L1 function (Class 3 mutations) is much more likely to cause severe hydrocephalus than the other types of mutations, L1 should play an important role in the evolution or maintenance of CSF passage patency. Although no indication of aqueductal stenosis was found in L1 knock-out mice, the aqueduct was significantly elongated and possibly more vulnerable to obstruction (Fransen et al., 1998a). The altered shape of the aqueduct may be a result of impaired neuronal migration that leads to increased accumulation of cells in inappropriate places, either near the aqueduct or at a distance that would produce distortion in the developing brain. A similar scenario could also apply to the third ventricle in XLH, where impaired neuronal migration produces fused thalami that sometimes obstruct the CSF passage (Sato, 1992; Yamasaki et al., 1995). Therefore, it is tempting to speculate that loss of L1 in developing axons causes mild ventricular dilatation either by loss of white matter or by increased brain

FIG. 3. A hypothetical model for the genesis of ventricular dilatation in XLH: implication of L1-mediated axon–axon adhesion in maintaining brain compliance. In the normal developing brain, interaxonal adhesion generates tension in the white matter that is sufficient to balance with hydrostatic pressure of the CSF (A). Loss of adhesion would allow axons to slide along each other, leading to increased brain compliance. As a result, the ventricular size is increased even with normal CSF pressure (B). In some cases, secondary stenosis of CSF passages produces progressive and high-pressure hydrocephalus (C).

54 compliance, while loss of L1 in migrating neurons results in severe hydrocephalus.

CONCLUSIONS The past several years have produced significant advances in understanding how nervous system malformations are caused by mutations in the L1 gene. There are a number of similarities between humans with L1 mutations and L1 knock-out mice. The results from the genetic analyses suggest certain molecular and cellular biological explanations for different aspects of XLH, but at the same time, they raise many questions. Is the L1CD critical for axon guidance but relatively unimportant in neuronal migration? Does loss of L1-mediated adhesion disrupt migration of specific groups of neuronal precursors? Are neuronal migration defects involved in the progression of hydrocephalus? Production of new mouse lines with targeted mutations in the L1 gene, rather than the knock-outs, should allow a test of whether or not axon growth and guidance defects and hydrocephalus are causally linked.

ACKNOWLEDGMENTS We are grateful to S. Burden-Gulley and A. W. Schaefer for helpful comments on the manuscript and to E. Fransen, A. J. W. Furley, and M. Yamasaki for providing pictures. Preparation of this manuscript was supported by grants from NEI and NINDS and the Roche Foundation to V.L. and M.L.H.

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