Mutant mouse models of insulin-like growth factor actions in the central nervous system

Neuropeptides (2002) 36(2±3), 209±220 Special Issue on Transgenics and Knockouts with Mutations in Genes for Neuropeptides and their Receptors. ß 2002 Elsevier Science Ltd. All rights reserved. doi: 10.1054/npep.2002.0893, available online at http://www.idealibrary.com on

Mutant mouse models of insulin-like growth factor actions in the central nervous system A. Joseph D'Ercole,1 Ping Ye,1 John R. O'Kusky2 1 Department of Pediatrics, Division of Endocrinology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599±7220 and 2Department of Pathology & Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada V5Z 1M9

Summary Insulin-like growth factor-I (IGF-I) and its cognate receptor, the type 1 IGF receptor (IGF1R), as well as highaffinity IGF binding proteins (IGFBP) that modulate IGF-I actions, are expressed throughout the course of brain development. These observations, taken together with studies in cultured neural cells demonstrating a variety of IGF-I growth-promoting activities, provide a strong argument for IGF-I having a central role in the growth and development of the CNS. This report reviews studies of brain development in mutant mice with alterations of IGF-I expression or action. Transgenic (Tg) mice overexpressing IGF-I postnatally exhibit brain overgrowth characterized by increased neuron and oligodendrocyte number, as well as marked increases in myelination. Mutant mice with ablated IGF-I and IGF1R expression, as well as those with overexpression of IGFBPs capable of inhibiting IGF actions, exhibit brain growth retardation with a variety of growth deficits. These studies confirm a role for IGF-I in neural development, and indicate that IGF-I stimulates neurogenesis and synaptogenesis, facilitates oligodendrocyte development, promotes neuron and oligodendrocyte survival, and stimulates myelination. Evidence from experiments in these mouse models also indicates that IGF-I has a role in recovery from neural injury. ß 2002 Elsevier Science Ltd. All rights reserved.

INTRODUCTION Accumulating evidence indicates a major role for insulinlike growth factor-I (IGF-I) in central nervous system (CNS) development [see reviews: (D'Ercole et al., 1996; Folli et al., 1996; Anlar et al., 1999)]. IGF-I stimulates increases in neuron and oligodendrocyte numbers by mechanisms that involve both the stimulation of proliferation and the promotion of survival. IGF-I also in¯uences neuron and oligodendrocyte differentiation and function, e.g., it stimulates neuritic outgrowth, synaptogenesis, and myelination. Many of the latter ®ndings come from studies of mice with genetic alterations in the expression of insulinlike growth factors (IGF), the type 1 IGF receptor (IGF1R),

Received 2 February 2002 Accepted 3 March 2002 Correspondence to: A. Joseph D'Ercole, M.D. Harry S. Andrews Professor. f Pediatrics Chief, Pediatric Endocrinology Dept. of Pediatrics, CB# 7220 University of North Carolina Chapel Hill, NC 27599±7220 Tel.: (919) 966 4435 229; Fax: (919) 966 2423; E-mail: [email protected]

and IGF binding proteins (IGFBP). This review addresses insights derived from these mutant mice. IGF-I is expressed in the CNS from early in neurogenesis [reviewed in (D'Ercole et al., 1996)]. The type 1 IGF receptor, the predominant, if not exclusive, mediator of IGF-I actions, is expressed by neural stem cells (Brooker et al., 2000) and subsequently it appears to be expressed in all neural cells. IGF-I actions are modulated by IGFBPs that can either inhibit or augment IGF action [see review: ( Jones and Clemmons, 1995)]. IGF-II, a homologue of IGF-I, also is expressed from early in embryogenesis, predominately in mesenchymal structures and neural crest derivatives, and latter in the choroid plexus. Given the proximity of IGF-II expression to the developing CNS, it could in¯uence CNS development, however, there is little evidence to support this. OVERVIEW OF IGF SYSTEM MUTANT MICE Multiple lines of mice with genomic alterations in IGF system proteins have been generated [reviewed in 209

210 D'Ercole et al.

(D'Ercole 1999; Efstratiadis 1998)]. Table 1 lists those mice that are known to manifest a change in brain growth and/ or phenotype. Table 2 reviews the somatic growth characteristics of mice with null mutations. IGF-I and IGF-II Overexpression IGF-I overexpressing mice were among the ®rst transgenic (Tg) mice generated (Mathews et al., 1988; Behringer et al., 1990). The initial line of Tg mice was created using a transgene driven by the mouse metallothionein (mMT-I) promoter and encoding a human IGF-I cDNA. The mice in this and subsequent lines generated with the same fusion gene express IGF-I in multiple tissues beginning at birth (Mathews et al., 1988; Ye et al., 1995a). Depending upon the tissues and magnitude of transgene expression among these lines of mice, postnatal somatic overgrowth begins

at 3±4 weeks of age and leads to a moderate increase in weight (30%) by early adulthood. The IGF-I Tg mice exhibit disproportional overgrowth of some organs, most markedly of the brain (Ye et al., 1995a). As with somatic overgrowth, organ overgrowth appears to depend upon the degree of organ speci®c transgene expression among the lines. For example, brains weights are increased from 25±85% in different MT-I/IGF-I Tg lines. Mice with the largest brain weights often do not survive past weaning, and then invariably breed poorly or not at all. The reason(s) for this is not clear, but it does not appear to be due to hydrocephalus or the development of tumors. Furthermore, mice with larger brain weights usually do not exhibit somatic overgrowth; rather they have modestly reduced adult body weight (15%) and similarly reduced serum IGF-I levels. We speculate that increased IGF-I expression in the pituitary results in decreased

Table 1 IGF System Protein Mutant Mouse Lines with Known Alterations in Brain Phenotype Protein

Description of Mutant Line

Abbreviation in Text

Brain Phenotype

IGF-I

m Metallothionein-I (mMT-I) driven hIGF-IA cDNA m IGF-II 5' flanking region

mMT-I/IGF-I

Increased postnatal brain growth Increased postnatal brain growth, especially the cerebellum

IGF1R

IGFBP-1

IGFBP-2

mIGF-II/IGF-I

Gene ablation by homologous recombination: Heterozygous ablation Homozygous ablation Partial gene ablation by homologous recombination Gene ablation by homologous recombination: Heterozygous ablation Homozygous ablation

IGF-I KO IGF-I ‡ / IGF-I / Midi-IGF-I

No abnormality Brain growth retardation Brain growth retardation

IGF1R KO

mMT-I promoter driven hIGFBP-1 cDNA m Phospoglycerate Kinase-1 promoter driven rIGFBP-1 gene ha-1 antitrypsin promoter driven hIGFBP-1 cDNA CMV promoter driven mIGFBP-2 cDNA

IGF1R ‡ / IGF1R /

No abnormality Brain growth retardation

mMT-I/hIGFBP-1

a-1AT/hIGFBP-1

Postnatal brain growth retardation Brain growth retardation from fetal life Brain abnormalities

CMV/IGFBP-2

Brain growth retardation

mPGK/rIGFBP-1

Table 2 Growth Characteristics of Null Mutant Mice mice [Data from references: (Liu et al., 1993; Baker et al., 1993; Ludwig et al., 1996; Accili et al., 1996; Louvi et al., 1997) Mutation

Onset of Growth Retardation

Birth weight (% Normal)

Perinatal Viability

Postnatal Growth

Single Mutations IGF-I IGF-II IGF1R InR

   

E13 E10.5 E10.5 E18.5

   

< 5 ±  70% Normal None None

Very poor:  25% of normal as adults Normal velocity, but no catch-up growth. None. None.

Multiple Mutations IGF-I & IGF-II IGF-I & IGF1R IGF-II & IGF1R InR & IGF1R

   

E10.5 E10.5 E10.5 E10.5

25±35  45  30  30

None None None None

None None None None

Neuropeptides (2002) 36(2±3), 209±220

60 60 45 90

ß 2002 Elsevier Science Ltd. All rights reserved.

Insulin-like Growth Factor Actions in the Central Nervous System 211

growth hormone (GH) secretion and consequent decreased expression of the native IGF-I gene in somatic tissues, that is not fully compensated by transgene IGF-I expression. Using a variety of promoters aimed at generating organ speci®c IGF-I overexpression, a number of other IGF-I Tg mice have been created [see review: (D'Ercole, 1999)]. Using an 11.3 kb fragment of the mouse IGF-II genomic regulatory region, we generated a line of IGF-I mice that expresses only in the CNS (Ye et al., 1996). While the transgene expresses throughout the brain, its expression is markedly increased in the cerebellum and results in a near doubling of cerebellar weight by adulthood. Most of our studies of IGF-I actions in the CNS have utilized one or more of these lines of IGF-I overexpressing mice. Currently, we are characterizing IGF-I overexpressing mice generated with a transgene driven by a regulatory regions derived from the nestin genome. Unlike other IGF-I Tg mice with the brain overexpression, the neural stems cells of these mice express the transgene beginning during embryonic life, and exhibit an increase in brain size prenatally. While a number of Tg mice have been created that overexpress IGF-II, none exhibit somatic overgrowth and no phenotypic alterations in the brain or nervous system have been reported [see review: (D'Ercole, 1999)]. Many of these IGF-II Tg mice have been created with fusion genes utilizing promoters that would not be expected to express in brain. On the other hand, no CNS consequences of IGF-II overexpression have been reported either in Tg with brain transgene expression (Van Buul-Offers et al., 1995; Rogler et al., 1994; Wolf et al., 1994) or in those with elevated serum IGF-II levels (Rogler et al., 1994; Wolf et al., 1994). The reasons for this apparent lack of an IGF-II in¯uence on the CNS are not clear. Given that higher IGF-II concentrations are often required to mimic IGF-I action, insuf®cient expression IGF-II transgene expression may explain the apparent lack of an alteration in the brain phenotype. IGF-I, IGF-II and IGF1R null mutant mice Generation and study of mice with IGF and IGF1R null mutations have provided direct evidence of the central role of IGF in somatic growth (Efstratiadis, 1998). Table 2 summarizes the nature of the somatic growth retardation that accrues when these genes are not expressed. A detailed discussion of the insights provided by these studies is beyond the scope of this chapter. Suf®ce it to say that in the mouse both IGF-I and IGF-II are necessary for normal in utero growth, while only IGF-I is necessary for normal postnatal growth. The IGF1R appears to mediate all IGF-I growth promoting actions, as well as much of IGF-II stimulated growth. The IGF-II and/or the ß 2002 Elsevier Science Ltd. All rights reserved.

insulin receptor, however, mediate a portion of IGF-II stimulation of somatic fetal growth. Mice that are homozygous for the disrupted IGF-I gene, i.e., knockout mice (IGF-I±/± or IGF-I KO mice), exhibit marked in utero and postnatal growth retardation, and depending upon the genetic background, have reduced survival past birth (Liu et al., 1993; Baker et al., 1993; Powell-Braxton et al., 1993). Disruption of a single IGF-I gene has little impact. Mice without IGF-I expression have birth weights that are  60% of normal, and continued poor postnatal growth such that they weigh about  25% of normal as adults (Baker et al., 1993). Knockout of the IGF-II gene also results in a similar degree of in utero growth retardation. The character of this in utero growth, however, is distinct from that of IGF-I KO mice, in that the reduction in growth rate begins earlier and is con®ned to in utero life (DeChiara et al., 1990). Homozygous IGF1R null mutants (IGF1R KO mice) exhibit more profound in utero growth retardation than either IGF-I KO or IGF-II KO mice. They reach only  45% of normal size by the end of gestation, and do not survive the perinatal period. The brains of IGF-I KO and IGF1R KO mice are small relative to controls, although they are not as growth retarded as body weight, and the brain phenotype in each appears to be identical (Liu et al., 1993; Baker et al., 1993). At birth the striking histologic feature of these mice is their increase in neuronal density (Liu et al., 1993). De®cits in speci®c neuronal populations have been identi®ed in another line of adult IGF-I KO mice (Beck et al., 1995). These IGF-I KO mice also have a paucity of myelin and an apparent decrease in oligodendrocyte number. No information on the brain of IGF-II KO mice has been reported. Mice with altered IGFBP expression A number of mice with altered expression of IGFBPs have been generated [see review: (Schneider et al., 2000)]. The ®rst of these were IGFBP-1 overexpressing mice (D'Ercole et al., 1994). IGFBP-1 is an inhibitor of IGF action when present in molar excess, and would be expected to inhibit the actions of endogenous IGF-I and IGF-II. Using different promoters, several groups have generated IGFBP-1 Tg mice, and each line exhibits somatic and brain growth retardation (D'Ercole et al., 1994; Ye et al., 1995a; Murphy et al., 1993; Gay et al., 1997). The time when the somatic and brain growth retardation occurs appears to depend upon the developmental time when the transgene promoter is activated. For example, brain growth retardation is apparent earlier and is more severe when the transgene is expressed during fetal life, as in mPGK/rIGFBP-1 Tg mice (Murphy et al., 1993), compared to mMT-I/hIGFBP-1 Tg mice (D'Ercole et al., 1994) (see Table 1). IGFBP-1 does not appear to be normally expressed in the brain. Both of the Neuropeptides (2002) 36(2±3), 209±220

212 D'Ercole et al.

above noted transgenes drive IGFBP-1 expression in the brain (as well as in other tissues), and therefore, IGFBP1 expression is ectopic and likely acts in brain to block IGF stimulated growth. Circulating IGFBP-1, however, may also affect the brain, because a line of IGFBP-1 Tg with no brain transgene expression may also exhibit CNS abnormalities (Doublier et al., 2000). Tg mice overexpressing IGFBP-2, -3, -4 and -5 also have been generated. IGFBP-2 Tg mice with brain transgene expression exhibit a modest reduction in brain weight at 5 weeks of age (Hoe¯ich et al., 2001). No change in brain growth or phenotype has been reported in IGFBP-3 Tg mice, made with a mMT-I-driven transgene (Murphy et al., 1995). While IGFBP-4 Tg mice have been generated, they have been created with promoters that do not drive CNS expression, and as expected no alteration in brain size was reported (Wang et al., 1998). IGFBP-5 overexpression from early in gestation also appears to inhibit somatic growth including that of the brain (Salih et al., 2001). When we used the MT-I promoter to generate Tg mice with postnatal brain IGFBP-5 overexpression, however, we noted a possible minimal increase in brain weight, consistent with the known capacity of IGFBP-5 to augment IGF actions (unpublished). The postnatal and apparent less marked IGFBP-5 overexpression in the later mice likely explains the differences in phenotype. To date mice with null mutations in IGFBP genes have not been found to have a brain phenotype (Pintar et al., 1995; Pintar et al., 1996), and J. Pintar, personal communication). Presumably, the absence of a single IGFBP is compensated by other IGFBPs. IGF ACTIONS ON NEUROGENESIS, SYNAPTOGENESIS AND CNS GROWTH Accumulating evidence from multiple lines of mice with genomic alterations in IGF system proteins indicates that IGF-I promotes CNS growth by increasing the total number of both neurons and synapses. Furthermore, regional increases in neuron number appear to result from both an increase in neuron proliferation and an inhibition of apoptosis during the phase of naturally occurring neuron death. Brain weight, regional volumes and CNS growth IGF-I acts to increase both the weight and volume of the developing brain. In various lines of MT-I/IGF-I Tg mice with increased expression of IGF-I during postnatal development, adult mice exhibit an increase in brain weight ranging from 22±91% with no signi®cant change in body weight (GutieÂrrez-Ospina et al., 1996). Morphometric analysis of the somatosensory cortex in one line revealed an 81% increase in the volume of Neuropeptides (2002) 36(2±3), 209±220

the cerebral cortex and a 68% increase in the volume of the somatosensory barrels in cortical layer IV of Tg mice (GutieÂrrez-Ospina et al., 1996). IGF-I transgene expression and elevated levels of IGF-I can only be detected In the brain of IGF-II/IGF-I mice (Ye et al., 1996). Expression is ®rst observed at embryonic day (E) 18 and gradually increase to plateau values by postnatal day (P) 20. Brain weight gradually increases after P7 with a 35% increase in adults. Although the transgene expresses throughout the brain in these mice, expression is markedly increased in the cerebellum. Weight of the cerebellum in young adults was found to increase by 90% in Tg mice relative to littermate controls, with a 92% increase in the volume of the internal granular layer of the cerebellar cortex (Ye et al., 1996). In IGF-II/IGF-I Tg mice at P30, total brain weight was found to be increased by 28%, with regional increases in the cerebellum (43%), hippocampus (34%), diencephalon (28%), brainstem (28%) and cerebral cortex (9%)(O'Kusky et al., 2000). The total volume of the medulla was found to be signi®cantly increased in Tg mice by 27% at P35 (Dentremont et al., 1999). Individual medullary nuclei exhibited differential increases in volume, including the nucleus of the solitary tract (59%), the dorsal motor nucleus of the vagus (84%), the hypoglossal nucleus (29%) and the facial nucleus (21%). In the hippocampal dentate gyrus, the volumes of both the granule cell layer and the molecular layer were signi®cantly greater in Tg mice after P7, exceeding controls by 55±66% at 130 days of age (O'Kusky et al., 2000). In IGF-II/IGF-I mice these regional differences in weight and volume may re¯ect regional differences in transgene expression, the caudal-to-rostral gradient in CNS development, or differential dependencies on IGF-I as a growth factor. Total IGF-I de®ciency due to targeted disruption of the IGF-I gene causes severe brain growth retardation. At two months of age mice homozygous for the null mutation (IGF-I ±/± mice) exhibit a 38% decrease in brain weight and a 74% decrease in body weight relative to wild type controls (Beck et al., 1995). Decreased tissue volumes were reported in the pyramidal cell layer of the hippocampus (38%), the granule cell layer of the dentate gyrus (59%), and the striatum (28%). Disproportionately greater decreases were detected in white matter regions, with a 69% decrease in the area of the anterior commissure and a 70% decrease in the thickness of the corpus callosum due to decreased numbers of axons and oligodendrocytes (Beck et al., 1995). This preferential involvement of white matter in reduced brain volumes has been reported in subsequent studies (Cheng et al., 1998). Null mutations of the gene encoding the IGF1R (IGF1R±/± mice) invariably die at birth due to respiratory failure and exhibit a 55% decrease in body weight compared to wild type controls (Liu et al., 1993). At E14 to E18, IGF1R±/± ß 2002 Elsevier Science Ltd. All rights reserved.

Insulin-like Growth Factor Actions in the Central Nervous System 213

mutants exhibit corresponding growth retardation of the brain, with reduced volume and increased density of cells in the mantle zone of the spinal cord and brainstem (Liu et al., 1993). These results suggest that growth retardation results predominantly from a reduction in the volume of neuropil in the CNS regions examined. IGFBP-1 inhibits the interactions of IGF-I and IGF-II with their cell surface receptors when present in molar excess. In lines of Tg mice with ectopic brain IGFBP-1 expression, brain weight was signi®cantly decreased (8±16%) by the second postnatal week (D'Ercole et al., 1994). This brain growth retardation was most obvious in the cerebral cortex, hippocampus and diencephalon, where brain weights were decreased by 18%, 20% and 12%, respectively (Ye et al., 1995a). Decreased density of myelinated axons was prominent in the cerebral cortex, anterior commissure, corpus callosum and diencephalon, although the cerebellum and brainstem were relatively spared (Ye et al., 1995a). In two lines of IGFBP-1 Tg mice, brain weight was reduced 22±24% by P90, with no corresponding change in body weight, (GutieÂrrez-Ospina et al., 1996). Morphometric analysis of the somatosensory cortex in these Tg mice revealed a 29% decrease in cortical volume and a 24% decrease in the volume of the somatosensory barrels in layer IV. In another line of IGFBP-1 Tg mice, the authors reported signi®cant decreases in body weight (12%), brain weight (40%), whole brain DNA content (16%) and total protein (23%) in adult mice (Ni et al., 1997). Cross-sectional areas of the hippocampus and the granule cell layer of the dentate gyrus on representative sections were found to be signi®cantly reduced by 55% and 72%, respectively, compared to a decrease of 33% for the brain as a whole. White matter and ®ber tracts were generally less well developed in Tg mice, and the thickness of the corpus callosum was decreased by 62. In a line of Tg mice with hepatic IGFBP-1 overexpression and increased circulating levels of IGFBP-1, there was signi®cant reduction in brain weight by two months of age (Doublier et al., 2000). Tg mice with increased expression of IGFBP-2 in the brain exhibit modest decreases in brain weight of 9±13% from postnatal weeks 5±15 (Hoe¯ich et al., 2001). IGF-I actions on neurogenesis Morphometric and stereological analyses of the developing brain in IGF-I overexpressing Tg mice have reported substantial increases in the total number of neurons in the cerebral cortex (GutieÂrrez-Ospina et al., 1996), cerebellar cortex (Ye et al., 1996), dentate gyrus of the hippocampus (O'Kusky et al., 2000), and selected brainstem nuclei (Dentremont et al., 1999). By contrast, in IGF-I null mutants (Beck et al., 1995; Camarero et al., 2001) and in IGFBP-1 Tg mice, in whom IGF actions are inhibited (GutieÂrrez-Ospina et al., 1996; Ni et al., 1997), signi®cant decreases in neuron ß 2002 Elsevier Science Ltd. All rights reserved.

number have been reported in the cerebral cortex, hippocampus, dentate gyrus, striatum, and cochlear nucleus. While in vitro studies have shown that IGF-I is essential for neuron proliferation and differentiation [(Arsenijevic and Weiss, 1998) and review: (D'Ercole et al., 1996)], most available evidence in mutant mice documents the antiapoptotic actions of IGF-I. Detailed morphometric studies have been performed largely in Tg mice carrying transgenes that are expressed postnatally, only after neuron precursor proliferation has occurred in most CNS regions. Morphometric analysis of the somatosensory cortex in MT-I/IGF-I mice revealed a 24% increase in the total number of neurons in somatosensory barrels in cortical layer IV by P90. In addition, there was a 39% decrease in the numerical density of neurons (NV, cells per unit volume) indicating an increase in the volume of neuropil separating individual cell bodies, and a 33% increase in mean neuronal pro®le area. Neurons in layer IV of the cerebral cortex in mouse are generated during prenatal development (Hicks and D'Amato, 1968), while apoptotic neuron death occurs predominantly from birth to P10 (Sprea®co et al., 1995; Verney et al., 2000). Given that the transgene in MT-I/IGF-I mice is ®rst expressed after birth (Ye et al., 1995a), it would appear that increased neuron number in these Tg adults results from decreased apoptosis during the regressive phase of neurogenesis. A similar analysis of the somatosensory cortex in IGFBP-1 Tg mice revealed a 15% decrease in the total number of neurons in somatosensory barrels and a 39% increase in the NV of neurons (GutieÂrrez-Ospina et al., 1996). Decreased cortical volume in these Tg mice resulted from a decrease in both the number of neurons and the volume of neuropil separating individual neuronal cell bodies. Since the transgene in these mice is ®rst expressed after birth (D'Ercole et al., 1994), decreased neuron number likely results from increased apoptosis during the regressive phase of neurogenesis. In the cerebellar cortex of IGF-II/IGF-I Tg mice at P50, the total number of Purkinje cells and granule cells increased by 20% and 82%, respectively (Ye et al., 1996). Intraperitoneal injections of bromo-2-deoxyuridine (BrdU) were employed to examine proliferation of granule cell progenitors in the external granule cell layer between P7 and P21. Results indicated no difference between Tg and control mice in the proportion of labeled cells, although the total number of labeled cells was increased in Tg mice by 38% at P15. Purkinje cells of the cerebellar cortex in mouse originate from mitotic neuroepithelial precursors from E11 to E13, while granule cells originate from E17 to P15 (Miale and Sidman, 1961; Mares et al., 1970). Given this protracted period of proliferation for granule cells and the increase in BrdU-labeled progenitors, it appears likely that elevated levels of IGF-I during early postnatal development act to increase the rate of mitosis Neuropeptides (2002) 36(2±3), 209±220

214 D'Ercole et al.

in the external granular layer. In a subsequent study, the anti-apoptotic effects of elevated IGF-I were investigated in the cerebellum of IGF-II/IGF-I Tg mice (Chrysis et al., 2001). Morphometric analysis of apoptotic cells in the cerebellum, detected by terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL), revealed a 47% decrease in Tg mice when compared to controls. Activities of procaspase-3 and caspase-3 were also decreased in Tg mice, accompanied by increased expression of the anti-apoptotic Bcl genes, Bcl-xL and Bcl-2. At P21 decreased expression of the pro-apoptotic genes Bax and Bad was observed in Tg mice. In another study Bcl-2 was found to be increased in immunohistochemical studies of the cerebellum in these Tg mice (Baker et al., 1999). These results provide direct evidence that elevated IGF-I acts to inhibit apoptosis during early postnatal development in a developmentally speci®c manner. Increased growth of the hippocampal dentate gyrus has been studied in IGF-II/IGF-I mice throughout postnatal development (O'Kusky et al., 2000). In control mice the total number of neurons in the granule cell layer increased by 113% from P7 to P35, with no additional increase in neuron number by P130. In these Tg mice, there was a 172% increase in neuron number from P7 to P35, with an additional signi®cant increase of 17% between P35 and P130, suggesting a protracted period of accelerated neurogenesis. Comparing Tg and control mice, the total number of neurons in the granule cell layer was signi®cantly greater in Tg mice by 56% at P35 and by 61% at P130. In IGFBP-1 Tg mice with inhibited IGF-I actions, BrdU labeling of proliferating cells in the granule cell layer of the dentate gyrus revealed a 41% decrease in the number of labeled cells in Tg mice at P3, as compared to controls (Ni et al., 1997). In the ventricular and subventricular zones of the lateral ventricle, BrdU-labeled cells were decreased by 19%. The number of TUNEL-labeled apoptotic cells throughout the hippocampus was found to be increased by 55% in Tg mice (Ni et al., 1997). These studies indicate that IGF-I acts to increase neuron proliferation while inhibiting apoptosis in a region of the brain where the progressive and regressive phases of neurogenesis are known to occur throughout the life of the organism. In the brainstem of IGF-II/IGF-I Tg mice at P35, the total number of neurons was signi®cantly increased in the nucleus of the solitary tract (50%) and in the dorsal motor nucleus of the vagus (53%), but not in the hypoglossal nucleus or the facial nucleus (Dentremont et al., 1999). Neuron proliferation in the mouse occurs from E9-E12 for the nucleus of the solitary tract and from E9-E10 for the dorsal motor nucleus of the vagus, the hypoglossal nucleus and the facial nucleus (Pierce, 1973). Given that transgene expression in these mice is very low prior to birth, increased rates of neuron proliferation are unlikely to account for increased neurons. An inhibition of Neuropeptides (2002) 36(2±3), 209±220

naturally occurring neuron death appears to be more likely. Apoptotic death of motor neurons in the hypoglossal nucleus occurs exclusively from E16 to E21 in the rodent (Friedland et al., 1995), as it does for motor neurons in the spinal cord (Oppenheim, 1986). Motor neurons in the facial nucleus are likely to undergo similarly early programmed cell death. Thus, the lack of effect of elevated IGF-I on neuron number in the hypoglossal and facial nuclei in these mice appear to stem from the age at which the transgene is expressed. Interestingly, although the total number of neurons did not differ signi®cantly between Tg and control mice in the hypoglossal and facial nuclei, the NV of neurons was signi®cantly decreased in both regions while the mean neuronal pro®le area was signi®cantly increased. Changes in these variables indicate an increased volume of neuropil and possibly more complex arborizations of the dendritic trees on individual neurons within these regions. In IGF-I±/± mutants immunohistochemical studies have reported signi®cant decreases in the number of parvalbumin-immunoreactive neurons in the striatum (52%), hippocampus (32%) and dentate gyrus (59%) (Beck et al., 1995). Interestingly, the numbers of cholinergic neurons in both the striatum and basal forebrain and dopaminergic neurons in the mesencephalon did not change, suggesting a differential susceptibility of neurotransmitter-speci®c neuron populations to the effects of IGF-I (Beck et al., 1995). Delayed maturation of the inner ear and neuron loss also have been reported in homozygous IGF-I±/± mutant mice during early postnatal development (Camarero et al., 2001). The volume of the cochlea and cochlear ganglion were reduced by 34% at P20, accompanied by a 19% loss of cochlear neurons and a 31% decrease in the mean volume of the cell body in surviving neurons. The number of apoptotic neurons, determined by TUNEL labeling, and caspase- 3-mediated apoptosis were increased in IGF-I±/± mice compared to wild type controls (Camarero et al., 2001). IGF-I actions on synaptogenesis The effects of elevated IGF-I on the progressive and regressive phases of synaptogenesis in the hypoglossal nucleus have been investigated using stereological analyses by light and electron microscopy in IGF-II/IGF-I mice (O'Kusky et al., 2000). In control mice the total number of synapses increased by 354% from P7 to peak values at P21, followed by a signi®cant decrease of 33% by P130. In Tg mice the total number of synapses increased by 522% from P7 to peak values at P21, followed by a signi®cant decrease of 28% by P130. The total number of synapses was signi®cantly greater in Tg mice than in controls by 42% and 52% on P21 and P130, respectively. Given that ß 2002 Elsevier Science Ltd. All rights reserved.

Insulin-like Growth Factor Actions in the Central Nervous System 215

the total number of hypoglossal neurons did not differ signi®cantly between Tg and control mice at any age, the synapse-to-neuron ration was found to be signi®cantly greater in Tg mice after P14. These results indicate that the increased in vivo expression of IGF-I during postnatal development augments the progressive phase of synaptogenesis, although it does not prevent synapse elimination during the regressive phase. In the hippocampal dentate gyrus of IGF-II/IGF-I Tg mice, signi®cant increases in the total number of synapses in the molecular layer were observed on P14 (61%), P21 (42%), P28 (105%), P35 (96%), and P130 (54%) (O'Kusky et al., 2000). The NV of synapses was signi®cantly greater in Tg mice only at P28 (36%) and P35 (21%). Interestingly, the synapse-to-neuron ratio was greater in Tg mice only at P28 (60%) and P35 (24%), returning to normal values by P130. Thus, the increase in synapse number in the dentate gyrus tended to re¯ect the increased number of neurons in the granule cell layer. Immunohistochemical studies using IGF-I±/± null mutants have reported abnormal synaptophysin expression in the organ of Corti of the null mutants at P20 (Camarero et al., 2001). The pattern of immunoreactivity in the cell bodies of cochlear ganglion neurons and sensory hair cells in IGF-I±/± mice at P20 more closely resembled controls at P5, indicating the persistence of an immature pattern of synapse distribution in the absence of IGF-I.

availability (IGF-I KO and IGFBP-1 Tg mice) or with IGF-I overexpression (Beck et al., 1995; Ni et al., 1997) their response to injury is altered by IGF-I expression. Ectopic brain expression of IGFBP-1, a protein that reduces IGF-I availability and thereby inhibits IGF-I action when in molar excess, impairs brain development and reduces astrocyte response to injury (Ni et al., 1997). Cuprizone, a copper chelator and neurotoxicant, is known to increase microglia/macrophages number in the corpus callosum (CC) and cause oligodencroyte and myelin damage. When IGF-I overexpressing Tg mice are subjected to cuprizone injury, there is a dramatic increase in the number of microglia/macrophages, despite the fact that no increase is apparent under homeostatic conditions (Mason et al., 2000). IGF Actions on oligodendrocytes and myelination Myelination is tightly controlled by neuronal in¯uences (Chen and DeVries, 1989; Barres and Raff, 1993; Dutly and Schwab, 1991) and multiple growth factors and hormones (Van der Pal et al., 1988; Ferret-Sena et al., 1990; Raff, 1989; Eccleston and Silberberg, 1985; Noble et al., 1988; Besnard et al., 1989; Legrand, 1980). Increasing evidence from recent studies using mutant mice strongly points to an important role for IGF-I in the process, including promotion of proliferation and maturation of cells in oligodendrocyte lineage and stimulation of myelination.

Summary Mutant mouse models of IGF actions in the developing CNS provide compelling evidence that IGF-I promotes growth of the brain by increasing neuron number, process outgrowth and synaptogenesis. IGF-I acts to increase neuron number by increasing the rate of neuron proliferation, while inhibiting apoptosis during the phase of naturally occurring neuron death. Some regions of the brain, notably the hippocampus and dentate gyrus, appear to have a greater dependency on the growth-promoting effects of IGF-I. IGF ACTIONS IN GLIOGENESIS IGF-I actions on oligodendrocytes and myelination have been well studied in mice with mutations of IGF system proteins. There are, however, few such studies on other types of glial cells, i.e. astrocytes, microglia, radial glial, and ependymal cells. Astrocytes and microglia While astrocytes and microglia/macrophages appear to develop normally in mice with either reduced IGF-I ß 2002 Elsevier Science Ltd. All rights reserved.

Oligodendrocyte number: proliferation versus enhanced survival Signi®cant evidence from IGF mutant mice indicates that IGF-I increases the number of oligodendrocytes and their precursors. Despite ®nding a dramatic four fold increase in brain myelin content, the ®rst studies to speci®cally address IGF-I-induced alterations in the brains of IGF-I Tg mice did not ®nd evidence of increased oligodendrocyte number, as judged by assessing the percentage of carbonic anhydrase II (CA-II)-positive cells (Carson et al., 1993). In latter studies using a different IGF-I Tg mouse line generated using the same IGF-I transgene, a moderate increase in the number of oligodendrocytes (Ye et al., 1995a; Mason et al., 2000) and oligodendrocyte precursors was observed (Mason et al., 2000) in cerebral cortex (CTX) and corpus collosum (CC). In these studies NG2 antibodies were used to identify precursors, and PLP in situ hybridization or antibodies against GST-p and CAII were used to identify mature oligodendrocytes. The different markers used are likely to explain the discrepancies in the above studies, although differences in the brain regions and age of mice studied could also have contributed. In another study IGF-I was found to increase the number of Neuropeptides (2002) 36(2±3), 209±220

216 D'Ercole et al.

oligodendrocytes and their precursors, even in the face of undernutrition during the suckling period ± a situation that signi®cantly impairs oligodendrocyte development and myelination (Ye et al., 2000). Conversely in mutant mice with reduced IGF-I availability, oligodendrocyte number and myelination are reduced. When studying IGF-I±/± mice, Beck et al., found a signi®cant reduction in the number of oligodendrocytes and their precursors in the AC and CC (Beck et al., 1995). Cheng et al., however, found no decrease in the apparent percentage of oligodendrocytes in adult IGF-I KO mice (Cheng et al., 1998). In another study of IGF-I±/± mice, the number of oligodendrocytes and the concentration of myelin-speci®c proteins were observed to be reduced during postnatal development. The concentrations of myelin-speci®c proteins, however, approached normal in adult IGF-I±/± mice (ms. in review). The abundance of IGFII also was increased in these mice, suggesting that in the absence of IGF-I expression IGF-II can compensate at least in part for IGF-I actions on myelination. Such an apparent compensatory increase in IGF-II is not unprecedented because an increase in serum IGF-II also was observed in the single patient identi®ed as having an IGF-I null gene mutation (Woods et al., 1996). Further evidence that IGF-II may in¯uence oligodendrocyte number and myelination comes from studies of IGFBP-1 Tg mice. This IGFBP interacts with IGF-I and IGF-II with equal af®nity, and thus, can inhibit the actions of both peptides. In Tg mice with ectopic brain IGFBP-1 expression, oligodendrocyte number is decreased and myelination is markedly reduced even in adult mice (Ye et al., 1995a; Ni et al., 1997). This can be taken to mean that when the actions of both IGFs are blocked throughout the time of postnatal development myelination is severely impaired. In vitro studies strongly indicate that IGF-I promotes proliferation of oligodendrocyte precursors and survival of oligodendrocytes and their precursors (McMorris et al., 1986; McMorris and Dubois-Dalcq, 1988; McMorris et al., 1993; Ye and D'Ercole, 1999). While Ni et al., (Ni et al., 1997) showed a signi®cant reduction in BrdU-labeled cells in the ventricular zone, a location where oligodendrocyte precursors develop, there is little in vivo data addressing this issue in IGF mutant mice. The paucity of data stems from (1) the relatively few stage-speci®c markers for cells in oligodendrocyte lineage, and (2) the dif®culty in ascertaining the identity apoptotic cells. For example, the cell membrane lipid, A2B5, is a well recognized oligodendrocyte precursor marker in culture. The rapid disintegration and removal of apoptotic cells abrogates the use of such a marker to determine the apoptotic cell type in vivo. Part of the problem likely also derives from the instability of lipid during the course of paraf®n embedding and other tissue preparation procedures (Vass et al., 1992). Neuropeptides (2002) 36(2±3), 209±220

While there are no studies in IGF system mutant mice addressing whether IGF-I acts to promote oligodendrocyte lineage cell proliferation or survival during development, several studies clearly demonstrate that IGF-I promotes survival of oligodendrocyte lineage cells during injury. Mason et al., have shown that cuprizone treated mice exhibit massive apoptosis of oligodendrocyte lineage cells in the CC followed by demyelination (Mason et al., 2000). When the same injury is induced in IGF-I Tg mice, demyelination occurs but oligodendrocyte apoptosis is limited, and the surviving oligodendrocytes retain the capacity to reinitiate axon myelination (Mason et al., 2000). Delivery of IGF-I by injection of IGF-I expressing COS cells has similar effects on axonectomy-induced oligodendrocyte death (Barres et al., 1993). Differentiation To address whether IGF-I in¯uences oligodendrocyte differentiation, the expression of myelin basic protein (MBP) and proteolipid protein (PLP) in IGF-I and IGFBP-1 Tg mice and in IGF-I KO mice (Ye et al., 1995a; Ye et al., 2000) was assessed. Because these two proteins are expressed only in mature oligodendrocytes and myelin, they provide an index of oligodendrocyte maturation. The abundance of these two proteins was found to be increased in IGF-I Tg mice and reduced in IGFBP-1 Tg mice during early development. Taken together with the data showing oligodendrocyte number is increased in the IGF-I Tg mice, these data suggest that IGF-I promotes oligodendrocyte development in vivo. Myelination Perhaps the best documented function of IGF-I in the brain is the stimulation of myelin synthesis. Carson et al. showed that myelin content of IGF-I Tg mice is increased four fold (Carson et al., 1993). This dramatic increase in myelin results from thicker myelin sheathes comprised of more sheathes wrapped around each axon, and myelination of relatively more small diameter axons (Ye et al., 1995a; 1995b). Conversely, mice with reduced IGF-I expression (IGF-I KO mice) and/or availability (IGFBP-1 Tg mice) exhibit evidence of decreased myelination, although as mentioned above, IGF-II expression may be capable of ameliorating the decrease in myelin in IGF-I KO mice (Beck et al., 1995; Ye et al., 1995a; Ni et al., 1997). As would be expected from these ®ndings, the expression of myelin-speci®c protein genes is increased in IGF-I Tg mice and reduced in mutant mice with reduced IGF-I availability (Carson et al., 1993; Ye et al., 1995a; 2000). Because the changes in expression of myelin-related protein genes can not be fully accounted for by the changes in oligodendrocyte number, it appears ß 2002 Elsevier Science Ltd. All rights reserved.

Insulin-like Growth Factor Actions in the Central Nervous System 217

that IGF-I speci®cally stimulates oligodendrocyte myelin synthesis. Furthermore, IGF-I may stimulate the initiation of myelination., because as mentioned above the number of ensheathed axons and the number of myelinated axons of small size are increased in IGF-I Tg mice (Ye et al., 1995b). IGF-I also protects oligodendrocyte and myelination from injury and promotes re-myelination after injury. In cuprizone-induced injury treatment causes apoptosis of oligodendrocyte and severe demyelination in the CC (Blakemore, 1972; Hiremath et al., 1998; Mason et al., 2000). While it is not able to inhibit cuprizone-induced demyelination, IGF-I signi®cantly enhances oligodendrocyte survival in cuprizone-treated mice. In contrast to failure of remyelination in cuprizone-treated WT mice, surviving oligodendrocytes remyelinate axons rapidly in cuprizone-treated IGF-I Tg mice. In addition, IGF-I overexpression in brain ameliorates undernutrition induced hypomyelination during development (Ye et al., 2000). These data suggest a possible therapeutic usage for IGF-I in the treatment of demyelinating diseases and injuries. Summary Studies of cultured oligodendrocyte lineage cells strongly suggest that IGF-I promotes oligodendrocyte precursor proliferation, differentiation and survival and mature oligodendrocyte survival and myelin synthesis. While studies of IGF-I system mutant mice are consistent with these roles for IGF-I in vivo, they do not provide direct evidence of each of these functions. Furthermore, these in vivo studies do not exclude the possibility that IGF-I actions are mediated by neuronal factors. CONCLUSIONS AND FUTURE DIRECTIONS Mutant mouse models of IGF action have provided unique opportunities for in vivo studies of the growth-promoting actions of IGF-I in the developing CNS. While a role for IGF-I has been established in the control of neurogenesis, synaptogenesis, oligodendrocyte ontogeny and myelination, much research remains. Detailed studies of cell cycle kinetics in neuronal precursors are required to understand the mechanism by which IGF-I controls neuronal proliferation and differentiation, as well as studies of the intracellular signaling mechanisms through which IGF-I controls these events. The cellular and molecular mechanisms underlying IGF-I anti-apoptotic actions and IGF-I's therapeutic potential in neurodegenerative diseases are prime objectives. Given the disproportionate increases in the volumes of white matter tracts in response to increased IGF-I expression, it's role in the control of axon outgrowth and axon remodeling to form speci®c neuronal projections needs to be elucidated in order to ß 2002 Elsevier Science Ltd. All rights reserved.

assess it's therapeutic potential in developmental disorders of the CNS. While the mouse models studied to date have provided major advances in our knowledge, each is limited in some way and each introduces potential confounding variables. Because of the limitations inherent in each mutant mouse line, a precise understanding of the relative contribution of IGF-I signaling to neurogenesis and synaptogenesis is lacking. Expression of the transgenes in each IGF-I Tg mouse line studied begins at about the time of birth, and thus, after the most neuron progenitors have ®nished proliferating. For the most part, therefore, the proliferative effects of IGF-I overexpression have not been evaluated. Study of IGF-I KO mice also does not adequately address IGF-I actions on neurogenesis, because IGF-II (and possibly insulin) could compensate in part for IGF-I de®ciency. While most, if not all, IGF-I actions are ablated in IGF-I KO mice, studies in these mice also are problematic. They invariable die at birth and they are markedly growth retarded. While their small in utero size makes detailed analyses of the neurogenesis dif®cult, the most signi®cant concern is whether the multiple growth de®cits and developmental delays that they exhibit introduce confounding variables. New mutant models offer hope to better address neurogenesis as well as other issues. For example, our early results in Tg mice created to overexpress IGF-I in neural stem cells (using with a nestin promoter driven IGF-I transgene) clearly show that IGF-I stimulates early neurogenesis. We now are investigating whether IGF-I increases neuron number by stimulation of proliferation or promotion of survival. Yet another approach underway in our laboratories is to ablate the IGF1R speci®cally in neural stem cells using the Cre/Lox recombinase system [see details of methodology in (Schwenk et al., 1995)]. It is hoped that these mutant mice will have greater viability and fewer somatic abnormalities. While the later two models are virtually certain to extend our knowledge, neither will be optimal to address the apparent multiple actions of IGF-I on neural cells. In each of the later models IGF-I activity will be altered from the onset of neurogenesis, and therefore, the entire course of neurogenesis likely will to be altered in some way. It may be inappropriate, therefore, to study these mutants in order to evaluate IGF-I actions later in development or to assess IGF-I functions in the adult brain. Furthermore, such studies would almost certainly yield results that are prone to misinterpretation. Questions regarding IGF-I action at different developmental stages can be addressed, but will require different mouse models. Using a combination of inducible and cell speci®c promoters, it is now possible to induce transgene overexpression or gene ablation not only in a speci®c cell type, but also at developmental times of our choice. If we match the Neuropeptides (2002) 36(2±3), 209±220

218 D'Ercole et al.

questions we ask to the models we design and create, then we will answer most questions. We are limited only by the quality of the questions and by our creativity and imagination. GRANT SUPPORT Much of the research reported was supported by grants RO1 HD08299 and NS3891 from NICHD and NIMH, respectively, of the US National Institutes of Health (to AJD), and grant MOP 37536 from Canadian Institutes of Health Research/Canadian Neurotrama Research Program Grant, and support from the Rick Hansen Institute, NeuroScience Canada Foundation, NeuroPartners Canada, Ontario Neurotrama Foundation, Alberta Paraplegic Foundation, and British Columbia Neurotrama Initiative (to JRO). REFERENCES Accili D, Drago J, Lee EJ, Johnson MD, Cool MH, Salvatore P, Asico LD, Jose PA, Taylor SI, Westphal H (1996) Early neonatal death in mice homozygous for a null allele of the insulin receptor gene. Nature Genet 12: 106±109. Anlar B, Sullivan KA, Feldman EL (1999) Insulin-like growth factor-I and central nervous system development. Horm Metabol Res 31: 120±125. Arsenijevic Y Weiss S (1998) Insulin-like growth factor-I is a differentiation factor for postmitotic CNS stem cell-derived neuronal precursors: Distinct actions from those of brainderived neurotrophic factor. J Neurosci 18: 2118±2128. Baker J, Hardy MP, Zhou J, Bondy C, Lupu F, Bellve AR, Efstratiadis A (1996) Effects of an Ig f1 gene null mutation on mouse reproduction. Mol Endocrinol 10: 903±918. Baker J, Liu J-P, Robertson EJ, Efstratiadis A (1993) Role of insulin-like growth factors in embryonic and postnatal growth. Cell 75: 73±82. Baker NL, Russo VC, Bernard O, D'Ercole AJ, Werther G A (1999) Interactions between Bcl-2 and the IGF system control apoptosis in the developing mouse brain. Dev Brain Res 118: 109±118. Barres BA, Jacobson MD, Schmid R, Sendnter M, Raff MC (1993) Does oligodendrocyte survival depend on axons. Curr Biol 3: 489±497. Barres BA, Raff MC (1993) Proliferation of oligodendrocyte precursor cells depends on electrical activity in axons. Nature 361: 258±260. Beck KD, Powell-Braxton L, Widmer H-R, Valverde J, Hefti F (1995) Igf1 gene disruption results in reduced brain size, CNS hypomyelination, and loss of hippocampal and striatal parvalbumin-containing neurons. Neuron 14: 717±730. Behringer RR, Lewin TM, Quaife CJ, Palmiter RD, Brinster RL, D'Ercole AJ (1990) Expression of insulin-like growth factor I stimulates normal somatic growth in growth hormone-deficient transgenic mice. Endocrinology 127: 1033±1040. Besnard F, Perraud F, Sensenbrenner M, Labourdette G (1989) Effects of acidic and basic fibroblast growth factors on proliferation and maturation of cultured rat oligodendrocytes. Int J Dev Neurosci 7: 401±409. Blakemore WF (1972) Observations on oligodendrocyte degeneration, the resolution of status spongiosus and

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