The Insulin-Like Growth Factor System and Its Pleiotropic Functions in Brain V.C Russo, P.D. Gluckman, E.L. Werther Endocrine Reviews (7): Journal Club Masahiro, Masuzawa

In 1957, Salmon and Daughaday ： "sulfation factor" 、 cartilage sulfation and longitudinal bone. Dulak and Temin ： the cell proliferative factors in serum and termed one such activity multiplication-stimulating activity. insulin-like activity not suppressible by antiinsulin antibodies [nonsuppressible insulin-like activity (NSILA) I and II] 、 similar biochemical structure to the ß-chain of insulin. later renamed as somatomedins. Shortly thereafter, two mammalian somatomedins were identified by protein sequence and cDNA data and their structural homology with proinsulin led to their current designation of IGF-I and IGF-II. In the middle 1990s, Sara and co-workers identified a brain-specific variant of IGF-I, des(1-3) or "truncated" IGF-I, which lacks the first three amino acids and is more potent than intact IGF-I in various cell culture systems, probably due to its lower affinity for IGF binding proteins (IGFBPs) IGF-1 mRNA, IGF-1 receptor mRNA, and IGF binding proteins were found expressed in postnatal rat brain. Historical Perspective

The IGFs: major growth factors, consist of A, B, C, and D domains.Large parts of the sequences within the A and B domains are homologous to the α- and ß-chain of the human proinsulin. This sequence homology is 43% for IGF-I and 41% for IGF-II. No sequence homology exists between the C domains of IGFs and the C peptide region of human proinsulin. The IGF peptides

Figure 1. Binding of Circulating IGFs to Target Cells. Three binding sites for the IGFs are expressed on the surface of target cells: the insulin receptor, the IGF-I receptor, and the IGF-II-mannose-6-phosphate receptor (IGF-II-M-6-PR). Insulin and IGF-I receptors are structurally homologous — both are tyrosine kinases and interact with various intracellular mediators. The IGF-II-M-6-PR differs from the insulin and IGF-I receptors in structure, and it has no known signaling action. Insulin binds to its own receptor and to the IGF-I receptor. Both IGFs bind to the insulin receptor. The relative affinities of ligands for the various receptors and binding proteins are indicated by the width of the arrows in the upper part of the diagram. Adapted with modifications from Ruderman et al., 9 with the permission of the author and the publisher. New Engl J Med 1997, (337): lysosomal enzyme trafficking, endocytosis, and lysosomal degradation of extracellular ligands, regulation of apoptotic/mitogenic effects

1) transport IGF in plasma and control its diffusion and efflux from the vascular space. 2) increase the half-life and regulate clearance of the IGFs. 3) provide specific binding sites for the IGFs in the extracellular and pericellular space. 4) modulate, inhibit, or facilitate interaction of IGFs with their receptors. IGFBP biological activity is regulated by posttranslational modifications such as glycosylation and phosphorylation and/or differential localization of the IGFBPs in the pericellular and extracellular space. The effects of the IGFBPs are further regulated by the presence of specific IGFBP proteases, which cleave the binding proteins, generating fragments with reduced or no binding affinity for the IGFs. Some IGFBPs, including IGFBP-2 and -3, can induce direct cellular effects independent of the IGFs. IGFBP-3, similar to IGFBP-5, contains sequences with the potential for nuclear localization and detection of IGFBP-3 in the nuclei of dividing cells. IGFBPs ( IGF binding proteins)

FluidMajor IGFBP SerumIGFBP-3 Amniotic fluidIGFBP-1 Follicular fluidIGFBP-3 CSFIGFBP-2, IGFBP-6 Table 2. Distribution of IGFBPs in biological fluids Endocrine Reviews 1997: 18 (6):

IG FB P IGF carrier function in serum Daily variatio n Major regulators Alterations during physiological situation Alterations during pathological situation 1MinorYesInsulinAgingDiabetes Glucagon 2MinorNoIGFs, GH, nutrition AgingUndernutrition, Tumor 3MajorNoIGFs, GH, glucocorticoids PubertyGH deficiency AgingAcromegaly 4MinorNot known PTHAgingOsteoporosis 5MinorNot known IGFs, glucocorticoids PubertyOsteoporosis Aging 6MinorNot known Retinoic acidNot known Table 5. Regulators of serum IGFBP levels

Figure 3. Mean Serum IGF-I Concentrations in Normal Subjects from Birth to Adulthood. New Engl J Med 1997, (337):

Figure 4. Concentrations of IGFs and IGFBPs in adult human serum. IGF-I, IGF-II, IGFBP-3, IGFBP-4, and IGFBP-5 values were determined in the author’s laboratory. Data for IGFBP-1, IGFBP-2, and IGFBP-6 were compiled from published literature. Values are mean ± SD.[Reproduced with permission from S. Mohan and D. J. Baylink: J Clin Endocrinol Metab 81:3817–3820, 1996 (149).

Figure 2. Proposed model of the forms in which IGFs circulate in human serum. The 150-kDa complex consists of 7.5 kDa IGF-I or IGF-II plus 38–43 kDa IGFBP-3 and a 80- to 90-kDa non-IGF-binding acid-labile component called ALS. The 50-kDa complex consists of IGF-I or IGF-II bound to one of the remaining five IGFBPs. Endocrine Reviews 1997: 18 (6):

Figure 6. Modulation of IGF bioavailability by IGFBP proteolysis. IGF proteases may regulate the availability of IGFs by controlling the transport of IGFs from the vascular space into tissue space. The majority of IGFs exist as ALS-IGFBP-3-IGF complex in the serum, which does not cross the vascular barrier. Since transportation of serum IGFs from the vasculature into the tissue space is necessary in order for IGFs to elicit hormonal growth-stimulating responses, the IGFs bound to IGFBP-3-ALS complex must be released first. This can be accomplished by the IGFBP-3 protease produced by vascular endothelial cells or by IGFBP-3 protease present in serum. Proteolysis of IGFBP-3 by IGFBP-3 protease results in disruption of this complex and release of IGFs. IGFs, thus released, may get transported into the tissue space or may bind to other IGFBPs such as IGFBP-4 and cross the endothelium. Because the small molecular mass IGFBPs are present in excess, this is likely to occur. The binding of IGFs to these small molecular mass IGFBPs may protect the IGFs from degradation and may also increase half-life in the circulation. Upon transport into the tissue space, IGFBP-4 protease produced by target cells may release the IGFs to bind to IGF receptor and exhibit a growth-promoting response. Thus IGFBP protease may play a role in controlling the transport of IGFs into the tissue space and regulating the availability of free IGFs in the tissue space.

IGFBPsExpression in the nervous systemActions IGFBP 1Not detectedInhibit somatic growth, weight gain, tissue growth, glucose metabolism IGFBP 2early in embryogenesis structures including the neural tube and neuroepithelium, epithelium of the choroid plexus, the floor plate, and the infundibulum. Adult olfactory bulb. Regulates IGF-I cellular responses by facilitating receptor targeting of IGF-I or modulating IGF-I bioavailability in the pericellular space. Act as a "linker" molecule allowing pericellular storage of IGF. IGFBP 3low level in the CNS, mainly in nonneuronal structures including epithelial cells. Promote either enhancement or inhibition of IGF-I action in brain cells Neuronal degeneration in AD IGFBP 4a very low level in the CNS meningeal cells, astrocytes, and fetal neuronal cells Local modulator of IGF action IGFBP 5highly abundant during brain development.The early expression of IGFBP-5 at embryonic d 10.5 indicates a key role of this IGFBP during embryogenesis. a modulator or determinant of IGF action. IGFBP 6tightly restricted to trigeminal ganglia and, relative to the rest of the embryo The highest levels of expression in the adult animal are in the hindbrain, spinal cord, cranial ganglia, and dorsal root ganglia. Preferential binding to the IGF-II ligand (relatively specific inhibitor of IGF-II actions). Maintenance of cells associated with the coordination of sensorimotor function in the cerebellum.

Expression in the nervous systemActions IGFsDuring embryogenesis, IGF-I mRNA expression is detectable in many brain regions, with its expression being particularly high in neuronal rich regions such as the spinal cord, midbrain, cerebral cortex, hippocampus, and olfactory bulb. The mitral and tufted cells of the olfactory bulb, which undergo constant cell renewal/turnover ; in these cells IGF-I expression persists at a high level throughout life. In the development of the nervous system, with demonstrated effects on many stages of brain development including cell proliferation, cell differentiation, and cell survival. Postnatal circulating IGF-I might exert neurogenic/survival activity IGF- Ⅰ receptor Expressed from early stages of embryogenesis and throughout life. High levels of expression detected in the developing cerebellum, midbrain, olfactory bulb, and in the ventral floorplate of the hindbrain. The level of IGF-IR decreases to adult levels soon after birth but remains relatively high in the choroid plexus, meninges, and vascular sheaths IGF- Ⅱ receptor the pyramidal cell layers of the hippocampus, the granule layer of the dentate gyrus, olfactory bulb, the choroid plexus, and in the cerebral vasculature, ependymal cells, retina, pituitary, brainstem, and spinal cord Transporting lysosomal enzymes. Participate in control of neuronal growth, differentiation, and repair, processes regulated by IGF-II/M6P receptor ligands including LIF, TGFß, and retinoic acid.

FIG. 1. The IGF system in the rat brain olfactory bulb. IGF-I is expressed and synthesized in distinct areas of the rat brain, adjacent to regions rich in IGF-I receptors and IGFBPs (i.e., olfactory bulb is shown). This provides strong evidence for an autocrine and paracrine action of the IGFs in the nervous system. IGF action is modulated by locally expressed IGFBPs. ONL, Olfactory nerve layer; GL, glomerular cell layer; EPL, external plexiform cell layer; MI, mitral cell layer; GRL, granular cell layer. Section A-A shows a hematoxylin-eosin staining of olfactory bulb. [Modified with permission from L. W. Swanson: Brain Maps: Structures of the Rat Brain, 2nd edition, Elsevier, Amsterdam, 1998/1999 (528 ).] Area in the square is enlarged and represented in the cartoon.

FIG. 2. Neuroendocrine cross-talk. In vivo, growth factors such as IGF-I do not exist in isolation. Hence, the presence of other growth factors may further modulate the biological activity and cellular responses of IGFs

Figure 8. IGF-1 immunostaining of the rat cerebral cortex after transient global cerebral ischemia. A, IGF-1 expression (red) was apparently restricted mainly to neurons at 4 d of recovery from cardiac arrest. B, Double staining for HIF-1 (green) showing colocalization with some IGF-1-positive neurons (arrows). Scale bar, 100 µm. The Journal of Neuroscience, October 15, 2002, 22(20):

Altered Expression of the IGF System in Response to CNS Injury Hypoxic/ischemic injured and stroke rat model:3 d after hypoxia, IGF mRNA induction. IGF- Ⅱ, IGFBP-2, IGFBP-3, IGFBP-5 genes are induced. In the periinfarcted regions, IGFBP-2 was highly expressed by reactive astrocytes that were juxtaposed to surviving neurons. Centrally administrated 3H-IGF-1 is rapidly translocated to neurons and glia ( colocalized IGFBP-2). The 3H-IGF-1 signal persist for up to 6 hours. → local storage of the IGF-1 molecule. IGFBP-3 is only, moderately induced in reactive microglia, and glial cells and is substantially decreased in neuronal cells of the region of injury. In the early stages of the injury response, IGFBP-3 expression increased rapidly in vascular endothelial cells. → role for IGFBP-3 as carrier/transporter of vascular IGF-1 into the brain tissue in the early phases of the injury response. IGFBP-5 is up-regulated after severe hypoxia-ischemic injury in the infant rat brain. → required to maximize the availability of IGF. After peripheral nerve injury, IGFBP-6 mRNA and its protein expression are strongly up-regulated in the spinal motoneurons. → regulates axonal regeneration.

FIG. 3. Altered expression of the IGF system in response to CNS injury. A role for endogenous IGF system in the injured brain is suggested by a number of studies showing the induction of components of the IGF system after transient unilateral hypoxic/ischemic injury and stroke in the rat model. IGF-I mRNA induction is seen within infarcted regions by 3–5 d after hypoxia/ischemic brain injury (d 5 after insult is shown). In addition, IGF-I receptor and IGFBPs genes are differentially induced in specific regions after hypoxic/ischemic injury in the same model, suggesting that they may modulate the actions of IGF-I in a spatiotemporal-specific manner (d 3 and 5 after insult are shown). In panels A–C, IGF-I, IGF-I receptor, IGFBP-3, IGFBP-5, and IGFBP-2 mRNA were detected by in situ hybridization (ISH; 5-µm paraffin sections). Immunohistochemical staining for glial fibrillary acid protein (GFAP) is shown in panels A and C, whereas staining for the 150-kDa neurofilament (150 kDa-NF) is shown in panel B. Immunoreactivity for IGFBP-2 is shown in panel C. CO, Cortex; AH, Ammon’s horn; DG, dentate gyrus; IHC, immunoistochemistry. Hematoxylin-eosin staining is shown in the left panels [obtained from L. W. Swanson: Brain Maps: Structures of the Rat Brain, 2nd edition, Elsevier, Amsterdam, 1998/1999 (528 ), with permission from Elsevier].

FIG. 4. A model for neurotrophic and neuroprotective actions of IGF-I in brain. Secreted IGF-I exerts local autocrine (1 ) or paracrine (2 ) trophic actions. A family of IGFBPs (3 ) modulates IGF-I bioavailability. IGFBP-2, the most abundant brain IGFBP, mediates pericellular storage of IGF-I via interaction with cell surface proteoglycans (PG) (4 ) or components of the extracellular matrix. Cell surface IGF-I/IGFBP-2/PG complexes (4 ) are suggested to play a role in targeting of IGF-I to its membrane receptors. IGFBP-2 mediated IGF-I receptor targeting at the cell surface, and this event might be further potentiated by the presence of a specific IGFBP-2 protease (5 ), which generates IGFBP-2 fragments that have reduced affinity for IGF-I. In response to a number of cerebral insults (i.e., hypoxia/ischemic brain injury) (6 ), IGFBP-2 proteolysis might also affect the level of pericelluar IGF-I (7 ), therefore augmenting its neuroprotective activity (8 ). Following cerebral insult activation and recruitment of specialized brain cells (9 ) might further contribute to modulate the local IGF system (9 ).

Altered Expression of the IGF System in Malignancies of the Nervous System Increased expression of IGF- Ⅰ, IGF- Ⅱ, IGF- Ⅰ R is present in a wide range of human cancer. IGF- Ⅰ R overexpression leads to cellular transformation, tumor cell proliferation, growth, migration, and invasion. Glioblastoma cell treatment with IGF- Ⅰ triple helix-forming DNA, antisense IGF- Ⅰ R, kinase-defective IGF- Ⅰ R, or IGF- Ⅰ R mutant dominant-negative constructs all induce growth suppression and /or apotosis. High IGF- Ⅱ expression occurs in meningiomas, and the IGF- Ⅱ /IGFBP-2 ratio, indicating free IGF- Ⅱ levels, correlates with tumor anaplastic histopathology. IGF- Ⅰ R overexpression protects neuroblastoma from apotosis.

IGF- Ⅰ Therapy in Nervous System Disease Models Intracerebroventricular administration of IGF- Ⅰ 2 h after injury was shown to be neuroprotective in a dose-dependent fashion. IGF- Ⅱ administered in conjunction with IFG- Ⅰ attenuates the neuroprotective effect of IGF- Ⅰ IGF- Ⅰ administration improved cognitive function after traumatic brain injury. In experimental autoimmune encephalomyelitis( preclinical model for multiple sclerosis), IGF- Ⅰ improve neurological and histological outcome. IGF- Ⅰ in vitro inhibits amyloid induced nueronal death, induces choline acetyl-transferase and affect CNS amyloid-β levels. IGF- Ⅰ increases Aβ clearance form brain by enphancing transport of Aβ carrier proteins into the brain through the choroid plexus. IGF prevents apoptosis in MN, glial cells, and muscles cells. IGF- Ⅰ serum levels are decreased in ALS patients. Expression IGF- Ⅰ receptors is increased in the spinal cord of ALS patients.

Figure 6. The signal transduction pathway that mediates the regulation of tau phosphorylation by insulin and IGF-1 in NT2N neurons. The diagram shows that GSK-3 phosphorylates tau in neuronal cells, and insulin and IGF-1 regulate tau phosphorylation by inhibition of GSK-3. Our data suggest that this effect is mediated through activation of the PI(3)K-PKB pathway but not the MAPK-MAPKAPK1 pathway (indicated by ×). The role of p70 S6K is also excluded (×). The American Society for Biochemistry and Molecular Biology, Volume 272, Number 31, Issue of August 1, 1997 pp

Figure 5. Proposed mechanism of extrahepatic tumor-induced hypoglycemia. The hypoglycemic state in patients with-non islet cell tumors is associated with an increase in serum level of pro-IGF-II, a decrease in the circulating level of the 150-kDa complex, and a corresponding increase in the circulating level of the 50-kDa complex. The altered distribution of IGFs between the 150-kDa and 50-kDa complexes is likely to be due to the failure of tumor-secreted pro-IGF-II to form a complex with ALS and IGFBP-3. The increase in 50-kDa IGF pool increases the bioavailability of IGFs (because 50-kDa and not 150- kDa IGF complex can cross the vascular endothelium) to produce insulin-like effects in the target tissues. The association between the decreased 150-kDa complex and glucose level in the serum of tumor-induced hypoglycemia emphasizes the central role of the 150-kDa IGF complex in glucoregulation (248–251, 253).

Figure 4. The Role of IGF-II in Tumor Hypoglycemia. Some tumors secrete large amounts of a prohormone form of IGF-II ("big IGF-II"). Big IGF-II directly stimulates the uptake of glucose by the tumor and by insulin-responsive tissues such as muscle and fat. Hepatic glucose production and insulin secretion are decreased by hypoglycemia and by the direct inhibitory effects of big IGF-II on the pancreatic beta cells. In addition, big IGF-II inhibits the secretion of pituitary growth hormone (GH), which in turn decreases the synthesis and secretion of IGF-I and IGF-binding protein 3 (IGF BP-3); decreased concentrations of IGF BP-3 enhance the effects of circulating IGF-II.New Engl J Med 1997, (337):

Figure 2. Micrograph demonstrating neuritic plaques in the neocortex of a patient with Alzheimer disease (silver stain; original magnification, x400).

Figure 3. Schematic diagram of the proteolytic processing of the amyloid precursor protein (APP). The numbers above the fragments indicate the amino acid sequence relative to the first amino acid of the A ß domain. A ß = ß -amyloid; α-secretase = the enzyme that cleaves APP within the A ß domain, preventing the formation of amyloid fibrils; γ-secretase = the enzyme that cleaves both sAPP-α and sAPP- ß to form the p3, C57/59, and A ß fragments; sAPP-α = soluble APP-α ; sAPP- ß = soluble APP- ß.

Figure 4. Micrograph demonstrating neurofibrillary tangles in the neocortex of a patient with Alzheimer disease (Bielschowsky stain; original magnification, x200).