Decreased Insulin-Like Growth Factor-I and Its Receptor Expression in the Hippocampus and Somatosensory Cortex of the Aged Mouse

Insulin-like growth factor-I (IGF-I) is a multifunctional polypeptide and has diverse effects on brain functions. In the present study, we compared IGF-I and IGF-I receptor (IGF-IR) immunoreactivity and their protein levels between the adult (postnatal month 6) and aged (postnatal month 24) mouse hippocampus and somatosensory cortex. In the adult hippocampus, IGF-I immunoreactivity was easily observed in the pyramidal cells of the stratum pyramidale in the hippocampus proper and in the granule cells of the granule cell layer of the dentate gyrus. In the adult somatosensory cortex, IGF-I immunoreactivity was easily found in the pyramidal cells of layer V. In the aged groups, IGF-I expression was dramatically decreased in the cells. Like the change of IGF-I immunoreactivity, IGF-IR immunoreactivity in the pyramidal and granule cells of the hippocampus and in the pyramidal cells of the somatosensory cortex was also markedly decreased in the aged group. In addition, both IGF-I and IGF-IR protein levels were significantly decreased in the aged hippocampus and somatosensory cortex. These results indicate that the apparent decrease of IGF-I and IGF-IR expression in the aged mouse hippocampus and somatosensory cortex may be related to age-related changes in the aged brain.     

Insulin-like growth factor-I activation of Akt survival cascade in neuronal cells requires the presence of its cognate receptor in caveolae

Insulin-like growth factor-I (IGF-I) plays important roles in survival of neurons. Caveolae, cholesterol-rich microdomains of plasma membrane, act as platforms for some neurotrophic factors. In this study, we examined a possible role of caveolae in IGF-I signal transduction in pheochromocytoma PC12 cells. IGF-I treatment attenuated serum withdrawal-induced apoptosis, which was reversed by treatment with methyl-beta-cyclodextrin (CD) that removes cholesterol from plasma membrane. Immunocytochemical and subcellular fractionation analyses revealed that IGF-I receptor (IGF-IR) was colocalized with caveolin-1, a major protein component in caveolae, and that CD treatment reduced IGF-IR contents in caveolae. Consistent with these findings, IGF-I phosphorylation of insulin receptor substrate-1 and Akt was impaired, and cholesterol supply restored the IGF-I action. Furthermore, experiments using small interfering RNA revealed that the reduction of caveolin-1 expression impaired the IGF-I action. In addition, the colocalization of IGF-IR with caveolin-1, and the caveolae-dependent IGF-I action were duplicated in primary culture of rat cerebellar granule neurons. These results demonstrate that the presence of IGF-IR in caveolae is required for the neuroprotective action of IGF-I.     

Localization of insulin-like growth factor-I mRNA in rat brain by in situ hybridization--relationship to IGF-I receptors

Recent evidence has demonstrated regional synthesis of insulin-like growth factor I (IGF-I) in rat brain, which is also known to contain widespread specific type I IGF receptors. In order to precisely define sites of IGF-I mRNA synthesis, and their relationship to IGF-I receptor sites, we have applied the techniques of in situ hybridization and in vitro receptor autoradiography in rat brain. Frozen sections of adult rat brain and liver were hybridized with 32P-labeled cDNA inserts for human IGF-I (780 base pairs) or a positive control transthyretin cDNA (1430 base pairs) probe, or a series of negative probes, followed by film or emulsion autoradiography. Receptor autoradiography was performed on similar sections using 125I-IGF-I in buffer, some chambers containing excess unlabeled IGF-I. Hybridization of IGF-I probe was clearly seen only in three major brain regions: the olfactory bulb, hippocampus and cerebellum, whereas transthyretin only hybridized to choroid plexus as expected, and other probes showed no hybridization. In olfactory bulb, hybridization was greatest in the internal granular and mitral cell layers, with lower levels in the glomerular layer, where IGF-I receptors were concentrated. In hippocampus, hybridization was to pyramidal cells of Ammon's horn in CA1 and CA2 layers and dentate gyrus, with some labeling in CA3. IGF-I receptors were most dense in CA2, CA3, CA4, and dentate gyrus. In cerebellum, hybridization was to the granule cell layer, with IGF-I receptors primarily in the adjacent molecular layer. We have clearly demonstrated precise sites of local IGF-I synthesis in adult rat brain, adjacent to, and sometimes overlapping sites of high density IGF-I receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histochemical localization of NADPH-diaphorase in the rat accessory olfactory bulb

The distribution of NADPH-diaphorase activity was examined inthe accessory olfactory bulb of the rat using a direct histochemicaltechnique. Labeled fibers and somata were found in all layersof the accessory olfactory bulb. The entire vomeronasal nerveand all vomeronasal glomeruli were strongly labeled, contraryto the main olfactory bulb, where only dorsomedial olfactoryglomeruli displayed NADPH-diaphorase activity. NADPH-diapborasepositive neurons were identified as periglomerular cells inthe glomerular layer and external plexiform layer, horizontalcells in the internal plexiform layer, and granule cells anddeep short-axon cells in the granule cell layer. The labeleddendrites of the granule cells formed a dense neuropile in thegranule cell layer, internal plexiform layer and external plexiformlayer. The staining pattern in the accessory olfactory bulbwas more complex than what has been previously reported, anddemonstrated both similarities and differences with the distributionof NADPH-diaphorase in the main olfactory bulb.     

Increased Tyrosine Kinase Activity but Not Calcium Mobilization Is Required for Ceramide-Induced Apoptosis

The insulin-like growth factors (IGFs) are capable of blocking apoptosis in many cell lines in vitro, potentially via activation of the IGF-I receptor (IGF-IR). We have previously shown that lower doses of the sphingolipid analogue C2-ceramide are required to induce apoptosis in IGF-IR-minus vs -positive murine fibroblasts, indicating a protective feedback loop in the latter and corroborating evidence that the IGF-IR functions as a survival receptor [1, 2]. Since, unexpectedly, C2-ceramide was capable of activating MAP kinase, phosphorylating the IGF-I receptor, and promoting entry into the G2 phase of the cell cycle, we wished to further determine the mechanisms involved. Using IGF-IR-positive fibroblasts we demonstrate here for the first time that ceramide is capable of activating a tyrosine kinase which acts at the level of the IGF-IR to increase cell death. We also demonstrate that in the presence of sodium orthovanadate, ceramide-induced death is increased, and the phosphorylation of a 75-kDa protein which associates with the IGF-I receptor is enhanced. Although the identity of this protein is not known, we speculate that it may link into the Raf kinase signaling pathway; indeed, inhibitors of MEKK reduce ceramide-induced apoptosis, thus substantiating this theory [1, 2]. Although calcium mobilization did cause apoptosis in these cells, it was not required as a mediator of ceramide-induced apoptosis. Finally, the potential hydrolysis of ceramide to sphingosine-1-phosphate was not the cause of increased MAP kinase activation, substantiating the role of an IGF-IR interacting tyrosine kinase, which may be involved in apoptosis.     

Hypertrophy of cultured adult rat ventricular cardiomyocytes induced by antibodies against the insulin-like growth factor (IGF)-I or the IGF-I receptor is IGF-II-dependent

Antibodies against the insulin-like growth factor-I (IGF-I) or the IGF-I receptor (IGF-IR) directly initiate a rapid (within 6 h) hypertrophy of isolated adult rat ventricular cardiomyocytes cultured in the absence of serum. Further, cardiomyocytes treated with either of these agonistic antibodies upregulate the expression of their genes for insulin-like growth factor-II (IGF-II) and the IGF-II receptor (IGF-IIR). Genistein, an inhibitor of the tyrosine kinase IGF-IR, also induces the cardiomyocytes to hypertrophy. Anti-IGF-II antibody inhibits the cardiomyocyte hypertrophy induced by anti-IGF-I and anti-IGF-IR antibodies or by genistein. Results are consistent with a model in which local production of IGF-II is upregulated when the IGF-IR signaling pathway is blocked and in which an IGF-II-mediated pathway, likely involving the IGF-IIR, then stimulates hypertrophy of the cardiomyocytes.     

The central pathway of primary olfactory axons is abnormal in mice lacking the N-CAM-180 isoform

Although N-CAM has previously been implicated in the growth and fasciculation of axons, the development of axon tracts in transgenic mice with a targeted deletion of the 180-kD isoform of the neural cell adhesion molecule (N-CAM-180) appears grossly normal in comparison to wild-type mice. We examined the organization of the olfactory nerve projection from the olfactory neuroepithelium to glomeruli in the olfactory bulb of postnatal N-CAM-180 null mutant mice. Immunostaining for olfactory marker protein revealed the normal presence of fully mature primary olfactory neurons within the olfactory neuroepithelium of mutant mice. The axons of these neurons form an olfactory nerve, enter the nerve fiber layer of the olfactory bulb, and terminate in olfactory glomeruli as in wild-type control animals. The olfactory bulb is smaller and the nerve fiber layer is relatively thicker in mutants than in wild-type mice. Previous studies have revealed that the plant lectin Dolichos biflorus agglutinin (DBA) clearly stains the perikarya and axons of a subpopulation of primary olfactory neurons. Thus, DBA staining enabled the morphology of the olfactory nerve pathway to be examined at higher resolution in both control and mutant animals. Despite a normal spatial pattern of DBA-stained neurons within the nasal cavity, there was a distorted axonal projection of these neurons onto the surface of the olfactory bulb in N-CAM-180 null mutants. In particular, DBA-stained axons formed fewer and smaller glomeruli in the olfactory bulbs of mutants in comparison to wild-type mice. Many primary olfactory axons failed to exit the nerve fiber layer and contribute to glomerular formation. These results indicate that N-CAM-180 plays an important role in the growth and fasciculation of primary olfactory axons and is essential for normal development of olfactory glomeruli. © 1997 John Wiley & Sons, Inc. J Neurobiol 32 : 643–658, 1997     

The Reciprocal Regulation of γ-Synuclein and IGF-I Receptor Expression Creates a Circuit That Modulates IGF-I Signaling

Insulin-like growth factor (IGF) system plays important roles in carcinogenesis and maintenance of the malignant phenotype. Signaling through the IGF-I receptor (IGF-IR) has been shown to stimulate the growth and motility of a wide range of cancer cells. γ-Synuclein (SNCG) is primarily expressed in peripheral neurons but also overexpressed in various cancer cells. Overexpression of SNCG correlates with tumor progression. In the present study we demonstrated a reciprocal regulation of IGF-I signaling and SNCG expression. IGF-I induced SNCG expression in various cancer cells. IGF-IR knockdown or IGF-IR inhibitor repressed SNCG expression. Both phosphatidylinositol 3-kinase and mitogen-activated protein kinase were involved in IGF-I induction of SNCG expression. Interestingly, SNCG knockdown led to proteasomal degradation of IGF-IR, thereby decreasing the steady-state levels of IGF-IR. Silencing of SNCG resulted in a decrease in ligand-induced phosphorylation of IGF-IR and its downstream signaling components, including insulin receptor substrate (IRS), Akt, and ERK1/2. Strikingly, SNCG physically interacted with IGF-IR and IRS-2. Silencing of IRS-2 impaired the interaction between SNCG and IGF-IR. Finally, SNCG knockdown suppressed IGF-I-induced cell proliferation and migration. These data reveal that SNCG and IGF-IR are mutually regulated by each other. SNCG blockade may suppress IGF-I-induced cell proliferation and migration. Conversely, IGF-IR inhibitors may be of utility in suppressing the aberrant expression of SNCG in cancer cells and thereby block its pro-tumor effects.     

The sox gene Dichaete is expressed in local interneurons and functions in development of the Drosophila adult olfactory circuit

In insects, the primary sites of integration for olfactory sensory input are the glomeruli in the antennal lobes. Here, axons of olfactory receptor neurons synapse with dendrites of the projection neurons that relay olfactory input to higher brain centers, such as the mushroom bodies and lateral horn. Interactions between olfactory receptor neurons and projection neurons are modulated by excitatory and inhibitory input from a group of local interneurons. While significant insight has been gleaned into the differentiation of olfactory receptor and projection neurons, much less is known about the development and function of the local interneurons. We have found that Dichaete, a conserved Sox HMG box gene, is strongly expressed in a cluster of LAAL cells located adjacent to each antennal lobe in the adult brain. Within these clusters, Dichaete protein expression is detected in both cholinergic and GABAergic local interneurons. In contrast, Dichaete expression is not detected in mature or developing projection neurons, or developing olfactory receptor neurons. Analysis of novel viable Dichaete mutant alleles revealed misrouting of specific projection neuron dendrites and axons, and alterations in glomeruli organization. These results suggest noncell autonomous functions of Dichaete in projection neuron differentiation as well as a potential role for Dichaete‐expressing local interneurons in development of the adult olfactory circuitry. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Expression of the insulin-like growth factor-I receptor in primary breast cancer and lymph node metastases: correlations with estrogen receptors alpha and beta.

Numerous laboratory studies and some epidemiological data have suggested the involvement of the insulin-like growth factor-I receptor (IGF-IR) in breast cancer development and progression. However, data on IGF-IR expression in human tissues, including breast cancer sections, are limited and often inconsistent. We therefore examined by immunohistochemistry the expression of IGF-IR in primary tumors and breast cancer metastases to lymph nodes, and correlated IGF-IR positivity with estrogen receptor (ER) status and selected clinicopathological features. We found that 1) IGF-IR was expressed in primary tumors as well as in lymph node metastases, but the expression in primary tumors was more frequent (56 % vs. 44.4 %); 2) IGF-IR expression in primary tumors was associated with negative node status (p < 0.033); 3) in node-negative primary tumors, IGF-IR positively correlated with ERbeta (p < 0.008; r = 0.538), but not with ERalpha, tumor size or grade; 4) both IGF-IR-positive and IGF-IR-negative primary tumors were found to produce IGF-IR-positive as well as IGF-IR-negative metastases; 5) in metastases, IGF-IR expression did not associate with ERalpha, ERbeta or any of the studied pathobiological markers. The results suggest that IGF-IR could become a viable pharmaceutical target in breast cancer therapy, but such therapy should be based on IGF-IR assessment in primary tumor and metastasis in each potential patient.     

Association of heterotrimeric G(i) with the insulin-like growth factor-I receptor. Release of G(betagamma) subunits upon receptor activation

The insulin-like growth factor-I receptor (IGF-IR) is a key regulator of cell proliferation and survival. Activation of the IGF-IR induces tyrosine autophosphorylation and the binding of a series of adaptor molecules, thereby leading to the activation of MAPK. It has been demonstrated that pertussis toxin, which inactivates the G(i) class of GTP-binding proteins, inhibits IGF-I-mediated activation of MAPK, and a specific role for G(betagamma) subunits in IGF-I signaling was shown. In the present study, we have investigated the role of heterotrimeric G(i) in IGF-IR signaling in neuronal cells. Pertussis toxin inhibited IGF-I-induced activation of MAPK in rat cerebellar granule neurons and NG-108 neuronal cells. G(alphai) and G(beta) subunits were associated with IGF-IR immunoprecipitates. Similarly, in IGF-IR-null mouse embryo fibroblasts transfected with the human IGF-IR, G(i) was complexed with the IGF-IR. G(alphas) was not associated with the IGF-IR in any cell type. IGF-I induced the release of the G(beta) subunits from the IGF-IR but had no effect on the association of G(alphai). These results demonstrate an association of heterotrimeric G(i) with the IGF-IR and identify a discrete pool of G(betagamma) subunits available for downstream signaling following stimulation with IGF-I.     

Evolution and ultrastructure of the bovine spermatogonia precursor cell line

We have used a cytochemical technique to investigate the distribution of acetylcholinesterase (AChE) activity in the deutocerebrum of the brain of the sphinx moth Manduca sexta. To distinguish between extra-and intracellular pools of the enzyme, some brains were treated prior to histochemical staining with echothiophate, an irreversible AChE inhibitor which penetrates cell membranes very slowly and, therefore, inhibits only extracellular AChE. In the antennal nerve, fascicles of presumably mechanosensory fibers show echothiophateinsensitive AChE activity. They bypass the antennal lobe and project to the antennal mechanosensory and motor center of the deutocerebrum. In the antennal lobe, fibers in the coarse neuropil, cell bodies in the lateral cell group, and all glomeruli exhibit AChE activity. In most ordinary glomeruli, echothiophate-sensitive AChE activity is concentrated in the outer cap regions, corresponding to the terminal arborizations of olfactory afferents. A previously unrecognized glomerulus in the ventro-median antennal lobe shows uniform and more intense AChE-specific staining that the other glomeruli. No AChE activity appeared to be associated with malespecific pheromone-sensitive afferents in the macro-glomerular complex. About 67 interneurons with somata in the lateral cell group of the antennal lobe show echo-thiophate-insensitive AChE activity. These neurous seem to be members of two types of antennal-lobe projection neurons with fibers passing through the outer-antenno-cerebral tract to the protocerebrum. AChE-stained arborizations of these neurons appear to invade all glomeruli, including three distinguishable subunits of the male-specific macroglomerular complex. In echothiophate-treated animals, the projections of one of these types of fiber form large terminals in the lateral horn of protocerebrum, which partly protrude into the adjacent glial cell layer. The results suggest that extracellularly accessible AChE is associated with ordinary olfactory receptor terminals but apparently not with pheromone-sensitive afferents. Intracellular AChE appears to be present in antennal mechanosensory fibers and in two types of olfactory projection neurons of the antennal lobe. The study provides further evidence for cholinergic neurotransmission of most antennal afferents. The AChE-containing interneurons might be cholinergic as well or use the enzyme for functions unrelated to hydrolysis of acetylcholine.Abbreviations ACh acetylcholine - AChE acetylcholinesterase - AL antennal lobe - AMMC antennal mechanosensory and motor center - ChAT choline acetyltransferase - IACT inner antenno-cerebral tract - MGC macroglomerular complex     

Olfactory marker protein during ontogeny: Immunohistochemical localization

Specific immunohistochemical staining for the olfactory marker protein (OMP) is first demonstrated in rat olfactory receptor neurons at embryonic day 18, at which age no OMP can be seen in the olfactory bulb or vomeronasal epithelium. At 21 days OMP staining in the olfactory epithelium is more extensive and is evident in the fibrous and glomerular layers of the bulb as well. Staining intensity increases progressively until the full adult pattern is seen by 1 month postnatally. In the vomeronasal organ, staining is not observed until the fourth postnatal day and, even then, only with higher antiserum concentrations. In mice, very similar results are obtained, except for a much earlier appearance of OMP, on embryonic day 14. Olfactory epithelium from 12- and 13-day rat embryos maintained in organ culture for up to 2 weeks did not exhibit OMP staining, nor did several neural or nonneural tissues from adult animals. The temporal and causal interrelationships between OMP and other indicators of olfactory receptor cell maturation are considered.     

The activated insulin-like growth factor I receptor induces depolarization in breast epithelial cells characterized by actin filament disassembly and tyrosine dephosphorylation of FAK, Cas, and paxillin.

Insulin-like growth factor I (IGF-I) promotes the motility of different cell types. We investigated the role of IGF-I receptor (IGF-IR) signaling in locomotion of MCF-7 breast cancer epithelial cells overexpressing the wild-type IGF-IR (MCF-7/IGF-IR). Stimulation of MCF-7/IGF-IR cells with 50 ng/ml IGF-I induced disruption of the polarized cell monolayer followed by morphological transition toward a mesenchymal phenotype. Immunofluorescence staining of the cells with rhodamine-phalloidin revealed rapid disassembly of actin fibers and development of a cortical actin meshwork. Activation of phosphatidylinositol (PI)3-kinase downstream of the IGF-IR was necessary for this process, as blocking PI 3-kinase activity with the specific inhibitor LY 294002 at 10 microM prevented disruption of the filamentous actin. In parallel, IGF-IR activation induced rapid and transient tyrosine dephosphorylation of focal adhesion proteins p125 focal adhesion kinase (FAK), p130 Crk-associated substrate (Cas), and paxillin. This process required phosphotyrosine phosphatase (PTP) activity, since pretreatment of the cells with 5 microM phenylarsine oxide (PAO), an inhibitor of PTPs, rescued FAK and its associated proteins Cas and paxillin from IGF-I-induced dephosphorylation. In addition, PAO-pretreated cells were refractory to IGF-I-induced morphological transition. Thus, our findings reveal a new function of the IGF-IR, the ability to depolarize epithelial cells. In MCF-7 cells, mechanisms of IGF-IR-mediated cell depolarization involve PI 3-kinase signaling and putative PTP activities.     

猫嗅球外丛层和僧帽细胞层年龄相关形态学变化

分别用Nissl法及免疫组织化学ABC法标记青、老年猫嗅球中嗅觉二级神经元和外丛层胶质细胞,显微镜下观察其分布并计数,对嗅觉二级神经元胞体直径和外丛层厚度进行测量,比较其年龄相关性变化,研究神经元与胶质细胞之间的关系,探讨老年性嗅觉功能衰退的相关神经机理。结果显示,老年猫嗅觉二级神经元胞体直径和分布密度均有不同程度的显著性下降(P<0.05);外丛层厚度变化不明显(P>0.05);外丛层胶质细胞特别是星形胶质细胞显著性增生(P<0.05)。表明在衰老过程中嗅觉二级神经元有丢失,并呈现功能下降,可能是老年性嗅觉功能衰退的原因之一。同时外丛层胶质细胞增生以进一步保护神经元,延缓其衰老。     

Mitogenic effect of the insulin analogue glargine in malignant cells in comparison with insulin and IGF-I.

The aim of the study was to investigate if the insulin analogue glargine, with an increased affinity for the IGF-I receptor (IGF-IR), affects the cell growth to a larger extent than human insulin in malignant cells expressing IGF-IRs. The breast cancer cell lines MCF-7 and SKBR-3, and the osteosarcoma cell line SaOS-2 were used. Gene expression was determined by real-time RT-PCR and receptor protein quantified by ELISAs. Receptor phosphorylation was assessed by immunoprecipitation and Western blot. Mitogenic effect was determined as (3)H-thymidine incorporation into DNA. The gene expression of insulin receptor (IR) varied between 4.3-7.5 x 10(-3) and the expression of IGF-IR between 7.7-147.7 x 10(-3) in relation to GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Insulin receptor and IGF-IR protein varied between 2.0-4.1 ng/mg protein and 2.0-40.4 ng/mg protein, respectively. The IGF-IR was phosphorylated by IGF-I at a concentration of 10(-10)-10(-9) M. All three polypeptides stimulated DNA synthesis in MCF-7, SKBR-3, and SaOS-2 cells. SaOS-2 cells were more sensitive to IGF-I than to insulin and glargine. MCF-7 cells were more sensitive to des(1-3)IGF-I than to IGF-I. In SKBR-3 and SaOS-2 cells, glargine tended to be more potent than human insulin to stimulate DNA synthesis. Our results suggest that glargine, compared to human insulin, has little or no increased mitogenic effect in malignant cells expressing IGF-IRs.     

Expression and regulation of mRNAs for insulin-like growth factor-I receptor and LH receptor in corpora lutea

Relationship between insulin-like growth factor-I receptor (IGF-IR) and luteinizing hormone receptor (LHR) mRNA expression as well as their regulation was determined in rat corpora lutea (CL) . In the CL of estrous cycle rat, LHR mRNA positive CL expressed high level of mRNA of IGF-IR. While the expression of LHR mRNA decreased on estrus, the CL still expressed relatively high level of IGF-IR mRNA. In pseudopregnant rat CL, the expression level of LHR mRNA was low on day 1, the most intense signals were detected on day 8, the signals of LHR mRNA became undetectable on day 14. In contrast to LHR expression, the high level of IGF-IR mRNA was observed in pseudopregnant CL of day 1, and thereafter its signals were detected from day 2 to day 14. Pregnant rat CL expressed both LHR and IGF-IR mRNAs. IGF-I stimulated LHR expression in CL. PGF2 inhibited expression of IGF-IR and LHR. PGE2 negated the inhibiting effects of PGF2. These data suggest that IGF-I may be involved in regulating CL function, and maintaining CL structure through changes in expression of its receptors. Inhibited expression of IGF-IR by PGF2 may be part of mechanisms for regression of CL.