Age-related functional and structural retinal modifications in the Igf1-/- null mouse.

BACKGROUND: Mutations in the gene encoding human insulin-like growth factor-I (IGF-I) cause syndromic neurosensorial deafness. To understand the precise role of IGF-I in retinal physiology, we have studied the morphology and electrophysiology of the retina of the Igf1(-/-) mice in comparison with that of the Igf1(+/-) and Igf1(+/+) animals during aging. METHODS: Serological concentrations of IGF-I, glycemia and body weight were determined in Igf1(+/+), Igf1(+/-) and Igf1(-/-) mice at different times up to 360days of age. We have analyzed hearing by recording the auditory brainstem responses (ABR), the retinal function by electroretinographic (ERG) responses and the retinal morphology by immunohistochemical labeling on retinal preparations at different ages. RESULTS: IGF-I levels are gradually reduced with aging in the mouse. Deaf Igf1(-/-) mice had an almost flat scotopic ERG response and a photopic ERG response of very small amplitude at postnatal age 360days (P360). At the same age, Igf1(+/-) mice still showed both scotopic and photopic ERG responses, but a significant decrease in the ERG wave amplitudes was observed when compared with those of Igf1(+/+) mice. Immunohistochemical analysis showed that P360 Igf1(-/-) mice suffered important structural modifications in the first synapse of the retinal pathway, that affected mainly the postsynaptic processes from horizontal and bipolar cells. A decrease in bassoon and synaptophysin staining in both rod and cone synaptic terminals suggested a reduced photoreceptor output to the inner retina. Retinal morphology of the P360 Igf1(+/-) mice showed only small alterations in the horizontal and bipolar cell processes, when compared with Igf1(+/+) mice of matched age. CONCLUSIONS: In the mouse, IGF-I deficit causes an age-related visual loss, besides a congenital deafness. The present results support the use of the Igf1(-/-) mouse as a new model for the study of human syndromic deaf-blindness.

[Neurotrophic action of insulin-like growth factor-I in the inner ear]. / Acciones neurotróficas del factor de crecimiento similar a la insulina de tipo I en el oído interno.

INTRODUCTION: Loss of hearing constitutes one of the most frequent disabling sensory impairments in the developed world. Different therapeutic approaches are currently being studied, including treatment with stem cells, genetic manipulation and pharmacological protection. AIM: To evaluate the role played by insulin-like growth factor-I (IGF-I) in the development, maintenance and repair of auditory functioning. DEVELOPMENT: Proper development of the inner ear is dependent on a suitable coordination of the cell processes of proliferation, differentiation, neurogenesis and programmed cell death, which are regulated by different factors, one of which is IGF-I. During the embryogenesis of the inner ear, this factor is expressed in abundance and is essential for cell survival and maintaining neuronal precursors. Studies conducted in Igf-1-/- null mice have highlighted its importance in the development and continued functioning of the inner ear. Mice with a deficit in this gene display morphological disorders that correspond to severe functional deficiencies, which are confirmed by analysing brainstem auditory evoked potentials. A deficit of IGF-I in humans is also accompanied by profound sensory hypoacusis. CONCLUSIONS: In a scenario like this, IGF-I appears as a key factor in the development of auditory functioning and a candidate for regenerative therapy of the inner ear.

IGF-1 deficiency causes atrophic changes associated with upregulation of VGluT1 and downregulation of MEF2 transcription factors in the mouse cochlear nuclei.

Insulin-like growth factor 1 (IGF-1) is a neurotrophic protein that plays a crucial role in modulating neuronal function and synaptic plasticity in the adult brain. Mice lacking the Igf1 gene exhibit profound deafness and multiple anomalies in the inner ear and spiral ganglion. An issue that remains unknown is whether, in addition to these peripheral abnormalities, IGF-1 deficiency also results in structural changes along the central auditory pathway that may contribute to an imbalance between excitation and inhibition, which might be reflected in abnormal auditory brainstem responses (ABR). To assess such a possibility, we evaluated the morphological and physiological alterations in the cochlear nucleus complex of the adult mouse. The expression and distribution of the vesicular glutamate transporter 1 (VGluT1) and the vesicular inhibitory transporter (VGAT), which were used as specific markers for labeling excitatory and inhibitory terminals, and the involvement of the activity-dependent myocyte enhancer factor 2 (MEF2) transcription factors in regulating excitatory synapses were assessed in a 4-month-old mouse model of IGF-1 deficiency and neurosensorial deafness (Igf1 (-/-) homozygous null mice). The results demonstrate decreases in the cochlear nucleus area and cell size along with cell loss in the cochlear nuclei of the deficient mouse. Additionally, our results demonstrate that there is upregulation of VGluT1, but not VGAT, immunostaining and downregulation of MEF2 transcription factors together with increased wave II amplitude in the ABR recording. Our observations provide evidence of an abnormal neuronal cytoarchitecture in the cochlear nuclei of Igf1 (-/-) null mice and suggest that the increased efficacy of glutamatergic synapses might be mediated by MEF2 transcription factors.

Strict regulation of c-Raf kinase levels is required for early organogenesis of the vertebrate inner ear.

Regulation of organogenesis involves a dynamic balance of the mechanisms regulating cell division, differentiation and death. Here we have investigated the pattern of expression of c-Raf kinase in the inner ear during early developmental stages and the consequences of manipulating c-Raf levels by misexpression of c-raf viral vectors in organotypic cultures of otic vesicle explants. We found that otic vesicles expressed c-Raf and its level remained constant during embryonic days 2 and 3 (E2-E3). c-Raf activity was increased in response to insulin like growth factor-I (IGF-I) and the activation by IGF-I of the c-Raf kinase pathway was a requirement to turn on cell proliferation in the otic vesicle. Overexpression of c-raf in E2.5 explants increased the proliferative response to low serum and IGF-I and blocked differentiation induced by retinoic acid. The increase in c-Raf levels also prevented nerve growth factor (NGF)-dependent induction of programmed cell death. Consistent with these results, the expression of a dominant negative c-Raf mutant potentiated retinoic acid action and decreased the rate of cell proliferation. We conclude that a strict control of c-Raf levels is essential for the co-ordination of the biological processes that operate simultaneously during early inner ear development.

c-Raf regulates cell survival and retinal ganglion cell morphogenesis during neurogenesis.

The signaling cascade Ras/Raf/mitogen-activated protein kinases modulates cell proliferation, differentiation, and survival, all key cellular processes during neural development. To better define the in vivo role of Raf during chick retinal neurogenesis, we interfered with Raf-dependent signaling during days 4.5 to 7.5 of embryonic development by expressing a dominant negative mutant of c-Raf (DeltaRaf), which blocks Ras-dependent Raf activation, and by overexpressing wild-type c-Raf. DeltaRaf expression induced an increase in cell death by apoptosis, whereas it did not affect overall cell proliferation and differentiation. In parallel, the number of Islet-1/2-positive and TUJ1-positive retinal ganglion cells were diminished in their definitive layer, whereas there was an increase in the number of mislocated Islet-1/2-positive cells. This disturbed morphogenesis correlated with a disruption of the optic fiber layer. Conversely, c-Raf overexpression caused moderate opposite effects on apoptosis. These results frame in vivo early neurogenesis processes in which c-Raf is essential.

Delayed inner ear maturation and neuronal loss in postnatal Igf-1-deficient mice.

Insulin-like growth factor-1 (IGF-1) has been shown to play a key role during embryonic and postnatal development of the CNS, but its effect on a sensory organ has not been studied in vivo. Therefore, we examined cochlear growth, differentiation, and maturation in Igf-1 gene knock-out mice at postnatal days 5 (P5), P8, and P20 by using stereological methods and immunohistochemistry. Mutant mice showed reduction in size of the cochlea and cochlear ganglion. An immature tectorial membrane and a significant decrease in the number and size of auditory neurons were also evident at P20. IGF-1-deficient cochlear neurons showed increased caspase-3-mediated apoptosis, along with aberrant expression of the early neural markers nestin and Islet 1/2. Cochlear ganglion and fibers innervating the sensory cells of the organ of Corti presented decreased levels of neurofilament and myelin P(0) in P20 mouse mutants. In addition, an abnormal synaptophysin expression in the somata of cochlear ganglion neurons and sensory hair cells suggested the persistence of an immature pattern of synapses distribution in the organ of Corti of these animals. These results demonstrate that lack of IGF-1 in mice severely affects postnatal survival, differentiation, and maturation of the cochlear ganglion cells and causes abnormal innervation of the sensory cells in the organ of Corti.

Role of glycosyl-phosphatidylinositol hydrolysis as a mitogenic signal for epidermal growth factor.

We have investigated the role of the hydrolysis of glycosyl-phosphatidylinositol (GPI) as one of the signalling pathways elicited after interaction of epidermal growth factor (EGF) with its specific plasma membrane receptor (EGFR). Endogenous GPI was characterized in both NIH 3T3 mouse fibroblast cells and in EGFR-transfected NIH 3T3 cells (designated EGFR T17). GPI molecules isolated from both cell lines were identical and they incorporated radioactivity from both sugar and fatty acid substrates. Incubation of EGFR T17 cells with EGF, produced a rapid and transient hydrolysis of GPI. Maximum hydrolysis occurred after a 1-min incubation with 50 nM EGF. No such effects of EGF were observed in the parental cell line. Both inositol phosphoglycan (IPG)- and EGF-induced cell proliferation was inhibited in the presence of an IPG-antibody to different extents. The relationship between GPI hydrolysis and the activity of the EGFR was studied using the tyrosine kinase inhibitors tyrphostin (RG50864) and genistein. These agents were able to significantly inhibit EGF-mediated cell proliferation, EGF-dependent hydrolysis of GPI and EGF-regulated autophosphorylation of the EGFR. It is concluded that GPI hydrolysis is one of the earliest intracellular events generated in response to EGF.

Insulin-like effects of inositol phosphate-glycan on messenger RNA expression in rat hepatocytes.

The ability of an inositol phosphate-glycan (IPG) to mimic the effects of insulin on regulation of the expression of specific mRNAs was studied in isolated hepatocytes from normal and diabetic rats. Incubation of normal liver cells with IPG (10 microM) during 90 min produced a 5-fold decrease in phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels, which had been previously increased about 10-fold by incubation with 8-bromo-cAMP (0.1 mM). The effect of IPG was dose dependent and could not be reproduced by galactose, glucosamine, or myo-inositol. IPG reduction of PEPCK mRNA is primarily due to a decrease in the rate of transcription of the gene, as judged by nuclear run-on transcription experiments performed in rat hepatoma H4IIE cells. In hepatocytes isolated from diabetic rats, treatment with 5 microM IPG for 15 min caused a 4-fold induction in the expression of alpha 2-microglobulin mRNA concomitantly with a 2.5-fold decrease in the level of PEPCK mRNA. Cleavage of IPG with nitrous acid abolished both the increase and the decrease in specific mRNAs levels. Glycosyl-phosphatidylinositol, the lipid precursor of IPG, did not modify either PEPCK or alpha 2-microglobulin mRNA levels. These data indicate that both positive and negative effects of insulin on the regulation of gene expression are mimicked by IPG.

Inositol-phosphoglycan inhibits calcium oscillations in hepatocytes by reducing calcium entry.

Inositol-phosphoglycan (IPG) is a putative mediator of insulin action that has been shown to affect numerous biochemical processes. IPG, prepared from liver membranes, promptly inhibited phenylephrine- or vasopressin-induced [Ca2+]i oscillations when perfused over Fura-2-dextran injected rat hepatocytes. An antibody to IPG suppressed the inhibitory effect of insulin on the [Ca2+]i oscillations. Measurement of the rate of quench of cytoplasmic Fura-2 by extracellular Mn2+ showed that Ca2+ entry occurred continuously in the unstimulated cell and was not affected by phenylephrine or vasopressin. IPG, specifically, almost completely abolished the Mn2+ quench rate. Elevated extracellular [Ca2+] reversed the inhibitory effect of IPG on [Ca2+]i oscillations. We conclude that IPG inhibits the hepatocyte Ca2+ oscillatory by reducing the continuous Ca2+ influx that is required to sustain oscillations in [Ca2+]i.

The role of glycosyl-phosphatidylinositol in signal transduction.

Glycosyl-phosphatidylinositol (GPI) lipids have a structural role as protein anchors to the cell surface. In addition, they are implicated in hormone, growth factor and cytokine signal transduction. Their phosphodiesteric hydrolysis mediated by an activated phospholipase results in the generation of water soluble oligosaccharide species termed the inositol phosphoglycan (IPG). This product has been demonstrated to possess biological properties when added exogenously to cells mimicking the biological effects of a variety of extracellular ligands. This may be accomplished since IPG is generic for a family of closely related species which are released in a tissue-specific manner and additionally have cell-specific targets. Micro-organic synthesis has recently been able to shed new light on this topic by the introduction of defined oligosaccharide analogues of IPG for the assessment of their biological activity. These have complemented the findings observed with purified IPG from biological sources thus strengthening the belief that the GPI/IPG signalling system represents a truly novel aspect of transmembrane signalling.

Insulin-like growth factor-I regulates cell proliferation in the developing inner ear, activating glycosyl-phosphatidylinositol hydrolysis and Fos expression.

The role of insulin-like growth factors (IGF) was investigated during the early development of the inner ear. IGF-I stimulated growth of otic vesicles that were isolated and cultured in vitro. IGF-I induced DNA synthesis, increased cell number, and mitotic rate in a dose-dependent manner at concentrations between 0.1-10 nM. IGF-II also induced growth but with a lower potency, whereas insulin had no effect. In the presence of IGF-I, otic vesicles developed from stage 18 to stage 21 in 24-h cultures, mimicking the normal mitotic pattern and morphogenesis in vivo. IGF-I also stimulated growth in the cochleovestibular ganglion. Binding of 125I-IGF-I to specific receptors occurred with high affinity. An autoradiographic study of sections from otic vesicles showed radiolabeled IGF-I in the epithelium. Immunoreactivity to IGF-I was detected in the otic vesicle and in the cochleovestibular ganglion. Intracellular signaling mechanisms of IGF were explored by studying the turnover of glycosylated phosphatidylinositols and the expression of Fos oncoprotein. IGF-I rapidly increased Fos levels in cultured otic vesicles. Furthermore, antisense oligonucleotides complementary to c-fos were able to inhibit IGF-I-induced growth. Both IGF-I-induced cell proliferation and Fos expression were blocked by an antiinositol phosphoglycan (alpha-IPG) antibody. This work suggests that IGF-I may be a candidate to regulate proliferative growth of the otic primordium during normal development and that this action requires the sequential modulation of glycosyl-phosphatidylinositol turnover and Fos expression.

Pattern of methionine adenosyltransferase isoenzyme expression during rat liver regeneration after partial hepatectomy.

The present report investigates the pattern of expression of liver-specific and extrahepatic methionine adenosyltransferase (MAT) isoenzymes in regenerating rat liver after partial hepatectomy. The results show that there is a switch in the expression of these isoenzymes that is coincident with maximal cell proliferation in the remaining liver lobes. Extrahepatic MAT levels increase about three times 4 h after hepatectomy, reaching a maximum 36 h later. This is accompanied by a rapid and transient increase in total MAT activity and levels of related metabolites S-adenosyl-L-methionine and S-adenosyl-L-homocysteine. Liver-specific MAT levels are reversibly down-regulated within 24-48 h post surgery. Our results indicate that MAT isoenzyme expression is tightly regulated during liver regeneration after two-third hepatectomy. The implications of these observations for evaluation of the degree of liver regeneration are briefly discussed.

Phospholipase cleavage of glycosylphosphatidylinositol reconstituted in liposomal membranes.

Glycosylphosphatidylinositol (GPI) purified from rat liver lipids was incorporated into lipid bilayers of defined compositions, in the form of large unilamellar vesicles. The GPI concentration in the bilayers was kept constant at 25 mole%, whereas the remaining lipids being phosphatidylcholine, phosphastidylethanolamine, sphingomyelin and/or cholesterol were varied. The resulting liposomes consisted of spherical vesicles, approximately 100 nm in diameter, that could keep their aqueous contents separated from the extravesicular medium. When these liposomes were treated with either Bacillus cereus phosphatidylinositol-phospholipase C, Trypanosoma brucei GPI-phospholipase C, or bovine serum GPI-phospholipase D, GPI was hydrolyzed at different rates, depending on the enzyme and the bilayer lipid composition. These observations open the way to biophysical and biochemical studies of enzymic GPI cleavage under defined conditions. Extensive GPI hydrolysis was observed in certain cases that could allow the use of these systems for the preparation of inositol phosphoglycans, proposed second messengers of a wide variety of hormones, cytokines and growth factors.

Towards the in vitro reconstitution of caveolae. Asymmetric incorporation of glycosylphosphatidylinositol (GPI) and gangliosides into liposomal membranes.

Large unilamellar vesicles consisting of phospholipids with or without cholesterol have been prepared containing GPI and/or gangliosides asymmetrically located in the outer leaflet of the bilayer. Such asymmetric distribution of GPI and gangliosides is found in 'rafts' and caveolae. Using these vesicles, GPI can be readily hydrolysed by phospholipases. Both cholesterol and ganglioside are seen to inhibit, in an additive way, the hydrolytic activity of GPI-specific phospholipase D.

Induction of cell growth by insulin and insulin-like growth factor-I is associated with Jun expression in the otic vesicle.

The present report investigates the cellular mechanisms involved in the regulation of cell proliferation by insulin and insulin-like growth factor-I (IGF-I) in the developing inner ear. The results show that insulin and IGF-I stimulate cell proliferation in the otic vesicle. This effect is associated with the induction of the expression of the nuclear proto-oncogene c-jun. The temporal profile of Jun expression coincided with the proliferative period of growth of the otic vesicle. IGF-I promoted the hydrolysis of a membrane glycosyl-phosphatidylinositol, which was characterised as the endogenous precursor for inositol phosphoglycan (IPG). Both purified IPG and a synthetic analogue, 6-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-D-myoinositol-1,2-cyclic phosphate (C3), were able to mimic the effects of IGF-I on Jun expression. Anti-IPG antibodies blocked the effects of IGF-I, which were rescued by the addition of IPG or its analogue. These results suggest that the sequence involving the hydrolysis of membrane glycolipids and the expression of c-jun and c-fos proto-oncogenes is part of the mechanism that activates cell division in response to insulin and IGF-I during early organogenesis of the avian inner ear. The implications of these observations for otic development and regeneration are briefly discussed.

Glycosyl phosphatidylinositol (GPI)/inositolphosphate glycan (IPG): an intracellular signalling system involved in the control of thyroid cell proliferation.

In porcine thyrocytes, TSH alone does not induce cell growth. Recently, it has been demonstrated that acute stimulation by TSH of porcine thyrocytes leads to release an inositolphosphate glycan (IPG) described as a putative second messenger for various growth factors in different cell types. IPG isolated from porcine thyrocytes induces proliferation of fibroblasts EGFR T17 and porcine thyrocytes. In porcine thyrocytes we have confirmed that cell growth requires the presence of both TSH and insulin. This effect is reproduced by 8-bromo cyclic AMP suggesting a mediation by intracellular cyclic AMP. Cooperative effects between 8-bromo cyclic AMP and IPG have also been evidenced and are in favour of a crosstalk between distinct signalling pathways.

Role of diffusible and transcription factors in inner ear development: implications in regeneration.

Organogenesis involves a dynamic balance of the mechanisms regulating cell division, differentiation and death. The development of the chicken embryo inner ear offers a well-characterised model at the morphological level to study which signals are implicated in the modulation of cellular activation and commitment. The early developmental decisions that control the origin of the inner ear elements are just beginning to be identified by complementary in vivo and in vitro studies. Insulin-like growth factor-I (IGF-I) and nerve growth factor (NGF) are among the best characterised diffusible factors acting during inner ear development. Although the cellular actions of these factors are beginning to be understood, the signalling pathways triggered by them still remain largely unknown. In this context, viral vehicles can be used to deliver genes and then analyse their functional roles during inner ear development. A model is proposed where the actions of IGF-I and NGF contribute to the combinatorial expression of Jun and Fos family members in particular domains of the otic vesicle. Some of these mechanisms may be also implicated in otic regeneration.

Synthesis and investigation of the possible insulin-like activity of 1D-4-O- and 1D-6-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-myo-inositol 1-phosphate and 1D-6-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-myo-inositol 1,2-(cyclic phosphate).

The synthesis of the glycosyl-myo-inositol 1-phosphates 1 and 2 and of the glycosyl-myo-inositol 1,2-(cyclic phosphate) 3, starting from previously synthesized intermediates, is reported. Compound 3 was found to display proliferative effects on the early developing inner ear of chick embryo.

Cell signalling by inositol phosphoglycans from different species.

The discovery of glycosyl-phosphatidylinositol (GPI) molecules and their products has given new insight into the field of signal transduction. In the last decade a novel mechanism of protein attachment to membranes has emerged, which involves a covalent linkage of the protein to the glycan moiety of a GPI. The discovery that GPI-anchored proteins are ubiquitous throughout the eukaryotes was followed by the observation that uncomplexed GPI molecules are implicated in signal transduction for a diversity of hormones and growth factors. The hydrolysis of free-GPI generates a novel second messenger: the inositol phosphoglycan (IPG). The aim of this article is to review the role of IPG and IPG-like molecules in signal transduction and to discuss future research directions.

Early otic development depends on autophagy for apoptotic cell clearance and neural differentiation.

Autophagy is a highly regulated program of self-degradation of the cytosolic constituents that has key roles during early development and in adult cell growth and homeostasis. To investigate the role of autophagy in otic neurogenesis, we studied the expression of autophagy genes in early stages of chicken (Gallus gallus) inner ear development and the consequences of inhibiting the autophagic pathway in organotypic cultures of explanted chicken otic vesicles (OVs). Here we show the expression of autophagy-related genes (Atg) Beclin-1 (Atg6), Atg5 and LC3B (Atg8) in the otocyst and the presence of autophagic vesicles by using transmission electron microscopy in the otic neurogenic zone. The inhibition of the transcription of LC3B by using antisense morpholinos and of class III phosphatidylinositol 3-kinase with 3-methyladenine causes an aberrant morphology of the OV with accumulation of apoptotic cells. Moreover, inhibition of autophagy provokes the misregulation of the cell cycle in the otic epithelium, impaired neurogenesis and poor axonal outgrowth. Finally, our results indicate that autophagy provides the energy required for the clearing of neuroepithelial dying cells and suggest that it is required for the migration of otic neuronal precursors. Taken together, our results show for the first time that autophagy is an active and essential process during early inner ear development.