Ana M. Fernandez

The many faces of insulin-like peptide signalling in the brain

Central and peripheral insulin-like peptides (ILPs), which include insulin, insulin-like growth factor 1 (IGF1) and IGF2, exert many effects in the brain. Through their actions on brain growth and differentiation, ILPs contribute to building circuitries that subserve metabolic and behavioural adaptation to internal and external cues of energy availability. In the adult brain each ILP has distinct effects, but together their actions ultimately regulate energy homeostasis — they affect nutrient sensing and regulate neuronal plasticity to modulate adaptive behaviours involved in food seeking, including high-level cognitive operations such as spatial memory. In essence, the multifaceted activity of ILPs in the brain may be viewed as a system organization involved in the control of energy allocation.

Dehydroepiandrosterone, pregnenolone and sex steroids down-regulate reactive astroglia in the male rat brain after a penetrating brain injury.

Astrocytes are a target for steroid hormones and for steroids produced by the nervoussystem (neurosteroids). The effect of gonadal hormones and several neurosteroids in theformation of gliotic tissue has been assessed in adult male rats after a penetrating wound of thecerebral cortex and the hippocampal formation. The hormones testosterone, 17β‐estradiol and progesterone and the neurosteroids dehydroepiandrosterone, pregnenolone andpregnenolone sulfate resulted in a significant decrease in the accumulation of astrocytes in theproximity of the wound and in a decreased bromodeoxyuridine incorporation in reactiveastrocytes. Of all steroids tested, dehydroepiandrosterone was the most potent inhibitor of gliotictissue formation. These findings suggest that neurosteroids and sex steroids may affect brainrepair by down‐regulating gliotic tissue.

Neurodegeneration Is Associated to Changes in Serum Insulin-like Growth Factors

Serum levels of insulin and insulin-like growth factors and their binding proteins (IGFs and IGFBPs, respectively) are changed in human neurodegenerative diseases of very different etiology, such as Alzheimers disease, amyotrophic lateral sclerosis, or cerebellar ataxia. However, the significance of these endocrine disturbances is not clear. We now report that in two very different inherited neurodegenerative conditions, ataxia-telangiectasia (AT) and Charcot-Marie-Tooth 1A (CMT-1A) disease, serum levels of IGFs are also altered. Both types of patients have increased serum IGF-I and IGFBP-2 levels, and decreased serum IGFBP-1 levels, while only AT patients have high serum insulin levels. Furthermore, serum IGFs are also changed in three different animal models of neurodegeneration: neurotoxin-induced motor discoordination, diabetic neuropathy, and hereditary cerebellar ataxia. In these three models, serum insulin levels are significantly decreased, serum IGF-I and IGFBP-1, -2, and -3 are decreased in diabetic and neurotoxin-injected rats, while serum IGFBP-1 is increased in hereditary ataxic rats. Altogether, these observations indicate that a great variety of neurodegenerative diseases show endocrine perturbations, resulting in changes in serum IGFs levels. These perturbations are disease-specific and are probably due to metabolic and endocrine derangements, nerve cell death, and sickness-related disturbances associated to the neurodegenerative process. Our observations strongly support the need to evaluate serum IGFs in other neurodegenerative conditions.

Calcineurin in Reactive Astrocytes Plays a Key Role in the Interplay between Proinflammatory and Anti-Inflammatory Signals

Maladaptive inflammation is a major suspect in progressive neurodegeneration, but the underlying mechanisms are difficult to envisage in part because reactive glial cells at lesion sites secrete both proinflammatory and anti-inflammatory mediators. We now report that astrocytes modulate neuronal resilience to inflammatory insults through the phosphatase calcineurin. In quiescent astrocytes, inflammatory mediators such as tumor necrosis factor-α (TNF-α) recruits calcineurin to stimulate a canonical inflammatory pathway involving the transcription factors nuclear factor κB (NFκB) and nuclear factor of activated T-cells (NFAT). However, in reactive astrocytes, local anti-inflammatory mediators such as insulin-like growth factor I also recruit calcineurin but, in this case, to inhibit NFκB/NFAT. Proof of concept experiments in vitro showed that expression of constitutively active calcineurin in astrocytes abrogated the inflammatory response after TNF-α or endotoxins and markedly enhanced neuronal survival. Furthermore, regulated expression of constitutively active calcineurin in astrocytes markedly reduced inflammatory injury in transgenic mice, in a calcineurin-dependent manner. These results suggest that calcineurin forms part of a molecular pathway whereby reactive astrocytes determine the outcome of the neuroinflammatory process by directing it toward either its resolution or its progression.

Neuroprotective actions of peripherally administered insulin‐like growth factor I in the injured olivo‐cerebellar pathway

Exogenous administration of insulin‐like growth factor I (IGF‐I) restores motor function in rats with neurotoxin‐induced cerebellar deafferentation. We first determined that endogenous IGFs are directly involved in the recovery process because infusion of an IGF‐I receptor antagonist into the lateral ventricle blocks gradual recovery of limb coordination that spontaneously occurs after partial deafferentation of the olivo‐cerebellar circuitry. We then analysed mechanisms whereby exogenous IGF‐I restores motor function in rats with complete damage of the olivo‐cerebellar pathway. Treatment with IGF‐I normalized several markers of cell function in the cerebellum, including calbindin, glutamate receptor 1 (GluR1), γ‐aminobutyric acid (GABA) and glutamate, which are all depressed after 3‐acetylpyridine (3AP)‐induced deafferentation. IGF‐I also promoted functional reinnervation of the cerebellar cortex by inferior olive (IO) axons. In the IO, increased expression of bax in neurons and bcl‐X in astrocytes after 3AP was significantly reduced by IGF‐I treatment. On the contrary, IGF‐I prevented the decrease in poly‐sialic‐acid neural cell adhesion molecule (PSA‐NCAM) and GAP‐43 expression induced by 3AP in IO cells. IGF‐I also significantly increased the number of neurons expressing bcl‐2 in brainstem areas surrounding the IO. Altogether, these results indicate that subcutaneous IGF‐I therapy promotes functional recovery of the olivo‐cerebellar pathway by acting at two sites within this circuitry: (i) by modulating death‐ and plasticity‐related proteins in IO neurons; and (ii) by impinging on homeostatic mechanisms leading to normalization of cell function in the cerebellum. These results provide insight into the neuroprotective actions of IGF‐I and may be of practical consequence in the design of new therapeutic approaches for neurodegenerative diseases.

Estrogen receptors and insulin-like growth factor-I receptors mediate estrogen-dependent synaptic plasticity

Previous studies have shown that estradiol induces a transient disconnection of axo-somatic inhibitory synapses in the hypothalamic arcuate nucleus of adult ovariectomized rats. The synaptic disconnection is accompanied by an increase in the levels of insulin-like growth factor-I (IGF-I) in the arcuate nucleus, suggesting that IGF-I signaling may be involved in the estrogen-induced synaptic plasticity. The role of estrogen receptors and IGF-I receptors in the synaptic changes has been studied by assessing the number of axo-somatic synapses in ovariectomized rats treated with intracerebroventricular administration of the estrogen receptor antagonist ICI 182,780 and the IGF-I receptor antagonist JB1 to ovariectomized rats. Estradiol administration resulted in a significant decrease in the number of axo-somatic synapses on arcuate neurons in control ovariectomized rats. Both the estrogen receptor antagonist and the IGF-I receptor antagonist blocked the estrogen-induced synaptic decrease. This finding suggest that estrogen-induced synaptic plasticity in the arcuate nucleus is dependent on the activation of both estrogen receptors and IGF-I receptors.

GLP-1(7-36)-amide and Exendin-4 Stimulate the HPA Axis in Rodents and Humans

Glucagon-like peptide-1 (GLP-1) is a potent insulinotropic peptide expressed in the gut and brain, which is secreted in response to food intake. The levels of GLP-1 within the brain have been related to the activity of the hypothalamic-pituitary-adrenal (HPA) axis, and hence, this peptide might mediate some responses to stress. Nevertheless, there is little information regarding the effects of circulating GLP-1 on the neuroendocrine control of HPA activity. Here, we have studied the response of corticoadrenal steroids to the peripheral administration of GLP-1 (7-36)-amide and related peptides [exendin (Ex)-3, Ex-4, and Ex-4(3-39)] in rats, mice, and humans. GLP-1 increases circulating corticosterone levels in a time-dependent manner, both in conscious and anaesthetized rats, and it has also increased aldosterone levels. Moreover, GLP-1 augmented cortisol levels in healthy subjects and diabetes mellitus (DM)-1 patients. The effects of GLP-1/Ex-4 on the HPA axis are very consistent after distinct means of administration (intracerebroventricular, iv, and ip), irrespective of the metabolic state of the animals (fasting or fed ad libitum), and they were reproduced by different peptides in this family, independent of glycaemic changes and their insulinotropic properties. Indeed, these effects were also observed in diabetic subjects (DM-1 patients) and in the DM-1 streptozotocin-rat or DM-2 muscle IGF-I receptor-lysine-arginine transgenic mouse animal models. The mechanisms whereby circulating GLP-1 activates the HPA axis remain to be elucidated, although an increase in ACTH after Ex-4 and GLP-1 administration implicates the central nervous system or a direct effect on the pituitary. Together, these findings suggest that GLP-1 may play an important role in regulating the HPA axis.

Insulin-like growth factor I modulates c-fos induction and astrocytosis in response to neurotoxic insult

Insulin-like growth factor I participates in the cellular response to brain insult by increasing its messenger RNA expression and/or protein levels in the affected area. Although it has been suggested that insulin-like growth factor I is involved in a variety of cellular responses leading to homeostasis, mechanisms involved in its possible trophic effects are largely unknown. Since activation of c-Fos in postmitotic neurons takes place both in response to insulin-like growth factor I and after brain injury, we have investigated whether this early response gene may be involved in the actions of insulin-like growth factor I after brain insult. Partial deafferentation of the cerebellar cortex by 3-acetylpyridine injection elicited c-Fos protein expression on both Purkinje and granule cells of the cerebellar cortex. This neurotoxic insult also triggered gliosis, as determined by an increased number of glial fibrillary acidic protein-positive cells (reactive astrocytes) in the cerebellar cortex. When 3-acetylpyridine-injected animals received a continuous intracerebellar infusion of either a peptidic insulin-like growth factor I receptor antagonist or an insulin-like growth factor I antisense oligonucleotide for two weeks through an osmotic minipump, c-Fos expression was obliterated while reactive gliosis was greatly increased. On the contrary, continuous infusion of insulin-like growth factor I significantly decreased reactive gliosis without affecting the increase in c-Fos expression. These results indicate that insulin-like growth factor I is involved in both the neuronal (c-Fos) and the astrocytic (glial fibrillary acidic protein) activation in response to injury.

Reduced brain insulin-like growth factor I function during aging

Peripheral insulin-like growth factor I (IGF-I) function progressively deteriorates with age. However, whereas deterioration of IGF-I function in the aged brain seems probable, it has not been directly addressed yet. Because serum IGF-I can enter into the brain through the cerebrospinal fluid (CSF), we examined this route of entrance in aged mice. To distinguish endogenous murine IGF-I from exogenously applied IGF-I, we used human IGF-I. We found that after intraperitoneous injection, CSF levels of human IGF-I were significantly higher in old mice (2 year-old) as compared to young ones (4-month-old). In spite of this increase capacity to take IGF-I from the circulation, brain and plasma IGF-I levels were reduced in naive old mice. Moreover, IGF-I signaling was deteriorated in the brain of aged animals. Basal as well as IGF-I-induced activation of the brain IGF-I receptor/Akt/GSK3 pathway was markedly reduced even though old mice have higher levels of brain IGF-I receptors. These data suggest that increases in brain IGF-I receptors and in the capacity to take up serum IGF-I result ineffective because IGF-I function is reduced and aged mice are cognitively impaired, a trait dependant on preserved serum IGF-I input to the brain.

The insulin-like growth factor I receptor regulates glucose transport by astrocytes

Previous findings indicate that reducing brain insulin‐like growth factor I receptor (IGF‐IR) activity promotes ample neuroprotection. We now examined a possible action of IGF‐IR on brain glucose transport to explain its wide protective activity, as energy availability is crucial for healthy tissue function. Using 18FGlucose PET we found that shRNA interference of IGF‐IR in mouse somatosensory cortex significantly increased glucose uptake upon sensory stimulation. In vivo microscopy using astrocyte specific staining showed that after IGF‐IR shRNA injection in somatosensory cortex, astrocytes displayed greater increases in glucose uptake as compared to astrocytes in the scramble‐injected side. Further, mice with the IGF‐IR knock down in astrocytes showed increased glucose uptake in somatosensory cortex upon sensory stimulation. Analysis of underlying mechanisms indicated that IGF‐IR interacts with glucose transporter 1 (GLUT1), the main facilitative glucose transporter in astrocytes, through a mechanism involving interactions with the scaffolding protein GIPC and the multicargo transporter LRP1 to retain GLUT1 inside the cell. These findings identify IGF‐IR as a key modulator of brain glucose metabolism through its inhibitory action on astrocytic GLUT1 activity. GLIA 2016;64:1962–1971