Neurotrophic uncoupling of IGF-1 in Alzheimer’s disease: translation into early diagnosis and involvement of lifestyle risk factors

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Anatomía, Histología y Neurociencia. Fecha de lectura: 20-02-2015Insulin-like growth factor 1 (IGF-1) is a circulating hormone that is mainly produced by the liver in response to the growth hormone from the adenohypophysis. Previous data from our laboratory have shown that, in the adult, IGF-1 is able to cross the blood-brain barrier (BBB) to enter the central nervous system (CNS) through a tonic (constitutive) and a phasic mechanism. The last process is coupled to neuronal activity, what means that active brain areas accumulate IGF-1 from the blood, and as a result it has been named “neurotrophic coupling”. On the other hand, Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease and a major cause of dementia among the worldwide population. Despite a century of research has elapsed, its etiology and pathogenesis are yet to be fully defined. As a result, it remains one of the biggest medical challenges of our time. Due to the recent failure of many clinical trials based on the amyloid cascade hypothesis, there is an unmet need to find new etiological hypothesis and to reach an earlier diagnosis of AD. Previous experiments done on our laboratory demonstrated that IGF-1 plays a protective role in AD by clearing amyloid beta (Aβ) away from the brain and resolving chronic neuroinflammation. Based on this and on abundant literature describing an impaired central insulin system in AD, we posited the existence of a dysfunction in IGF-1 access to the brain that would play a pathogenic role in AD and would be an early event in disease progression. With the project I hereby present as my PhD thesis, I intended to test this hypothesis and to try to translate the potential outcomes into an early diagnostic tool for AD. Environmental enrichment (EE) is an experimental protocol used for diffuse physiological stimulation of the brain and so susceptible of inducing IGF-1 input to the engaged brain areas. However, little was known about the regulation of the latter and, because of it, we decided to study it in more detail. Because cell signaling of growth factors is tightly regulated in time and IGF-1 is one of the most potent mitogens in the organism, we hypothesized that its entrance to the brain would be similarly limited in homeostasis. As a result, we found out that in wild type (WT) animals the response to IGF-1 was maximal after 2h of EE in the hippocampus, whereas mice with liver IGF-1 deficiency did not display any activation of the IGF-1 receptor (IGF-1R). At longer exposures of EE there were fluctuations in the levels of phosphorylated (active) IGF-1R, which ultimately led to the development of tolerance in the chronic stimulation of one month. When we analyzed the response of asymptomatic AD mice (APP and APP/PS1) to acute EE, we detected that their expected response was dramatically impaired as compared to WT controls. We discovered an intrinsic brain resistance to IGF-1 in AD mice that we considered the main cause of the observed disrupted transport of circulating IGF-1 into the CNS. Previous findings of the laboratory suggested that IGF-1 enhances neuronal excitability and others had reported that peripheral insulin modified the electroencephalogram (EEG) in humans. Taking this into account we proposed that systemically injected IGF-1 would affect brain electrical activity. We confirmed this in WT anaesthetized mice, in which IGF-1 was able to potentiate the fast component of the electrocorticogram (ECoG). As for AD mice, the injection of IGF-1 did not exert any effect on the ECoG (in APP/PS1) or it did it feebly (APP). Because this pattern perfectly matched that of the phospho-IGF-1R evoked by EE, we believe this test is an easily accessible and non-invasive way of determining brain IGF-1 sensitivity. If ECoG/EEG response to a peripheral injection of IGF-1 was diminished, this might be used as a biomarker of AD. Nonetheless, this ought to be done in concurrence with other biomarkers used for risk assessment of AD in healthy subjects, such as those detecting amyloidopathy or subtle neurodegeneration before the onset of clinical symptoms. To further explore the pathogenic implications of IGF-1 system in sporadic AD we have recently started using a diet-induced model of the metabolic syndrome (MetS), an established risk factor for dementia. So far we have identified that, after a short time, a high-fat diet (HFD) induces the entrance of IGF-1 in the cerebrospinal fluid (CSF) of WT mice. This may be related to the prolonged hippocampal IGF-1 signaling and the dendritic abnormalities seen in the dentate gyrus and CA1 regions of diet-induced obese (DIO) mice. Nevertheless, more work is still needed to assess the true role of IGF-1 brain signaling in these events, also including other pathogenic markers found in DIO brains such as the increase in mitochondria fission and tau abnormal phosphorylation.El factor de crecimiento insulínico de tipo 1 (IGF-1) es una hormona circulante que se produce principalmente en el hígado en respuesta a la hormona del crecimiento adenohipofisaria. Datos previos del laboratorio han demostrado que, en el adulto, IGF-1 es capaz de cruzar la barrera hematoencefálica para entrar al sistema nervioso central a través de un mecanismo tónico (constitutivo) y/o fásico. El último está asociado a la actividad neuronal, lo que implica que en áreas activas del cerebro se acumula IGF-1 de la sangre, y por ello es que se ha denominado “acoplamiento neurotrófico”. Por otra parte, la enfermedad de Alzheimer es la más prevalente de entre las neurodegenerativas y la mayor causa de demencia en la población mundial. A pesar de que ya ha pasado un siglo de investigación, su etiología y patogenia aún no están del todo definidas. Como consecuencia, continúa siendo uno de los mayores retos clínicos de nuestra era. Debido al reciente fracaso de muchos ensayos clínicos apoyados en la hipótesis de la cascada amiloide, existe una gran necesidad de encontrar nuevas hipótesis etiológicas y de adelantar el diagnóstico de la enfermedad de Alzheimer. Experimentos previos del laboratorio muestran que IGF-1 tiene un papel protector en esta patología a través del aclaramiento del péptido beta amiloide (Aβ) del cerebro así como de la resolución de la neuroinflamación crónica asociada a la enfermedad. Basándonos en esto y en la abundante literatura que describe la alteración central del sistema de insulina en el Alzheimer, postulamos la existencia de una disfunción temprana en el acceso de IGF-1 al cerebro y que ello podría jugar un rol patogénico en la misma. Con el proyecto que aquí presento como mi tesis doctoral, he intentado probar esta hipótesis y trasladar los posibles resultados hacia el desarrollo de una herramienta de diagnóstico temprano de la enfermedad de Alzheimer. El enriquecimiento ambiental es un protocolo experimental utilizado para la estimulación fisiológica difusa del cerebro y por lo tanto es susceptible de inducir el paso de IGF-1 desde la sangre hacia las zonas cerebrales activas. No obstante, se sabe poco de cómo esto se regula y, por esto, decidimos estudiarlo más en detalle. Debido a que la señalización de los factores de crecimiento se encuentra firmemente regulada en el tiempo y a que IGF-1 es uno de los más potentes mitógenos del organismo, hipotetizamos que su entrada al cerebro se encontraría similarmente controlada en la homeostasis. Mediante experimentos en animales silvestres o wild type (WT) encontramos que la respuesta al enriquecimiento fue máxima tras 2h y en el hipocampo, mientras que ratones sin producción hepática de IGF-1 no mostraban activación del receptor de IGF-1 (IGF-1R). En exposiciones más largas encontramos fluctuaciones en los niveles de IGF-1R fosforilado (activo, pIGF-1R), que en último lugar llevaron al desarrollo de tolerancia en la estimulación crónica de un mes. Cuando analizamos la respuesta al enriquecimiento agudo de los ratones de Alzheimer en etapas asintomáticas (APP y APP/PS1), detectamos que ésta se encontraba dramáticamente alterada en comparación con los controles WT. Descubrimos en estos ratones una resistencia cerebral intrínseca a IGF-1, que consideramos como la causa principal del trastocado transporte de IGF-1 circulante al cerebro. Anteriores hallazgos del laboratorio sugerían que IGF-1 aumenta la excitabilidad neuronal y otros investigadores habían descrito que la insulina periférica modifica el electroencefalograma (EEG) en humanos. Teniendo ambos hechos en cuenta, propusimos que la inyección sistémica de IGF-1 afectaría a la actividad eléctrica del cerebro. Esto fue confirmado en animales WT anestesiados, en los que IGF-1 potenció el componente rápido del electrocortigorama (ECoG). En animales transgénicos modelo de la enfermedad de Alzheimer, la administración intraperitoneal de IGF-1 o bien no tuvo ningún efecto sobre el ECoG (en APP/PS1) o bien fue muy débil (APP). Ya que este patrón encaja a la perfección con lo observado para el pIGF-1R en el enriquecimiento, creemos que esta prueba es una forma no invasiva y fácilmente accesible para la determinación de la sensibilidad cerebral a IGF-1. Si la respuesta del ECoG/EEG a la administración periférica de IGF-1 estuviera disminuida, se podría utilizar como biomarcador de la enfermedad de Alzheimer. Aun así, ello debería hacerse en concurrencia con otros biomarcadores utilizados para la evaluación del riesgo de Alzheimer en sujetos sanos, como por ejemplo aquellos enfocados hacia la detección de amiloidopatía o neurodegeneración sutil previos a la aparición de los síntomas clínicos. Con el objetivo de explorar más en profundidad la implicación patogénica del sistema IGF-1 en el Alzheimer esporádico, hemos comenzado a utilizar recientemente un modelo basado en la inducción de síndrome metabólico (un conocido factor de riesgo para la enfermedad de Alzheimer) a través de la dieta. Hasta ahora hemos encontrado que una dieta rica en grasas induce la entrada de IGF-1 hacia el líquido cefalorraquídeo en animales WT. Este hecho podría estar relacionado con la prolongada señal de IGF-1 hipocampal y las anormalidades dentríticas del giro dentado y CA1 que se observan en los ratones obesos. No obstante, aún es necesario más trabajo para asegurar el verdadero papel de la señalización central de IGF-1 en estos eventos, incluyendo otros marcadores patogénicos encontrados en el cerebro de los animales obesos tales como el incremento de la fisión mitocondrial y la anormal fosforilación de tau

Insulin-like growth factor I sensitization rejuvenates sleep patterns in old mice

Sleep disturbances are common during aging. Compared to young animals, old mice show altered sleep structure, with changes in both slow and fast electrocorticographic (ECoG) activity and fewer transitions between sleep and wake stages. Insulin-like growth factor I (IGF-I), which is involved in adaptive changes during aging, was previously shown to increase ECoG activity in young mice and monkeys. Furthermore, IGF-I shapes sleep architecture by modulating the activity of mouse orexin neurons in the lateral hypothalamus (LH). We now report that both ECoG activation and excitation of orexin neurons by systemic IGF-I are abrogated in old mice. Moreover, orthodromical responses of LH neurons are facilitated by either systemic or local IGF-I in young mice, but not in old ones. As orexin neurons of old mice show dysregulated IGF-I receptor (IGF-IR) expression, suggesting disturbed IGF-I sensitivity, we treated old mice with AIK3a305, a novel IGF-IR sensitizer, and observed restored responses to IGF-I and rejuvenation of sleep patterns. Thus, disturbed sleep structure in aging mice may be related to impaired IGF-I signaling onto orexin neurons, reflecting a broader loss of IGF-I activity in the aged mouse brain.This work was funded by a grant from Ciberned and is part of the project SAF2016-76462 funded by MCIN/AEI/https://doi.org/10.13039/501100011033. J.A. ZegarraValdivia acknowledges the fnancial support of the National Council of Science, Technology and Technological Innovation (CONCYTEC, Perú) through the National Fund for Scientifc and Technological Development (FONDECYT, Perú). J. Fernandes received a post-doc fellowship from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP: # 2017/14742–0; # 2019/03368–5)

Loss of serum IGF-I input to the brain as an early biomarker of disease onset in Alzheimer mice

Circulating insulin-like growth factor I (IGF-I) enters the brain and promotes clearance of amyloid peptides known to accumulate in Alzheimer's disease (AD) brains. Both patients and mouse models of AD show decreased level of circulating IGF-I enter the brain as evidenced by a lower ratio of cerebrospinal fluid/plasma IGF-I. Importantly, in presymptomatic AD mice this reduction is already manifested as a decreased brain input of serum IGF-I in response to environmental enrichment. To explore a potential diagnostic use of this early loss of IGF-I input, we monitored electrocorticogram (ECG) responses to systemic IGF-I in mice. Whereas control mice showed enhanced ECG activity after IGF-I, presymptomatic AD mice showed blunted ECG responses. Because nonhuman primates showed identically enhanced electroencephalogram (EEG) activity in response to systemic IGF-I, loss of the EEG signature of serum IGF-I may be exploited as a disease biomarker in AD patients.This work has been supported by grants from the Spanish Ministry of Economy (Explora Program and SAF2010/17036 to IT-A and BFU2012/36107 to AN). Ángel Trueba-Sáiz holds an FPU fellowship of the Spanish Ministry of Education (ref. FPU AP2009-0372).Peer Reviewe

Circulating Insulin-Like Growth Factor I is Involved in the Effect of High Fat Diet on Peripheral Amyloid β Clearance

© 2020 by the authors.Obesity is a risk factor for Alzheimer’s disease (AD), but underlying mechanisms are not clear. We analyzed peripheral clearance of amyloid β (Aβ) in overweight mice because its systemic elimination may impact brain Aβ load, a major landmark of AD pathology. We also analyzed whether circulating insulin-like growth factor I (IGF-I) intervenes in the effects of overweight as this growth factor modulates brain Aβ clearance and is increased in the serum of overweight mice. Overweight mice showed increased Aβ accumulation by the liver, the major site of elimination of systemic Aβ, but unaltered brain Aβ levels. We also found that Aβ accumulation by hepatocytes is stimulated by IGF-I, and that mice with low serum IGF-I levels show reduced liver Aβ accumulation—ameliorated by IGF-I administration, and unchanged brain Aβ levels. In the brain, IGF-I favored the association of its receptor (IGF-IR) with the Aβ precursor protein (APP), and at the same time, stimulated non-amyloidogenic processing of APP in astrocytes, as indicated by an increased sAPPα/sAPPβ ratio after IGF-I treatment. Since serum IGF-I enters into the brain in an activity-dependent manner, we analyzed in overweight mice the effect of brain activation by environmental enrichment (EE) on brain IGF-IR phosphorylation and its association to APP, as a readout of IGF-I activity. After EE, significantly reduced brain IGF-IR phosphorylation and APP/IGF-IR association were found in overweight mice as compared to lean controls. Collectively, these results indicate that a high-fat diet influences peripheral clearance of Aβ without affecting brain Aβ load. Increased serum IGF-I likely contributes to enhanced peripheral Aβ clearance in overweight mice, without affecting brain Aβ load probably because its brain entrance is reduced.This work was funded by a grant from Ciberned, by an Inter-CIBER project (PIE14/00061), and from SAF2013-40710-R (AEI/FEDER, UE). J.A.Z.-V. acknowledges the financial support of the National Council of Science, Technology and Technological Innovation (CONCYTEC, Perú) through the National Fund for Scientific and Technological Development (FONDECYT, Perú).Peer reviewe

Diet influences peripheral amyloid ß metabolism: a role for circulating IGF-I.

Obesity is a risk factor for Alzheimer´s disease (AD), but underlying mechanisms are not clear. We analyzed peripheral clearance of amyloid β (Aβ) in overweight mice because its systemic elimination may impact on brain Aβ load, a major landmark of AD pathology. Overweight mice showed increased peripheral Aβ clearance by the liver, the major site of elimination of systemic Aβ, but unaltered brain Aβ levels. Since circulating insulin-like growth factor I (IGF-I) modulates brain Aβ clearance, and is increased in serum of overweight mice, we determined whether it affects peripheral Aβ clearance. We found that Aβ uptake by hepatocytes is stimulated by IGF-I. Moreover, mice with low serum IGF-I levels show reduced peripheral Aβ clearance. In the brain, IGF-I favored association of its receptor (IGF-IR) with Aβ precursor protein (APP), and at the same time stimulated non-amyloidogenic processing of APP in astrocytes, as indicated by an increased sAPPα/sAPPβ ratio after IGF-I treatment. Since serum IGF-I enters into the brain in an activity-dependent manner, we analyzed in overweight mice the effect of brain activation by environmental enrichment (EE) on brain IGF-IR phosphorylation and its association to APP, as a readout of IGF-I activity. After EE, significantly less activation of brain IGF-IR phosphorylation and APP/IGF-IR association was found in overweight mice as compared to lean controls. Collectively, these results indicate that diet influences peripheral clearance of Aβ without affecting brain Aβ load. Increased serum IGF-I likely contributes to enhanced peripheral Aβ clearance in overweight mice, without affecting brain Aβ clearance probably because its brain entrance is reduced.We are thankful to M. Garcia for technical support. This work was funded by a grant from Ciberned, by an Inter-CIBER project (PIE14/00061), and from SAF2013-40710-R (AEI/FEDER, UE)