Abstract
The insulin superfamily of peptides is essential for homeostasis as well as neuronal plasticity, learning, and memory. Here, we show that insulin-like growth factors 1 and 2 (IGF1 and IGF2) are differentially expressed in hippocampal neurons and released in an activity-dependent manner. Using a new fluorescence resonance energy transfer sensor for IGF1 receptor (IGF1R) with two-photon fluorescence lifetime imaging, we find that the release of IGF1 triggers rapid local autocrine IGF1R activation on the same spine and more than several micrometers along the stimulated dendrite, regulating the plasticity of the activated spine in CA1 pyramidal neurons. In CA3 neurons, IGF2, instead of IGF1, is responsible for IGF1R autocrine activation and synaptic plasticity. Thus, our study demonstrates the cell type–specific roles of IGF1 and IGF2 in hippocampal plasticity and a plasticity mechanism mediated by the synthesis and autocrine signaling of IGF peptides in pyramidal neurons.
INTRODUCTION
The insulin superfamily of peptides, including insulin and insulin-like growth factors 1 and 2 (IGF1 and IGF2), plays essential roles in regulating cellular proliferation, differentiation, development, and metabolism in various tissues (1, 2). Recent studies suggest that these peptides are also critical for the development and plasticity of the central nervous system (CNS) (3, 4). Insulin and IGFs in the adult brain have two major sources. They are primarily secreted from hepatocytes in the liver and transported to the cerebral spinal fluid (CSF) or synthesized locally in the brain by neurons and glia (5, 6).
Insulin-like peptides signal through their corresponding receptors, insulin receptors (IR) and IGF1 and IGF2 receptors (IGF1R and IGF2R), which are highly expressed in the hippocampus, striatum, hypothalamus, cerebral cortex, and olfactory bulb (6). IGF1R is a receptor tyrosine kinase with α2β2 tetrameric structure (7). The binding of ligands to the extracellularly located α subunits results in a conformation change, which results in the autophosphorylation of intracellular β subunits (8). The autophosphorylation generates docking sites for various adaptor proteins, including IR substrate (IRS) and Phosphoinositide 3-kinases (PI3Ks), leading to canonical signaling pathways regulating gene transcription (9, 10). This pathway regulates many critical cellular processes such as glucose transportation, cell proliferation, cell survival, and synaptic remodeling (4, 11, 12). Both IGF1 and IGF2 primarily exert their effects through the IGF1R, while the affinity of IGF1 is ~10 times higher than IGF2 for the IGF1R. On the other hand, IGF2R does not have any obvious intracellular functional domain in its C-tail and has been thought to suppress IGF1-IGF1R signaling by scavenging IGF2 (13, 14).
Previous studies have documented the critical role of IGF1R signaling in learning and memory in various animals (15–17). Abnormal regulation of IGF1 in the brain is implicated in the cognitive decline in aging and brain diseases, including Alzheimer’s disease (AD) (18–20). The restoration of IGF1 to the normal level in patients with attention and memory deficits or in AD animal models is reported to result in improvements in short- and long-term memory (15, 16, 21–23). Hippocampal IGF2 is up-regulated during and after learning, whereas perturbation of IGF2 levels in the hippocampus bidirectionally affects memory formation and consolidation (15, 24, 25). Disruption of IR and IRS produces impaired protein synthesis–dependent memory in Drosophila (26). Intracerebroventricular administration of insulin into rats results in higher memory retention levels in a passive-avoidance task (27, 28). Moreover, mice trained with spatial memory tasks exhibit higher IR mRNA and insulin signaling molecules in the dentate gyrus and the CA1 area of the hippocampus (27). In addition, signaling via insulin-like peptides is found to modulate long-term synaptic plasticity (29, 30). However, the source and the regulation of insulin-like peptides in hippocampal synaptic plasticity at the single synapse level are largely unknown.
Here, we show that IGF1 and IGF2 are synthesized in and released from CA1 and CA3 pyramidal neurons, respectively, in the mouse hippocampus. Using a new fluorescence resonance energy transfer (FRET)–based sensor for IGF1R activity, we found that IGF1 synthesized in CA1 pyramidal neurons is released from their dendrites, likely from dendritic spines, in response to N-methyl-d-aspartate receptor (NMDAR) activation to trigger local autocrine activation of IGF1R signaling on the same spines. In CA3 pyramidal neurons, IGF2, instead of IGF1, is released from dendrites and activates IGF1R in an autocrine manner. The IGF-IGF1R signaling in dendritic spines plays an essential role in regulating the structural and electrophysiological plasticity.
RESULTS
CA1 pyramidal neurons synthesize, store, and secret IGF1
To explore the role of CNS-derived IGF1 in the hippocampus, we examined the expression pattern of IGF1 by immunostaining brain sections of juvenile mice (P20, postnatal day 20) with an anti-IGF1 antibody. To test the neuronal synthesis of IGF1, we used conditional knockout of IGF1 (Igf1fl/fl), so that we can delete Igf1 in a specific set of cells with Cre recombinase [Fig. 1, A to D, for conditional knockout of IGF1 (Igf1fl/fl) and fig. S1 for wild-type mice]. We found a punctuated pattern primarily in dendrites and soma, specifically for CA1 pyramidal neurons, but little for other types of cells, including CA3 pyramidal neurons (Fig. 1A and fig. S1). To verify the specificity of the antibody signal and IGF1 synthesis in CA1 pyramidal neurons, we removed Igf1 from a sparse subset of excitatory neurons in Igf1fl/fl mice by in utero injection of AAV-Camk2a-Cre and AAV-CAG-Flex-EGFP. We found that the IGF1 signals in Cre- and enhanced green fluorescent protein (EGFP)–expressing cells (Cre+) were statistically significantly lower than that in nearby cells without EGFP expression (Cre−) for CA1 pyramidal neurons (Fig. 1, B and E). EGFP expression with AAV-CAG-EGFP without Cre did not change IGF1 in CA1 pyramidal neurons (Fig. 1, C and E). In addition, wild-type mice injected with AAV-Camk2a-Cre and AAV-CAG-Flex-EGFP did not show a difference in IGF1 expression between Cre+ and Cre− cells (Fig. 1, D and E). These results indicate that CA1 pyramidal neurons synthesize and store IGF1, while other cell types in the hippocampus express much less IGF1.
