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. 2018 Jan 24;38(4):1015-1029.
doi: 10.1523/JNEUROSCI.2010-17.2017. Epub 2017 Dec 7.

Insulin-Like Growth Factor II Targets the mTOR Pathway to Reverse Autism-Like Phenotypes in Mice

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Free PMC article

Insulin-Like Growth Factor II Targets the mTOR Pathway to Reverse Autism-Like Phenotypes in Mice

Adam B Steinmetz et al. J Neurosci. .
Free PMC article

Abstract

Autism spectrum disorder (ASD) is a developmental disability characterized by impairments in social interaction and repetitive behavior, and is also associated with cognitive deficits. There is no current treatment that can ameliorate most of the ASD symptomatology; thus, identifying novel therapies is urgently needed. We used male BTBR T+Itpr3tf /J (BTBR) mice, a model that reproduces most of the core behavioral phenotypes of ASD, to test the effects of systemic administration of insulin-like growth factor II (IGF-II), a polypeptide that crosses the blood-brain barrier and acts as a cognitive enhancer. We show that systemic IGF-II treatments reverse the typical defects in social interaction, cognitive/executive functions, and repetitive behaviors reflective of ASD-like phenotypes. In BTBR mice, IGF-II, via IGF-II receptor, but not via IGF-I receptor, reverses the abnormal levels of the AMPK-mTOR-S6K pathway and of active translation at synapses. Thus, IGF-II may represent a novel potential therapy for ASD.SIGNIFICANCE STATEMENT Currently, there is no effective treatment for autism spectrum disorder (ASD), a developmental disability affecting a high number of children. Using a mouse model that expresses most of the key core as well as associated behavioral deficits of ASD, that are, social, cognitive, and repetitive behaviors, we report that a systemic administration of the polypeptide insulin-like growth factor II (IGF-II) reverses all these deficits. The effects of IGF-II occur via IGF-II receptors, and not IGF-I receptors, and target both basal and learning-dependent molecular abnormalities found in several ASD mice models, including those of identified genetic mutations. We suggest that IGF-II represents a potential novel therapeutic target for ASD.

Keywords: autism spectrum disorder; insulin-like growth factor II; insulin-like growth factor II receptor; mTOR; memory; mouse model.

Figures

Figure 1.
Figure 1.
IGF-II reverses social interaction deficits and enhances social novelty memory in BTBR mice. Experimental timeline is shown above graphs. In all experiments mice received a subcutaneous injection (↑) of either vehicle (Veh) or IGF-II, 20 min before the first test. All data are expressed as the mean percentage (±SEM). n = 7–9 per group. Two-way ANOVA followed by Tukey or Bonferroni post hoc tests. *p < 0.05, ***p < 0.001. A, Percentage exploration preference for Mouse 1 versus novel object (Test 1), novel Mouse 2 versus Mouse 1 (Test 2), and novel Mouse 3 versus Mouse 1 (Test 3) of B6 and BTBR mice injected with Veh or IGF-II. B, Percentage exploration preference for Mouse 1 versus novel object (Test 1), novel Mouse 2 versus Mouse 1 (Test 2), and novel Mouse 3 versus Mouse 1 (Test 3) of B6 (top) and BTBR (bottom) mice that received a bilateral dorsal hippocampal injections (red ↑) of IGF-IR blocker (JB1), IGF-IIR functionally blocking antibody (Anti), IgG or Veh immediately before a systemic Veh or IGF-II injection. Time spent exploring Mouse 1 versus novel object (Test 1), novel Mouse 2 versus Mouse 1 (Test 2), and novel Mouse 3 versus Mouse 1 (Test 3) of B6 and BTBR mice injected with Veh or IGF-II. Exploration times are reported in the Extended Data (Figs. 1-1 to 1-3. Detailed statistical analyses are reported in the Extended Data tables (Figs. 1-4 to 1-7).
Figure 2.
Figure 2.
IGF-II reverses memory deficits of BTBR mice via hippocampal IGF-IIR. Experimental timelines are shown above graphs. In all experiments mice received a subcutaneous injection of either vehicle (Veh) or IGF-II (↑) 20 min before either training or memory reactivation as indicated. All data are expressed as the mean (±SEM). n = 7–8 per group. Two-way ANOVA followed by Tukey post hoc tests. *p < 0.05, **p < 0.01, ***p < 0.001. See Extended Data table (Fig. 2-2) for detailed statistical analyses. A, B, Percentage exploration preference for a novel object compared with a familiar object during novel object recognition training (Train) and testing conducted 5 min (A), 4 h (B, Test 1), or 24 h (B, Test 2) after training of B6 and BTBR mice. C, Latency of B6 and BTBR mice injected with Veh or IGF-II prior IA training (Train). Mice were tested at 24 h after training (Test 1) and again 6 d later (Test 2). D, Percentage of time spent freezing before (Pre-US) or after (Post-US) the shock delivery during contextual fear conditioning training and testing at 24 h after training (Test) of B6 and BTBR mice injected with Veh or IGF-II. E, Percentage exploration preference for a novel object compared with a familiar object during novel object recognition of B6 and BTBR mice injected with Veh or IGF-II 20 min before reactivation, which consisted in a full test session given 4 h after training (Train). Test was conducted 24 h after the reactivation. F, IA latency of B6 and BTBR mice during training (Train), Test 1 conducted 24 h after reactivation and Test 2 conducted 6 d after Test 1. Reactivation consisted in 30 s exposure to the context. G, Percentage of time spent freezing during CFC testing (Test) of B6 and BTBR mice, which received a bilateral dorsal hippocampal injection (↑) of IGF-IR blocker (JB1), IGF-IIR functionally blocking antibody (Anti), Veh, or IgG immediately before a subcutaneous injection of either Veh or IGF-II. Test was conducted 24 h after training. Exploration times are shown in the Extended Data (Fig. 2-1), and detailed statistical analyses are reported in the Extended Data tables (Figs. 2-2 and 2-3).
Figure 3.
Figure 3.
IGF-II reverses high repetitive behaviors of BTBR mice. Experimental timelines are shown above graphs. In all experiments mice received injections (↑) 20 min before testing. All data are expressed as the mean (±SEM). n = 7–8 per group. Two-way ANOVA followed by Tukey post hoc tests. *p < 0.05, **p < 0.01, ***p < 0.001. A, Time spent burying marbles by B6 and BTBR mice injected with vehicle (Veh) or IGF-II, 20 min before the test. B, Total number of marbles buried by B6 and BTBR mice injected with Veh or IGF-II before test. C, Time spent grooming (Test) 20 min following IGF-II or Veh injections in B6 and BTBR mice. D, Percentage of correct alternations in a Y-maze of B6 and BTBR mice injected with vehicle (Veh) or IGF-II before testing (Test). For detailed statistical analysis see the Extended Data table (Fig. 3-1).
Figure 4.
Figure 4.
IGF-II does not change anxiety responses in BTBR mice. Experimental timelines are shown above graphs. In all experiments mice received a subcutaneous injection (↑) at the indicated time before behavioral assessment or euthanasia (Euth), as specified in each experiment. All data are expressed as the mean (±SEM). n = 7–8 per group. Two-way ANOVA followed by Tukey post hoc tests. *p < 0.05, **p < 0.01, ***p < 0.001. A, Percentage of center entries, (B) time spent in the center, and (C) total entries into the center of an open-field by B6 and BTBR mice following a subcutaneous vehicle (Veh) or IGF-II injection given 20 min before Test. D, Time spent in the open arms, (E) percentage of entries into the open arms, and (F) total entries into the open arms of an elevated plus maze by B6 and BTBR mice 20 min following Veh or IGF-II injection. G, H, Relative concentration of plasma corticosterone (normalized to the levels of B6 untrained mice) of untrained B6 and BTBR mice 20 min (G) or 3 h (H) following a subcutaneous injection of Veh or IGF-II. I, Relative concentration of plasma corticosterone (normalized to levels of B6 untrained mice) of untrained or CFC-trained B6 and BTBR mice, which received a subcutaneous injection of Veh or IGF-II and were killed (Euth) 20 min after training or at the matched time point for untrained mice. For detailed statistical analyses see the Extended Data table (Fig. 4-1).
Figure 5.
Figure 5.
Increased proIGF-II and decreased IGF-IIR in BTBR compared with B6 mice. Western blot analyses of dorsal hippocampus, mPFC, striatum, and cerebellum, total and SN protein extracts. Each relative value was normalized against β-actin detected on the same blot. Representative blots are shown above their respective grouped data. Left: 20 kDa pro-IGF-II and 14 kDa IGF-II; Right: IGF-IIR in BTBR and B6 mice. All data are expressed as the mean (±SEM) and normalized to the level of B6 naive mice. n = 4–8 per group. Independent t test. *p < 0.05, **p < 0.01, ***p < 0.001. For SN comparisons to total extracts for MAP2 and PSD-95 see the Extended Data (Fig. 5-1), and for detailed statistical analyses see the Extended Data table (Fig. 5-2).
Figure 6.
Figure 6.
IGF-II reduces the abnormally high levels of the mTOR-S6K pathway in the brain of BTBR mice. Experimental timelines are shown above graphs. In all experiments mice received a subcutaneous injection (↑) at the time specified in each panel. All data are expressed as the mean (±SEM) and normalized to the level of B6 vehicle-injected mice. n = 6–8 per group. Two-way ANOVA followed by Tukey post hoc tests. *p < 0.05, **p < 0.01, ***p < 0.001. Representative blots are shown beside their respective grouped data. A, Western blot analyses of mTOR, pmTOR, S6K, pS6K, AMPK, pAMPK, ULK1, and pULK1 of SN protein extracts obtained from dorsal hippocampi of untrained B6 and BTBR mice 80 min following a subcutaneous vehicle (Veh) or IGF-II injection. Each relative value was normalized to β-actin detected on the same blot. B, Western blot analyses of mTOR, pmTOR, S6K, pS6K, AMPK, pAMPK, ULK1, and pULK1 of SN protein extracts obtained from dorsal hippocampi of untrained and trained B6 and BTBR mice that received a subcutaneous vehicle (Veh) or IGF-II injection 20 min before training and were killed 1 h after training or at the matched time point for untrained controls. Each relative value was normalized to β-actin detected on the same blot. For Western blot analyses of LC3-BI, LC3-BII, and p62 from dorsal hippocampi of naive B6 and BTBR mice; see the Extended Data (Fig. 6-1). For detailed statistical analyses see the Extended Data tables (Figs. 6-2 and 6-3).
Figure 7.
Figure 7.
IGF-II reduces overactive translation in the hippocampus of BTBR mice. Experimental timelines are shown above graphs. Active translation was measured by puromycin incorporation, which was injected bilaterally into the dorsal hippocampus (yellow ↑). Immediately after the puromycin injection mice received a subcutaneous injection (↑) of IGF-II or vehicle (Veh; B) in the presence or absence of anti-IGF-II receptor blocking antibody (C). Representative blots are shown beside their respective grouped data. Each relative value was normalized against β-tubulin (Tu) detected on the same blot. All data are expressed as the mean (±SEM) of B6 naive (A), B6 vehicle-injected (B), or BTBR vehicle-injected (C) mice. n = 6–8 per group. −Control represents the labeling of control samples without puromycin. A, B, Two-way ANOVA followed by Tukey post hoc tests. C, One-way ANOVA followed by Tukey post hoc tests. *p < 0.05, **p < 0.01. A, Western blot analyses of puromycin (P) incorporation in SN (top) and total protein (bottom) extracts obtained from dorsal hippocampi of untrained B6 and BTBR mice killed 60 min after P injection. B, Western blot analyses of P incorporation in SN (top) and total protein (bottom) extracts obtained from dorsal hippocampus of B6 and BTBR mice injected subcutaneously with either Veh or IGF-II and killed 60 min after the injection. C, Western blot analyses of P incorporation in SN protein extracts obtained from dorsal hippocampi of BTBR mice injected bilaterally into the dorsal hippocampus with an IGF-IIR functionally blocking antibody (Anti; red ↑) or IgG immediately before a subcutaneous injection of Veh or IGF-II and killed 60 min after the subcutaneous injection. For detailed statistical analyses see the Extended Data table (Fig. 7-1).

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