Intranasal long R3 insulin-like growth factor-1 treatment promotes amyloid plaque remodeling in cerebral cortex but fails to preserve cognitive function in male 5XFAD mice
Matthew G Engel et al. J Alzheimers Dis. 2025 Jan.
Abstract
Background: Insulin-like growth factor-1 (IGF-1) promotes neurogenesis, cell survival, and glial function, making it a promising candidate therapy in Alzheimer's disease (AD).
Objective: Long arginine 3-IGF-1 (LR3-IGF-1) is a potent IGF-1 analogue. We sought to determine whether intranasal (IN) LR3 treatment would delay cognitive decline and pathology in 5XFAD mice.
Methods: Wildtype and 5XFAD male mice were treated for 7 months (3-10 months of age), with IN LR3-IGF-1 or IN Vehicle (Veh) (n = 19-27 mice/group). Behavior, memory, and brain imaging were assessed at 8-9 months of age and tissues collected at 10 months. A comprehensive amyloid-β (Aβ) profile and other pathologic features were conducted and supportive in vitro stimulation studies in BV-2 microglial cells were also performed.
Results: In male 5XFAD mice, IN LR3-IGF-1 treatment improved body composition, but did not significantly alter cognitive symptoms, as assessed by multiple assays. In cortex, LR3 treatment improved some facets of pathology, including a reduction in filamentous plaques, and increase in inert plaques, corresponding with a reduction in low molecular weight Aβ oligomers. In vitro, uptake of Aβ1-42 peptide by BV2 cells was enhanced by LR3-IGF-1, which was also found to promote gene pathways implicated in actin remodeling and endocytosis.
Conclusions: LR3 promotes favorable effects on Aβ plaque remodeling in cortex of male 5XFAD mice but fails to preserve aspects of behavior or memory. While these data do not support LR3 as a monotherapy per se, they do warrant further investigation into its potential for combinatorial formulations aimed at targeting the complexity of AD.
Keywords: Alzheimer's disease; IGF-1; LR3-IGF-1; amyloid; intranasal.
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Conflict of interest statement
Conflicts of Interest: The authors declare no conflicts of interest.
Figures Figure 1.. Experimental design, ex vivo LR3-IGF-1 stimulation, and body composition with long-term LR3-IGF-1 treatment.
(A) Experimental design for longitudinal intranasal (IN) treatments. Male C57BL/6J WT or 5XFAD mice were acclimated at 10 weeks of age, then treated intranasally with Vehicle or LR3-IGF-1 (4 x 50 μg weekly) from 3 months to 10 months of age. Behavior and memory were assessed from 8-9 months of age, and MRI imaging was acquired at 9 months of age. (B) Ex vivo stimulation of cerebral cortex and hippocampus of wild-type 8-month-old male C57BL/6J mice (n=10) with aCSF (vehicle), 10 nM LR3-IGF-1, or 10 nM insulin for 2 minutes. Phospho-Akt(T308) expression increased in both cortex and hippocampus treated with 10 nM LR3-IGF-1 relative to control. Image of the strain-free gel is included to confirm equal loading. (C) Body weight-change over the study duration. Mean weight change from 3-10 months was +3.9 g (WT-Veh), +4.6 g (WT-LR3), +2.4 g (5X-Veh), and +2.6 g (5X-LR3), with significant weight change over time (p<0.0001) modified by genotype (p<0.005). (D-F) Lean mass (p<0.005) and fat mass (p<0.05) were modified in 5XFAD mice compared with WT, with a trend toward reduced percentage body fat (fat mass / total body weight) in IN LR3-IGF-1 treated 5XFAD mice compared with those treated with IN Vehicle (p=0.0523). Data are shown as mean ± S.E.M. *p<0.05. Figure 2.. Protein Markers of canonical IGF-1 Signaling in WT and 5XFAD mice following long-term IN vehicle or LR3 treatment.
(A) IGF-1R expression was unaltered with longitudinal LR3-IGF-1 treatment and was not different between 10-month-old male WT and 5XFAD heterozygous mice (n=4-10 per group). (B-F) Immunoblotting for total and phosphorylated proteins downstream of IGF-1R was used to assess changes in basal signaling following 7 months of treatment, 24 hours after the final intranasal dose. Basal phosphorylation of p70S6K (n=9-12), GSK3β (n=9-12), and ERK1/2 (n=4-10) were not different among treatment groups. A significant interaction was observed for phosphorylation of Akt on Threonine 308 (p<0.05), but no significant differences were observed among planned comparisons (n=4-10). Phosphorylation of 4E-BP1 on Threonine 37 and 46 was modified by 5XFAD genotype but was not different among groups (n=9-12). Image of the strain-free gel is included to confirm equal loading. Data are shown as mean ± S.E.M. Figure 3.. β-Amyloid Phenotype in WT and 5XFAD mice following long-term IN vehicle or LR3 treatment.
(A) Diaminobenzidine (DAB) stain of β-amyloid (1:500, D54D2) in sagittal sections obtained from intranasally treated mice (n=4 per WT group, n=7-8 per 5XFAD group). Plaques are undetectable in WT mice but are abundant in both 5X-Veh and 5X-LR3 mice. Scale bar = 100 μm. (B,C) Size distribution analysis of D54D2+ plaques. A trend toward reduced plaques in the 40-60 um range was seen in cerebral cortex with IN LR3-IGF-1 treatment (p=0.0614). (D) Triple stain of D54D2+ β-amyloid, Iba1 (microglia marker), and methoxy-X04 to identify dense plaque cores, n=8 per group. Representative filamentous (D54D2+,MeX04 lo/−), compact (D54D2+,MeX04+), and inert (D54D2 lo/−,MeX04+) plaques are indicated with arrows. 20x magnification, scale bar = 100 μm. (E) (E-F) Percentage of each plaque type in cerebral cortex and hippocampus. The percentage of filamentous plaques is diminished (35% vs 29%, p<0.05) and of inert plaques is increased (13% vs 22%, p<0.01) in cerebral cortex with IN LR3-IGF-1 treatment, with similar, but non-significant, changes observed in the distribution of plaque types in hippocampus. (G) Normalized D54D2+ fluorescence in 5X-Veh compared with 5X-LR3. Cortical D54D2+ fluorescence is reduced in IN LR3-IGF-1 treated 5XFAD mice (1.0 vs 0.62, p<0.01) but is not significantly different in hippocampus (1.0 vs 0.77, p=0.170). (H-J) Representative immunoblot and quantification for RIPA-soluble β-amyloid (D54D2) in cerebral cortex. Image of the strain-free gel is included to confirm equal loading. Whereas only APP bands can be seen in WT control, diffuse D54D2 bands are seen in 5XFAD lysate, representing multimers and increasingly large aggregates. Low-molecular weight oligomers are observable between 10-50 kD, with higher-molecular weight oligomers in the >50 kD range. No significant change was observed in normalized Aβ monomer expression (cortex, 1.0 vs 0.82, p=0.267; hippocampus, 1.0 vs 0.96, p=0.642), but low molecular-weight Aβ oligomers were reduced in cerebral cortex (1.0 vs 0.84, p<0.05). N=16-18 per 5XFAD group. Data are shown as mean ± S.E.M. *p<0.05. Figure 4.. Behavioral and Memory Performance following long-term IN vehicle or LR3 treatment.
A series of behavioral and cognitive assays were performed in intranasally treated mice between 8-9 months of age, 5-6 months into intranasal treatment. (A) Y-maze did not detect any cognitive deficits in 5XFAD mice relative to WT nor was there any effect of treatment. (B) During the box maze, 5XFAD mice take significantly longer than WT mice to escape the arena (p<0.0001), but all groups reduce latency as trials progress (p<0.0001). (C-D) Treatment (p<0.05), but not genotype, led to altered activity in the open field test, but all groups spend a comparable amount of time in the central zone of the open field. (E) 5XFAD mice spend more time in the open arm of the zero maze relative to WT (p<0.005), but the percentage of time spent in the open arm is not significantly different among groups. (F) 5XFAD mice are less immobile in the forced swim test than WT (p<0.005), with 5X-LR3 mice tending to show reduced immobility relative to vehicle-treated groups (p=0.0788). (G) Latency to detect an olfactory stimulus is unchanged with intranasal LR3-IGF-1 treatment and does not differ among groups. (F) n=19-27 for all assays except box maze (n=13-17). Data are shown as mean ± S.E.M. Figure 5.. Structural MRI and cerebral blood flow.
(A,B) Neither total brain volume nor cerebral blood flow significantly varied among groups. (C) In the cortical frontal lobe, a significant interaction was observed (p<0.05) for T2*-weighted relaxation time and was found to decrease in 5X-Veh mice relative to WT (p<0.01). (D) In hippocampus, a significant interaction (p(interaction )<0.05) for T2* relaxation time was observed, but there were no differences among groups. (E,F) In the cortical parieto-temporal lobe, fractional anisotropy was lower in 5XFAD mice relative to WT (p<0.005), but axial diffusivity was unaltered. (G,H) In hippocampus, fractional anisotropy and axial diffusivity were both reduced by 5XFAD genotype (p<0.005). n=5-10 per group. Data are shown as mean ± S.E.M. **p<0.01. Figure 6.. LR3-IGF-1 Stimulation of Aβ(1-42) Peptide Uptake and Transcriptional Changes in BV-2 Cells.
(A-C) BV-2 cells were incubated with 200 nM HiLyte FluorTM 488 Aβ(1-42) for 1h with or without pre-treatment for 3h with 50 nM LR3-IGF-1 (n=7-8 per condition). LR3-IGF-1 pre-treatment did not significantly increase the number of puncta but did increase the %Aβ-fluorescent area (p<0.05). Scale bar = 100 μm. (D-F) Bulk transcriptomic analysis of BV-2 cells treated for 4h with 1 μm oligomeric Aβ (A) or LR3-IGF-1+Aβ (AR) (n=4 biological replicates per condition. (E)Volcano plot comparing AR vs A. The top 20 differentially expressed genes are shown. (F) Heatmap of AR vs A with hierarchical clustering of top 50 genes. (G) Normalized enrichment scores for top KEGG pathways positively or negatively enriched in AR vs A. Bar graphs and lines show results as mean ± S.E.M. *p<0.05.
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