Insulin resistance and exendin-4 treatment for multiple system atrophy

Abstract

See Stayte and Vissel (doi:10.1093/awx064) for a scientific commentary on this article. Multiple system atrophy is a fatal sporadic adult-onset neurodegenerative disorder with no symptomatic or disease-modifying treatment available. The cytopathological hallmark of multiple system atrophy is the accumulation of α-synuclein aggregates in oligodendrocytes, forming glial cytoplasmic inclusions. Impaired insulin/insulin-like growth factor-1 signalling (IGF-1) and insulin resistance (i.e. decreased insulin/IGF-1) have been reported in other neurodegenerative disorders such as Alzheimer's disease. Increasing evidence also suggests impaired insulin/IGF-1 signalling in multiple system atrophy, as corroborated by increased insulin and IGF-1 plasma concentrations in multiple system atrophy patients and reduced IGF-1 brain levels in a transgenic mouse model of multiple system atrophy. We here tested the hypothesis that multiple system atrophy is associated with brain insulin resistance and showed increased expression of the key downstream messenger insulin receptor substrate-1 phosphorylated at serine residue 312 in neurons and oligodendrocytes in the putamen of patients with multiple system atrophy. Furthermore, the expression of insulin receptor substrate 1 (IRS-1) phosphorylated at serine residue 312 was more apparent in inclusion bearing oligodendrocytes in the putamen. By contrast, it was not different between both groups in the temporal cortex, a less vulnerable structure compared to the putamen. These findings suggest that insulin resistance may occur in multiple system atrophy in regions where the neurodegenerative process is most severe and point to a possible relation between α-synuclein aggregates and insulin resistance. We also observed insulin resistance in the striatum of transgenic multiple system atrophy mice and further demonstrate that the glucagon-like peptide-1 analogue exendin-4, a well-tolerated and Federal Drug Agency-approved antidiabetic drug, has positive effects on insulin resistance and monomeric α-synuclein load in the striatum, as well as survival of nigral dopamine neurons. Additionally, plasma levels of exosomal neural-derived IRS-1 phosphorylated at serine residue 307 (corresponding to serine residue 312 in humans) negatively correlated with survival of nigral dopamine neurons in multiple system atrophy mice treated with exendin-4. This finding suggests the potential for developing this peripheral biomarker candidate as an objective outcome measure of target engagement for clinical trials with glucagon-like peptide-1 analogues in multiple system atrophy. In conclusion, our observation of brain insulin resistance in multiple system atrophy patients and transgenic mice together with the beneficial effects of the glucagon-like peptide-1 agonist exendin-4 in transgenic mice paves the way for translating this innovative treatment into a clinical trial.

Keywords: MSA; alpha-synuclein; movement disorders; neuroprotection.

Figure 1

Neurons are insulin resistant in MSA. Representative images of IRS-1pS312 and IRS-1pS616 staining in neurons of controls (n = 5) (A and E) and MSA patients (n = 7) (B and F). The number of IRS-1pS312 and IRS-1pS616 positive neurons is decreased in MSA (n = 7) compared to healthy controls (n = 5) (C and G). IRS-1pS312 staining intensity is increased in neurons of MSA patients (n = 7) compared to healthy controls (n = 5) (D), while no differences were observed for IRS-1pS616 staining intensity (H). Scale bar = 10 µm. A t-test was used to compare data between MSA patients and healthy controls. If data were not normally distributed, a Mann-Whitney test was used instead. Values are mean ± SEM. **P < 0.01 and ***P < 0.001 versus controls. A.U. = arbitrary units.

Figure 2

Oligodendrocytes are insulin resistant in MSA. Representative images of IRS-1pS312 and IRS-1pS616 staining in oligodendrocytes of controls (n = 5) (A and E) and MSA patients (n = 7) (B and F). The number of IRS-1pS312 and IRS-1pS616 positive oligodendrocytes was similar in both groups (C and G). IRS-1pS312 staining intensity is increased in oligodendrocytes of MSA patients (n = 7) compared to healthy controls (n = 5) (D). Quantification of IRS-1pS616 staining intensity showed no significant difference between groups (H). Scale bar = 10 µm. A t-test was used to compare data between MSA patients and healthy controls. If data were not normally distributed, a Mann-Whitney test was used instead. Values are mean ± SEM. *P < 0.05 versus controls. A.U. = arbitrary units.

Figure 3

IRS-1pS312 and IRS-1pS616 staining intensity is not different in astrocytes. Representative images of IRS-1pS312 and IRS-1pS616 staining in astrocytes of controls (n = 5) (A and E) and MSA patients (n = 7) (B and F). The number of IRS-1pS312 and IRS-1pS616 positive astrocytes is increased in MSA patients (n = 7) compared to healthy controls (n = 5) (C and G). Quantification of IRS-1pS312 and IRS-1pS616 staining intensity showed no significant differences between groups (D and H). Scale bar = 10 µm. A t-test was used to compare data between MSA patients and healthy controls. If data were not normally distributed, a Mann-Whitney test was used instead. Values are mean ± SEM. **P < 0.01 versus controls. A.U. = arbitrary units.

Figure 4

IRS-1pS312 and IRS-1pS616 staining intensity is not different in microglia. Representative images of IRS-1pS312 and IRS-1pS616 staining in microglia of controls (n = 5) (A and E) and MSA patients (n = 7) (B and F). The number of IRS-1pS312 and IRS-1pS616 positive microglia is not different in MSA patients (n = 7) compared to healthy controls (n = 5) (C and G). IRS-1pS312 and IRS-1pS616 staining intensity was not different between groups (D and H). Scale bar = 10 µm. A t-test was used to compare data between MSA patients and healthy controls. If data were not normally distributed, a Mann-Whitney test was used instead. Values are mean ± SEM. A.U. = arbitrary units.

Figure 5

Insulin resistance in PLP-SYN mice is reversed by exendin-4 treatment. (A) Representative image of IRS-1pS307 protein levels in the striatum of PLP-SYN mice and wild-type littermates. (B) Significant increase in striatal IRS-1pS307 protein levels in PLP-SYN mice (n = 7) compared to wild-type (WT) littermates (n = 7). (C) Representative image of striatal IRS-pS612 protein expression levels in the striatum of PLP-SYN mice and wild-type littermates. (D) No difference was observed in IRS-1pS612 protein levels between groups. (E and G) Representative images of IRS-1pS307 and IRS-1pS612 protein expression in the striatum of PLP-SYN mice treated with placebo (n = 9), 3.5 pmol/kg/min (n = 9) or 8.75 pmol/kg/min exendin-4 (n = 7). (F and H) Significant decrease in IRS-1pS307 and IRS-1pS612 protein expression in the 8.75 pmol/kg/min exendin-4 treated group (n = 7) compared to placebo PLP-SYN mice (n = 9). A t-test was used to compare striatal IRS-1pS307 and IRS-1pS612 protein expression levels between PLP-SYN and wild-type littermates. A one-way ANOVA was applied to compare treatment effects of exendin-4 between groups, followed by post hoc Holm-Sidak tests for multiple comparisons if appropriate. If data were not normally distributed, an ANOVA on ranks was performed instead, followed by Dunn’s multiple comparisons when appropriate. Values are mean ± SEM. *P < 0.05 versus placebo PLP-SYN, **P < 0.01 versus placebo PLP-SYN. A.U. = arbitrary units.

Figure 6

Exendin-4 treatment protects dopaminergic neurons in the SNc in PLP-SYN mice. (A) A trend for improvement of motor performance in the traversing beam task was observed in PLP-SYN mice receiving the higher dose exendin-4 treatment but the difference between groups did not reach significance. (B) Significant loss in tyrosine hydroxylase (TH) positive neurons in the SNc of placebo PLP-SYN mice (n = 9) compared to PLP-SYN mice treated with 8.75 pmol/kg/min exendin-4 (n = 7). (C) Significant loss in Nissl-stained neurons in the SNc of placebo PLP-SYN mice (n = 9) compared to PLP-SYN mice treated with either 3.5 pmol/kg/min (n = 9) or 8.75 pmol/kg/min exendin-4 (n = 7). (D–F) Representative SNc sections from placebo (D), 3.5 pmol/kg/min exendin-4 (E) and 8.75 pmol/kg/min exendin-4 treated PLP-SYN mice (F). Scale bar = 600 µm. A one-way ANOVA was applied to compare treatment effects of exendin-4 between groups, followed by post hoc Holm-Sidak tests for multiple comparisons if appropriate. If data were not normally distributed, an ANOVA on ranks was performed instead, followed by Dunn’s multiple comparisons when appropriate. Values are mean ± SEM. *P < 0.05 versus 8.75 pmol/kg/min exendin-4 PLP-SYN mice, ^$P < 0.05 versus 3.5 pmol/kg/min exendin-4 treated PLP-SYN mice.

Figure 7

Exendin-4 treatment decreases α-synuclein load in the striatum of PLP-SYN. mice. (A) Representative western blots of high molecular weight (Hmw) oligomeric and monomeric α-synuclein in the striatum. (B) High molecular weight α-synuclein levels were not different between placebo (n = 9), 3.5 pmol/kg/min (n = 9) and 8.75 pmol/kg/min exendin-4 treated PLP-SYN mice (n = 7). (C) A significant decrease in monomeric α-synuclein was observed in PLP-SYN mice treated with 8.75 pmol/kg/min exendin-4 (n = 7) compared to placebo PLP-SYN mice (n = 9). A one-way ANOVA was applied to compare treatment effects of exendin-4 between groups, followed by post hoc Holm-Sidak tests for multiple comparisons if appropriate. If data were not normally distributed, an ANOVA on ranks was performed instead, followed by Dunn’s multiple comparisons when appropriate. Values are mean ± SEM. **P < 0.01 versus placebo PLP-SYN. A.U. = arbitrary units.