IL-17 is up-regulated in APP/PS1 transgenic mice and induces neural toxicity.
Previous studies suggested critical roles for inflammatory cytokines in the pathogenesis of AD [11, 13, 28]. To analyze the expression of IL-1β, IL-6, TNF-α and IL-17 in amyloidosis-prone APP/PS1 mice, we performed ELASA test of 6-month-old APP/PS1 mice hippocampus. The expressions of IL-1β, IL-6, TNF-α (sFig. 1A-C) and IL-17 (Fig. 1A) were increased in hippocampus of APP/PS1 mice when compared to controls. Furthermore, analysis of western blotting from APP/PS1 mice hippocampus also showed significantly increased level of IL-17 (Fig. 1B, C). However, specific mechanism of IL-17 in neurodegenerative disease is still controversial. We then try to investigate the mechanism of IL-17 on AD-like pathology by observing the effect on neural in primary hippocampal neurons. The hippocampal primary neurons were divided into control group and IL-17 group (IL-17, 10 µg/mL). When compared with the control, IL-17 treatment resulted in an obvious decreased dendritic arborization complexity at all points farther than 60 um from the cell body (Fig. 1D-F). These data indicate that IL-17 may play a pathophysiological role in neural toxicity.
IL-17 results in cognitive and memory impairments and synaptic dysfunction accompanying with Aβ overproduction.
Thirty healthy wild type C57BL mice were adaptively domesticated for 7 days in animal laboratory. Then the mice were randomly divided into 2 groups (control and IL-17 group) of 15 each. The IL-17 group mice were injected with IL-17 for 1 week by intracerebroventricular surgery, and saline served as control for 1 week. We first carried open-field test after injection and the result showed that there was no significant difference in the total distance covered between the two groups (Fig. 2A), but the time of center duration was significantly reduced in IL-17 group compared with the control (Fig. 2B), implicating that IL-17 might reduce the ability of exploration. To further observe the exploration and short-term memory capacity in the mice, we employed novelty recognition experiments and found that the curiosity in IL-17 group was significant reduced, as the time spent for exploration of new object in 2h and 24h were significantly decreased (Fig. 2C, D). To examine the scene memory of the mice, we carried out the contextual fear conditioning test and found out that the scene memory was significantly impaired in the test group (Fig. 2E, F). Finally, we tested memory and learning abilities using MWM, and observed that there was no significant difference in the speed, but significantly increased latency to find the hidden platform between the two groups (Fig. 2G, H). On day 6, the spatial memory was checked by removing the platform. A remarkable decrease in the time spent in the target quadrant (Fig. 2I), as well as a decrease in the number of target platform crossing (Fig. 2J), was observed in the IL-17 group mice.
It is clear that IL-17 results in a series of cognitive and memory impairments in mice. Among these behavioral alterations, learning and memory impairments are commonly shared with other neurological disorders including neurodegenerative disease . And the above result showed that IL-17 level was up-regulated in APP/PS1 mice. The hippocampus is an integral part of the temporal limbic system and plays a critical role in memory and spatial location which is the reflection of synaptic plasticity [30, 31]. We wondered whether IL-17 might increase Aβ levels and then lead to synaptic dysfunction. Interestingly, the Aβ40 and Aβ42 in hippocampus were significantly higher in IL-17 group mice than in the control ones (Fig. 3A, B). We further investigated the levels of sAPPβ and pT668. The results showed that the levels of sAPPβ and pT668 in IL-17 group were increased compared with control group (sFig. 2A -C). Moreover, IL-17 dramatically caused the LTP impairment of mice (Fig. 3C, D), suggesting impaired synaptic function in IL-17 mice. To investigate the underlying mechanism, we further observed the ultrastructure of synapse in hippocampus with TEM, and results showed that the number of synapses per 100 um2 CA1 areas decreased significantly after IL-17 injection (Fig. 3E, F). We further examined the spine density of hippocampus neurons (Fig. 3G). The Golgi staining showed a significant decrease in the dendritic spine density of the IL-17 mice (Fig. 3H). In addition, we further performed western blotting and the result from the IL-17 groups showed a significant reduction in the synaptic protein levels of SYN and PSD95 (Fig. 3I, J). These results together imply that IL-17-induced cognitive impairments are associated with synaptic dysfunction accompanying with Aβ overproduction.
IL-17 inhibitor (Y-320) ameliorates Aβ 42 -induced neural toxicity in primary hippocampal neurons.
We have shown that IL-17 could induce cognitive impairment and synaptic dysfunction, an effect that may be mediated via increasing Aβ level. Aβ oligomers now widely regarded as instigating neuron damage leading to Alzheimer’s dementia [32, 33]. To further confirm this hypothesis, we performed an inhibitor of IL-17 (Y-320) to investigate whether it could ameliorate Aβ42-induced neural toxicity in primary hippocampal neurons at first. In the experiment, the hippocampal primary neurons were divided into three groups: control group, Aβ42 group (Aβ42, 2.5 µM), Aβ42 + IL-17 inhibitor group (Aβ42, 2.5 µM; Y-320, 100 nM). Y-320 resulted in a robust increase in the dendritic complexity at all points farther than 60µm from the cell body when compared with Aβ42 treated neurons (Fig. 4A -C). These data indicate that IL-17 inhibitor may ameliorate Aβ42-induced neural toxicity.
IL-17 inhibitor alleviates Aβ 42 -induced cognitive and memory Deficits in mice.
Studies using the Aβ42 human peptide showed impaired cognitive function early after a single intracerebroventricular injection [24, 34]. Based on the primary neuron results, we explored the effect of IL-17 inhibitor in Aβ42 model in vivo. We used 45 healthy C57BL mice randomly divided into 3 groups. Control group mice injected with saline (5 µL) in brain unilateral ventricle. Aβ42 mice model was established after injecting in brain unilateral ventricle with the solution Aβ42 (2.0 µg/µL, 5 µL), while the IL-17 inhibitor group were injected with Aβ42 (2.0 µg/µL, 5 µL), followed with oral gavage treatment of Y-320 (3 mg/kg, 7 days). Following the treatment, we again performed a couple of behavioral tests. In the open-field test, total distance showed no significant difference among three groups (Fig. 5A), indicating that the locomotion activity was not influenced by Aβ42 and Y-320 treatment. But the time of center duration was significantly increased in Aβ42 + IL-17 inhibitor group compared with the Aβ42 group. Next, the NORT showed that IL-17 inhibitor could significantly lengthen the time of new object spent by Aβ42 model mice in 2h and 24h (Fig. 5C, D). And then, the contextual fear conditioning test were performed and found out that the scene memory of mice was alleviated in Aβ42 + IL-17 inhibitor group compared with Aβ42 model group (Fig. 5E, F). Last, in MWM, the escape latency was significantly increased in the Aβ42 group compared with the control group on day 2, 3, 4 and 5, while supplementation with Y-320 attenuated Aβ42-induced learning deficits (Fig. 5H). In the probe trial on day 6, time was remarkably increased in the target quadrant (Fig. 5I) and the crossing number of target platform was increased (Fig. 5J) in Aβ42 + IL-17 inhibitor group. These behavioral tests results suggest that IL-17 inhibitor alleviates Aβ42-induced learning and memory deficits in mice.
IL-17 inhibitor rescues Aβ 42 -induced spine loss and synaptic dysfunction by reducing Aβ production.
We have previously found that IL-17 leads to synaptic dysfunction accompanying with up-regulation levels of Aβ40 and Aβ42 in hippocampus (Fig. 3). To further explore the relationship between IL-17 and Aβ, we supplied Y-320 in Aβ42 model mice. Western blotting and Elisa test showed the level of IL-17 was increased in Aβ42 model mice compared with control group, and reversed by IL-17 inhibitor treatment (Fig. 6A-C). We also tested Aβ40 and Aβ42 by Elisa and the results showed in hippocampus the levels were significant increased in Aβ42 model group and attenuated after supplementation with Y-320 (Fig. 6D, F), again suggesting that IL-17 inhibitor could be a protective element against the pathogenesis of Aβ toxicity. We further investigated the possible mechanism underlying the effect of IL-17 inhibitor on reducing Aβ production. The results showed that the levels of sAPPβ and pT668 in Aβ42 + IL-17 inhibitor group were decreased compared with Aβ42 group (sFig. 3A-C). In view of the above data, we wonder whether the IL-17 inhibitor plays a positive role in synaptic dysfunction induced by Aβ42. We therefore carried out electrophysiology experiments and found that IL-17 inhibitor enhanced the slope of fEPSP produced by mice in Aβ42 model group (Fig. 7A, 7B). In addition, we observed the number of synapses in hippocampus with TEM, and results showed that the number of synapses per 100 µm2 CA1 areas increased significantly after IL-17 inhibitor supplement compared with Aβ42 model group mice (Fig. 7C, D). And Aβ42 injection resulted in significant dendritic spine loss revealed in Golgi staining, and IL-17 inhibitor effectively reversed the spine loss (Fig. 7E, F). Consistent with these findings, the levels of SYN and PSD95 were reduced in Aβ42 group and partially recovered after supplementation with Y-320 (Fig. 7G-I). These results indicate that IL-17 inhibitor rescues the Aβ42–induced synaptic dysfunction probably by reducing Aβ generation.