Cerebral Semaphorin3D is a novel risk factor for age-associated cognitive impairment

Abstract

Background: We previously reported that miR-195 exerts neuroprotection by inhibiting Sema3A and cerebral miR-195 levels decreased with age, both of which urged us to explore the role of miR-195 and miR-195-regulated Sema3 family members in age-associated dementia.

Methods: miR-195a KO mice were used to assess the effect of miR-195 on aging and cognitive functions. Sema3D was predicted as a miR-195 target by TargetScan and then verified by luciferase reporter assay, while effects of Sema3D and miR-195 on neural senescence were assessed by beta-galactosidase and dendritic spine density. Cerebral Sema3D was over-expressed by lentivirus and suppressed by si-RNA, and effects of over-expression of Sema3D and knockdown of miR-195 on cognitive functions were assessed by Morris Water Maze, Y-maze, and open field test. The effect of Sema3D on lifespan was assessed in Drosophila. Sema3D inhibitor was developed using homology modeling and virtual screening. One-way and two-way repeated measures ANOVA were applied to assess longitudinal data on mouse cognitive tests.

Results: Cognitive impairment and reduced density of dendritic spine were observed in miR-195a knockout mice. Sema3D was identified to be a direct target of miR-195 and a possible contributor to age-associated neurodegeneration as Sema3D levels showed age-dependent increase in rodent brains. Injection of Sema3D-expressing lentivirus caused significant memory deficits while silencing hippocampal Sema3D improved cognition. Repeated injections of Sema3D-expressing lentivirus to elevate cerebral Sema3D for 10 weeks revealed a time-dependent decline of working memory. More importantly, analysis of the data on the Gene Expression Omnibus database showed that Sema3D levels were significantly higher in dementia patients than normal controls (p < 0.001). Over-expression of homolog Sema3D gene in the nervous system of Drosophila reduced locomotor activity and lifespan by 25%. Mechanistically, Sema3D might reduce stemness and number of neural stem cells and potentially disrupt neuronal autophagy. Rapamycin restored density of dendritic spines in the hippocampus from mice injected with Sema3D lentivirus. Our novel small molecule increased viability of Sema3D-treated neurons and might improve autophagy efficiency, which suggested Sema3D could be a potential drug target. Video Abstract CONCLUSION: Our results highlight the importance of Sema3D in age-associated dementia. Sema3D could be a novel drug target for dementia treatment.

Keywords: Aging; Autophagy; Cognition; Neurodegeneration; Sema3D; miR-195.

Fig. 1

miR-195a KO mice present cognitive impairments and neurodegenerative features. A-B Learning and spatial memory were evaluated by the Morris Water Maze test (MWM) in miR-195a KO and age-matched WT mice. A Longer escape latency to reach the hidden platform indicates lower learning ability. Significant results from one-way repeated measures ANOVA for each type of mice are indicated by pound signs (#), and from two-way repeated measures ANOVA for comparison between KO and WT mice indicated by asterisk signs (*). B Longer escape latency to reach the platform indicates worse memory. Two-way repeated measures ANOVA yielded that KO had worse memory than age-matched WT mice. C The Y-maze test assesses working memory by measuring the time to reach the arm of the novel toy. Quantitative data is in the right panel. D Locomotor function was measured by the open field test. The representative images (left panel) show the traveled tracks of WT and miR-195a KO mice in the open field. The quantitative data are in the right panel. E Senescence-associated ß-galactosidase (SA-ß-gal) stain in the hippocampus. The representative images in the left panel show hippocampal senescent cells (green). Scale bar = 100 μm (n = 3/each group). F miR-195a KO mice had reduced NSC population than did age-matched WT mice. Representative images show SOX2 + (red) NSCs in the hippocampal dentate gyrus (DG) of WT mice and miR-195a KO mice (n = 3/group). Magnification: 20X. Scale bar = 200 μm. G Decreased dendritic spine density in miR-195a KO mice. Representative images of apical dendritic shaft of CA1 pyramidal neurons from 4-month-old miR-195a KO mice and age-matched WT mice (n = 3 mice/group; 15 neurons/mice). Scale bar = 5 μm. Figure 1A: #p < 0.05 verse first-day escape latency of the same mice type. *p < 0.05, **p < 0.01, *** p < 0.001

Fig. 2

Increased Sema3D levels deteriorate cognitive functions. A Representative IHC stain of endogenous Sema3D protein in the brain of WT mice. Upper panel: whole-brain section; Lower panel: hippocampus. B-D Learning and spatial memory performance were examined by the MWM in control and Sema3D-overexpressing mice aged 4 months (n = 4 /group). Significant results from one-way repeated measures ANOVA for each type of mice as indicated by pound signs (#), and from two-way repeated measures ANOVA for comparisons between Sema3D-overexpressing and control mice as indicated by asterisk signs (*). B The control mice significantly improved their learning after 4-day practices but Sema3D-overexpressing mice failed to show significant improvement after 5-day practices. C Escape latency and D frequency were measured in the memory trials. These two types of mice had significant differences in memory over the 14-day test. E Sema3D-overexpressing mice significantly lost short-term memory on day 14 in the novel object recognition test (n = 4/group). F The Y-maze test was performed to measure spatial working memory in mice for 10 weeks (n = 14/each group). A lower percentage of alteration indicates worse spatial working memory. Data in Fig. 2B-F are presented as mean ± SEM. **p < 0.01 and ***p < 0.001. #p < 0.05 G The Y-maze test measuring spatial working memory was used to assess the effect of siRNA-Ctrl or siRNA-Sema3D injected into miR-195a KO mice aged 12 months (n = 7/each group). The tests for siRNA-Sema3D and siRNA-Ctrl mice were conducted on the same experiment day. Two-way repeated measures ANOVA showed siRNA-Sema3D significantly improved spatial memory (*p < 0.05). H Locomotor function was determined using the total travel distance, with longer distance indicating better locomotor function

Fig. 3

Sema3D affects neural stem cells and autophagy. A Survival curve of Sema2A-overexpressing flies and control flies (n = 300/ each group) was analyzed by the Gehan-Breslow-Wilcoxon test. The data showed Sema2A-overexpressing flies had shorter life span than the control flies. B Adult Sema2A-overexpressing flies show age-related locomotion decline by the negative geotaxis assay at 2, 3, and 4 weeks-post eclosion (n = 150/group; p = 0.020, Mann–Whitney test). C Representative images of neurosphere formation by human NSCs at 48 h after Sema3D treatment. Scale bar = 200 μm. Quantitative data are presented as mean ± SEM from three independent experiments. **p < 0.01 and ***p < 0.001. D Sema3D decreased the NSC population in a dose-dependent manner. Representative images of SOX2 + (red) NSCs in the hippocampal dentate gyrus (DG) of mice receiving Sema3D injection (left panel). Magnification: 20X. (Scale bar = 200 μm; n = 3/group). Quantification of SOX2 + cells is shown on the right (mean ± SEM) from three independent experiments. *p < 0.05 and **p < 0.01. E Lv.Sema3D or Lv.Ctrl was bilaterally injected into the hippocampus of 4-month-old WT mice. On Day14, Sema3D and autophagy-associated proteins in the hippocampus were measured by western blot (n = 3/group). F Rapamycin reversed the inhibitory effect of Sema3D on cell proliferation in human neurons (SY5Y) by simultaneous treatment of Sema3D and rapamycin for 72 h. The proliferation of neurons was determined using Ki67 staining. Representative fluorescence images of Ki67 and DAPI staining. Scale bar = 50 μm. Quantitative data are presented as mean ± SEM from three independent experiments. *p < 0.05 and **p < 0.01. G Lv.Sema3D with/without rapamycin (0.2 nmol/0.2 µL) or Lv.Ctrl was bilaterally injected into the hippocampus of 4-month-old WT mice. The dendritic spine density was measured on Day 14. Representative images of apical dendritic shaft of CA1 pyramidal neurons (n = 3 mice/group; 15 neurons/mice). Scale bar = 5 μm. **p < 0.01

Fig. 4

Novel Sema3D antagonist CHOV20191024. A Predicted binding site of Sema3D according to the critical residues of Sema3A and PlexinA2 interface. K108, H216, and R404 were on Sema3A; K122, S233, and K422 were on Sema3D. B Cytotoxicity of CHOV20191024 was assessed in human neurons (SY5Y) and the total cell number was calculated at 48 h-post-treatment. Quantitative data are presented as mean ± SEM from three independent experiments. C CHOV20191024 rescued cell viability of Sema3D-treated SY5Y cells that were simultaneously treated with Sema3D and CHOV20191024 for 48 h. The MTT assay was conducted to determine cell viability. Quantitative western blot data are on the right (mean ± SEM) from three independent experiments. N.S. no significance; *p < 0.05. D CHOV20191024 reversed Sema3D-induced signaling cascade in SY5Y cells treated with Sema3D and CHOV20191024 for 24 h. Phosphorylation of the PI3K/Akt/mTOR signaling pathway was measured by western blot. E CHOV20191024 rescued Sema3D-induced autophagy dysfunction in SY5Y cells treated with Sema3D and CHOV20191024 for 72 h. Autophagy-associated proteins including mTOR, p62, Beclin-1, and LC3-II / I were measured by western blot. For Fig. 4D and E, quantitative data are presented as mean ± SEM from three independent experiments. *p < 0.05 and **p < 0.01