Fig. 5

Primary cilia dysfunction contributed to axonal spheroid formation and Aβ deposition through vesicle accumulation. A Immunostaining of the 5xFAD mouse septum for nucleus (DAPI), Aβ, and MIF showed that MIF protein was located on DNs adjacent to Aβ plaques, suggesting that its failure of secretion and accumulation in DNs. Scale bars = 10 μm, green dashed lines: DN, white dashed lines: Aβ plaque, red dashed lines: MIF protein in DNs. B To examine the localization of MIF protein in the hippocampal neurons, MIF was immunostained using 5xFAD hippocampus. MIF protein was found at the basal end of ACIII-labeled primary cilia (yellow arrowhead) and co-localized with the ubiquitin-labeled proteasome system. Scale bar = 10 μm for ACIII and scale bar = 5 μm for ubiquitin staining. C Primary hippocampal neurons were harvested after siIft88 (40 nM) transfection for 48 h and oligomeric Aβ1-42 (1 μM) treatment for 6 h (siCon: scrambled short interfering RNA, siIft88: short interfering RNA for Ift88). Whole cell lysates (n = 3) were analyzed by LC–MS/MS. A total of 3546 proteins, with 3152 overlapping, 32 siCon-unique, and 362 siIft88-unique proteins, were identified. A scatter plot showed 527 up-regulated (log₂ fold-change > 0.25, adjusted p-value < 0.1) and 126 down-regulated proteins (log₂ fold-change < − 0.25, adjusted p-value < 0.1) as a result of the differentially expressed protein analysis. Shown are the fold changes in protein abundance between siIft88-transfected and siCon- transfected primary neurons and the weight value of this quantification. The position of the representative proteins selected for the pathway enrichment analysis is indicated. D Gene-set enrichment analysis based on GO Biological Process revealed 3 downregulated pathways related to vesicle-mediated transport and cilia assembly, and 16 upregulated pathways related to vesicle transport, endocytosis, and exocytosis. E Protein complexes were isolated from the primary neuron lysate (top) or the EV proteins collected from the conditioned media (bottom) followed by IP with the anti-CD63. Western blot analysis showed that Aβ was not complexed with CD63 in the lysate but was preferentially bound to CD63 in the control EVs. F Using hippocampal neurons derived from 5xFAD; Ift88-flox/flox mice grown in compartmentalized neuron culture platforms with Cre/loxP-mediated Ift88 deletion, the area of axon terminals was measured (left plot, axonal spheroid), and the co-localization coefficients of CD63 and Aβ in the axon terminals were measured (right plot). Primary neurons were derived from 5xFAD mice to induce intraneuronal Aβ accumulation without extracellular treatment. Both axon terminal areas and co-localization of CD63 and intraneuronal Aβ were significantly increased in primary neurons with Cre-induced knock-down of Ift88 expression (n = 3). Data are presented as the mean ± S.E.M. ****p < 0.001 as determined by the unpaired t-test (Ift88KD: knockdown of the Ift88 gene). G 5xFAD; Ift88-flox/flox; Ai6 (mut) and 5xFAD; Ift88flox/wt; Ai6 (het) mice were used for stereotaxic injection of AAV-Cre. AAV vectors were injected into the ventral hippocampus, and confocal imaging was conducted using the septum to measure the number of axonal spheroids surrounding the Aβ deposition (n = 3). Higher magnification images were taken from the box area of the upper panel. Data are presented as the mean ± S.E.M. *p < 0.05 as determined by the unpaired t-test. Scale bars = 50 μm (upper), 5 μm (lower)