Role of spindle pole body component 25 in neurodegeneration

Abstract

Background: Aberrant growth and polarization of microglia are critical for pathological initiation and progression of neurodegenerative conditions like Alzheimer's disease (AD). However, the molecular signals that govern the outgrowth of microglia have not yet been fully determined. Spindle pole body component 25 (SPC25) is an important part for forming NDC80 complex, which plays a key role in the assembly of the microtubule-binding domain of kinetochores. Nevertheless, the role of SPC25 in microglial growth during neurodegeneration has not been described before, and was thus addressed in the current study.

Methods: We generated an adeno-associated virus (AAV) serotype PHP.B carrying short hairpin RNA (shRNA) for SPC25 (shSPC25) under a microglia-specific TMEM119 promoter (AAV-pTMEM-shSPC25). Serotype PHP.B allowed the virus to cross blood-brain barrier, while TMEM119 promoter allowed specific targeting microglia in vitro and in vivo. We intravenously administrated AAV-pTMEM-shSPC25 to AD-prone APP/PS1 male and female mice and determined this effect on microglia proliferation and mouse behavior.

Results: Depletion of SPC25 did not alter polarization of microglia cell polarization in vitro. On the other hand, AD-prone APP/PS1 mice that had received AAV-pTMEM-shSPC25 significantly decreased SPC25 levels in microglia and attenuated microglia proliferation, resulting in significant improvement of the performance of the mice in behavior tests.

Conclusions: Specific depletion of SPC25 in microglia may prevent AD development through suppression of microglia outgrowth. SPC25 may be a promising novel target for preventing AD through microglia.

Keywords: Alzheimer’s disease (AD); Neurodegeneration; microglia; spindle pole body component 25 (SPC25).

Figure 1

Generation of AAV-pTMEM-shSPC25 capable of crossing BBB. (A) Schematic of generation of AAV-pTMEM-shSPC25 and control AAV-pTMEM-scramble, using serotype PHP.B. The viral promoter also drives an RFP reporter connected with transgene with a p2A connector. (B) Transduction of mouse microglia cell line HMC3, mouse neuronal cell line HCN-2, mouse macrophage cell line RAW264.7, and mouse fibroblast cell line 3T3 with AAV-pTMEM-scramble. (C,D) Viral RFP was detected by RT-qPCR in different organs (C), or by gross image (D), 5 days after tail vein injection of AAV-pTMEM-scramble. (E,F) Transduction of HMC3 by AAV-pTMEM-shSPC25 and control AAV-pTMEM-scramble. (E) Immunofluorescent staining for SPC25. (F) RT-qPCR for PSC25, iNOS and arginase. *P<0.05. NS: non-significant. N=5. Scale bars are 20 µm. AAV, adeno-associated virus; BBB, blood-brain barrier; RFP, red fluorescent protein; RT-qPCR, quantitative reverse transcription polymerase chain reaction.

Figure 2

Administration with AAV-pTMEM-shSPC25 attenuates behavior disorder in AD-prone mice. We performed single tail vein injection with AAV-pTMEM-shSPC25 or AAV-pTMEM-scramble at 3 months of age, and waited for another 3 months before evaluating the mice. (A-C) A Morris Water Maze test for assessment of path length in the visible platform (A), path length in the hidden platform (B) and mean target quadrant occupancy (C). (D-E) A motor assessment test of time crossing a narrow beam (D) and falling rate during crossing the beam (E). *P<0.05. N=6. AD, Alzheimer’s disease; AAV, adeno-associated virus.

Figure 3

Administration with AAV-pTMEM-shSPC25 attenuates Aβ deposition and p-Tau formation in APP/PS1 mouse brain. (A) Neuron loss in the CA1 region of mouse hippocampus. (B,C) The Aβ density in the CA1 region of mouse hippocampus was quantified at sacrifice, shown by representative images (B), and by quantification (C). (D) ELISA for Aβ levels in the CA1 region of mouse hippocampus at sacrifice. (E,F) The p-Tau density in the CA1 region of mouse hippocampus was quantified at sacrifice, shown by quantification (E), and by representative images (F). (G) ELISA for pTau levels in the CA1 region of mouse hippocampus at sacrifice. *P<0.05. N=6. Scale bars are 50 µm. AAV, adeno-associated virus; ELISA, enzyme-linked immunosorbent assay.

Figure 4

Administration with AAV-pTMEM-shSPC25 attenuates microglia outgrowth. (A) Immunostaining for a microglia-specific marker IBA-1 in the CA1 region of mouse hippocampus at sacrifice. (B) Representative flow charts for isolation of CD11b-positive, CD45-low microglia from the CA1 region of mouse hippocampus at sacrifice by flow cytometry. (C,D) Microglia percentage (C) and absolute number (D). *P<0.05. Scale bars are 50 µm. N=6. AAV, adeno-associated virus.