Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 4, 31
eCollection

Thrombospondin-1 Protects Against Aβ-induced Mitochondrial Fragmentation and Dysfunction in Hippocampal Cells

Affiliations

Thrombospondin-1 Protects Against Aβ-induced Mitochondrial Fragmentation and Dysfunction in Hippocampal Cells

Seokjo Kang et al. Cell Death Discov.

Abstract

Alzheimer's disease (AD) is often characterized by the impairment of mitochondrial function caused by excessive mitochondrial fragmentation. Thrombospondin-1 (TSP-1), which is primarily secreted from astrocytes in the central nervous system (CNS), has been suggested to play a role in synaptogenesis, spine morphology, and synaptic density of neurons. In this study, we investigate the protective role of TSP-1 in the recovery of mitochondrial morphology and function in amyloid β (Aβ)-treated mouse hippocampal neuroblastoma cells (HT22). We observe that TSP-1 inhibits Aβ-induced mitochondrial fission by maintaining phosphorylated-Drp1 (p-Drp1) levels, which results in reduced Drp1 translocation to the mitochondria. By using gabapentin, a drug that antagonizes the interaction between TSP-1 and its neuronal receptor α2δ1, we observe that α2δ1 acts as one of the target receptors for TSP-1, and blocks the reduction of the p-Drp1 to Drp1 ratio, in the presence of Aβ. Taken together, TSP-1 appears to contribute to maintaining the balance in mitochondrial dynamics and mitochondrial functions, which is crucial for neuronal cell viability. These data suggest that TSP-1 may be a potential therapeutic target for AD.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1. TSP-1 inhibits Aβ-induced mitochondrial fission in HT22 cells.
a HT22 cells were stained with MitoTracker green dye to visualize mitochondrial morphology. Magnified images in lower panels are the areas marked with white lines. Scale bar, 20 μm. b, c Mitochondrial length, represented as aspect ratio and mitochondrial circular shape, represented as form factor were analyzed with the Image J program software. TSP-1 treatment significantly recovered Aβ-induced mitochondrial fragmentation. Data were obtained from at least three replicates for each group, and 100–150 cells were analyzed for each group. Data are presented as mean ± SEM. *p < 0.05, ***p < 0.001 vs. vehicle (DMSO)-treated cells; #p < 0.05 vs. Aβ-treated cells. d The effect of TSP-1 on mitochondrial morphology was analyzed in EM images. Arrows indicate normal mitochondria. Arrow heads indicate fissioned mitochondria. Dashed circle indicates damaged (void) mitochondria. Scale bar, 1 μm. eg Average mitochondrial length, number of damaged (void) mitochondria per one cell, and the total number of mitochondria per one cell were quantified. TSP-1 treatment significantly recovered mitochondrial length, decreased number of damaged mitochondria, and the total number of mitochondria per one cell. Data were obtained from at least two replicates, and more than total 10 mitochondria were analyzed for each group. Data are presented as mean ± SEM. **p < 0.01, ***p < 0.001 vs. vehicle (DMSO)-treated cells; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. Aβ-treated cells
Fig. 2
Fig. 2. TSP-1 prevents Aβ-mediated mitochondrial dysfunction in HT22 cells.
a HT22 cells were stained with DCFDA dye to measure cellular ROS levels. b TSP-1 completely blocked Aβ-mediated ROS increase. Data were obtained from at least five replicates for each group. Data are presented as mean ± SEM. *p < 0.05 vs. vehicle (DMSO)-treated cells; #p < 0.05 vs. Aβ-treated cells. Scale bar, 50 μm. c MitoSOX reagent was used to detect mitochondrial specific ROS levels. d TSP-1 significantly inhibited Aβ-induced ROS increment. Data were obtained from at least five replicates for each group. Data are presented as mean ± SEM. **p < 0.01 vs. vehicle (DMSO)-treated cells; ##p < 0.01 vs. Aβ-treated cells. Scale bar, 50 μm. ei Seahorse assay using XF analyzer was performed to measure oxygen consumption rate (OCR) after both Aβ only and Aβ + TSP-1 treatment. In addition, basal respiration, ATP production, the maximal capacity of the OXPHOS system, and non-mitochondrial respiration were also measured by calculating the change of the OCR value by oligomycin, FCCP, rotenone, and antimycin A administration, with a seahorse XF analyzer. Data were obtained from at least nine replicates for each group for three independent experiments. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Aβ-treated cells
Fig. 3
Fig. 3. TSP-1 recovers neuronal cell viability against Aβ toxicity in HT22 cells and primary hippocampal neurons.
TSP-1 plays a protective role with regards to cell viability against Aβ toxicity. Aβ-mediated cell death in HT22 cells was completely blocked by TSP-1 treatment in a MTT and b Calcein-AM assays. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01 vs. vehicle (DMSO)-treated cells; #p < 0.05, ##p < 0.01 vs. Aβ-treated cells. c To visually confirm the protective effect of TSP-1 on cell viability, TUNEL assay was performed in the rat primary hippocampal neurons. TSP-1 treatment considerably decreased DNA fragmentation (shown as brown or black dots) in Aβ-treated primary neurons. Scale bar, 20 μm
Fig. 4
Fig. 4. TSP-1 restores the Aβ-induced decrease of p-Drp1 to Drp1 ratio.
a In HT22 cells, mitochondrial fission/fusion-related protein levels were determined by WB under Aβ- and/or TSP-1-treated condition. be Phospho-Drp1 (Ser637) to Drp1 ratio, which is important for mitochondrial dynamics was recovered by TSP-1 against Aβ toxicity while other fusion/fission proteins such as Mfn1, OPA1, and Fis1 were not changed. Data are presented as mean ± SEM. *p < 0.05, ***p < 0.001 vs. Aβ-treated cells; n.s. means statistically nonsignificant. Data were obtained from at least five replicates
Fig. 5
Fig. 5. α2δ1 receptor is important to mediate the regulating effect of TSP-1 for the ratio of pDrp-1 to Drp1.
a, b Gabapentin (100 μM), a blocker for TSP-1-α2δ1 interaction, suppressed the ability of TSP-1 to maintain p-Drp1 levels. Data are presented as mean ± SEM. ***p < 0.001 vs. Veh (DMSO)-treated cells; ##p < 0.01 vs. Aβ-treated cells; &&&p < 0.001 vs. GBP + TSP-1-treated cells; n.s. means statistically nonsignificant. Data were obtained from at least five replicates

Similar articles

See all similar articles

Cited by 2 PubMed Central articles

References

    1. Querfurth HW, LaFerla FM. Alzheimer’s disease. N. Engl. J. Med. 2010;362:329–344. doi: 10.1056/NEJMra0909142. - DOI - PubMed
    1. Chen JX, Yan SS. Role of mitochondrial amyloid-β in Alzheimer’s disease. J. Alzheimer’s Dis. 2010;20:S569–S578. doi: 10.3233/JAD-2010-100357. - DOI - PubMed
    1. Cha MY, et al. Mitochondria-specific accumulation of amyloid β induces mitochondrial dysfunction leading to apoptotic cell death. PLoS ONE. 2012;7:e34929. doi: 10.1371/journal.pone.0034929. - DOI - PMC - PubMed
    1. Lin MT, Beal MF. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature. 2006;443:787–795. doi: 10.1038/nature05292. - DOI - PubMed
    1. Archer SL. Mitochondrial dynamics—mitochondrial fission and fusion in human diseases. N. Engl. J. Med. 2013;369:2236–2251. doi: 10.1056/NEJMra1215233. - DOI - PubMed

LinkOut - more resources

Feedback