Mitochondrial Dysfunction Triggers Synaptic Deficits via Activation of p38 MAP Kinase Signaling in Differentiated Alzheimer's Disease Trans-Mitochondrial Cybrid Cells

Abstract

Loss of synapse and synaptic dysfunction contribute importantly to cognitive impairment in Alzheimer's disease (AD). Mitochondrial dysfunction and oxidative stress are early pathological features in AD-affected brain. However, the effect of AD mitochondria on synaptogenesis remains to be determined. Using human trans-mitochondrial "cybrid" (cytoplasmic hybrid) neuronal cells whose mitochondria were transferred from platelets of patients with sporadic AD or age-matched non-AD subjects with relatively normal cognition, we provide the first evidence of mitochondrial dysfunction compromises synaptic development and formation of synapse in AD cybrid cells in response to chemical-induced neuronal differentiation. Compared to non-AD control cybrids, AD cybrid cells showed synaptic loss which was evidenced by a significant reduction in expression of two synaptic marker proteins: synaptophysin (presynaptic marker) and postsynaptic density protein-95, and neuronal proteins (MAP-2 and NeuN) upon neuronal differentiation. In parallel, AD-mediated synaptic deficits correlate to mitochondrial dysfunction and oxidative stress as well as activation of p38 MAP kinase. Notably, inhibition of p38 MAP kinase by pharmacological specific p38 inhibitor significantly increased synaptic density, improved mitochondrial function, and reduced oxidative stress. These results suggest that activation of p38 MAP kinase signaling pathway contributes to AD-mediated impairment in neurogenesis, possibly by inhibiting the neuronal differentiation. Our results provide new insight into the crosstalk of dysfunctional AD mitochondria to synaptic formation and maturation via activation of p38 MAP kinase. Therefore, blockade of p38 MAP kinase signal transduction could be a potential therapeutic strategy for AD by alleviating loss of synapses.

Keywords: Alzheimer’s disease; cybrid cells; mitochondrial dysfunction; synaptic deficits.

Fig. 1

Comparison of differentiation status in neuronal processes between non-AD and AD cybrid cells during differentiation. A) Representative morphological images from non-AD and AD cybrid cells under undifferentiated conditions or induced by SAT (14 days after 10 nM staurosporine [SAT] treatment). Scale bar = 50 μm. B) Quantification of neuronal process length of cybrid cells using the image J program. Black bars denote the process length of cybrid cells following SAT treatment in both non-AD and AD groups. Open bars represent process lengths of undifferentiated cybrid cells without SAT treatment.

Fig. 2

Expression of neuronal markers in non-AD and AD cybrid cells during differentiation. Immunoblotting of protein extracts from non-AD and AD cybrid cells for MAP2 (A) and NeuN (B) in the indicated groups of cells. β-actin was used for protein loading control. The upper panel displays quantification of immunoreactive bands for the corresponding protein relative to β-actin. Data are expressed as fold increase relative to undifferenciated non-AD cybrid cells. Quantification of fluorescent intensity of MAP2 (C) and NeuN (D), and (E), representative images of immunofluorescent staining for MAP2 (green) and NeuN (red) in the indicated groups of cybrid cells. Scale bar = 50 μm.

Fig. 3

Expression of synaptic proteins in AD cybrid cells during differentiation. A) Immunoblotting of protein extracts from non-AD and AD cybrid cells for PSD95 and synaptophysin in the indicated groups of cells. β-actin was used for protein loading control. The upper panel displays quantification of immunoreactive bands for the corresponding protein relative to β-actin. Data are expressed as fold increase relative to undifferentiated non-AD cybrid cells. Quantification of fluorescent intensity of synaptophysin (B) and PSD95 (C). Representative images of immunofluorescent staining for synaptophysin (red) and PSD95 (green) in the indicated groups of cybrid cells (D). Scale bars = 50 μm.

Fig. 4

Effect of p38 signaling pathway and its inhibitor on neuronal differentiation and expression of synaptic proteins in AD cybrid cells during differentiation. A) Immunoblotting of protein extracts from non-AD and AD cybrid cells for p-p38 and t-p-38 in the indicated groups of cells. β-actin was used for protein loading control. The upper panel displays quantification of immunoreactive bands for the corresponding protein relative to β-actin. Data are expressed as fold increase relative to non-AD vehicle cybrid cells. Open bars represent the protein expression level of p-p38 in the indicated groups of cybrid cells without any treatment. Black bars denote the protein expression level of p-p38 in the indicated groups of cybrid cells with the treatment of SB203580. Scale bar = 50 μm. B) Representative morphological images of mitochondria in the processes of above differentiated non-AD and AD cybrid cells (I–IV). I and II: non-AD and AD cells under differentiation without any treatment. III and IV: non-AD and AD cybrid cells under differentiation with the addition of the inhibitor of P38, SB203580. C) Quantification of neuronal process length of cybrid cells using the image J program. D) Immunoblotting of protein extracts from differentiated non-AD and AD cybrid cells with or without SB203580 treatment for MAP2 and NeuN. β-actin was used for protein loading control. The upper panel displays quantification of immunoreactive bands for the corresponding protein relative to β-actin. Data are expressed as fold increase relative to non-AD vehicle cybrid cells. Open bars represent the protein expression level of MAP2 in the indicated groups of cybrid cells. Black bars denote the protein expression level of NeuN in the indicated groups of cybrid cells. E) Immunoblotting of protein extracts from differentiated non-AD and AD cybrid cells with or without SB203580 treatment for PSD95 and Synaptophysin. β-actin was used for protein loading control. The upper panel displays quantification of immunoreactive bands for the corresponding protein relative to β-actin. Data are expressed as fold increase relative to non-AD vehicle cybrid cells.

Fig. 5

Effect of p38 MAP kinase pathway and its inhibitor on mitochondrial function and ROS in AD cybrid cells during differentiation. Mitochondrial ROS levels were measured by MitoSox staining intensity (A and B) and generation of ROS in the indicated groups of cells was detected by electron paramagnetic resonance (EPR) spectra (C–D). Enzymatic activity of complex IV (E), and cellular ATP levels (F) were determined in cell lysates from differentiated AD cybrid cells with or without the addition of SB203580. Data were expressed as fold increase relative to AD cybrid cells without the addition of SB203580 (vehicle treatment). Mitochondrial membrane potential was measured by TMRM staining intensity (G and H). Quantifications of immunofluorescent intensity for MitoSox (A), the signal intensity of EPR (C), and TMRM (G) in mitochondria of the indicated cybrid cells. The representative EPR and staining images were shown for EPR (D), and red fluorescence for Mitosox (B) and TMRM (H). Data were collected from 20–25 processes of each AD cybrid cell line with different treatments. Scale bars = 25 μm.

Fig. 6

Schematic depiction of p38 MAP kinase pathway in response to elevated ROS levels and mitochondrial dysfunction, leading to arrested differentiation and synaptic deficits in AD cybrid cells. Meanwhile, activated p38 MAP kinase pathway in turn promotes ROS production and augments mitochondrial dysfunction, eventually exacerbates the impaired differentiation and synaptic deficits in AD cybrid cells. Thus, blocking p38 MAPK activation using a pharmacological inhibitor of p38 MAPK (SB203580) could reverses AD mitochondria-mediated detrimental effects by suppressing ROS production and accumulation, powering mitochondrial function, and maintaining normal differentiation and synaptic development.