Aβ-induced mitochondrial dysfunction in neural progenitors controls KDM5A to influence neuronal differentiation

Mitochondria in neural progenitors play a crucial role in adult hippocampal neurogenesis by being involved in fate decisions for differentiation. However, the molecular mechanisms by which mitochondria are related to the genetic regulation of neuronal differentiation in neural progenitors are poorly understood. Here, we show that mitochondrial dysfunction induced by amyloid-beta (Aβ) in neural progenitors inhibits neuronal differentiation but has no effect on the neural progenitor stage. In line with the phenotypes shown in Alzheimer’s disease (AD) model mice, Aβ-induced mitochondrial damage in neural progenitors results in deficits in adult hippocampal neurogenesis and cognitive function. Based on hippocampal proteome changes after mitochondrial damage in neural progenitors identified through proteomic analysis, we found that lysine demethylase 5A (KDM5A) in neural progenitors epigenetically suppresses differentiation in response to mitochondrial damage. Mitochondrial damage characteristically causes KDM5A degradation in neural progenitors. Since KDM5A also binds to and activates neuronal genes involved in the early stage of differentiation, functional inhibition of KDM5A consequently inhibits adult hippocampal neurogenesis. We suggest that mitochondria in neural progenitors serve as the checkpoint for neuronal differentiation via KDM5A. Our findings not only reveal a cell-type-specific role of mitochondria but also suggest a new role of KDM5A in neural progenitors as a mediator of retrograde signaling from mitochondria to the nucleus, reflecting the mitochondrial status.


Construction of pLentiSyn1.41g-mitoAβ
MitoAβ sequence was designed and constructed as previously described 20  In our routine preparation, the titers were ≒ 10 7 transduction units (TU) per mL without further concentration. For stereotaxic injection, the lentivirus particles were concentrated by ultracentrifugation on a 20 % sucrose cushion (2 hrs at 50,000 g) at 4 °C.

Mitochondria fractionation
In order to separate the cytoplasm and mitochondria, cells were homogenized in the solution with 20 mM HEPES, pH 7.5, 250 mM sucrose, 20 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride by using a glass Dounce homogenizer. Cell lysates were centrifuged at 600 g for 10 min at 4 °C to remove undissolved cell debris and nuclei. The supernatant was again centrifuged at 7,000 g for 10 min at 4 °C, and the resulting pellet was collected as a mitochondria-enriched fraction.

Chromatin immunoprecipitation (ChIP)
ReN CX cells or differentiated cells (after 5 days) were fixed with 1 % formaldehyde at room temperature for 10 min. After cross-linking reactions, the fixation was stopped by adding 1 M glycine solution to be at a final concentration of 125 mM. The solution was centrifuged at 3,000 rpm for 5 min, and the resulting pellet was then washed 3 times with ice-cold phosphate-buffered saline (PBS). Cell pellets were suspended with lysis buffer (sc-45000, Santa Cruz) and centrifuged at 3,000 rpm for 5 min.

ChIP sequencing library preparation and sequencing
The construction of library was performed using NEBNext ® Ultra TM DNA Library Prep Kit for Illumina (New England Biolabs, UK) according to the manufacturer's instructions. Briefly, the chipped DNA was ligated with adaptors. After purification, PCR reaction was done with adaptor-ligated DNA and index primer for multiplexing sequencing. Library was purified by using magnetic beads to remove all reaction components. The size of library was assessed by Agilent 2100 bioanalyzer (Agilent Technologies, Amstelveen, The Netherlands). High-throughput sequencing was performed as pairedend sequencing (101 bp) using HiSeq 2500 (Illumina, Inc., USA).

ChIP sequencing data analysis
The reads were trimmed and aligned using Bowtie2 (

Protein extraction
Collected hippocampal tissues from two control (LV-GFP) and two Aβ (LV-Mito-Aβ) groups were individually pulverized using Covaris CP02 Cryoprep device (Covaris, Woburn, MA). Briefly, each tissue piece was placed in a Covaris tissue bag (Covaris, 430487), and the bag was placed in a liquid nitrogen for 30 s. The frozen tissue was subsequently pulverized at impact level 2. We then added 500 µl of lysis buffer (4 % SDS in Tris-HCl pH 7.6 and phosphatase inhibitor) to the tissue powder, which were subsequently sonicated using a probe sonicator (Q55, QSONICA, Newtown, CT) at 30 W on ice for 30 s. The tissue lysate was centrifuged at 16,000 g for 10 min, and supernatants was transferred to a siliconized low-retention tube. Protein concentration was then measured using the BCA protein assay (Pierce, Rockford, IL).

Protein digestion
All hippocampal proteins were digested by the filter aided sample preparation (FASP) method 1

LC-MS/MS data analysis
The LC-MS/MS data from global proteome and phosphoproteome profiling experiments were processed with the PE-MMR (Post-experiment monoisotopic mass refinement) method as previously

Identification of differentially expressed peptides and proteins
The intensities of the iTRAQ reporter ions for hippocampus samples from two LV-GFP and two LV-Mito-Aβ injected mice were normalized using the quantile normalization method 9 . Using the normalized intensities of each peptide, we performed Student t-test and calculated log2-median-ratios (fold-changes) for LV-Mito-Aβ to LV-GFP groups. We next estimated the null hypothesis distributions of these values by performing random permutations of the samples and computing t-statistic values and log2-median-ratios as previously described 10 . An adjusted p value was calculated for a t-statistic value for each peptide by two sample test using the empirical distribution. Differentially expressed (DEpeptides) and phosphorylated peptides (DPpeptides) were then identified as the peptides with p < 0.05 from the two sample t-test and log2-median-ratios > 95 th percentiles in its empirical null distribution (1.32-and 1.58-fold for global and phosphoproteome profiles, respectively). The DEPs were identified as the proteins with at least two unique DEpeptides showing consistent up-or down-regulation in LV-mitoAβ injected mice. DPpeptides with more than two spectral counts were used for the subsequent analyses, and differentially phosphorylated proteins (DPPs) were defined as the proteins containing the DPpeptides.

GOBP association analysis.
We built a network model describing the links among the 586 GOBPs enriched by the three sets of proteins affected by LV-mitoAβ: 1) 281 upand 2) 218 down-regulated proteins and 3) 191 DPPs. For each pair of the 586 GOBPs (GOBP1 with n proteins, GOBP2 with m proteins, and k shared proteins between GOBP1 and 2), we computed the similarity score as 2k/(n+m) and connected the two GOBPs with the similarity score > 0.52 (99 % of the null distribution for the similarity score). This procedure resulted in 43 connected subnetworks, called functional modules. The importance (weighted degree) of GOBPs in each module was estimated by summing the similarity scores of its interactors in the corresponding subnetwork. The GOBPs were then ranked by their weighted degrees such that the topranked GOBP has the largest weighted degree (i.e., a hub-like term). Also, the key GOBPs should be highly enriched with up-and down-regulated proteins and DPPs. Thus, we evaluated the enrichment of the GOBPs in each module using EASE scores obtained from DAVID 11 . The GOBPs in each module was then ranked such that the top-ranked GOBP has the largest enrichment score. After summing the two ranks from the weighted degrees and the enrichment scores, finally, the key GOBP in each module was selected as the top-ranked one based on the summed ranks.  was used as a marker for mitochondria-enriched samples and caspase 7 was used as a marker for the cytoplasm. d Experimental procedure of making homogenous cells expressing mitoAβ. Before analyzing cellular respiration and transmission electron microscope imaging, mitoAβ was transduced into the HT22 cell line using a lentiviral system, and GFP + or GFP/mitoAβ + cells were collected by