FOXO3 directly regulates an autophagy network to functionally regulate proteostasis in adult neural stem cells

Abstract

Maintenance of a healthy proteome is essential for cellular homeostasis and loss of proteostasis is associated with tissue dysfunction and neurodegenerative disease. The mechanisms that support proteostasis in healthy cells and how they become defective during aging or in disease states are not fully understood. Here, we investigate the transcriptional programs that are essential for neural stem and progenitor cell (NSPC) function and uncover a program of autophagy genes under the control of the transcription factor FOXO3. Using genomic approaches, we observe that FOXO3 directly binds a network of target genes in adult NSPCs that are involved in autophagy, and find that FOXO3 functionally regulates induction of autophagy in these cells. Interestingly, in the absence of FOXO activity, aggregates accumulate in NSPCs, and this effect is reversed by TOR (target of rapamycin) inhibition. Surprisingly, enhancing FOXO3 causes nucleation of protein aggregates, but does not increase their degradation. The work presented here identifies a genomic network under the direct control of a key transcriptional regulator of aging that is critical for maintaining a healthy mammalian stem cell pool to support lifelong neurogenesis.

Fig 1. FOXO3 directly binds a network of autophagy genes in NSPCs.

(A) STRING analysis of the direct FOXO3 autophagy-related network in NSPCs based on ChIP-seq analysis of FOXO3 binding in this cell type. (B) A snapshot of the highly connected autophagic machinery network (red box in 1A). (C) Overlap between FOXO3 bound genes based on ChIP-seq analysis and autophagy genes. Left panel shows enrichment of autophagy genes among all FOXO3 targets and right panel depicts the FOXO3 targets within the entire autophagy network. p-value from Fisher’s exact test is shown. (D) Motif analysis of FOXO3 binding sites at the 151 direct autophagy targets. The top most significant motif enriched in the analysis is shown, which corresponds to the FOXO consensus binding motif.

Fig 2. FOXO transcription factors regulate autophagy genes in NSPCs.

(A) Examples of FOXO3 target genes that are induced by FOXO3. For each target, RT-qPCR analysis showing induction by CA-FOXO3 is on the left, and a snapshot of the genome browser showing FOXO3 binding enrichment (ChIP-seq in NSPCs) is shown on the right. (B) Ablation of FOXO transcription factor in NSPCs reduces expression of autophagy genes. For (A) and (B), n = 3 experiments; Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001. (C) Schematic diagram of the autophagy pathway showing direct FOXO3 targets identified in this study and their role in autophagy. Genes shown in red were also induced by FOXO3 in this study.

Fig 3. FOXO3 regulates autophagy in NSPCs.

(A) Schematic representation of the tandem LC3 reporter system. The processed LC3 reporter localizes to autophagosomes and fluoresces both red and green. Upon fusion with the acidic lysosome, the low pH of the autolysosome quenches the GFP fluorescence, but the mCherry fluorescence remains stable. Under starvation conditions (+HBSS), flux through the autophagic pathway increases, resulting in reduced GFP signal. (B) Example images of the tandem LC3 reporter in NSPCs under basal conditions (upper panels) and starvation conditions (two hours in HBSS; lower panels). Note the reduced GFP signal in the +HBSS condition. (C) Overexpression of FOXO3 further enhances autophagic flux under HBSS conditions, as indicated by a decrease in GFP signal. (D) Similar to wild type FOXO3, constitutively active FOXO3 enhances autophagic flux under starvation conditions. n = 3 experiments; *p < 0.05. (E) Overexpression of wild type or constitutive active FOXO3 increases endogenous LC3 levels as shown by western blot in NSPCs. (F) BafA treatment further enhances processed LC3 levels upon FOXO3 or CA-FOXO3 overexpression. One representative example of at least three replicates is shown for (E) and (F). (G) Overexpression of wild type FOXO3 increases autophagy induction. Fold change of LC3-GFP buildup in the presence of BafA compared to basal conditions is shown. n = 3 experiments; **p < 0.01.

Fig 4. FOXOs are required to maintain autophagy levels in NSPCs.

(A) Schematic representation of the LC3 tandem reporter system. Bafilomycin A disrupts proton transport in lysosomes and blocks fusion of the lysosome with the autophagosome. (B) Representative images of the LC3 reporter system under basal conditions and in response to Bafilomycin A. Note the increase in mCherry and GFP in the +Bafilomycin A conditions. (C) Ablation of FOXO transcription factors in NSPCs reduces autophagic flux. Quantification of FACS experiments detecting GFP intensity in control cells (empty vector Trifloxed cells) and FOXO-ablated cells (FOXO conditional KO cells) in the presence of Bafilomycin A. (D) FOXOs regulate the turnover of endogenous LC3 in NSPCs. Western blot showing levels of LC3 I and LC3 II in wild type and FOXO ablated NSPCs, with and without Bafilomycin A treatment to detect LC3 turnover. (E) Quantification of western blots shown in (D). LC3 turnover was reduced in the FOXO-ablated cells, indicating that FOXOs are required to maintain the endogenous level of autophagic turnover in NSPCs. n = 3 experiments for C-E; Student’s t-test; *p < 0.05, p** < 0.01.

Fig 5. FOXO3 promotes aggregate clearance in NSPCs.

(A) Protein aggregates are significantly increased in the absence of FOXO activity and this increase is reduced by TORC1 inhibition (24 hour rapamycin treatment). (B) Quantification of aggregate formation assay. TORC1 inhibition reverses the effect of FOXO ablation on aggregate levels. n = 3; Student’s t-test; ***p < 0.001, ****p < 0.0001. (C) Western blot showing no change in TOR activity in the absence of FOXOs in NSPCs, but reduced pS6 under starvation (HBSS) conditions. (D) Protein aggregates are increased in response to FOXO3 overexpression (example of n = 3 biological replicates).