Formaldehyde induces ferritinophagy to damage hippocampal neuronal cells

Abstract

Formaldehyde (FA) causes neurotoxicity and contributes to the occurrence of neurodegenerative diseases. However, the mechanism of FA-induced neurotoxicity has not been fully elucidated. Ferritinophagy, an autophagy process of ferritin mediated by the nuclear receptor coactivator 4 (NCOA4), is a potential mechanism of neurotoxicity. In this study, we explored whether ferritinophagy is associated with the neurotoxicity of FA. Our results showed that FA (50, 100, 200 μM; 24 h) exposure upregulated ferritinophagy in the mouse hippocampal neuronal HT22 cells, which was evidenced by the upregulated autophagic flux, the increased colocalizations of NCOA4 with ferritin heavy chain (FTH1) and NCOA4 with microtubule-associated protein 1 light chain-3B (LC3B), the augmented expression of NCOA4, and the reduced content of FTH1. We also found that FA (0.1, 1, and 10 μmol, i.c.v., 7d) administration boosted ferritinophagy in the hippocampus of Sprague-Dawley (SD) rats, which was demonstrated by the accumulated autophagosomes, the increased expressions of LC3II/I and NCOA4, and the decreased contents of p62 and FTH1 in the hippocampus. Further, we confirmed that inhibition of ferritinophagy by silencing the expression of NCOA4 decreased FA-induced toxic damage in HT22 cells. These results indicated that FA induces neurotoxicity by promoting ferritinophagy. Our findings suggest a potential mechanism insight into the FA-induced neurotoxicity, which in turn provides a new thought for the treatment of FA-related neurodegenerative diseases.

Keywords

Formaldehyde ferritinophagy neurotoxicity neurodegenerative diseases hippocampal neurons

Introduction

Formaldehyde (FA) is an important chemical substance with broad application in different walks of life. Unfortunately, FA causes acute or chronic damage to various human systems (Inci et al., 2013; Duong et al., 2011; Zhang et al., 2010), especially the nervous system (Songur et al., 2010). Studies revealed that FA exposure induces common characteristics of neurodegenerative diseases, such as hyperphosphorylation of tau protein (Lu et al., 2013), accumulation of amyloid-β (Liu et al., 2018b), and cognitive deficit (Zhai et al., 2018). Moreover, the accumulation of endogenous FA also contributes to the oligomerization of amyloid-β (Chen et al., 2006; Fei et al., 2020) and induces cognitive impairment (Tong et al., 2013, 2015) in neurodegenerative diseases. Although these studies have shown that FA-induced neurotoxicity promotes the development of neurodegenerative diseases, the underlying mechanism of FA-induced neurotoxicity is rather complicated, which makes the treatment of FA-related neurodegenerative diseases elusive. Hence, enhancing our knowledge on the potential mechanism of FA-induced neurotoxicity is of great significance to establish new avenues for the treatment of FA-relevant neurodegenerative diseases.

Ferritinophagy is a novel type of autophagy process by which the ferritin was degraded with the assistance of the nuclear receptor coactivator 4 (NCOA4) (Hou et al., 2016). Originally identified as an androgen receptor coactivator (Lim et al., 2001), NCOA4 is subsequently recognized as a cargo receptor that mediates ferritin lysosome-dependent degradation by directly interacting with ferritin heavy chain (FTH1) (Mancias et al., 2014; Dowdle et al., 2014). Mounting evidence has suggested that the activation of ferritinophagy is a mechanism of cytotoxicity (Qin et al., 2021; Dong et al., 2019). Importantly, a study found that the activated ferritinophagy mediates the neurotoxicity in the mouse hippocampal neuronal HT22 cells (Xiao et al., 2021). Previous studies have shown that autophagy can be triggered by FA (Liu et al., 2018a; Han et al., 2015). Therefore, we wonder whether FA activates ferritinophagy, thereby inducing neurotoxicity.

In this study, we identified that FA upregulated autophagic flux in HT22 cells and boosted ferritinophagy in HT22 cells. FA also caused the accumulation of autophagosomes and upregulated ferritinophagy in the hippocampus of SD rats. Furthermore, inhibiting ferritinophagy reduced FA-induced toxic damage in HT22 cells. Taking together, we found that FA promotes ferritinophagy to injure hippocampal neuronal cells, which contributes to a deeper understanding of FA-induced neurotoxicity and provides a potential target for the treatment of FA-related neurodegenerative diseases.

Materials and methods

Reagents and antibodies

Polyformaldehyde (#158127) and trypan blue (T6146) were purchased from Sigma Chemical Company (St. Louis, MO, USA). Anti-LC3A/B antibody (#4108S), anti-SQSTM1/p62 antibody (#5114S), and anti-FTHI antibody (#4393S) from Cell Signaling Technology (Danvers, MA, USA) and anti-NCOA4 antibody (SAB2108396) from Sigma were used for Western blot analysis. Anti-NCOA4 antibody (SAB1409837) offered by Sigma and anti-FTH1 antibody (ab65080) and anti-LC3A/B antibody (ab48394) offered by Abcam (Hong Kong, China) were used for co-location analysis. Malondialdehyde (MDA) kit was purchased from Uscn Life Science Inc (Wu Han, China). 4-Hydroxynonenal (4-HNE) kit was provided by Bio-Swamp Life Science (Wu Han, China). PE Annexin V Apoptosis Detection Kit was purchased from BD (Franklin Lakes, NJ, USA).

Cells culture and FA exposure

The mouse hippocampal HT22 cells, a gift from the Institute of Cardiovascular Diseases of the University of South China, were cultured in the Dulbecco’s Modified Eagle’s Medium containing 10% fetal bovine serum, 100 IU/mL of penicillin, and 100 mg/mL of streptomycin at 37°C and 5% CO₂ incubator. The HT22 cells were passaged once every three days. According to experimental requirements, the HT22 cells were exposed to FA (50, 100, and 200 μM) for 24 h, respectively.

Animals and FA treatment

Adult male Sprague-Dawley (SD) rats, weighted (275 ± 25) g, were provided by the Hunan SJA Laboratory Animal Center (Changsha, Hunan, China). They were individually kept in the cages and located in a house condition that temperature was at 22 ± 2°C and relative humidity was at 55 ± 5%. All of them enjoyed food and water freely and were managed with a light-dark cycle of 12/12 h. After a condition adaptation for 7 days, they were randomly divided into four groups: control group, 0.1 μmol FA group, 1 μmol FA group, and 10 μmol FA group (n = 10). The different groups of rats were individually exposed to 2.5 μl FA (0.1, 1, and 10 μmol) by intracerebroventricular injection (i.c.v.) for 7 days according to our previous study (Tang et al., 2013; Li et al., 2021). Then, the hippocampal tissues were immediately separated and preserved for analysis. All protocols were following the Guide for the Care and Use of Laboratory Animals published by the Ministry of Health of the People’s Republic of China and were approved by the Animal Use and Protection Committee of the University of South China.

Intracerebroventricular cannulation and FA injection

SD rats were fixed in a stereotaxic frame after sodium pentobarbital anesthesia (45 mg/kg, intraperitoneal injection). The micropump was implanted into the lateral ventricle at the coordinates that AP: −1mm; ML: 2 mm; and DV: 4 mm. 2.5 μl FA (0.1, 1, and 10 μmol) was injected into the lateral ventricle with the help of the micropump. After the injection of FA, additional 2 min should be waited before removing the needle to make sure full execution of the injection and balance of pressure in the lateral brain.

Cell cytotoxicity measurements

Cell viability was assessed by trypan blue assay. Briefly, HT22 cells were seeded in 24-well plates at 3 × 10³ cells/well and treated with FA (100 μM) or phosphate buffered saline (PBS) for 24 h. Then, HT22 cells were made into a suspension, mixed with 4% trypan blue solution, and then observed by an inverted microscope (XSZ-D2, Nikon, Japan). Cell viability was shown as the percentage of the number of living cells/(number of dead cells + number of living cells).

Cell death was measured by Annexin V-PE/7-AAD staining assay. In brief, HT22 cells were seeded in 6-well plates at 1 × 10⁶ cells/well and followed by FA (100 μM) or PBS for 24 h. Subsequently, cells were stained by PE Annexin V Apoptosis Detection kit following the manufacturer’s instructions. Finally, cells were detected by bivariate flow cytometry (FC-500, BD, USA). The number of dead cells was the sum of Q1, Q2, and Q3.

Autophagic flux analysis

For the detection of autophagic flux, HT22 cells were seeded at a density of 1 × 10⁵ cells/well in 6-well plates and transfected with mRFP-GFP-LC3 virus according to the manufacturer’s instructions for 24 h at 37°C. Cells were then treated with FA (50, 100, and 200 μM) for 24 h. After the treatment, the expression of mRFP-GFP-LC3 was observed by using the fluorescence microscope (TS2R-C-AL, Nikon, Japan). The numbers of autophagosomes (yellow puncta) and autolysosomes (red puncta) were analyzed by Image-Pro Plus software.

NCOA4 with FTH1/LC3B colocalization analysis

The colocalization of NCOA4 with FTH1/LC3B was presented via fluorescence microscopy after fluorescent double-labeling staining. Briefly, HT22 cells were plated at a density of 1 × 10⁶ cells/well in 6-well dishes. After FA (50, 100, and 200 μM) exposure for 24 h, cells were washed with PBS (2 × 3 min), fixed for 30 min by 4% paraformaldehyde, and permeabilized for 20 min in the 5% TritonX-100 (mixed with PBS) solution at room temperature. Cells were subsequently washed by PBS (3 × 3 min), blocked for 30 min with goat serum at room temperature, and incubated with primary antibodies (anti-NCOA4, anti-FTH1, and anti-LC3B, 1:1200) overnight at 4°C. After rinsed by PBST (3 × 3 min), goat anti-rabbit or goat anti-mouse secondary antibody was applied to cells for 1 h under a dark ambient condition. Finally, the cells were rinsed with PBST (3 × 3 min) and incubated with DAPI, followed by inverted fluorescence microscopy (TS2R-C-AL, Nikon, Japan). Image-Pro Plus software was used for fluorescence intensity analysis.

shRNA Interference experiment and RT-PCR analysis

The Negative-shRNA and NCOA4 shRNA were synthesized by China zorin biology Co., Ltd (Shanghai, China). The sequences were as follows:

NCOA4-RNAi-1-Mouse: Sense: 5′-GCTAATGGGCCAATTCAATTG-3′

Anti-sense: 5′-CAATTGAATTGGCCCATTAGC-3′

NCOA4-RNAi-2-Mouse: Sense: 5′-GGAAAGGACAAGAATGGAATG-3′

Anti-sense: 5′-CATTCCATTCTTGTCCTTTCC-3′

NCOA4-RNAi-3-Mouse: Sense: 5′-GCTCTTTGAAAGGGACTTTGT-3′

Anti-sense: 5′-ACAAAGTCCCTTTCAAAGAGC-3′

Negative-shRNA: Sense: 5′-CCACAGCTGAGAGGGAAATC-3′

Anti-sense: 5′-AAGGAAGGCTGGAAAAGAGC-3′

HT22 cells were transfected with NCOA4-RNAi-Mouse shRNA and negative-shRNA (served as the empty virus control group) according to the manufacturer’s agreements. After transfection, cells were cultured in the fresh culture medium containing puromycin until the uninfected cells were completely killed. Confirmation studies of the NCOA4 mRNA silencing were performed by reverse-transcription quantitative PCR (RT-qPCR) analysis. Roughly, total mRNA was abstracted using Trizol reagent (Proteintech, USA) according to the manufacturer’s protocol. The mRNA purity was assessed based on the ratio of OD260/OD280. The reverse-transcription system (RNase Free water, 20 μL; 5 × prime Script RT Master MIX, 4 μL; total RNA, 0.5 μg) was kept at 37°C for 15 min and 85°C for 5 s for cDNA synthesis. The SYBR Green RT-PCR kit (A25742, ABI, USA) was used for cDNA amplification and quantification following the manufacturer’s instructions. The results were analyzed by ABI Prism 7500 SDS Software.

Determination of MDA and 4-HNE contents

The contents of malondialdehyde (MDA) and 4-Hydroxynonenal (4-HNE) in HT22 cells were measured by enzyme-linked immunosorbent assay (ELISA). Briefly, cells were plated into 6-well dishes at a density of 1 × 10⁶ cells/well and exposed to FA (100 μM) for 24 h. Next, cells were rinsed twice with PBS and harvested into 1.5 mL EP tubes. Contents of MAD and 4-HNE were then determined using ELISA kits according to the manufacturer’s protocol, respectively. Absorbance values of samples at 450 nm were displayed by the microplate reader (ELX-800, Bio-TEK, USA).

Autophagosomes observation by transmission electron microscope

CA1 and CA3 regions of the rat hippocampus were diced (about 1 mm³) and fixed by 2% glutaraldehyde for 12 h. Then, tissue blocks were soaked in 1% osmium tetroxide for 4 h, and embedded into Spurr resin after dehydration by gradient alcohol. Next, tissue blocks were sectioned into ultrathin slices and undergone a stain of uranium acetate-lead citrate. The ultrastructural images of the CA1 and CA3 regions were presented by transmission electron microscope (JEM-1230, JEOL, Japan).

Western blot analysis

Expression levels of NCOA4, FTH1, LC3II/I, and sequestome-1 (also named p62) were assessed by Western blot analysis. Concisely, after FA treatment, proteins from HT22 cells or SD rat hippocampus were extracted by RIPA Lysis Buffer (Beyotime, Shanghai, China) containing PMSF on ice. The quantitation of protein concentrations was by using the bicinchoninic acid (BCA) Protein Assay Kit (CoWin Biosciences, Shanghai, China). Next, proteins were separated by electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) and were electro-transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked in 5% non-fat milk for 2 h and then incubated with diluted primary antibodies (anti-LC3II/I and anti-p62, 1:1000; anti-NCOA4 and anti-FTH1, 1:2000) overnight at 4°C. The next day, membranes were washed with TBST (5 × 5 min), incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000, Proteintech, SA00001-2) for 2 h, and washed with TBST (3 × 10 min). The proteins were visualized using the enhanced chemiluminescence detection system (BeyoECL Plus kit, P0018). The intensities of the protein bands were analyzed by Image J software 2200.

Statistical analysis

All data were analyzed with the assistant of SPSS 22.0 software and presented as mean ± SEM. Differences between groups were evaluated by one-way analysis of variance (one-way ANOVA) followed by the least significant difference test (LSD-t). p < 0.05 was identified as statistically significant.

Results

FA upregulates autophagic flux in HT22 cells

Ferritinophagy is a process of autophagy (Hou et al., 2016), so we first explored whether FA affects autophagy by detecting autophagic flux in HT22 cells. To detected the effect of FA on autophagic flux in HT22 cells, we determined the expressions of autophagic flux-related proteins LC3II/I and p62 after FA (50, 100, 200 μM) exposure for 24 h. The results showed that FA (100 μM) exposure upregulated autophagic flux, as evidenced by the upregulated expression of LC3II/I (Figure 1(a), p < 0.01) and the downregulated expression of p62 (Figure 1(b), p < 0.001). To further confirm the upregulation of autophagic flux caused by FA, we transfected HT22 cells with LC3 (mRFP-GFP) reporter (a tool for autophagic flux detection) before FA (50, 100, 200 μM, 24 h) exposure. The representative images showed that FA (100 μM) upregulated the autophagic flux (Figure 1(c)). Quantitative analysis showed that FA increased the numbers of autophagosomes (Figure 1(d), p < 0.001) and autolysosomes (Figure 1(e), p < 0.001). Taking together, these results suggested that FA enhances the autophagic flux in HT22 cells.

FA upregulates ferritinophagy in HT22 cells

Next, we further determined whether FA triggers ferritinophagy in HT22 cells. Immunofluorescence results showed that treatment of FA (50, 100, and 200 μM, 24 h) promoted the colocalization of NCOA4 with FTH1 (Figure 2(a)) and the colocalization of NCOA4 with LC3B (Figure 2(b)) in HT22 cells, respectively. In addition, FA administration (100 μM) increased the expression of NCOA4 protein (Figure 2(c), p < 0.001), and decreased the expression of FTH1 protein (Figure 2(d), p < 0.01) in HT22 cells. These results demonstrated that FA upregulates ferritinophagy in HT22 cells.

FA upregulates hippocampal ferritinophagy in vivo

To further support our discoveries above, we investigated whether FA strengthens ferritinophagy in the hippocampus of SD rats. As showed by the result of transmission electron microscopy, FA administration (0.1, 1, and 10 μmol, i.c.v.) promoted the accumulation of autophagosomes in the hippocampal CA1 region (Figure 3(a), p < 0.05) and CA3 region (Figure 3(b), p < 0.05). Besides, FA (1 μmol) exposure increased the expressions of LC3II/I (Figure 3(c), p < 0.001) and NCOA4 (Figure 3(e), p < 0.001), and decreased the expressions of p62 (Figure 3(d), p < 0.001) and FTH1 (Figure 3(f), p < 0.05) in the hippocampus of rats. Taking together, these results indicated that FA upregulates hippocampal ferritinophagy in vivo.

Silencing NCOA4 alleviates FA-induced toxic damage in HT22 cells

Given the vital function of NCOA4 in the autophagic process of ferritin (Mancias et al., 2014), we further explored whether silencing NCOA4 reverses FA-caused toxic damage in HT22 cells. The level of NCOA4 mRNA (Figure 4(a), p < 0.001) and the expression of NCOA4 protein (Figure 4(b), p < 0.001) were decreased more than 50% after shNCOA4-2 transfection in HT22 cells, suggesting that the shNCOA4-2 shows the highest efficiency of NCOA4 silence. Then, we transfected shNCOA4-2 in HT22 cells before FA (100 μM) exposure. Oxidative stress (Lu et al., 2008; Gurel et al., 2005) and cell death (Zararsiz et al., 2007; Sayyar et al., 2018) are two important manifestations of the neurotoxicity of FA. Therefore, we evaluated the FA-caused toxic damage by detecting oxidative stress and cell death in HT22 cells. We found that shNCOA4-2 transfection decreased the contents of MDA (Figure 4(c), p < 0.01) and 4-HNE (Figure 4(d), p < 0.01) in FA (100 μM)-exposed HT22 cells, suggesting that silence of NCOA4 reduces FA-induced oxidative stress. Furthermore, shNCOA4-2 administration restored the cell viability (Figure 4(e), p < 0.01) and lessened the cell death (Figure 4(f), p < 0.05) in FA (100 μM)-exposed HT22 cells. Tanking together, these results indicated that silence of NCOA4 reverses FA-induced toxic damage in HT22 cells.

Discussion

Although the wide application of FA has been approved by various industries, its toxicity cannot be disregarded, especially neurotoxicity. FA-induced neurotoxicity is one of the risk factors for neurodegenerative diseases (Tulpule and Dringen, 2013; Wang et al., 2019). Nevertheless, the mechanism of FA-induced neurotoxicity is still unclear and needs further exploration. Ferritinophagy, a process of autophagy-dependent ferritin degradation (Mancias et al., 2014), has been implicated as a potential mechanism of neurotoxicity (Xiao et al., 2021). In this study, we investigated whether the neurotoxicity of FA is bound up with ferritinophagy. The major accomplishments of this investigation are summarized as follows: (1) FA promoted autophagic flux in HT22 cells; (2) FA upregulated ferritinophagy in HT22 cells; (3) FA-induced excessive activation of hippocampal ferritinophagy in SD rats; and (4) silencing NCOA4 reversed FA-induced toxic damage in HT22 cells. Collectively, these results indicated that FA upregulates ferritinophagy to damage hippocampal neuronal cells and that the upregulated ferritinophagy is an underlying mechanism of FA-induced neurotoxicity.

Autophagy is a cellular life process for the degradation of proteins and organelles (Parzych and Klionsky, 2014). In this process, proteins and organelles are packaged by phagophore to form autophagosomes, and autophagosomes are subsequently transferred to lysosomes where proteins and organelles are degraded (Yu et al., 2018). Autophagic flux is a vivid depiction of this dynamic procedure (Barth et al., 2010). Given that ferritinophagy is a form of autophagy, we first monitored the autophagic flux for the investigation that whether FA affects autophagy in HT22 cells. We found upregulated autophagic flux in HT22 cells after FA administration, suggesting that FA promotes autophagy in HT22 cells. This is in line with the previous finding that FA is an inducer of autophagy (Liu et al., 2018a; Han et al., 2015). Previous studies have found that upregulation of autophagy promotes HT22 cell damage (Chang et al., 2021; Kim et al., 2009) while inhibition of autophagy alleviates HT22 cell damage (Chimeh et al., 2018; Chen et al., 2020). Therefore, upregulated autophagy is involved in the neurotoxicity of FA.

Autophagy for ferritin degradation is known as ferritinophagy (Mancias et al., 2014). Ferritinophagy depends on the function of autophagy-specific cargo receptor NCOA4 that mediates the degradation of ferritin by carrying ferritin to autophagosome through binding with FTH1 purposefully (Mancias et al., 2014; Dowdle et al., 2014). We found the increased colocalizations including NCOA4 with FTHI and NCOA4 with autophagosome membrane-associated protein LC3B, the increased expression of NCOA4, and the decreased content of FTH1 after FA exposure, suggesting that FA augments ferritinophagy in HT22 cells. To further certificate our finding that FA is a push hand of ferritinophagy, we exposed the hippocampus of SD rats to FA for 1 week and then ascertained the level of ferritinophagy in the hippocampus of SD rats. The key point of autophagy is the formation of autophagosomes, that is, cargo is surrounded by double-membrane (Yu et al., 2018). Our visualized result of hippocampus ultrastructure showed that FA exposure promoted the formation of autophagosomes and resulted in the accumulation of autophagosomes both in the hippocampal CA1 and CA3 regions. Simultaneously, FA promoted the degradation of ferritin through the NCOA4-mediated autophagy-dependent pathway. These results indicated that FA enhances ferritinophagy in the hippocampus of SD rats. It was reported that NCOA4-mediated ferritinophagy contributes to the death of PC-12 cells (a neural cell line) (Xiao et al., 2021). Therefore, FA may cause neuronal damage by enhancing ferritinophagy.

Because that NCOA4 is necessary for the implementation of ferritinophagy, ferritinophagy can be hindered by deleting or inhibiting NCOA4 (Hou et al., 2016; Ito et al., 2021). For more persuasiveness that NCOA4-dependent ferritinophagy is the mechanism of FA-induced neurotoxicity, we purposefully silenced NCOA4 in HT22 cells before FA treatment through shRNA interference technology to investigate whether inhibiting NCOA4-dependent ferritinophagy reverses toxic damage resulting from FA. Our result showed that FA-induced toxic damage was mitigated after NCOA4 silence, indicating that inhibiting ferritinophagy attenuates FA-induced neurotoxicity. Since FA-induced neurotoxicity can be attenuated by inhibiting ferritinophagy, the hypothesis that FA induces ferritinophagy to injure neurons was been confirmed. Notably, FA-induced neurotoxicity contributes to the initiation and progression of neurodegenerative diseases (Zhai et al., 2018) while inhibition of FA-caused neurotoxicity improves neurodegenerative diseases (Fei et al., 2020). Thus, restraining excessive ferritinophagy may be beneficial in treating neurodegenerative diseases associated with FA.

Overall, our present work found that FA induces ferritinophagy and that inhibition of ferritinophagy alleviates FA-induced neurotoxicity, which indicates that the upregulation of ferritinophagy is a mechanism behind the neurotoxicity of FA. This finding not only expands our understanding of FA-induced neurotoxicity but also suggests that astricting ferritinophagy is a potential strategy for the treatment of FA-related neurodegenerative diseases.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical approval

All protocols on animals in this study were following the Guide for the Care and Use of Laboratory Animals published by the Ministry of Health of the People’s Republic of China and were approved by the Animal Use and Protection Committee of the University of South China.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by National Natural Science Foundation of China (81771178).

References

Barth

Glick

Macleod

(2010) Autophagy: assays and artifacts. The Journal of Pathology 221(2): 117–124.

Chang

Wang

, et al. (2021) Silver nanoparticles induced cytotoxicity in HT22 cells through autophagy and apoptosis via PI3K/AKT/mTOR signaling pathway. Ecotoxicology and Environmental Safety 208: 111696.

Chen

Wang

Luo

, et al. (2020) Chd8 rescued TBI-induced neurological deficits by suppressing apoptosis and autophagy via wnt signaling pathway. Cellular and Molecular Neurobiology 40(7): 1165–1184.

Chen

Maley

(2006) Potential inplications of endogenous aldehydes in beta-amyloid misfolding, oligomerization and fibrillogenesis. Journal of Neurochemistry 99(5): 1413–1424.

Chimeh

Zimmerman

Gilyazova

, et al. (2018) B355252, A novel small molecule, confers neuroprotection against cobalt chloride toxicity in mouse hippocampal cells through altering mitochondrial dynamics and limiting autophagy induction. International Journal of Medical Sciences 15(12): 1384–1396.

Dong

Tan

Qin

, et al. (2019) Polybrominated diphenyl ethers quinone induces NCOA4-mediated ferritinophagy through selectively autophagic degradation of ferritin. Chemical Research in Toxicology 32(12): 2509–2516.

Dowdle

Nyfeler

Nagel

, et al. (2014) Selective VPS34 inhibitor blocks autophagy and uncovers a role for NCOA4 in ferritin degradation and iron homeostasis in vivo. Nature Cell Biology 16(11): 1069–1079.

Duong

Steinmaus

McHale

, et al. (2011) Reproductive and developmental toxicity of formaldehyde: a systematic review. Mutation Research 728(3): 118–138.

Fei

Zhang

Mei

, et al. (2020) Degradation of FA reduces Abeta neurotoxicity and Alzheimer-related phenotypes. Molecular Psychiatry 14: 121. 10.1038/s41380-020-00929-7.

10.

Gurel

Coskun

Armutcu

, et al. (2005) Vitamin E against oxidative damage caused by formaldehyde in frontal cortex and hippocampus: biochemical and histological studies. Journal of Chemical Neuroanatomy 29(3): 173–178.

11.

Han

Zhou

Lin

, et al. (2015) Formaldehyde exposure induces autophagy in testicular tissues of adult male rats. Environmental Toxicology 30(3): 323–331.

12.

Hou

Xie

Song

, et al. (2016) Autophagy promotes ferroptosis by degradation of ferritin. Autophagy 12(8): 1425–1428.

13.

Inci

Zararsiz

Davarci

, et al. (2013) Toxic effects of formaldehyde on the urinary system. Turkish Journal of Urology 39(1): 48–52.

14.

Ito

Omiya

Rusu

, et al. (2021) Iron derived from autophagy-mediated ferritin degradation induces cardiomyocyte death and heart failure in mice. Elife 10: 234.

15.

Kim

Choi

Ryu

, et al. (2009) Activation of autophagy during glutamate-induced HT22 cell death. Biochemical and Biophysical Research Communications 388(2): 339–344.

16.

Yang

, et al. (2021) Formaldehyde induces ferroptosis in hippocampal neuronal cells by upregulation of the Warburg effect. Toxicology 448: 152650.

17.

Lim

Hawkins

Hughes

(2001) Genetic evidence to exclude the androgen receptor co-factor, ARA70 (NCOA4) as a candidate gene for the causation of undermasculinised genitalia. Clinical Genetics 59(4): 284–286.

18.

Liu

Zhou

, et al. (2018a) Formaldehyde inhalation triggers autophagy in rat lung tissues. Toxicology and Industrial Health 13: 157. 10.1177/0748233718796347.

19.

Liu

Zhang

, et al. (2018b) Acute formaldehyde exposure induced early Alzheimer-like changes in mouse brain. Toxicology Mechanisms and Methods 28(2): 95–104.

20.

Miao

, et al. (2013) Formaldehyde induces hyperphosphorylation and polymerization of Tau protein both in vitro and in vivo. Biochimica et Biophysica Acta 1830(8): 4102–4116.

21.

Qiao

, et al. (2008) Effect of inhaled formaldehyde on learning and memory of mice. Indoor Air 18(2): 77–83.

22.

Mancias

Wang

Gygi

, et al. (2014) Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy. Nature 509(7498): 105–109.

23.

Parzych

Klionsky

(2014) An overview of autophagy: morphology, mechanism, and regulation. Antioxidants & Redox Signaling 20(3): 460–473.

24.

Qin

Zhang

Wang

, et al. (2021) Ferritinophagy is involved in the zinc oxide nanoparticles-induced ferroptosis of vascular endothelial cells. Autophagy: 1–20. Epub ahead of print 2021/04/13. DOI: 10.1080/15548627.2021.1911016.

25.

Sayyar

Yazdinezhad

Hassan

, et al. (2018) Protective effect of matricaria chamomilla ethanolic extract on hippocampal neuron damage in rats exposed to formaldehyde. Oxidative Medicine and Cellular Longevity 2018: 6414317.

26.

Songur

Ozen

Sarsilmaz

(2010) The toxic effects of formaldehyde on the nervous system. Reviews of Environmental Contamination and Toxicology 203: 105–118.

27.

Tang

Zhuang

Zhang

, et al. (2013) Formaldehyde impairs learning and memory involving the disturbance of hydrogen sulfide generation in the hippocampus of rats. Journal of Molecular Neuroscience 49(1): 140–149.

28.

Tong

Han

Luo

, et al. (2013) Accumulated hippocampal formaldehyde induces age-dependent memory decline. Age (Dordr) 35(3): 583–596.

29.

Tong

Han

Qiang

, et al. (2015) Age-related formaldehyde interferes with DNA methyltransferase function, causing memory loss in Alzheimer's disease. Neurobiology of Aging 36(1): 100–110.

30.

Tulpule

Dringen

(2013) Formaldehyde in brain: an overlooked player in neurodegeneration? Journal of Neurochemistry 127(1): 7–21.

31.

Wang

Chen

, et al. (2019) Formaldehyde, epigenetics, and Alzheimer's disease. Chemical Research in Toxicology 32(5): 820–830.

32.

Xiao

Zhang

, et al. (2021) Arsenite induces ferroptosis in the neuronal cells via activation of ferritinophagy. Food and Chemical Toxicology 151: 112114.

33.

Chen

Tooze

(2018) Autophagy pathway: cellular and molecular mechanisms. Autophagy 14(2): 207–215.

34.

Zararsiz

Kus

Ogeturk

, et al. (2007) Melatonin prevents formaldehyde-induced neurotoxicity in prefrontal cortex of rats: an immunohistochemical and biochemical study. Cell Biochemistry and Function 25(4): 413–418.

35.

Zhai

Rizak

Zheng

, et al. (2018) Alzheimer's disease-like pathologies and cognitive impairments induced by formaldehyde in non-human primates. Current Alzheimer Research 15(14): 1304–1321.

36.

Zhang

Freeman

Nakamura

, et al. (2010) Formaldehyde and leukemia: epidemiology, potential mechanisms, and implications for risk assessment. Environmental and Molecular Mutagenesis 51(3): 181–191.