Aging-associated excess formaldehyde leads to spatial memory deficits

Tong, Zhiqian; Han, Chanshuai; Luo, Wenhong; Li, Hui; Luo, Hongjun; Qiang, Min; Su, Tao; Wu, Beibei; Liu, Ying; Yang, Xu; Wan, You; Cui, Dehua; He, Rongqiao

doi:10.1038/srep01807

Download PDF

Article
Open access
Published: 09 May 2013

Aging-associated excess formaldehyde leads to spatial memory deficits

Zhiqian Tong^1,5^na1,
Chanshuai Han^1,4^na1,
Wenhong Luo²^na1,
Hui Li²^na1,
Hongjun Luo²^na1,
Min Qiang^1,4^na1,
Tao Su^1,4^na1,
Beibei Wu^1,4^na1,
Ying Liu¹^na1,
Xu Yang³^na1,
You Wan⁵^na1,
Dehua Cui⁵^na1 &
…
Rongqiao He^1,6^na1

Scientific Reports volume 3, Article number: 1807 (2013) Cite this article

4491 Accesses
87 Citations
5 Altmetric
Metrics details

Subjects

Abstract

Recent studies show that formaldehyde participates in DNA demethylation/methylation cycle. Emerging evidence identifies that neuronal activity induces global DNA demethylation and re-methylation; and DNA methylation is a critical step for memory formation. These data suggest that endogenous formaldehyde may intrinsically link learning-responsive DNA methylation status and memory formation. Here, we report that during spatial memory formation process, spatial training induces an initial global DNA demethylation and subsequent re-methylation associated with hippocampal formaldehyde elevation then decline to baseline level in Sprague Dawley rats. Scavenging this elevated formaldehyde by formaldehyde-degrading enzyme (FDH), or enhancing DNA demethylation by a DNA demethylating agent, both led to spatial memory deficits by blocking DNA re-methylation in rats. Furthermore, we found that the normal adult rats intrahippocampally injected with excess formaldehyde can imitate the aged-related spatial memory deficits and global DNA methylation decline. These findings indicate that aging-associated excess formaldheyde contributes to cognitive decline during aging.

Dnmt3a1 regulates hippocampus-dependent memory via the downstream target Nrp1

Article Open access 18 March 2024

Endogenous formaldehyde is a memory-related molecule in mice and humans

Article Open access 29 November 2019

Knockdown of METTL16 disrupts learning and memory by reducing the stability of MAT2A mRNA

Article Open access 28 October 2022

Introduction

Formaldehyde (FA) is present in all invertebrate and vertebrate cells^1,2. Using fluorescence-HPLC, we have previously shown that levels of normal physiological brain formaldehyde are about 0.2 ~ 0.4 mM, similar to levels determined by other groups using gas chromatography/mass spectrometry^3,4. Enzymatic degradation of formaldehyde is mainly regulated by alcohol dehydrogenase 3 (ADH3) and aldehyde dehydrogenase 2 (ALDH2)⁵. ADH3 (also known as formaldehyde dehydrogenase, FDH⁶), is distributed in the hippocampus and cortex and helps to defend the brain against neurodegenerative processes⁶. ADH3-knockout mice are sensitive to formaldehyde toxicity⁷ and only less than 20% of these mice can survive after postnatal day 6⁸. Moreover, knockout of ALDH2 also induces age-dependent memory loss in mice⁹. In our previous study, inhibition of ADH3 or ALDH2 activity results in hippocampal formaldehyde accumulation and cognitive impairments in rats¹⁰. However, over-expression of S-nitrosoglutathione reductase (GSNOR, a mammalian homolog of ADH3 that accelerates the degradation of formaldehyde¹¹) in Drosophila markedly induces visual memory loss¹². These data indicate that either an abnormal excess or a deficiency of endogenous formaldehyde leads to memory impairments.

However, under physiological condition, endogenous formaldehyde participates in DNA demethylation and methylation cycle. Recent studies have shown that DNA demethylation leads to the release of formaldehyde (as a methyl moiety) or methanol¹³ (methanol is reversibly transformed into formaldehyde by ADH1⁵). In addition, S-adenosylmethionine-dependent DNMTs catalyze reversible covalent addition of exogenous aliphatic aldehydes (for instance formaldehyde) to their target residues in DNA¹⁴, because DNA-5-methyl-cytosine is the product between formaldehyde and DNA-cytosine (methyl group acceptor)². Emerging evidence has shown that external stimuli (electroconvulsive stimulation), not only elicit initial transient DNA demethylation then re-methylation of memory-related genes¹⁵, but also induce initial global DNA demethylation and re-methylation in the hippocampus¹⁶. That is, DNA methylation (regulated by DNA methyltransferases, DNMTs¹⁷), is a critical step in neuro-epigenetic memory formation and remote memory maintenance^18,19. These diverse lines of evidence strongly suggest a possible link between endogenous formaldehyde and memory-related DNA methylation status.

Accumulating evidence has shown that environmental pollutions, diet, aging and genetic factors can induce endogenous formaldehyde accumulation¹⁰. Both exogenous and endogenous excess formaldehyde can induce animal and human cognitive impairments^20,21,22,23, especially in Alzheimer's disease (AD) transgenic mice and AD patients¹⁰. Recent studies have identified that DNA methylation plays a critical role in memory formation^18,19. However, DNMT activity and global DNA methylation are obviously decreased in brains of rats during aging²⁴. Moreover, global DNA methylation decline has been observed in autopsy brain samples from AD patients²⁵. Although we have demonstrated that chronic excess formaldehyde induces cognitive deficits in rats¹⁰, the relationship between the excess formaldehyde-memory deficits and global DNA methylation decline during aging has not been clarified.

Results

Changes in global DNA methylation status and formaldehyde levels during LTP formation

First, we investigated how external stimuli induces the dynamic changes in global DNA status and formaldehyde concentration. To explore the relationship between the elevated hippocampal formaldehyde levels and the intensity of external stimulation, an increase in the number of electrical stimuli trains or duration of spatial training were applied in this study. In anesthetized normal adult male SD rats, three trains of HFS (Three-HFS) treatments induced higher amplitudes of fEPSP than a single HFS treatment ( Fig. 1A, 1D ) and were associated with a higher elevation in formaldehyde level (~0.08 mM) than that of a single HFS treatment (~0.06 mM) after electrical stimuli 5 minutes ( Fig. 1B, 1E ). Markedly, three-HFS treatments induced a higher degree of global DNA demethylation (~3%, similar to previous report¹⁶) and formaldheyde generation (~0.08 mM) than a single HFS treatment after electrical stimuli 5 minutes ( Fig. 1C, 1F ). This result indicates that electrical stimuli induce initial global DNA demethylation and subsequent re-methylation associated with formaldheyde elevation then decline to baseline level.

Changes in global DNA methylation status and formaldehyde levels during spatial memory formation

By varying the duration of training in the Morris water maze, we observed whether spatial memory was formatted in rats. We did not observe that spatial training for one day induces the changes in global DNA methylation and hippocampal formaldehyde ( Fig. 2A–2C ). However, spatial training for 3 days obviously elicited global DNA demethylation associated with an elevation in hippocampal formaldehyde concentration (~0.03 mM) on day 3 and a validate spatial memory formation on day 4 ( Fig. 2A, 2D–2E ). Furthermore, spatial training for 6 days resulted in an obvious global DNA demethylation associated with an elevation in hippocampal formaldehyde concentration (~0.08 mM) on day 6 and a stable spatial memory formation associated with global DNA re-methylation on day 7 ( Fig. 2A, 2G–2H ). These results demonstrate that learning activity also induces a dynamic change in global DNA methylatin status and formaldheyde level as well as LTP formation .

Scavenging the elevated formaldehyde impairs LTP and spatial memory in rats

To explore whether this elevated formaldehyde (~0.08 mM) is indispensable for memory formation, a formaldehyde scavenger-formaldehyde dehydrogenase (FDH, a specific formaldehyde-degrading enzyme²⁶) was used in this study. After high frequency stimulation (HFS, 100 Hz), we observed an elevation in hippocampal formaldehyde level (: 0.06 mM), however, this elevation was suppressed in rats injected with FDH (i.c.v.). Injection with FDH also markedly impaired hippocampal LTP formation ( Fig. 3A, 3B ). Notably, FDH injection did not inhibit initial global DNA demethylation, but abolished subsequent global DNA re-methylation after HFS treatment 60 min ( Fig. 3C ).

Furthermore, intrahippocampal injection with FDH markedly not only impaired the ability of spatial learning on day 3, 4, 5 and 6, but also damaged the ability of memory retrieval on day 7 ( Fig. 3D, 3E ). Similarly, FDH injection did not inhibit the initial global DNA demethylation on day 6, but abolished global DNA re-methylation on day 7 ( Fig. 3F ). This suggests that the elevated formaldehyde may contribute to subsequent DNA re-methylation and the deficiency of DNA re-methylation impairs spatial memory.

Blocking DNA re-methylation impairs LTP and spatial memory in rats

To further confirm whether inhibition of DNA re-methylation leads to spatial memory deficits, 5-aza-2-deoxycytidine (5-AzA, a DNA demethylating agent by inhibiting DNMT activity^27,28), was used in this study. We found that injection with 5-AzA (i.c.v.) markedly suppressed hippocampal LTP formation ( Fig. 4A ). Notably, 5-AzA injection enhanced transient global DNA demethylation and formaldehyde accumulation after HFS treatment 5 min ( Fig. 4B ) and reduced global DNA re-methylation after HFS treatment 60 min ( Fig. 4C ). Furthermore, intrahippocampal injection with 5-AzA not only impaired the ability of spatial learning on day 3, 4, 5 and 6, but also damaged the ability of memory retrieval on day 7 ( Fig. 4D, 4E ). Similarly, 5-AzA enhanced global DNA demethylation on day 6 and obviously reduced global DNA re-methylation on day 7 ( Fig. 4F ). This result also indicates that blocking DNA re-methylation leads to memory deficits.

DNA demethylation elicits formaldehyde generation in the dividing cells and post-mitotic neurons

1) External stimulation induces DNA demethylation in vitro and in vivo

Previous studies have shown that neuronal activity induced by electroconvulsive stimulation and NMDA receptor-mediated synaptic activity, can trigger DNA demethylation^15,16,29. Furthermore, treatment with NMDA-receptor antagonist APV (DL-2-amino-5-phosphonovaleric acid) or Ca²⁺ chelator BAPTA (1,2-bis- (o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid) is known to inhibit DNA demethylation^29,30. These data imply that activation of the NMDA-receptor-Ca²⁺-pathway is necessary for external stimulation-induced DNA demethylation ( Fig. 5A ). To verify this hypothesis, we used NMDA (an excitatory neurotransmitter) to treat the cultured hippocampal neurons for 30, 60 and 180 minutes, respectively. There was an obvious time-dependently elevation in formaldehyde concentration in media of cultures and BAPTA markedly reduced formaldehyde accumulation in vitro ( Fig. 5B ). After HFS (200 Hz) stimulation, there was a significant decrease (about 2.5%) in global DNA methylation (DNA demethylaiton); BAPTA treatment obviously blocked HFS-induced global DNA demethylation in hippocampus of the normal SD rats ( Fig. 5C ). In vivo study, global DNA demethylation induced by external stimuli (WFS, HFS, Three-HFS) or spatial training was positively correlated (R = 0.930) with formaldehyde generation in hippocampus of the SD rats ( Fig. 5D ).

2) DNA demethylating agent induces DNA demethylation in human SY5Y cell lines

As mentioned above, that 5-AzA induced global DNA demethylation associated with hippocampal formaldehyde accumulation in vivo has been observed ( Fig. 4B ). To provide the direct evidence that DNA demethylation elicits formaldehyde generation, the authorized passive demethylating agent ( Fig. 5E ), 5-AzA, was applied in post-mitotic neurons (hippocampal neurons) and the dividing cells (Human SY5Y cell lines) in vitro. We found that 5-AzA treatment induced a time-dependent elevation in formaldehyde concentrations in the cultured medium of hippocampal neurons ( Fig. 5F ) and a dose-dependent elevation in formaldehyde levels in hippocampus by inhibiting DNMT activity in vivo ( Fig. 5G, 5H ). Similarly, 5-AzA treatment for several hours also resulted in a time-dependent elevation in formaldehyde concentrations and a decrease in global DNA methylation by inhibiting DNMT activity in the cultured SY5Y cell lines ( Fig. 5I–5K ). Global DNA methylation levels affected by 5-AzA were negatively correlated (R = 0.978) with formaldehyde generation in hippocampus. The data provide evidence that DNA demethylation induces formaldehyde generation^13,31.

Aging-associated with excess formaldehyde induces spatial memory decline

1) Aging induces excess formaldehyde accumulation in hippocampus

Age-related excess hippocampal formaldehyde accumulation has been observed in our previous study¹⁰. Then, to explore the relationship between global DNA methylation and formaldehyde accumulation during aging, the normal SD rats with 2, 16 and 32-months old were employed. We found that global DNA methylation levels were gradually decreased in the SD rats during aging ( Fig. 6A ). Hippocampal formaldehyde concentrations were significantly elevated to the pathological level (~0.5 mM) in the aged rats ( Fig. 6B ). This suggests that excess formaldehyde is associated with global DNA methylation decline (DNA demethylation) in hippocampus during aging.

2) Excess formaldehyde blocks DNA methylation by inhibiting DNMT activity

Then, we investigated how excess formaldehyde induces global DNA methylation decline during aging. Since inhibition of DNMT activity by 5-AzA induced formaldehyde generation, this suggests that the decrease in DNMT activity may be the cause of global DNA methylation decline during aging. Therefore, we examined whether formaldehyde regulates DNMT activity in vitro and in vivo. Administration of 0.5 mM formaldehyde (i.c.v.) markedly inhibited DNMT activity in the hippocampus in vivo. Excess formaldehyde (0.5 mM) treatment obviously inhibited DNMT activity in cultured hippocampal neurons ( Fig. 6C, 6D ). Approximately, 0.08 mM formaldehyde markedly increased the DNMT activity of cultured hippocampal neurons and hippocampus in vivo ( Fig. 6C, 6D ). These results indicate that ~0.08 mM formaldehyde enhances DNMT activity; but, age-related excess formaldehyde inhibits DNMT activity.

3) Intrahippocampal injection with excess formaldheyde induces memory deficits in adult rats

To examine whether excess formaldehyde induces age-related cognitive decline, the normal adult SD rats were intrahippocampally injected with excess formaldehyde (0.5 mM, the average concentration was detected in the aged rats), then spatial memory behaviors were observed. Excess formaldehyde injection (0.5 mM, i.c.v.) markedly suppressed hippocampal LTP formation ( Fig. 6E, 6F ). Excess formaldehyde injection induced hippocampal formaldehyde accumulation associated with the initial decline in global DNA methylation after HFS treatment 5 min and abolished subsequent global DNA re-methylation after HFS treatment 60 min ( Fig. 6G, 6H ).

Furthermore, intrahippocampal injection with excess formaldehyde (0.5 mM) obviously not only impaired the ability of spatial learning in the adult rats, but also damaged the ability of memory retrieval associated with a decrease in global DNA methylation on day 7. More importantly, the changes in formaldehyde and global DNA methylation in the adult rats injected with excess formaldheyde were similar to the result of the aged rats ( Fig. 6I–6L ). These data suggest that aging-associated excess formaldehyde impairs spatial memory by blocking DNA methylation. Finally, resveratrol³², a natural formaldehyde scavenger, was applied into hippocampus of the aged rats, we found that resveratrol obviously improved the behavious performance of spatial memory in the aged rats associated with an increase in global DNA methylation ( Fig. 6I–6L ).

Discussion

In the present study, we found that spatial training induced an initial global DNA demethylation and subsequent re-methylation and blocking DNA re-methylation led to spatial memory deficits. More importantly, aging-associated excess formaldehyde impairs spatial memory by blocking DNA methylation ( Fig. S1 ). Our results support that neuronal activity induces DNA demethylation and re-methylation during memory formation.

Emerging evidence has identified that both electroconvulsive stimulation and acute exercise induce a transient global DNA demethylation³³. Electrical stimulation can induce transient DNA to RNA transcription (such as c-fos) in several seconds and DNA demethylation is required for gene activation³⁴. This suggests that external stimuli can elicit a transient DNA demethylation. In this study, we also observed that external stimuli (WFS, HFS, Three-HFS and spatial training) elicit initial transient global DNA demethylation and formaldehyde generation in hippocampus ( Fig. 1 , 2 ). Recent studies suggest that neuronal activity induced by external stimulation can trigger DNA demethylation via NMDA-receptor pathway^15,16,29. Calcium chelator BAPTA can inhibit DNA demethylation^29,30. Similarly, we also found that there was a significant decrease in formaldehyde generation and a marked inhibition of global DNA demethylation after treatment with BAPTA ( Fig. 5B, 5C ). Previous studies have shown that inhibition of DNMT activity elicits DNA demethylation in the dividing cancer cells^27,28 and DNA demethylation leads to formaldehyde generation^13,31 ( Fig. S2A ). We also found that 5-AzA obviously induces DNA demethylation and formaldehyde accumulation in human SY5Y cell lines in several hours.

Whether DNA demethylation occurs in post-mitotic neurons is rarely reported. Recently, however, Levenson and colleagues have found that 5-AzA (a DNA demethylation reagent) obviously induces DNA demethylation of reelin gene (increasing unmethylated DNA and decreasing methylated DNA) in hippocampal slice after adminitration 40 minutes and this gene is expressed in hippocampal neurons³⁵. In the present study, 5-AzA treatment elicited global DNA demethylation in hippocampal neurons in vitro and hippocampus of rats in vivo ( Fig. 5E–5K ). These data indicate that external stimuli can induce DNA demethylation and formaldehyde generation in post-mitotic neurons. There are multiple pathways of DNA demethylation, such as: direct active process; demethylation; indirect active process: mismatch/excision repair; and passive loss of methyl group¹³. However, the exact mechanism that neuroal activity induces DNA demethylation is currently unclear³⁶.

Recent studies have shown that formaldehyde participates in DNA methylation, for DNA-5-methyl-cytosine is a product between formaldehyde and DNA-cytosine (methyl group acceptor)². S-Adenosylmethionine-dependent DNMTs catalyze reversible covalent addition of exogenous formaldehyde (as a methyl moiety) to their target residues in DNA, yielding corresponding 5-hydroxymethylcytosine (5 hmC) and 5-methylcytosine (5 mC)¹⁴ ( Fig. S2B ). DNA methylation is required for memory formation¹⁹. In the present study, we found that external stimuli elevate hippocampal formaldehyde concentration (~0.08 mM) ( Fig. 1 ). Formaldehyde at 0.08 mM signifcantly enhanced DNMT activity both in vitro and in vivo ( Fig. 6C, 6D ). Conversely, scavenging the elevated hippocampal formaldehyde by FDH treatment did not affect DNA demethylation, but obviously blocked global DNA re-methylation and impaired spatial memory in rats ( Fig. 3 ). Over-expression of FDH (ADH3) in Drosophila can induce visual memory loss¹². Especially, knockout of DNMT_1a is known to impair LTP and memory¹⁷. Our data along with these reports suggest that ~0.08 mM formaldehyde derived from the initial transient DNA demethyltion induced by external stimuli, may contribute to enhance subsequent DNA re-methylation at least in part via DNMTs.

Endogenous formaldehyde is kept a relative stable level in body, but there is a chronic accumulation of formaldehyde during aging. For example, a formaldehyde generating enzyme, semicarbazide-sensitive amine oxidase (SSAO), SSAO activity is increased in human plasma during aging³⁷ and associated with formaldehyde accumulation³⁸. A specific formaldehyde degrading enzyme³⁹, ADH3 (FDH), its activity is decreased in human during aging⁴⁰. Recent studies show that FDH can scavenge formaldehyde by transferring S-hydroxymenthyl which is a derivative between formaldehyde with GSH^5,41,42. In the present study, we also found that extracellular FDH reduced hippocampal formaldehyde ( Fig. 3 ). An unspecific formaldehyde degrading enzyme, ALDH2⁴³, its activity is also attenuated in human and diabetic rats during aging⁴⁴. We also have found that inhibiting ADH3 or ALDH2 leads to an abnormal high concentration of formaldehyde accumulated in hippocampus in rats¹⁰. Especially, over-expression of ADH3 (a specific formaldehyde degrading enzyme) leads to visual memory decline in Drosophila¹². Deficiency of ALDH2 (a unespecific formaldehyde degrading enzyme) induces memory loss in mice⁹. Age-related oxidative stress can increase in the content of reactive aldehydes⁴⁵, including formaldehyde⁴⁶. In this study, we found that age-related formaldehyde was over 0.5 mM in the aged rats ( Fig. 6B ). All these data support that endogenous formaldehyde is accumulated during aging.

In the present study, we found that endogenous formaldehyde participates in spatial memory formation, but during aging process, aging-associated excess formaldehyde blocks global DNA methylation and leads to memory decline. Other reports also show that DNMT activity and global DNA methylation are decreased in brains during aging²⁴. We found that formaldehyde was over 0.5 mM in the aged rats and formaldehyde at 0.5 mM (the pathological level) obviously inhibited the DNMTs activity in vitro and in vivo, but formaldehyde at 0.3 mM (the physiological level for endogenous formaldehyde) did not affect DNMT activity ( Fig. 6C, 6D ). More importantly, the normal adult rats intrahippocampally injected with this excess formaldehyde (0.5 mM) can imitate the aged-related spatial memory deficits associated with global DNA methylation decline and formaldehyde accumulation ( Fg. 6 ). These data suggest that age-related accumulated formaldehyde (over 0.5 mM) erasues global DNA methylation by inhibiting DNMT activity and restults in spatial memory deficits. Notably, a natural formaldehyde scavenger-resveratrol³², can rescue the behavior performance of spatial memory in the aged rats by decreasing hippocampal formaldehyde concentration ( Fig. 6I–6L ). Resveratrol has been considered as a therapeutic agent for neurodegenerative diseases⁴⁷. Dietary supplementation with resveratrol can reduce plaque pathology in a transgenic model of Alzheimer's disease⁴⁸. Our results along with these reports suggest that aging-associated excess formaldehyde is one of the critical pathological factors for cognitive decline in neurodegenerative disease.

Methods

Reagents

All reagents were from Sigma unless otherwise indicated.

Biological samples

All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Biological Research Ethics Committee of the Institute of Biophysics, Chinese Academy of Sciences (SYXK2010-127).

Intracerebroventricular formaldehyde injection

Sprague Dawley (SD) rats were intracerebroventricularly (i.c.v.) injected with formaldehyde (dissolved in normal saline, 0.01 and 0.08 mM, 5 μl, i.c.v. over a period of 5 min), or resveratrol (Res, 0.5 mM, 5 μl). Cannulae placements in brains: 1.4 mm anterior/posterior to bregma; 1.8 mm medial/lateral to the midsagittal suture; 3.0 mm dorsal/ventral from the brain surface.

Intrahippocampal formaldehyde injection

Sprague Dawley (SD) rats were intrahippocampally injected with formaldehyde (0.08 mM, 1 μl, over a period of 5 min), or resveratrol (Res, 0.5 mM, 1 μl) 30 min before the daily behavioral experiments. Cannulae placements in the dorsal hippocampus: 3.6 mm anterior/posterior to bregma; 2.5 mm medial/lateral to the midsagittal suture; and 2.5 mm dorsal/ventral from the brain surface.

Rat hippocampal neuron and human SY5Y cell lines cultures

We prepared primary hippocampal neuron cultures from 18-day old embryos and cultured human SY5Y cell lines as described previously⁴⁹. Mature hippocampal neurons or cultrued media after drugs treatment were collected in order to detect formaldehyde and the activity of DNMTs in vitro.

Detection of formaldehyde by Fluo-HPLC

Drug-treated medium from cultured hippocampal neurons was collected and immediately placed on ice before storing at −70°C. After centrifugation (3000 rpm, 4°C, 10 min), supernatant fractions of medium samples and brain homogenates (weight of brain tissue: ultrapure water = 1:4) were analyzed by high-performance liquid chromatography with fluorescence detection (Fluo-HPLC) as described¹⁰.

Electrophysiological studies in vivo

Electrophysiological recordings were performed according to our previous reports¹⁰. WFS-LTP in vivo LTP was induced using a weak frequency stimulation (WFS) protocol consisting of 20 pulses at 50 Hz.

HFS-LTP in vivo

LTP was induced using a high frequency stimulation (HFS) protocol consisting of 20 pulses at 100 Hz or 200 Hz.

L-LTP in vivo

Three pulses of HFS at 5 min intervals were applied to produce a robust late long-term potentiation (L-LTP).

Morris water maze behavioral test

Spatial memory was assessed by the Morris water maze test, as described previously¹⁰. Sprague Dawley (SD) rats were intrahippocampally injected with FDH (120 μM, 1 μl), or 5-aza-2-deoxycytidine (5-AzA, 60 μM, 1 μl, vehicle (0.001% CH₃COOH)), or excess formaldehyde (0.5 mM, 1 μl), or resveratrol (Res, 0.5 mM, 1 μl), 30 min before the daily behavioral experiment.

Quantification of global DNA methylation

A MethylampTM Global DNA Methylation Quantification Ultra Kit (Epigentek, USA) was used according to the manufacturer's instructions to quantify global DNA methylation of genomic DNA in the hippocampus of SD rats.

DNA methyltransferase (DNMT) assay

Nuclei were extracted from rat hippocampi using an EpiQuik™ nuclear extraction kit (Epigentek, USA). DNA methyltransferase activity was assayed using a DNMT assay kit (Epigentek, USA) according to the manufacturer's instructions.

Statistical analysis

We determined the statistical significance of in vivo data by analysis of variance (one-way ANOVA) followed by unpaired Student's t-tests; and a repeated measures ANOVA followed by post-hoc analysis was used to determine statistical significance for the Morris water maze experiments. The statistical significance level was set at P < 0.05. Data are reported as means ± standard errors.

References

Kalapos, M. P. A possible evolutionary role of formaldehyde. Exp Mol Med 31, 1–4 (1999).
CAS PubMed Google Scholar
Kalasz, H. Biological role of formaldehyde and cycles related to methylation, demethylation and formaldehyde production. Mini Rev Med Chem 3, 175–192 (2003).
CAS PubMed Google Scholar
Heck, H. D., White, E. L. & Casanova-Schmitz, M. Determination of formaldehyde in biological tissues by gas chromatography/mass spectrometry. Biomed Mass Spectrom 9, 347–353 (1982).
CAS PubMed Google Scholar
Tong, Z. Q. et al. Urine formaldehyde level is inversely correlated to mini mental state examination scores in senile dementia. Neurobiol Aging 32, 31–42 (2011).
CAS PubMed Google Scholar
Teng, S. et al. The formaldehyde metabolic detoxification enzyme systems and molecular cytotoxic mechanism in isolated rat hepatocytes. Chem Biol Interact 130–132, 285–296 (2001).
PubMed Google Scholar
Mori, O. et al. Histological distribution of class III alcohol dehydrogenase in human brain. Brain Res 852, 186–190 (2000).
CAS PubMed Google Scholar
Deltour, L., Foglio, M. H. & Duester, G. Metabolic deficiencies in alcohol dehydrogenase Adh1, Adh3 and Adh4 null mutant mice. Overlapping roles of Adh1 and Adh4 in ethanol clearance and metabolism of retinol to retinoic acid. J Biol Chem 274, 16796–16801 (1999).
CAS PubMed Google Scholar
Molotkov, A. et al. Stimulation of retinoic acid production and growth by ubiquitously expressed alcohol dehydrogenase Adh3. Proc Natl Acad Sci U S A 99, 5337–5342 (2002).
ADS CAS PubMed PubMed Central Google Scholar
Ohsawa, I. et al. Age-dependent neurodegeneration accompanying memory loss in transgenic mice defective in mitochondrial aldehyde dehydrogenase 2 activity. J Neurosci 28, 6239–6249 (2008).
CAS PubMed PubMed Central Google Scholar
Tong, Z. Q. et al. Accumulated hippocampal formaldehyde induces age-dependent memory decline. AGE [Epub ahead of print] (2012).
Greenwald, R. et al. Breath formate is a marker of airway S-nitrosothiol depletion in severe asthma. PLoS One 5, e11919 (2010).
ADS PubMed PubMed Central Google Scholar
Hou, Q. et al. Nitric oxide metabolism controlled by formaldehyde dehydrogenase (fdh, homolog of mammalian GSNOR) plays a crucial role in visual pattern memory in Drosophila. Nitric Oxide 24, 17–24 (2011).
CAS PubMed Google Scholar
Patra, S. K. et al. Demethylation of (Cytosine-5-C-methyl) DNA and regulation of transcription in the epigenetic pathways of cancer development. Cancer Metastasis Rev 27, 315–334 (2008).
CAS PubMed Google Scholar
Liutkeviciute, Z., Lukinavicius, G., Masevicius, V., Daujotyte, D. & Klimasauskas, S. Cytosine-5-methyltransferases add aldehydes to DNA. Nat Chem Biol 5, 400–402 (2009).
CAS PubMed Google Scholar
Ma, D. K. et al. Neuronal activity-induced Gadd45b promotes epigenetic DNA demethylation and adult neurogenesis. Science 323, 1074–1077 (2009).
ADS CAS PubMed PubMed Central Google Scholar
Guo, J. U. et al. Neuronal activity modifies the DNA methylation landscape in the adult brain. Nat Neurosci 14, 1345–1351 (2011).
CAS PubMed PubMed Central Google Scholar
Feng, J. et al. Dnmt1 and Dnmt3a maintain DNA methylation and regulate synaptic function in adult forebrain neurons. Nat Neurosci 13, 423–430 (2010).
CAS PubMed PubMed Central Google Scholar
Miller, C. A., Campbell, S. L. & Sweatt, J. D. DNA methylation and histone acetylation work in concert to regulate memory formation and synaptic plasticity. Neurobiol Learn Mem 89, 599–603 (2008).
CAS PubMed Google Scholar
Miller, C. A. et al. Cortical DNA methylation maintains remote memory. Nat Neurosci 13, 664–666 (2010).
CAS PubMed PubMed Central Google Scholar
Lu, Z. S. et al. Effect of inhaled formaldehyde on learning and memory of mice. Indoor Air 18, 77–83 (2008).
CAS PubMed Google Scholar
Malek, F. A., Möritz, K. U. & Fanghänel, J. A study on the effect of inhalative formaldehyde exposure on water labyrinth test performance in rats. Ann Anat 185, 277–285 (2003).
CAS PubMed Google Scholar
Kilburn, K. H. Neurobehavioral impairment and seizures from formaldehyde. Arch Environ Health 49, 37–44 (1994).
CAS PubMed Google Scholar
Perna, R. B., Bordini, E. J. & Deinzer-Lifrak, M. A case of claimed persistent neuropsychological sequelae of chronic formaldehyde exposure: clinical, psychometric and functional findings. Arch Clin Neuropsychol 16, 33–44 (2001).
CAS PubMed Google Scholar
Liu, L. et al. Insufficient DNA methylation affects healthy aging and promotes age-related health problems. Clin Epigenetics 2, 349–360 (2011).
PubMed PubMed Central Google Scholar
Mastroeni, D. et al. Epigenetic changes in Alzheimer's disease: decrements in DNA methylation. Neurobiol Aging 31, 2025–2037 (2010).
CAS PubMed Google Scholar
Thompson, C. M., Ceder, R. & Grafstrom, R. C. Formaldehyde dehydrogenase: beyond phase I metabolism. Toxicol Lett 193, 1–3 (2010).
CAS PubMed Google Scholar
Yamagata, Y. et al. Rapid turnover of DNA methylation in human cells. Epigenetics 7, 141–145 (2012).
CAS PubMed PubMed Central Google Scholar
Hamm, C. A. et al. Global demethylation of rat chondrosarcoma cells after treatment with 5-aza-2′-deoxycytidine results in increased tumorigenicity. PLoS One 4, e8340 (2009).
ADS PubMed PubMed Central Google Scholar
Nelson, E. D., Kavalali, E. T. & Monteggia, L. M. Activity-dependent suppression of miniature neurotransmission through the regulation of DNA methylation. J Neurosci 28, 395–406 (2008).
CAS PubMed PubMed Central Google Scholar
Collas, P. Modulation of plasmid DNA methylation and expression in zebrafish embryos. Nucleic Acids Res 26, 4454–4461 (1998).
CAS PubMed PubMed Central Google Scholar
Gerken, T. et al. The obesity-associated FTO gene encodes a 2-oxoglutarate-dependent nucleic acid demethylase. Science 318, 1469–1472(2007).
ADS CAS PubMed PubMed Central Google Scholar
Tyihak, E. et al. Formaldehyde cycle and the natural formaldehyde generators and capturers. Acta Biol Hung 49, 225–238 (1998).
CAS PubMed Google Scholar
Barres, R. et al. Acute exercise remodels promoter methylation in human skeletal muscle. Cell Metab 15, 405–411 (2012).
CAS PubMed Google Scholar
Ikeda, Y. & Kinoshita, T. DNA demethylation: a lesson from the garden. Chromosoma 118, 37–41 (2009).
CAS PubMed Google Scholar
Levenson, J. M. et al. Evidence that DNA (cytosine-5) methyltransferase regulates synaptic plasticity in the hippocampus. J Biol Chem 281, 15763–15773 (2006).
CAS PubMed Google Scholar
Wu, S. C. & Zhang, Y. Active DNA demethylation: many roads lead to Rome. Nat Rev Mol Cell Biol 11, 607–620 (2010).
CAS PubMed PubMed Central Google Scholar
del Mar Hernandez, M., Esteban, M., Szabo, P., Boada, M. & Unzeta, M. Human plasma semicarbazide sensitive amine oxidase (SSAO), beta-amyloid protein and aging. Neurosci Lett 384, 183–187 (2005).
PubMed Google Scholar
Conklin, D. J. et al. Vasoactive effects of methylamine in isolated human blood vessels: role of semicarbazide-sensitive amine oxidase, formaldehyde and hydrogen peroxide. Am J Physiol Heart Circ Physiol 286, H667–676 (2004).
CAS PubMed Google Scholar
Moreno, A., Pares, A., Ortiz, J., Enriquez, J. & Pares, X. Alcohol dehydrogenase from human stomach: variability in normal mucosa and effect of age, gender, ADH3 phenotype and gastric region. Alcohol Alcohol 29, 663–671 (1994).
CAS PubMed Google Scholar
Hines, L. M. et al. A prospective study of the effect of alcohol consumption and ADH3 genotype on plasma steroid hormone levels and breast cancer risk. Cancer Epidemiol Biomarkers Prev 9, 1099–1105 (2000).
MathSciNet CAS PubMed Google Scholar
Tulpule, K. et al. Formaldehyde induces rapid glutathione export from viable oligodendroglial OLN-93 cells. Neurochem Int 61, 1302–1313 (2012).
CAS PubMed Google Scholar
Tsuboi, S. et al. Purification and characterization of formaldehyde dehydrogenase from rat liver cytosol. J Biochem 111, 465–471 (1992).
CAS PubMed Google Scholar
Luo, H. R., Israel, Y., Tu, G. C., Eriksson, C. J. & Zhang, Y. P. Genetic polymorphism of aldehyde dehydrogenase 2 (ALDH2) in a Chinese population: gender, age, culture and genotypes of ALDH2. Biochem Genet 43, 223–227 (2005).
CAS PubMed Google Scholar
Wang, J. et al. Inhibition of aldehyde dehydrogenase 2 by oxidative stress is associated with cardiac dysfunction in diabetic rats. Mol Med 17, 172–179 (2011).
CAS PubMed Google Scholar
Maboudou, P., Mathieu, D., Bachelet, H., Wiart, J. F. & Lhermitte, M. Detection of oxidative stress. Interest of GC-MS for malondialdehyde and formaldehyde monitoring. Biomed Chromatogr 16, 199–202 (2002).
CAS PubMed Google Scholar
Yu, P. H., Lai, C. T. & Zuo, D. M. Formation of formaldehyde from adrenaline in vivo; a potential risk factor for stress-related angiopathy. Neurochem Res 22, 615–620 (1997).
CAS PubMed Google Scholar
Sun, A. Y., Wang, Q., Simonyi, A. & Sun, G. Y. Resveratrol as a therapeutic agent for neurodegenerative diseases. Mol Neurobiol 41, 375–383 (2010).
CAS PubMed PubMed Central Google Scholar
Karuppagounder, S. S. et al. Dietary supplementation with resveratrol reduces plaque pathology in a transgenic model of Alzheimer's disease. Neurochem Int 54, 111–118 (2009).
CAS PubMed Google Scholar
Nie, C. L., Wang, X. S., Liu, Y., Perrett, S. & He, R. Q. Amyloid-like aggregates of neuronal tau induced by formaldehyde promote apoptosis of neuronal cells. BMC Neurosci 8, 9 (2007).
PubMed PubMed Central Google Scholar

Download references

Acknowledgements

This research was supported by grants from the 973-Project (2012CB911000; 2010CB912303, 2013CB531000) and QCAS Biotechnology Fund No. GJHZ1131, the Chinese Postdoctoral Fund 20090460047 and 2012T50144, the Natural Scientific Foundation of China NSFC 31171080, 30970695.

Author information

Tong Zhiqian, Han Chanshuai and Luo Wenhong contributed equally to this work.

Authors and Affiliations

State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
Zhiqian Tong, Chanshuai Han, Min Qiang, Tao Su, Beibei Wu, Ying Liu & Rongqiao He
Central Laboratory, Shantou University Medical College, Guangdong, 515041, China
Wenhong Luo, Hui Li & Hongjun Luo
Laboratory of Environmental Sciences, Huazhong Normal University, Wuhan, 430079, China
Xu Yang
Graduate University of the Chinese Academy of Sciences, Beijing, 100049, China
Chanshuai Han, Min Qiang, Tao Su & Beibei Wu
Neuroscience Research Institute, Peking University, Beijing, 100191, China
Zhiqian Tong, You Wan & Dehua Cui
Key Lab of Mental Health, Institute of Psychology, Chinese Academy of Sciences, Beijing, 100101, China
Rongqiao He

Authors

Zhiqian Tong
View author publications
You can also search for this author in PubMed Google Scholar
Chanshuai Han
View author publications
You can also search for this author in PubMed Google Scholar
Wenhong Luo
View author publications
You can also search for this author in PubMed Google Scholar
Hui Li
View author publications
You can also search for this author in PubMed Google Scholar
Hongjun Luo
View author publications
You can also search for this author in PubMed Google Scholar
Min Qiang
View author publications
You can also search for this author in PubMed Google Scholar
Tao Su
View author publications
You can also search for this author in PubMed Google Scholar
Beibei Wu
View author publications
You can also search for this author in PubMed Google Scholar
Ying Liu
View author publications
You can also search for this author in PubMed Google Scholar
Xu Yang
View author publications
You can also search for this author in PubMed Google Scholar
You Wan
View author publications
You can also search for this author in PubMed Google Scholar
Dehua Cui
View author publications
You can also search for this author in PubMed Google Scholar
Rongqiao He
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

T.Z. conceived and designed the experiments, analyzed the data and prepared the manuscript; T.Z., H.C., L.W., L.H., L.J., Q.M., S.T., W.B., Y.X., L.Y. and W.Y. performed the experiments. C.D. revised the manuscript. H.R. supervised the project. All authors reviewed the manuscript. T.Z., H.C. and L.W. contributed equally to this work.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Electronic supplementary material

Supplementary Information

Supplementary information

Rights and permissions

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/

Reprints and permissions

About this article

Cite this article

Tong, Z., Han, C., Luo, W. et al. Aging-associated excess formaldehyde leads to spatial memory deficits. Sci Rep 3, 1807 (2013). https://doi.org/10.1038/srep01807

Download citation

Received: 05 March 2013
Accepted: 23 April 2013
Published: 09 May 2013
DOI: https://doi.org/10.1038/srep01807

This article is cited by

Gut microbiome, cognitive function and brain structure: a multi-omics integration analysis
- Xinxiu Liang
- Yuanqing Fu
- Ju-Sheng Zheng
Translational Neurodegeneration (2022)
Amino acid dependent formaldehyde metabolism in mammals
- Matthias Pietzke
- Guillermo Burgos-Barragan
- Alexei Vazquez
Communications Chemistry (2020)
Endogenous formaldehyde is a memory-related molecule in mice and humans
- Li Ai
- Tao Tan
- Zhiqian Tong
Communications Biology (2019)
Icariin protects SH-SY5Y cells from formaldehyde-induced injury through suppression of Tau phosphorylation
- Yi-xiang Song
- Jun-ye Miao
- Wei-wei Li
Chinese Journal of Integrative Medicine (2016)
Effect of Support on the Activity of Ag-based Catalysts for Formaldehyde Oxidation
- Jianghao Zhang
- Yaobin Li
- Hong He
Scientific Reports (2015)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.