Dnmt3a2/Dnmt3L Overexpression in the Dopaminergic System of Mice Increases Exercise Behavior through Signaling Changes in the Hypothalamus

Abstract

Dnmt3a2, a de novo DNA methyltransferase, is induced by neuronal activity and participates in long-term memory formation with the increased expression of synaptic plasticity genes. We wanted to determine if Dnmt3a2 with its partner Dnmt3L may influence motor behavior via the dopaminergic system. To this end, we generated a mouse line, Dnmt3a2/3L^Dat/wt, with dopamine transporter (DAT) promotor driven Dnmt3a2/3L overexpression. The mice were studied with behavioral paradigms (e.g., cylinder test, open field, and treadmill), brain slice patch clamp recordings, ex vivo metabolite analysis, and in vivo positron emission tomography (PET) using the dopaminergic tracer 6-[¹⁸F]FMT. The results showed that spontaneous activity and exercise performance were enhanced in Dnmt3a2/3L^Dat/wt mice compared to Dnmt3a2/3L^wt/wt controls. Dopaminergic substantia nigra pars compacta neurons of Dnmt3a2/3L^Dat/wt animals displayed a higher fire frequency and excitability. However, dopamine concentration was not increased in the striatum, and dopamine metabolite concentration was even significantly decreased. Striatal 6-[¹⁸F]FMT uptake, reflecting aromatic L-amino acid decarboxylase activity, was the same in Dnmt3a2/3L^Dat/wt mice and controls. [¹⁸F]FDG PET showed that hypothalamic metabolic activity was tightly linked to motor behavior in Dnmt3a2/3L^Dat/wt mice. Furthermore, dopamine biosynthesis and motor-related metabolic activity were correlated in the hypothalamus. Our findings suggest that Dnmt3a2/3L, when overexpressed in dopaminergic neurons, modulates motor performance via activation of the nigrostriatal pathway. This does not involve increased dopamine synthesis.

Keywords: DNA methylation; behavioral paradigms; dopaminergic system; positron emission tomography; transgenic mouse model.

Figure 1

Dnmt3a2/3L specifically overexpressed in dopaminergic neurons, and Dnmt3a2/3L^Dat/wt animals showed greater spontaneous activity and higher exercise performance. (A) DAB immunohistochemical staining showed that the mCherry reporter was specifically expressed in midbrain areas in Dnmt3a2/3L^Dat/wt mice compared to control littermates. (B) mCherry was expressed specifically in neurons double labelled for tyrosine hydroxylase (TH) in Dnmt3a2/3L^Dat/wt mice. (C) Dnmt3a2 expression level detected by western blot was increased in the olfactory bulb (OB), striatum (ST) and midbrain (MB) in Dnmt3a2/3L^Dat/wt mice. (D) qRT-PCR of Dnmt3a2 and Dnmt3L was shown an elevated expression in midbrain of Dnmt3a2/3L^Dat/wt mice. (E) 5mC staining showed stronger fluorescence intensity in TH positive cells in Dnmt3a2/3L^Dat/wt mice. (F) Quantification for the DNA methylation of TH positive cells was significantly higher in Dnmt3a2/3L^Dat/wt mice. (G) Dnmt3a2/3L^Dat/wt mice exhibited a higher spontaneous activity in the cylinder test in mixed gender and female groups, no significance but the same trend was found in the male group. (H) Dnmt3a2/3L^Dat/wt mice showed a higher exercise performance on the treadmill in mixed gender and male groups, no significance but the same trend occurred in the female group. Values are reported as the mean ± SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001. Student t-test has been used for comparison between Dnmt3a2/3L^wt/wt and Dnmt3a2/3L^Dat/wt.

Figure 2

Intrinsic electrophysiological properties of SNc DA neurons are changed in Dnmt3a2/3L^Dat/wt mice. (A–F) Electrophysiological parameters of SNc DA neurons in Dnmt3a2/3L^Dat/wt and Dnmt3a2/3L^wt/wt mice. Current clamp recordings were performed in the perforated patch clamp configuration. (A) DA neurons in the SNc were identified by their slow and regular firing (left) and the presence of a large I_H (hyperpolarization activated cation current)-dependent ‘sag’ potential (right). (B) Spontaneous activity. (C) Coefficiant of variation. (D) Cell input resistance. * p < 0.05, ** p < 0.01. p-values are for Welch’s unpaired Student’s t-tests (B,D) or unpaired t-tests (C). Excitability of DA neurons in Dnmt3a2/3L^Dat/wt and Dnmt3a2/3L^wt/wt mice. (E) Representative voltage responses to depolarizing current pulses. (F) Mean spike counts during current pulses (1.5 s) as a function of injected current from a holding potential of −70 mV. Mean values are represented as mean ± SEM. F(1,24) = 468.7; p < 0.0001.

Figure 3

Levels of neurotransmitters in the striatum. The level of dopamine was the same in both Dnmt3a2/3L^Dat/wt and Dnmt3a2/3L^wt/wt animals. The levels of DOPAC, 3MT, HVA were lower in Dnmt3a2/3L^Dat/wt animals. Abbreviations: DA: dopamine; 3MT: 3-methoxytyramine (extracellular metabolite); DOPAC: 3,4-dihydroxyphenyl acetic acid; HVA: homovanillic acid. Values are reported as the mean ± SEM. * p < 0.05. Student t-test has been used for comparison

Figure 4

Change in 6-[¹⁸F]FMT uptake in the brain of Dnmt3a2/3L^Dat/wt mice compared to control littermates. (A) Mean cerebral 6-[¹⁸F]FMT uptake in Dnmt3a2/3L^Dat/wt mice (n = 6). Image intensity was normalized to 6-[¹⁸F]FMT uptake in the occipital cortex. (B) Mean 6-[¹⁸F]FMT uptake of control littermates (n = 6). Both genders were used, and uptake took place under anaesthesia. (C) Student’s t-test (p < 0.05, controlled for multiple testing with a TFCE procedure) for comparison between control littermates and Dnmt3a2/3L^Dat/wt mice. Red voxels indicate significantly higher 6-[¹⁸F]FMT uptake in Dnmt3a2/3L^Dat/wt mice. Significantly higher uptake in control mice was not observed. (D) Comparison of global 6-[¹⁸F]FMT uptake between groups. (E) Comparison of striatal 6-[¹⁸F]FMT uptake between groups. Abbreviations: Am: amygdala, Hyp: hypothalamus, Pir: piriform cortex, S1/S2: primary and secondary sensory cortex, VP: ventral pallidum.

Figure 5

Change in cerebral [¹⁸F]FDG uptake during running and home cage stay in Dnmt3a2/3L^Dat/wt mice compared to control littermates.(A) Mean [¹⁸F]FDG uptake in the homecage (n = 14). Because the mice groomed and gnawed, head muscles were active, causing radioactivity spillover into the brain (sketched areas). (B) Mean [¹⁸F]FDG uptake during running on the treadmill (n = 14). Head muscles were relaxed, only some spillover (sketched areas) from the Harderian glands is visible. (C) Significant differences (p < 0.05, corrected for multiple testing) between [¹⁸F]FDG uptake during home cage and treadmill running across all animals. Red voxels: significantly higher [¹⁸F]FDG uptake during running. Blue voxels: significantly higher [¹⁸F]FDG uptake during home cages stay. (D) Comparison (p < 0.05, corrected for multiple testing) of [¹⁸F]FDG difference images (“treadmill minus home cage”) between Dnmt3a2/3L^Dat/wt mice and controls (n = 7 each). Red voxels: significantly higher difference between running and home cage in Dnmt3a2/3L^Dat/wt mice. Blue voxels: significantly higher difference between running and home cage in controls. (E) Significant differences (p < 0.05, corrected for multiple testing) of [¹⁸F]FDG uptake in the home cage between Dnmt3a2/3L^Dat/wt mice and controls (n = 7 each). Red voxels: significantly higher [¹⁸F]FDG uptake in Dnmt3a2/3L^Dat/wt mice. Blue voxels: significantly higher [¹⁸F]FDG uptake in control mice.(F) Significant differences of [¹⁸F]FDG uptake during treadmill running between Dnmt3a2/3L^Dat/wt mice and controls (n = 7 each).(G) Correlation (p < 0.05, corrected for multiple testing with TFCE) between [¹⁸F]FDG difference images (“treadmill minus home cage”) and [¹⁸F]FMT images with all animals pooled (n = 12). Red voxels: positive correlation between [¹⁸F]FMT uptake and [¹⁸F]FDG difference images. Blue voxels: negative correlation.Abbreviations: 2Cb, 9Cb: lobule 2 and 9 of the cerebellar vermis, AHyp: anterior hypothalamus, DCN: deep cerebellar nuclei, dHip: dorsal hippocampus, DMHyp: dorsomedial hypothalamus, Hb: habenula, LPO: lateral preoptic nucleus; M2: secondary motor cortex; MM: medial mammillary nucleus, MPO: medial preoptic nucleus; PAG: periaqueductal gray, PFl: paraflocculus, PrT: pretectum, RM: retromammillary nucleus, S1: primary sensory cortex, Sim: simple lobule of the cerebellum, Str: striatum, VDB: ventral diagonal band of Broca, vHip: ventral hippocampus, VMHyp: ventromedial hypothalamus; VTh: ventral thalamus.