Physical weight loading induces expression of tryptophan hydroxylase 2 in the brain stem

Abstract

Sustaining brain serotonin is essential in mental health. Physical activities can attenuate mental problems by enhancing serotonin signaling. However, such activity is not always possible in disabled individuals or patients with dementia. Knee loading, a form of physical activity, has been found to mimic effects of voluntary exercise. Focusing on serotonergic signaling, we addressed a question: Does local mechanical loading to the skeleton elevate expression of tryptophan hydroxylase 2 (tph2) that is a rate-limiting enzyme for brain serotonin? A 5 min knee loading was applied to mice using 1 N force at 5 Hz for 1,500 cycles. A 5-min treadmill running was used as an exercise (positive) control, and a 90-min tail suspension was used as a stress (negative) control. Expression of tph2 was determined 30 min - 2 h in three brain regions --frontal cortex (FC), ventromedial hypothalamus (VMH), and brain stem (BS). We demonstrated for the first time that knee loading and treadmill exercise upregulated the mRNA level of tph2 in the BS, while tail suspension downregulated it. The protein level of tph2 in the BS was also upregulated by knee loading and downregulated by tail suspension. Furthermore, the downregulation of tph2 mRNA by tail suspension can be partially suppressed by pre-application of knee loading. The expression of tph2 in the FC and VMH was not significantly altered with knee loading. In this study we provided evidence that peripheral mechanical loading can activate central tph2 expression, suggesting that physical cues may mediate tph2-cathalyzed serotonergic signaling in the brain.

Figure 1. Mechanical treatments and brain samples.

(A) Knee loading applied laterally to the mouse knee. (B) Unloading of hindlimbs through tail suspension. (C) Knee loading comprising cyclic compression of 1 N at 5 Hz for 1500 cycles. (D) Force-displacement relationship. (E) Division of a mouse whole brain, containing frontal cortex, FC, ventromedial hypothalamus, VMH, and raphe nuclei of caudal brain stem, BS. The dashed lines indicate approximate split for harvest. The olfactory bulb marked in arrow was excluded. The dissected tissue from three subdivisions is hatched as FC, VMH, and BS.

Figure 2. Effects of loading and unloading on mRNA levels of Col I, Col II, and NGFß.

(A) Knee loading-induced increases in Col I and unloading-induced decreases in Col I in the femur and tibia. (B) Knee loading-induced increases in Col II and unloading-induced decreases in Col II in cartilage. Dashed lines denote the mRNA levels for untreated controls; A to B. (C) Effects of knee loading on NGFß mRNA. Note that “cart., tread, knee, and tail” denote cartilage, treadmill, knee loading and tail suspension, respectively.

Figure 3. Effects of knee loading on the mRNA and protein levels of tph2 in mouse brain.

(A) mRNA levels of tph2 in the BS with knee loading, tail suspension, and knee loading followed by tail suspension. (B) Immunoblots displaying protein levels of tph2 and ß-actin in the BS of mice with knee loading. Next to immunoblots showing tph2 protein fold change. (C) Relative mRNA abundance of REST and Sim1 along with that of Pet 1, lhx8, and RGS4 in the brain in response to knee loading. Dash lines denote the mRNA levels of sham loaded controls. Note that “K+T” denotes knee loading plus tail suspension.

Figure 4. Immunohistochemistry demonstrating the elevated tph2 protein level in the BS with knee loading.

(A) Rectangular region of interest in caudal brain sections. Orientation of medial and dorsal corresponds to micrographs in B. (B) Representative micrographs from bregma −5.9±0.1 mm showing tph2 immunoreactivity in mice treated with sham, knee loading, and tail suspension. Nuclear counterstain by DAPI overlaid with tph2 in the lower panels. (C) The proposed role of knee loading and unloading in serotonin synthesis through tph2 in the brain.