A neuronal action of sirtuin 1 suppresses bone mass in young and aging mice

Abstract

The various functions of the skeleton are influenced by extracellular cues, hormones, and neurotransmitters. One type of neuronal regulation favors bone mass accrual by inhibiting sympathetic nervous system (SNS) activity. This observation raises questions about the transcriptional mechanisms regulating catecholamine synthesis. Using a combination of genetic and pharmacological studies, we found that the histone deacetylase sirtuin 1 (SIRT1) is a transcriptional modulator of the neuronal control of bone mass. Neuronal SIRT1 reduced bone mass by increasing SNS signaling. SIRT1 did so by increasing expression of monoamine oxidase A (MAO-A), a SIRT1 target that reduces brain serotonin levels by inducing its catabolism and by suppressing tryptophan hydroxylase 2 (Tph2) expression and serotonin synthesis in the brain stem. SIRT1 upregulated brain catecholamine synthesis indirectly through serotonin, but did not directly affect dopamine β hydroxylase (Dbh) expression in the locus coeruleus. These results help us to understand skeletal changes associated with selective serotonin reuptake inhibitors (SSRIs) and may have implications for treating skeletal and metabolic diseases.

Keywords: Bone Biology; Mouse models; Neuroendocrine regulation; Neuroscience; Osteoporosis.

Figure 1. Increased sympathetic tone and decreased bone mass in TgSirt1 mice.

(A) Number of osteoblasts per trabecular area (N.Ob/T.Ar) (/mm²); (B) osteoclast surface per bone surface (Oc.S/BS) (%); (C) bone volume over tissue volume (BV/TV) (%); and (D) BFR/BS (μm³/μm²/yr) of TgSirt1 mice (1.5 months: n = 6; 3 months: n = 8; 12 months: n = 5) versus WT controls (1.5 months: n = 3; 3 months: n = 6; 12 months: n = 5) at 1.5, 3, and 12 months of age. (E) Representative images of spines from TgSirt1 and WT control mice stained with von Kossa. (F) Relative expression levels of osteoblast and osteoclast differentiation marker genes in long bones of 3-month-old TgSirt1 mice (n = 6) versus WT controls (n = 6). (G) Urine epinephrine levels in 3-month-old TgSirt1 mice (n = 7) versus WT controls (n = 3). (H) Urine NE levels in 3-month-old TgSirt1 mice (n = 7) versus WT controls (n = 3). (I) Ucp1 expression levels in BAT of 3-month-old TgSirt1 mice (n = 6) versus WT controls (n = 6). (J) Relative expression levels of sympathetic tone target genes in long bones of 3-month-old TgSirt1 mice (n = 6) versus WT controls (n = 6). (K) N.Ob/T.Ar (/mm²); (L) Oc.S/BS (%); (M) BV/TV (%); and (N) BFR/BS (μm³/μm²/yr) of 3-month-old TgSirt1 and WT mice treated with propranolol (WT: n = 5; TgSirt1: n = 5; WT/propranolol: n = 5; TgSirt1/propranolol: n = 5). (O) Representative images of spines from 3-month-old TgSirt1 and WT mice treated with propranolol stained with von Kossa. Data are represented as mean ± SEM. (A–J) *P < 0.05, TgSirt1 versus WT by Student’s t test. (K–N) *P < 0.05, TgSirt1 treated with propranolol versus TgSirt1 by 1-way ANOVA.

Figure 2. Neuronal SIRT1 regulates SNS activity and controls bone mass.

(A) SIRT1 immunostaining in brain sections of WT mice including brain stem (left panel) and locus coeruleus (right panel). Scale bars: 100 μm. Bright field images on the left demonstrate the region of the brain under study and the coordinates in mouse brain atlas. (B) GFP immunostaining in hypothalamus, brain stem, and locus coeruleus sections of Adeno-CMV-Cre i.c.v. injected Sirt1^COIN/COIN (Sirt1_brain^–/–) mice. Scale bars: 100 μm. (C) Ucp1 expression levels in BAT of Adeno-CMV-Cre i.c.v. injected 3-month-old Sirt1^COIN/COIN (Sirt1_brain^–/–) mice (n = 4) versus Sirt1^COIN/COIN controls (n = 4). (D) BV/TV (%); (E) N.Ob/T.Ar (/mm²); (F) BFR/BS (μm³/μm²/yr); and (G) Oc.S/BS (%) of 3-month-old Sirt1_brain^–/– mice (n = 5) versus Sirt1^COIN/COIN controls (n = 5). (H) Representative images of spines from 3-month-old Sirt1_brain^–/– mice versus Sirt1^COIN/COIN controls stained with von Kossa. Data are represented as mean ± SEM. *P < 0.05 versus Sirt1^COIN/COIN by Student’s t test.

Figure 3. Decreased serotonin synthesis and increased Dbh and MAO-A expression in the brain of TgSirt1 mice.

(A) Tph2 expression levels in the brain stem (BS) of 3-month-old TgSirt1 mice (n = 5) versus WT controls (n = 5). (B) 5HT levels in brain stem of TgSirt1 mice (n = 6) versus WT controls (n = 4) measured by HPLC. (C) MAO-A expression levels in the rest of brain (ROB) of 3-month-old TgSirt1 mice (n = 4) versus WT controls (n = 4). (D) MAO-A activity (10³ RLU/μg protein/h) in the hypothalamus, brain stem, and rest of brain of TgSirt1 mice treated with phenelzine (n = 5) versus vehicle controls (n = 5). (E) 5HT and 5-HIAA levels in brain stem and rest of brain of TgSirt1 mice treated with phenelzine (n = 5) versus vehicle controls (n = 5) measured by HPLC. (F) BV/TV (%); (G) N.Ob/T.Ar (/mm²); (H) BFR/BS (μm³/μm²/yr); and (I) Oc.S/BS (%) of 3-month-old TgSirt1 mice treated with phenelzine (n = 5) versus vehicle (n = 6) and WT controls (n = 5). (J) Representative images of spines from 3-month-old TgSirt1 mice treated with phenelzine versus vehicle and WT controls stained with von Kossa. (K) Dbh expression levels in midbrain (MB) of 3-month-old TgSirt1 mice (n = 5) versus WT controls (n = 5). (L) Tph2 expression levels in brain stem of 3-month-old Sirt1_brain^–/– mice (n = 4) versus Sirt1^COIN/COIN controls (n = 4). (M) MAO-A expression levels in rest of brain of 3-month-old Sirt1_brain^–/– mice (n = 4) versus Sirt1^COIN/COIN controls (n = 4). (N) Dbh expression levels in MB of 3-month-old Sirt1_brain^–/– mice (n = 4) versus Sirt1^COIN/COIN controls (n = 4). Data are represented as mean ± SEM. (A–E and K–N) *P < 0.05, Student’s t test. (F–I) *P < 0.05, TgSirt1 mice treated with phenelzine versus vehicle by 1-way ANOVA.

Figure 4. Inactivation of Sirt1 in serotonergic and MAO-A–expressing neurons, but not in the locus coeruleus, increases bone mass in spines of male mice.

(A) BV/TV (%); (B) N.Ob/T.Ar (/mm²); (C) BFR/BS (μm³/μm²/yr); and (D) Oc.S/BS (%) of 3-, 12-, and 18-month-old male Sirt1_Syn^–/– mice (3 months: n = 9; 12 months: n = 6; 18 months: n = 5) versus Sirt1^COIN/COIN controls (3 months: n = 8; 12 months: n = 5; 18 months: n = 7). (E) Representative images of spines from 3-, 12-, and 18-month-old male Sirt1_Syn^–/– mice versus Sirt1^COIN/COIN controls, stained with von Kossa. (F) BV/TV (%); (G) N.Ob/T.Ar (/mm²); (H) BFR/BS (μm³/μm²/yr); and (I) Oc.S/BS (%) of 3-, 12-, and 18-month-old male Sirt1_Sert^–/– mice (3 months: n = 8; 12 months: n = 8; 18 months: n = 9) versus Sirt1^COIN/COIN controls (3 months: n = 6; 12 months: n = 5; 18 months: n = 8). (J) Representative images of spines from 3-, 12-, and 18-month-old male Sirt1_Sert^–/– mice versus Sirt1^COIN/COIN controls stained with von Kossa. (K) BV/TV (%); (L) N.Ob/T.Ar (/mm²); (M) BFR/BS (μm³/μm²/yr); and (N) Oc.S/BS (%) of 3- and 12- month-old male Sirt1_Dbh^–/– mice (3 months: n = 5; 12 months: n = 7) versus Sirt1^COIN/COIN controls (3 months: n = 5; 12 months: n = 5). (O) Representative images of spines from 3- and 12-month-old male Sirt1_Dbh^–/– mice versus Sirt1^COIN/COIN controls stained with von Kossa. Data are represented as mean ± SEM. *P < 0.05 versus Sirt1^COIN/COIN by Student’s t test.

Figure 5. Neuronal SIRT1 decreases bone mass by decreasing serotonin synthesis and enhancing its catabolism through its actions on serotonergic and MAO-A–expressing neurons.

(A) Ucp1 expression in BAT of Sirt1_Syn^–/– mice (n = 7) versus controls (n = 7). (B) Expression of SNS target genes in long bone of Sirt1_Syn^–/– mice (n = 5) versus controls (n = 5). (C) Tph2 expression in brain stem of Sirt1_Syn^–/– mice (n = 7) versus controls (n = 7). (D) MAO-A expression and (E) MAO-A activity in rest of brain of Sirt1_Syn^–/– mice (n = 5) versus controls (n = 5). (F) Dbh expression in MB of Sirt1_Syn^–/– mice (n = 5) versus controls (n = 5). (G) Bche expression in hypothalamus of Sirt1_Syn^–/– mice (n = 6) versus controls (n = 6). (H) Ucp1 expression in BAT of Sirt1_Sert^–/– mice (n = 4) versus controls (n = 8). (I) Expression of SNS target genes in long bone of Sirt1_Sert^–/– mice (n = 4) versus controls (n = 4). (J) Tph2 expression in brain stem of Sirt1_Sert^–/– mice (n = 4) versus controls (n = 6). (K) MAO-A expression in rest of brain of Sirt1_Sert^–/– mice (n = 4) versus controls (n = 8). (L) MAO-A activity in rest of brain of Sirt1_Sert^–/– mice (n = 5) versus controls (n = 5). (M) Dbh expression in MB of Sirt1_Sert^–/– mice (n = 4) versus controls (n = 5). (N) Bche expression in hypothalamus of Sirt1_Sert^–/– mice(n = 4) versus controls (n = 4). (O) Ucp1 expression in BAT of Sirt1_Dbh^–/– mice (n = 5) versus controls (n = 5). (P) Expression of SNS target genes in long bone of Sirt1_Dbh^–/– mice (n = 5) versus controls (n = 5). (Q) Tph2 expression in brain stem of Sirt1_Dbh^–/– mice (n = 4) versus controls (n = 5). (R) MAO-A expression in rest of brain of Sirt1_Dbh^–/– mice (n = 4) versus controls (n = 5). (S) MAO-A activity in rest of brain of Sirt1_Dbh^–/– mice (n = 5) versus controls (n = 5). (T) Dbh expression in MB and (U) Bche expression in hypothalamus of Sirt1_Dbh^–/– (n = 4) versus controls (n = 5). Data are represented as mean ± SEM. *P < 0.05 versus Sirt1^COIN/COIN by Student’s t test.

Figure 6. Central effects of SIRT1 are dominant over its peripheral direct effects in bone cells on the regulation of bone mass in the aging skeleton.

(A) BV/TV (%); (B) N.Ob/T.Ar (/mm²); (C) BFR/BS (μm³/μm²/yr); and (D) Oc.S/BS (%) of 18-month-old Adeno-CMV-Cre i.c.v. injected Sirt1_ob^–/– mice (n = 5) versus vehicle (n = 5) and Sirt1^COIN/COIN controls (n = 6). (E) Representative images of spines from 18-month-old Adeno-CMV-Cre i.c.v. injected Sirt1_ob^–/– mice versus vehicle and Sirt1^COIN/COIN controls stained with von Kossa. (F) BV/TV (%); (G) N.Ob/T.Ar (/mm²); (H) BFR/BS (μm³/μm²/yr); and (I) Oc.S/BS (%) of 18-month-old Adeno-CMV-Cre i.c.v. injected Sirt1_oc^–/– mice (n = 4) versus vehicle (n = 4) and Sirt1^COIN/COIN controls(n = 5). (J) Representative images of spines from 18-month-old Adeno-CMV-Cre i.c.v. injected Sirt1_oc^–/– mice versus vehicle and Sirt1^COIN/COIN controls stained with von Kossa. Data are represented as mean ± SEM. *P < 0.05, Adeno-CMV-Cre i.c.v. injected Sirt1_ob^–/– or Sirt1_oc^–/– mice versus vehicle by 1-way ANOVA.