Changes and function of the sympathetic nervous system in the skeleton during aging

Abstract

The sympathetic nervous system is a major efferent pathway through which the central nervous system controls peripheral organ function. Genetic and pharmacologic evidence in mice indicated that stimulation of the β2 adrenergic receptor (β2AR) in osteoblasts promotes bone loss. However, considerably less evidence exist to understand the putative impact of endogenous norepinephrine (NE), released by sympathetic nerves, on bone homeostasis. In this study, we investigated the in vivo expression and activity of the norepinephrine transporter (NET), a membrane pump known to actively uptake NE from the extracellular space in presynaptic neurons but also in differentiated osteoblasts in vitro. We were able to detect active NET-specific NE uptake in mouse cortical bone compartment in vivo. This NE uptake was the highest in young mice and decreased with age, whereas NE bone content increased with age. NET expression in adult mouse bones was detected in osteocytes via immunofluorescence, and qPCR analyses revealed a reduction of expression with aging, consistent with the observed decrease in NE uptake in aging mice. NET immunofluorescence was also detected in human bone cortical osteocytes. Taking advantage of tissue-specific fluorescent reporter mice, we used CLARITY imaging and light sheet microscopy to visualize the 3D distribution of sympathetic fibers in whole mount preparations of bone tissues. These analyses allowed us to detect tyrosine hydroxylase (TH)-positive sympathetic nerve fibers penetrating the cortical bone, where NET+ osteocytes reside. Together, these results support the existence of an extraneuronal and osteocytic function of NET that may buffer the bone catabolic action of endogenous NE released by sympathetic nerves. They also suggest that failure of this homeostatic system might contribute to age-related bone loss.