Extracellular norepinephrine clearance by the norepinephrine transporter is required for skeletal homeostasis.

Changes in bone remodeling induced by pharmacological and genetic manipulation of ß-adrenergic receptor (ßAR) signaling in osteoblasts support a role of sympathetic nerves in the regulation of bone remodeling. However, the contribution of endogenous sympathetic outflow and nerve-derived norepinephrine (NE) to bone remodeling under pathophysiological conditions remains unclear. We show here that differentiated osteoblasts, like neurons, express the norepinephrine transporter (NET), exhibit specific NE uptake activity via NET and can catabolize, but not generate, NE. Pharmacological blockade of NE transport by reboxetine induced bone loss in WT mice. Similarly, lack of NE reuptake in norepinephrine transporter (Net)-deficient mice led to reduced bone formation and increased bone resorption, resulting in suboptimal peak bone mass and mechanical properties associated with low sympathetic outflow and high plasma NE levels. Last, daily sympathetic activation induced by mild chronic stress was unable to induce bone loss, unless NET activity was blocked. These findings indicate that the control of endogenous NE release and reuptake by presynaptic neurons and osteoblasts is an important component of the complex homeostatic machinery by which the sympathetic nervous system controls bone remodeling. These findings also suggest that drugs antagonizing NET activity, used for the treatment of hyperactivity disorders, may have deleterious effects on bone accrual.

Memory deficits, gait ataxia and neuronal loss in the hippocampus and cerebellum in mice that are heterozygous for Pur-alpha.

Pur-alpha is a highly conserved sequence-specific DNA and RNA binding protein with established roles in DNA replication, RNA translation, cell cycle regulation, and maintenance of neuronal differentiation. Prior studies have shown that mice lacking Pur-alpha (-/-) display decreased neurogenesis and impaired neuronal differentiation. We sought to examine for the first time, the behavioral phenotype and brain histopathology of mice that are heterozygous (+/-) for Pur-alpha. Standardized behavioral phenotyping revealed a decreased escape response to touch, limb and abdominal hypotonia, and gait abnormalities in heterozygous Pur-alpha (+/-) mice, compared to wild-type (+/+) littermates. Footprint pattern analyses showed wider-based steps, increased missteps and more outwardly rotated hindpaws in heterozygous Pur-alpha (+/-) mice, suggestive of cerebellar pathology. The Barnes maze and novel object location testing revealed significant memory deficits in heterozygous Pur-alpha mice, suggestive of hippocampal pathology. Quantitative immunohistochemical assays of the vermal region of the cerebellum and CA1-3 regions of the hippocampus revealed reduced numbers of neurons in general, as well as reduced numbers of Pur-alpha+-immunopositive neurons and dendrites in heterozygous Pur-alpha mice, compared to wild-type littermates. Past studies have implicated mutations in Pur-alpha in several diseases of brain development and neurodegeneration. When combined with these new findings, the Pur-alpha heterozygous knockout mice may provide an animal model to study mechanisms of and treatments for Pur-alpha-related cognitive deficiencies and neuropathology.

Norepinephrine transporter variant A457P knock-in mice display key features of human postural orthostatic tachycardia syndrome.

Postural orthostatic tachycardia syndrome (POTS) is a common autonomic disorder of largely unknown etiology that presents with sustained tachycardia on standing, syncope and elevated norepinephrine spillover. Some individuals with POTS experience anxiety, depression and cognitive dysfunction. Previously, we identified a mutation, A457P, in the norepinephrine (NE; also known as noradrenaline) transporter (NET; encoded by SLC6A2) in POTS patients. NET is expressed at presynaptic sites in NE neurons and plays a crucial role in regulating NE signaling and homeostasis through NE reuptake into noradrenergic nerve terminals. Our in vitro studies demonstrate that A457P reduces both NET surface trafficking and NE transport and exerts a dominant-negative impact on wild-type NET proteins. Here we report the generation and characterization of NET A457P mice, demonstrating the ability of A457P to drive the POTS phenotype and behaviors that are consistent with reported comorbidities. Mice carrying one A457P allele (NET(+/P)) exhibited reduced brain and sympathetic NE transport levels compared with wild-type (NET(+/+)) mice, whereas transport activity in mice carrying two A457P alleles (NET(P/P)) was nearly abolished. NET(+/P) and NET(P/P) mice exhibited elevations in plasma and urine NE levels, reduced 3,4-dihydroxyphenylglycol (DHPG), and reduced DHPG:NE ratios, consistent with a decrease in sympathetic nerve terminal NE reuptake. Radiotelemetry in unanesthetized mice revealed tachycardia in NET(+/P) mice without a change in blood pressure or baroreceptor sensitivity, consistent with studies of human NET A457P carriers. NET(+/P) mice also demonstrated behavioral changes consistent with CNS NET dysfunction. Our findings support that NET dysfunction is sufficient to produce a POTS phenotype and introduces the first genetic model suitable for more detailed mechanistic studies of the disorder and its comorbidities.

Novel and functional norepinephrine transporter protein variants identified in attention-deficit hyperactivity disorder.

Attention-deficit hyperactivity disorder (ADHD) is a highly heritable disorder of impaired behavioral inhibition, increased motor activity, and inattention. The norepinephrine transporter (NET, SLC6A2) represents an important candidate gene for contribution to ADHD because it regulates catecholamine extracellular and tissue concentrations and contributes to executive functions disrupted in ADHD, and NET is a target for most effective ADHD therapeutics. We identified four NET coding single nucleotide polymorphisms (SNPs) in two ADHD sample sets; two SNPs produce protein variants (T283M, V245I), one of which, T283M, is a novel variant. Examination of the maternal family members through whom the T283M mutation was transmitted, provided no additional ADHD diagnoses. Given the previous identification of a NET mutation that contributes to a familial tachycardia syndrome, we examined autonomic function to reveal in the proband the highest standing-induced increase in heart rate among the ADHD subjects examined. We measured [3H]NE and [3H]dopamine transport for T283M, V245I, and a previously identified NET variant, T283R. T283M and V245I demonstrated decreased substrate transport, as did T283R, suggesting that the T283 residue is sensitive to mutation. Identification of polymorphic sites within NET, specifically those that produce functional consequences, is one critical step in elucidating the genetic variation contributing to the heritable component of diseases such as ADHD.