Norepinephrine transporter analog benzylguanidine-conjugated nanoparticles for the delivery of paclitaxel in neuroblastoma

Aim: We previously synthesized a polyethylene glycol-based norepinephrine transporter-targeted agent, BG-P-TAT, which has a benzylguanidine and a triazolyl-tetrac group. This targeted conjugate showed suppression of neuroblastoma tumor progression. In this study we aimed to synthesize nanoparticles to encapsulate the chemotherapeutic agent paclitaxel for targeting neuroblastoma tumors by using benzylguanidine so that it can compete with norepinephrine for uptake by neuroendocrine cells. Methods: Biocompatible poly(lactide-co-glycolic acid)-polyethylene glycol was chosen to prepare targeted nanoparticles for safe delivery of the chemotherapy agent paclitaxel. Result: Paclitaxel concentration was 60% higher in neuroblastoma tumors of mice treated with paclitaxel encapsulated in targeted nanoparticles than with non-targeted nanoparticles. Conclusion: These findings support the targeted delivery of paclitaxel as a chemotherapeutic agent for neuroblastoma.

Variable Response of Norepinephrine Transporter to Traumatic Stress and Relationship to Hyperarousal

The noradrenergic systems play a key role in stress triggered disorders such as post-traumatic stress disorder (PTSD). We hypothesized that traumatic stress will alter expression of norepinephrine transporter (NET) in locus coeruleus (LC) and its target brain regions which could be related to hyperarousal. Male Sprague-Dawley rats were subjected to single prolonged stress (SPS) and several weeks later the LC was isolated. NET mRNA levels in LC, determined by RT-PCR, displayed variable response with high and low responsive subgroups. In different cohort, acoustic startle response (ASR) was measured 2 weeks after SPS and levels of NET mRNA and protein in LC determined. The high NET responsive subgroup had greater hyperarousal. Nevertheless, NET protein levels, as determined by western blots, were lower than unstressed controls in LC, ventral hippocampus and medial prefrontal cortex and displayed considerable variability. Hypermethylation of specific CpG region in promoter of SLC6A2 gene, encoding NET, was present in the low, but not high, NET mRNA responsive subgroup. Taken together, the results demonstrate variability in stress elicited changes in NET gene expression and involvement of epigenetic changes. This may underlie mechanisms of susceptibility and resilience to traumatic stress triggered neuropsychiatric symptoms, especially hyperarousal.

Autoradiographic localization of [3H]-Nisoxetine binding sites in the CNS of male and female Japanese quail

Background In the central nervous system of mammals, transporters localized on the presynaptic nerve terminals regulate the reuptake of neurotransmitters. These transporters are selective for a specific neurotransmitter such as dopamine (DA) and norepinephrine (NE). Specifically in the synapse, the dopamine transporter (DAT) reuptakes DA and the norepinephrine transporter (NET) reuptakes NE. However previous research has found that avian species do not have a gene for DAT, and therefore, birds may be using the NET to clear both NE and DA from the synapse. The current study aimed to extend this finding by localizing NET expression in male and female Japanese quail (Coturnix japonica) brains using [3H]Nisoxetine, a selective NET blocker. Results High densities of binding sites were observed in the olfactory tubercle (OTu), the medial striatum (MSt), and the lateral striatum (LSt). Lower densities of binding sites were detected in the amygdala (AMY) and hypothalamus (Hyp), and low binding was found in the medial preoptic area (mPOA) and the pallium. Conclusion The areas with the highest densities of NET are also areas that previous research has shown to have high levels of DA activity but low levels of NE innervation (e.g. striatum). The distribution of this reuptake transporter is consistent with the theory that NET acts to clear both DA and NE from the synapse.

Neurokinin-1 Antagonism Distinguishes the Role of Norepinephrine Transporter from Dopamine Transporter in Mediating Amphetamine Behaviors

<b><i>Background:</i></b> Amphetamine (AMPH) and other psychostimulants act on the norepinephrine (NE) transporter (NET) and the dopamine (DA) transporter (DAT) and enhance NE and DA signaling. Both NET and DAT share anatomical and functional characteristics and are regulated similarly by psychostimulants and receptor-linked signaling pathways. We and others have demonstrated that NET and DAT are downregulated by AMPH and substance P/neurokinin-1 receptor (NK1R)-mediated protein kinase C pathway. <b><i>Objectives:</i></b> Since both NET and DAT are downregulated by AMPH and NK1R activation and share high sequence homology, the objective of the study was to determine the catecholamine transporter specificity in NK1R modulation of AMPH-induced behaviors. <b><i>Methods:</i></b> The effect of NK1R antagonism on AMPH-induced conditioned place preference (CPP) as well as AMPH-induced NET and DAT downregulation was examined using NET and DAT knockout mice (NET-KO and DAT-KO) along with their wild-type littermates. <b><i>Results:</i></b> Aprepitant (5 mg/kg i.p.) significantly attenuated AMPH (2 mg/kg i.p.)-induced CPP in the wild-type and DAT-KO but not in the NET-KO. Locomotor activity measured during the post-conditioning test (in the absence of AMPH) showed higher locomotor activity in DAT-KO compared to wild-type or NET-KO. However, the locomotor activity of all 3 genotypes remained unchanged following aprepitant. Additionally, in the ventral striatum of wild-type, the AMPH-induced downregulation of NET function and surface expression but not that of DAT was attenuated by aprepitant. <b><i>Conclusions:</i></b> The results from the current study demonstrate that aprepitant attenuates the expression of AMPH-induced CPP in DAT-KO mice but not in NET-KO mice suggesting a role for NK1R-mediated NET regulation in AMPH-induced behaviors.

The Role of the Second Extracellular Loop of Norepinephrine Transporter, Neurotrophin-3 and Tropomyosin Receptor Kinase C in T Cells: A Peripheral Biomarker in the Etiology of Schizophrenia

The neurobiology of schizophrenia is multifactorial, comprising the dysregulation of several biochemical pathways and molecules. This research proposes a peripheral biomarker for schizophrenia that involves the second extracellular loop of norepinephrine transporter (NEText), the tropomyosin receptor kinase C (TrkC), and the neurotrophin-3 (NT-3) in T cells. The study of NEText, NT-3, and TrkC was performed in T cells and plasma extracted from peripheral blood of 54 patients with schizophrenia and 54 healthy controls. Levels of NT-3, TrkC, and NET were significantly lower in plasma and T cells of patients compared to healthy controls. Co-immunoprecipitation (co-IPs) showed protein interactions with Co-IP NEText–NT-3 and Co-IP NEText–TrkC. Computational modelling of protein–peptide docking by CABS-dock provided a medium–high accuracy model for NT-3–NEText (4.6935 Å) and TrkC–NEText (2.1365 Å). In summary, immunocomplexes reached statistical relevance in the T cells of the control group contrary to the results obtained with schizophrenia. The reduced expression of NT-3, TrkC, and NET, and the lack of molecular complexes in T cells of patients with schizophrenia may lead to a peripheral dysregulation of intracellular signaling pathways and an abnormal reuptake of norepinephrine (NE) by NET. This peripheral molecular biomarker underlying schizophrenia reinforces the role of neurotrophins, and noradrenergic and immune systems in the pathophysiology of schizophrenia.

Predictors of the Pressor Response to the Norepinephrine Transporter Inhibitor, Atomoxetine, in Neurogenic Orthostatic Hypotension

We previously reported that the norepinephrine transporter inhibitor, atomoxetine, improved standing blood pressure and lightheadedness in patients with neurogenic orthostatic hypotension (nOH). The purpose of the present study was to determine the predictors of the pressor response to atomoxetine. Patients with nOH who participated in the clinical trials ( https://www.clinicaltrials.gov ; Unique identifiers: NCT00223691 and NCT01316666) were included in this retrospective analysis. All subjects underwent autonomic function testing, plasma norepinephrine, systolic, diastolic blood pressure, and symptoms assessments, whereas seated and standing, before, and 60 minutes after a single dose of atomoxetine 18 mg. A subset of 25 patients underwent iodine-123–labeled metaiodobenzylguanidine scanning to estimate the degree of cardiac sympathetic denervation. A total of 99 subjects with nOH (67±9 years old, 40 women) participated in the study, 35 with multiple system atrophy, 52 with pure autonomic failure, and 12 with Parkinson disease. The average orthostatic decrease in their systolic blood pressure/diastolic blood pressure was −52±26/−22±15 mm Hg. Supine plasma norepinephrine levels predicted the standing systolic blood pressure (adjusted R 2 was 0.12, F [3,80]=4.66, P =0.007) and diastolic blood pressure (adjusted R 2 was 0.18, F [3, 80]=7.04, P =0.001) in response to atomoxetine. The increase in systolic blood pressure after atomoxetine was associated with the decrease in nOH-related symptoms ( R 2 =0.14, F [1,44]=8.16 P =0.007). In conclusion, plasma norepinephrine was modestly associated with the pressor response to atomoxetine in patients with nOH. Additionally, the improvement in nOH-related symptoms was associated with the increase in the pressor response to atomoxetine.

Rare Variant in the SLC6A2 Encoding a Norepinephrine Transporter Is Associated with Elite Athletic Performance in the Polish Population

Numerous genetic factors have been shown to influence athletic performance, but the list is far from comprehensive. In this study, we analyzed genetic variants in two genes related to mental abilities, SLC6A2 (rs1805065) and SYNE1 (rs2635438) in a group of 890 athletes (320 endurance, 265 power, and 305 combat athletes) vs. 1009 sedentary controls. Genotyping of selected SNPs was performed using TaqMan SNP genotyping assays. SLC6A2 codes for norepinephrine transporter, a protein involved in modulating mood, arousal, memory, learning, and pain perception, while SYNE1 encodes protein important for the maintenance of the cerebellum—the part of the brain that coordinates complex body movements. Both SNPs (rs2635438 and rs1805065) showed no statistically significant differences between the frequencies of variants in the athletes and the sedentary controls (athletes vs. control group) or in the athlete subgroups (martial vs. control, endurance vs. control, and power vs. control). The rs1805065 T variant of SLC6A2 was found to be overrepresented in male high-elite martial sports athletes when compared to sedentary controls (OR = 6.56, 95%CI = 1.82–23.59, p = 0.010). This supports the hypothesis that genetic variants potentially affecting brain functioning can influence elite athletic performance and indicate the need for further genetic association studies, as well as functional analyses.