Darios ES, Winner BM, Charvat T, Krasinksi A, Punna S, Watts SW. The adipokine chemerin amplifies electrical field-stimulated contraction in the isolated rat superior mesenteric artery. Am J Physiol Heart Circ Physiol 311: H498 -H507, 2016. First published July 1, 2016; doi:10.1152/ajpheart.00998.2015.-The adipokine chemerin causes arterial contraction and is implicated in blood pressure regulation, especially in obese subjects with elevated levels of circulating chemerin. Because chemerin is expressed in the perivascular adipose tissue (PVAT) that surrounds the sympathetic innervation of the blood vessel, we tested the hypothesis that chemerin (endogenous and exogenous) amplifies the sympathetic nervous system in mediating electrical field-stimulated (EFS) contraction. The superior mesenteric artery, with or without PVAT and with endothelium and sympathetic nerve intact, was mounted into isolated tissue baths and used for isometric contraction and stimulation. Immunohistochemistry validated a robust expression of chemerin in the PVAT surrounding the superior mesenteric artery. EFS (0.3-20 Hz) caused a frequency-dependent contraction in isolated arteries that was reduced by the chemerin receptor ChemR23 antagonist CCX832 alone (100 nM; with, but not without, PVAT), but not by the inactive congener CCX826 (100 nM). Exogenous chemerin-9 (1 M)-amplified EFSinduced contraction in arteries (with and without PVAT) was blocked by CCX832 and the ␣-adrenergic receptor antagonist prazosin. CCX832 did not directly inhibit, nor did chemerin directly amplify, norepinephrine-induced contraction. Whole mount immunohistochemical experiments support colocalization of ChemR23 with the sympathetic nerve marker tyrosine hydroxylase in superior mesenteric PVAT and, to a lesser extent, in arteries and veins. These studies support the idea that exogenous chemerin modifies sympathetic nervemediated contraction through ChemR23 and that ChemR23 may be endogenously activated. This is significant because of the wellappreciated role of the sympathetic nervous system in blood pressure control. BECAUSE OF ITS LOCATION, the perivascular adipose tissue (PVAT) has the potential to affect the function of the blood vessels it encases. This can occur by secretion of adipokines that directly affect vascular tone, such as the relaxant adiponectin (25). We recently discovered the protein chemerin in PVAT and demonstrated that a shorter chemerin agonist, chemerin-9, caused direct arterial contraction through activation of the best-characterized chemerin receptor, ChemR23 (3,8,27,33,38,48). Chemerin is secreted from the liver and fat depots (10), functioning as an adipokine that regulates adipogenesis (12,31,32) and as an activator of inflammatory cells (13,50,52). Circulating levels of chemerin are positively associated with body mass index (1,4,9,16,36,37,40). Because of our long-term interest in understanding whether chemerin could play a role in obesity-associated hypertension, we turned our attention to a means by which arterial function could be m...

Botulinum neurotoxin (BoNT) serotype E is one of three serotypes that cause the preponderance of human botulism cases and is a Tier 1 Select Agent. BoNT/E is unusual among BoNT serotypes for its rapid onset and short duration of intoxication. Here we report two large panels of unique, unrelated camelid single-domain antibodies (VHHs) that were selected for their ability to bind to BoNT/E holotoxin and/or to the BoNT/E light chain protease domain (LC/E). The 19 VHHs which bind to BoNT/E were characterized for their subunit specificity and 8 VHHs displayed the ability to neutralize BoNT/E intoxication of neurons. Heterodimer antitoxins consisting of two BoNT/E-neutralizing VHHs, including one heterodimer designed using structural information for simultaneous binding, were shown to protect mice against co-administered toxin challenges of up to 500 MIPLD50. The 22 unique VHHs which bind to LC/E were characterized for their binding properties and 9 displayed the ability to inhibit LC/E protease activity. Surprisingly, VHHs selected on plastic-coated LC/E were virtually unable to recognize soluble or captured LC/E while VHHs selected on captured LC/E were poorly able to recognize LC/E coated to a plastic surface. This panel of anti-LC/E VHHs offer insight into BoNT/E function, and some may have value as components of therapeutic antidotes that reverse paralysis following BoNT/E exposures.

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This study focused on the solubility enhancement and the in vivo antidotal efficacy testing of a new potential cyanide (CN) countermeasure, dimethyl trisulfide (DMTS). Various FDA approved cyclodextrins (HPβCD, RMβCD, HPγCD), cosolvents (ethanol, polyethylene glycols, propylene glycol), surfactants (cremophor EL, cremophor RH 40, sodium cholate, sodium deoxycholate, polysorbate 80) and their combinations were applied. Based on the solubility enhancing potential of the tested systems, polysorbate 80 was chosen for further in vivo efficacy studies. A composition comprising 15% polysorbate 80 and 50 mg/ml DMTS with the applied DMTS dose of 100 mg/kg provided a therapeutic antidotal protection of 3.4 × LD. For comparison, the present therapy of sodium thiosulfate (TS) with the dose of 100 mg/kg provided only 1.1 × LD protection, and at the dose of 200 mg/kg, the LD was enhanced by 1.3 times. No difference in the therapeutic protection by DMTS was detected when the concentration of polysorbate 80 was increased to 20% (3.2 × LD protection). These data demonstrate the potential importance of DMTS as a CN countermeasure, and the formulation comprising polysorbate 80 provides the base of an injectable intramuscular dosage form that can later serve as a CN antidotal kit suitable for mass scenario.

Parkinson disease (PD) is prevalent in elderly individuals and is characterized by selective degeneration of nigrostriatal dopamine (NSDA) neurons. Interestingly, not all dopamine (DA) neurons are affected equally by PD and aging, particularly mesolimbic (ML) DA neurons. Here, effects of aging were examined on presynaptic DA synthesis, reuptake, metabolism and neurotoxicant susceptibility of NSDA and mesolimbic dopamine (MLDA) neurons and astrocyte DA metabolism. There were no differences in phenotypic markers of DA synthesis, reuptake or metabolism in NSDA or MLDA neurons in aged mice, but MLDA neurons displayed lower DA stores. Astrocyte metabolism of DA to 3-methoxytyramine (3-MT) in the striatum was decreased in aged mice, but was maintained in the nucleus accumbens. Despite diminished DA vesicular storage capacity in MLDA neurons, susceptibility to acute neurotoxicant exposure was similar in young and aged mice. These results reveal an age- and neurotoxicant-induced impairment of DA metabolic activity in astrocytes surrounding susceptible NSDA neurons as opposed to maintenance of DA metabolism in astrocytes surrounding resistant MLDA neurons, and suggest a possible therapeutic target for PD.

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A pathological hallmark of Parkinson disease (PD) is progressive degeneration of nigrostriatal dopamine (NSDA) neurons, which underlies the motor symptoms of PD. While there is severe loss of midbrain NSDA neurons, tuberoinfundibular (TI) DA neurons in the mediobasal hypothalamus (MBH) remain intact. In the present study, confocal microscopic analysis revealed that mitochondrial content and numbers of mitophagosomes were lower in NSDA neuronal cell bodies in the substantia nigra pars compacta (SNpc) compared to TIDA neuronal cell bodies in the arcuate nucleus (ARC) of C57BL/6J male mice. Mitochondrial respiration, mass, membrane potential and morphology were determined using bioenergetic, flow cytometric and transmission electron microscopic analyses of synaptosomes isolated from discrete brain regions containing axon terminals of NSDA and TIDA neurons. Maximum and spare respiratory capacities, and mitochondrial mass were lower in synaptosomal mitochondria derived from the striatum (ST) as compared with the MBH, which correlated with lower numbers of mitochondria per synaptosome in these brain regions. In contrast, there was no regional difference in mitochondrial basal, maximum or spare respirations following inhibition of Complex I activity with rotenone. These results reveal that higher numbers of viable mitochondria are correlated with more extensive autophagic mitochondrial quality maintenance in TIDA neurons as compared with NSDA neurons.

Botulinum neurotoxins (BoNTs) are highly potent, neuroparalytic protein toxins that block the release of acetylcholine from motor neurons and autonomic synapses. The unparalleled toxicity of BoNTs results from the highly specific and localized cleavage of presynaptic proteins required for nerve transmission. Currently, the only pharmacotherapy for botulism is prophylaxis with antitoxin, which becomes progressively less effective as symptoms develop. Treatment for symptomatic botulism is limited to supportive care and artificial ventilation until respiratory function spontaneously recovers, which can take weeks or longer. Mechanistic insights into intracellular toxin behavior have progressed significantly since it was shown that toxins exploit synaptic endocytosis for entry into the nerve terminal, but fundamental questions about host-toxin interactions remain unanswered. Chief among these are mechanisms by which BoNT is internalized into neurons and trafficked to sites of molecular toxicity. Elucidating how receptor-bound toxin is internalized and conditions under which the toxin light chain engages with target SNARE proteins is critical for understanding the dynamics of intoxication and identifying novel therapeutics. Here, we discuss the implications of newly discovered modes of synaptic vesicle recycling on BoNT uptake and intraneuronal trafficking.