In the last decades, there is an increasing interest in the use of biomolecules, such as peptides, as therapeutic agents. One important challenge inherent to peptides and proteins is the occurrence ...of aspecific adsorption to all kind of surfaces, hampering drug research and development. In recent years, a lot of research was already performed highlighting adsorption during dissolution, sample pretreatment and analysis. However, only limited data is available about the loss of peptide caused by adsorption to the in vivo administration setup. For this communication we assessed the adsorption of three neuropeptides in glass and plastic syringes by measuring the neuropeptide concentration with nanoUHPLC–MS/MS as a function of time, delivered by the syringe at a flow rate of 2 μL/min. To create a more fundamental approach, three different infusion solvents were examined. Our results clearly show that—although peptide dependent—an important influence of the kind of syringe material and perfusion solvent can be seen on peptide recovery. In addition, we discuss different strategies, and their corresponding in vivo difficulties, to increase peptide recovery during administration.
The neuromedin peptides are peripherally and centrally produced, but until now, it is generally believed that they only function as locally acting compounds without any quantitative knowledge about ...their blood-brain barrier (BBB) passage. Here, we characterize the transport kinetics of four neuromedins (NMU, NMN, NMB and NT) across the BBB, as well as their metabolization profile, and evaluate if they can act as endocrine hormones.
Using the in vivo mouse model, multiple time regression (MTR), capillary depletion (CD) and brain efflux studies were performed. Data was fitted using linear (NMU, NT and NMB) or biphasic modeling (NMU and NMN). Three of the four investigated peptides, i.e. NMU, NT and NMN, showed a significant influx into the brain with unidirectional influx rate constants of 1.31 and 0.75 μL/(g × min) for NMU and NT respectively and initial influx constants (K1) of 72.14 and 7.55 μL/(g × min) and net influx constants (K) of 1.28 and 1.36 × 10−16 μL/(g×min) for NMU and NMN respectively. The influx of NMB was negligible. Only NMN and NT showed a significant efflux out of the brain with an efflux constant (kout) of 0.042 min−1 and 0.053 min−1 respectively.
Our results indicate that locally produced neuromedin peptides and/or fragments can be transported through the whole body, including passing the BBB, and taken up by different organs/tissues, supporting the idea that the neuromedins could have a much bigger role in the regulation of biological processes than currently assumed.
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•The peptides NMU, NT and NMN show a clear influx from the blood to the brain.•The peptides that enter the brain capillaries continue to the brain parenchyma.•NMN and NT show an efflux from the brain to the blood.•The stability of all peptides was evaluated and the metabolites identified.
Neuromedin U (NMU) is an evolutionary conserved neuropeptide that has been implicated in multiple processes, such as circadian regulation, energy homeostasis, reward processing and stress coping. ...Although the central expression of NMU has been addressed previously, the lack of specific and sensitive tools has prevented a comprehensive characterization of NMU-expressing neurons in the brain. We have generated a knock-in mouse model constitutively expressing Cre recombinase under the
promoter. We have validated the model using a multi-level approach based on quantitative reverse-transcription polymerase chain reactions,
hybridization, a reporter mouse line and an adenoviral vector driving Cre-dependent expression of a fluorescent protein. Using the Nmu-Cre mouse, we performed a complete characterization of NMU expression in adult mouse brain, unveiling a potential midline NMU modulatory circuit with the ventromedial hypothalamic nucleus (VMH) as a key node. Moreover, immunohistochemical analysis suggested that NMU neurons in the VMH mainly constitute a unique population of hypothalamic cells. Taken together, our results suggest that Cre expression in the Nmu-Cre mouse model largely reflects NMU expression in the adult mouse brain, without altering endogenous NMU expression. Thus, the Nmu-Cre mouse model is a powerful and sensitive tool to explore the role of NMU neurons in mice.
Bidirectional communication between neurons and astrocytes shapes synaptic plasticity and behavior. D-serine is a necessary co-agonist of synaptic N-methyl-D-aspartate receptors (NMDARs), but the ...physiological factors regulating its impact on memory processes are scantly known. We show that astroglial CB
receptors are key determinants of object recognition memory by determining the availability of D-serine at hippocampal synapses. Mutant mice lacking CB
receptors from astroglial cells (GFAP-CB
-KO) displayed impaired object recognition memory and decreased in vivo and in vitro long-term potentiation (LTP) at CA3-CA1 hippocampal synapses. Activation of CB
receptors increased intracellular astroglial Ca
levels and extracellular levels of D-serine in hippocampal slices. Accordingly, GFAP-CB
-KO displayed lower occupancy of the co-agonist binding site of synaptic hippocampal NMDARs. Finally, elevation of D-serine levels fully rescued LTP and memory impairments of GFAP-CB
-KO mice. These data reveal a novel mechanism of in vivo astroglial control of memory and synaptic plasticity via the D-serine-dependent control of NMDARs.
The neurobiological effects of repetitive transcranial magnetic stimulation are believed to run in part through the dopaminergic system. Accelerated high frequency rTMS (aHF-rTMS), a new form of ...stimuli delivery, is currently being tested for its usefulness in treating human and canine mental disorders. However, the short-and long-term neurobiological effects are still unclear, including the effects on the dopaminergic system. In aHF-rTMS, multiple sessions are delivered within 1 day instead of one session per day, not only to accelerate the time to response but also to increase clinical efficacy. To gain more insight into the neurobiology of aHF-rTMS, we investigated whether applying five sessions in 1 day has direct and/or delayed effects on the dopamine transporter (DAT), and on dopamine metabolites of cerebrospinal fluid (CSF) in beagles.
Thirteen beagles were randomly divided into two groups: five active stimulation sessions (
= 9), and 5 sham stimulation sessions (
= 4). Using DaTSCAN, DAT binding indices (BI) were obtained at baseline, after 1 day, 1 month, and 3 months post stimulation. CSF samples were collected after each scan.
Active aHF-rTMS significantly reduced striatal DAT BI 1 day post-active stimulation session (
< 0.01), and the effect lasted to 1 month (
< 0.01). No significant DAT BI change was found in sham group. No significant changes in dopamine metabolites of CSF were found.
Although no significant effects on CSF dopamine metabolites were observed, five sessions of active aHF-rTMS significantly decreased striatal DAT BI after 1 day and up to 1 month post stimulation, indicating immediate and delayed effects on the brain dopaminergic system. Our findings in healthy beagles further substantiate the assumption that (a)HF-rTMS affects the brain dopaminergic system and it may pave the way to apply (a)HF-rTMS treatment in behaviorally disturbed dogs.
Visual cortical areas show enhanced tactile responses in blind individuals, resulting in improved behavioral performance. Induction of unilateral vision loss in adult mice, by monocular enucleation ...(ME), is a validated model for such cross-modal brain plasticity. A delayed whisker-driven take-over of the medial monocular zone of the visual cortex is preceded by so-called unimodal plasticity, involving the potentiation of the spared-eye inputs in the binocular cortical territory. Full reactivation of the sensory-deprived contralateral visual cortex is accomplished by 7 weeks post-injury. Serotonin (5-HT) is known to modulate sensory information processing and integration, but its impact on cortical reorganization after sensory loss, remains largely unexplored. To address this issue, we assessed the involvement of 5-HT in ME-induced cross-modal plasticity and the 5-HT receptor (5-HTR) subtype used. We first focused on establishing the impact of ME on the total 5-HT concentration measured in the visual cortex and in the somatosensory barrel field. Next, the changes in expression as a function of post-ME recovery time of the monoamine transporter 2 (vMAT2), which loads 5-HT into presynaptic vesicles, and of the 5-HTR1A and 5-HTR3A were assessed, in order to link these temporal expression profiles to the different types of cortical plasticity induced by ME. In order to accurately pinpoint which 5-HTR exactly mediates ME-induced cross-modal plasticity, we pharmacologically antagonized the 5-HTR1A, 5-HTR2A and 5-HTR3A subtypes. This study reveals brain region-specific alterations in total 5-HT concentration, time-dependent modulations in vMAT2, 5-HTR1A and 5-HTR3A protein expression and 5-HTR antagonist-specific effects on the post-ME plasticity phenomena. Together, our results confirm a role for 5-HTR1A in the early phase of binocular visual cortex plasticity and suggest an involvement of 5-HTR2A and 5-HTR3A but not 5-HTR1A during the late cross-modal recruitment of the medial monocular visual cortex. These insights contribute to the general understanding of 5-HT function in cortical plasticity and may encourage the search for improved rehabilitation strategies to compensate for sensory loss.
Repetitive transcranial magnetic stimulation (rTMS) is thought to partly exert its antidepressant action through the serotonergic system. Accelerated rTMS may have the potential to result in similar ...but faster onset of clinical improvement compared to the classical daily rTMS protocols, but given that delayed clinical responses have been reported, the neurobiological effects of accelerated paradigms remain to be elucidated including on this neurotransmitter system. This sham-controlled study aimed to evaluate the effects of accelerated high frequency rTMS (aHF-rTMS) over the left frontal cortex on the serotonin transporter (SERT) in healthy beagle dogs. A total of twenty-two dogs were randomly divided into three unequal groups: five active stimulation sessions (five sessions in one day,
= 10), 20 active stimulation sessions (five sessions/day for four days,
= 8), and 20 sham stimulation sessions (five sessions/day for four days,
= 4). The SERT binding index (BI) was obtained at baseline, 24 h post stimulation protocol, one month, and three months post stimulation by a
CDASB PET scan. It was found that one day of active aHF-rTMS (five sessions) did not result in significant SERT BI changes at any time point. For the 20 sessions of active aHF-rTMS, one month after stimulation the SERT BI attenuated in the sgACC. No significant SERT BI changes were found after 20 sessions of sham aHF-rTMS. A total of four days of active aHF-rTMS modified sgACC SERT BI one month post-stimulation, explaining to some extent the delayed clinical effects of accelerated rTMS paradigms found in human psychopathologies.
Neuromedin U (NMU) is a multifunctional neuropeptide which is characterized by a high conservation through all species. Herein, we describe the synthesis of a novel set of NMU-analogs based on the ...truncated NMU-8. Through combination of previously reported modifications, an elaborate structure–activity relationship study was performed aiming for the development of peptides with an increased selectivity toward NMU receptor 1 (NMUR1). Compound 7 possessed the highest NMUR1 selectivity (IC50 = 0.54 nM, selectivity ratio = 5313) together with an increased potency (EC50 = 3.7 nM), an 18% increase of the maximal effect at NMUR1, and a higher resistance against enzymatic degradation as compared to the native NMU-8. The development of a potent NMUR1 agonist with extended half-life could represent an attractive tool to further unveil the role of NMUR1 in NMU signaling.
•Peptides are a fast growing class of new drug molecules.•Developing bioanalytical methods for peptides remains complicated.•Proper solubilization is primordial.•Challenges and progresses in green ...analytical method development are discussed.
Peptide-based biopharmaceuticals represent one of the fastest growing classes of new drug molecules. New reaction types included in the synthesis strategies to reduce the rapid metabolism of peptides, along with the availability of new formulation and delivery technologies, resulted in an increased marketing of peptide drug products. In this regard, the development of analytical methods for quantification of peptides in pharmaceutical and biological samples is of utmost importance. From the sample preparation step to their analysis by means of chromatographic or electrophoretic methods, many difficulties should be tackled to analyze them. Recent developments in analytical techniques emphasize more and more on the use of green analytical techniques. This review will discuss the progresses in and challenges observed during green analytical method development for the quantification of peptides in pharmaceutical and biological samples.
Parkinson's disease (PD) is a neurodegenerative disorder, characterized by a loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Caloric restriction (CR) has been shown to ...exert ghrelin-dependent neuroprotective effects in the 1-methyl-4-phenyl-1,2,3,6-tetrathydropyridine (MPTP)-based animal model for PD. We here investigated whether CR is neuroprotective in the lactacystin (LAC) mouse model for PD, in which proteasome disruption leads to the destruction of the DA neurons of the SNc, and whether this effect is mediated via the ghrelin receptor. Adult male ghrelin receptor wildtype (WT) and knockout (KO) mice were maintained on an ad libitum (AL) diet or on a 30% CR regimen. After 3 weeks, LAC was injected unilaterally into the SNc, and the degree of DA neuron degeneration was evaluated 1 week later. In AL mice, LAC injection significanty reduced the number of DA neurons and striatal DA concentrations. CR protected against DA neuron degeneration following LAC injection. However, no differences were observed between ghrelin receptor WT and KO mice. These results indicate that CR can protect the nigral DA neurons from toxicity related to proteasome disruption; however, the ghrelin receptor is not involved in this effect.
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