scholarly journals hNGF Peptides Elicit the NGF-TrkA Signalling Pathway in Cholinergic Neurons and Retain Full Neurotrophic Activity in the DRG Assay

Biomolecules ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 216 ◽  
Author(s):  
Viviana Triaca ◽  
Elena Fico ◽  
Valentina Sposato ◽  
Silvia Caioli ◽  
Maria Teresa Ciotti ◽  
...  
Keyword(s):  
Dorsal Root ◽  
Cholinergic Neurons ◽  
Early Gene ◽  
Low Molecular Weight ◽  
Primary Neurons ◽  
Brain Delivery ◽  
Drg Neurons ◽  
Neurotrophic Activity ◽  
Mapk Signalling

In the last decade, Nerve Growth Factor (NGF)-based clinical approaches have lacked specific and efficient Tyrosine Kinase A (TrkA) agonists for brain delivery. Nowadays, the characterization of novel small peptidomimetic is taking centre stage in preclinical studies, in order to overcome the main size-related limitation in brain delivery of NGF holoprotein for Central Nervous System (CNS) pathologies. Here we investigated the NGF mimetic properties of the human NGF 1–14 sequence (hNGF1–14) and its derivatives, by resorting to primary cholinergic and dorsal root ganglia (DRG) neurons. Briefly, we observed that: 1) hNGF1–14 peptides engage the NGF pathway through TrkA phosphorylation at tyrosine 490 (Y490), and activation of ShcC/PI3K and Plc-γ/MAPK signalling, promoting AKT-dependent survival and CREB-driven neuronal activity, as seen by levels of the immediate early gene c-Fos, of the cholinergic marker Choline Acetyltransferase (ChAT), and of Brain Derived Neurotrophic Factor (BDNF); 2) their NGF mimetic activity is lost upon selective TrkA inhibition by means of GW441756; 3) hNGF1–14 peptides are able to sustain DRG survival and differentiation in absence of NGF. Furthermore, the acetylated derivative Ac-hNGF1–14 demonstrated an optimal NGF mimetic activity in both neuronal paradigms and an electrophysiological profile similar to NGF in cholinergic neurons. Cumulatively, the findings here reported pinpoint the hNGF1–14 peptide, and in particular its acetylated derivative, as novel, specific and low molecular weight TrkA specific agonists in both CNS and PNS primary neurons.

scholarly journals Spatial transcriptomics reveals unique molecular fingerprints of human nociceptors

2021 ◽  
Author(s):  
Diana Tavares-Ferreira ◽  
Stephanie Shiers ◽  
Pradipta R. Ray ◽  
Andi Wangzhou ◽  
Vivekanand Jeevakumar ◽  
...  
Keyword(s):  
Sex Differences ◽  
Sensory Neurons ◽  
Dorsal Root ◽  
Expression Profiles ◽  
Organ Donors ◽  
Drg Neurons ◽  
Pain Mechanisms ◽  
Molecular Fingerprints ◽  
Sensory Disorders

AbstractSingle-cell transcriptomics on mouse nociceptors has transformed our understanding of pain mechanisms. Equivalent information from human nociceptors is lacking. We used spatial transcriptomics to molecularly characterize transcriptomes of single dorsal root ganglion (DRG) neurons from 8 organ donors. We identified 10 clusters of human sensory neurons, 6 of which are C nociceptors, 1 Aβ nociceptor, 1 Aδ, and 2 Aβ subtypes. These neuron subtypes have distinct expression profiles from rodents and non-human primates and we identify new markers for each of these subtypes that can be applied broadly in human studies. We also identify sex differences, including a marked increase in CALCA expression in female putative itch nociceptors. Our data open the door to new pain targets and unparalleled molecular characterization of clinical sensory disorders.One Sentence SummaryThree A-fiber mechanoreceptor and seven nociceptor subtypes are identified, revealing sex differences and unique aspects of human DRG neurons.

scholarly journals Fixative Composition Alters Distributions of Immunoreactivity for Glutaminase and Two Markers of Nociceptive Neurons, Nav 1.8 and TRPV1, in the Rat Dorsal Root Ganglion

2009 ◽  
Vol 58 (4) ◽  
pp. 329-344 ◽  
Author(s):  
E. Matthew Hoffman ◽  
Ruben Schechter ◽  
Kenneth E. Miller
Keyword(s):  
Dorsal Root Ganglion ◽  
Dorsal Root ◽  
Picric Acid ◽  
Detection Sensitivity ◽  
Formaldehyde Concentration ◽  
Drg Neurons ◽  
Nociceptive Neurons ◽  
Drg Neuron ◽  
Neuron Populations

Most, if not all, dorsal root ganglion (DRG) neurons use the neurotransmitter glutamate. There are, however, conflicting reports of the percentages of DRG neurons that express glutaminase (GLS), the enzyme that synthesizes glutamate, ranging from 30% to 100% of DRG neurons. Defining DRG neuron populations by the expression of proteins like GLS, which indicates function, is routinely accomplished with immunolabeling techniques. Proper characterization of DRG neuron populations relies on accurate detection of such antigens. It is known intuitively that fixation can alter immunoreactivity (IR). In this study, we compared the effects of five formaldehyde concentrations between 0.25% and 4.0% (w/v) and five picric acid concentrations between 0.0% and 0.8% (w/v) on the IR of GLS, the voltage-gated sodium channel 1.8 (Nav 1.8), and the capsaicin receptor TRPV1. We also compared the effects of five incubation time lengths from 2 to 192 hr, in primary anti-serum on IR. Lowering formaldehyde concentration elevated IR for all three antigens, while raising picric acid concentration increased Nav 1.8 and TRPV1 IR. Increasing IR improved detection sensitivity, which led to higher percentages of labeled DRG neurons. By selecting fixation conditions that optimized IR, we found that all DRG neurons express GLS, 69% of neurons express Nav 1.8, and 77% of neurons express TRPV1, indicating that some previous studies may have underestimated the percentages of DRG neurons expressing these proteins. This manuscript contains online supplemental material at http://www.jhc.org . Please visit this article online to view these materials.

Biopolymers with Medical Applications Optimized method for obtaining chitosan-based delivery systems

2018 ◽  
Vol 69 (7) ◽  
pp. 1756-1759 ◽  
Author(s):  
Luminita Confederat ◽  
Iuliana Motrescu ◽  
Sandra Constantin ◽  
Florentina Lupascu ◽  
Lenuta Profire
Keyword(s):  
Drug Delivery ◽  
Drug Delivery Systems ◽  
Average Diameter ◽  
Delivery Systems ◽  
Cross Linking ◽  
Low Molecular Weight ◽  
Polymeric Systems ◽  
Chitosan Microparticles ◽  
Degree Of Swelling

The aim of this study was to optimize the method used for obtaining microparticles based on chitosan � a biocompatible, biodegradable, and nontoxic polymer, and to characterize the developed systems. Chitosan microparticles, as drug delivery systems were obtained by inotropic gelation method using pentasodiumtripolyphosphate (TPP) as cross-linking agent. Chitosan with low molecular weight (CSLMW) in concentration which ranged between 0.5 and 5 %, was used while the concentration of cross-linking agent ranged between 1 and 5%. The characterization of the microparticles in terms of shape, uniformity and adhesion was performed in solution and dried state. The size of the microparticles and the degree of swelling were also determined. The structure and the morphology of the developed polymeric systems were analyzed by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM).The average diameter of the chitosan microparticles was around 522 �m. The most stable microparticles were obtained using CSLMW 1% and TPP 2% or CSLMW 0.75%and TPP 1%. The micropaticles were spherical, uniform and without flattening. Using CSLMW in concentration of 0.5 % poorly cross-linked and crushed microparticles have been obtained at all TPP concentrations. By optimization of the method, stable chitosan-based micropaticles were obtained which will be used to develop controlled release systems for drug delivery.

scholarly journals Expression of Cholinergic Markers and Characterization of Splice Variants during Ontogenesis of Rat Dorsal Root Ganglia Neurons

2021 ◽  
Vol 22 (11) ◽  
pp. 5499
Author(s):  
Veronica Corsetti ◽  
Carla Perrone-Capano ◽  
Michael Sebastian Salazar Intriago ◽  
Elisabetta Botticelli ◽  
Giancarlo Poiana ◽  
...  
Keyword(s):  
Dorsal Root Ganglia ◽  
Dorsal Root ◽  
Splice Variants ◽  
Multiple Roles ◽  
Pcr Analysis ◽  
Drg Neurons ◽  
Embryonic Developmental Stage ◽  
Embryo Stage ◽  
Dorsal Root Ganglia Neurons ◽  
Cholinergic Markers

Dorsal root ganglia (DRG) neurons synthesize acetylcholine (ACh), in addition to their peptidergic nature. They also release ACh and are cholinoceptive, as they express cholinergic receptors. During gangliogenesis, ACh plays an important role in neuronal differentiation, modulating neuritic outgrowth and neurospecific gene expression. Starting from these data, we studied the expression of choline acetyltransferase (ChAT) and vesicular ACh transporter (VAChT) expression in rat DRG neurons. ChAT and VAChT genes are arranged in a “cholinergic locus”, and several splice variants have been described. Using selective primers, we characterized splice variants of these cholinergic markers, demonstrating that rat DRGs express R1, R2, M, and N variants for ChAT and V1, V2, R1, and R2 splice variants for VAChT. Moreover, by RT-PCR analysis, we observed a progressive decrease in ChAT and VAChT transcripts from the late embryonic developmental stage (E18) to postnatal P2 and P15 and in the adult DRG. Interestingly, Western blot analyses and activity assays demonstrated that ChAT levels significantly increased during DRG ontogenesis. The modulated expression of different ChAT and VAChT splice variants during development suggests a possible differential regulation of cholinergic marker expression in sensory neurons and confirms multiple roles for ACh in DRG neurons, both in the embryo stage and postnatally.

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