Presynaptic synthesis of acetylcholine (ACh) requires a steady supply of choline, acquired by a plasma membrane, hemicholinium-3-sensitive (HC-3) choline transporter (CHT). A significant fraction of synaptic choline is recovered from ACh hydrolyzed by acetylcholinesterase (AChE) after vesicular release. Although antecedent neuronal activity is known to dictate presynaptic CHT activity, the mechanisms supporting this regulation are unknown. We observe an exclusive localization of CHT to cholinergic neurons and demonstrate that the majority of CHTs reside on small vesicles within cholinergic presynaptic terminals in the rat and mouse brain. Furthermore, immunoisolation of presynaptic vesicles with multiple antibodies reveals that CHT-positive vesicles carry the vesicular acetylcholine transporter (VAChT) and synaptic vesicle markers such as synaptophysin and Rab3A and also contain acetylcholine. Depolarization of synaptosomes evokes a Ca2+-dependent botulinum neurotoxin C-sensitive increase in the Vmax for HC-3-sensitive choline uptake that is accompanied by an increase in the density of CHTs in the synaptic plasma membrane. Our study leads to the novel hypothesis that CHTs reside on a subpopulation of synaptic vesicles in cholinergic terminals that can transit to the plasma membrane in response to neuronal activity to couple levels of choline re-uptake to the rate of ACh release.
Vanilloid receptor 1 (TRPV1) has been proposed to be the principal heat-responsive channel for nociceptive neurons. The skin of both rat and mouse receives major projections from primary sensory afferents that bind the plant lectin isolectin B4 (IB4). The majority of IB4-positive neurons are known to be heat-responsive nociceptors. Previous studies suggested that, unlike rat, mouse IB4-positive cutaneous afferents did not express TRPV1 immunoreactivity. Here, multiple antisera were used to confirm that mouse and rat have different distributions of TRPV1 and that TRPV1 immunoreactivity is absent in heat-sensitive nociceptors. Intracellular recording in TRPV1 Ϫ/Ϫ mice was then used to confirm that TRPV1 was not required for detecting noxious heat. TRPV1 Ϫ/Ϫ mice had more heatsensitive neurons, and these neurons had normal temperature thresholds and response properties. Moreover, in TRPV1 Ϫ/Ϫ mice, 82% of heat-responsive neurons did not express immunoreactivity for TRPV2, another putative noxious heat channel.
Most, if not all, nociceptor sensory neurons are dependent on nerve growth factor (NGF) during early embryonic development. A large subpopulation of these sensory neurons loses NGF dependency between embryonic day 16 and postnatal day 14 and become responsive to glial cell line-derived growth factor (GDNF), a member of the transforming growth factor beta (TGF-beta) family. To examine the survival and phenotypic effects of GDNF on sensory neurons in vivo, we generated transgenic mice that overexpress GDNF in the skin. GDNF-overexpresser mice had increased numbers of small unmyelinated sensory neurons that express the tyrosine kinase receptor Ret and bind the plant isolectin B4 (IB4). Surprisingly, in wild-type and transgenic mice, few ( approximately 2%) IB4-positive neurons expressed the vanilloid receptor VR1, a heat-sensitive receptor expressed by many IB4-positive neurons of the rat. Thus, in mouse, GDNF-dependent IB4-positive neurons must use a non-VR1 heat receptor. In addition, the behavior of GDNF-overexpresser animals to noxious heat or mechanical stimuli was indistinguishable from wild-type animals, indicating that, on a behavioral level, peripherally applied GDNF does not alter the sensitivity of the somatosensory system.
Sensory neurons in aging mammals undergo changes in anatomy, physiology and gene expression that correlate with reduced sensory perception. In this study we compared young and aged mice to identify proteins that might contribute to this loss of sensation. We first show using behavioral testing that thermal sensitivity in aged male and female mice is reduced. Expression of sodium channel (Nav1.8 and Nav1.9) and transient receptor potential vanilloid (TRPV) channels in DRG and peripheral nerves of young and old male mice was then examined. Immunoblotting and RT-PCR assays showed reduced Nav1.8 levels in aged mice. No change was measured in TRPV1 mRNA levels in DRG though TRPV1 protein appeared reduced in the DRG and peripheral nerves. The GFRα3 receptor, which binds the growth factor artemin and is expressed by TRPV1-positive neurons, was also decreased in the DRG of aged animals. These findings indicate that loss of thermal sensitivity in aging animals may result from a decreased level of TRPV1 and Nav1.8 and decreased trophic support that inhibits efficient transport of channel proteins to peripheral afferents.
At least two classes of neciceptors can be distinguished based on their growth factor requirements: glial cell-line derived neurotrophic factor (GDNF)- and nerve growth factor (NGF)-dependent primary afferent neurons. Based on numerous anatomical and biochemical differences, GDNF- and NGF-dependent neurons have been proposed to be involved in the development of different types of persistent pain. To examine this hypothesis we used two lines of transgenic mice that contained a supernormal number of either NGF- or GDNF-dependent neurons (referred to as NGF-OE and GDNF-OE mice, respectively). These mice were tested in a model of inflammatory pain (induced by injection of complete Freund's adjuvant) and neuropathic pain (using a spinal nerve ligation protocol). Contrary to expectations, neither line of transgenic mice became more hyperalgesic following induction of persistent pain. In fact, NGF-OE mice recovered more rapidly and became hypoalgesic despite extensive paw swelling in the inflammatory pain model. In the neuropathic pain model, only wildtype mice became hyperalgesic. Real-time PCR analysis showed that the NGF-OE and GDNF-OE mice exhibited changes in neuronal-specific mRNAs in the dorsal root ganglia but not the spinal cord dorsal horn. These results indicate that increasing the number of nociceptors results in potent compensatory mechanisms that may begin with changes in the sensory neurons themselves.
The DNAJB1-PRKACA fusion transcript is the oncogenic driver in fibrolamellar hepatocellular carcinoma, a lethal disease lacking specific therapies. This study reports on the identification, characterization, and immunotherapeutic application of HLA-presented neoantigens specific for the DNAJB1-PRKACA fusion transcript in fibrolamellar hepatocellular carcinoma. DNAJB1-PRKACA-derived HLA class I and HLA class II ligands induce multifunctional cytotoxic CD8+ and T-helper 1 CD4+ T cells, and their cellular processing and presentation in DNAJB1-PRKACA expressing tumor cells is demonstrated by mass spectrometry-based immunopeptidome analysis. Single-cell RNA sequencing further identifies multiple T cell receptors from DNAJB1-PRKACA-specific T cells. Vaccination of a fibrolamellar hepatocellular carcinoma patient, suffering from recurrent short interval disease relapses, with DNAJB1-PRKACA-derived peptides under continued Poly (ADP-ribose) polymerase inhibitor therapy induces multifunctional CD4+ T cells, with an activated T-helper 1 phenotype and high T cell receptor clonality. Vaccine-induced DNAJB1-PRKACA-specific T cell responses persist over time and, in contrast to various previous treatments, are accompanied by durable relapse free survival of the patient for more than 21 months post vaccination. Our preclinical and clinical findings identify the DNAJB1-PRKACA protein as source for immunogenic neoepitopes and corresponding T cell receptors and provide efficacy in a single-patient study of T cell-based immunotherapy specifically targeting this oncogenic fusion.
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