Favorite Music Mediates Pain-related Responses in the Anterior Cingulate Cortex and Skin Pain Thresholds.

PURPOSE: Music therapy is widely used to enhance well-being, reduce pain, and distract patients from unpleasant symptoms in the clinical setting. However, the degree to which music modulates pain perception is unknown. The medial pain pathway including the limbic system is associated with emotion, but how music alters pathway activity is unclear. The aim of the study was to investigate pain thresholds and pain-related responses in the anterior cingulate cortex (ACC) and whether they were modulated when subjects listened to their favorite music genre. SUBJECTS AND METHODS: First, 30 subjects were examined for left forearm pain threshold using electrical stimulation with Pain Vision PS-2011N. The pain thresholds with and without music were compared. Second, when an 80-µA current from Pain Vision was applied to the left ankle of eight women, the pain-related responses of the ACC with and without music were observed with functional magnetic resonance device (fMRI). The changes in the pain-related activity in both parameters were discussed. RESULTS: The median pain threshold with favorite music was 38.9 µA, compared to 29.0 µA without, which was significantly different (p<0.0001). The men's thresholds were significantly higher than women's both with music (p<0.05) and without music (p<0.01). The pain threshold in women was more strongly affected by music than in men. The fMRI results showed that the pain-related response in the ACC in five of eight subjects was attenuated while they listened to their favorite music. No change was observed in the other three subjects. CONCLUSION: The present findings suggest that pain perception might be strongly affected by listening to favorite music, possibly through modulation of pain-related responses in the ACC.

Effect of single-nucleotide polymorphisms in TRPV1 on burning pain and capsaicin sensitivity in Japanese adults.

Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel that is expressed in the sensory neurons and responds to various noxious stimuli including heat and capsaicin. The molecular properties of TRPV1 have been clearly examined; however, there are obvious individual differences in human sensitivity to thermal stimuli and capsaicin. Here, we examined the possibility that different genome sequence of human TRPV1 caused the different sensitivity to heat or capsaicin. The sensitivities to burning pain and capsaicin of Japanese adult subjects were compared with their TRPV1 genome sequence, and we detected 6 single-nucleotide polymorphisms and 11 single-nucleotide polymorphisms related to burning pain and capsaicin sensitivity, respectively. In particular, homozygous I585V, a single-nucleotide polymorphism with amino acid substitution, significantly related to higher capsaicin sensitivity.

Distribution and Origin of VIP-, SP-, and Phospholipase Cß2 -Immunoreactive Nerves in the Tongue of the Bullfrog, Rana catesbeiana.

Previous studies have found a few intralingual ganglionic cells that were immunoreactive to vasoactive intestinal polypeptide (VIP) in the frog. A recent study reported a large number of such cells, and the possibility of the release of substance P (SP) from these. The aim of the present study was to investigate the distribution, origin, and colocalization of VIP- and SP- immunoreactive nerves in the tongue of the bullfrog, R. catesbeiana. In addition, the study also examined the colocalization of SP and phospholipase Cß2 (PLCß2 ) in the tongue and jugular ganglion. VIP immunoreactivity was seen in unipolar cells that were sparse in nerve bundles in the submucosal and muscle layers. The density of VIP-immunoreactive cells was approximately 4.8 cells/mm(3) . Their fibers terminated in the vicinity of the epithelial basal layer of the fungiform papillae. SP immunoreactivity was not seen in the VIP-immunoreactive cells, but was observed in pseudounipolar cells in the jugular ganglion. The SP fibers terminated close to the free surface, showing spindle- and button-like profiles. Transection of glossopharyngeal nerve resulted in the persistence of VIP-immunoreactive cells and the disappearance of SP-immunoreactive fibers in the tongue. SP immunoreactivity was co-expressed with PLCß2 in both the tongue and jugular ganglia. No PLCß2 immunoreactivity was seen in cells comprising the epithelial taste disk. These findings indicate that the origin of VIP nerve fibers are unipolar cells in the tongue, and SP and PLCß2 fibers originate from pseudounipolar cells that may be able to release SP primarily in the jugular ganglion. Anat Rec, 299:929-942, 2016. © 2016 Wiley Periodicals, Inc.

Alternation of gene expression in trigeminal ganglion neurons following complete Freund's adjuvant or capsaicin injection into the rat face.

The hyperexcitability of trigeminal ganglion (TG) neurons following inflammation or C-fiber stimulation is known to be involved in a variety of changes in gene expression in TG neurons, resulting in pain abnormalities in orofacial regions. We analyzed nocifensive behavior following complete Freund's adjuvant (CFA) or capsaicin injection into the maxillary whisker pad, and gene expression in the TG neurons using microarray analysis. The head-withdrawal latency to capsaicin injection or the head-withdrawal threshold to mechanical stimulation of the whisker pad skin in CFA-treated rats was significantly decreased compared to vehicle-treated rats. Many up-regulated and down-regulated genes in the TG neurons of each model were reported. Genes which have not been linked to peripheral inflammation or C-fiber activation were detected. Moreover, microarray chip containing a number of non-coding sequences was also up-regulated by C-fiber activation. These findings suggest that the diverse gene expressions in TG neurons are differentially involved in the inflammatory chronic pain and the acute pain induced by C-fiber activation, and the hyperexcitation of C-fibers are associated with the activation of certain non-coding RNAs.

Altered localization of amyloid precursor protein under endoplasmic reticulum stress.

Recent reports have shown that the endoplasmic reticulum (ER) stress is relevant to the pathogenesis of Alzheimer disease. Following the amyloid cascade hypothesis, we therefore attempted to investigate the effects of ER stress on amyloid-beta peptide (Abeta) generation. In this study, we found that ER stress altered the localization of amyloid precursor protein (APP) from late compartments to early compartments of the secretory pathway, and decreased the level of Abeta 40 and Abeta 42 release by beta- and gamma-cutting. Transient transfection with BiP/GRP78 also caused a shift of APP and a reduction in Abeta secretion. It was revealed that the ER stress response facilitated binding of BiP/GRP78 to APP, thereby causing it to be retained in the early compartments apart from a location suitable for the cleavages of Abeta. These findings suggest that induction of BiP/GRP78 during ER stress may be one of the regulatory mechanisms of Abeta generation.

Candidates for tumor-specific alternative splicing.

Gene expression can be regulated not only by transcription and post-transcriptional modifications, but also by splicing regulation. Recent genome-wide analyses have indicated that up to 70% of human genes may have alternatively spliced forms, suggesting that splicing regulation affects a wide range of gene expression. Tumor tissues show significantly altered protein expressions, and this is also thought to be affected by alternative splicing. Although some alternative splicing events have been reported to be cancer specific and others have been predicted from database analyses, the process of alternative splicing and its regulatory machinery are hardly understood. We searched for and detected alternative splicing events that alter protein splicing in all or a subset of tumor tissues. The results revealed tissue-specific alterations of splicing regulation by tumorigenesis, and regulatory cis-element analyses further suggested that multiple splicing regulatory machineries were affected by this process.

Identification of regulatory cis-acting elements for alternative splicing of presenilin 2 exon 5 under hypoxic stress conditions.

An alternatively spliced form of the presenilin 2 (PS2) gene lacking exon 5 (PS2V) was found in human brains with sporadic Alzheimer's disease. PS2V was induced by hypoxic stress in human neuroblastoma SK-N-SH cells, indicating that hypoxic stress affects the splicing machineries for PS2 exon 5. Here, we identified the critical cis-acting element (sec 2) on the PS2 pre-mRNA responsible for the aberrant splicing of PS2 exon 5 under hypoxic stress conditions. The element was composed of 23 nucleotides in exon 5 and RNA structural analyses showed a stem-loop structure in this sequence. Treatment with an antisense oligonucleotide directed toward the cis-acting element caused an increase in exon 5 inclusion. These results indicate that the sec 2 identified in this study is a novel regulatory element for exon 5 splicing under stress conditions and that trans-acting factors could specifically bind to the element to skip exon 5 of PS2.

Tra2 beta, SF2/ASF and SRp30c modulate the function of an exonic splicing enhancer in exon 10 of tau pre-mRNA.

Some of mutations in the tau gene, which were found in frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), affect alternative splicing of its exon 10 which encodes one of four microtubule-binding motifs. To examine the molecular mechanisms responsible for aberrant splicing of the tau gene containing mutations linked to FTDP-17, we performed Exon trapping and binding assay using tau exon 10 pre-mRNA and nuclear extracts of neuroblastoma cell lines and in vitro splicing using dsx-substrate. We determined that 5' site of tau exon 10 (nucleotides 12-45) possesses exonic splicing enhancer (ESE) activities in vitro splicing and the FTDP-17-linked mutations affect the ESE activities and alter the splicing patterns of tau exon 10. Tra2 beta directly and ASF/SF2 indirectly associated with the ESE of wild tau exon 10. The binding amounts of these SR proteins to tau exon 10 bearing N279K mutation increased and they enhanced splicing the mutant tau exon 10. SRp30c also enhanced the splicing of tau exon 10. These results suggest that mutations in tau exon 10 that are linked to FTDP-17 affect the ESE activities by altering the binding of some SR proteins to its pre-mRNA.

Characterization of mammalian synemin, an intermediate filament protein present in all four classes of muscle cells and some neuroglial cells: co-localization and interaction with type III intermediate filament proteins and keratins.

Using a monoclonal antibody, we have detected a high molecular weight muscle protein, co-localized and co-isolating with desmin. Searching a human cDNA database with partial amino acid sequences of the protein, we found a cDNA clone encoding a 1565-amino-acid polypeptide, identified as a mammalian (human) synemin, a member of the intermediate filament (IF) protein family. Immunoblotting showed the presence of a 180-kDa polypeptide in skeletal muscle and 180- and 200-kDa polypeptides in cardiac and smooth muscles. Interestingly, synemin was also found in myoepithelial cells, which have keratin filaments instead of desmin. Moreover, synemin was also found in astrocytes of optic nerves and non-myelin-forming Schwann cells, together with glial fibrillary acidic protein (GFAP) and vimentin. Blot overlays pointed to molecular interactions of synemin with desmin, vimentin, GFAP and keratin 5 and 6, but not with keratin 14. The experimental data also suggested a possible link with nebulin, a skeletal muscle protein. Purified synemin was coassembled with desmin in different molar ratios, and at 1:25, as typically found in vivo, IFs were formed which were comparable in length to desmin filaments. However, at molar ratios of 3:25 and 6:25, much shorter and irregular shaped filamentous polymers were generated. The fact that synemin is present in all four classes of muscle cells and a specific type of glial cells is indicative of important functions. Its incorporation may give structural and functional versatility to the IF cytoskeleton.

An intronic splicing enhancer element in survival motor neuron (SMN) pre-mRNA.

Spinal muscular atrophy is caused by the homozygous loss of survival motor neuron 1 (SMN1). SMN2, a nearly identical copy gene, differs from SMN1 only by a single nonpolymorphic C to T transition in exon 7, which leads to alteration of exon 7 splicing; SMN2 leads to exon 7 skipping and expression of a nonfunctional gene product and fails to compensate for the loss of SMN1. The exclusion of SMN exon 7 is critical for the onset of this disease. Regulation of SMN exon 7 splicing was determined by analyzing the roles of the cis-acting element in intron 7 (element 2), which we previously identified as a splicing enhancer element of SMN exon 7 containing the C to T transition. The minimum sequence essential for activation of the splicing was determined to be 24 nucleotides, and RNA structural analyses showed a stem-loop structure. Deletion of this element or disruption of the stem-loop structure resulted in a decrease in exon 7 inclusion. A gel shift assay using element 2 revealed formation of RNA-protein complexes, suggesting that the binding of the trans-acting proteins to element 2 plays a crucial role in the splicing of SMN exon 7 containing the C to T transition.

MDG1/ERdj4, an ER-resident DnaJ family member, suppresses cell death induced by ER stress.

BACKGROUND: Alterations in homeostasis after various cellular stresses, which prevent protein folding and cause an accumulation of misfolding or malfolding proteins in the endoplasmic reticulum (ER), have the potential to induce cellular damage, and are therefore a type of 'ER stress.' To understand the molecular events or cascades underlying the ER stress response regulated by gene transcription and mediated by stress transducers, it is crucial to identify the molecules induced during ER stress and to analyse the roles of these genes. RESULTS: We identified MDG1/ERdj4, a member of the DnaJ protein family, as an inducible gene during ER stress. MDG1/ERdj4 contains the J domain, which is essential for interacting with Hsp70s, at the N-terminal portion and just at the back of the transmembrane domain. Its trypsin digestion and glycosylation of a chimeric protein composed of MDG1/ERdj4 fused with the extracellular domain of the amyloid precursor protein at its C-terminus, showed that its C-terminal portion containing the J domain could be orientated to the ER lumen. Over-expression of it inhibited the cell death induced by ER stress. In contrast, its mutants with the J domain deleted showed no protective effects against cell death. CONCLUSIONS: MDG1/ERdj4 may play roles in stabilizing GRP78/BiP binding to unfolded substrate proteins in a J domain-dependent manner and prevent the accumulation of unfolded proteins in the ER, consequently protecting cells from ER stress.

A novel family of unconventional actins in volvocalean algae.

The unicellular green alga Chlamydomonas reinhardtii has two actin genes, one encoding a conventional actin (90% amino acid identity with mammalian actin), the other a highly divergent actin (64% identity) named novel actin-like protein (NAP). To see whether the presence of conventional and unconventional actins in a single organism is unique to C. reinhardtii, we searched for genomic sequences related to the NAP sequence in several other species of volvocalean algae. Here we show that Chlamydomonas moewusii and Volvox carteri also have, in addition to a conventional actin, an unconventional actin similar to the C. reinhardtii NAP. Analyses of the deduced protein sequences indicated that the NAP homologues form a distinct group derived from conventional actin.

FAD-linked presenilin-1 mutants impede translation regulation under ER stress.

FAD mutations in presenilin-1 (PS1) cause attenuation of the induction of the endoplasmic reticulum (ER)-resident chaperone GRP78/BiP under ER stress, due to disturbed function of IRE1, the sensor for accumulation of unfolded protein in the ER lumen. PERK, an ER-resident transmembrane protein kinase, is also a sensor for the unfolded protein response (UPR), causing phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) to inhibit translation initiation. Here, we report that the FAD mutant PS1 disturbs the UPR by attenuating both the activation of PERK and the phosphorylation of eIF2alpha. Consistent with the results of a disturbed UPR, inhibition of protein synthesis under ER stress was impaired in cells expressing PS1 mutants. These results suggest that mutant PS1 impedes general translational attenuation regulated by PERK and eIF2alpha, resulting in an increased load of newly synthesized proteins into the ER and subsequently increasing vulnerability to ER stress.

Identification of a cis-acting element for the regulation of SMN exon 7 splicing.

Spinal muscular atrophy results from the loss of functional survival motor neuron (SMN1) alleles. Two nearly identical copies of SMN exist and differ only by a single non-polymorphic C to T transition in exon 7. This transition leads to alteration of exon 7 splicing; that is, SMN1 produces a full-length transcript, whereas SMN2 expresses a low level of full-length transcript and predominantly an isoform lacking exon 7. The truncated transcript of SMN encodes a less stable protein with reduced self-oligomerization activity that fails to compensate for the loss of SMN1. In this paper, we identified a cis-acting element (element 1), which is composed of 45 bp in intron 6 responsible for the regulation of SMN exon 7 splicing. Mutations in element 1 or treatment with antisense oligonucleotides directed toward element 1 caused an increase in exon 7 inclusion. An approximately 33-kDa protein was demonstrated to associate with a pre-mRNA sequence containing both element 1 and the C to T transition in SMN exon 7 but not with the sequence containing mutated element 1, suggesting that the binding of the approximately 33-kDa protein plays crucial roles in the skipping of SMN exon 7 containing the C to T transition.

Novel alternative splicings of BPAG1 (bullous pemphigoid antigen 1) including the domain structure closely related to MACF (microtubule actin cross-linking factor).

BPAG1 (bullous pemphigoid antigen 1) was originally identified as a 230-kDa hemidesmosomal protein and belongs to the plakin family, because it consists of a plakin domain, a coiled-coil rod domain and a COOH-terminal intermediate filament binding domain. To date, alternatively spliced products of BPAG1, BPAG1e, and BPAG1n are known. BPAG1e is expressed in epithelial tissues and localized to hemidesmosomes, on the other hand, BPAG1n is expressed in neural tissues and muscles and has an actin binding domain at the NH(2)-terminal of BPAG1e. BPAG1 is also known as a gene responsible for Dystonia musculorum (dt) neurodegeneration syndrome of the mouse. Another plakin family protein MACF (microtubule actin cross-linking factor) has also an actin binding domain and the plakin domain at the NH(2)-terminal. However, in contrast to its high homology with BPAG1 at the NH(2)-terminal, the COOH-terminal structure of MACF, including a microtubule binding domain, resembles dystrophin rather than plakins. Here, we investigated RNAs and proteins expressed from the BPAG1 locus and suggest novel alternative splicing variants, which include one consisting of the COOH-terminal domain structure homologous to MACF. The results indicate that BPAG1 has three kinds of cytoskeletal binding domains and seems to play an important role in linking the different types of cytoskeletons.