Hyperbaric oxygen upregulates cochlear constitutive nitric oxide synthase

<p>Abstract</p> <p>Background</p> <p>Hyperbaric oxygen therapy (HBOT) is a known adjuvant for treating ischemia-related inner ear diseases. Controversies still exist in the role of HBOT in cochlear diseases. Few studies to date have investigated the cellular changes that occur in inner ears after HBOT. Nitric oxide, which is synthesized by nitric oxide synthase (NOS), is an important signaling molecule in cochlear physiology and pathology. Here we investigated the effects of hyperbaric oxygen on eardrum morphology, cochlear function and expression of NOS isoforms in cochlear substructures after repetitive HBOT in guinea pigs.</p> <p>Results</p> <p>Minor changes in the eardrum were observed after repetitive HBOT, which did not result in a significant hearing threshold shift by tone burst auditory brainstem responses. A differential effect of HBOT on the expression of NOS isoforms was identified. Upregulation of constitutive NOS (nNOS and eNOS) was found in the substructures of the cochlea after HBOT, but inducible NOS was not found in normal or HBOT animals, as shown by immunohistochemistry. There was no obvious DNA fragmentation present in this HBOT animal model.</p> <p>Conclusions</p> <p>The present evidence indicates that the customary HBOT protocol may increase constitutive NOS expression but such upregulation did not cause cell death in the treated cochlea. The cochlear morphology and auditory function are consequently not changed through the protocol.</p

Homozygosity for the C9orf72 GGGGCC repeat expansion in frontotemporal dementia

An expanded hexanucleotide repeat in the C9orf72 gene is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis (c9FTD/ALS). We now report the first description of a homozygous patient and compare it to a series of heterozygous cases. The patient developed early-onset frontotemporal dementia without additional features. Neuropathological analysis showed c9FTD/ALS characteristics, with abundant p62-positive inclusions in the frontal and temporal cortices, hippocampus and cerebellum, as well as less abundant TDP-43-positive inclusions. Overall, the clinical and pathological features were severe, but did not fall outside the usual disease spectrum. Quantification of C9orf72 transcript levels in post-mortem brain demonstrated expression of all known C9orf72 transcript variants, but at a reduced level. The pathogenic mechanisms by which the hexanucleotide repeat expansion causes disease are unclear and both gain- and loss-of-function mechanisms may play a role. Our data support a gain-of-function mechanism as pure homozygous loss of function would be expected to lead to a more severe, or completely different clinical phenotype to the one described here, which falls within the usual range. Our findings have implications for genetic counselling, highlighting the need to use genetic tests that distinguish C9orf72 homozygosity

Molecular mechanisms underlying haplotypespecific regulation of gene expression at the microtubule associated protein tau locus

Genome wide association studies (GWAS) have identified the H1 microtubule associated protein tau (MAPT) haplotype single nucleotide polymorphisms as leading common risk variants for Parkinson's disease (PD), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). Gene expression studies have demonstrated haplotype-specific increases in expression of MAPT exon 3-containing transcripts from the protective H2 allele compared to the H1. The difference in alternative splicing between the haplotypes likely contributes risk or protection in the absence of protein coding variants. Here, we investigate the regulation of MAPT exon 3 alternative splicing by common, risk-associated, non-coding, haplotype-specific single nucleotide polymorphisms (SNPs) through a combination of in silico analysis of the MAPT locus, in vitro gene expression and biochemistry studies. Comparative sequence analysis of whole-locus genomic H1 and H2 MAPT (143 kb) vectors showed they capture over 86&amp;percnt; of the MAPT sequence diversity. We generated and expressed haplotype-hybrid H1 and H2 MAPT vectors in a human neuroblastoma cell culture model and demonstrated that a functional SNP rs17651213 near the exon 3 5' splice site regulates exon 3 inclusion in a haplotype-specific manner. Using RNA-electrophoretic mobility shift assays (RNA-EMSA), we showed differential RNA-protein complex formation at the H1 and H2 sequence variants of SNP rs17651213. We further identified candidate trans-acting splicing factors interacting with functional SNP rs17651213 sequences by RNA-protein pull-down experiment and mass spectrometry. Finally, gene knockdown of candidate splice factors identified by mass spectrometry demonstrated a role for hnRNP F and hnRNP Q in the haplotype-specific regulation of exon 3 inclusion. In this study, we have dissected the MAPT locus to identify sequences regulating the allele-specific alternative splicing of exon 3 and provided mechanistic insights into how common non-coding H1/H2 MAPT haplotype-specific SNPs may contribute to the risk/protection of neurodegeneration at a complex genetic locus.</p

Molecular mechanisms underlying haplotypespecific regulation of gene expression at the microtubule associated protein tau locus

Genome wide association studies (GWAS) have identified the H1 microtubule associated protein tau (MAPT) haplotype single nucleotide polymorphisms as leading common risk variants for Parkinson's disease (PD), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). Gene expression studies have demonstrated haplotype-specific increases in expression of MAPT exon 3-containing transcripts from the protective H2 allele compared to the H1. The difference in alternative splicing between the haplotypes likely contributes risk or protection in the absence of protein coding variants. Here, we investigate the regulation of MAPT exon 3 alternative splicing by common, risk-associated, non-coding, haplotype-specific single nucleotide polymorphisms (SNPs) through a combination of in silico analysis of the MAPT locus, in vitro gene expression and biochemistry studies. Comparative sequence analysis of whole-locus genomic H1 and H2 MAPT (143 kb) vectors showed they capture over 86&percnt; of the MAPT sequence diversity. We generated and expressed haplotype-hybrid H1 and H2 MAPT vectors in a human neuroblastoma cell culture model and demonstrated that a functional SNP rs17651213 near the exon 3 5' splice site regulates exon 3 inclusion in a haplotype-specific manner. Using RNA-electrophoretic mobility shift assays (RNA-EMSA), we showed differential RNA-protein complex formation at the H1 and H2 sequence variants of SNP rs17651213. We further identified candidate trans-acting splicing factors interacting with functional SNP rs17651213 sequences by RNA-protein pull-down experiment and mass spectrometry. Finally, gene knockdown of candidate splice factors identified by mass spectrometry demonstrated a role for hnRNP F and hnRNP Q in the haplotype-specific regulation of exon 3 inclusion. In this study, we have dissected the MAPT locus to identify sequences regulating the allele-specific alternative splicing of exon 3 and provided mechanistic insights into how common non-coding H1/H2 MAPT haplotype-specific SNPs may contribute to the risk/protection of neurodegeneration at a complex genetic locus.</p

Haplotype-specific MAPT exon 3 expression regulated by common intronic polymorphisms associated with Parkinsonian disorders

Abstract Background Genome wide association studies have identified microtubule associated protein tau (MAPT) H1 haplotype single nucleotide polymorphisms (SNPs) as leading common risk variants for Parkinson’s disease, progressive supranuclear palsy and corticobasal degeneration. The MAPT risk variants fall within a large 1.8 Mb region of high linkage disequilibrium, making it difficult to discern the functionally important risk variants. Here, we leverage the strong haplotype-specific expression of MAPT exon 3 to investigate the functionality of SNPs that fall within this H1 haplotype region of linkage disequilibrium. Methods In this study, we dissect the molecular mechanisms by which haplotype-specific SNPs confer allele-specific effects on the alternative splicing of MAPT exon 3. Firstly, we use haplotype-hybrid whole-locus genomic MAPT vectors studies to identify functional SNPs. Next, we characterise the RNA-protein interactions at two loci by mass spectrometry. Lastly, we knockdown candidate splice factors to determine their effect on MAPT exon 3 using a novel allele-specific qPCR assay. Results Using whole-locus genomic DNA expression vectors to express MAPT haplotype variants, we demonstrate that rs17651213 regulates exon 3 inclusion in a haplotype-specific manner. We further investigated the functionality of this region using RNA-electrophoretic mobility shift assays to show differential RNA-protein complex formation at the H1 and H2 sequence variants of SNP rs17651213 and rs1800547 and subsequently identified candidate trans-acting splicing factors interacting with these functional SNPs sequences by RNA-protein pull-down experiment and mass spectrometry. Finally, gene knockdown of candidate splice factors identified by mass spectrometry demonstrate a role for hnRNP F and hnRNP Q in the haplotype-specific regulation of exon 3 inclusion. Conclusions We identified common splice factors hnRNP F and hnRNP Q regulating the haplotype-specific splicing of MAPT exon 3 through intronic variants rs1800547 and rs17651213. This work demonstrates an integrated approach to characterise the functionality of risk variants in large regions of linkage disequilibrium

Adefovir dipivoxil for wait-listed and post-liver transplantation patients with lamivudine-resistant hepatitis B : final long-term results

Wait-listed (n = 226) or post-liver transplantation (n = 241) chronic hepatitis B (CHB) patients with lamivudine-resistant hepatitis B virus (HBV) were treated with adefovir dipivoxil for a median of 39 and 99 weeks, respectively. Among wait-listed patients, serum HBV DNA levels became undetectable (<1,000 copies/mL) in 59% and 65% at weeks 48 and 96, respectively. After 48 weeks, alanine aminotransferase (ALT), albumin, bilirubin, and prothrombin time normalized in 77%, 76%, 60%, and 84% of wait-listed patients, respectively. Among posttransplantation patients, serum HBV DNA levels became undetectable in 40% and 65% at weeks 48 and 96, respectively. After 48 weeks, ALT, albumin, bilirubin, and prothrombin time normalized in 51%, 81%, 76%, and 56% of posttransplantation patients, respectively. Among wait-listed patients who underwent on-study liver transplantation, protection from graft reinfection over a median of 35 weeks was similar among patients who did (n = 34) or did not (n = 23) receive hepatitis B immunoglobulin (HBIg). Hepatitis B surface antigen was detected on the first measurement only in 6% and 9% of patients who did or did not receive HBIg, respectively. Serum HBV DNA was detected on consecutive visits in 6% and 0% of patients who did or did not receive HBIg, respectively. Treatment-related adverse events led to discontinuation of adefovir dipivoxil in 4% of patients. Cumulative probabilities of resistance were 0%, 2%, and 2% at weeks 48, 96, and 144, respectively. In conclusion, adefovir dipivoxil is effective and safe in wait-listed or posttransplantation CHB patients with lamivudine-resistant HBV and prevents graft reinfection with or without HBIg