Toshiki Takenouchi

Hypoxic regulation of the cerebral microcirculation is mediated by a carbon monoxide-sensitive hydrogen sulfide pathway

Enhancement of cerebral blood flow by hypoxia is critical for brain function, but signaling systems underlying its regulation have been unclear. We report a pathway mediating hypoxia-induced cerebral vasodilation in studies monitoring vascular disposition in cerebellar slices and in intact mouse brains using two-photon intravital laser scanning microscopy. In this cascade, hypoxia elicits cerebral vasodilation via the coordinate actions of H2S formed by cystathionine β-synthase (CBS) and CO generated by heme oxygenase (HO)-2. Hypoxia diminishes CO generation by HO-2, an oxygen sensor. The constitutive CO physiologically inhibits CBS, and hypoxia leads to increased levels of H2S that mediate the vasodilation of precapillary arterioles. Mice with targeted deletion of HO-2 or CBS display impaired vascular responses to hypoxia. Thus, in intact adult brain cerebral cortex of HO-2–null mice, imaging mass spectrometry reveals an impaired ability to maintain ATP levels on hypoxia.

Novel overgrowth syndrome phenotype due to recurrent de novo PDGFRB mutation

Using exome analysis, we identified a novel overgrowth syndrome arising from a mutation in PDGFRB, which plays a critical role in growth and differentiation. This entity is characterized by somatic overgrowth, distinctive facial features, hyperelastic and fragile skin, white matter lesions, and neurologic deterioration.

Therapeutic hypothermia for neonatal encephalopathy: JSPNM & MHLW Japan Working Group Practice Guidelines Consensus Statement from the Working Group on Therapeutic Hypothermia for Neonatal Encephalopathy, Ministry of Health, Labor and Welfare (MHLW), Japan, and Japan Society for Perinatal and Neonatal Medicine (JSPNM).

Neonatal encephalopathy (NE) secondary to intrapartum asphyxia remains a major cause of post-natal death and permanent neurological deficits worldwide. Supportive therapy has been the mainstay of the treatment until recent series of large clinical trials demonstrating benefit of therapeutic hypothermia (TH) in this high risk population. Now the International Liaison Committee on Resuscitation (ILCOR) recommends TH as a standard of care with the protocols used in the large clinical trials as tentative standard protocols. Our goal is to develop a nationwide consensus practice guideline not only consistent with the international standard protocols but also practical and compatible with the current medical system in Japan. In summary, TH should be offered to newborn infants born ≥36 weeks gestational age and birth weight ≥1800 g exhibiting clinical signs of moderate to severe NE as well as evidence of hypoxia-ischemia, i.e. 10 min Apgar score ≤5, a need for resuscitation at 10 min, blood pH<7.00, or base deficit ≥16 mmol/L. TH should be conducted in the NICUs capable of multidisciplinary care and under the standard protocols, i.e. utilization of cooling device, target (rectal or esophageal) temperatures at 33.5±0.5 and 34.5±0.5°C for whole body and selective head cooling respectively, duration of TH for 72 h, gradual rewarming not exceeding the rate of 0.5°C/h. Long term follow-up with multidisciplinary approach including standardized psychological assessment is warranted.

Gas biology: Tiny molecules controlling metabolic systems☆

It has been recognized that gaseous molecules and their signaling cascades play a vital role in alterations of metabolic systems in physiologic and pathologic conditions. Contrary to this awareness, detailed mechanisms whereby gases exert their actions, in particular in vivo, have been unclear because of several reasons. Gaseous signaling involves diverse reactions with metal centers of metalloproteins and thiol modification of cysteine residues of proteins. Both the multiplicity of gas targets and the technical limitations in accessing local gas concentrations make dissection of exact actions of any gas mediator a challenge. However, a series of advanced technologies now offer ways to explore gas-responsive regulatory processes in vivo. Imaging mass spectrometry combined with quantitative metabolomics by capillary-electrophoresis/mass spectrometry reveals spatio-temporal profiles of many metabolites. Comparing the metabolic footprinting of murine samples with a targeted deletion of a specific gas-producing enzyme makes it possible to determine sites of actions of the gas. In this review, we intend to elaborate on the ideas how small gaseous molecules interact with metabolic systems to control organ functions such as cerebral vascular tone and energy metabolism in vivo.

Severe congenital lipodystrophy and a progeroid appearance: Mutation in the penultimate exon of FBN1 causing a recognizable phenotype

Recently, three marfanoid patients with congenital lipodystrophy and a neonatal progeroid appearance were reported. Although their phenotype was distinct from that of classic Marfan syndrome, they all had a truncating mutation in the penultimate exon, i.e., exon 64, of FBN1, the causative gene for Marfan syndrome. These patients might represent a new entity, but the exact phenotypic and genotypic spectrum remains unknown. Here, we report on a girl born prematurely who exhibited severe congenital lipodystrophy and a neonatal progeroid appearance. The patient exhibited a characteristic growth pattern consisting of an accelerated growth in height with a discrepant poor weight gain. She had a characteristic facial appearance with craniosynostosis. A mutation analysis identified c.8175_8182del8bp, p.Arg2726Glufs*9 in exon 64 of the FBN1 gene. A review of similar, recently reported patients revealed that the cardinal features of these patients include (1) congenital lipodystrophy, (2) premature birth with an accelerated linear growth disproportionate to the weight gain, and (3) a progeroid appearance with distinct facial features. Lines of molecular evidence suggested that this new progeroid syndrome represents a neomorphic phenotype caused by truncated transcripts with an extremely charged protein motif that escapes from nonsense‐mediated mRNA decay, altering FBN1‐TGF beta signaling, rather than representing the severe end of the hypomorphic phenotype of the FBN1‐TGF beta disorder spectrum. We propose that this marfanoid entity comprised of congenital lipodystrophy, a neonatal progeroid appearance, and a peculiar growth profile and caused by rare mutations in the penultimate exon of FBN1, be newly referred to as marfanoid–progeroid syndrome.

Therapeutic hypothermia achieves neuroprotection via a decrease in acetylcholine with a concurrent increase in carnitine in the neonatal hypoxia-ischemia

Although therapeutic hypothermia is known to improve neurologic outcomes after perinatal cerebral hypoxia-ischemia, etiology remains unknown. To decipher the mechanisms whereby hypothermia regulates metabolic dynamics in different brain regions, we used a two-step approach: a metabolomics to target metabolic pathways responding to cooling, and a quantitative imaging mass spectrometry to reveal spatial alterations in targeted metabolites in the brain. Seven-day postnatal rats underwent the permanent ligation of the left common carotid artery followed by exposure to 8% O2 for 2.5 hours. The pups were returned to normoxic conditions at either 38°C or 30°C for 3 hours. The brain metabolic states were rapidly fixed using in situ freezing. The profiling of 107 metabolites showed that hypothermia diminishes the carbon biomass related to acetyl moieties, such as pyruvate and acetyl-CoA; conversely, it increases deacetylated metabolites, such as carnitine and choline. Quantitative imaging mass spectrometry demarcated that hypothermia diminishes the acetylcholine contents specifically in hippocampus and amygdala. Such decreases were associated with an inverse increase in carnitine in the same anatomic regions. These findings imply that hypothermia achieves its neuroprotective effects by mediating the cellular acetylation status through a coordinated suppression of acetyl-CoA, which resides in metabolic junctions of glycolysis, amino-acid catabolism, and ketolysis.

YY1 Haploinsufficiency Causes an Intellectual Disability Syndrome Featuring Transcriptional and Chromatin Dysfunction

Yin and yang 1 (YY1) is a well-known zinc-finger transcription factor with crucial roles in normal development and malignancy. YY1 acts both as a repressor and as an activator of gene expression. We have identified 23 individuals with de novo mutations or deletions of YY1 and phenotypic features that define a syndrome of cognitive impairment, behavioral alterations, intrauterine growth restriction, feeding problems, and various congenital malformations. Our combined clinical and molecular data define “YY1 syndrome” as a haploinsufficiency syndrome. Through immunoprecipitation of YY1-bound chromatin from affected individuals’ cells with antibodies recognizing both ends of the protein, we show that YY1 deletions and missense mutations lead to a global loss of YY1 binding with a preferential retention at high-occupancy sites. Finally, we uncover a widespread loss of H3K27 acetylation in particular on the YY1-bound enhancers, underscoring a crucial role for YY1 in enhancer regulation. Collectively, these results define a clinical syndrome caused by haploinsufficiency of YY1 through dysregulation of key transcriptional regulators.

The Use of Next-Generation Sequencing in Molecular Diagnosis of Neurofibromatosis Type 1: A Validation Study

AIMS We assessed the validity of a next-generation sequencing protocol using in-solution hybridization-based enrichment to identify NF1 mutations for the diagnosis of 86 patients with a prototypic genetic syndrome, neurofibromatosis type 1. In addition, other causative genes for classic genetic syndromes were set as the target genes for coverage analysis. RESULTS The protocol identified 30 nonsense, 19 frameshift, and 8 splice-site mutations, together with 10 nucleotide substitutions that were previously reported to be pathogenic. In the remaining 19 samples, 10 had single-exon or multiple-exon deletions detected by a multiplex ligation-dependent probe amplification method and 3 had missense mutations that were not observed in the normal Japanese SNP database and were predicted to be pathogenic. Coverage analysis of the genes other than the NF1 gene included on the same diagnostic panel indicated that the mean coverage was 115-fold, a sufficient depth for mutation detection. CONCLUSIONS The overall mutation detection rate using the currently reported method in 86 patients who met the clinical diagnostic criteria was 92.1% (70/76) when 10 patients with large deletions were excluded. The results validate the clinical utility of this next-generation sequencing-based method for the diagnosis of neurofibromatosis type 1. Comparable detection rates can be expected for other genetic syndromes, based on the results of the coverage analysis.

Progressive cognitive decline in an adult patient with cleidocranial dysplasia.

Cleidocranial dysplasia is a skeletal disorder characterized by a defective skull and defective clavicles caused by RUNX2, an activator of osteoblast differentiation. Consistent with the expression pattern of RUNX2, this disorder typically affects the skeletal system, but not the central nervous system. A 56-year-old man with the prototypic skeletal defects of cleidocranial dysplasia and a RUNX2 deletion presented with a progressive cognitive decline after the age of 40 years. After a failed cranioplasty during childhood, he had worn a protective helmet until young adulthood. His current neuroimaging studies revealed extensive cystic encephalomalacia beneath the defective skull, suggesting that his cognitive decline could likely be attributed to repetitive cerebral contusions. Late-onset progressive cognitive decline in the context of a defective skull accompanied by extensive cystic encephalomalacia illustrates the importance of natural calvarial protection against head injury. Since the majority of patients with cleidocranial dysplasia do not wear protective helmets beyond childhood, mainly for cosmetic reasons, a discussion of whether the social disadvantage outweighs the potential risk of brain parenchymal injury may be necessary.

Somatic CTNNB1 mutation in hepatoblastoma from a patient with Simpson-Golabi-Behmel syndrome and germline GPC3 mutation

Simpson–Golabi–Behmel syndrome is a rare overgrowth syndrome caused by the GPC3 mutation at Xq26 and is clinically characterized by multiple congenital abnormalities, intellectual disability, pre/postnatal overgrowth, distinctive craniofacial features, macrocephaly, and organomegaly. Although this syndrome is known to be associated with a risk for embryonal tumors, similar to other overgrowth syndromes, the pathogenetic basis of this mode of tumorigenesis remains largely unknown. Here, we report a boy with Simpson‐Golabi–Behmel syndrome who had a germline loss‐of function mutation in GPC3. At 9 months of age, he developed hepatoblastoma. A comparison of exome analysis results for the germline genome and for the tumor genome revealed a somatic mutation, p.Ile35Ser, within the degradation targeting box of β‐catenin. The same somatic mutation in CTNNB1 has been repeatedly reported in hepatoblastoma and other cancers. This finding suggested that the CTNNB1 mutation in the tumor tissue represents a driver mutation and that both the GPC3 and the CTNNB1 mutations contributed to tumorigenesis in a clearly defined sequential manner in the propositus. The current observation of a somatic CTNNB1 mutation in a hepatoblastoma from a patient with a germline GPC3 mutation supports the notion that the mutation in GPC3 may influence one of the initial steps in tumorigenesis and the progression to hepatoblastoma.