Optineurin deficiency impairs autophagy to cause interferon beta overproduction and increased survival of mice following viral infection.

BACKGROUND: Optineurin (OPTN) is associated with several human diseases, including amyotrophic lateral sclerosis (ALS), and is involved in various cellular processes, including autophagy. Optineurin regulates the expression of interferon beta (IFNß), which plays a central role in the innate immune response to viral infection. However, the role of optineurin in response to viral infection has not been fully clarified. It is known that optineurin-deficient cells produce more IFNß than wild-type cells following viral infection. In this study, we investigate the reasons for, and effects of, IFNß overproduction during optineurin deficiency both in vitro and in vivo. METHODS: To investigate the mechanism of IFNß overproduction, viral nucleic acids in infected cells were quantified by RT-qPCR and the autophagic activity of optineurin-deficient cells was determined to understand the basis for the intracellular accumulation of viral nucleic acids. Moreover, viral infection experiments using optineurin-disrupted (Optn-KO) animals were performed with several viruses. RESULTS: IFNß overproduction following viral infection was observed not only in several types of optineurin-deficient cell lines but also in Optn-KO mice and human ALS patient cells carrying mutations in OPTN. IFNß overproduction in Optn-KO cells was revealed to be caused by excessive accumulation of viral nucleic acids, which was a consequence of reduced autophagic activity caused by the loss of optineurin. Additionally, IFNß overproduction in Optn-KO mice suppressed viral proliferation, resulting in increased mouse survival following viral challenge. CONCLUSION: Our findings indicate that the combination of optineurin deficiency and viral infection leads to IFNß overproduction in vitro and in vivo. The effects of optineurin deficiency are elicited by viral infection, therefore, viral infection may be implicated in the development of optineurin-related diseases.

Analysis of genetic risk factors in Japanese patients with Parkinson's disease.

Parkinson's disease (PD) is caused by a combination of genetic and environmental factors. Notably, genetic risk factors vary according to ethnicity and geographical regions, and few studies have analyzed the frequency of PD causative genes in Japanese patients. Therefore, we performed genetic analyses of Japanese patients with PD. We recruited 221 participants, including 26 patients with familial PD. Genetic risk factors were evaluated by target sequencing and gene dosage analysis. We detected the genetic risk factors in 58 cases (26.2%) and classified patients into three groups to clarify the differences in genetic risk factors by age at onset (AAO). The early-onset group (AAO < 50 years) included 18 cases (44.7%), who tended to have a larger number of genetic risk factors than the later-onset groups. Regarding the AAO for each causative gene, patients with PRKN variants were significantly younger at onset than those bearing LRRK2 variants. LRRK2 variants showed similar frequency in each AAO group. Of note, we identified two novel variants. Patients with early-onset PD have more genetic risk factors than patients with late-onset PD. In Japanese patients with PD, PRKN, and LRRK2 were the major PD-related genes. Particularly, LRRK2 was a common genetic factor in all age groups because of the presence of the Asian-specific variant such as LRRK2 p.G2385R. Accumulation of genetic and clinical data can contribute to the development of treatments for PD.

Optineurin defects cause TDP43-pathology with autophagic vacuolar formation.

We previously showed that optineurin (OPTN) mutations lead to the development of amyotrophic lateral sclerosis. The association between OPTN mutations and the pathogenesis of amyotrophic lateral sclerosis remains unclear. To investigate the mechanism underlying its pathogenesis, we generated Optn knockout mice. We evaluated histopathological observations of these mice and compared with those of OPTN- amyotrophic lateral sclerosis cases to investigate the mechanism underlying the pathogenesis of amyotrophic lateral sclerosis caused by OPTN mutations. The Optn (-/-) mice presented neuronal autophagic vacuoles immunopositive for charged multivesicular body protein 2b, one of the hallmarks of granulovacuolar degenerations, in the cytoplasm of spinal cord motor neurons at the age of 8 months and the OPTN- amyotrophic lateral sclerosis case with homozygous Q398X mutation. In addition, Optn (-/-) mice showed TAR-DNA binding protein 43/sequestosome1/p62 -positive cytoplasmic inclusions and the clearance of nuclear TAR-DNA binding protein 43. The axonal degeneration of the sciatic nerves was observed in Optn (-/-) mice. However, we could not observe significant differences in survival time, body weight, and motor functions, at 24 months. Our findings suggest that homozygous OPTN deletion or mutations might result in autophagic dysfunction and TAR-DNA binding protein 43 mislocalization, thereby leading to neurodegeneration of motor neurons. These findings indicate that the Optn (-/-) mice recapitulate both common and specific pathogenesis of amyotrophic lateral sclerosis associated with autophagic abnormalities. Optn (-/-) mice could serve as a mouse model for the development of therapeutic strategies.

Aggressive periodontitis and NOD2 variants.

Aggressive periodontitis (AgP) occurs at an early age and causes rapid periodontal tissue destruction. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) encodes a protein with two caspase recruitment domains and eleven leucine-rich repeats. This protein is expressed mainly in peripheral blood leukocytes and is involved in immune response. NOD2 variants have been associated with increased susceptibility to Crohn's disease, and recently, NOD2 was reported as a causative gene in AgP. The present study aimed to identify potential NOD2 variants in an AgP cohort (a total of 101 patiens: 37 patients with positive family histories and 64 sporadic patients). In the familial group, six patients from two families had a reported heterozygous missense variant (c.C931T, p.R311W). Four patients in the sporadic group had a heterozygous missense variant (c.C1411T, p.R471C), with no reported association to the disease. Overall, two NOD2 variants, were identified in 10% of our AgP cohort. These variants were different from the major variants reported in Crohn's disease. More cases need to be investigated to elucidate the role of NOD2 variants in AgP pathology.

The first Japanese case of primary familial brain calcification caused by an MYORG variant.

Primary familial brain calcification (PFBC) is a hereditary neurological disorder characterized by idiopathic calcification of the bilateral basal ganglia and other areas of the brain. MYORG has been identified as the first causative gene of autosomal recessive PFBC in Chinese families. There have been several reports of PFBC associated with MYORG (MYORG-PFBC) in individuals of Middle Eastern, European, and Latin American ancestry but to date, there have been no reported Japanese cases. We report the first Japanese case of MYORG-PFBC. The patient was a 43-year-old Japanese woman who experienced mild headaches and cerebellar ataxia including dysarthria. Computed tomography showed calcification in the cerebral white matter, basal ganglia, cerebellum, and brainstem. Using exome sequencing, we identified a homozygous variant in the MYORG gene (NM_020702.4: c.794C>T,p.Thr265Met). Our patient presented dysarthria and extensive calcification affecting the pons, which are specific features of MYORG-PFBC. We report clinical symptoms and imaging findings of a case with p.Thr265Met variant.

Middle-age-onset cerebellar ataxia caused by a homozygous TWNK variant: a case report.

BACKGROUND: The TWNK gene encodes the twinkle protein, which is a mitochondrial helicase for DNA replication. The dominant TWNK variants cause progressive external ophthalmoplegia with mitochondrial DNA deletions, autosomal dominant 3, while the recessive variants cause mitochondrial DNA depletion syndrome 7 and Perrault syndrome 5. Perrault syndrome is characterized by sensorineural hearing loss in both males and females and gonadal dysfunction in females. Patients with Perrault syndrome may present early-onset cerebellar ataxia, whereas middle-age-onset cerebellar ataxia caused by TWNK variants is rare. CASE PRESENTATION: A Japanese female born to consanguineous parents presented hearing loss at age 48, a staggering gait at age 53, and numbness in her distal extremities at age 57. Neurological examination revealed sensorineural hearing loss, cerebellar ataxia, decreased deep tendon reflexes, and sensory disturbance in the distal extremities. Laboratory tests showed no abnormal findings other than a moderate elevation of pyruvate concentration levels. Brain magnetic resonance imaging revealed mild cerebellar atrophy. Using exome sequencing, we identified a homozygous TWNK variant (NM_021830: c.1358G>A, p.R453Q). CONCLUSIONS: TWNK variants could cause middle-age-onset cerebellar ataxia. Screening for TWNK variants should be considered in cases of cerebellar ataxia associated with deafness and/or peripheral neuropathy, even if the onset is not early.

Optineurin regulates osteoblastogenesis through STAT1.

A sophisticated and delicate balance between bone resorption by osteoclasts and bone formation by osteoblasts regulates bone metabolism. Optineurin (OPTN) is a gene involved in primary open-angle glaucoma and amyotrophic lateral sclerosis. Although its function has been widely studied in ophthalmology and neurology, recent reports have shown its possible involvement in bone metabolism through negative regulation of osteoclast differentiation. However, little is known about the role of OPTN in osteoblast function. Here, we demonstrated that OPTN controls not only osteoclast but also osteoblast differentiation. Different parameters involved in osteoblastogenesis and osteoclastogenesis were assessed in Optn-/- mice. The results showed that osteoblasts from Optn-/- mice had impaired alkaline phosphatase activity, defective mineralized nodules, and inability to support osteoclast differentiation. Moreover, OPTN could bind to signal transducer and activator of transcription 1 (STAT1) and regulate runt-related transcription factor 2 (RUNX2) nuclear localization by modulating STAT1 levels in osteoblasts. These data suggest that OPTN is involved in bone metabolism not only by regulating osteoclast function but also by regulating osteoblast function by mediating RUNX2 nuclear translocation via STAT1.

Biallelic mutation of HSD17B4 induces middle age-onset spinocerebellar ataxia.

OBJECTIVE: To determine the genetic underpinnings of slowly progressive spinocerebellar ataxia, autosomal recessive (SCAR), we performed exome analysis and examined the relationship between clinical severity and functional change induced by the mutation. METHODS: Homozygosity fingerprinting and exome sequencing were performed to identify causative mutations in 2 consanguineous families. We assessed the expression of D-bifunctional protein (DBP) and the amount of dimerized DBP in fibroblasts by immunoblot and quantitative reverse transcription PCR. The pathogenicity of the mutation was evaluated using the Combined Annotation-Dependent Depletion (CADD) scores; these results were compared with the scores of previously reported mutations. RESULTS: We identified a homozygous mutation as causative of middle age-onset SCAR: p.Ala175Thr, which is located in HSD17B4 that encodes peroxisomal DBP. The patients developed cerebellar ataxia, and the subsequent progression was slow. The symptoms presented were milder than those in previously reported cases. The messenger RNA expression levels were normal, but protein levels were diminished. Dimerization of DBP was also reduced. The CADD score of the identified mutation was lower than those of previously reported mutations. CONCLUSIONS: This is the report of middle age-onset DBP deficiency. Residual functional DBP caused relatively mild symptoms in the affected patients, i.e., slowly progressive ataxia and hearing loss. This study broadens the scope of DBP deficiency phenotypes and indicates that CADD scores may be used to estimate the severity of DBP deficiencies.

Genetic screening for potassium channel mutations in Japanese autosomal dominant spinocerebellar ataxia.

Spinocerebellar ataxia (SCA) is a genetically heterogeneous disease characterized by cerebellar ataxia. Many causative genes have been identified to date, the most common etiology being the abnormal expansion of repeat sequences, and the mutation of ion channel genes also play an important role in the development of SCA. Some of them encode calcium and potassium channels. However, due to limited reports about potassium genes in SCA, we screened 192 Japanese individuals with dominantly inherited SCA who had no abnormal repeat expansions of causative genes for potassium channel mutations (KCNC3 for SCA13 and KCND3 for SCA19/SCA22) by target sequencing. As a result, two variants were identified from two patients: c.1973G>A, p.R658Q and c.1018G>A, p.V340M for KCNC3, and no pathogenic variant was identified for KCND3. The newly identified p.V340M exists in the extracellular domain, and p.R658Q exists in the intracellular domain on the C-terminal side, although most of the reported KCNC3 mutations are present at the transmembrane site. Adult-onset and slowly progressive cerebellar ataxia are the main clinical features of SCA13 and SCA19 caused by potassium channel mutations, which was similar in our cases. SCA13 caused by KCNC3 mutations may present with deep sensory loss and cognitive impairment in addition to cerebellar ataxia. In this study, mild deep sensory loss was observed in one case. SCA caused by potassium channel gene mutations is extremely rare, and more cases should be accumulated in the future to elucidate its pathogenesis due to channel dysfunction.

The Commercial Antibodies Widely Used to Measure H3 K56 Acetylation Are Non-Specific in Human and Drosophila Cells.

Much of our understanding of the function of histone post-translational modifications in metazoans is inferred from their genomic localization and / or extrapolated from yeast studies. For example, acetylation of histone H3 lysine 56 (H3 K56Ac) is assumed to be important for transcriptional regulation in metazoan cells based on its occurrence at promoters and its function in yeast. Here we directly assess the function of H3 K56Ac during chromatin disassembly from gene regulatory regions during transcriptional induction in human cells by using mutations that either mimic or prevent H3 K56Ac. Although there is rapid histone H3 disassembly during induction of some estrogen receptor responsive genes, depletion of the histone chaperone ASF1A/B, which is required for H3 K56 acetylation, has no effect on chromatin disassembly at these regions. During the course of this work, we found that all the commercially available antibodies to H3 K56Ac are non-specific in human cells and in Drosophila. We used H3-YFP fusions to show that the H3 K56Q mutation can promote chromatin disassembly from regulatory regions of some estrogen responsive genes in the context of transcriptional induction. However, neither the H3 K56R nor K56Q mutation significantly altered chromatin disassembly dynamics by FRAP analysis. These results indicate that unlike the situation in yeast, human cells do not use H3 K56Ac to promote chromatin disassembly from regulatory regions or from the genome in general. Furthermore, our work highlights the need for rigorous characterization of the specificity of antibodies to histone post-translational modifications in vivo.

A mutation in the low voltage-gated calcium channel CACNA1G alters the physiological properties of the channel, causing spinocerebellar ataxia.

BACKGROUND: Spinocerebellar ataxia (SCA) is a genetically heterogeneous disease. To date, 36 dominantly inherited loci have been reported, and 31 causative genes have been identified. RESULTS: In this study, we analyzed a Japanese family with autosomal dominant SCA using linkage analysis and exome sequencing, and identified CACNA1G, which encodes the calcium channel CaV3.1, as a new causative gene. The same mutation was also found in another family with SCA. Although most patients exhibited the pure form of cerebellar ataxia, two patients showed prominent resting tremor in addition to ataxia. CaV3.1 is classified as a low-threshold voltage-dependent calcium channel (T-type) and is expressed abundantly in the central nervous system, including the cerebellum. The mutation p.Arg1715His, identified in this study, was found to be located at S4 of repeat IV, the voltage sensor of the CaV3.1. Electrophysiological analyses revealed that the membrane potential dependency of the mutant CaV3.1 transfected into HEK293T cells shifted toward a positive potential. We established induced pluripotent stem cells (iPSCs) from fibroblasts of the patient, and to our knowledge, this is the first report of successful differentiation from the patient-derived iPSCs into Purkinje cells. There was no significant difference in the differentiation status between control- and patient-derived iPSCs. CONCLUSIONS: To date, several channel genes have been reported as causative genes for SCA. Our findings provide important insights into the pathogenesis of SCA as a channelopathy.

Mutations in Twinkle primase-helicase cause Perrault syndrome with neurologic features.

OBJECTIVE: To identify the genetic cause in 2 families of progressive ataxia, axonal neuropathy, hyporeflexia, and abnormal eye movements, accompanied by progressive hearing loss and ovarian dysgenesis, with a clinical diagnosis of Perrault syndrome. METHODS: Whole-exome sequencing was performed to identify causative mutations in the 2 affected sisters in each family. Family 1 is of Japanese ancestry, and family 2 is of European ancestry. RESULTS: In family 1, affected individuals were compound heterozygous for chromosome 10 open reading frame 2 (C10orf2) p.Arg391His and p.Asn585Ser. In family 2, affected individuals were compound heterozygous for C10orf2 p.Trp441Gly and p.Val507Ile. C10orf2 encodes Twinkle, a primase-helicase essential for replication of mitochondrial DNA. Conservation and structural modeling support the causality of the mutations. Twinkle is known also to harbor multiple mutations, nearly all missenses, leading to dominant progressive external ophthalmoplegia type 3 and to recessive mitochondrial DNA depletion syndrome 7, also known as infantile-onset spinocerebellar ataxia. CONCLUSIONS: Our study identifies Twinkle mutations as a cause of Perrault syndrome accompanied by neurologic features and expands the phenotypic spectrum of recessive disease caused by mutations in Twinkle. The phenotypic heterogeneity of conditions caused by Twinkle mutations and the genetic heterogeneity of Perrault syndrome call for genomic definition of these disorders.

At the intersection of non-coding transcription, DNA repair, chromatin structure, and cellular senescence.

It is well accepted that non-coding RNAs play a critical role in regulating gene expression. Recent paradigm-setting studies are now revealing that non-coding RNAs, other than microRNAs, also play intriguing roles in the maintenance of chromatin structure, in the DNA damage response, and in adult human stem cell aging. In this review, we will discuss the complex inter-dependent relationships among non-coding RNA transcription, maintenance of genomic stability, chromatin structure, and adult stem cell senescence. DNA damage-induced non-coding RNAs transcribed in the vicinity of the DNA break regulate recruitment of the DNA damage machinery and DNA repair efficiency. We will discuss the correlation between non-coding RNAs and DNA damage repair efficiency and the potential role of changing chromatin structures around double-strand break sites. On the other hand, induction of non-coding RNA transcription from the repetitive Alu elements occurs during human stem cell aging and hinders efficient DNA repair causing entry into senescence. We will discuss how this fine balance between transcription and genomic instability may be regulated by the dramatic changes to chromatin structure that accompany cellular senescence.

Epigenetic inheritance of an inducibly nucleosome-depleted promoter and its associated transcriptional state in the apparent absence of transcriptional activators.

BACKGROUND: Dynamic changes to the chromatin structure play a critical role in transcriptional regulation. This is exemplified by the Spt6-mediated histone deposition on to histone-depleted promoters that results in displacement of the general transcriptional machinery during transcriptional repression. RESULTS: Using the yeast PHO5 promoter as a model, we have previously shown that blocking Spt6-mediated histone deposition on to the promoter leads to persistent transcription in the apparent absence of transcriptional activators in vivo. We now show that the nucleosome-depleted PHO5 promoter and its associated transcriptionally active state can be inherited through DNA replication even in the absence of transcriptional activators. Transcriptional reinitiation from the nucleosome-depleted PHO5 promoter in the apparent absence of activators in vivo does not require Mediator. Notably, the epigenetic inheritance of the nucleosome-depleted PHO5 promoter through DNA replication does not require ongoing transcription. CONCLUSION: Our results suggest that there may be a memory or an epigenetic mark on the nucleosome-depleted PHO5 promoter that is independent of the transcription apparatus and maintains the promoter in a nucleosome-depleted state through DNA replication.

Regulation of retinal cell fate specification by multiple transcription factors.

Retinal cell fate specification is strictly regulated by multiple transcription factors. Regarding regulation of cell proliferation and differentiation, basic helix-loop-helix (bHLH) type repressors and activators function in an antagonistic manner. Repressor-type bHLH factors maintain retinal progenitor cells, whereas activator-type bHLH factors promote neuronal cell fate determination. However, bHLH genes alone are not sufficient for acquiring proper neuronal subtype identity. Recent findings have shown that retinal cell fate specification is regulated by combinations of bHLH and homeobox genes. It is conceivable that homeobox genes confer positional identity whereas bHLH genes regulate neuronal determination and differentiation. Moreover, it has been shown that bHLH genes implicated in retinal cell fate determination regulate expression of other bHLH genes, implying that there is a complicated transcription network regulating retinal development.

Hes1 and Hes5 control the progenitor pool, intermediate lobe specification, and posterior lobe formation in the pituitary development.

The pituitary gland is composed of two distinct entities: the adenohypophysis, including the anterior and intermediate lobes, and the neurohypophysis, known as the posterior lobe. This critical endocrine organ is essential for homeostasis, metabolism, reproduction, and growth. The pituitary development requires the control of proliferation and differentiation of progenitor cells. Although multiple signaling molecules and transcription factors are required for the proper pituitary development, the mechanisms that regulate the fate of progenitor cells remain to be elucidated. Hes genes, known as Notch effectors, play a crucial role in specifying cellular fates during the development of various tissues and organs. Here, we report that mice deficient for Hes1 and Hes5 display severe pituitary hypoplasia caused by accelerated differentiation of progenitor cells. In addition, this hypoplastic pituitary gland (adenohypophysis) lacks the intermediate lobe and exhibits the features of the anterior lobe only. Hes1 and Hes5 double-mutant mice also lack the neurohypophysis (the posterior lobe), probably due to incomplete evagination of the diencephalon. Thus, Hes genes control not only maintenance of progenitor cells but also intermediate vs. anterior lobe specification during the adenohypophysis development. Hes genes are also essential for the formation of the neurohypophysis.

Sustained Notch signaling in progenitors is required for sequential emergence of distinct cell lineages during organogenesis.

Mammalian organogenesis results from the concerted actions of signaling pathways in progenitor cells that induce a hierarchy of regulated transcription factors critical for organ and cell type determination. Here we demonstrate that sustained Notch activity is required for the temporal maintenance of specific cohorts of proliferating progenitors, which underlies the ability to specify late-arising cell lineages during pituitary organogenesis. Conditional deletion of Rbp-J, which encodes the major mediator of the Notch pathway, leads to premature differentiation of progenitor cells, a phenotype recapitulated by loss of the basic helix-loop-helix (bHLH) factor Hes1, as well as a conversion of the late (Pit1) lineage into the early (corticotrope) lineage. Notch signaling is required for maintaining expression of the tissue-specific paired-like homeodomain transcription factor, Prop1, which is required for generation of the Pit1 lineage. Attenuation of Notch signaling is necessary for terminal differentiation in post-mitotic Pit1+ cells, and the Notch-repressed Pit1 target gene, Math3, is specifically required for maturation and proliferation of the GH-producing somatotrope. Thus, sustained Notch signaling in progenitor cells is required to prevent conversion of the late-arising cell lineages to early-born cell lineages, permitting specification of diverse cell types, a strategy likely to be widely used in mammalian organogenesis.