Four-repeat tauopathies and late-onset psychiatric disorders: Etiological relevance or incidental findings?

Argyrophilic grain disease (AGD), progressive supranuclear palsy (PSP) and corticobasal degeneration are four-repeat (4R) tauopathies that develop in the presenium or later. Whether these diseases are associated with the occurrence of late-onset psychiatric disorders remains unclear. To facilitate the accumulation of clinicopathological findings regarding this issue, we here present a selected series of 11 cases that clinically developed psychotic disorder (n = 7; age at onset: 41-75 years), depressive disorder (n = 1; 49 years), bipolar disorder (n = 2; 32 and 37 years) and somatoform disorder (n = 1; 88 years), and had at least one pathological hallmark of these tauopathies. The mean age at death was 74.3 years. No case showed dementia, at least in the early stage of the course. Nine cases had AGD. Granular fuzzy astrocytes in the amygdala were noted in all AGD cases and one non-AGD case. Two AGD cases had tufted astrocytes (TAs) in the amygdala but not in the frontal cortex and striatum. Three AGD and two non-AGD cases had TAs in the frontal cortex and/or striatum but not in the amygdala. One AGD case had a small number of astrocytic plaques in the frontal cortex, striatum and globus pallidus. Only one case was diagnosed as atypical PSP according to the NINDS-PSP neuropathological criteria. No case had high-level Alzheimer's disease pathology, Lewy body disease or limbic-predominant age-related TDP-43 encephalopathy. Two cases had mild neuronal loss in the hippocampus and substantia nigra, respectively. Clinicopathological studies focusing especially on early changes of 4R tauopathies, as well as the development of surrogate markers of these diseases, may be necessary for better understanding of the pathogenic backgrounds of late-onset psychiatric disorders.

Synthesis and physical and biological properties of 1,3-diaza-2-oxophenoxazine-conjugated oligonucleotides.

The artificial nucleobase 1,3-diaza-2-oxophenoxazine (tCO) and its derivative G-clamp strongly bind to guanine and, when incorporated into double-stranded DNA, significantly increase the stability of the latter. As the phenoxazine skeleton is a constituent of major pharmaceuticals, we hypothesized that oligonucleotides (ONs) containing phenoxazine bases would induce property changes related to intracellular uptake and migration in tissues. In this study, we designed and synthesized a novel G-clamp-linker antisense oligonucleotide (ASO) in which a G-clamp base with a flexible linker was introduced into the 5'-end of an ASO targeting mouse long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (mMALAT1). Compared to unconjugated ASO, the G-clamp-linker ASO induced significantly more effective knockdown of mMALAT1 in mouse skeletal muscle. The ASOs conjugated with 2'-deoxyribonucleotide(s) bearing a tCO nucleobase at the 5'-end exhibited a similar knockdown effect in skeletal muscle. Thus, it may be possible to improve therapeutic effects against skeletal muscle diseases, such as muscular dystrophy, by using ONs with incorporated phenoxazine nucleobases.

Crystallographic Structure of Novel Types of AgI -Mediated Base Pairs in Non-canonical DNA Duplex Containing 2'-O,4'-C-Methylene Bridged Nucleic Acids.

Metal-mediated base pairs have widespread applications, such as in DNA-metal nanodevices and sensors. Here, we focused on their sugar conformation in duplexes and observed the crystallographic structure of the non-canonical DNA/DNA duplex containing 2'-O,4'-C-methylene bridged nucleic acid in the presence of AgI ions. The X-ray crystallographic structure was successfully obtained at a resolution of 1.5 Å. A novel type of AgI -mediated base pair between the N1 positions of anti-conformation of adenines in the duplex was observed. In the central non-canonical region, a hexad nucleobase structure containing AgI -mediated base pairs between the N7 positions of guanines was formed. A highly bent non-canonical structure was formed at the origin of AgI -mediated base pairs in the central region. The bent duplex structure induced by the addition of AgI ions might become a powerful tool for dynamic structural changes in DNA nanotechnology applications.

2',4'-BNA/LNA with 9-(2-Aminoethoxy)-1,3-diaza-2-oxophenoxazine Efficiently Forms Duplexes and Has Enhanced Enzymatic Resistance*.

Artificial nucleic acids are widely used in various technologies, such as nucleic acid therapeutics and DNA nanotechnologies requiring excellent duplex-forming abilities and enhanced nuclease resistance. 2'-O,4'-C-Methylene-bridged nucleic acid/locked nucleic acid (2',4'-BNA/LNA) with 1,3-diaza-2-oxophenoxazine (BNAP (BH )) was previously reported. Herein, a novel BH analogue, 2',4'-BNA/LNA with 9-(2-aminoethoxy)-1,3-diaza-2-oxophenoxazine (G-clamp), named BNAP-AEO (BAEO ), was designed. The BAEO nucleoside was successfully synthesized and incorporated into oligodeoxynucleotides (ODNs). ODNs containing BAEO possessed up to 104 -, 152-, and 11-fold higher binding affinities for complementary (c) RNA than those of ODNs containing 2'-deoxycytidine (C), 2',4'-BNA/LNA with 5-methylcytosine (L), or 2'-deoxyribonucleoside with G-clamp (PAEO ), respectively. Moreover, duplexes formed by ODN bearing BAEO with cDNA and cRNA were thermally stable, even under molecular crowding conditions induced by the addition of polyethylene glycol. Furthermore, ODN bearing BAEO was more resistant to 3'-exonuclease than ODNs with phosphorothioate linkages.

Enhanced duplex- and triplex-forming ability and enzymatic resistance of oligodeoxynucleotides modified by a tricyclic thymine derivative.

We designed and synthesized an artificial nucleic acid, [3-(1,2-dihydro-2-oxobenzo[b][1,8]naphthyridine)]-2'-deoxy-D-ribofuranose (OBN), with a tricyclic structure in a nucleobase as a thymidine analog. Oligodeoxynucleotides (ODNs) containing consecutive OBN displayed improved duplex-forming ability with complementary single-stranded (ss) RNA and triplex-forming ability with double-stranded DNA in comparison with ODNs composed of natural thymidine. OBN-modified ODNs also displayed enhanced enzymatic resistance compared with ODNs with natural thymidine and phosphorothioate modification, respectively, due to the structural steric hindrance of the nucleobase. The fluorescence spectra of OBN-modified ODNs showed sufficient fluorescence intensity with ssDNA and ssRNA, which is an advantageous feature for fluorescence imaging techniques of nucleic acids with longer emission wavelengths than bicyclic thymine (bT).

Oligonucleotides Containing Phenoxazine Artificial Nucleobases: Triplex-Forming Abilities and Fluorescence Properties.

1,3-Diaza-2-oxophenoxazine ("phenoxazine"), a tricyclic cytosine analogue, can strongly bind to guanine moieties and improve π-π stacking effects with adjacent bases in a duplex. Phenoxazine has been widely used for improving duplex-forming abilities. In this study, we have investigated whether phenoxazine and its analogue, 1,3,9-triaza-2-oxophenoxazine (9-TAP), could improve triplex-forming abilities. A triplex-forming oligonucleotide (TFO) incorporating a phenoxazine component was found to show considerably decreased binding affinity with homopurine/homopyrimidine double-stranded DNA, so the phenoxazine system was considered not to function as either a protonated cytosine or thymine analogue. Alternatively, a 9-TAP-containing artificial nucleobase developed by us earlier as a new phenoxazine analogue functioned as a thymine analogue with respect to AT base pairs in a parallel triplex DNA motif. The fluorescence of the 9-TAP moiety was maintained even in triplex (9-TAP:AT) formation, so 9-TAP might be useful as an imaging tool for various oligonucleotide nanotechnologies requiring triplex formation.

1,3,9-Triaza-2-oxophenoxazine: An Artificial Nucleobase Forming Highly Stable Self-Base Pairs with Three AgI Ions in a Duplex.

Metal-mediated base pairs (MMBPs) formed by natural or artificial nucleobases have recently been developed. The metal ions can be aligned linearly in a duplex by MMBP formation. The development of a three- or more-metal-coordinated MMBPs has the potential to improve the conductivity and enable the design of metal ion architectures in a duplex. This study aimed to develop artificial self-bases coordinated by three linearly aligned AgI ions within an MMBP. Thus, artificial nucleic acids with a 1,3,9-triaza-2-oxophenoxazine (9-TAP) nucleobase were designed and synthesized. In a DNA/DNA duplex, self-base pairs of 9-TAP could form highly stable MMBPs with three AgI ions. Nine equivalents of AgI led to the formation of three consecutive 9-TAP self-base pairs with extremely high stability. The complex structures of 9-TAP MMBPs were determined by using electrospray ionization mass spectrometry and UV titration experiments. Highly stable self-9-TAP MMBPs with three AgI ions are expected to be applicable to new DNA nanotechnologies.

2'-O,4'-C-Methylene-Bridged Nucleic Acids Stabilize Metal-Mediated Base Pairing in a DNA Duplex.

The 2'-O,4'-C-methylene-bridged or locked nucleic acid (2',4'-BNA/LNA), with an N-type sugar conformation, effectively improves duplex-forming ability. 2',4'-BNA/LNA is widely used to improve gene knockdown in nucleic acid based therapies and is used in gene diagnosis. Metal-mediated base pairs (MMBPs), such as thymine (T)-HgII -T and cytosine (C)-AgI -C have been developed and used as attractive tools in DNA nanotechnology studies. This study aimed to investigate the application of 2',4'-BNA/LNA in the field of MMBPs. 2',4'-BNA/LNA with 5-methylcytosine stabilized the MMBP of C with AgI ions. Moreover, the 2',4'-BNA/LNA sugar significantly improved the duplex-forming ability of the DNA/DNA complex, relative to that by the unmodified sugar. These results suggest that the sugar conformation is important for improving the stability of duplex-containing MMBPs. The results indicate that 2',4'-BNA/LNA can be applied not only to nucleic acid based therapies, but also to MMBP technologies.

Synthesis and thermal stabilities of oligonucleotides containing 2'-O,4'-C-methylene bridged nucleic acid with a phenoxazine base.

We designed and synthesized a novel artificial 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA/LNA) with a phenoxazine nucleobase and named this compound BNAP. Oligodeoxynucleotide (ODN) containing BNAP showed higher binding affinities toward complementary DNA and RNA as compared to ODNs bearing 2',4'-BNA/LNA with 5-methylcytosine or 2'-deoxyribonucleoside with phenoxazine. Thermodynamic analysis revealed that BNAP exhibits properties associated with the phenoxazine moiety in DNA/DNA duplexes and characteristics associated with the 2',4'-BNA/LNA moiety in DNA/RNA duplexes.

Time-Dependent Alterations of Vancomycin-Induced Nephrotoxicity in Mice.

Vancomycin hydrochloride (VCM) is a glycopeptide antibiotic that is commonly used against methicillin-resistant, Gram-positive cocci despite the nephrotoxic side effects. VCM-induced nephrotoxicity has been reported in 5-28% of recipient patients. Therefore, renal failure induced by VCM has become an important clinical problem. However, the exceedingly complex mechanism of VCM-induced nephrotoxicity is not fully understood. Therefore, this study was designed to clarify time-dependent alterations of VCM-induced nephrotoxicity in mice as a step toward decreasing the risks of kidney injury associated with VCM therapy. VCM was injected intraperitoneally into mice at a dose of 400 mg/kg body weight at 24-h intervals for 3, 5, 7, and 14 d. At 24 h after the last injection, we examined histopathological alterations of the kidney as well as blood biochemistry. VCM administration resulted in a decrease of body weight and increase of kidney weight. Histological examination revealed renal damage such as dilated proximal tubules with occasional casts and interstitial fibrosis in VCM-treated mice. Furthermore, immunohistochemical staining with anti-CD10 and anti-single-stranded DNA antibodies highlighted damaged renal proximal tubules with marked dilatation as well as numerous apoptotic cells as early as day 4 of VCM-treatment. The severity of symptoms progressed until day 15. These results suggest that VCM-induced renal damage and incipient renal failure begin soon after the start of treatment and progressively worsen. This is the first report describing the time-dependence of VCM-induced nephrotoxicity in mice and depicting a model that clarifies the mechanisms of this tissue damage.

Ascorbic acid deficiency affects genes for oxidation-reduction and lipid metabolism in livers from SMP30/GNL knockout mice.

BACKGROUND: We sought to elucidate the effect of an ascorbic acid (AA) deficiency on gene expression, because the water soluble antioxidant AA is an important bioactive substance in vivo. METHODS: We performed microarray analyses of the transcriptome in the liver from senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which are unable to synthesize AA in vivo. RESULTS: Our microarray analysis revealed that the AA deficiency increased gene expression related to the oxidation-reduction process, i.e., the nuclear factor, erythroid derived 2, like 2 (Nrf2) gene, which is a reactive oxygen species-sensitive transcriptional factor. Moreover, this AA deficiency increased the expression of genes for lipid metabolism including the cytochrome P450, family 7, subfamily a, polypeptide 1 (Cyp7a1), which is a late-limiting enzyme of the primary bile acid biosynthesis pathway. Although an AA deficiency increased the Cyp7a1 protein level, bile acid levels in the liver and gallbladder decreased. Since Cyp7a1 has a heme iron at the active site, AA must function as a reductant of the iron required for the continuous activation of Cyp7a1. CONCLUSIONS: This experimental evidence strongly supports a role for AA in the physiologic oxidation-reduction process and lipid metabolism including bile acid biosynthesis. GENERAL SIGNIFICANCE: Although many effects of AA supplementation have been reported, no microarray analysis of AA deficiency in vivo is available. Results from using this unique model of AA deficiency, the SMP30/GNL-KO mouse, now provide new information about formerly unknown AA functions that will implement further study of AA in vivo.

Exome sequencing of senescence-accelerated mice (SAM) reveals deleterious mutations in degenerative disease-causing genes.

BACKGROUND: Senescence-accelerated mice (SAM) are a series of mouse strains originally derived from unexpected crosses between AKR/J and unknown mice, from which phenotypically distinct senescence-prone (SAMP) and -resistant (SAMR) inbred strains were subsequently established. Although SAMP strains have been widely used for aging research focusing on their short life spans and various age-related phenotypes, such as immune dysfunction, osteoporosis, and brain atrophy, the responsible gene mutations have not yet been fully elucidated. RESULTS: To identify mutations specific to SAMP strains, we performed whole exome sequencing of 6 SAMP and 3 SAMR strains. This analysis revealed 32,019 to 38,925 single-nucleotide variants in the coding region of each SAM strain. We detected Ogg1 p.R304W and Mbd4 p.D129N deleterious mutations in all 6 of the SAMP strains but not in the SAMR or AKR/J strains. Moreover, we extracted 31 SAMP-specific novel deleterious mutations. In all SAMP strains except SAMP8, we detected a p.R473W missense mutation in the Ldb3 gene, which has been associated with myofibrillar myopathy. In 3 SAMP strains (SAMP3, SAMP10, and SAMP11), we identified a p.R167C missense mutation in the Prx gene, in which mutations causing hereditary motor and sensory neuropathy (Dejerine-Sottas syndrome) have been identified. In SAMP6 we detected a p.S540fs frame-shift mutation in the Il4ra gene, a mutation potentially causative of ulcerative colitis and osteoporosis. CONCLUSIONS: Our data indicate that different combinations of mutations in disease-causing genes may be responsible for the various phenotypes of SAMP strains.

Ascorbic acid enhances the expression of type 1 and type 4 collagen and SVCT2 in cultured human skin fibroblasts.

Ascorbic acid (AA) is essential for collagen biosynthesis as a cofactor for prolyl and lysyl hydroxylase and as a stimulus for collagen gene expression. Many studies have evaluated the relationship between AA and collagen expression in short- and long-term effects on cells after a single administration of AA into the culture medium. However, no such study has monitored in detail the stability of AA in medium or the alterations of intracellular AA levels during a protracted interval. Therefore, we examined here intracellular AA levels and stability throughout its exposure to human skin fibroblasts in vitro. Moreover, we determined the effects on type 1 and type 4 collagen and sodium-dependent vitamin C transporter (SVCT) gene expression when medium containing 100 µM AA was replaced every 24h for 5 days to avoid depletion of AA. Throughout this long-term culture, intracellular AA levels remained constant; the expression of type 1 and type 4 collagens and SVCT2 mRNA was enhanced, and type 1 procollagen synthesis increased. Thus, these results indicate that human skin fibroblasts exposed to AA over time had rising levels of type 1/type 4 collagens and SVCT2 mRNA expression and type 1 procollagen synthesis.

Insufficient ascorbic acid intake during gestation induces abnormal cardiac dilation in fetal and neonatal SMP30/GNL knockout mice.

BACKGROUND: Despite the acknowledged importance of ascorbic acid (AA) in maintaining pregnancy and normal fetal development, its precise actions remain obscure. Therefore, we investigated the impact of maternal AA content on the growth of fetal mice during the gestation period using senescence marker protein-30/gluconolactonase (SMP30/GNL) knockout (KO) mice, which cannot synthesize AA in vivo. METHODS: SMP30/GNL KO mice gave birth after a gestation period under conditions of absent, low, or normal AA intake. AA was measured using high-performance liquid chromatography and electrochemical detection. Whole-body sections were stained with hematoxylin and eosin, Elastica van Gieson, and Azan. RESULTS: The mothers in the group absent AA intake failed to bear young because of incomplete fetal development. Offspring born under the low-AA condition generally died within a few days after birth. Morphological analysis revealed that the latter neonates of SMP30/GNL KO mothers whose intake of AA was low during gestation manifested abnormal cardiac dilation, congestion of the liver and lungs, incompletely expanded pulmonary alveoli, and impaired vertebral bodies. In contrast, a normal AA diet produced healthy progeny. CONCLUSION: A diet sufficiently replete with AA is essential during the gestational period for normal tissue development in the fetus and neonate.

Validation of the revised Addenbrooke's Cognitive Examination (ACE-R) for detecting mild cognitive impairment and dementia in a Japanese population.

BACKGROUND: Early detection of dementia will be important for implementation of disease-modifying treatments in the near future. We aimed to investigate the diagnostic validity and reliability of the Japanese version of the revised Addenbrooke's Cognitive Examination (ACE-R J) for identifying mild cognitive impairment (MCI) and dementia. METHODS: We translated and adapted the original ACE-R for use with a Japanese population. Standard tests for evaluating cognitive decline and dementing disorders were applied. A total of 242 subjects (controls = 73, MCI = 39, dementia = 130) participated in this study. RESULTS: The optimal cut-off scores of ACE-R J for detecting MCI and dementia were 88/89 (sensitivity 0.87, specificity 0.92) and 82/83 (sensitivity 0.99, specificity 0.99) respectively. ACE-R J was superior to the Mini-Mental State Examination in the detection of MCI (area under the curve (AUC): 0.952 vs. 0.868), while the accuracy of the two instruments did not differ significantly in identifying dementia (AUC: 0.999 vs. 0.993). The inter-rater reliability (ICC = 0.999), test-retest reliability (ICC = 0.883), and internal consistency (Cronbach's α = 0.903) of ACE-R J were excellent. CONCLUSION: ACE-R J proved to be an accurate cognitive instrument for detecting MCI and mild dementia. Further neuropsychological evaluation is required for the differential diagnosis of dementia subtypes.

Pick's disease.

Picks disease is a major clinicopathological disease having circumscribed atrophy in the frontotemporal lobe. Demented patients with frontotemporal atrophy are now clinically diagnosed as frontotemporal lobar degeneration (FTLD). Other underlying pathologies in patients with FTLD include FTLD with TDP-43-positive inclusions, corticobasal degeneration, progressive supranuclear palsy, basophilic inclusion body disease, neuronal intermediate filament inclusion disease and argyrophilic grain disease. In this chapter, recent findings regarding the distinct clinical and histopathological features of these pathological disease entities are presented including the discussion on the possibility of future antemortem diagnosis of patients with the disease.In this chapter, recent findings regarding the distinct clinical and histopathological features of these pathological disease entities are presented including the discussion on the possibility of future antemortem diagnosis of patients with the disease.

Defective glycosylation of α-dystroglycan contributes to podocyte flattening.

In addition to skeletal muscle and the nervous system, α-dystroglycan is found in the podocyte basal membrane, stabilizing these cells on the glomerular basement membrane. Fukutin, named after the gene responsible for Fukuyama-type congenital muscular dystrophy, is a putative glycosyltransferase required for the post-translational modification of α-dystroglycan. Chimeric mice targeted for both alleles of fukutin develop severe muscular dystrophy; however, these mice do not have proteinuria. Despite the lack of a functional renal defect, we evaluated glomerular structure and found minor abnormalities in the chimeric mice by light microscopy. Electron microscopy revealed flattening of podocyte foot processes, the number of which was significantly lower in the chimeric compared to wild-type mice. A monoclonal antibody against the laminin-binding carbohydrate residues of α-dystroglycan did not detect α-dystroglycan glycosylation in the glomeruli by immunoblotting or immunohistochemistry. In contrast, expression of the core α-dystroglycan protein was preserved. There was no statistical difference in dystroglycan mRNA expression or in the amount of nephrin and α3-integrin protein in the chimeric compared to the wild-type mice as judged by immunohistochemistry and real-time RT-PCR. Thus, our results indicate that appropriate glycosylation of α-dystroglycan has an important role in the maintenance of podocyte architecture.

Suicidal ideation among patients with gender identity disorder.

In this study, we tried to clarify the prevalence of suicidal ideation and self-mutilation including suicide attempts among patients with gender identity disorder (GID) and the relationship of those behaviors to demographic characteristics. A total of 500 consecutive Japanese GID patients without any other psychiatric comorbidity were evaluated at the outpatient GID Clinic of Okayama University Hospital. The lifetime rate of suicidal ideation was 72.0% of the total sample. There were no significant differences in the prevalence of suicidal ideation among groups divided by sex, age, age at onset or education. The lifetime prevalence of self-mutilation including suicide attempts was 31.8% of the total sample. Low level of education was significantly related to self-mutilation among both male-to-female and female-to-male GID patients. Younger age at onset was a significant factor affecting self-mutilation only among MTF GID patients. A lack of strategies to cope with severe distress among persons with lower education might induce a high frequency of self-mutilation including suicidal attempt. GID patients with a low level education might be at high risk of self-mutilation and should be watched with special attention to self-mutilation.

Validation of Addenbrooke's cognitive examination for detecting early dementia in a Japanese population.

There is a clear need for brief, but sensitive and specific, cognitive screening instruments for dementia. We assessed the diagnostic accuracy of the Japanese version of Addenbrooke's Cognitive Examination (ACE) in identifying early dementia in comparison with the conventional Mini-Mental State Examination (MMSE). Standard tests for evaluating dementia screening tests were applied. A total of 201 subjects (Alzheimer's disease (AD)=65, frontotemporal dementia (FTD)=24, vascular dementia=26, dementia with Lewy bodies=11, mild cognitive impairment (MCI)=13, and controls=62) participated in this study. The reliability of the ACE was very good (alpha coefficient=0.82). In our patient series, the sensitivity for diagnosing dementia with an ACE score of ≤74 was 0.889 with a specificity of 0.987, and the sensitivity of an ACE score of ≤80 was 0.984 with a specificity of 0.867. The Japanese version of the ACE is a very accurate instrument for the detection of early dementia, and should be widely used in clinical practice.

Kana Pick-out Test and brain perfusion imaging in Alzheimer's disease.

BACKGROUND: The Kana Pick-out Test (KPT), which was developed in Japan, is suitable for evaluating frontal lobe function and screening for mild dementia. However, the neural substrates involved remain to be elucidated. The aim of the present study was to identify the regional perfusion patterns in the brain associated with performance scores on the KPT in patients with mild Alzheimer's disease (AD), using brain perfusion assessed by single photon emission computed tomography (SPECT). METHODS: Twenty AD patients with high scores on the KPT and 20 age- and sex-matched AD patients with low scores were selected from 227 consecutive Japanese patients of the Memory Clinic of Okayama University Hospital. All 40 subjects underwent brain SPECT with 99mTc-ethylcysteinate dimer, and the SPECT images were analyzed by Statistical Parametric Mapping. RESULTS: With the exception of KPT scores, no significant differences were found between high and low scoring groups with respect to Addenbrooke's Cognitive Examination scores, Mini-mental State Examination scores, or the depression score of the Neuropsychiatric Inventory subscale. Compared to patients with high scores on the KPT, AD patients with low scores on the KPT showed significant hypoperfusion in the left subgenual cingulate gyrus (SGC) extending to the right SGC. CONCLUSIONS: Our results suggest that functional activity of the SGC is closely related to scores on the KPT. KPT might be a promising strategy to use in detecting early stages of AD with low SGC function.