Publications by authors named "Ikuo Tooyama"

143 Publications

Lactoferrin-like Immunoreactivity in Distinct Neuronal Populations in the Mouse Central Nervous System.

Acta Med Okayama 2021 Apr;75(2):153-167

Molecular Neuroscience Research Center, Shiga University of Medical Science.

Lactoferrin (Lf) is an iron-binding glycoprotein mainly found in exocrine secretions and the secondary granules of neutrophils. In the central nervous system (CNS), expression of the Lf protein has been reported in the lesions of some neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, as well as in the aged brain. Lf is primarily considered an iron chelator, protecting cells from potentially toxic iron or iron-requiring microorganisms. Other biological functions of Lf include immunomodulation and transcriptional regulation. However, the roles of Lf in the CNS have yet to be fully clarified. In this study, we raised an antiserum against mouse Lf and investigated the immunohistochemical localization of Lf-like immunoreactivity (Lf-LI) throughout the CNS of adult mice. Lf-LI was found in some neuronal populations throughout the CNS. Intense labeling was found in neurons in the olfactory systems, hypothalamic nuclei, entorhinal cortex, and a variety of brainstem nuclei. This study provides detailed information on the Lf-LI distribution in the CNS, and the findings should promote further understanding of both the physiological and pathological significance of Lf in the CNS.
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http://dx.doi.org/10.18926/AMO/61894DOI Listing
April 2021

Differential accumulation of tau pathology between reciprocal F1 hybrids of rTg4510 mice.

Sci Rep 2021 May 5;11(1):9623. Epub 2021 May 5.

Molecular Neuroscience Research Center, Shiga University of Medical Science, Seta Tsukinowa-Cho, Otsu, 520-2192, Japan.

Tau, a family of microtubule-associated proteins, forms abnormal intracellular inclusions, so-called tau pathology, in a range of neurodegenerative diseases collectively known as tauopathies. The rTg4510 mouse model is a well-characterized bitransgenic F1 hybrid mouse model of tauopathy, which was obtained by crossing a Camk2α-tTA mouse line (on a C57BL/6 J background) with a tetO-MAPT*P301L mouse line (on a FVB/NJ background). The aim of this study was to investigate the effects of the genetic background and sex on the accumulation of tau pathology in reciprocal F1 hybrids of rTg4510 mice, i.e., rTg4510 on the (C57BL/6 J × FVB/NJ)F1 background (rTg4510_CxF) and on the (FVB/NJ × C57BL/6 J)F1 background (rTg4510_FxC). As compared with rTg4510_CxF mice, the rTg4510_FxC mice showed marked levels of tau pathology in the forebrain. Biochemical analyses indicated that the accumulation of abnormal tau species was accelerated in rTg4510_FxC mice. There were strong effects of the genetic background on the differential accumulation of tau pathology in rTg4510 mice, while sex had no apparent effect. Interestingly, midline-1 (Mid1) was identified as a candidate gene associated with this difference and exhibited significant up/downregulation according to the genetic background. Mid1 silencing with siRNA induced pathological phosphorylation of tau in HEK293T cells that stably expressed human tau with the P301L mutation, suggesting the role of Mid1 in pathological alterations of tau. Elucidation of the underlying mechanisms will provide novel insights into the accumulation of tau pathology and is expected to be especially informative to researchers for the continued development of therapeutic interventions for tauopathies.
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http://dx.doi.org/10.1038/s41598-021-89142-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8100160PMC
May 2021

Gm14230 controls Tbc1d24 cytoophidia and neuronal cellular juvenescence.

PLoS One 2021 22;16(4):e0248517. Epub 2021 Apr 22.

Molecular Neuroscience Research Center (MNRC), Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, Shiga, Japan.

It is not fully understood how enzymes are regulated in the tiny reaction field of a cell. Several enzymatic proteins form cytoophidia, a cellular macrostructure to titrate enzymatic activities. Here, we show that the epileptic encephalopathy-associated protein Tbc1d24 forms cytoophidia in neuronal cells both in vitro and in vivo. The Tbc1d24 cytoophidia are distinct from previously reported cytoophidia consisting of inosine monophosphate dehydrogenase (Impdh) or cytidine-5'-triphosphate synthase (Ctps). Tbc1d24 cytoophidia is induced by loss of cellular juvenescence caused by depletion of Gm14230, a juvenility-associated lncRNA (JALNC) and zeocin treatment. Cytoophidia formation is associated with impaired enzymatic activity of Tbc1d24. Thus, our findings reveal the property of Tbc1d24 to form cytoophidia to maintain neuronal cellular juvenescence.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0248517PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8062039PMC
April 2021

Fluorine-19 Magnetic Resonance Imaging for Detection of Amyloid β Oligomers Using a Keto Form of Curcumin Derivative in a Mouse Model of Alzheimer's Disease.

Molecules 2021 Mar 4;26(5). Epub 2021 Mar 4.

Molecular Neuroscience Research Center, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu 520-2192, Japan.

Recent evidence suggests that the formation of soluble amyloid β (Aβ) aggregates with high toxicity, such as oligomers and protofibrils, is a key event that causes Alzheimer's disease (AD). However, understanding the pathophysiological role of such soluble Aβ aggregates in the brain in vivo could be difficult due to the lack of a clinically available method to detect, visualize, and quantify soluble Aβ aggregates in the brain. We had synthesized a novel fluorinated curcumin derivative with a fixed keto form, named as Shiga-Y51, which exhibited high selectivity to Aβ oligomers in vitro. In this study, we investigated the in vivo detection of Aβ oligomers by fluorine-19 (F) magnetic resonance imaging (MRI) using Shiga-Y51 in an APP/PS1 double transgenic mouse model of AD. Significantly high levels of F signals were detected in the upper forebrain region of APP/PS1 mice compared with wild-type mice. Moreover, the highest levels of Aβ oligomers were detected in the upper forebrain region of APP/PS1 mice in enzyme-linked immunosorbent assay. These findings suggested that F-MRI using Shiga-Y51 detected Aβ oligomers in the in vivo brain. Therefore, F-MRI using Shiga-Y51 with a 7 T MR scanner could be a powerful tool for imaging Aβ oligomers in the brain.
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http://dx.doi.org/10.3390/molecules26051362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7961357PMC
March 2021

Keto form of curcumin derivatives strongly binds to Aβ oligomers but not fibrils.

Biomaterials 2021 Mar 23;270:120686. Epub 2021 Jan 23.

Molecular Neuroscience Research Center, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, 520-2192, Japan. Electronic address:

The accumulation of β-amyloid (Aβ) aggregates in the brain occurs early in the progression of Alzheimer's disease (AD), and non-fibrillar soluble Aβ oligomers are particularly neurotoxic. During binding to Aβ fibrils, curcumin, which can exist in an equilibrium state between its keto and enol tautomers, exists predominantly in the enol form, and binding activity of the keto form to Aβ fibrils is much weaker. Here we described the strong binding activity the keto form of curcumin derivative Shiga-Y51 shows for Aβ oligomers and its scant affinity for Aβ fibrils. Furthermore, with imaging mass spectrometry we revealed the blood-brain barrier permeability of Shiga-Y51 and its accumulation in the cerebral cortex and the hippocampus, where Aβ oligomers were mainly localized, in a mouse model of AD. The keto form of curcumin derivatives like Shiga-Y51 could be promising seed compounds to develop imaging probes and therapeutic agents targeting Aβ oligomers in the brain.
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http://dx.doi.org/10.1016/j.biomaterials.2021.120686DOI Listing
March 2021

Seven-Day Pedometer-Assessed Step Counts and Brain Volume: A Population-Based Observational Study.

J Phys Act Health 2021 Jan 11;18(2):157-164. Epub 2021 Jan 11.

Background: To investigate the association between step counts and brain volumes (BVs)-global and 6 a priori selected cognition-related regions of interest-in Japanese men aged 40-79 years.

Methods: The authors analyzed data from 680 cognitively intact participants of the Shiga Epidemiological Study of Subclinical Atherosclerosis-a population-based observational study. Using multivariable linear regression, the authors assessed cross-sectional associations between 7-day step counts at baseline (2006-2008) and BVs at follow-up (2012-2015) for age-stratified groups (<60 y and ≥60 y).

Results: In the older adults ≥60 years, step counts at baseline (per 1000 steps) were associated with total BV at follow-up (β = 1.42, P = .022) while adjusted for potential covariates. Regions of interest-based analyses yielded an association of step counts with both prefrontal cortexes (P < .05) in older adults, while the left entorhinal cortex showed marginally significant association (P = .05). No association was observed with hippocampus, parahippocampal, cingulum, and cerebellum. No association was observed in younger adults (<60 y).

Conclusions: The authors found a positive association between 7-day step counts and BVs, including prefrontal cortexes, and left entorhinal cortex in apparently healthy Japanese men.
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http://dx.doi.org/10.1123/jpah.2019-0659DOI Listing
January 2021

Thioredoxin-Interacting Protein (TXNIP) with Focus on Brain and Neurodegenerative Diseases.

Int J Mol Sci 2020 Dec 8;21(24). Epub 2020 Dec 8.

Molecular Neuroscience Research Center, Shiga University of Medical Science, Seta-Tsukinowa, Otsu 525-0072, Shiga, Japan.

The development of new therapeutic approaches to diseases relies on the identification of key molecular targets involved in amplifying disease processes. One such molecule is thioredoxin-interacting protein (TXNIP), also designated thioredoxin-binding protein-2 (TBP-2), a member of the α-arrestin family of proteins and a central regulator of glucose and lipid metabolism, involved in diabetes-associated vascular endothelial dysfunction and inflammation. TXNIP sequesters reduced thioredoxin (TRX), inhibiting its function, resulting in increased oxidative stress. Many different cellular stress factors regulate TXNIP expression, including high glucose, endoplasmic reticulum stress, free radicals, hypoxia, nitric oxide, insulin, and adenosine-containing molecules. TXNIP is also directly involved in inflammatory activation through its interaction with the nucleotide-binding domain, leucine-rich-containing family, and pyrin domain-containing-3 (NLRP3) inflammasome complex. Neurodegenerative diseases such as Alzheimer's disease have significant pathologies associated with increased oxidative stress, inflammation, and vascular dysfunctions. In addition, as dysfunctions in glucose and cellular metabolism have been associated with such brain diseases, a role for TXNIP in neurodegeneration has actively been investigated. In this review, we will focus on the current state of the understanding of possible normal and pathological functions of TXNIP in the central nervous system from studies of in vitro neural cells and the brains of humans and experimental animals with reference to other studies. As TXNIP can be expressed by neurons, microglia, astrocytes, and endothelial cells, a complex pattern of regulation and function in the brain is suggested. We will examine data suggesting TXNIP as a therapeutic target for neurodegenerative diseases where further research is needed.
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http://dx.doi.org/10.3390/ijms21249357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7764580PMC
December 2020

Alcohol drinking and brain morphometry in apparently healthy community-dwelling Japanese men.

Alcohol 2021 02 3;90:57-65. Epub 2020 Dec 3.

Shiga University of Medical Science, Otsu, Shiga, Japan.

The clinical implications of alcohol consumption have been extensively examined; however, its effects on brain structures in apparently healthy community-dwellers remain unclear. Therefore, we investigated the relationship between alcohol consumption and brain gray matter volume (GMV) in community-dwelling Japanese men using voxel-based morphometry (VBM). We recruited cognitively intact Japanese men, aged 40-79 years, from a population-based cohort in Shiga, Japan. Brain magnetic resonance imaging was performed, on average, 2 years after demographic and medical information was obtained in 2010-2014. A multivariable linear regression analysis of 639 men was conducted to elucidate the relationship between the amount of alcohol consumed and GMV. VBM statistics were analyzed by threshold-free cluster enhancement with a family-wise error rate of <0.05. The results obtained demonstrated that the amount of alcohol consumed was associated with lower GMV. The VBM analysis showed lower GMV within the parahippocampal, entorhinal, cingulate, insular, temporal, and frontal cortices and cerebellum in very heavy drinkers (≥42 ethanol g/day) than in non-drinkers. Furthermore, alcohol consumption was associated with a higher white matter lesion volume. These results suggest subclinical structural changes similar to alcohol-related neurological diseases.
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http://dx.doi.org/10.1016/j.alcohol.2020.11.006DOI Listing
February 2021

Neuroepithelial cell competition triggers loss of cellular juvenescence.

Sci Rep 2020 10 22;10(1):18044. Epub 2020 Oct 22.

Molecular Neuroscience Research Center (MNRC), Shiga University of Medical Science, Seta Tsukinowa-Cho, Otsu, Shiga, 520-2192, Japan.

Cell competition is a cell-cell interaction mechanism which maintains tissue homeostasis through selective elimination of unfit cells. During early brain development, cells are eliminated through apoptosis. How cells are selected to undergo elimination remains unclear. Here we aimed to identify a role for cell competition in the elimination of suboptimal cells using an in vitro neuroepithelial model. Cell competition was observed when neural progenitor HypoE-N1 cells expressing RAS were surrounded by normal cells in the co-culture. The elimination through apoptosis was observed by cellular changes of RAS cells with rounding/fragmented morphology, by SYTOX blue-positivity, and by expression of apoptotic markers active caspase-3 and cleaved PARP. In this model, expression of juvenility-associated genes Srsf7 and Ezh2 were suppressed under cell-competitive conditions. Srsf7 depletion led to loss of cellular juvenescence characterized by suppression of Ezh2, cell growth impairment and enhancement of senescence-associated proteins. The cell bodies of eliminated cells were engulfed by the surrounding cells through phagocytosis. Our data indicates that neuroepithelial cell competition may have an important role for maintaining homeostasis in the neuroepithelium by eliminating suboptimal cells through loss of cellular juvenescence.
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http://dx.doi.org/10.1038/s41598-020-74874-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582913PMC
October 2020

A Mechanistic Evaluation of Antioxidant Nutraceuticals on Their Potential against Age-Associated Neurodegenerative Diseases.

Antioxidants (Basel) 2020 Oct 20;9(10). Epub 2020 Oct 20.

Department of Biochemistry, Faculty of Medicine, UKM Medical Centre, Universiti Kebangsaan Malaysia, Cheras, Kuala Lumpur 56000, Malaysia.

Nutraceuticals have been extensively studied worldwide due to its neuroprotective effects in in vivo and in vitro studies, attributed by the antioxidative properties. Alzheimer (AD) and Parkinson disease (PD) are the two main neurodegenerative disorders that are discussed in this review. Both AD and PD share the similar involvement of oxidative stress in their pathophysiology. Nutraceuticals exert their antioxidative effects via direct scavenging of free radicals, prevent damage to biomolecules, indirectly stimulate the endogenous antioxidative enzymes and gene expressions, inhibit activation of pro-oxidant enzymes, and chelate metals. In addition, nutraceuticals can act as modulators of pro-survival, pro-apoptotic, and inflammatory signaling pathways. They have been shown to be effective particularly in preclinical stages, due to their multiple mechanisms of action in attenuating oxidative stress underlying AD and PD. Natural antioxidants from food sources and natural products such as resveratrol, curcumin, green tea polyphenols, and vitamin E are promising therapeutic agents in oxidative stress-mediated neurodegenerative disease as they have fewer adverse effects, more tolerable, cheaper, and sustainable for long term consumption.
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http://dx.doi.org/10.3390/antiox9101019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7588884PMC
October 2020

Neuroprotective Effects of Casein-Derived Peptide Met-Lys-Pro (MKP) in a Hypertensive Model.

Front Neurosci 2020 14;14:845. Epub 2020 Aug 14.

Molecular Neuroscience Research Center, Shiga University of Medical Science, Otsu, Japan.

We have previously reported that casein hydrolysate, CH-3, from bovine milk and casein-derived tripeptide Met-Lys-Pro (MKP) has ACE inhibitory activity and reduces blood pressure. In this study, we investigated the therapeutic effects of MKP in a hypertensive rat model (7-week-old male SHRSP/Izm rats). For long term evaluation, rats were fed either a diet containing CH-3 or normal diet. The survival rate of SHRSP rats was significantly improved by intake of CH-3 for 181 days. For short term evaluation, rats were orally administered synthetic tripeptide MKP or distilled water for 4 weeks. MRI study demonstrated that hemorrhagic lesions were observed in two of five rats in the control group, while no hemorrhagic lesions were observed in the MKP group. Volumetric analysis using MRI revealed that MKP administration inhibited atrophy of diencephalic regions. Histological examinations revealed that hemorrhage areas and astrogliosis in the hippocampus and cerebral cortex were lower in the MKP group than in the control group. Gene expression analysis indicated that MKP administration reduced expression of genes related to cerebral circulation insufficiency such as immune responses ( and ), response to hypoxia (, , and ), reactive oxygen species metabolic process ( and ), and apoptotic process (, , and ), suggesting that MKP administration prevented cerebral ischemia associated with hypertension. In addition, some genes encoding responses to hormone stimulus (, , and ) were also downregulated. Serum aldosterone and corticosterone levels were also significantly decreased following MKP administration. The present study indicates that MKP shows neuroprotective effects in SHRSP rats by regulating cerebral circulation insufficiency and corticoid levels. MKP administration may therefore be a potential therapeutic strategy for hypertensive brain diseases such as cerebrovascular disease.
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http://dx.doi.org/10.3389/fnins.2020.00845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7457086PMC
August 2020

Novel fluorinated derivative of curcumin negatively regulates thioredoxin-interacting protein expression in retinal pigment epithelial and macrophage cells.

Biochem Biophys Res Commun 2020 11 7;532(4):668-674. Epub 2020 Sep 7.

Molecular Neuroscience Research Center, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, Shiga, 520-2192, Japan. Electronic address:

Thioredoxin-interacting protein (TXNIP) has multiple disease-associated functions including inducing oxidative stress by inhibiting the anti-oxidant and thiol reducing activity of thioredoxin (TRX), reducing cellular glucose transport, and is a component of the activated inflammasome complex. Increased expression of TXNIP is encountered in diabetic conditions of high glucose. Curcumin and chemical derivatives have multiple therapeutic properties as anti-inflammatories, anti-oxidants, amyloid aggregation inhibitors and modulate a number of cellular signaling pathways. Using a fluorinated-derivative of curcumin (designated Shiga-Y6), we showed significant inhibition of TXNIP mRNA and protein expression, and induction of TRX mRNA and protein in ARPE-19 retinal pigment epithelial cells and THP-1-derived macrophages, while the non-fluorinated structural equivalent (Shiga-Y52) and native curcumin did not show these same effects. Shiga-Y6 was effective in reducing high glucose, endoplasmic reticulum stress-induced TXNIP in ARPE-19 cells, and reducing lipopolysaccharide and endoplasmic stress-induced proinflammatory gene expression in THP-1 macrophages. Moreover, TXNIP-knockdown experiments showed that the anti-inflammatory effect of Shiga-Y6 in LPS-stimulated THP-1 macrophages was TXNIP-independent.
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http://dx.doi.org/10.1016/j.bbrc.2020.08.114DOI Listing
November 2020

A F-MRI probe for the detection of Fe(ii) ions in an aqueous system.

Org Biomol Chem 2020 Aug;18(30):5843-5849

Laboratory of Pharmaceutical and Medicinal Chemistry, Gifu Pharmaceutical University, 1-25-4, Daigaku-nishi, Gifu-shi, Gifu, 501-1196, Japan.

Iron deposits are often observed in the brains of patients with neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. This study outlines the development of F-Nox-1 as the first example of a 19F-MRI probe that can selectively detect Fe(ii) in aqueous solutions. The use of tetrafluoro-p-phenylenediamine (TFPDA) as a 19F signal emitter with an Fe(ii)-selective chemical switch, based on our previously reported N-oxide chemistry, yielded a readout of a symmetry-dependent 19F signal change in response to Fe(ii). The addition of Fe(ii) ions to F-Nox-1 triggered a 19F signal change, both in the chemical shift and signal intensity, and the response was highly selective to Fe(ii) over other biologically relevant metal ions. The probe could also detect Fe(ii) in serum containing various biological contaminants by 19F magnetic resonance imaging (19F-MRI). Imaging of soluble Fe(ii) species, which is the major component of water-soluble iron species, by 19F-MRI will potentially enable the direct monitoring of the elevation of Fe(ii) levels prior to the formation of iron deposits, which is a potential risk factor for neurodegenerative diseases.
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http://dx.doi.org/10.1039/d0ob00903bDOI Listing
August 2020

Effects of FTMT Expression by Retinal Pigment Epithelial Cells on Features of Angiogenesis.

Int J Mol Sci 2020 May 21;21(10). Epub 2020 May 21.

Molecular Neuroscience Research Center, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, Shiga 520-2192, Japan.

Aberrant angiogenesis is a pathological feature of a number of diseases and arises from the uncoordinated expression of angiogenic factors as response to different cellular stresses. Age-related macular degeneration (AMD), a leading cause of vision loss, can result from pathological angiogenesis. As a mutation in the mitochondrial ferritin (FTMT) gene has been associated with AMD, its possible role in modulating angiogenic factors and angiogenesis was investigated. FTMT is an iron-sequestering protein primarily expressed in metabolically active cells and tissues with high oxygen demand, including retina. In this study, we utilized the human retinal pigment epithelial cell line ARPE-19, both as undifferentiated and differentiated cells. The effects of proinflammatory cytokines, FTMT knockdown, and transient and stable overexpression of FTMT were investigated on expression of pro-angiogenic vascular endothelial growth factor (VEGF) and anti-angiogenic pigment epithelial-derived factor (PEDF). Proinflammatory cytokines induced FTMT and VEGF expression, while NF-κB inhibition significantly reduced FTMT expression. VEGF protein and mRNA expression were significantly increased in FTMT-silenced ARPE-19 cells. Using an in vitro angiogenesis assay with endothelial cells, we showed that conditioned media from FTMT-overexpressing cells had significant antiangiogenic effects. Collectively, our findings indicate that increased levels of FTMT inhibit angiogenesis, possibly by reducing levels of VEGF and increasing PEDF expression. The cellular models developed can be used to investigate if increased FTMT may be protective in angiogenic diseases, such as AMD.
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http://dx.doi.org/10.3390/ijms21103635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7279371PMC
May 2020

Generation of Transgenic Cynomolgus Monkeys Overexpressing the Gene for Amyloid-β Precursor Protein.

J Alzheimers Dis 2020 ;75(1):45-60

Molecular Neuroscience Research Center, Shiga University of Medical Science, Seta, Tsukinowa-cho, Otsu, Shiga, Japan.

Alzheimer's disease (AD) is the most common cause of dementia and understanding its pathogenesis should lead to improved therapeutic and diagnostic methods. Although several groups have developed transgenic mouse models overexpressing the human amyloid-β precursor protein (APP) gene with AD mutations, with and without presenilin mutations, as well as APP gene knock-in mouse models, these animals display amyloid pathology but do not show neurofibrillary tangles or neuronal loss. This presumably is due to differences between the etiology of the aged-related human disease and the mouse models. Here we report the generation of two transgenic cynomolgus monkeys overexpressing the human gene for APP with Swedish, Artic, and Iberian mutations, and demonstrated expression of gene tagged green fluorescent protein marker in the placenta, amnion, hair follicles, and peripheral blood. We believe that these nonhuman primate models will be very useful to study the pathogenesis of dementia and AD. However, generated Tg monkeys still have some limitations. We employed the CAG promoter, which will promote gene expression in a non-tissue specific manner. Moreover, we used transgenic models but not knock-in models. Thus, the inserted transgene destroys endogenous gene(s) and may affect the phenotype(s). Nevertheless, it will be of great interest to determine whether these Tg monkeys will develop tauopathy and neurodegeneration similar to human AD.
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http://dx.doi.org/10.3233/JAD-191081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7306892PMC
May 2021

Srsf7 Establishes the Juvenile Transcriptome through Age-Dependent Alternative Splicing in Mice.

iScience 2020 Mar 22;23(3):100929. Epub 2020 Feb 22.

Molecular Neuroscience Research Center (MNRC), Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, Shiga 520-2192, Japan. Electronic address:

The juvenile phase is characterized by continuously progressing physiological processes such as growth and maturation, which are accompanied by transitions in gene expression. The contribution of transcriptome dynamics to the establishment of juvenile properties remains unclear. Here, we investigated alternative splicing (AS) events in postnatal growth and elucidated the landscape of age-dependent alternative splicing (ADAS) in C57BL/6 mice. Our analysis of ADAS in the cerebral cortex, cardiomyocytes, and hepatocytes revealed numerous juvenile-specific splicing isoforms that shape the juvenile transcriptome, which in turn functions as a basis for the highly anabolic status of juvenile cells. Mechanistically, the juvenile-expressed splicing factor Srsf7 mediates ADAS, as exemplified by switching from juvenile to adult forms of anabolism-associated genes Eif4a2 and Rbm7. Suppression of Srsf7 results in "fast-forwarding" of this transcriptome transition, causing impaired anabolism and growth in mice. Thus, juvenile-specific AS is indispensable for the anabolic state of juveniles and differentiates juveniles from adults.
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http://dx.doi.org/10.1016/j.isci.2020.100929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7063262PMC
March 2020

Patterns of Expression of Purinergic Receptor P2RY12, a Putative Marker for Non-Activated Microglia, in Aged and Alzheimer's Disease Brains.

Int J Mol Sci 2020 Jan 20;21(2). Epub 2020 Jan 20.

Neurodegenerative Disease Research Center, Arizona State University, Tempe, AZ 85287, USA.

Neuroinflammation is considered a key pathological process in neurodegenerative diseases of aging, including Alzheimer's disease (AD). Many studies have defined phenotypes of reactive microglia, the brain-resident macrophages, with different antigenic markers to identify those potentially causing inflammatory damage. We took an alternative approach with the goal of characterizing the distribution of purinergic receptor P2RY12-positive microglia, a marker previously defined as identifying homeostatic or non-activated microglia. We examined the expression of P2RY12 by dual-color light and fluorescence immunohistochemistry using sections of middle temporal gyrus from AD, high plaque and low plaque non-demented cases in relation to amyloid beta (Aβ) plaques and phosphorylated tau, markers of pathology, and HLA-DR, IBA-1, CD68, and progranulin, microglial phenotype markers. In low plaque cases, P2RY12-positive microglia mostly had non-activated morphologies, while the morphologies of P2RY12-positive microglia in AD brains were highly variable, suggesting its expression could encompass a wider range of phenotypes than originally hypothesized. P2RY12 expression by microglia differed depending on the types of plaques or tangles they were associated with. Areas of inflammation characterized by lack of P2RY12-positive microglia around mature plaques could be observed, but many diffuse plaques showed colocalization with P2RY12-positive microglia. Based on these results, P2RY12 expression by microglia should not be considered solely a marker of resting microglia as P2RY12 immunoreactivity was identifying microglia positive for CD68, progranulin and to a limited extent HLA-DR, markers of activation.
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http://dx.doi.org/10.3390/ijms21020678DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7014248PMC
January 2020

Alcohol consumption and cognitive function in elderly Japanese men.

Alcohol 2020 06 7;85:145-152. Epub 2020 Jan 7.

Center for Epidemiologic Research in Asia, Shiga University of Medical Science, Otsu, Japan; Department of Public Health, Shiga University of Medical Science, Otsu, Japan.

Although heavy alcohol consumption has been identified as a risk factor for adverse cognitive functioning, it currently remains unclear whether moderate alcohol consumption exerts similar effects. Observational studies previously reported the potential benefits of moderate alcohol consumption on cognition, particularly in the elderly; however, these effects have not yet been demonstrated in Asian populations. The aim of the present study was to investigate the relationship between alcohol consumption levels and global and domain-specific cognitive functions in cognitively intact elderly Japanese men. Cross-sectional data from the Shiga Epidemiological Study of Subclinical Atherosclerosis (SESSA), an ongoing prospective, population-based study in Shiga, Japan, were used to examine the relationship between alcohol consumption and cognitive function. Men (n = 585) aged ≥65 years provided information on their weekly consumption of alcohol, and the data obtained were used to construct categories of never, ex- (quit before interview), very light (<14 g/day), light (14-23 g/day), moderate (>23-46 g/day), and heavy (>46 g/day) drinkers. Cognitive function was measured using the Cognitive Abilities Screening Instrument (CASI). A fractional logistic regression model adjusted for age, education, body mass index, smoking, exercise, hypertension, diabetes, and dyslipidemia showed that the CASI scores for global and domain-specific cognitive functions were not significantly different between all subgroups of current drinkers and never-drinkers. However, the CASI score of ex-drinkers (multivariable adjusted mean CASI score [SD]) was significantly lower than that of never-drinkers in the global [never vs. ex: 90.16 (2.21) vs. 88.26 (2.58)] and abstraction and judgment domain [never vs. ex: 9.48 (0.46) vs. 8.61 (0.57)]). The present results do not suggest any beneficial or adverse relationship between current alcohol consumption levels and cognitive functioning (both global and domain specific) in elderly Japanese men; however, low cognitive function among ex-drinkers warrants future investigations to identify the factors causing drinkers to quit.
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http://dx.doi.org/10.1016/j.alcohol.2020.01.001DOI Listing
June 2020

Characterization of lysosomal proteins Progranulin and Prosaposin and their interactions in Alzheimer's disease and aged brains: increased levels correlate with neuropathology.

Acta Neuropathol Commun 2019 12 21;7(1):215. Epub 2019 Dec 21.

Molecular Neuroscience Research Center, Shiga University of Medical Science, Seta, Otsu, Otsu, 520-2192, Japan.

Progranulin (PGRN) is a protein encoded by the GRN gene with multiple identified functions including as a neurotrophic factor, tumorigenic growth factor, anti-inflammatory cytokine and regulator of lysosomal function. A single mutation in the human GRN gene resulting in reduced PGRN expression causes types of frontotemporal lobar degeneration resulting in frontotemporal dementia. Prosaposin (PSAP) is also a multifunctional neuroprotective secreted protein and regulator of lysosomal function. Interactions of PGRN and PSAP affect their functional properties. Their roles in Alzheimer's disease (AD), the leading cause of dementia, have not been defined. In this report, we examined in detail the cellular expression of PGRN in middle temporal gyrus samples of a series of human brain cases (n = 45) staged for increasing plaque pathology. Immunohistochemistry showed PGRN expression in cortical neurons, microglia, cerebral vessels and amyloid beta (Aβ) plaques, while PSAP expression was mainly detected in neurons and Aβ plaques, and to a limited extent in astrocytes. We showed that there were increased levels of PGRN protein in AD cases and corresponding increased levels of PSAP. Levels of PGRN and PSAP protein positively correlated with amyloid beta (Aβ), with PGRN levels correlating with phosphorylated tau (serine 205) levels in these samples. Although PGRN colocalized with lysosomal-associated membrane protein-1 in neurons, most PGRN associated with Aβ plaques did not. Aβ plaques with PGRN and PSAP deposits were identified in the low plaque non-demented cases suggesting this was an early event in plaque formation. We did not observe PGRN-positive neurofibrillary tangles. Co-immunoprecipitation studies of PGRN from brain samples identified only PSAP associated with PGRN, not sortilin or other known PGRN-binding proteins, under conditions used. Most PGRN associated with Aβ plaques were immunoreactive for PSAP showing a high degree of colocalization of these proteins that did not change between disease groups. As PGRN supplementation has been considered as a therapeutic approach for AD, the possible involvement of PGRN and PSAP interactions in AD pathology needs to be further considered.
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http://dx.doi.org/10.1186/s40478-019-0862-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925443PMC
December 2019

Modulation of Proteome Profile in AβPP/PS1 Mice Hippocampus, Medial Prefrontal Cortex, and Striatum by Palm Oil Derived Tocotrienol-Rich Fraction.

J Alzheimers Dis 2019 ;72(1):229-246

Molecular Neuroscience Research Center, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, Japan.

Tocotrienol-rich fraction (TRF) is a mixture of vitamin E analogs derived from palm oil. We previously demonstrated that supplementation with TRF improved cognitive function and modulated amyloid pathology in AβPP/PS1 mice brains. The current study was designed to examine proteomic profiles underlying the therapeutic effect of TRF in the brain. Proteomic analyses were performed on samples of hippocampus, medial prefrontal cortex (mPFC), and striatum using liquid chromatography coupled to Q Exactive HF Orbitrap mass spectrometry. From these analyses, we profiled a total of 5,847 proteins of which 155 proteins were differentially expressed between AβPP/PS1 and wild-type mice. TRF supplementation of these mice altered the expression of 255 proteins in the hippocampus, mPFC, and striatum. TRF also negatively modulated the expression of amyloid beta A4 protein and receptor-type tyrosine-protein phosphatase alpha protein in the hippocampus. The expression of proteins in metabolic pathways, oxidative phosphorylation, and those involved in Alzheimer's disease were altered in the brains of AβPP/PS1 mice that received TRF supplementation.
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http://dx.doi.org/10.3233/JAD-181171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6839455PMC
November 2020

Microglial Phenotyping in Neurodegenerative Disease Brains: Identification of Reactive Microglia with an Antibody to Variant of CD105/Endoglin.

Cells 2019 07 23;8(7). Epub 2019 Jul 23.

Molecular Neuroscience Research Center, Shiga University of Medical Science, Otsu 520-2192, Japan.

Inflammation is considered a key pathological process in neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD), but there are still mechanisms not understood. In the brain, most microglia are performing essential homeostatic functions, but can also respond to pathogenic stimuli by producing harmful pro-inflammatory cytokines or free radicals. Distinguishing between damaging and homeostatic microglia in human diseased brain tissues is a challenge. This report describes findings using a monoclonal antibody to CD105/Endoglin (R&D Systems MAB1097) that identifies subtypes of activated microglia. CD105/Endoglin is a co-receptor for transforming growth factor beta (TGFβ) receptor that antagonizes TGFβ signaling. CD105/Endoglin is a marker for vascular endothelial cells, but was originally identified as a marker for activated macrophages. This antibody did not identify endothelial cells in brain sections, only microglia-like cells. In this study, we examined with this antibody tissue section from middle temporal gyrus derived from human brains from normal control subjects with low-plaque pathology, high-plaque pathology, and AD cases, and also substantia nigra samples from control and PD cases, in conjunction with antibodies to markers of pathology and microglia. In low-plaque pathology cases, CD105-positive microglia were mostly absent, but noticeably increased with increasing pathology. CD105-positive cells strongly colocalized with amyloid-beta plaques, but not phosphorylated tau positive tangles. In substantia nigra, strong microglial CD105 staining was observed in microglia associated with degenerating dopaminergic neurons and neuromelanin. In PD cases with few surviving dopaminergic neurons, this staining had decreased. By Western blot, this antibody identified polypeptide bands of 70 kDa in brain samples, and samples from microglia, macrophages, and brain endothelial cells. In comparison with other tested CD105 antibodies, this antibody did not recognize the glycosylated forms of CD105 on Western blots. Overall, the data indicate that this antibody and this marker could have utility for subtyping of microglia in pathologically-involved tissue.
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http://dx.doi.org/10.3390/cells8070766DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678308PMC
July 2019

Transcriptome analysis revealed bisphenol A and nonylphenol affect reproduction.

Reprod Toxicol 2019 09 6;88:39-47. Epub 2019 Jul 6.

Laboratory of Cell Engineering, Department of Pharmaceutical Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan. Electronic address:

Effects of endocrine disrupting chemicals (EDCs) on reproduction have not been fully explained comprehensively. In this study, we tried to validate the common effect of Bisphenol A (BPA) and Nonylphenol (NP) on the differentiation of embryonic stem (ES) cells and found that they modify the expression of germ cell specific genes. To elucidate functional significance on biological process, we performed Gene Ontology (GO)-based microarray analysis comparing with published GeneChip data of primordial germ cell development in vivo. Cluster analysis of gene expression profile revealed that EDC treatment and primordial germ cell (PGC) development shared characteristic cluster consists of GO terms related to "germ cell development" and "reproduction". In the GO term "reproduction", meiosis related genes showed high expression level by EDC exposure. These results suggest that BPA and NP affect not only some of the germ cell specific genes, but functionally interferes germ cell development and reproduction.
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http://dx.doi.org/10.1016/j.reprotox.2019.06.006DOI Listing
September 2019

Proteinuria and Reduced Estimated Glomerular Filtration Rate are Independently Associated With Lower Cognitive Abilities in Apparently Healthy Community-Dwelling Elderly Men in Japan: A Cross-sectional Study.

J Epidemiol 2020 Jun 25;30(6):244-252. Epub 2019 May 25.

Department of Public Health, Shiga University of Medical Science.

Background: The association of proteinuria and reduced estimated glomerular filtration rate (eGFR) with cognition needs more clarification. We cross-sectionally examined whether proteinuria and reduced eGFR, even in moderate stages, were independently associated with lower cognition in a community-based sample of elderly men.

Methods: Our cohort initially comprised 1,094 men aged 40-79 years from a random sample from Shiga, Japan in 2006-2008. Of 853 men who returned for the follow-up examination (2009-2014), we analyzed 561 who were ≥65 years, free of stroke, and completed the Cognitive Abilities Screening Instrument (CASI) at follow-up (higher CASI scores [range 0 to 100] indicate better cognition). Proteinuria was assessed via dipstick. eGFR was calculated according to the Chronic Kidney Disease Epidemiology Collaboration Equation. Participants were divided into three groups either by eGFR (≥60, 59-40, and <40 mL/min/1.73 m) or by proteinuria (no, trace, and positive), considered normal, moderate, and advanced, respectively. Using linear regression, we computed mean CASI score, with simultaneous adjustment for proteinuria and eGFR in addition to other potential confounders.

Results: Significant trends of lower cognition were observed across the groups of worse proteinuria and lower eGFR independently: multivariable-adjusted mean CASI scores were 90.1, 89.3, and 88.4 for proteinuria (P = 0.029), and 90.0, 88.5, and 88.5 for eGFR (P = 0.015) in mutual-adjustment model.

Conclusions: Proteinuria and reduced eGFR, even in their moderate stages, were independently associated with lower cognition in a community-based sample of elderly men. The results suggest the importance of proteinuria and low eGFR for early detection and prevention of cognitive decline.
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http://dx.doi.org/10.2188/jea.JE20180258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7217691PMC
June 2020

Elevated Fasting Blood Glucose Levels Are Associated With Lower Cognitive Function, With a Threshold in Non-Diabetic Individuals: A Population-Based Study.

J Epidemiol 2020 Mar 25;30(3):121-127. Epub 2019 May 25.

Department of Public Health, Shiga University of Medical Science.

Background: Cognitive dysfunction has been recognized as a diabetes-related complication. Whether hyperglycemia or elevated fasting glucose are associated with cognitive decline remains controversial. We aimed to investigate the relationship between fasting glucose levels and cognitive function in diabetic and non-diabetic individuals.

Methods: Participants were Japanese diabetic (n = 191) and non-diabetic (n = 616) men, aged 46-81 years, from 2010-2014. Blood samples were taken after a 12 h fast. The Cognitive Ability Screening Instrument (CASI), with a maximum score of 100, was used for cognitive assessment. Cognitive domains of CASI were also investigated. Fractional logit regression with covariate adjustment for potential confounders was used to model cross-sectional relationships between fasting blood glucose and CASI score.

Results: For diabetic individuals, CASI score was 0.38 (95% confidence interval: 0.66-0.12) lower per 1 mmol/L higher fasting glucose level. Short-term memory domain also exhibited an inverse association. For non-diabetic individuals, a reverse U-shaped relationship was observed between fasting glucose and cognitive function, identifying a threshold for highest cognitive performance of 91.8 CASI score at 3.97-6.20 mmol/L (71.5-111.6 mg/dL) fasting glucose. Language ability domain displayed a similar relationship with fasting glucose.

Conclusions: Elevated fasting glucose levels in diabetic men were associated with lower cognitive function, in which short-term memory was the main associated domain. Interestingly, in non-diabetic men, we identified a threshold for the inverse relationship of elevated fasting glucose with cognitive function. Contrastingly to diabetic men, language ability was the main associated cognitive domain among non-diabetic men.
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http://dx.doi.org/10.2188/jea.JE20180193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7025918PMC
March 2020

Anthropometric Obesity Indices were Stronger than CT-Based Indices in Associations with Carotid Intima-Media Thickness in Japanese Men.

J Atheroscler Thromb 2019 Dec 15;26(12):1102-1114. Epub 2019 May 15.

Department of Public Health, Shiga University of Medical Science.

Aim: Computed tomography (CT) can directly provide information on body compositions and distributions, compared to anthropometric indices. It has been shown that various obesity indices are associated with carotid intima-media thickness (IMT). However, whether CT-based obesity indices are stronger than anthropometric indices in association with atherosclerosis remains to be determined in a general population.

Methods: We cross-sectionally assessed carotid IMT using ultrasound in 944 community-dwelling Japanese men free of stroke and myocardial infarction. CT image at the L4-L5 level was obtained to compute areas of visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT). Anthropometric measures assessed included body mass index (BMI), waist circumference, and waist-to-hip ratio. Using multivariable linear regression, slopes of IMT per 20 to 80 percentile of each index were compared. We also compared the slope of index with simultaneous adjustment for BMI in the same model.

Results: Areas of VAT and SAT were positively associated with IMT, but not stronger than those of anthropometric indices in point estimates. Among all obesity indices, BMI was strongest in association with IMT after adjusting for age and lifestyle factors or further adjusting for metabolic factors. In simultaneous adjustment models, BMI, but not CT-based indices, remained significant and showed the strongest association.

Conclusions: In community-dwelling Japanese men, anthropometric obesity indices, BMI in particular, were more strongly associated with carotid atherosclerosis than CT-based obesity indices. The association of general obesity with carotid atherosclerosis was strong and adding CT-based obesity measure did not considerably influence in the association.
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http://dx.doi.org/10.5551/jat.47977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927809PMC
December 2019

A Novel Antiserum Against a Predicted Human Peripheral Choline Acetyltransferase (hpChAT) for Labeling Neuronal Structures in Human Colon.

Front Neuroanat 2019 16;13:37. Epub 2019 Apr 16.

Molecular Neuroscience Research Center, Shiga University of Medical Science, Otsu, Japan.

Choline acetyltransferase (ChAT), the enzyme synthesizing acetylcholine (ACh), has an exon-skipping splice variant which is expressed preferentially in the peripheral nervous system (PNS) and thus termed peripheral ChAT (pChAT). A rabbit antiserum previously produced against rat pChAT (rpChAT) has been used for immunohistochemistry (IHC) to study peripheral cholinergic structures in various animals. The present study was undertaken to develop a specific antiserum against a predicted human pChAT (hpChAT) protein. A novel mouse antiserum has been successfully raised against a unique 14-amino acid sequence of hpChAT protein. Our Western blot using this antiserum (termed here anti-hpChAT serum) on human colon extracts revealed only a single band of 47 kDa, matching the deduced size of hpChAT protein. By IHC, the antiserum gave intense staining in many neuronal cells and fibers of human colon but not brain, and such a pattern of staining seemed identical with that reported in colon of various animals using anti-rpChAT serum. In the antibody-absorption test, hpChAT-immunoreactive staining in human colon was completely blocked by using the antiserum pre-absorbed with the antigen peptide. Double immunofluorescence in human colon moreover indicated that structures stained with anti-hpChAT were also stained with anti-rpChAT, and vice versa. hpChAT antiserum allowed the identification of cell types, as Dogiel type cells in intramural plexuses, and fiber innervation of colon muscles and mucosae. The present results demonstrate the specificity and reliability of the hpChAT antiserum as a novel tool for immunohistochemical studies in human colon, opening venues to map cholinergic innervation in other human PNS tissues.
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http://dx.doi.org/10.3389/fnana.2019.00037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6476985PMC
April 2019

The juvenility-associated long noncoding RNA maintains cellular juvenescence.

J Cell Sci 2019 04 16;132(8). Epub 2019 Apr 16.

Molecular Neuroscience Research Center (MNRC), Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, Shiga 520-2192, Japan

Juvenile animals possess distinct properties that are missing in adults. These properties include capabilities for higher growth, faster wound healing, plasticity and regeneration. However, the molecular mechanisms underlying these juvenile physiological properties are not fully understood. To obtain insight into the distinctiveness of juveniles from adults at the molecular level, we assessed long noncoding RNAs (lncRNAs) that are highly expressed selectively in juvenile cells. The noncoding elements of the transcriptome were investigated in hepatocytes and cardiomyocytes isolated from juvenile and adult mice. Here, we identified 62 juvenility-associated lncRNAs (JAlncs), which are selectively expressed in both hepatocytes and cardiomyocytes from juvenile mice. Among these common (shared) JAlncs, is evolutionarily conserved and is essential for cellular juvenescence. Loss of impairs cell growth and causes cellular senescence. safeguards cellular juvenescence through recruiting the histone methyltransferase Ezh2 to , thereby repressing the functional role of in cellular senescence. Thus, we identify as a juvenility-selective lncRNA required to maintain cellular juvenescence.
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http://dx.doi.org/10.1242/jcs.227801DOI Listing
April 2019

Microglial Progranulin: Involvement in Alzheimer's Disease and Neurodegenerative Diseases.

Cells 2019 03 11;8(3). Epub 2019 Mar 11.

Molecular Neuroscience Research Center, Shiga University of Medical Science, Otsu 520-2192, Japan.

Neurodegenerative diseases such as Alzheimer's disease have proven resistant to new treatments. The complexity of neurodegenerative disease mechanisms can be highlighted by accumulating evidence for a role for a growth factor, progranulin (PGRN). PGRN is a glycoprotein encoded by the GRN/Grn gene with multiple cellular functions, including neurotrophic, anti-inflammatory and lysosome regulatory properties. Mutations in the GRN gene can lead to frontotemporal lobar degeneration (FTLD), a cause of dementia, and neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. Both diseases are associated with loss of PGRN function resulting, amongst other features, in enhanced microglial neuroinflammation and lysosomal dysfunction. PGRN has also been implicated in Alzheimer's disease (AD). Unlike FTLD, increased expression of PGRN occurs in brains of human AD cases and AD model mice, particularly in activated microglia. How microglial PGRN might be involved in AD and other neurodegenerative diseases will be discussed. A unifying feature of PGRN in diseases might be its modulation of lysosomal function in neurons and microglia. Many experimental models have focused on consequences of PGRN gene deletion: however, possible outcomes of increasing PGRN on microglial inflammation and neurodegeneration will be discussed. We will also suggest directions for future studies on PGRN and microglia in relation to neurodegenerative diseases.
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http://dx.doi.org/10.3390/cells8030230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468562PMC
March 2019

Differences in Gene Expression Profiles and Phenotypes of Differentiated SH-SY5Y Neurons Stably Overexpressing Mitochondrial Ferritin.

Front Mol Neurosci 2018 8;11:470. Epub 2019 Jan 8.

Molecular Neuroscience Research Center, Shiga University of Medical Science, Otsu, Japan.

Mitochondrial ferritin (FtMt) is an iron-transport protein with ferroxidase properties localized to mitochondria. Levels are generally low in all tissues, while increasing the expression of FtMt in neuronal-like cells has been shown to be protective. To determine whether FtMt has potential as a therapeutic approach, there remains the question of how much FtMt is protective. To address this issue, we transfected SH-SY5Y neuroblastoma cells with a FtMt expression plasmid and isolated cell lines with stable expression of FtMt at high, medium and low levels. Using these cell lines, we examined effects of FtMt on neuronal phenotype, neuroprotective activity and gene expression profiles. The phenotypic properties of high, medium and low FtMt expressors were compared with native untransfected SH-SY5Y cells after differentiation with retinoic acid to a neuronal phenotype. Overexpression of FtMt, even in low expressing cells, showed significant protection from oxidative stress induced by hydrogen peroxide or cobalt chloride. Higher levels of FtMt expression did not appear to offer greater protection, and did not have toxic consequences to cells, even though there were significantly more aggregated mitochondria in the highest expressing clone. The phenotypes differed between cell clones when assessed by cell growth, neurite outgrowth, and expression of neuronal proteins including those associated with neurodegenerative diseases. Microarray analysis of high, medium and negative FtMt-expressing cells identified different patterns of expression of certain genes associated with oxidative stress and neuronal development, amongst others. Validation of microarray analyses was carried out by real time polymerase chain reaction. The results showed significant differences in expression of thioredoxin-interacting protein (TXNIP) and microsomal glutathione transfer-1 (MGST-1), which can have critical roles in the regulation of oxidative stress. Differences in expression of calcitonin-related polypeptide alpha (CALCA), growth differentiation factor-15 (GDF-15) and secretogranin II (SCG2) were also observed. Our findings indicate that even low levels of increased FtMt expression can be protective possibly by alterations of some oxidative stress-related and growth factor genes, while high levels of expression did not appear to offer greater protection from oxidative stress or induce significant toxicity in cells. These experiments provide supporting data that increasing FtMt might be a feasible strategy for therapeutics in certain neurodegenerative and neurological diseases.
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http://dx.doi.org/10.3389/fnmol.2018.00470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331485PMC
January 2019