Disruption of a RAC1-centred network is associated with Alzheimer’s disease pathology and causes age-dependent neurodegeneration
Abstract
Abstract The molecular biological mechanisms of Alzheimer’s disease (AD) involve disease-associated crosstalk through many genes and include a loss of normal as well as a gain of abnormal interactions among genes. A protein domain network (PDN) is a collection of physical bindings that occur between protein domains, and the states of the PDNs in patients with AD are likely to be perturbed compared to those in normal healthy individuals. To identify PDN changes that cause neurodegeneration, we analysed the PDNs that occur among genes co-expressed in each of three brain regions at each stage of AD. Our analysis revealed that the PDNs collapsed with the progression of AD stage and identified five hub genes, including Rac1, as key players in PDN collapse. Using publicly available as well as our own gene expression data, we confirmed that the mRNA expression level of the RAC1 gene was downregulated in the entorhinal cortex (EC) of AD brains. To test the causality of these changes in neurodegeneration, we utilized Drosophila as a genetic model and found that modest knockdown of Rac1 in neurons was sufficient to cause age-dependent behavioural deficits and neurodegeneration. Finally, we identified a microRNA, hsa-miR-101-3p, as a potential regulator of RAC1more »
- Authors:
-
- Department of Genome Informatics, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan
- Department of Alzheimer’s Disease Research, Center for Development of Advanced Medicine for Dementia, National Center for Geriatrics and Gerontology, Aichi 474-8511, Japan, Department of Experimental Gerontology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Aichi 467-8603, Japan
- Department of Molecular Genetics, Brain Research Institute, Niigata University, Niigata 951-8585, Japan
- Department of Molecular Genetics, Brain Research Institute, Niigata University, Niigata 951-8585, Japan, Asahigawaso Medical-Welfare Center, Asahigawaso Research Institute, Okayama 703-8207, Japan
- Publication Date:
- Sponsoring Org.:
- USDOE Office of Electricity (OE), Advanced Grid Research & Development. Power Systems Engineering Research
- OSTI Identifier:
- 1607604
- Alternate Identifier(s):
- OSTI ID: 1599912
- Resource Type:
- Published Article
- Journal Name:
- Human Molecular Genetics
- Additional Journal Information:
- Journal Name: Human Molecular Genetics Journal Volume: 29 Journal Issue: 5; Journal ID: ISSN 0964-6906
- Publisher:
- Oxford University Press
- Country of Publication:
- United Kingdom
- Language:
- English
Citation Formats
Kikuchi, Masataka, Sekiya, Michiko, Hara, Norikazu, Miyashita, Akinori, Kuwano, Ryozo, Ikeuchi, Takeshi, Iijima, Koichi M., and Nakaya, Akihiro. Disruption of a RAC1-centred network is associated with Alzheimer’s disease pathology and causes age-dependent neurodegeneration. United Kingdom: N. p., 2020.
Web. https://doi.org/10.1093/hmg/ddz320.
Kikuchi, Masataka, Sekiya, Michiko, Hara, Norikazu, Miyashita, Akinori, Kuwano, Ryozo, Ikeuchi, Takeshi, Iijima, Koichi M., & Nakaya, Akihiro. Disruption of a RAC1-centred network is associated with Alzheimer’s disease pathology and causes age-dependent neurodegeneration. United Kingdom. https://doi.org/10.1093/hmg/ddz320
Kikuchi, Masataka, Sekiya, Michiko, Hara, Norikazu, Miyashita, Akinori, Kuwano, Ryozo, Ikeuchi, Takeshi, Iijima, Koichi M., and Nakaya, Akihiro. Thu .
"Disruption of a RAC1-centred network is associated with Alzheimer’s disease pathology and causes age-dependent neurodegeneration". United Kingdom. https://doi.org/10.1093/hmg/ddz320.
@article{osti_1607604,
title = {Disruption of a RAC1-centred network is associated with Alzheimer’s disease pathology and causes age-dependent neurodegeneration},
author = {Kikuchi, Masataka and Sekiya, Michiko and Hara, Norikazu and Miyashita, Akinori and Kuwano, Ryozo and Ikeuchi, Takeshi and Iijima, Koichi M. and Nakaya, Akihiro},
abstractNote = {Abstract The molecular biological mechanisms of Alzheimer’s disease (AD) involve disease-associated crosstalk through many genes and include a loss of normal as well as a gain of abnormal interactions among genes. A protein domain network (PDN) is a collection of physical bindings that occur between protein domains, and the states of the PDNs in patients with AD are likely to be perturbed compared to those in normal healthy individuals. To identify PDN changes that cause neurodegeneration, we analysed the PDNs that occur among genes co-expressed in each of three brain regions at each stage of AD. Our analysis revealed that the PDNs collapsed with the progression of AD stage and identified five hub genes, including Rac1, as key players in PDN collapse. Using publicly available as well as our own gene expression data, we confirmed that the mRNA expression level of the RAC1 gene was downregulated in the entorhinal cortex (EC) of AD brains. To test the causality of these changes in neurodegeneration, we utilized Drosophila as a genetic model and found that modest knockdown of Rac1 in neurons was sufficient to cause age-dependent behavioural deficits and neurodegeneration. Finally, we identified a microRNA, hsa-miR-101-3p, as a potential regulator of RAC1 in AD brains. As the Braak neurofibrillary tangle (NFT) stage progressed, the expression levels of hsa-miR-101-3p were increased specifically in the EC. Furthermore, overexpression of hsa-miR-101-3p in the human neuronal cell line SH-SY5Y caused RAC1 downregulation. These results highlight the utility of our integrated network approach for identifying causal changes leading to neurodegeneration in AD.},
doi = {10.1093/hmg/ddz320},
journal = {Human Molecular Genetics},
number = 5,
volume = 29,
place = {United Kingdom},
year = {2020},
month = {1}
}
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1093/hmg/ddz320
https://doi.org/10.1093/hmg/ddz320
Other availability
Search WorldCat to find libraries that may hold this journal
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.
Works referenced in this record:
Altered Expression of Diabetes-Related Genes in Alzheimer's Disease Brains: The Hisayama Study
journal, April 2013
- Hokama, Masaaki; Oka, Sugako; Leon, Julio
- Cerebral Cortex, Vol. 24, Issue 9
Neuropathological stageing of Alzheimer-related changes
journal, September 1991
- Braak, H.; Braak, E.
- Acta Neuropathologica, Vol. 82, Issue 4
Markers of microglia in post-mortem brain samples from patients with Alzheimer’s disease: a systematic review
journal, December 2017
- Hopperton, K. E.; Mohammad, D.; Trépanier, M. O.
- Molecular Psychiatry, Vol. 23, Issue 2
Ineffective phagocytosis of amyloid-β by macrophages of Alzheimer's disease patients
journal, June 2005
- Fiala, Milan; Lin, Justin; Ringman, John
- Journal of Alzheimer's Disease, Vol. 7, Issue 3
The Unraveling Truth About IRE1 and MicroRNAs in Diabetes
journal, December 2016
- Belmadani, Souad; Matrougui, Khalid
- Diabetes, Vol. 66, Issue 1
A Rac1/Phosphatidylinositol 3-Kinase/Akt3 Anti-apoptotic Pathway, Triggered by AlsinLF, the Product of the ALS2 Gene, Antagonizes Cu/Zn-superoxide Dismutase (SOD1) Mutant-induced Motoneuronal Cell Death
journal, December 2004
- Kanekura, Kohsuke; Hashimoto, Yuichi; Kita, Yoshiko
- Journal of Biological Chemistry, Vol. 280, Issue 6
Non-coding RNAs in the pathogenesis of COPD
journal, May 2015
- De Smet, Elise G.; Mestdagh, Pieter; Vandesompele, Jo
- Thorax, Vol. 70, Issue 8
Attenuated protein kinase C activity and translocation in Alzheimer's Disease brain
journal, May 1994
- Wang, Hoau-Yan; Pisano, Michael R.; Friedman, Eitan
- Neurobiology of Aging, Vol. 15, Issue 3
Rac1 Protein Rescues Neurite Retraction Caused by G2019S Leucine-rich Repeat Kinase 2 (LRRK2)
journal, March 2011
- Chan, Diane; Citro, Allison; Cordy, Joanna M.
- Journal of Biological Chemistry, Vol. 286, Issue 18
The Rac1 Polybasic Region Is Required for Interaction with Its Effector PRK1
journal, November 2007
- Modha, Rakhee; Campbell, Louise J.; Nietlispach, Daniel
- Journal of Biological Chemistry, Vol. 283, Issue 3
Serum microRNA miR-501-3p as a potential biomarker related to the progression of Alzheimer’s disease
journal, January 2017
- Hara, Norikazu; Kikuchi, Masataka; Miyashita, Akinori
- Acta Neuropathologica Communications, Vol. 5, Issue 1
Integrated Systems Approach Identifies Genetic Nodes and Networks in Late-Onset Alzheimer’s Disease
journal, April 2013
- Zhang, Bin; Gaiteri, Chris; Bodea, Liviu-Gabriel
- Cell, Vol. 153, Issue 3
Conserved brain myelination networks are altered in Alzheimer's and other neurodegenerative diseases
journal, October 2017
- Allen, Mariet; Wang, Xue; Burgess, Jeremy D.
- Alzheimer's & Dementia, Vol. 14, Issue 3
Innate immunity in Alzheimer's disease
journal, February 2015
- Heneka, Michael T.; Golenbock, Douglas T.; Latz, Eicke
- Nature Immunology, Vol. 16, Issue 3
Forgetting Is Regulated through Rac Activity in Drosophila
journal, February 2010
- Shuai, Yichun; Lu, Binyan; Hu, Ying
- Cell, Vol. 140, Issue 4
Divergence of human and mouse brain transcriptome highlights Alzheimer disease pathways
journal, June 2010
- Miller, Jeremy A.; Horvath, Steve; Geschwind, Daniel H.
- Proceedings of the National Academy of Sciences, Vol. 107, Issue 28
Involvement of the γ-Secretase-Mediated EphA4 Signaling Pathway in Synaptic Pathogenesis of Alzheimer's Disease: EphA4 Signaling in Alzheimer's Disease
journal, April 2012
- Matsui, Chiyuki; Inoue, Eiji; Kakita, Akiyoshi
- Brain Pathology, Vol. 22, Issue 6
starBase v2.0: decoding miRNA-ceRNA, miRNA-ncRNA and protein–RNA interaction networks from large-scale CLIP-Seq data
journal, December 2013
- Li, Jun-Hao; Liu, Shun; Zhou, Hui
- Nucleic Acids Research, Vol. 42, Issue D1
Identification of evolutionarily conserved gene networks mediating neurodegenerative dementia
journal, December 2018
- Swarup, Vivek; Hinz, Flora I.; Rexach, Jessica E.
- Nature Medicine, Vol. 25, Issue 1
Age-related alteration of PKC, a key enzyme in memory processes: Physiological and pathological examples
journal, February 1998
- Pascale, A.; Govoni, S.; Battaini, F.
- Molecular Neurobiology, Vol. 16, Issue 1
Integrative network analysis of nineteen brain regions identifies molecular signatures and networks underlying selective regional vulnerability to Alzheimer’s disease
journal, November 2016
- Wang, Minghui; Roussos, Panos; McKenzie, Andrew
- Genome Medicine, Vol. 8, Issue 1
Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer's disease
journal, July 2017
- Sims, Rebecca; van der Lee, Sven J.; Naj, Adam C.
- Nature Genetics, Vol. 49, Issue 9
Tissue specificity and the human protein interaction network
journal, January 2009
- Bossi, Alice; Lehner, Ben
- Molecular Systems Biology, Vol. 5, Issue 1
Regulation of phagocytosis by Rho GTPases
journal, April 2015
- Mao, Yingyu; Finnemann, Silvia C.
- Small GTPases, Vol. 6, Issue 2
MicroRNA-101 Regulates Amyloid Precursor Protein Expression in Hippocampal Neurons
journal, April 2010
- Vilardo, Elisa; Barbato, Christian; Ciotti, MariaTeresa
- Journal of Biological Chemistry, Vol. 285, Issue 24
Structure of the TPR Domain of p67phox in Complex with Rac·GTP
journal, October 2000
- Lapouge, Karine; Smith, Susan J. M.; Walker, Philip A.
- Molecular Cell, Vol. 6, Issue 4
Endosome function and dysfunction in Alzheimer's disease and other neurodegenerative diseases
journal, March 2005
- Nixon, Ralph A.
- Neurobiology of Aging, Vol. 26, Issue 3
Crystal Structure of the Rac1−RhoGDI Complex Involved in NADPH Oxidase Activation † , ‡
journal, August 2001
- Grizot, S.; Fauré, J.; Fieschi, F.
- Biochemistry, Vol. 40, Issue 34
Phosphatidylinositol 3-Kinase, Cdc42, and Rac1 Act Downstream of Ras in Integrin-Dependent Neurite Outgrowth in N1E-115 Neuroblastoma Cells
journal, January 2000
- Sarner, S.; Kozma, R.; Ahmed, S.
- Molecular and Cellular Biology, Vol. 20, Issue 1
Variant of TREM2 Associated with the Risk of Alzheimer's Disease
journal, January 2013
- Jonsson, Thorlakur; Stefansson, Hreinn; Steinberg, Stacy
- New England Journal of Medicine, Vol. 368, Issue 2
Discovery of tissue-specific exons using comprehensive human exon microarrays
journal, January 2007
- Clark, Tyson A.; Schweitzer, Anthony C.; Chen, Tina X.
- Genome Biology, Vol. 8, Issue 4
Incipient Alzheimer's disease: Microarray correlation analyses reveal major transcriptional and tumor suppressor responses
journal, February 2004
- Blalock, Eric M.; Geddes, James W.; Chen, Kuey Chu
- Proceedings of the National Academy of Sciences, Vol. 101, Issue 7
Hippocampal Activation of Rac1 Regulates the Forgetting of Object Recognition Memory
journal, September 2016
- Liu, Yunlong; Du, Shuwen; Lv, Li
- Current Biology, Vol. 26, Issue 17
A central role for the small GTPase Rac1 in hippocampal plasticity and spatial learning and memory
journal, July 2009
- Haditsch, Ursula; Leone, Dino P.; Farinelli, Mélissa
- Molecular and Cellular Neuroscience, Vol. 41, Issue 4
Neuroinflammation in Alzheimer's disease
journal, April 2015
- Heneka, Michael T.; Carson, Monica J.; Khoury, Joseph El
- The Lancet Neurology, Vol. 14, Issue 4
Genome-wide association study identifies variants at CLU and PICALM associated with Alzheimer's disease
journal, September 2009
- Harold, Denise; Abraham, Richard; Hollingworth, Paul
- Nature Genetics, Vol. 41, Issue 10
Biomarkers of inflammation and amyloid-β phagocytosis in patients at risk of Alzheimer disease
journal, January 2010
- Fiala, Milan; Veerhuis, Robert
- Experimental Gerontology, Vol. 45, Issue 1
Immune attack: the role of inflammation in Alzheimer disease
journal, May 2015
- Heppner, Frank L.; Ransohoff, Richard M.; Becher, Burkhard
- Nature Reviews Neuroscience, Vol. 16, Issue 6
RhoA Inhibits the Nerve Growth Factor-induced Rac1 Activation through Rho-associated Kinase-dependent Pathway
journal, March 2001
- Yamaguchi, Yoshiaki; Katoh, Hironori; Yasui, Hidekazu
- Journal of Biological Chemistry, Vol. 276, Issue 22
Decreased levels of protein kinase C in Alzheimer brain
journal, June 1988
- Cole, Gregory; Dobkins, Karen R.; Hansen, Lawrence A.
- Brain Research, Vol. 452, Issue 1-2
Potent degradation of neuronal mi RNA s induced by highly complementary targets
journal, February 2015
- la Mata, Manuel; Gaidatzis, Dimos; Vitanescu, Mirela
- EMBO reports, Vol. 16, Issue 4
Genome-wide meta-analysis identifies new loci and functional pathways influencing Alzheimer’s disease risk
journal, January 2019
- Jansen, Iris E.; Savage, Jeanne E.; Watanabe, Kyoko
- Nature Genetics, Vol. 51, Issue 3
Exploration, normalization, and summaries of high density oligonucleotide array probe level data
journal, April 2003
- Irizarry, Rafael A.; Hobbs, Bridget; Collin, Francois
- Biostatistics, Vol. 4, Issue 2, p. 249-264
The structural basis of Arfaptin-mediated cross-talk between Rac and Arf signalling pathways
journal, May 2001
- Tarricone, C.; Xiao, B.; Justin, N.
- Nature, Vol. 411, Issue 6834
INstruct: a database of high-quality 3D structurally resolved protein interactome networks
journal, May 2013
- Meyer, Michael J.; Das, Jishnu; Wang, Xiujuan
- Bioinformatics, Vol. 29, Issue 12
A Systems Level Analysis of Transcriptional Changes in Alzheimer's Disease and Normal Aging
journal, February 2008
- Miller, J. A.; Oldham, M. C.; Geschwind, D. H.
- Journal of Neuroscience, Vol. 28, Issue 6
C-terminal Src Kinase Gates Homeostatic Synaptic Plasticity and Regulates Fasciclin II Expression at the Drosophila Neuromuscular Junction
journal, February 2016
- Spring, Ashlyn M.; Brusich, Douglas J.; Frank, C. Andrew
- PLOS Genetics, Vol. 12, Issue 2
TREM2 Variants in Alzheimer's Disease
journal, January 2013
- Guerreiro, Rita; Wojtas, Aleksandra; Bras, Jose
- New England Journal of Medicine, Vol. 368, Issue 2
Altered neuronal gene expression in brain regions differentially affected by Alzheimer's disease: a reference data set
journal, April 2008
- Liang, Winnie S.; Dunckley, Travis; Beach, Thomas G.
- Physiological Genomics, Vol. 33, Issue 2
Prognostic serum miRNA biomarkers associated with Alzheimer’s disease shows concordance with neuropsychological and neuroimaging assessment
journal, October 2014
- Cheng, L.; Doecke, J. D.; Sharples, R. A.
- Molecular Psychiatry, Vol. 20, Issue 10
Spatio-temporal transcriptome of the human brain
journal, October 2011
- Kang, Hyo Jung; Kawasawa, Yuka Imamura; Cheng, Feng
- Nature, Vol. 478, Issue 7370
The Small GTPase RAC1/CED-10 Is Essential in Maintaining Dopaminergic Neuron Function and Survival Against α-Synuclein-Induced Toxicity
journal, February 2018
- Kim, Hanna; Calatayud, Carles; Guha, Sanjib
- Molecular Neurobiology, Vol. 55, Issue 9
MicroRNA-101 downregulates Alzheimer’s amyloid-β precursor protein levels in human cell cultures and is differentially expressed
journal, January 2011
- Long, Justin M.; Lahiri, Debomoy K.
- Biochemical and Biophysical Research Communications, Vol. 404, Issue 4
The NHGRI GWAS Catalog, a curated resource of SNP-trait associations
journal, December 2013
- Welter, Danielle; MacArthur, Jacqueline; Morales, Joannella
- Nucleic Acids Research, Vol. 42, Issue D1
Genome-wide association study identifies variants at CLU and CR1 associated with Alzheimer's disease
journal, September 2009
- Lambert, Jean-Charles; Heath, Simon; Even, Gael
- Nature Genetics, Vol. 41, Issue 10
A systematic integrated analysis of brain expression profiles reveals YAP1 and other prioritized hub genes as important upstream regulators in Alzheimer's disease
journal, September 2017
- Xu, Min; Zhang, Deng-Feng; Luo, Rongcan
- Alzheimer's & Dementia, Vol. 14, Issue 2
Labelling and optical erasure of synaptic memory traces in the motor cortex
journal, September 2015
- Hayashi-Takagi, Akiko; Yagishita, Sho; Nakamura, Mayumi
- Nature, Vol. 525, Issue 7569
Dissecting the pathological effects of human A 40 and A 42 in Drosophila: A potential model for Alzheimer's disease
journal, April 2004
- Iijima, K.; Liu, H. -P.; Chiang, A. -S.
- Proceedings of the National Academy of Sciences, Vol. 101, Issue 17
Transcriptional Alterations Related to Neuropathology and Clinical Manifestation of Alzheimer’s Disease
journal, November 2012
- Silva, Aderbal R. T.; Grinberg, Lea T.; Farfel, Jose M.
- PLoS ONE, Vol. 7, Issue 11
A Unique Microglia Type Associated with Restricting Development of Alzheimer’s Disease
journal, June 2017
- Keren-Shaul, Hadas; Spinrad, Amit; Weiner, Assaf
- Cell, Vol. 169, Issue 7
MicroRNA-101 Targets MAPK Phosphatase-1 To Regulate the Activation of MAPKs in Macrophages
journal, November 2010
- Zhu, Qing-Yuan; Liu, Qin; Chen, Jian-Xia
- The Journal of Immunology, Vol. 185, Issue 12
Variations in the transcriptome of Alzheimer's disease reveal molecular networks involved in cardiovascular diseases
journal, January 2008
- Ray, Monika; Ruan, Jianhua; Zhang, Weixiong
- Genome Biology, Vol. 9, Issue 10
Rac1 plays an essential role in axon growth and guidance and in neuronal survival in the central and peripheral nervous systems
journal, September 2015
- Hua, Zhong L.; Emiliani, Francesco E.; Nathans, Jeremy
- Neural Development, Vol. 10, Issue 1
ITIM-dependent endocytosis of CD33-related Siglecs: role of intracellular domain, tyrosine phosphorylation, and the tyrosine phosphatases, Shp1 and Shp2
journal, October 2007
- Walter, Roland B.; Raden, Brian W.; Zeng, Rong
- Journal of Leukocyte Biology, Vol. 83, Issue 1
Meta-analysis of 74,046 individuals identifies 11 new susceptibility loci for Alzheimer's disease
journal, October 2013
- Lambert, Jean-Charles; Ibrahim-Verbaas, Carla A.; Harold, Denise
- Nature Genetics, Vol. 45, Issue 12
Aβ42 Mutants with Different Aggregation Profiles Induce Distinct Pathologies in Drosophila
journal, February 2008
- Iijima, Koichi; Chiang, Hsueh-Cheng; Hearn, Stephen A.
- PLoS ONE, Vol. 3, Issue 2
Synaptic genes are extensively downregulated across multiple brain regions in normal human aging and Alzheimer's disease
journal, June 2013
- Berchtold, Nicole C.; Coleman, Paul D.; Cribbs, David H.
- Neurobiology of Aging, Vol. 34, Issue 6
Structural Basis of Guanine Nucleotide Exchange Mediated by the T-Cell Essential Vav1
journal, July 2008
- Chrencik, Jill E.; Brooun, Alexei; Zhang, Hui
- Journal of Molecular Biology, Vol. 380, Issue 5
Temporal Tracking of Microglia Activation in Neurodegeneration at Single-Cell Resolution
journal, October 2017
- Mathys, Hansruedi; Adaikkan, Chinnakkaruppan; Gao, Fan
- Cell Reports, Vol. 21, Issue 2
Metascape provides a biologist-oriented resource for the analysis of systems-level datasets
journal, April 2019
- Zhou, Yingyao; Zhou, Bin; Pache, Lars
- Nature Communications, Vol. 10, Issue 1
Protein interaction network of alternatively spliced isoforms from brain links genetic risk factors for autism
journal, April 2014
- Corominas, Roser; Yang, Xinping; Lin, Guan Ning
- Nature Communications, Vol. 5, Issue 1
Identification of Unstable Network Modules Reveals Disease Modules Associated with the Progression of Alzheimer’s Disease
journal, November 2013
- Kikuchi, Masataka; Ogishima, Soichi; Miyamoto, Tadashi
- PLoS ONE, Vol. 8, Issue 11
Control of Excitatory Synaptic Transmission by C-terminal Src Kinase
journal, April 2008
- Xu, Jindong; Weerapura, Manjula; Ali, Mohammad K.
- Journal of Biological Chemistry, Vol. 283, Issue 25
The DH and PH Domains of Trio Coordinately Engage Rho GTPases for their Efficient Activation
journal, May 2007
- Chhatriwala, Mariya K.; Betts, Laurie; Worthylake, David K.
- Journal of Molecular Biology, Vol. 368, Issue 5
Implication of the Immune System in Alzheimer's Disease: Evidence from Genome-Wide Pathway Analysis
journal, June 2010
- Lambert, Jean-Charles; Grenier-Boley, Benjamin; Chouraki, Vincent
- Journal of Alzheimer's Disease, Vol. 20, Issue 4