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• W2893664438 abstract "Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that primarily affects motor neurons in the cerebral cortex, brainstem, and spinal cord, leading to progressive paralysis and eventual death. Approximately 95% of all ALS cases are sporadic without known causes. Enteroviruses have been suspected to play a role in ALS because of their ability to target motor neurons and to cause muscle weakness and paralysis. In vitro enteroviral infection results in cytoplasmic aggregation and cleavage of transactive response DNA binding protein-43, a pathologic hallmark of ALS. However, whether enteroviral infection can induce ALS-like pathologies in vivo remains to be characterized. In this study, neonatal BALB/C mice were intracranially inoculated with either a recombinant coxsackievirus B3 expressing enhanced green fluorescent protein or mock-infected for 2, 5, 10, 30, and 90 days. Histologic and immunohistochemical analysis of brain tissues demonstrated sustained inflammation (microglia and astrogliosis) and lesions in multiple regions of the brain (hippocampus, cerebral cortex, striatum, olfactory bulb, and putamen) in parallel with virus detection as early as 2 days for up to 90 days after infection. Most notably, ALS-like pathologies, including cytoplasmic mislocalization of transactive response DNA binding protein-43, p62-, and ubiquitin-positive inclusions, were observed in the areas of infection. These data provide the first pathologic evidence to support a possible link between enteroviral infection and ALS. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that primarily affects motor neurons in the cerebral cortex, brainstem, and spinal cord, leading to progressive paralysis and eventual death. Approximately 95% of all ALS cases are sporadic without known causes. Enteroviruses have been suspected to play a role in ALS because of their ability to target motor neurons and to cause muscle weakness and paralysis. In vitro enteroviral infection results in cytoplasmic aggregation and cleavage of transactive response DNA binding protein-43, a pathologic hallmark of ALS. However, whether enteroviral infection can induce ALS-like pathologies in vivo remains to be characterized. In this study, neonatal BALB/C mice were intracranially inoculated with either a recombinant coxsackievirus B3 expressing enhanced green fluorescent protein or mock-infected for 2, 5, 10, 30, and 90 days. Histologic and immunohistochemical analysis of brain tissues demonstrated sustained inflammation (microglia and astrogliosis) and lesions in multiple regions of the brain (hippocampus, cerebral cortex, striatum, olfactory bulb, and putamen) in parallel with virus detection as early as 2 days for up to 90 days after infection. Most notably, ALS-like pathologies, including cytoplasmic mislocalization of transactive response DNA binding protein-43, p62-, and ubiquitin-positive inclusions, were observed in the areas of infection. These data provide the first pathologic evidence to support a possible link between enteroviral infection and ALS. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that primarily targets motor neurons of the central nervous system (CNS), but it may also result in secondary effects, such as muscle weakness and cognitive impairment.1Brown Jr., R.H. Al-Chalabi A. Amyotrophic lateral sclerosis.N Engl J Med. 2017; 377: 1602Crossref PubMed Scopus (816) Google Scholar, 2Hardiman O. Al-Chalabi A. Chio A. Corr E.M. Logroscino G. Robberecht W. Shaw P.J. Simmons Z. van den Berg L.H. Amyotrophic lateral sclerosis.Nat Rev Dis Primers. 2017; 3: 17085Crossref PubMed Scopus (101) Google Scholar, 3van Es M.A. Hardiman O. Chio A. Al-Chalabi A. Pasterkamp R.J. Veldink J.H. van den Berg L.H. Amyotrophic lateral sclerosis.Lancet. 2017; 390: 2084-2098Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar The prevalence of ALS in both Europe and North America is around three to five cases per 100,000 individuals each year, with the overall lifetime risk increasing with age and being higher in men than in women (1:350 versus 1:400).1Brown Jr., R.H. Al-Chalabi A. Amyotrophic lateral sclerosis.N Engl J Med. 2017; 377: 1602Crossref PubMed Scopus (816) Google Scholar, 3van Es M.A. Hardiman O. Chio A. Al-Chalabi A. Pasterkamp R.J. Veldink J.H. van den Berg L.H. Amyotrophic lateral sclerosis.Lancet. 2017; 390: 2084-2098Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar Currently, there is no cure for ALS. Two treatments approved by the Food and Drug Administration can only modestly delay disease progression and prolong the lifespan of the patients.4Martinez A. Palomo Ruiz M.D. Perez D.I. Gil C. Drugs in clinical development for the treatment of amyotrophic lateral sclerosis.Expert Opin Investig Drugs. 2017; 26: 403-414Crossref PubMed Scopus (17) Google Scholar ALS is typically grouped into two categories: familial and sporadic ALS. Familial ALS is responsible for approximately 5% of all cases and is associated with mutations in >30 genes that encode proteins involved in various cellular functions, including vesicle trafficking, RNA processing, and axonal integrity.5Al-Chalabi A. van den Berg L.H. Veldink J. Gene discovery in amyotrophic lateral sclerosis: implications for clinical management.Nat Rev Neurol. 2017; 13: 96-104Crossref PubMed Scopus (173) Google Scholar, 6Chia R. Chio A. Traynor B.J. Novel genes associated with amyotrophic lateral sclerosis: diagnostic and clinical implications.Lancet Neurol. 2018; 17: 94-102Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar Sporadic ALS, however, accounts for the majority of ALS cases (approximately 95%) without clear cause. A gene–environment interaction model that involves both genetic mutations and environmental risk factors, such as viral exposure, physical activity, smoking, heavy metals, and pesticides and chemicals, has been proposed to contribute to the development and progression of sporadic ALS.7Al-Chalabi A. Calvo A. Chio A. Colville S. Ellis C.M. Hardiman O. Heverin M. Howard R.S. Huisman M.H. Keren N. Leigh P.N. Mazzini L. Mora G. Orrell R.W. Rooney J. Scott K.M. Scotton W.J. Seelen M. Shaw C.E. Sidle K.S. Swingler R. Tsuda M. Veldink J.H. Visser A.E. van den Berg L.H. Pearce N. Analysis of amyotrophic lateral sclerosis as a multistep process: a population-based modelling study.Lancet Neurol. 2014; 13: 1108-1113Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar Two widely studied viruses in the context of ALS are human endogenous retrovirus (HERV) and enterovirus (EV). Increased gene expression of HERV-K and reverse transcriptase activity have been found in the blood and brain samples of ALS patients.8Kury P. Nath A. Creange A. Dolei A. Marche P. Gold J. Giovannoni G. Hartung H.P. Perron H. Human endogenous retroviruses in neurological diseases.Trends Mol Med. 2018; 24: 379-394Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar Moreover, transgenic mouse study reveals that expression of HERV-K in the neurons can lead to motor dysfunction, suggesting a role of HERV infection in ALS.9Li W. Lee M.H. Henderson L. Tyagi R. Bachani M. Steiner J. Campanac E. Hoffman D.A. von Geldern G. Johnson K. Maric D. Morris H.D. Lentz M. Pak K. Mammen A. Ostrow L. Rothstein J. Nath A. Human endogenous retrovirus-K contributes to motor neuron disease.Sci Transl Med. 2015; 7: 307ra153Crossref PubMed Scopus (264) Google Scholar However, despite great efforts for the discovery of EV genome in ALS samples, the clinical data are controversial,10Berger M.M. Kopp N. Vital C. Redl B. Aymard M. Lina B. Detection and cellular localization of enterovirus RNA sequences in spinal cord of patients with ALS.Neurology. 2000; 54: 20-25Crossref PubMed Google Scholar, 11Giraud P. Beaulieux F. Ono S. Shimizu N. Chazot G. Lina B. Detection of enteroviral sequences from frozen spinal cord samples of Japanese ALS patients.Neurology. 2001; 56: 1777-1778Crossref PubMed Scopus (36) Google Scholar, 12Nix W.A. Berger M.M. Oberste M.S. Brooks B.R. McKenna-Yasek D.M. Brown Jr., R.H. Roos R.P. Pallansch M.A. Failure to detect enterovirus in the spinal cord of ALS patients using a sensitive RT-PCR method.Neurology. 2004; 62: 1372-1377Crossref PubMed Scopus (39) Google Scholar, 13Swanson N.R. Fox S.A. Mastaglia F.L. Search for persistent infection with poliovirus or other enteroviruses in amyotrophic lateral sclerosis-motor neurone disease.Neuromuscul Disord. 1995; 5: 457-465Abstract Full Text PDF PubMed Scopus (35) Google Scholar, 14Vandenberghe N. Leveque N. Corcia P. Brunaud-Danel V. Salort-Campana E. Besson G. Tranchant C. Clavelou P. Beaulieux F. Ecochard R. Vial C. Broussolle E. Lina B. Cerebrospinal fluid detection of enterovirus genome in ALS: a study of 242 patients and 354 controls.Amyotroph Lateral Scler. 2010; 11: 277-282Crossref PubMed Scopus (19) Google Scholar, 15Walker M.P. Schlaberg R. Hays A.P. Bowser R. Lipkin W.I. Absence of echovirus sequences in brain and spinal cord of amyotrophic lateral sclerosis patients.Ann Neurol. 2001; 49: 249-253Crossref PubMed Scopus (51) Google Scholar, 16Woodall C.J. Riding M.H. Graham D.I. Clements G.B. Sequences specific for enterovirus detected in spinal cord from patients with motor neurone disease.BMJ. 1994; 308: 1541-1543Crossref PubMed Scopus (81) Google Scholar probably because of the differences in viral detection techniques and/or in the disease stage of sample collection, as well as the potential virus-triggered prion-like mechanism, in which an active viral infection may not be required for ALS progression.17Xue Y.C. Feuer R. Cashman N. Luo H. Enteroviral infection: the forgotten link to amyotrophic lateral sclerosis?.Front Mol Neurosci. 2018; 11: 63Crossref PubMed Scopus (44) Google Scholar EVs are a family of positive, single-stranded RNA viruses, which include poliovirus, coxsackievirus, echovirus, and EV (eg, EV-A71 and EV-D68).17Xue Y.C. Feuer R. Cashman N. Luo H. Enteroviral infection: the forgotten link to amyotrophic lateral sclerosis?.Front Mol Neurosci. 2018; 11: 63Crossref PubMed Scopus (44) Google Scholar EVs are known to infect the CNS and are responsible for various neurologic diseases, such as poliomyelitis, meningitis, encephalitis, and non-polio flaccid paralysis.18Huang H.I. Shih S.R. Neurotropic enterovirus infections in the central nervous system.Viruses. 2015; 7: 6051-6066Crossref PubMed Scopus (74) Google Scholar, 19Rhoades R.E. Tabor-Godwin J.M. Tsueng G. Feuer R. Enterovirus infections of the central nervous system.Virology. 2011; 411: 288-305Crossref PubMed Scopus (139) Google Scholar In addition to acute infection, EVs can also cause a persistent infection within the CNS, which may be reactivated years later.20Feuer R. Ruller C.M. An N. Tabor-Godwin J.M. Rhoades R.E. Maciejewski S. Pagarigan R.R. Cornell C.T. Crocker S.J. Kiosses W.B. Pham-Mitchell N. Campbell I.L. Whitton J.L. Viral persistence and chronic immunopathology in the adult central nervous system following Coxsackievirus infection during the neonatal period.J Virol. 2009; 83: 9356-9369Crossref PubMed Scopus (72) Google Scholar, 21Han J. Ma X.J. Wan J.F. Liu Y.H. Han Y.L. Chen C. Tian C. Gao C. Wang M. Dong X.P. Long persistence of EV71 specific nucleotides in respiratory and feces samples of the patients with hand-foot-mouth disease after recovery.BMC Infect Dis. 2010; 10: 178Crossref PubMed Scopus (55) Google Scholar, 22Julien J. Leparc-Goffart I. Lina B. Fuchs F. Foray S. Janatova I. Aymard M. Kopecka H. Postpolio syndrome: poliovirus persistence is involved in the pathogenesis.J Neurol. 1999; 246: 472-476Crossref PubMed Scopus (58) Google Scholar Although a causal relationship between chronic EV infection and ALS development remains to be established, our previous research in cultured cells demonstrates that EV infection induces ALS-like transactive response DNA-binding protein-43 (TDP-43) pathology, that is, cytoplasmic mislocalization, aggregation, and cleavage of TDP-43,23Fung G. Shi J. Deng H. Hou J. Wang C. Hong A. Zhang J. Jia W. Luo H. Cytoplasmic translocation, aggregation, and cleavage of TDP-43 by enteroviral proteases modulate viral pathogenesis.Cell Death Differ. 2015; 22: 2087-2097Crossref PubMed Scopus (37) Google Scholar indicating a possible involvement of EVs in ALS pathogenesis. Here, we investigated the neuropathologic phenotype of a persistent CNS infection of coxsackievirus B3 (CVB3) in mice. Our results reveal that EV infection induces a similar ALS-like pathology in vivo. Recombinant CVB3 expressing enhanced green fluorescent protein (eGFP-CVB3) was generated as described previously.24Feuer R. Mena I. Pagarigan R. Slifka M.K. Whitton J.L. Cell cycle status affects coxsackievirus replication, persistence, and reactivation in vitro.J Virol. 2002; 76: 4430-4440Crossref PubMed Scopus (148) Google Scholar In brief, eGFP (Clonetech, Palo Alto, CA) was inserted into pMKS1, a plasmid that includes a unique SfiI restriction enzyme site in the backbone of CVB3 clone pH3. The new pMKS1 was transfected into COS cells for 48 hours, and the cell culture was subjected to three cycles of freeze-thaw on dry ice to release viruses. After centrifugation, the supernatant was harvested and added to HeLa RW cell culture. After 48 hours, eGFP-CVB3 virus was collected and plaque purified. BALB/c mice were obtained from Harlan Sprague Dawley (Harlan Laboratories, San Diego, CA). The breeding pairs were monitored daily to identify pups within 24 hours of birth. The neonates at 2 to 3 days old were intracranially inoculated with either a nonlethal dose of eGFP-CVB3 (1 × 106 plague-forming units) or equal volume (25 μL) of Dulbecco's modified Eagle's medium (mock infection) as previously reported.20Feuer R. Ruller C.M. An N. Tabor-Godwin J.M. Rhoades R.E. Maciejewski S. Pagarigan R.R. Cornell C.T. Crocker S.J. Kiosses W.B. Pham-Mitchell N. Campbell I.L. Whitton J.L. Viral persistence and chronic immunopathology in the adult central nervous system following Coxsackievirus infection during the neonatal period.J Virol. 2009; 83: 9356-9369Crossref PubMed Scopus (72) Google Scholar All mice survived except for a few who died from the procedure of intracranial injection, independent of viral infection. All animal procedures were approved by the San Diego State University Institutional Animal Care and Use Committee and are in strict accordance with the Public Health Service Policy and United States Department of Agriculture Animal Welfare Regulations. At 2, 5, 10, 30, and 90 days after infection, mice were euthanized in a bell jar with isoflurane, followed by immediate decapitation. Mouse brains were harvested and fixed with 4% formaldehyde. Paraffin-embedded sections (4-μm thick) were deparaffinized first through xylene and then with a gradually decreasing concentration of isopropanol (100%, 90%, and 70%). Hematoxylin and eosin staining was conducted to evaluate virus-induced brain damage. The sections were also subjected to immunohistochemical (IHC) staining. Briefly, antigen retrieval was performed by heating the sections in citrate buffer pH 6.0 (005000; Life Technologies Carlsbad, CA) for 25 minutes at 121°C. Slides then underwent peroxidase blocking by using hydrogen peroxide (30 mg/mL), followed by washes with 1 × tris-buffered saline (0.05 mol/L Tris, 0.155 mol/L NaCl, pH 7.6). After blocking, sections were incubated with primary antibodies overnight at 4°C. The MACH4 Universal HRP-Polymer Detection System (BRI4012H; Biocare Medical, Pacheco CA) was then used to detect the staining according to the manufacture's procedure. At the end of the procedure, all slides were also counterstained with hematoxylin solution Gill II (GHS232; Sigma-Aldrich, St. Louis, MO). Mouse brain tissues were immunostained by using the follow primary antibodies diluted in tris-buffered saline/phosphate-buffered saline (1% bovine serum albumin, 1.5 mol/L; NaCl, 0.5 mol/L; pH 7.6): double-stranded RNA (dilution 1:1000; J2 mouse IgG2a monoclonal antibody; 10010200; Scicon, Szirák, Hungary), GFP (dilution 1:200; mouse monoclonal antibody; sc-9996; Santa Cruz Biotechnology, Santa Cruz, Dallas, TX), glial fibrillary acidic protein (GFAP; dilution 1:200; mouse monoclonal antibody; SMC-441; StressMarq, Victoria, BC, Canada), ionized calcium binding adaptor molecule 1 (Iba1; dilution 1:200; mouse monoclonal antibody; sc-32725; Santa Cruz Biotechnology), pSTAT3 (dilution 1:200; mouse monoclonal antibody; sc-8059; Santa Cruz Biotechnology), choline acetyltransferase (ChAT; dilution 1:500; mouse monoclonal antibody; sc-55557; Santa Cruz Biotechnology), RZ3 (pThr-231 Tau; dilution 1:300; mouse monoclonal antibody; courtesy of Dr. Peter Davies at the Feinstein Institute for Medical Research, New York, NY), TDP-43 (dilution 1:1000; rabbit polyclonal antibody; 10782-2-AP; Proteintech, Rosemont, IL), p62/sequestosome-1 (SQSTM1; dilution 1:200; mouse monoclonal antibody; sc-28359; Santa Cruz Biotechnology), and ubiquitin (dilution 1:2000; mouse monoclonal antibody; sc-8017; Santa Cruz Biotechnology). Images were taken by using the Aperio ScanScope AT (Digital slide scanner; Leica Biosystems Inc., Buffalo Grove, IL) or a Nikon Eclipse E600 microscope equipped with a SPOT Flex Model 15.2 64 Mp Shifting Pixel camera (Diagnostic Instrument Inc., Sterling Heights, MI). Quantification of immunohistochemistry images was performed by using ImageJ version 1.0 (NIH, Bethesda, MD) with the combination of Color Deconvolution Plugin version 1.5 (http://www.mecourse.com/landinig/software/cdeconv/cdeconv.html) to generate the optical density value based on the intensity of the staining as described.25Ruifrok A.C. Johnston D.A. Quantification of histochemical staining by color deconvolution.Anal Quant Cytol Histol. 2001; 23: 291-299PubMed Google Scholar The quantified results are presented as means ± SD for two separate brain regions (hippocampus and cerebral cortex) at the indicated time points after infection. Statistical analysis was conducted by one-way analysis of variance (ANOVA), followed by Bonferroni's multiple comparison test by using GraphPad Prism software version 6.0c (GraphPad Software, La Jolla, CA). P value of 0.05 was considered to be statistically significant. To determine the in vivo impacts of EV infection on the neuropathology, the neonates of BALB/c mice, a EV-susceptible strain, were intracranially inoculated with a nonlethal dose of eGFP-CVB3 or mock-infected with Dulbecco's modified Eagle's medium for 2, 5, 10, 30, or 90 days. Mouse brains were harvested for viral detection by IHC staining by using antibodies against GFP and double-stranded RNA, an intermediate form of the replicating viral genomic RNA. IHC staining of GFP (virally expressed protein) demonstrated focal infections located mainly in the hippocampus region, with the most active infection occurring between day 2 and day 5 post-infection (PI) (Figure 1). The GFP levels gradually decreased but remained detectable up to day 90 PI. The IHC signal for double-stranded RNA peaked at day 5 PI and then markedly reduced with only faint detection on days 30 and 90 PI. These results were consistent with the previous reports that viral RNA that peaked at day 2 to 5 PI can still be detected by real-time RT-PCR up to day 90 PI,26Ruller C.M. Tabor-Godwin J.M. Van Deren Jr., D.A. Robinson S.M. Maciejewski S. Gluhm S. Gilbert P.E. An N. Gude N.A. Sussman M.A. Whitton J.L. Feuer R. Neural stem cell depletion and CNS developmental defects after enteroviral infection.Am J Pathol. 2012; 180: 1107-1120Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar suggesting a persistent viral infection within the brain. In addition to the hippocampus, viral infections in other areas of the brain, that is, cerebral cortex (especially the neocortex) (Figure 1), olfactory bulb, striatum, and putamen (data not shown), were also observed. Corresponding to the peak infection time and areas, histologic examination by hematoxylin and eosin staining revealed massive immune cell infiltrations and substantial tissue damages between day 2 and day 10 PI within the regions of viral infection (Figure 2). Tissue vacuolization was observed at the later times (Figure 2). These results supported previous findings that CVB3 has a high tendency to infect actively replicating cells that usually reside within the neurogenic regions, such as the hippocampus, olfactory bulb, and lateral ventral regions of the CNS to establish persistent viral infections.27Feuer R. Mena I. Pagarigan R.R. Harkins S. Hassett D.E. Whitton J.L. Coxsackievirus B3 and the neonatal CNS: the roles of stem cells, developing neurons, and apoptosis in infection, viral dissemination, and disease.Am J Pathol. 2003; 163: 1379-1393Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 28Feuer R. Pagarigan R.R. Harkins S. Liu F. Hunziker I.P. Whitton J.L. Coxsackievirus targets proliferating neuronal progenitor cells in the neonatal CNS.J Neurosci. 2005; 25: 2434-2444Crossref PubMed Scopus (68) Google Scholar, 29Tsueng G. Tabor-Godwin J.M. Gopal A. Ruller C.M. Deline S. An N. Frausto R.F. Milner R. Crocker S.J. Whitton J.L. Feuer R. Coxsackievirus preferentially replicates and induces cytopathic effects in undifferentiated neural progenitor cells.J Virol. 2011; 85: 5718-5732Crossref PubMed Scopus (30) Google ScholarFigure 2Intracranial coxsackievirus B3 infection results in brain lesions. Hematoxylin and eosin staining was conducted on the collected mouse brain tissues. The left column of the whole tissue images illustrate the distribution of viral infection, and the middle and right columns show the corresponding magnified images of the infected regions. Within the area of viral infection, immune cell infiltration and tissue lesions can be observed. n = 3 individual mouse brain tissues at each time point post-infection (PI). Scale bars: 2 mm (left column, mock and Day 2 PI); 3 mm (left column, Day 5 PI, Day 10 PI, Day 30 PI, Day 90 PI); 60 μm (middle and right columns). CC, cerebral cortex; HIP, hippocampus.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because neuroinflammation plays an important role in ALS pathogenesis,30Philips T. Robberecht W. Neuroinflammation in amyotrophic lateral sclerosis: role of glial activation in motor neuron disease.Lancet Neurol. 2011; 10: 253-263Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar the effects of EV exposure on the pattern and extent of brain inflammation were studied. IHC staining was conducted to assess the level of astrocyte reactivity with anti-GFAP and anti-pSTAT3 antibodies, microglial activation using anti–Iba1 antibody, and natural killer (NK) cell maturation and neurogenesis with anti-ChAT antibody. Compared with mock infection, CVB3-infected mice at various time points after infection demonstrated increased immune responses. In the hippocampus region, IHC staining of GFAP, a reactive astrocyte marker,31Eng L.F. Ghirnikar R.S. GFAP and astrogliosis.Brain Pathol. 1994; 4: 229-237Crossref PubMed Scopus (711) Google Scholar displayed significant increases in expression intensity at day 5 to 10 and day 90 PI (Figure 3, A and B ). Likewise, another reactive astrocyte marker, pSTAT3,32Liddelow S.A. Barres B.A. Reactive astrocytes: production, function, and therapeutic potential.Immunity. 2017; 46: 957-967Abstract Full Text Full Text PDF PubMed Scopus (1051) Google Scholar also exhibited enhanced CNS astrocyte activation, in particular at day 5 PI. As a marker of microglia,33Ahmed Z. Shaw G. Sharma V.P. Yang C. McGowan E. Dickson D.W. Actin-binding proteins coronin-1a and IBA-1 are effective microglial markers for immunohistochemistry.J Histochem Cytochem. 2007; 55: 687-700Crossref PubMed Scopus (184) Google Scholar Iba1 staining revealed a continuous immune response (microgliosis) toward the viral infection with a peak expression at day 5 PI. IHC staining for ChAT, a marker for NK cell maturation and neurogenesis,34Paez-Gonzalez P. Asrican B. Rodriguez E. Kuo C.T. Identification of distinct ChAT(+) neurons and activity-dependent control of postnatal SVZ neurogenesis.Nat Neurosci. 2014; 17: 934-942Crossref PubMed Scopus (98) Google Scholar demonstrated a strong reaction at the early phase of viral infection, and the immunoreactivity remained detectable until the end of the experiment. Similarly, significantly increased GFAP staining was observed at day 5 and day 90 PI in the cerebral cortex region (Figure 3C). Together, these data indicated that CVB3 induced microgliosis, astrogliosis, and activation of NK cells in persistently infected mouse brains. Familial ALS and sporadic ALS share the pathologic feature of TDP-43, which has been identified in >95% of all ALS cases.35Mackenzie I.R. Bigio E.H. Ince P.G. Geser F. Neumann M. Cairns N.J. Kwong L.K. Forman M.S. Ravits J. Stewart H. Eisen A. McClusky L. Kretzschmar H.A. Monoranu C.M. Highley J.R. Kirby J. Siddique T. Shaw P.J. Lee V.M. Trojanowski J.Q. Pathological TDP-43 distinguishes sporadic amyotrophic lateral sclerosis from amyotrophic lateral sclerosis with SOD1 mutations.Ann Neurol. 2007; 61: 427-434Crossref PubMed Scopus (722) Google Scholar, 36Neumann M. Sampathu D.M. Kwong L.K. Truax A.C. Micsenyi M.C. Chou T.T. Bruce J. Schuck T. Grossman M. Clark C.M. McCluskey L.F. Miller B.L. Masliah E. Mackenzie I.R. Feldman H. Feiden W. Kretzschmar H.A. Trojanowski J.Q. Lee V.M. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.Science. 2006; 314: 130-133Crossref PubMed Scopus (4471) Google Scholar Mice with chronic CVB3 infection exhibited signs of flaccid tail tone and hind limb dysfunction.20Feuer R. Ruller C.M. An N. Tabor-Godwin J.M. Rhoades R.E. Maciejewski S. Pagarigan R.R. Cornell C.T. Crocker S.J. Kiosses W.B. Pham-Mitchell N. Campbell I.L. Whitton J.L. Viral persistence and chronic immunopathology in the adult central nervous system following Coxsackievirus infection during the neonatal period.J Virol. 2009; 83: 9356-9369Crossref PubMed Scopus (72) Google Scholar CVB3 infection resulted in cytoplasmic mislocalization and aggregation of TDP-43 in virus-infected HeLa cells23Fung G. Shi J. Deng H. Hou J. Wang C. Hong A. Zhang J. Jia W. Luo H. Cytoplasmic translocation, aggregation, and cleavage of TDP-43 by enteroviral proteases modulate viral pathogenesis.Cell Death Differ. 2015; 22: 2087-2097Crossref PubMed Scopus (37) Google Scholar and primarily isolated mouse neuronal cells (Y.C.X. et al., unpublished data). Here, we extended our previous work to examine the distribution and expression pattern of TDP-43 in CVB3-infected mouse brains.23Fung G. Shi J. Deng H. Hou J. Wang C. Hong A. Zhang J. Jia W. Luo H. Cytoplasmic translocation, aggregation, and cleavage of TDP-43 by enteroviral proteases modulate viral pathogenesis.Cell Death Differ. 2015; 22: 2087-2097Crossref PubMed Scopus (37) Google Scholar To better correlate virus-infected areas with TDP-43 alteration, two serial slides from mock and CVB3-infected mouse brains were stained for GFP and TDP-43, respectively. TDP-43 was predominantly localized in the nucleus in mock-infected neuronal cells, whereas in day 5 CVB3-infected brain within the hippocampus region, TDP-43 was mostly mislocalized to the cytoplasm, along with nuclear down-regulation (Figure 4A). Notably, at days 30 and 90 PI, TDP-43 pathology remained apparent in the survival cells within the infected areas (Figure 4B), suggesting a similar pathologic phenotype to ALS. To further characterize the cytoplasmic inclusions, brain tissues were stained for p62/SQSTM1 (a ubiquitin-binding protein), ubiquitin, and phosphorylated Tau (a microtubule-associated protein). Both p62 and ubiquitin were associated with protein aggregates detected in ALS.37Blokhuis A.M. Groen E.J. Koppers M. van den Berg L.H. Pasterkamp R.J. Protein aggregation in amyotrophic lateral sclerosis.Acta Neuropathol. 2013; 125: 777-794Crossref PubMed Scopus (355) Google Scholar Within the similar infected regions observed in TDP-43 pathology, p62/SQSTM1 and ubiquitin were stained positive at all infection time points, with the exception of the mock and the faintly positive day 2 PI samples (Figure 5). However, the immunoreactivity of phosphorylated Tau, which has been observed in ALS with cognitive impairment,38Yang W. Strong M.J. Widespread neuronal and glial hyperphosphorylated tau deposition in ALS with cognitive impairment.Amyotroph Lateral Scler. 2012; 13: 178-193Crossref PubMed Scopus (42) Google Scholar was not evident within the infected region until day 90 PI (Figure 5). To properly associate EV infection with the pathology of ALS in infected mouse brains, the areas of viral infection were confirmed, and ALS-associated pathologic phenotypes within the identified regions of infection were analyzed. CVB3 infections were focally localized in multiple areas of the brain, including the hippocampus and cerebral cortex, and caused massive tissue damages, that is, structural fragmentation and vacuolization at early and late infections, respectively. This pattern of infection is likely because of the susceptibility of migrating neuronal progenitor cells to CVB3 infection and/or the distribution of viral receptors (ie, coxsackievirus and adenovirus receptor, and its co-receptor decay-accelerating factor).20Feuer R. Ruller C.M. An N. Tabor-Godwin J.M. Rhoades R.E. Maciejewski S. Pagarigan R.R. Cornell C.T. Crocker S.J. Kiosses W.B. Pham-Mitchell N. Campbell I.L. Whitton J.L. Viral persistence and chronic immunopathology in the adult central nervous system following Coxsackievirus infection during the neonatal period.J Virol. 2009; 83: 9356-9369Crossref PubMed Scopus (72) Google Scholar, 27Feuer R. Mena I. Pagarigan R.R. Harkins S. Hassett D.E. Whitton J.L. Coxsackievirus B3 and the neonatal CNS: the roles of stem cells, developing neurons, and apoptosis in infection, viral dissemination, and disease.Am J Pathol. 2003; 163: 1379-1393Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 28Feuer R. Pagarigan R.R. Harkins S. Liu F. Hunziker I.P. Whitton J.L. Coxsackievirus targets proliferating neuronal progenitor cells in the neonatal CNS.J Neurosci. 2005; 25: 2434-2444Crossref PubMed Scopus (68) Google Scholar Virus-mediated damages of the hippocampus could lead to possible behavioral changes, such as poor long-term memory and spatial navigation, whereas impairments to the cerebral cortex, especially the neocortex (the newest evolutionary layer of cerebral cortex), might affect cognition, sensory perception, and motor function.39Lodato S. Arlotta P. Generating neuronal diversity in the mammalian cerebral cortex.Annu Rev Cell Dev Biol. 2015; 31: 699-720Crossref PubMed Scopus (194) Google Scholar Overall, the various possible consequences of EV infection appear to correlate with the symptoms associated with several neurodegenerative diseases, such as ALS and Alzheimer disease. Even though motor dysfunction was seen during the experimentation, more precise and robust behaviors and physiological tests, however, will be required to confidently link EV infection with clinical signs of neurodegenerative diseases. Within the infected regions, levels of GFAP and pSTAT3, which mark the activation of reactive astrocytes, were markedly increased with a peak at 5 to 10 days and remained high until 90 days PI. This result indicates an elevated reactive astrocyte response. In addition, an increase in infiltration/activation of microglia/macrophage within the infected regions, in particular the hippocampus region at early time points PI, was also observed through Iba1 immunostaining. Collectively, these data suggest a peak immune response toward an acute virus infection in the brain, and then a sustained low level of microglia/macrophage response to a chronic infection that is likely led by autoreactive T cells.40Sin J. Mangale V. Thienphrapa W. Gottlieb R.A. Feuer R. Recent progress in understanding coxsackievirus replication, dissemination, and pathogenesis.Virology. 2015; 484: 288-304Crossref PubMed Scopus (67) Google Scholar Of more interest, elevated stimulations were also observed in both NK cell maturation and neurogenesis at day 5 and day 90 PI, specifically in the hippocampus region, by ChAT staining. Activation of microglia/macrophages in this context could stimulate circulating NK cells and excite the release of cytokines and chemokines, such as IL-15 and IL-12, which are coincidentally elevated in both the serum and cerebral spinal fluid from EV-infected patients and ALS patients.19Rhoades R.E. Tabor-Godwin J.M. Tsueng G. Feuer R. Enterovirus infections of the central nervous system.Virology. 2011; 411: 288-305Crossref PubMed Scopus (139) Google Scholar, 41Rentzos M. Rombos A. Nikolaou C. Zoga M. Zouvelou V. Dimitrakopoulos A. Alexakis T. Tsoutsou A. Samakovli A. Michalopoulou M. Evdokimidis I. Interleukin-15 and interleukin-12 are elevated in serum and cerebrospinal fluid of patients with amyotrophic lateral sclerosis.Eur Neurol. 2010; 63: 285-290Crossref PubMed Scopus (32) Google Scholar Building on previous findings that active immune systems are likely the promoters for ALS-like neurodegeneration,1Brown Jr., R.H. Al-Chalabi A. Amyotrophic lateral sclerosis.N Engl J Med. 2017; 377: 1602Crossref PubMed Scopus (816) Google Scholar, 2Hardiman O. Al-Chalabi A. Chio A. Corr E.M. Logroscino G. Robberecht W. Shaw P.J. Simmons Z. van den Berg L.H. Amyotrophic lateral sclerosis.Nat Rev Dis Primers. 2017; 3: 17085Crossref PubMed Scopus (101) Google Scholar, 3van Es M.A. Hardiman O. Chio A. Al-Chalabi A. Pasterkamp R.J. Veldink J.H. van den Berg L.H. Amyotrophic lateral sclerosis.Lancet. 2017; 390: 2084-2098Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar we speculate that in addition to virus-induced direct damages to the CNS, injury secondary to host immune responses may drive the development of the disease even further. Intracellular protein inclusions are a pathologic hallmark of ALS neurodegeneration. TDP-43 inclusion is particularly important because it is identified in most ALS cases, contributing significantly to the pathogenesis of ALS.42Ling S.C. Polymenidou M. Cleveland D.W. Converging mechanisms in ALS and FTD: disrupted RNA and protein homeostasis.Neuron. 2013; 79: 416-438Abstract Full Text Full Text PDF PubMed Scopus (1046) Google Scholar Within EV-infected regions, typical TDP-43 pathology in the form of nuclear-to-cytoplasmic mislocalization could be seen. The degree/frequency of TDP-43 mislocalization appeared to be correlated well with the severity of the infection, in which day 5 PI, the peak of viral infection, has a larger area of TDP-43 abnormalities than later time points. This is likely because virus-infected cells die as a result of productive viral replication after the acute viremia period. However, the remaining survival cells in the infected areas retained abnormal TDP-43 phenotype. In addition to TDP-43, EV-infected regions showed strong positive staining for p62/SQSTM1 and ubiquitin, two proteins also known as major components of ALS-related inclusions.37Blokhuis A.M. Groen E.J. Koppers M. van den Berg L.H. Pasterkamp R.J. Protein aggregation in amyotrophic lateral sclerosis.Acta Neuropathol. 2013; 125: 777-794Crossref PubMed Scopus (355) Google Scholar Of interest, phosphorylation of tau protein was observed only at day 90 PI. Phosphorylated tau is not generally associated with ALS except for individuals with impaired cognition.38Yang W. Strong M.J. Widespread neuronal and glial hyperphosphorylated tau deposition in ALS with cognitive impairment.Amyotroph Lateral Scler. 2012; 13: 178-193Crossref PubMed Scopus (42) Google Scholar The mechanisms by which EV infection induces ALS-like proteinopathies are unclear. Expression of CVB3-encoded proteinase 2A causes cytoplasmic redistribution of TDP-43,23Fung G. Shi J. Deng H. Hou J. Wang C. Hong A. Zhang J. Jia W. Luo H. Cytoplasmic translocation, aggregation, and cleavage of TDP-43 by enteroviral proteases modulate viral pathogenesis.Cell Death Differ. 2015; 22: 2087-2097Crossref PubMed Scopus (37) Google Scholar suggesting a mechanism of EV-induced TDP-43 pathology through the disruption of nucleocytoplasmic trafficking by viral proteinase cleavage of nucleoporin proteins.43Gustin K.E. Inhibition of nucleo-cytoplasmic trafficking by RNA viruses: targeting the nuclear pore complex.Virus Res. 2003; 95: 35-44Crossref PubMed Scopus (71) Google Scholar Moreover, recent findings that EVs, including CVB3, poliovirus, and EV-D68, inhibit autophagic flux by preventing the formation of the soluble N-ethylmaleimide-sensitive factor activating protein receptor complex indicate that enhanced accumulation of p62 and ubiquitin may be a consequence of defective autophagy.44Corona A.K. Saulsbery H.M. Corona Velazquez A.F. Jackson W.T. Enteroviruses remodel autophagic trafficking through regulation of host SNARE proteins to promote virus replication and cell exit.Cell Rep. 2018; 22: 3304-3314Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 45Mohamud Y. Shi J. Qu J. Poon T. Xue Y.C. Deng H. Zhang J. Luo H. Enteroviral infection inhibits autophagic flux via disruption of the SNARE complex to enhance viral replication.Cell Rep. 2018; 22: 3292-3303Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar The present study demonstrates that CVB3 infection induces characteristics of TDP-43 pathology in virus-infected neural cells, accompanied by the presence of CNS lesions, microgliosis, and astrogliosis in persistently infected mouse brains. Even though human studies had failed to establish a conclusive relationship between ALS and EV infection,17Xue Y.C. Feuer R. Cashman N. Luo H. Enteroviral infection: the forgotten link to amyotrophic lateral sclerosis?.Front Mol Neurosci. 2018; 11: 63Crossref PubMed Scopus (44) Google Scholar animal models, unlike human models in which many external and internal factors cannot be controlled, will confirm the likelihood of this connection in a more controlled manner. Therefore, although animal studies like the one presented here may not lead to a solid conclusion, they can prompt more robust and specific clinical studies in the causational relationship between ALS patients and EV infection in which a more decisive result could be discovered." @default.
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• W2893664438 title "Enteroviral Infection Leads to Transactive Response DNA-Binding Protein 43 Pathology in Vivo" @default.
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